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Amiodarone has been demonstrated to form a cytochrome P-450Fe(II):metabolite complex. The administration of other agents which form this type of complex, such as troleandomycin, has been shown to deplete hepatic glutathione content. Depletion of glutathione will result in an increased synthesis of glutathione and an increased utilization of cysteine. Since inorganic sulfate and glutathione share cysteine as a common precursor, we postulated that amiodarone pretreatment may reduce the sulfation of drugs. To test this hypothesis, the effect of amiodarone pretreatment on acetaminophen disposition was examined in the rat. Acetaminophen (150 mg/kg) was administered to rats pretreated with amiodarone hydrochloride (100 mg/kg/d) or diluent for 5 d. There were no significant differences in the urinary recovery of acetaminophen sulfate or the partial clearance of acetaminophen to the sulfate metabolite between control and amiodarone-pretreated animals. There was a trend toward an increased urinary recovery of acetaminophen glucuronide in animals pretreated with amiodarone, but this did not reach statistical significance. Amiodarone pretreatment had no effect on the renal clearances of acetaminophen or its metabolites. These results suggest that amiodarone pretreatment does not alter the sulfation of drugs and that the formation of an amiodarone P-450Fe(II):metabolite complex is quantitatively insignificant.

In our attempt to design a method which would be more reliable than dietary survey interviews when estimating salt and protein intakes in a population survey, a urinalysis-related study was done on volunteers. Urinary sodium (Na), urea nitrogen (UN) and inorganic sulfate (SO4), all indices of dietary salt, protein and sulfoamino acids, respectively, were confirmed to reflect the nutritional condition. Interaction between salt and protein was not observed at least at the dietary levels used in the present study (for salt, 0.33 and 0.1 g/kg body weight/day; for protein, 1.6 and 0.7 g/kg body weight/day). Excretion of components was delayed several days or more after dietary ingestion, and nutritional estimation by urinalysis, therefore, may not be so much affected by daily variables in the diet intake. Further, partial urine samples proved to have a highly significant correlation with 24-hour (hr) urine, as for urinary Na, potassium (K), SO4, UN and their creatinine (Cr) ratios, thus indicating the availability of partial urine samples as substitutes for 24-hr urine specimens. Thus, urinalysis is a more readily facilitated, more scientific and more quantitative method for epidemiological nutritional surveys.

To investigate whether melatonin (aMT) can be metabolized to N-acetyl serotonin (NAS), a low dose of deuterated aMT was administered to four normal subjects, and their urine samples were analyzed for the presence of deuterated NAS and deuterated 6-hydroxymelatonin (6-HaMT). In one set of experiments, the urine samples were subjected to column chromatography to separate the glucuronide and sulfate conjugates for independent analysis. In another, an internal standard (NAS-sulfate) was used for quantification and total conjugate analysis. Measurement was by gas chromatography-mass spectrometry, and the molecular ions of deuterated and nondeuterated NAS and 6-HaMT were monitored. Deuterated aMT was metabolized to deuterated NAS and deuterated 6-HaMT. The proportion of NAS was less in the sulfate than in the glucuronide conjugates and, overall, represented 15% of the total. Since demethylation is not a pathway that occurs with other pineal methoxyindoles, even at a much larger dose, it seems to be a significant finding with regard to aMT. Thus, it may be important to elucidate the differential metabolism of aMT at different time points and in different age groups.

The metabolism of iv-administered 14C-cortisol (F) by pregnant baboons (107, 124 and 150 days gestation) was compared with that previously reported for nonpregnant animals and with that of animals examined 6-18 h after spontaneous vaginal delivery (178 plus or minus 4 days). Unconjugated, glucuronoside (beta-glucuronidase) and sulfate (H2SO4-ethyl acetate) fractions were extracted with ethyl acetate from urine containing more than 80% of injected 14C. Metabolites of interest were isolated by paper partition chromatography and purified by crystallization and derivative formation. Compared with nonpregnant animals, the following changes (P less than 0.05) were observed in pregnancy: (1) an increase in the percent urinary 14C in the unconjugated fraction and a decrease in the proportion of 14C appearing in the glucuronoside fraction; (2) an increase in excretion of metabolites more polar than the cortols; (3) a decrease in excretion of metabolites less polar than cortisone in the glucuronoside fraction; (4) an increase in unconjugated F excretion. Production rate of F (11.9 plus or minus 0.7 mg/day) estimated by isotope dilution and percent urinary 14C in tetrahydrocortisol and tetrahydrocortisone from the glucuronoside fraction were as in nonpregnant animals. With the exception of an increase in F production (22.7 plus or minus 0.8 mg/day), presumably the result of the stress of labor, F metabolism in the immediate postpartum period was strikingly similar to timals indicates that changes in the mother alone can account for the altered metabolic disposition of F in pregnancy and suggests that the fetus takes little part in metabolism of maternal circulating F.

A direct high-performance liquid chromatographic (HPLC) assay was developed to simultaneously quantitate diflunisal and its three known metabolites (i.e., the phenolic and acyl glucuronides and the sulfate conjugate) in human urine. Chromatographically pure standards of the diflunisal conjugates were isolated from urine of volunteers following ingestion of multiple doses of diflunisal (500 mg twice daily). Diflunisal, its three conjugates, and an internal standard (naproxen) were separated on a reversed-phase column using gradient elution. The column eluate was monitored fluorometrically (excitation: 258 nm; emission: 428 nm). Urine samples were diluted with phosphate buffer (pH 5.75) and injected onto the column. The limit of detection was approximately 1 microgram/mL for each conjugate and 0.1 microgram/mL for diflunisal. Due to the presence in most urine samples of significant concentrations of rearrangement products of the biosynthetic 1-O-acyl glucuronide of diflunisal, the acyl glucuronide could not be reliably quantitated by direct injection of diluted urine samples. Instead, diflunisal acyl glucuronide was quantitated indirectly following alkaline hydrolysis of the urine samples. The method has been successfully used to investigate the dose-dependent glucuronidation and sulfation of diflunisal in humans.

The excretion rates and precursors of the 3-sulfate and glucuronide conjugates of 16 beta-hydroxydehydroepiandrosterone (16 beta-OH DHEA) were measured in normotensive controls and in patients with normal and low renin essential hypertension. The hypertensive subjects, and to the greatest degree those of the low renin subgroup, excreted increased amounts of 16 beta-OH DHEA sulfate and glucuronide and lesser amounts of DHEA sulfate and glucuronide than the controls. The major precursor of the urinary 16 beta-OH DHEA sulfate in the hypertensives was circulating DHEA sulfate, whereas the major precursors of 16 beta-OH DHEA glucuronide were DHEA, DHEA sulfate and 17-OH pregnenolone, as determined from their specific activities. Furthermore, both subgroups of hypertensives had similarly elevated DHEA and DHEA sulfate secretory rates compared to the controls. The stimulus to this increased peripheral conversion of circulating 17-OH pregnenolone, DHEA and DHEA sulfate into 16 beta-OH DHEA conjugates in essential hypertension, especially of the low renin type, is unknown.

This is a new method for isolating and concentrating urinary organic acids before gas chromatography--mass spectrometry. Sulfate and phosphate anions are removed by precipitation with Ba(OH)2, and the urine pH is adjusted to 8-8.5. The sample is then applied onto small, disposable, strong-anion-exchange columns. Neutral and basic compounds are washed out with water, which is then removed by centrifugation and by rinsing the columns with methanol and diethyl ether. The organic acids are eluted with a 4/1 (by vol) mixture of an organic solvent--either n-butanol or ethyl acetate--and formic acid containing HSO4- (0.1 mol/L) as a highly selective counter-ion, and finally with methanol alone. Sulfate ions are retained, and the eluate is evaporated before trimethylsilyl derivatization. Analytical recoveries (about 100%) of organic acids compare favorably with those obtained with solvent extraction. This convenient procedure is selective and reproducible and is a suitable alternative to the more cumbersome diethylaminoethyl-Sephadex extraction method.

The urinary excretion of sulfur-containing compounds was studied in 11 burned patients during the first 10 days after injury. They were all given carbohydrate and fat by parenteral nutrition. Two patients also received large amounts of amino acids including methionine, whereas five patients were given amino acids including moderate amounts of methionine and cysteine. A sustained high excretion of mercaptolactate occurred in most patients. The excretion was not influenced by the type of parenteral nutrition, but related to the area of burned skin. A normal excretion of methionine, cyst(e)ine, inorganic sulfate, taurine, mercaptoacetate, thiosulfate, and thiocyanate was found at the end of the observation period in patients without amino acid therapy. Increased amounts of inorganic sulfate and methionine were excreted in patients receiving high doses of methionine, whereas an increased cyst(e)ine and taurine excretion was the most prominent finding in patients receiving moderate amounts of methionine and cysteine. These results indicate that burned patients have an unimpaired ability to metabolize sulfur amino acids. Patients not receiving amino acids or receiving moderate amounts of cysteine and methionine showed a negative sulfur balance, whereas patients given high doses of methionine maintained sulfur balance at near-zero levels. However, since signs of hepatic dysfunction appeared in the latter group, parenteral nutrition with amino acid mixtures containing high amounts of methionine is not recommended during the catabolic phase for burned patients.

A case of combined deficiency of sulphite-oxidase and xanthine-oxidase with a defect of the molybdenum cofactor, which is vital to the activity of sulphite-, xanthine- and aldehyde-oxidase, is reported here. Seven cases of combined deficiencies have been described with regard to both clinical and laboratory findings. The clinical, laboratory and anatomo-pathological features and, in particular, the central nervous system lesions of the present case correspond exactly to those in the case described Rosenblum in which an isolated deficiency in sulphite-oxidase was present. As the cerebral alterations in the present case are comparable to those described in Rosenblum's case, they probably result from the defect in sulphite-oxidase activity.

General medical examinations were performed in two rural areas of Shimane Prefecture in Japan, one was a farming village and the other a fishing village where incidences of death due to stroke differed, higher in the former and lower in the latter. Comparisons were made on salt and protein intake by analyzing the fasting single spot urine collected in the morning and by blood tests. Urine samples were analyzed for sodium (Na), potassium (K), urea nitrogen (UN), inorganic sulfate (SO4) and creatinine (Cr) and blood samples for cholesterol (Chl), triglyceride (TG), hematocrit (Ht) and hemoglobin (Hb). Blood pressure, Na/Cr, Na/K and Ht were higher in the farming village and K/Cr, UN/Cr, SO4/Cr, SO4/UN, Chl and TG were higher in the fishing village. These findings indicate the higher salt intake and lower intake of K, animal protein and fat in the farming village. This typical dietary pattern of the Japanese may explain the higher incidence of hypertension and stroke in the farming village.

The marine winter flounder, Pseudopleuronectes americanus, ingests seawater (SW) and excretes most of the resultant divalent ion load by renal tubule secretion. The site of secretion is generally thought to be the proximal tubule. Although sulfate clearance ratios (C ratio-sulfate clearance/polyethylene glycol clearance) have been reported as high as 12 in SW-acclimated animals, the present study shows that net secretion ceases after acclimation to 10% SW (SO4 free). Intravenous infusion of sulfate into the latter induced net secretion within 5 h (C ratio increased from 0.83 +/- 0.23 to 2.12 +/- 0.33). Increased sulfate secretion coincided with an increase in the magnitude of HCO3-SO4 exchange in brush-border membrane vesicles (BBMV) isolated from flounder renal tubules. Treatment of 10% SW-acclimated flounder with 60 micrograms dexamethasone/100 g body wt also caused an increase in the HCO3-SO4 exchange in BBMV to a level comparable to that of BBMV from SW fish. The glucocorticoid effect was further tested in flounder renal tubule primary monolayer cultures. Measurement of unidirectional sulfate fluxes (J) showed that in the presence of cortisol net secretion (serosal-to-mucosal flux) dominated (Js----m = 111 +/- 6.5, Jm----s = 9 +/- 4.3, Jnet = 102 +/- 2.2 nmol.h-1.cm-2). Removal of cortisol from the culture medium significantly reduced net sulfate secretion to one-third of control (Js----m = 39 +/- 13.9, Jm----s = 6 +/- 1.0, Jnet = 33 +/- 14.1 nmol.h-1.cm-2).

Excretion data and urinary metabolite patterns of di-, d-, and l-norgestrel were obtained from women who received a single, oral 1.5-mg dose of 14C-labeled racemic norgestrel (Ng) or one of its enantiomers. The average percentage of administered radioactivity +/- SD recovered in the urine after 7 days was 58.1 +/- 7.9% for dl-Ng, 44.8 +/- 8.9% for d-Ng, and 63.6 +/- 15.1% for l-Ng; in feces it was 23.4 +/- 7.7% (dl-Ng), 31.6 +/- 8.2% (d-Ng), and 24.8 +/- 10.7% (l-Ng). Different metabolite patterns were observed for each enantiomer in urine, and the pattern for the racemate appeared to be an approximate composite of the metabolite patterns of the two enantiomers. These differences in the metabolite pattern result from stereoselective transformations; notably 16 beta-hydroxylation of l-Ng and ring A reduction of d-Ng. Other stereoselective pathways noted were: 16 alpha- and 1 beta-hydroxylation as well as D-homoannulation of l-Ng and sulfate conjugation of l-16 beta-hydroxynorgestrel; 2 alpha-hydroxylation of d-Ng, formation of a labile neutral, polar compound which contained the norgestrel moiety in the d-form, and formation of a glucuronide of d-16 beta-hydroxynorgestre. The formation of phenolic derivatives occurred to a very minor degree from transformations of the biologically inactive l-enantiomer. With d-norgestrel, this formation occurred to an even lesser extent, if at all.

2-Butoxyethanol (BE) is a massively produced glycol ether of which more than 230 million pounds was produced in the United States in 1983. It is extensively used in aerosols and cleaning agents intended for household use. This creates a high potential for human exposure during its manufacturing and use. A single exposure of rats to BE causes severe hemolytic anemia accompanied by secondary hemoglobinuria as well as liver and kidney damage. Butoxyacetic acid (BAA) was earlier identified as a urinary metabolite of BE. In addition, we have recently identified two additional urinary metabolites of BE, namely, BE-glucuronide and BE-sulfate conjugates. The current studies were undertaken to investigate the metabolic basis of BE-induced hematotoxicity in male F344 rats. Treatment of rats with pyrazole (alcohol dehydrogenase inhibitor) protected rats against BE-induced hematotoxicity and inhibited BE metabolism to BAA. Pyrazole inhibition of BE metabolism to BAA was accompanied by increased BE metabolism to BE-glucuronide and BE-sulfate as determined by quantitative high-performance liquid chromatography analysis of BE metabolites in urine. There was approximately a 10-fold decrease in the ratio of BAA to BE-glucuronide + BE-sulfate in the urine of rats treated with pyrazole + BE compared to rats treated with BE alone. Pretreatment of rats with cyanamide (aldehyde dehydrogenase inhibitor) also significantly protected rats against BE-induced hematotoxicity and modified BE metabolism in a manner similar to that caused by pyrazole. Administration of equimolar doses of BE, the metabolic intermediate butoxyacetaldehyde, or the ultimate metabolite BAA caused similar hematotoxic effects. Cyanamide also protected rats against butoxyacetaldehyde-induced hematotoxicity. Further evidence of the involvement of metabolism in hematotoxicity was demonstrated by the administration of deuterium-labeled BE (1,1-dideuterio-2-BE) which resulted in a significant delay in the development of hematotoxicity. It is therefore concluded that: a) there is a strong correlation between the amount of BAA in the urine and BE-induced hematotoxicity; b) metabolic activation of BE via alcohol and aldehyde dehydrogenases is a prerequisite for the development of BE-induced hematotoxicity; and c) hematotoxicity induced by BE can be attributed to its metabolite BAA. Finally, the current studies may prove beneficial in the treatment of acute glycol ethers poisoning with alcohol dehydrogenase inhibitors.

These studies quantitat the significantly elevated sulfate excretion in the urine of children with classic renal tubular acidosis as compared to six weight-matched controls (1.4 +/-0.5 vs. 0.7 +/- 0.2 mEq/kg/day;p less than 0.05). This sulfate loss may result in a subclinical sulfate deficiency which may contribute to a chondroitin sulfate metabolic disorder.,and thus contribute to the growth failure in children with real tubular acidosis. Futhermore, elevated urinary sulfate excretion may be an early diagnostic finding in infancy prior to the manifestation of renal acidification defects. It is not known whether the sulfate loss is related to a primary renal tubular defect or secondary to metabolic acidosis, although the increased sulfate excretion persisting after correction of metabolic acidosis would tend to suggest a primary defect. However, the normal plasma sulfate concentration and the relatively short period of study permit no definitive answer at this time.

The blood serum fasting levels of inorganic sulfate increase with age, other differences are largely due to diet. The serum sulfate shows a circadian rhythm with a minimum before noon and a maximum in the afternoon and evening. Urinary excretion of sulfate disappears during a low-protein diet. The serum sulfate levels rise temporarily if oral intake of protein, water or inorganic sulfate in non-laxative doses is increased. From the decomposition of protein, sulfate is first excreted with alkali cations and sooner than other catabolites. Drinking water mobilizes sulfate from its tissue pool, which can be blocked by the addition of sodium chloride. The subsequent excretion of an oral sulfate dose is prolonged over one day.

Mucopolysaccharides were extracted from both normal and stone-forming urines, and those from the stone-forming samples showed a higher degree of sulfation than those from normal urines, as determined by sulfate analysis and electrophoretic measurement. The sulfated mucopolysaccharides from stone-forming urines formed insoluble calcium salts, whereas those from normal urines generally remained soluble in the presence of calcium ion. Rachitic rat cartilage was found to have more highly sulfated mucopolysaccharides than normal rat cartilage. Highly sulfated mucopolysaccharides appear to be a significant factor in calcium stone formations.

Merino wethers and Brahman x Shorthorn steers, offered lucerne or spear grass hay, were used to study the movements of sulphate through pools in plasma and ruminal liquor. The irreversible loss of sulphate from ruminal liquor was 60 and 76% of sulphur ingested for both species fed lucerne and spear grass respectively. The irreversible loss of sulphate from the plasma averaged 67 and 56% of sulphur ingested for animals fed lucerne and spear grass respectively. Daily recycling of sulphate to the rumen of sheep was 98 mg sulphur on the lucerne diet and 3.9 mg sulphur on the spear grass diet. Sulphate recycling in cattle fed lucerne was 533 mg sulphur; in cattle fed spear grass the value was 234 mg sulphur. Over 6 days following an intravenous injection of [35S]sulphate into sheep and cattle fed lucerne, 5-10% of the dose was excreted in the faeces and c. 10% was retained. Corresponding values for animals fed spear grass were 23-31% in faeces and 40-51% of the dose retained. After intraruminal injections of [35S]sulphate, animals fed lucerne excreted 15-18% of the dose in the faeces and retained 25-30% of the dose over 6 days. Values for animals fed spear grass were 22-26% in faeces and 62-70% retained. It was concluded that sulphate recycling to the rumen is a limiting factor in microbial synthesis for sheep fed low-quality roughage, and that secretion of endogenous sulphur into the postruminal tract of ruminants is of importance in the metabolism of sulphate.

1. Urinary 17-oxosteroid conjugates were measured by gas-liquid chromatography in five patients with hereditary coproporphyria. 2. Three patients were in an acute attack and showed significantly increased excretion of sulphate or glucuronide conjugates of aetiocholanolone. There was increased excretion of several other related steroids but no consistent pattern was apparent. 3. In the two patients in remission, excretion of urinary 17-oxosteroids was not increased. 4. The ratio of total urinary aetiocholanolone to androsterone (5beta:5alpha) was found to be significantly elevated for the three patients in an acute attack. Serial measurements were made in two of these patients and showed a highly significant linear correaltion between this ratio and the urinary content of delta-aminolaevulic acid and porphobilinogen. 5. These observations suggest the involvement of the 17-oxosteroids, espically aetiocholanolone, in the pathogenesis of hereditary coproporphyria.

Glucuronides of lithocholate, chenodeoxycholate and cholate were synthesized enzymatically and characterized by thin layer chromatography, column chromatography and specific enzymatic hydrolysis. Bile salt glucuronides were quantitatively analysed in thr urine of patients with intra- and extra- hepatic cholestasis and were found to be present in 19 out of 20 patients studied. Our patients with intrahepatic cholestasis excreted 8.9 mg non-sulphated and non-glucoronidated bile salts, 18.2 mg sulphated bile salts and 7.2 mg glucuronidated bile salts. The patients with extrahepatic cholestasis excreted 14.7 mg non-sulphated and non-glucuronidated bile salts, 20.7 mg sulphated bile salts and 4.7 mg glucuronidated bile salts. These findings indicate that glucuronidation of bile salts occurs in man and represents a metabolic pathway in patients with cholestasis.

Infusion of magnesium salts into the body cavity of freshwater-adapted rainbow trout led to elevated plasma magnesium concentrations and to stimulation of renal tubular secretion of magnesium. The majority of the infused load was excreted renally, no net branchial excretion being detected. Magnesium sulphate infusion led to increased tubular secretion of sulphate. Magnesium chloride infusion led to reduced tubular reabsorption of chloride. Magnesium could either be reabsorbed or secreted in control freshwater-adapted trout, apparently as a function of nutritional status. Fish could switch from reabsorption to secretion in response to magnesium loading. It is suggested that freshwater fish eliminate excess dietary magnesium renally.

To elucidate precise chemical nature of urinary glycosaminoglycans (GAG), GAG in the seven major acidic subfractions in a previous paper (Endo et al. 1980a) were analyzed for the chemical compositions and studied on the linkage between carbohydrate and peptide. All the subfractions examined contained galactosamine, hexuronic acid as the major constituent sugars, and xylose, galactose, L-fucose as the minor ones. Some of the subfractions contained additional glucosamine. Sulfate was also found in all the subfractions as their major constituent. The contents of total amino acids ranged from 1.20 to 17.9%. Serine, threonine, aspartic acid, glutamic acid, glycine, alanine and leucine were found as major amino acids. Pronase digestion did not significantly change their molecular size. The presence of O-xylosyl-serine linkage between carbohydrate chains and peptide moieties ws verified byan alkali treatment. Nevertheless, the presence of small portions of galactose and xylose residues at the reducing ends of the carbohydrate chains suggested a possibility of exertion of endo-beta-galactosidase and endo-beta-xylosidase activities for the linkage regions.

The purpose of this study was to determine if certain physiologic parameters (plasma bilirubin concentration and urinary excretion rate of D-glucaric acid) can be used to predict a newborn infant's ability to eliminate a phenolic drug, and particularly to predict the ability to conjugate that drug with glucuronic acid. Tweleve healthy 2- to 3-day-old full-term infants with plasma bilirubin concentrations of 1.0 to 11.6 mg/100 ml and D-glucaric acid excretion rates of 0.131 to 0.345 mg/kg/day received a single oral dose of acetaminophen, 12 mg/kg. Urine was collected serially for 48 hours and analyzed for acetaminophen, acetaminophen glucuronide, acetaminophen sulfate, and D-glucaric acid. The biologic half-life of acetaminophen was 3.5 plus or minus 0.85 hours (average plus or minus SD) as compared to average values of 1.9 to 2.2 hours observed in five reported studies on a total of 39 adults. The rate constant for acetaminophen glucuronide formation in neonates was considerably smaller, on the average, than in adults but the average rate constant for acetaminophen sulfate formation was somewhat larger than in adults. There is not statistically significant colucaric acid excretion. The results of this study suggest that the limited ability of neonates to conjugate phenolic drugs with glucuronic acid is compensated to a degree by a well-developed capability for sulfate conjugation.

The influence of continuous imbalanced high protein intake on the metabolism of minerals (calcium, magnesium, phosphorus) and renal function was the subject of a long-term experiment with rats. In the first part of the study particular attention was directed to the contribution of protein-induced endogenous acid production and renal excretion of hydrogen ions and sulphate to the development of hypercalciuria. For 61 weeks 200 male Wistar rats in eight groups were fed isocaloric diets, whose protein contents were increased from 13 to 26 and 40 J% at the expense of carbohydrate intake. The fat content of the diets was 40 J%. In two groups with 13 and 26 J% protein the effect of different kinds of animal protein was also studied, replacing casein by beef. Mineral contents were kept constant in these diets. To examine the excretion mechanisms of calcium and phosphorus especially under conditions of excessive protein intake, the ratio of calcium to phosphorus was varied in three diets with 40 J% protein by increasing both minerals alternatively or together from 0.6 to 1.2%. An increase in dietary protein content from 13 to 26 or 40 J% produced a sustained hypercalciuria and also hypermagnesiuria over a period of more than 400 days (after 58 weeks: 3.3, 5.9, and 6.8 mg calcium/day; 2.2, 3.3, and 3.4 mg magnesium/day; p less than or equal to 0.05). No adaptation to high protein intake occurred. Hypermagnesiuria, which equally hasn't been described before as a result of high protein intake, was accompanied by a reduced fecal excretion of magnesium. With increased protein intake (casein and beef) hypercalciuria and also hypermagnesiuria were positively correlated with an increased formation and renal excretion of hydrogen ions and sulphate, which resulted from protein catabolism. The dietary protein source influenced the extent of hypercalciuria, irrespective of a constant phosphorus intake. Although leading to equal increases in renal total acid and sulphate excretion, beef as the main protein source caused a lower calciuria than casein. High phosphorus intake caused the highest total acid excretion of all groups, but resulted in a reduced hypercalciuria and hypermagnesiuria and counteracted the influence of an increased protein intake.

Melatonin is metabolised by hydroxylation to form 6-hydroxy-melatonin and by demethylation to form N-acetyl-serotonin, which are excreted as sulphate and glucuronide conjugates. We required these metabolites as pure powders and therefore undertook their isolation and characterisation. Three volunteers ingested 1 g each of melatonin, and their urine was collected and pooled. For the sulphate conjugates, a Lichoprep column was used to concentrate the metabolites and to remove most of the urea. The sulphate conjugates were separated from the glucuronides on a Florisil column and further purified on a fractogel column. They were separated by high-performance liquid chromatography (HPLC) resulting in white powders of 6-hydroxy-melatonin sulphate (SaMT) and N-acetyl-serotonin sulphate (SNAS). For the glucuronide conjugates, an aliquot of the pooled urine was taken to dryness, the residue was dissolved in methanol, and the solution was filtered. The methanol filtrate was taken to dryness, and the residue was applied to a Florisil column. The isolated glucuronide conjugates were recrystallized prior to separation by HPLC, which gave pure white powders of N-acetyl-serotonin glucuronide (GNAS) and 6-hydroxy-melatonin glucuronide (GaMT). Characterisation was achieved by using infrared and ultraviolet spectroscopy, thin-layer chromatography (TLC), and gas chromatography-mass spectrometry (GCMS). These techniques unambiguously confirmed the assigned structures for SaMT and SNAS and fully supported the assigned structures for GNAS and GaMT. Three TLC solvent systems were used, and in each case the individual conjugated metabolite appeared as a discreet spot. Purity, as assessed by GCMS, was shown to be greater than 95% for SNAS, SaMT, and GaMT and to be 88% for GNAS.

In a broom manufacturing factory the authors performed microclimatic measurements, measurements of sulfur dioxide concentration and dust content. Workers (n = 190) were polled regarding discomforts characteristic of sulfur dioxide effects. Sulfates were determined in urine of 56 subjects, and methemoglobin and sulfhemoglobin were determined in blood. Sulfates were determined in 43 controls and methemoglobin and sulfhemoglobin were determined in 39 controls. Sulfur dioxide concentration in work environment ranged from 17.1 to 149.4 mg/m3 in winter and from 0 to 0.75 mg/m3 in summer. The exposed workers complained most often of coughing (94.2%), dyspnea (91.0%), burning in nose, eyes and throat (from 74.7 to 83.7%), substernal pain (75.3%), sore throat (74.7%), tearing (64.7%), etc. Sulfate concentrations were found to be statistically significantly higher (P less than 0.01) in urine of workers exposed to sulfur dioxide than in the controls. Methemoglobin concentrations were also significantly higher in blood of the exposed workers, whereas no difference was found in concentrations of sulfhemoglobin.

A method for the determination of propranolol and six of its metabolites, as well as their glucuronide and/or aryl sulfate conjugates in human urine is described. Propranolol and its basic and neutral metabolites are extracted into ether at pH 9.8, evaporated to dryness, reconstituted, separated on a reversed-phase, high-pressure liquid chromatographic system and quantitated using fluorescence detection. The aqueous urine aliquot is then made acidic and the acid metabolites extracted and measured using similar methods. The presence of 2% sodium metabisulfite in all urines collected is essential to ensure the stability of 4-hydroxy-propranolol during collection and storage. Preliminary data is presented from 24-h urine samples collected from three patients chronically receiving propranolol.

At the beginning of a Head Start and nursery school program in the fall, nursery school children had significantly higher hemoglobin and hematocrit levels and mean corpuscular volume than Head Start children. The latter had a higher mean corpuscular hemoglobin concentration and alpha 1 globulin level. In the spring, at the close of both preschool programs, nursery school children still had higher hemoglobin and hematocrit levels and mean corpuscular volume, and also a higher mean corpuscular hemoglobin and total iron binding capacity. Head Start children exhibiting signs of anemia in the fall were improving in iron status in the spring. Normal blood protein levels for both groups of children indicated adequate protein status. This condition was confirmed by urinary analysis of urea, creatinine, and inorganic sulfate sulfur.

Women who consume a diet low in fat are at lower risk for breast cancer than women whose diet is relatively high in fat. To investigate the effects of a low fat diet on estrogen metabolism, six normal young women were studied while eating a Western-style high fat diet and again after 2 months of consuming a defined low fat diet. Both studies involved the simultaneous administration of [3H]estradiol [( 3H]E2) orally and [14C]E2 iv and the subsequent collection of multiple blood samples and urine for 96 h. The blood samples were analyzed for radioactivity as estrone (E1), E2, their glucuronides, and E1 sulfate. An aliquot of the pooled 96-h urine was analyzed for radioactivity as the glucuronides and sulfates of E1, E2, estriol, 16 alpha-hydroxyestrone (16 alpha-OHE1), and the catechol estrogens, i.e. 2-hydroxy and 2-methoxy metabolites of E1 and E2. The low fat diet resulted in a consistent and significant (P less than 0.05) decrease in urinary excretion of both 16-hydroxylated metabolites, estriol and 16 alpha-OHE1, expressed as a percentage of administered dose of [3H]E2 and [14C]E2, and an increase in the excretion of the catechol estrogens. These changes in metabolite excretion were not, however, mirrored by changes in the MCRs or conversion ratios of either [3H]E2 or [14C]E2. Thus, while neither the clearance of E2 from the blood nor its absorption from the intestinal tract was altered by a relatively short term decrease in dietary fat, there was a shift in the pattern of urinary metabolites away from the purported carcinogenic estrogen (16 alpha-OHE1) and toward the less active catechol estrogens. This may represent an important mechanism whereby low fat diets decrease the risk of breast cancer.

A new mass-spectrometric technique relies on ionization during bombardment of the analyte (dissolved in a liquid matrix, usually glycerol) by an atom beam (e.g., Ar0, Xe0). This technique, termed "fast atom bombardment," is particularly useful in the characterization of polar charged molecules. A neutral beam is not essential, and a primary beam of cesium ions has been successfully used to produce spectra equivalent to those obtained by fast atom bombardment. In this communication I report data on the use of both ion and atom primary beams for producing secondary-ion mass spectra of conjugated steroids. In negative-ion spectra produced for steroid glucuronides and sulfates, the ion [M - H]- is invariably the major high-mass peak, and the lack of substantial fragmentation allows assay of relatively complex mixtures if the analytes differ in mass. I describe here the use of secondary-ion mass spectrometry for distinguishing, by urinary steroid analysis, patients with the four enzyme defects that can affect cortisol synthesis: defects in 17 alpha-hydroxylase, 3 beta-hydroxysteroid dehydrogenase/isomerase, 21-hydroxylase, and 11 beta-hydroxylase.

The urinary excretion of sulfur-containing compounds was studied before, on the third, and on the seventh day of fasting in 10 healthy subjects. The excretion of total sulfur, inorganic sulfate, ester sulfate, "non-sulfate sulfur", methionine, cystathionine, cysteine, N-acetylcysteine, taurine, thiosulfate and thiocyanate was decreased during fasting, whereas the excretion of mercaptoacetate was unaltered and that of mercaptolactate increased. The excretion of inorganic sulfate, taurine and thiocyanate was also decreased when calculated relative to that of total sulfur, suggesting that these compounds are derived mainly from dietary sulfur amino acids. The output of ester sulfate, methionine, cystathionine, cysteine and thiosulfate was unaltered in relation to that of total sulfur, indicating that these compounds are derived from both dietary and endogenous sulfur amino acids, liberated during protein catabolism. By contrast, the excretion of mercaptolactate and mercaptoacetate was increased relative to that of total sulfur, suggesting that these compounds are derived mainly from endogenous sulfur amino acids formed by the enhanced protein catabolism seen during fasting.

Ritodrine is a beta 2-adrenergic agonist that is used clinically for the management of preterm labor. The beta 2 activity of ritodrine produces the relaxation of smooth muscles and is believed to act directly on the beta 2-receptors of the myometrium. Reports in the literature suggest that ritodrine is inactivated by sulfate and glucuronide conjugation, but this has not been verified in humans. Studies on animal models indicate that the sulfate conjugate is a major urinary metabolite of ritodrine. Recent investigations of maternal and neonatal urinary excretion of ritodrine indicate that 80% to 90% of the drug is in the form of conjugates. The purpose of this study was to determine the nature of these conjugates. Our study indicates that both the mother and neonate excrete glucuronide and sulfate conjugates of ritodrine. The sulfate conjugate accounts for 45% of maternal excretion and 66% of neonatal excretion; the glucuronide conjugate accounts for 38% and 23% of maternal and neonatal excretion, respectively. Significantly different metabolic profiles suggest that the neonate may be capable of forming conjugated metabolites of ritodrine.

Rats fed with dietary beta- and gamma-isomers of hexachlorocyclohexane excreted higher amounts of phenolics and glucuronides in the urine as compared to controls. The excretion profile of urinary phenolics and glucuronides suggested a relatively higher turnover rate of the gamma-isomer in the body as compared to the beta-isomer. Excretion of ethereal sulfate was low in hexachlorocyclohexane fed animals.

Renal excretion of bile salts was studied in 17 patients with cirrhosis of the liver. The average quantity of bile salts in urine was 10.2 plus or minus 8.3 mg per 24 hr, 56% of which were sulfated. Of the individual urinary bile salts, 24% oithocholate were sulfated. In contrast, neither sulfated nor nonsulfated bile salts could be detected in urine from 2 normal subjects. Kinetics of bile salt metabolism was measured in 2 of the cirrhotic patients after oral administration of [14C] cholate and [3H] chenodeoxycholate. Approximately 3 to 12% of bile salts synthesized in liver were excreted in urine. Most urinary bile salts (76 to 80%) were sulfated, whereas only 4 to 5% of serum bile salts and 7 to 10% of biliary bile salts were sulfated. Renal clearance of cholate was more than 3 times greater than the clearance of chenodeosycholate or deoxycholate. Renal clearance of sulfated bile salts was 20 to 200 times greaterthan the clearance of the corresponding nonsulfated bile salts.