Riboflavin transporter deficiency mimicking mitochondrial myopathy caused by complex II deficiency.

Biallelic likely pathogenic variants in SLC52A2 and SLC52A3 cause riboflavin transporter deficiency. It is characterized by muscle weakness, ataxia, progressive ponto-bulbar palsy, amyotrophy, and sensorineural hearing loss. Oral riboflavin halts disease progression and may reverse symptoms. We report two new patients whose clinical and biochemical features were mimicking mitochondrial myopathy. Patient 1 is an 8-year-old male with global developmental delay, axial and appendicular hypotonia, ataxia, and sensorineural hearing loss. His muscle biopsy showed complex II deficiency and ragged red fibers consistent with mitochondrial myopathy. Whole exome sequencing revealed a homozygous likely pathogenic variant in SLC52A2 (c.917G>A; p.Gly306Glu). Patient 2 is a 14-month-old boy with global developmental delay, respiratory insufficiency requiring ventilator support within the first year of life. His muscle biopsy revealed combined complex II + III deficiency and ragged red fibers consistent with mitochondrial myopathy. Whole exome sequencing identified a homozygous likely pathogenic variant in SCL52A3 (c.1223G>A; p.Gly408Asp). We report two new patients with riboflavin transporter deficiency, caused by mutations in two different riboflavin transporter genes. Both patients presented with complex II deficiency. This treatable neurometabolic disorder can mimic mitochondrial myopathy. In patients with complex II deficiency, riboflavin transporter deficiency should be included in the differential diagnosis to allow early treatment and improve neurodevelopmental outcome.

Riboflavin and health: A review of recent human research.

There has lately been a renewed interest in Riboflavin owing to insight into its recognition as an essential component of cellular biochemistry. The knowledge of the mechanisms and regulation of intestinal absorption of riboflavin and its health implications has significantly been expanded in recent years. The purpose of this review is to provide an overview of the importance of riboflavin, its absorption and metabolism in health and diseased conditions, its deficiency and its association with various health diseases, and metabolic disorders. Efforts have been made to review the available information in literature on the relationship between riboflavin and various clinical abnormalities. The role of riboflavin has also been dealt in the prevention of a wide array of health diseases like migraine, anemia, cancer, hyperglycemia, hypertension, diabetes mellitus, and oxidative stress directly or indirectly. The riboflavin deficiency has profound effect on iron absorption, metabolism of tryptophan, mitochondrial dysfunction, gastrointestinal tract, brain dysfunction, and metabolism of other vitamins as well as is associated with skin disorders. Toxicological and photosensitizing properties of riboflavin make it suitable for biological use, such as virus inactivation, excellent photosensitizer, and promising adjuvant in chemo radiotherapy in cancer treatment. A number of recent studies have indicated and highlighted the cellular processes and biological effects associated with riboflavin supplementation in metabolic diseases. Overall, a deeper understanding of these emerging roles of riboflavin intake is essential to design better therapies for future.

The discovery and characterization of riboflavin.

The first observation of a pigment in milk with yellow-green fluorescence can be traced to the English chemist Alexander Wynter Blyth in 1872, but it was not until the early 1930s that the substance was characterized as riboflavin. Interest in accessory food factors began in the latter half of the 19th century with the discovery of the first vitamin, thiamin. Thiamin was water soluble and given the name vitamin B(1). However, researchers realized that there were one or more additional water-soluble factors and these were called the vitamin B-2 complex. The search to identify these accessory food factors in milk, whole wheat, yeast, and liver began in the early 1900s. As there is no classical nutritional disease attributable to riboflavin deficiency, it was the growth-stimulating properties of the food extracts given to young rats that provided the tool with which to investigate and eventually extract riboflavin. Riboflavin was the second vitamin to be isolated and the first from the vitamin B-2 complex; the essential nature of the vitamin as a food constituent for man was shown in 1939.

Riboflavin (Vitamin B2) Deficiency Induces Apoptosis Mediated by Endoplasmic Reticulum Stress and the CHOP Pathway in HepG2 Cells.

Riboflavin is an essential micronutrient and a precursor of flavin mononucleotide and flavin adenine dinucleotide for maintaining cell homeostasis. Riboflavin deficiency (RD) induces cell apoptosis. Endoplasmic reticulum (ER) stress is considered to induce apoptosis, and C/EBP homologous protein (CHOP) is a key pathway involved in this process. However, whether RD-induced apoptosis is mediated by ER stress and the CHOP pathway remains unclear and needs further investigation. Therefore, the current study presents the effect of RD on ER stress and apoptosis in the human hepatoma cell line (HepG2). Firstly, cells were cultured in a RD medium (4.55 nM riboflavin) and a control (CON) medium (1005 nM riboflavin). We conducted an observation of cell microstructure characterization and determining apoptosis. Subsequently, 4-phenyl butyric acid (4-PBA), an ER stress inhibitor, was used in HepG2 cells to investigate the role of ER stress in RD-induced apoptosis. Finally, CHOP siRNA was transfected into HepG2 cells to validate whether RD triggered ER stress-mediated apoptosis by the CHOP pathway. The results show that RD inhibited cell proliferation and caused ER stress, as well as increased the expression of ER stress markers (CHOP, 78 kDa glucose-regulated protein, activating transcription factor 6) (p < 0.05). Furthermore, RD increased the cell apoptosis rate, enhanced the expression of proapoptotic markers (B-cell lymphoma 2-associated X, Caspase 3), and decreased the expression of the antiapoptotic marker (B-cell lymphoma 2) (p < 0.05). The 4-PBA treatment and CHOP knockdown markedly alleviated RD-induced cell apoptosis. These results demonstrate that RD induces cell apoptosis by triggering ER stress and the CHOP pathway.

Adequate vitamin B12 and riboflavin status from menus alone in residential care facilities in the Lower Mainland, British Columbia.

Older adults have potential increased risk of nutrient deficiencies because of age-related decreased dietary intake and malabsorption; it is important to ensure nutrient needs are met to avoid adverse health outcomes. B vitamins are of particular interest: vitamin B12 deficiency can cause irreversible neurodegeneration; there is mandatory folic acid fortification in Canada; and suboptimal riboflavin status has been reported among older adults in the United Kingdom. In this exploratory secondary analysis study we assessed vitamin B12 and riboflavin biochemical status (via microparticle enzyme immunoassay and erythrocyte glutathione reductase activity coefficient (EGRac), respectively), and the vitamin B12, riboflavin, and folate content of menus served to a convenience sample of older adults (≥65 years) from 5 residential care facilities within the Lower Mainland of British Columbia, Canada. Diet was assessed from customized 28-day cycle meal plans. Participants (n = 207; 53 men and 154 women) were aged 86 ± 7 years, largely of European descent (92%), and nonsmokers (95%). The menus served had a low prevalence of inadequacy for vitamin B12 and riboflavin (only 4% and 1% of menus contained less than the estimated average requirement (EAR), respectively), but 93% contained less than the EAR for folate. Mean ± SD serum total vitamin B12 concentration was 422 ± 209 pmol/L, and EGRac was 1.30 ± 0.19. The majority of older adults in residential care were provided with adequate vitamin B12 and riboflavin menu amounts, and only 5% were vitamin B12 deficient (<148 pmol/L). However, 26% were riboflavin deficient (EGRac ≥ 1.4), which may warrant further investigation.

Impact and consequences of dietary riboflavin deficiency treatment on flesh quality loss in on-growing grass carp (Ctenopharyngodon idella).

Fish is among the cheapest and most promising sources of animal protein. The main edible portion of fish is muscle. This study explored the impact of dietary riboflavin on fish flesh quality and showed the possible role of muscle antioxidant defense in flesh quality in relation to dietary riboflavin. On-growing grass carp (initial average weight of 275.82 ± 0.57 g) were fed diets containing graded levels of riboflavin (0.63, 1.95, 3.98, 6.02, 7.96, and 10.04 mg kg-1 diet) for eight weeks. The results indicated that compared with the optimal riboflavin levels (3.98 and/or 6.02 mg riboflavin per kg diet), riboflavin deficiency treatment (0.63 mg riboflavin per kg diet) significantly reduced the muscle nutrients, including the protein, lipid, flavor amino acid, and total essential amino acid contents. Furthermore, the muscle shear force, pH value, and hydroxyproline concentration were reduced, while the muscle cooking loss and lactic acid content increased (P < 0.05). Compared with optimal riboflavin levels, the riboflavin deficiency treatment increased the reactive oxygen species (ROS), malondialdehyde (MDA), and protein carbonyl contents, while riboflavin treatments of 3.98-10.04 mg riboflavin per kg diet showed the lowest ROS and MDA contents (P < 0.05). Compared with the optimal riboflavin levels, the riboflavin deficiency treatment decreased the activities of copper/zinc superoxide dismutase (CuZnSOD), glutathione reductase (GR), catalase (CAT), and glutathione peroxidase (GPx), and reduced the glutathione (GSH) content (P < 0.05). Furthermore, the relative mRNA levels of antioxidant enzymes, including CuZnSOD, CAT, GR and GPx, and antioxidant-related signaling molecules, including NF-E2-related factor 2 (Nrf2) and casein kinase 2, were down-regulated, while those of Kelch-like ECH-associated protein 1b were up-regulated (P < 0.05). Collectively, the present study indicates that riboflavin deficiency treatment reduces the flesh quality, partly due to inhibition of the antioxidant defense through the Nrf2 signaling pathway, while optimal riboflavin levels reverse these negative effects.

Riboflavin-deficient and Trichinella spiralis-induced stresses on plasma corticosterone associated with spermatogenesis in male Wistar rats.

The objective of this study was to investigate the effects of riboflavin-deficient and Trichinella spiralis-induced stresses on corticosterone associated with spermatogenesis in male Wistar rats. Rats were allocated into 4 groups: Group 1: control; group 2: riboflavin-deficient diet; group 3: T. spiralis infection; group 4: riboflavin deficient diet with T. spiralis infection. This experiment lasted for 12 weeks. Plasma corticosterone was significantly enhanced when exposed to acute riboflavin deficiency and/or T. spiralis infection stress. When the rats were chronically subjected to such stresses, T. spiralis per se had prolonged effects, in a marked increase in corticosterone. T. spiralis per se tended to impact on such sperm characteristics as sperm motility, sperm count and daily sperm production, even defected seminiferous tubules. It was proposed that the Trichinella spiralis-induced stress probably had adverse effects on the level of adrenocortical-testicular axis whenever their habitats on muscle fibers were evident. However, riboflavin-deficient-induced stress had little implication in the adrenocortical-testicular axis.

Thiamin, riboflavin, and vitamins B-6 and C: impact of combined restricted intake on functional performance in man.

A double-blind study of combined restriction of thiamin, riboflavin, and vitamins B-6 and C was carried out with 23 healthy males. During 8 wk of low vitamin intake, 12 deficient subjects consumed daily a diet of normal food products, providing maximally 32.5% of the Dutch Recommended Dietary Allowances (RDA) for thiamin, riboflavin, vitamins B-6 and C. Other vitamins were supplemented at twice the RDA. Eleven control subjects consumed the same diet but with a supplementation of twice the RDA of all vitamins. In deficient subjects blood vitamin levels, urinary vitamin excretion, and erythrocytic enzyme activities decreased; in vitro enzyme stimulation increased. Vitamin depletion had no ill effects on health, physical activity, and mental performance. A significant decrease was observed in aerobic power (VO2max) and onset of blood lactate accumulation (p less than 0.001) of 9.8 and 19.6%, respectively. A combined restricted intake of thiamin, riboflavin, and vitamins B-6 and C causes a decrease in physical performance within a few weeks.

Riboflavin (vitamin B-2) and health.

Riboflavin is unique among the water-soluble vitamins in that milk and dairy products make the greatest contribution to its intake in Western diets. Meat and fish are also good sources of riboflavin, and certain fruit and vegetables, especially dark-green vegetables, contain reasonably high concentrations. Biochemical signs of depletion arise within only a few days of dietary deprivation. Poor riboflavin status in Western countries seems to be of most concern for the elderly and adolescents, despite the diversity of riboflavin-rich foods available. However, discrepancies between dietary intake data and biochemical data suggest either that requirements are higher than hitherto thought or that biochemical thresholds for deficiency are inappropriate. This article reviews current evidence that diets low in riboflavin present specific health risks. There is reasonably good evidence that poor riboflavin status interferes with iron handling and contributes to the etiology of anemia when iron intakes are low. Various mechanisms for this have been proposed, including effects on the gastrointestinal tract that might compromise the handling of other nutrients. Riboflavin deficiency has been implicated as a risk factor for cancer, although this has not been satisfactorily established in humans. Current interest is focused on the role that riboflavin plays in determining circulating concentrations of homocysteine, a risk factor for cardiovascular disease. Other mechanisms have been proposed for a protective role of riboflavin in ischemia reperfusion injury; this requires further study. Riboflavin deficiency may exert some of its effects by reducing the metabolism of other B vitamins, notably folate and vitamin B-6.

Riboflavin status during pregnancy.

The riboflavin status of 20 nonpregnant and 60 pregnant women (in the 2nd trimester, 3rd trimester, and early postpartum) was determined by the erythrocyte glutathione reductase activation test. None of the nonpregnant subjects but 26 pregnant subjects (21.7% in the 2nd trimester, 20.8% in the 3rd trimester, and 29.6% in early postpartum) had an activity coefficient greater than 1.20, indicative of biochemical deficiency of riboflavin. The deficiency developed at any of the three stages of pregnancy under study. Follow-up of individual cases revealed no progressive deterioration in riboflavin nutriture with advancement of pregnancy. The mean intake of riboflavin was higher than the recommended intake and revealed a significant negative correlation with activity coefficient values at the 3rd trimester. No significant correlation of riboflavin status with the outcome of pregnancy was noted. The effect of numbers of previous pregnancies, history of oral contraceptive usage, smoking, and alcohol showed no consistent effect on the percentage incidence of deficiency at all the three periods.

In vitro assay of pancreatic acinar-cell function of rats made chronically riboflavin deficient.

To determine the effect of riboflavin deficiency on the rat pancreas, one-third of a group of rats was fed a purified riboflavin-sufficient diet ad libitum and two-thirds were fed isocaloric amounts of riboflavin-deficient diet for 13 wk; one-half of the latter group was replenished with daily intraperitoneal injections of riboflavin for the last 3 wk. Body weight, pancreas weight, DNA, protein, amylase, chymotrypsinogen, and trypsinogen decreased in riboflavin-deficient animals. In vitro basal secretion of chymotrypsinogen decreased and basal and bethanechol-stimulated secretions of trypsinogen increased in riboflavin-deficient rats. These changes were considered to be caused by relative inanition resulting from decreased food consumption. On replenishment of riboflavin, amylase content reverted to that of animals fed ad libitum whereas increases in body weight, pancreas weight, DNA, protein, chymotrypsinogen, and trypsinogen were not statistically significant. Both basal- and bethanechol-stimulated secretions of chymotrypsinogen increased. These data indicate that riboflavin deficiency, which commonly accompanies chronic alcoholism, may contribute to the pancreatic injury in chronic alcoholism.

Accelerated development of riboflavin deficiency by treatment with chlorpromazine.

The present study was undertaken to determine whether treatment with chlorpromazine accelerates the depletion of tissue stores of flavin adenine dinucleotide during dietary riboflavin deficiency. These investigations derived their impetus from earlier findings that low doses of chlorpromazine in rats fed abundant riboflavin increase urinary riboflavin excretion and reduce hepatic flavin stores. From 6 to 10 days after beginning to feed on a riboflavin-deficient diet, rats treated with chlorpromazine, 2 mg/kg body weight twice daily, had approximately twice the urinary riboflavin excretion of that of pair-fed saline-treated controls. When the riboflavin-deficient diets and chlorpromazine treatments were extended for 3 weeks and the animals killed, FAD levels in liver, kidney, and heart were markedly lower in drug-treated than in saline-treated animals. When studies were extended for 7 weeks, tissue FAD levels in saline-treated animals declined further and were equal to those of chlorpromazine-treated rats after only 3 weeks of dietary deficiency. Thus, chlorpromazine treatment accelerated urinary riboflavin loss and accelerated tissue depletion of FAD levels during dietary riboflavin deficiency. Brain levels of FAD by contrast were relatively resistant to both dietary riboflavin withdrawal and treatment with chlorpromazine. Subsequent studies showed that urinary riboflavin excretion began to increase within 6 hr of treatment with chlorpromazine. It is concluded that significant riboflavin depletion occurs following treatment with low doses of chlorpromazine, both in animals fed a normal diet and in animals fed a riboflavin-deficient diet, particularly during the early stages of deficiency.

Hypotensive action of 5-HT receptor agonists in the vitamin B6-deficient hypertensive rat.

Feeding a vitamin B6-deficient diet to rats causes a moderate hypertension. The blood pressure responses to 5-HT1A receptor agonists were studied in conscious vitamin B6-deficient hypertensive rats. They were all effective in lowering blood pressure with the following rank order of potency: 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) > flesinoxan > 5-methylurapidil > urapidil. The putative 5-HT1A receptor antagonist spiroxatrine by itself, did not have any effect on the blood pressure at the doses used (0.01-1 mumol/kg). However, dose dependently, it antagonized the hypotensive effect of flesinoxan and urapidil. The alpha 1-adrenoreceptor antagonist prazosin, on prior treatment, did not change the hypotensive effect of either flesinoxan or urapidil. The alpha 2-adrenoreceptor agonist clonidine dose dependently (0.01-0.1 mumol/kg) reduced blood pressure. This effect of clonidine was unaffected by spiroxatrine, but was dose dependently antagonized by the alpha 2-adrenoreceptor antagonist yohimbine. Binding studies with [3H]8-OH-DPAT indicated that the affinity and Bmax of 5-HT1A receptors was increased in vitamin B6-deficient hypertensive rats. The results suggest that decreased synthesis of 5-HT in brain regions and the consequent alterations in 5-HT receptors in the vitamin B6-deficient rats may be the underlying cause of the hypertension seen in these animals.

The influence of riboflavin deficiency on Plasmodium berghei infection in rats.

Two experiments were done in which rats in various stages of riboflavin deficiency were infected with Plasmodium berghei. Various control groups were included to compare the influence of food restriction on the P. berghei infection with that of riboflavin deficiency, namely, pair-fed (PF), weight-matched (WM) and ad libitum-fed (C-AL) control groups. Riboflavin deficiency depressed maximum parasite counts by comparison with all control groups and the degree of depression was inversely related to the riboflavin status. Survival of animals with P. berghei infection was approximately 10 to 14 days and was not significantly influenced by any of the dietary regimens. Two possible mechanisms by which riboflavin deficiency might influence the growth and multiplication of P. berghei are discussed, namely, a depression of reticulocytosis and an effect on the synthesis of reduced glutathione in the parasite or red blood cell.

Alterations in the lenticular proteins of rats on riboflavin deficient diet.

Effect of feeding riboflavin deficient diet to rats on lens protein composition was investigated. Total proteins and profile of soluble and insoluble proteins in lenses from rats fed on a riboflavin deficient diet for seven weeks were found to be similar to that of the vitamin supplemented diet. Distribution of high molecular weight protein (above 4 X 10(6) daltons) isolated from the 9,900 g supernatant fraction was found to be significantly higher and gamma crystallin was significantly lower in riboflavin deficient group as compared to the normal lenses. However, the distribution of alpha and beta crystallins was not affected. Gel electrophoresis in sodium dodecyl sulfate of soluble lens proteins from riboflavin deficient animals had lower proportions of polypeptide species with molecular weight above 40,000 daltons while insoluble protein fraction had higher proportions of these polypeptide species as compared to control rats. These data suggest that the composition of lens proteins is altered in riboflavin deficiency.

The effect of riboflavin deficiency on cerebrum and cerebellum of developing rat brain.

After 21-day-old weanling rats were maintained on diets deficient in riboflavin the weights of their brains were 19.8% less than those of rats on control diets. In riboflavin deficiency, the myelin lipids, cerebrosides, and sphingomyelin, as well as phosphatidylethanolamine, a significant component of the myelin membrane, were considerably reduced in proportion. It is considered that riboflavin plays some role in the metabolism of essential fatty acids in brain lipids and the pathological effect of its deficiency is similar to that of essential fatty acid deficiency, causing a fast impairment to brain development and maturation.

[Studies on the influence of riboflavin intake to osteogenesis in rats].

Influence of an intake of the riboflavin that exerts upon osteogenesis in this research, was examined by a nutritional methods. It search the condition of femur in rats they reared and given three type riboflavin dosage, namely, sufficient riboflavin, few deficient riboflavin and administered sufficient after few deficient riboflavin. As a result, an influence of riboflavin dosage was not recognized about each of the body weight, food intake, and weight and length of femur. In other words, a deficiency symptom was not observed seemingly in the rat that given riboflavin deficiency diet. Also, the calcium contents in the femur decreased significantly by the deficiency of riboflavin. And, the low value was shown the even in the rat that administered sufficient fibroflavin later. Furthermore, even breaking force of the femur decreased by the deficiency of riboflavin.