This study compares total and direct-reacting bilirubin values in 40 serum samples from patients with various diagnoses, as measured by automated methods (Beckman Synchron CX-5, Beckman Astra 8, Kodak Ektachem 700) and HPLC and by a manual method for delta bilirubin. For total bilirubin, within-run CVs were less than 6%. The Ektachem 700 method underestimated bilirubin with serum samples from patients with Crigler-Najjar syndrome and from newborns in whom unconjugated bilirubin concentrations were increased but conjugated bilirubins were not present or were present only in small amounts. The Astra 8 and Synchron CX-5 methods were inaccurate with cholestatic serum samples, in which conjugated bilirubin concentrations were increased and other compounds such as bile acids could be expected to interfere. We conclude that each automated method examined provides reasonable estimates for total and direct-reacting bilirubin values for routine clinical use. The need for each laboratory to select the appropriate bilirubin method for its particular situation is obvious.

Localizing the bleeding site in pediatric patients with gastrointestinal hemorrhage may require invasive and costly diagnostic procedures. A simple index to discriminate upper and lower bleeding sources would be invaluable. We evaluated the reliability of the calculated blood urea nitrogen/creatinine (BUN/Cr) ratio in segregating upper from lower gastrointestinal bleeding sites in 40 children. For upper gastrointestinal hemorrhage, the calculated BUN/Cr ratios (mg/mg) ranged from 10 to 140, with a mean value of 34. For lower gastrointestinal bleeders, the BUN/Cr ratios ranged from 3.3 to 30, with a mean value of 16. All BUN/Cr ratios greater than 30 corresponded to patients with documented upper gastrointestinal bleeding sources. Calculation of the BUN/Cr ratio in the initial evaluation of gastrointestinal bleeding may prove useful in guiding the sequence of diagnostic procedures and examinations.

Tin-protoporphyrin (SnP) is actively being investigated for treatment of exaggerated neonatal hyperbilirubinemia. Because both bilirubin conjugation and excretion are immature in the human newborn, we investigated the effect of SnP on bilirubin-conjugating mechanisms and the efficacy of SnP in suppressing serum bilirubin levels in adult rats made cholestatic by surgical bile duct ligation. Male Sprague-Dawley rats received SnP (100 mumol/kg body weight) subcutaneously either 24 h before or 24 or 48 h after bile duct ligation. Serum and urine specimens were collected 72 h after bile duct ligation and analyzed for bilirubin and its conjugates. As compared to a control group that received bile duct ligation and a sodium phosphate buffer injection, all SnP-treated animals had a significant lowering of total serum bilirubin levels. No differences in the distribution of serum bilirubin mono- and diconjugates in serum or urine samples were observed. However, the concentrations of covalently linked bilirubinprotein conjugates were significantly higher in the control cholestatic rats when compared to the SnP-treated animals. SnP effectively lowers serum bilirubin levels in rats with an impaired biliary excretory pathway for SnP. There was no adverse effect on bilirubin conjugation and no observable toxicity.

The ubiquitous gas, carbon monoxide (CO), is of substantial biological importance, but apart from its affinity for reduced transition metals, particularly heme-iron, it is surprisingly nonreactive—as is the ferrous-carbonyl—in living systems. CO does form strong complexes with heme proteins for which molecular O2 is the preferred ligand and to which are attributed diverse physiological, adaptive, and toxic effects. Lately, it has become apparent that both exogenous and endogenous CO produced by heme oxygenase engender a prooxidant milieu in aerobic mammalian cells which initiates signaling related to reactive oxygen species (ROS) generation. ROS signaling contingent on CO can be segregated by CO concentration-time effects on cellular function, by the location of heme proteins, e.g., mitochondrial or nonmitochondrial sites, or by specific oxidation-reduction (redox) reactions. The fundamental responses to CO involve overt physiological regulatory events, such as activation of redox-sensitive transcription factors or stress-activated kinases, which institute compensatory expression of antioxidant enzymes and other adaptations to oxidative stress. In contrast, responses originating from highly elevated or protracted CO exposures tend to be nonspecific, produce untoward biological oxidations, and interfere with homeostasis. This brief overview provides a conceptual framework for understanding CO biology in terms of this physiological-pathological hierarchy.

Tin protoporphyrin (SnP), an inhibitor of heme oxygenase, has been propossd for the treatment of neonatal hyperbilirubinemia. The neonate is physiologically chcOestatic, thus normal excretion of SnP and bilirubin (BR) may be impaired. To investigate the effects of SnP on serum BR fractions during cholestasis, we administered a single dose of SnP (100 μmoles/g) to rats rendered cholestatic by bile duct legation. Four groups of rats were studied, 1 control group with vehicle injection at time of ligation, and 3 experimental groups, with varying time of SnP injections. Ligation of all rats was at T=0, and sacrifice at T=72 hrs. BR fractions were measured by HPLC. Covalent bound bUirubin protein conjugates (BP) were measured by solvent precipitation. There was no difference in the percent of total BR contributed by unconjugated and conjugated BR. Conclusions; 1. SnP does not appear to interfere with bilirubin conjugation while suppressing total BR levels. 2. Cholestasis does not inhibit SnP action. 3. The hyperbilirubinemia of cholestasis can be modified by SnP, suggesting clinical relevance.

We used a highly specific method, alkaline methanolysis-high performance liquid chromatography, for determining the concentration and patterns of the unconjugated and esterified bilirubin fractions in the sera of pediatric patients with hepatobiliary disease. Bilirubin-protein conjugates were assayed using a new method that selectively removes bilirubin reversibly bound to protein, allowing measurement of the tightly bound bilirubin-protein conjugates by use of a diazo method. Fifty-two serum samples from children with varying bilirubin concentrations and diagnoses were studied. Whereas no conjugated pigment was detectable in the serum samples of healthy children or in individuals with Gilbert syndrome or Crigler-Najjar syndrome, bilirubin monoester and diester conjugates and bilirubin-protein conjugates were present in the sera of children with cholestatic liver disease, and accounted for 69% +/- 15% of the total bilirubin in these samples. Bilirubin fractional analysis was incapable of differentiating extrahepatic biliary obstruction from hepatocellular disease, because of overlap between the groups. The presence of bilirubin-protein conjugates in serum always coincided with detection of bilirubin monoester and diester conjugates. The distribution of bilirubin and its conjugates in sera provides a sensitive, although nonspecific, measure of hepatic disease.