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Anticardiolipin antibody positivity in diabetic patients with and without diabetic foot
Journal of Diabetes and Its Complications 16 (2002) 172 – 175
Anticardiolipin antibody positivity in diabetic patients with and without diabetic foot Hakan Alago¨zlu¨a,*, Zahir Bakıcıb, Fu¨sun Gu¨ltekinc, Birdal Yıldırıma, Hafize Sezerd a
Department of Emergency Medicine, Division of Internal Medicine, Faculty of Medicine, Cumhuriyet University, 58140, Sivas, Turkey b Department of Microbiology, Faculty of Medicine, Cumhuriyet University, 58140, Sivas, Turkey c Department of Internal Medicine, Faculty of Medicine, Cumhuriyet University, 58140, Sivas, Turkey d Department of Medical Biostatistics, Faculty of Medicine, Cumhuriyet University, 58140, Sivas, Turkey Received 28 March 2001; received in revised form 22 May 2001; accepted 31 May 2001
Abstract Anticardiolipin (aCL) antibodies may play a role in the enhancement of platelet aggregation and/or progression of the macrovascular diabetic complications. Also, aCL antibodies may cause or promote ischemia and thrombosis. Therefore, we aimed to investigate IgG aCL and IgM aCL antibodies positivity in type 2 diabetic patients with and without ischemic diabetic foot. In this case-control study, we examined 40 diabetic patients without diabetic foot problem and 35 diabetic patients with ischemic diabetic foot. Forty diabetic patients (19 females, 21 males) without diabetic foot served as Group 1 and 35 diabetic patients (17 females, 18 males) who had ischemic diabetic foot served as Group 2. In the control group, 35 nondiabetic healthy subjects (18 females, 17 males) were included in Group 3. The groups were similar in age and sex, which is not statistically significant ( P > .05). There was no difference in the IgG aCL antibodies positivity between Groups 1 and 3 ( P > .05). However, IgG aCL antibodies positivity in Group 2 was significantly higher than those of the other groups ( P < .05). IgG aCL antibodies were found positive in 10% (4/40) of Group 1, 34.3% (12/35) of Group 2 and 8.6% (3/35) of Group 3. When Groups 1 and 2 were compared, the odds ratio adjusted for age, gender, hypertension, coronary artery disease history, cigarette smoking, duration of diabetes mellitus, cholesterol, and haemoglobin A1C (HbA1c) was 6.8 [95% confidence interval (CI), 1.41 – 32.66; P = .016] for aCL positivity. In conclusion, although available evidence does not prove a causal association between positivity of aCL and diabetic foot, we believe that a causal association is supported by the data obtained from this study. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Diabetic foot; Anticardiolipin antibody
1. Introduction Anticardiolipin (aCL) antibodies are part of a heterogeneous family of antibodies directed against phospholipids. aCL antibodies occur in patients with systemic lupus erythematosus (SLE) or lupus-like illnesses, other chronic illnesses (especially neoplastic diseases involving immunoglobulin producing cells), infectious diseases, drug-induced lupus syndromes, and with advanced age. Antibody levels do not change rapidly over time; serial measurement is therefore not generally useful. Antibodies associated with infections, drugs, or neoplasia tend to have IgM more than
* Corresponding author. Tel.: +90-346-2191010/2661; fax: +90-3462191284. E-mail address: [email protected] (H. Alago¨zlu¨).
IgG isotype. aCL antibodies are also associated with the antiphospholipid syndrome, which is characterized by venous and arterial thrombosis, thrombocytopenia, cerebral vascular accidents, and recurrent fetal miscarriage (Hughes, 1993; Khamashta & Hughes, 1995; Lockshin, 1994; Martini & Ravelli, 1997). There are several mechanisms whereby aCL antibodies may cause or promote ischemia and thrombosis in general, including functional alterations of protein C (Cariou, Tobelem, & Belluci, 1988; Freyssinet & Cazenave, 1987), fibrinolysis (Tsakaris, Marbet, Makris, Settas, & Duckert, 1989), antithrombin III (Cosgriff & Martin, 1981), prostacyclin (PGI2) activity (Carreras & Vermylen, 1982), platelet aggregability (Khamashta et al., 1988), and complement activation (Davis & Brey, 1992). Diabetes mellitus is associated with vascular and neurological complications. Diabetic foot is one of the most important of the complications. Diabetic foot and their
1056-8727/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 5 6 - 8 7 2 7 ( 0 1 ) 0 0 1 6 6 - 0
H. Alago¨zlu¨ et al. / Journal of Diabetes and Its Complications 16 (2002) 172–175
direct sequelae are one of the most common causes of admission to emergency service among diabetic patients (Foster & Edmonds, 1987). aCL antibodies may play a role in the enhancement of platelet aggregation and/or progression of the macrovascular diabetic complications. Therefore, we aimed to investigate aCL antibodies in type 2 diabetic patients with and without diabetic foot.
2. Material and methods In this case-control study, 40 type 2 diabetic patients without diabetic foot problem and 42 type 2 diabetic patients with diabetic foot problem who were admitted Division of Internal Medicine of Emergency Department in Cumhuriyet University Hospital were evaluated. Forty diabetic patients (19 females, 21 males) without diabetic foot served Group 1. The feet were carefully assessed and classified according to Wagner’s (1981) classification. Seven patients had neuropathic foot and 35 patients had ischemic foot. We excluded seven diabetics with neuropathic foot from this study. Therefore, remaining 35 diabetic patients (17 females, 18 males) with diabetic foot problem were investigated and served as Group 2. All patients were formerly diagnosed as type II diabetics whose glycaemic control was judged to be unsatisfactory [haemoglobin A1C (HbA1c) level>7.5%] for at least 3 months despite oral hypoglycaemic or insulin treatment, so these patients were accepted poor glycaemic. The diabetic patients used drugs irregularly or were not under periodic control of an internist doctor. Thirty-two (42.6%) out of the total 75 total diabetic patients had been taking oral antidiabetics. Others had been taking human insulin. The patients were matched for age and sex with 35 healthy nondiabetic individuals (18 females, 17 males) who were
Table 1 Characteristics of diabetic and control subjects
No Female Male Age (years) Known duration of diabetes (years) Known duration of diabetic foot (days) Cigarette smoking [n (%)] Hypertension history [n (%)] Coronary artery disease history [n (%)] HbA1c (%) Cholesterol (mg/dL)
Group 1
Group 2
Group 3
40 19 21 60.42 ± 0.81 4.6 ± 0.35
35 17 18 60.22 ± 1.02 10.37 ± 0.7**
35 18 17 60.11 ± 0.82 –
–
92 ± 9
–
5 (12.5)
6 (17.1)
5 (14.3)
7 (17.5)
8 (22.8)
–
5 (12.5)
5 (14.3)
–
8.03 ± 0.07 249.62 ± 7.01
8.28 ± 0.12 265.57 ± 6.89
3.77 ± 0.05* 198.17 ± 5.98*
* P < .05 vs. Groups 1 and 2. ** P < .05 vs. Group 1.
173
Table 2 aCL positivity in diabetic and control subjects
aCL-IgG (+) aCL-IgM (+)
Group 1 (n = 40)
Group 2 (n = 35)
Group 3 (n = 35)
4 (10%) 2 (5%)
12 (34.3%)* 3 (8.6%)
3 (8.6%) 1 (2.8%)
Values are number (percentage). * P < .05 vs. Groups 1 and 3.
the control group serving as Group 3. All patients gave informed consent. Patients were also evaluated regarding their habits, duration of diabetes and diabetic foot, lipid profiles, treatments they received and history of hypertension, and coronary artery disease (defined as previous myocardial infarction, angina, or a coronary revascularization procedure). Blood for aCL antibody levels was drawn by clean venipuncture into a standard clot tube. Specimens were centrifuged within 4 h of the blood draw and the serum was immediately frozen and stored at 70C until the day of experiment. aCL antibodies were measured in the sera by an enzyme-linked immunosorbent assay (ELISA) technique (Biomaster EIA, CLI Cardiolipin ELISA kit, Clark Laboratories, Jamestown, NY, USA) according to the procedure described by Loizou et al. (1985). Measurement of blood glucose, serum creatinine, total cholesterol, and triglyceride was done colorimetrically with an ILAB 1800 autoanalyzer. HbA1c was assayed in all the groups by turbidimetry (ILAB 1800). Cardiac status was determined by history, physical examination, and electrocardiography in all subjects. c2 analysis was used for comparison of categorical variables, and the student t test was used for comparison of continuous variables. The odds ratio and its 95% confidence interval (CI) were calculated using logistic regression with adjustment for the matched attributes (age and sex) as well as the other established risk factors (hypertension, coronary artery disease, cholesterol, cigarette smoking, and HbA1c). The results are expressed as mean ± S.E. Differences with P < .05 were considered significant. In Tables 1 and 2, ‘‘no’’ demonstrates the number of subjects and ‘‘mean’’ stands for the arithmetic mean; ‘‘S.E.,’’ standard error.
3. Results The groups were similar in age and sex, which is not statistically significant ( P > .05). Additionally, there were no statistical differences in terms of cigarette smoking, history of hypertension, and coronary artery disease in the three groups ( P > .05; Table 1). We have found the following results. There was no difference in the IgG aCL antibodies positivity between Groups 1 and 3 ( P > .05). However, IgG aCL antibodies positivity in Group 2 were significantly higher than those of the other groups ( P < .05). There was no difference in the
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IgM aCL antibodies positivity among all groups ( P > .05). IgG aCL antibodies were found positive in 10% (4/40) of Group 1, 34.3% (12/35) of Group 2, and 8.6% (3/35) of Group 3 (Table 2). When Groups 1 and 2 were compared with regard to aCL positivity, nonadjusted univariate odds ratio was 4.69 (95% CI, 1.35– 16.33; P = .01). The odds ratio adjusted for age, gender, hypertension, coronary artery disease history, cigarette smoking, duration of diabetes mellitus, cholesterol, and HbA1c was 6.8 (95% CI, 1.41 – 32.66; P = .016) for aCL positivity.
4. Discussion Antiphospholipid antibodies (aPL) include aCL antibody, glycoprotein I cardiolipin antibody (anti-b2 GPI CL antibody), lupus anticoagulant, and antiprothrombin antibodies (Hughes, 1993; Khamashta & Hughes, 1995; Lockshin, 1994). Several reports have focused on a new clinical entity characterized by the presence of antiphospholipid and by a tendency to venous and arterial thrombosis (de Godoy, de Godoy, Braile, & Torres, 2000; Eldrup-Jorgenson et al., 1989; Munakata et al., 2000; Nityanand et al., 1995). In addition to their thrombogenic effects, antiphospholipid antibodies may play a role in the impairment of the thromboresistant property of vascular endothelium and in the enhancement of platelet aggregation leading to the pathogenesis and/or progression of the diabetic macrovascular complications (Galtier-Dereure et al., 1998; Hendra et al., 1989; Triolo, Giardina, Scarantino, Seddio, & Bompiani, 1989). Diabetic foot is the most frequent cause of macrovascular complications and hospitalisation in diabetic patients. Its dramatic consequences may result in amputation. Ulcers in diabetics with neuropathy, but no vasculopathy, will heal if properly treated, whereas the risk of amputation arises in the presence of a vascular disease and increases in function of its severity (Apelqvist, Larsson, & Agardh, 1990, 1992). Antiphospholipid antibodies interfere with the normal function of blood vessels, both by causing narrowing and irregularity of the vessel (called ‘‘vasculopathy’’) and by causing clots in the vessel (called ‘‘thrombosis’’). These blood vessel problems can then lead to complications such as thromboembolic complications and miscarriage (Hughes, 1993; Khamashta & Hughes, 1995; Lockshin, 1994; Martini & Ravelli, 1997). These associations suggest that these antibodies, directed against negatively charged phospholipids, play a role in the pathogenesis of thromboembolic complications. One of the proposed mechanisms of action of these antibodies is an inhibition of the vascular PGI2 synthesis, possibly by interfering with the liberation of arachidonic acid from the membrane phospholipids. PGI2 is a strong vasodilator and is the most potent endogenous inhibitor of platelet aggregation known (Carreras & Vermylen, 1982; Esmon, Smirnov, & Esmon, 1997). On the other hand, the major platelet arachidonate metabolite, thrombox-
ane A2 (TxA2), has the opposite activity. The balance between synthesis of PGI2 and TxA2 has been proposed to be a very important regulator of haemostasis and vascular tone (Bunting, Moncada, & Vane, 1983). It has been speculated that aCL antibodies may activate platelets by binding to phospholipids in the platelet membrane, causing aggregation, thrombosis, and thrombocytopenia (Harris et al., 1985). In addition to an effect on platelets or through prostanoid production, aCL may also influence haemostasis by an interaction with endothelial thrombomodulin resulting in reduced synthesis of protein C (Cariou, Tobelem, Soria, & Caen, 1986). Also, an association between anti-b2-GPI antibodies and thrombosis has been reported (Munakata et al., 2000; Tanne, Triplett, & Levine, 1998). We did not measure anti-b2-GPI CL antibodies or assess the role of b2-GPI in aCL binding to cardiolipin. The significance of b2-GPI remains controversial, and a better understanding of its role will help to clarify the relationship between aCL and thrombogenicity. When aCL-associated thrombosis occurs in the arterial circulation, we can say that the foot can be the most often affected in type 2 diabetic patients according to our study. No previous study reported a significant association between aCL antibodies and diabetic foot in literature. In conclusion, although available evidence does not prove a causal association between positivity of aCL and diabetic foot, we believe that a causal association is supported by the data obtained from this study. Additionally, we can recommend thromboprophylaxis in type 2 diabetic patients with diabetic foot and positive aCL antibodies. Thus, thromboprophylaxis can reduce the percentage of amputation in these patients.
References Apelqvist, J., Larsson, J., & Agardh, C. D. (1990). The importance of peripheral pulses, peripheral oedema and local pain for the outcome of diabetic foot ulcers. Diabetes and Medicine, 7, 590 – 594. Apelqvist, J., Larsson, J., & Agardh, C. D. (1992). Medical risk factors in diabetic patients with foot ulcers and severe peripheral vascular disease and their influence on outcome. Journal of Diabetes and its Complications, 6, 167 – 174. Bunting, S., Moncada, S., & Vane, J. R. (1983). The prostacyclin – thromboxane A2 balance: pathophysiological and therapeutic implications. British Medical Bulletin, 39, 271 – 276. Cariou, R., Tobelem, G., & Belluci, S. (1988). Effect of lupus anticoagulant on antithrombogenic properties of endothelial cells: inhibition of thrombomodulin-dependent protein C activation. Thrombosis and Haemostasis, 60, 54 – 58. Cariou, R., Tobelem, G., Soria, C., & Caen, J. (1986). Inhibition of protein C activation by endothelial cells in the presence of lupus anticoagulant. New England Journal of Medicine, 314, 1193 – 1194. Carreras, L. O., & Vermylen, J. G. (1982). ‘‘Lupus’’ anticoagulant and thrombosis — possible role of inhibition of prostacyclin formation. Thrombosis and Haemostasis, 48, 38 – 40. Cosgriff, T. M., & Martin, B. A. (1981). Low functional and high antigenic antithrombin III level in a patient with the lupus anticoagulant and recurrent thrombosis. Arthritis & Rheumatism, 24, 94 – 96. Davis, W. D., & Brey, R. L. (1992). Complement activation in stroke
H. Alago¨zlu¨ et al. / Journal of Diabetes and Its Complications 16 (2002) 172–175 associated with antiphospholipid antibodies. Clinical and Experimental Rheumatology, 10, 455 – 460. de Godoy, J. M., de Godoy, M. F., Braile, D. M., & Torres, C. A. (2000). Prevalence of anticardiolipin antibodies in peripheral arterial thrombosis. Angiology, 51, 473 – 477. Eldrup-Jorgenson, J., Brace, L., Flanigan, D. P., Schwarz, T. H., Fritsma, G., Meyer, J. P., & Schuler, J. J. (1989). Lupus-like anticoagulants and lower extremity arterial occlusive disease. Circulation, 80, 54 – 58. Esmon, N. L., Smirnov, M. D., & Esmon, C. T. (1997). Lupus anticoagulants and thrombosis: the role of phospholipids. Haematologica, 82, 474 – 477. Foster, A., & Edmonds, M. E. (1987, September). Diabetic foot emergencies: a strategy for their care. Presented at the British Diabetic Association medical and scientific section meeting, Liverpool, England. Freyssinet, J. M., & Cazenave, J. P. (1987). Lupus-like anticoagulants, modulation of the protein C pathway and thrombosis. Thrombosis and Haemostasis, 58, 679 – 681. Galtier-Dereure, F., Biron, C., Vies, M., Bourgeois, V., Schved, J. F., & Bringer, J. (1998). Vascular complications of diabetes mellitus: what role for phospholipid-binding antibodies? Lupus, 7, 469 – 474. Harris, E. N., Asherson, R. A., Gharavi, A. E., Morgan, S. H., Derue, G., & Hughes, G. R. V. (1985). Thrombocytopenia in SLE and related autoimmune disorders: association with anticardiolipin antibody. British Journal of Haematology, 59, 227 – 230. Hendra, T. J., Baguley, E., Harris, E. N., Khamashta, M. H., Trembath, R. C., Hughes, G. R. V., & Yudkin, J. S. (1989). Anticardiolipin antibody levels in diabetic subjects with and without coronary artery disease. Postgraduate Medical Journal, 65, 140 – 143. Hughes, G. R. V. (1993). The antiphospholipid syndrome: ten years on. Lancet, 342, 341 – 344. Khamashta, M. A., Harris, E. N., Gharavi, A. E., Derue, G., Gil, A., Vazquez, J. J., & Hughes, G. R. (1988). Immune mediated mechanism for thrombosis: antiphospholipid antibody binding to platelet membranes. Annals of the Rheumatic Diseases, 47, 849 – 854.
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Khamashta, M. A., & Hughes, G. R. V. (1995). Antiphospholipid antibodies and antiphospholipid syndrome. Current Opinion in Rheumatology, 7, 389 – 394. Lockshin, M. D. (1994). Antiphospholipid syndrome. Rheumatic Diseases Clinics of North America, 20, 45 – 59. Loizou, S., McCrea, J. D., Rudge, A. C., Reynolds, R., Boyle, C. C., & Harris, E. N. (1985). Measurement of anti-cardiolipin antibodies by an enzyme-linked immunosorbent assay (ELISA): standardisation and quantitation of results. Clinical and Experimental Immunology, 62, 738 – 745. Martini, A., & Ravelli, A. (1997). The clinical significance of antiphospholipid antibodies. Annals of Medicine, 29, 159 – 163. Munakata, Y., Saito, T., Matsuda, K., Seino, J., Shibata, S., & Sasaki, T. (2000). Detection of complement-fixing antiphospholipid antibodies in association with thrombosis. Thrombosis and Haemostasis, 83, 728 – 731. Nityanand, S., Bergmark, C., de Faire, U., Swedenborg, J., Holm, G., & Lefvert, A. K. (1995). Antibodies against endothelial cells and cardiolipin in young patients with peripheral atherosclerotic disease. Journal of Internal Medicine, 238, 437 – 443. Tanne, D., Triplett, D. A., & Levine, S. R. (1998). Antiphospholipid-protein antibodies and ischemic stroke: not just cardiolipin any more. Stroke, 29, 1755 – 1758. Triolo, G., Giardina, E., Scarantino, G., Seddio, G., & Bompiani, G. (1989). Detection of anti-phospholipid (cardiolipin, phosphatidylserine) antibodies in the serum of patients with non insulin-dependent (type 2) diabetes mellitus and macroangiopathy. Coexistence of anti-platelet reactivity. Diabetes Research, 10, 63 – 67. Tsakaris, D. A., Marbet, G. A., Makris, P. E., Settas, L., & Duckert, F. (1989). Impaired fibrinolysis as an essential contribution to thrombosis in patients with lupus anticoagulant. Thrombosis and Haemostasis, 61, 175 – 177. Wagner, F. W. (1981). The dysvascular foot: a system for diagnosis and treatment. Foot and Ankle, 2, 64 – 67.
Anticardiolipin antibody positivity in diabetic patients with and without diabetic foot Hakan Alago¨zlu¨a,*, Zahir Bakıcıb, Fu¨sun Gu¨ltekinc, Birdal Yıldırıma, Hafize Sezerd a
Department of Emergency Medicine, Division of Internal Medicine, Faculty of Medicine, Cumhuriyet University, 58140, Sivas, Turkey b Department of Microbiology, Faculty of Medicine, Cumhuriyet University, 58140, Sivas, Turkey c Department of Internal Medicine, Faculty of Medicine, Cumhuriyet University, 58140, Sivas, Turkey d Department of Medical Biostatistics, Faculty of Medicine, Cumhuriyet University, 58140, Sivas, Turkey Received 28 March 2001; received in revised form 22 May 2001; accepted 31 May 2001
Abstract Anticardiolipin (aCL) antibodies may play a role in the enhancement of platelet aggregation and/or progression of the macrovascular diabetic complications. Also, aCL antibodies may cause or promote ischemia and thrombosis. Therefore, we aimed to investigate IgG aCL and IgM aCL antibodies positivity in type 2 diabetic patients with and without ischemic diabetic foot. In this case-control study, we examined 40 diabetic patients without diabetic foot problem and 35 diabetic patients with ischemic diabetic foot. Forty diabetic patients (19 females, 21 males) without diabetic foot served as Group 1 and 35 diabetic patients (17 females, 18 males) who had ischemic diabetic foot served as Group 2. In the control group, 35 nondiabetic healthy subjects (18 females, 17 males) were included in Group 3. The groups were similar in age and sex, which is not statistically significant ( P > .05). There was no difference in the IgG aCL antibodies positivity between Groups 1 and 3 ( P > .05). However, IgG aCL antibodies positivity in Group 2 was significantly higher than those of the other groups ( P < .05). IgG aCL antibodies were found positive in 10% (4/40) of Group 1, 34.3% (12/35) of Group 2 and 8.6% (3/35) of Group 3. When Groups 1 and 2 were compared, the odds ratio adjusted for age, gender, hypertension, coronary artery disease history, cigarette smoking, duration of diabetes mellitus, cholesterol, and haemoglobin A1C (HbA1c) was 6.8 [95% confidence interval (CI), 1.41 – 32.66; P = .016] for aCL positivity. In conclusion, although available evidence does not prove a causal association between positivity of aCL and diabetic foot, we believe that a causal association is supported by the data obtained from this study. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Diabetic foot; Anticardiolipin antibody
1. Introduction Anticardiolipin (aCL) antibodies are part of a heterogeneous family of antibodies directed against phospholipids. aCL antibodies occur in patients with systemic lupus erythematosus (SLE) or lupus-like illnesses, other chronic illnesses (especially neoplastic diseases involving immunoglobulin producing cells), infectious diseases, drug-induced lupus syndromes, and with advanced age. Antibody levels do not change rapidly over time; serial measurement is therefore not generally useful. Antibodies associated with infections, drugs, or neoplasia tend to have IgM more than
* Corresponding author. Tel.: +90-346-2191010/2661; fax: +90-3462191284. E-mail address: [email protected] (H. Alago¨zlu¨).
IgG isotype. aCL antibodies are also associated with the antiphospholipid syndrome, which is characterized by venous and arterial thrombosis, thrombocytopenia, cerebral vascular accidents, and recurrent fetal miscarriage (Hughes, 1993; Khamashta & Hughes, 1995; Lockshin, 1994; Martini & Ravelli, 1997). There are several mechanisms whereby aCL antibodies may cause or promote ischemia and thrombosis in general, including functional alterations of protein C (Cariou, Tobelem, & Belluci, 1988; Freyssinet & Cazenave, 1987), fibrinolysis (Tsakaris, Marbet, Makris, Settas, & Duckert, 1989), antithrombin III (Cosgriff & Martin, 1981), prostacyclin (PGI2) activity (Carreras & Vermylen, 1982), platelet aggregability (Khamashta et al., 1988), and complement activation (Davis & Brey, 1992). Diabetes mellitus is associated with vascular and neurological complications. Diabetic foot is one of the most important of the complications. Diabetic foot and their
1056-8727/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 5 6 - 8 7 2 7 ( 0 1 ) 0 0 1 6 6 - 0
H. Alago¨zlu¨ et al. / Journal of Diabetes and Its Complications 16 (2002) 172–175
direct sequelae are one of the most common causes of admission to emergency service among diabetic patients (Foster & Edmonds, 1987). aCL antibodies may play a role in the enhancement of platelet aggregation and/or progression of the macrovascular diabetic complications. Therefore, we aimed to investigate aCL antibodies in type 2 diabetic patients with and without diabetic foot.
2. Material and methods In this case-control study, 40 type 2 diabetic patients without diabetic foot problem and 42 type 2 diabetic patients with diabetic foot problem who were admitted Division of Internal Medicine of Emergency Department in Cumhuriyet University Hospital were evaluated. Forty diabetic patients (19 females, 21 males) without diabetic foot served Group 1. The feet were carefully assessed and classified according to Wagner’s (1981) classification. Seven patients had neuropathic foot and 35 patients had ischemic foot. We excluded seven diabetics with neuropathic foot from this study. Therefore, remaining 35 diabetic patients (17 females, 18 males) with diabetic foot problem were investigated and served as Group 2. All patients were formerly diagnosed as type II diabetics whose glycaemic control was judged to be unsatisfactory [haemoglobin A1C (HbA1c) level>7.5%] for at least 3 months despite oral hypoglycaemic or insulin treatment, so these patients were accepted poor glycaemic. The diabetic patients used drugs irregularly or were not under periodic control of an internist doctor. Thirty-two (42.6%) out of the total 75 total diabetic patients had been taking oral antidiabetics. Others had been taking human insulin. The patients were matched for age and sex with 35 healthy nondiabetic individuals (18 females, 17 males) who were
Table 1 Characteristics of diabetic and control subjects
No Female Male Age (years) Known duration of diabetes (years) Known duration of diabetic foot (days) Cigarette smoking [n (%)] Hypertension history [n (%)] Coronary artery disease history [n (%)] HbA1c (%) Cholesterol (mg/dL)
Group 1
Group 2
Group 3
40 19 21 60.42 ± 0.81 4.6 ± 0.35
35 17 18 60.22 ± 1.02 10.37 ± 0.7**
35 18 17 60.11 ± 0.82 –
–
92 ± 9
–
5 (12.5)
6 (17.1)
5 (14.3)
7 (17.5)
8 (22.8)
–
5 (12.5)
5 (14.3)
–
8.03 ± 0.07 249.62 ± 7.01
8.28 ± 0.12 265.57 ± 6.89
3.77 ± 0.05* 198.17 ± 5.98*
* P < .05 vs. Groups 1 and 2. ** P < .05 vs. Group 1.
173
Table 2 aCL positivity in diabetic and control subjects
aCL-IgG (+) aCL-IgM (+)
Group 1 (n = 40)
Group 2 (n = 35)
Group 3 (n = 35)
4 (10%) 2 (5%)
12 (34.3%)* 3 (8.6%)
3 (8.6%) 1 (2.8%)
Values are number (percentage). * P < .05 vs. Groups 1 and 3.
the control group serving as Group 3. All patients gave informed consent. Patients were also evaluated regarding their habits, duration of diabetes and diabetic foot, lipid profiles, treatments they received and history of hypertension, and coronary artery disease (defined as previous myocardial infarction, angina, or a coronary revascularization procedure). Blood for aCL antibody levels was drawn by clean venipuncture into a standard clot tube. Specimens were centrifuged within 4 h of the blood draw and the serum was immediately frozen and stored at 70C until the day of experiment. aCL antibodies were measured in the sera by an enzyme-linked immunosorbent assay (ELISA) technique (Biomaster EIA, CLI Cardiolipin ELISA kit, Clark Laboratories, Jamestown, NY, USA) according to the procedure described by Loizou et al. (1985). Measurement of blood glucose, serum creatinine, total cholesterol, and triglyceride was done colorimetrically with an ILAB 1800 autoanalyzer. HbA1c was assayed in all the groups by turbidimetry (ILAB 1800). Cardiac status was determined by history, physical examination, and electrocardiography in all subjects. c2 analysis was used for comparison of categorical variables, and the student t test was used for comparison of continuous variables. The odds ratio and its 95% confidence interval (CI) were calculated using logistic regression with adjustment for the matched attributes (age and sex) as well as the other established risk factors (hypertension, coronary artery disease, cholesterol, cigarette smoking, and HbA1c). The results are expressed as mean ± S.E. Differences with P < .05 were considered significant. In Tables 1 and 2, ‘‘no’’ demonstrates the number of subjects and ‘‘mean’’ stands for the arithmetic mean; ‘‘S.E.,’’ standard error.
3. Results The groups were similar in age and sex, which is not statistically significant ( P > .05). Additionally, there were no statistical differences in terms of cigarette smoking, history of hypertension, and coronary artery disease in the three groups ( P > .05; Table 1). We have found the following results. There was no difference in the IgG aCL antibodies positivity between Groups 1 and 3 ( P > .05). However, IgG aCL antibodies positivity in Group 2 were significantly higher than those of the other groups ( P < .05). There was no difference in the
174
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IgM aCL antibodies positivity among all groups ( P > .05). IgG aCL antibodies were found positive in 10% (4/40) of Group 1, 34.3% (12/35) of Group 2, and 8.6% (3/35) of Group 3 (Table 2). When Groups 1 and 2 were compared with regard to aCL positivity, nonadjusted univariate odds ratio was 4.69 (95% CI, 1.35– 16.33; P = .01). The odds ratio adjusted for age, gender, hypertension, coronary artery disease history, cigarette smoking, duration of diabetes mellitus, cholesterol, and HbA1c was 6.8 (95% CI, 1.41 – 32.66; P = .016) for aCL positivity.
4. Discussion Antiphospholipid antibodies (aPL) include aCL antibody, glycoprotein I cardiolipin antibody (anti-b2 GPI CL antibody), lupus anticoagulant, and antiprothrombin antibodies (Hughes, 1993; Khamashta & Hughes, 1995; Lockshin, 1994). Several reports have focused on a new clinical entity characterized by the presence of antiphospholipid and by a tendency to venous and arterial thrombosis (de Godoy, de Godoy, Braile, & Torres, 2000; Eldrup-Jorgenson et al., 1989; Munakata et al., 2000; Nityanand et al., 1995). In addition to their thrombogenic effects, antiphospholipid antibodies may play a role in the impairment of the thromboresistant property of vascular endothelium and in the enhancement of platelet aggregation leading to the pathogenesis and/or progression of the diabetic macrovascular complications (Galtier-Dereure et al., 1998; Hendra et al., 1989; Triolo, Giardina, Scarantino, Seddio, & Bompiani, 1989). Diabetic foot is the most frequent cause of macrovascular complications and hospitalisation in diabetic patients. Its dramatic consequences may result in amputation. Ulcers in diabetics with neuropathy, but no vasculopathy, will heal if properly treated, whereas the risk of amputation arises in the presence of a vascular disease and increases in function of its severity (Apelqvist, Larsson, & Agardh, 1990, 1992). Antiphospholipid antibodies interfere with the normal function of blood vessels, both by causing narrowing and irregularity of the vessel (called ‘‘vasculopathy’’) and by causing clots in the vessel (called ‘‘thrombosis’’). These blood vessel problems can then lead to complications such as thromboembolic complications and miscarriage (Hughes, 1993; Khamashta & Hughes, 1995; Lockshin, 1994; Martini & Ravelli, 1997). These associations suggest that these antibodies, directed against negatively charged phospholipids, play a role in the pathogenesis of thromboembolic complications. One of the proposed mechanisms of action of these antibodies is an inhibition of the vascular PGI2 synthesis, possibly by interfering with the liberation of arachidonic acid from the membrane phospholipids. PGI2 is a strong vasodilator and is the most potent endogenous inhibitor of platelet aggregation known (Carreras & Vermylen, 1982; Esmon, Smirnov, & Esmon, 1997). On the other hand, the major platelet arachidonate metabolite, thrombox-
ane A2 (TxA2), has the opposite activity. The balance between synthesis of PGI2 and TxA2 has been proposed to be a very important regulator of haemostasis and vascular tone (Bunting, Moncada, & Vane, 1983). It has been speculated that aCL antibodies may activate platelets by binding to phospholipids in the platelet membrane, causing aggregation, thrombosis, and thrombocytopenia (Harris et al., 1985). In addition to an effect on platelets or through prostanoid production, aCL may also influence haemostasis by an interaction with endothelial thrombomodulin resulting in reduced synthesis of protein C (Cariou, Tobelem, Soria, & Caen, 1986). Also, an association between anti-b2-GPI antibodies and thrombosis has been reported (Munakata et al., 2000; Tanne, Triplett, & Levine, 1998). We did not measure anti-b2-GPI CL antibodies or assess the role of b2-GPI in aCL binding to cardiolipin. The significance of b2-GPI remains controversial, and a better understanding of its role will help to clarify the relationship between aCL and thrombogenicity. When aCL-associated thrombosis occurs in the arterial circulation, we can say that the foot can be the most often affected in type 2 diabetic patients according to our study. No previous study reported a significant association between aCL antibodies and diabetic foot in literature. In conclusion, although available evidence does not prove a causal association between positivity of aCL and diabetic foot, we believe that a causal association is supported by the data obtained from this study. Additionally, we can recommend thromboprophylaxis in type 2 diabetic patients with diabetic foot and positive aCL antibodies. Thus, thromboprophylaxis can reduce the percentage of amputation in these patients.
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