A review of feline serum protein electrophoresis

A review of the current literature available on feline serum proteins is presented. Early studies concentrated on comparative aspects of species variations in the electrophoretic pattern. The feline electrophoretogram was divided into five basic regions: albumin, alpha-1, alpha-2, beta and gamma. Different subdivisions of these areas were recognized depending on the support medium used. Current papers have compared the relative migration distances of each globulin peak to the migration of albumin. This "Rf" value enables reliable peak identification. To date, no data exists identifying the individual proteins responsible for the peaks in the alpha and beta regions. The only feline globulin to be studied is haptolobin; however its precise location on the electrophoretic strip was not identified.     

Characterization of an intravenous lipopolysaccharide inflammation model in calves with respect to the acute-phase response

Our objective was to develop a lipopolysaccharide (LPS) inflammation model in calves to evaluate the acute-phase response with respect to the release of pro-inflammatory cytokines and acute-phase proteins, fever development and sickness behaviour. Fourteen 4-week-old male Holstein Friesian calves were included and randomly assigned to a negative control group (n = 3) and an LPS-challenged group (n = 11). The latter received an intravenous bolus injection of 0.5 μg of LPS/kg body weight. Blood collection and clinical scoring were performed at 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, 18, 24, 28, 32, 48, 54 and 72 h post LPS administration (p.a.). In the LPS group, the following clinical signs were observed successively: tachypnoea (on average 18 min p.a.), decubitus (29 min p.a.), general depression (1.75 h p.a.), fever (5 h p.a.) and tachycardia (5 h p.a.). Subsequent to the recovery from respiratory distress, general depression was prominent, which deteriorated when fever increased. One animal did not survive LPS administration, whereas the other animals recovered on average within 6.1 h p.a. Moreover, the challenge significantly increased plasma concentrations of tumour necrosis factor-α, interleukin 6, serum amyloid A and haptoglobin, with peaking levels at 1, 3.5, 24 and 18 h p.a., respectively. The present LPS model was practical and reproducible, caused obvious clinical signs related to endotoxemia and a marked change in the studied inflammatory mediators, making it a suitable model to study the immunomodulatory properties of drugs in future research.     

Concentrations of haptoglobin in bovine plasma determined by ELISA or a colorimetric method based on peroxidase activity

The objective was to compare different procedures for determination of haptoglobin in bovine plasma. Nine Angus steers were vaccinated against Mannheimia haemolytica to stimulate an acute‐phase response. Blood samples were collected immediately prior to vaccination (day 0), and on days 1, 3, 5, 7 and 10. Plasma samples were frozen in duplicates at ?80 °C. One set of the duplicates was analysed for haptoglobin concentrations using a commercial ELISA kit. A day effect was detected (p < 0.01) because haptoglobin peaked on day 3 and returned to baseline on day 7 relative to vaccination. The second duplicate was analysed using a procedure that measures haptoglobin–haemoglobin complexing by estimating differences in peroxidase activity (CPPA) with results expressed as optical density. Further, based on the ELISA results, the plasma sample with the greatest haptoglobin concentration was also serially diluted into a plasma sample with negligible haptoglobin concentration from the same steer (1:1 through 1:1024 dilution). These dilutions were used within the CPPA method to generate a standard curve and estimate plasma haptoglobin concentrations (CPPA + STD). A linear standard curve was generated (r² = 0.99). A day effect similar to the ELISA method was detected for the CPPA and CPPA + STD methods (p < 0.01). Results obtained from CPPA and ELISA methods were positively correlated (r = 0.97; p < 0.01). The values generated by the CPPA + STD procedure were similar (p = 0.38) compared to the values generated by the ELISA method. In conclusion, assessing concentrations of haptoglobin in bovine plasma using the CPPA and CPPA + STD methods generate highly correlated or similar results, respectively, compared to ELISA. Therefore, the CPPA + STD and CPPA methods can be used as a less expensive alternative to ELISA to determine concentrations or monitor changes in plasma haptoglobin in bovine samples.     

Acute phase proteins and biomarkers of oxidative status in feline spontaneous malignant mammary tumours

Acute phase proteins (APP) and biomarkers of oxidative status change in human and canine mammary tumours, however, they have not been studied in feline mammary tumours. The aims of this study were to investigate the APP and antioxidant responses in feline malignant mammary tumours, to evaluate their relation with tumour features, and to assess their prognostic value. Serum amyloid A (SAA), haptoglobin (Hp), albumin, butyrylcholinesterase (BChE), insulin‐like growth factor1 (IGF1), paraoxonase1 (PON1), total serum thiols (Thiol), glutathione peroxidase (GPox) and total antioxidant capacity determined by different assays, including trolox equivalent antioxidant capacity assessed by two different methodologies (TEAC1/2), ferric reducing ability of plasma (FRAP), and cupric reducing antioxidant capacity (CUPRAC), were determined in serum of 50 queens with spontaneous mammary carcinomas and of 12 healthy female cats. At diagnosis, diseased queens presented significantly higher SAA and Hp, and lower albumin, BChE, GPox, TEAC1, TEAC2 and CUPRAC than controls. Different tumour features influenced concentrations of APP and antioxidants. Increases in serum Hp, and decreases in albumin, Thiol and FRAP were significantly associated with neoplastic vascular emboli, metastasis in regional lymph nodes and/or in distant organs. Distant metastasis development during the course of the disease was associated with increases in SAA and TEAC1. At diagnosis, decreased albumin was associated with a longer survival, and BChE <1.15 μmoL/mL.minute was associated with a shorter survival time on multivariate analysis. Feline malignant mammary tumours are associated with an APP response and oxidative stress, and different tumour features influence the inflammatory response and the oxidative damage. Furthermore, some of these analytes proved to have prognostic value.     

Plasma haptoglobin and immunoglobulins as diagnostic indicators of deoxynivalenol intoxication

This study aimed to discover potential biomarkers for dioxynivalenol (DON) intoxication. B6C3F1 male mice were orally exposed to 0.83, 2.5 and 7.5 mg/kg body weight (bw) DON for 8 days and the differential protein expressions in their blood plasma were determined by SELDI - Time-of-Flight/Mass Spectrometry (TOF/MS) and the immunoglobulins (Igs) G, A, M and E in the serum were investigated. 11.7 kDa protein was significantly highly expressed according to DON administration and this protein was purified by employing a methyl ceramic HyperD F column with using optimization buffer for adsorption and desorption. The purified protein was identified as a haptoglobin precursor by peptide mapping with using LC/Q-TOF/MS and MALDI-TOF/MS and this was confirmed by western blotting and ELISA. IgG and IgM in serum were decreased in a dose-dependent manner and IgA was decreased at 7.5 mg/kg bw DON administration, but the IgE level was not changed. To compare the expressions of haptoglobin and the Igs patterns between aflatoxin B1 (AFB1), zearalenone (ZEA) and DON intoxications, rats were orally administered with AFB1 1.0, ZEA 240 and DON 7.5 mg/kg bw for 8 days. Haptoglobin was increased only at DON 7.5 mg/kg bw, while it was slightly decreased at ZEA 240 mg/kg bw and it was not detected at all at AFB1 1.0 mg/kg bw. IgG and IgA were decreased by DON, but IgG, IgA, IgM and IgE were all increased by AFB1. No changes were observed by ZEA administration. These results show that plasma haptoglobin could be a diagnostic biomarker for DON intoxication when this is combined with examining the serum Igs.     

Acute phase protein concentrations in retired racing Greyhounds

Background: Retired racing Greyhounds are popular as pets. Greyhounds have several differences in physiological values compared with other breeds, including lower serum α‐ and β‐globulin concentrations. We hypothesized that lower acute phase protein (APP) concentrations could contribute to lower α‐ and β‐globulin concentrations in this breed. Objectives: The purpose of this study was to compare serum concentrations of several APPs in Greyhounds with those of other dog breeds. Methods: We measured the serum concentrations of C‐reactive protein (CRP), haptoglobin (Hp), acid‐soluble glycoprotein (ASG), ceruloplasmin (CP), and serum amyloid A (SAA) in 15 clinically healthy retired racing Greyhounds and 11 age‐ and gender‐matched healthy nonGreyhound controls using previously validated methods. Results were compared by Student's t‐tests. Results: The concentration of Hp by both colorimetric and immunoturbidimetric methods was significantly lower in Greyhounds than in nonGreyhound dogs (P=.0009 and .019, respectively). The concentration of ASG was also significantly (P=.007) lower in Greyhounds, but CRP and CP concentrations were not significantly different between groups. SAA concentration was below the detection limit of the method in all dogs. Conclusions: The low serum concentrations of Hp and ASG should be taken into consideration when interpreting APP results in Greyhounds. Because both Hp and some ASG migrate in the α‐globulin fraction, these results may explain the low α‐globulin concentrations in Greyhounds.