Determination of plasma homocysteine by high-performance liquid chromatography with fluorescence detection

Donald W. Jacobsen, Vytenis J. Gatautis, Ralph Green

Research output: Contribution to journalArticle

88 Scopus citations

Abstract

Severe homocystinemia is frequently associated with vascular disease while the pathological consequences of moderate or slightly elevated plasma homocysteine are unknown. Cobalamin and folate deficiencies may result in an elevation of plasma homocysteine. A sensitive and reproducible assay for total plasma homocysteine has been developed. The essential steps in the assay include (i) conversion of homocysteine disulfides to free homocysteine with borohydride reduction; (ii) conjugation of homocysteine with monobromobimane; (iii) separation of homocysteine-bimane from other plasma thiol-bimane adducts by reverse-phase high-performance liquid chromatography; and (iv) detection and quantitation of homocysteine-bimane by fluorometry. The method has a sensitivity of 4.4. pmol of homocysteine and is highly reproducible (intra- and interassay co-efficients of variation = 4.97 and 4.53%, respectively). The mean concentration of total plasma homocysteine in nonfasting adult males (n = 12) and females (n = 12) was 15.8 (range, 7.0-23.7) and 16.5 nmol/ml (range, 8.6-20.7), respectively. Markedly elevated levels of homocysteine were found in patients with cobalamin and folate deficiency. Total plasma homocysteine represents approximately 4% of borohydride-generated thiol reactivity in the plasma of normal individuals.

Original languageEnglish (US)
Pages (from-to)208-214
Number of pages7
JournalAnalytical Biochemistry
Volume178
Issue number1
DOIs
StatePublished - 1989
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Fingerprint Dive into the research topics of 'Determination of plasma homocysteine by high-performance liquid chromatography with fluorescence detection'. Together they form a unique fingerprint.

  • Cite this