Journal of the American Association for Laboratory Animal Science Copyright 2016 by the American Association for Laboratory Animal Science
Vol 55, No 2 March 2016 Pages 137–146
Plasma Metabolomics of Common Marmosets (Callithrix jacchus) to Evaluate Diet and Feeding Husbandry Sophia A Banton,1 Quinlyn A Soltow,1,† Ken H Liu,1 Karan Uppal,1 Daniel E L Promislow,2 Michael L Power,3 Suzette D Tardif,4 Lynn M Wachtman,5,‡ and Dean P Jones1,* Common marmosets (Callithrix jacchus) are an important NHP model for the study of human aging and age-related diseases. However, the full potential of marmosets as a research model has not been realized due to a lack of evidence-based, standardized procedures for their captive management, especially regarding diet and feeding husbandry. In the present study, we conducted a high-resolution metabolomics analysis of plasma from marmosets from a 3-mo dietary crossover study to determine whether significant metabolic differences occur with a semisynthetic chemically defined (purified) diet as needed for controlled nutrition research. Marmosets were fed a standard, diverse-ingredient diet, followed by a semisynthetic purified diet, and then were switched back to the standard diet. The standard diet used in this analysis was specific to the animal facility, but it is similar in content to the diets currently used for other marmoset colonies. High-resolution metabolomics of plasma with liquid chromatography–mass spectrometry and bioinformatics was used to measure metabolic differences. The concentration of the essential amino acids methionine, leucine/isoleucine, lysine, and threonine were higher when marmosets were fed the purified diet. In contrast, phenylalanine concentrations were higher during exposure to the standard diet. In addition, metabolic pathway enrichment and analysis revealed differences among metabolites associated with dopamine metabolism and the carnitine shuttle. These results show that diet-associated differences in metabolism occur in marmosets and suggest that additional nutritional studies with detailed physiologic characterization are needed to optimize standard and purified diets for common marmosets.
The common marmoset (Callithrix jacchus) is a small New World NHP with average and maximum lifespans roughly 50% of those of Old World species, such as macaques. Its small size, short lifespan, and early maturation make this species a valuable NHP model for the study of aging and chronic disease.30,34 In addition, marmosets share several similarities with humans, making this NHP model suitable for understanding common metabolic processes. Like humans, the natural diet of marmosets is omnivorous. The full potential of marmosets as a research model for aging has not been realized due to a lack of evidence-based, standardized procedures for their captive management, especially regarding diet and feeding husbandry.1,2,18,35,36,38 Diverse foods (including fruits, legumes, dairy products, seeds, nuts, and insects) are used to ensure sufficient caloric intake.25,27 This practice leads to wide variation in carbohydrate, fat, protein, vitamin, and mineral intake. In addition, food sources and consistency vary (extruded pellet, canned, and purified or gelled diets), and commercial ‘base diets’ might comprise a smaller percentage of the daily nutrient intake in marmosets than in other research primates. These approaches result in high variaReceived: 26 Feb 2015. Revision requested: 27 Apr 2015. Accepted: 19 Jun 2015. 1Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, Clinical Biomarkers Laboratory, Emory University, Atlanta, Georgia; 2Department of Pathology, University of Washington, Seattle, WA; 3Nutrition Laboratory, Smithsonian Conservation Biology Institute, National Zoological Park, Washington, District of Columbia; 4Southwest National Primate Research Center, San Antonio, Texas; and 5New England Primate Research Center, Harvard University, Southborough, Massachusetts. *Corresponding author. Email: [email protected] Current addresses: †Amplyx Pharmaceuticals, San Diego, California, and ‡Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts
tion in the nutrient intake of marmosets both within and among colonies, thus perhaps contributing unnecessary variation in experimental outcomes. Marmosets have food preferences both in nature and in research facilities, and these preferences have been used in the formulation of the diets that are fed to marmosets in captivity.19,33 Although food preferences need to be considered when feeding laboratory animals, the diet must provide the appropriate mix of nutrients to support health and wellbeing. This requirement is not only an ethical obligation; it also enhances the reliability of biomedical studies and thus the value of the animal model. Laboratory diets fed to marmosets have been associated with diet-dependent early-onset weight gain and obesity.38 Compared with their counterparts in the wild, laboratory marmosets tend to maintain higher body weights, typically 350 to 400 g compared with the 320 to 340 g observed for marmosets in the wild.31 Similarly, inappropriate diet and nutrition might contribute to the intestinal disease that is common in marmoset colonies. Cumulatively, the manner in which marmosets are fed affects their growth, breeding, disease resistance, lifespan, and susceptibility to stress.5,38 High-resolution metabolomics methods allow the measurement of diverse markers of endogenous metabolism.14 For instance, a comparison of 7 mammalian species, including marmosets, revealed 1945 common metabolites, including amino acids, carbohydrates, lipids, and environmental chemicals.23 Other studies used this method to study essential and nonessential amino acids,28 fatty acid and branched-chain amino acid metabolism,7 bile acids, polyamines, complex lipids, and bacterial products associated with human disease3,4,20,22,29 and 137
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Figure 1. Food consumed by each marmoset on the baseline and NE diets.
Figure 2. Plasma metabolites in the marmoset that are significantly associated with a change to the purified diet from the baseline and NE diets. (A) Type 1 Manhattan plot showing the –log P for each metabolite (mass:charge feature) as a function of its mass:charge ratio (m/z). (B) Type 2 Manhattan plot showing the –log P for each metabolite as a function of chromatographic retention time. The 252 statistically significant features are shown in green above the dashed horizontal line (raw P less than 0.05).
immunity.15,26 Therefore, this platform provides a practical approach to study the potential effects of diet on metabolism in marmosets. The purpose of the current study was to test whether changes occur in the plasma metabolome of marmosets when they are switched from the standard diverse-ingredient diet with various natural foods to a previously described semisynthetic diet consisting of defined amounts of lactalbumin, dextrin, sucrose, soybean oil, cellulose, and a vitamin–mineral mix;33 this purified diet serves as part of the base diet for the marmoset colony at the Southwest National Primate Research Center. The ‘NE diet’ is the standard diet in the New England Primate Research Center, which is similar but not identical to that used in other marmoset facilities. We used a nutritional crossover design in which marmosets received the NE diet for 2 wk, followed by the purified diet for 2 wk, and then the NE diet again for 2 wk. A sample representing the baseline for the NE diet was
collected from each marmoset on study entry. High-resolution metabolomics, consisting of liquid chromatography combined with high-resolution mass spectrometry, was used to measure metabolites in plasma. Biostatistic, bioinformatic, and pathway enrichment methods were used to evaluate metabolic differences.
Materials and Methods
Animals. Common marmosets (Callithrix jacchus; age, 24 to 72 mo; 4 male and 4 female) from the New England Primate Research Center (Southborough, MA) were selected for the study, and each marmoset was exposed to all treatment conditions in a crossover experimental design. The marmosets were housed at the New England Primate Research Center and maintained in accordance with the Guide for the Care and Use of Laboratory Animals.12 The facility was AAALAC-accredited, and all work
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Figure 3. Principal component (PC) analysis of the marmoset plasma metabolome. Each point or circle represents a specific time point (0, 2, 2, and 2 wk) on a specific diet (baseline, NE diet, purified diet, and return to NE diet) for each marmoset (nos. 1 through 8; colored uniquely). The bar plot shows the R-squared (proportion of variation) explained by each principal component.
was approved by Harvard Medical School’s Standing Committee on Animals (IACUC). The study design involved the collection of plasma 1) at baseline (on study entry), 2) after feeding the NE diet for 2 wk, 3) after feeding the purified diet for 2 wk, and 4) after returning to and feeding the NE diet for another 2 wk. To prevent any stress due to changes in environment, the marmosets experienced no change in housing. Healthy marmoset pairs were selected, and the entire study was performed in their home cages; 4 marmosets were housed in male–female pairs, one pair of males was housed in a single cage, and 2 females were housed individually. The male marmosets housed in male–female pairs had been vasectomized previously to prevent productive breeding. The marmosets were checked twice daily by a single
animal-care technician, who reported no significant changes in appetite. In addition, the marmosets were checked at least daily by a research technician and clinical veterinarian to ensure that the animals were eating the supplied food. Body composition was evaluated biweekly by using EchoMRI (Houston, TX), and all marmosets maintained their body weight within 10% of their baseline weight for the duration of the study. The NE diet contained marmoset chow (New World Primate Chow 8791, Harlan Teklad, Indianapolis, IN) and a canned marmoset formula (Marmoset Diet Canned, ZuPreem, Shawnee, KS) and was supplemented with fresh fruits, vegetables, seeds, eggs, and mealworms (Figure 1). The purified diet was a base colony diet described previously33 and consisted of defined amounts of lactalbumin, dextrin, sucrose, soybean oil, cellulose, and a 139
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Figure 4. Metabolite intensities of the significant metabolites that were differentially expressed between the NE and purified diets. (A) The top 252 significant metabolites (raw P < 0.05). (B) Putative identification of the metabolites by using Mummichog15 (P < 0.05). The ordering of the marmosets is the same for each diet cluster. Blue hues indicate decreased intensities, and red hues indicate increased intensities. Confirmed metabolite identities include phenylalanine, carnitine, leucine and isoleucine, threonine, methionine, dopamine, taurine, and creatine.
vitamin–mineral mix.33 Water purified by reverse osmosis was provided free choice in glass bottles when the NE and purified diets were provided; during feeding of the baseline diet, marmosets had unrestricted access to city water in polycarbonate bottles. The husbandry of this marmoset colony has been described previously.32 Metabolomics. Plasma samples were collected into EDTA after marmosets were sedated with ketamine (20 mg/mL IM; Ketaset, Fort Dodge Animal Health, Fort Dodge, IA). Plasma samples were frozen, shipped on dry ice from Harvard University (Cambridge, MA) to Emory University (Atlanta, GA), and maintained at –80° until analysis. Thawed samples were processed and analyzed according to a previously described protocol.13 Briefly, each sample was run in duplicate by using a 10-µL injection volume, with separation by C18 reversephase chromatography (Higgins Analytical, Targa, 2.1 × 10 cm) over an acetonitrile gradient containing a mixture of 14 stable isotope internal standards,13 electrospray ionization, and detection at mass-to-charge ratio of 85 to 2000 and a resolution of 60,000 (LTQ-Velos Orbitrap, Thermo Scientific, Waltham, MA). Feature eetection and extraction. Data from liquid chromatography–high-resolution mass spectrometry were collected and preprocessed by using file-converter software (XCalibur, Thermo Scientific). apLCMS37 was used for feature extraction. A metabolic feature was defined as a specific mass-to-charge ratio coupled with its retention time and associated ion intensity;
14,799 features were detected. Data were log2-transformed, normalized by total ion intensity, and underwent quality assessment, including exclusion of data for technical replicates with overall Pearson correlation (r) < 0.70. Statistical testing and feature identification. The metabolite features were averaged for replicates and analyzed pairwise by using the Wilcoxon signed rank test, t test, or ANOVA (P < 0.05). For each comparison, the list of statistically significant features underwent pathway analysis with Mummichog16 to identify metabolic pathways and modules. The Mummichog software predicts functional activity directly from spectral feature tables without a priori identification of metabolites. The Mummichog analysis also produces tentative metabolite annotation, by combining prior metabolic knowledge and database record matching. The Kyoto Encyclopedia of Genes and Genomes (KEGG) identifiers from the Mummichog results were then used to classify metabolites with KEGG Brite and KEGG Pathway mapping (http://www.genome.jp/kegg/compound/). The collective metabolic reactions were visualized as network figures by using Cytoscape.17
Results
Effects of feeding the NE and purified diets. One-way ANOVA followed by post-hoc t tests revealed 252 metabolites that were differentially expressed (P < 0.05) across the dietary conditions (Figure 2). Statistical significance was preserved for 120 metabo-
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Figure 5. Micromolar concentrations of essential amino acids in marmoset plasma that were significantly (P < 0.05) affected by a change from the NE diet to the purified diet. With our methods, leucine and isoleucine coeluted with identical mass:charge ratios and therefore were indistinguishable. Error bars represent the standard error (σ/√n).
lites at a false-discovery rate of 0.2 and for 36 metabolites at a false-discovery rate of 0.1. Both the charge-to-mass ratio (Figure 2 A) and retention time (Figure 2 B) of the features were used for subsequent pathway analysis and metabolite annotation. Metabolomic profiles associated with the purified and NE diets. No differences in appetite or weight were apparent during any of the feeding periods. The consistency of response within individual marmosets was visualized by principal component analysis (Figure 3) and confirmed by ANOVA. The resulting plot shows each individual marmoset’s time points on the different diets grouped together closely for all but 1 of the 8 marmosets, suggesting that the animal-specific aspect to the response is minimal. A within-subjects ANOVA showed diet-associated metabolic differences (Figure 4 A); this finding was confirmed by t tests (P < 0.05) to be associated with the purified diet. Some metabolites increased from baseline levels when marmosets were fed the purified diet and decreased when they returned to the NE diet. The list of features was annotated by using Mummichog (Figure 4 B), and it includes metabolites from a range of biologic processes. Metabolites that were differentially expressed due to the change in diet included several essential amino acids (Figure 5). The micromolar concentration of each amino acid was estimated by using the reference standards that were analyzed alongside the samples. The plasma phenylalanine concentration was higher when marmosets were fed NE diet (baseline vs purified, P = 0.0273; NE vs purfied ,P = 0.0163; return to NE vs purified, P
= 0.0375), whereas the concentrations of methionine, threonine, leucine and isoleucine, and lysine were higher when marmosets were fed the purified diet (P < 0.05). Biologic pathways affected during feeding of the NE diet compared with the purified diet. Pathway analysis was completed with Mummichog by using the list of metabolic features differentially expressed (P < 0.05) between the baseline (first measure of the NE diet), purified, and NE diets for each pairwise comparison. When mapped to the KEGG database, the significant metabolites were enriched in several pathways (Figure 6). Enriched pathways included various forms of amino acid metabolism and the carnitine shuttle (Table 1). The P values in Table 1 are dependent on the pathway size (that is, the number of features detected in the known biologic pathways from the entire data set and the overlap size) and the number of statistically significant metabolites that belong to the pathway of interest. Biochemical networks altered during feeding of the NE diet compared with the purified diet. Using Mummichog revealed 3 prominent metabolic networks with strong statistical confidence (α = 0.05). The first of these networks (Figure 7 A) includes a core framework of mostly amino acids and their derivatives. The expression levels of these metabolites varied 6-fold, and the majority were more highly expressed in the purified diet. This amino acid network was first detected for the comparison between the baseline and purified diets and then confirmed in subsequent comparisons between the NE and purified diets (Figure 7 B and C). Each figure shows the chemical activity of 141
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Table 1. Pathway analysis of significant metabolite features for the baseline compared with purified diet, NE compared with purified diet, and return to NE compared with purified diet Overlap
Pathway
Enrichment
size
size
P
P
Glycine, serine, alanine, and threonine metabolism
10
27
0.1085
0.0030
Aspartate and asparagine metabolism
14
33
0.0188
0.0014
Lysine metabolism
8
16
0.0256
0.0016
Valine, leucine, and isoleucine degradation
6
17
0.2317
0.0087
Methionine and cysteine metabolism
5
18
0.4807
0.0373
Carnitine shuttle
7
14
0.0366
0.0019
Glycine, serine, alanine, and threonine metabolism
10
27
0.0085
0.0009
Aspartate and asparagine metabolism
8
33
0.1763
0.0042
Lysine metabolism
6
16
0.0381
0.0014
Valine, leucine and isoleucine degradation
7
17
0.0146
0.0010
Methionine and cysteine metabolism
5
18
0.1720
0.0055
Carnitine shuttle
4
14
0.1980
0.0083
Glycine, serine, alanine and threonine metabolism
11
27
0.0017
0.0017
Aspartate and asparagine metabolism
11
33
0.0104
0.0019
Lysine metabolism
6
16
0.0319
0.0027
Valine, leucine and isoleucine degradation
6
17
0.0429
0.0031
Methionine and cysteine metabolism
4
18
0.3313
0.0322
Carnitine shuttle
4
14
0.1785
0.0125
Baseline vs purified diet
Model
NE diet vs purified diet
Return to NE diet vs purified diet
Figure 6. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway mapping of marmoset plasma metabolites associated with a change from the NE diet to the purified diet. The black dots represent metabolites in the pathways that were identified by using Mummichog.15
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Figure 7. Plasma amino acid concentrations affected by changes between the baseline, NE, and purified diets. The metabolic network shown here represents output from Mummichog15 and occured in all 3 comparisons: (A) Baseline compared with purified diet. (B) NE diet compared with purified diet. (C) Return to NE diet compared with purified diet. Blue metabolites are increased in the purified diet, whereas red, green, and purple metabolites are increased in the baseline, NE, and return to NE diets, respectively.
Table 2. Carnitine metabolites that changed in concentration after feeding of the baseline or NE diet compared with the purified diet Relative to quantity in purified diet Carnitine
NE ↑, Baseline ↑
Propionylcarnitine (C3)
Baseline ↓
Octanoylcarnitine (C8)
NE ↓, Baseline ↓
Palmitoylcarnitine (C16:1)
Baseline ↑
Oleoylcarnitine (C18:1 [n-9])
Baseline ↑
Linoelaidyl carnitine (C18:2 [n-6])
Baseline ↑
Cervonyl carnitine (C22:6 [n-3])
Baseline ↑
↑, metabolite concentration higher than that in marmosets fed the purified diet; ↓, metabolite concentration lower than that in marmosets fed the purified diet.
the network under the various dietary conditions. In addition to the essential amino acids, carnitine metabolites (Table 2) were enriched in the pathway analyses conducted by using Mummichog. As components of the carnitine shuttle, these metabolites are key players in fatty acid metabolism. Notably, the carnitine metabolites that were detected among the paired comparisons with the purified diet differed between the NE and baseline diets, except for octanoyl carnitine, which was lower in both the NE and baseline diets. The third group of metabolites that was strongly enriched in the Mummichog analyses involves several chemicals that are involved in dopamine metabolism. The activity of the network was detected as a biologic module by using Mummichog in the pairwise comparisons (baseline compared with purified diet and NE diet compared with purified diet). The concentration of the 143
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Figure 8. Mean intensity of cortisol (charge-to-mass ratio = 363.215, retention time = 153.89 s) in marmoset plasma for each diet condition. Mean cortisol levels did not differ between diets, as determined by one-way ANOVA (F3,28 = 0.568, P = 0.64). Error bars represent the standard error (σ/√n).
neurotransmitter was lower in marmosets at baseline and after feeding the NE diet (baseline NE vs purified, P = 0.0275) and higher in marmosets fed the purified diet (Figure 4 B).
Discussion
Optimal nutrition is integral to maintaining the health of laboratory-housed common marmosets (Callithrix jacchus). Marmosets currently are used as a model for numerous human diseases and conditions, and a lack of health in these animals might skew or prompt skepticism regarding the findings of disease studies. As a statistical confounder, poor health might either exaggerate or diminish the significance of study outcomes. Our findings demonstrate that the metabolic profiles of the purified and NE diets differed in common marmosets. Specifically, our findings revealed that the plasma concentrations of some essential amino acids, some carnitines, and dopamine were higher in marmosets fed the purified diet than when marmosets received the NE diet. The most pronounced difference between the purified and NE diets was the differential expression of the essential amino acids. This finding was reinforced by both pathway analysis and module selection (Table 1 and Figure 7), which showed elevated levels of methionine, leucine and isoleucine, lysine, and threonine and decreased levels of phenylalanine in marmosets fed the purified diet compared with the NE diet. The high abundance of phenylalanine in the plasma of the common marmoset relative to the other essential amino acids has been reported previously in a metabolome-wide association study of phenylalanine in this species.6 Whether this particular profile of phenylalanine concentration in the plasma is specific to marmosets or an effect of the diets they are fed has yet to be determined. Secondary to this observation was the metabolic enrichment of carnitine, a metabolite essential for mitochondrial metabolism of fatty acids. Because marmosets are frequently used as an aging model for humans, the differences in the plasma concentrations of carnitine and the essential amino acids associated with the 2 diets are biologically noteworthy. Available evidence indicates that amino acid fluctuation contributes to the
response of lifespan to dietary restrictions.9 Similarly, a decline in mitochondrial function has been associated with aging and senescence in flies,10 mice,11 and humans.8,21 Carnitines are essential for the mitochondrial entry of fatty acids for initiation of the TCA cycle, and therefore the lower levels observed with the purified diet or the higher levels associated with the NE diet might inadvertently diminish or improve the overall health of laboratory marmosets during aging. A diet-associated effect also was noted for the neurotransmitter dopamine. We observed that dopamine levels were lower in marmosets when they were fed the NE diet and higher when they received the purified diet. This observation is integral not only for the present study but also for the body of work that has been produced by using marmosets as an animal model for diseases such as Parkinson disease. In Parkinson disease, dopamine levels are decreased due to the physical loss of the neurons that secrete the neurotransmitter. The role of dopamine in the mammalian system far exceeds its involvement in Parkinsonism and other neurologic diseases. Notably, dopamine is heavily involved in stress response mechanisms. Because the current methodology uses a peripheral measure, additional studies are necessary to determine whether the observed dopamine effect is central or peripheral in common marmosets. The present study was too brief to evaluate possible regression of the subjects toward the mean concentrations of the essential amino acids over time. This aspect might be evaluated in a comparable study during which the marmosets receive the 2 diets for longer than 2 wk. Similarly, the potential involvement of dopamine may be an artifact of the stress experienced by the marmosets when the diets were switched. However, no pronounced differences were apparent between the baseline and subsequent samples taken while the marmosets consumed the NE diet. Furthermore, targeted examination of cortisol, detected at a charge-to-mass ratio of 363.215 and retention time of 154 s (Figure 8), did not reveal any evidence for increased stress associated with change in diet. Finally, in the wild, common marmosets consume a substantial amount of gum, a β-linked complex polysaccharide, for which its digestive system has been specialized.24 Most primate research centers offer gums (for example, acacia gum) as an enrichment item, but these are not a main component of the diet. Therefore, laboratories are currently unable to feed marmosets gums in a way that mimics the natural diet. The future standardization of marmoset diets will need to address the absence of this critical nutrient. The present study makes use of new advances in high-resolution metabolomics, which permit the detection of thousands of potential metabolites in a single experiment.14 Although our focus was the outlining of the most suitable diet for marmosets in captivity, the ability to view the study within the framework of nutritional metabolomics should not be overlooked. The present study demonstrates the ability to profile the metabolomic changes of subjects who consume different diets at different times. For example, the heat map (Figure 4) follows each marmoset over time as it is introduced to the NE diet, switched to the purified diet, and then returned to the NE diet. For each of the annotated metabolites (Figure 4 B), we can track its ion intensity as the in vivo conditions under which it was measured changes. In addition to showcasing the effect of the purified diet on the health of laboratory marmosets, this study demonstrates the feasibility of providing personalized diets and nutrition forecasting and, with minor modifications, the personalization of medical and nutritional interventions.
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Acknowledgments
This research was supported by NIA grant AG038746 (DPJ, DEP, LW). The development of the purified diet was supported by grants RR02022 (ST) and DK077639 (ST and MP) and by the Southwest National Primate Research Center (P51 OD011133).
References
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19. Mitura A, Liebert F, Schlumbohm C, Fuchs E. 2012. Improving the energy and nutrient supply for common marmoset monkeys fed under long-term laboratory conditions. J Med Primatol 41:82–88. 20. Neujahr DC, Uppal K, Force SD, Fernandez F, Lawrence C, Pickens A, Bag R, Lockard C, Kirk AD, Tran V, Lee K, Jones DP, Park Y. 2014. Bile acid aspiration associated with lung chemical profile linked to other biomarkers of injury after lung transplantation. Am J Transplant 14:841–848. 21. Noland RC, Koves TR, Seiler SE, Lum H, Lust RM, Ilkayeva O, Stevens RD, Hegardt FG, Muoio DM. 2009. Carnitine insufficiency caused by aging and overnutrition compromises mitochondrial performance and metabolic control. J Biol Chem 284:22840–22852. 22. Osborn MP, Park Y, Parks MB, Burgess LG, Uppal K, Lee K, Jones DP, Brantley MA Jr. 2013. Metabolome-wide association study of neovascular age-related macular degeneration. PLoS One 8:e72737. 23. Park YH, Lee K, Soltow QA, Strobel FH, Brigham KL, Parker RE, Wilson ME, Sutliff RL, Mansfield KG, Wachtman LM, Ziegler TR, Jones DP. 2012. High-performance metabolic profiling of plasma from seven mammalian species for simultaneous environmental chemical surveillance and bioeffect monitoring. Toxicology 295:47–55. 24. Power ML. 2010.Nutritional and digestive challenges to being a gum-feeding primate, p 25–44. In: Burrows AM, Nash LT, editors. The evolution of exudativory in primates. New York (NY): Springer. 25. Power ML, Toddes B, Koutsos L. 2012. Chapter 10. nutrient requirements and dietary husbandry principles for captive nonhuman primates, p 269–286. In Abee CR, Mansfield K, Tardif S, Morris T. editors. Nonhuman primates in biomedical research: biology and management, 2nd ed. Boston (MA): Academic Press. 26. Ravindran R, Khan N, Nakaya HI, Li S, Loebbermann J, Maddur MS, Park Y, Jones DP, Chappert P, Davoust J, Weiss DS, Virgin HW, Ron D, Pulendran B. 2014. Vaccine activation of the nutrient sensor GCN2 in dendritic cells enhances antigen presentation. Science 343:313–317. 27. Rensing S, Oerke A-K. 2005. Husbandry and management of New World Species: marmosets and tamarins, p 145–162. In: Wolfe-Coote S, editor. The laboratory primate. Philadelphia (PA): Elsevier. 28. Roede JR, Park Y, Li S, Strobel FH, Jones DP. 2012. Detailed mitochondrial phenotyping by high-resolution metabolomics. PLoS One 7:e33020. 29. Roede JR, Uppal K, Park Y, Lee K, Tran V, Walker D, Strobel FH, Rhodes SL, Ritz B, Jones DP. 2013. Serum metabolomics of slow vs rapid motor progression Parkinson disease: a pilot study. PLoS One 8:e77629. 30. Ross CN, Davis K, Dobek G, Tardif SD. 2012. Aging phenotypes of common marmosets (Callithrix jacchus). J Aging Res 2012: 567143. 31. Ross CN, Power ML, Artavia JM, Tardif SD. 2013. Relation of food intake behaviors and obesity development in young common marmoset monkeys. Obesity (Silver Spring) 21:1891–1899. 32. Soltow QA, Strobel FH, Mansfield KG, Wachtman L, Park Y, Jones DP. 2011. High-performance metabolic profiling with dual chromatography–Fourier-transform mass spectrometry (DCFTMS) for study of the exposome. Metabolomics 9:S132–S143. 33. Tardif S, Jaquish C, Layne D, Bales K, Power M, Power R, Oftedal O. 1998. Growth variation in common marmoset monkeys (Callithrix jacchus) fed a purified diet: relation to care-giving and weaning behaviors. Lab Anim Sci 48:264–269. 34. Tardif SD, Mansfield KG, Ratnam R, Ross CN, Ziegler TE. 2011. The marmoset as a model of aging and age-related diseases. ILAR J 52:54–65. 35. Tardif SD, Power ML, Ross CN, Rutherford JN, Layne-Colon DG, Paulik MA. 2009. Characterization of obese phenotypes in a small nonhuman primate, the common marmoset (Callithrix jacchus). Obesity (Silver Spring) 17:1499–1505.
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36. Wachtman LM, Kramer JA, Miller AD, Hachey AM, Curran EH, Mansfield KG. 2011. Differential contribution of dietary fat and monosaccharide to metabolic syndrome in the common marmoset (Callithrix jacchus). Obesity (Silver Spring) 19: 1145–1156. 37. Yu T, Park Y, Johnson JM, Jones DP. 2009. apLCMS—adaptive processing of high-resolution LCMS data. Bioinformatics 25:1930– 1936. 38. Ziegler TE, Sosa ME, Peterson LJ, Colman RJ. 2013. Using snacks high in fat and protein to improve glucoregulatory function in adolescent male marmosets (Callithrix jacchus). J Am Assoc Lab Anim Sci 52:756–762.Figure 6. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway mapping of marmoset plasma metabolites associated with a change from the NE diet to the purified diet. The black dots represent metabolites in the pathways that were identified by using Mummichog.15
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Plasma Metabolomics of Common Marmosets (Callithrix jacchus) to Evaluate Diet and Feeding Husbandry Sophia A Banton,1 Quinlyn A Soltow,1,† Ken H Liu,1 Karan Uppal,1 Daniel E L Promislow,2 Michael L Power,3 Suzette D Tardif,4 Lynn M Wachtman,5,‡ and Dean P Jones1,* Common marmosets (Callithrix jacchus) are an important NHP model for the study of human aging and age-related diseases. However, the full potential of marmosets as a research model has not been realized due to a lack of evidence-based, standardized procedures for their captive management, especially regarding diet and feeding husbandry. In the present study, we conducted a high-resolution metabolomics analysis of plasma from marmosets from a 3-mo dietary crossover study to determine whether significant metabolic differences occur with a semisynthetic chemically defined (purified) diet as needed for controlled nutrition research. Marmosets were fed a standard, diverse-ingredient diet, followed by a semisynthetic purified diet, and then were switched back to the standard diet. The standard diet used in this analysis was specific to the animal facility, but it is similar in content to the diets currently used for other marmoset colonies. High-resolution metabolomics of plasma with liquid chromatography–mass spectrometry and bioinformatics was used to measure metabolic differences. The concentration of the essential amino acids methionine, leucine/isoleucine, lysine, and threonine were higher when marmosets were fed the purified diet. In contrast, phenylalanine concentrations were higher during exposure to the standard diet. In addition, metabolic pathway enrichment and analysis revealed differences among metabolites associated with dopamine metabolism and the carnitine shuttle. These results show that diet-associated differences in metabolism occur in marmosets and suggest that additional nutritional studies with detailed physiologic characterization are needed to optimize standard and purified diets for common marmosets.
The common marmoset (Callithrix jacchus) is a small New World NHP with average and maximum lifespans roughly 50% of those of Old World species, such as macaques. Its small size, short lifespan, and early maturation make this species a valuable NHP model for the study of aging and chronic disease.30,34 In addition, marmosets share several similarities with humans, making this NHP model suitable for understanding common metabolic processes. Like humans, the natural diet of marmosets is omnivorous. The full potential of marmosets as a research model for aging has not been realized due to a lack of evidence-based, standardized procedures for their captive management, especially regarding diet and feeding husbandry.1,2,18,35,36,38 Diverse foods (including fruits, legumes, dairy products, seeds, nuts, and insects) are used to ensure sufficient caloric intake.25,27 This practice leads to wide variation in carbohydrate, fat, protein, vitamin, and mineral intake. In addition, food sources and consistency vary (extruded pellet, canned, and purified or gelled diets), and commercial ‘base diets’ might comprise a smaller percentage of the daily nutrient intake in marmosets than in other research primates. These approaches result in high variaReceived: 26 Feb 2015. Revision requested: 27 Apr 2015. Accepted: 19 Jun 2015. 1Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, Clinical Biomarkers Laboratory, Emory University, Atlanta, Georgia; 2Department of Pathology, University of Washington, Seattle, WA; 3Nutrition Laboratory, Smithsonian Conservation Biology Institute, National Zoological Park, Washington, District of Columbia; 4Southwest National Primate Research Center, San Antonio, Texas; and 5New England Primate Research Center, Harvard University, Southborough, Massachusetts. *Corresponding author. Email: [email protected] Current addresses: †Amplyx Pharmaceuticals, San Diego, California, and ‡Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts
tion in the nutrient intake of marmosets both within and among colonies, thus perhaps contributing unnecessary variation in experimental outcomes. Marmosets have food preferences both in nature and in research facilities, and these preferences have been used in the formulation of the diets that are fed to marmosets in captivity.19,33 Although food preferences need to be considered when feeding laboratory animals, the diet must provide the appropriate mix of nutrients to support health and wellbeing. This requirement is not only an ethical obligation; it also enhances the reliability of biomedical studies and thus the value of the animal model. Laboratory diets fed to marmosets have been associated with diet-dependent early-onset weight gain and obesity.38 Compared with their counterparts in the wild, laboratory marmosets tend to maintain higher body weights, typically 350 to 400 g compared with the 320 to 340 g observed for marmosets in the wild.31 Similarly, inappropriate diet and nutrition might contribute to the intestinal disease that is common in marmoset colonies. Cumulatively, the manner in which marmosets are fed affects their growth, breeding, disease resistance, lifespan, and susceptibility to stress.5,38 High-resolution metabolomics methods allow the measurement of diverse markers of endogenous metabolism.14 For instance, a comparison of 7 mammalian species, including marmosets, revealed 1945 common metabolites, including amino acids, carbohydrates, lipids, and environmental chemicals.23 Other studies used this method to study essential and nonessential amino acids,28 fatty acid and branched-chain amino acid metabolism,7 bile acids, polyamines, complex lipids, and bacterial products associated with human disease3,4,20,22,29 and 137
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Figure 1. Food consumed by each marmoset on the baseline and NE diets.
Figure 2. Plasma metabolites in the marmoset that are significantly associated with a change to the purified diet from the baseline and NE diets. (A) Type 1 Manhattan plot showing the –log P for each metabolite (mass:charge feature) as a function of its mass:charge ratio (m/z). (B) Type 2 Manhattan plot showing the –log P for each metabolite as a function of chromatographic retention time. The 252 statistically significant features are shown in green above the dashed horizontal line (raw P less than 0.05).
immunity.15,26 Therefore, this platform provides a practical approach to study the potential effects of diet on metabolism in marmosets. The purpose of the current study was to test whether changes occur in the plasma metabolome of marmosets when they are switched from the standard diverse-ingredient diet with various natural foods to a previously described semisynthetic diet consisting of defined amounts of lactalbumin, dextrin, sucrose, soybean oil, cellulose, and a vitamin–mineral mix;33 this purified diet serves as part of the base diet for the marmoset colony at the Southwest National Primate Research Center. The ‘NE diet’ is the standard diet in the New England Primate Research Center, which is similar but not identical to that used in other marmoset facilities. We used a nutritional crossover design in which marmosets received the NE diet for 2 wk, followed by the purified diet for 2 wk, and then the NE diet again for 2 wk. A sample representing the baseline for the NE diet was
collected from each marmoset on study entry. High-resolution metabolomics, consisting of liquid chromatography combined with high-resolution mass spectrometry, was used to measure metabolites in plasma. Biostatistic, bioinformatic, and pathway enrichment methods were used to evaluate metabolic differences.
Materials and Methods
Animals. Common marmosets (Callithrix jacchus; age, 24 to 72 mo; 4 male and 4 female) from the New England Primate Research Center (Southborough, MA) were selected for the study, and each marmoset was exposed to all treatment conditions in a crossover experimental design. The marmosets were housed at the New England Primate Research Center and maintained in accordance with the Guide for the Care and Use of Laboratory Animals.12 The facility was AAALAC-accredited, and all work
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Effect of purified diet on marmoset plasma metabolomics
Figure 3. Principal component (PC) analysis of the marmoset plasma metabolome. Each point or circle represents a specific time point (0, 2, 2, and 2 wk) on a specific diet (baseline, NE diet, purified diet, and return to NE diet) for each marmoset (nos. 1 through 8; colored uniquely). The bar plot shows the R-squared (proportion of variation) explained by each principal component.
was approved by Harvard Medical School’s Standing Committee on Animals (IACUC). The study design involved the collection of plasma 1) at baseline (on study entry), 2) after feeding the NE diet for 2 wk, 3) after feeding the purified diet for 2 wk, and 4) after returning to and feeding the NE diet for another 2 wk. To prevent any stress due to changes in environment, the marmosets experienced no change in housing. Healthy marmoset pairs were selected, and the entire study was performed in their home cages; 4 marmosets were housed in male–female pairs, one pair of males was housed in a single cage, and 2 females were housed individually. The male marmosets housed in male–female pairs had been vasectomized previously to prevent productive breeding. The marmosets were checked twice daily by a single
animal-care technician, who reported no significant changes in appetite. In addition, the marmosets were checked at least daily by a research technician and clinical veterinarian to ensure that the animals were eating the supplied food. Body composition was evaluated biweekly by using EchoMRI (Houston, TX), and all marmosets maintained their body weight within 10% of their baseline weight for the duration of the study. The NE diet contained marmoset chow (New World Primate Chow 8791, Harlan Teklad, Indianapolis, IN) and a canned marmoset formula (Marmoset Diet Canned, ZuPreem, Shawnee, KS) and was supplemented with fresh fruits, vegetables, seeds, eggs, and mealworms (Figure 1). The purified diet was a base colony diet described previously33 and consisted of defined amounts of lactalbumin, dextrin, sucrose, soybean oil, cellulose, and a 139
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Figure 4. Metabolite intensities of the significant metabolites that were differentially expressed between the NE and purified diets. (A) The top 252 significant metabolites (raw P < 0.05). (B) Putative identification of the metabolites by using Mummichog15 (P < 0.05). The ordering of the marmosets is the same for each diet cluster. Blue hues indicate decreased intensities, and red hues indicate increased intensities. Confirmed metabolite identities include phenylalanine, carnitine, leucine and isoleucine, threonine, methionine, dopamine, taurine, and creatine.
vitamin–mineral mix.33 Water purified by reverse osmosis was provided free choice in glass bottles when the NE and purified diets were provided; during feeding of the baseline diet, marmosets had unrestricted access to city water in polycarbonate bottles. The husbandry of this marmoset colony has been described previously.32 Metabolomics. Plasma samples were collected into EDTA after marmosets were sedated with ketamine (20 mg/mL IM; Ketaset, Fort Dodge Animal Health, Fort Dodge, IA). Plasma samples were frozen, shipped on dry ice from Harvard University (Cambridge, MA) to Emory University (Atlanta, GA), and maintained at –80° until analysis. Thawed samples were processed and analyzed according to a previously described protocol.13 Briefly, each sample was run in duplicate by using a 10-µL injection volume, with separation by C18 reversephase chromatography (Higgins Analytical, Targa, 2.1 × 10 cm) over an acetonitrile gradient containing a mixture of 14 stable isotope internal standards,13 electrospray ionization, and detection at mass-to-charge ratio of 85 to 2000 and a resolution of 60,000 (LTQ-Velos Orbitrap, Thermo Scientific, Waltham, MA). Feature eetection and extraction. Data from liquid chromatography–high-resolution mass spectrometry were collected and preprocessed by using file-converter software (XCalibur, Thermo Scientific). apLCMS37 was used for feature extraction. A metabolic feature was defined as a specific mass-to-charge ratio coupled with its retention time and associated ion intensity;
14,799 features were detected. Data were log2-transformed, normalized by total ion intensity, and underwent quality assessment, including exclusion of data for technical replicates with overall Pearson correlation (r) < 0.70. Statistical testing and feature identification. The metabolite features were averaged for replicates and analyzed pairwise by using the Wilcoxon signed rank test, t test, or ANOVA (P < 0.05). For each comparison, the list of statistically significant features underwent pathway analysis with Mummichog16 to identify metabolic pathways and modules. The Mummichog software predicts functional activity directly from spectral feature tables without a priori identification of metabolites. The Mummichog analysis also produces tentative metabolite annotation, by combining prior metabolic knowledge and database record matching. The Kyoto Encyclopedia of Genes and Genomes (KEGG) identifiers from the Mummichog results were then used to classify metabolites with KEGG Brite and KEGG Pathway mapping (http://www.genome.jp/kegg/compound/). The collective metabolic reactions were visualized as network figures by using Cytoscape.17
Results
Effects of feeding the NE and purified diets. One-way ANOVA followed by post-hoc t tests revealed 252 metabolites that were differentially expressed (P < 0.05) across the dietary conditions (Figure 2). Statistical significance was preserved for 120 metabo-
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Effect of purified diet on marmoset plasma metabolomics
Figure 5. Micromolar concentrations of essential amino acids in marmoset plasma that were significantly (P < 0.05) affected by a change from the NE diet to the purified diet. With our methods, leucine and isoleucine coeluted with identical mass:charge ratios and therefore were indistinguishable. Error bars represent the standard error (σ/√n).
lites at a false-discovery rate of 0.2 and for 36 metabolites at a false-discovery rate of 0.1. Both the charge-to-mass ratio (Figure 2 A) and retention time (Figure 2 B) of the features were used for subsequent pathway analysis and metabolite annotation. Metabolomic profiles associated with the purified and NE diets. No differences in appetite or weight were apparent during any of the feeding periods. The consistency of response within individual marmosets was visualized by principal component analysis (Figure 3) and confirmed by ANOVA. The resulting plot shows each individual marmoset’s time points on the different diets grouped together closely for all but 1 of the 8 marmosets, suggesting that the animal-specific aspect to the response is minimal. A within-subjects ANOVA showed diet-associated metabolic differences (Figure 4 A); this finding was confirmed by t tests (P < 0.05) to be associated with the purified diet. Some metabolites increased from baseline levels when marmosets were fed the purified diet and decreased when they returned to the NE diet. The list of features was annotated by using Mummichog (Figure 4 B), and it includes metabolites from a range of biologic processes. Metabolites that were differentially expressed due to the change in diet included several essential amino acids (Figure 5). The micromolar concentration of each amino acid was estimated by using the reference standards that were analyzed alongside the samples. The plasma phenylalanine concentration was higher when marmosets were fed NE diet (baseline vs purified, P = 0.0273; NE vs purfied ,P = 0.0163; return to NE vs purified, P
= 0.0375), whereas the concentrations of methionine, threonine, leucine and isoleucine, and lysine were higher when marmosets were fed the purified diet (P < 0.05). Biologic pathways affected during feeding of the NE diet compared with the purified diet. Pathway analysis was completed with Mummichog by using the list of metabolic features differentially expressed (P < 0.05) between the baseline (first measure of the NE diet), purified, and NE diets for each pairwise comparison. When mapped to the KEGG database, the significant metabolites were enriched in several pathways (Figure 6). Enriched pathways included various forms of amino acid metabolism and the carnitine shuttle (Table 1). The P values in Table 1 are dependent on the pathway size (that is, the number of features detected in the known biologic pathways from the entire data set and the overlap size) and the number of statistically significant metabolites that belong to the pathway of interest. Biochemical networks altered during feeding of the NE diet compared with the purified diet. Using Mummichog revealed 3 prominent metabolic networks with strong statistical confidence (α = 0.05). The first of these networks (Figure 7 A) includes a core framework of mostly amino acids and their derivatives. The expression levels of these metabolites varied 6-fold, and the majority were more highly expressed in the purified diet. This amino acid network was first detected for the comparison between the baseline and purified diets and then confirmed in subsequent comparisons between the NE and purified diets (Figure 7 B and C). Each figure shows the chemical activity of 141
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Table 1. Pathway analysis of significant metabolite features for the baseline compared with purified diet, NE compared with purified diet, and return to NE compared with purified diet Overlap
Pathway
Enrichment
size
size
P
P
Glycine, serine, alanine, and threonine metabolism
10
27
0.1085
0.0030
Aspartate and asparagine metabolism
14
33
0.0188
0.0014
Lysine metabolism
8
16
0.0256
0.0016
Valine, leucine, and isoleucine degradation
6
17
0.2317
0.0087
Methionine and cysteine metabolism
5
18
0.4807
0.0373
Carnitine shuttle
7
14
0.0366
0.0019
Glycine, serine, alanine, and threonine metabolism
10
27
0.0085
0.0009
Aspartate and asparagine metabolism
8
33
0.1763
0.0042
Lysine metabolism
6
16
0.0381
0.0014
Valine, leucine and isoleucine degradation
7
17
0.0146
0.0010
Methionine and cysteine metabolism
5
18
0.1720
0.0055
Carnitine shuttle
4
14
0.1980
0.0083
Glycine, serine, alanine and threonine metabolism
11
27
0.0017
0.0017
Aspartate and asparagine metabolism
11
33
0.0104
0.0019
Lysine metabolism
6
16
0.0319
0.0027
Valine, leucine and isoleucine degradation
6
17
0.0429
0.0031
Methionine and cysteine metabolism
4
18
0.3313
0.0322
Carnitine shuttle
4
14
0.1785
0.0125
Baseline vs purified diet
Model
NE diet vs purified diet
Return to NE diet vs purified diet
Figure 6. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway mapping of marmoset plasma metabolites associated with a change from the NE diet to the purified diet. The black dots represent metabolites in the pathways that were identified by using Mummichog.15
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Figure 7. Plasma amino acid concentrations affected by changes between the baseline, NE, and purified diets. The metabolic network shown here represents output from Mummichog15 and occured in all 3 comparisons: (A) Baseline compared with purified diet. (B) NE diet compared with purified diet. (C) Return to NE diet compared with purified diet. Blue metabolites are increased in the purified diet, whereas red, green, and purple metabolites are increased in the baseline, NE, and return to NE diets, respectively.
Table 2. Carnitine metabolites that changed in concentration after feeding of the baseline or NE diet compared with the purified diet Relative to quantity in purified diet Carnitine
NE ↑, Baseline ↑
Propionylcarnitine (C3)
Baseline ↓
Octanoylcarnitine (C8)
NE ↓, Baseline ↓
Palmitoylcarnitine (C16:1)
Baseline ↑
Oleoylcarnitine (C18:1 [n-9])
Baseline ↑
Linoelaidyl carnitine (C18:2 [n-6])
Baseline ↑
Cervonyl carnitine (C22:6 [n-3])
Baseline ↑
↑, metabolite concentration higher than that in marmosets fed the purified diet; ↓, metabolite concentration lower than that in marmosets fed the purified diet.
the network under the various dietary conditions. In addition to the essential amino acids, carnitine metabolites (Table 2) were enriched in the pathway analyses conducted by using Mummichog. As components of the carnitine shuttle, these metabolites are key players in fatty acid metabolism. Notably, the carnitine metabolites that were detected among the paired comparisons with the purified diet differed between the NE and baseline diets, except for octanoyl carnitine, which was lower in both the NE and baseline diets. The third group of metabolites that was strongly enriched in the Mummichog analyses involves several chemicals that are involved in dopamine metabolism. The activity of the network was detected as a biologic module by using Mummichog in the pairwise comparisons (baseline compared with purified diet and NE diet compared with purified diet). The concentration of the 143
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Figure 8. Mean intensity of cortisol (charge-to-mass ratio = 363.215, retention time = 153.89 s) in marmoset plasma for each diet condition. Mean cortisol levels did not differ between diets, as determined by one-way ANOVA (F3,28 = 0.568, P = 0.64). Error bars represent the standard error (σ/√n).
neurotransmitter was lower in marmosets at baseline and after feeding the NE diet (baseline NE vs purified, P = 0.0275) and higher in marmosets fed the purified diet (Figure 4 B).
Discussion
Optimal nutrition is integral to maintaining the health of laboratory-housed common marmosets (Callithrix jacchus). Marmosets currently are used as a model for numerous human diseases and conditions, and a lack of health in these animals might skew or prompt skepticism regarding the findings of disease studies. As a statistical confounder, poor health might either exaggerate or diminish the significance of study outcomes. Our findings demonstrate that the metabolic profiles of the purified and NE diets differed in common marmosets. Specifically, our findings revealed that the plasma concentrations of some essential amino acids, some carnitines, and dopamine were higher in marmosets fed the purified diet than when marmosets received the NE diet. The most pronounced difference between the purified and NE diets was the differential expression of the essential amino acids. This finding was reinforced by both pathway analysis and module selection (Table 1 and Figure 7), which showed elevated levels of methionine, leucine and isoleucine, lysine, and threonine and decreased levels of phenylalanine in marmosets fed the purified diet compared with the NE diet. The high abundance of phenylalanine in the plasma of the common marmoset relative to the other essential amino acids has been reported previously in a metabolome-wide association study of phenylalanine in this species.6 Whether this particular profile of phenylalanine concentration in the plasma is specific to marmosets or an effect of the diets they are fed has yet to be determined. Secondary to this observation was the metabolic enrichment of carnitine, a metabolite essential for mitochondrial metabolism of fatty acids. Because marmosets are frequently used as an aging model for humans, the differences in the plasma concentrations of carnitine and the essential amino acids associated with the 2 diets are biologically noteworthy. Available evidence indicates that amino acid fluctuation contributes to the
response of lifespan to dietary restrictions.9 Similarly, a decline in mitochondrial function has been associated with aging and senescence in flies,10 mice,11 and humans.8,21 Carnitines are essential for the mitochondrial entry of fatty acids for initiation of the TCA cycle, and therefore the lower levels observed with the purified diet or the higher levels associated with the NE diet might inadvertently diminish or improve the overall health of laboratory marmosets during aging. A diet-associated effect also was noted for the neurotransmitter dopamine. We observed that dopamine levels were lower in marmosets when they were fed the NE diet and higher when they received the purified diet. This observation is integral not only for the present study but also for the body of work that has been produced by using marmosets as an animal model for diseases such as Parkinson disease. In Parkinson disease, dopamine levels are decreased due to the physical loss of the neurons that secrete the neurotransmitter. The role of dopamine in the mammalian system far exceeds its involvement in Parkinsonism and other neurologic diseases. Notably, dopamine is heavily involved in stress response mechanisms. Because the current methodology uses a peripheral measure, additional studies are necessary to determine whether the observed dopamine effect is central or peripheral in common marmosets. The present study was too brief to evaluate possible regression of the subjects toward the mean concentrations of the essential amino acids over time. This aspect might be evaluated in a comparable study during which the marmosets receive the 2 diets for longer than 2 wk. Similarly, the potential involvement of dopamine may be an artifact of the stress experienced by the marmosets when the diets were switched. However, no pronounced differences were apparent between the baseline and subsequent samples taken while the marmosets consumed the NE diet. Furthermore, targeted examination of cortisol, detected at a charge-to-mass ratio of 363.215 and retention time of 154 s (Figure 8), did not reveal any evidence for increased stress associated with change in diet. Finally, in the wild, common marmosets consume a substantial amount of gum, a β-linked complex polysaccharide, for which its digestive system has been specialized.24 Most primate research centers offer gums (for example, acacia gum) as an enrichment item, but these are not a main component of the diet. Therefore, laboratories are currently unable to feed marmosets gums in a way that mimics the natural diet. The future standardization of marmoset diets will need to address the absence of this critical nutrient. The present study makes use of new advances in high-resolution metabolomics, which permit the detection of thousands of potential metabolites in a single experiment.14 Although our focus was the outlining of the most suitable diet for marmosets in captivity, the ability to view the study within the framework of nutritional metabolomics should not be overlooked. The present study demonstrates the ability to profile the metabolomic changes of subjects who consume different diets at different times. For example, the heat map (Figure 4) follows each marmoset over time as it is introduced to the NE diet, switched to the purified diet, and then returned to the NE diet. For each of the annotated metabolites (Figure 4 B), we can track its ion intensity as the in vivo conditions under which it was measured changes. In addition to showcasing the effect of the purified diet on the health of laboratory marmosets, this study demonstrates the feasibility of providing personalized diets and nutrition forecasting and, with minor modifications, the personalization of medical and nutritional interventions.
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Effect of purified diet on marmoset plasma metabolomics
Acknowledgments
This research was supported by NIA grant AG038746 (DPJ, DEP, LW). The development of the purified diet was supported by grants RR02022 (ST) and DK077639 (ST and MP) and by the Southwest National Primate Research Center (P51 OD011133).
References
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