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.

The evolution and limitations of accuracy and precision standards.

Limits of maximum allowable analytical imprecision have been defined on the basis of normal range (Tonks), composite biological variation (Cotlove), intraindividual biological variation (Aspen Conference, College of American Pathologists), medical significance (Barnett, Skendzel), and a combination of medical significance and biological variation (Fritsche. Klee). Because error limits based on medical significance are less stringent than those based on biological variation, performance goals based on medical significance are more likely to be attained by laboratories than those defined by biological variation. Most clinical scientists realize that the goal to limit the analytical C.V. to one-half or less of the biological C.V. is arbitrary, and for the large majority of laboratory tests of no benefit to the patient, for the major component of total variation is not the analytical imprecision, but the intraindividual variation itself. Furthermore, the purpose of laboratory testing is to help physicians confirm or exclude potential diagnoses and monitoring therapy rather than detecting small deviations from normal values. Small changes in test values are quite often uninterpretable or lead to costly albeit fruitless investigations. In view of diminishing resources for health care we must establish the accuracy and precision required for each test. While improving the accuracy of some tests would be desirable, for most of them further improvement would be irrelevant to patient care because few tests are pathognomonic by themselves and quite often diagnosis is not made on the basis of a single laboratory result. If more accuracy is desirable, it must also be affordable and benefits should outweigh costs.

Serum and urine albumin: a progress report on their measurement and clinical significance.

For about 25 years, bromcresol green and bromcresol purple have been the basis for most of the measurements of serum albumin in the US and perhaps in the world. The longevity of the methods is due to their being simple, sensitive, specific, inexpensive and relatively free from interferences. The lack of change in the serum albumin methodology is balanced by two important developments. First, the recognition of the importance of serum albumin in the maintenance of good health, and the association of decreased concentrations with increased risk of morbidity and mortality. Second, the association of albuminuria with diabetic nephropathy, which without medical intervention could lead to end-stage renal disease. The development of accurate and precise methods for urinary albumin has provided a tool to physicians to extend the length and improve the quality of life of many diabetic individuals.

"Direct" and total bilirubin tests: contemporary problems.

In eight unique challenges mailed by The College of American Pathologists Comprehensive Chemistry Survey to participating laboratories within 3 years, results for direct-reacting bilirubin (DBIL) were highly variable among the 12 largest peer groups, and most of the results differed greatly from the values obtained by a referred method. Peer-group mean values for total bilirubin (TBIL) were in much better agreement with each other and with those obtained by the Reference Method for TBIL. From a review of the information on the assay of DBIL provided to us by the manufacturers, we conclude that among the major causes of the large variability and bias in DBIL assays are problems with calibration, lack of a serum blank measurement, inadequate concentrations of HCl in the reaction mixture, inappropriate use of bichromatic correction methods, and possibly the use of wetting agents or surfactants in the reagent. Within-group SDs were small and generally acceptable. The among-peer-group variability in DBIL values is attributable to bias, not imprecision. We recommend several simple changes that could improve the accuracy of DBIL determinations in clinical laboratories.

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.

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.

Molar absorptivities of bilirubin (NIST SRM 916a) and its neutral and alkaline azopigments.

Three laboratories in the U.S. and two in the Netherlands determined molar absorptivities (epsilon) of Standard Reference Material (SRM) 916a Bilirubin from the National Institute of Standards and Technology. In caffeine reagent the average epsilon values were 50,060 and 48, 980 L.mol-1.cm-1 at 432 and 457 nm, respectively. The epsilon value of the blue azopigment, obtained with the Reference Method for total serum bilirubin, was 76,490 L.mol-1.cm-1 at 598 nm. When the addition of alkaline tartrate was omitted, the molar absorptivity of the red azopigment was 56,600 L.mol-1.cm-1 at 530 nm.

Effects of enteric neural defunctioning on small bowel motility.

In this study, we investigated the role of intrinsic nerves of the small intestine on phase III migration of the migrating myoelectric complex. Fasting myoelectric activity was recorded from the small bowel in chronically instrumented dogs. Once control experiments were completed, the animals were divided into two groups and were reoperated. In the first group of five dogs, a 1.5-g/dl aqueous solution of cobaltous chloride (shown to induce degeneration of intestinal intrinsic nerves) was infused close intra-arterially to perfuse a 15-cm segment of jejunum. In the second group of dogs, a catheter was implanted in a branch of the superior mesenteric artery supplying a 15-cm segment of intestine. Tetrodotoxin (0.3-1 micrograms/kg) was infused through the catheter just before the arrival of phase III activity in the perfused segment. Subsequent to the fifth postcobalt perfusion day, phase III traversed but did not occur in the cobalt-treated segment. When tetrodotoxin was injected through the catheter, spontaneous phasic myoelectric and contractile activities in the perfused jejunal segment were inhibited, but phase III migration was not blocked. These findings suggest 1) acute or chronic defunctioning of enteric nerves does not interrupt phase III migration, but 2) phase III expression is dependent on the integrity of intrinsic nerves.

Chemical degeneration of intestinal nerves.

In 15 dogs, cobalt chloride solutions were infused close intra-arterially to perfuse a short segment of the jejunum. In an additional four dogs, the jejunum was perfused with the aqueous vehicle (perfusion control). All animals were killed after 1 mo and tissue samples from cobalt-treated and from nonperfused intestine (tissue comparison control) were obtained for electron microscopic and immunohistochemical studies. Segments infused with 0.25 g/dl cobalt solution showed minimal changes; the most striking feature was an increase of vasoactive intestinal polypeptide (VIP)- and substance P-containing neurosecretory granules. Cobalt chloride at higher concentrations (0.75-1.5 g/dl) induced degeneration of ganglion cells and axons in both the myenteric and submucosal plexi. In contrast, the smooth muscle and the mucosal cells of the cobalt-perfused intestine showed no histological abnormalities. Immunohistochemical staining of tissues treated with 0.75-1.5 g/dl cobalt solutions revealed absence of substance P, Met-enkephalin, and VIP immunoreactivity in all section studied; control segments showed the presence of all three peptides. Cobalt chloride in concentrations of 0.75-1.5 g/dl causes degeneration of intestinal intramural nerves and provides an experimental model suitable for studying the role of these nerves in small intestinal function.

Differences between values for plasma and serum in tests performed in the Ektachem 700 XR Analyzer, and evaluation of "plasma separator tubes (PST)".

We measured 25 analytes in plasma and serum from the same blood specimen, using the Kodak Ektachem 700 XR Analyzer. For 22 of the analytes, values for plasma were practically the same as those for serum; for the other three, differences between concentrations in plasma and serum were significant, both statistically and medically. Values for the same analytes in plasma from regular heparinized tubes were essentially indistinguishable from those for plasma obtained by use of Plasma Separator Tubes (PST).

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.

Proteinuria in health and disease assessed by measuring the urinary protein/creatinine ratio.

We measured daily excretion rates for urinary protein and the ratios of urinary protein to creatinine in 24-h urines and in untimed urines in 60 healthy adults, 30 patients with kidney disease, and 22 kidney-transplant recipients. The ratios for urinary protein/creatinine, mg/g, in untimed urines and in 24-h urines from the same subjects were closely correlated (r = 0.97) for rates of protein excretion ranging from normal (mean 44 mg/day) to nephrotic (maximum 19,300 mg/day). Because urinary protein/creatinine in healthy subjects never exceeded 100 mg/g, we propose that a ratio of less than 100 mg/g in untimed urines, obtained in the absence of exercise, fever, or other evidence of urinary tract disease, is a criterion of normal kidney function. Among patients with nephrotic syndrome (urinary protein excretion rate greater than or equal to 4000 mg/day), urinary protein/creatinine ratios always exceeded 2000 mg/g in both 24-h and untimed urines. Intermediate urinary protein/creatinine ratios (100 to 2000 mg/g) may reflect any type of kidney disease.

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.

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.