Restoration of thiamine status with white or whole wheat bread in a thiamine-depleted rat model.

Long-term thiamine deficiency has been largely documented, whilst little is known about effects of short-term depletion/repletion periods on thiamine vitamers status. Rats were submitted to short-term depletion (8 days) followed by different durations of repletion (3 or 14 days) with thiamine from bread (whole wheat bread or white bread, whole B and white B respectively) or corresponding controls. Short-term depletion drastically decreased plasma thiamine (-97%) and its urinary excretion (-77%). TDP (thiamine diphosphate) was strongly affected in liver (-67%) but less affected in cerebellum (-38%) or kidneys (-45%). Short-term repletion (3 days) with whole B diet or its control restored TDP at initial values in cerebellum and kidneys. A longer repletion (14 days) was required to restore liver TDP. Comparison of the diet groups indicates that thiamine status in tissues of rat fed whole B or white B diet was comparable to that of rats fed purified thiamine. Plasma thiamine concentration could not be restored at initial values in the bread groups or respective controls. In conclusion, thiamine in whole wheat bread appears effective in preventing marginal deficiencies and plasma thiamine is a less reliable indicator of thiamine status than tissue TDP levels.

Microbial metabolism of caffeic acid and its esters chlorogenic and caftaric acids by human faecal microbiota in vitro.

Caffeic acid and its esters, chlorogenic and caftaric acids, are major dietary polyphenols present in various foods and beverages. Although caffeic acid is easily absorbed in the small intestine, its esterification with quinic acid, as in chlorogenic acid, decreases its gut absorption and increases the quantities reaching the colon and its microbiota. The microbial conversion of caftaric acid, the tartaric acid ester of caffeic acid, has not been studied earlier. In this work we compared the direct action of a human faecal microbiota on the metabolism of caffeic, chlorogenic and caftaric acids in an in vitro fermentation model. All substrates disappeared quickly and none of the free acids (caffeic, quinic or tartaric acids) were detected after 2 hours of incubation. Two major microbial metabolites were identified by HPLC-ESI-MS-MS as 3-hydroxyphenylpropionic (3-HPP) and benzoic acids (BA). Maximal levels of 3-HPP were reached after 2 h of fermentation and accounted for 9-24% of the dose of caffeic acid and its esters. BA was formed steadily throughout the incubation, accounting for 4-5% of the initial dose of the substrates after 24 h of incubation. The similarities in the metabolic patterns observed for caffeic, chlorogenic and caftaric acids suggest that esterification does not influence the metabolism of caffeic acid by the gut microbiota.

Flavanone metabolism in healthy and tumor-bearing rats.

Flavanones, the main polyphenols of citrus fruits, are thought to contribute to the protective effects of these fruits against cardiovascular diseases and cancer. The metabolism of naringin (naringenin 7-O-neohesperidoside) is studied here in healthy (sham-operated, ShO) and tumor-bearing (TuB) rats. The tumor was induced by implanting Yoshida's sarcoma in hindlimb. Both groups received for 7 days a semi-synthetic diet containing 0.5% naringin in per feeding conditions. Flavanones were analyzed in plasma, liver, kidney and urine by tandem mass spectrometry. Naringenin conjugates (essentially glucuronides) accounted for up to 98% of the total flavanones in plasma. Low amounts of hesperetin (4'-O-methyl naringénine) and isosakuranetin (3'-hydroxy-4'-O-methylnaringenin) were also detected in all biological samples and accounted for 2% of the total flavanones in plasma. They were largely present as aglycones. The in vivo hydroxylation of flavanones is described here for the first time. Total concentrations of naringenin metabolites reached 17.3+/-2.7 microM in plasma 6 hours after the beginning of the meal in healthy rats and only 10.6+/-1.3 microM in TuB rats. The nature of metabolites was similar in both healthy and TuB rats and in plasma, tissues and urine. The lower concentration of flavanones in the TuB rats suggests that disease and more particularly cancer, may affect the bioavailability of flavonoids.

One-month exposure to soy isoflavones did not induce the ability to produce equol in postmenopausal women.

OBJECTIVE: As more and more postmenopausal women are taking soy isoflavone supplementation for relieving menopausal symptoms, we investigated the impact of chronic exposure on their bioavailability, with focus on achievable plasma concentrations and potential stimulation of the capacity to produce equol. SUBJECTS: A total of 12 Caucasian postmenopausal women. INTERVENTION: Volunteers ingested 100 mg isoflavones/day (aglycone equivalents, in cereal bars and yoghurts) for 1 month. Plasma concentrations of metabolites at 2, 4, 6, 8, 10, 12 and 24 h postdose, as well as urinary excretion in fractions over 36 h were compared between days 1 and 30. RESULTS: Similar plasma kinetic curves were obtained at day 1 and day 30 for genistein and daidzein. Maximum plasma concentrations were 1.68+/-0.68 micromol/l on day 1 compared to 2.27+/-0.76 micromol/l on day 30 for daidzein (P=0.056), and 3.88+/-1.50 micromol/l on day 1 compared to 5.30+/-2.38 micromol/l on day 30 for genistein (P=0.091). Urinary excretion of daidzein and genistein did not differ significantly between days 1 and 30. Maximum plasma concentration of equol increased significantly from 0.31+/-0.27 to 0.99+/-0.51 micromol/l for equol-producer volunteers (P=0.046). However, the seven volunteers who were classified as non-equol producers on day 1 did not acquire the ability to produce equol after 1-month exposure. CONCLUSIONS: Chronic exposure to isoflavones in postmenopausal women resulted in plasma concentrations as high as 2.5-5 micromol/l of each isoflavone, but did not induce the ability to produce equol.

Modulation of glucagon effects by changes in extracellular pH and calcium.

We have examined the influence of extracellular pH and calcium concentration on the action of glucagon on isolated rat hepatocytes, perfused liver or plasma membrane preparations. Incubation of rat hepatocytes with 10 nM glucagon at pH 7.4 caused an immediate increase in cAMP concentrations (8-fold), and this rise was almost 50% lower at acidic extracellular pH (6.9). This effect of pH could not be explained by an alteration of the hormone binding to its receptor for glucagon concentrations higher than 1 nM. The effect of acidosis on cAMP production was still present with non-hormonal effectors, such as 10 microM Gpp[NH]p, 30 microM forskolin or 10 mM NaF. This suggests a direct action of acidosis on the regulatory component Ns and/or on the catalytic subunit of adenylate cyclase. Acidic pH also depressed mitochondrial processes responsive to glucagon (NAD(P)H fluorescence, glutamine breakdown). Whatever the experimental model, calcium appeared to be required for maximal stimulation of cAMP production by glucagon. On perfused rat liver, glycogenolysis was depressed in the absence of extracellular calcium in the perfusate. In isolated hepatocytes, the stimulation of phosphorylase alpha activity by glucagon was modulated by extracellular calcium concentrations lower than 0.2 mM. This suggests that, although glucagon action is chiefly cAMP-mediated, its effect on calcium mobilization (affecting various cellular process, including cAMP production itself) should also be taken into account. This work also confirmed the importance of calcium in the stimulation of mitochondrial metabolism of glutamine by glucagon.

Propionate transport in rat liver cells.

Propionate extraction by liver is generally in the range of 95%, which could depend on a transport process across the cell membrane. The study reports conditions in which [14C]propionate uptake can be measured with minimal interferences from metabolism. Propionate uptake by isolated hepatocytes was mediated by two components: a low-affinity component of limited physiological interest and a high-affinity (apparent Km about 0.15 mM) component. This last component displayed a high capacity but was not Na+-dependent nor concentrative. Propionate transport was not markedly affected by acetate, butyrate or other C3 glucogenic compounds; it was inhibited by halogenated monocarboxylates, monochloroacetate and 2-chloropropionate being the most potent. Classical inhibitors of anion transport and of functional-SH groups were ineffective. Propionate uptake was responsive to external pH: stimulated by acidic and depressed by alkaline pH. Hepatic uptake of propionate in vivo was practically quantitative up to 0.8-1.0 mM in afferent plasma, in keeping with the measured capacity of the high-affinity component. It is suggested that propionate uptake is essentially carrier mediated but this process should not be rate limiting for hepatic utilization in physiological conditions.

Influence of glucogenic amino acids on the hepatic metabolism of threonine.

The supplementation of a low-protein diet with L-threonine leads to a marked accumulation of threonine in plasma and liver, whereas increasing dietary protein generally leads to an induction of threonine dehydratase in the liver, hence depressed availability for extrasplanchnic tissues. The aim of the present study was, thus, to further investigate the factors which control the utilization of threonine by the liver. Increasing the dietary supply of threonine led to parallel increases in the afferent and hepatic concentrations and in the rate of utilization by the liver; however, the fractional extraction tended to decrease. It appears that the addition of a mixture of glucogenic amino acids to the diet prevented the accumulation of threonine in plasma induced by exogenous threonine. The glucogenic amino acids increased the fractional hepatic uptake of threonine, and counteracted its accumulation in the liver. These effects reflect the fact that the glucogenic amino acids elicited a potent induction of the threonine dehydratase, whereas threonine alone was uneffective. Our results suggest that, besides the well-established effect of glucogenic conditions, the availability of some glucogenic amino acids is an important factor in the control of threonine catabolism.

Propionate and butyrate metabolism in rat or sheep hepatocytes.

The capacities of isolated hepatocytes to metabolize volatile fatty acids have been compared in rat and sheep hepatocytes. In both species, acetate utilization in vitro was quite limited. Significant species differences for propionate and butyrate consumption were found: propionate utilization by rat hepatocytes was relatively limited and plateaued at about 0.8-1.0 mM, whereas butyrate utilization was approx. 2-times higher. In contrast, ruminant hepatocytes exhibited a lower rate of butyrate utilization, but propionate metabolism was much more active than in rat liver cells. With relatively low concentrations of substrates (max. 2 mM), only propionate, compared to lactate or alanine, had a significant glucogenicity with hepatocytes from fed sheep. In both species, butyrate inhibited propionate consumption, although to a larger extent in sheep. The conversion of [2-14C]propionate to glucose by sheep hepatocytes was inhibited by 2 mM butyrate (60%) or ammonia (30%); 1 mM oleate or 10 mM glucose were ineffective. The basal rate of ammonia utilization by sheep hepatocytes was much lower than in rat and was unaffected upon addition of ornithine. Ammonia metabolism was markedly enhanced by butyrate and, in contrast to rat liver cells, also by propionate.

Fermentable carbohydrates exert a more potent cholesterol-lowering effect than cholestyramine.

The purpose of this work was to assess the respective role of bile acid excretion and of the end-products of cecal fermentations in the cholesterol-lowering effect of complex carbohydrates. The effects of two different fermentable carbohydrates (guar gum, beta-cyclodextrin), and sequestrant resin (cholestyramine) have been investigated in male Wistar rats. Guar gum and beta-cyclodextrin are broken down in the large bowel, with fermentation rich in propionic acid (37% against 26% for control), whereas cholestyramine did not enhance cecal fermentation. beta-Cyclodextrin and guar gum were less potent than cholestyramine to enhance bile acids and sterol excretion. Nevertheless, fermentable carbohydrates exerted a more potent cholesterol-lowering effect than cholestyramine. beta-Cyclodextrin also depressed triacylglycerol-rich lipoprotein (TGRLP). Fermentable carbohydrates lowered cholesterol of LDL and HDL1 fractions. The induction of hepatic HMG-CoA reductase was practically proportional to rate of fecal steroid excretion. Moreover, with beta-cyclodextrin, hepatic HMG-CoA reductase induction was concomitant to a decrease in fatty acid synthase (FAS) activity. Thus, the cholesterol-lowering effect of fermentable carbohydrates could be related to a depressed lipogenesis, as well as to an accelerated removal of HDL1, in relation to an elevated hepatic demand of cholesterol. In conclusion, fermentable carbohydrates could favour cholesterol elimination and have a general lipid-lowering effect by exerting more complex physiological effects than cholestyramine.

Determination of thiamine and its phosphate esters in rat tissues analyzed as thiochromes on a RP-amide C16 column.

A new reversed-phase chromatographic method is described for the separation and quantification of thiamine (T), thiamine monophosphate (TMP) and diphosphate (TDP) in rat tissues. Sample extraction with perchloric acid (HClO(4)) was found more suitable than extraction with trichloroacetic acid (TCA), as regards convenience and background fluorescence. Derivatization of thiamine vitamers to thiochromes was optimized and complete separation of TDP and TMP thiochromes was obtained on a RP-amide C16 column in isocratic elution, with T thiochrome eluting in less than 10 min. The precision and the accuracy of the HPLC procedure were assessed: ranging from 0.5 to 7.7% for intra-day and from 2.0 to 9.4% for inter-day precision, a recovery average of 101% was determined (range 90-111%). Mean values of recovery for TDP, TMP or T were 91, 96 and 90% for liver extracts, respectively. Analysis of vitamers in tissues of rat submitted to 8 days thiamin deficiency, followed by a 14 days repletion, showed a significant reduction of TPP after 8 days of depletion in liver (-67%), brains (-50%), kidneys (-60%), followed by a complete recovery upon repletion.

Rutin inhibits ovariectomy-induced osteopenia in rats.

Several studies suggest that polyphenols might exert a protective effect against osteopenia. The present experiment was conducted to observe the effects of rutin (quercetin-3-O-glucose rhamnose) on bone metabolism in ovariectomized (OVX) rats. Thirty 3-month-old Wistar rats were used. Twenty were OVX while the 10 controls were sham-operated (SH). Among the 20 OVX, for 90 days after surgery 10 were fed the same synthetic diet as the SH or OVX ones, but 0. 25% rutin (OVX + R) was added. At necropsy, the decrease in uterine weight was not different in OVX and OVX + R rats. Ovariectomy also induced a significant decrease in both total and distal metaphyseal femoral mineral density, which was prevented by rutin consumption. Moreover, femoral failure load, which was not different in OVX and SH rats, was even higher in OVX + R rats than in OVX or SH rats. In the same way, on day 90, both urinary deoxypyridinoline (DPD) excretion (a marker for bone resorption) and calciuria were higher in OVX rats than in OVX + R or SH rats. Simultaneously, plasma osteocalcin (OC) concentration (a marker for osteoblastic activity) was higher in OVX + R rats than in SH rats. High-performance liquid chromatography (HPLC) profiles of plasma samples from OVX + R rats revealed that mean plasma concentration of active metabolites (quercetin and isorhamnetin) from rutin was 9.46+/-1 microM, whereas it was undetectable in SH and OVX rats. These results indicate that rutin (and/or its metabolites), which appeared devoid of any uterotrophic activity, inhibits ovariectomy-induced trabecular bone loss in rats, both by slowing down resorption and increasing osteoblastic activity.

Bioavailability of rutin and quercetin in rats.

Quercetin is a powerful antioxidant which is widely distributed in edible plants, mainly as glycosides such as rutin. It has been reported to be absorbed in mammals, but its metabolism needs further investigation to evaluate its possible physiological effects. We compared the evolution of the absorption of quercetin and rutin in rats fed with supplemented diets. Rutin was absorbed more slowly than quercetin because it must be hydrolysed by the cecal microflora, whereas quercetin was absorbed from the small intestine. Conjugated derivatives of quercetin, and its methylated forms isorhamnetin and tamarixetin, were recovered in plasma from rats receiving the two kinds of experimental diets after the first meal, but after 10 days, no traces of tamarixetin were detected anymore. The rate of elimination of quercetin metabolites seems very low, and high plasma concentrations are easily maintained with a regular supply of quercetin or rutin in the diet.

Quercetin is recovered in human plasma as conjugated derivatives which retain antioxidant properties.

Quercetin is one of the most abundant flavonoids in the human diet. This study aimed to determine the plasma concentrations of quercetin in 10 healthy volunteers after the consumption of a complex meal rich in plant products. Quercetin was determined in plasma (2 h before, and 3, 7 and 20 h after the meal), and in a duplicated portion of the meal by HPLC analysis with an electrochemical detection. The amount of ingested quercetin was estimated to be 87 mg. Before the meal, quercetin concentration in hydrolyzed plasmas ranged from 28 to 142 nM. A marked increase was observed 3 h after the meal in all subjects, with a mean concentration of 373 nM (S.E.M. = 61). After 7 h, quercetin concentration in hydrolyzed plasmas decreased and after 20 h basal levels were found again. The antioxidant capacities of quercetin, 3'-O-methylquercetin, and of some of their conjugated derivatives were compared by the measurement of the conjugated dienes resulting from the Cu2+-induced oxidation of human LDL. 3'-O-Methylquercetin and conjugated derivatives of quercetin significantly prolonged the lag phase, but the magnitude of their effect was about half that of the aglycone.

Apolipoprotein B gene expression in rat intestine. The effect of dietary fiber.

The effect of the dietary fiber on apo B mRNA level was studied in the intestine of rats that were fed either fiber-free or high-fiber (30% sugar-beet fiber) low-fat diets for 3 weeks. The fiber diet studied does not affect jejunal apo B mRNA levels but decreases the level of ileal apo B mRNA. In the rat cecum, in both fiber-free and fiber groups, we failed to detect the apo B mRNA. The test fiber diet feeding markedly increased fecal bile salt and cholesterol excretions. We suggest that dietary fiber can modify apo B expression in the intestine. The increased fecal bile salt excretion might be involved in such a modification.

Comparison of the bioavailability of quercetin and catechin in rats.

Quercetin and catechin are present in noticeable amounts in human diet and these polyphenolic compounds are supposed to exert beneficial effects on human health. However, their metabolic fates in the organism have never been compared. In the present study, rats were fed a 0.25% quercetin or a 0.25% catechin diet. Quercetin and catechin metabolites were analyzed in plasma and liver samples by high-performance liquid chromatography coupled to an ultraviolet or a multielectrode coulometric detection. All plasma metabolites were present as conjugated forms, but catechin metabolites were mainly constituted by glucuronidated derivatives, whereas quercetin metabolites were sulfo- and glucurono-sulfo conjugates. Quercetin was more intensively methylated than catechin in plasma. The plasma quercetin metabolites are well maintained during the postabsorptive period (approximately 50 microM), whereas the concentration of catechin metabolites dropped dramatically between 12- and 24-h after an experimental meal (from 38.0 to 4.5 microM). In the liver, the concentrations of quercetin and catechin derivatives were lower than in plasma, and no accumulation was observed when the rats were adapted for 14 d to the supplemented diets. The hepatic metabolites were intensively methylated (90-95%), but in contrast to plasma, some free aglycones could be detected. Thus, it clearly appears that studies dealing with the biological impact of these polyphenols should take into account the feature of their bioavailability, particularly the fact that their circulating metabolites are conjugated derivatives.

Quercetin exerts a preferential cytotoxic effect on active dividing colon carcinoma HT29 and Caco-2 cells.

The effect of the naturally occurring flavonol, quercetin, was investigated on cell growth and metabolism of two human carcinoma cell lines, HT29 and Caco-2 cells, both during the exponentially growing phase and after confluence. Our results show clearly that, after a 48-h period of treatment, quercetin (in the range of concentration from 15 microM to 120 microM) exerted a preferential cytotoxic effect on active proliferating cells. This effect was dose dependent and was accompanied by a simultaneous inhibition of lactate release and a dramatic decrease of total cellular ATP content. In contrast, in confluent cells, quercetin failed to affect cell viability or lactate release, but led nevertheless to a depletion of cellular ATP level. In conclusion, the cytotoxicity of quercetin is markedly higher in actively growing cells in comparison with confluent cells.

Comparative effects of flavonoids on the growth, viability and metabolism of a colonic adenocarcinoma cell line (HT29 cells).

The aim of the present study was to compare the effect of five structural classes of flavonoids on the viability and metabolism of a colonic adenocarcinoma cell line (HT29 cells). The most prominent structural features of flavonoids favoring both their cytotoxic activity and their capacity to inhibit lactate release appear to be the desaturation of the 2, 3 bond and the position of attachment of the B ring. Indeed, flavonol and flavone are the most potent and, in both classes, the order of potency can be modulated by hydroxyl or methoxyl substituents. On the other hand, in our model, we did not find any correlation between flavonoid structure and their capacity to modulate cAMP level. This last point is discussed.

Role of fermentable carbohydrate supplements with a low-protein diet in the course of chronic renal failure: experimental bases.

During the past few years, considerable attention has been given to the impact of nutrition on kidney disease. The question arises of whether the effect of a moderate dietary protein restriction could be reinforced by enrichment of the diet with fermentable carbohydrates. Feeding fermentable carbohydrates may stimulate the extrarenal route of nitrogen (N) excretion through the fecal route. Such an effect has been reported in several species, including healthy humans and patients with chronic renal failure (CRF). Furthermore, studies of these subjects show that the greater fecal N excretion during the fermentable carbohydrate supplementation period was accompanied by a significant decrease in plasma urea concentration. In animal models of experimental renal failure, the consumption of diets containing fermentable carbohydrates results in a greater rate of urea N transfer from blood to the cecal lumen, where it is hydrolyzed by bacterial urease before subsequent microflora metabolism and proliferation. Therefore, this results in a greater fecal N excretion, coupled with a reduction in urinary N excretion and plasma urea concentration. Because elevated concentrations of serum urea N have been associated with adverse clinical symptoms of CRF, these results suggest a possible usefulness of combining fermentable carbohydrates with a low-protein diet to increase N excretion through the fecal route. Further investigations in this population of patients of whether fermentable carbohydrates in the diet may be beneficial in delaying or treating the symptoms and chronic complications of CRF will certainly emerge in the future. This should be realized without adversely affecting nutritional status and, as far as possible, by optimizing protein intake for the patients without being detrimental to renal function.

Relationship between flavonoid structure and inhibition of phosphatidylinositol 3-kinase: a comparison with tyrosine kinase and protein kinase C inhibition.

Depending on their structure, flavonoids display more or less potent inhibitory effects on the growth and proliferation of certain malignant cells in vitro, and these effects are thought to be due to inhibition of various enzymes. We investigated the inhibitory action of fourteen flavonoids of different chemical classes on phosphatidylinositol 3-kinase alpha (PI 3-kinase alpha) activity, an enzyme recently shown to play an important role in signal transduction and cell transformation. Of the fourteen flavonoids tested, myricetin was the most potent PI 3-kinase inhibitor (IC50 = 1.8 microM), while luteolin and apigenin were also effective inhibitors, with IC50 values of 8 and 12 microM, respectively. Fisetin and quercetin, as previously reported, were also found to significantly inhibit PI 3-kinase activity. The same flavonoids were also analyzed for inhibition of epidermal growth factor receptor (EGF-R), intrinsic tyrosine kinase and bovine brain protein kinase C (PKC). At elevated doses, some of these flavonoids were found to also cause significant inhibition of PKC and tyrosine kinase activity of EGF-R. A structure-activity study indicated that the position, number and substitution of the hydroxyl group of the B ring, and saturation of the C2-C3 bond are important factors affecting flavonoid inhibition of PI 3-kinase. They may also play a significant role in specificity of inhibition and could help to provide a basis for the further design of specific inhibitors of this lipid kinase. Finally, possible relationships between the antitumoral properties of these flavonoids and their biological activities are discussed.

Control of lactate utilization by extracellular pH in isolated rat liver cells.

This study reports the influence of external pH on lactate balance in hepatocytes isolated from fed and 24-hour-starved rats. The effects of changes in extracellular pH on the utilization of lactate by liver cells has been studied in conditions simulating metabolic acidosis (pH 7.15, 10 mmol/L bicarbonate). The addition of lactate to a suspension of liver cells from fed rats shifted the lactate balance from net release to net utilization; the threshold of this shift was about 3 mmol/L in the presence of 10 mmol/L glucose. In these cells, acidic external pH (7.15) played a crucial role in stimulating the lactate utilization as shown by (1) a diminished release of lactate in the absence of lactate addition (-60%); (2) a marked decrease of the threshold of lactate utilization down to 1.2 mmol/L; and (3) a net stimulation of the lactate utilization for concentrations in the physiologic range (2 to 3 mmol/L). The effect of acidosis was mediated by an inhibition of glycolysis (-40%). Besides that, at pH 7.45, the addition of 100 mumol/L AICA-riboside 5-amino-4-imidazolecarboxamide riboside, (an inhibitor of hepatic glycolysis) mimicked the effect of acidosis. Moreover, differences in lactate fluxes between the two pH conditions were decreased in the absence of glucose. In liver cells from starved rats, regardless of the concentration of added lactate, the lactate balance was always directed toward net utilization. Accordingly, a change in external pH from 7.45 to 7.15 had a lesser effect on lactate metabolism than in liver cells from fed rats.(ABSTRACT TRUNCATED AT 250 WORDS)