Development of a novel approach for the second-tier estimation of homocysteine in dried blood spot using tandem mass spectrometry (original) (raw)
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Clinical Chemistry
BackgroundClassical homocystinuria (HCU) results from deficient cystathionine β-synthase activity, causing elevated levels of Met and homocysteine (Hcy). Newborn screening (NBS) aims to identify HCU in pre-symptomatic newborns by assessing Met concentrations in first-tier screening. However, unlike Hcy, Met testing leads to a high number of false-positive and -negative results. Therefore, screening for Hcy directly in first-tier screening would be a better biomarker for use in NBS.MethodsDried blood spot (DBS) quality control and residual clinical specimens were used in analyses. Several reducing and maleimide reagents were investigated to aid in quantification of total Hcy (tHcy). The assay which was developed and validated was performed by flow injection analysis–tandem mass spectrometry (FIA-MS/MS).ResultsInterferents of tHcy measurement were identified, so selective derivatization of Hcy was employed. Using N-ethylmaleimide (NEM) to selectively derivatize Hcy allowed interferent...
Screening for Serum Total Homocysteine in Newborn Children
Clinical Chemistry, 2004
Background: Newborn screening for total homocysteine (tHcy) in blood may identify babies with vitamin B 12 (B 12 ) deficiency or homocystinuria, but data on the causes of increased tHcy in screening samples are sparse. Methods: Serum concentrations of tHcy, cystathionine, methionine, folate, and B 12 and the methylenetetrahydrofolate reductase (MTHFR) 677C>T polymorphism were determined in 4992 capillary blood samples collected as part of the routine screening program in newborn children. Methylmalonic acid (MMA), gender (SRY genotyping), and six cystathionine -synthase (CBS) mutations were determined in 20 -27% of the samples, including all samples with tHcy >15 mol/L (n ؍ 127), B 12 <100 pmol/L (n ؍ 159), or methionine >40 mol/L (n ؍ 154). Results: The median (5th-95th percentile) tHcy concentration was 6.84 (4.20 -12.83) mol/L. B 12 status, as determined by serum concentrations of B 12 , tHcy, and MMA, was moderately better in boys than in girls. tHcy concentrations between 10 and 20 mol/L were often associated with low B 12 , whereas tHcy >20 mol/L (n ؍ 43) was nearly always detected in babies with increased methionine. tHcy did not differ according to folate concentrations or MTHFR 677C>T genotypes. None of the babies had certain CBS deficiencies, but heterozygosity led to low cystathionine, increased methionine, but normal tHcy concentrations.
Total homocysteine is making its way into pediatric laboratory diagnostics
European Journal of Clinical Investigation, 2001
Objective. Cobalamin deficiency accompanied by bone marrow dysfunction and impaired central nervous system development has been reported in infants who were born to mothers with low cobalamin intake. We investigated the relation between cobalamin status in newborns and in their healthy mothers who consumed an omnivorous diet.
Homocysteine and methylmalonic acid in diagnosis and risk assessment from infancy to adolescence
The American journal of clinical nutrition, 2003
The concentration of total homocysteine (tHcy) in serum and plasma is elevated in both folate and cobalamin deficiencies, whereas methylmalonic acid (MMA) in serum, plasma, or urine is a specific marker of cobalamin function. The combined measurement of both metabolites is useful for the diagnosis and follow-up of these deficiency states. In addition, tHcy is elevated under various pathologic states (eg, renal failure), and hyperhomocysteinemia is associated with an increased risk of cardiovascular disease, cognitive dysfunction, and adverse pregnancy outcomes. The diagnostic utility of tHcy and MMA concentrations as markers of folate and cobalamin deficiencies in healthy and diseased children has been documented. This article briefly summarizes the biochemical background of tHcy and MMA and the associations of tHcy and MMA with various disease states and focuses on novel data obtained in infants, children, and adolescents, with emphasis on cobalamin status in infants. The utility o...
Comparison of Three Different Plasma Homocysteine Assays with Gas Chromatography Mass Spectrometry
1999
Background: Various methods are available to measure plasma total homocyst(e)ine (tHcy) concentrations, but whether plasma tHcy assays may be used interchangeably is not known. Methods: Results from three different methods [HPLC with fluorescence detection, enzyme immunoassay (EIA), and fluorescence polarization immunoassay (FPIA)] to determine fasting (n ؍ 163) and post-methionine load (n ؍ 80) plasma tHcy concentrations were compared with those obtained by gas chromatographymass spectrometry (GC-MS). Difference plots on nontransformed and log-transformed data were used to assess the agreement between HPLC and GC-MS, EIA and GC-MS, and FPIA and GC-MS. Results: The closest agreement between methods was observed between GC-MS and FPIA for fasting tHcy concentrations, with 95% of the FPIA values between 19% above and 24% below the corresponding GC-MS results. Post-methionine load tHcy concentrations measured by EIA showed the least agreement with GC-MS, with 95% of values measured by EIA ranging between 52% above and 16% below the GC-MS values. With respect to GC-MS, the above-mentioned methods showed a negative bias for fasting tHcy concentrations, but a positive bias for both immunoassays for postmethionine load tHcy concentrations.
Journal of inherited metabolic disease, 2015
Newborn screening (NBS) is justified if early intervention is effective in a disorder generally not detected early in life on a clinical basis, and if sensitive and specific biochemical markers exist. Experience with NBS for homocystinurias and methylation disorders is limited. However, there is robust evidence for the success of early treatment with diet, betaine and/or pyridoxine for CBS deficiency and good evidence for the success of early betaine treatment in severe MTHFR deficiency. These conditions can be screened in dried blood spots by determining methionine (Met), methionine-to-phenylanine (Met/Phe) ratio, and total homocysteine (tHcy) as a second tier marker. Therefore, we recommend NBS for cystathionine beta-synthase and severe MTHFR deficiency. Weaker evidence is available for the disorders of intracellular cobalamin metabolism. Early treatment is clearly of advantage for patients with the late-onset cblC defect. In the early-onset type, survival and non-neurological sym...
Simultaneous analysis of homocysteine and methionine in plasma
Journal of chromatography. B, Biomedical sciences and applications, 2001
The new isocratic cation exchange method separates up to eight different amino thiols. The separated sample components are detected electrochemically using a gold electrode and the integrated pulsed amperometry. The eluent composition is, for example, 0.15 M sodium perchlorate, 0.02 M perchloric acid and 5% acetonitrile. The report describes the optimization of chromatographic parameters such as column diameter and eluent composition. Quantitative performance is discussed for eight different amino thiols using standards. Also presented is a long term quantitative study for homocysteine and methionine in plasma samples. The preparation of plasma samples is simpler than with the previously reported version of the method. Only a reduction step is required, and neither column switching nor derivatization are necessary.
Biochemistry of homocysteine in health and diseases
Indian journal of biochemistry & biophysics, 2006
The amino acid homocysteine (Hcy), formed from methionine has profound importance in health and diseases. In normal circumstances, it is converted to cysteine and partly remethylated to methionine with the help of vit B12 and folate. However, when normal metabolism is disturbed, due to deficiency of cystathionine-beta-synthase, which requires vit B6 for activation, Hcy is accumulated in the blood with an increase of methionine, resulting into mental retardation (homocystinuria type I). A decrease of cysteine may cause eye diseases, due to decrease in the synthesis of glutathione (antioxidant). In homocystinurias type II, III and IV, there is accumulation of Hcy, but a decrease of methionine, thus, there is no mental retardation. Homocysteinemia is found in Marfan syndrome, some cases of type I diabetes and is also linked to smoking and has genetic basis too. In hyperhomocysteinemias (HHcys), clinical manifestations are mental retardation and seizures (type I only), ectopia lentis, s...