Agarose gel electrophoresis of alkaline phosphatase isoenzymes in the serum of hyperthyroid cats

Cats with hyperthyroidism [(increased serum thyroxine (T(4))] commonly have increased serum alkaline phosphatase (ALP) activity in addition to other serum biochemical abnormalities. Serum biochemical profiles were obtained from 10 hyperthyroid cats which had increased serum ALP. Agarose gel electrophoresis of serum from these cats was performed and stained for alkaline phosphatase activity. Alkaline phosphatase activity was calculated for each of the separate bands obtained, and the results were compared to those of tissue extracts, serum from normal cats, and serum from normothyroid cats with increased serum ALP activity. The hyperthyroid cats had increased ALP activity in bands corresponding to isoenzymes originating in the liver, bone, and an unidentified tissue source.     

Sporozoite proteome analysis of Cryptosporidium parvum by one-dimensional SDS-PAGE and liquid chromatography tandem mass spectrometry

Despite the development of new technologies, new challenges still remain for large scale proteomic profiling when dealing with complex biological mixtures. Fractionation prior to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis is usually the preferred method to reduce the complexity of any biological sample. In this study, a gel LC-MS/MS approach was used to explore the stage specific proteome of Cryptosporidium (C.) parvum. To accomplish this, the sporozoite protein of C. parvum was first fractionated using SDS-PAGE with subsequent LC-MS/MS analysis. A total of 135 protein hits were recorded from 20 gel slices (from same gel lane), with many hits occurring in more than one band. Excluding all non-Cryptosporidium entries and proteins with multiple hits, 33 separate C. parvum entries were identified during the study. The overall goal of this study was to reduce sample complexity by protein fractionation and increase the possibility of detecting proteins present in lower abundance in a complex protein mixture.     

Separation of serum glycated proteins by agarose gel electrophoresis and nitroblue tetrazolium staining in diabetic and normal cats

BACKGROUND: The total glycated protein (fructosamine) concentration in serum consists mainly of glycated albumin and lipoproteins. Measurement of fructosamine is used to diagnose and monitor diabetes mellitus in cats. OBJECTIVE: The aims of this study were to measure glycated proteins in diabetic and healthy (nondiabetic) cats using a semiquantitative technique and to determine whether measurement of any of the fractions of glycated protein could be potentially advantageous for the diagnosis and monitoring of diabetic cats. METHODS: Serum samples from 6 cats with diabetes mellitus and 10 clinically healthy adult cats were assayed for total glycated protein using a nitroblue tetrazolium (NBT) fructosamine assay. Serum proteins were separated by agarose gel electrophoresis and stained with NBT to identify individual glycated proteins within the bands. Gels were scanned by densitometry at 525 nm and the glycated protein content was calculated with reference to the total glycated protein content of the sample. RESULTS: Diabetic cats with increased total fructosamine concentrations had higher concentrations of glycated albumin and glycated alpha- and beta-lipoproteins compared with healthy cats. The concentration of glycated proteins in each of the fractions had a positive linear association with the total glycated protein content of serum, but there was large variation in the relative contributions of the 3 protein fractions to the total glycated protein concentration. CONCLUSIONS: Based on the results of this study, measurement of individual glycated fractions does not seem to offer any potential diagnostic advantage over measurement of total glycated protein (fructosamine) concentration alone. In some diabetic and healthy cats, glycated lipoproteins formed the major part of the total glycated protein, whereas in other cats albumin was the major contributor.     

Evaluation of serum haptoglobin and C‐reactive protein in dogs with mammary tumors

Background: In veterinary medicine, there is increasing interest in measuring acute phase proteins as a tool in the diagnosis and monitoring of neoplastic diseases. Although mammary neoplasms are the most common type of cancer in dogs, acute phase proteins have not been extensively evaluated in dogs with mammary tumors. Objectives: The aim of this study was to evaluate serum haptoglobin (Hp) and C‐reactive protein (CRP) concentrations in the dogs with mammary tumors and assess their potential association with malignancy. Methods: A retrospective study of dogs with mammary tumors was performed. Serum concentrations of CRP and Hp were determined in healthy control dogs (n=20) and dogs with mammary tumors before surgery (n=41). Mammary tumors were grouped as carcinomas (n=24), fibrosarcoma (n=1), malignant mixed tumors (n=7), benign mixed tumors (n=6), and adenomas (n=3). CRP and Hp concentrations were compared in dogs with different tumor types and were also compared based on tumor size, lymph node infiltration, skin ulceration, fixation to underlying tissue, and time between tumor identification and removal. Results: Hp concentration was significantly (P<.043) higher in dogs with mammary tumors (median 2.03 g/L, range 0.09–2.94 g/L) compared with controls (1.38 g/L, range 0.08–3.00 g/L), but the range of values overlapped considerably. CRP concentration was higher in dogs with carcinomas (4.70 mg/L, range 0.63–128.96 mg/L) vs controls (2.11 mg/L, range 0.25–6.57 mg/L) (P=.0008) and in dogs with ulcerated skin (14.8 mg/L, range 5.7–128.9 mg/L, n=3) compared with those without ulceration (2.4 mg/L, range 0.11–30.3 mg/L, n=38) (P=.048). Conclusions: Serum Hp and CRP do not appear to have value in diagnosing or predicting malignancy of mammary tumors in dogs. Higher CRP concentrations in dogs with mammary carcinoma suggest a role for inflammation in this tumor type.     

Relationship between serum acute phase protein concentrations and lesions in finishing pigs

To establish the relationship between serum levels of three acute phase proteins, haptoglobin (Hp), C-reactive protein (CRP) and serum amyloid A (SAA), and the occurrence and severity of lesions at slaughter, a study was carried out using 70 fattening pigs from a finishing unit. Pigs were divided into three groups: Group 1 (25 pigs with clinical signs of disease), Group 2 (25 apparently healthy pigs with lesions at slaughter) and Group 3 (20 apparently healthy pigs with no lesions at slaughter). Serum levels of CRP, SAA and Hp were significantly higher in pigs with clinical signs of disease than in apparently healthy animals. Additionally, in apparently healthy pigs, serum levels of Hp and CRP were significantly higher in animals with lesions than those without lesions. The extent and severity of lung lesions were related to serum levels of Hp.     

Agarose gel electrophoretic fractionation of serum proteins in adult cattle. II. A study of cows with different diseases.

In 283 dairy cows suffering from internal disorders the serum proteins were studied by agarose gel electrophoresis supplemented by total protein and albumin determinations. The clinical diagnoses could be grouped according to protein pattern. Group 1 (abomasal displacement and traumatic muscle injury) did not appreciably affect the serum protein concentrations and represented primarily non-inflammatory diseases or diseases of non-infectious origin. Group 2 (leukosis) occupied an exceptional position, with heavy lowering of albumin unaccompanied by a corresponding rise of total globulin. The γ-globulin concentration was significantly lowered. Group 3 (acute traumatic peritonitis) represented an acute inflammatory process with increase chiefly of the α-globulins, while the γ-globulin concentration was normal. Group 4 (chronic traumatic peritonitis, summer mastitis, chronic subclinical mastitis, chronic laminitis and polyarthritis, urinary tract infection, abscess, and sub-acute-chronic pneumonia) comprised diseases chiefly of chronic inflammatory character and with infectious origin. Especially characteristic was the heavy rise of globulins in general and of γ-globulin in particular. In most cases there was a large increase also of the α- and p-globulin fractions.     

Serum acute phase protein concentrations in dogs with hyperadrenocorticism with and without concurrent inflammatory conditions

Background: Acute phase proteins (APPs) are promising markers of inflammation in dogs, because they are more sensitive than WBC counts in detecting clinical and subclinical inflammation. Endogenous corticosteroids can mask an acute phase response and make it more difficult to identify underlying inflammatory disease. Objective: The purpose of this study was to evaluate the acute phase protein response in dogs with spontaneous hyperadrenocorticism (HAC) with and without concurrent inflammatory conditions. Methods: Serum concentrations of C‐reactive protein (CRP), haptoglobin (Hp), fibrinogen, and albumin were measured in 44 healthy adult dogs and 39 dogs with HAC; the HAC group was further divided into dogs with and without concurrent infection/inflammation. A fourth group of dogs with severe sepsis and without HAC was compared with the dogs with HAC and severe sepsis. Results: Dogs with uncomplicated HAC had significantly higher Hp and fibrinogen concentrations compared with healthy control dogs (P<.001). Dogs with HAC and severe inflammatory disease also had significantly higher CRP and lower albumin concentrations than control dogs and dogs with HAC without concurrent inflammation. Dogs with sepsis but without HAC had significantly higher CRP concentrations than dogs with HAC and sepsis. Conclusions: Dogs with HAC had increases in the moderate APPs (Hp and fibrinogen), and no significant changes in CRP and albumin compared with healthy dogs. Although concurrent HAC appeared to blunt the CRP response in dogs with sepsis, increased serum CRP concentration in dogs with HAC is likely indicative of severe concurrent inflammation.     

Evaluation of plasma folate and homocysteine concentrations in cats with and without oral squamous cell carcinoma

Feline oral squamous cell carcinoma (SCC) is a devastating disease with an extremely poor long‐term prognosis even with aggressive therapy. Folate and homocysteine derangements are identified in people diagnosed with head and neck SCC. The purpose of this study was to measure plasma folate and homocysteine concentrations in cats diagnosed with oral SCC (n = 13) and to compare these concentrations with those found in cats diagnosed with other tumour types (n = 25), cats with oral, non‐neoplastic disease (n = 6) and healthy cats (n = 24). The median plasma folate concentration in cats diagnosed with oral SCC was 14.7 ng mL^?1, while the median plasma homocysteine concentration was 2.61 μg mL^?1. These concentrations did not differ significantly from those of cats in the other groups. This suggests that different factors may contribute to the pathogenesis of this tumour in cats when compared with people, although evaluation of larger numbers of cats may still identify a difference between groups.     

Analysis of mouse, rat, dog, marmoset, and human serum proteins by capillary electrophoresis: comparison with agarose gel electrophoresis

BACKGROUND: Serum protein analysis in both humans and experimental animal species has so far been carried out by labor-intensive techniques, such as agarose gel electrophoresis (AGE). OBJECTIVE: The objective of this study was to evaluate capillary electrophoresis (CE) as an alternative technique to AGE for the analysis of serum proteins from healthy animals. METHODS: Blood samples were collected into tubes without anticoagulant from 6 fasted healthy male mice, rats, dogs, marmosets, and humans. Serum proteins were separated by CE using a technique standardized for the analysis of human proteins, and the results (efficiency, resolution, and precision) were compared with those obtained through AGE. RESULTS: Compared with AGE, CE resulted in narrower peaks and more peaks. The efficiency of protein separation by CE was significantly higher for all species, and resolution (R) was significantly higher in samples from dogs. Using rat serum, intraday reproducibility was lower for all protein fractions, and interday reproducibility was lower for most peaks, compared with AGE. CONCLUSIONS: We conclude that CE is a viable alternative to AGE for the determination of protein electrophoresis in a routine veterinary clinical pathology laboratory. The minimal sample requirement (2 microL), complete automation, and quantitative results make CE an especially valuable technique for protein analysis in experimental animal models.     

Pig-major acute phase protein and haptoglobin serum concentrations correlate with PCV2 viremia and the clinical course of postweaning multisystemic wasting syndrome

The aim of the present longitudinal study was to assess the evolution of two acute phase proteins (APPs), pig-major acute phase protein (pig-MAP) and haptoglobin (HPT), in serum from pigs that developed postweaning multisystemic wasting syndrome (PMWS) in comparison to healthy and wasted non-PMWS affected pigs. In addition, evidence of infection with other pathogens and its relation with variations in APPs concentrations was also assessed. Fourteen independent batches of 100–154 pigs were monitored from birth to PMWS outbreak occurrence in 11 PMWS affected farms. Pigs displaying PMWS-like signs and age-matched healthy controls were euthanized during the clinical outbreak. PMWS was diagnosed according to internationally accepted criteria and pigs were classified as: (i) PMWS cases, (ii) wasted non-PMWS cases and (iii) healthy pigs. At the moment of PMWS occurrence, pig-MAP and HPT concentration in PMWS affected pigs were higher than in healthy ones (p < 0.0001). No differences in APPs serum concentrations between subclinically PCV2-infected pigs and healthy non-PCV2-infected pigs (based on quantitative PCR on serum results) were detected. Results showed a significant correlation between PCV2 loads and both pig-MAP (R = 0.487–0.602, p < 0.0001) and HPT (R = 0.326–0.550, p < 0.05–0.0001) concentrations in serum of PMWS affected pigs, indicating that the acute phase response in PMWS affected pigs occurred concomitantly to PCV2 viremia. No other pathogen, apart from PCV2, was consistently related with variations in APPs concentrations. A ROC analysis, made to determine the capacity of discrimination of both APPs between PMWS affected and non-affected pigs, showed higher sensitivity and specificity values using pig-MAP compared to HPT. These results suggest that pig-MAP might be a better indicator of PMWS status than HPT. Moreover, the fact that APR occurred some weeks before the start of clinical signs suggests that APPs could provide valuable prognostic information for PMWS development.