Mitochondrial oxidative metabolism during respiratory infection in riboflavin deficient mice.

Studies in children and mice have shown that respiratory infection alters riboflavin metabolism, resulting in increased urinary loss of this vitamin. This could be due to mobilization of riboflavin from the liver to blood because liver Flavin adenine dinucleotide (FAD) levels were lowered in the mice during infection. To understand the functional implications of lowered hepatic FAD levels during respiratory infection, flavoprotein functions such as oxidative phosphorylation and beta-oxidation of the liver mitochondria were examined during infection in mice. Weanling mice were fed either riboflavin-restricted or control diet for 18 days and then injected with a sublethal dose of Klebsiella pneumoniae. During infection, the state 3 respiratory rate with palmitoyl-L-carnitine and glutamate were significantly lowered (27-29%) in the riboflavin-restricted group, whereas in the control group 10% reduction was observed with palmitoyl-L-carnitine as substrate. A 22% reduction in the respiratory control ratio with palmitoyl-L-carnitine as substrate was observed during infection in the riboflavin-restricted group. The beta-oxidation of palmitoyl-L-carnitine was significantly lowered (29%) in the riboflavin-restricted infected group. The results of the study suggest that the effects of infection on vital physiologic functions were more pronounced in the riboflavin-restricted mice than in the control mice.

Flavin metabolism during respiratory infection in mice.

Previous control studies carried out in children showed that respiratory infection alters riboflavin metabolism and leads to excessive urinary losses of the vitamin. In order to understand the nature of biochemical changes in riboflavin metabolism during respiratory infection, a study was carried out using the mouse as the experimental model, and Klebsiella pneumoniae as the infective organism. Mice were fed on either a low (0.5 mg/kg)- or high (13.3 mg/kg)-riboflavin semi-synthetic diet. Infection resulted in a 5-6-fold higher excretion of riboflavin in the urine of mice fed on the low-riboflavin diet. Higher erythrocyte FAD levels and lower liver FAD levels were also observed during infection. Of the four enzymes involved in the synthesis and breakdown of the flavin coenzymes studied, the activity of hepatic flavokinase (ATP: riboflavin 5'-phosphotransferase; EC 2.7.1.26) was significantly lower, and that of FAD synthetase (ATP: FMN adenylyltransferase; EC 2.7.7.2) was higher during riboflavin restriction and infection. The activity of FMN (acid) phosphatase (EC 3.1.3.2) was unchanged, whereas FAD (nucleotide) pyrophosphatase (EC 3.6.1.9) activity was significantly higher both with the low-riboflavin diet and during infection. Thyroid hormone is known to modulate flavokinase activity and, hence, thyroid status was assessed. Plasma triiodothyronine (T3) levels were not affected, but thyroxine levels were lower in the mice fed on the low-riboflavin diet. However, plasma T3 was significantly lower during infection, suggesting a mechanistic role for the hormone in the reduction of flavokinase activity.

The effect of exercise on the riboflavin status of adult men.

Six sedentary to moderately active men with biochemical signs of riboflavin deficiency were studied under metabolic ward conditions to examine the effects of physical activity on riboflavin status. All participants were subjected to additional exercise (EXER) for an 18 d period between two maintenance (M1 and M2) periods (16 and 13 d respectively) of habitual physical activity. Energy balance and riboflavin intake were maintained throughout the study. Riboflavin status, as judged by a significant reduction in erythrocyte glutathione reductase (EC 1.6.4.2) activation coefficient (EGR-AC), improved on changing from home (1.53 (SD 0.14)) to period M1 (1.36 (SD 0.21)) diets. The exercise period, however, resulted in a significant deterioration in riboflavin status (1.57 (SD 0.31)) which persisted in the subsequent period M2 (1.54 (SD 0.15)). There was a concomitant fall in the urinary excretion of riboflavin only in the EXER period, when results were expressed as a percentage of the dietary intake of riboflavin. These results suggest an increased demand for the vitamin for selective biochemical functions during exercise. However, the energy cost of walking (treadmill 4 km/h), 50 W and 100 W work-loads (bicycle ergometer) as well as delta mechanical efficiency (DME) did not change during the three metabolic periods. The urinary excretion of riboflavin was inversely related to DME (r -0.49; P < 0.05) and directly correlated with haemoglobin levels (r 0.63; P < 0.005). The present study suggests that riboflavin status further deteriorates during a short period of increased physical activity in individuals whose riboflavin status is marginal.

Interpretation of erythrocyte glutathione reductase activation test values for assessing riboflavin status.

The erythrocyte glutathione reductase activation test (EGR-AC) is considered to be the best method to assess riboflavin nutritional status. Riboflavin supplementation studies carried out in India have raised doubts about the validity of currently available interpretive guidelines for interpreting the EGR-AC test. Changes in EGR-AC values in response to graded doses of riboflavin supplementation were investigated in schoolchildren, aged 7-11 years, belonging to the low-income group. For comparison, unsupplemented well-to-do schoolchildren of similar age group were also examined. The results of the study based on the measurement of EGR-AC by the procedure of Bayoumi and Rosalki (Clinical Chemistry, 1976, Vol. 22, pp. 327-335) with an incubation period of 15 min suggest that the cut-off value for EGR-AC to discriminate between riboflavin-deficient and normal children cannot be less than 1.5.

In vitro stimulation of microsomal diethyl nitrosamine deethylase activity by vitamin K3 (menadione).

Vitamin K3 (menadione) has been found to stimulate diethyl nitrosamine (DEN)-deethylase activity in rat liver microsomes. The vitamin also takes care of the inhibitory effect of the anaerobic conditions as well as those of cytochrome poisons like sodium azide and sodium cyanide, possibly through production of active oxygen species. The enzyme was also stimulated by H2O2 and SOD and inhibited by catalase, thereby suggesting that H2O2 or some derivatives of it may be the active oxygen species involved in the reaction.

Effect of riboflavin or pyridoxine deficiency on inflammatory response.

Inflammatory response has been assessed in riboflavin or pyridoxine deficient rats. Edema was increased by 54% in pyridoxine deficiency as compared to weight-matched control rats. Food restriction per se reduced the volume of edema by 63%. In pyridoxine deficiency, concentrations of thiobarbituric acid reactive substances (which indicate the extent of lipid peroxidation) increase by 30 and 43% respectively in the edematous tissues of the paw as well as in the wounded skin. Both these parameters were not affected by riboflavin deficiency. Activities of NADPH oxidase and superoxide dismutase in elicited leukocytes from peritoneal cavity were reduced by 54 and 52%, respectively, in riboflavin deficiency but were unaltered in pyridoxine deficiency. Superoxide level and acid phosphatase activity were not influenced by either of the deficiencies, whereas hydrogen peroxide level was increased by 48% in riboflavin deficiency. Food restriction did not affect leukocyte enzymes or the levels of reduced oxygen species. The data suggest that inflammation is enhanced in pyridoxine deficiency but not in riboflavin deficiency.

Enzymatic evaluation of riboflavin status of infants.

Riboflavin status of solely breast-fed (SBF) infants aged 1-6 months (study 1) and solely or partially breast-fed (PBF) and weaned infants aged 6-24 months (study 2) was examined by the erythrocyte glutathione reductase activation test. Maternal riboflavin status and milk riboflavin content were also measured in study 1. Riboflavin status of the infants was significantly superior to that of their mothers. However, 35 per cent of SBF infants examined suffered from biochemical riboflavin deficiency as judged by the glutathione reductase test. Supplementary feeding of 6-8-month-old infants and weaning of older infants had positive effect on riboflavin status. There was no obvious relationship between riboflavin status and anthropometric status. Milk riboflavin concentration was comparable or only slightly lower than values reported for Western women, despite marked deficiency in the mothers.

PIP: Researchers compared the riboflavin status of 55 solely breast fed (SBF) 1-6 month old infants with 112 SBF, partially breast fed (PBF), or weaned 6-24 month old infants from low income families in India. They measured in vitro activation of erythrocyte glutathione reductase with its coenzyme FAD in blood samples from 6-12 month old infants and both the 1-6 month old infants and mothers to determine riboflavin status. They also analyzed the fore milk for riboflavin concentration. The riboflavin status of the 1-6 month old SBF infants was much greater than that of the mothers especially beginning with 3 month old infants (p.001-.02). Yet 35% of SBF 1-6 month old infants were biochemically deficient in riboflavin. In the 1st 2 months postpartum, however, the mothers had particularly good riboflavin status. The milk riboflavin level for SBF 1-6 month old infants peaked at 2 (26.9 mcg/dl) and 4 months (28.6 mcg/dl). It stood at 20 mcg/dl for 1 month old SBF infants. It fell from 18-14.3 mcg/dl between 5-6 months. The riboflavin status of 6-8 month old PBF infants stood greater than that of SBF 6-8 month old infants (p.001). Moreover 12 of the 29 (41%) 6-8 month old SBF infants were considered to be riboflavin deficient. No 6-8 month old infant was completely weaned. After 8 months, riboflavin status fell in PBF infants. In addition, the poorest infants (monthly family incomes of Rs500) had a much lower riboflavin status than the more privileged infants. The more privileged mothers (Rs 500) tended to begin supplemental feeding earlier than poorer mothers. The most significant finding was the beneficial effects of supplementary feeding on riboflavin status in 6-8 month old infants. Scientists suspect that riboflavin deficiency affects psychomotor functions and impairs collagen maturity and wound healing.

Spirulina as a source of vitamin A.

Experiments were carried out to assess spirulina fusiformis-a blue green algae as a source of vitamin A in rats. In one experiment, the control rats were fed synthetic vitamin A and the experimental rats spirulina as the sole source of vitamin A. The liver vitamin A concentration of spirulina-fed rats of both sexes was found to be significantly higher than that of the control rats. In another experiment the absorption of carotenes from the solvent extract of spirulina and their availability (vitamin A value) as judged by the levels of vitamin A and carotene in plasma and liver were compared with those of synthetic beta-carotene or vitamin A in male rats. The absorption of beta-carotene from spirulina extract tended to be lower than that of crystalline beta-carotene at doses of 550 and 1100 micrograms of beta-carotene. The difference became insignificant at lower beta-carotene dose of 275 micrograms. Spirulina carotene-fed rats did not show a strict dose related increase in the liver or serum vitamin A concentration. The liver vitamin A storage and plasma levels of vitamin A of spirulina carotene-fed rats was much higher than expected. The results of the two studies reported suggest that the algae spirulina can be a valuable source of vitamin A.

Effect of respiratory infection on tissue riboflavin and flavin enzymes in mice.

The effects of Klebsiella pneumoniae infection on red blood cell and liver riboflavin levels and a few flavin enzymes were examined to verify our earlier hypothesis based on human experience that upper respiratory infections result in mobilization of riboflavin from tissues into blood, resulting in a rise in red blood cell riboflavin and an increased saturation of erythrocyte glutathione reductase (EGR) with its coenzyme flavin adenine dinucleotide (FAD; reduction in EGR activation coefficient, AC, values). Thirty-six-day-old male mice fed a diet marginally sufficient in riboflavin were injected with a single sublethal dose of K. pneumoniae. Batches of control, deficient infected and deficient uninfected animals were killed during the peak period of infection and after recovery. Infection brought about a significant rise in basal EGR activity due to greater saturation with coenzyme FAD and a concomitant reduction in EGR-AC values. On recovery, basal EGR activity decreased, and EGR-AC values increased to levels beyond control values, suggesting riboflavin deficiency. In the liver, acyl-coenzyme A dehydrogenase showed a significant decrease and pyridoxamine-phosphate oxidase activity a significant increase. Both the enzymes normalized after recovery. D-amino acid oxidase activity did not change.

Skin wound healing in riboflavin deficiency.

Healing of excision and incision wounds was evaluated in riboflavin-deficient rats. The period taken for the epithelialization of excision wounds was 4 to 5 days longer in riboflavin-deficient animals compared to ad libitum-fed or food-restricted weight-matched control groups. Riboflavin deficiency as well as food restriction slowed the rate of wound contraction, the effect of riboflavin deficiency being of greater magnitude. The tensile strength of incision wounds in riboflavin deficiency was reduced to 42% of the ad libitum-fed control and 63% of the weight-matched control values. There was a decrease of 25% in total collagen content of incision wounds, in riboflavin deficiency and its maturity was drastically affected as indicated by a twofold increase in salt solubility (1 M NaCl) and a four-fold increase in the alpha/beta subunit ratio of salt-soluble collagen. Food restriction had similar effects but of lower magnitude. The data suggest that alteration in collagen content and maturity may be responsible for the lower tensile strength of incision wounds in riboflavin-deficient rats. This suggestion was supported by the results of a rehabilitation experiment.

Reduced anaerobic glycolysis in oral contraceptive users.

Oral contraceptives containing combinations of estrogens and progestogens are known to impair glucose tolerance. The biochemical mechanisms underlying this lesion are speculative. In the present study women treated with OC for periods exceeding 10 cycles showed significant reduction in the activity of the key glycolytic enzyme phosphofructokinase (40%) and the levels of lactate (42%) in the erythrocytes compared to controls. These observations in women are analogous to those made earlier in female rats.

PIP: Combined oral contraceptives (30 mg ethinyl estradiol, 150 mg d-norgestrel) are known to reduce glucose tolerance. To study the possible mechanisms of this impairment, 20 women were divided into 3 groups based on length of contraceptive use -- 3-5 months, 6-11 months, and 12-36 months. Blood samples were analyzed for phosphofructokinase activity and levels of glycolytic metabolites. Changes in glucose tolerance are seen within 3-6 months of oral contraceptive use, but only the blood taken from the 3rd group (12-36 months) showed significantly lower levels of phosphofructokinase activity and lowered levels of fructose-1,6-diphosphate, lactate, and pyruvate. It is suggested that impaired glucose tolerance is due to reduced glycolysis due to lower levels of phosphofructokinase synthesis, which cannot be detected for at least a year in erythrocytes, since they do not synthesize the enzyme.

Effects of female sex steroids on concanavalin A-mediated agglutination of hepatocytes from nonregenerating and regenerating rat liver and hepatic tumor marker enzymes.

Effect of treatment of female rats with an oral contraceptive agent (OCA), Ovulen-50, for 7 weeks on agglutination of hepatocytes with concanavalin A (con A) and activities of certain tumor marker enzymes were examined to find out if OCA treatment is related to preneoplastic or neoplastic processes. Hepatocytes from regenerating and nonregenerating livers of control female rats showed negligible agglutination with Con A, whereas hepatocytes from non regenerating but not from the regenerating livers of female rats treated with a combination of 5 micrograms ethinyl estradiol and 100 micrograms ethynodiol diacetate showed agglutination. Of the tumor marker enzymes such as hepatic glucose 6-phosphatase, gamma-glutamyl transpeptidase (gamma-GT), and arginase examined in the liver, only gamma-glutamyl transpeptidase showed a significant increase in activity in the steroid-treated rats. Plasma alkaline phosphatase activity was also higher in the treated animals. However, the magnitude of the changes observed was relatively small and perhaps unrelated to the neoplastic process.

Rise in plasma trimethyllysine levels in humans after oral lysine load.

Oral administration of 5 g of lysine to healthy male volunteers produced a marked and progressive rise in plasma trimethyllysine (TML) from 3 to 24 h after the lysine load. Urinary TML did not register a similar rise. Plasma and urinary carnitine also increased after the lysine load. A similar rise in TML or carnitine was not seen after a 5 g tryptophan load. This suggests that the effect is specific for lysine and is not a nonspecific consequence of amino acid load.

Urinary riboflavin excretion and erythrocyte glutathione reductase activity in preschool children suffering from upper respiratory infections and measles.

The urinary and blood levels of riboflavin and erythrocyte glutathione reductase (EGR) activity and its stimulation with FAD (EGR-AC) were examined in preschool children suffering from either measles or other upper respiratory infections and matched controls. Patients showed significantly higher levels of urinary riboflavin (per unit creatinine), erythrocyte riboflavin and EGR activity and lower EGR-AC values. This trend reversed after treatment. Mobilization of riboflavin from a labile tissue pool during infections may produce artefactual changes in the biochemical indices of riboflavin status. It would also prevent tissue saturation despite supplementation.