TY - JOUR
AU - Pencharz, P. B.
AB - ABSTRACT Carbon oxidation methods have been used as rapid and sensitive methods to determine whole-body AA requirements in multiple species. The objectives of the current studies were to validate complete CO2 recovery, determine the bicarbonate retention factor, and estimate the Phe requirement, in the presence of excess Tyr, in adult dogs using the direct oxidation technique. In this series of studies, 2 oxidation chambers were constructed and calibrated to ensure accurate collection of breath 13CO2. First, 104.6 ± 7.1% CO2 was recovered from chambers and suggests that the chambers were appropriately designed for complete and efficient CO2 recovery. Second, we determined bicarbonate retention in 5 dogs using repeated oral dosing of a bicarbonate tracer (NaH13CO3) with small meals. At isotopic and physiological steady state, 102.5 ± 2.6% of the delivered NaH13CO3 was recovered in breath. Third, the Phe requirement, when Tyr was supplied in excess, was determined by the rate of appearance of 13CO2 in the breath (F13CO2). Dogs (n = 5) were fed test diets with different concentrations of Phe ranging from deficient to excessive for 2 d prior to conducting the tracer studies. The mean Phe requirement (when Tyr was supplied in excess) was 0.535% of diet (upper 95% confidence interval = 0.645% diet) on an as-fed basis or 0.575% of diet (upper 95% confidence interval = 0.694% of diet) on a DM basis and was based on a calculated (modified Atwater calculation) dietary ME density of 3.73 Mcal/kg DM. These data support the use of carbon oxidation methods and oral dosing of isotope to measure whole-body requirements of indispensable AA in adult dogs and suggest the current recommendations may be low. INTRODUCTION Carbon oxidation methods for determining whole-body AA requirements are based on the oxidation of AA carbon (Pencharz and Ball, 2003), with the indicator AA oxidation (IAAO) method accepted as a least invasive method (Brunton et al., 1998; WHO/FAO/UNU, 2007). The IAAO method has been used to determine AA requirements in swine (Ball and Bayley, 1984), humans (Zello et al., 1995), chickens (Tabiri et al., 2002), horses (Mok, 2015), and cows (Ouellet et al., 2014). To our knowledge, indispensable AA requirements of the dog have not been quantified using carbon oxidation methods. Implementation of the IAAO in a new species requires a series of method development and verification steps. In the present study, oxidation chambers for dogs were constructed followed by validation of the breath collection system. We previously described oral and intravenous Phe kinetics in the dog (Gooding et al., 2013) and demonstrated that oral dosing of tracer and repeated meal feedings were a feasible approach to determine Phe oxidation and kinetics. In carbon oxidation studies conducted in other species, it has been well documented that a fraction of the CO2 pool is not recovered in breath but retained in the body (Tomera et al., 1982). The degree of bicarbonate retention will vary depending on the metabolic state and metabolic fuel selection of the animal. For example, laying hens use calcium carbonate in shell formation, resulting in greater bicarbonate retention values (Leslie et al., 2006). Measurement of the rate of 13CO2 expired following repeated delivery of NaH13CO3 allows for 13CO2 retention to be estimated (Hoerr et al., 1989; Wykes et al., 1994) and used as a correction for carbon oxidation experiments (House et al., 1998). Bicarbonate retention kinetics previously have been measured in fasting, conscious, and anesthetized adult dogs (Wolfe, 1984, 1992; Downey et al., 1986; Pouteau et al., 2002; Larsson et al., 2010, 2014a,b). Fasted dogs retain 0 to 28.2% of the administered bicarbonate regardless whether it was infused or fed. We wanted to investigate bicarbonate recovery of dogs in the fed state, during which the carbon oxidation experiments would be conducted. Finally, to use Phe as a tracer for IAAO experiments, an understanding of the population requirement for Phe in the presence of excess Tyr is needed to ensure future studies meet or exceed these physiological requirements. The objective of this experiment was to examine whether the direct oxidation method, with a short period of exposure to diets deficient in a single indispensable AA, could successfully be used to determine whole-body AA requirements of healthy dogs. Ultimately, our objective was to determine the Phe requirement, when excess dietary Tyr is provided, in adult, mixed-breed hounds using the direct oxidation method. MATERIALs AND METHODS Construction of Oxidation Chambers Two oxidation chambers (61 cm width, 83.8 cm depth, and 102.9 cm height) were constructed of plexiglass (P&A Plastics Inc., Hamilton, ON, Canada). These chambers were designed to fit a plastic-coated metal dog kennel (53.3 cm width, 76.2 cm depth, and 95.25 cm height) recommended for dogs weighing less than 32 kg. These dimensions resulted in a total volume of 526 L. The chambers provided sufficient room for the dogs to sit, stand, lie down, and turn around. The top of the oxidation chamber was fitted with a hinged opening to allow quick access to the dogs in the chamber and to allow feeding of the dogs in timed intervals for studies requiring repeated small meal feedings. The top edge of each chamber was fitted with a rubber gasket (1.2 cm. thick) to ensure air tightness during breath collection. The back of each chamber was also hinged to form an entry and exit door for the dog. A small amount of space (approximately 0.25 cm) was left at the bottom of the rear door to allow air to continuously be pulled through the chamber during oxidation studies. Figure 1 depicts the setup of the complete oxidation system. A rotary vane pump (model 0523-101Q-G588EDX; Gast Manufacturing, Inc., Benton Harbor, MI) is connected by a hose to an outlet placed on the front end of each chamber. As the pump withdraws air from the chambers, it creates negative pressure within the chamber, causing air to be pulled into the chamber through an opening at the bottom of the rear door. Mass flow was measured by a mass flow meter placed between the chamber and pump. A separate subsample of 300 mL of air was pulled from the main vacuum line by a small subsampling pump and pushed into a CO2 analyzer (Qubit Systems Inc., Kingston, ON, Canada) to measure CO2 concentrations. Real-time volume of CO2 data were collected using Qubit calorimetry software (Qubit C950-MCGES; Qubit Systems Inc.). In addition, a separate 300 mL of air was drawn off the main line by an additional pump and pushed through a Midget Bubbler (Fisher Scientific Company, Ottawa, ON, Canada) in which a CO2 absorber, 1 M NaOH (8 mL), was added to trap CO2 for subsequent determination of CO2 enrichment during the bicarbonate and Phe requirement studies. Carbon dioxide was continuously measured at the out port by a CO2 analyzer (Qubit Systems Inc.) to verify that all CO2 was trapped in the NaOH solution and none escaped the Midget bubbler system. Figure 1. View largeDownload slide Complete schematic of respiration system. The system was connected to a large-capacity vacuum pump (a) that pulls > 100 L/min. Mass flow for each chamber is set using a needle valve (b) and the mass flow (mL/min) is measured by the mass flow meter (MFM; c) and logged by the computer in real time. The needle valve controls the air being drawn from the chamber. Dotted lines below the main vacuum line represent subsampling lines connected to a small vacuum pump (d) that pull air from each main line, measured by the air flow meter (e) and then CO2 is measured by the CO2 analyzer (f) and then the CO2 trapped in 1 M NaOH (g) for subsequent analysis of breath enrichment. Figure 1. View largeDownload slide Complete schematic of respiration system. The system was connected to a large-capacity vacuum pump (a) that pulls > 100 L/min. Mass flow for each chamber is set using a needle valve (b) and the mass flow (mL/min) is measured by the mass flow meter (MFM; c) and logged by the computer in real time. The needle valve controls the air being drawn from the chamber. Dotted lines below the main vacuum line represent subsampling lines connected to a small vacuum pump (d) that pull air from each main line, measured by the air flow meter (e) and then CO2 is measured by the CO2 analyzer (f) and then the CO2 trapped in 1 M NaOH (g) for subsequent analysis of breath enrichment. Animals All study protocols were designed and conducted according to guidelines specified in the Guide for the Care and Use of Laboratory Animals (NRC, 1996) and approved by the Animal Care Committee at the University of Guelph. A total of 5 spayed female dogs (mixed-breed hounds) were obtained from a dog breeder and brought to the Central Animal Facility (CAF) at the University of Guelph, Guelph, ON, Canada. Dogs were medium size (21 ± 0.7 kg BW and 2.4 ± 0.2 yr of age) and deemed healthy by a basic veterinary exam. Animal Housing Dogs were housed in groups of 2 or 3 within the CAF. Pens housing 2 dogs measured 1.2192 m (4 feet) × 2.4384 m (8 feet) and pens housing 3 dogs measured 1.2192 m (4 feet) × 3.6576 m (12 feet). The animal room was maintained at a temperature of 21°C and the lighting schedule was 12:12 h light:dark. Pens had hammock beds for each dog and 1 doghouse per pen. Toys for environmental enrichment were provided and rotated on a weekly basis to maintain novelty. Water was provided ad libitum at all times and dogs were fed 13 g∙kg BW−1∙d−1 once daily. Exercise and Socialization All dogs were walked on leash for 2 d prior to their study day, for a minimum of 20 min and not exceeding 30 min. For the remaining 5 d a week, dogs received a short hand-walk and then a minimum of 40 min free run and play time in groups of 2 or 3 dogs. Each dog was consistently walked and exercised by the same 2 individuals at approximately the same time each day. Prior to the initiation of the trials, all dogs were adapted to eating in housing crates over a 4-wk period and then to being fed their full daily ration in crates throughout the study. After being adapted to eating in crates, the dogs were walked from the CAF to a separate testing site located within the Animal Science and Nutrition building. Each dog was placed in the oxidation chamber to simulate the testing environment. The chambers contained crates that were similar to those used in training to maintain familiarity and comfort for the dogs. Initially, dogs were placed in crates within the oxidation chamber for a 1-h period and fed 2 meals (each representing one-half of their total daily ration). Over a 4-wk period, dogs were gradually adapted to being fed in the oxidation chamber every 30 min over a 4-h period. Afterward, dogs were returned to the CAF. This acclimation protocol was designed to minimize potential stress to the dog that would affect whole-body metabolism and confound CO2 production. Then, the dogs were used to determine the air flow to maintain chambers under 0.8% CO2 (Objective 1), to determine fed state bicarbonate retention (Objective 3), and to determine the Phe requirement using the direct oxidation approach (Objective 4). Dogs were not required for Objective 2. Objective 1: CO2 Recovery Five separate tests were conducted in each chamber (n = 5 per chamber) using an airflow rate of 30 L/min. An initial CO2 measurement from each chamber was taken before CO2 was added to measure the background CO2 concentration. This background level represented the environmental contribution of CO2 to total CO2 recovered throughout a study and was used to correct the total CO2 recovery values. A canister of CO2 gas was weighed and placed within the chamber. Gas was released for approximately 10 min that was targeted to equate to a release of 30 g of CO2. The CO2 canister was weighed after each release to determine the actual amount of CO2 released into the chamber. The air pulled from the chamber was analyzed for volume of CO2 produced (VCO2) every 0.5 s, and VCO2 was quantified by area under the curve. The measured parameters from the CO2 analyzer were used to calculate grams of CO2 recovered using the following equation: g CO2 = flow/time × (difference/22,400 mL/mol) × 44 g/mol, in which flow/time is expressed in milliliters per 0.5 s, sample CO2/time is expressed in percent per 0.5 s, difference = sample CO2 − background value, 22,400 mL/mol is the volume of an ideal gas at standard temperature and pressure, and 44 g/mol is the molar mass of CO2. Objective 2: Bicarbonate Retention On the day of study and after a 20-h fast, each dog was placed into the crates/oxidation boxes. Prior to starting the pumps, CO2 analyzers were calibrated, mass flow meters were zeroed, and background CO2 readings were taken. The pumps were turned on and the rear and top doors were closed to allow the oxidation boxes to equilibrate and to keep CO2 concentrations constant and below 1%. Each dog's daily dietary ration (13 g food∙kg BW−1∙d−1) was determined according to individual BW, and this amount was divided into 12 equal portions before feeding. The diet was formulated to meet the dietary requirements as reported by the NRC (2006; Table 1). Dogs were fed every 30 min over a 2-h period before receiving a priming dose of NaH13CO3 (0.176 mg/kg of BW; Cambridge Isotope Laboratories, Inc., Woburn, MA). Dogs then received a NaH13CO3 dose (0.055 mg/kg) every 30 min in their allotted food portion starting at time 0 and with each subsequent 30-min period over a 4-h period. The tracer NaH13CO3 was accurately weighed and dissolved into known volumes of sterile water and used within 1 h of preparation. At time 0, the VCO2 measurements and CO2 collection began. Expired CO2 was collected by trapping subsampled air in 1 M NaOH over each 30-min period. The NaOH absorber was sampled and retained in 10-mL Vacutainers (Fisher Scientific Company) at room temperature for subsequent analysis by isotope ratio mass spectrometry. Measured VCO2 was averaged over the collection period to obtain a mean VCO2 for each dog. Bicarbonate retention was calculated from the plateau obtained in the rate of appearance of 13CO2 in the breath (F13CO2), which represents the ratio of 13CO2 to 12CO2. The isotopic plateau was defined as the absence of a significant slope and a CV < 5%. The difference between mean breath 13CO2 enrichments of the 3 baseline and 5 plateau samples was used to determine atom percent excess (APE) above baseline at isotopic steady state. Table 1. Ingredient composition and analyzed nutrient content of the basal and test diet (as-fed basis). Test diets for determination of the Phe requirements were the same except Phe was removed from the AA premix resulting in a Phe concentration of 0.43% and Tyr was added Ingredient, g/kg diet  Basal and test diet  Corn starch  473.84  Chicken fat  122.26  Chicken meal  111.80  Yellow corn  50.81  Brewer's rice  50.81  AA premix1  50.81  Beet pulp  30.49  Dicalcium phosphate  23.56  Chicken flavor  20.32  Potassium chloride  13.00  Sodium bicarbonate  10.16  Chicken liver flavor  5.08  Brewer's yeast  5.08  Ground flax  5.08  Choline chloride  4.49  Vitamin premix2  4.27  Hexametaphosphate  4.07  Calcium carbonate  3.54  Mineral premix3  3.45  Fish oil  2.90  Sodium chloride  1.87  Monosodium phosphate  1.80  Ethoxyquin  0.51  Ingredient, g/kg diet  Basal and test diet  Corn starch  473.84  Chicken fat  122.26  Chicken meal  111.80  Yellow corn  50.81  Brewer's rice  50.81  AA premix1  50.81  Beet pulp  30.49  Dicalcium phosphate  23.56  Chicken flavor  20.32  Potassium chloride  13.00  Sodium bicarbonate  10.16  Chicken liver flavor  5.08  Brewer's yeast  5.08  Ground flax  5.08  Choline chloride  4.49  Vitamin premix2  4.27  Hexametaphosphate  4.07  Calcium carbonate  3.54  Mineral premix3  3.45  Fish oil  2.90  Sodium chloride  1.87  Monosodium phosphate  1.80  Ethoxyquin  0.51  Analyzed nutrient contents  Basal diet  Test diet5  ME, kcal/kg (calculated)4  3,730  3,730  DM, %  93.00  93.00  CP, %  12.5  13.5  Arg, %  0.91  0.92  His, %  0.32  0.49  Ile, %  0.67  0.61  Leu, %  0.97  1.11  Lys, %  0.97  1.02  Met, %  0.56  0.56  Cys, %  0.67  0.62  Phe, %  0.74 (0.43)  0.435  Thr, %  0.69  0.83  Trp, %  0.31  0.37  Tyr, %  0.23 (0.564)  0.56  Val, %  0.65  0.77  Analyzed nutrient contents  Basal diet  Test diet5  ME, kcal/kg (calculated)4  3,730  3,730  DM, %  93.00  93.00  CP, %  12.5  13.5  Arg, %  0.91  0.92  His, %  0.32  0.49  Ile, %  0.67  0.61  Leu, %  0.97  1.11  Lys, %  0.97  1.02  Met, %  0.56  0.56  Cys, %  0.67  0.62  Phe, %  0.74 (0.43)  0.435  Thr, %  0.69  0.83  Trp, %  0.31  0.37  Tyr, %  0.23 (0.564)  0.56  Val, %  0.65  0.77  1Amino acid premix contained 15.36 kg corn starch, 3.0 kg L-Lys HCl, 2.91 kg L-Arg HCl, and 2.41 kg of L-Trp, L-His, L-Ile, L-Leu, L-Val, L-Thr, L-Cys, L-Phe, and L-Met. For test diets, Phe was removed and 2.41 kg of Tyr added. 2Vitamin premix contained, per kilogram, 6,650 K IU vitamin A, 365,000 IU vitamin D3, 100,400 IU vitamin E, 4,100 mg thiamine, 2,500 mg niacin, 2,000 mg pyridoxine, 7,750 mg D-pantothenic acid, 115 mg folic acid, 45 mg vitamin B12, 2,500 mg inositol, 13,750 mg vitamin C, and 1,200 mg β-carotene. 3Mineral premix contained, per kilogram, 150 mg cobalt, 4,500 mg copper, 900 mg iodine, 72,000 mg iron, 8,000 mg manganese (oxide), 5,800 mg manganese (sulfate), and 60,000 mg selenium. 4Calculated ME based on modified Atwater values. 5Test diet = Common test diet with deficient Phe and excess Tyr. View Large Table 1. Ingredient composition and analyzed nutrient content of the basal and test diet (as-fed basis). Test diets for determination of the Phe requirements were the same except Phe was removed from the AA premix resulting in a Phe concentration of 0.43% and Tyr was added Ingredient, g/kg diet  Basal and test diet  Corn starch  473.84  Chicken fat  122.26  Chicken meal  111.80  Yellow corn  50.81  Brewer's rice  50.81  AA premix1  50.81  Beet pulp  30.49  Dicalcium phosphate  23.56  Chicken flavor  20.32  Potassium chloride  13.00  Sodium bicarbonate  10.16  Chicken liver flavor  5.08  Brewer's yeast  5.08  Ground flax  5.08  Choline chloride  4.49  Vitamin premix2  4.27  Hexametaphosphate  4.07  Calcium carbonate  3.54  Mineral premix3  3.45  Fish oil  2.90  Sodium chloride  1.87  Monosodium phosphate  1.80  Ethoxyquin  0.51  Ingredient, g/kg diet  Basal and test diet  Corn starch  473.84  Chicken fat  122.26  Chicken meal  111.80  Yellow corn  50.81  Brewer's rice  50.81  AA premix1  50.81  Beet pulp  30.49  Dicalcium phosphate  23.56  Chicken flavor  20.32  Potassium chloride  13.00  Sodium bicarbonate  10.16  Chicken liver flavor  5.08  Brewer's yeast  5.08  Ground flax  5.08  Choline chloride  4.49  Vitamin premix2  4.27  Hexametaphosphate  4.07  Calcium carbonate  3.54  Mineral premix3  3.45  Fish oil  2.90  Sodium chloride  1.87  Monosodium phosphate  1.80  Ethoxyquin  0.51  Analyzed nutrient contents  Basal diet  Test diet5  ME, kcal/kg (calculated)4  3,730  3,730  DM, %  93.00  93.00  CP, %  12.5  13.5  Arg, %  0.91  0.92  His, %  0.32  0.49  Ile, %  0.67  0.61  Leu, %  0.97  1.11  Lys, %  0.97  1.02  Met, %  0.56  0.56  Cys, %  0.67  0.62  Phe, %  0.74 (0.43)  0.435  Thr, %  0.69  0.83  Trp, %  0.31  0.37  Tyr, %  0.23 (0.564)  0.56  Val, %  0.65  0.77  Analyzed nutrient contents  Basal diet  Test diet5  ME, kcal/kg (calculated)4  3,730  3,730  DM, %  93.00  93.00  CP, %  12.5  13.5  Arg, %  0.91  0.92  His, %  0.32  0.49  Ile, %  0.67  0.61  Leu, %  0.97  1.11  Lys, %  0.97  1.02  Met, %  0.56  0.56  Cys, %  0.67  0.62  Phe, %  0.74 (0.43)  0.435  Thr, %  0.69  0.83  Trp, %  0.31  0.37  Tyr, %  0.23 (0.564)  0.56  Val, %  0.65  0.77  1Amino acid premix contained 15.36 kg corn starch, 3.0 kg L-Lys HCl, 2.91 kg L-Arg HCl, and 2.41 kg of L-Trp, L-His, L-Ile, L-Leu, L-Val, L-Thr, L-Cys, L-Phe, and L-Met. For test diets, Phe was removed and 2.41 kg of Tyr added. 2Vitamin premix contained, per kilogram, 6,650 K IU vitamin A, 365,000 IU vitamin D3, 100,400 IU vitamin E, 4,100 mg thiamine, 2,500 mg niacin, 2,000 mg pyridoxine, 7,750 mg D-pantothenic acid, 115 mg folic acid, 45 mg vitamin B12, 2,500 mg inositol, 13,750 mg vitamin C, and 1,200 mg β-carotene. 3Mineral premix contained, per kilogram, 150 mg cobalt, 4,500 mg copper, 900 mg iodine, 72,000 mg iron, 8,000 mg manganese (oxide), 5,800 mg manganese (sulfate), and 60,000 mg selenium. 4Calculated ME based on modified Atwater values. 5Test diet = Common test diet with deficient Phe and excess Tyr. View Large Objective 3: Determination of the Phenylalanine Requirement of Adult Dogs using the Direct Oxidation Method Diets and Feeding. Dogs were adapted to being fed an extruded kibble basal diet (Table 1) over a 14-d period. Dogs were fed twice daily. Dogs were fed at individual ME intakes based on each dog's historical record of caloric intake that was known to maintain their individual BW. Dogs were provided water ad libitum via an automatic watering system. After the 14-d adaptation period, dogs were randomly assigned to 1 of 7 test diets solutions (Table 1) containing increasing levels of dietary Phe and a constant level of Tyr in excess of the recommended allowance (NRC, 2006) and added as a top-dress to the common test diet. The test diet was formulated to be mildly Phe deficient (0.40%) and had a resulting analyzed Phe concentration of 0.435% and a Tyr concentration of 0.56% (Table 1), and all other AA were similar between basal and test diets. The 6 Phe solutions also contained Ala to ensure isonitrogenous feeding conditions. The Phe-containing test solutions contained (g/L) 1) 0 Phe + 5.39 Ala, 2) 2.0 Phe + 4.31 Ala, 3) 4.0 Phe + 3.23 Ala, 4) 6.0 Phe + 2.20 Ala, 5) 8.0 Phe + 1.07 Ala, and 6) 10.0 Phe + 0 Ala. Each solution was top-dressed on the basal diet at 3.9 mL/kg BW to provide increasing levels of total dietary Phe (0.435, 0.50, 0.55, 0.61, 0.67, and 0.73%, as-fed basis) in combination with 13 g∙kg BW−1∙d−1 of the test diet to allow determination of the Phe requirement with a 2-phase linear regression model. The feeding and testing regimen consisted of a 2-d adaptation period to the assigned dietary Phe level for that test leg, which was followed by a single day to conduct the direct AA oxidation (DAAO) measures. After each test leg and collections, dogs returned to the basal diet for 4 d. This same feeding and collection regime was repeated in random order, with no dogs receiving the same order, for all subsequent Phe solutions until each dog was fed each of the 7 dietary Phe levels. A total of 6 wk was required to complete all 7 dietary Phe levels. Moehn et al. (2004) had previously validated that the 2-d adaptation period to a new dietary Phe level was sufficient before the next DAAO study could be conducted. Dogs were fed twice daily (0800 and 1300 h), except on the oxidation study days, when they received their daily ration divided into 13 separate and equal meals. The feeding and collection schedule is shown in Table 2. During the DAAO studies, dogs were initially fed every 10 min for 30 min starting at 0700 h to induce a fed state. They were then fed every 25 min thereafter to maintain the fed state. The quantity of diet was based on the BW of each dog, and that was determined on the morning of the oxidation study day following an overnight fast of 22 h from the last feeding. All dogs consumed their entire daily diet offerings throughout the study, negating the need to collect and measure any food refusals. Table 2. Feeding, isotope dosing, and sampling schedule on the day of each direct AA oxidation study. Symbols denote that dogs received that procedure during that time period   Fasting state  Fed state  Event  0  25  50  −95  −85  −75  −50  −25  0  25  50  75  100  125  150  175  200        Time, min    Food1        ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦    Isotope      Background phase      Isotope steady state phase    L-[1-13C]Phe2                ♣  ♣  ♣  ♣  ♣  ♣  ♣  ♣      Samples  Calorimetry3  ♠  ♠  ♠      ♠  ♠                      Breath4            4  4  4  4  4  4  4  4  4  4  4      Fasting state  Fed state  Event  0  25  50  −95  −85  −75  −50  −25  0  25  50  75  100  125  150  175  200        Time, min    Food1        ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦    Isotope      Background phase      Isotope steady state phase    L-[1-13C]Phe2                ♣  ♣  ♣  ♣  ♣  ♣  ♣  ♣      Samples  Calorimetry3  ♠  ♠  ♠      ♠  ♠                      Breath4            4  4  4  4  4  4  4  4  4  4  4    1The food offered to each dog consisted of a Phe-deficient basal diet top-dressed with 1 of 7 Phe-containing solutions to deliver total dietary Phe concentrations of 0.43, 0.50, 0.55, 0.61, 0.67, and 0.73% (as-fed basis). Each meal was isocaloric and isonitrogenous and represented one-thirteenth of each dog's daily food intake. 2Isotope: Priming dose of L[1-13C] phenylalanine was started with the sixth meal followed by continuous L[1-13C] phenylalanine dosing through the remaining 200 min of the study. Both priming and continuous isotope dosing solutions were top-dressed on the dry kibble meal. 3Three 25-min measures of respiratory gases were obtained prior to feeding to calculate resting volume of CO2 produced (VCO2). Starting at −75 min, VCO2 was measured in 25 min intervals for the duration of the study. 4Three background breath samples were obtained at −50, −25 and 0 min (fed state) before the isotope dosing. These samples represented total breath collection from −75 to −50, −50 to −25, and −25 to 0 min. Breath samples were then collected every 25 min for the duration of the study. View Large Table 2. Feeding, isotope dosing, and sampling schedule on the day of each direct AA oxidation study. Symbols denote that dogs received that procedure during that time period   Fasting state  Fed state  Event  0  25  50  −95  −85  −75  −50  −25  0  25  50  75  100  125  150  175  200        Time, min    Food1        ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦    Isotope      Background phase      Isotope steady state phase    L-[1-13C]Phe2                ♣  ♣  ♣  ♣  ♣  ♣  ♣  ♣      Samples  Calorimetry3  ♠  ♠  ♠      ♠  ♠                      Breath4            4  4  4  4  4  4  4  4  4  4  4      Fasting state  Fed state  Event  0  25  50  −95  −85  −75  −50  −25  0  25  50  75  100  125  150  175  200        Time, min    Food1        ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦  ♦    Isotope      Background phase      Isotope steady state phase    L-[1-13C]Phe2                ♣  ♣  ♣  ♣  ♣  ♣  ♣  ♣      Samples  Calorimetry3  ♠  ♠  ♠      ♠  ♠                      Breath4            4  4  4  4  4  4  4  4  4  4  4    1The food offered to each dog consisted of a Phe-deficient basal diet top-dressed with 1 of 7 Phe-containing solutions to deliver total dietary Phe concentrations of 0.43, 0.50, 0.55, 0.61, 0.67, and 0.73% (as-fed basis). Each meal was isocaloric and isonitrogenous and represented one-thirteenth of each dog's daily food intake. 2Isotope: Priming dose of L[1-13C] phenylalanine was started with the sixth meal followed by continuous L[1-13C] phenylalanine dosing through the remaining 200 min of the study. Both priming and continuous isotope dosing solutions were top-dressed on the dry kibble meal. 3Three 25-min measures of respiratory gases were obtained prior to feeding to calculate resting volume of CO2 produced (VCO2). Starting at −75 min, VCO2 was measured in 25 min intervals for the duration of the study. 4Three background breath samples were obtained at −50, −25 and 0 min (fed state) before the isotope dosing. These samples represented total breath collection from −75 to −50, −50 to −25, and −25 to 0 min. Breath samples were then collected every 25 min for the duration of the study. View Large Tracer Delivery and Breath Collections. After dogs entered the respiratory chambers, a 30-min gas equilibration period commenced before 3 fasting respiration calorimetry measurements were taken to establish resting VCO2 for each dog. Calorimetry data were collected automatically using Qubit calorimetry software (Customized Gas Exchange System and Software for Animal Respirometry; Qubit Systems Inc.). Each dog was then fed 3 of its individual meals in 10-min increments during the initial 30-min period and then fed every 25 min thereafter, with its final meal fed 25 min prior to the collection of the last breath measurement at 200 min. Background 13CO2 expiration was determined during 3 consecutive 25-min periods prior to dogs receiving their priming bolus of L-[1-13]Phe. The priming tracer dose was provided with their next meal corresponding to time 0. The remaining 6 meals contained the constant dose of tracer. The tracer, L-[1-13]Phe (99%; Cambridge Isotope Laboratories, Inc.), was accurately weighed and added to a premeasured amount of deionized water on a daily basis. Expired CO2 was collected over eight 25-min periods from 0 to 200 min. Although determination of Phe flux is warranted to correct for tracer dilution, we wanted to ascertain whether we could obtain an isoptic steady state in breath only and avoid using repeated blood samples. Analytical Procedures Concentrations of 13CO2 and 12CO2 in Breath Samples. Breath samples were collected as described above and kept at room temperature until analysis. The enrichment of 13C in breath CO2 was measured by continuous-flow isotope ratio mass spectrometry (20/20 isotope analyzer; PDZ Europa Ltd., Cheshire, UK). All samples were analyzed in triplicate. Breath samples collected during the background period and those collected during the isotope enrichment period were expressed in APE 13CO2 over a reference standard of compressed CO2 gas. Concentrations of Dietary CP and AA. Dietary nitrogen was analyzed and CP was calculated with a LECO analyzer (LECO Corporation, St. Joseph, MO), and dietary AA were measured using AOAC method 999.12 (AOAC International, 2000). Calculations. The rates of bicarbonate recovery and phenylalanine oxidation, where F13CO2 represents the rate of 13CO2 released by bicarbonate or phenylalanine tracer oxidation [(μmol 13CO2/(kg × h)], were calculated using the following equation:  in which F13CO2 is the CO2 production rate (mL/min), ECO2 is the 13CO2 enrichment in expired breath at isotopic steady state (APE), and BW is the weight of the dog (kg). The constant 44.6 μmol/mL converts the F13CO2 to micromoles per hour, and the factor 100 changes APE to a fraction. The factor 1.0 accounts for the fact that no CO2 is retained in the body due to bicarbonate fixation. Statistical Analyses Statistical analyses for CO2 recovery from the chambers and bicarbonate recovery from the dogs were conducted using GraphPad Prism version 3.00 for Windows (GraphPad Software, Inc., San Diego, CA). A 2-tailed, paired t test was used to compare CO2 recovery between chambers. If values were not different between chambers, then data were pooled and descriptive statistics were applied. For CO2 recovery and bicarbonate recovery, pooled data were assessed for normality and then tested for differences from 100% using the Wilcoxon signed rank test. Descriptive statistics were also used to describe bicarbonate retention values. A mixed linear model with dog as a random variable was used to analyze the effect of Phe intakes on APE and F13CO2 using PROC MIXED (SAS/STAT version 9.3; SAS Inst. Inc., Cary, NC). The estimate of the mean Phe requirement was derived by breakpoint analysis of the F13CO2 using a 2-phase linear regression model. This model selects for the minimum residual SE in a stepwise partitioning of data points between 2 regression lines. The first regression line has a slope and the second line is horizontal with minimal or no slope. The mixed model procedure accounts for correlations among observations made on the same dog and for possible heterogeneous variances among measurements made on the same dog over time. It is well suited for repeated measures study designs (Wang and Goonewardene, 2004). Different models containing 2 to 5 dietary intakes on the descending line were compared using the information criteria (Akaike information criteria, the finite sample-corrected Akaike criteria, and the Schwarz's Bayesian information criteria). The breakpoint estimate corresponding to the dietary Phe requirement was identified based on the final model that best fit the data to provide the lowest SE, lowest root mean square error, and the highest r2. The upper and lower 95% confidence intervals (CI) were calculated using Fieller's theorem (Seber, 1977). Briefly, 95% CI = breakpoint ± tdf, α/2 × SEM, in which SEM is the SEM of the breakpoint, df is associated with the residual mean square of the best fit model, and α is the 95% confidence level. The safe intake of Phe corresponds to the upper 95% CI, which is similar to the recommended allowance (NRC, 2006). Body weight was analyzed using a regression analysis in PROC MIXED of SAS (version 9.3; SAS Inst., Inc.) as a completely randomized study design with each dog receiving every dietary treatment in random order. The model was Yij = Wi + εij, in which Yij is the dependent variable, Wi is the dietary treatment (1, 2, 3, 4, 5, 6, and 7), and εij is the random residual error. Diet was considered a fixed effect and dog was considered a random effect. Treatment least squares means were compared using the pdiff multiple comparison procedure. Data are expressed as least squares means with a pooled SEM of the model. The results were considered statistically significant at P < 0.05, and no multiple comparison adjustments were made on the P-value. The covariance matrix having the smallest Akaike's information criterion value was chosen as the final model. RESULTS Objective 1: CO2 Recovery Quantified from a Controlled CO2 Release into Chambers to Determine if the Chambers Allowed for Complete CO2 Recovery Carbon dioxide recovery was determined by analyzing the area under the curve for VCO2 and comparing it with the volume of CO2 released into the empty chambers. The mean percentage weight of CO2 recovered from the chambers averaged 95 (SEM 8.9; n = 5) and 116 (SEM 9.4; n = 4) for chambers A and B, respectively. The CO2 recovery from chambers did not differ from each other (P > 0.05) or from 100% (P > 0.05). Based on this level of variation in CO2 recovery, a sample size of 39 individual CO2 releases would be required to detect differences of 5% at a power of 0.90 based on CO2 released as gas and not liberated from solution as Tabiri et al. (2002) demonstrated. However, this approach decreases the chance of Type II error but not Type I error. Because chambers did not differ, data were pooled to obtain the total mean percent CO2 recovery, which was 104.6 (SEM 7.1). This value was not different from 100%. These data demonstrate that these oxidation chambers are adequate for the complete recovery of 13CO2. Objective 2: Bicarbonate Retention When the bicarbonate was orally supplied with each 30-min meal, it resulted in a mean bicarbonate recovery of 102.5% (SEM 2.6). In addition, the percentage recovery of 13CO2 was not different from 100% as assessed by a Wilcoxon signed rank test. Based on this level of variation, a sample size of 7 dogs would be required to detect differences of greater than 5% bicarbonate retention between dogs at a power of 0.90. Subsequent direct and indirect AA oxidation studies will not use a correction factor, because these data indicate that no CO2 is retained by the fasting or fed dog. Leijssen and Elia (1996) reported that loss of labeled bicarbonate from syringes and tubing can range from 42 to 57% per day and that bicarbonate studies require bedside preparation of the bicarbonate solution. A loss of tracer causes a dilution of tracer and an overestimation of bicarbonate retention. As such, fresh bicarbonate solutions were mixed throughout the day and used within 1 h of preparation. Objective 3: Determination of the Phenylalanine Requirement of Adult Dogs using the Direct Oxidation Method Mean BW and VCO2 were not different (P > 0.05) throughout the Phe requirement study period, and there were no differences among dietary Phe intakes. All dogs reached isotopic steady state in APE and 13CO2 at every Phe intake. The oxidation of L-[1-13]Phe (F13CO2) was different between dietary Phe intakes (P < 0.05). The rate of acceptance of 13CO2 in the breath (F13CO2) remained low and constant from 0.435 to 0.55% of the diet (AFB) and then increased linearly. Two-phase linear regression crossover analysis of F13CO2 identified the breakpoint for the mean Phe requirement as 0.535% of diet (as-fed basis), with an upper 95% CI of 0.645% of diet (as-fed basis; Fig. 2). Figure 2. View largeDownload slide Effect of Phe intake on rate of production of 13CO2 from orally administered L-[1-13C] Phe in adult female dogs oxidation (F13CO2). All observations (n = 35) and all subjects (n = 5) are shown. The fitted breakpoint estimates the mean and upper estimates based on the use of a 2-phase linear crossoer regression crossover analyses to minimize the total sum of squares in error for the combined lines. The breakpoint of the 2-phase linear model represents a Phe requirement of 0.535 to 0.645 % (mean and upper 95% CI on an as fed basis) in the presence of excess Tyr. Figure 2. View largeDownload slide Effect of Phe intake on rate of production of 13CO2 from orally administered L-[1-13C] Phe in adult female dogs oxidation (F13CO2). All observations (n = 35) and all subjects (n = 5) are shown. The fitted breakpoint estimates the mean and upper estimates based on the use of a 2-phase linear crossoer regression crossover analyses to minimize the total sum of squares in error for the combined lines. The breakpoint of the 2-phase linear model represents a Phe requirement of 0.535 to 0.645 % (mean and upper 95% CI on an as fed basis) in the presence of excess Tyr. DISCUSSION The objective of the present series of experiments was to establish key parameters needed to use carbon oxidation methods for the determination of indispensable AA in dogs. As discussed in Tabiri et al. (2002), calibration and verification of breath collection parameters are required for the specific methodology and equipment to be used in future species-specific studies. At the flow rate of 30 L/min, we found 100% recovery of CO2 released into chambers, demonstrating complete collection of respiratory gases. Second, the adult mixed-breed hounds used in this study did not retain CO2, and this suggests there is no need for a CO2 retention correction factor when calculating F13CO2 from oral delivery of isotope. Finally, this study established the mean Phe requirement as 5.35 g/kg diet (as-fed basis) with an upper 95% CI of 6.45 g/kg (as-fed basis) diet when determined in the presence of excess Tyr and with a dietary energy density of 3.73 Mcal ME/kg DM. As such, future carbon oxidation studies using Phe as the isotopic tracer must provide dietary Phe at a concentration that meets or exceeds this level. The present results apply to adult mixed-breed hounds being fed a highly bioavailable diet and fed to energy balance and in a controlled experimental situation. Indeed, differences in any of these variables may result in a different estimate of the Phe requirement. Previously, we demonstrated that oral isotope dosing was a valid alternative to intravenous infusion of stable isotopes (Gooding et al., 2013). Furthermore, we wanted to avoid use of intravenous tracer infusions in subsequent AA studies for ethical reasons. As such, we used the less invasive approach of oral isotope dosing and repeated meal feedings to measure bicarbonate retention. The mean 102.5% (SEM 2.6) bicarbonate recovery from the dogs calculated in the present study represents the bicarbonate retention associated with intermittent feeding and isotope dosing (every 25 min) and the achievement of steady state 13CO2 conditions. Steady state assumes an equal rate of appearance from digestive absorption and disappearance into body cells (Tomera et al., 1982; Wolfe, 1992). Each dog achieved steady state with this regimen, indicating the success of this protocol. In their review of human bicarbonate studies, Leijssen and Elia (1996) reported larger variability in bicarbonate recovery estimates (50 to 100%) with continuous infusion studies compared with bolus studies. Similarly, greater bicarbonate recoveries were reported for humans in the fed state compared with those in a fasted state, and there was a large loss of bicarbonate through a number of vectors related to sample handling. For comparison with the present data in dogs, Pouteau et al. (2002) reported a bicarbonate recovery rate of 71.8% (SEM 3.1) in adult Beagles following an overnight fast and using intravenous tracer delivery with a primed constant infusion. Similarly, Downey et al. (1986) reported a bicarbonate recovery rate of 79% (SD 7) determined in fasting, anesthetized mongrel dogs using intravenous and arterial tracer delivery following a primed constant infusion. In a pilot study, Larsson et al. (2010) validated oral NaH13CO3 administration and observed bicarbonate recovery rates similar to those of intravenous administration. Larsson et al. (2014b) reported a bicarbonate recovery factor of 74% for adult large-breed dogs after an overnight fast and an oral bolus of bicarbonate tracer but without a repeated meal feeding and tracer dosing regimen. Furthermore, Larsson et al. (2014a) reported bicarbonate recovery factors of 72 and 94% for a group of calm and active dogs, respectively, when measured after an overnight fast. Our rates of recovery are greater than other reported estimates, but none of these previous estimates were conducted in the fed state and used an adapted prime constant approach to 13CO2 measurements, both of which are known to result in greater estimates of bicarbonate recovery. With the exception of Larsson et al. (2010, 2014a,b), no other studies provided oral bicarbonate tracer that resulted in greater 13CO2 recovery (Kriengsinyos et al., 2002). However, Larsson et al. (2010, 2014a,b) used a bolus delivery of the isotope, and this will also result in lower recovery estimates. Downey et al. (1986) is the only reference to comment on the need for the careful preparation of tracer solutions to minimize losses. Bicarbonate losses in the infusate result in lower bicarbonate retention estimates, because tracer enrichment is overestimated. Our estimate is similar to that reported in Wolfe (1984), but we could not find these results reported in a peer-reviewed manuscript. Our bicarbonate recovery estimate agrees with Wolfe (1984) but is greater than those of Downey et al. (1986), Larsson et al. (2010), Larsson et al. (2014a), and Pouteau et al. (2002) for reasons explained. Our future AA oxidation studies will not use a bicarbonate recovery factor based on these results. The use of stable isotopes represents an appealing, minimally invasive research tool to study whole-body metabolism, but further studies are warranted to more fully understand factors affecting bicarbonate recovery in dogs. The present study estimated the Phe requirement of adult, mixed-breed hounds as a mean of 0.535% (95% CI 0.645) on an as-fed basis in a diet matrix containing 8% moisture, excess Tyr, and an energy density of 3.73 Mcal/kg DM. There are relatively few studies reporting data on Phe requirements in dogs. To our knowledge, the only measurement of the Phe requirement has been reported by Milner et al. (1984), in which immature dogs were reported to require a diet containing 0.8% Phe (DM basis) to provide the total aromatic AA requirement (i.e., no Tyr present), with Tyr sparing 46% of that requirement (Milner et al., 1984). Their estimate of the requirement represents the mean requirement defined as the intersect of the broken line model, which corresponds to our estimate of the mean requirement and not the upper 95% CI. Taken together, the present estimate of the Phe requirement (mean), in the presence of excess Tyr, is 0.59% of diet (if expressed on DM basis), and that reported by Milner et al. (1984) equates to 0.432% diet when accounting for the contribution of Tyr. In contrast to the present study, Milner et al. (1984) used the nitrogen balance method and immature Beagles (2–6 wk after weaning) fed test diets comprising an agar gel containing 50% water and 50% DM. Each test diet was fed for 15 d, with urine and fecal collections on the last 5 d for nitrogen analyses. In general, carbon oxidation methods use a shorter dietary adaptation period, resulting in a more accurate estimate of the requirement in healthy adult animals and humans (Turner et al., 2006). Studies using carbon oxidation methods have found that the minimum rates of oxidative losses of indispensable AA are equal to, or considerably higher than, the estimates of upper range of AA requirements when determined using nitrogen balance and growth methods (Young et al., 1989). In addition, because we did not measure Phe flux, we cannot calculate absolute oxidation of phenylalanine and account for isotope dilution (Zello et al., 1990), and this may result in an overprediction of the requirement; however, blood sampling is a procedure that we wanted to avoid due to welfare considerations. Of greatest importance is that with this protocol of dosing and repeated small meals, we attained isotopic steady state in F13CO2 in all dogs and at all diet levels. Furthermore, it is generally expected that growing animals have greater indispensable AA requirements than adult animals due to the need for AA to support both maintenance and growth. As such, the use of nitrogen balance for adults at maintenance is not a favorable method due to the depletion of protein reserves at zero balance, which yields results that underestimate requirements. Nitrogen balance is a useful method when used to determine minimum requirements, but more accurate measurements are needed in which healthy body condition is maintained (Morris and Rogers, 1994; Waterlow, 1994; Pencharz and Ball, 2003). Finally, diets that are more elemental in their ingredient composition result in lower requirement estimates due to greater gastrointestinal digestibility and AA bioavailability compared with diets containing intact proteins. The NRC (2006) suggests a Phe recommended allowance of 0.45% of diet (DM basis). Our estimated 95% CI is 0.59% of diet (DM basis) and in greater than the recommended allowance suggested by the NRC that further assumes poorer protein and AA availability, although it is based on a dietary energy density of 4,000 kcal/kg diet. More studies are needed to fully understand the sparing effect of Tyr on the Phe requirement and to examine the AA requirements of growing puppies to provide a more direct comparison with Milner et al. (1984). In conclusion, the carbon recovery from the chambers and CO2 recovery results reported herein demonstrate that we can confidently move forward with this minimally invasive research capability that uses carbon oxidation methods to measure indispensable AA requirements of adult dogs. Our future studies will use orally delivered stable isotopes, no bicarbonate retention factors, a dietary Phe concentration that meets or exceeds the upper 95% CI or this population, and excess dietary Tyr to ensure Phe is shunted directly to oxidation. Finally, our estimate of the Phe requirement is greater than current recommendations and regulatory guidance for pet food manufacturing and deserves further investigation. LITERATURE CITED AOAC International 2000. Official methods of analysis. 16th ed. 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A.K.S. and G.M.D. have financial interest in Procter & Gamble due to previous employment. A.K.S. and G.M.D. also were employees of Mars Pet Care. American Society of Animal Science
TI - 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,,
JF - Journal of Animal Science
DO - 10.2527/jas.2017.1535
DA - 2017-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/calibration-and-validation-of-a-carbon-oxidation-system-and-AJ3k0hQQgN
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EP - 2927
VL - 95
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DP - DeepDyve
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