Does Modification of the Large Intestinal Microbiome Contribute to the Anti-Inflammatory Activity of Fermentable Fiber?

Fiber is an inadequately understood and insufficiently consumed nutrient. This review examines the possible causal relation between fiber-induced microbiome changes and the anti-inflammatory activity of fiber. To demonstrate the dominant role of fermentable plant fiber in shaping the intestinal microbiome, animal and human fiber-feeding studies are reviewed. Using culture-, PCR-, and sequencing-based microbial analyses, a higher prevalence of Bifidobacterium and Lactobacillus genera was observed from the feeding of different types of fermentable fiber. This finding was reported in studies performed on several host species including human. Health conditions and medications that are linked to intestinal microbial alterations likely also change the nutrient environment of the large intestine. The unique gene clusters of Bifidobacterium and Lactobacillus that enable the catabolism of plant glycans and the ability of Bifidobacterium and Lactobacillus to reduce the colonization of proteobacteria probably contribute to their prevalence in a fiber-rich intestinal environment. The fiber-induced microbiome changes could contribute to the anti-inflammatory activity of fiber. Although most studies did not measure fecal microbial density or total daily fecal microbial output (colon microbial load), limited evidence suggests that the increase in intestinal commensal microbial load plays an important role in the anti-inflammatory activity of fiber. Various probiotic supplements, including Bifidobacterium and Lactobacillus, showed anti-inflammatory activity only in the presence of fiber, which promoted microbial growth as indicated by increasing plasma short-chain fatty acids. Probiotics alone or pure fiber administered under sterile conditions showed no anti-inflammatory activity. The potential mechanisms that could mediate the anti-inflammatory effect of common microbial metabolites are reviewed, but more in vivo trials are needed. Future studies including simultaneous microbial composition and load measurements are also important.

Dietary or supplemental fermentable fiber intake reduces the presence of Clostridium XI in mouse intestinal microbiota: The importance of higher fecal bacterial load and density.

OBJECTIVES: Clostridium difficile infection is a public health concern. C. difficile was found in healthy human intestine as a member of Clostridium XI. Because soluble fermentable fiber ingestion affects intestinal microbiota, we used fiber-containing diets to determine the intestinal microbial condition that could reduce the presence of Clostridium XI. METHODS: Newly weaned male mice were assigned to three published diets: Control AIN-93G purified diet with only poorly fermented cellulose; Control plus 5% purified fermentable fiber inulin; Chow with wheat, soybean and corn that provide a mixture of unpurified dietary fibers. Methods were developed to quantify 24-hour fecal microbial load and microbial DNA density. The relative abundance of bacterial genera and the bacterial diversity were determined through 16S rRNA sequence-based fecal microbiota analysis. RESULTS: Mice adjusted food intake to maintain the same energy intake and body weight under these three moderate-fat (7% w:w) diets. Chow-feeding led to higher food intake but also higher 24-h fecal output. Chow-feeding and 1-8 wk ingestion of inulin-supplemented diet increased daily fecal microbial load and density along with lowering the prevalence of Clostridium XI to undetectable. Clostridium XI remained undetectable until 4 weeks after the termination of inulin-supplemented diet. Fermentable fiber intake did not consistently increase probiotic genera such as Bifidobacterium or Lactobacillus. Chow feeding, but not inulin supplementation, increased the bacterial diversity. CONCLUSIONS: Increase fecal microbial load/density upon fermentable fiber ingestion is associated with a lower and eventually undetectable presence of Clostridium XI. Higher bacterial diversity or abundance of particular genera is not apparently essential. Future studies are needed to see whether this observation can be translated into the reduction of C. difficile at the species level in at-risk populations.

Human and mouse microarrays-guided expression analysis of membrane protein trafficking-related genes in MDCK cells, a canine epithelial model for apical and basolateral differential protein targeting.

MDCK cells are widely used to study the differential targeting of membrane transporters to apical and basolateral membrane but its canine origin limited the commercial tools available for the analysis of protein trafficking machinery. Because apical and basolateral membranes are only found in differentiated epithelial cells, genes critical for differential targeting may be specifically up-regulated upon MDCK cell differentiation. To search for these genes, a cross-species screening strategy was used. We first analyzed the human microarray data for protein trafficking-related genes that were up-regulated in colon carcinoma Caco2 cells upon differentiation. The results of mouse 44K gene expression microarray analysis were then used to extract additional candidate genes that showed higher expression in normal colon epithelium compared to primary embryonic fibroblasts. Finally, NCBI genomic sequence information was used to design RT-PCR primers for 13 candidate and 10 negative control genes and used to analyze MDCK cells at 2, 13 and 17 days after seeding. To determine whether the gene up-regulation was specific in epithelial differentiation, we also performed RT-PCR on rat non-differentiating intestinal IEC-6 cells and mouse C2C12 cells, a differentiating myoblast model. Of the 13 candidate genes, 3 genes, SDCBP2, KIF12, KIF27, met all criteria of specific up-regulation in differentiated MDCK cells. In addition, KIF13A showed up-regulation in differentiated MDCK and C2C12 cells but not in IEC-6 cells cultured for the same duration. The functions of these genes need to be analyzed in the future. This cross-species screening strategy may be useful for other non-human, non-rodent cell models.

Gender Difference in Bacteria Endotoxin-Induced Inflammatory and Anorexic Responses.

Inflammation-related anorexic response has been observed in systemic diseases as well as in localized infection and is an important issue in patient care. We tested the hypothesis that upon the same endotoxin exposure, males have more severe inflammatory responses and thus suffer from more negative effect on appetite. Ten-week old male and female mice were compared in their plasma levels of pro-inflammatory cytokines after a body weight-based i.p. injection of bacterial endotoxin lipopolysaccharide. Male mice consistently showed significantly higher levels of IL6 and TNFα than female mice. The difference was observed starting at 3 hours after the systemic endotoxin exposure. It was independent of the level of endotoxin dosage and of the genotype of the anti-inflammatory cytokine, IL10. Interestingly, endotoxin-injected male mice also had significantly higher plasma IL10 levels compared to the female mice. Pre-puberty young mice showed no gender differences in the plasma levels of IL6, TNFα and IL10. Their cytokine levels were mostly between that of the adult males and females. Consistent with the higher inflammatory response in male mice, the endotoxin exposure also led to significantly more appetite loss in male mice at a range of doses in two strains of mice. Saline injection in the absence of endotoxin affected neither the cytokine levels nor the appetite. Although a direct mechanistic link between inflammation parameters and appetite was not addressed here, the results support that male gender could be a risk factor for higher pro-inflammatory cytokines and anorexic response after the endotoxin exposure.

Wild-type and IL10-null mice have differential colonic epithelial gene expression responses to dietary supplementation with synbiotic Bifidobacterium animalis subspecies lactis and inulin.

Prebiotic plus probiotic (synbiotic) supplementations promote fermentation and have shown anti-inflammatory activity in colonic epithelium. However, in many instances, patients with inflammatory bowel disease (IBD) have demonstrated adverse effects after prebiotic supplementation at a dose well tolerated by normal individuals. To test the hypothesis that the host inflammation affects the colonic epithelial response to increased fermentation, the gene expression of colonic epithelium was analyzed. In a 1-way experimental design to test the effect of supplements in wild-type mice using the standard diet formulated by the American Institute of Nutrition (AIN-93G) as the control diet, fermentable fiber inulin (5%) in the absence or presence of the probiotic Bifidobacterium animalis subspecies lactis (Bb12) (10(8) CFU/kg diet) showed limited effects on gene expression as determined by whole-genome microarray. Bb12 supplementation alone was known not to increase fermentation and here instead significantly upregulated genes in nucleic acid metabolic processes. The effects of the synbiotic diet were then determined in mice exposed to LPS-induced inflammation in a 2-way experimental design testing the effect of diet and LPS. The microarray and quantitative reverse transcription-polymerase chain reaction analyses on the wild-type mice revealed that LPS-induced changes in the colonic epithelium were 4- to 10-fold less in the synbiotic diet group compared with the control diet group. Unlike the wild-type mice, anti-inflammatory cytokine interleukin 10 (IL10)-null mice (susceptible to IBD) given the synbiotic diet, compared with those given the control diet, had 3- to 40-fold increased expression of inflammation-related genes such as Cxcl1 (chemokine C-X-C motif ligand 1) and S100a9 (S100 calcium binding protein A9) in the absence and presence of LPS exposure. These contrasting intestinal epithelial responses to increased fermentation in wild-type and IL10-null mice are similar to the difference between healthy human individuals and those with IBD, suggesting that the host disease/genetic background should be considered before prebiotic/probiotic supplementation.

The N-terminal basolateral targeting signal unlikely acts alone in the differential trafficking of membrane transporters in MDCK cells.

We have shown previously, using confocal imaging and transport assays, that the N-terminus of sodium-dependent vitamin C transporter 2 (SVCT2) can redirect apical SVCT1 to the basolateral membrane. Here, the SVCT model was used to further characterize the basolateral targeting peptide signal. Both the length (31 amino acids) and sequence accuracy of the N-terminus of SVCT2 were found to be important in basolateral targeting activity, suggesting a structural requirement. However, the N-terminal basolateral targeting sequence did not appear to act alone, based on analyses of heterologous chimeras. Although diverse N-terminal basolateral targeting signals from multipass membrane proteins can all redirect apical protein from the same gene family to the basolateral membrane, none of the N-terminal basolateral targeting signals can redirect the transmembrane and C-terminal regions from a different gene family. Instead, the presence of these heterologous N-terminal basolateral targeting signals affected the trafficking of otherwise apical protein, causing their accumulation in a stable tubulin-like non-actin structure. Nontargeting N-terminal sequences had no effect. Similar protein retention was observed previously and in this study when the C-terminus of apical or basolateral protein was mutated. These results suggest that the N- and C-termini interact, directly or indirectly, within each gene family for basolateral targeting. Circular dichroism and two-dimensional nuclear magnetic resonance analyses both found a lack of regular secondary structure in the conserved N-terminus of SVCT2, consistent with the presence of partner(s) in the targeting unit. Our finding, a departure from the prevailing single-peptide motif model, is consistent with the evolution of basolateral transporters from the corresponding apical genes. The interaction among the N-terminus, its partner(s), and the cellular basolateral targeting machinery needs to be further elucidated.

The effect of dietary prebiotics and probiotics on body weight, large intestine indices, and fecal bile acid profile in wild type and IL10-/- mice.

Previous studies have suggested roles of probiotics and prebiotics on body weight management and intestinal function. Here, the effects of a dietary prebiotic, inulin (50 mg/g diet), and probiotic, Bfidobacterium animalis subsp. lactis (Bb12) (final dose verified at 10(5) colony forming unit (cfu)/g diet, comparable to human consumption), were determined separately and in combination in mice using cellulose-based AIN-93G diets under conditions allowed for the growth of commensal bacteria. Continuous consumption of Bb12 and/or inulin did not affect food intake or body, liver, and spleen weights of young and adult mice. Fecal bile acid profiles were determined by nanoESI-MS/MS tandem mass spectrometry. In the presence of inulin, more bacterial deconjugation of taurine from primary bile acids was observed along with an increased cecal weight. Consumption of inulin in the absence or presence of Bb12 also increased the villus cell height in the proximal colon along with a trend of higher bile acid sulfation by intestinal cells. Feeding Bb12 alone at the physiological dose did not affect bile acid deconjugation and had little effect on other intestinal indices. Although interleukin (IL)10-null mice are susceptible to enterocolitis, they maintained the same body weight as the wild type mice under our specific pathogen-free housing condition and showed no signs of inflammation. Nevertheless, they had smaller cecum suggesting a mildly compromised intestinal development even before the disease manifestation. Our results are consistent with the notion that dietary factors such as prebiotics play important roles in the growth of intestinal microbiota and may impact on the intestinal health. In addition, fecal bile acid profiling could potentially be a non-invasive tool in monitoring the intestinal environment.

The interplay between fiber and the intestinal microbiome in the inflammatory response.

Fiber intake is critical for optimal health. This review covers the anti-inflammatory roles of fibers using results from human epidemiological observations, clinical trials, and animal studies. Fiber has body weight-related anti-inflammatory activity. With its lower energy density, a diet high in fiber has been linked to lower body weight, alleviating obesity-induced chronic inflammation evidenced by reduced amounts of inflammatory markers in human and animal studies. Body weight-unrelated anti-inflammatory activity of fiber has also been extensively studied in animal models in which the type and amount of fiber intake can be closely monitored. Fermentable fructose-, glucose-, and galactose-based fibers as well as mixed fibers have shown systemic and local intestinal anti-inflammatory activities when plasma inflammatory markers and tissue inflammation were examined. Similar anti-inflammatory activities have also been demonstrated in some human studies that controlled total fiber intake. The anti-inflammatory activities of synbiotics (probiotics plus fiber) were reviewed as well, but there was no convincing evidence indicating higher efficacy of synbiotics compared with that of fiber alone. Adverse effects have not been observed with the amount of fiber intake or supplementation used in studies, although patients with Crohn's disease may be more sensitive to inulin intake. Several possible mechanisms that may mediate the body weight-unrelated anti-inflammatory activity of fibers are discussed based on the in vitro and in vivo evidence. Fermentable fibers are known to affect the intestinal microbiome. The immunomodulatory role of the intestinal microbiome and/or microbial metabolites could contribute to the systemic and local anti-inflammatory activities of fibers.

Loss of IL-10 decreases mouse postpubertal mammary gland development in the absence of inflammation.

IL-10 is a pleiotrophic anti-inflammatory cytokine. Decreased IL-10 expression is associated with an increased breast cancer risk but the mechanism is not clear. This study was designed to test the hypothesis that the loss of IL-10 alters mammary development, even in the absence of inflammation. Wild-type and IL-10-/- mouse littermates were similar in growth, development, and breeding success. Using whole-mounts and paraffin sections, mammary glands from pre-pubertal mice (d21) were found to not be affected by the IL-10 null genotype. However, after the onset of estrous cycling, ductal structure, but not lymph nodes or adipocytes, of IL-10 knockout mice were found to moderately decrease at day 55, 80, and 150 of age. This phenotype was not rescued by lactogenesis. At day 2 of lactation, IL-10 null mice had reduced lobular complexity and glandular area with the retention of adipocytes. These results support the hypothesis that absence of IL-10 reduces glandular development during postnatal development, at maturity, and during the early stages of lactation. Although our study cannot distinguish between a direct IL-10 effect on the epithelial cells and an indirect systemic effect, epithelial cell responses to IL-10 should be considered in the therapeutic applications of cytokines or cytokine ablation.

Cellular phenotype-dependent and -independent effects of vitamin C on the renewal and gene expression of mouse embryonic fibroblasts.

Vitamin C has been shown to delay the cellular senescence and was considered a candidate for chemoprevention and cancer therapy. To understand the reported contrasting roles of vitamin C: growth-promoting in the primary cells and growth-inhibiting in cancer cells, primary mouse embryonic fibroblasts (MEF) and their isogenic spontaneously immortalized fibroblasts with unlimited cell division potential were used as the model pair. We used microarray gene expression profiling to show that the immortalized MEF possess human cancer gene expression fingerprints including a pattern of up-regulation of inflammatory response-related genes. Using the MEF model, we found that a physiological treatment level of vitamin C (10(-5) M), but not other unrelated antioxidants, enhanced cell growth. The growth-promoting effect was associated with a pattern of enhanced expression of cell cycle- and cell division-related genes in both primary and immortalized cells. In the immortalized MEF, physiological treatment levels of vitamin C also enhanced the expression of immortalization-associated genes including a down-regulation of genes in the extracellular matrix functional category. In contrast, confocal immunofluorescence imaging of the primary MEF suggested an increase in collagen IV protein upon vitamin C treatment. Similar to the cancer cells, the growth-inhibitory effect of the redox-active form of vitamin C was preferentially observed in immortalized MEF. All effects of vitamin C required its intracellular presence since the transporter-deficient SVCT2-/- MEF did not respond to vitamin C. SVCT2-/- MEF divided and became immortalized readily indicating little dependence on vitamin C for the cell division. Immortalized SVCT2-/- MEF required higher concentration of vitamin C for the growth inhibition compared to the immortalized wildtype MEF suggesting an intracellular vitamin C toxicity. The relevance of our observation in aging and human cancer prevention was discussed.

Hierarchal contribution of N- and C-terminal sequences to the differential localization of homologous sodium-dependent vitamin C transporters, SVCT1 and SVCT2, in epithelial cells.

Human sodium-dependent vitamin C transporters, SVCT1 and SVCT2, share 66% sequence identity yet localize in the apical and basolateral membranes of epithelial cells, respectively. This pair thus serves as a model for studying multipass membrane protein targeting. Domain swaps, deletions, insertions, and point mutations were performed on EGFP-tagged hSVCT1 and hSVCT2 plasmids. Mutant proteins stably expressed in MDCK cells were analyzed by confocal microscopy and Transwell ascorbate transport assays. These studies identified an SVCT2 basolateral targeting sequence (BTS) in the N-terminus, which is conserved among mammalian SVCT2 forms. The less conserved N-terminus of SVCT1 is not required for apical localization. The destruction of SVCT2 BTS led to apical localization of the protein in a manner independent of the C-terminal sequence. A C-terminal sequence present in both SVCTs appears to be required for plasma membrane incorporation and retention as its deletion led to an increased level of intracellular appearance of both apically and basolaterally targeted SVCTs in the absence or presence of BTS. Nevertheless, all C-terminal deletion mutants showed preferential apical transport activity, suggesting a greater importance of this sequence for basolateral targeting. Our results collectively suggested a default apical targeting of SVCT, which is consistent with the evolution-based prediction. The SVCT sorting model with a hierarchal contribution of N- and C-terminal sequences was compared to the observations made for other multipass membrane proteins. The involvement of both intracellularly localized termini of multipass membrane proteins in the sorting pathway suggests a more complex sorting mechanism compared to that for single-pass proteins.

Functional role of conserved transmembrane segment 1 residues in human sodium-dependent vitamin C transporters.

Sodium-dependent vitamin C transporters, SVCT1 and SVCT2, are the only two known proteins for the uptake of ascorbate, the active form of vitamin C. Little structural information is available for SVCTs, although a transport activity increase from pH 5.5 to 7.5 suggests a functional role of one or more conserved histidines (p K a approximately 6.5). Confocal fluorescence microscopy and uptake kinetic analyses were used here to characterize cells transfected with mutants of EGFP-tagged hSVCTs. Mutating any of the four conserved histidine residues (His51, 147, 210, or 354) in hSVCT1 to alanine did not affect the apical membrane localization in polarized MDCK cells. His51Ala (in putative transmembrane segment 1, TM1) was the only mutation that resulted in a significant loss of ascorbate transport and an increase in apparent Km with no significant effect on Vmax. The corresponding mutation in hSVCT2, His109Ala, also led to a loss of transport activity. Among eight other mutations of His51 in hSVCT1, significant sodium-dependent ascorbate transport activity was only observed with asparagine or tyrosine replacement. Thus, our results suggest that uncharged His51, directly or indirectly, contributes to substrate binding through the hydrogen bond. His51 cannot account for the observed pH dependence as neutral amino acid substitutions failed to abolish the pH-dependent activity increase. The importance of TM1 is further strengthened by the comparable loss of sodium-dependent ascorbate transport activity upon the mutation of adjacent conserved Gln50 and the apparent change in substrate specificity in the hSVCT1-His51Gln mutation, which showed a specific increase in sodium-independent dehydroascorbate transport.

Physiological levels of tea catechins increase cellular lipid antioxidant activity of vitamin C and vitamin E in human intestinal caco-2 cells.

Oxidative stress has been linked to the development of various chronic diseases. Vegetables and fruits, which contain polyphenols, were shown to have protective effects. (-)-Epigallocatechin-3-gallate (EGCG), a polyphenol abundant in tea, has been shown to have antioxidant activities in cell-free conditions and this study focused on the effect of cellular EGCG. Using an intestinal cell model to examine the oxidative stress induced by hydroxyl radicals, we report here that physiological concentrations (0.1-1 microM) of EGCG have dose- and incubation duration-dependent cell-associated lipid antioxidant activity (measuring malondialdehyde production). Vitamin E and vitamin C at 10-40 microM also showed cell-associated lipid antioxidant activities under shorter incubation durations. When EGCG was included in the incubation with vitamin E or C, more antioxidant activities were consistently observed than when vitamins were added alone. Catechin (widely present in fruits and vegetables) at 1 microM also significantly increased the antioxidant activity of vitamins E and C. Previous studies examining cell-associated activity of EGCG mainly focused on the 10-100 microM concentration range. Our results suggest that although the physiological level (0.1-1 microM) of dietary catechins is much lower than that of vitamins, they further contribute to the total antioxidant capacity even in the presence of vitamins.

Endotoxin increases ascorbate recycling and concentration in mouse liver.

Sublethal exposure to Escherichia coli endotoxin [lipopolysaccharide (LPS)] attenuates the lethal effects of subsequent insults associated with oxidative stress, such as higher LPS dose, septic peritonitis, and ischemia. Because administration of the antioxidant ascorbate protects against these same insults and injection of dehydroascorbic acid (DHAA) protects against ischemia, the hypothesis that sublethal LPS increases endogenous ascorbate concentration and recycling (i.e., synthesis from DHAA) was tested. Injection of LPS [5 x 10(6) endotoxin units/kg body weight, i.p.] in mice caused a temporary inhibition of food intake, which was significant by 20 h and recovered within 3 d. LPS increased ascorbate concentration in adrenal gland, heart, kidney, and liver. LPS had similar effects in wild-type and Slc23a2+/- mice despite the latter's deficiency in the ascorbate transporter SVCT2. In liver, the ascorbate response to LPS was not accompanied by change in glutathione concentration. LPS decreased gulonolactone oxidase activity, which is rate-limiting for de novo synthesis of ascorbate from glucose, but increased the rate of DHAA reduction to ascorbate. In conclusion, sublethal endotoxin increases ascorbate recycling in liver and ascorbate concentration in liver, adrenal gland, heart, and kidney. The enhanced rate of ascorbate production from DHAA may protect these organs against the reactive oxygen species produced by subsequent, potentially lethal challenges.

Polarized localization of vitamin C transporters, SVCT1 and SVCT2, in epithelial cells.

Messenger RNA of homologous sodium-vitamin C cotransporters, SVCT1 and SVCT2, were found in the intestine. Studies using cultured intestinal cells suggested an apical presence of SVCT1 but the function of SVCT2 was unknown. Here, we showed that enterocytes from heterozygous SVCT2-knockout mice had lower sodium-dependent vitamin C accumulation compared to those from the wildtype. Thus, SVCT2 appears to be functional in enterocytes. We then tested whether SVCT2 could have a redundant function as SVCT1 by constructing and expressing EGFP-tagged SVCTs in intestinal Caco-2 and kidney MDCK cells. In confluent epithelial cells, SVCT1 protein expressed predominantly on the apical membrane. SVCT2, in contrast, accumulated at the basolateral surface. Functionally, SVCT1 expression led to more transport activity from the apical membrane, while SVCT2 expression only increased the uptake under the condition when basolateral membrane was exposed. This differential epithelial membrane distribution and function suggests non-redundant functions of these two isoforms.

Dietary isoflavones suppress endotoxin-induced inflammatory reaction in liver and intestine.

Dietary isoflavone intake has been linked to cancer prevention and their anti-inflammation activity was examined. Intraperitoneal lipopolysaccharide (LPS) injection in mice led to a decrease in the liver antioxidant glutathione level but this decrease was prevented in mice fed with an isoflavone-containing diet. Similarly, isoflavone diet prevented the inflammation-associated induction of metallothionein (MT) in the intestine; and the induction of manganese superoxide dismutase (Mn-SOD) in the liver. Results from the intestinal cell studies suggest that isoflavones suppress the intestinal response to inflammation by modulating the action of pro-inflammatory cytokine, IL-6. IL-6 secretion and the STAT3 (signal transducer and activator of transcription protein 3) nuclear translocation in response to IL-6 were both decreased by genistein.

Gender and sodium-ascorbate transporter isoforms determine ascorbate concentrations in mice.

We evaluated the hypothesis that sodium-dependent vitamin C (ascorbate) transporters SVCT1 and SVCT2 (encoded by genes Slc23a1 and Slc23a2) regulate ascorbate concentrations in tissues of adult mice. Slc23a2+/- and Slc23a2+/+ mice were fed an ascorbate-free diet for 10-12 wk, and then segregated according to gender and genome, and were placed in groups of 3-4 in metabolic cages for 24-h urine collection. RT-PCR analysis showed that liver and kidney expressed mainly SVCT1, and brain, skeletal muscle, and spleen expressed predominantly SVCT2. Slc23a2+/- mice had low SVCT2 but normal SVCT1 messenger RNA (mRNA) levels, which did not differ between genders. Ascorbate concentrations were lower in Slc23a2+/- than Slc23a2+/+ mice in tissues where SVCT2 was the main isoform. Compared with males, females had lower ascorbate excretion and ascorbate:creatinine ratio in urine and had higher ascorbate concentrations in plasma and SVCT1-predominant tissues. SVCT2 contributed to a gender effect in spleen because males had higher spleen ascorbate concentration than females in wild-type but not in Slc23a2+/- mice. Hepatic gulonolactone oxidase mRNA and activity levels did not differ with genotype or gender, indicating no differences in ascorbate synthesis. We concluded that SVCT2 is a major determinant of ascorbate accumulation in tissues lacking SVCT1. The SVCT isoforms appear to function independently of one another because SVCT1 expression and ascorbate concentrations in SVCT1-predominant organs were not affected by SVCT2 deficiency. Additionally, lower ascorbate excretion in females may elevate the vitamin's concentrations in plasma and tissues expressing SVCT1 that, unlike SVCT2, is not saturated by plasma ascorbate concentrations.

Cellular vitamin C accumulation in the presence of copper.

Under the cell-free condition, copper is known to oxidize ascorbic acid (the active form of vitamin C) and the event leads to the loss of vitamin C. However, the biological consequence of this interaction was never examined in the presence of cells. We demonstrated in intestinal epithelial cells that dehydroascorbic acid (the oxidized form of ascorbic acid), when generated from ascorbic acid in the presence of copper, can be efficiently transported into the cells and reduced back to ascorbic acid. We also observed in other types of cells the transport and intracellular reduction of dehydroascorbic acid in the presence of copper. In the presence of iron, a metal that also oxidizes ascorbic acid, we observed similar oxidation-related accumulation in intestinal cells. Other metals that do not interact with ascorbic acid had little effect on vitamin C transport. A nonmetal pro-oxidant, hydrogen peroxide, is known to oxidize ascorbic acid and we observed that the oxidation is also accompanied by an increased intracellular accumulation of vitamin C. The efficient coupling between dehydroascorbic acid transport and intracellular reduction could help to preserve the important nutrient when facing oxidative metals in the intestine.

Flavonoid structure affects the inhibition of lipid peroxidation in Caco-2 intestinal cells at physiological concentrations.

The antioxidant activity of flavonoids in cell-free systems has been studied extensively. We compared flavonoids with different structural features on their abilities to protect live Caco-2 intestinal cells from lipid peroxidation due to hydrogen peroxide and Fe(2+) treatment. Flavonoids with o-dihydroxyl or vicinal-trihydroxyl groups, including quercetin, myricetin (flavonol), luteolin (flavone) and (-)-epigallocatechin gallate (EGCG; flavanol), when co-incubated with a mixture of 30 micro mol/L H(2)O(2) and 30 micro mol/L FeSO(4), prevented the formation of malondialdehyde (MDA) at 1 or 10 micro mol/L in at least one of two separate experiments. In experiments in which flavonoids were preincubated with cells but removed before the 30 micro mol/L H(2)O(2) and Fe(2+) treatment, quercetin at 0.1 micro mol/L, EGCG at 1 micro mol/L and luteolin at 10 micro mol/L exerted protective effects in at least one of two experiments. Kaempferol (flavonol) and the isoflavones, genistein and daidzein, did not prevent lipid peroxidation at 0.1-10 micro mol/L in either co- or preincubation experiments. None of the flavonoids tested at 0.1-10 micro mol/L increased H(2)O(2) and Fe(2+)-induced lipid peroxidation after co- or preincubation. In summary, these observations support the importance of plant-based food items such as vegetables, fruits and teas in the diet.