Calcium and protein kinetics in prepubertal boys. Positive effects of testosterone.

We investigated the effects of 4-6-wk administration of testosterone on calcium and protein metabolism in six healthy prepubertal short boys (mean age +/- SE = 12.9 +/- 0.6 yr). At baseline, subjects received a 4-h infusion of L-[1-13C]leucine and L-[2-15N]glutamine, and were given 42Ca intravenously, and 44Ca PO. Testosterone enanthate (approximately 3 mg/kg) was given I.M. 2 wk apart (two doses n = 5, three doses n = 1), and the study was repeated 4-5 d after the last injection. After testosterone therapy, there were significant increases in serum testosterone and mean peak and total growth hormone concentrations. Net calcium absorption (Va) and retention (Vbal) also increased (Va 13.3 +/- 2.3 vs 21.5 +/- 2.3; mg.kg-1.d-1, Vbal 8.0 +/- 2.1 vs 16.6 +/- 2.5, mg.kg-1.d-1, P < .05 both), as well as Ca's net forward flow into bone and total exchangeable pool (16 and 20%, respectively). The rate of appearance of leucine (an indicator of proteolysis) increased by 17.6 +/- 5.9%, P = 0.036. Leucine oxidation decreased by 48.6 +/- 8.0%, P = 0.004; thus, nonoxidative leucine disappearance, which estimates protein synthesis, increased significantly by 34.4 +/- 7.7%, P = 0.009. Glutamine's rate of appearance also increased (+32%), mostly through enhanced glutamine de novo synthesis (+42%). In conclusion, short term testosterone administration significantly increases calcium's retention and net forward flow into bone in prepubertal humans, as well as whole body estimates of protein and calcium anabolism. These effects may represent a pure androgen effect, an amplification of growth hormone's action or some combination of these factors.

One-day test using stable isotopes to measure true fractional calcium absorption.

True calcium absorption can only be measured by the time-consuming and expensive metabolic balance method and is not predicted well by measuring the fractional absorption (FA) of radiocalcium from a fixed calcium carrier. We describe here a 1 day method for measuring the actual fraction of calcium absorbed from the habitual diet (true fractional calcium absorption, TFCA) using stable isotopes of calcium. Oral and intravenous isotopes were administered with each of the three daily meals, and then the ratio of the two isotopes in the urine was measured by mass spectroscopy. In 12 subjects, TFCA determined from stable Ca correlated well with TFCA measured by the balance method (r = 0.71, P less than 0.01), and the mean values were not different (0.26 and 0.26). In contrast, no significant correlation was found between FA and TFCA. The weak relationship between FA and TFCA underscores the importance of tracing dietary calcium rather than a fixed calcium carrier in tests of calcium absorption. Using the new method, TFCA was inversely related to dietary calcium (r = -0.45, P less than 0.05), demonstrating that it could detect physiological changes in calcium absorption. Thus, this test has two important advantages: (1) it provides a simple way to measure TFCA and true fractional calcium absorption (the product of TFCA and dietary calcium), the physiologically relevant variables, and (2) because there is no radiation exposure, the test can be used in pregnant women and children, the isotopes can be prepared in advance, and several isotopes can be used simultaneously.

Demonstration of a difference in urinary calcium, not calcium absorption, in black and white adolescents.

Bone mineral density (BMD) of the forearm, lumbar spine, and femoral neck is greater in black than in white children. Studies were performed to determine whether differences in intestinal absorption of calcium or urinary calcium or both account for an assumed more positive calcium balance and greater bone mass in black children. Normal black and white boys and girls were admitted to a metabolic ward and given a constant daily diet containing 1000 mg calcium, 60% as calcium carbonate, for 2 1/2 days (study I) or 3 1/2 days (study II). Fasting blood and 24 h urine collections were obtained, and in study II, unidirectional fractional absorption of calcium (alpha) was determined with stable isotopes of calcium. It was found that (1) serum 25-hydroxyvitamin D (25-OHD) and urinary calcium were lower and serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] was higher in black than in white children, and (2) alpha was higher in boys than in girls with no racial difference, and (3) there were significant positive correlations between alpha and urinary calcium in the blacks and in the black and white children together. It is concluded that (1) alpha is higher in boys than in girls and (2) a lower urinary calcium, not increased intestinal absorption of calcium, is the means for a more positive calcium balance in blacks that accounts for the racial difference in BMD.

Interrelationship among vitamin D metabolism, true calcium absorption, parathyroid function, and age in women: evidence of an age-related intestinal resistance to 1,25-dihydroxyvitamin D action.

We studied the mechanism of impaired calcium absorption with aging in 51 healthy women whose ages ranged from 26 to 88 years. Serum concentrations of 1,25-dihydroxyvitamin D [1,25-(OH)2D, mean of four measurements per subject] increased with age by 22% (P less than 0.05) but, by split-point analysis, plateaued or decreased slightly after age 65. In a subset of 20 subjects, [3H]1,25-(OH)2D3 kinetic analysis showed that this increase with age resulted from both increased production and decreased metabolic clearance of 1,25-(OH)2D. Despite the increase in serum 1,25-(OH)2D concentration, true calcium absorption did not change with age. The expected inverse correlation between true fractional calcium absorption and dietary calcium intake, however, was easily demonstrated (r = 0.66, P less than 0.001). Serum intact parathyroid hormone (PTH) increased with age by 35% (P less than 0.02) and serum bone gla protein (BGP, osteocalcin) increased by 47% (P less than 0.001); the increases in serum PTH and serum BGP were directly correlated (r = 0.32, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Profound hypogonadism has significant negative effects on calcium balance in males: a calcium kinetic study.

The impact of estrogen deficiency on bone has been extensively studied in the female; however, the effects of androgen deficiency on calcium fluxes in males have been less well characterized. We investigated the effect of short-term, severe androgen deficiency on measures of calcium absorption and kinetics as well as on markers of bone turnover in males. To accomplish this, 11 healthy male volunteers were recruited (mean age 23.3 +/- 0.5 years [SEM], body mass index 25.3 +/- 0.8 kg/m2). They consumed a weight maintenance diet for at least 3 days prior to admission to our Research Unit, with a calcium intake of approximately 1200 mg/day. At baseline (D1), subjects received 42Ca intravenously as well as 44Ca PO mixed with milk or juice. A 29-h urine collection was begun and blood samples collected at frequent intervals for the measurement of the isotopic enrichment of 42Ca and 44Ca using thermal ionization mass spectrometry. Twice daily urine samples were collected for 5 days after the administration of the isotopes. A gonadotropin-releasing hormone agonist (Lupron) was given after D1, again 3 weeks later, and studies repeated identically 4 weeks (D2, n = 6) and 10 weeks from baseline (D3, n = 7) (two subjects completed three studies). Testosterone concentrations were markedly suppressed on both D2 and D3 (-95%, p < 0.006), whereas there were no detectable changes in growth hormone and insulin-like growth factor-1 concentrations. Urinary calcium excretion increased significantly after 4 weeks (43%, p = 0.0007) and 10 weeks (73%, p = 0.003) of sustained hypogonadism. Using a multicompartmental kinetic model, the contribution of oral calcium to the urinary losses was decreased by D3 (-41%, p = 0.01), yet the contribution of bone calcium to urine losses increased by 10 weeks (+11%, p = 0.01). There was a 21% decrease in bone calcium deposition (Vo+) by D3 (p < 0.05) with no significant change in bone resorption rates (Vo-). There was a significant correlation between the decrease in testosterone concentration and the increase in urinary calcium excretion, especially at 10 weeks (R2 = 0.84, p = 0.004). These kinetic changes were accompanied by a decrease in osteocalcin concentrations on D2, with improvements by D3. Urinary N telopeptide, a measure of bone resorption, also increased during the studies. In summary, profound hypogonadism in young males is associated with marked increases in urinary calcium losses, with a greater contribution of bone calcium to those losses and decreased kinetic markers of bone calcium deposition. We conclude that even short-term, severe deficiency in gonadal steroids can have profound negative effects on calcium and bone metabolism in males.

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.

Relationship between balance and dual tracer isotopic measurements of calcium absorption and excretion.

Calcium (Ca) absorption was measured by the balance method and the dual tracer isotopic method in 462 studies of adult women. Results for both fractional absorption and net Ca balance were similar using the 2 methods. Slightly higher values (mean difference, 0.031 +/- 0.092) were found for fractional absorption measured by the balance method than by the isotopic method. No differences in fractional absorption between the methods were seen at a fractional absorption less than 25%. Variability in the data was greater from the balance than the isotopic method. Endogenous fecal Ca excretion was directly related to Ca intake, with an estimated value of 70-80 mg/day for a Ca intake less than 200 mg/day. These findings support the usefulness and accuracy of isotope-based measures of mineral absorption. No evidence is found from these or previous data to suggest that the process of isotopic equilibration falsely increases estimates of absorption or endogenous excretion in tracer studies. Isotopic techniques allow studies of calcium absorption in diverse populations and evaluation of unique aspects of mineral metabolism not accessible through other techniques.

Relationship of intestinal calcium absorption to 1,25-dihydroxyvitamin D [1,25(OH)2D] levels in young versus elderly women: evidence for age-related intestinal resistance to 1,25(OH)2D action.

Intestinal calcium absorption decreases with aging, but it is unclear whether this is attributable to an age-related intestinal resistance to 1,25-dihydroxyvitamin D [1,25(OH)2D] action. Thus, we assessed the in vivo dose response of active intestinal calcium absorption to a broad range of circulating 1,25(OH)2D levels in elderly [age (mean +/- SD), 72.5+/-3.0 yr] vs. young women (age, 28.7+/-5.3 yr; n = 20 per group), who were stratified into 5 subgroups: group 1 was given a high calcium intake of 75 mmol/day, suppressing 1,25(OH)2D levels; group 2 was given a normal calcium diet of 15-30 mmol/day, representing basal 1,25(OH)2D levels; group 3 was given a low-calcium diet of 5 mmol/day to stimulate endogenous 1,25(OH)2D production; group 4 was given the low-calcium diet plus 1 microg/day 1,25(OH)2D; and group 5 was given a low-calcium diet plus 2 microg/day 1,25(OH)2D. After 7 days of diet and/or 1,25(OH)2D treatment, fasting fractional calcium absorption (FCA) was assessed by a double-tracer method using stable calcium isotopes. Serum 1,25(OH)2D and vitamin D-binding protein levels were measured concurrently, and the free 1,25(OH)2D index [molar ratio of 1,25(OH)2D to DBP] was calculated. FCA was significantly correlated with the free 1,25(OH)2D index in the young (R = 0.63, P = 0.003) but not in the elderly women (R = 0.27, P = 0.25). Moreover, the slope of the relationship between FCA and free 1,25(OH)2D index (representing intestinal sensitivity to 1,25(OH)2D) was significantly greater in the young (compared with the elderly) women [mean +/- SEM, 0.15+/-0.04 (young) vs. 0.03+/-0.02, elderly, P = 0.03]. Thus, using an experimental design that allowed us to assess FCA over a wide range of 1,25(OH)2D levels, we demonstrate that elderly women have a resistance to 1,25(OH)2D action that may contribute to their negative calcium balance, secondary hyperparathyroidism, and bone loss.

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.

Absent intestinal response to calciferols in hereditary resistance to 1,25-dihydroxyvitamin D: documentation and effective therapy with high dose intravenous calcium infusions.

We describe a patient with an absent intestinal response to 1,25-dihydroxyvitamin D [1,25-(OH)2D] and the beneficial effects of treatment with high dose iv calcium infusion. The patient presented with severe rickets despite therapy with extraordinarily high doses of 1 alpha-hydroxyvitamin D3 or 1,25-(OH)2D3. Unidirectional intestinal fractional calcium absorption when he was not treated with any calciferol was 14% (normal, 20-70%), as measured with stable calcium isotopes; no increase in calcium absorption occurred when serum 1,25-(OH)2D levels were more than 50-fold elevated. Cultured skin fibroblasts contained no detectable 25-hydroxyvitamin D3-24-hydroxylase activity in response to 1,25-(OH)2D3 (10(-9)-10(-6) mol/L). High dose iv calcium infusions and oral phosphorus supplementation for 135 days improved or normalized biochemical parameters and resulted in radiographic healing of the rachitic lesions. We conclude that 1) this patient had no response to 1,25-(OH)2D3 in vivo and in vitro; 2) long term parenteral calcium infusions were effective therapy in managing the patient's severe resistance to 1,25-(OH)2D; and 3) stable calcium isotopes are useful for measuring low levels of fractional calcium absorption.

Insulin-like growth factor I and growth hormone (GH) treatment in GH-deficient humans: differential effects on protein, glucose, lipid, and calcium metabolism.

We examined the effects of recombinant human (rh) insulin-like growth factor I (IGF-I) vs. rhGH in a variety of metabolic paths in a group of eight severely GH-deficient young adults using an array of contemporary tools. Protein, glucose, and calcium metabolism were studied using stable labeled tracer infusions of L-[1-13C]leucine, [6,6-2H2]glucose, and 42Ca and 44Ca; substrate oxidation rates were assessed using indirect calorimetry; muscle strength was determined by isokinetic and isometric dynamometry of the anterior quadriceps, as well as growth factors, hormones, glucose, and lipid concentrations in plasma before and after 8 weeks of rhIGF-I (60 microg/kg, sc, twice daily), followed by 4 weeks of washout, then 8 weeks ofrhGH (12.5 microg/kg-day, sc); the treatment order was randomized. In the doses administered, rhIGF-I and rhGH both increased fat-free mass and decreased the percent fat mass, with a more robust decrease in the percent fat mass after rhGH; both were associated with an increase in whole body protein synthesis rates and a decrease in protein oxidation. Neither hormone affected isokinetic or isometric measures of skeletal muscle strength. However, rhGH was more potent than rhIGF-I at increasing lipid oxidation rates and improving plasma lipid profiles. Both hormones increased hepatic glucose output, but rhGH treatment was also associated with decreased carbohydrate oxidation and increased glucose and insulin concentrations, indicating subtle insulin resistance. Neither hormone significantly affected bone calcium fluxes, supporting the concept that these hormones, by themselves, are not pivotal in bone calcium metabolism. In conclusion, rhIGF-I and rhGH share common effects on protein, muscle, and calcium metabolism, yet have divergent effects on lipid and carbohydrate metabolism in the GH-deficient state. These differences may allow for better selection of treatment modalities depending on the choice of desired effects in hypopituitarism.

Intestinal calcium absorption of women during lactation and after weaning.

We investigated whether intestinal calcium absorption and serum 1,25-dihydroxycholecalciferol (calcitriol) concentrations are higher in women during lactation and after weaning to compensate for calcium lost in breast milk. Measurements were obtained at 4.6 mo postpartum in 24 lactating women and 24 nonlactating women, at 9.6 mo postpartum in 24 lactating women (2.6 mo after complete weaning) and 24 nonlactating women. One-half of the women in each group were randomly assigned to receive 1 g supplemental Ca/d as calcium carbonate. Fractional calcium absorption was measured by using stable isotopic tracers 42Ca and 44Ca. Fractional absorption was 0.32+/-0.02 (+/-SEM) in both lactating and nonlactating women, but was higher in lactating women after weaning (0.37+/-0.02) compared with nonlactating postpartum control subjects (0.31+/-0.02). These effects were independent of calcium intake. Changes in serum calcitriol paralleled changes in fractional absorption. There were no differences in calcitriol concentrations between lactating and nonlactating women, but calcitriol was greater in women after weaning compared with postpartum control subjects. Lactating women who had resumed menses had higher fractional absorption and serum calcitriol than did lactating women who had not. Serum calcium and phosphorus concentrations were greater in lactating compared with nonlactating women; there were no differences between groups after weaning. We conclude that lactation stimulates increases in fractional calcium absorption and serum calcitriol, but the responses are only apparent after weaning or the resumption of menses.

Calcium kinetics in lactating women with low and high calcium intakes.

Absorption of calcium and its mobilization from bone during lactation are important for delivery of calcium to breast-feeding infants; whether calcium intake offsets bone resorption is not known. We hypothesized that calcium absorption is increased in lactation and greater in women on low calcium diets, resulting in similar rates of bone resorption and accretion. Calcium absorption and kinetic indexes were calculated by using two stable isotopic tracers in 8 women; 6 were studied both during lactation and nonlactation. Women consumed low calcium diets, with half receiving supplemental calcium. Intestinal absorption was related to serum 1,25-dihydroxyvitamin D and did not increase during lactation. Despite decreased urinary calcium excretion during lactation, especially in women with low calcium intake, net balance tended to be lower during lactation. Mean residence time decreased and bone resorption exceeded accretion in almost all lactating women. Calcium need for milk production appears to be met by decreased urinary excretion and increased bone resorption, and not by increased intestinal absorption.

Effect of growth hormone treatment on calcium kinetics in patients with osteogenesis imperfecta type III and IV.

Using a dual stable isotope technique, the effect of growth hormone (GH) on whole body calcium (Ca) metabolism was studied in children (ages 5-14 years) with type III (n = 9) and IV (n = 8) osteogenesis imperfecta. Each subject was studied twice: at baseline and following a GH (0.1-0.2 U/kg per day) treatment period of 1-1.5 years. Subjects were given 42Ca intravenously and 44Ca orally. The sera and urine 42Ca and 44Ca isotopic enrichments were followed over 7 days using thermal ionization mass spectrometry. The SAAM program was used to fit a three-compartment model to the tracer data. No significant differences were observed between: (1) children with type III and IV disease; or (2) baseline studies of boys and girls within each disease type. However, GH treatment significantly increased: (1) the exchangeable calcium pool (EP) in type III patients (2086 vs. 4422 mg/day, p = 0.02); and (2) the parameter associated with bone calcium accretion in type IV patients (Vo+: 973 vs. 1560 mg/day,p = 0.03) with boys responding with a significantly greater increase than girls (p = 0.008). Although not statistically significant, a trend toward an increase in Vo+ in type III patients and in EP in type IV was observed following treatment. Our observations imply that more Ca was available for bone mineralization following GH treatment in these subjects.

A three-hour measurement to evaluate bone calcium turnover.

It is well established that short-term clearance of an intravenous calcium load in vivo reflects bone uptake. Using results from isotope-dilution experiments with 42Ca, a 3-h test has been developed to measure a quantity, gamma, related to bone accretion. This test is proposed as a useful, clinically applicable measure of bone status. For early times, t, after a bolus of 42Ca, plasma tracer dilution was well approximated by t-gamma, where gamma is related to the fractional rate of loss of tracer, q, from blood into bone (1/q)(dq/dt) = -gamma/t). Gamma was evaluated from kinetic measurements on 91 normal female children, adolescents, and adult women in the age range 4-50 years. For t < or = 3 h, all clearance curves were well fit by a power function. Gamma was found to vary from 0.244 +/- 0.031 for adult premenopausal women (N = 22) to 0.392 +/- 0.056 for prepubertal children (N = 29). Using the Spearman rank-order correlation test, gamma was correlated with bone accretion measured from classic calcium kinetic studies with a correlation coefficient of 0.721, significant at p < 0.005. In those cases in which accretion and resorption remain tightly linked, gamma also provides information on the state of calcium loss from bone. Gamma was evaluated in 14 subjects with bone disease characterised by increased resorption (osteoporosis, Paget's disease) and in 27 subjects with decreased accretion (osteogenesis imperfecta, types I, III, IV; steroid-treated juvenile dermatomyositis). All subjects with Paget's disease and with osteoporosis showed increased gamma, consistent with high bone turnover. The osteoporotic patients furthermore exhibited gamma increasing monotonically by approximately 1% per year after age 55.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen therapy enhances calcium absorption and retention and diminishes bone turnover in young girls with Turner's syndrome: a calcium kinetic study.

Using stable tracers of calcium, we have previously shown a significant increase in calcium absorption and retention in prepubertal boys treated with exogenous testosterone. To investigate the effects of estrogen replacement on measures of calcium absorption, retention, and bone turnover, we studied a group of seven hypogonadal girls with Turner's syndrome (mean +/- SE age, 12.5 +/- 0.7 years). At baseline, 42Ca intravenously (IV) and 44Ca orally were administered, and blood and urine samples were collected for approximately 130 hours. Estrogen therapy was begun as oral ethinyl estradiol (4 or 20 micrograms/d) or intramuscular depot estradiol given over 4 weeks, after which an identical study was repeated. Analysis of calcium enrichment in blood and urine was performed using mass spectrometry methods. After estrogen therapy, there was a significant increase in calcium absorption ([Va] P = .03) and total calcium retention ([Vbal] P = .04), similar to the effects of testosterone in boys. Bone accretion (Vo+) decreased after estrogen therapy (P = .004), as did resorption ([Vo-] P = .004). The overall rate of whole-body calcium turnover (Vt) was significantly decreased after estrogen administration (P = .04). These findings were opposite of those observed in prepubertal boys treated with testosterone. The contribution of bone resorption to whole-body turnover (E) also decreased after estrogen therapy (P = .05). These changes were associated with increased levels of 1,25-dihydroxyvitamin D after therapy with estrogens (P = .05). We conclude that estrogen supplementation is significantly anabolic for calcium metabolism by markedly increasing calcium absorption and retention and diminishing the estimated whole-body calcium turnover in girls with severe hypogonadism and Turner's syndrome. Further studies assessing the dietary calcium and/or vitamin D intake and bone mineral density of hypogonadal girls whose estrogen replacement is intentionally delayed will further define the need for calcium or vitamin D supplements in the peripubertal years in this condition.

Pathophysiology of hypocalciuria in preeclampsia: measurement of intestinal calcium absorption.

OBJECTIVE: To analyze calcium absorption using stable isotopes in patients with preeclampsia and in normotensive controls. METHODS: Fifteen pregnant subjects were studied: eight with preeclampsia (hypertension and proteinuria) and seven normotensive controls. All patients were ingesting their normal diet. The subjects received two stable calcium isotopic tracers. An oral tracer (44Ca, 0.0124 mmol/kg) was given with milk, while an intravenous tracer (42Ca, 0.00249 mmol/kg) was infused over 7-10 minutes. Calcium concentration was determined by atomic absorption spectrophotometry, and isotope ratios by thermal ionization mass spectrometry from pooled 24-hour urine samples. RESULTS: No difference was noted in fractional intestinal absorption between preeclamptic subjects (0.282 +/- 0.051) and normotensive controls (0.306 +/- 0.079) (P = .49). However, the fraction of dietary calcium appearing in the urine differed significantly (0.06 for preeclamptic subjects and 0.087 for normotensive controls; P = .008). CONCLUSIONS: Despite the indirect evidence of others, calcium absorption does not appear to be impaired in patients with preeclampsia. The retention site of the unexcreted calcium is unidentified.

Valproylcarnitine: a novel drug metabolite identified by fast atom bombardment and thermospray liquid chromatography-mass spectrometry.

Urine samples from three children at different stages of chronic valproate therapy were partially purified using a cation exchange column. A signal consistent with either valproylcarnitine or octanoylcarnitine was observed in one of these extracts by direct fast atom bombardment-mass spectrometry analysis. These isomeric acylcarnitines were synthesized, separated and characterized by thermospray high performance liquid chromatography-mass spectrometry. This new technique was then employed to positively identify intact valproyl-carnitine in the patients' urine samples. The implications of this finding with regard to a mechanism to account for carnitine deficiency in patients receiving valproate are discussed.

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.

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.