A prospective study on hyperhomocysteinemia as an aggravating factor in chronic venous insufficiency
- Authors:
- Published online on: May 17, 2022 https://doi.org/10.3892/etm.2022.11376
- Article Number: 449
Abstract
Introduction
Clinical manifestation of chronic venous insufficiency (CVI) in the lower limbs is complex and varies from edema, venectasia and varicosities to ulcerations. CVI has a progressive evolution with a well-known etiopathogeny, prognosis and therapy (1). CVI prevalence increases with age, is more common in elderly females and has certain contributing factors (pregnancy, prolonged standing, menopause and hormone therapy, among others) (2). The reported prevalence of CVI in Romania has a variable value (68.4% in 2015 and 25% in 2020) (3,4), which is similar to the incidence rate in the rest of Europe (20-60%) (2). Etiopathogenic CVI can be congenital (Klippel-Trenaunay-Weber-Parks syndrome), primitive (hydrostatic varicose veins) and secondary (post-thrombotic, as a result of venous compression or external trauma) (5).
Vascular stenosis and valvular incompetence, as well as the procoagulant state represent essential factors for the initiation and evolution of the thrombotic process. The etiology of venous thrombosis is multifactorial (6). The process usually starts with an injury of the vascular endothelium, which is subsequently followed by activation and aggregation of thrombocytes at the lesion, initiating a procoagulant effect and the constitution of the thrombi (7).
Although at present antithrombotic prophylaxis is performed when necessary, an increase in the incidence of thromboses has been observed. This may be due to numerous associated thrombotic factors, including advanced age, invasive investigations, obesity, prolonged immobilization, birth control, hormonal therapy and neoplasia, which increase pressure on the vein wall (8). Furthermore, certain hematological diseases may induce a procoagulant status, such as leukemia and polycythemia verra, and certain medications (such as hydroxyurea) may favor the appearance of lower leg ulcers (9).
Over the last 25 years, hyperhomocysteinemia (HH) has been included as a risk factor for atherosclerosis and thromboembolisms, as well as for pregnancy and cardiovascular and cerebrovascular diseases (10-12). Homocysteine is an amino acid derived from methionine metabolism and is usually excreted renally. Plasmatic levels of homocysteine are normally <12 µmol/l. Increased levels can occur in congenital HH as a result of genetic deficiencies in cystathionine β synthase or methylenetetrahydrofolate reductase, enzymes which are responsible for the metabolism of homocysteine. In the acquired form, higher homocysteine levels can be a result of folate, vitamin B12 and vitamin B6 deficiencies, senescence, hypothyroidism, connective tissue diseases, nephropathies, neoplasia and drug intake (13,14).
Taking into account that venous thromboembolism (VTE) can appear during CVI and that the etiopathogenic implication of HH in the thrombotic process is recognized, the present prospective study investigated 166 patients with CVI and determined their homocysteine levels, observing their association with factors involved in CVI and VTE aggravation. The main aim of the present study was to determine the association between homocysteine plasmatic levels and the risk of VTE, in the presence or absence of anticoagulant treatments and venous ulcers in patients with CVI. The thrombogenic factors, which are more often linked to HH, including pregnancy, family and personal history of thrombosis, smoking and certain drug treatments, were also investigated. Furthermore, the link between the metabolic, inflammatory and procoagulant status of patients with HH and CVI was explored.
Materials and methods
Patients
Over a period of one year (June 2011 to May 2012), 166 patients with a mean age of 61.59 years (range, 29-89 years) with CVI who were admitted to the Clinic of Dermatology, County Emergency Hospital of Sibiu (Sibiu, Romania), were enrolled in the present study. Patients under the age of 18 were excluded, as well as patients with chronic venous disease stages, Clinical-Etiological-Anatomical-Pathophysiological classifications (CEAP) C0-C2 (C0, no clinical signs but with symptoms; C1, with telangiectasias; and C2, with varicose veins) (15). The experimental protocol was approved by the Ethics Committee of the County Emergency Hospital of Sibiu. All patients provided signed informed consent before participating in the present study.
The following information was collected for all patients: Demographic data, general associated pathology, personal and family history of thrombosis, procoagulant factors and any previous or current anticoagulant therapies. For every patient, the inflammatory, metabolic and procoagulant statuses [activated partial thromboplastin time (APTT) and prothrombin time (PT)] were analyzed, as well as the levels of homocysteine at the time of admission.
High-performance liquid chromatography (HPLC)
Homocysteine levels were determined using HPLC and a Homocysteine in Serum/Plasma HPLC kit (cat. no. 45000; Chromsystems Diagnostics). HPLC was carried out with a Homocysteine in Serum/Plasma kit (cat. no. 45000; Chromsystems Diagnostics) and column (cat. no. 39100, Chromsystems Diagnostics). Patient blood samples were drawn following fasting and the plasma obtained was centrifuged at ambient temperature for 30 min at 10,000 x g 4,000 rpm and then stored at 2-4˚C. A 100-µl sample volume was analyzed, and the flow rate was 1.5 ml/min. Levels over 12 µmol/l were considered to represent increased levels of homocysteine.
Statistical analysis
Data were analyzed using SPSS software (version 23.0; IBM Corp.). The nominal variables are expressed as numbers and percentages and the quantitative data as mean ± SD. A Shapiro-Wilk test was applied to evaluate the normal distribution. An ANOVA was used to compare the means of quantitative data with Gaussian distribution. For non-Gaussian distributed data, the Kruskal-Wallis test was used. As appropriate, associations between two categorical variables were analyzed with Pearson's χ2 test or Fisher's exact test. P<0.05 was considered to indicate a statistically significant difference.
Results
Demographic profile
The predominant demographic profile of the CVI patients taking part in the study was women (58.43%) of urban origin (55.42%), from the age group 46-75 years (73.49%; P=0.001).
Clinical forms of CVI
Approximately one-half of the patients were stage CEAP C6 of CVI with active ulcers (42.17%; P=0.118). Almost one-quarter of the cases (22.29%) presented with thrombophlebitis at the time of diagnosis (associated with venous ulcer, 16.07%; without ulcer, 6.22%). In less than one-half of the cases (42.77%) the disease was older than 3 years.
Post-thrombotic syndrome was present in 24.09% of patients (superficial thrombophlebitis, 12.04%; profound thrombophlebitis, 10.25%; and pulmonary thromboembolis, 1.80%) and family antecedent of thrombosis in 8.43% of cases.
Procoagulant factors
Procoagulant factors identified in the study group were smoking (21.67%), surgical interventions (16.27%) and prolonged immobilization (3.61%). For the female subgroup, abortions (31.93%) and hormonal treatments (5.42%) were also identified as procoagulant factors.
Blood coagulation, metabolic and inflammatory profiles
The blood coagulation profile demonstrated low values of PT in 1.20% of cases and APTT in 5.42% of the cases. In approximately one-half of the cases the metabolic profile was altered, with hyperglycemia (22.89%; P=0.176), hypercholesterolemia (42.77%; P=0.876), hypertriglyceridemia (28.92%; P=0.752) and hyperlipidemia (14.46%; P=0.05) (Table I) being present. Less than one-quarter of the patients with CVI presented a proinflammatory profile, with increased inflammation marker levels (erythrocyte sedimentation rate, 23.50%; C-reactive protein, 16.27%; and fibrinogen, 13.86%).
Homocysteine levels
In 54.22% of cases (90 patients), increased plasmatic levels of homocysteine were identified. In patients with HH, the average homocysteine level was 17.33±5.75 µmol/l. Moderate forms of HH, with values of 12-20 µmol/l (42.77%) and 20-30 µmol/l (10.24%) and intermediary forms, with values over 30 µmol/l but lower than 100 µmol/l (1.20%) (Fig. 1) were mainly identified. Comparative analysis between average homocysteine levels in patients with CVI and anticoagulant therapy (17.31±4.89 µmol/l) compared with patients without anticoagulant therapy displayed no significant differences.
Homocysteine levels were linked to a CVI diagnosis with ulcers (15.24±7.02 µmol/l) and a CVI diagnosis with ulcers and thrombophlebitis (14.13±4.08 µmol/l), compared with 13.12±5.81 µmol/l for patients with CVI CEAP C3-5 (P=0.138) (Table II). Homocysteine levels were also linked to pulmonary thromboembolism (15.33±4.07 µmol/l) and deep vein thrombosis (DVT; 14.08±5.97 µmol/l) (P=0.819) (Table II). The average homocysteine levels presented the highest levels for patients >75 years of age (15.03 µmol/l) and the age group 61-75 years (14.53 µmol/l).
 | Table IIMean ± SD homocysteine levels in the study group, associated with the diagnosis of CVI, disease age, personal thrombosis history and obesity. |
The age of CVI was linked to HH (P=0.217). Average homocysteine levels varied between 11.44±3.23 µmol/l in patients with a CVI disease age of <1 year and 15.03±6.49 µmol/l in patients with CVI >20 years (P=0.148) (Table II). The differential analysis of homocysteine on subgroups of patients presenting with CVI without anticoagulant therapies, compared with patients with anticoagulant therapies, demonstrated that for a disease age ≤10 years, 56.40% compared with 35.70% of the cases presented with normal homocysteine levels, and for a disease age of >30 years, 87.50% of the cases compared with 71.40% of cases presented with increased homocysteine levels (P=0.050). The average homocysteine levels in patients with obesity compared with patients of normal weight was similar (13.77±4.07 µmol/l vs. 13.33±7.19 µmol/l; P=0.931) (Table II).
For patients with HH, no statistically significant associations were demonstrated between homocysteine levels and the inflammatory, metabolic and blood coagulation profiles, as well as certain thrombogenic factors, including prolonged immobilization, recent surgical interventions and hormone therapies (Table I). Among the procoagulant factors, smoking was statistically linked to HH in patients with CVI without anticoagulant therapy (P=0.088) compared with patients with CVI with chronic anticoagulant therapy.
Discussion
In CVI of the lower limbs, an increase in the intraluminal pressure determines alterations in endothelial structures with secondary cutaneous lesions. The dysfunction of the endothelium results in endothelial adhesion molecule expression, producing a chemotactic effect for leukocytes. The activated leukocytes release cytokines, including transforming growth factor-β, vascular endothelial growth factor, interleukin-1 (IL-1), tumor necrosis factor α and proteinases, which consequently initiate and maintain the inflammatory signaling cascade resulting in a procoagulant and proatherosclerotic state (16). The procoagulant state is a key etio-pathogenic factor of VTE and indirectly of venous and post-thrombotic ulcers. The interaction between chronic inflammation and thrombosis serves an essential role in post-thrombotic chronic ulceration (16). Matrix metalloproteinases (MMPs), whose activity is stimulated by chronic inflammation, serve an important role in the occurrence of ulcers (17,18). Excess MMP results in changes in fibroblast function alongside dermal fibrosis, proliferation of dermal capillaries and deterioration of extracellular matrix and glycocalyx (19).
Deficiencies in anticoagulant factors (such as anti-thrombin 3, S protein, C protein, factor V) or mutations of certain antithrombotic genes (such as prothrombin gene G20210A), activated C protein resistance and HH, can induce a congenital procoagulant state. Numerous diseases have also previously been determined to induce acquired hypercoagulant states, including neoplasia, cardiovascular diseases, cerebrovascular diseases, venous and hepatic insufficiency, diabetes mellitus, dyslipidemia, connective tissue diseases and HH, as well as tobacco smoking, prolonged immobilization and the long use of oral contraceptives.
As a factor with procoagulant value, HH can occur via congenital deficiencies, including cystathionine β synthase deficiency, methylenetetrahydrofolate reductase deficiency or innate deficiencies in the metabolism of cobalamin. However, HH can also be acquired, such as in folate, vitamin B12 and vitamin B6 metabolism disorders, chronic renal insufficiency, hypothyroidism and neoplasia (breast, ovarian and pancreatic neoplasms, and leukemia), as well as a result of certain treatments (for example, methotrexate, phenytoin, theophylline and phosphodiesterase inhibitors), advanced age and smoking (13,14,20-24). In the present study, a hyper-coagulant state induced by CVI, mainly via HH (54.22%), and the general associated pathology (86.14% of cases presented with associated cardiovascular pathology) were identified.
Advanced age is a risk factor for hypercoagulability. This is a consequence of endothelial modifications, which occur during young adulthood and middle age as a result of cardiovascular risk factors (25). Increases in the concentrations of certain procoagulant factors, such as fibrinogen, factor VII, VIII and X and the reduction of certain anticoagulant factors, such as plasminogen, antithrombin III and tissular activator of plasminogen, also contribute towards endothelial modifications (16). In the present study, HH occurred more often in individuals >60 years old (64.45%), with an average homocysteine level of 17.93 µmol/l, compared with that in individuals <60 years old (35.55%), with an average homocysteine level of 15.55 µmol/l (P=0.024).
Blood coagulation and fibrinogen profiles have also been demonstrated to be linked to thrombosis risk. Therefore, low APTT and elevated fibrinogen levels are associated with an increased risk of DVT and pulmonary thromboembolism. Low PT and international normalized ratio also serve a minor role in determining the procoagulant state (16). In the present study, a small number of patients displayed elevated fibrinogen (13.86%) and low APTT (5.42%) levels.
HH is a risk factor in arteriosclerotic cardiovascular and cerebrovascular diseases, as it is a promoter of endothelial oxidative stress and stimulates the production of reactive oxygen species. Furthermore, HH favors leukocyte inflow mediated by endothelial adhesion molecules, with the infiltration of leukocytes in the vascular wall and the release of chemokines inducing a proinflammatory response (26). In the present study group, in patients with CVI, HH was associated with pulmonary thromboembolism (100%), the presence of ulcers (61.50%), ulcers associated with thrombophlebitis (61.40%; P=0.003) and profound thrombophlebitis (58.80%; P=0.260). Alterations of lipid status (with the implication of adipokines and adiponectin) results in a proinflammatory effect over time. Chronic inflammation of the vascular wall associated with HH can influence the hypercoagulant state (27). In the present study, the analysis of the metabolic profile (glucidic and lipid) in patients with HH compared with patients with normal levels of homocysteine, was not significant. HH also contributes to venous and arterial thrombotic diseases by increasing the expression of tissular factors, alleviating anticoagulant processes, improving the thrombocyte activity, increasing thrombin production, intensifying the activity of factor V, altering the fibrinolytic potential and favoring vascular injuries (28). HH is a recognized thrombotic risk factor and also serves a role in certain complex diseases, such as Klippel-Trenaunay-Weber syndrome (29,30).
Homocysteine treatments are useful, as HH is one of the few post-thrombotic risk factors that can be corrected by administering folic acid and vitamins B6 and B12. However, although treatment using folic acid and vitamin B supplements can lower the homocysteine level by 25%, it does not prevent venous thrombosis from reappearing (31). The prevalence of HH in patients with CVI in the present study was 21.18%. In a meta-analysis published by Ray (32), the prevalence of HH in patients with VTE varied between 5.7 and 34.8% compared with 0-7.1% in healthy individuals. This study also reported that an increase of 5 µmol/l homocysteine can lead to 2-3-fold increase in the risk of VTE. Compared with the study by Ray (32), the present study determined that HH prevalence is relatively similar for patients with CVI and VTE.
HH can be moderate (<30 µmol/l), intermediary (30-100 µmol/l) and severe (>100 µmol/l) (33). In the present study, 54.22% of patients with CVI presented with HH. Almost all patients with HH presented with moderate HH (97.79%) and 2.22% presented with intermediary HH, with no cases of severe HH. These results indicated that moderate and intermediary HH may be an aggravating factor in CVI evolution with DVT and venous ulcer risk.
Increases in plasmatic levels of homocysteine were also demonstrated in the present patients with CVI with ulcers, thrombophlebitis and pulmonary thromboembolic episodes, in elderly patients, in patients with advanced disease age and in smokers.
In conclusion, HH is a well-known risk factor in arterial and venous thrombotic diseases and also serves an important role in CVI, which increases the thrombogenic risk in these patients, especially in the elderly and those with an advanced venous disease age. Treatment of moderate HH in CVI by administration of folic acid, vitamin B6 and vitamin B12 can lead to the alleviation of symptoms, reducing the risk of VTE and their complications and implicitly slowing down the progression of CVI.
Acknowledgements
Not applicable.
Funding
Funding: The present study was supported in part by a grant (no. 221) from ‘Lucian Blaga’ University of Sibiu and Advanced Medical Services.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
MR and GMI created and designed the present study, analyzed and interpreted the patient data, and drafted and revised the manuscript for important intellectual content. IB was responsible for data acquisition, analysis and interpretation of patient data, manuscript drafting and design. All authors read and approved the final manuscript. All authors confirm the authenticity of all the raw data.
Ethics approval and consent to participate
The study was approved by the Ethics Committee of the County Emergency Hospital of Sibiu (Sibiu, Romania; approval no. 356) and all patients provided written inform consent regarding participation in the study.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Authors' information
IOANA BALDOVIN is Ioana Baldovin née Rădulescu.
References
|
Nicolaides AN, Allegra C, Bergan J, Bradbury A, Cairols M, Carpentier P, Comerota A, Delis C, Eklof B, Fassiadis N, et al: Management of chronic venous disorders of the lower limbs guidelines according to scientific evidence. Int Angiol. 27:1–59. 2008.PubMed/NCBI | |
|
Zolotukhin IA, Seliverstov EI, Shevtsov YN, Avakiants IP, Nikishkov AS, Tatarintsev AM and Kirienko AI: Prevalence and risk factors for chronic venous disease in the general Russian population. Eur J Vasc Endovasc Surg. 54:752–758. 2017.PubMed/NCBI View Article : Google Scholar | |
|
Feodor T, Baila S, Mitea I, Branisteanu D and Vittos O: Epidemiology and clinical characteristics of chronic venous disease in Romania. Exp Ther Med. 17:1097–1105. 2019.PubMed/NCBI View Article : Google Scholar | |
|
Rabe E, Regnier C, Goron F, Salmat G and Pannier F: The prevalence, disease characteristics and treatment of chronic venous disease: An international web-based study. J Comp Eff Res. 9:1205–1218. 2020.PubMed/NCBI View Article : Google Scholar | |
|
Patel SK and Surowiec SM: Venous Insufficiency. In: StatPearls [Internet]. StatPearls Publishing, Treasure Island, FL, 2021. | |
|
Ashorobi D, Ameer MA and Fernandez R: Thrombosis. In: StatPearls [Internet]. StatPearls Publishing, Treasure Island, FL, 2021. | |
|
Yau JW, Teoh H and Verma S: Endothelial cell control of thrombosis. BMC Cardiovasc Disord. 15(130)2015.PubMed/NCBI View Article : Google Scholar | |
|
Hotoleanu C: Association between obesity and venous thromboembolism. Med Pharm Rep. 93:162–168. 2020.PubMed/NCBI View Article : Google Scholar | |
|
Iancu GM, Ocneanu A and Rotaru M: Hydroxyurea-induced superinfected ulcerations: Two case reports and review of the literature. Exp Ther Med. 20(191)2020.PubMed/NCBI View Article : Google Scholar | |
|
Fortin LJ and Genest J Jr: Measurement of homocyst(e)ine in the prediction of arteriosclerosis. Clin Biochem. 28:155–162. 1995.PubMed/NCBI View Article : Google Scholar | |
|
Tinelli C, Di Pino A, Ficulle E, Marcelli S and Feligioni M: Hyperhomocysteinemia as a risk factor and potential nutraceutical target for certain pathologies. Front Nutr. 6(49)2019.PubMed/NCBI View Article : Google Scholar | |
|
Cattaneo M: Hyperhomocysteinemia and venous thromboembolism. Semin Thromb Hemost. 32:716–723. 2006.PubMed/NCBI View Article : Google Scholar | |
|
Brustolin S, Giugliani R and Felix TM: Genetics of homocysteine metabolism and associated disorders. Bra J Med Biol Res. 4:1–7. 2010.PubMed/NCBI View Article : Google Scholar | |
|
Son P and Lewis L: Hyperhomocysteinemia. In: StatPearls [Internet]. StatPearls Publishing, Treasure Island, FL, 2020. | |
|
Zegarra TI and Tadi P: CEAP Classification of Venous Disorders. In: StatPearls [Internet]. StatPearls Publishing, Treasure Island, FL, 2021. | |
|
Hantusch B: Morphological and functional characteristics of blood and lymphatic vessels. In: Fundamentals of Vascular Biology. Learning Materials in Biosciences. Geiger M (ed). Springer, Cham, pp1-43, 2019. | |
|
Herouy Y: The role of matrix metalloproteinases (MMPs) and their inhibitors in venous leg ulcer healing. Phebolymphology. 44:31–267. 2004. | |
|
Barbu A, Neamțu B, Zăhan M, Iancu GM, Bacila C and Mireșan V: Current trends in advanced alginate-based wound dressings for chronic Wounds. J Pers Med. 11(890)2021.PubMed/NCBI View Article : Google Scholar | |
|
Ali MM, Mahmoud AM, Le Master E, Levitan I and Phillips SA: Role of matrix metalloproteinases and histone deacetylase in oxidative stress-induced degradation of the endothelial glycocalyx. Am J Physiol Heart Circ Physiol. 316:H647–H663. 2019.PubMed/NCBI View Article : Google Scholar | |
|
Brattström L and Wilcken DE: Homocysteine and cardiovascular disease: Cause or effect? Am J Clin Nutr. 72:315–323. 2000.PubMed/NCBI View Article : Google Scholar | |
|
Sule AA, Chin TJ and Khien LH: Recurrent unprovoked venous thrombembolism in a young female patient with high levels of homocysteine. Int J Angiol. 21:95–98. 2012.PubMed/NCBI View Article : Google Scholar | |
|
Bolal M, Ates I, Demir BF, Altay M, Turhan T and Yilmaz N: The relationship between homocysteine and autoimmune subclinical hypothyroidism. Int J Med Biochem. 3:1–7. 2020. | |
|
Hasan T, Arora R, Bansal AK, Bhattacharya R, Sharma GS and Sigh LR: Disturbed homocysteine metabolism in associated with cancer. Exp Mol Med. 51:1–13. 2019.PubMed/NCBI View Article : Google Scholar | |
|
Lovčić V, Klobučić M, Bašić-Jukić N and Lovčić P: Is hyperhomocysteinemia approaching traditional risk factors for cardiovascular diseases? Acta Clin Croat. 45 (Suppl 1):S65–S72. 2006. | |
|
Elian V, Cioca G, Pantea-Stoian A, Dobjanschi C and Serafinceanu C: Metabolic syndrome and chronic kidney disease: Pathogenic, clinical, and therapeutic correlations. In: Proceedings of the 1st International Conference on Interdisciplinary Management of Diabetes Mellitus and its Complications (INTERDIAB), Bucharest, 2015. | |
|
Papatheodorou L and Weiss N: Vascular oxidant stress and inflammation in hyperhomocysteinemia. Antioxid Redox Signal. 9:1941–1958. 2007.PubMed/NCBI View Article : Google Scholar | |
|
Esfahani M, Movahedian A, Baranchi M and Goodarzi MT: Adiponectin: An adipokine with protective features against metabolic syndrome. Iran J Basic Med Sci. 18:430–442. 2015.PubMed/NCBI | |
|
Undas A, Brozek J and Szczeklik A: Homocysteine and thrombosis: From basic science to clinical evidence. Thromb Haemost. 94:907–915. 2005.PubMed/NCBI View Article : Google Scholar | |
|
Samonakis DN, Oustamanolakis P, Manousou P, Kouroumalis EA and Burroughs AK: Klippel-Trenaunay syndrome, pregnancy and the liver: An unusual interplay. Ann Gastroenterol. 25:365–367. 2012.PubMed/NCBI | |
|
Rotaru M and Iancu GM: Klippel-Trenaunay-Weber syndrome. Acta Medica Transilvanica. 2:265–267. 2010. | |
|
den Heijer M, Willems HPJ, Blom HJ, Gerrits WBJ, Cattaneo M, Eichinger S, Rosendaal FR and Bos GMJ: Homocysteine lowering by B vitamins and the secondary prevention of deep vein thrombosis and pulmonary embolism: A randomized, placebo-controlled, double-blind trial. Blood. 109:139–144. 2007.PubMed/NCBI View Article : Google Scholar | |
|
Ray JG: Meta-analysis of hyperhomocysteinemia as a risk factor for venous thromboembolic disease. Arch Intern Med. 158:2101–2106. 1998.PubMed/NCBI View Article : Google Scholar | |
|
Ganguly P and Alam SF: Role of homocysteine in the development of cardiovascular disease. Nutr J. 14(6)2015.PubMed/NCBI View Article : Google Scholar |
