The relationship between vitamin D status and leukocytes in hospitalised cats
Titmarsh, H.F,1 Cartwright J.A1,Kilpatrick, S.1, Gaylor, D1., Milne E 1 Berry, J2., Bommer, N1., Gunn-Moore, D1., Reed, N1., Handel, I. 1, Mellanby, R.J1. *
- Royal (Dick) School of Veterinary Studies and The Roslin Institute, The University of Edinburgh, Roslin, Midlothian, United Kingdom
- Specialist Assay Laboratory (Vitamin D), Clinical Biochemistry, Manchester Royal Infirmary, Manchester, United Kingdom
Corresponding author:
Helen Titmarsh BVetMed(Hons)MRCVS
Hospital for Small Animals
Royal (Dick) School of Veterinary Studies
Easter Bush Campus
The University of Edinburgh
Roslin
Midlothian
0131 650 7650
Keywords:25 hydroxyvitamin D,neutrophils, vitamin D, cat
Abstract
Objectives: Vitamin D deficiency, as assessed by serum 25 hydroxyvitamin D (25(OH)D) concentrations, has been linked to markers of systemic inflammation in human and canine medicine. However, the relationship between vitamin D status and inflammation has not been previously investigated in cats. The aim of this study was to examine the relationship between serum 25(OH)D concentrations and leukocyte counts in hospitalised sick cats.
Methods: Serum 25(OH)D concentrations, and haematology profiles were measured in 170consecutive hospitalised sick cats. A binary logistical regression model examined the relationship between serum 25(OH)D concentration, age, sex, breed and neutrophil, monocyte, eosinophil and lymphocyte counts.
Results: Cats with a neutrophilia had lower serum 25(OH)D concentrations than cats with neutrophil concentrations below the upper limit of the reference range. There were no differences in serum 25(OH)D concentrations in cats with monocyte, lymphocyte or eosinophil counts above their respective reference interval compared to cats with counts below the upper limit of the reference interval.
Conclusions andrelevance: Hospitalised cats with a neutrophil count above the reference range had lower vitamin D status. There is a need to establish whether lower vitamin D status is a cause or consequence of increased neutrophil counts.
Introduction
The actions of vitamin D have been classically understood in terms of its role in regulating calcium homeostasis and bone metabolism. However, more diverse physiological roles of vitamin Dhave been demonstrated in recent years and numerous cell types have been shown to express the vitamin D receptor.[1, 2]Low serum vitamin D status, most commonly assessed bymeasuring serum 25-hydroxvitamin D (25(OH)D) concentrations, are commonly reported in a number of human and canine diseases including hypertension[3], diabetes mellitus, [4]cardiovascular disease, [5, 6] cancer [7, 8] autoimmune conditions [9], chronic enteropathies[10, 11] and infectious diseases.[12-16] In feline medicine low vitamin D status has been observed in cats with mycobacterial infections[15] and in cats with inflammatory bowel disease or small cell gastrointestinal lymphoma.[17] Importantly, serum 25(OH)D concentrations are also predictive of short-term mortality in hospitalised cats.[18] Similarly,low serum 25(OH)D concentrations have also been found to bepredictive of mortality in people[19] and horses.[20]
Despite the clear association between serum 25(OH)D concentrations and the incidence and outcome of many diseases,it remains unclear whether vitamin D plays a mechanistic role in poor health outcomes or is a surrogate marker of ill-health. It has been argued that vitamin D status may influence health outcomes through its ability to modulate the immune system. Most leukocytes, including, antigen presenting cells, T-lymphocytes and B-lymphocytes[21], express the vitamin D receptor (VDR). Vitamin D can promote immune tolerance by inhibiting pro-inflammatory immune responses and by increasing regulatory T cell populations.[22-28]
In humans serum 25(OH)D concentrations have been inversely associated with markers of inflammation including acute phase proteins and, pro-inflammatory cytokines. This has been demonstrated in a number of diseases includinginflammatory polyarthritis,[29] diabetes mellitus,[30] autoimmune diseases,[31, 32]inflammatory bowel disease[33, 34]and human immunodeficiency virus.[35] Inflammatory markers are also increased in elderly and healthy people with low serum concentrations of vitamin D.[36-38]In addition, low vitamin D status has been associated with markers of inflammation in dogs with haemoabdomens[8] and dogs with chronic enteropathies.[39]
Despite a number of studies having investigated the association between serum 25(OH)D concentrations and inflammation, surprisingly few studies have looked at the relationship between serum vitamin D concentrations and leukocyte populations in either human or veterinary patients.[40]One study which investigated the association between white blood cell concentrations and serum 25(OH)D found an inverse relationship between these two parameters in smokers.[40]The lack of studies which have examined the relationship between leukocytes and vitamin D status in humans resulted in Lee et al (2015) concluding that ‘evaluation of different forms of inflammatory cells, not just total white blood cell count, may be a useful method to identify the association of vitamin D and inflammation’[41].
Despite the growing body of work investigating the relationship between serum 25(OH)D concentrations and illness in humans and dogs, the association between 25(OH)D and inflammation has not been previously investigated in cats. Therefore, the objective of this study was to measure serum concentrations of 25(OH)D, alongside whole blood neutrophil, lymphocyte, monocyte and eosinophil numbers in a population of hospitalised sick cats. The hypothesis of the study was that there would be a negative relationship between serum 25(OH)D concentrations and leukocyte counts.
Material and Methods
Cats consecutively examined at the Hospital for Small Animals, Royal Dick School of Veterinary Studies, were considered for inclusion in the study. Clinical records were reviewed for each cat enrolled. The age, sex, breed and final diagnosis was recorded for each cat. In order to be eligible for inclusion the following clinical data needed to be available; total white blood cell count, segmented and band neutrophil, monocyte, eosinophil and lymphocyte counts. In additionresidual stored serum samples had to be available for 25(OH)D quantification.
Following handling of blood samples for routine diagnostic procedures on the day of admission, serum samples were initally stored at -20°C and later moved to -80oC for longer term storage until 25(OH)D concentrations were measured in batches.Vitamin D is extremely stable even when stored at -20oC.[42] Serum concentrations of 25(OH)D2 and 25(OH)D3 were determined by liquid chromatography tandem mass spectrophotometry (LC-MS/MS) using an ABSciex 5500 tandem mass spectrophotometer (Warrington, UK) and the Chromsystems (Munich, Germany) 25OHD kit for LC-MS/MS following the manufacturers’ instructions (intra- and inter-assay CV 3.7% and 4.8% respectively). This Supraregional Assay Service laboratory is accredited by CPA UK (CPA number 0865) and has been certified as proficient by the international Vitamin D Quality Assurance Scheme (DEQAS). Total 25(OH)D is defined as the sum of 25(OH)D2 and 25(OH)D3.
Haematology variables were measured on an ADVIA(r) 2120i System with Autoslide (Siemens Medical Solutions Diagnostics Ltd California, USA). A manual white blood cell differential count was undertaken on at least 100 leukocytesto establish the concentrations of neutrophils, monocytes, lymphocytes, eosinophils and basophils. Blood smearsfrom all cases were evaluated under the direct supervision of a Board-certified veterinary clinical pathologist. Total calcium concentrations were measured using an ILab650 biochemistry analyser (Diamond Diagnostics, USA).
The relationship between serum 25(OH)D concentrations and neutrophils, lymphocytes, monocytes and eosinophilswas initially examined using dot plotswhichplotted leukocyte counts against serum 25(OH)D concentrations. The relationship between 25(OH)D concentrations in segmented neutrophils, lymphocytes, monocytes and eosinophils populations which were above or below the upper limit of the reference interval wereexamined using a Mann Whitney U test. In the four leukocyte populations, the relationship between 25(OH)D concentrations, age, breed and sex was investigated bya binarylogistical regression model. Co-variates were removed from the initial model to minimise Akaike Information Criterion (AIC), a parameter penalised measure of best fit, to give a final parsimonious model. The relationship between calcium and serum 25(OH)D concentrations was assessed by Spearman’s rank correlation test.
The study was approved by the University of Edinburgh’s Veterinary Ethical Review Committee. Informed consent for the storage and subsequent use of residual clinical blood samples for research purposes was obtained at admission for each cat enrolled.
Results
One hundred and seventy catswere included in the study. The median age of the cats was 108 months (2.5-264 months). There were 4 entire males, 102 neutered males, 1entire female and 63 neutered female cats. Breeds included in the study were 110 Domestic Short Hair cats, 15 Domestic Long Hair cats, 8 Maine Coons, 3Oriental Short Hairs, 8 Burmese, 7 Bengals, 6 Siamese, 2 Ragdolls and one of each of the following breeds; Chinchilla, Abyssinian, Persian, British Blue, Manx, Siberian, Russian Blue, Egyptian, NorwegianForest Cat, Burmilla and Tonkinese.There was a significant positive correlation between total calcium and 25(OH)D concentration (r=0.21, p<0.01).
Dot plots of serum 25(OH)D concentrations from cats with haematology parameters above or below the upper referenceinterval are shown in figure 1. Serum 25(OH)D concentrations were significantly lower in cats with a segmented neutrophil count above the upper reference interval compared to cats with a segmented neutrophil count below the upper reference range (p=0.04). This significant difference remained even after removal of the high outlying result in the <12.8x109/l group. Twelve cats had band neutrophils. The median serum concentration of 25(OH)D from cats with band neutrophils was 59.8nmol/l compared to cats with band neutrophils was 97.4nmol/l. This difference approached significance (p=0.06). There was no significant difference in 25(OH)D concentrations in cats with eosinophil, lymphocyte and monocyte counts above upper reference interval compared to cats with leukocyte counts below the upper end of the reference interval. The lower serum 25(OH)D concentrations observed in cats with a segmented neutrophil count above the upper reference interval was not confounded by age, breed or sex as thefinal logistic regression model only included 25(OH)D concentrations as a predictor of increased neutrophil counts (p=0.021). The odds of having a neutrophil count within the reference interval increased by 1.32 (1.04-1.72 95% CI) per 25nmol/l increase in serumconcentrations of 25(OH)D.
Discussion
The main finding of this study was that cats with a neutrophilia have lower vitamin D status than cats with neutrophil counts below the upper reference interval. This observation is consistent with studies in human and canine patients together with experimental models of inflammation. For example, a study of over 1300 children found a negative correlation between neutrophil count and 25(OH)D concentrations.[43]Furthermore, vitamin D supplementation has been shown to reduce the proportion of neutrophils in a murine model of allergic airway disease.[44]The finding of a negative relationship between 25(OH)D concentrations and segmented neutrophils is also consistent with our recent work in dogs with intestinal inflammation.[39]This study demonstrated that vitamin D status was negatively correlated with neutrophil counts, together with several other markers of systemic and intestinal inflammation, in dogs with a chronic enteropathy.
It is presently unclear if low 25(OH)D concentrations are a cause or consequence of inflammation. It has been hypothesised that serum 25(OH)D may act as a negative acute phase protein.[45]Evidence in support of this hypothesis include the demonstration that serum 25(OH)D concentrations decrease alongside parallel increases in inflammatory markers in patients whom have undergone elective knee surgeries.[46, 47] Decreases in serum 25(OH)D concentrations have also been reported with spontaneously occurring inflammatory conditions such as acute pancreatitis.[48]Alterations in vitamin D status may also occur due the effect of inflammation on vitamin D binding proteins. For example, serum concentrations of vitamin D binding protein concentration decrease in acute inflammation.[45]In contrast, other investigators have demonstrated that patients with lower 25(OH)D concentrations are more likely to develop an inflammatory response following immune stimulation. For example, the acute phase response following administration of parenteral bisphosphonates was greater in patients with low vitamin D status, suggesting that hypovitaminosis D may predispose people to acute inflammatory reactions.[49]In addition, other studies suggest that vitamin D may not decrease acutely in all inflammatory conditions. For example, no significant decreases in 25(OH)D concentrations were reported secondary to myocardial infarction.[50] Similarly, there was no association between hypovitaminosis D and markers of an acute phase response in humans with pulmonary tuberculosis.[51]
Investigating the potential relationship between vitamin D and inflammation in a number of species is important asvitamin D supplementation couldbea beneficial treatment for patients with inflammatory diseases. In experimental animal models, vitamin D and vitamin D analogues have demonstrated anti-inflammatory properties in a number of conditions including cerebral malaria[52],experimental models of colitis [53], models of cognitive dysfunction[54], acute kidney injury[55] and interstitial cystitis[56]. There is evidence that supplementing vitamin D can reduce inflammatory markersin systemic lupus erythematosus,[57] cystic fibrosis[58] and patients with type II diabetes.[59] However, vitamin D supplementation does not universally reduce markers of inflammation.[60, 61]Therefore, the role of vitamin D in the initiation, perpetuation and resolution of inflammation is worthy of further investigation
Our study also demonstrated a significant correlation between total calcium and 25(OH)D. This is consistent with our findings in dogs with a CE [11]and in humans. [62]Our finding of an association between total calcium and 25(OH)Dis consistent with the known effects of vitamin D,which includes increasing serum calcium concentrations by increasing dietary calcium absorption in the intestines, and acting in the kidney to promote calcium reabsorptionand promote calcium resorption from bone. [63]
There are some limitations with this study. Firstly, leukocyte numbers can be altered by processes other than inflammation. For example, neutrophil numbers will transiently increase due to the release of adrenaline in response to stressful stimuli such as hospitalisation or blood sampling. Other limitations of this study include that cats come from a referral population. This makes it difficult to fully assessa number ofconfounding variables that could affect both serum vitamin D concentrations and leukocyte numbers. For example, the cats involved in this study may have been treated with a number of medications prior to admission to the hospital,which may have influenced haematological values. It is not known ifmedications influence serum 25(OH)D concentrations in cats. Although glucocorticoids may influence vitamin D concentrations in people, short term, anti-inflammatory doses of glucocorticoids do not significantly alter vitamin D metabolism in dogs.[64] As information regarding the influence of drugs on vitamin D homeostasis in cats is lacking, no cases were excluded based on previous medical therapy. However, it must be acknowledged that pre-treatment with drugs may have influenced the results of this study and future work investigating the effect of drugs such as steroids on serum 25(OH)D concentrations of cats would be valuable. Diet could also influence serum 25(OH)D in cats and a further limitation of this study is that the cats didn’t receive the same diets. However, it is standard practice in our hospital to record the diets fed to cats at the time of admission. All but one cat in this study was fed commercial cat food. As commercial cat foods are standardly supplementedwith vitamin D within recommended limits,[65] variation in vitamin D content of the dietsconsumed by cats enrolled in the study was unlikely to have been a significant confounding variable
In conclusion, the study has demonstrated that cats with a neutrophilia have lower vitamin D status than cats with a neutrophil count below the upper limit of the reference range. This study demonstrates the need to further clarify the relationship between inflammation and vitamin D status in cats.
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