Research Notes: Riboflavin

From Treatment of Mitochondrial Cytopathies (Medscape Pediatrics)

Riboflavin (vitamin B2) is a precursor to flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are cofactors of ETC complexes I and II, respectively. Riboflavin has been proposed to act therapeutically by one of several potential mechanisms including inhibition of the breakdown of complex I by providing more resistance to proteolysis or stabilizing the mitochondrial membrane. It has been used with some success in some patients with mitochondrial disease without any apparent side effects.

A 33-year-old patient with MELAS and complex I deficiency was administered a combination of riboflavin (100 mg three times per day) and nicotinamide (1 g four times per day) in a double-blinded, randomized fashion. In conjunction with this treatment, there was cessation of this patient's encephalopathic and myopathic symptoms. The spectroscopic findings were equivocal: resting PCr/Pi did not show any treatment effect, whereas high-energy phosphate recovery deteriorated upon withdrawal of nicotinamide (but not riboflavin). Sural nerve sensory testing revealed a drop in amplitude once therapy was withdrawn with recovery following the reinitiation of treatment (P = 0.00007 right, 0.017 left).[52]

A 10-month-old infant girl with a partial defect of complex I was treated with increasing doses of riboflavin (beginning at 3 mg per kg). It was observed that lactate levels normalized and muscle weakness improved at the maximal dose of 13 mg per kg.[53] Riboflavin was given to five patients with mitochondrial myopathy due to complex I deficiency in dosages ranging from 9 to 60 mg per day. Patients presented with either pure myopathic symptoms or encephalomyopathy. One patient had stabilization of the previously regressive disease and three showed clinical improvement, especially in the myopathic component. There was normalization of complex I activity in three of the patients in addition to improved lactate levels or muscle histopathology.[54] A 13-year-old girl with progressive exercise intolerance, severe lactic acidosis, and complex I deficiency was treated with 100 mg of oral riboflavin daily. There was remarkable and persistent clinical improvement with an increase in exercise tolerance. Exercise parameters (maximal work capacity, O2 uptake, and base excess) also improved.[55] A 6-year-old boy with a defect of complex I and myopathy presenting as slowly progressive weakness was successfully treated with riboflavin and carnitine. Clinically, muscle strength and motor conduction velocities were improved. In addition, the complex I activity and carnitine levels normalized 7 months after the start of treatment.[56] More recently, Ogle et al[57] report on a 21/2-year-old patient with complex I deficiency, mitochondrial DNA mutation, and myopathy and who had persistent response to riboflavin therapy (20 to 25 mg twice daily) over a 3-year period. Despite persistent lactic acidosis, this patient demonstrated improvements in terms of overall muscle strength and endurance.

Riboflavin is not always effective, as demonstrated by the case of a 4-month-old infant with severe congenital lactic acidosis and complex I deficiency, who underwent a trial of riboflavin at a dose of 100 mg daily. The child's serum lactate and ratio to pyruvate remained significantly elevated, and the patient continued to demonstrate features of severe myopathy and cardiomyopathy until his death.[58] Four patients with MELAS in association with complex I deficiency treated with riboflavin, other supplements, and the ketogenic diet did not show improvement.[59] Another large trial of riboflavin in combination with other supplements found no significant therapeutic success.[33]

The results of treatment with riboflavin, with a large variation in doses and treatment duration in this diverse population, are not uniform but demonstrate that those with complex I deficiency and pure myopathy may benefit from supplemental riboflavin, with or without other supplements. Of course, the clinical course of these diseases remains variable such that any improvement observed may not be due to therapeutic interventions.

Chem Biol Interact. 2006 Oct 27.
Mitochondrial function and toxicity: role of the B vitamin family on mitochondrial energy metabolism.
Depeint F, Bruce WR, Shangari N, Mehta R, O'Brien PJ.
Department of Pharmaceutical Sciences, University of Toronto, Canada; Department of Nutritional Sciences, University of Toronto, Canada.

The B vitamins are water-soluble vitamins required as coenzymes for enzymes essential for cell function. This review focuses on their essential role in maintaining mitochondrial function and on how mitochondria are compromised by a deficiency of any B vitamin. Thiamin (B1) is essential for the oxidative decarboxylation of the multienzyme branched-chain ketoacid dehydrogenase complexes of the citric acid cycle. Riboflavin (B2) is required for the flavoenzymes of the respiratory chain, while NADH is synthesized from niacin (B3) and is required to supply protons for oxidative phosphorylation. Pantothenic acid (B5) is required for coenzyme A formation and is also essential for alpha-ketoglutarate and pyruvate dehydrogenase complexes as well as fatty acid oxidation. Biotin (B7) is the coenzyme of decarboxylases required for gluconeogenesis and fatty acid oxidation. Pyridoxal (B6), folate and cobalamin (B12) properties are reviewed elsewhere in this issue. The experimental animal and clinical evidence that vitamin B therapy alleviates B deficiency symptoms and prevents mitochondrial toxicity is also reviewed. The effectiveness of B vitamins as antioxidants preventing oxidative stress toxicity is also reviewed.

Mol Genet Metab. 2004 Apr.
Cofactor treatment improves ATP synthetic capacity in patients with oxidative phosphorylation disorders.
Marriage BJ, Clandinin MT, Macdonald IM, Glerum DM.
Department of Medicine, University of Alberta, Edmonton, Alberta, Canada.

Marked progress has been made over the past 15 years in defining the specific biochemical defects and underlying molecular mechanisms of oxidative phosphorylation disorders, but limited information is currently available on the development and evaluation of effective treatment approaches. Metabolic therapies that have been reported to produce a positive effect include coenzyme Q(10) (ubiquinone), other antioxidants such as ascorbic acid and vitamin E, riboflavin, thiamine, niacin, vitamin K (phylloquinone and menadione), and carnitine. The goal of these therapies is to increase mitochondrial ATP production, and to slow or arrest the progression of clinical symptoms. In the present study, we demonstrate for the first time that there is a significant increase in ATP synthetic capacity in lymphocytes from patients undergoing cofactor treatment. We also examined in vitro cofactor supplementation in control lymphocytes in order to determine the effect of the individual components of the cofactor treatment on ATP synthesis. A dose-dependent increase in ATP synthesis with CoQ(10) incubation was demonstrated, which supports the proposal that CoQ(10) may have a beneficial effect in the treatment of oxidative phosphorylation (OXPHOS) disorders.

J Am Diet Assoc. 2003 Aug.
Nutritional cofactor treatment in mitochondrial disorders.
Marriage B, Clandinin MT, Glerum DM.
Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada.

Mitochondrial disorders are degenerative diseases characterized by a decrease in the ability of mitochondria to supply cellular energy requirements. Substantial progress has been made in defining the specific biochemical defects and underlying molecular mechanisms, but limited information is available about the development and evaluation of effective treatment approaches. The goal of nutritional cofactor therapy is to increase mitochondrial adenosine 5'-triphosphate production and slow or arrest the progression of clinical symptoms. Accumulation of toxic metabolites and reduction of electron transfer activity have prompted the use of antioxidants, electron transfer mediators (which bypass the defective site), and enzyme cofactors. Metabolic therapies that have been reported to produce a positive effect include Coenzyme Q(10) (ubiquinone); other antioxidants such as ascorbic acid, vitamin E, and lipoic acid; riboflavin; thiamin; niacin; vitamin K (phylloquinone and menadione); creatine; and carnitine. A literature review of the use of these supplements in mitochondrial disorders is presented.

No To Hattatsu. 2000 Mar.
A case of riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency (glutaric aciduria type II). [Article in Japanese]
Tojo M, Gunji T, Yamaguchi S, Shimizu N, Koga Y, Nonaka I.
Department of Pediatrics, Niigata Prefecture Hamagumi Medical Rehabilitation Center for Handicapped Children.

We reported a male infant with multiple acyl CoA dehydrogenase deficiency, probably due to electron transfer flavoprotein dehydrogenase deficiency. He was noted to have severe muscle weakness, a high serum creatine kinase (CK) level up to 6920 IU/L, lipid storage myopathy and fatty liver at 6 months of age. A GC/MS analysis of urinary organic acids showed excess excretion of dicarboxylic acids, including glutaric, 2-hydroxyglutaric, adipic, suberic, sebacic, malonic, ethylmalonic and methylsuccinic acids. On a urinary acylglycine analysis, hexanoylglycine and suberylglycine were increased, but not isovalerylglycine, in amount. No ketosis was noted. The muscle pathology showed increased oil-red O positive lipid droplets of various sizes indicative of lipid storage myopathy. There was diffuse decrease in the activity of cytochrome c oxidase. No ragged-red fibers were noted. His clinical symptoms improved remarkably after the administration of riboflavin (100 mg/day) and L-carnitine (1000 mg/day). He was then diagnosed as having probable riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency. The glutaryl CoA dehydrogenase activity in lymphocytes was normal, as were the alpha- and beta-subunits of electron transfer flavoprotein. These findings led us to suspect electron transfer flavoprotein dehydrogenation deficiency. Although he had several episodes of short-term deterioration in clinical and laboratory findings, he developed normally with normal intelligent till 10 years of age.

Eur J Pediatr. 1997 Oct.
Stridor as the major presenting symptom in riboflavin-responsive multiple acyl-CoA dehydrogenation deficiency.
Sperl W, Geiger R, Lehnert W, Rhead W.
Department of Paediatrics, University of Innsbruck, Austria.

Inspiratory stridor of unknown origin was the leading clinical symptom in an 11-month-old boy. The stridor increased over a period of 4 weeks, and assisted ventilation became necessary. Selective urinary screening by gas chromatography/mass spectrometry analysis revealed excretion of ethylmalonic and 3-OH-isovaleric acid and of N-isobutyryl-, N-2-methylbutyryl-, N-isovaleryl-, N-hexanoyl- and N-suberylglycine. Neither hypoglycaemia nor metabolic acidosis were noticed. Treatment with 200 mg of riboflavin per day led to a dramatic clinical improvement with restoration of normal respiration and an increase in muscular tone within 2 months. During this period, metabolite excretion in urine completely normalized. Riboflavin-sensitive multiple acyl-CoA dehydrogenation deficiency was confirmed in cultured fibroblasts. With riboflavin supplementation, the development of the child has been favourable, with normal school attendance now at an age of 9 years. CONCLUSION: As respiratory symptoms might precede other symptoms in disorders of mitochondrial oxidation, we propose determination of urinary organic acids in all cases of unexplained laryngeal stridor.

Pediatr Res. 1993 Feb.
Multiple acyl-coenzyme A dehydrogenation disorder responsive to riboflavin: substrate oxidation, flavin metabolism, and flavoenzyme activities in fibroblasts.
Rhead W, Roettger V, Marshall T, Amendt B.
Department of Pediatrics, University of Iowa, Iowa City.

Multiple acyl-CoA dehydrogenation disorders result from generalized defects in intramitochondrial acyl-CoA dehydrogenation. Fibroblasts from a riboflavin-responsive multiple acyl-CoA dehydrogenation disorder patient catabolized 14C-butyrate, -octanoate, and -leucine normally after culture in riboflavin-supplemented medium (2 mg/L). After culture in riboflavin-depleted medium (< or = 1.4 micrograms/L), his cells oxidized the same substrates poorly at 20 to 33% of control (p < 0.05). Patient cells incubated in a wide range of D-[2-14C]riboflavin concentrations (3, 31.4, and 100 micrograms/L) synthesized 14C-flavin mononucleotide and 14C-flavin adenine dinucleotide (FAD) normally and had normal cytosolic 14C-flavin mononucleotide and 14C-FAD contents, which argues against defects in cellular riboflavin uptake and conversion to flavin mononucleotide and FAD. After culture in 31.4 micrograms 14C-riboflavin/L for 2 wk, 14C-FAD specific radioactivities plateaued and were similar in patient and control cells. However, culturing these uniformly labeled cells in riboflavin-depleted medium for 2 wk lowered the patient's cellular 14C-FAD content to only 23% of control levels. Similarly, after incubation in low 14C-riboflavin concentrations (4.4 micrograms/L), the patient's mitochondrial 14C-FAD content was only 51% of control after 1 h and 29% of control at 4 h. After a 4-h incubation in a high physiologic concentration of 14C-riboflavin (31.4 micrograms/L), which raised the patient's cellular 14C-FAD levels 3- to 4-fold, his mitochondrial 14C-FAD content rose to normal; control values did not change. We also investigated possible defective FAD binding to flavoenzymes essential for acyl-CoA dehydrogenation.(

J Inherit Metab Dis. 1985.
Riboflavin-responsive defects of beta-oxidation.
Gregersen N.

The key reaction in the beta-oxidation of fatty acids is the acyl-CoA dehydrogenation, catalyzed by short chain, medium chain, and long chain acyl-CoA dehydrogenases. Acyl-CoA dehydrogenation reactions are also involved in the metabolism of the branched chain amino acids, where isovaleryl-CoA and 2-methylbutyryl-CoA dehydrogenases are involved and in the metabolism of lysine, 5-hydroxylysine and tryptophan, where glutaryl-CoA dehydrogenase functions. In all of these dehydrogenation systems reducing equivalents are transported to the main respiratory chain by electron transfer flavoprotein (ETF) and electron transfer flavoprotein dehydrogenase (ETFDH), which are common to all the dehydrogenation systems. The acyl-CoA dehydrogenation enzymes are dependent on flavin adenine dinucleotide (FAD) as coenzyme, for which riboflavin is the precursor. Patients with multiple acyl-CoA dehydrogenation deficiencies have been found in whom the defect has been located to ETF and/or ETFDH. A few patients with multiple acyl-CoA dehydrogenation deficiencies have been described, in whom no defects in acyl-CoA dehydrogenases, ETF or ETFDH have been found but who respond clinically and biochemically to pharmacological doses of riboflavin. This indicates a defect related to the metabolism of FAD. An uptake defect of riboflavin or a synthesis defect of FAD from riboflavin have been excluded by in vivo and in vitro studies. A mitochondrial transport defect of FAD or a defect in the binding FAD to ETF and/or ETFDH remains possible.

Pediatr Res. 1982 Oct.
C6-C10-dicarboxylic aciduria: investigations of a patient with riboflavin responsive multiple acyl-CoA dehydrogenation defects.
Gregersen N, Wintzensen H, Christensen SK, Christensen MF, Brandt NJ, Rasmussen K.

The abnormal metabolites - adipic, suberic, and sebacic acids - were detected in large amounts in the urine of a boy during a Reye's syndrome-like crisis. Substantial amounts of 5-OH-caproic acid, caproylglycine, glutaric acid, and 3-OH-butyric acid and moderately elevated amounts of ethylmalonic acid, methylsuccinic acid, 3-OH-isovaleric acid, and isovalerylglycine were also found. These metabolites were consistently present in urine samples collected in the boy's habitual condition after the attack. 1-[14C]-Palmitic acid was oxidized at a normal rate, whereas U-[14C]-Palmitic acid was oxidized at a reduced rate in cultured skin fibroblasts from the patient, thus indicating a defect at the level of medium- and/or short-chain fatty acid oxidation. Riboflavin medication (100 mg three times a day) significantly reduced the excreted amounts of pathologic metabolites, suggesting a flavineadeninedinucleotide-related acyl-CoA dehydrogenation defect as the cause of the disease. Carnitine in plasma was low in the patient (6 mumole/liter, controls 26-74 mumole/liter), suggesting carnitine deficiency as a secondary effect of the acyl-CoA dehydrogenation deficiency. The present patient, who presented with a Reye's syndrome-like attack, suffers from impaired dehydrogenation of acyl-CoA resulting in accumulation of acyl-CoA in the cells. Attacks with similar symptoms are seen in other acyl-CoA dehydrogenation deficiencies, such as glutaric aciduria types I and II, other types of C6-C10-dicarboxylic acidurias and isovaleric acidemia. Reduced flow through the acyl-CoA dehydrogenation steps may therefore be an etiologic factor in Reye's syndrome. Several of the accumulated acyl-CoA's are toxic and may be responsible for some of the symptoms. The low carnitine level in plasma and the elevated esterified carnitine excretion in the present patient indicate that acyl-CoA accumulation may cause a functional carnitine deficiency by sequestration of carnitine as acyl-carnitines. As the inborn defect, systemic carnitine deficiency may exhibit symptoms like those of Reye's syndrome, it may be speculated whether functional carnitine deficiency in patients with accumulated acyl-CoA is another causal factor in the development of the symptoms during attacks.