Determination of hepatic cysteinesulfinate decarboxylase activity in fish by means of OPA‐prelabeling and reverse‐phase high‐performance liquid chromatographic separation

Taurine is known as one of the final metabolites of sulfur amino acids in mammals. In mammals, this sulfur-containing substance is considered to be involved in physiological functions such as membrane protection, detoxification, and antioxidation. Although it is generally believed that fish contain large amounts of taurine in their tissues, there is little information available on the capacity of taurine biosynthesis in fish. Only Yokoyama et al. have reported that rainbow trout are able to synthesize a considerable amount of taurine in their liver via the same metabolic pathways as mammals. However, Park et al. have reported that the supplementation of taurine to the diet of juvenile flounder improves their growth performance. Takagi et al. have also demonstrated that the addition of taurine to the substituted protein diet of red sea bream reduces the incidence of green liver and elevates feed efficiency. These data suggest the necessity of extensive studies on the ability of taurine biosynthesis in fish. It seems that marine fish cannot synthesize sufficient amounts of taurine in its pure form. In the present report, we developed an analytical method for determining the cysteinesulfinate decarboxylase [EC 4.1.1.29] activity in fish by means of the OPA-prelabeling and reverse-phase high performance liquid chromatography (HPLC) because this enzyme is known to play a regulatory role in the biosynthesis of taurine in mammals. After the oxidation of hypotaurine to taurine, which is formed by the enzyme reaction, we measured the taurine content and expressed this content as the enzyme activity. Freshly isolated liver was minced and homogenized in 2.5-fold volumes of 0.25 M sucrose containing 10 mM sodium phosphate buffer (pH 7.4). The supernatant obtained after centrifugation at 1500 ¥g for 5 min was dialyzed against 10 mM phosphate buffer for 4 h to remove the endogenous taurine. This operation was carried out at a temperature of 4∞C or below. The crude enzyme solution was used for the enzyme assay. Total incubation volume was 1.0 mL and the incubation mixture consisted of 100 mM sodium phosphate buffer (pH 7.2), 1.0 mM cysteinesulfinate, 0.2 mM pyridoxal 5¢-phosphate, 4 mM 2mercaptoethanol, and 0.4–1.0 mg protein of the crude enzyme solution. Protein content was estimated by the colorimetric method described by Lowry et al. The reaction was started by adding the enzyme solution and incubation was continued at 35∞C for 60 min. The reaction was terminated by heating the mixture at 70∞C for 3 min, and b-Alanine (0.2 mmole) was then added as an internal standard. During incubation, hypotaurine formed by the enzymatic reaction is thought to be oxidized to taurine by either enzymatic and/ or non-enzymatic reactions. Thus, the formed hypotaurine was converted into taurine by the addition of 200 mL of 31% H2O2 and 29% NH4OH (1 : 1, v/v) mixture. After standing overnight, an aliquot of the solution was dried under a stream of Short Paper