GC-MS analysis of S-nitrosothiols after conversion to S-nitroso-N-acetyl cysteine ethyl ester and in-injector nitrosation of ethyl acetate.

S-Nitrosothiols from low-molecular-mass and high-molecular-mass thiols, including glutathione, albumin and hemoglobin, are endogenous potent vasodilators and inhibitors of platelet aggregation. By utilizing the S-transnitrosation reaction and by using the lipophilic (pK(L) 0.78) and strong nucleophilic synthetic thiol N-acetyl cysteine ethyl ester (NACET) we have developed a GC-MS method for the analysis of S-nitrosothiols and their (15)N- or (2)H-(15)N-labelled analogs as S-nitroso-N-acetyl cysteine ethyl ester (SNACET) and S(15)NACET or d(3)-S(15)NACET derivatives, respectively, after their extraction with ethyl acetate. Injection of ethyl acetate solutions of S-nitrosothiols produced two main reaction products, compound X and compound Y, within the injector in dependence on its temperature. Quantification was performed by selected-ion monitoring of m/z 46 (i.e., [NO(2)](-)) for SNACET and m/z 47 (i.e., [(15)NO(2)](-)) for S(15)NACET/d(3)-S(15)NACET for compound X, and m/z 157 for SNACET and m/z 160 for d(3)-S(15)NACET for compound Y. In this article we describe the development, validation and in vitro and in vivo applications of the method to aqueous buffered solutions, human and rabbit plasma. Given the ester functionality of SNACET/S(15)NACET/d(3)-S(15)NACET, stability studies were performed using metal chelators and esterase inhibitors. The method was found to be suitable for the quantitative determination of various S-nitrosothiols including SNACET externally added to human plasma (0-10microM). Nitrite contamination in ethyl acetate was found to interfere. Our results suggest that the concentration of endogenous S-nitrosothiols in human plasma does not exceed about 200nM in total. Oral administration of S(15)NACET to rabbits (40-63micromol/kg body weight) resulted in formation of ALB-S(15)NO, [(15)N]nitrite and [(15)N]nitrate in plasma.

Mechanism of the insulin-releasing action of alpha-ketoisocaproate and related alpha-keto acid anions.

Alpha-ketoisocaproate directly inhibits the ATP-sensitive K(+) channel (K(ATP) channel) in pancreatic beta-cells, but it is unknown whether direct K(ATP) channel inhibition contributes to insulin release by alpha-ketoisocaproate and related alpha-keto acid anions, which are generally believed to act via beta-cell metabolism. In membranes from HIT-T15 beta-cells and COS-1 cells expressing sulfonylurea receptor 1, alpha-keto acid anions bound to the sulfonylurea receptor site of the K(ATP) channel with affinities increasing in the order alpha-ketoisovalerate < alpha-ketovalerate < alpha-ketoisocaproate < alpha-ketocaproate < beta-phenylpyruvate. Patch-clamp experiments revealed a similar order for the K(ATP) channel-inhibitory potencies of the compounds (applied at the cytoplasmic side of inside-out patches from mouse beta-cells). These findings were compared with the insulin secretion stimulated in isolated mouse islets by alpha-keto acid anions (10 mM). When all K(ATP) channels were closed by the sulfonylurea glipizide, alpha-keto acid anions amplified the insulin release in the order beta-phenylpyruvate < alpha-ketoisovalerate < alpha-ketovalerate approximately alpha-ketocaproate < alpha-ketoisocaproate. The differences in amplification apparently reflected special features of the metabolism of the individual alpha-keto acid anions. In islets with active K(ATP) channels, the first peak of insulin secretion triggered by alpha-keto acid anions was similar for alpha-ketoisocaproate, alpha-ketocaproate, and beta-phenylpyruvate but lower for alpha-ketovalerate and insignificant for alpha-ketoisovalerate. This difference from the above orders indicates that direct K(ATP) channel inhibition is not involved in the secretory responses to alpha-ketoisovalerate and alpha-ketovalerate, moderately contributes to initiation of insulin secretion by alpha-ketoisocaproate and alpha-ketocaproate, and is a major component of the insulin-releasing property of beta-phenylpyruvate.