Antiphospholipid Antibodies in Patients with Type 2 Diabetes Mellitus

Patients with diabetes mellitus type 2 have a greater risk of accelerated atherosclerosis. Antiphospholipid antibodies(aPL)are associated with greater risk for thrombosis. To demonstrate the possible role of anticardiolipin(aCL) and anti-β2 glycoprotein(β2 GP1) antibodies in such patients, we investigate the presence of these antibodies in a group of type 2 diabetic patients. Objectives: 1. To investigate the presence of anticardiolipin IgM and IgG antibodies and anti-β2 GP1 IgG antibodies in diabetic patients and compare them with a control group. 2. To analyze their potential implication in the occurrence of vasculopathy in such patients. Patients and Methods: Fifty patients with type 2 diabetes mellitus and 33 healthy subjects were included in the study. Each blood sample was tested for IgM, IgG aCL antibodies and for anti-β2 GP1 IgG antibodies. Results: Seven patients were positive for aCL IgM, 6 were positive for aCL IgG and 4 patients were positive for anti-β2 GP1 IgG antibodies. There was no differences in the means of IgM, IgG aCL and anti-β2 GP1 IgG antibodies titers in patients with complicated and uncomplicated diabetes mellitus. Conclusion: The aCL and β2 GP1 antibodies positive titer means among type2 diabetics were significantly higher than non-diabetic subjects. Positive but low titers of aCL and β2 GPI antibodies could suggest that these autoantibodies may play a minor role in the pathogenesis of atherosclerosis. Low titers of aCL and β2 GPI antibodies were seen in complicated and non-complicated diabetic populations that probably lessen the importance of these autoantibodies as effective contributors in the pathogenesis of diabetic vasculopathy. Keyword: Diabetes, antiphospholipid antibodies, anticardiolipin antibodies, vasculopathy. ةداضملا ماسجلاا يركسلا ىضرم ىدل ةٌتافسوفلا موحشلل ًناثلا عونلا نم **ليضف حلاص يلع .د ** نيسح ذيؤم ذمحا .د .*ىسح اللهذبع يذن .د ةيرهجملا ءايحلاا عرف* ، يىنين بط ةيلك ، ،لصىملا ** ,يىنين بط ةيلك ،بطلا عرف لصىملا

Many studies showed controversy about the occurrence of antiphospholipid antibodies in type 1 and type 2 diabetic patients.
We studied the presence of IgM, IgG anticardiolipin antibodies and IgG antibodies to β 2 GP1 in a group of patients with type 2 diabetes mellitus.
We aim to investigate the presence of antibodies to phospholipid and the phospholipid associated proteins in type 2 diabetic patients and to verify their possible implication in the development of thrombotic events in such patients

PATIENTS AND METHODS
The study was conducted from March 2013 to December 2013.It included a group of 50 patients with type 2 diabetes mellitus attending Diabetic Clinic in Al-Salam Teaching Hospital.They were 9 males and 41 females.Their ages range between 40 and 64 years with a mean age of 51.72± 9.26 years.These patients were of different duration of disease.Criteria for exclusion were co-existent autoimmune disorders.Vascular complications were assessed by the presence or absence of retinopathy, nephropathy and polyneuropathy.
The comparison group was included 33age and gender-matched healthy people.They were 6 males and 27 females.They have age range of 36-58 years and a mean age of 47.30±11.16years.
All sera of both patients and comparison groups were tested for the presence of anticardiolipin IgM and IgG isotypes and for β2GP1 IgG using ELISA technique.The titer of aCL was measured in MPL and GPL international units.Positivity of aCL was defined as a titer of IgM aCL higher than 15 MPL and a titer of IgG aCL higher than 10 GPL.A titer higher than 20 SGU was considered positive for β2GP1 IgG according to the recommendation of the kitsꞌs manufacturers (IMMUNOSPEC Corporation).

Statistical Analysis
Standard statistical methods were used to estimate the mean and standard deviation.Paired t-test and two sample t-test were used for comparing the results of various parameters among the studied groups.
The clinical characteristics of patients and controls are shown in Table 1.
As shown in Table 2, only one diabetic patient (2%) had a moderately elevated titer of IgM aCL, while 6 patients (12%) had low positive titers of IgM aCL.Six patients (12%) had low positive titers of IgG aCL (Table 2).
Two patients (4%) had highly positive titers of IgG β2GP1 and 1 patient (2%) had moderately elevated titer of IgG β2GP1 antibodies while the low positive titer of IgG β2GP I antibodies was found in 1 patient (2%)Tables 3, 4 and 5 showed the presence of aCL of IgM and IgG types with IgG β2GP1 antibodies among type 2 DM patients was statistically significant (p<0.0001).
Tables 6, 7 and 8 demonstrated the statistically significant higher levels of titers of IgM aCL titer, IgG aCL titer and IgG β2GP1antibodies among type 2 DM patients in correlation to the nondiabetic group.

DISCUSSION
This study showed that the presence of moderate to high aCL antibody titers and β 2 GP I antibodies in patients with type 2 DM was infrequent.Similar studies such as that of Tarkun et al found that no case had an IgG aCL titer more than 20 GPL units 12 .Palomo et al showed that only four out of 100 patients had moderate level of aCL 13 , the same findings were detected by Calvo-Romero who found low aCL titers in diabetic patients 14 .On the contrary, Galtier et al demonstrated that an IgG aCL titer more than 15 GPL Units was found in 9.5% of cases 11 .Our findings of frequent low positive titers of aCL antibodies could suggest that these autoantibodies may be blameless in the pathogenesis of diabetic atherosclerosis.
Our patients were mostly of old age, and the frequency of low aCL titers might be explained by the immunosenescence theory which suggest that immune dysfunction with aging can lead to increase autoantibody production 15 .This is in accordance with the study by Fields et al, whereby IgG and IgM aCL were detected in 12% of the healthy elderly and in 2% of younger adults 16 .
On the contrary, another study showed that aCL positive results in the elderly were reported to be insignificant and similar to younger populations 17 .It was found that the presence of β 2GP1 is a crucial requirement for antibody-phospholipid interaction, indicating that bound β 2GP1 forms the antigen to which aPL antibodies are directed.However the frequency and pathogenic role of these antibodies in atherosclerosis have been a matter of discussion 18 .
Although there was significant differences in the titers of IgM, IgG aCL and IgG β 2GP1 antibodies in the diabetic group compared to the comparison group, it seemed to be of no clinical importance as the mean titers of both of them is considered relatively low.
Low titers of aCL and β2 GP1 antibodies were seen in both complicated and non-complicated diabetic patients.These results, although limited by the small sample, do not favor a pathogenic effect of aCL and β2GPI antibodies in type 2 diabetic macrovasculopathy.Our data are established by those reported by Tarkun et al., which recede aCL and β2 GP1 antibodies from vascular complications of type 2 diabetes 12 , and also by Copetti who concluded that anticardiolipin antibodies do not mediate macrovascular complication of type 2 diabetes and that aCL positivity rates were similar in diabetic patients with and without vasculopathy 10 .

CONCLUSION
1.The aCL and β2-glycoprotein-I antibodies positive titer means among type 2 diabetics are significantly higher than the non-diabetics.2.Positive but low titers of aCL and β2 GPI antibodies could suggest that these autoantibodies may play a minor role in the pathogenesis of atherosclerosis.3.Low titers of aCL and β2 GP1 antibodies were seen in both complicated and non-complicated diabetic populations that probably lessen the importance of these autoantibodies as effective contributors in the pathogenesis of diabetic vasculopathy.

RECOMMENDATIONS
Prospective studies of large populations with follow up for complications are essential to reveal this association further.
Inclusion of IgA aCL in the future studies to be tested alongside the currently studied antibodies has to be considered.

F
-Test was used for equal variance estimation when appropriate.Some values expressed as Mean±SD and p value of <0.05 was considered statistically significant.The statistical tests were conducted by usage of SPSS version 19 and MedCalc version 13.1.

Table 1 :
Patients and non-diabetic group haracteristics.

Table 2 :
Distribution of Anticariolipin and Anti-β 2 Glycoprotein 1 antibodies in diabetic group.