Synthesis of triheptanoin and formulation as a solid diet for rodents

Triheptanoin-enriched diets have been successfully used in the experimental treatment of various metabolic disorders. Maximal therapeutic effect is achieved in the context of a ketogenic diet where triheptanoin oil provides 30-40% of the daily caloric intake. However, pre-clinical studies using triheptanoin-rich diets are hindered by the difficulty of administering to laboratory animals as a solid foodstuff. In the present study, we successfully synthesized triheptanoin to the highest standards of purity from glycerol and heptanoic acid, using sulfonated charcoal as a catalyst. Triheptanoin oil was then formulated as a solid, stable and palatable preparation using a ketogenic base and a combination of four commercially available formulation agents: hydrophilic fumed silica, hydrophobic fumed silica, microcrystalline cellulose, and talc. Diet compliance and safety was tested on C57Bl/6 mice over a 15-week period, comparing overall status and body weight change. Practical applications: This work provides a complete description of (i) an efficient and cost-effective synthesis of triheptanoin and (ii) its formulation as a solid, stable, and palatable ketogenic diet (triheptanoin-rich; 39% of the caloric intake) for rodents. Triheptanoin-rich diets will be helpful on pre-clinical experiments testing the therapeutic efficacy of triheptanoin in different rodent models of human diseases. In addition, using the same solidification procedure, other oils could be incorporated into rodent ketogenic diet to study their dosage and long-term effects on mammal health and development. This approach could be extremely valuable as ketogenic diet is widely used clinically for epilepsy treatment.

Single amino acid charge switch defines clinically distinct proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1)-associated inflammatory diseases.

BACKGROUND: Hyperzincemia and hypercalprotectinemia (Hz/Hc) is a distinct autoinflammatory entity involving extremely high serum concentrations of the proinflammatory alarmin myeloid-related protein (MRP) 8/14 (S100A8/S100A9 and calprotectin). OBJECTIVE: We sought to characterize the genetic cause and clinical spectrum of Hz/Hc. METHODS: Proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1) gene sequencing was performed in 14 patients with Hz/Hc, and their clinical phenotype was compared with that of 11 patients with pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome. PSTPIP1-pyrin interactions were analyzed by means of immunoprecipitation and Western blotting. A structural model of the PSTPIP1 dimer was generated. Cytokine profiles were analyzed by using the multiplex immunoassay, and MRP8/14 serum concentrations were analyzed by using an ELISA. RESULTS: Thirteen patients were heterozygous for a missense mutation in the PSTPIP1 gene, resulting in a p.E250K mutation, and 1 carried a mutation resulting in p.E257K. Both mutations substantially alter the electrostatic potential of the PSTPIP1 dimer model in a region critical for protein-protein interaction. Patients with Hz/Hc have extremely high MRP8/14 concentrations (2045 ± 1300 μg/mL) compared with those with PAPA syndrome (116 ± 74 μg/mL) and have a distinct clinical phenotype. A specific cytokine profile is associated with Hz/Hc. Hz/Hc mutations altered protein binding of PSTPIP1, increasing interaction with pyrin through phosphorylation of PSTPIP1. CONCLUSION: Mutations resulting in charge reversal in the y-domain of PSTPIP1 (E→K) and increased interaction with pyrin cause a distinct autoinflammatory disorder defined by clinical and biochemical features not found in patients with PAPA syndrome, indicating a unique genotype-phenotype correlation for mutations in the PSTPIP1 gene. This is the first inborn autoinflammatory syndrome in which inflammation is driven by uncontrolled release of members of the alarmin family.

Altered nitrogen balance and decreased urea excretion in male rats fed cafeteria diet are related to arginine availability

Hyperlipidic diets limit glucose oxidation and favor amino acid preservation, hampering the elimination of excess dietary nitrogen and the catabolic utilization of amino acids.We analyzed whether reduced urea excretion was a consequence of higherNO ; (nitrite,nitrate, and other derivatives) availability caused by increased nitric oxide production in metabolic syndrome. Rats fed a cafeteria diet for 30 days had a higher intake and accumulation of amino acid nitrogen and lower urea excretion.There were no differences in plasma nitrate or nitrite. NO and creatinine excretion accounted for only a small part of total nitrogen excretion. Rats fed a cafeteria diet had higher plasma levels of glutamine, serine, threonine, glycine, and ornithinewhen comparedwith controls,whereas arginine was lower. Liver carbamoyl-phosphate synthetase I activity was higher in cafeteria diet-fed rats, but arginase I was lower. The high carbamoyl-phosphate synthetase activity and ornithine levels suggest activation of the urea cycle in cafeteria diet-fed rats, but low arginine levels point to a block in the urea cycle between ornithine and arginine, thereby preventing the elimination of excess nitrogen as urea. The ultimate consequence of this paradoxical block in the urea cycle seems to be the limitation of arginine production and/or availability.

Potentiometric and conductometric studies on the system acid-isopolytungstate and the formation of lanthanum tungstates

The stoichiometry of the polyanions formed by the action of nitric acid on sodium tungstate (< 0.01M) has been studied by means of electrometric techniques involving pH-potentiometric and conductometric titrations. The well defined inflections and breaks in the titration curves provide evidence for the existence of the polyanions, para-W12O41(10-) and meta-W12O39(6-) corresponding to the ratio of H+:WO4(2-) as 7:6 and 9:6 in the pH ranges 5.7-6.0 and 3.6-4.1, respectively. The interaction of lanthanum nitrate with sodium tungstate solutions, at specific pH levels 8.0, 5.9 and 4.0 was also studied by pH and conductometric titrations, in aqueous and alcoholic media, with each of the reagents alternatively used as titrant. The electrometric experiments provide definite evidence of the formation of normal-La2O3.3WO3, para-5La2O3.36WO3 and meta-La2O3.12WO3 tungstates in the vicinity of pH 6.3, 5.0 and 4.2, respectively. Analytical investigations on the precipitates formed confirm the results of the electrometric study.

Kainic acid-induced seizures: inflammation and excitotoxic neuronal damage in the developing rat hippocampus

Epileptic seizures are harmful to the developing brain. During epileptic seizures, overactivation of glutamate receptors (GluR) leads to neuronal degeneration, defined as excitotoxicity. The hippocampus is especially vulnerable to excitotoxic neuronal death, but its mechanism has remained incompletely known in the developing brain. Recently, signs of activation of inflammatory processes after epileptic seizures have been detected in the hippocampus. The purpose of this thesis was to study the inflammatory reaction and death mechanisms in excitoxic neurodegeneration induced by the glutamate analogue kainic acid (KA) in the developing hippocampus. Organotypic hippocampal slice cultures (OHCs), prepared from 6-7-day-old rats (P6-7) and treated with KA, served as an in vitro model. KA-induced status epilepticus in P9 and P21 rats was used as an in vivo model. The results showed that the pyramidal cell layers of the hippocampus were the most susceptible to irreversible and age-specific neurodegeneration, which occurred in the juvenile (P21), but not in the immature (P9), rat hippocampus. The primary death mechanism was necrosis as there were no significant changes in the expression of selected apoptosis markers and morphological cellular features of necrosis were found. Inflammatory response was similarly age-dependent after KA treatment as a rapid, fulminant and wide response was detected in the juvenile, but not in the immature, rat brain. An anti-inflammatory drug treatment, given before KA, was not neuroprotective in OHCs, possibly because of the timing of the treatment. In summary, the results suggest that KA induces an age-dependent inflammatory response and necrotic neurodegeneration, which may cause disturbances in hippocampal connectivity and promote epileptogenesis.

Comparison of methods for determination of nutrient digestibility of a dry kibble diet for ocelots

In this experiment, methods of total fecal collection (TFC) and internal markers (acid-insoluble ash - AIA, crude fiber - CF, and acid-detergent fiber - ADF) were compared for determination of the coefficients of apparent digestibility (CAD) for dry matter (DM), crude protein (CP), ether extract (EE), nitrogen-free extracts (NFE), and gross energy (GE) of commercial feline dry kibble for ocelots (Leopardus pardalis). Six adult animals, weighing 12.45±1.37 kg, gradually received experimental kibble in their usual diet until the beginning of the experiment and were submitted to an adaptation period ten days prior to the collection period. CAD obtained by TFC, AIA, CF, and ADF were, respectively, 73.7, 76.83, 62.01, and 46.03% for dry matter; 81.9, 84.8, 75.8, and 63.8% for crude protein; 85, 86.7, 78.5, and 69.1% for ether extract; 78.52, 79.55, 69.11, and 53.04% for nitrogen-free extracts; and 80.5, 82.2, 71.4, and 58.4% for gross energy. The AIA method showed to be efficient in determining coefficients of apparent digestibility and may contribute to investigations on the digestibility of diets for wild felines. In comparison to the items of ocelot's usual diet, the kibble used in this paper provided an adequate nutritional supply with reduced daily costs per animal.

Selenium and vitamin E enriched diet increases NK cell cytotoxicity in cattle

A number of studies has shown that antioxidants, fatty acids and trace minerals may modulate different immune cell activities, and that their deficiency may be associated with diseases and impaired immune responses. In innate immunity, natural killer (NK) cells have a central role, killing virally infected and cancerous cells, and also secreting cytokines that shape adaptive immune responses. Thus, the aim of this study was to evaluate the effect of enriched diets in selenium plus vitamin E and/or canola oil on complete blood count and on NK cell cytotoxicity from blood lymphocytes of Nellore bulls. Bulls that received selenium plus vitamin E had (P=0.0091) higher NK cell cytotoxicity than control bulls. This result positively correlated with serum selenium levels. To the best of our knowledge, this is the first study that showed immunostimulatory effects of selenium plus vitamin E on NK cell cytotoxicity of Nellore bulls.

Evaluation of the addition of ascorbic acid to the ration of cultivated Piaractus mesopotamicus (Characidae) on the infrapopulation of Anacanthorus penilabiatus (Monogenea)

Sixty Piaractus mesopotamicus Holmberg, 1887 (pacu) fry fed a diet containing 0, 50, 100 and 200 mg ascorbic acid/kg dry feed were studied to evaluate the effect on parasitic infestation by the monogenean Anacanthorus penilabiatus Boeger, Husak and Martins, 1995 (Monogenea: Dactylogyridae) for a period of 24 weeks. The temperature of the aquaria was measured daily and remained between 28 and 31oC. At the beginning of the experiment, fish showed 6.15 ± 0.33 cm standard length and 8.64 ± 1.62 g average body weight. A sample of fish was examined and showed 43 ± 17 monogeneans per fish. At the end of the experiment, the gills of control and vitamin C-treated fish were collected for parasite counts. Control fish had 42.5 parasites per fish, a significantly higher number (P<0.05) when compared with fish fed vitamin C, that showed 16.5 parasites per fish. Ascorbic acid fortification in the food promoted an increase in fish resistance to parasites. It is suggested that an optimum level of 139 mg/kg vitamin C supplementation either elicited better nutritional conditions by stimulating the appetite of the fish or improved the immune response.

Increase in skeletal muscle protein content by the ß-2 selective adrenergic agonist clenbuterol exacerbates hypoalbuminemia in rats fed a low-protein diet

This investigation examined how the nutritional status of rats fed a low-protein diet was affected when the animals were treated with the ß-2 selective agonist clenbuterol (CL). Males (4 weeks old) from an inbred, specific-pathogen-free strain of hooded rats maintained at the Dunn Nutritional Laboratory were used in the experiments (N = 6 rats per group). CL treatment (Ventipulmin, Boehringer-Ingelheim Ltd., 3.2 mg/kg diet for 2 weeks) caused an exacerbation of the symptoms associated with protein deficiency in rats. Plasma albumin concentrations, already low in rats fed a low-protein diet (group A), were further reduced in CL rats (A = 25.05 ± 0.31 vs CL = 23.64 ± 0.30 g/l, P<0.05). Total liver protein decreased below the level seen in either pair-fed animals (group P) or animals with free access to the low-protein diet (A = 736.56 ± 26 vs CL = 535.41 ± 54 mg, P<0.05), whereas gastrocnemius muscle protein was higher than the values normally described for control (C) animals (C = 210.88 ± 3.2 vs CL = 227.14 ± 1.7 mg/g, P<0.05). Clenbuterol-treated rats also showed a reduction in growth when compared to P rats (P = 3.2 ± 1.1 vs CL = -10.2 ± 1.9 g, P<0.05). This was associated with a marked decrease in fat stores (P = 5.35 ± 0.81 vs CL = 2.02 ± 0.16 g, P<0.05). Brown adipose tissue (BAT) cytochrome oxidase activity, although slightly lower than in P rats (P = 469.96 ± 16.20 vs CL = 414.48 ± 11.32 U/BAT x kg body weight, P<0.05), was still much higher than in control rats (C = 159.55 ± 11.54 vs CL = 414.48 ± 11.32 U/BAT x kg body weight, P<0.05). The present findings support the hypothesis that an increased muscle protein content due to clenbuterol stimulation worsened amino acid availability to the liver and further reduced albumin synthesis causing exacerbation of hypoalbuminemia in rats fed a low-protein diet.

Effect of all-trans retinoic acid on newly diagnosed acute promyelocytic leukemia patients: results of a Brazilian center

Thirty-seven patients with acute promyelocytic leukemia (APL) were treated with all-trans retinoic acid (ATRA). Patients received 45 mg m-2 day-1 po of ATRA until complete remission (CR) was achieved, defined as: a) presence of less than 5% blasts in the bone marrow, with b) white blood cells >103/mm3, c) platelets >105/mm3 and d) hemoglobin concentration >8 g/dl, with no blood or platelet transfusions. Thirty-one (83.7%) patients achieved CR by day 50, and 75% of these before day 30. Correction of the coagulopathy, achieved between days 2 and 10 (mean, 3 days), was the first evidence of response to treatment. Only one patient had been previously treated with chemotherapy and three had the microgranular variant M3 form. Dryness of skin and mucosae was the most common side effect observed in 82% of the patients. Thrombosis, hepatotoxicity and retinoid acid syndrome (RAS) were observed in 7 (19%), 6 (16%) and 4 (11%) patients, respectively. Thirteen (35%) patients had to be submitted to chemotherapy due to hyperleukocytosis (above 40 x 103/mm3) and six of these presented with new signs of coagulopathy after chemotherapy. Four (11%) patients died secondarily to intracerebral hemorrhage (IH) and two (5.4%) dropped out of the protocol due to severe ATRA side effects (one RAS and one hepatotoxicity). RAS and IH were related strictly to hyperleukocytosis. The reduced use of platelets and fresh frozen plasma probably lowered the total cost of treatment. We conclude that ATRA is an effective agent for inducing complete remission in APL patients.

Obese women on a low energy rice and bean diet: effects of leucine, arginine or glycine supplementation on protein turnover

This study examined if leucine, arginine or glycine supplementation in adult obese patients (body mass index of 33 ± 4 kg/m²) consuming a Brazilian low energy and protein diet (4.2 MJ/day and 0.6 g protein/kg) affects protein and amino acid metabolism. After four weeks adaptation to this diet, each subject received supplements of these amino acids (equivalent to 0.2 g protein kg-1 day-1) in random order. On the seventh day of each amino acid supplementation, a single-dose 15N-glycine study was carried out. There were no significant differences in protein flux, synthesis or breakdown. The protein flux (grams of nitrogen, gN/9 h) was 55 ± 24 during the nonsupplemented diet intake and 39 ± 10, 44 ± 22 and 58 ± 35 during the leucine-, glycine- and arginine-supplemented diet intake, respectively; protein synthesis (gN/9 h) was 57 ± 24, 36 ± 10, 41 ± 22 and 56 ± 36, respectively; protein breakdown (gN/9 h) was 51 ± 24, 34 ± 10, 32 ± 28 and 53 ± 35, respectively; kinetic balance (gN/9 h) was 3.2 ± 1.8, 4.1 ± 1.7, 3.4 ± 2.9 and 3.9 ± 1.6. There was no difference in amino acid profiles due to leucine, arginine or glycine supplementation. The present results suggest that 0.6 g/kg of dietary protein is enough to maintain protein turnover in obese women consuming a reduced energy diet and that leucine, arginine or glycine supplementation does not change kinetic balance or protein synthesis.

A leucine-supplemented diet improved protein content of skeletal muscle in young tumor-bearing rats

Cancer cachexia induces host protein wastage but the mechanisms are poorly understood. Branched-chain amino acids play a regulatory role in the modulation of both protein synthesis and degradation in host tissues. Leucine, an important amino acid in skeletal muscle, is higher oxidized in tumor-bearing animals. A leucine-supplemented diet was used to analyze the effects of Walker 256 tumor growth on body composition in young weanling Wistar rats divided into two main dietary groups: normal diet (N, 18% protein) and leucine-rich diet (L, 15% protein plus 3% leucine), which were further subdivided into control (N or L) or tumor-bearing (W or LW) subgroups. After 12 days, the animals were sacrificed and their carcass analyzed. The tumor-bearing groups showed a decrease in body weight and fat content. Lean carcass mass was lower in the W and LW groups (W = 19.9 ± 0.6, LW = 23.1 ± 1.0 g vs N = 29.4 ± 1.3, L = 28.1 ± 1.9 g, P < 0.05). Tumor weight was similar in both tumor-bearing groups fed either diet. Western blot analysis showed that myosin protein content in gastrocnemius muscle was reduced in tumor-bearing animals (W = 0.234 ± 0.033 vs LW = 0.598 ± 0.036, N = 0.623 ± 0.062, L = 0.697 ± 0.065 arbitrary intensity, P < 0.05). Despite accelerated tumor growth, LW animals exhibited a smaller reduction in lean carcass mass and muscle myosin maintenance, suggesting that excess leucine in the diet could counteract, at least in part, the high host protein wasting in weanling tumor-bearing rats.

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.

Paradoxical effect of a pequi oil-rich diet on the development of atherosclerosis: balance between antioxidant and hyperlipidemic properties

Pequi is the fruit of Caryocar brasiliense and its oil has a high concentration of monounsaturated and saturated fatty acids, which are anti- and pro-atherogenic agents, respectively, and of carotenoids, which give it antioxidant properties. Our objective was to study the effect of the intake of a cholesterol-rich diet supplemented with pequi oil, compared to the same diet containing soybean oil, on atherosclerosis development, and oxidative stress in atherosclerosis-susceptible LDL receptor-deficient mice (LDLr-/-, C57BL/6-background). Female mice were fed a cholesterol-rich diet containing 7% soybean oil (Soybean group, N = 12) or 7% pequi oil (Pequi group, N = 12) for 6 weeks. The Pequi group presented a more atherogenic lipid profile and more advanced atherosclerotic lesions in the aortic root compared to the Soybean group. However, the Pequi group presented a less advanced lesion in the aorta than the Soybean group and showed lower lipid peroxidation (Soybean group: 50.2 ± 7.1; Pequi group: 30.0 ± 4.8 µmol MDA/mg protein) and anti-oxidized LDL autoantibodies (Soybean group: 35.7 ± 9.4; Pequi group: 15.6 ± 3.7 arbitrary units). Peritoneal macrophages from the Pequi group stimulated with zymosan showed a reduction in the release of reactive oxygen species compared to the Soybean group. Our data suggest that a pequi oil-rich diet slows atherogenesis in the initial stages, possibly due to its antioxidant activity. However, the increase of serum cholesterol induces a more prominent LDL migration toward the intimae of arteries, increasing the advanced atherosclerotic plaque. In conclusion, pequi oil associated with an atherogenic diet worsens the lipid profile and accelerates the formation of advanced atherosclerotic lesions despite its antioxidant action.

Lipoic acid, but not tempol, preserves vascular compliance and decreases medial calcification in a model of elastocalcinosis

Vascular calcification decreases compliance and increases morbidity. Mechanisms of this process are unclear. The role of oxidative stress and effects of antioxidants have been poorly explored. We investigated effects of the antioxidants lipoic acid (LA) and tempol in a model of atherosclerosis associated with elastocalcinosis. Male New Zealand white rabbits (2.5-3.0 kg) were fed regular chow (controls) or a 0.5% cholesterol (chol) diet+104 IU/day vitamin D2 (vitD) for 12 weeks, and assigned to treatment with water (vehicle, n=20), 0.12 mmol·kg-1·day-1 LA (n=11) or 0.1 mmol·kg-1·day-1 tempol (n=15). Chol+vitD-fed rabbits developed atherosclerotic plaques associated with expansive remodeling, elastic fiber disruption, medial calcification, and increased aortic stiffness. Histologically, LA prevented medial calcification by ∼60% and aortic stiffening by ∼60%. LA also preserved responsiveness to constrictor agents, while intima-media thickening was increased. In contrast to LA, tempol was associated with increased plaque collagen content, medial calcification and aortic stiffness, and produced differential changes in vasoactive responses in the chol+vitD group. Both LA and tempol prevented superoxide signals with chol+vitD. However, only LA prevented hydrogen peroxide-related signals with chol+vitD, while tempol enhanced them. These data suggest that LA, opposite to tempol, can minimize calcification and compliance loss in elastocalcionosis by inhibition of hydrogen peroxide generation.