Analysis of homocysteine in human urine using high-performance liquid chromatography with electrochemical detection.

PIP: This report describes the modifications made to the analytical system prior to determination of homocysteine in human urine. It also contains data which compare the homocysteine concentrations in the urine of males and females as well as the results of a study in which the effect of oral contraceptive (OC) use by a group of women upon their urinary concentrations was compared to a control group. Furthering this work, the effect of cigarette smoking on the urinary homocysteine concentrations of OC users as compared to control was assessed as were the urinary homocysteine concentrations over a 28-day collection period for both an OC-using female and a male volunteer. 2 M perchloric acid (AR grade, Ajax Chemicals) was used to prevent thiol group oxidation. Approximately 50 ml of urine flow from volunteers participating in this study were collected into vessels containing 10 ml of 2M perchloric acid. In all cases the samples were obtained in the morning immediately upon waking, and for the comparison of OC users with controls, the urine samples were obtained on the 21st day of their cycle. Homocysteine was quantified by the standard addition technique using peak-height measurement, the determination being carried out in triplicate. The homocysteine concentration then was expressed per mg creatinine. All the study participants were apparently healthy with an average age in the mid-20s for the femles and the late 20s for the males. The significantly higher excretion rate of homocysteine for the males compared with the females was of interest since the risk of myocardial infarction for males is up to 6 times greater than for females in the under 40 age bracket. Neither OC formulation caused an increase in the excreted level of homocysteine in the urine of users as compared to the control group. It seems that the increased incidence of thrombotic episodes observed among women using OCs as compared to controls was due to a cause other than an elevated homocysteine level caused by the synthetic steroid components of the OC formulation. The similarity of homocysteine excretion levels in the urine of smoking and nonsmoking OC users suggests that smoking most likely increases the risks associated with OC, including that of developing thrombosis. Other results show that any homocysteine urinary excretion changes which might follow the estrogen pattern of a triphasic OC formulation during a cycle would appear to be impossible to detect. This is because dietary effects, among other things, may cause sufficiently large changes in the level of homocysteine excreted in the urine to mask any effects upon the excretion rate that hormonal changes might produce.^ieng

Changes in urinary homocysteine following synthetic steroidal estrogen and progestogen administration to rats.

The present work involved the administration of both ethynyl estradiol and levonorgestrel to groups of rats, followed by determination of the homocysteine excretion rate in urine. The results indicate that a statistically significant difference exists between the excreted levels of homocysteine in the urine of both control and levonorgestrel-treated rats and the levels shown by rats treated with ethynyl estradiol. The implications of these results are discussed, especially with respect to observations which indicate that homocysteine may be a precipitating factor in the development of thrombosis. Also included in this paper is a study which confirms the identity of the HPLC peak as being homocysteine by forming a radioactive derivative of this particular sulphydryl-containing amino acid, and then analysing the resulting mixture by TLC.