Ascorbic acid, carnitine and fatigue

Med. Sci..Res., 1988; 15,'12l-,723 FOfUm Ascorbic acid, carnitine and fatigue R. Elwyn Hughes Department of Applied Biologt, University of l4lales Institute of Science and TÞchnologt, CardifÍ, Wales This paper reviews the evidence that ascorbic acid (vitamin C) may have a contributory role in the prevention of fatigue in humàns. The biochemical history of carnitine (B-hydroxy-7-Ntrimethylammonium butyrate) is characterised by alternating periods of activity and dormancy. The re-emergence ofinterest during the past 15 years has been characterised by three main areas of emphasis - the endogenous biosynthesis of carnitine from lysine, the involvement of carnitine as a co-factor in the metabolism of long-chain fatty acids, and the identifrcation of the comparatively rare, but metabolically interesting, camitine deficiency diseases [1 - 4]. Biosynthesis of c¡rnitine Two essential amino acids - methionine and lysine - are involved in the biosynthesis of carnitine. Lysine is methylated to produce the protein-bound trimethyl-lysine which is then hydroxylated to form trimethylaminobutyrate (3=hydroxyltrimethylJysine) ; in a second hydroxylation step trimethylaminobutyrate (7-butyrobetaine) is converted into carnitine [3]. These two hydroxylations are catalysed by separate ferrous-requiring oxygenases and both are stimulated in vitroby the addition of ascorbic acid; in the case of the trimethyl-lysine hydroxylase the ascorbic acid is partially replaceable by other reductants but the requirement of the 7-butyrobetaine hydroxylase for ascorbic acid would appear to be specihc [5, 61. More recently, the earlier in vitro work has been extended to whole animal studies. A number of reports have indicated that the formation of carnitine is impaired in guinea-pigs deprived of dietary ascorbic acid [7-10]. In perfusion studies it was shownrthat 'scorbutic' guinea-pig livers had a significantly reduced capacity for converting 7-butyrobetaine to carnitine and that this defect could be corrected by prior perfusion with ascorbic acid [l l]. These and other aspects of the relationship between ascorbic acid and carnitine biosynthesis have been recently reviewed t12, l3]; England and Seifter [l3J in their survey of the fìeld concluded that there is in vitro stimulation of both hydroxylases by ascorbic acid, that there are variable decreases in tissue carnitine concentrations when guinea-pigs are deprived of ascorbic acid, and that the evidence, on balance, suggests that there is a reduced activity of liver 7-butyrobetaine hydroxylase in avitaminotic C guinea-pigs. It is diffìcult to quantify the amount (or proportion) of ascoibic acid required for the carnitine biosynthetic pathway. Dietary carnitine significantly prolonged the life span of scorbutic guinea-pigs, but only by some l0%, which would suggest that the requirement for ascorbic' acid för carnitine biosynthesis is small when compared with its main metabolic iole in the formation of collagen [14]. Metabolic role of carnitinè The essential role of carnitine in both man and animals is that it is required for the'transport of long-chain fatty acids into the mitochondrial matrix where, by B-oxidation, they may be used as a source of energy [31. Fatty acid:CoA esters are transesterifîed by carnitine acyl (palmitoyl)transferase I 72r and the fatty acyl-carnitine esters are transported across the inner mitochondrial membrane by a carnitine-acyl translocase. Fatty acid-CoA esters are released by a carnitine acyl (palmitoyl)transferase II on the inner membrane and the cycle is repeated [3, 15]. A lack of carnitine, or a diminished or defective activity of one or more of the transport system enzymes, will reduce the availability of fatty acids as a source of energy. This, apart from producing a generally reduced 'energy status', could be of critical significance in situations where the bulk of the energy metabolism is believed to be derived from fatty acid metabolism - such as in the newly-born infant [16] or in the metabolism of normal cardiac muscle U7l. It has been suggested that endogenous production of carnitine is impaired in the newly-born infant and that consequently there is a requirement for a supply of dietary carnif.ine to maintain the necessary tissue concentrations. The ratelimiting factor in carnitine production is the ascorbic aciddependent 7-butyrobetaine hydroxylase [18] and the ascorbic acid intake of a newly-born child would, of course, be low compared with that of a developing child on a mixed diet, as human milk contains only 3 mg ascorbic acid per 100 ml. However, in a recent publication, Olson and Rebouche [19] have indicated that 7-butyrobetaine hydroxylase is not rate-limiting for carnitine formation in the human infant. Carnitine dise¡ses The identification and characterisation of the various carnitine discases have produced useful information about the carnitineenergy relationship. Two types of primary carnitine deficiency have been recognised - the muscle or myopathic form which is largely confined to the skeletal muscle system, and a systemic form rqhich is a more generalised disease [151. The causes of carnitine deficiency in man are, theoretically, multiple and no sinþ,le biochemical lesion has been defltned. A common feature of the carnitine deficiencies is the progressively increasing muscle weakness and fatigue - a point of central importance to the main thesis of this review [20]. The clinical notes for the ltrst reported case of systemic carnitine deltciency carry the observation "... a gradual development of muscle weakness ... he could no longer run .'. he became short of breath after walking half a block .'. he was always fatigued" [21]. Similarly, a 2O yeat-old male who presented with myopathic carnitine deficiency was "... able to walk only 100 yards on level ground and had great difficulty in climbing stairs ... there was generalised muscular weakness..." [221. These descriptions bear a similarity to the clinical descriptions of the earli'features of scurvy (avitaminosis C) in man. Fetiguein human scurvy References to the early emergence in scurvy of fatigue and lassitude were ìnvariable features of the earliest clinical descriptions of the disease [23]. Eugalenus in 1658 spoke of "spontaneous debility" [24], Lister, in 1696, wrote of "weakness of limbs and considerable fatigue" [25] and Sydenham in 1742 of "spontaneous lassitude and difficulty of breathing after exercise" [261. Naval surgeons with first hand 722 experience ofscurvy were equally clear in.their descriptions: "The signes of the Scurvie are many, namely a general lazinesse ... shortnesse and difficultie ofbreathing, especially when they moove themsçlves" commented Woodall in 1639 I27l and Lind, over a century later, wrote: 'i... this lassitude, with a breathlessness upon motion, are observed to be among the most common concomitants of the distemper" [28]. Practising 'land physicians' in the last century made similar observations. Shapter, a careful clinical observer, describing an outbreak ofscurvy in Exeter in I 847. perhaps put the matter most clearly: "... the spongy and swollen gum appears to me to have been erroneously estimated as amongst the primary and most obvious manifestations of the scurvy ... I am inclined to say there is a class of well-marked symptoms preceding this... The first or initiatory stage ... has appeared to me to be characterised by ... debility ... weakness, listlessness and a disinclination to exercise" [291. More recent cases of scurvy have also underlined the early emergence of fatigue. In 1952 it was noted in a çase history that the patient had, during the year before admission, ',become increasingly weak and easily fatigued" [30] and reports of experimentally induced sçurvy in human volunteers similarly drew attention to the early emergence of fatigue [3]-33]. Crandon, who placed himself on a scorbutogenic dief, commented that a feeling of fatigue developed from the beginning of the 3rd month of dehciency, a full 6 to 8 weeks before the emergence of the traditional'overt'signs of scurvy such as perifollicular hyperkeratotic papules, petechiae, poor wound healing and softening of the gums [34] . It will be noted that the fatigue of scurvy, like the fall in muscle carnitine in hypovitaminotic C guinea-pigs [7], evidences itselfbefore the traditional overt signs ofscurvy and it has been suggested that it reflects an impairment of the endogenous biosynthesis of camitine in the absence of adequate ascorbic acid [23]. The pathological features customarily associated with scurvy are all, theoretically, amenable to reductionist treatment in terms of the hydroxylation of lysyl and prolyl residues in the formation of collagen. Fatigue bears no identihable relationship to collagen formation, and this is possibly the reason why this fêature of incipient scurvy has been generally ignored by students ofthe disease. Dietarysignificance It has been estimated that over three quarters of the body carnitine in the adult rat and in the normal human is produced endogenously by the biosynthetic pathway from lysine and methionine [2], 351. Vegetarians, with a negligible intake of preformed carnitine, maintain normal plasma concentrations [36] and by the same token, systemic carnitine deficiency is exacerbated by a vegetarian (i.e., a low carnitine) diet [37]. Any dietary influence on carnitine status in the normal adult is, therefore, most likely to be mediated via the availability of the precursor molecules and co-factors necessary for the biosynthesis. The most likely candidates in this respect are lysine (and, to a lesser extent, methionine) and ascorbic acid. (Theoretically, iron, nicotinic acid and pyridoxine could also be involved [2] but theyfall outside thescope ofthis reviewand are, in any case, less likely to be limiting factors in the average diet). There is evidence that modifications to the dietary lysine (or to lysine-rich proteins) result in changes in the carnitine status [38]. Carnitine formation is reduced by lysinedeficient diets [39] and urinary carnitine is decreased in persons receiving non-optimal diets with rice as the main component [40]. A¿utt Wistar rats receiving a diet to which protein contributed 370/o of R. Elwyn Hughes the total calories, excreted two and a half times as much free urinary carnitine as a corresponding group where the protein accounted for only 8.50/o of the calories [41]. Ascorbic acidr carnitine and fatigue In contrast to the influence of dietary protein on urinary carnitine in rats, changing the ascorbic acid status of guineapigs from hypovitaminosis C (produced by giving 0.2 mg ascorbic acid per 100 g body weight daily) to tissue saturation did not produce any resultant change in the excretion of carnitine [,11] and in young and elderly women no relationship was found between plasma carnitine and leucocyte ascorbic acid although thirteen of the elderly women included in the study had a leucocyte ascorbic acid concentration below l5 ¡r,g/10 cells, customarily regarded as the 'scurvy risk' cut off point [421. This would appear to suggest that protein is a more powerful determinant of carnitine biosynthesis than ascorbic acid. Nevertheless, there is some evidence that in man a relationship exists between ascorbic acid status and carnitine formation; in elderly men and women a highly significant positive correlation was found between urinary free carnitine and leucocyte ascorbic acid [42]. These are findings difficult to reconcile but there are indications both from accounts of carnitine deficiency diseases and from studies with normal adults that the relationship between serum carnitine and tissue carnitine concentrations is an equally brratic one [20, 43]. There is therefore no direct evidence that muscle carnitine in man is related to vitamin C status - and confounding factors such as dietary protein intake reduce the possibility ofan easy resolution of.the question. Nor does ihe plasma carnitine concentrati,on present the type of correlation with age and sex found for ascorbic acid 144, 451. Table 1: Ascorbic acid. intake and self-assessed fatigue in adult females: combined results for two areas in Wales (means t SEM) (SJ. Gruffudd and R.E. Hughes, 1988, unpublished results) Forum Ascorbic acid status Mean ascorbic acid intake (mglday) Fatigue nscofe < 30mg/day 5t > lOOmg/day 54 Random sample 76 l9.l -t0.86 142.6'+ 5.5 34.3 -+ 7e 25.3 + 2b 30.3 É l.0c 69.9 t 4.7 Sígnificance of di.fference between meanfatigue scorcs: a and b, p 1 0.001; a and c,p < 0.05; b and c,p < 0.002. An alternative approach would be to examine the relationship between fatigue and ascorbic acid status by determiriing the degree of correlation between leucocyte ascorbic acid (generally accepted as an index ofascorbic acid status) and a measurement of physical fatigue. A less direct, and less satisfactory, method would be to substitute psychological self-assessment of fatigùe for actual physical methods. Dietary-personality studies of this type have, hitherto, been little used in vitamin C work. ln a recent preliminary study of this type the relationship between ascorbic acid intake (assessed by a frequency-portion size questionnaire) and self-assessed fatigue [46] was examined in 500 randomly selectcd adult females in two areas of \Yales. There was a negative correlation between estimated ascorbic acid intake and the score for self-assessed fatigue. The fatigue score for intakes below 30 mg a day (the Recommended Daily Amount) was significantly greater than for intakes of over 100 mg (approaching tissue saturation) and both values were individually significantly different from a random sample of the total participants (Table l). Diet-personality studies of this type are of course fraught Ascorbic acid, carnitine and fatigue with diffìculties and are justihably unpopular with nutritional scientists [47]; more than any other rype of scientific exercise, they are open to spurious interpretations. The results of the study described above could be interpreted as impl)¡ing that fatigue results in a more restricted choice of diet with a resultant reduction in vitamin C intake - instead of vice versa. The variables and imponderables present reduce the validity of a simple 'causal' explanation. Nevertheless, the general negative correlation found between ascorbic acid intake and fatigue score is consistent with three reasonably well-established findings: (i) ascorbic acid deficiency reduces the formation of carnitine; (ii) carnitine iS necessary for the production of energy from fatty acids and impairment of this function results in fatigue;and (iii) fatigue is an invariable feature of incipient avitaminosis C (scurvy) in humans. The current Recommended Daily Allowance (RDA) for ascorbic acid in the United Kingdom is 30 mg daily. This is based essentially on a single experiment done over 40 years ago on a small number of non-representative members of the population when the role ofascorbic acid was assessed solely in terms of the overt ('collagen defect') signs of scurvy [45]. The presence of fatigue, and its relationship to the ascorbic acid intake, was not studied. Both animal studies [7] and observations on human scurvy [23] appear to indicate that the maintenance of a functionally-appropriate level of carnitine and the prevention of physical fatigue may require an intake of ascorbic acid greater than that necessary to 'protect'against the traditional overt signs ofscurvy. As the ascorbic acid intake ofa substantial proportion of the population of the United Kingdom is currently below the RDA further studies on the relationship between ascorbic acid, carnitine and fatigue would be useful. l. Frenkel, R.A. and McGarry, J.D. (eds), (1980) in Carnitine Biosynthesß, Metabolism and Func¡ozs, Academic Press, London, New York 2. Borum, P.R. (1983) Annu. Rev. Nutr.,3,233-259 3. Bremer, J. (1983) Physiol. Rev,,63,7420-1480 4. Rebouch, C.R. and Pôulson, D.J. (1986) Annþ. Rev. Nutn,6,4l-66 5. Henderson, L.M., Hulse, J.D. and Henderson, L.L. 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