Association of animal and plant protein intakes with biomarkers of insulin and insulin-like growth factor axis.

BACKGROUND & AIMS: Insulin and insulin-like growth factor (IGF)-1 signaling is a proposed mechanism linking dietary protein and major chronic diseases. However, it is unclear whether animal and plant proteins are associated with biomarkers of insulin and IGF axis. METHODS: We analyzed a total of 14,709 participants from Nurses' Health Study and Health Professionals Follow-up Study who had provided a blood sample. Detailed dietary information was assessed using validated food frequency questionnaires. We assessed C-peptide, insulin, IGF-1, and IGF binding proteins (BP). Multivariable-adjusted linear regressions were used to examine associations of animal and plant protein intake with biomarkers after adjusting for confounders. RESULTS: The medians (5th-95th percentiles) of animal and plant protein intake (% of total energy) were 13% (8-19%) and 5% (4-7%), respectively. Compared to participants in the lowest quintile, those in the highest quintile of animal protein had 4.8% (95% CI: 1.9, 7.9; P-trend<0.001) higher concentration of IGF-1 and -7.2% (95% CI: -14.8, 1.1; P for trend = 0.03) and -11.8% (95% CI: -20.6, -1.9; P-trend<0.001) lower concentration of IGFBP-1 and IGFBP-2, respectively, after adjustment for major lifestyle factors and diet quality. In contrast, no association was observed between animal protein intake and C-peptide, insulin and IGFBP-3. The associations were restricted to participants with at least one unhealthy lifestyle risk factor (i.e., overweight/obese, physical inactivity, smoking, and heavy alcohol intake). Plant protein tended to be strongly associated with numerous biomarkers in age-adjusted analyses but these became largely attenuated or non-significant in multivariable adjustment. Plant protein intake remained positively associated with IGF-1 (P-trend = 0.002) and possibly IGFBP-1 (P-trend = 0.02) after multivariable adjustment. Substitution of plant protein with animal protein sources was associated with lower IGFBP-1. In additional analysis, IGF-1 and IGFBPs were estimated to mediate approximately 5-20% of the association between animal protein and type 2 diabetes. CONCLUSIONS: Higher animal protein intake was associated with higher IGF-1 and lower IGFBP-1 and IGFBP-2, whereas higher plant protein intake was associated with higher IGF-1 and IGFBP-1.

Integrated Single-cell and Plasma Proteomic Modeling to Predict Surgical Site Complications: A Prospective Cohort Study.

OBJECTIVE: The aim of this study was to determine whether single-cell and plasma proteomic elements of the host's immune response to surgery accurately identify patients who develop a surgical site complication (SSC) after major abdominal surgery. SUMMARY BACKGROUND DATA: SSCs may occur in up to 25% of patients undergoing bowel resection, resulting in significant morbidity and economic burden. However, the accurate prediction of SSCs remains clinically challenging. Leveraging high-content proteomic technologies to comprehensively profile patients' immune response to surgery is a promising approach to identify predictive biological factors of SSCs. METHODS: Forty-one patients undergoing non-cancer bowel resection were prospectively enrolled. Blood samples collected before surgery and on postoperative day one (POD1) were analyzed using a combination of single-cell mass cytometry and plasma proteomics. The primary outcome was the occurrence of an SSC, including surgical site infection, anastomotic leak, or wound dehiscence within 30 days of surgery. RESULTS: A multiomic model integrating the single-cell and plasma proteomic data collected on POD1 accurately differentiated patients with (n = 11) and without (n = 30) an SSC [area under the curve (AUC) = 0.86]. Model features included coregulated proinflammatory (eg, IL-6- and MyD88- signaling responses in myeloid cells) and immunosuppressive (eg, JAK/STAT signaling responses in M-MDSCs and Tregs) events preceding an SSC. Importantly, analysis of the immunological data obtained before surgery also yielded a model accurately predicting SSCs (AUC = 0.82). CONCLUSIONS: The multiomic analysis of patients' immune response after surgery and immune state before surgery revealed systemic immune signatures preceding the development of SSCs. Our results suggest that integrating immunological data in perioperative risk assessment paradigms is a plausible strategy to guide individualized clinical care.

Differential effects of protein intake versus intake of a defined oligopeptide on FGF-21 in obese human subjects in vivo.

BACKGROUND: FGF-21 is described as a powerful metabolic regulator with beneficial effects including glucose-lowering and improvement of insulin sensitivity without hypoglycaemia. On the other hand, FGF-21 is activated when muscle and other tissues are stressed by external effects or internal cellular pathogens that lead to shortcomings in metabolic balance. Previous results suggested that FGF-21 could be a promising target to develop future metabolic therapeutics. PURPOSE: The present study was performed to gain deeper insight into the regulation of FGF-21 by protein metabolism in obese human subjects. METHODS: FGF-21 serum concentrations were measured in a cohort of n = 246 obese humans ± type 2 diabetes mellitus (T2DM) (median age 53.0 [46.0; 60.0] years and BMI 40.43 [35.11; 47.24] kg/m2) and related to the nutritional protein intake. In addition, the effect of a novel oligopeptide purified from a ß-casein hydrolysate on FGF-21 was examined in vitro in liver cells and in vivo in a human intervention study with the main focus on metabolic inflammation including 40 mainly obese subjects (mean age 41.08 ± 9.76 years, mean BMI 38.29 ± 9.4 kg/m2) in a randomized 20 weeks double-blind cross-over design. MAIN FINDINGS: In the cohort analysis, FGF-21 serum concentrations were significant lower with higher protein intake in obese subjects without T2DM but not in obese subjects with T2DM. Furthermore, relative methionine intake was inversely related to FGF-21. While global protein intake in obesity was inversely associated with FGF-21, incubation of HepG2 cells with a ß-casein oligopeptide increased FGF-21 expression in vitro. This stimulatory effect was also present in vivo, since in the clinical intervention study treatment of obese subjects with the ß-casein oligopeptide for 8 weeks significantly increased FGF-21 serum levels from W0 = 23.86 pg/mL to W8 = 30.54 pg/mL (p < 0.001), while no increase was found for placebo. CONCLUSION: While the total nutritional protein intake is inversely associated with FGF-21 serum levels, a purified and well characterised oligopeptide is able to induce FGF-21 serum levels in humans. These findings suggest a differential role of various components of protein metabolism on FGF-21, rather than this factor being solely a sensor of total nutritional protein intake.

Diet, Physical Activity and Adiposity as Determinants of Circulating Amino Acid Levels in a Multiethnic Asian Population.

Profiles of circulating amino acids have been associated with cardiometabolic diseases. We investigated the associations between dietary protein intake, physical activity and adiposity and serum amino acid profiles in an Asian population. We used data from 3009 male and female participants from the Singapore Prospective Study Program cohort. Dietary and physical activity data were obtained from validated questionnaires; anthropometric measurements were collected during a health examination; and fasting concentrations of 16 amino acids were measured using targeted LC-MS. The association between lifestyle factors and amino acid levels was modeled using multiple linear regression with adjustment for other sociodemographic and lifestyle factors and correction for multiple testing. We observed significant associations between seafood intake (ß-coefficient 0.132, 95% CI 0.006, 0.257 for a 100% increment), physical activity (ß-coefficient -0.096, 95% CI -0.183, -0.008 in the highest versus lowest quartile) and adiposity (BMI ß-coefficient 0.062, 95% CI 0.054, 0.070 per kg/m2; waist circumference ß-coefficient 0.034, 95% CI 0.031, 0.037 per cm) and branched-chain amino acid levels (expressed per-SD). We also observed significant interactions with sex for the association between meat and seafood and total intakes and BCAA levels (P for interaction 0.007), which were stronger in females than in males. Our findings suggest novel associations between modifiable lifestyle factors and amino acid levels in Asian populations.

Postprandial inflammatory responses after oral glucose, lipid and protein challenges: Influence of obesity, sex and polycystic ovary syndrome.

BACKGROUND & AIMS: Most evidence linking the polycystic ovary syndrome (PCOS) with chronic low-grade inflammation has been obtained in the fasting state. We have studied the postprandial inflammatory response to oral glucose, lipid and protein challenges and the possible influences of obesity, sex and PCOS on these responses. METHODS: On alternate days, we submitted 17 women with PCOS (9 non-obese, 8 obese), 17 control women (9 non-obese, 8 obese) and 19 control men (10 non-obese, 9 obese) to isocaloric (300 Kcal) oral macronutrient loads. We assayed serum for TNF-α, IL-6, IL-18, IL-10, pentraxin-3 and galectin-3 concentrations and leukocytes for expression of TNF, IL6, IL10 and their receptors TNFRSF1B, IL6R and IL10RA. RESULTS: Circulating IL-6 levels decreased after glucose and protein ingestion but slightly increased after oral lipid intake. Leukocyte IL6 expression did not change after the ingestion of any macronutrient yet IL6R expression increased during all macronutrient challenges, the largest increase being observed after glucose ingestion. Serum TNF-α similarly decreased during either macronutrient load, whereas TNF expression increased after macronutrient ingestion, the highest increase observed after oral glucose. TNFRSF1B expression also increased after glucose intake but not after lipid or protein ingestion. No global effect of obesity or group on postprandial circulating IL-6, TNF-α, or IL6, IL6R, TNF and TNFRSF1B expression was found. Circulating IL-18 concentrations decreased during all oral challenges, whereas in case of galectin-3 and pentraxin-3 only the protein load caused a reduction in its concentrations. Of the genes studied here, IL10 showed the largest increase in expression throughout all the postprandial curves, particularly after glucose. Obesity blunted the increase in IL10 expression. IL10RA expression decreased after glucose ingestion but remained unchanged during lipid and protein loads. CONCLUSIONS: Glucose ingestion, as opposed to lipid and protein intake, results into the largest increase in leukocyte gene expression of inflammatory mediators. The expression of the anti-inflammatory cytokine IL10 was the largest observed here, suggesting a compensatory mechanisms against postprandial inflammation that may be blunted in obesity. However, these responses did not translate into the circulating concentrations of these inflammatory mediators during the immediate postprandial phase.

Hydrolyzed proteins from herring and salmon rest raw material contain peptide motifs with angiotensin-I converting enzyme inhibitors and resulted in lower urine concentrations of protein, cystatin C and glucose when fed to obese Zucker fa/fa rats.

The use of angiotensin-I converting enzyme (ACE) inhibitors is a common strategy for treating kidney disease. Several amino acid sequences with ACE inhibiting activity are identified in filet and rest raw material from various species of fish, and fish protein hydrolysates could be of interest for possible treatment or prevention of kidney disease. Therefore, we hypothesized that protein hydrolysates from rest raw material from herring and salmon contained ACE inhibiting motifs, and could beneficially affect typical markers for kidney function in an obesity rat model prone to developing renal failure. We identified 81 and 49 peptide sequences with known ACE inhibiting activity in herring and salmon protein hydrolysates from rest raw material, respectively. To investigate the effects of fish protein hydrolysates on markers of kidney function, obese Zucker fa/fa rats consumed diets with 25% of protein from herring (HER) or salmon (SAL) protein hydrolysate from rest raw material and 75% of protein from casein/whey, or 100% protein from casein/whey (CAS) for 4 weeks. Rats fed HER or SAL diets had lower urine concentrations (relative to creatinine) of protein, cystatin C and glucose when compared to rats fed CAS diets, with no differences between groups for serum concentrations of protein, creatinine and cystatin C. To conclude, protein hydrolysates from herring and salmon rest raw material contained several peptide sequences with known ACE inhibiting activities, and resulted in lower urine concentrations of proteins, cystatin C and glucose when fed to obese Zucker rats.

Is additional enrichment of diet in branched-chain amino acids or glutamine beneficial for patients receiving total parenteral nutrition after gastrointestinal cancer surgery?

OBJECTIVES: Total Parenteral Nutrition (TPN) is necessary in patients unable to receive oral or enteral feeding for a period of at least 7 days. Branched-chain amino acids (BCAA): valine (Val), leucine (Leu), and isoleucine (Ile) are essential amino acids, which are important regulators in protein metabolism. They are also the main nitrogen source for glutamine synthesis in muscles. In this process they undergo irreversible degradation and cannot be reutilised for protein synthesis. In catabolic states, like cancers, glutamine demand increases and therefore also its utilisation, which can decrease the level of BCAA required for Gln synthesis. The purpose of this study was to evaluate the necessity of BCAA or glutamine-enriched TPN in patients after gastrointestinal cancers surgery. MATERIAL AND METHODS: Our aim was to investigate changes of plasma BCAA and glutamine concentrations in patients operated for colorectal, small intestine or pancreatic cancer and who are either receiving TPN or not in the postoperative period. Free amino acids plasma concentrations were determined by the ion-exchange chromatography. RESULTS: Surgery in the control group caused a decrease in Val, Ile and Leu concentrations in the postoperative period. In TPN patients this depression was inhibited beginning from the third day after surgery, except for Val and Leu in colorectal cancer group. In control and TPN patient groups, Gln concentration decreased after the surgery and subsequently increased beginning from the third day after the operation. CONCLUSIONS: Gastrointestinal cancer patients' surgery results in decrease in BCAA concentrations. Standard TPN exerts a beneficial effect on the BCAA level in patients with pancreatic and small intestine cancer. In colorectal cancer such TPN should be enriched with Leu and Val.

Influence of protein intake from haem and non-haem animals and plant origin on inflammatory biomarkers among apparently-healthy adults in Greece.

Intake of different types of protein may be associated with differences in biomarkers among various populations. This work investigated the influence of protein intake from haem and non-haem animals as well as protein from plants on haematological and biochemical parameters in inflammation among apparently-healthy adults living in Greece, a Mediterranean country. Four hundred and ninety apparently-healthy subjects (46 +/- 16 years, 40% men), who consecutively visited Polykliniki General Hospital for routine examinations, voluntarily agreed to participate in the study (participation rate 85%). Demographic, anthropometric and lifestyle characteristics were recorded. Participants completed a valid, semi-quantitative food frequency questionnaire. Protein intake was classified into three sources: protein from haem animals, protein from non-haem animals, and protein from plant origin. Fasting blood samples were taken from all participants; uric acid, creatinine, lipids, cystatin C, haptoglobin, haemoglobin, haematocrit, iron, ferritin, white blood cells, monocytes, platelets, and C-reactive protein were measured. Protein intake from only haem animals was associated with increased haemoglobin and haematocrit levels (p < 0.05) whereas intake of protein from non-haem animals and plant origin was not associated with the investigated haematological and biochemical markers of low-grade chronic inflammation when lifestyle factors and overall dietary habits were taken into account. Intake of protein from only haem animals seems to be consistently associated with haematological markers. The confounding role of dietary habits and lifestyle variables on the tested parameters deserves further attention in future research.

T-type amino acid transporter TAT1 (Slc16a10) is essential for extracellular aromatic amino acid homeostasis control.

The uniporter TAT1 (Slc16a10) mediates the facilitated diffusion of aromatic amino acids (AAAs) across basolateral membranes of kidney, small intestine and liver epithelial cells, and across the plasma membrane of non-epithelial cells like skeletal myocytes. Its role for body AA homeostasis has now been investigated using newly generated TAT1 (Slc16a10) defective mice (tat1(-/-)). These mice grow and reproduce normally, show no gross phenotype and no obvious neurological defect. Histological analysis did not reveal abnormalities and there is no compensatory change in any tested AA transporter mRNA. TAT1 null mice, however, display increased plasma, muscle and kidney AAA concentration under both normal and high protein diet, although this concentration remains normal in the liver. A major aromatic aminoaciduria and a smaller urinary loss of all substrates additionally transported by l-type AA antiporter Lat2-4F2hc (Slc7a8) were revealed under a high protein diet. This suggests an epithelial transport defect as also shown by the accumulation of intravenously injected (123)I-2-I-l-Phe in kidney and l-[(3)H]Phe in ex vivo everted gut sac enterocytes. Taken together, these data indicate that the uniporter TAT1 is required to equilibrate the concentration of AAAs across specific membranes. For instance, it enables hepatocytes to function as a sink that controls the extracellular AAAs concentration. Additionally, it facilitates the release of AAAs across the basolateral membrane of small intestine and proximal kidney tubule epithelial cells, thereby allowing the efflux of other neutral AAs presumably via Lat2-4F2hc.

High-carbohydrate/low-protein-induced hyperinsulinemia does not improve protein balance in children after cardiac surgery.

OBJECTIVE: In pediatric cardiac surgery, fluid-restricted low-protein (LoProt) diets account for cumulative protein deficits with increased morbidity. In this setting, we aimed to inhibit proteolysis by a high-carbohydrate (HiCarb)-intake-induced hyperinsulinemia and improve protein balance. METHODS: The effect of a HiCarb/LoProt (glucose 10 mg · kg(-1) · min(-1)/protein 0.7 g · kg(-1) · d(-1)) versus a normal-carbohydrate (NormCarb)/LoProt (glucose 7.5 mg · kg(-1) · min(-1)/protein 0.3 g · kg(-1) · d(-1)) enteral diet on whole-body protein breakdown and balance was compared in a prospective, randomized, single-blinded trial in 24 children after cardiac surgery. On the second postoperative day, plasma insulin and amino acid concentrations, protein breakdown (endogenous rate of appearance of valine), protein synthesis (non-oxidative disposal of valine), protein balance, and the rate of appearance of urea were measured by using an isotopic infusion of [1-(13)C]valine and [(15)N(2)]urea. RESULTS: The HiCarb/LoProt diet led to a serum insulin concentration that was three times higher than the NormCarb/LoProt diet (596 pmol/L, 80-1833, and 198 pmol/L, 76-1292, respectively, P = 0.02), without differences in plasma glucose concentrations. There were no differences in plasma amino acid concentrations, non-oxidative disposal of valine, and endogenous rate of appearance of valine between the groups, with a negative valine balance in the two groups (-0.65 µmol · kg(-1) · min(-1), -1.91 to 0.01, and -0.58 µmol · kg(-1) · min(-1), -2.32 to -0.07, respectively, P = 0.71). The serum cortisol concentration in the HiCarb/LoProt group was lower compared with the NormCarb/LoProt group (204 nmol/L, 50-544, and 532 nmol/L, 108-930, respectively, P = 0.02). CONCLUSION: In children with fluid restriction after cardiac surgery, a HiCarb/LoProt diet compared with a NormCarb/LoProt diet stimulates insulin secretion but does not inhibit proteolysis further and therefore cannot be advocated for this purpose.

Diurnal variation of phenylalanine concentrations in tyrosinaemia type 1: should we be concerned?

BACKGROUND: Tyrosinaemia type 1 (HT1) is treated with a tyrosine and phenylalanine-restricted diet, amino acids free of phenylalanine and tyrosine, and nitisinone (NTBC). Treatment guidelines recommend plasma tyrosine between 200-400 µm and phenylalanine at least >30 µm. There is little information on the diurnal variation of plasma tyrosine or phenylalanine in HT1. Low plasma phenylalanine <30 µm may be associated with poor growth and cognitive delay. The present study aimed to document diurnal variation of tyrosine and phenylalanine plasma concentrations and growth in children with HT1. METHODS: Median tyrosine and phenylalanine plasma concentrations were reviewed retrospectively over 3 years in 11 subjects (median age 4 years) with HT1. Subjects routinely collected morning fasting blood samples but afternoon nonfasted samples were taken in the clinic (<10% of samples). Growth Z-scores were calculated. RESULTS: The percentage of all plasma phenylalanine concentrations <30 µm was 8.6% and <40 µm was 13.6%. Only 2% of fasting morning phenylalanine concentrations were <30 µm, compared to 83% of nonfasting afternoon samples. All but one child had a height Z-score <0. CONCLUSIONS: Blood phenylalanine concentrations were consistently lower in the afternoon. Taking blood samples at variable time points in the day may lead to variation in interpreting dietary control. A detailed study is necessary to examine the 24-h diurnal variation of plasma phenylalanine and tyrosine in HT1. It is possible that phenylalanine concentrations may be very low for a substantive time over 24 h and the potential impact that this may have on cognitive development and growth in children is unknown.

Acute reduction of serum 8-iso-PGF2-alpha and advanced oxidation protein products in vivo by a polyphenol-rich beverage; a pilot clinical study with phytochemical and in vitro antioxidant characterization.

BACKGROUND: Measuring the effects of the acute intake of natural products on human biomarker concentrations, such as those related to oxidation and inflammation, can be an advantageous strategy for early clinical research on an ingredient or product. METHODS: 31 total healthy subjects were randomized in a double-blinded, placebo-controlled, acute pilot study with post-hoc subgroup analysis on 20 of the subjects. The study examined the effects of a single dose of a polyphenol-rich beverage (PRB), commercially marketed as "SoZo(®)", on serum anti-inflammatory and antioxidant markers. In addition, phytochemical analyses of PRB, and in vitro antioxidant capacity were also performed. RESULTS: At 1 hour post-intake, serum values for 8-iso-PGF2-alpha and advanced oxidation protein products decreased significantly by 40% and 39%, respectively. Additionally, there was a trend toward decreased C-reactive protein, and increased nitric oxide levels. Both placebo and PRB treatment resulted in statistically significant increases in hydroxyl radical antioxidant capacity (HORAC) compared to baseline; PRB showed a higher percent change (55-75% versus 23-74% in placebo group), but the two groups did not differ significantly from each other. CONCLUSIONS: PRB produced statistically significant changes in several blood biomarkers related to antioxidant/anti-inflammatory effects. Future studies are justified to verify results and test for cumulative effects of repeated intakes of PRB. The study demonstrates the potential utility of acute biomarker measurements for evaluating antioxidant/anti-inflammatory effects of natural products.

Dietary carbohydrate-to-protein ratio affects TOR signaling and metabolism-related gene expression in the liver and muscle of rainbow trout after a single meal.

Most teleost fish are known to require high levels of dietary proteins. Such high-protein intake could have significant effects, particularly on insulin-regulated gene expression. We therefore analyzed the effects of an increase in the ratio of dietary carbohydrates/proteins on the refeeding activation of the Akt-target of rapamycin (TOR) signaling pathways in rainbow trout and the effects on the expression of several genes related to hepatic and muscle metabolism and known to be regulated by insulin, amino acids, and/or glucose. Fish were fed once one of three experimental diets containing high (H), medium (M), or low (L) protein (P) or carbohydrate (C) levels after 48 h of feed deprivation. Activation of the Akt/TOR signaling pathway by refeeding was severely impaired by decreasing the proteins-to-carbohydrates ratio. Similarly, postprandial regulation of several genes related to glucose (Glut4, glucose-6-phosphatase isoform 1), lipid (fatty acid synthase, ATP-citrate lyase, sterol responsive element binding protein, carnitine palmitoyltransferase 1, and 3-hydroxyacyl-CoA dehydrogenase), and amino acid metabolism (serine dehydratase and branched-chain α-keto acid dehydrogenase E2 subunit) only occurred when fish were fed the high-protein diet. On the other hand, diet composition had a low impact on the expression of genes related to muscle protein degradation. Interestingly, glucokinase was the only gene of those monitored whose expression was significantly upregulated by increased carbohydrate intake. In conclusion, this study demonstrated that macro-nutrient composition of the diet strongly affected the insulin/amino acids signaling pathway and expression pattern of genes related to metabolism.

Lunasin is prevalent in barley and is bioavailable and bioactive in in vivo and in vitro studies.

Lunasin, a unique 43-amino acid peptide found in a number of seeds, has been shown to be chemopreventive in mammalian cells and in a skin cancer mouse model. To elucidate the role of cereals in cancer prevention, we report here the prevalence, bioavailability, and bioactivity of lunasin from barley. Lunasin is present in all cultivars of barley analyzed. The liver and kidney of rats fed with lunasin-enriched barley (LEB) show the presence of lunasin in Western blot. Lunasin extracted from the kidney and liver inhibits the activities of HATs (histone acetyl transferases), yGCN5 by 20% and 18% at 100 nM, and PCAF activity by 25% and 24% at 100 nM, confirming that the peptide is intact and bioactive. Purified barley lunasin localizes in the nuclei of NIH 3T3 cells. Barley lunasin added to NIH 3T3 cells in the presence of the chemical carcinogen MCA activates the expression of tumor suppressors p21 and p15 by 45% and 47%, decreases cyclin D1 by 98%, and inhibits Rb hyperphosphorylation by 45% compared with the MCA treatment alone. We conclude that lunasin is prevalent in barley, bioavailable, and bioactive and that consumption of barley could play an important role of cancer prevention in barley-consuming populations.

Muscle protein breakdown has a minor role in the protein anabolic response to essential amino acid and carbohydrate intake following resistance exercise.

Muscle protein breakdown (MPB) is increased following resistance exercise, but ingestion of carbohydrate during postexercise recovery can decrease MPB with no effect on muscle protein synthesis (MPS). We sought to determine whether a combination of essential amino acids (EAA) with low carbohydrate or high carbohydrate could effectively reduce MPB following resistance exercise and improve muscle protein net balance (NB). We hypothesized that higher levels of carbohydrate and resulting increases in circulating insulin would inhibit MPB and associated signaling, resulting in augmented NB. Thirteen male subjects were assigned to one of two groups receiving equivalent amounts of EAA (approximately 20 g) but differing carbohydrate levels (low = 30, high = 90 g). Groups ingested nutrients 1 h after an acute bout of leg resistance exercise. Leg phenylalanine kinetics (e.g., MPB, MPS, NB), signaling proteins, and mRNA expression were assessed on successive muscle biopsies using stable isotopic techniques, immunoblotting, and real-time quantitative PCR, respectively. MPB tended to decrease (P < 0.1) and MPS increased (P < 0.05) similarly in both groups following nutrient ingestion. No group differences were observed, but muscle ring finger 1 (MuRF1) protein content and MuRF1 mRNA expression increased following resistance exercise and remained elevated following nutrient ingestion, while autophagy marker (light-chain 3B-II) decreased after nutrient ingestion (P < 0.05). Forkhead box-O3a phosphorylation, total muscle atrophy F-box (MAFbx) protein, and MAFbx and caspase-3 mRNA expression were unchanged. We conclude that the enhanced muscle protein anabolic response detected when EAA+carbohydrate are ingested postresistance exercise is primarily due to an increase in MPS with minor changes in MPB, regardless of carbohydrate dose or circulating insulin level.

Identification of food-derived bioactive peptides in blood and other biological samples.

Recent studies have demonstrated the presence of food-derived peptides in human blood after ingestion of enzymatic hydrolysates of food proteins, while most peptides in food are degraded into amino acids during digestion and absorption. To capture and clarify the food-derived peptides in blood, solid-phase extraction (SPE) using a mini-spin column packed with a strong cation exchanger was developed. This technique allows the use of a nonvolatile acid such as trichloroacetic acid, a strong protein denaturant, for the deproteinizing procedure. To improve resolution of hydrophilic peptide and increase specificity and sensitivity in the detection of peptide by reversed-phase high-performance liquid chromatography (RP-HPLC) after subfractionation by size-exclusion chromatography (SEC), peptides are derivatized with phenyl isothiocyanate. The resultant phenyl thiocarbamyl (PTC)-peptides can be resolved with high resolution and sensitivity by RP-HPLC. By comparing chromatograms of PTC derivatives from blood before and after ingestion of a peptide sample, food-derived peptide can be detected. The isolated PTC-peptide can be applied to a peptide sequencer based on the Edman degradation reaction.

Does maternal knowledge impact blood phenylalanine concentration in Turkish children with phenylketonuria?

The aim of this study was to compare the level of maternal knowledge and the blood phenylalanine (Phe) control in phenylketonuria (PKU; OMIM 261600). The study was conducted on 144 children (81 boys, 63 girls) with PKU, aged between 1 and 15 years, at Hacettepe University Ihsan Dogramaci Children's Hospital, Metabolism and Nutrition Unit. All subjects were treated with a low-Phe diet using a Phe-exchange system. A 20-question multiple-choice questionnaire was applied to the mothers to determine their knowledge about PKU and its dietary treatment. Questions in the test consisted of the knowledge about the disease (6 questions), general dietary knowledge (14 questions) and knowledge about specific exchange within the dietary questions (6 questions). The median blood Phe concentration for the previous 3-year period was used as an indicator of metabolic control. Eighty-seven children had a median blood Phe concentration above the MRC Working Party Guidelines. There was a negative correlation between maternal knowledge about exchange and median blood Phe concentration in the child (p<0.05). Maternal knowledge about a standard 15 mg Phe exchange system is correlated with dietary compliance as measured by blood Phe concentrations in our subjects. We would like to implement an easier method of measuring Phe exchanges to improve dietary knowledge in the mothers.

Inadequate protein intake affects skeletal muscle transcript profiles in older humans.

BACKGROUND: Inadequate dietary protein intake causes adverse changes in the morphology and function of skeletal muscle. These changes may be reflected in early alterations in muscle messenger RNA levels. OBJECTIVE: This study assessed whether inadequate protein intake differentially affects skeletal muscle transcript concentrations and expression profiles in older adults. DESIGN: Twenty-one older men and women (aged 55-80 y) consumed controlled diets that provided 1.2 g protein x kg(-1) x d(-1) (adequate protein) for 1 wk and then were randomly assigned to consume either 0.5 g protein x kg(-1) x d(-1) [inadequate protein (IP) group; n=11] or 1.2 g protein x kg(-1) x d(-1) (control group; n=10) for a second week. RNA was isolated from fasting-state vastus lateralis biopsy samples obtained at the end of each period, and transcript levels in the IP group were measured by using microarray analysis. Changes in selected transcript levels were confirmed by real-time polymerase chain reaction in both groups. RESULTS: Analysis of variance showed 529 differentially expressed transcripts (P<0.05) after inadequate protein intake. Using the false discovery rate (FDR) correction to adjust for multiple comparisons, we observed that 85 transcripts were differentially expressed: 54 were up-regulated and 31 were down-regulated. The differentially expressed transcripts were in functional classes for immune, inflammatory, and stress responses (predominantly up-regulated); contraction, movement, and development (up-regulated); extracellular connective tissue (up-regulated); energy metabolism (down-regulated); protein synthesis (down-regulated); and proliferation (down-regulated). Diet-related differences in the expression of 9 transcripts were cross-validated by using real-time polymerase chain reaction. CONCLUSION: The results document changes in skeletal muscle transcript levels induced by short-term inadequate protein intakes in older humans that might precede adverse metabolic, functional, and structural events, including muscle wasting.

Improvement in isolation and identification of food-derived peptides in human plasma based on precolumn derivatization of peptides with phenyl isothiocyanate.

For the isolation and detection of food-derived peptides in blood, an approach based on the derivatization of peptides with phenyl isothiocyanate (PITC) was developed. This approach allows hydrophilic peptides to be resolved and specifically detected by reversed-phase (RP) HPLC. For the rapid capturing and clarification of peptides in human plasma, solid-phase extraction by using a mini spin column (5 mmx5 mm) packed with a strong cation exchanger was used. The clarified peptide fraction was further fractionated by size-exclusion chromatography (SEC). The peptides in the SEC fractions were derivatized with PITC, and the derivatives were resolved by RP-HPLC by using an ammonium acetate buffer or a trifluoroacetic acid system. An automatic peptide sequencer based on Edman degradation with a modified program can directly analyze the resolved derivatives. Some synthetic peptides and food-derived peptides in human plasma were successfully isolated and identified by this approach.

Dietary habits are major determinants of the plasma antioxidant status in healthy elderly subjects.

Previous studies indicate that regular consumption of a diet rich in fruits and vegetables is associated with a lower risk for age-related diseases. The aim of the present study was to evaluate whether the often-reported age-related decrease of plasma antioxidants in man depends on differences in dietary intake or on other age- and gender-related factors. In this observational case-control study, thirty-nine community-dwelling healthy subjects aged 65 years and older consuming high intakes of fruits and vegetables daily (HI) and forty-eight healthy subjects aged 65 and older consuming low intakes of fruit and vegetables daily (LI) were enrolled. Plasma levels of retinol, tocopherols, carotenoids and malondialdehyde (MDA) as well as content of protein carbonyls in Ig G were measured. Plasma levels of retinol, tocopherols and carotenoids were significantly higher in group HI than in group LI subjects independent of age and gender. MDA levels were inversely correlated with vitamin A and alpha-carotene. Protein carbonyls were inversely correlated with gamma-tocopherol. In the elderly, a higher daily intake of fruits and vegetables is associated with an improved antioxidant status in comparison to subjects consuming diets poor in fruits and vegetables. Modification of nutritional habits among other lifestyle changes should be encouraged to lower prevalence of disease risk factors in later life.