New values for the molar extinction coefficients of NADH and NADPH for the use in routine laboratories (author's transl)]

Extensive re-investigations with regard to the molar extinction coefficients of NADH and NADPH proved that in future, calculations in routine work can be performed with the following much more accurate epsilon-values: 6.15 x 10(3) 1 x mol-1 x cm-1 at Hg 334 nm (NADH and NADPH), 6.3 X 10(3) 1 X mol-1 x cm-1 at 340 nm (NADH and NADPH), 3.4 X 10(3) 1 X mol-1 X Cm-1 (NADH) and 3.5 x 10(3) 1 x mol-1 x cm-1 (NADPH) at Hg 365 nm, respectively. The safest measurement is performed at Hg 334 nm, because here epsilon is identical for both coenzymes and deviations of the epsilon-value caused by temperature, pH and ionic strength are less than 0.5%.     

Molar absorptivities of beta-NADH and beta-NAD at 260 nm.

To determine the molar absorptivities of beta-NADH and beta-NAD at 260 nm, we purified the reduced form of the coenzyme by repeated anion-exchange chromatography. A NADH preparation was so obtained for which the 260 nm/340 nm absorbance ratio was 2.265. When calculated with epsilon 340 beta-NADH = 6.22 times 10-3 for beta-NADH at 260 nm and 25 degrees C, a molar absorptivity of 14.1 times 10-3 liter - mol minus 1 - cm minus 1 resulted from this quotient. By use of the lactate dehydrogenase or glycerol-3-phosphate dehydrogenase assay, respectively, and referring to the new absorption coefficient for NADH at 260 nm, the molar absorptivity of beta-NAD at 260 nm and 25 degrees C was established to be 17.4 times 10-3 liter - mol minus 1 - cm minus 1. The values observed are lower than those reported thus far.     

Determination of the molar absorptivity of NADH.

The molar absorptivity of NADH at 340 nm has been determined by an indirect procedure in which high-purity glucose is phosphorylated by ATP in the presence of hexokinase, coupled to oxidation of the glucose-6-phosphate by NAD+ in the presence of glucose-6-phosphate dehydrogenase. The average value from 85 independent determinations is 6317 liter mol-1 cm-1 at 25 degrees C and pH 7.8. The overall uncertainty is -4.0 to +5.5 ppt (6292 to 6352 liter mol-1 cm-1), based on a standard error of the mean of 0.48 ppt and an estimate of systematic error of -2.6 to +4.1 ppt. Effects of pH, buffer, and temperature on the molar absorptivity are also reported.     

Interlaboratory proficiency, intermethod comparison, and calibrator suitability in assay of serum aspartate aminotransferase activity.

Sources of variation in assays of aspartate aminotransferase (EC 2.6.1.1) activity were examined in an interlaboratory survey and through an examination of materials used as calibration materials in these assays. Four highly stable lyophilized specimens containing human cytoplasmic enzyme, with activities of 0, 22, 46, and 96 U/liter at 30 degrees C and optimal substrate concentrations, were assayed by 319 laboratories. Mean values obtained on these specimens by laboratories using 2,4-dinitrophenylhydrazine kits varied among manufacturers and deviated from values expected from this procedure. The average coefficient of variation (CV) with these kits was greater than 20%. Automated continuous-flow procedures with use of diazonium salt showed the best precision (av CV, less than 10%). However, the automated continuous-flow malate dehydrogenase/NADH coupled method produced an average CV greater than 20%. Results from each of the automated methods were related to a reference malate dehydrogenase/NADH coupled continuous kinetic assay method by temperature relationships alone. Mean values from manual diazonium salt procedures were 1.7-fold greater than similar reference values (av CV was 18%). The higher results were attributed to the use of poorly-defined units and to an artifact caused by chromophore stabilizers in this procedure when aqueous samples are used. The average CV in continuous kinetic methods varied among kit manufacturers, ranging from 6 to 28% for the specimen of highest activity. Variations in results were much larger at 366 nm than at 340 nm than at 340ity. Variations in results were much larger at 366 nm than at 340 nm. Interassay relationships of these methods are presented. Concentrations of pyruvate in commercially available calibration materials differed between manufacturers, varied in stability, and deviated from the expected concentration. For some colorimetric assays the precision attained on reported absorbance values for the enzyme specimens was of the same order of magnitude as that for pyruvate standards. Other sources of error are revealed by the interlaboratory survey. The value of commercially available sources of enzyme activity as calibration or control materials was assessed by evaluating the following properties: activity at suboptimal concentrations of L-aspartate or 2-oxoglutarate, temperature effects, preincubation lability owing to aspartate and phosphate, pyridoxal phosphate saturation, contamination with glutamate dehydrogenase, and manufacturer's rated activity. These properties are compared to those of human cytoplasmic enzyme in a human serum matrix.     

Formation and properties of lactate dehydrogenase inhibitors in NADH.

We describe some characteristics of the mode of formation of inhibitors of lactate dehydrogenase from commercial NADH. Inhibitor formation is time- and concentration-dependent and also varies with the commercial source of NADH. At least two inhibitory components can form in concentrated NADH solutions. One of these can be separated from NADH by chromatography on either diethylaminoethyl-celluose or diethylaminoethyl-Sephadex; the second cannot. The NADH-associated inhibitor appeared to be present in each of the three commercial NADH preparations studied. The 260 nm/340 nm absorbance ratio was of no help in locating this inhibitor during chromatography.     

Lactate dehydrogenase inhibitors in NADH preparations.

The presence of a new lactate dehydrogenase inhibitor on the trailing edge of the NADH peak from chromatography on diethylaminoethyl-celluose [Loshon et al., Clin. Chem., this issue] was verified. It was resolved from the NADH by high-performance liquid chromatography on muBondapak C18. When the new inhibitor was present in a reaction mixture to the extent that, of the initial 260-nm absorbance, about 5% was contributed by the inhibitor, the rate of NADH oxidation by lactate dehydrogenase decreased by 65%. The inhibitor absorbs at 260 and 340 nm, and is different from the Strandjord-Clayson inhibitor [J. Lab. Clin. Med. 67, 144 (1966)] by both types of chromatography. Because this new inhibitor has ultraviolet properties similar to those of NADH and chromatographs with the NADH on DEAE-cellulose, the high-performance liquid chromatographic method must be used to ensure its absence in preparations of NADH used for clinical assay.     

Automated enzymatic assay for plasma ammonia.

An enzymatic method for determining plasma ammonia with the Du Pont Automatic Clinical Analyzer (aca) is described. The assay requires a sample volume of 500 muL for a kinetic ammonia measurement. The reaction is initiated with glutamate dehydrogenase and the rate of depletion of NADPH is monitored with two measurements, 17 s apart, at 340 nm. Reaction conditions have been optimized for maximum sensitivity through both one-factor-at-a-time and multiple variable response surface optimization techniques. Linearity to 1000 mumol of ammonia per liter of plasma has been achieved. No significant interferences were observed from anticoagulants or endogenous blood components, including pyruvate and oxalacetate. Use of the coenzyme NADPH (instead of NADH) in this aca procedure eliminates the lengthy pre-incubation otherwise required for endogenous dehydrogenase reactions.     

Interaction between pyridine nucleotide coenzymes and heme proteins as a possible source of error in assay of activities of coenzyme-linked enzyme

The ultraviolet absorbance spectra of pyridine nucleotide coenzymes change in the presence of heme-containing proteins. The positions of each of the two main absorbance peaks of NADH are shifted progressively towards shorter wavelengths in the presence of increasing concentrations of hemoglobin, and the third peak, at 220 nm, disappears altogether. Similar changes are seen in the spectra of NAD+ and NADPH, and similar effects on these spectra are produced by myoglobin and cytochrome c, but not by comparable concentrations of albumin. The spectral shifts are generally accompanied by a decreased peak height. This finding may help explain problems reported by previous workers in the measurement of the activity of enzymes such as transketolase or lactate dehydrogenase in erythrocyte hemolysates. Errors may be considerable if allowance is not made for this effect, especially if the concentration of heme protein in the spectrophotometer cuvette much exceeds 1 g/L. The interaction appears to indicate some form of bonding, occurring generally between pyridine nucleotide coenzymes and the heme group in proteins. We relate the findings to measurement of activities of pyridine nucleotide-linked enzymes in erythrocyte lysates and in plasma containing myoglobin after muscle breakdown.     

New ultraviolet (340 nm) method for assay of uric acid in serum or plasma

We propose a novel enzymatic method for assay of uric acid at 340 nm, which eliminates several disadvantages of both the colorimetric and enzymatic methods now in common use. Here, uric acid is catalytically oxidized to allantoin and hydrogen peroxide. The peroxide is reacted with ethanol in the presence of catalase to form acetaldehyde and water, and the acetaldehyde is reduced by NADH in the presence of alcohol dehydrogenase to ethanol. The decrease in absorbance at 340 nm caused by oxidation of NADH is directly proportional to the concentration of uric acid in the sample. Measurement of the change in absorbance between 20 and 200 s eliminates the need for a serum blank measurement. Absorbance and concentration are linearly related to 120 mg of uric acid per liter. The new method was compared with the uricase method in which decomposition of uric acid at 293 nm is directly measured. The results for the 47 patients' sera so examined can be expressed by the linear equation y340 = 1.0078x293 + 0.122 (r = 0.9984).     

Evaluation of the UV-340 spectrophotometric determination for pseudocholinesterase activity (EC 3.1.1.8) in human serum

A pseudocholinesterase catalytic activity assay using p-hydroxybenzoylcholine as substrate and measuring the decrease of NADPH at 340 nm was compared with a colorimetric method using acetylthiocholine as substrate. The assay is simple, uses 50 microliters serum and is performed at 37 degrees C. Precision of the UV-340 method was good except at low ranges. The catalytic activity was depressed by the anticoagulants citrate and fluoride but not by EDTA or heparin. The reference values obtained with the evaluated UV-340 method are somewhat higher than those with the colorimetric method. As the results with both methods are comparable, the choice of procedure will depend on the local facilities.     

Enzymatic assay of inorganic phosphate with use of sucrose phosphorylase and phosphoglucomutase.

We developed a new enzymatic method for the assay of inorganic phosphate (Pi) by using sucrose phosphorylase (SP; EC 2.4.1.7) and phosphoglucomutase (PGM; EC 5.4.2.2). Pi is transferred to sucrose by SP, producing alpha-D-glucose 1-phosphate (G1P) and alpha-D-fructose. G1P is transphosphorylated by PGM in the presence of alpha-D-glucose 1,6-bisphosphate to form alpha-D-glucose 6-phosphate, which is oxidized by NAD+ and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) to form 6-phosphogluconate (6PG) and NADH. Finally, the oxidation of 6PG by NAD+, catalyzed by 6-phosphogluconic dehydrogenase (EC 1.1.1.44), yields D-ribulose 5-phosphate and NADH. Thus two molecules of NADH are formed for each molecule of Pi, and the reaction is monitored at 340 nm. The Km values of SP for Pi and sucrose were 4.44 and 5.31 mmol/L, respectively. The best buffer was 1,4-piperazinediethanesulfonic acid (PIPES) at 50 mmol/L and pH 6-7. Implementing this method with a Cobas-Bio centrifugal analyzer allowed us to measure Pi accurately and precisely.     

A kinetic procedure for the estimation of arginine in serum using arginine kinase

A method for estimation of arginine in 50 microliters serum was developed using commercially available arginine kinase (EC 2.7.3.3). The assay is based on the transformation of arginine and ATP into phospho-arginine and ADP by the enzyme. ADP is measured by two coupling reactions involving pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.27) with measurement of NADH consumption at 340 nm. The method involves preincubation of serum in the reaction medium without arginine kinase to eliminate side reactions and a kinetic rate protocol with measurements of absorbance at 60 s and 180 s. Reaction temperature is 30 degrees C. The reaction is linear up to at least 3 mmol/l of arginine. Within-batch CV is less than 3% for arginine levels above 0.75 mmol/l and the between-batch CV is 6.5% or less. The method correlates well with an automatic amino acid analyzer procedure (r = 0.983). The reference range derived from sera of 40 blood donors has been determined to be 0.06-0.20 mmol/l.     

Automated enzymatic measurement of lecithin, sphingomyelin, and phosphatidylglycerol in amniotic fluid

We describe methods for automated enzymatic measurement of lecithin, sphingomyelin, and phosphatidylglycerol in amniotic fluid. Phospholipase C (EC 3.1.4.3) and sphingomyelin phosphodiesterase (EC 3.1.4.12) are reacted with lecithin and sphingomyelin, respectively, to liberate phosphocholine. Phosphocholine is then reacted with alkaline phosphatase, choline oxidase, peroxidase, and 4-aminoantipyrine to form a colored complex, for which the absorbance at 500 nm is measured with a centrifugal analyzer. Phosphatidylglycerol is hydrolyzed by phospholipase D (EC 3.1.4.4) to form glycerol, which is subsequently reacted with ATP and NAD+ in the presence of glycerol kinase and glycerol-3-phosphate dehydrogenase to yield NADH. The absorbance of the NADH formed is measured at 340 nm. These methods provide a simple, rapid, and accurate alternative to thin-layer chromatography for determination of phospholipids in amniotic fluid for assessment of fetal lung maturity.     

Comparisons of 17 lots of 2-oxoglutarate, and specifications for use of this substrate in reference methods

We examined 17 lots of 2-oxoglutarate (seven acid forms, three K salt forms, and seven Na salt forms), obtained from eight commercial suppliers, for suitability for measuring aspartate aminotransferase (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2) in human serum. Measurements of the catalytic activity concentrations of these two aminotransferases with each of these 17 preparations were not sufficiently sensitive to distinguish good from poor-quality material. Thus, we ranked these lots for purity, by specific analysis with glutamate dehydrogenase and by liquid chromatography, and determined the water content, acid content, and spectral characteristics of each. On the basis of a 2-oxoglutarate assay value by glutamate dehydrogenase of 98% or greater, we considered seven of the preparations acceptable and 10 unacceptable. The molar absorptivities (L X mol-1 X cm-1, mean +/- SD) of the seven acceptable lots in 1 mol/L HCl were: epsilon 325 nm = 9.12 +/- 0.02 (CV = 0.2%), epsilon 279 nm = 2.63 +/- 0.23 (CV = 9.9%), and epsilon 245 nm = 37.9 +/- 4.1 (CV = 10.9%). Use of these spectrophotometric limits alone unambiguously distinguished the inferior lots of 2-oxoglutarate. We urge the inclusion of detailed spectrophotometric specifications for 2-oxoglutarate in Reference Methods for aminotransferase measurements.     

Purification and analysis of the purity of NADH.

We developed an analytical reverse-phase high-performance liquid chromatographic procedure for rapid assessment of the purity of NADH. The method completely separates adenosine monophosphate and adenosine diphosphoribose from NADH. By use of this analytical technique we found that preparative chromatography on DEAE-cellulose gives NADH that is free from adenine nucleotides as well as other impurities that commonly are present in NADH. The absorbance ratio at 260 and 340 nm of the purified NADH in 1.8 mmol/liter ammonium carbonate is 2.261 +/- 0.002 (+/- 1 SD).     

Detection of inhibitors in reduced nicotinamide adenine dinucleotide by kinetic methods.

Methods are described for detection of lactate dehydrogenase (LDH) inhibitors in preparations of reduced nicotinamide adenine dinucleotide. They are (a) comparison of values by kinetic methods with those measured for highly purified NADH and (b) examination of Lineweaver-Burk plots. Chromatographic inhomogeneities are correlated with deviant values for the kinetic constants of NADH preparations. Lineweaver-Burk plots that curve upward at the high concentrations or have a larger or smaller than normal slope may indicate the presence of inhibitor. As determined in bicarbonate buffer (0.11 mol/liter, pH 7.9) by use of 0.600 mmol/liter pyruvate and NADH freshly separated from impurities by chromatography on diethyl-aminoethyl-cellulose, the Km (apparent) of NADH at 25 degrees C has the value 8.11 +/- 0.71 mumol/liter (SD, n = 28) with LDH-1 (pig heart, 2.48 +/- 0.05 U per milliliter of reaction mixture, or 41.3 +/- 0.8 nmol/liter per second). Under similar conditions, the Km (apparent) of NADH has the value of 8.57 +/- 1.58 mol/liter (SD, n = 21) with LDH-5 (pig muscle, 1.77 +/- 0.03 U/ml of reaction mixture), or 29.4 +/- 0.6 nmol/liter per second). At infinite substrate concentrations with the same pH, buffer, and temperature, the Km (apparent) for NADH was 26.0 +/- 0.63 mumol/liter with LDH-1 and 23.2 +/- 4.6 mumol/liter with LDH-5.     

Stability of NADPH: effect of various factors on the kinetics of degradation

Seeking to minimize degradation of NADPH during storage, reagent preparation, and assays, we investigated the effects of pH, temperature, and ionic strength as well as the effects of phosphate and acetate. NADH was also included for comparison. Our results indicate that the rate of degradation of NADPH is proportional most importantly to temperature and concentrations of hydronium ion, but also to concentrations of phosphate and acetate. The degradation rate decreased with increasing ionic strength at neutral pH, but increased slightly at lower pH. NADPH generally is less stable than NADH under the same conditions. The reaction orders with respect to hydronium ion and anions were near 1 for NADH degradation reactions, about 0.5 for NADPH. Rate constants for NADH and NADPH differed more at higher pH and lower phosphate and acetate concentrations.     

Determination of serum catalase activity on a centrifugal analyzer by an NADP/NADPH coupled enzyme reaction system.

We developed a simple, kinetic method for the determination of catalase activity in which i) the enzyme catalyzes the peroxidation of ethanol by hydrogen peroxide to acetaldehyde and water, and ii) the acetaldehyde so formed is rapidly oxidized to acetic acid and NADPH by the addition of an excess of NADP+ and aldehyde dehydrogenase. The rate of NADPH production was monitored at 340 nm in a COBAS centrifugal analyzer. The reaction was linear to 800 U/L or a delta A of 0.020/min. Using human serum pools containing 80 and 460 U/L of peroxidase activity, the within-run coefficients of variation (CV) were 1.9 and 1.3%, respectively. Between-run CV values were 5% for both pools. The reference range for sera from 72 males and 52 females was 23 to 158 U/L (mean + 2 SD) by log normal transformation. The activity in red cells was 600 U/g hemoglobin but did not change the reference range appreciably provided that serum without visible hemolysis was used. Preliminary observations on sera from nine patients with various pancreatic disorders showed a poor correlation between the activities of catalase (peroxidase) and amylase in serum. The reasons for this discrepancy are under investigation.     

Influence of salts on Michaelis-constant values for NADH.

We previously observed [Clin. Chem. 22, 1648 (1976)] that values of the Michaelis constant for NADH for the conversion of pyruvate to lactate with lactate dehydrogenase (EC 1.1.1.27) in the presence of 0.1 mol/liter buffers at 25 degrees C showed first-order dependence on enzyme concentration. This is now recognized to be the result of an inhibitory influence exerted by buffers [NH4HCO2, tris(hydroxymethyl)aminomethane, and phosphate] and salts [(NH4)2SO4 and NaCl] present in the reaction mixtures. Inhibition constants for the enzyme/inhibitor complexes formed with these substances are about 0.3 mol/liter for competition of NH4HCO3 with NaOH and 0.4 mol/liter for competition of NH4HCO3 with pyruvate; they are 0.6 mol/liter for NaCl, 1.0 mol/liter for sodium phosphate, 0.3 mol/liter for (NH4)2SO4, and 0.8 mol/liter for tris(hydroxymethyl)aminomethane when these substances compete with NADH. Because of the large molar ratio of buffer to substrate (about 10(9):1) in enzymatic assays, the buffer concentration significantly influences the Michaelis constant, despite the large value for the inhibition constant. Attention to the concentrations of these substances may be required for decreasing variability in clinical assays in which lactate dehydrogenase and possible other enzymes are used.     

用肌酐亚氨水解酶偶联谷氨酸脱氢酶的酐酶法鉴定

目的 建立适合于自动化分析的人血清和尿肌酐亚氨水解酶偶联谷氨酸脱氢酶指示系统的酶促动力学测定方法。方法 在反应体系中加入异柠檬酸脱氢酶及其作用底物异柠檬酸，再生由主反应前氨消耗的NADPH和α－酮戊二酸，其后加入镁离子络合剂CyDTA和肌亚氨水解酶的启动试剂启动反应，在此同时NADPH和α－酮戊二酸的再生反应被终止，测定NADPH于340nm处吸光度的变化，计算样品中肌酐的浓度。结果 本法测定线性范围为40－2000μmol／L，批内和常规条件下的变异系数均小于5％，回收率为98．5％，与高效液相色谱法具有良好的相关性（r=0.9930)。乳糜、黄疸、溶血、酮体、乳酸盐、临床常用的治疗药物和高至2mmol/L的氨对测定没有干扰。结论 本法操作简便、精确，推荐作为常规测定方法。