patient-oriented and epidemiological research
Serum bilirubin levels in familial hypercholesterolemia:
a new risk marker for cardiovascular disease?
Pernette R.W. de Sauvage Nolting,* D. Meeike Kusters,† Barbara A. Hutten,§ and
John J. P. Kastelein1,† for the ExPRESS study group
Cardiology Centers of The Netherlands,* Rotterdam, The Netherlands; and Departments of Vascular
Medicine† and Clinical Epidemiology, Biostatistics, and Bioinformatics,§ of the Academic Medical Center,
Amsterdam, The Netherlands
Abstract Low concentrations of bilirubin are associated
with an increased risk for cardiovascular disease (CVD).
Possibly, bilirubin exerts its effect through the protection
of LDL from oxidation. Therefore, we examined whether
low bilirubin might also be a risk marker for CVD in pa-
tients with familial hypercholesterolemia (FH) and whether
statins influence serum bilirubin levels. Patients with FH
were recruited from 37 lipid clinics. After a washout pe-
riod of 6 weeks, all patients were started on monotherapy
with simvastatin 80 mg for a period of two years. A total of
514 patients were enrolled. Bilirubin at baseline was in-
versely associated with the presence of CVD, also after ad-
justment for age, gender, presence of hypertension, and
HDL cholesterol levels. Moreover, bilirubin levels were
significantly raised, by 7%, from 10.0 to 10.8 ␮mol/L after
treatment with simvastatin 80 mg. We hypothesize first that
high bilirubin levels might protect patients with FH from
CVD. Furthermore, bilirubin levels were significantly in-
creased after treatment with simvastatin 80 mg, indepen-
dent of changes in liver enzymes, which might confer
additional protection against CVD. Whether this is also
true for lower doses of simvastatin or for other statins re-
mains to be investigated.—de Sauvage Nolting, P. R. W.,
D. M. Kusters, B. A. Hutten, and J. J. P. Kastelein. Serum
bilirubin levels in familial hypercholesterolemia: a new
risk marker for cardiovascular disease? J. Lipid Res. 2011.
52: 1755–1759.
Supplementary key words atherosclerosis • drug therapy • oxidized
lipids • statins
Serum total bilirubin concentrations have been shown
to be inversely associated with the risk for cardiovascular
disease (CVD) (1–10). The explanation for this associa-
tion is not fully understood. In contrast, bilirubin was for
a long time regarded as cytotoxic, in particular for its role
The ExPRESS study was sponsored by Merck, Sharp, and Dohme, The
Netherlands.
Manuscript received 26 November 2010 and in revised form 22 June 2011.
Published, JLR Papers in Press, June 23, 2011
DOI 10.1194/jlr.P013193
Copyright © 2011 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
in neonatal jaundice. It is only since the end of the 1980s
that a physiological role for bilirubin functioning has
emerged as a potent antioxidant (11, 12). In fact, in vitro
evidence suggests that LDL can be protected from oxida-
Downloaded from www.jlr.org by guest, on December 23, 2013
tion by bilirubin (13). Therefore, low bilirubin concen-
trations could be a reflection of a heightened oxidative
state and increased consumption of bilirubin. Furthermore,
bilirubin has been shown to have anti-inflammatory prop-
erties (14). Taken together, these results point to potential
beneficial effects of bilirubin toward the chronic inflam-
matory state we currently associate with atherosclerosis.
Familial hypercholesterolemia (FH) is an autosomal
dominant disorder of lipoprotein metabolism and af-
fects approximately 1 in 400 people in The Netherlands
(15). The underlying defect consists of mutations in the
gene encoding for the LDL receptor protein or in its li-
gand, apo B-100. These mutations result in markedly
elevated plasma cholesterol levels, predisposing FH
patients to premature atherosclerosis and CVD (16).
Statins, or HMG-CoA reductase inhibitors, are currently
considered the preferred lipid-lowering agents in these
patients, since they have been proven to be safe and well-
tolerated agents that reduce LDL cholesterol (LDL-C)
levels as well as the incidence of coronary artery disease
(CAD).
To the best of our knowledge, it has not been examined
before whether low bilirubin is associated with the pres-
ence of CVD in FH patients and whether statins can raise
serum bilirubin levels. We therefore set out to study the
role of bilirubin in these patients and the effect of simva-
statin therapy on bilirubin levels. Here we present our
results.
Abbreviations: ALAT, alanine-amino transferase; ASAT, aspartate-
amino transferase; BMI, body mass index; CAD, coronary artery dis-
ease; CVD, cardiovascular disease; ECG, electrocardiogram; FH,
familial hypercholesterolemia; HDL-C, HDL cholesterol; HO-1, heme
oxygenase 1; LDL-C, LDL cholesterol; MI, myocardial infarction; TG,
triglyceride.
1
To whom correspondence should be addressed.
e-mail: j.j.kastelein@amc.uva.nl
Journal of Lipid Research Volume 52, 2011 1755
 METHODS
Study design and subjects
Data for the present analysis were derived from the database
of the ExPRESS FH (Examination of Probands and Relatives in
Statin Studies with Familial Hypercholesterolemia) study, in
which the two year efficacy and safety of simvastatin 80 mg were
evaluated in 526 heterozygous FH patients (17). For this open-
label study, subjects were recruited from 37 lipid clinics in The
Netherlands. Patients were included if they met the following
criteria: all patients had to have either a molecular diagnosis for
FH or were diagnosed with definite FH and had to have six or
more points, according to an algorithm (to allow standardization
of the diagnosis of FH based on clinical findings, personal and
familial clinical history, and biochemical parameters) (18); at
least 18 years of age; patients with a history of myocardial infarc-
tion (MI), coronary artery bypass graft, or percutaneous translu-
minal coronary angioplasty could be included if the physician
thought it was medically allowed for the patient to have a wash-
out period. Patients were excluded if they were pregnant or nurs-
ing women, or premenopausal women not using adequate
contraceptives; had acute liver disease, hepatic dysfunction, or
persistent elevations of serum transaminases; had hypersensitiv-
ity or intolerance to simvastatin or any of its components; had
hyperlipidemia type I, III, IV, or V or homozygous FH; had a
recent history of alcohol or drug abuse; had secondary hyper-
cholesterolemia due to any cause; had inadequately controlled
diabetes, unstable angina, intermediate coronary syndrome,
clinically significant ventricular arrhythmia at study entry, MI
within the past three months; were on concurrent use of erythro-
mycin and similar drugs affecting the cytochrome P450 enzyme,
or had a history of cancer.
The ethics institutional review boards committees of all 37
centers approved the protocol, and written informed consent
was obtained from all participants. The investigation was con-
ducted according to the principles outlined in the Declaration of
Helsinki.
After a six week washout period, patients started monotherapy
with simvastatin 80 mg for the duration of two years. No other
lipid-lowering medication was allowed. Medical history, physical
examination, and additional risk factors for CVD, as well as labo-
ratory analysis of lipid and lipoprotein levels and routine safety
parameters, were obtained in all patients.
CVD
CVD was considered to be present if subjects met one of the
following criteria: subjects who had 1) an MI, proven by elec-
trocardiogram (ECG) abnormalities and enzyme changes; 2)
an ischemic stroke; 3) a diagnosis of clinically documented an-
gina pectoris; 4) a history of intermittent claudication docu-
mented by ultrasound; 5) coronary bypass surgery or percutaneous
coronary interventions; 6) a clinically significant stenosis on
coronary angiogram; or 7) an unequivocally positive exercise
ECG.
Biochemical analysis
Blood samples were taken in the morning after an overnight
fast. Total plasma cholesterol (TC), HDL cholesterol (HDL-C),
and triglycerides (TGs) were routinely determined in the differ-
ent laboratories and standardized by a virtual central laboratory.
LDL-C was calculated using the Friedewald formula (19). Total
serum bilirubin was routinely measured in the different laborato-
ries by spectrophotometry. All results were harmonized to one
level according to the standardized Jendrassik-Grof method by
the virtual central laboratory (20).
1756 Journal of Lipid Research Volume 52, 2011
Statistical analysis
Mean values in lipids between subgroups were compared
using the independent sample t-test. Other parameters (TG and
bilirubin) were compared by the nonparametric Mann-Whitney
U test because they had a skewed distribution. ␹2 tests were ap-
plied for comparing distributions of dichotomous data (gender,
smoker, presence of hypertension or diabetes, and bilirubin lev-
els (>17 µmol/L versus ⭐17 µmol/L). The association between
presence of CVD and bilirubin levels at baseline was evaluated
using a logistic regression model. We adjusted for potential con-
founders, i.e., age, gender, hypertension, diabetes, body mass
index (BMI), HDL-C, and TG by means of stepwise backward
elimination. Mean values in lipids before and after treatment
were compared using the paired sample t-test. TG, bilirubin,
alanine-amino transferase (ALAT) and aspartate-amino transferase
(ASAT) levels were compared by the nonparametric Wilcoxon
test because they had a skewed distribution. Pearson correlations
were applied to evaluate the correlation between absolute
changes in bilirubin, ASAT, and ALAT. All statistical analyses
were performed using the SPSS package (version 15.0; SPSS,
Inc., Chicago, IL). A P value of less than 0.05 was considered to
be statistically significant.
Downloaded from www.jlr.org by guest, on December 23, 2013
RESULTS
Study population
Among the 526 FH patients who participated in the
ExPRESS FH study, baseline total bilirubin levels were
available for 514 patients, and these patients comprised
our study population. Age ranged from 18 to 80 years with
a mean age of 47.4 years [standard deviation (SD) ± 13.2],
and 188 (37%) patients were known to have CVD. Base-
line demographic and clinical characteristics of patients
with and without CVD are summarized in Table 1. Patients
with CVD were older and had, on average, higher values of
BMI, in addition to a higher prevalence of hypertension
and diabetes compared with those without CVD. Fewer
current smokers were seen in the CVD group. Further-
more, mean HDL-C was lower in patients with CVD,
whereas the median TG level was significantly higher.
FH patients and bilirubin levels
Median baseline serum bilirubin level in all FH patients
was 10.0 µmol/L [interquartile range (IQ): 7.8 to 12.8]. In
patients with CVD, the median bilirubin level was signifi-
cantly lower, compared with patients without CVD [9.7
(IQ: 7.3–11.7) versus 10.5 (7.8–13.5) µmol/L, respectively;
P = 0.006]. A significantly lower proportion of patients
with elevated bilirubin levels (i.e., bilirubin >17 µmol/L)
was observed in those with CVD compared with patients
without CVD (3.7% versus 9.8%, respectively; P = 0.01).
Association between CVD and bilirubin
We evaluated the association between bilirubin levels
and CVD in a logistic regression model with CVD as the
response variable and bilirubin as the explanatory vari-
able. Levels of bilirubin were negatively associated with
CVD (OR, 0.94; 95% CI, 0.90–0.98; P = 0.005). By means of
multiple regression models, we further explored the role of
potential confounders. Backward hierarchical elimination
 TABLE 1. Baseline characteristics of FH patients with and without CVD

FH with CVD FH without CVD

n = 188 n = 326 P

Age (years) 55.5 ± 9.8 42.7 ± 12.6 <0.0001

Male gender, n (%) 110 (58.5) 177 (54.3) 0.36
Current smoking, n (%) 34 (18.0) 102 (31.3) 0.001
Hypertension, n (%) 51 (27.0) 29 (8.9) <0.0001
Diabetes, n (%) 9 (4.8) 1 (0.3) <0.0001
2
BMI (kg/m ) 26.7 ± 3.4 25.4 ± 3.5 <0.0001
TC (mmol/L) 10.65 ± 2.36 10.36 ± 2.05 0.16
LDL-C (mmol/L) 8.45 ± 2.31 8.28 ± 2.02 0.40
HDL-C (mmol/L) 1.18 ± 0.31 1.25 ± 0.36 0.02
TG (mmol/L) 1.90 (1.40–2.78) 1.60 (1.10–2.30) <0.0001
Total bilirubin (␮mol/L) 9.7 (7.2–11.7) 10.5 (7.8–13.5) 0.006
Bilirubin >17 ␮mol/L 7 (3.7) 32 (9.8) 0.01

Except where given as percentages, all values are given as mean levels ± SD. Only TGs and bilirubin are given
as median with IQR between brackets.

strategy was used to identify the final model, and, subse-
quently, TG (OR, 0.96; 95% CI, 0.79–11.79; P = 0.719),
presence of diabetes (OR, 6.49; 95% CI, 0.68–61.96, P =
0.104) and BMI (OR, 1.06; 95% CI, 0.97–1.14, P = 0.062)
dropped out of the model. In the final model adjusted for
and change in ASAT (r = 0.05; P = 0.26), ALAT (r = ⫺0.02;
P = 0.63), or BMI (r = 0.032, P = 0.51).
DISCUSSION

Downloaded from www.jlr.org by guest, on December 23, 2013

age (OR, 1.11; 95% CI, 1.08–1.13; P < 0.0001), male gen-
der (OR, 1.85; 95% CI, 1.14–3.01; P = 0.01), presence of
hypertension (OR, 1.96; 95% CI, 1.11–3.46; P = 0.02),
HDL-C (OR, 0.33; 95% CI, 0.16–0.65; P = 0.002), and bili-
rubin (OR, 0.92; 95% CI, 0.88–0.97; P = 0.004) remained
significantly associated with CVD. Additionally, we per-
formed the same analyses with bilirubin as a dichotomous
variable (cutoff at 17 µmol/L). In the univariate analysis as
well as in the multiple analyses adjusted for age, gender,
hypertension, and HDL-C, bilirubin was significantly asso-
ciated with CVD (OR, 0.36; 95% CI, 0.15–0.82; P = 0.02
and OR, 0.27; 95% CI, 0.08–0.73; P = 0.01, respectively).
Treatment with simvastatin and bilirubin
In Table 2 treatment effects of simvastatin 80 mg on lip-
ids, bilirubin, and liver enzymes are given. TC, LDL-C, and
TG levels were reduced by 39.2%, 48.0%, and 26.3%, re-
spectively, whereas HDL-C levels were elevated by 12.7%.
Median bilirubin levels were significantly raised after treat-
ment with 80 mg simvastatin with 7% from 10.0 [IQR: 7.8;
12.8] to 10.8 [IQR: 7.8; 13.7] µmol/L. This increase was
more pronounced in the patients with CVD compared
with those without CVD [1.40 (IQR: ⫺0.6; 3.1) µmol/L
versus 0.40 (IQR: ⫺2.0; 2.5) µmol/L; P = 0.008]. Notably,
no correlation was observed between change in bilirubin
Baseline bilirubin and CVD
The current study is the first to show that serum biliru-
bin levels in patients with FH were independently and in-
versely associated with the presence of CVD. These findings
indicate that bilirubin levels at the low end of the normal
range are associated with a significantly higher prevalence
of CVD and suggest cardiovascular protection from ele-
vated serum bilirubin levels in these high-risk patients.
Our results are consistent with findings of both previous
retrospective (2–4) and prospective (5–9) studies. In these
studies, similar inverse associations have been shown not
only between serum bilirubin concentrations and CVD,
but also between bilirubin and peripheral vascular disease,
carotid intima-media thickness, and stroke (21, 22). Fur-
thermore, a meta-analysis by Novotny and Vitek (10) of 11
studies has shown an inverse and dose-dependent relation-
ship between serum bilirubin and different types and se-
verities of CVD. It was found that a serum bilirubin level of
10 µmol/L is the cut-point for discrimination of cardiovas-
cular risk. Accordingly, in our study with a much smaller
study cohort, the median level of bilirubin levels in
patients with and without CVD was just below and above
10 µmol/L, respectively. However, women were not in-
cluded in this meta-analysis, and it is therefore unclear

TABLE 2. Treatment effects of simvastatin 80 mg in FH patients

Baseline After Two Years

a
n = 514 n = 436 % Change P

TC (mmol/L) 10.52 ± 2.17 6.30 ± 1.41 ⫺39.2 ± 11.8 <0.0001

LDL cholesterol (mmol/L) 8.38 ± 2.14 4.29 ± 1.31 ⫺48.0 ± 13.5 <0.0001
HDL cholesterol (mmol/L) 1.23 ± 0.35 1.36 ± 0.36 12.7 ± 21.8 <0.0001
Triglycerides (mmol/L) 1.80 (1.20 to 2.40) 1.20 (0.90 to 1.70) ⫺26.3 (⫺46.2 to ⫺5.6) <0.0001
Total bilirubin (␮mol/L) 10.0 (7.8 to 12.8) 10.8 (7.8 to 13.7) 7.0 (⫺14.1 to 31.1) <0.0001
ASAT (U/l) 20 (17 to 24) 23 (19 to 27) 14 (0 to 32) <0.0001

All values are given as median with IQR between brackets; only TC, LDL-C, and HDL-C levels are given as mean
levels ± SD.
a
Baseline versus after two years.

Serum bilirubin levels in familial hypercholesterolemia 1757

whether these findings can be extrapolated to our study
population. As for the strength of the found association,
Hopkins et al. (2) found that bilirubin levels were compa-
rable to HDL-C in terms of CVD protection. Similarly, in
our study, bilirubin levels above 17.0 ␮mol/L were associ-
ated with CVD protection comparable to a 1 mmol/L in-
crease in HDL-C levels in FH patients.
Some methodological aspects of our study merit discus-
sion. First of all, because of the cross-sectional design, we
could not establish a causal relation between serum biliru-
bin levels and occurrence of CVD, i.e., that high levels of
bilirubin prevent future CVD. However, a considerable
number of prospective cohort studies that have shown an
inverse relationship between bilirubin and CVD provide
direct support that bilirubin may have a causal role in ath-
erosclerotic vascular disease (5–9). Moreover, several stud-
ies have shown that bilirubin acts as an antioxidant and
suppresses lipid oxidation (12, 23, 24), which is known to
prevent plaque formation and atherosclerosis.
Due to the observational nature of our data, the asso-
ciation between CVD and bilirubin could possibly be ex-
plained by undiscovered confounding. Aspirin use could
be a potential confounder since it has been suggested
that aspirin raises bilirubin levels through heme oxyge-
nase 1 (HO-1) activity induction, whereas it is also asso-
ciated with CVD (25, 26). However, despite the use of
aspirin in most of the CVD patients, we found that biliru-
bin levels were significantly lower in these patients,
which actually suggests an underestimation of the found
association. Furthermore, when we adjusted for use of
aspirin in a stepwise multiple regression analysis to eval-
uate the relation between bilirubin levels and CVD, aspi-
rin use did not emerge as a significant confounder.
Another potential confounder could be smoking. Not
only is smoking a well-known risk factor for CVD, it is
also inversely associated with bilirubin concentrations
(27). Strikingly, in our study cohort with FH patients,
fewer smokers were seen in the group of patients with
CVD. This discrepancy may be explained by a high num-
ber of former smokers in the CVD group, who stopped
smoking after their first cardiovascular event. Unfortu-
nately, we did not collect any data on previous smoking
habits. Because current smoking would not accurately
reflect previous exposure to smoking, we have chosen
not to include this variable in the multiple regression
analyses. Seasonal variation in bilirubin could also be an
influence (28); however, subjects were recruited for the
study throughout the whole year, so we assume that this
phenomenon played a minor role. Finally, the associa-
tion between CVD and bilirubin could possibly be con-
founded by patients with concomitant elevated liver
enzymes (5, 10). In our study, one of the exclusion cri-
teria was acute liver disease, hepatic dysfunction, or per-
sistent elevations of serum transaminases. To avoid
confounding, we also performed all analyses in patients
in which liver enzymes were below certain strict limits
(ASAT < 40 U/l and ALAT < 45 U/l). This yielded 458
patients at baseline and 335 patients after two years of
therapy. However, results in this subgroup were similar
1758 Journal of Lipid Research Volume 52, 2011
to the results obtained in the whole study cohort (data
not shown).
Effect of simvastatin on bilirubin
We observed that bilirubin levels were increased by 7%,
from 10.0 to 10.8 µmol/L, in patients with heterozygous
FH, after two years of treatment with simvastatin 80 mg.
This increase was more pronounced in the patients with
CVD as compared with those without CVD. Although liver
enzymes were also slightly increased, changes in ASAT or
ALAT levels were not correlated with change in bilirubin.
No other reports are available with regard to the effects
of statins on bilirubin or HO-1 levels in clinical studies.
The effect of statins on bilirubin levels and HO-1 protein
levels were measured in in vitro and in vivo studies (29–32)
that showed raising of bilirubin levels by statins. These re-
sults may further explain the pleiotropic, antioxidant,
anti-inflammatory, and antiatherogenic actions of statins.
Unfortunately, we did not measure HO-1 activity, and de-
velopment of a serial monitoring system of HO-1 activity
for use in clinical setting is desirable. At present, there is
no specific and sensitive marker for HO-1 activity in vivo.
Downloaded from www.jlr.org by guest, on December 23, 2013
An increase of 0.8 µmol/L of bilirubin levels is modest,
but in the multiple regression analysis, an increase of
1 µmol/L was associated with an 8% decrease in CVD.
This is in line with Hunt et al. (33), who found in 328 pa-
tients with early CAD a 7% increase of CAD prevalence for
each 1 µmol/L decrease in serum bilirubin. Whether this
bilirubin increase by simvastatin confers additional benefit
over and above cholesterol reduction cannot be answered
by our study, both because of small numbers of events and
the likely overwhelming effect of LDL-C lowering.
The observation that the bilirubin increase was more
pronounced in the patients with CVD as compared with
those without CVD can possibly be explained by the fact
that the groups with and without CVD were moderately
heterogeneous with regard to factors such as sex, age, and
smoking status. In fact, we did observe a trend toward a
greater increase in bilirubin in males and in subjects >50
years, which are both more represented in the CVD group.
For the subjects who did not smoke, a significantly greater
increase in bilirubin was seen, as compared with the sub-
jects who did smoke (1.1 ± 4.4 vs. ⫺0.2 ± 3.1 ␮mol/L, re-
spectively; P = 0.007). Because there were more nonsmokers
in the CVD group, this could have contributed to the ob-
served difference in bilirubin increase as well. Neverthe-
less, more studies will be needed to confirm our results
and delineate the role of bilirubin and HO-1 in atherogen-
esis and CVD.
In summary, we found significantly lower serum total
bilirubin levels in FH patients known to have CVD as com-
pared with FH patients without CVD. On the basis of these
findings, we hypothesize that high bilirubin levels might
protect FH patients from CVD. Furthermore, bilirubin lev-
els were significantly increased after two years of treatment
with simvastatin 80 mg, independent of changes in liver
enzymes, which might confer additional protection against
CVD. Whether this is also true for lower doses of simvasta-
tin or for other statins remains to be investigated.
 REFERENCES
1. Schwertner, H. A., and L. Vitek. 2008. Gilbert syndrome,
UGT1A1*28 allele, and cardiovascular disease risk: possible protec-
tive effects and therapeutic applications of bilirubin. Atherosclerosis.
198: 1–11.
2. Hopkins, P. N., L. L. Wu, S. C. Hunt, B. C. James, G. M. Vincent,
and R. R. Williams. 1996. Higher serum bilirubin is associated with
decreased risk for early familial coronary artery disease. Arterioscler.
Thromb. Vasc. Biol. 16: 250–255.
3. Levinson, S. S. 1997. Relationship between bilirubin, apolipo-
protein B, and coronary artery disease. Ann. Clin. Lab. Sci. 27:
185–192.
4. Schwertner, H. A., W. G. Jackson, and G. Tolan. 1994. Association
of low serum concentration of bilirubin with increased risk of coro-
nary artery disease. Clin. Chem. 40: 18–23.
5. Breimer, L. H., G. Wannamethee, S. Ebrahim, and A. G. Shaper.
1995. Serum bilirubin and risk of ischemic heart disease in middle-
aged British men. Clin. Chem. 41: 1504–1508.
6. Djousse, L., D. Levy, L. A. Cupples, J. C. Evans, R. B. D’Agostino,
and R. C. Ellison. 2001. Total serum bilirubin and risk of cardiovas-
cular disease in the Framingham offspring study. Am. J. Cardiol. 87:
1196–1200.
7. Troughton, J. A., J. V. Woodside, I. S. Young, D. Arveiler, P.
Amouyel, J. Ferrieres, P. Ducimetiere, C. C. Patterson, F. Kee, J.
W. Yarnell, et al. 2007. Bilirubin and coronary heart disease risk
in the Prospective Epidemiological Study of Myocardial Infarction
(PRIME). Eur. J. Cardiovasc. Prev. Rehabil. 14: 79–84.
8. Temme, E. H., J. Zhang, E. G. Schouten, and H. Kesteloot. 2001.
Serum bilirubin and 10-year mortality risk in a Belgian population.
Cancer Causes Control. 12: 887–894.
9. Hunt, S. C., L. L. Wu, P. N. Hopkins, and R. R. Williams. 1996.
Evidence for a major gene elevating serum bilirubin concentration
in Utah pedigrees. Arterioscler. Thromb. Vasc. Biol. 16: 912–917.
10. Novotny, L., and L. Vitek. 2003. Inverse relationship between se-
rum bilirubin and atherosclerosis in men: a meta-analysis of pub-
lished studies. Exp. Biol. Med. (Maywood). 228: 568–571.
11. Stocker, R., Y. Yamamoto, A. F. McDonagh, A. N. Glazer, and B. N.
Ames. 1987. Bilirubin is an antioxidant of possible physiological
importance. Science. 235: 1043–1046.
12. Stocker, R., A. N. Glazer, and B. N. Ames. 1987. Antioxidant ac-
tivity of albumin-bound bilirubin. Proc. Natl. Acad. Sci. USA. 84:
5918–5922.
13. Wu, T. W., K. P. Fung, and C. C. Yang. 1994. Unconjugated biliru-
bin inhibits the oxidation of human low density lipoprotein better
than Trolox. Life Sci. 54: 477–481.
14. Nakagami, T., K. Toyomura, T. Kinoshita, and S. Morisawa. 1993.
A beneficial role of bile pigments as an endogenous tissue protec-
tor: anti-complement effects of biliverdin and conjugated bilirubin.
Biochim. Biophys. Acta. 1158: 189–193.
15. Lansberg, P. J., S. Tuzgol, M. A. van de Ree, J. C. Defesche, and
J. J. Kastelein. 2000. [Higher prevalence of familial hypercholester-
olemia than expected in adult patients of four family practices in
Netherlands] Ned. Tijdschr. Geneeskd. 144: 1437–1440.
16. Brown, M. S., and J. L. Goldstein. 1986. A receptor-mediated path-
way for cholesterol homeostasis. Science. 232: 34–47.
17. De Sauvage Nolting, P. R., R. J. Buirma, B. A. Hutten, and J. J.
Kastelein. 2002. Two-year efficacy and safety of simvastatin 80 mg
in familial hypercholesterolemia [the Examination of Probands
Serum bilirubin levels in familial hypercholesterolemia
and Relatives in Statin Studies with Familial Hypercholesterolemia
(ExPRESS FH)]. Am. J. Cardiol. 90: 181–184.
18. Defesche, J. C. 2000. Familial hypercholesterolaemia. In Lipids and
Vascular Disease. D. J. Betteridge, editor. Dunitz, London. 65–76.
19. Friedewald W. T., R. I. Levy, D. S. Fredrickson. 1972. Estimation of
the concentration of low-density lipoprotein cholesterol in plasma,
without use of the preparative ultracentrifuge. Clin. Chem. 18:
499–502.
20. Perry, B. W., B. T. Doumas, D. D. Bayse, T. Butler, A. Cohen, W.
Fellows, C. C. Garber, B. Howell, T. Koch, S. Krishnamurthy, et al.
1983. A candidate reference method for determination of bilirubin
in serum. Test for transferability. Clin. Chem. 29: 297–301.
21. Vitek, L., L. Novotny, M. Sperl, R. Holaj, and J. Spacil. 2006. The
inverse association of elevated serum bilirubin levels with subclini-
cal carotid atherosclerosis. Cerebrovasc. Dis. 21: 408–414.
22. Erdogan, D., H. Gullu, E. Yildirim, D. Tok, I. Kirbas, O. Ciftci, S.
T. Baycan, and H. Muderrisoglu. 2006. Low serum bilirubin levels
are independently and inversely related to impaired flow-mediated
vasodilation and increased carotid intima-media thickness in both
men and women. Atherosclerosis. 184: 431–437.
23. Wu, T. W., K. P. Fung, J. Wu, C. C. Yang, and R. D. Weisel. 1996.
Antioxidation of human low density lipoprotein by unconjugated
and conjugated bilirubins. Biochem. Pharmacol. 51: 859–862.
24. Neuzil, J., and R. Stocker. 1994. Free and albumin-bound biliru-
bin are efficient co-antioxidants for alpha-tocopherol, inhibiting
plasma and low density lipoprotein lipid peroxidation. J. Biol. Chem.
269: 16712–16719.
25. Grosser, N., A. Abate, S. Oberle, H. J. Vreman, P. A. Dennery, J.
Downloaded from www.jlr.org by guest, on December 23, 2013
C. Becker, T. Pohle, D. S. Seidman, and H. Schroder. 2003. Heme
oxygenase-1 induction may explain the antioxidant profile of aspi-
rin. Biochem. Biophys. Res. Commun. 308: 956–960.
26. Ebong, P. E., E. U. Eyong, and E. O. Udosen. 1998. Effects of as-
pirin (acetylsalicylic acid) and Cataflam (potassium diclofenac)
on some biochemical parameters in rats. Afr. J. Med. Med. Sci. 27:
243–246.
27. Schwertner, H. A. 1998. Association of smoking and low serum bili-
rubin antioxidant concentrations. Atherosclerosis. 136: 383–387.
28. Miyake, K., N. Miyake, S. Kondo, Y. Tabe, A. Ohsaka, and T. Miida.
2009. Seasonal variation in liver function tests: a time-series analysis
of outpatient data. Ann. Clin. Biochem. 46: 377–384.
29. Grosser, N., K. Erdmann, A. Hemmerle, G. Berndt, U. Hinkelmann,
G. Smith, and H. Schroder. 2004. Rosuvastatin upregulates the an-
tioxidant defense protein heme oxygenase-1. Biochem. Biophys. Res.
Commun. 325: 871–876.
30. Grosser, N., A. Hemmerle, G. Berndt, K. Erdmann, U. Hinkelmann,
S. Schurger, N. Wijayanti, S. Immenschuh, and H. Schroder. 2004.
The antioxidant defense protein heme oxygenase 1 is a novel target
for statins in endothelial cells. Free Radic. Biol. Med. 37: 2064–2071.
31. Hsu, M., L. Muchova, I. Morioka, R. J. Wong, H. Schroder, and D.
K. Stevenson. 2006. Tissue-specific effects of statins on the expres-
sion of heme oxygenase-1 in vivo. Biochem. Biophys. Res. Commun.
343: 738–744.
32. Lee, T. S., C. C. Chang, Y. Zhu, and J. Y. Shyy. 2004. Simvastatin in-
duces heme oxygenase-1: a novel mechanism of vessel protection.
Circulation. 110: 1296–1302.
33. Hunt, S. C., F. Kronenberg, J. H. Eckfeldt, P. N. Hopkins, R. H.
Myers, and G. Heiss. 2001. Association of plasma bilirubin with
coronary heart disease and segregation of bilirubin as a major gene
trait: the NHLBI family heart study. Atherosclerosis. 154: 747–754.
1759