Enrichment Of Amino Acids Research Articles

Direct evidence for embryonic uptake of paternally-derived nutrients in two pipefishes (Syngnathidae: Syngnathus spp.)

Seahorses, sea dragons and pipefishes of the teleost family Syngnathidae are unique in that embryos develop within specialized brooding structures of the male. We enriched brooding Syngnathus fuscus and Syngnathus floridae males with injections of L-lysine-[(15)N(2)] and 16:0-palmitic acid 1-[(13)C] to demonstrate embryonic uptake of paternally-derived nutrients. While all embryos demonstrated amino acid enrichment, late stages showed significantly higher [(15)N], indicating greater utilization of paternal resources as yolk reserves diminished and embryonic energy demands increased. Limited embryonic [(13)C] uptake, defined as less than 10% of adult enrichment, in 75 and 81% of S. floridae and S. fuscus respectively signified rapid lipid metabolism and thus the need for greater enrichment. Interspecific differences in embryonic uptake of paternally-derived nutrients were not demonstrated. However, interspecific differences in egg nutrient reserves and fry size but comparable fry nutrient levels along with data from a published paternal exposure study indicate paternal transfer in S. fuscus most likely compensates for the comparative egg nutrient deficiency. This study is the first to our knowledge to provide direct evidence for the functional significance of the brood pouch in nutrient provisioning. These results add comparative information on the diversity of Syngnathid paternal care and further our understanding of paternal influence on development.

Contribution of gut microbial lysine to liver and milk amino acids in lactating does

The contribution of microbial amino acids through caecotrophy to tissue protein metabolism was investigated in lactating does. Attempts were made to vary microbial supply through a dietary antibiotic, Zn bacitracin, and to vary tissue demand through manipulation of litter size. Three groups of eight New Zealand does were fed different experimental diets from day 28 of pregnancy to day 26 of lactation. The control group received the basal diet formulated to meet requirements with grass hay, wheat, soybean meal and barley grain. The second (no antibiotic) group and the third (bacitracin; BAC) group ingested the basal diet supplemented with ammonium sulfate (5 g/kg), initially unlabelled (day 1 to day 8) then labelled with 15N (day 9 to day 30), while the BAC diet was also supplemented throughout with antibiotic (Zn bacitracin; 100 mg/kg). From just after birth each group of does was subdivided into two groups, each of four females, with the litter size either five (LS5) or nine (LS9) pups. The 15N enrichment in liver, milk and caecal bacteria amino acids was determined by GC-combustion-isotope ratio MS. All amino acids in bacterial protein were enriched with the (15 NH 4)2SO4 treatment, with lysine 15N enrichment significantly greater in caecal bacteria (0.23 (SE 0.0063) atom % excess (ape)) than in liver (0.04 (SE 0.0004) ape) or milk protein (0.05 (SE 0.0018) ape), confirming the double origin (bacterial and dietary) of tissue lysine. The contribution of microbes to tissue lysine was 0.23 (SE 0.006) when milk protein was used as reference.

홍국균 발효가 콩의 GABA와 유리아미노산 함량에 미치는 영향

콩 기질에 대한 홍국균의 접종 발효는 원료 콩에 함유된 GABA와 유리아미노산의 함량을 강화시키는 유효한 발효기법으로 나타났다. 선별된 5가지 균주(M. pilosus IFO 480, M. anka IFO 873, M. kaoliang ATCC 592, M. purpurea IFO 482, M. kaling ATCC 598)로 발효시킨 홍국 발효콩은 비발효콩에 비해 GABA의 함량이 2배 이상 증가되는 것으로 나타났으며, 가장 활성이 강한 균은 Monascus pilosus IFO 480으로 나타났다. Monascus pilosus IFO 480으로 30℃에서 30일간 발효시킨 홍국 발효콩의 발효기간 중 GABA(발효 20일)와 유리아미노산(발효 10일) 함량의 최대변화는 비발효콩에 비해 각각 2.5배와 5.6배 증가되었다. 특히 발효 20일 경에 GABA의 생성량은 건조 발효콩 100 g당 78.5 ㎎으로 나타나 비발효콩의 31.5 ㎎에 비해 149% 증가된 것으로 비교되었다. 또한 홍국 발효콩에 함유된 필수아미노산의 함유율은 비발효콩에 비해 3.6배 증가되었으며 특히 threonine, leucine, lysine 등의 증가율은 비발효콩에 비해 12～24배까지 두드러져 홍국 발효콩은 이들 아미노산의 강화에 매우 유효한 발효기법으로 나타났다. 따라서 GABA와 필수아미노산의 함량이 강화된 홍국 발효콩은 다양한 약리학적 소재뿐 아니라 영양학적 소재류에서도 매우 활용가능성이 높은 복합기능성의 신소재로 평가할 수 있다.

Culture nutrition and physiology impact the inhibitor tolerance of the yeastPichia stipitisNRRL Y‐7124

Pichia stipitis NRRL Y-7124 is one of the natural yeasts best able to utilize biomass because it is able to ferment hexoses and the pentose, xylose, to economically recoverable concentrations of ethanol. To test the impact of culture conditions on inhibitor tolerance, inhibitors were spiked to growing or stationary-phase P. stipitis supplied either glucose or xylose and varying nitrogen and mineral compositions; then the ensuing specific death rate response was measured. Resistance of glucose- or xylose-grown cells to inhibitors was generally greater in stationary-phase cells than log-phase cells, despite a greater exposure of stationary cells to ethanol. Consistent with this, the specific productivity of detoxification products, furan methanol or furan-2,5-dimethanol, from respective spikes of furfural or HMF increased as cultures progressed into stationary phase. However, when xylose was the substrate, ethanol resistance behaved uniquely and was greater for log- than stationary-phase cells. Amino acid enrichment of the growth medium significantly enhanced ethanol tolerance if xylose was the carbon source, but had no impact if glucose supplied carbon. Regardless of the carbon source, amino acid enrichment of the culture medium enhanced the ability of cells to resist furfural and HMF exposure. Mineral compositions tested had little impact on inhibitor resistance except stationary-phase xylose-grown cells were more susceptible to inhibitor exposure when magnesium sulfate was excessive. Observed tolerance optimization based on specific death rate as a function of culture physiological state, carbon source, nitrogen source and mineral composition provides new knowledge supporting process designs to convert biomass to ethanol using P. stipitis.

Total sulfur amino acid requirement and metabolism in parenterally fed postsurgical human neonates

BackgroundExcept for tyrosine, the amino acid requirements of human neonates receiving parenteral nutrition (PN) have not been experimentally derived. ObjectivesThe objectives were to determine the total sulfur amino acid (TSAA) requirement (methionine in the absence of cysteine) of postsurgical, PN-fed human neonates by using the indicator amino acid oxidation (IAAO) technique with l-[1-13C]phenylalanine as the indicator. DesignFifteen postsurgical neonates were randomly assigned to receive 1 of 18 methionine intakes ranging from 10 to 120 mg · kg−1 · d−1, delivered in a customized, cysteine-free amino acid solution. Breath and urine samples were collected for the measurement of 13CO2 and amino acid enrichment. Blood samples were collected at baseline and after the test methionine infusion for the measurement of plasma methionine, homocysteine, cystathionine, and cysteine concentrations. ResultsBreakpoint analysis determined the mean TSAA requirements to be 47.4 (95% CI: 38.7, 56.1) and 49.0 (95% CI: 39.9, 58.0) mg · kg−1 · d−1 with the use of oxidation and F13CO2, respectively. ConclusionsThis is the first study to report the TSAA requirement of postsurgical, PN-fed human neonates. The estimated methionine requirement expressed as a proportion of the methionine content of current commercial pediatric PN solutions was 90% (range: 48–90%) of that found in the lowest methionine-containing PN solution.

Elimination of KATP Channels in Mouse Islets Results in Elevated [U-13C]Glucose Metabolism, Glutaminolysis, and Pyruvate Cycling but a Decreased γ-Aminobutyric Acid Shunt

Pancreatic beta cells are hyper-responsive to amino acids but have decreased glucose sensitivity after deletion of the sulfonylurea receptor 1 (SUR1) both in man and mouse. It was hypothesized that these defects are the consequence of impaired integration of amino acid, glucose, and energy metabolism in beta cells. We used gas chromatography-mass spectrometry methodology to study intermediary metabolism of SUR1 knock-out (SUR1(-/-)) and control mouse islets with d-[U-(13)C]glucose as substrate and related the results to insulin secretion. The levels and isotope labeling of alanine, aspartate, glutamate, glutamine, and gamma-aminobutyric acid (GABA) served as indicators of intermediary metabolism. We found that the GABA shunt of SUR1(-/-) islets is blocked by about 75% and showed that this defect is due to decreased glutamate decarboxylase synthesis, probably caused by elevated free intracellular calcium. Glutaminolysis stimulated by the leucine analogue d,l-beta-2-amino-2-norbornane-carboxylic acid was, however, enhanced in SUR1(-/-) and glyburide-treated SUR1(+/+) islets. Glucose oxidation and pyruvate cycling was increased in SUR1(-/-) islets at low glucose but was the same as in controls at high glucose. Malic enzyme isoforms 1, 2, and 3, involved in pyruvate cycling, were all expressed in islets. High glucose lowered aspartate and stimulated glutamine synthesis similarly in controls and SUR1(-/-) islets. The data suggest that the interruption of the GABA shunt and the lack of glucose regulation of pyruvate cycling may cause the glucose insensitivity of the SUR1(-/-) islets but that enhanced basal pyruvate cycling, lowered GABA shunt flux, and enhanced glutaminolytic capacity may sensitize the beta cells to amino acid stimulation.

Analysis of 13C labeling enrichment in microbial culture applying metabolic tracer experiments using gas chromatography–combustion–isotope ratio mass spectrometry

The applicability of gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) for the quantification of 13C enrichment of proteinogenic amino acids in metabolic tracer experiments was evaluated. Measurement of the 13C enrichment of proteinogenic amino acids from cell hydrolyzates of Corynebacterium glutamicum growing on different mixtures containing between 0.5 and 10% [1- 13C]glucose shows the significance of kinetic isotope effects in metabolic flux studies at low degree of labeling. We developed a method to calculate the 13C enrichment. The approach to correct for these effects in metabolic flux studies using δ 13C measurement by GC–C–IRMS uses two parallel experiments applying substrate with natural abundance and 13C-enriched tracer substrate, respectively. The fractional enrichment obtained in natural substrate is subtracted from that of the enriched one. Tracer studies with C. glutamicum resulted in a statistically identical relative fractional enrichment of 13C in proteinogenic amino acids over the whole range of applied concentrations of [1- 13C]glucose. The current findings indicate a great potential of GC–C–IRMS for labeling quantification in 13C metabolic flux analysis with low labeling degree of tracer substrate directly in larger scale bioreactors.

Liquid and gas chromatography coupled to isotope ratio mass spectrometry for the determination of 13C–valine isotopic ratios in complex biological samples

On-line gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is commonly used to measure isotopic ratios at natural abundance as well as for tracer studies in nutritional and medical research. However, high-precision (13)C isotopic enrichment can also be measured by liquid chromatography-isotope ratio mass spectrometry (LC-IRMS). Indeed, LC-IRMS can be used, as shown by the new method reported here, to obtain a baseline separation and to measure (13)C isotopic enrichment of underivatised amino acids (Asp, Thr-Ser, Glu, Pro, Gly, Ala, Cys and Val). In case of Val, at natural abundance, the SD(delta(13)C) reported with this method was found to be below 1 per thousand . Another key feature of the new LC-IRMS method reported in this paper is the comparison of the LC-IRMS approach with the conventional GC-C-IRMS determination. To perform this comparative study, isotopic enrichments were measured from underivatised Val and its N(O, S)-ethoxycarbonyl ethyl ester derivative. Between 0.0 and 1.0 molar percent excess (MPE) (delta(13)C= -12.3 to 150.8 per thousand), the calculated root-mean-square (rms) of SD was 0.38 and 0.46 per thousand and the calculated rms of accuracy was 0.023 and 0.005 MPE, respectively, for GC-C-IRMS and LC-IRMS. Both systems measured accurately low isotopic enrichments (0.002 atom percent excess (APE)) with an SD (APE) of 0.0004. To correlate the relative (delta(13)C) and absolute (atom%, APE and MPE) isotopic enrichment of Val measured by the GC-C-IRMS and LC-IRMS devices, mathematical equations showing the slope and intercept of the curves were established and validated with experimental data between 0.0 to 2.3 MPE. Finally, both GC-C-IRMS and LC-IRMS instruments were also used to assess isotopic enrichment of protein-bound (13)C-Val in tibial epiphysis in a tracer study performed in rats. Isotopic enrichments measured by LC-IRMS and GC-C-IRMS were not statistically different (p>0.05). The results of this work indicate that the LC-IRMS was successful for high-precision (13)C isotopic measurements in tracer studies giving (13)C isotopic enrichment similar to the GC-C-IRMS but without the step of GC derivatisation. Therefore, for clinical studies requiring high-precision isotopic measurement, the LC-IRMS is the method of choice to measure the isotopic ratio.

Citrulline directly modulates muscle protein synthesis: evidence from in vitro study

Introduction : The ability of citrulline (CIT) to stimulate muscle protein synthesis has been recently reported. The question of how CIT can stimulate muscle protein synthesis thus arises. The aim of this work is to evaluate the direct effect of CIT on muscle protein synthesis rate using a model of incubated skeletal muscle. The study was performed in healthy and malnourished rats.Material and methods : 3 month‐old rats were submitted to dietary restriction (50 % of the spontaneous food intake with a 5% protein diet) during 6 weeks (R, n=10) or fed ad libitum(AL, n=10). Epitrochlearismuscles were removed and were placed in a tube containing 3mL of appropriate medium (Krebs‐Ringer). Muscles were first incubated for 30 min at 37°C with 95%O2:5%CO2. Muscles were then incubated for 2 hours in medium containing insulin (0.01 U/ml) and [ring‐13C6] L‐phenylalanine (1mM) with (left) or without (right) CIT (2.5 mM) under the conditions described above. At the end of the incubation period, muscles and media were frozen and stored at −80°C. The absolute synthesis rate (ASR) was determined using the [ring‐13C6]‐phe enrichment of intracellular free amino acids and proteins by mass spectrometry.Results:Conclusions: this study demonstrates for the first time that CIT has a direct effect on muscle protein synthesis and that action is not related to the generation of other amino acids.This work was supported by funds from the Ministry of Research and Technology

Determination of protein synthesis in vivo using labeling from deuterated water and analysis of MALDI-TOF spectrum

This paper describes a method of determining protein synthesis and turnover using in vivo labeling of protein with deuterated water and analysis of matrix-assisted laser desorption time-of-flight mass spectrometer (MALDI-TOF) spectrum. Protein synthesis is calculated using mass isotopomer distribution analysis instead of precursor to product amino acid enrichment ratio. During protein synthesis, the incorporation of deuterium from water changes the mass isotopomer distribution (isotope envelop) according to the number of deuterium atoms (0, 1, 2, 3, etc.) incorporated, and the distribution of the protein with 0, 1, 2, 3,... atoms of deuterium follows a binomial distribution. A mathematical algorithm by which the distribution of deuterium isotopomers can be extracted from the observed MALDI-TOF spectrum is presented. Since deuterium isotopomers are unique to newly synthesized proteins, the quantitation of their distribution provides a method for the quantitation of newly synthesized proteins. The combined use of postsource decay sequence identification and mass isotopomer distribution analysis makes the use of in vivo labeling with deuterated water a precise method to determine specific protein synthesis.

Competitive Inhibition of Amino Acid Uptake Suppresses Chlamydial Growth: Involvement of the Chlamydial Amino Acid Transporter BrnQ

Chlamydiaceae are obligate intracellular bacterial pathogens that strictly depend on host metabolites, such as nucleotides, lipids, and amino acids. Depletion of amino acids in cell culture media results in abnormal chlamydial development in vitro. Surprisingly, enrichment of certain amino acids also retards chlamydial growth. Our experiments revealed that the antichlamydial effects are largely independent of changes in the host cell transcriptome or proteome and in the major signal transduction pathway modulated by amino acids, the mTOR (mammalian target of rapamycin) pathway. Furthermore, the chlamydial growth inhibition induced by leucine, isoleucine, methionine, or phenylalanine was completely reversed by concomitant addition of valine. In contrast, the growth inhibition induced by serine, glycine, or threonine was not reversed by valine addition. Functional characterization of the only predicted chlamydial transporter for branched-chain amino acids, BrnQ, revealed that it can be blocked by leucine, isoleucine, methionine, or phenylalanine but not by serine, glycine, or threonine. This chlamydial transporter is the only known BrnQ homolog possessing specificity for methionine, suggesting a unique strategy for methionine uptake among gram-negative bacteria. The antichlamydial effects of leucine, isoleucine, methionine, and phenylalanine could be explained as competitive inhibition of the BrnQ transporter and subsequent valine starvation.

Close Coupling between Astrocytic and Neuronal Metabolisms to Fulfill Anaplerotic and Energy Needs in the Rat Brain

Carbon metabolism in the rat brain was studied in animals anesthetized with a light dose of pentobarbital and in awake animals under morphine, which were infused with either [1-13C]glucose+acetate or glucose+[2-13C]acetate for various periods of time. Brain amino-acid enrichments in tissue extracts were determined by nuclear magnetic resonance (NMR) spectroscopy and their time evolution was analyzed by automatic fitting. Acetyl-coenzyme A C2 enrichment and ratio between pyruvate carboxylase and pyruvate dehydrogenase activity (PC/PDH) were determined from glutamate and glutamine labeling. The following results were obtained: (i) amino-acid enrichment patterns implied metabolic compartmentation and occurrence of the glutamate-glutamine cycle; (ii) kinetics of aspartate, GABA, and glutamate labeling from [1-13C]glucose and of glutamine labeling from [2-13C]acetate indicated a twofold higher metabolic activity in awake than in anesthetized rat brain; (iii) evaluation of the contributions of the astrocytic and neuronal metabolisms to glutamine synthesis in both groups of rats indicated a coupling between neuronal tricarboxylic acid (TCA) cycle, glutamate-glutamine cycle and glial TCA cycle; and (iv) analyzing the extrapolations back to time zero and the steady-state values of PC/PDH indicated a close coupling between PC activity and both astrocytic and neuronal TCA cycles. All these results suggest a cooperative-like behavior of astrocytic and neuronal metabolisms to fulfill the anabolic and energy needs linked to brain activation.

Alternative equations for whole-body protein synthesis and for fractional synthetic rates of proteins

In a constant infusion study of a mass isotope of leucine, two alternative equations are commonly available to calculate amino acid oxidation rate and, thence, whole-body protein synthesis. One, developed by Matthews et al (Am J Physiol Endocrinol Metab. 1980;238:E473-E479), is shown here to require assuming a tracee steady state (TSS), namely, that tracee (unlabeled) amino acid concentrations and fluxes (rates of oxidation and incorporation into protein) are unaltered compared with the preinfusion state. The other, developed by Garlick and coworkers (Melville et al, Metabolism 1989;38:248-255), stems from a protein steady state (PSS) assumption, namely, that protein synthesis is unaffected by the tracer infusion. We derive here a simple expression for the relative difference in whole-body protein synthesis computed from the two assumptions, and a simple test of the validity of TSS in the form of an equality that must be satisfied by plasma measurements at all times. We also propose two experiments to discriminate between the two assumptions. Theoretical reasons and experimental evidence from the literature are offered to support PSS. The two assumptions result in different expressions for fractional synthetic rates (FSRs) of individual or organ proteins—TSS requires the use of tracer-to-tracee ratios and PSS the use of enrichments. An expression is derived here for the relative difference in FSR with TSS vs PSS. For both whole-body synthesis and for FSR, the TSS assumption consistently results in an underestimate, the relative bias roughly equal to the precursor amino acid enrichment.

The Route of Administration (Enteral or Parenteral) Affects the Conversion of Isotopically Labeled L‐[2‐15N]Glutamine Into Citrulline and Arginine in Humans

Glutamine exhibits numerous beneficial effects in experimental and clinical studies. It has been suggested that these effects may be partly mediated by the conversion of glutamine into citrulline and arginine. The intestinal metabolism of glutamine appears to be crucial in this pathway. The present study was designed to establish the effect of the feeding route, enteral or parenteral, on the conversion of exogenously administered glutamine into citrulline and arginine at an organ level in humans, with a focus on gut metabolism. Sixteen patients undergoing upper gastrointestinal surgery received an IV or enteral (EN) infusion of L-[2-(15)N]glutamine. Blood was sampled from a radial artery and from the portal and right renal vein. Amino acid concentrations and enrichments were measured, and net fluxes of [(15)N]-labeled substrates across the portal drained viscera (PDV) and kidneys were calculated from arteriovenous differences and plasma flow. Arterial [(15)N]glutamine enrichments were significantly lower during enteral tracer infusion (tracer-to-tracee ratio [labeled vs unlabeled substrate, TTR%] IV: 6.66 +/- 0.35 vs EN: 3.04 +/- 0.45; p < .01), reflecting first-pass intestinal metabolism of glutamine during absorption. Compared with IV administration, enteral administration of the glutamine tracer resulted in a significantly higher intestinal fractional extraction of [(15)N]glutamine (IV: 0.15 +/- 0.03 vs EN: 0.44 +/- 0.08 micromol/kg/h; p < .01). Furthermore, enteral administration of the glutamine tracer resulted in higher arterial enrichments of [(15)N]citrulline (TTR% IV: 5.52 +/- 0.44 vs EN: 8.81 +/- 1.1; p = .02), and both routes of administration generated a significant enrichment of [(15)N]arginine (TTR% IV: 1.43 +/- 0.12 vs EN: 1.68 +/- 0.18). This was accompanied by intestinal release of [(15)N]citrulline across the PDV, which was higher with enteral glutamine (IV: 0.38 +/- 0.07 vs EN: 0.72 +/- 0.11 micromol/kg/h; p = .02), and subsequent [(15)N]arginine release in both groups. In humans, the gut preferably takes up enterally administered glutamine compared with intravenously provided glutamine. The route of administration, enteral or IV, affects the quantitative conversion of glutamine into citrulline and subsequent renal arginine synthesis in humans.

A GC/MS method for the assessment of 15N and 13C incorporation into soil amino acid enantiomers

Identifying the transformation process of amino acid enantiomers was essential to probe into the fate, turnover and aging of soil nitrogen due to their important roles in the biogeochemical cycling. If this can be achieved by differentiating between the newly biosynthesized and the inherent compounds in soil, then the isotope tracer method can be considered most valid. We thereby developed a gas chromatography/mass spectrometry (GC/MS) method to trace the 15N or 13C isotope incorporation into soil amino acid enantiomers after being incubated with 15NH 4 + or U- 13C-glucose substrates. The most significant fragments (F) as well as the related minor ions were monitored by the full scan mode and the isotope enrichment in amino acids was estimated by calculating the atom percentage excess (APE). 15NH 4 + incorporation was evaluated according to the relative abundance increase of m/ z F+1 to F for neutral and acidic amino acids and F+2 to F (mass 439) for lysine. The assessment of 13C enrichment in soil amino acids was more complicated than that of 15N due to multi-carbon atoms in amino acid molecules. The abundance ratio increment of m/ z F+ n to F ( n is the original skeleton carbon number in each fragment) indicated the direct conversion from the added glucose to amino acids, but the total isotope incorporation from the added 13C can only be calculated according to all target isotope fragments, i.e. the abundance ratio increment summation from m/ z (Fa+1) through m/ z (Fa+ T) represented the total incorporation of the added 13C (Fa is the fragment containing all original skeleton carbons and T is the carbon number in the amino acid molecule). This method has a great advantage especially for the evaluation of high-abundance isotope enrichment in organic compounds compared with GC/C/IRMS. And in principle, this technique is also valid for amino acids besides enantiomers if stereoisomers are not concerned. Our assessment approach could shine a light on investigating the biochemical mechanism of microbial transformation of N and C in soils of terrestrial ecosystem.

Regulation of in vivo phenylalanine hydroxylation by dietary tyrosine using enrichment in Apo‐B100

We compared estimates of phenylalanine hydroxylation (Phe-OH), across a range of tyrosine intakes, determined using plasma and ApoB-100 phenylalanine and tyrosine enrichments, with a measurement of in vivo change in protein synthesis. Six, healthy, male subjects receiving 4.54 μmol.kg−1.h−1 of phenylalanine, were randomized to receive each of the 7 test tyrosine intakes (3, 4.5, 6.0, 7.5, 9.0, 10.5 and 12 mg.kg−1.d−1) for 3-d. Studies were conducted at each level using the tracers of L-[15N] phenylalanine and L-[3,3-2H2] tyrosine. Blood samples were collected at baseline and isotopic steady state. Repeated ANOVA using mixed model was performed to assess the effect of tyrosine intake on phenylalanine hydroxylation. Using ApoB-100 as the sampling site, estimates of phenylalanine hydroxylation were lower than those from plasma phenylalanine and tyrosine. Apob-100 enrichment followed a linear plateau pattern with a break point at a tyrosine intake of 6.8 mg.kg−1.d−1. We conclude that ApoB-100 is a more suitable method than plasma for estimating intracellular enrichment of amino acids in the liver. (CIHR supported). Table 1. Effect of Tyrosine intake on Phe-OH (μmol.kg−1.h−1) in plasma and ApoB-100 protein

Supplementation of soy protein with branched-chain amino acids alters protein metabolism in healthy elderly and even more in patients with chronic obstructive pulmonary disease

Background:It is often suggested that chronic wasting diseases [eg, chronic obstructive pulmonary disease (COPD)] may benefit from branched-chain amino acid (BCAA) administration via improved protein metabolism.Objective:The aim was to examine whether adding BCAAs to a soy protein meal would enhance protein anabolism in COPD patients and in healthy elderly persons.Design:Eight normal-weight COPD patients and 8 healthy control subjects were examined on 2 test days. Simultaneous continuous intravenous infusion of l-[ring-2H5]phenylalanine (Phe) and l-[ring-2H2]tyrosine tracers was done postabsorptively and at 2 h of ingestion of a maltodextrin soy or maltodextrin soy + BCAA protein meal (rate of ingestion: 0.02 g protein·kg body weight−1·20 min−1) in a crossover design. Together with the meal, oral ingestion of 1-[13C]Phe was performed to measure first-pass Phe splanchnic extraction (SPEPhe). The endogenous rate of Phe appearance [reflecting whole-body protein breakdown (WbPB)], whole-body protein synthesis (WbPS), and net WbPS (WbPS − WbPB) were calculated. Arterialized venous blood was sampled for amino acid enrichment and concentration analyses.Results:Soy feeding induced a reduction in WbPB and an increase in WbPS. BCAA supplementation of soy protein resulted in a significantly higher (P< 0.05) increase in WbPS than did soy protein alone in COPD patients but not in the healthy elderly. BCAA supplementation did not significantly alter the change in WbPB or net WbPS. Furthermore, BCAA supplementation decreased (absolute) SPEPhe(P< 0.05) but did not change the percentage Phe hydroxylation in the splanchnic area, which indicates a BCAA-related reduction in splanchnic protein synthesis.Conclusion:BCAA supplementation to soy protein enhances WbPS in patients with COPD and alters interorgan protein metabolism in favor of the peripheral (muscle) compartment in healthy elderly and even more in COPD patients.

Metabolomic analysis of plasma from hibernating and active ground squirrels

Mammals that hibernate survive seasonal food shortages by using extended periods of torpor to conserve energy. The hibernation phenotype is associated with resilience to several physiological challenges, such as hypoxia, ischemia/reperfusion injury, and obesity. Thirteen-lined ground squirrels (Spermophilus tridecemlineatus) during interbout arousals (IBA) within the hibernation season have enhanced resistance to a hypovolemic challenge relative to summer-active (SA) squirrels or rats at the same body temperature. To better understand the molecular mechanisms of this resistance and provide insight into the hibernation phenotype in general, plasma from IBA and SA squirrels was analyzed by LC-MS. Using accurate mass and chromatographic retention time information, thousands of features were characterized. Several identified features differed significantly between IBA and SA squirrels. These include certain peroxidized lipids that were enriched in IBA relative to SA, whereas SA squirrels had higher enrichment of certain amino acids, vitamins and other nutrients. This approach permits a molecular characterization of the hibernation phenotype in ground squirrels and will provide candidate molecules responsible for hibernation-induced protection. Supported by DARPA W81XWH-05-02-0016.

Enrichment of Amino Acids by Ultrasonic Atomization

Dilute aqueous solutions of two amino acids, tryptophan (Trp) and phenylaramine (Phe), were atomized ultrasonically, and it was found that the amino acids were enriched in the collected mist droplets. The enrichment ratio increased with decreasing initial concentration of amino acids. An enrichment model was proposed and was verified with experimental results. The mechanism is analogous to that of nonfoaming adsorptive bubble separation (NFBS) reported previously. The model explained well the experimental results, and the specific surface area of the mist droplets was estimated. In the case of ultrasonic atomization, the estimated specific surface areas for both Trp and Phe were larger than those of NFBS. This suggests that smaller droplets will be formed by ultrasonic atomization. The present method will be available to separate gas−liquid interfaces where surface-active substances adsorb and to enrich them from dilute aqueous solution.

Effect of branched chain amino acid enrichment of total parenteral nutrition on nitrogen sparing and clinical outcome of sepsis and trauma: A prospective randomized double blind trial

Administration of extra branched chain amino acids (BCAA) has been associated with a nitrogen sparing effect in septic and traumatized patients. Whether nitrogen sparing is associated with decreased morbidity and mortality rates is unknown. We therefore undertook a prospective, randomized, double blind trial investigating the effects of BCAA enrichment of a total parenteral nutrition (TPN) regimen on nitrogen balance, 3-methylhistidine excretion, morbidity as evidenced by disturbances in organ function, severity of sepsis and mortality. One hundred and one patients entered the study; 52 received a standard TPN solution and 49 a BCAA-enriched solution. Both groups received 30 kcal kg-1 body-weight, 15 per cent fat calories and 0.17 g nitrogen kg-1 body-weight. In the BCAA-enriched group, patients received 0.56 g BCAA kg-1 body-weight (50.2 per cent BCAA). Standard group patients received 0.18 g BCAA kg-1 body-weight (15.6 per cent BCAA). Nitrogen balances and 3-methylhistidine excretion were not significantly different between groups. Although morbidity scores tended to decrease during the study no difference was observed between groups. Mortality (early or late), sepsis or stress-related, did not differ significantly between groups. We were not able to confirm the reported beneficial effects of BCAA-enriched TPN solutions for use in septic and traumatized patients.