PhD Thesis defense by Blanca Pérez Montero at the VISAVET Centre of the Complutense University of Madrid

April 26^th, 2024

Oxidative stress, defined as an imbalance between oxidizing agents and the antioxidant forces of the body, has been associated with multiple pathologies in the canine species. Among the most commonly used biomarkers are malondialdehyde (MDA), a metabolite of lipid peroxidation, 8-hydroxy-2`-deoxyguanosine (8-OHdG), a product of DNA oxidation, and the 2,2`-azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS) assay as an indicator of total antioxidant status (TAS). Although these biomarkers have demonstrated clinical value in dogs, the absence of established reference intervals (RIs) is noteworthy. Therefore, the objectives of this work were: to determine the RI of serum MDA, 8-OHdG, and TAS concentrations in dogs, evaluate the influence of biological variation sources and multiple analytical parameters, and determine endogenous interference by hemolysis, icterus, and lipemia (HIL) in the assays.

A population of 190 clinically healthy dogs from three sources was selected: private guardians, the National Police, and an Animal Protection Center (APC). After performing a hematological and biochemical profile, the value of MDA was determined using the thiobarbituric acid reactive substances (TBARS) assay, that of 8-OHdG using a competitive ELISA, and that of TAS using the ABTS colorimetric assay. Various international guidelines were followed for establishing RIs and determining HIL interference limits. To evaluate the influence of biological variation sources and analytical parameters, a univariable analysis was performed using non-parametric tests and a multivariable analysis was conducted by constructing linear regression models.

The final reference population comprised 144 dogs (57 private, 43 police, and 44 from the APC). The established serum RIs were 1.85 to 14.51 μM for MDA; 0.06 to 0.75 ng/mL for 8-OHdG; and 1.41 to 2.27 mmol/L for TAS. After considering statistical and clinical criteria, RI partitioning was performed in subgroups for TAS (from 1.37 to 1.98 mmol/L for private, from 1.72 to 2.47 for police dogs, and from 1.39 to 2.31 mmol/L for the APC).

The regression model for MDA evidenced its association with the value of 8-OHdG, in line with the biochemical mechanisms of oxidative stress; with cholesterol, attributed to in vivo or in vitro peroxidation phenomena; and with monocyte count, possibly related to phagocytosis of MDA adducts. The 8-OHdG model confirmed its association with MDA value and revealed its association with urea value, possibly linked to renal excretion. The TAS model identified a higher value in police dogs, which could be explained by genetic and environmental factors; a positive association with protein fractions (albumin and globulins), consistent with the assay`s biochemical characteristics and the antioxidant functions of the analytes; and a positive influence of cholesterol due to chemical interference phenomena or lipoprotein antioxidant action. No direct relationships were found between biomarkers and age or animal size, possibly due to the lack of linearity between aging and oxidative stress or the demographic heterogeneity of the population. The interference study revealed that all three interferents (HIL) induce a significant deviation from TBARS and TAS, obeying various causes of chemical and spectral interference, urging the discarding of samples with such alterations.

This would be the first work to establish RIs of MDA, 8-OHdG, and TAS in dogs, as well as to determine endogenous interferences and characterize their serum values according to multiple biological and analytical factors, thus favoring their interpretation and implementation in clinical practice.