Every day an adult produces about 250–400 mg of bilirubin, most of which (>80%) comes from the catabolism of hemoglobin and a small amount from the hepatic catabolism of other hemoproteins (proteins containing the heme group). In particular, the bilirubin produced by the catabolism of hemoglobin can be divided into a hemo-cateretic portion (70%), which comes from the physiological destruction of red blood cells in the reticuloendothelial system of the spleen, and an erythropoietic portion (30%) from ineffective erythropoiesis in the bone marrow (catabolism of heme molecules not used in erythropoiesis and destruction of erythroblasts, reticulocytes, and newly formed red blood cells).

Bilirubin is formed by the sequential catalytic degradation of heme mediated by two enzymes, heme oxygenase and biliverdin reductase. Heme oxygenase initiates the opening of the porphyrin ring of heme, leading to the formation of the green pigment, known as biliverdin, which, subsequently, will be reduced by biliverdin reductase to a yellow-orange pigment, namely the bilirubin (Fig. 1).

Fig1. Bilirubin metabolism. (Copyright EDISES 2021. Reproduced with permission)

The bilirubin synthesized, called indirect or unconjugated, is poorly soluble in water at physiological pH and, being liposoluble, is responsible for the toxic effects of bilirubin. This form of bilirubin circulates in the plasma bound to proteins, mainly albumin and, to a much lesser extent, to high-density lipoproteins; the binding to lipoproteins becomes significant in severe hypoalbuminemia. Binding to albumin keeps bilirubin in the vascular space, thus preventing its deposition in extra-hepatic tissues, including the brain, and minimizing its filtration at the glomerular level. Albumin is also vital in transporting indirect bilirubin to the sinusoidal surface of hepatocytes, where the pigment dissociates from albumin and enters the hepatocytes by facilitated diffusion. Defects in genes encoding for bilirubin transporters may result in hyperbilirubinemia. The passage of bilirubin across the membrane of the sinusoidal surface of hepatocytes is bidirectional. In order to prevent the escape of bilirubin from hepatocytes, bilirubin, as well as other organic anions, is bound to the cytosolic protein glutathione S-transferase (GST), also known as ligandin, which mediates its transport to the endoplasmic reticulum (ER). In the ER, indirect bilirubin undergoes a process of glucuronidation that converts it to a water-soluble form, the direct bilirubin (Fig. 1). The glucuronidation of bilirubin, as well as of various compounds, both endogenous (steroid hormones, thyroid hormones, and catecholamines) and exogenous (drugs, toxins, carcinogens), is mediated by a family of enzymes known as uridine diphosphate glucuronosyltransferases (UGT). The resulting glucuronides are water-soluble and, therefore, readily excreted in bile and urine. Glucuronidation is one of the most important detoxification mechanisms in our body. Among isoforms of the UGT family, only UGT1A1 is physiologically crucial in bilirubin metabolism. Following glucuronidation, mono- and di- glucuronide bilirubin molecules are formed and directed toward the apical canalicular membrane and then transported into the bile canaliculus. Conjugated bilirubin, also called direct bilirubin, is secreted by active transport against a con centration gradient mediated by an ATP-dependent pump, known as MRP-2 (Multidrug Resistance Protein) or ABC- C2, expressed on the membrane of the biliary canaliculi of hepatocytes. Most of the bilirubin (approximately 80%) excreted in bile is present in the form of diglucuronide. In subjects with reduced glucuronidation activity, the bilirubin diglucuronide rate decreases, and the bilirubin monoglucuronide rate increases. Inhibitors of hepatic UGT1A1 may be present in breast milk, leading to neonatal jaundice, or in maternal plasma and cross the placental barrier, reaching the fetus (Lucey Discroll syndrome). A UGT1A1 deficiency can be found in newborns, in patients with chronic hepatitis, and in patients with some hereditary disorders, such as Gilbert’s syndrome or Crigler–Najjar syndrome. Biliary excretion of conjugated bilirubin may be impaired in several acquired conditions, such as viral or alcoholic hepatitis and cholestasis in pregnancy, or congenital conditions, such as Dubin Johnson syndrome and Rotor syndrome.

Conjugated bilirubin, produced at the hepatic level, after a brief stay in the gallbladder, reaches through the bile in the small intestine where, being water-soluble, it is not absorbed through the membrane of the epithelial cells. In the intestine, beta-glucuronidase enzymes mediate a process inverse to the hepatic one, removing glucuronic acid from bilirubin. Bilirubin is further metabolized by enzymes produced by the bacterial flora that mediate its reduction to urobilinogen (Fig. 1). A small fraction of urobilinogen is reabsorbed and returns to the circulation to be subsequently picked up in the liver or eliminated in urine, where urobilinogen under goes further oxidation to urobilin. Urobilin is the molecule that gives urine its characteristic golden-yellow or straw- yellow color. Most of the urobilinogen (80%) in the colon is further processed by the bacterial flora into stercobilinogen and eliminated in the feces, where it undergoes further oxidation to stercobilin. Stercobilin gives the characteristic color to the feces.