Measurement Of Total Homocysteine Research Articles

Elevated Homocysteine after Elevated Propionylcarnitine or Low Methionine in Newborn Screening Is Highly Predictive for Low Vitamin B12 and Holo-Transcobalamin Levels in Newborns.

Early diagnostics and treatment of vitamin B12 deficiency (B12D) in infants, mainly maternally conditioned, is crucial in preventing possible developmental delay and neurological deficits. Currently, B12D is rarely listed in regular newborn screening panels and mostly regarded as an incidental finding. The aim of this study was to evaluate a targeted newborn screening strategy for detection of suspected B12D. A decision strategy based on the primary parameters propionylcarnitine and methionine for selection of samples to be analyzed for total homocysteine by mass spectrometry was established. Therefore, 93,116 newborns were initially screened. Concentrations of vitamin B12 and holotranscobalamin in serum were obtained from clinical follow-up analyses of recalled newborns. Moreover, an extremely sensitive mass spectrometric method to quantify methylmalonic acid from the dried blood spots was developed. Overall, 0.15% of newborns were screened positive for suspected B12D, of which 64% had vitamin B12 concentrations below 148 pM. We also determined a cutoff value for methylmalonic acid in dried blood spots indicative for B12D in infants. Overall, we calculated a prevalence of 92/100,000 for suspected B12D in the Austrian newborns. In conclusion, we present a screening algorithm including second-tier measurement of total homocysteine that allows detection of low B12 serum concentrations with a high detection rate and low false-positive rate.

Newborn screening for remethylation disorders and vitamin B12 deficiency-evaluation of new strategies in cohorts from Qatar and Germany.

Newborn screening is a precondition for early diagnosis and successful treatment of remethylation disorders and classical homocystinuria (cystathionine-ß-synthase deficiency). Newborn screening for classical homocystinuria using total homocysteine measurement in dried blood spots has been very successfully performed for many years for newborns from Qatar. A new optimized newborn screening strategy for remethylation disorders and homocystinuria was developed and evaluated for newborns from Qatar using total homocysteine measurement as first-tier and methionine, methionine-phenylalanine-ratio and propionylcarnitine as second-tiers. Proposed cut-offs were also retrospectively evaluated in newborn screening samples of 12 patients with remethylation disorders and vitamin B12 deficiency from Qatar and Germany. Over a 12 months period, the proposed strategy led to a decrease in the recall rate in homocysteine screening for Qatar from 1.09% to 0.68%, while allowing for additional systematic inclusion of remethylation disorders and vitamin B12 deficiency into the screening panel for Qatar. In the evaluated period the applied strategy would have detected all patients with classical homocystinuria identified by the previous strategy and in addition 5 children with maternal nutritional vitamin B12 deficiency and one patient with an isolated remethylation disorder. Additional retrospective evaluation of newborn screening samples of 12 patients from Germany and Qatar with remethlyation disorders or vitamin B12 deficiency showed that all of these patients would have been detected by the cut-offs used in the proposed new strategy. In addition, an adapted strategy for Germany using methionine, methionine-phenylalanine-ratio and propionylcarnitine as first-tier, and homocysteine as a second-tier test was also positively evaluated retrospectively. The proposed strategy for samples from Qatar allows inclusion of remethylation disorders and vitamin B12 deficiency in the screening panel, while lowering the recall rate. An adapted second-tier strategy is presented for screening in Germany and will be prospectively evaluated over the next years in a pilot project named "Newborn Screening 2020".

A case of homocystinuria due to CBS gene mutations revealed by cerebral venous thrombosis

BackgroundHomocystinuria caused by cystathionine beta synthase (CBS) deficiency is most often diagnosed in childhood and has a variable expressivity. The most frequent abnormalities include intellectual disability, ectopia lentis, myopia, skeletal abnormalities or thromboembolism. ObjectiveTo report a case of homocystinuria unraveled by cerebral venous thrombosis (CVT). ObservationA 17 year old female was admitted in our department of neurology for subacute headache and presented seizures in the emergency room. Cerebral imaging revealed CVT. Severe hyperhomocysteinemia was found and led to the diagnosis of homocystinuria due to composite heterozygous mutations in the CBS gene. Further investigations disclosed lens subluxation in association with myopia, mild scoliosis and osteopenia. The patient was treated by heparin followed by warfarin, vitamin therapy and dietary methionine restriction. Total homocysteine and methionine levels became normal in a few weeks and the patient had a complete recovery. ConclusionIn patients with CVT, plasma total homocysteine measurement as part of the etiologic work up may reveal severe hyperhomocysteinemia due to CBS or remethylation defects that require specific treatment and management including perhaps protein-restricted diet and/or vitamin therapy for life.

Congenital heart defects and maternal genetic, metabolic, and lifestyle factors

The purpose of this study was to identify metabolic, genetic, and lifestyle factors that discriminate between women who have pregnancies affected by congenital heart defects (CHDs) from those who have unaffected pregnancies. We analyzed the concentrations of 13 serum biomarkers, 3 functional genetic variants, and 4 lifestyle factors among 417 women with CHD-affected pregnancies and 250 controls. To identify risk factors that discriminated between cases and controls, we used logistic regression followed by recursive partitioning to identify non-linear interactions. A receiver operating characteristic (ROC) curve was constructed to evaluate the discriminatory accuracy of the final model. A combination of risk factors discriminated women who had pregnancies affected by CHDs from those who had unaffected pregnancies. Among 21 possible determinants, serum concentrations of homocysteine and methionine, and reduced:oxidized glutathione ratios (GSH:GSSG) had the greatest discriminatory power. Recursive partition modeling resulted in five terminal nodes each illustrating the interplay of these three biomarkers. Women with elevated homocysteine and low GSH:GSSG had the highest risk of having CHD-affected pregnancy, whereas women with low homocysteine, high methionine, and high GSH:GSSG had the lowest risk. The corresponding area under the ROC curve was 81.6% (95% confidence interval [CI], 78.1-85.2%), indicating high ability to discriminate between cases and controls. High homocysteine, low methionine, and a reduced GSH:GSSG ratio were the strongest discriminating factors between cases and controls. Measurement of total homocysteine, methionine, and total and reduced glutathione in reproductive aged women may play a role in primary prevention strategies targeted at CHDs.

A fast liquid chromatographic tandem mass spectrometric method for the simultaneous determination of total homocysteine and methylmalonic acid

A liquid chromatography(LC)/electrospray ionization tandem mass spectrometry (MS) method for the quantitative determination of total homocysteine and methylmalonic acid and the monitoring of methionine, homocystine and succinic acid in plasma has been developed. The analytes are determined under the presence of the deuterated internal standards methylmalonic acid-d (3) and homocystine-d (8). Although methylmalonic acid can be determined directly, a reduction step has to be carried out to ensure the measurement of total homocysteine. Ultrafiltration was applied afterwards to deproteinize the samples prior to LC/MS injection. LC/MS analysis is carried out isocratically using a mobile phase consisting of 5% methanol and 95% of a 0.06 M formic acid solution on a reversed-phase C18 column at a flow rate of 0.5 mL/min. The MS measurement was separated into several periods: homocysteine, homocystine and methionine were determined in the positive-ion mode, whereas the determinations of methylmalonic acid and succinic acid were carried out in the negative-ion mode. The intraday coefficients of variation (CVs) were 2.9% or less and 3.2% or less for homocysteine and methylmalonic acid, respectively. Interday CVs ranged from 3.8 to 5.9% for homocysteine and from 3.5 to 6.3% for methylmalonic acid. Analyte concentrations could reliably be determined, also far below the reference values. Furthermore, the linearity was determined and a correlation study with respect to the existing homocysteine and methylmalonic acid methods at Medisch Spectrum Twente Hospital was carried out.

Hyperhomocysteinemia and central retinal vein occlusion in Iranian population

To evaluate total plasma homocysteine level during the acute phase of central retinal vein occlusion (CRVO) in the Iranian population and determine whether hyperhomocysteinemia is also a risk factor for CRVO. Fifty-four patients with recently diagnosed CRVO were studied. Their fasting total plasma homocysteine level was compared with a matched control group of 51 patients evaluated in the same clinic for a non-retinal disease diagnosis. The mean total plasma homocysteine level was 14.76+/-7.67 micromol/l in cases, and 11.42+/-3.74 micromol/l in control subjects. It showed a significant difference (P=0.005) in mean plasma homocysteine level between cases and control group. Odds ratio of CRVO for individuals with hyperhomocysteinemia was 2.88 (95% CI=1.08-7.71 and P=0.03). The overall multivariable-adjusted odds of CRVO in participants with plasma homocysteine level above 15 micromol/l was 4.71 (95% CI=1.46-15.19 and P=0.009) Hyperhomocysteinemia was not statistically different in each age group (<60 years: 27%, 61-70 years: 33.3%, 71-80 years: 31.6%, >81 years: 33.3%, Chi-square test, P=0.98). Elevated total plasma homocysteine level is an independent risk factor for CRVO in Iranian population. In addition to an evaluation of all conventional cardiovascular risk factors, measurement of total homocysteine for evidence of hyperhomocysteinemia may be important in the initial investigation and management of patients with CRVO.

Clinical validation of a new blood collection tube for the accuracy of total homocysteine measurement by different methods

Objective: To evaluate the clinical use of Homocysteine–Primavette®, a new blood collection medium for total homocysteine (tHcy) assay. Methods: The agreement between baseline tHcy and tHcy in stabilized samples (40 h) was assessed for FPIA, HPLC, GC-MS, LC-MS, and ICL. Results: tHcy concentrations in whole blood were stable for 40 h in Hcy–Primavette tubes. Conclusion: Primavette tubes are a good alternative for the accurate tHcy measurement and no readjustment of reference intervals is needed.

Hyperhomocysteinemia, Enzyme Polymorphism and Thiobarbituric Acid Reactive System in Children with High Coronary Risk Family History

Objectives: To investigate both the frequency and the genetic background of hyperhomocysteinemia and the frequency of increased plasma thiobarbituric acid reactive system (TBARS) levels in children and adolescents whose parents had premature coronary heart disease (CHD).Methods: The study was performed on children and adolescents aged 4–18 years (105 offspring of parents with CHD before age 45 and 74 referents from families without any evidence of premature atherosclerosis). Fasting serum total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), and total triglyceride (TG) levels were measured by enzymatic methods. Low density lipoprotein cholesterol (LDL-C) level was calculated by the Friedewald formula. Plasma total homocysteine (THCy) level was measured by fluorescence polarisation immunoassay. Plasma TBARS level was determined by fluorimetric method. 5,10-methylenetetrahydrofolate reductase (MTHFR) enzyme polymorphism was analyzed by polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP).Results: Hyperhomocysteinemia was found in 32 cases and in 4 controls. Increased plasma THCy level was found in 10 children and adolescents from 12 cases homozygous for the C677T polymorphism of the MTHFR gene. No similar high frequency was observed in heterozygous subjects. Elevated fasting plasma TBARS levels were found in 38 cases and in 8 controls. The frequency differences were significant (p < 0.01). Allele frequency of the MTHFR polymorphism among cases and controls was similar. Significant correlation (r = 0.53, p < 0.02) was detected between plasma THCy and TBARS levels. One child had high serum TC level, 5 had low serum HDL-C level and all other children had normal serum TC, LDL-C, HDL-C and TG levels from children with hyperhomocysteinemia and/or high plasma TBARS levels. A significant correlation (r = 0.64, p < 0.01) was observed between plasma THCy levels of parents and children in the case group.Conclusion: The measurement of plasma THCy and TBARS levels may contribute to the detection of the risk of children and adolescents with high CHD risk family history.

Facts and recommendations about total homocysteine determinations: an expert opinion.

Measurement of plasma total homocysteine has become common as new methods have been introduced. A wide range of disorders are associated with increased concentrations of total homocysteine. The purpose of this review is to provide an international expert opinion on the practical aspects of total homocysteine determinations in clinical practice and in the research setting and on the relevance of total homocysteine measurements as diagnostic or screening tests in several target populations. Published data available on Medline were used as the basis for the recommendations. Drafts of the recommendations were critically discussed at meetings over a period of 3 years. This review is divided into two sections: (a) determination of homocysteine (methods and their performance, sample collection and handling, biological determinants, reference intervals, within-person variability, and methionine loading test); and (b) risk assessment and disease diagnosis (homocystinuria, folate and cobalamin deficiencies, cardiovascular disease, renal failure, psychiatric disorders and cognitive impairment, pregnancy complications and birth defects, and screening of elderly and newborns). Each of these subsections concludes with a separate series of recommendations to assist the clinician and the research scientist in making informed decisions. The review concludes with a list of unresolved questions.

Variation in plasma cystathionine and its relation to changes in plasma concentrations of homocysteine and methionine in healthy subjects during a 24-h observation period

Plasma cystathionine measurement may be a useful complement to total homocysteine measurement in the assessment of B vitamin status. Information on the within-person variation in cystathionine is currently sparse. The goal was to study the daily variation in plasma cystathionine concentrations in healthy subjects. Twelve subjects (aged 22-29 y) were followed for 24 h. During the observation period, the subjects received a breakfast (containing 15-18 g protein) at 0900 and a beef dinner (containing approximately 50 g protein) at 1500. Multiple blood samples for metabolite analyses were collected during the day, and a final sample was obtained the next morning. The results are expressed as medians and interquartile ranges. All subjects had normal fasting cystathionine concentrations [0.120 (0.100-0.160) micro mol/L]. Cystathionine concentrations increased significantly after breakfast, reached a maximum after 4 h of 142.4% (100.0-170.3%) of the fasting concentration, and then declined to fasting concentrations before dinner. After dinner, plasma cystathionine started to increase within 0.5 h and reached a maximum after 6 h [281.3% (194.1-351.4%) of the concentration measured before dinner]. The changes in plasma methionine and total homocysteine concentrations during the day were less pronounced. Food intake, even of foods with low protein content, causes an increase in plasma cystathionine concentrations that is more pronounced than the concomitant changes in total homocysteine and methionine. In studies including plasma cystathionine measurement, blood sampling in the fasting state should be considered.

Comparison of isotope dilution mass spectrometry methods for the determination of total homocysteine in plasma and serum.

Two independent methods have been critically evaluated and applied to the measurement of total homocysteine in serum and plasma: solid-phase anion extraction (SPAE) gas chromatography/mass spectrometry (GC/MS) and protein precipitation liquid chromatography/tandem mass spectrometry (LC/MS/MS). In addition, analysis of samples prepared by SPAE was accomplished by liquid chromatography/mass spectrometry (LC/MS) and LC/MS/MS. These methods have been used to determine total homocysteine levels in several existing serum-based Standard Reference Materials (SRMs) from the National Institute of Standards and Technology and in patient plasma samples provided by the Centers for Disease Control and Prevention. The precision of the homocysteine measurements in serum and plasma was critically evaluated, and method comparisons were carried out using Bland-Altman plots and bias analysis. On the basis of the excellent precision and close agreement of the mass spectrometric (MS) methods, the MS-based methods will be used for certification of a serum-based SRM for homocysteine and folates.

Hyperhomocysteinemia and B-vitamin deficiencies in infants and children.

Measurement of total homocysteine (tHcy) in healthy and diseased children has documented the utility of this marker in pediatric research and diagnostics. This article focuses on novel data obtained in infants, children and adolescents, with emphasis on cobalamin status in infants. In children, determinants of plasma tHcy are similar to those established in adults, and include age, gender, nutrition, B-vitamin status, and some drugs interfering with B-vitamin function. In infants (age < 1 year), tHcy is moderately elevated and related to serum cobalamin, whereas in older children and throughout childhood, plasma tHcy is low (about 60% of adult levels), and folate status becomes a strong tHcy determinant. As in adults, hyperhomocysteinemia in childhood is a risk factor for stroke, and folate-responsive hyperhomocysteinemia has been detected in children with renal failure. tHcy seems to be a sensitive indicator of folate deficiency in children on a poor diet, in HIV-infected children, and in children treated with anti-folate drugs. In children at increased risk of cobalamin deficiency, which includes children born to vegetarian mothers or children in developing countries on a poor diet, tHcy and methylmalonic acid are responsive indicators of a deficiency state. In newborns and infants born to mothers with an adequate nutrition, there are consistent observations of low cobalamin, elevated tHcy and methylmalonic acid, and reduction of both metabolites by cobalamin supplementation. These data have raised the question whether cobalamin deficiency may be widespread and undetected in babies born to non-vegetarian women on a Westernized diet.

Optimal Time Interval between Repeated Blood Sampling for Measurements of Total Homocysteine in Healthy Individuals

Many studies have shown that increased plasma concentrations of total homocysteine (tHcy) are associated with cardiovascular disease (1). Like most biological variables, plasma tHcy fluctuates within an individual around a long-term mean (2)(3). tHcy measurements often are based on a single blood sample. Taking the mean of two or more repeated measurements will reduce the error attributable to within-subject biological fluctuations and reflect more closely a person’s true mean tHcy value within a certain period. However, as the time interval between two repeated measurements becomes shorter, the values become more similar and the error reduction diminishes. In that case, the benefit of taking the mean of two measurements is not fully used. On the other hand, intervals that are too long (several weeks) may compromise the compliance of study participants and allow long-term seasonal changes (4)(5). Several studies on the weekly and monthly biological variation, as expressed by the CV, in plasma tHcy concentrations have been published (Table 1⇓ ). From these studies, no conclusions can be drawn about the optimal time interval between two repeated samples to obtain a sufficiently precise estimate of the true tHcy concentration within a predefined period. The optimal time interval is reached when the interval-specific CV does not increase further with increasing time intervals. The aim of this study was to determine how many days between two samplings within 1 week are needed to reach the maximum biological variation in tHcy. Our findings may be useful in studies involving estimates of an individual’s true mean concentration of tHcy. The participants in this study were six male and nine female healthy volunteers, ages 19–46 years. Use of medication or dietary supplements known to affect Hcy or folate metabolism were contraindications for participation. We applied strict criteria related to folate …

Effect of anticoagulants on plasma homocysteine determination

Background: Waiting temperature before centrifugation and anticoagulants used, markedly effect total homocysteine concentrations. The aim of this study was to investigate the effect of different anticoagulants and temperature on plasma homocysteine levels. Methods: We studied total homocysteine concentrations in 23 healthy subjects. Blood was drawn in K 3EDTA, sodium citrate- or sodium fluoride-containing tubes, and kept at 0°C or 22°C for 3 h. Total homocysteine measurements were performed with fluorescence polarization immunoassay (FPIA) method. We compared all results with baseline EDTA values (samples put on crushed ice and centrifuged immediately) recommended in literature for reference handling. Results: At 22°C, the tubes containing sodium citrate and sodium fluoride showed significantly higher total homocysteine concentrations than their respective baseline values ( p=0.000). However, sodium fluoride tubes were not significantly different than baseline EDTA levels. Waiting 3 h at 0°C did not effect sodium citrate and EDTA plasma total homocysteine concentrations when compared to baseline EDTA, but sodium fluoride-containing plasma levels were significantly decreased ( p=0.000). Conclusions: According to our results, the most available and practical temperature and anticoagulant for total homocysteine determination is sodium fluoride at room temperature up to 3 h.

The history of folic acid.

The history of folic acid.

Determination of biologically active low-molecular-mass thiols in human blood: III. Highly sensitive narrow-bore isocratic reversed-phase high-performance liquid chromatography with fluorescence detection

The fast isocratic narrow-bore reversed-phase high-performance liquid chromatographic method employing fluorescence detection is described for the precise reproducible simultaneous measurement of total homocysteine, cysteine and glutathione in human blood. Sample preparation involves conversion of disulfides to free thiols with triphenylphosphine, precipitation of proteins with sulfosalicylic acid, and conjugation of thiols with monobromobimane. Optimized sample preparation conditions as well as chromatographic conditions allowed to obtain reliable quantitative results within the concentration range corresponding to the levels of these thiols in human blood in norm and pathology. The detection limit was ∼70 amol for all labeled aminothiols. The proposed method for these compounds analysis includes simple sample preparation, high selectivity, good linearity ( r 2>0.998), high reproducibility (within-run precision for derivatized aminothiol peaks area RSD<1.8% for three times consequently injected sample); high reliability and the small sample volume (1 μl) required for analysis make it suitable for clinical studies.

Is there a role of cyclosporine A on total homocysteine export from human renal proximal tubular epithelial cells?

Immunosuppressive therapy may influence homocysteine metabolism in allograft recipients. We examined whether cyclosporine A influences the in vitro formation of homocysteine as determined by the measurement of total homocysteine (tHcy) concentrations in supernatants of human renal proximal tubule epithelial cells (hRPTEC), an important site of homocysteine metabolism. Cells were incubated with and without vitamins in the presence of low or high methionine concentrations at different cyclosporine A concentrations for 24, 48 and 72 hours (N = 7 for each experiment). The concentration of tHcy in culture supernatants was measured by a fluorescence polarization immunoassay. Data were analyzed by four-way ANOVA, three-way ANOVA and t tests. The Hcy export from hRPTEC (tHcy in the culture supernatant) was 2.69 micromol/L during standard cell culture conditions at time point 24 hours and increased by 28.3% at 48 hours and by 44.6% at 72 hours. Comparisons of tHcy levels in culture supernatants over time by four way ANOVA showed that cyclosporine A at 200 or 800 ng/mL had no influence on tHcy export from hRPTEC (P = 0.67991). In contrast, the presence of vitamins in the medium (P = 0.000001), in vitro methionine loading (P < 0.000001), and prolonged incubation time (P < 0.000001) were associated with an increase of tHcy export from hRPTEC. Significant interactions in this analysis were "vitamins x methionine" (P = 0.001804), "vitamins x time" (P = 0.001478), "methionine x time" (P < 0.000001), and "vitamins x methionine x time" (P = 0.018128), pointing to a combined effect of vitamins in the presence of high methionine concentrations at the later time points. Our study shows that hRPTEC export Hcy into the cell culture medium, an effect that is enhanced by in vitro methionine loading and modulated by the presence of vitamins. Cyclosporine A had no major influence on Hcy export from tubule cells. Therefore, our findings do not support the assumption that cyclosporine A elevates total homocysteine plasma levels in organ transplant patients.

Determination of biologically active low-molecular-mass thiols in human blood: I. Fast qualitative and quantitative, gradient and isocratic reversed-phase high-performance liquid chromatography with photometric and fluorescence detection

The fast isocratic and gradient reversed-phase high-performance liquid chromatographic methods employing photometric and/or fluorescence detection are described for the precise reproducible simultaneous measurement of total homocysteine, cysteine, and glutathione in human blood. Sample preparation involves conversion of disulfides to free thiols with triphenylphosphine, precipitation of proteins with sulfosalicylic acid, and conjugation of thiols with monobromobimane. The aminothiol assay is optimized by reduction and derivatization step conditions (pH, temperature and time of reactions), as well as by chromatographic conditions to obtain reliable quantitative results within the concentration range corresponding to the levels of these thiols in human blood in norm and pathology. Its sensitivity allows the detection of aminothiol quantities >2 pmol.

Total homocysteine measurement in clinical practice.

This review will primarily address methodological aspects of homocysteine measurement, highlighting changes since the subject was comprehensively reviewed in 1993, and will bring together practical information of relevance to laboratory planning or to providing an analytical service including homocysteine measurement. Because the physiological state of the patient during blood collection is often neglected, and because it has become apparent that the bloodsampling procedure may contribute considerably more variation to measurement of homocysteine than the total variation of the analytical procedures, the review will pay particular attention to the critical roles of controlled blood collection and sample processing.

Qualitative and quantitative determination of biologically active low-molecular-mass thiols in human blood by reversed-phase high-performance liquid chromatography with photometry and fluorescence detection

The reversed-phase high-performance liquid chromatographic method employing photometry and fluorescence detection is described for the precise reproducible simultaneous measurement of total homocysteine (tHcy), cysteine (Cys), and glutathione (GSH) in human blood. Sample preparation involves conversion of disulfides to free thiols with triphenylphosphine, precipitation of proteins with trichloroacetic acid, conjugation of the thiols with monobromobimane (mBrB). The aminothiol assay is optimized by reduction and derivatization step conditions (pH, temperature and time of reactions) to obtain reliable quantitative results within the concentration range corresponding to normal and pathological levels of these thiols in human blood.