Amplifier system for microanalysis - of nicotinamide adenosine di:nucleotide and its reduced form

摘要

Amplifier system for determining microquantities of NAD and NADH comprises (a) a single enzyme (I) specific for converting NAD to NADH as well as NADH to NAD; (b) an 18 or 19C steroid substrate (II) having DELTA 5 or DELTA 5(10) or DELTA' or DELTA'(10) double bonds and 3 beta-OH and 17-keto functional gps. and nc) an isomerase (III) which converts the enzymatic oxido-redn. prods. of DELTA 5 and DELTA 5(10) steroids to a 3-keto-DELTA 4-chromophore. Also new is a kit comprising separate tubes contg. na) 40 mu g/ml. (II); (b) 10m IU/ml. (Ia) and (c) 100m IU/ml. (III). Esp. (I) is a 3 beta-17 beta-hydroxysteroid dehydrogenase (Ia) esp. purified to remove any NAD, and (III) is esp. a DELTA 5-3-keto-steroid isomerase (IIIa) used at 10-1000 m I.U. Used in analytical and clinical assays e.g. of bile acids, steroid hormones or prostaglandins. The system improves sensitivity for NAD and NADH permitting analysis of quantities down to 50 pmoles to 200 pmoles with good precision.