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Investigation of Asymmetric and Symmetric Dimethylarginine Levels after Iloprost Treatment in Patients with Buerger's Disease
Eur J Vasc Endovasc Surg (2017) 53, 439e442
Investigation of Asymmetric and Symmetric Dimethylarginine Levels after Iloprost Treatment in Patients with Buerger’s Disease S. Senol a b
a,*
, A. Senol
b
Department of Cardiovascular Surgery, Educational and Research Hospital, Elazig, Turkey Department of Infectious Diseases and Clinical Microbiology, Educational and Research Hospital, Elazig, Turkey
WHAT THIS PAPER ADDS The study results suggest that iloprost treatment decreases ADMA and SDMA levels, which are associated with endothelial dysfunction in patients with Buerger’s disease.
Objective: The aim of this study was to compare the levels of acetyl-dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and the L-arginine/ADMA ratio before and after iloprost treatment in patients with Buerger’s disease. Methods: Between January 2011 and December 2015, data from 44 patients (36 males, 8 females, mean age 48.7 18.1 years) with the diagnosis of Fontaine Stage IIIeIV Buerger’s disease were included. Iloprost infusion was administered intravenously through the forearm veins for 7 days at a dose of 0.5e1.5 ng/kg/min over 16 h. Blood samples were collected before and after treatment for measurement of ADMA, SDMA, and L-arginine. ADMA, SDMA, L-arginine levels were measured using high performance liquid chromatography (HPLC). Results: After iloprost treatment, ADMA and SDMA levels significantly decreased (p ¼ .001). The increase in the Larginine levels was not significant (p ¼ .16). However, the L-arginine/ADMA ratio increased significantly (p ¼ .001). Conclusion: Iloprost treatment decreases ADMA and SDMA, which are associated with endothelial dysfunctions in patients with Buerger’s disease. Of note, the still higher than normal range of SDMA levels after iloprost treatment suggests that treatment should continue until SDMA levels are within the normal range in this patient population. Ó 2016 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved. Article history: Received 8 March 2016, Accepted 6 December 2016, Available online 28 January 2017 Keywords: Buerger’s disease, Iloprost, Asymmetric dimethylarginine, Symmetric dimethylarginine, L-Arginine
INTRODUCTION Buerger’s disease (thromboangiitis obliterans) is one of the non-atherosclerotic, segmental, inflammatory, peripheral obstructive vascular diseases involving medium sized and small arteries.1 Although the disease is directly related to smoking and tobacco use, genetic factors, hypercoagulability, vascular endothelial structure, and immunological mechanisms have also been reported to play a role in the etiology of the disease.1,2 One of the interesting features of Buerger’s disease is its geographical distribution in the world. It is more frequently seen in the Middle and Far East than in the United States. The prevalence of Buerger’s disease is less than 1% among other peripheral arterial diseases in the United States. Its prevalence is 0.5e5.6% in Western Europe, and approximately 10% in Turkey; however, it is 50% in the Far East.1 Prostacyclin analogues and * Corresponding author. E-mail addresses: [email protected] (S. Senol). 1078-5884/Ó 2016 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejvs.2016.12.017
anti-aggregants are the most frequently used agents in the treatment of this frequently seen disease.1,2 Iloprost, which is a prostaglandin analogue, is widely used in the treatment of other ischaemic conditions, such as peripheral vascular disease, diabetic foot, and pulmonary arterial hypertension.3 It has a direct effect on dilated arteries and veins, inhibiting platelet aggregation and release of endothelin-1, and inhibiting tumour necrosis factor-a and soluble adhesion molecules, namely intercellular adhesion molecule and vascular cell adhesion molecule-1. E-selectin inhibits production of profibrotic growth factors, namely transforming growth factor and connective tissue growth factor.3e5 Nitric oxide (NO) is produced from an amino acid, L-arginine, by endothelial nitric oxide synthase (NOS) and it regulates vascular tone and plays an important role in the maintenance of vascular homeostasis.6 The formation of acetyldimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) is carried out by the addition of a class of enzymes and methyl groups to residues of the protein arginine.7 ADMA is the endogenous inhibitor of endothelial NOS. SDMA does not inactivate the NOS enzyme; however, it has
440
an indirect effect on NO production, inhibiting the cellular entry with arginine and ADMA.8 ADMA is the endogenous competitive inhibitor of endothelial NOS and it decreases production and bioavailability of endothelial NOS.9 In vascular diseases, ADMA levels increase before the onset of clinical symptoms. The levels are also associated with endothelial dysfunction, and ADMA has been shown to be a better indicator for endothelial injury than cholesterol levels.10,11 The major pathway for the maintenance of endothelial function is the presence of NO produced by NOS.11 ADMA was previously considered to be the main molecule in this pathway, being at the junction of the effects of all risk factors.11,12 Several previous studies have demonstrated that the Arg/ADMA ratio was associated with impaired organ function, cardio- and cerebrovascular diseases, and mortality.13 Therefore, the Arg/ADMA ratio was examined in the present study. In this study, the aim was to compare the levels of ADMA, SDMA, and the L-arginine/ ADMA ratio before and after iloprost treatment in patients with Buerger’s disease. MATERIAL AND METHODS Between January 2011 and December 2015, data from 44 patients (36 males, 8 females, mean age 48.7 18.1 years) with a diagnosis of Fontaine Stage IIIeIV Buerger’s disease were included in the study. An iloprost (Ilomedin, Bayer Schering Pharma AG, Berlin, Germany) infusion was administered intravenously at a dose of 0.5 ng/kg/min over 16 h through the forearm veins.14 On the first day of treatment, the dose was titrated up to 1.5 ng/kg/min with 0.5 ng/kg/min increments every 30 min. Probable side effects were closely monitored. The drug was then given for 7 days at a dose of 1.5 ng/kg/min depending on the tolerability profile of the patient. Blood samples were collected on Day 8 following treatment. The diagnosis of Buerger’s disease was made on the basis of Shionaya’s criteria, which include clinical symptoms and pathognomonic angiographic findings of the collateral vessels. Patients with Buerger’s disease who might need a major amputation without any history of vascular reconstruction or lumen opening interventions (angioplasty) or fibrinolytic therapy and had not smoked within the past 2 weeks were included. All people included in this study smoked cigarettes. Those with myocardial infarction within the past month, with acute coronary syndrome, kidney or liver dysfunction, or any known systemic disease, cardiovascular risk factors (hypertension, obesity, hyperlipidemia [> 2.5 g/L], and diabetes mellitus) and those who were unable to tolerate iloprost treatment were excluded. Substance abusers were also excluded. A single study protocol was applied to all patients during treatment. The patients were informed about the study and informed consent was obtained from each patient. The study protocol was approved by the local ethics committee. All patients underwent a detailed physical examination and their demographic characteristics were recorded.
S. Senol and A. Senol Table 1. Patient demographics. Age, years Sex Male Female Clinical stage Fontaine stage III Fontaine stage IV Ischaemic wounds Upper limb Lower limb Level of amputation Distal phalanx Lower extremity (metatarsal) Smokers
n 48.7 18.1
%
36 8
81.8 18.1
34 10
77.2 22.7
4 40
9 90.9
2 8 44
4.5 18.1 100
ADMA, SDMA, and L-arginine Blood samples collected from the antecubital veins before and after treatment were put into pre-cooled test tubes containing ethylenediaminetetraacetic acid (EDTA), and the tubes were put into ice. The samples were centrifuged at 3,000 g for 10 min. Then, serum and plasma were separated and kept at 80 C until analysis. On the day of analysis, serum and plasma were thawed promptly. The levels of ADMA, SDMA, and L-arginine were measured in thawed plasma samples using high performance liquid chromatography (HPLC) (Shimadzu 10 AVP, Japan) and appropriate commercial kits according to the manufacturers’ instructions.15 Statistical analysis Statistical analysis was performed using SPSS (Statistical Packages for the Social Sciences; SPSS Inc., Chicago, IL, USA) v12.0 software. The data were presented as mean standard deviation. The paired samples t test was used to compare pre- and post-treatment variables. A p value < .05 was considered statistically significant. RESULTS The demographic and clinical characteristics of the patients are shown in Table 1. Among the patients with Fontaine Stage IIIeIV Buerger’s disease, 77.2% were Fontaine Stage III and 22.7% were Stage IV. All patients had ischaemic trophic changes (the skin is pale and hairless), most of them in the lower extremity (90.9%), and the minority in the upper extremity (9.1%). The findings were limited to the fingers and toes in all patients. Ten (22.7%) patients who had tissue necrosis and Gram negative infection before iloprost administration had amputations after treatment. Necrosis was the main determinant for the decision to do amputation. All 44 patients included in the study completed their therapy at a pre-specified dose and duration. Sixteen patients (36.4%) had adverse effects during treatment. The most frequent adverse effects were headache in eight (18.2%) cases and nausea in four (9.1%). The adverse effects
Investigation of Asymmetric and Symmetric Dimethylarginine Levels Table 2. Adverse effects of iloprost treatment. Adverse effects Headache Phlebitis Hypotension Nausea Flushing Rash
n 8 2 0 4 0 2
% 18.1 4.5 0 9 0 4.5
did not necessitate dose reduction or interruption of treatment. The adverse effects are given in Table 2. In the 34 patients who did not undergo amputation, iloprost infusion relieved the pain. In addition, before iloprost treatment, the plasma levels of ADMA, SDMA, and L-arginine, were 2.78 1.01 mmol/L, 3.23 1.14 mmol/L, and 77.6 21.7 mmol/L, respectively, and the L-arginine/ADMA ratio was 35 9.7%. After iloprost infusion, these values were 1.3 0.41 mmol/L, 1.5 0.38 mmol/L, and 86.1 32.1 mmol/L, and 49 17.6% (Table 3). Fourteen of the patients had a watery discharge. Culture studies of these patients are shown in Table 4. The most common organisms were mixed type (aerobes and anaerobes). In this study, ADMA levels (2.78 1.01 vs. 1.30 0.41) and SDMA levels (3.23 1.14 vs. 1.5 0.38) significantly decreased, and the L-arginine/ADMA ratio significantly increased (49 17.6 vs. 35 9.7) (p ¼ .001) after treatment. However, there was no significant difference in the pre- and post-treatment arginine levels (77.6 21.7 vs. 86.1 32.1) (p ¼ .13). DISCUSSION Iloprost has been widely used in the treatment of critical ischaemia, relieving resting pain, reducing the need for analgesics, resolving trophic lesions, increasing walking distance, and halting the progression to amputation in the long term.14 There are no known studies available in the literature investigating ADMA, SDMA, and L-arginine in patients with Buerger’s disease before and after iloprost. One study investigated ADMA levels after iloprost treatment in occlusive arterial disease and reported low ADMA and serotonin levels after treatment.17 Similar to the Blardi et al. study,16 ADMA levels decreased after treatment in the current study. ADMA is an endogenous inhibitor of NOS, but SDMA does not have any inhibitory effect on NOS. However, it has been suggested that it indirectly affects the NO production rate, influencing cellular entry of arginine and ADMA.10 In the current study, a significant decrease in SDMA levels after iloprost treatment (p ¼ .001) was found. Table 3. ADMA, SDMA, and L-arginine (mmol/L) levels in patients. Pre-iloprost ADMA 2.78 1.01 SDMA 3.23 1.14 L-Arginine 77.6 21.7 L-Arg/ADMA 35 9.7 ADMA ¼ acetyl-dimethylarginine; dimethylarginine.
Post-iloprost 1.3 0.41 1.5 0.38 86.1 32.1 49 17.6 SDMA ¼
p .001 .001 .13 .001 symmetric
441 Table 4. Micro-organisms produced in discharged tissue (specimens obtained from amputee or debrided tissues). n 7 4 2 1
% 50 28.6 14.3 7.1
Micro-organism type Mixed (aerobes and anaerobes) Anaerobes Gram (þ) Streptococcus pyogenes Gram () E.coli and Proteus species
Vasodilatation induced by iloprost was neither NO nor endothelin-1 dependent and occurred mainly in the microcirculatory bed.3 However, lower ADMA and SDMA levels were found after iloprost treatment in patients with Buerger’s disease, which exerts a direct or indirect effect on NO.8,9 In contrast, in another study, ADMA levels decreased in patients with Buerger’s disease.2 However, despite lower ADMA levels, normal values were reached. The difference between the studies can be attributed to the use of different kits. Recently, enzyme linked immunosorbent assay (ELISA) was reported to be compatible with HPLC and many centres have adopted these HPLC compatible ELISA kits. However, a number of studies have demonstrated that HPLC has a higher sensitivity and specificity than the ELISA.17 Furthermore, there is a growing number of studies suggesting that the Arg/ADMA ratio has a critical role and is a reliable method.13 Therefore, the Arg/ADMA ratio before and after iloprost treatment was compared and found significant compared with ADMA levels. Moreover, normal ranges of ADMA and SDMA have been reported to be 0.41e0.96 mmol/L and 0.27e0.67 mmol/L in healthy individuals, as confirmed by several large scale studies.2,13,18 One of the most interesting findings of the present study was the greater increase in SDMA levels than ADMA levels. SDMA levels still remained higher than normal range after iloprost treatment (1.5 0.38). This can be explained by the fact that SDMA may indirectly play a more critical role in endothelial dysfunction than ADMA in patients with Buerger’s disease. However, there is a limitation to this study. Small sample size could prevent establishing a definite conclusion. Therefore, further large scale studies are required to increase the statistical power and reach a robust conclusion. In conclusion, iloprost treatment decreases ADMA and SDMA, which are associated with endothelial dysfunction in patients with Buerger’s disease. Of note, higher than normal SDMA levels after iloprost treatment suggest continuing the treatment until SDMA levels are within the normal range. If another group of patients had been available showing the changes in these parameters only after quitting smoking in Buerger’s disease which is highly related with smoking, the effect of iloprost may have been shown more clearly. However, further large scale studies are required to reach a conclusion. CONFLICT OF INTEREST None. FUNDING None.
442
REFERENCES 1 Bozkurt AK, Cengiz K, Arslan C, Mine DY, Oner S, Deniz DB, et al. Stable prostacyclin analogue (iloprost) in the treatment of Buerger’s disease: a prospective analysis of 150 patients. Ann Thorac Cardiovasc Surg 2013;19(2):120e5. 2 Tuygun AK, Tuygun A, Yurtseven N, Sensöz Y, Günay R, Keser M, et al. Asymmetric dimethylarginine levels in Buerger’s disease. Ann Vasc Surg 2011;25(4):547e54. 3 Caliskan A, Yavuz C, Karahan O, Yazici S, Guclu O, Demirtas S, et al. Iloprost reduces myocardial edema in a rat model of myocardial ischemia reperfusion. Perfusion 2014;29(3): 260e4. 4 Coppolino G, Buemi A, Bolignano D. Perioperative iloprost and endothelial progenitor cells in uremic patients with severe limb ischemia undergoing peripheral revascularization. J Surg Res 2009;157:129e35. 5 Di Stefano R, Barsotti MC, Melillo E. The prostacyclin analogue iloprost increases circulating endothelial progenitor cells in patients with critical limb ischemia. Thromb Haemost 2008;100:871e7. 6 Landmesser U, Drexler H. The clinical significance of endothelial dysfunction. Curr Opin Cardiol 2005;20:547e51. 7 Clarke S. Protein methylation. Curr Opin Cell Biol 1993;5:977e 83. 8 Ogawa T, Kimoto M, Sasaoka K. Purification and properties of a new enzyme. NG, NG-dimethylarginine dimethylaminohydrolase from rat kidney. J Biol Chem 1989;264:10205e9. 9 Can F, Ziyrek M, Erdem S¸, Civan M, Görmüs¸ U, S¸ahin S, et al. The association between coronary atherosclerotic burden and asymmetric dimethylarginine, carotis intima media thickness and endothelial function. Turk Kardiyol Dern Ars 2014;42(8): 701e9.
S. Senol and A. Senol 10 Lentz SR, Rodinov RN, Dayal S. Hyperhomocysteinemia, endothelial dysfunction, and cardiovascular risk: the potential role of ADMA. Atheroscler Suppl 2003;4:61e5. 11 Tran CT, Leiper JM, Vallance P. The DDAH/ADMA/NOS pathway. Atheroscler Suppl 2003;4:33e40. 12 Vallance P, Leiper J. Cardiovascular biology of the asymmetric dimethylarginine: dimethylarginine dimethylaminohydrolase pathway. Arterioscler Thromb Vasc Biol 2004;24:1023e30. 13 Notsu Y, Yano S, Shibata H, Nagai A, Nabika T. Plasma arginine/ ADMA ratio as a sensitive risk marker for atherosclerosis: Shimane CoHRE study. Atherosclerosis 2015;239(1):61e6. 14 Lessiani G, Vazzana N, Cuccurullo C, Di Michele D, Laurora G, Sgrò G, et al. Inflammation, oxidative stress and platelet activation in aspirin-treated critical limb ischaemia: beneficial effects of iloprost. Thromb Haemost 2011;105(2):321e8. 15 Teerlink T. Measurement of asymmetric dimethylarginine in plasma: methodological considerations and clinical relevance. Clin Chem Lab Med 2005;43:1130e8. 16 Blardi P, de Lalla A, Pieragalli D, De Franco V, Meini S, Ceccatelli L, et al. Effect of iloprost on plasma asymmetric dimethylarginine and plasma and platelet serotonin in patients with peripheral arterial occlusive disease. Prostaglandins Other Lipid Mediat 2006;80(3e4):175e82. 17 Valtonen P, Karppi J, Nyyssönen K, Valkonen VP, Halonen T, Punnonen K. Comparison of HPLC method and commercial ELISA assay for asymmetric dimethylarginine (ADMA) determination in human serum. J Chromatogr B Analyt Technol Biomed Life Sci 2005;828(1e2):97e102. 18 Atzler D, Schwedhelm E, Nauck M, Ittermann T, Böger RH, Friedrich N. Serum reference intervals of homoarginine, ADMA, and SDMA in the study of health in Pomerania. Clin Chem Lab Med 2014;52(12):1835e42.
Investigation of Asymmetric and Symmetric Dimethylarginine Levels after Iloprost Treatment in Patients with Buerger’s Disease S. Senol a b
a,*
, A. Senol
b
Department of Cardiovascular Surgery, Educational and Research Hospital, Elazig, Turkey Department of Infectious Diseases and Clinical Microbiology, Educational and Research Hospital, Elazig, Turkey
WHAT THIS PAPER ADDS The study results suggest that iloprost treatment decreases ADMA and SDMA levels, which are associated with endothelial dysfunction in patients with Buerger’s disease.
Objective: The aim of this study was to compare the levels of acetyl-dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and the L-arginine/ADMA ratio before and after iloprost treatment in patients with Buerger’s disease. Methods: Between January 2011 and December 2015, data from 44 patients (36 males, 8 females, mean age 48.7 18.1 years) with the diagnosis of Fontaine Stage IIIeIV Buerger’s disease were included. Iloprost infusion was administered intravenously through the forearm veins for 7 days at a dose of 0.5e1.5 ng/kg/min over 16 h. Blood samples were collected before and after treatment for measurement of ADMA, SDMA, and L-arginine. ADMA, SDMA, L-arginine levels were measured using high performance liquid chromatography (HPLC). Results: After iloprost treatment, ADMA and SDMA levels significantly decreased (p ¼ .001). The increase in the Larginine levels was not significant (p ¼ .16). However, the L-arginine/ADMA ratio increased significantly (p ¼ .001). Conclusion: Iloprost treatment decreases ADMA and SDMA, which are associated with endothelial dysfunctions in patients with Buerger’s disease. Of note, the still higher than normal range of SDMA levels after iloprost treatment suggests that treatment should continue until SDMA levels are within the normal range in this patient population. Ó 2016 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved. Article history: Received 8 March 2016, Accepted 6 December 2016, Available online 28 January 2017 Keywords: Buerger’s disease, Iloprost, Asymmetric dimethylarginine, Symmetric dimethylarginine, L-Arginine
INTRODUCTION Buerger’s disease (thromboangiitis obliterans) is one of the non-atherosclerotic, segmental, inflammatory, peripheral obstructive vascular diseases involving medium sized and small arteries.1 Although the disease is directly related to smoking and tobacco use, genetic factors, hypercoagulability, vascular endothelial structure, and immunological mechanisms have also been reported to play a role in the etiology of the disease.1,2 One of the interesting features of Buerger’s disease is its geographical distribution in the world. It is more frequently seen in the Middle and Far East than in the United States. The prevalence of Buerger’s disease is less than 1% among other peripheral arterial diseases in the United States. Its prevalence is 0.5e5.6% in Western Europe, and approximately 10% in Turkey; however, it is 50% in the Far East.1 Prostacyclin analogues and * Corresponding author. E-mail addresses: [email protected] (S. Senol). 1078-5884/Ó 2016 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejvs.2016.12.017
anti-aggregants are the most frequently used agents in the treatment of this frequently seen disease.1,2 Iloprost, which is a prostaglandin analogue, is widely used in the treatment of other ischaemic conditions, such as peripheral vascular disease, diabetic foot, and pulmonary arterial hypertension.3 It has a direct effect on dilated arteries and veins, inhibiting platelet aggregation and release of endothelin-1, and inhibiting tumour necrosis factor-a and soluble adhesion molecules, namely intercellular adhesion molecule and vascular cell adhesion molecule-1. E-selectin inhibits production of profibrotic growth factors, namely transforming growth factor and connective tissue growth factor.3e5 Nitric oxide (NO) is produced from an amino acid, L-arginine, by endothelial nitric oxide synthase (NOS) and it regulates vascular tone and plays an important role in the maintenance of vascular homeostasis.6 The formation of acetyldimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) is carried out by the addition of a class of enzymes and methyl groups to residues of the protein arginine.7 ADMA is the endogenous inhibitor of endothelial NOS. SDMA does not inactivate the NOS enzyme; however, it has
440
an indirect effect on NO production, inhibiting the cellular entry with arginine and ADMA.8 ADMA is the endogenous competitive inhibitor of endothelial NOS and it decreases production and bioavailability of endothelial NOS.9 In vascular diseases, ADMA levels increase before the onset of clinical symptoms. The levels are also associated with endothelial dysfunction, and ADMA has been shown to be a better indicator for endothelial injury than cholesterol levels.10,11 The major pathway for the maintenance of endothelial function is the presence of NO produced by NOS.11 ADMA was previously considered to be the main molecule in this pathway, being at the junction of the effects of all risk factors.11,12 Several previous studies have demonstrated that the Arg/ADMA ratio was associated with impaired organ function, cardio- and cerebrovascular diseases, and mortality.13 Therefore, the Arg/ADMA ratio was examined in the present study. In this study, the aim was to compare the levels of ADMA, SDMA, and the L-arginine/ ADMA ratio before and after iloprost treatment in patients with Buerger’s disease. MATERIAL AND METHODS Between January 2011 and December 2015, data from 44 patients (36 males, 8 females, mean age 48.7 18.1 years) with a diagnosis of Fontaine Stage IIIeIV Buerger’s disease were included in the study. An iloprost (Ilomedin, Bayer Schering Pharma AG, Berlin, Germany) infusion was administered intravenously at a dose of 0.5 ng/kg/min over 16 h through the forearm veins.14 On the first day of treatment, the dose was titrated up to 1.5 ng/kg/min with 0.5 ng/kg/min increments every 30 min. Probable side effects were closely monitored. The drug was then given for 7 days at a dose of 1.5 ng/kg/min depending on the tolerability profile of the patient. Blood samples were collected on Day 8 following treatment. The diagnosis of Buerger’s disease was made on the basis of Shionaya’s criteria, which include clinical symptoms and pathognomonic angiographic findings of the collateral vessels. Patients with Buerger’s disease who might need a major amputation without any history of vascular reconstruction or lumen opening interventions (angioplasty) or fibrinolytic therapy and had not smoked within the past 2 weeks were included. All people included in this study smoked cigarettes. Those with myocardial infarction within the past month, with acute coronary syndrome, kidney or liver dysfunction, or any known systemic disease, cardiovascular risk factors (hypertension, obesity, hyperlipidemia [> 2.5 g/L], and diabetes mellitus) and those who were unable to tolerate iloprost treatment were excluded. Substance abusers were also excluded. A single study protocol was applied to all patients during treatment. The patients were informed about the study and informed consent was obtained from each patient. The study protocol was approved by the local ethics committee. All patients underwent a detailed physical examination and their demographic characteristics were recorded.
S. Senol and A. Senol Table 1. Patient demographics. Age, years Sex Male Female Clinical stage Fontaine stage III Fontaine stage IV Ischaemic wounds Upper limb Lower limb Level of amputation Distal phalanx Lower extremity (metatarsal) Smokers
n 48.7 18.1
%
36 8
81.8 18.1
34 10
77.2 22.7
4 40
9 90.9
2 8 44
4.5 18.1 100
ADMA, SDMA, and L-arginine Blood samples collected from the antecubital veins before and after treatment were put into pre-cooled test tubes containing ethylenediaminetetraacetic acid (EDTA), and the tubes were put into ice. The samples were centrifuged at 3,000 g for 10 min. Then, serum and plasma were separated and kept at 80 C until analysis. On the day of analysis, serum and plasma were thawed promptly. The levels of ADMA, SDMA, and L-arginine were measured in thawed plasma samples using high performance liquid chromatography (HPLC) (Shimadzu 10 AVP, Japan) and appropriate commercial kits according to the manufacturers’ instructions.15 Statistical analysis Statistical analysis was performed using SPSS (Statistical Packages for the Social Sciences; SPSS Inc., Chicago, IL, USA) v12.0 software. The data were presented as mean standard deviation. The paired samples t test was used to compare pre- and post-treatment variables. A p value < .05 was considered statistically significant. RESULTS The demographic and clinical characteristics of the patients are shown in Table 1. Among the patients with Fontaine Stage IIIeIV Buerger’s disease, 77.2% were Fontaine Stage III and 22.7% were Stage IV. All patients had ischaemic trophic changes (the skin is pale and hairless), most of them in the lower extremity (90.9%), and the minority in the upper extremity (9.1%). The findings were limited to the fingers and toes in all patients. Ten (22.7%) patients who had tissue necrosis and Gram negative infection before iloprost administration had amputations after treatment. Necrosis was the main determinant for the decision to do amputation. All 44 patients included in the study completed their therapy at a pre-specified dose and duration. Sixteen patients (36.4%) had adverse effects during treatment. The most frequent adverse effects were headache in eight (18.2%) cases and nausea in four (9.1%). The adverse effects
Investigation of Asymmetric and Symmetric Dimethylarginine Levels Table 2. Adverse effects of iloprost treatment. Adverse effects Headache Phlebitis Hypotension Nausea Flushing Rash
n 8 2 0 4 0 2
% 18.1 4.5 0 9 0 4.5
did not necessitate dose reduction or interruption of treatment. The adverse effects are given in Table 2. In the 34 patients who did not undergo amputation, iloprost infusion relieved the pain. In addition, before iloprost treatment, the plasma levels of ADMA, SDMA, and L-arginine, were 2.78 1.01 mmol/L, 3.23 1.14 mmol/L, and 77.6 21.7 mmol/L, respectively, and the L-arginine/ADMA ratio was 35 9.7%. After iloprost infusion, these values were 1.3 0.41 mmol/L, 1.5 0.38 mmol/L, and 86.1 32.1 mmol/L, and 49 17.6% (Table 3). Fourteen of the patients had a watery discharge. Culture studies of these patients are shown in Table 4. The most common organisms were mixed type (aerobes and anaerobes). In this study, ADMA levels (2.78 1.01 vs. 1.30 0.41) and SDMA levels (3.23 1.14 vs. 1.5 0.38) significantly decreased, and the L-arginine/ADMA ratio significantly increased (49 17.6 vs. 35 9.7) (p ¼ .001) after treatment. However, there was no significant difference in the pre- and post-treatment arginine levels (77.6 21.7 vs. 86.1 32.1) (p ¼ .13). DISCUSSION Iloprost has been widely used in the treatment of critical ischaemia, relieving resting pain, reducing the need for analgesics, resolving trophic lesions, increasing walking distance, and halting the progression to amputation in the long term.14 There are no known studies available in the literature investigating ADMA, SDMA, and L-arginine in patients with Buerger’s disease before and after iloprost. One study investigated ADMA levels after iloprost treatment in occlusive arterial disease and reported low ADMA and serotonin levels after treatment.17 Similar to the Blardi et al. study,16 ADMA levels decreased after treatment in the current study. ADMA is an endogenous inhibitor of NOS, but SDMA does not have any inhibitory effect on NOS. However, it has been suggested that it indirectly affects the NO production rate, influencing cellular entry of arginine and ADMA.10 In the current study, a significant decrease in SDMA levels after iloprost treatment (p ¼ .001) was found. Table 3. ADMA, SDMA, and L-arginine (mmol/L) levels in patients. Pre-iloprost ADMA 2.78 1.01 SDMA 3.23 1.14 L-Arginine 77.6 21.7 L-Arg/ADMA 35 9.7 ADMA ¼ acetyl-dimethylarginine; dimethylarginine.
Post-iloprost 1.3 0.41 1.5 0.38 86.1 32.1 49 17.6 SDMA ¼
p .001 .001 .13 .001 symmetric
441 Table 4. Micro-organisms produced in discharged tissue (specimens obtained from amputee or debrided tissues). n 7 4 2 1
% 50 28.6 14.3 7.1
Micro-organism type Mixed (aerobes and anaerobes) Anaerobes Gram (þ) Streptococcus pyogenes Gram () E.coli and Proteus species
Vasodilatation induced by iloprost was neither NO nor endothelin-1 dependent and occurred mainly in the microcirculatory bed.3 However, lower ADMA and SDMA levels were found after iloprost treatment in patients with Buerger’s disease, which exerts a direct or indirect effect on NO.8,9 In contrast, in another study, ADMA levels decreased in patients with Buerger’s disease.2 However, despite lower ADMA levels, normal values were reached. The difference between the studies can be attributed to the use of different kits. Recently, enzyme linked immunosorbent assay (ELISA) was reported to be compatible with HPLC and many centres have adopted these HPLC compatible ELISA kits. However, a number of studies have demonstrated that HPLC has a higher sensitivity and specificity than the ELISA.17 Furthermore, there is a growing number of studies suggesting that the Arg/ADMA ratio has a critical role and is a reliable method.13 Therefore, the Arg/ADMA ratio before and after iloprost treatment was compared and found significant compared with ADMA levels. Moreover, normal ranges of ADMA and SDMA have been reported to be 0.41e0.96 mmol/L and 0.27e0.67 mmol/L in healthy individuals, as confirmed by several large scale studies.2,13,18 One of the most interesting findings of the present study was the greater increase in SDMA levels than ADMA levels. SDMA levels still remained higher than normal range after iloprost treatment (1.5 0.38). This can be explained by the fact that SDMA may indirectly play a more critical role in endothelial dysfunction than ADMA in patients with Buerger’s disease. However, there is a limitation to this study. Small sample size could prevent establishing a definite conclusion. Therefore, further large scale studies are required to increase the statistical power and reach a robust conclusion. In conclusion, iloprost treatment decreases ADMA and SDMA, which are associated with endothelial dysfunction in patients with Buerger’s disease. Of note, higher than normal SDMA levels after iloprost treatment suggest continuing the treatment until SDMA levels are within the normal range. If another group of patients had been available showing the changes in these parameters only after quitting smoking in Buerger’s disease which is highly related with smoking, the effect of iloprost may have been shown more clearly. However, further large scale studies are required to reach a conclusion. CONFLICT OF INTEREST None. FUNDING None.
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REFERENCES 1 Bozkurt AK, Cengiz K, Arslan C, Mine DY, Oner S, Deniz DB, et al. Stable prostacyclin analogue (iloprost) in the treatment of Buerger’s disease: a prospective analysis of 150 patients. Ann Thorac Cardiovasc Surg 2013;19(2):120e5. 2 Tuygun AK, Tuygun A, Yurtseven N, Sensöz Y, Günay R, Keser M, et al. Asymmetric dimethylarginine levels in Buerger’s disease. Ann Vasc Surg 2011;25(4):547e54. 3 Caliskan A, Yavuz C, Karahan O, Yazici S, Guclu O, Demirtas S, et al. Iloprost reduces myocardial edema in a rat model of myocardial ischemia reperfusion. Perfusion 2014;29(3): 260e4. 4 Coppolino G, Buemi A, Bolignano D. Perioperative iloprost and endothelial progenitor cells in uremic patients with severe limb ischemia undergoing peripheral revascularization. J Surg Res 2009;157:129e35. 5 Di Stefano R, Barsotti MC, Melillo E. The prostacyclin analogue iloprost increases circulating endothelial progenitor cells in patients with critical limb ischemia. Thromb Haemost 2008;100:871e7. 6 Landmesser U, Drexler H. The clinical significance of endothelial dysfunction. Curr Opin Cardiol 2005;20:547e51. 7 Clarke S. Protein methylation. Curr Opin Cell Biol 1993;5:977e 83. 8 Ogawa T, Kimoto M, Sasaoka K. Purification and properties of a new enzyme. NG, NG-dimethylarginine dimethylaminohydrolase from rat kidney. J Biol Chem 1989;264:10205e9. 9 Can F, Ziyrek M, Erdem S¸, Civan M, Görmüs¸ U, S¸ahin S, et al. The association between coronary atherosclerotic burden and asymmetric dimethylarginine, carotis intima media thickness and endothelial function. Turk Kardiyol Dern Ars 2014;42(8): 701e9.
S. Senol and A. Senol 10 Lentz SR, Rodinov RN, Dayal S. Hyperhomocysteinemia, endothelial dysfunction, and cardiovascular risk: the potential role of ADMA. Atheroscler Suppl 2003;4:61e5. 11 Tran CT, Leiper JM, Vallance P. The DDAH/ADMA/NOS pathway. Atheroscler Suppl 2003;4:33e40. 12 Vallance P, Leiper J. Cardiovascular biology of the asymmetric dimethylarginine: dimethylarginine dimethylaminohydrolase pathway. Arterioscler Thromb Vasc Biol 2004;24:1023e30. 13 Notsu Y, Yano S, Shibata H, Nagai A, Nabika T. Plasma arginine/ ADMA ratio as a sensitive risk marker for atherosclerosis: Shimane CoHRE study. Atherosclerosis 2015;239(1):61e6. 14 Lessiani G, Vazzana N, Cuccurullo C, Di Michele D, Laurora G, Sgrò G, et al. Inflammation, oxidative stress and platelet activation in aspirin-treated critical limb ischaemia: beneficial effects of iloprost. Thromb Haemost 2011;105(2):321e8. 15 Teerlink T. Measurement of asymmetric dimethylarginine in plasma: methodological considerations and clinical relevance. Clin Chem Lab Med 2005;43:1130e8. 16 Blardi P, de Lalla A, Pieragalli D, De Franco V, Meini S, Ceccatelli L, et al. Effect of iloprost on plasma asymmetric dimethylarginine and plasma and platelet serotonin in patients with peripheral arterial occlusive disease. Prostaglandins Other Lipid Mediat 2006;80(3e4):175e82. 17 Valtonen P, Karppi J, Nyyssönen K, Valkonen VP, Halonen T, Punnonen K. Comparison of HPLC method and commercial ELISA assay for asymmetric dimethylarginine (ADMA) determination in human serum. J Chromatogr B Analyt Technol Biomed Life Sci 2005;828(1e2):97e102. 18 Atzler D, Schwedhelm E, Nauck M, Ittermann T, Böger RH, Friedrich N. Serum reference intervals of homoarginine, ADMA, and SDMA in the study of health in Pomerania. Clin Chem Lab Med 2014;52(12):1835e42.