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Keywords:

  • Calcidiol;
  • Dog;
  • Neoplasia;
  • Vitamin D

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments
  8. References

Background

Spirocercosis in dogs is characterized by esophageal nodules that can undergo neoplastic transformation. Hypovitaminosis D has been associated with neoplasia formation. We hypothesized hypovitaminosis D in neoplastic spirocercosis and that it could be a risk factor for neoplastic transformation.

Objective

To measure and compare vitamin D status, assessed by serum 25-hydroxyvitamin D [25(OH)D] concentrations in non-neoplastic (n = 25) and neoplastic (n = 26) spirocercosis client-owned dogs and healthy dogs (n = 24).

Animals

Twenty-five non-neoplastic dogs, 26 neoplastic dogs, and 24 healthy dogs.

Methods

Fifty-one dogs were randomly selected from 119 dogs diagnosed with spirocercosis presenting to our hospital, and further divided into non-neoplastic or neoplastic groups. Exclusion criteria included dogs less than 1 year old, with concurrent diseases, received corticosteroids, or treated prophylactically for spirocercosis. Serum 25(OH)D concentration was measured by high-performance liquid chromatography. Spirocercosis dogs' appetites were graded and compared.

Results

Serum 25(OH)D concentrations were significantly different among all groups (P < .001). 25-Hydroxyvitamin D concentrations were significantly lower in neoplastic group (median 30.7 nmol/L [range 14.7–62.2]) compared to non-neoplastic (median 52.7 nmol/L [range 19.1–129.7, P < .05]) and healthy groups (median 74.6 nmol/L [range 37.4–130.5, P < .005]). 25-hydroxyvitamin D concentrations were significantly lower in non-neoplastic spirocercosis dogs compared to healthy ones (P < .05). Neoplastic and non-neoplastic spirocercosis dogs had similar appetite scores (P = 1.0). 25-Hydroxyvitamin D concentrations were not significantly different between dogs with normal (P = .087) and abnormal (P = .125) appetites within neoplastic and non-neoplastic spirocercosis groups.

Conclusions and Clinical Importance

Further studies are warranted to determine potential use of vitamin D treatment in spirocercosis and explore role of hypovitaminosis D in pathogenesis of malignant transformation.

Abbreviations
1,25(OH)2D

1,25-dihydroxyvitamin D or calcitriol

1α(OH)ase

1α-hydroxylase

24(OH)ase

24-hydroxylase

25(OH)ase

25-hydroxylase

25(OH)D

25-hydroxyvitamin D or calcidiol

CBC

complete blood count

CT

computed tomography

DBP

vitamin D-binding protein

FGF23

fibroblast growth factor 23

HPLC

high-performance liquid chromatography

IBD

inflammatory bowel disease

ISO

International Organization for Standarization

RI

reference interval

VDR

vitamin D receptor

Spirocercosis in dogs is caused by the nematode Spirocerca lupi and characterized by esophageal nodules which can undergo neoplastic transformation[1, 2] and form an osteosarcoma, fibrosarcoma, or undifferentiated sarcoma.[1-4]

Spirocercosis diagnosis is based on the detection of S. lupi eggs on fecal evaluation,[4] pathognomonic thoracic radiographic or computed tomography (CT) findings (spondylitis, esophageal mass and aortic aneurysm),[5] esophagoscopy,[5] response to the treatment, histopathology of the nodules,[6] or combinations. Confirmation of concomitant neoplastic transformation, however, is challenging and requires invasive or expensive diagnostic tools.

The antineoplastic vitamin D effect relates to its capacity to inhibit cell proliferation and angiogenesis and stimulate cell differentiation and apoptosis,[7] with the activity of 1,25-dihydroxyvitamin D [1,25(OH)2D], being far more active in initiating this response than 25-hydroxyvitamin D [25(OH)D]. This is initiated and maintained by growth factors that lead to vitamin D activation/inhibition of genomic functions[8] and subsequent formation of transcription factors, inhibiting tumor growth and metastatic disease.[9]

There are many studies linking hypovitaminosis D to a higher neoplastic risk in humans[7, 9-12] and some studies in dogs.[13, 14] There is an antineoplastic effect of vitamin D in dogs.[14-16] Therefore, vitamin D might potentially play a role in spirocerca-induced neoplastic transformation. We hypothesized that hypovitaminosis D is present in spirocercosis and that it may potentially lead to neoplastic transformation. The objective of this study was to measure and compare serum 25(OH)D concentrations among dogs with non-neoplastic and neoplastic spirocercosis and healthy dogs.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments
  8. References

Selection of Cases

Fifty-one client-owned dogs diagnosed with spirocercosis at the Onderstepoort Veterinary Academic Hospital in South Africa were included in the study with owner consent. Twenty-five of these dogs were diagnosed with non-neoplastic spirocercosis and 26 with neoplastic spirocercosis. Twenty-four healthy dogs were used as a control group. The spirocercosis cases were selected randomly from a total of 119 spirocercosis cases (39 neoplastic and 80 non-neoplastic) that presented to our hospital over a 3-year period.

Inclusion criteria for all dogs diagnosed with spirocercosis were a complete blood count (CBC), serum biochemistry, urinalysis, fecal analysis, modified centrifugal fecal flotation, thoracic radiographs, thoracic computer tomography (CT) and upper-gastrointestinal endoscopy, or combinations. The differentiation between the non-neoplastic and neoplastic groups was based on macroscopic findings during endoscopy; positive responses to doramectin therapy in suspected non-neoplastic cases with subsequent endoscopic confirmation of clinical cure denoted by absent esophageal nodules post-treatment. The non-neoplastic dogs had a follow-up esophagoscopy performed at days 48–56 postdiagnosis and treatment to assess clinical response and cure. If no nodules were detected, clinical cure was assumed and prophylactic therapy instituted. If the nodules were still present, yet smaller or reduced in number, treatment was continued for another 6 weeks and esophagoscopy performed a second time to assure clinical cure.

Neoplastic dogs were diagnosed by thoracic CT with evidence of esophageal mass(es) and compatible lung metastatic lesions; and histopathology of nodules/masses removed either surgically or obtained by endoscopic biopsy or during postmortem examination. Neoplastic spirocercosis dogs were offered treatment by surgical excision with or without chemotherapy, symptomatic therapy (antiulcerative and antiemetic therapy, application of a percutaneous endoscopic gastrotomy feeding tube, or both) or euthanasia followed by a postmortem and histopathology. Surgery was performed in 1/26 dogs but not followed by chemotherapy, 2/26 dogs were given symptomatic therapy for a total 1–2 months, and remaining 23/26 dogs were euthanized within a few days postdiagnosis.

In the healthy dogs, anamnesis, CBC, serum biochemistry, urinalysis, fecal analysis, modified centrifugal fecal flotation, and thoracic radiographs were performed to rule out spirocercosis or any other systemic disease.

Exclusion criteria for all dogs of all groups included dogs less than 1 year of age or with evidence of liver or renal disease based on clinical signs, serum biochemistry, urinalysis, abdominal ultrasound, and postmortem. Dogs with concurrent diseases, treated with medications that could influence vitamin D concentrations (corticosteroids, anticonvulsants, calcium channel blockers, and diuretics)[17] during the past month or treated prophylactically for spirocercosis in the past 6 months were also excluded. The prophylactic therapy for spirocercosis was the only exclusion criterion not considered in the healthy dog group.

Appetite Scoring

The appetite of all dogs diagnosed with spirocercosis was scored as normal or abnormal (decreased appetite or total anorexia) based on information obtained from the owners. The type of diet (commercial, home cooked or mixed) was also questioned in all dogs.

The appetite scores were compared between the non-neoplastic and neoplastic spirocercosis groups and then further compared to the serum 25(OH)D concentrations of each spirocercosis group.

Test Method

Serum samples from all the 3 groups of dogs for 25(OH)D analysis were frozen within 1 hour of collection and stored at −70°C before being sent to the laboratory for analysis on dry ice. Serum concentrations of 25(OH)D were measured and validated as described elsewhere.[18, 19] Briefly, samples were extracted using acetonitrile and applied to C18 Silica Sep-paks.a Metabolites were separated by straight phase high-performance liquid chromatographyb using a Hewlett-Packard Zorbax-Sil Columnc eluted with hexane : propan-2-ol : methanol (92 : 4 : 4). Serum 25(OH)D2 and 25(OH)D3 were measured separately by application to a second Zorbax-Sil,d column eluted with hexane : propan-2-ol (98 : 2) and quantified by UV absorbance at 265 nm (radioimmunoassay) and corrected for recovery (sensitivity 5 nmol/L, intra- and interassay coefficients of variation 3.0 and 4.2%, respectively).[20] Results were expressed as total 25(OH)D. The Specialist Assay Laboratory CSB3e is accredited to International Organization for Standarization (ISO) 9001:2008 and ISO 13485:2003 and participates successfully in the Vitamin D quality assurance scheme.

Statistical Analyses

Kruskall–Wallis test was performed to compare 25(OH)D serum concentrations, ages, and serum albumin concentrations among all groups and a post hoc Dunn's multiple comparison test to assess differences between the S. lupi-infected groups. Fisher's exact test was used to compare the appetite scores between the two spirocercosis groups. A Mann–Whitney U-test was used to compare 25(OH)D concentrations and the appetite scores in each spirocercosis group. This statistical analysis was performed with a commercial software package (GraphicPad Prism 5f).

To evaluate the influence of age and serum albumin concentration on serum 25(OH)D concentrations in all groups, the homogeneity of slopes test was performed followed by an analysis of covariance (ANCOVA). Statistica 10g was used for this statistical analysis.

Breeds, and sex and spay/neuter status were compared among the 3 groups using the chi-square test (Microsoft Excel 2010h). Body weight was evaluated for normality (Kolmogorov–Smirnov test) and statistically compared among the groups (Kruskall–Wallis test) using GraphicPad Prism 5.f A multivariable linear regression analysis was performed using an adequate commercial software package (GraphicPad Prism 5f). All 75 dogs were combined as 1 dataset, and the interaction and significance of sex and spay neuter status, body weight, and appetite score (independent variables) with serum 25(OH)D concentrations (dependent variable) were assessed.

A P value of <.05 was considered statistically significant in all statistical tests performed.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments
  8. References

25(OH)D serum concentrations

A statistically significant difference in serum 25(OH)D concentrations was observed among the 3 groups (P < .001) (Fig 1). Post-test analysis demonstrated that the serum 25(OH)D concentrations were significantly lower in the neoplastic spirocercosis group (median 30.7 nmol/L [range 14.7–62.2, n = 26]) compared to the non-neoplastic spirocercosis (median 52.7 nmol/L [range 19.1–129.7, n = 25] [P < .05]) and healthy groups (median 74.6 nmol/L [range 37.4–130.5, n = 24] [p < .005]) (Fig 1). A significant difference was also observed in the 25(OH)D concentrations between the healthy and the non-neoplastic spirocercosis groups (P < .05).

image

Figure 1. Box plot of the comparison between the serum 25(OH)D concentrations in the healthy (n = 24), non-neoplastic spirocercosis (n = 25), and neoplastic spirocercosis (n = 26) groups. The box incorporates the middle 50% of the observations with the line inside the box as the median. The whiskers extend to the smallest and largest observations indicating the range of the data. The median and range of serum 25(OH)D concentrations in nmol/L are stated below the box plot.

Download figure to PowerPoint

Appetite Scores in Dogs with Spirocercosis

No significant differences in appetite scoring were seen between the 2 spirocercosis groups using the Fisher's exact test (P = 1.0).

All dogs were fed a mixture of home cooked and commercial diet (various types of grocery store diets). The proportion of each diet could not be accurately assessed. Therefore, calculation of total dietary vitamin D content ingested was not possible.

25(OH)D Serum Concentrations and Appetite Scoring

No significant differences were observed in the serum 25(OH)D concentrations of dogs with normal or abnormal appetite, either in the non-neoplastic spirocercosis (P = .125) or in the neoplastic spirocercosis groups (P = .0869).

Age, Serum Albumin, and 25(OH)D Concentrations in Dogs with Spirocercosis

There was a significant difference in the ages among all groups (P < .001). Post-test analysis demonstrated that the ages were significantly higher in the neoplastic spirocercosis group (median 72 months [range 36–132 months, n = 26]) compared to the non-neoplastic spirocercosis (median 38.25 months [range 16–150 months, n = 25] [P < .01]) and healthy groups (median 30 months [range 12–120 months, n = 24] [P < .01]). No significant difference was observed in the ages between the healthy and the non-neoplastic spirocercosis groups (P = 1).

The serum albumin concentration was significantly different among all groups (P < .001). Serum albumin concentration was significantly lower in the neoplastic spirocercosis group (median 22.1 g/L [range 12.1–35.9, n = 26]) compared to the non-neoplastic spirocercosis (median 32.1 g/L [range 23.1–37.4, n = 25] [P < .001]) and the healthy groups (median 35.2 g/L [range 24.5–39.2, n = 24] [P < .001]). From the non-neoplastic spirocercosis group, 5/25 dogs had evidence of soft feces, whereas 9/26 neoplastic spirocercosis dogs had diarrhea, possibly contributing to the hypoalbuminemia. No significant difference was observed in the serum albumin concentration between the non-neoplastic spirocercosis and the healthy groups (P > .05).

The ANCOVA demonstrated a significant difference in serum 25(OH)D concentrations among the 3 groups of dogs that was independent of either albumin or age (P < .05).

Breed, Body Weight, Sex, Spay Neuter Status, and Serum 25(OH)D Concentration

No statistical significance was detected for breed (P = .84), sex (P = .32), and spay neuter status (P = .58) among the 3 groups (Table 1). Breeds varied within all groups (total 26 dog breeds), including small, medium, large, and giant breed dogs.

Table 1. Body weight, sex, and spay neuter status of dogs diagnosed with spirocercosis and healthy control dogs
 Weight (kg) Median and RangesSex and Spay Neuter Status
MaleFemale
IntactNeuteredIntactNeutered
Non-neoplastic spirocercosis dogs (n = 25)30.2 (6.9–78) 64 96
Neoplastic spirocercosis dogs (n = 26)23.2 (5.4–50)122 75
Healthy dogs (n = 24)29.7 (5.4–70) 44115

The non-neoplastic spirocercosis dogs had a median body weight of 30.2 kg (range 6.9–78), followed by the healthy ones with 29.7 kg (range 5.4–70), and the neoplastic spirocercosis dogs with 23.2 kg (range 5.4–50) (Table 1). No statistical significant difference was observed in the median body weight among the 3 groups (P = .27).

Multivariable linear regression analysis demonstrated that serum 25(OH)D concentration was significantly different among all groups (P < .001, r = 0.35), that all dependent variables were independent of each other, and that there was no linear relationship with either body weight (P = .08, r = 0.24) or appetite score (P = .16, r = −0.49). Neutered and female dogs had higher 25(OH)D concentrations than intact and male dogs.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments
  8. References

This study has shown that serum 25(OH)D is low in dogs with spirocercosis and that it is significantly lower in the neoplastic form of the disease compared to the non-neoplastic form. A reduction in serum 25(OH)D concentrations with progression of the disease from a non-neoplastic to a neoplastic state was also evident in this study. These results were independent of appetite, age, and serum albumin and the low vitamin D status could thus potentially play a role in the neoplastic transformation of spirocercosis in dogs.

Causes of reduced serum 25(OH)D concentrations could be attributed to anorexia, alterations in hepatic function, or overexpression of FGF23 and 24(OH)ase activity.[10, 12, 21, 22] Firstly, our study showed that appetite was unlikely to account for the difference in serum 25(OH)D concentrations among the spirocercosis groups. However, it is possible that dogs that had inflammatory esophageal disease or neoplasia were eating less commercial dog food and more table foods (that are typically low in vitamin D) than control dogs, which may have contributed to the vitamin D deficiency in the former group of dogs. No linear relationship was also found between either body weight or appetite score and 25(OH)D concentrations. Secondly, only dogs without evidence of liver disease based on serum liver enzymes evaluation, abdominal ultrasonographic findings, histopathology, or a combination were included in the study. Therefore, hepatic disease was also an unlikely cause of the reduced 25(OH)D concentrations. Thirdly, we speculate that the pathophysiologic mechanism(s) leading to the low serum 25(OH)D concentrations in spirocercosis could relate to increased catabolism caused by FGF23 overexpression and increase 24-OHase activity,[10, 11, 22] genetic mutations leading to reduced 25(OH)D synthesis,[23] or chronic inflammation.[24, 25] Neoplastic and non-neoplastic spirocercosis nodules have been previously shown to overexpress FGF, with higher expressions in the neoplastic compared to the non-neoplastic cases.[26] This could potentially contribute to the low vitamin D status detected in spirocercosis compared to healthy dogs. Genetic mutations leading to the overexpression of FGF or genetic polymorphisms in genes encoding VDR, vitamin D-binding protein, 1α(OH)ase (CYP27B1), 24(OH)ase (CYP24A1), and 25(OH)ase (CYP2R1) remain possible,[23] although further studies to prove this association are required. Chronic inflammation caused by parasitic infections is recognized as an important risk factor for neoplastic development.[25] Inflammatory mediators that are produced can induce DNA damage in tumor suppressor genes, leading to post-translational modifications of proteins involved in cellular apoptosis, DNA repair, and cell cycle checkpoints.[25, 27] There is an association between neoplasia development, chronic inflammation,27 and vitamin D demonstrated in in vitro human[9, 12, 15] and animal[9, 16] studies, and in human epidemiologic investigations.[10, 12] Chronic inflammation leads to lipid peroxidation and potentially to genetic mutations.[27] If these mutations accumulate in key host cell regulatory genes they can eventually change the cell phenotype and lead to neoplasia.[27] Vitamin D seems to modulate the immune system,[28] preventing neoplasia by suppressing inflammation that facilitates tumorigenesis and by activating receptors of cells of the adaptive immune system in the presence of abnormal cells or antigens.[9] Induced endotoxemic dogs have low serum vitamin D concentrations.[24] Hypovitaminosis D in sepsis results from an impaired activity of the 1αOHase, reduced DBP, and loss of urinary 25(OH)D.[24] In our study, hypovitaminosis D in non-neoplastic spirocercosis seems to be an effect of chronic inflammation and a potential risk factor for neoplastic transformation.

Reduction in serum vitamin D concentrations has been associated with increasing age.[29] The 25(OH)D concentrations were lower in the non-neoplastic group versus the healthy group, yet the ages were similar, supporting the fact that age is unlikely to account for the differences in vitamin D. The relationship between age, vitamin D, and neoplasia remains questionable, because many exposures and events accumulate with age, potentially leading to genetic mutations and epigenetic changes associated with neoplasia development.[9]

Some of the dogs in this study had low albumin and evidence of soft feces or diarrhea. Gastrointestinal albumin loss has been shown to correlate with serum 25(OH)D concentrations in dogs with intestinal bowel disease (IBD).[30] Intestinal loss of DBP was postulated as one of the potential causes of hypovitaminosis D in dogs with IBD and hypoalbuminemia.[30-33] In this study, although the serum albumin was significantly lower in the neoplastic group, the hypovitaminosis D was independently lower, and therefore it can only be regarded as a contributing factor to the low vitamin D concentrations. This phenomenon, although valid, would still not explain the hypovitaminosis D detected in the remaining dogs without evidence of diarrhea, soft feces or hypoalbuminemia. The cause of the hypoalbuminemia detected in our study could relate to loss from the ulcerated neoplastic lesion, malnutrition, parasitism, or chronic inflammation (albumin is a negative acute phase protein).

The antineoplastic treatment properties of calcitriol that leads to tumor regression have been shown in in vitro and in vivo neoplasias in dogs[13, 15, 16, 34] (transitional cell carcinomas,[15] mast cell tumors,[13] osteosarcoma,[34] hemangiosarcoma,[34] and carcinomas[34]) and in humans.[9-11] Treatment of neoplastic cells with 1,25(OH)2D may inhibit cell tube formation and tumor growth by repressing vascular endothelial growth factor (VEGF) and IL-8.[11] Dogs with neoplastic spirocercosis overexpress IL-8[35] and VEGF,[23] therefore the use of 1,25(OH)2D as an adjuvant therapeutic agent in the neoplastic spirocercosis could be considered. Its use in non-neoplastic spirocercosis could also be beneficial for its anti-inflammatory effects on COX-2 expression and the prostaglandin pathway,[7] potentially reducing the risk of neoplastic transformation. The major in vivo limitation for calcitriol supplementation/treatment is the potential hypercalcemic effects, especially when used for its antineoplastic effects (dose-dependent effect), because high doses would be required.[13] Further studies are warranted to evaluate calcitriol treatment and to determine whether it would decrease the incidence of neoplastic transformation. The therapeutic use of calcitriol should be accompanied by close monitoring of serum calcium concentrations.

Limitations of this study include a lack of quantification of the proportion of home cooked diet versus commercial diet, and a lack of body condition scoring. Body condition scoring (although subjective) would have been a helpful piece of information for our understanding of whether food intake was a factor influencing the 25(OH)D concentrations among the groups, as it is thought less subjective than appetite scoring by owners. Furthermore, as this was not a breed-matched study, the use of body weight was a poor proxy for either hyporexia or adiposity.

Conclusion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments
  8. References

This study demonstrated low serum 25(OH)D concentration in dogs with spirocercosis with lower concentrations in the neoplastic group. The hypovitaminosis D detected in the non-neoplastic spirocercosis could relate to FGF overexpression or chronic inflammation and immune system suppression, and could potentially be a risk factor for neoplastic transformation. Further studies are required to evaluate vitamin D as a potential adjuvant therapeutic agent for its antiproliferative, apoptotic, and antiangiogenic effects and to elucidate its role in the pathogenesis of the neoplastic transformation of dogs diagnosed with spirocercosis.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments
  8. References

Conflict of Interest Declaration: Authors disclose no conflict of interest.

Footnotes
  1. 1

    C18 Silica Sep-paks; Waters Ltd, Elstree, UK

  2. 2

    High-performance liquid chromatography; Waters Associates, Milford, CT

  3. 3

    Hewlett-Packard Zorbax-Sil Column; Hichrom, Reading, UK

  4. 4

    Zorbax-Sil; Agilent Technologies, Stockport, UK

  5. 5

    Specialist Assay Laboratory CSB3; Manchester Royal Infirmary, Manchester, UK

  6. 6

    GraphicPad Prism 5, GraphPad Software Inc, La Jolla, CA

  7. 7

    Statistica 10, StatSoft Inc, Ramat Hasharon, Israel

  8. 8

    Microsoft Excel 2010, Microsoft, Bryanston, Johannesburg, RSA

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgments
  8. References