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Results of Blood Inflammatory Markers Are Associated More Strongly With ToeBrachial Index Than With Ankle-Brachial Index in Patients With Type 2 Diabetes YOSHIMASA ASO, MD1 KI-ICHI OKUMURA, MD1 TERUO INOUE, MD2 RIKA MATSUTOMO, MD1

NOBORU YOSHIDA, MD1 SADAO WAKABAYASHI, MD1 KOHZO TAKEBAYASHI, MD1 TOSHIHIKO INUKAI, MD1

OBJECTIVE — Three blood markers of inflammation (high-sensitivity C-reactive protein [hsCRP], interleukin [IL]-6, and fibrinogen) were compared with markers of atherosclerotic cardiovascular disease (CVD) (history of stroke or cardiac ischemia and measured toe-brachial index [TBI]) to determine whether inflammatory markers are associated with atherosclerosis in patients with type 2 diabetes. RESEARCH DESIGN AND METHODS — Of 103 patients with type 2 diabetes, 26 had CVD. TBI was plethysmographically determined in both great toes. Serum hsCRP was immunonephelometrically determined. Plasma IL-6 was measured by an enzyme immunoassay. RESULTS — Both ABI and TBI were lower in diabetic patients with CVD than in those without CVD (1.05 ⫾ 0.19 vs. 1.14 ⫾ 0.09, P ⬍ 0.05, and 0.75 ⫾ 0.20 vs. 0.95 ⫾ 0.21, P ⬍ 0.001, respectively). By linear regression, right TBI but not right ABI showed a significant negative correlation with serum hsCRP (r ⫽ ⫺0.372, P ⬍ 0.01) and plasma fibrinogen (r ⫽ ⫺0.224, P ⬍ 0.05). Serum hsCRP was also negatively correlated with lower TBI, but not lower ABI. We found no significant correlation between plasma IL-6 and ABI or TBI. CONCLUSIONS — TBI was strongly associated with CVD, serum hsCRP, and plasma fibrinogen. Of these inflammatory markers, serum hsCRP may be the most promising marker for vascular inflammation. Diabetes Care 27:1381–1386, 2004

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nkle-brachial index (ABI) is a simple, useful method for assessing peripheral vascular disease (PVD) (1– 3). Furthermore, an ABI value ⬍0.9 is an independent risk factor for an episode of cardiovascular disease (CVD), for recurrent CVD, and for mortality in elderly adults (4). However, application of this index to diabetic patients is considered

questionable given the prevalence of medial arterial calcification, which falsely elevates the ABI (5,6). Therefore, in diabetic patients with advanced nephropathy or severe autonomic neuropathy, index measurements involving toe and brachial systolic pressure (toe-brachial index [TBI]) are advocated (7) because medial

arterial calcification is less frequent in the toe than the ankle. Atherosclerosis is a chronic low-grade inflammatory disease (8). Accumulations of inflammatory cells, such as macrophages and T-cells, are prominent in atheromatous plaques (9). Interleukin (IL)-6 is the main proinflammatory cytokine that induces acute-phase responses (10). An increased plasma IL-6 concentration is a powerful predictor of myocardial infarction in healthy individuals (11). Fibrinogen, an acute-phase reactant, participates in early atherosclerotic plaque formation and in thrombus formation by conversion of fibrinogen to fibrin through the action of thrombin (12). Elevated concentrations of plasma fibrinogen have been associated with cardiovascular morbidity and mortality (13). Although several plasma markers of inflammation have been evaluated as possible predictors of CVD, evidence is accumulating that serum concentrations of C-reactive protein (CRP) determined by a high-sensitivity method (high-sensitivity CRP [hsCRP]) are particularly associated with future coronary events (14). In a prospective clinical study of apparently healthy men, baseline serum CRP predicted future risk of symptomatic PVD (15). However, no reports have assessed the relationship between serum hsCRP and TBI or ABI in patients with type 2 diabetes. The present study was undertaken in type 2 diabetic patients to investigate whether TBI or ABI was more strongly associated with multiple blood markers of inflammation such as hsCRP, IL-6, and fibrinogen.

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From the 1Department of Internal Medicine, Koshigaya Hospital, Dokkyo University School of Medicine, Koshigaya, Japan; and the 2Department of Cardiology, Koshigaya Hospital, Dokkyo University School of Medicine, Koshigaya, Japan. Address correspondence and reprint requests to Yoshimasa Aso, MD, Department of Internal Medicine, Dokkyo University School of Medicine, Koshigaya Hospital, Saitama, Japan. E-mail: yaso @dokkyomed.ac.jp. Received for publication 15 August 2003 and accepted in revised form 21 February 2004. Abbreviations: ABI, ankle-brachial index; CRP, C-reactive protein; CVD, cardiovascular disease; hsCRP, high-sensitivity CRP; IL, interleukin; PVD, peripheral vascular disease; TBI, toe-brachial index. © 2004 by the American Diabetes Association.

DIABETES CARE, VOLUME 27, NUMBER 6, JUNE 2004

RESEARCH DESIGN AND METHODS — We studied 103 type 2 diabetic patients (47 women and 56 men). The diagnosis of type 2 diabetes was made according to the criteria of the World Health Organization. All patients who fulfilled the following inclusion criteria were considered for study: no epi1381

TBI and hsCRP in type 2 diabetes

Table 1 —Demographic, clinical, and laboratory data for type 2 diabetic patients with or without CVD

n (M/F) Age (years) BMI (kg/m2) Diabetes duration (years) FPG (mmol/l) HbA1c (%) Total cholesterol (mmol/l) Triglycerides (mmol/l) HDL cholesterol (mmol/l) C-peptide (nmol/l) Fibrinogen (mg/dl) hsCRP (log10 ng/ml) IL-6 (pg/ml) ABI TBI Hypertension Treatment (diet alone/OHA/insulin)

No CVD

CVD

77 (41/38) 57.4 ⫾ 12.6 23.6 ⫾ 3.7 10.0 (6.0–15.0) 10.2 ⫾ 3.5 9.7 ⫾ 2.0 5.27 ⫾ 1.14 1.92 (1.32–2.66) 1.25 ⫾ 0.32 0.72 ⫾ 0.41 381 ⫾ 87 2.74 ⫾ 0.65 2.25 (1.50–3.760) 1.14 ⫾ 0.10 0.95 ⫾ 0.21 30 (39) 23/42/12

26 (16/10) 65.4 ⫾ 8.0* 24.3 ⫾ 4.1 10.0 (6.0–14.0) 11.3 ⫾ 5.5 9.2 ⫾ 2.0 5.53 ⫾ 2.32 2.01 (1.59–2.92) 1.22 ⫾ 0.36 0.71 ⫾ 0.44 444 ⫾ 120† 3.30 ⫾ 0.66† 3.90 (3.00–5.25)† 1.05 ⫾ 0.19* 0.75 ⫾ 0.20‡ 19 (73)† 5/13/8

Data are means ⫾ SD, median (interquartile range), or n (%). *P ⬍ 0.05; †P ⬍ 0.01; ‡P ⬍ 0.001. FPG, fasting plasma glucose; OHA, oral hypoglycemic agent.

sodes of ketoacidosis, diagnosis of diabetes after age ⬎30 years, and insulin therapy, if any, started after at least 5 years of known disease. CVD was defined as coronary artery disease and/or stroke and/or PVD. Coronary artery disease was defined as a history of myocardial infarction, coronary artery bypass grafting, or an abnormal coronary angiogram. Stroke was defined as a history of ischemic stroke confirmed by cerebral computed tomography or nuclear magnetic resonance imaging. Although PVD was defined as symptoms of intermittent claudication, a history of peripheral artery reconstruction, or the amputation of a foot, patients with lower extremity vascular surgery were excluded. Twenty-six patients had CVD. Forty-nine of the diabetic patients had hypertension, which was defined as systolic blood pressure ⬎140 mmHg and/or diastolic blood pressure ⬎90 mmHg or alternatively as treatment with antihypertensive agents. These medications included an ACE inhibitor (15 patients), a calcium channel blocker (24 patients), and/or an angiotensin-receptor blocker (7 patients). All patients gave informed consent. The study was approved by the local ethics committee. Measurements of ABI and TBI All measurements were performed in a room maintained at a temperature of 23– 1382

25°C after at least 30 min of acclimatization, with the subject in a supine position. The ABI was determined as the ratio of ankle systolic blood pressure to the brachial systolic blood pressure, with both determined using an automatic device (form PWV/ABI; AT, Komaki, Aichi, Japan). This automatic device simultaneously measured bilateral brachial and ankle blood pressure with the modified oscillometric pressure sensor method. The ABI of this device is highly correlated with that of the Doppler method (16). As brachial pressure, the higher of left and right pressures was used for calculating ABI and TBI. To measure toe pressure, a 25-mm digital cuff was placed around the base of the great toe and a photo-transducer was taped to the pulp of the toe. The cuff was inflated to a maximum of 200 mmHg and then slowly deflated. Using a plethysmograph (EC-5R plethysmograph; DE Hokanson, Bellevue, WA), the point at which the arterial waveform reappeared was defined as the toe systolic blood pressure. The toe pressure was reported as the mean of three measurements. Similarly to ABI, TBI was calculated as the ratio of toe systolic blood pressure to brachial systolic blood pressure. All measurements were performed by an appropriately trained physician (K.O.). We evaluated the reproducibility of toe pressure measurements. The measurements were performed on

two different days in a group of 10 healthy control subjects. We found a highly linear correlation between the results of the first day and those of the repeat day (r ⫽ 0.914). Venous blood was obtained between 6:00 and 7:00 A.M. after an overnight fast. Serum concentrations of hsCRP were determined by an immunonephelometric assay (N-High-Sensitivity CRP; Dade Behring, Marburg, Germany), and the intraand interassay coefficients of variation were 1.72 and 2.80%, respectively. Plasma concentrations of IL-6 were measured by a chemiluminescent enzyme assay (Human IL-6 CLEIA; Fujirebio, Tokyo, Japan), and the intra- and interassay coefficients of variation were 5.24 and 6.83%, respectively. Plasma concentrations of fibrinogen were determined by the Claus method. To evaluate the relationship between insulin resistance and atherosclerosis, fasting plasma C-peptide, an index of insulin resistance, was determined by radioimmunoassay. Urinary albumin excretion (concentrations in a 24-h collection specimen) was measured with an immunoturbidimetric assay. Statistical analysis Data are expressed as the mean ⫾ SD or the median and interquartile ranges. Differences between groups were analyzed by a Student’s paired t test or an unpaired t test. For nonparametric data, differences between groups were analyzed by the Mann-Whitney U test. Correlation was determined by linear regression analysis. A P value ⬍0.05 was accepted as indicating statistical significance. Statistical analyses were carried out using SPSS 8.0 J software (SPSS, Tokyo, Japan). RESULTS — We divided the diabetic patients into two groups according to presence of CVD. As shown in Table 1, we found no difference in duration of diabetes, glycemic control, or blood lipid profile between diabetic patients with and without CVD. Plasma concentrations of fibrinogen were significantly higher in patients with than in those without CVD (P ⬍ 0.01). Furthermore, hsCRP and IL-6 were significantly higher in patients with than in those without CVD (P ⬍ 0.01, respectively). Although ABI was significantly lower in patients with than in those without CVD (P ⬍ 0.05), TBI was profoundly lower in patients with than in those without CVD (P ⬍ 0.001). HyperDIABETES CARE, VOLUME 27, NUMBER 6, JUNE 2004

Aso and Associates

Table 2 —Univariate analysis of relationships between ABI or TBI and characteristics of patients with type 2 diabetes ABI Variable Age (years) BMI (kg/m2) Diabetes duration (years) FPG (mmol/l) HbA1c (%) Total cholesterol (mmol/l) Triglycerides (mmol/l) HDL cholesterol (mmol/l) C-peptide (nmol/l) UAE (log10 mg/24 h) Fibrinogen (mg/dl) IL-6 (pg/ml)

TBI

r

P

r

P

⫺0.163 ⫺0.130 ⫺0.199 ⫺0.158 0.020 ⫺0.021 ⫺0.054 ⫺0.068 0.020 0.054 ⫺0.0395 0.054

0.1323 0.2317 0.0841 0.1499 0.8543 0.8487 0.6214 0.5323 0.8544 0.6217 0.7196 0.6264

⫺0.204 ⫺0.160 ⫺0.052 ⫺0.126 0.001 ⫺0.065 ⫺0.005 0.168 ⫺0.058 ⫺0.064 ⫺0.224 0.044

0.0614 0.1528 0.6650 0.2625 0.9926 0.5647 0.8870 0.1221 0.6150 0.5778 0.0462 0.7031

FPG, fasting plasma glucose; UAE, urinary albumin excretion.

variate analysis controlling for age, diabetes duration, blood pressure, blood lipid concentrations, glycemic control, and renal function. In a model that explained 51.1% of the variation of TBI, ABI (␤ ⫽ 0.464, P ⫽ 0.0005) and hsCRP (␤ ⫽ ⫺0.273, P ⫽ 0.020) were independent determinants of TBI in patients with type 2 diabetes (data not shown). CONCLUSIONS — T h e p r e s e n t study demonstrated that in patients with type 2 diabetes, serum hsCRP concentrations show significant negative correlation with TBI but not with ABI. We also found a negative correlation between TBI and plasma fibrinogen, an acute-phase reactant, in diabetic patients. No significant correlation was evident between ABI and plasma fibrinogen. Several markers of inflammation are available for predicting

tension was more prevalent in patients with CVD (P ⬍ 0.01). In all patients with type 2 diabetes, TBI was negatively correlated with plasma fibrinogen (r ⫽ ⫺0.224, P ⫽ 0.0462) (Table 2). TBI also tended to correlate negatively with age. We found no significant correlation between plasma IL-6 concentrations and ABI or TBI (Table 2). Serum fasting C-peptide, a possible index of insulin resistance, showed no significant correlation with ABI or TBI. Right ABI was positively correlated with left ABI (r ⫽ 0.670, P ⬍ 0.0001) (Fig. 1A). Right TBI showed a stronger positive correlation with left TBI (r ⫽ 0.724, P ⬍ 0.0001) (Fig. 1B). We found no significant correlation between serum hsCRP and right, left, or lower ABI (r ⫽ ⫺0.141, P ⫽ 0.2041; r ⫽ ⫺0.165, P ⫽ 0.1433; and r ⫽ ⫺0.128, P ⫽ 0.2574, respectively) (Fig. 2). On the other hand, serum concentrations of hsCRP correlated negatively with right and lower TBI values (r ⫽ ⫺0.372, P ⫽ 0.0010 and r ⫽ ⫺0.315, P ⫽ 0.0055) (Fig. 3). Left TBI tended to correlate negatively with serum hsCRP (r ⫽ ⫺0.215, P ⫽ 0.0633). In diabetic patients with CVD, serum hsCRP showed no significant correlation with ABI (r ⫽ 0.012, P ⫽ 0.9602) or TBI (r ⫽ ⫺0.188, P ⫽ 0.4278). In diabetic patients without CVD, serum hsCRP was negatively correlated with TBI (r ⫽ ⫺0.276, P ⫽ 0.0415) but not with ABI (r ⫽ ⫺0.146, P ⫽ 0.2672). To determine independent influences on TBI, we performed a stepwise multi-

Figure 1—Correlation between left and right ABI (A) or between left and right ABI (B) in all patients with type 2 diabetes


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Figure 2—Correlation between serum concentrations of CRP by high-sensitivity determinations and right (A), left (B), and lower (C) ABI in patients with type 2 diabetes

the development of CVD because chronic low-grade inflammation plays an important role in the pathogenesis of atherosclerosis. Among these, hsCRP is the most promising so far because several studies have demonstrated that baseline CRP is associated with future cardiovascular 1384

events (17,18). Elevated CRP thus may be a strong independent predictor of coronary episodes. Furthermore, a prospective study (15) demonstrated that in apparently healthy men, elevated hsCRP predicts risk of subsequent symptomatic PVD. Plasma fibrinogen is also an estab-

lished predictor of CVD (13). A previous study (19) identified plasma fibrinogen as an independent determinant of PVD in the elderly. The present study also indicated that both hsCRP and fibrinogen concentrations were significantly higher in patients with CVD than in those without CVD, suggesting that chronic inflammation may be an important component of atherosclerosis. On the basis of our findings, TBI may be more strongly associated than ABI with the development of atherosclerotic disease, including PVD, as estimated by markers of vascular inflammation in patients with type 2 diabetes. We found a significant difference in age between diabetic patients with and without CVD. The patients with CVD were older than those without CVD. However, there were no significant differences in duration of diabetes and metabolic profiles between the patients with and without CVD. These results suggest that advancing age, a nonreversible factor, is a major risk factor for atherosclerotic vascular disease, irrespective of whether the subject has diabetes. In fact, there is a growing body of evidence (20) that age-associated vascular changes, including increased large artery thickening and stiffness and endothelial dysfunction, predict a higher risk for developing clinical atherosclerosis. A low ABI (ⱕ0.9), which is a useful diagnostic tool for detecting PVD, is also considered a strong predictor of cardiovascular morbidity and mortality in older adults (4). In short, a low ABI may reflect systemic atherosclerosis. However, because 5–10% of diabetic patients have medial arterial calcification, these patients have stiffened, noncompressible peripheral vessels (21). This yields a falsely high ABI value. Most patients with an ABI ⬎1.3 have been shown to have such calcification (7). Instead of ABI, assessment of TBI should be used to diagnose PVD in diabetic patients, especially when the ABI is ⱖ1.3 (22). Because the present study showed no significant correlation between ABI values and serum hsCRP or fibrinogen when all diabetic subjects were considered, ABI does not have a straightforward association with PVD or systemic vascular inflammation in type 2 diabetes. Unfortunately, we did not obtain radiographs of the feet, so we could not assess numbers of patients with medial arterial calcification among diabetic patients. However, falsely high ABI values may acDIABETES CARE, VOLUME 27, NUMBER 6, JUNE 2004

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that plasma IL-6 was higher in patients with CVD than in those without CVD, supporting an association of plasma IL-6 with atherosclerosis. However, TBI showed a significant negative correlation with serum hsCRP but not with plasma IL-6. This suggests that CRP may be a more promising marker for atherosclerosis. Ridker et al. (17) demonstrated that in a recent analysis from the Women’s Health Study, CRP was the strongest predictor of cardiovascular events of all of the inflammatory markers, including IL-6. One possible explanation for the lack of association of IL-6 with ABI or TBI is that, unlike IL-6, CRP has a long half-life, affording stability levels with no circadian variation (23). Another possibility is that CRP per se has several direct effects on the pathogenesis of atherosclerosis (9), resulting in a significant correlation between TBI and hsCRP. Certain limitations of the present study should be noted. As mentioned, one major limitation is the lack of radiographs of the feet to confirm the presence of medial arterial calcification in our diabetic patients. Another major limitation is the cross-sectional nature of the study design. The causal relationship cannot be proven by cross-sectional data. A prospective study should be undertaken to confirm causality between high ABI and the development of atherosclerotic disease. Acknowledgments — We thank S. Moriyama of SRL for his kind assistance.

Figure 3—Correlation between serum concentrations of CRP by high-sensitivity determinations and right (A), left (B), and lower (C) TBI in patients with type 2 diabetes

count for the absence of a significant correlation between hsCRP and ABI in our diabetic patients. In these patients, TBI is a better marker of and for generalized atherosclerosis than ABI and should be substituted. In the present study, we found no significant correlation between plasma IL-6 DIABETES CARE, VOLUME 27, NUMBER 6, JUNE 2004

concentration and TBI or ABI in patients with type 2 diabetes. IL-6 is the main proinflammatory cytokine that induces acute-phase responses, including CRP and fibrinogen (10). An increased plasma IL-6 concentration is a powerful predictor of myocardial infarction in healthy individuals (11). The present study showed

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