Simultaneous and accurate determination of water- and fat-soluble vitamins in multivitamin tablets by using an RP-HPLC method

In the present study, a reversed-phase high-performance liquid chromatographic (RP-HPLC) procedure was developed and validated for the simultaneous determination of seven water-soluble vitamins (thiamine, riboflavin, niacin, cyanocobalamin, ascorbic acid, folic acid, and p-aminobenzoic acid) and four fat-soluble vitamins (retinol acetate, cholecalciferol, α-tocopherol, and phytonadione) in multivitamin tablets. The linearity of the method was excellent (R² > 0.999) over the concentration range of 10 - 500 ng mL-1. The statistical evaluation of the method was carried out by performing the intra- and inter-day precision. The accuracy of the method was tested by measuring the average recovery; values ranged between 87.4% and 98.5% and were acceptable quantitative results that corresponded with the label claims.

Selection of microorganisms producer of lipase for fat removal from biodiesel purification water

The objective this study has been the selection of lipase productor microorganism, for removal of oils and grease, in the pre-treatment of biodiesel wastewater washing. For this, analyses of the physicist-chemistries characteristics had been made with the wastewater of the biodiesel washing, and then it had been isolated and chosen, by means of determinations of the lipase activity. Following, it was made a test of fat biodegradation, in the conditions: pH (5.95), temperature (35 ºC), rotation (180 rpm) and ammonium sulfate as nitrogen source (3 g L-1) and establishing as variable the two microorganism preselected and the time (24; 48; 72; 96 and 120 h). The biodiesel purification wastewater had presented high potential of environmental impact, presenting a concentration of O of 6.76 g L-1. From the six isolated microbiological cultures, two microorganisms (A and B) had been selected, with enzymatic index of 0.56 and 0.57, respectively. The treatment of the wastewater using the isolated microorganism (Klebsiella oxytoca) had 80% of the fatty removal in 48 h.

Immunization by subcutaneous implants of polyester-polyurethane sponges coupled with antigen

A new protocol is described for immunization of outbred Swiss mice. The procedure is based on subcutaneous implantation of antigen-coupled polyester-polyurethane sponges cut into disks of 10 mm in diameter vs 2 mm in thickness. Antigen coupling was performed by overnight incubation of the sponge with a solution of ovalbumin (Ova) (2 mg/ml) diluted in sodium carbonate buffer, pH 9.6. The amount of ovalbumin that was taken up by the sponge was between 71.4 to 82.5 µg. This was estimated by comparing the Ova absorbance at 280 nm in coating buffer solutions before and after incubation. To compare the efficiency of the proposed method, experimental groups immunized with the antigen in the presence of adjuvants (10 µg in Al(OH)3 or 100 µg in complete Freund's adjuvant (CFA)) were run in parallel. The data obtained after the 3rd week of immunization indicate that both cellular and humoral immune responses were achieved. These were assayed by antigen-induced footpad swelling and ELISA (specific antibodies), respectively. The levels of both immune responses elicited were similar to the responses observed in mice immunized with ovalbumin in the presence of Al(OH)3. The method might represent an advantage when immunizing with pathogenic antigens. Preliminary experiments have suggested that the antigen remains immobilized or bound to the sponge for a long period of time, since there is an increment on the cell population inside the sponges after boosting the animals. If so, the undesirable effects of immunization would be reduced.

Effect of dietary (n-3) highly unsaturated fatty acids on growth and survival of fat snook (Centropomus parallelus, Pisces: Centropomidae) larvae during first feeding

The effect of rotifers, Brachionus rotundiformis (S-type), fed three different diets: A (rotifer fed Nannochloropsis oculata), B (rotifer fed N. oculata and baker's yeast, 1:1), and C (rotifer fed N. oculata and baker's yeast, 1:1, and enriched with Selcoâ), was evaluated based on the survival, growth and swim bladder inflation rate of fat snook larvae. Rotifers of treatment A had higher levels (4.58 mg/g dry weight) of eicosapentaenoic acid (EPA) than B (1.81 mg/g dry weight), and similar levels (0.04 and 0.06 mg/g dry weight, respectively) of docosahexaenoic acid (DHA). Rotifers of treatment C had the highest levels of EPA (13.2 mg/g dry weight) and DHA (6.08 mg/g dry weight). Fat snook eggs were obtained by spawning induction with human chorionic gonadotropin. Thirty hours after hatching, 30 larvae/liter were stocked in black cylindric-conical tanks (36-liter capacity). After 14 days of culture, there were no significant differences among treatments. Mean standard length was 3.13 mm for treatment A, 3.17 mm for B, and 3.39 mm for C. Mean survival rates were very low (2.7% for treatment A, 2.3% for B, and 1.8% for C). Swim bladder inflation rates were 34.7% for treatment A, 27.1% for B, and 11.9% for C. The lack of differences in growth and survival among treatments showed that the improvement of the dietary value of rotifer may not have been sufficient to solve the problem of larval rearing. Some other factor, probably pertaining to the quality of the larvae, may have negatively influenced survival.

Effect of dietary fat saturation on lipid metabolism, arachidonic acid turnover and peritoneal macrophage oxidative stress in mice

We investigated the effects of a saturated fat diet on lipid metabolism and arachidonic acid (AA) turnover in mouse resident peritoneal macrophages. The pro-oxidative effect of this diet was also studied. Female C57BL/6 mice were weaned at 21 days of age and assigned to either the experimental diet containing coconut oil (COCO diet), or the control diet containing soybean oil as fat source (10 mice per group). The fat content of each diet was 15% (w/w). Mice were fed for 6 weeks and then sacrificed. The concentration of total lipids, triglycerides, (LDL + VLDL)-cholesterol, thiobarbituric acid-reactive substances (TBARS) and reduced glutathione were increased in the plasma of mice fed the COCO diet, without changes in phospholipid or total cholesterol concentrations compared to control. The concentrations of total cholesterol, free and esterified cholesterol, triglycerides, and TBARS were increased in the macrophages of COCO-fed mice, while the content of total phospholipids did not change. The phospholipid composition showed an increase of phosphatidylcholine and a decrease of phosphatidylethanolamine. The [³H]-AA distribution in the phospholipid classes showed an increase in phosphatidylcholine and phosphatidylethanolamine. Incorporation of [³H]-cholesterol into the macrophages of COCO-fed mice and into the cholesterol ester fraction was increased. The COCO diet did not affect [³H]-AA uptake but induced an increase in [³H]-AA release. The COCO diet also enhanced AA mobilization induced by lipopolysaccharide. These results indicate that the COCO diet, high in saturated fatty acids, alters the lipid metabolism and AA turnover of peritoneal macrophages in female mice and also produces a significant degree of oxidative stress.

Ectopic development of skeletal muscle induced by subcutaneous transplant of rat satellite cells

The present study analyzes the ectopic development of the rat skeletal muscle originated from transplanted satellite cells. Satellite cells (10(6) cells) obtained from hindlimb muscles of newborn female 2BAW Wistar rats were injected subcutaneously into the dorsal area of adult male rats. After 3, 7, and 14 days, the transplanted tissues (N = 4-5) were processed for histochemical analysis of peripheral nerves, inactive X-chromosome and acetylcholinesterase. Nicotinic acetylcholine receptors (nAChRs) were also labeled with tetramethylrhodamine-labeled alpha-bungarotoxin. The development of ectopic muscles was successful in 86% of the implantation sites. By day 3, the transplanted cells were organized as multinucleated fibers containing multiple clusters of nAChRs (N = 2-4), resembling those from non-innervated cultured skeletal muscle fibers. After 7 days, the transplanted cells appeared as a highly vascularized tissue formed by bundles of fibers containing peripheral nuclei. The presence of X chromatin body indicated that subcutaneously developed fibers originated from female donor satellite cells. Differently from the extensor digitorum longus muscle of adult male rat (87.9 ± 1.0 µm; N = 213), the diameter of ectopic fibers (59.1 µm; N = 213) did not obey a Gaussian distribution and had a higher coefficient of variation. After 7 and 14 days, the organization of the nAChR clusters was similar to that of clusters from adult innervated extensor digitorum longus muscle. These findings indicate the histocompatibility of rats from 2BAW colony and that satellite cells transplanted into the subcutaneous space of adult animals are able to develop and fuse to form differentiated skeletal muscle fibers.

Body fat and cholecalciferol supplementation in elderly homebound individuals

Vitamin D deficiency, observed mainly in the geriatric population, is responsible for loss of bone mass and increased risk of bone fractures. Currently, recommended doses of cholecalciferol are advised, but since there are few studies evaluating the factors that influence the serum levels of 25-hydroxyvitamin D (25(OH)D) following supplementation, we analyzed the relationship between the increase in serum 25(OH)D after supplementation and body fat. We studied a group of 42 homebound elderly subjects over 65 years old (31 women) in order to assess whether there is a need for adjustment of the doses of cholecalciferol administered to this group according to their adipose mass. Baseline measurements of 25(OH)D, intact parathyroid hormone and bone remodeling markers (osteocalcin and carboxy-terminal fraction of type 1 collagen) were performed. Percent body fat was measured by dual-energy X-ray absorptiometry. The patients were divided into three groups according to their percent body fat index and were treated with cholecalciferol, 7,000 IU a week, for 12 weeks. The increases in serum levels of 25(OH)D were similar for all groups, averaging 7.46 ng/mL (P < 0.05). It is noteworthy that this increase only shifted these patients from the insufficiency category to hypovitaminosis. Peak levels of 25(OH)D were attained after only 6 weeks of treatment. This study demonstrated that adipose tissue mass does not influence the elevation of 25(OH)D levels following vitamin D supplementation, suggesting that there is no need to adjust vitamin D dose according to body fat in elderly homebound individuals.

Coronary fat content evaluated by morphometry in patients with severe atherosclerosis has no relation with serum lipid levels

The relationship between lipid serum levels and coronary atherosclerotic plaque fat content was studied in 51 necropsy patients. Serum lipids were measured by standard techniques, during life, in the absence of lipid-lowering drugs. Intima, intimal fat and media areas were measured using a computerized system in cryosections of the odd segments of the right, anterior descending and circumflex coronary arteries stained with Sudan-IV. Mean intimal and lipid areas were 5.74 ± 1.98 and 1.22 ± 0.55 mm² (22.12 ± 8.48%) in 26 cases with high cholesterol (³200 mg/dL) and 4.98 ± 1.94 and 1.16 ± 0.66 mm² (22.75 ± 9.06%) in 25 cases with normal cholesterol (<200 mg/dL; P > 0.05). Patients with high levels of low-density lipoprotein (³130 mg/dL, N = 15) had a higher intima/media area ratio than those with normal levels of low-density lipoprotein (<130 mg/dL, N = 13, P < 0.01). No significant difference in the morphometrical variables was found in groups with high or low serum levels of triglycerides (³200 mg/dL, N = 13 vs <200 mg/dL, N = 36) or high-density lipoprotein (³35 mg/dL, N = 11 vs <35 mg/dL, N = 17). The association between the morphological measurements and serum levels of cholesterol, its fractions, and triglycerides was also tested and the correlation coefficients were low. Although high cholesterol is a risk factor, we show here that in patients with severe atherosclerosis blood cholesterol and triglyceride levels seem to have little influence on coronary lipid content, indicating that other factors may contribute to arterial lipid deposition and plaque formation.

Long-term aerobic swimming training by rats reduces the number of aberrant crypt foci in 1,2-dimethylhydrazine-induced colon cancer

We determined the effect of long-term aerobic swimming training regimens of different intensities on colonic carcinogenesis in rats. Male Wistar rats (11 weeks old) were given 4 subcutaneous injections (40 mg/kg body weight each) of 1,2-dimethyl-hydrazine (DMH, dissolved in 0.9% NaCl containing 1.5% EDTA, pH 6.5), at 3-day intervals and divided into three exercise groups that swam with 0% body weight (EG1, N = 11), 2% body weight (EG2, N = 11), and 4% body weight of load (EG3, N = 10), 20 min/day, 5 days/week for 35 weeks, and one sedentary control group (CG, N = 10). At sacrifice, the colon was removed and counted for tumors and aberrant crypt foci. Tumor size was measured and intra-abdominal fat was weighed. The mean number of aberrant crypt foci was reduced only for EG2 compared to CG (26.21 ± 2.99 vs 36.40 ± 1.53 crypts; P < 0.05). Tumor incidence was not significantly different among groups (CG: 90%; EG1: 72.7%; EG2: 90%; EG3: 80%). Swimming training did not affect either tumor multiplicity (CG: 2.30 ± 0.58; EG1: 2.09 ± 0.44; EG2: 1.27 ± 0.19; EG3: 1.50 ± 0.48 tumors) or size (CG: 1.78 ± 0.24; EG1: 1.81 ± 0.14; EG2: 1.55 ± 0.21; EG3: 2.17 ± 0.22 cm³). Intra-abdominal fat was not significantly different among groups (CG: 10.54 ± 2.73; EG1: 6.12 ± 1.15; EG2: 7.85 ± 1.24; EG3: 5.11 ± 0.74 g). Aerobic swimming training with 2% body weight of load protected against the DMH-induced preneoplastic colon lesions, but not against tumor development in the rat.

Beneficial effects of exercise training (treadmill) on insulin resistance and nonalcoholic fatty liver disease in high-fat fed C57BL/6 mice

C57BL/6 mice develop signs and symptoms comparable, in part, to the human metabolic syndrome. The objective of the present study was to evaluate the effects of exercise training on carbohydrate metabolism, lipid profile, visceral adiposity, pancreatic islet alterations, and nonalcoholic fatty liver disease in C57BL/6 mice. Animals were fed one of two diets during an 8-week period: standard (SC, N = 12) or very high-fat (HF, N = 24) chow. An exercise training protocol (treadmill) was then established and mice were divided into SC and HF sedentary (SC-Sed, HF-Sed), exercised groups (SC-Ex, HF-Ex), or switched from HF to SC (HF/SC-Sed and HF/SC-Ex). HF/HF-Sed mice had the greatest body mass (65% more than SC/SC-Sed; P < 0.0001), and exercise reduced it by 23% (P < 0.0001). Hepatic enzymes ALP (+80%), ALT (+100%) and AST (+70%) were higher in HF/HF mice than in matched SC/SC. Plasma insulin was higher in both the HF/HF-Sed and HF/SC-Sed groups than in the matched exercised groups (+85%; P < 0.001). Pancreatic islets, adipocytes and liver structure were greatly affected by HF, ultimately resulting in islet β-cell hypertrophy and severe liver steatosis. The HF group had larger islets than the SC/SC group (+220%; P < 0.0001), and exercise significantly reduced liver steatosis and islet size in HF. Exercise attenuated all the changes due to HF, and the effects were more pronounced in exercised mice switched from an HF to an SC diet. Exercise improved the lipid profile by reducing body weight gain, visceral adiposity, insulin resistance, islet alterations, and fatty liver, contributing to obesity and steatohepatitis control.

Effects of chronic treadmill training on body mass gain and visceral fat accumulation in overfed rats

This study evaluated the effects of chronic treadmill training on body mass gain and visceral fat accumulation in overfed rats. Overfeeding was induced by reducing the litter size to 3 male pups per mother during the suckling period. The litter size of control rats was adjusted to 10 male pups per mother. Seven weeks after birth overfed and normally fed rats were selected and assigned to a sedentary protocol or to a low-intensity treadmill training protocol (60 min, 5 times/week, for 9 weeks). Four groups (overfed sedentary, N = 23; normally fed sedentary, N = 32; overfed exercised, N = 18, and normally fed exercised, N = 18) were evaluated at 18 weeks. Data are reported as means ± SEM. Initial body weight was similar in control and overfed rats [8.0 ± 0.2 g (N = 42) vs 8.0 ± 0.1 g (N = 50); P > 0.05] and body weight gain during the suckling period was higher in the overfed rats (30.6 ± 0.9 vs 23.1 ± 0.3 g; P < 0.05). Exercise attenuated the body weight gain of overfed compared to sedentary rats (505 ± 14 vs 537 ± 12 g; P < 0.05). The sedentary overfed rats showed higher visceral fat weight compared to normally fed animals (31.22 ± 2.08 vs 21.94 ± 1.76 g; P < 0.05). Exercise reduced visceral fat by 36.5% in normally fed rats and by 35.7% in overfed rats. Exercise attenuated obesity in overfed rats and induced an important reduction of visceral fat.

Anthropometric midarm measurements can detect systemic fat-free mass depletion in patients with chronic obstructive pulmonary disease

Our objective was to determine whether anthropometric measurements of the midarm (MA) could identify subjects with whole body fat-free mass (FFM) depletion. Fifty-five patients (31% females; age: 64.6 ± 9.3 years) with mild/very severe chronic obstructive pulmonary disease (COPD), 18 smokers without COPD (39% females; age: 49.0 ± 7.3 years) and 23 never smoked controls (57% females; age: 48.2 ± 9.6 years) were evaluated. Spirometry, muscle strength and MA circumference were measured. MA muscle area was estimated by anthropometry and MA cross-sectional area by computerized tomography (CT) scan. Bioelectrical impedance was used as the reference method for FFM. MA circumference and MA muscle area correlated with FFM and biceps and triceps strength. Receiver operating characteristic curve analysis showed that MA circumference and MA muscle area cut-off points presented sensitivity and specificity >82% to discriminate FFM-depleted subjects. CT scan measurements did not provide improved sensitivity or specificity. For all groups, there was no significant statistical difference between MA muscle area [35.2 (29.3-45.0) cm²] and MA cross-sectional area values [36.4 (28.5-43.3) cm²] and the linear correlation coefficient between tests was r = 0.77 (P < 0.001). However, Bland-Altman plots revealed wide 95% limits of agreement (-14.7 to 15.0 cm²) between anthropometric and CT scan measurements. Anthropometric MA measurements may provide useful information for identifying subjects with whole body FFM depletion. This is a low-cost technique and can be used in a wider patient population to identify those likely to benefit from a complete body composition evaluation.

Effect of silybin on high-fat-induced fatty liver in rats

Silybin, a natural antioxidant, has been traditionally used against a variety of liver ailments. To investigate its effect and the underlying mechanisms of action on non-alcoholic fatty liver in rats, we used 60 4-6-week-old male Sprague-Dawley rats to establish fatty liver models by feeding a high-fat diet for 6 weeks. Hepatic enzyme, serum lipid levels, oxidative production, mitochondrial membrane fluidity, homeostasis model assessment-insulin resistance index (HOMA-IR), gene and protein expression of adiponectin, and resistin were evaluated by biochemical, reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. Compared with the model group, silybin treatment (26.25 mg·kg-1·day-1, started at the beginning of the protocol) significantly protected against high-fat-induced fatty liver by stabilizing mitochondrial membrane fluidity, reducing serum content of alanine aminotransferase (ALT) from 450 to 304 U/L, decreasing hepatic malondialdehyde (MDA) from 1.24 to 0.93 nmol/mg protein, but increasing superoxide dismutase (SOD) and glutathione (GSH) levels from 8.03 to 9.31 U/mg protein and from 3.65 to 4.52 nmol/mg protein, respectively. Moreover, silybin enhanced the gene and protein expression of adiponectin from 215.95 to 552.40, but inhibited that of resistin from 0.118 to 0.018. Compared to rosiglitazone (0.5 mg·kg-1·day-1, started at the beginning of the protocol), silybin was effective in stabilizing mitochondrial membrane fluidity, reducing SOD as well as ALT, and regulating gene and protein expression of adiponectin (P < 0.05). These results suggest that mitochondrial membrane stabilization, oxidative stress inhibition, as well as improved insulin resistance, may be the essential mechanisms for the hepatoprotective effect of silybin on non-alcoholic fatty liver disease in rats. Silybin was more effective than rosiglitazone in terms of maintaining mitochondrial membrane fluidity and reducing oxidative stress.

Effect of high-fat diets on body composition, lipid metabolism and insulin sensitivity, and the role of exercise on these parameters

Dietary fat composition can interfere in the development of obesity due to the specific roles of some fatty acids that have different metabolic activities, which can alter both fat oxidation and deposition rates, resulting in changes in body weight and/or composition. High-fat diets in general are associated with hyperphagia, but the type of dietary fat seems to be more important since saturated fats are linked to a positive fat balance and omental adipose tissue accumulation when compared to other types of fat, while polyunsaturated fats, omega-3 and omega-6, seem to increase energy expenditure and decrease energy intake by specific mechanisms involving hormone-sensitive lipase, activation of peroxisome proliferator-activated receptor α (PPARα) and others. Saturated fat intake can also impair insulin sensitivity compared to omega-3 fat, which has the opposite effect due to alterations in cell membranes. Obesity is also associated with impaired mitochondrial function. Fat excess favors the production of malonyl-CoA, which reduces GLUT4 efficiency. The tricarboxylic acid cycle and beta-oxidation are temporarily uncoupled, forming metabolite byproducts that augment reactive oxygen species production. Exercise can restore mitochondrial function and insulin sensitivity, which may be crucial for a better prognosis in treating or preventing obesity.