The setup and evaluation of a high performance liquid chromatography procedure for plasma homocysteine analysis and its application in determining typical values and age- and sex- related differences in a sample of apparently healthy local Chinese subjects

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The setup and evaluation of a high performance liquid chromatography procedure for plasma homocysteine analysis and its application in determining typical values and age- and sex- related differences in a sample of apparently healthy local Chinese subjects

 

Author: Chu, Chau-hung
Title: The setup and evaluation of a high performance liquid chromatography procedure for plasma homocysteine analysis and its application in determining typical values and age- and sex- related differences in a sample of apparently healthy local Chinese subjects
Degree: M.Sc.
Year: 2010
Subject: Hong Kong Polytechnic University -- Dissertations
Homocysteine -- Pathophysiology
Liquid chromatography
Blood plasma
Cardiovascular system -- Diseases -- Risk factors.
Department: Dept. of Health Technology and Informatics
Pages: xiii, 125 leaves : ill. ; 31 cm.
InnoPac Record: http://library.polyu.edu.hk/record=b2355960
URI: http://theses.lib.polyu.edu.hk/handle/200/5842
Abstract: Background: Plasma homocysteine (Hcy) is an independent emerging cardiovascular disease (CVD) risk biomarker. Its measurement together with well-established traditional CVD risk biomarkers such as blood cholesterol is clinically useful in better assessing CVD risk. Therefore, a sensitive and specific method is needed for Hcy measurement. Aims: To 1. set up and evaluate of a high performance liquid chromatography (HPLC) procedure for plasma homocysteine (Hcy) analysis. 2. determine typical values and investigate of age- and sex- related differences of plasma Hcy in a sample of apparently healthy local Chinese subjects. Results: An HPLC with fluorescence detection (HPLC-FL) for plasma total homocysteine (tHcy) analysis was set up. After pre-treatment procedures, injection of a 10 μL derivatized sample onto the HPLC-FL system was performed. The analysis time per sample is 10 minutes. The method proved to be linear from 0 to 100 μmol/L (r² = 0.9999). The within-run and between-run coefficient of variations were 0.5%-0.8% and 2.8%-4.6% respectively at each of 3 different Hcy levels (5, 10 and 50 μmol/L, n = 6 for each). The detection reproducibility of plasma tHcy at 4℃ over 15 hours was 0.7%-2.0% (n = 11) across 3 different Hcy levels (5, 10 and 50 μmol/L). The mean recovery was 94.5%-102.8% across 3 different Hcy levels (5, 10 and 50 μmol/L, n = 6 for each). The limit of detection and the limit of quantitation in plasma matrix were 0.36 μmol/L and 0.73 μmol/L respectively. The range of plasma tHcy in 197 apparently healthy adults was 3.6 to 50.7 μmol/L. The median, mean (SD) and range of plasma tHcy for men (n = 86) were found to be 9.7, 10.9 (5.8) and 5.3-50.7 μmol/L respectively. The median, mean (SD) and range of plasma tHcy for women (n = 111) were found to be 7.2, 7.2 (1.6) and 3.6-12.0 μmol/L respectively. The plasma tHcy in men with a median of 9.7 μmol/L was significantly higher (p < 0.0001) than that of women with a median plasma tHcy of 7.2 μmol/L. The median, mean (SD) and range of plasma tHcy for the age group 20-35 years (n = 57) were found to be 8.6, 10.3 (6.9) and 3.6-50.7 μmol/L respectively. The median, mean (SD) and range of plasma tHcy for the age group 36-50 years (n = 63) were found to be 7.6, 8.2 (2.5) and 4.5-20.7 μmol/L respectively. The median, mean (SD) and range of plasma tHcy for the age group 51-65 years (n = 77) were found to be 7.7, 8.2 (2.6) and 4.5-17.5 μmol/L respectively. The medians of plasma tHcy of 8.6, 7.6 and 7.7 μmol/L for the different age groups were not significantly different (p = 0.0777) from each other. The range covering 95% of results was 5.3-17.3 μmol/L for men and 3.6-10.2 μmol/L for women.
Conclusion: A rapid, reliable, sensitive and specific HPLC-FL for plasma tHcy analysis was developed. It gives a documented evaluated methodology for plasma tHcy measurement in our Health Technology and Informatics Biomedical Science reference laboratory. In a sample of 197 apparently healthy adults in our local population, men had higher plasma tHcy levels than women. Interestingly, taking a cut-off of "desirable" plasma tHcy of 10 μmol/L, 39 (45%) men and 7 (6%) women had high levels, indicating elevated CVD risk. Eight (9%) men but no women had clearly elevated plasma tHcy levels (> 15 μmol/L), indicating significantly elevated CVD risk. Results indicate that health promotion for CVD risk reduction in the general population is needed.

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