Determination of total homocysteine in dried blood spots using high performance liquid chromatography for homocystinuria newborn screening

Febriani, Andi Dwi Bahagia; Sakamoto, Akiko; Ono, Hiroaki; Sakura, Nobuo; Ueda, Kazuhiro; Yoshii, Chiyoko; Kubota, Miho; Yanagawa, Junko

doi:10.1111/j.1442-200x.2004.01825.x

Cited by 21 publications

(22 citation statements)

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“…This reduced the number of variables measured, and lack of serum often prevented retesting of samples with results that were unexpected or outside the measurement range. Another problem was that we had no clinical data, and we could therefore not examine the effects of other factors that may influence tHcy and B vitamin status in the newborn baby, including maternal B vitamin status (14,15 ), gestational age (36,37 ), breastfeeding vs other types of nutrient intake (13 ), or the use of nitrous oxide during delivery (38,39 ). Although the metabolite profile points to the site of a defect, it rarely provides firm evidence of the underlying cause or the clinical consequences.…”

Section: Methodsmentioning

confidence: 99%

Screening for Serum Total Homocysteine in Newborn Children

et al. 2004

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Results:The median (5th-95th percentile) tHcy concentration was 6.8 (4.2-12.8) 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 explained by increased methionine. tHcy did not differ according to folate concentrations or MTHFR 677C>T genotypes. None of the babies had definite CBS deficiencies, but heterozygosity led to low cystathionine, increased methionine, but normal tHcy concentrations.

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Section: Methodsmentioning

confidence: 99%

Screening for Serum Total Homocysteine in Newborn Children

et al. 2004

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“…Increased variability may be attributable to the process of blood-spot preparation, the small sample volume, elution of homocysteine from the blood spot, and the greater sample dilution. The assay may not be able to quantify DBS homocysteine in all individuals because the lower limit of detection is above the concentrations found in some healthy neonates (7,12 ).…”

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confidence: 98%

“…A recent report assessed stability in screening cards that were stored at 4°C, which does not reflect routine practice (6 ). Another investigated the stability of DBS samples, using whole blood to which large concentrations of aqueous homocysteine calibrator had been added, thus potentially masking any increase in homocysteine attributable to release from erythrocytes (7 ). The lack of information on the stability of homocysteine in DBS as applied to neonatal screening prompted the following investigation.…”

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confidence: 99%

“…Vitamin D deficiency can cause secondary hyperparathyroidism and, if severe, osteomalacia. Both of these conditions are associated with loss of bone density, and both are reversible by repletion of vitamin D (7)(8)(9)(10). Recently, the association of vitamin D deficiency with an increased risk for a broader range of diseases, such as prostate cancer (11,12 ) and colon cancer (13 ), has come to attention.…”

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Blood Spot Homocysteine: A Feasibility and Stability Study

Bowron¹,

Barton

Scott

et al. 2005

Clinical Chemistry

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Homocystinuria is an autosomal recessive disorder usually caused by deficiency of cystathionine ␤-synthase, leading to grossly increased plasma and urine concentrations of homocysteine. There is considerable evidence that early detection and treatment can prevent the clinical consequences of the enzyme deficiency (1, 2 ); therefore, screening for the disorder has been advocated (1 ). Many cases of homocystinuria have secondary hypermethioninemia. Neonatal screening for homocystinuria by measurement of increased methionine concentrations in dried blood spots (DBS) has been performed in some centers but has poor sensitivity. Approximately 20% of cases are missed, partly because of low methionine concentrations in breast milk and some infant formulas (3 ). The measurement of homocysteine in DBS has not been used as a screen for homocystinuria, partly because of uncertainty about the suitability of DBS samples. Homocysteine concentrations are unstable in whole blood stored at room temperature and increase by ϳ1.0 mol/L per hour (4 ). This is attributable to in vitro erythrocyte transmethylation reactions that lead to continuous production and release of homocysteine (5 ). It is not known whether homocysteine is released from erythrocytes in blood that has been spotted on filter paper and dried, either during the spotting and drying process or during storage at room temperature. A recent report assessed stability in screening cards that were stored at 4°C, which does not reflect routine practice (6 ). Another investigated the stability of DBS samples, using whole blood to which large concentrations of aqueous homocysteine calibrator had been added, thus potentially masking any increase in homocysteine attributable to release from erythrocytes (7 ). The lack of information on the stability of homocysteine in DBS as applied to neonatal screening prompted the following investigation.Blood samples were collected from adult inpatients into tubes containing potassium EDTA. Samples were mixed gently, and blood was spotted on filter paper cards (standard neonatal screening cards) and dried in air. The cards were stored at room temperature until analysis. Immediately after the blood spots were prepared, the remaining samples were centrifuged, and the plasma was removed and stored at Ϫ20°C until analysis. Ethical approval was obtained from the UK South West Local Research Ethics Committee.We measured total homocysteine in DBS by HPLC, using a modification of a method for plasma total homocysteine (8 ). We punched 6-mm DBS into flat-bottomed tubes, added 80 L of 2.16 mol/L cysteamine (internal standard) and 15 L of 100 mL/L tributylphosphine in dimethylformamide to the tubes, and incubated the tubes at 4°C for 30 min. We used 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfuric acid to derivatize the thiols. Separation was performed by reversed-phase HPLC with fluorometric detection (Shimadzu RF-10AXL). The method was standardized by use of 15 L of an aqueous homocysteine calibrator (20 mol/L) in place of DBS. The DBS method was linear u...

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“…According to a report published by the Center for Disease Control and Prevention, as of 2001, public health laboratories screened more than 95% of all newborns in the United States for inborn metabolic disorders (Mei et al 2001) using the DBS technique. Over last 47 years the DBS sampling technique has been used for both the qualitative and quantitative screening of wide varieties of diagnostic compounds and biomarkers (De Jesus et al 2009;Chalcraft and Britz-McKibbin 2009;Al-Dirbashi et al 2008;de Wilde et al 2008;Searles et al 2008;Oglesbee et al 2008;Wang et al 2005;Bowron et al 2005;Febriani et al 2004;Constantinou et al 2004;O'Broin and Gunter 1999;Chace et al 1993;Spence et al 1993).…”

Section: Introductionmentioning

confidence: 99%

The Use of Dried Blood Spots for Concentration Assessment in Pharmacokinetic Evaluations

Majumdar¹,

Howard

2011

Pharmacokinetics in Drug Development

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This chapter reviews the technology and application of Dried Blood Spot (DBS) sampling techniques. In part due to its relative simplicity, DBS has begun to receive increased use in the pharmaceutical industry, particularly where blood or plasma sample volumes are small, are difficult to collect, store, process, or transport. This chapter presents methods and techniques, highlights applications, and discusses benefits and drawbacks associated with its use in pharmacokinetics.

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Determination of total homocysteine in dried blood spots using high performance liquid chromatography for homocystinuria newborn screening

Abstract: The present method, which is rapid, user friendly and reliable, seems applicable to newborn screening of HCU in place of methionine measurement.

Cited by 21 publications

References 14 publications

Screening for Serum Total Homocysteine in Newborn Children

Screening for Serum Total Homocysteine in Newborn Children

Blood Spot Homocysteine: A Feasibility and Stability Study

The Use of Dried Blood Spots for Concentration Assessment in Pharmacokinetic Evaluations

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