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.

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.

Preservation of nutrients during long-term storage of source-separated yellowwater.

Source separation of human urine (yellowwater) enhances the sustainability of wastewater management and efficiency of nutrient recovery and recycling. Storage of source-separated yellowwater is recommended prior to agronomic reuse. At this point, it is of immense interest to determine the effect of storage time on quality of yellowwater. Therefore, this study focused on examining changes in some chemical properties of raw, undiluted, freshly collected, source-separated yellowwater stored for a period of 1 year under different temperature regimes: cold (4 °C), mild (10 °C) and warm (22 °C). Chemical parameters (biochemical oxygen demand (BOD(5)), N-tot, N-NO(2), N-NO(3), N-NH(4), P-tot, K, S, and pH), with the main focus on fertiliser nutrient compounds intended for agricultural utilisation, were tested. The outcomes revealed that both nitrification and denitrification processes took place in the stored yellowwater, and an increase in the pH level of up to pH greater than 9 was observed. The study found that the main macronutrients can be well preserved in yellowwater, as there were no substantial changes in the contents of these elements over a 1 year storage period at the three temperatures tested.

Urinary Excretion of Sulfur Metabolites and Risk of Cardiovascular Events and All-Cause Mortality in the General Population.

Aims: Thiosulfate and sulfate are metabolites of hydrogen sulfide (H2S), a gaseous signaling molecule with cardiovascular (CV) protective properties. Urinary thiosulfate excretion and sulfate excretion are associated with favorable disease outcome in high-risk patient groups. We investigated the relationship between urinary excretion of sulfur metabolites, and risk of CV events and all-cause mortality in the general population. Results: Subjects (n = 6839) of the Prevention of Renal and Vascular End-stage Disease (PREVEND) study were followed prospectively. At baseline, 24-h urinary excretion of thiosulfate and sulfate was determined. Median urinary thiosulfate and sulfate excretion values were 1.27 (interquartile range [IQR] 0.89-2.37) µmol/24 h and 15.7 (IQR 12.0-20.3) mmol/24 h, respectively. Neither thiosulfate nor sulfate excretion showed an independent association with risk of CV events. Sulfate, but not thiosulfate, was inversely associated with risk of all-cause mortality, independent of potential confounders (hazard ratio 0.73 [95% confidence interval 0.63-0.84], p < 0.001). This association appeared most pronounced for normolipidemic subjects (pinteraction = 0.019). Innovation: The strong association between sulfate excretion and mortality in the general population emphasizes the (patho)physiological importance of sulfate or its precursor H2S. Conclusion: We hypothesize that urinary sulfate excretion, which is inversely associated with all-cause mortality in the general population, holds clinical relevance as a beneficial modulator in health and disease. Antioxid. Redox Signal. 30, 1999-2010.

Studies of a urinary biomarker of dietary inorganic sulphur in subjects on diets containing 1-38 mmol sulphur/day and of the half-life of ingested 34SO4(2-).

OBJECTIVE: Sulphites are widely used food additives that may damage health, hence limits are set on their use. They are excreted in urine as sulphate, along with sulphate derived from sulphur amino acids. Dietary intakes of sulphites are hard to determine, so we have tested the utility of urinary nitrogen:sulphate ratio as a biomarker of inorganic sulphur (IS) intake. Additionally we determined the half-life of ingested (34)SO(4)(2-) from its urinary excretion. SUBJECTS: Twenty healthy adult subjects were recruited by poster advertisement, for a 24-h study where they ate specified foods, which were high in IS, in addition to their normal diet. The half-life of ingested (34)SO(4)(2-) was assessed in five healthy volunteers, given 5.9 mmols of Na(2)(34)SO(4) as a single dose and collecting all urine specimens for 72-96 h. Urine and duplicate diets from three previously conducted studies were analysed for nitrogen and sulphate content, thus expanding the range of IS intakes for evaluation. METHODS: Duplicate diets were analysed for IS content by ion exchange chromatography, while IS intake was predicted from urinary sulphate (g/day S)-(urinary nitrogen (g/day)/18.89). (32)S:(34)S ratios were determined by liquid chromatography mass spectrometry/mass spectrometry. RESULTS: The range of IS intake was 1.3-37.5 mmol S/day. Actual and predicted IS intakes were mmol/day+/-s.e. 9.2+/-0.65 and 7.0+/-0.45, respectively, and were correlated r=0.60 (n=108). The mean half-life of ingested (34)SO(4)(2-) was 8.2 h. CONCLUSIONS: From a 24-h urine collection, IS intake from the habitual diet can be determined for groups of individuals. To predict individual intakes of IS, which may include high sporadic amounts from beer and wine, at least 48 h of urine collection would be required.

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.

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.

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.

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.

Relationship between the sulfur content of total parenteral nutrition and sulfoester excretion in low-birthweight infants.

Inorganic sulfate is an end product of sulfur amino acid metabolism but it is also the cosubstrate for the biosynthesis of a wide array of complex sulfoesters. In vitro studies have shown that SO4 availability may be the primary determinant of sulfoconjugation rates for specific substrates but the relationship between S intake and sulfoester formation in vivo is not known. By substituting MgCl2 for MgSO4 in an amino acid infusate for parenteral nutrition, we were able to examine prospectively the effect of an altered SO4 load on S metabolism. In comparing 21 low-birthweight infants on the experimental MgC2 infusate with 14 subjects on the control MgSO4 infusate, we observed a 40% decrease in urinary excretion of free SO4 and a 31% decrease in excretion of total acid-labile sulfoesters. There was a significant correlation (r = 0.44; p less than 0.02) between total S intake and sulfoester excretion, suggesting that S intake influences sulfoconjugation in the low-birthweight infant requiring total parenteral nutrition.

Intra-abdominal actinomycosis presenting with sulfur granules in the urine.

Intra-abdominal actinomycosis is rarely suspected and is difficult to diagnose. A 46-year-old woman with intra-abdominal actinomycosis is described in whom the condition was first suspected when sulfur granules were found in her urine. The infection had involved her bladder but not her kidneys.

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.

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.

Distribution of sulfur in urine of patients with cystathioninuria before and during administration of pyridoxine.

During administration of 400 mg daily of vitamin B6 as pyridoxine hydrocholoride to two patients with homozygous cystathioninuria, cystathionine excretion fell in a characteristic manner. Inorganic sulfate excretion did not change significantly when considered by itself. Total sulfur excretion, determined as sulfate after oxidation, decreased slightly in both patients and this decrease was statistically significant in one. The ratio of sulfate to total sulfur excretion increased significantly in both patients during vitamin B6 administration. The difference between total sulfur and free plus bound or ethereal sulfate is largely accounted for by cystathionine and lesser amounts of N-acetyl-cystathionine. The results indicate that cystathionine sulfur is not excreted other than as sulfate during vitamin B6 administration and support the hypothesis that cystathionase activity is enhanced by pyridoxine, the resultant cysteine, alpha-ketobutyrate, and ammonia being added to large body pools.

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.

[Effects of the combination of a pesticide, lindane, and a sulfur depleted diet on pregnancy in the rat]. / Effects de l'association d'un pesticide, le lindane, à un régime pauvre en soufre sur le déroulement de la gestation chez la ratte.

Effects of assocation of lindane ingestion and sulfur depletion on pregnancy in the rat. Sulfoconjugation stands as one of the main processes of foreign compound detoxication in mammals. When feeding conditions are adequate, sulfate ions necessary to form sulfoconjugates are provided by the sulfur aminoacids of the diet. The feeding to pregnant rats of a diet containing lindane, when sulfur aminoacids are limiting factors, should lead to competition between their utilisation for anabolic or detoxifying processes. The balance between stimulation of glucuroconjugation and sulfoconjugation seems to depend on which of the 2 compounds necessary for detoxification is the most limiting factor. Glucuroconjugation spares sulfur aminoacids for foetal growth; sulfoconjugation spares glucose for foetal utilisation. The foetal needs appear to modulate the orientation of the maternal detoxication processes; foetal anabolism is predominent over detoxication.

Urinary sulfur containing metabolites after administration of ethanol, acetaldehyde and formaldehyde to rats.

Acetaldehyde and formaldehyde react in vitro with cysteine to form a product, probably a thiazolidine derivative, which eluted as a single peak in cation exchange chromatography. The reactivity of formaldehyde was much higher than that of acetaldehyde. Rats were injected with single dose of 7.6, 6.2 and 2.5 mmol, respectively of ethanol, acetaldehyde and formaldehyde, and urine was analysed for alkali-hydrolysable thiol groups. Acetaldehyde caused a significant increase in urinary alkali-hydrolysable thiols. Ethanol and acetaldehyde treatment stimulated the ability of the urine to catalyse the iodine-azide reaction suggesting the presence of an excess of compounds containing C-S-atoms.

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.

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.

Re-evaluation of the "week-end effect" data: possible role of urinary copper and phosphorus in the pathogenesis of renal calculi.

Early morning urinary concentrations of 10 elements which had demonstrated a "week-end effect" in a previous study, were subjected to a normalization procedure thereby allowing a re-assessment of their potential role in urolithiasis. After transformation of each concentration to a weighted proportion of the total concentration on each day, only Cu and P values were significantly different for kidney stone formers and healthy controls on all three days indicating that these elements may play a role in the pathogenesis of renal calculi. The results obtained in this study demonstrate that a more meaningful picture of the possible differences in the urinary concentrations of stone formers and normal controls might emerge if "proportional" rather than "raw" concentrations are compared.