Phe Oxidation Research Articles

Chicken meal is not an appropriate reference protein for estimating protein quality of ingredients used in extruded diets intended for dogs.

The indicator amino acid oxidation (IAAO) method has been used to determine metabolic availability (MA) of amino acids in feedstuffs for pigs, humans, and preliminarily for cats. Peas are a commonly used protein source in grain-free extruded dog diets. However, peas have a poor sulfur amino acid (AA) ratio (methionine [Met]:cysteine) with Met being the first limiting AA. Furthermore, little is known about the MA of Met in peas fed to dogs. Therefore, our objective was to compare the MA of Met in peas to chicken meal (CM), as a gold-standard reference protein. The study was done as a replicated 5 × 5 complete Latin square design. Ten neutered male mixed-breed dogs (1.5 years old; 26.0 kg ± 2.4kg body weight; BW) fed to maintain ideal BW received all dietary treatments: BAS: lamb-based diet (deboned lamb and lamb meal) providing Met at 50% of its requirement (0.27g/100g dry matter [DM]), CHK: CM and lamb-based diet, and PEA: ground dried pea and lamb-based diet both providing Met at 68% of its requirement (0.35 and 0.37g/100g DM, respectively). Two other treatments were created by blending BAS with PEA (BAP) and the BAS with CHK (BAC) to create diets with Met at 59% of requirement (0.32 and 0.31g/100g DM, respectively). This resulted in three graded levels of Met for both CM and peas to allow for a slope-ratio assay approach to quantify MA with the BAS diet as the common first point. All other AAs were provided to meet at least 120% of the AAFCO recommendations for adult dogs. The BAS diet, with supplemental DL-Met, was fed for a 2-wk wash-in period. After 2 d of diet adaptation IAAO was performed. Dogs were fed 13 small meals where meal 6 contained a priming dose (9.4mg/kg BW) of L-[1-13C]-phenylalanine (Phe; 99%) as well as a constant dose (2.4mg/kg BW) in meals 6-13. Breath samples were collected and enrichment of 13CO2 was measured using isotope-ratio mass spectrometry to calculate the rate of Phe oxidation (F13CO2 umol/kg BW/h). Oxidation was analyzed via SAS using PROC GLIMMIX with dog and period as random effects, and diet, %Met, and their interaction as fixed effects. Unexpectedly, the slope of Phe oxidation, in response to increasing Met intake, from CM was 31% of that of peas, indicating a lower MA for Met in CM as compared to peas. This finding may be due to damage of AAs during rendering. At this time, CM in extruded diets is not an acceptable reference protein to determine MA of AAs in dogs, and the MA of Met from peas cannot be confidently assessed.

106 Minimum Methionine Requirement in Adult Cats Using the Indicator Amino Acid Oxidation Technique

Abstract Despite the importance of Met for protein synthesis and a wide range of metabolic functions, little is known about its requirement in adult cats. Current recommendations for adult cats are based solely on studies using kittens and growth or nitrogen balance as the outcomes. Thus, this study aimed to determine the minimum Met requirement in mature cats using the indicator amino acid oxidation (IAAO) technique. Four adult neutered male cats (4.90 + 0.77 kg BW) were used in a Latin rectangle design. Cats were fed a semi-synthetic diet sufficient in Met for 2 weeks prior to being randomly allocated to one of the eight dietary Met treatments (0.12%, 0.17%, 0.21%, 0.28%, 0.35%, 0.42%, 0.50% and 0.57% dry-matter). Cys was provided in excess to spare the Met requirement and meet the Cys requirement. After 2d of adaptation, individual IAAO studies were performed. During each IAAO study day, cats were placed in indirect calorimetry chambers and fed 75% of their daily ration (12.38g/kg BW) split in 13 ½-hourly small meals. A priming dose of NaH13CO3 (0.44mg/kg) and L-[1-13C]-Phe (4.8mg/kg) were provided in the 4th and 5th meal, respectively, following by a constant dose (1.04mg/kg) of L-[1-13C]-Phe in the next meals. Breath samples were collected ½-hourly to measure 13CO2 enrichment to quantify 13CO2 and calculate F13CO2 during isotopic steady state (Phe oxidation). Competing models, broken-line linear (BLL) ascending model, and a broken-line quadratic (BLQ) ascending model, were fitted on F13CO2 to determine the minimum requirement of Met (breakpoint). The BLL yielded the best model with mean minimum Met requirement estimated at 0.305% (dry-matter), and the 95% confidence interval estimated at 0.521% (dry-matter) for adult cats. In conclusion, the estimated min Met requirement in this study is greater than those currently recommended by the NRC (2006; 0.17% dry-matter) and AAFCO (0.2% dry-matter).

Net absorption and liver metabolism of amino acids and heat production of portal-drained viscera and liver in multiparous sows during transition and lactation

BackgroundDetermination of nutrient requirements in the late gestating and lactating sows is essential to optimize sow productivity. The objectives of the present study were to quantify amino acid (AA) fluxes and heat production across portal-drained viscera (PDV) and liver in multiparous sows during transition and lactation.MethodsEight second parity sows were fitted with indwelling catheters in the femoral artery and in the mesenteric, portal and hepatic veins. Eight hourly sets of blood samples were taken starting 0.5 h before feeding at − 10, − 3, + 3, and + 17 d in milk (DIM). Blood gases, plasma metabolites and apparent total tract digestibility (ATTD) of nutrients were measured.ResultsFeed intake, the ATTD of DM, energy, nitrogen, fat and crude fiber changed with DIM (P < 0.001). Except for Glu, O2, and urea, all net portal fluxes were positive, and all were affected by DIM (P < 0.05) and by sampling time (P < 0.01). Compared with pre partum levels, net portal uptake of AA was 3-63% lower at + 3 DIM but 40-100% higher at + 17 DIM. Net portal fluxes of AA peaked at 1.5 to 2.5 h after feeding except for Glu, and they were positively correlated with changes in sow feed intake across DIM. The net portal recovery was low for Met (49%), Thr (54%), and His (54%) and high for the remaining essential AA (63-69%) and none of them differed across DIM. Net hepatic uptake (i.e. hepatic oxidation) of Lys, Thr, Ile, Leu and Phe peaked at 0.5 to 2.5 h after feeding, whereas uptake of Trp, Val, and His was constant, while that of Met was close to zero.ConclusionThe net portal recovery was substantially lower for Met, Thr, and His than the remaining essential AA. Hepatic AA oxidation peaks 0.5 to 2.5 h after feeding. The heat production in PDV and liver was approximately two-fold higher at peak lactation compared to other stages. The study suggests that lysine was the limiting AA in peak lactation but not in early lactation.

PSI-B-40 Late-Breaking: Determination of lysine requirements of young and senior Labrador retrievers using the indicator amino acid oxidation technique

Abstract In order improve the health and longevity of canines, the need to determine amino acid requirements for aging populations is particularly important. In this experiment, a total of six young (&amp;lt; 1.5 yrs) and six old (&amp;gt;8 yrs) Labrador retrievers were studied in order to determine the lysine (Lys) requirement through the indicator amino acid oxidation (IAAO) technique. All dogs were supplied with constant dietary Phe in the control and test diets. The control diet was fed for two days, followed by a day in which the test diet was fed, a tracer amino acid was supplied, and breath samples were collected. On test day, a priming dose of L-[1-13C]phenylalanine (Cambridge Isotope Laboratories, Inc.) based on the subject’s body weight was first supplied, followed by [1-13C]Phe doses every thirty minutes, spanning a four hour period. A respiration mask was placed on each subject every thirty minutes (Oxymax, Columbus Instruments), 13CO2 was collected, and enrichment was determined by isotope ratio mass spectrometry (IRMS). Results for IRMS were converted to atom percent excess (APE) and analyzed using a broken-line model of best fit (JMP Pro 14.1). The [1-13C] Phe oxidation results showed that the minimal requirement of the young adult and senior dogs was 1.08 ± 0.59 and 0.83 ± 0.12 (mean ± 2SD) g/1000 kcal ME, respectively. The lower lysine requirements for the senior dogs is due to the seniors having an average of 987g less lean mass per dog than the young adults.

Probing the radical chemistry and the reaction zone during the sono-degradation of endocrine disruptor 2-phenoxyethanol in water

Sonochemical degradation at 600 kHz of 2-phenoxyethanol (PhE), an endocrine disrupting compound, was performed in the presence of several organic additives, namely: 2-propanol, Triton X-100 and sucrose, of different volatilities to obtain detailed information on the reaction zone and the oxidation pathway of this priority emerging water contaminate. It was found that sonication at 600 kHz and 120 W completely remove PhE (10 mg L−1) from aerated solutions within 100 min of irradiation. Very little removal of PhE (∼7%) and low accumulation of H2O2 took place in the presence of adequate amount of 2-propanol, indicating that reaction with OH radical outside the bubble is the major degradation pathway of PhE. Addition of the hydrophobic surfactant Triton X-100, as an OH-probe for the interfacial region, at 10 and 100 mM reduced the degradation event by 57% and 72% and resulted in more than 50% decrease in the yield of H2O2, confirming that PhE degradation occurs mainly at the bubble/solution interface with hydroxyl radical attack. Addition of the hydrophilic substrate glucose at high doses decreased slightly (∼7%) the degradation of PhE and the formation rate of H2O2, meaning that the bulk of the solution participate marginally in the degradation of the pollutant. Finally, analyzing the degradation rates at various initial PhE concentrations (2–400 mg L−1) with a heterogeneous Langmuir type mechanism underlined the predominance of interfacial radical reactions during the oxidation of PhE, particularly at high initial pollutant concentrations.

An Efficient and Rapid Method to Monitor the Oxidative Degradation of Protein Pharmaceuticals: Probing Tyrosine Oxidation with Fluorogenic Derivatization

The loss of potency of protein therapeutics can be linked to the oxidation of specific amino acid residues leading to a great variety of oxidative modifications. The comprehensive identification of these oxidative modifications requires high-resolution mass spectrometry analysis, which requires time and expensive resources. Here, we propose a fluorogenic derivatization method of oxidized Tyr and Phe yielding benzoxazole derivatives, as an orthogonal technique for the rapid screening of protein oxidation. Four model proteins, IgG1, human growth hormone (hGH), insulin and bovine serum albumin (BSA) were exposed to oxidation via peroxyl radicals and metal-catalyzed reactions and efficiently screened by fluorogenic derivatization of Tyr and Phe oxidation products. Complementary LC-MS analysis was done to identify the extent of methionine oxidation in oxidized proteins. The Fluorogenic derivatization technique can easily be adapted to a 96-well plate, in which several protein formulations can be screened in short time. Representatively for hGH, we show that the formation of benzoxazole parallels the oxidation of Met to methionine sulfoxide which enables estimation of Met oxidation by just recording the fluorescence. Our rapid fluorescence based screening allows for the fast comparison of the stability of multiple formulations.

Multiple Quality Control Pathways Limit Non-protein Amino Acid Use by Yeast Cytoplasmic Phenylalanyl-tRNA Synthetase

Non-protein amino acids, particularly isomers of the proteinogenic amino acids, present a threat to proteome integrity if they are mistakenly inserted into proteins. Quality control during aminoacyl-tRNA synthesis reduces non-protein amino acid incorporation by both substrate discrimination and proofreading. For example phenylalanyl-tRNA synthetase (PheRS) proofreads the non-protein hydroxylated phenylalanine derivative m-Tyr after its attachment to tRNA(Phe) We now show in Saccharomyces cerevisiae that PheRS misacylation of tRNA(Phe) with the more abundant Phe oxidation product o-Tyr is limited by kinetic discrimination against o-Tyr-AMP in the transfer step followed by o-Tyr-AMP release from the synthetic active site. This selective rejection of a non-protein aminoacyl-adenylate is in addition to known kinetic discrimination against certain non-cognates in the activation step as well as catalytic hydrolysis of mispaired aminoacyl-tRNA(Phe) species. We also report an unexpected resistance to cytotoxicity by a S. cerevisiae mutant with ablated post-transfer editing activity when supplemented with o-Tyr, cognate Phe, or Ala, the latter of which is not a substrate for activation by this enzyme. Our phenotypic, metabolomic, and kinetic analyses indicate at least three modes of discrimination against non-protein amino acids by S. cerevisiae PheRS and support a non-canonical role for SccytoPheRS post-transfer editing in response to amino acid stress.

Tryptophan requirement of pregnant sows1

Amino acid requirements of sows may change from early to late gestation due to the accelerated growth of products of conception after d 70 of pregnancy. The objective of this study was to determine the Trp requirement, Phe kinetics, and energy expenditure in early (d 35 to 53) and late (d 92 to 111) pregnancy using the indicator amino acid oxidation method and indirect calorimetry. The same 6 second parity sows were fed 6 diets in a Latin square design in both early and late pregnancy. The diets based on corn, corn starch, and sugar provided 20 to 120% of the current recommended Trp intake (2.5 g/d) in early pregnancy and 60 to 180% in late pregnancy. Feed allowance was constant for each sow at 2.41 kg/d (SE 0.029). Expired air and blood were collected every 30 min for 5 1/2 h. After three 30-min periods to determine background (13)C enrichment in expired CO2 and plasma Phe, L[1-(13)C]Phe was given orally at a rate of 2 mg/(kg BW · h) with 8 1/2 hourly meals. Expired air and plasma were analyzed for (13)CO2 and (13)C-Phe enrichment, respectively. Requirements were determined as the breakpoint of 2-phase linear models. Sows grew from 167.7 kg (SE 3.93) at breeding to 211.9 kg (SE 5.18) post-farrowing and had litters of 14.5 piglets (SE 0.43) weighing 19.0 kg (SE 1.41) at birth. The Trp requirement was 1.7 g/d (SE 0.29, P = 0.001) in early pregnancy and 2.6 g/d (SE 0.37, P = 0.013) in late pregnancy, or 0.7 g/kg and 1.1 g/kg diet of total Trp, respectively, for a feed allowance of 2.4 kg/d. The Trp requirement in late pregnancy tended (P = 0.056) to be greater than in early pregnancy. Quantitative Phe kinetics were not affected by Trp intake except for a quadratic response of Phe oxidation and retention (P < 0.1) to Trp intake in early pregnancy. In late pregnancy, sows oxidized less Phe and retained more Phe (P = 0.001) than in early pregnancy, indicating that young, growing pregnant sows increase the efficiency of utilizing AA in late pregnancy to maintain protein synthesis in both maternal and fetal tissues. Oxidation and body protein breakdown contributed less to Phe flux in late than early pregnancy while protein synthesis contributed more (P < 0.01). Heat production and energy retention were not affected by Trp level or stage of gestation. To meet both energy and AA requirements in late gestation, a phase feeding program with 2 diets is recommended. The feed allowance in late pregnancy should be greater than in early pregnancy to account for the increased energy expenditure.

(Fe3O4)-graphene oxide as a novel magnetic nanomaterial for non-enzymatic determination of phenylalanine

Fe3O4-graphene oxide (GO) modified glassy carbon (GC) electrode was used as a new magnetic nanosensor for determination of phenylalanine (Phe). It was found that Fe3O4-GO has been stably absorbed on GC electrode modified by simple technique. The cyclic voltammograms of the modified electrode in an aqueous solution displayed a pair of well-defined, stable and irreversible reductive/oxidation redox systems. The apparent electron transfer rate constant (ks) and transfer coefficient (α) were determined by cyclic voltammetry and were approximately 9.3s−1 and 0.67, respectively. The modified electrode showed excellent catalytic activity towards the oxidation of Phe at an unusually positive potential in buffer solution. This nanosensor also displayed fast response time, high sensitivity, low detection limit and had a remarkably positive potential oxidation of Phe that decreased the effect of interferences in analysis.

Protein turnover in pregnant pigs when feeding limiting amounts of amino acids

Experiments to determine the amino acid (AA) requirements of pigs for threonine, lysine, tryptophan and isoleucine in early and late pregnancy were evaluated to determine which factors caused changes in protein turnover during pregnancy in pigs when the test AA intake was below requirements. In all experiments, each of 6 to 7 sows received 6 diets with graded levels of test AA in both early and late gestation. Protein turnover was determined based on the 13C enrichment in expired CO2 and plasma free phenylalanine (Phe) after oral L[1–13C]Phe dosing using a stochastic model. The observations (max n=188) were evaluated using mixed models (SAS). The final models were correlated to the data with r2 = 0.82 (protein synthesis, S) to r2 = 0.92 (Phe oxidation, Ox, g/d). Increasing AA intake decreased Ox (P=0.001) and increased Phe retention (RPhe, P=0.001) but did not affect S, breakdown (B) or Phe flux. Increasing BW and gestational age decreased Phe flux and Ox, S and B (P&lt;0.02) and increased RPhe (P=0.001). In parity 3, values for Phe kinetics were greatest (P &lt; 0.05), except for RPhe, which decreased linearly (P = 0.001) with sow age. Increasing litter size increased B (P=0.001) but did not affect other Phe kinetic parameters. Phe kinetics in pregnant pigs given limiting AA intakes were more affected by physical attributes, i.e. sow age (parity), BW, weight gain and gestational age than dietary attributes.

Fluorogenic Tagging Methodology Applied to Characterize Oxidized Tyrosine and Phenylalanine in an Immunoglobulin Monoclonal Antibody

Metal-catalyzed oxidation (MCO) of proteins is of primary concern in the development of biotherapeutics as it represents a prominent degradation pathway with potential undesired biological and biotherapeutic consequences. We developed a fluorogenic derivatization methodology to study the MCO of IgG1 using a model oxidation system, CuCl2/L-ascorbic acid. Besides the oxidation of Met, Trp and His residues, we detected significant oxidation of Phe and Tyr in IgG1. The fluorogenic derivatization method provides an alternative approach for the rapid detection of oxidized Tyr and Phe as their benzoxazole derivatives by fluorescence spectrometry and size exclusion chromatography coupled to fluorescence detection.

Dietary lysine requirement of sows increases in late gestation1

Current AA recommendations for sows are to provide a fixed amount of AA intake throughout gestation; however, the demand for nutrients changes from maternal lean tissue in early gestation (EG) to fetal and mammary growth in late gestation (LG). The objective of this study was to determine the Lys requirement in EG (d 24 to 45) and LG (d 86 to 110) using the indicator AA oxidation method with simultaneous determination of heat production. Each of 7 Large White × Landrace sows received 6 diets in random order in both EG and LG. Three semisynthetic diets (14.0 MJ ME/kg) based on corn were formulated and mixed to produce a basal diet (60% of 1998 NRC Lys requirement) and high diets for EG and LG (150% and 185% of 1998 NRC Lys requirements, respectively). The 6 test diets provided Lys intakes of 7.5 to 19.3 g/d in EG and 8.1 to 23.7 g/d in LG. Sows were placed in respiration chambers, and expired air and blood were collected every 30 min for 5.5 h. The tracer AA, l-[1-(13)C]Phe, was given orally at a rate of 2 mg/(kg BW ⋅ h) over the last 4 h, divided into 8, 0.5-h meals. Expired air was measured for (13)CO(2) enrichment, and plasma was measured for l-[1-(13)C]Phe enrichment and free Lys concentration. Background (13)CO(2) was subtracted from plateau (13)CO(2) enrichment. Requirements were determined using a 2-phase nonlinear model. Mean maternal BW gain in gestation (43.7 kg; pooled SE, 1.2 kg), litter size (14.6 total born piglets; pooled SE, 0.8), and litter weight (19.4 kg; pooled SE, 0.9 kg) did not differ between parities. Sow weight gain and BW was greater (P = 0.001) in LG than EG. Lysine requirement was 9.4 and 17.4 g/d in EG and LG, respectively. Phenylalanine retention in LG was maximized at a Lys intake of 17.7 g/d. Heat production was more (P = 0.069) and energy retention less (P = 0.019) in LG than EG. Energy retention in LG was not different from 0. Quantitative Phe kinetics in EG were not affected by Lys intake. In LG, Phe retention increased with Lys intake (P = 0.004), whereas Phe oxidation decreased (P = 0.005). The Lys requirement was determined to be less than current recommendations in EG and more than current recommendations in LG. To meet the change in requirements, diets with increased lysine content are needed in LG. Increasing the feed allowance in LG is necessary to maintain a positive energy balance throughout gestation.

TRIENNIAL LACTATION SYMPOSIUM: Mammary metabolism of amino acids in dairy cows1,2

Although in dairy cows the mammary gland (MG) is the major net user of essential AA (EAA) supply, milk protein synthesis from absorbed EAA is not a straightforward process. Early studies identified 2 groups of EAA based on different pattern of mammary utilization: group 1 [Met, Phe (+Tyr), Trp], where MG uptake was similar to secretion in milk protein, and group 2 (Arg, Ile, Leu, Lys, Thr, and Val), where uptake exceeded milk protein output. This review examines the validity of this classification under variable protein supply through a meta-analysis, with the outcomes then explained with studies in which the fates of individual EAA were monitored using isotope approaches. For the meta-analysis, the Fick principle, based on stoichiometric transfer of Phe+Tyr uptake to milk protein, was used to estimate mammary plasma flow across all studies. This approach was judged acceptable because doubling Phe supply did not result in mammary oxidation of Phe+Tyr and either limited or no contribution of peptides to Phe and Tyr mammary supply could be detected. The AA content of proteins synthesized by the MG was estimated from milk protein composition, and the uptake-to-output ratio (U:O) for individual AA was re-calculated based on these assumptions. Analysis of individual samples by isotopic dilution resulted in reduced variance compared with analysis on pooled samples performed with an AA analyzer. Globally, the U:O of His and Met is maintained close to unity under variable protein supply. The group 2 AA could be subdivided. First, the U:O for group "2v" AA (Ile, Leu, Val, and Lys) is greater than 1 and varied with protein supply. Accordingly, the increased U:O of Leu, induced by duodenal casein infusion, led to extra-mammary Leu oxidation. Decreasing Lys supply decreased Lys U:O and the associated transfer of N to non-EAA, mainly to Glx, Asx, Ser, and Ala. Second, the U:O of group "2nv" AA, Arg and Thr, does not vary with protein supply. The Arg U:O averages 2.5, whereas the Thr U:O, albeit averaging 1.2, does not differ from unity. Excess of both these AA is probably directed toward the synthesis of non-EAA rather than energy supply. Overall, the ability of the MG to use excess EAA-N supply offers alternative sources of N and C for energy provision, lactose synthesis and non-EAA synthesis. The latter function spares dietary non-EAA for other necessary processes, such as gluconeogenesis and energy supply, in other tissues to support lactation.

Novel pharmacological chaperones that correct phenylketonuria in mice

Phenylketonuria (PKU) is caused by inherited phenylalanine-hydroxylase (PAH) deficiency and, in many genotypes, it is associated with protein misfolding. The natural cofactor of PAH, tetrahydrobiopterin (BH(4)), can act as a pharmacological chaperone (PC) that rescues enzyme function. However, BH(4) shows limited efficacy in some PKU genotypes and its chemical synthesis is very costly. Taking an integrated drug discovery approach which has not been applied to this target before, we identified alternative PCs for the treatment of PKU. Shape-focused virtual screening of the National Cancer Institute's chemical library identified 84 candidate molecules with potential to bind to the active site of PAH. An in vitro evaluation of these yielded six compounds that restored the enzymatic activity of the unstable PAHV106A variant and increased its stability in cell-based assays against proteolytic degradation. During a 3-day treatment study, two compounds (benzylhydantoin and 6-amino-5-(benzylamino)-uracil) substantially improved the in vivo Phe oxidation and blood Phe concentrations of PKU mice (Pah(enu1)). Notably, benzylhydantoin was twice as effective as tetrahydrobiopterin. In conclusion, we identified two PCs with high in vivo efficacy that may be further developed into a more effective drug treatment of PKU.

Complete restoration of phenylalanine oxidation in phenylketonuria mouse by a self‐complementary adeno‐associated virus vector

Classical phenylketonuria (PKU) arises from a deficiency of phenylalanine hydroxylase (PAH) that catalyses phenylalanine oxidation in the liver. Lack of PAH activity causes massive hyperphenylalaninemia and consequently severe brain damage. Preclinical studies showed that conventional adeno-associated virus (AAV) vectors could correct hyperphenylalaninemia in a mouse model of PKU, although limitations such as very large dose requirement and relative inefficiency in female animals were recognized. An AAV8-pseudotyped vector was constructed with a self-complementary AAV (scAAV) genome for efficient liver transduction and expression. Following vector injection to PKU mice, blood Phe was periodically measured by an enzymatic fluorometric assay. In vivo Phe oxidation was evaluated by a non-invasive breath test using [1-(13) C]Phe. Vector copy number in the host tissues was determined by quantitative polymerase chain reaction. A single injection of 1 × 10(11) -1 × 10(12) particles of the scAAV8 vector resulted in a reduction of blood Phe to normal or near-normal levels for more than 1 year in both genders. The treated animals showed normal level of in vivo Phe oxidation. The presence of > 1 copy of vector DNA per diploid genome in the liver was associated with normal blood Phe in the AAV-treated PKU mice. Complete phenotypic correction of PKU mice was achieved by the scAAV8 vector for the longest duration reported to date. The vector overcame the female-specific disadvantage in AAV-mediated liver transduction; thus, it offers a promising platform of long-lasting gene therapy for PKU.

Using whole‐body phenylalanine kinetics to measure the effects of amino acid supplementation in mature horses

Stable isotopes have not been used in horses to measure whole‐body protein kinetics or to assess the adequacy of a diet's amino acid profile. The objective of this study was to determine whether supplementing a control diet (CON) with equal (0.38 mmol/kg/d) amounts of either glutamate (+GLU), leucine (+LEU), lysine (+LYS) or phenylalanine (+PHE) would affect whole‐body PHE kinetics in mature horses. 12 mature male horses were each studied 3 times: once while receiving the CON diet (n=12) and while receiving 2 of the 4 supplemented diets (n=6/diet). Following 7d of adaptation, horses received two 6h primed, constant infusions each period: [1‐13C]PHE (d8) and [13C]bicarbonate (d9). Breath samples were collected to measure the rate of 13CO2 appearance, to calculate PHE oxidation (d8) and total CO2 production (d9). Blood was sampled (d8) to calculate PHE flux and kinetics using a stochastic model. Horses receiving the +LEU, +LYS and +PHE diets had higher plasma concentrations of their supplemented amino acid than when they received the CON diet (P &lt; 0.05). Relative to CON, horses receiving +PHE had a greater PHE flux (+20%) and rate of PHE oxidation (+111%) (P &lt; 0.05). There were no differences in PHE use for protein synthesis relative to CON for any of the supplemented diets (P &gt; 0.05). The isotope methodology was sensitive enough to detect differences in PHE oxidation between diets. Amino acid supplementation of the CON diet did not appear to increase whole‐body protein synthesis in mature horses. Funded by WALTHAM Centre for Pet Nutrition.

An estimate of the methionine requirement and its variability in growing pigs using the indicator amino acid oxidation technique1

Although AA requirements for the mean of a population of growing pigs have been established using traditional methods, there are no estimates of the variability within the population and whether this variation differs among AA. With the increased use of supplemental Lys in pig diets, there will be an increased need to supplement Met, commonly the second or third limiting AA in corn-soybean diets. The indicator AA oxidation method allows repeated measurements in a short period of time so that the AA requirement can be determined for individual pigs at a similar physiological stage. The objective of this study was to determine the mean Met requirement in individual gilts and to estimate the related variability. Six individually housed female pigs (initial BW = 8.8 kg, SD 1.5) each received diets providing 6 levels of dl-Met. The isonitrogenous and isoenergetic diets contained 0.187, 0.250, 0.290, 0.320, 0.350, and 0.377% Met (analyzed, as-fed basis). Cysteine (0.48%) and Lys (1.44%) concentrations were similar for all diets. Pigs were adapted for 6 d to the basal corn-soybean meal diet (0.187% Met), which was offered at 95 g/kg(0.75) of BW to ensure complete consumption of the test diets. During 4-h oxidation studies, 313.4 kBq, (SD 35.6) of L-[1-(14)C]Phe was mixed with each of 8 half-hourly meals, and expired CO(2) was collected. The breakpoint in Phe oxidation, representing the Met requirement, and its variability, was determined using 2-phase linear regression. Phenylalanine oxidation decreased as the Met content increased from 0.187 to 0.29%. Phenylalanine oxidation was not different (P > 0.2) for diets ranging from 0.320 to 0.377% Met. The dietary Met requirement varied from 0.320 to 0.373% for individual pigs. The mean Met requirement for individual pigs was determined to be 0.340% of diet (SD = 0.024%, CV= 7.1%), with 0.340, 0.364, and 0.388% covering the requirement of 50, 66, and 95% of the population, respectively. The present mean population estimate was similar to the recommended dietary Met concentration of 0.325% for pigs of this BW and feed intake. To maximize profitability, Met levels in starter pig diets should be determined, depending on the cost of crystalline Met and the fraction of the population whose requirement is to be met.

Estimate of the variability of the lysine requirement of growing pigs using the indicator amino acid oxidation technique1

Although AA requirements for the mean in a population of growing pigs are well established, there are no direct estimates of their variability within the population. The indicator AA oxidation method allows repeated measurements in a short period of time so that the AA requirement can be determined for individual pigs. The objective was to determine the Lys requirement in individual pigs to derive a first estimate of the population mean requirement and its variability. Nine individually housed barrows (15 to 18 kg) were surgically implanted with venous catheters for isotope infusion. Pigs were offered, in random order, isonitrogenous and isoenergetic diets with one of seven Lys concentrations (4.8 to 15.5 g of Lys/kg diet, as-fed basis). The pigs were fed twice daily, except for study days when they received one-half of the daily allowance in eight equal hourly meals. After a validated minimum adaptation period, indicator (Phe) oxidation was determined for each dietary Lys level during a 4-h primed, constant infusion of L-[1-(14C)]Phe at a rate of 464 kBq/h. The Lys requirement was calculated using a two-phase linear regression crossover analysis within individual pigs. For each pig, Phe oxidation decreased linearly (P < 0.02) as the dietary Lys concentration increased until the requirement was reached; thereafter, Phe oxidation was not different. The true ileal digestible Lys requirement ranged from 7.5 to 10.6 g/kg of diet (as-fed basis) for the nine animals. The mean requirement for all pigs was 9.1 g/d (CV, 11.6%) or 93.9% (CV, 9.8%) of the predicted (NRC, 1998) requirement based on each pig's mean BW and energy intake. The measured and predicted requirements did not differ. The indicator AA oxidation method gave values for Lys requirement similar to conventional methods. The short (< 3 wk) experimental period allows, for the first time, the estimate of population variability, which provides for more accurate calculation of the effect of altering Lys intake on herd performance and production economics. This method is suitable to use with all dietary indispensable AA.

Phenylalanine 4-Monooxygenase and the S-Oxidation of S-Carboxymethyl-L-cysteine

The identity of the enzyme responsible for the S-oxidation of the mucolytic S-substituted L-cysteine drug, S-carboxymethyl-L-cysteine (SCMC), has been actively investigated for the last 10 years. A genetic polymorphism exists in the oxidation of the thioether moiety that has been identified as a disease susceptibility factor in a number of degenerative diseases. This polymorphism has also been implicated in the wide variation in clinical response to SCMC therapy in man. To date little is known about the molecular enzymology of this reaction but a previous investigation revealed that rat activated phenylalanine 4-monooxygenase (PAH) could S-oxidise both Met- and S-methyl-L-cysteine (SMC) to their S-oxide metabolites. We have investigated the hypothesis that SCMC was also a substrate for activated PAH in the cytosolic faction of the Wistar rat. 1. Substrate and inhibitor investigation revealed that SCMC was a substrate for activated PAH activity in vitro. 2. The large aromatic amino acid hydroxylase monoclonal antibody and the Fe3+ chelator, deferoxamine, completely inhibited both Phe and SCMC oxidation to their respective metabolites. 3. Analysis of the Dixon plots revealed that both Phe and SCMC competitively inhibited each other's oxidation. 4. Correlation studies showed that the rate of production of Tyr was positively correlated to the production of both SCMC and SMC S-oxides in 20 female Wistar rat hepatic cytosolic fractions. These results strongly support the hypothesis that PAH is the enzyme responsible for SCMC S-oxidation in the rat.

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LETTERS TO THE EDITORREPLYJosef VogtJosef VogtUniversitätsklinik Ulm Klinik für Anästhesiologie 89070 Ulm, Germany 10.1152/ajpendo.00173.2003Published Online:01 Aug 2003https://doi.org/10.1152/ajpendo.00173.2003MoreSectionsPDF (40 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat The main aim of our study (2) was to explore the impact of initial Phe tracer distribution processes, Phe turnover, and whole body CO2 production rates on [13C]Phe breath test values. If these factors were considered, the breath test would give an oxidation rate for cirrhotic patients that, compared with healthy controls, was even more reduced than estimates based on a “simplistic” interpretation of 13CO2 production data. There were some open questions, such as varying retention of 13CO2 between cirrhotic patients and healthy volunteers, which could have influenced our final conclusion. Therefore we also looked at the Pheto-Tyr (Phe/Tyr) conversion rate (Qtp), as it does not depend on metabolic 13CO2 binding or release. These results were interpreted as another “indicator for Phe oxidation,” and were used to confirm our finding of a potential underestimation for the intergroup difference. We further stated that “any interpretation beyond that is difficult, because the determination of the Phe-to-Tyr conversion is subject to a large measurement error.” Hence, we never used Qpt as a “hard number,” nor did we state anywhere that this could be done, because it was calculated from assumed Phe/Tyr protein ratios. For readers who might overlook these confines, the letter of Dr. Short may be helpful.Dr. Short mentions that there is no valid basis for choosing the appropriate Tyr/Phe protein ratio. We used for both groups a Tyr/Phe protein ratio of 0.68. Considering a higher Tyr/Phe protein ratio for cirrhotic patients, i.e., 0.9, would decrease the intergroup difference in the estimated Qpt values. Generally, an underestimation of the Tyr/Phe protein ratio for cirrhotic patients would lead to an underestimation of Qpt, which would be in favor of larger intergroup differences. However, even for the extreme case of a Phe/Tyr ratio of 0.9 in cirrhotic patients, their estimated conversion rates would increase from 0.7 ± 0.3 to 1.0 ± 0.4 μmol · kg-1 · h-1 and would still be much lower than the control values of 3.0 ± 0.4 μmol · kg-1 · h-1.Indeed, we missed the study of Moller et al. (1), indicating a substantial Phe/Tyr conversion rate in the kidney. How would a conversion in the kidney influence the interpretation of our data? With the assumption of an equal contribution of liver and kidney, the conversion rate results in ∼1.5 μmol · kg-1 · h-1 for both organs in healthy volunteers. Patient data regarding the distribution of the Phe/Tyr conversion between liver and kidney are, to our knowledge, still missing. In our case, the whole body Phe/Tyr conversion in cirrhotic patients is lower than the estimated kidney conversion for controls. Because kidney function in our cirrhotic group was not impaired, as based on creatinine concentrations, the renal Phe/Tyr conversion should not be affected. This, however, would imply that Qpt in the liver would be reduced even more. Generally, as soon as we admit some kidney conversion, the estimate for the hepatic conversion decreases. Hence, even if the underlying metabolic processes are more complex than those we considered, our main interpretation of the data remains valid: the observed reduction of the Phe/Tyr conversion confirms the 13CO2-derived reduction of Phe oxidation. It is tempting to speculate that, in cirrhotic patients, part of the Phe/Tyr conversion is taken over by the kidney and that hepatic conversion would be not sufficient to produce some excess Tyr that escapes into the circulation. Dr. Short recommends using more elaborate tracer protocols to obtain a more reliable estimate for the whole body Qpt conversion rate. However, the gain in reliability does not help if we cannot infer hepatic processes from whole body data. One would have to specify organ conversion rates from tracer-to-tracee balances, which require an invasive catheterization of hepatic and kidney veins combined with blood flow measurements. References 1 Moller N, Meek S, Bigelow M, Andrews J, and Nair KS. The kidney is an important site for the in vivo phenylalanine-totyrosine conversion in adult humans: a metabolic role of the kidney. Proc Natl Acad Sci USA 97: 1242–1246, 2000.Crossref | PubMed | ISI | Google Scholar2 Tugtekin I, Wachter U, Barth E, Weidenbach H, Wagner DA, Adler G, Georgieff M, Radermacher P, and Vogt JA. Phenylalanine kinetics in healthy volunteers and liver cirrhotics: implications for the phenylalanine breath test. Am J Physiol Endocrinol Metab 283: E1223–E1231, 2002.Link | ISI | Google Scholar Download PDF Previous Back to Top FiguresReferencesRelatedInformation More from this issue > Volume 285Issue 2August 2003Pages E448-E448 Copyright & PermissionsCopyright © 2003 by American Physiological Societyhttps://doi.org/10.1152/ajpendo.00173.2003History Published online 1 August 2003 Published in print 1 August 2003 Metrics