A kinetic determination of ammonia in plasma

A simple method is described for the determination of the ammonia concentration in plasma using the enzyme glutamic dehydrogenase.The measuring principle is a kinetic one on a LKB Reaction Rate Analyzer. In this way the unreliable determination of the absorbance at the beginning and at the endpoint of the reaction, needed in other enzymatic methods, can be avoided.     

Enzymatic determination of ammonia in blood plasma

A kinetic method is described for the determination of the ammonia content of blood plasma using glutamate dehydrogenase and giving satisfactory accuracy and precision. The reaction mixture is composed of 2.2 ml of 0.05 M diethanolamine buffer pH 8.4, 0.1 ml of 4.2 mM reduced nicotinamide adenine dinucleotide, 0.1 ml of 0.1 M 2-oxoglutarate and 0.5 ml of fresh blood plasma. One tenth ml of glutamate dehydrogenase suspension containing 400 U of the enzyme is added after preincubation for 25 min at 20° and the initial velocity of the enzyme reaction measured by reading or recording the decrease in absorbance at 340 nm due to the oxidation of reduced nicotinamide adenine dinucleotide during glutamate formation from the ammonia present. The ammonia concentration in plasma is obtained from a calibration curve or by calculation. Possible effects of endogenous enzymes are discussed.     

Two-point determination of plasma ammonia with the centrifugal analyzer.

A simple, convenient, and rapid method for determining ammonia in plasma by the glutamate dehydrogenase reaction is described for the centrifugal analyzer. The measuring principle is fixed-time, with NADH as the coenzyme. ADP is added to stabilize glutamate dehydrogenase and prevent interference from endogenous plasma ADP. The reaction is linear to 400 mumol of ammonia per liter. The plasma sample volume is 100 microliter and the whole procedure takes only 25 min, including the 15-min preincubation. The normal range for venous plasma was 44 +/- 13.5 (SD) mumol of ammonia per liter.     

The fluorimetric determination of ammonia in protein-free filtrates of human blood plasma.

Ammonia has been determined in filtrates of human plasma after precipitation of the proteins by perchloric acid. After restoration of the pH to around 7.5, addition of 2-oxoglutarate, NADH and glutamate dehydrogenase (GDH) convers the ammonia to L-glutamate with oxidation of the NADH to NAD. This latter reaction was utilised in two ways. In the first, reduction of native NADH fluorescence under the conditions of the GDH reaction provided a measure of ammonia concentration. In the second, residual NADH was destroyed by acid treatment, and the fluorescent product generated from NAD under strongly alkaline conditions was assayed. The optimal requirements for both methods were defined, their linearity and precision ascertained, and their relative merits compared. The first method was convenient for "one-off" estimations, and the second for larger batches. Ammonia concentration increased in plasma and in acid protein-free filtrates of plasma irrespective of the conditions of storage; however when the latter were neutralised, storage at -20 degrees C was effective. The distribution of plasma ammonia concentration in healthy subjects was log-normal. The range for males was 21-58 mumol/1 and for females 17-51 mumol/1; this difference was statistically significant (P less than 0.01).     

A modified, optimized kinetic photometric assay for the determination of blood coagulation factor XIII activity in plasma

BACKGROUND: Blood coagulation factor XIII (FXIII) is a zymogen that is transformed into an active transglutaminase by thrombin and Ca(2+). FXIII plays an essential role in fibrin stabilization and in the protection of fibrin from proteolytic degradation. No convenient method has been available for the measurement of FXIII activity in plasma. The aim of the present study was to improve and optimize a kinetic photometric FXIII assay originally developed in our laboratory. METHODS: In the assay, FXIII was activated by thrombin and Ca(2+). Fibrin polymerization was prevented by an inhibitory tetrapeptide. Glycine-ethyl ester and a glutamine residue of a synthetic dodecapeptide served as acyl acceptor and acyl donor transglutaminase substrates, respectively. The amount of ammonia released during the reaction was monitored using glutamate dehydrogenase and NADPH. RESULTS: The use of a new glutamine substrate and optimization of activator and substrate concentrations increased sensitivity. Substitution of NADPH for NADH and introduction of an appropriate blank eliminated systemic overestimation of FXIII activity. The recovery of FXIII was 96%, the assay was linear up to 470 U/L, the detection limit was 1 U/L, and the imprecision (CV) was <8% even at very low FXIII activities. A reference interval of 108-224 U/L (69-143%) was established. The results correlated well with results obtained by an immunoassay specific for plasma FXIII. CONCLUSIONS: The optimized FXIII assay is a simple, rapid method for the diagnosis of inherited or acquired FXIII deficiencies and increased FXIII concentrations. It can be easily adapted to clinical chemistry analyzers.     

Ultrafiltrate analysis confirms the specificity of the selected method for plasma ammonia determination.

The specificity of the direct enzymatic determination of plasma ammonia has hitherto not been unequivocally confirmed, because a suitable comparison method was lacking. Therefore a method variant was elaborated, which includes ultrafiltration to eliminate the high-molecular-mass components regarded as potential sources of unspecificity in the direct measurement procedure according to Rattliff, C.R. & Hall, F. F. (Select. Meth. Clin. Chem. 9, 85-90 (1982)). As the distribution of ammonia during plasma ultrafiltration is markedly influenced by pH and protein concentration, plasma pH is adjusted to 5.5 where the distribution ratio is 1 and nearly independent of actual protein concentration. Acidification significantly diminishes the spontaneous increase of ammonia in plasma at 2-4 degrees C, and the plasma ultrafiltrate is virtually stable. Taking into consideration the slow ammonia formation during sample preparation, excellent agreement was found between ammonia concentrations measured in plasma and in plasma ultrafiltrate, using samples with an apparently normal matrix (n = 30), dysproteinaemia (n = 32) or paraproteinaemia (n = 8). Our data show that the protein matrix of the sample does not cause significant unspecificity in the direct "endpoint" procedure for ammonia determination nor does it affect imprecision. In samples with added bilirubin (up to 252 mumol/l), haemolysate (haemoglobin up to 3.87 g/l) or lipid emulsion (triacylglycerol up to 3.86 mmol/l) ammonia values determined directly in plasma differed maximally by 4% from ultrafiltrate values. A simplified procedure for the ultrafiltration of plasma may be used routinely in clinical service in cases of grossly icteric, haemolytic or turbid samples.     

Fixed-time kinetic assay of plasma ammonia, with NADPH as cofactor, with a centrifugal analyzer.

We describe a fixed-time, enzymatic, reaction-rate procedure for determining plasma ammonia with a centrifugal analyzer (Rotochem IIA/36; American Instrument Co., silver Spring, MD 20910), with NADPH as cofactor. The reaction is based on that of da Fonseca-Wollheim's modification [J. Clin. Chem. Clin. Biochem. 11, 421 (1973)] of the Kirstein reaction, which depends on the catalytic amination of alpha-ketoglutarate by the action of glutamate dehydrogenase with NADPH as the cofactor instead of NADH. Use of NADPH minimizes interference from endogenous reactions such as that between lactate dehydrogenase and pyruvate. This method permits shortened preincubation time and thus improves both specificity and precision. This assay requires 100 microliter of freshly collected heparinized plasma, gives quantitative analytical recovery, and the standard curve is linear to 430 mumol/L. Data are presented comparing results with those by two other enzymatic ammonia procedures.     

Faster determination of clottable fibrinogen in human plasma: an improved method and kinetic study

Clottable fibrinogen in human plasma was determined by measuring spectrophotometrically the increase in turbidity with time due to the fibrinogen-fibrin conversion with thrombin. From the maximal absorbance, Amax, at 450 nm obtained 2 min or less after thrombin as added to plasma, we estimated the fibrinogen concentration in plasma of normal subjects and patients. Analysis of the rate of the absorbance increase yielded the Km value, 1.6 X 10(-5) mol/liter, which closely agrees with the Km of 1.2 X 10(-5) mol/liter obtained by analysis of the fibrinopeptides released from fibrinogen.