Clinical usefulness of a HPLC method for simultaneous quantitation of plasma homocysteine and cysteine.

OBJECTIVE: Homocysteine (Hcy) is a risk for vascular occlusion. It is metabolized via remethylation to methionine and transsulfuration to cysteine which has also been related to vascular occlusion. Simultaneous determination of Hcy and cysteine has additional clinical usefulness in providing a presumptive clue to the nature of hyperhomocysteinemia. MATERIAL AND METHOD: A manual HPLC method has been worked out for simultaneous determination of plasma Hcy and cysteine. Concentrations of Hcy were validated with the widely used automated Abbott AxSYM assay. Its usefulness was tested in 87 omnivores and 111 vegans. RESULTS: Excellent correlation between the values of Hcy was found between the manual HPLC method and the automated Abbott assay. The vegans had significantly higher levels of Hcy but lower levels of cysteine than the omnivores (mean +/- SD, micromol/L 23.6 +/- 18.0 vs. 8.8 +/- 2.1 p < 0.001, 225 +/- 30 vs. 245 +/- 34 p < 0.001, respectively). In contrast, the vegans had significantly lower levels of serum vitamin B12 and plasma vitamin B6 than the omnivores (median values 186 vs 565 pg/ml, p < 0.001; 37.4 vs. 47.4 nmol/L, p < 0.001 respectively). These findings indicate that the hyperhomocysteinemia in the vegans results from impairment of both remethylation and transsulfuration pathways of Hcy secondary to inadequacy of vitamins B12 and B6 respectively. Thus simultaneous determination of Hcy and cysteine is more useful than determination of only Hcy in that it provides a clue to the nature of hyperhomocysteinemia. CONCLUSION: The manual HPLC method and the Abbott assay gave comparable Hcy values, and thus can be used interchangeably. The HPLC method is economical, useful for hospitals with less demand for determination of Hcy, and capable of simultaneously determining cysteine which has implication in clinical practice.

Comparison of colorimetry and electrothermal atomic absorption spectroscopy for the quantification of non-transferrin bound iron in human sera.

This paper describes a comparison of two analytical techniques, one employing bathophenanthrolinedisulfonate (BPT), a most commonly-used reagent for Fe (II) determination, as chromogen and an electrothermal atomic absorption spectroscopy (ETAAS) for the quantification of non-transferrin bound iron (NTBI) in sera from thalassemic patients. Nitrilotriacetic acid (NTA) was employed as the ligand for binding iron from low molecular weight iron complexes present in the serum but without removing iron from the transferrin protein. After ultrafiltration the Fe (III)-NTA complex was then quantified by both methods. Kinetic study of the rate of the Fe (II)-BPT complex formation for various excess amounts of NTA ligand was also carried out. The kinetic data show that a minimum time duration (> 60 minutes) is necessary for complete complex formation when large excess of NTA is used. Calibration curves given by colorimetric and ETAAS methods were linear over the range of 0.15-20 microM iron (III). The colorimetric and ETAAS methods exhibited detection limit (3sigma) of 0.13 and 0.14 microM, respectively. The NTBI concentrations from 55 thalassemic serum samples measured employing BPT as chromogen were statistically compared with the results determined by ETAAS. No significant disagreement at 95% confidence level was observed. It is, therefore, possible to select any one of these two techniques for determination of NTBI in serum samples of thalassemic patients. However, the colorimetric procedure requires a longer analysis time because of a slow rate of exchange of NTA ligand with BPT, leading to the slow rate of formation of the colored complex.