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Primary hyperoxaluria type I (PH1; McKusick 259900) is a disorder of glyoxylate metabolism characterized by the excessive synthesis and excretion of oxalate, eventually leading to renal failure. The underlying cause of the disease is a deficiency of the hepatic peroxisomal enzyme alanine:glyoxylate aminotransferase (AG; EC 2.6.1.44), as first shown by Danpure and Jennings (1986). Correct diagnosis of hyperoxaluria type I rests upon determination of the enzyme activity in liver biopsy specimens, since the enzyme is expressed in liver only. The enzyme activity is usually measured radiochemically (Allsop et al 1987; Toone and Applegarth 1991), although spectrophotometric assays have also been described (Danpure and Jennings 1988;Wanders et al 1990). However, both methods are rather laborious and suffer from several drawbacks. Elaborating on our earlier-described method we now describe a simple, direct method for measurement of hepatic alanine : glyoxylate aminotransferase. The method is based on the use of Tris to terminate the reaction by forming a complex with one of the substrates, i.e. glyoxylate, and lactate dehydrogenase to estimate the amount of pyruvate formed. M E T H O D SThe liver samples were homogenized in 100mmol/L potassium phosphate (pH 8.0) plus 0.1% (w/v) Triton X-100 in a glass Potter-Elvehjem microhomogenizer with a tight-fitting pestle to give a 1% (w/v) homogenate. This was followed by sonication at 0°C for three periods of 15 s (8 W absorbed power) at 45 s intervals. The assay for alanine:glyoxylate aminotransferase activity was carried out in a Cobas Fara centrifugal analyser (Hoffman-La Roch, Basel, Switzerland): 5 #g of human liver homogenate protein was incubated in 50 #1 of a medium containing 200 mmol/L potassium phosphate (pH = 8.0), 100/~mol/L pyridoxal-5-phosphate, 0.1% (w/v)

SUMMARY. In this paper we studied the glyoxy1ate-dependent transamination of Lalanine and L-glutamate in human liver homogenates in order to develop a reliable method for the determination of true alanine:glyoxylate aminotransferase activity in liver homogenates from patients suspected to suffer from hyperoxaluria type 1. Measurements were made according to two protocols described in literature in control human liver homogenates which were either untreated or treated with an antiserum raised against purified alanine:glyoxylate aminotransferase. The results obtained show that enzyme activity can best be determined at pH 8.0 as compared to pH 7.4 since the former leads to a higher sensitivity of the method. Alanine:glyoxylate aminotransferase activities measured at pH 8.0 are approximately 50070 higher compared to the enzyme activities measured at pH 7.4. Accordingly, it is proposed to measure alanine:glyoxylate aminotransferase activity at pH 8.0 using the newly determined correction factor as described in this paper.