Measurement Of Direct Bilirubin Research Articles

Evaluation of Newborn Direct Bilirubin As Screening for Cholestatic Liver Disease.

Biliary atresia (BA) remains the most common indication for pediatric liver transplantation. Early diagnosis is essential for a favorable long-term prognosis for patients with BA. Preliminary data suggests that measurement of direct bilirubin (DB) in newborns may be an effective screening tool for neonatal cholestasis, particularly BA, allowing for early referral and diagnosis. The objective of our study was to establish a cutoff DB value to predict diagnosis of cholestatic liver disease (CLD) with high sensitivity and specificity, as well as, to evaluate whether newborns with elevated DB received appropriate follow-up in our health system. Baseline data were collected on infants born between 2016 and 2019 who had serum total bilirubin and DB drawn in the nursery, and who continued to follow in our health system. Sensitivity, specificity, and positive and negative predictive values were examined using cutoff values of 0.5, 0.6, and 0.7 mg/dL for identifying infants at risk for CLD. Patients' charts were reviewed to note whether they had follow-up levels drawn by their pediatrician or by the hepatology team within 2 months of age and whether they were diagnosed with CLD. Serum total bilirubin and DB levels were drawn from 11 965 infants during their hospitalizations. Three infants from this cohort were diagnosed with CLD: 2 with BA and 1 with Alagille syndrome. DB cutoff values of 0.5, 0.6, and 0.7 mg/dL had sensitivity of 100% and specificity of 96.83% (95% confidence interval [CI], 96.69%-97.53%), 99.08% (95% CI, 98.81%-99.30%), and 99.63% (95% CI, 99.4%-99.7%), respectively. Given that a DB of 0.6 mg/dL had a sensitivity of 100% and specificity of 99%, this value was chosen as the cutoff value to monitor for DB follow-up and diagnosis of CLD. Out of 60 infants who met criteria for DB ≥0.6 mg/dL, only 15 (25%) had a repeat level drawn after nursery discharge; 3 (5%) were eventually diagnosed with CLD. A DB cutoff value of 0.6 mg/dL yielded high sensitivity and specificity for identifying patients with CLD. All 3 patients diagnosed with CLD had elevated DB at hospital discharge. The data revealed that the majority (75%) of eligible newborns did not receive follow-up for their elevated DB in the outpatient setting.

Quantitative effects of bilirubin structural photoisomers on the measurement of direct bilirubin via the vanadate oxidation method.

Exposing blood serum samples to ambient white light-emitting diode (WLED) light may accelerate bilirubin photoisomer production. We previously demonstrated the quantitative effect of bilirubin configurational isomers (BCI) on direct bilirubin (DB) value using the vanadate oxidation method. However, the effects of bilirubin structural photoisomers (BSI) remain unclear. In Study 1, the relationship between WLED irradiation time and BSI production was examined. Serum samples from five neonates were irradiated with WLED light for 0, 10, 30, 60 and 180 min. Bilirubin isomer concentration and BSI production rates were calculated. In Study 2, we performed quantitative investigation of BSI effect on DB values: Differences in DB, BCI and BSI values before and after irradiation were calculated as ⊿DB, ⊿BCI and ⊿BSI, respectively. Assuming the coefficient of BCI affecting DB values was 'a', relational expression was ⊿DB = a*⊿BSI + 0.19*⊿BCI. Serum samples from 15 neonates were irradiated with green LED light for 10 and 30s. The respective bilirubin isomer levels were measured, and the coefficient was derived. In Study 1, the median BSI production rate was 0.022 mg/dL per min in specimens with an unconjugated bilirubin concentration of 10.88 mg/dL. In Study 2, assuming that ⊿DB-0.19*⊿BCI was Y and ⊿BSI was X, the relational expression was Y = 0.34X-0.03 (R2 = 0.87; p < .01) and a = 0.34. Under ambient WLED light, serum sample generated 1.3 mg/dL BSIs in 1 h. Approximately 34% (0.44mg/dL) of BSI concentrations was measured as DB when using the vanadate oxidation method according to the above equation.

Effects of bilirubin configurational photoisomers on the measurement of direct bilirubin by the vanadate oxidation method.

Direct-reacting bilirubin concentrations measured using vanadate chemical oxidation method do not exactly match the conjugated bilirubin concentration. One of the causes is the effect of bilirubin photoisomers. However, the quantitative evaluation of the effects of these photoisomers has not been sufficiently conducted. In particular, the influence of bilirubin configurational isomers on direct bilirubin is the most critical factor. Sixteen residual serum samples were used. For quantitative analysis based on the change in direct bilirubin and bilirubin configurational isomer, samples were irradiated via blue light-emitting diodes to suppress the production of bilirubin structural isomers. Total bilirubin and direct bilirubin concentrations were measured using the vanadate chemical oxidation method. Concentrations of 4Z,15Z-bilirubin IXα and its photoisomers were measured using high-performance liquid chromatography. The sum of 4Z,15E-bilirubin IXα and 4E,15Z-bilirubin IXα was notated as bilirubin configurational isomer, and the differences between the measured values of the irradiated and non-irradiated samples were calculated and notated as ΔDB and ΔBCI. In non-irradiated and irradiated samples, total bilirubin and direct bilirubin concentrations were 10.73 mg/dL with significant a decrease to 10.60 mg/dL and 0.69 mg/dL with a significant increase to 0.78 mg/dL, while bilirubin configurational isomer values were 1.00 mg/dL and 1.52 mg/dL, respectively. The linear regression equation revealed a significant positive correlation of Y = 0.187X-0.006 between ΔDB (Y) and ΔBCI (X). Applying the vanadate chemical oxidation method affected approximately 19% of the bilirubin configurational isomer concentration for direct bilirubin. Extreme caution is necessary when interpreting the measured values of samples indicative of unconjugated hyperbilirubinaemia.

Association of Early Direct Bilirubin Levels and Biliary Atresia Among Neonates

Biliary atresia (BA) is a fibrosclerosing cholangiopathy resulting in obstruction of the biliary tree. Diagnosis and early performance of the Kasai hepatic portoenterostomy improves outcomes.1 Current guidelines recommend measurement of direct bilirubin (DB) or conjugated bilirubin levels in neonates who are still jaundiced at age 2 to 3 weeks.2,3 However, recent studies have documented the elevation of DB or conjugated bilirubin levels within the first days after birth in neonates with BA,4,5 suggesting that BA is a developmental cholangiopathy associated with a genetic, prenatal, or developmental event. To test this hypothesis, this case series evaluated DB levels in the first days after birth among neonates who were subsequently diagnosed with BA.

Irreversible electroporation of malignant liver tumors: Effect on laboratory values.

Liver cancer is often associated with chronic liver diseases. Treatment with percutaneous irreversible electroporation (IRE) may preserve liver function. In the present study, the clinical data of 29 patients with liver tumors between July 2015 and December 2016, all of whom underwent liver IRE at Fuda Cancer Hospital, Guangzhou, China was retrospectively reviewed. All the patients survived the treatment. Of the 29 patients, 7 were positive for hepatitis B, 15 had hepatocellular carcinoma (HCC) and 7 had pancreatic cancer with liver metastases. All patients survived IRE. Despite liver-protective treatment prior to IRE, the mean alanine transaminase (ALT) and aspartate transaminase (AST) levels were significantly elevated 1-2 days after IRE, to 540 and 712 U/l, respectively; however they had returned to the preoperative values by 2 weeks following IRE. Prior to IRE, the mean total bilirubin and direct bilirubin measurement levels were normal; however, 8-10 days after IRE, they had increased to 24 U/l and 12 µmol/l, respectively, and had returned back to the preoperative levels by 2 weeks after IRE. This first group included all patients. The result of the 4 subgroups of cancer patients demonstrated a variation between different measurement days and recovery with patients positive for the hepatitis B virus taking the longest duration to recover (17±3 days) meanwhile patients with pancreatic cancer with liver metastases took the shortest time to achieve recovery (10.78±2 days). The findings of the present study indicate that hepatic injury caused by IRE is transient and self-limiting in patients with liver tumors.

Effects of bilirubin photoisomers on the measurement of direct bilirubin by the bilirubin oxidase method.

Background We occasionally encounter increases in direct bilirubin value on reanalysis of the surplus serum collected in the past from a neonate with indirect hyperbilirubinemia. But the details of this phenomenon are unclear. We evaluated the change of direct bilirubin and the relation of bilirubin photoisomer of the serum exposed to room light. Methods Surplus serum samples from neonates with indirect hyperbilirubinemia were exposed to room light for 24 h. The bilirubin fraction assay of samples was performed by the bilirubin oxidase method (Nescauto and Aqua-auto Kainos reagent) and high-performance liquid chromatography. Results Direct bilirubin increased significantly from 0.61 to 2.36 mg/dL. The respective ratios of bilirubin photoisomers before and after exposure were as follows: cyclobilirubin (0.007 to 0.29) and (EZ)-bilirubin (0.018 to 0.041) increased significantly, (ZZ)-bilirubin decreased 0.84 to 0.55 significantly. The difference of the cyclobilirubin concentration was most closely associated with those of the direct bilirubin concentration. Conclusion Direct bilirubin value was increased after exposure to the room light, and increase in direct bilirubin was significantly correlated by cyclobilirubin increase in the serum samples from neonates with indirect hyperbilirubinemia.

Reactivity of bilirubin photoisomers on the measurement of direct bilirubin using vanadic acid method.

Objective Investigation of the reactivity of fractions of bilirubin photoisomers with the vanadic acid oxidation method. Methods Bilirubin photoisomers were prepared by irradiating a bilirubin/human serum albumin solution with blue light emitting diode. Direct bilirubin and bilirubin fractions were measured using the vanadic acid oxidation method and high-performance liquid chromatography in the sample before and after irradiation. Results Direct bilirubin was increased in the solution containing bilirubin photoisomers. ( EE)-/( EZ) -cyclobilirubin-IXα and ( ZE)-/( EZ)-bilirubin-IXα completely disappeared after the addition of vanadic acid. Conclusion Bilirubin photoisomers reacted as direct bilirubin in the vanadic acid oxidation method.

Operational impact of using a vanadate oxidase method for direct bilirubin measurements at an academic medical center clinical laboratory

ObjectivesThe aim of this study was to compare the operational impact of using vanadate oxidase versus diazo direct bilirubin assays for an academic medical center patient population. Design and methodsRetrospective study was done over an approximately 3.5 year period. The main automated chemistry instrumentation was a Roche Diagnostics cobas 8000 line. The Roche Direct Bilirubin assay was compared to Diazyme Laboratories Direct Bilirubin Assay and Randox Laboratories Direct Bilirubin assay using manufacturer's guidelines for hemolysis index, lipemia index, and analytical measurement range (AMR). ResultsRetrospective data was analyzed for 47,333 serum/plasma specimens that had clinical orders for direct bilirubin. A total of 5943 specimens (12.6%) exceeded the hemolysis index limit for the Roche method compared to only 0.2% and 0.05% of specimens for the Diazyme and Randox methods, respectively. The impact was particularly large on patients less than 2 years old, for which 51.3% of specimens exceeded the hemolysis index for the Roche method. A total of 1671 specimens (3.5%) exceeded the lipemia index limit for the Roche method compared to less than 0.1% for the Randox method. Lastly, 988 (2.1%) of specimens had direct bilirubin concentrations exceeding the upper AMR limit of 10mg/dL [171µmol/L] for the Roche assay compared to less than 1% of specimens for the vanadate oxidase methods. ConclusionsVanadate oxidase direct bilirubin methods offer advantages over diazo methods in terms of less interference by hemolysis and lipemia, as well as wider AMR. The advantages are particularly evident for neonatal and infant populations.

Development of an equation to correct for hemolysis in direct bilirubin measurements

BackgroundDirect bilirubin is measured for the investigation of pediatric and adult jaundice. Package inserts suggest that hemolysis decreases direct bilirubin measurements, but no published studies have adequately described the extent of interference. MethodsThe influence of hemolysis on direct bilirubin quantification (Beckman AU680) was evaluated by titrating increasing amounts of hemoglobin into specimens with variable starting concentrations of direct bilirubin. An equation was derived to predict the nominal interference-free concentration of direct bilirubin as a function of measured concentration and hemolysis-index. ResultsHemolysis decreased the direct bilirubin concentration reported by the AU680. The extent of interference is a function of both the interference-free concentration of direct bilirubin and the degree of hemolysis. ConclusionsThe concentration of direct bilirubin in hemolyzed specimens can be predicted.

Perspective on Neonatal Hyperbilirubinemia

Jaundice in newborns provides a different response from the parents whencompared to jaundice in older children and adults. Physiologic hyperbilirubinemia occurs commonly in term newborn infants in the absence of any underlying pathologic cause. Yet, the jaundice itself is commonly regarded as a problem in the transition to extrauterine life.In Neonatal hyperbilirubinemia (NHB) the total bilirubin level is greater than 15mg/dL in 15 day or less old neonates and 2mg/dL in neonates above 15 days of age. Estimation of total bilirubin is preferred in the routine analyses for NHB compared to measurement of direct bilirubin. If certain conditions like sepsis, hepatic infections and other liver diseasesare present it may be prudent to use direct bilirubin measurement. Yet contrary to the usual assumption of pathology, there are several lines of evidence supporting an adaptive role for neonatal hyperbilirubinemia. First, experimental and clinical evidence indicate that neonatal enzyme systems are not yet mature at birth; bilirubin has been demonstrated toscavenge potentially toxic oxygen free radicals that in later life are removed by the mature antioxidant enzyme system. Second, presence of bilirubin in mammals, similar patterns of expression of neonatal hyperbilirubinemia in nonhuman primates, and significant interpopulation variation in newborn serum bilirubin levels among humans all suggest that bilirubin production, metabolism, and excretion are under genetic control. Therefore bilirubin metabolism and its understanding may help improve its diagnosis and prognosis.

Oxyhaemoglobin and protein interference on spectrophotometric detection of cerebrospinal fluid bilirubin

Aim Diagnosis of subarachnoid haemorrhage (SAH) may involve the spectrophotometric analysis of cerebrospinal fluid for net bilirubin absorbance (NBA) and net oxyhaemoglobin absorbance (NOA). NBA>0.007 AU is supportive of a diagnosis of SAH. A limitation is that oxyhaemoglobin and total protein (TP) can interfere with NBA measurement such that according to published guidelines, results may be deemed inconclusive if NOA>0.1 AU or TP>1000 μg/L. The aim of this study was to assess the interference of oxyhaemoglobin and TP on NBA measurement. Method Increasing amounts of haemolysate was added to samples with a NBA of 0.007, 0.14 or 0.028 AU. Increasing amounts of human albumin was added to samples with a NBA of 0.01 AU and NOA 0.09 AU. Precision was also evaluated. Results At an initial NBA = 0.007 AU, increasing NOA to 0.025 and 0.094 AU decreased NBA by –29% and –77%, respectively. At an initial NBA = 0.014 and 0.028 AU, a significant decrease occurred at NOA = 0.269 AU (–36%) and NOA = 0.253 (–26%), respectively. Increasing TP to 1400 μg/L decreased NBA by – 0.003AU (–30%). Precision was satisfactory. Conclusion Oxyhaemoglobin significantly interferes with a NBA close to the decision point at a level lower than suggested by the guidelines. Samples with a higher NBA are more resistant to oxyhaemoglobin interference. TP up to 1400 μg/L would only significantly interfere if NBA was close to the decision point. These interferences are not seen with direct bilirubin measurement. Re-evaluation of the guidelines may be indicated. Diagnosis of subarachnoid haemorrhage (SAH) may involve the spectrophotometric analysis of cerebrospinal fluid for net bilirubin absorbance (NBA) and net oxyhaemoglobin absorbance (NOA). NBA>0.007 AU is supportive of a diagnosis of SAH. A limitation is that oxyhaemoglobin and total protein (TP) can interfere with NBA measurement such that according to published guidelines, results may be deemed inconclusive if NOA>0.1 AU or TP>1000 μg/L. The aim of this study was to assess the interference of oxyhaemoglobin and TP on NBA measurement. Increasing amounts of haemolysate was added to samples with a NBA of 0.007, 0.14 or 0.028 AU. Increasing amounts of human albumin was added to samples with a NBA of 0.01 AU and NOA 0.09 AU. Precision was also evaluated. At an initial NBA = 0.007 AU, increasing NOA to 0.025 and 0.094 AU decreased NBA by –29% and –77%, respectively. At an initial NBA = 0.014 and 0.028 AU, a significant decrease occurred at NOA = 0.269 AU (–36%) and NOA = 0.253 (–26%), respectively. Increasing TP to 1400 μg/L decreased NBA by – 0.003AU (–30%). Precision was satisfactory. Oxyhaemoglobin significantly interferes with a NBA close to the decision point at a level lower than suggested by the guidelines. Samples with a higher NBA are more resistant to oxyhaemoglobin interference. TP up to 1400 μg/L would only significantly interfere if NBA was close to the decision point. These interferences are not seen with direct bilirubin measurement. Re-evaluation of the guidelines may be indicated.

Haemolysis and turbidity influence on three analysis methods of quantitative determination of total and conjugated bilirubin on ADVIA 1650

Plasma bilirubin testing is crucial to prevent the occurrence of neonatal kernicterus. Haemolysis may occur during sampling and interfere with bilirubin determination. Moreover, lipidic infusions may induce plasma lipemia and also interfere with bilirubin measurement. We evaluated the interference of haemolysis and lipemia with three methods of total and direct bilirubin measurement adaptated on an Advia 1650 analyser (Siemens Medical Solutions Diagnostics) : Synermed (Sofibel), Bilirubin 2 (Siemens) and Bilirubin Auto FS (Diasys). The measurement of total bilirubin was little affected by haemolysis with all three methods. The Bilirubin 2 (Siemens) method was the less sensitive to haemolysis even at low bilirubin levels. The measurement of conjugated bilirubin was significantly altered by low heamoglobin concentrations for Bilirubin Auto FS(R) (30 microM or 0,192 g/100 mL haemoglobin) and for Synermed (60 microM or 0,484 g/100 mL haemoglobin). In marked contrast, we found no haemoglobin interference with the Direct Bilirubin 2 reagent which complied with the method validation criteria from the French Society for Biological Chemistry. The lipemia up to 2 g/L of Ivelip did not affect neither the measurement of total bilirubin for all three methods nor the measurement of conjugated bilirubin with the Diasys and Siemens reagents. However, we observed a strong interference starting at 0,5 g/L of Ivelip with the Synermed reagent. Our data suggest that both Siemens and Diasys methods allow to measure accurately total and conjugated bilirubin in hemolytic and lipemic samples, nevertheless, the Siemens methodology is less affected by these interferences.

Artifactual hyperbilirubinemia due to paraprotein interference.

Paraprotein interference in automated chemistry is uncommon. We describe 2 patients with paraproteinemia and elevated total bilirubin levels measured erroneously using the Roche total bilirubin assay. To explain the mechanism of this artifactual hyperbilirubinemia and to determine its frequency in patients with monoclonal or increased immunoglobulins. The assay was performed manually using serum from 2 index patients and from control patients (without M proteins). Total bilirubin was also determined using another manufacturer's assay. A prospective study was then undertaken using serum from 100 consecutive patients with various monoclonal gammopathies and from 13 patients with polyclonal hypergammaglobulinemia and cryoglobulins. For all patients, serum immunoglobulin (Ig) G, IgA, IgM, total and direct bilirubin, creatinine, and a direct spectrophotometric assessment of icterus were measured. After the addition of assay reagents, a white precipitate formed in the reaction mixtures containing serum from the index patients, but not in other samples. This turbidity, rather than the expected color change to pink, increased the absorbance and falsely elevated the total bilirubin value. Serum from both index patients was anicteric, their direct bilirubin measurements were unaffected, and total bilirubin measured using an alternate assay was normal. Among the 113 patients studied, no additional spurious total bilirubin values were detected. Paraprotein interference with the Roche automated total bilirubin assay is caused by precipitate formation. This interference is rare and probably idiosyncratic. Spurious hyperbilirubinemia from paraprotein interference may cause clinical confusion. If artifactual elevation of total bilirubin is suspected, the laboratory should examine the specimen for icterus (manually or by spectrophotometry) or measure total bilirubin using a different method.

Effects of bilirubin and its photoisomers on direct bilirubin measurement using bilirubin oxidase

We examined the reactivity of human serum albumin-bound bilirubin and its photoisomers as substrates for a direct bilirubin assay using bilirubin oxidase. The reduction of (EZ)-cyclobilirubin reached 100% 5 min after addition of the enzyme at any pH tested (3·5–7·4) in 0·1 mol/L phosphate buffer, whereas the reduction of (ZE)-bilirubin or (ZZ)-bilirubin reached 100% only below pH 4·5 or 5·5, respectively. (ZZ)-Bilirubin and its photoisomers did not react in citrate-lactate buffer at pH 3·7. The circular dichroism spectrum of (ZZ)-bilirubin in this buffer did not show a positive Cotton effect. These results indicate that a three-dimensional structure surrounding the reaction site of bilirubin is important for the reactivity with bilirubin oxidase.

Effects of bilirubin and its photoisomers on direct bilirubin measurement using bilirubin oxidase.

We examined the reactivity of human serum albumin-bound bilirubin and its photoisomers as substrates for a direct bilirubin assay using bilirubin oxidase. The reduction of (EZ)-cyclobilirubin reached 100% 5 min after addition of the enzyme at any pH tested (3.5-7.4) in 0.1 mol/L phosphate buffer, whereas the reduction of (ZE)-bilirubin or (ZZ)-bilirubin reached 100% only below pH 4.5 or 5.5, respectively. (ZZ)-Bilirubin and its photoisomers did not react in citrate-lactate buffer at pH 3.7. The circular dichroism spectrum of (ZZ)-bilirubin in this buffer did not show a positive Cotton effect. These results indicate that a three-dimensional structure surrounding the reaction site of bilirubin is important for the reactivity with bilirubin oxidase.

Glucose-6-Phosphate Dehydrogenase Deficiency: A Worldwide Potential Cause of Severe Neonatal Hyperbilirubinemia

After completing this article, readers should be able to: 1. Explain methods of preventing the hyperbilirubinemia caused by glucose-6-phosphate dehydrogenase (G6PD) deficiency. 2. Compare and contrast the correlation between hemolysis and serum bilirubin levels in healthy infants and those who are G6PD-deficient. 3. Describe the role of deficient bilirubin conjugation in the pathogenesis of G6PD deficiency-associated neonatal hyperbilirubinemia. 4. Define the risk for neonatal hyperbilirubinemia in female G6PD-deficient heterozygotes, G6PD-deficient homozygotes, and hemizygote males. Glucose-6-phosphate dehydrogenase (G6PD) deficiency, a commonly occurring X-linked genetic enzyme defect, is notorious for its association with acute hemolytic crises occurring in response to a frequently identifiable trigger (favism). Another potentially devastating danger of this condition is severe neonatal hyperbilirubinemia with its accompanying bilirubin encephalopathy, kernicterus, and death. Far from being a condition limited to historical time epochs and developing countries, G6PD deficiency-associated kernicterus still is seen in modern times. Indeed, among 80 infants from 21 states in the United States documented in a pilot Kernicterus Registry between 1984 and 1998, 18 (22.5%) were reported to have G6PD deficiency. Furthermore, Maisels recently listed G6PD deficiency among the 10 factors that are associated most commonly with an increased risk of nonhemolytic jaundice. Because of the association with favism, G6PD-associated hyperbilirubinemia traditionally has been regarded as hemolytic in origin. However, recent research has shown that although acute hemolysis does play a role, its contribution in many cases may be smaller than previously thought. The emphasis now has been placed on decreased bilirubin conjugation, with promoter polymorphism for the gene for the bilirubin conjugating enzyme, UDP glucuronoslytransferase 1A1 (UGT1A1), being a major factor in production of the icterus. In this review we highlight key aspects of the pathogenesis of this type of hyperbilirubinemia, explain the relevance of the condition, and describe a particularly problematic group that has a recently recognized high incidence of …

Determination of the Sum of Bilirubin Sugar Conjugates in Plasma by Bilirubin Oxidase

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. 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. 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. 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.

Direct Bilirubin Measurements in Jaundiced Term Newborns

To investigate the usefulness of measuring direct bilirubin in jaundiced term newborns, we reviewed the outcome of 5255 such measurements on 2877 term (37 weeks' gestation) newborns in two hospitals. Direct bilirubin tests were ordered 15 times as often per infant at the University of California, San Francisco, as at Stanford (Calif) University, and the reported results were more than twice as high. In most of the 149 infants with high (greater than 95th percentile) direct bilirubin levels, the high levels remained unexplained (52% of cases) or were due to apparent laboratory errors (21% of cases). Forty infants (27%) had conditions sometimes associated with high direct bilirubin levels. Elevation of direct bilirubin levels contributed to the diagnosis in only four of these infants. All had minor laboratory abnormalities that resolved spontaneously. Because of their low yield and poor specificity, direct bilirubin tests are seldom helpful in evaluating jaundice in term newborns.

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.

Measuring bile-salt concentrations lacks clinical value for detecting hepatic dysfunction in infants receiving parenteral nutrition.

Concentrations of conjugated cholate, chenodeoxycholate, direct bilirubin, and alanine aminotransferase (ALT, EC 2.6.1.2) were measured in plasma of 122 low-birthweight infants receiving parenteral nutrition. Eighteen (15%) of them developed hepatic dysfunction. We observed two distinct biochemical patterns in these infants. In the Type A pattern (12 infants), concentrations of direct-reading bilirubin and bile salts increased with no change in ALT activity. In the Type B pattern (six infants), increases in the concentrations of bile salt and direct bilirubin were followed by increases in ALT activity. Hepatic dysfunction persisted significantly longer in infants who developed the Type B pattern. The two patterns did not differ significantly in the times at which values for bile salts or direct bilirubin in plasma became abnormal or became normal at resolution, nor did maximal concentrations of bile salts in plasma differ significantly. Maximal concentrations of direct bilirubin were higher in the Type B infants. We conclude that, in such infants, measurement of bile-salt concentrations in plasma offers no advantages for detecting hepatic dysfunction over the more conventional measurement of direct bilirubin in plasma.