Intestinal flora and bilirubin
Authors: Claudio Tiribelli, J. Donald Ostrow
Journal: Journal of Hepatology (2004)

In an adult human weighing 70 kg, the bilirubin conjugates formed in the liver and secreted into the bile are normally derived from two sources: 260–300 mg of unconjugated bilirubin (UCB) produced from catabolism of hemes, of which 80–85% comes from destruction of senescent erythrocytes in the spleen, and a relatively small amount of UCB recirculating from the intestine [[1]Berk P.D. Noyer C.M. Bilirubin metabolism and the hereditary hyperbilirubinemias.Semin Liver Dis. 1994; 14: 321-394Google Scholar]. The bile pigments secreted into the intestine are eventually eliminated almost exclusively in the feces, mainly as metabolites of UCB [1Berk P.D. Noyer C.M. Bilirubin metabolism and the hereditary hyperbilirubinemias.Semin Liver Dis. 1994; 14: 321-394Google Scholar, 2Billing B.H. Intestinal and renal metabolism of bilirubin including enterohepatic circulation.in: Ostrow J.D. Bile pigments and jaundice; molecular, metabolic and medical aspects. Marcel Dekker, New York1986: 255-269Google Scholar]. The secreted bilirubin conjugates are deconjugated in the intestine by β-glucuronidases derived from bacteria and enterocytes [3Saxerholt H. Skar V. Midtvedt T. HPLC separation and quantification of bilirubin and its glucuronide conjugates in faeces and intestinal contents of germ-free rats.Scand J Clin Lab Invest. 1990; 50: 487-495Google Scholar, 4Saxerholt H. Carlstedt-Duke B. Hoverstad T. Lingaas E. Norin K.E. Steinbakk M. et al.Influence of antibiotics on the faecal excretion of bile pigments in healthy subjects.Scand J Gastroenterol. 1986; 21: 991-996Google Scholar]. The limited hydrolytic activity in the small intestine comes primarily from coliforms in older infants and adults and enterocytes in neonates [[2]Billing B.H. Intestinal and renal metabolism of bilirubin including enterohepatic circulation.in: Ostrow J.D. Bile pigments and jaundice; molecular, metabolic and medical aspects. Marcel Dekker, New York1986: 255-269Google Scholar]. Most of the deconjugation occurs in the distal ileum and colon where anaerobic Clostridia sp. and Bacteroides fragilis not only hydrolyze the bilirubin conjugates but also reduce (hydrogenate) the resultant UCB to colorless urobilinoids [5Troxler R.F. Dawber N.H. Lester R. Synthesis of urobilinogen by broken cell preparations of intestinal bacteria.Gastroenterology. 1968; 54: 568-574Google Scholar, 6Fahmy K. Gray C.H. Nicholson D.C. The reduction of bile pigments by faecal and intestinal bacteria.Biochim Biophys Acta. 1972; 264: 85-97Google Scholar]. It is probable that anaerobic bacteria can synthesize urobilinoids only from UCB, so that deconjugation is an obligatory preceding step [5Troxler R.F. Dawber N.H. Lester R. Synthesis of urobilinogen by broken cell preparations of intestinal bacteria.Gastroenterology. 1968; 54: 568-574Google Scholar, 6Fahmy K. Gray C.H. Nicholson D.C. The reduction of bile pigments by faecal and intestinal bacteria.Biochim Biophys Acta. 1972; 264: 85-97Google Scholar, 7Gustafsson B.E. Lanke L.S. Bilirubin and urobilins in germfree, ex-germfree and conventional rats.J Exp Med. 1960; 112: 975-980Google Scholar]. Since measured urobilinoids in the stool average 120–150 mg daily, it has been assumed that the remaining half of the bile pigments secreted with the bile are reabsorbed by the intestine, mainly at the level of the colon. The reabsorbed UCB and urobilinoids return via the portal blood to the liver, where they are in part extracted by the hepatocytes and re-excreted into bile, the enterohepatic circulation (EHC) of bile pigments [[2]Billing B.H. Intestinal and renal metabolism of bilirubin including enterohepatic circulation.in: Ostrow J.D. Bile pigments and jaundice; molecular, metabolic and medical aspects. Marcel Dekker, New York1986: 255-269Google Scholar]. About 5–10 mg/day of the reabsorbed urobilinoids are excreted by the kidneys into the urine, a route not available to UCB [[2]Billing B.H. Intestinal and renal metabolism of bilirubin including enterohepatic circulation.in: Ostrow J.D. Bile pigments and jaundice; molecular, metabolic and medical aspects. Marcel Dekker, New York1986: 255-269Google Scholar]. The crucial role of intestinal flora in the intestinal catabolism of UCB has been established by studies of germ-free rats, whose fecal bile pigments consist almost entirely of conjugated bilirubin with no detectable urobilinoids [7Gustafsson B.E. Lanke L.S. Bilirubin and urobilins in germfree, ex-germfree and conventional rats.J Exp Med. 1960; 112: 975-980Google Scholar, 8Vítek L. Kotal P. Jirsa M. Malina J. Cerna M. Chmelar D. et al.Intestinal colonization leading to fecal urobilinoid excretion may play a role in the pathogenesis of neonatal jaundice.J Pediatr Gastroenterol Nutr. 2000; 30: 294-298Google Scholar, 9Saxerholt H. Skar V. Midtvedt T. HPLC separation and quantification of bilirubin and its glucuronide conjugates in faeces and intestinal contents of germ-free rats.Scand J Clin Lab Invest. 1990; 50: 487-495Google Scholar]; recolonization of the gut with exogenous bacteria restores a more normal pattern of fecal bile pigments. Among the more than 400 bacterial strains present in the gut of a healthy adult man [[10]Holdeman L.V. Good I.J. Moore W.E. Human fecal flora: variation in bacterial composition within individuals and a possible effect of emotional stress.Appl Environ Microbiol. 1976; 31: 359-375Crossref Google Scholar], only 4 strains so far have been found capable of deconjugating bilirubin and reducing the UCB to urobilinoids: Clostridiun ramosum [[7]Gustafsson B.E. Lanke L.S. Bilirubin and urobilins in germfree, ex-germfree and conventional rats.J Exp Med. 1960; 112: 975-980Google Scholar], Clostridium perfringens and difficile [[8]Vítek L. Kotal P. Jirsa M. Malina J. Cerna M. Chmelar D. et al.Intestinal colonization leading to fecal urobilinoid excretion may play a role in the pathogenesis of neonatal jaundice.J Pediatr Gastroenterol Nutr. 2000; 30: 294-298Google Scholar], and Bacteriodes fragilis [[6]Fahmy K. Gray C.H. Nicholson D.C. The reduction of bile pigments by faecal and intestinal bacteria.Biochim Biophys Acta. 1972; 264: 85-97Google Scholar]. Escherichia coli, which is a dominant organism in the more proximal small bowel shows a β-glucuronidase activity more that 30 times lower than C. perfringens [[11]Leung J.W. Liu Y.L. Leung P.S. Chan R.C. Inciardi J.F. Cheng A.F. Expression of bacterial beta-glucuronidase in human bile: an in vitro study.Gastrointest Endosc. 2001; 54: 346-350Google Scholar]. Eradication of relevant Clostridia sp. would increase the unmetabolized UCB in the gut, enhancing the EHC of bilirubin. By contrast, eradicating E. coli should not increase the EHC of bilirubin because bilirubin conjugates are minimally absorbed [12Lester R. Schmid R. Intestinal absorption of bile pigments. I. The enterohepatic circulation of bilirubin in the rat.J Clin Invest. 1963; 42: 736-746Google Scholar, 13Lester R. Schmid R. Intestinal absorption of bile pigments. II. Bilirubin absorption in man.N Engl J Med. 1963; 269: 178-182Google Scholar], and are efficiently converted to UCB and urobilinogens by colonic anerobes [5Troxler R.F. Dawber N.H. Lester R. Synthesis of urobilinogen by broken cell preparations of intestinal bacteria.Gastroenterology. 1968; 54: 568-574Google Scholar, 6Fahmy K. Gray C.H. Nicholson D.C. The reduction of bile pigments by faecal and intestinal bacteria.Biochim Biophys Acta. 1972; 264: 85-97Google Scholar]. Since the fractional uptake of UCB by the liver is only 30% of the load, any increase in the formation or EHC of UCB must engender a proportional increase in plasma bilirubin levels. The unconjugated hyperbilirubinemia resulting from increased bilirubin production (e.g. due to hemolysis) has been long appreciated clinically [[14]Berk P.D. Martin J.F. Blaschke T.F. Scharschmidt B.F. Plotz P.H. Unconjugated hyperbilirubinemia; physiologic evaluation and experimental approaches to therapy.Ann Intern Med. 1975; 82: 552-570Google Scholar], whereas the role of an increased EHC of bilirubin has been disregarded in recent years, even though its potential importance in neonatal jaundice was suggested decades ago [15Brodersen R. Hermann L.S. Intestinal reabsorption of unconjugated bilirubin. A possible contributing factor to neonatal jaundice.Lancet. 1963; : 1242-1243Google Scholar, 16Poland R.L. Odell G.B. Physiologic jaundice: the enterohepatic circulation of bilirubin.N Engl J Med. 1971; 284: 1-6Google Scholar]. The paper by Vitek et al. in this issue of the Journal [[17]Vítek L. Zelenka J. Zadinová M. Malina J. The impact of intestinal microflora on serum bilirubin levels.J Hepatol. 2005; 42: 238-243Abstract Full Text Full Text PDF Scopus (44) Google Scholar] re-emphasizes the importance of EHC in the overall balance of bilirubin metabolism. Four days of treatment of congenitally hyperbilirubinemic Gunn rats with clindamicin/neomycin, which are non-absorbable antibiotics against the anaerobic intestinal flora that can convert UCB to urobilinogens, resulted in virtual disappearance of fecal urobilinogens and a consistent increase in plasma bilirubin levels. By contrast, administration of co-trimethoxazole, to which C. perfringens is insensitive, did not affect either fecal urobilinoid or serum bilirubin levels. Oral administration of C. perfringens to the clindamycin/neomycin-treated rats resulted in partial restoration of fecal urobilinoids and decrease in serum bilirubin toward control values. Since the intestinal bacterial content was not quantitatively assessed, it cannot be determined whether the partial response indicated incomplete recolonization, or that factors other than intestinal flora are involved in the changes in serum bilirubin level and fecal bilirubin elimination. Recolonization with C. pasteurianum, a bacterium reported by the authors to be unable to reduce UCB to urobilinoids, neither reversed the antibiotic-induced increase in plasma bilirubin nor restored fecal excretion of urobilinoids. Similar observations, on the effects on intestinal bile pigment metabolism of the eradication of gut flora with antibiotics and their restoration by recolonization, have been reported previously in rats [[7]Gustafsson B.E. Lanke L.S. Bilirubin and urobilins in germfree, ex-germfree and conventional rats.J Exp Med. 1960; 112: 975-980Google Scholar] and humans [[18]Saxerholt H. Carlstedt-Duke B. Hoverstad T. Lingaas E. Norin K.E. Steinbakk M. et al.Influence of antibiotics on the faecal excretion of bile pigments in healthy subjects.Scand J Gastroenterol. 1986; 21: 991-996Google Scholar]. There are a few quantitative aspects in the study by Vitek et al. that require comment. In the steady state, the fecal output of bile pigments should balance the rate of bilirubin production [[19]Bloomer J.R. Berk P.D. Howe R.B. Waggoner J.G. Berlin N.I. Comparison of fecal urobilinogen excretion with bilirubin production in normal volunteers and patients with increased bilirubin production.Clin Chim Acta. 1970; 29: 463-471Google Scholar], yet the measured fecal output of urobilinoids+UCB was less than 25% of the expected bilirubin production rate in Gunn rats [[20]Cohen A.N. Kapitulnik J. Ostrow J.D. Zenone E.A. Cochrane C. Celic L. et al.Effects of phenobarbital on bilirubin metabolism and its response to phototherapy in the jaundiced Gunn rat.Hepatology. 1985; 5: 310-316Google Scholar]. In humans, turnover studies with 14C-UCB have similarly found that fecal excretion of urobilinoids+UCB is only about half the production rate [[21]Bloomer J.R. Berk P.D. Howe R.B. Waggoner J.G. Berlin N.I. Comparison of fecal urobilinogen excretion with bilirubin production in normal volunteers and patients with increased bilirubin production.Clin Chim Acta. 1970; 29: 463-471Google Scholar]. That study showed that an average of 92% of the 14C-bilirubin turnover was recovered in the feces, and concluded that the difference had to be accounted for by other catabolites of UCB, for example a variety of dipyrroles formed by oxidation of UCB, some of which are colored and account for the brown color of stool [[2]Billing B.H. Intestinal and renal metabolism of bilirubin including enterohepatic circulation.in: Ostrow J.D. Bile pigments and jaundice; molecular, metabolic and medical aspects. Marcel Dekker, New York1986: 255-269Google Scholar]. Thus, one cannot calculate the amount of bilirubin+urobilinoids absorbed from the intestine by simply subtracting the chemically measured fecal output of these bile pigments from total bilirubin production. Vitek et al. calculated the variations in bilirubin absorption on different regimens from the change in the fecal bile pigment pool, and averred that this matched the change in the plasma pool of UCB. However, this indirect calculation ignores the extravascular pool of bilirubin, which is almost equal to the plasma pool in the Gunn rat. Unmentioned also were the absorption of UCB [12Lester R. Schmid R. Intestinal absorption of bile pigments. I. The enterohepatic circulation of bilirubin in the rat.J Clin Invest. 1963; 42: 736-746Google Scholar, 13Lester R. Schmid R. Intestinal absorption of bile pigments. II. Bilirubin absorption in man.N Engl J Med. 1963; 269: 178-182Google Scholar] and urobilinogens [[22]Lester R. Schmid R. Intestinal absorption of bile pigments. III. The enterohepatic circulation of urobilinogen in the rat.J Clin Invest. 1965; 44: 722-730Google Scholar], and the hydrolysis of bilirubin conjugates that would occur in the small intestine of normal rats [[2]Billing B.H. Intestinal and renal metabolism of bilirubin including enterohepatic circulation.in: Ostrow J.D. Bile pigments and jaundice; molecular, metabolic and medical aspects. Marcel Dekker, New York1986: 255-269Google Scholar]. Despite these methodological and interpretative problems, the data of Vitek et al. clearly show, for the first time that, in the Gunn rat, which is congenitally unable to conjugate UCB, the presence of bilirubin-reducing flora in the gut can play a significant role in minimizing the EHC of bilirubin and, consequently, the plasma bilirubin levels. There is potential clinical applicability to newborn infants, in whom the uptake, conjugation and biliary secretion of bilirubin is immature [[23]Gourley G.R. Bilirubin metabolism and kernicterus.Adv Pediatr. 1997; 44: 173-229Google Scholar], and there are few anaerobic bacterial flora to convert intestinal UCB to urobilinoids [[24]Midtvedt A.C. Carlstedt-Duke B. Norin K.E. Saxerholt H. Midtvedt T. Development of five metabolic activities associated with the intestinal microflora of healthy infants.J Pediatr Gastroenterol Nutr. 1988; 7: 559-567Google Scholar]. This results in a much higher EHC of UCB from the gut to the plasma, where the pigment accumulates due to the poorly-developed hepatic excretory processes [[23]Gourley G.R. Bilirubin metabolism and kernicterus.Adv Pediatr. 1997; 44: 173-229Google Scholar]. In healthy, term neonates, serum bilirubin levels usually peak below 170 μM at about one week of life and return to normal over the next 1–2 weeks. This neonatal 'physiological jaundice' may be aggravated by different causes, including hemolysis and an augmented EHC of UCB [[23]Gourley G.R. Bilirubin metabolism and kernicterus.Adv Pediatr. 1997; 44: 173-229Google Scholar]. Increases in serum UCB levels lead to greater entry of UCB into the central nervous system (CNS), which may cause neurotoxicity and bilirubin encephalopathy [[25]Ostrow J.D. Pascolo L. Brites D. Tiribelli C. Molecular basis of bilirubin-induced neurotoxicity.Trends Mol Med. 2004; 10: 65-70Google Scholar]. It is, therefore, important to keep the serum bilirubin level in newborns as low as possible. Currently, this is usually managed with phototherapy to convert UCB to photoisomers which can be excreted in bile without conjugation and/or with metalloporphyrin inhibitors of heme oxygenase to decrease UCB formation [[26]Ostrow J.D. Therapeutic amelioration of jaundice.Hepatology. 1988; 8: 683-689Google Scholar]. Past attempts to decrease the extensive EHC of UCB have involved oral administration of agents that either trap UCB in the intestinal lumen by adsorption to non-absorbable solids (e.g. agar [[27]Odell G.B. Gutcher G.R. Whitington P.F. Yang G. Enteral administration of agar as an adjunct to phototherapy of neonatal hyperbilirubinemia.Pediatr Res. 1983; 17: 810-814Google Scholar], activated charcoal [[28]Amitai Y. Regev M. Arad I. Peleg O. Boehnert M. Treatment of neonatal hyperbilirubinemia with repetitive oral activated charcoal as an adjunct to phototherapy.J Perinat Med. 1993; 21: 189-194Google Scholar] or cholestyramine [[29]Nicolopoulos D. Hadjigeorgiou E. Malamitsi A. Kalpoyannis N. Karli I. Papadakis D. Combined treatment of neonatal jaundice with cholestyramine and phototherapy.J Pediatr. 1978; 93: 684-688Google Scholar]), or form insoluble bilirubinate salts Ca++ [[30]van der Veere C.N. Schoemaker B. Bakker C. Van der M.R. Jansen P.L. Elferink R.P. Influence of dietary calcium phosphate on the disposition of bilirubin in rats with unconjugated hyperbilirubinemia.Hepatology. 1996; 24: 620-626Google Scholar] or Zn++ [[31]Mendez-Sanchez N. Roldan-Valadez E. Flores M.A. Cardenas-Vazquez R. Uribe M. Zinc salts precipitate unconjugated bilirubin in vitro and inhibit enterohepatic cycling of bilirubin in hamsters.Eur J Clin Invest. 2001; 31: 773-780Google Scholar]. All these agents have been effective in animal models of unconjugated hyperbilirubinemia, especially if combined with phototherapy. However, the non-absorbable agents have given disappointing and variable results in humans, and there is scanty data available on the clinical efficacy or toxicity of Ca++ or Zn++ salts in human neonates. More recently, orlistat, a lipase inhibitor, that increases intestinal and fecal fat content, has been shown to enhance fecal bilirubin excretion and decrease plasma bilirubin levels in Gunn rats [[32]Nishioka T. Hafkamp A.M. Havinga R. vn Lierop P.P. Velvis H. Verkade H.J. Orlistat treatment increases fecal bilirubin excretion and decreases plasma bilirubin concentrations in hyperbilirubinemic Gunn rats.J Pediatr. 2003; 143: 327-334Google Scholar]; its efficacy and safety in human neonates has not yet been reported. The approach reported in the paper of Vitek et al. opens the possibility to modulate the intestinal flora of the newborns to decrease the EHC of UCB by introducing bacteria that can reduce UCB to urobilinoids that are eliminated with the feces or non-toxic if absorbed. In summary, the data of Vitek et al. point to a role of intestinal reabsorption of unconjugated bilirubin (formed by the deconjugation of pigment by the intestinal bacteria and enterocytes) in overall bilirubin balance, especially in Gunn rats and probably in neonates with severely impaired conjugation of UCB. It is expected that the greater the deconjugation by bacteria, the higher the reabsorption and bilirubin concentration in the circulation, particularly in the portal venous blood. It remains to be seen whether, as postulated by Vitek et al., this phenomenon would significantly influence serum bilirubin levels in adults with less severe impairment of hepatic removal of the unconjugated bilirubin recycling from the gut. Caution is advised in extrapolating this approach to human subjects, especially to newborns, until possible problems, related to colonization of the intestine by exogenous anerobic bacteria, have been fully clarified. This work was supported by grants from the Italian Ministry of University and Research (FIST), Fondazione Cassa di Risparmio di Trieste (FCRT 01/02) and Fondo Studi Fegato-ONLUS.