Gender and age differences in plasma carnitine, muscle strength, and exercise tolerance in haemodialysis patients
Dumitru Constantin-Teodosiu,
Stephanie Young,
Fiona Wellock,
Anthony H. Short,
Richard P. Burden1,
Anthony G. Morgan1 and
Paul L. Greenhaff
School of Biomedical Sciences, Medical School, Queen's Medical Centre, Nottingham, UK and
1 Department of Renal Medicine, Nottingham City Hospital, Nottingham, UK
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Abstract
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Background. Haemodialysis patients have impaired exercise tolerance that may be related to impaired carnitine availability and/or alterations in carnitine metabolism. As carnitine metabolism is sex- and age-related, we examined plasma free and esterified carnitine concentrations, muscle strength, and estimated exercise tolerance in female (n=51) and male (n=63) patients of different ages (1886 years).
Methods. Concentrations of free carnitine and acetylcarnitine were determined in the plasma of patients. Isometric handgrip strength was measured using a dynamometer while exercise tolerance was predicted from scores for self-reported walking distance on the level and on an incline.
Results. Plasma total carnitine concentration in the females was significantly lower than that of males (35.4±1.3 and 42.4±1.4 µmol/l, respectively, P<0.01). This was almost entirely due to the lower plasma free carnitine concentration of females when compared with the males (20.6±0.9 and 26.3±1.1 µmol/l, respectively, P<0.05). Furthermore, plasma free carnitine concentrations were negatively correlated with age in females (r=-0.45, P<0.001) even when the linear effects of haemoglobin, albumin, body weight, time on dialysis, and muscle strength were removed from the regression analysis (partial correlation coefficient; pcc=-0.43, P=0.018), but not in the males (r=0.03, P>0.05). Isometric handgrip strength and estimated exercise tolerance on the level were lower in females than males (138.9±10.9 and 259.0±15.2 N, P<0.001; and 3.5±0.2 and 4.3±0.2, P<0.05, respectively). Isometric handgrip strength and estimated exercise tolerance (walking distance on the level and on an incline) were positively correlated with plasma free carnitine concentrations in females, but not in the males.
Conclusions. There was a strong relationship between plasma free carnitine concentrations and age in female patients, but not in males. It is unlikely that the reduction in plasma free carnitine in females was a direct causative factor in their reduced exercise tolerance, but probably reflects greater muscle de-conditioning/atrophy with age in female patients.
Keywords: carnitine; fatigue; haemodialysis; renal insufficiency; sex
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Introduction
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Carnitine is a naturally occurring compound that is synthesized mainly in the liver and kidney in man, or can be acquired from dietary sources. It is an essential factor in long-chain fatty acid oxidation because it facilitates the translocation of long-chained fatty acyl groups across the inner mitochondrial membrane. Carnitine is also involved in energy metabolism in a second fundamental way because by buffering excess acyl group formation it regulates the cytosolic/mitochondrial acyl-CoA/CoASH ratio during conditions of impaired energy metabolism or increased energy metabolism, and by doing so prevents the inhibition of many cellular reactions which depend on the existence of a viable pool of CoASH [1].
Renal failure patients undergoing chronic haemodialysis have severely impaired exercise tolerance. This intolerance has been reported to be as low as 50% of that measured in age-matched normal subjects [2]. Several factors have been suggested to be responsible for the impairment in exercise tolerance, including the loss of muscle cross-sectional area, renal anaemia, inactivity, malnutrition, impaired muscle energy production or fatty acid oxidation, and carnitine deficiency [3].
Carnitine has become of interest in renal failure since it has been suggested that renal failure patients undergoing haemodialysis may develop metabolic and functional disturbances due to acute removal of carnitine during dialysis [4]. Thus, it has been shown that patients on haemodialysis have chronic sub-normal plasma and muscle free carnitine and/or elevated acylcarnitine concentrations [4], although the decline in plasma and muscle carnitine concentration has not been consistently confirmed [5]. In these previous studies, however, patients were neither sex- nor age-segregated, which would have been desirable as plasma carnitine is known to be influenced by both these factors in normal subjects [69].
The aims of the present study, therefore, were first, to investigate the relationship between plasma carnitine concentrations and the sex and age of patients undergoing haemodialysis. Secondly, to ascertain the relationship between plasma carnitine status and muscle strength and exercise tolerance in female and male renal failure patients using three different measures of muscle functional capacity.
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Subjects and methods
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One hundred and fourteen chronic renal failure patients on haemodialysis, whose demographic data are presented in Table 1
, participated in the present study. All patients were informed of the purpose and nature of the experiment before their voluntary consent was obtained. Any patient with clinical evidence of concurrent illness or malnutrition was excluded. The study was approved by the Ethics Committee of Nottingham City Hospital, Nottingham, UK.
Clinical information
The most commonly identified causes of chronic renal failure were diabetic nephropathy (17%), glomerulonephritis (13%), and adult polycystic kidney disease (8%). Intravenous iron was given regularly aiming at a plasma ferritin concentration of at least 100 µg/l to prevent iron deficiency. Dialysis adequacy was assessed by the urea reduction ratio method. Dialysers were not re-used. Body mass index was calculated as the ratio of body weight (kg)/height (m2).
Blood sampling and analysis
A 10-ml blood sample was obtained at least 24 h after a haemodialysis session, and at least 7 h post-prandially, from an antecubital vein and was mixed with lithium heparin. Following centrifugation (10 min at 3000 r.p.m.), the plasma was snap-frozen in liquid nitrogen and stored at -80°C. Aliquots of plasma were extracted with chloroform/methanol (3/2, v/v). After evaporation, the residue was dissolved in 0.1 mol/l KOH, incubated at 50°C for 2 h and, subsequent to neutralization with 0.5 mol/l HCl, used for determination of total carnitine with an enzymatic assay containing radioisotopic substrate, as described previously [10]. Free carnitine was determined by dissolving the residue in water. Concentrations of acetylcarnitine were measured in deproteinized plasma (0.5 mol/l perchloric acid) using an enzymatic assay containing radioisotopic substrate, as described previously [10]. All measurements were performed in duplicate. Total acylcarnitine concentrations were obtained by subtracting free carnitine from total carnitine concentrations. Medium- and long-chain acylcarnitine concentrations were obtained by subtracting free carnitine and acetylcarnitine from total carnitine concentrations.
Serum urea, sodium, potassium, calcium, alkaline phosphatase, ferritin, and haemoglobin were measured by standard laboratory techniques. Serum albumin concentrations were determined using the bromcresol purple dye method for which the reference range is 3052 g/l.
Measurements of muscle strength
Isometric handgrip strength study was measured using a dynomometer devised by Bassey et al. [11]. The patients were familiarized with the use of the dynomometer before measurements were made. Briefly, patients were asked to place their dominant hand on the centre of a gripping bar and then to squeeze the instrument as hard as possible for 3 s. Verbal encouragement was given to each patient throughout. Each patient made five attempts, with a rest of at least 1 min between attempts. The highest recording was taken as the measurement of handgrip strength.
Estimations of exercise tolerance
Exercise tolerance was predicted from scores for self-reported walking distance on the level and on an incline. The self-rating scale questionnaire that was used has been shown previously to strongly correlate with exercise tolerance assessed using the treadmill walking exercise (r=0.90, P<0.001) [12]. The questionnaire asked the patient to identify statements most appropriate to them using a six-point scale with respect to their ability to walk on the level and on an incline. The statements concerned distance, speed, perceived difficulty, intensity of pain, and frequency of performing walking tasks.
Statistics
Differences in plasma carnitine concentrations between the two patients groups, i.e. females and males, were analysed (SPSS Base 10.1) using one-way analysis for variance (ANOVA). For the analysis of relationships between plasma carnitine concentrations and age, haemoglobin, albumin, body weight, length of time on dialysis, and handgrip strength within each group, a simple linear regression (bivariate) was used. When correlations between variables were significant, a second partial correlation test was used in order to provide a new measure of correlation between two variables by removing or adjusting for linear effects of one or more control variables. For the analysis of relationships between plasma free carnitine concentrations and estimated exercise tolerance (on the level and on an incline) the non-parametric Spearman rank-order correlation was used because of the non-ordinate nature of the exercise tolerance questionnaire scores. Differences in exercise tolerance estimates between the two patient groups were analysed using the non-parametric MannWhitney test (P<0.05). Values in the tables and figures represent means±SEM.
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Results
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There was no significant difference between the female and male renal failure patients in terms of age, time on dialysis, nor body mass indexalthough body weight was significantly lower in females than males (65±2 and 72±2 kg, respectively, P<0.01) (Table 1
).
Blood biochemistry data are presented in Table 2
. In 52% of the patients the haemoglobin concentration was less than 10 g/dl, the mean haemoglobin concentration and the red cell counts being slightly, but significantly lower in females compared with males (P<0.01). However, the mean ferritin concentration was slightly (but significantly) greater in females than males (Table 2
). The ferritin concentration was lower than 100 µg/l in only two patients. There was no difference in urea reduction ratio between females and males (Table 2
).
Plasma total carnitine concentrations in the female patients were significantly lower than that of the male patients (35.4±1.3 and 42.4±1.4 µmol/l, respectively; P<0.001; Figure 1
). This difference was solely accounted for by the lower plasma free carnitine concentration in the female group (20.6±0.9 and 26.2±1.1 µmol/l in females and males, respectively; P<0.001; Figure 1
). Comparison of plasma medium- and long-chain acylcarnitine concentration revealed no difference in female patients compared with male patients (9.3±0.5 and 8.7±0.7 µmol/l, respectively; P>0.05; Figure 1
). Similarly, the total plasma acyl (acetyl-, medium-, and long-chain acylcarnitine) to free carnitine ratio was not significantly different in the female patients compared with male patients (0.82±0.09 and 0.68±0.04, respectively, P>0.05). There was a significant negative correlation between the plasma free carnitine concentration and age in the female patients (r=-0.45, P<0.001, n=51, Figure 2
) even when the linear effects of haemoglobin, albumin, body weight, dialysis time, and muscle strength were removed from the regression analysis (partial correlation coefficient; pcc=-0.43, P=0.018). This was not the case in the males (r=0.03, P>0.05, n=63).

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Fig. 1. Plasma total, free, acetyl-, medium-, and long-chain acylcarnitine concentrations in female and male renal failure patients on haemodialysis. Values represent means±SEM. ***P<0.001, significantly different from male patients.
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Fig. 2. Relationship between plasma free carnitine concentrations and age in female and male renal failure patients undergoing haemodialysis. Each point represents an individual patient. Plasma free carnitine concentrations was negatively correlated with age in the female patient group (y=31.64-0.19 x; r=-0.45, P<0.001), but not in male patients (y=25.44+0.01 x; r=0.03, P>0.05).
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Handgrip strength in all patients in the present study was 208.3±11.7 N. The females in our sample population were weaker than the males (138.9±10.9 and 259.0±15.2 N, respectively; P<0.001). Similarly, estimates of exercise tolerance on the level and on an incline (means±SEM (median)) were lower in female patients than in male patients (3.5±0.2 (3) and 4.3±0.2 (4), P<0.05, respectively, and 3.0±0.2 (3) and 3.6±0.2 (4), P>0.05, respectively, Figure 3
). In the female patients, handgrip strength and estimates of exercise tolerance on the level and on an incline were positively correlated with plasma free carnitine concentrations (r=0.36, P<0.05; r=0.41, P<0.05; r=0.45, P<0.01, respectively; Table 3
). Furthermore, the positive correlation between plasma free carnitine and handgrip strength in the female patients was maintained when the linear effects of body weight and time on dialysis were removed from the regression analysis (pcc=0.33, P<0.05), but not when the effect of age was also removed (pcc=0.15, P=0.40). No such correlations were founded in the male patients (r=0.23, P>0.05; r=0.04, P>0.05; and r=0.14, P>0.05, respectively; Table 3
).

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Fig. 3. Box plots of exercise tolerance (walking on the level and on an incline) scores in female and male renal failure patients undergoing haemodialysis. *P<0.05, significantly different from male patients (MannWhitney test). Each abbreviation represents: M, median; UQ, upper quartile; LQ, lower quartile.
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Table 3. Relationship between plasma free carnitine and age, body weight, duration on haemodialysis, handgrip strength and exercise tolerance questionnaire score (walking distance on the level and an incline) in chronic renal female (F) and male (M) patients on haemodialysis
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Discussion
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The major findings of the present study were, first, that female renal failure patients had a significantly lower plasma free carnitine concentration (20.6±0.9 µmol/l) compared with their male counterparts (26.3± 1.1 µmol/l). Secondly, plasma free carnitine concentration was negatively correlated with age in female patients, which was not the case in male patients. Finally, the plasma free carnitine concentration in female patients was positively correlated with handgrip strength and two indicators of exercise tolerance, but these relationships were not evident in male patients.
In a previous study we showed that total carnitine concentration in continuous ambulatory peritoneal dialysis (CAPD) patients was similar to the concentration measured in age- and sex-matched healthy controls (42.8±1.6 and 43.1±2.3 µmol/l, respectively) [13]. Nevertheless, plasma free carnitine concentration in CAPD patients was significantly lower than that of healthy controls (28.5±1.4 and 36.2±2.5 µmol/l, respectively; P<0.05). The difference between total and free carnitine was accounted by the difference in the concentration of acyl-carnitine. Interestingly, plasma free carnitine concentrations in CAPD female and male patients were similar (28.4±4.1 and 28.6±0.7 µmol/l, respectively) and represented 88 and 68% of plasma free carnitine concentrations measured in their sex-matched healthy controls (32.2±2.0 and 42.2±3.9 µmol/l in females and males, respectively). In the present study, the concentrations of plasma free carnitine in female and male patients on haemodialysis were even lower representing 64 and 62% of plasma free carnitine concentrations measured in their corresponding healthy controls [13].
Factors such as sex, age [69], nutrition [14], and disease [4,15] have been shown to affect plasma carnitine concentrations in humans. A decline in plasma carnitine concentrations has been observed in patients receiving chronic haemodialysis [4]. However, to our knowledge there has been no attempt to examine plasma carnitine concentrations over a large age span in both female (2586 years) and male (1881 years) renal failure patients, and to correlate these concentrations with sex, age, muscle strength, and indicators of exercise tolerance.
Plasma carnitine concentrations are known to be lower in young healthy female subjects when compared with age-matched male subjects [6,7,9]. Similar sex-related differences in plasma free carnitine were also observed in the present study. Furthermore, this sex-related difference has been reported to disappear in age groups over 40 years, when plasma carnitine concentrations have been reported to increase in healthy females [7]. One proposed explanation for this finding is the decline in the plasma estradiol concentration in females over 40 years of age [7,9]. However, in contradiction to the report of Borum [7], and others [6,8,9], the present findings showed a strong negative correlation between plasma free carnitine concentrations and age over the range of 2486 years in renal failure female patients on haemodialysis (P<0.001; Figure 2
). It is important to note that this correlation was maintained even when the linear effects of haemoglobin, albumin, body weight, time on dialysis, and handgrip strength were removed from regression analysis (ppc=-0.43, P=0.018), suggesting that the effect was entirely due to the relationship between age and plasma free carnitine. This observation contradicts previous findings that showed a steady rise in plasma free carnitine with increasing age in healthy female subjects [69]. It is difficult to explain this age-dependent decline in plasma free carnitine concentrations in female renal failure patients in the present study, as there is no evidence to suggest that differences in plasma estradiol levels exist between patients and healthy females.
Patients with end-stage renal disease receiving chronic haemodialysis have severely impaired exercise tolerance. This is thought to be a result of impairments in both cardiorespiratory function and skeletal muscle strength. Thus, renal failure patients on haemodialysis have lower peak heart rates, lower peak ventilation rates, lower peak blood lactate concentrations [2], and lower peak values for oxygen consumption during exercise when compared with healthy age- and sex-matched untrained subjects [16,17]. It would appear that the decline in skeletal muscle strength is a principal factor responsible for the impairment of exercise tolerance in this patient group [16]. However, the specific reasons for the decline in skeletal muscle strength are still largely unresolved. Several candidates have been suggested including a loss of muscle cross-sectional area, anaemia, inactivity, malnutrition, an impairment in muscle energy production and fatty acid oxidation, and carnitine deficiency [3,4].
With respect to the latter, carnitine is present in plasma in free and esterified forms. The ratio of esterified to free is normally 0.150.20 [13]. However, a redistribution of plasma free carnitine to its esterified form has been reported to occur in disease [13] and during exercise [1]. This is thought to result from carnitine's ability to buffer increases in tissue acyl group levels [1]. In agreement with this proposed function, the present study showed that a similar redistribution of plasma free carnitine into an esterified form had occurred in plasma of both female and male patients (0.82±0.09 and 0.68±0.04, respectively). These abnormal ratios have been cited as a possible cause of a relative carnitine deficiency [18]. However, a novel finding of the present study was that this apparent deficiency was further exacerbated in the female patients, as indicated by their lower plasma free carnitine concentrations, despite them having the same plasma acylcarnitine concentration as the male patients (Figure 1
). Given the important role of carnitine in cellular energy metabolism, it is possible that these deficiencies (lower carnitine availability and/or abnormal amounts of carnitine acylation) may contribute to the premature muscular fatigue and weakness that renal failure patients experience, especially female patients. Indeed, in the present study the female patients consistently performed worse than the male patients in measures and predictors of muscle function. This of course could simply be a reflection of the lower body (and therefore muscle) weight in the female patients (Table 1
). Nevertheless, there were positive correlations between plasma carnitine availability and all three measures/predictors of exercise tolerance in the female patients (Table 3
). Furthermore, the positive correlation between plasma free carnitine and handgrip strength was maintained when the linear effects of body weight and dialysis time were removed from the regression analysis (pcc=0.33, P<0.05), but not when the effect of age was also removed (pcc=0.15, P=0.40). It would appear, therefore, that rather than being causative, these associations merely reflect greater muscle de-conditioning/disuse atrophy in the female patients with age.
Haemoglobin concentrations in both female and male patients were reduced and, therefore, their capacity to transport oxygen to contracting muscle must have been impaired. A previous study by Mayer et al. [19] concluded that impairments of aerobic and anaerobic capacity in renal failure patients were significantly correlated with the severity of renal anaemia. However, these findings have not been consistently replicated by other studies, which have shown a poor correlation between haemoglobin concentration and endurance performance [17]. Furthermore, relatively young renal failure patients (average age 41 years) have a greater capacity to produce ATP per muscle unit, probably due to an adaptation of their mitochondria to the lower oxygen availability and transport [20]. Whether this adaptation is extended to older renal failure patients remains to be established.
In conclusion, the present study demonstrated that female renal failure patients had a significantly lower plasma free carnitine concentration compared with their male counterparts. Furthermore, plasma free carnitine concentrations in the female patients were negatively correlated with age, which was not apparent in male patients. Plasma free carnitine concentrations in female patients were positively correlated with handgrip strength and two indicators of exercise tolerance. It is unlikely that the reduction in plasma free carnitine concentration in females was a direct causative factor in their reduced exercise tolerance, but probably reflect greater muscle de-conditioning/atrophy with age in female patients.
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Notes
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Correspondence and offprint requests to: D. Constantin-Teodosiu, PhD, School of Biomedical Sciences, Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK. Email: tim.constantin{at}nottingham.ac.uk 
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References
|
---|
- Constantin-Teodosiu D, Carlin JI, Cederblad G, Harris RC, Hultman, E. Acetyl group accumulation and pyruvate dehydrogenase activity in human muscle during incremental exercise. Acta Physiol Scand1991; 143: 367372[ISI][Medline]
- Barnea N, Drory Y, Iaina A et al. Exercise tolerance in patients on chronic hemodialysis. Isr J Med Sci1980; 16: 1721[ISI][Medline]
- Fahal IH, Bell GM, Bone JM, Edwards RHT. Physiological abnormalities of skeletal muscle in dialysis patients. Nephrol Dial Transplant1997; 12: 119127[Abstract]
- Moorthy AV, Rosenblum M, Rajaram R, Shug AL. A comparison of plasma and muscle carnitine levels in patients on peritoneal or hemodialysis for chronic renal failure. Am J Nephr1983; 3: 205208
- Rogerson ME, Rylance PB, Wilson R et al. Carnitine and weakness in haemodialysis patients. Nephrol Dialysis Transplant1989; 4: 366371[Abstract]
- Cederblad, G. Plasma carnitine and body composition. Clin Chim Acta1976; 67: 207212[ISI][Medline]
- Borum PR. Plasma carnitine compartment and red blood cell carnitine compartment of healthy subjects. Am J Clin Nutr1987; 46: 437441[Abstract]
- Costell M, O'Connor JE, Grisolía S. Age-dependent decrease of carnitine content in muscle of mice and humans. Biochem Biophys Res Commun1989; 161: 11351143[ISI][Medline]
- Takiyama N, Matsumoto K. Age- and sex-related differences of serum carnitine in a Japanese population. J Am Coll Nutr1998; 17: 7174[Abstract/Free Full Text]
- Cederblad G, Carlin JI, Constantin-Teodosiu D, Harper P, Hultman E. Radioisotopic assays of CoASH and carnitine and their acetylated forms in human skeletal muscle. Anal Biochem1990; 185: 274278[ISI][Medline]
- Bassey EJ, Dudley BR, Harries UJ. A new portable stain-gauged handgrip dynamometer. J Physiol1986; 373: 6P
- Drakes AH. Exercise testing in patients with chronic renal failure. PhD thesis, Nottingham University, 1996; 175
- Constantin-Teodosiu D, Kirby PJD, Short AH, Burden RP, Morgan AG, Greenhaff PL. Concentrations of free and esterified carnitine in continuous ambulatory peritoneal dialysis patients. Kidney Int1996; 49: 158162[ISI][Medline]
- Kahn-Siddiqui L, Bamji MS. Plasma carnitine levels in adult males in India: effects of high cereals, low fat diet, fat supplementation, and nutrition status. Am J Clin Nutr1980; 33: 12591263[Abstract]
- Böhmer T, Rydning A, Solberg HE. Carnitine levels in human serum in health and disease. Clin Chim Acta1974; 57: 5561[ISI][Medline]
- Diesel AH, Noakes TD, Swanepoel C, Lambert M. Isokinetic muscle strength predicts maximum exercise tolerance in renal failure patients on chronic hemodialysis. Am J Kidney Dis1990; 2: 109114
- Painter P, Messer-Rehak D, Hansson P, Zimmerman SW, Glass NR. Exercise capacity in hemodialysis, CAPD, and renal transplant patients. Nephron1986; 42: 4751[ISI][Medline]
- Winter SC, Szabo-Aczel S, Curry CJR, Hucthinson HT, Hogue R, Shug A. Plasma carnitine deficiency. Am J Dis Child1987; 141: 660665[Abstract]
- Mayer G, Thum J, Graf H. Anaemia and reduced exercise capacity in patients on chronic haemodialysis. Clin Sci1989; 76: 265268[ISI][Medline]
- Barany P, Wibom R, Hultman E, Bergström J. ATP production in isolated muscle mitochondria from haemodialysis patients: effects of correction of anaemia with erythropoietin. Clin Sci1991; 81: 646653
Received for publication: 31. 7.01
Accepted in revised form: 17. 5.02