Bilirubin is a metabolic byproduct of the breakdown of hemoglobin degradation which itself must be metabolized for appropriate excretion. High levels of bilirubin are associated with decreased risk of coronary heart disease (CHD) and cardiovascular disease (CVD) [1]. While the full spectrum by which bilirubin acts to protect against CVD is not fully understood, there has been evidence of protecting against oxidative stress by reducing reactive oxygen species and possibly having additional anti-atherogenetic properties [2, 3]. Previous studies have reported associations of serum bilirubin levels to cardiovascular disease risk factors, including total cholesterol and blood pressure [4–6]. Serum bilirubin levels have also been associated with socioeconomic and behavioral CVD risk factors such as smoking and alcohol intake [7, 8]. However, the nature of these associations is unclear, including the potential for residual confounding among serum bilirubin levels and the associated CVD risk factors due to factors measured poorly or not measured at all. Notably, the temporal ordering of variation in these variables is hard to determine, raising challenges in separating 'cause' from 'effect'.

Genotype is uniquely unaffected by most other epidemiologic risk factors. If one accepts the notion that genotypes are in a sense "randomly assigned" during gamete formation, then using genetic variation as an independent variable is attractive. Relationships between genotype and outcomes have only limited susceptibility to confounding and in particular are generally not subject to temporal misspecification of 'cause' and 'effect' (i.e., reverse causality). Thus, genetic variation can be used to aid in inferring causality between a gene product and some outcome of interest. If a genetic variant is strongly causal of a measured exposure, the variant can be used as a proxy for exposure during modeling. This approach, known as Mendelian randomization, can provide evidence of causal inference between two variables [9]. Mendelian randomization has particular utility for evaluation of biomarkers that are highly influenced by a single gene, are of interest in a disease process, and are highly subject to confounding.

The gene UGT1A1 codes for a liver-specific glucoronosyl transferase that converts bilirubin into a more water-soluble form the body is better able to excrete. Homozygosity at the polymorphic promoter repeat locus UGT1A1*28 leads to decreased ability of the UGT1A enzyme to metabolize bilirubin and subsequent mild hyperbilirubinuria (also known as Gilbert's syndrome) [10–12]. Due to UGT1A1*28's strong causal link to bilirubin, use of the UGT1A1*28 genotype to more precisely assess bilirubin - CVD risk factor associations is an attractive and informative application of the Mendelian randomization approach.

In this study, we describe use of the Mendelian randomization approach to evaluate relations of serum bilirubin levels with CVD risk factors and subclinical atherosclerosis in a sample of relatively healthy Old Order Amish individuals from Lancaster County, PA. We subsequently contrast the observed crude estimates with those obtained utilizing Mendelian randomization to illustrate its use. Separating causal from non-causal relations provides evidence of bilirubin's mechanistic role in reducing CVD.

Associations between serum bilirubin and multiple CVD risk factors have been previously reported, but attribution of causality has been challenged by the potential for confounding and uncertainty regarding temporal ordering [24]. Importantly, Lin et. al. [1] utilized genotype at the UGT1A1*28 locus to suggest a causal role for bilirubin in cardiovascular and coronary heart diseases. Most notably, using longitudinal data over 24 years of follow up, they demonstrated a striking unconfounded association with CVD events. While that study supports the hypothesis that increased bilirubin levels cause a decreased risk of CVD, it did not provide estimates of the causal relation of bilirubin with markers of cardiovascular disease burden and hence did not help identify the underlying pathway by which bilirubin might protect against CVD events. In this study, using Mendelian randomization, we attempt to extend previous studies by exploring the association between bilirubin and sub-clinical markers of vascular reactivity and CVD burden in "healthy" subjects, to help delineate potential early mechanistic pathways related to bilirubin. Of note, we find an association between bilirubin and brachial artery diameter and cold pressure reactivity, while utilizing genotype as an instrumental variable per Mendelian Randomization. This association between bilirubin and brachial artery diameter was not present in our data using the traditional regression approach, most likely due to the presence of confounding factors. In this case, it appears that confounding acts to hide or underestimate a potentially causal association. If one believes UGT1A1*28 locus is a reliable instrument of bilirubin levels, these data provide evidence that bilirubin may act on CVD via mechanism associated with brachial artery diameter.

While baseline brachial artery diameter had traditionally been measured to derive brachial flow mediated dilation measures as a surrogate of endothelial function, several studies have demonstrated an independent association between brachial artery diameter with coronary artery calcification [25], serum uric acid [26], blood pressure, serum lipids, smoking and glucose [27, 28], carotid IMT [29], pregnancy [30], severity of chronic heart failure [31], angiographic measures of coronary artery disease [32] and CVD events in a cohort of over 2500 subjects [33]. Brachial artery size is also a determinant not only of both FMD and time to peak FMD, but also of non-endothelial related vasodilatory capacity [34]. These studies clearly demonstrate repeatable and significant associations between brachial artery diameter and both CVD risk factors, outcomes and measures of vascular disease, and suggest that in certain populations, it may be more predictive of CVD than FMD measures. As such, our finding of smaller brachial artery diameter in hyperbilirubinemic subjects is consistent with the significant decrease in CVD disease noted in subjects with Gilbert's syndrome [1, 35] and raises a potential new mechanism associated with the CVD protective effects of bilirubin, separate from its described antioxidant and anti-inflammatory properties [24, 36]. The CPT, by measuring blood pressure change in response to cold stimulus, has been used as a measure of global sympathetic activity and/or response. Our study shows a modest association between bilirubin and increased CPT reactivity. While several studies have shown that increased reactivity in healthy populations may be associated with increased probability for hypertension, the results have not always been consistent, notably in healthy cohorts such as in this study [37, 38]. One reason for this could be that increased CPT reactivity may reflect either increased sympathetic activity, which is likely not protective of CVD or may be due to increased reactivity of blood vessels to sympathetic stimulus, which may be a reflection of healthy vasculature and less CVD. Indeed, ageing and obesity are associated with decreased vascular response to sympathetic activity and norepinephrine [39–41]. As such, it is plausible that higher bilirubin levels may be associated with increased vascular response capacity as opposed to an increase in sympathetic neural activity. The lack of association between the UGT1A1*28 genotype and heart rate also suggests no direct increase in baseline sympathetic activity. Mechanistic pathways for the well noted observation of smaller brachial artery diameter with decreased measures of CVD have not yet been elucidated. We did not find an association between the UGT1A1*28 genotype and markers of more advance vascular pathology such as PWV or IMT. This could be secondary to the absence of any such association or also because we studied a relatively young and healthy population with a low CVD disease burden.

In conclusion, we have utilized Mendelian randomization techniques to address confounding in the estimation of bilirubin's causal relationship with known CVD risk factors. Our data suggest that many of the associations from observational epidemiology studies reported in the literature are subject to un-accounted for confounding and/or reverse causality. Of the tested risk factors, brachial artery diameter and to a lesser degree CPT reactivity were significantly associated with genotype at the UGT1A1*28 locus, providing compelling evidence that bilirubin affects CVD through pathways associated with artery size such as vasomotor tone, reactivity and possibly arterial wall structure.