Long-term dietary supplementation with cystathionine improves tissue glutathione in ageing rats.

BACKGROUND: Ageing is associated with decrease in tissue glutathione that can be reduced by food fortification with the amino acid cysteine. However, cysteine is not stable in solution and generates bad taste. Cystathionine, the direct precursor of cysteine, could be a valuable alternative. AIMS: This study aimed to determine whether long-term dietary supplementation with cystathionine induces an increase in glutathione pools. METHODS: Aged rats (20.5-month-old) were fed ad libitum during 29 weeks with either a cystathionine-supplemented diet (7.3 g/kg, n = 90 rats) or a control iso-nitrogenous alanine-supplemented diet (2.9 g/kg, n = 90 rats). RESULTS: Cystathionine was detected in the plasma of the cystathionine-supplemented rats but not in the control alanine-supplemented rats. Cystathionine increased glutathione concentrations in liver, small intestine and gastrocnemius muscle (P < 0.03). No adverse effect was observed. CONCLUSION: Cystathionine supplementation being able to increase moderately glutathione in healthy old rats could be considered as a candidate for nutritional supports aiming to revert the stronger glutathione depletions occurring in unhealthy elderly.

Effect of slow-release ß-alanine tablets on absorption kinetics and paresthesia.

Oral ß-alanine (ßA) doses larger than 800 mg commonly result in unpleasant sensory symptoms (paresthesia). However, the association of form (pure vs. slow-release) with side-effects has not been fully described. The aim of this single-blinded, randomized three-arm clinical trial was to compare plasma kinetics and symptoms following ßA bolus administration in solution or in slow-release tablet form. Eleven healthy adults ingested 1.6 g of a pure ßA reference solution (REF), 1.6 g in slow-release ßA tablets (TAB) or a placebo (PLA) after an overnight fast. During the next 6 h, urinary and plasma ßA concentrations were measured and questionnaires about intensity, nature (pins and needles, itching, flushing, irritation, numbness, soreness), and spatial distribution of unusual sensations were filled in. TAB resulted in a smaller peak plasma concentration than REF (82 vs. 248 µmol L(-1), p<0.001), delayed time to peak (1.0 vs. 0.5 h, p<0.01) no difference in area under the curve, reduced loss in urine (202 vs. 663 µmol, p<0.0001), and improved retention (98.9 vs. 96.3%, p<0.001). Symptoms described as "pins and needles" were perceived rapidly on the skin of the arms and trunk after REF (Tmax=15 min) and their time course nearly mimicked plasma concentrations. Maximum intensity scores were weaker with TAB ("very low") than with REF ("low", p<0.001), while TAB and PLA did not differ with respect to side-effects. In summary, ingesting 1.6 g ßA in slow-release tablets rather than pure in solution results in slower absorption kinetics, improved whole body retention and sensory side-effects that cannot be differentiated from PLA.

A diet containing whey protein, free glutamine, and transforming growth factor-beta ameliorates nutritional outcome and intestinal mucositis during repeated chemotherapeutic challenges in rats.

Anticancer chemotherapy often induces side effects such as mucositis. Recent data suggest that a diet, Clinutren Protect (CP), containing whey proteins, glutamine, and transforming growth factor-beta (TGFbeta)-rich casein limits intestinal mucositis and improves recovery after a single methotrexate (MTX) challenge in rats. Chemotherapy consists of alternating periods of treatment and rest. Thus, our study evaluated the effects of CP on nutritional outcome and intestinal mucositis in rats receiving repeated chemotherapeutic challenges. Thirty-six Sprague-Dawley rats received 3 cycles of MTX at 8-d intervals. Rats had free access to CP or control diet (Co) from 7 d before the first MTX injection until the end of the experiment at d 27. In Co, whey proteins and TGFbeta-rich casein were replaced by TGFbeta-free casein. L-Glutamine was replaced by L-alanine. Body composition was assessed by dual energy X-ray absorptiometry. Before MTX challenges, food intake and body weight were similar in both groups but became higher during MTX challenges in CP (P < 0.05). Fat mass decreased similarly in both groups. In contrast, the decrease of fat free mass between d -1 and d 27 was less pronounced in the CP group (-9.5 g) than in the Co group (-57.2 g) (P < 0.05). The intestinal damage score was lower in the CP group (0.6 +/- 0.3 vs. 2.1 +/- 0.6; P < 0.05). Fecal IgA increased over time in the CP group (P < 0.05) but not in the Co group. A diet containing whey proteins, glutamine, and TGFbeta improves nutritional outcome by limiting the reduction of fat free mass and reduces intestinal mucositis during repeated chemotherapeutic challenges in rats.

A diet containing whey protein, glutamine, and TGFbeta modulates gut protein metabolism during chemotherapy-induced mucositis in rats.

BACKGROUND: Mucositis, a common side effect of chemotherapy, is characterized by compromised digestive function, barrier integrity and immune competence. AIMS: Our aim was to evaluate the impact of a specifically designed diet Clinutren Protect (CP), which contains whey proteins, TGFbeta-rich casein, and free glutamine, on mucositis in rats. METHODS: Mucositis was induced by three consecutive injections (day 0, day 1, day 2) of methotrexate (2.5 mg/kg). Rats had free access to CP or placebo diets from days -7 to 9. In the placebo diet, whey proteins and TGFbeta-rich casein were replaced by TGFbeta-free casein and glutamine by alanine. Intestinal parameters were assessed at day 3 and 9. Values, expressed as mean +/- SEM, were compared using two-way ANOVA. RESULTS: At day 3, villus height was markedly decreased in the placebo (296 +/- 11 microm) and CP groups (360 +/- 10 microm) compared with controls (464 +/- 27 microm), but more markedly in the placebo as compared to CP group. The intestinal damage score was also reduced in the CP compared with the placebo group. Glutathione content increased in the CP compared with the placebo group (2.2 +/- 0.2 vs. 1.7 +/- 0.2 micromol/g tissue). Gut protein metabolism was more affected in the placebo than in the CP group. The fractional synthesis rate was decreased in the placebo group (93.8 +/- 4.9%/day) compared with controls (121.5 +/- 12.1, P < 0.05), but not in the CP group (106.0 +/- 13.1). In addition, at day 9, rats exhibited improved body weight and food intake recovery in the CP compared to the placebo group. CONCLUSIONS: Clinutren Protect feeding reduces intestinal injury in the acute phase of methotrexate-induced mucositis in rats and improves recovery.

Ingestion of a Pre-bedtime Protein Containing Beverage Prevents Overnight Induced Negative Whole Body Protein Balance in Healthy Middle-Aged Men: A Randomized Trial.

Age related muscle wasting leads to overall reductions of lean body mass, reduced muscle strength, and muscle function resulting in compromised quality of life. Utilizing novel nutritional strategies to attenuate such losses is of great importance in elderly individuals. We aimed to test if a complete dietary supplement containing 25 g of milk proteins and ingested in the evening before bed would improve protein metabolism in terms of whole body protein balance over a 10 h overnight period following ingestion of the test drink in healthy middle-aged male subjects. In addition we also assessed the rates of muscle protein synthesis during the second half of the night in order to see if previously reported extended amino acidemia during sleep results in increased rates of muscle protein synthesis. Seventeen healthy middle-aged male subjects (59.4 ± 3.2 year) consumed a dietary supplement drink at 21:00 containing either 25 g milk protein concentrate, 25 g maltodextrin, 7.75 g canola oil (treatment group), or an isocaloric protein void drink (placebo group). Muscle protein synthesis was assessed from a muscle biopsy following the continuous intravenous infusion of 13C-phenylalanine for 5 h (from 03:00 to 08:00). Whole body protein balance was greater in the treatment group (-0.13 ± 11.30 g prot/10 h) compared to placebo (-12.22 ± 6.91 g prot/10 h) (P ≤ 0.01). In contrast, no changes were observed on rates of muscle protein synthesis during the second half of the night. Ingestion of a dietary supplement containing 25 g of milk proteins significantly reduced the negative protein balance observed during the night. Therefore, pre-bedtime protein ingestion may attenuate overnight losses of lean tissue in healthy elderly men. Despite increases in aminoacidemia during the second part of the night, no changes were observed in the rates of muscle protein synthesis during this time. Clinical Trial Registration: www.ClinicalTrials.gov, identifier: NCT02041143.

Chemotherapy-induced mucositis is associated with changes in proteolytic pathways.

Mucositis, a common toxic side effect of chemotherapy, is characterized by an arrest of cell proliferation and a loss of gut barrier function, which may cause treatment reduction or withdrawal. Gut integrity depends on nutritional and metabolic factors, including the balance between protein synthesis and proteolysis. The effects of methotrexate (MTX; a frequently used chemotherapeutic agent) on intestinal proteolysis and gut barrier function were investigated in rats. Male Sprague-Dawley rats received 2.5 mg/kg of MTX subcutaneously during 3 days and were euthanized at Day 4 (D4) or Day 7 (D7). We observed at D4 that MTX induced mucosal damage and increased intestinal permeability (7-fold) and the mucosal concentration of interleukin (IL)-1beta and IL-6 (4- to 6-fold). In addition, villus height and glutathione content significantly decreased. Intestinal proteolysis was also affected by MTX as cathepsin D activity increased at D4, whereas chymotrypsin-like proteasome activity decreased and calpain activities remained unaffected. At D7, cathepsin D activity was restored to control levels, but proteasome activity remained reduced. This disruption of proteolysis pathways strongly contributed to mucositis and requires further study. Lysosomal proteolytic activity may be considered the main proteolytic pathway responsible for alteration of mucosal integrity and intestinal permeability during mucositis, as cathepsin D activity was found to be correlated with mucosal atrophy and intestinal permeability. Proteasome regulation could possibly be an adaptive process for survival. Future investigation is warranted to target proteolytic pathways with protective nutritional or pharmacological therapies during mucositis.

Kinetics of creatine ingested as a food ingredient.

The aim of the present study was to test if the consumption of creatine incorporated in food bars modifies creatine plasma kinetics, erythrocyte retention and loss in urine and in feces when compared with its consumption in the form of an aqueous solution (AS). Seventeen healthy young men ingested 2 g creatine either in the form of AS, or incorporated in a protein (PP)- or in a beta-glucan (BG)-rich food bar. Kinetics of plasma creatine was measured for 8-h duration and urinary excretion for 24 h. Then, the subjects received the same treatment thrice a day for 1 week at the end of which creatine contents were determined in erythrocytes and in feces (n = 4 for feces). The three crossover treatments were interspaced by a 40 +/- 1.2-day wash-out. Absorption of creatine was slowed down by 8-fold in the presence of BG (P < 0.001) and by 4-fold with PP (P < 0.001) whereas the velocity rate constant of elimination and the area under the curve were not modified. Urinary loss of creatine in the first 24 h following ingestion was 15 +/- 1.9% in AS and 14 +/- 2.2% in PP conditions (NS), whereas it was only 8 +/- 1.2% with BG (P = 0.004). Increase in creatine concentration in erythrocyte was similar in whatever form the creatine was ingested. Creatine seems to be totally absorbed since no creatine or creatinine was detectable in feces. No side effects were reported. In conclusion, ingestion of creatine combined with BG facilitates its retention by slowing down its absorption rate and reducing its urinary excretion.

Effect of an enteral diet supplemented with a specific blend of amino acid on plasma and muscle protein synthesis in ICU patients.

BACKGROUND & AIM: Polytrauma patients are characterized by a negative nitrogen balance and muscle wasting. Standard nutrition is relatively inefficient to improve muscle protein turnover. The aim of this study was to investigate the effect of enteral nutrition (EN) supplemented with specific amino acids on protein metabolism in polytrauma patients. METHODS: In a double blind study, 12 polytrauma patients were randomized to receive EN supplemented with either a mixture of cysteine, threonine, serine and aspartate (AA patients) or alanine at isonitrogenous levels (Ala patients). An intravenous infusion of l-[1-(13)C]-leucine was performed in the fed state between day 9 and 12 post-injury (Df) in patients and in a group of healthy volunteers (n=8) (EN+Ala) to measure whole body leucine kinetics, plasma and muscle protein synthesis rates. Nitrogen balance, 3-methyl histidine excretion were measured from day 3 to Df. RESULTS: The contribution of total plasma proteins to whole body protein synthesis was greatly increased, from 11% in healthy volunteers to about 25% in polytrauma patients. AA supplementation had no effect on nitrogen balance, leucine kinetics or plasma protein synthesis in patients. In contrast, the urinary excretion of 3-methyl histidine tended to decrease along the study in the AA supplemented group compared to an increase in the Ala group. Muscle protein synthesis tended to be higher in the AA group than in the Ala group (46%, P=0.065). CONCLUSION: During injury, an increased supply of cysteine, threonine, serine and aspartate could be able to better cover the specific amino requirements, thus resulting in improved muscle protein synthesis without impairment of acute phase protein synthesis.

A comparative study of proteolysis methods for the measurement of 3-nitrotyrosine residues: enzymatic digestion versus hydrochloric acid-mediated hydrolysis.

A common approach for the quantification of 3-nitrotyrosine (NY) in routine analyses relies on the cleavage of peptide bonds in order to release the free amino acids from proteins in tissues or fluids. NY is usually monitored by either GC-MS(/MS) or LC-MS/MS techniques. Various proteolysis methods have been employed to combine digestion efficiency with prevention of artifactual nitration of tyrosine. However, so far, no study was designed to compare the HCl-based hydrolysis method with enzymatic digestion in terms of reliability for the measurement of NY. The present work addresses the digestion efficiency of BSA using either 6M HCl, pronase E or a cocktail of enzymes (pepsin, pronase E, aminopeptidase, prolidase) developed in our laboratory. The HCl-based hydrolysis leads to a digestion yield of 95%, while 25 and 75% are achieved with pronase E and the cocktail of enzymes, respectively. These methods were compared in terms of NY measurement and the results indicate that a prior reduction of the disulfide bonds ensures a reliable quantification of NY. We additionally show that the enzyme efficacy is not altered when the digestion is carried out in the presence of BSA with a high content of NY.

Quantitative determination of dityrosine in milk powders by liquid chromatography coupled to tandem mass spectrometry using isotope dilution.

An analytical method to quantify dityrosine (DiTyr) in milk powder samples is presented. The assay is based on isotope dilution liquid chromatography coupled to electrospray ionisation tandem mass spectrometry (LC-ESIMS/MS). The sample preparation entails acid hydrolysis of milk proteins followed by a solid phase-extraction (SPE) step. Neither artifactual formation nor degradation of DiTyr were observed during the proteolysis step. Mass spectral detection was performed in the positive ion mode by recording five transition reactions for DiTyr, in order to unambiguously confirm the presence of DiTyr by correct ion ratios. Under the analytical conditions used, the limit of detection (LOD) and limit of quantification (LOQ) for DiTyr were estimated at ca. 2 and 6 micromol DiTyr per mol of Tyr (using ca. 500 microg of milk proteins), with a mean recovery of ca. 90%. Quantification was conduced in eight different commercial milk powder samples, and the level of DiTyr ranged from below the LOQ up to 393.0 +/- 9.1 micromol DiTyr per mol of Tyr.

Methotrexate induces intestinal mucositis and alters gut protein metabolism independently of reduced food intake.

One of the main secondary toxic side effects of antimitotic agents used to treat cancer patients is intestinal mucositis. This one is characterized by compromised digestive and absorptive functions, barrier integrity, and immune competence. At the same time, food intake is decreased, which may induce intestinal damages per se. The aim of the study was to characterize which alterations are specific to methotrexate, independently of the anorexic effect of the drug. Male Sprague-Dawley rats received subcutaneously saline solution as control group or 2.5 mg/kg of methotrexate during 3 days (D0-D2). Methotrexate-treated rats were compared with ad libitum and pair-fed controls. Histological examinations and specific markers of the immune and nonimmune gut barrier function were assessed at D4 or D7. Compared with ad libitum and pair-fed controls, methotrexate induced at D4 villus atrophy associated with epithelial necrosis. Mucosal protein synthesis rate and mucin contents of methotrexate treated rats were reduced. At the same time, cathepsin D proteolytic activity was increased compared with ad libitum and pair-fed controls, whereas calpain activity was increased when compared with the only pair-fed controls. These intestinal lesions were associated with various metabolic disturbances such as increased TNF-alpha level and inflammation score in the jejunum but also disturbances of amino acid concentrations in the duodenum and plasma. At D7, these alterations were partially or completely normalized. In addition to the consequences of a low food intake, methotrexate further impairs different biological processes leading to a dramatic loss of gut homeostasis. Targeted nutritional management of chemotherapy receiving patients should be set up to prevent or limit such alterations.

Threonine utilization for synthesis of acute phase proteins, intestinal proteins, and mucins is increased during sepsis in rats.

We hypothesized that the dietary threonine demand for the anabolic response may be increased more than that of other essential amino acids during sepsis. Using a flooding dose of either L-[1 -13C]valine or L-[U -13C]threonine, we measured valine and threonine utilization for syntheses of plasma proteins (minus albumin), and wall, mucosal, and mucin proteins of the small intestine in infected (INF; d 2 and d 6 of postinfection) and control pair-fed (PF) rats. At d 2, the protein absolute synthesis rate (ASR) of INF rats was 21% (mucins) to 41% (intestinal wall) greater than that of PF when measured using valine as tracer, and 45% (mucosa) to 113% (mucins) greater than that of PF when measured with threonine as tracer. Plasma protein ASR was higher in INF than in PF rats, reaching 5- to 6-fold the value of PF. The utilization of both amino acid tracers for the protein synthesis was significantly increased by the infection in all compartments studied. The daily increased absolute threonine utilization for protein synthesis in gut wall plus plasma proteins was 446 micromol/d compared with 365 micromol/d for valine, and it represented 2.6 times the dietary threonine intake of rats at d 2. Most changes in protein ASR and threonine utilization observed at d 6 of postinfection were limited. In conclusion, sepsis increased the utilization of threonine for the anabolic splanchnic response. Because this threonine requirement is likely covered by muscle protein mobilization, increasing the threonine dietary supply would be an effective early nutritional management for patients with sepsis.

Specific amino acids increase mucin synthesis and microbiota in dextran sulfate sodium-treated rats.

During the anabolic response associated with inflammation, mucin synthesis and colonic protection may be compromised by the limited availability of specific amino acids. We therefore determined the effect of dietary amino acid supplementation on the microbiota, mucin status, and mucosal damage in dextran sulfate sodium (DSS)-treated rats. From 8 d before to 28 d after colitis induction, male Sprague-Dawley rats (10 mo old, n = 8/group) were fed a control diet supplemented or not with 2 different doses of an amino acid cocktail containing L-threonine, L-serine, L-proline, and L-cysteine. All diets were isonitrogenous (adjusted with L-alanine). The higher dose of amino acids increased the number of Muc2-containing goblet cells in the surface epithelium of the ulcerated area, stimulated mucin production in the colon, and restored the mucin amino acid composition and mucosal content to healthy, control values. The colonic mucin synthesis rate was specifically stimulated by 95%, whereas the protein turnover was unchanged. All bacterial populations, markedly altered by the DSS treatment, were promoted. In conclusion, in inflammatory situations, an increase in threonine, serine, proline, and cysteine dietary supply can promote mucin synthesis, reequilibrate the gut microbiota, and thus favor colonic protection and mucosal healing.

Artifactual nitration controlled measurement of protein-bound 3-nitro-L-tyrosine in biological fluids and tissues by isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry.

A sensitive and selective method is presented to accurately determine the level of protein-bound 3-nitro-L-tyrosine (NTyr) in rat plasma and kidney samples. This assay is based on isotope dilution liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The sample preparation entails protein precipitation, acid hydrolysis with 6 N HCl, and solid-phase extraction (using reverse and aminopropyl phase cartridges) prior to the determinative step. For kidney samples, NTyr is converted into its butyl ester to improve sensitivity. The potential formation of artifactual NTyr during the acid hydrolysis step was carefully followed and determined by supplementation of the samples with (13)C-labeled L-tyrosine (Tyr) prior to protein digestion. Hence, the concomitant measurement of formation of (13)C-enriched NTyr enabled the accurate determination of artifactual NTyr. This approach was employed to measure the basal level of protein-bound NTyr in rat plasma and kidney samples, revealing levels in the range of 4-18 micromol/mol of Tyr and 50-68 micromol/mol of Tyr, respectively. No artifactual nitration of Tyr was observed in kidney proteins, whereas in the case of plasma the contribution of the artifactual response ranged from 16 to 40%. This method allows the analysis of protein-bound NTyr with a full control of the artifactual nitration of tyrosine during the proteolysis and/or sample preparation. Reliable detection of NTyr in proteins may allow insight into the role of nitric oxide-derived oxidants under various pathological conditions.

Short-term dietary conjugated linoleic acid supplementation does not enhance the recovery of immunodepleted dexamethasone-treated rats.

BACKGROUND: Conjugated linoleic acid (CLA) has been reported to decrease fat deposition, and increase lean body mass. This has been broadly inferred to mean that CLA alters protein turnover. However, data to test the effects of CLA on protein turnover are lacking. An enhancement in immune responses by CLA has also been demonstrated. AIM OF THE STUDY: The objective of this study was to determine the potential for dietary CLA and protein intervention to improve nutritional and functional recovery in an animal model of catabolic stress and immunodepletion. METHODS: Diets varying in their protein levels in the presence or absence of CLA were tested for their effects on the recovery of glucocorticoid (intraperitoneal injection of dexamethasone, 120 mg/kg) treated rats. Following steroid injection, rats were fed 4 dietary treatments for 4 d. The diets contained 10 or 20 g/100 g protein with or without 0.5 g/100 g CLA. RESULTS: Dexamethasone treatment resulted in a decreased food intake and loss of weight, independent of dietary treatment. A higher number of blood monocytes occurred in rats fed the high CLA diets. The protein fractional synthesis rate in spleens of rats fed the diets containing either high proteins or CLA were higher compared to those fed diets with low protein content or without CLA, respectively. CLA, consumed post-dexamethasone treatment, did not improve protein turnover in the other tissues studied, including gut mucosa, liver, muscle and thymus. CONCLUSIONS: The present study was performed to determine the effect of CLA in acute conditions, as opposed to a preventive approach, on the recovery from a catabolic stress with immunodepletion. Overall, no effect of short-term feeding CLA on the recovery from dexamethasone-mediated immunodepletion was observed.