Determination of the sum of bilirubin sugar conjugates in plasma by bilirubin oxidase.

BACKGROUND: A reliable indicator of cholestasis is the presence of abnormal concentrations of bilirubin mono- and diglucuronide [conjugated bilirubin (CB)] in blood. A routine assay of CB is available only to those who possess a certain type of clinical analyzer. We describe a two-point manual method for CB that could be adapted as a rate assay to automated clinical analyzers. METHODS: The measurement of CB is based on its oxidation to biliverdin by bilirubin oxidase. The resulting decrease in absorbance at 460 nm is proportional to the CB concentration. The assay is calibrated with solutions of ditaurobilirubin in human serum. RESULTS: Under the conditions of the assay (0.1 mol/L glycine buffer, pH 10.0; reaction time, 2 min), only 5% of unconjugated bilirubin is oxidized and delta-bilirubin is not oxidized at all. Results obtained with the bilirubin oxidase method agreed well with those obtained by HPLC. The long-term CVs at CB concentrations of 6 and 63.4 mg/L were 20% and 2.6%, respectively. The reference values, established by analyzing 51 plasma specimens from healthy adults, were 0.0-1.2 mg/L, with a mean value of 0.2 mg/L. CONCLUSIONS: The proposed method for CB has good analytical specificity and obviates the requirement for HPLC or a dry chemistry analyzer. The measurement of CB in blood is superior to the measurement of direct bilirubin because an abnormal concentration of direct bilirubin does not necessarily indicate the presence of cholestasis.

Relationship between nutrition, weight change, and fluid compartments in preterm infants during the first week of life.

This study was conducted to investigate the redistribution of fluid compartments and to examine the factors contributing to the variability of early weight loss in premature infants. Fourteen preterm infants (mean +/- SD: birth weight, 1473 +/- 342 gm; gestational age, 30.7 +/- 2.4 weeks) were studied at 1 and 7 days of age. Total body water was measured by deuterium oxide dilution, extracellular volume by bromide dilution, and intracellular volume by the difference between total body water and extracellular volume. There were significant changes in body fluid distribution per concurrent weight from birth to age 1 week. Extracellular volume decreased by 11%, and intracellular volume increased by 8.5% with no change in total body water. Infants were then grouped according to postnatal weight loss (group 1 (n = 7) > 10% and group 2 (n = 7) < 5% of birth weight). In group 1 there was a significant loss of both weight (mean +/- SD: 15.6% +/- 3.7%) and extracellular volume (15.9% +/- 9% of birth weight), with no change in intracellular volume. In group 2 there was no significant weight loss (1.4% +/- 1.8%), but a significant loss of extracellular volume (13.0% +/- 5.4% of birth weight) and a significant increase in intracellular volume. Other differences between the groups were a lower energy intake in group 1 than in group 2 (mean +/- SD: 177 +/- 46 vs 269 +/- 45 kilojoules/kg per day; p < 0.005) and a higher physiologic stability index in group 1 (p < 0.05). We conclude that significant postnatal weight loss as a result of the contraction of the extracellular compartment occurs only in less stable infants whose energy intake is inadequate. With adequate energy intake, weight loss is minimal because of the expansion of the intracellular compartment, which may be related to the onset of growth.

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.

Delta bilirubin: absorption spectra, molar absorptivity, and reactivity in the diazo reaction.

Delta bilirubin (B delta), isolated from serum, has an absorption maximum near 440 nm and a molar absorptivity of 72,000 L mol-1cm-1 in either Tris HCl (0.1 mol/L, pH 8.5) or phosphate (0.13 mol/L, pH 7.4) buffer. This absorptivity exceeds by approximately 50% and 59%, respectively, that of unconjugated bilirubin in the same buffers. This finding suggests that substantial errors can be incurred in direct spectrophotometry of bilirubins in serum. In the total diazo (TBIL) assay (Clin Chem 1985;31:1779-89), the color yield from B delta increases by 10% as the final diazo concentration is increased from 0.27 to 0.81 mmol/L. In the direct (DBIL) assay, if done in HCl (50 mmol/L), B delta yields approximately 15% more color as the diazo concentration is increased from 0.51 to 1.53 mmol/L, whereas in acetate buffer (0.4 mol/L, pH 4.7) the corresponding color yield is 25% greater. However, the absolute color yield for the reaction in HCl exceeds that in acetate buffer. In both the TBIL and the DBIL assay, B delta reacts slowly, nearly complete reaction requiring 10 min. Thus, B delta may be seriously underestimated in diazo (especially DBIL) methods in which short reaction times (20 s to 1 min) are used.

Candidate reference method for determination of total bilirubin in serum: development and validation.

This candidate Reference Method for measuring total bilirubin in serum is based on the Jendrassik-Gróf principle (Clin Chem 29: 297-301, 1983). Standard Reference Material no. 916 bilirubin (National Bureau of Standards) is used as the standard. Bilirubin standard solutions may be prepared either in human serum or in 40 g/L albumin solution (human or bovine), because we found the molar absorptivity of the azopigment at 598 nm to be identical in these media. The absorptivities of the unconjugated and conjugated azopigments appear to be identical, but the conjugated azopigment is completely hydrolyzed in the final reaction mixture. Bilirubin added to serum from adults or neonates was quantitatively accounted for. Interference by hemoglobin (up to 2 g/L), ascorbic acid (up to 20 mg/L), or zinc (at physiological concentrations) is negligible. Of the therapeutic drugs we tested, only L-dopa and alpha-methyldopa interfere. We established normal adult reference values for total bilirubin and examined the intraindividual variation in 19 subjects.

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.

Pitfalls in the American Monitor kit methods for determination of total and "direct" bilirubin.

We evaluated the American Monitor Corporation kit for total and direct-reacting bilirubin and found that it suffers serious deficiencies, which lead to inaccurate and imprecise results. The main problem with the total-bilirubin procedure is that the short reaction time (2 min) is inadequate for completion of the reaction. The poor precision of the direct-bilirubin method is due to the short reaction time and the inability of the "stabilizer" (hydroxylamine sulfate) to completely destroy the diazo reagent. Depending on when Fehling's reagent is added, the reaction time may vary from 2 min to 7 min. Values for direct bilirubin at 7 min exceed those obtained at 2 min by 17 to 29%. The short reaction time makes color development temperature dependent, an additional source of imprecision. The suboptimal concentration of the diazo reagent results in underestimation of direct-reacting bilirubin. We recommend changes that improve both precision and accuracy of the kit procedures.

Effect of light and temperature on the stability of creatine kinase in human sera and controls.

Commercial control preparations were exposed to light at room temperature for as long as 24 h. Decreases in creatine kinase activity in seven of the nine controls ranged from 28.8 to 83.3%. When these controls were protected from light and stored at various temperatures, the decrease in activity was either eliminated or substantially reduced, indicating that the decreased activity in the presence of light was not due entirely to thermal inactivation. In the absence of oxygen, light had little effect on creatine kinase suggesting that light inactivation is a light-catalyzed oxidative process. The decreased activity observed when some of the controls were exposed to light was not completely restored by incubating with dithiothreitol before analysis. However, increases in creatine kinase activity ranging from about 37 to 176% were observed when freshly prepared controls were pre-incubated with dithiothreitol. Neither light nor dithiothreitol had any effect on the creatine kinase activity in two of the nine controls. When human sera were exposed to light for 24 h, the largest decrease in activity was about 15%. Incubation of fresh human sera with dithiothreitol before analysis caused an average increase in activity of approximately 10%.

Performance of the Du Pont aca ammonia method.

We evaluated the performance of the Du Pont aca ammonia procedure with regard to (a) linearity, (b) precision, (c) interferences, and (d) effect of anticoagulants. Linearity extends to 2,000 mumol/L. The SD of the method was essentially constant (2 to 3 mumol/L) and independent of the NH3 concentration. Hemoglobin, bilirubin, and lipemia do not interfere. Either EDTA or heparin is suitable as anticoagulant. Recovery of NH3 added to plasma samples averaged 102% (range: 97--107%). We established normal values by measuring NH3 in 188 plasma samples from apparently healthy individuals. The 95% confidence range is from 10 to 35 mumol/L. The aca ammonia method compares very well with that of Kingsley and Tager but correlates less strongly with that of Reinhold and Chung. We describe a protein-based solution with stable NH3 concentration that is suitable as a control material.

Lactate dehydrogenase activities in serum and plasma.

We investigated lactate dehydrogenase activity in serum and plasma because of the conflicting data found in the literature. We assayed serum, platelet-rich, and platelet-poor plasma by two colorimetric endpoint methods and by an ultraviolet kinetic procedure. Platelet-poor plasma and serum had essentially the same activities by all three methods, whereas the activity in platelet-rich plasma plasma averaged fourfold that in platelet-poor plasma or serum when the assay was performed under conditions that result in lysis of platelets and release of their lactate dehydrogenase. When measurements were performed in platelet-rich plasma under conditions that prevented lysis of platelets, all three types of specimens gave the same results. This occurred when the osmolality of the reaction mixture was about 240 mOsm/kg of water. At an osmolality of about 120 mOsm, the activity of platelet-rich plasma was substantially lower than that of platelet-poor plasma or serum. If plasma must be used, the sample must first be centrifuged (3000 X g, 15 min) to provide a platelet-free plasma.