Factors influencing vitamin B6 status in domestic cats: age, body condition score, and inflammation

doi:10.21203/rs.3.rs-3338209/v1

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Factors influencing vitamin B6 status in domestic cats: age, body condition score, and inflammation

https://doi.org/10.21203/rs.3.rs-3338209/v1

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published 23 Jan, 2024

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Limited studies are available on the vitamin B6 status in domestic cats. To this end, we evaluated glutamate-oxaloacetate transaminase activity in hemolysates with and without pyridoxal 5’-phosphate supplementation in two feline populations: a cohort of 60 domestic (sexually intact and specific pathogen-free) cats maintained under strictly controlled conditions with optimal diets (Feline Nutrition and Pet Care Center) and a cohort of 57 cats randomly selected between December 2022 to January 2023 that visited the Veterinary Medicine Teaching Hospital to seek care under different circumstances. We analyzed the data based on the animal's age, sex, body condition score, and diagnosis to elucidate the main factors driving vitamin B6 deficiencies in domestic cats. Based on the age and body condition scores, most cats with vitamin B6 deficiency had a low body condition score (41.7%), suggesting low food intake and/or malassimilation, as most were of advanced age (≥7 y old; 66.7%). This result is consistent with the increased energy requirements and compromised digestibility in older cats linked to a higher proportion of underweight elderly cats. When the data of vitamin B6-deficient cats were analyzed in terms of medical diagnosis, most (58.4%) had a condition associated with an increased inflammatory response, whether through an infection (feline coronavirus; 25%) or via diseases associated directly or indirectly with an inflammatory response (33.4%). Thus, our study supports that vitamin B6 supplementation may be indicated in older animals, especially those undergoing an active inflammation process.

Health sciences/Biomarkers/Diagnostic markers

Biological sciences/Physiology/Ageing

Biological sciences/Physiology/Metabolism

The term vitamin B6 refers to six interconvertible compounds that share a 2-methyl-3-hydroxypyridine structure with variable substituents at positions C4 and C5, i.e., pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL), and their phosphorylated derivatives pyridoxine 5′-phosphate (PNP), pyridoxamine 5′-phosphate (PMP) and pyridoxal 5′-phosphate (PLP).

PLP is the coenzyme form of vitamin B6 that is a cofactor in > 160 different catalytic functions, including transamination. Most PLP-dependent enzymes are involved in amino acid metabolism, organic acids, glucose, sphingolipids, and fatty acids, with an essential role in the metabolism of neurotransmitters, such as dopamine, serotonin, glycine, glutamate, γ-aminobutyric acid (GABA)^1,2.

In humans, overt vitamin B6 deficiency caused by dietary insufficiency is rare in developed countries since it is present in most foods. Consequently, B6 deficiency occurs in combination with other B vitamins or is linked to some lifestyle habits (contraceptive drugs³, smoking^4–7, alcoholism^8–11) and diseases (celiac disease¹², diabetes^13,14 or inflammatory conditions^15–18). Secondary vitamin B6 deficiency may result from mutations causing defects in B6 salvage pathways^19,20, inborn errors causing accumulation of intermediates that react with PLP, and intake of drugs that reduce the availability of PLP ²¹.

In addition, epidemiological human studies reported age-dependent changes in vitamin B6 status, which may reflect differences in B6 metabolism ^22–25. Low vitamin B6 intake in humans is associated with an increased risk of age-related diseases⁷ (e.g., cardiovascular disease^26–30 and cancer ^3,31,32); however, conflicting results have been reported on B6 intake and disease risk^33,34. Despite the wealth of studies in humans, no reports are available on B6 status in domestic pets, including cats, and more importantly, as it relates to age.

As the life expectancy of domestic cats increases, the occurrence of behavioral problems seen in senior cats, such as cognitive dysfunction syndrome, is significant. While the cause of this syndrome is unclear, it has been suggested that it may result from age-related neurodegeneration and defects at the neurotransmission levels, in which vitamin B6 plays a crucial role. However, to our knowledge, no thorough vitamin B6 status assessment in cats is available, and less so factors that may contribute to vitamin B6 deficiency. This knowledge gap undermines the resources pet owners and clinicians need to make informed decisions regarding dietary supplements and pet food manufacturers' formulation of senior diets.

To this end, our first goal was to evaluate vitamin B6 status using cat blood. Erythrocyte PLP content is a more relevant marker of vitamin B6 status than plasma PLP content because the former serves as an intracellular enzymatic cofactor ^6,35. It is directly correlated with vitamin B6 intake^36–38, plasma PLP ³⁹, PL^40,41 and PA³⁷, urinary PA excretion ³⁷, and degree of PLP saturation of the erythrocyte aspartate transaminase, responding within weeks to vitamin B6 depletion and repletion ^37,38,42. Furthermore, erythrocyte PLP content seems a more reliable marker under conditions and diseases associated with inflammation ³⁹, altered alkaline phosphatase, and low albumin ⁴¹. However, assessing the total content of PLP does not address the binding of PLP to the apoenzymes to form a functionally active holoenzyme. The functional category includes evaluating PLP-dependent transaminase activities in red blood cells. Red blood cells are rich in the PLP-dependent glutamate-oxaloacetate transaminase (GOT)⁴³, whose activity responds to changes in vitamin B6 status and in-vitro-supplied PLP^44,45. The specific test includes assessing GOT's activity and that induced by the in vitro addition of saturating concentrations of PLP. The results are the primary activation ratio (PAR⁴⁶) and the specific activity ratio obtained with and without PLP addition. Thus, a higher PAR reflects a lower vitamin B6 status. Advantages of evaluating this ratio include overcoming differences related to various methods and subject variability^45,47, in addition to serving as a long-term indicator of vitamin B6 status about the life span of the erythrocytes³⁵ and vitamin B6 intake ⁴⁸. Most importantly, the GOT activity test is not associated with albumin, alkaline phosphatase activity ^49,50, some immune indices⁵¹ and kidney function.

We evaluated GOT activity in hemolysates with and without PLP supplementation in two feline populations: a cohort of 60 domestic (sexually intact and specific pathogen-free) cats maintained under strictly controlled conditions with optimal diets (Feline Nutrition and Pet Care Center School of Veterinary Medicine, the University of California-Davis, named hereafter as Cat Colony) and a cohort of 57 cats randomly selected between December 2022 to January 2023 that were brought to the Veterinary Medicine Teaching Hospital at the University of California-Davis (VMTH) to seek care under different circumstances with available blood samples. Our second goal was to analyze the data based on the animal's age, sex, body condition score, and diagnosis to elucidate the main factors driving vitamin B6 deficiencies in domestic cats.

Vitamin B6 status in domestic research cats

Blood samples were collected from 47 mostly female, specific-pathogen-free, sexually intact cats (41 females, 6 males) ranging from 1 to 17 y old (Fig. 1A). This cohort had an almost equal representation of junior (1–2 y; n = 13), adults (3 to 6 y; n = 11), and mature cats (7–10 y; n = 15) with less representation of seniors (11–14 y; n = 7) and geriatric cats (≥15 y; n = 1).

To assess the overall fitness status, we used the body condition score. This parameter estimates adipose tissue, similar to the human body mass index⁵². The scale used in this study span from 1 (emaciated) to 9 (grossly obese), with a score of 5 being optimal⁵². The average body condition of this cohort indicated that most cats had a healthy body weight (mean ± SD = 5 ± 1). Notably, for those with scores available, the body condition score showed a positive and statistically significant correlation with age (Fig. 1B). The higher body condition score with age may be linked to various medical conditions such as atopic dermatitis, hypertension, diabetes, asthma, ophthalmic diseases, and allergies⁵³; however, all cats at the time of blood withdrawal were healthy with no signs of any of these conditions.

The specific glutamate-oxaloacetate transaminase activity (expressed as units/mg protein) was assessed in all blood samples with and without the supplementation of saturating PLP concentrations. The GOT activity without PLP addition declined steadily with age from 1 to 17 y (Fig. 2A; P = .008), whereas upon PLP supplementation, the significance of the correlation was lost (Fig. 2B).

As the degree of saturation of erythrocytic GOT by PLP has been used as a status indicator of B6 ^54,55, we calculated the primary activation ratio (the ratio of GOT activity with and without PLP supplementation). The PAR increased linearly with age (Fig. 2C; P < .0001), following a trend similar to that reported for humans (i.e., high in the newborn and gradually decreasing throughout the lifespan ^56,57). The effect of PLP addition on GOT activity in samples from older animals is between 2 and 3 times greater than on the GOT in the younger ones (10 vs. 1 y old). These results are consistent with the incubation of hemolysates with PLP from older subjects, resulting in a greater activation of GOT than in samples from younger subjects, reflecting lower vitamin B6 levels in the blood of elders ^58–60. These reports and the results presented here indicate that PLP is present in red blood cells in suboptimal concentrations for maximal GOT activity.

Considering that the PAR values increased with age, the mean PAR values corrected for age will result in cut-off values that could be used to identify marginal vitamin B6 deficiency. Since the PAR values normalized to age followed a normal distribution (Supplementary information), 1.65 x SD results in a tail that gives a probability of 5% of the data being excluded from the distribution. If this value is added to the mean, anything above this value has a < 5% probability of being significant. Accordingly, values > 1.24 of the age-adjusted mean indicated vitamin B6 deficiency. Under this condition, 6 cats (5 females, one male) were identified as having abnormally high PAR values normalized to age. The age distribution of these 6 cats indicated that the vitamin B6 deficiency was widely spread with no apparent pattern, with two juniors (1 and 2 y), one adult (3 y), two mature (7 and 8 y), and one senior (14 y).

Vitamin B6 status in domestic client-owned cats

Blood samples were collected from 54 male (n = 35) and female (n = 19) cats that visited the VMTH for a medical consultation between December 2022 and January 2023, from which blood samples were available ranging from 3 months to 17 y old of age (Fig. 3A). This cohort had more males (male-to-female ratio = 1.8 from the VMTH vs. 0.15 from the Cat Colony) and a more comprehensive representation of ages than those at the Cat Colony: junior (1–2 y; n = 7), adults (3 to 6 y; n = 13), mature cats (7–10 y; n = 14), seniors (11–14 y; n = 14) and geriatric cats (≥15 y; n = 6). In contrast with the correlation between body condition scores and age obtained with the Cat colony (Fig. 1B), the VMTH cohort did not show a statistically significant correlation (Fig. 3B).

As described before, the specific GOT activity was assessed in all blood samples with and without the supplementation of saturating PLP concentrations. The GOT activity without or with PLP addition shows a statistically significant correlation with age (Fig. 4A-B), but the PAR (as obtained before; Fig. 2C) showed a positive correlation (Fig. 4C).

The PAR values were corrected for age to establish a cut-off value to indicate vitamin B6 deficiency. After cleaning for outliers and verifying a normal distribution (Supplementary information), the cut-off was set at 1.23, similar to that obtained with the Cat Colony. Under this condition, 6 cats (3 females, 3 males) were identified as having abnormally high PAR values normalized to age. The age distribution of these 6 cats indicated that the vitamin B6 deficiency was mainly seen at older ages with one adult (4 y), three mature (7, 9, and 10 y old), and two seniors (11 and 12 y old).

Contributors to vitamin B6 status in cats

Whether the relatively higher PAR effect in elderly cats from both the Cat Colony and VMTH reflected poorer vitamin B6 status, lower plasma phosphatase activity (activity required to release pyridoxal from PLP for tissue uptake), or lower hepatic albumin secretion (needed for PLP transport) as suggested for humans^58–61, is not known. To ascertain whether PLP or other factors were contributors to this effect, we presented the data from the Cat Colony and the VMTH (n = 101) in terms of a specific activity ratio (i.e., average specific GOT activity from junior divided by the average specific GOT activity from each age group, namely adults, mature, seniors, and geriatric). It was found that the initial specific activity ratio of 2.4 ± 0.2 decreased with the addition of PLP to 1.2 ± 0.2 (Fig. 5A). As the GOT activity from older animals with PLP supplementation was similar to that of younger animals (ratio close to 1), it is surmised that PLP accounts for most differences in enzymatic activities between younger and older animals, precluding a significant role for additional factors (86% for PLP, 14% to other factors). This result is similar to that reported for humans in which PLP status had a major contribution (77%); but, in humans, the role of factors other than B6 that account for the residual difference in GOT activity was more robust, and as such, it could not be excluded (23%)⁶¹.

To explore the putative contributors to the vitamin B6 status in domestic cats, we gathered more information on the 12 cats identified as vitamin B6 deficient (6 from the Cat Colony and 6 from the VMTH) as judged by the age-normalized PAR threshold. Based on the age and body condition scores (Fig. 5B), most cats with vitamin B6 deficiency had a low body condition score (41.7%), suggesting low food intake and/or malassimilation, and they were of advanced age (7 y old or older; 66.7%). This result is consistent with previously reported data, in which the increased energy requirements and compromised digestibility in older cats ^62–64 are linked to a higher proportion of underweight elderly cats with lower body condition scores⁶⁵. In contrast, two cross-sectional studies performed with human adults indicated that vitamin B6 concentrations were lower in obese subjects and patients with metabolic syndrome than in control subjects ^66,67.

Changes in vitamin B6 status have been linked to numerous human diseases and conditions. For instance, GOT apoenzyme contents increase in diseases related to necrotic processes, while decreases had been reported following alcohol intake ^45,47. Low plasma PLP in older adults is not explained by low dietary vitamin B6 or low protein intake ^24,68, deficit in absorption, impaired synthesis or retention of PLP in erythrocytes or liver ⁶⁹. However, some ⁷⁰ but not all ^69,71 studies suggest increased catabolism related or not to age-dependent decreases in albumin and, in particular, with an increase in alkaline phosphatase^50,69,72. At the same time, the rise in plasma PA (which may be taken wrongly as increased catabolism) in both older men and women may partly be explained by impaired renal function ⁷². When the data of 12 vitamin B6-deficient cats were analyzed in terms of medical diagnosis (Fig. 5C), most (58.4%) had a condition associated with an increased inflammatory response, whether through an infection (feline coronavirus; 25%) or via diseases associated directly or indirectly with an inflammatory response (33.4%). None of the cats residing at the Cat Colony were asymptomatic for feline coronavirus, but they were tested as a part of the routine panel.

Concluding Remarks

In this study, and for the first time, a functional biomarker of vitamin B6 status has been obtained from domestic cats and established a clear path to define vitamin B6 deficiency. For the first time, we also identified key contributors to vitamin B6 deficiency: older age, low body weight, and inflammation.

In humans, various diseases have been associated with low levels of plasma PLP⁷³, including rheumatoid arthritis, inflammatory bowel disease, cardiovascular disease, deep vein thrombosis, diabetes, and cancer ^{1,14,15,74–81}. An inverse relationship has been found between the inflammatory marker C-reactive protein and plasma PLP status^82,83, the acute-phase protein alpha1-acid glycoprotein, tumor necrosis factor-α, and the proinflammatory cytokine interleukin-6 in rheumatoid arthritis and rheumatoid arthritis and inflammatory bowel disease^15,78,84. While the inverse association between vitamin B6 and inflammatory diseases may be interpreted as low dietary intake, and while this is true in some instances (e.g., Fig. 5B; 1 of 12; most of them 7 y old and older with putative lower food digestibility), in most human studies, there is no correlation between vitamin B6 intake and plasma PLP concentration with no indication of inadequate dietary intake of vitamin B6. Indeed, the cats from the Cat Colony are fed a diet with optimal nutrient levels (Purina Cat Chow Complete Formula), and no indication of dietary issues was recorded in the VMTH records for the cohort of cats tested. Furthermore, lower PLP concentration observed in inflammatory diseases seems linked to the inherent inflammation process, suggesting higher B6 is needed by the immune system during active inflammation⁷³. Indeed, 58.4% of the cats identified in our study as vitamin B6 deficient were undergoing some inflammatory process (Fig. 5C). Thus, our study supports that vitamin B6 supplementation may be indicated in older and underweight animals, especially those undergoing an active inflammation process.

Ethics statement

The ethics of this study was approved by the IACUC (protocol number 21780). All experimental procedures were strictly performed per the ethical requirements of IACUC at the University of California Davis.

ARRIVE guidelines

This study was fully aligned with the ARRIVE 2.0 guidelines⁸⁵, ensuring transparent and comprehensive reporting of the research methods utilized in this study and its outcomes. Adhering to these guidelines enhances research quality and reproducibility, underscoring our commitment to upholding rigorous standards and promoting transparency in our findings. We confirm that all experiments were performed in accordance with relevant guidelines and regulations.

Animal subjects

The experimental protocol adhered to the Guide for the Care and Use of Laboratory Animals (NRC 1985) and was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California-Davis. The study population included 60 sexually intact, specific-pathogen-free domestic shorthair cats (Felis catus) aged 1 to 17 y from the Feline Nutrition and Pet Care Center School of Veterinary Medicine, the University of California-Davis. Pathogens tested in this cohort included feline calici, herpes, leukemia, and immunodeficiency viruses (all negative). All animals present in the colony between March and July 2021 were included in this study, and these criteria were established a priori. All cats underwent a veterinary examination and were determined to be healthy and able to participate in the study.

Cats in this facility are housed in large group enclosures with enrichment. Cats were group-housed in large wire cages (2.5×2.5×2.5 m) in humidity- and temperature-controlled rooms (21 ± 2°C) with a light: dark cycle of 14 hours:10 hours. The cats had habitual free access to tap water and a commercially available, balanced, dry expanded diet formulated for all life stages (Purina Cat Chow Complete Formula, Nestle Purina PetCare Company, St. Louis, MO; minimum 1 mg pyridoxine/1000 kcal⁸⁶). Staff veterinarians evaluated the body condition score with a scale from 1 to 9 and a score of 5 as optimal⁵².

The other study population included 54 cats aged 3 months to 17 years old brought to the Veterinary Medical Teaching Hospital at the University of California, Davis, between December 2022 and January 2023 to seek care under different circumstances. A designated veterinarian performed a veterinary examination, determined the body condition score, and gave a diagnosis.

Sample collection

Blood collection across subjects from the Cat Colony was done randomly, and the number of subjects bled/day varied depending on the technician's schedule and workload. Blood collection was adequately performed for most cats while the animal was awake using the appropriate restraint. Restraint was necessary to prevent movement resulting in blood vessel or other organ lacerations and serious complications. Only trained staff performed this procedure by following the criteria to determine the maximum, safe amount of blood to withdraw. The approximate single blood draw was < 5 ml for a 5 kg cat, following the University's IACUC guidelines. This volume represents an estimate depending on the animal's size, health, and hydration status. Cats were not returned to their enclosure until complete hemostasis (i.e., no more blood coming from the collection site as determined using gauze and direct pressure). Up to several minutes of pressure sometimes was required following jugular vein puncture. Collecting blood from the medial saphenous, cephalic, and jugular is recommended for cats. We chose to use the latter site because it does not require anesthesia, and the trained staff have ample experience with this procedure.

Blood samples collected at the VMTH followed standard and approved protocols.

All blood samples from both populations were collected using disposable syringes and needles; samples were transferred into 2-ml tubes containing K₂EDTA (Sarstedt, Nümbrecht, Germany). For measurement of enzymatic activities and protein concentrations, 6 ml of blood was aliquoted into 3 EDTA tubes (2 mL/tube). Blood samples were stored at 4°C until analysis and were analyzed 7–8 hours after phlebotomy. Blood samples were collected in the morning to avoid any putative diurnal cycles. After centrifugation at 3000 x g at 4°C, plasma was removed, and the equivalent 1 ml of packed red blood cells was washed in phosphate-buffered saline three times and suspended in 1 ml of a 1% solution of Triton X‐100 double density, peroxide‐free detergent (Sigma, St. Louis, MO, USA). If needed, washed and permeabilized red cells were stored at − 30°C for a maximum of 3 months or at − 70°C for up to 18 months. After homogenization and incubation at 25°C for 10‐min, the hemolysate was centrifuged at 13,000g for 10 min at 4˚C.

GOT activity assessment

Blood hemolysates (10 µl) were homogenized and diluted 1/100 with 50 mM phosphate buffer (pH 7.5) containing 0.2% bovine albumin and 2 mM K₂EDTA. 1/20 with phosphate saline buffer. Each sample was run in duplicates. If values exceeded a coefficient of variation higher than 10%, they were re-assessed. Each sample was tested without and with 20 µM PLP addition. For each of these conditions, a blank and a complete system were run (for a total of 8 wells/sample) in which the "blank" contained 0.1 M Tris-HCl, 0.5 EDTA, 0.2 mM freshly prepared NADH, 10 µM L-aspartate (pH 8.0), and 10 mU malate dehydrogenase. In contrast, the complete system had 4–5 microliters of diluted hemolysates. The decrease in absorbance at 340 nm was followed for 15 min in a Tecan Microplate reader at 37˚C. The blank rates were subtracted from those rates with hemolysates. All reagents were of analytical grade and obtained from Sigma Chemical Company. The assay of this enzyme followed essentially that published by Buetler ⁸⁷. The following parameters and ratios were calculated for each blood sample: the GOT activity with and without supplementation of PLP was expressed in units of enzymatic activity per mg protein (1 U = 1 µmol x min-1) and calculated as [ΔAbs 340 nm/min x (6.22 µmol/ml)⁻¹] = µmol/min x (ml)⁻¹ x (mg of protein x ml^− 1)⁻¹ = U/mg. The primary activation ratio (PAR) is the ratio of the activity in the presence of saturating PLP to that in the absence of added PLP. Protein determination was performed using the bicinchoninic acid method (Pierce™BCA protein assay kit, catalog #23225).

Statistical analyses-Enzymatic activities were assessed in technical triplicates. Descriptive statistics were generated for each parameter and analyzed for normality by the Kolmogorov-Smirnov test. For all analyses, P ≤ .05 was considered significant. Data were analyzed by use of JMP software.

Acknowledgments: The study was funded by the Center for Companion Animal Health Award to CG; the STAR Program at UC Davis funded Ms. Chu.

Author Contributions: Conceptualization, methodology, formal analysis: VC and CG; data resources: AJF, and JAL; data curation: CG; writing—original draft preparation: CG; collection of blood samples and records: MM and VC; writing—review and editing: all authors; funding acquisition: CG. All authors have agreed on the final version of this study.

Animal Review Board Statement: The University of California, Davis Institutional Review Board for the Protection of Animal Welfare approved this study (#21780).

Financial Disclosures: The funder (CCAH) had no role in the study's design, in the collection, analysis, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. No potential financial and non-financial competing interests that could directly or indirectly undermine the objectivity, integrity, and value of this publication through a potential influence on the judgments and actions of authors regarding objective data presentation, analysis, and interpretation were found. Jennifer A. Larsen (JAL) is an investigator in clinical trials sponsored by Royal Canin and Nestlé Purina PetCare. She develops educational materials for Brief Media, Mark Morris Institute, and Healthy Pet magazine. She participates as a speaker or attendee in continuing education events sponsored or organized by Royal Canin, Nestlé Purina PetCare, Nature's Variety, and Hill's Pet Nutrition. Andrea J. Fascetti (AJF) is the Scientific Director of the Feline Nutrition and PetCare Center and Amino Acid Laboratory at the University of California, Davis, providing research support fee-for-service. None of these activities lead to a conflict of interest or influence the collection or interpretation of results. AJF advised Synergy Food Ingredients, Clorox, and received a grant from Nutro, remuneration for lectures or as an advisor on behalf of Nestlé Purina PetCare, Mars Petcare, and the Pet Food and Mark Morris Institutes. A nutrition resident received funds from Hill's Pet Nutrition Resident Clinical Study Grants program at the time of the study. The Veterinary Medical Teaching Hospital at UCD receives partial support for a Nutrition Technician from Nestlé Purina PetCare and its veterinary nutrition program from Nestlé Purina, Mars Petcare, and Hill's Pet Care. C.G. is an Editorial Board Member of Scientific Reports (Nature Publishing Company). She received compensation as a Field Chief Editor for Frontiers in Molecular Biosciences and honoraria from participating in NIH peer review meetings. The rest of the authors declare no conflict of interest.

Data availability: All data generated and analyzed in this study are included in the published article.

Funding: This study was funded by a grant to CG from the Center for Companion Animal Health (School of Veterinary Medicine, University of California Davis). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the Center for Companion Animal Health or the School of Veterinary Medicine.

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Competing interest reported. Financial Disclosures: The funder (CCAH) had no role in the study's design, in the collection, analysis, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. No potential financial and non-financial competing interests that could directly or indirectly undermine the objectivity, integrity, and value of this publication through a potential influence on the judgments and actions of authors regarding objective data presentation, analysis, and interpretation were found. Jennifer A. Larsen (JAL) is an investigator in clinical trials sponsored by Royal Canin and Nestlé Purina PetCare. She develops educational materials for Brief Media, Mark Morris Institute, and Healthy Pet magazine. She participates as a speaker or attendee in continuing education events sponsored or organized by Royal Canin, Nestlé Purina PetCare, Nature's Variety, and Hill's Pet Nutrition. Andrea J. Fascetti (AJF) is the Scientific Director of the Feline Nutrition and PetCare Center and Amino Acid Laboratory at the University of California, Davis, providing research support fee-for-service. None of these activities lead to a conflict of interest or influence the collection or interpretation of results. AJF advised Synergy Food Ingredients, Clorox, and received a grant from Nutro, remuneration for lectures or as an advisor on behalf of Nestlé Purina PetCare, Mars Petcare, and the Pet Food and Mark Morris Institutes. A nutrition resident received funds from Hill's Pet Nutrition Resident Clinical Study Grants program at the time of the study. The Veterinary Medical Teaching Hospital at UCD receives partial support for a Nutrition Technician from Nestlé Purina PetCare and its veterinary nutrition program from Nestlé Purina, Mars Petcare, and Hill's Pet Care. C.G. is an Editorial Board Member of Scientific Reports (Nature Publishing Company). She received compensation as a Field Chief Editor for Frontiers in Molecular Biosciences and honoraria from participating in NIH peer review meetings. The rest of the authors declare no conflict of interest.

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Factors influencing vitamin B6 status in domestic cats: age, body condition score, and inflammation

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Vitamin B6 status in domestic research cats

Vitamin B6 status in domestic client-owned cats

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