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

A fluorometric flow-injection method for determining carnitine with use of immobilized enzymes carnitine dehydrogenase (EC 1.1.1.108) and diaphorase (EC 1.8.1.4) was developed and applied to the assay of carnitine in serum of patients treated with valproic acid. After fractionation and hydrolysis of carnitines in serum samples by perchloric acid and potassium hydroxide, liberated carnitine was converted to resorufin by immobilized carnitine dehydrogenase and diaphorase in the presence of beta-NAD+ (1.0 mmol/L), resazurin (12.5 mumol/L), and Tris acetate (0.6 mol/L, pH 9.0) at 37 degrees C. The fluorescence intensity of resorufin was monitored at lambda Ex 560 nm and lambda Em 580 nm. The calibration curve was linear for carnitine amounts from 0.1 to 1.0 nmol. Quantitative analytical recovery and satisfactory within- and between-run imprecision of carnitine in each carnitine fraction were obtained. Interference by bilirubin, serum albumin, and hemoglobin was negligible. Carnitine deficiencies were detected in about 20% of the valproic acid-treated patients (n = 198). The present method should be useful for monitoring carnitine deficiencies in clinical laboratories.
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PMID:Fluorometric determination of carnitine in serum with immobilized carnitine dehydrogenase and diaphorase. 225 48

Cyclic ADP-ribose (cADPR) is an intracellular messenger that triggers the release of calcium ions from intracellular stores in a variety of cell types. The fluorometric cycling assay has become the preferred method for measuring cADPR due to its high level of sensitivity (in the sub-nanomolar range) and its use of commercially available reagents. Additionally, the assay is performed in multiwell plates, making it suitable for high throughput screening using a fluorescence plate reader. The findings reported in this paper present several problems that may be encountered during various stages of the assay, and provide solutions to these problems. Modifications to the assay address reduced recovery of sample and cADPR with removal of perchloric acid (PCA) using organic solvent, reduction in diaphorase activity with heat treatment, and effects on resorufin fluorescence by pH range. Using these modifications, we report an increase of approximately 15% in recovery of brain cADPR, and show that between-subject variability is greatly reduced. We hope that these observations will encourage more widespread application of this valuable assay.
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PMID:Modifications to increase the efficiency of the fluorometric cycling assay for cyclic ADP-ribose. 1701 83