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*
Waltham Centre for Pet Nutrition, Waltham on the Wolds, Melton Mowbray, Leics LE14 4RT, UK and the
Department of Clinical Biochemistry, University of Cambridge, Addenbrookes Hospital, Cambridge2 2QR, UK
2To whom correspondence should be addressed. E-mail: kim.russell{at}eu.effem.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • indirect calorimetry • fat oxidation • amino acid catabolism • carnivore • cats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Such flexibility in oxidation has been demonstrated in humans and mice (2
–5
) and enables a variety of species to adopt widely differing dietary strategies to satisfy their energy requirements (6
).
Cats have a high requirement for dietary protein compared with omnivores and herbivores (7
), and this has been attributed to a lack of metabolic flexibility (8
). A classic study in vitro indicated that feline hepatic enzymes involved in amino acid catabolism were permanently set to a high level and could not be down-regulated to accommodate lower protein diets (8
). Although subsequent work challenged these conclusions (9
–11
), support for Rogers et al. (8
) has come from a more recent study in vivo, which reported that feline protein oxidation did not respond to manipulation of dietary protein intake (12
). If cats cannot regulate protein oxidation, this would suggest that, unlike other species, the fuel mix oxidized would not adapt to the dietary macronutrient profile. Such a situation could provide an evolutionary disadvantage in times of variation in food supply, when body proteins would be rapidly depleted on lower protein diets. However, if protein oxidation in cats is regulated, an alternative hypothesis for the high protein requirement of this species may be required.
The objective of the current study was to determine whether protein oxidation in cats does fully respond in vivo to changes in dietary protein level. Cats were studied after adaptation to two levels of dietary protein intake. Measurement of protein oxidation in cats is not straightforward. Protein oxidation is normally estimated from urinary nitrogen excretion (6
,12
) or using tracers, for example the exhalation of [13C] from a labeled amino acid on the breath (13
,14
). However, these techniques for measuring protein oxidation have their limitations (15
,16
). The use of urinary nitrogen depends on complete collection that is often difficult in animals and actually reflects the amount of deaminated amino acids. It is not possible to distinguish between urinary nitrogen arising from oxidation and other pathways, and as such, this method is susceptible to changes in nitrogen flux, for example, variation in urea pool size. Tracer techniques require assumptions to be made about equilibration of the label within the body pool, the contribution of the single amino acid to overall amino acid oxidation, and retention of label in the body.
In this study, protein oxidation was assessed from measurements of respiratory gas exchange by whole-body indirect calorimetry. The oxidation of macronutrients results in the consumption of oxygen and the production of carbon dioxide. Each macronutrient has a characteristic energy release per liter of O2 consumed and a characteristic ratio of CO2 production to O2 consumption, the respiratory exchange ratio (RER)3
. Thus, from respiratory O2 and CO2 exchange measurements, the oxidation rates of any two macronutrients can be derived if the remaining macronutrient oxidation rates are independently measured or are zero. Most commonly, fat and carbohydrate oxidation are estimated from respiratory gas exchange (5
,12
,16
). For this study, expressions were derived for protein and fat oxidation while carbohydrate oxidation was minimized by maintaining dietary carbohydrate at a small (<1%) and constant proportion of food intake.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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Six adult domestic short-hair cats (WALTHAM center for Pet Nutrition, Melton Mowbray, UK) were studied. Three were females and three males, all neutered, mean age, 4.0 ± 0.35 y and mean body weight, 5.0 ± 0.45 kg.
Two diets, of moderate (MP) and high protein (HP) content, were fed in a crossover design, each phase lasting 50 d. After a 14-d adaptation to the allocated diet, six replicate 18-h indirect calorimetry measurements were completed for each cat for each diet. Cats were measured in rotation. Calorimetry measurements commenced at 1500 h and a 12-hour light-dark cycle was maintained with lights turned on at 0700 h. Part of phase 1 was repeated (after another 14-d adaptation) at the end of the trial due to earlier technical difficulties, and data from this were substituted for three of the original calorimeter measurements. One measurement was lost (MP diet) due to technical difficulty.
During periods of food intake data collection, cats were housed individually in purpose built, environmentally enriched accommodation. Food was offered continuously and renewed twice daily. When not scheduled for data collection, the cats were group housed and socialized, being returned to individual housing for feeding, which occurred for two generous 1-h meals each day (i.e., cats were fed to appetite). For the 30 h before and during each calorimetry measurement, the cats were given continuous access to the allocated diet. Water was freely available at all times. Body weight was measured once weekly and additionally on entry to and exit from the calorimeter. The WALTHAM ethical review committee approved the study.
Diet details.
Two minimal carbohydrate, canned diets were used (Pedigree Masterfoods, Melton Mowbray, UK): a MP diet and an HP diet. The MP diet was diluted immediately before feeding by the addition of water (204 g/kg) to make the two diets near equally energy-dense when fed (Table 2)
. Food intake was assessed gravimetrically (TS2KV balance; Ohaus UK Ltd., Leicester, UK).
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Respiratory gas exchange measurements were made by whole-body indirect calorimetry. The calorimeter consisted of a chamber 1.0 x 0.6 x 0.6 m. One side of the chamber was a Plexiglass door, sealed when closed by compressible foam rubber. Before the study, the cats were familiarized with the chamber. Food, water, a bed and a litter tray containing sawdust were provided within the chamber. Room temperature was maintained at 19°C.
The calorimeter was of open circuit, flow-through ventilated design, with room air drawn through the chamber at 10 L/min. The flow rate was measured with a Hastings mass flowmeter (Teledyne Hastings Instruments, Los Angeles, CA). Samples of ingoing and chamber air were dried by columns of calcium chloride and analyzed for O2 and CO2 by a Xentra 4100 gas purity analyser (Servomex International Ltd., Crowborough, UK). The composition of ingoing air was measured every 30 min, and chamber air every 15 s then averaged for each minute. Data from the mass flow meter and gas analysers were digitized by a multi-channel digital multi-meter type 34970A (Agilent Technolgies, Palo Alto, CA) and logged by a PC using bespoke software written in LabView (National Instruments, Austin, TX). Calibration was effected before each test and checked at hourly intervals throughout, using 100% N2 to set or record the analyzer zero readings and 20.95% O2 + 1% CO2 in N2 for the analyzer spans. The calorimeter was validated before the study by determination of apparent O2 consumption during constant, measured infusion of 100% N2, and by O2 and CO2 production during infusion of 20% CO2 in N2 (all gasses by Air Products PLC, Crewe, UK). Expected recoveries were calculated as described previously (17
) and observed values were within ± 2.5% of expectation. These tests were repeated regularly throughout the study. The combustion of 99.7–100% ethanol (Sigma Aldrich Co. Ltd., Gillingham, UK) showed that the measurement of RER, 0.68 ± 0.01 (n = 8), was close to but consistently higher than the expected value of 0.67 (16
). This difference was attributed to analysis tolerances in the calibration gas mixtures, and appropriate correction was applied to CO2 production for all results.
Calculation of substrate oxidation from gas exchanges.
Oxygen consumption and carbon dioxide production were calculated using the expressions derived previously for a pull calorimeter (18
).
Oxidation rates of protein and fat were calculated from the gas exchanges using a model derived in a similar way to that published for a two-substrate mixture by Elia and Livesey (16
). The contribution due to the oxidation of dietary carbohydrate was accounted for as a constant 0.83 (MP) or 0.85 (HP) percentage of the diet as fed. Other constants adopted in the derivation were as follows:
RER of fat oxidation = 0.71
RER of protein oxidation = 0.835
Consumption of O2 by fat oxidation = 2.01 L/g
Consumption of O2 by protein oxidation = 0.952 L/g
Energy produced by fat oxidation = 39.33 kJ/g
Energy produced by protein oxidation = 18.56 kJ/g
The resulting expressions were:
Protein oxidation (g) = -5.966 x VO2 + 8.403 x VCO2 - 1.818 x CHO
and
Fat oxidation (g) = 3.323 x VO2 -3.979 x VCO2 + 0.490 x CHO,
where VO2 and VCO2 are the standard temperature and pressure volumes of O2 consumed and CO2 produced during an analysis period, and CHO is the mass of carbohydrate (g), expressed as monosaccharide, oxidized in the period.
The sensitivity of the expression for protein oxidation to uncertainty in carbohydrate oxidation was examined by analysis of errors. For a carbohydrate intake of 2 g, variability in CHO oxidation with a CV of 30% of intake and SD of 0.6 g would result in uncertainty in protein oxidation of 1.2 g. However, the carbohydrate content of the diets was assessed by difference and not directly analyzed, and this may introduce some uncertainty in calculating carbohydrate intake.
The data were assessed for normality and homogeneity of variance. Those that could be considered approximately normally distributed and homogenous were analyzed by multifactor ANOVA (with cat as the second factor), the remainder analyzed by Kruskal-Wallis. In all cases, P < 0.05 was considered significant. Data are expressed as means ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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Food intake during the 18-h calorimetry period did not produce any difference in energy intake (Table 1
; ANOVA, P = 0.96). The cats were in slightly negative energy balance throughout each test, although this was similar for both diets (Table 1)
. Losses of body weight were -1.8 ± 0.3% (MP) and -1.7 ± 0.3% (HP), which in absolute terms were equivalent to -84.1 ± 10.6 g (MP) and -82.5 ± 12.2 g (HP). The majority of cats urinated immediately upon entry to the chamber, which may account for some weight lost while in the chamber.
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), more protein (ANOVA, P < 0.001) and less fat (Kruskal-Wallis, P < 0.001) were eaten during the 18-h calorimetry period for the HP diet than for the MP diet. Carbohydrate intakes from both diets were very low, although higher for the HP diet (ANOVA, P < 0.001; Table 3
).
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).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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,8
). Protein oxidation is a term used to describe the release of energy from the carbon skeleton of amino acids after deamination. Deamination is effected by amino acid catabolic enzymes, and decreasing the activities of these enzymes is an important mechanism to conserve nitrogen when low protein diets are fed. Similarly, enzyme activity is increased when HP diets are fed to prevent adverse effects of toxic concentrations of certain amino acids. Changes in the activity of enzymes may occur at several levels, from alterations in substrate availability, allosteric regulation and altered phosphorylation-state to changes in gene expression resulting in altered amounts of enzyme protein.
The high protein requirement of cats has been attributed to the apparent inability of the hepatic ureagenic, gluconeogenic and catabolic enzymes of this species to adapt to dietary protein intake (7
,8
). In their classic study in vitro, Rogers et al. (8
) assessed the activity of several amino acid catabolic enzymes in liver biopsies taken from cats fed either low or high protein diets. The authors concluded that the hepatic catabolic enzymes of cats seemed to be permanently set to a very high level and failed to adapt to low dietary protein as in other species (19
–23
). However, it should be noted that the enzyme assays used in this study were those optimized for rats, without validation for cats, and this could possibly have affected the estimation of absolute activities. In addition, tests of enzyme activity in vitro reflect maximal activity and may not reflect activity under physiological conditions.
Further support for the hypothesis was provided by a study in vivo that reported no change in protein oxidation in cats fed diets differing in protein energy (12
). However, in this study fat and carbohydrate oxidation were measured by indirect calorimetry, while protein oxidation was calculated indirectly from urinary nitrogen excretion, and so may reflect nitrogen flux rather than oxidation of the carbon skeleton (15
,16
). Furthermore, the energy value ascribed to urinary nitrogen is also affected by differences in nitrogen partitioning within the urine (16
), a possible outcome of alterations in dietary protein intake.
However, the observations above (8
,12
) were not supported by other work in vitro (9
–11
), and the current study supports these findings, with increased protein oxidation in cats fed a high protein diet. If feline hepatic enzymes do not genetically adapt to dietary protein level (8
) and are set to an intermediate or high level, other mechanisms may be more important for nitrogen conservation by cats. Thus, catabolic enzyme activity may be controlled largely by substrate supply in cats. If this is viewed in terms of reactive control (substrate supply) and adaptive control (enzyme adaptation), then it may be that cats react, rather than adapt, to dietary protein levels, but with the same net result.
Increased protein oxidation in response to increased protein intake would be expected, because there is no capacity in the body to store amino acids above those required for protein synthesis, as demonstrated in rats and humans (24
,25
). Protein oxidation would also be expected to decrease during fasting, to limit the use of body protein stores for energy, confirmed in mink (13
) and humans (14
).
The use of indirect calorimetry for the assessment of oxidation measures net oxidation (substrate disappearance) irrespective of pathway. This means that protein oxidation, as measured by indirect calorimetry, will include not only straightforward amino acid oxidation, but also protein that undergoes gluconeogenesis and is subsequently oxidized as glucose. Similarly, net fat oxidation not only includes direct oxidation, but also oxidation of ketone bodies and oxidation of glucose formed from glycerol by gluconeogenesis. This is particularly relevant for this study, which incorporated very low levels of dietary carbohydrate. The cats would have been operating a high level of gluconeogenesis to maintain blood glucose (26
), and this glucose oxidation would be included in the estimation of both fat and protein oxidation, accurately attributed to its source substrate. However, the absolute requirement for glucose would be similar when cats were fed the two diets studied (26
). This methodology also does not distinguish between hepatic amino acid catabolism and catabolism in other tissues, for example, branched chain amino acid catabolism in skeletal muscle.
The greatest potential for error in estimation of protein oxidation by indirect calorimetry arises from the assumption that carbohydrate oxidation is in close equilibrium with the dietary intake. The sensitivity of protein oxidation measurement to diet-derived carbohydrate oxidation is such that an unaccounted gram of carbohydrate oxidation will induce an error of 2 g in protein oxidation. Carbohydrate intake was estimated to be close to 2 g, and allowing a 30% CV in oxidation could produce uncertainty of SD 1.2 g in protein oxidation. The six repeated measures reduced the impact of this to 0.49 g per treatment, a very small level of uncertainty in the context of the 7.4-g difference in protein oxidation between diets (Table 4)
.
A further dietary consideration in the current study is the relatively high protein level of both diets, which were above the maintenance requirement of cats (27
). The original hypothesis for the high protein requirement of cats was based on the permanently high level of hepatic enzymes (7
,8
). This study finds that there is adaptability in oxidative capacity above the maintenance protein requirement but did not test this at or below the minimum 10% protein energy. If there were a lower limit to the adaptability of protein oxidation, this might still represent the reason for this species’ high protein requirement. This would be difficult to test because low protein diets are often poorly accepted by cats (28
).
This study has shown that net protein oxidation in cats responds to dietary protein intake as in other species. It is not possible using this methodology to differentiate between the different metabolic pathways that result in oxidation, and so it is not clear how much amino acid was converted to glucose before oxidation when cats consumed the two diets. However, by adapting the amino acid catabolic enzymes to dietary protein intake, and after confirmation of the adaptability of ureagenesis to dietary protein intake (9
,29
), it is unclear why cats should require such a high level of dietary protein.
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FOOTNOTES |
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3 Abbreviations used: CHO, carbohydrate; HP, higher protein; MP, moderate protein; RER, respiratory exchange ratio; VCO2, volume of carbon dioxide produced; VO2, volume of oxygen consumed.
Manuscript received 31 July 2001. Initial review completed 29 August 2001. Revision accepted 10 December 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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