Brain, Vol. 122, No. 12, 2401-2411,
December 1999
© 1999 Oxford University Press
Riboflavin therapy
Biochemical heterogeneity in two adult lipid storage myopathies
1 Neuromuscular Center, Department of Neurological Science, 2 Department of Pediatrics, 3 Department of Pharmacology, University of Padova, Padova, 4 Department of Biochemistry and Molecular Biology, University of Bari and Study Center for Mitochondria and Energy Metabolism, Italian Council for Research, Bari, Italy and 5 Department of Molecular Biology, University of Amsterdam, Amsterdam, The Netherlands
Correspondence to:
Lodovica Vergani, Dipartimento di Scienze Neurologiche e Psichiatriche, Via Giustiniani 5, 35128 Padova, Italy E-mail: Ivergani{at}ux1.unipd.it
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Abstract |
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Two unrelated adult males, aged 36 (patient 1) and 25 (patient 2) years, presented with subacute carnitine-deficient lipid storage myopathy that was totally and partly responsive to riboflavin supplementation in the two patients, respectively. Plasma acyl-carnitine and urinary organic acid profiles indicated multiple acyl coenzyme A dehydrogenase deficiency, which was mild in patient 1 and severe in patient 2. The activities of short-chain and medium-chain acyl coenzyme A dehydrogenases in mitochondrial fractions were decreased, especially in patient 2. This was in agreement with Western blotting results. Flavin-dependent complexes I and II were studied by immunoblotting and densitometric quantification of two-dimensional electrophoresis with comparable results. Complex I was present in normal amounts in both patients, whereas complex II was decreased only in the pretherapy muscle of patient 2. Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) concentrations in muscle and isolated mitochondria, and the activity of mitochondrial FAD pyrophosphatase, showed that patient 1 had low levels of FAD (46%) and FMN (49%) in mitochondria, with a significant increase (P < 0.01) in mitochondrial FAD pyrophosphatase (273%) compared with controls. Patient 2 had similar low levels of FAD and FMN in both total muscle (FAD and FMN 22% of controls) and mitochondria (FAD 26%; FMN 16%) and normal activity of mitochondrial FAD pyrophosphatase. All of these biochemical parameters were either totally or partly corrected after riboflavin therapy.
acyl-carnitine profile; flavo-coenzyme; mitochondrial FAD pyrophosphatase; multiple acyl coenzyme A dehydrogenase deficiency; OXPHOS enzymes
CoA = coenzyme A; FAD = flavin adenine dinucleotide; FMN = flavin mononucleotide; OXPHOS = oxidative phosphorylation; MAD = multiple acyl coenzyme A dehydrogenase deficiency; MCAD = medium-chain acyl coenzyme A dehydrogenase; SCAD = short-chain acyl coenzyme A dehydrogenase
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Introduction |
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Riboflavin is the precursor for essential flavo-coenzymes [flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD)]. FMN and FAD are the prosthetic groups of numerous enzymes that catalyse the various electron transfer reactions that occur in energy-producing, biosynthetic, detoxifying and electron-scavenging pathways. Most cell flavoproteins are found in mitochondria (McCormick, 1989
). As riboflavin cannot be synthesized by mammals and there is no major tissue storage pool known other than that of riboflavin-derived cofactors associated with specific enzymes, the supplies of this vitamin must come from food (Bates, 1993).
An inadequate dietary intake of riboflavin, tissue growth or increasing metabolic demand, and also fasting, which causes increased excretion of riboflavin (Windmüeller et al., 1964), may induce a riboflavin-depleted status, as a consequence of which certain flavo-enzymes may decrease and stop functioning. The homeostasis of riboflavin and its coenzymes may also be altered by other factors, such as reduced intestinal adsorption and cellular transport, defective FMN and/or FAD synthesis, increased catabolism and altered mitochondrial metabolism and transport (Ross and Hansen, 1992).
In the intracellular metabolism of riboflavin, besides the known cytoplasmic enzymes that interconvert riboflavin, FMN and FAD (McCormick, 1989), new mitochondrial-specific enzymes have been discovered in both the synthetic and the catabolic pathway of flavin cofactors (Barile et al., 1993, 1997), further elucidating the mechanisms by which the mitochondrial level of riboflavin-derived cofactors is maintained.
Certain riboflavin-responsive patients present with defects in different single flavo-apoenzymes: complex 1 (Bernsen et al., 1993; Ogle et al., 1997), pyruvate dehydrogenase (Scholte et al., 1992), electron transfer flavoprotein (Bell et al., 1990) and short-chain acyl coenzyme A (CoA) dehydrogenase (SCAD) (Kmoch et al., 1995). In these cases, the beneficial effects of therapy may be due to the stabilization and maintenance of polymorphic flavoprotein, with consequent increased activity.
Several other patients, who respond clinically and biochemically to pharmaceutical doses of riboflavin, have been described with defects of ß-oxidation (Gregersen, 1985; De Visser et al., 1986; Gilkeson and Caldwell, 1988; Turnbull et al., 1988; DiDonato et al., 1989; Peluchetti et al., 1991; Triggs et al., 1992; Antozzi et al., 1994; Araki et al., 1994; Vergani et al., 1996; Elias et al., 1997). According to their clinical-biochemical features, riboflavin-responsive patients have been described as suffering from glutaric aciduria type II, a severe form of multiple acyl CoA dehydrogenase deficiency (MAD) or ethylmalonic-adipic aciduria, a mild form of MAD. Defect(s) related to the metabolism of FAD or FMN in these patients have been regarded as theoretically possible but have never been ascertained. Uptake defects of riboflavin and altered cytoplasmic metabolism of FAD or FMN have been excluded by in vitro studies (Gregersen, 1985; Antozzi et al., 1994), but a defect in the maintenance of mitochondrial FAD was found in cultured cells from a riboflavin-responsive patient (Rhead et al., 1993).
In the present study, two adult male patients suffering from MAD were investigated. They showed a positive response to riboflavin therapy, although they had different degrees of clinical and biochemical recovery. In order to obtain more insight into their metabolic dysfunction(s), we measured (i) the presence and level of oxidative phosphorylation (OXPHOS) enzymes, (ii) FAD and FMN in total muscle extracts and muscle mitochondria before and after riboflavin therapy, and (iii) the activity of a novel mitochondrial FAD pyrophosphatase in isolated muscle mitochondria.
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Patients and methods |
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Patient 1
This patient, a 36-year-old male, was in good health until he developed progressive generalized muscle weakness. In 8 months he became virtually tetraplegic and was admitted to hospital in November 1993. The following serum enzymes were elevated: creatine phosphokinase, 2700 U/l (normally <190 U/l); lactic dehydrogenase, 848 U/l (normally <220 U/l); aldolase, 21.9 U/l (normally <7.6 U/l); aspartate transferase, 313 U/l (normally <45 U/l); and alanine transferase, 191 U/l (normally <55 U/l). Uric acid was 0.56 mM (normally 0.21–0.42 mM). Abnormally high ketone body levels were found in the urine. Electrocardiography showed tachycardia, whereas an electromyograph was myopathic. Since the patient's masticatory muscles were particularly weak, he had fasted for a long period, with a consequent weight loss of 14 kg. His faeces were positive for Strongyloides stercolaris, and this worm infection was treated. A muscle biopsy presented a lipid storage myopathy, type 1 fibre atrophy and mild mitochondrial alterations. Muscle and mitochondrial fractions were collected and used for biochemical studies (pretherapy sample). Total and free muscle carnitine concentrations were 4.3 and 1.8 nmol/mg non-collagen protein respectively (normal range: total, 10.5–29.5; free, 8.3–24.3 nmol/mg) with a five-fold increase in the acyl : free ratio (patient, 1.41; normal range, 0.09–0.78). The patient's clinical condition improved rapidly after therapy with carnitine (4 g/day), riboflavin (100 mg/day) and prednisolone (75 mg/day). The steroids were tapered off when (i) his creatine phosphokinase decreased, and both (ii) his muscle strength and (iii) body weight returned to normal. The urine organic acid profile, studied by liquid chromatography after 2 months of treatment, was normal. In August 1994, the patient was rehospitalized for cholecystectomy. During this period, he fasted again and was on parenteral nutrition for 2 weeks; riboflavin therapy was suspended. Six weeks later he again complained of fatigue and muscle weakness, and his condition progressively deteriorated until March 1995. Riboflavin supplementation was started again and his symptoms disappeared in 2 weeks. In June 1996, while the patient was still under riboflavin therapy and he was in good clinical condition, a second muscle biopsy was carried out and mitochondria were isolated. Muscle specimens were analysed (post-therapy sample). The morphological parameters and carnitine concentration of the second biopsy were normal.
Patient 2
This patient, a 25-year-old male, had been in good health until 1993, when he had a depressive episode associated with loss of appetite, heavy drinking, nausea and vomiting. He lost 11 kg (from 83 to 62 kg), complained of muscle pain, fatigue and difficulty in climbing stairs, and was able to walk only for a short distance (1 km). Shortly afterwards, he had difficulty in raising his arms and keeping his head upright. He was hospitalized in July 1993. Neurological examination revealed decreased strength of the suprascapular and periscapular muscles, and the deltoid, biceps, neck flexor and ileopsoas muscles were also slightly weak. He had mainly scapulohumeral and pelvic muscle involvement and facial asymmetry; there was slight scoliosis. Electromyography showed myopathic motor units and a few fibrillations. Creatine phosphokinase was 478 U/l, lactic dehydrogenase 1000 U/l, aldolase 4.9 U/l, aspartate transferase 90 U/l, alanine transferase 64 U/l and uric acid 0.4 mM. Serum carnitine showed an abnormal acyl : free carnitine ratio. The first muscle biopsy (July 1993) showed a vacuolar myopathy with lipid storage and evident mitochondrial alterations (subsarcolemmal rims, myofibrillar disorganization with mitochondrial proliferation) and carnitine deficiency: total carnitine was 2.0 and free carnitine 1.3 nmol/mg non-collagen protein. Treatment consisting of carnitine (3 g/day) and coenzyme Q (120 mg/day) was started, but the patient did not improve with carnitine supplementation alone. Two months later he was again hospitalized, and a second muscle biopsy was carried out, and mitochondrial fractions were prepared. Muscle carnitine fractions were 6.4 (total) and 2.9 (free) nmol/mg non-collagen protein. Ultrastructural examination confirmed the mitochondrial abnormalities observed in the first biopsy, and showed mitochondrial hyperplasia with paracrystalline inclusions. This muscle sample was also used for biochemical studies (pretherapy). Riboflavin (100 mg/day) was added to carnitine in August 1993 for 6 months. There was some improvement, and the patient had less difficulty in walking and raising his arms, although he still complained of fatigue and muscle pain. As upper girdle muscle weakness persisted, in February 1994 a third muscle biopsy was performed, and again mitochondria pellets were collected. This muscle sample was used for post-therapy studies of SCAD and medium-chain acyl CoA dehydrogenase (MCAD), OXPHOS enzymes and total FAD and FMN quantification. Histochemical and ultrastructural examination showed partial regression of abnormalities, and muscle carnitine concentrations rose to 11.6 (total) and 8.2 (free) nmol/mg non-collagen protein. Medium-chain triglyceride oil was added to the therapy. This treatment led to definite improvement for 2 years and the patient's weakness disappeared. The resting lactate concentration, measured in November 1995, was 2.1 mM (normally 1.0–1.8 mM) and after 5 and 10 min of aerobic exercise it reached 9.5 and 7.8 mmol/l respectively. In January 1997, the patient again presented with increasing muscle weakness and pain in the upper girdle muscles. Neurological examination showed asymmetric shoulder girdle weakness, with predominant weakness of the left infrascapular and deltoid muscles. Creatine phosphokinase was 229 U/l and urine 3-hydroxybutyric acid was 41 mM/M creatinine (normally 0.1–5.8 nM/M). At that time, a fourth muscle biopsy was carried out, and mitochondria were isolated and used for post-therapy quantification of mitochondrial FAD, mitochondrial FMN and mitochondrial FAD pyrophosphatase. The patient was treated with methyl prednisone (8 mg/day), medium-chain triglyceride oil, riboflavin (100 mg/day) and L-carnitine (3 g/day). His condition rapidly improved and steroids were tapered off. In conclusion, this patient had fluctuating myopathy involving the scapulohumeral girdle, and also complained of intermittent leg weakness. He is now doing well under strict dietary control.
Preparation of mitochondrial fractions
Skeletal muscle samples (vastus lateralis) were obtained by open biopsy from the two patients before and after riboflavin therapy and from informed-consent subjects undergoing orthopaedic surgery. Mitochondrial fractions were prepared from fresh skeletal muscle as described previously (Zerbetto et al., 1997).
Analytical procedures
Urine organic acids were tested by gas chromatography/mass spectrometry of trimethylsilyl derivates after extraction with diethyl ether on a Hewlett Packard 5780 apparatus (Helwett Packard, Palo Alto, USA). Plasma acyl-carnitine profiles were determined by electrospray-ion/tandem mass spectrometry (Quattro II; Micromass, Manchester, UK) according to a method described previously (Rashed et al., 1995). Muscle carnitine was assayed as described elsewhere (Vergani et al., 1996). Concentrations of FAD and FMN in muscle extracts and mitochondrial fractions were obtained by HPLC (Beckman, Fullerton, USA) as described previously (Batey and Eckhert, 1990), using a reverse-phase column (ultrasphere ODS 5µ, 4.6 mm x 25 cm; Beckman). Protein concentrations were determined by Bradford's method (Bradford, 1976).
Enzyme activity assays
SCAD and MCAD activities were evaluated in muscle by the ferricenium method (Lehman et al., 1990).
The activity of mitochondrial FAD pyrophosphatase was measured in muscle mitochondria according to the original method described by Barile and colleagues (Barile et al., 1997). Briefly, FAD (10 µM) was suspended at 25°C in 1.5 ml of a standard medium consisting of 0.25 M sucrose, 20 mM Tris–HCl, pH 8.5, and 1 mM MgCl2, and the FAD hydrolysis reaction was started by the addition of Triton X-100 solubilized mitochondria (0.01–0.17 mg protein). The fluorescence of the suspension was continuously monitored by means of a spectrophotofluorimeter (LS50, Perkin Elmer, Norwalk, USA) with excitation and emission wavelengths set at 450 and 520 nm, respectively. Since in the experimental conditions used here the FAD fluorescence constant proved to be ~10 times lower than that of FMN, the rate of FAD hydrolysis was calculated from the rate of fluorescence increase and was expressed as pmol FAD hydrolysed/min/mg protein, as previously described (Barile et al., 1997). When the hydrolysis of endogenous FAD was measured, no addition of FAD to the mitochondrial suspension was made.
Immunoblotting
SCAD and MCAD
Proteins in mitochondrial fractions were separated by SDS–PAGE (sodium dodecyl sulphate–polyacrylamide gel electrophoresis) (Laemmli, 1970) using 10% polyacrylamide gels, transferred to nitrocellulose membrane (Towbin et al., 1979) and probed with polyclonal antibodies against acyl CoA dehydrogenases (a gift from Professor D. M. Turnbull, Newcastle-upon-Tyne, UK).
OXPHOS complexes
Complexes were extracted from 15 mg samples of skeletal muscle with 10% dodecyl maltoside, resolved by blue native PAGE (Zerbetto et al., 1997) and blotted onto nitrocellulose filters (Towbin et al., 1979). The nitrocellulose membrane was cut into three strips, corresponding to the various complexes that were resolved: complex I, complex V and complex II. Each strip was probed with the specific polyclonal antibodies against subunit 24 of complex I (a gift from Professor J. E. Walker, Cambridge, UK), complex V (Nijtmans et al., 1995) and the FAD covalently bound to the flavoprotein subunit of complex II (Barber et al., 1987) (a gift from Professor R. Brandsch, Freiburg, Germany), respectively. Immunoreactive materials were visualized by ECL.
Quantification of OXPHOS complexes
Fifteen milligrams of skeletal muscle was detergent-extracted twice and used as the starting material for two-dimensional (2D) electrophoresis. In the first dimension, separation of complexes by blue native PAGE occurred according to molecular mass (Zerbetto et al., 1997), and on SDS–PAGE in the second dimension the individual subunits of the complexes were resolved as described previously (Schägger et al., 1994). The 2D gels were stained with Coomassie Blue and, while still wet, were subjected to quantitative evaluation by densitometric analysis (HP 3p Scanjet, Hewlett Packard, Palo Alto, USA). OXPHOS complexes were quantified by integration of the areas belonging to the individual subunits of each complex, using a specific program (Sigma plot-Jandel, Sigma, St Louis, USA), and were expressed as a ratio of the value obtained for complex V, which was considered as the internal standard (Schägger et al., 1994). The reproducibility of the method was checked by linear regression analysis between whole stained areas of complexes I, III and IV and complex V, which turned out to be highly significant (r = 0.9, 0.89 and 0.97, respectively). For complex II, a good linear correlation was obtained only for the area of the 70 kDa subunit (r = 0.81). In fact, in the first-dimension blue native PAGE run, many impurities were co-run with complex II and, after 2D resolution, no correlation was found between the integral area of whole complex II and quantified complex V, used as the internal standard.
Statistical analysis
Statistical evaluation was carried out according to the confidence limit method for normal distributions.
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Results |
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Organic acids and acyl-carnitine
Organic acid analysis was measured in both patients. In patient 1, urine was collected during treatment and did not show any abnormality. In patient 2, urine collected during the acute phase and on carnitine therapy alone showed increases in ethylmalonic, methylsuccinic, isovaleryglycine, hexanoylglycine, glutaric, adipic, suberic and sebacic acids. This metabolic profile is indicative of MAD. Riboflavin therapy normalized the organic acid profile.
The powerful technique of electrospray-ion tandem mass spectrometry detected abnormal profiles of acyl-carnitine even in plasma samples collected during riboflavin therapy in both patients. Patient 1 had increased C4, C6 and C8 acyl-carnitine profiles, indicating mild MAD. In patient 2, the presence of C4, C6, C8, C10, C12 and C14 acyl-carnitines indicated the severe form of MAD (Fig. 1).
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Mitochondrial flavoproteins
SCAD and MCAD
In the mitochondria of patient 1 before therapy, SCAD and MCAD activities were moderately decreased to 51 and 76% (P < 0.05) of control values (Table 1
). ß-Oxidation was measured in fresh muscle homogenate from a post-therapy muscle sample using labelled substrates. Octanoate and butyrate oxidations were in the normal range (data not shown). Patient 2 had a greater reduction in these enzyme activities before therapy, when SCAD and MCAD were 34 and 53% (P < 0.05) of control values, respectively (Table 1). Acyl CoA dehydrogenases increased partly or totally to normal values in the mitochondrial samples after therapy.
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In order to understand whether the observed defects in SCAD and MCAD activity corresponded to decreased amounts of enzyme proteins, evaluation of the corresponding cross-reacting materials was performed by Western blotting. Figure 2
shows the results of blots, immunostained using anti-SCAD and anti-MCAD polyclonal antibodies, from the two patients and controls. SCAD- and MCAD-cross-reacting materials were normal in both biopsies of patient 1. In contrast, SCAD- and MCAD-cross-reacting materials were markedly reduced in the muscle biopsy of patient 2 before therapy, but returned to normal levels after therapy.
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OXPHOS complexes
The two patients were also different with regard to the enzymatic aspect of flavin-dependent OXPHOS complexes. In the muscle of both patients before therapy, NADH nicotinamide adenine dinucleotide) : ubiquinone oxidoreductase (complex I) activity was 47 and 59% of control, but only patient 2 showed reduced activity (30%) of succinate dehydrogenase (complex II). These deficiencies were totally or partly corrected by therapy (Zerbetto et al., 1997
).
In order to characterize the defects leading to impaired activity of OXPHOS flavoenzymes, complexes I, II and V, the last acting as the internal standard, were analysed by Western blotting (Fig. 3). Surprisingly, complex I was found in normal amounts in the biopsies of both patients before therapy. In good agreement with the measured enzymatic activities (Zerbetto et al., 1997), flavinylated succinate dehydrogenase-cross-reacting material (i.e. the amount of FAD covalently bound to succinate dehydrogenase) was found at normal levels in both biopsies of patient 1, whereas it was present in lower amounts than in controls in patient 2 before therapy, and returned to normal after therapy (Fig. 3).
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In order to confirm these results and to ascertain whether the reduced amount of flavinylated peptide was actually due to a reduction in the apoenzyme, densitometric quantification of all OXPHOS complexes was performed following 2D electrophoresis. These analyses were carried out on the muscle biopsies of both patients before and after therapy and were compared with those from 10 control subjects. Figure 4
shows the results obtained, expressed as the ratio of the amount of complex V in each gel.
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Complex I was present in normal amounts before and after therapy in both patients, confirming our Western blotting data. As expected from immunoblot analysis, complex II was normal in patient 1 before and after therapy. Interestingly, the 70 kDa subunit of succinate dehydrogenase in the muscle sample of patient 2 before therapy was 40% (statistically different from controls, P < 0.01), and increased to 160% of the control mean after therapy. Complexes III and IV were present in normal ratios in both patients and were substantially unaffected by therapy.
FAD and FMN contents
In order to understand whether a correlation existed between the observed reduction of activity and the amounts of mitochondrial flavoproteins and flavin cofactor availability, the concentrations of FAD, FMN and riboflavin in total muscle and in isolated muscle mitochondria from both patients before and after therapy were evaluated. Riboflavin was not detectable in any of the samples. In patient 1 before therapy (Table 2), no significant variation was found in the amounts of FAD and FMN in total muscle, whereas the mitochondrial concentrations of FAD and FMN had decreased to 46 and 49% (P < 0.05) of control values, respectively (Table 2). Both values returned to normal after therapy. Severe coenzyme depletion was found in the samples of patient 2 before therapy, muscle FAD and FMN concentrations being 22% (P < 0.05) and 22% of normal values, respectively. The reduction was of the same degree in the mitochondrial FAD and FMN, which were 27 and 17% (P < 0.05) of control values, respectively. After therapy, the amounts of FMN were totally restored, while FAD increased but did not reach normal values, either in muscle or mitochondria (Table 2).
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Mitochondrial FAD catabolism
In order to evaluate whether the reduction in flavin cofactors was actually linked to a possible alteration in the activity of one of the enzymes involved in maintaining the flavin cofactor pool, we investigated whether human muscle mitochondria could accomplish FAD catabolism and measured the activity of mitochondrial FAD pyrophosphatase fluorimetrically, as described in Patients and methods. A mean rate of 64 ± 20 pmol FAD hydrolysed/min/mg protein was calculated in five different experiments carried out using muscle mitochondria from various control subjects (Table 3
). This activity is ~10-fold lower than that measured in rat liver mitochondria in the same experimental conditions (Barile et al., 1997). Consistently, no FAD hydrolysis was found when 1 mM AMP, a non-competitive inhibitor of rat liver mitochondrial FAD pyrophosphatase, was added together with FAD (data not shown).
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The ability of human muscle mitochondria to catabolize endogenous FAD was also tested. Fluorimetric measurements of the rate of hydrolysis of endogenous FAD, carried out with various mitochondrial preparations from control subjects, gave a mean rate of 0.57 ± 0.18 pmol FAD hydrolysed/min/mg protein (Table 3
). In order to characterize the products of endogenous FAD hydrolysis, HPLC measurements of the amounts of FAD, FMN and riboflavin in mitochondrial samples were made at different times during the increase in fluorescence. These experiments show that mitochondrial FAD was hydrolysed to FMN, whereas no increase in riboflavin was found in these experimental conditions; in both fluorescence and HPLC assays, externally added AMP was found to prevent FAD hydrolysis (data not shown).
The activity of mitochondrial FAD pyrophosphatase measured in muscle mitochondria from patient 2 was in the normal range (Table 3), whereas it was significantly increased (P < 0.01) in the mitochondrial samples from patient 1 (Table 3), as measured both with endogenous (192–257%) and exogenous (273–373%) FAD.
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Discussion |
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TopAbstractIntroductionPatients and methodsResultsDiscussionReferences |
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Our two patients suffered from subacute carnitine-deficient lipid storage myopathy. Although they both showed improvement following riboflavin supplementation, they were heterogeneous in most of their biochemical parameters as well as in clinical recovery. Clinically, after oral riboflavin therapy, patient 1 totally regained his strength twice within a short period, whereas patient 2 only partly recovered his muscle strength and suffered from fluctuating myopathy that definitely improved after medium-chain triglyceride oil supplementation. In patient 2, long-chain acyl-carnitines (Fig. 1
) were also detectable during riboflavin supplementation, indicating the presence of toxic long-chain fatty acids. The beneficial effects of medium-chain triglyceride oil were probably due to the reduction in long-chain fatty acids. In fact, through their toxic intermediates, long-chain fatty acids may impair glucose utilization and high-energy nucleotide metabolism (Hendrickson et al., 1997). Medium-chain triglyceride oil may also provide optimal nutritional management during fasting or catabolic conditions with glucose and protein-sparing (Parsons and Dias, 1991).
Patient 1 had a biochemically milder form of MAD compared with patient 2, i.e. higher muscle carnitine (20% of normal), presence of short-chain plasma acyl-carnitine profile, less reduced activity of SCAD and MCAD, and normal contents of SCAD- and MCAD-cross-reacting material. His metabolic profile was indicative of mild MAD (Fig. 1). He had high levels of ketone bodies during a long fasting period, probably due to a mechanism similar to that observed in riboflavin-deficient animals (Ross and Hoppel, 1987). Only mitochondrial FAD and FMN showed reduced quantities (48 and 52% of controls, respectively), and these returned to normal values after therapy (Table 2). The striking abnormality in patient 1 was the significantly higher activity of mitochondrial FAD pyrophosphatase (Table 3), which may have been responsible for the dysfunction of mitochondrial homeostasis of flavin coenzymes (Barile et al., 1997). Mitochondrial FAD pyrophosphatase in the pretherapy sample could not be measured. Nevertheless, the increase cannot have been due to the riboflavin treatment, because normal activity of this enzyme was found in patient 2 after riboflavin treatment. Rhead and colleagues (Rhead et al., 1993) reported derangement of mitochondrial FAD and FMN homeostasis in cultured fibroblasts from the first-described riboflavin-responsive patient. The alteration in that patient could be ascertained only in vitro, using a vitamin-depleted medium, whereas low levels of riboflavin occurred in vivo in our patient 1, but this was probably due to an intestinal worm infection and prolonged fasting on the second relapse (Windmüeller et al., 1964). In mouse liver, cytoplasmic FAD pyrophosphatase activity is significantly elevated during respiratory infections, with increased excretion of riboflavin in mice fed on a low-riboflavin diet (Brijlal et al., 1996). We have previously shown in rat liver mitochondria that the homeostasis of flavin cofactors is maintained by a multistep pathway (Fig. 5), in which, in physiological conditions, mitochondrial FAD pyrophosphatase is the rate-limiting step in the route of FAD to riboflavin breakdown. Presumably, an alteration in this enzyme activity causes an increase in the rate of the overall process. This enzyme has only recently been discovered and characterized (Barile et al., 1997), together with newly characterized translocators and enzymes, pertaining to a mitochondrial pathway shown in Fig. 5. However, the factors and mechanisms influencing mitochondrial FAD pyrophosphatase activity are still unknown, and further investigation is needed to clarify this issue, if it is due to increased protein content or to increased specific catalytic constant.
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Patient 2 showed a more severe biochemical picture. The presence of a characteristic organic acid profile indicating abnormal fatty acid and branched-chain oxidations as well as medium- and long-chain acyl-carnitine patterns were consistent with MAD. Muscle carnitine was <10% that of controls but was partly restored after therapy. A severe decrease in FAD and FMN concentrations in pretherapy muscle and mitochondria samples, reaching only ~20% of control values, probably caused his severe phenotype. In fact, all the flavoproteins tested except complex I (SCAD, MCAD and complex II) had greatly reduced activity and cross-reacting material compared with patient 1. After therapy, the amounts of FAD and FMN were partly or totally restored to normal values in both muscle and mitochondria. The flavin depletion was probably due to a biochemical defect at the cellular level in the availability of FAD. This statement is supported both by the similar amounts of FAD and FMN found in total muscle and in mitochondria before therapy and by the fact that 100 mg/day of riboflavin therapy increased his FMN to normal, whereas FAD did not even reach the lower value of the normal range, but was only 48 and 71% of the control value in muscle and mitochondria, respectively, after therapy (Table 2
). Cellular riboflavin uptake, cytoplasmic FMN and/or FAD synthesis or degradation are the most probable defects in flavin metabolism, although other, unknown mechanisms involved in regulating tissue-specific flavin concentrations in patient 2 cannot be excluded at the moment. It is interesting to emphasize that normal activity of mitochondrial FAD pyrophosphatase was recorded in this patient.
In order to better understand mitochondrial involvement in the pathogenesis of these patients, OXPHOS complex levels were measured by means of Western blotting and densitometric quantification of 2D electrophoresis. Complex I immunoblot detection was carried out using antibodies against the 24 kDa subunit belonging to the subcomplex of flavoprotein, together with 9 and 51 kDa subunits, to which FMN is bound (Yamaguchi and Hatefi, 1993). Quantification of 2D gel was carried out by measuring the staining intensity of the following different selected subunits: 75 kDa, unresolved 51 and 49 kDa, and 39 kDa subunits of NADH-dehydrogenase (Bentlage et al., 1995).
Data obtained from these two different approaches and techniques gave similar results: although the activity of complex I was reduced in muscle samples from both patients before therapy (Zerbetto et al., 1997), normal amounts of cross-reacting material of normally sized complex I (Fig. 3) and normal quantities (Fig. 4) were present.
We therefore conclude that, in both patients, the low catalytic activity of complex I was due to low mitochondrial levels of FMN (Table 2), which does not appear to influence the assembly and stability of complex 1. Schägger obtained similar results in Parkinson's disease patients, in whom no correlation between complex I activities and quantities was observed (Schägger, 1995), whereas good agreement between its activities and quantities was detected in patients suffering from MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and strokelike episodes), and encephalomyopathy (Bentlage et al., 1995).
A different relationship between cofactor availability, enzyme activity and protein amount was observed in the case of complex II. Western blotting and 2D electrophoretic approaches to complex II in the pretherapy muscle of patient 2 gave similar results, indicating that a decrease in enzymatic rate (Zerbetto et al., 1997) corresponds to a decrease in cross-reacting material in holo-complex II (Fig. 3) and in the 70 kDa subunit (Fig. 4). Our experimental data demonstrate that 75% less mitochondrial FAD (Table 2) reduces the covalent flavinylation of complex II, with a consequent increase in proteolytic degradation of the succinate dehydrogenase apoenzyme (Brandsch et al., 1989; Robinson et al., 1994; Robinson and Lemire, 1996), whereas in patient 1 a 50% reduction of mitochondrial FAD prevented the catabolic fate of succinate dehydrogenase and did not affect its activity or the amounts of apo- and holoproteins. In riboflavin-deficient animals, reduced activity of complex II was found (25% of that of controls), and it increased after riboflavin supplementation (Addison and McCormick, 1978).
A correlation among reductions in mitochondrial FAD and SCAD and MCAD was observed. In patient 1, a smaller decrease in mitochondrial FAD diminished the activity of SCAD and MCAD but did not affect their stability. Instead, in patient 2, the higher decrease in the flavin cofactor was associated with a reduction in both the activity and the apoenzyme amounts of SCAD and MCAD.
In conclusion, our biochemical data confirm heterogeneity in two adult patients with lipid storage myopathies responsive to riboflavin.
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Acknowledgments |
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We wish to thank Professor S. Passarella for his helpful critical revision, Dr G. Guidetti for valuable guidance in the statistical analysis of the data and Dr A. Giordano for helpful collaboration. This study was supported by CNR grant (97.04000.CT04) to M.B. and by Telethon grant no. 618 to L.V.
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References |
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Received April 29, 1999. Accepted May 26, 1999.
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