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Keywords:

  • total bilirubin;
  • inflammation;
  • endothelial function;
  • HIV infection

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Objectives

Enhanced inflammation is evident in HIV infection, even with virological suppression. Outside HIV infection, studies show an independent association between higher total bilirubin and better endothelial function as well as a lower prevalence of coronary heart disease, possibly as a consequence of the anti-inflammatory and antioxidant effect of bilirubin. The aim of this study was to determine whether such an association exists in HIV-infected individuals.

Methods

A cross-sectional study was performed in HIV-1-infected adults on stable antiretroviral therapy (ART) to determine if a relationship exists between total bilirubin and endothelial function [flow-mediated dilation (FMD) of the brachial artery], inflammation [interleukin-6 (IL-6), soluble tumour necrosis factor receptors, C-reactive protein, and adhesion molecules], coagulation markers (fibrinogen and D-dimer) and oxidative stress (F 2-isoprostanes). Endpoints were compared based on total bilirubin levels and atazanavir status using distributionally appropriate, two-sample tests. Correlation coefficients were determined between total bilirubin and endpoints. Linear regression was used to model the relationship between total bilirubin (and atazanavir status) and FMD.

Results

A total of 98 adults were included in the study. Total bilirubin was higher in the atazanavir group when compared to the non-atazanavir group [median (interquartile range) 1.8 (1.1–2.6) vs. 0.6 (0.4–1.4) mg/dL; P < 0.01] as were insulin, the homeostasis model assessment of insulin resistance (HOMA-IR) and fibrinogen. Total bilirubin was positively correlated with fibrinogen and was not correlated with other outcomes. After adjustment, neither total bilirubin nor atazanavir status was associated with FMD.

Conclusions

In virologically suppressed, HIV-infected adults on stable ART, neither total bilirubin nor atazanavir use was associated with improved endothelial function as measured using FMD, inflammation or oxidative stress as measured using biomarkers.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The important role of inflammation in atherosclerosis and atherothrombosis is increasingly recognized [1], and in HIV-infected patients, it may be the principal driver of increased risk of subclinical atherosclerosis [2] and cardiovascular events [3]. This has spurred interest in the development of anti-inflammatory therapeutics to reduce cardiovascular risk.

Bilirubin, an endogenous product of haemoglobin catabolism, has antioxidant and anti-inflammatory properties that attenuate endothelial activation and dysfunction in response to pro-inflammatory stress [4]. It has been shown to prevent oxidation of low-density lipoproteins and to inhibit vascular cell adhesion molecule-1 (sVCAM-1)-dependent migration of leucocytes into the endothelium [5]. Epidemiological studies in HIV-uninfected populations have associated elevated serum bilirubin levels with better endothelial function [6] and lower prevalences of coronary heart disease [7], stroke [8] and lower-extremity peripheral arterial disease [9].

Gilbert's syndrome is a common cause of mildly elevated indirect bilirubin which occurs in patients with decreased activity of hepatic bilirubin uridine diphosphate-glucuronosyltransferase (UGT1A1). A specific mutation in this gene (UGT1A1*28) has been associated with a lower risk of cardiovascular disease [10]. The protease inhibitor (PI) atazanavir (ATV) inhibits UGT1A1 activity, which results in mild hyperbilirubinaemia, similar to Gilbert's syndrome. As such, ATV may have a beneficial effect on inflammation, oxidative stress and cardiovascular risk which is independent of its favourable metabolic profile.

Studies have been conflicting with regard to the effect of ATV on endothelial function. In a small, randomized, placebo-controlled trial in patients with diabetes, 3 days of ATV 300 mg twice daily improved endothelial function measured using venous occlusion plethysmography [11]. However, in another small, randomized, placebo-controlled trial in healthy adult men, 4 weeks of ATV 400 mg daily did not affect methacholine-induced endothelium-dependent vasodilation of the femoral artery [12]. In HIV infection, two randomized trials that switched patients to unboosted [13] or boosted [14] ATV failed to show short-term improvements in endothelial function measured using flow-mediated dilation (FMD) of the brachial artery. These two studies focused on whether improvement in lipid profiles would restore endothelial function. There is no report on the relationship between serum bilirubin and endothelial function.

The primary objective of our study was to examine the relationship between total bilirubin level and endothelial function measured using FMD of the brachial artery among ATV users and nonusers. We additionally assessed the relationship between total bilirubin and markers of inflammation, coagulation, oxidative stress and lipid levels.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Study design

This was a retrospective, cross-sectional study designed to evaluate the relationship between total bilirubin levels and FMD of the brachial artery as well as markers of inflammation, coagulation and oxidative stress and lipid levels. All HIV-1-infected adults on stable antiretroviral therapy (ART) for at least 12 weeks with HIV-1 RNA < 400 HIV-1 RNA copies/mL who had FMD of the brachial artery performed using ultrasound as part of entry into a study through the HIV Metabolic Research Center at Case Western Reserve University were eligible for inclusion in this study. Exclusion criteria were active infection, an inflammatory condition or malignancy, uncontrolled diabetes mellitus, creatinine clearance <50 mL/min, alanine aminotransferase (ALT) or aspartate aminotransferase (AST) > two times the upper limit of normal within 6 months, pregnancy, lactation, regular use of anti-inflammatory or antioxidant medication, injecting drug use or daily alcohol use. No selection criteria regarding specific ART regimens were imposed for any of the studies. Participants evaluated for enrolment into the HIV Metabolic Research Center studies were recruited from the John T. Carey Special Immunology Unit at University Hospitals Case Medical Center in Cleveland, OH. All individuals provided written informed consent to participate in the HIV Metabolic Research Center trials and also to have their blood stored for use in future HIV-related metabolic research. This study was approved by the University Hospital Case Medical Center Institutional Review Board with a waiver for further informed consent.

All data collected, demographics, HIV and cardiovascular characteristics, laboratory values and stored samples were obtained on the date on which FMD was performed. The primary outcome of this study was endothelial function determined using FMD of the brachial artery. Secondary outcomes of interest included markers of inflammation [interleukin-6 (IL-6), soluble tumour necrosis factor receptors I and II (sTNFR-I and -II), high-sensitivity C-reactive protein (hs-CRP), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1)], coagulation (D-dimer and fibrinogen), oxidative stress (F2-isoprostanes), lipoprotein levels and insulin resistance estimated using the homeostasis model assessment of insulin resistance (HOMA-IR).

Evaluation of endothelial function

Endothelial function was evaluated by measuring FMD of the brachial artery with ultrasound [15] as previously described [16]. Participants were instructed to come fasting, to not take anti-hypertensive medications and not to use tobacco or caffeine-containing products for 12 h before the study. All studies were performed by a single technologist (CW) using a Phillips iU22 Ultrasound and an L10-7 MHz linear array transducer (Phillips Healthcare, Bothell, WA, USA) and a 5-min occlusion time. Images were read using Brachial Artery Analyzer software (Medical Imaging Applications LLC, Coralville, IA, USA), a semi-automated, border-interfacing program. For FMD determination, brachial artery diameters before and after confirmed reactive hyperaemia were measured in triplicate and averaged from a 1-cm segment of the artery. FMD is expressed as a percentage change from baseline brachial artery diameter to brachial artery diameter after reactive hyperaemia.

Evaluation of biomarkers

Plasma from each participant was previously stored at −70°C immediately after processing. Stored samples were then batched and tested for the markers of inflammation, coagulation and oxidative stress outlined above. IL-6, sTNFR-I and -II, sICAM-1 and sVCAM-1 were determined by quantitative sandwich enzyme-linked immunosorbent assays (ELISAs) (R&D Systems, Minneapolis, MN, USA). Interassay variability was 2.02–15.36%, 3.66–5.77%, 2.13–3.79%, 3.43–7.37% and 4.76–8.77%, respectively. hs-CRP and fibrinogen were determined using particle enhanced immunonephelometric assays on a BNII nephelometer (Siemens, Indianapolis, IN, USA). Interassay variability was 3.01–6.46% and 3.42–7.59%, respectively. D-dimer was determined using an immunoturbidometric assay on a STA-R Coagulation Analyzer (Diagnostica Stago, Parsippany, NJ, USA). Interassay variability was 1.54–9.03%. All the above biomarker assays were performed at the Laboratory for Clinical Biochemistry Research under the direction of Dr Russell Tracy, Department of Pathology, University of Vermont. F2-isoprostanes were measured in the Eicosanoid Core Laboratory at Vanderbilt University. Briefly, F2-isoprostanes were quantified using gas chromatography–mass spectrometry after Sep-Pak (Waters Corporation, Milford, MA, USA) and thin layer chromatography purification as pentafluorobenzyl ester and trimethylsilyl ether derivatives utilizing stable isotope dilution techniques with [2H4]-15-F2t-IsoP (Cayman Chemical, Ann Arbor, MI, USA) as an internal standard. The precision of this assay is ±4%, the accuracy is ±95% and the interassay variability is <8%.

Data analysis

Important demographic, HIV and cardiovascular factors are described for the group overall, by ATV status (currently taking ATV vs. not) and by total bilirubin level (≥75th percentile vs. <75th percentile). The median and interquartile range (IQR) are reported for continuous variables and the frequency and percentage for categorical variables. All demographic, HIV and cardiovascular factors, as well as endpoints, were compared based on ATV status and total bilirubin level using unpaired t-tests or Wilcoxon rank sum tests as distributionally appropriate for continuous variables, and χ2 tests, Fisher's exact tests or Pearson exact χ2 tests as appropriate for categorical variables.

Spearman correlation coefficients were determined between total bilirubin as a continuous variable and endpoints. All above statistical tests were two-sided and considered significant with P < 0.05. No corrections for multiple comparisons were made in this exploratory study.

Next, in order to explore the relationship between FMD and total bilirubin in this sample, univariable followed by multivariable linear regressions were performed. In the univariable analysis, all demographic, HIV and cardiovascular factors, and inflammation, coagulation and oxidative stress markers as well as ATV status and total bilirubin as a dichotomized variable by ≥75th percentile compared with <75th percentile and a continuous variable were modelled with FMD as the outcome. In the first multivariable modelling approach, those variables with P < 0.25 were included in three separate multivariable models with ATV status or total bilirubin, as a categorical or continuous variable, as the independent variable of interest. In addition, a second multivariable modelling approach including clinically relevant variables regardless of statistical association was undertaken. Age, sex, race, body mass index (BMI), CD4 cell count, HIV-1 RNA level, whether on an anti-hypertensive or cholesterol-lowering medication, smoking status and brachial artery diameter were included in three separate models with ATV status or total bilirubin, as a categorical or continuous variable, as the independent variables of interest as above. In the multivariable models, variables with P < 0.05 were considered statistically significant. Final models were checked to ensure that the assumptions of linear regression were met.

All analyses were performed using sas v. 9.2 (SAS Institute, Cary, NC).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

A total of 98 individuals met the eligibility criteria. Table 1 shows demographic, cardiovascular and HIV characteristics overall, by ATV status (ATV vs. no ATV) and by total bilirubin level (≥75th percentile vs. <75th percentile). Comparing participants on ATV with those not on ATV, the groups were similar except for total bilirubin level, insulin and HOMA-IR. Total bilirubin was higher in the ATV group [median (IQR) 1.8 (1.1–2.6) vs. 0.4 (0.3–0.5) mg/dL for those not on ATV; P < 0.01], as expected. Insulin level and HOMA-IR were also higher in the ATV group [10 (6–17) vs. 7 (4–14) μIU/mL; P = 0.05 and 2.1 (1–4) vs. 1.4 (0.9–2.8); P = 0.05, respectively]. More patients in the highest quartile of total bilirubin were on PIs compared with those in the lowest three quartiles (96 vs. 37%, respectively; P < 0.01). For all other characteristics the groups were similar.

Table 1. Demographic, cardiovascular and HIV characteristics of patients by atazanavir use and by total bilirubin level
VariableOverall (N = 98)Atazanavir (n = 36)No atazanavir (n = 62)P* Total bilirubin ≥1.4 mg/dL (n = 25)Total bilirubin <1.4 mg/dL (n = 73) P *
  1. Continuous variables are reported as median (interquartile range); categorical variables are reported as frequency (column percentage).

  2. BP, blood pressure; HTN, hypertension; HDL, high-density lipoprotein; HOMA-IR, homeostasis model of insulin resistance; PI, protease inhibitor; NNRI, nonnucleoside reverse transcriptase inhibitor.

  3. *Unpaired t-test or Wilcoxon rank sum test as distributionally appropriate for continuous variables; χ2 test, Fisher's exact test or Pearson exact χ2 test as appropriate for categorical variables.

  4. **HOMA-IR was calculated using the following formula: HOMA-IR = fasting glucose (mg/dl) x fasting insulin (μU/ml)/405.

Age (years)47.5 (43–52)47 (44–51.5)48 (43–53)0.749 (45–53)47 (43–52)0.44
Gender (male)86 (88)31 (86)55 (89)0.7523 (92)63 (86)0.73
Race   0.75  0.94
Caucasian41 (42)13 (36)28 (45) 10 (40)31 (42)
African American52 (53)21 (58)31 (50) 14 (56)38 (52)
Latino4 (4)2 (6)2 (3) 1 (4)3 (4)
Other1 (1)0 (0)1 (2) 0 (0)1 (1)
Smoking status   0.42  0.38
Never smoked30 (31)8 (22)22 (35) 6 (24)24 (33)
Current smoker46 (47)19 (53)27 (44) 11 (44)35 (48)
Past smoker22 (22)9 (25)13 (21) 8 (32)14 (19)
Systolic BP (mmHg)120 (112–128)116.5 (112–122)120.5 (106–130)0.3115 (112–120)120 (106–130)0.33
Diastolic BP (mmHg)80 (74–86)80 (76–86)80 (72–85)0.6878 (76–85)80 (74–86)0.65
On anti-HTN drug29 (30)10 (28)19 (31)0.766 (24)23 (32)0.48
Total cholesterol (mg/dL)178 (151–209)165.5 (147–190.5)186.5 (156–210)0.24154 (139–214)181 (156–208)0.61
HDL cholesterol (mg/dL)40.5 (36–51)40 (35.5–46.5)41.5 (36–55)0.2940 (36–45)41 (35–52)0.87
Non-HDL cholesterol (mg/dL)131.5 (110–166)123.5 (103–154)139 (116–170)0.36119 (102–166)134 (116–165)0.57
Triglycerides (mg/dL)124.5 (84–198)137.5 (94–211.5)115.5 (81–198)0.27126 (87–182)123 (83–202)0.78
On lipid-lowering drug26 (27)6 (17)20 (32)0.096 (24)20 (27)0.74
Glucose (mg/mL)84 (78–90)86 (78.5–92)84 (78–89)0.1984 (78–92)84 (78–90)0.62
Insulin (μIU/mL)8 (5–15)10 (6–17)7 (4–14)0.058 (6–15)8 (4–15)0.61
HOMA-IR** 1.8 (0.9–3.2)2.1 (1–4)1.4 (0.9–2.8)0.051.8 (1–3.2)1.9 (0.9–3)0.6
Body mass index (kg/m2)26 (23.3–30)28.3 (23.2–30.6)25.2 (23.3–29)0.127.8 (23.2–30)25.5 (23.3–29.6)0.68
Total bilirubin (mg/dL)0.6 (0.4–1.4)1.8 (1.1–2.6)0.4 (0.3–0.5)<0.01
AST (U/L)22 (17–29)20.5 (16–30)22 (17–28)0.6221 (16–28)22 (17–29)0.77
ALT (U/L)40 (32–51)39 (32–53.5)40 (32–50)0.8540 (36–50)39 (30–51)0.41
CD4 count (cells/μL)578.5 (431–789)556 (451.5–739.5)588 (413–834)0.9529 (431–667)608 (455–864)0.22
Nadir CD4 count (cells/μL)130 (32–238)114 (32–231)140 (34–240)0.47130 (12–200)130 (38–246)0.24
HIV infection duration (years)11.3 (7.2–16.2)11.3 (8.8–15.2)10.1 (6.5–17.5)0.8611.3 (8.8–14.3)11.2 (7.2–17.5)0.81
On PI51 (52)24 (96)27 (37)<0.01
On NNRTI47 (48)1 (4)46 (63)<0.01

Results of FMD analysis and inflammation, coagulation and oxidation marker levels overall, by ATV use (ATV vs. no ATV) and by total bilirubin level (≥75th percentile vs. <75th percentile), are shown in Table 2. There were no differences between groups with regard to the baseline brachial artery diameter. Median (IQR) FMD for the overall group was low, 3.29% (1.58–6.17%), compared with healthy adults [17] and there were no between-group differences with regard to FMD in this study. There were no significant differences between groups divided by ATV use or by bilirubin level with regard to inflammation markers, D-dimer or F2-isoprostanes. However, fibrinogen was higher in the ATV group. Total bilirubin level as a continuous variable was not correlated with FMD or any inflammation or oxidation markers, with the exception of fibrinogen (Spearman correlation coefficient was 0. 2285; P = 0.02).

Table 2. Flow-mediated dilation (FMD), inflammation, coagulation and oxidation markers overall, by atazanavir use and by total bilirubin level
VariableOverall (N = 98)Atazanavir (n = 36)No atazanavir (n = 62) P * Total bilirubin ≥1.4 mg/dL (n = 25)Total bilirubin <1.4 mg/dL (n = 73) P *
  1. All variables are reported as median (interquartile range).

  2. IL-6, interleukin-6; sTNFR-I, soluble tumour necrosis factor receptor-I; sTNFR-II, soluble tumour necrosis factor receptor-II; hs-CRP, high-sensitivity C-reactive protein; sICAM-1, soluble intercellular adhesion molecule-1; sVCAM-1, soluble vascular cell adhesion molecule-1; F2-isoPs, F2-isoprostanes.

  3. *Unpaired t-test or Wilcoxon rank sum test as distributionally appropriate.

Brachial artery diameter (mm)4.68 (4.09–5.01)4.61 (4.08–4.98)4.7 (4.28–5.01)0.574.58 (4.13–5.02)4.7 (4.05–5)0.94
FMD (%)3.29 (1.58–6.17)3.15 (0.69–5.91)3.33 (1.8–6.25)0.672.79 (0.66–4.82)3.7 (2.06–6.41)0.08
IL-6 (pg/mL)2.44 (1.52–4.08)2.82 (1.79–5.34)2.2 (1.37–3.59)0.182.57 (1.65–4.29)2.36 (1.52–3.78)0.64
sTNFR-I (pg/mL)1286.19 (1074.2–1581.1)1273.24 (1067.51–1556.79)1296.71 (1074.2–1581.1)0.821303.4 (1052.32–1686)1284.48 (1076.29–1459.62)0.81
sTNFR-II (pg/mL)2694.95 (2269.6–3291.7)2673.55 (2335.2–3389.55)2707.96 (2198.2–3050.67)0.832717.9 (2152–3672.67)2682.5 (2296.5–3027.99)0.76
hs-CRP (μg/mL)1.33 (0.8–3.8)1.78 (0.56–4.65)1.28 (0.86–2.98)0.81.08 (0.47–2.58)1.34 (0.86–3.8)0.20
sICAM-1 (ng/mL)226.89 (150.27–310.65)254.32 (153.75–317.82)223.34 (150.27–296.37)0.51251.87 (145.15–310.65)225.67 (161.5–296.37)0.88
sVCAM-1 (ng/mL)687.41 (566.83–884.36)694.3 (555.19–879.79)677.61 (574.47–891.21)0.76756.62 (629.98–884.36)669.17 (566.83–836.11)0.4
D-dimer (μg/mL)0.17 (0.11–0.28)0.18 (0.13–0.29)0.14 (0.11–0.25)0.190.14 (0.11–0.31)0.17 (0.11–0.25)>0.99
Fibrinogen (mg/dL)372.5 (322–439)411.5 (354.5–501.5)350.5 (300–418)<0.01406 (331–493)365 (318–421)0.06
F2-isoPs (ng/mL)0.05 (0.03–0.21)0.06 (0.04–0.20)0.04 (0.03–0.22)0.260.06 (0.04–0.21)0.05 (0.03–0.2)0.23

In univariable analysis, total bilirubin, age, BMI, AST, HIV-1 RNA, IL-6, D-dimer and brachial artery diameter had P < 0.25 and were entered into the first multivariable model. Neither total bilirubin (as a categorical or continuous variable) nor ATV status was independently associated with FMD in this multivariable model. In a second modelling approach adjusting for clinically relevant variables, i.e. age, sex, race, BMI, CD4 cell count, HIV-1 RNA level, whether on an anti-hypertensive or cholesterol-lowering medication, smoking status and brachial artery diameter, did not change this result. Parameter estimates and significance levels for the variables of interest are shown in Table 3 for both univariable and multivariable analyses.

Table 3. Univariable and multivariable analyses with flow-mediated dilation (FMD) as the dependent variable
 Parameter estimate95% confidence interval P
  1. *Parameter estimates shown are the variables of interest adjusted for all variables with P < 0.25 in univariable analaysis, i.e. age, body mass index (BMI), aspartate aminotransferase (AST), HIV-1 RNA level, interleukin (IL)-6, D-dimer and brachial artery diameter.

  2. **Parameter estimates shown are the variables of interest adjusted for clinically relevant variables, i.e. age, sex, race, BMI, CD4 cell count, HIV-1 RNA level, whether on an anti-hypertension or lipid-lowering medication, smoking status and brachial artery diameter, regardless of statistical associations of these variables with the outcome, FMD.

Univariable approach
Atazanavir use (yes = 1)−0.2898−1.6206–1.0410.67
Total bilirubin (>75th percentile = 1)−1.2987−2.7482–0.15080.08
Total bilirubin (mg/dL)−0.4366−1.1165–0.24340.21
First multivariable approach*
Atazanavir use (yes = 1)−0.686−1.9342–0.56230.28
Total bilirubin (>75th percentile = 1)−1.2697−2.604–0.0650.06
Total bilirubin (mg/dL)−0.3099−0.9464–0.33660.34
Second multivariable approach**
Atazanavir use (yes = 1)−0.5744−1.9078–0.75890.39
Total bilirubin (>75th percentile = 1)−1.329−2.7427–0.08480.07
Total bilirubin (mg/dL)−0.3531−1.0284–0.32210.3

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

To our knowledge this is the first study to explore the relationship between total bilirubin and endothelial function, inflammation, coagulation and oxidation in virologically suppressed, HIV-1-infected adults on stable ART. With regard to our primary endpoint, neither total bilirubin nor ATV status was significantly associated with FMD of the brachial artery. This is consistent with both the study by Flammer et al. and the SABAR (Switch to Atazanavir and Brachial Artery Reactivity) study by Murphy et al., which showed that switching to ATV from another PI, whether boosted with ritonavir or not, did not improve endothelial function measured using FMD after 24 weeks, despite significant improvement in lipid levels [13, 14]. It is conceivable that the effect of a modest increase in bilirubin in this population is masked by the ongoing heightened inflammation resulting from chronic HIV infection. Indeed, with potent ART, inflammation and endothelial dysfunction do improve [18, 19], but not to normal levels, when compared with HIV-uninfected individuals [2, 19]. Further, participants included in this study did not have extremely elevated total bilirubin levels (median 1.8 mg/dL; IQR 1.1–2.6 mg/dL; minimum 0.3 mg/dL and maximum 5 mg/dL). Although seeing an effect with extreme hyperbilirubinaemia would be mechanistically intriguing, this would have uncertain clinical relevance. Another consideration is that the antioxidant effect of elevated bilirubin was outweighed by the oxidative stress induced by ART [20].

Although a different method for measuring endothelial function was used, our results are incongruent with the study by Dekker et al., in which ATV-induced hyperbilirubinaemia did improve endothelium-dependent vasodilation measured using forearm blood flow response to acetylcholine in participants with type II diabetes mellitus after 3 days [11]. In our study, perhaps an effect would have been seen if FMD had been measured earlier after ATV was initiated [median (IQR) duration on ATV in our study was 28.5 (16.8–47.7) months]. The clinical implication of a solely transient acute effect would also be questionable, however. Another consideration is that endothelial dysfunction is more pronounced in subjects with diabetes mellitus than in our HIV-infected population and is why an effect was seen in this potentially higher risk group. To better assess whether the degree of endothelial dysfunction played a role in the association between total bilirubin level and FMD in our study, the correlation between total bilirubin level and FMD in those with the lowest FMD (FMD less than the median FMD of 3.3%) was determined. Total bilirubin was not correlated with FMD in this subgroup (Spearman correlation coefficient = 0.13; P = 0.38).

With regard to our secondary endpoints, neither total bilirubin nor ATV status was associated with markers of inflammation, coagulation or oxidation, with the exception of fibrinogen. Fibrinogen levels were higher among participants taking ATV. This result is consistent with a study by Madden et al., where PI use was associated with elevated fibrinogen levels.[18] In our study, median (IQR) fibrinogen was 411 (49–411) mg/dL in those on a PI vs. 334 (294–334) mg/dL in those not on a PI (P < 0.01). Because most participants in our study on a PI were on ATV (36/51), it stands to reason that one is a marker for the other.

The strength of this study is the large number of participants, allowing for adequate power to address the study question. There are limitations, however. Because ART was not randomized in this study, unmeasured confounding or confounding by indication could be the reason for the results obtained. Cardiovascular risk may have contributed to the decision to prescribe an ATV-based regimen. If this were true, FMD may have been impaired to a greater extent in patients receiving ATV and may have masked the effect of bilirubin. However, cardiovascular risk factors were balanced between the participants, including those not modifiable, i.e. age, sex and race. Also, adjusting for cardiovascular risk factors did not change the results qualitatively. In addition, we were unable to control for time on ATV or prior ART exposure. As suggested above, an effect may have been seen if participants had recently been started on ATV; however, the clinical benefit of a transient effect of this intervention would be questionable. Another limitation is the lack of adjustment for multiple statistical tests, which could have increased the likelihood of finding statistical significance from chance alone. Finally, because of the cross-sectional design, it is not possible to attribute cause to effect. Given the negative results of this study, these last two points are less important, but should be taken into account in the design of future studies.

In conclusion, neither ATV use nor higher total bilirubin levels were statistically associated with better endothelial function or lower inflammation and oxidation in virologically suppressed, HIV-1-infected adults on stable ART. It is possible that the antioxidant and/or the anti-inflammatory effect of bilirubin is transient or is observed only with very high levels of bilirubin, or that it is not sufficiently potent to overcome other causes of endothelial dysfunction in this population.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors would like to thank the patients who participated in this research. This work was funded by the National Institute of Health (NR012642), Bristol-Myers Squibb and the Campbell Foundation and received support from the Case Center for AIDS Research (NIH Grant Number: A136219). COH has received research grant support from Bristol-Myers Squibb. CTL has received research grant support from Bristol-Myers Squibb. TLC serves on the DSMB of Prairie Education and Research Cooperative, has received research grant support from Baxter, Inc. and is on the speaker's bureau for Sanofi-Aventis. GAM has received research grant support and serves as a consultant for GlaxoSmithKline, Bristol-Myers Squibb, Gilead Sciences, and Tibotec and currently serves as the DMC Chair for a Pfizer-sponsored clinical trial. All other authors have no conflicts.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • 1
    Libby P, Ridker PM, Hansson GK. Inflammation in atherosclerosis: from pathophysiology to practice. J Am Coll Cardiol 2009; 54: 21292138.
  • 2
    Ross AC, Rizk N, O'Riordan MA et al. Relationship between inflammatory markers, endothelial activation markers, and carotid intima-media thickness in HIV-infected patients receiving antiretroviral therapy. Clin Infect Dis 2009; 49: 11191127.
  • 3
    Phillips AN, Carr A, Neuhaus J et al. Interruption of antiretroviral therapy and risk of cardiovascular disease in persons with HIV-1 infection: exploratory analyses from the SMART trial. Antivir Ther 2008; 13: 177187.
  • 4
    Kawamura K, Ishikawa K, Wada Y et al. Bilirubin from heme oxygenase-1 attenuates vascular endothelial activation and dysfunction. Arterioscler Thromb Vasc Biol 2005; 25: 155160.
  • 5
    Keshavan P, Deem TL, Schwemberger SJ, Babcock GF, Cook-Mills JM, Zucker SD. Unconjugated bilirubin inhibits VCAM-1-mediated transendothelial leukocyte migration. J Immunol 2005; 174: 37093718.
  • 6
    Erdogan D, Gullu H, Yildirim E et al. 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 2006; 184: 431437.
  • 7
    Djousse L, Levy D, Cupples LA, Evans JC, D'Agostino RB, Ellison RC. Total serum bilirubin and risk of cardiovascular disease in the Framingham offspring study. Am J Cardiol 2001; 87: 11961200.
  • 8
    Perlstein TS, Pande RL, Creager MA, Weuve J, Beckman JA. Serum total bilirubin level, prevalent stroke, and stroke outcomes: NHANES 1999-2004. Am J Med 2008; 121: 781788.
  • 9
    Perlstein TS, Pande RL, Beckman JA, Creager MA. Serum total bilirubin level and prevalent lower-extremity peripheral arterial disease: National Health and Nutrition Examination Survey (NHANES) 1999 to 2004. Arterioscler Thromb Vasc Biol 2008; 28: 166172.
  • 10
    Lin JP, O'Donnell CJ, Schwaiger JP et al. Association between the UGT1A1*28 allele, bilirubin levels, and coronary heart disease in the Framingham Heart Study. Circulation 2006; 114: 14761481.
  • 11
    Dekker D, Dorresteijn MJ, Pijnenburg M et al. The bilirubin-increasing drug atazanavir improves endothelial function in patients with type 2 diabetes mellitus. Arterioscler Thromb Vasc Biol 2011; 31: 458463.
  • 12
    Dube MP, Shen C, Greenwald M, Mather KJ. No impairment of endothelial function or insulin sensitivity with 4 weeks of the HIV protease inhibitors atazanavir or lopinavir-ritonavir in healthy subjects without HIV infection: a placebo- controlled trial. Clin Infect Dis 2008; 47: 567574.
  • 13
    Flammer AJ, Vo NT, Ledergerber B et al. Effect of atazanavir versus other protease inhibitor-containing antiretroviral therapy on endothelial function in HIV-infected persons: randomised controlled trial. Heart 2009; 95: 385390.
  • 14
    Murphy RL, Berzins B, Zala C et al. Change to atazanavir/ritonavir treatment improves lipids but not endothelial function in patients on stable antiretroviral therapy. AIDS 2010; 24: 885890.
  • 15
    Corretti MC, Anderson TJ, Benjamin EJ et al. Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force. J Am Coll Cardiol 2002; 39: 257265.
  • 16
    Hileman CO, Carman TL, Storer NJ, Labbato DE, White CA, McComsey GA. Omega-3 Fatty Acids Do Not Improve Endothelial Function In Virologically Suppressed HIV-Infected Men: a Randomized Placebo-Controlled Trial. AIDS Res Hum Retroviruses 2011 [Epub ahead of print].
  • 17
    Celermajer DS, Sorensen KE, Gooch VM et al. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet 1992; 340: 11111115.
  • 18
    Torriani FJ, Komarow L, Parker RA et al. Endothelial function in human immunodeficiency virus-infected antiretroviral-naive subjects before and after starting potent antiretroviral therapy: the ACTG (AIDS Clinical Trials Group) Study 5152s. J Am Coll Cardiol 2008; 52: 569576.
  • 19
    Regidor DL, Detels R, Breen EC et al. Effect of highly active antiretroviral therapy on biomarkers of B-lymphocyte activation and inflammation. AIDS 2011; 25: 303314.
  • 20
    Mondal D, Pradhan L, Ali M, Agrawal KC. HAART drugs induce oxidative stress in human endothelial cells and increase endothelial recruitment of mononuclear cells: exacerbation by inflammatory cytokines and amelioration by antioxidants. Cardiovasc Toxicol 2004; 4: 287302.