The physiology of bilirubin: health and disease equilibrium

Bilirubin has several physiological functions, both beneficial and harmful. In addition to reactive oxygen species-scavenging activities, bilirubin has potent immunosuppressive effects associated with long-term pathophysiological sequelae. It has been recently recognized as a hormone with endocrine actions and interconnected effects on various cellular signaling pathways. Current studies show that bilirubin also decreases adiposity and prevents metabolic and cardiovascular diseases. All in all, the physiological importance of bilirubin is only now coming to light, and strategies for increasing plasma bilirubin levels to combat chronic diseases are starting to be considered. This review discusses the beneficial effects of increasing plasma bilirubin, incorporates emerging areas of bilirubin biology, and provides key concepts to advance the field.     

Inverse relationship between serum bilirubin and atherosclerosis in men: a meta-analysis of published studies

Bilirubin, a major intravascular product of heme catabolism, is a potent antioxidant compound. Numerous studies have been published showing the relationship between serum bilirubin levels and atherosclerosis. In the present investigation all the epidemiological studies available on the effect of serum bilirubin levels and atherosclerotic disease were analyzed. Studies on the epidemiology of atherosclerotic diseases in relation to serum bilirubin levels were searched in the MEDLINE database. Selected studies were subdivided according to serum bilirubin levels and severity of atherosclerotic disease. Because of the limited number of females involved in the studies, only males were included into meta-analysis. Associations for ordered categorical variables (bilirubin and natural history of graded atherosclerosis) were assessed to find correlation and linear trend between analyzed variables. A stratified analysis was conducted to compare risks of clinical outcomes. Eleven relevant studies were used for analysis. A close negative relationship was found between serum bilirubin levels and severity of atherosclerosis (Spearman rank coefficient r = -0.31,P < 0.0001). The linear trend was confirmed in analysis of proportions with x(2) values for both disease conditions to be very significant (P < 0.0001). Unambiguous inverse relationship between serum bilirubin levels and atherosclerosis was demonstrated in this preliminary meta-analytic study. These results indicate the importance of hem oxygenase-related products in the prevention of oxidative stress-mediated diseases.     

Liquor Bilirubin Levels and False Prediction of Severity in Rhesus Haemolytic Disease

Liquor bilirubin levels gave a false prediction of outcome for the fetus in 80 out of 716 rhesus-sensitized women referred for treatment during 1965-9. Trauma caused by amniocentesis seemed to be responsible for an increase in the severity of immunization in a significant proportion of cases. In addition, contamination of liquor samples by plasma, particularly fetal plasma, completely invalidated liquor bilirubin estimations. Errors in estimation of gestational length were also found to be associated with misleading results and a poor fetal prognosis.     

Plasma levels of soluble ST2, but not IL‐33, correlate with the severity of alcoholic liver disease

Alcoholic liver disease (ALD) is a complication that is a burden on global health and economy. Interleukin‐33 (IL‐33) is a newly identified member of the IL‐1 cytokine family and is released as an “alarmin” during inflammation. Soluble suppression of tumourigenicity 2 (sST2), an IL‐33 decoy receptor, has been reported as a new biomarker for the severity of systemic and highly inflammatory diseases. Here, we found the levels of plasma sST2, increased with the disease severity from mild to severe ALD. Importantly, the plasma sST2 levels in ALD patients not only correlated with scores for prognostic models (Maddrey's discriminant function, model for end‐stage liver disease and Child‐Pugh scores) and indexes for liver function (total bilirubin, international normalized ratio, albumin, and cholinesterase) but also correlated with neutrophil‐associated factors as well as some proinflammatory cytokines. In vitro, lipopolysaccharide‐activated monocytes down‐regulated transmembrane ST2 receptor but up‐regulated sST2 mRNA and protein expression and produced higher levels of tumour necrosis factor‐α (TNF‐α). By contrast, monocytes pretreated with recombinant sST2 showed decreased TNF‐α production. In addition, although plasma IL‐33 levels were comparable between healthy controls and ALD patients, we found the IL‐33 expression in liver tissues from ALD patients was down‐regulated at both RNA and protein levels. Immunohistochemical staining further showed that the decreased of IL‐33‐positive cells were mainly located in liver lobule area. These results suggested that sST2, but not IL‐33, is closely related to the severity of ALD. Consequently, sST2 could be used as a potential biomarker for predicting the prognosis of ALD.     

Combined Effects of Smoking and Bilirubin Levels on the Risk of Lung Cancer in Korea: The Severance Cohort Study

Background

Smoking is a major risk factor for lung cancer. Bilirubin, an antioxidant, is inversely associated with the risk of diseases related to oxidative stress. This study was conducted to determine the influence of smoking and bilirubin levels on the risk of lung cancer in the Severance cohort study.

Methods

This study included 68,676 Korean who received a health examination at Severance Health Promotion Center from 1994 to 2004. Serum bilirubin measurements within normal range were divided into tertiles whereas smoking states were divided as never-smokers, former smokers and current smokers. A diagnosis of lung cancer was coded as occurring based on the report from the National Cancer Registry. Hazard ratios (HRs) and 95% confidence intervals (95% CIs) were calculated using Cox proportional hazards model.

Results

At the end of the study period, 240 patients (men: 181, women: 59) developed lung cancer. Compared to those with bilirubin levels ≥1.0 mg/dL, HRs (95% CI) for lung cancer were 2.8 (1.8–4.2) for subjects having bilirubin levels from 0.2 to 0.7 mg/dL in men. When we stratified our analysis by smoking status, bilirubin consistently showed a protective effect on the risk of lung cancer on both never- and current smokers. Current smokers having bilirubin levels from 0.2 to 0.7 mg/dL had a risk of lung cancer by 6.0-fold higher than never-smokers with bilirubin levels ≥1.0 mg/dL in men.

Conclusion

In this large prospective study, higher baseline bilirubin level in the normal range was associated with low risk of lung cancer. Smoking and low bilirubin levels were cumulatively associated with a higher risk of lung cancer.     

Degradation of plasma bilirubin by a bilirubin oxidase derivative which has a relatively long half-life in the circulation

To enhance degradation of unconjugated bilirubin in hyperbilirubinemic subjects, we synthesized a bilirubin oxidase (EC 1.3.3.5) (BO) derivative (PEGBO) by covalently linking (2,4-bis[O-methoxy(polyethyleneglycol)]-6-chloro-s-triazine) (PEG) to the enzyme. Intravenously injected BO in rats disappeared from the circulation with a half-life of 2.5 min; the half-life of PEGBO was 190 min. Intravenously injected BO minimally and transiently decreased plasma bilirubin levels in jaundiced Gunn rats and in bile-duct-ligated jaundiced rats. In contrast, PEGBO rapidly and substantially decreased plasma bilirubin levels and the effect persisted for longer than 3 h. Renal dysfunction often occurs in patients with liver diseases. To study the role of bilirubin toxicity for the kidney, functions of transtubular transport for organic anions was measured in bile-duct-ligated jaundiced animals before and after treatment with PEGBO. Bile duct ligation decreased urinary excretion of phenolsulfophthalein (PSP), an organic anion used for renal function test. Treatment of the jaundiced animals with PEGBO increased the rate of PSP disappearance from the circulation and normalized its urinary excretion. Thus, PEGBO might be useful for the study of bilirubin toxicity in jaundiced animals.     

Two polymorphisms of the FVII gene and their impact on the risk of myocardial infarction in poles under 45 years of age

High levels of coagulation factor VII (FVII) in plasma have been associated with the increased risk of myocardial infarction (MI) in some studies. Both environmental and genetic factors are responsible for different levels of FVII in plasma. In the FVII gene there are two common polymorphisms (−323A1/A2 and IVS7) which are related to the level of FVII. The purpose of this study was to evaluate the influence of these polymorphisms on the risk of acute myocardial infarction in Poles under 45 years of age. We performed a case-control study of 266 patients with the history of MI. All patients had the first incidence of MI before 45 years of age. The control group consisted of 137 healthy individuals older than 45 years. Carriers of the A2 allele (−323A1/A2 polymorphism) have a lower risk of MI than non-carriers (OR = 0.40, 95% CI = 0.20−0.80). The IVS7 polymorphism was shown not to be related to MI in this study. Our findings suggest that the −323A1/A2 polymorphism of the FVII gene is related to the risk of MI in Polish individuals. We pointed that plasma cholesterol (OR = 1.11, 95% CI = 1.03−1.18), arterial hypertension (OR = 3.84, 95% CI = 1.99−7.43) and family history (OR = 2.72, 95% CI = 1.57−4.73) are significant predictors of acute myocardial infarction.     

Endogenous nitric oxide formation correlates negatively with circulating matrix metalloproteinase (MMP)-2 and MMP-9 levels in black subjects

Deficient formation of endogenous nitric oxide (NO) contributes to cardiovascular diseases, and this may be associated with increased circulating levels of matrix metalloproteinase-9 (MMP-9), as previously shown in white subjects. Because interethnic differences exist with respect to risk factors, prevalence, and severity of cardiovascular diseases, we designed this study to examine whether the circulating levels of nitrites (a marker of endogenous NO formation) are associated with the plasma levels of MMP-9 and MMP-2 in healthy black subjects. We studied 198 healthy subjects self-reported as blacks not taking any medications. Venous blood samples were collected and plasma and whole blood nitrite levels were measured using an ozone-based chemiluminescence assay. Plasma MMP-2 and MMP-9 levels were determined by gelatin zymography. We found a positive correlation between plasma MMP-9 and MMP-2 levels (P < 0.0001, rs = 0.556). Interestingly, we found a negative relationship between the plasma MMP-9 levels and the plasma or whole blood nitrites levels (P = 0.04, rs = -0.149; and P < 0.0001, rs = -0.349, respectively). In parallel, we found similar negative relationships between plasma MMP-2 levels and plasma or whole blood nitrites levels (P = 0.02, rs = -0.172; and P < 0.0001, rs = -0.454, respectively). This is the first study to show that endogenous nitric oxide formation correlates negatively with the circulating levels of both MMP-2 and MMP-9 in black subjects. Our findings suggest a mechanistic link between deficient NO formation and increased MMPs levels, which may promote cardiovascular diseases.     

A correlative study on serum cholylglycine levels in hepatobiliary disease.

The serum cholylglycine (CG), alanine aminotransferase (ALT) and total bilirubin levels were studied in 210 patients with hepatobiliary disease and in 70 healthy subjects. Serum CG concentrations in all the hepatobiliary diseases were found to be significantly higher than those of their controls. Patients with abnormal increases in ALT and bilirubin levels also showed raised CG concentrations; however, some patients with normal ALT and bilirubin levels, still had markedly elevated CG values. Patients with hepatic cirrhosis had high serum CG levels, followed, in descending order, by chronic active hepatitis and chronic persistent hepatitis. In the cholecystitis and cholelithiasis cases, their CG levels were significantly higher than those of the controls but lower than the values in hepatic disease patients; however, more cholecystitis cases had abnormally high serum bilirubin levels than CG. The results also show that serum CG concentrations vary in the different hepatobiliary diseases, and that serial CG measurements are more sensitive than measuring ALT and bilirubin levels in the diagnosis of hepatic diseases. Serum CG can be used as an index for evaluating the activity of chronic hepatitis; it can also be employed as a diagnostic tool in cholecystitis and cholelithiasis.     

Oxidative damage of albumin in advanced liver disease

Albumin has a number of biological functions and the serum albumin level is related to prognosis in advanced liver disease. Oxidative stress is believed to play an important role in the pathogenesis of liver failure. The aim of the present study was to characterize oxidative modification of albumin in patients with various degrees of liver failure and to investigate implications for its binding function. Patients with liver cirrhosis (n=10), acute-on-chronic liver failure (n=8) and healthy controls (n=15) were included in the study. Three fractions of albumin were separated by HPLC according to the redox state of cysteine-34 and detected by fluorescence as well as UV absorption. Carbonyl groups were measured as a marker of oxidative modification in plasma proteins and, by western blotting, on albumin. Progressive oxidative modification of albumin was found with increasing severity of liver failure indicated by an increased content of carbonyl groups and oxidation of cysteine-34. Fluorescence properties of albumin were altered by oxidation and, in patients with acute-on-chronic liver failure, by high plasma levels of bilirubin. This alteration of albumin fluorescence by bilirubin provides evidence for a preferred binding of bilirubin to the fully reduced form of albumin.     

神经内科患者跌倒相关因素分析及护理对策

目的：探讨影响神经内科患者跌倒的原圜及影响因素,以便医护人员采取有效的护理措施预防患者跌倒,为神经内科防控住院患者跌倒提供切实可行的依据。方法：本研究采用回顾性调查分析的方法,对我院2009年10月-2010年10神经内科住院患者跌倒原因进行调查分析。结果：研究显示,调查的2307例患者中,发生跌倒79例,跌倒发生率为3．42％,影响神经内科患者跌倒的危险因素包括患者自身的疾病、年龄、药物原因、运动能力障碍、视听力障碍和认知能力障碍。患者自身的疾病、年龄和药物原因为主要影响因素。结论：神经内科患者发生跌倒与其年龄、基础疾病严重程度及使用药物等有关,护理人员可依据致使患者发生跌倒的危险因素,有针对性的采取防控措施,降低神经内科患者跌倒发生率,有效保障患者安全,提高护理质量。     

Lycopene,atherosclerosis, and coronary heart disease

Diets rich in fruits and vegetables containing carotenoids have been of interest because of their potential health benefit against chronic diseases such as cardiovascular diseases (CVD) and cancer. Interest particularly in lycopene is growing rapidly following the recent publication of epidemiological studies that have associated high lycopene levels with reductions in CVD incidence. Two studies were conducted. In the first one, we examined the role of lycopene as a risk-lowering factor with regard to acute coronary events and stroke in the prospective Kuopio Ischemic Heart Disease Risk Factor (KIHD) Study. The subjects were 725 middle-aged men free of coronary heart disease and stroke at the study baseline. In a Cox's proportional hazards' model adjusting for covariates, men in the lowest quartile of serum levels of lycopene had a 3.3-fold (P < 0.001) risk of the acute coronary event or stroke as compared with others. In the second study, we assessed the association between plasma concentration of lycopene and intima-media thickness of the common carotid artery wall (CCA-IMT) in a cross-sectional analysis of the Antioxidant Supplementation in the Atherosclerosis Prevention (ASAP) study data in 520 asymptomatic men and women. In a covariance analysis adjusting for common cardiovascular risk factors, low plasma levels of lycopene were associated with an 18% increase of IMT in men as compared with men in whom plasma levels were higher than median (P = 0.003 for difference). In women, the difference did not remain significant after the adjustments. On the basis of these works, it is evident that the circulating levels of lycopene play some role with regard to cardiovascular health in Finland, at least in men. We conclude that circulating levels of lycopene, a biomarker of tomato-rich food, may play a role in early stages of atherogenesis and may have clinical and public health relevance.     

Prediction of Severe Neonatal Hyperbilirubinemia Using Cord Blood Hydrogen Peroxide: A Prospective Study

Background

We hypothesized that cord blood hydrogen peroxide (H₂O₂) could be utilized to predict the severity of neonatal hyperbilirubinemia.

Methods

We prospectively enrolled term or near-term healthy neonates. Cord blood and capillary blood at three days of age were measured for hydrogen peroxide and bilirubin concentrations. For newborns with hyperbilirubinemia, further blood samples were obtained at five and seven days of age. Newborns were divided into severe or less severe hyperbilirubinemic groups (peak bilirubin ≥17 mg/dL or not). The sensitivity, specificity, and negative predictive values were determined.

Results

There were 158 neonates enrolled. The incidence of neonatal hyperbilirubinemia was 30.5% for a concentration ≥15 mg/dl. The rising patterns were similar among bilirubin concentrations and hydrogen peroxide levels during the first few days of life. There was a strong positive correlation between bilirubin concentrations and hydrogen peroxide levels after correlation analysis. The rate of severe hyperbilirubinemia was 13.3%. It revealed that a cord blood hydrogen peroxide signal level of 2500 counts/10 seconds was an appropriate cut-off for predicting severe hyperbilirubinemia. Sensitivity and the negative predictive value were 76.2% and 93.3%, respectively.

Conclusions

Our findings confirm that hydrogen peroxide levels and bilirubin concentrations in cord and neonatal blood are closely related. A cord blood hydrogen peroxide level above 2500 counts/10 seconds associated with a high predictive value for severe hyperbilirubinemia. This method provides information about which neonate should be closely followed after discharge from the nursery.     

Apolipoproteins and atherosclerosis. Apolipoprotein E and apolipoprotein(a) as candidate genes of premature development of atherosclerosis

Apolipoprotein E (apoE) is a plasma lipoprotein which plays a basic role in the degradation of particles rich in cholesterol and triglycerides. It is able to bind to LDL receptors, but also to receptors for chylomicron remnants. There are three major apoE isoforms, E2, E3, and E4. Their role in lipoprotein metabolism is related to their affinity for receptors. Allele E3 is predominant and apoE3 affects metabolism of lipoproteins in a standard way. When compared to allele E3, allele E2 is associated with lower LDL levels, whereas allele E4 with higher LDL levels. This has an impact on the progression of atherosclerosis. Allele E2 exhibits a protective role, whereas allele E4 is associated with a high risk factor. Lipoprotein(a) [Lp(a)] is a plasma lipoprotein, consisting of apolipoprotein(a), linked by a covalent bond with the LDL particle. Increased Lp(a) levels are associated with an increased incidence of diseases based on atherosclerosis, namely the ischemic heart disease. Another effect of Lp(a) is its competition with plasminogen, resulting in a decrease of fibrinolysis and thrombogenic activity. ApoE and Lp(a) are independent risk factors for premature development of atherosclerosis and therefore can be considered as candidate genes of premature atherosclerosis.     

Genetic Variation and Atherosclerosis

A family history of atherosclerosis is independently associated with an increased incidence of cardiovascular events. The genetic factors underlying the importance of inheritance in atherosclerosis are starting to be understood. Genetic variation, such as mutations or common polymorphisms has been shown to be involved in modulation of a range of risk factors, such as plasma lipoprotein levels, inflammation and vascular calcification. This review presents examples of present studies of the role of genetic polymorphism in atherosclerosis.     

Protein Content of Liquor Amnii in Prediction of Severity of Haemolytic Disease of Newborn

Two series of cases of Rh isoimmunization were subjected to liquor examination for bilirubin and protein level. Series 1 comprised 298 cases for the years 1962 and 1963. Series 2 comprised 179 consecutive cases for 1967 in which preliminary selection for liquor examination had been made on the basis of previous history and maternal antibody titre.Bilirubin was measured as the liquor bilirubin ratio, and protein levels were estimated in series 1 by the Folin and Ciocalteau technique and in series 2 by a modified biuret method.Correlations with severity of haemolytic disease in the foetus was made, taking into account the stage of gestation of liquor examination. Both bilirubin and protein levels correlate with severity, but bilirubin is superior to protein. Interrelation of these measurements as bilirubin/protein ratio was inferior to bilirubin level as a method of forecasting severity.     

Biochemical predictors for Sars-Cov-2 severity

It is of interest to assess the inflammatory marker profile in SARS-CoV-2 patients and to correlate the levels of systemic inflammatory biomarkers such as neutrophil-to-lymphocyte ratio (NLR), C-Reactive Protein CRP, Ferritin, Creatine kinase (CK), Lactate dehydrogenase (LDH) and liver function analytes total serum proteins, albumin, total bilirubin, direct bilirubin, alkaline phosphatase, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) with the severity of SARS-CoV-2 infections. A total of 1000 COVID-19 positive patient''s data were collected. Laboratory assessments consisted of NLR (neutrophil-lymphocyte ratio) by cell counter, C Reactive Protein (CRP) by immunoturbidimetry, Ferritin by electrochemiluminescence (ECLIA) and Creatine Kinase (CK), Lactate Dehydrogenase (LDH), Alkaline phosphatase (ALP), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Total Bilirubin, Direct Bilirubin, Total Protein and Albumin by spectrophotometry. The mean plasma CRP levels, NLR, ferritin, CK and LDH levels were higher in severe cases than in non-severe cases, and the difference was statistically significant (p<0.05). All liver function tests such as the total and direct bilirubin, AST, ALT, ALP, total protein and albumin were higher in severe patients than non-severe patients and the difference was statistically significant (p<0.05). Data indicate that NLR, CRP, Ferritin, CK, LDH and liver function analytes have a crucial role as prognostic markers for SARS-CoV-2 infections and hence should be routinely recommended for risk assessment and stratification of the patients to reduce the associated morbidity and mortality.     

Effect of cucurbitacins on bilirubin-albumin binding in human plasma

The aim of this study is to investigate the effect of three cucurbitacins (Cuc) E, D and I on the bilirubin-albumin binding, both in human serum albumin (HSA) and in plasma. Bilirubin-HSA solution and plasma free of cucurbitacins were prepared as well as others containing serial concentrations of cucurbitacins. The concentration of unbound bilirubin was determined in bilirubin-HSA solution and the direct and total bilirubin concentrations were measured in plasma (with normal or elevated bilirubinemia) by Jendrassik and Grof method. In the conditions we adopted Cuc E and D (to a lesser extent), decreased the levels of unbound bilirubin in bilirubin-HSA solution and decreased direct bilirubin concentration and total bilirubin concentration in plasma in a dose-dependent manner while Cuc I had no effect. The effect of Cuc is related to the presence of native HSA. Thus, when albumin was absent or has been denatured by heating or by urea, Cuc E did not modify bilirubin levels, suggesting that the native structure of albumin is essential for such activity. The interaction of HSA with Cuc E was investigated by fluorescence spectroscopy. Cuc E increased the intrinsic fluorescence of the protein and the magnitude of fluorescence intensity of bilirubin-albumin complex. We concluded that Cuc E and D produced a rearrangement in the structure of albumin, particularly in the domain-II, resulting in an increase in the binding of bilirubin to albumin regardless to whether it's conjugated to glucuronic acid or unconjugated.     

Fasting hyperbilirubinemia in normal squirrel monkeys.

The plasma of Bolivian squirrel monkeys, unlike that of Brazilian squirrel monkeys, is markedly yellow due to unconjugated hyperbilirubinemia after an overnight fast. The fasting hyperbilirubinemia in Bolivian squirrel monkeys is likely due to two mechanisms. First, a twofold increase in the bilirubin turnover/production rate occurs during a 24-hour fast. A second mechanism is the decreased hepatic conjugation potential for bilirubin due to the presence of a higher bilirubin UDP-glucuronosyltransferase UDPGAKm and a lower Vm; this results in higher steady-state plasma and hepatic bilirubin levels during a fast when hepatic UDP-glucuronic acid levels are low. The Bolivian squirrel monkey provides an excellent animal model for human Gilbert's syndrome type I in which to study rate-limiting mechanisms in the movement of bilirubin from plasma to bile.     

Genetic determinants of mortality. Can findings from genome-wide association studies explain variation in human mortality?

Twin studies have estimated the heritability of longevity to be approximately 20–30 %. Genome-wide association studies (GWAS) have revealed a large number of determinants of morbidity, but so far, no new polymorphisms have been discovered to be associated with longevity per se in GWAS. We aim to determine whether the genetic architecture of mortality can be explained by single nucleotide polymorphisms (SNPs) associated with common traits and diseases related to mortality. By extensive quality control of published GWAS we created a genetic score from 707 common SNPs associated with 125 diseases or risk factors related with overall mortality. We prospectively studied the association of the genetic score with: (1) time-to-death; (2) incidence of the first of nine major diseases (coronary heart disease, stroke, heart failure, diabetes, dementia, lung, breast, colon and prostate cancers) in two population-based cohorts of Dutch and Swedish individuals (N = 15,039; age range 47–99 years). During a median follow-up of 6.3 years (max 22.2 years), we observed 4,318 deaths and 2,132 incident disease events. The genetic score was significantly associated with time-to-death [hazard ratio (HR) per added risk allele = 1.003, P value = 0.006; HR 4th vs. 1st quartile = 1.103]. The association between the genetic score and incidence of major diseases was stronger (HR per added risk allele = 1.004, P value = 0.002; HR 4th vs. 1st quartile = 1.160). Associations were stronger for individuals dying at older ages. Our findings are compatible with the view of mortality as a complex and highly polygenetic trait, not easily explainable by common genetic variants related to diseases and physiological traits.