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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 (Abs max ) 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 Abs max in healthy males (R 2  = 0.439, P = 0.000), females (R 2  = 0.245, P = 0.000), female stroke patients (R 2  = 0.187, P = 0.000), but not in male stroke patients (R 2  = 0.008, P = ns). Fibrin CLT correlated with age in healthy females but not males while fibrin Abs max 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 Abs max , while urinary (u)HTL, uCysGly, and pCysGly were significantly associated with fibrin CLT. In healthy individuals, uHTL and uGSH were significantly associated with fibrin Abs max , while pGSH, and CBS T833C 844ins68 polymorphism were associated with fibrin CLT. In logistic regression, uHTL, uHcy, pCysGly, pGSH, MTHFR C677T polymorphism, and Abs max 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., (© 2024. The Author(s).)

Two cheap, simple and reproducible methods for the electrophoretic determination of homocysteine thiolactone (HTL) in human urine have been developed and validated. The first method utilizes off-line single drop microextraction (SDME), whereas the second one uses off-line SDME in combination with field amplified sample injection (FASI). The off-line SDME protocol consists of the following steps: urine dilution with 0.2 mol/L, pH 8.2 phosphate buffer (1:2, v/v ), chloroform addition, drop formation and extraction of HTL. The pre-concentration of HTL inside a separation capillary was performed by FASI. For sample separation, the 0.1 mol/L pH 4.75 phosphate buffer served as the background electrolyte, and HTL was detected at 240 nm. A standard fused-silica capillary (effective length 55.5 cm, 75 μm id) and a separation voltage of 21 kV (~99 μA) were used. Electrophoretic separation was completed within 7 min, whereas the LOD and LOQ for HTL were 0.04 and 0.1 μmol/L urine, respectively. The calibration curve in urine was linear in the range of 0.1-0.5 μmol/L, with R 2 = 0.9991. The relative standard deviation of the points of the calibration curve varied from 2.4% to 14.9%. The intra- and inter-day precision and recovery were 6.4-10.2% (average 6.0% and 6.7%) and 94.9-102.7% (average 99.7% and 99.5%), respectively. The analytical procedure was successfully applied to the analysis of spiked urine samples obtained from apparently healthy volunteers.

Aminothiols are closely related to chronic kidney disease, but little is known regarding levels of related aminothiols in the urine of immunoglobulin A vasculitis with nephritis (IgAVN) patients. Herein, a well-defined core-shell Zr-based metal-organic framework (Zr-MOF) composite SiO 2 @50Benz-Cys was constructed as a mercury ion affinity material via a solvent-assisted ligand exchange strategy for the selective extraction and enrichment of low-concentration aminothiols in IgAVN patient urine. SiO 2 @50Benz-Cys was competent to enrich the total glutathione (GSH) and total homocysteine (Hcy) in virtue of the excellent affinity after chelation with mercury ions. The extraction efficiencies were closely related to the pH, dithiothreitol amount, and the dose of functional Zr-MOF. Coupled with HPLC-MS/MS in optimized conditions, GSH and Hcy were determined with low detection limits of 0.5 and 1 nmol L -1 , respectively. The recoveries of GSH and Hcy for the urine sample at three spiked levels were in the range of 85.3-105% and 79.5-103%, which showed good precision and accuracy. Benefiting from the matrix interference elimination in the process of extraction, the simultaneous detection of aminothiols in the urine of the healthy group and immunoglobulin A vasculitis (IgAV) and IgAVN patients was successfully carried out, suggesting that the Zr-MOF and the robust method together provided a potential application in the analysis of urinary biomolecules. The analysis of variance (ANOVA) showed that the levels of GSH and Hcy had significant differences between the patients and the control. This work is very valuable as it provides a better understanding of concentration alterations of GSH and Hcy in urine involved with IgAVN for clinical research.

Background: Hyperhomocysteinemia is considered a possible contributor to the complex pathology of Alzheimer's disease (AD). For years, researchers in this field have discussed the apparent detrimental effects of the endogenous amino acid homocysteine in the brain. In this study, the roles of hyperhomocysteinemia driven by vitamin B deficiency, as well as potentially beneficial dietary interventions, were investigated in the novel App NL-G-F knock-in mouse model for AD, simulating an early stage of the disease., Methods: Urine and serum samples were analyzed using a validated LC-MS/MS method and the impact of different experimental diets on cognitive performance was studied in a comprehensive behavioral test battery. Finally, we analyzed brain samples immunohistochemically in order to assess amyloid-β (Aβ) plaque deposition., Results: Behavioral testing data indicated subtle cognitive deficits in App NL-G-F compared to C57BL/6J wild type mice. Elevation of homocysteine and homocysteic acid, as well as counteracting dietary interventions, mostly did not result in significant effects on learning and memory performance, nor in a modified Aβ plaque deposition in 35-week-old App NL-G-F mice., Conclusion: Despite prominent Aβ plaque deposition, the App NL-G-F model merely displays a very mild AD-like phenotype at the investigated age. Older App NL-G-F mice should be tested in order to further investigate potential effects of hyperhomocysteinemia and dietary interventions.

This review presents clinical patterns that should trigger homocysteine measurement in blood, as well as the further diagnostic work-up focused on inborn errors of metabolism and disorders of vitamin B12 (cobalamin) absorption and supply. The numerous conditions (e.g. cardiovascular disease, Alzheimer's disease) for which mild-to-moderate hyperhomocysteinaemia caused by genetic polymorphisms or acquired reasons is considered a risk factor are beyond the scope of this review.Homocysteine is a sulphur-containing amino acid, which is derived from the amino acid methionine. Homocysteine is either trans-sulphurated to form cystathionine and then cysteine, or re-methylated to methionine. The trans-sulphuration reaction depends on the enzyme cystathionine beta synthase and its cofactor vitamin B6. The re-methylation reaction not only involves the enzymes methionine synthase and methionine synthase reductase but also depends on the cofactor cobalamin and on the provision of methyl groups from the folate cycle. Because the homocysteine-methionine cycle provides for the vast majority of methyl groups in the body, it is central to numerous pathways that depend on methyl group supply, such as creatine synthesis or DNA methylation. Based on this premise, the severity of clinical presentations of inborn errors of metabolism, such as classical homocystinuria or the cobalamin C (cblC) defect, affecting this pathway is unsurprising.

Homocysteine thiolactone (HTL) is a chemically reactive thioester that has been implicated in cardiovascular disease. So far, its presence has been documented in human and mouse plasma and urine. Here, using a new method, we show that HTL is present in human saliva. The assay involves chloroform-methanol extraction of HTL, lyophilization, and derivatization with N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA) and trimethylchlorosilane (TMCS). The method is based on a gas chromatography coupled with mass spectrometry (GC-MS) and quantifies HTL in a linear range from 0.05 to 1 µmol L -1 saliva and urine. The limit of quantification (LOQ) was 0.05 µmol L -1 . With respect to saliva specimen, the accuracy was 98.7-112.6%, and 90.2-100.5%, while the precision was 7.1-13.5% and 12.5-15.0% for the intra- and inter-day variation, respectively. In relation to urine samples, the accuracy was 91.9-110.9% and 91.2-103.3%, while the precision varied from 2.2% to 14.5% and 7.4% to 14.3% for intra- and inter-day measurements, respectively. Using this method, we show that in apparently healthy individuals (n = 18), HTL levels in saliva are not positively correlated with urinary HTL levels. Undoubtedly, larger population should be investigated to get more meaningful results., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A microfluidic paper-based analytical device integrating carbon dot (CDs) is fabricated and used for a fluorometric off-on assay of biothiols. Vinyl sulfone (VS) click immobilization of carbon dots (CDs) on paper was accomplished by a one-pot simplified protocol that uses divinyl sulfone (DVS) as a homobifunctional reagent. This reagent mediated both the click oxa-Michael addition to the hydroxyl groups of cellulose and ulterior covalent grafting of the resulting VS paper to NH 2 -functionalized CDs by means of click aza-Michael addition. The resulting cellulose nanocomposite was used to engineer an inexpensive and robust microfluidic paper-based analytical device (μPAD) that is used for a reaction-based off-on fluorometric assay of biothiols (GSH, Cys, and Hcy). The intrinsic blue fluorescence of CDs (with excitation/emission maxima at 365/450 nm) is turned off via the heavy atom effect of an introduced iodo group. Fluorescence is turned on again due to the displacement of iodine by reaction with a biothiol. The increase in fluorescence is related to the concentration over a wide range (1 to 200 μM for GSH and 5-200 μM for Cys and Hcy, respectively), and the assay exhibits a low detection limit (0.3 μM for GSH and Cys and 0.4 μM for Hcy). The method allows for rapid screening and can also be used in combination with a digital camera readout. Graphical abstract Schematic representation of a μPAD based on click immobilized carbon dots and used for a reaction-based fluorometric off-on assay of biothiols. The intrinsic blue fluorescence of carbon dots is turned off via the heavy atom effect of an introduced iodo group and turned on by the displacement of this atom by reaction with a biothiol.

A simple, fast, sensitive and reproducible capillary zone electrophoresis (CZE) method with single drop microextraction (SDME) for determination of homocysteine thiolactone (HTL) in human urine has been developed and validated. The method is characterized by good precision, high accuracy, short analysis time and low consumption of reagents. The procedure consists only of few steps: urine sample centrifugation, dilution with phosphate buffer and methanol, chloroform addition onto the top of donor phase, on-line SDME in CE system, sample separation by CZE and ultraviolet detection of HTL at 240 nm. The background electrolyte was 0.1 M pH 4.75 phosphate buffer. Effective separation was achieved within 6.04 min under the separation voltage of 24 kV (~110 μA). The LOQ and LOD for HTL were 50 and 25 nM urine, respectively. The calibration curve in urine showed linearity in the range of 50-200 nM, with R 2 0.9995. The intra- and inter-day precision and recovery were 4.0-14.5% (average 8.7% and 9.3%) and 92.7-115.5% (average 103.6% and 104.8%), respectively. The procedure was successfully applied to analysis of urine samples., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Classical homocystinuria (HCU) is a genetic disorder caused by mutations in the cystathionine beta synthase gene, which results in impaired metabolism of the sulfur-bearing amino acid homocysteine and its accumulation in blood and tissues. Classical HCU can be detected via newborn screening in the United States, but the test is widely acknowledged to miss many patients. While severely elevated homocysteine levels (>100 μmol /L) frequently lead to a classical HCU diagnosis, intermediate levels (>30 to 100 μmol /L), though linked to many of the known complications of HCU, are not always recognized as associated with HCU. We aimed to identify and describe potentially undiagnosed classical HCU patients using a nationally-representative database of administrative claims and laboratory results. We estimated the national prevalence of patients with homocysteine >30 μmol /L, and compared their demographic and clinical characteristics to those of patients with homocysteine levels ≤30 μmol/L. Among 57,580 patients with a homocysteine test result, 1.8% had a value >30 μmol /L. Patients with homocysteine >30 μmol /L were more frequently diagnosed with hypothyroidism (39.2% vs. 20.7%, p < .001) and renal disease (9.7% vs. 5.5%, p < .001), and were more likely to have a prescription for an anxiolytic/antidepressant (44.5% vs. 38.9%), opioid (58.4% vs. 53.1%), steroid (46.4% vs. 42.5%), or thyroid hormone (38.8% vs. 18.8%), compared to patients with homocysteine ≤30 μmol /L (all p < .05). Both groups were equally likely to have a diagnosis of homocystinuria or another disorder of sulfur-bearing amino acid metabolism (3.8% vs. 4.0%, p = .752). The age-adjusted national prevalence of homocysteine >30 μmol /L was estimated at 33,068 (95% CI: 1033 - 35,104). These findings suggest that thousands of people in the US may be living with intermediate to severely elevated homocysteine levels and may require further evaluation for the presence of classical HCU., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

In this study, the levels of concentration of homocysteine thiolactone (HTL), cysteine (Cys), and cysteinylglycine (CysGly) in the urine of autistic and non-autistic children were investigated and compared. HTL has never been analyzed in autistic children. The levels of low molecular weight sulfur compounds in the urine of both groups were determined by validated methods based on high-performance liquid chromatography with spectrofluorometric and diode-array detectors. The statistical data show a significant difference between the examined groups. Children with autism were characterized by a significantly higher level of HTL (p = 5.86 × 10 -8 ), Cys (p = 1.49 × 10 -10 ) and CysGly (p = 1.06 × 10 -8 ) in urine compared with the control group. A difference in the p-value of <0.05 is statistically significant. Higher levels of HTL, Cys, and CysGly in the urine of 41 children with autism, aged 3 to 17, were observed. The obtained results may indicate disturbances in the metabolism of methionine, Cys, and glutathione in some autistic patients. These preliminary results suggest that further research with more rigorous designs and a large number of subjects is needed.

While the targeted analysis of mercapturic acid (MA) metabolites in human urine is used to assess exposure to selected chemicals, this compound class has only rarely been addressed in non-target screening utilizing diagnostic neutral loss liquid chromatography tandem mass spectrometry (LC-MS/MS). Additionally, this type of analysis is severely affected by matrix effects (MEs) causing poor comparability of samples and distortion of signal intensities. However, MEs have been neglected in urinary MA non-target screening so far. Therefore, we developed a non-target screening method relying on neutral loss scanning for MAs using post column infusion of an isotope-labelled standard. For signal correction, we synthesized a structural analogue to MAs, N-acetyl-S-methyl-homocysteine-D 3 , lacking the characteristic neutral loss of the MAs. For method development, 16 structurally different model MA compounds and 20 spiked urine samples were used. Twelve out of the 16 model compounds could be analysed by the developed method. We found severe matrix effects (largely signal suppression) for the spiked model compounds, with only 34% of all peaks' intensities changing by less than a factor of two. This could be compensated by the post column internal standard infusion with now 68% of all peaks' intensities changing by less than a factor of two. For three compounds, an over-compensation was observed resulting in an increase of signal of up to a factor of 16. In the 20 urine samples, altogether 558 native MAs (between 74 and 175 per sample) could be detected after ME compensation. These results indicate that a large number of so far uncharacterized MAs are present in urine, which yield a potential for biomarker discovery and pattern characterisation. Graphical Abstract.

Biothiols play important roles in regulating redox balance in biological systems, but their discrimination is challengeable. In this work, a colorimetric nanosensing array for biothiols was established, which was composed of gold nanorods (AuNRs) and metal ions (Hg 2+ , Pb 2+ , Cu 2+ , Ag + ). By employing label-free AuNRs as the colorimetric probe, and the color and spectral changes of AuNRs as the output signal, principal component analysis (PCA) was applied to processing the signal and generating a clustering map. Due to the different binding affinity between biothiols and metal ions, AuNRs exhibited a unique pattern to form a fingerprint-like colorimetric array, which was able to discriminate five biothiols by the naked eyes. This strategy combines PCA and sensor array to achieve rapid and accurate discrimination and detection of biothiols. In addition, the method shows the great potential in analysis of biothiols in human urine samples., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The role of homocysteic acid (HCA) in severe diseases like Alzheimer's disease is under discussion and some recent studies correlate elevated HCA concentrations with the diagnosis of Alzheimer's. However, non-selective and insufficiently sensitive methods have been used to quantitate HCA and results of different studies show large differences in the determined HCA concentration in samples from patients and controls, and therefore non-comparable results. An accurate and precise quantitation method for the determination of HCA in human serum, urine and CSF has been developed by using a combination of protein precipitation and solid phase extraction for sample preparation followed by an LC-MS/MS analysis using a combination of a HILIC separation and tandem mass spectrometry. The developed method has been fully validated in accordance with the guidelines provided by the US Food and Drug administration FDA and the European Medicines Agency EMA. Furthermore, the method has demonstrated its ability to determine the endogenous HCA concentration in serum and urine samples from healthy volunteers., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Postpartum depression affects about 10-20% of newly delivered women, which is harmful for both mothers and infants. However, the current diagnosis of postpartum depression depends on the subjective judgment of a practitioner, which may lead to misdiagnosis. Hence, an appended objective diagnosis index may help the practitioner to improve diagnosis. A metabolomic study can find biomarkers as an objective index to facilitate disease diagnosis. Forty-nine postpartum depressed patients and 50 healthy controls were recruited into this study. The metabolites in urine were scanned with LC-Q-TOF-MS. The metabolomic data were analyzed with a multivariate statistical analysis method. Data from 40 patients and 40 controls were used for partial least square-discriminate analysis (PLS-DA). The urine metabolomic profiles of patients were different from those of controls. The PLS-DA model was validated by a permutation test, and the model could accurately classify the other 9 patients and 10 controls in T-prediction. Ten differentiating metabolites were found as main contributors to this difference, which are involved in amino acid metabolism, neurotransmitter metabolism, bacteria population, etc. Some of these potential biomarkers, such as 4-hydroxyhippuric acid, homocysteine, and tyrosine, showed relatively high sensitivities and specificities. The metabolic profile alteration induced by postpartum depression was found, and some of the differentiating metabolites may serve as biomarkers to facilitate the diagnosis of postpartum depression.

Objectives: No individual homocysteine (Hcy) metabolite has been studied as a risk marker for coronary artery disease (CAD). Our objective was to examine Hcy-thiolactone, a chemically reactive metabolite generated by methionyl-tRNA synthetase and cleared by the kidney, as a risk predictor of incident acute myocardial infarction (AMI) in the Western Norway B-Vitamin Intervention Trial., Design: Single centre, prospective double-blind clinical intervention study, randomized in a 2 × 2 factorial design., Subjects and Methods: Patients with suspected CAD (n = 2049, 69.8% men; 61.2-year-old) were randomized to groups receiving daily (i) folic acid (0.8 mg)/vitamin B 12 (0.4 mg)/vitamin B 6 (40 mg); (ii) folic acid/vitamin B 12 ; (iii) vitamin B 6 or (iv) placebo. Urinary Hcy-thiolactone was quantified at baseline, 12 and 38 months., Results: Baseline urinary Hcy-thiolactone/creatinine was significantly associated with plasma tHcy, ApoA1, glomerular filtration rate, potassium and pyridoxal 5'-phosphate (positively) and with age, hypertension, smoking, urinary creatinine, plasma bilirubin and kynurenine (negatively). During median 4.7-years, 183 patients (8.9%) suffered an AMI. In Cox regression analysis, Hcy-thiolactone/creatinine was associated with AMI risk (hazard ratio = 1.58, 95% confidence interval = 1.10-2.26, P = 0.012 for trend; adjusted for age, gender, tHcy). This association was confined to patients with pyridoxic acid below median (adjusted HR = 2.72, 95% CI = 1.47-5.03, P = 0.0001; P interaction = 0.020). B-vitamin/folate treatments did not affect Hcy-thiolactone/creatinine and its AMI risk association., Conclusions: Hcy-thiolactone/creatinine ratio is a novel AMI risk predictor in patients with suspected CAD, independent of traditional risk factors and tHcy, but modified by vitamin B 6 catabolism. These findings lend a support to the hypothesis that Hcy-thiolactone is mechanistically involved in cardiovascular disease., (© 2018 The Authors Journal of Internal Medicine published by John Wiley & Sons Ltd on behalf of Association for Publication of The Journal of Internal Medicine.)

It is well established that homocysteine thiolactone (HTL) is associated with some health disorders, including cardiovascular diseases. HTL is a by-product of sulfur metabolic cycle. So far, its presence has been confirmed in human plasma and urine. It has been also shown that a vast majority of HTL is removed from human body through kidney. Thus, the aim of the current investigations has been the identification, separation and quantification of HTL in urine samples. For the first time a cheap, reliable and robust GC-MS method was developed for the determination of HTL in human urine in the form of its volatile isobutyl chloroformate derivative. Separation of the analyte and internal standard (homoserine lactone (HSL)) was achieved in 15 min followed by mass spectrometry detection (MS). Isocratic elution was accomplished with helium at a flow rate of 1 mL min -1 and a gradient of the column temperature was concomitant with the analysis. The mass spectrometer was set to the electron impact mode at 70 eV. The ion source, quadrupole and MS interface temperatures were set to 230 °C, 150 °C and 250 °C, respectively. Elaborated analytical procedure allows quantification of analyte in a linear range of 0.01-0.20 nmol mL -1 urine. The LOQ and LOD values were 0.01 and 0.005 nmol mL -1 , respectively. The method accuracy ranged from 98.0% to 103.2%, while precision varied from 6.4% to 9.5% and from 10.7% to 16.9% for intra- and inter-day measurements, respectively. Finally, the method has been successfully implemented in the analysis of 12 urine samples donated by apparently healthy volunteers. Concentration of HTL ranged from -1 urine (0.51 to 13.1 μmol mol -1 Crn)., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We report a new approach to create metal-binding site in a series of metal-organic frameworks (MOFs), where tetratopic carboxylate linker, 4',4'',4''',4''''-methanetetrayltetrabiphenyl-4-carboxylic acid, is partially replaced by a tritopic carboxylate linker, tris(4-carboxybiphenyl)amine, in combination with monotopic linkers, formic acid, trifluoroacetic acid, benzoic acid, isonicotinic acid, 4-chlorobenzoic acid, and 4-nitrobenzoic acid, respectively. The distance between these paired-up linkers can be precisely controlled, ranging from 5.4 to 10.8 Å, where a variety of metals, Mg 2+ , Al 3+ , Cr 3+ , Mn 2+ , Fe 3+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , Ag + , Cd 2+ and Pb 2+ , can be placed in. The distribution of these metal-binding sites across a single crystal is visualized by 3D tomography of laser scanning confocal microscopy with a resolution of 10 nm. The binding affinity between the metal and its binding-site in MOF can be varied in a large range (observed binding constants, K obs from 1.56×10 2 to 1.70×10 4  L mol -1 ), in aqueous solution. The fluorescence of these crystals can be used to detect biomarkers, such as cysteine, homocysteine and glutathione, with ultrahigh sensitivity and without the interference of urine, through the dissociation of metal ions from their binding sites., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background and Objective: Homocysteine (Hcy) is a non-protein α-amino acid, which plays several important roles in human physiology and in the central nervous system. Although Hcy has several known biological properties in one-carbon metabolism, its overproduction might be harmful, and could add to the pathophysiology associated with ASD. We reviewed the current evidence about changes in Hcy concentration in ASD and tried to correlate its changes with the clinical profile Discussion: The concentration of the amino acid in biological fluids (blood and urine) in children/ youngs with ASD is increased in the majority of studies when comparing to typically developing control subjects. Some report demonstrated a significant association between the severity of the disorder and the abnormalities in Hcy levels., Conclusion: Further research is needed to correlate the increase in Hcy with specific symptoms/ deficits in ASD and to evaluate the clinical impact of strategies that can reduce Hcy concentration in ASD., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

A rapid and selective method has been developed for highly sensitive determination of total cysteine and homocysteine levels in human blood plasma and urine by capillary electrophoresis (CE) coupled with liquid-liquid extraction. Analytes were first derivatized with 1,1'-thiocarbonyldiimidazole and then samples were purified by chloroform-ACN extraction. Electrophoretic separation was performed using 0.1 M phosphate with 30 mM triethanolamine, pH 2, containing 25 μM CTAB, 2.5 μM SDS, and 2.5% polyethylene glycol 600. Samples were injected into the capillary (with total length 32 cm and 50 μm id) at 2250 mbar*s and subsequent injection was performed for 30 s with 0.5 M KОН. The total analysis time was less than 9 min, accuracy was 98%, and precision was <2.6%. The LOD was 0.2 μM for homocysteine and 0.5 μM for cysteine. The use of liquid-liquid extraction allowed the precision and sensitivity of the CE method to be significantly increased. The validated method was applied to determine total cysteine and homocysteine content in human blood plasma and urine samples obtained from healthy volunteers and patients with kidney disorders., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A fast and simple HPLC-based assay has been developed for the simultaneous determination of homocysteine (Hcy) and methionine (Met) in plasma and urine samples, utilizing as small volume of sample as 10μL. The assay uses on-column derivatization with o-phthaldialdehyde. The separation of Hcy and Met was achieved in 14min on a reversed phase C-18 column, followed by fluorescence detection (excitation at 348nm and emission at 438nm for Met; excitation at 370nm and emission at 480nm for Hcy). Linearity of the detector response was observed in the range of 2-60 μmol L -1 for Met and 2-40 μmol L -1 for Hcy. The method was successfully applied for Met and Hcy quantification in human and mouse plasma and urine samples from cystathionine β-synthase deficient and unaffected individuals., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Homocysteine (Hcy) and its metabolites Hcy-thiolactone, N-Hcy-protein, and S-Hcy-protein are implicated in vascular and neurological diseases. However, quantification of these metabolites remains challenging. Here I describe streamlined assays for these metabolites based on their conversion to Hcy-thiolactone. Free Hcy-thiolactone is extracted from the urine with chloroform/methanol. Total Hcy is converted to Hcy-thiolactone in the presence of 1 N HCl. Major urinary protein (MUP)-bound S-linked Hcy is liberated from the protein by reduction with dithiothreitol and converted to Hcy-thiolactone. Acid hydrolysis of MUP with 6 N HCl liberates N-linked Hcy as Hcy-thiolactone, which is then extracted with chloroform/methanol. Ferritin is used as an N-Hcy-protein standard and an authentic Hcy-thiolactone is used to monitor the efficiency of extraction. Hcy-thiolactone (free, derived from total Hcy, or from MUP-bound N-linked or S-linked Hcy) is separated by a cation exchange high-performance liquid chromatography, post-column derivatized with o-phthaldialdehyde, and quantified by fluorescence. Using these assays with as little as 2-20 μL of urine I show that MUP carry N-linked and S-linked Hcy and that N-Hcy-MUP and S-Hcy-MUP and Hcy-thiolactone are severely elevated in cystathionine β-synthase-deficient mice. These assays will facilitate examination of the role of protein-related Hcy metabolites in health and disease., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The development of sensitive fluorescence probes to detect biothiols such as cysteine and homocysteine has attracted great attention in recent times. Herein, we described the design and synthesis of coumarin based long-wavelength fluorescence probe, Bromo-2-benzothiazolyl-3-cyano-7-hydroxy coumarin (BBCH, 2) for selective detections of cysteine and homocysteine. The probe is rationally designed in such a way that both sulfhydryl and adjacent amino groups of thiols are involved in sensing process. Only cysteine/homocysteine able to react with BBCH to release fluorescence reporter (BCH, 1); while, glutathione and other amino acids unable to react with BBCH due to the absence of adjacent amino groups. In presence of cysteine, the color of BBCH is turns from colorless to red and thus BBCH is a naked eye fluorescence indicator for cysteine. Besides, BBCH can discriminate cysteine and homocysteine based on color changes and different reaction rates. The described sensing platform showed good sensing performances to detect cysteine and homocysteine with detection limits of 0.87 and 0.19 μM, respectively. Practical applicability was verified in biological and pharmaceutical samples.

When the biosynthesis of homocysteine (Hcy), an amino acid containing thiol, exceeds its elimination, plasma Hcy concentration increases and results in hyperhomocysteinemia (HHC). Most of the Hcy in plasma covalently binds to the cysteine residues of albumin by disulfide bonds. Meanwhile tiopronin (TP), a thiol-containing compound, is used for treatment of cysteinuria to decrease the amount of insoluble cystine in urine, by replacing a cysteine molecule in cystine with TP to form soluble TP-cysteine complexes. The present study investigated the effect of TP on the total (protein-unbound and protein-bound) Hcy concentration and the ratio of protein-unbound Hcy to total Hcy in plasma, and the urinary excretion of Hcy. Methionine was administered orally to rats to induce temporary hyperhomocysteinemia, and then TP was administered and the plasma and urine were collected. The amount of Hcy excreted in urine was higher but the plasma concentration of Hcy was lower in the TP group than in the control group until 3 h after TP administration. In addition the ratio of protein-unbound Hcy in plasma tended to be increased by TP administration. These results demonstrated that TP enhanced the urinary excretion of Hcy, which might cause the decrease of its plasma concentration in rats., (Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Autism spectrum disorders (ASDs) are a major health problem because of their high prevalence in the general population. The pathophysiology of ASD remains unclear, although genetic defects may be detected in 10-20% of affected patients. Among these cases, the prevalence of inherited metabolic disorders (IMD) has not been extensively evaluated. IMDs responsible for ASDs are usually identified via clinical manifestations such as microcephaly, dysmorphic features, convulsions, and hepatosplenomegaly. Infrequently, patients with no additional clinical symptoms suggestive of an IMD may be diagnosed as having an idiopathic ASD. High consanguinity rates have resulted in an increased prevalence of IMDs in the Turkish population. The aim of this study was to explore the benefits of systematic screening for IMD among Turkish patients with ASDs. In our study, data were retrospectively collected for 778 children with ASDs. In all cases, the metabolic investigations included an arterial blood gas analysis, serum ammonia and lactate levels, a quantitative plasma amino acid analysis, a whole blood acylcarnitine profile via tandem mass spectrometry and a urine organic acid profile. Urinary glycosaminoglycan levels and homocysteine levels were screened in selected cases; 300 of the 778 patients with ASDs whose physical and metabolic investigations were complete and met this study's criteria were enrolled. Among the 300 children with autism, IMD were diagnosed in nine patients as follows: two patients were diagnosed with phenylketonuria, and one patient was diagnosed with partial biotinidase deficiency; one patient was diagnosed with mucopolysaccharidosis type III, and one patient was diagnosed with classical homocystinuria; one patient was diagnosed with glutaric acidemia type 1, and one patient was diagnosed with short chain acyl-CoA dehydrogenase deficiency; one patient was diagnosed with argininemia, and one patient was diagnosed with L-2-hydroxyglutaric aciduria., (© 2015 International Society for Autism Research, Wiley Periodicals, Inc.)

The clinical significance of high levels of homocysteine in autism spectrum disorder (ASD) is unknown. An experimental study was conducted in order to evaluate the concentration of homocysteine in children with ASD and typically developing children and to analyse any relationships with the severity of core symptoms of ASD and other clinical features (drugs, co-morbidities, gender, age, diet). Core symptoms of autism were evaluated by DSM-IV criteria. Homocysteine, glutathione, methionine, 3-nitrotyrosine were measured in urine. The increase in homocysteine concentration was significantly and directly correlated with the severity of the deficit in communication skills, but was unrelated to deficit in socialisation or repetitive/restricted behaviour. Urinary homocysteine concentration may be a possible biomarker for communication deficits in ASD and a potential diagnostic tool useful to evaluate new treatment options since no treatment for core symptoms of ASD are available., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: Limited data are available from controlled studies on biomarkers of maternal vitamin B-12 status., Objective: We sought to quantify the effects of pregnancy and lactation on the vitamin B-12 status response to a known and highly controlled vitamin B-12 intake., Methods: As part of a 10-12 wk feeding trial, pregnant (26-29 wk gestation; n = 26), lactating (5 wk postpartum; n = 28), and control (nonpregnant, nonlactating; n = 21) women consumed vitamin B-12 amounts of ∼8.6 μg/d [mixed diet (∼6 μg/d) plus a prenatal multivitamin supplement (2.6 μg/d)]. Serum vitamin B-12, holotranscobalamin (bioactive form of vitamin B-12), methylmalonic acid (MMA), and homocysteine were measured at baseline and study-end., Results: All participants achieved adequate vitamin B-12 status in response to the study dose. Compared with control women, pregnant women had lower serum vitamin B-12 (-21%; P = 0.02) at study-end, whereas lactating women had higher (P = 0.04) serum vitamin B-12 throughout the study (+26% at study-end). Consumption of the study vitamin B-12 dose increased serum holotranscobalamin in all reproductive groups (+16-42%; P ≤ 0.009). At study-end, pregnant (vs. control) women had a higher holotranscobalamin-to-vitamin B-12 ratio (P = 0.04) with ∼30% (vs. 20%) of total vitamin B-12 in the bioactive form. Serum MMA increased during pregnancy (+50%; P < 0.001) but did not differ by reproductive state at study-end. Serum homocysteine increased in pregnant women (+15%; P = 0.009) but decreased in control and lactating women (-16-17%; P < 0.001). Despite these changes, pregnant women had ∼20% lower serum homocysteine than the other 2 groups at study-end (P ≤ 0.02)., Conclusion: Pregnancy and lactation alter vitamin B-12 status in a manner consistent with enhanced vitamin B-12 supply to the child. Consumption of the study vitamin B-12 dose (∼3 times the RDA) increased the bioactive form of vitamin B-12, suggesting that women in these reproductive states may benefit from vitamin B-12 intakes exceeding current recommendations. This trial was registered at clinicaltrials.gov as NCT01127022., (© 2015 American Society for Nutrition.)

This study investigated the relationship between the urine homocysteine/creatinine (uHcy/Cr) ratio and the levels of natural anticoagulants, proinflammatory cytokines, and nitric oxide (NOx) metabolites in cerebrovascular diseases. No significant differences were observed in protein C, protein S, and antithrombin III levels among subjects with serum Hcy (sHcy) >15.0 μmol/L and ≤15.0 μmol/L. However, subjects with a uHcy/Cr ratio >14.8 μmol/g Cr showed significant differences in the levels of the corresponding parameters than those with uHcy/Cr ratio ≤14.8 μmol/g Cr. The sensitivity and specificity of the sHcy level at a cutoff of 15.0 μmol/L were 32.6% and 85.7%, respectively, with a positive predictive value of 69.5%. In contrast, those values for the uHcy/Cr ratio at a cutoff of 14.8 μmol/g Cr were 55.1% and 91.4% with a positive predictive value of 86.5%. The uHcy/Cr ratio correlated more closely with protein C, antithrombin III, TNF-α, and NOx levels than did sHcy concentrations. In short, the uHcy/Cr ratio has a significant relationship with anticoagulation- and inflammation-related parameters. A measurement of the uHcy/Cr ratio may provide helpful information for assessing patients with cerebrovascular diseases., (© 2014 by the Association of Clinical Scientists, Inc.)

Low-molecular-weight biothiols such as homocysteine, cysteine, and glutathione are metabolites of the sulfur cycle and play important roles in biological processes such as the antioxidant defense network, methionine cycle, and protein synthesis. Thiol concentrations in human plasma and blood are related to diseases such as cardiovascular disease, neurodegenerative disease, and cancer. The concentrations of homocysteine, cysteine, and glutathione in plasma samples from healthy human subjects are approximately in the range of 5-15, 200-300, and 1-5 μM, respectively. Glutathione concentration in the whole blood is in the millimolar range. Measurement of biothiol levels in plasma and blood is thought to be important for understanding the physiological roles and biomarkers for certain diseases. This review summarizes the relationship of biothiols with certain disease as well as pre-analytical treatment and analytical methods for determination of biothiols in human plasma and blood by using high-performance liquid chromatography and capillary electrophoresis coupled with ultraviolet, fluorescence, or chemiluminescence detection; or mass spectrometry., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Biologically active low-molecular-mass thiols, mainly including glutathione (GSH), cysteine (Cys), homocysteine (Hcy), and cysteinylglycine (Cys-Gly), are important physiological components in biological fluids, and their analytical methods have gained continuous attention over recent years. We developed and validated a novel HPLC method for the quantification of GSH, Cys, Hcy, and Cys-Gly in human plasma, urine, and saliva using 4-chloro-3,5-dinitrobenzotrifluoride as the derivatization reagent. Analyses were linear from 0.15 to 500 μM with the coefficient regression range of 0.9987-0.9994. Detection limits ranged from 0.04 to 0.08 μM (S/N=3). The developed method was applied to quantification of four thiols in human biological fluids collected from five donors with the concentration range of 2.50-124.25 μM, 0-72.81 μM, and 0-4.25 μM for plasma, urine, and saliva, respectively. The present method seemed to be an attractive choice for the determination of thiols in plasma, urine, and saliva.

Homocysteine thiolactone (Hcy-thiolactone), an intramolecular thioester, easily acylates free-amino groups in proteins, which impairs or alters the protein's biological function. Here, we describe new capillary electrophoresis assay for the determination of Hcy-thiolactone in human urine based on a field amplified sample injection and sweeping MEKC with UV detection. The two steps procedure relies on sample liquid-liquid extraction followed by CE separation and UV detection at 240nm. The Hcy-thiolactone standard added to the urine before the extraction step shows that the response of the detector is linear within the range studied, from 0.1 to 1μmolL(-1) urine. The intra- and interday precision and recovery were 3.2-14.4% (average 5.1% and 9.3%) and 92.5-112.6% (average 99.8% and 99.1%), respectively. The lower limit of quantification was 0.09nmol Hcy-thiolactone in 1mL of urine. The proposed method was applied for the analysis of 15 urine samples donated by apparently healthy volunteers. The average concentration of the analyte was 0.170±0.029μmolL(-1)., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: We evaluated the role of hyperhomocysteinemia as a risk factor for retinal vein occlusion (RVO) in Indian patients., Type of Study: Matched case control type of longitudinal study was conducted in 2006-2007., Materials and Methods: Two medical retina specialists examined the eyes having an event of RVO in the last 15 days. A similar number of eyes without RVO were also examined. The serum and urine homocysteine levels of these persons were tested. Matched pair analysis was carried out to determine the risk of RVO among those with hyperhomocysteinemia., Results: We included 20 cases of RVO and 20 age- and sex-matched persons without RVO. The risk of RVO was significantly higher in persons with hyperhomocysteinemia [difference of mean 31.62 μmol/L (95% Confidence Interval 16.60-47 . 86), P = 2.1 × 10-13 ]. The mean urine homocysteine level among cases and controls was not statistically significant. There were 12 persons with hypertension in both cases and control groups., Conclusion: Hyperhomocysteinemia is a risk factor for RVO. Any list of investigations for a case of RVO should include total plasma homocysteine (tHcy) levels.

A 20-year-old man was hospitalized for malignant hypertension, mechanical hemolysis, and kidney failure. Kidney biopsy confirmed glomerular and arteriolar thrombotic microangiopathy. Etiologic analyses, which included ADAMTS13 activity, stool culture, complement factor proteins (C3, C4, factor H, factor I, and MCP [membrane cofactor protein]), anti-factor H antibodies, HIV (human immunodeficiency virus) serology, and antinuclear and antiphospholipid antibodies, returned normal results. Malignant hypertension was diagnosed. Ten months later, we observed a relapse of acute kidney injury and mechanical hemolysis. Considering a diagnosis of complement dysregulation-related atypical hemolytic uremic syndrome (HUS), we began treatment with eculizumab. Despite the efficient complement blockade, the patient's kidney function continued to decline. We performed additional analyses and found that the patient's homocysteine levels were dramatically increased, with no vitamin B12 (cobalamin) or folate deficiencies. We observed very low plasma methionine levels associated with methylmalonic aciduria, which suggested cobalamin C disease. We stopped the eculizumab infusions and initiated specific treatment, which resulted in complete cessation of hemolysis. MMACHC (methylmalonic aciduria and homocystinuria type C protein) sequencing revealed compound heterozygosity for 2 causative mutations. To our knowledge, this is the first report of adult-onset cobalamin C-related HUS. Considering the wide availability and low cost of the homocysteine assay, we suggest that it be included in the diagnostic algorithm for adult patients who present with HUS., (Copyright © 2013 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

We have demonstrated for the first time the suitability of fluorosurfactant-capped spherical gold nanoparticles as HPLC postcolumn colorimetric reagents for the direct assay of cysteine, homocysteine, cystine, and homocystine. The success of this work was based on the use of an on-line tris(2-carboxyethyl)phosphine reduction column for cystine and homocystine. Several parameters affecting the separation efficiency and the postcolumn colorimetric detection were thoroughly investigated. Under the optimized conditions, cysteine, homocysteine, cystine, and homocystine in human urine and plasma samples were determined. Detection limits for cysteine, homocysteine, cystine, and homocystine ranged from 0.16-0.49 μM. The accuracy in terms of recoveries ranged between 94.0-102.1%. This proposed method was rapid, inexpensive, and simple., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Increased circulating concentrations of homocysteine (HCY) and asymmetric dimethylarginine (ADMA) are associated with vascular disease and vascular risk factors. HCY has been shown to inhibit the activity of endothelial dimethylaminohydrolase (DDAH), causing the accumulation of ADMA and the inhibition of nitric oxide synthesis. The concentrations of HCY and ADMA in biological fluids are used in the clinical diagnosis of cardiovascular diseases and this necessitates the development of a rapid and sensitive method for simultaneous determination of HCY and ADMA. A rapid, simple and sensitive method for simultaneous determination of HCY and ADMA by liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with electro spray ionization (ESI) in human urine was reported here. The methodology designed here was used to estimate these molecules in urine samples collected from patients reported to Cardiology Department of our hospital. Chromatographic separation was performed on Atlantis HILIC silica (100mm×2.1mm, 5μm, Waters). Positive multiple reactions monitoring (MRM) mode was chosen for quantification of each analyte and cystamine dihydrochloride (CYA) was used as the internal standard (IS) for the assay. The intra-assay precision and accuracy were in the range of 2.4-4.8 and -1.8% to 3.1%, respectively. The inter-assay precision and accuracy were in the range of 3.0-4.2% and -1.2% to 3.2%, respectively. The recoveries were between 94.9% and 101.4%. Our approach is simple, rapid and could be extended to routine urine assay., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: There is not much information on established standard therapy for patients with severe methionine adenosyltransferase (MAT) I/III deficiency., Case Presentation: We report a boy with MAT I/III deficiency, in whom plasma methionine and total homocysteine, and urinary homocystine were elevated. Molecular genetic studies showed him to have novel compound heterozygous mutations of the MAT1A gene: c.191T>A (p.M64K) and c.589delC (p.P197LfsX26). A low methionine milk diet was started at 31 days of age, and during continuing dietary methionine restriction plasma methionine levels have been maintained at less than 750 μmol/L. He is now 5 years old, and has had entirely normal physical growth and psychomotor development., Conclusions: Although some severely MAT I/III deficient patients have developed neurologic abnormalities, we report here the case of a boy who has remained neurologically and otherwise normal for 5 years during methionine restriction, suggesting that perhaps such management, started in early infancy, may help prevent neurological complications., (© 2013 Elsevier B.V. All rights reserved.)

In this work, with the assistance of the additivity of surface plasmon resonance band of nonionic fluorosurfactant-stabilized gold nanoparticles, we developed a new colorimetric assay approach of cysteine (Cys) and homocysteine (Hcy) in biological fluids, requiring no use of separation techniques. The detection limits of Cys and Hcy can be as low as 0.4 μM. The applicability of the method was validated by spiking known amount of Cys and Hcy in human urine and plasma samples. Recoveries were in the range of 97.2-106.7%. The present approach is simple, high selective and reproducible. In addition, the universality of the additivity of surface plasmon resonance band was demonstrated by virtue of silver nanoparticles. Therefore, the proposed method has a great potentiality in disease diagnosis associated with Cys and Hcy., (© 2013 Elsevier B.V. All rights reserved.)

Herein, a new postcolumn resonance light scattering (RLS) detection approach coupled with high-performance liquid chromatography (HPLC) was developed to detect cysteine and homocysteine. In the established system, the fluorosurfactant-capped gold nanoparticles (AuNPs) were first employed as postcolumn RLS reagents. The detection principle was based on the enhancement of RLS intensity of AuNPs upon the addition of cysteine/homocysteine. The RLS signals were detected by a common fluorescence detector at λ(EX)=λ(EM)=560 nm. The linear ranges for both cysteine and homocysteine were in the range of 5.0-50 μM. The detection limits were 5.9 pmol for cysteine and 12 pmol for homocysteine at a signal-to-noise ratio of 3. HPLC separation and RLS detection conditions were optimized in detail. The applicability of the proposed method has been validated by detecting cysteine and homocysteine in human urine samples. Recoveries from spiked urine samples were 95.0-103.0%., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Folate status, as reflected by red blood cell (RCF) and plasma folates (PF), is related to health and disease risk. Folate degradation products para-aminobenzoylglutamate (pABG) and para-acetamidobenzoylglutamate (apABG) in 24 hour urine have recently been shown to correlate with blood folate., Aim: Since blood sampling and collection of 24 hour urine are cumbersome, we investigated whether the determination of urinary folate catabolites in fasted spot urine is a suitable non-invasive biomarker for folate status in subjects before and during folic acid supplementation., Study Design and Methods: Immediate effects of oral folic acid bolus intake on urinary folate catabolites were assessed in a short-term pre-study. In the main study we included 53 healthy men. Of these, 29 were selected for a 12 week folic acid supplementation (400 µg). Blood, 24 hour and spot urine were collected at baseline and after 6 and 12 weeks and PF, RCF, urinary apABG and pABG were determined., Results: Intake of a 400 µg folic acid bolus resulted in immediate increase of urinary catabolites. In the main study pABG and apABG concentrations in spot urine correlated well with their excretion in 24 hour urine. In healthy men consuming habitual diet, pABG showed closer correlation with PF (rs = 0.676) and RCF (rs = 0.649) than apABG (rs = 0.264, ns and 0.543). Supplementation led to significantly increased folate in plasma and red cells as well as elevated urinary folate catabolites, while only pABG correlated significantly with PF (rs = 0.574) after 12 weeks., Conclusion: Quantification of folate catabolites in fasted spot urine seems suitable as a non-invasive alternative to blood or 24 hour urine analysis for evaluation of folate status in populations consuming habitual diet. In non-steady-state conditions (folic acid supplementation) correlations between folate marker (RCF, PF, urinary catabolites) decrease due to differing kinetics.

There are many reports about the significant roles of some amino acids in neurobiology and treatment of autism. This is a critical review of amino acids levels in autism. No published review article about the level of amino acids in autism was found. The levels of glutamate and homocystein are increased in autism while the levels of glutamine and tryptophan are decreased. Findings regarding the plasma levels of taurine and lysine are controversial. The urinary levels of homocysteine and essential amino acids in both the untreated and treated autistic children are significantly less than those in the controls. The current literature suffers from many methodological shortcomings which needed to be considered in future studies. Some of them are age, gender, developmental level, autism symptoms severity, type of autism spectrum disorders, medical comorbidities, intelligent quotient, diet, concomitant medications, body mass index, and technical method of assessment of amino acids.

Homocysteinuria is a rare inborn error of metabolism known to be associated with an increased risk of vascular events. A 36-year-old Caucasian man presented with a 2 day history of epigastric discomfort associated with nausea and sweating. He has a history of homocysteinuria and had been poorly compliant with treatment. An ECG showed ST-segment elevation and Q-waves in anterior leads. Blood tests showed markedly elevated high-sensitivity troponin and high homocysteine levels. He had a failed primary percutaneous coronary intervention due to extensive thrombus in the left anterior descending artery, which was aspirated and he received integrelin infusion for 48 h. Echocardiogram showed mild-to-moderate impairment of left ventricular function with apical akinesis extending to the mid-portion of anteroseptal walls consistent with anterior myocardial infarction. He was started on homocysteine-lowering treatment with betaine and folic acid. He is now on follow-up with clinical chemistry and cardiac rehabilitation.

Genetic or nutritional disorders in homocysteine (Hcy) metabolism elevate Hcy-thiolactone and cause heart and brain diseases. Hcy-thiolactone has been implicated in these diseases because it has the ability to modify protein lysine residues and generate toxic N-Hcy-proteins with auto-immunogenic, pro-thrombotic, and amyloidogenic properties. Bleomycin hydrolase (Blmh) has the ability to hydrolyze L-Hcy-thiolactone (but not D-Hcy-thiolactone) to Hcy in vitro, but whether this reflects a physiological function has been unknown. Here, we show that Blmh (-/-) mice excreted in urine 1.8-fold more Hcy-thiolactone than wild-type Blmh (+/+) animals (P = 0.02). Hcy-thiolactone was elevated 2.3-fold in brains (P = 0.004) and 2.0-fold in kidneys (P = 0.047) of Blmh (-/-) mice relative to Blmh (+/+) animals. Plasma N-Hcy-protein was elevated in Blmh (-/-) mice fed a normal (2.3-fold, P < 0.001) or hyperhomocysteinemic diet (1.5-fold, P < 0.001), compared with Blmh (+/+) animals. More intraperitoneally injected L-Hcy-thiolactone was recovered in plasma in Blmh (-/-) mice than in wild-type Blmh (+/+) animals (83.1 vs. 39.3 μM, P < 0.0001). In Blmh (+/+) mice injected intraperitoneally with D-Hcy-thiolactone, D,L-Hcy-thiolactone, or L-Hcy-thiolactone, 88, 47, or 6.3%, respectively, of the injected dose was recovered in plasma. The incidence of seizures induced by L-Hcy-thiolactone injections (3,700 nmol/g body weight) was higher in Blmh (-/-) than in Blmh (+/+) mice (93.8 vs. 29.5%, P < 0.001). Using the Blmh null mice, we provide the first direct evidence that a specific Hcy metabolite, Hcy-thiolactone, rather than Hcy itself, is neurotoxic in vivo. Taken together, our findings indicate that Blmh protects mice against L-Hcy-thiolactone toxicity by metabolizing it to Hcy and suggest a mechanism by which Blmh might protect against neurodegeneration associated with hyperhomocysteinemia and Alzheimer's disease.

Background: Homocystinuria is a rare metabolic disorder characterized by excess homocysteine in the urine. Vitamin B12 deficiency has diverse cutaneous, nervous and ophthalmic manifestations., Objective: To report a case of homocystinuria masquerading as vitamin B 12 deficiency., Case: We hereby are presenting an interesting case of a 4 year old boy who was being treated for Vitamin B 12 deficiency on the basis of history of delayed milestone, abdominal pain and hyperpigmentation of skin which was diagnosed as homocystinuria., Conclusion: It is important to carry out ophthalmological examination in every case of megaloblastic anemia if associated with blurring of vision and mental retardation., (© NEPjOPH.)

Objective: Methylmalonic aciduria is the most common disorder of organic acidurias in the mainland of China. It is also the one of treatable metabolic disorders. The clinical spectrum of the patients varies from severe neonatal-onset forms with neonatal brain injury and high mortality to milder forms with adult-onset. The clinical manifestations of neonates with methylmalonic aciduria are non-specific. Early diagnosis and adequate treatment contribute a lot to improving the prognosis of the patients. In this study, the abnormal clinical and laboratory findings in neonatal period of 160 Chinese patients with early-onset methylmalonic aciduria were investigated., Method: From 1996 to 2011, a total of 398 patients with methylmalonic aciduria were diagnosed in our hospital; 286 (71.9%) patients had early-onset before 1 year of age. Among 286 patients, 160 (55.9%) presented symptoms in neonatal period. Their urine organic acids were analyzed by gas chromatography-mass spectrometry. Blood amino acids and acylcarnitine profiles were determined by liquid chromatography tandem mass spectrometry. Serum and urine total homocysteine were measured using a fluorescence polarization immunoassay. In some patients, gene analysis was performed. Based on the disease types and general condition, individual dietary and medical interventions were started soon after diagnosis., Result: Out of the 160 patients, 131 (81.9%) had combined methylmalonic aciduria and homocysteinemia. Isolated methylmalonic aciduria was found in 29 cases (18.1%). The common presentations in neonatal period were feeding difficulty, seizures, lethargy and dyspnea. Megaloblastic anemia, liver dysfunction, hyperammonemia and metabolic acidosis were the frequent findings in the routine laboratory test. The most common initial clinical diagnosis was suspected hypoxic-ischemic encephalopathy. Even in 36 cases with abnormal family history, only 3 patients were admitted with suspected inborn errors of metabolism. Five cases (3.1%) were diagnosed by postmortem metabolic examination; 7 cases (4.4%) were detected by newborn screening. In 148 cases (92.5%), the diagnosis was much delayed to the age of one month to 8 years and 5 months (mean 13 months). Methylmalonic aciduria combined with homocysteinemia (MMACHC) gene analyses were performed in 31 cases with combined methylmalonic aciduria. CblC defect was confirmed. The patients with isolated methylmalonic aciduria were treated with protein-restricted diet, cobalamin and L-carnitine. The patients of methylmalonic aciduria combined with homocysteinemia were treated with cobalamin, L-carnitine, calcium folinate, betaine and common diet. Seven patients died without treatment. Clinical improvement was observed in 153 patients. Only 2 patients detected by newborn screening had normal mental and physical development. Mild to severe psychomotor retardation was observed in 151 cases., Conclusion: High mortality and disability rates were observed in the patients with early-onset methylmalonic aciduria. Combined methylmalonic aciduria and homocysteinemia is the common type of methylmalonic aciduria. The clinical manifestation in neonatal period of the patients with early-onset methylmalonic aciduria is complex. Feeding difficulty, seizures, lethargy and dyspnea are the common symptoms in neonatal period of the patients. Megaloblastic anemia, liver dysfunction, hyperammonemia and metabolic acidosis were the frequent laboratory findings.

Homocysteine (Hcy)-thiolactone is toxic, induces epileptic seizures in rodents, and has been implicated in Alzheimer's disease. Paraoxonase 1 (Pon1), a component of high-density lipoprotein, hydrolyzes Hcy-thiolactone in vitro. Whether this reflects a physiological function and whether Pon1 can protect against Hcy-thiolactone toxicity was unknown. Here we show that Hcy-thiolactone was elevated in brains of Pon1-/- mice (1.5-fold, p = 0.047) and that Pon1-/- mice excrete more Hcy-thiolactone than wild type animals (2.4-fold, p = 0.047). The frequency of seizures induced by intraperitoneal injections of L-Hcy-thiolactone was significantly higher in Pon1-/- mice compared with wild type animals (52.8% versus 29.5%, p = 0.042); the latency of seizures was lower in Pon1-/- mice than in wild type animals (31.8 min versus 41.2 min, p = 0.019). Using the Pon1 null mice, we provide the first direct evidence that a specific Hcy metabolite, Hcy-thiolactone, rather than Hcy itself is neurotoxic in vivo. Our findings indicate that Pon1 protects mice against Hcy-thiolactone neurotoxicity by hydrolyzing it in the brain, and suggest a mechanism by which Pon1 can protect against neurodegeneration associated with hyperhomocysteinemia and Alzheimer's disease.

Homocysteic acid (HA) has been suggested as a pathogen in a mouse model of Alzheimer's disease (AD), 3xTg-AD. However, it is not established whether HA is involved in humans. We investigated the relationship between urinary HA levels and Mini-Mental State Examination (MMSE) scores in AD patients (n = 70) and non-AD controls (n = 34). We found a positive, statistically significant relationship between the two variables (the urinary HA level and MMSE score) (r = 0.31, p = 0.0008, n = 70). This relationship was stronger in females than males (r = 0.43, p = 0.005, n = 44 in females; r = 0.48, p = 0.02, n = 22 in males). The urinary HA levels were significantly different in AD patients than controls (AD: 8.7 ± 7.5, n = 70; non-dementia control: 13.3 ± 9.4, n = 34, p < 0.01). In addition, aging and smoking were found as lowering factors for urinary HA levels. Our preliminary study showed a negative, statistically significant relationship between blood HA (micromole) and urine HA levels (r = -0.6, p = 0.007, n = 19), and between blood HA levels and MMSE scores (r = -0.79, p = 0.0000518, n = 19). On the basis of these results, we speculate that reduced urinary excretion induces elevated HA levels in blood, resulting in cognitive dysfunctions. This study also suggests that HA may be a candidate of neurotoxins for uremic encephalopathy. Since amyloid-β increases HA toxicity and HA is an agonist of N-methyl-D-aspartic acid (NMDA) receptor, we speculate that elevated blood HA affects the brain cognitive function through NMDA receptor-mediated toxicity in AD.

This study describes the use of 14 nm nonionic fluorosurfactant-capped gold nanoparticles (FSN-capped AuNPs) for the simultaneous detection of cysteine (Cys) and homocysteine (Hcy) using colorimetric method, requiring no use of separation techniques. It was found that the kinetics of Cys/Hcy-induced aggregation of the 14 nm FSN-capped AuNPs strongly depends on the pH value of gold colloidal solution. At a pH of 6.5, the Cys-induced aggregation kinetics of the FSN-capped AuNPs was almost identical to that induced by Hcy, facilitating simultaneous detection of total Cys and Hcy up to a concentration as low as 0.15 μM; while at pH 12.0, the kinetics of Cys-induced aggregation was much faster than that inducted by Hcy, leading to selective detection of Cys at concentration as low as 1.0 μM in the presence of Hcy. The applicability of the method was validated by spiking known amount of Cys and Hcy in human urine and plasma samples, obtaining a recovery of 95.4-105.5%. The present approach is simple, high selective and provides high reproducibility, and has a great potentiality in disease diagnosis., (Copyright © 2011 Elsevier B.V. All rights reserved.)