Impact of dietary protein content on uncoupling protein mRNA abundance in swine

The present study was designed to determine if dietary protein can alter uncoupling protein (UCP) expression in swine, as has been shown in rats, and attempt to identify the mechanism. Eight pigs (~ 50 kg body mass) were fed an 18% crude protein (CP) diet while another eight pigs were switched to a diet containing 12% crude protein (CP) and fed these diets until 110 kg body mass. The outer (OSQ) and middle (MSQ) subcutaneous adipose tissues, liver, leaf fat, longissimus (LM), red portion of the semitendinosus (STR) and the white portion of the ST (STW) were analyzed for gene expression by real-time PCR. Feeding of 12% CP did not alter growth or carcass composition, relative to 18% CP (P > 0.05). Serum growth hormone, non-esterified fatty acids, triglycerides and urea nitrogen were reduced with the feeding of 12% CP (P < 0.05). The UCP2 mRNA abundance was reduced in LM, STR, MSQ and OSQ with feeding of 12% CP (P < 0.05), as was UCP3 mRNA abundance in MSQ and STW (P < 0.01). Peroxisome proliferation activated receptor α (PPARα) and PPARγ were reduced in MSQ and STR (P < 0.05) with feeding 12% CP as was the PPARα regulated protein, acyl CoA oxidase (ACOX, P < 0.05). These data suggest that feeding 12% CP relative to 18% CP reduces serum NEFA, which reduces PPARα and PPARγ expression and consequently reduces UCP2 lipoperoxidation in OSQ and STR and also reduced UCP3 associated fatty acid transport in MSQ and STW.     

Effect of high-intensity exercise and high-fat diet on lipid metabolism in the liver of rats

[Purpose]

This study investigated the effects of high-intensity exercise (Ex) and high dietary fat intake on lipid metabolism in the liver of rats.

[Methods]

Male Sprague-Dawley rats were randomly assigned to one of the four groups (n=10 per group) that were maintained on a normal diet (ND) or high-fat diet (HFD) consisting of 30% fat (w/w), with or without exercise on a treadmill at 30 m/min and 8% grade) for 4 weeks (i.e., ND, ND+Ex, HFD, and HFD+Ex groups).

[Results]

Body weight (p<.001), total plasma cholesterol (TC) (p<.001), triglyceride (TG) (p<.05), and liver TG levels (p<.05) were increased in the HFD group relative to the ND groups, and serum glucose (p<.05), insulin (p<.05), homeostatic model assessment of insulin resistance (HOMA-IR) (p<.01), and liver TG levels (p<.01) were also higher in the HFD group compared to the ND+Ex group. Plasma free fatty acid was elevated in the HFD+Ex group compared to the HFD group (p<.01). With the exception of acetyl coenzyme A carboxylase, the expression of lipid metabolism-related genes in the liver was altered in the Ex groups compared to the control group (p<.05), with genes involved in lipolysis specifically up regulated in the HFD+Ex group compared to the other groups.

[Conclusion]

Vigorous exercise may increase glucose utilization and fat oxidation by activating genes in the liver that are associated with lipid metabolism compared to that in animals consuming a HFD without exercise. Therefore, high intensity exercise can be considered to counter the adverse effects of high dietary fat intake.     

Effects of Dietary Pantothenic Acid on Growth,Intestinal Function,Anti-Oxidative Status and Fatty Acids Synthesis of Juvenile Blunt Snout Bream Megalobrama amblycephala

Four groups of juvenile Megalobrama amblycephala were fed three times daily with six semi-purified diets containing 3.39 (PA unsupplied diet), 10.54, 19.28, 31.04, 48.38 and 59.72 mg kg^-1 calcium D-pantothenate. The results showed that survival rate, final weight, specific growth rate, protein efficiency ratio and nitrogen retention efficiency all increased significantly (P<0.01) as dietary PA levels increased from 3.39 to 19.28 mg kg^-1, whereas the opposite was true for feed conversion ratio. Whole-body crude protein increased as dietary PA levels increased, while the opposite pattern was found for the crude lipid content. Intestinal α-amylase, lipase, protease, Na⁺-K⁺-ATPase, alkaline phosphatase and gamma-glutamyl transferase activities were all elevated in fish fed PA-supplemented diets. Hepatic catalase activities improved with increases in dietary PA, while the opposite was true for malondialdehyde contents. The liver PA concentration and coenzyme A content rose significantly (P<0.01), up to 31.04 mg kg^-1, with increasing dietary PA levels and then plateaued. The percentage of hepatic saturated fatty acids increased significantly (P<0.01) as dietary PA levels increased, while the percentages of monounsaturated fatty acids and polyunsaturated fatty acid (PUFA) decreased as dietary PA increased. Fish fed diets containing 19.28 and 31.04 mg kg^-1 PA exhibited higher (P<0.01) docosahexaenoic acid and PUFA percentages in muscle than those fed with other diets. The expression of the gene encoding pantothenate kinase was significantly up-regulated (P<0.01) in fish fed PA-supplemented diets. Hepatic Acetyl-CoA carboxylase α, fatty acid synthetase, stearoyl regulatory element-binding protein 1 and X receptor α genes all increased significantly (P<0.01) as dietary PA levels increased from 3.39 to 31.04 mg kg^-1. Based on broken-line regression analyses of weight gain, liver CoA concentrations and PA contents against dietary PA levels, the optimal dietary PA requirements of juvenile blunt snout bream were estimated to be 24.08 mg kg^-1.     

Divergent effects of eicosapentaenoic and docosahexaenoic acid ethyl esters, and fish oil on hepatic fatty acid oxidation in the rat

The physiological activity of fish oil, and ethyl esters of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) affecting hepatic fatty acid oxidation was compared in rats. Five groups of rats were fed various experimental diets for 15 days. A group fed a diet containing 9.4% palm oil almost devoid of n-3 fatty acids served as a control. The test diets contained 4% n-3 fatty acids mainly as EPA and DHA in the form of triacylglycerol (9.4% fish oil) or ethyl esters (diets containing 4% EPA ethyl ester, 4% DHA ethyl ester, and 1% EPA plus 3% DHA ethyl esters). The lipid content of diets containing EPA and DHA ethyl esters was adjusted to 9.4% by adding palm oil. The fish oil diet and ethyl ester diets, compared to the control diet containing 9.4% palm oil, increased activity and mRNA levels of hepatic mitochondrial and peroxisomal fatty acid oxidation enzymes, though not 3-hydroxyacyl-CoA dehydrogenase activity. The extent of the increase was, however, much greater with the fish oil than with EPA and DHA ethyl esters. EPA and DHA ethyl esters, compared to the control diet, increased 3-hydroxyacyl-CoA dehydrogenase activity, but fish oil strongly reduced it. It is apparent that EPA and DHA in the form of ethyl esters cannot mimic the physiological activity of fish oil at least in affecting hepatic fatty acid oxidation in rat.     

Highly purified eicosapentaenoic acid prevents the progression of hepatic steatosis by repressing monounsaturated fatty acid synthesis in high-fat/high-sucrose diet-fed mice

Eicosapentaenoic acid (EPA) is a member of the family of n-3 polyunsaturated fatty acids (PUFAs) that are clinically used to treat hypertriglyceridemia. The triglyceride (TG) lowering effect is likely due to an alteration in lipid metabolism in the liver, but details have not been fully elucidated. To assess the effects of EPA on hepatic TG metabolism, mice were fed a high-fat and high-sucrose diet (HFHSD) for 2 weeks and were given highly purified EPA ethyl ester (EPA-E) daily by gavage. The HFHSD diet increased the hepatic TG content and the composition of monounsaturated fatty acids (MUFAs). EPA significantly suppressed the hepatic TG content that was increased by the HFHSD diet. EPA also altered the composition of fatty acids by lowering the MUFAs C16:1 and C18:1 and increasing n-3 PUFAs, including EPA and docosahexaenoic acid (DHA). Linear regression analysis revealed that hepatic TG content was significantly correlated with the ratios of C16:1/C16:0, C18:1/C18:0, and MUFA/n-3 PUFA, but was not correlated with the n-6/n-3 PUFA ratio. EPA also decreased the hepatic mRNA expression and nuclear protein level of sterol regulatory element binding protein-1c (SREBP-1c). This was reflected in the levels of lipogenic genes, such as acetyl-CoA carboxylase α (ACCα), fatty acid synthase, stearoyl-CoA desaturase 1 (SCD1), and glycerol-3-phosphate acyltransferase (GPAT), which are regulated by SREBP-1c. In conclusion, oral administration of EPA-E ameliorates hepatic fat accumulation by suppressing TG synthesis enzymes regulated by SREBP-1 and decreases hepatic MUFAs accumulation by SCD1.     

Serum levels of appetite-regulating hormones and pro-inflammatory cytokines are ameliorated by a CLA diet and endurance exercise in rats fed a high-fat diet

[Purpose]

This study examined whether conjugated linoleic acid (CLA) supplementation and endurance exercise affect appetite-regulating hormones and pro-inflammatory cytokines in rats.

[Methods]

Seven-week-old male Sprague-Dawley rats were divided randomly into the high-fat diet sedentary group (HS, n=8), the 1.0% CLA supplemented high-fat diet sedentary group (CS, n=8), and the 1.0% CLA supplemented high-fat diet exercise group (CE, n=8). Rats in the CE group swam 60 min/day, 5 days/week for 4 weeks.

[Results]

Leptin and insulin levels in the CS and CE groups were significantly lower than those in the HS group (p<0.001), whereas leptin (p<0.01) and insulin (p<0.05) levels decreased significantly in the CE compared to those in the CS group. Interleukin (IL)-1β (p<0.001) and IL-6 (p<0.01) levels in the CS and CE groups decreased significantly compared to those in the HS group. Leptin (IL-1β: r=0.835, p<0.001), IL-6 (r=0.607, p<0.05), insulin (IL-1β: r=0.797, p<0.01), and IL-6 (r=0.827, p<0.01) levels were positively related with pro-inflammatory cytokine levels.

[Conclusion]

Endurance exercise may play an important role during CLA supplementation of rats on a high-fat diet.     

High Fat Feeding Induces Hepatic Fatty Acid Elongation in Mice

Background

High-fat diets promote hepatic lipid accumulation. Paradoxically, these diets also induce lipogenic gene expression in rodent liver. Whether high expression of these genes actually results in an increased flux through the de novo lipogenic pathway in vivo has not been demonstrated.

Methodology/Principal Findings

To interrogate this apparent paradox, we have quantified de novo lipogenesis in C57Bl/6J mice fed either chow, a high-fat or a n-3 polyunsaturated fatty acid (PUFA)-enriched high-fat diet. A novel approach based on mass isotopomer distribution analysis (MIDA) following 1-¹³C acetate infusion was applied to simultaneously determine de novo lipogenesis, fatty acid elongation as well as cholesterol synthesis. Furthermore, we measured very low density lipoprotein-triglyceride (VLDL-TG) production rates. High-fat feeding promoted hepatic lipid accumulation and induced the expression of lipogenic and cholesterogenic genes compared to chow-fed mice: induction of gene expression was found to translate into increased oleate synthesis. Interestingly, this higher lipogenic flux (+74 µg/g/h for oleic acid) in mice fed the high-fat diet was mainly due to an increased hepatic elongation of unlabeled palmitate (+66 µg/g/h) rather than to elongation of de novo synthesized palmitate. In addition, fractional cholesterol synthesis was increased, i.e. 5.8±0.4% vs. 8.1±0.6% for control and high fat-fed animals, respectively. Hepatic VLDL-TG production was not affected by high-fat feeding. Partial replacement of saturated fat by fish oil completely reversed the lipogenic effects of high-fat feeding: hepatic lipogenic and cholesterogenic gene expression levels as well as fatty acid and cholesterol synthesis rates were normalized.

Conclusions/Significance

High-fat feeding induces hepatic fatty acid synthesis in mice, by chain elongation and subsequent desaturation rather than de novo synthesis, while VLDL-TG output remains unaffected. Suppression of lipogenic fluxes by fish oil prevents from high fat diet-induced hepatic steatosis in mice.     

Enantioselective ester hydrolase from Sphingobacterium sp. 238C5 useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis

A novel enzyme, β-phenylalanine ester hydrolase, useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis was characterized. The enzyme purified from the cell free-extract of Sphingobacterium sp. 238C5 well hydrolyzed β-phenylalanine esters (S)-stereospecifically. Besides β-phenylalanine esters, the enzyme catalyzed the hydrolysis of several α-amino acid esters with l-stereospecificity, while the deduced 369 amino acid sequence of the enzyme exhibited homology to alkaline d-stereospecific peptide hydrolases from Bacillus strains. Escherichia coli transformant expressing the β-phenylalanine ester hydrolase gene exhibited an about 8-fold increase in specific (S)-β-phenylalanine ethyl ester hydrolysis as compared with that of Sphingobacterium sp. 238C5. The E. coli transformant showed (S)-enantiomer specific esterase activity in the reaction with a low concentration (30 mM) of β-phenylalanine ethyl ester, while it showed both esterase and transpeptidase activity in the reaction with a high concentration (170 mM) of β-phenylalanine ethyl ester and produced β-phenylalanyl-β-phenylalanine ethyl ester. This transpeptidase activity was useful for β-phenylalanine β-peptide synthesis.     

Dietary n-3 LCPUFA from fish oil but not α-linolenic acid-derived LCPUFA confers atheroprotection in mice

The atheroprotective potential of n-3 α-linolenic acid (ALA) has not yet been fully determined, even in murine models of atherosclerosis. We tested whether ALA-derived, n-3 long chain polyunsaturated fatty acids (LCPUFA) could offer atheroprotection in a dose-dependent manner. Apolipoprotein B (ApoB)^100/100LDLr^−/− mice were fed with diets containing two levels of ALA from flaxseed oil for 16 weeks. Fish oil- and cis-monounsaturated-fat-enriched diets were used as positive and negative controls, respectively. The mice fed cis-monounsaturated fat and ALA-enriched diets exhibited equivalent plasma total cholesterol (TPC) and LDL-cholesterol (LDL-c) levels; only mice fed the fish-oil diet had lower TPC and LDL-c concentrations. Plasma LDL-CE fatty acid composition analysis showed that ALA-enriched diets lowered the percentage of atherogenic cholesteryl oleate compared with cis-monounsaturated-fat diet (44% versus 55.6%) but not as efficiently as the fish-oil diet (32.4%). Although both ALA and fish-oil diets equally enriched hepatic phospholipids with eicosapentaenoic acid (EPA) and ALA-enriched diets lowered hepatic cholesteryl ester (CE) levels compared with cis-monounsaturated-fat diet, only fish oil strongly protected from atherosclerosis. These outcomes indicate that dietary n-3 LCPUFA from fish oil and n-3 LCPUFA (mostly EPA) synthesized endogenously from ALA were not equally atheroprotective in these mice.     

Carnitine synthesis and uptake into cells are stimulated by fasting in pigs as a model of nonproliferating species

In rodents, fasting increases the carnitine concentration in the liver by an up-regulation of enzymes of hepatic carnitine synthesis and novel organic cation transporter (OCTN) 2, mediated by activation of peroxisome proliferator-activated receptor (PPAR) α. This study was performed to investigate whether such effects occur also in pigs which like humans, as nonproliferating species, have a lower expression of PPARα and are less responsive to treatment with PPARα agonists than rodents. An experiment with 20 pigs was performed, which were either fed a diet ad-libitum or fasted for 24 h. Fasted pigs had higher relative mRNA concentrations of the PPARα target genes carnitine palmitoyltransferase 1 and acyl-CoA oxidase in liver, heart, kidney, and small intestinal mucosa than control pigs, indicative of PPARα activation in these tissues (P<.05). Fasted pigs had a higher activity of γ-butyrobetaine dioxygenase (BBD), enzyme that catalyses the last step of carnitine biosynthesis in liver and kidney, and higher relative mRNA concentrations of OCTN2, the most important carnitine transporter, in liver, kidney, skeletal muscle, and small intestinal mucosa than control pigs (P<.05). Fasted pigs moreover had higher concentrations of free and total carnitine in liver and kidney than control pigs (P<.05). This study shows for the first time that fasting increases the activity of BBD in liver and kidney and up-regulates the expression of OCTN2 in various tissues of pigs, probably mediated by PPARα activation. It is concluded that nonproliferating species are also able to cover their increased demand for carnitine during fasting by an increased carnitine synthesis and uptake into cells.     

Rapid ester biosynthesis screening reveals a high activity alcohol‐O‐acyltransferase (AATase) from tomato fruit

Ethyl and acetate esters are naturally produced in various yeasts, plants, and bacteria. The biosynthetic pathways that produce these esters share a common reaction step, the condensation of acetyl/acyl‐CoA with an alcohol by alcohol‐O‐acetyl/acyltransferase (AATase). Recent metabolic engineering efforts exploit AATase activity to produce fatty acid ethyl esters as potential diesel fuel replacements as well as short‐ and medium‐chain volatile esters as fragrance and flavor compounds. These efforts have been limited by the lack of a rapid screen to quantify ester biosynthesis. Enzyme engineering efforts have also been limited by the lack of a high throughput screen for AATase activity. Here, we developed a high throughput assay for AATase activity and used this assay to discover a high activity AATase from tomato fruit, Solanum lycopersicum (Atf‐S.l). Atf1‐S.l exhibited broad specificity towards acyl‐CoAs with chain length from C₄ to C₁₀ and was specific towards 1‐pentanol. The AATase screen also revealed new acyl‐CoA substrate specificities for Atf1, Atf2, Eht1, and Eeb1 from Saccharomyces cerevisiae, and Atf‐C.m from melon fruit, Cucumis melo, thus increasing the pool of characterized AATases that can be used in ester biosynthesis of ester‐based fragrance and flavor compounds as well as fatty acid ethyl ester biofuels.     

Effects of long-term supplementation with omega-3 fatty acids on longitudinal changes in bone mass and microstructure in mice

A diet rich in omega-3s has previously been suggested to prevent bone loss. However, evidence for this has been limited by short exposure to omega-3 fatty acids (FAs). We investigated whether a diet enriched in eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) for the entire adult life of mice could improve bone microstructure and strength. Thirty female mice received a diet enriched in DHA or EPA or an isocaloric control diet from 3 to 17 months of age. Changes in bone microstructure were analyzed longitudinally and biomechanical properties were analysed by a three-point bending test. Bone remodelling was evaluated by markers of bone turnover and histomorphometry. Trabecular bone volume in caudal vertebrae was improved by EPA or DHA at 8 months (+26.6% and +17.2%, respectively, compared to +3.8% in controls, P=.01), but not thereafter. Trabecular bone loss in the tibia was not prevented by omega-3 FAs (BV/TV −94%, −93% and −97% in EPA, DHA and controls, respectively). EPA improved femur cortical bone volume (+8.1%, P<.05) and thickness (+4.4%, P<.05) compared to controls. EPA, but not DHA, reduced age-related decline of osteocalcin (−70% vs. −83% in controls, P<.05). EPA and DHA increased leptin levels (7.3±0.7 and 8.5±0.5 ng ml⁻¹, respectively, compared to 4.5±0.9 ng ml⁻¹ in controls, P=.001); however, only EPA further increased IGF-1 levels (739±108 ng ml⁻¹, compared to 417±58 ng ml⁻¹ in controls, P=.04). These data suggest that long-term intake of omega-3 FA, particularly EPA, may modestly improve the structural and mechanical properties of cortical bone by an increase in leptin and IGF-1 levels, without affecting trabecular bone loss.     

The effects of a diet rich in fish oil on human neutrophils: Identification of leukotriene B5 as a metabolite

Diets that are enriched with fish oil have been shown to alter arachidonic acid metabolism via the cyclooxygenase pathway. Recently it has been shown that one of the major component fatty acids of fish oil, eicosapentaenoate (EPA), is a substrate for the leukotriene B (LTB) pathway when added exogenously to human neutrophils . We fed a diet that contained 8–10 gm/day of EPA to four human subjects for three weeks and compared the arachidonate metabolism of their neutrophils to the same functions while the subjects were on their usual diet. The fish oil-supplementation increased neutrophil EPA content from undetectable levels to 7.4 ± 2.4% (p<0.01, expressed as % of total fatty acid), and decreased arachidonate from 15.4 ± 2.3% to 12.8 ± 2.3% (p<0.05). Leukotriene B₅ was identified as a metabolite during the fish oil-diet by its chromatographic profile and mass spectrum. During the experimental diet LTB₄ decreased from 160 ± 37 ng/10⁷ neutrophils to 120 ± 12 (p<0.05), and LTB₅ increased from 0 to 39 ± 9 ng/10⁷ neutrophils (p<0.005). The diet had no effect on neutrophil aggregation or adherence to nylon fibers.     

Timed high-fat diet in the evening affects the hepatic circadian clock and PPARα-mediated lipogenic gene expressions in mice

A long-term high-fat diet may result in a fatty liver. However, whether or not high-fat diets affect the hepatic circadian clock is controversial. The objective of this study is to investigate the effects of timed high-fat diet on the hepatic circadian clock and clock-controlled peroxisome proliferator-activated receptor (PPAR) α-mediated lipogenic gene expressions. Mice were orally administered high-fat milk in the evening for 4 weeks. The results showed that some hepatic clock genes, such as Clock, brain-muscle-Arnt-like 1 (Bmal1), Period 2 (Per2), and Cryptochrome 2 (Cry2) exhibited obvious changes in rhythms and/or amplitudes. Alterations in the expression of clock genes, in turn, further altered the circadian rhythm of PPARα expression. Among the PPARα target genes, cholesterol 7α-hydroxylase (CYP7A1), 3-hydroxy-3-methylglutaryl-coenzyme A reductase, low-density lipoprotein receptor, lipoprotein lipase, and diacylglycerol acyltransferase (DGAT) showed marked changes in rhythms and/or amplitudes. In particular, significant changes in the expressions of DGAT and CYP7A1 were observed. The effects of a high-fat diet on the expression of lipogenic genes in the liver were accompanied by increased hepatic cholesterol and triglyceride levels. These results suggest that timed high-fat diets at night could change the hepatic circadian expressions of clock genes Clock, Bmal1, Per2, and Cry2 and subsequently alter the circadian expression of PPARα-mediated lipogenic genes, resulting in hepatic lipid accumulation.     

Nonalcoholic Fatty Liver Disease: Correlation of the Liver Parenchyma Fatty Acid with Intravoxel Incoherent Motion MR Imaging–An Experimental Study in a Rat Model

PurposeTo prospectively evaluate the changes in fatty acid concentration after administrating a 60% high-fat diet to a non-alcoholic fatty liver disease rat model and to perform a correlation analysis between fatty acid with molecular diffusion (D_true), perfusion-related diffusion (D_fast), and perfusion fraction (P_fraction).ResultsThe highest mean TL value was at week 8 (0.278 ± 0.10) after the administration of the 60% high-fat diet, followed by weeks 6, 4, 2, and 0. The concentration level (16.99±2.29) of TSFA at week 4 was the highest. No significant differences in the concentrations of TUSFA, TUSB, or PUSB were observed in different weeks.ConclusionAfter the administration of the 60% high-fat diet in nonalcoholic fatty liver disease model, TL and TSFA depositions had significant changes. The mean concentrations of TUSFA, TUSB, PUSB did not significantly change. Total unsaturated fatty acid and polyunsaturated bond showed positive correlations with D_true and P_fraction.     

l-arginine supplementation of milk liquid or dry diets fed to pigs after weaning has a positive effect on production in the first three weeks after weaning at 21 days of age

A total of 48, 21-day-old weaned pigs, was used in a 2 × 2 factorial arrangement of treatments with the factors being diet type (milk liquid replacer vs. dry feed) and l-arginine (ARG) supplementation (0 vs. 6 g ARG/kg) to test the hypothesis that dietary supplementation with ARG would increase performance of pigs after weaning. Pigs were fed the experimental diets for 10 d (days) after weaning and then transitioned over a 3-d period to a dry Phase II diet fed in meal form devoid of supplemental ARG. The study ended at d 21. There were five replicates (pens) per treatment (a total of 12 pigs per treatment). Blood samples were collected from two pigs per replicate on d 7 and 16 of the experiment, and free amino acids (AA) and plasma urea nitrogen (PUN) levels analysed. Milk-fed pigs outperformed (P<0.001) dry-fed pigs for the first 10 d of the experiment as well as for the total 21-d period. At d 7, milk-fed pigs had higher (P<0.05) levels of most free indispensable and dispensable amino acids in their plasma. In both the milk-fed and dry-fed pigs supplemented with ARG, average daily feed intake (ADFI, P<0.05) and average daily gain (ADG, P<0.05) were increased during the dietary transition period (d 11–14), when pigs were being changed to the Phase II diet. The difference in production in the transition period caused a tendency for ARG-supplemented pigs to eat more feed (P<0.1) and grow faster (P<0.5) over the 21-d experimental period. Pigs supplemented with ARG had higher plasma ARG levels (P<0.05) at d 7 after weaning and lower plasma urea levels (P<0.05) at both d 7 and 16 after weaning. These data show the benefits of feeding a milk liquid diet as well as of ARG supplementation after weaning on production indices.     

Ichthyotoxicity of Chattonella marina (Raphidophyceae) to damselfish (Acanthochromis polycanthus): the synergistic role of reactive oxygen species and free fatty acids

This investigation aimed to elucidate the relative roles of putative brevetoxins, reactive oxygen species and free fatty acids as the toxic principle of the raphidophyte Chattonella marina, using damselfish as the bioassay. Our investigations on Australian C. marina demonstrated an absence or only very low concentrations of brevetoxin-like compounds by radio-receptor binding assay and liquid chromatography–mass spectroscopy techniques. Chattonella is unique in its ability to produce levels of reactive oxygen species 100 times higher than most other algal species. However, high levels of superoxide on their own were found not to cause fish mortalities. Lipid analysis revealed this raphidophyte to contain high concentrations of the polyunsaturated fatty acid eicosapentaenoic acid (EPA; 18–23% of fatty acids), which has demonstrated toxic properties to marine organisms. Using damselfish as a model organism, we demonstrated that the free fatty acid (FFA) form of EPA produced a mortality and fish behavioural response similar to fish exposed to C. marina cells. This effect was not apparent when fish were exposed to other lipid fractions including a triglyceride containing fish oil, docosahexaenoate-enriched ethyl ester, or pure brevetoxin standards. The presence of superoxide together with low concentrations of EPA accelerated fish mortality rate threefold. We conclude that the enhancement of ichthyotoxicity of EPA in the presence of superoxide can account for the high C. marina fish killing potential.     

The relationship between micronuclei in human lymphocytes and selected micronutrients in vegetarians and non-vegetarians

A vegetarian diet results in higher intake of vitamins and micronutrients, which – although providing antioxidant defence – may lead to deficiency in other micronutrients involved in DNA metabolism and stability (such as vitamins belonging to the B group). The principal difference among various vegetarian diets is the extent to which animal products are avoided. We have performed a pilot study to determine the relationship between the micronucleus frequency in lymphocytes and diet, and we compared the levels of Vitamins C and E, β-carotene, B₁₂, folic acid, homocysteine and total antioxidant capacity in healthy vegetarians and non-vegetarians. The vegetarian group, consisting of 24 volunteers (13 women and 11 men), were matched for age and sex with 24 volunteers (12 women and 12 men) with a traditional dietary habit. Among the vegetarians were 13 lacto-ovo-vegetarians with average duration of vegetarian diet 10.8 years (ranging from 5 to 26 years) and 11 lacto-vegetarians with average duration of vegetarian diet 8.2 years (ranging from 3 to 15 years). Homocysteine, Vitamins C and E and β-carotene levels in plasma were assayed by HPLC, and serum folate and Vitamin B₁₂ were determined with Elecsys Immunoassay tests. The total antioxidant capacity of plasma was estimated by measuring the ferric-reducing activity in a spectrophotometric assay. Micronuclei were measured in cytokinesis-blocked lymphocytes. Vegetarians had significantly higher levels of Vitamin C and β-carotene (but not Vitamin E) in plasma compared with non-vegetarians (P < 0.001). There were no significant differences in serum levels of folic acid and Vitamin B₁₂ between the monitored groups. Levels of folic acid in vegetarians correlated with length of vegetarianism (r = 0.62, P = 0.001, N = 24). Vegetarians had elevated levels of homocysteine compared with non-vegetarians (P = 0.007), as did vegetarian women compared with non-vegetarian women (P = 0.031). We did not find any differences in total antioxidant capacity or in micronucleus frequency between the groups. Micronuclei correlated with age (r = 0.62, P < 0.001, N = 48), women having higher frequencies than men. Multifactorial regression analysis showed significant effects of age, sex and total antioxidant capacity on micronucleus frequency (N = 48, P < 0.001).