The effect of dietary olive polyphenols on markers of inflammation and bodyweight in senior domestic cats (2024)

Biochemistry heath screen (Feline)

Blood samples collected on days 0, 28, and 56 were sent for biochemistry health screening. This included measurement of symmetric dimethylarginine (SDMA), creatine kinase (CK), aspartate aminotransferase (AST), alkaline phosphatase (ALP), bilirubin, total protein, albumin, globulin, urea, creatinine, phosphate, calcium, cholesterol, sodium, potassium, and chloride.

Complete blood count (differential)

Blood samples collected on days 0, 28, and 56 were sent for complete blood count (CBC) analyses. This included measurement of red blood cell concentration (RBC), haemoglobin concentration (HB), haematocrit or packed cell volume (HCT/PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet concentration (PLAT), absolute reticulocyte count (absolute retic), reticulocyte haemoglobin (rHb), and white blood cell concentration (WBC).

Cytokines and chemokines

A comprehensive cytokine and chemokine assay (Millipore Feline Cytokine Panel, 19-plx; MilliporeSigma, Burlington, MA, USA) was conducted on all blood samples (i.e. days 0, 28, and 56) by Massey University Nutrition Laboratory (School of Food and Advanced Technology, Massey University, Palmerston North, New Zealand). The following cytokines and chemokines were assessed: apoptosis antigen 1 (i.e. Fas or CD95), Fms- like tyrosine kinase 3 ligand (Flt-3L), granulocyte macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-γ), platelet-derived growth factor (PDGF- BB), keratinocytes-derived chemokine (KC or CXC-L1), stromal cell-derived factor 1 (SDF-1 or CXC-L12), chemokine ligand 5 (CCL5 or RANTES), stem cell factor (SCF), monocyte chemoattractant protein 1 (MCP-1), tumour necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-12, IL-13, and IL-18.

General health of the cats on day 0:

On day 0, none of the cats had any biochemistry or haematology results that showed either acute or chronic illness. Renal function appeared to be normal in all cats. Three of the cats had slightly elevated SDMA (15, 16, and 19 ug/dl; normal range 0–15 ug/dl), which could be transient or indicative of early renal disease. Since SDMA concentrations were only slightly elevated and both urea and creatinine concentrations were normal, renal degeneration or disease is unlikely. In addition, markers of liver function (e.g. ALT, ALB, BR, and TP) were normal in all cats.

Red blood cells were present at normal concentrations (RBC count) and proportions (haematocrit) in all cats on day 0. In addition, the volume of RBC (MCV) and haemoglobin contents of each RBC (MCHC) of each cat were normal. The absolute reticulocyte count was low in most cats. This can indicate decreased erythropoiesis due to anaemia-causing conditions such as chronic kidney disease (Cowgill et al., 2003). However, the RBC did were normal for all cats and the diet did not affect the absolute reticulocyte count or RBC of the cats. One cat had a mildly elevated WBC count (36.9×10⁹ cells/l; normal range 5.5–19.5×10⁹ cells/l) on day 0, which suggested that the cat may have been experiencing an infection or inflammation. The WBC count of this cat remained slightly above the normal range throughout the study. There were no other signs of illness in this individual’s biochemistry or CBC panel, thus inflammation was the most likely cause.

The effect of polyphenol treatment on blood parameters

The provision of a diet supplemented with 0.1% olive polyphenols for 56 days had a minimal effect on the parameters assessed in the biochemistry health panel and haematology of the cats (Table 2). Serum urea, phosphate, and potassium concentrations were all significantly higher on day 56 than on day 0, and 28 (Table 2). All serum urea and phosphate concentration values were within the normal range for cats (Table 2). As such, the physiological significance of the increases in these parameters is unclear and should not be over-interpreted. The mean potassium concentrations on day 56 were just above the normal range (Table 2), but slightly elevated potassium alone is of minimal significance in a clinical context. There was a tendency (P<0.1) for AST to increase from day 0 to 56 (Table 2), but all AST concentrations were within normal ranges.

In terms of haematology, none of the parameters in the cats differed significantly over time (Table 2), which suggested that the polyphenol treatment had no effect. The mean values of all haematology parameters were within the normal ranges throughout the study with the exception of elevated WBC in one cat and a low absolute reticulocyte count on day 0, 28, and 50 for most cats (Table 2). As mentioned previously, the low absolute reticulocyte count is unlikely to be clinically significant as RBC counts were normal for all tests.

Cytokines and chemokines

Reference ranges are lacking for most cytokines in domestic cats, making it difficult to determine whether the concentrations were within normal ranges. Troia et al. (2020) examined the concentrations of several cytokines and chemokines in the plasma of 40 clinically healthy cats, and these results are presented in Table 3. The concentrations of many cytokines (Flt-3L, IL-6, IL-8, IL-12, MCP-1, PDGF-BB, RANTES, SDF-1, IL-4, and IL-13) in the present study were markedly higher than previously reported (Troia et al., 2020; Table 3). This was possible because a cohort of cats selected for this trial was older/senior (>10 years), although this was difficult to confirm as Troia et al. (2020) did not report the age of the cats used. Nonetheless, several conditions that are more prominent in older cats (e.g. kidney or liver disease and joint disease) are known to increase cytokine concentrations and oxidative stress (Clarke et al., 2005; Prahl et al., 2007). While there were no clear signs of these conditions in the biochemistry and haematology panels for the cats, it is worth noting that these parameters have a low diagnostic sensitivity and may not be able to detect such conditions until mid to late-onset. It is possible that the senior cats in this study had early onset conditions that led to these increases in cytokine concentrations, but this could not be confirmed. In any case, cytokine concentrations are well-known to differ with various factors (e.g. age, breed, sex, disease, etc.), hence why each cat in the present trial was used as its own control.

A comparison of the concentrations of the assessed cytokines at the start (day 0) and end (day 56) of dietary supplementation with 0.1% polyphenols is presented in Table 3. There were no clear differences in the circulating cytokine concentrations of the cats from the start to the end of the trial, except for Flt-3L, which had a tendency (P=0.09) to decrease over the treatment period (Table 3). However, a post-hoc test yielded no significant differences. The main role of Flt-3L is to promote haematopoiesis, that is, the production of new blood cells (Gilliland et al., 2002). The decrease in Flt-3L could have been associated with the low absolute reticulocyte count observed in this study (Table 2). The clinical relevance of the decreased Flt-3L concentrations remains unclear and should not be overinterpreted, as it was only a statistical trend. It was worth noting that Flt-3L concentrations at the end of the study were still well above those previously reported by (Troia et al., 2020). None of the other cytokines or chemokines exhibited either statistical or visual differences over time.

Overall, there were no clear and consistent differences in cytokine and chemokine concentrations as a result of dietary supplementation with 0.1% olive polyphenols (Table 3). This lack of difference was somewhat surprising, based on past literature in other species such as rats, which suggested that dietary polyphenol supplementation decreases the concentrations of several pro-inflammatory cytokines, increase the concentrations of several anti-inflammatory concentrations, and decreases reactive oxygen species (i.e. oxidative stress) (Molina et al., 2015; Shen et al., 2012). It may be that the dose of 0.1% of the diet was insufficient to elicit a clear response to the polyphenol treatment.

Similar concentrations (0.1%) of citrus polyphenol extract have been found to have physiological effects and decrease IFN-γ and IL-2 mRNA levels in the peripheral mononuclear blood cells of cats (Leray et al., 2011). However, Leray et al. (2011) did not assess circulating cytokine and chemokine concentrations, as in the present study, making direct comparison difficult. It is possible that olive polyphenols are required in higher concentrations than citrus polyphenols, but this requires confirmation. Based on the present data, the dose given could be increased, although caution is needed when giving dietary polyphenols as excessive doses can result in a plethora of adverse effects including death (Kapetanovic et al., 2009). In addition, there is a risk of adversely affecting the palatability of the diet as polyphenols are considered bitter, especially given the initial aversion or low intake of the diet observed in this study. Alternatively, there may be more sensitive parameters (e.g. cytokine mRNA levels in the peripheral mononuclear blood cells or markers of oxidative stress) that could be used to investigate the effects of polyphenols in cats.

It is worth noting the Millipore Feline Cytokine Panel, which was selected as it can be used to analyse 19 cytokines from a single low-volume blood sample, did not include some of the major anti-inflammatory cytokines (e.g. IL-10 and transforming growth factor β (TGF-β). While major pro-inflammatory cytokines were not significantly affected by the polyphenol treatment, changes in these key anti-inflammatory cytokines may have occurred and warrants further investigation. There is evidence that dietary polyphenols can decrease oxidative stress in cats, which has a number of health benefits for older cats in which oxidative stress is more prominent (Jeusette et al., 2010). Thus, it may be worthwhile investigating whether dietary polyphenol supplementation decreased markers of oxidative stress of the cats (e.g. urinary isoprostane or blood reactive oxygen species concentrations); stored urine and serum samples from the present study could be used to assess this.