The influence of pH and various anions on the distribution of NH4+ in human blood.

The pH-dependent distribution of ammonia between blood cells and plasma was investigated with oxygenated blood samples from healthy subjects at 37 degrees C. Blood pH was varied between 6.95 and 7.65 by equilibration with different CO2 mixtures. Plasma ammonia concentrations were measured directly with a specific enzymatic method. Ammonia concentrations within blood cells were calculated from a) the concentration changes of ammonia in plasma after addition of 87.7 mumol/l NH4Cl to whole blood and b) the pH-dependent haematocrit. The pH-dependency of the distribution ratio(ammonia) = P-ammonia/cell ammonia (substance concentrations in water spaces) is described by the equation distribution ratio(ammonia) = 3.095 - 0.342 x pHplasma (r = 0.928, n = 36) in good agreement with available literature data on the distribution of H+. A quantitative figure to describe the actual NH4+ concentration in oxygenated whole blood at defined values of P-NH4+, P-pH and haematocrit is given. Values of distribution ratio(ammonia) at pH 7.4 (0.57 or 0.75, ammonia concentrations corrected/not corrected for water content) are higher than those assumed so far in the literature. Addition of non-permeating anions (citrate, EDTA) to whole blood results in a shift of NH4+ from the intra- to the extracellular compartment. In contrast, chaotropic anions like iodide or thiocyanate lower distribution ratio(ammonia). To avoid medically important bias in the measurement of plasma ammonia concentration, the changes in pH or in the ionic composition of the blood sample following pretreatment with anticoagulants or preservatives should not exceed certain limits. Citrate is not a suitable anticoagulant.

Haemoglobin interference in the bichromatic spectrophotometry of NAD(P)H at 340/380 nm.

The negative bias observed in the NADPH-based bichromatic measurement of glucose in haemolysates (da Fonseca-Wollheim, F., Heinze, K.-G. & Liss, E. (1992) Temperature-dependent matrix effect in the direct enzymatic measurement of blood glucose, this journal 30, 371-375) is caused by shifts in the UV absorbance of haemoglobin which affect the absorbance difference delta A340/380 nm. In model experiments with haemoglobin solutions, spectral changes resulting in decreases of the absorbance at 340 nm and/or increases at 380 nm were found to occur for the following three reasons: 1. Oxidation of haemoglobin-O2 with formation of Fe(III) derivatives. Methaemoglobin formation is accelerated by lowering the pH, raising the temperature from 25 to 37 degrees C or by adding organic phosphates (inositol hexakisphosphate, ATP). At pH 6, addition of plasma increases the rate of methaemoglobin formation, while at pH values > 7, haemoglobin-O2 is stabilised. The oxidation of haemoglobin-O2 in the presence of sodium lauryl sulphate is also accompanied by a decrease of delta A340/380 nm. The haemichromes formed in this reaction exhibit stable UV light absorptivity. 2. Increase in the temperature of the haemoglobin-O2 solution. It is shown that the temperature-induced shifts in the haemoglobin-O2 absorptivity are reversible and that similar changes occur with the chemically more stable cyanomethaemoglobin. 3. Deoxygenation of haemoglobin-O2 at low pO2. Theoretically, the variation of factors influencing the pO2 (0.5) such as temperature, pH and allosteric effectors can also lead to changes in delta A340/380 nm.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of pCO2 on the rate of ammonia formation in blood.

The influence of the sample pCO2 on the rate of ammonia formation was studied with gas equilibrated blood samples, using different gas mixtures for the equilibration. The rate of increase in plasma ammonia concentration at a mean pCO2 of 62 mm Hg = 8.2 kPa (mean pH = 7.282) was significantly lower than at 36 mm Hg = 4.8 kPa (pH = 7.438). In CO2-depleted blood (pH > 8) ammonia formation was strongly accelerated. This was reversible by readjusting the pH to 7.4 by addition of Tris-HCl solution. In stoppered containers with or without enclosed atmospheric air, a decrease of blood pCO2 or an increase of pH values was not observed during storage over 15 minutes at 0 or 20 degrees C. Although this study confirms that the pCO2 (or rather the pH) is an important analytical influence quantity in the determination of plasma ammonia, strictly anaerobic processing of the blood samples is not necessary; the usual technique of transporting and preprocessing blood samples in partially filled and stoppered containers appears to be adequate. Mainly due to the deamination of intracellular AMP (1), the ammonia1) concentration in blood increases continuously after sampling. Rates of increase in plasma ammonia concentration have recently been investigated thoroughly with blood samples from healthy probands to define the maximum delay between sampling and separation of the blood cells that can be tolerated if the in vivo existing plasma ammonia concentration has to be measured (2). Strictly speaking, the guidelines for handling the blood samples (2) apply to blood stored under anaerobic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Which is the appropriate coenzyme for the measurement of ammonia with glutamate dehydrogenase?

The determination of ammonia in plasma, using glutamate dehydrogenase, is complicated by non-specific oxidation of the coenzyme, promoted by components of the sample matrix. Measurements performed with appropriate plasma blanks show that 2'-phosphorylated coenzymes (NADPH, deamino-NADPH) are much less oxidized than NADH. By adding lactate dehydrogenase, NADH oxidation by endogenous pyruvate can be completed within a short time. Considerable consumption of coenzyme occurs, however, and endogenous L-alanine aminotransferase also represents a possible source of interference. The results of ammonia determinations using deamino-NADPH (y) or NADPH (x) were identical (a = 0.0 mumol/l, b = 1.00; r = 0.996, n = 62). With NADH as the coenzyme, the method displays adequate specificity only at high sample dilution, e.g. in the measurement of urea after conversion to ammonia.

Temperature-dependent matrix effect in the direct enzymatic measurement of blood glucose.

The influence of the high-molecular-mass sample matrix in the direct enzymatic measurement of glucose in haemolysate was investigated by a comparison study using ultrafiltered haemolysate for reference. Haemolysate was obtained by 1:21 dilution of whole blood with a solution of digitonin and maleinimide. It was shown that at low protein concentration glucose distributes in a 1:1 ratio during ultrafiltration. With a hexokinase/glucose-6-phosphate dehydrogenase procedure excellent agreement was found between values measured in haemolysate (y) and ultrafiltrates (x), when incubation was performed at 25 degrees C (a = 0.047 mmol/l; b = 0.99; r = 0.999, n = 37); at 37 degrees C, however, the same procedure resulted in a non-tolerable systematic deviation in the direct analysis of haemolysate (a = -0.426 mmol/l; b = 1.00, r = 0.997, n = 37). The precision of measurements in haemolysate and ultrafiltrate was similar (CV 1.0-1.2%). Since stable reference material with an appropriate matrix is not available, it is important to evaluate haemolysate procedures carefully by comparison studies with patient samples. For reduction of experimental error in such studies we recommend the use of ultrafiltered haemolysate, since this can be analysed side by side with haemolysate in the same run.

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.

Preanalytical increase of ammonia in blood specimens from healthy subjects.

The course and magnitude of spontaneous increase in ammonia concentration in plasma on standing were investigated with EDTA-treated blood specimens from 36 healthy subjects with use of a sensitive and precise enzymic method. Over 90 min, the rates of increase were virtually constant at fixed temperature. The mean (and SE) rates at 0, 20, and 37 degrees C were 3.9 (0.23), 5.2 (0.23), and 25.2 (0.59) mumol/L per hour, respectively. At these temperatures, the plasma contributed at most 7%, 15%, and 10%, respectively, to the formation of ammonia in whole blood. In view of the medical needs and the measured rates of ammonia increase, an interval of 15 min between blood sampling and the start of centrifugation may be tolerated at a specimen temperature of 0 degree C. Rates of ammonia increase showed significant correlations with erythrocyte and platelet count as well as with the plasma activities of gamma-glutamyltransferase (EC 2.3.2.2) and alanine aminotransferase (EC 2.6.1.2).

Deamidation of glutamine by increased plasma gamma-glutamyltransferase is a source of rapid ammonia formation in blood and plasma specimens.

Owing to increased enzymatic hydrolysis of glutamine, additional ammonia is formed in blood and plasma specimens with increased gamma-glutamyltransferase activity (gamma-GT, EC 2.3.2.2.). The close correlation between gamma-GT and glutaminase (EC 3.5.1.2) activities (r = 0.97) in plasma, the inhibition with 6-diazo-5-oxo-L-norleucin or borate plus serine, and the activation with maleate clearly show that gamma-GT itself is the glutamine-deamidating enzyme in plasma. Under pathological conditions, increased gamma-GT activity can increase the rate of ammonia formation in plasma more than 30-fold the mean values of healthy subjects. Increased in vitro formation of ammonia caused by increased gamma-GT activity is an important source of false-positive findings in ammonia determination. Because of the high prevalence of pathological gamma-GT activities in clinical populations, blood specimens for ammonia analysis should be preserved by addition of borate plus serine.

Serum ultrafiltration for the elimination of endogenous interfering substances in creatinine determination.

Serum, at neutral pH, was submitted to a simple filtration, using centrifugation in the disposable Centrisart. The ultrafiltrate was similar to serum in its creatinine content but was virtually free from proteins, including protein-bound bilirubin, haemoglobin and lipoproteins. The creatinine concentrations of anicteric serum specimens and the corresponding ultrafiltrates as determined with Jaffé and enzymic procedures show a high correlation and are convertible. With icteric sera the negative interference effect of bilirubin in a particular analytical procedure can be quantified using ultrafiltrate as the reference. It is suggested that ultrafiltration is useful in selected cases for eliminating elevated concentrations of bilirubin, haemoglobin and turbidity, which would interfere in the direct creatinine determination. Relative to the continuous flow methods with dialysis of the analyte, direct methods for creatinine are more susceptible to interference by endogenous factors like hyperbilirubinaemia, hypertriglyceridaemia and haemolysis (1). The negative interference by bilirubin is of special importance, since it interferes in some modifications of the kinetic Jaffé method (2) and in the chromogenic enzymatic method (3). As a simple alternative, we evaluated the use of serum ultrafiltrate for the accurate determination of creatinine by the Jaffé and enzymatic methods, free from interfering by the high-molecular serum matrix and compounds bound to it.