BilR is a gut microbial enzyme that reduces bilirubin to urobilinogen.

Metabolism of haem by-products such as bilirubin by humans and their gut microbiota is essential to human health, as excess serum bilirubin can cause jaundice and even neurological damage. The bacterial enzymes that reduce bilirubin to urobilinogen, a key step in this pathway, have remained unidentified. Here we used biochemical analyses and comparative genomics to identify BilR as a gut-microbiota-derived bilirubin reductase that reduces bilirubin to urobilinogen. We delineated the BilR sequences from similar reductases through the identification of key residues critical for bilirubin reduction and found that BilR is predominantly encoded by Firmicutes species. Analysis of human gut metagenomes revealed that BilR is nearly ubiquitous in healthy adults, but prevalence is decreased in neonates and individuals with inflammatory bowel disease. This discovery sheds light on the role of the gut microbiome in bilirubin metabolism and highlights the significance of the gut-liver axis in maintaining bilirubin homeostasis.

The Combined Escherichia coli Nissle 1917 and Tryptophan Treatment Modulates Immune and Metabolome Responses to Human Rotavirus Infection in a Human Infant Fecal Microbiota-Transplanted Malnourished Gnotobiotic Pig Model.

Human rotavirus (HRV) is a major cause of childhood diarrhea in developing countries where widespread malnutrition contributes to the decreased oral vaccine efficacy and increased prevalence of other enteric infections, which are major concerns for global health. Neonatal gnotobiotic (Gn) piglets closely resemble human infants in their anatomy, physiology, and outbred status, providing a unique model to investigate malnutrition, supplementations, and HRV infection. To understand the molecular signatures associated with immune enhancement and reduced diarrheal severity by Escherichia coli Nissle 1917 (EcN) and tryptophan (TRP), immunological responses and global nontargeted metabolomics and lipidomics approaches were investigated on the plasma and fecal contents of malnourished pigs transplanted with human infant fecal microbiota and infected with virulent (Vir) HRV. Overall, EcN + TRP combined (rather than individual supplement action) promoted greater and balanced immunoregulatory/immunostimulatory responses associated with greater protection against HRV infection and disease in malnourished humanized piglets. Moreover, EcN + TRP treatment upregulated the production of several metabolites with immunoregulatory/immunostimulatory properties: amino acids (N-acetylserotonin, methylacetoacetyl-CoA), lipids (gamma-butyrobetaine, eicosanoids, cholesterol-sulfate, sphinganine/phytosphingosine, leukotriene), organic compound (biliverdin), benzenoids (gentisic acid, aminobenzoic acid), and nucleotides (hypoxathine/inosine/xanthine, cytidine-5'-monophosphate). Additionally, the levels of several proinflammatory metabolites of organic compounds (adenosylhomocysteine, phenylacetylglycine, urobilinogen/coproporphyrinogen) and amino acid (phenylalanine) were reduced following EcN + TRP treatment. These results suggest that the EcN + TRP effects on reducing HRV diarrhea in neonatal Gn pigs were at least in part due to altered metabolites, those involved in lipid, amino acid, benzenoids, organic compounds, and nucleotide metabolism. Identification of these important mechanisms of EcN/TRP prevention of HRV diarrhea provides novel targets for therapeutics development. IMPORTANCE Human rotavirus (HRV) is the most common cause of viral gastroenteritis in children, especially in developing countries, where the efficacy of oral HRV vaccines is reduced. Escherichia coli Nissle 1917 (EcN) is used to treat enteric infections and ulcerative colitis while tryptophan (TRP) is a biomarker of malnutrition, and its supplementation can alleviate intestinal inflammation and normalize intestinal microbiota in malnourished hosts. Supplementation of EcN + TRP to malnourished humanized gnotobiotic piglets enhanced immune responses and resulted in greater protection against HRV infection and diarrhea. Moreover, EcN + TRP supplementation increased the levels of immunoregulatory/immunostimulatory metabolites while decreasing the production of proinflammatory metabolites in plasma and fecal samples. Profiling of immunoregulatory and proinflammatory biomarkers associated with HRV perturbations will aid in the identification of treatments against HRV and other enteric diseases in malnourished children.

Hemolysis during and after 21 days of head-down-tilt bed rest.

Hemoconcentration is observed in bed rest studies, descent from altitude, and exposure to microgravity. Hemoconcentration triggers erythrocyte losses to subsequently normalize erythrocyte concentration. The mechanisms of erythrocyte loss may involve enhanced hemolysis, but has never been measured directly in bed rest studies. Steady-state hemolysis was evaluated by measuring two heme degradation products, endogenous carbon monoxide concentration [CO] and urobilinogen in feces, in 10 healthy men, before, during, and after two campaigns of 21 days of 6° head-down-tilt (HDT) bed rest. The subjects were hemoconcentrated at 10 and 21 days of bed rest: mean concentrations of hemoglobin (15.0 ± 0.2 g/L and 14.6 ± 0.1 g/L, respectively) and erythrocytes (5.18 ± 0.06E6/µL and 5.02 ± 0.06E6/µL, respectively) were increased compared to baseline (all Ps < 0.05). In contrast, mean hemoglobin mass (743 ± 19 g) and number of erythrocytes (2.56 ± 0.07E13) were decreased at 21 days of bed rest (both Ps < 0.05). Indicators of hemolysis mean [CO] (1660 ± 49 ppb and 1624 ± 48 ppb, respectively) and fecal urobilinogen concentration (180 ± 23 mg/day and 199 ± 22 mg/day, respectively) were unchanged at 10 and 21 days of bed rest compared to baseline (both Ps > 0.05). A significant decrease in [CO] (-505 ppb) was measured at day 28 after bed rest. HDT bed rest caused hemoconcentration in parallel with lower hemoglobin mass. Circulating indicators of hemolysis remained unchanged throughout bed rest supporting that enhanced hemolysis did not contribute significantly to erythrocyte loss during the hemoconcentration of bed rest. At day 28 after bed rest, decreased hemolysis accompanied the recovery of erythrocytes, a novel finding.

Biochemical intestinal parameters in germ-free minipigs and rats and in ex-germ-free minipigs and rats monoassociated with Escherichia coli.

Intestinal contents of newborn and young germ-free minipigs and germ-free rats were investigated for the following biochemical parameters - conversion of cholesterol to coprostanol, degradation of beta-aspartylglycine, level of tryptic activity, formation of urobilinogen and the profile of short-chain fatty acids. Additionally, germ-free minipigs and germ-free rats were monoassociated with non-pathogenic strains of Escherichia coli and were investigated for the same biochemical parameters. The conversion of cholesterol to coprostanol, degradation of beta-aspartylglycine, tryptic activity and the short-chain fatty acid profile were similar to those found in previous studies in germ-free animals. Slightly higher amounts of urobilinogen than in the other species investigated so far were found in samples from germ-free and monoassociated minipigs. Except for the total amount of short-chain fatty acids in rats, monoassociation with E. coli did not alter any of the parameters either in the minipigs or in the rats.

Isolation and characterization of a urobilinogenoidic chlorophyll catabolite from Hordeum vulgare L.

A new type of chlorophyll catabolite was isolated from extracts of de-greened primary leaves of barley (Hordeum vulgare cv. Lambic). Its constitution was elucidated by one-dimensional and two-dimensional [(1)H,(13)C]-homo- and heteronuclear NMR spectroscopic techniques and by high resolution mass spectroscopy. The isolated catabolite, a water-soluble, colorless, and nonfluorescent linear tetrapyrrole, resembles urobilinogen in which one of the propionic side chains forms a five membered isocylic ring system, indicating its origin from the chlorophylls.

Influence of bacitracin on microbial functions in the gastrointestinal tract of horses.

This study investigated the influence of zinc bacitracin on the intestinal flora of horses. The functionally active intestinal flora was examined in 6 horses during treatment with zinc bacitracin. Utilising gas chromatography, spectrophotometry, gel electrophoresis and paper chromatography, samples were analysed on biochemical markers reflecting the action of parts of the intestinal flora. The following 5 flora-related functions were studied in faecal samples and intestinal samples from different sections of the hindgut: conversion of cholesterol to coprostanol and of bilirubin to urobilinogens, degradation of mucin and of beta-aspartylglycine and inactivation of tryptic activity. Conversion to coprostanol, conversion to urobilinogens and degradation of mucin were affected by treatment of zinc bacitracin and conversion to coprostanol was most sensitive. All functions were normalised in a short time, in contrast to man and rats. Differences in environmental exposures are probably the reason for a more rapid normalisation of the intestinal flora functions in horses.

Ursodeoxycholic acid and cholesterol induce enterohepatic cycling of bilirubin in rodents.

BACKGROUND & AIMS: Oral administration of ursodeoxycholic acid (UDCA) and cholesterol causes bile salt malabsorption; the former by competition for and the latter by down-regulation of ileal bile acid transporters. Because ileectomy in rats induces enterohepatic cycling of bilirubin, the hypothesis that dietary steroids might have the same effect was tested. METHODS: Male inbred C57L/J mice and Sprague-Dawley rats were fed low doses of UDCA, chenodeoxycholic acid (CDCA), or cholesterol added to laboratory chow with simultaneous chow-fed controls. After 1 week (mice) or 2 weeks (rats), indices of bile salt malabsorption and enterohepatic cycling of bilirubin were measured, including bilirubin secretion rates into bile, serum and intestinal bilirubin and bile salt levels, and urobilinogen levels in cecum, large intestine, and feces. RESULTS: Dietary UDCA and cholesterol, but not CDCA, significantly increased bilirubin secretion rates into bile. In UDCA-fed mice, gallbladder biles contained increased levels of bilirubin conjugates and unconjugated bilirubin, and in 60%, granules of amorphous calcium bilirubinate precipitated. Dietary cholesterol and bile acids, particularly UDCA, increased cecal bile salt levels, unconjugated bilirubin and urobilinogen concentrations, and decreased fecal bilirubin outputs, consistent with colonic absorption. CONCLUSIONS: By causing bile salt malabsorption, dietary UDCA and cholesterol induce enterohepatic cycling of bilirubin.

Reconstitution of the holoenzyme form of Escherichia coli porphobilinogen deaminase from apoenzyme with porphobilinogen and preuroporphyrinogen: a study using circular dichroism spectroscopy.

Porphobilinogen deaminase (PBG-D), an early enzyme of the tetrapyrrole biosynthetic pathway, catalyzes the formation of a tetrapyrrole chain, preuroporphyrinogen, from four molecules of porphobilinogen (PBG). The PBG-D apoenzyme is responsible for the autocatalytic synthesis and covalent attachment of a dipyrromethane cofactor at its active site. In this paper an efficient method for the purification of Escherichia coli PBG-D apoenzyme using an affinity chromatography resin is reported. Circular dichroism (CD) spectra of apoenzyme and holoenzyme were recorded and significant differences in both the backbone and aromatic region of the spectra were observed. The differences in the spectra allowed the reconstitution of holoenzyme from purified apoenzyme with PBG and preuroporphyrinogen in solution to be monitored separately by CD. Apoenzyme incubated with preuroporhyrinogen gave a CD spectrum that was much more like the CD spectrum of holoenzyme than apoenzyme incubated with PBG. The results showed clearly that the cofactor was generated much more rapidly from preuroporphyrinogen than from PBG. Changes in the CD spectrum associated with the aromatic side-chain region, in particular the contribution assigned to phenylalanine-62, were found to correlate well with the activity of the reconstituted enzyme. Phenylalanine-62 is located in close proximity to the cofactor and acts as a sensitive probe to active-site changes. The stability of the holoenzyme and apoenzyme were compared with respect to both heat and susceptibility to proteolysis. The results were consistent with a model for the apoenzyme in which, in the absence of the cofactor, the three domains of the protein are held less rigidly together, thereby making the protein more susceptible to heat denaturation and proteolysis. The CD spectrum of the holoenzyme was found to be similar at both pH 5.1 and 7.4, suggesting that the crystal structure, determined at pH 5.1, is likely to be similar at physiological pH values.

Fasting-related hyperbilirubinemia in rats: the effect of decreased intestinal motility.

BACKGROUND & AIMS: Fasting increases serum bilirubin levels in both humans and rats. Because the pathogenesis of fasting hyperbilirubinemia is not fully understood, the effect of fasting on disposition of bile pigments was investigated in rats. METHODS: Bilirubin and urobilinogen were determined in excreta, bile, plasma, and liver tissues of fasted Gunn and Wistar rats. RESULTS: Fasting increased the intestinal transit time of Wistar rats. As a result, the fecal output of bile pigments was decreased by food deprivation. In contrast, the intestinal content of total bile pigments was augmented in both Wistar and Gunn rats. This finding was paralleled by the increase of serum bilirubin concentration in both rat strains. A similar increment of serum bilirubin levels was observed after injection of bilirubin into the cecum of Wistar rats. Furthermore, biliary efflux of bilirubin in Wistar rats was increased after 48 hours of fasting. Intubation of nonabsorbable bulk to fasted Wistar rats prevented the increase of serum bilirubin levels during a 48-hour period of food deprivation. CONCLUSIONS: Fasting decreases intestinal motility and elimination of bile pigments. Accumulation of bilirubin in the intestine during fasting allows enhanced enterohepatic circulation and results in an increased reflux to plasma. This seems to be a major factor involved in fasting-induced hyperbilirubinemia.

Urobilinogen-i is a major derivative of bilirubin in bile of homozygous Gunn rats.

Gunn rats lack bilirubin UDP-glycosyltransferases, but diazo-negative derivatives of bilirubin have been described in their bile. In order to investigate this alternative disposal of bilirubin, crude bile samples from Gunn and Wistar rats were directly analysed by h.p.l.c. Besides bilirubin (in Gunn rats) or its glycosides (in Wistar rats), two major compounds were detected. A yellow one corresponded to the previously documented vitamin B-2 and was equally prominent in Gunn rats or Wistar-rat bile. The other compound was colourless, but on standing in contact with air it was spontaneously oxidized to a pinkish-yellow pigment. It was far more prominent in Gunn-rat bile. Analysis of bile obtained after intravenous injection of [14C]bilirubin to Gunn rats demonstrated that this compound was highly labelled. Freezing and thawing of the bile resulted in the formation of a series of diazo-negative derivatives, demonstrating that the original compound was quite labile. Spectral (adsorption and fluorescent) and chromatographic (h.p.l.c., t.l.c. and paper chromatography) analysis of the oxidized form of the labelled compound allowed its identification as urobilin-i. The colourless compound secreted in bile was urobilinogen-i. Administration of neomycin and bacitracin to Gunn rats or gut resection suppressed the biliary excretion of urobilinogen and thus confirmed its intestinal origin. Urobilinogen seems thus to represent the major bilirubin derivative present in Gunn-rat bile. Its breakdown products might represent the so-far-unidentified diazo-negative polar bilirubin derivatives. Since only a small amount of bilirubin is present in Gunn-rat bile, the urobilinogen formed in the intestinal lumen seems to be derived from bilirubin reaching the gut via routes other than the biliary one.

Establishment of a biochemically active intestinal ecosystem in ex-germfree rats.

A time course study for the establishment of some biochemical microbial intestinal functions was undertaken in ex-germfree rats conventionalized, i.e., colonized with conventional flora, in three different ways: untreated (group 1); contact with visitor rats (group 2); inoculated with intestinal contents from conventional rats (group 3). The first two groups of rats were inoculated with the intestinal contents from conventional rats after being out of the germfree isolators for 4 weeks. The biochemical parameters studied were degradation of mucin, inactivation of tryptic activity, conversion of cholesterol to coprostanol and of bilirubin to urobilinogen, degradation of beta-aspartylglycine, and formation of short-chain fatty acids. The results showed that the way in which the microbes were introduced and the microbial biochemical functions themselves were of importance. In several cases, social contacts, i.e., contact with visitor rats, were just as effective for the functionally adequate establishment of microbial intestinal functions as was inoculation with intestinal contents from conventional rats. Some of the biochemical parameters studied were established after a few days, whereas the establishment of others was markedly delayed. When inoculated after 4 weeks, all rats in the first two groups were colonized with conventional flora within 1 week. The results indicate that the model system described is suitable when studying buildup mechanisms in intestinal ecosystem(s).

Urinalysis.

When properly performed and interpreted, urinalysis is one of the most useful tests available to the emergency physician. This article reviews procedures for urine collection and analysis, emphasizing inexpensive and noninvasive chemical, macroscopic, and microscopic techniques.

Some aspects of bile acid and urobilinogen excretion and fecal elimination in men given a rural Guatemalan diet and egg formulas with and without added oat bran.

Six healthy men were fed a formula diet with and without oat bran and a natural food diet typical of rural Guatemala. No significant difference in dye transit time was found between diets but the Guatemalan diet significantly decreased dye retention time and increased stool frequency. Serum cholesterol and triglyceride levels showed no significant differences among dietary treatments. Excretion of fecal bile acids significantly increased on the Guatemalan and oat bran diets, but fecal bile acid concentration was significantly lower only on the Guatemalan diet. Urinary urobilinogen excretion and fecal urobilinogen concentration were significantly lower with the Guatemalan diet.

New approaches to the pathogenesis of peptic ulcer based on the protective action of saliva with special reference to roughage, vegetable fibre and fermented milk products.

Epidemiologic studies have shown differences in the prevalence of peptic ulcer disease among various communities to be related to variation in diet and eating patterns. Results of studies of the effect of diet and pattern of eating on saliva, on gastric juice, and on the amount of bile are reviewed. It is suggested that bile, and not hydrochloric acid, plays the causative role in pathogenesis of peptic ulceration. The role of saliva in the prevention of peptic ulcer is emphasized. The salivary mucus swallowed with food is protective because it decreases the flow rate of bile which, when held in the gall bladder longer, loses its alkalinity and therefore its ability to damage the mucous cells. Roughage, cellulose and vegetable fibres and fermented milk products act in a similar manner. When food is well masticated, containing plenty of vegetable fibres and fermented milk products such as ghee and yoghurt, resulting in an increase in the amount of salivary mucus, peptic ulceration may be prevented and cured and relapses may be prevented. This does not require a big change in the pattern of diet but only a change in the manner of eating so that hasty eating is avoided and good is chewed well. The pattern of diet, especially the relative dietary preponderance of short-chain fatty acids, such as those present in milk, yoghurt and other fermented milk products, have a protective action; the short-chain fatty acids retard gall bladder contraction and thus diminish the amount of bile entering the duodenal lumen.

Bovine protoporphyria: the first nonhuman model of this hereditary photosensitizing disease.

Protoporphyria, a photosensitizing disease documented only in humans, was transmitted as a recessive trait to seven female calves. Cutaneous lesions were extensive, and erythrocyte and fecal protoporphyrin concentrations exceeded by far those of human protoporphyria. Average ferrochelatase activity was decreased to one-half of normal in the liver of carriers, and to about one-tenth of normal in liver, kidney, heart, spleen, lung, and marrow of protoporphyrics.