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Serum bilirubin as a predictor of oxidative stress-mediated diseases

Bilirubin, the principle bile pigment, is the end product of heme catabolism. For many years, bilirubin was thought to have no physiological function other than that of a waste product of heme catabolism – useless at best and toxic at worst. Although hyperbilirubinemia in neonates has been shown to be neurotoxic, studies performed during the past decade have found that bilirubin has a number of new and interesting biochemical and biological properties. In addition, there is now a strong body of evidence suggesting that bilirubin may have a beneficial role in preventing oxidative changes in a number of diseases including atherosclerosis and cancer, as well as a number of inflammatory, autoimmune and degenerative diseases. 

The results also suggest that activation of the heme oxygenase and heme catabolic pathway may have beneficiary effects on disease prevention either through the action of bilirubin or in conjunction with bilirubin. If so, it may be possible to therapeutically induce heme oxygenase, increase bilirubin concentrations, and lower the risk of oxidative stress-related diseases. Serum bilirubin has been shown to be protective not only against cardiovascular and PVD and there is some evidence that it may be protective against some forms of cancer. A number of studies indicate that serum bilirubin may be associated with a lower prevalence of several other diseases and that higher serum bilirubin concentrations may lessen the harmful effects of a number of medical conditions. It was, for example, found that hyperbilirubinemic infants had a significantly lower incidence of neonatal complications including circulatory failure, neonatal depression/asphyxia, aspiration syndromes, and proven sepsis. In another study, a protective role of bilirubin in several oxygen radical diseases in preterm infants was demonstrated. In those studies, they found a lower incidence of oxygen radical-mediated injuries such as necrotizing enterocolitis, bronchopulmonary dysplasia, and intraventricular hemorrhage.

Several studies have shown an inverse relationship between serum bilirubin levels and retinopathy of prematurity. The inverse relationship between serum bilirubin and retinopathy in premature infants, however, was not found in several other studies. There is also some preliminary evidence to indicate that low serum bilirubin concentrations are inversely related to rheumatoid arthritis, systemic lupus erythematosus and Wegener granulomatosis. These results indicate that serum bilirubin may protect against rheumatologic oxidative stress-mediated diseases. A possible beneficial role of bilirubin has also been described in several interesting case reports. In one of the case reports, transient relief of asthma was found to occur possibly as a result of hepatitis B-induced hyperbilirubinemia. Likewise, a patient with exacerbation of idiopathic pulmonary fibrosis was resolved probably due to the coexisting hyperbilirubinemia elicited by biliary tract obstruction.

Low serum bilirubin concentrations may be associated also with mental illnesses. Oren and colleagues, for example, found that patients with winter depressions had lower nocturnal bilirubin concentrations compared to the controls and suggested that bilirubin may serve as an important chronobiological photoreceptor involved in human circadian rhythms. Low serum bilirubin concentrations have also been recently found to be related to an increased risk of amyotrophic lateral sclerosis. A similar inverse relationship between serum bilirubin levels and schizophrenia has also been found in our recent study on schizophrenic patients who have Gilbert’s genotype and phenotype. In our study, we found that the prevalence rates of homozygotes for two common GS polymorphisms including UGT1A1*28 were markedly lower in schizophrenic patients than in the controls. These results indicate that individuals with GS should have some protection against schizophrenia, whereas those with low bilirubin levels might be predisposed to the development of this psychotic disease. Recent data also suggest that an increased HO-1 expression is associated with protection against oxidative stress.

There are also many human studies demonstrating that a low expression of HO-1 gene due to its promoter polymorphism is associated with various pathologic conditions. The HO-1 gene promoter contains a (GT)n repeat responsible for HO-1 gene transcription that is highly polymorphic. Subjects with the large size of a (GT)n (n=30) in the HO-1 gene promoter categorized as a class L allele, for example, were shown to have 8 times higher transcriptional activity than those with the (GT)n=22. Indeed, the presence of the class L allele was shown to be associated with susceptibility to chronic pulmonary emphysema as well as longevity in the Japanese population, although the link to the decline of lung functions in smokers with class L allele was not confirmed in a Canadian study. Furthermore, Austrian investigators recently observed a low frequency of short (GT)n repeats (categorized as class S allele) in HO-1 gene promoter in patients with abdominal aortic aneurysm as compared to healthy controls [61]. In other studies, patients with short (GT)n repeats were show to have an attenuated inflammatory response to balloon injury and a reduced rate of restenosis after balloon angioplasty.

The same group also found a reduced risk for cerebrovascular events in individuals with normal plasma lipid levels and with short repeats in the HO-1 gene promoter. Similarly, patients with longer GT repeats on either allele had significantly more frequent angiographic restenoses. In a study by Endler et al., carriers of short allele in the promoter of HO-1 gene had higher bilirubin and high-density lipoprotein (HDL) levels, although differences in CHD prevalence were not detected in this study. The Austrian group also reported in their recent prospective study of almost 500 patients with advanced peripheral artery disease, that carriers of class S allele had more than twice reduced adjusted hazard ratio for coronary events as compared to non-carriers. Patients with hypercholesterolemia, diabetes or smoking and shorter GT repeats in HO-1 gene promoter were also less likely to have CHD than those with longer GT repeats. Similar results were described by Chen et al. in a group of Taiwanese patients with type 2 diabetes. Those patients with longer GT repeats in HO-1 gene promoter were more susceptible to developing CHD. The same authors also described in their prospective study of 289 patients with CHD an almost four fold increased risk of angiographic restenoses after coronary stenting and adverse cardiac events during follow-up in class L allele carriers.

Similar results were also described in a recent prospective study on 199 Austrian patients. Interestingly the link of class L allele carrier state to the increased serum levels of CRP as well as IL-6 was described recently providing further mechanism, by which increased HO-1 activity might protect against atherosclerotic diseases. In an interesting study by Baan and colleagues involving renal transplant patients, graft survival was found to be associated with donor HO-1 gene polymorphism. Patients who received a kidney from L class homozygotes lost their graft significantly more often to chronic allograft nephropathy than carriers with the S-alleles. In contrast, the risk for graft failure was reduced two-fold in kidneys with S-alleles. Better renal functions of class S allele renal allograft recipients were observed also in another study, although class S vs. L allele recipients did not differ significantly with respect to the incidence of allograft rejection. However, controversial finding was observed in another recent retrospective study on heart transplantation. In this study the frequency and severity of heart cardiac allograft vasculopathy was not different between class S allele recipients and non-recipients. Interestingly, different single nucleotide promoter polymorphisms in HO-1 gene promoter [T(-413)A] has been shown to affect the activity of HO-1. In fact, 8-fold higher HO-1 activity was described in A allele promoter variant as compared to T allele variant. AA genotype was found to be associated with decreased occurrence of CHD in Japanese study groups.

This genotype, however, was found to be associated with increased incidence of arterial hypertension in women with no clear explanation of this observation. While many studies have shown a relationship between HO-1 and cardiovascular disease, the role of HO-1 in cancerogenesis seems to be more uncertain. HO-1 has been shown to exert a potent antiproliferative effect on vascular smooth muscle cells and on kidney epithelial and pulmonary cells. According to one recent paper, the proportion of class L allele frequencies was found to be significantly higher in patients with lung adenocarcinoma than in the control subjects. In this regard, it is interesting to note the results of a Japanese study that found the HO-1 expression to be inversely associated with the grade and stage of oral squamous cell carcinoma as well as tongue squamous cell carcinoma. Similar results were found by Taiwanese investigators. In this study, they found the L-allele in HO-1 promoter to be associated with an increased risk of oral squamous cell carcinoma. On the other hand, HO-1 overexpression has been observed in various tumor cells including hepatoma, glioblastoma, melanoma, pancreatic, lung, renal, and prostate cell carcinoma and HO-1 inhibitors were suggested as potential novel antitumor agents. These studies indicate that the role of HO-1 in cancerogenesis is more complex and further studies of possible contributing molecular mechanisms are needed. Of interest with this respect is a recent study by Abraham et al. who demonstrated that HO-1 overexpression resulted in modulation of numerous genes associated with cell cycle progression. Several lines of evidence suggest that biopyrrins may be a general marker of oxidative stress.

Biopyrrin concentrations have been shown to correlate with levels of 8-hydroxydeoxyguanosine, an oxidative DNA damage marker, acrolein-lysine adducts, a marker of lipid peroxidation and with nitrites, which are markers of nitric oxide production. Recent studies have also found a negative association between serum bilirubin and serum pentosidine and carboxy-methyllysine. Such an association would be expected as pentosidine and carboxy-methyllysine belong to the group of advanced glycation-end products and are markers of protein oxidation and glycation. Bilirubin concentrations have been found to be elevated in extensively training adults and this increase is associated with a resistance to protein oxidation further corroborating the importance of bilirubin in the defense against oxidative stress. These data are consistent with the in vitro and in vivo bilirubin-related decreases in protein oxidation discussed above. It is remains to be determined if bilirubin is the major protective factor in the heme catabolic pathway, if it is a surrogate for other factors, or with it is working in concert with other protective factors. Since CO is a vasodilator, it is possible that bilirubin and CO may be providing protection through different mechanisms. Even though prospective studies show an inverse relationship between bilirubin and cardiovascular disease and cardiovascular mortality in men, further studies are needed to more firmly establish a causal link between HO and CO and the various diseases and metabolic conditions. The results of the retrospective and prospective studies indicate that low bilirubin concentrations, particularly those below 10 μmol/l, are associated with an increased prevalence of several diseases as well as disease mortality.

If these finding are confirmed in other studies, then bilirubin concentrations below 10 μmol/l or in the lowest quartile for men and women may be considered as “abnormal”. In the past, serum bilirubin concentrations in the normal range were indicative of health. The current evidence appears to suggest otherwise. With regard to diagnostics, serum bilirubin is equal to HDL cholesterol, smoking, and blood pressure. When combined with cholesterol, LDL-cholesterol, HDL-cholesterol, it improves the prediction of coronary artery disease over that achieved with LDL-cholesterol or cholesterol/HDL-cholesterol ratios alone. Combining serum bilirubin with hs-CRP and lipids might further improve the prediction of disease. Other diagnostic applications for serum bilirubin are possible and are currently being examined. A potentially profitable area of research is in finding ways to increase serum bilirubin concentrations in individuals with low or moderate serum bilirubin levels. Cigarette smoking is known to decrease serum bilirubin concentrations and smoking cessation should result in increased serum bilirubin concentrations. The effects of both antioxidants and oxidants in food, physical fitness, and aerobic capacity on HO activity, CO, and bilirubin need to be examined. Low serum bilirubin concentrations also could be due to the bodies over all oxidative state or to a low levels and activities of HO and BVR.

Certainly, drugs which induce HO and drugs which decrease or increase serum bilirubin levels will enhance our understanding of the major protective factors in the heme metabolic pathway. Further studies need to be performed to evaluate existing drugs as well as new drugs to determine if they can induce HO and raise serum bilirubin and CO concentrations. If such drugs are found, clinical studies need to be performed to see if the drugs can actually improve the health of individuals with low and moderate serum bilirubin concentrations. Aspirin and the statins are two drugs that need to be studied in more detail as they are safe and appear to have an effect on HO and on serum bilirubin. In addition, drugs which induce HO and bilirubin synthesis or those which inhibit bilirubin clearance are all viable and attractive therapeutic candidates.

Many of the existing longitudinal studies have collected data on serum bilirubin concentrations and can be used to further examine the importance of bilirubin and the heme metabolic pathway. Drugs which induce HO, increase bilirubin synthesis, or inhibit bilirubin clearance could result in moderate increases in serum bilirubin concentrations sufficient to offer protection against cardiovascular disease, certain forms of cancer, and other diseases and age-related conditions associated with chronic oxidative stress. While low serum bilirubin concentrations are clearly associated with the prevalence of many diseases, the protective effects of elevated serum bilirubin concentrations such as found in GS are less certain. The risk of CVD in women with the UGT1A1*28 mutation, for example, was not found to be lower in this group despite the moderately elevated serum bilirubin concentrations.

Some of the reasons for a lack of protection in individuals with UGT1A1*28 mutation and in women have been presented in this review. The weight of the evidence, however, indicates that bilirubin is cytoprotective, although a causal link between serum bilirubin and a specific disease has not been established. In this respect, both drug and genetic studies involving the heme catabolic pathway may provide such a link. The interaction of bilirubin with various regulatory receptors also needs to be examined in greater detail. Most of the information on serum bilirubin and disease have been derived from chronic studies. If bilirubin is an effective antioxidant and anti-inflammatory agent it should have a role in ameloriating the acute medical conditions. Lastly, the effects of the heme catabolic pathway in acute medical conditions need to be examined in more detail.

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