Minimum methionine requirement in adult cats as determined by indicator amino acid oxidation

Abstract

There is a lack of empirical data on the dietary Met requirement, in the presence of Cys or cystine, in adult cats. Thus, the aim of this study was to determine the Met requirement, in the presence of excess Cys, in adult cats at maintenance using the indicator amino acid oxidation (IAAO) technique. Six adult neutered male cats were initially selected and started the study. Cats were adapted to the basal diet sufficient in Met (0.24% dry matter, DM) for 14 d prior to being randomly allocated to one of eight dietary levels of Met (0.10%, 0.13%, 0.17%, 0.22%, 0.27%, 0.33%, 0.38%, and 0.43% DM). Different dietary Met concentrations were achieved by supplementing the basal diet with Met solutions. Alanine was additionally included in the solutions to produce isonitrogenous and isoenergetic diets. Cats underwent a 2-d adaptation period to each experimental diet prior to each IAAO study day. On IAAO study days, 13 meals were offered corresponding to 75% of each cat’s daily food allowance. The remaining 25% of their daily food intake was offered after each IAAO study. A bolus dose of NaH¹³CO₃ (0.44 mg kg⁻¹) and l-[1-¹³C]-phenylalanine (¹³C-Phe; 4.8 mg kg⁻¹) were provided in fifth and sixth meals, respectively, followed by a constant dose of ¹³C-Phe (1.04 mg kg⁻¹) in the next meals. Breath samples were collected and total production of ¹³CO₂ was measured every 25 min through respiration calorimetry chambers. Steady state of ¹³CO₂ achieved over at least three breath collections was used to calculate oxidation of ¹³C-Phe (F¹³CO₂). Competing models were applied using the NLMIXED procedure in SAS to determine the effects of dietary Met on ¹³CO₂. Two cats were removed from the study as they did not eat all meals, which is required to achieve isotopic steady. A breakpoint for the mean Met requirement, with excess of Cys, was identified at 0.24% DM (22.63 mg kg⁻¹) with an upper 95% confidence limit of 0.40% DM (37.71 mg·kg⁻¹), on an energy density of 4,164 kcal of metabolizable energy/kg DM calculated using the modified Atwater factors. The estimated Met requirement, in the presence of excess of Cys, is higher than the current recommendations proposed by the National Research Council’s Nutrient Requirement of Dogs and Cats, the Association of American Feed Control Officials, and the European Pet Food Industry Federation.

The minimum methionine requirement estimated in our study was higher than the current recommendations set by American and European regulatory bodies.

Introduction

Methionine is a sulfur amino acid (AA) that is considered dietary indispensable for mammalian species, including the domestic cat. Besides the role of Met in initiation of translation and incorporation into protein, Met is remarkably involved in the physiology of the whole organism (Stipanuk et al., 1986). Methionine can react with adenosine triphosphate to generate S-adenosylmethionine (SAM), which functions as the universal methyl donor as most cellular methylation reactions, including the deoxyribonucleic acid (DNA), occur via provision of methyl units by SAM. After donating its methyl group, SAM is converted to S-adenosylhomocysteine (SAH) and homocysteine (Hcys), which can be remethylated to Met or be irreversibly oxidized to Cys (Du Vigneud et al., 1994). It is important to note that due to the sparing effect of Cys on Met, around 50% of the total sulfur AA requirement (Met + Cys; wt:wt) in mammals can be furnished by Cys (Finkelstein and Mudd, 1967; Shannon et al., 1972; Shoveller et al., 2003; Humayun et al., 2006); consequently, the amount of Met that cannot be replaced by Cys to meet the requirement of total sulfur AA is defined as the minimum Met requirement (reviewed by Ball et al., 2006). The importance of Cys is not only due to its sparing effect on Met. Through the transsulfuration pathway, Cys can be further utilized to produce secondary metabolites, such as glutathione and taurine (Larsson et al., 1983), that are involved in the maintenance of cellular redox balance and regulation of the immune system. Together, the role of Met and interaction with methyl compounds and as a precursor of Cys require careful dietary control when determining the minimum Met requirement. Further to this, differences in food intake will result in different pool sizes of all these products, and thus, food intake needs to be equal among treatments when determining the requirement for Met.

Although the importance of Met and its secondary metabolites are well known, Met is also one of the most toxic AA and may be detrimental to health when consumed in excess (reviewed by Baker, 2006). Furthermore, high dietary intake of Met is linked to hyperhomocysteinemia, which may play a role in the pathogenicity of some disorders (reviewed by Mutairi, 2020). As both deficiency and excess intake of Met have deleterious effects, it is important to define the minimum Met requirement to ensure its adequate supply in order to optimize feline nutrition and support overall health. However, despite its importance, the current recommendations for dietary Met and total sulfur AA for adult cats at maintenance presented by the National Research Council’s Nutrient Requirement of Dogs and Cats (NRC, 2006) are based solely on one, non-peer-reviewed report (Burger and Smith, 1987). In this report, the total sulfur AA requirement in adult cats was determined using the nitrogen balance technique, which is insensitive in adult animals and underestimates requirements (reviewed by Elango et al., 2008). As such, the Met and total sulfur AA recommendations proposed for adult cats by NRC (2006) may be inaccurate, and thus, it is necessary to apply more sensitive methodologies to more empirically determine the requirement of Met in mature cats to support optimization of formulation.

The indicator AA oxidation (IAAO) is a noninvasive and sensitive technique that has been widely applied to determine AA requirements in different species (reviewed by Elango et al., 2008), including the dog (Shoveller et al., 2017; Templeman et al., 2019; Mansilla et al., 2020a, 2020b; Sutherland et al., 2020). Due to its high sensitivity and short dietary adaptation periods required, especially with diets deficient in the test AA, estimates of AA requirement derived from IAAO studies have been consistently greater than those determined using the nitrogen balance methodology (reviewed by Elango et al., 2012). The IAAO methodology, however, has never been utilized in the domestic cat to determine the requirement of AA. Therefore, we have recently developed a semi-synthetic diet to be used in IAAO studies in cats (Pezzali et al., 2021, 2023a) and adapted the IAAO protocol used in dog studies to allow the successful application of this technique to determine the requirement of indispensable AA in the domestic cat (Pezzali et al., 2023b, 2023c). Thus, the aim of this study was to determine the minimum Met requirement, when excess of Cys is provided, in adult cats at maintenance using the IAAO technique. We hypothesized that the minimum Met requirement derived using the IAAO technique is greater than those presented by NRC (2006) and the American and European regulatory bodies.

Materials and Methods

This study was conducted according to the guidelines for animal care and use provided by the Canadian Council on Animal Care. All the procedures were approved by the University of Guelph Animal Care Committee (AUP #4640).

Animals and housing

Six adult neutered male cats (Marshall’s BioResources, Waverly, NY, USA) initially started the study. All cats were deemed healthy prior to the start of the study based on physical examination performed by a veterinarian, and on serum biochemistry and complete blood count analyses. The cats were group housed in an indoor free-living environment (7.1 × 5.8 m) located in the Animal Biosciences Department at the University of Guelph. The room was approved for cat inhabitation by the Chief Veterinary Inspector of the Ontario Ministry of Agriculture, Food, and Rural Affairs under the Animals for Research Act prior to the arrival of the cats.

Animals were kept in individual crates only during feeding. Water was available ad libitum throughout the study from both standing water bowls and free-flowing water. The light (12 h light:12 h cycle), temperature (20 °C), and humidity (40% to 60%) were controlled and monitored daily. Toys, scratching posts, perches, beds, climbing apparatuses, and hidden boxes were provided to enrich the environment. Additionally, cats were socialized with a familiar person for 2 h daily, 5 d a week.

Body composition determination

Prior to the study, dual-energy x-ray absorptiometry scans were performed by a trained person 24 h after the last meal to determine lean soft tissue mass (LSTM). Cats were sedated using intramuscular dexmedetomidine hydrochloride (Dexdomitor, Zoetis, Kirkland, QC, Canada; 0.5 mg/mL) (0.015 mg kg⁻¹). Total body mass, fat mass, LSTM, and bone mass were measured through duplicate scans on small animal mode with the thin setting using a fan-beam dual-energy x-ray absorptiometry (Prodigy Advance GE Healthcare, Madison, WI, USA). Cats were positioned in dorsal recumbency with forelimbs extended cranially and repositioned as necessary between scans, which were completed in ~10 min. Estimates for LSTM were obtained from the system software (enCORE Version 16; GE Healthcare, Madison, WI, USA). Results from both scans were averaged. Sedation was reversed by 0.015 mg kg⁻¹ atipamezole (Antisedan, Zoetis, Kirkland, QC, Canada) after the completion of the second scan.

Diets and study design

A basal diet was formulated with inclusion of intact ingredients (Table 1) to meet and exceed all nutrient requirements (recommended allowance; RA) for adult cats at maintenance according to NRC (2006; Table 2), with the exception of Met. The basal diet was formulated in a similar manner as previously reported by Pezzali et al. (2023a); however, poultry meal and black soldier fly larvae meal were replaced by pork meal and lamb meal due to ingredient supply and to limit the inclusion of Met. Excess Cys was supplied to meet the total sulfur AA requirement, and thus, avoid the metabolism of Met through the transmethylation and trans-sulfuration pathway to meet the Cys requirement. Similarly, Tyr was provided in excess to ensure Phe (tracer and the tracee) was shunted to protein synthesis and oxidation, not to Tyr production (Shiman and Gray, 1998). The basal diet was prepared as described by Pezzali et al. (2023a). Briefly, dry ingredients were mixed prior to the start of the study and stored in a closed container. Water and fat were mixed daily prior to each feeding using a commercial mixer (KitchenAid, St Joseph, MI, USA). Cats were transitioned from a commercial dry food (T11 Nutram Total Grain-Free, Nutram, Canada) to the experimental diet during a 6-d period. The cats were offered 75% of their daily energy requirement as kibble and 25% as basal diet for 2 d, followed by 50% kibble and 50% basal diet for 2 d, and finally 25% kibble and 75% basal diet for the last 2 d. Cats then underwent a 14-d adaptation period to the basal diet where they were fed to maintain body weight (BW) based on historical feeding records. Food was provided in two equal feedings (0730 and 1600 hours) throughout the study. During the transition and adaptation periods, the basal diet was top dressed with a dl-Met solution (2.29 g L⁻¹) at 7.2 mL kg-BW⁻¹ to provide 140% of the Met RA (0.24%; NRC, 2006). After the adaptation period, cats were randomly assigned to one of the eight experimental diets. The experimental diets were developed by supplementing the basal diet with one of eight dl-Met solutions (0, 0.42, 0.85, 1.52, 2.20, 2.88, 3.56, and 4.23 g L⁻¹) at 7.2 mL kg-BW⁻¹. Additionally, l-Ala (≥99% l-Alanine, Sigma-Aldrich, St. Louis, MO) was included in the solutions (2.53, 2.27, 2.02, 1.62, 1.21, 0.81, 0.40, and 0 g L⁻¹ for solutions 1 to 8, respectively) to produce isonitrogenous and isoenergetic diets. The final content of Met in the test diets after addition of the supplemental solutions was 0.10%, 0.13%, 0.17%, 0.22%, 0.27%, 0.33%, 0.38%, and 0.43% on a dry matter (DM) basis. The cats underwent the following 3-d feeding regimen: (1) days 0 to 1: 2 d adaptation period to the experimental diet where cats then fed 16 g/kg BW (maintenance of BW) in their regular feeding regimen as described above and (2) day 2: IAAO study day. This 3-d feeding regimen was repeated eight times so all cats were offered all dietary treatments. It is important that on adaptation days and IAAO study days, every cat is supplied the same amount of diet kg BW⁻¹ to account for inter-animal variability.

Table 1.

Ingredient composition of the basal diet deficient in methionine

Ingredient	%, as-fed basis
Wheat starch1	34.16
Water	21.00
Poultry fat	13.50
Amino acid premix2	12.56
Lamb meal	4.50
Pork meal	4.10
Cellulose3	2.32
Sodium bicarbonate	1.70
Dicalcium phosphate	1.54
Palatant4	1.30
Potassium chloride	1.00
Brewer’s yeast	0.70
Calcium carbonate	0.60
Choline chloride, 60%	0.40
Salt	0.39
Mineral premix5	0.12
Vitamin premix6	0.12

¹PAYGEL 290 Pregelatinized Wheat Starch, Archer Daniels Midland Company, USA.

²Provides per 100 g of final diet: 2.62 g of l-glutamic acid, 1 g of l-tyrosine, 0.80 of l-alanine and glycine each, 0.90 g of l-leucine, 0.70 g of l-arginine and proline each, 0.60 g of l-threonine and l-serine each, 0.50 g of l-lysine, l-isoleucine, and l-phenylalanine each, 0.45 g of l-valine, 0.40 g of l-asparagine H₂O, 0.35 g of taurine, 0.23 g of l-histidine HCl H₂O, and 0.11 g of tryptophan.

³Arbocel BWW40 Natural Cellulose Fibers, J. Rettenmaier USA LP, USA.

⁴PALASURANCE®C45-140 Dry, Kemin Nutrisurance, Inc., USA.

⁵Mineral composition of the premix: 1.55% calcium, 0.0348% phosphorus, 0.4658% magnesium, 13.92% potassium, 0.2% sodium, 12.93% chloride, 11.90% sulfur, 15,493.9 ppm copper, 92,272.1 ppm iron, 9,397.4 ppm manganese, 361 ppm selenium, 104,105.8 ppm zinc, and 1,953 ppm iodine.

⁶Vitamin composition of the premix: 105.83 mg/kg biotin, 1,573 mg/kg folic acid, 91,700 mg/kg niacin, 7,020 mg/kg pantothenic acid, 6,350 mg/kg pyridoxine, 4,930 mg/kg riboflavin, 10,100 mg/kg thiamin, 24.94 mg/kg vitamin B12, 13,079,000 IU/kg vitamin A, 412,000 IU/kg vitamin D3, 46,718 IU/kg vitamin E.

Table 2.

Nutrient composition of the basal diet deficient in methionine

Nutrient content	Analyzed content	NRC1 MR	NRC1 RA
ME, kcal/kg as-fed (calculated)2	3,172
Dry matter (DM), %	76.18
Crude protein, % DM	21.66	16.00	20.00
Acid-hydrolyzed fat, % DM	19.36
Ash, % DM	8.69
Predicted crude fiber, % DM	2.93
Amino acids, % DM
Alanine	1.69
Arginine	1.34		0.77
Aspartic acid	1.12
Glutamic acid	4.18
Glycine	1.87
Histidine	0.43		0.26
Isoleucine	0.82		0.43
Leucine	1.61		1.02
Lysine	1.09	0.27	0.34
Methionine	0.10	0.14	0.17
Methionine + Cysteine	1.15	0.27	0.34
Phenylalanine	0.96		0.40
Phenylalanine + Tyrosine	2.44		1.53
Proline	1.11
Serine	0.92
Threonine	1.06		0.52
Valine	0.86		0.51
Tryptophan	0.18		0.13

¹NRC, National Research Council (NRC, 2006); MR, Minimal requirement; RA, Recommended allowance.

²Metabolizable energy (ME) = 3.5 kcal/g × crude protein (%) + 8.5 kcal/g × acid-hydrolyzed fat (%) + 3.5 kcal/g × nitrogen free extract (%); nitrogen free extract (%) = 100% − (crude protein % + acid-hydrolyzed fat % + crude fiber % + ash %).

IAAO studies

The morning of each IAAO study day, BW was measured to ensure accurate delivery of experimental diets and isotope dosages. Cats were then placed in individual respiration chambers where they stayed for 30 min with the main rotary pumps off to ensure increase in respiratory gases. A similar IAAO feeding, isotope, and breath collection protocol as described by Pezzali et al. (2023c) was applied. Briefly, three fasting respiration measurements occurred and breath samples, for determination of background ¹³CO₂, were collected. Thirteen meals were offered corresponding to 75% of each cat’s food allowance; after completion of each IAAO, cats were fed the remaining 25% of their daily food intake. Accordingly, top dressing solutions were proportionally reduced to 75% of their original supply. The first three meals were fed every 10 min (0, 10, and 20 min) to achieve fed state and the following meals were fed every 25 min. A priming dose of NaH¹³CO₃ (0.44 mg kg⁻¹; 99%, Cambridge Isotope Laboratories, Inc., Tewsbury, MA) and l-[1-¹³C]-Phe (4.8 mg kg⁻¹; 99%, Cambridge Isotope Laboratories, Inc.) was provided in the fourth and fifth meals, respectively, followed by a constant dose (1.04 mg kg⁻¹) of l-[1-¹³C]-Phe in the next meals. Breath samples were collected every 25 min, to measure enrichment of ¹³CO₂, for the duration of the study. Detailed description of the protocol is described in Pezzali et al. (2023c).

Sample collection and analysis

Diet analysis

The basal diet was analyzed for moisture (AOAC 930.15), crude protein (AOAC 990.03), hydrolyzed fat (AOAC 945.16), and ash (AOAC 942.05) at a commercial laboratory (SGS Agri-food Laboratories, Guelph, CA). Dietary AA concentrations were analyzed by Ajinomoto Animal Nutrition North America, Inc. (Chicago, IL) (total AA [AOAC 994.12], Trp [IAO 13904:2005 E], and free AA [AOAC 994.13]).

Breath samples and calorimetry data

Samples of CO₂ produced before and after the start of the tracer protocol began were collected by trapping subsamples of CO₂ in 8 mL of 1 M NaOH over each 25 min period. The samples were transferred and retained in a 10-mL vacutainer tube (#366430 BD) that was evacuated to prevent dilution of ¹³CO₂ and stored at −20 °C until later analysis. Expired ¹³CO₂ enrichment was measured with a continuous-flow isotope ratio mass spectrometer. Enrichments were expressed above background samples (atom percent excess, APE). Fasting volume of CO2 (VCO₂) was collected automatically using Qubit calorimetry software (Customized Gas Exchange System and Software for Animal Respirometry; Qubit Systems Inc.) and used to calculate the rate of l-[1-¹³C]-Phe oxidation.

Calculations

The rate of appearance of ¹³CO₂ in breath flux of 13CO2 (F¹³CO₂, mmol kg⁻¹ h⁻¹), which represents l-[1-¹³C]-Phe oxidation, was calculated as follows:

$${\displaystyle \begin{array}{l}{\displaystyle {\mathrm{F}}^{13}\mathrm{C}{\mathrm{O}}_2=\frac{(\mathrm{V}\mathrm{C}{\mathrm{O}}_2\times \mathrm{A}\mathrm{P}{\mathrm{E}}_{\mathrm{C}{\mathrm{O}}_2}\times 44.6\times 60)}{(\mathrm{B}\mathrm{W}\times 100\times 1)}}\end{array}}$$ (1)

where VCO₂ is the average production of CO₂ during fasting state (mL min⁻¹), APE_CO2 represents the APE of ¹³CO₂ during isotopic steady state (%), BW is the cat’s BW expressed as total BW (kg) or LSTM (kg), 44.6 (mmol mL^-−1) and 60 (min h⁻¹) convert the VCO₂ to micromoles per hour, the factor 100 changes APE to a fraction, and 1.0 is the retention factor of CO₂ due to bicarbonate fixation based on previous dog report (Shoveller et al., 2017). Fasting VCO₂ was used because of the increased variability associated with fed-state VCO₂ in free-living animals.

Statistical analysis

The study was designed and conducted as 8 × 6 Latin rectangle design. The effect of dietary Met content on F¹³CO₂ was analyzed using the PROC GLIMMIX procedure of SAS (v. 9.4; SAS Institute Inc., Cary, NC) with diet as a fixed effect and cat as a random effect. Additionally, the effect of dietary Met content on F¹³CO₂ was fitted using competing statistical models, namely broken-line linear and broken-line quadratic, using the NLMIXED procedure of SAS where diet was treated as a fixed effect and cat as a random effect. The estimated mean breakpoint parameter and its corresponding 95% confident interval were determined in each model. Models were then compared based on the Bayesian Information Criterion, where the lowest the value, the best the fit.

Results

Cats maintained BW and consumed all their daily food during the adaptation period (data not shown). During the experimental period, two out of the six cats consumed all dietary treatments, maintained food intake and BW, and completed all IAAO study days (eight in total). Two other cats started to decrease their food intake after the second week of the experimental period; however, they successfully completed a minimum of four IAAO studies (six and four IAAO study days in total for each cat, respectively), and thus, their data were included in the analysis. Finally, the other two cats decreased their food intake during the first week of the experimental period and did not successfully complete at least four IAAO study days; thus, they were removed from the study and data generated were not used. Only data from cats that maintained BW among IAAO studies that were successfully completed and that reached isotopic steady state of ¹³CO₂ in breath were used to calculate F¹³CO₂ and included in the statistical analysis (Table 3). A sample size of four cats was achieved for diets containing 0.13%, 0.17%, 0.22%, and 0.27 % Met (DM basis), of three cats for diets containing 0.10% and 0.38% Met (DM basis), and of two cats for diets containing 0.33% and 0.43% Met (DM basis).

Table 3.

Characteristics of the cats and experimental diets consumed by each subject to determine the minimum methionine requirement, in the presence of excess cysteine, using the indicator amino acid oxidation technique

Cat1	Experimental diets consumed2	Age, yr	Body weight3, kg	Lean soft tissue mass, %
Ace	All	1.83	4.20 ± 0.03	87
Theo	All	2.83	4.56 ± 0.04	85
Sam	0.10%, 0.13%, 0.17%, 0.22%, 0.27%, and 0.38%	1.83	6.16 ± 0.06	76
Tintin	0.13%, 0.17%, 0.22%, and 0.27%	2.75	4.73 ± 0.03	85

¹Neutered male American shorthair.

²Diets successfully consumed during the adaptation period and the indicator amino acid oxidation study day.

³Mean body weight ± standard derivation.

The model that best fit the effect of dietary Met on F¹³CO₂ was the broken-line linear, both when F¹³CO₂ was expressed as a function of total BW or LSTM. A breakpoint for the mean Met requirement with excess of Cys, when F¹³CO₂ as a function of total BW (mmol kg⁻¹ h⁻¹) was used as the outcome of interest, was identified at 0.24 % (22.63 mg kg⁻¹) with an upper 95% confidence limit (CL) of 0.40% (37.71 mg kg⁻¹) on a DM basis (Figure 1; Table 4), on an energy density of 4,164 kcal metabolizable energy (ME)/kg DM calculated using the modified Atwater factors. When expressed on energy density of 4,000 kcal ME/kg DM, the CL would equate to 0.38% on a DM basis. When F¹³CO₂ as a function of LSTM (mmol kg-LSTM⁻¹ h⁻¹) was used as the outcome of interest, a breakpoint for the mean Met requirement was found at 0.20% with an upper 95% CL of 0.39% on a DM basis (Figure 1) on an energy density of 4,164 kcal ME/ kg DM calculated using the modified Atwater factors. When expressed on energy density of 4,000 kcal ME/kg DM, the CL would be 0.37% on a DM basis.

Figure 1.

Influence of dietary methionine (Met) on the production of ¹³CO₂ from phenylalanine oxidation (F¹³CO₂) on adult cats using the indicator amino acid oxidation technique. The F¹³CO₂ is presented as a function of total body weight (A) and lean soft tissue mass (B). The breakpoint (dashed arrows) represents the estimated mean  Met requirement when cysteine is supplied in excess (0.24% in A and 0.21% in B). Solid arrows represent the upper 95% confidence limit for Met requirement (0.40% in A and 0.39% in B). A sample size of n = 4 was achieved for diets containing 0.13%, 0.17%, 0.22%, and 0.27% Met (dry matter [DM] basis), n = 3 for diets containing 0.10% and 0.38% Met (DM basis) and n = 2 for diets containing 0.33% and 0.43% Met (DM basis). The experimental diets had an energy density of 4,164 kcal of metabolizable energy/kg DM calculated using the modified Atwater factors.

Table 4.

Dietary minimum methionine requirement for adult cats at maintenance recommended by AAFCO, FEDIAF, NRC, and the present study

Units	AAFCO1	FEDIAF2	NRC3 MR4	NRC RA	Present study
					MR	CL
g/100 g DM5	0.20	0.23/0.17	0.135	0.17	0.23	0.38
g/Mcal ME	0.50	0.57/0.43	0.34	0.43	0.58	0.96
mg/kg BW					22.63	37.71
						(24.86)6

¹Association of American Feed Control Officials Manual (2023).

² European Pet Food Industry Federation (2023) Nutritional guidelines for complete and complementary pet food for cats and dogs. Values depend on maintenance energy requirements of 75 kcal/kg^0.67 or 100 kcal/kg^0.67, respectively.

³Nutrient requirements of dogs and cats. National Research Council, 2006.

⁴MR, minimal requirement; RA, recommended allowance; CL, upper 95% confidence limit.

⁵Values for g/100 g DM are determined assuming a dietary energy density of 4,000 kcal ME/kg.

⁶Values in parentheses represent NRC recommendation for methionine requirement for adult cats at maintenance converted from mg/kg BW^0.67 to mg/kg BW using the average BW of cats in the present study.

Discussion

To the best of our knowledge, this is the first study to empirically determine the Met requirement, when Cys is provided in excess, in healthy adult neutered male cats using the IAAO technique. First, it is important to note that AA requirements are a function of BW and should be expressed accordingly. This exemplifies the need to provide all cats with the same amount of diet on IAAO days, calculated as a function of BW. In the current study, the minimum Met requirement is presented as mg kg⁻¹ d⁻¹ and also as percentage of diet to compare with the recommendations presented by NRC (2006) and other regulatory agencies and when dietary energy is in surfeit. NRC (2006) additionally presents the requirement in a metabolic BW basis (mg kg^0.67), even though protein deposition, and thus AA requirements, are a function of total BW, not metabolic BW. When F¹³CO₂ was expressed as a function of BW, the population-safe (upper 95% CI) Met requirement (0.38% DM when expressed on energy density of 4,000 kcal ME/ kg DM for direct comparison with current recommendations) was 123% higher than the RA (0.17% DM) presented by NRC (2006). We additionally considered the Met requirement as a function of LSTM due to the metabolic activity the lean body tissue has with AA (Laflamme and Hannah, 2013). The population-safe (upper 95% CL) requirement, however, did not differ when expressed as BW or LSTM considering the cats used in the study were within optimal body condition (Laflamme, 1997). The higher estimates determined herein are in agreement with our hypothesis as the presented recommendation for the Met requirement in adult cats by NRC (2006) is derived from only one dose–response study published as a proceedings paper, not a peer-reviewed manuscript, using the nitrogen balance technique (Burger and Smith, 1987).

The insensitive nature of the nitrogen balance technique to determine AA requirements in mature animals owes to the fact that the N pool is in equilibrium, and thus, changes in the urea pool due to different AA intake are minimal and difficult to capture. Additionally, the nitrogen balance technique does not capture all AA fates (e.g., skin) and subjects will “accommodate” after the long adaptation period needed to equilibrate the body’s urea pool (Scrimshaw and Young, 1989), becoming more efficient in protein metabolism, and consequently, confounding the effects of AA intake on nitrogen balance. Supporting our findings, the population-safe (upper 95% CL) Met requirement estimated in dogs using the IAAO technique (Mansilla et al., 2020b) was 46% higher than the RA for adult dogs at maintenance presented by NRC (2006). Similarly, the total sulfur AA requirement found in humans by Di Buono et al. (2001) was 60% higher than the population-safe intake presented by FAO/WHO/UNU (1985). Our estimate, however, is 135% higher than the recommendation presented by NRC (2006). This may be due to a higher utilization of Met for non-protein demands in the cat (carnivorous animal) compared to omnivores that were previously not captured in studies using the nitrogen balance technique. Although cats cannot synthetize taurine in sufficient amounts via Cys and cysteinesulfinic acid due to low activity of cysteine dioxygenase and cysteinesulfinic decarboxylase (Knopf et al., 1978), Cys is metabolized to at least two other pathways involving desulphydration and transamination of Cys to release pyruvate, and sulfur, which may be upregulated in the domestic cat. Cats might have a higher methyl demand to support the synthesis of SAM, and thus, production of choline to export lipids out of the liver due to the high fat content of the diet of their ancestors (Verbrugghe and Bakovic, 2013). Additionally, a higher methyl demand may be driven to synthetize creatine to support hunting behavior that is associated with bouts of intense exercise.

Although the IAAO method captures the net result of AA metabolism, it likely does not capture the epigenetic effects of dietary Met intake, which is important to consider if health and longevity are the most important objective of a dietary intervention. As precursor of SAM, a ubiquitous methyl donor, Met can impact the production of methylation reactions products, such as creatine, choline, and L-carnitine. As a result, the metabolism of these compounds is interconnected and can impact the availability of one another. For example, Met can reduce the choline requirement via provision of methyl donors in the cat (Anderson et al., 1979). The contrary is also true. In neonatal piglets fed a Met-restricted diet, the supply of methyl donors was able to contribute to 10% of the Met requirement (Robinson et al., 2016). More recently, Verbrugghe et al. (2021) and Rankovic et al. (2021) reported an increase in plasma concentrations of Met in the fasted state in cats fed a high choline diet. The interplay between methyl donors on Met availability, and consequently, the requirement deserves further investigation in the domestic cat. To ensure that Met intake was the only factor driving the minimum Met requirement in this study, the dietary concentration of methyl donors and co-factors of the Met cycle were kept constant among dietary treatments and provided above the requirement for adult cats (NRC, 2006), although choline requirements may in fact be greater than current recommendations (Rankovic et al., 2022). Besides donating its methyl group to generate many metabolites, SAM methylates DNA and specific residues in histone proteins, and thus, plays a major role in epigenetic gene regulation (reviewed by Mentch and Locasale, 2016; Zhang, 2018). Unfortunately, the serum concentrations of SAM and SAH were not measured in this study as the cats used herein require sedation to ethically and calmly collect blood samples. When methyl donors were supplied in a Met deficient diet for 8 d, the hepatic SAM:SAH ratio (i.e., “methylation index”) increased compared to neonatal piglets fed a Met deficient diet alone (Robinson et al., 2016), indicating a higher methyl group availability. The authors, however, did not evaluate methylome and transcriptome as outcomes. The IAAO technique likely captures the interconnectivity of enzymatic regulations in the Met and one-carbon cycle; however, the short adaptation period likely does not capture its effects on methylome and transcriptome, which would likely take much longer. A recent study (Stone et al., 2021) reported that changes in transcriptome, ribonucleic acid biology, and proteome occur quickly, within 6 h in mice consuming a Met deficient diet. However, concentrations of some metabolites involved in the Met cycle and the expression of some genes differ between 6 h and 11 wk after consumption of a Met-deficient diet (Stone et al., 2021). In European seabass (Dicentrarchus labrax), changes in gene expressions due to Met intake differed after 2 and 12 wk supplementation, with the later supporting an immune enhancing role of Met (Machado et al., 2020). As such, although the metabolism quickly responds to differences in Met intake, long-term transcriptional, behavioral, and physiological responses to Met intake may differ and deserve further investigation in cats. It is important to note, though, that a short adaptation period is still ideal when the goal is to determine the requirement of indispensable AA. As discussed, feeding AA-deficient diets for prolonged periods would have led to metabolic “accommodation”, masking the effects of the test AA intake on protein synthesis, and thus, the requirement of AA (Scrimshaw and Young, 1989). Additionally, feeding AA-deficient diets for long periods would be considered ethically unacceptable in some species, such as dogs and cats. The short adaptation period required to change the transfer ribonucleic acid expression is beneficial as it also allows the application of the IAAO technique in vulnerable populations and in species, such as the cat, that do not voluntarily consume semi-synthetic diets for long periods. Long-term studies should be applied after the Met requirement is determined via sensitive techniques (e.g., IAAO) to determine the ideal Met inclusion to optimize specific metabolic outcomes. As such, future studies should investigate the long-term effects of Met intake (at and above the requirement) on additional metabolic outcomes, methylome, and transcriptome in the domestic cat.

Due to the unpalatable nature of the semi-synthetic diet used in our study, a sample size of four subjects was not achieved for all dietary treatments as originally aimed for. The intact protein sources used in the semi-synthetic diet previously developed (Pezzali et al., 2021, 2023a), poultry meal and black soldier fly larvae meal, were replaced by pork meal and lamb meal in this study due to the supply of ingredients and differences in AA composition within the same protein sources acquired at different times of the year. This may have decreased the palatability of the semi-synthetic diet, which resulted in the removal of cats throughout the study. However, it is important to note that the breakpoint found in the statistical model was between dietary levels that had a sample size of four cats. As such, we do not believe that the smaller sample size in some treatments had a significant impact on the breakpoint. It is also important to note that the substantially higher population-safe (upper 95% CI) Met requirement compared to the mean minimum Met requirement (0.40 vs. 0.24 % DM, respectively) is in accordance with previous reports in the literature among different species. For example, when six men were used to determine the minimum Met requirement by Di Buono et al., (2001), the authors found differences between the mean minimum and the safe-population intake even higher (4.5 and 10.1 mg kg⁻¹ d⁻¹, respectively) than ours. The higher population-safe (upper 95% CI) Met requirement observed in our study is likely due to the expected variation found between individuals. However, the limited sample size may have prevented us from identifying the true population variability of the individual factors, which may alter the prediction of the population-safe requirement. To avoid limitations due to sample size in future studies, we recommend a decrease in the adaptation time to reduce the overall length of the study, and consequently, decrease the exclusion of cats. Although a 14-d adaptation period to the basal diet has been used in IAAO studies in dogs (Shoveller et al., 2017; Mansilla et al., 2018, 2020a, 2020b; Templeman et al., 2019; Sutherland et al., 2020) as used herein, an adaptation period of only 2 d is necessary to adapt to different dietary protein intakes in humans (Hoerr et al., 1993; Motil et al., 1994; Thorpe et al., 1999), and growing and adult pigs (Moehn et al., 2004). Thus, we suggest that future IAAO studies in cats decrease the adaptation period to the diet from 14 to 7 d. A more conservative adaptation period (7 vs. 2 d) is recommended as the effects of different protein intake on flux of ¹³CO₂ after oral delivery of l -[1-¹³C]-Phe have not been evaluated in the domestic cat. Additionally, this longer adaptation period will allow the gastrointestinal tract to adapt to the diet. Regarding the adaptation to the test AA intake, we recommend a reduction from 2 to 1 d as it has been demonstrated that only 8 h is necessary in humans (Elango et al., 2009). Indeed, an adaptation prior to protein intake for 2 d followed by study day adaptation to the test AA intake is the standard protocol used to determine protein and AA requirements in humans of different life stages and health status (Bross et al., 2000; Mager et al., 2003; Tang et al., 2014; Rafii et al., 2015). Thus, we believe that the adaptation period of 7 d to protein intake and of 1 d to test AA intake will not interfere with Phe kinetics and can be used in IAAO studies in the domestic cat. This proposed protocol was successfully used in our subsequent study in which the minimum Phe requirement was determined using four adult cats via the direct AA oxidation method (Lambie et al., 2023). It is worth mentioning that the dietary concentrations of Phe used in the present study were above the estimate derived by Lambie et al. (2023), which is imperative to prevent the use of l-[1-¹³C]-Phe to meet the metabolic needs of Phe. Finally, it would be worth applying this shorter protocol to estimate again the minimum Met requirement in adult cats using the IAAO technique to avoid the sample size limitation encountered in the present study, and thus, to better capture the true population variability. The estimates derived from this study, however, are still important to build on our limited knowledge on AA requirements in adult cats. All predictions are imperfect, and we should bear that in mind when we apply imperfect estimates to propose the nutritional requirements to a heterogenous population.

Lastly, additional factors not assessed in this study may impact the Met requirement in cats. In this species, Met and Cys are precursors to felinine (2-amino-7-hydroxy-5,5-dimethyl-4-thiaheptanoic acid), a putative precursor to a pheromone (Hendriks et al., 2001). The excretion of felinine is regulated by testosterone (Tarttelin et al., 1998); thus, intact male cats excrete larger amounts of felinine compared to neutered males and mature female cats (Hendriks et al., 1995). As such, the minimum Met requirement in intact male cats may be higher than the estimate determined in our study due to a higher utilization of Met and Cys for felinine production. On the other hand, the determined Met requirement may overestimate the requirement of hairless cats due to a lack of utilization of sulfur AA for this fate. Other factors, such as immune status, may also impact the requirement of Met. Immune system stimulation appears to increase the Met and total sulfur AA (Litvak et al., 2013; Rakhshandeh et al., 2014) requirements in pigs likely due to a higher utilization of Met for production of Cys and subsequently, glutathione, and other components involved in the immune response. The impact of the immune system on the Met requirement has yet to be investigated in the domestic cat. Finally, it is noteworthy that our recommendations may underestimate the recommended Met intake of cats eating commercial diets due to lower bioavailability in the latter compared to the experimental diets used in our study, which were supplemented with a highly bioavailable Met source (dl-Met).

Conclusion

The results of this study suggest that the minimum Met requirement, when Cys is provided in excess, for neutered male adult cats at maintenance is higher (upper 95% CL: 0.40% DM, on an energy density of 4,164 kcal ME/ kg DM calculated using the modified Atwater factors) than the current recommendations proposed by NRC (2006; RA: 0.17% DM), the Association of American Feed Control Officials (AAFCO, 2023; 0.20% DM), and the European Pet Food Industry Federation (FEDIAF, 2023; 0.23% DM). An adaptation period to the basal diet of 7 d followed by 1 d adaptation period to the test AA intake should be used in future IAAO studies in cats to potentially increase the sample size and better capture the true population variability.

Glossary

Abbreviations

AA

amino acid

AAFCO

American Association of Feed Control Officials

AI

adequate intake

APE

atom percent excess

BW

body weight

CL

confidence limit

DM

dry matter

F¹³CO₂

flux of ¹³CO₂

FEDIAF

European Pet Food Industry Federation

IAAO

indicator amino acid oxidation

LSTM

lean soft tissue mass

ME

metabolizable energy

MR

minimal requirement

NRC

National Research Council

RA

recommended allowance

VCO₂

volume of CO₂

Funding

This work was supported by funds from the Natural Sciences and Engineering Research Council of Canada Discovery Research Program (#401522).

Author Contributions

Júlia Guazzelli Pezzali: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Validation, Visualization, Writing—original draft. Jocelyn G. Lambie: Investigation, Writing—review & editing. Adronie Verbrugghe: Investigation, Resources, Writing—review & editing. Anna K. Shoveller: Conceptualization, Funding Acquisition, Methodology, Resources, Supervision, Validation, Writing—review & editing.

Conflict of interest statement

The authors declare that there is no conflict of interest regarding the publication. However, it is noteworthy that A.K.S. is the Champion Petfoods Chair in Canine and Feline Nutrition, Physiology, and Metabolism and additionally consults for Champion Petfoods. A.K.S. was previously employed by P&G and Mars Pet Care and has received honoraria and research funding from various commodity groups, pet food manufacturers, and ingredient suppliers.