MATERIALS AND METHODS

In 2007, 122 consecutive patients (61 men, 61 women) who presented with diabetic cardiovascular disease were enrolled in this study. These patients were recruited from Alghadir Hospital (Tehran-Iran) for participation in this trial. In control group (50 cardiovascular patients) Hp genotyping was determined.

Blood samples were collected in the presence of EDTA and the plasma was stored at -70 °C. Genomic DNA was extracted from whole blood using DNG^TM plus solution (Fermentas). Two sets of polymerase chain reaction (PCR) were performed (Koch et al., 2002):

Statistical analysis: Chi-square test was used to determine association between Hp allele and diabetic CVD and CVD diseases and gender effect on distribution Hp genotypes in those patients. Odds ratio were calculated together with their 95% confidence intervals. The proportion test was used to compare percentage of two genotypes in diabetic cardiovascular disease. For all statistical analyses, p<0.05 was considered significant (the calculations were manual).

RESULTS

After electrophoresis of the reaction products in 1% agarose gel, Hp genotyping-specific banding patterns were obtained.With primers A and B, Hp 1-1 and Hp 2-2 genotypes were characterized by single bands representing the 1757 and 3481 bp products, respectively and Hp 2-1 genotype was characterized by the presence of both the 1757 and 3481 bp products (Fig. 1). Primers C and D were used for detecting the 349 bp Hp allele two-specific product (Fig. 2).

The distribution of three major Hp genotypes in diabetic cardiovascular patients was 1.6% (1-1), 52% (2-1), 45% (2-2) in the men and 3.27% (1-1), 54.09% (2-1), 42.62% (2-2) in the women. These prevalence were shown to be in Hardy-Weinberg equilibrium. In the no diabetic Iranian cardiovascular patients these prevalence were respectively 36% (1-1), 62% (2-1), 2% (2-2) (data not shown).

The Chi-square test determined the association between Hp allele and diabetic cardiovascular diseases (χ² = 52.98 p<0.001) and this finding was independent of gender (χ² = 0.39 p>0.05). Odds ratio shows that the chance of having Hp 2-2 phenotype in diabetic patients (men) with CVD were estimated about 504 (95% CI, 29.6 to 8577.7) times more than those with Hp 1-1 phenotype ( p<0.0001) and 27.12 (95% CI, 3.47 to 211.73) times more than those with Hp 2-1 phenotype (p = 0.0008). The chance of having Hp 2-1 phenotype in the same patients were calculated about 18.58 (95% CI, 2.33 to 147.74) times more than those with Hp 1-1 phenotype (p = 0.02). The chance of having Hp 2-2 phenotype in diabetic patients (women) with CVD were 234 (95% CI, 19.7 to 2779) times more than those with Hp 1-1 phenotype (p<0.0001) and 24.42 (95% CI, 3.12 to 190) times more than those with Hp 2-1 phenotype (p = 0.001). The chance of having Hp 2-1 phenotype in the same patients were calculated about 9.58 (95% CI, 2.05 to 44.7) times more than those with Hp 1-1 phenotype (p = 0.0019). The alterations of odds ratio in both sex were homogenous (Table 1).

Fig. 1:	Haptoglobin genotyping with primer A and B. (Lane 1) DNA size marker (Lane 2) genotype Hp 1-1 (Lane 3) genotype Hp 2-2 (Lane 4) genotype Hp 2-1

Fig. 2:	Haptoglobin genotyping with primer C and D. (Lane 1) DNA size marker, (Lanes 2 and 3) Hp 2-specific amplification product

Table 1:	The relationship between diabetic cardiovascular disorder and Hp genotypes

It is concluded that there is no difference between percentage of Hp 2-2 and Hp 2-1 phenotype in diabetic patients with cardiovascular diseases (Z = 1, p>0.05).

DISCUSSION

The oxidative modification hypothesis of atherosclerosis states that oxidative modification of low-density lipoprotein (LDL) or other oxidative events within the blood vessel wall promotes the development and progression of atherosclerotic lesion (Brown and Goldstein, 1983; Steinberg and Witzum, 2002). Hemoglobin (Hb) is an oxidant of LDL by the Fenton reaction (Kristiansen et al., 2001; Mowri et al., 2000). Hb is released from red blood cells with intravascular hemolysis in endothelial cells and injury may enter it into the sub endothelial space (Fernandez et al., 2001).

The amount of hemoglobin penetrating into the blood vessel wall is increased in diabetes because of an increased turnover of red blood cells (Venerando et al., 2002) and an increase in endothelial cell dysfunction and injury (Laight et al., 2000).

So the generation of reactive oxygen species has an important role in the development of diabetic vascular complication (Melamed-Frank et al., 2001; Suzuki et al., 1999). Haptoglobin is an antioxidant that is able to prevent hemoglobin-driven oxidation (Langlois and Delanghe, 1996; Melamed-Frank et al., 2001). The mechanism by which haptoglobin serves as an antioxidant is believed to be via stabilization of the heme moiety within the hemoglobin protein (Quaye, 2008). Ability of haptoglobin to protect against hemoglobin-driven oxidative stress is lost when hemoglobin becomes heavily glycosylated. Glycosylated Hb is markedly increased in the diabetic state (Asleh et al., 2003). In diabetic white, South Asian and Afro-Caribbean patients have been shown that Hp 2-2 increases the risk of developing CVD (UK Prospective Diabetes Study Group, 1998; Carter and Worwood, 2007).

This study for the first time in Iran suggests that Hp genotype may contribute to developing CVD in diabetic patients and we showed that the presence of Hp allele two in diabetic individuals increases the risk of developing CVD.

ACKNOWLEDGMENTS

The authors wish to thank Alghadir Hospital and Alzahra University for their grateful support of this work.