Diabetologia

, Volume 56, Issue 4, pp 724–736 | Cite as

Plasma total bilirubin levels predict amputation events in type 2 diabetes mellitus: the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study

  • K. H. Chan
  • R. L. O’Connell
  • D. R. Sullivan
  • L. S. Hoffmann
  • K. Rajamani
  • M. Whiting
  • M. W. Donoghoe
  • M. Vanhala
  • A. Hamer
  • B. Yu
  • R. Stocker
  • M. K. C. Ng
  • A. C. Keech
  • on behalf of the FIELD study investigators
Article

Abstract

Aims/hypothesis

Bilirubin has antioxidant and anti-inflammatory activities. Previous studies demonstrated that higher bilirubin levels were associated with reduced prevalence of peripheral arterial disease (PAD). However, the relationship between bilirubin and lower-limb amputation, a consequence of PAD, is currently unknown. We hypothesised that, in patients with type 2 diabetes, bilirubin concentrations may inversely associate with lower-limb amputation.

Methods

The relationship between baseline plasma total bilirubin levels and amputation events was analysed in 9,795 type 2 diabetic patients from the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study. The analysis plan was pre-specified. Lower-limb amputation was adjudicated blinded to treatment allocation. Relevant clinical and biochemical data were available for analyses. Amputation was a pre-specified tertiary endpoint.

Results

Bilirubin concentrations were significantly inversely associated with lower-limb amputation, with a greater than threefold risk gradient across levels. Individuals with lower bilirubin concentrations had a higher risk for first amputation (HR 1.38 per 5 μmol/l decrease in bilirubin concentration, 95% CI 1.07, 1.79, p = 0.013). The same association persisted after adjustment for baseline variables, including age, height, smoking status, γ-glutamyltransferase level, HbA1c, trial treatment allocation (placebo vs fenofibrate), as well as previous PAD, non-PAD cardiovascular disease, amputation or diabetic skin ulcer, neuropathy, nephropathy and diabetic retinopathy (HR 1.38 per 5 μmol/l decrease in bilirubin concentration, 95% CI 1.05, 1.81, p = 0.019).

Conclusions/interpretation

Our results identify a significant inverse relationship between bilirubin levels and total lower-limb amputation, driven by major amputation. Our data raise the hypothesis that bilirubin may protect against amputation in type 2 diabetes.

Keywords

Amputation Bilirubin Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study Peripheral arterial disease Type 2 diabetes mellitus 

Abbreviations

CVD

Cardiovascular disease

FIELD

Fenofibrate Intervention and Event Lowering in Diabetes

GGT

γ-Glutamyltransferase

HMOX-1

Haem oxygenase-1

IRR

Incidence rate ratio

PAD

Peripheral arterial disease

UGT1A1

Uridine diphosphate glucuronosyltransferase-1 family polypeptide A1

Introduction

Non-traumatic lower-limb amputations are often the end-stage clinical events in intractable limb ischaemia. Despite modern therapy, peripheral arterial disease (PAD) remains a major burden on the healthcare system, with at least one amputation due to diabetes occurring every 30 s worldwide, up to eight million patients in the USA being devastated by immobility and significant morbidity, and an annual cost exceeding US$1.6 billion in 2001 [1, 2]. Furthermore, attention to classic vascular risk factors has failed to substantially reduce the risk of amputation [3], highlighting the need to find novel predictors and biomarkers for PAD and amputation events that may help to identify new therapeutic targets [4, 5, 6].

Bilirubin, a product of haem degradation, may confer vascular protective effects [7, 8], and is therefore a possible candidate biomarker for predicting amputation events. Experimental studies have reported that bilirubin possesses potent antioxidant and anti-inflammatory properties in vitro and in vivo [9, 10, 11, 12, 13, 14]. Given that atherosclerosis and ischaemia are characterised by a state of heightened inflammation and oxidation [15, 16], it is conceivable that bilirubin may confer beneficial effects through these known activities. Consistent with this notion, case–control studies have reported that individuals with elevated bilirubin levels caused by Gilbert’s syndrome have a decreased incidence of atherosclerotic disease compared with normal controls [12, 14]. Also, previous studies reported an inverse relationship between bilirubin levels and PAD prevalence [17, 18, 19, 20]. However, the association between bilirubin and the hard clinical endpoint of amputation events has not been reported, to our knowledge. We therefore hypothesised that plasma bilirubin concentrations may inversely associate with lower-limb amputation. We studied this association in a longitudinal cohort of individuals with type 2 diabetes, as these patients have an eightfold higher amputation risk compared with non-diabetic patients [21].

Methods

Study participants

The relationship between baseline plasma total bilirubin levels and non-traumatic amputation was analysed in 9,795 type 2 diabetic patients from the FIELD (Fenofibrate Intervention and Event Lowering in Diabetes) study in a subsidiary analysis. In brief, patients in the FIELD study were randomised to either fenofibrate or placebo treatment between February 1998 and November 2000, and were followed up for a median duration of 5 years [22]. All patients were aged 50–75 years and had a diagnosis of type 2 diabetes according to WHO criteria [23]. Individuals with renal impairment, chronic liver disease, symptomatic gallbladder disease, or those who had experienced a cardiovascular event within the 3 months before recruitment were excluded. Non-traumatic amputation was a pre-specified tertiary endpoint. All amputations that occurred during study follow-up (on-study amputations) were adjudicated blinded to treatment allocation by two clinicians separately, and any discrepancies were resolved by mutual agreement. Pre-study amputations were adjudicated in the same fashion. Major amputations were defined as those above the ankle and minor amputations as those below the ankle [3]. All patients provided written informed consent. This study had ethics committee approval in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines. The original trial was registered with the International Standard Randomised Controlled Trial Number (ISRCTN) 64783481.

Laboratory measurements

Early-morning fasting baseline blood specimens were obtained in all individuals prior to study randomisation. Plasma (EDTA) levels of total bilirubin, HbA1c and γ-glutamyltransferase (GGT) were determined using an automated analyser (Hitachi 917, Roche Diagnostics, Basel, Switzerland). Total bilirubin was measured as the plasma concentration expressed in μmol/l using the Diazo method [24].

Baseline patient variables

A medical history was obtained to determine whether the patient had been diagnosed with or experienced any of: claudication or PAD; prior amputation; diabetic skin ulcer; diabetic retinopathy; neuropathy; or cardiovascular disease (CVD). Age was categorised into <65 years or ≥65 years. Previous PAD was present if the patient was diagnosed with or experienced claudication or PAD, and/or previous peripheral revascularisation. Prior non-PAD CVD was defined as any history of CHD or stroke. Neuropathy was present if the patient was diagnosed with or experienced diabetic neuropathy, and/or the patient’s foot had absent sensation on monofilament testing. Nephropathy was defined by the presence of albuminuria as previously described [3].

Statistical analyses

All analyses were performed on an intention-to-treat basis. Baseline characteristics were analysed with χ2 tests for categorical variables, t tests for continuous variables or, if the distribution of the data was non-normal, the Wilcoxon rank-sum test. Cox proportional hazards regression was used to compute HRs and 95% CIs to assess the relationship between total bilirubin levels and time to first amputation. As there were 190 amputations occurring in 115 patients, a multiple-event analysis was performed using Poisson regression modelling for all amputation events and adjusting for months of observation and overdispersion, using the Pearson method. The computed HR (or incidence rate ratio [IRR] for the Pearson method) for the bilirubin effect was expressed as the increased risk per 5 μmol/l decrease in bilirubin (≈1 SD, a statistically relevant difference in bilirubin concentrations).

A multivariable analysis was also performed adjusting for the following baseline covariates: age; height; smoking status; HbA1c; history of previous PAD; amputation or diabetic skin ulcer; neuropathy; nephropathy; diabetic retinopathy; and trial treatment allocation (placebo vs fenofibrate). These covariates (which were determined by use of backwards selection and then confirmed by exhaustive search methods) were selected as they have been reported to be significant predictors of first amputation in the FIELD study [3]. Other prior CVD (non-PAD) was also included as a covariate, as this is a well-recognised risk factor for PAD and amputation [25]. Liver function test variables (alanine transaminase and GGT) and alcohol intake were also considered as covariates, but GGT was the only significant predictor of amputation and was therefore retained as a covariate in the multivariable model. The possibility of over-fitting because of the large number of potential predictors assessed and the small number of events was examined by calculating the heuristic shrinkage factor. The shrinkage factor of 0.92 indicates that the degree of over-fitting was negligible. A test of interaction was conducted between bilirubin level and treatment allocation. The cumulative risk curves of time to first amputation across five ordered groups of bilirubin level (categorised according to 5 μmol/l [≈1SD] increments in bilirubin concentration, i.e. 0–5, 6–10, 11–15, 16–20 and ≥21 μmol/l) was calculated using the Kaplan–Meier method, and the p value computed using the logrank test of trend. The cumulative rate of amputation across the five ordered groups of bilirubin levels was analysed using the Cochran–Armitage trend test (after testing for linearity). The test for deviation from linearity was conducted by fitting a model including both a linear and a categorical version of the variable-grouped bilirubin level. For this method, the test of the overall effect of the individual categories assesses the significance of a non-linear component [26]. These tests were performed for both a logistic regression (Cochran–Armitage trend test) and a Cox model (logrank trend test) and indicated no deviation from linearity.

Post hoc analyses were also undertaken according to quintile of bilirubin concentration. A test of trend was performed by fitting a Cox model including a discrete variable derived using the median bilirubin level of each quintile (as the ranges of bilirubin level within each quintile were not equal); the level of significance was based on the score test provided by the model. Deviation from linearity was tested using the method described above. A Cox model was also fitted that included the bilirubin quintile group as a categorical variable, both unadjusted and adjusted for the variables mentioned above. The lowest bilirubin quintile was used as the reference category. Analyses were also performed separately for major and minor amputations, sex and smoking status, but in each case, there were insufficient events in at least one subgroup to perform adjusted analyses.

The stability of bilirubin level over time was assessed by calculation of the reliability ratio in a subset of 923 individuals, with repeat measures at year 1. The reliability ratio indicates the proportion of overall variation which is true variation as opposed to true plus random variation (i.e. it gives the ratio of the variance of true to observed bilirubin concentration). Hypotheses and the substudy plan were specified prior to data analyses. The substudy analysis plan stipulated the use of the same statistical methods as in the earlier FIELD amputation study [3], and that the five ordered groups of bilirubin were categorised according to ≈1 SD increments in bilirubin concentration. As only five patients, none of whom had undergone amputation, had any missing data, no statistical adjustment was made for this. A two-sided p value <0.05 was considered to indicate statistical significance.

Results

The FIELD study enrolled 9,795 individuals, with only 22 being lost to follow-up and nine withdrawing from the study. The mean age of the participants was 62 ± 7 years, with a median diabetes duration of 5 years (interquartile range 2–10 years). There were 190 amputations occurring in 115 patients, with 93 out of 6,045 (1.5%) men and 22 out of 3,750 (0.6%) women suffering from ≥1 amputation. A total of 35 patients experienced major amputations only, 65 minor amputations only, and 15 one or more of both categories. Baseline patient characteristics according to bilirubin level, sex, and amputation classification are shown in Tables 1 and 2 and electronic supplementary material (ESM) Table 1, respectively. Bilirubin levels were well balanced by study treatment group. The mean baseline plasma total bilirubin concentration was 9.7 μmol/l (median 9.0, SD 4.6, range 1–70 μmol/l). In a subset of 923 patients, bilirubin concentration measured 1 year apart did not change significantly, with a mean difference of only 0.3 μmol/l between measurements and a reliability ratio of 0.73.
Table 1

Baseline characteristics according to bilirubin levels (5 μmol/l increments) and sex

Variable

Men (n = 6,045)

Women (n = 3,750)

Bilirubin (μmol/l)

p valuea

Bilirubin (μmol/l)

p valuea

1–5

6–10

11–15

16–20

≥21

1–5

6–10

11–15

16–20

≥21

Age ≥65 years

162 (37)

1360 (41)

744 (43)

187 (44)

118 (45)

0.019

205 (32)

925 (39)

185 (39)

40 (38)

27 (52)

0.003

Height (cm, median)

173

175

175

175

176

<0.001b

161

161

162

162

160

0.142b

Hypertension

247 (56)

1716 (52)

907 (53)

207 (48)

137 (52)

0.137

403 (63)

1521 (64)

301 (63)

69 (65)

34 (65)

0.689

Hypercholesterolaemia

106 (24)

770 (23)

305 (18)

67 (16)

38 (14)

0.000

229 (36)

901 (38)

156 (33)

21 (20)

11 (21)

0.001

Smoker

96 (22)

383 (12)

120 (7)

29 (7)

10 (4)

0.000

69 (11)

184 (8)

28 (6)

3 (3)

0 (0)

0.000

HbA1c >7% (53 mmol/mol)

241 (55)

1514 (46)

727 (42)

156 (36)

93 (35)

0.000

277 (43)

1,042 (44)

221 (46)

42 (40)

20 (38)

0.962

Nephropathy

173 (39)

966 (29)

456 (26)

115 (27)

65 (25)

0.000

140 (22)

459 (19)

98 (21)

23 (22)

9 (17)

0.594

Previous PAD

55 (13)

298 (9)

118 (7)

30 (7)

23 (9)

0.003

53 (8)

147 (6)

33 (7)

5 (5)

2 (4)

0.118

Previous non-PAD CVD

110 (25)

596 (18)

293 (17)

80 (19)

52 (20)

0.122

105 (16)

320 (13)

72 (15)

12 (11)

9 (17)

0.489

Prior amputation or diabetic skin ulcer

24 (5)

120 (4)

41 (2)

9 (2)

8 (3)

0.003

20 (3)

76 (3)

13 (3)

2 (2)

2 (4)

0.672

Diabetic neuropathy

114 (26)

606 (18)

310 (18)

76 (18)

46 (17)

0.017

91 (14)

353 (15)

72 (15)

15 (14)

5 (10)

0.811

Diabetic retinopathy

50 (11)

295 (9)

150 (9)

32 (7)

28 (11)

0.385

46 (7)

172 (7)

34 (7)

6 (6)

0 (0)

0.211

Fenofibrate treatment

230 (52)

1,595 (49)

882 (51)

219 (51)

144 (54)

0.121

332 (52)

1,167 (49)

247 (52)

54 (51)

24 (46)

0.775

Results are expressed as n (%)

To convert μmol/l to mg/dl, divide by 17.1

aDifferences between categories were analysed using the Cochran–Armitage trend test

bLinear regression of height vs bilirubin group variable

Table 2

Baseline characteristics according to bilirubin levels (quintiles) and sex

Variable

Men (n = 6,045)

Women (n = 3,750)

Bilirubin (μmol/l)

p valuea

Bilirubin (μmol/l)

p valuea

1–6

7–8

9–10

11–12

≥13

1–6

7–8

9–10

11–12

≥13

Age ≥65 years

363 (37)

578 (41)

581 (43)

402 (41)

647 (45)

0.000

431 (34)

433 (39)

266 (42)

117 (40)

135 (39)

0.008

Height (cm, median)

174

174

175

175

175

<0.001b

161

161

161

162

161

0.054b

Hypertension

541 (56)

715 (51)

707 (52)

504 (51)

747 (52)

0.326

797 (63)

698 (63)

429 (68)

180 (62)

224 (65)

0.211

Hypercholesterolaemia

245 (25)

349 (25)

282 (21)

184 (19)

226 (16)

0.000

481 (38)

410 (37)

239 (38)

102 (35)

86 (25)

0.000

Smoker

194 (20)

175 (13)

110 (8)

75 (8)

84 (6)

0.000

123 (10)

93 (8)

37 (6)

17 (6)

14 (4)

0.000

HbA1c >7% (53 mmol/mol)

507 (52)

671 (48)

577 (43)

428 (43)

548 (38)

0.000

567 (45)

477 (43)

275 (44)

129 (44)

154 (45)

0.963

Nephropathy

345 (35)

413 (30)

381 (28)

260 (26)

376 (26)

0.000

271 (21)

215 (19)

113 (18)

61 (21)

69 (20)

0.445

Previous PAD

107 (11)

134 (10)

112 (8)

69 (7)

102 (7)

0.000

95 (7)

71 (6)

34 (5)

21 (7)

19 (6)

0.151

Previous non-PAD CVD

213 (22)

247 (18)

246 (18)

164 (17)

261 (18)

0.118

199 (16)

148 (13)

78 (12)

49 (17)

44 (13)

0.210

Prior amputation or diabetic skin ulcer

46 (5)

57 (4)

41 (3)

21 (2)

37 (3)

0.001

42 (3)

35 (3)

19 (3)

8 (3)

9 (3)

0.455

Diabetic neuropathy

215 (22)

262 (19)

243 (18)

179 (18)

253 (18)

0.023

182 (14)

177 (16)

85 (14)

43 (15)

49 (14)

0.787

Diabetic retinopathy

102 (10)

129 (9)

114 (8)

84 (9)

126 (9)

0.224

91 (7)

76 (7)

51 (8)

25 (9)

15 (4)

0.318

Fenofibrate treatment

472 (49)

715 (51)

638 (47)

507 (51)

738 (52)

0.210

627 (49)

554 (50)

318 (51)

155 (53)

170 (49)

0.637

Results are expressed as n (%)

To convert μmol/l to mg/dl, divide by 17.1

aDifferences between categories were analysed using the test of trend using the median bilirubin level of each quintile

bLinear regression of height vs bilirubin (median bilirubin level of each quintile)

Association between baseline plasma total bilirubin concentrations and amputation events

Baseline plasma total bilirubin concentrations were significantly inversely associated with lower-limb amputation rate (HR 1.38 per 5 μmol/l decrease in bilirubin concentration, 95% CI 1.07, 1.79, p = 0.013; Table 3). The same association persisted after adjustment for baseline covariates including age, height, smoking status, GGT, HbA1c, trial treatment allocation (placebo vs fenofibrate), and previous PAD, non-PAD CVD, amputation or diabetic skin ulcer, neuropathy, nephropathy and diabetic retinopathy (HR 1.38 per 5 μmol/l decrease in bilirubin concentration, 95% CI 1.05, 1.81, p = 0.019). There was also no interaction between bilirubin concentration and treatment allocation (p = 0.74). Likewise, the multiple-event analysis revealed a similar relationship (IRR 1.38 per 5 μmol/l decrease in bilirubin concentration, 95% CI 1.02, 1.86, p = 0.035). This association also persisted after adjustment for baseline covariates (IRR 1.37 per 5 μmol/l decrease in bilirubin concentration, 95% CI 1.08, 1.73, p = 0.009). Patients with only one amputation and multiple (≥2) amputation events had similar bilirubin concentrations (median 8 μmol/l for both, p = 0.29).
Table 3

Association between baseline plasma total bilirubin concentration and risk of first amputation event, and total amputation events

Variable

Risk of amputation (HR per 5 μmol/l decrease in bilirubin, 95% CI)

p value

First amputation event

 Unadjusted

1.38 (1.07, 1.79)

0.013

 Adjusteda

1.38 (1.05, 1.81)

0.019

Total amputation eventsb

 Unadjusted

1.38 (1.02, 1.86)

0.035

 Adjusteda

1.37 (1.08, 1.73)

0.009

To convert μmol/l to mg/dl, divide by 17.1

aAdjusted for the following baseline patient variables: age, height, smoking status, GGT, HbA1c, and history of previous PAD, non-PAD CVD, amputation or diabetic skin ulcer, neuropathy, nephropathy and diabetic retinopathy, as well as trial treatment allocation (placebo vs fenofibrate)

bPoisson method: the IRR, analogous to the HR, is shown

When the bilirubin levels were categorised into 5 μmol/l (≈1 SD) increments in concentration, the cumulative risk of amputation over the study duration was highest in patients with the lowest (0–5 μmol/l) bilirubin concentrations, and lowest (with no amputation events) in patients with the highest bilirubin (≥21 μmol/l) concentrations (p = 0.025; Fig. 1a). Figure 1b shows the cumulative rate of amputation by sex and smoking status. If the patients with bilirubin ≥21 μmol/l were excluded from the analysis, the relationship was no longer significant (p = 0.11).
Fig. 1

Amputation risk according to plasma total bilirubin concentration (5 μmol/l increments). (a) Cumulative risk curves (% events over time) to first amputation event. Logrank test of trend, p = 0.025. (b) Cumulative 5 year amputation rates by sex and smoking status classification. Black bars, male smokers; white bars, male non-smokers; diagonally hatched bars, female smokers; horizontally hatched bars, female non-smokers. To convert μmol/l to mg/dl, divide by 17.1

Analyses according to bilirubin quintiles also demonstrated a similar inverse association between bilirubin and amputation. A bilirubin level <6 μmol/l carries a significantly greater (twofold higher) risk than that for individuals with bilirubin >12 μmol/l (HR 0.51 for highest quintile, 95% CI 0.27, 0.94, p = 0.031; Table 4 and Fig. 2a). This association also persisted after adjusting for baseline covariates (HR 0.50, 95% CI 0.27, 0.95, p = 0.035). Figure 2b shows the cumulative rate of amputation by sex, smoking status and bilirubin quintile.
Table 4

Association between baseline plasma total bilirubin concentration (according to quintiles) and risk of first amputation event

Bilirubin (μmol/l)

Risk of first amputation (HR, 95% CI)

p value

Unadjusted analysis

 ≤6

1 (reference)

 7–8

0.76 (0.47, 1.24)

0.269

 9–10

0.71 (0.42, 1.21)

0.206

 11–12

0.71 (0.38, 1.32)

0.284

 ≥13

0.51 (0.27, 0.94)

0.031

Adjusted analysisa

 ≤6

1 (reference)

 7–8

0.74 (0.45, 1.22)

0.244

 9–10

0.67 (0.39, 1.16)

0.158

 11–12

0.76 (0.39, 1.45)

0.400

 ≥13

0.50 (0.27, 0.95)

0.035

To convert μmol/l to mg/dl, divide by 17.1

aAdjusted for the following baseline patient variables: age, height, smoking status, GGT, HbA1c, and history of previous PAD, non-PAD CVD, amputation or diabetic skin ulcer, neuropathy, nephropathy and diabetic retinopathy, as well as trial treatment allocation (placebo vs fenofibrate)

Fig. 2

Amputation risk according to plasma total bilirubin concentration (quintiles). (a) Cumulative risk curves (% events over time) to first amputation event. Logrank test of trend (median method), p = 0.031. (b) Cumulative 5 year amputation rates by sex and smoking status classification. Black bars, male smokers; white bars, male non-smokers; diagonally hatched bars, female smokers; horizontally hatched bars, female non-smokers. To convert μmol/l to mg/dl, divide by 17.1

Interestingly, the inverse association between bilirubin concentration and amputation event was separately significant for major (HR 1.83 per 5 μmol/l decrease in bilirubin concentration, 95% CI 1.17, 2.87, p = 0.009), but not minor (HR 1.22 per 5 μmol/l decrease in bilirubin concentration, 95% CI 0.92, 1.61, p = 0.18) amputation (ESM Fig. 1). Analyses according to bilirubin quintile also revealed a similar inverse association (ESM Table 2 and ESM Fig. 1).

Analysis according to sex

Consistent with previous studies [4, 5, 19, 27, 28], our study showed that women had lower median bilirubin concentrations than men (8 vs 10 μmol/l). We found that the inverse relationship between bilirubin concentrations and risk of first amputation was highly significant for men (HR 1.60 per 5 μmol/l decrease in bilirubin concentration, 95% CI 1.19, 2.14, p = 0.0017), but not women (HR 1.90 per 5 μmol/l decrease in bilirubin concentration, 95% CI 0.84, 4.33, p = 0.126; p for interaction by sex = 0.71; Fig. 3a–c). Analyses according to bilirubin quintile also demonstrated a similar inverse association for men (HR 0.38 for highest quintile, 95% CI 0.19, 0.75, p = 0.005; Table 5 and Fig. 3d–f), but not women. The amputation rate among individuals ≥65 years old was higher than in younger individuals of both sexes, with cumulative first amputation rates of 2.1% vs 1.1% for men and 1.1% vs 0.3% for women, respectively.
Fig. 3

Association by sex classification. (a, b) Cumulative risk curves (% events over time) to first amputation event according to baseline plasma total bilirubin concentration (5 μmol/l increments) in (a) men, and (b) women. Logrank test of trend, p = 0.0045 for men and p = 0.125 for women. (c) Cumulative 5 year amputation rates according to baseline plasma total bilirubin concentration (5 μmol/l increments) in men and women. Cochran–Armitage trend test, p = 0.0041 for men and p = 0.131 for women. Black bars, men; white bars, women. (d, e) Cumulative risk curves (% events over time) to first amputation event according to baseline plasma total bilirubin concentration (quintiles) in (d) men, and (e) women. Logrank test of trend (median method), p = 0.0024 for men and p = 0.136 for women. (f) Cumulative 5 year amputation rates according to baseline plasma total bilirubin concentration (quintiles) in men and women. Cochran–Armitage trend test (median method), p = 0.0023 for men and p = 0.136 for women. Black bars, men; white bars, women. To convert μmol/l to mg/dl, divide by 17.1

Table 5

Association between baseline plasma total bilirubin concentration (quintiles) and risk of first amputation event, by sex

Bilirubin (μmol/l)

Risk of first amputation (HR, 95% CI)

p value

Men

 ≤6

1 (reference)

 7–8

0.78 (0.44, 1.36)

0.377

 9–10

0.56 (0.30, 1.03)

0.062

 11–12

0.56 (0.28, 1.10)

0.092

 ≥13

0.38 (0.19, 0.75)

0.005

Women

 ≤6

1 (reference)

 7–8

0.37 (0.12, 1.16)

0.089

 9–10

0.67 (0.22, 2.07)

0.483

 11–12

0.37 (0.05, 2.83)

0.337

 ≥13

0.30 (0.04, 2.34)

0.253

To convert μmol/l to mg/dl, divide by 17.1

Analysis according to smoking status

Consistent with previous studies [19, 29], our study showed that current smokers had lower median bilirubin levels compared with non-smokers (8 vs 9 μmol/l). We found that the inverse relationship between bilirubin concentration and risk of first amputation was significant for non-smokers (HR 1.34 per 5 μmol/l decrease in bilirubin concentration, 95% CI 1.01, 1.77, p = 0.04), but not for smokers (HR 1.24 per 5 μmol/l decrease in bilirubin concentration, 95% CI 0.66, 2.33, p = 0.513; p for interaction by smoking status = 0.81; Fig. 4a–c). The same analyses according to bilirubin quintile were not statistically significant for either non-smokers or smokers, although there was a trend towards an inverse association in non-smokers (Table 6 and Fig. 4d–f).
Fig. 4

Association according to smoking status. (a, b) Cumulative risk curves (% events over time) to first amputation event according to baseline plasma total bilirubin concentration (5 μmol/l increments) in (a) smokers, and (b) non-smokers. Logrank test of trend, p = 0.96 for smokers and p = 0.034 for non-smokers. (c) Cumulative 5 year amputation rates according to baseline plasma total bilirubin concentration (5 μmol/l increments) in smokers and non-smokers. Cochran–Armitage trend test, p = 0.98 for smokers and p = 0.032 for non-smokers. Black bars, smokers; white bars, non-smokers. (d, e) Cumulative risk curves (% events over time) to first amputation event according to baseline plasma total bilirubin concentration (quintiles) in (d) smokers, and (e) non-smokers. Logrank test of trend (median method), p = 0.58 for smokers and p = 0.095 for non-smokers. (f) Cumulative 5 year amputation rates according to baseline plasma total bilirubin concentration (quintiles) in smokers and non-smokers. Cochran–Armitage trend test, p = 0.56 for smokers and p = 0.09 for non-smokers. Black bars, smokers; white bars, non-smokers. To convert μmol/l to mg/dl, divide by 17.1

Table 6

Association between baseline plasma total bilirubin concentration (quintiles) and risk of first amputation event, by smoking status

Bilirubin (μmol/l)

Risk of first amputation (HR, 95% CI)

p value

Smoker

 ≤6

1 (reference)

 7–8

0.92 (0.34, 2.48)

0.875

 9–10

0.50 (0.11, 2.31)

0.375

 11–12

1.58 (0.49, 5.14)

0.445

 ≥13

0.36 (0.05, 2.82)

0.328

Non-smoker

 ≤6

1 (reference)

 7–8

0.75 (0.43, 1.32)

0.319

 9–10

0.80 (0.45, 1.43)

0.455

 11–12

0.63 (0.30, 1.31)

0.217

 ≥13

0.57 (0.30, 1.12)

0.103

To convert μmol/l to mg/dl, divide by 17.1

Discussion

Our results identify a significant inverse relationship between baseline plasma total bilirubin levels and lower-limb amputation in patients with type 2 diabetes. This association was observed between bilirubin and total amputations, as well as separately for major but not minor amputations. The relationship with total (major plus minor) amputation remains robust even after adjusting for numerous other classic vascular and known amputation risk factors, liver function tests (GGT) and study treatment allocation, suggesting that it is likely to be independent of these other factors. Our data are therefore consistent with the hypothesis that bilirubin may be a useful biomarker for amputation in patients with type 2 diabetes. Potentially, this association is of major clinical importance, as the natural range of bilirubin levels seen here covers a striking, greater than threefold, risk gradient in amputation rates over 5 years.

Previous studies focusing on the relationship between bilirubin and PAD also reported a similar inverse association. The largest of these studies analysed 7,075 individuals from the National Health and Nutrition Examination Survey, and reported that increased bilirubin levels were associated with a reduced prevalence of PAD [19]. However, hard clinical endpoints were not explored in that and other similar smaller case–control studies [17, 18, 19, 20]. Our study therefore for the first time extends these findings to amputation events. This inverse association is further supported by studies showing a similar relationship between bilirubin levels and CHD, ischaemic stroke, and surrogate markers of atherosclerosis including endothelial dysfunction and carotid intima-media thickness [12, 14, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42]. A number of prospective studies found a U- or reversed J-shaped relationship rather than an inverse relationship between bilirubin levels and CHD [4, 5, 27, 28, 30, 33, 34, 37]. The differences between our study and these earlier studies are that they concentrated on CHD and mainly enrolled men, although whether these differences could plausibly explain the differing relationships reported is unclear. Nonetheless, our results, together with the previously published studies, support the possibility that bilirubin may be protective against atherosclerotic disease.

It is well established that atherosclerosis and its consequence, tissue ischaemia, are characterised by a state of heightened inflammation and oxidative stress [15, 16]. Studies have shown that bilirubin possesses antioxidant and anti-inflammatory activities in vitro [9, 11]. These experimental studies are supported by human studies reporting that individuals with elevated bilirubin levels due to Gilbert’s syndrome have higher antioxidant capacity and lower proinflammatory markers compared with control individuals [13, 14, 42]. Moreover, in a rat model of vascular injury, hyperbilirubinaemic Gunn rats had reduced neo-intima formation after balloon injury compared with control wild-type animals, suggesting that bilirubin itself may prevent the development of intimal hyperplasia [43]. How relevant these findings are, with much higher bilirubin levels in Gunn rats compared with humans, is still debated. The potential anti-atherogenic properties of bilirubin could also be consistent with our findings that the inverse association with amputation events was significant for major (which may more accurately reflect large-vessel atherosclerotic disease) but not minor amputation.

Our results are consistent with the previous study by Perlstein et al [19], showing a significant inverse relationship between bilirubin levels and amputation in men but not women. The lack of significant association in women may simply reflect the lower amputation rates in women compared with men, and it should be noted that there was no significant interaction by sex, so we were unable to conclude that the relationship between bilirubin and amputation risk truly differs by sex. Similarly (in contrast to the study by Perlstein et al), although we found a significant inverse relationship in non-smokers (when analysed according to 5 μmol/l bilirubin increments), but not smokers, again there was no significant interaction by smoking status, and only 9% of the cohort were current smokers. An alternative explanation for this disparate result may be that our study cohort exclusively comprised patients with type 2 diabetes, whereby the adverse combination of hyperglycaemia and smoking might negate any antioxidative benefits that bilirubin might otherwise confer in amputation prevention.

Plasma bilirubin concentrations are determined by the relative activities of the enzymes that form and remove bilirubin. Therefore, a better understanding of the pathways that regulate bilirubin concentrations may lead to new therapeutic targets for the treatment of atherosclerotic disease [7]. This is of particular importance to PAD, in which progressive disease leads to amputation at rates that have not changed significantly over the last three decades despite advances in vascular-targeted therapies [2]. The enzymes most widely studied to date include haem oxygenase-1 (HMOX-1) and uridine diphosphate glucuronosyltransferase-1 family polypeptide A1 (UGT1A1) [6]. HMOX-1 catalyses the rate-limiting step of haem degradation to carbon monoxide, ferrous iron and biliverdin [44]. Biliverdin is then rapidly reduced to bilirubin via biliverdin reductase. Although initially known for its metabolic role in haem catabolism, it is now well recognised that HMOX-1 may confer vascular protective effects, and this may be mediated, in part, via its haem catabolism by-products, carbon monoxide and bilirubin [7, 8]. Supporting this, Kawamura et al reported that both HMOX-1 induction and the addition of bilirubin (but not carbon monoxide) conferred anti-inflammatory effects on human endothelial cells in vitro, suggesting that the anti-inflammatory properties of HMOX-1 may be mediated through the action of bilirubin [9].

In humans, it is thought that the length of the (GT)n repeat is associated with variation in the HMOX1 gene activity, with shorter (GT)n repeats having higher HMOX1 transcriptional activity and expression compared with longer (GT)n repeats [8]. Studies exploring the association between HMOX1 polymorphism, bilirubin levels and vascular events have yielded conflicting results [6, 38, 45, 46]. One such study reported that longer (GT)n repeats (and hence lower HMOX1 activity) were associated with lower bilirubin levels and higher risk of CHD [38]. After adjusting for bilirubin, the effect of HMOX1 polymorphism on risk of CHD was no longer present, suggesting that the effect of HMOX1 polymorphism might be mediated through its influence on bilirubin levels [38]. However, other studies have not found such an association between HMOX1 polymorphism and bilirubin [45, 46]. Likewise, when comparing FIELD participants who developed on-study amputation with those who did not, there was no significant difference in the presence of the L allele (i.e. longer [GT]n repeats) between these two groups of patients (see the ESM Methods, ESM Figs 2 and 3 and ESM Tables 36). These results suggest that carbon monoxide, (i.e. an HMOX1-derived product other than biliverdin/bilirubin), may not be responsible for the decrease in amputation rate associated with high bilirubin levels.

It is increasingly thought that hepatic UGT1A1, rather than HMOX-1, plays a greater role in regulating bilirubin levels [6]. The link between UGT1A1 polymorphism, bilirubin and vascular disease was first noted with the low CHD prevalence in patients with Gilbert’s syndrome, a hereditary unconjugated hyperbilirubinaemia secondary to UGT1A1 deficiency [14]. Subsequently, a prospective study involving 1,780 individuals from the Framingham Heart Study Offspring cohort found that UGT1A1 polymorphism resulting in higher bilirubin levels was associated with lower risk of cardiovascular events [36]. Again, however, some other smaller studies have failed to show the same association between UGT1A1 polymorphism and CVD [18, 34, 47]. It is possible that these smaller studies lack the statistical power to detect this association. Nevertheless, clear associations between UGT1A1 and bilirubin have been demonstrated in most studies, suggesting that UGT1A1 may be a more important factor than HMOX1 in regulation of bilirubin levels [6]. In our patient cohort, it is likely that the 317 (out of 9,795) patients with bilirubin concentration >20 μmol/l have Gilbert’s syndrome, which is consistent with the estimated prevalence of 3–5% in the general population, although we do not have the results of UGT1A1 polymorphism for confirmation. Therefore, future studies exploring the link between UGT1A1 polymorphism and amputation in the FIELD study cohort will also be important to confirm the results from these previous studies.

There are several limitations of this study. First, bilirubin was, in most cases, only measured once at baseline. It is possible that bilirubin measurements vary significantly for an individual over time. However, random measurement error may result in an underestimation of a true association. Furthermore, in a subset of patients who had their bilirubin remeasured after 1 year, mean bilirubin concentrations had not changed significantly and the individual’s levels over 1 year correlated strongly. Second, our study cohort included only patients with type 2 diabetes, and our findings cannot necessarily be generalised to the non-diabetic population. Third, bilirubin levels are inversely associated with glucose levels [48, 49], thereby potentially confounding the results from our study exclusively comprising patients with type 2 diabetes. However, reassuringly, the inverse relationship between bilirubin level and amputation survives adjustment for glycaemic control, and fasting glucose levels were measured at all time points. Fourth, our study does not provide a proven mechanism for the observed association between bilirubin and amputation. Last, the association reported here is not necessarily causal, and could be a false-positive result arising from multiple comparisons, as the FIELD study is exploring risk factors for CHD in diabetes as well as for each of the microvascular complications of diabetes. Therefore, it is possible that the association of bilirubin with amputations is overestimated, and that bilirubin may be a marker rather than an agent directly protective against amputation. Randomised studies raising bilirubin would be better suited to establishing the causal link. This last point is relevant as uric acid, although originally proposed as an antioxidant, is now generally accepted as a biomarker for lifestyle factors rather than a directly protective antioxidant [5, 50].

Despite these limitations, the strengths of the current study include its large sample size, longitudinal nature and the very well-characterised nature of the patient cohort. In fact, the inverse association between bilirubin level and amputation persisted despite adjusting for a wide range of known risk factors for diabetic complications and amputation (though this did not negate the striking effects of fenofibrate treatment itself to reduce amputation events even after bilirubin adjustment [HR 0.64, 95% CI 0.44, 0.94, p = 0.02]).

In summary, our study reports for the first time a significant association between higher bilirubin levels and reduced risk of non-traumatic lower-limb amputation in individuals with type 2 diabetes. Furthermore, this association is not explained by other risk factors for amputation in the FIELD study. Our data raise the hypothesis that bilirubin may be a useful biomarker for, and may protect against, amputation in type 2 diabetes. Ultimately, a better understanding of the genes regulating bilirubin level and the potentially protective mechanisms may result in new therapeutic targets for the treatment of PAD and the prevention of amputation in the future.

Notes

Acknowledgements

The authors acknowledge the advice of S. Curtis (School of Veterinary Sciences, University of Bristol, Bristol, UK) on genotyping techniques. We also thank R. Pike (National Health and Medical Research Council Clinical Trials Centre, University of Sydney, Sydney, NSW, Australia) for her assistance with preparation of the figures and tables.

Funding

K. H. Chan is supported by the National Heart Foundation postgraduate scholarship (PC 08S 4127). L. S. Hoffmann was awarded the Feodor-Lynen Fellowship from the Alexander von Humboldt-Foundation. Her work was funded in part by a Program Grant from the National Health and Medical Research Council of Australia to R. Stocker. R. Stocker is supported by a National Health and Medical Research Council Senior Principal Research Fellowship. Laboratoires Fournier (now part of Abbott Pharmaceuticals) and the National Health and Medical Research Council funded the FIELD study.

Duality of interest

Some authors have been reimbursed by the pharmaceutical industry for the costs of participating in scientific meetings, contributing to advisory boards, or doing other research: D. R. Sullivan (Abbott Pharmaceuticals, AstraZeneca, Merck Sharp and Dohme, Pfizer, sanofi-aventis, Roche and Amgen) and K. Rajamani (Abbott Pharmaceuticals). All other authors declare that there is no duality of interest associated with this manuscript.

Contribution statement

KHC, LSH, MV, AH, RS, MKCN and ACK were responsible for the conception and design of the study. KHC, RO’C, DRS, LSH, KR, MW, MKCN and ACK acquired the data; KHC, RO’C, DRS, KR, MWD, BY, MKCN and ACK analysed the data. KHC, RO’C, DRS, KR, MWD, MV, AH, BY, RS, MKCN and ACK interpreted the data. KHC, RO’C, LSH, RS, MKCN and ACK were responsible for drafting the article. All authors revised the manuscript and approved the final version.

Supplementary material

125_2012_2818_MOESM1_ESM.pdf (90 kb)
ESM Methods(PDF 89 kb)
125_2012_2818_MOESM2_ESM.pdf (90 kb)
ESM Table 1(PDF 90 kb)
125_2012_2818_MOESM3_ESM.pdf (75 kb)
ESM Table 2(PDF 74 kb)
125_2012_2818_MOESM4_ESM.pdf (86 kb)
ESM Table 3(PDF 85 kb)
125_2012_2818_MOESM5_ESM.pdf (9 kb)
ESM Table 4(PDF 8.94 kb)
125_2012_2818_MOESM6_ESM.pdf (73 kb)
ESM Table 5(PDF 72 kb)
125_2012_2818_MOESM7_ESM.pdf (73 kb)
ESM Table 6(PDF 72.7 kb)
125_2012_2818_MOESM8_ESM.pdf (112 kb)
ESM Fig. 1(PDF 112 kb)
125_2012_2818_MOESM9_ESM.pdf (37 kb)
ESM Fig. 2(PDF 36 kb)
125_2012_2818_MOESM10_ESM.pdf (47 kb)
ESM Fig. 3(PDF 46 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • K. H. Chan
    • 1
    • 2
    • 3
    • 4
  • R. L. O’Connell
    • 4
  • D. R. Sullivan
    • 4
    • 5
  • L. S. Hoffmann
    • 6
  • K. Rajamani
    • 1
    • 4
  • M. Whiting
    • 7
  • M. W. Donoghoe
    • 4
  • M. Vanhala
    • 8
  • A. Hamer
    • 9
  • B. Yu
    • 2
    • 10
  • R. Stocker
    • 6
  • M. K. C. Ng
    • 1
    • 2
    • 3
  • A. C. Keech
    • 1
    • 2
    • 4
  • on behalf of the FIELD study investigators
  1. 1.Department of CardiologyRoyal Prince Alfred HospitalSydneyAustralia
  2. 2.Sydney Medical SchoolSydneyAustralia
  3. 3.The Heart Research InstituteSydneyAustralia
  4. 4.National Health and Medical Research Council Clinical Trials Centre, Level 6, Medical Foundation Building, K25University of SydneySydneyAustralia
  5. 5.Department of BiochemistryRoyal Prince Alfred HospitalSydneyAustralia
  6. 6.Center for Vascular Research, School of Medical Sciences (Pathology) and Bosch InstituteUniversity of SydneySydneyAustralia
  7. 7.Department of Medical BiochemistryFlinders Medical CentreAdelaideAustralia
  8. 8.Unit of Family PracticeCentral Finland Central HospitalJyväskyläFinland
  9. 9.Department of CardiologyNelson HospitalNelsonNew Zealand
  10. 10.Department of Molecular and Clinical GeneticsRoyal Prince Alfred HospitalSydneyAustralia

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