UK guidance on the initial evaluation of an infant or an adolescent with a suspected disorder of sex development.

It is paramount that any child or adolescent with a suspected disorder of sex development (DSD) is assessed by an experienced clinician with adequate knowledge about the range of conditions associated with DSD. If there is any doubt, the case should be discussed with the regional team. In most cases, particularly in the case of the newborn, the paediatric endocrinologist within the regional DSD team acts as the first point of contact. The underlying pathophysiology of DSD and the strengths and weaknesses of the tests that can be performed should be discussed with the parents and affected young person and tests undertaken in a timely fashion. This clinician should be part of a multidisciplinary team experienced in management of DSD and should ensure that the affected person and parents are as fully informed as possible and have access to specialist psychological support. Finally, in the field of rare conditions, it is imperative that the clinician shares the experience with others through national and international clinical and research collaboration.

Diet, environmental factors, and lifestyle underlie the high prevalence of vitamin D deficiency in healthy adults in Scotland, and supplementation reduces the proportion that are severely deficient.

Vitamin D deficiency has recently been implicated as a possible risk factor in the etiology of numerous diseases, including nonskeletal conditions. In humans, skin synthesis following exposure to UVB is a potent source of vitamin D, but in regions with low UVB, individuals are at risk of vitamin D deficiency. Our objectives were to describe the prevalence of vitamin D deficiency and to investigate determinants of plasma 25-hydroxyvitamin D (25-OHD) concentrations in a high northern latitude country. Detailed dietary, lifestyle, and demographic data were collected for 2235 healthy adults (21-82 y) from Scotland. Plasma 25-OHD was measured by liquid chromatography-tandem MS. Among study participants, 34.5% were severely deficient (25-OHD <25 nmol/L) and 28.9% were at high risk of deficiency (25-40 nmol/L). Only 36.6% of participants were at low risk of vitamin D deficiency or had adequate levels (>40 nmol/L). Among participants who were taking supplements, 21.3% had a May-standardized 25-OHD concentration >50 nmol/L, 54.2% had 25-50 nmol/L, and 24.5% had <25 nmol/L, whereas this was 15.6, 43.3, and 41%, respectively, among those who did not take supplements (P < 0.0001). The most important sources of vitamin D were supplements and fish consumption. Vitamin D deficiency in Scotland is highly prevalent due to a combination of insufficient exposure to UVB and insufficient dietary intake. Higher dietary vitamin D intake modestly improved the plasma 25-OHD concentration (P = 0.02) and reduced the proportion of severely deficient individuals (P < 0.0001). In regions with low UVB exposure, dietary and supplement intake may be much more important than previously thought and consideration should be given to increasing the current recommended dietary allowance of 0-10 µg/d for adults in Scotland.

Confirmation of congenital adrenal hyperplasia by adrenal steroid profiling of filter paper dried blood samples using ultra-performance liquid chromatography-tandem mass spectrometry.

BACKGROUND: The specificity of screening for congenital adrenal hyperplasia by direct measurement of 17-hydroxyprogesterone in filter paper dried blood spot samples by immunoassay is low and has a high false-positive rate. In order to reduce the false-positive rate of this test, we developed a rapid, robust, specific confirmatory procedure in which cortisol, 4-androstene-3,17-dione and 17-hydroxyprogesterone were measured simultaneously by ultra-performance liquid chromatography-tandem mass spectrometry. METHODS: After extraction, samples were analysed by ultra-performance liquid chromatography-tandem mass spectrometry and 17-hydroxyprogesterone was quantified accurately. Other steroids were determined using stable deuterated internal standards. In total, 25 patient blood spot samples and 92 control samples were analysed. RESULTS: The assay was linear for 17-hydroxyprogesterone, with a coefficient of determination >0.997 and imprecision ≤ 6.5%. An upper limit of normal for 17-hydroxyprogester-one of 4.45 nmol/L was established by analysing a cohort of samples from unaffected newborns. In addition, a cut-off of 3.5 for the peak areas ratio (17-hydroxyprogesterone+4-androstene-3,17-dione)/cortisol, allows confirmation of the affected steroidogenic enzyme. CONCLUSIONS: A high throughput method for the detection of steroids related to congenital adrenal hyperplasia has been developed, allowing the false-positive rate associated with screening for 17-hydroxyprogesterone by immunoassay to be determined.

UK Food Standards Agency Workshop Consensus Report: the choice of method for measuring 25-hydroxyvitamin D to estimate vitamin D status for the UK National Diet and Nutrition Survey.

The consensus workshop, organised on behalf of the Food Standards Agency, was convened to recommend the most appropriate and secure method for measuring vitamin D status in the UK. Workshop participants (the Expert Panel) were invited on the basis of expertise in current 25-hydroxyvitamin D (25OHD) assays, or expertise in vitamin D nutrition and metabolism or detailed knowledge and experience in the National Diet and Nutrition Survey (NDNS). A decision support matrix, which set out the particular criteria by which the different options were scored and evaluated, was used to structure the discussion. The Expert Panel agreed that five methods for measuring 25OHD should be evaluated according to eleven criteria, selected on the basis of their relevance to the NDNS. All three of the evaluating subgroups of the Expert Panel produced similar total scores over the eleven criteria for the different methods; they scored LC-MS/MS and HPLC-UV similarly highly, while the scores for the immunoassay methods were lower. The Expert Panel recommended that an LC-MS/MS method should be the preferred method for the NDNS. A detailed specification for the method will be required to ensure comparability between NDNS and the National Health and Nutrition Examination Survey in the US facilitating future comparisons. The Expert Panel also recommended that the method should be carried out in a laboratory with appropriate expertise, competency and history of records of good performance. The method should be standardised against the National Institute of Standards and Technology SRM 972. If the recommended LC-MS/MS is adopted, the Expert Panel indicated that the method should be able to discriminate the C-3 epimer of 25OHD(3), especially if used to measure 25OHD in young infants in the forthcoming Diet and Nutrition Survey of Infants and Young Children, who are known to have high circulating concentrations of the C-3 epimer.

A simple automated solid-phase extraction procedure for measurement of 25-hydroxyvitamin D3 and D2 by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Measurement of 25-hydroxyvitamin D(3) (25OHD(3)) and D(2) (25OHD(2)) is challenging. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been described but they are often complex and difficult to automate. We have developed a simplified procedure involving an automated solid-phase extraction (SPE). METHODS: Internal standard (hexadeuterated 25-hydroxyvitamin D(3)) was added to serum or plasma followed by protein precipitation with methanol. Following centrifugation, a robotic instrument (CTC PAL [Presearch] for ITSP SPE [MicroLiter Analytical Supplies, Inc]) performed a six-step SPE procedure and the purified samples were injected into the LC-MS/MS. Quantification of 25OHD(3) and 25OHD(2) was by electrospray ionization MS/MS in the multiple-reaction monitoring mode. RESULTS: The lower limit of quantitation was 4.0 nmol/L for 25OHD(3) and 7.5 nmol/L for 25OHD(2). Within- and between-assay precision was below 10% over the concentration range of 22.5-120 nmol/L for D(3) and 17.5-70 nmol/L for D(2) (n = 10). The calibration was linear up to 2500 nmol/L (r = 0.99). Recoveries ranged between 89% and 104% for both metabolites and no ion suppression was observed. The results obtained compared well (r = 0.96) with the IDS-OCTEIA 25-hydroxyvitamin D enzyme immunoassay for samples containing less than 125 nmol/L, at higher concentrations the immunodiagnostic system (IDS) method showed positive bias. CONCLUSIONS: Our simplified sample preparation and automated SPE method is suitable for the measurement of 25OHD(3) and D(2) in a routine laboratory environment. The system can process up to 300 samples per day with no cumbersome solvent evaporation step and minimal operator intervention.

Adiponectin and coronary heart disease: a prospective study and meta-analysis.

BACKGROUND: There is uncertainty about the association between circulating concentrations of adiponectin and coronary heart disease (CHD) risk. We report new data from a prospective study in the context of a meta-analysis of previously published prospective studies. METHODS AND RESULTS: We measured baseline adiponectin levels in stored serum samples of 589 men with fatal CHD or nonfatal myocardial infarction and in 1231 controls nested within a prospective study of 5661 men (aged 40 to 59 years) recruited during 1978-1980, as well as in paired samples obtained 4 years apart from 221 of these participants. Baseline adiponectin concentrations correlated (P < 0.0001) positively with HDL cholesterol (r = 0.33) and inversely with C-reactive protein (r = -0.11) and BMI (r = -0.21), and the year-to-year consistency of adiponectin values was comparable to those of blood pressure and total cholesterol levels. No significant difference between median adiponectin levels at baseline was observed between cases and controls (10.2 versus 10.8 microg/mL; P = 0.5), despite the fact that body mass index, HDL, and C-reactive protein were all significant predictors of events in this cohort. The odds ratio for CHD was 0.89 (95% CI, 0.67 to 1.18) in a comparison of men in the top third of adiponectin concentrations compared with those in the bottom third, similar to a meta-analysis (including the present study) of 7 prospective studies involving a total of 1318 CHD cases (odds ratio, 0.84 [95% CI, 0.70 to 1.01]). CONCLUSIONS: In contrast to the strong associations previously reported between adiponectin levels and risk of type 2 diabetes, any association with CHD risk is comparatively moderate and requires further investigation.

Plasma leptin: associations with metabolic, inflammatory and haemostatic risk factors for cardiovascular disease.

BACKGROUND AND AIM: Leptin, an adipocyte-derived protein, regulating food intake and metabolism has been implicated in the development of coronary heart disease. We have examined the relationship between leptin and vascular risk factors including insulin resistance, metabolic, inflammatory and haemostatic risk factors. METHODS AND RESULTS: The study was carried out in 3640 non-diabetic men aged 60-79 years drawn from general practices in 24 British towns and who were not on warfarin. Leptin was strongly positively correlated with waist circumference (r=0.58; p<0.0001). Leptin concentrations decreased significantly with increasing physical activity and were lowered in cigarette smokers and elevated in men with pre-existing coronary heart disease and stroke; alcohol intake showed no association with leptin concentration. After adjustment for waist circumference and these lifestyle factors, increased leptin was independently associated with significant increases in insulin resistance, triglycerides, inflammatory markers (interleukin-6, C-reactive protein, fibrinogen, plasma viscosity), coagulation factor VIII, endothelial markers von Willebrand factor, tissue plasminogen activator, and fibrin D-dimer levels; and a decrease in HDL-cholesterol. No association was seen between leptin and blood pressure, total cholesterol, glucose or white cell count after adjusting for waist circumference. Further adjustment for insulin resistance abolished the relationships between leptin and triglycerides and HDL-cholesterol, weakened the associations with the haemostatic factors although they remained significant, but made minor differences to the associations with inflammatory markers. CONCLUSION: Plasma leptin is associated with insulin resistance, inflammation and disturbances in haemostasis independent of waist circumference, suggesting possible pathways by which leptin may influence risk of cardiovascular disease.

Benefits of salmon eating on traditional and novel vascular risk factors in young, non-obese healthy subjects.

AIM: The present clinical study tested the hypothesis that oil-rich fish consumption improves CHD risk factors. METHODS: Forty-eight (16 men) non-obese, healthy adults aged 20-55, consumed 125 g/day of salmon for a 4-week period followed by a 4-week period with no-fish (41 completers). Subjects were instructed to maintain dietary and physical activity patterns during the period of study. Blood pressure, anthropometric, body composition and dietary information with fasting blood samples to determine traditional and novel CHD risk markers and plasma fatty acids were obtained before and after each period. RESULTS: Compared to no-fish, eating salmon significantly decreased systolic, diastolic and mean arterial blood pressure by 4%, triglycerides by 15%, and LDL-cholesterol by 7%, and significantly increased HDL-cholesterol by 5% (P<0.05). The changes in blood pressure and lipids alone with salmon intake predict around a 25% reduction in CHD risk based on the PROCAM risk calculator. Plasma adiponectin demonstrated a trend towards improvement (8.39 micromol/L with salmon and 7.52 with no-fish; P=0.086) but no significant changes were found either in plasma leptin, glucose or insulin after salmon consumption. CONCLUSIONS: Daily consumption of salmon improves traditional risk predictors of CHD in non-obese subjects. Adiponectin may be involved but the impact on novel risk factors needs study in high-risk subjects.

Strikingly low circulating CRP concentrations in ultramarathon runners independent of markers of adiposity: how low can you go?

OBJECTIVE: This study was undertaken to evaluate to what extent C-reactive protein (CRP) can be reduced by exercise by examining its circulating concentrations in male ultramarathon runners and to determine if low leptin as a robust circulating marker of fat mass could account for low CRP in such men. METHODS AND RESULTS: Sixty-seven male ultramarathon runners and 63 sedentary male controls of similar age and body mass index were recruited. CRP and leptin were measured by ELISA and radioimmunoassay, respectively. Median CRP concentration in lean (body mass index <25 kg/m2) marathon runners was less than half control median (0.4 [0.2 to 0.9] mg/L versus 0.9 [0.5 to 2.7] mg/L, P=0.0013) and, more strikingly, in nonlean runners was approximately 26% of control median (0.4 [0.3 to 0.8] mg/L versus 1.5 [0.9 to 2.5] mg/L, P=0.0002). Circulating leptin levels were also substantially lower in lean (45% less) and nonlean (63% less, both P<0.0001) ultramarathon runners. However, interleukin-6 levels were not different. Furthermore, leptin adjustment only minimally attenuated the case-control difference in CRP, suggesting that mechanisms other than fat mass reduction contribute to low concentrations of CRP in marathon runners. CONCLUSIONS: This study suggests that circulating CRP concentrations can be markedly suppressed, independently of total adiposity or indeed fat mass, by intense regular physical exercise.

Evidence for tissue selectivity of the synthetic androgen 7 alpha-methyl-19-nortestosterone in hypogonadal men.

The potent synthetic androgen 7 alpha-methyl-19-nortestosterone (MENT) is resistant to 5 alpha-reductase but is a substrate for aromatase. It may therefore offer selective sparing of the prostate gland while supporting other androgen-dependent tissues. MENT acetate implants were administered for 24 wk to 16 hypogonadal men, randomly allocated to 1 or 2 implants (groups I and II, respectively; releasing approximately 400 microg/d x implant). Hemoglobin concentration and hematocrit were maintained during MENT treatment. Prostate volume fell in group I and to a small, but statistically nonsignificant, degree in group II; the level of prostate-specific antigen fell significantly in both. Lumbar spine bone mineral density decreased in both groups. Sexual behavior and erectile function declined in group I, but were maintained in group II. Thus, overall, one MENT implant appeared to provide subphysiological androgen replacement. The 2-implant dose of MENT was able to maintain most androgen-dependent functions, except bone mass, and there was evidence to support selective sparing of the prostate gland. These results demonstrate for the first time in humans the selectivity of MENT in tissues dependent on 5 alpha-reductase. In addition, our data are consistent with the importance of adequate estrogenicity as part of the necessary spectrum of activity of an androgen for replacement therapy in men.

Maternal obesity is associated with dysregulation of metabolic, vascular, and inflammatory pathways.

Obesity is increasing in prevalence worldwide and in all age groups. In nonpregnant individuals, obesity is associated with dyslipidemia; hyperinsulinemia; vascular dysfunction; and, more recently, low-grade chronic inflammation. However, whether such effects are sustained during pregnancy has been sparsely investigated but is important to establish, given the association of maternal obesity with numerous adverse metabolic and vascular consequences. We consecutively recruited 47 healthy women in the third trimester of pregnancy and divided the participants into 2 groups, lean [n = 24; median body mass index (BMI), 22.1 kg/m(2)] and obese (n = 23; median BMI, 31.0 kg/m(2)) around the median first trimester BMI. The age, parity, and smoking history were comparable in both groups. A detailed panel of metabolic and inflammatory parameters was measured and an in vivo assessment of endothelial-dependent and -independent microvascular function made using laser doppler imaging. Although low-density lipoprotein cholesterol and glycosylated hemoglobin were similar, fasting triglyceride concentrations were higher [2.70 (interquartile range, 2.3-3.21) vs. 2.20 (IQ range, 2.0-2.6) mmol/liter, P = 0.02] and high-density lipoprotein concentrations were lower [1.55 (IQ range, 1.1-1.7) vs. 1.72 (IQ range, 1.4-2.0) mmol/liter, P = 0.02] in the obese group. Leptin [55.6 (range, 45-64.4) ng/ml vs. 23.8 (range, 13.2-35.2) ng/ml, P < 0.0001] and fasting insulin [14.5 (range, 11.4-27.3) vs. 6.5 (range, 4.6-9.7) mU/liter, P < 0.0001] levels were more than double. Similarly, levels of inflammatory parameters, IL-6 [3.15 (range, 2.4-3.5) vs. 2.1 (range, 1.73-2.85) pg/ml, P = 0.003], and sensitive C-reactive protein [4.45 (range, 2.9-6.6) vs. 2.25 (range, 0.92-3.65) mg/ml, P = 0.0015] were also substantially elevated. Both endothelial-dependent and -independent vasodilatory responses were significantly reduced in the obese group (P = 0.0003 and P = 0.02, respectively, ANOVA) and systolic blood pressure was higher (P = 0.01). Metabolic factors, C-reactive protein (r = 0.289, P = 0.049), and insulin (r = 0.339, P = 0.02) were related inversely to endothelial-dependent function. These comprehensive data demonstrate that, as in nonpregnant obese individuals, obesity in pregnancy is associated not only with marked hyperinsulinemia (without necessarily glucose dysregulation) and dyslipidemia but also impaired endothelial function, higher blood pressure, and inflammatory up-regulation. Such a spectrum of risk factors may contribute to maternal complications in obese women and, as a result, influence fetal programming of adult vascular disease. Clearly, these data provide further rationale to examine the potential benefits of preconceptual weight loss and antenatal exercise.

Ovarian function and metabolic factors in women with oligomenorrhea treated with metformin in a randomized double blind placebo-controlled trial.

Women with oligomenorrhea and polycystic ovaries show a high incidence of ovulation failure perhaps linked to insulin resistance and related metabolic features. A number of reports show that the biguanide metformin improves ovarian function. However, in these trials the quality of evidence supporting ovulation is suboptimal, and few studies have been placebo-controlled. The aim of our study was to use a double-blind, placebo-controlled approach with detailed assessment of ovarian activity (two blood samples per week) to assess the validity of this therapeutic approach in this group of women. Of the 94 patients randomized, 2 withdrew before treatment commenced, 47 received placebo, and 45 received metformin (850 mg, twice a day). The numbers discontinuing the study prematurely were higher in the treatment group (n = 15) than the placebo group (n = 5; P < 0.05). The ovulation frequency assessed by the ratio of luteal phase weeks to observation weeks was significantly (P < 0.01) higher in the treated group (23%) compared with the placebo (13%), and the time to first ovulation was significantly (P < 0.05) shorter [23.6 d; 95% confidence interval (CI), 17, 30; compared with 41.8 d; 95% CI, 28, 56]. The proportion of patients failing to ovulate during the placebo-treatment period was higher (P < 0.05) in the placebo group, and the majority of ovulations were characterized by normal progesterone concentrations in both groups. The effect of metformin on follicular maturation was rapid, because the E2 circulating concentration increased over the first week of treatment only in the metformin group. Significant (P < 0.01) weight loss (and leptin reduction) was recorded in the metformin group, whereas the placebo group actually increased weight (P < 0.05). A significant increase in circulating high-density lipoprotein was observed only in the metformin-treated group. Metabolic risk factor benefits of metformin treatment were not observed in the morbidly obese subgroup of patients (body mass index > 37). No change in fasting glucose concentrations, fasting insulin, or insulin responses to glucose challenge was recorded after 14-wk metformin or placebo therapy. There was an inverse relationship between body mass and treatment efficacy. We show in a large randomized placebo-controlled trial that metformin treatment improves ovulation frequency in women with abnormal ovarian function and polycystic ovaries significantly but to a modest degree, and protracted treatment improves cardiovascular risk factors. These data support a beneficial effect of metformin in improving ovarian function in women with oligomenorrhea and polycystic ovaries.

Studies on the origin of circulating 18-hydroxycortisol and 18-oxocortisol in normal human subjects.

18-Hydroxycortisol (18-OHF) and 18-oxocortisol (18-oxoF) are derivatives of cortisol found in primary aldosteronism but whose origin and regulation in normal subjects are uncertain. 18-OHF can be synthesized by zona fasciculata 11-beta hydroxylase; 18-oxoF can only be produced by zona glomerulosa aldosterone synthase (AS). Stably transfected cell lines expressing either CYP11B1 (11beta-hydroxylase) or CYP11B2 (AS) were incubated with cortisol and other substrates over a range of concentrations. Both enzymes could synthesize 18-OHF from cortisol, but only AS could synthesize 18-oxoF. AS was more efficient than 11beta-hydroxylase at 18-hydroxylation. The apparent Michaelis-Menten constant (K(m)) of AS for cortisol was estimated to be 2.6 microm. In five patients with adrenal insufficiency maintained on hydrocortisone, urinary free cortisol and cortisone levels were high; 18-oxoF was detectable in all patients and 18-OHF in three. It is likely that the 18-oxygenated steroids were synthesized from circulating cortisol, either in the zona glomerulosa or at extraadrenal sites. In eight male volunteers, dexamethasone treatment decreased urinary excretion rates of free cortisol, cortisone, 18-OHF, and 18-oxoF, confirming dependence of 18-oxygenated steroid levels on cortisol availability. In both groups, hydrocortisone administration resulted in detectable levels of 18-OHF and raised levels of 18-oxoF. There was close correlation between 18-oxoF and cortisol excretion during hydrocortisone administration in normal subjects (r = 0.86; P < 0.001). These data show, for the first time, that 18-OHF and 18-oxoF can be synthesized from circulating cortisol. The close correlation between 18-oxoF and cortisol suggests that 18-oxoF is normally produced by the action of AS using circulating cortisol as a substrate. Although 18OHF can be synthesized using circulating cortisol as substrate, our data suggest this is normally produced in the zona fasciculata by 11beta-hydroxylase from locally available cortisol.

Divergent metabolic and vascular phenotypes in pre-eclampsia and intrauterine growth restriction: relevance of adiposity.

OBJECTIVE: Pre-eclampsia (PE) and intrauterine growth restriction (IUGR) may both arise secondary to inadequate trophoblast invasion. Maternal vascular disease is evident only in PE. Little mechanistic evidence exists to explain this dichotomy. METHODS: We employed laser Doppler imaging (LDI) to examine microvascular function in 15 women with PE and 30 healthy pregnant women matched for body mass index (BMI). We also examined 16 women with IUGR. Other factors examined included indices of inflammation, lipoproteins, leptin and insulin concentrations. RESULTS: Women with PE had double the concentration of leptin and 30% higher triglyceride than controls. Vascular cell adhesion molecule (VCAM)-1 and interleukin (IL)-6 were also higher in women with PE, with both factors correlating with leptin independently of BMI. No difference in microvascular reactivity was observed between controls and women with PE. Women with IUGR had a four-unit smaller BMI than women with PE. When compared with controls, they also had lower low-density lipoprotein cholesterol (LDL-C) concentrations and systemic inflammatory measures were not elevated. CONCLUSIONS: The technique of LDI is not sensitive to the vascular dysfunction of PE. However, circulating endothelial-derived factors are elevated in association with markedly elevated leptin levels. Therefore, women with IUGR may demonstrate a protective role for their 'leanness' with regard to maternal systemic inflammatory effect.

The relationship between leptin concentrations, the systemic inflammatory response and illness severity in surgical patients admitted to ITU.

BACKGROUND & AIMS: The relationship between circulation leptin concentrations, mediators of the systemic inflammatory response and illness severity in critically ill patients remains unclear. The aim of the present study was to examine these relationships in critically ill surgical patients admitted to the intensive therapy unit (ITU). PATIENTS AND METHODS: Patients (n = 38) who had undergone surgery and subsequently admitted to ITU were prospectively entered into a cross-sectional study. Circulating concentrations of leptin, cortisol, growth hormone, interleukin-6, C-reactive protein and albumin were measured. Sex and age matched controls (n = 14) were also studied. RESULTS: On day 1, the critically ill group had lower BMI and leptin concentrations and a pronounced systemic inflammatory response compared with controls. There was a weak positive correlation between leptin concentrations and BMI in the critically ill patients (r = 0.42, P<0.10). In contrast, leptin was inversely correlated with C-reactive protein (r = -0.59, P<0.05) but not with cortisol, growth hormone, interleukin-6, APACHE II or predicted mortality. Leptin concentrations did not alter with the day of admission to ITU. CONCLUSIONS: On ITU admission, leptin concentrations appeared to be low for BMI, related to the magnitude of the systemic inflammatory response but did not appear to be regulated by proposed mediators in critically ill surgical patients.

Analysis of biological samples by gas chromatography-mass spectrometry without a reference standard: measurement of urinary 18-hydroxytetrahydro-11-dehydrocorticosterone excretion rate in human subjects.

Reference standards for some minor urinary steroid metabolites are sometimes unavailable. We describe a novel procedure to quantitate a urinary steroid metabolite of known structure and mass spectrum, using as a standard a compound which produces ions in common with it and has a similar retention time in gas chromatography-mass spectrometry. The steroid of interest was 18-hydroxy-11-dehydrotetrahydrocorticosterone (18-OH-THA), the major urinary metabolite of 18-hydroxycorticosterone (18-OH-B), a putative intermediate in the conversion of 11-deoxycorticosterone to aldosterone. The steroid used as an alternative to the authentic 18-OH-THA standard was beta-cortol which, like 18-OH-THA, produces a fragmentation ion at m/z 457. Allo-tetrahydrodeoxycorticosterone (5alpha-THDOC) was used as the internal standard. beta-Cortolone also has the fragmentation ion at m/z 449 (in common with beta-cortol) and an authentic standard is available commercially. To validate the procedure, we quantitated beta-cortolone urinary excretion rate against this alternative standard and also against authentic beta-cortolone standards. Both methods produced similar results (adjusted R(2): 0.998, P<0.001). The method was then used to measure urinary excretion of 18-OH-THA rate in healthy volunteers. The reference range obtained was 20-204 microgram/24 h (n=32). This is similar to the few results available by conventional assay. Method performance was also similar to other assays of urinary steroids. This procedure could be generally applicable for assays when authentic standards are not available but mass spectra are known or can be predicted.

Comparison of simple acid-ethanol precipitation with gel exclusion chromatography for measuring leptin binding in serum of normal subjects and cancer patients.

BACKGROUND: In humans, leptin circulates in a free form and is also bound to macromolecules. The aim of the present study was to compare a rapid acid-ethanol precipitation (AEP) method of measuring bound leptin with the more laborious gel exclusion chromatography (GEC) reference procedure. Serum samples collected from healthy subjects and cancer patients were used in this comparison. METHODS: AEP and GEC methods for measuring leptin binding in serum (from 14 healthy volunteers and 14 patients with advanced non-small cell lung cancer) were adapted from previously published procedures. RESULTS: Intra- and inter-assay precision of the AEP method were 6% (n = 10) and 8% (n = 10), respectively. Bland-Altman analysis of results obtained from the AEP and GEC methods indicated no significant difference in healthy controls. However, significantly higher results were obtained by the AEP method in the cancer patients. CONCLUSIONS: Evaluation of the AEP method revealed that on examination of normal subjects the method was less precise than had previously been reported. Moreover, the method gave differing results in the cancer patients when compared with the GEC method. This study indicates that careful evaluation of any new method for measuring leptin binding requires comparison with a GEC method using the sample matrix of interest.

Morning salivary cortisol versus short Synacthen test as a test of adrenal suppression.

BACKGROUND: The short Synacthen test (SST) is the most commonly used test for the assessment of adrenal suppression. We investigated the potential of a simpler and more cost-effective procedure [morning salivary cortisol (MSC)] as an outpatient screening tool to detect adrenal suppression in patients using topical intranasal corticosteroids for rhinosinusitis. METHOD: Forty-eight patients who were using topical corticosteroids underwent adrenal function assessment by way of SST and MSC measurement. RESULTS: Sixteen of the 48 patients had impaired MSCs. Of these 16 patients, 15 had an impaired SST (sensitivity 100%) and one had a normal SST. All patients with normal MSCs also had normal SSTs (specificity 97%). CONCLUSION: The morning salivary cortisol measurement is a useful screening tool for adrenal suppression in this setting.