Direct determination of human urinary cortisol metabolites by HPLC/CRIMS.

Feasibility of using high performance liquid chromatographic input to the chemical reaction interface mass spectrometry system was assessed by measuring the profile of hydrolyzed urinary metabolites of [9,12,12-2H3] cortisol in six human subjects with no preparation other than hydrolysis and solid phase extraction. Relative amounts of tetrahydrocortisol, tetrahydrocortisone, and cortolones (as the sum of alpha- and beta-) were 0.417 +/- 0.047, 0.523 +/- 0.036 and 0.059 +/- 0.019, respectively. The constant reproducibility of the measurements coupled with a profile consistent with that observed by other workers shows that the technique represents an important tool in the determination of metabolites of endogenous molecules.

Mineral balance and bone turnover in adolescents with anorexia nervosa.

We evaluated seven female adolescents with anorexia nervosa to determine whether calcium metabolism was affected by their disorder. We measured calcium absorption, urinary calcium excretion, and calcium kinetics, using a dual-tracer, stable-isotope technique during the first weeks of an inpatient nutritional rehabilitation program. Results were compared with those from a control group of seven healthy adolescent girls of similar ages. The percentage of absorption of calcium was lower in subjects with anorexia nervosa than in control subjects (16.2% +/- 6.3% vs 24.6% +/- 7.2%; p < 0.05). Urinary calcium excretion was greater in subjects with anorexia nervosa than in control subjects (6.4 +/- 2.5 vs 1.6 +/- 0.7 mg.kg-1 x day-1; p < 0.01) and was associated with bone resorption rather than calcium hyper-absorption. Calcium kinetic studies demonstrated a decreased rate of bone formation and an increased rate of bone resorption. These results suggest marked abnormalities in mineral metabolism in patients with anorexia nervosa. From these results, we hypothesize that improvement in bone mineralization during recovery from anorexia nervosa will require resolution of hormonal abnormalities, including hypercortisolism, in addition to increased calcium intake.

Developmental changes in calcium kinetics in children assessed using stable isotopes.

Total exchangeable calcium pool size (TEP) and bone calcium accretion rate (Vo+) were measured using stable isotopes in healthy children and young adults. 42Ca or 46Ca was given intravenously to 10 children aged 10 months to 14 years and 3 women aged 23-33 years. Calcium kinetic parameters were determined using a two- or three-exponential curve of the resultant serum and urine tracer excesses. These data were compared with previously reported (radiotracer) kinetic studies of 21 children and 5 adults without known bone disease. Current results are comparable to those previously obtained, and the data from all studies were analyzed together. Total Vo+ was significantly greater in children aged 3-16 years than in adults (2.8 +/- 1.6 versus 0.7 +/- 0.2 g/day, p less than 0.01). Both TEP and Vo+ were significantly correlated to age independently of variations in body weight (p less than 0.01 for each). The ratio ko+ = Vo+/TEP was greater in children than adults (0.36 +/- 0.15 versus 0.12 +/- 0.03 day-1, p less than 0.001). These data demonstrate increased bone flow of calcium associated with increases in exchangeable calcium pools in children compared to adults. Vo+ and TEP may be maximum in early adolescence, associated with peak rates of net calcium accretion. The use of stable isotopes permits the safe evaluation of calcium kinetics in patients of all ages.

Dual tracer stable isotopic assessment of calcium absorption and endogenous fecal excretion in low birth weight infants.

Using a dual tracer (44Ca orally and 46Ca i.v.) stable isotope technique, true dietary Ca absorption, endogenous fecal Ca excretion, and net Ca retention were measured in 12 low birth weight (1426 +/- 260 g) infants fed a high Ca-containing formula. Endogenous fecal Ca excretion averaged 7.2 +/- 4.1% of intake, and exceeded 10% of intake in three infants. Net Ca retention, 103 +/- 38 mg/kg/d, was consistent with previous studies of Ca retention obtained using mass balance techniques and correlated closely (r = 0.98, p less than 0.001) with true Ca absorption but not with endogenous fecal excretion (r = -0.40, p = 0.19). Although endogenous fecal excretion may represent a significant source of Ca loss for some low birth weight infants, these data suggest that net Ca retention in low birth weight infants fed a high Ca-containing formula is primarily determined by the total dietary Ca absorbed.

Stable isotopic measurement of endogenous fecal calcium excretion in children.

Using a stable isotopic technique in which 42Ca was administered via a bolus injection, we measured endogenous fecal calcium excretion, Vf, in five healthy children, aged 3-14 years. The Vf averaged 1.4 +/- 0.4 mg/kg/day, and was lower than urinary Ca excretion (Vu) in four of the five children. These results for Vf are consistent with previously reported results for Vf in healthy adults and much lower than those reported in premature infants. These results may be useful in understanding developmental changes in Ca metabolism and in interpreting dual tracer Ca isotope studies in children.

Daily cortisol production rate in man determined by stable isotope dilution/mass spectrometry.

Growth retardation as well as the development of Cushingoid features in adrenally insufficient patients treated with the currently accepted replacement dose of cortisol (33-41 mumol/day.m2; 12-15 mg/m2.day) prompted us to reevaluate the cortisol production rate (FPR) in normal subjects and patients with Cushing's syndrome, using a recently developed thermospray liquid chromatography-mass spectrometry method. The stable isotope [9,12,12-2H3]cortisol was infused continuously for 31 h at about 5% of the anticipated FPR. Blood samples were obtained at 20-min intervals for 24 h, spun, and pooled in 4-h groups. Tracer dilution in plasma was determined by liquid chromatography/mass spectrometry. The method was validated with controlled infusions in 6 patients with adrenal insufficiency. Results from 12 normal volunteers revealed a FPR of 27.3 +/- 7.5 mumol/day (9.9 +/- 2.7 mg/day) or 15.7 mumol/day.m2; 5.7 mg/m2. day). A previously unreported circadian variation in FPR was observed. Patients with Cushing's syndrome demonstrated unequivocal elevation of FPR and cortisol concentration correlated during each sample period in normal volunteers, indicating that cortisol secretion, rather than metabolism, is mainly responsible for changes in plasma cortisol. Our data suggest that the FPR in normal subjects may be lower than previously believed.

Cortisol production rate in childhood and adolescence.

We studied the daily cortisol production rate in 33 normal children and adolescents, using a stable isotope-dilution technique employing high-performance liquid chromatography-mass spectrometry. Two indwelling intravenous catheters were inserted and tracer 9,12,12-2H3-cortisol (deuterated cortisol) was infused continuously for 30 hours. After 6 hours of tracer infusion to allow for equilibration, blood was obtained every 20 minutes for 24 hours. The mean (+/- SD) cortisol production rate was 9.5 +/- 2.5 mg/day (6.8 +/- 1.9 mg/m2/day). Cortisol production rate did not vary with sex or pubertal stage. These results suggest that the cortisol production rate in children and adolescents is significantly lower than previously estimated.

Cortisol production rates measured by liquid chromatography/mass spectrometry.

Cortisol production rates (FPRs) in physiologic and pathologic states in humans have been investigated over the past 30 years. However, there has been conflicting evidence concerning the validity of the currently accepted value of FPRs in humans (12 to 15 mg/m2/d) as determined by radiotracer methodology. The present study reviews previous methods proposed for the measurement of FPRs in humans and discusses the applications of the first method for the direct determination of 24-hour plasma FPRs during continuous administration of a stable isotope, using a thermospray high-pressure liquid chromatography-mass spectrometry technique. The technique is fast, sensitive, and, unlike gas chromatography-mass spectrometry methods, does not require derivatization, allowing on-line detection and quantification of plasma cortisol after a simple extraction procedure. The results of determination of plasma FPRs by stable tracer/mass spectrometry are directly in units of mass/time and, unlike radiotracer methods, are independent of any determination of volume of distribution or cortisol concentration. Our methodology offers distinct advantages over radiotracer techniques in simplicity and reliability since only single measurements of isotope ratios are required. The technique was validated in adrenalectomized patients. Circadian variations in daily FRPs were observed in normal volunteers, and, to date, results suggest a lower FRP in normal children and adults than previously believed.

Stable isotope dilution method using thermospray liquid chromatography/mass spectrometry for quantification of daily cortisol production in humans.

A thermospray high-performance liquid chromatography/mass spectrometry method for the separation and quantification of cortisol in human serum has been developed. The technique does not require derivatization, allows for both qualitative and quantitative determinations, provides increased specificity not available from conventional high-performance liquid chromatography, and has a detection limit of 5 pmol on-column. This isotope dilution mass spectrometry method, using d3-cortisol as an internal standard, allows precise determinations even at low isotopic ratios (2%-5 mole%). Evidence that this technique can be applied to the quantification of serum cortisol and to the determination of daily cortisol production in humans is presented.

Metabolic kinetics and quantitative analysis by isotope dilution thermospray LC/MS.

The use of thermospray LC/MS for quantitative analysis using isotope dilution is reviewed. Assays for acetylcholine, glucose, sorbitol, cortisol, testosterone and 1,25-(OH)2-vitamin D in biological fluids are discussed.