Alterations in gut microbiota associated with a cafeteria diet and the physiological consequences in the host.

OBJECTIVE: Gut microbiota have been described as key factors in the pathophysiology of obesity and different components of metabolic syndrome (MetS). The cafeteria diet (CAF)-fed rat is a preclinical model that reproduces most of the alterations found in human MetS by simulating a palatable human unbalanced diet. Our objective was to assess the effects of CAF on gut microbiota and their associations with different components of MetS in Wistar rats. METHODS: Animals were fed a standard diet or CAF for 12 weeks. A partial least square-based methodology was used to reveal associations between gut microbiota, characterized by 16S ribosomal DNA gene sequencing, and biochemical, nutritional and physiological parameters. RESULTS: CAF feeding resulted in obesity, dyslipidemia, insulin resistance and hepatic steatosis. These changes were accompanied by a significant decrease in gut bacterial diversity, decreased Firmicutes and an increase in Actinobacteria and Proteobacteria abundances, which were concomitant with increased endotoxemia. Associations of different genera with the intake of lipids and carbohydrates were opposed from those associated with the intake of fiber. Changes in gut microbiota were also associated with the different physiological effects of CAF, mainly increased adiposity and altered levels of plasma leptin and glycerol, consistent with altered adipose tissue metabolism. Also hepatic lipid accretion was associated with changes in microbiota, highlighting the relevance of gut microbiota homeostasis in the adipose-liver axis. CONCLUSIONS: Overall, our results suggest that CAF feeding has a profound impact on the gut microbiome and, in turn, that these changes may be associated with important features of MetS.

Grape seed proanthocyanidin supplementation reduces adipocyte size and increases adipocyte number in obese rats.

OBJECTIVES: White adipose tissue (WAT) expands through hypertrophy (increased adipocyte size) and/or hyperplasia (increased adipocyte number). Hypertrophy has been associated with insulin resistance and dyslipidemia independently of body composition and fat distribution. In contrast, hyperplasia protects against metabolic alterations. Proanthocyanidins, which are the most abundant flavonoids in the human diet, improve metabolic disturbances associated with diet-induced obesity without reducing body weight or adiposity. The aim of this study was to determine whether grape seed proanthocyanidin extract (GSPE) can modulate WAT expandability. Because GSPE also contains gallic acid, we also studied the capacity of gallic acid to remodel WAT. DESIGN: Male Wistar rats were fed a standard chow diet (n=6) or a cafeteria diet (CAF) for 11 weeks. After 8 weeks, the CAF-fed animals were supplemented with 25 mg GSPE/kg body weight (n=6), 7 mg gallic acid/kg body weight (n=6) or the vehicle (n=6) for 3 weeks. Histological analyses were performed in the retroperitoneal (rWAT) and inguinal (iWAT) WAT to determine adipocyte size and number. Specific markers for adipogenesis and WAT functionality were analysed in rWAT using quantitative RT-PCR. RESULTS: GSPE or gallic acid supplementation did not reduce weight gain or reverse and adiposity. However, GSPE reduced adipocyte size significantly in rWAT and moderately in iWAT and tripled the adipocyte number in rWAT. Gallic acid slightly reduced adipocyte size in rWAT and iWAT and doubled the adipocyte number in both WATs. In accordance with this adipogenic activity, Pref-1 and PPARγ tended to be overexpressed in rWAT of rats supplemented with GSPE. Moreover, GSPE supplementation increased Plin1 and Fabp4 expression and restored adiponectin expression completely, indicating a better functionality of visceral WAT. CONCLUSIONS: GSPE supplementation has anti-hypertrophic and hyperplasic activities in rats with established obesity, mainly in visceral WAT inducing a healthier expansion of WAT to match the surplus energy provided by the cafeteria diet.

Grape seed procyanidin supplementation to rats fed a high-fat diet during pregnancy and lactation increases the body fat content and modulates the inflammatory response and the adipose tissue metabolism of the male offspring in youth.

OBJECTIVE: Procyanidins are polyphenolic bioactive compounds that exert beneficial effects against obesity and its related diseases. The aim of this study was to evaluate whether supplementation with low doses of a grape seed procyanidin extract (GSPE) to rats during pre- and postnatal periods provides biological effects to their offspring in youth. DESIGN: The metabolic programming effect of GSPE was evaluated in the 30-day-old male offspring of four groups of rats that were fed either a standard diet (STD) or a high-fat diet (HFD) and that were supplemented with either GSPE (25 mg kg(-1) of body weight per day) or vehicle during pregnancy and lactation. RESULTS: Significant increases in the adiposity index and in the weights of all the white adipose tissue depots studied (retroperitoneal, mesenteric, epididymal (EWAT) and inguinal) were observed in the offspring of rats that were fed a HFD and that were treated with GSPE (HFD-GSPE group) compared with the offspring of rats that were fed the same diet but that did not receive the procyanidins (HFD group). The HFD-GSPE animals also exhibited a higher number of cells in the EWAT, a sharp decrease in the circulating levels of monocyte chemoattractant protein-1 (MCP-1) and a moderate decrease in the plasma glycerol levels. The transcriptomic analysis performed in the EWAT showed 238 genes that were differentially expressed between the HFD and the HFD-GSPE animals, most of which were associated with the immune function and the inflammatory response, in addition to genes associated with adipose tissue remodeling and function, lipid and glucose homeostasis and the metabolism of methyl groups. CONCLUSION: The GSPE treatment in rats that were fed an HFD during pregnancy and lactation induced a clear metabolic programming effect in the offspring, increasing adiposity, decreasing the circulating levels of MCP-1 and changing the gene expression in the EWAT toward a better inflammatory profile.

Mapping of the circulating metabolome reveals α-ketoglutarate as a predictor of morbid obesity-associated non-alcoholic fatty liver disease.

BACKGROUND: Obesity severely affects human health, and the accompanying non-alcoholic fatty liver disease (NAFLD) is associated with high morbidity and mortality. Rapid and non-invasive methods to detect this condition may substantially improve clinical care. METHODS: We used liquid and gas chromatography-quadruple time-of-flight-mass spectrometry (LC/GC-QTOF-MS) analysis in a non-targeted metabolomics approach on the plasma from morbidly obese patients undergoing bariatric surgery to gain a comprehensive measure of metabolite levels. On the basis of these findings, we developed a method (GC-QTOF-MS) for the accurate quantification of plasma α-ketoglutarate to explore its potential as a novel biomarker for the detection of NAFLD. RESULTS: Plasma biochemical differences were observed between patients with and without NAFLD indicating that the accumulation of lipids in hepatocytes decreased ß-oxidation energy production, reduced liver function and altered glucose metabolism. The results obtained from the plasma analysis suggest pathophysiological insights that link lipid and glucose disturbances with α-ketoglutarate. Plasma α-ketoglutarate levels are significantly increased in obese patients compared with lean controls. Among obese patients, the measurement of this metabolite differentiates between those with or without NAFLD. Data from the liver were consistent with data from plasma. Clinical utility was assessed, and the results revealed that plasma α-ketoglutarate is a fair-to-good biomarker in patients (n=230). Other common laboratory liver tests used in routine application did not favourably compare. CONCLUSION: Plasma α-ketoglutarate is superior to common liver function tests in obese patients as a surrogate biomarker of NAFLD. The measurement of this biomarker may potentiate the search for a therapeutic approach, may decrease the need for liver biopsy and may be useful in the assessment of disease progression.

Detection and characterization of silver nanoparticles and dissolved species of silver in culture medium and cells by AsFlFFF-UV-Vis-ICPMS: application to nanotoxicity tests.

A methodology based on Asymmetric Flow Field-Flow Fractionation (AsFlFFF) coupled with UV-Vis absorption spectrometry and ICP mass spectrometry (ICPMS) has been developed and applied to the study of silver nanoparticles (AgNPs) and dissolved species of silver in culture media and cells used in cytotoxicity tests. The effect of a nano-silver based product (protein stabilized silver nanoparticles ca. 15 nm average diameter) on human hepatoma (HepG2) cell viability has been studied. UV-Vis absorption spectrometry provided information about the nature (organic vs. nanoparticle) of the eluted species, whereas the silver was monitored by ICPMS. A shift towards larger hydrodynamic diameters was observed in the AgNPs after a 24 hour incubation period in the culture medium, which suggests a "protein corona" effect. Silver(I) associated with proteins present in the culture medium has also been detected, as a consequence of the oxidation process experimented by the AgNPs. However, the Ag(I) released into the culture medium did not justify the toxicity levels observed. AgNPs associated with the cultured HepG2 cells were also identified by AsFlFFF, after applying a solubilisation process based on the use of tetramethylammonium hydroxide (TMAH) and Triton X-100. These results have been confirmed by transmission electronic microscopy (TEM) analysis of the fractions collected from the AsFlFFF. The effect of AgNPs on HepG2 cells has been compared to that caused by silver(I) as AgNO3 under the same conditions. The determination of the total content of silver in the cells confirms that a much larger mass of silver as AgNPs with respect to AgNO3 (16 to 1) is needed to observe a similar toxicity.

Low doses of grape seed procyanidins reduce adiposity and improve the plasma lipid profile in hamsters.

OBJECTIVE: Procyanidins are polyphenolic compounds with beneficial effects on health in relation to cardiovascular disease and metabolic syndrome. In this study, we evaluated the potential beneficial effects of low doses of a grape seed procyanidin extract (GSPE) on body weight and fat deposition. DESIGN: Four groups of hamsters were fed either a standard diet (STD) or a high-fat diet (HFD) for 30 days and supplemented with either GSPE at 25 mg per kg of body weight per day (STD-GSPE and HFD-GSPE groups) or vehicle (STD and HFD groups) during the last 15 days of the study. RESULTS: A significant decrease in body weight gain was observed in both GSPE-treated animals at the end of the experiment. GSPE treatment significantly reduced the adiposity index and the weight of all the white adipose tissue depots studied (retroperitoneal (RWAT), mesenteric (MWAT), epididymal (EWAT) and inguinal (IWAT)) in both GSPE-treated groups. GSPE administration reversed the increase in plasma phospholipids induced by the HFD feeding. In the RWAT, GSPE treatment increased the mRNA expression of genes related to ß-oxidation and the glycerolipid/free fatty acid (GL/FFA) cycle, mainly in HFD-GSPE animals. In the MWAT, the effects of GSPE at the transcriptional level were not as evident as in the RWAT. Moreover, GSPE treatment induced heparin-releasable lipoprotein lipase activity in the RWAT and MWAT depots. The alterations in the lipid metabolic pathways induced by GSPE were accompanied by lower FFA levels in the plasma and decreased lipid and triglyceride accumulation in the MWAT. CONCLUSION: The use of GSPE at low doses protects against fat accumulation and improves the plasma lipid profile in hamsters. We suggest that GSPE exerts these effects in part through the activation of both ß-oxidation and the GL/FFA cycle, mainly in the RWAT.

Atherosclerosis prevention by nutritional factors: a meta-analysis in small animal models.

BACKGROUND AND AIMS: Atherosclerosis prevention in small laboratory models has been used as a preclinical stage in the development of functional foods with claimed antiatherogenic properties. However, a high heterogeneity of experimental atherosclerosis models as well as species-specific differences in lipoprotein metabolism could limit the usefulness of these developments. To solve this, we have performed a meta-analysis on the effects of nutritional complements (i.e. less than 2% of diet) with potential antiatherogenic properties in mice, rabbits and hamsters, and compared the outcomes with those obtained in humans. METHODS AND RESULTS: A meta-analysis comprising works dealing with dietary prevention of experimental atherosclerosis (i.e. macroscopic and/or pathological evidences of atheromatosis in aorta) has been performed (n = 110 works). Quality criteria were applied resulting in selection of 16 works comprising 511 animals. Despite high heterogeneity, there is a significant effect of nutritional interventions reducing atheroma globally (mean effect 24.38% (95% CI: 13.24-35.51%) of prevention). In mouse studies (20.64% (95% CI: 8.38-32.90%)) and in rabbits (40.48% (95% CI: 6.73-74.23%)) this effect was significant, in contrast with hamster-based works (95% CI: 13.66-49.48%). Meta-regression showed that reduction of atheroma plaque formation was not linked to changes either in total circulating cholesterol or LDL cholesterol levels. CONCLUSION: Nutritional addition of selected compounds significantly prevents experimental atheromatosis, but the reproduction of positive effects observed in humans was very limited. These analyses reinforce the need for adequate standardization of atherosclerosis studies in preclinical models and for human intervention trials.

Relative absorption of silicon from different formulations of dietary supplements: a pilot randomized, double-blind, crossover post-prandial study.

The purpose of the present study was to compare the relative absorption of a new powder presentation of silicon (Si) as orthosilicic acid with maltodextrin (Orgono Powder) compared to usual Si liquid presentations as orthosilicic acid with Equisetum arvense and Rosmarinus officinalis (G5 Siliplant) and orthosilicic acid with aloe vera (G7 Aloe). All dietary supplements were administered at the same Si oral dose (21.6 mg) in a randomized, double-blind, crossover post-prandial study conducted in 5 healthy men. Urine was collected at baseline and over the 6-h post-dose period in 2 separate 3-h collections for the analysis of Si concentration, which was conducted by inductively coupled plasma optical emission spectrometry as the gold standard method. No significant differences in total urinary Si excretion were found after the intake of these 3 dietary supplements; 34.6%, 32.4% and 27.2% of the ingested Si from G7 Aloe, G5 Siliplant and Orgono Powder, respectively, was excreted in urine over the 6-h follow-up period. The 3 different oral Si formulations tested, in powder and liquid presentations, provide highly bioavailable Si and present an equivalent relative absorption in healthy humans.

Effects of Hesperidin Consumption on the Cardiovascular System in Pre- and Stage 1 Hypertensive Subjects: Targeted and Non-Targeted Metabolomic Approaches (CITRUS Study).

SCOPE: The aim of the present work is to determine new biomarkers of the biological effects of hesperidin in orange juice (OJ) applying a non-targeted metabolomics approach validated by targeted metabolomics analyses of compliance biomarkers. METHODS AND RESULTS: Plasma/serum and urine targeted (HPLC-MS/MS) and untargeted (1 H-NMR) metabolomics signatures are explored in a subsample with pre- and stage-1 hypertension subjects of the CITRUS study (N = 159). Volunteers received 500 mL day-1 of control drink, OJ, or hesperidin-enriched OJ (EOJ) for 12-weeks. A 6-h postprandrial study is performed at baseline. Targeted analyses reveals plasma and urine hesperetin 7-O-ß-d-glucuronide as the only metabolite differing between OJ and EOJ groups after 12-weeks consumption, and in urine is correlated with a decreased systolic blood pressure level. The non-targeted approach shows that after single dose and 12-weeks consumption of OJ and EOJ change several metabolites related with an anti-inflammatory and antioxidant actions, lower blood pressure levels and uremic toxins. CONCLUSIONS: Hesperetin 7-O-ß-d-glucuronide can be a candidate marker for distinguishing between the consumption of different hesperidin doses at 12-weeks consumption as well as a potential agent mediating blood pressure reduction. Moreover, changes in different endogenous metabolites can explain the mechanisms of action and the biological effects of hesperidin consumption.

Grape seed proanthocyanidins correct dyslipidemia associated with a high-fat diet in rats and repress genes controlling lipogenesis and VLDL assembling in liver.

OBJECTIVE: To determine whether proanthocyanidins can protect against dyslipidemia induced by a high-fat diet (HFD) and to address the mechanisms that underlie this hypolipidemic effect. DESIGN AND MEASUREMENTS: Female Wistar rats were fed on a HFD for 13 weeks. They were divided into two groups, one of which was treated with a grape seed proanthocyanidin extract (25 mg kg(-1) of body weight) for 10 days. Plasma and liver lipids were measured by colorimetric and gravimetric analysis. Liver, muscle and adipose tissue were used to study the expression of genes involved in the synthesis and oxidation of fatty acids and lipoprotein homeostasis by real-time RT-PCR. RESULTS: The administration of proanthocyanidins normalized plasma triglyceride and LDL-cholesterol (both parameters significantly increased with the HFD) but tended to decrease hypercholesterolemia and fatty liver. Gene expression analyses revealed that proanthocyanidins repressed both the expression of hepatic key regulators of lipogenesis and very low density lipoprotein (VLDL) assembling such as SREBP1, MTP and DGAT2, all of which were overexpressed by the HFD. CONCLUSION: These findings indicate that natural proanthocyanidins improve dyslipidemia associated with HFDs, mainly by repressing lipogenesis and VLDL assembly in the liver, and support the idea that they are powerful agents for preventing and treating lipid altered metabolic states.

Distribution and evaluation of sense of coherence among older immigrants before and after a health promotion intervention - results from the RCT study promoting aging migrants' capability.

BACKGROUND: The migration process can be a threat to a person's sense of coherence (SOC) and to their ability to experience life as comprehensible, manageable, and meaningful. Seen from a salutogenic perspective, this may have a negative impact on the experience of health. PURPOSE: We describe the distribution of SOC and its components among older persons with an immigrant background now aging in Sweden. In addition, we evaluated whether a group-based health promotion program with a person-centered approach could support the SOC among older persons in this group. MATERIALS AND METHODS: A randomized controlled trial with postintervention follow-ups at 6 and 12 months was conducted with 131 independently living persons aged ≥70 years from Finland and the Balkan Peninsula. Participants were randomly allocated to an intervention group (4 weeks of group intervention and one follow-up home visit) and a control group (no intervention but access to ordinary health care services). The outcome measure was the SOC measured by SOC-13. Chi-square and ORs were calculated. RESULTS: There was a significant improvement in total SOC scores for the intervention group at 6-month follow-up. Also, the ORs for the SOC components were higher in the person-centered intervention group. However, we found no significant between-group differences nor did the effect last until the 12-month follow-up. CONCLUSION: Persons who have lived a long time in a host country after migration seem to have a SOC similar to native-born persons. Interventions with a person-centered approach could support the SOC by capturing individual life situations. Such interventions could support older persons by making everyday life more comprehensible and manageable and helping them to cope with challenges in daily life caused by aging.

COCOA (Theobroma cacao) Polyphenol-Rich Extract Increases the Chronological Lifespan of Saccharomyces cerevisiae.

BACKGROUND: Saccharomyces cerevisiae is a model organism with conserved aging pathways. Yeast chronological lifespan experiments mimic the processes involved in human non-dividing tissues, such as the nervous system or skeletal muscle, and can speed up the search for biomolecules with potential anti-aging effects before proceeding to animal studies. OBJECTIVE: To test the effectiveness of a cocoa polyphenol-rich extract (CPE) in expanding the S. cerevisiae chronological lifespan in two conditions: in the stationary phase reached after glucose depletion and under severe caloric restriction. MEASUREMENTS: Using a high-throughput method, wild-type S. cerevisiae and its mitochondrial manganese-dependent superoxide dismutase null mutant (sod2Δ) were cultured in synthetic complete dextrose medium. After 2 days, 0, 5 and 20 mg/ml of CPE were added, and viability was measured throughout the stationary phase. The effects of the major components of CPE were also evaluated. To determine yeast lifespan under severe caloric restriction conditions, cultures were washed with water 24 h after the addition of 0 and 20 mg/ml of CPE, and viability was followed over time. RESULTS : CPE increased the chronological lifespan of S. cerevisiae during the stationary phase in a dose-dependent manner. A similar increase was also observed in (sod2Δ). None of the major CPE components (theobromine, caffeine, maltodextrin, (-)-epicatechin, (+)-catechin and procyanidin B2) was able to increase the yeast lifespan. CPE further increased the yeast lifespan under severe caloric restriction. CONCLUSION: CPE increases the chronological lifespan of S. cerevisiae through a SOD2-independent mechanism. The extract also extends yeast lifespan under severe caloric restriction conditions. The high-throughput assay used makes it possible to simply and rapidly test the efficacy of a large number of compounds on yeast aging, requiring only small amounts, and is thus a convenient screening assay to accelerate the search for biomolecules with potential anti-aging effects.

In vitro glucose and 2-aminoisobutyric acid uptake by rat interscapular brown adipose tissue.

The dependence upon substrate and insulin concentrations, as well as on sodium and potassium concentrations in the medium of the uptake of glucose and 2-aminoisobutyric acid, was determined for fragments of brown and white adipose tissues incubated in vitro. Brown adipose tissue showed a high capacity for glucose uptake at high glucose concentrations, this uptake being dependent on both glucose and insulin concentration. White adipose tissue showed much more limited uptake capabilities. The presence of Na+ and K+ had little effect on the uptake. The uptake of 2-aminoisobutyric acid was similar in both adipose tissues, being enhanced by physiological levels of insulin and depressed by ouabain. This amino acid transport was dependent on Na+ and K+ concentrations, and the overall transporting capability was two to three orders of magnitude lower than that for glucose. It was concluded that amino acids could not play a significant role as bulk thermogenic substrates for brown adipose tissue, as their transporters lack the plasticity of response to high substrate and insulin concentrations which characterize brown adipose tissue uptake of glucose.

Metabolic fate of glucose on 3T3-L1 adipocytes treated with grape seed-derived procyanidin extract (GSPE). Comparison with the effects of insulin.

In this paper we investigate the effects of a grape seed procyanidin extract (GSPE) on the metabolic fate of glucose in adipocytes. Differentiated 3T3-L1 cells were treated with 140 mg/L GSPE or 100 nM insulin for a short period (1 h, acute treatment) or for a long period (15 h, chronic treatment). 2-Deoxy-[1-(3)H]glucose uptake and [1-(14)C]glucose incorporation into cells, glycogen, and lipid were measured. We found that GSPE mimicked the anabolic effects of insulin but there were several important differences. GSPE stimulated glycogen synthesis less than insulin. After chronic exposure, GSPE induced a higher incorporation of glucose into lipid, mainly due to the increase in glucose directed to glycerol synthesis. Our main conclusions, therefore, are that GSPE has insulinomimetic properties and activates glycogen and lipid synthesis. However, the differences between the effects of GSPE and the effects of insulin indicate that GSPE uses mechanisms complementary to those of insulin signaling pathways to bring about these effects.

Chronic consumption of dietary proanthocyanidins modulates peripheral clocks in healthy and obese rats.

Circadian rhythm plays an important role in maintaining homeostasis, and its disruption increases the risk of developing metabolic syndrome. Circadian rhythm is maintained by a central clock in the hypothalamus that is entrained by light, but circadian clocks are also present in peripheral tissues. These peripheral clocks are trained by other cues, such as diet. The aim of this study was to determine whether proanthocyanidins, the most abundant polyphenols in the human diet, modulate the expression of clock and clock-controlled genes in the liver, gut and mesenteric white adipose tissue (mWAT) in healthy and obese rats. Grape seed proanthocyanidin extracts (GSPEs) were administered for 21 days at 5, 25 or 50 mg GSPE/kg body weight in healthy rats and 25 mg GSPE/kg body weight in rats with diet-induced obesity. In healthy animals, GSPE administration led to the overexpression of core clock genes in a positive dose-dependent manner. Moreover, the acetylated BMAL1 protein ratio increased with the same pattern in the liver and mWAT. With regards to clock-controlled genes, Per2 was also overexpressed, whereas Rev-erbα and RORα were repressed in a negative dose-dependent manner. Diet-induced obesity always resulted in the overexpression of some core clock and clock-related genes, although the particular gene affected was tissue specific. GSPE administration counteracted disturbances in the clock genes in the liver and gut but was less effective in normalizing the clock gene disruption in WAT. In conclusion, proanthocyanidins have the capacity to modulate peripheral molecular clocks in both healthy and obese states.

Grape seed-derived procyanidins have an antihyperglycemic effect in streptozotocin-induced diabetic rats and insulinomimetic activity in insulin-sensitive cell lines.

Flavonoids are functional constituents of many fruits and vegetables. Some flavonoids have antidiabetic properties because they improve altered glucose and oxidative metabolisms of diabetic states. Procyanidins are flavonoids with an oligomeric structure, and it has been shown that they can improve the pathological oxidative state of a diabetic situation. To evaluate their effects on glucose metabolism, we administered an extract of grape seed procyanidins (PE) orally to streptozotocin-induced diabetic rats. This had an antihyperglycemic effect, which was significantly increased if PE administration was accompanied by a low insulin dose. The antihyperglycemic effect of PE may be partially due to the insulinomimetic activity of procyanidins on insulin-sensitive cell lines. PE stimulated glucose uptake in L6E9 myotubes and 3T3-L1 adipocytes in a dose-dependent manner. Like insulin action, the effect of PE on glucose uptake was sensitive to wortmannin, an inhibitor of phosphoinositol 3-kinase and to SB203580, an inhibitor of p38 MAPK. PE action also stimulated glucose transporter-4 translocation to the plasma membrane. In summary, procyanidins have insulin-like effects in insulin-sensitive cells that could help to explain their antihyperglycemic effect in vivo. These effects must be added to their antioxidant activity to explain why they can improve diabetic situations.

Determination of plasma amino acids in small samples with the use of Dansyl-chloride.

A radiochemical method based on dansylation of plasma samples with Dansyl-chloride and thin alyer chromatography on polyamide sheets is presented for the quantitative individual determination of plasma amino acids in very small blood samples. The results are first corrected with a norvaline internal standard and secondly with specific recovery factors for each amino acid. The results agree slosely with other methods and with already published plasma normal amino acids values in healthy adult rats.

Respiratory toxicity of copper.

Respiratory toxicity of copper was tested in Wistar rats by spraying copper sulfate (330 g/l spray) for daily periods of 1 hr in a self-contained chamber for up to 10 days. The respiratory toxicity was compared with that from intraperitoneal administration of 1 mg Cu/mg body weight and with adequate control rats. Analysis of tissue Cu and Zn was done in lung, liver, kidney, and plasma by using atomic absorption spectrophotometry. Similar organ and subcellular distribution of both elements were found between the two treated groups, and only statistically significant higher levels of Cu were found in plasma and liver. After exposure, Cu and Zn were basically associated with a low-molecular-weight component, which eluted as metallothionein in the postmicrosomal fractions.

Nickel-induced hyperglycaemia: the role of insulin and glucagon.

Glucagon and insulin changes were measured in acute nickel-treated rats. Also, several parameters related to glucose homeostasis were evaluated. Nickel treatment caused an important and transitory rise in plasma glucose levels. These changes occurred simultaneously to hyperglucagonemia and hypoinsulinemia, leading to a drastic drop in the insulin/glucagon plasma ratio. In such a catabolic situation, hepatic and muscular glycogen levels remained almost unaltered. Hepatic fructose-2,6-bisphosphate (an indicator of gluconeogenic/glycolytic state) was drastically reduced a short time after nickel injection. Such events suggested that it was mainly gluconeogenesis and not glycogenolysis, which contributes to enhanced plasma glucose. Animals treated with large doses of glucagon did not mimic the hyperglycaemic responses induced by nickel, due to counteracting effects of insulin on plasma glucose. When diabetic rats were treated with nickel, the hyperglucagonemic response still remained, but plasma glucose levels did not increase at the same extent as when nickel was applied to control animals. Overall results suggest that both, glucagon and insulin changes are essential in the development of nickel-induced hyperglycaemia. Also, the lack of glycogenolytic response insinuates a direct or indirect inhibition of this process mediated by nickel and will need further investigation.

Effects of acute nickel toxicity upon plasma and liver metal homeostasis as a function of sex.

The effects of an acute dose of nickel chloride (4 mg Ni/kg body wt) upon liver and plasma essential metal homoeostasis were studied in male and female rats. Total levels of copper and zinc in the tissues and in the fractions of chromatographic profiles (Sephadex G-100 and G-150) were determined. Plasma and liver levels of both metals rose significantly. The higher levels of copper in plasma are associated with increased ceruloplasmin activity and the initial increase of zinc in plasma is due to higher zinc content in the plasma albumin fraction. In the liver, the higher levels of both metals similarly affected all the metal-containing chromatographic fractions, although a significant increase is only observed in metallothionein-containing fractions, which agrees with previous reports on increased levels of metallothionein after nickel treatment. Regarding the sex-dependent changes, both sexes showed the same alterations, yet males recovered faster than females from all the nickel-induced changes in metal homoeostasis.