Bilirubin, the principal tetrapyrrole, bile pigment and catabolite of haem, is an emerging biomarker of disease resistance, which may be related to several recently documented biological functions. Initially believed to be toxic in infants, the perception of bilirubin has undergone a transformation: it is now considered to be a molecule that may promote health in adults. Data from the last decade demonstrate that mildly elevated serum bilirubin levels are strongly associated with reduced prevalence of chronic diseases, particularly cardiovascular diseases (CVDs), as well as CVD-related mortality and risk factors. Recent data also link bilirubin to other chronic diseases, including cancer and Type 2 diabetes mellitus, and to all-cause mortality. Therefore, there is evidence to suggest that bilirubin is a biomarker for reduced chronic disease prevalence and a predictor of all-cause mortality, which is of important clinical significance. In the present review, detailed information on the association between bilirubin and all-cause mortality, as well as the pathological conditions of CVD, cancer, diabetes and neurodegenerative diseases, is provided. The mechanistic background concerning how bilirubin and its metabolism may influence disease prevention and its clinical relevance is also discussed. Given that the search for novel biomarkers of these diseases, as well as for novel therapeutic modalities, is a key research objective for the near future, bilirubin represents a promising candidate, meeting the criteria of a biomarker, and should be considered more carefully in clinical practice as a molecule that might provide insights into disease resistance. Clearly, however, greater molecular insight is warranted to support and strengthen the conclusion that bilirubin can prevent disease, with future research directions also proposed.

INTRODUCTION

The formation, metabolism and excretion of bile pigments, including orange-coloured bilirubin and green biliverdin, have been comprehensively reported and discussed in the literature for some decades. These reports have led to important medical advancements, and their measurement is currently used to diagnose various hepatobiliary diseases and minimize the risk of infants experiencing central nervous system (CNS) toxicity. However, closer examination of bile pigments physiological importance has occurred recently, which is likely to provide advances within medical and clinical science in the short to medium term.

Unconjugated bilirubin (UCB) is neurotoxic at pathological concentrations that exceed the binding capacity of plasma albumin [13]. Therefore, UCB was initially and exclusively considered to be a potentially harmful waste product of haem catabolism [4]. However, a potential physiological role for bilirubin was proposed in 1987, which has formed the basis of a paradigm shift with regard to the importance of bilirubin in human biology [5]. Nowadays, bile pigments are also discussed in the context of health promotion and disease prevention (reviewed by Bulmer et al. [6]). Due to growing evidence of bile pigments' anti-mutagenic and antioxidant effects [5,713], and an increasingly evident anti-atherogenic action [6,1419], bilirubin is emerging as a ‘health-promoting’ molecule (in the absence of hepatic/haematological disease), and therefore its role as a novel biomarker for chronic disease prediction requires clearer definition. Mildly elevated circulating UCB levels could represent a promising target for prevention or reduction of the prevalence of coronary heart disease (CHD) [2024] and other oxidative-stress-mediated disorders, including Type 2 diabetes and cancer [2528]. Therefore, the role of bilirubin as a biological predictor in the risk assessment of chronic disorders, with increasing worldwide prevalence, is of great medical and economic importance, because heart diseases, stroke and cancer represent the main causes of mortality and burden of disease [29]. However, the underlying mechanisms of bilirubin's action remain vastly unknown and an understanding of the physiological importance of (moderately elevated) plasma bilirubin on a cellular/molecular level, with reference also to metabolic diseases and the ageing process, remains elusive. This emphasizes the significance and need to explore bilirubin further as a reliable novel biomarker in disease prediction.

The present review summarizes the existing knowledge on bilirubin and its relationship to the major chronic diseases, and specifically highlights whether bilirubin represents a biomarker or, more importantly, a predictive factor in the context of chronic disease prevention. In addition, this review identifies the current gaps in our understanding and topics in the bilirubin research field that need to be addressed in the next 5 years.

INSIGHTS INTO BILIRUBIN METABOLISM

Bile pigments are tetrapyrrolic dicarboxylic acids, derived from the porphyrin family of molecules. They colourfully reflect the enzymatic breakdown of haem, taking its course in the mononuclear phagocyte system [30]. Under the catalytic activity of a series of enzymes, bilirubin is produced in a multistep pathway (Figure 1).

Bile pigment metabolism–from haem to bilirubin

Figure 1
Bile pigment metabolism–from haem to bilirubin

Haemoglobin is cleaved to yield globin and haem (red). Haem is enzymatically converted to biliverdin (green) by liberating iron, via oxidation of its α-methene bridge, with loss of a carbon atom (CO). This opens the porphyrin ring, and forms the open-chain linear tetrapyrrole biliverdin, which yields bilirubin (orange) after enzymatic reduction of biliverdin's central methene bond. In the liver, bilirubin is conjugated to enable excretion, requiring the enzyme UGT1A1. A redox cycle between bilirubin and biliverdin has been proposed [246248].

Figure 1
Bile pigment metabolism–from haem to bilirubin

Haemoglobin is cleaved to yield globin and haem (red). Haem is enzymatically converted to biliverdin (green) by liberating iron, via oxidation of its α-methene bridge, with loss of a carbon atom (CO). This opens the porphyrin ring, and forms the open-chain linear tetrapyrrole biliverdin, which yields bilirubin (orange) after enzymatic reduction of biliverdin's central methene bond. In the liver, bilirubin is conjugated to enable excretion, requiring the enzyme UGT1A1. A redox cycle between bilirubin and biliverdin has been proposed [246248].

Within splenic macrophages, haemoglobin (Hb) is released from senescent red blood cells and non-erythroid haemoproteins, representing the two major sources of organic haem available for bile pigment production [31]. Haem is oxidatively degraded by the rate-limiting isoenzyme complex of haem oxygenase (HMOX) 1/2, which generates intracellular UCB [32]. In this reaction, equimolar amounts of carbon monoxide, iron and biliverdin are liberated. Biliverdin is used as a substrate, fuelling enzymatic bilirubin production by biliverdin reductase (BLVRA), which is highly expressed in the cytosol of most eukaryotic cells [33].

Due to UCB's poor aqueous solubility threshold of approximately 70 nM [33], most of the UCB formed is transported in the plasma tightly bound to albumin [34,35], to be taken up into the hepatocytes [36]. Bilirubin is then transported to the endoplasmic reticulum by the α-isoform of GST B (ligandin, protein Y), or fatty-acid-binding protein 1 (FABP1, protein Z) [37], in which glucuronosides are formed by bilirubin UDP-glucuronosyltransferase (UGT1A1) [30,38]. The formation of bilirubin glucuronosides is impaired in the benign condition of Gilbert's syndrome, leading to accumulation of UCB within the blood [6]. On conjugation, multidrug-resistance protein 2 (MRP2) transports bilirubin, dependent on ATP, into the bile [39], where bilirubin conjugates are released into the duodenum via the biliary tract. In the intestinal tract, conjugated bilirubin glucuronosides are cleaved by β-glucuronidase originating from either intestinal bacteria or intestinal epithelial cells. Part of the resulting UCB is excreted, with small amounts reabsorbed via the enterohepatic recirculation [4042]. Bilirubin remaining within the intestines undergoes a series of reduction steps involving the intestinal microflora, leading to the formation of urobilinoids [43,44]. These metabolites are found predominantly in the intestinal tract [30], providing the distinct coloration of faeces. Bilirubin plasma levels usually range between 0.2 and 1 mg/dl (3 and 17 μM) [30]. Daily endogenous bilirubin production approximates 4 mg/kg of body weight, and an estimate of 300 mg of UCB and/or its degradation products is excreted mainly via the faeces every day [45,46].

BILIRUBIN AND MORTALITY WITH REFERENCE TO NON-COMMUNICABLE DISEASES

A multitude of interesting epidemiological studies investigating bilirubin and its association with various endpoint markers, including mortality from cardiovascular disease (CVD) or cancer and all-cause mortality, have been published recently (Table 1).

Table 1
Summary of studies linking bilirubin and mortality

BIRNH, Belgian Interuniversity Research on Nutrition and Health; CVE, cardiovascular event; MI, myocardial infarction; NA, not available; RR, relative risk. Only total bilirubin measured in all studies; RR calculated from values given in original papers according to the following equation after Sistrom and Garvan [244]: RR=[a/(a+b)]/[c(c+d)]; proportion of people: a endpoint+risk factor [high bilirubin or (TA)7/7]; b endpoint−risk factor; c no endpoint+risk factor; d no endpoint−risk factor.

Source Main parameters Outcome Number of participants Age (years) Study design Bilirubin levels Relative risk (RR) Genotyped 
Temme et al. (2001), Belgium [25Total bilirubin in connection to all-cause, cardiovascular and cancer mortality Men: RR for all-cause and cancer mortality of men of higher normal bilirubin levels relative to the low-bilirubin group were 0.73 and 0.42. Cancer mortality risk linearly decreased as bilirubin increased, especially with reference to non-lung cancers. No significant correlation between bilirubin and cardiovascular mortality found for either gender Men: 5460; women: 4843; representative of the Belgian population 25–74 Prospective population study; 10-year follow-up mortality data from BIRNH study Median serum bilirubin: 7.48 μM in men and 5.95 μM in women.
Classification: high normal (≥10.2 μM) vs low normal bilirubin levels (≤3.4 μM) 
High vs low bilirubin: 0.34 (men), 1.13 (women).
Calculated per 1000 person-years 
No 
Fulks et al. (2009), U.S.A. [47Total bilirubin and all-cause mortality Below-midpoint bilirubin levels were associated with significantly increased mortality only in men (U-shaped) 2 million participants (insurance applicants) NA Social Security Death Master File was used. Median follow-up of death was 12 years NA NA No 
Chen et al. (2011), Taiwan [55Total bilirubin in chronic haemodialysis and all-cause mortality/CVEs) After adjustment, individuals with bilirubin in the upper tertile had a hazard ratio of 0.32 for CVEs and 0.48 for all-cause mortality, compared with those in the lower tertile
Individuals homozygous for (TA)7/7 had significantly higher bilirubin levels than those with (TA)6/6 and (TA)7/6 genotypes
Individuals with (TA)7/7 [vs (TA)6/6] had approximately one-tenth of the risk for CVEs and one-quarter of the risk for all-cause mortality
(TA)7/7 had strong effects on bilirubin levels and might have an important lowering effect on CVEs and death 
Men: 335; women: 326; total: 661 58±14 Haemodialysis patients prospectively followed up for 12 years Total bilirubin (μM): 13.3 (±2.6).
Classification into tertiles:
upper, 16.8 (±4.3);
middle, 12.9 (±0.7);
lower, 10 (±1.5) 
NA No 
Ajja et al. (2011), U.S.A. [49Total bilirubin and measures of cardiorespiratory fitness, in relation to overall mortality During follow-up, 698 deaths, 253 (36%) of which due to CVD. Men in highest vs lowest bilirubin quartiles: significantly lower risk for all-cause mortality. Men in the moderate- to high-fitness quartiles in the low- and high-bilirubin groups: all-cause mortality and CVD mortality significantly lower Men: 1279 30–82 Based on data from a prospective, nested, case-control study obtained from the Aerobics Center Longitudinal Study (ACLS); follow-up period of 17 years Bilirubin quartiles (μM):
Q1: 1.7–8.4
Q2: 8.5–11.7
Q3: 11.8–15.2
Q4: 15.5–44.2 
High vs low bilirubin: 0.83 (men) No 
Horsfall et al. (2012), U.K. [51Total bilirubin all-cause mortality and coronary events Incidence rates for CVD, CHD, MI and death tended to decrease with increasing bilirubin decile categories in both genders. Participants with lower bilirubin levels had a significantly higher all-cause mortality risk (linear relationship); the connection of bilirubin to CVD and events of MI was U-shaped Men: 69 209; women: 60 843; total: 130 000 participants (all statin-treated) 58–65 Prospective study follow-up of 43 months Median total bilirubin levels (μM) were:
11 in men, and 9 in women.
Lowest bilirubin decile category: 1–6.
Highest decile: 19–40.
Total bilirubin: measured 3 months before statin treatment and recorded in the U.K. primary care database 
High vs low bilirubin: 0.83 (men), 0.73 (women) No 
Horsfall et al. (2013), U.K. [50Total bilirubin and all-cause mortality rates in GS and non-GS participants GS group: 24 deaths per 10 000 person-years.
Non-GS group: 50 deaths per 10 000 person-years 
GS: n=4266.
Non-GS: n=21 968 (67% males) 
35–61 Cohort study: 9-year follow-up ≤17 (10±3) μM
≥17 (29±5) μM 
GS vs control: 1.63 (men and women).
Calculated per 10 000 person-years 
No 
Cox et al. (2013), U.S.A. [27SNPs encoding bilirubin levels related to measures of subclinical CVD (vascular calcified plaque), impaired glucose regulation and mortality Within the UGT1A family, 18 SNPs associated with total bilirubin were detected. The study's findings support a potential role of UGT genetic variants (UGT1A8 and -A10) that potentially increase bilirubin levels, with all-cause mortality risk in Type 2 diabetes; no effect on measures of subclinical CVD (vascular calcified plaque) was found 1220 individuals, from 475 families (Diabetes Heart Study participants), 53.5% of whom were women Mean age 62.1  Mean level 13.9 (±5.1) μM NA Yes 
Vasovic et al. (2014), Serbia [52Bilirubin as novel biomarker for CVD mortality; develop a new inflammatory–malnutrition–renal involvement score (IMRIS) Bilirubin levels were significantly higher (11.9 μM) in CVD survivors vs non-survivors (10.2 μM).
Significantly increased risk score in participants with bilirubin levels <10.5 μM.
Bilirubin was found to be a significant uni- and multi-variate predictor of RR 
253 community-dwelling elderly participants, 78% women 65–99 2.6-year follow-up Mean total bilirubin level 11.1 (±3.9) μM NA No 
Ong et al. (2014), U.S.A. [48Total bilirubin and overall mortality Bilirubin levels were significantly negatively correlated with mortality rates and total mortality. Mortality was significantly lower as bilirubin >17.1 μM 4303 participants from NHANES study ≥60 4.5-year follow-up Bilirubin levels ≤11.9 μM.
After the follow-up: mean serum bilirubin (μM):
survivors, 11.2;
non-survivors, 10.5 
High vs low bilirubin: 0.65 (men and women) No 
Source Main parameters Outcome Number of participants Age (years) Study design Bilirubin levels Relative risk (RR) Genotyped 
Temme et al. (2001), Belgium [25Total bilirubin in connection to all-cause, cardiovascular and cancer mortality Men: RR for all-cause and cancer mortality of men of higher normal bilirubin levels relative to the low-bilirubin group were 0.73 and 0.42. Cancer mortality risk linearly decreased as bilirubin increased, especially with reference to non-lung cancers. No significant correlation between bilirubin and cardiovascular mortality found for either gender Men: 5460; women: 4843; representative of the Belgian population 25–74 Prospective population study; 10-year follow-up mortality data from BIRNH study Median serum bilirubin: 7.48 μM in men and 5.95 μM in women.
Classification: high normal (≥10.2 μM) vs low normal bilirubin levels (≤3.4 μM) 
High vs low bilirubin: 0.34 (men), 1.13 (women).
Calculated per 1000 person-years 
No 
Fulks et al. (2009), U.S.A. [47Total bilirubin and all-cause mortality Below-midpoint bilirubin levels were associated with significantly increased mortality only in men (U-shaped) 2 million participants (insurance applicants) NA Social Security Death Master File was used. Median follow-up of death was 12 years NA NA No 
Chen et al. (2011), Taiwan [55Total bilirubin in chronic haemodialysis and all-cause mortality/CVEs) After adjustment, individuals with bilirubin in the upper tertile had a hazard ratio of 0.32 for CVEs and 0.48 for all-cause mortality, compared with those in the lower tertile
Individuals homozygous for (TA)7/7 had significantly higher bilirubin levels than those with (TA)6/6 and (TA)7/6 genotypes
Individuals with (TA)7/7 [vs (TA)6/6] had approximately one-tenth of the risk for CVEs and one-quarter of the risk for all-cause mortality
(TA)7/7 had strong effects on bilirubin levels and might have an important lowering effect on CVEs and death 
Men: 335; women: 326; total: 661 58±14 Haemodialysis patients prospectively followed up for 12 years Total bilirubin (μM): 13.3 (±2.6).
Classification into tertiles:
upper, 16.8 (±4.3);
middle, 12.9 (±0.7);
lower, 10 (±1.5) 
NA No 
Ajja et al. (2011), U.S.A. [49Total bilirubin and measures of cardiorespiratory fitness, in relation to overall mortality During follow-up, 698 deaths, 253 (36%) of which due to CVD. Men in highest vs lowest bilirubin quartiles: significantly lower risk for all-cause mortality. Men in the moderate- to high-fitness quartiles in the low- and high-bilirubin groups: all-cause mortality and CVD mortality significantly lower Men: 1279 30–82 Based on data from a prospective, nested, case-control study obtained from the Aerobics Center Longitudinal Study (ACLS); follow-up period of 17 years Bilirubin quartiles (μM):
Q1: 1.7–8.4
Q2: 8.5–11.7
Q3: 11.8–15.2
Q4: 15.5–44.2 
High vs low bilirubin: 0.83 (men) No 
Horsfall et al. (2012), U.K. [51Total bilirubin all-cause mortality and coronary events Incidence rates for CVD, CHD, MI and death tended to decrease with increasing bilirubin decile categories in both genders. Participants with lower bilirubin levels had a significantly higher all-cause mortality risk (linear relationship); the connection of bilirubin to CVD and events of MI was U-shaped Men: 69 209; women: 60 843; total: 130 000 participants (all statin-treated) 58–65 Prospective study follow-up of 43 months Median total bilirubin levels (μM) were:
11 in men, and 9 in women.
Lowest bilirubin decile category: 1–6.
Highest decile: 19–40.
Total bilirubin: measured 3 months before statin treatment and recorded in the U.K. primary care database 
High vs low bilirubin: 0.83 (men), 0.73 (women) No 
Horsfall et al. (2013), U.K. [50Total bilirubin and all-cause mortality rates in GS and non-GS participants GS group: 24 deaths per 10 000 person-years.
Non-GS group: 50 deaths per 10 000 person-years 
GS: n=4266.
Non-GS: n=21 968 (67% males) 
35–61 Cohort study: 9-year follow-up ≤17 (10±3) μM
≥17 (29±5) μM 
GS vs control: 1.63 (men and women).
Calculated per 10 000 person-years 
No 
Cox et al. (2013), U.S.A. [27SNPs encoding bilirubin levels related to measures of subclinical CVD (vascular calcified plaque), impaired glucose regulation and mortality Within the UGT1A family, 18 SNPs associated with total bilirubin were detected. The study's findings support a potential role of UGT genetic variants (UGT1A8 and -A10) that potentially increase bilirubin levels, with all-cause mortality risk in Type 2 diabetes; no effect on measures of subclinical CVD (vascular calcified plaque) was found 1220 individuals, from 475 families (Diabetes Heart Study participants), 53.5% of whom were women Mean age 62.1  Mean level 13.9 (±5.1) μM NA Yes 
Vasovic et al. (2014), Serbia [52Bilirubin as novel biomarker for CVD mortality; develop a new inflammatory–malnutrition–renal involvement score (IMRIS) Bilirubin levels were significantly higher (11.9 μM) in CVD survivors vs non-survivors (10.2 μM).
Significantly increased risk score in participants with bilirubin levels <10.5 μM.
Bilirubin was found to be a significant uni- and multi-variate predictor of RR 
253 community-dwelling elderly participants, 78% women 65–99 2.6-year follow-up Mean total bilirubin level 11.1 (±3.9) μM NA No 
Ong et al. (2014), U.S.A. [48Total bilirubin and overall mortality Bilirubin levels were significantly negatively correlated with mortality rates and total mortality. Mortality was significantly lower as bilirubin >17.1 μM 4303 participants from NHANES study ≥60 4.5-year follow-up Bilirubin levels ≤11.9 μM.
After the follow-up: mean serum bilirubin (μM):
survivors, 11.2;
non-survivors, 10.5 
High vs low bilirubin: 0.65 (men and women) No 

In a large Belgian retrospective population study, Temme et al. [25] investigated the association between serum bilirubin and all-cause, cardiovascular and cancer mortality in 5460 men and 4843 women, using 10-year follow-up mortality data from the Belgian Interuniversity Research on Nutrition and Health (BIRNH) study. The adjusted relative risks for all-cause and cancer mortality of men within the group with higher bilirubin levels (≥0.6 mg/dl, 10.3 μM) were 0.73 and 0.42, respectively, when compared with the low-bilirubin group (≤0.2 mg/dl, 3.4 μM). The cancer mortality risk linearly decreased as bilirubin increased, especially with reference to non-lung cancers. The effects were retained in the female group, but were non-significant.

Another large study by Fulks et al. [47] included almost 2 million participants (insurance applicants) and investigated all-cause mortality associated with bilirubin levels, using the Social Security Death Master File. In this study, below-midpoint bilirubin levels were associated with significantly increased mortality in men (U-shaped); however, this effect was again not present in women [47]. A very recent report from the U.S. National Health and Nutrition Examination Survey (NHANES) study supports the negative relationship between bilirubin and all-cause mortality. The group's investigations included a nationally representative sample (4303 participants) of older adults, aged ≥60 years, who had participated in the NHANES study, in which participants were followed up for an average of 4.5 years. Although bilirubin levels did not exceed 0.7 mg/dl (12 μM), there was a significant negative correlation with all-cause mortality [48].

Ajja et al. [49] investigated the utility of serum bilirubin concentrations and measures of cardiorespiratory fitness (treadmill time) as predictors of overall mortality: 1279 men aged between 30 and 82 years participated in the study between 1974 and 1997. During the average 17-year follow-up period, 698 deaths occurred, 253 (36%) being due to CVDs. Men in the highest bilirubin quartile (≤2.6 mg/dl, 44.5 μM) had a significantly reduced risk of all-cause mortality when compared with men in the lowest bilirubin quartile (≤0.49 mg/dl, 8.4 μM). All-cause mortality and CVD mortality were significantly reduced in men in the high-bilirubin groups in both the low- and the high-fitness quartiles. In conclusion, serum bilirubin levels had a strong and independent negative association with all-cause and CVD mortality [49].

Also focusing on CVDs, Horsfall et al. [50] prospectively investigated a statin-treated cohort including approximately 130 000 participants. Total bilirubin levels were measured 3 months before statin treatment, with a follow-up of 43 months. Participants with lower bilirubin levels had a significantly greater all-cause mortality risk (linear relationship), whereas the relationship between bilirubin and CVD/myocardial infarction events was U-shaped [50]. In a second investigation, the same group analysed mortality rates (9-year follow-up) in participants diagnosed with Gilbert's syndrome (GS; n=4266) and controls (non-GS; n=21 968). Data mining was completed using the Health Improvement Network (THIN) database, which represents the general practice population in the U.K. After accounting for multiple potential confounding factors, the GS and control groups experienced 24 and 50 deaths per 10 000 person-years, respectively [51]. These data are profound because they suggest that a previously considered benign syndrome confers a significant (statistically and clinically) effect on mortality.

Recently, the specific relevance of bilirubin, as a novel biomarker to predict cardiovascular mortality, was assessed. A Serbian study included 253 participants (aged ≥65 years, 78% women), who were observed for 32 months. Cut-off points of multivariate predictors, including bilirubin, were used to establish a cardiovascular risk score system. Among other variables, a significantly increased risk score was observed in those participants with bilirubin levels <10.5 μM [52].

With reference to impaired blood-glucose regulation and CVD, Cox et al. [27] studied 1220 individuals enrolled on the Diabetes Heart Study to investigate the relationship of single nucleotide polymorphisms (SNPs) regulating bilirubin levels (i.e. within the UGT1A gene family) and CVD/all-cause mortality. Within the UGT1A family, 18 SNPs associated with total bilirubin were detected. The study's findings support a potential role of UDP-glucuronosyltransferase (UGT) genetic variants that increase bilirubin levels in influencing risk of mortality in patients with Type 2 diabetes. No effect on the measures of subclinical CVD (vascular calcified plaque) was found [27]. Importantly, bilirubin exerts its beneficial role only when liver function is preserved. This explains bilirubin's U-shaped relationship with cardiovascular risk in men participating in the British Regional Heart Study reported by Breimer et al. [53]. Furthermore, the effect of impaired liver function, which increases total bilirubin and mortality from CVD risk, was fully elucidated in a meta-analytical study by Novotny and Vitek [54], and should be carefully considered when investigating the role of bilirubin in disease prevention (see below). Chen et al. [55] have also shown that patients on haemodialysis (prospectively followed for 12 years) with higher bilirubin plasma levels (0.99 mg/dl, approximately 17 μM) had a significantly reduced incidence of CVD and all-cause mortality compared with patients with low and normal circulating bilirubin levels.

Based on these findings, an important protective role of bilirubin against (disease-/age-related) all-cause mortality seems evident. However, a molecular explanation concerning how bilirubin protects against these diseases must be revealed in order to understand why and how certain groups of the population are less likely to develop chronic diseases. Another important issue to be explored concerns the dimorphic effect of bilirubin in men and women, because in females bilirubin is apparently less effective in protecting against oxidative-stress-mediated diseases [25,56,57].

BILIRUBIN AND CVDs

The role of bilirubin as a biomarker or predictive factor of atherosclerosis has been a subject of many comprehensive reviews published over the last few years [18,19,5862]. However, a great body of novel data on the role of bilirubin in the pathogenesis of many diseases has revealed new and intriguing hypotheses concerning protection from overlapping and occasionally unrelated pathologies.

Bilirubin was first conclusively shown to exhibit antioxidant activity in 1987 by Stocker et al. [5]. Since then, multiple experimental and clinical studies have been published, the vast majority of which demonstrate a clear, negative, linear relationship between systemic bilirubin concentrations and CVD (reviewed in [18,19,58,59,63]). The antioxidant potential of bilirubin is important, in the context of CVD, because the oxidation of low-density lipoprotein (LDL) (which bilirubin inhibits) represents one important hypothesis of CVD pathogenesis. Despite this, it should be acknowledged that there are now a number of new hypotheses about how bilirubin protects against CVDs and these should also be considered (Table 2).

Table 2
Factors contributing to atherogenesis modulated by bilirubin

iNOS, inducible nitric oxide synthase; MAPK, mitogen-activated protein kinase; PTEN, phosphatase and tensin homologue deleted on chromosome 10.

Atherogenic mechanism Link to bilirubin 
Oxidative stress ↑ total antioxidant capacity 
 ↓ LDL oxidation 
 ↓ production of advanced glycation end-products (AGE) 
 ↓ production of superoxide via NADPH oxidase inhibition [107,108
Inflammation ↓ production of proinflammatory cytokines [61,111
 ↓ high-sensitivity CRP levels [61,110,145147
 T-regulatory cell differentiation [143
Blood lipid profile ↓ LDL cholesterol 
 ↓ triacylglycerols 
 ↑ HDL-C 
Glucose metabolism Preserves glucose homoeostasis (for a review, see Vitek [28]) 
 ↑ insulin sensitivity [126
 ↓ insulin resistance [127
Blood pressure ↓ blood pressure [51
Obesity Negative association [120,121
Platelet function and haemostasis ↓ platelet activation [150,245
 ↓ mean platelet volume [153
 ↑ activated partial thromboplastin time [154
NO production ↓ TNF-α-stimulated iNOS induction [158
Cellular adhesion ↓ TNF-α-induced expression of VCAM-1/ICAM-1/E-selectin [156,157
 ↓ VCAM-1-mediated transendothelial leucocyte migration [159
Intracellular signalling ↓ MAPK pathway [162,163
 ↓ NADPH oxidase pathway [107
 ↑ PTEN [166
 ↓ TNF-α-stimulated NF-κB translocation [157
Vascular dysfunction ↓ arterial stiffness [168,169
 ↑ coronary microvascular function [147
 ↑ aortic elastic properties [171
 Preserves coronary vasoreactivity [173
Atherogenic mechanism Link to bilirubin 
Oxidative stress ↑ total antioxidant capacity 
 ↓ LDL oxidation 
 ↓ production of advanced glycation end-products (AGE) 
 ↓ production of superoxide via NADPH oxidase inhibition [107,108
Inflammation ↓ production of proinflammatory cytokines [61,111
 ↓ high-sensitivity CRP levels [61,110,145147
 T-regulatory cell differentiation [143
Blood lipid profile ↓ LDL cholesterol 
 ↓ triacylglycerols 
 ↑ HDL-C 
Glucose metabolism Preserves glucose homoeostasis (for a review, see Vitek [28]) 
 ↑ insulin sensitivity [126
 ↓ insulin resistance [127
Blood pressure ↓ blood pressure [51
Obesity Negative association [120,121
Platelet function and haemostasis ↓ platelet activation [150,245
 ↓ mean platelet volume [153
 ↑ activated partial thromboplastin time [154
NO production ↓ TNF-α-stimulated iNOS induction [158
Cellular adhesion ↓ TNF-α-induced expression of VCAM-1/ICAM-1/E-selectin [156,157
 ↓ VCAM-1-mediated transendothelial leucocyte migration [159
Intracellular signalling ↓ MAPK pathway [162,163
 ↓ NADPH oxidase pathway [107
 ↑ PTEN [166
 ↓ TNF-α-stimulated NF-κB translocation [157
Vascular dysfunction ↓ arterial stiffness [168,169
 ↑ coronary microvascular function [147
 ↑ aortic elastic properties [171
 Preserves coronary vasoreactivity [173

The first clinical study reporting an intriguing CVD protective association for bilirubin was published in 1994 [64]. Among many other studies, individuals with GS had a reduced prevalence of CHDs compared with the general population (2% vs 12%), and a similar relationship was observed in a 3-year follow-up study [65]. In our meta-analysis focusing on the association between CVD and bilirubin, and involving almost 15 000 men, each 1-μM increase in serum bilirubin was associated with a 6.5% decrease in CVD risk [54]. It is interesting that men with GS were found to have a 25-year delay in a clinically relevant manifestation of carotid atherosclerosis compared with individuals with normo-bilirubinaemia [67].

More recent data continue to support a protective role of bilirubin against CVD with dose-dependent bilirubin concentrations being negatively associated with coronary atherosclerosis and calcified plaques (cross-sectional study conducted in almost 3000 healthy men undergoing routine coronary computed tomography). In this study, individuals in the highest bilirubin quartile (>1.2 mg/dl, 20.6 μM) had a 41% reduced risk of coronary atherosclerosis compared with individuals in the lowest bilirubin quartile (<0.8 mg/dl, 13.7 μM) [68]. Coronary artery calcification, a marker of subclinical atherosclerosis, has also been negatively associated with systemic bilirubin levels in several other studies [69,70].

The beneficial effect of the mildly elevated serum bilirubin seen in patients with GS on heart rate, Q–T interval dispersion and P-wave abnormalities, indicative of atrial enlargement, was also observed in an observational Turkish study [71], suggesting anti-arrhythmic effects of bilirubin. These data are corroborated by other groups that observed anti-arrhythmic and cardioprotective effects of bezoar bovis (pulverized bovine pigment gallstones, used in traditional Chinese medicine) containing large amounts of bilirubin [72] on bufadienolide-induced cardiotoxicity [73,74].

A similar negative association between serum/plasma bili-rubin concentrations was found not only for coronary artery diseases (CADs) but also for peripheral atherosclerosis (for a review see Schwertner and Vitek [18]). Indeed, patients with carotid and peripheral vascular disease have reduced bilirubin concentrations [18]. Concordantly, patients with GS have a reduced risk of peripheral artery disease and stroke, of about 50% [75,76]. In fact, a negative association between systemic levels of bilirubin and the intima–media thickness of carotid arteries has been reported in several studies, indicating that bili-rubin exerts its effect early in the process of atherogenesis [67,77,78].

Further evidence to support the above findings was published by Chen et al. [79], who reported, in a prospective study on patients undergoing chronic haemodialysis, a significantly decreased risk of future cardiovascular events and all-cause mortality in patients demonstrating mildly elevated bilirubin concentrations (versus individuals with low concentrations). Furthermore, individuals homozygous for (TA)7/7 (UGT1A1*28 GS genotype) had one-tenth of the risk of future cardiovascular events (and half the risk of future all-cause mortality) compared with wild-type UGT1A1 genotype carriers [(TA)6/6; UGT1A1*1] [79]. Data published by Kim et al. [80] further support these results, revealing a negative association between serum bilirubin levels and the Framingham risk score. These data are consistent with recent experimental results demonstrating, in rat models, a protective effect of intraperitoneal bilirubin administration on ischaemic left ventricular function and infarct size [81]. Importantly, these findings were recently confirmed in a rodent model of endogenous hyperbilirubinaemia, which documented improved post-ischaemic cardiac function, in association with decreased phospholamban and superoxide dismutase gene expression, and an increase in glutathione peroxidase gene expression. These data suggest that bilirubin influences gene expression, and has the potential to further enhance cellular antioxidant defences, reduce basal cardiac contractility and improve post-ischaemic systolic pressure development [82].

It is very important to note that negative associations between serum bilirubin concentrations for numerous heterogeneic, inflammatory, autoimmune and degenerative diseases, such as chronic obstructive pulmonary disease [83], inflammatory bowel disease [84], systemic lupus erythematosus [85], rheumatoid arthritis [86], psoriasis [77], schizophrenia [87], multiple sclerosis [88], osteoporosis [89] and pre-eclampsia [90], have also been reported. Surprisingly, all of these conditions have been associated with an increased risk of CVDs [9199], indicating a possible common protective molecular mechanism for bilirubin's action.

What factors are responsible for bilirubin's atheroprotective effects?

Although it is generally assumed that bilirubin exerts atheroprotective effects by inhibiting oxidative stress, there appear to be many other factors contributing to its atheroprotective effects (see Table 2).

Bilirubin and oxidative stress

The traditional view based on the potent antioxidant effects of bilirubin reported by Stocker et al. [100] links the antiatherogenic effect of bilirubin to its ability to prevent oxidative lipoprotein modification (for a review see Stocker [45]), which increases during the development of atherosclerosis [58]. Indeed, bilirubin was shown to effectively inhibit LDL-cholesterol (LDL-C) oxidation in vitro [101,102], and was negatively associated with oxysterol levels in a human clinical study [103]. The decreased production of oxidative stress markers in individuals with GS [104106] is probably the result of augmented antioxidant capacity in these individuals [20,65]. Bilirubin, similar to other bile pigments, is also a potent inhibitor of mitochondrial superoxide production via suppression of mitochondrial NADPH oxidase activity [107,108]. This property is believed to have implications for the amelioration of oxidative stress, in addition to affecting multiple intracellular pathways regulated by reactive oxygen species (ROS).

Bilirubin and lipid metabolism

LDL hypercholesterolaemia is a well-established risk factor for atherosclerotic diseases. In view of the atheroprotective effects of bilirubin, it is not surprising that serum bilirubin levels are also negatively associated with LDL-C [65,109] (for a comprehensive review, see Bulmer et al. [60]). The association between serum bilirubin and individual LDL-C subfractions, including small dense LDLs [110113] and oxidatively (ox) modified LDLs [106,110], was also recently reported, although these data are not completely conclusive and require further investigation [60]. The same negative association is also true for very-low-density lipoprotein (VLDL) and intermediate-density lipoprotein (IDL) subfractions [113], and, as expected, for apolipoprotein B (Apo-B), as well as the Apo-B/Apo-A1 ratio [101], which are also independent predictors of atherosclerotic disease. Furthermore, in a very recent study, low serum bilirubin levels predicted incident LDL hypercholesterolaemia [114].

Nevertheless, the effect of bilirubin on lipid metabolism seems to be complex, because there is also a negative association between serum bilirubin and serum triacylglycerol [106,115], as well as remnant lipoprotein cholesterol levels [115], the importance of which as predictors of CVD morbidity/mortality has been increasingly acknowledged [116119]. In fact, a negative relationship between serum triacylglycerols and serum bili-rubin has been reported in numerous studies (for a review see Bulmer et al. [60]). This link seems to be extremely important because elevated serum triacylglycerols belong to the principal diagnostic and pathogenic criteria of the metabolic syndrome.

However, significant associations between individual parameters (i.e. bilirubin and LDLs), and linking such relationships to protection from disease, is not always appropriate, given the complexity of disease processes. In a cross-sectional study, various serum lipid, lipoprotein and bilirubin combinations were assessed for their ability to predict existing CAD in middle-aged men. These relationships were then compared with the prediction of CVD using established lipid and lipoprotein risk factors only. The traditional risk factors of cholesterol, high-density lipoprotein (HDL)-C, cholesterol/HDL-C ratios, triacylglycerols, age, cigarette smoking and systolic blood pressure were tested by discriminant analysis, as were various cholesterol/bilirubin, cholesterol/(HDL-C + bilirubin) and LDL-C/(HDL-C + bilirubin) ratios. Each of the bilirubin-containing ratios was found to be a better risk predictor of disease prediction than using traditional risk factors only. When the LDL-C/(HDL-C + bilirubin) ratio was included with the traditional risk predictors, prediction of severe CAD was improved from 28.4% to 35.3% and the overall correct classification of CAD increased from 68.3% to 71.1%. When the 75th percentile was used as a cut-off point, the diagnostic sensitivities obtained with cholesterol/(HDL-C +bilirubin) ratios (52.1%) and LDL-C/(HDL-C + bilirubin) ratios (51.7%) were better than those obtained with cholesterol/HDL-C ratios (40.4%) (P=0.033 and 0.048, respectively). LDL-C/(HDL-C + bilirubin) ratios also improved the prediction of severe CAD over prediction scores obtained with LDL-C/HDL-C ratios alone (43.4%). The authors of that study concluded that serum bilirubin levels, when combined with other lipid and lipoprotein data, could help identify those people at risk of CAD and those who should undergo further testing [109].

Bilirubin and the metabolic syndrome, and diabetes

With reference to the above reports, it is not surprising that similar negative associations are well documented between serum bilirubin and indices of the metabolic syndrome including waist circumference, body mass index (BMI; as reviewed in [28,60]) and obesity [120,121], as well as elevated blood pressure (for a review see Boon et al. [61]), diabetes [28,122] and the presence of non-alcoholic steatohepatitis [123125]. Elevated bilirubin is associated with improved insulin sensitivity [126], decreased insulin resistance [127] and increased levels of adiponectin [128], which is an insulin-sensitizing agent [129]. Increased concentrations of adiponectin were also observed in patients with GS who possess less epicardial adipose tissue (a marker of coronary atherosclerosis), when compared with normobilirubinaemic individuals [130].

In addition, although not present in all studies, there is a positive association between serum bilirubin and HDL-C levels [60], further corroborating the importance of bilirubin as a key marker and potentially protective agent against these conditions. It is also important to note the relationship between weight reduction and serum bilirubin. In individuals undergoing weight loss, each 1% decrease in weight had a highly significant association with a linear increase in serum bilirubin concentration (∼0.21±0.03 μM in men and 0.11±0.02 μM in women) [120].

The papers discussed above consistently indicate that serum bilirubin is a predictor of incident metabolic syndrome, as recently reported in a large 4-year retrospective study of Korean men [131], with the same association also found for incident diabetes (31% decreased risk of incident diabetes in the highest compared with the lowest bilirubin quartiles after multiple adjustment for potentially confounding factors) [132]. Although not all studies confirm a protective association between CVD and bilirubin in women, this is not the case for the metabolic syndrome, with a negative association also reported for females [133]. It is important to indicate that the first report on the association between bilirubin and diabetes was published in 2007 by Inoguchi et al. [122], who reported a reduced prevalence rate of vascular complications in patients with diabetes together with GS compared with normobilirubinaemic, hyperglycaemic individuals. It is of interest that the prevalence rate of GS in this large cohort of patients with diabetes was at least three times lower than what would be expected in the general Japanese population. In another large study on patients with Type 2 diabetes, plasma bilirubin concentrations were negatively associated with the risk of lower limb amputation [134]. These data are consistent with our observation of a reduced prevalence of diabetes in individuals with the (TA)7/7 promoter variant in UGT1A1 [135], as well as with low levels of advanced glycation end-products (AGEs) in patients with GS [105]. Interestingly, serum bilirubin is also negatively correlated with the pre-diabetic state [136], giving further support to the role of bilirubin as a biomarker of future diabetes.

All of these clinical observations fit well with experimental data demonstrating a protective role of HMOX induction on glucose metabolism and insulin sensitivity [137]. However, the metabolic interplay of bilirubin is far more complex. Bilirubin, for instance, was demonstrated to correlate positively with thyroid hormone concentrations, which can contribute to insulin resistance [138].

Bilirubin and inflammation

Inflammation is believed to represent a key regulatory process that links multiple risk factors for atherosclerosis to altered arterial biology [139]. As bilirubin was demonstrated to affect inflammatory processes at several stages, the anti-inflammatory effects of bilirubin may contribute to protection against atherosclerosis. The first report on bilirubin's effects on the immune system was published as early as 1937 [140]. Since then, dozens of experimental and clinical reports have convincingly documented the anti-inflammatory effects of bilirubin. Bilirubin substantially inhibits complement induction by inhibiting the interaction between C1q and immunoglobulins, thus inhibiting initial complement activation through the classic pathway [141]. In addition, bilirubin modulates the activity of cytotoxic T-lymphocytes [142], is responsible for T-regulatory cell (Treg) expansion [143] and also inhibits the production of proinflammatory cytokines, such as interleukin (IL)-1β or IL-6 [62,111]. Cytokines are responsible for the production of C-reactive protein (CRP) by liver tissue [144], so it is not surprising that serum bilirubin levels are negatively associated with CRP concentrations [62,110,145147], which may also represent a pathogenic factor for atherosclerosis [148].

It should be noted that bilirubin may also act as an immuno-modulator, which can heighten inflammatory responses in the presence of inflammatory mediators, e.g. when human blood is incubated with lipopolysaccharide, UCB is positively associated with the IL-1 receptor antagonist (IL-1RA) and interferon-γ gene expression responses of leucocytes, which may assist in combating infection [149]. In this context, it would be interesting to determine whether extreme hyperbilirubinaemia promotes inflammation in jaundiced newborns and patients with the Crigler–Najjar syndrome.

Bilirubin, platelet function and haemostasis

Very recent data also show that bilirubin might contribute to atheroprotection by modulating platelet function [150] and inhibiting haemostasis. In addition, mean platelet volume, which is an independent risk factor for atherosclerosis and ischaemic heart disease [151], and a marker of increased platelet reactivity [152], was reported to be significantly reduced in individuals with GS compared with normobilirubinaemic individuals [153]. It is interesting that higher bilirubin levels prolong activated partial thromboplastin time in jaundiced newborns [154]. These data further emphasize potential roles of bilirubin in preventing atherothrombosis.

Bilirubin, intercellular adhesion and nitric oxide homoeostasis

Communication between cells within the endothelial compartment is extremely important for proper vascular function, with impairment in endothelial function contributing importantly to the process of atherogenesis [155]. Recent evidence also indicates that endothelial function may be modulated by UCB, e.g. in vitro studies show that UCB inhibits leucocyte adhesion to endothelial cells by preventing tumour necrosis factor α (TNF-α)-induced over-expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), as well as E-selectin [156], via suppression of the nuclear factor κB (NF-κB) pathway [157]. The same mechanism seems to be involved in the inhibitory effect of bilirubin on TNF-α-stimulated induction of inducible nitric oxide synthase (iNOS) [158]. Bilirubin also inhibits VCAM-1-mediated transendothelial leucocyte migration [159]. These data were confirmed in a clinical study demonstrating a negative correlation between serum bilirubin and P-selectin, as well as the CD40 ligand [160].

Bilirubin and intracellular signalling pathways

In addition to the above-mentioned intracellular mechanisms, bilirubin potently modulates intracellular phosphorylation cascades, a factor implicated not only in its presumably anti-cancer effects [161], but also in the proliferation of vascular smooth muscle cells (VSMCs). Bilirubin attenuates the mitogen-activated protein kinase (MAPK) pathway with subsequent hypophosphorylation of retinoblastoma tumour-suppressor protein, leading to cell cycle arrest [162,163]. It also markedly influences the cyclin-dependent kinase (CDK) inhibitor p53 [164], pointing to multiple effects of bilirubin on the cell cycle. Furthermore, bilirubin is a strong inhibitor of NADPH oxidase, which generates superoxide [107], an important signalling molecule for VSMC migration and proliferation [165]. Finally, bilirubin also up-regulates the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) tumour suppressor [166], the activity of which inhibits VSMC migration and proliferation [167].

Bilirubin and vascular dysfunction

The above-mentioned mechanisms may contribute to the beneficial effects of bilirubin on endothelial and microvascular function. Indeed, serum bilirubin is negatively correlated with the brachial–ankle pulse-wave velocity (a marker of arterial stiffness and CVD) in healthy Chinese men [168], in men and women with established CAD [56], as well as in hypertensive individuals [169]. Total bilirubin is also negatively associated with brachial artery flow-mediated dilatation, a marker of endothelial dysfunction [170] in healthy individuals, as well as with coronary microvascular dysfunction [147] and aortic elastic properties [171]. Moreover, in patients with acute coronary occlusion, higher bilirubin concentrations are associated with better coronary collateral development [172]. Similarly, serum bilirubin has been found to correlate with coronary vasoreactivity in Japanese patients [173]. A proof-of-principle study on the role of bilirubin in protecting against endothelial dysfunction was reported by Dekker et al. [174], who induced moderate hyperbilirubinaemia in patients with Type 2 diabetes using atazanavir, a partial inhibitor of UGT1A1, after which a significant improvement in plasma antioxidant capacity and endothelium-dependent vasodilatation, as well as decreased levels of von Willebrand's factor, a marker of endothelial dysfunction, was reported. Consistent with these data, and as mentioned above, bilirubin concentrations significantly correlated with the risk of lower limb amputations in patients with Type 2 diabetes [134].

Further supporting evidence demonstrates a >5-fold increased risk of leukoaraiosis (cerebral white matter hyperintensity due to chronic ischaemia of the cerebral arterioles [175], and a risk factor for atherosclerosis [176] and the metabolic syndrome [177]) in Korean women (but not men) reporting a bilirubin level <10 μmol/l compared with those in the upper bilirubin tertile [178]. It is also interesting to note that the risk of leukoaraio-sis is positively associated with the mean platelet volume [179], a factor related to bilirubin concentrations (see above), further revealing interrelationships of multiple pathological conditions.

Medication for CVD prevention and bilirubin

Many drugs that are also used for CVD prevention may have the potential to modulate bilirubin levels, and thus may represent potential treatment strategies, e.g. statins may induce mild elevations in circulating bilirubin by influencing hepatic bilirubin transport or by inducing bilirubin formation via increased HMOX1 transcription activity [180]; atorvastatin, fluvastatin and rosuvastatin inhibit hepatocyte bilirubin uptake in in vitro studies, which could increase circulating concentrations [181]. Furthermore, statins, particularly atorvastatin [180], may induce HMOX1 transcription and thus increase bilirubin concentrations as documented in numerous clinical studies, e.g. minor increases in total bilirubin concentrations are documented after daily administration of 40 mg of simvastatin or 10 mg of rosuvastatin, increasing bilirubin by 1.6 μM and 0.7 μM, respectively [182]. However, there is also evidence to the contrary from a study investigating the NHANES cohort, which concluded that statin usage was associated with decreased bilirubin concentrations [183]. Statins vary in their HMOX1-inducing effect and this may differentially affect circulating bilirubin concentrations [180]. Further confounding issues are highlighted, in that hyperlipidaemia and the presence of CVD are associated with reduced bilirubin concentrations and are treated with statins. Therefore, bilirubin levels could have been lower in statin users because of underlying CVD/hyperlipidaemia [184]. Additional preliminary data support a non-significant increase (∼0.48 μM) in bilirubin in individuals prescribed atorvastatin at 10–40 mg and/or simvastatin at 20–40 mg daily for 4–8 weeks [184]. However, it is possible that a high dose (i.e. 80 mg daily simvastatin) and prolonged usage (i.e. 2 years) are necessary to increase bilirubin levels (by 0.7 μM) [185].

Furthermore, the administration of specific protease inhibitors is associated with pronounced hyperbilirubinaemia in patients with HIV, e.g. atazanavir and indinavir inhibit the bilirubin conjugation activity of UGT1A1 [186], so their administration is associated with increased unconjugated bilirubin concentrations. For example, non-infected patients with Type 2 diabetes who took 300 mg of atazanavir twice daily increased their total bilirubin concentrations from 7 μM to 64 μM [174]. Importantly, the dose of atazanavir prescribed in that study was greater than the recommended dosage for HIV patients of 400 mg daily (or 300 mg daily of atazanavir boosted with 100 mg daily of ritonavir). Therefore, HIV patients undergoing protease inhibitor treatment do not generally experience such pronounced hyperbilirubinaemia [187]. Unfortunately, protease inhibitors induce a variety of side effects [188], so their use would not represent a target for increasing bilirubin in the medium to long term.

Novel experimental testing indicates that a new class of Morpholino antisense oligonucleotides, which selectively bind to RNA and block protein translation/activity, may represent a ‘new generation’ of UGT1A1 modulators [189]. Thus far, such an approach has been used only in animal studies with an intravenous injection (16 μg/kg) of a UGT1A1-targeted Vivo-Morpholino, increasing total bilirubin concentrations from 3.4 μM to 20.5 μM, 24 h after antisense administration [190]. Clinical trials of Morpholino administration are currently under way for Duchenne muscular dystrophy, the results of which may provide a precedent for their use in other conditions [191].

In conclusion, targeting UGT1A1 for inhibition would probably represent an effective approach to mildly increasing circulating bilirubin to concentrations that might induce therapeutic effects (i.e. +2–5 μM above normal values). Furthermore, administration of UGT1A1 inhibitors would not affect iron and carbon monoxide production, as HMOX1 inducers would, so inducing more specific effects related to altered bilirubin metabolism.

An important question that remains to be answered includes whether low bilirubin levels in the circulation are simply a biomarker of increased oxidative stress accompanying atherosclerosis and many other diseases, or whether they are genetically encoded and predispose to atherosclerosis, a typical problem of reverse causality [192].

BILIRUBIN AND CANCER

Bilirubin and its link to cancer risk is a comparably young field and therefore only a few, although important, observations have been published (Table 3). Although, in the clinical environment, total bilirubin is measured routinely rather than UCB, higher bilirubin plasma levels or the underlying UGT1A1 promoter gene variation [(TA)7/7] are negatively associated with cancer mortality [25]. This is particularly true in terms of colorectal cancer (CRC) [26,161], and lung [21] and breast cancer [193].

Table 3
Summary of studies linking bilirubin and cancer

GI-cancer, gastrointestinal cancer; NA, not available; NS, not significant. RR calculated from values given in original papers according to the following equation after Sistrom and Garvan [244]: RR=a/(a+b)/c(c+d); proportion of people: a endpoint+risk factor [high bilirubin or (TA)7/7)]; b endpoint−risk factor; c no endpoint+risk factor; d no endpoint−risk factor.

Source Type of cancer and outcome Number of participants Age (years) Study design Bilirubin levels Relative risk (RR) Genotyped Limitations 
Ko et al. (1994), U.S.A. [200CRC (no association) Total: 236.
Cases: 118;
controls: 118 
Range: <25–75 Case–control Lower bilirubin levels among cancer patients NA No Low number of cases, only total bilirubin measured 
Guillemette et al. (2001), U.S.A. [194UGT and breast cancer (no association)
OR 1.28 in (TA)7/7 (NS) 
Total: 1064.
Cases: 455;
controls: 609 
Range: 30–55 Case–control nurses' health NA (TA)7/7 vs (TA)6/6: 0.92
(TA)7/7 vs (TA)6/6+(TA)6/7: 0.94 (men and women) 
Yes No total bilirubin/UCB measured 
Temme et al. (2001), Belgium [25Men
Total cancer: bilirubin ≥10.3 μM, RR 0.31/0.42 [P=0.0001/0.004 (unadjusted/adjusted)].
Lung cancer: bilirubin ≥10.3 μM, RR 0.27/0.40 (P 0.0034/0.0898).
Non-lung cancer: total bilirubin ≥10.3 μM, RR 0.34/0.43 (P=0.0008/0.0195)
Women
Total cancer, non-lung cancer, lung cancer: total bilirubin ≥8.55 μM, RR no sign 
Total: 10 303 Men: 5460; women: 4843 Range: 25–74 Prospective including 10-year follow-up Bilirubin (μM):
men: 7.5, range: 1.7–64.6;
women: 10, range: 1.7–64.6 
Highest vs lowest bilirubin group (RR: calculated per 1000 person-years).
Men: total cancer: 0.20; lung cancer: 0.17; non-lung cancer: 0.21.
Women: total cancer: 0.92 
No Detection method: only total bilirubin measured, smoking 
Ching et al. (2001), Australia [193Breast cancer (OR 0.5 in highest vs lowest bilirubin quartile) (>4.1 μM/>7.5 μM), reduced risk.
Only women 
Total: 304.
Cases: 153;
controls: 151 
Range: 30–84 Case–control NA Highest vs lowest bilirubin group.
Breast cancer: 0.74 
No Low number of participants
No total bili-rubin/UCB levels of cases vs controls 
Zucker et al. (2004), U.S.A. [26CRC cancer: 17.1 μM increase associated with a lower prevalence for CRC (OR 0.257):
Men: OR 0.295
Women: OR 0.186
Non-GI cancers (OR 0.809):
Men: OR:1.619
Women: OR 0.901 
Total: 16 865 Men: 7892; women: 8973 Range: ≥17 Cross-sectional study Bilirubin (μM) 10.6±0.1.
Range: 0–135.1.
Women: 8.9±0.1.
Men: 12.3±0.1 
NA No Wide bilirubin range; possible underlying liver pathology due to wide total bilirubin range 
Lacko et al. (2010), The Netherlands [202Head and neck cancer: OR 1.57
(TA)6/6 compared with (TA)7/7.
Logistical regression: more (TA)6/6 among the patients; (TA)6/6 is associated with increased risk of cancer 
Total: 838 (cases/controls).
Men: 333/323;
women: 88/94 
Cases: 61 (23–91);
controls: 57 (36–91) 
Case–control NA (TA)7/7 vs (TA)6/6: 0.78 (TA)7/7 vs (TA)6/6+(TA)6/7: 0.83 (men and women) Yes Only total bilirubin measured 
Horsfall et al. (2011), U.K. [21Lung cancer.
Men: incidence in first decile (3.1–5.8 μM): 5.0 with last decile (19.0–40.0 μM): 1.5;
woman: incidence in first decile (3.1–4.8 μM): 3.0 with last decile (15.1–29.9 μM): 0.8 
Total: 504 206.
Men: 218 727;
women: 285 479 
Men: 54±16;
women: 55±19 
Cohort study, follow-up 8 years Bilirubin: 10 μM (median) in men and 9 μM in women NA No Method: only total bilirubin measured, early cut-off (exclusion of men >40 μM and women >30 μM) 
Jirásková et al. (2012), Czech Republic [161CRC negatively associated with (TA)7/7. (TA)7/7 less frequent in CRC patients total: OR 0.80; men: OR 0.75. Lower bilirubin levels in cases. A decrease of 1 μM of bilirubin was associated with a 7% decrease of CRC risk Total: 1763 (cases/controls).
Men: 453/571;
women: 324/415 
Cases: 61.8±11;
controls: 49.2±19 
Case–control Bilirubin (μM) in cases: 9.8;
men: 10.1;
women: 9.2.
Bilirubin (μM) in controls: 11.4;
men: 12.8;
women: 10.2 
(TA)7/7 vs (TA)6/6: 0.89; (TA)7/7+(TA)7/6 vs (TA)6/6: 0.88;
(TA)7/7 vs (TA)6/6+(TA)6/7: 0.95 (men and women) 
Yes Only total bilirubin measured
Smoking status not reported 
Bajro et al. (2012), Macedonia [201(TA)7/7 more frequent in CRC patients [OR 1.55, (TA)6/60]. Only in men trend to higher cancer (OR 2.67, P=0.08). No difference in women Cases: 168; controls: 96 Cases: 61.6;
controls: 78.5 
Case–control NA (TA)7/7 vs (TA)6/6: 1.29 (men and women). Men: (TA)7/7 vs (TA)6/6: 1.63 (TA)7/7 vs 6/6+6/7: 1.35 Yes Low number of participants, no total bilirubin/UCB measured, and no adjustment for age, meat intake, smoking, heterogeneous groups 
Karakosta et al. (2014), Greece [203(TA)7/7 polymorphism in chronic lymphocytic leukaemia (CLL).
Positive association of (TA)7/7 carrier and CLL-specific abnormalities, no difference in distribution of (TA)7/7 genotype between cases and controls 
Cases: 109;
controls: 108 
NA Case–control NA NA Yes Low number of participants.
No total bilirubin/UCB measured 
Grant et al. (2004), U.S.A. [206Cancer-associated biomarker:
MNi, in smokers with high genotype [(TA)7/7; (TA)8/8]; positive association among kinetochore-positive MNi (low number) 
Total: 101 Smokers: 34.2;
Non-smokers: 30.5 
Cohort study, community-based subsample NA NA Yes Low number of participants.
No total bili-rubin/UCB measured 
Wallner et al. 2012 Austria; [205No difference in MNi formation and chromosomal damage in GS vs controls Total: 76.
GS: 38;
controls: 38 
GS: 32.3;
controls: 31.9 
Case–control study/cross-sectional design UCB (μM):
GS: 32±13.6;
controls: 10.3±3.31 
NA No Low number of participants, young population 
Source Type of cancer and outcome Number of participants Age (years) Study design Bilirubin levels Relative risk (RR) Genotyped Limitations 
Ko et al. (1994), U.S.A. [200CRC (no association) Total: 236.
Cases: 118;
controls: 118 
Range: <25–75 Case–control Lower bilirubin levels among cancer patients NA No Low number of cases, only total bilirubin measured 
Guillemette et al. (2001), U.S.A. [194UGT and breast cancer (no association)
OR 1.28 in (TA)7/7 (NS) 
Total: 1064.
Cases: 455;
controls: 609 
Range: 30–55 Case–control nurses' health NA (TA)7/7 vs (TA)6/6: 0.92
(TA)7/7 vs (TA)6/6+(TA)6/7: 0.94 (men and women) 
Yes No total bilirubin/UCB measured 
Temme et al. (2001), Belgium [25Men
Total cancer: bilirubin ≥10.3 μM, RR 0.31/0.42 [P=0.0001/0.004 (unadjusted/adjusted)].
Lung cancer: bilirubin ≥10.3 μM, RR 0.27/0.40 (P 0.0034/0.0898).
Non-lung cancer: total bilirubin ≥10.3 μM, RR 0.34/0.43 (P=0.0008/0.0195)
Women
Total cancer, non-lung cancer, lung cancer: total bilirubin ≥8.55 μM, RR no sign 
Total: 10 303 Men: 5460; women: 4843 Range: 25–74 Prospective including 10-year follow-up Bilirubin (μM):
men: 7.5, range: 1.7–64.6;
women: 10, range: 1.7–64.6 
Highest vs lowest bilirubin group (RR: calculated per 1000 person-years).
Men: total cancer: 0.20; lung cancer: 0.17; non-lung cancer: 0.21.
Women: total cancer: 0.92 
No Detection method: only total bilirubin measured, smoking 
Ching et al. (2001), Australia [193Breast cancer (OR 0.5 in highest vs lowest bilirubin quartile) (>4.1 μM/>7.5 μM), reduced risk.
Only women 
Total: 304.
Cases: 153;
controls: 151 
Range: 30–84 Case–control NA Highest vs lowest bilirubin group.
Breast cancer: 0.74 
No Low number of participants
No total bili-rubin/UCB levels of cases vs controls 
Zucker et al. (2004), U.S.A. [26CRC cancer: 17.1 μM increase associated with a lower prevalence for CRC (OR 0.257):
Men: OR 0.295
Women: OR 0.186
Non-GI cancers (OR 0.809):
Men: OR:1.619
Women: OR 0.901 
Total: 16 865 Men: 7892; women: 8973 Range: ≥17 Cross-sectional study Bilirubin (μM) 10.6±0.1.
Range: 0–135.1.
Women: 8.9±0.1.
Men: 12.3±0.1 
NA No Wide bilirubin range; possible underlying liver pathology due to wide total bilirubin range 
Lacko et al. (2010), The Netherlands [202Head and neck cancer: OR 1.57
(TA)6/6 compared with (TA)7/7.
Logistical regression: more (TA)6/6 among the patients; (TA)6/6 is associated with increased risk of cancer 
Total: 838 (cases/controls).
Men: 333/323;
women: 88/94 
Cases: 61 (23–91);
controls: 57 (36–91) 
Case–control NA (TA)7/7 vs (TA)6/6: 0.78 (TA)7/7 vs (TA)6/6+(TA)6/7: 0.83 (men and women) Yes Only total bilirubin measured 
Horsfall et al. (2011), U.K. [21Lung cancer.
Men: incidence in first decile (3.1–5.8 μM): 5.0 with last decile (19.0–40.0 μM): 1.5;
woman: incidence in first decile (3.1–4.8 μM): 3.0 with last decile (15.1–29.9 μM): 0.8 
Total: 504 206.
Men: 218 727;
women: 285 479 
Men: 54±16;
women: 55±19 
Cohort study, follow-up 8 years Bilirubin: 10 μM (median) in men and 9 μM in women NA No Method: only total bilirubin measured, early cut-off (exclusion of men >40 μM and women >30 μM) 
Jirásková et al. (2012), Czech Republic [161CRC negatively associated with (TA)7/7. (TA)7/7 less frequent in CRC patients total: OR 0.80; men: OR 0.75. Lower bilirubin levels in cases. A decrease of 1 μM of bilirubin was associated with a 7% decrease of CRC risk Total: 1763 (cases/controls).
Men: 453/571;
women: 324/415 
Cases: 61.8±11;
controls: 49.2±19 
Case–control Bilirubin (μM) in cases: 9.8;
men: 10.1;
women: 9.2.
Bilirubin (μM) in controls: 11.4;
men: 12.8;
women: 10.2 
(TA)7/7 vs (TA)6/6: 0.89; (TA)7/7+(TA)7/6 vs (TA)6/6: 0.88;
(TA)7/7 vs (TA)6/6+(TA)6/7: 0.95 (men and women) 
Yes Only total bilirubin measured
Smoking status not reported 
Bajro et al. (2012), Macedonia [201(TA)7/7 more frequent in CRC patients [OR 1.55, (TA)6/60]. Only in men trend to higher cancer (OR 2.67, P=0.08). No difference in women Cases: 168; controls: 96 Cases: 61.6;
controls: 78.5 
Case–control NA (TA)7/7 vs (TA)6/6: 1.29 (men and women). Men: (TA)7/7 vs (TA)6/6: 1.63 (TA)7/7 vs 6/6+6/7: 1.35 Yes Low number of participants, no total bilirubin/UCB measured, and no adjustment for age, meat intake, smoking, heterogeneous groups 
Karakosta et al. (2014), Greece [203(TA)7/7 polymorphism in chronic lymphocytic leukaemia (CLL).
Positive association of (TA)7/7 carrier and CLL-specific abnormalities, no difference in distribution of (TA)7/7 genotype between cases and controls 
Cases: 109;
controls: 108 
NA Case–control NA NA Yes Low number of participants.
No total bilirubin/UCB measured 
Grant et al. (2004), U.S.A. [206Cancer-associated biomarker:
MNi, in smokers with high genotype [(TA)7/7; (TA)8/8]; positive association among kinetochore-positive MNi (low number) 
Total: 101 Smokers: 34.2;
Non-smokers: 30.5 
Cohort study, community-based subsample NA NA Yes Low number of participants.
No total bili-rubin/UCB measured 
Wallner et al. 2012 Austria; [205No difference in MNi formation and chromosomal damage in GS vs controls Total: 76.
GS: 38;
controls: 38 
GS: 32.3;
controls: 31.9 
Case–control study/cross-sectional design UCB (μM):
GS: 32±13.6;
controls: 10.3±3.31 
NA No Low number of participants, young population 

However, reports concerning breast cancer are contradictory. Guillemette et al. [194] could not reveal an association between the (TA)7/7 genotype and breast cancer in women. Limitations of this study were, however, the lack of bilirubin measurement and missing age information for the participants. It is interesting that a Russian study revealed a higher rate of the (TA)7/7 genotype [odds ratio (OR) 1.79] distribution among breast cancer patients compared with the (TA)6/6 wild type [195]. Contradictory results that have been reported might be due to gender differences in metabolism. Females generally have lower bilirubin levels than males, and a potential ‘oestrogen effect’ should be considered. The complex (feedback) effects of male and female (oestrogen) sex hormones on UGT activity are currently poorly understood [196]. UGT1A1 contributes to the biotransformation of oestrogen, which plays a substantial role in breast cancer carcinogenesis [197], based on the hypothesis that oestrogen excretion is lower in hyper- than in normo-bilirubinaemic women. Although the protective role of oestrogen is believed to account for the differences in cardiovascular risk between males and females [198], the factors responsible for the differences in the UGT1A1 promoter variants and cancer risk in males and females are less clear and require further careful investigation. The involvement of UGT1A1 variants in the metabolism of sex hormones and xenobiotics [199], as well as their feedback effects on UGT1A1 activity [196], might, however, contribute to the gender differences observed.

The importance of delineating gender effects is necessary because the association with CRC for each (17 μM) 1 mg/dl increase in serum bilirubin is more pronounced in women than in men (OR women 0.186, men 0.295, total 0.257). However, this does mean that individuals with phenotypic GS, independent of gender, have an approximately four times reduced risk of CRC compared with non-GS individuals [26]. In a very recent study, TA(7/7) carrier status was associated with a 20% reduction in CRC risk (OR 0.80; P<0.022) after age adjustment. This effect was, however, more substantial in males (OR 0.75) than females (OR 0.88) [161].

No significant association between CRC and bilirubin was reported in a study by Ko et al. [200], although these authors observed a trend towards reduced bilirubin levels in cancer patients. Contradictory results were published by the Macedonian authors Bajro et al. [201], with a higher frequency of homozygous [(TA)7/7]+heterozygous [(TA)6/7] carriers compared with wild-type [(TA)6/6] in patients with CRC. However, when homozygous individuals [(TA)7/7] were compared with heterozygous [(TA)6/7]+wild-type [(TA)6/6], there was no significant difference in the frequency of cases. The same effect was detected when only men were considered and no differences were found for women [201]. Nevertheless, limitations in these analyses were clear in that adjustment for age, smoking and red meat intake was missing from these reports. The analysis of bilirubin in investigated cohorts is frequently missing, but is critical to understanding whether bilirubin itself is an important factor in protection. Therefore reporting of bilirubin levels and genotype in observational studies is recommended.

In a prospective study, bilirubin concentrations were of borderline significance in predicting lung cancer incidence, although the non-lung cancer incidence was associated with bilirubin after adjustment for risk factors (e.g. age, smoking, BMI, cholesterol) in men but not in women [25]. In contrast, a retrospective study of 504 206 participants revealed a significant negative association between bilirubin and lung cancer in both sexes (incidence rate ratio: men 0.92 and women 0.89) after adjustment for multiple, potentially confounding factors, such as age, BMI, blood pressure, smoking and alcohol. The incidence of lung cancer was lowest in the highest decile of bilirubin level in both sexes (men: 1.11–2.34 mg/dl, 19–40 μM; women: 0.88–1.75 mg/dl, 15–29.9 μM) [21]. A case–control study in patients with head and neck cancer revealed a higher risk for such cancers in homozygous carriers of the (TA)6/6 (wild-type) genotype compared with the homozygous carriers of the (TA)7/7 genotype (OR 1.57 vs 1) [202]. In contrast, the (TA)7/7 genotype has been positively associated with chronic lymphocytic leukaemia, although no significant differences in genotype distribution between cases and controls were observed [203].

Studies on DNA damage (SCGE/Comet Assay) and micronuclei (MNi) formation, as biomarkers for future cancer prevalence [204], have not revealed reduced counts of cellular abnormalities in human cross-sectional studies in individuals with GS versus age- and gender-matched controls. This might have been limited by the fairly young age of the participants (GS patients 32.3 years vs controls 31.9 years) and healthy study population [205]. The (TA)7/7 polymorphism status of individuals should be considered very carefully because a higher frequency of cancer-associated, kinetochore-positive MNi has been reported in homozygous carriers, but only in smokers [206]. In this context, the condition of reduced UGT1A1 activity in individuals with GS may reduce detoxification of phytochemicals, drugs and other toxic substances, and could be responsible for some of the positive associations between GS and cancer that have been reported [195,201,203].

There are almost no in vivo experimental data available to address the mechanism of bilirubin's possible anti-carcinogenic effects. The translation of mechanistic animal studies is complicated because there is only one rat model of unconjugated hyperbilirubinaemia, which exhibits a homozygous UGT1A1 mutation with almost no UGT1A1 enzyme activity and, therefore, potentially neurotoxic concentrations of UCB are produced. In geno-toxicity studies using UGT1A1-deficient rat skin fibroblasts, UGT1A1 status may act in a genoprotective manner. In a recent study, only peripheral blood mononuclear cells, but not colonocytes and hepatocytes, of Gunn rats had higher DNA strand breaks compared with normobilirubinaemic controls [207]. An explanation for genotoxicity might include no or a late metabolic clearance of drugs by phase II enzymes [208,209], based on a complete absence of UGT1A1 from these animals [210].

Anti-genotoxic/chemopreventive action of bilirubin and bile pigments

The interaction of tetrapyrroles with pro-mutagens/mutagens was first suggested several years ago [8, 211], with direct 3D co-ordination including stacking or non-covalent/covalent complex formation proposed. Interactions with the synthetic mutagen 2,4,7-trinitro-9-fluorenone, as has been confirmed for a series of porphyrins and tetrapyrrole compounds, may account for the important anti-mutagenic effects of bile pigments [213,214]. Furthermore, bilirubin and its derivatives induce cell cycle arrest [215], apoptosis and cytostasis in multiple cancer cell lines [216,217], which indicates a possible anti-carcinogenic effect of bilirubin. Bilirubin may also protect against cancer by interfering with pro-carcinogenic signalling pathways [via activation of extracellular-signal-regulated kinase (ERK)], and inhibit tumour cell proliferation [216]. Furthermore, bilirubin induces mitochondrial depolarization in colon cancer cells, and provokes apoptosis in human gastric cancer cells [215]. Anti-genotoxic activity (DNA damage) seems to be present in malignant cells only [4,217]. In a recent study, investigating the anti-genotoxic effects of intestinally abundant tetrapyrroles (urobilin, stercobilin, protoporphyrin), DNA-damaging effects were reported in HepG2 and Caco2 cells. Furthermore, correlations between tetrapyrrole concentration and apoptosis and cell cycle arrest were observed. These results suggest a possible chemopreventive effect of tetrapyrroles within the liver and intestine [217]. In terms of dietary pro-mutagens/mutagens and intestinal protection, intestinally abundant tetrapyrroles counteract aflatoxin B1 and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine mutagenesis, which emphasizes the importance of these molecules for health [11].

In conclusion, the above reports concerning in vitro anti-genotoxic activity of tetrapyrroles support a conclusion that the compounds may possess an additional physiological role as anti-mutagens and possibly as chemopreventives. The relationship between bilirubin and cancer prevalence is not as strong as that with CVD. This emphasizes the important need for further research to understand the present results and build on them, and to document what role bilirubin might have in preventing cancer.

BILIRUBIN AND NEURODEGENERATIVE DISEASES

The brain is particularly sensitive to oxidative stress due to its increased content of oxidizable lipids, reliance on oxidative metabolism and a relatively poor antioxidant defence system. The last in particular is present soon after birth (in the undeveloped CNS) [218] or in elderly people [219]. The antioxidant and anti-inflammatory properties of bilirubin could, therefore, be important in the manifestation of diseases affecting the CNS. Few epidemiological data describe the correlation between bilirubin and neurological diseases, but there is a consistently reduced serum bilirubin level reported in patients with neurological conditions.

In Alzheimer's disease, despite being activated, the bilirubin system does not seem to be sufficiently active to overcome the pathological mechanism driven by the accumulation of amy-loid β-peptide (Aβ). Alzheimer's disease is one of the most common neurodegenerative diseases, involving the hippocampus, amygdala and frontal cortex, and thus leading to cognitive and memory deficits. In Alzheimer's disease, the insertion of Aβ in the cell bilayer causes oxidative stress, inflammation, glutathione depletion, mitochondrial dysfunction with bioenergetic crisis and apoptosis [220,221]. Despite being up-regulated, the HMOX1-BLVRA complex is insufficiently active to counteract the relevant oxidative/nitrosative challenge that characterizes Alzheimer's disease. Paradoxically HMOX1 activation might increase carbon monoxide- and iron-induced cellular stress, and consume the biliverdin/UCB initially produced by the synergic BLVRA up-regulation. In line with the initial up-regulation of the HMOX1/BLVRA cascade is an increased concentration of bilirubin in the cerebrospinal fluid of patients with Alzheimer's disease [222]. Thereafter, when exposed to the oxidative/nitrosative milieu, BLVRA undergoes post-translational modification (hypophosphorylation of serine/threonine/tyrosine residues) inhibiting its activity (despite the increased protein levels detected in patient tissue samples), leading to reduced bilirubin production and the dysregulation of the ERK1/2 signalling (reviewed by Barone et al. [223]).

A similar observation may apply to Parkinson's disease, another neurological condition in which free radicals are widely considered to be responsible for the onset of disease. In Parkinson's disease, HMOX1 staining reveals intracellular aggregates (Lewy bodies) in the affected dopaminergic neurons of the substantia nigra. In the same region, the activation of astroglia is characterized by increased HMOX1 expression, probably triggered by molecules released from dying neurons. Increased HMOX1 activity promotes iron accumulation and electron chain transport deficit in mitochondria [224]. This is a paradox where the over-expression of HMOX (especially in astroglia) rather than an adaptive response to stress “is a common pathway leading to pathological iron deposition and oxidative mitochondrial damage in a host of human neurological disorders” [224], and a marker of cellular damage.

Interestingly, patients with Parkinson's disease who have been exposed to L-dopa possess higher plasma bilirubin levels than untreated patients with Parkinson's disease or controls. It has been supposed that L-dopa both enhances ROS production and acts as an antioxidant itself. The increased bilirubin level observed in L-dopa-treated individuals may indicate an improved ability of the UCB system to combat ROS formation [225].

Silent cerebral infarction (SCI) is usually discovered during routine brain imaging in the absence of clinical symptoms. SCI may precede and thus predict major diseases such as ischaemia, dementia, stroke and CVD. In a large study including 2865 participants, the 343 SCI cases had lower total plasma bilirubin levels. After adjustment for conventional vascular risk factors, a very strong and significant association between higher plasma bilirubin and reduced risk of SCI was detected (OR 0.925; P<0.001) [226]. These data are consistent with the observation of a negative association between serum bilirubin and risk of leukoaraiosis, as discussed above [178].

A correlation between reduced concentrations of UCB and a pathology duration of >2 years was also reported in patients with multiple sclerosis; however, a clear cause–effect role of bilirubin was not demonstrated [227]. A reduced plasma bilirubin concentration was also detected in patients with mild clinical amyotrophic lateral sclerosis despite HMOX up-regulation, suggesting that UCB-mediated defence may be compromised in the degeneration of motor neurons caused by oxidative damage [228].

The role of bilirubin as a biomarker or predictor of schizophrenia is less clear. Although some studies suggest that bilirubin is a risk factor for the development of schizophrenia [229,230], others reported the opposite association [231,232]. In our own study, in which serum bilirubin concentrations were studied together with UGT1A1 genotypes in patients with schizophrenia, substantially reduced bilirubin concentrations were observed in patients across all genotypes, suggesting consumption of this important endogenous antioxidant by the increased oxidative stress accompanying schizophrenia [233].

Low nocturnal bilirubin concentrations are associated also with the risk of winter depression (seasonal affective disorder) [234], due to a presumed effect of bilirubin on the circadian rhythm [235].

Despite being considered ‘constitutive’, HMOX2 is regulated by adrenal glucocorticoids, glutamate and nitric oxide (NO), as well as by CNS drugs such as morphine and glucocorticoids [32]. Its main role seems mostly related to the maintenance of the cell biliverdin levels required to generate UCB [236]. For this reason its contribution to diseases merits further attention.

If a general rule should be deduced in the context of neurodegenerative disease, the up-regulation of the HMOX1 enzyme does not seem to be beneficial. On the other hand, lower serum bilirubin concentrations are detected in affected individuals. An explanation for this apparent contradiction may reside in better comprehension of the ‘bilirubin system’ and the CNS's specific susceptibility to oxidative stress. Both clinical and experimental data agree that, under sustained stress, the protective activation of the HMOX1/BLVRA system is switched and becomes deleterious. In this respect, the low serum bilirubin level observed should be interpreted as a sustained consumption of bilirubin during its activity as a scavenger of ROS. Low levels of serum UCB may indicate a sustained pro-oxidant situation, indicating increased consumption compared with production/regeneration. Theoretically, the supply of UCB (alone, without the concomitant production of iron) within the brain may be helpful in the prevention or progression of neurological diseases. Convincing epidemiological studies on the relationship between neurological conditions and chronic unconjugated hyperbilirubinaemia (such as in patients with GS or Crigler–Najjar syndrome) are scarce and future studies are required to identify clear relationships.

BIOMARKERS FOR CHRONIC DISEASES OR ALL-CAUSE MORTALITY–A ROLE FOR BILIRUBIN?

There are several definitions for the term ‘biomarker’, one of them by the U.S. National Institutes of Health, which defines it as: “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention” [237].

Numerous biomarkers are in use, but many of them fail to meet the definition due to various limitations concerning the marker itself, the system used for its assessment (in vivo vs in vitro–at present around 90% of antioxidative experiments are conducted in vitro with established cell lines or subcellular fractions but not in humans or animals), the matrix used for its assessment (food, cells, blood, plasma, urine, DNA) or the complexity of the measurement technique (e.g. ELISA, enzyme activities, HPLC, GC, MS coupling, -omics techniques), or simply due to the inappropriate study designs used to assess their prognostic potential [238].

For chronic diseases, a plethora of markers is currently proposed, but only a few are generally accepted. Examples of biomarkers that have been incorporated into clinical practice, and shown to have value in addition to traditional cardiovascular risk factor analysis, include N-terminal pro-B-type natriuretic peptide (NT-proBNP) for heart failure [239], glycated haemoglobin (HbA1c) for glycaemic control in diabetes [240], high-sensitivity troponin I [241] and high-sensitivity CRP for cardiovascular risk prediction [242].

In addition, ‘classic’ markers are accepted such as LDL-C, e.g. from the European Food Safety Authority to establish a disease reduction claim [243]. Furthermore, the transition ‘from bench to bedside’ for each new biomarker discovered must be associated with concurrent advances in the characterization of analytical features and development of routine assays. Not only the person performing the tests, but also mainly the physician/clinician, should be appropriately trained in the use of the biomarker in order to be able to request the test accurately and interpret the results correctly. Moreover, the implementation of recently described biomarkers into clinical practice calls for a co-ordinated and concerted effort of clinicians, biochemists, epidemiologists and technology experts to translate the new insights into improved diagnosis and/or management of patients. Bilirubin has become a molecule of interest in this respect and has strong potential as a biomarker. We believe that the present review has indicated the potential of bilirubin as a biomarker of many chronic diseases, in the absence of hepatobiliary pathology and haemolytic conditions.

As published data on this topic remain heterogeneous (in respect of the conditions against which bilirubin may protect), it remains inappropriate to consider total bilirubin as a confident biomarker for cancer and neurological conditions. However, it is acknowledged that total bilirubin probably represents a strong candidate for prediction of future and existing CVD and related mortality. Additional mechanistic and epidemiological data are clearly required to confidently conclude whether bilirubin can predict future cancer (particularly for individual cancers) and neurological disease; however, it should be noted that existing data indicate a promising role for this molecule in future.

STEPS NEEDED IN FUTURE BILIRUBIN RESEARCH

The summary of data in the present review is promising and, in the context of CVD, can be somewhat convincing that bilirubin represents a viable biomarker. Further research is needed in the field of cancer biology and neurophysiology to determine what, if any, role bilirubin and related compounds might have in predicting and modulating disease processes. These conclusions justify the need for investment and engagement in bilirubin research in the short and medium term.

Also due to improvements in measurement techniques and analytical tools, new fields, including -omics-based platforms, will assist in generating new hypotheses to rapidly progress the field and break new ground in bilirubin research. We propose the following recommendations for focused research in the near future:

  • Investigation of the interaction of gender, oestrogen and lipid metabolism in individuals with or without GS.

  • Completion of genomic, metabolomic and proteomic profiling of individuals with GS vs controls.

  • Focused exploration of the effects of UCB on different cancer cell types and animal models of dietary-induced malignancy.

  • Improved understanding of the link between various cancer types and total bilirubin/UCB in prospective studies.

  • Deeper mechanistic insight and better differentiation of which factors, besides antioxidant effects, are responsible for CVD and total mortality protection.

  • Exploration of the role of bilirubin in the aging process.

Clinical Perspectives

Chronic disease prevalence is increasing worldwide and the need for appropriate biomarkers in the clinical setting to predict the risk of important pathologies such as CVD, cancer, diabetes and all-cause mortality is evident.

One very promising marker in this respect appears to be total bilirubin or, even better, UCB, which is strongly associated with a reduced prevalence of CVD and is thought to be linked to reduced prevalence of CVD, cancer, diabetes and all-cause mortality. The total bilirubin concentration (as part of standard liver function tests) can easily be detected and interpreted in the clinical environment either in the blood or by polymorphism analysis.

The studies reviewed here significantly contribute to a topic that has so far been under-appreciated in clinical research and practice. Compared with other biomarkers such as cholesterol, bilirubin research indicates a new and promising direction to explore for the prevention of disease (CVD, cancer, neurodegenerative disease). This emphasizes the importance of studying people with a benign condition (GS) that affects millions of people worldwide, who may be protected from disease.

FUNDING

This work was supported by the Czech Ministry of Education [grant numbers LH11030 and PRVOUK 4102280002], and the Czech Ministry of Health [grant number RVO VFN64165 (to L.V.)]. Furthermore, this work was supported by the Austrian Science Fund [grant number P21162-B11 (to K.-H.W., C.M., M.W. and A.B.)] and the Research Platform Active Ageing (to K.-H.W.). The financial support of an intramural grant from the Italian Liver Foundation (to S.G. and C.T.) is also acknowledged.

Abbreviations

     
  • amyloid β-peptide

  •  
  • BLVRA

    biliverdin reductase

  •  
  • BMI

    body mass index

  •  
  • C

    cholesterol

  •  
  • CAD

    coronary artery disease

  •  
  • CHD

    coronary heart disease

  •  
  • CNS

    central nervous system

  •  
  • CRC

    colorectal cancer

  •  
  • CRP

    C-reactive protein

  •  
  • CVD

    cardiovascular disease

  •  
  • ERK

    extracellular-signal-regulated kinase

  •  
  • GS

    Gilbert's syndrome

  •  
  • HDL

    high-density lipoprotein

  •  
  • HMOX

    haem oxygenase

  •  
  • ICAM-1

    intercellular adhesion molecule-1

  •  
  • IL

    interleukin

  •  
  • LDL

    low-density lipoprotein

  •  
  • MNi

    micronuclei

  •  
  • MRP2

    multidrug-resistance protein 2

  •  
  • NF-κB

    nuclear factor κB

  •  
  • NHANES

    National Health and Nutrition Examination Survey

  •  
  • OR

    odds ratio

  •  
  • ROS

    reactive oxygen species

  •  
  • SCI

    silent cerebral infarction

  •  
  • SNP

    single nucleotide polymorphism

  •  
  • TNF-α

    tumour necrosis factor α

  •  
  • UCB

    unconjugated bilirubin

  •  
  • UGT

    UDP-glucuronosyltransferase

  •  
  • UGT1A1

    bilirubin UDP-glucuronosyltransferase

  •  
  • VCAM-1

    vascular cell adhesion molecule-1

  •  
  • VSMC

    vascular smooth muscle cell

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