Diabetes Mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. It is a serious illness with multiple complications and premature mortality, accounting for at least 10% of total health expenditure in many countries (1,2). The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of kidneys, nerves, blood vessels resulting in foot ulcers (1).
Diabetic foot ulcers and lower-limb amputation account for morbidity, mortality and healthcare expenditures among diabetic patients and will occur in 5-10% of diabetic population and, up to 3% will have a lower extremity amputation(3). Ulceration is the most common precursor to amputation and is identified in more than two-thirds of lower limb amputations (4). Diabetic foot ulcer development involves several mechanisms, such as neuropathy, increased biomechanical stress, external trauma and peripheral artery disease (5).
Homocysteine is a non-protein amino acid containing sulphur and is an intermediate in the metabolism of methionine (6). In 1969, Mc Cully first described the association of homocysteine with vascular disease (7). In the follow-up period several studies showed that homocysteine levels are associated with wound healing, endothelial dysfunction, insulin resistance, prothrombotic states, macroangiopathy, nephropathy and neuropathy (6,8-10).
In this study, we evaluated the relationship of homocysteine with the development of diabetic foot ulcer.
Materials and Methods
Between October 2007 and May 2008, 51 patients with diabetic foot ulcers, 35 diabetic patients without foot ulcers and 60 healthy controls were included in this study. Patients were recruited from visitors to an outpatient clinic. Group sample sizes of 35 and 51 achieve 98% post-hoc power for homocysteine.
Exclusion criteria were: presence of Type 1 diabetes mellitus, serious hepatic disease, cardiac or renal failure, diseases with acute inflammation, malignancy, thyroid disorders and psychiatric disorders. In addition, medications (metformin, fenofibrate) and/or vitamin supplements (vitamin B6, 12 and folic acid) were not allowed, so that homocysteine values could not be influenced. Local Ethics Committee approved the study protocol. Informed consent was obtained from each participant.
Blood samples were taken in the morning after an overnight fast. Serum total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, uric acid, creatinine, vitamin B12, folic acid, thyroid stimulating hormone, glycolized hemoglobin, high sensitivity C-reactive protein, erythrocyte sedimentation rate, complete blood count, microalbuminuria, and homocysteine levels were measured. Biochemical parameters were studied using a Roche/Hitachi Modular autoanalyzer, complete blood count was studied using a Roche Symex autoanalyzer.
Blood samples were drawn into tubes containing tripotassium EDTA. Plasma was separated and frozen at -80 0C until assay. Plasma homocysteine levels were measured by florescence polarization method in Abbott AxSYM system using AxSYM Homocysteine reagent (Abbott, Wiesbaden, Germany).
Foot ulcer is defined as full-thickness skin defect that do not heal within 14 days (11). Wagner classification was used for diabetic foot ulcers. Infection was defined as at least two of the following: purulent discharge, local heat increment, local erythema, lymphangitis, edema, fever and bad odour (4,5).
Retinopathy was diagnosed by ophthalmologic evaluation (12).
Diabetic nephropathy (microalbuminuria) was diagnosed when albumin excretion rate (AER), measured by radioimmunoassay (RIA), was 30-300 mg/24-hours in at least two out of three 24-hours urine collections over a three-month period. Creatinine clearance was calculated by Cockroft-Gault formula (GFR) [GFR=(140-age) x weight (kg)/ Plasma creatinine x 72 (in women x 0,85)](13).
Macrovascular disease was defined as presence of ischemic heart disease, stroke, transient ischemic attack or peripheral artery disease (4,12). Peripheral artery disease was diagnosed by absence of both feet pulses and/or ankle-brachial pressure index (ABPI) lower than 0,90 (4,5,11,14). ABPI measurement was made using a hand-held Doppler ultrasound (Hadeco ES-101EX, 8 Mhz).
Peripheral sensory neuropathy was defined as insensitivity to the 5.07 (10-g) Semmes-Weinstein monofilament at any one of ten sites on either foot (dorsal midfoot, plantar aspect of foot including pulp of the first, third, and fifth digits, the first, third and fifth metatarsal heads, the medial and lateral midfoot and the calcaneus). No other methods were employed in order to diagnose patients with peripheral diabetic neuropathy.
Statistical analysis was made by using SPSS 11.0 for Windows. Pearson’s Chi-Square test was used for categorical data and the Mann-Whitney U test was used for assessing numeric data. Logistic multiple regression analysis was used for evaluating peripheral neuropathy and serum homocysteine values as independent risk factors for diabetic foot ulcer.
A total of 86 patients and 60 controls were enrolled in the study. There were 25 females (71.4%), 10 males (28.6%) in the diabetic group without foot ulcer; 26 males (51 %), 25 females (49%) in the diabetic group with foot ulcers; 29 females (48.3%), 31 males (51.7%) in the control group.
The median age was 60.0±10.5 years (Range: 38-85 years) in the diabetic group with foot ulcer, 57.8±9.6 years (range: 41-74 years) in the diabetic group without foot ulcer and 55.6±6.8 years (range: 40-70 years) in the control group (p=0.062). No significant difference was observed between the groups in terms of age.
In the diabetic group with foot ulcers, 14 patients (27.5%) had grade 1 ulcer, 14 (27.5%) - grade 2, 9 (17.6%) - grade 3, 11 (21.6%) - grade 4 and 3 patients (5.9%) had grade 5 ulcer.
Anthropometric and clinical characteristics of the diabetic patients are shown in Table 1, laboratory parameters are shown in Table 2. Significant differences were seen in diabetes duration, retinopathy, neuropathy, macrovascular disease and infection between the patient groups. (Table 1). HbA1c, HDL-cholesterol, Folic Acid, Vitamin B12, CRP and ESR were significantly different between patient groups ( Table 2).
The median plasma homocysteine levels were 10.7 (7/22) μmol/l in the control group, 13.38(7/23.6) μmol/l in the diabetic group without foot ulcers and 16.27 (8.1/31.8) μmol/l in the diabetic group with foot ulcers and were significantly different (p<0.001, Figure 1). There was no difference in homocysteine levels according to diabetic foot stages in the diabetic foot group (Table 3).
Regression analyses showed that homocysteine levels and neuropathy were (b=0.171 and b=2.357, respectively) related and were independent risk factors (p=0.013 and p=0.008 respectively) for the development of diabetic foot ulcer. Patients who had hyperhomocysteinemia (OR:1.186; 95% CI:1.0-1.3) and neuropathy (OR:10.558; 95% CI:1.8-60.7) were likely to develop diabetic foot ulcers (Table 4).
Serum homocysteine differences between the groups according to gender and folic acid values were adjusted and covariance analysis was used. The effect of homocysteine levels on diabetic foot development was found to be rather meaningful (p= 0.004).
Diabetic foot ulceration is one of the serious complications of diabetes causing major extremity amputations with considerable economic and public health implications (15,16).
In the development of diabetic foot ulceration, diabetic neuropathy and peripheral artery disease are the major risk factors where as microvascular disease, biomechanical abnormalities, limited joint movements, and infection are the minor ones. Diabetic foot ulceration is seen frequently in the sixth decade of life. Clinically, 45-60% of ulcerations are pure neuropathic, 10% are pure ischemic and 25-45% are neuroischemic (15).
Diabetic peripheral neuropathy is observed in 55-70% of diabetic patients and 90% of patients with diabetic foot ulcers. Similarly, peripheral neuropathy incidence was 62.9% in diabetic patients and 96.1% in diabetic foot ulcer group in our study (p<0.001). In a diabetic patient with neuropathy, foot ulceration development is more likely (OR: 10.5 ; CI:95%, 1.8-60.7) to develop. This suggests a strong relationship of neuropathy with foot ulceration.
The other major risk factor is peripheral artery disease and accompanies 60% of nonhealing diabetic foot ulcers (17). We found that macrovascular disease is seen in 62.7% of diabetic patients with foot ulcers and in 40% of patients with diabetes. This difference between the groups was significant (p=0.038). There was no significant difference between macrovascular disease and diabetic foot ulcer development with regression analysis which can be due to insufficient number of patients (p=0.493).
In the literature, infection is observed in 35-50 % of diabetic foot ulcers which was 49% in our study (5).
The median age was 60.0±10.5 years (Range: 38-85 years) in the diabetic patients with foot ulcers which is consistent with other studies. This result showed that, although no significant difference was seen between the groups, diabetic foot ulcers were seen in the elderly patients. Male gender (51%) is related with diabetic foot ulcers, but no significant relationship was observed in our study. In the diabetic foot ulcer group, retinopathy, neuropathy and macrovascular disease were seen more frequently than in the diabetic group (p<0.05). This may be due to the long duration of diabetes years (mean:13 years, range:1-30 years versus mean:9 years, range: 2-25, [p=0.048]) in the diabetic foot ulcer group.
It is well known that, increased plasma homocysteine level is a risk factor for cardiovascular disease (18,19,20,21-23). We designed this study to investigate the relationship of homocysteine levels with chronic complications of diabetes.
Diverse results are seen between homocysteine levels and diabetes (20). Elevated homocysteine levels are seen in patients with nephropathy, neuropathy, macrovascular diseases, and retinopathy in some studies (8,18,20,22,23). We found elevated homocysteine levels in the diabetic patients without foot ulcers and diabetic foot ulcer group compared to to controls [13.38 (7/23.6) μmol/l, 16.27 (8.1/31.8) μmol/l, 10.7 (7/22) μmol/l, respectively (p<0.05)]. Similarly with the literature, this significance was also observed in the diabetic foot ulcer patients which chronic complications are seen more frequently (p<0.05).
Some studies showed that elevated homocysteine levels are related to poor ulcer healing (9,10). Majors et al. (10) reported that homocysteine may alter normal thrombosis and delay or interfere with wound healing by impairing the interaction of fibronectin with fibrin.
Boykin et al. (9) reported a diabetic patient with intractable bilateral venous ulcerations, who had an elevated homocysteine level, which was healed after multivitamin therapy.
Similarly, we found that homocysteine levels were elevated in diabetic foot ulcer group. Also hyperhomocysteinemia had a linear relation with ulcer formation in the diabetic foot ulcer group. In a diabetic patient with hyperhomocysteinemia, foot ulceration development is more likely (OR:10.5; CI:95%, 1.8-60.7) to develop. Although not as strong as neuropathy, hyperhomocysteinemia could be related to the development of diabetic foot ulcer. Serum homocysteine differences between the groups according to gender and folic acid values were adjusted and covariance analysis was used. The effect of homocysteine levels on the development of diabetic foot was found to be rather meaningful (p= 0.004).
Hemoglobin A1c (HBA1c) is one of the most important risk factors for the development of complications. In our study, the HBA1c levels in the non-ulcer patients were statistically higher than the ulcer patients. Lower HbA1c levels in patients with foot ulcer were due to the intensive therapy in this group, which was regarded as a temporary characteristic of our study group. On the other hand, the homocystein effect could have been far more meaningful if both groups had similar HbA1c levels. This situation was a flaw of our study.
Hyperhomocysteinemia is not uncommon in diabetic patients and it can aggravate cardiovascular diseases. The mechanism of this increased hyperhomocysteinemia prevalence is vague but it is suggested that, insulin plays a role in the regulation of plasma homocysteine and, insulin resistance causes hyperhomocysteinemia (18). Besides, the mechanisms that cause peripheral and/or autonomic neuropathy are complex and are not yet fully understood. Hypothetically, homocysteine can also contribute to the neuropathy development through neurovascular disruption or through direct toxic effect (8). In some studies in this field, it has been shown that there could be a link between hyperhomocysteinemia and autonomic or peripheral neuropathy (8,24). According to recent data, hyperhomocysteinemia can be a significant risk factor for cardiovascular diseases in the diabetic population (18,25). The relationship between hyperhomocysteinemia and cardiovascular diseases in patients with type 2 diabetics is 1.6 times stronger than in non-diabetic patients (18). Another important factor is the link between nitric oxide (NO) and homocysteine. Nitric oxide (NO) is a free radical gas which has a critical role in wound healing. Homocysteine hinders the NO production in various ways, such as the inhibition of arginine transport, the inhibition of the destruction of NO inhibitor asymmetric dimethylarginine (ADMA), and pro-oxidant behaviours (9). We think that, all the above mentioned mechanisms, combined with the presence of hyperhomocysteinemia, cause the development of foot ulcer in diabetic patients.
As a result, serum homocysteine values are associated with diabetic foot ulcer. It is safe and simple to measure homocysteine levels. In the cases of hyperhomocysteinemia, a cheap treatment could lower the homocysteine levels in most patients. Therefore, in practice, homocysteine levels as well as other efficient parameters (metabolic control such as HBA1c) should be measured routinely before the development of diabetic foot ulcer. We think it will be cost-effective to measure homocysteine levels. However, further studies are needed on this subject.
Address for Correspondence: Mehmet Erdogan MD, Ege University Medical School, Department of Endocrinology and Metabolism Disease, Bornova, Izmir, Turkey
Phone: +90 232 390 35 24 Fax: +90 232 373 77 01 E-mail: email@example.com Recevied: 24.01.2011 Accepted: 19.08.2011
1. Diagnosis and Classification of Diabetes Mellitus. American Diabetes Association. Diabetes Care 2006;29:43-8.
2. Roglic G, Unwin N, Bennett PH, et al. The Burden of Mortality Attributable to Diabetes: realistic estimates for the year 2000. Diabetes Care 2005;28:2130-5.
3. Boyko EJ, Ahroni JH, Stensen V, et al. A Prospective Study of Risk Factors for Diabetic Foot Ulcer. The Seattle Diabetic Foot Study. Diabetes Care 1999;22:1036-42.
4. Oyibo SO, Jude EB, Tarawneh I, et al. A Comparison of Two Diabetic Foot Ulcer Classification Systems: The Wagner and The University of Texas Wound Classification Systems. Diabetes Care 2001;24:84-8.
5. Prompers L, Huijberts M, Apelqvist J, et al. High Prevalence of Ischemia, Infection and Serious Comorbidity in Patients with Diabetic Foot Disease in Europe. Diabetologia 2007;50:18-25.
6. Ndrepepa G, Kastrati A, Braun S, et al. Circulating Homocysteine Levels in Patients with Type 2 Diabetes Mellitus. Nutr Metab Cardiovasc Dis 2008;18:66-73.
7. Schini-Kerth VB. Homocysteine, A Proinflammatory and Proatherosclerotic Factor: Role of Intracellular Reactive Oxygen Species. Circ Res 2003;93:271-3.
8. Ambrosch A, Dierkes J, Lobmann R, et al. Relation Between Hyperhomocysteinemia and Diabetic Neuropathy in Patients with Type 2 Diabetes Mellitus. Diabet Med 2001;18:185-92.
9. Boykin JV Jr, Baylis C, Allen SK, et al. Treatment of Elevated Homocysteine to Restore Normal Wound Healing: A Possible Relationship Between Homocysteine, Nitric Oxide, and Wound Repair. Adv Skin Wound Care 2005;18:297-300.
10. Majors AK, sengupta S, Willard B, et al. Homocysteine Binds to Human Plasma Fibronectin and Inhibits Its Interaction with Fibrin. Arterioscler Thromb Vasc Biol 2002;22:1354-9.
11. Adler AI, Boyko EJ, Ahroni JH, Smith DG. Lower-Extremity Amputation in Diabetes. The Independent Effects of Peripheral Vascular Disease, Sensory Neuropathy, and Foot Ulcers. Diabetes Care 1999;22:1029-35.
12. Tan KC, Chow WS, Ai VH, et al. Hyperhomocysteinemia and Impaired vasomotor Function in Type 2 Diabetes Mellitus. Eur J Clin Invest 2002;32:328-34.
13. Buysschaert M, Jamart J, Dramais AS, Wallemacq P, Hermans MP. Micro- and Macrovascular Complications and Hyperhomocysteinemia in Type 1 Diabetic Patients. Diabets Metab 2001;27:655-9.
14. Kravitz SR, McGuire J, Shanahan SD. Physical Assessment of The Diabetic Foot. Adv Skin Wound Care 2003;16:68-75.
15. Rathur HM, Boulton AJM. The Diabetic Foot. Clin Dermatol 2007;25:109-20.
16 Boulton AJ, Meneses P, Ennis WJ. Diabetic Foot Ulcers: A Framework for Prevention and Care. Wound Repair Regen 1999;7:7-16.
17. Gibbons GW. The Diabetic Foot. Becker KL. Principles and Practice of Endocrinology and Metabolism, 3. Ed, Philadelphia: A Walter Kluwer Company, 2001;1435-8.
18. Fonseca V, Guba SC, Fink LM. Hyperhomocysteinemia and Endocrine System: Implications for Atherosclerosis and Thrombosis. Endocr Rev 1999;20:738-59.
19. Perry DP. Hyperhomocysteinemia. Bailliere’s Clinical Haematology 1999;12:451-77.
20. Elias AL, Eng S. Homocysteine Concentrations in Patients with Diabetes Mellitus-relations to Microvascular and Macrovaskular Disease. Diabetes Obes Metab 2005;7:117-21.
21. de Luis DA, Fernandez N, Arranz ML, et al. Total Homocysteine Levels Relation with Chronic Complications of Diabetes, Body Composition, and Other Cardiovascular Risk Factors in A Population of Patients with Diabetes Mellitus Type 2. J Diabetes Complications 2005;19:42-6.
22. Agullo-Ortuno MT, Albaladejo MD, Parra S, et al. Plasmatic Homocysteine Concentration and Its Relationship Associated to Diabetes Mellitus. Clin Chim Acta 2002;326:105-12.
23. van Guldener C, Stehouwer CD. Diabetes Mellitus and Hyperhomocysteinemia. Semin Vasc Med 2002;2:87-95.
24. Smulders YM, Rakic M, Slaats EH, et al. Fasting and post-methionine homocysteine levels in NIDDM. Determinants and correlations with retinopathy, albuminuria, and cardiovascular disease. Diabetes Care 1999;22:125-32.
25. Huijberts MS, Becker A, Stehouwer CD. Homocysteine and Cardiovascular Disease in Diabetes: A Double Hit? Clin Chem Lab Med 2005;45:993-1000.