Comparison of hypothalamus-pituitary-adrenal axis suppression from superpotent topical steroids by standard endocrine function testing and gas chromatographic mass spectrometry.

We evaluated 38 males who had psoriasis vulgaris for evidence of hypothalamus-pituitary-adrenal axis suppression (HPAS) during treatment with superpotent topical glucocorticosteroids. All men were treated with 49 g per week of either Betamethasone Diproprionate in an optimized vehicle or Clobetasol Proprionate ointment. Three methods used to assess HPAS were compared. Classic 8 a.m. plasma cortisol measurements, urinary-free cortisol, and 17-hydroxycorticosteroid determinations and gas chromatograph-mass spectrometry (GCMS) quantitation of urinary cortisol metabolites were compared. Values for all methods were obtained just prior to therapy and at days 4, 7, 14, and 21 during therapy and at day 28 after treatment was stopped for 7 d. Plasma cortisol measurements correlated well with other measures of HPAS. GCMS determination of urinary cortisol metabolites was slightly more sensitive at detecting HPAS than the other two methods. Persistent HPAS after day 7 was only appreciated by GCMS. Urinary-free cortisol and 17-hydroxycortisol was the least sensitive of the three methods. Analysis of urinary cortisol metabolites by GCMS may be most useful in the monitoring of HPAS resulting from use of topical glucocorticosteroid preparations.

Placental transport of leucine, phenylalanine, glycine, and proline in intrauterine growth-restricted pregnancies.

L-[1-13C]Leucine, [1-13C]glycine, L-[1-13C]phenylalanine, and L-[1-13C]proline were infused as a bolus into the maternal circulation of seven appropriate for gestational age at 30.3 +/- 3.0 wk and 7 intrauterine growth-restricted pregnancies at 26.5 +/- 1.0 wk gestation to investigate placental transport in vivo. Umbilical venous samples were obtained at the time of in utero fetal blood sampling at 450 +/- 74 sec from the bolus injection. In normal pregnancies the fetal/maternal (F/M) enrichment ratios for leucine (0.76 +/- 0.06) and phenylalanine (0.77 +/- 0.06) were higher (P < 0.01) than the F/M ratios for glycine (0.18 +/- 0.04) and proline (0.22 +/- 0.02). This suggests that these two essential amino acids rapidly cross the placenta in vivo. Compared with the essentials, both glycine and proline had significantly lower F/M enrichment ratios, which were not different from each other. The results support the hypothesis that amino acids with high affinity for exchange transporters cross the placenta most rapidly. In intrauterine growth-restricted pregnancies, the F/M enrichment ratio was significantly lower (P < 0.01) for L-[1-13C]leucine (0.76 +/- 0.06 vs. 0.48 +/- 0.07) and for L-[1-13C]phenylalanine (0.77 +/- 0.06 vs. 0.46 +/- 0.07) compared with appropriate for gestational age pregnancies reflecting impaired transplacental flux. The F/M enrichment ratio did not differ for [1-13C]glycine (0.18 +/- 0.04 vs. 0.17 +/- 0.03), and L-[1-13C]proline (0.22 +/- 0.02 vs. 0.18 +/- 0.04).

Trolox C, a lipid-soluble membrane protective agent, attenuates myocardial injury from ischemia and reperfusion.

The lipophilic antioxidant Trolox C, a vitamin E analog, was administered to isolated, buffer-perfused rabbit hearts subjected to 25 min of global stop-flow ischemia and 30 min of reperfusion. In six hearts, Trolox C (200 microM) was infused for 15 min immediately prior to ischemia and for the first 15 min of reperfusion. Six control hearts received only vehicle. Gas chromatography analysis confirmed that effective myocardial levels of Trolox were attained. At 30 min reperfusion, the recovery of left ventricular developed pressure was 56 +/- 3% of baseline in control hearts versus 70 +/- 4% in Trolox-treated hearts (p < .01). There was also significant improvement in recovery of Trolox-treated hearts in diastolic pressure and both maximum and minimum values of the first derivative of left ventricular pressure (dP/dt). Creatine phosphokinase release into the coronary effluent at 30 min of reperfusion was 16.5 +/- 8.4 IU/min in untreated and 6.3 +/- 1.0 IU/min (p < .05) in Trolox-treated hearts. Thus Trolox C, a lipophilic antioxidant, attenuated myocardial injury during stop-flow ischemia and reperfusion.

The analysis of stable isotopes in urine to determine the fractional absorption of zinc.

We measured isotopic enrichment in urine after oral and intravenous administration of stable isotopes of zinc to determine fractional absorption (FA). 68Zn and 70Zn were administered orally and intravenously to four normal adults. Subsequently, urine and fecal samples were collected for 7 and 14 d, respectively, ashed, and passed through ion-exchange columns to separate zinc from other elements. Samples were analyzed by fast-atom-bombardment mass spectrometry. From 32 h onwards the enrichment of 68Zn and 70Zn in urine declined proportionately so that FA could be determined as follows: FA = enrichment (oral/iv) x dose (iv/oral). FA determinations from urine and feces (cumulative excretion) were, respectively, for subject ZK1, urine 0.79 +/- 0.03 and feces 0.70 +/- 0.01; ZK2, 0.79 +/- 0.05 and 0.69 +/- 0.02; ZK3, 0.26 +/- 0.01 and 0.25 +/- 0.01; and ZK4, 0.41 +/- 0.02 and 0.37 +/- 0.02. ZK1 and ZK2 received the oral isotope while fasting whereas ZK3 and ZK4 received the oral isotope with meals. FA of zinc can be determined by measurement of isotope enrichment in urine.

Influence of a meal and incremental doses of zinc on changes in zinc absorption.

The objective of this study was to determine the effect of increasing quantities of zinc, administered with and without a meal, on zinc absorption. Fractional absorption of incremental quantities of zinc in four normal adults was determined by measuring fecal excretion of unabsorbed isotope on 3 consecutive days by using three different stable isotopes of zinc (67Zn, 68Zn, and 70Zn). Isotopes were administered in the post-absorptive state and, on a subsequent occasion, with a standard zinc-free breakfast. In the postabsorptive state, fractional absorption was not affected by the quantity of zinc ingested until this exceeded 5 mg. When the zinc was administered with a meal, however, fractional absorption of 3 and 5 mg was less than for 1 mg. These results are compatible with the hypothesis that exogenous dietary zinc has to compete for absorption with endogenous zinc that has been secreted into the lumen of the gastrointestinal tract in response to a meal.

Protein balance in the first week of life in ventilated neonates receiving parenteral nutrition.

BACKGROUND: Protein intake is frequently delayed in ill neonates because of concerns about their ability to metabolize substrates. OBJECTIVE: We aimed to determine the factors affecting protein balance in ventilated, parenterally fed newborns during the first week of life. DESIGN: Leucine kinetic studies were performed in 19 neonates by using the [1-(13)C]leucine tracer technique after 24 h of a stable total parenteral nutrition (TPN) regimen. TPN intakes were prescribed by rotating attending physicians, enabling assessment of protein metabolism over a range of clinically used nutrient intakes. RESULTS: Mean (+/-SD) birth weight was 1.497 +/- 0.779 kg, gestational age at birth was 30.3 +/- 4.0 wk, and age at study was 3.9 +/- 1.4 d. Amino acid intakes (AAIs) ranged from 0.0 to 2.9 g x kg(-1) x d(-1). Based on leucine kinetic data, protein balance was calculated as the difference between protein synthesis and catabolism. By multiple regression analysis, AAI was the only predictor associated independently with protein balance (P < 0.01); energy intake, lipid intake, glucose intake, birth weight, and gestational age were not. Both leucine oxidation and nonoxidative leucine disposal rates were significantly correlated with leucine intake (P < 0.0005 and P < 0.01, respectively). Of the 12 infants with AAIs > 1 g x kg(-1) x d(-1), only 1 infant was significantly catabolic (protein balance <-1 g x kg(-1) x d(-1)). There was no evidence of protein intolerance as determined by elevated creatinine (69 +/- 31 micromol/L), plasma urea nitrogen (6.7 +/- 2.53 mmol/L), or metabolic acidosis (pH: 7.36 +/- 0.05). CONCLUSIONS: Ill neonates can achieve a positive protein balance in the first days of life without laboratory evidence of protein toxicity.

A multiple infusion start time (MIST) protocol for stable isotope studies of fetal blood.

A new approach utilizing multiple infusion start times for two stable isotopes of leucine was applied to seven pregnancies in order to assess equilibration times for isotopic studies when a single fetal blood sample is available. Two infusates, one containing l -[1-(13)C]-leucine and the other l -[5,5,5-D3]-leucine, were given as a primed constant infusion in the maternal circulation at fetal blood sampling (FBS). In five patients l -[1-(13)C]-leucine infusion was started at time zero (T(0)) whereas l -[5,5,5-D3]-leucine infusion began 30 min later, and both were continued until the umbilical sample was obtained at 149.7+/-8.8 min. In order to assure non-steady state conditions, in two patients the first infusion started at T(0)and the second 17 and 6 min before FBS was performed at 115 and 154 min, respectively. The fetal/maternal ratio for l -[5,5,5-D3]-leucine over the fetal/maternal ratio for l -[1-(13)C]-leucine was 0.98+/-0.03, indicating steady state conditions for both infusions for the first six patients. In the last patient the ratio was 0.51, indicative of non-steady state conditions for the shortest infusion time. Our results show that a single fetal sample can provide data for fetal amino acid enrichments reflecting multiple time points. Leucine steady state is achieved 20 min after a primed continuous infusion both in the maternal and fetal circulations.

An evaluation of fetal glucogenesis in intrauterine growth-retarded pregnancies.

The presence of fetal glucogenesis was evaluated in nine patients with pregnancies complicated by intrauterine growth retardation (IUGR) at the time of fetal blood sampling (FBS) between 29 and 35 weeks of pregnancy. Eight were singleton pregnancies and one was a twin pregnancy in which blood samples were obtained from both twins. A maternal primed-constant infusion of D(U-13C]glucose was performed, and the presence of fetal glucogenesis was assessed by a comparison of steady-state maternal and fetal glucose enrichments. No significant difference was present between maternal and fetal molar percent excess ([MPE] P = .97), and the mean fetal to maternal (F/M) MPE ratio (0.99 +/- 0.01) was not significantly different from 1 (P = .76). F/M MPE ratio was independent of the time of FBS and umbilical venous glucose and lactate concentrations. Thus fetal glucogenesis is not demonstrable in a group of fairly severe growth-retarded fetuses after an overnight fast with this relatively noninvasive approach.

Estrogens in intrahepatic cholestasis of pregnancy.

OBJECTIVE: To determine whether estrogen production and excretion are impaired in gravidas with intrahepatic cholestasis. METHODS: Plasma and urine samples were collected from 13 women from the United States and Chile at 35-38 weeks' gestation with mild (n = 9) or severe (n = 4) intrahepatic cholestasis of pregnancy. Urinary and plasma steroid levels from women with cholestasis were compared with levels from 27 normal pregnant women within the same gestational age range. Urinary concentrations of dehydroepiandrosterone (DHEA), estrone (E1), estradiol (E2), estriol (E3), estetrol, progesterone, and 16-hydroxy-pregnenolone were measured by gas chromatography mass spectrometry, and plasma concentrations of DHEA sulfate, progesterone, unconjugated E1, unconjugated E2, unconjugated E3, sulfated E3 derivatives, glucuronidated E3 derivatives, and total E3 were measured by radioimmunoassay. RESULTS: Compared with normal pregnant women, women with cholestasis had significantly lower plasma levels of estrogens and DHEA sulfate, the precursor to placental estrogen production synthesized by the fetal adrenal gland (Hotelling-Lawley trace = 0.81; F4,19 = 3.9; P = .02). The mean plasma DHEA sulfate, unconjugated E2, unconjugated E3, and total E3 concentrations were 0.271, 10.21, 9.80, and 99.53 ng/mL, respectively, in women with cholestasis compared with 0.802, 18.98, 16.28, and 145.07 ng/mL for controls. CONCLUSION: Fetal adrenal production of DHEA sulfate, and in response, downstream placental production of estrogens, was compromised by intrahepatic cholestasis of pregnancy.

Acylglycines. the gas chromatograph/mass spectrometric identification and interpretation of their spectra.

The gas chromatographic retention time (methylene units) and mass spectra of a series of acylglycine compounds as their trimethylsilyl (TMS) derivatives are presented. A general scheme for the interpretation of these compounds is developed from the mass spectra of known compounds and is shown to be in agreement with data derived from other sources (e.g. stable isotope and high resolution mass spectra). These data should provide information for the identification of these compounds in physiological fluid of both normal and diseased patients. In addition, the general interpretation scheme should provide the investigator with an approach to the interpretation of the spectra of unknown acylglycines.

Determination of steroid profiles in healthy and diseased states: identification and quantitation of a block of 17 alpha-hydroxylase.

The steroid metabolic profile on a patient with a suspected block in steroid biosynthesis was analyzed by gas chromatography and gas chromatography-mass spectrometry. The results of this work led to an interpretation of a block at the 17 alpha-hydroxylase step. Although the steroid metabolic profile was complex, we could not detect any steroids with a hydroxy moiety at position C-17. The use of gas chromatography-mass spectrometry, in this case, was the method of choice for a final diagnosis because of the confusion that resulted from other, more classical, forms of analysis. Data from our patient's sample are reported and compared to normals and to other cases where a block in 17 alpha-hydroxylase have been reported.

4-hydroxycyclohexane-1-carboxylic acid: an unusual compound isolated from the urine of children with suspected disorders of metabolism.

1. An unknown compound has been isolated in the acidic fraction of urine samples taken from several children suspected of having metabolic disorders. 2. This unknown has been characterized using a gas chromatograph/mass spectrometer/computer system. Both the high and low resolution mass spectra have been determined and a structure proposed. 3. Authentic samples were synthesized and compared to the unknown and a final proof of structure is presented. The compound, 4-hydroxycyclohexane-1-carboxylic acid, is suspected to come from a dietary source but the actual genesis will be determined in future work.

Methylmalonic/beta-hydroxy-n-valeric aciduria due to methylmalonyl-CoA mutase deficiency.

A patient with methylmalonic and beta-hydroxy-n-valeric aciduria, apparently due to deficiency of methylmalonyl-CoA mutase, is described. The excretion of beta-hydroxy-n-valerate did not parallel that of beta-hydroxypropionate and methylmalonate but was observed, together with beta-keto-n-valerate, only during ketosis. beta-Hydroxy-n-valerate excretion thus correlates primarily not with the pool size of propionyl-CoA but with that of acetyl-CoA, and may occur during ketosis in any disorder causing accumulation of propionyl-CoA.

The complete structure of the capsular polysaccharide from Streptococcus sanguis 34.

A complete structure for the capsular polysaccharide of Streptococcus sanguis 34, which is responsible for coaggregation of this bacterium with Actinomyces viscosus T14V, an important step in the formation of dental plaque, is proposed, based partly on the 1H-n.m.r. spectrum, which was assigned by 2-dimensional COSY, homonuclear Hartmann-Hahn spectroscopy, and nuclear Overhauser effects. A phosphoric diester linkage was identified from the 31P-n.m.r. spectrum, and the linkage was determined from long range 1H-31P correlation spectroscopy. The proposed structure is supported both by methylation analysis before and after dephosphorylation and by g.l.c.-m.s. of the phosphorylated monosaccharides as their trimethylsilyl derivatives, isolated by partial hydrolysis of the polysaccharide. The structure is composed of repeating linear hexasaccharide units joined by a phosphoric diester linkage, i.e., [----PO4(-)----6)-alpha-D-GalpNAc-(1----3)-beta-L-Rhap-(1----4)-be ta -D-Glcp- (1----6)-beta-D-Galf-(1----6)-beta-D-GalpNAc-(1----3)-alpha-D-Galp -(1----]n.

Structure of a new hexasaccharide from the coaggregation polysaccharide of Streptococcus sanguis 34.

The major constituent of a coaggregation polysaccharide from Streptococcus sanguis 34 is a hexasaccharide, isolated as the alditol. The proposed structure is alpha-D-GalpNAc-(1----3)-beta-L-Rhap-(1----4)-beta-D-Glcp-(1----6) -beta-D-Galf- (1----6)-beta-D-GalpNAc-(1----3)-D-Galol, based upon g.l.c.-m.s. of alditol acetates and partially methylated alditol acetates, f.a.b.-m.s., 1H-n.m.r. spectroscopy, g.l.c.-m.s. of trimethylsilylated (+)- and (-)-2-butyl glycosides, and cleavage by alpha-N-acetylgalactosaminidase. The structural deduction was facilitated by cleavage of the hexasaccharide at the furanoside linkage by 48% hydrogen fluoride, and reduction of the product, to yield alpha-D-GalpNAc-(1----3)-beta-L-Rhap-(1----4)-beta-D-Glcp-(1----6) -D-Galol.

Methandrostenolone metabolism in humans: potential problems associated with isolation and identification of metabolites.

Methandrostenolone dose (amount and duration) and methods of isolation from urine can influence the identification and quantitation of methandrostenolone metabolites. Long-term use of methandrostenolone at high dosages led to the appearance of unmetabolized drug in the urine and contributed to the identification of a previously unreported metabolite, 3 beta, 6 section, 17 beta-trihydroxy-17 alpha-methyl-5 section-1-androstene. Exposure of methandrostenolone in vitro to acid conditions induced a retropinacol rearrangement in the D-ring of the methandrostenolone molecule, causing the formation of 18-nor-17,17-dimethyl-1,4,13(14)-androstatrien-3-one in large amounts. The same acidic conditions led to the addition of a hydroxyl at the 6 position of the B-ring of either the retropinacol rearrangement products or native methandrostenolone resulting in the formation of 6 beta-hydroxy-18-nor-17,17-dimethyl-1,4,13(14)-androstatrien-3-one, 6 alpha- hydroxy-18-nor-17,17-dimethyl-1,4,13(14)-androstatrien, 6 beta-17 alpha-methyl-1,4-androstadien-3-one and 6 alpha,17 beta-dihydroxy-17 alpha-methyl-1,4-androstadien-3-one. Hydroxylation of native methandrostenolone at the 6 position also occurs endogenously. However, no evidence of an endogenous retropinacol rearrangement was found. Silylating agents alone can induce the formation of small amounts of 6 beta-17 beta-dihydroxy-17 alpha-methyl-1,4-androstadien-3-one. Discrepancies between previously published reports on methandrostenolone metabolism in man are discussed and compared with an animal model.

Inhibition of oxidative metabolism by propionic acid and its reversal by carnitine in isolated rat hepatocytes.

The present study was designed to study the interaction of propionic acid and carnitine on oxidative metabolism by isolated rat hepatocytes. Propionic acid (10 mM) inhibited hepatocyte oxidation of [1-14C]-pyruvate (10 mM) by 60%. This inhibition was not the result of substrate competition, as butyric acid had minimal effects on pyruvate oxidation. Carnitine had a small inhibitory effect on pyruvate oxidation in the hepatocyte system (210 +/- 19 and 184 +/- 18 nmol of pyruvate/60 min per mg of protein in the absence and presence of 10 mM-carnitine respectively; means +/- S.E.M., n = 10). However, in the presence of propionic acid (10 mM), carnitine (10 mM) increased the rate of pyruvate oxidation by 19%. Under conditions where carnitine partially reversed the inhibitory effect of propionic acid on pyruvate oxidation, formation of propionylcarnitine was documented by using fast-atom-bombardment mass spectroscopy. Propionic acid also inhibited oxidation of [1-14C]palmitic acid (0.8 mM) by hepatocytes isolated from fed rats. The degree of inhibition caused by propionic acid was decreased in the presence of 10 mM-carnitine (41% inhibition in the absence of carnitine, 22% inhibition in the presence of carnitine). Propionic acid did not inhibit [1-14C]palmitic acid oxidation by hepatocytes isolated from 48 h-starved rats. These results demonstrate that propionic acid interferes with oxidative metabolism in intact hepatocytes. Carnitine partially reverses the inhibition of pyruvate and palmitic acid oxidation by propionic acid, and this reversal is associated with increased propionylcarnitine formation. The present study provides a metabolic basis for the efficacy of carnitine in patients with abnormal organic acid accumulation, and the observation that such patients appear to have increased carnitine requirements ('carnitine insufficiency').

Chemoreceptively active compounds in secretions, excretions and tissue extracts of marine mammals.

Semen, prostate glandular extract, perianal glandular secretion, urine, feces and blood from Atlantic bottlenose dolphins, Tursiops truncatus, were analyzed chemically by gas chromatography. A fecal sample from a California sea lion, Zalophus californianus, was analyzed similarly. Acids, esters, amino acids, amines, steroids, esters, sugars and alcohols were among the compounds revealed in the analyses. Twenty-two identified compounds can be detected gustatorily in aqueous solutions of sufficient concentration by humans. The chemical compositions of samples from Tursiops are similar to those from humans and other mammals.