-Ketoglutarate (a-KG) is used in the determination of transaminases, malate dehydrogenase, glutamate dehydrogenase, urea and ammonia.

1. 0.1M Triethanolamine buffer/substrate, pH 7.6: 1.86 g TEA• HCl with 80 ml distilled water. Adjust pH to 7.6 with 1M NaOH-Na₂. Adjust volume to 100 ml with distilled water.
2. 14 mM NADH: 10 mg NADH-Na₂ with 1 ml distilled water.
3. 3 M Ammonium chloride: 16.05 g NH₄Cl with 100 ml distilled water.
4. Glutamate dehydrogenase, from beef liver: 20 mg protein/ml (120 U/mg).

1. Dissolve 25 mg Ketoglutarate in 25 ml distilled water in a volumetric flask.
2. Set spectrophotometer (equipped with strip chart recorder and temperature control) at 340 nm and 25° C.
3. Into a cuvette, pipette the following:

		BLANK	SAMPLE
Buffer	(1)	2.80 ml	2.80 ml
NADH	(2)	0.05 ml	0.05 ml
NH4Cl	(3)	0.15 ml	0.15 ml
distilled H2O		0.05 ml	- - -
sample		- - -	0.05 ml

Mix and read absorbance A₁.
4. Start the reaction by adding 0.01 ml of GLDH to both the blank and sample. Mix and read absorbance A₂.

A = (A₁ - A₂)_sample - (A₁ - A₂)_blank
Concentration of -KG = X A (mg/ml sample solution)
Concentration of -KG-Na₂ H₂• 3H₂ O = X A (mg/ml sample solution)
% -KG = X 100
Total Vol. = 3.06 ml
190.1 = MW of -KG
Sample Vol. = 0.05ml