Determination of Free L-Carnitine Levels in Type II Diabetic Women with and Without Complications

ORIGINAL ARTICLE

Determination of free L-carnitine levels in type II diabetic women with and without complications

A Poorabbas1, F Fallah1, J Bagdadchi2, R Mahdavi2, A Aliasgarzadeh3, Y Asadi2, H Koushavar2 and M Vahed Jabbari2

1Ilam Medical University, Ilam, Iran; 2Faculty of Nutrition, Tabriz Medical University, Tabriz, Iran and 3Tabriz Medical University, Tabriz, Iran

Background: Studies on the determination of carnitine levels and nutritional status in patients of type II diabetes. Objective: We designed this study to determine changes of serum-free L-carnitine in type II diabetic women. Design: A cross-sectional study (case–control study). Setting: Clinical of Endocrinology and Metabolism, Sina Hospital, Pharmacological Research Center, Tabriz Medical university, Iran. Patients and methods: Taking into account the importance of the control of diabetes, in the present case-control study, the levels of serum-free L-carnitine, blood glucose and lipids, systolic and diastolic blood pressure, body mass index (BMI) and nutritional status assessed in the case and control groups which were selected by the simple sampling method. The control group (n ¼ 18) included patients with no complications and the case group (n ¼ 33) was grouped into three subgroups including patients with retinopathy, hyperlipidemia and neuropathy. Results: Study results indicated that the mean serum-free L-carnitine concentration in the case group was significantly lower than its mean concentration level in the control group, 39.6378.99 vs 53.4270.93 mmol/l, respectively (Po0.001). Serum-free L-carnitine in retinopathy, hyperlipidemia and neuropathy case subgroups were 39.0379.89, 39.6378.99 and 40.44712.50 mmol/l, respectively (P40.05). No significant difference was found between the serum-free carnitine levels of the case subgroups. The mean blood glucose, triglycerides, total cholesterol, low-density lipoprotein cholesterol, very low- density lipoprotein cholesterol levels and systolic and diastolic blood pressure were significantly higher in the case group than in the control group. Conclusion: In this study, the mean serum-free L-carnitine levels in diabetic patients with complications was almost 25% lower than in diabetic patients with no complications. On the basis of the study results, carnitine supplementation in diabetic patients, especially in patients with diabetes complications, might be useful. European Journal of Clinical Nutrition (2007) 61, 892–895; doi:10.1038/sj.ejcn.1602594; published online 21 February 2007

Keywords: free carnitine; diabetes complications

Introduction renal diseases), the relation of glycemia to macrovascular disease in type II diabetes is more modest (ADA, 2003). There Diabetes mellitus is often accompanied by dyslipidemia and are studies that have reported abnormal carnitine metabo- dysfunction in glucose and protein metabolism (Marc et al., lism in diabetes (Williamson et al., 1992). 2005). Type II diabetes is associated with a two- to fourfold The synthesis of L-carnitine requires two essential amino excess risk of coronary heart disease (CHD). Although the acids (lysine and methionine), iron (Fe2 þ ), and vitamins C, degree of glycemia in diabetic patients is strongly related to B6 and niacin in the form of nicotinamide dinucleotide the risk of microvascular complications (retinopathy and (NAD) (Seim et al., 2001). Carnitine is synthesized primarily in the liver and also in the kidney, and must be transported Correspondence: A Poorabbas or F Fallah, Ilam Medical University, Ilam Ms 98, to other tissues. It is most concentrated in tissues that use Iran. fatty acids as their primary dietary fuel, such as skeletal and E-mail: [email protected] Received 15 September 2005; revised 2 November 2006; accepted 3 cardiac (heart) muscle (Arrigoni-Martelli and Caso, 2001). November 2006; published online 21 February 2007 Carnitine acyltransferases have crucial functions in fatty acid L-carnitine level in Type II diabetic women A Poorabbas et al 893 metabolism. Members of this enzyme family show distinctive pathy. Diagnosis of retinopathy was carried out by fundo- substrate preferences for short-, medium- or long-chain fatty scopic examination of the eye, neuropathy with acids. The molecular mechanism for this substrate selectivity is examination with current methods. All the patients were not clear because so far only the structure of carnitine screened for lipid profile. Serum total cholesterol, high- acetyltransferase has been determined (Gerwald et al., 2005). density lipoprotein (HDL), triglycerides (TG), low-density Carnitine deficiency may be primary or secondary. In all cases, lipoprotein (LDL) and glucose concentrations were measured secondary carnitine deficiencies are characterized by decreased using enzymatic methods. availability of free L-carnitine.Insuchcases,totalcarnitinelevels The sample was transported on dry ice to the laboratory may be normal, but free L-carnitine levels are decreased. where serum was separated from the cells. Serum was stored Factors such as sex, age, nutritional status, fasting and at À201C before batch analysis. Free L-carnitine concentra- disease have been cited as influencing plasma carnitine tion in serum was estimated by using the Boehringer- levels in man (Cederblad, 1987). Plasma carnitine levels have Manheim kit. This kit utilizes the enzymatic UV test been found to be normal or decreased in studies on carnitine described by Weiland (1985). metabolism in diabetes mellitus patients (Okuda et al., 1987; The results are presented as mean7standard deviation Winter et al., 1987; Pregnant et al., 1993). (s.d.). The cutoff level for statistical significance was set at Diabetes in animals and human lead to deterioration Po0/05. Unpaired Student’s t-test (two-tailed) and analysis of carnitine and lipid metabolism (Depalo et al., 1981; of variance (ANOVA) test were used to determine differences Cederblad et al., 1982; Hoppel and Genuth, 1982; Stearns, between independent groups. 1983; Brooks et al., 1985; Aoshima et al., 1993). It is well known that streptozotocin-induced diabetic rats develop myocardial carnitine deficiency (Vary and Neely, 1982; Results Brooks et al., 1985; Aoshima et al., 1993). In several studies, decreased carnitine concentrations in diabetic animals have According to the study results, increase in age was positively been observed (Vary and Neely, 1982; Aoshima et al., 1993). related with increasing BMI. Total dietary fiber consumption Acute hypercarnitinemia stimulates nonoxidative glucose was lower than normal levels (25 g/day). Table 1 shows the disposal during euglycemic hyperinsulinemic clamp in concentrations of serum glucose and plasma lipids profile. healthy volunteers. Similar results were obtained in type II Fasting blood glucose (FBG), TG, total cholesterol, LDL-c and diabetic patients (Cattolica et al., 2004). L-carnitine, which is VLDL-c levels were significantly higher in the case group required for mitochondrial free fatty acid (FFA) transport/ than in the control group (Table 2). oxidation, has been reported to improve vascular function in Study results indicated that the mean serum-free L- subjects with diabetes and heart disease (Shankar et al., carnitine concentration in the case group was significantly 2004). The above studies suggest a relationship between lower than its mean concentration (39.6378.99 vs diabetes and FFA and plasma carnitine. 53.4270.93 mmol/l, respectively, Po0.001 level) in the The aim of the present experimental study was to control group (Table 3). Free carnitine (FC) levels of the investigate the content of free L-carnitine in type 2 diabetic groups with hyperlipidemia, and polyneuropathy and women with and without complications. retinopathy were not significantly different when compared with each other (P40.05) (Table 3).

Methods Table 1 Characteristics of anthropometrics, blood pressure, FBS and In the present case – control study, the levels of serum-free lipid profiles L-carnitine, blood glucose and lipids, systolic and diastolic Case Control P value blood pressure, body mass index (BMI) and nutritional factors participating in carnitine biosynthesis (iron, vitamins Age 54.4578.97 5278.71 0.350 Weight (kg) 73.7379.50 58.5672.59 0.001 B6, niacin and vitamin C) were determined in 33 diabetic o Height (m) 155.55 75.75 154.0675.13 0.363 women with complications (case group) and 18 diabetic BMI (kg/m2) 29.4774.32 23.6671.74 o0.001 women without complications (control group) of the same Systolic BP (mm Hg) 155.61728.61 13578.74 0.005 age (30–65 years), who were selected by the simple sampling Diastolic Bp (mm Hg) 91.8278.91 85.8377.91 0.021 method. FBS (mg/dl ) 212.48786.40 147.89723.06 0.003 Total-cholesterol (mg/dl ) 238.52757.12 135.06710.71 0.001 In this study, patients with CHD, anemia, chronic renal o LDL-c (mg/dl ) 160.03720.63 75.67718.63 o0.001 failure, heart failure, liver cirrhosis, antiepileptic drug users VLDL-c (mg/dl ) 48.5677.20 19.5074.82 o0.001 and those who receive carnitine treatment were excluded TG (mg/dl ) 298.307116.50 120.17722.59 o0.001 from the study. The patients were grouped into four groups, HDL-c (mg/dl ) 36.6778.34 39.6176.62 0.201 the control group included the patients with no complica- Abbreviations: BMI, body mass index; FBS, fasting blood sugar; HDL-c, high- tions and the case group was divided into three subgroups, density lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol; TG, patients with retinopathy, hyperlipidemia and polyneuro- triglycerides; VLDL-c, very low-density lipoprotein cholesterol.

European Journal of Clinical Nutrition L-carnitine level in Type II diabetic women A Poorabbas et al 894 Table 2 Characteristics of nutrition status and factors affected in the In several studies, decreased carnitine concentrations in synthesis of carnitine both plasma and myocardium in diabetic animals have been Case group N ¼ 33 Control group N ¼ 18 P value observed. Inokuchi reported that the combination of impaired glucose tolerance (IGT) with simple obesity may Calories (kcal) 1616.30.7397.66 1710.287208.35 0.356 trigger the acceleration of hepatic ketogenosis in conjunc- Protein (g) 58.98717.65 85.85745.18 0.004 tion with an elevated SCAC (short-chain acylcarnitine) and Carbohydrate (g) 209.18761.32 264.06760.65 0.004 an increased AC/FC ratio (acylcarnitine/free carnitine) Dietary fiber (g) 10.5074.08 14.9978.18 0.012 Lysine (mg) 585.417407.87 509.717151.66 0.453 (Inokuchi et al., 1995). Susa C found a relation between the Methionine (mg) 291.457122.26 393.997216.08 0.035 levels of urine glucose and ketones and the level of 2 þ Iron (Fe ) (mg) 16.7675.34 28.0179.43 o0.001 acylcarnitine. They also showed that carnitine deficiency 7 7 Vitamin C (mg) 38.25 17.35 49.54 721.94 0.049 and relative insufficiency may be an overlooked component Vitamin B3 (mg) 13.4974.19 23.2578.13 o0.001 in the management of diabetes (Winter et al., 1987). Keller Vitamin B6 (mg) 0.7070.20 0.9770.50 0.008 et al. (1998) suggested that acute L-carnitine supplementa- Values are X7s.d. tion significantly improves the recovery of the ischemic myocardium in diabetic and euglycemic rats. The results Table 3 Free L-carnitine levels of experimental groups showed that the total and free carnitine levels are low in diabetes mellitus complications (Tamamogullart et al., 1999). Groups N L-carnitine P value Irat et al. (2003) suggested that the beneficial effects of R etinopathy 20 39.0379.89 L-carnitine treatment partially improve vascular reactivity Hyperlipidemia 33 39.6378.99 and antioxidant property beyond its reduction of plasma Neuropathy 20 40.44712.50 40.05 lipids and it may have an important therapeutic approach in 7 Case 33 39.63 8.99 the treatment of diabetic vascular complications. Control 18 53.4273.95 o0.001 In our study, the mean serum-free Lcarnitine levels in N, patient number. Values are X7s.d. diabetic patients with complications was almost 25% lower than diabetic patients with no complications. It could be supposed that this difference might be due to Statistical regression analysis test showed a positive increased carnitine acylation, increased acyl-L-carnitine ex- significant relationship between serum-free L-carnitine and cretion or its decreased renal reabsorption. This difference the percent of protein intake (r ¼ 0.35, P ¼ 0.04), and also the may be the result of increased carnitine requirement in lysine intake (r ¼ 0.34, P ¼ 0.04), in the case group. diabetic patients with complications also. Steiber et al. (2004) reported that plasma carnitine concentration positively correlates with the dietary intake Discussion of carnitine. This study showed the possible role of protein and lysine Carnitine is necessary to maintain normal cardiac function. in carnitine biosynthesis (in the case group). However, these Decreased levels of carnitine in cardiac tissue may impair relationships were not observed in the control group. myocardial function, as it has been shown that carnitine No significant relationship was found between serum-free deficiency can produce cardiomyopathy (Akisu et al., 2002). L-carnitine levels and the other factors participating in the It has been thought that there is a relation between carnitine biosynthesis cycle, including methionine, ascorbic insufficient control of metabolism and complications in acid, niacin, pyrodoxin and iron intakes, in either of the diabetes (Morabito et al., 1993). Also, elevated FFA levels groups. diminish glucose-induced insulin secretion by a glucose– Also, no significant relationship was observed between fatty acid cycle in which fatty acid oxidation inhibits glucose serum-free L-carnitine and blood glucose, lipid profile, BMI oxidation by decreasing pyruvate dehydrogenase (PDH) and systolic and diastolic blood pressure in any of the activity and increasing PDH kinase activity (Zhou et al., groups. 1996). It has been thought that carnitine plays an important On the basis of the study results, carnitine supplementa- role in diabetes and its complications (Tamamogullart et al., tion in diabetic patients, especially in patients with diabetes 1999). De grandis acetyl-L-carnitine was effective in improv- complications, might be useful. ing neurophysiological parameters and in reducing pain in patients with diabetic neuropathy. Our study indicated that free L-carnitine concentration in the case group was References significantly lower than its mean concentration level in the control group. The mean blood glucose, TG, total cholester- Akisu M, Kultursay N, Coker I, Huseyinov A (2002). Myocardial and ol, LDL-c, VLDL-c levels and systolic and diastolic blood hepatic free carntine concentrations in pups of diabetic female rats. Ann Nutr Metab 46, 45–48. pressure were significantly higher in the case group than in American Diabetes Association (2003). Management of dyslipidemia the control group. in adults with diabetes. Diabetes Care 26, S83–S86.

European Journal of Clinical Nutrition L-carnitine level in Type II diabetic women A Poorabbas et al 895 Aoshima S, Fujisawa S, Kobayayashi A (1993). Changes in the Morabito E, Serafini S, Corsico N, Martelli AE (1993). Acetyl-L- subcellular distribution of free carnitine and its acyl derivatives in carnitine effect on nerve conduction velocity in streptozotocin – diabetic rat hearts following treatment with L-carnitine. Jpn Heart J diabetic rats. Drug Res 43, 343–346. 34, 763–772. Okuda Y, Kawi K, Murayama Y, Yamashita K (1987). Postprandial Arrigoni-Martelli E, Caso V (2001). Carnitine protects mitochondria changes in plasma ketone body and carnitine levels in normal and and removes toxic acyls from xenobiotics. Drugs Exp Clin Res 27, non-insulin –dependent diabetic subjects. Endocrinol Japan 34, 15–22. 27–49. Pregnant P, Kaiser E, Schernthaner G (1993). Noeffect of insulin Brooks SD, Bahl JJ, Bressler R (1985). Carnitine in thestreptozotocin – treatment or glycemic improvement on plasma carnitine levels in diabetic rat. J Nutr 115, 1267–1273. type 2 diabetic patients. Clin Invest 71, 610–612. Cattolica CS, Gemelli LA (2004). Carnitine in type 2 diabetes. Ann NY Seim H, Eichler K, Kleber H (2001). L(-)-Carnitine and its precursor, Acad Sci 1033, 99–107. gamma-butyrobetaine. In: Kramer K, Hoppe P, Packer L (eds) Cederblad G (1987). Effects of diet on plasma carnitine levels and Nutraceuticals in Health and Disease Prevention. New York: Marcel urinary carnitine excretion in humans. Am J Clin Nutr 45, Dekker, Inc. pp 217–256. 720–729. Shankar SS, Mirzamohammadi B, Walsh JP, Steinberg HO (2004). Cederblad G, Hermansson G, Ludvigsson J (1982). Plasma and urine L-carnitine may attenuate free fatty acid-induced endothelial carnitine in children with diabetes mellitus. Clin Chim Acta 125, dysfunction. Ann NY Acad Sci 1033, 189–197. 207–217. Stearns SB (1983). Carnitine content of skeletal and cardiac muscle De Grandis D, Minardi C (2002). Acetyl-L-carnitine (levacecarnine) in from genitically diabetic and control mice. Biochem Med 29, 57–63. the treatment of diabetic neuropathy. A long-term, randomised, Steiber A, Kerner J, Hoppel CL (2004). Carnitine: a nutritional, double-blind, placebo-controlled study. Drugs RD 3, 223–231. biosynthetic, and functional perspective. Mol Aspects Med 25, Depalo E, Gatti R, Sicolo N, Padovan D, Vetor R, federspil G (1981). 455–473. Plasma and urine free L-carnitine in human diabetes mellitus. Acta Tamamogullart N, Silig Y, Icagasioglu S, Atalay A (1999). Carnitine Diabetol Lat 18, 91–95. deficiency in diabetes mellitus complications. J Diab Camp (5/6), Donath MY, Ehses JA, Maedler K, Schumann DM, Ellingsgaard H, 251–253. Eppler E et al. (2005). Mechanisms of b-Cell Death in Type 2 Vary TC, Neely JR (1982). A mechanism for reduced myocardial Diabetes. Diabetes 54, S108–S113. carnitine content in diabetic animals. Am J Physiol 243, Gerwald J, Yu-Shan H, Liang T (2005). Crystal structure of mouse H154–H158. carnitine octanoyltransferase and molecular determinants of Weiland OH (1985). Enzymatic uv test for the determination of substrate selectivity. J Biol Chem 280, 744–783. L-carnitine. In: Bergmeyer HU (ed) 3rd edn, vol VIII, Methods of Hoppel CL, Genuth SM (1982). urinary excretion of acetyl carnitine enzymatic analysis. VCH Verlagsgesellschaft, Weinheim, Deerfield during human diabetic and fasting ketosis. AM J Physiol 243, Beach. pp 481–488. E168–E172. Williamson JR, Arrigoni ME, Martelli E (1992). The roles of Inokuchi T, Imamura K, Nomura K, Nomoto K, Isogai S (1995). glucosoinduced metabolic hypoxia and imbalances in carnitine Changes in carnitine metabolism with ketone body production in metabolism in mediating diabetes – induced vascular dysfunction. obese glucose -intolerant patients. Diab Res Clin Prac 30, 1–7. Int J Clin Pharmacol Res 12, 247–252. Irat AM, Aktan F, Ozansoy G (2003). Effects of L-carnitine treatment Winter CS, Simon M, Zorn ME (1987). Relative carnitine insuffi- on oxidant/antioxidant state and vascular reactivity of streptozo- ciency in children with type I diabetes mellitus. AJDC 143, tocin-diabetic rat aorta. J Pharm Pharmacol 55, 1389–1395. 1337–1339. Keller VA, Toporoff B, Raziano RM, Pigott JD, Mills NL (1998). Zhou YP, Berggren PO, Grill V (1996). A fatty acid- induced decrease Carnitine supplementation improves myocardial function in in pyruvate dehydrogenase activity is an important determinant hearts from ischemic diabetic and euglycemic rats. Ann Thorac of b- cell dysfunction in the obese diabetic db/db mouse. Diabetes Surg 66, 1600–1603. 45, 580–586.

European Journal of Clinical Nutrition