Clustering analysis of lipoprotein profiles to identify subtypes of hypertriglyceridemia in Miniature Schnauzers.

BACKGROUND: Hypertriglyceridemia (HTG) is prevalent in Miniature Schnauzers, predisposing them to life-threatening diseases. Varied responses to management strategies suggest the possibility of multiple subtypes. HYPOTHESIS/OBJECTIVE: To identify and characterize HTG subtypes in Miniature Schnauzers through cluster analysis of lipoprotein profiles. We hypothesize that multiple phenotypes of primary HTG exist in this breed. ANIMALS: Twenty Miniature Schnauzers with normal serum triglyceride concentration (NTG), 25 with primary HTG, and 5 with secondary HTG. METHODS: Cross-sectional study using archived samples. Lipoprotein profiles, generated using continuous lipoprotein density profiling, were clustered with hierarchical cluster analysis. Clinical data (age, sex, body condition score, and dietary fat content) was compared between clusters. RESULTS: Six clusters were identified. Dogs with primary HTG were dispersed among 4 clusters. One cluster showed the highest intensities for triglyceride-rich lipoprotein (TRL) and low-density lipoprotein (LDL) fractions and also included 4 dogs with secondary HTG. Two clusters had moderately high TRL fraction intensities and low-to-intermediate LDL intensities. The fourth cluster had high LDL but variable TRL fraction intensities with equal numbers of NTG and mild HTG dogs. The final 2 clusters comprised only NTG dogs with low TRL intensities and low-to-intermediate LDL intensities. The clusters did not appear to be driven by differences in the clinical data. CONCLUSIONS AND CLINICAL IMPORTANCE: The results of this study support a spectrum of lipoprotein phenotypes within Miniature Schnauzers that cannot be predicted by triglyceride concentration alone. Lipoprotein profiling might be useful to determine if subtypes have different origins, clinical consequences, and response to treatment.

Quantitative bioimaging of copper in frozen liver specimens from cats using laser ablation-inductively coupled plasma-mass spectrometry.

OBJECTIVES: The aim of this study was to assess laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) as a tool for measuring concentrations and determining accumulation of copper in frozen liver specimens from cats. METHODS: Six frozen liver specimens were evaluated by qualitative copper staining and quantitative flame atomic absorption spectroscopy. Tissue specimens were cryo-sectioned and quantitative bioimaging of copper was performed using LA-ICP-MS. Results were compared with those obtained using conventional methods. RESULTS: Of the six specimens, only one showed positive staining for copper with rhodanine. Using flame atomic absorption spectroscopy (FAAS), one specimen showed a deficient copper level (<100 µg/g dry weight), two specimens had copper within the reference interval (RI; 150-180 µg/g) and three specimens had copper concentrations above the RI. Bioimaging from LA-ICP-MS showed inhomogeneous distribution of hepatic copper. The areas with dense copper accumulation were represented as hotspots in the liver specimens. Hepatic copper quantification by LA-ICP-MS correlated well with copper quantified by FAAS (r = 0.96, P = 0.002). CONCLUSIONS AND RELEVANCE: Our findings suggest that quantitative bioimaging by LA-ICP-MS could be used to demonstrate the distribution and concentration of copper in frozen liver specimens from cats. The distribution of copper in these specimens was inhomogeneous with dense accumulation represented as hotspots on tissue sections. A positive correlation of hepatic copper concentrations determined by LA-ICP-MS and FAAS was found. Further studies to establish an RI for hepatic copper using this technique and to further determine its clinical utility are warranted.

Immunohistochemical Expression of Oxidative Stress and Apoptosis Markers in Archived Liver Specimens from Dogs with Chronic Hepatitis.

Chronic hepatitis (CH) in dogs is histologically characterized by an inflammatory infiltration of the liver accompanied by hepatocellular apoptosis or necrosis, varying degrees of fibrosis and regeneration. Oxidative stress has been suggested to play a role in the pathogenesis of various liver diseases, including CH. This study assessed the immunohistochemical expression of markers of oxidative stress (4-hydroxynonenal [4-HNE] and malondialdehyde [MDA]) and apoptosis (active caspase-3 [casp-3]) in 35 surplus archived formalin-fixed paraffin-embedded liver biopsies from 25 dogs with CH and 10 control dogs that had no significant hepatic changes. Correlations between immunohistochemical markers and necroinflammatory, fibrosis and histological copper scores, and hepatic copper concentrations were also determined. There were no significant differences in 4-HNE expression between the two groups. Control dogs had lower hepatic MDA scores than dogs with CH. MDA scores were positively correlated with copper scores as well as hepatic copper concentrations. There was no significant difference in casp-3-positive hepatocytes between groups. However, a positive correlation between casp-3 immunoreactivity and copper scores, as well as hepatic copper concentrations, was identified. Necroinflammatory and fibrosis scores were positively correlated with immunoreactivity for MDA and casp-3. MDA and casp-3 are expressed in canine liver and both markers are correlated with necroinflammatory scores, fibrosis scores and hepatic copper accumulation. Our results suggest the utility of immunolabelling for MDA and casp-3 for assessment of hepatic oxidative stress and apoptosis, respectively, in dogs with CH.

Assessment of the intracellular distribution of copper in liver specimens from cats.

The intracellular distribution of copper in the liver has been investigated in dogs and humans. However, this has not been reported in cats. This study aimed to assess the intracellular copper distribution in liver specimens from cats with a range of hepatic copper concentrations. Twenty-nine frozen liver specimens from cats were included. Each liver specimen was divided into two pieces for overall copper quantification and tissue fractionation. The copper concentrations in liver specimens and liver fractions were measured by flame atomic absorption spectroscopy. Five specimens had copper concentrations < 100 µg/g dry weight, eight had copper concentrations between 100 and 180 µg/g, 14 had copper concentrations between 181 and 700 µg/g, and two had copper concentrations >700 µg/g. Only one specimen had positive copper staining. Regardless of the overall concentrations, copper was mostly found in the cytosolic fraction followed by the nuclear, large granule, and microsomal fractions. Our findings indicate that similarly to other species, intracellular copper is predominantly found in the cytosolic and nuclear fractions in cats. The distribution in cats with copper-loaded conditions, such as primary copper hepatopathy, was not assessed but warrants evaluation.

Assessment of copper accumulation in archived liver specimens from cats.

OBJECTIVES: The aim of this study was to assess hepatic copper concentrations and zonal distribution in cat liver specimens. METHODS: For this study, 121 archived, formalin-fixed, paraffin-embedded liver specimens from cats were used. Tissue sections were stained for copper with rhodanine and scored from 0 (no copper accumulation) to 5 (panlobular copper accumulation). The tissue specimens were then deparaffinized and hepatic copper concentrations were measured using flame atomic absorption spectroscopy. RESULTS: Tissue samples were categorized into four groups based on histopathologic findings: (1) no significant histopathologic hepatic changes (n = 66); (2) hepatic steatosis (n = 18); (3) inflammatory or infectious disease (n = 24); and (4) neoplasia (n = 13). Of the 121 specimens, 13 (11%) stained positive for copper, with three having a score ⩾3. Thirty-seven specimens (31%) had copper concentrations above the reference interval ([RI] <180 µg/g dry weight liver). Copper concentrations in cats with hepatic inflammatory or infectious disease were significantly higher than cats with hepatic steatosis (P = 0.03). Copper-staining score and concentration were positively correlated (rs = 0.46, P <0.001). CONCLUSIONS AND RELEVANCE: Despite the fact that 31% of specimens had copper concentrations above the RI, only 11% showed positive copper staining and only 2.5% had a score ⩾3. Our findings suggest that hepatic copper concentrations greater than the upper limit of the RI are relatively common in cats. Further studies to determine the factors that influence hepatic copper staining in cats and to establish contemporary RIs for hepatic copper in healthy cats are warranted.