- Email: [email protected]
Relation of Haptoglobin Phenotype to Early Vascular Changes in Patients With Diabetes Mellitus
Relation of Haptoglobin Phenotype to Early Vascular Changes in Patients With Diabetes Mellitus Marina Shor, MDa,b, Mona Boaz, PhDc, Dov Gavish, MDa,b,e, Julio Wainshtein, MDd, Zipora Matas, PhDb, and Marina Shargorodsky, MDb,d,e,f,* Haptoglobin (Hp) is an antioxidant protein and the major susceptibility gene for atherosclerosis in diabetic patients. The effect of Hp phenotype on arterial compliance and metabolic and inflammatory parameters was investigated. Patients were divided into 3 groups according to Hp phenotype of Hp 2-2, Hp 2-1, and Hp 1-1. Arterial elasticity of large and small arteries was evaluated using the pulse-wave contour analysis method. The large-artery elasticity index (LAEI) was lower in patients with Hp 2-2 compared with Hp 1-1 (8.4 ⴞ 2.3 vs 12.6 ⴞ 4.1 ml/mm Hg ⴛ 100; p <0.0001). The difference in LAEIs between the Hp 2-1 and Hp 1-1 groups was also significant (9.9 ⴞ 2.6 vs 12.6 ⴞ 4.1 ml/mm Hg ⴛ 100; p ⴝ 0.025). The Hp 2-2 and Hp 2-1 groups did not differ from one another. The small-artery elasticity index (SAEI) was significantly lower in patients with Hp 2-2 compared with Hp 1-1 (2.8 ⴞ 1.0 vs 4.4 ⴞ 1.9 ml/mm Hg ⴛ 100; p ⴝ 0.004). Differences in SAEIs between patients with Hp 2-1 and Hp 1-1, as well as those with Hp 2-1 and Hp 2-2, were not detected. Systemic vascular resistance differed significantly across groups, driven by the difference between patients with Hp 2-2 and Hp 1-1. In conclusion, LAEI and SAEI were significantly lower and systemic vascular resistance was higher in homozygotes for the 2 allele (Hp 2-2) compared with patients with Hp 2-1 or Hp 1-1 phenotypes. Differences in arterial elasticity were detected despite the lack of by-phenotype differences in glycemic control, blood pressure, or presence of cardiovascular risk factors. © 2007 Elsevier Inc. All rights reserved. (Am J Cardiol 2007;100:1767–1770)
Recent technological progress has permitted reliable measurement of vascular compliance, a means of assessing the early stages of vascular disease. One such technique, arterial pulse-wave contour analysis, involves quantifying the amount of arterial stiffening and can be regarded as a valid marker of generalized atherosclerosis.1–3 Estimation of vascular compliance may serve as a surrogate for cardiovascular morbidity and mortality risk,4 – 6 as well as an indicator of treatment benefit.7,8 The present study was designed to evaluate the effect of haptoglobin (Hp) phenotype on arterial compliance, specifically whether Hp phenotype predicted early vascular changes in diabetic patients, determined using arterial pulse-wave contour analysis. Methods The study group consisted of 64 Caucasian subjects (40 women, 24 men) with type 2 diabetes mellitus. Study patients were classified as diabetic if fasting plasma glucose level was ⱖ126 mg/dl on ⱖ2 blood samples or they were treated with antidiabetic medications. Additional cardiovascular risk factors were defined using the National Cholesa Department of Internal Medicine, bThe Brunner Institute for Cardiovascular Research, cEpidemiology and Research Unit, and dDiabetes Unit, E. Wolfson Medical Center; eSackler School of Medicine, Tel Aviv University; and fEndocrinology Unit, E. Wolfson Medical Center, Tel Aviv, Israel. Manuscript received April 22, 2007; revised manuscript received and accepted July 1, 2007. *Corresponding author: Tel: 972-3-502-8614; fax: 972-3-503-2693. E-mail address: [email protected] (M. Shargorodsky).
0002-9149/07/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.amjcard.2007.07.052
terol Education Program risk-factor categories9 of current cigarette smoking, hypertension (systolic blood pressure [BP] ⱖ140 mm Hg and/or diastolic BP ⱖ90 mm Hg and/or antihypertensive medication use), hypertriglyceridemia (triglycerides ⱖ150 mg/dl), low high-density lipoprotein cholesterol (⬍40 mg/dl in men and ⬍50 mg/dl in women). All patients underwent blood chemistry testing, including fasting plasma glucose, hemoglobin A1c, lipid profile, C-reactive protein, and complete blood count; Hp phenotyping; and arterial compliance measurements. Patients were divided into 3 groups according to Hp phenotype. Group 1 included diabetic patients homozygous for the 2 allele (Hp 2-2), group 2 included patients heterozygous at the Hp locus (Hp 2-1), and group 3 included patients homozygous for the 1 allele (Hp 1-1). Allele frequency distributions in the Israeli population, as well as in Europe and the United States, were 9% for Hp 1-1, 49% for Hp 2-2, and 42% is Hp 2-1.10 At the beginning of the study, this allele frequency distribution was similar to that in the general population at 12% for Hp 1-1, 38% for Hp 2-1, and 49% for Hp 2-2. To increase study power, intensive recruitment of patients with Hp 1-1 was performed, and final frequency distributions were 27 patients with Hp 2-2 (42.2%; group 1), 21 patients with Hp 2-1 (32.8%; group 2), and 16 patients with Hp 1-1 (25%; group 3). Hp phenotyping was performed using polyacrylamide gel electrophoresis according to established methods.11 Arterial compliance measurements were performed between 8 and 11 A.M. in a quiet temperature-controlled laboratory. Radial arterial waveforms were recorded for 30 www.AJConline.org
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Table 1 Demographic and clinical characteristics by haptoglobin (Hp) phenotype Variable Women/men Age (yrs) Body mass index (kg/m2) Diabetes duration (yrs) Hypertension Dyslipidemia* Smoker Diabetes medications Oral Insulin Combination Aspirin use ACE-inhibitor/ARB use Calcium channel blocker use Statin use Fasting plasma glucose (mg/dl) Hemoglobin A1c (%) Total cholesterol (mg/dl) Low-density lipoprotein cholesterol (mg/dl) High-density lipoprotein cholesterol (mg/dl) Triglycerides (mg/dl) High-sensitivity C-reactive protein (mg/dl)
Hp 2-2 (n ⫽ 27)
Hp 2-1 (n ⫽ 21)
Hp 1-1 (n ⫽ 16)
14/13 66 ⫾ 9 29 ⫾ 5 5.8 (2–10) 22 (81.5%) 20 (74.1%) 10 (37.0%)
13/8 64 ⫾ 9 30 ⫾ 5 5.2 (3–14) 14 (66.7%) 15 (71.4%) 7 (33.3%)
11/5 61 ⫾ 10 28 ⫾ 4 5.8 (3–14) 9 (56.3%) 12 (75.0%) 5 (31.3%)
15 (55.6%) 12 (57.1%) 7 (43.85) 5 (18.52%) 4 (19.1%) 4 (25%) 7 (25.9%) 5 (23.8%) 5 (31.3%) 22 (81.5%) 17 (81.0%) 13 (81.3%) 19 (70.4%) 16 (76.2%) 13 (81.3%) 5 (18.5%) 5 (23.8%) 4 (25%) 21 (77.8%) 15 (71.4%) 11 (68.8%) 165.4 ⫾ 66.0 162.8 ⫾ 72.5 163.6 ⫾ 59.3 8.1 ⫾ 1.3 8.0 ⫾ 0.9 196.1 ⫾ 38.6 185.6 ⫾ 32.3 121.0 ⫾ 41.9 123.6 ⫾ 37.5 43.3 ⫾ 11.7
44.2 ⫾ 14.3
7.6 ⫾ 1.0 189.3 ⫾ 36.8 122.8 ⫾ 22.5 42.1 ⫾ 8.5
182.6 ⫾ 89.3 183.7 ⫾ 105.1 181.6 ⫾ 50.8 0.7 ⫾ 0.4 0.7 ⫾ 0.2 0.7 ⫾ 0.3
Values expressed as mean ⫾ SD, mean (range), or number (percent). By-group comparisons were made using 1-way analysis of variance simultaneously comparing all 3 phenotypes. No significant differences were detected in any variable. * Dyslipidemia defined as triglycerides ⱖ150 mg/dl and/or high-density lipoprotein cholesterol ⬍40 mg/dl in men and ⬍50 mg/dl in women or lipid-lowering agent use. ACE ⫽ angiotensin-converting enzyme; ARB ⫽ angiotensin receptor blocker.
seconds with the subject in a supine position. The pressure transducer amplifier system was connected to a specially designed device (model CR-2000; Hypertension Diagnostics Inc., Eagen, Minnesota). The passive transient response of the arterial vasculature to the initial loading conditions was determined by analyzing the diastolic portion of the pressure pulse-wave form. This technique, which was recently validated and used extensively,1,12–14 was performed using a simple noninvasive radial pulse-wave recording and computer analysis of the diastolic decay, providing separate assessment of large-artery or capacitive compliance and small-artery reflective or oscillatory compliance. Systemic vascular resistance (SVR) was calculated as mean arterial pressure divided by cardiac output. Mean arterial pressure was derived from waveform analysis, integrating the area under the curve and calculating the mean area of recordings during 30 seconds. Arterial compliance measurements were performed with no knowledge of the patient’s Hp phenotype. Analysis of data was carried out using SPSS, version 9.0, statistical analysis software (SPSS Inc., Chicago, Illinois). For continuous variables, descriptive statistics were calculated and reported as mean ⫾ SD. Normalcy of distribution
of continuous variables was assessed using the Kolmogorov-Smirnov test (cut-off at p ⬍0.01). All variables were normally distributed and were compared across Hp categories using 1-way analysis of variance followed by Bonferroni’s pairwise comparisons. All tests were 2 sided and considered significant at p ⬍0.05. Results Characteristics of the 3 study groups are listed in Table 1. As listed, all 3 groups were similar in terms of age, gender distribution, body mass index, presence of concomitant cardiovascular risk factors, and prescribed medications. As listed in Table 2, systolic and diastolic BPs were similar in the 3 groups. The large-artery elasticity index (LAEI) was significantly lower in patients homozygous for the 2 allele (Hp 2-2) compared with patients homozygous for the 1 allele (Hp 1-1). Likewise, LAEI was significantly lower in group 2 (Hp2-1) than group 3 (Hp1-1). LAEI values for groups 1 (Hp 2-2) and 2 (Hp 1-2) did not differ from 1 another (Figure 1). The small-artery elasticity index (SAEI) was also significantly lower in patients with Hp 2-2 patients compared with Hp 1-1. Although the SAEI was 32.5% higher in patients with Hp 2-1 compared with Hp 2-2, this difference was not statistically significant. A difference in SAEI between patients with Hp 1-2 and Hp 1-1 was not detected (Figure 1). SVR differed significantly across groups, driven by the difference between patients with Hp 2-2 and Hp 1-1. Although SVR was 9.5% lower in patients with Hp 2-1 compared with Hp 2-2, this difference was not statistically significant. A difference in SVR between patients with Hp 1-2 and Hp 1-1 was not detected (Figure 1). Metabolic and inflammatory parameters, including fasting plasma glucose, hemoglobin A1c, total cholesterol, and high-sensitivity C-reactive protein, did not differ significantly among the 3 groups (Table 1). Discussion In the present study, LAEI and SAEI were significantly lower and SVR was significantly higher in diabetic patients homozygous for the 2 allele (Hp 2-2) compared with patients with the Hp 2-1 or Hp 1-1 phenotypes. The Hp 2-2 phenotype was associated with an adverse effect on arterial elasticity despite a lack of by-phenotype differences in glycemic control, BP, or presence of cardiovascular risk factors. Findings of the present study concur with those of previous studies that identified increased microvascular and macrovascular complications, including diabetic nephropathy, diabetic retinopathy, and cardiovascular disease, in diabetic patients homozygous for the Hp 2 allele compared with patients homozygous for the Hp 1 allele, whereas heterozygotes had an intermediate risk.15–17 In patients with diabetes, arterial stiffening was consistently observed across all age groups and may contribute in part to the excess cardiovascular morbidity and mortality observed in these patients.2,6 – 8 Several preexisting risk factors, including hypertension, dyslipidemia, cigarette smoking, and increased adiposity, may further increase the car-
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Table 2 Hemodynamic and arterial compliance measurements by haptoglobin (Hp) phenotype
Systolic BP (mm Hg) Diastolic BP (mm Hg) LAEI (ml/mm Hg ⫻ 100) SAEI (ml/mm Hg ⫻ 100) SVR (dyne ⫻ s ⫻ cm)
Hp 2-2 (n ⫽ 27)
Hp 2-1 (n ⫽ 21)
Hp 1-1 (n ⫽ 16)
p Value
141 ⫾ 20 72 ⫾ 12 8.4 ⫾ 2.3 2.8 ⫾ 1.0 1,751.8 ⫾ 344.9
140 ⫾ 26 71 ⫾ 14 9.9 ⫾ 2.6 3.7 ⫾ 1.8 1,598.3 ⫾ 310.2
139 ⫾ 25 72 ⫾ 10 12.6 ⫾ 4.1 4.4 ⫾ 1.9 1,442.8 ⫾ 367.7
NS NS ⬍0.0001 ⬍0.004 ⬍0.01
The p values were calculated using 1-way analysis of variance, simultaneously comparing all 3 phenotypes.
14.0
4.5
p<0.0001
4.0 3.5
10.0
ml/mmHg*100
ml/mmHg*10
12.0
8.0 6.0 4.0
3.0 2.5 2.0 1.5 1.0
2.0
0.5 0.0
0.0 Hp2-2
A
p=0.004
Hp2-1
Hp1-1
Hp2-2
B
Haptoglobin phenotype
Hp2-1 Haptoglobin phenotype
Hp1-1
p=0.01
1800.0
dyne*sec*cm
1600.0 1400.0 1200.0 1000.0 800.0 600.0 400.0 200.0 0.0 Hp2-2
C
Hp2-1
Hp1-1
Haptoglobin phenotype
Figure 1. (A) LAEI, (B) SAEI, and (C) SVR according to Hp phenotype. Group 1 included diabetic patients homozygous for the 2 allele (Hp 2-2), group 2 included patients heterozygous at the Hp locus (Hp 2-1), and group 3 included patients homozygous for the 1 allele (Hp 1-1).
diovascular risk associated with hyperglycemia, leading to excess cardiovascular disease risk at an earlier age. Hp appears to be a major susceptibility gene for atherosclerosis in diabetic patients.17,18 There are a number of mechanisms by which Hp might affect arterial stiffening in both the short and long term. Hp is an antioxidant protein binding free hemoglobin with very high affinity and stability. Hemoglobin binding inhibits hydroxyl free radical formation and prevents the development of the oxidative tissue damage that contributes to vascular complications in patients with diabetes.19 The Hp-hemoglobin complex prevents the dissociation of heme iron, which is responsible for oxidation of lowdensity lipoproteins. In this way, the complex decreases lipid peroxidation damage of vascular endothelial cells.20 Because free hemoglobin inhibits endothelium-derived nitric oxide and decreases endothelium-dependent vascular relaxation, binding of Hp to hemoglobin may have an important regulatory role on vascular endothelial function.21 In addition, Hp may influence the development of
vascular complications by modulating immune and inflammatory reactions.10 In concurrence with previous studies, the present study did not detect by-phenotype differences in glycemic control, lipids, or BP.22 However, an association between Hp 2-2 and increased serum total and free cholesterol was also reported, without significant differences in high-density lipoprotein cholesterol, apolipoprotein E, lipoprotein(a), fibrinogen, or C-reactive protein. Age, body mass index, smoking, alcohol intake, and BP were similar among the 3 Hp phenotypes.23 Because vascular changes inflicted by multiple environmental and genetic factors develop years before an event, detection of vascular damage can serve as a predictor of future cardiovascular complications. Thus, estimation of vascular elasticity can serve as a surrogate end point for prediction of morbid events and estimation of success of treatment. In the present study, Hp phenotype was a significant predictor of early vascular adverse changes detected using arterial pulse-wave analysis.
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1. Cohn JN, Finkelstein S, McVeigh G, Morgan D, LeMay I, Robinson J, Mock J. Non-invasive pulse wave analysis for the detection of arterial vascular disease. Hypertension 1995;26:503–508. 2. Salomaa V, Riley W, Kark JD, Nardo C, Folsom AR. Non-insulindependent diabetes mellitus and fasting glucose and insulin concentration are associated with arterial stiffness indexes. The ARIC Study. Atherosclerosis Risk in Communities Study. Circulation 1995;91: 1432–1443. 3. van Popele NM, Grobbee DE, Bots ML, Asmar R, Topouchian J, Reneman RS, Hoeks AP, van der Kuip DA, Hofman A, Witteman JC. Association between arterial stiffness and atherosclerosis: the Rotterdam Study. Stroke 2001;32:454 – 460. 4. Blacher J, Guerin AP, Pannier B, Marchais SJ, Safar ME, London GM. Impact of aortic stiffness on survival in end-stage renal disease. Circulation 1999;99:2434 –2439. 5. Laurent S, Boutouyrie P, Asmar R, Gautier I, Laloux B, Guize L, Ducimetiere P, Benetos A. Aortic stiffness is an independent predictor of all-cause and cardiovascular mortality in hypertensive patients. Hypertension 2001;37:1236 –1241. 6. Cruickshank K, Riste L, Anderson SG, Wright JS, Dunn G, Gosling RG. Aortic pulse-wave velocity and its relationship to mortality in diabetes and glucose intolerance. An integrated index of vascular function? Circulation 2002;106:2085–2090. 7. Zieman SJ, Melenovsky V, Kass DA. Mechanisms, pathophysiology, and therapy of arterial stiffness. Arterioscler Thromb Vasc Biol 2005; 25:932–943. 8. Giannattasio C, Mancia G. Arterial distensibility in humans. Modulating mechanisms, alterations in diseases and effects of treatment. J Hypertens 2002;20:1889 –1899. 9. Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults. Executive summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults. JAMA 2001;285:2486 –2497. 10. Langllois MR, Delanghe JR. Biological and clinical significance of haptoglobin polymorphism in humans. Clin Chem 1996;42:1589 – 1600. 11. Smithies O. Zone electrophoresis in starch gels: group variations in the serum proteins of normal human adults. Biochemistry 1955;61:629 – 641. 12. Shargorodsky M, Leibovitz E, Lubimov L, Gavish D, Zimlichman R. Prolonged treatment with the AT1 receptor blocker, valsartan, in-
13.
14.
15.
16.
17.
18.
19. 20.
21.
22.
23.
creases small and large artery compliance in uncomplicated essential arterial hypertension: Am J Hypertens 2002;15:1087–1091. McVeigh GE, Burns DE, Finkelstein SM, McDonald KM, Mock JE, Feske W, Carlyle PF, Flack J, Grimm R, Conh JN. Reduced vascular compliance as a marker for essential hypertension. Am J Hypertens 1991;4:245–257. Zimlichman R, Shargorodsky M, Boaz M, Duprez D, Rahn KH, Rizzoni D, Payeras AC, Hamm C, McVeigh G. Determination of arterial compliance using blood pressure waveform analysis with the CR-2000 system. Am J Hypertens 2005;18:65–71. Levy AP, Roguin A, Marsh S, Nakhoul FM, Herer P, Hochberg I, Skorecki K. Haptoglobin phenotype and vascular complications in diabetes. N Engl J Med 2000;343:969 –970. Nakhoul F, Marsh S, Hochberg I, Leibu R, Miller BP, Levy AP. Haptoglobin phenotype and diabetic retinopathy. JAMA 2000;284: 1244 –1245. Levy AP, Hochberg I, Jablonski K, Resnic HE, Lee ET, Best L, Howard BV. Haptoglobin phenotype is an independent risk factor for cardiovascular disease in individuals with diabetes: the Strong Heart Study. J Am Coll Cardiol 2002;40:1984 –1990. Suleiman M, Aronson D, Asleh R, Kapeliovich MR, Roguin A, Meisel SR, Shochat M, Sulieman A, Reisner SA, Markiewicz W, et al. Haptoglobin polymorphism predicts 30-day mortality and heart failure in patients with diabetes and acute myocardial infarction. Diabetes 2005;54:2802–2806. Guigliano D, Ceriello A, Paolisso G. Oxidative stress and diabetic vascular complications. Diabetic Care 1996;19:257–267. Gutteridge JM. The antioxidant activity of haptoglobin towards hemoglobin-stimulated lipid peroxidation. Biochem Biophys Acta 1987;917: 219 –223. Collins P, Burman J, Chung H, Fox K. Hemoglobin inhibits endothelium-dependent relaxation to acetylcholine in human coronary arteries in vivo. Circulation 1993;87:80 – 85. Delanghe J, Duprez D, De Buyzere M, Bergez B, Callens B, LerouxRoels G, Clement D. Haptoglobin phenotypes and complications in established essential arterial hypertension. J Hypertens 1993;11:861– 867. Braeckman L, De Bacquer D, Delanghe J, Claeys L, De Backer G. Association between haptoglobin polymorphism, lipids, lipoproteins and inflammatory variables. Atherosclerosis 1999;143:383–388.
Recent technological progress has permitted reliable measurement of vascular compliance, a means of assessing the early stages of vascular disease. One such technique, arterial pulse-wave contour analysis, involves quantifying the amount of arterial stiffening and can be regarded as a valid marker of generalized atherosclerosis.1–3 Estimation of vascular compliance may serve as a surrogate for cardiovascular morbidity and mortality risk,4 – 6 as well as an indicator of treatment benefit.7,8 The present study was designed to evaluate the effect of haptoglobin (Hp) phenotype on arterial compliance, specifically whether Hp phenotype predicted early vascular changes in diabetic patients, determined using arterial pulse-wave contour analysis. Methods The study group consisted of 64 Caucasian subjects (40 women, 24 men) with type 2 diabetes mellitus. Study patients were classified as diabetic if fasting plasma glucose level was ⱖ126 mg/dl on ⱖ2 blood samples or they were treated with antidiabetic medications. Additional cardiovascular risk factors were defined using the National Cholesa Department of Internal Medicine, bThe Brunner Institute for Cardiovascular Research, cEpidemiology and Research Unit, and dDiabetes Unit, E. Wolfson Medical Center; eSackler School of Medicine, Tel Aviv University; and fEndocrinology Unit, E. Wolfson Medical Center, Tel Aviv, Israel. Manuscript received April 22, 2007; revised manuscript received and accepted July 1, 2007. *Corresponding author: Tel: 972-3-502-8614; fax: 972-3-503-2693. E-mail address: [email protected] (M. Shargorodsky).
0002-9149/07/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.amjcard.2007.07.052
terol Education Program risk-factor categories9 of current cigarette smoking, hypertension (systolic blood pressure [BP] ⱖ140 mm Hg and/or diastolic BP ⱖ90 mm Hg and/or antihypertensive medication use), hypertriglyceridemia (triglycerides ⱖ150 mg/dl), low high-density lipoprotein cholesterol (⬍40 mg/dl in men and ⬍50 mg/dl in women). All patients underwent blood chemistry testing, including fasting plasma glucose, hemoglobin A1c, lipid profile, C-reactive protein, and complete blood count; Hp phenotyping; and arterial compliance measurements. Patients were divided into 3 groups according to Hp phenotype. Group 1 included diabetic patients homozygous for the 2 allele (Hp 2-2), group 2 included patients heterozygous at the Hp locus (Hp 2-1), and group 3 included patients homozygous for the 1 allele (Hp 1-1). Allele frequency distributions in the Israeli population, as well as in Europe and the United States, were 9% for Hp 1-1, 49% for Hp 2-2, and 42% is Hp 2-1.10 At the beginning of the study, this allele frequency distribution was similar to that in the general population at 12% for Hp 1-1, 38% for Hp 2-1, and 49% for Hp 2-2. To increase study power, intensive recruitment of patients with Hp 1-1 was performed, and final frequency distributions were 27 patients with Hp 2-2 (42.2%; group 1), 21 patients with Hp 2-1 (32.8%; group 2), and 16 patients with Hp 1-1 (25%; group 3). Hp phenotyping was performed using polyacrylamide gel electrophoresis according to established methods.11 Arterial compliance measurements were performed between 8 and 11 A.M. in a quiet temperature-controlled laboratory. Radial arterial waveforms were recorded for 30 www.AJConline.org
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Table 1 Demographic and clinical characteristics by haptoglobin (Hp) phenotype Variable Women/men Age (yrs) Body mass index (kg/m2) Diabetes duration (yrs) Hypertension Dyslipidemia* Smoker Diabetes medications Oral Insulin Combination Aspirin use ACE-inhibitor/ARB use Calcium channel blocker use Statin use Fasting plasma glucose (mg/dl) Hemoglobin A1c (%) Total cholesterol (mg/dl) Low-density lipoprotein cholesterol (mg/dl) High-density lipoprotein cholesterol (mg/dl) Triglycerides (mg/dl) High-sensitivity C-reactive protein (mg/dl)
Hp 2-2 (n ⫽ 27)
Hp 2-1 (n ⫽ 21)
Hp 1-1 (n ⫽ 16)
14/13 66 ⫾ 9 29 ⫾ 5 5.8 (2–10) 22 (81.5%) 20 (74.1%) 10 (37.0%)
13/8 64 ⫾ 9 30 ⫾ 5 5.2 (3–14) 14 (66.7%) 15 (71.4%) 7 (33.3%)
11/5 61 ⫾ 10 28 ⫾ 4 5.8 (3–14) 9 (56.3%) 12 (75.0%) 5 (31.3%)
15 (55.6%) 12 (57.1%) 7 (43.85) 5 (18.52%) 4 (19.1%) 4 (25%) 7 (25.9%) 5 (23.8%) 5 (31.3%) 22 (81.5%) 17 (81.0%) 13 (81.3%) 19 (70.4%) 16 (76.2%) 13 (81.3%) 5 (18.5%) 5 (23.8%) 4 (25%) 21 (77.8%) 15 (71.4%) 11 (68.8%) 165.4 ⫾ 66.0 162.8 ⫾ 72.5 163.6 ⫾ 59.3 8.1 ⫾ 1.3 8.0 ⫾ 0.9 196.1 ⫾ 38.6 185.6 ⫾ 32.3 121.0 ⫾ 41.9 123.6 ⫾ 37.5 43.3 ⫾ 11.7
44.2 ⫾ 14.3
7.6 ⫾ 1.0 189.3 ⫾ 36.8 122.8 ⫾ 22.5 42.1 ⫾ 8.5
182.6 ⫾ 89.3 183.7 ⫾ 105.1 181.6 ⫾ 50.8 0.7 ⫾ 0.4 0.7 ⫾ 0.2 0.7 ⫾ 0.3
Values expressed as mean ⫾ SD, mean (range), or number (percent). By-group comparisons were made using 1-way analysis of variance simultaneously comparing all 3 phenotypes. No significant differences were detected in any variable. * Dyslipidemia defined as triglycerides ⱖ150 mg/dl and/or high-density lipoprotein cholesterol ⬍40 mg/dl in men and ⬍50 mg/dl in women or lipid-lowering agent use. ACE ⫽ angiotensin-converting enzyme; ARB ⫽ angiotensin receptor blocker.
seconds with the subject in a supine position. The pressure transducer amplifier system was connected to a specially designed device (model CR-2000; Hypertension Diagnostics Inc., Eagen, Minnesota). The passive transient response of the arterial vasculature to the initial loading conditions was determined by analyzing the diastolic portion of the pressure pulse-wave form. This technique, which was recently validated and used extensively,1,12–14 was performed using a simple noninvasive radial pulse-wave recording and computer analysis of the diastolic decay, providing separate assessment of large-artery or capacitive compliance and small-artery reflective or oscillatory compliance. Systemic vascular resistance (SVR) was calculated as mean arterial pressure divided by cardiac output. Mean arterial pressure was derived from waveform analysis, integrating the area under the curve and calculating the mean area of recordings during 30 seconds. Arterial compliance measurements were performed with no knowledge of the patient’s Hp phenotype. Analysis of data was carried out using SPSS, version 9.0, statistical analysis software (SPSS Inc., Chicago, Illinois). For continuous variables, descriptive statistics were calculated and reported as mean ⫾ SD. Normalcy of distribution
of continuous variables was assessed using the Kolmogorov-Smirnov test (cut-off at p ⬍0.01). All variables were normally distributed and were compared across Hp categories using 1-way analysis of variance followed by Bonferroni’s pairwise comparisons. All tests were 2 sided and considered significant at p ⬍0.05. Results Characteristics of the 3 study groups are listed in Table 1. As listed, all 3 groups were similar in terms of age, gender distribution, body mass index, presence of concomitant cardiovascular risk factors, and prescribed medications. As listed in Table 2, systolic and diastolic BPs were similar in the 3 groups. The large-artery elasticity index (LAEI) was significantly lower in patients homozygous for the 2 allele (Hp 2-2) compared with patients homozygous for the 1 allele (Hp 1-1). Likewise, LAEI was significantly lower in group 2 (Hp2-1) than group 3 (Hp1-1). LAEI values for groups 1 (Hp 2-2) and 2 (Hp 1-2) did not differ from 1 another (Figure 1). The small-artery elasticity index (SAEI) was also significantly lower in patients with Hp 2-2 patients compared with Hp 1-1. Although the SAEI was 32.5% higher in patients with Hp 2-1 compared with Hp 2-2, this difference was not statistically significant. A difference in SAEI between patients with Hp 1-2 and Hp 1-1 was not detected (Figure 1). SVR differed significantly across groups, driven by the difference between patients with Hp 2-2 and Hp 1-1. Although SVR was 9.5% lower in patients with Hp 2-1 compared with Hp 2-2, this difference was not statistically significant. A difference in SVR between patients with Hp 1-2 and Hp 1-1 was not detected (Figure 1). Metabolic and inflammatory parameters, including fasting plasma glucose, hemoglobin A1c, total cholesterol, and high-sensitivity C-reactive protein, did not differ significantly among the 3 groups (Table 1). Discussion In the present study, LAEI and SAEI were significantly lower and SVR was significantly higher in diabetic patients homozygous for the 2 allele (Hp 2-2) compared with patients with the Hp 2-1 or Hp 1-1 phenotypes. The Hp 2-2 phenotype was associated with an adverse effect on arterial elasticity despite a lack of by-phenotype differences in glycemic control, BP, or presence of cardiovascular risk factors. Findings of the present study concur with those of previous studies that identified increased microvascular and macrovascular complications, including diabetic nephropathy, diabetic retinopathy, and cardiovascular disease, in diabetic patients homozygous for the Hp 2 allele compared with patients homozygous for the Hp 1 allele, whereas heterozygotes had an intermediate risk.15–17 In patients with diabetes, arterial stiffening was consistently observed across all age groups and may contribute in part to the excess cardiovascular morbidity and mortality observed in these patients.2,6 – 8 Several preexisting risk factors, including hypertension, dyslipidemia, cigarette smoking, and increased adiposity, may further increase the car-
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Table 2 Hemodynamic and arterial compliance measurements by haptoglobin (Hp) phenotype
Systolic BP (mm Hg) Diastolic BP (mm Hg) LAEI (ml/mm Hg ⫻ 100) SAEI (ml/mm Hg ⫻ 100) SVR (dyne ⫻ s ⫻ cm)
Hp 2-2 (n ⫽ 27)
Hp 2-1 (n ⫽ 21)
Hp 1-1 (n ⫽ 16)
p Value
141 ⫾ 20 72 ⫾ 12 8.4 ⫾ 2.3 2.8 ⫾ 1.0 1,751.8 ⫾ 344.9
140 ⫾ 26 71 ⫾ 14 9.9 ⫾ 2.6 3.7 ⫾ 1.8 1,598.3 ⫾ 310.2
139 ⫾ 25 72 ⫾ 10 12.6 ⫾ 4.1 4.4 ⫾ 1.9 1,442.8 ⫾ 367.7
NS NS ⬍0.0001 ⬍0.004 ⬍0.01
The p values were calculated using 1-way analysis of variance, simultaneously comparing all 3 phenotypes.
14.0
4.5
p<0.0001
4.0 3.5
10.0
ml/mmHg*100
ml/mmHg*10
12.0
8.0 6.0 4.0
3.0 2.5 2.0 1.5 1.0
2.0
0.5 0.0
0.0 Hp2-2
A
p=0.004
Hp2-1
Hp1-1
Hp2-2
B
Haptoglobin phenotype
Hp2-1 Haptoglobin phenotype
Hp1-1
p=0.01
1800.0
dyne*sec*cm
1600.0 1400.0 1200.0 1000.0 800.0 600.0 400.0 200.0 0.0 Hp2-2
C
Hp2-1
Hp1-1
Haptoglobin phenotype
Figure 1. (A) LAEI, (B) SAEI, and (C) SVR according to Hp phenotype. Group 1 included diabetic patients homozygous for the 2 allele (Hp 2-2), group 2 included patients heterozygous at the Hp locus (Hp 2-1), and group 3 included patients homozygous for the 1 allele (Hp 1-1).
diovascular risk associated with hyperglycemia, leading to excess cardiovascular disease risk at an earlier age. Hp appears to be a major susceptibility gene for atherosclerosis in diabetic patients.17,18 There are a number of mechanisms by which Hp might affect arterial stiffening in both the short and long term. Hp is an antioxidant protein binding free hemoglobin with very high affinity and stability. Hemoglobin binding inhibits hydroxyl free radical formation and prevents the development of the oxidative tissue damage that contributes to vascular complications in patients with diabetes.19 The Hp-hemoglobin complex prevents the dissociation of heme iron, which is responsible for oxidation of lowdensity lipoproteins. In this way, the complex decreases lipid peroxidation damage of vascular endothelial cells.20 Because free hemoglobin inhibits endothelium-derived nitric oxide and decreases endothelium-dependent vascular relaxation, binding of Hp to hemoglobin may have an important regulatory role on vascular endothelial function.21 In addition, Hp may influence the development of
vascular complications by modulating immune and inflammatory reactions.10 In concurrence with previous studies, the present study did not detect by-phenotype differences in glycemic control, lipids, or BP.22 However, an association between Hp 2-2 and increased serum total and free cholesterol was also reported, without significant differences in high-density lipoprotein cholesterol, apolipoprotein E, lipoprotein(a), fibrinogen, or C-reactive protein. Age, body mass index, smoking, alcohol intake, and BP were similar among the 3 Hp phenotypes.23 Because vascular changes inflicted by multiple environmental and genetic factors develop years before an event, detection of vascular damage can serve as a predictor of future cardiovascular complications. Thus, estimation of vascular elasticity can serve as a surrogate end point for prediction of morbid events and estimation of success of treatment. In the present study, Hp phenotype was a significant predictor of early vascular adverse changes detected using arterial pulse-wave analysis.
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