title: The determination of plasma oxalate concentrations using an enzyme/bioluminescent assay. 2. Co-immobilisation of bioluminescent enzymes and studies of in vitro oxalogenesis.
abstract: An inexpensive, continuous flow assay for the determination of oxalate in plasma is described. The assay is based on the bioluminescent determination of NADH, a product of the degradation of oxalate by oxalate decarboxylase and formate dehydrogenase, using bioluminescent enzymes immobilized on cyanogen bromide-activated sepharose. The detection limit of the assay is 0.8 mumol/l. Intra-batch CV values of 5.2 and 3.8% were obtained at oxalate concentrations of 18 and 60 mumol/l. Recovery of added oxalate averaged 100.7%. Plasma oxalate ranged from less than 0.8 to 2 mumol/l in 14 healthy subjects, and from 6 to 134 mumol/l in 125 patients with renal disease treated by continuous ambulatory peritoneal dialysis. Ascorbic and dehydroascorbic acid did not directly interfere in the assay. In vitro oxalogenesis was observed in blood from 12 healthy subjects, but only after samples had stood at room temperature for more than 6 h. No significant oxalate generation occurred in blood from 24 patients with impaired renal function, even after standing at room temperature for 24 h. Oxalate generation was inhibited by the addition of oxalate to plasma, but the addition of urea and creatinine was without effect.
authors.affiliation: Department of Clinical Biochemistry, University of Newcastle upon Tyne, New Medical School, UK.
authors.forename: I S
authors.initials: IS
authors.lastname: Parkinson
authors.forename: S M
authors.initials: SM
authors.lastname: Channon
authors.forename: C R
authors.initials: CR
authors.lastname: Tomson
authors.forename: L R
authors.initials: LR
authors.lastname: Adonai
authors.forename: M K
authors.initials: MK
authors.lastname: Ward
authors.forename: M F
authors.initials: MF
authors.lastname: Laker
mesh_terms: D003404:Creatinine; D004797:Enzyme-Linked Immunosorbent Assay; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000378:metabolism*; D009243:NAD / Q000032:analysis; D010070:Oxalates / Q000097:blood* / Q000378:metabolism; D012016:Reference Values; D014508:Urea
chemical_list: D004800:Enzymes, Immobilized; D008164:Luminescent Proteins; D010070:Oxalates; D009243:NAD; D014508:Urea; D003404:Creatinine