Development and evaluation of a liquid chromatography tandem mass spectrometry method for the simultaneous quantitation of methylmalonic acid and homocysteine in plasma, and study of their relationships with other biomarkers of vitamin B12 deficiency

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Development and evaluation of a liquid chromatography tandem mass spectrometry method for the simultaneous quantitation of methylmalonic acid and homocysteine in plasma, and study of their relationships with other biomarkers of vitamin B12 deficiency

 

Author: Pang, Wing-hang
Title: Development and evaluation of a liquid chromatography tandem mass spectrometry method for the simultaneous quantitation of methylmalonic acid and homocysteine in plasma, and study of their relationships with other biomarkers of vitamin B12 deficiency
Degree: M.Sc.
Year: 2010
Subject: Hong Kong Polytechnic University -- Dissertations
Vitamin B12 deficiency
Blood plasma -- Measurement
Homocysteine
Methyl groups
Malonic acid
Department: Dept. of Health Technology and Informatics
Pages: 123 leaves : ill. ; 31 cm.
InnoPac Record: http://library.polyu.edu.hk/record=b2355942
URI: http://theses.lib.polyu.edu.hk/handle/200/5864
Abstract: Introduction: Vitamin B 12 deficiency is generally diagnosed based on plasma levels of total B12. However, normal total B12 levels may not mean adequate intake, as over deficiency may not manifest itself until 2 to 5 years of a diet supplying inadequate amounts of vitamin B12. A more sensitive biomarker of early stages of vitamin B12 deficiency is needed. Background: Recent studies indicated that the determination of plasma homocysteine (Hcy) and methylmalonic acid (MMA) may be more sensitive and specific vitamin B12 deficiency biomarkers than total B12 and/or active B12, especially for the early stage of vitamin B 12 deficiency. With this in mind, a novel LC MS MS method for simultaneous quantitation of MMA and Hcy in plasma was developed and validated, and used to measure plasma Hcy and MMA in a group of apparently healthy Chinese subjects. Interrelationships between these and total and active B12 levels were examined. Study design: This study consists of two parts: I) analytical study that included method development and evaluation; II) clinical study to investigate the relationships between biomarkers of vitamin B12 deficiency (total and active B12 and Hcy and MMA in plasma). Method: Plasma samples (500 μl) were treated with a reductant (dithiothreitol) and ultracentrifugation. The treated samples were analyzed by LC MS MS for simultaneous measurement of Hcy and MMA. The remaining plasma samples were analyzed using immunoassay methods for measurement of total B12 and active B12.
Results: The novel method reached analytic goals. Detection limits (signal-to-noise ratio: 3:1) were 39.4 nmol/l (MMA) and 9.7 nmol/l (Hcy), respectively. The assay was linear to at least 10,000 nmol/l for both MMA and Hcy. The intra-assay precision CVs ranged from 0.34% to 0.86% (Hcy: n = 8, MMA: n = 10), and the inter-assay precision CVs ranged from 4% to 8% (Hcy and MMA: n = 8). The recovery was between 75% and 111 % for the different analytes at different concentrations. Expected values of external quality control materials compared with obtained results showed very good agreement (MMA: r = 0.9989, p<0.0001 and Hcy: r = 0.9983, p<0.0001). Fasting plasma samples from a total of 144 apparently healthy Chinese volunteers were measured for Hcy level and the median was 23 μmol/l (range 2.0 μmol/l -108 μmol/l), with the majority (62%) with Hcy concentration > 15 μmol/l, the accepted upper threshold of desirable Hcy. Of the 128 subjects who had MMA measured, the median was 306 nmol/l (range 2 nmol/l-1,441 nmol/l); 48 (38%) had MMA concentration >400 nmol/l. Among 66 subjects with total and active B12 measured, one subject showed vitamin B12 deficiency (total B12 = 132 pmol/l and active B12 = 30 pmol/l). Twenty two subjects had borderline low for both or one of B12 markers Results showed a significant correlation between total B12 and active B12 (r = 0.65; p<0.0001). No statistically significant correlation between total B12 and Hcy (r=-0.1325; P=0.3129; n=60) or between active B12 and Hcy (r=0.0608; P=0.6445; n=60). Inverse correlation was seen between active B12 and MMA (r=-0.3048; P=0.0179; n=60) and total B12 and MMA (r=-0.2471; P=0.0570; n=60). Conclusion: A novel LC MS MS method that met analytical goals was developed for the simultaneous quantitation of MMA and Hcy in plasma. The method can be used in further study investigating if MMA and Hcy are useful in screening for early detection of B12 deficiency. The significant correlation of B12 status with MMA but not with Hcy indicates that plasma MMA could be considered a more sensitive or specific biomarker of early stage vitamin B12 deficiency.

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