Characteristics of the heme catabolic pathway in mild unconjugated hyperbilirubinemia and their associations with inflammation and disease prevention

Abstract

Heme catabolism exerts physiological functions that impact health through depressing inflammation. Upon reactive pathway progression, as in Gilbert's Syndrome (GS; UGT1A1*28 polymorphism), aggravated health effects have been determined. Based on lower inflammation and improved metabolic health reported for GS, inter-group differences in heme catabolism were explored. Therefore, a case-control study including 120 fasted, healthy, age- and gender matched subjects with/without GS, was conducted. Genetic expressions of HMOX-1 and BLVRA were measured. Additionally participants were genotyped for those polymorphisms that are known (UGT1A1*28) or likely (HMOX-1 microsatellites) to impact bilirubinemia. Intracellular interleukins (IL-6, IL-1β, TNFα), circulatory C-reactive protein (CRP), serum amyloid A (SAA) and haptoglobin (Hpt) were analysed as inflammatory markers. To assess intracellular heme oxygenase 1 (HO-1) isolated PBMCs were used. In GS vs. C, inflammation markers were significantly decreased. This was supported by an altered heme catabolism, indirectly reflecting in elevated unconjugated bilirubin (UCB; main phenotypic feature of GS) and iron, decreased hemopexin (Hpx) and Hpt and in up-regulated biliverdin reductase (BLVRA) gene expressions. Moreover, HMOX (GT)_n short alleles were non-significantly more prominent in female GS individuals. Herewith, we propose a concept to elucidate why GS individuals encounter lower inflammation, and are thus less prone to oxidative-stress mediated diseases.

Figure 1

Differences in the inflammatory status between GS and C individuals (all subjects). Figure 1 (a–d) illustrates inter-group differences in the inflammatory parameters of intracellular IL-6, IL-1β, TNFα and circulating CRP (Phenotype: GS: dark grey, C: light grey). Abbreviations: IL-6/IL-1β: intracellular interleukins 6/1β; TNFα: intracellular tumor necrosis factor; CRP: circulatory C-reactive protein; GS: Gilbert’s syndrome; C: control subjects. *Indicates significant difference on a 5% level of error.

Figure 2

(a–d) Histograms showing frequency of HMOX-genotypes (classified as SS, SM, SL, MM, ML) stated in the BiliHealth study population. Figure 2 shows statistical distribution and frequency of respective HMOX-genotypes, expressed as (GT)_n allelic mean, between the study groups. (a and b) Male and female (all) subjects (Phenotype: GS: dark grey; C: light grey). (c and d) Female subjects only (Phenotype: GS: dark grey; C: light grey). Data distribution proposes a non-significant higher abundance of SM alleles in GS individuals, whereas the MM variant appeared to be more prominent in C subjects. This non-significant observation suggests a (gender-specific) higher frequency of shorter GT-repeats in GS individuals, which would be worth pursuing in a larger study collective. Classification of GT-fragment lengths (S < 26, L > 30) was performed following Chang et al..

Figure 3

Graphical summary of inter-variable connections for the entire BiliHealth study population. Figure 3 illustrates statistical connections of variables of interest. Bivariate correlations were calculated for the entire study population using the model of Spearman’s rho. R coefficients and p-values (p ≤ 0.05) are presented in the box and are summarised in a graphical model. (f): effect valid for female subjects only; (m): effect valid for male subjects only. Abbreviations: UCB: unconjugated bilirubin; UGT1A1: UGT1A1 genotype; IL-6/IL-1β: interleukins 6 and 1β; BLVRA expr.: genetic expression of biliverdin reductase; HMOX expr.: genetic expression of heme oxygenase; HMOX (GT)_n: heme oxygenase GT fragment lengths expressed as genotype (short, medium, long) or as mean fragment length of the two alleles; Hpt: haptoglobin; Hpx: hemopexin; CRP: C-reactive protein; SAA: serum amyloid A; BV: biliverdin IXα; CO: carbon monoxide; Fe²⁺: ferrous iron; BMI: body mass index.

Figure 4

Intracellular levels of interleukins are related to circulating UCB and underlying molecular measures of heme catabolism. Figure 4 shows explanatory powers of parameters that are involved in the heme catabolic pathway for variation in inflammatory markers. Inflammation appears to be directly influenced by UCB on the one hand, and by HMOX/BLVRA genetic expressions on the other. As far as UCB was concerned, a further connection with markers of inflammation was found that was mainly established through the variables of body composition (BMI, BF). Variation in the unspecific inflammatory marker of CRP is merely explained by the demographic variable age as well as body composition. The interconnection of iron and UCB emphasizes a potential feedback-loop of UCB on heme catabolism. Stepwise linear regression analysis was performed to assess inter-variable dependence and explanatory power (%), based on corrected R² values and a p-value of ≤0.05 (Supplementary Table S4). ^CEffect valid for control subjects only. Abbreviations: UCB: unconjugated bilirubin; UGT1A1: UGT1A1 genotype; IL-6/IL-1β: intracellular interleukins 6 and 1β; BLVRA expr.: genetic expression of biliverdin reductase; HMOX expr.: genetic expression of heme oxygenase; Hpt: haptoglobin; CRP: C-reactive protein; SAA: serum amyloid A.