Development and validation of an LC-MS/MS assay for the quantification of allopregnanolone and its progesterone-derived isomers, precursors, and cortisol/cortisone in pregnancy.

Neuroactive steroids are potent neuromodulators that play a critical role in both maternal and fetal health during pregnancy. These stress-responsive compounds are reportedly low in women with perinatal depression and may be associated with poor pregnancy outcomes in animal models. Chronic stress is a risk factor for adverse birth outcomes. Simultaneous quantification of neuroactive steroids, in combination with stress hormones cortisol/cortisone, provides an opportunity to investigate the synergistic relationship of these analytes within the convenience of one assay. A simple, reliable, and sensitive method for quantifying these endogenous compounds is necessary for further research with the potential to advance clinical diagnostic tools during pregnancy. Analytes were extracted from serum with a simple protein precipitation using methanol and then separated and quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). After online extraction, analytes were separated using an Agilent Poroschell 120, 50 × 4.6 mm, 2.7 µm particle size, EC-C18 analytical column. The reliable quantification range was from 0.78 to 1000 ng/mL. QC sample inter- and intraday trueness was between 90 and 110% while inter- and intraday imprecision was less than 10%. Extracted samples were stable up to 7 days at 4 °C and extraction recovery was above 95%. Serum samples from 54 women in pregnancy were analyzed using this method. Here, we provide a validated, fast, and specific assay with sufficient sensitivity that allows for simultaneous quantification of blood serum concentrations of allopregnanolone (3α-hydroxy-5α-pregnan-20-one), pregnanolone (3α-hydroxy-5ß-pregnan-20-one), epipregnanolone (3ß-hydroxy-5ß-pregnan-20-one), pregnenolone, progesterone, cortisol, and cortisone in pregnancy for clinical study samples and clinical diagnostics.

Long-term exposure to PM1 and PM2.5 is associated with serum cortisone level and meat intake plays a moderation role.

BACKGROUND: Although short-term exposure to particulate matter (PM) was associated with increased glucocorticoids (GCs) levels, available evidence on associations of long-term exposure to PM and GCs levels is still scant. Previous studies has showed that meat intake is associated with sex hormones levels, but it is unknown whether meat intake is associated with GCs levels. Furthermore, the role of meat intake in the associations between PM and GCs levels remains unclear. AIMS: The aims of this study were to explore the associations of long-term exposure to PM and GCs levels among Chinese rural adults, and the role of meat intake in these associations. MATERIALS AND METHODS: A total of 6223 subjects were recruited from the Henan Rural Cohort Study. Serum GCs levels were measured with liquid chromatography-tandem mass spectrometry. The concentrations of PM (PM1 and PM2.5) for each subject were assessed with machine learning algorithms. The food frequency questionnaire (FFQ) was used to obtain each participant' information on meat intake. The effects of PM and meat intake on GCs levels were assessed using generalized linear models. In addition, modification analyses were performed to identify the role of meat intake played in the associations of PM with serum GCs levels. RESULTS: Per 1 µg/m3 increment in PM1 or PM2.5 concentration was associated with a 0.364 ng/ml (95% confidence interval (CI): 0.234, 0.494) or 0.227 ng/ml (95%CI: 0.110, 0.343) increase in serum cortisone, respectively. In addition, the moderation effects of total meat intake and red meat intake on the associations of long-term exposure to PM1 or PM2.5 with serum cortisone were observed (P < 0.05), indicating that individuals who had high levels of PM1 or PM2.5 and meat intake were more susceptible to have a higher state of serum cortisone. CONCLUSIONS: Our findings suggested that long-term exposure to PM1 or PM2.5 was associated with serum cortisone. Moreover, meat intake was found to be a significant moderator in the association of PM1 or PM2.5 with serum cortisone levels.

Chemical sympathectomy reduces peripheral inflammatory responses to acute and chronic sleep fragmentation.

Sleep loss contributes to the development of cardiovascular, metabolic, and neurological disorders by promoting a systemic proinflammatory phenotype. The neuroendocrine-immune mechanisms contributing to such pathologies are poorly understood. The sympathetic nervous system (SNS) regulates immunity and is often activated following sleep disturbances. The aims of this study were to determine 1) the effect of SNS inhibition on inflammatory responses to sleep fragmentation (SF) and 2) whether homeostasis can be restored after 1 wk of recovery sleep. We measured stress responses (norepinephrine and corticosterone), gene expression levels of pro- and anti-inflammatory cytokines in peripheral (heart, liver, and spleen) tissues, and protein levels of cytokines and chemokines in serum of female mice that were subjected to acute SF for 24 h, chronic SF for 8 wk, or 7 days of recovery after chronic SF. In each experiment, SF and control mice were chemically sympathectomized with 6-hydroxydopamine (6-OHDA) or injected with vehicle. Both acute and chronic SF elevated mRNA and protein levels of cytokines in peripheral tissues. Changes in inflammatory responses mirrored stress-axes activation, with increased corticosterone and norepinephrine in SF mice. 6-OHDA treatment significantly alleviated SF-induced inflammation, thus providing evidence of SNS regulation of peripheral inflammation from SF. Effects of chronic SF were more severe than acute SF, and 1 wk of recovery from SF sufficiently alleviated peripheral inflammatory responses but not NE responses.

The contribution of serum cortisone and glucocorticoid metabolites to detrimental bone health in patients receiving hydrocortisone therapy.

BACKGROUND: Glucocorticoid therapy is the most common cause of iatrogenic osteoporosis. Less is known regarding the effect of glucocorticoids when used as replacement therapy on bone remodelling in patients with adrenal insufficiency. Enhanced intracellular conversion of inactive cortisone to active cortisol, by 11 beta-hydroxysteroid dehydrogenase type 1(11ß-HSD1) and other enzymes leading to alterations in glucocorticoid metabolism, may contribute to a deleterious effect on bone health in this patient group. METHODS: Study design: An open crossover prospective study randomizing ten hypopituitary men, with severe ACTH deficiency, to three commonly used hydrocortisone dose regimens. MEASUREMENTS: Following 6 weeks of each regimen, patients underwent 24-h serum cortisol/cortisone sampling, measurement of bone turnover markers, and a 24-h urine collection for measurement of urinary steroid metabolites by gas chromatography-mass spectrometry (GC-MS). Serum cortisone and cortisol were analysed by liquid chromatography-mass spectrometry (LC-MS). RESULTS: Dose-related and circadian variations in serum cortisone were seen to parallel those for cortisol, indicating conversion of ingested hydrocortisone to cortisone. The median area under the curve (AUC) of serum cortisone was significantly higher in patients on dose A (20 mg/10 mg) [670.5 (IQR 621-809.2)] compared to those on dose C (10 mg/5 mg) [562.8 (IQR 520.1-619.6), p = 0.01]. A negative correlation was observed between serum cortisone and bone formation markers, OC [1-49] (r = - 0.42, p = 0.03), and PINP (r = - 0.49, p = 0.01). There was a negative correlation between the AUC of night-time serum cortisone levels with the bone formation marker, OC [1-49] (r = - 0.41, p = 0.03) but there were no significant correlations between day-time serum cortisone or cortisol with bone turnover markers. There was a negative correlation between total urinary cortisol metabolites and the bone formation markers, PINP (r = - 0.39, p = 0.04), and OC [1-49] (r = - 0.35, p = 0.06). CONCLUSION: Serum cortisol and cortisone and total urinary corticosteroid metabolites are negatively associated with bone turnover markers in patients receiving replacement doses of hydrocortisone, with nocturnal glucocorticoid exposure having a potentially greater influence on bone turnover. TRIAL REGISTRATION: Irish Medicines Board Clinical Trial Number - CT900/459/1 and EudraCT Number - 2007-005018-37 . Registration date: 07-09-2007.

Determination of estrone sulfate, testosterone, androstenedione, DHEAS, cortisol, cortisone, and 17α-hydroxyprogesterone by LC-MS/MS in children and adolescents.

Quantitation of endogenous steroids and their precursors is essential for diagnosis of a wide range of endocrine disorders. Usually, these analyses have been carried out using immunoassays. However, immunoassays often overestimate concentrations due to assay interference by other endogenous steroids, especially for low concentrations. Mass spectrometry based methods offer superior specificity, accuracy, and sensitivity. We therefore present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with automated sample preparation for determination of 17α-hydroxyprogesterone (17OHP), cortisol, cortisone, dehydroepiandrosterone sulfate (DHEAS), androstenedione (A4), testosterone (T), and estrone sulfate (E1S). Samples were prepared using protein precipitation and 96-well filter plates, fully automated in a pipetting robot and analyzed by LC-MS/MS. Serum samples from 187 healthy children and adolescents aged 5-18 years were used to study hormone changes in relation to sex and pubertal stage. Lower limit of quantification for 17OHP was 0.7 nmol/L, for cortisol 11 nmol/L, for cortisone 2 nmol/L, for DHEAS 0.1 µmol/L, and for A4, T, and E1S, 0.2 nmol/L. This study showed a general increase in 17OHP, DHEAS, A4, T and E1S in both genders during puberty. In boys, A4 and T increased significantly throughout pubertal development. Girls had significantly higher A4 and E1S concentrations, while boys had higher T concentrations. No sex- or puberty-specific differences were seen in cortisol or cortisone concentrations. To the best of our knowledge, this is the first presentation of changes in serum E1S concentrations during pubertal development in healthy children.

Family-transmitted stress in a wild bird.

Recent data suggest that, in animals living in social groups, stress-induced changes in behavior have the potential to act as a source of information, so that stressed individuals could themselves act as stressful stimuli for other individuals with whom they interact repeatedly. Such form of cross-over of stress may be beneficial if it enhances adaptive responses to ecological stressors in the shared environment. However, whether stress can be transferred among individuals during early life in natural populations remains unknown. Here we tested the effect of living with stressed siblings in a gull species where, as in many vertebrates, family represents the basic social unit during development. By experimentally modifying the level of stress hormones (corticosterone) in brood mates, we demonstrate that the social transfer of stress level triggers similar stress responses (corticosterone secretion) in brood bystanders. Corticosterone-implanted chicks and their siblings were faster in responding to a potential predator attack than control chicks. In gulls, fast and coordinated reactions to predators may increase the chances of survival of the whole brood, suggesting a beneficial fitness value of cross-over of stress. However, our data also indicate that living with stressed brood mates early in life entails some long-term costs. Near independence, fledglings that grew up with stressed siblings showed reduced body size, high levels of oxidative damage in lipids and proteins, and a fragile juvenile plumage. Overall, our results indicate that stress cross-over occurs in animal populations and may have important fitness consequences.

Effects of dietary γ-aminobutyric acid supplementation on antioxidant status, blood hormones and meat quality in growing-finishing pigs undergoing transport stress.

γ-Aminobutyric acid (GABA) is a natural nonprotein amino acid distributed in animals, plants and microbes. GABA is an inhibiting neurotransmitter which takes great effect in mammalian central nervous system. We carried out the research to study the influence of GABA on blood hormone concentrations, antioxidant status and meat quality in fattening pigs after transportation. The 72 pigs with a starting weight of approximately 32.67 ± 0.62 kg were randomly allocated to 2 groups based on dietary treatments, containing 6 replicates with 6 pigs in each. The pigs were fed dietary supplementation of GABA (0 or 30 mg/kg of diets) for 74 days. Twelve pigs were randomly selected from each group and assigned to the either 1 hr of transport (T group) or no transport (N group), resulting in two-factor factorial design. Compared to the control, GABA supplementation increased average daily gain (ADG) (p < .01) and decreased feed-gain ratio (F/G) (p < .05). The pH45 min was lower and drip loss was greater in the longissimus muscles (LM) of post-slaughter of transported pigs (p < .05). The pH45 min of 0/T group (group with 0 mg/kg GABA and transport) was significantly lower than the pH45 min of the 30/T group (diet × transport; p < .05). GABA supplementation significantly increased serum glutathione peroxidase (GSH-Px) concentration (p < .05) before transportation. Following transport, pigs fed GABA had decreased concentrations of serum malonaldehyde (MDA), adrenal cortical hormone and cortisol (p < .05). The results indicate that feeding GABA significantly increased the growth performance of growing-finishing pigs. The transportation model negatively impacted meat quality, antioxidant indexes and hormone parameters, but dietary supplementation of GABA could suppress the rise of drip loss of LM, ACTH and COR and suppress the drop of pH45 min of LM after transportation stress in growing-finishing pigs. Feeding GABA alleviated transportation stress in pigs.

Stress biomarkers in minimally invasive and conventional colorectal resections.

INTRODUCTION: Any surgical procedure develops a stress situation for the patient, which can modulate the individual outcome. At present, there is only limited information about stress response in colorectal resections by laparoscopic compared to conventional surgery. Therefore, our objectives were the feasibility and the investigation of stress biomarkers including copeptin and steroid hormones before, during and after colorectal surgery. METHODS: Eleven patients underwent minimally invasive and ten patients conventionally open colorectal surgery. Blood samples were collected before, during and 24 h after surgery and copeptin, NT-proBNP, cortisol, cortisone, interleukin-6 and glucose were analyzed. RESULTS: Both, minimally invasive and conventional-open colorectal surgery caused a fast but heterogeneous response of stress biomarkers. However, the postoperative decrease of cortisol, cortisone and glucose differed between both groups. The stress biomarkers decreased faster down to baseline after minimally invasive surgery, while in open surgery cortisol, cortisone and glucose did not return to baseline within 24 h after operation. CONCLUSIONS: We show in this feasibility study for the first time an increase of copeptin in combination with glucocorticoids as stress biomarkers by open surgery compared to minimally invasive procedures in patients undergoing colorectal surgery. Exceeding an individual threshold of 'stress burden' may have unfavorable effects on the long-time clinical outcome.

Particulate Matter Exposure and Stress Hormone Levels: A Randomized, Double-Blind, Crossover Trial of Air Purification.

BACKGROUND: Exposure to ambient particulate matter (PM) is associated with a number of adverse health outcomes, but potential mechanisms are largely unknown. Metabolomics represents a powerful approach to study global metabolic changes in response to environmental exposures. We therefore conducted this study to investigate changes in serum metabolites in response to the reduction of PM exposure among healthy college students. METHODS: We conducted a randomized, double-blind crossover trial in 55 healthy college students in Shanghai, China. Real and sham air purifiers were placed in participants' dormitories in random order for 9 days with a 12-day washout period. Serum metabolites were quantified by using gas chromatography-mass spectrometry and ultrahigh performance liquid chromatography-mass spectrometry. Between-treatment differences in metabolites were examined using orthogonal partial least square-discriminant analysis and mixed-effect models. Secondary outcomes include blood pressure, corticotropin-releasing hormone, adrenocorticotropic hormone, insulin resistance, and biomarkers of oxidative stress and inflammation. RESULTS: The average personal exposure to PMs with aerodynamic diameters ≤2.5 µm was 24.3 µg/m3 during the real purification and 53.1 µg/m3 during the sham purification. Metabolomics analysis showed that higher exposure to PMs with aerodynamic diameters ≤2.5 µm led to significant increases in cortisol, cortisone, epinephrine, and norepinephrine. Between-treatment differences were also observed for glucose, amino acids, fatty acids, and lipids. We found significantly higher blood pressure, hormones, insulin resistance, and biomarkers of oxidative stress and inflammation among individuals exposed to higher PMs with aerodynamic diameters ≤2.5 µm. CONCLUSIONS: This study suggests that higher PM may induce metabolic alterations that are consistent with activations of the hypothalamus-pituitary-adrenal and sympathetic-adrenal-medullary axes, adding potential mechanistic insights into the adverse health outcomes associated with PM. Furthermore, our study demonstrated short-term reductions in stress hormone following indoor air purification. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02712333.

Pulsatility of glucocorticoid hormones in pregnancy: Changes with gestation and obesity.

OBJECTIVE: Hypothalamic-pituitary-adrenal axis (HPA) activity is decreased in obese pregnancy and associates with increased foetal size. Pulsatile release of glucocorticoid hormones regulates their action in target tissues. Glucocorticoids are essential for normal foetal growth, but little is known about glucocorticoid pulsatility in pregnancy. We aimed to investigate the ultradian rhythm of glucocorticoid secretion during obese and lean pregnancy and nonpregnancy. DESIGN: Serum cortisol, cortisone, corticosterone and 11-dehydrocorticosterone were measured by LC-MS/MS from samples obtained at 10-minute intervals between 08.00-11.00 hours and 16.00-19.00 hours, from 8 lean (BMI <25 kg/m2 ) and 7 obese (BMI > 35 kg/m2 ) pregnant women between 16-24 weeks gestation and again at 30-36 weeks), and nonpregnant controls (lean n = 3, obese n = 4) during the luteal phase of their menstrual cycle. Interstitial fluid cortisol was measured by ELISA, from samples obtained using a portable microdialysis and automated collection device at 20-minute intervals over 24 hours. RESULTS: Serum cortisol AUC, highest peak and lowest trough increased significantly with gestation in lean and obese pregnant compared with nonpregnant subjects. Pulsatility of cortisol was detected in interstitial fluid. In pregnant subjects, interstitial fluid pulse frequency was significantly lower with advancing gestation in obese, but not in lean. CONCLUSIONS: We demonstrate cortisol pulsatility in interstitial fluid. Pulse frequency is altered with increased gestation and BMI. This may be a novel mechanism to explain decreased HPA activity in obese pregnancy.

Use of salivary cortisol and cortisone in the high- and low-dose synacthen test.

CONTEXT: Salivary cortisone reflects serum cortisol levels, is more sensitive than salivary cortisol at lower values of serum cortisol and is noninvasive. OBJECTIVE: To investigate the relationship between serum cortisol and salivary cortisol and cortisone following low- and high-dose synacthen. DESIGN AND SETTING: Prospective pharmacodynamic studies in clinical research facilities. PARTICIPANTS AND INTERVENTION: Thirty-five dexamethasone-suppressed, healthy adult males underwent an intravenous synacthen test: N = 23 low-dose (1 mcg), N = 12 high-dose (250 mcg). Paired serum and salivary samples were taken at 15 sampling points over 120 minutes. MAIN OUTCOME MEASURE: Serum cortisol and salivary cortisol and cortisone were analysed for correlations and by a mixed-effects model. RESULTS: At baseline, the correlation between serum cortisol and salivary cortisol was weak with many samples undetectable (r = .45, NS), but there was a strong correlation with salivary cortisone (r = .94, P < .001). Up to 50 minutes following synacthen, the correlation coefficient between serum cortisol and salivary cortisol and cortisone was <0.8, but both had a stronger correlation at 60 minutes (salivary cortisol r = .89, P < .001, salivary cortisone r = .85, P < .001). The relationship was examined excluding samples in the dynamic phase (baseline to 60 minutes). Salivary cortisol and cortisone showed a close relationship to serum cortisol. Salivary cortisone showed the stronger correlation: salivary cortisol r = .82, P < .001, salivary cortisone r = .96, P < .001. CONCLUSION: Following synacthen, both salivary cortisol and cortisone reflect serum cortisol levels, but there is a lag in their rise up to 60 minutes. The results support further research for possible future use of a 60-minute salivary cortisone measurement during the synacthen test.

Plasma Nervonic Acid Is a Potential Biomarker for Major Depressive Disorder: A Pilot Study.

Background: Diagnostic biomarkers of major depressive disorder, bipolar disorder, and schizophrenia are urgently needed, because none are currently available. Methods: We performed a comprehensive metabolome analysis of plasma samples from drug-free patients with major depressive disorder (n=9), bipolar disorder (n=6), schizophrenia (n=17), and matched healthy controls (n=19) (cohort 1) using liquid chromatography time-of-flight mass spectrometry. A significant effect of diagnosis was found for 2 metabolites: nervonic acid and cortisone, with nervonic acid being the most significantly altered. The reproducibility of the results and effects of psychotropic medication on nervonic acid were verified in cohort 2, an independent sample set of medicated patients [major depressive disorder (n=45), bipolar disorder (n=71), schizophrenia (n=115)], and controls (n=90) using gas chromatography time-of-flight mass spectrometry. Results: The increased levels of nervonic acid in patients with major depressive disorder compared with controls and patients with bipolar disorder in cohort 1 were replicated in the independent sample set (cohort 2). In cohort 2, plasma nervonic acid levels were also increased in the patients with major depressive disorder compared with the patients with schizophrenia. In cohort 2, nervonic acid levels were increased in the depressive state in patients with major depressive disorder compared with the levels in the remission state in patients with major depressive disorder and the depressive state in patients with bipolar disorder. Conclusion: These results suggested that plasma nervonic acid is a good candidate biomarker for the depressive state of major depressive disorder.

Relationship between adrenal steroid hormones in cord blood and birth weight: The Sapporo Cohort, Hokkaido Study on Environment and Children's Health.

OBJECTIVES: We investigated the relationship between steroid hormone levels in cord blood and birth weight. METHODS: Among 514 participants in a prospective birth cohort study in Sapporo, the following hormone levels were measured in 294 stored cord blood samples from 135 males and 159 females: androstenedione, dehydroepiandrosterone (DHEA), cortisol, and cortisone. Birth weight information was obtained from medical records. RESULTS: Androstenedione/DHEA was significantly higher in males than in females, while DHEA was significantly higher in females. Birth weight was significantly higher in males than in females. Regarding cortisone, androstenedione/DHEA, and cortisone/cortisol, a correlation was observed with birth weight in males but not in females. CONCLUSIONS: Prenatal adrenal steroids as well as converting enzymes such as 11ß-hydrosteroid dehydrogenase type 2 and 3ß-hydrosteroid dehydrogenase may have an impact on prenatal physical development.

In Vivo 11ß-Hydroxysteroid Dehydrogenase Inhibition in Posaconazole-Induced Hypertension and Hypokalemia.

We describe a case of apparent mineralocorticoid excess (AME) secondary to posaconazole therapy and suggest the biochemical mechanism. Clinical and laboratory investigation confirmed 11ß-hydroxysteroid dehydrogenase inhibition and withholding therapy led to a resolution of all clinical and laboratory abnormalities. Posaconazole was later restarted at a lower dose and prevented recurrence of this syndrome. Additional studies are necessary to determine the frequency of posaconazole-induced AME and whether other azole antifungals can be associated with this phenomenon.

Effects of dietary L-tryptophan supplementation on intestinal response to chronic unpredictable stress in broilers.

Stress has been recognized as a critical risk factor for gastrointestinal diseases in both humans and animals. However, nutritional strategies to attenuate stress-induced intestinal barrier function and underlying mechanisms remain largely unknown. This study tested the hypothesis that L-tryptophan enhanced intestinal barrier function by regulating mucosal serotonin metabolism in chronic unpredictable stress-exposed broilers. One-day-old male broilers (Arbor Acres) were fed a basal diet supplemented with or without L-tryptophan in the absence or presence of chronic unpredictable stress. Feed intake, body weight gain, plasma corticosterone and 5-hydroxytryptamine (5-HT), intestinal permeability, mucosal secretory IgA (sIgA), and mRNA levels for tryptophan hydroxylase 1 (TPH1), IL-1ß, IL-6, TNF-α, IL-10, protein abundance for claudin-1, occludin, and ZO-1 were determined. Stress exposure led to elevated plasma corticosterone (P < 0.05), increased intestinal permeability (P < 0.05), reduced growth performance (P < 0.05), and decreased sIgA secretion compared with the controls. These effects were largely reversed (P < 0.05) by L-tryptophan supplementation. Western blot analysis showed that stress exposure resulted in decreased protein abundance for occludin, claudin-1, and ZO-1, which was attenuated by L-tryptophan. mRNA levels for IL-1ß, IL-6, and TNF-α were increased, but those for IL-10 were decreased, in the jejunal tissue of broilers subjected to stress. This effect of stress on cytokine expression was abolished by L-tryptophan treatment. The effects of stress were associated with decreased plasma concentration of 5-HT (P < 0.05), and reduced (P < 0.05) mRNA levels for TPH1. L-Tryptophan supplementation markedly attenuated stress-induced alterations in 5-HT and TPH1 mRNA level in jejunal tissues of broilers. Collectively, these results indicate that L-tryptophan supplementation alleviates chronic unpredictable stress-induced intestinal barrier dysfunction by regulating 5-HT metabolism in broilers.

Prediction of tacrolimus metabolism and dosage requirements based on CYP3A4 phenotype and CYP3A5(*)3 genotype in Chinese renal transplant recipients.

AIM: To examine how the endogenous CYP3A4 phenotype and CYP3A5(*)3 genotype of Chinese renal transplant recipients influenced the dose-corrected trough concentration (C0/D) and weight-corrected daily dose (D/W) of tacrolimus. METHODS: A total of 101 medically stable kidney transplant recipients were enrolled, and their blood and urine samples were gathered. The endogenous CYP3A4 phenotype was assessed by the ratio of 6ß-hydroxycortisol and 6ß-hydroxycortisone to cortisol and cortisone in urine. CYP3A5(*)3 genotype was determined using PCR-RELP. RESULTS: In overall renal transplant recipients, a multiple regression analysis including the endogenous CYP3A4 phenotype, CYP3A5(*)3 genotype and post-operative period accounted for 60.1% of the variability in C0/D ratio; a regression equation consisting of the endogenous CYP3A4 phenotype, post-operative period, body mass index, CYP3A5(*)3 genotype, gender, total bilirubin and age explained 61.0% of the variability in D/W ratio. In CYP3A5(*)3/(*)3 subjects, a combination of the endogenous CYP3A4 phenotype, post-operative period and age was responsible for 65.3% of the variability in C0/D ratio; a predictive equation including the endogenous CYP3A4 phenotype, post-operative period, body mass index, gender and age explained 61.2% of the variability in the D/W ratio. Base on desired target range of tacrolimus trough concentrations, individual daily dosage regimen was calculated, and all the observed daily doses were within the predicted range. CONCLUSION: This study provides the equations to predict tacrolimus metabolism and dosage requirements based on the endogenous CYP3A4 phenotype, CYP3A5(*)3 genotype and other non-genetic variables.

Term neonates with infection and shock display high cortisol precursors despite low levels of normal cortisol.

AIM: Neonatal therapy-resistant septic shock is a common problem in middle and low-income countries. We investigated whether newborn infants with infection and therapy-resistant hypotension showed evidence of abnormal levels of cortisol or cortisol precursors. METHODS: A total of 60 term or near term neonates with evidence of infection were enrolled after informed consent. Of these, 30 had an infection and refractory shock and 30 had an infection without shock. There were no detectable differences between the groups in the length of gestation, birth weight or gender distribution. Serum was obtained during days four and 14 after birth. Cortisol and cortisol precursor concentrations were analysed using liquid chromatography-tandem mass spectrometry. RESULTS: The cortisol concentrations were low considering the expected responses to stress and they did not differ between the groups. The infants with infection and shock had higher serum dehydroepiandrosterone (DHEA) levels than those without shock (319.0 ± 110.3 µg/dL, versus 22.3 ± 18.3 µg/dL; p < 0.0001) and they also had higher 17-hydroxy-pregnenolone, pregnenolone and progesterone concentrations. There were no detectable differences in the levels of 17-hydroxy-progesterone, 11-deoxy-cortisol, cortisol or cortisone. CONCLUSION: Septic newborn infants with therapy-resistant hypotension had very high DHEA levels, suggesting that 3-beta-hydroxysteroid dehydrogenase activity limited the rate of cortisol synthesis.

[THE TECHNIQUE OF HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY FOR SIMULTANEOUS DIAGNOSTIC OF INHERENT HYPERPLASIA OF ADRENAL GLANDS TYPE I AND II].

The article considers the technique of high-performance liquid chromatography making it possible simultaneously detect cortisol, cortisone and secondary steroids in serum for consequent analysis of common reversed-phase high-performance liquid chromatography with ultraviolet under 240 nm. The liquid-liquid extraction from alkaline medium in diethyl ether The separation using column of 150x4.6 size ODS 3.5 mkm in isocratic mode. The eluent acetonitrile--0.02 M phosphate buffer pH 8.0--isopropanol (40:60:1). The application of proposed technique managed to separate cortisol, cortisone, dexamethasone, corticosterone, 11-desoxicortisol, testosterone, desoxicorticosterone, 17α-gidroxiprogesterone and androstendion in 20 minutes. The simplicity, reproducibility and sufficient selectivity and sensitivity of technique permit implement it in clinical practice for simultaneous diagnostic of inherent hyperplasia of adrenal glands type I and II.

The effect of different degrees of feed restriction on heat shock protein 70, acute phase proteins, and other blood parameters in female broiler breeders.

The aim of the current study was to determine the physiological response to feed restriction in female broiler breeders using a range of conventional and novel indicators. One hundred female breeders were subjected to one of five feeding regimens from d 28 to 42 as follows (i) ad libitum feeding (AL), (ii-v) 75, 60, 45, and 30% of ad libitum feed intake. Blood heterophil to lymphocyte ratio (HLR), and plasma circulating corticosterone (CORT), ghrelin (GHR), serotonin (5-HT), and dopamine (DA) and serum acute phase proteins (APP) concentrations together with brain heat shock protein (HSP) 70 level were measured. The results showed a significant effect of feed restriction on blood HLR and plasma CORT, GHR, 5-HT, DA, and brain HSP 70 levels. However, feed restriction had no effect on serum levels of APP of alpha-1 acid glycoprotein, ovotransferin, and ceruloplasmin. Serum levels of 5-HT and GHR varied curvilinearly with the feed restriction level. The relationship between brain HSP 70 and level of feed restriction was negligible. However, significant linear relationships between HLR, CORT, DA, and the level of feed restriction were noted. Thus, these 3 parameters appear to represent a straight forward relation with severity of feed restriction.