Donaby Henton

Cholestatic Effect of Intraperitoneal Administration of Tryptophan to Suckling Rat Pups

Summary: The potential cholestatic effect of amino acids and metabolites of tryptophan were evaluated by use of seven daily intraperitoneal injections to suckling and weanling rat pups. Of the amino acids present in parenteral nutrition solutions, only tryptophan (given at a dose of 4 mM/kg) produced a significant (p < 0.01) elevation of serum cholylglycine (12.8 ± 1.0 μM/liter) as determined by radioimmunoassay, compared to 4.9 ± 0.4 μM/liter in salinetreated control animals. Total serum conjugates of cholic acid, as determined by radioimmunoassay, were similarly elevated, as was serum alanine aminotransferase. Tryptophan injection resulted in elevated cholylglycine concentrations only at doses of 3 mM/kg/day or higher. Animals more than 2 weeks old did not demonstrate elevation of serum cholylglycine. Injection of light-exposed tryptophan in suckling animals caused a greater elevation of cholylglycine (39.0 ± 8.6 μM/liter) than freshly prepared tryptophan solutions (p < 0.005). Tryptophan and its spontaneous degradation products could contribute to the cholestatic liver changes observed during parenteral nutrition therapy.

Correlation of carbon monoxide and bilirubin production by tissue homogenates

The production of carbon monoxide (CO) has been used as an index of bilirubin production in animals and man [l]. The validity of this concept is based on the characteristics of the heme degradation pathway. Heme oxygenase (EC 1.14.99.3), with the consumption of O2 and reduced nicotinamide-adenine dinucleotide phosphate (NADPH ), oxidizes heme into equimolar amounts of CO and biliverdin [ 21. The latter is reduced directly to bilirubin with the aid of biliverdin reductase and the consumption of a second molecule of NADPH [ 31. The heme oxygenase reaction is reported to be the rate-limiting step when the tissue preparations are not extensively purified [4]. Due to improved analytical techniques, we are now able to determine in vitro simultaneously the CO produced by the heme oxygenase reaction and bilirubin, the end-product of the heme degradation pathway. CO was determined in the reaction vessel by headspace gas chromatography (CC) [5,61, and bilirubin was extracted from the reaction mixture with chloroform and quantified by high-performance liquid chromatography (HPLC ) [71.

A subacute rabbit model for hepatobiliary dysfunction during total parenteral nutrition.

Total parenteral nutrition (TPN) in children is associated with the complicating syndromes of cholestasis and cholelithiasis. The causes of these syndromes are not completely clear. Gastrointestinal hypomotility associated with enteral fasting may be involved in the pathogenesis of both syndromes. We compared weanling rabbits maintained solely on TPN with chow pair-fed and free-fed controls over a 10-day period. Gastrointestinal transit time, assessed with a solid marker technique, was significantly greater in the TPN-treated animals. No difference in intestinal or biliary bacterial flora was demonstrated by aerobic or anaerobic cultures. Gallbladder bile contained a higher percentage of lithocholic acid, unconjugated bilirubin, and total calcium in the TPN-treated animals. Markers of hepatic dysfunction were elevated in the serum of the TPN-treated animals. Mild steatosis and edema were the only histologic differences in the livers of the TPN-treated animals. We conclude that gastrointestinal hypomotility associated with enteral fasting plays a role in the pathophysiologic changes leading to TPN-associated hepatobiliary dysfunction. This dysfunction may be mediated by an increase in the absolute and relative concentrations of lithocholic acid in the bile of TPN-treated animals.

The BUN/creatinine ratio in localizing gastrointestinal bleeding in pediatric patients

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.

Distribution of serum bilirubin conjugates in pediatric hepatobiliary diseases

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.

The use of fecal alkaline phosphatase as an indicator of intestinal damage.

Fecal alkaline phosphatase excretion was evaluated as a marker for intestinal damage in rats. Animals received either intraperitoneal bleomycin or saline. Controls were pair-fed with animals in the bleomycin group throughout the study. Both groups demonstrated similar patterns of fecal alkaline phosphatase excretion. There was, however, marked daily variability of fecal enzymatic activity. Fecal alkaline phosphatase excretion was largely composed of intestinal alkaline phosphatase, which was characterized by enzymatic inhibition with L-phenylalanine. Dietary intake as well as daily fecal output and protein excretion were reduced immediately following bleomycin injections and gradually increased to baseline values by 7 days. It appeared that both the direct toxic effects of bleomycin and dietary intake influenced fecal alkaline phosphatase excretion. Routine clinical application of this assay may be limited because of the number of factors which may affect its excretion.

Vitamin E measurement in patients receiving intravenous lipid emulsions.

Two methods for the determination of plasma vitamin E--high-pressure liquid chromatography and spectrophotofluorometry--were compared on samples from four groups of pediatric patients: children and infants receiving lipid emulsion as part of their parenteral nutrition regimen, neonates receiving parenteral nutrition who were not receiving lipid emulsion at the time of blood sampling, and short admission surgery control subjects. In control subjects and patients not receiving lipid emulsions, both methods yielded similar results for vitamin E as alpha-tocopherol. In contrast, in patients receiving lipid emulsion, the fluorometric method yielded values ranging from 200% to 300% greater than did high-pressure liquid chromatography. The source of the discrepancy is most probably the presence of naturally occurring non-alpha-tocopherol isomers in the lipid products, which add to the fluorescent measurement but are resolved by high-pressure liquid chromatography. This study confirms clinically that fluorescent measurement of vitamin E is no longer the method of choice for monitoring tocopherol status in intensive care nurseries.

The Effect of Tin-Protoporphyrin on Bilirubin Conjugation and Production in Cholestatic Rats

ABSTRACT: 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 click-static by surgical bile duct ligation. Male Sprague-Dawley rats received SnP (100 μmol/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 bilirubin-protein 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.

SERUM BILIRUBIN FRACTIONS IN CHOLESTATIC RATS TREATED WITH TIN PROTOPORPHYRIN

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.