Studies on the biosynthesis of N-acetylneuraminic acid in cultured arterial wall cells

( a ) Case I . (b) Case 2 . 0, Urine specimen, disc method; 0, urine specimen, direct method; .. urine specimen, amino acid analyser; A. blood specimen. amino acid analyser. Results are shown as a pcrcentage of the first day value under each condition. enzyme, and attempted to determine the level of homocysteine in patients with homocystinuria by our newly developed method for quantifying homocysteine in body fluids. The procedures consist of the condensation of homocysteine in body fluids with adenosine, quantitative determination of the product, adenosyl homocysteine, by h.p.l.c., and calculation of the level of homocysteine based on the above determination. The conditions for h.p.1.c. analysis on adenosyl homocysteine are described in a previous report (Shimizu et al. 1986). Urine samples obtained from two clinical cases of homocystinuria were examined. Case 1 was for a 15-year-old girl, who was being treated with vitamin B, and showed rather stabilized cljnical signs for several months (Fig. la) . Case 2 was a 1 I-year-old boy, who did not respond well to treatment with vitamin B, and showed large variations in the level of homocystine in his blood (Fig. Ib). The results of analyses of the urine and blood samples of the patients are summarized in Fig. 1 . They are shown as a percentage of the first day value. The direct method refers to the enzymic determination of homocystine without any pretreatment of urine samples, whereas the disc method refers to the enzymic determination of homocystineine in urine samples dried on a filter paper and extracted from the paper. Amino acid analysis was performed on aliquots of urine and blood without drying on paper. The patterns of variation shown in Fig. 1 indicate that the difference in changeability of homocystine levels in urine and blood between cases 1 and 2 as determined by amino acid analysis are replicated by the results of simple enzymic analysis of the urine, either by the direct method or by the disc method. The enzymic disc method allows collection of samples for mass screening by post, and is much simpler and more rapid than the methods using radioisotopes (Refsum et al., 1985) or an amino acid analycer.