A sensitive method for estimation of oxaloacetate

Abstract

The colorimetric method for estimation of acetoacetate by reaction with diazotized-p-nitroaniline has been adapted for measurements of oxaloacetate in amounts of 0.003-0.03 umole. To each of 2 glass-stoppered test tubes containing 2 ml of supernatant from a 20% tissue homogenate (solutions A and B) is added 0.4 ml of 0.25% NaNO2 (to remove ascorbic acid). Exactly 30 seconds later 2 ml of [image]-sodium acetate-acetic acid buffer, pH 5.2, is added, followed immediately by 3 ml of freshly prepared cold diazo reagent. After incubation for 20 minutes at 25[degree], the tubes are placed in an ice-bath for 3 minutes. The color due to the reaction of the diazo reagent with both acetoacetate and oxaloacetate is determined with solution A by adding 6 ml of ethyl acetate and then 6 ml of 5 [image] HC1. The color is immediately and quantitatively extracted into ethyl acetate by shaking the tubes 30-35 times. Not more than 7.5 seconds should elapse between the addition of acid and the extraction. With solution B the color produced by oxaloacetate is destroyed by adding 6 ml of 5 [image] HCl and placing the tube for exactly 8 minutes in an ice-bath. The remaining color is then extracted with 6 ml of ethyl acetate. A reagent blank containing 2 ml of 3% trichloro-acetic acid instead of the tissue extract is subjected to each of the above procedures. For solution C both oxaloacetate and acetoacetate are destroyed by heating 2 ml of the extract at 100[degree] for 5 minutes. The solution is men cooled rapidly and treated in the same way as solution B and read against the appropriate reagent blank. The optical density of the ethyl acetate layer is read at 450 m[mu] in a 4 cm cell (model SP. 500 Unicam spectrophotometer). The bottom of the cell is raised, and the cell carrier appropriately masked, so that readings can be made with a 5-6 ml sample. The difference in optical density between solution A and solution B is taken to represent the color produced by reaction of the diazo reagent with oxaloacetate, and between solutions B and C that with acetoacetate. The steady-state level of oxaloacetate in the livers of normal and fasted rats was found to be about 4 umoles/100 g dry weight of tissue. The level was essentially unchanged in starvation when the level of acetoacetate increased tenfold.