Higher Net Protein Balance following the Ingestion of Free Range Reindeer compared to Commercial Beef

Wild game consumption has been associated with health benefits, but the influence on protein metabolism remains unknown. We compared the feeding-induced response to free-range reindeer (FR) versus commercial beef (CB) using stable isotope methodology. Seven male and female participants (age: 38±12 years; body mass index: 24±3 kg/m2) completed two studies using a randomized, crossover design in which they ingested 2 oz of FR or CB. L-[ring 2H5]phenylalanine & L-[ring 2H2]tyrosine were delivered via primed, continuous intravenous infusion. Blood samples were collected during the basal period and following consumption of FR or CB. Feeding-induced changes in whole body protein synthesis (PS), protein breakdown (PB), and net protein balance (NB) were determined via analysis of plasma samples for phenyalanine and tyrosine enrichment by gas chromatography mass spectrometry; plasma essential amino acid concentrations were determined by liquid chromatography-electrospray ionization-mass spectrometry. Plasma post-prandial essential amino acid (EAA) concentrations were higher with the ingestion of FR compared to CB (P=0.02). The acute feeding-induced response in PS was not different in either trial, but PB was reduced with the ingestion of FR compared to CB (P<0.0001). The difference in PB contributed to a superior level of NB (P<0.0001). When protein kinetics were normalized relative to the amino acids ingested, PB/EAAs and total amino acids ingested were reduced (P<0.01 and 0.001, respectively) in FR compared to CB; contributing to greater NB/total amino acid ingested (P<0.0001) between FR and CB. We conclude that the nutrient profiles of FR may have a more favorable benefit on protein metabolism Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 20 February 2020 doi:10.20944/preprints202002.0284.v1

compared to CB. These data support the potential health benefits of wild game in the preservation of whole-body protein.

Introduction
Alaska's aging indigenous people are growing in number, and their functional disabilities are disproportionately greater than those experienced by other populations 1,2,3 . In Alaska, the factors responsible for increased functional disabilities are complex due to limited economic competition, higher profit margins, expensive medical infrastructure, and unique environmental and socio-demographic elements 4 .
Such inherent difficulties already contribute to the most extreme healthcare costs in the United States, 2.5 times the national average 5 . Instead of addressing these challenges after individuals reach the older stages of life, healthy aging strategies throughout the lifespan are necessary to counteract the risks of sarcopenia, disability, and rising healthcare costs 6 . Sarcopenia is defined as an age-related decline in muscle mass (reflected by loss of lean tissue), strength, and function 7 . In order to maintain a healthy state of dynamic equilibrium with regard to lean tissue, rates of whole body protein synthesis (PS) and protein breakdown (PB) are modulated by dietary intake and/or physical activity. For PS to occur in response to nutrient ingestion, essential amino acids (EAA's) must be available in sufficient amounts 8 . Consumption of proteins providing EAA's is therefore necessary for PS to exceed PB, and primarily indicates the net balance of muscle protein 9 .
The quality of a dietary protein is reflected by the digestible indispensable amino acid score (DIAAS), and is a function of the amount and profile of EAAs as well as digestibility of that protein 11 . Much research has been done concerning protein quality and quantitiy 10 , but practical questions remain concerning the potential for greater benefits relative to serving in wild game. Free range cervids contain a more desirable balance of ω-6:ω-3 fatty acid ratios 12 . Relevant to protein metabolism, amino acidenriched medical formulas fortified with a balanced provision of omega-6 (ω-6) to omega-3 (ω-3) fatty acid ratios have demonstrated a favorable influence on protein metabolism 13 .
To our knowledge, the acute influence of the same amount of wild game meat compared commercial meat ingestion on protein kinetics in humans has not been evaluated. We have investigated the acute response of human protein metabolism to consumption of of 2 ounces of FR and commercial beef (CB).. FR is similar to the caribou found in Alaska Native traditional diets 14 , and is USDA or state-approved for consumption. We chose 70% lean/30% fat CB for comparison, as it is the mostpurchased meat in the US by millions of pounds 15 . We hypothesized that NBwould be higher in FR compared to CB; due to existing differences in the total amount of protein in FR. If our data support the superior efficacy of FR consumption per ounce on NB, it is our assertion that traditional amino and fatty acid sources such as wild red meats may augment the retention of whole body protein largely through existing differences in total protein intake/serving size.

Materials and Methods
Eight male and female participants were recruited from the Fairbanks, Alaska area through a combination of local newspaper advertisements and flyers posted around the University of Alaska Fairbanks (UAF) campus. All testing occurred at the Clinical Research and Imaging Facility (CRIF) except baseline blood sampling for medical screening performed at Labcorp, Inc (1626 30th Ave, Fairbanks, AK 99701). All participants were properly consented, which included a comprehensive verbal and written review of all procedures, and copies of the consent forms were provided.
Participants completed clinical assessments to determine eligibility during the initial consent and screening visit. Exclusion criteria included a creatinine level of >1.4 and/or a serum glutamate pyruvate transaminase >2 times normal; a resting blood pressure above 160/90 mmHg; previously diagnosed diabetes (fasting blood glucose ≥ 126 mg/dl; history of kidney or liver disease; heart disease as indicated by interventional procedures; recent history of alcoholism; and/or active cancer. Volunteers with a chronic inflammatory condition or taking corticosteroids were not eligible. Volunteers were excluded from the study if the study physician recognized a medical condition or medication that in his opinion represented an unacceptable risk. Participant confidentiality was ensured for all participants. Protected records were kept in a locked file cabinet behind a locked door in a facility with restricted swipe card access. The study was reviewed and approved by the UAF Institutional Review Board (IRB) under protocol 986801-17.
One participant dropped out due to circumstances unrelated to the study.
Eligible participants visited the CRIF at UAF on three separate occasions: a) consent and determination of eligibility status, b) feeding/ tracer study #1, and c) feeding/tracer study #2. The feeding/tracer studies were performed in a randomized, double blind fashion. Using stable isotope methodology on participant visits #1 and #2, the response to the ingestion of equivalent 2 ounce pre-cooked servings of randomly assigned FR or 70% lean/30% fat CB was evaluated. These amounts are consistent with the variable portion sizes listed in the Dietary Guidelines 2015-2020 16 . The nutritional profiles for reindeer and beef are listed in Table 1. A dual-energy X-ray absorptiometry (GE iDXA) scan for the determination of body composition was also performed. with ExpressHT Ultra LC (Eksigent Div.; AB Sciex) after derivatization with 9-fluoren-9methoxycarbonyl, and concentrations were measured using the internal standard method 20 .
Statistical Methods. A one-way ANOVA with repeated measures was used to examine overall differences in plasma essential amino acid concentrations between FR and CB; a Bonferroni post-test was used to detect potential differences in the plasma concentrations of essential amino acids at individual time points. A two-way ANOVA was utilized to evaluate differences in PS, PB, and NB in response to the ingestion of FR or CB. All data were analyzed using the Graphpad Prism 6 for Mac (Graphpad Software, Inc. La Jolla CA), and presented as mean ± SD.

Results
Eight participants (40.4±14.0 years of age) with a body mass index (BMI) of 24.0±2.9 kg/m 2 were recruited and enrolled in the study ( Table 2). All participants provided their own transportation to the CRIF at UAF. One participant dropped out due to a personal matter completely unrelated to the study. Therefore, seven participants completed all aspects of the study; reflected in Table 2. Plasma concentrations of essential amino acids were higher with FR compared to CB at t=240 and 270 min (Figure 1). The EAA and branched chain amino acids area under the curve (AUC) during the fed state were also greater following FR consumption (P=0.009 and P=0.04, respectively), but there were no differences in leucine AUC (data not shown). Basal post-absorptive rates of PS, PB, and NB were not different in FR and CB. As previously described in feeding studies using identical stable isotope methodologies 9,13 , we have calculated and expressed PS, PB, and NB relative to their changes from the fasted to the fed state. While there was a similar increase in PS after FR and CB ingestion, the heightened suppression of PB (P<0.0001) contributed to a substantially greater enhancement of NB after ingestion of FR (P<0.0001) compared to CB ( Figure 2). We also calculated the rates of PS, PB, and NB relative to the ingested amounts of EAAs and total amino acids. There was a greater reduction in PB/EAAs ingested in FR compared to CB (P<0.01), and there were no significant differences between PS and NB when expressed relative to the amount of EAAs ingested ( Figure   2). PB, with respect to the total amino acids, was suppressed significantly greater after consumption of FR compared to CB (P<0.001); contributing to higher NB/total amino acids ingested with FR as well (P<0.0001) (Figure 2).

Discussion
In this study, we examined the responses of whole body protein kinetics to the dietary ingestion of 2 ounces of FR and CB on whole body protein kinetics in humans.
We demonstrated significantly greater increases in plasma EAAs after consumption of FR compared to CB. Despite these differences in plasma concentrations of EAAs, the PS response to FR was not greater than that to CB. Increased suppression of PB contributed to superior differences in NB after ingestion of FR compared to CB. The profile of amino acid composition was apparently linked to greater whole body protein retention, as the NB after FR consumption was greater than CB even when normalized for the amount of total amino acids ingestedTo our knowledge, these data represent the first acute feeding studies to illustrate the superior benefits of free-range red meat on direct changes in protein metabolism.
Modulation of PS occurs in conjunction with alterations in PB during fasting and fed conditions and/or physical activity 21 . For the most part, changes in PS and PB reflect feeding or activity-based changes in muscle protein synthesis and breakdown 21 .
Consumption of nutrients providing EAA's is necessary in order to maintain optimal NB 22 . Differences in the total amount of amino acids ingested were linked to higher plasma EAA concentrations following ingestion of FR, and may have contributed to the suppression of PB 9,13 . It is particularly interesting that in another recent study, the suppression of PB was even greater in response to 4 ounces and 11 ounces of red meat ingested, respectively 23 . Therefore, the influence of the total amount of amino acids ingested seems to remain consistent across the dose response; supporting the rationale behind the importance of the amount of dietary protein in the modulation of NB 9 .
It is well accepted that EAAs are largely responsible for the stimulation of muscle protein synthesis 24 . Non-essential amino acids are not primary factors in the stimulation of muscle protein synthesis 8 . That being said, significant correlations have been reported between overall protein intake and NB (R 2 =0.765; P<0.0001) 25 . This correlation indicates the potential importance of non-essential amino acids and overall amino acid intake on the suppression of PB. This assertion is further supported by the consensus that the delivery of 0.24 g of amino acids/kg/meal in 2 oz of FR equates to the maximal response of PS relative to the overall influence on NB 26 . While isolating the individual contributions of each non-essential amino acid is beyond the scope of the current investigation, the ingestion of arginine, glutamine, glutamate, proline, and glycine have all been implicated in the preservation of lean tissue 27 . Recommendations for amino acid take (essential and non-essential) should consider variations in growth rates, reproductive status, and aging, 28 and that the "indispensable" characterization of non-essential amino acids may represent an oversimplification 29 . In addition to the importance of amino acids in the modulation of protein kinetics, the beneficial influence of ω-3 fatty acids on protein metabolism has also gained some attention 30,31 . Many of the benefits of ω-3 fatty acids are derived from potential improvements in the sensitivity of mTOR and downstream signaling targets that positively affect muscle protein synthesis 32 . In our study, there were no differences in PS between FR and CB. Even though the combined ω-3 fatty acids eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA) was just 51 mg from FR, CB provided neither one. The relatively small amount of ω-3 fatty acids may not have produced a sufficient stimulus to augment PS. Nonetheless, substantial evidence suggests that EPA may mitigate protein degradation 33 , potentially through its inhibitory influence on the NF-κB pathway 34 . DHA has also been demonstrated to reduce protein degradation through the PPARγ/ NF-κB pathway 34 . Data from these mechanistically-oriented studies corresponds with favorable findings from clinical studies that support the role of ω-3 fatty acids on maintenance of lean tissue mass and physical function 35,36 . Therefore, differences in the amount of DHA and EPA may have contributed to the differences in NB between FR and CB.
The interactive combination of amino acids and ω-3 fatty acids is particularly interesting given that the consumption of ω-6 to ω-3 fatty acids has progressively increased from the 1:1 ratio of our hunter/gatherer ancestors to 20:1 or more in our modern, Westernized diets [37][38] . Alterations in ω-6 to ω-3 fatty acid ratios have increased in conjunction with the widespread availability of highly processed foods, and comingled with a reduced consumption of wild and natural proteins with higher overall protein quality. Transformations in these nutritive proportions coincide with gradual and deleterious declines in metabolic health, as progressive elevations in ω-6 to ω-3 fatty acid ratios have been linked to increases in obesity and other chronic illnesses 39