Determination of a steady-state isotope dilution protocol for carbon oxidation studies in the domestic cat

Abstract

The present study aimed to develop an isotope protocol to achieve equilibrium of ¹³CO₂ in breath of cats during carbon oxidation studies using L-[1-¹³C]-Phenylalanine (L-[1-¹³C]-Phe), provided orally in repeated meals. One adult male cat was used in two experiments. In each experiment, three isotope protocols were tested in triplicate using the same cat. During carbon oxidation study days, the cat was offered thirteen small meals to achieve and maintain a physiological fed state. In experiment 1, the isotope protocols tested (A, B and C) had a similar priming dose of NaH¹³CO₃ (0⋅176 mg/kg; offered in meal 6), but different priming [4⋅8 mg/kg (A) or 9⋅4 mg/kg (B and C); provided in meal 6] and constant [1⋅04 mg/kg (A and B) or 2⋅4 mg/kg (C); offered in meals 6-13] doses of L-[1-¹³C]-Phe. In experiment 2, the isotope protocols tested (D, E and F) had similar priming (4⋅8 mg/kg; provided in meal 5) and constant (1⋅04 mg/kg; provided in meals 5-13) doses of L-[1-¹³C]-Phe, but increasing priming doses of NaH¹³CO₃ (D: 0⋅264, E: 0⋅352, F: 0⋅44 mg/kg; provided in meal 4). Breath samples were collected using respiration chambers (25-min intervals) and CO₂ trapping to determine ¹³CO₂:¹²CO₂. Isotopic steady state was defined as the enrichment of ¹³CO₂, above background samples, remaining constant in at least the last three samples. Treatment F resulted in the earliest achievement of ¹³CO₂ steady state in the cat's breath. This feeding and isotope protocol can be used in future studies aiming to study amino acid metabolism in cats.

Keywords: Amino acid metabolism; Amino acid oxidation technique; Bicarbonate metabolism; Carnivore.

Fig. 1.

Feeding, isotope and sample protocol (treatment F in pilot trial 2) proposed to be utilised in IAAO studies in cats. ^aEach meal represented one-thirteenth of half of the daily food intake for the cat. ^bPriming dose of NaH¹³CO₃ was top-dressed on the fourth meal (time: 45 min). ^cPriming dose of L-[1-¹³C]-Phenylalanine (L-[1-¹³C]-Phe) was top-dressed on the fifth meal. The continuous dose of L-[1-¹³C]-Phe started on the fifth meal with the priming dose, followed by continuous supply through the remaining meals. ^dIC: indirect calorimetry. Three 25-min measures of respiratory gases were obtained prior to feeding to obtain the resting volume of CO₂ produced (VCO₂). Starting at 45 min, VCO₂ was measured in 25-min intervals for the duration of the study. ^eThree 25-min breath samples collection for ¹³CO₂ background were obtained at −50, −25 and 0 min (fasted state) before food and isotope provision. One breath sample was collected at time −45 min before isotope provision for determination of ¹³CO₂ background during fed state. Breath samples were then collected every 25 min for the duration of the study.

Fig. 2.

Pilot trial 1: (a) visual inspection (values are x̄ ± sd) and (b) fitted broken-line linear model for ¹³CO₂ expressed as atoms percent excess (APE) as a function of meal (25-min intervals). Isotope was provided orally over small meals. The priming dose (0⋅176 mg/kg) of NaH¹³CO₃ remained similar among treatments (Trt), while the priming and constant doses of L-[1-¹³C]-Phe varied as follows. Trt A: priming dose: 4⋅8 mg/kg; constant dose: 1⋅04 mg/kg. Trt B: priming dose: 9⋅4 mg/kg; constant dose: 1⋅04 mg/kg. Trt C: priming dose: 9⋅4 mg/kg; constant dose: 2⋅4 mg/kg.

Fig. 3.

Pilot trial 1: (a) visual inspection (values are x̄ ± sd) and (b) fitted broken-line linear (purple) or broken-line quadratic (blue) model for ¹³CO₂ expressed as atoms percent excess (APE) as a function of meal (25-min intervals). Isotope was provided orally over small meals. The priming (4⋅8 mg/kg) and constant (1⋅04 mg/kg) doses of L-[1-¹³C]-Phe remained similar among treatments (Trt). The priming dose of NaH¹³CO₃ varied across Trt D (0⋅264 mg/kg), Trt E (0⋅352 mg/kg) and Trt F (0⋅44 mg/kg).

Fig. 4.

Major metabolic fates of ¹³CO₂ derived from oxidation of L-[1-¹³C]-phenylalanine.