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Serum concentration of high density lipoproteins (HDLs) in leishmaniotic dogs
Research in Veterinary Science 98 (2015) 89–91
Contents lists available at ScienceDirect
Research in Veterinary Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r v s c
Serum concentration of high density lipoproteins (HDLs) in leishmaniotic dogs Fabrizio Ibba a,b, Gabriele Rossi a, Sara Meazzi a, Alessia Giordano a,c, Saverio Paltrinieri a,c,* a b c
Department of Veterinary Sciences and Public Health, University of Milan, Via Celoria 10, 20133, Milan, Italy Veterinary Clinic Poggio di Pini, Capoterra (CA), Italy Clinical Pathology Laboratory, Veterinary Teaching Hospital, University of Milan, Via dell’Università 6, 26900, Lodi, Italy
A R T I C L E
I N F O
Article history: Received 11 July 2014 Accepted 14 November 2014 Keywords: Clinical pathology Parasitology Companion animal medicine Canine leishmaniasis Inflammation
A B S T R A C T
In order to assess whether the concentration of high density lipoproteins (HDLs) changes in leishmaniotic dogs before and after treatment, HDL cholesterol (HDL-Chol and HDL%), C reactive protein (CRP) and activity of the antioxidant enzyme paraoxonase (PON-1) were measured in sera from 10 controls and 10 leishmaniotic dogs. Seven of these latter were sampled also 3, 7, 14, 21 and 28 days after treatment with antimonials and allopurinol. HDL-chol, and PON-1 were low in leishmaniotic dogs at admission and increased after treatment. HDL-chol and HDL% correlated positively with PON-1 and negatively with CRP suggesting that HDLs decrease through an oxidative mechanism. Therefore, HDLs may be used to monitor the magnitude of oxidation associated with inflammation in leishmaniotic dogs. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction High density lipoproteins (HDLs), produced in the liver, distribute cholesterol and triglycerides to peripheral tissues and have antioxidant properties (Bruss, 2008). In people, HDL levels negatively correlate with the severity of clinical signs and with the concentration of cytokines in several diseases (Chait et al., 2005). During inflammation, HDLs undergo structural changes: the acute phase proteins serum amyloid A and ceruloplasmin adhere to HDLs and apolipoprotein-A, esterified cholesterol and associated enzymes, such as paraoxonase-1 (PON-1), are replaced by free cholesterol, triglycerides and free fatty acids, reducing the antioxidative activity of HDLs (Van Lenten et al., 2006). Canine leishmaniasis (CL) is characterized by oxidative stress (Almeida et al., 2013). The activation of phagocytes in tissues increases the oxidative metabolism and decreases antioxidant compounds, including PON-1 (Rossi et al., 2014). Therefore, oxidative stress may affect also HDL concentrations. In turn, changes of HDL may affect the course of leishmaniasis as demonstrated in people in vitro, where HDLs decrease the production of TNF-α by infected macrophages (Soares et al., 2010).
Abbreviations: CL, canine leishmaniasis; Chol, total cholesterol; CKD, chronic kidney disease; CRP, C-reactive protein; HDL, high density lipoprotein; HDL-chol, HDLcholesterol (mg/ml); HDL%, percentage of HDL-chol on total chol; PON-1, paraoxonase-1 * Corresponding author. Department of Veterinary Sciences and Public Health, University of Milan, Via Celoria 10, 20133, Milan, Italy. Tel.: +39 02 50318103; fax: +39 03 50318095. E-mail address: [email protected] (S. Paltrinieri). http://dx.doi.org/10.1016/j.rvsc.2014.11.011 0034-5288/© 2014 Elsevier Ltd. All rights reserved.
The aim of the study was to investigate the changes in serum HDL concentration in leishmaniotic dogs before and after treatment and the correlation between HDL and PON-1. 2. Materials and methods Serum was collected from 10 clinically healthy control dogs (6 female, 4 male, age range 3–7 years, median: 6 years), living in a non-endemic area and seronegative for leishmania and from 10 dogs with CL (7 female, 3 male, age range 2–8 years, median: 5 years), on which the activity of PON-1 and the concentration of C reactive protein (CRP) were recorded in a previous study (Rossi et al., 2014). Dogs were sampled during wellness visits or for diagnostic/ monitoring purposes under informed consent of the owners and therefore a formal approval from the ethical committee was not required by our Institution. As specified by Rossi et al. (2014), CL was diagnosed according to current guidelines (Paltrinieri et al., 2010), by positive serology (immunofluorescence test with antibody titers fourfold higher than the positive threshold of the laboratory) and by positive cytology or PCR in lymph nodes or bone marrow of dogs with clinical signs of visceral leishmaniasis. Leishmaniotic dogs were classified as sick (n = 7) or severely sick (n = 3) based on the criteria established by the guidelines. Samples from the seven sick dogs were collected also on day 3, 7, 14, 21 and 28 after the beginning of treatment with meglumine antimoniate (100 mg/kg q24h for 4 weeks) and allopurinol (10 mg/kg, q12h, for 6 months) (Oliva et al., 2010). The concentration of total cholesterol (Chol – cholesterol oxidase method) and HDL cholesterol (immunoseparation method) was measured on all the dogs in an ILAB300-plus spectrophotometer
90
F. Ibba et al./Research in Veterinary Science 98 (2015) 89–91
Fig. 1. Results obtained at admission in controls and in dogs with leishmaniasis. White dots indicate dogs with severe leishmaniasis. Black asterisks indicate significant differences (**P < 0.01; ***P < 0.001) compared with controls.
(Instrumentation Laboratory) while CRP concentrations (immunoturbidimetric method) and PON-1 activity (enzymatic method) were measured in controls using a Cobas Mira spectrophotometer (Roche diagnostic) as previously described (Rossi et al., 2014). HDL cholesterol was expressed in mg/dL (HDL-chol) and as percentage of total cholesterol (HDL%). Using a specific software (Analyse-it Software Ltd), a Mann–Whitney U test was used to compare the results obtained at admission in dogs with and without CL, results of sequential samplings were compared with each other using a Friedman test followed by a Wilcoxon signed rank test to assess the differences from day 0, and the correlation between the analytes was assessed using the Spearman test.
3. Results At admission, Chol, HDL-chol, and PON-1 activity were significantly lower in dogs with CL than in controls (Fig. 1). However, a high individual variability was found in dogs with leishmaniasis. After treatment, the concentration of Chol and of HDL% did not change over time. Conversely, HDL-chol increased over time, with significant differences from day 21 (Fig. 2). Both HDL-chol and HDL% positively correlated with PON-1 (P = 0.002; r = 0.55 and P = 0.019; r = 0.44, respectively) and negatively with CRP (P = 0.049, r = −0.37 and P = 0.014, r = −0.47,
Fig. 2. Concentration of HDL-Chol recorded after treatment. Asterisks indicate a significant difference (P < 0.05) compared with day 0. The boxes indicate the I–III interquartile range (IQR), the horizontal line indicates median values, whiskers extend to further observation within the I quartile minus 1.5 × IQR or within the III quartile plus 1.5 × IQR. The symbol “+”indicates near outliers (values exceeding the I quartile minus 1.5 × IQR).
F. Ibba et al./Research in Veterinary Science 98 (2015) 89–91
respectively), which in turn negatively correlated with PON-1 (P = 0.000, r = −0.60). 4. Discussion This study investigated the serum concentration of HDLs in dogs with leishmaniasis, since this condition is characterized by inflammation and oxidative stress (Almeida et al., 2013), and HDLs have antioxidant properties (Bruss, 2008). To this aim, we selected from a previous study (Rossi et al., 2014) samples from leishmaniotic dogs with complete information on the presence of inflammation (high CRP levels) on which the activity of the antioxidant enzyme PON-1 had already been determined. Another advantage of using samples from the previous study is that the dogs that recovered after treatment were repeatedly sampled over time, using a sampling schedule difficult to repeat in field that allows the detection of early changes induced by treatments. Conversely, the disadvantage of this selection is that the number of cases included in the study was low, although sufficient for a preliminary evaluation of the role of HDL in the pathogenesis of CL and in monitoring CL over time. In particular, the number of dogs with severe leishmaniasis was too low to permit a reliable statistical comparison with other groups. However, despite the low number of dogs, this study demonstrated that HDLs show evident changes in dogs with CL and a rapid restoration of HDL values after treatment. Chol was variable in leishmaniotic dogs, likely due to the hypercholesterolemia that characterizes the nephrotic syndrome affecting leishmaniotic dogs (Bruss, 2008). Despite the high Chol, HDL-chol was significantly lower in leishmaniotic dogs, suggesting the presence of oxidative stress. As a further support to this hypothesis, PON-1 activity was significantly lower in leishmaniotic dogs (especially in those with severe leishmaniasis), as expected (Rossi et al., 2014). The changes of HDL-chol after treatment were similar to those recorded in the same dogs for PON-1 and reported in Rossi et al. (2014). Several factors may induce the normalization of HDL, PON-1 and CRP after treatment. CRP increases in dogs with chronic kidney disease (CKD) (Rossi et al., 2013) and PON-1 activity decreases in people with CKD (Ak et al., 2002). It is thus possible that the improvement of renal function contributed to the normalization of these analytes. Conversely, it is unlikely that PON-1 and HDLs increased due to the recovery of appetite and food consumption, as reported in people (Bruss, 2008; Ferré et al., 2003), since anorexia and weight loss were not reported in dogs sampled over time. Moreover, dietary factors usually need a longer time to influence blood analytes. Therefore, the changes of HDL and PON-1 likely reflect the reduced rate of oxidation associated with decreased inflammatory stimuli. This hypothesis is supported by the earlier normalization of PON-1 (1 week) compared with HDL-chol (3 weeks). This may depend on the fact that during inflammation PON-1 consumption increases and PON-1 synthesis decreases (Feingold et al., 1998). It is therefore possible that hepatic synthesis increased in the first week after treatment, but consumption associated with
91
oxidation, extended until day 21, as demonstrated by the longer normalization time of CRP and HDL-chol. Results of Spearman test, that showed a positive correlation between HDL and PON-1 activity, and a negative correlation between both HDLs and PON-1 activity and CRP levels, further support the hypothesis that changes in HDLs depend on oxidative stress associated with inflammation. In conclusion, this study demonstrated that the serum concentration of HDLs decreases in CL likely due to the oxidative stress and inflammation. After treatment, the concentration of HDLs increases, likely due to the decrease of oxidation/inflammation, although the increase of HDLs occurs later than that of PON-1. Therefore, HDLs may be used as additional markers in association with CRP or PON-1, to identify and monitor over time the magnitude of oxidation and inflammation in dogs with leishmaniasis.
Acknowledgements The Authors are grateful to Dr. Pierangelo Moretti for the technical support.
References Ak, G., Ozgönül, M., Sözmen, E.Y., Aslan, S.L., Sözmen, B., 2002. Renal cortical thickness and PON1 activity both decrease in chronic renal failure. Journal of Nephrology 15, 144–149. Almeida, B.F., Narciso, L.G., Melo, L.M., Preve, P.P., Bosco, A.M., Lima, V.M.F., et al., 2013. Leishmaniasis causes oxidative stress and alteration of oxidative metabolism and viability of neutrophils in dogs. The Veterinary Journal 198, 599–605. Bruss, M.L., 2008. Lipids and ketones. In: Kaneko, J.J., Harvey, J.W., Bruss, M.L. (Eds.), Clinical Biochemistry of Domestic Animals, sixth ed. Elsevier, San Diego, CA, pp. 81–116. Chait, A., Han, C.Y., Oram, J.F., Heinecke, J.W., 2005. Thematic review series: the immune system and atherogenesis. Lipoprotein-associated inflammatory proteins: markers or mediators of cardiovascular disease? Journal of Lipid Research 46, 389–403. Feingold, K.R., Memon, R.A., Moser, A.H., Grunfeld, C., 1998. Paraoxonase activity in the serum and hepatic mRNA levels decrease during the acute phase response. Atherosclerosis 139, 307–315. Ferré, N., Camps, J., Fernández-Ballart, J., Arija, V., Murphy, M.M., Ceruelo, S., et al., 2003. Regulation of serum paraoxonase activity by genetic, nutritional, and lifestyle factors in the general population. Clinical Chemistry 49, 1491–1497. Oliva, G., Roura, X., Crotti, A., Maroli, M., Castagnaro, M., Gradoni, L., et al., 2010. Guidelines for treatment of leishmaniasis in dogs. Journal of the American Veterinary Medical Association 236, 1192–1198. Paltrinieri, S., Solano-Gallego, L., Fondati, A., Lubas, G., Gradoni, L., Castagnaro, M., et al., 2010. Guidelines for diagnosis and clinical classification of leishmaniasis in dogs. Journal of the American Veterinary Medical Association 236, 1184–1191. Rossi, G., Giordano, A., Breda, S., Lisi, C., Roura, X., Zatelli, A., et al., 2013. Big-endothelin 1 (Big ET-1) and homocysteine in the serum of dogs with chronic kidney disease. The Veterinary Journal 198, 109–115. Rossi, G., Ibba, F., Meazzi, S., Giordano, A., Paltrinieri, S., 2014. Paraoxonase activity as a tool for clinical monitoring of dogs treated for canine leishmaniasis. The Veterinary Journal 199, 143–149. Soares, N.M., Leal, T.F., Fiúza, M.C., Reis, E.A., Souza, M.A., Dos-Santos, W.L., et al., 2010. Plasma lipoproteins in visceral leishmaniasis and their effect on Leishmaniainfected macrophages. Parasite Immunology 32, 259–266. Van Lenten, B.J., Reddy, S.T., Navab, M., Fogelman, A.M., 2006. Understanding changes in high density lipoproteins during the acute phase response. Arteriosclerosis, Thrombosis, and Vascular Biology 26, 1687–1688.
Contents lists available at ScienceDirect
Research in Veterinary Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r v s c
Serum concentration of high density lipoproteins (HDLs) in leishmaniotic dogs Fabrizio Ibba a,b, Gabriele Rossi a, Sara Meazzi a, Alessia Giordano a,c, Saverio Paltrinieri a,c,* a b c
Department of Veterinary Sciences and Public Health, University of Milan, Via Celoria 10, 20133, Milan, Italy Veterinary Clinic Poggio di Pini, Capoterra (CA), Italy Clinical Pathology Laboratory, Veterinary Teaching Hospital, University of Milan, Via dell’Università 6, 26900, Lodi, Italy
A R T I C L E
I N F O
Article history: Received 11 July 2014 Accepted 14 November 2014 Keywords: Clinical pathology Parasitology Companion animal medicine Canine leishmaniasis Inflammation
A B S T R A C T
In order to assess whether the concentration of high density lipoproteins (HDLs) changes in leishmaniotic dogs before and after treatment, HDL cholesterol (HDL-Chol and HDL%), C reactive protein (CRP) and activity of the antioxidant enzyme paraoxonase (PON-1) were measured in sera from 10 controls and 10 leishmaniotic dogs. Seven of these latter were sampled also 3, 7, 14, 21 and 28 days after treatment with antimonials and allopurinol. HDL-chol, and PON-1 were low in leishmaniotic dogs at admission and increased after treatment. HDL-chol and HDL% correlated positively with PON-1 and negatively with CRP suggesting that HDLs decrease through an oxidative mechanism. Therefore, HDLs may be used to monitor the magnitude of oxidation associated with inflammation in leishmaniotic dogs. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction High density lipoproteins (HDLs), produced in the liver, distribute cholesterol and triglycerides to peripheral tissues and have antioxidant properties (Bruss, 2008). In people, HDL levels negatively correlate with the severity of clinical signs and with the concentration of cytokines in several diseases (Chait et al., 2005). During inflammation, HDLs undergo structural changes: the acute phase proteins serum amyloid A and ceruloplasmin adhere to HDLs and apolipoprotein-A, esterified cholesterol and associated enzymes, such as paraoxonase-1 (PON-1), are replaced by free cholesterol, triglycerides and free fatty acids, reducing the antioxidative activity of HDLs (Van Lenten et al., 2006). Canine leishmaniasis (CL) is characterized by oxidative stress (Almeida et al., 2013). The activation of phagocytes in tissues increases the oxidative metabolism and decreases antioxidant compounds, including PON-1 (Rossi et al., 2014). Therefore, oxidative stress may affect also HDL concentrations. In turn, changes of HDL may affect the course of leishmaniasis as demonstrated in people in vitro, where HDLs decrease the production of TNF-α by infected macrophages (Soares et al., 2010).
Abbreviations: CL, canine leishmaniasis; Chol, total cholesterol; CKD, chronic kidney disease; CRP, C-reactive protein; HDL, high density lipoprotein; HDL-chol, HDLcholesterol (mg/ml); HDL%, percentage of HDL-chol on total chol; PON-1, paraoxonase-1 * Corresponding author. Department of Veterinary Sciences and Public Health, University of Milan, Via Celoria 10, 20133, Milan, Italy. Tel.: +39 02 50318103; fax: +39 03 50318095. E-mail address: [email protected] (S. Paltrinieri). http://dx.doi.org/10.1016/j.rvsc.2014.11.011 0034-5288/© 2014 Elsevier Ltd. All rights reserved.
The aim of the study was to investigate the changes in serum HDL concentration in leishmaniotic dogs before and after treatment and the correlation between HDL and PON-1. 2. Materials and methods Serum was collected from 10 clinically healthy control dogs (6 female, 4 male, age range 3–7 years, median: 6 years), living in a non-endemic area and seronegative for leishmania and from 10 dogs with CL (7 female, 3 male, age range 2–8 years, median: 5 years), on which the activity of PON-1 and the concentration of C reactive protein (CRP) were recorded in a previous study (Rossi et al., 2014). Dogs were sampled during wellness visits or for diagnostic/ monitoring purposes under informed consent of the owners and therefore a formal approval from the ethical committee was not required by our Institution. As specified by Rossi et al. (2014), CL was diagnosed according to current guidelines (Paltrinieri et al., 2010), by positive serology (immunofluorescence test with antibody titers fourfold higher than the positive threshold of the laboratory) and by positive cytology or PCR in lymph nodes or bone marrow of dogs with clinical signs of visceral leishmaniasis. Leishmaniotic dogs were classified as sick (n = 7) or severely sick (n = 3) based on the criteria established by the guidelines. Samples from the seven sick dogs were collected also on day 3, 7, 14, 21 and 28 after the beginning of treatment with meglumine antimoniate (100 mg/kg q24h for 4 weeks) and allopurinol (10 mg/kg, q12h, for 6 months) (Oliva et al., 2010). The concentration of total cholesterol (Chol – cholesterol oxidase method) and HDL cholesterol (immunoseparation method) was measured on all the dogs in an ILAB300-plus spectrophotometer
90
F. Ibba et al./Research in Veterinary Science 98 (2015) 89–91
Fig. 1. Results obtained at admission in controls and in dogs with leishmaniasis. White dots indicate dogs with severe leishmaniasis. Black asterisks indicate significant differences (**P < 0.01; ***P < 0.001) compared with controls.
(Instrumentation Laboratory) while CRP concentrations (immunoturbidimetric method) and PON-1 activity (enzymatic method) were measured in controls using a Cobas Mira spectrophotometer (Roche diagnostic) as previously described (Rossi et al., 2014). HDL cholesterol was expressed in mg/dL (HDL-chol) and as percentage of total cholesterol (HDL%). Using a specific software (Analyse-it Software Ltd), a Mann–Whitney U test was used to compare the results obtained at admission in dogs with and without CL, results of sequential samplings were compared with each other using a Friedman test followed by a Wilcoxon signed rank test to assess the differences from day 0, and the correlation between the analytes was assessed using the Spearman test.
3. Results At admission, Chol, HDL-chol, and PON-1 activity were significantly lower in dogs with CL than in controls (Fig. 1). However, a high individual variability was found in dogs with leishmaniasis. After treatment, the concentration of Chol and of HDL% did not change over time. Conversely, HDL-chol increased over time, with significant differences from day 21 (Fig. 2). Both HDL-chol and HDL% positively correlated with PON-1 (P = 0.002; r = 0.55 and P = 0.019; r = 0.44, respectively) and negatively with CRP (P = 0.049, r = −0.37 and P = 0.014, r = −0.47,
Fig. 2. Concentration of HDL-Chol recorded after treatment. Asterisks indicate a significant difference (P < 0.05) compared with day 0. The boxes indicate the I–III interquartile range (IQR), the horizontal line indicates median values, whiskers extend to further observation within the I quartile minus 1.5 × IQR or within the III quartile plus 1.5 × IQR. The symbol “+”indicates near outliers (values exceeding the I quartile minus 1.5 × IQR).
F. Ibba et al./Research in Veterinary Science 98 (2015) 89–91
respectively), which in turn negatively correlated with PON-1 (P = 0.000, r = −0.60). 4. Discussion This study investigated the serum concentration of HDLs in dogs with leishmaniasis, since this condition is characterized by inflammation and oxidative stress (Almeida et al., 2013), and HDLs have antioxidant properties (Bruss, 2008). To this aim, we selected from a previous study (Rossi et al., 2014) samples from leishmaniotic dogs with complete information on the presence of inflammation (high CRP levels) on which the activity of the antioxidant enzyme PON-1 had already been determined. Another advantage of using samples from the previous study is that the dogs that recovered after treatment were repeatedly sampled over time, using a sampling schedule difficult to repeat in field that allows the detection of early changes induced by treatments. Conversely, the disadvantage of this selection is that the number of cases included in the study was low, although sufficient for a preliminary evaluation of the role of HDL in the pathogenesis of CL and in monitoring CL over time. In particular, the number of dogs with severe leishmaniasis was too low to permit a reliable statistical comparison with other groups. However, despite the low number of dogs, this study demonstrated that HDLs show evident changes in dogs with CL and a rapid restoration of HDL values after treatment. Chol was variable in leishmaniotic dogs, likely due to the hypercholesterolemia that characterizes the nephrotic syndrome affecting leishmaniotic dogs (Bruss, 2008). Despite the high Chol, HDL-chol was significantly lower in leishmaniotic dogs, suggesting the presence of oxidative stress. As a further support to this hypothesis, PON-1 activity was significantly lower in leishmaniotic dogs (especially in those with severe leishmaniasis), as expected (Rossi et al., 2014). The changes of HDL-chol after treatment were similar to those recorded in the same dogs for PON-1 and reported in Rossi et al. (2014). Several factors may induce the normalization of HDL, PON-1 and CRP after treatment. CRP increases in dogs with chronic kidney disease (CKD) (Rossi et al., 2013) and PON-1 activity decreases in people with CKD (Ak et al., 2002). It is thus possible that the improvement of renal function contributed to the normalization of these analytes. Conversely, it is unlikely that PON-1 and HDLs increased due to the recovery of appetite and food consumption, as reported in people (Bruss, 2008; Ferré et al., 2003), since anorexia and weight loss were not reported in dogs sampled over time. Moreover, dietary factors usually need a longer time to influence blood analytes. Therefore, the changes of HDL and PON-1 likely reflect the reduced rate of oxidation associated with decreased inflammatory stimuli. This hypothesis is supported by the earlier normalization of PON-1 (1 week) compared with HDL-chol (3 weeks). This may depend on the fact that during inflammation PON-1 consumption increases and PON-1 synthesis decreases (Feingold et al., 1998). It is therefore possible that hepatic synthesis increased in the first week after treatment, but consumption associated with
91
oxidation, extended until day 21, as demonstrated by the longer normalization time of CRP and HDL-chol. Results of Spearman test, that showed a positive correlation between HDL and PON-1 activity, and a negative correlation between both HDLs and PON-1 activity and CRP levels, further support the hypothesis that changes in HDLs depend on oxidative stress associated with inflammation. In conclusion, this study demonstrated that the serum concentration of HDLs decreases in CL likely due to the oxidative stress and inflammation. After treatment, the concentration of HDLs increases, likely due to the decrease of oxidation/inflammation, although the increase of HDLs occurs later than that of PON-1. Therefore, HDLs may be used as additional markers in association with CRP or PON-1, to identify and monitor over time the magnitude of oxidation and inflammation in dogs with leishmaniasis.
Acknowledgements The Authors are grateful to Dr. Pierangelo Moretti for the technical support.
References Ak, G., Ozgönül, M., Sözmen, E.Y., Aslan, S.L., Sözmen, B., 2002. Renal cortical thickness and PON1 activity both decrease in chronic renal failure. Journal of Nephrology 15, 144–149. Almeida, B.F., Narciso, L.G., Melo, L.M., Preve, P.P., Bosco, A.M., Lima, V.M.F., et al., 2013. Leishmaniasis causes oxidative stress and alteration of oxidative metabolism and viability of neutrophils in dogs. The Veterinary Journal 198, 599–605. Bruss, M.L., 2008. Lipids and ketones. In: Kaneko, J.J., Harvey, J.W., Bruss, M.L. (Eds.), Clinical Biochemistry of Domestic Animals, sixth ed. Elsevier, San Diego, CA, pp. 81–116. Chait, A., Han, C.Y., Oram, J.F., Heinecke, J.W., 2005. Thematic review series: the immune system and atherogenesis. Lipoprotein-associated inflammatory proteins: markers or mediators of cardiovascular disease? Journal of Lipid Research 46, 389–403. Feingold, K.R., Memon, R.A., Moser, A.H., Grunfeld, C., 1998. Paraoxonase activity in the serum and hepatic mRNA levels decrease during the acute phase response. Atherosclerosis 139, 307–315. Ferré, N., Camps, J., Fernández-Ballart, J., Arija, V., Murphy, M.M., Ceruelo, S., et al., 2003. Regulation of serum paraoxonase activity by genetic, nutritional, and lifestyle factors in the general population. Clinical Chemistry 49, 1491–1497. Oliva, G., Roura, X., Crotti, A., Maroli, M., Castagnaro, M., Gradoni, L., et al., 2010. Guidelines for treatment of leishmaniasis in dogs. Journal of the American Veterinary Medical Association 236, 1192–1198. Paltrinieri, S., Solano-Gallego, L., Fondati, A., Lubas, G., Gradoni, L., Castagnaro, M., et al., 2010. Guidelines for diagnosis and clinical classification of leishmaniasis in dogs. Journal of the American Veterinary Medical Association 236, 1184–1191. Rossi, G., Giordano, A., Breda, S., Lisi, C., Roura, X., Zatelli, A., et al., 2013. Big-endothelin 1 (Big ET-1) and homocysteine in the serum of dogs with chronic kidney disease. The Veterinary Journal 198, 109–115. Rossi, G., Ibba, F., Meazzi, S., Giordano, A., Paltrinieri, S., 2014. Paraoxonase activity as a tool for clinical monitoring of dogs treated for canine leishmaniasis. The Veterinary Journal 199, 143–149. Soares, N.M., Leal, T.F., Fiúza, M.C., Reis, E.A., Souza, M.A., Dos-Santos, W.L., et al., 2010. Plasma lipoproteins in visceral leishmaniasis and their effect on Leishmaniainfected macrophages. Parasite Immunology 32, 259–266. Van Lenten, B.J., Reddy, S.T., Navab, M., Fogelman, A.M., 2006. Understanding changes in high density lipoproteins during the acute phase response. Arteriosclerosis, Thrombosis, and Vascular Biology 26, 1687–1688.