Discrepancy between use of lean body mass or nitrogen balance to determine protein requirements for adult cats

Abstract

This study was undertaken to contrast the minimum protein intake needed to maintain nitrogen balance or lean body mass (LBM) in adult cats using a prospective evaluation of 24 adult, neutered male cats fed one to three different diets. Following a 1-month baseline period during which all cats consumed a 34% protein diet, cats were fed a 20% (LO), 26% (MOD) or 34% (HI) protein diet for 2 months. During the baseline period and following the 2-month feeding period, nitrogen balance was assessed using a 96-h complete collection of urine and feces, and LBM was assessed using dual energy X-ray absorptiometry. Weight loss increased in a linear manner with decreasing protein intake (P <0.01), despite no significant difference in calorie intake. Linear regression of the data indicated that approximately 1.5 g protein/kg (2.1 g/kg^0.75) body weight is needed to maintain nitrogen balance, while 5.2 g protein/kg (7.8 g/kg^0.75) body weight is needed to maintain LBM. This study provides evidence that nitrogen balance studies are inadequate for determining optimum protein requirements. Animals, including cats, can adapt to low protein intake and maintain nitrogen balance while depleting LBM. Loss of LBM and an associated reduction in protein turnover can result in compromised immune function and increased morbidity. Current Association of American Feed Control Officials (AAFCO) and National Research Council (NRC) standards for protein adequacy may not provide adequate protein to support LBM. The minimum daily protein requirement for adult cats appears to be at least 5.2 g/kg (7.8 g/kg^0.75) body weight, well in excess of current AAFCO and NRC recommendations. Further research is needed to determine the effect, if any, of body condition, age and gender on protein requirements.

Introduction

Adequate dietary protein is required to provide essential amino acids and nitrogen for the synthesis of other amino acids, endogenous proteins and nitrogenous compounds. However, what constitutes ‘adequate dietary protein’ depends, in part, on the method of assessment. The requirements for growing animals are typically based on maximizing growth rates. While this may be a useful assay for growing animals, it is not a suitable assay for adult animals. In adults, protein requirements have frequently been equated with the minimum amount that will maintain nitrogen balance.^{1 –3} Nitrogen balance is defined as nitrogen intake equal to losses, and is determined as the difference between total nitrogen intake and nitrogen loss. In practice, only urine and fecal losses are measured, while skin, hair, sweat and other minor losses are ignored or estimated.^{1,2,4 –7} Less frequently, maintenance of protein turnover or maintenance of lean body mass (LBM) have been used as indicators of protein adequacy.^{4,8 –10}

LBM, especially skeletal muscle, provides the protein reserves to support protein turnover. ¹¹ Protein turnover, which refers to the constant and highly regulated process of catabolism and synthesis of endogenous proteins, provides a means of continuous redistribution of amino acids into proteins essential for life at that moment. ⁹ The daily flux of amino acids that constitutes protein turnover is quantitatively large, involving about 5–10 times the protein provided from the daily dietary protein requirements. ⁹

Most species adapt to reduced protein intake by reducing protein turnover and amino acid oxidation, and utilizing LBM for essential protein synthesis. Proteins from the blood, liver and intestinal cells are utilized during acute protein deprivation, while muscle and skin are the major sources of amino acids during chronic protein deprivation.^9,11 Thus, inadequate protein intake over extended periods can result in a reduction of LBM, despite reduced protein turnover. This can be clinically significant as it appears to leave the subject in a compromised state with reduced ability to respond to stressors, such as trauma, toxins or infection.^6,8,10

Homeostatic adaptation to dietary protein intake allows most adult animals to maintain nitrogen balance at a wide range of protein intakes.^5,11,12 However, it has been shown previously that cats have a reduced capacity to regulate the activity of transaminases and urea cycle enzymes; thus, they have a limited ability to adapt to low protein intakes and to conserve protein nitrogen. ¹³ Therefore, one would expect cats fed inadequate dietary protein to be in negative nitrogen balance and to demonstrate a loss of LBM, while cats fed adequate diets should maintain a neutral or positive balance and maintain LBM.

Many of the published studies conducted to determine the amino acid and protein requirements of cats have been performed using purified diets or diets supplemented with crystalline amino acids.^1,14 Crystalline amino acids have a greater bioavailability than amino acids in natural proteins. Thus, minimum requirements determined using these diets and nitrogen balance studies, suggested to be 16% of the dry matter, ¹⁴ may not apply readily to commercial cat foods. The current recommendation by the Association of American Feed Control Officials (AAFCO) for protein in adult cat foods is 26% of the diet dry matter or 6.5 g protein/100 kcal metabolizable energy (ME). ¹⁵ However, the criteria used to reach this recommendation are unclear.

Therefore, the objectives of this study were to determine and compare the amount of protein required to maintain nitrogen balance or LBM in adult cats fed protein sources typical of commercial cat foods.

Materials and methods

Animals

Twenty-four adult neutered male domestic shorthair cats were blocked by age, weight and nine-point body condition score, ¹⁶ and randomly assigned to one of three dietary treatment groups. Cats were housed individually within environmentally controlled rooms and maintained under identical conditions. Cats were allowed to eat to appetite with access to their assigned diets for at least 16 h daily. Water was available ad libitum.

Diets

Diets were formulated to be as similar as possible except for protein and amino acid content. Protein sources were exchanged for carbohydrate sources to maintain similar energy contents. All diets were extruded, dry diets with a moisture content less than 10%. Diets were formulated to contain 20% (LO), 26% (MOD) and 34% (HI) dietary crude protein, on an as-fed basis (21.9, 28.2 and 36.6% on dry basis and 5.6, 7.3 and 9.5 g protein/100 kcal diet respectively), and to maintain essential amino acids in consistent proportions across the diets while minimizing the use of crystalline amino acids (Table 1).

Table 1.

Nutrient analysis of experimental diets

	LO	MOD	HI	LO	MOD	HI
Nutrient analysis	Percent of dry matter			g/100 kcal ME
Protein	21.9	28.2	36.6	5.6	7.3	9.5
Fat	14.6	14.7	13.6	3.7	3.8	3.5
Crude fiber	1.4	1.9	1.8	0.4	0.5	0.5
Carbohydrate *	55.0	46.5	40.2	14.0	12.0	10.5
Metabolizable energy (ME) (kcal/g)	3.93	3.86	3.84
Essential amino acids, † percent of total amino acids	43	45	44	2.4	3.3	4.2

^*Carbohydrate determined by difference as nitrogen-free extract

^†Excludes tryptophan and taurine in analysis

Experimental procedures

All cats were fed the high protein diet during a 1-month adaptation period, during which initial evaluations were made. Following this, cats were fed their assigned diets for a 2-month treatment period, after which final evaluations were conducted. The attending veterinarian and technicians performing the evaluations were blinded to the specific dietary treatments. Initial and final evaluations included physical examination by the attending veterinarian, serum biochemical profile, complete blood count, nitrogen balance using a 96-h complete collection of urine and feces, and LBM assessment using dual energy X-ray absorptiometry (DEXA) (Lunar Corp, Madison, WI, USA). The DEXA technique has a coefficient of variance for LBM of 0.6% in our laboratory. Body weight was recorded weekly and food intake was recorded daily throughout the adaptation and feeding periods. Dietary, urinary and fecal nitrogen were measured by Kjeldahl analysis, and nitrogen balance was calculated as the difference between total intake and total fecal plus urinary excretion, with no adjustment for minor losses, such as in skin and hair.

Statistical analysis

Analysis of variance was used to evaluate differences among the treatment groups. Where significant differences were found, a Student–Newman–Keuls multiple comparison test was used to detect treatment differences. Linear contrasts were used to evaluate the effect across protein intakes on body weight, serum biochemical profiles and hematology. Regression analysis was used to determine zero balance for nitrogen and LBM versus protein intake. Differences were considered significant at a P-value of <0.05.

Results

Three cats were removed from the study owing to medical conditions unrelated to dietary treatments and one cat was removed from the study owing to excess weight loss secondary to poor intake of the low protein diet. None of these cats were included in the data analysis. The mean age, initial body weight and body condition scores of the 20 cats completing the study were 4.9 years (range, 2–8 years), 5.3 kg (range, 4.2–7.2 kg) and 5.7 (range, 5–8), respectively. Physical examination of all remaining cats indicated good health, although slight declines in body condition, skin and haircoat condition were noticed in cats in the LO group by the end of the study.

All values on the serum biochemical profiles and cell counts remained within normal ranges. The only treatment effect was on serum urea nitrogen. There was a linear decrease across treatment groups (P <0.01) and the mean value was significantly lower in cats fed the LO diet than cats fed the HI diet (mean ± SD 6.03 ± 1.08 vs 8.03 ± 0.89 mmol/l, P <0.01).

ME intake did not differ (P >0.05) among treatment groups throughout the study, although food intake was significantly (P <0.05) less during the treatment period for cats in the MOD group compared with the other dietary groups (Table 2). Although neither initial nor final mean body weights differed between groups, change in body weight was affected by diet. Percent weight loss increased in a linear manner with decreasing protein intake (P <0.01) and the change in body weight was different (P <0.05) between the LO and HI treatment groups (Table 2). Weight loss was greatest for the LO cats despite maintaining food intake during the treatment period equivalent to the adaptation period. Protein intake was reduced in a linear fashion across treatments (P <0.01), but mean protein intake differed only between the HI treatment group and the other groups (P <0.05) (Table 2).

Table 2.

Body weights and intake of cats fed diets with different protein concentration. Data are means ± standard deviation

	LO	MOD	HI
Number of cats	6	8	6
Body weight, initial (kg)	5.0 ± 0.6	5.2 ± 0.6	5.3 ± 0.8
Body weight, final (kg)	4.6 ± 0.8	4.9 ± 0.7	5.3 ± 0.9
Body weight change (%)	–7.5 ± 6.3 a	–4.4 ± 4.1 ab	0.0 ± 3.6 b
Food intake (g/kg), adaptation period *	16.1 ± 2.4	14.5 ± 2.5	15.9 ± 1.9
Food intake (g/kg), treatment period †	16.9 ± 2.3 a	13.7 ± 1.7 b	15.9 ± 1.7 ab
Food intake (g/kg), metabolism period ‡	14.0 ± 4.6	14.6 ± 3.0	14.2 ± 2.8
Protein intake (g/kg), adaptation period	5.5 ± 0.8	4.9 ± 0.8	5.4 ± 0.6
Protein intake (g/kg), treatment period	3.5 ± 0.5 a	3.6 ± 0.5 a	5.4 ± 0.6 b
Protein intake (g/kg), metabolism period	2.9 ± 0.9 a	3.8 ± 0.8 ab	4.8 ± 0.9 b
Metabolizable energy (ME) intake (kcal/kg), adaptation period	59.7 ± 8.8	53.8 ± 9.2	58.8 ± 6.8
ME intake (kcal/kg), treatment period	57.3 ± 7.7	51.5 ± 6.5	58.6 ± 6.3
ME intake (kcal/kg), metabolism period	47.6 ± 15.6	55.7 ± 11.3	52.5 ± 10.3

^a,bValues within a row with different superscripts differ, P <0.05

^*During the adaptation period, all cats were fed the HI protein diet

^†Treatment period refers to entire period between baseline and final dual energy x-ray absorptiometry analysis

^‡Metabolism period refers to the period during the 4-day nitrogen balance study

Regression analysis on nitrogen balance and maintenance of LBM was performed only on data from cats that maintained weight within 10% of initial body weight. Due to this, final LBM assessment was available for only four cats in the LO group. Cats in LO and MOD treatment groups lost LBM, while cats in the HI group gained LBM; however, the difference was significant (P <0.05) only between the MOD and HI cats (Table 3). Regression analysis of protein intake indicated that 1.5 g protein /kg (2.1 g/kg^0.75) body weight would be needed to maintain nitrogen balance (Figure 1), while a mean intake of 5.2 g protein/kg (7.8 g/kg^0.75) body weight was needed to maintain LBM (Figure 2). Regression analysis of protein intake on LBM was repeated after excluding four cats with body fat in excess of 33%. Results of this analysis suggested that lean cats required a mean intake of 5.42 g protein/kg (8.12 g/kg^0.75) body weight to maintain LBM.

Table 3.

Body composition of cats fed different amounts of dietary protein

	LO	MOD	HI
Number of cats	4	8	6
LBM, initial (g)	4233 ± 411	3857 ± 322	4001 ± 208
LBM, final (g)	4213 ± 436	3725 ± 361	4034 ± 248
LBM, change (g)	−20 ± 91 ab	−133 ± 112 a	33 ± 102 b
Fat mass, initial (g)	1298 ± 513	1650 ± 666	1705 ± 664
Fat mass, final (g)	1267 ± 572	1453 ± 599	1641 ± 753
Fat mass, change (g)	−31 ± 216	−196 ±229	−65 ± 167
Bone mineral content, initial (g)	174 ± 17	159 ± 19	162 ± 15
Bone mineral content, final (g)	176 ± 13	158 ± 17	162 ± 17
Bone mineral content, change (g)	1.5 ± 5.5	−0.6 ± 3.9	0.6 ± 3.2
Total body mass, initial (g)	5531 ± 535	5507 ± 742	5706 ± 816
Total body mass, final (g)	5480 ± 343	5178 ± 689	5675 ± 929
Total body mass, change (g)	−0.1 ± 0.2	−0.3 ± 0.3	0.0 ± 0.2

Regression analysis on nitrogen balance and maintenance of lean body mass (LBM) was performed only on data from cats that maintained weight within 10% of initial body weight. Owing to this, final LBM assessment was available for only four cats in the LO group. Data are means ± standard deviations

^a,bValues within a row with different superscripts differ, P <0.05

Figure 1.

Estimation of protein intake needed to maintain nitrogen balance. Data include only cats that maintained body weight within 10% of initial weight. Data are shown by dietary treatment group (○ LO; ● MOD; ▼ HI), but graphed by average daily protein intake per kilogram of body weight during the metabolism study. The predicted average daily protein intake to maintain nitrogen balance is 1.5 g protein/kg body weight: y = −0.134 + 0.090X; r² = 0.325; SE = 0.130.0; P <0.05

Figure 2.

Estimation of protein intake needed to maintain lean body mass (LBM). Data include only cats that maintained body weight within 10% of initial weight. Data are shown by dietary treatment group (○ LO; ● MOD; ▼ HI), but graphed by average daily protein intake per kilogram of body weight during the entire time between baseline and final measures of LBM. The average daily protein intake needed to maintain LBM is 5.2 g protein/kg body weight: y = −372.088 + 71.155X; r² = 0.226; SE = 113.0; P <0.05.

Discussion

The dietary requirement is typically defined as the minimal intake that maintains nitrogen balance.^1–3 By the end of the 8-week period, all cats completing this study had adapted and were able to maintain nitrogen balance. Based on regression of the data, a protein intake of 1.5 g protein/kg (2.1 g/kg^0.75) body weight should be sufficient to maintain nitrogen balance. These results are similar to those reported by Earle ¹ and Hendriks, ¹⁷ who showed adult cats maintained nitrogen balance or had minimal endogenous nitrogen loss at about 1.4–1.7 g protein/kg body weight. There are limits to cats’ abilities to maintain nitrogen balance at low protein intake. Green et al ¹⁸ documented that cats do adjust protein oxidation to various concentrations of dietary protein, but were unable to adapt sufficiently when protein intake fell too low. In their study, cats were able to maintain nitrogen balance when consuming 2.5 g protein/kg body weight or more, but not when fed about 1 g protein/kg body weight. ¹⁸

There are numerous limitations to nitrogen balance studies.^2,11,19 For example, nitrogen balance can be affected by a number of factors, including protein quality, energy intake and prior nutritional status.^2,11 In addition, nitrogen balance does not take into account changes in body composition.

Protein quality was controlled across diets by proportionally increasing the protein-supplying ingredients, and all diets were formulated to provide amino acids at an optimal ratio of essential to total amino acids. ²⁰ Apparent digestibility of the protein was approximately 85% (data not shown), although it was lower in the LO diet owing, most likely, to a relative increase in the contribution of endogenous and bacterial proteins to fecal nitrogen. The protein requirement might be expected to be even higher if diets with lesser amounts of one or more essential amino acids, or protein with lower digestibility, were used.

Energy intake can influence apparent protein requirements.^2,11 Animals, including cats, will oxidize carbohydrates or fats for energy when protein intake is low. ¹⁸ Abundant dietary energy intake from carbohydrates or fats will spare protein, while subjects in negative energy balance will utilize more protein as an energy source, altering the results of nitrogen balance studies to suggest a higher protein requirement. For example, healthy children with marginal protein intake (0.7 g/kg body weight) maintained positive nitrogen balance when energy intake was moderate-to-high, but were in negative nitrogen balance when energy intake was inadequate, despite the same protein intake. ²¹ Food intake did not decrease when cats in this study were fed the lower protein diets, and did not differ between diets during the nitrogen balance study. However, many cats did lose weight during the study, so energy balance may have influenced the apparent protein requirement determined in this study. If so, the actual protein requirement for maintaining nitrogen balance may be lower than the projected 1.5 g/kg body weight.

Energy intake may also influence the percent of protein required to maintain LBM. For example, heavy or obese cats generally need or consume fewer calories per kilogram of body weight.^14,22 Assuming their actual protein requirement does not differ from leaner cats, they would require a greater proportion of calories from protein to meet their needs. However, when obese cats were removed from the dataset in this study regarding the requirement for protein to maintain LBM, the results suggested that obese cats may have slightly lower protein needs (5.42 g protein/kg body weight for lean cats vs 5.23 g protein/kg body weight for all cats in the study). Unfortunately, it is not clear if this minor difference is related truly to body composition or metabolic effects, or simply to a somewhat smaller and different dataset. Further research will be needed to address this question.

A major limitation to nitrogen balance is that the intakes that maintain nitrogen balance are not necessarily adequate for optimum health,^10,23,24 and the assay cannot differentiate between animals maintained in a relatively depleted state or in a condition in which the tissue proteins are maximal, so may be considered an inadequate measure of protein requirements.^6,8 In this study, although regression of the nitrogen balance data indicated that 1.5 g protein/kg (2.1g/kg^0.75) body weight should be adequate to maintain nitrogen balance, more than three times that amount was needed to maintain LBM in adult cats.

When subjects are first fed protein deficient diets, they will respond by utilizing endogenous proteins and by decreasing protein turnover. Protein oxidation will decrease, while oxidation of fats and carbohydrates will increase. ¹⁸ If deficiency is temporary, the subject will achieve a new steady state and nitrogen balance, at a reduced LBM.^11,12 If protein depletion continues, LBM will continue to decline, despite reduced protein turnover. It is possible, as all cats were in positive nitrogen balance at the end of this study period, that all had achieved a new steady state at the lower LBM and that further deterioration would not occur. However, it is equally likely that nitrogen balance was being maintained by a continued drain on LBM, and that LBM would continue to be depleted in cats fed the LO and MOD protein diets. This can be clinically significant as there is growing recognition that loss of LBM or reduced protein turnover can have significant adverse health effects.^{6,8,10,25 –27}

The current recommendation by the AAFCO for protein in adult cat foods is 26% of the diet dry matter or 6.5 g protein/100 kcal ME. ¹⁵ Assuming an average calorie intake of 50–60 kcal ME/kg body weight for adult cats, this would equate to approximately 3.25–3.9 g protein/kg body weight. The National Research Council (NRC) guidelines indicate a minimum daily protein requirement and a recommended daily protein allowance of 2.5 and 3.13 g protein/kg body weight, respectively. ¹⁴ The results of the current study indicate that a daily protein intake of 1.5 g/kg (2.1 g/kg^0.75) should be adequate to maintain nitrogen balance, but an average of 5.2 g/kg (7.8 g/kg^0.75) was needed to maintain LBM. Most cats consuming the 34% protein (HI: 9.5 g protein/100 kcal ME) diet maintained LBM, whereas most cats consuming the 20% or 26% protein (LO and MOD: 5.6 g and 7.3 g protein/100 kcal ME, respectively) diets lost LBM. Thus, the current AAFCO and NRC recommendations may be adequate to support nitrogen balance, but appear to be inadequate to support protein turnover and LBM.

There are several limitations of this study, some of which have been alluded to previously. Many cats lost weight during the study so were in obvious negative energy balance. This can affect and falsely elevate the apparent protein requirement, as protein may be used as an energy source. This effect was minimized by using LBM data only from cats that maintained their body weight within 10% of initial weight. This study used only neutered male cats. The protein requirements for female cats and geriatric cats may differ from those of young, neutered male cats. The cats involved in the study covered a broad range of body conditions, but insufficient cats were evaluated to determine if there is an effect from body composition on protein requirements. The study period lasted only 2 months. It is possible that cats would have lost more LBM had the study gone on longer, or cats may have adapted to the diets and stabilized at their somewhat reduced level of LBM. Further research is needed to address these latter points.

Conclusions

This study provides further evidence that nitrogen balance studies are an inadequate means for determining optimum protein requirements. Animals, including cats, can adapt to low protein intake and maintain nitrogen balance despite depletion in LBM. The minimum daily protein requirement for adult cats appears to be at least 5.2 g/kg (7.8 g/kg^0.75) body weight. With a typical calorie intake of 50–60 kcal/kg body weight, this represents approximately 30–40% of calories. For cats with lower energy requirements, a greater proportion of calories as protein may be required. Further research is needed to determine the effect, if any, of body condition, age and gender on protein requirements of cats.