Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.99.3 (diaphorase)
 5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe a highly sensitive and accurate automated continuous-flow method for determining bile acids in serum. The bile acids are first liberated from serum protein by dialysis at alkaline pH and then measured fluorometrically after the following enzymic reaction. Bile acids are converted to 3-oxo bile acids with 3alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) with concomitant reduction of NAD+ to NADH. The hydrogen in the generated NADH is transferred by diaphorase (EC 1.6.4.3) to resazurin to yield resorfin, the fluorophore. Only 100 microliter of serum is required and 40 determinations can be done per hour. The CV for 20 replicate determinations in serum with a mean bile acid concentration of 9.8 mumol/liter was 2.6%. The CV for day-to-day variation for another serum on 27 successive days was 3.0% (mean concentration, 10.0 mumol/liter). We applied this method to 826 sera from various diseases; 29% exceeded the upper limit of normal, 10 mumol/liter, and abnormally high values (greater than 20 mumol/liter) were almost exclusively limited to sera from hepatobiliary and enteric disorders.
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PMID:Continuous-flow determination of bile acids in serum, and its clinical application. 65 94

A simple and sensitive method was developed for the quantification of serum total 3alpha-hydroxy bile acids. 0.1 ml of serum was mixed with tris(hydroxymethyl) aminomethane hydrochloric acid buffer and heated at 67 degrees C for 30 min. To the solution were added 3alpha-hydroxysteroid : oxidoreductase (EC 1.1.1.50; 3alpha-HSD), NAD, diaphorase (EC 1.6.4.3) and resazurin. The mixture was incubated at 20 degrees C for 1 h. The resultant fluorescence of resorfin was measured at 580 nm with the excitation at 560 nm. The blank value was obtained after the same treatment of another 0.1 ml of the same serum without 3alpha-HSD. A linear relationship was obtained between the amount of bile acids and the fluorescence intensities in the range of 1 to 150 mumol/1. The recovery of bile acids added to the serum was 81.4 +/- 2.5 (S.D.)% for cholate, chenodeoxycholate and deoxycholate. The bile acid content in the serum was 48.8 mumol/1 with a standard deviation of +/- 0.42 and a coefficient of variation of +/- 0.87% in 10 replicate determinations. The mean bile acid content of normal fasting male sera was 8.0 mumol/1 (3.6-12.6 mumol/1, n = 12) and of female sera 6.8 mumol/1 (3.2-12.7 mumol/1, n = 13).
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PMID:A simple and sensitive assay of total serum bile acids. 94 25

In this simple and direct method for determining total bile acids in serum, the serum was mixed with sodium pyruvate, a lactate dehydrogenase blocker, and bile acids were then measured spectrophotometrically after the following enzyme reaction. Bile acids are converted to 3-oxo bile acids with 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) with concomitant reduction of NAD+ to NADH. The hydrogen in the NADH generated is transferred by diaphorase (EC 1.6.4.3) to nitrotetrazolium blue to yield diformazan 540 nm). Analytical recovery of the various bile acids in serum averaged 96.2%. The CV for the day-to-day variation was 4.3%. Normal values are less than 7 mumol/L. Total serum bile acids were estimated by this method in 118 fasting patients with various liver diseases. This determination is clearly shown to be useful as a liver-function test.
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PMID:Direct spectrophotometry of total bile acids in serum. 689 53