Development of an underivatized LC-MS/MS method for quantitation of 14 neurotransmitters in rat hippocampus, plasma and urine: Application to CUMS induced depression rats.

Sensitive and comprehensive measurement of systemic metabolites of tryptophan, phenylalanine and glutamate metabolism in biological samples is effective for understanding the pathogenesis of depression and other neurological diseases. Therefore, this study developed an underivatized liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneous monitoring the 3 components of glutamate metabolism in rat hippocampus and 11 components of tryptophan and phenylalanine metabolism in rat hippocampus, plasma and urine, and applied it to investigate their changes in rats induced by chronic unpredictable mild stress (CUMS). The investigated analytes are as follows: tryptophan, serotonin, 5-hydroxyindoleacetic acid, kynurenine, kynurenic acid, xanthurenic acid, 3-hydroxyanthranilic acid, quinolinic acid, phenylalanine, tyrosine, tyramine, glutamate, glutamine and gamma-aminobutyric acid. The method was verified to be sensitive and effective with satisfactory linearity, accuracies in the range of 78.2%-120.4%, and precisions less than 17.8% for all identified analytes. A series of significant changes in CUMS-induced rats can be detected: tryptophan, serotonin and tyrosine levels decreased and quinolinic acid increased in both hippocampus and plasma. In addition, the kynurenine/tryptophan ratios increased in hippocampus and plasma, the kynurenic acid/quinolinic acid ratios of plasma and urine were significantly reduced. These findings demonstrated that the CUMS procedure could lead to the central and peripheral imbalances of tryptophan and phenylalanine metabolism. In conclusion, a LC-MS/MS method for simultaneous measurement of several neurotransmitters in rat hippocampus, plasma and urine was developed and successfully applied to investigation of the central and peripheral changes in CUMS-induced rats. The method would be expected to provide applicability to the study of the mechanisms of depression and other related diseases associated with these neurotransmitters.

LC-MS/MS based studies on the anti-depressant effect of hypericin in the chronic unpredictable mild stress rat model.

ETHNOPHARMACOLOGICAL RELEVANCE: St John׳s Wort (Hypericum perforatum, SJW) is a widely used herbal medicine in western countries but also an important Uygur drug in China. Hypericin (HY) is the main components in SJW extracts, which is used to treat fatigue, weakness, and mild depression. The aim of this study was to investigate the anti-depression effects of HY on chronic unpredictable mild stress (CUMS) model rats and identify the possible mechanisms. MATERIALS AND METHODS: In this study, the protective effects of HY on CUMS-induced depression in rats were investigated by using a combination of behavioral assessments and urinary metabolites analysis. Urinary metabolites analyses were performed using LC-MS/MS in conjunction with principal components analysis (PCA) after oral administration of either HY or Venlafaxine (VF) for 27 days. During the procedure of experiment, food consumption, body weight, adrenal gland, thymus and spleen indices, behavior scores, sucrose consumption, and stress hormone levels were measured. RESULTS: Changes in the classic behavioral tests and pharmacological biochemical indices reflected that HY alleviated the symptoms of depression in a shorter period than VF, which was used as positive control for antidepression. Metabolites analysis of urine revealed that HY affected excitatory amino acids and monoamine neurotransmitter metabolites. Remarkably, urinary valine was increased remarkably by HY, even much higher than CUMS group. These results provide important mechanistic insights into the protective effects of HY against CUMS-induced depression and metabolic dysfunction. CONCLUSION: As the most important active ingredient in SJW extracts, HY possesses the better protective effect against CUMS-induced depression symptoms and metabolic disturbances.

A simple LC-MS/MS method for quantitative analysis of underivatized neurotransmitters in rats urine: assay development, validation and application in the CUMS rat model.

Many amino acid neurotransmitters in urine are associated with chronic stress as well as major depressive disorders. To better understand depression, an analytical LC-MS/MS method for the simultaneous determination of 11 underivatized neurotransmitters (4-aminohippurate, 5-HIAA, glutamate, glutamine, hippurate, pimelate, proline, tryptophan, tyramine, tyrosine and valine) in a single analytical run was developed. The advantage of this method is the simple preparation in that there is no need to deconjugate the urine samples. The quantification range was 25-12,800 ng mL(-1) with >85.8% recovery for all analytes. The nocturnal urine concentrations of the 11 neurotransmitters in chronic unpredictable mild stress (CUMS) model rats and control group (n = 12) were analyzed. A series of significant changes in urinary excretion of neurotransmitters could be detected: the urinary glutamate, glutamine, hippurate and tyramine concentrations were significantly lower in the CUMS group. In addition, the urinary concentrations of tryptophan as well as tyrosine were significantly higher in chronically stressed rats. This method allows the assessment of the neurotransmitters associated with CUMS in rat urine in a single analytical run, making it suitable for implementation as a routine technique in depression research.

Capsaicin pretreatment increased the bioavailability of cyclosporin in rats: involvement of P-glycoprotein and CYP 3A inhibition.

Capsaicin (CAP), the main ingredient responsible for the hot pungent taste of chilli peppers. This study investigated the effect of CAP on the pharmacokinetics of Cyclosporin A (CyA) in rats and the mechanism of this food-drug interaction. The results indicated that after 7 days of low or middle dose of CAP (0.3 or 1.0 mg/kg), the blood concentration of CyA was not significantly changed compared with that of vehicle-treated rats, whereas the blood concentration of CyA in high dose group (3.0 mg/kg) was significantly increased. The total clearance (CL/F) of CyA was decreased, and the bioavailability was significantly increased to about 1.44-fold of that in vehicle-treated rats after 7 days of high dose CAP treatment. At this time, the P-gp and CYP3A1/2 in the liver and intestine were decreased at both the mRNA and protein levels. These results demonstrated that chronic ingestion of high doses of CAP will increase the bioavailability of CyA to a significant extent in rats and the food-drug interaction between CAP and CyA appears to be due to modulation of P-gp and CYP3A gene expression by CAP, with differential dose-dependence.

Comparative Bioavailability and Tolerability of a Single 2-mg Dose of 2 Repaglinide Tablet Formulations in Fasting, Healthy Chinese Male Volunteers: An Open-Label, Randomized-Sequence, 2-Period Crossover Study.

BACKGROUND: Repaglinide, an oral insulin secretagogue, was the first meglitinide analogue to be approved for use in patients with type 2 diabetes mellitus. OBJECTIVE: In our study, the bioavailability and tolerability of the proposed generic formulation with the established reference formulation of repaglinide 2 mg were compared in a fasting, healthy Chinese male population. METHODS: This 2-week, open-label, randomized-sequence, single-dose, 2-period crossover study was conducted in 22 healthy native Han Chinese male volunteers. Eligible subjects were randomly assigned in a 1:1 ratio to receive a single 2-mg dose of the test or reference formulation, followed by a 7-day washout period and administration of the alternate formulation. After an overnight fast, subjects received a single oral dose of repaglinide (2 mg). Blood samples were drawn at predetermined time points (0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, and 6.0 hours). All plasma concentrations of repaglinide were measured by LC-MS/MS. The observed Cmax, Tmax, t1/2, and AUC were assessed. The formulations were to be considered bioequivalent if the ln-transformed ratios of Cmax and AUC were within the predetermined bioequivalence range of 80% to 125% established by the State Food and Drug Administration of the People's Republic of China. Tolerability was assessed throughout the study via subject interview, vital signs, and blood sampling. RESULTS: The mean (SD) age of the subjects was 24.2 (2.3) years; their mean (SD) weight was 62.6 (5.8) kg, their mean (SD) height was 172 (5.7) cm, and their mean (SD) body mass index was 21.0 (1.1). The mean (SD) Cmax for repaglinide with the test and reference formulations were 20.0 (5.1) and 18.7 (8.7) ng/mL. The AUC0-t for the test formulation was 46.3 (15.1) and AUC0-∞ was 47.9 (16.5) ng(•)h/mL. With the reference formulation, the corresponding values were 46.4 (26.1) and 49.0 (31.3) ng(•)h/mL. The mean (SD) Tmax values with the test and reference formulations were 1.2 (0.7) hours and 1.5 (0.8) hours and the mean (SD) values t1/2 values were 1.0 (0.3), and 0.9 (0.3) hours, respectively. The ln-transformed ratios of Cmax, AUC0-t, and AUC0-∞ were 113.6:1, 105.6:1, and 104.7:1. The corresponding 90% CIs were 99.8 to 129.2, 93.4 to 119.5, and 91.8 to 119.5, respectively. CONCLUSIONS: This single-dose study found that the test and reference formulations of repaglinide met the regulatory criteria for bioequivalence in these fasting, healthy Chinese male volunteers. Both formulations appeared to be well tolerated. ClinicalTrials.gov identifier: 2012L01684.

Comparative bioavailability and tolerability of single and multiple doses of 2 diclofenac sodium sustained-release tablet formulations in fasting, healthy chinese male volunteers.

BACKGROUND: Diclofenac is a nonsteroidal anti-inflammatory drug used for the treatment of patients with osteoarthritis. OBJECTIVES: Our primary objective was to compare bioavailability and tolerability of a generic sustained-release tablet with the established reference sustained-release tablet of diclofenac sodium in a fasting, healthy Chinese male population. METHODS: A randomized, open-label, single- and multiple-dose study design was used. After the single dose, volunteers received diclofenac sodium sustained-release tablet once daily for 5 days. In the single-dose phase, blood samples were collected from 0 to 36 hours after drug administration. In the multiple-dose phase, samples were obtained before drug administration at 8:00 am on Days 3 and 4 to determine Cmin,ss of diclofenac sodium; on Day 5, samples were collected from 0 to 36 hours. Adverse events were monitored via subject interview, vital signs, and blood sampling. RESULTS: Twenty-four Chinese male volunteers were enrolled. The pharmacokinetic parameters (mean [SD]) for diclofenac after single dose of 75 and 100 mg were: Cmax 473.5 [179.5] and 546.6 [154.9] ng/mL; AUC0-∞ 3841.2 [1402.3], and 5019.1 [2,314.0] ng·h/mL; Tmax 4.9 [2.4], and 4.3 [2.2] hours; t1/2 5.9 [2.5], and 6.0 [2.2] hours. Mean [SD] values after multiple doses of 75 and 100 mg were: Cmax,ss 525.6 [127.4] and 650.5 [167.0] ng/mL, Cmin,ss 33.9 [20.9] and 62.9 [34.9] ng/mL, AUCss 4316.3 [633.0] and 5335.1 [1291.9] ng·h/mL, Cav,ss 179.8 [26.4] and 222.3 [53.8] ng/mL, Tmax 5.1 [1.8] and 4.5 [0.9] hours and t1/2 5.2 [2.9] and 5.5 [2.8] hours, respectively. CONCLUSIONS: This diclofenac sodium 75 mg tablet has features compatible with the 100 mg sustained-release tablet and appeared to be well tolerated. ClinicalTrials.gov identifier: 2010L01969.

Food-drug interactions: effect of capsaicin on the pharmacokinetics of simvastatin and its active metabolite in rats.

Capsaicin (trans-8-methy-N-vanilly-6-nonenamide, CAP), the main ingredient responsible for the hot pungent taste of chilli peppers. However, little is known about the metabolic interactions between CAP and clinically used drugs. This study attempted to investigate the effect of CAP on the pharmacokinetics of simvastatin (SV), a cytochrome P450 (CYP) 3A substrate and an important cholesterol-lowering agent. CAP (3, 8 or 25 mg/kg), ketoconazole, dexamethasone or 5% CMC-Na was given to rats for seven consecutive days and on the seventh day SV (80 mg/kg) was administered orally. The results showed that when a single dose of SV was administered to rats fed with CAP over one week, AUC(0→∞), C(max) of SV and its acid metabolite was significantly decreased in comparison to the control treatment. Pretreatment of rats with CAP resulted in an decrease in the AUC(0-∞) of SV of about 67.06% (CAP 3 mg/kg, P<0.05), 73.21% (CAP 8 mg/kg, P<0.01) and 77.49% (CAP 25 mg/kg, P<0.01) compared with the control group. The results demonstrate that chronic ingestion of high doses of CAP will decrease the bioavailability of SV to a significant extent in rats.

Food-drug interactions: effect of capsaicin on the pharmacokinetics of galantamine in rats.

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide, CAP) is a naturally occurring alkaloid extracted from the fruit of Capsicum plant family. It represents an important ingredient in spicy foods consumed throughout the world. However, little is known about the metabolic interactions between CAP and clinically used drugs. This study attempted to investigate the effect of CAP on the pharmacokinetics of galantamine, a competitive and reversible cholinesterase inhibitor. CAP, dexamethasone or sodium salt of carboxymethyl cellulose (CMC-Na) was given to rats for seven consecutive days and on the seventh day galantamine (10 mg/kg) was administered orally. Dexamethasone was used as a CYP inducer and CMC-Na was used as a vehicle. The results showed that the pretreatment of rats with CAP resulted in a decrease in the AUC(0-∞) of galantamine of about 49.70% (p < 0.01) compared with the control group. After oral administration of galantamine (10 mg/kg), the apparent oral clearance of galantamine was raised by 2.05-fold by pretreatment with CAP (p < 0.05). These results demonstrate that the chronic ingestion of high doses of CAP will decrease the bioavailability of galantamine to a significant extent in rats.

[Leptin level in azoospermic patients and its clinical value].

OBJECTIVE: To detect the levels of seminal plasma leptin (SPL) and serum leptin (SL) in patients with azoospermia, and to explore the methods of using SPL and SL alone or the combination of SPL, SL and follicle stimulating hormone (FSH) for the differential diagnosis of obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). METHODS: We enrolled in this study 45 patients with diagnosed OA, 41 with unexplained NOA and 30 men with normal semen parameters as controls. The azoospermia patients underwent percutaneous aspiration from the epididymis (PESA) or aspiration/extraction from the testis (TESA/TESE), and all the subjects were detected for the levels of serum FSH, SPL and SL. Individual and multiple indexes were evaluated by Fisher's discriminant analysis combined with ROC curve analysis. RESULTS: There were no significant differences in the body mass index (BMI) among the three groups. Compared with the normal control, the OA patients showed an obviously elevated level of SPL (P = 0.048), and the NOA patients remarkably increased levels of FSH (P = 0.000), SL (P = 0.000) and SPL (P = 0.000). In comparison with the OA group, the levels of FSH (P = 0.000), SL (P = 0.006) and SPL (P = 0.033) were significantly increased in the NOA group. For the differential diagnosis of OA and NOA, the areas under the ROC curve of SPL and SL were 0.658 (P = 0.014) and 0.702 (P = 0.002) , respectively, both significantly greater than 0.5, while that of the combination of SPL, SL and FSH was the greatest (0.953). In addition, with 0.026 x SPL +0.05 x SL +0.106 x FSH -2.197 as the combined indicator value and -0.289 as the cut-off value (> or = cut-off value for NOA), the sensitivity and specificity of the combination were 0.878 and 0.902, respectively, both reached the maximum. CONCLUSION: Both the levels of SPL and SL are valuable for the differential diagnosis of OA and NOA, but the joint consideration of SPL, SL and FSH may provide better indicators.

Leptin and varicocele-related spermatogenesis dysfunction: animal experiment and clinical study.

The objective of this study was to explore the relationships between varicocele-related spermatogenesis dysfunction and the expression of leptin and leptin receptors. In rats with experimental varicocele, the function of spermatogenesis, the expression of leptin and leptin receptors in testes were analysed; and in patients with varicocele-related male infertility, serum and seminal plasma levels of leptin, gonadal hormones and semen parameters were evaluated. In the testes of rats, leptin was expressed in seminiferous tubules and intersitium, leptin receptor was predominantly expressed in interstitium. The expression of leptin and its receptor in the testis of rats was not related to the weight of rat, but was inversely related to the weight of testis (r = -0.408, p = 0.009 and r = -0.433, p = 0.005, respectively), the Johnsen scores (r = -0.916, p = 0.000 and r = -0.863, p = 0.000, respectively), the seminiferous tubules diameter (r = -0.853, p = 0.000 and r = -0.870, p = 0.000, respectively) and the thickness of seminiferous epithelium (r = -0.929, p = 0.000 and r = -0.948, p = 0.000, respectively). In varicocele patients (N = 40), the sperm concentration and motility were significantly lower (p = 0.000) than those in the control group (N = 25), and the leptin level in seminal plasma was significantly higher (p = 0.000) than that in the control group. The leptin in serum and seminal plasma was positively related (r = 0.223, p = 0.002). The seminal plasma leptin level was inversely related to sperm concentration (r = -0.632, p = 0.000) and motility (r = -0.635, p = 0.000). There was no significant relation between serum leptin and seminal parameters and between leptin and gonadal hormone values. The dysfunction of spermatogenesis in varicocele-related infertile male is associated with increase in leptin and leptin receptors. Leptin may have local effects on the function of testis and spermatogenesis.

[Jinleng method is effective and safe for the treatment of oligospermia and asthenospermia].

OBJECTIVE: The Jinleng method is based on the principle of lowered temperature and diathermic action on the testis. The aim of this study is to investigate the effect and safety of the Jinleng method on oligospermia and asthenospermia. METHODS: We treated 39 infertile males with oligospermia or asthenospermia with Jinleng underpants (Jinleng method) bid for 3 months, observed the changes in the sperm parameters of the patients after the treatment and recorded the pregnancy outcomes of their wives. RESULTS: Of the 36 patients who accomplished the treatment, 31 showed significantly improvement in semen volume, sperm concentration, forward sperm motility, total sperm motility, total sperm count and total motile sperm count (P < 0.05), with an effectiveness rate of 86.1%. Five of the patients wives achieved pregnancy in the 2-month follow-up. Adverse effects were found in none of the patients. CONCLUSION: Jinleng method is effective and safe for the treatment of infertile males with oligospermia and asthenospermia.

Sexual and reproductive health service needs of university/college students: updates from a survey in Shanghai, China.

AIM: To promote the provision of reproductive health services to young people by exploring the attitudes and perceptions of university students in Shanghai, China, toward reproductive health. METHODS: From July 2004 to May 2006, 5 243 students from 14 universities in Shanghai took part in our survey. Topics covered the demands of reproductive health-care services, attitudes towards and experience with sex, exposure to pornographic material, and knowledge on sexual health and sexually transmitted infections (STIs)/AIDS. RESULTS: Of the 5 067 students who provided valid answer sheets, 50.05% were female and 49.95% were male, 14.86% were medical students, and 85.14% had non-medical backgrounds. A total of 38.4% of respondents had received reproductive health education previously. The majority of students supported school-based reproductive health education, and also acquired information about sex predominantly from books, schoolmates, and the Internet. Premarital sexual behavior was opposed by 17.7% of survey participants, and 37.5% could identify all the three types of STIs listed in the questionnaire. Although 83.7% knew how HIV is transmitted, only 55.7% knew when to use a condom and 57.8% knew that the use of condoms could reduce the risk of HIV infection. CONCLUSION: The reproductive health service is lagging behind current attitudes and demands of university students. Although students' attitudes towards sexual matters are liberal, their knowledge about reproductive health and STIs/AIDS is still limited. It is therefore necessary to provide effective and confidential reproductive health services to young people.

[The feeder layer of human embryonic fibroblasts supports the growth of human spermatogonial stem cells].

OBJECTIVE: To investigate the methods and conditions for the isolation, purification and culture of human spermatogonial stem cells (SSCs) on the feeder layer cells of human embryonic fibroblasts (hEFs). METHODS: SSCs isolated and purified from normal human fetal testicular tissues by sequential two-step enzyme digestion and Percoll uncontinuous density gradient centrifugation were cultured on the feeder layer cells of hEFs isolated from 5-9 weeks old human embryos. The surface markers SSEA-1 and OCT4 of the SSCs were detected by immunohistochemistry; the alkaline phosphatase (AKP) activity of the SSC clones measured; and the expressions of the SSC-related genes determined by RT-PCR. RESULTS: SSCs survived, proliferated and formed colonies on the feeder layers, and the colonies were highly positive for SSEA-1 and OCT4, with strong AKP activity and high expressions of the SSC-related genes. CONCLUSION: The feeder layer of hEFs supports the growth of human spermatogonial stem cells.

[IIEF-5 cannot be used as a tool for differential diagnosis of vasculogenic erectile dysfunction].

OBJECTIVE: To evaluate the 5-item version of the international index of erectile function (IIEF-5) as a method to differentiate the causes of vasculogenic erectile dysfunction (ED). METHODS: In all, 103 ED patients (mean age 46.8 +/- 18.7) were reviewed by IIEF-5. Penile blood flow was also assessed in each patient after an intracavernosal injection (ICI) and audio-visual sex stimulation by duplex Doppler ultrasonography. The 99mTc-(113m)In dual radioisotope test was performed to confirm specific vascular causes in the vasculogenic ED cases. Kruskal-Wallis TEST was employed to compare the scores of IIEF-5 with the results of ICI, duplex Doppler ultrasonography and the 99mTC-(113m)In dual radioisotope test. RESULTS: Of the total number of ED cases, 37 (37/103, 35.9%) were nonvasculogenic, 18 (18/103, 17.5%) arteriogenic, 35 (35/103, 34.0%) venogenic and 13 (13/103, 12.6%) combined vasculogenic. There was no significant difference in the IIEF-5 scores either between the vasculogenic group and the non-vasculogenic one (P = 0.253) or among different groups of the vasculogenic ED patients. CONCLUSION: IIEF-5 cannot be used as a tool for differential diagnosis of vasculogenic ED, or to compare its specific vascular causes, nor can the scores of IIEF-5 reflect penile vascular conditions.

[Update of PDE5 inhibitors as treatment of ED].

Erectile dysfunction is a common ailment in middle-aged and old men. The management of ED has entered a new stage since sildenafil was used to treat ED in 1998. Sildenafil became the first-line treatment for its efficacy and safety. In recent years, new PDE5 inhibitors--vardenafil and tadalafil came into market in succession, providing more options available for oral therapy. This review is about the development of preclinical and clinical medicine research on the three PDE5 inhibitors, and provide information for clinical choices.