J Endocrinol Invest DOI 10.1007/s40618-013-0023-z
ORIGINAL ARTICLE
Lipid and non-lipid cardiovascular risk factors in postmenopausal type 2 diabetic women with and without coronary heart disease G. T. Russo • A. Giandalia • E. L. Romeo • M. Marotta • A. Alibrandi • C. De Francesco K. V. Horvath • B. Asztalos • D. Cucinotta
•
Received: 17 July 2013 / Accepted: 17 November 2013 Ó Italian Society of Endocrinology (SIE) 2013
Abstract Background Coronary heart disease (CHD) is the leading cause of death in diabetic women. In addition to hyperglycemia, other factors may contribute to the excessive cardiovascular risk. Aim In this study we evaluated common and emerging risk factors in a selected group of postmenopausal type 2 diabetic women with (n = 36) and without CHD (n = 59), not taking lipid-lowering medications. Methods Clinical and lifestyle data were collected, and metabolic and lipid profile, as well as fasting plasma levels of total homocysteine (tHcy), folate, vitamin B12, C-reactive protein (hsCRP), interleukin 6 (IL-6), and vascular cell adhesion molecule-1 (VCAM-1) were measured in all participants. Results Age, menopause and diabetes duration, family history for cardiovascular disease, prevalence of hypertension and current insulin use were greater in diabetic women with than without CHD (P \ 0.05 for all comparisons). CHD women also showed higher levels of triglycerides, small dense LDL (sdLDL), remnant-like particle cholesterol, tHcy, and VCAM-1, and a lower creatinine G. T. Russo (&) A. Giandalia E. L. Romeo M. Marotta C. De Francesco D. Cucinotta Department of Clinical and Experimental Medicine, University of Messina, Messina, Italy e-mail: [email protected] A. Alibrandi Department of Statistic Sciences, Universityof Messina, Messina, Italy K. V. Horvath B. Asztalos Lipid Metabolism Laboratory, JM-USDA-Human Nutrition Research Center on Aging at Tufts University, Boston, MA, USA
clearance (P \ 0.05 all). Conversely, the two groups were comparable for BMI, waist circumference, smoking habit, fasting plasma glucose, HbA1c, total cholesterol, low-density lipoprotein cholesterol (LDL-C), HDL cholesterol, folate, vitamin B12, hsCRP and IL-6 levels. At multivariate analysis, lower creatinine clearance (OR = 0.932, P = 0.017) and higher sdLDL serum concentration (OR = 1.224, P = 0.037) were the strongest risk factors associated with CHD in this population, whereas no significant association was noted with LDL-C. Conclusions Our data suggest that beyond LDL-C, a lower creatinine clearance and more subtle alterations of LDL particles, together with a constellation of several well known and emerging cardiovascular risk factors, are stronger contributors to the high CHD risk of diabetic women. Keywords Creatine clearance SdLDL Coronary heart disease Type 2 diabetes Women
Introduction Cardiovascular disease is the leading cause of death, also in women [1]. Women generally have a *10 year-delay in the onset of cardiovascular events as compared to men; however, diabetes seems to eliminate this ‘gender-advantage’, with diabetic women showing even a higher risk for coronary heart disease (CHD) than diabetic men [1–5]. Beyond hyperglycemia, a number of other factors such as dyslipidemia, hypertension, as well as low-grade systemic inflammation and endothelial damage, may contribute to the atherosclerosis process associated with diabetes. High plasma levels of low-density lipoprotein cholesterol (LDL-C) are the major CHD risk factor also in
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diabetic subjects [6]; however, lipid-lowering trials have largely demonstrated that a substantial residual risk often remains after reaching LDL-C goals [7, 8]. In particular, the presence of atherogenic dyslipidemia, i.e., low levels of HDL cholesterol (HDL-C) and an increase in triglycerides and in small dense LDL (sdLDL) concentrations may be partly responsible for this high CHD risk [9]. Among non-lipid risk factors, systemic inflammation certainly plays a crucial role in the development of atherosclerotic lesions [10], and several inflammatory markers, such as interleukin 6 (IL-6) and C-reactive protein (hsCRP), have been consistently associated with type 2 diabetes, insulin resistance [11, 12] and CHD [13, 14]. Similarly, other endothelial dysfunction markers, such as the adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) could help to define CHD residual risk [15]. Also moderate hyperhomocysteinemia may contribute to endothelial damage, although its independent role as CHD risk factor appears to be still controversial, also in diabetic subjects [16–18]. Notably, gender differences have been reported in the distribution, control and management of several of these risk factors [19, 20], with women being particularly exposed to diabetes and hypertension [3]. Furthermore, diabetic women show a higher prevalence of all major CHD risk factors, including dyslipidemia [4], and a lower rate of success in reaching the recommended targets for most of them [19–21]. The hormonal modifications accompanying menopause may modulate some of these risk factors [22], adding complexity to the frame. Although an increasing number of evidences point to a different impact of CHD risk factors in the female gender, their role has not been adequately evaluated in a high-risk population, such as diabetic women. The purpose of this study was therefore to investigate the role of several common and emerging risk factors in a group of postmenopausal type 2 diabetic women with and without CHD.
as defined by amenorrhea for at least 12 months before the study entry in women with an intact uterus, or hysterectomy, considering the date of surgery as the beginning of menopause. Exclusion criteria, valid for all participants, were current use of hormonal replacement therapy (HRT), multivitamin supplementation, current treatment with b-blockers, fibrates, statins, omega 3 fatty acids, niacin, glitazones, FANS, antiplatelet drugs and corticosteroids, fasting serum creatinine [1.2 mg/dl, macroalbuminuria (Albustix positive), renal, hepatic, cardiac failure, current cancer diagnosis or cancer remission \5 years, untreated thyroid disease or inflammatory systemic diseases, as well as any other major medical condition in the last 6 months preceding the study. At the enrollment visit, all participants underwent a clinical questionnaire, a complete physical examination and fasting blood sampling for the measurement of study parameters. Subjects were defined as no-smokers or current smokers, including in the latter category also those who had quit within 1 year. BMI and blood pressure were measured according to standard procedures. Hypertension in diabetic women was defined as a systolic and/or diastolic blood pressure values C130/80, and/or current use of antihypertensive medications. All patients were on dietary therapy, oral hypoglycemic agents, insulin or a combination between them, whereas none of them was on acarbose and/or incretins at the time of the study. Only after the enrollment visit, participants who did not meet the recommended targets for LDL-C, as well as those who had a specific indication because of their CHD history, were initiated to specific therapies, i.e. statins, betablockers, etc., as properly indicated by current guidelines [23]. All the participants gave their informed consent and the study was approved by the local ethical committee.
Patients and methods
Measurement of metabolic parameters
Study subjects
After a 12–14 h fasting, blood samples were collected from all participants for the determination of the study parameters. Blood was drown in a 10-ml tube containing EDTA (0.15 % final concentration) and in a regular 10-ml tube. After collection, plasma and serum were immediately separated at 2,500 rpm for 30 min at 4 °C, and aliquots were stored at -80 °C until analysis. Fasting plasma glucose and serum creatinine levels were measured with standard automated laboratory methods (Roche Diagnostics, Milan, Italy). Creatinine clearance was calculated with the Cockcroft-Gault formula [24].
Ninety-five type 2 diabetic postmenopausal women, who were consecutively referred, for the first time, to the Metabolic Disease outpatient clinic at the Department of Clinical and Experimental Medicine of the Messina University Hospital and fulfilled all the following inclusion and exclusion criteria, were included into the study. Inclusion criteria were a diagnosis of type 2 diabetes according to ADA criteria [23], and postmenopausal status,
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Glycated hemoglobin (HbA1c) was measured using an automated high-performance liquid chromatography (HPLC) analyzer (Diamat; Bio-Rad Laboratories, Milan, Italy); normal range values in our laboratory are 4–6 %. Fasting insulin concentration was measured by radioimmunoassay (Diagnostic Corporation, Los Angels, CA, USA). Insulin resistance was calculated by the homeostasis model assessment (HOMAIR) [25]. Measurement of lipid parameters and sdLDL All lipid and lipoprotein measurements were performed at the Lipid Metabolism Laboratory, Tufts University (Boston, MA). Plasma total cholesterol (TC) and triglycerides levels were measured by automated enzymatic assays. HDL-C was measured directly with a kit from Roche Diagnostics (Indianapolis, IN). Plasma remnant-like particle cholesterol (RLP-C) levels were measured using an immunoseparation technique (Polymedco, Cortlandt Manor, NY) [26]. This technique utilizes a monoclonal anti-apo A-I antibody to remove HDL particles and an anti-apo B antibody that does not recognize partially hydrolyzed lipoprotein remnants to remove both large nascent VLDL and LDL particles. The cholesterol content of the remaining remnant lipoproteins was then measured. Homogenous direct LDL cholesterol and sdLDL cholesterol were measured by automated standardized enzymatic analysis on a Hitachi 911 analyzer (Hitachi Corporation, Tokyo, Japan) using kits provided by Denka-Seiken Corp. Tokyo, Japan [27]. Measurement of inflammatory and endothelial dysfunction markers Serum levels of hsCRP were assayed with a high-sensitivity test (Dade Behring Inc., Deerfield, Illinois); those of IL-6, resistin and VCAM-1 were determined by ELISA (R&D Systems, Minneapolis, Minnesota). tHcy plasma concentration was measured with HPLC technique (BioRad Laboratories, Milan, Italy; CV 2.9 %), while the plasma levels of folate and vitamin B12 were assayed with radioimmunoassay technique (Bio-Rad Laboratories, Milan, Italy; CV 3.8 and 5.9 %, respectively). Assessment of long-term diabetes complications Diabetic retinopathy was diagnosed or excluded on the basis of direct ophthalmoscopy performed by an expert ophthalmologist within 1 year before the study. Peripheral neuropathy was assessed by questioning patients about symptoms of neuropathy, and confirmed by measurement of nerve conduction velocities. Retinopathy and neuropathy were diagnosed in 15 and 30 % of diabetic participants, respectively.
Carotid intima-media thickness (IMT) and carotid plaques were diagnosed on the basis of the ultrasound examination of the common carotid arteries, the carotid bifurcations, and the origin of the internal carotid arteries, with the B-mode system (Ultramark 9 HDI-High Definition Imaging) with a 5- to 10-MHz sounding. Carotid plaque was defined as local thickening of the cIMT of [50 % compared to the surrounding vessel wall, an IMT [1.5 mm, or local thickening [0.5 mm. CHD was defined as chronic ischemic heart disease, myocardial infarction, coronary-artery bypass, or coronary angioplasty, as documented by cardiologist medical records and/or hospital discharge. Statistical analysis Statistical analysis was performed using the SPSS program, version 11.0 for Windows (SPSS Inc. Chicago, IL). Data are given as mean ± SD. We used v2 test to compare categorical measures, and the analysis of variance (ANOVA) for continuous measures. Bivariate associations were estimated using the Pearson’s correlation coefficient and linear regression models were determined using a stepwise selection procedure. In particular, a logistic regression model to verify the dependence of CHD from creatinine clearance has been firstly estimated. Then, because of the numerous significant predictors at univariate analysis, a correlation matrix has been estimated to evaluate co-linearity among the several variables of the model. This procedure led to the exclusion of diabetes duration, menopause duration, VCAM-1, LDLC from the multivariate model. Then, the linear regression models were determined using a stepwise selection procedure for all the remaining factors, entering the continuous variables with the use of appropriate transformations. All statistical comparisons are two-tailed and they were considered significant at the P \ 0.05 level.
Results Clinical features of postmenopausal diabetic women participating in the study, according to the presence of CHD, are shown in Table 1. Diabetic women with CHD were older, with longer menopause and diabetes duration, and a greater prevalence of family history for cardiovascular disease, as compared to those without CHD (P \ 0.05 all). The two groups were overall obese and comparable for anthropometric measures and for mean values of systolic and diastolic blood pressure, although the percentage of subjects with hypertension was significantly greater in the CHD group (P \ 0.01). The percentage of insulin-treated subjects was significantly
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J Endocrinol Invest Table 1 Clinical features of type 2 diabetic women with and without coronary heart disease
Data are n %, mean ± SD Only significant P values are shown. P1 = age-adjusted P value CHD coronary heart disease, CVD cardiovascular disease, BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure, OHAs oral hypoglycemic agents
Type 2 diabetic women without CHD
P
P1
n (%)
59 (62.1)
36 (37.9)
Age (years)
62.20 ± 8.12
70.5 ± 7.49
\0.01
Menopause duration (years)
11.54 ± 8.37
21.92 ± 9.19
\0.01
– 0.04
Diabetes duration (years)
7.21 ± 10.06
12.25 ± 8.54
Family history of CVD n (%)
13 (22.03)
20 (55.5)
\0.01
–
Family history of diabetes n (%)
27 (45.8)
19 (52.8)
–
–
0.02
–
Smokers n (%)
7 (11.9)
1 (2.8)
–
–
Weight (kg)
74.87 ± 15.41
73.53 ± 11.11
–
–
BMI (kg/m2)
30.72 ± 5.75
30.75 ± 4.93
–
–
Waist circumference (cm)
101.00 ± 11.63
101.69 ± 10.79
–
–
SBP (mmHg) DBP (mmHg)
138.47 ± 18.71 79.32 ± 9.26
138.06 ± 14.16 77.64 ± 9.45
– –
– –
Hypertension n (%)
38 (64.4)
34 (94.4)
\0.01
OHAs treatment n (%)
49 (83.05)
27 (75 %)
–
–
Metformin treatment n (%)
41 (69.5)
20 (55.6)
–
– \0.01
Insulin therapy n (%)
1 (1.7)
8 (22.2)
\0.01
Carotid intimal thickening n (%)
34 (57.62)
24 (66.7)
–
–
Carotid plaques n (%)
18 (30.51)
13 (36.1)
–
–
higher among CHD women (P \ 0.01), while that of patients treated with metformin or other hypoglycemic agents was comparable between the two groups. Since the CHD group had a significant older age than women without CHD, an age-adjusted analysis was performed to compare all the study variables between the two groups. After an age-adjustment, menopausal duration (P = 0.04) and insulin therapy (P \ 0.01) remained significantly different between the two groups. No differences were noted in the occurrence of carotid thickness and/or carotid plaques between women with and without CHD (Table 1), and in the frequency of the other long-term diabetes complications (retinopathy and neuropathy; data not shown). Lipid and non-lipid risk factors according to the presence of CHD in type 2 diabetic women As shown in Table 2, lipid profile was different between the two groups, and these differences were still significant, although attenuated, after an age-adjusted analysis. CHD women showed higher serum concentrations of triglycerides (P \ 0.01; age-adjusted P = 0.02), RLP-C (P = 0.04, age-adjusted P = 0.06) and sdLDL (P = 0.02; age-adjusted P = 0.01). Conversely, no differences were noted in fasting blood glucose and HbA1c levels, as well as in those of TC, LDL-C and HDL-C between the two groups. Also HOMAIR values were similar in the two
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Type 2 diabetic women with CHD
groups, although they are of limited significance in diabetic subjects under glucose-lowering medications. Furthermore, women with CHD had significantly higher serum levels of creatinine (P \ 0.01; age-adjusted P = 0.05) and a significantly lower creatinine clearance (P \ 0.01) as compared to those without CHD. When systemic inflammation and endothelial damage markers were compared (Table 2), CHD women had significantly higher circulating levels of VCAM-1 (P = 0.048; age-adjusted P [ 0.05), whereas IL-6 levels were comparable between the two groups. Also serum hsCRP levels were overall high and similar in diabetic women with and without CHD (mean values of 5.02 and 4.90 mg/l, respectively; unadjusted and age-adjusted P [ 0.05). Circulating levels of tHcy were significantly higher in diabetic women with CHD (P \ 0.01; ageadjusted P [ 0.05), although those of vitamin B12 and folate, the two major cofactors implicated in tHcy metabolism, were similar in the two groups. Lipid and non-lipid risk factors associated with CHD in type 2 diabetic women As shown in Table 3, many of the examined risk factors, i.e., age, creatinine clearance, insulin therapy, high blood pressure, family history of cardiovascular disease, duration of menopause, tHcy, RLP-C, sdLDL, VCAM-1 concentrations, duration of diabetes, and triglycerides levels were all significantly associated with CHD occurrence (P \ 0.05), although
J Endocrinol Invest Table 2 Lipid and non-lipid risk factors in type 2 diabetic women with and without coronary heart disease
Fasting blood glucose (mg/dl)
Data are n %, mean ± SD
CHD coronary heart disease, LDL-C LDL cholesterol, HDLC HDL cholesterol, RLP-C remnants-associated cholesterol, SdLDL small dense LDL, tHcy total homocysteine levels, hsCPR C-reactive protein, VCAM-1 soluble vascular cell adhesion molecule-1 a
Since creatinine clearance formula already included age, it does not require a further adjustment
tHcy total homocysteine levels, VCAM-1 soluble vascular cell adhesion molecule-1, CVD cardiovascular disease, SdLDL small dense LDL, RLP-C remnants-associated cholesterol
P
P1
160.12 ± 45.65
163.06 ± 54.35
–
–
5.77 ± 5.11
7.97 ± 6.52
–
–
HbA1c (%)
7.60 ± 1.49
7.56 ± 1.53
–
–
0.87 ± 0.13
1.02 ± 0.29
\0.01
81.21 ± 24.91
63.24 ± 20.00
\0.01
Creatinine clearance (ml/min)a
0.05
Total cholesterol (mg/dl)
188.53 ± 29.16
178.51 ± 27.26
–
–
LDL-C (mg/dl)
117.32 ± 26.81
112.81 ± 25.09
–
–
HDL-C (mg/dl)
49.83 ± 13.66
48.29 ± 13.54
–
–
102.36 ± 40.80
134.80 ± 57.63
\0.01
\0.01
7.22 ± 3.96
9.31 ± 5.93
0.04
0.06
8.67 ± 7.85 123.63 ± 17.84
12.41 ± 7.23 122.06 ± 19.99
0.02 –
0.01 –
12.67 ± 5.43
16.33 ± 7.37
Triglycerides (mg/dl) RLP-C (mg/dl) SdLDL (mg/dl) Apo A I (mg/dl) tHcy (mmol/l) Folate (ng/ml) Vitamin B12 (pg/ml)
\0.01
–
4.97 ± 2.52
4.92 ± 1.93
–
–
396.59 ± 181.79
409.48 ± 203.98
–
–
hsCRP (mg/l)
5.02 ± 7.34
4.90 ± 4.55
–
–
IL-6 (pg/ml)
3.04 ± 3.84
3.35 ± 2.03
–
–
748.18 ± 194.80
940.11 ± 481.97
0.048
–
VCAM-1 (ng/ml)
Table 3 Factors associated with coronary heart disease (CHD) in type 2 diabetic women
Only significant associations are shown
Diabetic women with CHD
HOMA-IR Creatinine (mg/dl)
Only significant P values are shown. P1 = age-adjusted P value
Diabetic women without CHD
Univariate associations
Multivariate associations
B
B
P
OR (95 % CI)
-0.70
0.017
0.932 (0.880–0.987)
0.037
1.224 (1.012–1.492)
P
OR (95 % CI)
Age
0.126
0.001
1.134 (1.067–1.204)
Menopause duration
0.131
0.001
1.140 (1.063–1.223)
Creatinine clearance
-0.038
0.001
0.963 (0.941–0.985)
Hypertension
2.24
0.004
9.395 (2.050–43.058)
Triglycerides
0.014
0.004
Creatinine
4.19
0.006
65.777 (3.420–1,264.983)
Insulin therapy
2.81
0.01
16.571 (1.975–139.059)
tHcy
0.09
0.02
1.098 (1.017–1.186)
VCAM Diabetes duration
0.002 0.055
0.02 0.04
1.002 (1.000–1.004) 1.056 (1.002–1.113)
CVD family history
1.306
0.04
3.692 (1.052–12.957)
1.014 (1.004–1.023)
SdLDL
0.067
0.04
1.069 (1.003–1.139)
RLP-C
0.090
0.05
1.094 (0.998–1.199)
with a different statistical power; conversely, LDL-C serum concentration was not associated with CHD in this cohort. At multivariate analysis (Table 3), using a stepwise procedure, a lower creatinine clearance (B = -0.70; P = 0.017; OR 0.932; 95 % CI 0.880–0.987) and the presence of higher sdLDL concentration (B = 0.206; P = 0.037; OR 1.229; 95 %CI 1.012–1.492) were the only variables independently associated with CHD in our population.
0.206
Discussion Type 2 diabetes is associated with a very high CHD risk, and this risk has been reported to be greater in diabetic women than in men [2–5]. Although the reasons underlying these gender differences have not been fully elucidated yet, it is possible that common cardiovascular risk factors may have a different
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impact in the two sexes [5, 28]. In a large Italian diabetes cohort study, the risk of the first CHD event depended on sex, age, diabetes duration and geographic location, but the higher triglycerides/lower HDL-C phenotype and the presence of microvascular complications were independent risk factors only in diabetic women [29]. In addition to hyperglycemia, diabetes-associated cardiovascular disease has been attributed to several traditional risk factors, such as hypertension, dyslipidemia, obesity, smoking and, particularly, to high LDL-C serum levels. Although LDL-C represents the major target for CHD primary and secondary prevention also in type 2 diabetes [6, 30], numerous intervention trials have demonstrated that a considerable number of coronary events still occur despite reaching LDL-C goals [31, 32]. Thus, a mounting evidence suggests that measuring LDL-C alone may not be sufficient for identifying all individuals at high CHD risk [7, 8] and other risk factors, such as atherogenic dyslipidemia may play a crucial role. Diabetic dyslipidemia is characterized by high triglycerides and low values of HDL-C, as well as by more subtle alterations in lipoproteins metabolism and composition, with LDL-C levels often in the normal range [9, 33]. In our cohort, serum concentrations of triglycerides, RLP-C and sdLDL were all significantly higher in CHD women, but sdLDL circulating levels were the only lipid variable independently associated with CHD risk at multivariate analysis. Conversely, LDL-C did not emerge as a significant risk factor in our dataset. Our diabetic women had LDL-C values only moderately above the recommended targets [30], and these values were similar in women with and without CHD (mean LDL-C 117 and 112 mg/dl, respectively), although all participants were free from lipid-lowering medications at the time of recruitment. As a matter of fact, the finding of a high percentage of CHD patients who are still not treated with statins, although disappointing, is in line with data coming from a recent Italian Survey on diabetes [19, 34]. CHD women also showed a *9 % higher prevalence of carotid intimal media thickness and a *6 % higher prevalence of carotid plaques. Although this modest difference in carotid atherosclerosis between CHD and non-CHD women may be partially puzzling, it could be explained by the not excessively increased LDL-C levels in the two groups. Furthermore, these results seem to support the emerging thesis that different risk factors, i.e. a lower creatinine clearance and sdLDL as in our cohort, may preferentially affect the coronary than the peripheral or cerebral district [21]. Also epidemiological data support a major role for increased sdLDL levels in defining CHD risk [35], especially in diabetes, where they are associated with a
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threefold increased risk [36, 37], and in postmenopausal women, since aging and menopause seem to delay their clearance [38]. Similar to our data, in the Framingham Offspring Study, sdLDL-C levels were higher in women with than without CHD, who had comparable LDL-C levels, whereas these differences were not evident in men [39]. However, despite these consistent epidemiological data, the great analytical variability in measuring sdLDL concentrations still limits their clinical usefulness. In addition to lipid abnormalities, a lower creatinine clearance was independently associated with CHD in our cohort. The importance of renal function in defining CHD risk in diabetic subjects has been largely documented, and recent reports have confirmed that a mild-moderate reduction of glomerular filtration rate (GFR) is an independent risk factor for cardiovascular disease in these subjects [21, 40]. Notably, an isolated reduction of GFR, which is a common finding in women [41], seems to be preferentially associated to a high risk of developing coronary events, as recently reported in an Italian diabetic population [21]. Our results, demonstrating that a reduced GFR was the strongest risk factor associated with CHD in diabetic women, are in line with these observations. Our data also revealed that other non-lipid abnormalities were associated with CHD in diabetic women. Among them, hypertension, family history of cardiovascular disease, age, menopause and diabetes duration were all associated with CHD occurrence, although with a graded statistical power. Many of these risk factors, such as hypertension, have been demonstrated to contribute to the development of coronary atherosclerosis, especially in diabetic women [42]. However, with the exception of menopause duration, these factors were no longer associated with CHD after age-adjustment. Furthermore, since these factors did not enter the multivariate model, it is likely that an age- and diabetes-related decrease of renal function may account for most of them. The greater number of subjects on insulin therapy in the CHD group observed in our study has been previously reported also in larger diabetic cohorts, and specifically in diabetic women, although this association seems to be weak and not always confirmed [29, 43, 44]. The finding of a higher percentage of insulin-treated subjects among CHD women in our study is probably not causal, and it is related to the longer diabetes duration and/or to the indications for insulin therapy after a CHD event. Furthermore, a recent large trial does not support a causal role for insulin therapy on cardiovascular disease [45]. In our analysis, also tHcy and VCAM-1 serum levels showed significant associations with CHD, although with a lower statistical weight, suggesting that the impact of these still-debated [15–18] CHD risk factors is of limited value.
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Although hsCRP levels are consistently associated with CHD [46], in our population, both hsCRP and IL-6 were well above the recognized higher-risk values [46], and comparable in diabetic women with and without CHD. These results suggest that in subject with an already high cardiovascular burden, hsCRP plasma levels may be not always able to further discriminate those individuals who are at higher risk, as also reported by the Emerging Risk Factors Collaboration (ERFC) group [47]. Although our study was limited by the cross-sectional design, the small sample size, and by the unavailability of microalbuminuria measurements, it should be noted that our results were obtained in a well-characterized group of postmenopausal type 2 diabetic women, who were free of any medication (i.e., lipid-lowering and anti-inflammatory drugs, glitazones and/or ERT) potentially influencing the examined lipid and non-lipid variables, until they came to our attention. Despite the growing body of evidence indicating that CHD is a multifactorial disease, clinicians still focus only on LDL-C; our data indicate that, especially when LDL-C levels are not high, atherogenic sdLDL and renal function are stronger contributors to CHD in women with diabetes. If confirmed in larger cohorts of patients and by longitudinal and/or intervention studies, our results suggest the need to focus on and to prevent/treat renal dysfunction also to reduce the associated CHD risk in diabetic women, and leave the open question whether it is useful to measure lipoprotein subpopulations to better define CHD risk in the clinical practice. Conflict of interest The authors G. T. Russo, A. Giandalia, E. L. Romeo, M. Marotta, A. Alibrandi, C. De Francesco, K. V. Horvath, B. Asztalos, and D. Cucinotta declare that they have no conflict of interest.
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ORIGINAL ARTICLE
Lipid and non-lipid cardiovascular risk factors in postmenopausal type 2 diabetic women with and without coronary heart disease G. T. Russo • A. Giandalia • E. L. Romeo • M. Marotta • A. Alibrandi • C. De Francesco K. V. Horvath • B. Asztalos • D. Cucinotta
•
Received: 17 July 2013 / Accepted: 17 November 2013 Ó Italian Society of Endocrinology (SIE) 2013
Abstract Background Coronary heart disease (CHD) is the leading cause of death in diabetic women. In addition to hyperglycemia, other factors may contribute to the excessive cardiovascular risk. Aim In this study we evaluated common and emerging risk factors in a selected group of postmenopausal type 2 diabetic women with (n = 36) and without CHD (n = 59), not taking lipid-lowering medications. Methods Clinical and lifestyle data were collected, and metabolic and lipid profile, as well as fasting plasma levels of total homocysteine (tHcy), folate, vitamin B12, C-reactive protein (hsCRP), interleukin 6 (IL-6), and vascular cell adhesion molecule-1 (VCAM-1) were measured in all participants. Results Age, menopause and diabetes duration, family history for cardiovascular disease, prevalence of hypertension and current insulin use were greater in diabetic women with than without CHD (P \ 0.05 for all comparisons). CHD women also showed higher levels of triglycerides, small dense LDL (sdLDL), remnant-like particle cholesterol, tHcy, and VCAM-1, and a lower creatinine G. T. Russo (&) A. Giandalia E. L. Romeo M. Marotta C. De Francesco D. Cucinotta Department of Clinical and Experimental Medicine, University of Messina, Messina, Italy e-mail: [email protected] A. Alibrandi Department of Statistic Sciences, Universityof Messina, Messina, Italy K. V. Horvath B. Asztalos Lipid Metabolism Laboratory, JM-USDA-Human Nutrition Research Center on Aging at Tufts University, Boston, MA, USA
clearance (P \ 0.05 all). Conversely, the two groups were comparable for BMI, waist circumference, smoking habit, fasting plasma glucose, HbA1c, total cholesterol, low-density lipoprotein cholesterol (LDL-C), HDL cholesterol, folate, vitamin B12, hsCRP and IL-6 levels. At multivariate analysis, lower creatinine clearance (OR = 0.932, P = 0.017) and higher sdLDL serum concentration (OR = 1.224, P = 0.037) were the strongest risk factors associated with CHD in this population, whereas no significant association was noted with LDL-C. Conclusions Our data suggest that beyond LDL-C, a lower creatinine clearance and more subtle alterations of LDL particles, together with a constellation of several well known and emerging cardiovascular risk factors, are stronger contributors to the high CHD risk of diabetic women. Keywords Creatine clearance SdLDL Coronary heart disease Type 2 diabetes Women
Introduction Cardiovascular disease is the leading cause of death, also in women [1]. Women generally have a *10 year-delay in the onset of cardiovascular events as compared to men; however, diabetes seems to eliminate this ‘gender-advantage’, with diabetic women showing even a higher risk for coronary heart disease (CHD) than diabetic men [1–5]. Beyond hyperglycemia, a number of other factors such as dyslipidemia, hypertension, as well as low-grade systemic inflammation and endothelial damage, may contribute to the atherosclerosis process associated with diabetes. High plasma levels of low-density lipoprotein cholesterol (LDL-C) are the major CHD risk factor also in
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diabetic subjects [6]; however, lipid-lowering trials have largely demonstrated that a substantial residual risk often remains after reaching LDL-C goals [7, 8]. In particular, the presence of atherogenic dyslipidemia, i.e., low levels of HDL cholesterol (HDL-C) and an increase in triglycerides and in small dense LDL (sdLDL) concentrations may be partly responsible for this high CHD risk [9]. Among non-lipid risk factors, systemic inflammation certainly plays a crucial role in the development of atherosclerotic lesions [10], and several inflammatory markers, such as interleukin 6 (IL-6) and C-reactive protein (hsCRP), have been consistently associated with type 2 diabetes, insulin resistance [11, 12] and CHD [13, 14]. Similarly, other endothelial dysfunction markers, such as the adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) could help to define CHD residual risk [15]. Also moderate hyperhomocysteinemia may contribute to endothelial damage, although its independent role as CHD risk factor appears to be still controversial, also in diabetic subjects [16–18]. Notably, gender differences have been reported in the distribution, control and management of several of these risk factors [19, 20], with women being particularly exposed to diabetes and hypertension [3]. Furthermore, diabetic women show a higher prevalence of all major CHD risk factors, including dyslipidemia [4], and a lower rate of success in reaching the recommended targets for most of them [19–21]. The hormonal modifications accompanying menopause may modulate some of these risk factors [22], adding complexity to the frame. Although an increasing number of evidences point to a different impact of CHD risk factors in the female gender, their role has not been adequately evaluated in a high-risk population, such as diabetic women. The purpose of this study was therefore to investigate the role of several common and emerging risk factors in a group of postmenopausal type 2 diabetic women with and without CHD.
as defined by amenorrhea for at least 12 months before the study entry in women with an intact uterus, or hysterectomy, considering the date of surgery as the beginning of menopause. Exclusion criteria, valid for all participants, were current use of hormonal replacement therapy (HRT), multivitamin supplementation, current treatment with b-blockers, fibrates, statins, omega 3 fatty acids, niacin, glitazones, FANS, antiplatelet drugs and corticosteroids, fasting serum creatinine [1.2 mg/dl, macroalbuminuria (Albustix positive), renal, hepatic, cardiac failure, current cancer diagnosis or cancer remission \5 years, untreated thyroid disease or inflammatory systemic diseases, as well as any other major medical condition in the last 6 months preceding the study. At the enrollment visit, all participants underwent a clinical questionnaire, a complete physical examination and fasting blood sampling for the measurement of study parameters. Subjects were defined as no-smokers or current smokers, including in the latter category also those who had quit within 1 year. BMI and blood pressure were measured according to standard procedures. Hypertension in diabetic women was defined as a systolic and/or diastolic blood pressure values C130/80, and/or current use of antihypertensive medications. All patients were on dietary therapy, oral hypoglycemic agents, insulin or a combination between them, whereas none of them was on acarbose and/or incretins at the time of the study. Only after the enrollment visit, participants who did not meet the recommended targets for LDL-C, as well as those who had a specific indication because of their CHD history, were initiated to specific therapies, i.e. statins, betablockers, etc., as properly indicated by current guidelines [23]. All the participants gave their informed consent and the study was approved by the local ethical committee.
Patients and methods
Measurement of metabolic parameters
Study subjects
After a 12–14 h fasting, blood samples were collected from all participants for the determination of the study parameters. Blood was drown in a 10-ml tube containing EDTA (0.15 % final concentration) and in a regular 10-ml tube. After collection, plasma and serum were immediately separated at 2,500 rpm for 30 min at 4 °C, and aliquots were stored at -80 °C until analysis. Fasting plasma glucose and serum creatinine levels were measured with standard automated laboratory methods (Roche Diagnostics, Milan, Italy). Creatinine clearance was calculated with the Cockcroft-Gault formula [24].
Ninety-five type 2 diabetic postmenopausal women, who were consecutively referred, for the first time, to the Metabolic Disease outpatient clinic at the Department of Clinical and Experimental Medicine of the Messina University Hospital and fulfilled all the following inclusion and exclusion criteria, were included into the study. Inclusion criteria were a diagnosis of type 2 diabetes according to ADA criteria [23], and postmenopausal status,
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Glycated hemoglobin (HbA1c) was measured using an automated high-performance liquid chromatography (HPLC) analyzer (Diamat; Bio-Rad Laboratories, Milan, Italy); normal range values in our laboratory are 4–6 %. Fasting insulin concentration was measured by radioimmunoassay (Diagnostic Corporation, Los Angels, CA, USA). Insulin resistance was calculated by the homeostasis model assessment (HOMAIR) [25]. Measurement of lipid parameters and sdLDL All lipid and lipoprotein measurements were performed at the Lipid Metabolism Laboratory, Tufts University (Boston, MA). Plasma total cholesterol (TC) and triglycerides levels were measured by automated enzymatic assays. HDL-C was measured directly with a kit from Roche Diagnostics (Indianapolis, IN). Plasma remnant-like particle cholesterol (RLP-C) levels were measured using an immunoseparation technique (Polymedco, Cortlandt Manor, NY) [26]. This technique utilizes a monoclonal anti-apo A-I antibody to remove HDL particles and an anti-apo B antibody that does not recognize partially hydrolyzed lipoprotein remnants to remove both large nascent VLDL and LDL particles. The cholesterol content of the remaining remnant lipoproteins was then measured. Homogenous direct LDL cholesterol and sdLDL cholesterol were measured by automated standardized enzymatic analysis on a Hitachi 911 analyzer (Hitachi Corporation, Tokyo, Japan) using kits provided by Denka-Seiken Corp. Tokyo, Japan [27]. Measurement of inflammatory and endothelial dysfunction markers Serum levels of hsCRP were assayed with a high-sensitivity test (Dade Behring Inc., Deerfield, Illinois); those of IL-6, resistin and VCAM-1 were determined by ELISA (R&D Systems, Minneapolis, Minnesota). tHcy plasma concentration was measured with HPLC technique (BioRad Laboratories, Milan, Italy; CV 2.9 %), while the plasma levels of folate and vitamin B12 were assayed with radioimmunoassay technique (Bio-Rad Laboratories, Milan, Italy; CV 3.8 and 5.9 %, respectively). Assessment of long-term diabetes complications Diabetic retinopathy was diagnosed or excluded on the basis of direct ophthalmoscopy performed by an expert ophthalmologist within 1 year before the study. Peripheral neuropathy was assessed by questioning patients about symptoms of neuropathy, and confirmed by measurement of nerve conduction velocities. Retinopathy and neuropathy were diagnosed in 15 and 30 % of diabetic participants, respectively.
Carotid intima-media thickness (IMT) and carotid plaques were diagnosed on the basis of the ultrasound examination of the common carotid arteries, the carotid bifurcations, and the origin of the internal carotid arteries, with the B-mode system (Ultramark 9 HDI-High Definition Imaging) with a 5- to 10-MHz sounding. Carotid plaque was defined as local thickening of the cIMT of [50 % compared to the surrounding vessel wall, an IMT [1.5 mm, or local thickening [0.5 mm. CHD was defined as chronic ischemic heart disease, myocardial infarction, coronary-artery bypass, or coronary angioplasty, as documented by cardiologist medical records and/or hospital discharge. Statistical analysis Statistical analysis was performed using the SPSS program, version 11.0 for Windows (SPSS Inc. Chicago, IL). Data are given as mean ± SD. We used v2 test to compare categorical measures, and the analysis of variance (ANOVA) for continuous measures. Bivariate associations were estimated using the Pearson’s correlation coefficient and linear regression models were determined using a stepwise selection procedure. In particular, a logistic regression model to verify the dependence of CHD from creatinine clearance has been firstly estimated. Then, because of the numerous significant predictors at univariate analysis, a correlation matrix has been estimated to evaluate co-linearity among the several variables of the model. This procedure led to the exclusion of diabetes duration, menopause duration, VCAM-1, LDLC from the multivariate model. Then, the linear regression models were determined using a stepwise selection procedure for all the remaining factors, entering the continuous variables with the use of appropriate transformations. All statistical comparisons are two-tailed and they were considered significant at the P \ 0.05 level.
Results Clinical features of postmenopausal diabetic women participating in the study, according to the presence of CHD, are shown in Table 1. Diabetic women with CHD were older, with longer menopause and diabetes duration, and a greater prevalence of family history for cardiovascular disease, as compared to those without CHD (P \ 0.05 all). The two groups were overall obese and comparable for anthropometric measures and for mean values of systolic and diastolic blood pressure, although the percentage of subjects with hypertension was significantly greater in the CHD group (P \ 0.01). The percentage of insulin-treated subjects was significantly
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J Endocrinol Invest Table 1 Clinical features of type 2 diabetic women with and without coronary heart disease
Data are n %, mean ± SD Only significant P values are shown. P1 = age-adjusted P value CHD coronary heart disease, CVD cardiovascular disease, BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure, OHAs oral hypoglycemic agents
Type 2 diabetic women without CHD
P
P1
n (%)
59 (62.1)
36 (37.9)
Age (years)
62.20 ± 8.12
70.5 ± 7.49
\0.01
Menopause duration (years)
11.54 ± 8.37
21.92 ± 9.19
\0.01
– 0.04
Diabetes duration (years)
7.21 ± 10.06
12.25 ± 8.54
Family history of CVD n (%)
13 (22.03)
20 (55.5)
\0.01
–
Family history of diabetes n (%)
27 (45.8)
19 (52.8)
–
–
0.02
–
Smokers n (%)
7 (11.9)
1 (2.8)
–
–
Weight (kg)
74.87 ± 15.41
73.53 ± 11.11
–
–
BMI (kg/m2)
30.72 ± 5.75
30.75 ± 4.93
–
–
Waist circumference (cm)
101.00 ± 11.63
101.69 ± 10.79
–
–
SBP (mmHg) DBP (mmHg)
138.47 ± 18.71 79.32 ± 9.26
138.06 ± 14.16 77.64 ± 9.45
– –
– –
Hypertension n (%)
38 (64.4)
34 (94.4)
\0.01
OHAs treatment n (%)
49 (83.05)
27 (75 %)
–
–
Metformin treatment n (%)
41 (69.5)
20 (55.6)
–
– \0.01
Insulin therapy n (%)
1 (1.7)
8 (22.2)
\0.01
Carotid intimal thickening n (%)
34 (57.62)
24 (66.7)
–
–
Carotid plaques n (%)
18 (30.51)
13 (36.1)
–
–
higher among CHD women (P \ 0.01), while that of patients treated with metformin or other hypoglycemic agents was comparable between the two groups. Since the CHD group had a significant older age than women without CHD, an age-adjusted analysis was performed to compare all the study variables between the two groups. After an age-adjustment, menopausal duration (P = 0.04) and insulin therapy (P \ 0.01) remained significantly different between the two groups. No differences were noted in the occurrence of carotid thickness and/or carotid plaques between women with and without CHD (Table 1), and in the frequency of the other long-term diabetes complications (retinopathy and neuropathy; data not shown). Lipid and non-lipid risk factors according to the presence of CHD in type 2 diabetic women As shown in Table 2, lipid profile was different between the two groups, and these differences were still significant, although attenuated, after an age-adjusted analysis. CHD women showed higher serum concentrations of triglycerides (P \ 0.01; age-adjusted P = 0.02), RLP-C (P = 0.04, age-adjusted P = 0.06) and sdLDL (P = 0.02; age-adjusted P = 0.01). Conversely, no differences were noted in fasting blood glucose and HbA1c levels, as well as in those of TC, LDL-C and HDL-C between the two groups. Also HOMAIR values were similar in the two
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Type 2 diabetic women with CHD
groups, although they are of limited significance in diabetic subjects under glucose-lowering medications. Furthermore, women with CHD had significantly higher serum levels of creatinine (P \ 0.01; age-adjusted P = 0.05) and a significantly lower creatinine clearance (P \ 0.01) as compared to those without CHD. When systemic inflammation and endothelial damage markers were compared (Table 2), CHD women had significantly higher circulating levels of VCAM-1 (P = 0.048; age-adjusted P [ 0.05), whereas IL-6 levels were comparable between the two groups. Also serum hsCRP levels were overall high and similar in diabetic women with and without CHD (mean values of 5.02 and 4.90 mg/l, respectively; unadjusted and age-adjusted P [ 0.05). Circulating levels of tHcy were significantly higher in diabetic women with CHD (P \ 0.01; ageadjusted P [ 0.05), although those of vitamin B12 and folate, the two major cofactors implicated in tHcy metabolism, were similar in the two groups. Lipid and non-lipid risk factors associated with CHD in type 2 diabetic women As shown in Table 3, many of the examined risk factors, i.e., age, creatinine clearance, insulin therapy, high blood pressure, family history of cardiovascular disease, duration of menopause, tHcy, RLP-C, sdLDL, VCAM-1 concentrations, duration of diabetes, and triglycerides levels were all significantly associated with CHD occurrence (P \ 0.05), although
J Endocrinol Invest Table 2 Lipid and non-lipid risk factors in type 2 diabetic women with and without coronary heart disease
Fasting blood glucose (mg/dl)
Data are n %, mean ± SD
CHD coronary heart disease, LDL-C LDL cholesterol, HDLC HDL cholesterol, RLP-C remnants-associated cholesterol, SdLDL small dense LDL, tHcy total homocysteine levels, hsCPR C-reactive protein, VCAM-1 soluble vascular cell adhesion molecule-1 a
Since creatinine clearance formula already included age, it does not require a further adjustment
tHcy total homocysteine levels, VCAM-1 soluble vascular cell adhesion molecule-1, CVD cardiovascular disease, SdLDL small dense LDL, RLP-C remnants-associated cholesterol
P
P1
160.12 ± 45.65
163.06 ± 54.35
–
–
5.77 ± 5.11
7.97 ± 6.52
–
–
HbA1c (%)
7.60 ± 1.49
7.56 ± 1.53
–
–
0.87 ± 0.13
1.02 ± 0.29
\0.01
81.21 ± 24.91
63.24 ± 20.00
\0.01
Creatinine clearance (ml/min)a
0.05
Total cholesterol (mg/dl)
188.53 ± 29.16
178.51 ± 27.26
–
–
LDL-C (mg/dl)
117.32 ± 26.81
112.81 ± 25.09
–
–
HDL-C (mg/dl)
49.83 ± 13.66
48.29 ± 13.54
–
–
102.36 ± 40.80
134.80 ± 57.63
\0.01
\0.01
7.22 ± 3.96
9.31 ± 5.93
0.04
0.06
8.67 ± 7.85 123.63 ± 17.84
12.41 ± 7.23 122.06 ± 19.99
0.02 –
0.01 –
12.67 ± 5.43
16.33 ± 7.37
Triglycerides (mg/dl) RLP-C (mg/dl) SdLDL (mg/dl) Apo A I (mg/dl) tHcy (mmol/l) Folate (ng/ml) Vitamin B12 (pg/ml)
\0.01
–
4.97 ± 2.52
4.92 ± 1.93
–
–
396.59 ± 181.79
409.48 ± 203.98
–
–
hsCRP (mg/l)
5.02 ± 7.34
4.90 ± 4.55
–
–
IL-6 (pg/ml)
3.04 ± 3.84
3.35 ± 2.03
–
–
748.18 ± 194.80
940.11 ± 481.97
0.048
–
VCAM-1 (ng/ml)
Table 3 Factors associated with coronary heart disease (CHD) in type 2 diabetic women
Only significant associations are shown
Diabetic women with CHD
HOMA-IR Creatinine (mg/dl)
Only significant P values are shown. P1 = age-adjusted P value
Diabetic women without CHD
Univariate associations
Multivariate associations
B
B
P
OR (95 % CI)
-0.70
0.017
0.932 (0.880–0.987)
0.037
1.224 (1.012–1.492)
P
OR (95 % CI)
Age
0.126
0.001
1.134 (1.067–1.204)
Menopause duration
0.131
0.001
1.140 (1.063–1.223)
Creatinine clearance
-0.038
0.001
0.963 (0.941–0.985)
Hypertension
2.24
0.004
9.395 (2.050–43.058)
Triglycerides
0.014
0.004
Creatinine
4.19
0.006
65.777 (3.420–1,264.983)
Insulin therapy
2.81
0.01
16.571 (1.975–139.059)
tHcy
0.09
0.02
1.098 (1.017–1.186)
VCAM Diabetes duration
0.002 0.055
0.02 0.04
1.002 (1.000–1.004) 1.056 (1.002–1.113)
CVD family history
1.306
0.04
3.692 (1.052–12.957)
1.014 (1.004–1.023)
SdLDL
0.067
0.04
1.069 (1.003–1.139)
RLP-C
0.090
0.05
1.094 (0.998–1.199)
with a different statistical power; conversely, LDL-C serum concentration was not associated with CHD in this cohort. At multivariate analysis (Table 3), using a stepwise procedure, a lower creatinine clearance (B = -0.70; P = 0.017; OR 0.932; 95 % CI 0.880–0.987) and the presence of higher sdLDL concentration (B = 0.206; P = 0.037; OR 1.229; 95 %CI 1.012–1.492) were the only variables independently associated with CHD in our population.
0.206
Discussion Type 2 diabetes is associated with a very high CHD risk, and this risk has been reported to be greater in diabetic women than in men [2–5]. Although the reasons underlying these gender differences have not been fully elucidated yet, it is possible that common cardiovascular risk factors may have a different
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impact in the two sexes [5, 28]. In a large Italian diabetes cohort study, the risk of the first CHD event depended on sex, age, diabetes duration and geographic location, but the higher triglycerides/lower HDL-C phenotype and the presence of microvascular complications were independent risk factors only in diabetic women [29]. In addition to hyperglycemia, diabetes-associated cardiovascular disease has been attributed to several traditional risk factors, such as hypertension, dyslipidemia, obesity, smoking and, particularly, to high LDL-C serum levels. Although LDL-C represents the major target for CHD primary and secondary prevention also in type 2 diabetes [6, 30], numerous intervention trials have demonstrated that a considerable number of coronary events still occur despite reaching LDL-C goals [31, 32]. Thus, a mounting evidence suggests that measuring LDL-C alone may not be sufficient for identifying all individuals at high CHD risk [7, 8] and other risk factors, such as atherogenic dyslipidemia may play a crucial role. Diabetic dyslipidemia is characterized by high triglycerides and low values of HDL-C, as well as by more subtle alterations in lipoproteins metabolism and composition, with LDL-C levels often in the normal range [9, 33]. In our cohort, serum concentrations of triglycerides, RLP-C and sdLDL were all significantly higher in CHD women, but sdLDL circulating levels were the only lipid variable independently associated with CHD risk at multivariate analysis. Conversely, LDL-C did not emerge as a significant risk factor in our dataset. Our diabetic women had LDL-C values only moderately above the recommended targets [30], and these values were similar in women with and without CHD (mean LDL-C 117 and 112 mg/dl, respectively), although all participants were free from lipid-lowering medications at the time of recruitment. As a matter of fact, the finding of a high percentage of CHD patients who are still not treated with statins, although disappointing, is in line with data coming from a recent Italian Survey on diabetes [19, 34]. CHD women also showed a *9 % higher prevalence of carotid intimal media thickness and a *6 % higher prevalence of carotid plaques. Although this modest difference in carotid atherosclerosis between CHD and non-CHD women may be partially puzzling, it could be explained by the not excessively increased LDL-C levels in the two groups. Furthermore, these results seem to support the emerging thesis that different risk factors, i.e. a lower creatinine clearance and sdLDL as in our cohort, may preferentially affect the coronary than the peripheral or cerebral district [21]. Also epidemiological data support a major role for increased sdLDL levels in defining CHD risk [35], especially in diabetes, where they are associated with a
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threefold increased risk [36, 37], and in postmenopausal women, since aging and menopause seem to delay their clearance [38]. Similar to our data, in the Framingham Offspring Study, sdLDL-C levels were higher in women with than without CHD, who had comparable LDL-C levels, whereas these differences were not evident in men [39]. However, despite these consistent epidemiological data, the great analytical variability in measuring sdLDL concentrations still limits their clinical usefulness. In addition to lipid abnormalities, a lower creatinine clearance was independently associated with CHD in our cohort. The importance of renal function in defining CHD risk in diabetic subjects has been largely documented, and recent reports have confirmed that a mild-moderate reduction of glomerular filtration rate (GFR) is an independent risk factor for cardiovascular disease in these subjects [21, 40]. Notably, an isolated reduction of GFR, which is a common finding in women [41], seems to be preferentially associated to a high risk of developing coronary events, as recently reported in an Italian diabetic population [21]. Our results, demonstrating that a reduced GFR was the strongest risk factor associated with CHD in diabetic women, are in line with these observations. Our data also revealed that other non-lipid abnormalities were associated with CHD in diabetic women. Among them, hypertension, family history of cardiovascular disease, age, menopause and diabetes duration were all associated with CHD occurrence, although with a graded statistical power. Many of these risk factors, such as hypertension, have been demonstrated to contribute to the development of coronary atherosclerosis, especially in diabetic women [42]. However, with the exception of menopause duration, these factors were no longer associated with CHD after age-adjustment. Furthermore, since these factors did not enter the multivariate model, it is likely that an age- and diabetes-related decrease of renal function may account for most of them. The greater number of subjects on insulin therapy in the CHD group observed in our study has been previously reported also in larger diabetic cohorts, and specifically in diabetic women, although this association seems to be weak and not always confirmed [29, 43, 44]. The finding of a higher percentage of insulin-treated subjects among CHD women in our study is probably not causal, and it is related to the longer diabetes duration and/or to the indications for insulin therapy after a CHD event. Furthermore, a recent large trial does not support a causal role for insulin therapy on cardiovascular disease [45]. In our analysis, also tHcy and VCAM-1 serum levels showed significant associations with CHD, although with a lower statistical weight, suggesting that the impact of these still-debated [15–18] CHD risk factors is of limited value.
J Endocrinol Invest
Although hsCRP levels are consistently associated with CHD [46], in our population, both hsCRP and IL-6 were well above the recognized higher-risk values [46], and comparable in diabetic women with and without CHD. These results suggest that in subject with an already high cardiovascular burden, hsCRP plasma levels may be not always able to further discriminate those individuals who are at higher risk, as also reported by the Emerging Risk Factors Collaboration (ERFC) group [47]. Although our study was limited by the cross-sectional design, the small sample size, and by the unavailability of microalbuminuria measurements, it should be noted that our results were obtained in a well-characterized group of postmenopausal type 2 diabetic women, who were free of any medication (i.e., lipid-lowering and anti-inflammatory drugs, glitazones and/or ERT) potentially influencing the examined lipid and non-lipid variables, until they came to our attention. Despite the growing body of evidence indicating that CHD is a multifactorial disease, clinicians still focus only on LDL-C; our data indicate that, especially when LDL-C levels are not high, atherogenic sdLDL and renal function are stronger contributors to CHD in women with diabetes. If confirmed in larger cohorts of patients and by longitudinal and/or intervention studies, our results suggest the need to focus on and to prevent/treat renal dysfunction also to reduce the associated CHD risk in diabetic women, and leave the open question whether it is useful to measure lipoprotein subpopulations to better define CHD risk in the clinical practice. Conflict of interest The authors G. T. Russo, A. Giandalia, E. L. Romeo, M. Marotta, A. Alibrandi, C. De Francesco, K. V. Horvath, B. Asztalos, and D. Cucinotta declare that they have no conflict of interest.
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