Butz and Du Vigneaud at the University of Illinois discovered homocysteine about 70 years ago. By heating methionine in sulfuric acid these investigators isolated a homolog of cystine, bis-(γ-amino-γ-carboxypropyl) disulfide, that they called homocystine since it had the structure of the “next higher symmetrical homolog of cystine.”1 Since its original discovery, homocysteine has received increasing attention as a risk factor for atherosclerosis2 and recently, evidence is accumulating that it may be involved in the pathogenesis of alcoholic liver injury (reviewed by Ji and Kaplowitz.3) Homocysteine plays a pivotal role in the metabolism of the essential amino acid methionine, which is the precursor of S-adenosylmethionine (SAMe), the methyl donor in hundreds of methylation reactions including RNA, DNA, proteins, and lipids. Homocysteine is formed upon demethylation of SAMe and subsequent hydrolysis of S-adenosylhomocysteine (SAH) and lies at the junction of two intersecting pathways, the transsulfuration pathway — the pathway that converts the sulfur atom of methionine to cysteine and glutathione, as well as its ultimate removal as sulfate — and the remethylation pathway —the pathway that conserves homocysteine as methionine, and that is coupled to cobalamin, folate and betaine metabolism (Fig. 1) (reviewed by Finkelstein4). It has long been recognized that methionine metabolism is altered in patients with liver disease. In 1947 Kinsell et al. demonstrated marked impairment in methionine clearance following a methionine load in patients with liver injury, thereby establishing the central role of the liver in methionine metabolism.5 Later, Horowitz et al.6 provided evidence for impairment of the transsulfuration pathway in cirrhosis, and in 1988 we demonstrated that the marked impairment of methionine metabolism in patients with cirrhosis was due to a striking reduction in the rate of hepatic SAMe synthesis.7
Homocysteine belongs to a group of molecules known as cellular thiols. The most abundant cellular thiols are glutathione (the intracellular concentration of glutathione is about 1 to 10 mmol/L) and cysteine (plasma total cysteine ranges from 200 to 300 μmol/L). These two thiols are considered the “good thiols,” because their functions include maintaining intracellular and extracellular redox homeostasis, facilitate the removal of toxic compounds, and are also part of the cellular antioxidant defense system.8 Homocysteine is normally found at much lower concentrations than glutathione and cysteine. The concentration of homocysteine within the cell is around 1 μmol/L, and in plasma ranges from 5 to 15 μmol/L. Different from glutathione and cysteine, homocysteine is considered a “bad thiol” because of its association with a variety of health conditions including cardiovascular disease,2 end-stage renal disease,9 neural tube defects,10 and cognitive dysfunctions including Alzheimer disease.11 Recently, homocysteine has also been implicated in the pathogenesis of alcoholic liver injury.12
One of the most common mutations, or polymorphisms, that is associated with a mild increase in plasma homocysteine (hyperhomocysteinemia) is the 677C→T substitution (an alanine to valine change) in the enzyme methylenetetrahydrofolate reductase (MTHFR). Because MTHFR catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the primary circulating form of folate and the one-carbon donor for homocysteine remethylation to methionine (Fig. 1), a disruption of MTHFR activity is expected to increase homocysteine levels. The frequency of this variant in the homozygous state varies from 0% to1% in African Americans to 25% in Hispanic Americans, ranging for most populations (Canada and United States, Europe, Asia, and Australia) between 10% to15%.13 In this issue of HEPATOLOGY, Adinolfi et al. report that mild hyperhomocysteinemia and MTHFR 677C→T polymorphism is associated with steatosis and fibrosis in chronic hepatitis C (CHC) patients.14 These investigators have examined 116 CHC patients for histology activity index, fibrosis, steatosis grade, hyperhomocysteinemia, and MTHFR 677C→T polymorphism. As expected, hyperhomocysteinemia is associated with TT genotype of MTHFR, and plasma levels of homocysteine in CC, CT, and TT genotypes were 9.3, 12.2, and 18.6 μmol/L, respectively. Steatosis also correlated with MTHFR polymorphism and hyperhomocysteinemia. Thus, prevalence and high grade (>20%) of steatosis were 41% and 11% in CC genotype, 61% and 49% in CT genotype, and 79% and 64% in TT genotype, respectively. In the literature the association between mutant genotype and mild hyperhomocysteinemia has been observed predominantly in individuals with lower plasma folates15 and subjects with the TT genotype are quite responsive to exogenous folate administration with respect to homocysteine lowering.13 Thus, it would have been desirable to have data about the folate status of these patients with CHC. Nevertheless, the results of this study may have important implications for liver disease as it suggests that the MTHFR 677C→T polymorphism and/or hyperhomocysteinemia may facilitate the development of steatosis, which in its turn may accelerate the progression of liver fibrosis in CHC.
The results of Adinolfi et al. agree with earlier work showing that when rats and mice are fed a diet deficient in methyl groups (choline, methionine, folate, and vitamin B12) the liver develops steatosis within a few days.16 If the diet continues, the liver develops nonalcoholic steatohepatitis (NASH), fibrosis and cirrhosis, with some animals developing hepatocellular carcinoma (HCC). Moreover, numerous nutritional studies have shown that dietary methyl deficiency causes a decrease in the hepatic content of SAMe, an increase in the concentration of SAH and an elevation of plasma homocysteine levels. It has been demonstrated, for example, that mice deficient in hepatic SAMe synthesis have hepatic hyperplasia and spontaneously develop NASH and HCC;17, 18 and that mice lacking MTHFR develop severe steatosis and have elevated plasma homocysteine as well as increased hepatic content of SAH and reduced SAMe.19 Since steatosis can develop from decreased hepatic SAMe alone,17 it would have been insightful in the work of Adinolfi et al. to include hepatic levels of SAMe and SAH. Altogether, due to its pivotal role in methionine metabolism, plasma homocysteine may be a good indicator of the status of liver function, and the common 677C→T MTHFR polymorphism may be taken into consideration as a novel risk factor to develop steatosis. Consistent with these observations, we have recently identified that MTHFR gene expression is downregulated in patients with early stage NASH (Mato JM and Lu SC, unpublished).
Although a variety of epidemiological studies have linked mild hyperhomocysteinemia with a variety of diseases,2, 9–11 the molecular mechanisms involved are unclear. Three mechanisms have been proposed, namely oxidative stress, endoplasmic reticulum (ER) stress, and activation of proinflammatory factors.3 Hyperhomocysteinemia has been associated with oxidative stress, a hypothesis that is favored by Adinolfi et al. in their paper. Many in vitro studies have demonstrated the ability of homocysteine to enhance the production of oxygen free radicals and impair endothelial nitric oxide availability.3, 20 However, although the reduced thiol group of homocysteine is capable of undergoing the same oxidation and reduction reactions as glutathione and cysteine, it is unlikely to function as a major oxidant because its cellular concentration is too low. On the other hand, it has been shown to inhibit the expression of a wide range of antioxidant enzymes.3, 20 Thus, homocysteine may act to sensitize the cell to the cytotoxic effect of agents or conditions that generate oxidative stress. Homocysteine has been shown to induce ER stress in a variety of in vitro studies3 and hyperhomocysteinemia correlated with development of ER stress in an animal model of alcoholic liver injury.12 ER stress is a condition under which unfolded and misfolded proteins accumulate, which then triggers the unfolded protein response (UPR), resulting in the activation of a number of transcription factors, including the sterol regulatory element-binding proteins (SREBPs).3 Early on, these responses have the effect of increasing lipid synthesis needed for generation of more ER membranes and reducing protein load to the ER. However, prolonged UPR will lead to activation of apoptosis.3 Homocysteine is believed to cause ER stress by disrupting disulfide bond formation and causing misfolding of proteins.3 ER stress can then lead to fatty liver as SREBPs induce genes involved in the cholesterol/triglyceride biosynthesis and uptake pathways.21 Finally, homocysteine has been shown in in vitro studies to activate proinflammatory cytokines, possibly related to its ability to activate nuclear factor kappa B.3
There are, however, two other biochemical reactions specific of homocysteine that may be involved in homocysteine-mediated liver injury. The first is the synthesis of SAH, a potent inhibitor of methyltransferases22 in equilibrium with homocysteine (Fig. 1). Consequently, increased homocysteine may result in inhibition of multiple methylation reactions, a key component of many signaling pathways. The second reaction is the formation of homocysteine thiolactone (Fig. 1), a molecule first synthesized by Baernstein in 193423 that can react with primary amines of proteins such as lysine. Thiolactone formation, which is produced by an intramolecular condensation reaction between the thiol and the carboxylic acid of homocysteine, is unique to this amino acid (cysteine has only a single carbon atom within the side chain whereas homocysteine has two). Homocysteine thiolactone can react with lysine residues and free amine groups on numerous cellular proteins, leading to altered biological activity and premature degradation.24 All of these aforementioned mechanisms may be involved in mediating the adverse effect of hyperhomocysteinemia.
Although there are multiple potential mechanisms to explain the association of hyperhomocysteinemia and degree of steatosis, it is less clear how higher degree of steatosis accelerates the progression of liver fibrosis in CHC. Potential mechanisms proposed by Lonardo et al include enhanced sensitivity of the steatotic livers to oxidative stress, cytokines and steatosis-related hepatic insulin resistance.25 There is also the possibility of direct profibrogenic effect of homocysteine as it has been shown to induce α1(I) procollagen and tissue inhibitor of metalloproteinase-1 expression in a hepatic stellate cell line.26 Future work examining this possibility will be of interest. In brief, the work of Adinolfi et al. identified MTHFR polymorphism as a contributing factor in the progression of CHC and support the concept that strategies aimed at lowering the “bad thiols” homocysteine, SAH, and homocysteine thiolactone (i.e., by supplementation with betaine, folic acid, and SAMe) (Fig. 1) may reduce the progression of liver diseases including CHC.