Capillary gas chromatography measurement of oxalate in plasma and urine

Abstract

Oxalic acid can be measured reliably in urine by colorimetric methods [1] and is useful for the diagnosis of hyperoxaluric states that result in nephrolithiasis. The measurement of plasma oxalic acid has been far more difficult due to its very low concentration in normal plasma and a variety of methodologic problems. Normal plasma oxalic acid, measured by in vivo isotopic dilution, has been reported in the range of —l mo1es/liter (-9 g/dl) [2—4]. Far higher values, obtained by most published methods [5, 6] suggest a lack of specificity that would obscure variations in normal levels. The difficulty in preserving specimens, the lack of a way of monitoring interference, and the need for very large sample volumes makes enzymatic methods [7] less than ideal. Recently, Wolthers and Hayer [81 reported a capillary gas chromatographic method for measuring plasma oxalic acid that gave a normal range closer to that obtained by in vivo isotopic dilution [41. We have adapted and verified this method for measuring urinary and plasma oxalic acid and applied the method to normal adults and patients with primary hyperoxaluria. These patients have been studied during renal insufficiency, during hemodialysis, and with various levels of renal function after renal transplantation. Methods. Urine specimens were collected for 24 hr on ice, acidified to a pH of less than 2.0 with HCI (1:100 v/v), and frozen at —20°C or —70°C until analysis. To triplicate 0.5 ml samples we added malonic acid (15 pg), a further two drops of 6 N HCI, and 1 g NaC1. Heparinized blood was collected on ice, the plasma separated (X2000 g, 20 mm) within 1 hr of collection, and 0.5-ml aliquots acidified with two drops of HC1 and frozen (—70°C) in microfuge tubes (Sarsted). Samples were partially thawed to transfer of the frozen/precipitated sample, containing 2 g of malonic acid and 1 g NaC1, with one rinse of 0.5 ml of 0.1 N HCI. For either urine or plasma aliquots, a 4-mm vigorous solvent extraction with 6 ml of ethyl acetate was performed twice. Ethyl acetate was evaporated at 35°C under anhydrous N2, placed under P205 in vacuo for 30 mm and organic acids derivatized in the same tube with 50 to 150 (urine) or 30 tl (plasma) of 30:1 (v/v) mixture of bis-trimethylsilyltrifluoroacetamide (BSTFA) (Regis Chemical Co., Morton