Radioimmunoassay Procedure Research Articles

The Association Between Vitamin D Level and Chest Pain, Anxiety, and Fatigue in Patients With Coronary Artery Disease.

This study sought to assess the association between vitamin D deficiency (VDD) and chest pain, anxiety, and fatigue in patients with coronary artery disease (CAD). A cross-sectional study was conducted to collect data from a sample of 90 participants with CAD. Serum 25OHD3 was measured using a radioimmunoassay procedure. Chest pain was assessed using the Numeric Pain Rating Scale, anxiety using the Anxiety Subscale, and fatigue by the General Fatigue subscale. The majority of participants (80%) had VDD. Results showed that the level of vitamin D was significantly negatively associated with chest pain (r = -.43, p = .00); anxiety (r = -.466, p = .00); and fatigue (r = -.25, p = .018). Findings suggest the role of VDD in heightened central sensitivity in cardiac-related symptoms such as chest pain, anxiety, and fatigue. Patients with CAD should be monitored for VDD so that the condition can be corrected.

Abstract C027: Racial variation in umbilical cord blood vitamin D concentrations and telomere length: Implications for cancer risk

Abstract Background: Non-Hispanic Blacks (NHBs) experience a markedly higher cancer burden and cancer mortality compared to non-Hispanic Whites (NHWs). Several factors account for this disparity, including suboptimal or deficient vitamin D status. Vitamin D has an important role in cancer risk, disease/cancer progression and telomere biology. Given the documented cancer disparities between these groups, it is important to examine vitamin D concentrations and leukocyte telomere length in utero, a window of susceptibility for disease and cancer risk later in life. We investigated racial differences in neonate umbilical cord blood vitamin D concentrations, and whether differences in vitamin D concentrations are associated with umbilical cord blood telomere length by race and gender. Methods: In 2006-2007, pregnant women were recruited for the Expanded Hormones in Umbilical Cord Blood Study (EHUB) and followed to postpartum. Venous umbilical cord blood samples, along with maternal and birth characteristics, were collected in 39 NHB and 65 NHW full-term neonates. 25(OH)D and 1,25(OH)2D levels were assayed using an equilibrium radioimmunoassay procedure, and relative telomere length was measured by qPCR in leukocyte DNA. Geometric mean plasma concentrations of 25(OH)D and 1,25(OH)2D were calculated. Linear regression analysis was used to examine associations between neonatal cord blood 25(OH)D and 1,25(OH)2D concentrations and leukocyte telomere length by race and gender. Results: Compared to NHW mothers, NHBs were younger, had higher parity, were more likely to be overweight and obese prepregnancy, and more likely to be exposed to secondhand smoke. NHB neonates had lower birth lengths (19.4 versus 20.2 inches; p<.0001) and lower birth weights (3287 vs. 3425 gm) than NHW neonates. Compared to NHW neonates, NHBs had lower adjusted mean 25(OH)D (6.4, 95% CI: 5.4-7.6 ng/mL versus 16.4, 95% CI: 14.2-18.9 pg./mL; p<.0001) and lower 1,25(OH)2D concentrations (51.1, 95% CI: 43.3-60.4 pg./mL versus 71.7, 95% CI: 62.6-82.0 pg./mL; p<.0003). After adjusting for race and gender, 25(OH)D and 1, 25(OH)2D concentrations were not associated with telomere length. There was an interaction between race and vitamin D, but not gender and vitamin D on telomere length. Compared to NHWs, NHB neonates had shorter telomere lengths per increase in vitamin D concentrations (-0.83 per unit of 25(OH)D and -0.79 per unit of 1,25(OH)2D). The interaction persisted after adjusting for maternal and birth characteristics. Conclusion: We did not find an association between umbilical cord blood vitamin D concentrations and telomere length. Instead, our findings suggest an interaction between race and umbilical cord blood vitamin D on telomere length. Further research is warranted to understand whether the effect of in utero exposure to vitamin D concentration on telomere length can inform the underlying mechanisms that are associated with cancer disparities in adulthood. Citation Format: Tanya Agurs-Collins, John Barber, Jessica Bienstock, Paige Green, Christopher Heaphy, Jiayun Lu, Alan Meeker, Anthony Rizzo, Sabine Rohrmann, Elizabeth Platz. Racial variation in umbilical cord blood vitamin D concentrations and telomere length: Implications for cancer risk [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C027.

Effect of prepartum exercise on lying behavior, labor length, and cortisol concentrations

The objective of this study was to assess the effect of exercise and pasture turnout on lying behavior, labor length, and cortisol concentrations around the time of parturition in dairy cows. Twenty-nine primiparous and 31 multiparous, pregnant, nonlactating Holstein (n = 58) and Jersey × Holstein cross (n = 2) dairy cows were assigned to control (n = 20), exercise (n = 20), or pasture (n = 20) treatments at dry-off using rolling enrollment. Control cows remained in the dry cow group pen. Exercise cows were removed from the dry cow group pen 5 times per week and walked for 1.4 ± 0.1 h at 1.88 ± 0.58 km/h. Pasture cows were moved to an outdoor paddock 5 times per week for 1.8 ± 0.3 h/d. Cows were housed in deep-bedded sand freestalls in a naturally ventilated, 4-row freestall barn. Cows were moved into maternity pens on the day of projected calving or when cows displayed signs that calving was imminent (restlessness, raised or lifted tail, ruptured amniotic sac, or swollen vulva), and treatments were discontinued. Cameras continuously recorded cows from entry into the pen until farm staff noted a calf, and one observer continuously watched video for two visually observable periods throughout the calving process: time from initial observation of amniotic sac to initial observation of calf's feet, and time from initial observation of calf's feet to full expulsion of calf. Assisted calvings were excluded. Accelerometers were attached to the rear fetlocks of cows 3 d before dry-off and removed 14 d postpartum. Activity was summarized by day for the 7 d before and after delivery time recorded from video observation into lying time (hours per day), lying bout frequency (bouts per day), lying bout duration (minutes per bout), and steps (number per day). Plasma total cortisol concentration was measured on d 0 and 3 postpartum and determined by a radioimmunoassay procedure using a commercially available kit. Data were analyzed using mixed linear model. During calving, time from appearance of the amniotic sac to appearance of the calf's feet was longer for pasture cows compared with control. Control cows engaged in fewer lying bouts and less overall lying time compared with pasture and exercise cows. Cortisol concentrations were higher on the day of calving compared with 3 d later, regardless of treatment. Understanding the effects of lying alterations around calving and increases in labor period length may allow for physical activity recommendations for late-gestation dairy cows.

Optimization of reagent concentration for radioiodination of rat C-peptide II in development of radioimmunoassay procedure for rats

Rat C-peptide is a polypeptide molecule made up of 31 amino acids and secreted from pancreas into circulation in two isoforms I and II. Quantification of rat C-peptide II in rat serum is important as it is directly related to the diagnosis of carbohydrate metabolism abnormalities, pancreatic performance analysis, monitoring of hypoglycemia, and diabetes-related illness in rat model. The aim of the present work is to develop a tracer by chloramine-T method for radioimmunoassay (RIA) procedure and to determine the optimum amount of chloramine-T required for the preparation of stable radioiodinated product with a specific activity of around 24.97 MBq/μg, corresponding to 1 125I atom per molecule of the peptide. Tyrosylated rat C-peptide II was selected for the radioiodination procedure as rat C-peptide II does not contain either tyrosine or histidine which is mandatory for the incorporation of 125I atom to the rat C-peptide II. Tyrosylated rat C-peptide II was subjected to radioiodination by chloramine-T method with different concentrations of chloramine-T and sodium metabisulfite (MBS) to obtain a stable radiolabeled compound. Optimized reaction conditions relating to the concentration of chloramine-T (10 μg) and MBS (20 μg) yielded a stable 125I-rat C-peptide II with specific activity of 21.01 MBq/μg corresponding to 0.84 125I atoms per molecule of the peptide. Preparation of high integrity tracer of rat C-peptide II was achieved by combining one molecule of oxidant (chloramine-T) and two molecule of reductant (MBS).

Effect of smoking on acute phase reactants, stress hormone responses and vitamin C in pulmonary tuberculosis.

Chronic inflammation, possibly exacerbated by cigarette smoking, is considered to be the primary cause of pulmonary damage in patients with tuberculosis (TB). However, the mechanisms which underpin these harmful inflammatory responses, have not been well documented. The current study was undertaken to determine possible associations between systemic biomarkers of inflammation (acute phase reactants, stress hormones, leukocyte vitamin C) and smoking status in patients (n=71, 20 smokers) with newly-diagnosed pulmonary TB presenting at a tertiary hospital, Johannesburg, South Africa. Plasma concentrations of C-reactive protein (CRP), ferritin, cortisol, epinephrine, norepinephrine, dopamine and leukocyte vitamin C were measured using a combination of immunonephelometric, radioimmunoassay, immunochromatographic and spectrophotometric procedures. Demographic, clinical and laboratory data was captured and analysed by parametric and non-parametric analyses where appropriate. Smokers were predominantly males (P<0.0001), of older age (P<0.0003) with a significantly lower body mass index (P<0.03). Plasma levels of CRP, ferritin and dopamine were higher in the group of smokers in the setting of lower levels of epinephrine, and leukocyte vitamin C, with CRP and vitamin C attaining statistical significance (P<0.04 and P<0.02 respectively). Those of cortisol and norepinephrine were comparable to those of non-smokers, as were radiographic changes and clinical indices of disease activity. Cigarette smoking is associated with an exaggerated systemic inflammatory response in pulmonary TB in the setting of decreased concentrations of leukocyte vitamin C. Although no significant associations with radiographic changes and most clinical indices of disease activity were evident on presentation, these pro-inflammatory interactions may have prognostic significance.

Association of testosterone levels and future suicide attempts in females with bipolar disorder

BackgroundConsiderable evidence suggests that testosterone may play a role in the pathophysiology of mood disorders in females. This is the first prospective study to examine whether blood testosterone levels predict suicide attempts in females with bipolar disorder. MethodsFemales with a DSM-IV diagnosis of a bipolar disorder in a depressive or mixed episode with at least one past suicide attempt were enrolled. Demographic and clinical parameters were assessed and recorded. Plasma testosterone was assayed using a double antibody radioimmunoassay procedure. Patients were followed up prospectively for up to 2.5 years. ResultsAt baseline, testosterone levels positively correlated with the number of previous major depressive episodes and suicide attempts. Cox proportional hazards regression analysis found that higher baseline testosterone levels predicted suicide attempts during the follow-up period. LimitationsA limitation of the study is that the sample size is modest. Another limitation is that we did not have a bipolar nonattempter or healthy volunteer control group for comparison. ConclusionTestosterone levels may predict suicidal behavior in women with bipolar disorder.

Potential drug interactions with melatonin

Possible interactions of melatonin with concurrently administered drugs were investigated in in vitro studies utilising human hepatic post-mitochondrial preparations; similar studies were conducted with rat preparations to ascertain whether rat is a suitable surrogate for human. Drugs were selected based not only on the knowledge that the 6-hydroxylation of exogenous melatonin, its principal pathway of metabolism, is mainly mediated by hepatic CYP1A2, but also on the likelihood of the drug being concurrently administered with melatonin. Hepatic preparations were incubated with either melatonin or 6-hydroxymelatonin in the presence and absence of a range of concentrations of interacting drug, and the production of 6-sulphatoxymelatonin monitored using a radioimmunoassay procedure. Of the drugs screened, only the potent CYP1A2 inhibitor 5-methoxypsoralen impaired the 6-melatonin hydroxylation at pharmacologically relevant concentrations, and is likely to lead to clinical interactions; diazepam, tamoxifen and acetaminophen (paracetamol) did not impair the metabolic conversion of melatonin to 6-sulphatoxymelatonin at concentrations attained following therapeutic administration. 17-Ethinhyloestradiol appeared not to suppress the 6-hydroxylation of melatonin but inhibited the sulphation of 6-hydroxymelatonin, but this is unlikely to result in an interaction following therapeutic intake of the steroid. Species differences in the inhibition of melatonin metabolism in human and rat hepatic post-mitochondrial preparations were evident implying that the rat may not be an appropriate surrogate of human in such studies.

Serum Vitamin D and C-Reactive Protein Levels Are Independently Associated With Diastolic Dysfunction

Background and AimVitamin D deficiency is common and may contribute to cardiovascular diseases. We hypothesized that serum 25-hydroxyvitamin D [25(OH)D] levels would be inversely associated with inflammation and with diastolic...

Impact of automatic tube current modulation on CT dose and image quality from a view of nationwide survey

Impact of automatic tube current modulation on CT dose and image quality from a view of nationwide survey

Sex effects on plasma leptin concentrations in newborn and postnatal beef calves

Changes in neonatal plasma leptin play a central role in regulating development of the hypothalamic appetite control centers in rodents. In lambs, a postnatal leptin surge has just been reported, and maternal obesity induced by overfeeding initiated before conception and continued throughout gestation blocks this leptin surge in newborn lambs. To investigate the presence, timing, and duration of neonatal leptin profiles, nulliparous cows carrying bull (n = 6) and heifer (n = 6) calves were selected. Cows exhibited no complications during parturition and received no assistance during delivery were chosen for this experiment. A blood sample from the jugular vein was obtained within 2 h of birth, then daily until d 8, and then every other day until d 18 of age at 0700 h. Plasma was collected and analyzed for glucose, insulin, cortisol, and leptin concentrations via validated colorimetric and radioimmunoassay procedures. Plasma hormone and metabolite values were analyzed as repeated measures. Bull calves exhibited elevated plasma leptin concentrations compared with heifers (P = 0.01). Furthermore, plasma leptin concentrations increased from birth until d 2 and then decreased until d 16 of age (P < 0.01). Plasma cortisol was elevated (P < 0.01) at birth and then decreased over the next 5 d. Bull calves had greater (P = 0.04) plasma insulin than did heifer calves. We conclude that there is a postnatal change in plasma leptin with differences due to calf sex and may affect the appetite centers of the hypothalamus influencing appetite and BW gain.

Characterization of the prolactin response to prolonged endurance exercise

This study characterized the blood prolactin responses to a prolonged endurance exercise bout in comparison to a resting, control period with no exercise. Six healthy exercise trained males completed both a 90 minute cycle exercise (70% VO2max) and a rest-control experimental session under standardized conditions. Blood samples were collected at – 15, 0 (exercise start), 15, 30, 45, 60, 75, 90 (exercise end), 105, and 120 minute time points in the exercise and rest-control sessions. Prolactin concentrations were analyzed using radioimmunoassay procedures and tested for significant changes with ANOVA analysis. In the exercise session, prolactin concentrations from 45 to 120 minutes were significantly greater than the 15 minute concentration before exercise (p &lt; 0.01). Furthermore, the exercise concentrations at 45 to 120 minutes were also significantly greater than the concentrations observed at the comparable rest-control time points (p &lt; 0.01; approximately 300% elevation). The frequent blood sampling protocol used in this study clearly portrays the magnitude, timeline, and extend of the prolactin response to prolonged endurance activity. The mechanism and role for the prolactin response was not the focus of this study, but relative to the latter, it is speculated the hormonal change could pertain to signaling energy usage-status within the body and, or prompting immune system activation.

Testosterone levels in suicide attempters with bipolar disorder

ObjectiveThe best known neurobehavioral effects of testosterone are on sexual function and aggression. However, testosterone and other androgens may be involved in the pathophysiology of mood disorders and suicidal behavior. This is the first study to examine whether there is a relation between testosterone levels and clinical parameters in bipolar suicide attempters. MethodsPatients with a DSM-IV diagnosis of a bipolar disorder (16 males and 51 females), in a depressive or mixed episode with at least one past suicide attempt were enrolled. Demographic and clinical parameters, including lifetime suicidal behavior, were assessed and recorded. Plasma testosterone was assayed using a double antibody radioimmunoassay procedure. ResultsThe number of major depressive episodes, the maximum lethality of suicide attempts, and the testosterone levels were higher in men compared to women. Current suicidal ideation scores were higher in women compared to men. Controlling for sex, we found that testosterone levels positively correlated with the number of manic episodes and the number of suicide attempts. ConclusionOur findings are consistent with previous observations of the association between testosterone levels and parameters of mood and behavior. This study suggests that testosterone levels may be related to the course of bipolar disorder and suicidal behavior. Further studies of the role of testosterone in the neurobiology of mood disorders and suicidal behavior are merited.

Sustained-Release Delivery of Octreotide from Biodegradable Polymeric Microspheres

The study reports on the drug release behavior of a potent synthetic somatostatin analogue, octreotide acetate, from biocompatible and biodegradable microspheres composed of poly-lactic-co-glycolic acid (PLGA) following a single intramuscular depot injection. The serum octreotide levels of three Oakwood Laboratories formulations and one Sandostatin LAR(®) formulation were compared. Three formulations of octreotide acetate-loaded PLGA microspheres were prepared by a solvent extraction and evaporation procedure using PLGA polymers with different molecular weights. The in vivo drug release study was conducted in male Sprague-Dawley rats. Blood samples were taken at predetermined time points for up to 70 days. Drug serum concentrations were quantified using a radioimmunoassay procedure consisting of radiolabeled octreotide. The three octreotide PLGA microsphere formulations and Sandostatin LAR(®) all showed a two-phase drug release profile (i.e., bimodal). The peak serum drug concentration of octreotide was reached in 30 min for all formulations followed by a decline after 6 h. Following this initial burst and decline, a second-release phase occurred after 3 days. This second-release phase exhibited sustained-release behavior, as the drug serum levels were discernible between days 7 and 42. Using pharmacokinetic computer simulations, it was estimated that the steady-state octreotide serum drug levels would be predicted to fall in the range of 40-130 pg/10 μL and 20-100 pg/10 μL following repeat dosing of the Oakwood formulations and Sandostatin LAR(®) every 28 days and every 42 days at a dose of 3 mg/rat, respectively.

Hamstring Stiffness and Knee Laxity Across the Menstrual Cycle in Females with ACL Reconstruction

Previous research in healthy, uninjured females has been equivocal as to whether knee flexor stiffness (FLEX) changes across the menstrual cycle (MC). However, once an athlete sustains an ACL injury, they are 6 times more likely to suffer a second ACL rupture to either the contra or ipsliateral limb. Given this information, females with a history of ACL injury are at greater risk of rupture compared to the general population, which creates a strong rationale for utilizing them in research. If reproductive hormones influence ACL injury risk by altering the properties of tissue, then females that have suffered a non-contact ACL injury may be more sensitive to hormonal fluctuations than females from the general, uninjured population. PURPOSE: To determine if FLEX and knee laxity (LAX) change across the menstrual cycle (MC) in ACL reconstructed females. METHODS: 20 females with noncontact, unilateral ACL rupture completed the study. Each subject was tested twice across the MC at periods associated with low (3-5 days post menses) and high (within 3 days post ovulation) estrogen concentrations. MC phase testing order was randomly assigned and the PI was blind to MC phase at the time of testing. The uninjured limb was used for testing. FLEX was assessed by quantifying the damping effect imposed by the knee flexors on oscillatory knee flexion/extension induced by perturbation. LAX was assessed via the KT 1000 knee arthrometer in the anterior direction at 133N of force. Blood hormone levels (estrogen [E], progesterone [P], and free testosterone [FT]) were assessed with radioimmunoassay procedures. Separate paired t-tests were performed with MC phase as the within subject factor (α <.05). RESULTS: [E] and [P] were higher at ovulation than post-menses ([E]: 31.1±13.7 vs 70.3±54.7 pg/mL, p=.009, [P]: 0.51±0.3 vs 3.92±4.27 ng/mL, p=.003). LAX (6.6±1.4 vs 7.3±1.3 mm, p=.03), and FLEX (178.6±35.7 vs 199.0±37.9 Nm/rad·kg-1, p=.03) were also increased at ovulation. [FT] was equal at both time periods (0.8±0.3 vs 0.9±0.2 ng/mL, p=.41). CONCLUSIONS: Increased knee flexor stiffness at ovulation may represent an effort to increase knee joint stability in compensation for increased anterior knee laxity. These factors change across the MC in females with a history of ACL injury and may increase the risk of a second injury. Supported by the NASM.

Relationships between circulating androgens, aggressive behaviour and breeding tubercles in males of the common bream Abramis brama L. in an aquarium environment

In this study, relationships between circulating androgens, aggressive behaviour and breeding tubercles in males of common bream Abramis brama were examined in an aquarium environment. The breeding tubercles of fish were counted, the number of attacks was quantified by male status and circulating rates of testosterone and 11-ketotestosterone from blood plasma were analysed using radioimmunoassay procedures. The results revealed that no significant differences were found between circulating testosterone and 11-ketotestosterone in territorial and nonterritorial males. Furthermore, no significant correlations were found between circulating androgens, androgens and aggression, androgens and tubercles and breeding tubercles and aggression in common bream by male status. However, territorial fish displayed a significantly higher level of aggressive behaviour and breeding tubercles than nonterritorial fish. In natural environments, the occurrence of breeding tubercles during the spawning season could contribute to identifying the behavioural status of common bream males.

DEVELOPMENT AND VALIDATION OF A TWO-STEP FREE THYROXINE RADIOIMMUNOASSAY BASED ON ANTIBODY COATED TUBES

We describe a two-step radioimmunoassay procedure (RIA) for the measurement of free thyroxine (fT4) in human serum. A commercial antibody with the affinity of 5.5 × 109 L/M was used for this study. The anti-T4 antibody was immobilized on the inner walls of the polystyrene tubes by passive adsorption through normal rabbit IgG and anti-rabbit IgG as immunobridges. The method developed uses very small amounts of antibody with minimum dilution of the sample. The assay covers the range of 0 to 87.5 pmol/L with sensitivity of 0.9 pmol/L. The intra-assay precisions were 6.3% and 7% at 6.7 and 20.7 pmol/L, respectively, for 15 replicates. The inter-assay precision at 6.4 and 24.4 pmol/L were 13% and 11.6%, respectively. The normal range established by analyzing 54 healthy volunteers was 15.3–24.7 pmol/L and that of 69 ambulatory subjects was 14.8–23.3 pmol/L. Progressive dilution of euthyroid and hypothyroid samples up to 50-fold yielded virtually constant values, whereas a registered decrease in free T4 was observed with hyperthyroid samples. Free T4 estimated for 75 samples, including samples from 16 pregnant female subjects, correlated well with the values obtained by a reputed commercial kit with the linear correlation coefficient of 0.84.

Influence of dietary carbohydrate intake on the free testosterone: cortisol ratio responses to short-term intensive exercise training

This study examined the effect of dietary carbohydrate (CHO) consumption on the free testosterone to cortisol (fTC) ratio during a short-term intense micro-cycle of exercise training. The fTC ratio is a proposed biomarker for overreaching-overtraining (i.e., training stress or imbalance) in athletes. The ratio was studied in two groups, control-CHO (approximately 60% of daily intake, n = 12) and low-CHO (approximately 30% of daily intake, n = 8), of male subjects who performed three consecutive days of intensive training (approximately 70-75% maximal oxygen consumption, 60 min per day) with a dietary intervention (on the day before and during training). Resting, pre-exercise blood samples were collected under standardized-controlled conditions before each day of training (Pre 1, 2, 3) and on a fourth day after the micro-cycle (Rest). Bloods were analyzed for free testosterone and cortisol via radioimmunoassay procedures. Subjects performed no additional physical activity other than prescribed training. Statistical analysis (ANCOVA) revealed the fTC ratio decreased significantly (p < 0.01) from pre-study resting measurement (Pre 1) to the final post-study resting measurement (Rest) in the low-CHO group (-43%), but no change occurred (p > 0.05) in the control-CHO group (-3%). Findings suggest if the fTC ratio is utilized as a marker of training stress or imbalance it is necessary for a moderately high diet of CHO to be consumed to maintain validity of any observed changes in the ratio value.

The Relationship Between Changes in Aerobic Power and Cortisol in Youth Undergoing Weight Status Alteration

PURPOSE: To determine if changes in aerobic power and cortisol are related in youth who underwent weight status alterations. METHODS: Data were collected from 60 youth at baseline (t1) and two years later (t2), (mean age t1 = 9.6 yr). Participants were selected from a larger study (CHIC III) and divided into two groups based upon their weight status: N-O (normal weight at t1 and overweight at t2), and O-N (overweight at t1 and normal weight at t2). Normal weight was defined as >5th and <85th BMI percentile, while overweight was ≥ 85th BMI percentile. Aerobic power was estimated using the submaximal PWC195, and expressed per kg of fat free mass (VO2/kgffm). Blood was taken in the morning (standardized) at t1 and t2, and cortisol was analyzed via radioimmunoassay procedures. The relationship between changes in VO2/kgffm and cortisol was examined using Pearson correlations. Multiple regression analyses were also computed to determine the influence of changes in VO2/kgffm and BMI percentile, race and gender on change in cortisol. RESULTS: When both groups were analyzed together, there was a correlation (r=0.32, p<0.02) between the two-year changes in VO2/kgffm and cortisol. In the overall multiple regression model, we found that changes in VO2/kgffm and BMI percentile each accounted for 11% of the variance in change in cortisol (Total Rfs = 0.21, p<0.02). When the groups were analyzed separately, the relationship between changes in VO2/kgffm and cortisol was only significant for the O-N group (r=0.45, p<0.03). Similarly, in the regression model for each of the O-N and N-O groups, change in VO2/kgffm was the only predictor of cortisol change, although it was only significant for the O-N group, accounting for 20% of the variance. Change in BMI percentile was not a significant predictor of change in cortisol for either group. CONCLUSIONS: Changes in cortisol and VO2/kgffm are related in youth who underwent weight status alterations, independent of changes in BMI percentile. The influence of VO2/kgffm on cortisol change is stronger in youth who were overweight and became normal weight, compared to youth who increased their weight status by becoming overweight. Supported by NINR #NR01-1837.

Molecular diagnosis of 5α-reductase-2 gene mutation in two Indian families with male pseudohermaphroditism

To identify the genotype of two Indians with male pseudohermaphroditism. Standard radioimmunoassay procedure was used for estimating hormonal levels. Conventional cytogenetic analysis was carried out for diagnosing the genetic sex in these subjects with genital ambiguity. Molecular analysis was carried out by standard polymerase chain reaction procedure using different sets of primers and reaction conditions specific for the 5alpha-reductase type 2 gene (SRD5A2) gene. Direct sequencing was carried out using the ABI Prism dye terminator sequencing kit and the ABI 310 sequencing apparatus. We found an SRD5A2 gene mutation in exon 5, where arginine is substituted with glutamine (R246Q), in two males with pseudohermaphroditism and ambiguous genitalia from unrelated families. This is the first time this mutation has been reported in individuals from India. Identification of the R246Q mutation of the SRD5A2 gene from two unrelated Indian families possibly extends the founder gene effect.

Acute allograft rejection and immunosuppression: influence on endogenous melatonin secretion

Melatonin displays a dose-dependent immunoregulatory effect in vitro and in vivo. Exogenous high-dose melatonin therapy exerted an immunosuppressive effect, abrogating acute rejection (AR), significantly prolonging transplant survival. Endogenous melatonin secretion, in response to heterotopic rat cardiac allograft transplantation (Tx), was investigated during the AR response and under standardized immunosuppressive maintenance therapy with cyclosporin A (CsA) and rapamycin (RPM). Recipients of syngeneic transplants, and recipients of allogeneic grafts, either untreated or receiving immunosuppressive therapy constituted the experimental groups. Endogenous circadian melatonin levels were measured at 07:00, 19:00, and 24:00 hr, using a novel radioimmunoassay (RIA) procedure, under standardized 12-hr-light/dark-conditions (light off: 19:00 hr; light on: 07:00 hr), before and after Tx. Neither the operative trauma, nor the challenge with a perfused allograft or the AR response influenced endogenous melatonin peak secretion. Immunosuppressive therapy with CsA led to a significant increase in peak secretion, measured for days 7 (212 +/- 40.7 pg/mL; P < 0.05), 14 (255 +/- 13.9 pg/mL; P < 0.001), and 21 (219 +/- 34 pg/mL; P < 0.01) after Tx, as compared with naïve animals (155 +/- 25.8 pg/mL). In contrast, treatment with RPM significantly decreased the melatonin peak post-Tx up to day 7 (87 +/- 25.2 pg/mL; P < 0.001), compared with naïve animals (155 +/- 25.8 pg/mL). These findings imply a robust nature of the endogenous circadian melatonin secretion kinetics, even against the background of profound allogeneic stimuli. Immunosuppressive maintenance therapy with CsA and RPM modulated early melatonin secretion, indicating a specific secondary action of these drugs. Further studies are necessary to disclose the long-term effect of immunosuppressive therapy on circadian melatonin secretion in transplant recipients.