Novel role of the nutraceutical bioactive compound berberine in lectin-like OxLDL receptor 1-mediated endothelial dysfunction in comparison to lovastatin.

BACKGROUND AND AIMS: Oxidized LDL (oxLDL) or pro-inflammatory stimuli lead to increased oxidative stress linked to endothelial dysfunction and atherosclerosis. The oxLDL receptor-1 (LOX1) is elevated within atheromas and cholesterol-lowering statins inhibit LOX1 expression. Berberine (BBR), an alkaloid extracted from plants of gender Berberis, has lipid-lowering and anti-inflammatory activity. However, its role in regulating LOX1-mediated signaling is still unknown. The aim of this study was to investigate the effect of BBR on oxLDL- and TNFα-induced endothelial dysfunction in human umbilical vein endothelial cells (HUVECs) and to compare it with that of lovastatin (LOVA). METHODS AND RESULTS: Cytotoxicity was determined by lactate dehydrogenase assay. Antioxidant capacity was measured with chemiluminescent and fluorescent method and intracellular ROS levels through a fluorescent dye. Gene and protein expression levels were assayed by qRT-PCR and western blot, respectively. HUVECs exposure to oxLDL (30 µg/ml) or TNFα (10 ng/ml) for 24 h led to a significant increase in LOX1 expression, effect abrogated by BBR (5 µM) and LOVA (5 µM). BBR but not LOVA treatment abolished the TNFα-induced cytotoxicity and restored the activation of Akt signaling. In spite of a low direct antioxidant capacity, both compounds reduced intracellular ROS levels generated by treatment of TNFα but only BBR inhibited NOX2 expression, MAPK/Erk1/2 signaling and subsequent NF-κB target genes VCAM and ICAM expression, induced by TNFα. CONCLUSIONS: These findings demonstrated for the first time that BBR could prevent the oxLDL and TNFα - induced LOX1 expression and oxidative stress, key events that lead to NOX, MAPK/Erk1/2 and NF-κB activation linked to endothelial dysfunction. CHEMICAL COMPOUNDS STUDIED IN THIS ARTICLE: Berberine (PubChem CID: 2353); Lovastatin (PubChem CID: 53232).

Restricted intraindividual urinary iodine concentration variability in nonfasting subjects.

OBJECTIVE: Individual urinary iodine concentration (UIC) reflects iodine intake over a short time prior to sampling. Since eating habits are relatively constant in single subjects, UIC should be relatively constant in a given individual. The aim of our study was to verify this hypothesis by assessing UIC in repeated single urine samples from a group of healthy subjects. DESIGN AND SETTING: A prospective sequential investigation was performed in 131 volunteer health workers or students recruited in our University hospital. INTERVENTIONS: Single urine samples were taken in a nonfasting state, between 0900 and 1100 hours. Group 1 was composed by 131 subjects who collected one urine sample. Group 2 was composed by 11 subjects of the group 1, who collected multiple repeated urine samples (as a whole 158 urine samples, mean 14 samples each). UIC mean+/-s.d., median and coefficient of variation (CV%) was measured in both groups. RESULTS: Interindividual UIC variation was wide, UIC ranging from 21 to 382 microg/l, mean 136+/-84 microg/l, median 124 microg/l, CV 62%. Also in the 11 subjects repeatedly sampling there were considerable differences among individual UIC average levels (ranging from 37+/-15 to 221+/-91 microg/l). However, in this second group, the intraindividual variation was considerably restricted (CV% 36). CONCLUSIONS: The present study shows that in a nonfasting state in mid-morning UIC is more stable from day to day in a single subject, depending on his eating habits, than in various subjects. Thus, a single urine sample even in nonfasting state may give some rough information about the individual's iodine status.

The effect of oxygen radicals on rat thymocyte glucose transport is independent of the site of their generation.

We studied the relationship between the site of production of oxygen radicals and their effect on a rat thymocyte functional activity, the glucose transport, measured using a radioactive analogue of glucose, 2-deoxy-glucose. We compared the effects of a hydrophilic thermolabile azo compound, mimicking a radical attack outside the cell, with the lipid-soluble cumene hydroperoxide, which initiates lipid peroxidation in cell membranes. Our results show that a low grade oxidative stress stimulated glucose uptake rapidly, independently of the site of radical generation. In the presence of the azocompound, glucose uptake increased smoothly, attaining its maximum extent within 1 h. In thymocytes treated with cumene hydroperoxide the rate of glucose transport increased suddenly and remained constant over 1 h. The effects of the radical donors on TBARS production and protein sulfhydryl groups content were also evaluated. In thymocytes treated with the azo derivative no lipid peroxidation was observed, but a slow decrease of protein thiol groups occurred; after the addition of cumene hydroperoxide sulfhydryl groups did not change and TBARS increased significantly. The water-soluble antioxidant Trolox was able to remove the glucose uptake increase induced by the hydrophilic initiator and to delay the loss of membrane integrity.

Antioxidants and total peroxyl radical-trapping ability of olive and seed oils.

The presence of the oxidized and reduced forms of ubiquinones Q(9) and Q(10) was determined in commercial extra virgin olive and seed oils, where the amounts of alpha- and gamma-tocopherols and beta-carotene were also quantitated. Very high concentrations of ubiquinones were found in soybean and corn oils. Furthermore, the total antioxidant capability of each oil was evaluated by measuring total radical-trapping antioxidant parameters (TRAP) in tert-butyl alcohol and using egg lecithin as the oxidizable substrate. These values decreased in the order sunflower > corn > peanut > olive; the highest TRAP, which was found in sunflower oil, was related to the very high amount of alpha-tocopherol. Olive oil, because of the low content of alpha-tocopherol, exhibited a TRAP value approximately one-third that of sunflower oil. TRAP values of corn and soybean oils, in which low amounts of alpha-tocopherol but very high contents of gamma-tocopherol and reduced ubiquinones were present, were intermediate. gamma-Tocopherol exhibited a poor ability of trapping peroxyl radicals in tert-butyl alcohol. This behavior was probably due to the effects of the solvent on the rate of hydrogen abstraction from this phenol.

Development of a chemiluminescent urease activity assay for Helicobacter pylori infection diagnosis in gastric mucosa biopsies.

A chemiluminescent urease activity assay has been developed and optimized using the chemiluminescent pH indicator phthalhydrazidylazoacetylacetone. This compound is stable at pH

Methylprednisolone administration in primary biliary cirrhosis increases cholic acid turnover, synthesis, and deoxycholate concentration in bile.

As immunosuppressive agents, corticosteroids may be considered an appropriate treatment for primary biliary cirrhosis, even if bone loss and other side effects may occur. We studied biliary lipid metabolism in 10 nonicteric patients, with histologically proven primary biliary cirrhosis (stage I-IV). We administered methylprednisolone (24 mg daily) for 30 days to ascertain its effects on biliary lipid metabolism, which are largely still unknown. All patients underwent a 30-day drug-washout period before entering the trial. The following parameters were studied before and after methylprednisolone treatment: serum biochemistry; cholic acid pool size, kinetics and synthesis; biliary lipid secretion; biliary bile acid pattern; biliary lipid molar percentage; and cholesterol saturation index. Methylprednisolone induced a statistically significant (Wilcoxon rank test) increase in cholic acid turnover (from 0.26+/-0.04 to 0.50+/-0.05 K/day, P = 0.005) and synthesis (from 0.42+/-0.12 to 0.78+/-0.11 mmol/day, P = 0.04), and in bile deoxycholic acid molar percentage (from 19.4+/-2.7 to 30.6+/-4.4% molar, P = 0.01). On the other hand, a significant decrease in biliary cholesterol molar percentage (from 7.9+/-0.7 to 6.4+/-0.5% molar, P = 0.005), cholesterol saturation index (from 1.11+/-0.11 to 0.95+/-0.07, P = 0.05), and biliary cholesterol secretion (from 64.7+/-5.4 to 53.0+/-4.5 micromol/hr, P = 0.005) was observed. These findings show that short-term administration of methylprednisolone in patients with primary biliary cirrhosis does not induce expansion of the cholic acid pool but increases cholic acid synthesis and turnover, as well as intestinal production of deoxycholic acid. If long-term treatment is considered, the beneficial immunosuppressive effects of corticosteroids have to be weighed against the hepatotoxic properties of deoxycholic acid.

Polyamines directly induce release of cytochrome c from heart mitochondria.

Cytochrome c release from mitochondria to the cytosol represents a critical step in apoptosis, correlated to the activation of the caspase cascade. In this report, we show that addition of micromolar concentrations of polyamines to isolated rat heart mitochondria induces the release of cytochrome c. Spermine, which is effective at concentrations of 10-100 microM, is more potent than spermidine, whereas putrescine has no effect up to 1 mM. The release of cytochrome c caused by spermine is a rapid, saturable and selective process that is independent of mitochondria damage. Spermine, unlike polylysine, is able to release a discrete amount of cytochrome c from intact, functional mitochondria. The cytochrome c-releasing power of spermine is not affected by cyclosporin A, differently from the effect of permeability transition inducers. In a cardiac cell-free model of apoptosis, the latent caspase activity of cytosolic extracts from cardiomyocytes could be activated by cytochrome c released from spermine-treated heart mitochondria. These data indicate a novel mechanism of cytochrome c release from the mitochondrion, and suggest that prolonged and sustained elevation of polyamines, characteristic of some pathologies such as heart hypertrophy, could be involved in the development of apoptosis.

Milk represents an important source of iodine in schoolchildren of the Veneto region, Italy.

The aim of the present study is to evaluate the relationships between urinary iodine concentration (UIC) and the intake of milk and other foods, in a group of school children of the Veneto region, in North East Italy. A questionnaire, concerning the daily intake of milk, yoghurt, cheese and other animal foodstuffs, was distributed to 233 schoolchildren aged between 11 and 15 yr. The use of iodized salt was also investigated. UIC was measured in a casual urine sample of all children investigated. The iodine content of 28 samples of milk and of 13 samples of yoghurt, bought during the summer in shops of the same area, was measured. UIC values ranged between 25 and 436 microg/l, median value was 140 microg/l, mean value 149+/-78 microg/l. The median iodine content of milk and yoghurt were 278 microg/l and 216 microg/l, respectively. With regard to dietary habits, about 70% of the children took 200 ml of milk or more per day, which corresponds to a daily intake of iodine ranging between 50 and 100 microg a day. About 30% of schoolchildren used iodized salt. A highly significant correlation between UIC and milk intake was observed (p=0.0005), while the relationship was poor or absent in the case of both intake of other foodstuffs and use of iodized salt (p=0.38). In conclusion, the results of the study document the very important role of cows' milk as a source of iodine in childhood in the Veneto region, Italy.

Detection of phospholipid oxidation in oxidatively stressed cells by reversed-phase HPLC coupled with positive-ionization electrospray [correction of electroscopy] MS.

Measurement of lipid peroxidation is a commonly used method of detecting oxidative damage to biological tissues, but the most frequently used methods, including MS, measure breakdown products and are therefore indirect. We have coupled reversed-phase HPLC with positive-ionization electrospray MS (LC-MS) to provide a method for separating and detecting intact oxidized phospholipids in oxidatively stressed mammalian cells without extensive sample preparation. The elution profile of phospholipid hydroperoxides and chlorohydrins was first characterized using individual phospholipids or a defined phospholipid mixture as a model system. The facility of detection of the oxidized species in complex mixtures was greatly improved compared with direct-injection MS analysis, as they eluted earlier than the native lipids, owing to the decrease in hydrophobicity. In U937 and HL60 cells treated in vitro with t-butylhydroperoxide plus Fe(2+), lipid oxidation could not be observed by direct injection, but LC-MS allowed the detection of monohydroperoxides of palmitoyl-linoleoyl and stearoyl-linoleoyl phosphatidylcholines. The levels of hydroperoxides observed in U937 cells were found to depend on the duration and severity of the oxidative stress. In cells treated with HOCl, chlorohydrins of palmitoyloleoyl phosphatidylcholine were observed by LC-MS. The method was able to detect very small amounts of oxidized lipids compared with the levels of native lipids present. The membrane-lipid profiles of these cells were found to be quite resistant to damage until high concentrations of oxidants were used. This is the first report of direct detection by LC-MS of intact oxidized phospholipids induced in cultured cells subjected to oxidative stress.

Normal values for thyroid ultrasonography, goiter prevalence and urinary iodine concentration in schoolchildren of the Veneto Region, Italy.

Goiter prevalence in school-age children and median urinary iodine concentration (UIC) are the main indicators of iodine deficiency in a population. In areas of mild iodine deficiency, where goiters are small, ultrasound is preferable to physical examination to estimate goiter prevalence. The World Health Organization (WHO) has adopted thyroid volume ultrasonography results from a survey of European schoolchildren as an international reference, but these values have recently been questioned. The aims of the study were: a) to determine regional normal echographic reference values of thyroid volume in children aged between 11 and 14 yr in the Veneto Region, in North-East Italy; b) to determine goiter prevalence by physical and ultrasonographic examination; c) to determine UIC in this section of the population. A cross-sectional study was carried out on 1730 schoolchildren, aged between 11 and 14, living in towns in low-lying areas, in the valleys of the pre-Alps and in the mountains between 600 and 1200 m. Thyroid volume was evaluated by inspection and palpation using the WHO criteria. In 560 children thyroid volume was determined by ultrasound. UIC was measured in 1368 children. On physical examination a grade I goiter was found in 7.5% of children. No goiter grade II or grade III was found. The regional thyroid volume reference values by ultrasonography were similar, or slightly lower (5-20%), to the corresponding WHO reference values. Mean UIC was 148 +/- 110 microg/l, with no difference between lowlands and uplands; UIC values less than 100 microg/l were found in about 30-35% of the children. UIC was higher in children using iodized salt than in non-users. No correlation was found between thyroid volume by ultrasonography and UIC. Thyroid volume was found to be bigger in upland children than in those in low-lying areas, probably because of low iodine intake in people living in the mountains in previous generations. This data show that Veneto is not a iodine-deficient area, with no presence of endemic goiter. However, the great number of children with a UIC of less than 100 microg/l also suggests the use of iodized salt in the Veneto Region.