A novel quantification method for sulfur-containing biomarkers of formaldehyde and acetaldehyde exposure in human urine and plasma samples.

A novel method for the quantification of the sulfur-containing metabolites of formaldehyde (thiazolidine carboxylic acid (TCA) and thiazolidine carbonyl glycine (TCG)) and acetaldehyde (methyl thiazolidine carboxylic acid (MTCA) and methyl thiazolidine carbonyl glycine (MTCG)) was developed and validated for human urine and plasma samples. Targeting the sulfur-containing metabolites of formaldehyde and acetaldehyde in contrast to the commonly used biomarkers formate and acetate overcomes the high intra- and inter-individual variance. Due to their involvement in various endogenous processes, formate and acetate lack the required specificity for assessing the exposure to formaldehyde and acetaldehyde, respectively. Validation was successfully performed according to FDA's Guideline for Bioanalytical Method Validation (2018), showing excellent performance with regard to accuracy, precision, and limits of quantification (LLOQ). TCA, TCG, and MTCG proved to be stable under all investigated conditions, whereas MTCA showed a depletion after 21 months. The method was applied to a set of pilot samples derived from smokers who consumed unfiltered cigarettes spiked with 13C-labeled propylene glycol and 13C-labeled glycerol. These compounds were used as potential precursors for the formation of 13C-formaldehyde and 13C-acetaldehyde during combustion. Plasma concentrations were significantly lower as compared to urine, suggesting urine as suitable matrix for a biomonitoring. A smoking-related increase of unlabeled biomarker concentrations could not be shown due to the ubiquitous distribution in the environment. While the metabolites of 13C-acetaldehyde were not detected, the described method allowed for the quantification of 13C-formaldehyde uptake from cigarette smoking by targeting the biomarkers 13C-TCA and 13C-TCG in urine.Graphical abstract.

Novel sulphur-containing imatinib metabolites found by untargeted LC-HRMS analysis.

Untargeted metabolite profiling using high-resolution mass spectrometry coupled with liquid chromatography (LC-HRMS), followed by data analysis with the Compound Discoverer 2.0™ software, was used to study the metabolism of imatinib in humans with chronic myeloid leukemia. Plasma samples from control (drug-free) and patient (treated with imatinib) groups were analyzed in full-scan mode and the unknown ions occurring only in the patient group were then, as potential imatinib metabolites, subjected to multi-stage fragmentation in order to elucidate their structure. The application of an untargeted approach, as described in this study, enabled the detection of 24 novel structurally unexpected metabolites. Several sulphur-containing compounds, probably originating after the reaction of reactive intermediates of imatinib with endogenous glutathione, were found and annotated as cysteine and cystine adducts. In the proposed mechanism, the cysteine adducts were formed after the rearrangement of piperazine moiety to imidazoline. On the contrary, in vivo S-N exchange occurred in the case of the cystine adducts. In addition, N-O exchange was observed in the collision cell in the course of the fragmentation of the cystine adducts. The presence of sulphur in the cysteine and cystine conjugates was proved by means of ultra-high resolution measurements using Orbitrap Elite. The detection of metabolites derived from glutathione might improve knowledge about the disposition of imatinib towards bioactivation and help to improve understanding of the mechanism of its hepatotoxicity or nephrotoxicity in humans.

High sulfur content in corn dried distillers grains with solubles protects against oxidized lipids by increasing sulfur-containing antioxidants in nursery pigs.

Some sources of corn dried distillers grains with solubles (DDGS) contain relatively high amounts of oxidized lipids produced from PUFA peroxidation during the production process. These oxidized lipids may impair metabolic oxidation status of pigs. The objective of this study was to understand the effects of feeding corn-soybean meal diets (CON) or diets containing 30% highly oxidized DDGS with 1 of 3 levels of supplemental vitamin E (dl-α-tocopheryl acetate), none, the 1998 NRC level (11 IU/kg), and 10x the 1998 NRC level (110 IU/kg), on oxidative status of nursery pigs. The DDGS source used in this study contained the greatest thiobarbituric acid reactive substances (TBARS) value, peroxide value, and total S content (5.2 ng/mg oil, 84.1 mEq/kg oil, and 0.95%, respectively) relative to 30 other DDGS sources sampled (mean values = 1.8 ng/mg oil, 11.5 mEq/kg oil, and 0.50%, respectively). Barrows (n = 54) were housed in pens and fed the experimental diets for 8 wk after weaning and transferred to individual metabolism cages for collection of feces, urine, blood, and liver samples. Total S content was greater in DDGS diets than in CON (0.39 vs. 0.19%). Dietary inclusion of 30% DDGS improved apparent total tract digestibility of S (86.8 vs. 84.6%; P < 0.001) and S retained (2.94 vs. 2.07 g/d; P < 0.01) compared with CON. Although pigs were fed highly oxidized DDGS in this study, serum TBARS were similar between DDGS and CON treatments. There was an interaction between DDGS and dietary vitamin E level for serum concentrations of α-tocopherol. Serum α-tocopherol concentrations were greater (P < 0.001) in pigs fed DDGS diets than those fed CON when dl-α-tocopheryl acetate was not provided or provided at the NRC level but were similar when dl-α-tocopheryl acetate was supplemented at the 10x NRC level. Pigs fed DDGS diets had greater serum concentrations of S-containing AA, particularly Met (P < 0.001) and taurine (P = 0.002), compared with those fed CON. Liver glutathione concentration was greater in pigs fed DDGS diets than CON (56.3 vs. 41.8 nmol/g). Dietary inclusion of DDGS (P < 0.001) and vitamin E (P = 0.03) increased enzyme activity of glutathione peroxidase. The elevated concentrations of S-containing antioxidants (Met, taurine, and glutathione) in vivo may protect pigs against oxidative stress when feeding highly oxidized DDGS. Therefore, the increased S content in DDGS may be beneficial, and increasing concentrations of vitamin E in diets may not be necessary to protect pigs against metabolic oxidative stress when feeding high S and highly peroxidized DDGS.

Sulphur tracer experiments in laboratory animals using 34S-labelled yeast.

We have evaluated the use of (34)S-labelled yeast to perform sulphur metabolic tracer experiments in laboratory animals. The proof of principle work included the selection of the culture conditions for the preparation of sulphur labelled yeast, the study of the suitability of this labelled yeast as sulphur source for tracer studies using in vitro gastrointestinal digestion and the administration of the (34)S-labelled yeast to laboratory animals to follow the fate and distribution of (34)S in the organism. For in vitro gastrointestinal digestion, the combination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and high-performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICP-MS) showed that labelled methionine, cysteine and other low molecular weight sulphur-containing biomolecules were the major components in the digested extracts of the labelled yeast. Next, in vivo kinetic experiments were performed in healthy Wistar rats after the oral administration of (34)S-labelled yeast. The isotopic composition of total sulphur in tissues, urine and faeces was measured by double-focusing inductively coupled plasma mass spectrometry after microwave digestion. It was observed that measurable isotopic enrichments were detected in all samples. Finally, initial investigations on sulphur isotopic composition of serum and urine samples by HPLC-ICP-MS have been carried out. For serum samples, no conclusive data were obtained. Interestingly, chromatographic analysis of urine samples showed differential isotope enrichment for several sulphur-containing biomolecules.

Profiling biological samples using ultra performance liquid chromatography-inductively coupled plasma-mass spectrometry (UPLC-ICP-MS) for the determination of phosphorus and sulfur-containing metabolites.

In this preliminary study UPLC-ICP-MS has been utilized to profile a range of different bio-fluids and tissue extracts for sulfur and phosphorus-containing metabolites. Particular attention has been given to the livers, plasma and urine from lean and obese Zucker rats, with a view to differentiating between them based solely on their respective sulfur or phosphorus profiles and/or their total sulfur and phosphorus content. In addition, bile and tumour extracts have been analysed to observe the nature of their profiles. To the best of our knowledge this is the first time ICP-MS has been used in a non-targeted metabonomic study. Results have shown lower limits of quantification for sulfur and phosphorus methods of 0.25 and 0.15 ng on column with CVs of 14.7% and 10.9% respectively. Total phosphorus analysis of the Zucker rat aqueous liver extracts, plasma and urine has shown the pattern of phosphorus concentrations to be statistically significantly different in the lean and obese Zucker rats. Chromatographic separation of the Zucker rat organic liver extracts and plasma allowed further differentiation between the lean and obese rats using their phosphorus profiles alone. In conclusion, this preliminary study has shown the potential of UPLC-ICP-MS to quantitatively discriminate between different species biofluids, fluids and tissues based solely on their phosphorus or sulfur concentrations and/or metabolomes.

Urinary sulfur excretion and the nitrogen/sulfur balance ratio reveal nonprotein sulfur amino acid retention in piglets.

We evaluated the use of urinary sulfur (S) excretion as a measure of sulfur amino acid (SAA) catabolism and the nitrogen/sulfur (N/S) molar balance ratio as an indicator of nonprotein SAA storage in growing piglets. After confirming that an intravenous dose of sulfate is fully recovered in urinary sulfate, we measured urinary S recovery after an intravenous dose of methionine in 6 piglets fed an adequate protein (AP) diet and 6 piglets fed a low protein (LP) diet with normal energy provision. As measured over 48 h, recoveries of the methionine load as urinary total S was 106% in the AP group but only 69% in the LP group (P < 0.05). On the baseline diets the N/S balance ratio in the AP group was 36, whereas that in the LP group was 30 (P < 0.05); immediately after the methionine load, this ratio remained constant in the AP group but decreased further, to 26 (P < 0.05) in the LP group. These results indicate that protein-deficient piglets accumulate relatively more S than N from their diet, and under these conditions a significant portion of the S derived from a methionine load is retained in nonprotein compounds. Urinary S excretion, a simple nontracer measurement, can provide an accurate measure of SAA catabolism, and the N/S balance ratio is a potentially useful indicator of changes in nonprotein SAA stores of growing piglets.

Massive loss of sulfur in HIV infection.

Skeletal muscle tissue from SIV-infected macaques was previously found to contain abnormally high sulfate and low glutathione levels indicative of an excessive cysteine catabolism. We now confirm the peripheral tissue as a site of massive cysteine catabolism in HIV infection and have determined the urinary loss of sulfur per time unit. The comparison of the sulfate concentrations of the arterial and venous blood from the lower extremities of 16 symptomatic HIV+ patients and 18 HIV- control subjects (study 1) revealed (1) that the peripheral tissue of HIV+ patients with or without highly active antiretroviral therapy (HAART) releases large amounts of sulfate and (2) that plasma sulfate, thioredoxin, and interleukin-6 levels are elevated in these patients. A complementary investigation of 64 asymptomatic HIV+ patients and 65 HIV- subjects (study 2) revealed increased plasma sulfate levels in the asymptomatic patients. The analysis of the daily urinary excretion of sulfate and urea of another group of 19 HIV+ patients and 22 healthy HIV- subjects (study 3) confirmed (1) that HIV+ patients experience a massive loss of sulfur and (2) that this loss is not ameliorated by HAART. The sulfur loss of asymptomatic patients was equivalent to a mean loss of about 10 g of cysteine per day. If extrapolated, this would correspond to an alarming negative balance of approximately 2 kg of cysteine per year under the assumption that the normal sulfate excretion equivalent to approximately 3 g of cysteine per day is balanced by a standard Western diet. The abnormally high sulfate/urea ratio suggests that this process drains largely the glutathione pool.

Some sulfur-containing metabolites of tri-n-butyltin chloride in male rats.

In an attempt to elucidate metabolic destination of TBTO, sulfur-containing metabolites were investigated in the urine. Tri-n-butyltin chloride (TBTC), tri-n-butyltin oxide (TBTO), and their in vitro metabolites in rat liver microsomal enzyme systems, di-n-butyl(3-hydroxybutyl)tin chloride (T3OH), di-n-butyl(3-oxobutyl)tin chloride (T3CO), dibutyltin dichloride (DBTC), and monobutyltin trichloride (MBTC), were intraperitoneally administered to rats. In particular, administration of T3OH and T3CO gave higher amounts of mercapturic acid derivatives, such as N-acetyl-S-(3-oxobutyl)-L-cysteine (3CO-MA) and N-acetyl-S-(3-hydroxybutyl)-L-cysteine (3OH-MA), than TBTC or TBTO. On the other hand, DBTC and MBTC did not yield measurable amounts of 3CO-MA and/or 3OH-MA. The appearance of organotin metabolites in urine indicates that T3OH, T3CO, and hypothesized secondary metabolites, such as n-butyl(3-hydroxybutyl)(3-oxobutyl)tin chloride, n-butyl(3-hydroxybutyl)(4-hydroxybutyl)tin chloride, etc., are subject to the action of glutathione S-transferase to give mercapturic acid derivatives. These sulfur-containing metabolites (3CO-MA and 3OH-MA) were also found in control rat urine.

The effect of dietary sulfur-containing amino acids on calcium excretion.

The relationships between dietary protein and sulfur amino acid (methionine and cystine or taurine) intakes and urinary calcium excretion were examined both in animals and in young men. Thirty-two adult Wistar rats were divided into 4 groups, i.e., basal diet (group I), supplemented with albumin (II), methionine and cystine (III), or taurine (IV). During the 5-week feeding period, food consumption was recorded and 48 h urine samples were collected 4 times for each rat. Urinary calcium, creatinine and sulfate were measured. The results showed that the calcium and sulfate excretion in rats in group II and III were significantly higher than rats in the basal diet group. In contrast, supplementing a basal diet with taurine did not increase sulfate excretion and failed to induce hypercalciuria. The same result was also observed in the study carried out in Chinese young men. An increase in protein intake from 67 g to 107 g caused an increase in urinary calcium and sulfate. Supplementation with methionine and cystine in an amount to simulate those in the high protein diet had a similar effect. Adding taurine to the diet had no effect on urinary calcium and sulfate excretion. About 60 percent of the supplemented taurine in the diet was detected in the urine.

Hypercysteinemia and delayed sulfur excretion in cirrhotics after oral cysteine loads.

Biosynthesis of cysteine from methionine via the hepatic transsulfuration pathway is impaired in some cirrhotic patients, who therefore might require cysteine in the diet. However, because further metabolism of cysteine also occurs primarily in the liver, the metabolic clearance of this amino acid could be impaired in cirrhosis. We administered oral loads of L-cysteine to cirrhotic patients and healthy volunteers. Plasma cyst(e)ine (free and protein-bound cysteine, and 1/2 cystine) and urinary sulfur-containing constituents were measured at various times postload. Cirrhotic subjects exhibited a greater maximal plasma cyst(e)ine concentration and plasma elimination half-life (t1/2) and a delayed excretion of metabolic end products after an oral L-cysteine load. The postload increase in total plasma cyst(e)ine was accounted for primarily by an increase in the disulfide form (cystine). These studies show that cirrhotics have an impaired ability to clear cyst(e)ine from the plasma.

Nutritional status of men under hypokinesia.

Hypokinesia (diminished muscular activity) elicits substantial changes in energy and nutritional requirements in humans. The objective of this investigation was to determine the nutritional status of six physically healthy men aged 19 to 21 years under 95 days of hypokinesia without the use of any preventive measures. For the simulation of the hypokinetic effect the men were placed under 95 days of an ad libitum bed rest regimen. During the background period, that is, prior to exposure to hypokinesia, the caloric value of the diet was 3124 kcal per day; under hypokinesia, the caloric value was 2745 kcal per day. In calculating the nutritional requirements of men under hypokinesia, several biochemical parameters were measured. Effects of hypokinesia demonstrated included certain changes in the enzymatic activity of glands; increased excretion of nitrogenous compounds in the urine; increased blood cholesterol content; changes in the amount of blood sugar; changes in acid-base balance; increased elimination of fluid, calcium, and phosphorus; impaired supply of vitamins to the organism; and increased energy expenditure. It was concluded that the nutritional status of men underwent substantial changes under conditions of diminished muscular activity.

Poor sulphoxidation ability in patients with food sensitivity.

Patients with well defined reactions to foods were examined for their ability to carry out both sulphur and carbon oxidation reactions by using carbocisteine and debrisoquine as probe compounds. The proportion of poor sulphoxidisers (58 of 74) was significantly greater than that of a previously determined normal control population (67 of 200; p less than 0.005). The proportion of poor carbon oxidisers was not significantly different from the controls. Metabolic defects may play a part in the pathogenesis of adverse reactions to foods.

Transsulfuration in an adult with hepatic methionine adenosyltransferase deficiency.

We investigated sulfur and methyl group metabolism in a 31-yr-old man with partial hepatic methionine adenosyltransferase (MAT) deficiency. The patient's cultured fibroblasts and erythrocytes had normal MAT activity. Hepatic S-adenosylmethionine (SAM) was slightly decreased. This clinically normal individual lives with a 20-30-fold elevation of plasma methionine (0.72 mM). He excretes in his urine methionine and L-methionine-d-sulfoxide (2.7 mmol/d), a mixed disulfide of methanethiol and a thiol bound to an unidentified group X, which we abbreviate CH3S-SX (2.1 mmol/d), and smaller quantities of 4-methylthio-2-oxobutyrate and 3-methylthiopropionate. His breath contains 17-fold normal concentrations of dimethylsulfide. He converts only 6-7 mmol/d of methionine sulfur to inorganic sulfate. This abnormally low rate is due not to a decreased flux through the primarily defective enzyme, MAT, since SAM is produced at an essentially normal rate of 18 mmol/d, but rather to a rate of homocysteine methylation which is abnormally high in the face of the very elevated methionine concentrations demonstrated in this patient. These findings support the view that SAM (which is marginally low in this patient) is an important regulator that helps to determine the partitioning of homocysteine between degradation via cystathionine and conservation by reformation of methionine. In addition, these studies demonstrate that the methionine transamination pathway operates in the presence of an elevated body load of that amino acid in human beings, but is not sufficient to maintain methionine levels in a normal range.

Sulfur amino acid metabolism in juvenile-onset nonketotic and ketotic diabetic patients.

Sulfur amino acid metabolism was studied in non-fasting nonketotic and ketotic juvenile-onset diabetic children and the results were compared to age-matched healthy children on an ordinary diet. An increased excretion of total sulfur and inorganic sulfate was found in diabetic children, probably a result of a decreased protein-serum synthesis and/or increased endogenous protein catabolism, although as a result of hyperglycemia a decreased tubular reabsorption may also have contributed. All diabetics showed a normal excretion of methionine. For cyst(e)ine and taurine an increased excretion was seen in ketotic diabetics, probably also a consequence of an increased endogenous protein degradation. As a sign of the latter, an increased output of 3-methylhistidine was also observed, a confirmation of earlier reports. The increased output of mercaptolactate and mercaptoacetate found in ketotic patients, was probably also a result of enhanced endogenous protein degradation. An increased urinary excretion of N-acetylcysteine was seen in diabetic children, which may reflect an enhanced availability to acetyl coenzyme A.

[Principles of pharmacotherapy in geriatrics]. / Prinzipien der Pharmakotherapie in der Geriatrie.

This article examines the pharmacokinetics and pharmacodynamics of drugs under the aspects of senile changes in organs and systems in the neurohumoral regulation and in metabolism. It gives reasons for the inadequacy of polypharmacy in the treatment of elderly and old people and for the need of a strict individualization. The retarded reactivity of the organism of old people, their increased susceptibility and reduced tolerance of the effects of drugs necessitate smaller dosages of drugs in geriatrics in comparison with the generally usual dosages, especially at the beginning of treatment. The multitude of exogenous and endogenous changes in the organism, that go hand in hand with the process of aging, leads in old people to an increased susceptibility to more frequent and pronounced secondary effects caused by drugs. Of special importance in the prevention of a possible intoxication by drugs are, in the case of elderly people, the proper composition of food, the water balance and the electrolyte metabolism.

The excretion of sulfur compounds in the urine of newborn infants.

The excretion of sulfur-containing compounds was determined on the third and sixth day of life in male infants and the results were compared with those previously obtained on fed and fasting men. The output of total sulfur and inorganic sulfate was very low on the third day of life but had increased by the sixth day to levels found in the fasting men, whereas the excretion of mercaptolactate in the newborns decreased from the third to the sixth day of life. These results may be explained by the initial fasting state of neonates followed by an anabolic phase. Neonates excreted acid-labile ester sulfate and mercaptoacetate at levels similar to those found in adults, but the neonatal urine also contained sulfate esters (probably steroid sulfates) that required more drastic acid conditions for hydrolysis. Raised concentrations of sulfur-containing amino acids (methionine, cystathionine, cyst(e)ine and taurine) were found in neonatal urine in confirmation of earlier reports. The excretion of thiosulfate could only be determined in newborns on the sixth day and was low in comparison with that of adults. High urinary thiocyanate concentrations were found in newborns fed by tobacco-smoking mothers, whereas the excretion of thiocyanate was low in other newborns. The possible medical hazards from the exposure of neonates to thiocyanate are emphasized.

Purge and trap flame photometric gas chromatography technique for the speciation of trace organotin and organosulfur compounds in a human urine standard reference material (SRM).

Ultratrace levels of organotin species and an organosulfur compound were detected in a National Bureau of Standards (NBS) human urine Standard Reference Material, SRM 2670, and a previously certified urine SRM 2672, using a purge and trap system coupled to a gas chromatograph equipped with a flame photometric detector. samples of the SRM were treated with sodium borohydride to form volatile tin hydrides. Species detected included dimethyltin (1.04 ng/ml), butyltin (0.03 ng/ml), and dimethyl-disulfide (2.73 ng/ml) in the new stock of freeze dried human urine SRM 2670 being prepared for issue by NBS and methyltin (1.0 ng/ml), butyltin (1.5 ng/ml), and inorganic tin (28.1 ng/ml) in the old stock of SRM 2672. This analytical technique should have useful applications in studies that are needed to develop a toxicological data base and monitoring programs for human organotin exposure.

Sulfur balances in intravenously fed infants: effects of cysteine supplementation.

Sulfur balances were completed in newborn infants parenterally fed with or without cysteine. In both groups, the preservative, potassium metabisulfite, accounted for the majority of sulfur intake (32 mg S/kg/day), while methionine intakes provided an additional 27 sulfate losses accounted for approximately 95% of the sulfur excretion, with the remainder contained in amino acids. Balance data accounted for over 99% of the sulfur infused in the unsupplemented group, but only 90% of that given to the cysteine-supplemented group. Thus, urinary excretion of sulfate generally reflects input from either inorganic or amino acid sources. Of the sulfur retained in the supplemented group, 75% was calculated to be retained in lean tissue and in increases in total body sulfate, but the distribution of the remaining 25% remains unknown. The failure to account fully for the sulfate provided to the cysteine-supplemented group, however, may be due to errors in the balance technique or due to an accumulation of cysteine or sulfate in body pools undefined by this study.

Urinary bivalent sulfur metabolites of phenanthrene excreted by the rat and guinea pig.

After the administration of phenanthrene (50 mg/kg, ip) to young adult male rats and guinea pigs, a series of bivalent sulfur urinary metabolites were isolated and characterized by gas chromatography and gas chromatography-mass spectrometry. Seven methylthio metabolites were isolated from the neutral fraction of hydrolyzed rat urine, whereas only two were detected in guinea pig urine. The major methylthio metabolite excreted by each species was 9-hydroxy-10-methylthio-9, 10-dihydrophenanthrene. This was observed as a second-day metabolite in the rat, and its appearance was accompanied by 9-phenanthrol. In addition to the methylthio compounds, which were excreted predominantly as glucuronides, six acidic bivalent sulfur metabolites were isolated from hydrolyzed rat urine and identified by GC/MS; five were present in hydrolyzed guinea pig urine. The major acidic metabolite in hydrolyzed rat urine was the hydroxydihydromercapturic acid N-acetyl-S-(9-hydroxy-9, 10-dihydro-10-phenanthryl)-L-cysteine. The major acidic metabolite in guinea pig urine was the mercaptoacetic acid S-(9-hydroxy-9, 10-dihydro-10-phenanthryl)mercaptoacetic acid, but the hydroxydihydromercapturic acid was also present.