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Relation of Plasma Glucose and Endothelial Function in a Population-BasedMultiethnic Sample of Subjects Without Diabetes Mellitus

Carlos J. Rodriguez, MD, MPHa,d,*, Yumiko Miyake, MDa, Cairistine Grahame-Clarke, MDa,Marco R. Di Tullio, MDa, Robert R. Sciacca, EngScDa, Bernadette Boden-Albala, DrPHb,e,

Ralph L. Sacco, MD, MSb,c, and Shunichi Homma, MDa

To determine whether endothelial dysfunction precedes the clinical diagnosis ofdiabetes mellitus, we investigated the relation of endothelial flow-mediated dilation(FMD) with fasting plasma glucose among a multiethnic population-based cohort of579 nondiabetic subjects without previous myocardial infarction or stroke enrolled inthe Northern Manhattan Study (age 66 � 9 years; 41% men, 16% white, 15% black,and 68% Hispanic). Impaired fasting glucose or prediabetic status, defined as afasting glucose level of 100 to 125 mg/dl, was present in 95 subjects (16%). Endothelialfunction was determined using FMD during reactive hyperemia. Multiple linearregression analyses were used to assess the relation between plasma glucose andendothelial function after adjustment for potential confounders. FMD was signifi-cantly lower (4.9 � 3.8% vs 6.1 � 3.7%, p � 0.003) in those with impaired fastingglucose than in subjects with normal fasting glucose. Prediabetic status was signifi-cantly associated with impaired FMD (odds ratio 1.9, 95% confidence interval 1.1 to3.1, p � 0.02). After adjustment for age, gender, body mass index, and hypertensivestatus, a higher fasting glucose was significantly associated with a lower FMD (� ��0.024 � 0.012, p � 0.04) in a continuous linear relation. Thus, for each 10-mg/dlincrease in plasma glucose, a 0.24% decrease occurred in FMD. Impaired FMD waspresent among prediabetics. An elevated fasting plasma glucose level is associatedwith impaired endothelial function among nondiabetics. These results further supportthe role of hyperglycemia in the pathogenesis of vascular dysfunction at differentstages of diabetes development and the role of impaired fasting glucose as a riskfactor for macrovascular disease. © 2005 Elsevier Inc. All rights reserved. (Am J

Cardiol 2005;96:1273–1277)

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t is well known that endothelial dysfunction precedes thenset of overt cardiovascular disease.1 Because the hyper-lycemic diagnosis of impaired fasting glucose (IFG) pre-edes the onset of frank diabetes, nitric oxide synthesisould also be impaired in the prediabetic state. Thus, wenvestigated whether hyperglycemia influences endothelialunction among nondiabetic subjects.

• • •he Northern Manhattan Study (NOMAS) was designed asprospective cohort study of stroke risk factors among a

ri-ethnic (white, black, and Hispanic) urban population.he methods of subject recruitment and enrollment in theOMAS have been described in previous publications.2,3 In

Departments of aMedicine and bNeurology and cSergievsky Center,olumbia University College of Physicians and Surgeons; and dDepart-ent of Epidemiology and eDepartment of Sociomedical Science, Colum-

ia University Mailman School of Public Health, New York, New York.anuscript received February 11, 2005; revised manuscript received and

ccepted June 20, 2005.This project was supported in part by Grant R0I NS 29993 from the

ational Institute of Neurological Disorders and Stroke, Bethesda, Mary-and.

* Corresponding author: Tel: 212-342-4523; fax: 212-342-2904.

E-mail address: cjr10@columbia.edu (C.J. Rodriguez).

002-9149/05/$ – see front matter © 2005 Elsevier Inc. All rights reserved.oi:10.1016/j.amjcard.2005.06.070

rief, random digit dialing of approximately 25,000 house-olds was performed by Audits and Surveys (Langhorne,ennsylvania). Community participants were enrolled in theOMAS if they had never been diagnosed with stroke, were40 years old, and had resided in Northern Manhattan for3 months in a household with a telephone. The institu-

ional review board at Columbia University Medical Centerpproved the study.

Trained research assistants collected information aboutisk factors through standardized interviews, and the studyhysicians performed physical and neurologic examina-ions. Standard techniques were used to measure bloodressure, height, weight, fasting lipid panels, and fastinglucose, as described in previous publications.2,3 Hyperten-ion was defined as a systolic blood pressure of �140 mmg or a diastolic blood pressure of �90 mm Hg or theatient’s self-report of a history of hypertension or antihy-ertensive use. Diabetes mellitus was indicated by a fastinglasma glucose (FPG) value �126 mg/dl, the subject’s self-eport of such a history, or insulin or oral hypoglycemic med-cation use. The most recent American Diabetes Associationriteria for the diagnosis of IFG or prediabetes included theew cut-off points of FPG �100 and �126 mg/dl.4

Arterial endothelial function was noninvasively assessed as

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1274 The American Journal of Cardiology (www.AJConline.org)

he brachial artery response to flow-mediated dilation (FMD)uring reactive hyperemia using high-resolution-mode ultrasonography.5 Subjects were evaluated in a quiet,

emperature-controlled room. Electrocardiographic leads wereonnected, and the signal was displayed on a monitor. Therachial artery was longitudinally imaged 6 cm above thentecubital fossa using a 15-MHz linear array probe (Hewlett-ackard Sonos 5500, Hewlett-Packard, Andover, Massachu-etts). Magnified images were gated to the R wave of thelectrocardiographic QRS complex, and the flow velocities athe mid-artery were estimated using pulsed Doppler. Forearmschemia was induced by interrupting the arterial blood supplyith a cuff inflated to suprasystolic pressures (approximately50 mm Hg) for 3 minutes. Reactive hyperemia was inducedy the quick release of the cuff while images of the brachialrtery were recorded on S-VHS tapes for 30 seconds beforend 90 seconds after cuff deflation. Immediately after the cuffelease, arterial flow velocity was measured for 10 seconds toonfirm induction of increased flow velocity. An automaticuff inflator/deflator and a blood pressure monitor (Hokanson20 rapid cuff inflator, Bellvue, Washington; AG101 inflatorir source, and Critikon Dinamap vital sign monitor, Critikon,ampa, Florida) were used to minimize the variability in therocedure associated with cuff inflation/deflation.

All studies were analyzed offline by a reader blinded tohe subjects’ clinical status. End-diastolic frames were dig-tized at baseline and 60 seconds after cuff deflation by aigital frame grabber. The arterial diameter was then mea-ured using a digital caliper at a comparable site at baselinend after cuff release (model LG3, Scion, Frederich, Mary-and). Five points along the brachial artery anterior wallmedia–adventitia interface) and posterior wall were man-ally chosen by the reader. Three consecutive cardiac cyclesere analyzed for the baseline and hyperemia studies of

ach subject and the measurements averaged. FMD wasetermined as the maximal percentage of change of theost-occlusion arterial diameter relative to the mean base-ine measurement. The percentage of change in the vesseliameter after reactive hyperemia was calculated accordingo the following algorithm: [(brachial artery diameter atyperemia � brachial artery diameter at baseline)/brachialrtery diameter at baseline] � 100. Intra- and interobserverariabilities in endothelial reactivity measurements are.3% and 2.7%, respectively (n � 15).

For subjects with IFG and a normal fasting glucose,eans were calculated for continuous variables and propor-

ions for categorical variables. Univariate comparisons wereerformed using t tests for continuous data and the chi-quare test for categorical data. Simple and multiple linearegression analyses were then used to analyze the associa-ion between blood glucose and endothelial reactivity beforend after adjusting for potential confounding demographicnd medical variables, including age, gender, body massndex, hypertensive status, current cigarette smoking, andotal cholesterol, high-density cholesterol, triglyceride, and

ow-density lipoprotein levels. Analysis of variance was h

erformed to investigate the effect of differing glucosealues on FMD within the IFG and normal groups. FMDelow the upper tertile (�7.5%) was associated with aigher risk of myocardial infarction, stroke, and vasculareath in our cohort.6 Thus, abnormal or impaired FMD wasefined as �7.5% for categorical analysis.

Statistical significance was determined at the � � 0.05evel using 2-sided tests. Statistical analyses were con-ucted using Statistical Analysis Systems, version 8.2, com-uter software (SAS Institute, Cary, North Carolina).

Endothelial function measurements were obtained in 909ubjects, 64 of whom were excluded from the analysisecause of a previous myocardial infarction. Of the remain-ng 846, 187 were diabetic and 80 were not fasting at thelucose blood draw, leaving 579 nondiabetic subjectshose fasting blood glucose was available for analysis.The mean age of the total cohort was 66 � 9 years; 59%

ere women, and 68% were Hispanic, 15% black, and 16%hite. Most had hypertension (62%), and the mean bodyass index was 27.4 � 4.7 kg/m2. Table 1 lists the patient

haracteristics and laboratory results by glucose category.otal cholesterol and triglyceride levels were significantly

able 1ohort demographics

Fasting Glucose p Value

Normal(n � 484)

Impaired(n � 95)

ge (yrs) 66 � 9 66 � 8 0.80omen/men 58%/42% 62%/38% 0.57

ody mass index (kg/m2) 27.4 � 4.7 28.2 � 4.8 0.13ypertension 61% 71% 0.08igarette smoking 15% 17% 0.79otal cholesterol (mg/dl) 204 � 39 214 � 37 0.03ow-density lipoprotein (mg/dl) 132 � 36 137 � 39 0.21igh-density lipoprotein (mg/dl) 46 � 14 46 � 16 0.66riglycerides (mg/dl) 129 � 74 156 � 119 0.004asting plasma glucose (mg/dl) 83 � 9 108 � 7 �0.0001aseline arterial diameter (mm) 3.8 � 0.7 3.8 � 0.6 0.51

able 2egression coefficients of univariate linear regression analysis betweenascular function and clinical variables

ariable � Estimate SE p Value

ge (yrs) �0.062 0.012 0.0004ender* �0.38 0.32 0.234ody mass index �0.10 0.03 0.002igarette smoking† 0.032 0.43 0.94ypertension‡ �1.69 0.31 �.0001otal cholesterol (mg/dl) 0.0006 0.0040 0.88ow-density lipoprotein (mg/dl) 0.0005 0.0043 0.91igh-density lipoprotein (mg/dl) 0.018 0.011 0.10riglycerides (mg/dl) �0.978 0.749 0.192

* Men were the reference group.† Nonsmokers were the reference group.‡ Nonhypertensives were the reference group.

igher among subjects with IFG. Age, gender, body mass

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1275Preventive Cardiology/Endothelial Function and Glucose in Nondiabetics

ndex, hypertensive status, current cigarette smoking, base-ine brachial artery diameter, low-density lipoprotein level,nd high-density cholesterol level were not significantlyifferent, irrespective of glucose category.

Table 2 lists the associations of FMD with the otherariables tested by univariate linear regression analysis.ge, body mass index, and hypertensive status correlated

ignificantly with FMD. Fasting glucose showed a modest,et statistically significant, continuous linear inverse asso-iation with FMD (Figure 1). For example, for every 25g/dl increase in fasting glucose (approximately 2 SDs), a

.8% decrease occurred in FMD. Figure 2 shows an evidentrend in risk of impaired FMD by fasting glucose level.

To assess the independent association of fasting bloodlucose or hyperglycemia with FMD, multivariate linearegression analysis was used. Multivariate analysis control-ing for all the univariate predictors of FMD (age, bodyass index, and hypertensive status) revealed that higher

asting blood glucose independently predicted decreasedMD (� � �0.024, p � 0.042; Table 3).

To analyze the effect of glucose category on the percentagef change in brachial artery diameter in response to reactive

Figure 1. Relation of plasma glucose to endothelial reactivity.

igure 2. Odds ratios for abnormal flow-mediated brachial artery dilationy fasting glucose level. A fasting glucose category of �90 mg/dl was theeferent. CI � confidence interval.

yperemia, we compared the mean FMD by glucose category. F

he mean FMD was significantly higher (6.1% � 3.7%) in the78 subjects with normal fasting glucose levels than in the 95rediabetic subjects (4.9% � 3.8%, p � 0.003) with IFG.hus, prediabetic status was associated with a 25% decrease inndothelial function compared with subjects with a normalasting glucose. Prediabetic status also significantly increasedhe risk of having abnormal FMD (odds ratio 1.9, 95% confi-ence interval 1.1 to 3.1, p � 0.02). These associations re-ained significant on multivariate analysis, including age, gen-

er, hypertensive status, total cholesterol level, low-densityipoprotein level, high-density cholesterol level, triglycerideevel, and body mass index (p � 0.01 and p � 0.04, respec-ively). Within the glucose range of 110 to 125 mg/dl, FMDas significantly reduced compared with a glucose level of90 or 90 to 99 mg/dl (p �0.05; Figure 3). FMD within the

lucose range of 100 to 109 mg/dl was not significantlyeduced.

• • •he results of this study demonstrate that in a multiethnicondiabetic population free of overt cardiovascular disease,

igure 3. Endothelial reactivity according to FPG category. Comparison ofMD levels among different FPG categories (�90, 90 to 99, 100 to 109,nd 110 to 125 mg/dl), with significant (p �0.05) differences between 110o 125 and �90 and 90 to 99 mg/dl.

able 3redictors of endothelial dysfunction—multiple linearegression analysis*

ariable � Estimate SE p Value

ge (yrs) �0.062 0.017 0.0003ender† �0.62 0.31 0.044ody mass index �0.11 0.03 0.0014ypertension‡ �1.33 0.32 �0.0001lucose (mg/dl) �0.024 0.012 0.043

* Variables used for the multivariate model included age, gender, and allignificant (p �0.05) univariate predictors; when low-density lipoprotein,igh-density lipoprotein, and triglycerides (log transformed) were includedn model, effect of glucose failed to reach significance (p � 0.068).

† Men were the reference group.‡ Nonhypertensives were the reference group.

PG has an inverse, linear, and independent correlation with

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1276 The American Journal of Cardiology (www.AJConline.org)

ndothelial function as measured by FMD. Thus, elevatedPG confers risk on a continuous scale. In particular, pre-iabetics as defined by the new American Diabetes Asso-iation criteria had significantly worse endothelial functionhan those with normal FPG levels. Previous studies ofmall, selected populations have also suggested that patientsith abnormal, although nondiabetic, glucose homeostasisave worsened endothelial function.7–9 Our study, however,s the largest to measure FMD systematically and correlate itith fasting glucose levels in a multiethnic, population-based

ohort of nondiabetic subjects, and the first to do this using theew American Diabetes Association criteria for IFG.

It is becoming clear that diabetes mellitus is just the tipf the iceberg, with prediabetic patients sharing an increasedisk of cardiovascular disease.10 In 1 study, prediabetictatus was a powerful predictor of outcome, with 7-yearurvival rates similar to that of patients with overt diabe-es.11 We found that endothelial dysfunction exists amongrediabetics. Prediabetic and diabetic subjects alike haveubclinical atherosclerotic disease likely related to pro-onged and repeated exposure of the vessel wall to hy-erglycemia, which may be present for many years, thusontributing to the risk of subsequent macrovascularomplications.12,13

Recently, the American Diabetes Association changed itsriteria for defining IFG, lowering the fasting glucose cutoffrom 110 to 100 mg/dl.4 By doing so, the number of pre-iabetic patients in the United States has substantially in-reased. Using the National Health and Nutrition Examina-ion Survey data set (total population of 182 million) tovaluate the affect,14 IFG prevalence has increased from.7% to 24.1% (an increase equivalent to �30 millionatients). Endothelial dysfunction has been shown to beredictive of cardiovascular events.15,16 Our findings furthermphasize that prediabetic patients should be focused onnd targeted for lifestyle interventions (e.g., diet and exer-ise) to control glycemia and possibly normalize endothelialunction and stop progression to frank diabetes and cardio-ascular disease. A recent study demonstrated that amongverweight nondiabetic subjects, the decrease in plasma glu-ose concentration induced by weight loss was associated withn improvement in endothelial function.17 Another studyhowed that using thiazolidinediones in prediabetic subjectsecreased levels of asymmetric dimethylarginine, an nitricxide synthase inhibitor, improving endothelial function.18

hus, pharmacologic intervention among prediabetics maylso be of benefit. Furthermore, monitoring endothelialunction may have clinical use to guide risk reduction ther-py in these patients. Enhanced endothelial function (byequential testing of FMD) in response to an interventionas identified a group of patients who have an improvedardiovascular prognosis.19 This needs to be studied furthermong prediabetics.

Given that our study was cross-sectional, the chronologicequence of events could not be assessed. However, it is

nlikely that the outcome (FMD) caused the predictor (hyper-

lycemia). Although it is possible that another process causesMD and hyperglycemia separately, we attempted to adjust forotential confounders in our multivariate model. Neither aistory of ever smoking nor current smoking was related toMD in our cohort. Biochemical measurements to determine

obacco use may have been useful, given a possible bias ornaccuracy in smoking status determined by subject recall.nother limitation of our study was that serum measurementsf C-reactive protein, insulin levels, and oxidative stress (e.g.,hiobarbituric acid-reactive substances) were not obtained. Al-hough the role of plasma glucose in endothelial dysfunctionay be direct, glucose may also be a marker for these other

oncomitant abnormalities.In this large, multiethnic population-based cohort, hyper-

lycemia was independently associated with endothelial dys-unction among nondiabetics with IFG. Prediabetic subjectsad significantly worse endothelial function than subjects withormal fasting glucose levels. This further supports the rolef hyperglycemia in the pathogenesis of vascular dysfunc-ion at different stages of the development of diabetes,uggesting that exposure of the endothelium to hyperglyce-ia plays an important role in the development of athero-

clerosis even among prediabetics.

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