Bioconversion of polyphenols and organic acids by gut microbiota of predigested Hibiscus sabdariffa L. calyces and Agave (A. tequilana Weber) fructans assessed in a dynamic in vitro model (TIM-2) of the human colon.

The present work aimed at understanding gut microbiota bioconversion of phenolic compounds (PC) and organic acids in predigested Hibiscus sabdariffa (Hb) calyces and the mixture of Hb and Agave (Agave tequilana Weber) fructans (AF). With this purpose, dried Hb and Hb/AF were predigested with enzymatic treatment, and then fermented in a dynamic in vitro model of the human colon (TIM-2). After HPLC-ESI-QToF-MS analysis of samples taken at 0, 24, 48 and 72 h of fermentation, it was observed that hydroxycinnamic acids, flavanols, flavonols, and anthocyanins were mainly transformed into derivatives of hydroxyphenylpropionic, hydroxyphenylacetic and hydroxybenzoic acids. Moreover, organic acids, such as hydroxycitric and hibiscus acids, were formed along with unidentified lactones and reduced compounds. Interestingly, no differences were observed between microbial-derived metabolites formed after the fermentation of Hb and Hb/AF. In conclusion, colonic fermentation of polyphenol-rich Hb yields a wide range of microbial phenolic metabolites with potential effects on health.

Experimental confounding factors affecting stability, transport and metabolism of flavanols and hydroxycinnamic acids in Caco-2 cells.

Studying bioavailability of polyphenols is essential to understand the health effects of these compounds. Human epithelial cells are commonly used in intestinal absorption and transport experiments but the changes polyphenols undergo during incubation, due to their chemical instability under the cell culture conditions, are scarcely known and might lead to inaccurate conclusions. Based on abundance of flavanols and hydroxycinnamic acids in the diet, epicatechin, epicatechin-3-gallate and procyanidin B2 as flavanols along with 5-caffeoylquinic and 3,5-dicaffeoylquinic acids as hydroxycinnamic acids were selected to comparatively evaluate their absorption and metabolism using an in vitro Caco-2 cell model. Special emphasis was paid to the structure-stability relationship of these phenolic compounds in Dulbecco's Modified Eagle's Medium (DMEM) under the cell culture conditions. The tested compounds were scarcely absorbed and minimally metabolized by the intestinal epithelium cells. The cell transport study showed prevalent efflux for flavanols opposite to absorption for hydroxycinnamates. Intestinal metabolism revealed that hydroxycinnamates were preferentially hydrolyzed and subsequently methylated, whereas hydrolysis of flavanols could not be confirmed, being mostly conjugated to sulfate, methyl- and methyl-sulfate derivatives. It is noteworthy that methyl derivatives of procyanidin-B2 were detected inside Caco-2 cells, confirming its absorption. In addition, culture medium influenced phenol isomerization to a higher extent than cells. In conclusion, hydroxycinnamates were better absorbed than flavanols although their bioavailability was limited in this intestinal cell model.

Antiproliferative and cytotoxic effects of green coffee and yerba mate extracts, their main hydroxycinnamic acids, methylxanthine and metabolites in different human cell lines.

This work aimed at studying the effects of green coffee bean (GCBE) and yerba mate (YME) extracts, their main phenolic components (5-caffeoylquinic acid, 5-CQA; 3,5-dicaffeoylquinic acid, 3,5-DCQA) and metabolites (ferulic acid, FA; caffeic acid, CA; dihydrocaffeic acid, DHCA; and dihydroferulic acid, DHFA) along with caffeine (CAF) on the viability and proliferation of different human cell lines. Extracts (10-1000 µg/mL) and standards (10-1000 µM) were assayed in colon (Caco-2), lung (A549), oesophageal (OE-33), urinary bladder (T24) human carcinoma cells, and a non-cancer cell line (CCD-18Co). YME significantly reduced viability of cancer cells at all assayed concentrations, the higher doses also reducing cell proliferation. GCBE effects on cell viability were more effective at 100 and 1000 µg/mL, showing modest effects on cell proliferation. The highest doses of 5-CQA and 3,5-DCQA reduced cell viability and proliferation in all cell lines, whereas FA, DHCA and DHFA had lower and variable effects. Caffeine had no effect. Dietary-attainable concentrations (0.1, 1 and 10 µg/mL) of YME were tested for cytotoxicity and reactive oxygen species generation, showing no cytotoxic effect. Low concentrations of all tested compounds were non-cytotoxic to CCD-18Co cells. CONCLUSION: YME and to a lower degree GCBE, their phenolic components and metabolites may decrease cancer cell viability and proliferation.

A phenolic extract from grape by-products and its main hydroxybenzoic acids protect Caco-2 cells against pro-oxidant induced toxicity.

Grape/wine industry produces large amounts of by-products, however knowledge on their health-promoting qualities is limited. This study investigated the effects of a grape phenolic extract (GPE) and its phenolic compounds, gallic acid (GA) and syringic acid (SA) on human intestinal Caco-2 cells, directly or after cytotoxicity induced by tert-butylhydroperoxide (t-BOOH). Direct treatment with 0.1-10 µg/mL GPE, or 0.1-10 µM GA and SA produced no major cytotoxic effect, either changes in antioxidant defences (glutathione content, glutathione peroxidase and reductase activities) or protein damage (carbonyl groups). However, 10 µg/mL GPE, 1 and 10 µM GA and 10 µM SA decreased reactive oxygen species (ROS) production. Pre-treatment with GPE, SA and GA at the same concentrations for 20 h showed that 10 µg/mL GPE and 10 µM GA or SA significantly counteracted ROS increase induced by t-BOOH. 10 µg/mL GPE and 1-10 µM GA or 10 µM of SA significantly reduced pro-oxidant-induced cytotoxicity. 1-10 µg/mL GPE, 1-10 µM GA and 10 µM SA significantly recovered both depleted glutathione and enhanced glutathione reductase and peroxidase activities, and reduced protein oxidative damage. Therefore, treatment with realistic concentrations of GPE and its main hydroxybenzoic acids protected Caco-2 cells against induced oxidative stress.

Hydroxytyrosyl ethyl ether exhibits stronger intestinal anticarcinogenic potency and effects on transcript profiles compared to hydroxytyrosol.

The anticarcinogenic activity of hydroxytyrosyl ethyl ether (HTy-Et) compared to its precursor hydroxytyrosol (HTy) has been studied in human Caco-2 colon adenocarcinoma cells. 451 and 977 genes were differentially expressed in Caco-2 cells exposed to HTy or HTy-Et for 24h, respectively, compared with untreated cells (P<0.005; FDR=0), using Affymetrix microarrays. Results showed that both HTy and HTy-Et inhibited cell proliferation and arrested the cell cycle by up-regulating p21 and CCNG2 and down-regulating CCNB1 protein expression. HTy and HTy-Et also altered the transcription of specific genes involved in apoptosis, as suggested by the up-regulation of BNIP3, BNIP3L, PDCD4 and ATF3 and the activation of caspase-3. Moreover, these polyphenols up-regulated xenobiotic metabolizing enzymes UGT1A10 and CYP1A1, enhancing carcinogen detoxification. In conclusion, these results highlight that HTy and its derivative HTy-Et modulate molecular mechanisms involved in colon cancer, with HTy-Et being more effective than HTy.

Intake and dietary sources of haem and non-haem iron in Flemish preschoolers.

BACKGROUND/OBJECTIVES: In the absence of biochemical data on iron status in preschoolers, data on the adequacy of iron intake may be used to assess the possible risk of iron deficiency in this population group. Therefore, this study aims to investigate iron intake and its food sources in Flemish preschoolers. SUBJECTS/METHODS: A total of 661 Flemish preschoolers 2.5-6.5 years old were recruited via a random cluster sampling design, using schools as primary sampling units. Three-day estimated diet records were used to assess dietary intakes. The contribution to iron intake (haem and non-haem) of 57 food groups was computed by summing the amount provided by the food group for all individuals divided by the total intake for all individuals. RESULTS: Mean total iron intake (s.d.) was 7.4 (±2.3) and 6.7 (±2.8) mg/day for boys and girls, respectively. In all 65% of the children <4 years old and 45% of those 4-6.5 years old presented adequate iron intakes. The food groups with the highest mean proportional contribution to total iron intake were bread, meat and meat products, breakfast cereals and sweet snacks (in that order). Children from small families whose mother had a low educational level had higher iron intakes. CONCLUSION: Iron intakes were similar for boys and girls and almost half of the Flemish preschoolers do not comply with the dietary iron recommendations.

Increased lung neutrophil apoptosis and inflammation resolution in nonresponding pneumonia.

Neutrophil activation state and its relationship with an inflammatory environment in community-acquired pneumonia (CAP) remain insufficiently elucidated. We aimed to evaluate the neutrophil apoptosis and cytokine pattern in CAP patients after 72 h of treatment, and their impact on infection resolution. Apoptosis of blood and bronchoalveolar lavage (BAL) neutrophils was measured in nonresponding CAP (NCAP), in responding CAP (blood only) and in patients without infection (control). Pro-inflammatory (interleukin (IL)-6, IL-8) and anti-inflammatory (IL-10) cytokines were measured. Main outcomes were clinical stability and days of hospitalisation. Basal neutrophil apoptosis was higher in the BAL and blood of NCAP, whereas spontaneous apoptosis (after 24 h culture) was lower. Cytokines in NCAP were higher than in responding CAP and control: IL-6 was increased in BAL and blood, IL-8 in BAL and IL-10 in blood. An increased basal apoptosis (≥20%) in BAL of NCAP was associated with lower systemic IL-10 (p<0.01), earlier clinical stability (p=0.05) and shorter hospital stay (p=0.02). A significant correlation was found for systemic IL-6 and IL-10 with days to reach stability and length of stay. After 72 h of treatment, an increased basal alveolar neutrophil apoptosis might contribute to downregulation of inflammation and to faster clinical stability.

Iron status biomarkers in iron deficient women consuming oily fish versus red meat diet.

Specific recommendations for anemic individuals consist in increasing red meat intake, but the population at large is advised to reduce consumption of red meat and increase that of fish, in order to prevent the risk of developing cardiovascular disease. This study aimed to determine the effects of consuming an oily fish compared to a red meat diet on iron status in women with low iron stores. The study was designed attending the Consolidated Standards of Reporting Trials (CONSORT) statement guidelines. It was a randomised crossover dietary intervention study of two 8-week periods. Twenty-five young women with low iron stores completed the study. Two diets containing a total of 8 portions of fish, meat and poultry per week were designed differing only in their oily fish or red meat content (5 portions per week). At the beginning and the end of each period blood samples were taken and hemoglobin, hematocrit, serum ferritin, serum iron, serum transferrin, serum transferrin receptor-2 and the Zn-protoporphyrin/free-protoporphyrin ratio were determined. Food intake and body weight were monitored. During the oily fish diet, PUFA intake was significantly higher (p=0.010) and iron intake lower (mean+/-SD, 11.5+/-3.4 mg/day vs. 13.9+/-0.1 mg/day, p=0.008), both diets providing lower mean daily iron intake than recommended for menstruating women. Although there were no significant differences after 16 weeks, serum ferritin moderately decreased and soluble transferrin receptor increased with the oily fish, while changes with the red meat diet were the opposite. In conclusion, an oily fish diet compared to a red meat diet does not decrease iron status after 8 weeks in iron deficient women.

Marcadores del estado de hierro en mujeres con deficiencia de hierro que consumen una dieta de pescado azul frente a una dieta de carne roja / Iron status biomarkers in iron deficient women consuming oily fish versus red meat diet

Specific recommendations for anemic individuals consist in increasing red meatintake, but the population at large is advised to reduce consumption of red meat andincrease that of fish, in order to prevent the risk of developing cardiovascular disease.This study aimed to determine the effects of consuming an oily fish compared to ared meat diet on iron status in women with low iron stores. The study was designedattending the Consolidated Standards of Reporting Trials (CONSORT) statementguidelines. It was a randomised crossover dietary intervention study of two 8-weekperiods. Twenty-five young women with low iron stores completed the study. Twodiets containing a total of 8 portions of fish, meat and poultry per week weredesigned differing only in their oily fish or red meat content (5 portions per week).At the beginning and the end of each period blood samples were taken and hemoglobin,hematocrit, serum ferritin, serum iron, serum transferrin, serum transferrinreceptor-2 and the Zn-protoporphyrin/free-protoporphyrin ratio were determined.Food intake and body weight were monitored. During the oily fish diet, PUFAintake was significantly higher (p=0.010) and iron intake lower (mean±SD, 11.5±3.4mg/day vs. 13.9±0.1 mg/day, p=0.008), both diets providing lower mean daily ironintake than recommended for menstruating women. Although there were no significantdifferences after 16 weeks, serum ferritin moderately decreased and solubletransferrin receptor increased with the oily fish, while changes with the red meat dietwere the opposite. In conclusion, an oily fish diet compared to a red meat diet doesnot decrease iron status after 8 weeks in iron deficient women(AU)

Las recomendaciones nutricionales dirigidasa personas con anemia consisten generalmenteen aumentar el consumo de carne roja,mientras que las recomendaciones para lapoblación general están enfocadas a la reduccióndel consumo de esta carne y aumentar elconsumo de pescado, con el fin de reducir elriesgo de desarrollar enfermedades cardiovasculares.El presente estudio se diseñó parainvestigar los efectos del consumo de una dietabasada en pescado azul frente a una de carneroja sobre el estado de hierro de mujeres conbajas reservas de hierro. Este estudio se planteóde acuerdo con la guía CONSORT (patronesconsolidados para la publicación de ensayos).Se trató de una intervención nutricional cruzada,aleatorizada, con 2 periodos de 8 semanascada uno. Veinticinco mujeres finalizaron elestudio. Se diseñaron dos dietas que contenían8 raciones de pescado, carne y aves a la semana.Sólo se diferenciaban en el contenido de pescadoazul o carne roja (4 raciones semanales). Alinicio y final de cada periodo se obtuvieronmuestras de sangre y se analizó la concentraciónde hemoglobina, hematocrito, ferritina,hierro sérico, transferrina, receptor-2 de latransferrina y el cociente Zn-protoporfirina/protoporfirina libre. El peso y la ingesta de alimentosse controlaron durante el estudio.Durante la dieta de pescado azul la ingesta deácidos grasos poliinsturados (AGP) fue significativamentemayor (p=0,010) y la ingesta dehierro se redujo (media±SD, 11,5±3,4 frente a13,9±0,1 mg/día, p=0.008), siendo el aporte dehierro menor al recomendado para esta población(AU)

Aunque no se encontraron diferenciassignificativas durante 16 semanas, la ferritina descendió ligeramente y la concentración del receptor de transferrina aumentó con la dieta de pescado azul, mientras que los cambios observados con la dieta rica en carne roja fueron los opuestos. En conclusión, una dieta basada en pescado azul comparada con una dieta rica en carne roja, no provoca un descenso en el estado de hierro de mujeres con deficiencia de hierro después de 8 semanas(AU)

Does bicarbonated mineral water rich in sodium change insulin sensitivity of postmenopausal women?

AIM: To study the effects of drinking 0.5 L of two sodium-rich bicarbonated mineral waters (BMW-1 and 2), with a standard meal, on postprandial insulin and glucose changes. And to determine, if the effects vary depending on insulin resistance, measured by homeostasis model assessment (HOMA). METHODS: In a 3-way randomized crossover study, 18 healthy postmenopausal women consumed two sodium-rich BMWs and a low-mineral water (LMW) with a standard fat-rich meal. Fasting and postprandial blood samples were taken at 30, 60 and 120 min. Serum glucose, insulin, cholesterol and triacylglycerols were determined. Insulin resistance was estimated by HOMA and insulin sensitivity was calculated by quantitative insulin sensitivity check index (QUICKY). RESULTS: Glucose levels did not change. HOMA and QUICKY values were highly inversely correlated (r = -1,000; p < 0.0001). Insulin concentrations showed a significant time effect (p < 0.0001) and a significant water x time interaction (p < 0.021). At 120 min insulin levels with BMW-1 were significantly lower than with LMW (p = 0.022). Postprandial insulin concentrations showed significantly different patterns of mineral water intake depending on HOMA n-tiles (p = 0.016). CONCLUSION: Results suggests an increase in insulin sensitivity after BMWs consumption. This effect is more marked in the women, who have higher HOMA values. These waters should be considered part of a healthy diet in order to prevent insulin resistance and cardiovascular disease.

Does bicarbonated mineral water rich in sodium change insulin sensitivity of postmenopausal women? / ¿Modifica el agua mineral bicarbonatada rica en sodio la sensibilidad a la insulina de las mujeres postmenopáusicas?

Aim: To study the effects of drinking 0.5 L of two sodium-rich bicarbonated mineral waters (BMW-1 and 2), with a standard meal, on postprandial insulin and glucose changes. And to determine, if the effects vary depending on insulin resistance, measured by homeostasis model assessment (HOMA). Methods: In a 3-way randomized crossover study, 18 healthy postmenopausal women consumed two sodiumrich BMWs and a low-mineral water (LMW) with a standard fat-rich meal. Fasting and postprandial blood samples were taken at 30, 60 and 120 min. Serum glucose, insulin, cholesterol and triacylglycerols were determined. Insulin resistance was estimated by HOMA and insulin sensitivity was calculated by quantitative insulin sensitivity check index (QUICKY). Results: Glucose levels did not change. HOMA and QUICKY values were highly inversely correlated (r = –1,000; p < 0.0001). Insulin concentrations showed a significant time effect (p < 0.0001) and a significant water x time interaction (p < 0.021). At 120 min insulin levels with BMW-1 were significantly lower than with LMW (p = 0.022). Postprandial insulin concentrations showed significantly different patterns of mineral water intake depending on HOMA n-tiles (p = 0.016). Conclusion: Results suggests an increase in insulin sensitivity after BMWs consumption. This effect is more marked in the women, who have higher HOMA values. These waters should be considered part of a healthy diet in order to prevent insulin resistance and cardiovascular disease

Objetivo: Estudiar los efectos de la ingesta de 0.5L de dos aguas minerales bicarbonatadas ricas en sodio (BMW-1 y 2), junto con una comida estándar, sobre los cambios en la insulina y la glucosa postprandial; y determinar si los posibles efectos varían en función de la resistencia a la insulina evaluada a través del modelo homeostático (HOMA). Métodos: 18 mujeres postmenopáusicas sanas participaron en un estudio triple cruzado aleatorizado, en el que bebieron 2 aguas minerales bicarbonatadas ricas en sodio (BMW-1 y 2) y un agua mineral débil (LMW) junto con una comida estándar rica en grasa. Se tomaron muestras de sangre en ayunas y postprandiales a los 30, 60 y 120 min. Se determinó glucosa, insulina, colesterol y triglicéridos en suero. La resistencia a la insulina fue estimada a través del HOMA y la sensibilidad a la insulina se calculó mediante el índice de sensibilidad cuantitativa a la insulina (QUICKY). Resultados: Los niveles de glucosa no presentaron cambios. Los valores de HOMA y QUICKY presentaron una fuerte correlación inversa (r = –1,000; p < 0,0001). Las concentraciones de insulina mostraron un efecto significativo en el tiempo (p < 0,0001) y una interacción agua x tiempo significativa (p < 0,021). A los 120 min los niveles de insulina fueron significativamente inferiores con BMW1 respecto a LMW (p = 0,022). Las concentraciones postprandiales de insulina mostraron patrones significativamente distintos según el tipo de agua que se bebía dependiendo de los n-tiles del HOMA (p = 0,016). Conclusión: Los resultados sugieren un aumento de la sensibilidad a la insulina tras el consumo de las dos aguas minerales bicarbonatadas ricas en sodio. Este efecto es más marcado en las mujeres que tienen unos valores de HOMA más altos. Este tipo de aguas deberían ser consideradas como parte de una dieta saludable con objeto de prevenir la resistencia a la insulina y las enfermedades cardiovasculares

Reticulated platelets interfere with flow cytometric reticulocyte counts.

As part of an iron absorption study, we needed to accurately count reticulocytes in the peripheral blood of healthy human volunteers before measuring their enrichment with stable iron isotopes given in an oral dose. Recent studies have suggested the usefulness of reticulocyte counting by flow cytometry, through a combination of differential light scatter and measurement of the stoichiometric binding of thiazole orange (TO) to RNA within the maturing erythrocyte. Using this method we set out to improve the precision of our quantitative analysis by counting more cells, as reticulocytes normally comprise <2% of the red cell population. To ensure exclusion of other cell types, we identified WBCs and platelets with CD16+CD45- allophycocyanin and CD61- phycoerythrin, respectively. After removal of CD16(+) CD45(+) TO(+) WBCs and CD61(+) TO(-) platelets from analysis, the remaining cells were a combination of CD61(-) TO(-) erythrocytes, CD61(-) TO(+) reticulocytes and CD61(+) TO(+) reticulated platelets. Reticulocyte counts were lower after exclusion of CD61(+) TO(+) cells from analysis. They were similarly lower when erythrocyte precursors were positively identified through their glycophorin A expression and TO uptake. We conclude that it is necessary to exclude reticulated platelets from flow cytometric reticulocyte analysis.

A comparative study of iron bioavailability from cocoa supplemented with ferric pyrophosphate or ferrous fumarate in rats.

BACKGROUND: Food iron fortification can be a good strategy to prevent iron deficiency. Iron bioavailability from cocoa powder enriched with ferric pyrophosphate encapsulated in liposomes or ferrous fumarate was assessed in rats. METHODS: Three groups of rats consumed during 28 days either a control diet or two diets prepared with ferric pyrophosphate- or ferrous fumarate-enriched cocoa powder as the unique source of iron. Body weight and food intake were monitored and last-week feces were collected. On day 28, animals were sacrificed and livers and spleens were removed. Hemoglobin and total iron binding capacity (TIBC) were determined. RESULTS: There were no significant differences in body weight and food intake. Apparent iron absorption and % absorption/intake were significantly lower in rats consuming enriched cocoa compared to the control group, without significant differences due to the iron form. Enriched cocoa groups showed significantly lower spleen iron content and concentration than the control. Liver iron was lower in the ferric pyrophosphate group compared to the other two groups. Hemoglobin and TIBC values showed a deficient iron status in ferric pyrophosphate rats. CONCLUSION: Cocoa powder is a good vehicle for iron fortification when enriched with ferrous fumarate compared to ferric pyrophosphate encapsulated in liposomes.

Relaxation of the isolated human internal anal sphincter by sildenafil.

BACKGROUND: Hypertonicity of the internal anal sphincter (IAS) appears to be involved in the pathogenesis of anal fissure. The relaxant effects of sildenafil, a selective phosphodiesterase 5 (PDE5) inhibitor, on isolated human IAS were investigated. METHODS: The efficacy (maximal effect, E(max)) and potency (-log IC(50), where IC(50) is half-maximal inhibitory concentration) of the PDE5 inhibitors, sildenafil and zaprinast, and of nitric oxide donors, sodium nitroprusside and glyceryl trinitrate, as relaxants of histamine (0.1 mmol/l)-induced tone were examined in IAS strips under isometric contraction. The presence of PDE5 isoenzymes and changes in intracellular calcium and cyclic nucleotide levels in IAS muscle were tested by real-time reverse transcriptase-polymerase chain reaction, epifluorescence microscopy and enzyme immunoassay respectively. RESULTS: Sildenafil produced a concentration-related inhibition of the mean(s.e.m.) histamine-induced tone (E(max) 83(2) per cent, - log IC(50) 7.04(0.05); n = 12). Zaprinast produced relaxation to similar degree, but with lower potency. Nitric oxide donors also relaxed IAS. Sildenafil (1 micromol/l) produced a 1.8-fold increase in guanosine 3',5'-cyclic monophosphate content, with no change in adenosine 3',5'-cyclic monophosphate levels. Sildenafil markedly depressed the peak intracellular calcium increase evoked by histamine. PDE5A1, PDE5A2 and PDE5A3 transcripts were expressed in IAS muscle. CONCLUSION: Sildenafil relaxes the augmented tone of human IAS in vitro. These results support the potential use of this PDE5 inhibitor in the treatment of chronic anal fissure.

Phosphodiesterase 4 inhibition decreases MUC5AC expression induced by epidermal growth factor in human airway epithelial cells.

BACKGROUND: A common pathological feature of chronic inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease (COPD) is mucus hypersecretion. MUC5AC is the predominant mucin gene expressed in healthy airways and is increased in asthmatic and COPD patients. Recent clinical trials indicate that phosphodiesterase type 4 (PDE4) inhibitors may have therapeutic value for COPD and asthma. However, their direct effects on mucin expression have been scarcely investigated. METHODS: MUC5AC mRNA and protein expression were examined in cultured human airway epithelial cells (A549) and in human isolated bronchial tissue stimulated with epidermal growth factor (EGF; 25 ng/ml). MUC5AC mRNA was measured by real time RT-PCR and MUC5AC protein by ELISA (cell lysates and tissue homogenates), Western blotting (tissue homogenates) and immunohistochemistry. RESULTS: EGF increased MUC5AC mRNA and protein expression in A549 cells. PDE4 inhibitors produced a concentration dependent inhibition of the EGF induced MUC5AC mRNA and protein expression with potency values (-log IC(50)): roflumilast (approximately 7.5) > rolipram (approximately 6.5) > cilomilast (approximately 5.5). Roflumilast also inhibited the EGF induced expression of phosphotyrosine proteins, EGF receptor, and phospho-p38- and p44/42-MAPK measured by Western blot analysis in A549 cells. In human isolated bronchus, EGF induced MUC5AC mRNA and protein expression was inhibited by roflumilast (1 microM) as well as the MUC5AC positive staining shown by immunohistochemistry. CONCLUSION: Selective PDE4 inhibition is effective in decreasing EGF induced MUC5AC expression in human airway epithelial cells. This effect may contribute to the clinical efficacy of this new drug category in mucus hypersecretory diseases.

Estimation of iron absorption in humans using compartmental modelling.

OBJECTIVE: To evaluate whether a compartmental model could estimate iron absorption as accurately as the well-validated technique of plasma area under the curve using labelled test meals. DESIGN: The study is a randomised cross-sectional intervention. SETTING: The study was carried out at the Human Nutrition Unit at the Institute of Food Research, Norwich, UK. SUBJECTS: A total of nine female volunteers, aged 33+/-8 y. INTERVENTIONS: Volunteers were given an oral dose (approximately 5 mg) of Fe-57 as iron sulphate in an orange juice test drink and simultaneously infused Fe-58 (approximately 200 microg) as iron citrate over 90 min. Multiple blood samples were taken for the following 6 h. The samples were analysed by mass spectrometry and iron absorption was estimated using a mathematical model based on the appearance of Fe isotopes in plasma and the area under the curve technique. RESULTS: The geometric mean (-1 s.d., +1 s.d.) absorption of the model estimate is 16% (9, 31) and the area under curve estimate is 18% (8, 29). CONCLUSIONS: Results indicate that a compartmental model can be used to estimate labelled iron absorption although it is unlikely that this new method will be used in favour of an existing one. Further studies are now needed with unlabelled iron to assess whether the technique could have application in the assessment of total (haem+nonhaem) iron absorption from food.

Zinc and iron bioavailability in a powder or in-bottle-sterilized infant formula estimated by in vitro and in suckling rats.

Maillard reaction and lactose isomerization may be induced during the processing involved in the manufacture of infant formulas. The effects of dehydratation and sterilization in an infant formula on iron and zinc bioavailability were studied. A powder (PIF), previously reconstituted, and an in-bottle-sterilized liquid infant formula (LIF), from the same manufacturer, were evaluated using an in vitro method and in suckling rats. After in vitro digestion the dialyzed and non-dialyzed soluble, and insoluble fractions of iron and zinc were separated. Two-week-old rat pups were fed PIF or LIF in a drinking bottle for 7 days. Infant formula intake (I), body weight and the fecal and urinary excretions were monitored and the following parameters calculated: apparent absorption (A), retention (R), and the coefficients %A/I, %R/A and %R/I. Soluble iron (dialyzed) and zinc (non-dialyzed) were higher (p < 0.001) in LIF than PIF after in vitro digestion. Insoluble iron was similar in both infant formulas but insoluble zinc was lower (p < 0.05) in LIF than PIF. Food intake (p = 0.045) and body weight on day 4 (p < 0.05) and on day 7 (p < 0.001) were lower in LIF compared to PIF. A, R (p < 0.05 for both minerals), %A/I, and %R/I (p < 0.001 and p < 0.05 for iron and zinc, respectively) were significantly lower in rats fed LIF. Similarly, the %R/A of iron was lower (p < 0.001) in this group. Hematocrit and hemoglobin did not show significant differences. Iron and zinc levels in liver, spleen and erythrocytes were similar in both groups, but skin iron concentration was higher in LIF. Therefore, in contrast with the in vitro results, consumption of the in-bottle-sterilized formula determines lower iron and zinc bioavailability compared to the reconstituted powder infant formula.

Does processing of a powder or in-bottle-sterilized liquid infant formula affect calcium bioavailability?

Processing of infant formulas can induce Maillard reaction or lactose isomerization, among other changes. These reactions were evaluated with furosine and lactulose, respectively. Protein alteration was assessed with sodium dodecylsulfate-polyacrylamide gel electrophoresis. Repercussions on calcium bioavailability in powder and in-bottle-sterilized liquid infant formulas were studied. Lactulose, advanced Maillard-reaction products, and denatured proteins were higher in liquid infant formula. After in vitro digestion, soluble non-dialyzed calcium was significantly higher in liquid than in powder infant formula, but there were no differences in dialyzed insoluble calcium. Two-week-old rat pups drank the powder or liquid infant formula for 7 d. Food intake and final body weight were significantly lower in those fed liquid formula. Accordingly, the intake, apparent absorption, and retention of calcium were measured; the percentages of retention versus absorption and retention versus intake were significantly lower, although calcium digestibility (percentage of absorption versus intake) was higher. These results show that, although calcium in the sterilized infant formula was available in vitro and was absorbed more efficiently in vivo, it was poorly used by suckling rats. The low acceptability of this formula and the interaction of calcium with lactulose and advanced but absorbable Maillard-reaction products might explain the results. Thus, for calcium bioavailability, we recommend the powder instead of the conventional sterilized infant formula.

Effects of phorbol 12,13-diacetate on human isolated bronchus.

Protein kinase C appears to be involved in the regulation of airway contractility. Phorbol 12,13-diacetate (PDA; 0.01-10 microM), a protein kinase C activator, produced a transient relaxation followed by a sustained contraction of human isolated bronchus. Different protein kinase C inhibitors (calphostin C, staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine) (H-7), nifedipine (NIF; 1 microM) or incubation with Ca(2+)-free medium, inhibited the spasmogenic response to phorbol, while ouabain (10 microM) suppressed only the initial relaxation. These results indicate that the initial relaxation, in response to PDA, is related to the activation of Na(+)/K(+)-ATPase, while the ensuing contraction depends on extracellular Ca(2+) entry.Incubation with PDA (1-5 microM) depressed the maximal relaxation to theophylline and caffeine obtained at 37 degrees C but augmented the spasmogenic responses to methylxanthines (10 mM) obtained in cooled preparations. These effects do not result apparently from increased extracellular entry of Ca(2+), but instead, from facilitation of the release of Ca(2+) from intracellular stores.

Halothane inhibits endothelium-dependent relaxation elicited by acetylcholine in human isolated pulmonary arteries.

This study examined whether a clinically relevant concentration of the volatile anaesthetic halothane modifies the endothelium-dependent relaxation produced by acetylcholine (3 nM-10 microM), histamine (1 pM-0.1 microM) and anti-human immunoglobulin E (1:1000) in human isolated pulmonary arteries submaximally precontracted with noradrenaline. An inhibitor of nitric oxide formation, N(G)-nitro-L-arginine (100 microM), attenuated acetylcholine-induced relaxation but failed to inhibit histamine- and anti-human immunoglobulin E-induced relaxation. Indomethacin (2.8 microM, a cyclooxygenase inhibitor) preferentially reduced the relaxation to histamine and anti-human IgE. Halothane (2%) significantly attenuated the relaxation to acetylcholine but had no significant effect on the relaxation elicited by histamine and anti-human IgE. Halothane (2%) enhanced the basal release of prostaglandin I2 by human pulmonary arteries (control 0.31 +/- 0.04 ng mg(-1); treated tissues 0.50 +/- 0.06 ng mg(-1); n = 5; P < 0.05). Halothane (2%) did not alter the responsiveness and sensitivity of preparations to relaxants acting through activation of adenylyl cyclase (forskolin) or guanylyl cyclase (sodium nitroprusside) or by the opening of K(ATP) channels (cromakalim). In conclusion, halothane inhibits the endothelium-dependent relaxation of human pulmonary arteries to acetylcholine by interfering with the nitric oxide pathway at a site before activation of soluble guanylyl cyclase in vascular smooth muscle.