Abstract
Background: Hypercoagulability is an important risk factor for thrombosis and its complications in hemodialysis patients. This study was designed to investigate the effects of l-carnitine supplement on plasma coagulation and anticoagulation factors in hemodialysis patients. Methods: Thirty-six hemodialysis patients were randomly assigned to either a carnitine or a placebo group. Patients in the carnitine group received 1000 mg/day oral l-carnitine for 12 weeks, whereas patients in the placebo group received a corresponding placebo. At baseline and the end of week 12, 5 mL blood was collected after a 12- to 14-hour fast and plasma fibrinogen concentration, activity of plasma protein C, coagulation factors V, VII, IX, and serum concentrations of tissue plasminogen activator (tPA), plasminogen activator inhibitor type-1 (PAI-1), free carnitine, and C-reactive protein (CRP) were measured. Results: In the carnitine group, mean serum free carnitine concentration increased significantly to 150% of baseline (p < 0.001), whereas plasma fibrinogen and serum CRP had 98 mg/dL (p < 0.01) and 41% (p < 0.01) significant decreases, respectively, at the end of week 12 compared with baseline. The reductions were significant compared with the placebo group (p < 0.05). No significant differences were observed between the two groups with regard to mean changes of the activity of plasma protein C, coagulation factors V, VII, IX, and serum PAI-1 to tPA ratio. Conclusion: l-Carnitine supplement reduces serum CRP, a marker of systemic inflammation, and plasma fibrinogen, an inflammation-related coagulation factor, in hemodialysis patients. Therefore, l-carnitine may play an effective role in preventing cardiovascular diseases in these patients.
INTRODUCTION
Cardiovascular disease (CVD) is common in hemodialysis patients and accounts for approximately 50% of mortality in these patients.Citation1 Conventional risk factors for CVDs such as lipid abnormalities, hypertension, and diabetes cannot fully explain the increased mortality in these patients.Citation2 Some studies have indicated that hypercoagulability occurs frequently in dialysis patientsCitation3–5 because of decreased plasma tissue plasminogen activator (tPA) concentration and the activity of protein C, and increased plasma concentrations of fibrinogen, plasminogen activator inhibitor type-1 (PAI-1), PAI-1 to tPA ratio, and the activity of coagulation factor VII.Citation4–14 Hypercoagulability is an important risk factor for thrombosis and its complications such as ischemic heart disease, stroke, and vascular access failure in hemodialysis patients and contributes substantially to high mortality in this population.Citation3–5,Citation15
l-Carnitine has largely been studied and is also frequently used in hemodialysis patients, but according to the available literature, no study investigated the effects of l-carnitine supplement on plasma coagulation (including fibrinogen, coagulation factors V, VII, IX, and PAI-1) and anticoagulation (such as protein C and tPA) factors in hemodialysis patients. Only one study in patients with peripheral vascular diseases showed that l-carnitine supplement could cause an increase in plasma tPA and a decrease in plasma PAI-1.Citation16 Therefore, the present study was designed to investigate the effects of l-carnitine supplement on plasma coagulation and anticoagulation factors in hemodialysis patients.
MATERIALS AND METHODS
This study was a randomized, double-blind, placebo-controlled trial. The minimum sample size estimation for each group was 16 at a power (1-β) of 90% and α = 0.05 for a two-arm parallel study with two-tailed testing to detect a difference of 6.5 ng/mL in serum PAI-1 concentration with a pooled standard deviation of 5.8 ng/mL, obtained from Pola et al.’s study.Citation16 Thirty-six adult hemodialysis patients (15 men and 21 women) in the age range of 20–74 years were recruited from Soodeh hemodialysis center in Islamshahr, Iran. Patients enrolled in the study did not have inflammatory diseases, diabetes, infectious diseases including hepatitis, and none of them received l-carnitine, ω3-fatty acids supplement, vitamin E and/or C supplements, steroidal and/or nonsteroidal anti-inflammatory drugs, anticoagulant drugs, and valproate sodium as an antiepileptic drug. All participating patients had an arteriovenous fistula and were treated with hemodialysis three times a week (4 hours per session) using bicarbonate dialysate and polysulfone capillary dialyzers, except three patients with two times per week and four patients with four times per week. Heparin sodium, as an anticoagulant, was infused through the catheter transporting the blood into the dialysis machine at a dose of 2500 IU at the beginning of hemodialysis and followed by another infusion of 2500 IU 2 hours after the initiation of hemodialysis. During the study, the hemodialysis procedure, dose of heparin, and type of dialyzer did not alter for each patient.
The study protocol was approved by the Ethics Committee of the National Nutrition and Food Technology Research Institute of Iran. The study was in adherence with the Declaration of Helsinky. Written informed consent was obtained from all patients.
Patients were randomly allocated to either a carnitine or a placebo group. Subjects in the carnitine group received orally a vial containing 1000 mg l-carnitine daily for 12 weeks, whereas the placebo group received a corresponding placebo vial. Subjects were advised not to change their dietary habits, physical activities, and drug regimens. At baseline and the end of week 12, blood samples were drawn from each patient after a 12- to 14-hour fast before hemodialysis. Blood samples were divided into two test tubes. The first tubes, containing 3.2% sodium citrate, and the second tubes, without any anticoagulant, were centrifuged at 1000 g for 15 minutes, to separate plasma and sera, respectively.
The concentration of plasma fibrinogen was determined by the Clauss method, and the activity of plasma protein C and coagulation factors V, VII, and IX was determined by various clotting methods, using commercial kits (Diagnostica Stago, Asnieres, France) and a STA Compact Coagulation Analyzer (Diagnostica Stago). The coefficient of variation (CV) for plasma fibrinogen, protein C, and coagulation factors V, VII, and IX was 3.3%, 4.3%, 3.8%, 7.3%, and 6.4%, respectively. The serum PAI-1 and tPA were measured using enzyme-linked immunosorbent assay (ELISA) kits (Hyphen BioMed, Neuville-sur-Oise, France). The CVs for serum PAI-1 and tPA were 4.6% and 5.6%, respectively.
The serum CRP concentration was determined using ELISA kits (Diagnostics Biochem Canada (dbc), London, Canada), with a CV of 5%. The serum free carnitine concentration was measured enzymatically using commercial kits (Roche Applied Science, Penzberg, Germany), with a CV of 3%.
Patients were weighed after hemodialysis at baseline and the end of weeks 6 and 12. In addition, the dietary intakes of subjects were assessed using a 2-day dietary recall (1 dialysis day and 1 non-dialysis day) at baseline and the end of weeks 6 and 12. Patients' diets were analyzed using Nutritionist IV software (N Squared Computing, San Bruno, CA, USA).
At baseline and weeks 6 and 12, dialysis adequacy based on Kt/V index was determined for each patient by a Kt/V calculator software using information recorded in patient files, including predialysis blood urea nitrogen (BUN) concentration, postdialysis BUN, dialysis session length, postdialysis weight, and ultrafiltration volume.Citation17
Statistical analysis
Statistical analysis of the data was performed using Statistical Package for the Social Sciences (SPSS, Inc., Chicago, IL, USA) for windows version 15.0. A χ2 test was used to compare qualitative variables between the two groups. Because all quantitative parameters according to the Kolmogorov–Smirnov test had normal distributions, we used t-test and paired t-test to compare parameters between and within groups, respectively. In addition, because dietary and anthropometric parameters, and dialysis adequacy were measured three times during the study, analysis of variance for repeated measurements was used to compare data between various times. The results are expressed as mean ± SD, and differences were considered significant at p ≤ 0.05.
RESULTS
The baseline characteristics of patients did not differ significantly between the two groups ().
Table 1. Baseline characteristics of hemodialysis patients in the l-carnitine and placebo groups
The anthropometric and the dietary factors, and the hemodialysis adequacy had no significant differences between the two groups at the baseline and the end of weeks 6 and 12. In addition, these factors did not significantly change within each group during the study ().
Table 2. Dialysis adequacy, and anthropometric and dietary factors in the l-carnitine and the placebo groupsFootnotea
Serum free carnitine concentration increased significantly in the carnitine group at the end of week 12 compared with baseline (p < 0.001), whereas no significant change was observed in the placebo group. The increase of serum free carnitine concentration in the carnitine group was significant in comparison with the placebo group (p < 0.001; ).
Table 3. Serum concentrations of free carnitine, CRP, and PAI-1/tPA, plasma fibrinogen concentration and activity of protein C and coagulation factors in the l-carnitine and the placebo groupsFootnotea
Plasma fibrinogen concentration decreased significantly in the carnitine group (p < 0.001) and the placebo group (p < 0.05) at the end of week 12 compared with baseline. The reduction of plasma fibrinogen concentration in the carnitine group was significantly more than the placebo group (p < 0.05; ).
The plasma activity of protein C and coagulation factors V and VII did not significantly change within each group during the study ().
The percentage of plasma coagulation factor IX activity decreased significantly in the carnitine group (p < 0.01) and the placebo group (p < 0.05) at the end of week 12 compared with baseline. However, the reduction in the percentage of plasma coagulation factor IX activity was not significantly different between the two groups ().
Serum PAI-1 to tPA ratio decreased significantly in the carnitine group (p < 0.01) and the placebo group (p < 0.01) at the end of week 12 compared with baseline. However, the reduction in serum PAI-1 to tPA ratio was not significantly different between the two groups ().
Serum CRP concentration decreased significantly in the carnitine group at the end of week 12 compared with baseline (p < 0.01), but no significant change was evident in the placebo group. The reduction of serum CRP concentration in the carnitine group was significant in comparison with the placebo group (p < 0.05; ).
DISCUSSION
Mean serum free carnitine concentration in both groups was in the range of 20.5–22 μmol/L, and this is lower than the range of 29–64 μmol/L reported in healthy subjects.Citation18,Citation19 This low concentration is caused possibly by reduced dietary intake, impaired de novo carnitine renal synthesis, and loss of free carnitine from body during hemodialysis.Citation18 Carnitine deficiency can result in muscle weakness, muscle cramps, myopathy, cardiomyopathy, cardiac arrhythmia, loss of body protein, cachexia, insulin resistance, altered plasma lipid profile, anemia, and resistance to erythropoietin.Citation20 In our study, the administration of l-carnitine significantly increased mean serum free carnitine to 150% of baseline level over a period of 12 weeks. This result is in agreement with previous studies.Citation18,Citation21,Citation22
Hypercoagulability has been reported to occur frequently in dialysis patients.Citation3–5 This is due to increased plasma concentrations of fibrinogen, PAI-1, PAI-1 to tPA ratio, and the activity of coagulation factor VII, and decreased plasma tPA concentration and the activity of protein C.Citation4–14
Hypercoagulability is a major cause of complications in hemodialysis patients, leading not only to possibly fatal complications such as ischemic heart disease or stroke, but also to thrombus formation in arteriovenous fistulas.Citation3–5,Citation15
In our study, l-carnitine supplement cause a 98 mg/dL decrease in plasma fibrinogen concentration and this reduction was significant as compared to the placebo group. We found no research in available literature about the effect of l-carnitine supplement on plasma fibrinogen concentration to be compared with the results of our study.
Fibrinogen is an acute phase protein that is produced in the liver. The synthesis is largely regulated by inflammatory cytokines, especially interleukin-6 (IL-6), and is increased in systemic inflammation state.Citation23 Therefore, the decrease of plasma fibrinogen concentration in the carnitine group may be because of the reduction in systemic inflammation by l-carnitine supplement. In this study, serum CRP concentration, a systemic inflammation marker, decreased significantly by 41% to confirm the above-mentioned mechanism. Few studies on the effect of l-carnitine supplement on serum CRP are available, and their results are in agreement with the result of our study.Citation24,Citation25 A meta-analysis on 31 prospective studies of major CVDs and nonvascular mortality showed that the hazard ratios per 100 mg/dL (or 1 g/L) increase in usual fibrinogen concentration for coronary heart disease and stroke were 2.4 and 2, respectively.Citation26 Therefore, the reduction of plasma fibrinogen concentration by l-carnitine supplement may play an effective role in reducing the risk of CVD and stroke in hemodialysis patients.
In this study, the percentage of activity of coagulation factors V, VII, IX, and protein C, as an anticoagulation factor, did not significantly change in the carnitine group compared with the placebo group. To date, no studies have examined the effects of l-carnitine supplement on the activity of factors V, VII, IX, and protein C to be compared with the results of our study.
The ineffectiveness of l-carnitine supplement on the plasma activity of the above-mentioned factors may involve the fact that these factors in contrast to fibrinogen are not related to inflammation.
In hemodialysis patients not only the possibility of blood coagulation and fibrin clot formation are increased, but also fibrinolysis is reduced because of increased serum PAI-1 concentration and PAI-1 to tPA ratio.Citation8–11,Citation27 In our study, serum PAI-1 to tPA ratio was significantly reduced in the two groups. This result shows that the observed decrease in the carnitine group was not attributable to l-carnitine supplement, because it was also evident in the placebo group. The only probable explanation for this reduction is that serum PAI-1 to tPA ratio undergoes some changes over the time. Pola et al. showed, in a quasi-experimental study, that the daily administration of 3 g propionyl l-carnitine supplement to non-renal failure patients, for 20 days, increased plasma tPA concentration and decreased plasma PAI-1 concentration and PAI-1 to tPA ratio.Citation16 Pola et al.’s results are not in accordance with findings of our study. This disparity may involve the fact that Pola et al.’s study had no placebo group and was conducted in non-renal failure patients.
In conclusion, l-carnitine supplement reduces serum CRP, a marker of systemic inflammation, and plasma fibrinogen, an inflammation-related coagulation factor, in hemodialysis patients, whereas it has no effect on coagulation and anticoagulation factors, which are not related to inflammation. Therefore, l-carnitine may play an effective role in preventing CVDs in these patients.
Acknowledgments
This study was supported by National Nutrition and Food Technology Research Institute of Iran.
The authors thank the staff of Soodeh Hemodialysis Center for their invaluable assistance, the staff of the hematology laboratory of Iranian Blood Transfusion Organization, and the staffs of the research laboratory of Research Institute for Endocrine Sciences and the research laboratory of National Nutrition and Food Technology Research Institute for their technical assistance. The authors also gratefully acknowledge the cooperation of the participating patients, without whom this investigation would not have been possible.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
REFERENCES
- Jungers P, Khoa TN, Massy ZA, Incidence of atherosclerotic arterial occlusive accidents in predialysis and dialysis patient: A multicentric study in the Ile de France district. Nephrol Dial Transplant. 1999;14:898–902.
- Stenvinkel P. Inflammation in end-stage renal failure: Could it be treated? Nephrol Dial Transplant. 2002;17(Suppl. 8):33–38.
- Ambuhl PM, Wuthrich RP, Korte W, Schmid L, Krapf R. Plasma hypercoagulability in hemodialysis patients: Impact of dialysis and anticoagulation. Nephrol Dial Transplant. 1997;12:2355–2364.
- Smits JHM, Liden J, Blankestijn PJ, Rabelink TJ. Coagulation and hemodialysis access thrombosis. Nephrol Dial Transplant. 2002;15:1755–1760.
- Song IS, Yang WS, Kim SB, Lee JH, Kwon TW, Park JS. Association of plasma fibrinogen concentration with vascular access failure in hemodialysis patients. Nephrol Dial Transplant. 1999;14:1137–1141.
- Vaziri ND, Gonzales EC, Wang J, Said S. Blood coagulation, fibrinolytic, and inhibitory proteins in end-stage renal disease: Effect of hemodialysis. Am J Kidney Dis. 1994;23:828–835.
- Oda H, Ohno M, Ohashi H. Coagulation and fibrinolysis factors in dialysis patients with and without ischemic heart disease. Adv Perit Dial. 2000;16:152–155.
- Segarra A, Chacon P, Martinez-Eyarre C, Circulating levels of plasminogen activator inhibitor type-1, tissue plasminogen activator, and thrombomodulin in hemodialysis patients: Biochemical correlations and role as independent predictors of coronary artery stenosis. J Am Soc Nephrol. 2001;12:1255–1263.
- Lottermoser K, Petras S, Poge U, The fibrinolytic system in chronic renal failure. Eur J Med Res. 2001;6:372–376.
- Molino D, De Santo NG, Marotta R, Anastasio P, Mosavat M, De Lucia D. Plasma levels of plasminogen activator inhibitor type 1, factor VIII, prothrombin activation fragment 1+2, anticardiolipin, and antiprothrombin antibodies are risk factors for thrombosis in hemodialysis patients. Semin Nephrol. 2004;24:495–501.
- Trimarchi H, Duboscq C, Genoud V, Plasminogen activator inhibitor-1 activity and 4G/5G polymorphism in hemodialysis. J Vasc Access. 2008;9:142–147.
- Deguchi K, Izumi K, Noguchi M, Coagulation and fibrinolysis in patients with chronic renal failure on maintenance hemodialysis (abstr). Nippon Ketsueki Gakkai Zasshi. 1989;52:140–146.
- Vaziri ND, Toohey J, Paule P, Alikhani S, Hung E. Coagulation abnormalities in patients with end-stage renal disease treated with hemodialysis. Int J Artif Organs. 1984;7:323–326.
- Uchida Y. Long-term hemodialysis and changes in variables of coagulation and fibrinolysis (abstr). Fukuoka Igaku Zasshi. 1991;82:576–585.
- Erdem Y, Haznedaroglu IC, Celik L, Coagulation, fibrinolysis and fibrinolysis inhibitors in hemodialysis patients: Contribution of arteriovenous fistula. Nephrol Dial Transplant. 1996;11:1299–1305.
- Pola P, De Martini D, Gerardino L, De Rossi S, Tondi P. The action of propionyl-L-carnitine on the vasal endothelium: Increased t-PA synthesis and a decrease in the activity of PAI-1. A preliminary study. Drugs Exp Clin Res. 1992; 18:343–348.
- Daugirdas JT, Stone JCV. Physiologic principles and urea kinetic modeling. In: Daugirdas JT, Blake PG, Ing TS, eds. Handbook of Dialysis. 3rd ed., Philadelphia: Lippincott Williams & Wilkins; 2001:15–45.
- Evans A. Dialysis-related carnitine disorder and levocarnitine pharmacology. Am J Kidney Dis. 2003;41(Suppl. 4):S13–S26.
- Alberty R, Albertyova D. Biological variation of free and total carnitine in serum of healthy subjects. Clin Chem. 1997;43:2441–2443.
- Guarnieri G, Biolo G, Vinci P, Massolino B, Barazzoni R. Advances in carnitine in chronic uremia. J Ren Nutr. 2007;17:23–29.
- Elisaf M, Bairaktari E, Katopodis K, Effect of L-carnitine supplementation on lipid parameters in hemodialysis patients. Am J Nephrol. 1998;18:416–421.
- Guarnieri GF, Ranieri F, Toigo G, Lipid lowering effect of carnitine in chronically uremic patients treated with maintenance hemodialysis. Am J Clin Nutr. 1980;33:1489–1492.
- Libby P, Simon DI. Inflammation and thrombosis. Circulation. 2001;103:1718–1720.
- Savica V, Santoro D, Mazzaglia G, L-carnitine infusions may suppress serum C-reactive protein and improve nutritional status in maintenance hemodialysis patients. J Ren Nutr. 2005;15:225–230.
- Duranay M, Akay H, Yilmaz FM, Senes M, Tekeli N, Yücel D. Effects of L-carnitine infusions on inflammatory and nutritional markers in hemodialysis patients. Nephrol Dial Transplant. 2006;21:3211–3214.
- Danesh J, Lewington S, Thompson SG, Plasma fibrinogen level and the risk of major cardiovascular diseases and nonvascular mortality: An individual participant meta-analysis. J Am Med Assoc. 2005;294:1799–1809.
- Undas A, Kolarz M, Kopec G, Tracz W. Altered fibrin clot properties in patients on long-term hemodialysis: Relation to cardiovascular mortality. Nephrol Dial Transplant. 2008;23: 2010–2015.