Homocysteine thiolactone and other sulfur-containing amino acid metabolites are associated with fibrin clot properties and the risk of ischemic stroke.

Homocysteine (Hcy) and Hcy-thiolactone (HTL) affect fibrin clot properties and are linked to cardiovascular disease. Factors that influence fibrin clot properties and stroke are not fully understood. To study sulfur-containing amino acid metabolites, fibrin clot lysis time (CLT) and maximum absorbance (Absmax) in relation to stroke, we analyzed plasma and urine from 191 stroke patients (45.0% women, age 68 ± 12 years) and 291 healthy individuals (59.7% women, age 50 ± 17 years). Plasma and urinary levels of sulfur-containing amino acid metabolites and fibrin clot properties were significantly different in stroke patients compared to healthy individuals. Fibrin CLT correlated with fibrin Absmax in healthy males (R2 = 0.439, P = 0.000), females (R2 = 0.245, P = 0.000), female stroke patients (R2 = 0.187, P = 0.000), but not in male stroke patients (R2 = 0.008, P = ns). Fibrin CLT correlated with age in healthy females but not males while fibrin Absmax correlated with age in both sexes; these correlations were absent in stroke patients. In multiple regression analysis in stroke patients, plasma (p)CysGly, pMet, and MTHFR A1298C polymorphism were associated with fibrin Absmax, while urinary (u)HTL, uCysGly, and pCysGly were significantly associated with fibrin CLT. In healthy individuals, uHTL and uGSH were significantly associated with fibrin Absmax, while pGSH, and CBS T833C 844ins68 polymorphism were associated with fibrin CLT. In logistic regression, uHTL, uHcy, pCysGly, pGSH, MTHFR C677T polymorphism, and Absmax were independently associated with stroke. Our findings suggest that HTL and other sulfur-containing amino acid metabolites influence fibrin clot properties and the risk of stroke.

Therapeutic paracetamol treatment in older persons induces dietary and metabolic modifications related to sulfur amino acids.

Sulfur amino acids are determinant for the detoxification of paracetamol (N-acetyl-p-aminophenol) through sulfate and glutathione conjugations. Long-term paracetamol treatment is common in the elderly, despite a potential cysteine/glutathione deficiency. Detoxification could occur at the expense of anti-oxidative defenses and whole body protein stores in elderly. We tested how older persons satisfy the extra demand in sulfur amino acids induced by long-term paracetamol treatment, focusing on metabolic and nutritional aspects. Effects of 3 g/day paracetamol for 14 days on fasting blood glutathione, plasma amino acids and sulfate, urinary paracetamol metabolites, and urinary metabolomic were studied in independently living older persons (five women, five men, mean (±SEM) age 74 ± 1 years). Dietary intakes were recorded before and at the end of the treatment and ingested sulfur amino acids were evaluated. Fasting blood glutathione, plasma amino acids, and sulfate were unchanged. Urinary nitrogen excretion supported a preservation of whole body proteins, but large-scale urinary metabolomic analysis revealed an oxidation of some sulfur-containing compounds. Dietary protein intake was 13% higher at the end than before paracetamol treatment. Final sulfur amino acid intake reached 37 mg/kg/day. The increase in sulfur amino acid intake corresponded to half of the sulfur excreted in urinary paracetamol conjugates. In conclusion, older persons accommodated to long-term paracetamol treatment by increasing dietary protein intake without any mobilization of body proteins, but with decreased anti-oxidative defenses. The extra demand in sulfur amino acids led to a consumption far above the corresponding population-safe recommendation.

Fluorosurfactant-prepared triangular gold nanoparticles as postcolumn chemiluminescence reagents for high-performance liquid chromatography assay of low molecular weight aminothiols in biological fluids.

Our recent study demonstrates the synthesized triangular gold nanoparticles (AuNPs) by trisodium citrate reduction of HAuCl(4) in the presence of nonionic fluorosurfactant (FSN) could display stronger catalytic activity towards luminol-chemiluminescence (CL) than spherical AuNPs. Ultratrace aminothiols may cause a great decrease in CL intensity of the triangular AuNPs-luminol CL system. In this article, we utilize the as-prepared triangular AuNPs as novel postcolumn CL reagents to explore a simple high-performance liquid chromatography (HPLC)-CL method for the determination of low molecular weight aminothiols (i.e., cysteine, homocysteine, glutathione, cysteinylglycine and glutamylcysteine). The as-prepared triangular AuNPs were easier to synthesize, stable at a wider pH range and high ionic strength, and highly selective and sensitive towards reduced aminothiols. The detection limits at a signal-to-noise ratio of 3 for cysteine, homocysteine, glutathione, cysteinylglycine and glutamylcysteine were 0.016, 0.08, 0.1, 0.04 and 0.1pmol, respectively. Recoveries from spiked urine and plasma samples were 95.7-104.3%. The applicability of the proposed method has been validated by determining these low molecular weight aminothiols in human urine and plasma samples with satisfactory results, and thus it will have great potential application in clinical diagnosis.

Selective enrichment of aminothiols using polysorbate 20-capped gold nanoparticles followed by capillary electrophoresis with laser-induced fluorescence.

In this article, we report a simple method for selective enrichment of aminothiols using Tween 20-capped gold nanoparticles (AuNPs) prior to capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF). Compared to citrate-capped AuNPs, Tween 20-capped AuNPs exhibit the ability to disperse in a highly saline solution and selectively extract aminothiols through the formation of Au-S bonds. After extraction and centrifugation, 1mM thioglycollic acid (TGA) was utilized to remove aminothiols that attached to the NP surfaces. After a solution of 8.0 mL aminothiols were extracted using 2x AuNPs (200 microL), the extracted aminothiols derivatized with o-phthalaldehyde at pH 12.0 were detected by CE-LIF. As a result, the limits of detection at a signal-to-noise ratio of 3 for homocysteine (HCys), glutathione (GSH), and gamma-glutamycysteine (Glu-cys) are 4013.2, 79.8, and 382.8 pM, respectively. The use of this probe provided approximately 11-, 282-, and 21-fold sensitivity improvements for HCys, GSH, and Glu-cys, respectively. A practical analysis of HCys, GSH, and Glu-cys in human urine sample has been accomplished by this present method.

Manifestation of hawkinsinuria in a patient compound heterozygous for hawkinsinuria and tyrosinemia III.

Mutations in the gene for 4-hydroxyphenylpyruvic acid dioxygenase (HPD) cause either autosomal recessive tyrosinemia type III or autosomal dominant hawkinsinuria. We report a 6-month-old Indian infant who is compound heterozygous for both alleles and who has hawkinsinuria but not tyrosinemia type III based on biochemical investigations. The HPD gene was directly sequenced in the proband and both parents. The mechanistic model of the enzymatic function was built using the known structure of rat HPD. We identified a novel hawkinsinuria mutation, Asn241Ser, and a known tyrosinemia type III mutation, Ile335Met, in trans configuration. The structural analysis of the active site revealed that the IIe335Met mutation is situated in the close vicinity of one of the two highly conserved Phe rings which stack with the phenol ring of the substrate. The Asn241Ser mutation is situated further away from the 4-hydroxyphenylpyruvate binding pocket. Assuming that Asn241Ser causes hawkinsinuria, we propose positioning the dioxygen molecule in the HPD-catalyzed reaction as a novel role for the Asn residue. The IIe335Met allele is equivalent to a null mutation while the Asn241Ser allele results in a partially active enzyme with an uncoupled turnover causing hawkinsinuria.

Effect of glycine and vitamin supplementation on sulphur amino acid utilization by growing cattle.

Effects of glycine (Gly) and B-vitamins on sulphur amino acid (AA) utilization were studied in growing steers maintained under conditions where methionine (Met) was first limiting. Conditions were generated by limit feeding a diet low in ruminally non-degraded protein and abomasally infusing an AA mixture limiting in Met. Retained N tended (p = 0.07) to improve when steers received 10 mg folate, 10 mg vitamin B6, and 0.10 mg vitamin B12 daily. Hepatic vitamin B12 (p = 0.08) and folate (p = 0.05) concentrations increased with vitamin supplementation. In another trial, factorial treatments were 2 or 5 g/day L-Met and 0 or 50 g/day Gly infused abomasally. Retained N increased (p < 0.05) in response to Met, and responses were numerically larger in the presence of supplemental Gly. In a different trial, factorial treatments were 0 or 2.4 g/day L-cysteine (Cys) and 0 or 40 g/day Gly. Retained N was not affected by Cys in the absence of Gly, but was increased by Cys when Gly was supplemented (interaction, p = 0.01). B-vitamin status may affect sparing of Met by Cys. Supplemental Gly improved responses to supplemental Met and Cys.

High-throughput capillary electrophoretic method for determination of total aminothiols in plasma and urine.

Increased interest in the analysis of aminothiols in body fluids during the last years results in a request for high-throughput analytical methods for their determination. We report here a novel, high-throughput method for the determination of total concentrations of biogenous aminothiols - homocysteine, cysteine, glutathione, cysteinylglycine, gamma-glutamylcysteine, and of penicilamine, mercaptopropionylglycine, and cysteamine, three compounds used to treat disorders of aminothiol metabolism in plasma and urine. Samples were reduced with tris(carboxyethyl)phosphine and labeled with 5-(bromomethyl)fluorescein. Capillary electrophoretic separations were performed in 60 mmol/L borate - 15 mmol/L sodium dodecyl sulfate - 2-amino-2-methyl-1-propanol, pH 10.0, with laser-induced fluorescence detection. Analysis time was less than 2 min. The assay is linear (r > 0.999) up to 500 micromol/L. Reproducibilities of migration times (coefficient of variation, CV) were < 0.5%. Interassay repeatabilities (CV, n = 10) were 5.08% and 6.09% for 5 micromol/L addition of homocysteine and 0.60% and 3.78% for 100 micromol/L addition of cysteine in plasma and urine, respectively. Recovery values were within 94-106% and sensitivity was better than 0.19 micromol/L for all analyzed compounds. Results agreed well with a standard high-performance liquid chromatography (HPLC) method. The diagnostic usefulness of the method has been proven on 79 samples of cystinuric patients and 12 samples of homocystinuric patients. We report here a novel method for the determination of aminothiols in body fluids by capillary electrophoresis (CE). Determination is fast and sensitive enough for diagnostic purposes.

Mutations in the 4-hydroxyphenylpyruvic acid dioxygenase gene are responsible for tyrosinemia type III and hawkinsinuria.

The enzyme 4-hydroxyphenylpyruvic acid dioxygenase (HPD) catalyzes the reaction of 4-hydroxyphenylpyruvic acid to homogentisic acid in the tyrosine catabolism pathway. A deficiency in the catalytic activity of HPD may lead to tyrosinemia type III, an autosomal recessive disorder characterized by elevated levels of blood tyrosine and massive excretion of tyrosine derivatives into urine. It has been postulated that hawkinsinuria, an autosomal dominant disorder characterized by the excretion of 'hawkinsin,' may also be a result of HPD deficiency. Hawkinsin is a sulfur amino acid identified as (2-l-cystein-S-yl, 4-dihydroxycyclohex-5-en-1-yl)acetic acid. Patients with hawkinsinuria excrete this metabolite in their urine throughout their life, although symptoms of metabolic acidosis and tyrosinemia improve in the first year of life. We performed analyses of the HPD gene in a patient with tyrosinemia type III and two unrelated patients with hawkinsinuria. A homozygous missense mutation predicting an Ala to Val change at codon 268 (A268V) in the HPD gene was found in the patient with tyrosinemia type III. A heterozygous missense mutation predicting an Ala to Thr change at codon 33 (A33T) was found in the same HPD gene in the two patients with hawkinsinuria. These findings support the hypothesis that alterations in the structure and activity of HPD are causally related to two different metabolic disorders, tyrosinemia type III and hawkinsinuria.

[Sulphur amino acids, vitamin B12 and folic acid in children with chronic renal failure]. / Profil amininokwasów siarkowych, witaminy B12 i kwasu foliowego we krwi u dzieci z przewlekla niewydolnoscia nerek.

UNLABELLED: Aim of the study was to: 1) estimate plasma profile of sulphur AA in children with chronic renal failure (CRF) and in children on hemodialysis (HD), and 2) to evaluate any correlation with serum folic acid (FA) and vitamin B12. PATIENTS: 32 pts with CRF: 9 with GFR > 20 ml/min/1.73 m2 (group 1), 9 with GFR < 20 ml/min/1.73 m2 (group 2), and 14 pts on HD (group 3). METHODS: plasma homocysteine (Hcys), methionine (Met), cysteine (Cys), serine (Ser) were measured with gas chromatography. Serum FA and vit. B12 were measured using MEIA method. RESULTS: median Hcys concentrations were the lowest in group 1: 5 mumol/l vs 9 mumol/l (group 2) and 20 mumol/l (group 3) (p = 0.03). Similarly, the lowest Met levels were observed in group 1--26 mumol/l, vs 66 mumol/l (group 2) and 281 mumol/l (group 3) (p = 0.001). Median Cys level in group 1 was 98 mumol/l vs 54 mumol/l (group 2), and 122 mumol/l (group 3) (p = 0.02). No differences were found in median Ser levels: 153 mumol/l (group 1) vs 239 mumol/l (group 2) and 240 mumol/l (group 3). The median concentrations of FA were 6.3 ng/ml (group 1) vs 8 ng/ml (group 2) and 15 ng/ml (group 3) (NS). Median concentrations of vit. B12 were 256 pg/ml (group 1) vs 379 pg/ml (group 2) and 322 pg/ml (group 3) (NS). There were no correlation between sulphur AA and FA and vit. B12 levels. The only difference between pts with Hcys levels remaining in lower and upper quartile concerned Met concentration (38 vs 263 mumol/l, p < 0.02) and GFR (p < 0.01). CONCLUSIONS: Hyperhomocysteinemia develops already in moderate CRF. In pts on HD levels of Met and Cys are also raised. FA and vit. B12 concentrations are normal and do not correlate with plasma concentrations of sulphur AA.

Identification of an oxidation product of aminoethylcysteine ketimine dimer.

In continuation of our previous work dedicated to the detection of the oxidation products of aminoethylcysteine ketimine dimer by oxygen reactive species, we give here data for the identification of the alpha, beta unsaturated sulfoxide as the main product of interaction of the dimer with H2O2. Identification has been done on the basis of mass spectrometry and NMR analyses of the product isolated by preparative chromatography.

Long-term follow up of a new case of hawkinsinuria.

UNLABELLED: Hawkinsinuria is a rarely diagnosed autosomal dominantly transmitted inborn error of tyrosine metabolism with impaired conversion of 4-hydroxyphenylpyruvate to homogentisate. As a consequence of the defective 4-hydroxyphenylpyruvate dioxigenase activity, large amounts of the unusual, ninhydrin-positive amino acid hawkinsin and later on in life 4-hydroxycyclohexylacetic acid are formed and excreted. Clinically the disease is characterised mainly by chronic metabolic acidosis and severe growth retardation as a result of protein overload. As the ability to form 4-hydroxycyclohexylacetic acid and thereby to cope with the still not very well defined reactive and toxic intermediates increases, clinical symptoms vanish. We report here a new patient with hawkinsinuria having experienced a series of admissions because of unclear hepatopathy, growth retardation, and renal tubular acidosis. CONCLUSION: Prolonged tyrosyluria in the newborn and young baby should cause the clinical chemist not only to exclude tyrosinaemia, galactosaemia, and fructose intolerance but also to look carefully for hawkinsin in the aminoacid chromatogram.

Fully automated assay for total homocysteine, cysteine, cysteinylglycine, glutathione, cysteamine, and 2-mercaptopropionylglycine in plasma and urine.

We describe a 6-min HPLC method to measure the total concentrations of the most important thiols in plasma and urine--cysteine, homocysteine, cysteinylglycine, and glutathione--as well as the concentrations in plasma and urine, respectively, of cysteamine and 2-mercaptopropionylglycine, two compounds used to treat disorders of cysteine metabolism. Precolumn derivatization with bromobimane and reversed-phase HPLC were performed automatically by a sample processor. Throughput was up to 100 samples in 24 h. The within-run CV ranged from 0.9% to 3.4% and the between-run CV ranged from 1.5% to 6.1%. Analytical recovery was 97-107%, with little difference between plasma and urine samples. The detection limit was approximately 50 nmol/L for all the analytes studied. Thiol concentrations were determined in the plasma of 206 healthy donors and in the urine of 318 healthy donors distributed for age and sex. Mean values of plasma cysteine and homocysteine were significantly lower in infants (ages, <1 y) compared with other age groups (P <0.005). In adults, mean plasma homocysteine values were higher in males than in females (9.2 vs 6.7 micromol/L, P <0.0001) and in the 6- to 10-year-old group (P <0.05). Mean values for glutathione and cysteinylglycine were not sex- and age-dependent. In urine, both cysteine and homocysteine showed a wide range of variation.

Evidence of taurine depletion and accumulation of cysteinesulfinic acid in chronic dialysis patients.

Methionine, taurine and cysteinesulfinic acid (CSA) were determined by reversed-phase high-performance liquid chromatography (RP-HPLC) in plasma from ten patients treated with hemodialysis (HD) and eight patients treated with continuous ambulatory peritoneal dialysis (CAPD). The patients' data were compared with data obtained from ten healthy controls. Significant reductions in plasma taurine levels were observed in the HD patients (34 +/- 13 mumol/liter, mean +/- SD) and the CAPD patients (47 +/- 12 mumol/liter) compared to the controls (66 +/- 5 mumol/liter), while the CSA levels were markedly higher in the HD patients (9.1 +/- 2.8 mumol/liter) and the CAPD patients (9.1 +/- 2.4 mumol/liter) than in the controls (0.79 +/- 0.15 mumol/liter). A single HD treatment significantly reduced the plasma taurine and CSA concentrations (P < 0.01 and P < 0.001), respectively. The plasma methionine levels were normal in both patient groups. The finding of a low plasma taurine level and a large accumulation of CSA suggests that the metabolic conversion of CSA to taurine is impaired in uremic patients and this metabolic abnormality may cause taurine depletion.

Aminoethylcysteine ketimine decarboxylated dimer detected in normal human urine by gas-liquid chromatography, selected-ion monitoring and mass spectrometry.

Aminoethylcysteine ketimine is a biochemical product known to be converted spontaneously in the decarboxylated dimer. Since the ketimine has been detected in a mammalian brain, it was assumed that also the dimer could be present in the mammalian body and eventually excreted in the urine. Using human urine as the biological source, an extract was prepared which, submitted to gas-liquid chromatography, selected-ion monitoring and mass spectrometry, indicated the presence of the dimer.

Twenty-four hour felinine [corrected] excretion patterns in entire and castrated cats.

The purpose of this study was to determine the 24 h urinary excretion of a sulphur containing amino acid called felinine in entire and castrated cats of both sexes. Entire male cats excreted (mean +/- SEM) 122 +/- 23.6 mmol of felinine per kg bodyweight per day with castrated males, entire females and spayed females excreting 41 +/- 8.4, 36 +/- 7.3 and 20 +/- 3.8 mmol, respectively. There was an overall significant difference between groups in the amounts of felinine excreted in 24 h [F(3, 24) = 11.8, p < 0.0001] with there being significant differences between entire males and castrated males (p < 0.001) and castrated males and spayed females (p < 0.05). There was no difference in excretion between entire and spayed females. Urine volumes were not significantly different for the 24 h period. The differences in excretion levels were caused by different concentrations of felinine in the urine with entire male cats excreting (mean +/- SEM) 2.0 +/- 0.55 g of felinine per litre of urine. The data obtained in the present study support the concept that felinine, which has been found in Felidae species only, may be testosterone dependent. Felinine may be involved in territorial marking.

Hawkinsinuria in two families.

Hawkinsinuria, a disorder of tyrosine metabolism has been documented in two families in the United States, in one of which there was clear evidence of autosomal dominant inheritance. Metabolic acidosis and failure to thrive appear to be confined to infancy. Tyrosyl metabolites and 5-oxoproline are also found only in infancy, while 4-hydroxycyclohexylacetic acid was present only with time. The disease may be detected by organic acid analysis or by staining an electropherogram for sulfur containing compounds.

Identification of NAc-HCPC and NAc-beta-CEC, and qualitative analyses of sulphur amino acids in the urine of a patient with cystathioninuria using liquid chromatography/atmospheric pressure ionization mass spectrometry.

Standard sulphur amino acids and various cystathionine metabolites in the urine of a patient with cystathioninuria were analysed using liquid chromatography/mass spectrometry with an atmospheric pressure ionization interface system. Very intense quasi-molecular ions ([M + H]+) of synthetic cystathionine, N-monoacetylcystathionine, perhydro-1,4-thiazepine-3,5-dicarboxylic acid, S-(3-hydroxy-3-carboxy-n-propyl)cysteine, S-(2-carboxyethyl) cysteine, S-(2-hydroxy-2-carboxyethyl)homocysteine, S-(carboxymethyl)homocysteine, N-acetyl-S-(3-hydroxy-3-carboxy-n-propyl)cysteine and N-acetyl-S-(2-carboxyethyl)cysteine were observed by this method. Quasi-molecular ions ([M + H]+) of these sulphur amino acids were observed in the urine sample of the patient with cystathioninuria, and N-acetyl-HCPC and N-acetyl-beta-CEC as N-substituted sulphur amino acids were also identified in the urine of the same patient.

Influence of diet on cystathionine ketimine and lanthionine ketimine content in human urine.

A simple HPLC procedure for the routine analyses of Cystathionine ketimine (CK) and Lanthionine ketimine (LK) content in human urine has been developed. The values obtained in morning urine of fifteen healthy subjects (both sexes, 25-45 years old) on a common mixed diet are 330-2480 micrograms/g creatinine (mean 1110) of CK and 100-420 micrograms/g creatinine (mean 200) of LK. Quantitation of the two ketimines in urine of subjects on strictly vegetarian diet indicate that while the excretion of LK is independent of the diet, the excretion of CK is significantly decreased in conditions of vegetarian diet.

A therapeutic trial with N-acetylcysteine in subjects with hereditary glutathione synthetase deficiency (5-oxoprolinuria).

In a therapeutic trial, the effect of short-term low-dosage N-acetylcysteine supplementation on glutathione metabolism was investigated in two patients with hereditary glutathione deficiency (5-oxoprolinuria). Clinical and neurophysiological examinations of the patients indicated progressive neurological damage. The pretreatment concentrations of total and free glutathione in leukocytes were 15-20% of normal, whereas the corresponding gamma-glutamylcysteine levels were increased. In plasma, the glutathione concentrations were similarly decreased, but no gamma-glutamylcysteine was detected. Total glutathione in erythrocytes was markedly decreased. Low urinary excretion of cysteinylglycine, cyst(e)ine, taurine, N-acetylcysteine, mercaptolactate and mercaptoacetate and reduced leukocyte taurine levels constituted additional evidence of decreased intracellular availability of cysteine, i.e. glutathione. Oral supplementation with N-acetylcysteine (5 mg/kg x 3/day) had no effect on acid-base balance, erythrocyte glutathione levels or 5-oxoproline concentrations in plasma and urine. In leukocytes, the glutathione concentrations were increased by 20-30%, whereas the gamma-glutamylcysteine levels were essentially unaltered. In parallel, the urinary excretion of cysteinylglycine was increased and the leukocyte levels and urinary outputs of sulphur amino acids were restored. No side-effects of the treatment were noted. The results indicate that N-acetylcysteine may be of value in increasing the low intracellular glutathione concentrations and cysteine availability in patients with hereditary glutathione synthetase deficiency.