Determination Of Homocysteine Research Articles

Simultaneous determination of total homocysteine, methionine, methylmalonic acid and 2-methylcitric acid in dried blood spots by ultra-performance liquid chromatography–tandem mass spectrometry

Homocysteine, methionine, methylmalonic acid and 2-methylcitric acid are clinically relevant markers in the methionine, propionate, and cobalamin metabolism. This study aimed to develop and validate an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneously determining total homocysteine, methionine, methylmalonic acid and 2-methylcitric acid in dried blood spots. Three 3.2 mm discs were punched from each calibrator, quality control, and sample dried blood spot into a 96-well U-plate. Each sample was spiked with internal standards and extracted. Then the supernatant was transferred to another 96-well U-plate. After nitrogen drying, the dried residues were reconstituted, centrifuged, and the resulting supernatant was transferred to another 96-well plate for analysis. The method was performed using UPLC-MS/MS within 3 min, validated according to guidance documents, and applied to 72 samples from confirmed patients with methionine, propionate, and cobalamin metabolism disorders. The UPLC-MS/MS method provided satisfactory separation of the four analytes. The R2 values were ≥ 0.9937 for all analytes. The recoveries ranged from 94.17 to 114.29 %, and the coefficients of variation for intraday and interday precision were 0.19 % to 5.23 % and 1.02 % to 6.89 %, respectively. No significant carry-over was detected for the four analytes, and most of confirmed samples exhibited biomarker patterns characteristic of the relevant disorders. A simple and fast UPLC-MS/MS method was successfully developed, validated, and applied to clinical samples for the simultaneous determination of total homocysteine, methionine, methylmalonic acid, and 2-methylcitric acid in dried blood spots.

Sensitive Electrochemical Biosensor Using Gold and Manganese (IV) Oxide Nanomaterials on Multiwalled Carbon Nanotubes for the Determination of Homocysteine With a Portable Potentiostat

Serum homocysteine (Hcy) is considered to be a reliable biomarker of cardiovascular, cerebrovascular and neurological diseases. Therefore, rapid, specific and sensitive measurement of homocysteine is crucial for prevention and diagnosis. Electrochemical methods, which benefit from simplicity, low-cost and quick response, have been widely utilized in the determination of targeted biomolecules. In this work, based upon a portable mini-potentiostat, a homocysteine electrochemical biosensor has been successfully developed combined with the modification of gold and manganese (IV) nanomaterials on multiwalled carbon nanotubes (MWCNTs) (denoted as Au@MnO2/MWCNTs) on screen-printed electrodes (SPEs). Due to the excellent electrochemical activity and catalytic capability of Au@MnO2/MWCNTs, the proposed electrochemical biosensor exhibited wide-linear range (5–125 μmol L−1), high sensitivity (0.01110 μA (μmol L−1)−1) and low detection limit (0.6173 μmol L−1) for homocysteine, requiring only 400 s to complete detection. It was effectively applied in the determination of serum homocysteine, and the results were consistent with those of an automated clinical chemistry analyzer. Furthermore, the simple and portable electrochemical device offers a promising manner for on-site analysis and home health monitoring.

Polymorphic substitutions in folate cycle genes as predictors of hyperhomocysteinemia in children

Introduction. Mutant alleles of genes of folate cycle enzymes can lead to the significant deterioration of its function and varying severity of pathology. Several defects in these genes lead to severe hyperhomocysteinemia, the most common form of which is a deficiency of cystathionine beta-synthase B. 
 Aim: to establish polymorphic substitutions in the genes of folate cycle enzymes that contribute to the formation of hyperhomocysteinemia in children.
 Materials and methods. Two hundred seventy one children aged of 13–18 years were examined. The analysis of genetic polymorphisms of the folate cycle was carried out using a molecular genetic method. Quantitative determination of the blood homocysteine and folic acid level was performed by chemiluminescent immunoassay on microparticles. Statistical data processing was carried out using Statistica 6.1 application programs (StatSoft Inc., USA). 
 Results. The frequency of the T allele of the MTHFR 677 gene was revealed to be higher in adolescents of the main group compared with the control (p = 0.043). The frequency of the homozygous genotype 66 AA of the MTRR gene in children of the comparison group was significantly higher (p = 0.049), however, the heterozygous genotype 66 AG of the MTRR gene was significantly more often detected in adolescents of the main group (p = 0.008). The average concentrations of homocysteine in children of the main group were 11.6 mmol/L, in adolescents of the control group 9.3 mmol/L (p = 0.021). Hyperhomocysteinemia in children of the main group was detected in 217 (80.1%) adolescents, and in 57 (49.6%) children of the control group (p < 0.001). The baseline serum folate level was determined in the children of the main group. The average amount of vitamin B9 in the blood of children of the main group was 3.7 ng/ml, and in 145 (53.5%) children this indicator was significantly reduced.
 Conclusion. Low levels of folic acid contribute to an increase in homocysteine in blood plasma. Taking vitamin B9 and vitamin folate complexes significantly reduces the level of homocysteine in blood plasma (p < 0.001).

Determination of homocysteine in human plasma with a silver nanopaste-based microfluidic device

Elevated levels of homocysteine (Hcys) in plasma correlate with vascular disease, and accurate detection is crucial to maintaining good health and well-being. However, the specific detection of Hcys without interference from methionine and cysteine remains a challenge. In this study, we present a microfluidic method based on a convex-shaped design and electrochemical detection for the specific determination of Hcys in human plasma using a silver nanopaste-coated paper. The Hcys assays utilize the recombinant enzyme methionine α,γ-lyase (γMETase), which is applied onto the paper to selectively react with Hcys and generate H2S. This reaction subsequently interacts with the silver nanopaste, forming Ag-S bonds that lead to an increase in the paper’s electrical resistance. The paper-based analytical device exhibits a low detection limit of 1.59 × 10-8 M and a wide linear range of 0.1 μM to 32.5 μM. It also demonstrates good anti-interference properties, higher stability, and high selectivity compared with other nanoparticle and paper-based sensors. Finally, the biosensor has been successfully applied to the determination of Hcys in human plasma.

Photoluminescent and electrochemiluminescent detection of homocysteine based on bifunctional cyclometalated iridium(III) complex

A photoluminescent (PL) and electrochemiluminescent (ECL) chemodosimeter for homocysteine (Hcy) determination was developed based on a bifunctional cyclometalated iridium(III) complex (pba)2Ir(dcbpy). (pba)2Ir(dcbpy) was prepared by using 4′-(2-pyridyl)-benzaldehyde (pba) as main ligand and 2,2′-bipyridyl-4,4′-dicarboxylic acid (dcbpy) as ancillary ligand. The existence of aldehyde group on the main ligand and dicarboxylic acid groups on the ancillary ligand endows (pba)2Ir(dcbpy) good water solubility. Importantly, the aldehyde group on the main ligand also furnishes the chemodosimetric active site, which could react with Hcy highly in neutral buffer solution based on a unique cyclization reaction with aminoalkylthiol group to produce a binding adduct, enhancing both PL and ECL signal. The photophysical and ECL properties of (pba)2Ir(dcbpy) in the absence and presence of Hcy were investigated and the response mechanism of the luminescence enhancement was confirmed by mass spectrum. Both PL and ECL measurements exhibited good linear relationships to the Hcy with the detection of limit at micro-molar level. More significantly, (pba)2Ir(dcbpy) possessed a relatively high discriminating ability to differentiate Hcy from structurally related molecule (cysteine) under neutral buffer conditions. The PL and ECL chemodosimeter based on cyclometalated iridium(III) complex with specific active site is universal to the analysis of other targets, thus opening a new avenue for the application of cyclometalated iridium(III) complex in PL and ECL field.

One-pot sample preparation procedure for the determination of protein N-linked homocysteine by HPLC-FLD based method

The report presents robust and high throughput method, based on liquid chromatography coupled with fluorescence detection (HPLC-FLD), for the determination of total protein N-linked homocysteine (Hcy) in human plasma. The assay involves simultaneous proteins precipitation with perchloric acid and removal of any other form of Hcy, except protein N-linked Hcy, via disulfides reduction with tris(2-carboxyethyl)phosphine (TCEP) and plasma protein pellet wash with perchloric acid followed by liberation of N-linked Hcy from proteins by hydrochloric acid hydrolysis, drying under vacuum and residue reconstitution in diluted hydrochloric acid. The chromatographic separation of resulting in this way Hcy-thiolactone (HTL) is achieved within 3 min at room temperature on PolymerX RP-1 (150 × 4.6 mm, 5.0 µm) column using isocratic elution with eluent, consisted of o-phthaldialdehyde (OPA) in sodium hydroxide and acetonitrile (ACN), delivered at a flow rate 1 mL/min. The analyte is quantified by monitoring fluorescence at 480 nm using excitation at 370 nm, in a linear range from 0.25 to 10 µmol/L in plasma, while the limit of quantification (LOQ) equals 0.25 µmol/L. The method was successfully applied to plasma samples delivered by fifteen apparently healthy donors showing that the HPLC-FLD assay is suitable for screening of human plasma.

Manipulating the monomer-dimer transformation of a heptamethine cyanine ligand: near infrared chromogenic recognition of biothiols.

A novel absorbance recovery method has been developed for the determination of biothiols with a near-infrared reagent. This method employs a two-reagent system composed of cation heptamethine cyanine (CyL) and Hg2+. The absorbance of CyL, with a maximum peak at 760nm, was decreased due to addition of Hg2+, but recovered when biothiols were added. Under optimal conditions, the reciprocal extent of recovered absorbance was proportional to the concentration of biothiols. The calibration curves are linear over the range of (0.3-7.0) × 10-6M for cysteine, (1.0-10.0) × 10-6M for homocysteine and (1.0-9.0) × 10-6M for glutathione. Because of the specific affinity of Hg2+for biothiols, there is minimal interference from other amino acids. This method has been successfully applied to the determination of homocysteine in human urine samples with satisfactory results.

Extraction-assisted voltammetric determination of homocysteine using magnetic nanoparticles modified with molecularly imprinted polymer

Amagnetic graphite-epoxy composite (m-GEC) electrochemical sensor is presentedbased on magnetic imprinted polymer (mag-MIP) to determine homocysteine (Hcy). Mag-MIP was synthesized via precipitation polymerization, using functionalized magnetic nanoparticles (Fe3O4) together with the template molecule (Hcy), the functional monomer 2-hydroxyethyl methacrylate (HEMA), and the structural monomer trimethylolpropane trimethacrylate (TRIM). For mag-NIP (magnetic non-imprinted polymer), the procedure was the same in the absence of Hcy. Morphological and structural properties of the resultant mag-MIP and mag-NIP were examined usingTEM, FT-IR, and Vibrating Sample Magnetometer. Under optimized conditions, the m-GEC/mag-MIP sensor showed a linear range of 0.1-2µmol L-1, with alimit of detection (LOD) of 0.030µmol L-1. In addition, the proposed sensor responded selectively to Hcy compared to several interferents present in biological samples. The recovery values determined by differential pulse voltammetry (DPV) were close to 100% for natural and synthetic samples, indicating good method accuracy. The developed electrochemical sensor proves to be a suitable device for determining Hcy, with advantages related to magnetic separation and electrochemical analysis.

Determination of Homocysteine and Vitamin B12 level in blood Patients Rheumatoid Arthritis in KirKuk Governorate

Many of the clinical studies have clearly demonstrated the direct link between the high levels of homocystien in serum and Rheumatoid Arthritis and homocystien shows its impact by imbalance self- oxidized when high concentration in blood, which leads to a group of deeply rooted Free radicals and Rheumatoid Arthritis RA of Disease Fades inflammatory caused by free roots which cause injury to the damage or the total deficit circumstantial there are some features may occur to the formation of a contract of rheumatic smal
 Objective:. The aim of this study to assess the level of homocystien and vitamin B12patients suffering from Rheumatoid Arthritis compared with healthy people and operate in order relationship with vitamin B12 and its impact causing high levels of homocystien disease Rheumatoid Arthritis
 The way:- collecting blood samples (25) people healthy people (Control) (15) of the males and 10 females aged from (25-65) and (50) people of the patients (25) of the males and (25) of the females aged from (25-65) and those suffering from Rheumatoid Arthritis and measurement of the level of homocystien and vitamin B12.
 The findings and conclusions:- found that there is a moral rise in the level of homocystien when a group of patients suffering from Rheumatoid Arthritis compared with healthy group at the level of contingent liabilities (P≤0001) found moral decline in the level of vitamin B12 when a group of patients suffering from Rheumatoid Arthritis compared to a healthy and at the level of contingent liabilities (P≤0.0005).
 Word: homocystien , vitamin B12 , Rheumatoid Arthritis

O-phthalaldehyde assisted surface enhanced Raman spectroscopy selective determination of trace homocysteine in serum

High plasma homocysteine (Hcy) levels may indicate cardiovascular disease. However, sensitive and selective determination of Hcy remains a major challenge. Herein, we present a sensing strategy for Hcy by surface enhanced Raman scattering (SERS) method along with a specific reaction of o-phthalaldehyde (OPA) and Hcy. The obtained adduct 2-(1-carboxyl-3-thiopropyl)-1-isoindolinone (Hcy-OPA) can be directly detected by SERS using gold nanoparticles (Au NPs) as the substrate. The developed SERS method displays superior sensitivity (low detection limit of 2.50 × 10−12 mol L−1) with a broad linear range (5.00 × 10−10 −5.00 × 10−6 mol L−1). As a proof of real application, it can be used to detect Hcy in bovine serum samples with a concentration as low as 5.00 × 10−9 mol L−1, which is free from the interference of the other amino acids and glutathione.

Electrochemical Determination of Homocysteine Using Self-Assembled 6-Ferrocenylhexanethiol on a Molybdenum Disulfide Nanoparticle Modified Glassy Carbon Electrode (GCE)

The determination of homocysteine (Hcy) is important because high levels are associated with a greater risk of developing heart disease or stroke. Herein, an electrochemical sensor of homocysteine was constructed based upon the self-assembly of 6-ferrocenylhexanethiol (FcC6SH) on a MoS2 nanoparticles-modified electrode with a Nafion coating. The sensor showed a favorable electrocatalytic activity for oxidation of homocysteine since 6-ferrocenylhexanethiol acted as a good electron transfer mediator. Moreover, the MoS2 nanoparticles served as modifiers to covalently immobilize FcC6SH and enhance the electrochemical active surface area. Highly sensitive determination of homocysteine was achieved with a linear range from 2.0 to 100 µmol L−1 and a minimum detectable concentration of 0.30 µmol L−1 by chronoamperometry. The constructed sensor was applied to the analysis of immunoglobulin G (IgG) samples with satisfactory results.

Simultaneous estimation of total homocysteine and methylmalonic-acid using LC-MS/MS: Clinical application in adult and pediatric

Quantitative determination of biomarkers homocysteine (Hcy) and methylmalonic acid (MMA), the regulators of cobalamin (Cbl) and folate levels, together used as a biomarkers to diagnose chemical insufficiency/deficiency of Cbl and folate. We report simultaneous clinical estimation of total Hcy and MMA with efficient clean-up, sensitive and selective LC-MS/MS method. Efficient sample clean-up was achieved by a two-step extraction protocol with 100 μL serum. The validated method was applied to 893 clinical samples from 2 cohorts including pediatrics and mothers, respectively, for identifying their Cbl and folate status. The method shows excellent order of linearity for Hcy (22.2nM-3.7 μM) and MMA (42.34 nM - 5.92 μM), respectively. Complete method validation was performed where intraday-interday accuracy-precision and mean stability recovery data were found within ±15%. The validated method was extended for the quantification of serum total Hcy-MMA levels in clinical samples. The efficient extraction with negligible matrix-effect (ME) has reduced LC-MS/MS chocking and clean-up downtime. The rapid, sensitive and robust LC-MS/MS method has been successfully validated for simultaneous estimation of total Hcy and MMA using only 100 μL serum. The method was applicable to large number of clinical samples and was found to be good throughput with low contamination of mass detector, high sensitivity and selectivity.

A Label-Free Molecularly Imprinted Electrochemical Sensor Based on MXene Nanosheets Modified by Gold Nanoparticles for Sensitive and Selective Detection of Homocysteine

A label-free molecularly imprinted electrochemical sensor (MIECS) based on electropolymerized molecularly imprinted polymer (MIP) was developed for the determination of homocysteine (Hcy) in serum for the first time. MXene@AuNPs with layered structure was synthesized on the electrode by modifying MXene with gold nanoparticles (AuNPs). MIP based on dopamine hydrochloride (DA) were electropolymerized onto the surface of MXene@AuNPs modified electrode by molecular docking and quantum chemical calculations for specific recognition of Hcy. MXene@AuNPs as a carrier for immobilizing MIP steadily enhances the conductivity of the electrode (about 4.2-fold) and plays a crucial part in improving the detection sensitivity of MIECS. The results showed that the current response linearly decreased with the increasing concentration of Hcy in the detection range from 1 × 10−13 to 1 × 10−5 mol l−1, the limit of detection (LOD) and limit of quantification (LOQ) of 11.81 fmol l−1 and 39.49 fmol l−1, respectively. With favorable selectivity, stability, reproducibility and ruggedness, the developed MIECS was applied to the determination of Hcy in human serum samples with recoveries of 87.83%–92.58%. The proposed strategy has potential application for disease surveillance.

M024 Molecularly imprinted electrochemical sensor for the determination of homocysteine

M024 Molecularly imprinted electrochemical sensor for the determination of homocysteine

Simultaneous determination of serum homocysteine, cysteine, and methionine in patients with schizophrenia by liquid chromatography-tandem mass spectrometry.

Schizophrenia is a debilitating psychiatric disorder affecting approximately 1% of the population worldwide. Disturbances in the homocysteine metabolism are an important factor in the pathophysiology of schizophrenia. In this research, a novel validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification procedure was developed to investigate three significant compounds of homocysteine metabolism: homocysteine, cysteine, and methionine in patients with schizophrenia and healthy controls. Sample preparation involved a reduction with dithiothreitol followed by protein precipitation, and the chromatographic runtime was 2min. The LC-MS/MS method was validated according to CLSI C62-A and the Chinese Guidance for Liquid Chromatography and Mass Spectrometry Clinical Application. The performance of the method was excellent with a coefficient of variation for precision in the range of 0.5-6.9%, an accuracy of 90.4-101.6%. In addition, the practical applicability of the method was demonstrated by applying it in the routine sample analysis for a schizophrenic patient. Increased homocysteine levels and decreased cysteine levels were observed in the patient with schizophrenia. These results indicate that the activity of the transsulfuration pathway may play a key role in the pathogenesis of schizophrenia.

Single-Step Hydrolysis and Derivatization of Homocysteine Thiolactone Using Zone Fluidics: Simultaneous Analysis of Mixtures with Homocysteine Following Separation by Fluorosurfactant-Modified Gold Nanoparticles.

Herein, we report a new automated flow method based on zone fluidics for the simultaneous determination of homocysteine and homocysteine thiolactone using fluorimetric detection (λext = 370 nm/λem = 480 nm). Homocysteine thiolactone is hydrolyzed on-line in alkaline medium (1 mol L−1 NaOH) to yield homocysteine, followed by reaction with o-phthalaldehyde in a single step. Derivatization is rapid without the need of elevated temperatures and stopped-flow steps, while specificity is achieved through a unique reaction mechanism in the absence of nucleophilic compounds. Mixtures of the analytes can be analyzed quantitatively after specific separation with fluorosurfactant-capped gold nanoparticles that are selectively aggregated by homocysteine, leaving the thiolactone analogue in solution. As low as 100 nmol L−1 of the analyte(s) can be quantified in aqueous solutions, while concentrations > 2 μmol L−1 can be analyzed in artificial and real urine matrix following 20-fold dilution. The percent recoveries ranged between 87 and 119%.

Fluorescent determination of cysteine and homocysteine via adjustable synthesis of flower-shaped covalent organic frameworks

Detection of cysteine (Cys) and homocysteine (Hcy) is very important for the early diagnosis and prevention of related diseases. Herein, we propose a facile, green, room-temperature controllable approach for the synthesis of fluorescent covalent organic frameworks (COFs) with high specific surface areas, high crystallinity, good morphology, excellent chemical and thermal stability by using 2,5-dimethoxyterephthaldehyde and 1,3,5-tris(4-aminophenyl)benzene as precursors. Interestingly, we found that the size and morphology of COFs could be controlled by the amount of acetic acid. With increasing numbers of acetic acid (HAc) molecules, the morphology of COFs gradually changed from spherical to flower-shaped, and the particle size gradually decreased from micrometer to nanometer. Furthermore, we were surprised to find that the reaction time influenced the uniform dispersion and crystallinity of flower-shaped COFs. With increasing the reaction time, the boundary around COFs became blurred, hierarchical flower-shaped COFs gradually appeared, and the flower-shaped gradually became clear and uniform. On this basis, the flower-shaped COFs were further used for highly sensitive determination of Cys and Hcy, with detection limits as low as 11.4 nM and 6.7 nM, respectively. The possible quenching mechanism was deduced by the Dmol3 package provided by Materials Studio and DFT calculations of Gaussian 09 software. This strategy could provide us a new insight into the controllable preparation of flower-shaped fluorescent COFs and broaden the application range of COFs for biomarker detection.

Importance of homocysteine determination in differential diagnosis of rare causes of macrocytic anaemia

Importance of homocysteine determination in differential diagnosis of rare causes of macrocytic anaemia

Analysis of homocysteine and thiol-containing compounds using the heterocyclic disulfides

Biological low-molecular aminothiols, such as homocysteine, are characterized by the weak absorption in the UV – visible range. Therefore, aminothiols analysis requires using thiol-specific chemosensors or derivatization procedure. Heterocyclic disulfides belong to a large group of derivatizing agents. However, the disulfides widely used in the analytical practice, such as 5,5'-dithiobis-(2-nitrobenzoic acid), have several disadvantages: limited acceptable pH range, low sensitivity, susceptibility to hydrolisys. Hence, the search for the effective derivatizing agents continues. In the current work, 2,2'-dithiobis[benzoxazole] and 8,8'-dithiobis-quinoline disulfides were used for the analysis of homocysteine. Precise and sensitive methods (homocysteine detection limit in the range of 1.1·10-6–7.8·10-6 M, relative standard deviation in the range of 1.5–4.6%) for the homocysteine determination have been developed, including spectrophotometric method, capillary electrophoresis (CE), high-performance liquid chromatography (HPLC) and kinetic method with the spectrophotometric detection. The proposed disulfides have also been successfully used in the analysis of sulfhydryl groups content in the sample of human urine.

Nanozyme based on graphene oxide modified with Fe3O4, CuO, and cucurbit[6]uril for colorimetric determination of homocysteine.

A nanozyme based on graphene oxide modified with Fe3O4 NPs, CuO NPs, and cucurbit[6]uril has beensuccessfully fabricated by a simple sonochemical technique. By employing CB[6] as a specific binding pocket and Fe3O4@CuO-GO as a peroxidase mimic, this novel nanozyme (BN I) is equipped with molecular recognition ability and enhanced peroxidase-like activity. On the basis of the inhibition effect of homocysteine (Hcy) towards the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) catalyzed by BN I, a simple colorimetric method is established for the sensitive and selective determination of Hcy. This proposed method displays a good linear response in the range 5-200μM with a detection limit of 1.8μM. In thepractical assay of human plasma samples, the relative standard deviations (RSD) are lower than 11% and the recoveries are between 98.0 and 104.9%. In theassay of human urine samples, the RSD are below 9.0% and the recoveries range from 94.0 to 103.5%. Thecolorimetric method presentedoffers a convenient and accurate way for the determination of biomarkers in point-of-care testing (POCT).