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Objective: Aim: The aim of the study was evaluation of the methionine-enkephalin in patients with severe COVID-19 with various activities of the renin-aldosterone system in comparison with COVID-19 patients with pre-existing comorbidities (renal cell cancer, critical limb ischemia) and adverse pregnancy outcomes.., Patients and Methods: Materials and Methods: To test our hypothesis, this case-control study consisted of 20 healthy donors (control group); 49 patients with a positive diagnosis of COVID-19 according to PCR analysis; 15 patients with a positive diagnosis of COVID-19 in combination with renal cell cancer; 29 patients with a positive diagnosis of COVID-19 in combination with critical limb ischemia, 10 pregnant womens with COVID-19. Methionine-enkephalin, plasma renin activity, plasma aldosterone was measured by radioimmunoassay. Statistical and graphical analyses were done using Statistica 7.0 StatSoft software and using MedCalc., Results: Results: As our results have shown, hyperreninemia against the background of hypoaldosteronism and inhibition of enkephalinergic activity is fatal for COVID-19 patients. In COVID-19 patients with renal cell carcinoma, met-enkephalin, aldsterone, and plasma renin activity also showed a high predictive value for mortality. Whereas in patients with critical limb ischemia the strongest predictor biomarkers of mortality were only renin plasma activity. In conditions of physiological hyperreninemia during pregnancy, met-enkephalin acts as a biomarker for preterm birth, and in pregnant patients with COVID-19, plasma renin activity acts as such a biomarker., Conclusion: Conclusions: We found that in different categories of patients with COVID-19, the severity and outcome depend on the different activity of the renin-angiotensinaldosterone and enkephalinergic systems.

The opioid growth factor (OGF)-OGF receptor (OGFr) pathway is present in the ocular surface and functions to maintain homeostasis of the epithelium. The OGF-OGFr pathway has been reported to be dysregulated in diabetic individuals and animal models, and is reflected in elevations of the inhibitory growth factor, OGF, chemically termed [Met 5 ]-enkephalin. Recently, our laboratory reported elevated levels of OGF and OGFr in the serum and corneal epithelium of type 1 diabetic rats, suggesting that dysregulation of the OGF-OGFr axis may lead to dry eye, abnormal corneal surface sensitivity, and delayed re-epithelialization. Blockade of OGF-OGFr pathway using naltrexone, a potent opioid receptor antagonist, reverses dry eye symptoms and restores corneal surface sensitivity in diabetic rats when used as a therapy. Based on the evidence that both OGF and OGFr are elevated in type 1 diabetic rats, this study examined whether systemic or topical naltrexone treatment initiated at the time of induction of hyperglycemia could protect against the development of diabetic ocular surface complications. Diabetic male Sprague-Dawley rats treated systemically or topically with naltrexone had a delayed onset of dry eye and altered corneal surface sensitivity, and an improved healing rate for corneal wounds, that were comparable to non-diabetic rats. Serum levels of OGF were normal for rats receiving systemic naltrexone, and OGF tissue levels were normal for type 1 diabetic rats receiving twice daily naltrexone drops. OGFr levels remained elevated. These data support the role of the OGF-OGFr axis in regulation of ocular surface complications, and suggest that naltrexone therapy may be beneficial for pre-diabetic and early diabetic individuals.

Multiple sclerosis is a chronic progressive neurological disorder that has few distinctive biomarkers associated with disease progression or response to therapy. This research investigated whether non-invasive imaging correlated with animal behavior and morphological indicators of disease in response to serum levels of [Met 5 ]-enkephalin. Using the experimental autoimmune encephalomyelitis (EAE) model, adult female C57BL/6 J mice were randomized to receive daily injections of 0.1 mg/kg naltrexone (NTX) (= low dose naltrexone, LDN), 10 mg/kg Opioid Growth Factor (OGF) (chemically termed [Met 5 ]-enkephalin) or saline beginning at the time of disease induction. Daily composite behavior scores were recorded over a 30-day period based on tail tone, gait, righting reflex, and limb strength. Prior to disease onset (day 7), and at peak disease (day 18), mice were imaged and tissues (blood and spinal cord) collected at day 30 for serum analyses of OGF and morphology. Serum OGF levels of EAE mice treated with saline were significantly reduced from baseline and from normal mice. Longitudinal cohort data demonstrated an increase in fractional anisotropy in all cohorts by day 18. There was a significant decrease in radial diffusivity in the saline group seen at day 18 whereas the axial diffusivity was not altered amongst treatment groups. Treatment with OGF or LDN resulted in mean diffusivity rates that were comparable to baseline (normal) levels at days 7 and 18. Luxol fast blue staining of the lumbar spinal cords demonstrated a 16 % reduction in myelin staining in saline treated EAE animals when compared to OGF and LDN treated EAE mice. Immunohistochemistry with Olig2 (pan-oligodendrocyte marker) and myelin basic protein (MBP) revealed that OGF and LDN treatment restored the area (%) of MBP and number of oligodendrocytes to that of normal spinal cord (∼75 %). Saline treated EAE mice had more demyelination and fewer oligodendrocytes than normal mice. Collectively, these data suggest that a panel of biomarkers including imaging, serum biomarker levels, and behavior correlate with progression of disease, and may begin to validate use of specific non-invasive markers for MS., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Introduction: Bariatric surgery, as the only effective treatment of obesity, has strong effects on the metabolism, and nervous and endocrine systems. Thus, based on the different opinions about the efficaciousness of morbid obesity treatments, the aim of the present study was to estimate the association of serum ghrelin and Met-enkephalin (native, five amino acids and cryptic, precursor of enkephalin) concentrations with body mass index (BMI) value in bariatric patients within 30 postoperative days., Material and Methods: The study was performed on 38 female patients divided into two groups: I - BMI lower than 40 kg/m² (n = 18) and II - BMI higher than 40 kg/m² (n = 20). Blood was taken before (-24 h), and 72 h and 30 days after the sleeve gastrectomy. Routine haematological, anthropometric, and metabolic parameters as well as thyroid-stimulating hormone (TSH), ghrelin, and Met-enkephalin values were measured in all patients., Results: There were statistically significant differences between the two groups before the surgery in terms of TSH, both forms of Metenkephalin, triglycerides concentrations, and activity of alanine transaminase (ALT), gamma glutamyltransferase (GGTP), and C-reactive protein (CRP). After 72 h, the serum levels of cryptic Met-enkephalin and CRP, and activity of enzymes varied between the two groups of patients. Thirty days after the surgery, some metabolic and immune parameters were still different in both female groups in favour of patients with lover BMI. However, significant differences were noticed in the levels of ghrelin (increase), and native (decrease) and cryptic Met-enkephalins (increase)., Conclusions: The activity of endogenous peptides in bariatric patients is connected with the degree of obesity. Ghrelin level increases are negatively correlated with native Met-enkephalin changes shortly after bariatric surgery. The interplay of ghrelin and opioids might be considered as a predictor of postoperative weight loss success.

Remote ischaemic preconditioning (RIPC) has been employed as a non-invasive protective intervention against myocardial ischaemia-reperfusion injury in animal studies. However, the underlying mechanisms are incompletely defined in humans and its clinical efficacy has been inconclusive. As advanced age, disease, and drugs may confound RIPC mechanisms in patients, our aim is to measure whether RIPC evokes release of adenosine, bradykinin, met-enkephalin, nitric oxide, and apolipoproteins in healthy young adults. Healthy subjects (n = 18, 9 males, 23 ± 1.5 years old; 9 females, 23 ± 1.8 years old) participated after informed consent. RIPC was applied using a blood pressure cuff to the dominant arms for four cycles of 5-minute cuff inflation (ischaemia) and 5-minute cuff deflation (reperfusion). Blood was sampled at baseline and immediately after the final cuff deflation (Post-RIPC). Baseline and Post-RIPC plasma levels of adenosine, bradykinin, met-enkephalin, apolipoprotein A-1 (ApoA-1), apolipoprotein D (ApoD), and nitric oxide (as nitrite) were measured via ELISA and high-performance liquid chromatography. Mean (±SD) baseline levels of adenosine, bradykinin, met-enkephalin, ApoA-1, ApoD, and nitrite in healthy young adults were 13.8 ± 6.5 ng/mL, 2.6 ± 1.9 μg/mL, 594.1 ± 197.4 pg/mL, 3.0 ± 0.7 mg/mL, 22.2 ± 4.0 μg/mL, and 49.8 ± 13.4 nmol/L, respectively. Post-RIPC adenosine and nitrite levels increased (59.5 ± 37.9%, P < .0001; 32.2 ± 19.5%, P < .0001), whereas met-enkephalin and ApoD levels marginally decreased (5.3 ± 14.0%, P = .04; 10.8 ± 20.5%, P = .04). Post-RIPC levels were not influenced by sex, age, blood pressure, waist circumference, or BMI. RIPC produces increased levels of adenosine and nitrites, and decreased met-enkephalin and ApoD in the plasma of young healthy adults., (© 2019 John Wiley & Sons Australia, Ltd.)

Background: Inflammatory bowel diseases (IBD) are a group of chronic and recurrent gastrointestinal disorders that are difficult to control. Recently, a new IBD therapy based on the targeting of the endogenous opioid system has been proposed. Consequently, due to the fact that endogenous enkephalins have an anti-inflammatory effect, we aimed at investigating the degradation of serum enkephalin (Met- and Leu-enkephalin) in patients with IBD., Methods: Enkephalin degradation in serum of patients with IBD was characterized using mass spectrometry methods. Calculated half-life (T 1/2 ) of enkephalins were compared and correlated with the disease type and gender of the patients. Additionally, statistical analysis was used to examine the dynamics of changes in terms of inhibition of enkephalins degradation within research groups., Results: Our research indicates that the degree of enkephalins degradation depends on the gender of the patients. The difference is most evident for the rate of Met-enkephalin degradation between men (mean T 1/2  = 13.61 min) and women (mean T 1/2  = 21.84 min) with Crohn's disease (CD)., Conclusions: The most significant alternation of enkephalins degradation in serum samples of IBD patients, compared to control group, were observed in both Crohn's disease and ulcerative colitis (UC) female patients. We suggest that the differences observed between the genders in IBD patients may be explained by regulation of enkephalinases activity by estradiol., (Copyright © 2018 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.)

The aim of this work was to assess the influence of preanalytical variables on the stability of two endogenous opioid peptides (Methionine-Enkephalin and Leucine-Enkephalin) in human plasma. For this purpose, first a sensitive LC-MS/MS analytical method was developed and validated for the simultaneous quantitative analysis of these two peptides. The methodology consisted of a simple protein precipitation step followed by UPLC separation and MRM quantitative analysis using a stable isotope labelled Methionine-Enkephalin as internal standard. The method with a limit of quantitation of 10 pg/mL showed good reproducibility with excellent accuracy and precision, and was linear up to 2000 pg/mL. An extensive evaluation of the pre-analytical stability of these peptides in human blood was carried out to ensure an adequate sample collection procedure to obtain reliable results in the analysis of clinical samples., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The endogenous neuropeptide opioid growth factor, chemically termed [Met 5 ]-enkephalin, has growth inhibitory and immunomodulatory properties. Opioid growth factor is distributed widely throughout most tissues, is autocrine and paracrine produced, and interacts at the nuclear-associated receptor, OGFr. Serum levels of opioid growth factor are decreased in patients with multiple sclerosis and in animals with experimental autoimmune encephalomyelitis suggesting that the OGF-OGFr pathway becomes dysregulated in this disease. This study begins to assess other cytokines that are altered following opioid growth factor or low-dose naltrexone modulation of the OGF-OGFr axis in mice with experimental autoimmune encephalomyelitis using serum samples collected in mice treated for 10 or 20 days and assayed by a multiplex cytokine assay for inflammatory markers. Cytokines of interest were validated in mice at six days following immunization for experimental autoimmune encephalomyelitis. In addition, selected cytokines were validated with serum from MS patients treated with low-dose naltrexone alone or low-dose naltrexone in combination with glatiramer acetate (Copaxone®). Experimental autoimmune encephalomyelitis mice had elevated levels of 7 of 10 cytokines. Treatment with opioid growth factor or low-dose naltrexone resulted in elevated expression levels of the IL-6 cytokine, and significantly reduced IL-10 values, relative to saline-treated experimental autoimmune encephalomyelitis mice. TNF-γ values were increased in experimental autoimmune encephalomyelitis mice relative to normal, but were not altered by opioid growth factor or low-dose naltrexone. IFN-γ levels were reduced in opioid growth factor- or low-dose naltrexone-treated experimental autoimmune encephalomyelitis mice relative to saline-treated mice at 10 days, and elevated relative to normal values at 20 days. Validation studies revealed that within six days of immunization, opioid growth factor or low-dose naltrexone modulated IL-6 and IL-10 cytokine expression. Validation in human serum revealed markedly reduced IL-6 cytokine levels in MS patients taking low-dose naltrexone relative to standard care. In summary, modulation of the OGF-OGFr pathway regulates some inflammatory cytokines, and together with opioid growth factor serum levels, may begin to form a panel of valid biomarkers to monitor progression of multiple sclerosis and response to therapy. Impact statement Modulation of the opioid growth factor (OGF)-OGF receptor (OGFr) alters inflammatory cytokine expression in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Multiplex cytokine assays demonstrated that mice with chronic EAE and treated with either OGF or low-dose naltrexone (LDN) had decreased expression of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and the anti-inflammatory cytokine IL-10 within 10 days or treatment, as well as increased serum expression of the pro-inflammatory cytokine IL-6, relative to immunized mice receiving saline. Multiplex data were validated using ELISA kits and serum from MS patients treated with LDN and revealed decreased in IL-6 levels in patients taking LDN relative to standard care alone. These data, along with serum levels of OGF, begin to formulate a selective biomarker profile for MS that is easily measured and effective at monitoring disease progression and response to therapy.

Low-dose naltrexone is a widely used off-label therapeutic prescribed for a variety of immune-related disorders. The mechanism underlying low-dose naltrexone's efficacy for fatigue, Crohn's disease, fibromyalgia, and multiple sclerosis is, in part, intermittent blockade of opioid receptors followed by upregulation of endogenous opioids. Short, intermittent blockade by naltrexone specifically blocks the opioid growth factor receptor resulting in biofeedback events that increase production of the endogenous opioid growth factor (OGF) (chemically termed [Met 5 ]-enkephalin) facilitating interactions between opioid growth factor and opioid growth factor receptor that ultimately, result in inhibited cell proliferation. Preclinical studies have reported that enkephalin levels are deficient in animal models of experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. Our hypothesis is that serum enkephalin levels are diminished in humans with multiple sclerosis and experimental autoimmune encephalomyelitis mice, and that change in serum opioid growth factor levels may serve as a reasonable candidate biomarker for the onset of experimental autoimmune encephalomyelitis and response to therapy. To address this, we designed a two-part study to measure endogenous opioids in multiple sclerosis patients, and to investigate the temporal pattern of decline in serum enkephalin concentrations in mice with chronic progressive experimental autoimmune encephalomyelitis and treated with low-dose naltrexone. For comparison, we investigated whether low-dose naltrexone exposure in normal mice also resulted in altered enkephalin levels. In both animal models, we monitored tactile and heat sensitivity, as well as differential white blood cell counts as indicators of inflammation. Serum [Met 5 ]-enkephalin levels were lower in humans with multiple sclerosis relative to non-multiple sclerosis patients, and low-dose naltrexone restored their levels. In experimental autoimmune encephalomyelitis mice, [Met 5 ]-enkephalin levels were depressed prior to the appearance of clinical disease, and were restored with low-dose naltrexone treatment. Low-dose naltrexone therapy had no effect on serum [Met 5 ]-enkephalin or β-endorphin in normal mice. Thus, [Met 5 ]-enkephalin (i.e. opioid growth factor) may be a reasonable candidate biomarker for multiple sclerosis, and may signal new pathways for treatment of autoimmune disorders. Impact statement This report presents human and animal data identifying a novel biomarker for the onset and progression of multiple sclerosis (MS). Humans diagnosed with MS have reduced serum levels of OGF (i.e. [Met 5 ]-enkephalin) relative to non-MS neurologic patients, and low-dose naltrexone (LDN) therapy restored their enkephalin levels. Serum OGF levels were reduced in mice immunized with MOG 35-55 prior to any clinical behavioral sign of experimental autoimmune encephalomyelitis, and LDN therapy restored their serum OGF levels. β-endorphin concentrations were not altered by LDN in humans or mice. Thus, blood levels of OGF may serve as a new, selective biomarker for the progression of MS, as well as response to therapy.

Methionine enkephalin ([Met 5 ]-enkephalin, Opioid growth factor (OGF)) is a small neuropeptide with growth-related as well as immunomodulatory properties. OGF is distributed widely throughout the body, is both autocrine and paracrine produced, and has a very short half-life in serum. In addition to its neurotransmitter functions, OGF inhibits cell replication of a wide variety of cells involved in the autoimmune process. In this preclinical study, mice were immunized with myelin oligodendrocytic glycoprotein (MOG 35-55 ) to establish a chronic progressive form of autoimmune encephalomyelitis (EAE), and serum enkephalin levels were assessed throughout the disease as well as in response to OGF therapy in order to determine whether OGF may be a biological marker for EAE and multiple sclerosis. Immunized mice were randomly assigned to groups receiving daily 10mg/kg OGF (n=24) or saline (n=25) beginning at the time of established disease and clinical behavior. Open field activity, rearing, forced swimming, and novel object tests were monitored. Serum levels of peptide were measured prior to immunization, before clinical symptoms were observed, and at the onset and peak period of disease. Spinal cord neuropathology was evaluated 40days after immunization. EAE disease onset occurred on day 9 post immunization when the mean clinical score was 1.5. Peak disease scores for saline-injected EAE mice reached a mean of 5.7 on day 18, whereas mice receiving OGF had a peak clinical score of 2.5. Behavioral tests conducted 5days post-immunization (and before clinical signs of EAE) revealed that EAE mice had reduced serum enkephalin levels related to elevated clinical disease scores. Serum levels of enkephalin collected at peak disease and after 40days correlated with clinical scores. Disease status was associated with activity in the open field, rearing, time associating with a novel object, and pain sensitivity. Clinical signs of EAE correlated with levels of enkephalins such that as behavioral scores increased, serum [Met 5 ]-enkephalin levels decreased. Thus, [Met 5 ]-enkephalin is a novel biomarker that is associated with disease onset and progression, as well as response to therapy in a mouse model of EAE, and may provide new insight into MS., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Due to the important interactions of proenkephalin fragments (e.g., proenkephalin [107-140] Peptide F) to enhance activation of immune cells and potentially combat pain associated with exercise-induced muscle tissue damage, we examined the differential plasma responses of Peptide F to different exercise training programs. Participants were tested pre-training (T1), and after 8 weeks (T2) of training. Fifty-nine healthy women were matched and then randomly assigned to one of four groups: heavy resistance strength training (STR, n=18), high intensity endurance training (END, n=14), combined strength and endurance training (CMB, n=17), or control (CON, n=10). Blood was collected using a cannula inserted into a superficial vein in the antecubital fossa with samples collected at rest and immediately after an acute bout of 6 X 10 RM in a squat resistance exercise before training and after training. Prior to any training, no significant differences were observed for any of the groups before or after acute exercise. With training, significant (P≤0.95) elevations were observed with acute exercise in each of the exercise training groups and this effect was significantly greater in the CMB group. These data indicate that in untrained women exercise training will not change resting of plasma Peptide F concentrations unless both forms of exercise are performed but will result in significant increases in the immediate post-exercise responses. Such findings appear to indicate adrenal medullary adaptations opioid production significantly altered with exercise training., (Copyright © 2017. Published by Elsevier Inc.)

To determine if exercise training alters the pattern and magnitude of plasma concentrations of proenkephalin Peptide F and epinephrine, plasma proenkephalin [107-140] Peptide F(ir) and catecholamines were examined pre-training (T-1), and after 4- (T-2), 8- (T-3), and 12-weeks (T-4) of training. 26 healthy men were matched and randomly assigned to one of three groups: heavy resistance strength training (Strength, n=9), high intensity endurance training (Endurance, n=8), or both training modalities combined (Combined, n=9). Blood was collected using a syringe with a cannula inserted into a superficial arm vein with samples collected at rest, after each 7 min stage and 5 and 15 min into recovery. With training, all groups observed shifted plasma Peptide F responses to graded exercise, where significant increases were observed at lower exercise intensities. Increases in plasma epinephrine with exercise were observed in all groups. The Combined group saw increases at 25% at T-3 and for 50% at T-2, T-3, and T-4 which was higher than T-1. The Endurance group demonstrated increases for 50% at T-1, T-2, T-3 but not at T-4. The plasma epinephrine response to graded exercise was reduced in the Strength group. Increases in plasma norepinephrine above rest were observed starting at 50% . The Strength group demonstrated a significant reduction in norepinephrine observed at 100% at T-3 and T-4. Peptide F and catecholamines responses to graded exercise can be altered by different types of physical exercise training. Simultaneous high intensity training may produce adrenal medulla exhaustion when compared to single mode training., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Eighteen male cluster headache (CH) inpatients within a CH series participated in this research. Blood samples were drawn from patients at least 6-hour pain-free after the last acute CH episode and then shortly prior (SP), during, and soon after (SA) a new acute CH attack. Three healthy male, age-comparable drug-free volunteers served as controls; 5 samples were obtained from each of these individual over a 24-hour period. Individual patient's methionine-enkephalin (MET) plasma concentration showed significant changes, and in some subjects, dramatic changes, during the different phases of a single CH episode. Peptide levels followed a general pattern of higher plasma concentration SP to an acute CH attack, followed by decreased levels during the attack itself, and falling even further SA the acute episode. Consistently, 16 of the 18 patients tested showed pre-CH peptide levels significantly higher (arbitrarily the authors considered values 20% or more as "significant") than their own values obtained during the acute CH pain phase, with observed differences reaching 80% or more in 7 of these individuals. For about half of these patients, peptide concentration during the acute CH episode was significantly above the control's range (68.2-87.6 pg MET/mL; control's circulating MET concentration remaining essentially unchanged during a 24-hour period). MET levels were further decreased in essentially all of the post-CH samples, with values falling within (n = 6) or even further below than those in the control's range (n = 11). Neither age, time of CH occurrence, nor patient's use of a number of medications known for failing to influence plasma MET degradation kinetics seemed to significantly influence MET levels. These results might help in the biochemical characterization of the actual phases of a CH episode. Developing drugs modulating MET bioavailability could lead to novel antinociceptive agents useful for the treatment of CH's associated pain.

Purpose: To evaluate the impact of slow-tempo music listening periods in mechanically ventilated intensive care unit patients., Methods: A randomized crossover study was performed in a 16-bed, adult critical care unit at a tertiary care hospital. Still-sedated patients, mandating at least 3 more days of mechanical ventilation, were included. The intervention consisted in two 1-hour daily periods of music-vs-sham-MP3 listening which were performed on Day 1 or 3 post-inclusion, with a Day 2 wash-out. "Before-after" collection of vital signs, recording of daily sedative drug consumption and measurement of stress and inflammatory blood markers were performed., Results: Of 55 randomized patients, 49 were included in the final analyses. Along with music listening, (i) vital signs did not consistently change, whereas narcotic consumption tended to decrease to a similar sedation (P = .06 vs sham-MP3); (ii) cortisol and prolactin blood concentrations decreased, whereas Adreno Cortico Trophic Hormone (ACTH)/cortisol ratio increased (P = .02; P = .038; and P = .015 vs sham-MP3, respectively), (iii) cortisol responders exhibited reversed associated changes in blood mehionine (MET)-enkephalin content (P = .01)., Conclusions: In the present trial, music listening is a more sensitive stress-reliever in terms of biological vs clinical response. The hypothalamus-pituitary adrenal axis stress axis is a quick sensor of music listening in responding mechanically ventilated intensive care unit patients, through a rapid reduction in blood cortisol., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Proenkephalin Peptide F [107-140] is an enkephalin-containing peptide found predominantly within the adrenal medulla, co-packaged with epinephrine within the chromaffin granules. In vivo studies indicate that Peptide F has classic opioid analgesia effects; in vitro studies suggest potential immune cell interactions. In this investigation we examined patterns of Peptide F concentrations in different bio-compartments of the blood at rest and following sub-maximal cycle exercise to determine if Peptide F interacts with the white blood cell (WBC) bio-compartment during aerobic exercise. Eight physically active men (n=8) performed sub-maximal (80-85% V˙O2peak) cycle ergometer exercise for 30 min. Plasma Peptide F and WBC Peptide F immunoreactivity were examined pre-exercise, mid-exercise and immediately post-, 5-min post-, 15-min post-, 30-min post- and 60-min post-exercise and at similar time-points during a control condition (30 min rest). Peptide F concentrations significantly (p<0.05) increased at 5 and 60 min post-exercise, compared to pre-exercise concentrations. No significant increases in Peptide F concentrations in the WBC fraction were observed during or after exercise. However, a significant decrease was observed at 30 min post-exercise. An ultradian pattern of Peptide F distribution was apparent during rest. Furthermore, concentrations of T cells, B cells, NK cells, and total WBCs demonstrated significant changes in response to aerobic exercise. Data indicated that Peptide F was bound in significant molar concentrations in the WBC fraction and that this biocompartment may be one of the tissue targets for binding interactions. These data indicate that Peptide F is involved with immune cell modulation in the white blood circulatory biocompartment of blood., (Copyright © 2013. Published by Elsevier Inc.)

Results from a longitudinal study (blood drawn at days 29, 64, 89,124, 142, and 182 of the protocol) shows that the concentration of platelet-poor plasma (PPP) methionine(5)-enkephalin (MET) in healthy, drug-free, white male individuals (n = 5) remains within a relatively narrow range, well within the experimental error of the analytical procedures used. Interindividual differences fail to reach statistical significance [x ± SD and range (MET picograms per mL of PPP) of 91.2 ± 15.1, 67.1-113.5; 69.6 ± 7.5, 66.1-90.1; 76.6 ± 12.6, 58.5-93.1; 86.8 ± 10.9, 76.3-107.4; and 84.5 ± 11.4, 68.9-103.4; for subjects 1-5, respectively]. MET levels were similar to those recorded from single samples obtained from a group of 24 white male, age-comparable, drug-free healthy volunteers [x ± SD and range (picograms of MET per mL of PPP) of 83.3 ± 15.1 and 57.4-119.1]. The controls' range for all the subjects (n = 29) was 57.4-119.1 pgMET/mL PPP. Compared with the controls, individual patients with cluster headache (CH) show a much wider variation in PPP MET levels (blood drawn at different time intervals, at least 10 samples per patient, over a period of 221-298 days), with many (slightly over half) of single values below the controls range; no single MET level was above the controls range [x ± SD and range (picograms of MET per mL of PPP) of 56.4 ± 27.7, 6.1-100.5; 72.6 ± 20.5, 43.0-113.0; 46.0 ± 28.5, 10.0-92.6; 53.6 ± 27.5, 13.0-101.0; 52.0 ± 26.1, 17.5-83.6; 63.5 ± 22.3, 21.7-91.3 for individuals A-F, respectively]. Although interindividual differences within the patients' group were not statistically significant, their peptide levels were significantly lower than those of controls. Neither the presence of unspecified "headaches between clinic visits" and "daily headaches" (patients E and F, respectively), nor the use of a number of drugs known to lack inhibitory activity upon the aminopeptidase-MET degradation reaction, seemed to significantly influence MET concentration. The results could lead to a better understanding of the etiology of the pain associated with CH, with the relative changes in plasma peptide perhaps reflecting the patients' vulnerability to such a condition. Pharmacological modulation of MET function may prove useful in the treatment of CH-associated pain, whether the development of such drugs could find useful pharmacological applications remains to be explored.

Background: Postoperative pain management is a critical aspect of patient care. The inflammatory state of the post-sternotomy surgical wound sensitizes nerve endings, causing pain. Unrelieved or improperly managed pain compromises wound healing. Peripheral opioid receptors play a major role in analgesia, particularly under inflammatory conditions where both opioid receptor expression and efficacy are increased. Leukocytic opioid peptides include β-endorphin (END), met-enkephalin (ENK), and dynorphin-A (DYN), with END and ENK being predominant., Methodology/principal Findings: This work represents the first study of inflammatory cells collected from post-sternotomy wounds of patients undergoing cardiac surgery including coronary artery bypass grafting (CABG). Wound fluid (WF) and cells were collected from sternal wounds using a JP Blake drain at 24, 48, and 72 hours post sternum closure. Anti-CD15 staining and flow cytometry revealed that polymorphonuclear neutrophils (PMN) are the predominant cells present in wound fluid collected post-surgery. Compared to peripheral blood (PB) derived PMN, significant increases in CD177+/CD66b+ PMN were observed suggesting activation of wound-site PMN. Such activation was associated with higher levels of opioid peptide expression in PMN derived from WF. Indeed, increased level of opioid peptides in sternal wound environment was noted 72 h post-surgery. We demonstrate that WF contains factors that can significantly induce POMC transcription in human PMNs. IL-10 and IL-4 were abundant in WF and both cytokines significantly induced POMC gene expression suggesting that WF factors such as IL-10 and IL-4 contribute towards increased opioid peptide expression in wound-site PMN., Conclusions/significance: This approach provided a unique opportunity to study the cross-talk between inflammation and opioid peptides in PMN at a sternotomy wound-site. Wound-site PMN exhibited induction of END and ENK. In addition, sternal wound fluid significantly induced END expression in PMN. Taken together, these data constitute first clinical evidence that human wound-site PMNs are direct contributors of opioids at the sternal wound-site.

Purpose: Opioid peptides provide a link between the neuroendocrine and immune systems. They modify the inflammatory process through their effect on the synthesis and secretion of cytokines and on the proliferation of leukocytes to the inflammatory lesion. The evaluation analyzed changes in free met-enkephalin concentration values in the serum and colon mucosal biopsy specimens of patients with inflammatory bowel disease (IBD)., Material and Methods: In serum and colon mucosal biopsy specimens, free met-enkephalin levels were determined in 43 patients with ulcerative colitis (UC) and 38 individuals with Crohn's disease (CD). The evaluation analyzed the effect of disease activity, inflammatory lesions of the colon and laboratory parameters, on the level of the investigated marker. The control group consisted of 45 healthy volunteers., Results: Serum free met-enkephalin levels were depressed in patients with CD (85.4pg/ml) and UC (101.5pg/ml) as compared to the controls (119.4pg/ml). Met-enkephalin levels in colonic biopsies collected from inflammatory lesions in IBD patients were significantly higher as compared to sections without inflammatory lesions (6.59pg/mg vs. 2.89pg/mg, p < 0.01 in the CD group and 6.12pg/mg vs. 3.47pg/mg, p < 0.05 in the UC group) and their level correlated with disease activity., Conclusions: The present investigation is the first study that demonstrates changes in free met-enkephalin levels in IBD that may play a role in the pathogenesis and course of the disease. Further studies are necessary to assess the anti-inflammatory effect of opioid peptides.

Objective: Met- and leu-enkephalins are endogenous opioid neuropeptides with potent analgesic, vasoactive, immunomodulatory and anti-apoptotic properties. We hypothesised that clinical or immunological variables of early systemic sclerosis (SSc) might be correlated to plasma enkephalin levels., Methods: Plasma samples were collected at study entry of the Genetics versus Environment in Scleroderma Outcomes Study (GENISOS) cohort (early SSc, n=116). Plasma met-enkephalin and leu-enkephalin levels (microg/ml) were measured by high performance liquid chromatography (HPLC) and correlated to clinical and laboratory parameters in the GENISOS database. Statistical analyses were performed by nonparametric Wilcoxon rank sum tests and Pearson correlation coefficients., Results: Significantly lower plasma met-enkephalin levels were associated with anti-topoisomerase-I seropositivity (6+8.3 vs. 14.9+22.8 microg/ml, p=0.02). Plasma leu-enkephalin levels were significantly higher in SSc patients with digital pulp loss (95.6+130 vs. 64.9+101 microg/ml, p=0.02). Lower mean plasma met-enkephalin levels and inversely higher leu-enkephalin levels were noted in SSc patients with Raynaud's phenomena (p=NS)., Conclusion: The associations of plasma enkephalin levels to immunologic or clinical pathologies may underscore their vasogenic or fibrogenic significance and potential as therapeutic targets in early SSc.

Delayed corneal reepithelialization is a complication of diabetes, and may lead to ulcers and erosions, which cause ocular morbidity and visual loss. This study examined the efficacy of naltrexone (NTX), a long-acting, potent opioid antagonist, applied topically, to facilitate the repair of standardized corneal abrasions in diabetic (alloxan-induced) New Zealand White rabbits (glucose levels>450 mg/dL). NTX at a concentration of 10(-4)M, or sterile vehicle (SV), was administered topically 4 times per day for 7 days to the abraded eye of uncontrolled Type 1 diabetic (DB), insulin-controlled Type 1 diabetic (DB-IN), or non-diabetic (Normal) rabbits. Wound healing was monitored, and non-invasive (tonopen, pachymeter, hand-held slit lamp, and retinal camera) and invasive (histopathology) measurements evaluated. Corneal reepithelialization in the uncontrolled DB rabbits was significantly enhanced (up to a 47% reduction in wound area) following treatment with NTX relative to both Normal SV and DB SV rabbits at 24, 48, and 56 h following surgery. At 72 h, DB NTX rabbits had residual defects that were 64-82% smaller than Normal and DB SV animals. NTX treated DB-IN rabbits had residual defects that were 9-37% smaller than DB-IN rabbits receiving SV, and 6-40% smaller than Normal rabbits. No signs of toxicity from topical applications were noted. These data confirm and extend those documented in rats that demonstrated a lack of toxicity of NTX at a wide range of dosages, as well as efficacy for enhanced corneal epithelialization. These studies set the stage for clinical trials using NTX as a therapy for diabetic keratopathy., (Copyright 2009 Elsevier Inc. All rights reserved.)

To address the role of the opioid system in the pathogenesis of hepatic encephalopathy (HE) we measured plasma met-enkephalin, beta-endorphin and leu-enkephalin in patients with different grades of HE compared to control subjects and patients with cirrhosis. Plasma met-enkephalin levels were significantly higher in patients with cirrhosis and all grades of HE than controls. Plasma beta-endorphin levels were similar in the 3 groups. Plasma leu-enkephalin levels were significantly higher in HE grades II, III and IV than in controls, patients with cirrhosis and HE grade I patients. Our results support data on the involvement of met-enkephalin and leu-enkephalin in the pathogenesis of HE and provide a rationale for the use of opioid receptor antagonists in the treatment of HE.

Background: Remote preconditioning is known to be cardioprotective, but the exact mechanism has not been fully elucidated. The objective of the current study was to investigate the role of kappa-opioid receptors in cardioprotection by remote preconditioning and reveal possible underlying mechanisms., Methods: Remote preconditioning was induced in anesthetized male Sprague-Dawley rats by three cycles of 5 min of right femoral artery occlusion followed by 5 min of reperfusion. Myocardial ischemia-reperfusion was achieved by ligation of the left anterior descending coronary artery for 30 min and then reperfusion for 120 min. Infarct size was determined by 2,3,5-triphenyltetrazolium chloride staining. Levels of lactate dehydrogenase, dynorphin, and met-enkephalin in plasma were measured. The opening of the mitochondrial permeability transition pore was monitored with fluorescent calcein in isolated ventricular myocytes., Results: Both remote preconditioning and U-50,488H (10 mg/kg intravenous), a kappa-opioid receptor agonist, significantly decreased the infarct size and plasma lactate dehydrogenase level induced by ischemia-reperfusion, and these effects were attenuated by nor-binaltorphimine (10 mg/kg intravenous), a kappa-opioid receptor antagonist, and atractyloside (5 mg/kg intravenous), a mitochondrial permeability transition pore activator. However, administration of naltrindole (5 mg/kg), a delta-opioid receptor antagonist, had no effect on the cardioprotection by remote preconditioning. The dynorphin plasma level was increased after remote preconditioning treatment, but the met-enkephalin level did not change. In isolated ventricular myocytes loaded with calcein, U-50,488H (300 microM) decreased the mitochondrial permeability transition pore opening induced by calcium (200 microM), and this effect was attenuated by cotreatment with nor-binaltorphimine (5 microM) or atractyloside (20 microM)., Conclusion: Activation of cardiac kappa-opioid receptors is involved in the cardioprotection induced by remote preconditioning, and the mitochondrial permeability transition pore may participate in the postreceptor pathway.

Opioid growth factor (OGF) is an endogenous opioid peptide ([Met(5)]-enkephalin) that interacts with the OGF receptor (OGFr). OGF serves as a constitutively expressed and tonically active negative growth factor in neoplasia, and the OGF-OGFr axis contributes to the maintenance of an equilibrium in cell replication by targeting cyclin-dependent inhibitory kinase pathways. In a previous study, OGFr binding activity was found to decrease in concert with progression of human squamous cell carcinoma of the head and neck (SCCHN). To investigate the relationship of OGFr to advancement of human pancreatic and colon cancers, tumor cells were transplanted into nude mice, and small, medium, and large neoplasias were assessed for OGFr number and affinity by receptor binding analysis, and for gene expression of OGFr mRNA by Northern blot analysis. In addition, OGF levels were monitored in plasma. OGFr binding affinity and capacity, as well as transcriptional activity of OGFr, were not influenced by the size or state of differentiation of pancreatic or colon tumors. Plasma levels of OGF were 3.5- to 7.9-fold less in animals with pancreatic or colon cancers than in nude mice not receiving xenografts, and no differences in OGF values were recorded between small and large tumors. These data on human pancreatic and colon cancers, along with information in earlier studies on SCCHN, indicate that alterations in the OGF receptor are dependent on tumor type and that the integrity of the OGF-OGFr axis insofar as tumorigenesis needs to be evaluated for each type of neoplasm. This information will be relevant in the design of therapeutic modalities, the diagnosis and prognosis of neoplasia, as well as understanding of the processes and mechanisms of carcinogenesis.

This study was the first study to examine the three circulatory biocompartments (plasma, white blood cell layer (WBC) and red blood cell layer (RBC)) and determine PF concentrations before and after exercise. Proenkephalin peptide F (PF) is an enkephalin-containing peptide found predominantly within the adrenal medulla. PF is co-packaged with epinephrine, and both can be co-secreted in response to similar stimuli. PF and epinephrine have shown immunomodulating properties. Ten healthy resistance trained men performed six sets of 10 RM squats with 2 min rest periods between sets and 10 healthy active men were matched and served as resting controls. Blood samples were obtained pre-exercise, immediately post-exercise and 15 min post-exercise and were analyzed for lactate, cortisol, epinephrine and biocompartmentalized PF. There was no change in resting control values measured across time within respective PF biocompartments and endocrine profile, indicating stability and technique validity of peptide F across the time period measured. As expected, the acute resistance exercise protocol caused an increase in lactate at 15 min post-exercise. Circulating epinephrine increased immediately post-exercise and returned to baseline during 15 min into recovery. Plasma PF increased immediate post-exercise and 15 min post-exercise, while WBC-PF and RBC-PF only increased at 15 min into recovery. For all time points tested, resting and exercise WBC-PF and RBC-PF concentrations were lower than plasma PF thus indicating a concentration difference across the three different biocompartments within the same blood sample. The presence of PF within all three biocompartments of whole blood may indicate the potential for biological transport and interactions with other cells in other biocompartments of the blood.

Nonaqueous capillary electrophoresis with electrochemical detection (NACE-ED) was applied to the analysis of enkephalin peptides. The effect of different buffer compositions on the electrophoretic behavior of methionine enkephalin, leucine enkephalin, and [D-Ala2]-leucine enkephalin was studied. Separation of the protonated and the deprotonated peptides was obtained using ACN/methanol-based electrolyte systems. The electrochemical behavior of the enkephalins was studied by the capillary batch injection analysis technique. NACE-ED yielded well-defined signals in the oxidation mode only for the negatively charged analytes. The optimized BGE for the counterelectroosmotic separation consisted of 10 mM ammonium acetate in ACN/methanol (3:1 v/v). Using a platinum microdisk electrode set to an actual potential of +0.65 V detection limits in the submicromolar range were observed which are about one order of magnitude lower compared to UV detection. Problems concerning EOF instability and electrode fouling caused by water and other neutral sample impurities transported by the EOF can be avoided in the EOF-inverted mode using poly(ethylene glycol)-coated capillaries and an actual working electrode potential of +1.0 V. For the quantification of the enkephalins [D-Ala2]leucine enkephalin was used as internal standard. The practical utility for the determination of enkephalins in spiked plasma samples after SPE was demonstrated.

Proenkephalin peptide F [107-140] is an enkephalin-containing peptide found predominantly within the adrenal medulla and is co-packaged with epinephrine within adrenal medullary chromaffin granules. Peptide F has been shown to have the classic opioid analgesia effects along with immune cell interactions. This is only the second peptide F study in women, and in it we compare the responses of peptide F to a maximal cycle exercise test and recovery values over the follicular and luteal phases of the menstrual cycle. Eight untrained (directly documented in this study) women who were eumenorrheic performed a progressive maximal exercise test to volitional exhaustion on a cycle ergometer, once during the follicular phase, and once during the luteal phases of the menstrual cycle. Blood was obtained pre-exercise, immediately post-exercise and at 0, 15, and 30 min into recovery. Typical exercise changes in response to the cycle tests were observed with blood lactate increases that remained elevated 30 min into recovery. No significant exercise-induced elevations were observed for peptide F concentrations with exercise nor were any differences observed between the two menstrual phases. Thus, the effects of the menstrual cycle on peptide F concentrations appear to be minimal under the conditions of this investigation. With high concentrations of peptide F observed at rest (approx. 0.2-0.3 pmol ml(-1)) pre-exercise arousal mechanisms may have obviated any exercise-induced response. In addition, inhibition via elevated epinephrine may have inhibited any post-exercise increases and finally adrenal medullary capacity for circulatory concentrations of peptide F may have been reached in such untrained women. Pre-exercise arousal mechanisms potentially related to analgesia may also be involved to prepare untrained women for the stress of maximal exercise.

Purpose: Patients in the ICU after long-term administration of an opioid/hypnotic often develop delirium. To assess the nature of this phenomenon, patients in a surgical ICU following ventilatory support and sedation with an opioid/hypnotic/sedative were studied., Methodology: Following sufentanil/midazolam (group 1; n =14) or sufentanil/propofol (group 2; n =15) sedation, patients were evaluated for changes in mean arterial blood pressure and heart rate, the activity of the central nervous system (sensory evoked potentials, spectral edge frequency of EEG), and the endogenous opioids plasma concentrations (beta-endorphin, met-enkephalin). Data obtained were correlated with the individual intensities of withdrawal symptoms 6-, 12-, and 24 h following sedation., Results: Following a mean duration of ventilation of 7.7 days (+/-3.6 SD) in groups 1 and 3.5 (+/-1.7 SD) in group 2, withdrawal intensities peaked within the 6th hour after cessation. Plasma beta-endorphin and met-enkephalin levels were low during sedation, and only the sufentanil/midazolam group demonstrated a postinhibitory overshoot. Withdrawal symptom intensities demonstrated an inverse correlation with beta-endorphin and met-enkephalin levels, a direct linear correlation with amplitude height of the evoked potential, and blood pressure and heart rate changes. Withdrawal intensities did not correlate with EEG power spectral edge frequency., Conclusion: The endorphinergic system is suppressed when a potent exogenous opioid like sufentanil is given over a long period of time. Following sedation, abstinence symptoms seem to be related to postinhibitory increased endorphin synthesis. This is mostly seen in the combination of sufentanil/midazolam. In addition, an increase in the amplitude of the sensory-evoked potential suggests a postinhibitory excitatory state within the nociceptive system.

The aim of the study was to investigate the effect of repeated maternal separation (MS; 4 hr per day) during postnatal Days 1 to 15 on emotionality and voluntary ethanol intake in adult male and female Wistar rat offspring relative to controls exposed to a brief (5-min) daily handling procedure. Brain immunoreactive opioid peptide levels and plasma levels of corticosterone also were measured. There were mainly no alterations in any of the tested behaviors (i.e., fleeing and freezing responses, exploratory behavior, spontaneous and amphetamine-induced locomotor activity and competitive behavior), ethanol intake, or immunoreactive opioid peptide levels in MS offspring, either in males or females, compared to their respective controls nor were there any differences in plasma corticosterone between groups. In addition, the dams' retrieval behavior of the pups also was studied, showing that MS dams spent more time in the nest with the pups after the 4-hr separation period compared to control dams. With respect to the used protocol of the MS procedure in the present study, our results do not provide support for the suggestion that this procedure is a relevant model for studying development of psychopathology and vulnerability to drug abuse., ((c) 2004 Wiley Periodicals, Inc.)

Background: Opiate peptides are involved in the physical dependence on ethanol. Levels of methionine enkephalin (MEnk), for example, are affected by ethanol. No study on the effect of ethanol on endomorphin, the endogenous ligand for the mu-opiate receptor, has yet been conducted., Methods: We examined the effect of ethanol ingestion on serum endomorphin (EM)-1 and MEnk levels. We also determined the effect of antisense directed at MEnk on serum levels of EM-1 and MEnk., Results: Serum EM-1 levels steadily decreased about 20% during 56 days of ethanol ingestion in liquid feed, whereas a similar decrease in serum MEnk levels was not statistically significant. Serum MEnk levels decreased about 20% by 48 hr after antisense injection and then returned to baseline, whereas serum EM-1 levels increased by about 80% and remained elevated for about 2 weeks. In mice not treated with antisense or alcohol, there was no correlation between the serum levels of EM-1 and MEnk., Conclusions: These results show that serum levels of EM-1 are decreased by physical dependence on ethanol and that this effect is not directly mediated through MEnk.

Introduction: . Proenkephalin peptide F [107-140] is related to the enhancement of immune function, while microgravity has been shown to cause immuno-suppression. We investigated the physiological response of proenkephalin peptide F to microgravity., Methods: There were 12 Fischer 344 female rats, ovariectomized at 10.5 wk of age, used to determine plasma concentrations of peptide F in response to a 14-d flight aboard the Columbia Space Shuttle mission STS-62. There were 36 other such rats that served as ground-based controls to separate the effects of microgravity from those of thermal stress, flight stress, and crowded habitats. Control groups of 12 rats each were kept under the following conditions: 1) 22 degrees C vivarium, 2) 28 degrees C vivarium, and 3) variable (Var) to mimic flight. The flight and control groups were housed in animal enclosure modules 21 d prior to flight and for the duration of the study. The rats were sacrificed within 4-5 h after landing, at which time blood samples were obtained., Results: Body weights were obtained prior to sacrifice; mean values were flight, 199 g; 22 degrees C, 193 g; 28 degrees C, 192 g; and Var, 194 g. The flight group produced a significantly greater (p < or = 0.05) level of plasma peptide F (0.056 pmol x ml(-1)) compared with the controls (0.016, 0.022, and 0.016 pmol x ml(-1) for 22 degrees C, 28 degrees C, and Var, respectively). Flight animals demonstrated higher corticosterone concentrations and reduced T and B cell splenocyte counts than controls., Conclusions: These data indicate that the increases in proenkephalin peptide F observed with exposure to microgravity may present an adrenal-medullary response to cope with the decreased immune function and increased stress experienced during spaceflight and landing.

Opioid growth factor (OGF), [Met5]-enkephalin, interacts with the OGF receptor (OGFr) to inhibit the growth of human squamous cell carcinoma of the head and neck (SCCHN) in vitro. Administration of OGF by daily intraperitoneal injection (i.p.) to animals with xenografts of CAL-27, a poorly differentiated SCCHN, is known to repress tumorigenic events. In this study, the ubiquity of OGF action on SCCHN was investigated by examination of OGF activity on SCC-1 tumors; this human cell line is well-differentiated and highly invasive. Mice receiving daily i.p. injections (10 mg/kg) of OGF had more than a 3-day delay in tumor appearance, and decreases in tumor volume ranging from 51 to 64% in comparison to controls throughout the experimental period. Receptor binding analysis for OGFr showed that binding capacity (Bmax) was 2.2-fold greater than control values, but binding affinity (Kd) was comparable. Plasma OGF levels did not vary between OGF and control groups. Mice receiving OGF by continuous infusion using minipumps, or by daily intratumoral injection, had characteristics of tumorigenicity similar to their corresponding control animals, although the OGF levels in mice receiving the OGF by minipump were elevated 18-fold greater than the control group. These data indicate that: i) the inhibitory action of OGF may be ubiquitous for SCCHN, ii) OGF treatment alters the characteristics of the OGF receptor but not of plasma OGF levels, and iii) the magnitude of effects of OGF on SCCHN is dependent on the route of administration.

Most evidence agrees that levels of methionine enkephalin (Met-Enk) in brain are inversely correlated with ethanol drinking and withdrawal seizures. One area of discrepancy is the effect of chronic ethanol administration on the level of immunoactive Met-Enk in brain, with some authors reporting increased and others reporting decreased levels. These reports differed greatly in terms of method of ethanol administration, species used, length of time ethanol was administered, and the region of brain examined. We found that all studies could be resolved by considering only length of time ethanol was administered, with Met-Enk levels first increasing and then decreasing. We tested this finding by determining the effect of 4-56 days of ethanol delivered in liquid feed on levels of brain Met-Enk. We found that brain levels of Met-Enk peaked after 7 days of ethanol ingestion and declined to levels lower than control by 28 days. Exposure to ethanol abolished a correlation between brain and serum levels of Met-Enk which occurred in controls. HPLC showed that whereas 100% of immunoactivity eluted in the position of Met-Enk in controls, only about 50% eluted as Met-Enk in mice exposed to ethanol. These results support the hypothesis that exposure to ethanol alters brain Met-Enk in a way consistent with the reinforcement of physical dependence.

The local increase in Met-enkephalin level during zymosan-induced peritonitis may be related to its local production by exudatory leukocytes (as evidenced by our previous experiments) and influx from distal centers, as its level is significantly decreased in blood, inguinal lymph nodes, and some brain areas (striatum, hypothalamus, and pituitary gland).

A number of drugs with the phenothiazine molecule in their chemical structure inhibit in a dose-dependent manner human plasmatic aminopeptidase leucine(5)-enkephalin (LEU) metabolism. Half-life peptide degradation was significantly increased by thioridazine > fluphenazine > As-1397 [10-(alpha-diethylaminopropionyl)phenothiazine] >/= promethazine >/= chlorpromazine (final drug conc. 10(-4) M); t1/2 (+/- SD) 21.2 +/- 1.1, 19.6 +/- 1.0, 17.2 +/- 0.9, 17.1 +/- 1.0, and 17.1 +/- 1.1 min, respectively. Control and bacitracin (known aminopeptidase inhibitor) values were 11.8 +/- 1.0 and 31.3 +/- 1.7 min, respectively. These drugs significantly decreased (listed in the same order) LEU degradation initial velocity; Iv (+/- SD) 0.77 +/- 0.2, 0.82 +/- 0.2, 0.92 +/- 0.3, 0.93 +/- 0.2, 0.94 +/- 0.3 pg LEU/min, respectively. Control and bacitracin 1.10 +/- 0.3 and 0.20 +/- 0.1 pg LEU/min, respectively. Values represent results from 5 samples, each obtained by pooling 6 individual plasmas (4 male and 2 female; n = 30 healthy, drug-free volunteers). However, neither the phenothiazines ethopropazine, methotrimeprazine, prochlorperazine and trifluoperazine nor the various commonly used heterocyclic antipsychotics tested, e.g., molindone, loxapine, clozapine, haloperidol, sulpiride and thiothixene inhibited plasma LEU degradation kinetics. Our results failed to show correlations between chemical structure, antipsychotic properties and ability to inhibit plasmatic aminopeptidase LEU degradation. Whereas, presence of the phenothiazine molecule appears to be necessary for enzyme inhibition, only five out of nine substituted phenothiazines tested exhibited this effect. Furthermore, there was a lack of correlation between phenothiazines antipsychotic properties and their capacity to inhibit aminopeptidase activity, a property shown by promethazine (antihistaminic) and As-1397 (selective butyrylcholinesterase inhibitor) but lacking in prochlorperazine and trifluoperazine. Our results provide information which could lead to the rational design of agents capable to modulate the bioavailability of enkephalin and other endogenous aminopeptidase-degraded peptides believed to be involved in the etiology and/or pathophysiology associated with various disease conditions. Whether their development could find useful pharmacological applications remains to be explored., (Copyright 2003 S. Karger AG, Basel)

Unlabelled: Met-enkephalin is a pentapeptide belonging to the opioid system and like other opioid peptides is involved in phenomena associated with modulated pain perception, regulation of memory and emotional conditions, food and liquid consumption, regulation of immunological system and it has an impact upon digestive system motility, gastric and pancreatic secretion, metabolism of carbohydrates. Met-enkephalin concentration changes in liver tissue and plasma during liver disorders. It has been revealed that Met-enkephalin takes part in the etiopathogenesis of ascites and pruritus. Primary biliary cirrhosis (PBC) is a chronic inflammatory liver disease, its course involves the destruction of small and medium-sized bile ducts as a result of the process of granulation tissue growth developing in such diseases as fibrosis, liver cirrhosis development, and liver insufficiency. During the ending period of the PBC, clinical and biochemical symptoms of decompensation of liver cirrhosis occur. The objective of the study and paper was to make a comparison between the Met-enkephalin concentration rates in plasma and its content in liver tissue with the progress stage of PBC, which was assessed using clinical symptoms. The study was carried out in 40 female patients with PBC. PBC has been found by means of biochemical, serological investigations as well as histopathological liver bioptate estimation. The patietns with different causes of liver disorders have been excluded. Met-enkephalin concentration in plasma and content in liver tissue have been indicated in each patient. The control group consisted of 30 female patients treated in the Department of Gastroenterology with normal activity of indicative and cholestatic enzymes, and without any organic disorders. In the control group, the Met-enkephalin concentration rates in plasma were determined. Statistically significant difference has been indicated in Met-enkephalin plasma concentration between two patients groups: 23 patients with pruritus and 17 patients without it where (p = 0.0476). Statistically significant difference in Met-enkephalin concentration level in plasma and liver tissue in the group of 19 patients with liver failure symptoms (stadium IV and V according to Schaffner and Popper classification) and in the group of 21 patients without those symptoms (stadium I and II according to Schaffner and Popper classification) has been stated. For Met-enkephalin plasma concentration p = 0.002 and for Met-enkephalin liver tissue concentration p = 0.0214., Conclusions: 1. The Met-enkephalin concentration rates in serum of patients with the PBC is higher than in the control group; 2. In PBC patients' pruritus appears together with the increase of Met-enkephalin plasma concentration. 3. In PBC patients, when liver failure symptoms appear Met-enkephalin concentration increases in plasma, and it decreases in the liver tissue.

Adrenal gland as a major source of enkephalins on the periphery can be affected by a rare adrenal gland tumor, adrenal pheochromocytoma. It has been demonstrated that this tumor might be associated with altered concentration of enkephalin-like peptides. The effect of these peptides can be either prolonged or abbreviated by two neutrophil membrane bound enzymes; aminopeptidase N (APN) and neutral endopeptidase (NEP). We assumed that altered enkephalin level in pheochromocytoma patients (but not in patients with non-functional adenomas or tumors of different origin) might result in differently regulated APN and/or NEP activity. We measured APN and NEP activity on surface of neutrophils, level of lipid peroxidation (LPO) in plasma and enkephalin concentration in plasma in patients with pheochromocytomas, non-functional adenomas, malignant renal tumors and healthy controls. Catheholamines and vanyllmandelic acid (VMA) were measured in 24-h urine of pheochromocytoma patients. NEP and APN activity on neutrophils from all pheochromocytoma patients was significantly increased as compared with healthy controls, non-functional adenomas and malignant renal tumors. In all pheochromocytoma patients NEP activity was reduced almost to the control level after surgery. At the same time APN activity was in some patients up- and in others down-regulated. In comparison, elevated levels of cateholamines and VMA were found after multiple determinations in 6 out of 10 pheochromocytoma patients. Although preliminary, this study has shown specifically and consistently up-regulated NEP activity on neutrophils from pheochromocytoma patients, and uniformly decreased NEP activity in these patients after adrenalectomy.

Background: Previous studies have indicated that solar and artificial ultraviolet (UV) radiation have a positive influence on psychological variables such as mood and emotional state. Circulating opioid peptides have been suggested as being important in this effect., Objectives: To investigate in a controlled trial the influence of UVA radiation on opioid peptide levels., Methods: We determined plasma levels of beta-endorphin immunoreactive material (IRM) and met-enkephalin in UV-exposed (n = 35) and non-exposed (n = 9) healthy volunteers. On the first day of the study, blood samples were taken from the volunteers (time A). UVA irradiation was subsequently administered with an air-conditioned tanning device. During the UV exposures the volunteers wore opaque goggles. Twenty minutes after UV exposure, blood samples were collected again (time B). Within the following 3 weeks the volunteers had a series of five UV exposures. On the last day of the study (24 h after the sixth UV exposure) blood samples were collected (time C). The cumulative UVA doses were 96 J cm-2 for skin type II and 126 J cm-2 for skin type III. The controls had no UV exposures. Plasma beta-endorphin IRM and met-enkephalin levels were determined using radioimmunoassays., Results: At all times of blood collection (A, B, C), there were no significant differences in plasma levels of beta-endorphin IRM and met-enkephalin between UV-exposed and non-exposed volunteers (P > 0.05)., Conclusions: UVA irradiation does not significantly elevate plasma levels of beta-endorphin IRM and met-enkephalin. Therefore we suggest that psychological benefits claimed to occur after UV exposure are unlikely to be mediated by the types of circulating opioid peptides measured in this study.

Background: Cyclosporine A (CsA) and morphine have neurotoxic and psychiatric side effects, respectively. Endogenous opiatelike peptides can elicit a number of behavioral responses that mimic the symptoms of psychiatric illness. The purpose of this study was to quantitiate the changes of Met-enkephalin (ME) and beta-endorphin (BE) after administration of CsA and morphine in surgery and to assess the antinociceptive effect., Patients and Materials: Pain sensitivity, an antinociceptive indicator in rats, was determined with the hotplate test. Plasma ME and BE levels were measured with radioimmunoassays., Results: In normal unoperated rats, CsA induced a profound analgesic effect concomitant with an increased plasma ME level on day 1. Morphine produced an analgesic effect on days 1 and 2, with decreased ME levels on days 2 and 3. Coadministration of CsA and morphine prolonged the analgesia from days 1 to 4 and increased the plasma ME level on day 1. No change in plasma BE level was found. In surgically operated rats, CsA induced an analgesic effect and higher ME levels than those in unoperated rats. Interestingly, the combined use of CsA and morphine prolonged the analgesia and increased plasma ME levels from days 1 to 4, with no significant change in plasma BE levels., Conclusions: Our results showed that CsA can induce antinociception and increase plasma ME levels. This induction can be potentiated by the addition of morphine. Acute neuropsychiatric manifestations in the early posttransplant period might, therefore, be due to induction of ME after coadministration of CsA and morphine.

After hyperventilation, systolic and diastolic blood pressure (BP) significantly decreased in 14 hypertensive patients (group 1), did not change in 9 (group 2) and increased in 8 (group 3). Basal BP, norepinephrine and dynorphin B levels were higher in group 1 than in groups 2 and 3. The decrease in BP after hyperventilation was associated with a decrease in plasma norepinephrine, Met-enkephalin and dynorphin B and an increase in beta-endorphin. Naloxone abolished the hyperventilation-induced BP and norepinephrine decreases. Our findings indicate that hyperventilation may select hypertensive patients with different sympatho-adrenergic activity and that the increase in beta-endorphin reduces BP response to hyperventilation in patients with high sympatho-adrenergic tone.

Met-enkephalin plasma levels were evaluated in 20 cardioischemic diabetic patients. All the patients had ECG ischemic signs. Ten patients with diabetic autonomic neuropathy, experienced no pain during myocarial ischemia. Met-enkephalin levels in the diabetic patients with silent myiocardial ischemia were significantly lower compared to those in the symptomatic patients. This demonstrates that the absence of myocardial ischemic pain in neuropathic diabetic patients is not accounted for by met-enkephalin action.

Antibacterial peptides, found in both invertebrates and vertebrates, represent a potential innate defense mechanism against microbial infections. However, it is unknown whether this process occurs in humans during surgery. We looked for evidence of release of antibacterial peptides during coronary artery bypass grafting (CABG). We used immunological techniques and antibacterial assays combined with high-performance gel-permeation chromatography, reverse-phase HPLC, N-terminal sequencing and comparison with synthetic standards to characterize the peptide B/enkelytin. We show the presence of anionic antibacterial peptide, the peptide B/enkelytin which correspond to the C-terminal part of proenkephalin A, from the plasma of patients undergoing CABG. Our studies show that peptide B/enkelytin is initially present at low levels in plasma and is then released in increased amounts just after skin incision. Antibacterial assays confirmed that the peptides specifically target gram-positive bacteria. We also demonstrate that peptide B/enkelytin is metabolized in vivo to the opioid peptides methionine-enkephalin-Arg-Phe and methionine-enkephalin, peptides that we show have granulocyte chemotactic activity. These findings suggest that in humans, surgical incision leads to the release of antibacterial peptides. Furthermore, these antibacterial peptides can be metabolized into compounds that have immune-activating properties.

After hyperventilation, systolic blood pressure (SBP) significantly decreased in 10 subjects (group 1), did not change in eight (group 2) and increased in 15 (group 3). Diastolic blood pressure and heart rate increased in all groups. The decrease in SBP was associated with a decrease in plasma catecholamines and increase in beta-endorphin, whereas the increase in SBP was accompanied by an increase in catecholamine and Met-enkephalin levels. Naloxone abolished the hyperventilation-induced SBP and catecholamine decrease only in group 1. These findings show an activation of the endogenous opioid system after hyperventilation and the role of beta-endorphin in reducing SBP in response to the test.

Opioid growth factor (OGF, [Met5]-enkephalin) is an endogenous peptide that regulates the growth of human pancreatic cancer. To evaluate whether human subjects with pancreatic cancer have alterations in plasma levels of OGF, fasting blood samples were obtained from 15 patients with histologically confirmed pancreatic adenocarcinoma. Forty-five subjects with other malignancies, 20 patients with acute pancreatitis, and 30 aged-matched patients without cancer served as control populations. Individuals with pancreatic cancer had OGF values, as determined by radioimmunoassay, that were up to 7.3-fold greater than control subjects. No differences were found between OGF values obtained from patients with other malignancies, acute pancreatitis, or subjects without cancer. The sensitivity and specificity of OGF for pancreatic cancer were greater than either CA 19-9 or CEA. These data indicate that pancreatic cancer is associated with a marked increase in plasma OGF levels and suggest that this peptide may serve as a useful diagnostic tool in the screening for this disease.

The authors have described the connections between nervous and immune systems. Endogenous opioids are one of the factors linking both systems. Endogenous and exogenous opioids can modify the function of interferons, humoral factors, antibody production and lymphocytes' activation. Concurrently, some cytokines can modify the endogenous opioid system. The aim of this study was to asses whether the subcutaneous administration of IL-2 influences the Met-Enkephalins concentration in serum after IL-2 single administration due to renal cancer. The Met-Enk level was estimated with RIA method. The IL-2 single administration results in a significant decrease of Met-Enk in serum.

Weight-trained men [OT; n = 11; age = 22.0 +/- 0.9 (SE) yr] resistance trained daily at 100% one-repetition maximum (1-RM) intensity for 2 wk, resulting in 1-RM strength decrements and in an overtrained state. A control group (Con; n = 6; age = 23.7 +/- 2.4 yr) trained 1 day/wk at a low relative intensity (50% 1 RM). After 2 wk, the OT group exhibited slightly increased exercise-induced testosterone (preexercise = 26.5 +/- 1.3 nmol/l, postexercise = 29.1 +/- 5.9 nmol/l) and testosterone-to-cortisol ratio (preexercise = 0. 049 +/- 0.007 nmol/l, postexercise = 0.061 +/- 0.006 nmol/l) and decreased exercise-induced cortisol (preexercise = 656.1 +/- 98.1 nmol/l, postexercise = 503.1 +/- 39.7 nmol/l). Serum concentrations for growth hormone and plasma peptide F [preproenkephalin (107-140)] were similar for both groups throughout the overtraining period. This hormonal profile is distinctly different from what has been previously reported for other types of overtraining, indicating that high-relative-intensity resistance exercise overtraining may not be successfully monitered via circulating testosterone and cortisol. Unlike overtraining conditions with endurance athletes, altered resting concentrations of pituitary, adrenal, or gonadal hormones were not evident, and exercise-induced concentrations were only modestly affected.

Background/aims: Opioid peptides may contribute to some of the manifestations of hepatic encephalopathy. To address the role of the opioid system in the pathogenesis of hepatic encephalopathy, three representative opioid ligands were measured in plasma and cerebrospinal fluid of patients with hepatic encephalopathy., Methods: Plasma and cerebrospinal fluid were obtained in three groups of patients: group 1: patients with hepatic encephalopathy; group 2: patients with lumbar back pain; group 3: healthy controls. Met-enkephalin, leu-enkephalin and beta-endorphin levels were measured in extracted plasma and cerebrospinal fluid samples by radioimmunoassay., Results: Plasma met-enkephalin levels were 656% (p<0.05) and 301% (p<0.05) and cerebrospinal fluid met-enkephalin levels were 1481% (p<0.01) and 645% (p<0.05) higher when compared to healthy control and pain control patients, respectively. Although plasma and cerebrospinal leu-enkephalin levels were elevated in patients with hepatic encephalopathy, the increases were not statistically significant. Plasma and cerebrospinal beta-endorphin levels were similar in the three study groups., Conclusions: The results of this study support accumulating data on the role of the delta opioid receptor ligand met-enkephalin in the pathogenesis of hepatic encephalopathy, and provide a rationale for the use of opioid receptor antagonists in the treatment of hepatic encephalopathy.