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In isolated rat enterocytes, both normoenergized (normal) and de-energized with rotenone, riboflavin intracellular metabolic processes, operating in association with a membrane-specific transport mechanism, were investigated. The contents of unlabeled (endogenous) and labeled (exogenous) flavins [riboflavin (RF), flavin mononucleotide (FMN), flavin adenindinucleotide (FAD)] were determined by HPLC before and after incubation with tritiated RF. In normoenergized enterocytes, total labeled RF content (i.e., total uptake, the sum of RF membrane transport and intracellular metabolism) increased steadily to a plateau after 20 min incubation; FMN and FAD contents reached a plateau between 3 and 20 min, whereas free RF content increased constantly. The phosphorylated forms prevailed over the free form ( approximately 60% of total flavins). In de-energized enterocytes, RF total uptake was significantly lower than in normoenergized enterocytes and reached a plateau after only 3 min incubation. FMN and FAD contents were significantly lower than in normoenergized enterocytes, and free RF represented the prevailing form of flavins (70% of total RF ). In both normoenergized and de-energized enterocytes, the contents of unlabeled total RF, FMN and FAD decreased significantly after 20 min incubation, whereas free RF increased significantly only in normoenergized enterocytes. After 20 min incubation, the RF structural analog 8-dimethyl-amino-8-demethyl-RF caused a significant decrease of all flavin contents, whereas 5'-deoxy-RF decreased only the total and free RF contents. Results directly confirmed the leading role of metabolic processes such as phosphorylation in RF transport by isolated small intestinal enterocytes.

The effect of riboflavin alpha-D-glucoside, isolated from rat liver, on the uptake of riboflavin was studied using freshly isolated rat liver cells. The transport characteristics and metabolic fate of the glucoside were also determined using the radioactive compound. The initial (1-min) uptake of 1 mumol/L [3H]riboflavin glucoside (2.90 +/- 0.29 pmol/10(6) cells) was higher than that of 1 mumol/L [3H]riboflavin (1.35 +/- 0.30 pmol/10(6) cells). However, the accumulation of glucoside after 60 min was significantly lower than that of riboflavin. The presence of up to 30 mumol/L glucoside had no significant effect on the initial uptake of [3H]riboflavin (3 mumol/L, 10(9) cells/L). Measurement of the kinetic parameters for glucoside gave an apparent Kt value of 83.4 +/- 12.4 mumol/L and a Vmax of 208.6 +/- 17.9 pmol/(10(6) cells.min). Decreases in the temperature of incubation decreased uptake rate. Replacement of Na+ with other monovalent cations did not affect uptake. The presence of D-glucose (1 mumol/L to 5.5 mmol/L) had no inhibitory effect on uptake of 1 mumol/L [3H]riboflavin glucoside. The results indicate that the transport of riboflavin glucoside may not involve the transport mechanisms for riboflavin or D-glucose. Metabolic studies with isolated hepatocytes showed that the glucoside was hydrolyzed to yield riboflavin upon entry into the cell. The vitaminic efficiency of this compound was tested by feeding it to growing male rats. These experiments indicate that the glucoside and free riboflavin are comparable sources of the vitamin.

Although milk is one of the primary sources of riboflavin in the North American diet, the amounts of riboflavin and other flavin derivatives in milk have not been accurately quantified. In this study, a comprehensive assessment of milk was performed to identify and quantify greater than 95% of the flavins in this food. Riboflavin and flavin adenine dinucleotide (FAD) were the predominant flavins in all milk samples; 10-(2'-hydroxyethyl)flavin was next most common. The latter is potentially an antivitamin that may exert its effect at either the absorption or utilization level. Therefore, the presence of this analog should be considered when evaluating the riboflavin efficiency of milk. The large quantity of FAD indicates that precautions against underestimating FAD are warranted when determining total flavin content by fluorescence. This is particularly important because the fluorescence of FAD at pH 7.0 is only 10-20% of that for riboflavin. The presence of 7 alpha-hydroxyriboflavin (7-hydroxymethylriboflavin) in milk was ascertained by extensive testing. Traces of 8 alpha-hydroxyriboflavin (8-hydroxymethylriboflavin) were also detected. These metabolites may serve as indicators of cellular biochemical activity.

D-Amino acid oxidase and xanthine oxidase, two enzymes possessing ionically bound flavin coenzymes have been studied with their flavin coenzymes derived from either 7-ethyl-8-methyl-flavin or 7-methyl-8-ethyl-flavin, vitamin-like homologues of riboflavin. 7-Ethyl-8-methyl-flavin caused a significant reduction of both D-amino acid oxidase and xanthine oxidase in the liver, but not in the kidney. 7-Methyl-8-ethyl-flavin caused a significant reduction of D-amino acid oxidase in both the liver and kidney, a significant reduction of xanthine oxidase in the liver, but a large and significant increase of the latter enzyme in the kidney. An improved procedure for the assay of xanthine oxidase has been described.

The effect of riboflavin status on uptake was investigated in hepatocytes isolated from control, riboflavin-sufficient and riboflavin-deficient rats. The uptake exhibited biphasic characteristics with an initial rapid phase [13.2 +/- 1.8 pmol/(10(6) cells X minute)] for the first couple of minutes followed by a second slower phase which continued for over an hour. The accumulation of riboflavin at near equilibrium conditions was 2.5- and 5.2-fold greater than external concentration in control and riboflavin-deficient cells, respectively. An apparent Km of 12 +/- 1.3 microM and Vmax of 82.3 +/- 9.1 pmol/(10(6) cells X minute) were obtained for control and riboflavin-sufficient rats while a similar Km but higher Vmax were obtained with deficient animals. Correspondence of the Km to that of flavokinase for riboflavin suggested the possibility that uptake of the vitamin may occur via metabolic trapping, i.e., phosphorylation. As substantiation of this, the rate of uptake was decreased by lumiflavin and 2'-hydroxyethylflavin, which are competitive inhibitors, and by 7,8-dichloroflavin, a substrate for flavokinase. Furthermore, the uptake was found to be temperature-dependent and studies with carbonylcyanide-p-trifluoro-methoxyphenylhydrazone (FCCP) and ethionine indicated a requirement for ATP. These results showed that overall, entry of riboflavin into hepatocytes occurs predominantly by a facilitated diffusion process followed by rapid trapping by flavokinase-catalyzed phosphorylation to FMN.

The influence of 7-ethyl-8-methylflavin and 7-methyl-8-ethylflavin, vitamin-like homologues of riboflavin, on rat brain mitochondrial monoamine oxidase (MAO) was studied using tyramine as substrate. While riboflavin deficiency caused the enzyme activity to fall to 80% of norma, when 7-ethyl-8-methylflavin replaced riboflavin as the precursor of its coenzyme, it caused essentially complete loss of the enzyme activity. We showed that while 7-ethyl-8-methylflavin can serve as a coenzyme for MAO, 7-methyl-8-ethylflavin cannot serve as coenzyme for this enzyme.

Several analogs of riboflavin differing with respect to the substituent at C7, C8 or C10 were examined for their ability to replace riboflavin and to act as riboflavin antagonists in inhibiting growth of Tetrahymena pyriformis. Generally, the analogs with altered substituents at C7 or C8 (the 8-ethyl, 7,8-diethyl, 7-chloro and 8-chloro analogs) supported some growth but inhibited riboflavin-supported growth. However, the 8-bromo analog had no biological activity. The 7-ethyl analog with a bis(2-hydroxyethyl)aminoethyl side chain was the most potent antagonist. These results were compared with those observed in Lactobacillus casei and the rat.

Weanling mice were fed a riboflavin-deficient diet or the same diet with added galactoflavin. Both diets produced changes in hepatic mitochondrial morphology, the most striking of which was the development of giant mitochondria. The livers from these animals were fractionated, and the nuclear and mitochondrial fractions were examined by electron microscopy. The nuclear fraction contained giant mitochondria; the mitochondrial fraction contained the remaining normal to moderately enlarged mitochondria. Oxidative studies were carried out on the mitochondrial fractions. It was found that both experimental diets resulted in a marked reduction in fatty acid oxidation by the mitochondria. In addition, the mitochondria of mice with advanced riboflavin deficiency (induced simply by a riboflavin-free diet) showed a severely decreased state 3 (ADP-stimulated) respiration and depressed respiratory control ratios, but normal ADP/O ratios. In contrast, mitochondrial performance (aside from fatty acid oxidation) in galactoflavin-supplemented, riboflavin-deficient mice was related to the gross appearance, i.e., color, of the liver from which these organelles were derived. In mice fed this diet, the livers were either red or yellow. Mitochondria from yellow livers showed normal oxidative phosphorylation. Mitochondria from red livers showed a serious reduction in state 3 oxidation. This study demonstrates that in the mouse, riboflavin deficiency, however produced, not only results in altered mitochondrial morphology but also results in significantly impaired mitochondrial function.

Male rats were fed a riboflavin-deficient diet for 25 days and then fed [2-14C]riboflavin to replete their riboflavin pool(s). During a second depletion period, urine and feces were collected and 14C excretion monitored. The urine was chromatographed on R-15 resorcinol resin and 14C was measured in all fractions. The 14C compounds in the individual fractions were shown to be different by thin layer chromatography. The amount of 14C recovered as riboflavin increased after acid hydrolysis of the urine indicating the presence of riboflavin in conjugated forms. All R-15 fractions were found to support growth in a microbiological (L. casei) assay for riboflavin. This suggested that other derivatives, in addition to riboflavin and flavin nucleotides, are biologically active. However, the conjugated compounds were apparently less active, since acid hydrolysis of the urine enhanced its growth-promoting ability of L. casei. One 14C metabolite was identified as urea, thus providing evidence for degradation of the riboflavin molecule. This study has shown that the metabolic fates of riboflavin in the rat include conjugation and extensive degradation.

Detailed studies of the biological activity of several analogues of riboflavin in the riboflavin-deficient chick embryo were undertaken to extend our knowledge of the comparative biochemistry of these flavins. The riboflavin-deficient eggs were produced by hens homozygous for an autosomal recessive gene (rdrd). 7-Ethyl-8-methyl-flavin was found to be an adequate replacement for riboflavin in che chick embryo, a finding which mimics its effect in the riboflavin-deficient rat. 7,8-Diethyl-flavin was found to be an antagonist in the chick embryo as had been found to be true in the rat. 7-Methyl-8-ethyl-flavin can not be used as a replacement for riboflavin in the chick embryo and further, it inhibits the utilization of riboflavin in normal eggs. 7-Methyl-8-ethyl-flavin had been found to be an adequate replacement for riboflavin in the riboflavin-deficient rat. The classes mammalia and aves are clearly different in respect to the utilization of this flavin.

Groups of normal 5-day-old chicks of the Single Comb White Leghorn strain were fed diets containing no riboflavin, riboflavin, or one of several analogues of riboflavin, to explore differences between the classes Mammalia and Aves. It was found that 7-ethyl-8-methyl-flavin was a suitable replacement for the vitamin. 7-Methyl-8-ethyl-flavin permitted early growth of the chicks, but it did not allow survival when fed in place of riboflavin. The inability to utilize 7-methyl-8-ethyl-flavin represents a real difference in the utilization of flavin between the classes Mammalia and Aves. Other flavin analogues supported some growth, but the chicks did not survive. All groups showed better growth responses than was shown by the riboflavin-deficient group.

Changes in hepatic mitochondrial oxidative metabolism were examined during the development of severe riboflavin deficiency in mice, and during recovery from this deficiency. There was a marked reduction in oxidative rates for all substrates tested, with the decline being most pronounced with palmitoyl-1-carnitine. These effects were not enhanced by addition of galactoflavin to the riboflavin-deficient diet. Treatment of the deficient mice with riboflavin restored hepatic mitochondrial oxidation to normal within 24 hours in those mice fed a simple riboflavin-deficient diet, but required 72 hours in galactoflavin-supplemented mice. These metabolic changes in hepatic mitochondria appear to be temporally independent of the striking morphological changes occurring in these organelles during ariboflavinosis and recovery.