Noncompliance to iodine supplementation recommendation is a risk factor for iodine insufficiency in Portuguese pregnant women: results from the IoMum cohort.

PURPOSE: After a recommendation for iodine supplementation in pregnancy has been issued in 2013 in Portugal, there were no studies covering iodine status in pregnancy in the country. The aim of this study was to assess iodine status in pregnant women in Porto region and its association with iodine supplementation. METHODS: A cross-sectional study was conducted at Centro Hospitalar Universitário São João, Porto, from April 2018 to April 2019. Pregnant women attending the 1st trimester ultrasound scan were invited to participate. Exclusion criteria were levothyroxine use, gestational age < 10 and ≥ 14 weeks, non-evolutive pregnancy at recruitment and non-signing of informed consent. Urinary iodine concentration (UIC) was measured in random spot urine by inductively coupled plasma-mass spectrometry. RESULTS: Median UIC was 104 µg/L (IQR 62-189) in the overall population (n = 481) of which 19% had UIC < 50 µg/L. Forty three percent (n = 206) were not taking an iodine-containing supplement (ICS) and median UIC values were 146 µg/L (IQR 81-260) and 74 µg/L (IQR 42-113) in ICS users and non-users, respectively (p < 0.001). Not using an ICS was an independent risk factor for iodine insufficiency [adjusted OR (95% CI) = 6.00 (2.74, 13.16); p < 0.001]. Iodised salt use was associated with increased median iodine-to-creatinine ratio (p < 0.014). CONCLUSIONS: A low compliance to iodine supplementation recommendation in pregnancy accounted for a mild-to-moderately iodine deficiency. Our results evidence the need to support iodine supplementation among pregnant women in countries with low household coverage of iodised salt. Trial registration number NCT04010708, registered on the 8th July 2019.

Predictors of nutritional and inflammation risk in hemodialysis patients.

BACKGROUND: Malnutrition and chronic inflammation are prevalent complications in hemodialysis (HD) patients. Different nutritional assessment tools are used to identify patients at risk. A composite and comprehensive malnutrition inflammation score (MIS) has been correlated with morbidity and mortality, and appears to be a robust and quantitative tool. OBJECTIVES: Determine malnutrition risk profile in a sample of portuguese HD patients; determine the association of clinical and laboratory factors with MIS, and the impact of each parameter on MIS. METHODS AND RESULTS: We performed, between September 15th of 2015 and January 31st of 2016, a cross sectional analysis of 2975 patients, representing 25% of portuguese HD patients. 59% were men (66.7 ± 14.8 years); 31% diabetic; 79% and 21% performed, respectively, high-flux HD and HDF. A MIS >5 was considered to indicate higher risk and was present in 1489 patients (50%). Amongst all parameters, comorbilities/dialysis vintage, transferrin, functional capacity, changes in body weight and decreased fat stores showed the higher impact, while albumin had one of the lowest impact on the nutritional risk. MULTIVARIABLE ANALYSIS: Higher age (>75 years, OR 1.71, p < 0.001), diabetes (OR 1.25, p = 0.026), lower P levels (OR 1.57,p = 0.001), higher Ca levels (OR 1.51, p < 0.001), higher ERI (OR 1.05, p < 0.001), higher Kt/V (OR 2.14, p < 0.001) and higher CRP (OR 1.01, p < 0.001) were independently associated with a higher risk of MIS>5; higher nPNA (OR 0.29, p < 0.001) and higher Pcreat (OR 0.88, p < 0.001) were associated with a risk reduction of MIS>5 (95% CI). CONCLUSIONS: Routine clinical and analytic parameters were found to be associated with MIS range that might indicate higher risk, and may represent a simple alert sign for the need of further assessments.

The Fatty Acid Profile in Patients with Newly Diagnosed Diabetes: Why It Could Be Unsuspected.

CONTEXT: Several studies have shown a link between proinflammatory activity and the presence or deficit of some fatty acids. Inflammation is associated with several diseases including diabetes. OBJECTIVE: To characterize and compare the fatty acids profiles in children with inaugural type 1 diabetes, diabetic children (at least 1 year after diagnosis), and healthy children. DESIGN: Plasma fatty acids profiles in children with inaugural diabetes, children with noninaugural diabetes, and controls, all of whom were prepubescent with a BMI < 85th percentile, were evaluated. RESULTS: Omega-3 fatty acid levels were higher in recently diagnosed subjects with diabetes than in controls. The ratio of omega-6/omega-3 fatty acids was higher in the control population. Omega-6 fatty acid levels were higher in the nonrecent diabetic subjects than in the children with recently diagnosed diabetes, and the levels were higher in the nonrecent diabetes group compared to the control group. CONCLUSION: Our findings showed higher levels of alpha-linolenic acid, EPA, and DHA, as well as mono- and polyunsaturated fatty acids, in diabetic children. These findings reinforce the importance of precocious nutritional attention and intervention in the treatment of diabetic children.

Pharmacokinetics of table and Port red wine anthocyanins: a crossover trial in healthy men.

This study was designed to evaluate the pharmacokinetics of Port and table red wine anthocyanins in healthy men. Volunteers were recruited to drink 250 mL of a table red wine (221 mg of anthocyanins) and 150 mL of young Port red wine (49 mg of anthocyanins). Venous blood was collected from participants at 0, 15, 30, 60 and 120 min after wine ingestion. Urine samples were collected at baseline and at 120 min. Anthocyanins and anthocyanin metabolites in plasma and urine samples were quantified by HPLC-DAD and tentatively identified by LC-MS. Red wine anthocyanins were detected in their intact forms in both plasma and urine samples, but the glucuronylated metabolites of peonidin and malvidin (PnGlucr and MvGlucr) were the two main derivatives detected after both red wine consumptions. For the first time, and supported by the synthesis of Mv3Glucr, the main pathway followed by Mv3glc after absorption was described and involves anthocyanidin conjugation with glucuronic acid after glucose removal. Despite the lower total content of anthocyanins ingested when volunteers drank Port wine, no differences were observed in the plasma Cmax of MvGlucr and PnGlucr after table and Port red wine consumption. The relative bioavailability of anthocyanins in Port wine was 96.58 ± 5.74%, compared to the anthocyanins present in red wine. In conclusion, both Port and table red wines are good sources of bioavailable anthocyanins.

Anthocyanin extraction from plant tissues: A review.

Anthocyanins have gathered the attention of the scientific community mostly due to their vast range of possible applications. They have been the center point of the research in many different fields, among which is food development, where their innate coloring, antioxidant capacity, and biological potential open interesting venues to the development of new food additives and functional foodstuffs. As the range of application grows, so does the necessity to obtain these compounds, and since they are naturally occurring, the most common way to obtain anthocyanins is to extract them from different plant sources, such as fruits and flowers. Several efforts have been made to develop methods that allow for better extraction yields and higher purification rates therefore this review aims to compile the information regarding extraction and purification procedures in a comprehensive manner.

Antimicrobial, antiadhesive and antibiofilm activity of an ethanolic, anthocyanin-rich blueberry extract purified by solid phase extraction.

AIMS: The present work aimed to characterize the impact of an anthocyanin-rich blueberry extract upon the growth, adhesion and biofilm formation of several pathogens including some multiresistant bacteria. METHODS AND RESULTS: A group comprised of reference strains and clinical multiresistant isolates of Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, Acinetobacter baumannii and Staphylococcus aureus, were used to screen for antimicrobial activity. Microbial growth was determined through the measurement of the optical density while adhesion and biofilm formation was determined using the standard crystal violet staining procedure. The results showed that, while blueberry extract was only effective in hindering the growth of Staph. aureus and E. coli, it was capable of significantly inhibiting biofilm formation and bacterial adhesion for all micro-organisms tested. CONCLUSIONS: The extract demonstrated a considerable potential as a natural, alternative antimicrobial capable of either interfering with microbial growth or hamper the adhesion to surfaces, with Staph. aureus proving to be the most susceptible micro-organism. SIGNIFICANCE AND IMPACT OF THE STUDY: The overall study demonstrates the potential of anthocyanin extracts as natural effective alternative antimicrobial agents. Additionally, the extract's capacity to reduce adhesion without reducing bacterial growth reduces the likeliness of resistance development while reducing the probability of infection.

Beyond central adiposity: liver fat and visceral fat area are associated with metabolic syndrome in morbidly obese patients.

BACKGROUND: Despite its widespread clinical use, both body mass index (BMI) and waist circumference have been reported as inaccurate methods to measure abdominal obesity. The main objective of this study was to determine the relation between visceral fat area and fatty liver infiltration with the expression of metabolic syndrome (MS) in morbidly obese patients. METHODS: We recruited a random selection of 100 morbidly obese patients on pre-operative evaluation for bariatric surgery. A pre-operative CT slice at L4-L5 level, was performed to measure visceral fat and at T12 level to measure hepatic attenuation. RESULTS: Patients with MS had lower hepatic attenuation values (median 49.9 vs 55.5HU; p = .018) and had more VAT (242 vs 172 cm(2);p = .001). Conventional measures (BMI: p = .729 and waist circumference: p = .356), were not useful in discriminating morbidly obese patients with MS. By multivariable logistic regression, fatty liver infiltration (OR = 5.3; p = .03) and age (OR = 1.08; p = .04) were the only factors independently related to the presence of MS. MS prevalence was 100%, 71% and 55%, respectively for patients with both fatty liver and visceral adiposity; one; or none of this findings (AUC - .715; p = .016). CONCLUSION: CT scan seems to measure 2 important markers of MS: visceral adiposity and hepatic fatty infiltration. In morbidly obese patients, both visceral adiposity and hepatic fatty infiltration increase the risk for the presence of MS.

Helicobacter pylori antimicrobial resistance rates in the central region of Portugal.

Helicobacter pylori resistance to antimicrobial agents is steadily increasing. It is extremely important to be aware of the local prevalence of antibiotic resistance so as to adjust treatment strategies. During this single-centre, prospective study, we aimed to determine primary and secondary resistance rates of H. pylori to antibiotics as well as host and bacterial factors associated with this problem. Overall, 180 patients (131 female; mean age 43.4±13.5 years; primary resistance 103; secondary resistance 77) with positive (13) C-urea breath test were submitted to upper endoscopy with gastric biopsies. Helicobacter pylori was cultured and antimicrobial susceptibility was determined by Etest and molecular methods. Clinical and microbiological characteristics associated with resistance were evaluated by logistic regression analysis. Among the 180 isolates 50% were resistant to clarithromycin (primary 21.4%; secondary 88.3%), 34.4% to metronidazole (primary 29.1%; secondary 41.6%), 33.9% to levofloxacin (primary 26.2%; secondary 44.2%), 0.6% to tetracycline and 0.6% to amoxicillin. Being female was an independent predictor of resistance to clarithromycin and metronidazole. Previous, failed, eradication treatments were also associated with a decrease in susceptibility to clarithromycin. History of frequent infections, first-degree relatives with gastric carcinoma and low education levels determined increased resistance to levofloxacin. Mutations in the 23S rRNA and gyrA genes were frequently found in isolates with resistance to clarithromycin and levofloxacin, respectively. This study revealed that resistance rates to clarithromycin, metronidazole and levofloxacin are very high and may compromise H. pylori eradication with first-line and second-line empiric triple treatments in Portugal.

Effects of extracts of selected medicinal plants upon hepatic oxidative stress.

Aqueous extracts of a few medicinal plants traditionally used in Portugal have been assayed for their effects upon hepatic oxidative stress in mice. Previous in vitro studies had allowed characterization of agrimony, sage, savory, and raspberry in terms of overall antioxidant capacity and phenolic content. In the present study, the antioxidant effect and safety of these four plants were evaluated in vivo. For this purpose, mice ingested extracts in aqueous form (or water, used as the control) for 4 weeks; damage to lipids, proteins, and DNA was evaluated by oxidative cell biomarkers by the end of that period. Levels of hepatic glutathione and activities of enzymes involved in metabolism thereof were also determined. Finally, catalase and superoxide dismutase (SOD) activities were quantified, as these enzymes play a crucial role in antioxidant defense. When compared with the control, both raspberry and savory produced significant lipid protection; however, protein damage was significantly lower only in raspberry-treated animals. On the other hand, DNA damage was prevented only by savory. All plants led to a decrease in catalase activity, whereas all but sage also produced a decrease in SOD activity. With regard to glutathione levels and activities of enzymes involved in its metabolism, the aforementioned extracts exhibited different effects. In general, raspberry appeared to be the most promising extract, followed by savory, sage, and agrimony, sorted by decreasing performance in protection; the latter was even slightly toxic. Hence, the plants tested possess compounds with interesting biological activities that may support eventual inclusion in food or feed as functional additives.

Impact of cellular folate status and epidermal growth factor receptor expression on BCRP/ABCG2-mediated resistance to gefitinib and erlotinib.

The effect of folate status on breast cancer resistance protein (BCRP)-mediated drug resistance to epidermal growth factor receptor (EGFR)-targeted drugs, such as gefitinib and erlotinib, was investigated in two human colon cancer cell lines, WiDr and Caco-2, of which the latter displayed greater sensitivity to these drugs due to high EGFR expression. Caco-2 LF/LV cells, growing under low-folate (LF) conditions, showed increased BCRP protein expression compared with the high-folate (HF) counterpart, which was associated with 1.8-fold resistance to gefitinib. Of note, the BCRP-specific inhibitor Ko143 completely reverted this phenotype. WiDr LF cells also showed slightly increased BCRP expression compared with the HF cells, but no differences in gefitinib sensitivity were observed. Both Caco-2 LF/LV and WiDr LF cells showed 2.4- and 2.3-fold resistance to erlotinib, respectively, compared with their HF counterparts, which mechanistically seemed BCRP unrelated, as Ko143 had no effect on erlotinib activity. In conclusion, our data suggest that in EGFR-expressing Caco-2 cells, BCRP is one of the determinants of gefitinib resistance but not of erlotinib resistance. Beyond this, folate depletion can provoke an additional decrease in gefitinib and erlotinib activity by mechanisms dependent or independent of BCRP modulation.

Team approach to ERCP-directed single-brush cytology for the diagnosis of malignancy.

OBJECTIVE: To evaluate the clinical usefulness of single-brush cytology performed at ERCP as initial method for detecting pancreatobiliary malignancy, ensuring a very close relationship between endoscopists, cytotechnicians, and cytopathologists. STUDY DESIGN: All 125 cytodiagnoses considered in this study correspond to the first brushing for each patient, collected by one of the three members of a fixed team of endoscopists in the presence of the same cytotechnician. Smears were fixed immediately with Merckofix spray, stained with Papanicolau, and analyzed by the same cytopathologist in a laboratory exclusively devoted to gastrointestinal cytopathology located at the endoscopy unit. RESULTS: Of 125 cytological diagnoses 94 were considered benign, 4 suspicious, and 27 malignant. These findings were compared to the final diagnosis of 45 malignant and 80 benign lesions obtained either by surgical pathology or after at least one year of clinical follow-up. The comparison yielded 30 true positives, 78 true negatives, 1 false positive and 16 false negative results, which corresponds to a sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of 65.2, 98.7, 96.8, 83, and 86.4%, respectively. CONCLUSION: Results seem to confirm the usefulness of an effective team approach to ERCP-directed brush cytology for the diagnosis of pancreatobiliary malignancy. However, sensitivity continues to be rather low.

Characterization of rat heart alkaline phosphatase isoenzymes and modulation of activity.

Alkaline phosphatase (ALP) is important in calcification and its expression seems to be associated with the inflammatory process. We investigated the in vitro acute effects of compounds used for the prevention or treatment of cardiovascular diseases on total ALP activity from male Wistar rat heart homogenate. ALP activity was determined by quantifying, at 410 nm, the p-nitrophenol released from p-nitrophenylphosphate (substrate in Tris buffer, pH 10.4). Using specific inhibitors of ALP activity and the reverse transcription-polymerase chain reaction, we showed that the rat heart had high ALP activity (31.73 +/- 3.43 nmol p-nitrophenol.mg protein-1.min-1): mainly tissue-nonspecific ALP but also tissue-specific intestinal ALP type II. Both ALP isoenzymes presented myocardial localization (striated pattern) by immunofluorescence. ALP was inhibited a) strongly by 0.5 mM levamisole, 2 mM theophylline and 2 mM aspirin (91, 77 and 84%, respectively) and b) less strongly by 2 mM L-phenylalanine, 100 mL polyphenol-rich beverages and 0.5 mM progesterone (24, 21 to 29 and 11%, respectively). beta-estradiol and caffeine (0.5 and 2 mM) had no effect; 0.5 mM simvastatin and 2 mM atenolol activated ALP (32 and 36%, respectively). Propranolol (2 mM) tended to activate ALP activity and corticosterone activated (18%) and inhibited (13%) (0.5 and 2 mM, respectively). We report, for the first time, that the rat heart expresses intestinal ALP type II and has high total ALP activity. ALP activity was inhibited by compounds used in the prevention of cardiovascular pathology. ALP manipulation in vivo may constitute an additional target for intervention in cardiovascular diseases.

Characterization of rat heart alkaline phosphatase isoenzymes and modulation of activity

Alkaline phosphatase (ALP) is important in calcification and its expression seems to be associated with the inflammatory process. We investigated the in vitro acute effects of compounds used for the prevention or treatment of cardiovascular diseases on total ALP activity from male Wistar rat heart homogenate. ALP activity was determined by quantifying, at 410 nm, the p-nitrophenol released from p-nitrophenylphosphate (substrate in Tris buffer, pH 10.4). Using specific inhibitors of ALP activity and the reverse transcription-polymerase chain reaction, we showed that the rat heart had high ALP activity (31.73 ± 3.43 nmol p-nitrophenol·mg protein-1·min-1): mainly tissue-nonspecific ALP but also tissue-specific intestinal ALP type II. Both ALP isoenzymes presented myocardial localization (striated pattern) by immunofluorescence. ALP was inhibited a) strongly by 0.5 mM levamisole, 2 mM theophylline and 2 mM aspirin (91, 77 and 84 percent, respectively) and b) less strongly by 2 mM L-phenylalanine, 100 mL polyphenol-rich beverages and 0.5 mM progesterone (24, 21 to 29 and 11 percent, respectively). â-estradiol and caffeine (0.5 and 2 mM) had no effect; 0.5 mM simvastatin and 2 mM atenolol activated ALP (32 and 36 percent, respectively). Propranolol (2 mM) tended to activate ALP activity and corticosterone activated (18 percent) and inhibited (13 percent) (0.5 and 2 mM, respectively). We report, for the first time, that the rat heart expresses intestinal ALP type II and has high total ALP activity. ALP activity was inhibited by compounds used in the prevention of cardiovascular pathology. ALP manipulation in vivo may constitute an additional target for intervention in cardiovascular diseases.

Modulation of MPP+ uptake by tea and some of its components in Caco-2 cells.

The entry of most xeno/endobiotics into the organism is limited by their intestinal absorption. The interference of certain foods with the therapeutic efficacy of drugs or with chemical toxicity is becoming evident and growing attention is being given to these subjects. The aim of this work was to study the effect of green tea (GT) and black tea (BT), as well as some of their components, on the transport of organic cation molecules. For this purpose, 3H-MPP+ (radiolabeled 1-methyl-4-phenylpyridinium) was used as a model organic cation and Caco-2 cells were used as an intestinal epithelial model. Our results showed that both GT and BT significantly increased 3H-MPP+ absorption in these cells. Additionally, we studied the effect of epigallocatechin-3-gallate (EGCG), myricetin, caffeine, and theophylline. Whereas EGCG (2 mM) increased, myricetin (50 microM) and caffeine (1 mM) decreased, and theophylline (1 mM) had no effect on the uptake of 3H-MPP+ into Caco-2 cells. When GT was supplemented with caffeine or theophylline, we observed a partial loss of its effect. When BT was supplemented with EGCG, its ability to increase 3H-MPP+ uptake was much more pronounced than that observed with BT alone. In conclusion, this study showed that GT and BT might interfere with the absorption of the model organic cation MPP+ by the intestinal epithelium. Since important compounds are organic cations, the consequences of this interference may have an impact on human health. Although this constitutes only preliminary work and further studies are needed, tea should be included in the growing list of foodstuffs that have the potential to be involved in food-drug interactions.

Intestinal uptake of MPP+ is differently affected by red and white wine.

It is becoming increasingly evident that ingested products, such as wine, may have profound effects on the therapeutic efficacy of certain drugs. As various xeno- and endobiotics are organic cations, the purpose of our study was to examine the modulation of organic cations intestinal apical uptake by red (RW) and white wine (WW). For this purpose, we used RW, WW, the same alcohol-free wines, phenolic compounds and ethanol. The uptake of the organic cation 1-methyl-4-phenylpyridinium (MPP+) was evaluated in Caco-2 cells, an intestinal epithelial cell model. RW and alcohol-free RW increased 3H-MPP+ apical uptake, although the effect of alcohol-free RW was less pronounced. On the other hand, WW and alcohol-free WW decreased the organic cation uptake but the effect of alcohol-free WW was more pronounced. Our results show that the total content in phenolic compounds was 7 times higher, and the dialysis index was about 4 times higher in RW compared to WW. Ethanol, in the same concentration found in wine, caused a significant decrease in 3H-MPP+ apical uptake. The solution containing high molecular weight compounds from dialyzed RW increased 3H-MPP+ apical uptake. In conclusion, the results suggest that RW may increase and WW may reduce the intestinal absorption of organic cations present in the diet, such as drugs or vitamins (e.g. thiamine and riboflavin). As ethanol alone decreased the uptake of MPP+, and alcohol-free RW and WW had a lower potency than intact wine upon the transport, the presence of ethanol is probably important for the solubilisation/bioavailability of the components endowed with the transport modulating activity.

Modulation of uptake of organic cationic drugs in cultured human colon adenocarcinoma Caco-2 cells by an ecto-alkaline phosphatase activity.

Alkaline phosphatase (ALP) refers to a group of nonspecific phosphomonoesterases located primarily in cell plasma membrane. It has been described in different cell lines that ecto-ALP is directly or indirectly involved in the modulation of organic cation transport. We aimed to investigate, in Caco-2 cells, a putative modulation of 1-methyl-4-phenylpyridinium (MPP(+)) apical uptake by an ecto-ALP activity. Ecto-ALP activity and (3)H-MPP(+) uptake were evaluated in intact Caco-2 cells (human colon adenocarcinoma cell line), in the absence and presence of a series of drugs. The activity of membrane-bound ecto-ALP expressed on the apical surface of Caco-2 cells was studied at physiological pH using p-nitrophenylphosphate as substrate. The results showed that Caco-2 cells express ALP activity, characterized by an ecto-oriented active site functional at physiological pH. Genistein (250 micro M), 3-isobutyl-1-methylxanthine (1 mM), verapamil (100 micro M), and ascorbic acid (1 mM) significantly increased ecto-ALP activity and decreased (3)H-MPP(+) apical transport in this cell line. Orthovanadate (100 micro M) showed no effect on (3)H-MPP(+) transport and on ecto-ALP activity. On the other hand, okadaic acid (310 nM) and all trans-retinoic acid (1 micro M) significantly increased (3)H-MPP(+) uptake and inhibited ecto-ALP activity. There is a negative correlation between the effect of drugs upon ecto-ALP activity and (3)H-MPP(+) apical transport (r = -0.9; P = 0.0014). We suggest that apical uptake of organic cations in Caco-2 cells is affected by phosphorylation/dephosphorylation mechanisms, and that ecto-ALP activity may be involved in this process.

Regulation of [(3)H]MPP(+) transport by phosphorylation/dephosphorylation pathways in RBE4 cells: role of ecto-alkaline phosphatase.

The aim of this study was to investigate the role of phosphorylation/dephosphorylation mechanisms at the blood-brain barrier (BBB) in the uptake of organic cations. The experiments were performed using RBE4 cells, an immortalized, rat brain microvessel endothelial cell line, an in vitro model of the BBB. The modulation of the uptake of 1-methyl-4-phenylpyridinium (MPP(+)), a model organic cation, at the apical membrane of RBE4 cells was studied. Agents that stimulate protein kinase A, but not protein kinase C, produced a moderate inhibition (approximately 18% reduction) of uptake of [(3)H]MPP(+) by RBE4 cells. Okadaic acid, an inhibitor of protein serine/threonine phosphatase, did not affect uptake of (3)H-MPP(+), but the alkaline phosphatase (ALP) inhibitor levamisole markedly reduced (3)H-MPP(+) uptake. The activity of membrane-bound ALP expressed on the apical surface of RBE4 cells was studied at pH 7.4 using p-nitrophenylphosphate as substrate. Kaempferol, progesterone, 3-isobutyl-1-methylxanthine, all- trans-retinoic acid and iron stimulated ecto-ALP activity and uptake of [(3)H]MPP(+) in RBE4. Orthovanadate (a protein tyrosine phosphatase inhibitor) markedly inhibited both ecto-ALP activity and uptake of [(3)H]MPP(+) by RBE4 cells. In conclusion, these results suggest that apical transporter(s) of MPP(+) in RBE4 cells may be under the control of phosphorylation/dephosphorylation mechanisms, being active in the dephosphorylated state. A physiological role for ALP in the modulation of organic cation transport in the BBB is suggested.

Modulation of insulin transport in rat brain microvessel endothelial cells by an ecto-phosphatase activity.

The physiological function of alkaline phosphatase (ALP) remains controversial. It was recently suggested that this membrane-bound enzyme has a role in the modulation of transmembranar transport systems into hepatocytes and Caco-2 cells. ALP activity expressed on the apical surface of blood-brain barrier cells, and its relationship with (125)I-insulin internalization were investigated under physiological conditions using p-nitrophenylphosphate (p-NPP) as substrate. For this, an immortalized cell line of rat capillary cerebral endothelial cells (RBE4 cells) was used. ALP activity and (125)I-insulin internalization were evaluated in these cells. The results showed that RBE4 cells expressed ALP, characterized by an ecto-oriented active site which was functional at physiological pH. Orthovanadate (100 microM), an inhibitor of phosphatase activities, decreased both RBE4-ALP activity and (125)I-insulin internalization. In the presence of L-arginine (1 mM) or adenosine (100 microM) RBE4-ALP activity and (125)I-insulin, internalization were significantly reduced. However, D-arginine (1 mM) had no significant effect. Additionally, RBE4-ALP activity and (125)I-insulin internalization significantly increased in the presence of the bioflavonoid kaempferol (100 microM), of the phorbol ester PMA (80 nM), IBMX (1 mM), progesterone (200 microM and 100 microM), beta-estradiol (100 microM), iron (100 microM) or in the presence of all-trans retinoic acid (RA) (10 microM). The ALP inhibitor levamisole (500 microM) was able to reduce (125)I-insulin internalization to 69.1 +/- 7.1% of control. Our data showed a positive correlation between ecto-ALP activity and (125)I-insulin incorporation (r = 0.82; P < 0.0001) in cultured rat brain endothelial cells, suggesting that insulin entry into the blood-brain barrier may be modulated through ALP.