Systemic carnitine deficiency

Abstract: The effect of haemodialysis on peripheral blood lymphocyte levels of total carnitine (TC), free carnitine (FC) and acylcarnitine (AC), and on the ratio of AC to FC, is investigated in 20 healthy subjects (13 men and seven women) and 27 patients (10 men and 17 women) with chronic pyelonephritis. The mean predialysis peripheral lymphocyte levels of TC, FC and AC were not significantly different from those in the healthy controls (P >0.OS). However, after haemodialysis, peripheral lymphocyte levels of TC, FC and AC decreased significantly, compared with either predialysis (TC: 40%, FC: 37%, AC: 52%) or healthy controls (TC: 61%, FC: 63%, AC: 50%) (P

Key words: Carnitine. Hemodialysis. Kidney. Lymphocytes. Pyelonephritis.

Introduction

Carnitine (L-3-hydroxy-4-N-trimethylaminobutyrate) is a quaternary amine synthesised from lysine and methionine in the liver, kidney and brain. It is an essential cofactor and plays an important role in fatty acid metabolism.1 Since acyl-coenzyme A (acyl-CoA) esters cannot penetrate the inner mitochondrial membrane, they are transesterified to fatty acylcarnitine (AC) esters by carnitine palmitoyltransferase (EC2.31.12) and translocated across the inner mitochondrial membrane by carnitine:palmitoylcarnitine translocase into the mitochondrial matrix, where they undergo Beta-oxidation.2 Carnitine also helps to maintain mitochondrial acetyl-CoA/CoASH homeostasis;3 a function of great significance as CoASH is an intermediary in several metabolic pathways. Another established function of carnitine is the detoxification of certain poorly metabolised branched-chain acyl-CoAs that are generated from amino acid catabolism inside mitochondria. 1,2

Carnitine occurs widely in nature and is believed to be present in all animals and in many plants and microorganisms. However, its concentration varies widely between species and tissues.1 The highest levels of carnitine are reported in tissues that require large amounts of energy, such as skeletal and heart muscle.3,4 Carnitine exists either in free (FC) form or esterified as AC. Many physiological and pathological conditions affect its serum and urine levels, and its tissue distributions in both humans and animals; these include diabetes, renal and hepatic diseases, and some metabolic disorders.5-9

Carnitine is of interest to nephrologists because of reports that patients with end-stage renal disease (ESRD) on haemodialysis (HD) have abnormalities of carnitine metabolism.6-8 Carnitine deficiency may adversely affect the oxidation of fatty acids in tissues, and further aggravate abnormal lipid metabolism in patients with ESRD.9

Peripheral lymphopenia is well recognised in patients with chronic renal failure (CRF)10 and in those on HD." Recent investigation of the pathophysiological mechanisms of uraemia-associated immunodeficiency has focused on the dual activation versus deficiency state of immunocompetent cells. Despite major advances in HD treatment, significant improvement in the immune status of patients with CRF has not been achieved.12 Carnitine involvement in energy metabolism has been studied extensively in liver, skeletal muscle,4 heart,13 kidney14 and brain;15 however, its importance in the immune system remains unclear.

Carnitine deficiency syndromes can cause serious health problems16 and HD can reduce serum levels of total carnitine (TC), FC and AC in patients with chronic pyelonephritis (CPN), compared with either healthy controls or predialysis patients.7 In this study, the influence of HD on carnitine levels in peripheral lymphocytes in Saudi patients with CPN is assessed.

Materials and methods

L-carnitine hydrochloride, tris-(hydroxymethyl) aminomethane, 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), ethylenediaminetetraacetic acid disodium salt (EDTA), the lithium salt of acetyl coA, carnitine acetyltransferase (CAT) (EC 2.3.1.7) and Histodenz gradient solution (14%) were obtained from the Sigma Chemical Company, St Louis, MO, USA. Catalase (EC 1.1 .6) was obtained from Winlab, Maidenhead, UK. Other reagents were of analytical grade and glassdistilled water was used throughout.

All subjects were Saudis. The normal group of 20 healthy volunteers comprising 13 males (age: 31 +/- 11) and seven females (age: 30.9 +/- 2.4), and showed no chronic active disease, were not on medication, and had a normal physical examination. Twenty-seven patients with CPN comprised 10 males (age: 47.5 +/- 12.5) and 17 females (age: 36.7 +/- 13), and attended the Riyadh Armed Forces Hospital, Riyadh, Saudi Arabia. All subjects were not known to be smokers or alcoholics, and none of the female subjects had taken the oral contraceptive pill for at least three months. Other characteristics were described previously.7 All subjects participating in this study gave informed consent.

Chronic pyelonephritis is thought to start in early childhood and result from a combination of vesicoureteric reflux and urinary infection.17 It is characterised by enlargement and distortion of the calices and scarring of the overlying renal cortex.18 The criteria on which the diagnosis of CPN was based are described elsewhere.19 All patients had the typical morphological changes of caliectasis and scarring on intravenous urography.Those with a history of analgesic abuse, diabetes mellitus, papillary necrosis, renal tuberculosis, renal calculi or other systemic renal disease were excluded from the study.

Patients had not been treated previously with carnitine, and those with evidence of hypothyroidism, pregnancy or uncontrolled arterial hypertension were excluded from the study. At the time of enrolment, all patients were in good clinical condition and had stable haematological parameters for at least three months before entry into the study. Clinically stable patients were defined as those who were generally compliant with their haemodialysis regimens, diet and fluid intake. All patients suffered from CPS and no other selection criteria were used, as the purpose of this study was to evaluate carnitine levels in peripheral lymphocytes in patients with fundamental characteristics in common (type of dialysis, equipment used and absence of concomitant pathology [diabetes mellitus, severe liver disease or other diseases]) that might have a major influence on carnitine metabolism. Patients were encouraged not to modify their eating habits during the study.

The patients' current medications (mainly antihypertensive drugs, digitoxin, Beta-blockers, antacids, antiarrhythmic drugs or phosphate binders) were not changed during this study . Patients were not excluded from the study if they were receiving drugs which may elevate plasma lipids, such as Beta-adrenergic blocking agents. None had hepatitis-associated antigenaemia or active symptomatic bacterial or viral infection, and none was receiving immunosuppressive drugs.

Patients were on four-hour acetate HD (35 mmol/L), three times per week, using a Fresenius machine (Fresenius, Hamburg, Germany) and Fresenius polysulphone Hi-flux-filter F 60 dialyser of a size suited to the surface area of the patient. The acetate HD dialysate contained sodium (138 mmol/Il), potassium (1 mmol/I.), calcium (1.75 mmol/L), chloride (109.1 mmol/L), magnesium (0.75 mmol/L), acetate (35 mmol/I,) and dextrose monohydrate (2.2 g/L). Dialysis flow rate was 500-800 mL/min, with blood flow rate of 200-300 mL/min. These two rates did not change during the observation period. As anticoagulant, 2000 u heparin were used at the start of HD, followed by continuous heparin administration at a rate of 500-1000 u/h. The required infusion rate was based on both the patient's sensitivity to heparin and the heparin half-life.

Sample collection

Blood samples for lymphocyte isolation were drawn from patients' fistulas prior to dialysis (after overnight fasting) and after dialysis. Samples from healthy controls were obtained from an antecubital vein after an overnight fast. All samples were used for cell counts and routine determination of electrolytes, blood urea, creatinine, glucose, cholesterol, triglycerides, haemoglobin concentration, hematocrit and albumin using a Technicon autoanalyser as reported previously.7

Isolation of peripheral lymphocytes

Lymphocytes were isolated by the modified method of Boyum,20 using Histodenz gradient solution (14% [w/v]) containing Nycodenz (0.44%/i) [w/v]) and 5 mol/L tricine (pH 7.2, density 1.078 g/mL) (Sigma Chemical Company). Anticoagulated whole blood was mixed with an equal volume of saline solution for immediate processing. Diluted blood (6 mL) was overlaid onto the Histodenz solution (3 mL) and centrifuged at 800 x g for 25 min at room temperature. Lymphocytes formed a white laver at the interface between the plasma and Histodenz solution. Lymphocytes were collected, washed with saline solution and centrifuged at 400 xg for 10 min at room temperature to remove trace amounts of contaminating platelets. They were resuspended in saline solution and a sample taken for lymphocyte count, using a Coulter counter (Coulter STKS, Coulter Electronics Ltd, Luton, England). Each cell suspension was adjusted to 10^sup 6^ cells/mL.

Carnitine analysis

FC extraction was performed as described previously,21 whereas TC extraction was modified as follows: a sample of cell suspension was added to 0.5 mol/L KOH (pH>13) and homogenised in a stainless steel OmniMixer (Omni International, Gainesville, VA, LTS USA). The homogenate was incubated at 65oC for I h. Icecold perchloric acid (PCA, 2 mol/L) was added (pH

A sample of PCA supernatant was neutralised carefully with I mol/L K^sub 2^CO^sub 3^ in an ice bath. Prior to measuring carnitine, free thiol groups in the neutralised sample extract were oxidised with hydrogen peroxide reagent (100 mmol/L Tris [pH 7.8], 1.25 mmol/L EDTA [pH 8], 3l?o hydrogen peroxide). The mixture was incubated for 10 min at room temperature and then excess peroxide was destroyed by adding catalase (5 U).

Carnitine was measured enzymatically,23 and details of the optimisation, linearity, specificity, precision and reproducibility of the method were described previously.13 The method is based on the reaction of carnitine with acetyl-CoA, catalysed by CAT (EC 2.3.1.7), forming CoASH, which is detected by reaction with DT\B. Typically, the assay contained hydrogen peroxide-oxidised extract (1 mL), 0.13 mol/I. DTNB (pH 7.8) and 0.15 mmol/L acetyl-CoA. 'I`he reaction was initiated by adding CAT (0.9 U). Increase in absorbance was measured at 412 nm using an Ultrospec 2000 UV/visible spectrophotometer (Pharmacia Biotech Ltd, Cambridge, UK). Carnitine concentration was calculated with reference to absorbances obtained with standard carnitine solutions.

Statistical analysis

Samples were run in duplicate and results expressed as mean +/- SD (nmol/mg protein). The analysis of variance (one-way ANOVA) was used for between-group comparison. For multiple group comparison, P value was adjusted using the Newman-Keuls multiple comparisons test. Bartlett's test was used for homogeneity of variances. Spearman correlation analysis was used to examine the association between variables. Means were considered significant if P

Results

Levels of TC, FC and AC in peripheral lymphocytes, and the AC/FC ratio are shown in Fig. 1. Predialysis levels were not significantly different from healthy control means (P>0.05). However, after HD, levels declined significantly compared with those in healthy controls (FC: 63%, AC: 50"o and TC: 61%o, P

The AC/FC ratio was not significantly different in predialysis patients compared to those in healthy controls (P >0.05). However, after HD, this ratio was significantly higher than in either predialysis or healthy controls (control: 38%, predialysis: 41%, P

Discussion

The current study confirmed previous findings that peripheral lymphocytes from healthy subjects contain significant amounts of carnitine.24 This is surprising because these cells lack mitochondria and are energetically independent of fatty acid oxidation; therefore, there is no demand for classical carnitine-mediated fatty acid transport. The role of carnitine in peripheral lymphocytes remains unclear; however, as suggested by Cress et al.,24 it is possible that carnitine acts as a reservoir of immediately accessible activated acyl groups for metabolism.

The results presented here are consistent with previous findings6-8 that blood carnitine levels decline significantly after HD, compared with those in either predialysis or healthy controls. In a preliminary study, samples were obtained from seven patients (two males, five females) at 1-, 2-, 4-, 6-, 8- and 10-h intervals after haemodialysis. Results showed that carnitine levels did not differ significantly up to four hours after dialysis; however, there was a slight increase after six hours, but this was not statistically significant compared to postdialysis levels.

The mechanism(s) by which HD causes this depletion is being investigated, but it may be due to the fact that carnitine is a small, water-soluble molecule which leaks out during HD,6-8 and a recent study demonstrated the amount of carnitine released into dialysis fluid (TC: 5.84 +/- 0.56; FC: 3.43 +/- 0.18, AC: 2.36 +/- 0.13 nmol/mL).25

It has been suggested that the AC/FC ratio mav provide a useful marker of changes in carnitine metabolism.26 The present study showed that the ratio was significantly higher after HD, compared with either predialysis or healthy controls. The increased AC/FC ratio in plasma has been ascribed to increased formation of acylcarnitine in pathological disorders.26 Indeed, a recent study found that acetate LID fluid significantly increased this ratio in whole blood, plasma and erythrocytes, whereas bicarbonate HD fluid did not.27 The rise in AC levels in peripheral lymphocytes further supports the contention that HD affects the distribution of carnitine in tissues and/or changes the activity of carnitine-dependent enzymes such as carnitine acetyltransferase (EC 2.3.1.7)28,29 or carnitine palmitoyltransferase (EC 2.3.1.12).2

In conclusion, the findings showed that carnitine levels in peripheral lymphocytes were reduced after HD treatment, compared with those in predialysis and healthy controls. In addition, the AC/FC ratio was significantly higher. These abnormalities in carnitine distributions may be exacerbated by long-term HD treatment.

The results presented here raise several questions. For example, does carnitine supplementation help to restore normal levels in patients undergoing HD, and do different dialyser membranes affect carnitine distributions in the same way? These are being investigated currently in this laboratory.

Acknowledgements

This work was supported by grant Bio/1418/01 from the Research Center, College of Science, King Saud University, Riyadh, Saudi Arabia. The author would like to thank Mr William F. Popovich, Senior Dialysis Technician, Clinical Head of Dialysis Unit at Riyadh Armed Forces Hospital, for his valuable assistance. The author is indebted to the staff of Department of Haematology at Riyadh Armed Forces Hospital who performed blood counts.

References

1 Bieber LI.. Carnitine. Ann Rev Biochem 1988; 57:261-83.

2 McGarry JD, Mills SE, Long CS, Foster DU . Observations on the affinity for carnitine, and malonyl-CoA sensitivity, of carnitine palmitoyltransferase I in animal and human tissues. Biochem J 1983; 214:21-8.

3 Lysiak W, Toth PP, Swelter CH, Bieber LL. Quantitation of the afflux of acetylcarnitine from rat heart, brain and liver mitochondria. J Biol Chem 1986; 261:13698-703.

4 Alhomida AS, Duhaiman AS, Al-Jafari AA, Junaid MA. Determination of L-carnitine, acylcarnitine and total carnitine levels in plasma and tissues of camel (Camelus dromedarius). Comp Biochem Physiol 1995; 111B:441-5.

5 Alhomida AS, Al-Jafari AA, Junaid SIA, Al-Whaiby SA, Duhaiman AS. Age, sex and diabetes-related changes in total, free and acetylcarnitine in human plasma. Med Sci Res 1995; 23:167-9.

6 Alhomida AS, Duhaiman AS, Al-fI Jafari AA, Sobki S, AlSulaiman M, Al-khader A. Serum total, free and acyl carnitine concentrations in chronic glomerulonephritis patients. Med Sci Res 1996; 24:495-8.

7 Alhomida AS. Effect of chronic renal haemodialysis on serum total, free and acyl carnitine concentrations in adult chronic pyelonephritis patients. Arch Med Res 1997; 28:101-7.

8 Alhomida AS, Sobki SES, AI-Sulaiman MH, Al-Khadar AA. Influence of gender and chronic haemodialysis treatments on total, free and acyl carnitine concentrations in human serum. Int Urol Nephrol 1997; 29:479-87.

9 Penn D, Schmidt-Sommerfeld E. Creatinine and carnitine esters in plasma and adipose tissue of chronic uremic patients undergoing chronic hemodialysis. MetabolisM 1983; 32:806-9.

10 Descamps-Latscha B, Chatenoud L. T cells and B cells in chronic renal failure. Semin Nephrol 1996; 16:183-91.

11 Crowley JP, Valeri CR, Metzger JB, Pono I., Chazan JA. Lymphocyte subpopulations in long-term dialysis patients: a case-controlled study of the effects of blood transfusion. Transfusion 1990; 30:644-7.

12 Descamps-Latscha B, Herbelin A. Long-term dialysis and cellular immunity. A critical survey. Kidney It 1993; 43: S135-42.

13 Alhomida AS. Study of the effects of theophylline-related changes in total, free, short-chain acyl and long-chain acyl carnitine concentrations in rat heart. Toxicology 1997; 121: 205-13.

14 Alhomida AS. Investigation of the effect of theophylline administration on total, free, short-chain acyl and long-chain acS I carnitine distributions in rat renal tissues. Cell Biochem Func 1998; 16:165-il.

15 Netecz KA, Natecz ;NIJ. Carnitine- a known compound, a novel function in neural cells. Acta Neurobiol Exp 1996; 56:597-609.

16 Bohmer T, Bergrem H, Eikhid K. Carnitine deficiency induced during intermittent chronic haemodialysis fi)r renal failure. Lancet 1978; i:126-8.

17 Smellie J, Edwards D, Hunter N, Normand ICS, Prescod N. Vesico-ureteric reflux and renal scarring. Kidney Int 1975; 8:S65-72.

18 Arze RS, Ramos JM, Owen JP, Morley AR, Elliot RW, Wilkinson R et al. The natural history of chronic pyelonephritis in the adult. QJ Med 1982; 204:396-410.

19 Hodson CJ. Discussion on pyelonephritis. Proc R Soc.+fed 1959; 52:662-72.

20 Boyum A, Isolation of lymphocytes, granulocytes and macrophages. Scand J Immunol 1976; 5:9-15.

21 Al-Kholaifi AM, Alhomida AS. Evaluation of theophyllineinduced changes on plasma total, free, short-chain acl and long-chain acyl carnitine concentrations in rats. Ivied Sci Res 1997; 25:31-4.

22 Markwell MAK, Haes SM, Tolbert NE, Bieber L.L. Protein determination in membrane and lipoprotein samples: manual and automated procedures. Method Enzymol 1981; 72:269-303.

23 Alhomida AS. Total, free, short-chain, long-chain acyl carnitine levels in Arabian camel milk (Camelus dromedarius). Ann Nutr Metabol 1996; 40:221-6.

24 Cress AP, Fraker PJ, Bierber LL. Carnitine and acylcarnitine levels of human peripheral blood lymphocytes and mononuclear phagocytes. Biochim Biophys . li ta 1989; 992:135-9.

25 Alhomida AS. Influence of acetate and bicarbonate dialysate on blood short- and long-chain acl carnitine in adult pyelonephritis patients. :Inn Clin Biochem 1999; 36:48-5.

26 Winter SC, Zon EM, Vance WH. Creatinine deficiency. Lancet 1909: i:981-2.

27 Alhomida AS.I Comparative effects of acetate and bicarbonate haemodialysis on the distribution of whole blood, plasma and erythrocyte total, free, short-chain acyl and long-chain acyl carnitine in patients with chronic renal failure. lied Sci Res 1998; 26:367-71 .

28 Alhomida AS. Investigations of the effects of theophylline administration on carnitine acetyltransferase activity of rat heart. J Enz Inhibit 1997; 12:291-302.

29 Alhomida AS, Al-Jafari AA, Duhaiman AS, Rabbani N, Junaid MA. Kinetic properties of purified carnitine acetyltransferase from the skeletal muscle of the Arabian camel (Camelus dromedarius). Biorhemie 1996; 78:204-8.

A. S. ALHOMIDA

Department of Biochemistry, King Saud University, College of Science. PO Box 2455, Riyadh 11451, Saudi Arabia

(Accepted 19 February 1999)

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