7 Defects of fatty-acid oxidation in muscle CORRADO


INTRODUCTION Long-chain fatty acids (LCFA) are oxidized preferentially by muscle mitochondria after their transport from the cytosol and their activation by a thiokinase. Intracellular fatty-acid-binding proteins (FABPs) have been isolated both from liver and heart. The major role assigned to FABPs is the facilitation of the intracellular transport of fatty acids and acyl derivatives




SB8 TronsJocose





Acyl-CoA ~__j ,~-Oxldation

C4/ ~~)=

f Acyl-CoA ~x Molonyl-CoA TritonX

Figure 1. Mechanism of mitochondrial transfer of an activated long-chain fatty acid (AcylCoA). SB8, sulfobetamine, MCoA, malonyl-CoA. Bailli&e's Clinical Endocrinology and Metabolism-56 l Vol. 4, No. 3, September 1990 Copyright © 1990, by Bailli6re Tindall ISBN 0-7020-1464--8 All rights of reproduction in any form reserved



(Glatz and Veerkamp, 1985). Although the involvement of FABPs in intracellular fatty-acid transport and utilization is generally accepted, the massive amount of research into these proteins has investigated only their physiological role; there is only scanty evidence for their involvement in a pathological dysfunction (Vergani et al, 1990a). Carnitine, two forms of carnitine palmitoyltransferase (CPT-I and CPT-II) and a carnitine acylcarnitine translocase associated with the mitochondrial membrane (Figure 1) are required in mammalian skeletal muscles to transfer acyl CoAs across the mitochondrial membrane for B-oxidation (Bremer, 1983). Medium- and short-chain fatty acids can freely cross the mitochondrial membrane to be oxidized by B-oxidation enzymes, which include three FAD-dependent dehydrogenases (long-chain, mediumchain and short-chain acyl CoA-dehydrogenases or LCAD, MCAD and SCAD, respectively), an enoylCoA-hydratase, an NAD-dependent 3-hydroxyacylCoA dehydrogenase, and two 3-ketoacylCoA thiolases. The electron equivalents produced by the three FAD-linked dehydrogenases are conveyed to coenzyme Q by an electron-transferring flavoprotein (ETF) and then by iron-sulphur flavoprotein ETF-dehydrogenase (ETF-dh). The oxidation of fatty acids is essential for prolonged muscle exercise (Felig and Wahren, 1975); genetic and acquired defects of B-oxidation are therefore expected to cause myopathies. PATHOGENETIC MECHANISM

LCFA are good respiratory substrates for mitochondria. On the other hand, if they accumulate in muscle or other tissues they may be inhibitory. This chain of events leads to a muscle dysfunction with 'lipid storage myopathy' or to a Reye-like syndrome when liver and brain are involved. In fact, LCFA and their CoA-esters have an uncoupling effect on mitochondria, a detergent-like action, an atractyloside-like action, inhibiting the ADP-ATP exchange, and an oligomycin-like role; they are also dynamic traps for free CoA as acyl-CoAs (Dynnik and Djafarov, 1986). As a result of a partial or complete defect of LCFA transport or oxidation, these substrates may inhibit their own oxidation and block oxidative phosphorylation in mitochondria. The mechanism of self-inhibition is particularly evident under conditions that stress lipid metabolism (cold, fasting, long-chain fatty-acid diets) in genetically predisposed subjects, in which either carnitine palmitoyl transferase deficiency or carnitine defects (primary or secondary) are present. This effect of LCFA is explained by both a regulatory role of CPT and by the fact that a long-chain acylcarnitine (palmitoylcarnitine) and medium-chain acids (capric or caproic acid) can produce a fourfold reduction in respiratory rate in liver mitochondria; this effect can be reversed by succinate. HUMAN MYOPATHIES

Whereas B-oxidation has long been studied as an important fundamental



process, awareness of human inborn errors of fatty oxidation have burgeoned in the last 18 -'~ars, since the identification of muscle carnitine deficiency syndrome ant F deficiency in 1973. Several other lipid storage myopathies have been further identified that are due to impaired transport of acyl-CoAs or to defects in B-oxidation enzymes: glutaric aciduria was recognized in 1976, non-ketotic C6-C10 dicarboxylic aciduria in 1980, hepatic CPT deficiency in 1981, and acylCoA dehydrogenase deficiencies in 1983-1985. Other syndromes attributable to defects of ETF or ETF dehydrogenase were recognized in 1985. Table 1 summarizes those defects of fatty-acid metabolism that have been recognized in the last t w o decades as causing either a generalized disease or a disorder with prominent expression in skeletal muscle. The discovery of numerous clinical syndromes associated with defects of fatty-acid utilization has proved in recent years to be a fruitful field of investigation. Table 1. Genetic defects of fatty acid transfer and g-oxidation. 1. Defects in transfer of AcylCoAs (a) Carnitine deficiency (b) Carnitine palmitoyltransferase deficiency (CPT-I, CPT-II) 2. Defects in flavoprotein dehydrogenases (a) Long-chain acylCoA dehydrogenase (LCAD) deficiency (b) Medium-chain acylCoA dehydrogenase (MCAD) deficiency (c) Short-chain acylCoA dehydrogenase (SCAD) deficiency 3. Defect in electron-transferring flavoprotein (Multiple acylCoA dehydrogenase deficiency) (a) Glutaric aciduria type II due to ETF deficiency (b) Glutaric aciduria type H due to ETF dehydrogenase deficiency 4. Other defects of LCFA oxidation

(a) 3-HydroxyacylCoA dehydrogenase deficiency (b) g-Ketothiolase deficiency (c) Multisystem triglyceride storage disorder

Defects of LCFA oxidation can be classified as follows: 1.



Defects of fatty acid transfer in mitochondria: carnitine deficiency syndrome, presenting as muscle weakness, cardiomyopathy or Reye's attacks; carnitine palmitoyltransferase deficiency, often associated with myoglobinuria. Defects of acyl CoA-dehydrogenases medium-, short-chain) represent a common cause of non-ketotic hypoglycaemia in children and possibly a cause of 'cot-death' or 'sudden infant death syndrome' (SIDS). Defect of electron-transfer flavoprotein (ETF) and EFT dehydrogenase, which cause neonatal disorders with congenital anomalies and polycystic kidney, or a later-onset syndrome with organic aciduria ('ethylmalonic-adipic aciduria' or glutaric aciduria type II).




CARNITINE DEFICIENCIES Carnitine synthesis and transport Endogenous carnitine is derived in man from e-N-trimethyllysine (Figure 2), which is formed by the methylation of lysine residues in proteins such as myosin, actin and histones; therefore, carnitine is formed from e-Ntrimethyllysine after its liberation by breakdown of proteins. Most animal tissues, including brain and muscle, contain the enzymes necessary to conLYSINE Protein synthesis


kysine protein

zB Adomet Mes kysine-protein e-N-TM L


fl-Hydroxy-e-N-TM L

7-Trimethylam ino butyraldehyde




L-Carnitine Figure 2. Pathway of carnitine biosynthesis in man. TML, trimethyllysine.

vert trimethyllysine to butyrobetaine, but the hydroxylation of butyrobetaine to carnitine occurs only in liver and kidney; interorgan transport of butyrobetaine and carnitine therefore occurs in man. There is also intestinal absorption and transport of exogenous carnitine, as the plasma level is about 10.50-fold less than the concentration in muscle, heart and other tissues. An active uptake of carnitine must take place in vivo; this uptake, however, proceeds at greatly different rates in different tissues and the distribution of carnitine between tissues and the rate of uptake into a given tissue are controlled by growth and a number of hormones, particularly in liver and muscle (Bremer, 1983). Carnitine uptake has been studied in fibroblast tissue culture, in heart myocytes, in muscle myoblasts at various stages of maturation and after innervation (Table 2). The properties of the system for active uptake of carnitine are rather different in various tissues. The Km values reported for L-carnitine vary for different muscle preparations (60-585/xM) but all are lower than the Km found in liver cells (4200-



Table 2. Carnitineuptake in adult tissues. Turnover (hrs) Heart Muscle

21 105

Kidney cortex Small intestine Brain cortex Liver

0.4 --1.3


IXM) 24 60 585 90 206-316 2850 4200 5600

From B r e m e r (1983).

5600/ZM). This agrees with turnover time, which is 100-200 times longer for muscle than for liver. In heart myocytes the capacity for carnitine uptake increases when the cells are grown in the presence of prednisolone. This additive effect may explain why patients with lipid storage myopathy due to low muscle carnitine level, show improvement in treatment with carnitine and prednisolone. L-Carnitine transport into muscle cells has been studied in our laboratory (Vergani et al, 1990b) in human myoblasts at 10, 20 and 30 days of culture. We found two different active transports: the first operates at a 0.5-25/ZM concentration of L-carnitine and is called 'high affinity transport', and its Km and Vmaxdo not change during muscle maturation; the second operates at a 25-200/XM concentration of L-carnitine, is called 'low affinity transport' and its Km and Vmax change during muscle maturation. At 10 days gm is 244 + 85, V m a x = 224 + 75 pM/mg/h; at 20 days Km is 146 + 98, V m a x 85 +---40; at 30 days of muscle culture Km is 62 + 32, Vmax 85 + 40. These results support a modification related to maturation of 'low affinity' L-carnitine transport system.

Myopathic carnitine deficiency Carnitine deficiency was first described by Engel and Angelini (1973) in a woman with recurrent muscular weakness and a lipid storage myopathy, on the basis of the observation that [1-14C]palmitate and [1-14C]oleate oxidation returned to normal on the exogenous addition of 1.5 mM DL-carnitine in the 600g supernatant of a muscle homogenate. This implies that in muscle, after carnitine addition, g-oxidation proceeded normally and that a hypothetical carnitine carrier in the sarcolemmal membrane was abnormal. Since then, several patients with progressive or recurrent muscular weakness and wasting, accumulation of lipid droplets in type ! muscle fibres and low carnitine levels in skeletal muscle, with normal or borderline low levels of carnitine in plasma or liver, have been described. Carnitine esters are normal and there is no abnormal excretion of organic acids in the urine (Table 3). Oral L-carnitine treatment brings about a clinical improvement but is unable to modify the low carnitine content in muscle (Sholte and De Jonge, 1987).



Similar oxidative studies have not been repeated in all cases of lipid storage myopathies with carnitine deficiency, which were subsequently classified as 'muscle carnitine deficiency' (MCD) 'systemic carnitine deficiency' (SCD) or 'mixed carnitine deficiency' (MxCD) only on the basis of the reduced (below 20%) level of carnitine in muscle, plasma and liver (for SCD) and the association of other oxidative enzyme defects (MxCD). Laboratory data on free carnitine and carnitine fractions pooled from the literature show a differential distribution of free carnitine and short-chain acyl and long-chain acylcarnitines in MCD, SCD and MxCD (Table 3), suggesting a different aetiology of these forms and in agreement with the hypothesis that, in secondary MxCD, acylcarnitines accumulate as a result of a B-oxidation or other metabolic block.

Table 3. Laboratory test results in patients with carnitine deficiency. Carnitine deficiency Primary Controls a

Muscle type (%)b

Systemic type (%)b

Secondary with organic acidurias (%)b

43-85 56-135

11-21 10-65

3-97 30-I15

Plasma carnitine flxmol/litre) Free c Total c

(41) 42.8 _+ 8.5 51.1 + 9.4

Muscle carnitine (ixmol/gww) d Free c Short-chain acyl c Long-chain acyl c Total

1.99 0.40 0.11 2.52

_+ 0.68 _+ 0.21 _+ 0.07 _+ 0.76

2-20 32-52 133-463 13-18

1-15 31-111 53-325 14-24

8-52 64-507 75-625 16-73

Liver carnitine 0xmol/gww) d Free c Short-chain acyl c Long chain acyl. c Total c

0.66 0.21 0.03 0.78

(6) _+ 0.19 _+ 0.11 _+ 0.02 _+ 0.22

51-84 78-112 450-475 92-106

1-39 13-48 100-1000 22-36

21-42 28-95 0-188 17-49

(17) 260 ___145 166 d- 62 428 ___188

5-110 125 16-116

18-20 38-100 38-57

2-278 158-1430 58-898

Urine carnitine 0xmol/24 h) Free c Short-chain acyl c TotaY


Urinary organic acids


Succinic acid

Short dicarboxylic acids

Specific pattern

Tissue [1-14C]palmitate oxidation - Carnitine + Carnitine

Normal Normal

Decreased Normal

Decreased Normal

Decreased Decreased

Control data are the m e a n values + SD from our laboratory, n u m b e r of cases in parentheses. b Per cent values were obtained from patients reported in the literature, compared with their own controls. "Free' = free carnitine; 'acyl' = acyl-carnitines; "total' = total carnitine. d gww = gram wet weight.



Systemic carnitine deficiency Typical patients with this disorder suffer from episodes of Reye-like hepatic encephalopathy (Karpati et al, 1975); in some early-onset patients, muscle involvement can be restricted to hypotonia. Total carnitine content is low in plasma, muscle, liver and heart. Some of these cases may be associated with medium-chain acylCoA dehydrogenase deficiency (Zierz et al, 1988). A differential diagnosis between MCD and SCD is based on the earlier age of onset of the latter; SCD is also usually more rapidly evolving and potentially lethal. Nevertheless, the clinical features of patients categorized as having either type of carnitine deficiency are heterogeneous and they have shown a variable response to carnitine supplementation or to LCFA restriction. While muscle carnitine deficiency (Angelini et al, 1987b) is often associated with fluctuating weakness (17/20 cases), respiratory insufficiency (5/20 cases), myalgia and, in two cases, myoglobinuria, systemic carnitine deficiency presents with episodes of Reye-like syndrome (15/26 cases), cardiomyopathy and early age of onset (9f26 cases).

Primary genetic carnitine deficiency and cardiomyopathy Among SCD patients a subgroup is characterized by a progressive cardiomyopathy, which may be fatal (Waber et al, 1982). The disease is familial with autosomal recessive inheritance; carnitine content is low in muscle, heart, liver and plasma (Tripp et al, 1981). In some cases the cardiac dysfunction and hypotonia was so prominent that a 'cardiomyopathic subtype' of SCD with hypoglycaemia, hepatic dysfunction, muscle weakness and Reye-like episodes was proposed (Chapoy et al, 1980). A transport defect has been found recently in a 3~/a-year-old female infant and in a 4-year-old child with this type of cardiomyopathy (Erikson et al, 1988; Treem et al, 1988). In fibroblast cultures the defect of high-affinity uptake of carnitine has been clearly demonstrated. There is now firm evidence for the existence of primary carnitine deficiency (Table 4). It is of interest that in Table 4, Genetic carnitine deficiency • • • • •

Skeletal muscle weakness Cardiomyopathy Episodes of non-ketotic hypoglycaemia Low carnitine in plasma, liver, muscle Defect of high-affinity transport in fibroblasts

these patients the level of plasma and urine carnitine falls suddenly after carnitine supplementation is discontinued. In the cardiomyopathic case described by Chapoy et al (1980) a puzzling feature was that carnitine supplementation normalized the content of carnitine in the liver but not the muscle; after carnitine loading there was a rapid loss in the urine. This may suggest that the generalized defect can be recognized more easily on carnitine loading. Similarly, in the case of a 1-year-old boy with congestive cardiomyopathy described by Rodriguez Pereira et al (1988), after carnitine



loading there was a rise in plasma carnitine followed by a rapid loss in the urine and the faeces, suggesting a deficit in the brush border carnitine transport system of the kidneys and small intestine. Primary autosomic recessive genetic carnitine deficiency therefore seems to be an easily recognizable clinical entity characterized by hypotonia, cardiomyopathy, episodes of coma and hypoglycaemia during fasting (Stanley et al, 1990). The defect is detectable by measuring carnitine uptake in fibroblasts, and the demonstration of an impairment in high-affinity transport. These data suggest a common defect in the high-affinity transport system for carnitine that exists in human kidney, fibroblasts, muscle and possibly intestinal mucosa.

Secondary carnitine deficiency In mixed carnitine deficiencies, occurring in a number of 13-oxidation defects, the total CoA of mitochondria is constant, and so excess acyl-CoA in an enzymatic defect can be rectified only by the addition of carnitine. The overproduction of acylcarnitines results in excessive excretion of these compounds in the urine (Ohtani et al, 1984) and in loss of total carnitine from tissues. This mechanism is particularly evident in the secondary carnitine-deficiency syndromes associated with defects of B-oxidation (Table 5A). Less clear is the pathogenesis of the secondary carnitine deficiency states in respiratory chain defects. In addition, numerous conditions are known in which a low protein intake associated with defective synthesis is responsible for several secondary carnitine deficiency states (Table 5B). As carnitine biosynthesis is initiated by the methylation of lysine, and the trimethylysine formed is converted to butyrobetaine in all tissues but hydroxylated to carnitine only in liver and kidney (Bremer, 1983), it is not surprising that liver failure through cirrhosis results in a state of carnitine Table 5. Secondary carnitine deficiency syndromes A. Secondary to defects of oxidation

Long-chain acyl-CoA dehydrogenase deficiency Medium-chain acyl-CoA dehydrogenase deficiency Short-chain acyl-CoA dehydrogenase deficiency Multiple acyl-CoA dehydrogenase deficiency Isovaleryl-CoA dehydrogenase deficiency Propionyl-CoA carboxylase deficiency Methylmalonyl-CoA mutase deficiency f3-Hydroxy-f3-methylglutaric-CoAlyase deficiency B. Secondary to other syndromes

Idiopathic Reye's syndrome Valproate therapy Renal Fanconi syndrome Chronic renal failure treated by haemodialysis Total parenteral nutrition of premature infants Kwashiorkor Cirrhosis with cachexia Chronic severe myopathies Myxoedema, hypopituitarism, and adrenal insufficiency Pregnancy



deficiency. A peculiar condition in which this also occurs is total parenteral nutrition in premature infants or dialysis in children, in which lack of proper nutrition is combined with insufficient endogenous synthesis and exogenous intake of carnitine. Olson et al (1989), studying the effect of dietary carnitine in variable states of lipid metabolism of human infants, have concluded that lack of dietary carnitine definitely affects the lipid metabolism of infants in the first 4 months of life.

Carnitine palmitoyltransferase (CPT) deficiency Carnitine palmitoyltransferase (CPT; EC catalyses the transfer of LCFA through the mitochondrial inner membrane. It is generally assumed that there are two enzyme forms associated with the outer membrane and the inner surface of the mitochondrial membrane, called CPT-I and CPT-II respectively. Figure 3 depicts hypothetical models of the association of six subunits of inner and outer CPT with the mitochondrial membrane. Current data indicate a different oligomeric molecular weight for beef heart and rat liver mitochondria. A regulatory subunit is also postulated (Bieber, 1988).


~~.. P~ C




I Figure 3. Models of outer CPTo





CPT/ IM ._x

Model4 9((~

(or CPT-I) and inner CPTi (or CPT-II) in mitochondria. In Models 1 and 2 the two CPTs m a y or m a y not be identical. In Model 3 a regulatory subunit is added. In Model 4 the oligomeric subunits of the two C P T are different. IM = inner mitochondrial m e m b r a n e ; O M = outer mitochondrial m e m b r a n e . F r o m Bieber (1988).



Malonyl-CoA is a potent physiological inhibitor of CPT-I in liver, heart, skeletal muscle and other tissues. The mechanism by which malonyl-CoA interacts with CPT-I is not completely understood: inhibition of malonyl-CoA is competitive with longchain acyl-CoAs and requires membrane association of the enzyme. The binding site for malonyl-CoA appears to be distinct from the catalytic site of the enzyme (Figure 1).

Measurement of CPT Because of the bifunctional action of CPT, this enzyme can be measured with a simple isotope-exchange reaction, or by a forward assay (i.e. producing palmitoylcarnitine), or by a reverse reaction (i.e. producing palmitoyl CoA). However, none of these reactions is able to distinguish between CPT-I and CPT-II and therefore specific inhibitors (malonyl-CoA) or detergents such as Triton X are added for this purpose.

Muscle CPT deficiency Since the original description by Di Mauro and Di Mauro (1973) more than 50 cases with exercise-induced pigmenturia, pain and rhabdomyolysis have been reported. The disease is inherited in an autosomal recessive fashion (Angelini et al, 1981). Carriers demonstrate 50% activity in muscle and platelets as estimated by the isotope exchange method. In the literature there is a predominance of male patients reported, probably because environmental and hormonal factors influence the expression of the disease. In a typical patient the myoglobinuric attack is triggered by prolonged exercise or fasting (Trevisan et al, 1987). Cold, fever, anaesthesia or other stresses may induce the crisis: recently, Katsuya et al (1988) reported a patient with postanaesthetic acute renal failure due to CPT deficiency.

Pathogenesis and consequences of CPT deficiency Since the original description of muscle carnitine and CPT deficiency in 1973, the following problems have been the subject of debate: 1. 2. 3. 4.

Why do patients with CPT deficiency suffer from intermittent attacks of rhabdomyolysis whereas patients with carnitine deficiency suffer from a remitting or progressive muscular weakness? Why is there so little lipid accumulation in muscle of CPT-deficient patients? Why do cold or fasting trigger the rhabdomyolysis? Do one or two CPT isozymes exist and are they selectively mutated in different tissues (muscle, liver)?

An answer to some of these questions has come from a study by Zierz and Engel (1985) that demonstrated altered regulatory properties of CPT in CPT deficiency: the enzyme is abnormally inhibited by increasing substrate/



product concentration and by malonyl-CoA. This suggests that the patient's enzyme is more vulnerable when lipid metabolism is stressed (cold, fasting, infections) and LCFA are mobilized. The hypothesis that anaesthetics may be a precipitating factor for rhabdomyolysis in patients with CPT deficiency is supported by the observation by Zierz and Schmitt (1989) that the malonyl-CoA inhibition of the forward reaction of CPT in normal muscle is much greater after general anaesthesia (80%) than after local anaesthesia. Further clues are given by the inhibition of carnitine palmitoyltransferase by chlorpromazine or detergents, which is greater in patients than in controls, as shown by the isotope-exchange reaction (Zierz and Neumann-Schmitt, 1989). The amphiphilic character of chlorpromazine, a drug that interacts with phospholipid membranes, enables it to exhibit detergent-like properties. Abnormal inhibition of carnitine palmitoyltransferase activity in the muscle of patients with CPT deficiency has been shown also by the detergent Triton X-100, which solubilizes mostly CPT-I, the outer enzyme. A clear demonstration of two different CPT enzymes in man is still lacking, although McGarry et al (1990) have been able to show that CPT-I has different molecular sizes in various rat tissues (liver and muscle forms have molecular weights of 94 kD and 86 kD respectively). The inner CPT-II, however, seems to be more uniform in size and molecular weight in mammalian tissues (68 kD). The varying clinical presentation of CPT-deficient patients may be attributed to different types of mutation of these enzymes or to differences in their location and attachment to the mitochondrial membrane; defects in muscle CPT may ultimately be interpreted as an altered interaction between the enzyme and its membranous environment (Angelini et al, 1987a).

Hepatic CPT defect Bougn6res et al (1981) reported an 8-month-old female infant who had fasting hypoglycaemia, coma and seizures associated with deficient ketogenesis. The patient had minimal hepatomegaly, and hepatic CPT activity (measured by an 'isotope exchange' assay) was greatly reduced. Demaugre et al (1989) have made detailed studies in fibroblasts of 'hepatic' and 'muscle' CPT in four fibroblast cell lines. The first 'hepatic' cell line was derived from the original patient of Bougn6res et al (1981); the second was from a second 'hepatic' CPT patient, a 1-year-old who also presented, after 19 hours of fasting with hypoglycaemic coma and lack of ketogenesis; the other two fibroblast lines were from patients who presented with muscle cramps and rhabdomyolysis, i.e. with the classic 'muscular' symptoms. Overall, CPT activity was deficient in the fibroblasts of patients with the hepatic presentation. The 'hepatic' patients' fibroblasts displayed a CPT1 deficiency, which resulted in impaired long-chain fatty-acid oxidation. In contrast, CPT1 activity and palmitate oxidation were normal in fibroblasts of 'muscular' patients, where the maturation presumably involved CPT2 activity. Demaugre et al (1988) demonstrated this using three different assays for CPT activity and oxidation of LCFA in cultures.



The overall data are consistent with the fact that CPT deficiency is biochemically heterogeneous and is the result of two different mutations, producing two clinical patterns. DEFECTS OF ACYL CoA DEHYDROGENASE Common features Most of the genetic defects of g-oxidation have a fairly constant clinical presentation: episodes of lethargy in children leading to non-ketotic, hypoglycaemic coma, preceded by nausea and vomiting. These critical episodes may be fatal; in most cases of sudden infant death syndrome (SIDS), therefore, screening for g-oxidation defects is mandatory (Emery et al, 1988). A neonatal form characterized by failure to thrive may also be seen, while in children with defects of later onset, intellectual impairment and hypotonia gradually become manifest. Frequently, the metabolic attacks are triggered by fasting and/or intercurrent infectious diseases.

Long-chain acyl-CoA dehydrogenase(LCAD) The first description of three patients with LCAD deficiency was by Hale et al (1985); the patients were infants who presented episodes of non-ketotic hypoglycaemia and a specific involvement of cardiac muscle consisting of a progressive hypertrophic cardiomyopathy. Skeletal muscle was also involved, leading to chronic muscle weakness. Subsequently, 11 further cases have been reported, with age of onset variable between 2 days and 96 months (mean age: 5 months) (Table 6). The Table6. Long-chainacylCoA-dehydrogenase deficiency. • Children • Age 2 days- 96 months • Progressivestupor, vomiting, fever • Hypotonia • Hepatomegaly • Cardiomegaly:biventricular hypertrophy • Laboratory: Hypoglycaemia Hypocarnitinaemia Liver biopsy: macrovesicularfat clinical syndrome is characterized by progressive stupor, vomiting and fever after oral intake of food (Hale et al, 1990). Hypoglycaemia, hypotonia, hepatomegaly 6/15 cases) and cardiomegaly are also a common feature. On liver biopsy, macrovesicular fat is found. In urine the most common excretory products are adipic and suberic acid. Analysis of organic acids in urine reveals a characteristic pattern of C6-C14 dicarboxylic aciduria. In the patients described, LCAD activity was reduced to less than 10% of the control values in leukocytes, cultured fibroblasts and liver tissue. There are



low plasma carnitine and increased esterified acylcarnitine levels in plasma and urine. In familial studies some cases of SIDS have been observed. The differential diagnosis between LCAD and MCAD is given by a younger age of onset in the first defect and by the occurrence of a more profound cardiac involvement. However, a milder form of LCAD deficiency, as reported by Naylor et al (1985), may be difficult to distinguish from MCAD deficiency.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency The medium-chain acyl-CoA dehydrogenase or 'general acyl-CoAdehydrogenase' has a very important role: in fact, several cases that previously were attributed to systemic carnitine deficiency are now classified as MCAD (Zierz et al, 1988). Children affected by MCAD can be normal between the attacks; Reye-like episodes are precipitated by fasting or illness, when a dramatic clinical picture is presented. Typical signs include intolerance to prolonged fasting, nausea, vomiting, lethargy (Table 7) characterized by laboratory findings of non-ketotic hypoglycaemia and elevated

• • • • •

Table 7. Medium-chain acylCoA dehydrogenasedeficiency. Reye-likesyndrome Coma, bradycardia Non-ketotichypoglycaemia Frequent SIDS Laboratory: Low free carnitine (plasma, urine) Aeylglycineexcretion Aeylcarnitine excretion (L-carnitine loading test: octanoylcarnitine, hexanoylcarnitine)

dicarboxylic acid levels. The hypoglycaemia is due to the defective ketogenesis and exhaustion of gluconeogenic substrates. In the urine, increased excretion of medium-chain dicarboxylic acids, octanoyl-carnitines and acylglycines is found. The Reye-like episode can be fatal and 25% of these patients may succumb at the first episode. As a possible treatment is glucose administration and restriction of dietary fats, screening for MCAD is mandatory in families with SIDS by studying acylcarnitines and acylglycines in plasma and urine. Oxidation of labelled octanoic acid in fibroblasts (Rhead, 1990) is also useful in suspected cases, as MCAD deficiency is the most frequent condition arising from g-oxidation defect. So far, over 100 MCAD deficiency cases have been reported since the original description by Kolvras et al (1982) and MCAD is considered to be the commonest genetic cause of SIDS in infants. A useful method for the diagnosis of MCAD deficiency, and to investigate the percentage of SIDS that are actually due to MCAD deficiency, has been developed by Rinaldo et al (1988), who studied acylglycine estimation in urine samples using stable isotope dilution analysis. This test is very accurate for diagnostic purposes because MCAD patients excrete large amounts of suberyglycine, hexanoylglycine and 3-phenylpropionylglycine in the urine. In MCAD patients the activity is reduced in liver, skeletal muscle and cultured fibroblasts to levels 2-10% of the mean of controls. As far as the



molecular aspect of MCAD deficiency is concerned, the clinical phenotypes are probably attributable to a variety of molecular lesions, such as point mutations affecting the kinetic properties of the enzyme. Short-chain acyl-CoA dehydrogenase (SCAD) deficiency

Molecular cloning of the cDNA encoding human SCAD has been achieved by Naito et al (1989) and the gene has been mapped on chromosome 12. This enzyme defect can be observed in a generalized form in infants with poor weight gain and psychomotor retardation, or in adults as a myopathic later-onset disorder. There has been a considerable increase in knowledge over the last few years about the myopathic form of SCAD deficiency. Turnbull et al (1984) described a 46-year-old woman with proximal muscle weakness and muscle pain. She had glutaric aciduria type II, lipid storage myopathy and low plasma carnitine, and low activity of dehydrogenation of short-chain acyl-CoA esters in muscle. SCAD activity in cultured fibroblasts from this patient was normal and there was a large ketotic response to fasting indicating that the liver enzyme was also normal. Some patients with muscle SCAD deficiency are riboflavin responsive; it is therefore important to recognize this disorder (Table 8). Table 8. Short-chain acyl CoA-dehydrogenase

(SCAD) deficiency. A. Generalized infantile Poor weightgain Psychomotorretardation Fatal B. Myopathic form

Adult onset Lipid storage myopathy Low free carnitine,increasedacyl-carnitine 132responsive, SCAD reappears

Generalized SCAD deficiency Amendt et al (1987) described SCAD deficiency in two unrelated neonates that presented failure to thrive, vomiting, delay in developmental milestones, hypotonia and metabolic acidosis, and died at 20 and 30 months. The two children exhibited an abnormal organic aciduria including ethylmalonic and methylsuccinic acid, and butyrylglycine. The activity of the enzyme in the patients' cultured fibroblasts was 1-11% of that of controls. Coates et al (1988) subsequently described another female child with failure to grow, muscle weakness, mild muscle lipid storage and low free carnitine in muscle (50% of controls) that was 75% in the esterified form. SCAD was found to be deficient in the cultured fibroblasts after inhibition of a residual MCAD activity with an antibody; the parents had intermediate levels consistent with autosomal recessive inheritance.



Muscle SCAD deficiency Turnbull et al (1984) described a 46-year-old woman affected by a lipid storage myopathy. Over an 8-month period the patient developed persistent weakness of the left arm and both legs and after exertion mild myalgia and dyspnoea. She had low carnitine levels, but increased short- and long-chain acylcarnitine levels in muscle and palmitoyl-CoA oxidation in the presence of carnitine, palmitoyl carnitine and octanoate oxidation was only 20% in muscle mitochondria. The patient excreted increased amounts of ethyl malonic and methylsuccinic acids in the urine. Ketone body turnover was elevated. SCAD deficiency was found in the muscle mitochondria. Di Donato et al (1986b) described another adult patient with short-chain acyl-CoA dehydrogenase deficiency with secondary carnitine deficiency. It has been suggested that both these patients with muscle SCAD deficiency may suffer from a form of riboflavin-responsive multiple acyl-CoA dehydrogenase-deficient myopathy (Di Donato et al, 1989).

Riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency Patients with this condition are similar to those with mild ETF or ETF dehydrogenase defects (Gregersen, 1985) and to muscle SCAD patients. This clinical phenotype is also reminiscent of muscle or systemic carnitine deficiency, with a myopathy involving proximal limb and axial muscle and a clear lipid storage in muscle with low plasma and tissue carnitine. Occasional patients have, in addition, episodes of nausea, vomiting, metabolic acidosis and lethargy (De Visser et al, 1986). Carroll et al (1980) described a patient with muscle carnitine deficiency, who did not respond to carnitine supplementation in Vivo; furthermore, exogenous added carnitine was not able to correct the fatty acid oxidation defect in vitro. However, this patient responded to riboflavin supplementation. In similar cases reported by De Visser et al (1986) and Di Donato et al (1989), the authors have also observed normalization of organic acids in urine, resolution of metabolic attacks and increased muscle strength. Di Donato et al (1989) also found low SCAD and MCAD activity in a 12-year-old girl with lipid storage myopathy. Increase in muscle bulk and strength upon treatment was associated with the 'reappearance' of SCAD, implying a role of riboflavin in synthesis or catabolism of these enzymes.

DEFECTS OF THE ELECTRON-TRANSFER FLAVOPROTEIN (ETF) AND ETF DEHYDROGENASE ETF is a small dimeric protein consisting of an c~ and 13 subunit. A cDNA corresponding to the full-length transcript of the a subunit of ETF has been characterized recently (Finocchiaro et al, 1988); the human gene has been mapped to chromosome 15 (Finocchiaro et al, 1987). Electron-transfer flavoprotein can be prepared from pig liver for the assay of acyl-CoA dehydrogenase by the method of Husain and Steenkamp (1983) using a



series of columns (Figure 4), and then identified by electrophoresis, its characteristic absorbance at 436 nm (Table 9) or by its fluorescent spectrum. Different ETF preparations show slightly different mobilities of subunits and peak absorbances. Heterogeneous clinical syndromes have been described (Table 10): 1.

Neonatal form with congenital anomalies (polycystic kidney). This form is a rapidly fatal disorder due to cardiomyopathy (in this form both and 6 subunits of ETF are absent). Pig liver, 5 kg Step A Mitochondria

Step B Sonic supernatant, 1200 mL

(Fraction 1)

DEAE sephacel column (5x3Ocm) Hepes 25 mM, pH 7.8 Step C Crude ETF, 120mL

(Fraction l-F)


Step D

CM-sephadex (C25) column (30x60 cm) Hepes I0 mM/lO% ethylene glycol pH 7.8 Step E Partially purified ETF, 60mL

(Fraction TIT)

Step F Hydroxyapatite column (3x2Ocm) IOmM K-phosphate/lO% ethylene glycol pH 7.1 Step G Purified ETF, 40.19mg




Visible spectra

Fluorescence spectra

Figure 4. Preparation of a purified ETF from pig liver. ETF was then examined by electrophoresis, visible and fluorescent spectra (see Table 9).


DEFECTS OF FATTY-ACID OXIDATION IN MUSCLE Table 9. Comparison of ETF preparations. Property Absorbance characteristics

Present study Husain and Steenkamp (1983)

Peak (nm)


MR subunit (kD)

375,436,460 375,436,460

0.93 : 1.0 : 0.86 0.90 : 1.0:0.86

non-identical 35-29 non-identical 38-32

Table 10. ETF deficiency. Clinical heterogeneity 1. Neonatal with congenital anomalies (polycystic kidney) 2. Neonatal onset: severe cardiomyopathy 3. Later onset: ethylmalonic-adipic aciduria, glutaric aciduria type II, low carnitine



Neonatal form without congenital anomalies. These neonates exhibit hypoglycaemia, severe acidosis, lethargy and a peculiar odour ('sweaty feet odour'). They excrete in the urine large amounts of glutaric, ethylmalonic, isovaleric, isobutyric saturated and unsaturated dicarboxylic acids, sarcosine and glycine conjugates of organic acids. A later-onset form termed 'ethylmalonic-adipic aciduria' or late-onset glutaric aciduria type II, so called because of the pattern of organic acids found in the urine, the most prominent peak seen in the urine being glutaric acid. The organic acid profile reflects the block in oxidation of fatty acids, lysine and tryptophan as well as leucine, isoleucine and valine, all FAD-dependent substrates through ETF and ETF dehydrogenase.

A similar spectrum of disorders is found also in EFT dehydrogenase deficiency (Frerman and Goodman, 1985) and it is difficult to differentiate between the two forms unless specific antibodies against ETF are used to distinguish the two proteins in tissues. A partial deficiency of ETF has been reported in patients with the milder form of multiple acyl-CoA deficiency. Patients with the severe forms have marked reduction of oxidation rates in cultured fibroblasts. A late-onset adult form of glutaric aciduria type II associated with recurrent hypoglycaemia and progressive lipid storage myopathy has been reported by Dusheiko et al (1979). They described a 19-year-old woman who had metabolic attacks similar to Reye's syndrome, progressive weakness and wasting of skeletal muscle, a lipid storage myopathy and low carnitine. A similar case, of an 8-year-old boy who died of attacks of hypoglycaemic acidosis and coma, has been described by Di Donato et al (1986a): at autopsy, lipid storage and low carnitine was found in muscle, heart and liver; a marked deficiency of ETF dehydrogenase was found in cultured fibroblasts.







This is an autosomal recessive disorder characterized by the abnormal accumulation of triglycerides in muscle, liver, leukocytes (Jordan's anomaly), gastrointestinal cells and cultured skin fibroblasts. The spectrum of clinical signs includes ichthyosis, hepatosplenomegaly, vacuolated granulocytes in bone marrow, psychomotor delay and progressive myopathy (Angelini et al, 1980). The cultured cells show typical lipid storage droplets and accumulate increased [1J4C]fatty acids while their oxidation is decreased (Figure 5). [I-I¢C] Palmitote 40.E Controls ~.. 5O .b-

Patient fibroblosts

20 (O


o IO E 8




6 Time (h)



Figure 5. The oxidationof palmitate by fibroblastsis decreasedm a patient with multisystem triglyceridestorage disease. Over 10 similar cases have now been reported, including two sisters of Japanese origin born from a consanguineous marriage (Ibayashi et al, 1988). On electron microscopy the vacuoles are found to lack a limiting membrane and are adjacent to mitochondria. Despite the strong suggestion of impaired LCFA metabolism there is no evidence of carnitine deficiency or defective uptake in the cell or of a CPT defect. A 50% impaired oxidation of fatty acids was found on measuring the rate of production of 14CO2 or acidsoluble [14C]metabolites from [1-14C]palmitate in the cases described by Angelini et al (1980) and lbayashi et al (1988). Normal long-chain fatty-acid esterase activities were found in patients' leukocytes at pH 4.0 and 8.0. Conversely, Di Donato et al (1988) found normal oxidation of shortchain, medium-chain and long-chain fatty acids. A specific impairment in the degradation of endogenously synthesized fatty acids has been postulated. Although the underlying metabolic defect has not yet been elucidated, the disease seem to be an autosomal recessive inherited disorder of



systemic triglyceride storage, presumably attributable to impaired regulation of endogenous fatty-acid oxidative catabolism.

SUMMARY Long-chain fatty acids (LCFA) are oxidized by muscle mitochondria after transport in the cytosol by fatty-acid-binding protein(s) and their activation by a thiokinase. Carnitine, two forms of carnitine palmitoyltransferase(s) and carnitine acylcarnitine translocase are involved in L C F A gating. A primary genetic carnitine deficiency occurs in children with dilated cardiomyopathy, hypoglycaemia and low carnitine content in plasma, liver and muscle, owing to a defect in a c o m m o n high-affinity transport system. This high-affinity transport in muscle differs from a low-affinity transport that has modifications during muscle maturation. The genetic enzyme defects of g-oxidation (long-chain acyl-CoA dehydrogenase, medium- and short-chain acyl-CoA-dehydrogenase) present with Reye-like attacks that may lead to non-ketotic hypoglycaemia, coma and sudden infant death syndrome. T h e r e is elevated urinary excretion of dicarboxylic acids, acylcarnitines and acylglycines. Secondary carnitine deficiency m a y occur. E T F and E T F dehydrogenase deficiencies may present in a neonatal form with congenital anomalies, or in a later-onset form with ethylmalonic adipic aciduria. A still-unidentified defect leads to L C F A accumulation in fibroblasts, bone marrow, liver and muscle cells in a multisystem triglyceride disorder.

Acknowledgements This paper has been supported by the Muscular Dystrophy Association and by a CNR bilateral contract. Ms Annalisa Leone typed the manuscript. Doctors Zierz, Rosa, Carrozzo and Vergani collaborated in ETF purification.

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Defects of fatty-acid oxidation in muscle.

Long-chain fatty acids (LCFA) are oxidized by muscle mitochondria after transport in the cytosol by fatty-acid-binding protein(s) and their activation...
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