In phase 1 reactions, xenobiotics are converted to more polar, hydroxylated derivatives. In phase 2 reactions, these derivatives are conjugated with molecules such as glucuronic acid, sulfate, or glutathione. This renders them even more water-soluble, facilitating their eventual excretion in the urine or bile.
Glucuronidation Is the Most Frequent Conjugation Reaction
The glucuronidation of bilirubin is discussed in Chapter 31; xenobiotics are glucuronidated in the same way, using UDP glucuronic acid along with a variety of glucuronosyltransferases, present in both the endoplasmic reticulum and cytosol. Molecules such as 2-acetylaminofluorene (a carcinogen), aniline, benzoic acid, meprobamate (a tranquilizer), phenol, and many steroid hormones are excreted as glucuronides. The glucuronide may be attached to oxygen, nitrogen, or sulfur groups of the substrates.
Some Alcohols, Arylamines, and Phenols Are Sulfated
The sulfate donor in these and other biologic and sulfation reactions (eg, sulfation of steroids, glycosaminoglycans, glycolipids, and glycoproteins) is “active sulfate”—adenosine 3′-phosphate-5′-phosphosulfate (PAPS).
Glutathione Is Required for Conjugation of Electrophilic Compounds
The tripeptide glutathione (γ-glutamylcysteinylglycine) is important in the phase II metabolism of electrophilic com pounds, forming glutathione S-conjugates that are excreted in urine and bile. The reaction catalyzed by glutathione S-transferases is:
R + GSH →R−S−G
where R is an electrophilic compound.
There are four classes of cytosolic glutathione S-transferase and two classes of membrane-bound microsomal enzymes, as well as a structurally distinct kappa class that is found in mitochondria and peroxisomes. Glutathione S-transferases exist as homo- or heterodimers constructed from at least seven different types of subunit, and different subunits are induced by different xenobiotics.
Because glutathione S-transferases also bind a number of ligands that are not substrates, including bilirubin, steroid hormones, and some carcinogens and their metabolites, they are sometimes known as ligandin. Glutathione S-transferase binds bilirubin at a site distinct from the catalytic site. The complex transits from the bloodstream to the liver, then to the endoplasmic reticulum for conjugation of bilirubin with glucuronic acid, and excretion in the bile (see Chapter 31). Binding of carcinogens sequesters them, so preventing their actions on DNA.
The liver exhibits a very high level of glutathione S-transferase activity; in vitro the entire pool of glutathione can be depleted within minutes on exposure to a xenobiotic substrate. The activity of glutathione S-transferase is upregulated in many tumors, leading to resistance to chemotherapy.
Glutathione conjugates may be transported out of the liver, where they are substrates for extracellular γ-glutamyl transpeptidase and some dipeptidases. The resultant cysteine S-conjugates are taken up by other tissues (especially the kidney) and N-acetylated to yield mercapturic acids (N-acetyl cysteine S-conjugates), which are excreted in the urine. Some hepatic glutathione S-conjugates enter the bile canniculi, where they are broken down to cysteine S-conjugates that are then taken up into the liver for N-acetylation, and re-excreted in the bile.
In addition to its role in phase 2 metabolism, glutathione has a number of other roles in metabolism:
1. It provides the reductant for the reduction of hydrogen peroxide to water in the reaction catalyzed by glutathione peroxidase.
2. It is an important intracellular reductant and antioxidant, helping to maintain essential SH groups of enzymes in their reduced state.
3. A metabolic cycle involving GSH as a carrier has been implicated in the transport of some amino acids across membranes in the kidney. The first reaction of the cycle is catalyzed by γ-glutamyltransferase (GGT):
Amino acid + GSH → γ-Glutamyl amino acid + Cysteinylglycine
This reaction transfers amino acids across the plasma membrane as dipeptides, which are subsequently hydrolyzed, releasing the amino acid and glutamate and the GSH being resynthesized from cysteinylglycine. GGT is present in the plasma membrane of renal tubular cells and bile ductule cells, and in the endoplasmic reticulum of hepatocytes. It is released into the blood from hepatic cells in various hepatobiliary dis eases, providing an early indication of liver damage.
