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juniperpublishers-crdoj · 2 years ago

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Omega-3 Polyunsaturated Fatty Acids, Metabolic Syndrome and Diabetes Mellitus

Authored by Victoria Serhiyenko

Abstract

Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) are increasingly being used to prevent cardiovascular diseases (CVD), and cardiac societies recommend the intake of 1g/day of the two ω-3 PUFAs eicosapentaenoic and docosahexaenoic acid for primary and secondary prevention of CVD. Clinical trials clearly suggest beneficial effects of ω-PUFAs consumption on lipid metabolism profile, their anti-inflammatory actions; on endothelial activation, which are likely to improve vascular function; antithrombotic and antiatherosclerotic properties. Experimental studies demonstrate direct antiarrhythmic effects, which have been challenging to document in humans. By targeting arterial stiffness and endothelial dysfunction administration of ω-3 PUFAs may prevent atherosclerosis and CVD development. A synergistic interplay showed by ω-3 PUFAs prescription suggest the potential to beneficially impact on fundamental steps involved in the development of preclinical atherosclerosis. We reviewed available evidence of the benefits of ω-PUFAs administration, especially to patients with CVD, metabolic syndrome and type 2 diabetes mellitus, including their effects on potential molecular pathways, effects on glucose and lipids metabolism parameters, thrombocyte aggregation parameters and haemostasis, endothelial function, antioxidant/anti-inflammation and antiarrhythmic properties.

Keywords: Omega-3 polyunsaturated fatty acids; Coronary heart disease, atherosclerosis; Diabetes mellitus; Glucose, lipids; Inflammation; Platelets; Haemostasis; Endothelium; Heart rate variability; Arrhythmias; Arterial stiffness

Abbrevations: ω-3 and ω-6 PUFAs: Ω-3 and ω-6 Polyunsaturated Fatty Acids; MetS: Metabolic Syndrome; T2DM: Type 2 Diabetes Mellitus; CVD: Cardiovascular Diseases; DLP: Dyslipoproteinemia; OS: Oxidative Stress

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Introduction

Numerous studies report salutary effects of ω-3 polyunsaturated fatty acids (ω-PUFAs), i.e. eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) on cardiovascular diseases (CVD) risk factors. These effects include lowering of serum triglyceride (TG) by reducing of hepatic TG production; lowering of blood pressure (BP) by improving of endothelial cell functution; decreasing of platelet aggregation by reducing of prothrombotic prostanoids; decreasing inflammation via reduction in 4-series leukotrienes (LT) production; protection from arrhythmias by modulation of electrophysiological properties of cardiac myocytes. Systematic meta analysis suggests that high doses of ω-3 PUFAs (~3g/day) produce a small, but significant decrease in systolic blood pressure (SBP) in older and hypertensive subjects [1,2]. The aim of this study was to review the latest evidence about the ω-PUFAs, metabolic syndrome (MetS) and type 2 diabetes mellitus (T2DM).

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Discussion

Ω-3 and ω-6 PUFAs are essential fatty acids, as they cannot be synthesized de novo in humans. There are limited data available regarding the exact amount of dietary ω-3 PUFAs consumed by the general population. It is reported that the total daily intake of dietary ω-3 PUFAs in the US is approximately 1.6g. Of this α-linolenic acid (α-LLA) accounts for approximately 1.4g/q.d, and only 0.1–0.2g/q.d. comes from EPA and DHA. The conversion rate from α-LLA to EPA and DHA is variable (0.2-15%). Therefore, in general, the total amount of EPA and DHA available to the body from current dietary patterns is well below the recommended amounts. EPA and DHA didn’t show a significant negative effect on glucose metabolism [3].

Several experimental studies have shown that long-chain ω-PUFAs inhibit the absorption of cholesterol in the intestine and its synthesis in the liver, lead to increased clearance of lipoproteins in the blood, prevent the development of insulin resistance (IR) in experimental diabetes, increase the level of glucose transporter 4 in skeletal muscles, have a positive effect on age related decrease of blood flow in the brain and improve glucose utilization under stress; there isn’t any influence on the development of hypertension (HT) and MetS. Ω-3 PUFAs decrease level of BP, dose-dependent prevent the development of T2DM, IR, contribute to positive changes of blood coagulation parameters; enhance endothelial cell migration and inhibits the proliferation of smooth muscle cells [4]. A meta-analysis of 18 studies found a significant effect of fish oil to lower TG concentrations and increase high-density lipoprotein cholesterol (HDL-C) in the blood; while there were no statistically significant changes in preprandial glucose, glycated hemoglobin A1c, total cholesterol, low density-lipoprotein cholesterol levels. Ω-3 PUFAs may affect the IR and glucose homeostasis by inhibition of IR in the muscle tissue >adipose tissue >>liver, inhibition of insulin secretion, which defer the development of T2DM; and on the state of lipid metabolism (in particular, reduce the concentration of TG, very low density-lipoprotein cholesterol (VLDL-C), increase of HDL-C, improve lipid profile by mixed hyperlipidaemia (HLP), slightly decrease BP, improve endothelial function, have an positive impact on the antioxidant status and inflammatory reactions [5]. Ω-3 PUFAs decrease VLDL assembly and secretion, resulting in diminished TG production, through a decreased sterol receptor element binding protein-1c activity [6,5].

The highly concentrated pharmaceutical preparation Omacor™ (Pronova Biocare, Lysaker, Norway), known as Lovaza™ (Glaxo Smith Kline, St Petersberg, FL, US) in North America is approved by the FDA as an adjunct to diet to reduce very high TG levels (≥500 mg•dL-1) in adults. Each 1-g capsule of ω-3-acid ethyl esters contains ethyl esters of EPA (0.465 g) and DHA (0.375g). Patients take a q.d. dose of 4-g or two 2-g doses (two capsules b.i.d.) [7]. Clinical trials have shown that administration of 4 g•day-1 of Lovaza™ results in a decrease in TG levels of 30-50%; does not affect the efficacy of statins [8,5]. In patients with combined HLP, co-administration of Lovaza™ with statins was a safe and effective means of lowering serum TG, despite the persistent high TG levels when the patients received statins alone [9,5].

The anti-inflammatory actions of marine ω-3 PUFAs are [10]: reduced leucocyte chemotaxis (via decreased production of some chemoattractants (e.g. leukotriene B4 down-regulated expression of receptors for chemoatttactants); reduced adhesion molecule expression and decreased leucocyte-endothelium interaction (via down-regulated expression of adhesion molecule genes [via the nuclear factor kappa B (NF-kB) (i.e. peroxisome proliferator-activated receptor-ɣ (PPAR-ɣ) etc.); decreased production of eicosanoids from arachidonic acid (AA) (via lowered membrane content of AA; inhibition of AA metabolism); decreased production of AA containing endocannabinoids (via lowered membrane content of AA); increased production of ‘weak’ eicosanoids from EPA (via increased membrane content of EPA); increased production of anti-inflammatory EPA and DHA containing endocannabinoids (via increased membrane content of EPA and DHA); increased production of pro-resolution resolvins and protectins (via increased membrane content of EPA and DHA); decreased production of inflammatory cytokines (via down-regulated expression of inflammatory cytokine genes (via NF-kB, i.e. PPAR-ɣ etc.); decreased T cell reactivity (via disruption of membrane rafts (via increased content of EPA and DHA in specific membrane regions).

Ω-3 PUFAs may decrease the risk of atherothrombosis by affecting platelet aggregation and haemostasis. The antithrombotic properties of EPA and DHA have been attributed to the incorporation into platelet phospholipids at the expense of the ω-6 PUFAs, such as AA. An important set of pathways clearly influenced by changes in the ω-3/ω-6 ratio are those for synthesis of eicosanoids. These include the cyclooxygenase (COX), lipoxygenase and cytochrome P450 epoxygenase pathways, for which EPA and DHA compete with AA as a substrate, inhibiting the production of the proaggregatory thromboxane A2 (TXA2) originating from AA. Indeed, the production of TXA2 from platelets stimulated by a variety of agonists decreased by between 60% and 80% after fatty acid supplementation both in vitro and in vivo [11,5]. The mechanism by which ω-3 PUFAs influence endothelial function is mediated by their incorporation into biological membrane phospholipids; this allows modulation of membrane composition and fluidity. The reason lies in the fact that endothelial cell membrane houses caveolae and lipid rafts where several receptors and signaling molecules crucial for cell function are concentrated [12]. Caveolae-associated receptormediated cellular signal transduction includes important pathways such as the, the nitric oxide (NO)/cyclic guanosine monophosphate signaling pathway, the nicotinamide adenine dinucleotide phosphate oxidase and tumor necrosis factor-α/ NF-kB induced COX-2 and prostaglandin E2 activation pathway. By modulating the composition of caveolae, as described for other classes of lipids ω-3 PUFAs may exert their beneficial effects, which include increased NO production and reduced production of proinflammatory mediators [13,12]. In addition to increasing NO production, ω-3 PUFAs decrease oxidative stress.

The incorporation of ω-3 PUFAs in synaptic membranes could potentially influence the autonomic control of the heart. Both nervous tissue and heart tissue have a high content of ω-3 PUFAs (especially DHA) and this may be consistent with the finding that this marine ω-3 PUFAs may modulate cardiac autonomic function as assessed by heart rate variability (HRV) [14]. Thus, ω-3 PUFAs may modulate HRV both at the level of the autonomic nervous system and the heart. Most of the data support that ω-3 PUFAs beneficially modulates cardiac autonomic control thereby possibly reducing the risk of arrhythmias. Accumulating evidence from in vivo and in vitro experiments has demonstrated that ω-3 PUFAs exert antiarrhythmic effects through modulation of myocyte electrophysiology. Ω-3 PUFAs reduce the activity of membrane Na+ channels in cardiomyocytes, thus increasing the threshold for membrane potential depolarization. EPA and DHA also modulate the activity of L-type Ca2+ channels, leading to a reduction in free cytosolic Ca2+ ion, which stabilizes myocyte electrical excitability to prevent fatal arrhythmia. EPA blocks the Na+/Ca2+ channel; however, a single amino-acid point mutation in this channel attenuated the inhibitory effect of EPA. These findings suggested that the cardioprotective effect of ω-3 PUFAs is mediated by direct interaction with membrane ion channels [15].

Ω-3 PUFAs intake has shown to reduce BP especially in HT by interacting with several mechanisms of BP regulation: reduction of stroke volume and heart rate; improvement of left ventricular (LV) diastolic filling; reduction of peripheral vascular resistances; improvement of endothelial-dependent and endothelial-independent vasodilation (stimulation of NO production; reduction of the asymmetric di-methyl-arginine; reduction of endothelin-1; relaxation of vascular smooth muscle cells; metabolic effects on perivascular adipocytes; endothelial regeneration. Mechanisms of HT-related organ damage protection: anti-inflammatory, antioxidant, and antithrombotic effects; reduction of arterial stiffness; experimental effects on LV hypertrophy and abnormal gene expression; effects on atherosclerotic plaque progression and stability [7]. Ω-3 PUFAs offer a scientifically supported means of reducing arterial stiffness and this may account for some of the purported cardioprotective effects of ω-3 PUFAs [16,17].

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Conclusion

The antiarrhythmic effects of ω-3 PUFAs, which occur by blocking various ion channels, are encouraging. So, cardiovascular benefits of ω-3 PUFAs [7,18] are: antidysrhythmic effects (reduced sudden death; possible prevention of atrial fibrillation; possible protection against pathologic ventricular arrhythmias; improvement in HRV; antiatherogenic effects (reduction in non- HDL-C levels; reduction in TG and VLDL-C levels; reduction in chylomicrons; reduction in VLDL and chylomicron remnants; increase in HDL-C levels; plaque stabilization; antithrombotic effects (decreased platelet aggregation; improved blood rheologic flow); anti-inflammatory and endothelial protective effects (reduced endothelial adhesion molecules and decreased leukocyte adhesion receptor expression; reduction in proinflammatory eicosanoids and LT’s; vasodilation); decreased SBP and diastolic BP. Thus, further research to understand the mechanism of action and confirm the beneficially effect of ω-3 PUFAs on BP profile, artery stiffness and HRV parameters in patiens with MetS, T2DM is needed.

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ourhaileydavies · 2 years ago

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Expect the Unexpected with Erector Spinae Plane Block in Spine Surgery - Plan for the Worst and Hope for the Best: An Anesthesiologist Perspective-Juniper Publishers

Abstract

Spine surgery is associated with multiple postoperative complications, ranging from simple nausea and vomiting to devastating complications leading to postoperative morbidity or mortality. The postoperative neurological impairment, especially in the neurologically intact patient, is a dreadful event that makes it difficult for the surgeon to perform technically challenging or high-risk spine surgeries. Preoperative or intraoperative factors that can influence the postoperative neurological status include nature and the severity of the pathology, comorbid conditions of the patient, preexisting neurological symptoms, multiple levels involved, complex surgery or instrumentation, surgical blood loss, neurological monitoring, hemodynamic parameters, polypharmacy, and total duration of the surgery.

In addition to several known contributing factors (fixation failure, epidural hematoma, spinal cord edema, and ischemia-reperfusion injury), the role of the erector spinae plane block (ESPB) has recently been cited as a potential cause of postoperative transient paralysis after spine surgery. ESPB is considered a simple and safe regional anesthesia technique that may have an advantage in success rate and analgesic efficacy when used as an adjunct to general anesthesia in spine surgeries. Despite varied patterns of the drug spread, ESPB has been showing promising results due to consistent involvement of dorsal rami that supply all pain generators of the spine surgeries.

The potential role of ESPB in causing postoperative transient neurological complications is a diagnosis of exclusion that requires thorough clinical assessment and step-by-step evaluation using imaging modalities. Before administering ESPB in spine surgery, essential knowledge includes anatomical and technical considerations, drug distribution patterns, safe and effective volumes/types of local anesthetics, and possible associated complications. This review article describes the possible roles of all factors that lead to postoperative neurological impairment and suggests some tips and tricks for using ESPB in spine surgeries to prevent or manage such serious complications appropriately.

Keywords: Transient paraplegia; Erector spinae plane block; ESP block complications; ESP block in spine surgery; Paraplegia due to RA

Keywords: RA: Regional anesthesia; GA: General anesthesia; ESPB: Erector spinae plane block; ERAS: Enhanced recovery after surgery; LA: Local anesthetics; CT: Computed tomography; MRI: Magnetic resonance imaging; ESM: Erector spinae muscles; TP: Transverse process; SMPB: Sacral multifidus plane block; RLB: Retrolaminar block

Introduction

The occurrence of perioperative complications may be inevitable, but their prevention and management are always a shared responsibility of all team members involved. Thorough evaluation of such complications will help develop strategies to prevent and manage the same in the future. A systematic and stepwise approach is warranted before categorizing it as a surgical or anesthetic complication. Several interventions have been introduced in the surgical and anesthetic techniques to improve patient safety and satisfaction. Application of regional anesthesia (RA) alone or as an adjunct to general anesthesia (GA) is one such advance that helps reduce many polypharmacy-related side effects or complications. If a particular complication-reduction modality is inherently causing complications, it requires a comprehensive understanding of the situation and its contributing factors.

An erector spinae plane block (ESPB), a safe and simple RA technique, has shown promising results as an adjunct to multimodal analgesia in various orthopedic, general, thoracic, abdominal, obstetrics, and spine surgeries. In addition to its superior postoperative analgesic profile in spine surgeries at various levels, ESPB reduces hospitalization costs and the possible side effects of extensive anesthetic use. Since opioids have been linked to tumor recurrence [1,2], ESPB also reduces the risk of spine tumor recurrences by significantly reducing its consumption. ESPB meets all criteria suitable for enhanced recovery after surgery (ERAS) protocol [3] by facilitating early discharge and mobilization of patients. Being a novel RA technique, not many complications have been reported so far except for some anecdotal reports of bilateral quadriceps weakness, transient apathy or aphasia, minor neurological complications due to inadvertent intravascular injection of local anesthetics (LA) [4].

Recently, it has been described as a potential cause of transient paralysis after spine surgeries [5]. Therefore, it is essential to understand the differential diagnoses of postoperative neurological impairment, follow the step-by-step approach to rule them out one by one, determine the possible role of ESPB in their development, and learn the tricks for safely administering ESPB during spine surgery. This review article elaborates the essential background knowledge required before and after the administration of ESPB in spine surgeries.

Discussion

Postoperative neurological impairment after spine surgery in a neurologically intact patient is always daunting for the operating surgeon and the patient. Several common theories on neurological deterioration after decompressive spine surgeries include vascular compromise, hypotension, ischemia, direct trauma, or stretching of the neural elements. The major contributing factors of acute paralysis following spine surgery include fixation failure, epidural hematoma, spinal cord edema, and ischemia‑reperfusion injury [6].

Contributory factors

Neurons in the spinal cord are susceptible to ischemia and hypoxia. The mechanisms of spinal cord ischemia are multi-factorial and multi-channel. The pathogenesis of spinal cord lesions after spine surgeries is usually mechanical (pressure) damage via extensive hematoma or edema, resulting in pressure on the spinal cord leading to ischemic damage [7]. An altered cerebrospinal fluid flow dynamic may also cause cord compression [8]. In either case, the ultimate pathogenic cause is a secondary cellular injury due to the disruption of ionic homeostasis, development of free radicals, lipid oxidation, and degeneration of the cytoskeleton [7]. White cord syndrome, an imaging feature of spinal cord ischemia [9], is diagnosed as high intramedullary signal changes on sagittal T2 weighted MRI scans and is often seen in surgeries on the cervical spine.

The spinal infarct is one of the leading causes of paraplegia or quadriplegia in patients with preexisting vascular pathologies (thrombosis) or embolic events during surgery [10]. The anterior spinal cord has a higher risk of ischemia due to fewer anterior spinal artery feeding vessels [10] than the highly vascular posterior spinal cord due to anastomotic pial vessels. The sparing of the posterior column leads to unchanged intraoperative somatosensory evoked potentials [11]. The ischemia-reperfusion injury occurs upon restoring the blood flow to previously ischemic tissues and organs. Increased inflammatory cytokines such as TNF α and IL 1β may be considered vital indicators for evaluating decompression-associated spinal cord ischemia-reperfusion injury [12,13]. Its reported incidence is 2-5.7% following cervical and 14.5% following posterior thoracic decompression surgeries [14, 15].

Transient paralysis is one such complication that manifests itself as a temporary (up to 72 hours) loss of sensations, movements, anal reflexes, and sphincter function below the affected spinal segments [16]. It can occur after vertebroplasty, laminectomy, or thoracic decompressive procedures [17,18]. The longer duration of symptoms, multiple compression sites, and the high degree of preoperative stenosis are considered poor prognostic factors [18].

Who is the culprit?

The exact cause of the postsurgical neurological impairment is a diagnosis of exclusion requiring thorough clinical evaluation and imaging guidance to rule out each contributing factor (Table 1) in a step-by-step manner. Postoperative radiographic studies like computed tomography (CT) scan and magnetic resonance imaging (MRI) can help detect changes suggestive of misplaced implants, hematomas, edema, compressive lesions, white cord syndrome, or direct trauma to the spinal cord. Symptoms due to spinal cord edema typically occur at 48-72 hours post-surgery and may be relieved by anti-edema measures like fluid restriction [19].

The occurrence and severity of ischemia-reperfusion injury correlate with tissue ischemia time, the extent of ischemic tissue, and the oxygen requirement of the affected tissue [20]. The presence of deep tendon and superficial reflexes may rule out the possibility of hysterical paraplegia [18]. After excluding all contributing factors that may cause postoperative neurological impairment, the possible role of ESPB and LA can be considered and further evaluated. It requires an understanding of the anatomical and technical aspects, mechanism of drug spread, factors favoring neuraxial spread, and measures to avoid such incidents in the future [21].

Role of ESPB

ESPB involves depositing the local anesthetic solution between the erector spinae muscles (ESM) and the transverse process (TP) under ultrasound guidance. The ESM consists of three muscles: iliocostalis, longissimus, and spinalis. They arise from and insert into various bony components of the vertebral column [22] and form a paraspinal column that extends from the sacrum to the base of the skull. It gradually tapers upwards in the paravertebral groove on either side of the spinous processes. The retinaculum (thoracolumbar fascia in the lumbar region) that envelops this muscular column also facilitates the LA spread to several thoracic and lumbosacral levels [23]. The diverse multilayered fascial arrangement deep to the ESM may cause the inconsistent LA spread, resulting in multisegmented sensory block mainly involving dorsal rami with sometimes ventral rami.

This Para neuraxial block, when given bilaterally in spine surgery, can be advantageous in success rate and analgesic efficacy [24]. The absence of risks such as hypotension, vascular spread, or pneumothorax makes ESPB relatively safer than epidural anesthesia or paravertebral block. Bilateral ESPB offers effective perioperative analgesia without influencing the hemodynamic parameters. It significantly reduces the perioperative opioid requirements in spine surgeries at various levels (cervical, thoracic, and lumbar, and sacral) [25-32]. Its outcome depends on the volume and concentration of LA used, drug spread, and the anesthesiologist’s experience in selecting and locating the correct level of the TP.

The exact mechanism of action of the ESP block and pattern of the drug spread is still unclear. It has been suggested to anesthetize the spinal nerves by passing through the costotransverse foramen of Cruveilhier, accompanying the dorsal ramus and artery to the paravertebral space [33]. The deposited drug can spread in any direction, such as craniocaudal, anterior-posterior, and lateral-medial planes to reach the paravertebral space, neural foramina, epidural space, or sympathetic chain [34-38]. Fluoroscopic, CT, and MR imaging in living subjects have similarly confirmed the injectate tracking to the paravertebral area, intervertebral foramina, and epidural space following lumbar ESPB [39-42]. There is also a possibility of LA diffusion through the microscopic gaps in the mostly acellular architecture of interlinked collagen fibers of the fascia covering the erector spinae muscle [43].

ESPB at various spine levels

The anatomical differences at the various spine levels can cause varied drug spread and ultimately affect the outcomes of ESPB. Cervical ESPB is technically challenging due to the difficulty in identifying the tips of the cervical transverse processes due to their shorter length. It is mainly given at the C6 or C7 vertebral level. The probe needs to be kept anterolaterally rather than posteriorly to see the cervical TPs [44]. It may not be safe due to its proximity to the neuraxis (shorter transverse processes) and the possibility of bilateral phrenic nerve involvement [45-48].

Thoracic ESPB at the upper vertebral levels (T2 orT3) can be preferred in cervical spine surgery by inserting the needle from caudal-to-cranial direction to achieve the desired LA spread and avoid technical difficulties and complications associated with cervical ESPB. Thoracic ESPB can provide multilevel analgesia even with the small volumes of LA due to rigid boundaries of the thoracic paravertebral spaces that facilitate drug spread at several levels involving ventral and dorsal rami. Lower thoracic level ESPB is mainly performed for lumbar spine surgeries by inserting the needle from cranial-to-caudal direction to achieve the desired LA spread and avoid technical difficulties associated with lumbar ESPB [49,50].

The lumbar ESPB can also be technically challenging due to the increased thickness of the ESMs with their tendinous attachment to the TPs [51, 52] and increased corresponding depth of the intermuscular plane in the lumbar region. The psoas muscle is also closely adherent to the vertebral bodies and the anterior surface of the TPs. The anterior drug spread to include ventral rami may be compromised due to the lack of clear boundaries of lumbar paravertebral spaces [53]. There is a communication through the fat-filled plane between the ESM and TP with the fat-filled psoas compartment containing lumbar nerve roots and plexuses. The spread of LA to the epidural space is possible through this communication [54]. The compressed lamina and the ligaments of the lumbar spine favor LA spread more into the epidural space [55, 56]. Thus, the lumbar ESPB may result in either lumbar plexus block or epidural anesthesia. The resultant weakness in the quadriceps or lower extremity muscles depends on the LA concentration and volume used in ESPB.

Sacral ESPB is mainly described for gender reassignment surgery or perineal surgery [57-61]. Its application for lower lumbar or sacral spine surgery is yet to be determined. The sacral multifidus plane block (SMPB), one of the variants of the paraspinal block, involves the deposition of LA in the plane under the multifidus muscle and bony area between the median and intermediate crests of the sacrum. The possible mechanism of action of SMPB includes blocking the dorsal rami and medial cluneal nerves directly by LA deposition and ventral rami by anterior LA spread through dorsal and ventral sacral foramina. The SMPB may also block the pudendal nerve (S2–S4), lumbosacral plexus, and sciatic nerve via the anterior and cranial LA spread [61, 62].

The role of LA

The possible role of the LA used in ESPB in causing postoperative neurological compromise depends on its inadvertent spread into either the epidural or subarachnoid space. It can be determined based on the occurrence and recovery pattern of the neurological symptoms. Distal-to-proximal and motor-before-sensory recovery patterns are the hallmarks of the differential blockade of the LA [23]. Inadvertent spread of LA into the subarachnoid space can lead to severe hypotension and bradycardia, resulting in unstable intraoperative hemodynamics. The consequences of the epidural spread depend on the density of LA around the spinal nerves, which could be compromised in a subsequent surgical dissection affecting the potentiality of the epidural space.

The concentration of LA, which determines the mass of the drug, also affects the efficacy of any block. The deliberate use of LA in low concentrations can result in a preferred motor-sparing analgesic effect of such high-volume blocks [63, 64]. Bupivacaine and ropivacaine are the most commonly used LAs for bilateral ESPB. Both LA agents consistently display preferential blockade of C-fibres (slow pain) > A-delta fibers (fast pain) > A-beta fibers (touch/pressure) in both preclinical and clinical studies [64-66]. With the increasing concentration, these agents may result in loss of proprioception and loss of motor function. Lipid solubility and higher pKa of LA facilitate intraneural diffusion and ion channel blockade. Ropivacaine exhibits a relative motor-sparing effect due to its lower lipid solubility than bupivacaine [67]. Twenty milliliters of 0.375% ropivacaine is recommended for each side of the bilateral ESPB in adults [68, 69].

Technical aspects of ESPB

Unexpected outcomes like a neurological compromise can be correlated with possible technical errors while administrating ESPB. The first technical aspect is identifying the correct landmark under ultrasound depending on the surgical extent and the desired level of the block. It may further depend on the sonoanatomy quality and the experience of the anesthetist. Sometimes misidentifying the lamina as the tip of the TP can lead to the retrolaminar block (RLB), another variant of the paraspinal block. In RLB, the needle insertion is slightly medial, targeting the lamina of the vertebra instead of the tip of the TP. It works via diffusion of LA into the paravertebral space through the soft tissue gaps between adjacent vertebrae [70]. Both RLB and ESPB were consistently associated with the posterior spread of injectate to the back muscles and fascial layers [37].

Fluoroscopic-guided ESPB can lead to RLB due to the inability to see the tip of TP clearly like under ultrasound, resulting in deposition of the LA solution over the lamina. The proximity of the RLB to the neuraxis can lead to a high probability of epidural spread, which carries the risk of motor weakness. The second important aspect is the ergonomics associated with bilateral ESPB. Administering the bilateral ESPB by standing on only one side of the patient may result in deviation from the ideal needle trajectory on one side compared to the other. Therefore, technical considerations should focus on stabilizing the needle by one person, injecting LA by another person, and performing such bilateral blocks while standing on either side.

The third important aspect includes technical modifications such as keeping an ultrasound probe in a transverse view to help differentiate intramuscular drug spread from the effective linear drug spread between ESM and TP [71]. The fourth aspect is finding alternatives that involve dorsal rami consistently without causing drug spread to other unwanted areas. The thoracolumbar interfacial plane block is one such alternative that targets only the dorsal rami of the spinal nerve. Thus, it can provide more focused dermatomal coverage of the back required for thoracic and lumbar spine surgeries [72, 73]. However, its efficacy in spine surgeries is yet to be determined. We have suggested some tips and tricks for using ESPB in spine surgeries (Table 2), keeping all technical aspects in mind.

Conclusion

Postoperative neurological impairment following spine surgery is a serious concern for the operating surgeon and the patient. The role of ESPB in causing such complications is the diagnosis of exclusion made after a thorough evaluation of clinical symptoms and radiological studies. For that, understanding of various mechanisms involved in ESPB leading to neurological impairment is essential. It should encourage the anesthetists to take extreme precautions while administering this novel block, considering the anatomical differences at various spine levels. Surgeons should anticipate and explain the possibility of neurological deterioration while explaining the risks and benefits of the proposed surgical intervention. Intraoperatively, real-time neurophysiological monitoring is recommended as a useful tool to avoid further neurological deterioration, especially in extensive and multilevel surgeries or in high-risk and neurologically compromised patients.

After identifying or diagnosing such complications, intensive care and regular checking of spinal function are of great importance, along with simultaneous radiological workups to rule out various causative factors. Once paralysis occurs, early diagnosis and early intervention are essential in restoring spinal function. Despite the rare possibility of such complications, ESPB is still a promising option for ensuring effective perioperative analgesia in spine surgeries. It helps reduce postoperative morbidity by keeping the hemodynamic parameters stable and significantly reducing intraoperative blood loss. It can also avoid postoperative complications that lead to delay in mobility and discharge by significantly reducing the need for opioids and polypharmacy. However, further studies are needed to determine the safe concentration and volume of the LA solution used in ESPB, the exact surgery-specific vertebral level to cover desired surgical innervations, and the accurate LA deposition site to prevent spread to undesired areas.

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juniperpublishersanatomy-blog · 2 years ago

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Class Attendance and the Performances in Physiology Board Examinations: A Study in a Caribbean Medical School-Juniper Publishers

Abstract

Objective: Many faculty members in medical schools encourage their students to attend classes regularly emphasizing that some studies have reported strong positive correlation between the performances in the National Board of Medical Examiners (NBME) examination with their class attendance. The objective of our study was to assess the association of the physiology class attendance and NBME physiology scaled score.

Methods: For this study, 93 medical students who completed two terms of medical physiology at Trinity School of Medicine (TSOM) wereselected. They had their first attempt of NBME physiology examination from summer 2014 to fall 2015. Their physiology class attendance andNBME physiology scaled score were tabulated in Microsoft Excel. The correlation of the percentage of physiology class attendance and NBME physiology scaled score was determined by a Pearson correlation coefficient and regression analysis, using the SPSS version 24.

Results: The class attendance was a significant predictor of performance in physiology NBME subject examination (R2 (91) = 0.295, p = .000). A significant positive correlation was found between the class attendance (%) and NBME physiology score (r(91) = .543, p < 0.001).

Conclusion: The class attendance (%) was significantly correlated with the NBME physiology score. The physiology class attendance appears to be related to NBME physiology scaled score. However, the impacts of other factors such as study habits, environment, cultural habits, gender difference, and personal, familial, and socioeconomic stresses need to be assessed in further studies.

Keywords: NBME; Physiology; Scaled Scores; Class Attendance; Caribbean Medical School

Abbreviations: NBME: National Board of Medical Examiners; TSOM: Trinity School of Medicine; SAT: Scholastic Assessment Test; HSGPA: High School Grade Point Average; USMLE: United States Medical Licensing Examination

Introduction

Many faculty members in medical schools encourage their students to attend classes regularly emphasizing that regular class attendance facilitates learning and enhances their performance in examinations. Class attendance appears to be a better predictor of college grades than any other known predictor of college grades - including Scholastic Assessment Test (SAT) scores, High School Grade Point Average (HSGPA), studying skills, and the amount of time spent studying [1]. The efforts to increase the class attendance rates among college students helped to achieve dramatic improvements in average grades [2].

The National Board of Medical Examiners (NBME) provides subject examinations in the basic and clinical sciences to medical schools and other institutions that educate physicians or other health professionals. The NBME subject examinations assess the educational achievement of medical students in specific subject areas. These examinations are used at virtually all allopathic medical schools and many osteopathic medical schools in the United States and Canada, and approximately 25 international schools in the Caribbean, Mexico, Europe, Middle East, and Asia. The NBME examinations are primarily used as final examinations after courses, clerkships, or other units of instruction. However, the scores achieved on NBME examinations cannot be used by examinees for credit toward the United States Medical Licensing Examination (USMLE) [3]. Some studies reported that the NBME performance has a strong positive correlation with the class attendance [4-6]. Hence, we aimed to assess the correlation of physiology class attendance (%) with the NBME physiology scaled score.

Material and Methods

The present study is an analytical comparative study. The study was conducted in Trinity School of Medicine (TSOM), St. Vincent and the Grenadines, West Indies. At Trinity, Medical Physiology is taught to students in their first and second terms each of which comprises 15 weeks. The two terms of medical physiology are designed to give the students sufficient mastery over basic physiology and its application in clinical contexts to prepare them for the physiology sections of the USMLE Step 1. The students take part in NBME physiology at the end of their second term.

The medical students who studied two terms of medical physiology and took their NBME physiology subject examination from summer 2014 to fall 2015 at TSOM were included into this study. The students who dropped their physiology course before taking the NBME physiology and continued the same next term, those who did not study their two terms of medical physiology at TSOM, those transferred from other schools to TSOM, and those with low levels of attendance due to medical leave of absence and other extenuating circumstances were excluded from the study. Ethical clearance for the study was obtained from the research ethical committee of TSOM.

Three physiology faculty members utilized sign-in sheets, which were passed out in each class period to record the class attendance. Students were informed that the class attendance was being recorded for informational purposes and that there were no consequences regarding their absences. The students’ cooperation was good. At the end of the second term, their first attempt of NBME physiology scaled score and class attendance were tabulated in the Microsoft Excel. The correlation between the class attendance and NBME physiology scaled score was determined by a Pearson correlation coefficient and regression analysis, using the SPSS version 24. A p value of 0.05 or less was considered as statistically significant.

Results

A total of 93 students were included into the study of whom 42 were female and 51 were male. The students’ physiologyclass attendance was measured by the overall attendance percent (mean = 85.1%, SD = 11.9%) (Table 1). The students’ performance was assessed by the NBME physiology scaled score (mean = 45.9, SD = 9.1) (Table 1). There was wide variation in the class attendance (%) and NBME physiology scaled score of individual students.

The correlation coefficient (adjusted r2) of our study was 0.287 (Table 2) and significance F was <0.001. A significant positive correlation was found between the class attendance (%) and NBME physiology scaled score (r(91) = .543, p < 0.001) (Table 2). The regression model significantly predicted 29.5% (R2 = 0.295) of the NBME physiology scaled score of the students (Table 2).

Simple linear regression analysis between the class attendance (%) and NBME physiology scaled score showed that the class attendance contributed 26.9% (confidence interval 0.283, 0.552) of the variation in NBME physiology scaled score (Table 3). The regression model showed that for each 1.0% increase in class attendance, the NBME physiology scaled score is expected to increase by 0.417 (Table 3). The equation obtained from the regression model is: NBME physiology scaled score = 10.423 + 0.417 x class attendance (%). Hence, according to regression model equation, a student is expected to have 94.9% of class attendance to achieve a scaled score of 50, which is the national average in physiology NBME subject examination (Figure 1).

Discussion

The principal result of our study was a significant positive correlation between the class attendance (%) and NBME physiology scaled score (r = .543, p < 0.001). The result of our study is in line with the meta-analytical review done by Crede et al. [2] who found a strong relationship between the class attendance and college grades. Hence, they suggested increasing class attendance rates among college students to achieve dramatic improvements in average grades [2]. In a study of relationship between class attendance and NBME part I examination done by Fogleman et al. [4] analysed the results of survey via a 2 X 2 chi-square for each item and scores on the NBME examination. It showed that only class attendance were significantly different between students scoring above the mean (511.2) and those below the mean on the NBME part I examination (chi-square = 4.766; p <0.05) [4].

Similarly, Hammnen et al. [7] indicated a negative correlation between grades and number of absences from class. However, their correlation was weak, indicating that other factors were involved as most of the studies, including theirs, did not have matched experimental and control groups. Hence, it is impossible to state whether regular attendance caused slightly higher scores or whether the better students attend the classes more frequently [7]. Millis et al. [8] demonstrated that a significant correlation (r = 0.203, P < 0.05) between the attendance and students’ grade averages at the end of their second year. They found increased grade averages as attendance increased. Students may assume that the self-directed study and distance learning are equivalent to the class activities. The risk of poor performance in a significant number of first-year medical students may be because of their belief that the internet- and classroom-based instructions in basic medical science courses are equivalent [8].

In a study done by Subramaniam et al. [9] in a medical college, they found a significant correlation between the attendance and the students who passed the University examinations. The number of students passing the examination was maximum (>90%) compared to those getting distinction and failing the exam after they made attendance mandatory for the medical course [9]. A moderate to strong negative correlation between absenteeism and academic achievement suggested that the class attendance is very critical for learning and important in improving the knowledge and academic achievement [10]. Bamuhair et al. [11] reported the positive effect of attendance on the academic performances with a stronger effect for lectures compared to other teaching modalities. They suggested that the lecture attendance is critical for learning even in nontraditional methods of education [11].

In a study of association between lecture attendance and grade outcomes done by Horton et al. [12] they reported the positive correlation of exam grades with the lecture attendance in male students (r = 0.29, P < 0.04) and overall (r = 0.21, P < 0.02) but not for female students considered separately (r = 0.10, NS). They also found that the overall grades were correlated positively with lecture attendance in male students (r = 0.35, P < 0.01) and overall (r = 0.31, P < 0.001) but not when female students were considered separately (r = 0.20, NS) [12].

However, Cohall et al. [13] in their study found no significant association between the improvement in attendance and improved academic performances in the examinations. Their findings suggested that the other factors are more critical to academic success [13]. Therefore, even though the present study suggests that the higher percentage of class attendance add value to academic performance in board examinations, the other factors such as quality of lectures and the study habits may also have an equal impact in academic performance of the students in board examinations.

Conclusion and Recommendation

According to our study, the physiology class attendance (%) has a strong positive correlation with the scaled score in physiology NBME subject examination. It means that the performance of students in physiology NBME subject examination improves with increase in their class attendance (%). Therefore, we recommend that the faculty members should encourage the students to attend the classes regularly by implementing incentives such as points for class attendance or by applying academic activities during the class such as discussing comprehensive questions at the end of the lecture. Implementation of class attendance policies might enhance the performance of medical students in board examinations too.

We conducted this study only in the subject of Physiology. The study that includes a larger sample size from multiple basic medical science subjects and study samples from more than one medical school might further strengthen the results. Further studies should address the quality of lectures, other ways to increase intrinsic motivation for attending lectures, whether the relationship is causal, and whether the improvement in attendance percentage can improve the NBME physiology performances. In addition, the impacts of other factors such as study habit, environment, cultural habit, gender difference, and personal, familial, and socioeconomic stresses need to be assessed in further studies.

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juniperpublisherscellscienc-blog · 2 years ago

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Exosomal Consignment in Renal Allograft Injury

Abstract

Exosomes are small mobile endocytic vesicles (30-120nm), shredded by every cell to conduct trafficking of cell generated cargo. They are found in almost all body fluids (blood, csf, saliva, urine). These include proteins, lipids, DNA, mi(cro)RNAs etc. In multicellular organisms, they are packaged into numerous vesicles mainly in exosomes to conduct their transport for various cellular activities which can be exploited clinically. Presently the survival of renal allograft is monitored by mostly invasive methods (tissue biopsy, Creatinine, GFR) where curving the injury is quite difficult. Hence potency of molecular markers like proteins and then circulating miRNAs came to picture for early detection of renal injury (Acute Kidney Injury-AKI and Chronic Kidney Disease-CKD). However, due to lack of specificity of circulating miRNAs lose their feasibility and the discovery of these exosomal cargos in cellular communication has become an efficient tool for treatment of various complicated clinical condition including renal allograft injury.

Keywords: micro RNAs,exosome, Renal Allograft Injury

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Introduction

Exosomal world: a prologue

Exosomes are membrane bound mobile vehicles that are found in almost all circulating body fluids like- blood, CSF, saliva, urine, etc. These are responsible for transport of respective cellular cargo to extracellular target sites [1]. Recent studies with exosomes have revealed that exosomal cargo delivery has many important biological, physiological and pathological significance thus, can be an effective diagnostic tool for various diseases [2]. Exosomes are small circulating units of 30-120 nm in diameter, generating from late endosomal compartments of cells by its cell membrane invagination or budding or released as shedding vesicles. Cellular cargos include proteins, lipids, DNAs, mRNAs, miRNAs, etc [1]. The exosomal cell membrane mainly constitute a limiting lipid bilayer, transmembrane proteins and a hydrophilic core containing proteins, mRNAs and microRNAs (miRNAs).

Exsosomes were first discovered by Pan and Johnstone in 1983 [3] when they found that the release of transferrin receptors into extracellular space during sheep reticulocyte maturation was released inside a type of small vesicles. In 1989 Johnstone regarded these mammalian cargo delivering vesicles as exosomes [1-5]. Valadi et al. in 2007 first described about the composition of exosomes that apart from proteins and lipids these also contains DNAs and RNAs [6] which are recorded in ExoCarta database [7,8] . The exosomal cargo delivery requires stimulation of target cell which may be direct by receptor mediated interactions or may aid in transport from cell of origin to various bioactive molecules e.g. membrane receptors, proteins, lipids, mRNAs, miRNAs, etc [7]. When exosomes deliver its contents into the respective target sites the property and behavior of these cells changes to a great extent [8]. It is also understood from various studies done in last couple of years that miRNA composition of parent cell and exosomal components vary a lot [8] and of all the components, miRNAs have drawn the attention due to their regulatory role in gene expression as these are protected against RNAase-dependent degradation [1-8]. Thus exosomal cell-to-cell communication influence both physiological as well as pathological environment of the body. These play important roles in immune reactions, tumorigenesis and in neurodegenerative disorders [1]. e.g. in prostate cancer, ovarian cancers, lymphoma glioblastoma, etc [1].

Biogenesis

Exosomes are formed from late endosomal compartments of cells through endosomal sorting complex required for transport (ESCRT-that recognizes ubiquitylated proteins) to deliver the cargo to target cell or to fuse with lysosomes to degrade the undesired contents [1]. Earlier these exosomes were only considered to be “garbage bags” as their diversified capabilities were unknown then. But now these are the most emerging field of research. The way of formation and secretion of these vesicles from mutlivesicular bodies (MVBs) are of two types [9]:

Microvesicles, which are directly shed from cell membrane.

Exosomes, which are released by exocytosis when MVBs fuse with plasma membrane.

Exosomes can be identified by transmission microscopy as a cup-shaped morphology with negative staining [10-12]. These can be concentrated in 1.10-1.21 g/ml section of a sucrose density gradient [10-12]. Exosomes can be identified by various protein markers e.g. tetraspanin proteins- CD63, CD9, CD81, HSP70 and HSP90, etc [1, 8]. ExoQuick (a one-step precipitation procedure for exosome extraction), Immuno affinity capture, Immunobead (EpCAM), combination of EpCAM and ultracentrifugation, size exclusion chromatography and EpCAM and followed by Quantitative PCR, Microarray techniques for extraction and quantification of exosomes [1,8,13].

Exosomes formation and secretion requires enzymes and ATP. First the cell membrane is internalized to produce endosomes. Subsequently, many small vesicles are formed within endosomes by invagination of its cell membranes [8, 14]. Such endosomes are called MVBs. Finally, the MVBs fuse with endosomal cell membranes to release intraluminal vesicles into extracellular space which become exosomes [14].

The secretion or cell-to-cell communication of exosomes requires certain regulatory factors which were first identified by Ostrowski et al. who observed that Rab27a and Rab27b were associated with exosomal secretion [8]. It was also found that effectors of Rab27- SYTL4 and EXPH5 could also inhibit exosomal secretion in HeLa cells [15]. Also Yu et al. discovered that tumor repressor protein p53 and its downstream effector TSAP6 were required for influencing exosome secretion [16]. Another working group, Baietti et al. observed the importance of syndecan-syntenin which directly interact with ALIX protein via Leu-Tyr-Pro-X(n)-Leu motif in secrection of exosomes by endosomal budding [17]. Koumangoye et al. found that disruption of lipid rafts in exosomal membranes could inhibit its internalization in breast cell carcinoma cell line [18]. Trafficking of exosomes to target sites occurs in following mechanisms:

The transmembrane proteins of exosomes directly interact with signaling receptors of target cell membranes [19].

The exosomal fusion with plasma membrane of recipient cells to deliver the cargo into their cytosol [20].

The exosomes internalization into recipient cells have two fates[21].

in one, some exosomes are engulfed by the cell and may merge with the cell’s endosome and undergo transcytosis

in other case, engulfed exosomes fuse with endosomes and mature into lysosomes for degradation.

As per ExoCarta database records, of all the components proteins and miRNAs have been exploited for various research to correlate some application with different diseased state that could render some remedy. Due to the regulatory role of miRNAs in gene expression these are used as recent area of research as diagnostic tool [8,22]. Goldie et al. demonstrated that among small RNAs, the percentage of miRNAs is higher in exosomes than in parent cells [23]. Studies done with exosomalmiRNAs shows there are specific sorting mechanisms for miRNAs into exosomes. These are:

The neural sphingomyelinase 2 (nSMase-2)-dependent pathway by Kosaka et al. [24].

The miRNA motif and sumoylated heterogeneous nuclear ribonucleoproteins (hnRNPs)-dependent pathway by Villarroya- Beltri et al. [25].

The 3’ end of the miRNA sequence-dependent pathway by Koppers-Lalic et al. [26].

The miRNA induced silencing complex (miRISC)-related pathway and human AGO2 protein [27].

In short there are specific sequence in miRNAs as well as enzymes and proteins that guide them for their sorting into exosomes [8]. Exosomes are shed by cells during both normal as well as pathological conditions. Thus several studies have been made with exosomes in diseased states.

A brief sketch on therapeutic exosomal cargos:

Exosomal miRNA: miRNAs are the recent findings in the field of clinical research which are thought to be crucial in depicting human health and diseases. These biomarkers can also be an indicator for rejection onset of transplanted allograft. miRNAs are a class of small 18-25 nucleotide (nt) long endogenous, noncoding RNAs which play an important role in regulating gene expression [28,29]. A single miRNA has the ability to regulate expression (mostly silencing) of hundreds of mRNAs and have been known to play important role in many cellular functions that include induction of post-translational repression, mRNA degradation/silencing and transcriptional inhibition, cell development, differentiation, proliferation and functional regulation of immune response among others [28-31].

The mystery behind the functional maturation of miRNAs has been solved by research in last couple of years. Similar to mRNAs, miRNAs are also initially transcribed in the nucleus [32]. miRNAs are at first transcribed in nucleus as primary transcript by RNA polymerase II called pri-miRNA [32-35]. This pri-miRNA has a hairpin stem-loop structure (~80nt length) that contains the mature miRNAs [36]. The pri-miRNA processing include recognition of the stem loop followed by its cleavage by DROSHA (a class 2 ribonuclease III) and DGCR8 (a miRNA-processing multiprotein complex) to release pre-miRNA [32-35]. Pre-miRNA is then recognized by Exportin-5 which allows its exports to cytosol for further maturation into 19-25 nucleotide strands by RNA endonuclease III called Dicer [32- 35, 37]. Cleavage of this pre-miRNA by Dicer result in double stranded (ds) RNA molecule of which one of the single strand with more unstable 5’ base pairing is selected and transferred to an Argonaute (AGO) protein and the other strand is degraded [35,38,39]. The selected strand induces silencing of mRNAs through RNA Induced Silencing Complex(RISC) thus affecting various cellular functions like cell differentiation, proliferation as well as development and functional regulation of immune system [32-35,40]. In normal tissues, RISC remain as a low molecular weight entity with reduced regulatory activity while under stressed or replicating conditions these become high molecular weight units with intensified regulatory activity when bound to mRNA [36]. Thus mRNA silencing by miRNAs results in lower protein levels in the body [36,41].

ExosomalProteins: Proteins are the building blocks of life in all living organisms. These are amino acid chains linked by peptide bonds. They are exquisite necessity in every cellular events, may it be the formation of new cells or cell repair. Thus, can be an important biomarker in depicting biological changes. Emerging research have exploited this idea and conducted various proteomic studies. A more burning concept is ofexosomal proteins. The work done and data obtained shows that besides miRNAs another important exosomal load isexosomal proteins. TrairakPisitkun et al had worked on urinary biomarkers and found that urinary exosomal proteins can also be an efficient protein biomarker in reporting renal tubulopathies and other renal disorders [42]. Exosomes normally found in urine accounts for around 3% of the total urinary protein contents and isolation of these exosomes can result in very large enrichment of urinary proteins derived from renal tubular epithelial cells [42]. The exosomal packaging occurs when the apical membrane proteins undergo endocytosis and packaged into MVBs. These MVBs undergo encapsulation of cytosolic proteins into small vesicles. Finally outer membrane of MVBs fuse with apical plasma membrane releasing exosomes containing both membrane and cytosolic proteins [42]. The proteomics study with LC-MS/MS coupled with upstream one dimensional SDS-PAGE separation experiments had disclosed a number of proteins associated with exosome biogenesis like class E vacuolar protein sorting (VPS), ALIX, Aquaporin 1, Aquaporin 2, ESCRT, etc [43]. A total of 295 proteins of urinary exosomeswere found to be associated with renal diseases and hypertension. These have been enlisted in Urinary Exosome Protein Database [42]. In another experiment where polypeptides were considered reflect that β2- microglobulin could be an indicator of damage of renal proximal tubule cells [42,44]. The techniques used to evaluate exosomal protein change is carried out by two dimensional difference in gel electrophoresis and change proteins are identified by mass spectroscopy and validated by Western Blotting [45]. Zhou et al worked with Fetuin-A, a protein of liver as an important exosomal protein that can indicate occurrence of AKI(Acute Kidney Injury) [45].

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Early Molecular Biomarkers for Renal allograft status

Years of research with renal allograft injury for either Acute Kidney Injury (AKI) or Chronic Kidney Disease (CKD) suggest that instead of invasive detection of allograft status there are scopes for early and non-invasive detection of injury through molecular markers. The studies made at the molecular level have disclosed the fact that acute and chronic rejections to a transplanted graft at preliminary stage can be ascertained by alteration in levels as well as expressions of various molecular markers involved in signaling of graft injury. These can be measured from blood/urine samples of patients. In acute rejection the early pathological change is defined by Ischemia-Reperfusion Injury (IRI) where altered expression of various miRNAs [46] is observed 3-7 days post-injury [47]. At later stage when rejection is in progress changes in levels of miR-210,-10a and -10b as well as some proteins (like perforin, granzyme A andB mRNA, FAS Ligand, FOXP3, etc) are observed [48]. Chronic rejection in early graft injury is generally associated with Interstitial Fibrosis and Tubular Atrophy (IF/TA). Pathophysiology of IF/ TA is the deposition of Extracellular matrix (ECM) proteins and Epithelial-Mesenchymal Transition (EMT) which can be stimulated by Transforming Growth Factor beta (TGF-β)/Smad signaling cascades. Ample of literature suggest that TGF-β/ Smad signaling can cause up-regulation and down-regulation of various miRNAs (miR-21,-192 & miR-29 and -200 families under IF/TA conditions) [49,50]. Even though these biomarkers have provided fruitful information but they lack specificity and their cellular source is unknown since they circulate. So to get a much clearer picture of a particular injured cell research at molecular level have unrevealed the next generation biomarker –exosomalmiRNAs for early, specific and non-invasive detection. Moreover their cellular source is also defined so they can deliver exact status of a particular cell [1,8].

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Urinary Exosomal proteins and miRNAs in renal allograft injury as Next Gen Molecular Biomarkers

Studies done with renal diseases is pretty less and still a burning area of research that reveals the fact that urinary exosomal proteins as well as miRNAs can be a potential therapeutic tool for kidney and associated diseases [1,8].

The urinary exosomal proteins can be easily attainable by noninvasive means for diagnosis of ESRD as well as Urinary Tract Infection (UTI) [1]. In 2006 Zhou et al. reported that urinary exosomal protein- fetuin A was found to be increased in AKI (Acute Kidney Injury) patients in ICU than AKI patients in normal care [1,41,45]. In 2008, same group discovered that activating transcription factor-3 (ATF-3) was found in exosomes isolated from AKI patients than CKD patients or control [41,45,51]. Thus suggesting urinary exosomal proteins could be a diagnostic tool. Moreover, a reduced level of urinary exosomal aquaporin-1 has been observed in ischemia-reperfusion injury in rats [7]. ExosomalmiRNAs demonstrate potential diagnostic biomarker for renal fibrosis [8]. MiR-29c and CD2APmRNA [52,53] were observed in urinary exosomes of renal fibrosis patients. The findings by Stefano Gatti1 et al. showed that bone marrow derived Mesenchymal Stem Cells (MSC) Microvesicles (MV) when administered immediately after IR injury can prevent AKI and CKD in rats [8,54] through their paracrine actions. Tara K Sigdel et al have described that in AKI patients with perturbation exosomal proteins like CLCA1, PROS1, KIAA0753 were observed. In addition to that exosomal ApoM is more than soluble ApoM [55]. M.W. Welker group found that in patients with chronic Hepatitis C serum soluble exosomal CD 81, a surface protein marker is elevated [56]..

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Future Prospective and limitations

Lots of work have been done with circulating miRNAs but due to their less specificity with the exact status of injured tissues and accuracy in determining role of a miRNA and its cellular source, still more feasible molecular markers have been searched and scientists have found that the circulating vehicles of cells-circulating exosomes that carry respective cellular cargo to the target sites to conduct cell-to-cell communication can be an option. These can be more proficient in delivering the most specific information on the status of any cell, may it is normal or injured cells. The molecular composition of exosomes that has been found till date is being recorded in the ExoCarta database. By exploiting these data in different pathological diseases scientists have done lots of research with carcinomas. In renal diseases also these exosomal miRNAs are being used to find out a means for noninvasive early detection of graft rejection. The conclusion drawn from these studies that proteins like fetuin-A and activating transcription factor-3 (ATF-3) can be used as marker in acute kidney disease and miR-29c and CD2AP mRNA are identified from urinary exosomes in renal fibrosis patients.

Thus, the various convergent studies made in the field of transplantation have led to the discovery of potential therapeutic targets- non-invasive urinary exosomal miRNAs and proteins which can be used to investigate and confirm the injury of transplanted allograft at an early stage. Though the data obtained define exosomes as an appropriate marker when compared with mRNAs, still it has few limitations:

Diverse isolation procedures that can affect exosomal contents,

Exosomes containing large number of miRNAs that affect the signaling of the cell together but not itself alone and

TDifficultly in measuring the exact quantity of a particular miRNAs in a exosome when miRNA is in low concentration.

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Conclusion

The exosome cell-to-cell communication mechanisms’ experiments are still at its infant stage. There is the need for development of more sophisticated techniques to detect the exact amount of specific functional exosomal proteins and miRNAs and their proper signaling pathways. Thus more investigation are still required to exploit exosomes in clinical fields as therapeutic targets.

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juniperpublishers-chemistry · 2 years ago

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Is US Patent Policy Strong Enough to Withstand the Winds of Change: A Study of the Need to Change United States Patent Policy

Author by Kent R Acheson

Abstract

The purpose of this case study was to learn how US patent policy requirements differ for the Software and Pharmaceutical Industries, specifically if United States Patent Policy adequately protects intellectual property rights [1] for two divergent industries while still encouraging research and development (R & D) investment sufficient to increase profits and innovation. Data for this study consisted of 38 witness testimonies delivered to US Congress between the years 2005 and 2010 by experts representing the two industries of interest: pharmaceutical and software. Key findings from the data analysis of these 38 testimonies revealed both within industry differences and between industry differences in patent law protection. Within industry differences showed variance based on size of the company and the degree to which they relied on their own R & D. Between industry differences reflected divergent ‘products’ with Pharmaceutical Industries needing long-term protection to recover R & D expenditures that include expenses for human trials research to proceed from patent application to market. Software industry, on the other hand, uses follow-on innovation of others to continue technological advancement by constantly improving upon existing software. The data show that these two industries use patent policy protection in different ways for different reasons. This information will enable Policymakers to develop another form of product protection in lieu of the current patent law to better meet the needs of these two industries rather than try to modify the existing law.

Introduction

Patent law was developed in parts, building on one another with a single purpose in mind of protecting all innovations in a society and this created the basis for patent laws imposed on the current and future generations. Bessen [2,3] stressed that patents may not be valuable in protecting innovation [4-6] but are used solely to diffuse new ideas in the public. Bessen and Maskin [7] had previously highlighted that little research and development (R&D) in the Software Industry is used to gain patent protections and the enforcement issue with patents is difficult, as many patents are issued for products that are not new. Levin [8] and others found much earlier that patents were rated weak at protecting the returns on innovation, far behind the protection gained through lead time and learning curve advantages.

Patent’s role in different industries

The purpose of this qualitative case study was to explore the different requirements for patent policy for the Software and Pharmaceutical Industries. All transcripts from testimonies from the spokespersons representing the two industries introduced to either House between the years 2005 to 2010 concerning the U.S. Patent Reform Bills were collected and analyzed to answer the research question in this case study. The findings could be useful in further adjusting patent policy to encourage innovation for diverse industries, or suggest the creation of another form of idea protection.

A similar problem may be in the type of intellectual property protection that a company chooses to obtain to avoid the constraints of getting a patent and extend the time frame for protection, such as copyright protection that extends protection from the 20 years for a patent to 120 years. Apple Inc. obtained a copyright protection for their popular iPhone [9], which recently lost in a suit against the Federal Government. The landmark decision helps to control copyright creep. Initially when buying an iPhone, Apple Inc. limited the service provider to AT&T and applications had to be bought from the Official Apple Store. Now, however, through a hack on the iPhone, users can choose a different service provider and load other, unofficial, applications not supported by Apple Inc., and hackers are not in violation of Copyright Law.

Policy Makers can use the findings of this study to explore new directions for the United States Patent Policy to optimize advancement of technology in the Software and Pharmaceutical Industries. Historically in the United States, there has been one patent policy. Scholars, academicians, and the United States Government still do not know the ideal amount of IPRs mainly because the objective has been to uphold one uniform policy. This study clarified if further changes were needed for patent policy through a Patent Reform Act, which enables Policy Makers to understand the needs of the Software Industry, or design another form of protection designed specifically for the Software Industry.

Crowe [10] and others stated that a case study design is most appropriate when little is known of a phenomenon in its natural context. A case study is “used to generate an in-depth, multifaceted understanding of a complex issue in its real-life context” (p. 1). The Pharmaceutical Industry has a profitable track record using the existing Patent Law to protect their R&D investments. The Software Industry is comparatively new and therefore their issues are only just now becoming obvious. Case law is outside the boundaries of this study.

The multiple dimensions of the phenomenon of the nature of protecting intellectual property rights in the Software Industry property and the Pharmaceutical Industry are worthy of study to allow all voices to be heard, including large corporations from both software and pharmaceutical companies, generic drug companies, and smaller software startups. After carefully examining all relevant transcripts, these diverse opinions can be given venue to state their needs.

Methodology and main results

The research question addressed in this study was: How do the patent policy requirements differ for the Software and Pharmaceutical Industries? From the Software and Pharmaceutical Industries’ objectives and needs for the United States Patent Policy to address, the questions spotlighted the sufficiency and effectiveness of the United States Patent Policy.

The focus of this study has two parts, they are:

1. What is the evidence United States Patent Policy adequately protects Intellectual Property Rights (IPRs) for both the Software and Pharmaceutical Industries?

2. How does the United States Patent System encourage companies to make R&D investment in the Software and the Pharmaceutical Industry?

The first research question dealt with the effectiveness of the United States Patent Policy in protecting IPRs in two industries: software and pharmaceutical. The second research question related to how companies invest in R&D with support of the United States Patent Policy. The study explored the ability of the United States Patent Policy to foster innovation with satisfactory IPR protection to encourage R&D spending focusing on two specific industries. The Software Industry experiences a sequential and complementary nature of innovations, building on previous discoveries; and may not use the patent policy in effect in the United States. If patent policy does not consider the different requirements within the Pharmaceutical Industry and is too lax then enough R&D spending will not be invested and technological advancements, including new medications, may come to the market slower or not at all.

The scope of the study is to understand how the Software and Pharmaceutical Industries use the patent system and how better to adjust the patent system to optimize technological advancement. As discussed in assumptions, because of the nature of the source of data and the possible bias that was not fully known, the assumptions may or may not have had a credible or dependable basis and may therefore have biased the findings. Qualitative designs such as the case study have inherent limitations that may threaten validity, they may lack rigor and they may not be generalizable. These limitations may be mitigated with transparency in data analysis and reporting. Crowe 5 and others explains on page 8 “seeking potential, alternative explanations, and being explicit about how interpretations and conclusions were reached, helped readers to judge the trustworthiness of the case study report.

Evidence from various sources highlight the United States Patent system does not work as intended and needs a solution to continue or increase innovative activity. The principal problem deals with innovative activity that is sequential in nature and innovative activity that involves much R&D investment to bring a product to market. Sequential inventions build on previous breakthroughs and do not require much R&D investment. Secrecy would hinder follow-on discoveries of sequential innovative products.

This study used a content analysis of witness [11] testimonies to Congress on the Software and Pharmaceutical Industries from the years 2005 to 2010, and the possibility to develop more than one patent policy to accommodate different sectors of the economy. The study concentrated on software and pharmaceutical companies, as these two industries are most at odds with each other, and have prevented the passage of the Patent Reform Act of 2005 through 2010. The Patent Reform Act of 2010 [12,13] is the result of non-passage of the 2009 Act, as was each successive year from 2005. The stance of the Software and Pharmaceutical Industries remained relatively unchanged in their requirements, but the patent reform acts changed to incorporate the majority opinion of industry. The most important recommendations of the Federal Trade Commission (FTC 11) and National Academies of Sciences (NAS) studies that were first introduced in 2005 by Senator [14] Lamar Smith were considered.

The purpose of this descriptive analysis was to examine the current United States Patent Policy and the proposed changes to United States Patent Policy, and answer the research question – How do the patent policy requirements differ for the Software and Pharmaceutical Industries? This study will help decide if the Software and Pharmaceutical Industries effectively use the U.S. Patent Policy through protecting Intellectual Property Rights (IPRs) and encouraged investment research and development (R&D). The qualitative case study was the most suitable approach to study the issues and answer the research questions because it explored real-life experiences of industries looking to patent Intellectual Property (IP).

Data and Sample Statistics

Data were collected and analysis began using the Content Analysis Guide developed for this study. The testimonies of the BSA representatives, other computer software witnesses, Computing Technology Industry Association, PhRMA representatives, other generic pharmaceutical representatives, and the Generic Pharmaceutical Association, Biotechnology Industry Association (BIO), Intellectual Property Owners Association (IPO) [15-18], and venture capital organizations were included in this study. The IPO was included because IPO members represent 30% of patent applications at the USPTO and include members from the Software and Pharmaceutical Industries, among others. The study included Venture capitalists because some members of BSA [19] and other smaller software companies began with venture capital dollars. Each data point was examined for inclusion of any reference to R&D, including duration and support for R&D, the need for patent protections [20,21], and future needs for patent policy.

The 38 documents submitted to the congressional hearings were analyzed. Documents relating to software and pharmaceutical companies reviewed were not ambiguous but very clear and straight forward following a consistent format, so that anyone conducting another study would reach the same conclusions. They all stated who authored the document, who the document represented, who presented opinion to Congress, their position on the patent reform act, and agreements and disagreements with specific points of the patent reform act. No ambiguity existed and no information required subjective judgments to interpret the information reported. The nature of the data supported the reliability of the findings.

Cisco, Hewlett-Packard, and other big high-tech companies began pushing for reform legislation to limit the number of patent infringement lawsuits and therefore the amounts paid in damages. The United States Patent and Trademark Office’s (USPTO) proceedings’ transcript from the public hearings showed the patent policy needs for BSA’s principle member and founder Microsoft. The public hearing titled Use of the Patent System to Protect Software Related Inventions took place in 1994 at the San Jose Convention Center, California, and at the Crystal Forum in Arlington, Virginia. A brief summary of Microsoft’s speech follows. Microsoft (BSA) recommended that patent protection allow an accused infringer to identify readily the activity forbidden under the claim. The success of a particular claim in meeting these objectives may depend less on the form and more on claim substance and the supporting details.

BSA represents a large base of computer software and hardware companies in the United States. Phelps (2005) from Microsoft Corporation stated that BSA does not want the patent holder to have automatically injunctive relief. Injunctive relief occurs when the courts rule an infringement occurred and automatically issue a ruling to stop the infringer from continuing operations. From the congressional hearing in 2005 on harmonization and other matters, Phelps for BSA supports publication in 18 months. Phelps [39] expressed support for establishing a post grant opposition procedure and supported third-party opportunity to alert USPTO to questionable patents during review. Phelps also supported allowing third parties the opportunity to suggest relevant prior art to examiner during review, supported a limit on damages for willful infringement to include only egregious behavior, and supported limiting damages to only the contributing, patented piece of the invention and not the market value of the whole product, as it is now.

In a congressional hearing in 2005, Simon [40] from Intel , a BSA representative, stated the patent system is difficult to maneuver because of many pieces that comprise computers and software contain “potentially hundreds of patents [that] may be relevant to a particular computer or software technology” [40]. The primary way to challenge a patent under current law is through costly litigation. Intel suggests Congress create a balanced post grant opposition enabling third parties to challenge issued patents that includes a post grant opposition of 2 years from patent grant or 1 year from receiving notice of patent infringement. Simon also encouraged Congress to create a second window to make the post grant review meaningful. Simon suggested a limit on patent application continuations and for the court not to issue a continuation on any claim broader than the broadest claim previously published or issued. BSA suggested a stay on the lower court’s decision in interlocutory appeals before final determination by the Federal Circuit Court of Appeals. Micron Technology, Inc., a non-BSA member, suggested the same patent law reforms as BSA.

In a congressional hearing in 2006, Chandler [41] of Cisco (BSA) suggested a second window triggered by receipt of an infringement complaint. During the first window, the patent issues with thousands or millions of parts making the effectiveness of the patent examination questionable. Chandler (2006) encouraged Congress to make changes to remove venue shopping, and prevent suits from worldwide damages in United States Courts like the Microsoft and AT&T case. The only patent policy need described on the BSA website dated 1994 had no updates, which is understandable because United States Patent Policy has not changed significantly for more than 50 years and the proposed changes have not made it into law. The agreement with the Patent Reform Act was from the most influential voice for the Software Industry; nevertheless, there were disagreements within the Software Industry mainly arising from smaller companies and individual inventors. Software companies wanted patent reform by Congress but differences remained among large software companies and smaller organizations. An overhaul of the patent system and other measures to promote tech development efforts are top priorities of the Business Software Alliance, Cisco, Hewlett-Packard, and other big high-tech companies . BSA members began pushing for reform legislation to limit the number of patent infringement lawsuits, and therefore, the amounts paid in damages.

In an article in PC World dated March 9, 2008, patent reform leads a list of five legislative priorities expressed by BSA in 2008. The opinion article stated that BSA members want Congress to approve the Patent Reform Act but the legislation stalled in the United States Senate because of objections from inventors, pharmaceutical companies and some small tech (computer software) firms. In addition the article proclaimed, more than 170 California businesses and organizations oppose the Patent Reform Act in its current form. They mention that research to stay competitive is both expensive and risky, but strong protections from patent policy attract the necessary investments to commercialize a new product. This is especially the case for the hundreds of smaller, venture capital-backed firms in the state, of which many spun from California’s world-class research universities and private research institutes. According to GlaxoSmithKline, California Wireless and Mi5 Networks in paragraph eight on page one of Gross [42] (2008), the Patent Reform Act “would increase costs to obtain and maintain patents, undermine patent certainty, incentivize infringement, and weaken the enforceability of patent rights and intellectual property protections.”

Dr. Myhrovold [43-45] started Dynamical Systems, a software company, in 1984 that Microsoft bought in 1986. He worked with Microsoft from 1986 to 2000 (14 years). Myhrovold retired from Microsoft in 2000 to start another company, Intellectual Ventures, which files more than 300 patents a year making it the 25th largest inventing organization in America. Dr. Myhrovold stated “[Software is] a complex topic…and it’s all about company culture and how companies use patents” (Perspectives on Patents [46,47]. Continuing Dr. Myhrovold stated “…for most tech companies patents have never been important; they have never been a way to make money” (p. 76, para. 2), and “…patents are, at best, a distraction and most tech companies have made a deliberate decision to ignore the patent system” (p. 76, para. 5). Many other non-BSA members agreed with Myhrovold.

Defensive patenting by software companies explains if a company holds enough patents then this company can steal another product company’s ideas with impunity, but the problem enters when the other entity does not create a product to attack (Myhrovold, 2006, p. 77, para 3). These are the battle lines in the patent reform debate with universities, small inventors and pharmaceutical companies whose lifeblood is the patent system on one side, and companies who decide to infringe or at least do not care about infringing on the other side. Dr. Myhrovold is a witness from the vantage point of a Microsoft senior executive in the 1990s who discussed this role with other firms in the earlier rounds of patent reform debate.

Technology companies exaggerate the problem when over the last 20 years patents have remained in last place of lawsuits for the three forms of idea protection: trademark, copyright, and patents. A study of four high-tech companies that are active in the patent reform debate paid out $3.7 billion in patent lawsuit settlement from 1993 to 2005, but those same four companies earned $1.4 trillion in revenue over the same period making the sums for infringement only 0.26% of revenues on average. The company with the highest number of lawsuits experienced sums for infringement at only 0.51% of revenues. “Patent trolls” are companies that do not market a product but only the idea for a product. Companies that do not produce a product comprise only 2% of the patent infringement lawsuits. Software companies like to blame an innocuous group of patent troll companies when they themselves perform the same litigious practices blamed on trolls. Myhrovold stated the need to embrace the trend to make the alternate resolutions more like a court trial by creating a separate Patent Court, much like the Tax Court, Bankruptcy Court, or Divorce Court to try only specific cases.

Inter Digital is a technology and software company that disagrees with BSA’s proposed changes to patent law. Inter Digital’s Bernstein summarized the differences in the Software industry on page 220 last paragraph at the 2007 congressional hearings: “…the IT industry is absolutely not united in its support for mandatory apportionment, post grant opposition, expansive USPTO rulemaking authority, and interlocutory appeals fall outside the realm of patent ‘reform’.” Bernstein continues by expressing how such an action would degrade patent rights and increase litigation for smaller innovators. The weakening of legitimate patents would protect a few corporate giants and increase the number of lawsuits Bernstein (2007), [48,49].

An article by Mc Dougall [50] and Chabrow (2006), [51,52] in InformationWeek explains the problems as they perceive them with the Patent Reform Act from other software and computer companies. Hans Hxu, founder of online gift registry Felicite.com, says the industry’s large players want the appearance of IP openness but do not practice it. “IBM patents almost everything they do, and then they sit on it, which does not encourage innovation” (Microsoft Agenda, para. 3) says Hxu, a McKinsey consultant although other critics suggest the sellers’ moves cement their advantages when they face rising [53] competition from startups. In an August 2005 essay, Harvard Law School professor and tech entrepreneur James Moore argued the collaborative patent review proposed by IBM, Microsoft, and others would result in fewer patents issued because it would give examiners more ammunition to shoot down patent applications. “If fewer patents are issued, but existing patents are not revoked, IBM and Microsoft win because they already possess vast existing portfolios,” Moore writes (Microsoft Agenda, para. 4). Some Web 2.0 companies dismiss IBM’s argument that business-method patents protect obvious ideas. “Everything is obvious after someone has done it,” says a spokesperson for online movie renter Netflix (Microsoft Agenda, para. 5), which has patents on its queue-ordering system--and is suing Blockbuster for allegedly copying the system.

Small tech companies are taking matters into their own hands, forming patent cooperatives through which they share IPRs. Search company Wink shares in Creative Commons, a group that encourages sharing of copyrights and open source licenses, but there is a line between sharing and protecting intellectual property that creates competitive advantage, says Wink’s Chief Executive Officer (CEO) Michael [54,55] Tanne. “When companies have invested in the development of technologies, they really ought to be able to protect it,” Tanne says (Microsoft Agenda, para. 6). Resolving these issues will influence developing and commercializing tech innovations. Too many lengthy and expensive legal battles will persuade IT departments to stick with familiar technology, and this is something tech vendors should consider as they take one another to court.

The largest and best known pharmaceutical companies in the Pharmaceutical Industry represented by Pharmaceuticals Researchers and Manufacturers of America (PhRMA), Biotechnology Industry Organization (BIO), and the Professional Inventors Alliance disagree with the weakening of patent protection and the long, time frame proposed for patent reexamination. High R&D characterizes these industries and the Pharmaceutical Industry realizes a shortened patent protection because patent protection begins before FDA approval. This shortens patent protection to commercialize the product to the remaining years.

On September 17, 2007, The Professional Inventors Alliance expressed through a letter to President Bush the flaws in the Patent Reform Act of 2007. The Patent Reform Act of 2007 did not pass the United States Senate because of the opposition from PhRMA, small inventors, and small tech firms . The letter from the Professional Inventors Alliance expressed that if the Patent Reform Act of 2007 passed into law it would harm the United States’ innovative character because of the inability to enforce patents and would reduce the royalties associated with a patented technology. In 1980, PHRMA’s members invested $2 billion in R&D for new medicines; although, nearly 30 years later (in 2009), PHRMA’s members invested $50.3 billion in R&D out of the $65.2 billion industry-wide total. Pharmaceutical companies rely on government-granted patents to protect their substantial investments in researching and developing new drugs. It takes 10-15 years and costs $800 million on average to bring a new medicine to market. The Pharmaceutical Research and Manufacturers of America (PhRMA) represents the country’s leading pharmaceutical research and biotechnology companies.

Without patents to protect all the inventions necessary to develop a drug for a limited time, others could simply copy the drugs immediately, offering their versions at a reduced price because they did not incur the high costs to develop the drug. This would seriously affect the pharmaceutical companies’ ability to recoup their costs and reinvest in other research projects. PhRMA stated in 2010 that “a strong patent system is crucial to our economic [56,57] competitiveness, especially in these economically trying times” (PhRMA’s website, 2001, p. 1). The companies in favor and against the Patent Reform Act of 2010 divided into the companies that have favored and opposed the previous patent reform acts, that is, computer software favoring patent reform and pharmaceutical companies and biotechnology companies opposing patent reform. Those opposing and in favor of the patent reform acts through the six years in this study have not changed their needs but, instead, Congress changed trying to create a patent policy agreeable to most patent users.

The large pharmaceutical companies also known as the name brand pharmaceutical companies and the smaller, generic pharmaceutical companies were in general agreement on most issues. Both wanted strong patent protection and both sides were against the Patent Reform Bill [58] of 2005 and 2006 as stated in the congressional hearings on patent reform. The firstinventor- to-file patent system while harmonizing with the large United States trading partners also poses some difficulties and disagreements with United States patentees. The problems lay in the grace period of 1-year and the best mode requirement in the patent application. Harmonizing with other countries’ patent systems as currently written, such as Japan and Europe, would remove the United States grace period of 1 year to file a patent application and would remove the best mode requirement when filing a patent application. The best mode requirement is the descriptive part of the patent application the inventor has to include the inventor’s idea of how best to use or combine the chemicals for complete effectiveness.

The differences between the brand name and generic pharmaceutical companies lay in eliminating the best mode factor of the patent application and the inequitable conduct defense. Brand name pharmaceutical companies say the best mode provision of the patent law is subjective, and therefore should be removed. The generic pharmaceutical companies believe the best mode provision should remain because they cannot copy the patented medication without the recipe or the “best mode” of making the drug. By removing the inequitable conduct defense, brand name pharmaceutical companies will misuse the patent system to the harm of the public and generic pharmaceutical companies. Differences exist between the brand name pharmaceuticals and the generic pharmaceuticals. One example is the issue of patent quality: Best mode. Generic pharmaceuticals want to keep the “best mode” in the patent law language because it lowers cost of medications by allowing generic companies to copy name brand drugs more easily. Ely Lilly [59,60] and PhRMA want to remove the best mode language . The Generic Pharmaceutical Association also has qualms with weakening the inequitable conduct saying that weakening this provision gives brand-name pharmaceutical companies incentive to misrepresent their inventions.

Together the Case Lawre presented the most comprehensive line of court-led patent reforms, which makes patent reform substantially different in 2010 than 2005. Patent lawyers and the law association, AIPLA [63,64], believe that legislation is not necessary and the court system will eventually find a solution for compromise for the different users of the patent system and will define patent law through successive Case Law. Larger, more market capitalized firms make more noise and are heard more clearly than smaller, less capitalized companies or individual inventors, including companies that specialize in innovation but do not concurrently produce a product, also known as patent trolls. More innovation comes from smaller firms and individual inventors than large entities. The larger software enterprises that often infringe on patents held by companies that do not produce a product (patent trolls) behave similarly to the patent trolls. IBM and Microsoft sit on patents without an accompanying product, when another company discovers something similar the patent surprises the unsuspecting company, and a licensing or royalty agreement can avoid costly litigation. IBM earned over a billion dollars in 2005 solely from license agreements and royalties. Licensing and royalty agreements are another possible direction that companies take to avoid patent infringement suits; however, their use threatens other companies to ransom licensing or royalty agreements but is cheaper and the outcome more certain than litigation.

The Pharmaceutical Industry appreciates the current patent policy and is leery of any changes that would disrupt the current manner in which they use the patent system to optimize patent protection; also the Pharmaceutical Industry like the Software Industry makes the best of the current patent policy . Although pharmaceutical firms have to wait until after drug trials and resulting FDA approval to market the medication, which includes the 20-year patent term and drug approval sometimes lasts as much as 10 years, they too have found ways to evade current patent law to extend the patent length. The Pharmaceutical Industry commonly increases the shortened patent length by adding a known chemical to the patent protected drug therapy, and adds another patent protection term of 20 years by increasing the number of patents on a drug. One specific drug therapy created by a name-brand pharmaceutical firm that a generic company was exploring to copy had patent protection by more than 200 patents spanning 40 years.

Discussion and Conclusions

The specific research questions that framed this qualitative case study were 1. What is the evidence United States Patent Policy adequately protects Intellectual Property Rights [65] (IPRs) for both the Software and Pharmaceutical Industries? 2. How does the United States Patent Policy encourage companies to make research and development (R&D) investment in both the Software Industry and in the Pharmaceutical Industry? Based on the differences on how patent policy should read, issues of effectiveness of the United States Patent Policy to both protect and encourage IPRs and R&D investment should be considered. Patent policy in the United States has remained unchanged for the last 55 years, and has been effective in protecting IPRs and encouraging R&D investment. Pharmaceutical firms have been around many years and have flourished in the current patent policy environment. Only with the creation of the personal computer have software companies entered the scene and have expressed concern for the patent policy changes to reduce the software company’s purposeful infringement. In a few words, the large software companies want to weaken patent protections and reduce their costs to defend against patent infringement lawsuits because big software companies do not care about patents or patent infringement.

Three important findings from this study are

1. The Pharmaceutical and Software Industries use patent policy differently

2. BSA explicitly states they want a strong patent policy, but, in effect, want to weaken the current patent policy, and

3. Differences exist within each industry. Congress has attempted to improve patent law 6 years without success because there is not agreement pleasing all industries, but the principle differences embodied the Software and Pharmaceutical Industries.

Firstly, pharmacy and software use patent policy differently: Pharmacy to protect R&D and Software for defensive purposes. Software Industry (BSA) does not use the patent policy as designed to protect R&D, but to defend against the threat of patent infringement lawsuits. The testimonies to Congress provided evidence to answer my research question of how the patent policy requirements differ between the Software and Pharmaceutical Industries. The testimonies to Congress were clear and straightforward. I did not have to infer the meaning or needs of the witnesses. They clearly stated their position and what they wanted in patent policy. Many people in the Pharmaceutical Industry and smaller software companies specifically stated that larger software and computer companies began calling for patent reform to limit the many patent infringement suits against them. Myhrovold shared his experience working for Microsoft in the late 90s stating that large software companies are not concerned with infringing on another’s patents and the only reason they care at all about patents is to defend against patent infringement lawsuits.

Secondly, the data from congressional testimonies clearly showed that the Software Industry (BSA) verbalized they want a strong patent policy but, instead, they want to weaken the rights of patent holders. This weakening is from: An unlimited post patent review period, placing the burden of proof for infringement on the patent holder (instead of the offender), and limiting the damage awards for infringement to only the infringing part of an innovation. The testimonies clearly stated their position and what they wanted. The previous list clearly communicated to Congress what the Software Industry (BSA) wanted in a patent policy, and refuted by other expert testimonies in the Software Industry.

All BSA representatives stated they wanted strong patent protection, and continued with the above reasons, which amount to weakening a patent holders’ legal rights to their Intellectual Property Rights (IPRs). Many testimonies contrary to BSA stated specifically the reasons BSA wants to limit a patent holders’ IPRs is to stave off patent infringement lawsuits. Myhrovold (2006) shared that patent policy did not enter into Microsoft’s and other BSA members’ culture. Patents are not how software companies protect innovation, but, rather, secrecy, and lead time or economies of scale are more effective to protect innovation in a short product lifecycle industry. Thirdly, the entire Software Industry is not united with BSA, and the entire Pharmaceutical Industry is not united with PhRMA. Differences exist between the two industries and differences exist within each industry, such as difference between larger companies and smaller companies in Software Industry and brand name pharmaceutical versus generic pharmaceutical. Each expert clearly stated what they wanted, why they wanted it, and differences within their respective industries. The witnesses to the congressional hearings succinctly stated that the BSA or PhRMA did not represent the entire industry, and the industry was not united in its desires for patent policy. Siwik [66] said in the exact words that the Pharmaceutical Industry is not united, and based on the non-BSA members’ testimonies with them vehemently disagreeing with BSA’s stance, anyone would reach the same conclusions that BSA is far from united too.

The evidence suggests the two industries use patent policy in different ways. For instance, The Software Industry does not use the patent system to protect intellectual property but rather use the patent system for defensive purposes not so much to protect innovation but to defend against infringement lawsuits. Pharmaceutical industry relies heavily on a patent protection to recover large R&D spending. The evidence was found in examples of how each industry effectively uses the patent system. Based on research of the patent system and the evidence of how each industry uses the patent system, the data would suggest agreement with many of the pharmaceutical, biotechnology, and other industries that use the patent system effectively to protect research and development dollars that the system does not need major change. Research shows the answer to the question of how the United States Patent System encourages R&D and promotes innovation; the patent system performs well according to its design. It protects ideas. The current patent policy is effective in protecting innovation and encouraging research and development spending.

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juniperpublishers-endocrinology · 2 years ago

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Aldosterone and Parathyroid Hormone: Evidence for a Clinically Relevant Relationship-Juniper Publishers

JUNIPER PUBLISHERS-OPEN ACCESS JOURNAL OF ENDOCRINOLOGY AND THYROID RESEARCH

Introduction

A ‘new endocrine axis’, involving the bi-directional relationship between the parathyroid hormone (PTH) and the renin–angiotensin–aldosterone system (RAAS) has been established recently. Individually these have long been recognized, although it is only in recent times that we are realizing the interplay between the two and the corresponding effects this has on the physiological and pathological roles within the body. Other calciotropic hormones such as Vitamin D are also impacting on this relationship [1]. This report aims to highlight the cyclic nature of these relationships, through the physiological pathways, which will then lead into pathological disease in multiple areas such as heart failure, cardiovascular health and bone homeostasis. It is through this it is hoped to highlight this new bi-directional relationship.

Physiological effects of Calcitrophic Hormones on the RAAS

PTH is thought to influence aldosterone secretion in the zona glomerulosa of the adrenal glands [2]. This was demonstrated by Mazzocchi et al. [3], who found that PTH exerts a significant stimulatory action on aldosterone secretion by adrenocortical cells, through the PTH/PTH-RP receptors. PTH is also thought to directly stimulate renin release in the myoepithelial cells of the preglomerular arterioles [4]. Increased activity of the RAAS, and raised aldosterone levels, have been demonstrated in patients with primary hyperparathyroidism. Parathyroidectomy, and its associated reduction in PTH, has been shown to result in normalization of the RAAS system and aldosterone levels.

Physiological Effects of RAAS on the Calciotrophic Hormones and Calcium Homoestasis

Recent observational studies in individuals with primary hyperaldosteronism suggest that excess aldosterone may result in hyperparathyroidism. A better understanding of the normal physiologic relationship between the RAAS and PTH is of clinical relevance since inappropriate activity of both PTH and the RAAS may negatively impact cardiovascular and skeletal health [5]. Hyperaldosteronism is associated with a reversible secondary hyperparathyroidism due to increased renal and faecal calcium excretion. PTH is increased as a result of the MR (mineralocorticoid receptor) mediated calciuretic and magnesiuretic effects, with a trend towards hypocalcemia, hypomagnesemia and the direct effects of aldosterone on parathyroid cells via binding to the MR. Moreover, the angiotensin II receptor is expressed by human parathyroid tissue, and angiotensin may therefore directly stimulate PTH secretion [6].

RAAS and Vitamin D

Aldosterone acts through the mineralocorticoid receptor, which belongs to the same superfamily of nuclear receptors as the vitamin D receptors (VDR). Therefore, cross talk between these receptors and their agonists could potentially exist. [7]. Vitamin D has been shown to have an inverse relationship with renin levels [8]. Tomaschitz et al. [9] demonstrated that vitamin D deficient patients had significantly higher levels of renin, angiotensin 2 and aldosterone. In a subsequent study, Vaidya et al. [10] found that higher levels of Vitamin D were associated with lower plasma renin activity under both salt conditions, supporting the hypothesis that vitamin D acts as an inhibitor of renin production.

Bi-directional interplay between PTH and RAAS in Different Systemic Diseases

Neurohormonal activation is a hallmark of chronic heart failure (HF). Low perfusion pressure due to impaired left ventricular function results in the activation of the sympathetic nervous system and the RAAS. Stimulation of adrenal aldosterone synthesis in chronic HF occurs despite sodium and fluid retention. The hyperadrenergic state leads to translocation of cations into the intracellular compartment and the increased aldosterone secretion stimulates faecal and urinary loss of cations. The resulting hypocalcemia and hypomagnesaemia stimulate PTH secretion which tends to restore extracellular calcium and magnesium homeostasis. The PTH promotes mitochondrial Ca2+ excess in the myocardium which leads to oxidative stress and necrotic cell death which, in the long term, causes or amplifies myocardial fibrosis thus aggravating systolic and diastolic HF [11].

PTH also directly stimulates aldosterone synthesis by binding to the PTH/PTH-rP receptor, voltage-gated calcium channels, and the adrenocorticotropic hormone receptor, thus triggering a vicious cycle of mutually reinforcing aldosteronism and hyperparathyroidism. Relative aldosterone excess causes sodium retention and oxidative stress, thus increasing CV morbidity and mortality [2]. PTH also contributes to the development of arterial stiffness, arterial hypertension, and cardiac hypertrophy via binding to the PTH/PTH-rP receptor, which is expressed in vascular smooth muscle cells and cardiomyocytes. In addition, aldosterone excess exerts genomic (by binding to the MR), and non-genomic profibrotic and proinflammatory effects on blood vessels and the myocardium [12].

Aldosterone-Parathyroid Hormone Interactions in Primary Hyperparathyroidism

Early evidence for a physiological and pathophysiological bidirectional link between aldosterone and PTH in humans had initially been derived mainly from case reports. Subsequent studies documented the presence of secondary-hyperaldosteronism in patients with primary-hyperparathyroidism, subsequently a marked reduction in plasma-aldosterone, angiotensin II and renin-activity was noted after parathyroidectomy [13]. Thus, in patients with hyperparathyroidism, the concomitant hyperaldosteronism can aggravate arterial-stiffness, endothelialdysfunction, blood pressure-elevation, vascular-remodelling, cardiac hypertrophy/failure and CVD.

Several studies revealed that patients with PA have alterations in calcium and increased PTH levels [14]. Treatment of PA with either MR-antagonist or adrenalectomy was found to decrease PTH levels [15]. PTH levels were significantly higher in aldosterone-producing adenomas compared to bilateral adrenal-hyperplasia, suggesting that the severity of aldosteronism correlated with severity of hyperparathyroidism. The concomitant hyperparathyroidism could contribute to skeletal diseases and compound CV-risk (beyond the CV-risk associated with PA) [2].

Aldosterone-Parathyroid Hormone Interactions in Bone Diseases

In addition to reversible elevation in PTH levels, reversible PTH mediated bone loss as evidenced by reduction in bonemineral density (BMD), osteoporosis and vertebral fractures were reported in patients with PA. Studies have demonstrated that surgery or medical treatment of PA reduced PTH levels and increased BMD on follow up. Though, bone loss is mostly mediated by PTH, additional mechanisms have been proposed, including a direct effect of aldosterone on bone (MR-expression has been identified in osteoblasts and osteoclasts). Spironolactone has been shown to lower fracture-risk in HF and RAAS-inhibitors lower PTH in CKD.

Primary and Secondary Hyperaldosteronism

Primary aldosteronism (PA) represents a major cause of secondary hypertension with high cardiovascular morbidity and mortality. Secondary hyperaldosteronism (SA) indicates a bidirectional functional link between the adrenal and parathyroid glands. PA patients have been shown to have a significant increase of primary hyperparathyroidism (PPTH) [16]. Conversely, undetected hyper-functioning of the parathyroid gland can contribute to maintaining PA [17].

Hyperaldosteronism and hyperparathyroidism represent pathophysiologic conditions that correlate with each other; aldosterone regulates parathyroid hormone causing adverse skeletal complications and PTH regulates aldosterone and associates with adverse cardiovascular complications [18]. PTH stimulates in vitro the secretion of aldosterone from human adrenocortical cells in a concentration dependent manner which suggests that PTH acts as an aldosterone secretagogue that may cause human PA [3].

High aldosterone levels are associated with high PTH levels [14,19]. Brunaud L et al [13], observed that elevated aldosterone levels, positively correlated with PTH levels in patients with primary hyperparathyroidism and PTH levels >100 ng/L were an independent predictor of abnormally elevated plasma aldosterone concentration. So, primary hyperparathyroidism is associated with high aldosterone levels and primary aldosteronism is associated with high PTH levels. It is also suggested that PA contributes to secondary hyperparathyroidism [20].

Effect of Therapeutic Interventions on the CalciCalciotrophic and Renin Angiotensin Aldosterone Systems

Role of PTH surgery on Hyperaldosteronism and CVD Risk Factors

Chronic heart failure, along with hypercalciuria, hypocalcaemia and secondary hyperparathyroidism with subsequent intracellular calcium overload is frequently found in patients with low-renin hypertension and primary aldosteronism. Interplay between the hormones regulating calcium homeostasis and the RAAS contribute to the pathogenesis of arterial hypertension. Brunaud et al. [13] reported significantly decreased aldosterone, angiotensin II, renin activity and blood pressure levels after parathyroidectomy with primary hyperparathyroidism.

Effect of RAAS Blockage and Adrenalectomy on Hyperparathyroidism and Vitamin D Levels

Drugs affecting the RAAS and surgical treatment is associated with an increase in ionised calcium and a decrease in PTH (Rossi E). Adrenalectomy or MR blockers both decrease PTH secretion, arterial blood pressure as well as bone resorption. Animal studies show strong support for vitamin D3 mediated downregulation of renin expression and RAS activity via its interaction with vitamin D receptor [21]. Appendix 1 shows the influence untreated low Vitamin D levels can have on cardiovascular health, via the RAAS and PTH pathways [22-24].

Conclusion

As shown, increasing evidence is showing a bi-directional interplay between calciotrophic hormones, such as PTH, Vitamin D and the RAAS system. Calcitrophic hormones, particularly PTH and Vitamin D, have a cyclic nature on the RAAS system, affecting each at different receptor and cellular levels. As discussed, there are multiple pathways to consider. For example, the role of PTH’s direct stimulatory action on aldosterone, or hyperaldosteronism’s association with hyperparathyroidism. Or how Vitamin D shares the same receptor as aldosterone, and in vitamin D deficient patients the direct effect on the RAAS, through significantly higher levels of renin, angiotensin 2 and aldosterone. PTH and aldosterone both influence different pathological factors. As shown, it is this relationship between these calciotrophic hormones and the RAAS system that can adversely affect the disease states such as CVD, HF, PA & SA. It is through the recognition of this new ‘endocrine axis’ that there is a clinically relevant relationship between these hormones. From this, surgical and therapeutic interventions can be tailored with this relationship in mind, to ensure optimum treatment outcomes.

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laceyspencer · 2 years ago

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The Crisis of our Time and the End of the New “History”-Juniper Publishers

Introduction

The time has come to understand that we are facing an anthropological and not economic crisis as it is reductively defined; it is the failure of a socio-cultural model that has erased the fundamental human rights inscribed in 1948. The response to the crisis [1] as anthropological is in understanding the cultural and historical path that has brought us to chaos, overthrowing the dominant paradigm, to place man and society at the centre of our interests as an end and to bring economics back to its natural role as a means. The technical culture, master of the world, as defined by Emanuele Severino, has unnaturally transformed economics as a social science into an exact science; in the exact sciences we study the relationships between measurable things to define universal laws, but in the social sciences, such as economics, we study relationships between men where human subjectivity does not allow defining universal laws [2-4].

Von Hayek in his Nobel acceptance speech in 1974 denounced the serious mistake that would pave the way for rational finance and speculative markets distant from the real world; but he remained unheard. The extreme financialization of the real economy became deeply rooted since 1971 when Nixon unilaterally declared the end of the dollar’s convertibility into gold. Let us look at the sequence of events. In 1945, after the drama of war, rules were defined to stabilize markets and exchange rates; the printing of paper money that has no value was tied to a defined quantity of a real good and the “gold exchange standard” was launched - 36 $ for every ounce of gold. In that period that lasted until the beginning of the 70s, we had a fixed exchange rate system that favoured unprecedented economic and social growth [5-7]. In 1971, the point of maximum social cohesion in the US coincided exactly with the point of lower inequality in income, providing evidence that the two aspects are closely related and that only a cohesive and just society can lead to solid economic development [8] (Figures 1 & 2).

All the positive curves of the “golden age” have an inflection and deteriorated between 1971 and 1974 as a result of Nixon’s decision; from the “gold exchange standard” we would go to the “petrodollar exchange standard” and to a system of flexible exchange rates, and we would suffer terrible inflation that went from 4.4% to 24%, paving the way for today’s drama of uncontrolled finance, as we see in following graphs (Figures 3-6).

Italy would suffer the influx of petrodollars, which began to drain the efforts made in the post-war period to bring the country among those with the highest growth rate; the price of oil per barrel went from $ 1.40 to $ 40, and the dollar/lira exchange rate after 25 years of the fixed exchange rate of 624/5 lira for every dollar to 2450 lira for a dollar in just 9 years: it was to be the first financial war, but in reality a third World War not fought with traditional means but with the power of the financial markets [9- 11] (Figures 7 & 8).

The separation of paper money from the real finite created two incompatible systems: the infinite and non-measurable of currency that would be totally deregulated, and the finite and measurable of the real world that became fictitiously subordinate to the former [12,13]. Everything changes, and the neoliberal model taken as an end justifies unlimited personal accumulation and the legitimisation of human aggression. Everything becomes finance and pure speculation in virtue of a paper currency that, detached from finite bonds and the real world, becomes infinite and would turn into “macro-usury” capable of keeping companies and entire countries in check [14,15]. Infinite finance without constraints can be studied with exact mathematical models generating the false idea that financial markets are rational and never err in the allocation of wealth. The study of financeeconomics severs the ties with the humanistic sciences, becoming a pure arithmetic calculation far from reality but assumed as incontrovertible truth thanks to the many Nobel prizes assigned to economics, unfounded but serving higher interests [16-18].

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The Role of the Humanistic Culture that the Academy has Lost

Since the late 60s, in awarding the prizes for literature, economics, and peace - the three prizes with the most obvious contradictions - the anomalies have become more apparent, favouring a cultural model and its interests that have brought us to the true crisis of our time, the anthropological crisis that we still refuse to see. Since 1969, when the first prize for economics was awarded, American scholars have won the lion’s share [19- 21]. In the 44 years of Nobel prizes in economics, one or more than one has been awarded the prize 41 times: a monoculture without inconsistency and change 41 times out of 44. Only in three years did they not win: 1969, 1974, and 1988.

The trend was accentuated after the fall of the Berlin Wall when the awards rained down on economics scholars who defined the financial markets as rational and exact without possibility of error [22]. Finance has become a sort of hegemonic weapon over States able to exert pressure on the policies of individual States and global choices. Wealth is thus created without States and States without wealth, a model of an individualistic and conflictual society in which moral sense is subservient to personal interests and the strongest command. Yet, is the soul of this cultural model able to inspire feelings such as kindness, altruism, solidarity, respect for humankind, in short, the ideals that Alfred Nobel sought?

The answer can be found with disarming evidence in the prizes awarded for literature. In fact, since the end of the 60s, the United States that seemed omnipotent has only won one real prize in literature. Morrison, in 94, expressed the racial pain of coloured minorities, now majorities; Bellow in 76 and Singer in 78, were the expression of the European culture where they had lived for a long time before moving to the United States [23-25]. The other awards over the years have been divided among different countries where the type of wellbeing expressed by the economy was absent or irrelevant - for example, Ireland, Peru, Chile, Saint Lucia, Poland, Romania, Greece... The two cultural models are opposed, without the possibility of dialogue and sharing because the interests of economics and finance put the maximization of self-interest in first place and not the “common good”, exactly what Alfred Nobel wanted to avoid. The legitimacy of the single thought has suffocated the imagination and the universal values of freedom, equality and solidarity. In the words of Pascal, “l’esprit de finesse” was finally separated from the “esprit de geometrie”, but rational man arrived last in the race. Everyone is responsible, albeit in different ways, because everyone contributed, even in silence, to ascribing the value of incontrovertible truth to these positions [26].

The technical-rational culture of the post-modern era prepared by the field of speculation since the Enlightenment with Kant, Hegel, and then Marx transformed economics into an exact science by studying only what is measurable. The materialistic objectives promoted by capitalism and liberalism assumed as an end and not a means contributed to the creation of a society aimed at achieving self-interest at the expense of the common good and the normalization of unlawful behavior [27]. This has increasingly forced dominant interests to legitimize such studies with the Nobel Prizes that elevated their achievement to ultimate truth but not the real sciences, eventually disrupting the system of social relations, because the dogma has become ‘live to earn’ and not vice versa. This year once again, in rewarding the disrupting sociocultural model of human society, the Academy has deeply betrayed the noble intentions of Alfred Nobel.

Understanding the deviations of economic studies transformed from a social and moral science into a merely quantitative, exact, and positive science requires understanding the causes that favour the interests of the few at the expense of everyone else. At the end of World War II, the global dramas led to defining the universal and “inalienable” rights of man and the technical operational rules of economics in such a way as to guide global policymakers to restore the dignity of man as a person and not as he is in fact today.

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Literature Review

Everything becomes finance and everything is justified as “strictly scientific” by the Nobel Prize in Economic Sciences; the pursuit of maximum personal gains justifies the normalization of illicit behaviours, everything is played in a short or very short term logic in an infinite exchange at high speed where computers decide according to algorithms based on the nothingness of infinite finance, without anything underlying it. Stock exchanges become infinite casinos without rules or maximum profit in an amoral logic because those deciding in finance never pose themselves the problem of the consequences of their actions, and since it is always man who engages in illicit behaviours they become a practice of life that extends to young people who lose their identity in the confusion of value, becoming plankton at the mercy of the waves. Society without values and a humanistic culture become territories in which it is difficult to live and the individualism pursued as an end eradicates the first impulse that dictates the human spirit, that of aggression and lack of attention to others; “societas” understood as an alliance is transformed into the monster of the “bellum contra omnes”, the war of all against all, and everything becomes lawful [28].

The search for “maximum shareholder value” to reduce costs leads to the savage delocalisation that separates capital from labour by making it subordinate to the former, but also by countries, because the tax system allows leaving the taxes of blacklist countries by the wayside (Figures 9 & 10). We passively forget our history and become victims of this finance by transforming ourselves from the best creative artisans in the world into losing and checkmated financiers; the social system disintegrates and fragments, social ills explode, and the old solidarity of our people becomes a war of all against all with growing moral degradation because it justifies living to earn and not vice versa. Locust finance must be brought under strict control, eliminating the speculative derivatives that have emptied the reserves, redefining the boundary between business and commercial banks, but also with a return to the convertibility of currency into gold, as China, Russia, and other emerging countries close to creating an alternative financial system to the dollar are doing, because the new balances of power allow it [29].

The challenge of our time is to rebuild a cohesive social system, recovering the solidarity of our familial roots and the small and medium-sized businesses that constitute the backbone of our nation over time, and recover the pride of our history and autonomy that allows us to decide without having a gun pointed at our heads. History, forgotten by a society that lives only in the present, teaches over the millennia that a society can live only on solid family roots and maintain a trade-off between the agricultural world that generates solidarity and the urban world that creates individualism. Will we be able to understand the lessons of history? This is the enigma we have before us [30-31] (Figure 11).

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juniperpublishersoceanography · 2 years ago

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Valuation of Nutrient Utilization, Protein Requirement and Proximate Assessment of an Ecotype Tilapine ‘Wesafu’ Fingerlings Reared in Earthen Pond

Authored by: Albert O Amosu

Abstract

This study was conducted to evaluate the nutrient utilization, protein requirement and proximate analysis of Wesafu from fry to fingerlings in an earthen pond. The experimental fish were monitored for comparative analysis and growth performance using commercial feed (Coppens® 0.2mm) and wheat flour in formulating feed with 30%, 35%, 40% and 45% crude protein levels. Fry were fed thrice daily at 8am, 12 pm and 4pm with 5% body weight for 8 weeks. Samples of experimental diet and fingerlings were analyzed for proximate composition while growth performance and nutrient utilization of diet were evaluated using growth indices such as Weight Gain (WG), Percentage Weight Gain (%WG), Specific Growth Rate (SGR), Food Conversion Ratio (FCR), Gross Feed Conversion Efficiency (GFCE), Protein Intake (PI), Protein Efficiency Ratio (PER). The results revealed that the highest cumulative weight gain (10.58 ± 0.12) was recorded in fry fed 45% crude protein diet while the lowest (6.58 ± 0.21) was observed in the 30% crude protein diet .There were no significant differences (p>0.05) in specific growth rate, food intake, food conversion ratio, gross food conversion efficiency and protein efficiency ratio while there were significant differences (DNMRT; ANOVA; df = (n-1); p < 0.05) in weight gain, protein intake. This study indicated that a diet containing 45% crude protein appear to be suitable for rearing Wesafu fry to fingerlings in earthen ponds.

Keywords: Aquaculture; Cichlids; Diet; Ecotype; Fingerlings; Growth; Nutrient; Wesafu

Abbreviations: WG: Weight Gain; FCR: Food Conversion Ratio; GFCE: Gross Feed Conversion Efficiency; PI: Protein Intake; PER: Protein Efficiency Ratio; SGR: Specific Growth Rate; LSD: Least Significance Difference; DNMRT: Duncan’s New Multiple Range Test; SE: Standard Error

Introduction

As the global population continues to rise, the need for sustainable alternative sources of protein also increases [1]. Research estimated that the worldwide requirement for food will increase up to 50 % by 2030 [2]. Juxtaposing the production input efficiencies of aquaculture versus several of fisheries and terrestrial agriculture systems shows that aquaculture is among the world’s most efficient mass producer of protein [3]. Protein is the most important constituents of fish and also the most expensive constituent of fish feed and global expenditure exceeds (7.05 million MT) €1bn per annum [4,5]. Aquaculture production is growing at a rate of nearly 9% per annum [3,6]. As wild fish stocks decline, the aquaculture industry faces a massive challenge to identify cost-effective and environmentally friendly alternatives to fish production on which it is so heavily reliant [1,7]. Cichlid aquaculture has the potential to provide a solution to this problem as it is relatively underexploited in Nigeria and can be cultured in a sustainable manner [8]. Cichlids are one of the most diverse fish species and widely cultivated fish families in the world, though their natural distribution was confined to North America, Central America, South America, Africa and the Mid East [9]. The family has been introduced into various continents including Australia [10]. Wesafu is an indigenous ecotype cichlid, very important specie of the fisheries of Lagos coastal waters in Nigeria [11-12]. The diversity of an unidentified cichlid of great abundant in Epe Lagoon commonly referred to as Wesafu and the large size cum weight it attains in the wild influence the drive for possible domestication, culture and exact identification and naming of this specie [13-14]. Several research studies have been conducted on this indigenous specie such as Age and growth, Aquaculture system, Characterization, food and feeding habits, nutrition, meristic and morphometric characters among others [11-22].

Tilapines are Cichlids with fast growth, resistant to diseases and handling, easy to reproduce and are able to tolerate a wide range of environmental conditions. They are widely cultured in tropical and subtropical regions of the world and constitute the third largest group of farmed fin fish with an annual growth of about 11.5% [3,23-25]. Proper feeding management is therefore a necessary tool for successful tilapia culture. Nutrition and feeding play important role in sustainable cichlid aquaculture therefore, feed resources as well as costs continue to dominate aquaculture needs. Feed accounts for 40-60% of the total production costs in aquaculture, with protein sources accounting for a significant proportion of this cost [1,3,22-24]. Frys and fingerlings require diets higher in protein, lipids, vitamins, minerals and lower in carbohydrate as they are developing muscles, internal organs and bones with rapid growth. Adult fish needs more calories of fats and carbohydrate for basal metabolism and a smaller percent of protein for growth [19]. The energy needs of the fish can be met by less expensive lipid and carbohydrate sources. The protein requirement of tilapia was estimated to be from 25% to 45% of diet [26,29-34]. Under natural condition, Tilapia is predominantly an herbivore and a detritus feeder. This means that they can provide high quality protein, suitable for human consumption from less protein sources [35]. Inabilities to develop suitable commercial and improved strain of tilapias that will grow to table size in good time are few of the problems militating against a viable tilapia industry in Nigeria [14]. The problem of precocious sexual maturity and unwanted reproduction has long been accepted as a major constraint to further development and expansion of tilapia culture in Nigeria. In addition, unwanted reproduction which leads to excessive recruitment (overpopulation), particularly in ponds, resulting in competition for available food and space resources as well as the ease of reproduction represents the principal problem in the optimization of yield in tilapia culture. Therefore, this research was geared towards determining the growth performance, dietary protein requirement and nutrient utilization of this economic important ecotype cichlid. The increased intensification of culture method for warm water fish such as tilapia has necessitated the provision of balance ration to satisfy the dietary requirement. Despite the commercial values of Wesafu, a tilapia highly priced fish in Lagos, Nigeria due to its tasty flesh and large size of over 1500g in the wild, little information exists on its nutritional requirements, in cultural practices yet it has culture potential in the country.

Materials and Methods

Experimental fish

One thousand four hundred and forty (1440) fry of average weight of 0.93 ± 0.16 were used in determining the protein requirement of Wesafu. The fish was raised from fry to fingerlings stage at Seg farm a private aquaculture farm in Topo village (6°25’0’’ N; 2°55’59’’ E) (Figure 1) on the West Coast of Badagry, Lagos, Nigeria. Fry were weighed and stocked in hapas. Prior to feeding trials, the experimental fish were starved for a day (24h) to ensure that their guts were emptied.

Experimental Design

Twelve (12) hapas (1 × 2 ×1.5m) were placed in earthen pond were used which was conducted in four stages:

a) Fry fed with 30% crude protein represent A in triplicate

b) Fry fed with 35%crude protein represent B in triplicate

c) Fry fed with 40% crude protein represent C in triplicate

d) Fry fed with 45% crude protein represent D in triplicate The fish were divided into one hundred and twenty (120) fry, stocked per in twelve different hapas in three replicates of 30%, 35%, 40% and 45% crude protein level and fed at 5% body weight. The 5 % the daily ratio was divided into 3 equal parts and fed at 8a.m, the second portion at 12p.m and the third portion at 4p.m. Each unit of experiment lasted for 8 weeks. Coppen 0.2mm feed was used with a protein content of 56% (Manufactured by Alltech Coppens Aqua Center, Germany) and wheat flour of 12% to formulate the diets using Pearson square method. All these hapas were placed in earthen pond in the farm and were covered with net to prevent the fish from escaping.

Determination of growth performance and nutrient utilization

Weight gain (wg)

The weight gain by fish was calculated from the differences between the final mean and the initial mean weight that is the final mean weight of fish at week eight subtracted from the initial mean weight of fish at week zero [36].

Weight gain (WG) = final weight (W2) – initial weight (W1) WG = (W2) – (W1)

Where:

W2 = Final mean body weight (g)

W1 = Initial mean body weight (g)

Percentage Weight Gain (%WG)

The percentage weight gain was calculated from the formula according to [14].

% weight gain = (X2 ) – (X1)×100 / (X1)

where:

X2 = Final mean body weight (g)

X1 = Initial mean body weight (g)

Specific Growth Rate (SGR)

Specific growth rate was calculated according to [36] as

SGR = Loge W2 – Loge W1 / T2 – T1

where:

W2 = Weight of fish at time T2 in days

W1 = Weight of fish at time T1 in days

Loge = Natural log of base e

Food Conversion Ratio (FCR)

Food conversion ratio according to[36] as FCR, expressed as the proportion of dry food fed per unit live weight gain of fish:

FCR =Weight of dry fed (g) / Live weight gain (g)

Gross Food Conversion Efficiency (GFCE)

The gross food conversion efficiency was calculated according to [36] as the percentage of the reciprocal of feed conversion ratio.

gFCR = 1×100 / FCR

Protein Intake (PI ) = Total feed intake × % protein in the diet Protein Efficiency Ratio (PER) = weight gain / Protein intake

Nutrient Evaluation of Experimental feed and fish

Samples of experimental feeds and fish were analyzed for their proximate composition according to the methods of [37].

Moisture content

The moisture content of the different fry samples were determined

% Moisture content = M1 − M2 / M1 − M0 × 100

where

M0 = Weight in g of fish and lid

M1 = Weight of g of dish, lid and sample before drying

M2 = Weight in g of dish, lid and sample after drying

M1 − M0 =Weight of sample prepared for drying

% dry matter content = 100 − % moisture content

Crude protein

Determination of crude protein was done using total kjeldahl nitrogen method.

% Nitrogen = 0.0075× A / B ×C

where

A = Mg /L reading displayed

B = g – sample digested

C = ML digest analysed

Determination of crude fat or lipid

The measure fat content of all the soluble materials present was determined according to [38].

% Fat = W3 –W2 /W1

where

W3= Weight of the cup with the extracted oil.

W2 =Weight of the empty cup

W1 =Weight of the sample.

Determination of the total ash

The percent of ash was calculated as follows:

Percentage (%) of ash = (weight of ash / weight of sample)×100 % Ash = (W2 /W1)×100

where,

W1 = Weight of sample (g)

W2 = Weight of ash (g)

Determination of crude fibre

The amount of the crude fibre content in the sample was determined using the acid/base digestion process [39].

% Crude fiber =Weight A −Weight B / Sample Weight

Where

Weight A = Weight of crucible + dried residue

Weight B = weight of Crucible + residue ashed

Statistical Analysis

All data collected were presented as mean values of each determination ± standard error (SE). Analysis of variance was performed using one way ANOVA procedure. Differences between the mean values of the treatments were determined by the least significance difference (LSD) test and the Duncan’s New Multiple Range Test (DNMRT). The significance was defined at p < 0.05.

Results

The result of the experiment shows that there was increase in the weekly growth rate, weekly feed intake, and food conversion ratio, protein intake while there was decrease in the weekly Specific Growth Rate, Gross Food Conversion Efficiency and the Protein Efficiency Ratio. It was observed that the weekly feed intake consumed increased per week in the following sequence Diet 1 > Diet 2 > Diet 3 > Diet 4 (Table 1). The weekly mean of total feed consumed showed a progressive increase throughout the experiment and there was significance (p<0.05) difference throughout the experiment. The weekly mean weight in Figure 1 shows increase trend from week one to week eight and there was significance difference (p< 0.05) between the crude protein levels throughout the experiment.

The weekly mean of total feed consumed showed a progressive increase throughout the experiment and there was significance (p<0.05) difference throughout the experiment.

No significance difference (p > 0.05) observed in weight gain for week 6 and 7 and significance difference was observed throughout the remaining weeks. Also, there was no significance difference (p > 0.05) observed between specific growth rates of the dietary protein level in week five and seven and there was significance in the rest of the week. The weekly specific growth rate decreases throughout the duration of the experiment (Figure 2).

No significance difference occurred (p > 0.05) in week 3, 6, 7 and 8 and significance difference was observed in week 1, 2, 4 and 5 (Figure 3). The food conversion ratio increases throughout the experiment. It was reveal that Gross Conversion Efficiency decreases throughout the duration of the experiment and significance difference (p < 0.05) was observed from week seven to week 8 whereas no significance difference observed from week 1 to week 6 (Figure 3).

There was significance difference (p < 0.05) throughout the experiment and the protein intake increases throughout the experiment. Significance difference (p < 0.05) was observed in the protein efficiency ratio expect for week four where there is no significance difference in the fry fed with the four experimental diet (Figure 4). No significant differences (p > 0.05) in specific growth rate, food intake, food conversion ratio, gross food conversion efficiency and protein efficiency ratio while significant difference (p < 0.05) in the weight gain, protein intake (Table 2).

Table 3 shows proximate analysis of feed Percentage crude protein had the highest mean value in each Diet 34.37 ± 0.12, 28.47 ± 0.25, 26.86 ± 0.26, 24.04 ± 0.48, the lowest value was observed in Fat content for Diet 1, Diet 2, Diet 3 and Diet 4 had 7.31± 0.12, 5.22± 0.05, 5.16 ± 0.05 and 4.81 ± 0.07 respectively. The proximate analysis of fish sample in each Diet; the percentage crude fiber had the highest mean value of 40.18 ± 4.79, 47.52±0.55, 45.76±0.08, and 43.95±0.39 and fat content has the lowest Diet of 2.91 ± 0.04, 2.69 ± 0.04, 2.19 ± 0.08 and 2.24 ± 0.09 (Table 3). The experimental earthen pond water temperature, pH, dissolved oxygen and total alkalinity ranged from 27-28.5°C, 6.8-7.5, 5.8- 6.4ppm and 120-128ppm, respectively during the entire rearing period.

Discussion

The highest cumulative weight gain was recorded in fry fed on Diet 1(45%) with 10.58 ± 0.12 while the lowest was observed in fry fed with diet 4(30%) with 6.58 ± 0.21, similar findings have been reported by different authors for different tilapia species that the dietary protein requirements of several species of tilapia have been estimated to range from 20% to 56% [33,34,40-42]. The specific growth rate decreased throughout the duration of the experiment. The highest food conversion ratio was observed in the fry feeds with Diet 2 crude protein with the value of 0.32 ± 0.57 cumulative. The other feeds 30%, 35% and 45% crude protein recorded 0.25 ± 0.04, 0.32 ± 0.57 and 0.25 ± 0.04 respectively which were similar to [43]. The highest cumulative gross food conversion efficiency was recorded in the fry fed with 532.14 ± 131.12 and the lowest gross food conversion efficiency was recorded in fry fed with 35% crude protein with the value of 412.48 ±105.57 corresponding to the observations of [41,42]. The fry fed on Diet 2 crude protein recorded the highest protein efficiency ratio and fry fed with Diet 1 crude protein diet recorded the lowest protein efficiency ratio. PER, in the present study, was significantly affected by protein levels and manifests that protein utilization was obtained at low protein level. The decrease of PER with increasing dietary protein level have also been reported by different authors for different tilapia species [44,45]. This was mainly because more dietary protein is used as energy when high protein Diets are fed to fish [30,40]. The highest cumulative protein intake was recorded in the fry fed on on Diet1, having a value of 8.75 ± 1.43, this was followed by Diet 2 with the value of 7.40 ± 1.34. Frys fed with diet 3 with the value of 5.17 ± 0.91 and this was observed to the lowest experimental Diet which was fry fed with Diet 4 which recorded 3.74 ± 0.71. The proximate analyses of sample feed fed fry of Wesafu showed that the percentage crude protein was highest in the feed with 45% having a value of 34.37 ± 0.12 and the lowest was observed in feed with 30% with value of 24.04 ± 0.48. The formulated experimental feed with 40% recorded 28.47± 0.25 while 35% recorded 26.86 ± 0.26. Fry on proximate analysis recorded better productive protein values in the 45% feed with the value of 19.99 ± 0.35, 35% feed with value of 18.26 ± 0.12 followed by 40% feed with 17.61 ± 0.08 and 30% with 17.71 ± 0.17.

Conclusion

This study clearly indicated that diet containing 45% crude protein bodes well for cichlid aquaculture industry and appear to be suitable for rearing Wesafu fry to fingerlings in earthen pond with the potential to be a successful feed and alternative replacement for coppens in tilapine feed formulation.

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juniperpublishers-agriculture · 2 years ago

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Monitoring of Positive List System on Residual Pesticides Analysis of Agricultural Products in Southwest of Korea

Abstract

Pesticide residues must be managed for food safety and regulations are being strengthened worldwide. In Korea, the Positive List System (PLS) was introduced to prevent the misuse and manage unregistered pesticides. The PLS is a system that applies 0.01 mg/kg to crops for which maximum residue limits is not set. A more stringent regulatory system could lead to an increase in agricultural products that exceed standards. Therefore it is necessary to confirm the change in the determination rate of agricultural products exceeding the previous pesticide residue standards. In terms of climate change, temperature increase and changes in precipitation patterns are the main pest and pathogen infection determinants. Complex interactions and climate variability will lead to more frequent spraying of pesticides and eventually affect consumer exposure at the end of the food chain. Therefore, we confirmed the relationship between temperature and precipitation, which are climate factors and pesticide residues. Agricultural products (n=5,560) distributed at wholesale market (SeoBu, GakHwa) in the southwest Korea were collected and 311 kind of residual pesticides were analyzed using GC-MS/MS and LC-MS/MS. This study could serve as basic data for pesticide residue prevention, management, control and monitoring in countries seeking to tighten pesticide standards.

Keywords: Pesticideresidues/analysis/management; Agricultural products/vegetables; Positive List System; Maximum Residue Limit; Climate change; Consumer product safety

Abbreviations: PLS: Positive list system; MRLs: Maximum residue limits; GC: Gas chromatography; LC: Liquid chromatography; MS: Mass spectrometry; GC-MS/MS: Gas chromatography tandem mass spectrometry; LC-MS/MS: Liquid chromatography tandem mass spectrometry; LOD: Limit of detection; LOQ: Limit of quantitation; RSD: Relative standard deviation

Introduction

Pesticides are historically long-established commodities used in agriculture [1]. They include a wide range of compounds such as insecticides, fungicides, herbicides, and rodenticides [2]. Pesticides play an important role in reducing damage from weeds, diseases, and pests, and improving crop yield worldwide [2], but adversely impact the environment. The pesticides not only contaminate soil, surface water, and groundwater, but also remain in trace amounts in crops, threatening human health [2]. In humans, pesticides can be carcinogenic and cytotoxic and can cause bone marrow and nerve disorders, infertility, and immune and respiratory diseases [3]. The presence of large amounts of pesticide residues in food is a serious risk to consumers. In general, pesticides are applied directly to crops and they can remain on them. Infants, children, and adults can be exposed to these pesticides by consuming pesticide-contaminated food [4]. Residues of chemical pesticides have been detected more in vegetables and fruits than in other food items4. As agricultural product intake is a major pathway by which humans are exposed to pesticides, there is an increasing demand for safe food management from pesticide residues worldwide.

The European Commission, Environmental Protection Agency, Food and Agriculture Organization/World Health Organization have set limits on pesticide residues in food to protect people from the toxic effects of pesticide exposure [4]. Acceptable levels of pesticide residues in food are defined by setting a maximum residue limit (MRL) for each component. An MRL represents the highest concentration of a pesticide residue legally permitted in food when a pesticide is used correctly on an agricultural product. In Korea, the pesticide residue monitoring program was started in 1968 [5] and as a part of strengthening the food safety management, the introduction of a pesticide Positive List System (PLS) was planned in 2011 [6]. The PLS is a system that applies a uniform standard MRL of 0.01 mg/kg of chemical residues to crops that do not have pesticide residue tolerance standards. Priority was given to nuts, seeds, and tropical fruits in December 2016 [6]. Since January 2019, it has been fully expanded to domestic and imported agricultural products6. In other words, the PLS system was introduced to strengthen the pesticides management within the allowable daily intake range by uniformly applying the default MRL of 0.01 mg/kg. This system can ensure food safety for consumers by preventing in advance the use of unregistered pesticides and excessive spraying of pesticides.

However, there are concerns about the introduction of the PLS system in agricultural sites due to the insufficient number of registered pesticides, the problem of unintended pesticide residues, the problem of scattered contamination during pesticide application, residual problem of succeeding crop and soil residues [7]. A more stringent regulatory system, PLS, could lead to an increase in agricultural products that exceed standards. Therefore, after the expansion of the PLS to the assessment of all agricultural products, it is necessary to confirm that the change in the determination rate of agricultural products exceeds the earlier standard for pesticide residues. In some studies, the introduction of the PLS, which was expanded since 2019, did not affect the assessment result (conformity/nonconformity) for agricultural products exceeding the pesticide residue standard [7,8]. However, there may be differences according to the cultivation area of crops, and extensive studies have not been conducted. In terms of improving food safety and agricultural practices and minimizing economic losses, the pesticide residue monitoring program in food is very important, and the expanded application of PLS system has the potential to affect the cultivation and distribution of agricultural products, so comprehensive studies are required.

Several studies have found a correlation between the pesticide use and climate change [9-11]. Pesticide use is directly and indirectly related to climate change11. However, there have only been a few studies on establishing the relationship between pesticide residues and climate change for more than 5,000 agricultural products of various types. Despite tremendous improvements in technology and crop yield potential, food production remains highly dependent on climate, because solar radiation, temperature, and precipitation are the main drivers of crop growth [12]. In terms of climate change, temperature increase and changes in precipitation patterns are the main pest and pathogen infection determinants [12]. Complex interactions and climate variability will lead to more frequent spraying of pesticides and eventually affect consumer exposure at the end of the food chain [11]. As the impact of climate change grows, ensuring food safety while improving agricultural productivity through the use of pesticides is a huge challenge. Accordingly, we attempted to confirm the correlation between pesticide residues and climatic factors. The purpose of this study was to monitor pesticide residues in agricultural products in the southwest region of Korea and to investigate whether the introduction of a PLS system affects decisions of conformity and nonconformity. Furthermore, the relationship with pesticide residues between temperature and precipitation, which are climatic factors, was examined for more than 5,000 agricultural products of various types.

Material and Methods

Sample collection

This study was conducted at the Health and Environment Research Institute (Agro-Fishery Products Inspection Center) in Gwangju metropolitan city under a surveillance program of the Gwangju metropolitan government. A total of 5,560 agricultural products were collected between March 2020 and September 2021 from the largest wholesale market in the southwest of Korea, and all samples were analyzed within 24 h of collection. The types of agricultural products analyzed in this study are listed in Table 1.

Pesticide reference standard and Reagents

The certified pesticide reference standards were purchased from AccuStandard (New Haven, USA) or Kemidas (Suwon-si, Republic of Korea). A total of 311 pesticides that can be analyzed simultaneously were selected for analysis and are listed in Table 2. The reagents and solvents used for monitoring the pesticide residues, viz., acetonitrile, sodium chloride, acetone, and water containing 0.1% (v/v) formic acid, were procured from Merck (Darmstadt, Germany). Anhydrous sodium sulfate, n-hexane, and dichloromethane were purchased from Wako (Osaka, Japan), and methanol was purchased from Avantor (USA).

Sample preparation

The overall analysis was conducted using multi-class pesticide multi-residue method No. 2 for pesticide residues, in accordance with the Korean Food Code guidelines. The samples (1-2 kg of each agricultural product) were first pulverized using a blender (Robotcoupe, South Perkins, Ridgeland, USA) to obtain a homogeneous mixture. Then, 50 g of each sample was extracted with 100 mL of acetonitrile for 3 min. The sample/acetonitrile mixture was homogenized for 3 min and filtered using a Buchner funnel into a bottle containing 10 g of sodium chloride. Thereafter, the extract and sodium chloride were vortexed and the layers were separated. Then, 10 mL of the acetonitrile layer was transferred to a test tube and evaporated to dryness in a water bath using a stream of air at 40 °C or lower. For the purification of the pesticide extract, gas chromatography (GC) and liquid chromatography (LC) were performed independently.

For GC analysis, Florisil cartridges (Phenomenex, Torrance, CA, USA) were activated and conditioned by flowing 5 mL of hexane and 5 mL of 20% acetone/hexane. The sample extract was dissolved in 4 mL of 20% acetone/hexane and loaded into the Florisil cartridge, and the cartridge was eluted with 5 mL of 20% acetone/hexane. The eluate was collected in a tube and the solvent was evaporated under gentle air flow. The residue was then dissolved in 2 mL of acetone, filtered through a membrane filter (PTFE, 0.45 μm), and used as the test solution for GC analysis. For LC analysis, aminopropyl cartridges (Phenomenex, Torrance, CA, USA) were first activated and conditioned with 5 mL of dichloromethane. Then, the extracted solution was loaded and eluted with 4 mL of 1% methanol/dichloromethane, and the eluate was collected in a tube. The cartridge was eluted again with 7 mL of 1% methanol/dichloromethane. The combined solution of first elution and second elution was evaporated under gentle air flow at 40 °C in a water bath. The dried extract was dissolved in 2 mL of acetonitrile and filtered through a membrane filter (PTFE, 0.2 μm) for use as a test solution for LC analysis.

Pesticide residue analysis conditions

GC- MS (GC-tandem MS (GC-MS/MS), Agilent 7000B, California, USA) and LC-MS (LC- tandem MS (LC-MS/MS), AB SCIEX, TQ 4500, CA, USA) were used for the qualitative analysis of the pesticide residues. The quantitative analysis of each pesticide was carried out using a GC-MS/MS system, LC-MS/MS system, GC system equipped with a 63Ni electron capture detector (ECD), and GC system equipped with a nitrogen phosphorous detector (NPD), according to the Food Code set by the Korea Food and Drug Administration. An Agilent 7890 series GC instrument equipped with an NPD and ECD was used for GC analysis. LC analysis, electrospray ionization was performed in positive and negative modes, and the data were obtained in multiple reaction monitoring mode. The qualitative and quantitative instrument analysis conditions are presented in Table 3 & 4.

Measurement of recovery, LOD and LOQ

Method validation was performed based on SANCO/12571/2013 [13] and ICH/2005/Q2/R1 [14]. To a blank matrix sample of lettuce in which no residual pesticide was detected, each pesticide standard was added so that the sample content was 0.5 mg/kg, and the recovery was measured by repeating it three times. The limit of detection (LOD) and limit of quantitation (LOQ) were calculated as the standard deviation of the slope and residual through the regression line presented by the International Conference on Harmonization. It was repeated five times for each concentration using a standard solution and measured according to the following formula.

σ = standard deviation of the response

S = slope of calibration curve

Investigation of the impact of nonconformity due to the introduction of PLS

The detection and nonconformity assessment of each pesticide/item in the pesticide residues of agricultural products were conducted using Excel 2016 (Microsoft, Redmond, WA, USA). In order to determine the change in the nonconformity of the pesticide residues under the PLS, the data of nonconforming agricultural products were evaluated for suitability by applying the pesticide residue standards used before the introduction of the PLS. This method was applied according to the method specified in the Korean Food Code. The standard application sequence is as follows:

a. Application of Codex standards to agricultural products.

b. Application of the standards in the order of agricultural products, sub-categories, and major categories of the pesticide residue standards of the Food Code.

c. Application of the lowest standard among the pesticide standards for the pesticide compound in the Food Code.

Statistical Analyses

All statistical analyses were performed in SPSS version 22.0 software (IBM Corp., Armonk, NY, USA) and P-values less than 0.05 were considered statistically significant. Log transformation of data was performed where appropriate. Data analysis was calculated as mean ± standard deviation and all tests were twosided. Normal distribution of the variables was confirmed with a Kolmogorov–Smirnov (KS) test. And then we calculated the Pearson linear correlation coefficient in order to seek to determine the strength of association between data sets. The following points are the accepted guidelines for interpreting the correlation coefficient, and the results were interpreted based on this : perfect (|r| = 1), strong (0.7 ≤ |r| < 1.0), moderate (0.3 ≤ |r| < 0.7), weak (0.0 < |r| < 0.3), No correlation (|r| = 0) [15].

Results and Discussion

Validity of analytical methods

For the validation of the method, forty kinds of pesticides were selected based on the detection of more than twenty times in agricultural products. The method was validated for various parameters such as accuracy, precision, linearity, limit of detection and quantification (Table 5). The adequacy of the sample pretreatment was evaluated based on the recovery, and the analytical method was validated based on ICH guidelines [14]. The recovery was between 79.13 and 115.05% and the relative standard deviation (%RSD) values were less than 6%. A linear correlation between each pesticide concentrations and peak areas was obtained in the range of 0.9971 to 1.000. The limit of detection (LOD) were 0.001 to 0.019 mg/kg for GC-MS/MS and 0.001 to 0.004 mg/kg for LC-MS/MS. The limit of quantification (LOQ) were 0.004 to 0.058 mg/kg for GC-MS/MS and 0.002 to 0.013 mg/kg for LC-MS/MS. The validated parameters were found to be within acceptable ranges, suggesting that this method is suitable for use in the pesticide analysis of agricultural products.

Climate factors and pesticide residues of agricultural products

In this study, 5,560 agricultural products were monitored. Pesticide residues were detected in 51.1% of the agricultural products and the pesticide residues in 2.0% of the agricultural products were found to exceed the MRLs. The detection and nonconformity rates were categorized according to the four seasons in Korea, and the detection and nonconformity rates for 2020 and 2021 are shown in Figure 1. On average, the detection rate (nonconformity rate) was as follows: spring 46.4% (1.9%), summer 52.7% (1.6%), autumn 56.8% (2.1%), and winter 50.0% (3.2%). It was thus confirmed that the detection rate was the highest in autumn, while the nonconformity rate was the highest in winter. Different climates occur according to the season, and they have a major impact on the cultivation of agricultural products. Therefore, the relationship between climatic factors and pesticide residues in agricultural products was assessed. The climatic factors (temperature and precipitation) were obtained from the statistical data of the Korea Meteorological Administration [16]. It was analyzed using the average values of climate factors in Gwangju, Jeollabuk-do and Jeollanam-do, which are representative regions of the southwest region and account for most of the wholesale market products. The normality of experimental results is an important premise for the use of parametric statistical tests such as correlation analysis. If the assumption of normality is not confirmed by relevant tests, interpretation and inference from any statistical test may not be reliable or valid [17].

Normal distributions of all variables such as temperature, precipitation, detection rate, and nonconformity rate were confirmed by the Kolmogorov-Smirnov (KS) test, and all variables satisfied the normality with a significance probability p=0.200 Figure 2a & b. shows the relationship between the temperature and pesticide residues. Pearson correlation coefficient (r) between temperature and detection rate was 0.314 (p = 0.190). There was no significant change in the detection rate of the pesticide residues according to the temperature change. However, the nonconformity rate showed an opposite trend with respect to the temperature. Pearson correlation coefficient (r) between temperature and nonconformity rate was -0.765 (p = 0.000). It can be seen that there is a high negative correlation between temperature and nonconformity rate as high as 76.5%. Most winter agricultural products are grown in facility and the density of phytopathogenic fungi increases rapidly due to low temperature and high humidity, insufficient sunlight and poor ventilation. Therefore, it is judged that the frequent use of pesticides increased the nonconformity rate [18]. Moreover, several studies have shown that pesticide residue levels were higher in greenhouse-grown produce than in open field-grown produce Figure 2c & d [19,20]. shows the relationship between the precipitation and pesticide residues. Pearson correlation coefficient (r) between precipitation and detection rate was 0.200 (p = 0.411). Precipitation did not have a significant effect on the detection rate. On the other hand, As the correlation coefficient R= -0.701 (p = 0.001), it can be seen that there is a high negative correlation between precipitation and nonconformity rate as high as 70.1%. When there was a large amount of precipitation in July and August of 2020, the nonconformity rate decreased at first and then increased. Ilse Delcour confirmed that high precipitation causes pesticide dissipation, leading to a general tendency of increased pesticide use to overcome the loss [11]. According to our study results, the increase in the detection of pesticide residues after a large amount of precipitation can be considered as the corresponding effect. Considering the results of the study, it suggests that caution is needed in managing pesticides and consuming agricultural products during autumn (high detection rate), winter (high nonconformity rate), and after periods of consecutive precipitation (high nonconformity rate). In a previous study conducted in the USA, the usage of pesticides in the cultivation of each agricultural product was investigated according to the climate change to discover that the usage pattern of pesticides for each agricultural product differed according to the climate [9]. This suggests that the relationship between climate and pesticide residues may be different for each agricultural product. Therefore, in order to accurately identify the relationship between climate and pesticide residues, additional climate-related studies according to cultivated agricultural products and cultivation methods are needed. Our study was conducted for a short duration, and a longer study duration is necessary to adequately assess the relationship between temperature, precipitation, and residual pesticide detection. But, our research can be considered representative because more than 5,000 agricultural products were monitored.

Monitoring of pesticide residues in agricultural products

The detection rates of residual pesticides in the agricultural products (51.1% of the total number of products studied) are as follows: chili pepper (74.1%), crown daisy (73.0%), chwinamul (67.9%), perilla leaves (67.6%), and chives (66.7%). On the other hand, none of 102 cases of king oyster mushroom were detected (Table 6). A high detection rate of pesticide residues has been observed for chili pepper cultivated in several regions, including Saudi Arabia [21], Cameroon [22], and Southeast Asia [23]. Of the 311 pesticides monitored, 126 pesticides were detected, and 20 types of frequently detected pesticides are shown in Figure 3. The most frequent pesticides were fluopyram (n = 408), chlorfenapyr (n = 383), chlorantraniliprole (n = 320), fludioxonil (n = 287), and thiamethoxam (n = 287). Five pesticides detected with high frequency corresponded to three compounds in an insecticide and two compounds in a fungicide. In the case of fluopyram, lettuce (n = 81), chives (n = 70), crown daisy (n = 45), and cucumber (n = 33) had the highest frequencies. Chlorfenapyr was detected in chives (n = 66) and chili pepper (n = 51), while chlorantraniliprole was detected in young radish (n = 55) and winter cabbage (n = 43). Fludioxonil was detected at a high frequency in chives (n = 61), pimpinella brachycarpa (n = 41), while thiamethoxam was detected in young radish (n = 49) and crown daisy (n = 43). The MRLs of fluopyram have been set for 74 commodity standards in the Korean Food Code. In Korea, among agricultural products with a high non-conformity rate, a temporary residual limit of fluopyram is enforced for chives and crown daisy. If these standards are eliminated, and agricultural products are regulated according to the PLS, the nonconformity rates may increase. This also applies to other pesticide residues; that is, the introduction of PLS can lead to a stricter management of pesticide residues.

Monitoring of the nonconforming agricultural products

In our study, a total of 112 agricultural products of 31 types were found to exceed the 118 residual pesticide standard, including duplicate detection (Table 7). There were many nonconforming agricultural products in the order of crown daisy (n = 20), chives (n = 10), chwinamul (n = 9), pimpinella brachycarpa (n = 8), and lettuce (n = 6). A comparison of nonconformity rate of each agricultural product revealed that agricultural products exceeding the MRLs were the most frequent in the order of Sonchus-leaf crepidiastrum (12.5%), Korean cabbage (10.6%), crown daisy (8.7%), Silver divaricata (8.3%), and butterbur leaf (7.7%) Figure 4. shows 32 pesticide components that were deemed to be unsuitable in agricultural products. Some agricultural products contained two pesticides at concentrations exceeding the pesticide residue standards. Fluopyram (n = 17), diazinon, procymidone (n = 11), flubendiamide (n = 10), and chlorothalonil, diniconazole (n = 7) are representative pesticides that exceeded the pesticide residue standards. The fluopyram compound with the highest inconsistency was found in crown daisy (n = 7), chives (n = 5), pimpinella brachycarpa (n = 2), and germinated buckwheat/silver divaricata/spinach in one case each. Agricultural products with a high Nonconformity rate largely included many leafy vegetables/ stalks and stem vegetables. This is related to the permeability of the pesticide and the leaching of the pesticide from the surface of leafy products owing to its solubility in water [24].

Investigation of the impact of the introduction of the PLS on the evaluation of nonconforming agricultural products

Among the agricultural products determined to contain pesticides at levels exceeding the pesticide residue standard, there were 37 agricultural products to which the PLS standard (0.01 mg/kg) was applied, and a total of 20 pesticides were identified 39 times. Diazinon (n = 8) and chlorothalonil (n = 4) are repive pesticides that were determined to be unsuitable at the 0.01 mg/kg standard. According to Method 2.6, the effects of the introduction of the PLS and nonconformity assessment were investigated. A total of 18 agricultural products would not have been deemed as nonconforming if the PLS was not introduced (Table 8). When the standards before the introduction of the PLS system were applied, It was found that the agricultural products belonged to the suitable category in the order of lettuce (n = 3), chwinamul, cucumber and sweet potato stalk (n = 2). In the case of lettuce, which occupied the most number of times, it was found to be suitable when the standard for leaf vegetables, a similar agricultural product, was applied. Therefore, it can be seen that is being managed more strictly with the introduction of PLS, suggesting that lettuce producer education is necessary. Chwinamul, sweet potato stalk and shepherd’s purse are representative minor crops. For minor crops cultivated over a small area, the registration of available pesticides does not meet the demand [25]. There are many types of minor crops and various pests, and accordingly, the registration of pesticides for small-area crops has not been activated, which is a global issue [26]. This causes difficulties in the production of crops and leads to a trend of high pesticide detection rates and nonconformity rates. The expansion of the PLS application implies that as the risk assessment standard of each agricultural product becomes stricter, the concerned agricultural product can be deemed as a nonconforming product. This suggests that the development of harmless pesticides by related organizations is of urgency, and that it is necessary to develop new pesticides to prevent damage to farmers and consumers. In addition, to improve the conformity based on the PLS standards, it is necessary to educate farmers on contamination owing to unintentional mixing of pesticides and appropriate use of suitable pesticides. The results of this study can be used as fundamental data for pesticide management, system supplementation of agricultural products, and reinforcement of field management.

Conclusion

In this study it was confirmed that temperature and precipitation, which are climatic factors, affect the concentration of pesticide residues. According to correlation analysis results, a high detection rate in autumn, high nonconformity rate in low temperature and low precipitation were confirmed. Therefore, we propose the necessity of pesticide management and caution on agricultural products consumption in consideration of climatic factors (autumn, winter, after heavy rain). Pesticide residues were detected in 51.1% and exceeded the MRL in 2.0% of agricultural products. As a result of monitoring the residual pesticide list of agricultural products, the most frequent pesticides were fluopyram, chlorfenapyr and chlorantraniliprole. The most frequent unsuitable agricultural products were daisy, chives, chwinamul, pimpinella brachycarpa and lettuce.

As a result of investigating the effect of the introduction of the PLS standard on the evaluation of non-conforming agricultural products, 18 agricultural products were found to be in the suitable when the previous criteria were applied. Despite the short research period, it was found that food safety could be secured as the standards were strengthened. Therefore farmers should use only registered crops and spray targets for each pesticide, and must comply with the timing and frequency of use. In addition, it is proposed to conduct education of PLS system for various stakeholders such as local government officials, farmers, and pesticide dealers. This study could serve as basic data for pesticide residue prevention, management, control and monitoring in countries seeking to tighten pesticide standards.

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juniperpublishers-nutrition · 2 years ago

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Antiproliferative Effect of Green Papaya on Lymphocytic Leukemic Cells

Abstract

Numerous edible plants have been reported to interfere with the carcinogenic process, and therefore, the regular consumption of these plant products may reduce the risk of developing cancer. We investigated the effect of papaya fruit and leaves on the cell proliferation of Jurkat T-lymphocytic and Daudi B-lymphocytic leukemia cells. Cells were treated with aqueous or methanolic extracts from leaves, skin, pulp, and seeds from green papaya. The papaya fractions were tested for total phenolic content, total flavonoids content, and anti-oxidation activity using chemical assays. Cell proliferation was measured using a WST-1 assay. Our data indicate that methanol and water extracts of seeds and leaves contained higher concentrations of total phenolic compounds and higher anti-oxidation activity than that of extracts from skin and pulp.

Both methanol and water extracts from leaves and skin potently inhibited the proliferation of leukemic Jurkat T-cells and Daudi B-cells. However, the effect was more potent on Jurkat T-cells, and the leaf extracts were more effective than that of skin extracts. None of the pulp or seed extracts showed inhibitory activity on leukemic cell proliferation. Although papaya leaves are not consumed as a food, leaf extracts have been used for the treatment of various conditions, including dengue and malaria fevers, gastric ulcers, low platelet counts, and cancers of the breast, lung, and cervix. Our data suggest that the consumption of papaya leaf extracts may also be beneficial in preventing and/or treating lymphocytic cancer. Isolation of active compounds from papaya leaves will also help in developing new drugs for cancer treatment.

Keywords: Polyphenols; Antioxidants; Anticancer; Jurkat cells; Daudi cells; Leukemia

Introduction

Leukemia is a cancer of the blood or bone marrow, as described by an anomalous multiplication of immature clonal hematopoietic cells. Leukemia is one of the cancers that can affect all races in the United States. The American Cancer Society estimates that there will be around 60,530 new cases of leukemia in the United States in 2020, resulting in 23,100 deaths [1]. Leukemia is classified based on its onset (acute or chronic), the affected blood cell type (lymphoblastic/lymphocytic or myeloid/myelogenous), the maturity stage of the blood cell, and phenotypic expression of the disease [2]. Leukemia, like other forms of cancer, can be treated by using a number of methods, including chemotherapy, radiation therapy, targeted therapy, or stem cell therapy, though it is easier to treat acute leukemia than chronic leukemia [3]. Although leukemia remains a common medical issue, there has been a decrease in incidence due to preventive measures, and death rates have also declined since 1991 due to recent advances in treatment strategies [4]. The 5-year survival rate for children with acute lymphocytic leukemia has greatly increased over time to about 90% [5]. Although there are improved treatment strategies and survival rates, the cost of such therapies remains an issue that hinders access for all patients [6]. There is a need for less expensive alternative strategies to increase treatment access and survival outcomes for all patients.

Recent advances in alternative and complementary medicine have shown that various herbs, fruits, and vegetables have compounds that could act as inhibitors of cancer formation, blockers of carcinogen interaction, and suppressors of tumor progression. For example, Korean red ginseng extract treatment inhibited the growth of U937 leukemia cells by downregulating the expression of human telomerase reverse transcriptase [7]. Another study confirmed that Kenaf seed oil causes the death of human leukemia cells HL60 and K562 by induction of apoptosis [8]. Studies demonstrated that a hot water extract of Euphorbia formosana (EFW) restrained the growth of THP-1 human leukemic cells by inducing apoptosis through caspase-dependent cell death via Fas and mitochondrial pathways [9]. Water, ethanol, and methanol extracts of garlic have exhibited lethal cytotoxic effects of 50-75% on cells harvested from 3 patients with acute lymphoblastic leukemia (ALL) and three patients with acute myeloid leukemia (AML) [10]. Typhonium flagelliforme (TF), a herbal plant in Malaysia, exhibited cytotoxic effects in P388 [11] and WEHI-3 [12] murine leukemia cells. Further, in vivo studies showed a significant decline in the cell count of immature monocytes and immature granulocytes, when the TF extract was orally administered for 28 days in a BALB/c leukemic mouse model [12]. Studies with grape seed extracts reported induction of apoptosis in Jurkat leukemic cells through a process that involves sustained JNK activation and Cip1/p21 up-regulation, culminating in caspase activation [13].

Papaya fruit has also been studied for its anticancer activities for colorectal [14], prostate [15,16], cervical [17], breast [18- 20], and skin cancers [21]. Papaya also effectively inhibits cancers of hematopoietic cell lines, including T cell lymphoma (Jurkat), plasma cell leukemia (ARH77), Burkitt’s lymphoma (Raji), anaplastic large cell lymphoma (Karpas-299), and human promyelocytic leukemia (HL-60) [22-23]. A study demonstrated that Benzyl isothiocyanate (BITC), a promising chemo preventive agent found in papaya fruits, when given followed by cisplatin (BITC/cisplatin) caused significant death in HL-60 cells [24]. Furthermore, a recent study reported that papaya leaf extract improved bone marrow blast counts and stabilized the hematological parameters of a 76-year-old male with untreated chronic myelomonocytic leukemia [25].

It is well Known that fruits, vegetables, and other medicinal plants contain polyphenols and flavonoids that exhibit various biological and pharmacological properties [26], which are often mediated by their anti-oxidation activity [27]. Based on these earlier studies, we evaluated water and methanol extracts from leaves, skin, pulp, and seeds of green papaya on T-lymphocytic leukemia and B-cell lymphocytic leukemia using Jurakt and Daudi cell lines, respectively, and hypothesized that the effect of papaya fractions on cell proliferation of leukemic cells is correlated to their phenolic contents and/or anti-oxidation activity.

Materials and Methods

Materials

The Jurkat T-cell line and Daudi B-cell line were purchased from ATCC (Manassas, VA). RPMI 1640 medium was purchased from Gibco (Grand Island, NY). Fetal Bovine Serum (FBS) was purchased from RAMBIO (Missoula, MT). Penicillin and streptomycin antibiotics (BP2959) and Phosphate Buffered Saline (PBS; BP399-550) were purchased from Fisher Scientific (Fair Ln, NJ). Folin Ciocalteu, sodium carbonate, aluminum chloride, Diphenyl-1-picrylhydrazyl (DPPH), quercetin, gallic acid, and trolox were purchased from Sigma Chemical Co. (St Louis, MO). WST-1 (MK400) was purchased from Takara (Kusatsu, Shiga, Japan). Green papaya was obtained from Randolph Farm at Virginia State University, Petersburg, VA.

Isolation of papaya leaves, skin, pulp, and seeds

The papaya samples were isolated as described previously [28]. Briefly, the papaya was washed with distilled water and blotted dry with a paper towel. The skin was peeled off using a kitchen peeler. An unskinned papaya was cut into half to remove seeds, and then the pulp was cut into small pieces. The leaves and seeds were washed with distilled water. All fractions (leaves, skin, pulp, and seeds) were freeze-dried. The dried leaves, skin, pulp, and seeds were ground to a fine powder using a mortar and pestle with liquid nitrogen added to keep the powder frozen. The dried powder was flash-frozen with liquid nitrogen and stored at -80 ℃ until used.

Preparation of extracts from papaya leaves, skin, pulp and seeds

The methanol and water extracts of papaya fractions were prepared as described previously [28]. Briefly, a known quantity (5g) of different fractions of freeze-dried papaya powder was homogenized with 200ml of 80% methanol or distilled water. The mixtures were placed on a shaker at room temperature overnight. The next day, the mixture was centrifuged at 2000 x g for 20 minutes using a Thermo Scientific centrifuge (Waltham, MA). The supernatants were collected, and the residues were washed two times by resuspending in the respective solutions, mixing, and placing on a shaker overnight. The collected supernatants were pooled, and the insoluble residues were discarded. The methanol extracts were dried in a nitrogen evaporator (Organomation Associates, Inc, Berlin, MA), whereas the water extract was freezedried. The dried extracts were stored in a -20℃ freezer. The dried extract was suspended into DMSO at a concentration of 250mg/ ml and was further diluted into media for cellular studies.

Characterization of anti-oxidation activity of papaya extracts

The oxidative properties of papaya were determined by assaying their total polyphenolic content (TPC), total flavonoid content (TFC), and anti-oxidation capacity (DPPH) as previously described [29].

Cell culture

Jurkat T-cells and Daudi B-cells were maintained in RPMI 1640 media supplemented with penicillin (100 units/ml), streptomycin (100μg/ml) and 10% FBS. All cell cultures were incubated in a humidified incubator at 37 °C and 5% CO2. Media was changed every three days, and cells were subcultured when they became confluent.

Cell proliferation assay

The effects of papaya leaf, skin, pulp, and seed extracts on cell proliferation were determined using a WST-1 assay as per manufacturer instructions and described previously [28]. Briefly, cells were isolated from a confluent flask, and about 10,000 cells were placed in each well of a 96 well plate and incubated in a CO2 incubator for 24 hours. The next day, media was removed, and cells were incubated in serum-free media containing varying concentrations of papaya leaf, skin, pulp, or seed extract for 24 hours. After treatment with papaya extract, 10 μl of WST-1 was added in each well, and the plate was read at 420 nm in a 96 well plate reader for the background reading. The plate was then incubated for 3 hours at 37 ℃ in a CO2 incubator, and the reduction of WST-1 dye due to mitochondrial oxidation was recorded at 420 nm. The change in absorbance was calculated by subtracting the initial reading from the final reading. The results are expressed as % change from control (untreated cells).

Data analysis

The data are expressed as mean ± SD for at least three replicates. All comparisons were made by one-way ANOVA with Tukey’s -HSD-post-hoc test using SPSS Statistics 20 software (IBM, Armonk, NY). All significant differences are reported as P < 0.05 and indicated by “*” whereas non-significant differences are not marked.

Results

Characterization of papaya extract for anti-oxidation activity

The anti-oxidation potentials of papaya extracts were determined by assessing their total polyphenol content, total flavonoid content, and by assaying their anti-oxidation capacity. The data showing the total phenolic contents are presented in Table 1 and reported previously [28]. The highest amounts of polyphenols were found in the seed extracts that ranged 14 -16 mg/g dry weight. There was no significant difference in total phenolic contents between water and methanol extracts from papaya seeds. The leaves were second highest in total phenolic content but had a significantly lower amount of total phenolic content than that of seeds (P < 0.05). The leaves contained 1- 4 mg/g dry weight total phenolic content. Water extracts of leaves contained less total phenolic content (~ 1 mg/g dry weight) compared to that of methanol extracts. The skin extracts contained total phenolic content ~ 0.5 - 1 mg/g dry weight, whereas the pulp extracts have very small amounts of total phenolic content (0.05 - 0.5 mg/g dry weight).

The data for the total flavonoid contents in various papaya fractions are presented in (Table 2) and reported previously [28]. The water extract of seeds contained the highest amounts of total flavonoid content (5 mg/g dry weight), whereas the methanol extracts contained lower amounts (2-2.5 mg/g dry weight, P < 0.05). In leaves, the methanol extract contained more total flavonoid content (~ 5mg/g dry weight) than that of water (~2 mg/g dry weight). The total flavonoid contents in pulp and skin were less than those of seeds and leaves. In the skin, higher total flavonoid contents were present in the methanol extracts (~ 3.2 - 3.5 mg/g dry weight) than in water extracts (1 mg/g dry weight). The pulp contained the lowest total flavonoid contents compared to other fractions. The total flavonoid content in pulp ranged from 1 - 1.5 mg/g dry weight.

The anti-oxidation capacities of papaya fractions were measured by assaying the inhibition of DPPH oxidation and are shown in (Table 3) and reported previously [28]. The seeds and leaves contained significantly more (P < 0.05) anti-oxidation capacity than that of the skin and pulp. The methanol fractions of seeds and leaves contained more anti-oxidation activity than water extracts. The methanol fractions of seeds and leaves inhibited DPPH oxidation by 75 - 85%, whereas the water extracts of these fractions inhibited DPPH oxidation by 50 - 70%. The skin and pulp inhibited DPPH oxidation from 25 - 35%. There were no significant differences between water or methanol extracts of skin and pulp.

Effect of papaya water extracts on T- cell leukemia

The effects of papaya water extracts from different fractions are presented in Figure 1. The treatment with leaf extract caused a dose-dependent decrease in cell viability. A concentration of the extract as low as 50 μg/ml caused significant cell death by 30% (P < 0.05). Upon increasing the extract concentration more cells died, reaching a plateau of almost 70% cell death (P < 0.05) at 150 μg/ml. Additional increases to 250 μg/ml concentration resulted in a very small change in cell viability (Figure 1).

The treatment with water extract from papaya seeds caused a dose-dependent decrease in cell viability. Extract concentrations as low as 150 μg/ml caused a significant 20% increase in cell death (P < 0.05). On further increasing the extract concentration, no further decrease in cell viability occurred, which reached a plateau at 250 μg/ml with 25% cell death (P < 0.05) (Figure 1)

The water extract of papaya skin started to significantly (P < 0.05) inhibit leukemic cell proliferation at 25 μg/ml, causing about 30% cell death. However, the leukemic cells progressively died in a dose-dependent manner reaching to about 80% cell death at 250 μg/ml of the dried papaya skin extract.

The water extract from papaya pulp was unable to cause significant cell death of Jurkat leukemic T-cells.

Effect of papaya methanol extracts on T- cell leukemia

The effects of papaya methanol extracts from different fractions are presented in Figure 2. The methanolic extract of papaya leaves appeared to be less effective than water extract. At 25 μg/ml a significant (P < 0.05) 30% decrease in cell viability resulted; however, the cell viability was decreased by only 60% (P < 0.05) on further increasing the methanolic extract concentration to 250 μg/ml. Similarly, the methanolic extract of papaya seeds appeared to be less effective than water extract. At 200 μg/ml, a significant P < 0.05) decrease in cell viability resulted (10%); however, the further decrease in cell viability was not significant at 250 μg/ml. The methanolic extract of skin also caused significant cell death by 25% at 25 μg/ml (P < 0.05). Further increase of the extract concentration resulted in a dose-dependent response reaching 80% cell death at 250 μg/ml .

The methanol extract from papaya pulp caused a modest significant effect by decreasing cell viability by 20% at 50 μg/ml ; however, on increasing the concentration of pulp extract to 250 μg/ml , there was no further increase in cell death.

Effect of papaya water extracts on B- cell leukemia

The effects of water extract from different papaya fractions on Daudi leukemia B-cells are presented in Figure 3. Treatment with leaf extract caused a dose-dependent decrease in cell viability. Concentrations as low as 25 μg/ml caused a significant 20% cell death (P < 0.05). On further increasing the extract concentration, significantly more cells (P < 0.05) died in a dosedependent manner, with approximately 50% cells dead at the highest concentration of 250 μg/ml. The water extracts from seeds and pulp have no significant effect on cell viability at any concentration. The extract of papaya skin started to significantly (P < 0.05) inhibit leukemic cell proliferation at 25 μg/ml causing about 25% cell death. However, on further increasing the extract concentration, no change in cell death was observed

Effect of papaya methanol extracts on B- cell leukemia

The effects of methanol extracts from different papaya fractions on Daudi leukemia B-cells are presented in Figure 4. The methanolic extract of papaya leaves appeared to affect Daudi B-cells, similar to water extracts. At 50 μg/ml, a significant 30% (P < 0.05) of cells died and the cell death was further increased in a dose-dependent manner to 80% (P < 0.05) at 250 μg/ml. Consistent with the water extract, the methanol extract of papaya pulp has no effect on Daudi B-cells. The methanolic extract of skin had a similar effect on Daudi B-cells as the water extract. It caused significant cell death by 25% at 25 μg/ml (P < 0.05). Increasing the concentration of extract resulted in a dose-dependent effect, reaching 50% cell death at 250 μg/ml.

Discussion

The present study was carried out to investigate the effect of water and methanol extracts of papaya leaves, skin, pulp, and seeds on Jurkat leukemia T- cells and Daudi leukemia B-cells. Water and methanol were used because phenolic compounds have different chemical characteristics and polarities, and because of these properties, compounds in different fractions of papaya may or may not be soluble [30]. We initially tested these fractions for their phenolic contents and their anti-oxidation activity. Most of the anti-oxidation activity in fruits and vegetables is due to their total polyphenolic contents [31]. Our data indicate that papaya seeds have higher amounts of polyphenols than that of other fractions (Table 1). Papaya leaves also contained substantial amounts of total phenolic contents but were 1/3 that of seeds. Other fractions have very little total phenolic content. In seeds, the amount of total phenolic content was similar in both extracts, but the leaf water extract has less total phenolic content than that of methanol. However, the water extract of skin contained almost twice the polyphenols than that of the methanol extract. Organic solvents are generally used to extract bioactive agents from fruits and vegetables. The most suitable solvents for the extraction of polyphenols are aqueous mixtures containing ethanol, methanol, acetone, and ethyl acetate. Ethanol has been known as a good solvent for polyphenol extraction and is safe for human consumption. Methanol has been generally found to be more efficient in the extraction of lower molecular weight polyphenols, whereas aqueous acetone is good for the extraction of higher molecular weight flavonoids [32]. Currently, over 8000 phenolic structures have been identified in fruits and vegetables [33], and flavonoids are one of the major phenolic classes comprising almost 4000 compounds present in different edible plants [33]. We also determined the total flavonoids in the papaya extracts. Our data show that water extracts of seeds and methanol extracts of leaves contained the highest amount of total flavonoid content, which represented about 30- 40% of total phenolic compounds in seeds and leaves (Table 2). The data suggest that most of the polyphenols in seeds are water-soluble flavonoids, whereas a higher proportion of polyphenols in leaves are less water-soluble flavonoids.

Next, the anti-oxidation activities were determined in these fractions. The leaves and seeds possess a higher anti-oxidation activity compared to that of other fractions (Table 3). Although leaves and seeds have different phenolic and flavonoid contents, they have similar profiles for their anti-oxidation activity. It was interesting to note that methanol extracts exhibited higher antioxidation activity than the water extracts. This difference could be due to the different natures of polyphenols.

Next, we analyzed the anticancer properties of papaya fractions on Jurkat cells and Daudi cells. The Jurkat cells are an immortalized line of human T lymphocyte cells that are often used to study acute T cell leukemia. The Jurkat cell line was established in the late 1970s from the peripheral blood of a 14-year-old boy with T cell leukemia [34] whereas the Daudi cell line is a wellcharacterized B lymphoblast cell line which was derived from a 16-year-old black male with Burkitt’s lymphoma [35].

A large number of phytochemicals from vegetables, fruits, and grains have been reported to possess anticancer properties and are promoted for cancer prevention and treatment [36-40]. Recently, multiple scientific reports have emerged about the medicinal benefits of papaya [16,41-43].

Our data indicated that none of the extracts from seeds or pulp possess significant inhibitory activity for cell proliferation in Jurkat leukemic T-cells or Daudi leukemic B-cells. The extracts from leaves and skin, however, showed significant inhibition of both Jurkat leukemic T-cells and Daudi B-cell growth. This effect was stronger on Jurkat T-cells and modest on Daudi B-cells. During the present study, we found that papaya seeds contained the highest amounts of phenolic compounds; however, none of the extracts from papaya seeds have any inhibitory activity for Jurkat leukemic cells. Our data clearly indicated that the inhibitory activity of these extracts was neither dependent on their phenolic contents, nor was it dependent on their anti-oxidation activity. It appears from this observation that the inhibitory compounds in papaya leaves and skin extracts acted on cellular mechanisms linked to cell growth. For example, phytochemicals have been shown to interfere with the stabilization of the microtubule structure, thereby inhibiting mitosis and cancer cell propagation [44]. Vincristine and vinblastine, isolated from the leaves of Madagascar periwinkle, were the first phytochemicals to be used clinically in combination with other anticancer agents in lymphomas, leukemias, and breast, lung, and ovarian cancers [45,46].

Epigallocatechin-3-gallate (EGCG), a polyphenol from the leaves of Camellia sinensis (processed to green tea), possesses anti-oxidation activity but it limited cancer cell proliferation by reducing DNA methylation through the inhibition of DNA methyltransferase together with reactivation of the silenced tumor suppressor genes [47]. Curcumin (diferuloylmethane), a polyphenol isolated from the rhizome of the turmeric plant, has also shown therapeutic efficacy on numerous disorders, including cancer [48]. Curcumin is reported to inhibit NF-κB signaling that triggers the intracellular inflammatory response as well as cellcycle- associated genes. By arresting the cell cycle and inducing apoptosis through the relaying pathways, curcumin interferes with angiogenesis and reduces tumor invasion.

An additional group of anticancer phytochemicals functions as inhibitors of topoisomerase I or II, which are the nuclear enzymes that control DNA supercoiling, eliminate tangles in the chromatin structure, and allow DNA to be replicated and transcribed. Thus, topoisomerase inhibitors can act as anticancer agents by inducing a delay of the cell cycle, followed by cell death [36]. β-Lapachone from the bark of the Lapacho (Handroanthus impetiginosus) plant, camptothecin from the bark/stem of Camptotheca acuminata (the Chinese happy tree), and podophyllotoxin from the root of the Mayapple (Podophyllum peltatum) plant are examples of phytochemicals inhibiting topoisomerases in cancer cells [49- 51]. We have not yet characterized the chemical composition of aqueous and methanolic extracts of leaves and skin or explored the molecular mechanisms that may be involved in inhibition of Jurkat leukemic cell growth. Clearly, additional experiments are required for isolation and identification of active compounds in papaya leaves and skin to determine the cellular mechanisms of their anticancer activity.

Our data suggest that consumption of papaya leaves or skin may be beneficial for preventing and/or treating leukemia, particularly T-cell leukemia. Historically, papaya leaves have been used in different cultures around the world, which suggests that the consumption of leaves extract is not toxic [52]. It is possible that anticancer compounds from leaves can be extracted by boiling them in water. However, the skin is not typically consumed because of its bitter taste. The skin can be dried and made into a powder that can be consumed in the form of pills. Alternatively, the active compounds from leaves and skin can be extracted and isolated in pure form, which can be the basis for new therapeutic leukemia drugs. In conclusion, this study suggests that leaves and skin of papaya contained anticancer activity for leukemic cells.

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juniperpublishers-jdvs · 2 years ago

Text

Biological Efficient Dairy Cows in Grazing Systems

Abstract

The availability of indicators that would help to avoid the overvaluation of one of the characteristics involved in the assessment of a good dairy cow over others that are important as well would also allow identifying the most adapted biotypes to the different environments existing at the place of the evaluation This article aims to discuss the need to use several productive and reproductive indicators when measuring the biological efficiency of a dairy cow in grazing systems. It was used retrospective data corresponding to the lactations of 300 primiparous and multiparous cows of the Holstein breed - American-Canadian biotype, along with records of their entire productive life, from their incorporation into the system until their sale or death. The animals were divided into two categories: pure cows (PC, n = 120) and cows in the breeding record (CBR, n = 180). It is observed that there are two different strategies in some variables that achieve the same biological efficiency, where the CBRs live longer, produce less and have smaller first delivery intervals. There are no significant differences in the milk index but in the fat index. It is concluded that the greater individual production does not guarantee a greater production at the end of the productive life of the cow, nor a greater productive efficiency when considering the time involved to produce a certain amount of liters. In grazing systems, the contribution of other variables included in the milk index - longevity, rearing efficiency and reproductive behavior - should be considered while searching for an aggregate indicator that tends to achieve greater productive efficiency.

Keywords: Dairy cows; Indicators; Efficiency; Grazing Systems

Introduction

The notable increase in the productive performance and size of these modern high-production cows has been made possible by the repeated and asymmetric use of a selection based exclusively on milk production. Although this process has been accompanied by changes in the nutritional area, these have not been sufficient to prevent vital function deterioration such as reproduction and survival. It becomes increasingly difficult to provide a non-limiting environment, being almost impossible during the initial phase of lactation [1]. The efficiency of a productive system is one of the most important factors from an economic and social point of view. And the most used modality to evaluate it is assessing indicators of biological and economic productivity. However, when producing in conditions where resources are scarce and expensive (grazing systems), not only products or outputs but also inputs should be considered when evaluating efficiency. In the particular case of high production dairy cows, the traits associated with biological efficiency or fitness (reproduction and longevity) have deteriorated despite their importance for the viability of the company [2,3].

Suggest that the sustainability of dairy systems depends, to a large extent, on the availability of a biotype adapted to the handling conditions and that it is capable of efficiently transforming food into good quality milk. This biotype must have a good reproductive performance, being the main goal of the system to maximize an efficient productive response per unit area [4]. The search for the maximization of the value of a single productive variable disregarding the remaining variables can alter the equilibrium and deteriorate the overall efficiency of productive systems [5]. Every open system processes the inputs received and generates outputs. In productive systems, the concept of efficiency refers to the most appropriate way to use resources with existing technology and products.

As a result of this positioning, it is considered that a production process is efficient if the maximum output is obtained with the lowest possible inputs [6]. In dairy production, the expression “maximize outputs” may have different connotations: maximize individual production during lactation or maximize production considering the entire life of the cow, reproductive success should be considered in the analysis. The amount of milk produced by a cow can be considered the most important indicator in intensive systems, even though, this indicator alone is not the most appropriate to make operational a complex variable like productive efficiency, when the goal is to make the most out of grazing systems. In these cases, it should be complemented, or even replaced, by other more aggregated indicators that constitute alternatives as a more comprehensive measure to assess the behavior of production in those systems in which pasture is the basic component of the diet.

The availability of indicators of this nature would help to avoid the overvaluation of one of the characteristics involved in the assessment of a good dairy cow over others that are important as well. It would also allow identifying the most adapted biotypes to the different environments existing at the place of the evaluation [7]. This article aims to discuss the need to use several productive and reproductive indicators when measuring the biological efficiency of a dairy cow in grazing systems.

What Is Efficiency

Efficiency is the relationship between an income and an expense, between an input and an output or between a resource and a product [8]. When measuring efficiency, it is necessary to specify exactly which elements are used to evaluate the result through a relation of its values. And to define the units used to measure the values of these elements. The concept of efficiency refers to a relationship between elements and that the circumstances in which the relationship is established have a high specificity. As a consequence, the term itself is very relative, and any value that can be considered as good or low is even more so [8].

How to Measure Efficiency

The advantages of grazing systems are sought within the framework of this approach, in which the cow is provided directly with the necessary input to meet its food requirements, without the need for transportation, processing or distribution of rations [9]. Although it is the most widespread modality, the amount of milk produced by a cow does not represent the most appropriate indicator to make operational a complex variable as productive efficiency. As such, it should be complemented or replaced by other more aggregated indicators creating alternatives for a more comprehensive production measurement to assess their performance in grazing systems. The availability of indicators of this nature would help to avoid the overvaluation of one of the characteristics involved in the assessment of a good dairy cow over others that are important as well. It would also allow identifying the most adapted biotypes to the different environments existing at the place of the evaluation [10].

It was used retrospective data corresponding to the lactations of 300 primiparous and multiparous cows of the Holstein breed - American-Canadian biotype, along with records of their entire productive life, from their incorporation into the system until their sale or death. The animals were divided into two categories: pure cows (PC, n = 120) and cows in the breeding record (CBR, n = 180). The existence of significant differences in the assessed time between groups was studied by applying variance analysis to a classification criterion. JMP 5.0 for Windows (JMP®, SAS Institute, 2003) was used for statistical analysis Table 1.

The following variables were analyzed: a. Number of births b. Milk production (PL by its initials in Spanish): liters produced per cow adjusted to 305 days c. Age at first birth (PPE by its initials in Spanish) in days d. Total Butyrose Fat production in kg: GB, ΣGBi where “i” are the kilograms produced in the j-th lactation e. Total milk production (liters) = pl, Σpli, where “i” are the liters produced in the j-th lactation f. Milk index (milk production per day of life) in liters = IL, by its initials in Spanish, (il: LT / e e: age in days at the end of the last lactation): il = pl / age [11] g. Fat Index (production of Butyrose fat per day of life) in kg = IG by its initials in Spanish, (ig: Total GB / e e: age in days at the end of the last lactation): ig = kg GB / age [10] h. First delivery interval - delivery in days (IPP by its initials in Spanish): Σipp, where “i” are the days between deliveries / number of deliveries [12].

Pure cows are pregnant at an age closer to optimal, showing a difference of 60 days with cows in the breeding record. They produce in their five lactations 6516 liters on average against the 5933 liters in six lactations of cows in the breeding record, achieving a total of 41767 liters in its life against the 39653 liters of pure cows. Additionally, the pure cow takes for each new delivery 51 days (total 306 days (6 deliveries * 51 days)) more than the cow in the breeding record. In conclusion, the advantage in days they have because of its first birth during its life is lost in a single IPP, as well as producing almost for a year in the less efficient part of the lactation curve.

It is observed that there are two different strategies in some variables that achieve the same biological efficiency, where the CBRs live longer, produce less and have smaller IPP. There are no significant differences in the milk index but in the fat index. Although considering each variable separately is relevant, when reference values are available and allow these cows to be positioned in particular in the framework for milk production of the Holstein breed, the characterization of the efficiency of a productive system requires a joint analysis of all of them instead of individual consideration [5].

Conclusion

It is concluded that the greater individual production does not guarantee a greater production at the end of the productive life of the cow (LT) nor a greater productive efficiency when considering the time involved to produce a certain amount of liters (il). In grazing systems, the contribution of other variables included in the milk index - longevity, rearing efficiency and reproductive behavior - should be considered while searching for an aggregate indicator that tends to achieve greater productive efficiency.

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juniperpublishers-crdoj · 2 years ago

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Anti-hyperglycemic Effect and Regulation of Carbohydrate Metabolism by Phenolic Antioxidants of Medicinal Plants against Diabetes

Authored by HP Gajera

Introduction

Background

Diabetes mellitus is a carbohydrate metabolism disorder of endocrine system due to an absolute or relative deficiency of insulin secretion, action, or both [1]. The disorder affects more than 100 million people worldwide and it is predicted to reach 366 million by 2030. The non-insulin dependent diabetes mellitus (NIDDM, type 2) is the most prevalent form globally which is associated with elevated postprandial hyperglycemia. The occurrence of NIDDM 2 has been shown an alarming increase during the last decade (http//www.who.Int/diabetes/en/).

Plant derivatives with reported hypoglycaemic properties have been used in folk medicine and traditional healing systems. Very few of these traditional antidiabetic plants have received proper scientific or medical scrutiny despite recommendations by WHO. Ayurveda and other Indian traditional approaches have described more than 800 plants in the Indian subcontinent, known to possess antidiabetic potentials. These require to be effectively studied and in fact only few of them have been characterized for their mechanistic action [2,3]. Pancreatic α-amylase, is a key enzyme in the digestive system and catalyses the initial step in hydrolysis of starch to maltose and finally to glucose. Degradation of this dietary starch proceeds rapidly and leads to elevated post prandial hyperglycemia. It has been shown that activity of HPA in the small intestine correlates to an increase in post-prandial glucose levels, the control of an important aspect in treatment of diabetes [4]. Hence, retardation of starch digestion by inhibition of enzymes such as α- amylase would play a key role in the control of diabetes.

The discovery of specific high-affinity inhibitors of pancreatic α-amylase for the development of therapeutics has remained elusive. Inhibitors currently in clinical, use for example, acarbose, miglitol, and voglibose, are known to inhibit a wide range of glycosidases such as α-glucosidase and α-amylase. Because of their non specificity in targeting different glycosidases, these hypoglycemic agents have their limitations and are known to produce serious side effects. Therefore, the search for more safer, specific, and effective hypoglycemic agents has continued to be an important area of investigation with natural extracts from readily available traditional medicinal plants offering great potential for discovery of new antidiabetic drugs [5]. Ponnusamy et al. [6] studied on antidiabetic medicinal plants for human pancreatic amylase inhibitory effect in vitro and found that pancreatic α-amylase lower the levels of post perandial hyperglycemia via control of starch breakdown. The probable mechanism of action of the above fractions is due to their inhibitory action on HPA, thereby reducing the rate of starch hydrolysis leading to lowered glucose levels. Phytochemical analysis revealed the presence of alkaloids, proteins, tannins, cardiac glycosides, flavonoids, saponins and steroids as probable inhibitory compounds.

Anti-hyperglycemic effect of natural phenolic antioxidants

Advanced molecular studies showed that methanol extract of black jamun plant modulate the expression of glucose transporter (Glut-4), peroxisome proliferator activator receptor gamma (PPARγ) and phosphatidylinositol-3-kinase (PI3 kinase) comparable with insulin and rosiglitazone [7]. Evaluation of black jamun containing antidiabetic poly herbal formulation in alloxan induced diabetic rats also showed significant hypoglycemic activity, positive glucose tolerance activity and reduced lipid peroxidation in various organs compared to that of the diabetic control animals [8]. Meshram et al. [9] studied on hypoglycaemic action of black jamun seeds. The possible mechanism by which extracts bring about its may be by affecting the activity of glucoamylase or by increasing the glycogen biosynthesis. Thus, the significant inhibition of glucoamylase suggests that the active hypoglycaemic compound present in methanolic extracts of jamun seeds does not necessarily require the presence of functioning of β-cells for its favourable action seen in type-I. It means the methanol extracts of black jamun seeds may act in a variety of diabetic conditions with or without functioning of pancreatic β-cells.

Hasan et al. [10] studied DPPH radical scavenging activity of black jamun seed extracted in methanol. It has been determined that the antioxidant effect of plant products is mainly due to radical scavenging activity of phenolic compounds such as flavonoids, polyphenols, tannins, and phenolic terpenes [11]. Liang & Yi [12] identified hydrolysable tannins (ellagitannins) extracted from black jamun fruit showed a very good DPPH radical scavenging activity and ferric reducing/antioxidant power. The results are promising and indicating the utilization of the fruit of black jamun as a significant source of natural antioxidants.

Stanely et al. [13] evaluated the protective effects of gallic acid on brain lipid peroxidation products, antioxidant system, and lipids in streptozotocin induced type II diabetes mellitus. The results showed the beneficial effects of gallic acid on brain metabolism in streptozotocin induced type II diabetic rats. A diet containing gallic acid may be beneficial to type II diabetic patients. Meguro et al. [14] investigated the effects of continuous ingestion of a catechin rich beverage in patients with type 2 diabetes. The significant increase in insulin level was observed to patients fed with green tea containing the catechin. Rizvi et al. [15] evaluated the effect of tea catechins (epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG) and epicatechin (EC)) on markers of oxidative stress in erythrocytes from type 2 diabetics. The relative effectiveness of individual catechins are in the order of EGCG>ECG>EGC>EC. Higher intake of catechin rich food by diabetic patients may provide some protection against the development of long term complications of diabetes. Chlorogenic acid is a major component of coffee that may provide more of an explanation for coffee’s effect on risk for type 2 diabetes. Chlorogenic acid proposed beneficial effects on glucose metabolism. The chlorogenic acid may delay glucose absorption in the intestine through inhibition of glucose-6-phosphate translocase 1 and reduction of the sodium gradient driven apical glucose transport. In vitro studies and animal studies showed that chlorogenic acid derivates can be decreased hepatic glucose output through inhibition of glucose- 6-phospatase [16].

Jung et al. [17] investigated the blood glucose lowering effect and antioxidant capacity of caffeic acid in mice. Caffeic acid induced a significant reduction of the blood glucose and glycosylated hemoglobin levels than the control group. Increased plasma insulin by caffeic acid was attributable to an antidegenerative effect on the islets. Caffeic acid also markedly increased glucokinase activity and its mRNA expression and glycogen content and simultaneously lowered glucose-6- phosphatase and phosphoenol pyruvate carboxykinase activities and their respective mRNA expressions, accompanied by a reduction in the glucose transporter 2 expression in the liver. Zhi et al. [18] investigated the antioxidant activity of black jamun leaf extracts. Leaf extracts contained phenolic compounds, such as ferulic acid and catechin, responsible for their antioxidant activity.

Diabetes, when uncontrolled, causes dyslipidemia often followed by atherogenic abnormalities. Balasubashini [19] examined role of ferulic acid (flavonoid) in diabetes induced dyslipidemia. Study demonstrates that ferulic acid lowers the lipid levels in diabetic rats and hence prevents further complications. It has been documented that ferulic acid may lower blood sugar level of Type 1 and Type 2 diabetic mice by enhancing insulin secretion [20]. Diabetic mice was given rice derived ferulic acid for 17 days and results showed that plasma insulin level increased while blood sugar level decreased significantly compared with control [21]. Ferulic acid may be beneficial in Type 2 diabetic and for the management of diabetic complications. Hussain et al. [22] indicated that quercetin can decrease postprandial glucose level after disaccharides loading, which may be mainly attributed to inhibition of α-glucosidase as one of the expected mechanisms for the reduction of plasma glucose. This effect subsequently leads to suppression of postprandial hyperglycemia. Thus, quercetin can be considered as a potential candidate for the management of type 2 diabetes mellitus. Medicines that reduce postprandial hyperglycemia by suppressing the absorption of carbohydrates are shown to be effective for prevention and treatment of non-insulin dependent diabetes mellitus [23]. Quercetin inhibited in vitro the intestinal α-glucosidase activity [24]. It has been also assumed that quercetin activates tyrosine kinase. Phosphorylation of the specific region of the subunit in insulin receptor (including Tyr- 1158, Tyr-1161 and Tyr-1162) correlates with receptor tyrosine kinase activation and the propagation of the biological actions of the hormone [25].

Correlations between antidiabetic, antiradical and phenolic compounds

Our previous study Gajera et al. [26,27] suggested that antidiabetic activity of fruit parts of black jamun landraces was positively correlated with free radical scavenging activity, nutraceuticals profile and individual phenolic constituents. Total phenols and individual phenolics are positively correlated with antidiabetic and antiradical activities but vary with different level of significances. Individual phenolics - gallic, catechin, ellagic and ferulic acids are highly positively correlated (P0.001) with antidiabetic and free radical scavenging activity. The positive correlation (P0.01) was established for caffeic and chlorogenic acids to scavenge free radicals and α amylase inhibitory activity (antidiabetic) for methanolic extract of black jamun fruit parts. The quercetin was found only in seed and its part kernel fraction of BJLR-6 (very small size fruits) and found to be positively correlated (P0.05) with antidiabetic activity. Among the fruit parts of black jamun land races, seed exhibited maximum seven individual phenolics and total phenols, particularly in their kernel parts. Among the individual phenolics, gallic acid was most diverse phenolic constituents which significantly positively correlated (P0.001) with inhibition of α amylase activity and DPPH radical scavengers followed by catechin, ellagic and ferulic acids in different fruit parts of black jamun land races. The study explained correlation of individual phenolics including flavonoids (ferulic) with α amylase inhibition and free radical scavenging activities in fruit parts of indigenous black jamun landraces.

Muniappan et al. [28] reviewed the black jamun as an antidiabetic plant which contained ellagic acid, glycosides, anthocynine, kampferol, marcein and isoquarcetine; and halt the diastolic conversation of starch into sugar. The phenolic constituents may be contributed directly to the antioxidative action. Consequently, the antioxidant activities of plant/ herb extracts are often explained by their total phenolics and flavonoid contents with good correlation.

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ourhaileydavies · 2 years ago

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Skin Closure with Barbed Sutures: An Early Evaluation of Cosmesis and Complications

Author By: Vinay Kumar Tiwari

Abstract

Introduction: Barbed sutures have the potential advantage of decreased operative time and better wound cosmesis due to bidirectional fixation of wound. Present study evaluates the complications and scar cosmesis after skin closure with barbed sutures.

Material and Methods: This was a prospective, observational study. Patients underwent subcuticular skin suturing in surgically created clean wounds. Half of the wound closure was done with barbed absorbable suture and other half with non-barbed absorbable sutures. Comparison of scar cosmesis and wound complications was done.

Results: Mean time taken for suturing per cm of wound was lower in barbed suture group. Suture extrusion rate was higher in barbed suture group. No statistically significant difference was found between cosmesis of scar and rates of infection between the two groups.

Conclusion: The current study did not find any added advantage of using barbed sutures over and above conventional sutures in terms of cosmesis of scar. The only advantage was that of decreased operative time.

Keywords: Barbed sutures; Wound cosmesis; Operative time; Scar; Suture extrusion

Introduction

Barbed sutures have been in use since many decades. They provide effective wound closure due to bidirectional fixation within the wound. The presence of barbs leads to distribution of tension across the wound and also eliminates the need for knots. A barbed suture prevents backward slippage of the sutures, and as a result it does not gape in areas of tension allowing for an aesthetic subcuticular closure. Some clinical studies have shown a better resultant scar.

These sutures allow for a running closure of the wound, with fewer preliminary buried sutures leading to saving of one third to half of the time taken in suturing which can be their greatest benefit [1,2]. This study was done in Indian population where previously no such study has been reported. This study was done by using barbed and non-barbed suture in the same wound by dividing it into two halves thus removing all the confounding factors.

Material and Methods

This prospective, observational study includes 50 patients and was conducted from February 2018 to August 2019. Patients of all age groups were included in the study. The study population consisted of all the patients coming to plastic surgery department at our institute. All patients undergoing primary wound closure in a wound length of greater or equal to 5cm were included in the study. The study included patients with surgically created wounds and included simple surgical incisions and excisional wounds.

Patients with uncontrolled diabetes mellitus, collagen vascular disease, irradiated skin, immunodeficient states, past history of keloid formation, active cutaneous or systemic infection at the time of surgery, chronic renal or hepatic failure were excluded from the study. Preoperatively, patient’s wounds were marked, and dimensions were noted by using Vernier Callipers. Wound was divided into two equal halves and marked. Subcuticular continuous skin closure of one half of the wound was done by conventional polydioxanone sutures while the other half was sutured by barbed polydioxanone sutures. The wound closure was done by the same surgeon. Time taken for surgical closure was noted.

Post operatively various parameters were monitored to evaluate outcome and complications of patients. All patients were followed up for 2 weeks, 1 month, and 3 months after surgery and scar assessment was done by an independent and blinded observer. Scar cosmesis was compared by using POSAS and Vancouver scar scale. Width of the scar, time taken for surgical closure, median scar width and wound infection rates were compared. Wound infection was defined as wound erythema, tenderness or pus discharge from the wound.

Data was entered in Microsoft Excel spreadsheet. Statistical analysis was performed using SPSS (version 18.0). Categorical variables were summarized as frequencies and percentage. Continuous variables were presented as mean and standard deviation or median and inter quartile range based on the normality of data. Normality was assessed using Kolmogorov mirnov test. In case of non-normal or asymmetric distribution, non-parametric test was performed to assess statistical significance.

The following statistical tests were applied

(1) Quantitative variables were compared using Unpaired t-test/Mann-Whitney Test (when the data sets were not normally distributed) between the two groups.

(2) Qualitative variables were compared using Chi-Square test /Fisher’s exact test. A p value of <0.05 was considered to be statistically significant.

Results

A total of 50 patients were studied for a period of 18 months. There were 16 (32 %) women and 34 (68%) men. Maximum (54%) of patients were in the age group of 20-39 years (Table 1). The wounds involved head and neck in 7 patients, trunk in 23 patients, upper extremity in 5 patients, and lower extremity in 25 patients. The width of the scar was evaluated at 2 weeks, one month and 3 months post-operative period. Median scar width (in mm) was compared. Mann-whitney test was performed (Figure 1, 2 and 3).

There was no statistically significant difference in the width of the resultant scars between barbed and non-barbed sutures (Table 2). Time taken in suturing per cm of wound between barbed and nonbarbed suture groups was compared. In our study the time taken for barbed suture was less (23.38 seconds per cm of wound) than conventional absorbable sutures (25.16 seconds per cm of wound) and the difference was statistically significant with a p value of 0.001.

Comparison of Vancouver scar scorewas done at 2 weeks, one month and 2 months’ time period. The median scar score was higher for barbed sutures at one-month postoperative period. Mann-whitney test was performed and the difference was not statistically significant (Table 3). POSAS score (patient) was compared at 2 weeks, one month and 2 months’ time period (Figure 4, 5 and 6). Mann-whitney test was performed and the difference was not statistically significant. The median score was same for both the groups at 2 weeks follow up period. The score was higher for barbed suture group as compared with non-barbed suture group at 1 month and 3 months follow up period. But this difference was not statistically significant (Table 4).

POSAS score (observer) was compared at 2 weeks, one month and 2 months’ time period. The score was higher for barbed suture group as compared with non-barbed suture group at 2 weeks and 1 month follow up period. Mann-Whitney test was performed. The difference was not statistically significant (Table 5). Overall, no significant difference was found in wound cosmesis between barbed and nonbarbed suture group as evaluated by Vancouver scar scale and POSAS observer and patient scar scale. The wounds were evaluated for surgical site complications. Comparison of suture extrusion between groups was done.

In barbed suture group suture extrusion was seen in 9 cases over a period of 3 months. 7 cases were with wounds involving lower extremity and 2 patients with wounds in upper extremity. Only one case of suture extrusion was present in nonbarbed suture group which occurred in upper extremity. Chi square test was performed, p value was <0.01 and the difference was considered as statistically significant. Comparison of surgical site infection at two weeks, one month and 3 months’ time period was done. Chi square test was performed. Higher rate of infection was seen with barbed sutures but the difference was not statistically significant (Table 6).

Discussion

Scarring is an inevitable result of any surgery. Since time immemorial surgeons have been searching for techniques to reduce postoperative starring. Barbed sutures were introduced as tool to reduce scarring. The presence of barbs leads to better tissue fixation and lesser scarring. Theoretically barbed sutures lead to bidirectional fixation of wound which leads to lesser wound gaping and decreased width of resultant scar [1,2]. This was not seen in our study and there was no statistically significant difference in width of the resultant scars between the barbed and non- barbed suture groups.

Decreased width of scar leads to a better aesthetic outcome. A study by Koide et al displayed a significantly better aesthetic outcome in the barbed suture group than nonbarbed suture group [3]. But in our study, there was no statistically significant difference in scar cosmesis as compared by Vancouver scar scale and POSAS (observer and patient scar scale) between the barbed and the non-barbed suture groups. Our findings were similar to studies by Kristen Aliano et al, Amy P Murtha et al, Rubin et al and Grigoryants et al [4-7]. As our study evaluated the scar for only three months, a prolonged follow up is further required to evaluate the scar after remodelling. The time taken per cm of wound was evaluated for both barbed and non-barbed side.

In our study the time taken for barbed suture was less than conventional absorbable sutures and the difference was statistically significant. Similar results were seen in studies done by Koide et al, Kristen Aliano et al,Grigoryants et al, Jeremy P. Warner et al and Blacam et al [3,4,7-9]. Jandali et al, found that using barbed sutures reduced the operative time of unilateral breast reconstruction significantly, but no significant difference was seen in the operative time of bilateral breast reconstruction [10]. The only study showing increased operative time was that by Murtha et al, but the results were not statistically significant [5]. The decreased time taken in barbed suture group in our study is likely because there is no need to put a knot in barbed suture at the ends of the suture line. Also, the handling of suture is improved as the tissue gets fixed with each stitch with a barbed suture.

Our study evaluated the wound for surgical site infection. It was seen that barbed sutures were associated with a higher rate of surgical site infection, but the difference was not statistically significant. Overall, it was seen that the patients having lower extremity wounds had more surgical site infection. This may be due to a greater number of patients having wounds in lower extremity. Also, lower extremity skin is less lax hence wounds are usually closed under more tension leading to wound ischemia and more wound infection. Similar results were seen in studies done by Murtha et al, Jandali et al, where more infections were seen in barbed suture group, but it was not statistically significant [5,10].

Roberto Cortez et al, studied the complications associated with barbed sutures. Their retrospective study found that barbed sutures were associated with significantly higher rates of minor wound complications [11]. The increased incidence of infections in the barbed suture group may be due to the fact that the presence of barbs in the suture leads to colonization of bacteria and resultant higher rates of infection. In our study suture extrusion was seen in 9 out of 50 patients in the barbed suture site as compared to only one patient in the conventional sutures group. The difference was statistically significant. Suture extrusions or stitch abscesses were noted in a total of 10 patients, a probable consequence of the superficial placement of the sutures during subcuticular closure of dermal and subdermal layers.

Similar results were seen in a study done by Rubin et al, which showed increased suture extrusion on the barbed suture side than on the smooth suture side [6]. These findings were consistent with the findings of our study. In a study by Murtha et al, suture extrusion was seen in 10.2 percent of barbed suture subjects as compared with 19.7 percent control suture subjects [5]. The placement of sutures in our study was superficial dermal which might have led to the higher extrusion rate. Further study is warranted to evaluate the extrusion rate of sutures placed in deeper dermis. We used delayed absorbable sutures in our study which could also have contributed the higher rate of extrusion of sutures.

As Grigoryants et al, compared extrusion rate of slow absorbing and rapidly absorbing barbed sutures and found that rapidly absorbing barbed suture had lesser extrusion rate than delayed absorbing barbed sutures [7]. Overall, the complication rate was higher for the barbed suture group. These findings were in contradiction to a study done by Blacam et al, comparing abdominal closure with barbed and non-barbed sutures which found that the complications associated with barbed sutures were lesser. But the follow up period in this study was one month as compared to three months in our study which could have led to the difference in the findings [9].

Conclusion

Barbed sutures lead to decreased operative time but there was no advantage in terms of better scar cosmesis. Though there was an increased rate of surgical site infection barbed sutures, the difference was not statistically significant. The suture extrusion rate with barbed sutures was higher and the difference was statistically significant. Probably study over a larger sample size will give us a better picture. Barbed sutures are costlier than conventional sutures, but their use is less time consuming. So further studies in terms of cost analysis in relation to decreased operative time are warranted to fully evaluate the cost of using these sutures.

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Flaxseed- A Shield against Diseases?-Juniper Publishers

Abstract

Modernisation has made our lives comfortable but with an added baggage of problems. One of them is poor health which is because of increased incidences of modern day diseases like obesity, metabolic syndrome, diabetes, cardiovascular diseases, polycystic ovarian disease, allergies of different sorts and some type of cancers. Dietary modification by addition of a functional food, as one of the lifestyle modifications can help us to win our battle against this assortment of diseases. Flaxseed is being labelled as one of the functional foods. This review looks into the different research works and data to see if flaxseed fulfils the criteria of a functional food and can be used as a shield to protect us against these modern day diseases. We came to the conclusion that flaxseed is one of the richest sources of a-linolenic acid and lignan, in addition to being a good dietary source of fibre. Thus, flaxseed a natural source because of the presence of these bioactive compounds can help us in prevention and provide us protection against plethora of diseases. It is recommended that flaxseed a great functional food should be added as a dietary supplement in different forms and worldwide people should be educated about its health benefits.

Keywords:Flaxseed; Functional Food; a-Linolenic Acid (Ala); Lignans; Omega-6 Fatty Acids; Fibre

Abbreviations: SDG: Secoisolariciresinol Di-Glucoside; PUFA: Poly-Unsaturated Fatty Acids; DHA: Decosa-Hexanoic Acid; EPA: Eicosa-Pentaenoic Acid

Introduction

We have progressed in the modern world making our lives comfortable with the help of industrialisation, urbanisation, and globalisation, social and economic developments. These plethoras of events have led to rapid alterations in diets and lifestyles like inappropriate diet, poor quality of nutrition, lack of physical activity and much other behaviour known to be detrimental to health. Thus, large population of people have poor health because of the increased incidences of diseases like obesity, metabolic syndrome, diabetes, cardiovascular diseases, polycystic ovarian disease, allergies of different sorts and some types of cancers. Living a healthy life is a challenge for all of us in the modern day world as we are always being threatened by several degenerate lifestyle diseases. Indigenous populations tradionally used natural products to treat a broad range of diseases and ailments [1,2].

In recent times, many drug discovery experiments have natural products as their main component and because of their chemical diversity have led the researchers to study plant materials in hope of finding their medicinal properties [3,4]. Our quest for wellness and increasing awareness regarding the role of proper diet has made us search for foods that can work like medicine. The answer to this query is ‘Functional foods’ or ‘neutriceuticals’ which are basically foods or dietary compounds which not only provide basic nutrition but may also provide a health benefit .i.e., they give a health punch in addition to their traditional nutrient content [5]. Flaxseed is being labelled as a functional food because of its nutritional composition which has positive effects on disease prevention providing health beneficial components [6].

Discussion

Flaxseed is the seed from the flax plant, Latin name being Linum usitatissmum which means very useful [7] and is called as flaxseed/linseed, depending on whether it is being used for human consumption or for industrial uses respectively [8,9].

Canada is the major exporter of flaxseed and flaxseed products, approximately 80% of the global trade [8]. An analysis of brown Canadian flax averaged 41% fat, 20% protein, 28% total dietary fibre, 7.7% moisture and 4% Ash which is the mineral residue left after samples are burned [10,11]. Of all the lipids in flaxseed 53% are a-linolenic acid (ALA), 17% linoleic acid (LA), 19% oleic acid, 3% steric acid and 5% palmitic acid and provides an excellent n-6:n-3 fatty acid ratio of approximately 0.3:1[12]. The three components of flaxseed which are generally associated with improved cardiovascular health are [13]:

The omega-3 polyunsaturated a-linolenic acid (ALA, 20% of dry weight),

The plant lignan: secoisolariciresinol diglucoside (SDG, 1% of dry weight),

Soluble fibre (6% of dry weight).

Researchers are trying to find if omega-3 fatty acid: ALA present in flaxseed, which is one of its richest source [14] may help in protecting against certain infections and in treating diseases like ulcers, migraine headaches, eating disorders, attention deficit/hyperactivity disorders, emphysema, psoriasis, preterm labour, glaucoma, Lyme’s disease, lupus and panic attacks [15]. Many studies [16-19] done over a period of time have gathered data from large populations and reported that ALA levels are inversely correlated with primary cardiovascular events. Omega-3 fatty acids because of their biological effects have become useful in managing chronic conditions such as type-2 diabetes, kidney disease, hypertension, rheumatoid arthritis, stroke, Alzheimer disease, alcoholism and certain types of cancers [20]. After ingestion, ALA is metabolically converted into omega-3 fatty acids, eicosapentaenoic acid (EPA) and decosahexanoic acid (DHA) [15] and multiple clinical trials have demonstrated that the long chain polyunsaturated fatty acids (PUFA), EPA and DHA can reduce the risk of cardiac death [21- 23]. Fish is one of the sources of ALA but due to limited intake of fish worldwide, plant based PUFA: ALA may be an important alternative source and can be obtained from different types of nuts, seeds and seed oils, mainly flaxseed, walnut, canola oil and soybean oil [15]. Flaxseed is one of the most important oilseed crop because we can almost use plant’s every part commercially [8,9].

Dupasquier et al. [24] demonstrated that dietary flaxseed can inhibit atherosclerosis by decreasing the levels of circulating cholesterol and also via its anti-proliferative and anti-inflammatory actions at the cellular level [24]. Increased cardiovascular disease, obesity, diabetes, neurodegenerative declines and cancers are linked with chronic inflammation and oxidative stress [25]. High omega 6/omega 3 ratio in diet has found to promote the pathogenesis of many chronic diseases and increased dietary intake of linoleic acid, an omega-6 fatty acid leads to oxidation of low density lipoproteins (LDL), platelet aggregation and interferes with the incorporation of essential fatty acids (EFA) in cell membrane phospholipids. Omega-3 fatty acids exert strong anti-inflammatory effects by suppressing interleukin 1b, tumour necrosis factor-a and interleukin-6 while omega-6 fatty acids are pro-inflammatory [26,27]. Calguiri et al. [28] in their study on patients of peripheral arterial disease, using 30g of milled flaxseed/day for 6 months reported significant decrease in blood pressure, both systolic as well as diastolic and suggested that this might have occurred via soluble epoxide hydrolase inhibition by ALA of flaxseed, which altered oxylipin concentration.

Flaxseed also has bioactive compound lignin [29,30], the main lignan precursor being secoisosolariciresinol diglucoside (SDG) [31], after ingestion this plant SDG is converted to mammalian lignans by bacteria in the human colon [32]. SDG first undergoes hydrolysis to plant lignan secoisolariciresinol (SECO) which is further converted to enterolignans: enterodiol (ED) and enterolactone (EL), then these metabolites (phytoesterogens) are absorbed and exert their health benefits [33,34]. Thompson et al. [35] in their study on breast cancer patients found that daily intake of 25g of flaxseed significantly decreased cell proliferation, increased apoptosis and affect cell signalling by reducing c-erbB2(HER2) expression and proposed that may be lignans by competing with oestradiol for binding to oestradiol receptors act as anti-oestrogens. The mammalian lignan enterolactone has shown to decrease endogenous oestrogen synthesis by inhibiting aromatase activity [35,36]. Szewczyk et al. [37] used the compounds from the extracts of roots, leaves and stems of flax on the human breast cancer cell lines MCF7 (estrogen receptor positive) and BT20 (estrogen receptor negative) for their phytoestrogenic properties and suggested the role of plant extract for prevention of hormone sensitive carcinoma as they observed significant inhibition of cell vitality and proliferation in the MCF7 cell line.

In a study by Farzana et al. [38] on patients of polycystic ovarian disease(PCOS), supplementation with flaxseed powder, led to significant decrease in ovarian volume, number of follicles and improvement in frequency of menstrual cycles, reasoning it to be due to flaxseed causing reduction in testosterone, circulating oestrogen, luteinising hormone, insulin levels and increased follicle stimulating hormone contributing to follicular maturation and the anti-inflammatory actions to the reduction in ovarian volume. A diet of crushed 40g of flaxseeds was compared with the hormonal therapy for alleviating the menopausal symptoms; it was found to have the same effects but also decreased the glucose and insulin levels [39].

A variety of in-vitro and in-vivo studies have reported that the flaxseed lignan and its mammalian metabolites induce protective effects on many chronic diet related disease through variable mechanisms including phytoestrogenic and antioxidant effects [40-42]. Another study concluded that flaxseed plant lignans SDG and SECO are effective against oxidative stress as they exert strong antioxidant effects [33]. The fibre content of flaxseeds is twice that of high fibre beans and varies between 22% to 26% in the form of soluble (mainly mucilage gums) and insoluble (cellulose and lignins) dietary fibres in proportion varying between 20:80 and 40:60 [33,43,44]. It was found that flaxseed dietary fibres have a role in regulating the body weight by suppressing hunger and nutrient absorption, as the gel formed by them when mixed with water delays gastric emptying and decreases blood glucose level [34,45]. It also lowers cholesterol, as the gel surrounds it and doesn’t let it to be absorbed and favours its faecal excretion34. Thakur et al. [46] advised the use of flaxseed mucilage in combination with other mucilage providing added benefits for controlling Type 2 Diabetes Mellitus because when they used flaxseed mucilage to reduce the clinical symptoms of diabetes mellitus associated with lipidaemia there was decrease in fasting blood sugar, total cholesterol and LDL. In their study Kristensen et al. [45] found that both flax drink and flax bread reduced plasma total cholesterol, LDL, and increased fat excretion, so they opinioned that viscous flaxseed dietary fibres may be useful for lowering blood cholesterol and potentially play a role in energy balance.

When 55 hypercholesterolemic subjects were given 300/60mg/d of dietary SDG from flaxseed extract for 8 weeks, it significantly decreased total cholesterol, LDL-cholesterol and glucose concentrations in a dose dependent manner [47]. Flaxseed supplementation in postmenopausal women reduced serum total cholesterol, LDL cholesterol, triglycerides, apolipoproteinA-1 and apolipoprotein B concentrations the known risk factors of coronary heart disease [48]. Ingestion of flaxseed in hypertensive individuals produced significant reduction in blood pressure: both systolic and diastolic, with circulating a-linolenic acid levels correlating with systolic and diastolic blood pressures and lignan levels with changes in diastolic blood pressure [49]. Cassani et al. [50] found improvement in the blood levels of total cholesterol, LDL, uric acid, and adiponectin associated with weight loss, decreased systolic blood pressure and also reduction in inflammatory markers: C-reactive proteins (CRP) and tumour necrosis factor-  F-) and were in favour of adding flaxseed to a weight loss diet as an important tool to reduce the inflammatory markers CRP and TNF-a. Flaxseed intake decreased fasting glucose with increased insulin sensitivity in overweight or obese individuals with pre-diabetes but with no significant effect on fructosamine, CRP, adiponectin and Interleukin-6(IL-6) [51].

Flax is low in carbohydrates (sugars and starches) so can be advised to persons’ with specific diseases as it provides only 1g/100g contributing very little to total carbohydrate intake [34]. If we compare the amino acid pattern of flax protein with that of soybean which is considered as one of the most nutritious of the plant protein it is the same [52] and is rich in essential amino acids that are required for protein synthesis that have their role in maintenance and repair of cells, tissues and organs [34]. Calmodulin is a co-factor involved in the production of nitric oxide responsible for several neurodegenerative diseases and flaxseed protein hydrolysate induces a change in calmodulin’s secondary and tertiary structures [53]. It also inhibits angiotensin converting enzyme thus reducing angiotensin a vasoconstrictor which causes an increase in blood pressure [54]. Protein in flaxseed meal was observed to be more effective in reducing proteinuria and renal histologic abnormalities than soyprotein [55].

It has been found that 30g portion of the flaxseed constitutes 7% to 30% of the Recommended Dietary Allowances (RDA) for calcium, magnesium and phosphorous [11]. When its potassium content is compared with the recommended sources such as banana, its high [56] and high potassium intake has seen to be inversely associated with stroke incidences, platelet aggregation [57].

Conclusion and Recommendation

Modernisation along with making our lives comfortable has pushed us into a web of modern diseases like obesity, metabolic syndrome, diabetes, cardiovascular diseases, polycystic ovarian disease, allergies of different sorts and some type of cancers. Recent scientific research works have suggested that adding a functional food as a dietary modification can be one of the lifestyle modifications and play an important role in preventing and protecting us against these chronic diseases. Insight into multiple in-vitro and in-vivo research studies and data on flaxseed give us strong indication that flaxseed is one of the richest source of a-linoleinic acid and lignan in addition to being rich in dietary fibre. Flaxseed as one of the functional foods can act as protective shield to combat this variety of diseases. It being a good source of ALA can easily replace the fish and other marine sources of ALA whose use is limited worldwide. People should be educated about the health benefits of flaxseed and encouraged to incorporate it in their diet as a supplement in different forms.

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