Uptake of ascorbic acid by human granulocytes

The uptake of ascorbic acid by isolated human granulocytes is investigated under different conditions. The rate of uptake depends on the concentration of ascorbic acid in the incubation medium as well as on temperature, with a maximum at 40 degrees C. At 0 degrees C no uptake can be observed. N-formylated peptides being known to stimulate human granulocytes, considerably increase ascorbic acid uptake, but the non-formylated methionyl-leucyl-phenylalanine is without effect. Glucose is a strong inhibitor of ascorbate uptake with a Ki of 3.7 mM and a stoichiometry of 1:1. Fructose does not inhibit at all and galactose only at elevated concentrations. Phlorizin, a known inhibitor of glucose transport, inhibits the uptake of ascorbic acid to the same extent as that of glucose. Elevated glucose levels do not induce a release of ascorbic acid out of the cells. It is concluded that ascorbic acid is actively accumulated in human granulocytes and that stimulated cells respond with a pronounced increase of ascorbate uptake. The site of ascorbic acid transport across the membrane is probably the same as of glucose.     

Phagocytosis and leukocyte enzymes in ascorbic acid deficient guinea pigs.

Enzymes related to bactericidal activities of leukocytes were studied in ascorbic acid deficient guinea pig leukocytes. The activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were not affected either under resting or phagocytizing conditions in ascorbic acid deficiency. Granule bound NADPH-oxidase activity of resting leukocytes also was not altered in ascorbic acid deficiency. However, the extent of stimulation in NADPH-oxidase activity under phagocytizing condition was found to be significantly lower in ascorbic acid deficient leukocytes than that in control leukocytes. Similary, the extent of release of acid phosphatase from lysosomes during phagocytosis was also low in ascorbic acid deficient leukocytes. Ascorbic acid deficiency did not influence the activities of glutathione reductase and myeloperoxidase of leukocytes. The significance of these enzyme changes is discussed in relation to the decreased phagocytic and bactericidal activities of leukocytes in ascorbic acid deficiency.     

Intracellular accumulation of ascorbic acid is inhibited by flavonoids via blocking of dehydroascorbic acid and ascorbic acid uptakes in HL-60, U937 and Jurkat cells

In HL-60, U937 and Jurkat cells, the intracellular accumulation of ascorbic acid occurred via uptakes of both dehydroascorbic acid (an oxidized metabolite of ascorbic acid) and ascorbic acid (vitamin C). Dehydroascorbic acid and ascorbic acid were transported into cells by sodium-independent glucose transporters (GLUT 1 and GLUT 3) and sodium-dependent ascorbic acid transporters, respectively. Flavonoids inhibited the intracellular accumulation of ascorbic acid by blocking dehydroascorbic acid and ascorbic acid uptakes in the transformed cells. At flavonoid concentrations of 10-70 micromol/L, approximately 50% of dehydroascorbic acid uptake was inhibited in the cells. In Jurkat cells, two potent flavonoids (myricetin and quercetin) competitively inhibited dehydroascorbic acid uptake, and K(i) values were approximately 14 and 15 micromol/L, respectively. Because GLUT 1 and GLUT 3 transport dehydroascorbic acid, the inhibition of dehydroascorbic acid uptake by flavonoids was investigated by using Chinese hamster ovary cells overexpressing rat GLUT 1 or human GLUT 3. Myricetin at concentrations of 22 and 18 micromol/L, respectively, inhibited half of dehydroascorbic acid uptake in the cells overexpressing GLUT 1 and GLUT 3. Myricetin also inhibited ascorbic acid uptake; inhibition was noncompetitive with K(i) = 14 micromol/L in Jurkat cells. These data indicate that flavonoids inhibit both ascorbic acid and dehydroascorbic acid uptake but do so by different mechanisms. These data may contribute to new understanding of the biological effect of flavonoids on the intracellular accumulation of ascorbic acid in human cells.     

Glycolytic, hexose monophosphate shunt and bactericidal activities of leukocytes in ascorbic acid deficient guinea pigs.

It is well known that glycolytic and hexose monophosphate shunt activities of leukocytes increase during phagocytosis. The relevance of these metabolic changes to particle uptake and particle destruction is also well established. In the present study, these metabolic activities were studied to assess the phagocytic function of leukocytes isolated from ascorbic acid deficient guinea pigs. Glycolytic activity which provides the necessary energy for particle uptake was found to be decreased in both resting and phagocytizing leukocytes for ascorbic acid deficient guinea pigs. The direct oxidation of glucose through the hexose monophosphate shunt (HMS) was stimulated to a significantly lesser extent during phagocytosis in ascorbic acid deficient leukocytes. There was a progressive decline in phagocytosis induced shunt activity of leukocytes as the deficiency of ascorbic acid progressed. These findings show that particle uptake (as indicated by glycolytic activity) as well as particle destruction (as indicated by HMS activity) by leukocytes are impaired in ascorbic acid deficiency. Bactericidal capacity of leukocytes against Escherichia coli was also found to be low in ascorbic acid deficient guinea pigs as compared to those in the pair-fed control group.     

Effects of antacid or ascorbic acid on tissue accumulation and urinary excretion of chromium

Absorption of trivalent chromium is low. Unbound chromium can form insoluble complexes in the gastrointestinal tract. In this study, effects of a calcium-based antacid or ascorbic acid on ⁵¹chromium from chromium chloride were investigated. Rats were dosed with 1 mL of test substance (containing 150 mg calcium carbonate or 10 mg ascorbic acid) or 1 mL water followed by 20 uCi ⁵¹chromium chloride. Twenty-four hours after dosing, ⁵¹chromium in cumulative urine was higher (p<0.02) in the group dosed with ascorbic acid than in the groups dosed with antacid or water. Accumulation of ⁵¹chromium in the kidney, testes, and spleen was lower (p<0.05) in rats dosed with antacid than in those dosed with ascorbic acid or water. These data confirm that absorption of chromium chloride is low and suggest that antacids have a negative effect on chromium absorption from chromium chloride.     

Differences in ethanol ingestion between cholecystokinin-A receptor deficient and -B receptor deficient mice

AIMS: Cholecystokinin (CCK) modulates dopamine release in the nucleus accumbens through the CCK-A receptor (CCK-AR). The dopaminergic neurotransmission between the ventral tegmental area and the limbic forebrain is a critical neurobiological component of alcohol and drug self-administration. Based on the evidence of interaction between CCK and dopamine, we had found previously that the CCK-AR gene -81A/G polymorphism was associated with alcohol dependence. Since the precise mechanism underlying this association has not been elucidated, the role of CCK-AR in ethanol ingestion was examined using CCK-AR gene deficient (-/-) mice and compared with those of CCK-BR(-/-) and wild-type mice. METHODS: The two-bottle choice protocol was conducted and the righting reflex was examined in these three genotypes. Furthermore, the protein level of dopamine 2 receptor (D2R) in the nucleus accumbens was determined by western blotting. RESULTS: CCK-AR(-/-) mice consumed more ethanol than CCK-BR(-/-) and wild-type mice, and showed no aversion to high concentrations of ethanol solution. However, the difference was actually in the total fluid consumption and alcohol preference remained unchanged, indicating that the differences were not specific to alcohol. Behavioral sensitivity to ethanol, examined using the righting reflex, did not differ significantly between the groups. D2R expression in the nucleus accumbens was significantly lower in the CCK-BR(-/-) mice and was significantly higher in CCK-AR(-/-) mice than in wild-type mice. CONCLUSIONS: Voluntary ingestion of ethanol differed between CCK-AR(-/-) and CCK-BR(-/-) mice. The difference might be attributable in part to the different levels of D2R expression in the nucleus accumbens.     

Urinary ascorbic acid levels following the withdrawal of large doses of ascorbic acid in guinea pigs

Male guinea pigs received sodium ascorbate solution [equivalent to 1 g ascorbic acid/(kg body weight.d)] by intraperitoneal injection for 4 wk. During the ascorbic acid treatment period, plasma and urinary ascorbic acid levels rose markedly. Three weeks after the ascorbic acid treatment was withdrawn, mean urinary ascorbic acid levels were significantly lower than their corresponding basal levels. At both 2 and 5 wk after withdrawal of ascorbic acid treatment, mean plasma ascorbic acid levels were below normal. The results indicate that these animals had experienced a transient withdrawal effect after administration of large doses of ascorbic acid that lasted about 1 wk. This, in turn, indicates that the rate of ascorbic acid turnover was probably increased during treatment, and this effect persisted even after the ascorbic acid was withdrawn. Examination of data from each individual experimental animal revealed that the pattern of urinary ascorbic acid excretion after the withdrawal of large doses of ascorbic acid varied from animal to animal. Among the twelve experimental guinea pigs, seven had abnormally low urinary ascorbic acid levels 2-4 wk after the withdrawal of the large doses of ascorbic acid.     

Effect of dietary ascorbic acid, cholesterol and PCB on cholesterol and bile acid metabolism in a rat mutant unable to synthesize ascorbic acid

The effect of acute or chronic ascorbic acid deficiency on the activity of hepatic cholesterol 7 alpha-hydroxylase and fecal excretion of bile acids was investigated in ODS-od/od (OD) rats (a rat mutant unable to synthesize ascorbic acid) fed a purified basal diet or purified diets containing either cholesterol (2%) or polychlorinated biphenyl (PCB) (200 mg/kg). In OD rats, the dietary requirement of ascorbic acid to maintain normal growth and normal levels of cholesterol in serum and liver is about 300 mg of ascorbic acid/kg diet. In OD rats fed the basal diet, acute or chronic ascorbic acid deficiency did not affect the activity of hepatic cholesterol 7 alpha-hydroxylase and fecal excretion of bile acids. However, in OD rats fed diets containing either cholesterol or PCB, acute ascorbic acid deficiency caused a higher level of serum cholesterol, a lower activity of hepatic cholesterol 7 alpha-hydroxylase and a lower excretion of fecal bile acids than in OD rats fed a basal diet containing an adequate level of ascrobic acid. It is concluded that acute ascorbic acid deficiency causes a hypercholesterolemia due to the depression of bile acid synthesis in OD rats fed a purified diet with cholesterol or PCB.     

On the absorption of ascorbic acid in man

The daily intake of ascorbic acid with the diet was strongly restricted in seven healthy non-smoking males and 90 to 180 mg of ascorbic acid were orally supplemented. After an equilibration period of three weeks about 5 muCi of (1-14C) ascorbic acid was orally administered with carrier (30 mg or 60 mg). The daily intake was then increased to 4 X 1000 mg and the cumulative excretion of radioactivity in the urine measured over a period of 10 days. The average absorption was estimated to amount to 80-90% of the dose.