Effect of vitamin E and vitamin C on the DNA synthesis of human umbilical arterial endothelial cells

Summary Background Endothelial cell growth and reendothealization after vascular injury protect the vessel wall against endothelial dysfunction which is believed to play a major role in the pathogenesis of atherosclerosis. Aim of the study To investigate a possible protective role of antioxidant vitamins in the present study, the effect of vitamin E (α-tocopherol) alone and in combination with vitamin C on the DNA synthesis of human umbilical arterial endothelial cells (HUAEC) was examined. Furthermore, because oxidized low-density lipoprotein (ox-LDL) is thought to be involved in atherogenesis, the combined effect of vitamin E and vitamin C with ox-LDL and the influence of vitamin-pretreated LDL on HUAEC proliferation were investigated. Methods DNA-synthesis was determined by measurement of [³H]thymidine incorporation into the cell DNA. Results Vitamin E alone and in combination with vitamin C resulted in an increase in [³H]thymidine incorporation into cell DNA, especially in the presence of basic fibroblast growth factor (bFGF). All vitamin-pretreated LDL samples and ox-LDL led to a nearly complete inhibition of endothelial DNA-synthesis. The ox-LDL-induced effect could not be prevented by vitamin E alone nor in combination with vitamin C. Conclusions It seems that once LDL oxidation is in process, vitamin E alone and in combination with vitamin C is ineffective to exert its antioxidative capacity under the conditions used. Thus, vitamin E alone and combined with vitamin C may act as antiatherogens by inducing endothelial cell growth. Received: 3 January 2001, Accepted: 17 July 2001     

Dual isotope test for assessing beta-carotene cleavage to vitamin A in humans

Summary. Background: The ability of β-carotene to deliver bioactive retinoids to tissues is highly variable. A clearer understanding of the environmental and genetic factors that modulate the vitamin A potential of β-carotene is needed. Aim of study: Assess the vitamin A value of orally administered β-carotene relative to a co-administered reference dose of preformed vitamin A. Methods: Equimolar doses (30 μmol) of hexadeuterated D₆β-carotene and D₆ retinyl acetate were orally co-administered in an emulsified formulation to a male subject. The plasma concentration time courses of D₆ retinol (derived from D₆ retinyl acetate) and bioderived D₃ retinol (from D₆β-carotene) were determined for 554 h postdosing using gas chromatography/mass spectrometry. Intact D₆β-carotene plasma concentrations were determined by high-pressure liquid chromatography. The ratio of the two forms of vitamin A, D₆ retinol/D₃ retinol, at any single time point is postulated to reflect the quantity of vitamin A derived from β-carotene relative to preformed vitamin A. Additionally, a minute amount of ¹⁴C β-carotene (50 nCi; 0.27 μg) was included in the oral dose and cumulative 24-h stool and urine samples were collected for two weeks to follow absorption and excretion of the b-carotene. The ¹⁴C nuclide was detected using accelerator mass spectrometry (AMS). Results During the absorption/distribution phase (3–11 h) the D₆/D₃ ratio of the two retinols was not stable and ranged between a value of 3 and 16. Between 11 and 98 h postdosing the ratio was relatively stable with a mean value of 8.5 (95 % CI: 7.5, 8.7). These data suggest that in this subject and under these conditions, 8.5 moles of β-carotene would provide a vitamin A quantity equivalent to 1 mole of preformed vitamin A. On a mass basis, 15.9 μg of β-carotene was equivalent to 1 μg of retinol. The total administered β-carotene was found to be 55 % absorbed by AMS analysis of cumulative stool. Conclusion: The co-administration of D₆β-carotene and D₆ retinyl acetate provides a technique for assessing individual ability to process β-carotene to vitamin A. The results indicate that a single time point taken between 11–98 h after dose administration may provide a reliable value for the relative ratio of the two forms of vitamin A. However, results from more subjects are needed to assess the general utility of this method. Received: 29 November 2001, Accepted: 3 June 2002     

Effects of insulin and amino acids on leucine metabolism in young and middle-aged humans

Summary Background Aging is characterized by loss of muscle mass. In healthy subjects this process is associated with hormone and nutritional changes which take place over many decades. Aim of the study To investigate the effects of insulin and amino acids on amino acid metabolism in middle-aged humans. Methods We evaluated leucine kinetics by means of the intravenous infusion of [1-¹⁴C]leucine, in 8 young (age 24±2 yr, BMI 21±2 kg/m²) and in 6 middle-aged (age 53±4 yr, BMI 26±1 kg/m²) healthy subjects. Studies were performed under fasting conditions (basal), and after 180 min of euglycemic hyperinsulinemic clamp (study I), or 180 min of euglycemic hyperinsulinemia in combination with an intravenous amino acid infusion (study II). Results In the basal state endogenous leucine flux (ELF, an index of proteolysis), normalized for IBW, averaged 1.71±0.12 and 1.66±0.14 μmol/kg·min in young and middle-aged subjects, respectively. Basal leucine oxidation (0.22±0.03 vs 0.28±0.03 μmol/kg·min, p < 0.05) was lower in middle-aged with respect to young subjects. Non-oxidative leucine disposal (NOLD, an index of protein synthesis: 1.44±0.11 vs 1.43±0.11 μmol/kg·min) was similar in young and middle-aged subjects, respectively. In response to insulin (study I) the absolute and percent decline of ELF and LOX were similar in young and middle-aged subjects: ELF declined to 1.05±0.06 μmol/kg·<,min (−M39±5 %) and 1.07±0.14 μmol/kg·min (−36±4 %), in young and middle-aged, respectively (both p < 0.01 vs basal); LOX declined to 0.21±0.02 μmol/kg·min (−35±3 %), and 0.18±0.05 μmol/kg·min (−28±3 %, p < 0.05 vs basal) in young and middle-aged individuals respectively (both p < 0.01 vs basal). In contrast, insulin-mediated whole-body glucose uptake was lower in middle-aged subjects (6.6±1.4 mg/kg·min) with respect to young individuals (8.1±1.7 mg/kg·min, p < 0.05). During study II (insulin plus AA) a significant rise in NOLD was obtained in both young (1.72±0.10 μmol/kg·min, p < 0.01 vs basal) and middle-aged subjects (1.76±0.25 μmol/kg·min, p < 0.01 vs basal). Similarly, net leucine balance rose significantly in both young (+0.62±0.13 vs −0.25±0.02 μmol/kg·min, p < 0.01 vs basal) and middle-aged subjects (+0.37±0.08 vs −0.22±0.03 μmol/kg·min, p < 0.01 vs basal) suggesting that the anabolic response to amino acids is preserved in middle-aged subjects. Conclusions In middle-aged subjects we observed 1) a moderate decline in basal leucine oxidation; 2) a normal antiproteolytic response to insulin and a reduction in glucose uptake; and 3) a normal anabolic response to AA plus insulin. In conclusion, the data provide evidence for a normal regulation of protein anabolism and an early dissociation between the metabolic effects of insulin on glucose uptake and proteolysis in middle-aged subjects. Received: 6 September 2000, Accepted: 10 July 2001     

The status of plasma homocysteine and related B-vitamins in healthy young vegetarians and nonvegetarians

Summary. Background: Exclusion of animal products and having only plant protein in vegetarian diets may affect the status of certain B-vitamins, and further cause the elevation of plasma homocysteine concentration. Aim: The purpose of this study was to assess the status of homocysteine and related B-vitamins in vegetarians and nonvegetarians. The effects of biochemical parameters of B-vitamins and dietary protein on plasma homocysteine were also examined. Methods: The study was performed at the Chung Shan Medical University, Taichung, in the central part of Taiwan. Thirty-seven vegetarians (28.9 ± 5.5 y) and 32 nonvegetarians (22.9 ± 1.6 y) were recruited. Nutrient intake was recorded using 3-day dietary records. Fasting venous blood samples were obtained. Plasma homocysteine, folate and vitamin B-12 were measured. Vitamin B-6 status was assessed by direct measures [plasma pyridoxal 5'-phosphate (PLP) and urinary 4-pyridoxic acid (4-PA)] and indirect measures [erythrocyte alanine (EALT-AC) and aspartate (EAST-AC) aminotransaminase activity coefficient]. Results: There was no significant difference in vitamin B-6 intake between the two groups, although the vegetarian group had a significantly lower vitamin B-12 intake than the nonvegetarian group. Vegetarian subjects had significantly lower mean plasma PLP and vitamin B-12 concentrations than did nonvegetarian subjects (p < 0.05); however, a significantly higher mean plasma folate concentration was found in the vegetarian group. Vegetarian subjects had a significantly higher mean plasma homocysteine concentration than nonvegetarian subjects (13.2 ± 7.9 vs. 9.8 ± 2.2 μmol/L). Negative correlations were seen between plasma homocysteine and vitamin B-12 concentrations in the vegetarian (p = 0.004), nonvegetarian (p = 0.026), and pooled (p < 0.001) groups. From best subsets regression analyses, the plasma homocysteine concentration could be significantly predicted by total protein intake (p = 0.027) and plasma vitamin B-12 concentration (p = 0.005) in the pooled group. When the intake of protein is not considered, vitamin B-12 concentration is still a strong predictor of plasma homocysteine concentration (p = 0.012). Conclusions: Vitamin B-12 intake and mean plasma vitamin B-12 concentration were lower for vegetarian subjects than for nonvegetarian subjects, leading to an increase in plasma homocysteine concentration. Vitamin B-6 and folate had little effect on plasma homocysteine concentration when individuals had adequate vitamin B-6 and folate status. Received: 15 July 2002, Accepted: 24 October 2002 The study was supported by National Science Council (NSC 89–2320-B-040–046), Taiwan. Correspondence to: Y. C. Huang     

Faecal elastase-1 and fat-soluble vitamin profiles in patients with cystic fibrosis in Western Norway

Summary. Background: Exocrine pancreatic insufficiency is a major clinical manifestation of cystic fibrosis (CF). Almost nine of ten patients develop signs and symptoms of maldigestion and malabsorption, which often deteriorates nutritional status and therefore worsens the prognosis. Human faecal elastase-1 (FE-1) has shown promising results to assess exocrine pancreatic insufficiency, and this test has been used at Haukeland University Hospital since 1996. Aim of the study: To evaluate FE-1 values and fat-soluble vitamin profiles in patients with CF and to correlate exocrine pancreatic function as measured as FE-1 to fat-soluble vitamin profiles. Moreover, we wanted to assess if there are differences between fat-soluble vitamin profiles in patients with impaired versus patent exocrine pancreatic function, and thirdly, if fat-soluble vitamin deficiency at diagnosis is effectively treated by supplementation. Methods: Consecutive analyses (N = 212) of fat-soluble vitamin profiles and 35 analyses of FE-1 were investigated in 35 patients with CF. In 17 out of 35 patients fat-soluble vitamin profiles were also assessed at diagnosis. Results Mean value of FE-1 for all CF patients was 256.9 μg/g faeces (median 24.1 μg/g faeces). CF patients considered to have maldigestion (N=24) showed a mean value of 19.9 μg/g faeces (median 18.7 μg/g faeces), those without pancreas affection had a mean value of 773.9 μg/g faeces (median 728.9 μg/g faeces, p < 0.01). There was no difference in fat-soluble vitamin profiles among patients with or without exocrine pancreatic insufficiency while on appropriate supplementation. Median value for vitamin E in patients with exocrine pancreatic insufficiency at diagnosis was low (3.6 mg/L). Supplementation of pancreatic enzymes and vitamins normalised profiles in this group at follow-up. There was no significant correlation between exocrine pancreatic function as measured as FE-1 and fat-soluble vitamin profiles, neither in patients with impaired nor in those with patent pancreatic function. Conclusions: Severe degree of exocrine pancreatic insufficiency is common in patients with cystic fibrosis. There was no correlation of faecal elastase-1 levels to fat-soluble vitamin status. Fat-soluble vitamins (A, D, E) given in appropriate dosages combined with pancreatic enzymes ensured normal profiles in our patients with CF and malabsorption. Officially recommended supplementation of vitamin A and D in Norway during infancy and childhood may explain why so few patients had vitamin deficiencies at diagnosis. Received: 20 March 2002, Accepted: 11 June 2002     

Non-esterified plant sterols solubilized in low fat milks inhibit cholesterol absorption--a stable isotope double-blind crossover study

Summary. Background: The cholesterol absorption inhibiting properties of plant sterols in milks are unknown. The milk fat globule membrane components may enhance the absorption of cholesterol and could make plant sterols less efficient in this complex matrix. Aim of the study: To evaluate in hypercholesterolemic men the cholesterol absorption inhibiting properties of verified properly solubilized, non-esterified plant sterols in partly vegetable oil containing milks. Methods: The plant sterols in milk were determined to be properly solubilized, and to have effective in vitro functionality. Sixteen hypercholesterolemic adult men (initial total cholesterol 5.8–8.6 mM) then consumed milk containing sterols (1.8 g of non-esterified pure plant sterols/d) and control milk, alternatively, during two 6-day periods in a double blind cross over design. During the trial, cholesterol absorption was evaluated from the ratio of plasma isotopic enrichment of [26, 26, 26, 27, 27, 27–²H₆]cholesterol from oral intake (35.6 ± 0.2 μmol, ± SEM) over enrichment of [23, 24, 25, 26, 27–¹³C₅]cholesterol from intravenous injection (77.9 ± 0.5 μmol). Results: Plant sterols in low fat milks contained very few crystals > 11 μm in the presence and absence of bile salts and lysophospholipids, and inhibited cholesterol uptake in Caco-2 cell. This assured that the sterols were properly solubilized prior to the clinical trial. In the clinical study, compliance of volunteers was excellent. After tracer injections (72 h), the plasma [²H] and [¹³C] isotopic enrichments changed from 0.024 ± 0.001 and 0.072 ± 0.003 MPE (control) to 0.015 ± 0.001 and 0.074 ± 0.002 MPE during sterol treatment, respectively. Cholesterol absorption was reduced from 70.1 ± 4.2 % with control to 41.1 ± 4.0 % with milks containing plant sterol (P < 0.001). Conclusions: These results demonstrate that properly solubilized non-esterified plant sterols in milks significantly inhibit cholesterol absorption in mildly hypercholesterolemic men. Received: 10 October 2002, Accepted: 8 January 2003 Correspondence to: Etienne B. Pouteau     

Vitamin A equivalence of β-carotene in a Woman as determined by a stable isotope reference method

Summary Background: Quantitative information on conversion of β-carotene to vitamin A in humans is limited. Aim of the study: Our laboratory has developed a stable isotope method for studying the conversion of β-carotene (β-C) to vitamin A. Methods: Two dosage levels (a pharmacological dose, 126.0 mg β-C-d ₈, and a physiological dose, 6.0 mg β-D-d ₈) were used 2.5 y apart in an adult female volunteer to study dose effects on the conversation of β-C to vitamin A. Blood samples were collected over 21 d. β-C and retinol were extracted from serum and isolated by high performance liquid chromatography. The retinol fraction was derivatized to a trimethylsilyl ether which was analyzed by gas chromatograph/mass spectrometry with electron capture negative chemical ionization. Results: The retinol-d ₄ response in the circulation peaked at 24 hours after the β-C-d ₈ dose, with a higher percent enrichment after the pharmacological dose than after the physiological dose. By using retinyl acetate-d ₈ as the vitamin A reference, the retinol-d ₄ formed from 6 mg of β-C-d ₈ (11.2 μmol) was calculated to be equivalent to 1.6 mg of retinol (i. e., 3.8 mg of β-C was equivalent to 1 mg of retinol). However, the retinol-d ₄ formed from 126 mg of β-C-d ₈ (235 μmol) was equivalent to 2.3 mg of retinol (i. e., 55 mg β-C was equivalent to 1 mg retinol). Conclusion: These results provide evidence that it is feasibile to use stable isotope reference method to study retinol equivalence of β-C and that there may be a dose-dependence on bioconversion of β-carotene to retinol. Received: 30 April 1999, Accepted: 7 February 2000     

Tocopherol metabolites 2, 5, 7, 8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (α-CEHC) and 2, 7, 8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman (γ-CEHC) in human serum after a single dose of natural vitamin E

Summary Backgroundα- and γ-Tocopherol are vitamin E compounds in human blood and tissues. α-CEHC (2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman) and γ-CEHC (2,7,8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman) have been identified as water-soluble metabolites which are excreted with the urine in humans. Aim of the study To assess over-time changes of serum levels of α- and γ-CEHC in humans after a single dose of vitamin E from a natural source. Methods Twenty-one healthy subjects ingested a single dose of vitamin E (306 mg of RRR-α-tocopherol and 1.77 mg of γ-tocopherol). Blood was collected before (baseline) and 2, 6, 12, 24, 35, 50, and 74 h after ingestion. Serum was separated and levels of α- and γ-tocopherol and α- and γ-CEHC were determined by HPLC. Results After vitamin E ingestion, a statistically significant increase was observed for α-tocopherol and α-CEHC. Maximum serum levels for both compounds were measured 12 h after application (33.3 ± 11.1 μmol α-toco-pherol /L and 42.4 ± 18.3 nmol α-CEHC /L); baseline values were reached again after 72 h. While γ-tocopherol levels decreased during the study period, an increase in the metabolite γ-CEHC was observed. The optical isomer formed in the metabolism of RRR-α-tocopherol was assigned as S-α-CEHC. Conclusionsα-CEHC levels increase after administration of a single dose of natural vitamin E in humans. The appearance of the metabolite in blood parallels that of the parent compound. The γ-tocopherol analog appears to be metabolized more efficiently than α-tocopherol. Received: 14 November 2001, Accepted: 5 April 2002     

Spinach and tomato consumption increases lymphocyte DNA resistance to oxidative stress but this is not related to cell carotenoid concentrations

Summary Background The increased consumption of fruit and vegetables has been linked to protection against different chronic diseases, but the dietary constituents responsible for this association have not been clearly identified. Aim of the study We evaluated the effect of spinach and spinach+tomato puree consumption on cell DNA resistance to an oxidative stress. Methods To this aim, in a dietary controlled intervention study, 9 healthy female volunteers consumed a basal diet low in carotenoids (< 600 μg/day) enriched with daily portions (150 g) of spinach (providing about 9 mg lutein, 0.6 mg zeaxanthin, 4 mg β-carotene) for 3 weeks (from day 0 to day 21) followed by a 2 week wash-out period (basal diet) and finally another 3 weeks (from day 35 to day 56) of diet enriched with daily portions of spinach (150 g) + tomato puree (25 g, providing about 7 mg lycopene, 0.3 mg β-carotene). At the beginning and the end of each period of vegetable intake, blood samples were collected for lymphocyte separation. Carotenoid concentrations of lymphocytes were determined by HPLC and DNA damage was evaluated by the comet assay following an ex vivo treatment with H₂O₂. Results During the first period of spinach consumption, lymphocyte lutein concentration did not increase significantly (from 1.6 to 2.2 μmol/10¹² cells) while lycopene and β-carotene concentrations decreased significantly (from 1.0 to 0.1 μmol/10¹² cells, P < 0.001, and from 2.2 to 1.2 μmol/10¹² cells, P < 0.05, respectively). Lutein and lycopene concentrations increased after spinach+tomato puree consumption (from 1.2 to 3.5 μmol/10¹² cells, P < 0.01, and from 0.1 to 0.7 μmol/10¹² cells, P < 0.05, respectively). The increase may be attributed to the addition of tomato puree to spinach; however, the different concentrations of carotenoids in lymphocytes registered at the beginning of the two intervention periods may have affected the results. DNA resistance to H₂O₂ insult increased significantly after both the enriched diets (P < 0.01); however, no “additive effect” was seen after spinach + tomato puree consumption. In the spinach + tomato intervention period an inverse correlation was observed between lymphocyte lycopene concentration and DNA damage, but this seems not able to explain the protection observed. Conclusions The consumption of carotenoid-rich foods even for a short period of time gives protection against oxidative stress. The results obtained seem to suggest that this protective role is not specifically related to carotenoids. However they may contribute together with other substances present in vegetables to lymphocyte resistance to oxidative damage. Received: 20 September 2001, Accepted: 18 February 2002     

Vitamin E reduces lipid peroxidation in experimental hepatotoxicity in rats

Background and aims Lipid peroxidation is believed to be involved in the pathophysiology of a number of diseases and in the process of aging. This study investigates the effects of dietary supplementation with vitamin E (20 g/kg diet of all-rac-α-tocopheryl succinate for 3 weeks) on both non-enzymatic and enzymatic lipid peroxidation in experimental rats with carbon tetrachloride (CCl₄)-induced hepatotoxicity (2.5 mL/kg body). Methods Plasma, urine and liver samples from control rats (n = 6), CCl₄-treated rats (n = 6), and rats supplemented with vitamin E prior to CCl₄ treatment (n = 8) were collected. Non-enzymatic lipid peroxidation induced by free radicals was investigated by measurement of a major F₂-iso-prostane, 8-iso-prostaglandin F_2α (8-iso-PGF_2α). Cyclooxygenase-catalyzed enzymatic lipid peroxidation was measured with a major PGF_2α metabolite, 15-kto-13,14-dihydro-prostaglandin F_2α (15-K-DH-PGF_2α). Malondialdehyde and antioxidants in plasma were also quantified. Results CCl₄ treatment alone resulted in significantly higher levels of plasma, urinary and liver 8-iso-PGF_2α, and of plasma and urinary 15-K-DH-PGF_2α compared to controls. Rats supplemented with vitamin E prior to CCl₄ treatment had significantly lower levels of urinary and liver 8-iso-PGF_2α, urinary 15-K-DH-PGF_2α, and plasma malondialdehyde than rats treated with CCl₄ alone. However, plasma 8-iso-PGF_2α and plasma 15-K-DH-PGF_2α were not affected by vitamin E supplementation. Conclusion Thus, both non-enzymatic and enzymatic lipid peroxidation during experimental hepatic oxidative injury were suppressed by dietary vitamin E supplementation in rats. Received: 29 May 2000, Accepted: 16 November 2000     

Addition of vitamin E to long-chain polyunsaturated fatty acid-enriched diets protects neonatal tissue lipids against peroxidation in rats

Summary Background: Tissue 10:4(n-6) and 22:6(n-3) status have been correlated with neonatal development and growth. Artificial formulas for neonates have been supplemented with long chain polyunsaturated fatty acids (LCP) from animal and marine sources which may enhance sensitivity of cellular membranes to oxidative damage. Diet-derived antioxidants like vitamin E play a key role in the protection of tissue lipids against oxidation. Aim of the study: We seek to determine the influence of dietary vitamin E on tissue sensitivity to oxidative stress in rats fed for 4 weeks on diets enriched in (n-3) and (n-6) long-chain polyunsaturated fatty acids. Methods: Weanling rats received 10% fat diets that provided 18:1(n-9), 18:2(n-6) and 18:3(n-3) in a similar ratio to that of rat milk (group A), supplemented with fish oil (groups B and B+E) and supplemented with (n-6) and (n-3) LCP from an animal phospholipid concentrate (groups C and C+E). Vitamin E (500 mg vitamin E/kg fat) was added to diets B+E and C+E. Tissue fatty acid content and the activities of catalase, superoxide dismutase, glutathione transferase und glutathione peroxidase in liver and brain were measured. Glutathione status, vitamin E and the production of thiobarbituric acid reactive substances (TBARs) after incubation of erythrocyte, liver and brain lipids with inducers of enzymatic or non-enzymatic lipid peroxidation was measured. Results: Group B registered significantly lower total superoxide dismutase acitvity than group B+. Catalase activity was significantly higher in group C than in group C+E. Hepatic total and reduced glutathione levels were decreased in vitamin E supplemented groups compared to unsupplemented ones. TBARs production in erythrocyte lipids was significantly higher in groups B and C compared to vitamin E supplemented groups B+E and C+E. Conclusions: This study shows that the addition of vitamin E protected erythrocyte and liver microsome lipids enriched in (n-3) and (n-6) LCP from lipid peroxidation during the postnatal development of rats. The protection was more effectively in group C+E than in group B+E. Received: 14 January 1999, Accepted: 14 June 1999     

Short-chain fatty acid (SCFA) uptake into Caco-2 cells by a pH-dependent and carrier mediated transport mechanism

Summary The short-chain fatty acids, acetate, propionate, and butyrate, are the most abundant organic anions in the human colon. SCFA play a pivotal role in maintaining homeostasis in the colon. Particularly butyrate induces cell differentiation and regulates growth and proliferation of colonic mucosal epithelial cells, whereas it reduces the growth rate of colorectal cancer cell. Previous studies by several groups, including our own, using isolated membrane vesicles have demonstrated that the uptake of butyrate is at least in part mediated by a non-electrogenic SCFA⁻/HCO₃ ⁻ antiporter. The purpose of the present study was to determine (1) whether Caco-2 cells could serve as an experimental model to assess the mechanisms of SCFA transport, and (2) whether monocarboxylate transporters could play a role in SCFA transport in these cells. Caco-2 cells were found to transport ¹⁴C-butyrate in a concentration and time dependent manner. The uptake was sodium independent, but was stimulated by lowering extracellular pH. The uptake of 500μM butyrate was reduced by 49.6% ± 3.3% in the presence of propionate and by 57.2% ± 4.8% in the presence of 10 mM L-lactate. The addition of 1 mM α-cyano-4-hydroxycinnamate and phloretin, both known to be potent inhibitors of MCT1, decreased the uptake of 500 μM ¹⁴C-butyrate by 59.4% ± 4.1% and 48.9% ± 3.3%, respectively, whereas similar concentrations of DIDS did not have any effect. These data suggest that the uptake of butyrate in Caco-2 cells occurs via a carrier mediated transport system specific for monocarboxylic acids, which is in accordance with characteristics of the MCT1. Received: 28 February 2000, Accepted: 4 May 2000     

Chronic vitamin E inadequacy and thermally treated oils affect the synthesis of hepatic metallothionein isoforms

Summary Background: Metallothionein (MT)^# synthesis can be stimulated in many organs not only by various metals such as cadmium, zinc, and copper, but also by many nonmetalic compounds or experimental conditions such as oxidative stress. The latter lead to the hypothesis that MT is induced in response to free radicals formed in tissues and lipid peroxidation. Aims of the study: Whether the relationship between lipid peroxidation amd MT synthesis is a common phenomenon also valid for lipid peroxidation induced by dietary factors such as chronic vitamin E inadequacy and autoxidation products of polyenoic fatty acids derived from thermally oxidized oil was investigated in the presence study. Methods: The relationship between the induction of metallothionein isoforms I and II (MT-I and MT-II) in response to diet-induced lipid peroxidation using a rat model system in which lipid peroxidation was examined in vivo by chronic vitamin E inadequacy or by administration of lipid peroxidation products from a thermally treated polyenoicrich oil with either basal (dietary zinc concentration: 48 mg/kd; experiment 1) or Zn-stimulated MT levels (dietary zinc concentration: 305 mg/kd; experiment 2) was studied. In both experiments, growing male rats were fed diet containing either a fresh or a thermally treated soybean oil with deficient of sufficient amounts of vitamin E (14 and 11 vs. 648 and 560 mg α-tocopherol equivalents per kg diet) over 40 days according to a bifactorial experimental design. Plasma and liver concentrations of tocopherols and hepatic levels of thiobarbituric acid-reacitve substances (TBARS) were measured by high performance liquid chromatography. MT isoform concentrations in rat liver were isolated and quantified by ion-exchange high performance liquid chromatography and atomic absorption spectrometry. Results: Irrespective of the zinc supply, rats receiving inadequate amounts of vitamin E with the diet had markedly lower plasma and liver concentrations of α-tocopherol and total tocopherols than vitamin E-sufficient rats. ANOVA also revealed an interaction between the diet factors vitamin E and oil on tocopherols in plasma and liver of rats from both experiments. In experiment 1, where rats received normal amounts of dietary zinc, ingestion of the thermally treated oil impaired the tocopherol status compared to the treatment with the fresh oil, although this effect was only obvious in the vitamin E-deficient groups. In experiment 2, where rats received excessive amounts of zinc, the thermally treated oil did not contribute to a reduction of the tocopherol status in plasma and liver. In both experiments a significant increase in TBARS level, indicative of lipid peroxidation, was observed in the liver at chronic vitamin E inadequacy, but no effect of the oil was observed. Here, we show that the dietary treatment had some effects on the synthesis of liver metallothionein isoforms. In groups, receiving normal amounts of zinc, there was a significant interaction between the dietary treatments on the levels of MT-I and MT-II in liver. Chronic vitamin E inadequacy which was accompanied by diminisched tocopherol levels in liver induced the synthesis of MT-I and MT-II. When vitamin E inadequacy was combined with the ingestion of a thermally treated polyenoic acid-rich oil hepatic levels of MT-I and MT-II remained low. In experiment 2, where rats were fed the high zinc diet, vitamin E inadequacy caused an increase of hepatic MT-I level just as in experiment 1, although this MT stimulating effect was irrespective of the oil. For MT-II there was a 43% increase in the vitamin E-deficient group fed the fresh oil compared to all the other groups, although this effect was not statistically significant. The liver MT isoform response to stress was similar in rats with basal MT levels and Zn-induced liver MT levels. The failing effect of the thermally treated oil on MT levels which were stimulated by vitamin E deficiency in experiment 2 was possibly due to the low oxidation grade of the thermally treated oil. Conclusion: The present results are strongly indicative of an apparent induction of MT isoform synthesis in response to an impaired antioxidant defence system in the lipid regions of liver cells induced by vitamin E inadequacy. In contrast, thermally treated polyenoic-rich oils with a certain oxidation grade seem to restrain the induction of MT isoform synthesis under the present experimental conditions. Received: 10 January 2000, Accepted: 27 April 2000     

Plasma concentration response to drinks containing beta-carotene as carrot juice or formulated as a water dispersible powder

Summary. Background: Bioavailability of β-carotene is highly variable and depends on the source, the formulation and other nutritional factors. Objective: It was the aim of the study to compare β-carotene plasma response to b-carotene dosing with two commercially available drinks, containing β-carotene from carrot juice or as water dispersible β-carotene powder. Design In a randomized, parallel group study design, 4 volunteers per group received daily β-carotene doses of 6–7 or 18–22 mg of either drink over 6 weeks. Blood samples for determination of carotenoid and vitamin A plasma concentrations were collected before supplementation and over the dosing period. Results: Apparent steady-state β-carotene concentrations were attained after 40 days of supplementation. Consumption of the beverage containing β-carotene as a water dispersible powder resulted in a higher response of β-carotene plasma concentrations with increments of 3.84 ± 0.60 μmol/L (p < 0.05, dose: 7.2 mg/d) and 5.04 ± 0.72 μmol/L (p < 0.05, dose: 21.6 mg/d), respectively, in comparison to the carrot juice-based drink with increments of 0.42 ± 0.33 μmol/L (dose: 6 mg/d) and 1.71 ± 0.55 μmol/L (dose: 18 mg/d), respectively. β-carotene was cleared from the plasma with an apparent half-life of 6–11 days. Plasma concentrations of α-carotene, β-cryptoxanthin, lutein, zeaxanthin, and lycopene remained almost unchanged, whereas retinol plasma concentrations increased slightly. By contrast, with the exception of elevated 13-cis-retinoic acid in one group (21.6 mg/d, water dispersible powder), the concentrations of all-trans-retinoic acid, and the oxo-derivatives or retinoic acid were not significantly affected by b-carotene supplementation. Conclusions: The results confirm that the relative bioavailability of β-carotene depends largely on the source of b-carotene and demonstrate the superior bioavailability of β-carotene powder in comparison to that in carrot juice. Received: 7 May 2002, Accepted: 22 August 2002 This work was supported by a grant from F. Hoffmann-La Roche Ltd., Vitamins and Nutrition Research, Basle, Switzerland. Correspondence to: Prof. Dr. med. Petra A. Thürmann     

GLV supplements increased plasma β–carotene, vitamin C, zinc and hemoglobin in young healthy adults

Summary Background Green leafy vegetables (GLV) are rich sources of β–carotene, iron and other micronutrients. Our in vitro studies have demonstrated good antioxidant potential in GLV. Moreover linkages of GLV intakes with plasma retinol and ascorbic acid were seen in apparently healthy Indians. Aim of the study To investigate the effect of GLV as a natural fortificant of multiple micronutrients through a prospective human trial. Methods Short–term (0–4 h) response (AUC) of single dose of 7.9 mg β–carotene and 130 mg ascorbic acid through a spinach–carrot meal against the standard meal without GLV plus10 mg β–carotene and 150 mg ascorbic acid tablets was studied in two groups of 4 young volunteers each. In the second trial of 3 weeks' supplementation, 5 groups of young adults (n = 40) were given either 100 g GLV/day alone or with tablets of vitamin E (100 mg/day), or C (100 mg/day) or more oil (5 g/day) or non–GLV meal with tablet of β–carotene (10 mg/day). Hemoglobin (Hb), plasma β–carotene, zinc, vitamin C, glucose, and triglycerides were measured. Results In a postprandial response, AUC were comparable in both GLV and standard meals for β–carotene and ascorbic acid. In case of triglycerides and glucose AUC the GLV meal showed a better recovery to the baseline value after 4 hours than the standard meal. Three weeks' supplementation of GLV with more oil resulted in significant increase of plasma β–carotene (51%) and Hb (9%). GLV with vitamin E showed a significant increase in plasma β–carotene (40%), Hb (8%) and plasma vitamin C (6%). Supplementing β–carotene without GLV significantly increased Hb (11%), plasma zinc (14%) in addition to β–carotene. Multiple regression analyses weighted for energy intake indicated a significant association of percent increase in Hb with intakes of iron, riboflavin, folic acid, β–carotene, copper, phytate and fiber (p < 0.01), percent change in plasma zinc with intakes of zinc, β–carotene, vitamin C, riboflavin, copper, iron, and thiamin (p < 0.01), percent change in vitamin C with intakes of vitamin C, vitamin E, niacin, riboflavin, thiamin, β–carotene, zinc, phytate and fiber (p < 0.05) and percent change in plasma β–carotene with intakes of β–carotene, thiamin, folic acid, zinc, phytate and tannins (p < 0.05). Conclusion Using 100 g GLV/day with 10 g oil could be a single moderate strategy for supplementation of iron, β–carotene, ascorbic acid and zinc.     

Study of wheat breakfast rolls fortified with folic acid. The effect on folate status in women during a 3-month intervention

Summary. Background: Folate has come into focus due to its protective role against child birth defects such as neural tube defects (NTD). Swedish authorities recommend all fertile women to increase their folate intake to 400 μg/day by eating folate-rich foods. Because not all women follow these recommendations, there is a discussion today about whether Sweden should introduce folic acid fortification in wheat flour and sifted rye flour. This decision needs knowledge about the bioavailability of folic acid from fortified foods. Aim of the study: To investigate effects of two folic acid fortification levels on folate status in healthy female volunteers and to study the folic acid stability during the baking procedure and storage of the fortified breakfast rolls. Method: Twenty-nine healthy women were recruited. Folic acid-fortified wheat breakfast rolls were baked with the purpose to contain 200 μg folic acid/roll (roll L) and 400 μg folic acid/roll (roll H). Fourteen women were given one roll/day of roll L (group L) and 15 one roll/day of roll H (group H) during 12 weeks of intervention. Fasting venous blood samples were collected on days 0, 30, 60 and 90. Serum homocysteine concentrations were determined using an immunoassay. Serum and erythrocyte folate concentrations were analysed using a protein-binding assay with fluorescent quantification. The folic acid concentration in the breakfast rolls was analysed by HPLC on days 0, 30, 60 and 90. Total folate concentration was measured with microbiological assay on day 45. Results: Group L Group L had initially an average erythrocyte folate concentration of 577 ± 93 nmol/L. After 90 days of intervention, an increase of 20 % (p < 0.05) was observed. At day 0, mean serum folate concentrations were 16.9 ± 4.3 nmol/L. The mean serum folate concentrations increased by 30 % (p < 0.001) after 90 days. At day 0, mean serum homocysteine concentrations were 9.1 ± 2.0 μmol/L, which decreased by 20 % (p < 0.01) after 30 days. Group H Group H had an initial erythrocyte folate concentration of 784 ± 238 nmol/L. After 90 days, an increase of 26 % (p < 0.05) was observed. Serum folate increased at least 22 % after 30 days, from a level of 18.7 ± 4.8 nmol/L at day 0. Thereafter, all women of group H had serum concentrations at or above the upper limit of quantification (23 nmol/L). At day 0, mean serum homocysteine concentrations were 8.4 ± 1.7 μmol/L, which decreased by 16 % (p < 0.05) after 30 days. The baking procedure resulted in 20–25 % loss of fortified folic acid in the rolls used in the present study. The size of the rolls affected the retention of folic acid during baking. No significant loss was seen in folic acid concentration in the rolls during the intervention period. Conclusion: The present study showed that in healthy women, subjected to a 12-week intervention with breakfast rolls fortified with either 166 μg or 355 μg folic acid, serum homocysteine concentration decreased (p < 0.05) and erythrocyte folate increased (p < 0.05). The lower level of fortification seems to be sufficient to improve the folate status. Together with the average daily intake of natural folates, these women reach the recommended intake of 400 μg/day. Folic acid is stable in fortified bread for 90 days storage at −20 °C. Received: 22 April 2002, Accepted: 24 October 2002 Correspondence to: Madelene Johansson     

Red palm oil in the maternal diet increases provitamin A carotenoids in breastmilk and serum of the mother-infant dyad

Background Despite vitamin A supplementation programs, vitamin A deficiency in children remains a public health concern in Honduras. Aim of the study We investigated the effectiveness of short-term dietary supplementation of mothers with red palm oil as a strategy for improving the vitamin A status of the mother-infant dyad. Methods Lactating mothers in Colonia Los Pinos, a barrio of Tegucigalpa, Honduras, consumed a total of 90-mg β-carotene as red palm oil (n = 32) supplements (n = 36) or placebo (n = 18) in six equal doses over 10 days. Carotenoids and retinol in maternal and infant serum, and breastmilk carotenoids and retinol were measured before and after supplementation. Maternal diet was evaluated by 24-hour recall. Results Maternal serum α-carotene and β-carotene concentrations were increased 2 fold by palm oil compared with 1.2 fold by β-carotene supplements. Changes were significantly different in infant serum α-carotene but not β-carotene among the three experimental groups. Increases in breastmilk β-carotene were greater for the palm oil group (2.5 fold) than for the β-carotene supplement group (1.6 fold) and increases in milk α-carotene concentrations (3.2 fold) were slightly greater than those of β-carotene. There were also small but significant changes among groups in breastmilk lutein and lycopene. Breastmilk retinol was not significantly different among the groups over the treatment period. Conclusions Red palm oil in the maternal diet increases provitamin A carotenoids in breastmilk and serum of the mother-infant dyad. The use of dietary red palm oil to improve the vitamin A status of this population should be further investigated. Received: 24 August 2000, Accepted: 12 January 2001     

Hyperhomocysteinemia,and low intakes of folic acid and vitamin B12 in urban North India

Summary Background and Aim An adverse coronary risk profile has been reported amongst rural-to-urban migrant population living in urban slums undergoing stressful socio-economic transition. These individuals are likely to have low intakes of folic acid and vitamin B12, which may have an adverse impact on serum levels of homocysteine (Hcy). To test this hypothesis, we studied serum levels of Hcy in subjects living in an urban slum of North India and healthy subjects from urban non-slum area. Methods Group I consisted of 46 subjects (22 males and 24 females) living in an urban slum, while group II consisted of healthy subjects (n = 26, 13 males and 13 females) living in the adjacent non-slum area. Anthropometric measurements, biochemical profile (fasting blood glucose, total cholesterol, serum triglycerides, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol) and fasting serum levels of Hcy were measured. Dietary intakes of folic acid, vitamin B12, vitamin B1, and iron were calculated by the 24-hour dietary recall method. Serum levels of Hcy were correlated with dietary intakes of nutrients, anthropometry, and metabolic variables. Results Sex-adjusted serum levels of Hcy in mmol/L (Mean ± SD) were high, though statistically comparable, in both the groups (group I: 20.8 ± 5.9 and group II: 23.2 ± 5.9). Overall, higher than normal serum levels of Hcy (> 15 μmol/L) were recorded in 84 % of the subjects. A substantial proportion of subjects in both groups had daily nutrient intakes below that recommended for the Asian Indian population (folic acid: 93.4 % in group I and 96.7 % in group II, vitamin B12: 76.1 % in group I and 88.4 % in group II). However, between the two groups, average daily dietary intakes of both the nutrients were statistically comparable. As compared to non-vegetarians, vegetarians showed lower intakes of folic acid (p < 0.01) and vitamin B12 (p < 0.01) in both groups. On multivariate linear regression analysis with serum Hcy as the response variable and vegetarian/non-vegetarian status and sex (male/female) as predictor variables, higher serum levels of Hcy were observed in vegetarians vs non-vegetarians (β = 4.6, p < 0.05) and males vs females (β = 5.3, p < 0.01). Conclusions Low intakes of folic acid and vitamin B12, and hyperhomocysteinemia, in both the healthy population living in urban slums and adjacent urban non-slum areas, are important observations for the prevention of nutritional and cardiovascular diseases in the Indian subcontinent. Received: 30 October 2001, Accepted: 14 January 2002     

Relationship between dietary intake, antioxidant status and smoking habits in female Austrian smokers

Summary Background Previous studies have shown that cigarette smoke contains many oxidants and free radicals, which can increase lipid peroxidation. Aim of the study The association between smoking, food pattern, especially vitamin intake and plasma concentrations of important antioxidants, as well as lipid peroxidation products was assessed in this cross-sectional study. Subjects and methods Sixty Austrian women aged 18–40 y were enrolled in the study. Twenty-nine women were allocated to the smoking group; thirty-one women served as nonsmoking controls. Plasma concentrations of α- and γ-tocopherol, α- and β-carotene, lycopene, cryptoxanthin, retinol, ascorbate and malondialdehyde were determined by HPLC; dietary intake and food pattern had been assessed by four 24-h dietary intake recalls and one food frequency questionnaire. Results Generally, food intake patterns were not different between smoking and nonsmoking women. But, a significantly higher intake of alcohol was observed in the smoking group (P < 0.05). Plasma ascorbic acid concentration of the smoking group did not differ from the nonsmoking women. Despite the increased utilization because of the oxidative stress in smokers, this result might be explained by the high dietary intake of vitamin C in our smoking group. Significantly lower plasma concentrations of α-, β-carotene and lycopene have been partly ascribed to the enhanced metabolic turnover resulting from smoking-induced oxidative stress. Our results confirm that smoking had no effects on plasma tocopherol and plasma retinol concentrations. Conclusions The poor supply with the carotenoids α-, β-carotene and lycopene may result from the increased metabolism of antioxidants caused by oxidative stress and may be responsible for significantly higher levels of lipid peroxidation products in smokers compared to nonsmokers (P < 0.05). Received: 17 August 2000 / Accepted: 2 May 2001     

Progress in the science of probiotics: from cellular microbiology and applied immunology to clinical nutrition

Summary Background Bioavailability of fat–soluble vitamins from conventional oral supplements is insufficient in some conditions in which fat digestion and absorption are chronically impaired (e. g. cystic fibrosis). Aim of the study We used a water–soluble form of fatsoluble vitamin E (AQUANOVA^? solubilisate) to create a nutritional supplement (NS) in the form of vitaminized gummi bears (with micellised water–soluble α–tocopheryl acetate (100 IU) and 400 mg crystalline vitamin C). We assessed the bioavailability of the NS in comparison to conventional preparations. Methods The trial consisted of three study days (d0: NS sucked; d10: NS swallowed; d20: reference products swallowed). A total of 14 subjects (6 male/8 female), aged 25.3 (22.7–35.3) years, BMI 24.3 (19.0–31.7) kg/m² participated in the study. They had blood samples drawn after fasting for ≥12 hours and then 1, 5, 15, 30, 60, 120, 180, 240, 300 and 320 minutes after ingesting the vitamins. HPLC and a colorimetric method were used to determine vitamin E and vitamin C, respectively. Areas under the curve (AUC_0–320min) and maximum increases in plasma concentrations (Δ concentration) were calculated to assess bioavailability. Results The AUCs_0–320min of α–tocopherol from d0 were significantly larger (p = 0.016) when compared to d20. Moreover, the maximum increase in α–tocopherol plasma concentrations was significantly higher for d0 (p = 0.023) and d10 (p = 0.002) when compared to d20. Conclusions Short–term bioavailability of AQUANOVA^? micellised fat–soluble vitamin E from our NS was significantly higher than from regular supplements. The NS will now be tested for its clinical efficacy in a randomized double–blind controlled intervention trial with CF patients.