Rapid determination of free d-serine with chicken d-serine dehydratase

We have developed a simple, rapid, and inexpensive method of measuring the concentration of intrinsic free d-serine in tissue samples. This method uses chicken d-serine dehydratase in an enzymatic reaction to produce pyruvate, which is detected spectrophotometrically. Pyridoxal 5′-phosphate (PLP), a cofactor of d-serine dehydratase, increased pyruvate formation by 28%. The presence of Zn2+ or ethylenediaminetetraacetic acid (EDTA) did not have any effect on pyruvate formation under the present assay conditions. In addition, this method was not affected by the presence of a large excess of l-serine, nor by the presence of tissue extracts, and accurately determined concentrations of 2–30 μM (200 pmol–3 nmol) of d-serine. The entire assay requires only 60 min. With this method, we determined the concentration of d-serine in various silkworm tissues. The results were in agreement with high performance liquid chromatography measurements. We found high concentrations of d-serine in silkworm larvae at day 3 of the fifth instar; specifically, 509 nmol g−1 wet tissue in the midgut, 434 nmol g−1 in the ovary, and 353 nmol g−1 in the testis.