Measurement of total bilirubin by use of bilirubin oxidase

We used an enzymatic method for measuring total bilirubin in serum. Results by this method varied linearly with bilirubin concentrations to at least 300 mg/L. The day-to-day precision (CV) of the method ranged from less than 1% to about 11% at bilirubin concentrations of 183 and 12 mg/L, respectively. Commonly used anticoagulants, serum preparation materials, and selected drugs had no effect on the apparent bilirubin concentration, but turbidity caused a slight increase and hemoglobin concentrations of 2 g/L resulted in lower values, by as much as 17 mg/L at a bilirubin concentration of 95 mg/L. Patients' results obtained with this enzymatic method were slightly lower than those obtained with methods based on the Jendrassik-Grof principle. The largest differences, seen in samples with high "direct" bilirubin concentrations, can be decreased by measuring the absorbance at 425 nm instead of at 465 nm as recommended by the supplier of the bilirubin oxidase method.     

Measurement of direct bilirubin by use of bilirubin oxidase

We developed an enzymatic method for measuring direct-reacting bilirubin (DBIL) in serum. At pH 4.5, bilirubin oxidase (BOX) oxidizes mono-conjugated bilirubin, di-conjugated bilirubin, and most of the delta-bilirubin to biliverdin. The resulting decrease in absorbance at 460 nm is linearly related to the concentration of DBIL in serum. Mean DBIL values in the 51 patients' sera examined by the BOX method and a diazo procedure (Clin Chem 1982;28:2305) were 45.4 and 42.8 mg/L, respectively. For the same samples, mean values for DBIL and conjugated bilirubin by the Kodak "Ektachem" methods were 50.2 and 24.8 mg/L, respectively. Hemoglobin, up to 1.5 g/L, does not interfere. Unconjugated bilirubin reacts negligibly. Day-to-day CVs were 2.2% and 2.4% at DBIL concentrations of 37 and 74 mg/L, respectively.     

A new assay method of gamma-glutamyltransferase with 4-aminobenzoate hydroxylase from Agaricus bisporus as a coupling enzyme.

We have developed a new ultraviolet spectrophotometric method for measurement of serum gamma-glutamyltransferase activity. The principle of the method is as follows. Using L-gamma-glutamyl-3-chloro-4-aminobenzoate as the donor substrate, 3-chloro-4-aminobenzoate formed upon transfer of the gamma-glutamyl moiety from the donor substrate to the acceptor substrate glycylglycine is stoichiometrically converted to 3-chloro-4-hydroxyaniline by 4-aminobenzoate hydroxylase from Agaricus bisporus, coupled with the oxidation of NADH to NAD+, and the resulting decrease in absorbance at 340 nm is monitored. The results obtained indicate that there is a good possibility to establish an ultraviolet spectrophotometric method for measurement of serum gamma-glutamyltransferase activity using L-gamma-glutamyl-3-chloro-4-aminobenzoate as the donor substrate and 4-aminobenzoate hydroxylase from Agaricus bisporus as a coupling enzyme.     

Determination of conjugated and total bilirubin in serum of neonates, with use of bilirubin oxidase

A new, simple, and rapid assay for conjugated bilirubin that does not require serum-matrix standards was developed by using the enzyme bilirubin oxidase (EC 1.3.3.5). This procedure can also be modified to measure total bilirubin. Measurements from these assays were compared with results obtained with the Sigma 605 (Jendrassik-Gróf method), Sigma 550/551 (Walters-Gerarde method), and Kodak Ektachem (BuBc) bilirubin assays. The one-year study involved serum specimens from 283 infants younger than 30 days. Linear-regression analysis of data for conjugated bilirubin collected by this assay and by the Kodak Ektachem assay yielded a slope of 0.975, an intercept of 0.088, an Syx of 1.47 mg/L, and a correlation coefficient of 0.94 for a total of 49 specimens. Correlation was also good (r = 0.95) between results for total bilirubin by this assay and both the Sigma 605 and the Kodak Ektachem methods.     

Measurement of activation energy of gamma-glutamyltransferase as a marker for enzyme heterogeneity

In order to detect differences between various multiple forms of gamma-glutamyltransferase, the activation energy was measured. In the serum of patients with liver diseases, activation energy was measured. In the serum of patients with liver diseases, activation energy of the serum enzyme is higher than in normal individuals (41.9 +/- 1.2 vs. 38.9 +/- 1.5 kJ/mol, p less than 0.05). Neuraminidase treatment resulted in a reduction of activation energy. Various multiple forms of serum gamma-glutamyltransferase, as prepared by lectin affinity chromatography (concanavalin A, Ricinus communis I and II, wheat germ agglutinin) showed activation energy differences between binding and nonbinding fractions. Similar results were observed in seminal plasma gamma-glutamyltransferase, when patients with accessory gland infection were compared with a reference population. Our results suggest that the activation energy depends upon differences in the carbohydrate part of the enzyme. The low gamma-glutamyltransferase activation energy of tissue extracts increased significantly after butanol extraction and was then comparable with serum activation energy values, which suggests that lipid-binding is a factor in activation energy variation. In most cases, gamma-glutamyltransferase activities measured at a certain temperature can be easily converted to a corresponding activity at another temperature, but in severe liver disease significant errors may be introduced when simple temperature conversion factors are used.     

总胆红素氧化酶法检测系统的性能评价

目的评价氧化酶法测定血清总胆红素（TBil）的性能。方法用氧化酶法测定血清TBil,探讨氧化酶法的精密度、线性、分析灵敏度、抗干扰性和回收率,并与改良J-G法进行比较。结果 TBil的高、低浓度样本批内CV分别为0.38%、0.78%,日间CV分别为1.78%、2.73%;平均回收率为101.2%;分析灵敏度为1.23μmol/L;线性范围为1.23～574.40μmol/L;2种方法相关性良好,直线回归方程为Y=1.105X-1.11,r=0.998。血红蛋白、维生素C和脂肪乳分别为4.8 g/L、500 mg/L和5%时对氧化酶法测定血清TBil不产生干扰。结论氧化酶法具有良好的精密度、线性范围和抗干扰能力,与改良J-G法具有很好的一致性,适用于临床常规检测。     

Enzymatic assay for conjugated bilirubin (Bc) in serum using bilirubin oxidase (BOD).

We described an automated enzymatic assay for conjugated bilirubin (Bc) in serum using the Iatro D-Bil kit, with bilirubin oxidase (EC 1.3.3.5 BOD) from Myrothecium species. The specificity of the enzyme in the Iatro D-Bil kit was examined by analyzing unconjugated bilirubin (Bu), delta bilirubin (Bdelta), and Bc with high-performance liquid chromatography (HPLC), before and after the enzymatic reaction using BOD. The within-assay coefficients of variation (CV) of this method were 0.58 to 5.00% (n = 20) at 1.4 to 155.8 micromol/L. Day-to-day Cvs ranged from 1.61 to 7.14% at 1.2 to 182.1 micromol/L. The analytical recovery was 96 to 101%. The presence of ascorbic acid, reduced glutathione, L-cysteine, uric acid, urea, creatinine, glucose, lipemic material, anticoagulants, hemoglobin, or human serum albumin did not affect this assay system. The correlation coefficient between values obtained with the Iatro D-Bil kit (y) and HPLC method as reference for conjugated fractions (x) was; r = 0.983, y = 0.952x + 8.851 micromol/L, Sy/x = 11.97 (n = 56). We studied serum Bc levels, not including Bu and Bdelta, in patients with hepatic diseases or autoimmune hemolytic anemia. Levels of Bc obtained by the proposed method changed more rapidly than did those of direct bilirubin (D-Bil) obtained by diazo-dye method during the course of the diseases.     

A kinetic colorimetric assay of gamma-glutamyltransferase

We have explored a kinetic colorimetric method for measuring gamma-glutamyltransferase (EC 2.3.2.2) activity in serum, using L-gamma-glutamyl-3,5-dibromo-4-hydroxyanilide and glycylglycine as donor and acceptor substrates. The released product, 3,5-dibromo-4-hydroxyaniline, reacts with 2,5-dimethylphenol to produce a blue quinone monoimine in the presence of ascorbate oxidase (EC 1.10.3.3). This dye has peak absorption at 610 nm, whereas the donor substrate shows negligible absorption throughout the visible spectrum. The reaction can be run with all the reagents in a single working solution with serum as starter, or with the substrate solution as starting reagent. The sample/reagent volume ratio is 1:24. Adaptation of the method to several automated instruments gave good precision in all cases. Comparison with a method in which L-gamma-glutamyl-3-carboxy-4-nitroanilide is the donor substrate showed good correlation of results (r greater than or equal to 0.987). The dynamic range of the method exceeds the upper limits of the reference intervals for men (9-33 U/L) and women (8-25 U/L) by at least 18-fold.     

Activation energy and lectin affinity chromatography of gamma-glutamyltransferase as a marker for enzyme heterogeneity

Lectin affinity chromatography of gamma-glutamyl transferase (GGT,EC 2.3.2.2) is able to detect differences in the carbohydrate moiety of the enzyme. Binding of tissue GGT towards lectins is significantly different from serum GGT, showing increased galactosylation in tissue forms. Kidney GGT is less glycosylated than GGT from other tissues (liver, pancreas, prostate, vesiculae seminales). Increases in sialic acid content of GGT are associated with an increase in the activation energy of the catalyzed reaction. Differences in galactose, fucose and N-acetylhexosamine content induce much smaller effects on activation energy. In liver diseases, serum GGT is characterized by an altered affinity against lectins recognizing galactose, fucose and N-acetyglucosamine and by increased activation energy. In patients with liver disease, use of fixed temperature conversion factors can lead to erroneous calculations of serum GGT enzyme activity (errors up to 13.3%).     

Kinetic properties of gamma-glutamyltransferase from human liver

We studied the kinetic properties of purified "heavy" form of human liver gamma-glutamyltransferase (EC 2.3.2.2) in the presence and absence of the acceptor substrate glycylglycine under Vmax conditions and as a function of pH. gamma-Glutamyl carboxynitroanilide was used as the donor substrate. Our data suggest that hydrolysis of donor substrate is the major pathway in the absence of acceptor. Autotransfer, if it occurs, is not important. Hydrolysis and transfer reactions have different pH profiles both for Vmax and Km. The pH dependency of Vmax and Km for both the hydrolase and the transferase reactions most probably reflects a change in the rate-limiting step: deacylation of the enzyme at acidic pH and acylation at alkaline pH. Negative cooperativity, observed for both donor and acceptor substrates near neutral pH, is interpreted in terms of more than one active site per dimer for each substrate.     

Kinetic assay of human pepsin with albumin-bromphenol blue as substrate

A novel substrate, albumin complexed with bromphenol blue, has been developed for the assay of human gastric juice pepsin by a kinetic method in the Cobas centrifugal analyzer. The action of pepsin on the complex degrades the albumin and releases the dye. The change in the color of the substrate is a zero-order reaction. Human and porcine pepsin have different Km's with the new substrate. This kinetic method has a throughput of 28 tests in approximately 10 min and good precision (CV = 2.0%). Other advantages are analysis in homogeneous solution (thereby eliminating the need to separate substrate and products), lack of interference from bilirubin or phenol red, and the expression of pepsin activity in IUB enzyme units.     

D-Glucaric acid and gamma-glutamyltransferase as indices of hepatic enzyme induction in pregnancy.

Increased activity of hepatic microsomal enzymes can be evaluated by measuring D-glucaric acid excretion in urine and gamma-glutamyltransferase (EC 2.3.2.2) activity in serum. Aside from diverse foreign compounds, endogenous steroid hormones have been shown to be normal substrates of the microsomal enzyme system. Because there is an increase in steroid production in pregnancy, we sought to determine whether these indices of induction increase during pregnancy. In 90 women in various stages of pregnancy, all with normal kidney function, we measured glucaric acid excretion in urine and activity of the transferase in serum and in urine. Glucaric acid increased markedly during pregnancy, from 14.4 +/- 2.1 in the first trimester to 23.5 +/- 2.8 mumol of D-glucaro-1,4-lactone per gram of creatinine in the third trimester. We saw no correlation between glucaric acid excretion and the transferase activity in serum or urine. Activity of gamma-glutamyl-transferase remains within normal limits throughout pregnancy, which leaves doubt as to the value of this measurement in evaluating enzyme induction owing to endogenous steroids.     

Kinetic enzymic assay for D(-)-lactate, with use of a centrifugal analyzer

The D(-) isomer of lactic acid appears to cause a form of metabolic encephalopathy experienced by patients who have had jejunoileal bypass for morbid obesity. However, analysis for D(-)-lactate is not routinely available in clinical or reference laboratories. We describe an enzymic centrifugal-analyzer assay for D(-)-lactate in plasma or serum, with use of D(-)-lactate dehydrogenase. The method involves two-point kinetic calibration and preincubation of specimen and NAD+, thus eliminating the need for specimen-blanking or protein-precipitating pretreatment. This rapid, accurate, and precise assay should be helpful in evaluating patients with "short-bowel syndrome" who display confusion, lethargy, ataxia, or other central nervous-system disturbances that may be ascribable to D(-)-lactic acidosis.     

Colorimetric method for assay of serum gamma-glutamyltransferase activity with some L-gamma-glutamyl-carboxyanilides

A simple method for the assay of serum gamma-glutamyltransferase activity with soluble L-gamma-glutamyl-carboxyanilides is presented. It is based on transformation of aminobenzoic acid liberated by the enzyme into colour complex with 4-dimethyl-aminobenzaldehyde or with 4-dimethylaminocinnamaldehyde in acid medium. Among three carboxyanilides studied the highest enzyme activity was noted with L-gamma-glutamyl-3-carboxyanilide and no activity with L-gamma-glutamyl-2-carboxyanilide. A good correlation was demonstrated for serum gamma-glutamyltransferase activity determined by the new colorimetric method and by the standard one with L-gamma-glutamyl-3-carboxy-4-nitroanilide.     

A sensitive fluorometric assay for serum monoamine oxidase with kynuramine as substrate

The detailed procedure of a new sensitive fluorometric assay for human serum monoamine oxidase (MAO) with kynuramine as substrate is described. The data on its reproducibility, stability, correlation with another method, and serum MAO levels for 150 healthy subjects and 205 patients with various diseases, are presented to support the usefulness of this method. Since our method is much simpler and more sensitive than other methods conventionally used, we can recommend it for routine clinical investigation.     

Fluorometric assay of serum gamma-glutamyltransferase in solution and on a semi-solid surface.

We describe an enzymatic, fluorometric method for measuring the activity of serum gamma-glutamyltransferase (EC 2.3.2.2) in solution and on silicone rubber pads. N-gamma-L-Glutamyl-alpha-naphthylamide in tris(hydroxymethyl)aminomethane buffer is used as substrate. In solution, measurements are performed at 37 degrees C with use of a reaction volume of 3.0 ml, in Pyrex cuvets. Measurements on silicone rubber pads are also made at 37 degrees C, after establishing a stable substrate film by lyophilizing the reagent for the gamma-glutamyltransferase assay on the surface of the pads. Only 10-50 mul of buffered substrate and serum containing the enzyme to make a total volume of 60 mul are necessary for each assay. The rate of appearance of alpha-naphthylamine fluorescence (lambda ex equals 344 nm; lambda em equals 445 nm), liberated from N-gamma-L-glutamyl-alpha-naphthylamide by the enzymatic action of gamma-glutamyl-transferase, is measured and equated to its activity in serum. Calibration plots of the change in fluorescence per min vs. enzyme concentration for measurements in solution and on pads show a good proportionality in the range of 26.5-265 U/liter, and indicate the usefulness of these methods.     

Kinetic rate assay of urinary N-acetyl-beta-D-glucosaminidase with 2-chloro-4-nitrophenyl-N-acetyl-beta-D-glucosaminide as substrate

In this new procedure for determining the activity of N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30; NAG) in urine, a new synthetic substrate, 2-chloro-4-nitrophenyl-N-acetyl-beta-D-glucosaminide is used. No sample blank is required because the activity is determined kinetically. Very high precision is possible, with CVs of less than 3.8% both within-run and between-run. Glucose, ascorbic acid, bilirubin, bromosulfonphthalein, and phenosulfonphthalein do not interfere. The reference interval (mean +/- 2 SD) for healthy subjects was 1.9-11.1 U per gram of creatinine. NAG activity can be determined over a wide range (2-500 U/L) with high sensitivity. The method is suitable for use with various automated analyzers, and it should be useful for screening for renal diseases.     

Application of response surface methodology to the assay of gamma-glutamyltransferase

Response surface methodology (RSM) offers an empirical approach to the study of clinical enzyme assays. Variables such as pH, which are difficult to characterize by using theoretical enzyme kinetics, are easily included in RSM formulations. In this investigation, we studied with RSM the change in the measured activity of gamma-glutamy-transferase (EC 2.3.2.2) as a function of changes in concentrations of donor (gamma-glutamyl-3-carboxy-4-nitroanilide) acceptor (glycylglycine), and pH. The study defined large ranges for these variables over which maximum enzyme activity is obtained: donor 6.6 to 10.2 mmol/L, acceptor 129 to 250 mmol/L, and pH 7.8 to 8.5. The RSM regression polynomial was as accurate as a previously determined enzyme kinetic equation for predicting the transferase activity from given reagent substrate concentrations. Although not yielding a mechanistic understanding of an enzyme assay, RSM studies do produce an operational understanding of how an assay functions.