The measurement of bilirubin fractions in serum.

Bilirubin fractions are measured by (1) the direct diazo reaction, (2) high-performance liquid chromatography (HPLC), (3) direct spectrophotometry, and (4) enzymatic methods. HPLC, which effects separation and quantitation of the four bilirubin fractions, is the method of choice, but impractical for routine use. A special application of direct spectrophotometry allows the measurement of unconjugated bilirubin and the sum of bilirubin conjugates. This approach, which provides essentially the same information as HPLC, unfortunately is available only in one clinical analyzer. The direct diazo reaction measures bilirubin conjugates plus delta-bilirubin, albeit not very accurately. Direct diazo methods that measure unconjugated bilirubin as direct could obscure the clinical diagnosis. At acid pH, enzymatic methods measure all direct reacting bilirubins, while at pH 10 only conjugated bilirubins are measured. Because the measurement of conjugated bilirubins is clearly more helpful than that of direct bilirubin in the differential diagnosis of jaundice, direct diazo methods should be replaced by methods specific for bilirubin conjugates.     

Inaccurate values for direct bilirubin with some commonly used direct bilirubin procedures

We compared five methods for the determination of total and direct bilirubins in serum samples from normal controls, subjects with Gilbert's syndrome, and serum pools containing about 50 and 150 mg of total bilirubin per liter. The Kodak Ektachem method and a diazotized sulfanilic acid method with 0.15 mmol/L sodium nitrite concentrations are the only methods that gave accurate direct bilirubin values, as judged by liquid-chromatographic results. The aca method that involved p-nitrobenzene diazonium tetrafluoroborate and another diazotized sulfanilic acid method with a higher concentration of sodium nitrite (0.8 mmol/L) yielded falsely high values for direct bilirubin, which could lead to clinical confusion. The more recently introduced diazotized sulfanilic acid method of the aca gave substantially better results than the p-nitrobenzene diazonium tetrafluoroborate method but was still inaccurate. Systematic investigation of these procedures revealed that the overestimation of direct bilirubin by the diazotized sulfanilic acid method was related to the amount of unconjugated bilirubin present and its ability to react as direct bilirubin in the presence of higher concentrations of sodium nitrite. Inherent properties of p-nitrobenzene diazonium tetrafluoroborate appeared to be responsible for inaccuracies in that method, which could not be corrected by varying reagent concentration or the reaction conditions.     

Chemical nature of a synthetic bilirubin conjugate and its reactivities in the total and direct reactions by the Jendrassik-Gróf method

Using liquid chromatography, nuclear magnetic resonance spectroscopy, and elemental analyses, we verified that a commercially available synthetic bilirubin conjugate is predominantly a ditaurobilirubin (DTB) disodium salt. The Jendrassik-Gróf total bilirubin (TBIL) method quantitatively measures unconjugated bilirubin (Bu) and the Bu-equivalent content in DTB, from which we infer that the azopigments of Bu and DTB have identical molar absorptivities, which do not change in the presence of either serum or serum albumin of human or bovine origin. However, based on the ratio of direct bilirubin (DBIL) to TBIL, the DBIL reaction in dilute HCI is incomplete (even up to 20 min), with lower yields in a matrix of bovine serum albumin than in human serum. By contrast, the DBIL reaction in acetate buffer (pH 4.75) is quantitative for DTB in human serum (or its albumin), but less so in bovine serum (or its albumin). DTB is water soluble, is relatively stable when lyophilized with human serum, and is determined with such high precision in the above-mentioned endpoint assays that it may be a suitable surrogate calibrator for conjugated bilirubin.     

In vitro effects of light on serum bilirubin subfractions measured by high-performance liquid chromatography: comparison with four routine methods.

The effects of light on serum bilirubin subfractions in vitro were investigated by HPLC and four routine methods for bilirubin analysis. By HPLC, the rate of photodegradation of unconjugated bilirubin (Bu) was nearly twice that of monoconjugated bilirubin (mBc) and threefold that of diconjugated bilirubin (dBc); delta bilirubin (Bd) was most stable against photoirradiation. In the diazo method, the rate of photodegradation of direct bilirubin was almost the same as that of the sum of mBc, dBc, and Bd determined by the HPLC method. However, the rate of photodegradation of indirect bilirubin was significantly lower (P < 0.001) than that obtained by HPLC, because approximately 30% of the bilirubin photoproducts reacted with the diazo reagent as indirect bilirubin. The rate of photodegradation of total bilirubin determined by the direct spectrometric method was lower than that determined by the diazo method, but equal to that of the total peak areas of HPLC. In the Ektachem method, bilirubin photoproducts affected total bilirubin negligibly, and Bc and Bu positively, so that the value of Bd decreased. In the bilirubin oxidase method, bilirubin photoproducts were oxidized enzymatically by both the total and direct bilirubin reagents. We re-emphasize the importance of shielding serum from light to avoid generating bilirubin photoproducts that interfere with the accurate determination of serum bilirubin subfractions. We also recommend HPLC analysis as a standard method for bilirubin measurement.     

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.     

Amperometric determination of total cholesterol in serum, with use of immobilized cholesterol ester hydrolase and cholesterol oxidase.

We describe an electrochemical method for simple, rapid, and economical assay of total serum cholesterol with use of immobilized cholesterol esterase (EC 3.1.1.13) and cholesterol oxidase (EC 1.1.3.6). A rotating porous cell was specially designed to hold the immobilized enzymes firmly and to allow the reaction mixture to pass through the enzyme layer easily, thus catalyzing the enzymatic transformation quickly. Hydrogen peroxide resulting from a catalytic reactions was measured amperometrically at +0.60 V cs. a standard calomel electrode. The calibration curve for total serum cholesterol was linear from 0 to 5.00 g/liter. The method is specific, precise, and inexpensive. Our results correlate well with those obtained by the method of Abell et al. [Stand. Methods Clin. Chem. 2, 26 (1958)], the correlation coefficient being 0.992. Ascorbic acid or bilirubin in concentrations up to 700 mg/liter do not interfere. The immobilized enzymes are stable, and the same immobilized-enzyme stirrer can be used for at least 200 accurate, reproducible assays.     

Derivatives of 1-naphthyl ethylenediamine dihydrochloride for standardization of direct-bilirubin assays done with the Technicon "SMAC".

The Jendrassik--Groff assay for direct bilirubin was adapted for analysis rates of 150/h on the Technicon "SMAC" continuous-flow analyzer. This requires development of a standard that is compatible with the other 19 channels on this analyzer. N-1-Naphthyl ethylenediamine dihydrochloride has been used for standardization of direct bilirubin assays, but we found it to be unsuitable because values for potassium are falsely elevated when potassium is determined with a valinomycin ion-selective electrode. This interference can be eliminated by alkylating the aliphatic amine group in the standard. The resulting compounds undergo the coupling reaction in the same way as the original compound and function equally well as standards for the direct bilirubin reaction. The only limitation of these analogs is their decreased solubility at physiological pH in some cases. Thus only certain alkyl groups are suitable.     

蓝光照射法消除血清尿酸测定中胆红素负干扰

目的：建立以波长为420～460nm的蓝光预先照射高浓度胆红素血清样本一定时间后再测定血清尿酸的新方法．以消除血清高浓度胆红素对尿酸酶-过氧化物酶偶联Trinder反应法测定尿酸的负干扰。方法：使用20W蓝光灯管对含不同浓度胆红素的血清样本进行一定时间的照射后．在全自动生化分析仪上测定尿酸浓度，对比光照前后的变化，并进行重复性实验和回收实验。结果：蓝光照射后的血清样本胆红素浓度明显下降，尿酸浓度明显上升．重复性实验结果批内CV为2．7％，批间为7．6％。回收实验的回收率原法为55．9％～70．5％．新方法为89．8％～113．4％。结论：蓝光照射法可有效消除血清尿酸测定中胆红素负干扰，其方法简单，结果准确可靠。     

Effects of serum bilirubin on determination of uric acid by the uricase-peroxidase coupled reaction.

We examined the possible effect of different bilirubin species, including unconjugated (Bu), sugar-conjugated (Bc), and authentic delta bilirubin (Bd) isolated from serum, on the direct enzymatic measurement of uric acid by using the uricase (EC 1.7.3.3) and peroxidase (EC 1.11.1.7) coupled reaction. Bc, isolated from human bile, produced the most interference with uric acid determination. Although the presence of human serum albumin reduced interference by Bu and Bc, albumin-bound Bc complex still produced greater interference than Bu. Interference with the peroxidase reaction by Bc covalently bound to serum albumin (Bd) was less than that by Bu. We examined this phenomenon by using serum-isolated Bd obtained by ammonium sulfate precipitation and ultrafiltration and by using commercially available ditaurobilirubin (DTB) and chemically synthesized Bd (via Woodward's reagent, WBd) as surrogates for bile-isolated Bc and natural Bd, respectively. DTB produced the same amount of interference as natural Bc, but the interference produced by DTB in the presence of serum albumin was not the same as that produced by natural Bc with albumin. Our synthetic Bd appears to be a reliable surrogate for natural Bd in testing for bilirubin interference.     

Use of total patient data for indirect estimation of reference intervals for 40 clinical chemical analytes in Turkey.

In the present study we used patient data to calculate laboratory-specific indirect reference intervals. These values were compared with reference intervals obtained for a healthy group according to recommendations of the International Federation of Clinical Chemistry and Laboratory Medicine and manufacturer suggestions. Laboratory results (422,919 records) from all subjects of 18-45 years of age over a 1-year period were retrieved from our laboratory information system and indirect reference intervals for 40 common analytes were estimated using a modified Bhattacharya procedure. Indirect reference intervals for most of the biochemical analytes were comparable, with small differences in lower [alkaline phosphatase (ALP) (male), alanine aminotransferase (ALT), creatine kinase, iron (male), total iron-binding capacity, folic acid, calcium (female), lactate dehydrogenase (LDH), lipoprotein (a) [Lp(a)], thyroid-stimulating hormone (TSH), total triiodothyronine (T(3)), direct bilirubin, apolipoprotein A-I (apoA-I), glucose, homocysteine, total cholesterol, ferritin, total protein, ceruloplasmin, sodium, blood urea nitrogen (BUN) and uric acid (female)] and/or upper limits [albumin, ALP (male), amylase, apoA-I, creatine kinase-MB (CK-MB), total iron-binding capacity, phosphorus, glucose, total cholesterol, gamma-glutamyltransferase (gamma-GT), magnesium, total protein, high-density lipoprotein cholesterol (HDL-C), total T(3), ALP (male), ALT, aspartate aminotransferase (AST) (male), direct bilirubin (male), creatine kinase, iron, folic acid (female), Lp(a), uric acid and triglycerides], to the reference intervals determined for healthy subjects in our laboratory. The indirect reference intervals, with the exception of a few parameters (creatinine, direct total bilirubin, calcium, BUN and potassium), were not similar to the reference intervals suggested by the manufacturers. We conclude that laboratory-specific reference intervals can be determined from stored data with a relatively easy and inexpensive method. Indirect reference intervals derived from stored data may be particularly suitable for the evaluation of results for the presenting population.     

Enzymic measurement of primary bile acids and the primary bile acid ratio in serum with the IL-Multistat III Fluorescence Light-Scattering Centrifugal Analyzer

Enzymic fluorimetric methods are described for the determination of primary bile acids and of chenodeoxycholic acid (CDC) and cholic acid (C) in serum. Bile acids are extracted from 0.3 mL of serum in a simple 5-min step with use of Sep-Pak C cartridges. Total primary bile acids are measured by an equilibrium technique after reaction with beta-NAD in the presence of 7 alpha-hydroxysteroid dehydrogenase. Chenodeoxycholic acid (and its conjugates) is measured by a reaction-rate technique employing the same reaction as above but under different experimental conditions. A small contribution of cholic acid (and its conjugates) to the reaction rate is eliminated by simple calculations. Cholic acid is calculated by difference of the two determinations. In both assays NADH fluorescence is measured with the Multistat centrifugal analyzer. Absolute recovery of bile acids from serum was about 87%. Day-to-day standard deviations for CDC and C were 1.6 and 2.0 mumol/L at serum concentrations of 22.1 and 24.1 mumol/L respectively. Comparison data with a cholylglycine RIA procedure gave the following correlation coefficients (x = RIA, y = proposed method): r = 0.980 (RIA vs total primary bile acids), r = 0.918 (RIA vs CDC) and r = 0.989 (RIA vs C). The methods described appear more practical for use on a routine basis than methods in the literature for the calculation of the primary bile acid ratio.     

A new enzymatic approach for estimating total and direct bilirubin

We designed an enzymatic assay for total (TBil) and direct bilirubin (DBil), the principle of which involves measuring the decrease in absorbance at 450 nm produced by bilirubin oxidase from Myrothecium verrucaria. Since TBil and DBil are oxidized at pH 7.2 and 3.7, respectively, the degree of bilirubin oxidation is measurable in each case. An analysis of bilirubin by high-performance liquid chromatography, before and after the enzymatic reaction with bilirubin oxidase, verified the specificity of the enzyme. The results obtained using this method varied linearly with TBil and DBil concentrations up to at least 250 mg/L and 150 mg/L, respectively. Reducing substances, commonly used anticoagulants and hemoglobin showed no apparent interference. The degree of day-to-day precision (CV) for TBil and DBil ranged from 1.2% (206.2 mg/L) to 10.6% (3.5 mg/L) and from 1.8% (84.3 mg/L) to 12.4% (2.1 mg/L), respectively. Values measured using this new method correlated well with those obtained by Malloy-Evelyn's method and the slide method employing the Kodak Ektachem analyzer.     

Enzymatic determination of bilirubin fractions in serum

Novel enzymatic methods using bilirubin oxidase from Myrothecium verrucaria are described for the determination of bilirubin fractions in serum. These fractions include an unconjugated form (Bu), a conjugated form (Bc), and a delta fraction of bilirubin that reacts with direct diazo reagent and is irreversibly bound to serum albumin (Bδ). The determination is based on the different reactivities of the enzyme to bilirubin fractions at different pH in the presence or absence of anionic detergent such as sodium dodecyl sulfate (SDS) or sodium cholate. Thus, in the absence of detergents, Bc and Bδ are oxidized at acidic pH, while Bc is oxidized at alkaline pH; Bu is not oxidized at either acidic or alkaline pH.

The first approach is based on measuring the decreased absorbance of bilirubin colour at 450 nm caused by the enzymatic oxidation. Total bilirubin is measured at pH 8.0 in the presence of SDS. Direct bilirubin is measured at pH 3.7 and Bc is measured at pH 10.0 in the absence of SDS, respectively.

The second approach is made by coupling the diazo reaction with the enzyme reaction. Total and direct bilirubin are determined by a conventional diazo method without prior treatment by the enzyme. Bδ is determined with a direct diazo method after the serum sample is treated at pH 10.0 by the enzyme to oxidize the Bc fraction. The precision and the accuracy of these methods were compared with a precision and the accuracy of these methods were compared with a diazo method, an HPLC method and a slice method, and good results were obtained.     

以血浆作透析液在肝移植术前的应用

目的以人体新鲜冰冻血浆作透析液行血液透析(PHD)后继续进行连续性静脉-静脉血液滤过(CVVH),观察其对肝移植术前高胆红素血症及血浆细胞因子水平的影响。方法4例拟行肝移植手术的肝功能衰竭患者行PHD治疗6 h后,应用同一滤器(AV600)继续行CVVH治疗24 h。分别检测治疗前后血清胆红素(TB、DB、IB)、总胆汁酸(TBA)、血氨(BA)及细胞因子TNF-a,IL-6和IL-8的水平。结果PHD治疗6 h后患者总胆红素(TB)、直接胆红素(DB)、间接胆红素(IB)和总胆汁酸(TBA)分别下降(24.38±4.89)%(、26.23±2.67)%(、25.02±0.01)%、(27.38±8.59)%(P<0.05);PHD后继续行CVVH治疗24 h,总胆红素(TB)仍有所下降(10.61±0.32)%;CVVH在清除血氨,纠正电解质和酸碱失衡方面比PHD更有效(P<0.05);PHD及CVVH治疗后TNF-aI、L 6、IL-8较治疗前明显下降(P<0.05)。结论对肝功能衰竭患者,PHD联合CVVH治疗能显著降低血清胆红素、总胆汁酸、血氨及炎性细胞因子水平,调节水、电解质和酸碱平衡。     

Determination of serum bilirubin concentration during phototherapy of newborns and in vitro: results compared by the direct spectrometric method and the diazo method

During direct illumination of a serum bilirubin solution the bilirubin concentration decreased markedly, both as determined by the direct spectrometric method and (even more so) by the diazo method. In contrast, I found the same values for serum bilirubin concentrations as determined by these two methods for serum from untreated, "single light", and "double light" treated full-term infants with neonatal hyperbilirubinemia without blood type immunization. The same was true for untreated and "single light" treated premature infants with this disease. Furthermore, no difference was found in the above-mentioned relationship between "single light" treated infants with the same disease, and untreated infants with neonatal hyperbilirubinemia without immunization, all born at term. This is important, because the direct spectrometric method is simpler and requires less serum than does the diazo method.     

An automated assay for measuring serum ascorbic acid with use of 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy, free radical and o-phenylenediamine

We developed a novel, cost-effective, and automated assay for ascorbic acid (AsA) in serum using a COBAS MIRA S analyzer (Roche Diagnostic System). Our method has a wide dynamic range and covers AsA concentrations from well below the lower reference interval to well above it. AsA is oxidized by 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy, free radical (TEMPO) to dehydroascorbic acid (DAsA). The latter condenses with o-phenylenediamine (OPDA) to form a quinoxaline derivative that absorbs light at 340 nm. The change in absorbance at 340 nm is proportional to the concentration of AsA in the specimen. The automated system permitted the assay of 65 specimens per hour at a cost of approximately US$ 0.01 per specimen for reagents. The assay can be applied directly to serum specimens (direct method) and also to sera with a prior deproteinization step with metaphosphoric acid. The detection limit for the direct serum assays is 0.8 vs. 0.4 mg/l with the deproteinization method. The recovery of AsA from a supplemented serum pool was of >95% for both procedures. We used four distinct methods on 66 patients sera. The direct method for AsA correlated well with an HPLC method (r=0.964, P<0.001); the direct method also correlated well with a method that uses AsA oxidase (r=0.975, P<0. 001). The deproteinization method correlated well with HPLC (r=0.981, P<0.001), and with the AsA oxidase procedure (r=0.994, P<0.001). Ten within-day determinations on a serum pool gave a C.V. <4.3% for both the direct and deproteinization procedures. The between-day assays of the same serum pool over 10 days gave a C.V. of <6.7% by both methods.     

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.     

对钒酸氧化法测定血清中胆红素的评价

目的:评价钒酸氧化法测定血清中总胆红素和直接胆红素。方法:钒酸氧化法的精密度、准确度实验及其与重氨法相关和干扰因素实验。结果:批内CV总、直接胆红素分别为0.8％与1.3％。日间CV为1.8％与1.7％,平均回收总胆红素为101.1％,直接胆红素100.1％,与重氮试剂法相关(J-G法)总胆为y(本法)=1.015x(重氮法)-0.119,r=0.998,直接胆红素为y(本法)1.101x(重氮法)+0.81,r=0.952。相关良好,血红蛋白浓度和脂血分别高达3g/L和9.0mmol/L不干扰。结论:该法简单、快速、稳定,而且对溶血、乳糜等干扰因素的影响比重氮法小。     

Spectrophotometric study of bilirubin interference in the huang reaction for cholesterol

The difficult problem of ascribing the measure of bilirubin interference in a direct Liebermann-Burchard reaction (LB) for serum cholesterol has been investigated. Various parameters in this reaction have been described. The effect on the reaction temperatures of the addition of an aqueous sample and non-aqueous standard to the reagent solution indicates that this factor may be uncontrolled. The results obtained on studying the different initial starting temperatures has been shown to influence the final absorbance values variably at the two reported wavelengths of measurement. Spectra showing the effect of varying time and temperatures on the reactions of cholesterol, bilirubin and cholesterol—bilirubin mixtures with the Huang modification of the L—B reagent are detailed, along with the consideration of the choice of wavelength of measurement on cholesterol to bilirubin absorbance ratios. The validity or non-validity of suggested blanks is shown spectrophotometrically. The conclusion arrived at is that a correction for bilirubin interference in this direct reaction is quite difficult. For comparison purposes the reactions of p-toluene sulfonic acid and ferric chloride reagents with bilirubin are shown.     

对钒酸氧化法测定血清中胆红素的评价

目的 :评价钒酸氧化法测定血清中总胆红素和直接胆红素。方法 :钒酸氧化法的精密度、准确度实验及其重氮法相关和干扰因素实验。结果 :批内 CV总、直接胆红素分别为 0 .8%与 1 .3%。日间 CV为1 .8%与 1 .7% ,平均回收总胆红素为 1 0 1 .1 % ,直接胆红素 1 0 0 .1 %。与重氮试剂法相关 ( J- G法 )总胆为 Y(本法 ) =1 .0 1 5X(重氮法 ) - 0 .1 1 9,r=0 .998,直接胆红素为 Y(本法 ) =1 .1 0 1 x(重氮法 ) 0 .81 ,r=0 .952。相关良好 ,血红蛋白浓度和脂血分别高达 3g/L和 9.0 mmol/L不干扰。结论 :该法简单、快速、稳定 ,而且对溶血、乳糜等干扰因素的影响比重氮法小