Comparison of the precursor, amino acid oxidation, and end-product methods for the evaluation of protein turnover in senior dogs

Letícia G. Pacheco; Camila Goloni; Ludmilla G. Di Santo; Lucas B. Scarpim; Débora A. Eugênio; Ariel de Castro; Vladimir E. Costa; Aulus C. Carciofi

doi:10.1371/journal.pone.0305073

Abstract

Stable isotope methods have been used to study protein metabolism in humans; however, there application in dogs has not been frequently explored. The present study compared the methods of precursor (13C-Leucine), end-products (15N-Glycine), and amino acid oxidation (13C-Phenylalanine) to determine the whole-body protein turnover rate in senior dogs. Six dogs (12.7 ± 2.6 years age, 13.6 ± 0.6 kg bodyweight) received a dry food diet for maintenance and were subjected to all the above-mentioned methods in succession. To establish 13C and 15N kinetics, according to different methodologies blood plasma, urine, and expired air were collected using a specifically designed mask. The volume of CO2 was determined using respirometry. The study included four methods viz. 13C-Leucine, 13C-Phenylalanine evaluated with expired air, 13C-Phenylalanine evaluated with urine, and 15N-Glycine, with six dogs (repetitions) per method. Data was subjected to variance analysis and means were compared using the Tukey test (P<0.05). In addition, the agreement between the methods was evaluated using Pearson correlation and Bland-Altman statistics. Protein synthesis (3.39 ± 0.33 g.kg-^0,75. d^-1), breakdown (3.26 ± 0.18 g.kg^-0.75.d^-1), and flux estimations were similar among the four methods of study (P>0.05). However, only 13C-Leucine and 13C-Phenylalanine (expired air) presented an elevated Pearson correlation and concordance. This suggested that caution should be applied while comparing the results with the other methodologies.

Citation: Pacheco LG, Goloni C, Di Santo LG, Scarpim LB, Eugênio DA, de Castro A, et al. (2024) Comparison of the precursor, amino acid oxidation, and end-product methods for the evaluation of protein turnover in senior dogs. PLoS ONE 19(6): e0305073. https://doi.org/10.1371/journal.pone.0305073

Editor: Taimoor Hassan Farooq, Central South University of Forestry and Technology, CHINA

Received: September 26, 2023; Accepted: April 7, 2024; Published: June 20, 2024

Copyright: © 2024 Pacheco et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data, including all raw calculation results in dogs are within the manuscript or in the Supporting Information files.

Funding: The author(s) received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

Abbreviations: B, breakdow; F¹³CO₂, fraction excretion of ¹³CO₂; I, intake; O, oxidation; Q, flux; S, synthesis; VCO₂, volume produced of CO₂; δ^E, relative difference between the isotopes in δ^E (‰); χ^(E), excess atom fraction

Introduction

Stable isotopes are powerful tools that allow the study of protein metabolism without exposing animals to restriction and suffering, and without health or environmental contamination risks [1–3]. They have been routinely used in human nutrition [3–5]; however, although not new, the use of stable isotopes remains poorly explored in studies about dog nutrition [6].

Many nutritional, physiological, and pathological processes can interfere with protein turnover in adult animals, including diet composition [7], resistance exercise or hypertrophy [8,9], disease, and aging [10,11]. This process can induce changes in muscle mass, in turn, affecting health, physical activity, and disease resistance [10].

[12] published one of the first studies about protein metabolism in senior dogs and found an increase of approximately 50% in protein requirements of old beagles compared to adult beagles, although the nitrogen balance remained similar between compared age groups. These results were obtained using invasive markers of protein turnover such as muscle biopsy and changes in lean body mass. If metabolic tracers could be incorporated in such studies, valuable information about kinetics may be obtained. Therefore, turnover and metabolism of a particular nutrient of interest could be performed in a minimally invasive, precise, and “friendly” way, in turn, becoming a tool to better elucidate physiological issues such as the effect of ageing on protein metabolism [13].

Methods for precursor, end-product, and amino acid oxidation are well established in animal models to determine the rates of turnover, synthesis, and overall protein breakdown [14–16]. These methods can also be used to study body demand of amino acids [17–19]. The precursor method uses stable isotope ¹³C-Leucine. In addition to other essential amino acids that are metabolized in the liver, leucine is primarily catabolized in the muscle, well-known body protein reserve and participates in muscle protein synthesis, breakdown, and oxidative metabolism [20]. The final product method involves the use of ¹⁵N-Glycine, the first isotope tracer used to determine body protein turnover in vivo [21]. Some potential limitations to applying this method to dogs include the need for total urine collection. In addition, ¹⁵N in the tracer is incorporated differently based on various amino acids, and different turnover rates are observed when using other ¹⁵N-labelled amino acids [3,22,23].

Finally, an indirect method of amino acid oxidation, with ¹³C-Phenylalanine as a tracer, was developed with the aim of obtaining a minimally invasive model to study amino acid kinetics and its requirements, including studies in high-risk groups such as pregnant women, nursing mothers, the elderly, and newborns [2]. This approach involves the association of the precursor and final product methods, wherein urine is collected to quantify oxidation instead of plasma [2]. The isotope delivery can be either intravenous or oral, as the results were consistent in both delivery methods even though the route of administration is known to affect the tracer kinetics [24]. This method was originally developed to study amino acid requirements, but it may be used to obtain general values of protein flow, and determination of protein turnover. The rates of ¹³C-Phenylalanine incorporation into body reserves or oxidation in the postprandial period can be assessed using urine or expired air [25]; however, both approaches should be evaluated for advantages or disadvantages.

Considering that few studies are currently available that demonstrate usefulness in dogs of the methods of final products, indirect amino acid oxidation, and a radioactive isotope alternative for the precursor method [6,26,27], and that a direct comparison of results between them is not available, the present study compared the methods of precursor, end-product, and amino acid oxidation to determine the protein turnover rate in senior dogs that were fed a kibble diet for maintenance. In addition to comparing the results, the methods were standardized for their practical aspects related to feasibility and their practical application in studies with laboratory or privately owned dogs.

Material and methods

The study was conducted at the Research Laboratory of Nutrition and Nutritional Diseases of Dogs and Cats “Prof. Dr. Flávio Prada”, Universidade Estadual Paulista (UNESP), Jaboticabal, Brazil. All procedures were approved by the Ethics Committee on the Use of Animals (CEUA) at the institution (protocol number 009537/18).

Animals, housing and experimental design

The health of all dogs was previously verified by a veterinarian through physical examination, haemogram, and serum biochemical analyses, and all dogs were considered healthy. Six beagles (12.7 ± 2.6 years age and 13.6 ± 0.6 kg bodyweight) were fed with an extruded diet in controlled amounts to maintain a constant body weight (Natural Senior, Guabi Nutrição e Saúde Animal LTDA, Campinas, Brazil. Analysed chemical composition: 26% crude protein, 13% crude fat; 4.5% crude fiber; 1.92% leucine; 0.99% phenylalanine; 0.74% tyrosine; 1.83% glycine (DM basis); 16.1 kJ.g^-1 of metabolisable energy). Dogs received the food for 35 days and first days i.e., days 1 to 10, were considered for adaptation to the diet. Food amount was adjusted to animals maintain a constant body weight, to this the energy requirement was stablished based on the intake records of each individual, and the amount provided established considering the food metabolizable energy content. Dogs were then weekly weighted at fasting in the morning, and the amount supplied adjusted if necessary. The indirect amino acid oxidation method, the end-products method, and the precursor method were conducted on day 11, day 21, and on day 31, respectively. On Day 35, the volume of CO₂ produced by each dog was measured using a respirometer. The enrichment and sampling protocols used for the three methods are illustrated in Fig 1.

Download:

Fig 1. Enrichment and sampling protocol adopted in the indirect amino acid oxidation (¹³C-Phenylalanine), end-products (¹⁵N-Glycine) and precursor (¹³C-Leucine) methods.

https://doi.org/10.1371/journal.pone.0305073.g001

¹³C-Phenylalanine

For this test, dogs were housed individually in metabolic cages equipped with an apparatus to separate feces and urine for collection, measuring 0.9 m × 0.9 m × 0.9 m. All dogs were previously trained to stay in the cages. They underwent behavioral enrichment while in the cages, and one person stayed in the cage room most of the day. After 24 h fasting, the animals were fed their daily meal, but this total daily amount was divided into five equal portions, and each portion was offered at intervals of 30 minutes (Fig 1). So, the daily food amount was divided into five similar meals fed at each 30 min. Only dogs that consumed all the offered food in all the five meals were tested. Dogs that did not complete a meal were repeated in the following day.

Immediately after completing the fifth meal, dogs received a priming oral dose of 0.7 mg.kg^-1of L-[1-¹³C]-Phenylalanine and 0.2 mg.kg^-1 of NaH¹³CO₃ (Cambridge Isotope Laboratories, Tewksbury, US). For administration, both isotopes were previously conditioned in gelatin capsules. After this prime dose, the dogs were orally supplied for four times, at every 30 minutes, gelatin capsules with 1.2 mg.kg^-1 of L-[1-¹³C]-Phenylalanine.

Expired air samples were obtained both before the enrichment (basal state, collected before any isotope administration) and at specific intervals of time computed after the priming dose, namely: after 60, 90, 120, 150, 180, 210, and 240 minutes. During each sampling time, expired air was collected for a minimum of 2 minutes. The collection was conducted using a specially designed mask for the study, directly into a glass vial attached to the mask. The collected samples were then stored at room temperature for subsequent analysis.

The masks were developed by the researchers and designed to cover the entire nose and mouth of the dogs, to prevent expired air from escaping. A rubber membrane at the end attached the mask to animals’ skin, to the air cannot escape or entry from the bottom of the mask (Fig 2). All dogs were previously trained to breathe inside the mask without apparent discomfort. For this purpose, dogs were encouraged to voluntarily place their nose and mouth on the mask using wet food and positive reinforcement. When they lost fear of wearing a mask, they were further adapted for the necessary time breathing for data collection without signs of discomfort.

Download:

Fig 2. Illustration of the mask designed to collect breathed air samples in the study.

A. Illustration of the rubber membrane, the mask, and the glass vial. B. Illustration of the mask positioned in the dogs’ nose, completely covering the mouth of the animal. C. Front view of the mask positioned during the air sampling procedure.

https://doi.org/10.1371/journal.pone.0305073.g002

Expired air collection through masks has been previously validated. To this, after trained in the use of the mask, dogs was enrichment with 1.2 mg.kg^-1 of L-[1-¹³C]-Phenylalanine. Expired air samples of the dogs was collected before and after body enrichment. The masks and collection procedures were considered sufficiently adequate when clear enrichment, plateau, and reduction in ¹³CO₂/¹²CO₂ fraction enrichment were observed in the expired air samples. This procedure was repeated in several dogs until the protocol e equipment was considered adequate.

Urine samples were collected prior to enrichment, and after 8h, 12 h, 24 h, and 36 h of the fifth meal and priming dose, and frozen at -20°C until analysis. Urine samples were quantitatively collected in plastic containers placed under the collecting funnel of the metabolic cages, with 4 mL of 6N HCl as a preservative (Synth, LABSYNTH, Diadema, Brazil). Before analysis, urine samples were thawed and lyophilized (Thermo VLP200, Thermo Fisher, Massachusetts, USA).

¹⁵N-Glycine

The dogs were individually housed in metabolic cages as previously described. After 24 h fasting, the animals were fed their meal and allowed to eat for 20 min. Immediately after completing the meal, the animals were administered with a single dose of 20 mg.kg^-1 [¹⁵N] glycine (Cambridge Isotope Laboratories, Tewksbury, US). The labelled compound was orally administered in gelatin capsules. Only dogs that ate all the offered food were tested; animals that did not complete the meal were evaluated the following day.

Urine samples were collected prior to enrichment (basal state, collected before any isotope administration), and after 8 h, 12 h, 24 h, and 36 h of the meal and frozen at -20°C until analysis (Fig 1). The samples were quantitatively collected in plastic containers placed under the collecting funnel of the metabolic cages, with 4 mL of 6N HCl as a preservative (Synth, LABSYNTH, Diadema, Brazil).

For analysis, urine samples were thawed at room temperature and the incorporation of ¹⁵N were assessed in both, urinary ammonia, and urea. Briefly, ammonia and urea in the urine samples were separated using cation exchange resin (AG50W-X8, Bio-Rad Laboratories, California, USA), as described previously [28,29]. In the method, ammonia was immobilized into the resin in a neutral solution. The urea remains in the supernatant and was then converted into ammonia using urease (Sigma Aldrich, Missouri, USA), that was subsequently also incorporated into the resin. Ammonia was extracted from the resin using a procedure previously described [28,29], lyophilized, weighed into tin capsules, and subjected to N isotopic analysis.

¹³C-Leucine

On the test day, the cephalic vein was catheterized (Descarpack, São Paulo, Brasil) and a priming dose of a combination of 2 mg.kg^-1 of L-[1-¹³C] Leucine and 0.1 mg.kg^-1 of [13C] NaH¹³CO₃ (Cambridge Isotope Laboratories, Tewksbury, US) was infused in dogs after 24 h fasting. After five minutes of this priming dose, a bolus infusion of 0.7 mg.kg^-1 of L-[1-¹³C] Leucine was given four times through the catheter at every 20 min for, for a total period of 80 min.

Samples of expired air and blood plasma were collected prior to enrichment (basal state, collected before any isotope administration) and at 60, 80, 100, 120, 140, 160, and 180 min after prime dose enrichment. At each sampling, expired air was collected, for at least 2 min, directly into a glass vial coupled to the same mask as previously described and stored at room temperature for later analysis. Blood samples were collected using the catheter and a syringe, deposited in heparinized tubes, centrifuged for plasma separation and the plasma was kept frozen at -20°C. Immediately before analysis, plasma samples were lyophilized (Thermo VLP200, ThermoFisher, Massachusetts, USA).

Determination of CO₂ volume in an indirect calorimetry system

A respirometry system (Animal Respirometry System; Sable System International, Las Vegas, USA) was used to determine the volume of CO₂ (VCO₂) for each dog. The dogs were housed in hermetically sealed chambers, with dimensions of 1 m x 1 m x 1 m, with internal temperature and moisture control (ALB 1000 CG Respirometry Chamber; Inbras Equipamentos para Saúde, Ribeirão Preto, Brazil). The chamber air temperature and relative humidity were adjusted to 26±1°C and 55% to 60%, respectively. The dogs were previously trained to stay and feed inside the chambers for several days. They were subjected to behavioral enrichment and toys while inside the chambers, and one person stayed in the room the entire time while the dogs were in the respirometry system.

On the day of the experimentation, the dogs were fasted for 24 h, placed in the chambers, and fed their standardized meal. Water was provided ad libitum. Each experiment lasted 8 h. Based on previous data from pilot studies, 2 h were allowed to equilibrate the gases inside the chambers. Ambient air flows were introduced into the chambers using a negative pressure system with an average flow rate of 10 L min^-1, with control and adjustments provided by a mass-flow pump with a maximum flow capacity of 100 L min^-1 (Sable Systems International, Las Vegas, Nevada, USA). The air flow volumes were established to maintain CO₂ saturations below 1.0% (average CO₂: 0.3±0.1% and average O₂: 20.4±0.2%) inside the chambers. Channel air sampling was performed using independent pressure gauges (RM-8 gas-flow multiplexer; Sable Systems International, Las Vegas, Nevada, USA). The air samples were routed by a subsampler (SS-4 Gas Analyzer Subsampler, Sable Systems International, Las Vegas, Nevada, USA) to a humidity meter (RH-300 Relative Humidity & Dew Point Analyzer, Sable Systems International, Las Vegas, Nevada, USA). Oxygen concentrations were measured using a paramagnetic analyzer (PA-10, Sable System International, Las Vegas, Nevada, USA), and CO₂ concentrations were measured using a carbon dioxide analyzer (CA-10, Sable System International, Las Vegas, USA). Before measuring the gas concentrations, moisture was removed from the air using anhydrous magnesium perchlorate (Exodus Científica, Sumaré, São Paulo, Brazil). The control and gauging of the gas meters were performed using a standard air mixture (CO₂: 1.0031% and O₂: 21.00%; White Martins, Vinhedo, São Paulo, Brazil). The data obtained using a mass flow meter and gas analyzer were collected using an ExpeData system (Sable System International, Las Vegas, Nevada, USA).

The VCO₂ produced for each dog was determined using the following equation [30]: (1) where FR_e is the excurrent flow rate, F_e is the fractional concentration of gas in the excurrent airstream, and F_i is the concentration of gas in the incurrent airstream.

Indirect calorimetry system validation

Before and at the end of the evaluations, the chambers and respirometry system were validated for their gas recovery efficiency by burning ethanol (Sigma-Aldrich, Missouri, USA) for 430 min, and using the combustion values proposed [31]. The percentage of recovery was established considering the volume of burned ethanol calculated (measuring in a scale its initial and final weight), the recorded inflow and outflow of air and the volumes of O² and CO₂ detected by the device [32].

Isotopic analysis

The isotopic analyses were performed at the Stable Isotope Centre, Universidade Estadual Paulista (UNESP), Botucatu, Brazil. All samples were analyzed using continuous-flow isotope ratio mass spectrometry (CF-IRMS). The CO₂ samples were analyzed using an automated breath ¹³C analyzer isotope ratio mass spectrometer (ABCA2; SerCon, Cheshire, UK). Briefly, the system extracts the sample from the tube, the Helium flow carries the sample to the CF-IRMS, and the R(¹³C/¹²C) isotopic ratio is determined and expressed as an atomic fraction x(¹³C) (%), according to the general equation described by [33]: (2) where R(ⁱE/ⁱE) isotopic ratio was determined and expressed as the atomic fraction x(ⁱE) (%).

The results were normalized using a single-point anchorage: standard versus working gas [34] using NBS-22 standards. The ABCA-IRMS uncertainty was estimated at ±0.16‰, according to [35]. The difference between x(¹³C)_final and x(¹³C)_initial was used to calculate the excess atom fraction x^E(¹³C) (S1 Table).

Urine and plasma samples were weighed in 5 x 8 mm tin capsules (PN 24006400, Thermo Scientific, Germany) and analyzed using an elemental analyzer (EA)-type CF-IRMS. An IRMS (Delta V, Thermo Scientific, Germany) coupled with an EA (Flash EA, Thermo Scientific, Germany) and a gas interface (ConFlo IV, Thermo Scientific, Germany) were used.

The EA-IRMS uncertainty was estimated at ±0.15‰ and ±0.20‰ for δ¹³C and δ¹⁵N, respectively. The results were normalized by two-point anchorage (Paul et al., 2007) using IAEA-N-1 and USGS90 for δ¹⁵N. To calculate the excess atom fraction x^E(¹⁵N), the difference between x(¹⁵N)_final and x(¹⁵N)_initial was utilized.

Whole body protein turnover

The flux of the labelled compounds and the rates of protein synthesis and breakdown were calculated according to the model proposed by [21]. (3) where Q is tracer flux (g.kg^-0.75. d^-1), O is the measured amino acid oxidation evaluated by expired gas (not considered when expired ¹³C was not measured), S is the protein synthesis rate (g.kg^-0.75. d^-1), I is the dietary N intake (g.kg^-1. d^-1), and B is the protein breakdown rate (g.kg^-0.75. d^-1).

First, the rate of ¹³CO₂ released by the oxidation of the tracer, represented by the excreted fraction of ¹³CO₂ (F¹³CO₂: g.kg^-1. day^-1) was calculated as follows: (4) where VCO₂ is the rate of CO₂ production of the animal as determined in respirometry chambers (mL.min^-1), ECO₂ is the ¹³CO₂ enrichment plateau in the expired gas, BW is the body weight of the animal (kg). The constants 41.6 μmol.ml^-1 and 60 min.h^-1 convert VCO₂ to mol.h^-1 and the factor 100 changes the plateau to a fraction. MW is the molar weight of the tracer, converted into g by the multiplication of μg to 10⁶. The factor, 24, transforms hour in the day as required in the equation (Adapted from [20]).

Subsequently, the oxidation (O) of ¹³C-Phenylalanine (using expired air) and ¹³C-Leucine (using expired air) was calculated using the following equation: (5) where x^E(¹³C)_tracer is the excess atom fraction of the tracer, x^E(¹³C)_sample is the excess atom fraction of the sample, and represents enrichment in the steady state.

The oxidation (O) calculated with urine samples for ¹⁵N-Glycine and ¹³C-Phenylalanine tracers equals the flux (Q), provided the other parameters are calculated in the enrichment plateau [20]. The flux (Q) of ¹³C-Phenylalanine, ¹³C-Leucine (using plasma) and ¹⁵N-Glycine were calculated as: (6) (7) where D is the tracer infusion rate (μmol.kg^-1. h^-1), a is a constant with value 1.33 for plasma and 1.0 for other samples, x^E(¹⁵N)_Glycine is the excess atom fraction of ¹⁵N-Glycine, x^E(¹⁵N)_urine is the excess atom fraction of a harmonic mean from urea and ammonia [36], and MW is the molar weight of the tracer. Unit μmol is transformed to g by multiplication by the MW and 10⁶ and hours in a day are accounted for by multiplication with 24 (Adapted from [20]).

Protein synthesis (S) for ¹³C-Leucine or ¹³C-Phenylalanine was calculated using the flux (Q) and oxidation (O). Considering the equation developed by [21], protein synthesis for ¹⁵N-Glycine may be considered equal to the flux as oxidation is not calculated. Protein breakdown (B) was calculated for glycine or phenylalanine (when the animals were fed), based on the difference between the flux (Q) of ¹⁵N-Glycine or ¹³C-Phenylalanine and the intake of the amino acids (I). In the case of ¹³C-Leucine, in fasting animals, the I equals the isotope dose [20].

Statistical analysis

The results were evaluated using the equations, and protein flux, synthesis, and breakdown were calculated. After confirming the normality of the residue and variance homoscedasticity, the data were subjected to ANOVA in a completely randomized design, with four methods (treatments) and six experimental units (dogs) per treatment. Means were compared using Tukey’s test for verifying differences obtained with F test. The confidence intervals were calculated for assessing agreement between the results of the different methods as biases (mean difference ± 1.96 SD), according to [37], and the mean values were compared using the Pearson correlation, as a complementary statistic. Bland-Altman statistics and Pearson correlation were performed using R Software (PBC, Boston, MA, 2020), and the remaining analyses were performed using SAS software 9.1 using Proc MIXED (SAS Institute, Cary, NC, USA, 2003). P values of 0.05 were considered significant and P<0.1 was utilized as trend.

Results

Validation of the indirect calorimetry system with ethanol produced a mean CO₂ recovery rate of 96±6%, indicating accuracy. Therefore, the obtained VCO₂ values can be considered reliable, with a mean value of 75.4±6.0 ml of CO₂.min^-1 for the experimental group. The mean O₂ recovery rate was 82±5% and a mean energy expenditure (EE) of 406.6 ± 14.6 kJ.kg^-0.75. d^-1 was calculated for senior dogs using the observed VCO₂ and VO₂. The mean protein intake of the dogs for the study was 6.96 ± 0.26 g.kg^-0.75. d^-1, calculated using the food intake records and protein content of the diet.

The isotope enrichment based on the different methods and biological samples is illustrated in Fig 3. As visible, it is possible to observe the enrichment curve for all the isotopes from which the tracer flux was calculated.

Download:

Fig 3. Fractional enrichment curve for each method of analysis: y = X^E (¹³C or ¹⁵N in %); x = sampling time in hours or minutes.

A. ¹³C-Phenylalanine method; ¹³CO₂/¹²CO₂ evaluated along 200 min in expired air samples. B. ¹³C-Phenylalanine method; ¹³CO₂/¹²CO₂ evaluated along 36 hours in urine samples. C. ¹³C-Leucine method; ¹³CO₂/¹²CO₂ evaluated along 180 min in expired air samples. D. ¹³C-Leucine method; ¹³CO₂/¹²CO₂ evaluated along 180 min in plasma samples. E. ¹⁵N-Glycine method; ¹⁵N/¹⁴N evaluated along 36 hours in urinary ammonia samples. F. ¹⁵N-Glycine method; ¹⁵N/¹⁴N evaluated along 36 hours in urinary urea samples.

https://doi.org/10.1371/journal.pone.0305073.g003

The F test of analysis of variance for all methods and biological samples resulted in similar estimates of protein synthesis, breakdown, and flux (P>0.05), showing comparable results between them, as presented in Table 1. The adopted procedure to collect samples of expired air, masks, and storage of the air samples in glass vials may be considered adequate, as the results calculated with plasma (¹³C-Leucine) and urine (¹⁵N-Glycine and ¹³C-Phenylalanine) were similar to those calculated with expired air samples (P>0.05). This indicates equally efficient rates of isotope recovery, or at least recoveries that are precise enough to result in similar estimations of the synthesis, breakdown, and flux of body proteins. The Bland–Altman statistics and Pearson correlation comparison between the rates of synthesis and breakdown of body proteins are presented in Table 2.

Download:

Table 1. Protein flux, synthesis, and breakdown rates in old beagle dogs obtained with different traces and biological samples.

https://doi.org/10.1371/journal.pone.0305073.t001

Download:

Table 2. Pearson correlation and Bland & Altman statistics of the results obtained with the different methods and biological samples studied to estimate whole body protein synthesis and breakdown in dogs.

https://doi.org/10.1371/journal.pone.0305073.t002

Elevated Pearson correlation values were obtained for protein synthesis and breakdown, calculated using expired air or urine in the ¹³C-Phenylalanine method (P<0.05), with high concordance correlation coefficients, bias correction factors, and R² values. This is expected as the same enrichment protocol and tracer were used. In addition, it confirms that both biological samples, the expired air and urine, were equally adequate for the procedure, in turn, validating the adopted collection protocol. The ¹³C-Leucine and ¹³C-Phenylalanine (expired air) methods also presented elevated Pearson correlation values for protein synthesis and breakdown (P<0.05), with elevated concordance correlation coefficients, bias correction factors, and R² values, suggesting that both methods are interchangeable, and the selection of the most appropriate method depends on the experimental conditions and the moment (fasting or postprandial) at which protein metabolism will be studied. For the other methods, only a moderate Pearson correlation with a tendency of similar results (P<0.1) and moderate concordance correlation coefficient, bias correction factor, and R² values were observed between ¹³C-Leucine and ¹⁵N-Glycine. Although a similar mean value was observed in the F-test, no Pearson correlation was observed (P>0.05) for the remaining methods.

Discussion

The mean rates of protein synthesis, breakdown, and flux in the present study were consistent between the tested methods. The observed values were close to those for protein synthesis reported by [39] i.e., 3.08±0.13 g of protein.kg^-0.75. d^-1 in dogs fed a normal protein diet (15.1 g of crude protein per megajoule [MJ]). The diet used in the present study contained 16 g of crude protein per MJ, comparable to the diet used by [39], therefore justifying similar results. That authors evaluated protein metabolism through continuous infusion of ¹³C-Leucine in the blood as well as using plasma and expired air as sampling materials. Continuous intravenous infusion of ¹³C-Leucine for approximately two hours, with an enrichment plateau defined by arterial leucine, is the standard protocol for this tracer [20]. However, the comparison of [39] findings and those of the present study suggest that the protocol adopted for bolus enrichment with ¹³C-Leucine is adequate and comparable. Although bolus enrichment and frequent blood sampling necessitate the placement of a peripheral catheter, the procedure is easier and more “friendly” than a continuous infusion, which requires higher restriction and monitoring of dogs. In the authors’ experience, the proposed ¹³C-Leucine method is suitable for use in ambulatory conditions, with privately owned dogs after some dog training for blood sampling and collection of expired air.

The ¹³C-Leucine is considered the gold standard for determining protein turnover because of its true representation of free amino acid incorporation [40]. The calculated flux of labelled leucine was converted into whole-body turnover using the average leucine content of whole body protein (8 g/ 100 g protein, [20]). The difference between ¹³C-Leucine and the other two tracers is that the animals were fasting in the former, and although normal conditions of the animal’s daily life are not reflected, the measurements are adequate and reliable for most physiological conditions [3].

Whole-body protein turnover in adult and senior dogs was estimated by [6] using the ¹⁵N-Glycine tracer. Although the results varied considerably among diets and age groups, mean arbitrary values for dogs fed with 24% crude protein diet were 4.75 g and 4.35 g of protein degradation and synthesis per kg^-0.75 per day, respectively. The authors found no significant difference between the age groups of dogs, suggesting that the measurements were influenced more by protein intake than by the physiological condition of the animals. The ¹⁵N-Glycine method was popular in the 1980s because of its non-invasiveness and easy application, as well as requirement of only a single dose of tracer and urine collection [22,41–43]. However, the application of this method presents some challenges outside the laboratory. Urine collection cannot be well-controlled and it may not be adequately sensitive for use in different physiological and pathological conditions [3]. While in humans, individuals may be requested to urinate, empty bladder before enrichment, and then urinate at the correct collection times, this is not possible in dogs, and the animals may have a residual amount of urine in the bladder before enrichment, resulting in dilution of the first point of the plateau (the first spontaneous voided urine of the animal). Additionally, dogs do not urinate at the appointed times but when they have an urge to, and the sampling time does not correspond with the required protocol. This issue has already been discussed in cats using the doubly labelled water method with urine as the sampling fluid [44] but, as in the present study, the final values were comparable, supporting the use of this tracer in dogs. To increase its precision, placing a urinary catheter to collect urine samples from dogs could be attempted; however, this is very invasive and beyond the scope of the present study.

One limitation of the use of ¹⁵N as a tracer for studies on protein metabolism is the assumption that all amino acids integrate into a single nitrogen compartment and simultaneously achieve the same enrichment [45]. However, this is not entirely true because amino acids may have specific and independent pathways of use and degradation [23]. Nitrogen breaks down into ammonia and urea, and the harmonic mean of ¹⁵N incorporation in the two compounds must be included for calculation [22,42], as adopted in the current study.

In the present study, the mean of synthesis, breakdown, and flux were similar between ¹⁵N-Glycine and the other two tracers, and the method showed a moderate correlation with ¹³C-Leucine, supporting the use of the proposed methodology to evaluate protein metabolism in dogs. Similar results were seen in a previous study in elderly humans which compared protein metabolism using ¹⁵N-Glycine and ¹³C-Leucine as metabolic tracers. The authors found slightly higher values of synthesis and degradation with ¹³C-Leucine in comparison to ¹⁵N-Glycine, but as in the present study, the differences were statistically insignificant [46].

The method utilizing ¹³C-Phenylalanine was originally developed to study amino acid requirements in a minimally invasive and precise approach [2,47,48]. Previous publications have studied amino acid requirements in various animals [49,50] and dogs [26,51]. The post-prandial oxidation or incorporation of ¹³C-Phenylalanine was determined in the fed state by measuring the fractional enrichment of ¹³CO₂ (F¹³CO₂) an animal enriched with the tracer. For the calculation, only F¹³CO₂ is necessary, usually expressed as atoms percent in excess. The application of the method requires an equal body pool of phenylalanine and tyrosine in animals i.e., consuming equal amounts of these amino acids in excess of the requirement [52]. The diet used in the present study contained 0.99% phenylalanine and 0.74% tyrosine, well above the minimum requirements for these amino acids in dogs [53]. The authors decided to apply the tracer to estimate the protein flux and calculate the rates of whole protein synthesis and breakdown. To the best of our knowledge, this is the first publication that includes these data. Therefore, the basic parameter of F¹³CO₂ was applied to the standard equations proposed for the other tracers, and whole-body protein metabolism was estimated. These values were found to be similar to those of the other tracers. The results of ¹³C-Phenylalanine calculated with expired air and ¹³C-Leucine showed high Pearson correlation, with low bias and a high concordance correlation coefficient and R². Considering that most authors consider ¹³C-Leucine as a gold standard, results of the present study support high accuracy and precision of ¹³C-Phenylalanine (expired air) as a tracer to study protein metabolism, and not only to study amino acid requirements to animals, the original purpose for this method development.

The route of ¹³C-Phenylalanine delivery and the sample used to study ¹³C incorporation in animals were of concern, as they may interfere with the results [27,47]. [2] developed a 1-day protocol for protein metabolism studies comparing intravenous and oral infusion of ¹³C-Phenylalanine and analyzing the results in urine or expired air. Similar to the present study, the authors did not find any differences between the results of the tracer delivery route and the use of urine or expired air to assess ¹³C incorporation. However, although statistically similar, the results obtained for urine were slightly lower than those obtained for expired air in the present study. Additionally, no Pearson correlation, elevated bias, low concordance correlation coefficient, or R² values were observed between ¹³C-Leucine and ¹³C-Phenylalanine using urine. Taking this into account and considering the previous discussion on the difficulty of collecting urine in a proper moment, caution is required when applying the ¹³C-Phenylalanine method with urine as a sampling material.

Conclusions

The adopted procedures for precursor (¹³C-Leucine), end-products (¹⁵N-Glycine), and amino acid oxidation (¹³C-Phenylalanine) allowed similar results for protein synthesis, breakdown, and flux in senior dogs and can be used for research purposes in the future. The sampling procedures for expired air and urine were considered adequate, allowing the estimation of similar values for protein metabolism. However, only the results for ¹³C-Leucine and ¹³C-Phenylalanine (expired air) methods presented elevated Pearson correlation and concordance, suggesting caution when comparing the results of other methodologies. The selection of the most suitable method will depend on the experimental conditions, as ¹³C-Leucine evaluates animals in a fasting state while ¹³C-Phenylalanine and ¹⁵N-Glycine evaluate animals in fed state. In addition, the feasibility of total urine collection will depend on the structure of the research and dog population of the study.

Supporting information

S1 Table. Raw data to determine the whole-body protein turnover.

Enrichment of samples in basal and plateau time, flux, synthesis and breakdown of animals.

https://doi.org/10.1371/journal.pone.0305073.s001

(XLSX)

Acknowledgments

The authors thank Archer Daniels Midland (ADM), BRF PetFood Brazil and Adimax Pet for supporting of the Laboratory of Research in Nutrition and Nutritional Diseases of Dogs and Cats, and FAPESP for the scholarship to Letícia G Pacheco (number 2019/00790-8).

References

1. Waterlow J. C. (1984). Protein turnover with special reference to man. Quarterly Journal of Experimental Physiology: Translation and Integration, 69(3), 409–438.
- View Article
- Google Scholar
2. Bross R., Ball R. O., & Pencharz P. B. (1998). Development of a minimally invasive protocol for the determination of phenylalanine and lysine kinetics in humans during the fed state. The Journal of Nutrition, 128(11), 1913–1919. pmid:9808642
- View Article
- PubMed/NCBI
- Google Scholar
3. Wagenmakers A. J. (1999). Tracers to investigate protein and amino acid metabolism in human subjects. Proceedings of the Nutrition Society, 58(4), 987–1000. pmid:10817167
- View Article
- PubMed/NCBI
- Google Scholar
4. Liu Z., & Barrett E. J. (2002). Human protein metabolism: its measurement and regulation. American Journal of Physiology-Endocrinology and Metabolism, 283(6), E1105–E1112. pmid:12424100
- View Article
- PubMed/NCBI
- Google Scholar
5. Nair K. S., Schwartz R. G., & Welle S. T. E. P. H. E. N. (1992). Leucine as a regulator of whole body and skeletal muscle protein metabolism in humans. American Journal of Physiology-Endocrinology And Metabolism, 263(5), E928–E934. pmid:1443126
- View Article
- PubMed/NCBI
- Google Scholar
6. Williams C. C., Cummins K. A., Hayek M. G., & Davenport G. M. (2001). Effects of dietary protein on whole-body protein turnover and endocrine function in young-adult and aging dogs. Journal of Animal Science, 79(12), 3128–3136. pmid:11811469
- View Article
- PubMed/NCBI
- Google Scholar
7. Gaine P. C., Pikosky M. A., Martin W. F., Bolster D. R., Maresh C. M., & Rodriguez N. R. (2006). Level of dietary protein impacts whole body protein turnover in trained males at rest. Metabolism, 55(4), 501–507. pmid:16546481
- View Article
- PubMed/NCBI
- Google Scholar
8. Rodriguez N. R., Vislocky L. M., & Gaine P. C. (2007). Dietary protein, endurance exercise, and human skeletal-muscle protein turnover. Current Opinion in Clinical Nutrition & Metabolic Care, 10(1), 40–45.
- View Article
- Google Scholar
9. Phillips S. M., Tipton K. D., Ferrando A. A., & Wolfe R. R. (1999). Resistance training reduces the acute exercise-induced increase in muscle protein turnover. American Journal of Physiology-Endocrinology And Metabolism. pmid:9886957
- View Article
- PubMed/NCBI
- Google Scholar
10. Laflamme D. P. (2008). Pet food safety: dietary protein. Topics in companion Animal Medicine, 23(3), 154–157. pmid:18656844
- View Article
- PubMed/NCBI
- Google Scholar
11. Rennie M. J. (1985). Muscle protein turnover and the wasting due to injury and disease. British Medical Bulletin, 41(3), 257–264. pmid:3896381
- View Article
- PubMed/NCBI
- Google Scholar
12. Wannemacher R. W. Jr, & McCoy J. R. (1966). Determination of optimal dietary protein requirements of young and old dogs. The Journal of Nutrition, 88(1), 66–74. pmid:5900634
- View Article
- PubMed/NCBI
- Google Scholar
13. Kim I. Y., Suh S. H., Lee I. K., & Wolfe R. R. (2017). Applications of stable, nonradioactive isotope tracers in in vivo human metabolic research. Experimental & Molecular Medicine, 48(1), e203.
- View Article
- Google Scholar
14. Claydon A. J., Thom M. D., Hurst J. L., & Beynon R. J. (2012). Protein turnover: measurement of proteome dynamics by whole animal metabolic labelling with stable isotope labelled amino acids. Proteomics, 12(8), 1194–1206. pmid:22577021
- View Article
- PubMed/NCBI
- Google Scholar
15. Duggleby S. L., & Waterlow J. C. (2005). The end-product method of measuring whole-body protein turnover: a review of published results and a comparison with those obtained by leucine infusion. British journal of Nutrition, 94(2), 141–153. pmid:16115347
- View Article
- PubMed/NCBI
- Google Scholar
16. Millward D. J., & Garlick P. J. (1972). The pattern of protein turnover in the whole animal and the effect of dietary variations. Proceedings of the Nutrition Society, 31(3), 257–263. pmid:4579348
- View Article
- PubMed/NCBI
- Google Scholar
17. Mansilla W. D., Fortener L., Templeman J. R., & Shoveller A. K. (2020). Adult dogs of different breed sizes have similar threonine requirements as determined by the indicator amino acid oxidation technique. Journal of Animal Science, 98(3), skaa066. pmid:32108874
- View Article
- PubMed/NCBI
- Google Scholar
18. Templeman J. R., Mansilla W. D., Fortener L., & Shoveller A. K. (2019). Tryptophan requirements in small, medium, and large breed adult dogs using the indicator amino acid oxidation technique. Journal of Animal Science, 97(8), 3274–3285.
- View Article
- Google Scholar
19. Mansilla W. D., Gorman A., Fortener L., & Shoveller A. K. (2018). Dietary phenylalanine requirements are similar in small, medium, and large breed adult dogs using the direct amino acid oxidation technique. Journal of Animal Science, 96(8), 3112–3120. pmid:29846616
- View Article
- PubMed/NCBI
- Google Scholar
20. Matthews D. E., Motil K. J., Rohrbaugh D. K., Burke J. F., Young V. R., & Bier D. M. (1980). Measurement of leucine metabolism in man from a primed, continuous infusion of L-[1-3C] leucine. American Journal of Physiology-Endocrinology and Metabolism, 238(5), E473–E479.
- View Article
- Google Scholar
21. Picou D., & Taylor-Roberts T. (1969). The measurement of total protein synthesis and catabolism and nitrogen turnover in infants in different nutritional states and receiving different amounts of dietary protein. Clinical science, 36, 283–296. pmid:5772104
- View Article
- PubMed/NCBI
- Google Scholar
22. Fern E. B., Garlick P. J., McNurlan M. A., & Waterlow J. C. (1981). The excretion of isotope in urea and ammonia for estimating protein turnover in man with [15N]Glycine. Clinical Science, 61(2), 217–228. pmid:7261545
- View Article
- PubMed/NCBI
- Google Scholar
23. Garlick PJ & Fern EB (1985) Whole body protein turnover: theoretical considerations. In Substrate and Energy Metabolism, pp. 7–15 [JS Garrow and Halliday D, editors]. London: John Libbey.
24. Elango R., Ball R. O., & Pencharz P. B. (2008). Indicator amino acid oxidation: concept and application. The Journal of Nutrition, 138(2), 243–246. pmid:18203885
- View Article
- PubMed/NCBI
- Google Scholar
25. Darling P. B., Bross R., Wykes L. J., Ball R. O., & Pencharz P. B. (1999). Isotopic enrichment of amino acids in urine following oral infusions of L-[1-13C] phenylalanine and L-[1-13C] lysine in humans: confounding effect of D-[13C] amino acids. Metabolism, 48(6), 732–737. pmid:10381147
- View Article
- PubMed/NCBI
- Google Scholar
26. Shoveller A. K., Danelon J. J., Atkinson J. L., Davenport G. M., Ball R. O., & Pencharz P. B. (2017). Calibration and validation of a carbon oxidation system and determination of the bicarbonate retention factor and the dietary phenylalanine requirement, in the presence of excess tyrosine, of adult, female, mixed-breed dogs. Journal of Animal Science, 95(7), 2917–2927. pmid:28727110
- View Article
- PubMed/NCBI
- Google Scholar
27. Hoerr R. A., Yu Y. M., Wagner D. A., Burke J. F., & Young V. R. (1989). Recovery of 13C in breath from NaH13CO3 infused by gut and vein: effect of feeding. American Journal of Physiology-Endocrinology and Metabolism, 257(3), E426–E438. pmid:2551178
- View Article
- PubMed/NCBI
- Google Scholar
28. Read W. W. C., Harrison R. A., & Halliday D. (1982). A resin-based method for the preparation of molecular nitrogen for 15N analysis from urinary and plasma components. Analytical Biochemistry, 123(2), 249–254. pmid:7125200
- View Article
- PubMed/NCBI
- Google Scholar
29. Preston T., & McMillan D. C. (1988). Rapid sample throughput for biomedical stable isotope tracer studies. Biomedical & Environmental Mass Spectrometry, 16(1–12), 229–235. pmid:3149535
- View Article
- PubMed/NCBI
- Google Scholar
30. Lighton J. R. (2018). Measuring metabolic rates: a manual for scientists. Oxford University Press.
31. Elia M., & Livesey G. (1992). Energy expenditure and fuel selection in biological systems: the theory and practice of calculations based on indirect calorimetry and tracer methods. Metabolic Control of Eating, Energy Expenditure and the Bioenergetics of Obesity, 70, 68–131. pmid:1292242
- View Article
- PubMed/NCBI
- Google Scholar
32. Gerrits W., & Labussière E. (Eds.). (2015). Indirect calorimetry: Techniques, computations and applications. Wageningen Academic Publishers.
33. Coplen T. B. (2011). Guidelines and recommended terms for expression of stable‐isotope‐ratio and gas‐ratio measurement results. Rapid Communications in Mass Spectrometry, 25(17), 2538–2560. pmid:21910288
- View Article
- PubMed/NCBI
- Google Scholar
34. Paul D., Skrzypek G., & Fórizs I. (2007). Normalization of measured stable isotopic compositions to isotope reference scales–a review. Rapid Communications in Mass Spectrometry: An International Journal Devoted to the Rapid Dissemination of Up‐to‐the‐Minute Research in Mass Spectrometry, 21(18), 3006–3014. pmid:17705258
- View Article
- PubMed/NCBI
- Google Scholar
35. Carlos A. B. M., de Oliveira C. V., Rodrigues M. A. M., & Costa V. E. (2019). Is breath testing without 13 C-labelled external urea able to detect Helicobacter pylori infection?. Analytical and Bioanalytical Chemistry, 411, 5641–5645. pmid:31201459
- View Article
- PubMed/NCBI
- Google Scholar
36. Pfrimer K., Marchini J. S., Moriguti J. C., & Ferriolli E. (2009). Fed State Protein Turnover in Healthy Older Persons under a Usual Protein‐Rich Diet. Journal of Food Science, 74(4), H112–H115. pmid:19490328
- View Article
- PubMed/NCBI
- Google Scholar
37. Bland J. M., & Altman D. (1986). Statistical methods for assessing agreement between two methods of clinical measurement. The Lancet, 327(8476), 307–310. pmid:2868172
- View Article
- PubMed/NCBI
- Google Scholar
38. Waterlow J. C. (Ed.). (2006). Protein turnover. CABI.
39. Humbert B., Bleis P., Martin L., Dumon H., Darmaun D., & Nguyen P. (2001). Effects of dietary protein restriction and amino acids deficiency on protein metabolism in dogs. Journal of Animal Physiology and Animal Nutrition, 85(7‐8), 255–262. pmid:11686798
- View Article
- PubMed/NCBI
- Google Scholar
40. Matthews D. E., Schwarz H. P., Yang R. D., Motil K. J., Young V., & Bier D. M. (1982). Relationship of plasma leucine and α-ketoisocaproate during a L-[1-13C] leucine infusion in man: a method for measuring human intracellular leucine tracer enrichment. Metabolism, 31(11), 1105–1112.
- View Article
- Google Scholar
41. van Waardenburg D. A., Deutz N. E., Hoos M. B., Jansen N. J., van Kreel B. K., Vos G. D.,… & Forget, P. P. (2004). Assessment of whole body protein metabolism in critically ill children: can we use the [15N] glycine single oral dose method?. Clinical Nutrition, 23(2), 153–160. pmid:15030954
- View Article
- PubMed/NCBI
- Google Scholar
42. Fern E. B., Garlick P. J., & Waterlow J. C. (1985). Apparent compartmentation of body nitrogen in one human subject: its consequences in measuring the rate of whole-body protein synthesis with 15N. Clinical Science, 68(3), 271–282. pmid:3918824
- View Article
- PubMed/NCBI
- Google Scholar
43. Garlick P. J., Clugston G. A., & Waterlow J. C. (1980). Influence of low-energy diets on whole-body protein turnover in obese subjects. American Journal of Physiology-Endocrinology and Metabolism, 238(3), E235–E244. pmid:7369354
- View Article
- PubMed/NCBI
- Google Scholar
44. Goloni C., Peres F. M., Senhorello I. L., Di Santo L. G., Mendonça F. S., Loureiro B. A.,… & Carciofi, A. C. (2020). Validation of saliva and urine use and sampling time on the doubly labelled water method to measure energy expenditure, body composition and water turnover in male and female cats. British Journal of Nutrition, 124(4), 457–469. pmid:32172691
- View Article
- PubMed/NCBI
- Google Scholar
45. Assimon S. A., & Stein T. P. (1992). 15N-glycine as a tracer to study protein metabolism in vivo. In Modern methods in protein nutrition and metabolism (pp. 275–309). Academic Press.
46. Pannemans D. L., Wagenmakers A. J., Westerterp K. R., Schaafsma G., & Halliday D. (1997). The effect of an increase of protein intake on whole-body protein turnover in elderly women is tracer dependent. The Journal of Nutrition, 127(9), 1788–1794. pmid:9278561
- View Article
- PubMed/NCBI
- Google Scholar
47. Kriengsinyos W., Wykes L. J., Ball R. O., & Pencharz P. B. (2002). Oral and intravenous tracer protocols of the indicator amino acid oxidation method provide the same estimate of the lysine requirement in healthy men. The Journal of Nutrition, 132(8), 2251–2257. pmid:12163671
- View Article
- PubMed/NCBI
- Google Scholar
48. Pillai R. R., Elango R., Ball R. O., Kurpad A. V., & Pencharz P. B. (2015). Lysine requirements of moderately undernourished school-aged Indian children are reduced by treatment for intestinal parasites as measured by the indicator amino acid oxidation technique. The Journal of Nutrition, 145(5), 954–959. pmid:25761501
- View Article
- PubMed/NCBI
- Google Scholar
49. Kim K. I., Elliott J. I., & Bayley H. S. (1983). Oxidation of an indicator amino acid by young pigs receiving diets with varying levels of lysine or threonine, and an assessment of amino acid requirements. British Journal of Nutrition, 50(2), 391–399. pmid:6412743
- View Article
- PubMed/NCBI
- Google Scholar
50. Chamruspollert M., Pesti G. M., & Bakalli R. I. (2002). Determination of the methionine requirement of male and female broiler chicks using an indirect amino acid oxidation method. Poultry Science, 81(7), 1004–1013. pmid:12162337
- View Article
- PubMed/NCBI
- Google Scholar
51. Coon C. N., Varney J. L., Fowler J. W., & Weil J. T. (2019). Determination of amino acid requirements of lysine and tryptophan of 14 wk old growing Labrador retriever puppies using the indicator amino acid oxidation technique. Journal of Animal Science, 97, 59–60.
- View Article
- Google Scholar
52. Gooding M. A., Cant J. P., Pencharz P. B., Davenport G. M., Atkinson J. L., & Shoveller A. K. (2013). Oral and intravenous l‐[1‐13C] phenylalanine delivery measure similar rates of elimination when gastric emptying and splanchnic extraction are accounted for in adult mixed hounds1–4. Journal of Animal Physiology and Animal Nutrition, 97(1), 181–189.
- View Article
- Google Scholar
53. National Research Council. (2006). Nutrient requirements of dogs and cats. National Academies Press.

[ref1] 1. Waterlow J. C. (1984). Protein turnover with special reference to man. Quarterly Journal of Experimental Physiology: Translation and Integration, 69(3), 409–438.
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Bross R., Ball R. O., & Pencharz P. B. (1998). Development of a minimally invasive protocol for the determination of phenylalanine and lysine kinetics in humans during the fed state. The Journal of Nutrition, 128(11), 1913–1919. pmid:9808642
View Article
PubMed/NCBI
Google Scholar

[5] View Article

[6] PubMed/NCBI

[7] Google Scholar

[ref3] 3. Wagenmakers A. J. (1999). Tracers to investigate protein and amino acid metabolism in human subjects. Proceedings of the Nutrition Society, 58(4), 987–1000. pmid:10817167
View Article
PubMed/NCBI
Google Scholar

[9] View Article

[10] PubMed/NCBI

[11] Google Scholar

[ref4] 4. Liu Z., & Barrett E. J. (2002). Human protein metabolism: its measurement and regulation. American Journal of Physiology-Endocrinology and Metabolism, 283(6), E1105–E1112. pmid:12424100
View Article
PubMed/NCBI
Google Scholar

[13] View Article

[14] PubMed/NCBI

[15] Google Scholar

[ref5] 5. Nair K. S., Schwartz R. G., & Welle S. T. E. P. H. E. N. (1992). Leucine as a regulator of whole body and skeletal muscle protein metabolism in humans. American Journal of Physiology-Endocrinology And Metabolism, 263(5), E928–E934. pmid:1443126
View Article
PubMed/NCBI
Google Scholar

[17] View Article

[18] PubMed/NCBI

[19] Google Scholar

[ref6] 6. Williams C. C., Cummins K. A., Hayek M. G., & Davenport G. M. (2001). Effects of dietary protein on whole-body protein turnover and endocrine function in young-adult and aging dogs. Journal of Animal Science, 79(12), 3128–3136. pmid:11811469
View Article
PubMed/NCBI
Google Scholar

[21] View Article

[22] PubMed/NCBI

[23] Google Scholar

[ref7] 7. Gaine P. C., Pikosky M. A., Martin W. F., Bolster D. R., Maresh C. M., & Rodriguez N. R. (2006). Level of dietary protein impacts whole body protein turnover in trained males at rest. Metabolism, 55(4), 501–507. pmid:16546481
View Article
PubMed/NCBI
Google Scholar

[25] View Article

[26] PubMed/NCBI

[27] Google Scholar

[ref8] 8. Rodriguez N. R., Vislocky L. M., & Gaine P. C. (2007). Dietary protein, endurance exercise, and human skeletal-muscle protein turnover. Current Opinion in Clinical Nutrition & Metabolic Care, 10(1), 40–45.
View Article
Google Scholar

[29] View Article

[30] Google Scholar

[ref9] 9. Phillips S. M., Tipton K. D., Ferrando A. A., & Wolfe R. R. (1999). Resistance training reduces the acute exercise-induced increase in muscle protein turnover. American Journal of Physiology-Endocrinology And Metabolism. pmid:9886957
View Article
PubMed/NCBI
Google Scholar

[32] View Article

[33] PubMed/NCBI

[34] Google Scholar

[ref10] 10. Laflamme D. P. (2008). Pet food safety: dietary protein. Topics in companion Animal Medicine, 23(3), 154–157. pmid:18656844
View Article
PubMed/NCBI
Google Scholar

[36] View Article

[37] PubMed/NCBI

[38] Google Scholar

[ref11] 11. Rennie M. J. (1985). Muscle protein turnover and the wasting due to injury and disease. British Medical Bulletin, 41(3), 257–264. pmid:3896381
View Article
PubMed/NCBI
Google Scholar

[40] View Article

[41] PubMed/NCBI

[42] Google Scholar

[ref12] 12. Wannemacher R. W. Jr, & McCoy J. R. (1966). Determination of optimal dietary protein requirements of young and old dogs. The Journal of Nutrition, 88(1), 66–74. pmid:5900634
View Article
PubMed/NCBI
Google Scholar

[44] View Article

[45] PubMed/NCBI

[46] Google Scholar

[ref13] 13. Kim I. Y., Suh S. H., Lee I. K., & Wolfe R. R. (2017). Applications of stable, nonradioactive isotope tracers in in vivo human metabolic research. Experimental & Molecular Medicine, 48(1), e203.
View Article
Google Scholar

[48] View Article

[49] Google Scholar

[ref14] 14. Claydon A. J., Thom M. D., Hurst J. L., & Beynon R. J. (2012). Protein turnover: measurement of proteome dynamics by whole animal metabolic labelling with stable isotope labelled amino acids. Proteomics, 12(8), 1194–1206. pmid:22577021
View Article
PubMed/NCBI
Google Scholar

[51] View Article

[52] PubMed/NCBI

[53] Google Scholar

[ref15] 15. Duggleby S. L., & Waterlow J. C. (2005). The end-product method of measuring whole-body protein turnover: a review of published results and a comparison with those obtained by leucine infusion. British journal of Nutrition, 94(2), 141–153. pmid:16115347
View Article
PubMed/NCBI
Google Scholar

[55] View Article

[56] PubMed/NCBI

[57] Google Scholar

[ref16] 16. Millward D. J., & Garlick P. J. (1972). The pattern of protein turnover in the whole animal and the effect of dietary variations. Proceedings of the Nutrition Society, 31(3), 257–263. pmid:4579348
View Article
PubMed/NCBI
Google Scholar

[59] View Article

[60] PubMed/NCBI

[61] Google Scholar

[ref17] 17. Mansilla W. D., Fortener L., Templeman J. R., & Shoveller A. K. (2020). Adult dogs of different breed sizes have similar threonine requirements as determined by the indicator amino acid oxidation technique. Journal of Animal Science, 98(3), skaa066. pmid:32108874
View Article
PubMed/NCBI
Google Scholar

[63] View Article

[64] PubMed/NCBI

[65] Google Scholar

[ref18] 18. Templeman J. R., Mansilla W. D., Fortener L., & Shoveller A. K. (2019). Tryptophan requirements in small, medium, and large breed adult dogs using the indicator amino acid oxidation technique. Journal of Animal Science, 97(8), 3274–3285.
View Article
Google Scholar

[67] View Article

[68] Google Scholar

[ref19] 19. Mansilla W. D., Gorman A., Fortener L., & Shoveller A. K. (2018). Dietary phenylalanine requirements are similar in small, medium, and large breed adult dogs using the direct amino acid oxidation technique. Journal of Animal Science, 96(8), 3112–3120. pmid:29846616
View Article
PubMed/NCBI
Google Scholar

[70] View Article

[71] PubMed/NCBI

[72] Google Scholar

[ref20] 20. Matthews D. E., Motil K. J., Rohrbaugh D. K., Burke J. F., Young V. R., & Bier D. M. (1980). Measurement of leucine metabolism in man from a primed, continuous infusion of L-[1-3C] leucine. American Journal of Physiology-Endocrinology and Metabolism, 238(5), E473–E479.
View Article
Google Scholar

[74] View Article

[75] Google Scholar

[ref21] 21. Picou D., & Taylor-Roberts T. (1969). The measurement of total protein synthesis and catabolism and nitrogen turnover in infants in different nutritional states and receiving different amounts of dietary protein. Clinical science, 36, 283–296. pmid:5772104
View Article
PubMed/NCBI
Google Scholar

[77] View Article

[78] PubMed/NCBI

[79] Google Scholar

[ref22] 22. Fern E. B., Garlick P. J., McNurlan M. A., & Waterlow J. C. (1981). The excretion of isotope in urea and ammonia for estimating protein turnover in man with [15N]Glycine. Clinical Science, 61(2), 217–228. pmid:7261545
View Article
PubMed/NCBI
Google Scholar

[81] View Article

[82] PubMed/NCBI

[83] Google Scholar

[ref23] 23. Garlick PJ & Fern EB (1985) Whole body protein turnover: theoretical considerations. In Substrate and Energy Metabolism, pp. 7–15 [JS Garrow and Halliday D, editors]. London: John Libbey.

[ref24] 24. Elango R., Ball R. O., & Pencharz P. B. (2008). Indicator amino acid oxidation: concept and application. The Journal of Nutrition, 138(2), 243–246. pmid:18203885
View Article
PubMed/NCBI
Google Scholar

[86] View Article

[87] PubMed/NCBI

[88] Google Scholar

[ref25] 25. Darling P. B., Bross R., Wykes L. J., Ball R. O., & Pencharz P. B. (1999). Isotopic enrichment of amino acids in urine following oral infusions of L-[1-13C] phenylalanine and L-[1-13C] lysine in humans: confounding effect of D-[13C] amino acids. Metabolism, 48(6), 732–737. pmid:10381147
View Article
PubMed/NCBI
Google Scholar

[90] View Article

[91] PubMed/NCBI

[92] Google Scholar

[ref26] 26. Shoveller A. K., Danelon J. J., Atkinson J. L., Davenport G. M., Ball R. O., & Pencharz P. B. (2017). Calibration and validation of a carbon oxidation system and determination of the bicarbonate retention factor and the dietary phenylalanine requirement, in the presence of excess tyrosine, of adult, female, mixed-breed dogs. Journal of Animal Science, 95(7), 2917–2927. pmid:28727110
View Article
PubMed/NCBI
Google Scholar

[94] View Article

[95] PubMed/NCBI

[96] Google Scholar

[ref27] 27. Hoerr R. A., Yu Y. M., Wagner D. A., Burke J. F., & Young V. R. (1989). Recovery of 13C in breath from NaH13CO3 infused by gut and vein: effect of feeding. American Journal of Physiology-Endocrinology and Metabolism, 257(3), E426–E438. pmid:2551178
View Article
PubMed/NCBI
Google Scholar

[98] View Article

[99] PubMed/NCBI

[100] Google Scholar

[ref28] 28. Read W. W. C., Harrison R. A., & Halliday D. (1982). A resin-based method for the preparation of molecular nitrogen for 15N analysis from urinary and plasma components. Analytical Biochemistry, 123(2), 249–254. pmid:7125200
View Article
PubMed/NCBI
Google Scholar

[102] View Article

[103] PubMed/NCBI

[104] Google Scholar

[ref29] 29. Preston T., & McMillan D. C. (1988). Rapid sample throughput for biomedical stable isotope tracer studies. Biomedical & Environmental Mass Spectrometry, 16(1–12), 229–235. pmid:3149535
View Article
PubMed/NCBI
Google Scholar

[106] View Article

[107] PubMed/NCBI

[108] Google Scholar

[ref30] 30. Lighton J. R. (2018). Measuring metabolic rates: a manual for scientists. Oxford University Press.

[ref31] 31. Elia M., & Livesey G. (1992). Energy expenditure and fuel selection in biological systems: the theory and practice of calculations based on indirect calorimetry and tracer methods. Metabolic Control of Eating, Energy Expenditure and the Bioenergetics of Obesity, 70, 68–131. pmid:1292242
View Article
PubMed/NCBI
Google Scholar

[111] View Article

[112] PubMed/NCBI

[113] Google Scholar

[ref32] 32. Gerrits W., & Labussière E. (Eds.). (2015). Indirect calorimetry: Techniques, computations and applications. Wageningen Academic Publishers.

[ref33] 33. Coplen T. B. (2011). Guidelines and recommended terms for expression of stable‐isotope‐ratio and gas‐ratio measurement results. Rapid Communications in Mass Spectrometry, 25(17), 2538–2560. pmid:21910288
View Article
PubMed/NCBI
Google Scholar

[116] View Article

[117] PubMed/NCBI

[118] Google Scholar

[ref34] 34. Paul D., Skrzypek G., & Fórizs I. (2007). Normalization of measured stable isotopic compositions to isotope reference scales–a review. Rapid Communications in Mass Spectrometry: An International Journal Devoted to the Rapid Dissemination of Up‐to‐the‐Minute Research in Mass Spectrometry, 21(18), 3006–3014. pmid:17705258
View Article
PubMed/NCBI
Google Scholar

[120] View Article

[121] PubMed/NCBI

[122] Google Scholar

[ref35] 35. Carlos A. B. M., de Oliveira C. V., Rodrigues M. A. M., & Costa V. E. (2019). Is breath testing without 13 C-labelled external urea able to detect Helicobacter pylori infection?. Analytical and Bioanalytical Chemistry, 411, 5641–5645. pmid:31201459
View Article
PubMed/NCBI
Google Scholar

[124] View Article

[125] PubMed/NCBI

[126] Google Scholar

[ref36] 36. Pfrimer K., Marchini J. S., Moriguti J. C., & Ferriolli E. (2009). Fed State Protein Turnover in Healthy Older Persons under a Usual Protein‐Rich Diet. Journal of Food Science, 74(4), H112–H115. pmid:19490328
View Article
PubMed/NCBI
Google Scholar

[128] View Article

[129] PubMed/NCBI

[130] Google Scholar

[ref37] 37. Bland J. M., & Altman D. (1986). Statistical methods for assessing agreement between two methods of clinical measurement. The Lancet, 327(8476), 307–310. pmid:2868172
View Article
PubMed/NCBI
Google Scholar

[132] View Article

[133] PubMed/NCBI

[134] Google Scholar

[ref38] 38. Waterlow J. C. (Ed.). (2006). Protein turnover. CABI.

[ref39] 39. Humbert B., Bleis P., Martin L., Dumon H., Darmaun D., & Nguyen P. (2001). Effects of dietary protein restriction and amino acids deficiency on protein metabolism in dogs. Journal of Animal Physiology and Animal Nutrition, 85(7‐8), 255–262. pmid:11686798
View Article
PubMed/NCBI
Google Scholar

[137] View Article

[138] PubMed/NCBI

[139] Google Scholar

[ref40] 40. Matthews D. E., Schwarz H. P., Yang R. D., Motil K. J., Young V., & Bier D. M. (1982). Relationship of plasma leucine and α-ketoisocaproate during a L-[1-13C] leucine infusion in man: a method for measuring human intracellular leucine tracer enrichment. Metabolism, 31(11), 1105–1112.
View Article
Google Scholar

[141] View Article

[142] Google Scholar

[ref41] 41. van Waardenburg D. A., Deutz N. E., Hoos M. B., Jansen N. J., van Kreel B. K., Vos G. D.,… & Forget, P. P. (2004). Assessment of whole body protein metabolism in critically ill children: can we use the [15N] glycine single oral dose method?. Clinical Nutrition, 23(2), 153–160. pmid:15030954
View Article
PubMed/NCBI
Google Scholar

[144] View Article

[145] PubMed/NCBI

[146] Google Scholar

[ref42] 42. Fern E. B., Garlick P. J., & Waterlow J. C. (1985). Apparent compartmentation of body nitrogen in one human subject: its consequences in measuring the rate of whole-body protein synthesis with 15N. Clinical Science, 68(3), 271–282. pmid:3918824
View Article
PubMed/NCBI
Google Scholar

[148] View Article

[149] PubMed/NCBI

[150] Google Scholar

[ref43] 43. Garlick P. J., Clugston G. A., & Waterlow J. C. (1980). Influence of low-energy diets on whole-body protein turnover in obese subjects. American Journal of Physiology-Endocrinology and Metabolism, 238(3), E235–E244. pmid:7369354
View Article
PubMed/NCBI
Google Scholar

[152] View Article

[153] PubMed/NCBI

[154] Google Scholar

[ref44] 44. Goloni C., Peres F. M., Senhorello I. L., Di Santo L. G., Mendonça F. S., Loureiro B. A.,… & Carciofi, A. C. (2020). Validation of saliva and urine use and sampling time on the doubly labelled water method to measure energy expenditure, body composition and water turnover in male and female cats. British Journal of Nutrition, 124(4), 457–469. pmid:32172691
View Article
PubMed/NCBI
Google Scholar

[156] View Article

[157] PubMed/NCBI

[158] Google Scholar

[ref45] 45. Assimon S. A., & Stein T. P. (1992). 15N-glycine as a tracer to study protein metabolism in vivo. In Modern methods in protein nutrition and metabolism (pp. 275–309). Academic Press.

[ref46] 46. Pannemans D. L., Wagenmakers A. J., Westerterp K. R., Schaafsma G., & Halliday D. (1997). The effect of an increase of protein intake on whole-body protein turnover in elderly women is tracer dependent. The Journal of Nutrition, 127(9), 1788–1794. pmid:9278561
View Article
PubMed/NCBI
Google Scholar

[161] View Article

[162] PubMed/NCBI

[163] Google Scholar

[ref47] 47. Kriengsinyos W., Wykes L. J., Ball R. O., & Pencharz P. B. (2002). Oral and intravenous tracer protocols of the indicator amino acid oxidation method provide the same estimate of the lysine requirement in healthy men. The Journal of Nutrition, 132(8), 2251–2257. pmid:12163671
View Article
PubMed/NCBI
Google Scholar

[165] View Article

[166] PubMed/NCBI

[167] Google Scholar

[ref48] 48. Pillai R. R., Elango R., Ball R. O., Kurpad A. V., & Pencharz P. B. (2015). Lysine requirements of moderately undernourished school-aged Indian children are reduced by treatment for intestinal parasites as measured by the indicator amino acid oxidation technique. The Journal of Nutrition, 145(5), 954–959. pmid:25761501
View Article
PubMed/NCBI
Google Scholar

[169] View Article

[170] PubMed/NCBI

[171] Google Scholar

[ref49] 49. Kim K. I., Elliott J. I., & Bayley H. S. (1983). Oxidation of an indicator amino acid by young pigs receiving diets with varying levels of lysine or threonine, and an assessment of amino acid requirements. British Journal of Nutrition, 50(2), 391–399. pmid:6412743
View Article
PubMed/NCBI
Google Scholar

[173] View Article

[174] PubMed/NCBI

[175] Google Scholar

[ref50] 50. Chamruspollert M., Pesti G. M., & Bakalli R. I. (2002). Determination of the methionine requirement of male and female broiler chicks using an indirect amino acid oxidation method. Poultry Science, 81(7), 1004–1013. pmid:12162337
View Article
PubMed/NCBI
Google Scholar

[177] View Article

[178] PubMed/NCBI

[179] Google Scholar

[ref51] 51. Coon C. N., Varney J. L., Fowler J. W., & Weil J. T. (2019). Determination of amino acid requirements of lysine and tryptophan of 14 wk old growing Labrador retriever puppies using the indicator amino acid oxidation technique. Journal of Animal Science, 97, 59–60.
View Article
Google Scholar

[181] View Article

[182] Google Scholar

[ref52] 52. Gooding M. A., Cant J. P., Pencharz P. B., Davenport G. M., Atkinson J. L., & Shoveller A. K. (2013). Oral and intravenous l‐[1‐13C] phenylalanine delivery measure similar rates of elimination when gastric emptying and splanchnic extraction are accounted for in adult mixed hounds1–4. Journal of Animal Physiology and Animal Nutrition, 97(1), 181–189.
View Article
Google Scholar

[184] View Article

[185] Google Scholar

[ref53] 53. National Research Council. (2006). Nutrient requirements of dogs and cats. National Academies Press.

Figures

Abstract

Introduction

Material and methods

Animals, housing and experimental design

13C-Phenylalanine

15N-Glycine

13C-Leucine

Determination of CO2 volume in an indirect calorimetry system

Indirect calorimetry system validation

Isotopic analysis

Whole body protein turnover

Statistical analysis

Results

Discussion

Conclusions

Supporting information

S1 Table. Raw data to determine the whole-body protein turnover.

Acknowledgments

References

¹³C-Phenylalanine

¹⁵N-Glycine

¹³C-Leucine

Determination of CO₂ volume in an indirect calorimetry system