Spectrophotometric assays for evaluation of Reactive Oxygen Species (ROS) in serum: general concepts and applications in dogs and humans.

Reactive oxygen species (ROS) are reactive compounds derived from oxygen. In biological systems, an excessive amount of ROS can cause oxidative damage to biological macromolecules being involved in different diseases. Several assays have been developed in the last 30 years for ROS evaluation. The objective of this article will be to provide an update about the spectrophotometric methods currently used in the assessment of ROS in serum. The chemical basis of four different techniques will be reviewed, and examples of their possible applications will be provided. A particular emphasis about the practical applications of these assays in the dog will be made, but selected information about their use in humans will also be presented for comparative purposes, following a One-Health approach. The information about the spectrophotometric assays presented in this paper should be interpreted with caution once limited information about them is available yet, and further studies should be performed to clarify what they measure and their clinical application. Ideally, when applied to evaluate a sample's oxidative status, they should be incorporated in a panel of analytes where other oxidants, antioxidants, and biomarkers of inflammation were also included.

Haptoglobin and its association with naturally occurring diseases in Holstein heifer calves / [Haptoglobina e sua associação com doenças ocorridas naturalmente em bezerras da raça Holandesa]

The present study evaluated the use of haptoglobin (Hp) as an indicator of health and performance in 166 Holstein heifer calves reared in an intensive production system. Calves were evaluated at D6-9; D10-13; D20-23; D35-38 and D65-68, corresponding to the days of life. The absence or presence of diseases was evaluated by physical examination and classification of scores. The performance parameters evaluated were body weight, height at withers and hind width. Hp was measured by spectrophotometric technique. The highest prevalence of diarrhea (59.4%; 98/165) was observed in D10-13, bovine respiratory disease (BRD) was on D35-38 (25.8%; 42/163), and umbilical inflammations in D6-D9 (7.8%; 13/166). Highest values of Hp were observed in animals with diarrhea (P=0.02), and umbilical inflammation (P=0.057), in comparison with the group of healthy calves. A significant negative correlation was observed between Hp and performance index. This protein presented an important relation with diarrhea and performance of the calves, opening perspectives on its utilization as a biomarker of diseases.(AU)

O presente estudo avaliou o uso da haptoglobina (Hp) como indicadora de sanidade e desempenho em 166 bezerras Holandesas criadas em um sistema de produção intensivo. As bezerras foram avaliadas nos momentos D6-9; D10-13; D20-23; D35-38 e D65-68, sendo estes correspondentes aos dias de vida. A ausência ou a presença de doenças foi avaliada por meio do exame físico e da classificação por escores. Os parâmetros de desempenho avaliados foram peso corporal, altura de cernelha e largura de garupa. A Hp foi mensurada por técnica espectrofotométrica. A maior prevalência de diarreia (59,4%; 98/165) foi observada em D10-13, doença respiratória bovina (DRB) ocorreu em D35-38 (25,8%; 42/163) e inflamações umbilicais em D6-D9 (7,8%; 13/166). O valor de Hp foi maior nos animais que apresentaram diarreia (P=0,02) e inflamações umbilicais (P=0,057), em comparação ao grupo de bezerras saudáveis. Houve correlação negativa significativa entre a Hp e os índices de desempenho. Essa proteína apresentou uma importante relação com a diarreia e com o desempenho das bezerras, abrindo perspectivas sobre a sua utilização como biomarcadora de doenças.(AU)

The applicability of spectrophotometry for the assessment of blood meal volume inartificially fed Culicoides imicola in South Africa.

The volume of the blood meal of haematophagous insects will determine the number of infective particles taken up during feeding and may as such denote the minimum dose needed to infect a competent vector. Culicoides midges resort among the smallest of haematophagous vectors and determining and comparing their blood meal volumes may be challenging. Collected Culicoides imicola females were fed on defibrinated bovine blood through a Parafilm® membrane using a Hemotek® system. After feeding, the weight of pools of 10 engorged females was compared to that of 10 unfed females to determine the volume of blood imbibed. After weighing, the pools were homogenized and their absorbance read at 410 nm. Spectrophotometer readings were then converted to blood meal volumes using calibration curves, obtained by the dilution of known volumes of blood used for feeding. Although the mean blood meal volumes determined spectrophotometrically (0.06 µL), differed significantly (P < 0.01) from those obtained by weighing (0.07 µL), the range in blood meal volumes determined spectrophotometrically (0.03-0.08 µL) and by weighing (0.01-0.11 µL) was positively correlated (r = 0.7; P < 0.01). Both methods can be used to determine the blood meal volume.

Effect of heat stress on superoxide anion production in native and crossbred cattle under in vitro whole blood culture model.

Impact of global warming on the dairy industry has gained attention due to huge economic losses through low production and fertility caused by heat stress. Exposure to hyperthermia provokes a series of complex responses in mammals which are been related to morphological and physiological alterations including the production of reactive oxygen species (ROS). A quantitative spectrophotometric based nitroblue tetrazolium (NBT) reduction assay was used to estimate the superoxide anion (•O2-) level in heat stressed (at 42 °C) whole blood cultures of native and crossbred bulls (Sahiwal and Frieswal), in vitro. The breed effect in the kinetics of •O2- production at different time periods of continual heat stress was analyzed by repeated measures ANOVA. Comparison between different time periods in reference to 37 °C was analyzed by paired t-test. The •O2- level was significantly different (p < 0.05) between cells at 37 °C and 42 °C at different periods of incubation. Kinetics study showed increment of •O2- production on the acute phase of stress followed by a reduction in both Sahiwal and Frieswal breeds. In Sahiwal breed, the inflated superoxide level continued abated till 4 h and raised again at 6 h, while in Frieswal •O2- level reverted to raise sooner with in 2 h of incubation itself. Contrarily, kinetic of •O2- level in plasma showed a significant reduction (p < 0.001) at 30 min of 42 °C incubation followed by increment of •O2- level. Further, the breed variation was significant (p < 0.05) and a significant high reduction of •O2- level was observed in Sahiwal breed. Our finding indicates that, a better and longer •O2- production homeostasis and higher plasma scavenging ability of native breed may be one of the reasons for the higher thermal tolerance of these breeds in tropical climate.

Technical note: Rapid field test for the quantification of vitamin E, ß-carotene, and vitamin A in whole blood and plasma of dairy cattle.

Fast and easy tests for quantifying fat-soluble vitamins such as vitamin E and vitamin A, as well as ß-carotene, in whole blood without a need to preprocess blood samples could facilitate assessment of the vitamin status of dairy cattle. The objective of this study was to validate a field-portable fluorometer/spectrophotometer assay for the rapid quantification of these vitamins in whole blood and plasma of dairy cows and calves. We measured the concentrations of vitamin E and ß-carotene in whole blood and plasma from 28 dairy cows and 11 calves using the iCheck test (BioAnalyt GmbH, Teltow, Germany) and compared the results with the current analytical standard (HPLC) in 2 independent laboratories, one at the University of Potsdam (Germany) and at one at DSM Nutritional Products Ltd. (Kaiseraugst, Switzerland). For vitamin A, the HPLC measurements were done only in the laboratory in Germany. The whole-blood concentrations of vitamin E as determined by iCheck (blood-hematocrit-corrected) ranged from 1.82 to 4.99 mg/L in dairy cows and 0.34 to 3.40 mg/L in calves. These findings were moderately correlated (R2 = 0.66) with the values assessed by HPLC in dairy cattle (cows + calves). When calves were excluded, the correlation was higher (R2 = 0.961). The ß-carotene and vitamin A values obtained by the reference method HPLC were highly correlated with the iCheck methods in whole blood (R2 = 0.99 and 0.88, respectively). In plasma, we observed strong correlations between the concentrations assessed by iCheck and those of HPLC for vitamin E (R2 = 0.97), ß-carotene (R2 = 0.98), and vitamin A (R2 = 0.92) in dairy cattle (cows + calves). For vitamin E, ß-carotene, and vitamin A, we compared the relationship between the differences obtained by the iCheck assay and the HPLC measurements, as well as the magnitude of measurements, using Bland-Altman plots to test for systematic bias. For all 3 vitamins, the differences values were not outside the 95% acceptability limits; we found no systematic error between the 2 methods for all 3 analytes.

Collagen, glycosaminoglycans and matrix metalloproteinase-2 and metalloproteinase-9 in the cervix of the ewe during prepubertal development.

The tortuous nature of the ovine cervix restricts the transcervical passage of the cannula, and many studies have aimed to understand the endocrine mechanism of the remodelling of cervical tissue in adult ewe. However, little is known about the remodelling of the cervical tissue during the prepubertal development of the lambs. To obtain histochemical and biochemical evidence about the nature of the prepubertal development of the cervix of the ewe, cervices of Corriedale lambs obtained at 0, 1, 2, 4, 6 and 8 months of age (n = 5 to 6 in each) were processed. Neutral and acidic glycosaminoglycans (by PAS-Alcian stain) were weakly in the cervical stroma and not shown change during the development, whereas the percentage volume of fibrillar collagen (by van Gieson stain) increases throughout the experimental period in the superficial fold stroma and deep wall stroma (p < 0.05). The relative cervical weight (g/kg of body weight) and the collagen concentration (by spectrophotometry, mg/mg wet tissue) showed an early decreasing phase from months 0 to 4 and a later increasing phase from months 4 to 8 (p < 0.05). The latent form of matrix metalloproteinase-2 (MMP-2) detected by gelatin zymography (ng/mg protein) decreased from months 0 to 2 and increased from months 4 to 8, whereas the activated form decreased from months 0 to 2, remained low until month 6 and then recovered on month 8 (p < 0.0001). Data suggest that the relative cervical weight biphasic pattern during the development is related to MMP-2-dependent changes in the collagen content.

Validation of a spectrophotometric method for GGT measurement in canine urine and determination of the urine GGT-to-creatinine ratio reference interval and biological variation in 41 healthy dogs.

The urine gamma-glutamyl transferase (GGT)-to-creatinine ratio has been used to monitor patients at risk of acute renal injury. We validated the spectrophotometric quantification of GGT in urine in a commercial biochemistry analyzer. The assay was precise, accurate, and linear. Intra-assay precision was 3.59% in 4 samples, with GGT concentrations of 47-195 U/L. Inter-assay precision in 3 samples with activities of 11-51 U/L was 7.74%. Accuracy was 97.3%, with an absolute bias of 2.7 U/L. Urine GGT was unaffected by hematuria, hemoglobinuria, or bacteriuria. Urine GGT was stable at 20°C and 4°C for up to 3 d. Storage by freezing at -20°C resulted in a significant reduction in enzyme activity. A pH outside the range of 6.5-8 resulted in reduced GGT activity. The biological variation of urine GGT-to-creatinine ratio provided an index of individuality of 1.6, indicating that a population-based reference interval (RI) can be used. The reference change value was calculated, and an increase in consecutive measurements >43% is required to be regarded as significant. The urine GGT-to-creatinine ratio RI obtained in a population of 41 healthy dogs was 8.5-28.5 U/g.

Does lipidomic serum analysis support the assessment of digestive efficiency in chickens?

The increasing cost of conventional feedstuffs used in poultry diets has bolstered interest in genetic selection for digestive efficiency (DE) to improve the adaptation of the birds to various alternative feedstuffs. However, DE measurement through AMEn is time-consuming and constraining. To simplify selection for DE, the potential of serum composition to predict AMEn was evaluated based on 40 birds from two broiler lines (D+ and D-) divergently selected on the fecal AMEn of a difficult-to-digest wheat-based diet. Differences in serum coloration were suspected between the two lines, and thus a spectrophotometric analysis was carried out, revealing a significant difference in absorption between 430 nm and 516 nm, corresponding to the signature of orange-red lipophilic pigments such as xanthophylls. To go further, the liposoluble fraction of the serum was explored for its lipidome by mass spectrometry. Discriminant analysis revealed that a pattern of 10 metabolites, including zeaxanthin/lutein, can explain 82% of the lipidomic differences between the two lines. Colorimetry combined with lipidomics studies confirmed the relationship between digestive efficiency and serum composition, which opens up new possibilities for using it as a quick and easy proxy of digestive efficiency.

Changes in alpha-amylase activity, concentration and isoforms in pigs after an experimental acute stress model: an exploratory study.

BACKGROUND: Salivary alpha-amylase (sAA) is considered a non-invasive biomarker of acute stress that can be evaluated by changes in activity and concentration, and also by changes in its isoforms, although this last way of evaluation has never been used in veterinary medicine. This research evaluated the changes of sAA by three different ways in which sAA can be evaluated in an experimental acute stress model in six pigs based in a technique of temporarily restraining. These ways of evaluation were 1) activity by a spectrophotometric assay, 2) concentration by a fluorometric assay, and 3) isoforms of the enzyme by a Western blot. RESULTS: Although salivary cortisol significantly increased due to the stimulus of stress and all the pigs manifested signs of stress by high-pitched vocalization, sAA activity showed an increase of different degree in the six pigs after the stress stimulus, while sAA concentration showed decreases in four of the six pigs. sAA activity did not correlate with sAA concentration or salivary cortisol, and a low correlation was observed between sAA concentration and salivary cortisol (r = 0.48, p = 0.003). The inter-individual variability was higher in sAA activity than in sAA concentration and salivary cortisol. Finally, three possible isoforms of sAA at 154-160 kDa, 65-66 kDa and 59-60 kDa were observed that showed different dynamics after the stress induction. CONCLUSIONS: Although this pilot study's results should be taken with caution due to the low sample size, it reveals a different behavior between sAA activity and concentration in pig after an acute stressful stimulus leading to evident external signs of stress by high-pitched vocalization, and opens a new field for the evaluation of possible selected isoforms of sAA as potential biomarkers of stress.

Measurement of urea and creatinine in saliva of dogs: a pilot study.

BACKGROUND: Urea and creatinine in saliva have been reported to be possible markers of chronic kidney disease (CKD) in humans. The aim of this study was to assess if urea and creatinine could be measured in canine saliva, and to evaluate their possible changes in situations of CKD. RESULTS: The spectrophotometric assays for urea and creatinine measurements in saliva of dogs showed intra- and inter-assay imprecision lower than 12% and coefficients of correlation close to 1 in linearity under dilution tests. Healthy dogs showed median salivary concentrations of urea of 39.6 mg/dL and creatinine of 0.30 mg/dL, whereas dogs with CKD showed median salivary urea of 270.1 mg/dL and creatinine of 1.86 mg/dL. Positive high correlations were found between saliva and serum activities of the two analytes (urea, r = 0.909; P < 0.001; creatinine, r = 0.819; P < 0.001). CONCLUSIONS: Urea and creatinine concentrations can be measured in canine saliva with commercially available spectrophotometric assays. Both analytes showed higher values in saliva of dogs with CKD compared with healthy dogs and their values were highly correlated with those in serum in our study conditions.

A novel enolase from Taenia solium metacestodes and its evaluation as an immunodiagnostic antigen for porcine cysticercosis.

Cysticercosis is a worldwide parasitic disease of humans and pigs principally caused by infection with the larvae of the pork tapeworm Taenia solium. Through the use of the recently-made-available T. solium genome, we identified a gene within a novel 1448 bp ORF that theoretically encodes for a 433 amino acid-long protein and predicted to be an α-enolase closely related to enolases of other flatworms. Additional bioinformatic analyses revealed a putative plasminogen-binding region on this protein, suggesting a potential role for this protein in pathogenesis. On this basis, we isolated the mRNA encoding for this presumptive enolase from T. solium metacestodes and reverse-transcribed it into cDNA before subsequently cloning and expressing it in both E. coli (rEnoTs) and insect cells (rEnoTsBac), in a 6xHis tagged manner. The molecular weights of these two recombinant proteins were ∼48 and ∼50 kDa, respectively, with the differences likely attributable to differential glycosylation. We used spectrophotometric assays to confirm the enolase nature of rEnoTs as well as to measure its enzymatic activity. The resulting estimates of specific activity (60.000 U/mg) and Km (0.091 mM) are quite similar to the catalytic characteristics of enolases of other flatworms. rEnoTs also exhibited high immunogenicity, eliciting a strong polyclonal antibody response in immunized rabbits. We subsequently employed rEnoTsBac for use in an ELISA aimed at discriminating between healthy pigs and those infected with T. solium. This diagnostic assay exhibited a sensitivity of 88.4% (95% CI, 74.92%-96.11%) and a specificity of 83.7% (95% CI: 69.29%-93.19%). In conclusión, this study reports on and enzymatically characterizes a novel enolase from T. solium metacestode, and shows a potential use as an immunodiagnostic for porcine cysticercosis.

Comparative interpretation of lactate measurement by point of care spectrophotometric and ELISA methods in transition cows.

Early recognition of altered lactate levels is considered a useful prognostic indicator in dis- ease detection for both human beings and animals. It is reasonable therefore to hypothesize that a portable, point of care (POC) spectrophotometric device for analysis of lactate levels, may have an application for field veterinarians across a range of conditions and diagnostic procedures. In this study, a total of 72 cattle in the transition period underwent POC spectrophotometric lactate measurement with a portable device (The Vet Photometer) in the field, with a small portion of blood used for comparative ELISA evaluation. Lactate measurements were compared using a of Passing-Bablok regression analysis and Bland-Altman plots. The Vet Photometer lactate mea- surement results were in agreement with those generated by the ELISA method. Values for the agreement were derived, in a 95% CI between -1.3 and 0.99, and a positive correlation (r=0.71) between the two measurements. The equation y= 0.68x + 0.60 was achieved using a Pass- ing-Bablok regression analysis. There were no statistical differences in mean values between the measurement methods. In conclusion, a novel veterinary POC spectrophotometric device "Vet Photometer" is an accurate device for evaluation of lactate levels in healthy transition cows.

Spectrophotometry and Ultracentrifugation for Measurement of Plasma Lipids in Dogs with Diabetes Mellitus.

BACKGROUND: There are conflicting reports of plasma lipoprotein lipid content in dogs with diabetes mellitus (DM). OBJECTIVES: To determine lipoprotein lipid content of plasma of dogs with DM by spectrophotometry and ultracentrifugation; to compare lipoprotein lipid content in diabetic and healthy dogs; and to quantify apolipoprotein B-100 (ApoB) in dogs with DM. ANIMALS: 22 dogs with DM and 9 healthy dogs. METHODS: Cross-sectional study. Triglyceride (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) concentrations were measured by spectrophotometry. Very low-density lipoprotein cholesterol (VLDL-C) and low-density lipoprotein cholesterol (LDL-C) concentrations were calculated after ultracentrifugation. Non-HDL-C cholesterol was calculated by subtracting HDL-C from TC. ApoB was quantified by ELISA. The Mann-Whitney test was used for comparison of median lipoprotein concentrations, and Spearman's correlation was used to assess associations between ApoB and lipoprotein fractions. RESULTS: All values are reported in mg/dL. Median TG (122), TC (343.5), HDL-C, (200), VLDL-C, (27) LDL-C (68), non-HDL-C (114), and ApoB (320) were significantly higher in dogs with DM, compared to healthy dogs (57, 197, 168, 12, 16, 31, and 258, respectively, P-values 0.0079, <0.001, 0.029, 0.011, <0.001, <0.001, 0.025, respectively). A significant association was found between ApoB and LDL-C (Spearman's rho = 0.41, P = 0.022) and between ApoB and non-HDL-C (Spearman's rho = 0.40, P = 0.027). CONCLUSIONS AND CLINICAL IMPORTANCE: Dyslipidemia of dogs with DM is characterized by pronounced increases in LDL-C and non-HDL-C concentrations, although all lipoprotein fractions are significantly increased. Knowledge of specific lipoprotein fraction alterations in dogs with DM can enhance treatment options for diabetic dyslipidemia in dogs.

Feline Toxoplasmosis: Tumor Necrosis Factor, Nitric Oxide, and Free Radicals in Seropositive Cats.

Toxoplasma gondii is a cosmopolitan protozoan that causes disease in several species, including humans. In cats, these infections are usually asymptomatic, but in other species they can lead to high levels of inflammatory and cell damage markers, causing cellular damage. Therefore, the aim of this study was to measure levels of tumor necrosis factor (TNF-α), reactive oxygen species (ROS), and nitric oxide (nitrite/nitrate-NOx) in the serum of cats seropositive for T. gondii. Initially, we investigated the presence of antibodies against T. gondii in cats in the city of Concordia, Santa Catarina, Brazil, with the use of indirect immunofluorescence (IFA), and found 30 cats seropositive for T. gondii and 30 seronegative cats. In this study, seropositive cats showed higher levels of TNF-α, ROS, and NOx compared to seronegative cats. Although cats do not show clinical signs of disease, constant inflammatory response can cause cell damage, which over time may adversely affect the animal.

Adulterants interference on Fourier Transform Infrared analysis of raw milk / Interferência de adulterantes na análise do leite cru por espectroscopia pela transformada de Fourier

O objetivo deste trabalho foi analisar as leituras de composição do leite cru por meio de espectrofotometria FTIR, utilizando-se curva de regressão PLS, bem como as contagens de células somáticas e bacteriana total por citometria de fluxo, após adição de amido e sacarose. O leite cru foi adulterado com três concentrações de amido e sacarose (0,1%, 0,5% e 1%), colocado em frascos contendo bronopol ou azidiol, os quais foram armazenados em duas temperaturas (7±2°C e 25±2°C ). As análises foram realizadas após zero, três, 24, 48, 72 e 168 horas de armazenamento. O modelo de regressão linear múltipla foi utilizado para análise estatística. A adição de amido e sacarose resultou em mudança significativa (P<0,05) para todas as variáveis dependentes. O leite adulterado com amido resultou em aumento nas leituras de gordura, proteína, lactose, sólidos totais (ST), sólidos não gordurosos (SNG), CCS e CBT e em diminuição das leituras de caseína, do nitrogênio ureico do leite (NUL) e do ponto de congelamento. O leite adulterado com sacarose resultou no aumento das leituras da lactose, ST, SNG e NUL, enquanto as leituras de proteína, caseína, ponto de congelamento e CCS diminuíram. Este trabalho evidencia a importância do monitoramento de adulterantes reconstituintes no leite por afetarem os resultados analíticos da qualidade do leite, obtidos por métodos eletrônicos.(AU)

Use of the spectrophotometric color method for the determination of the age of skin lesions on the pig carcass and its relationship with gene expression and histological and histochemical parameters.

The presence of lesions on the pig carcass is an indicator of poor animal welfare and has economic impact as it downgrades the carcass value. The assessment of the age of lesions on the carcass may help identify risk factors and ultimately prevent their occurrence. The aim of this study was to assess the age of lesions on pig carcasses through spectrophotometric color evaluation and to relate the results with gene expression and histological and histochemical parameters. A total of 96 barrows were mixed 4 times over 3 d before slaughter and 80 lesions were selected after skin lesion observations to define 4 age categories: < 7 h (T1), 7-25 h (T2), 25-30 h (T3), and 49-54 h (T4). A nonlesioned skin area was used as a control. At slaughter, 3 biopsies per lesion and control skin were taken immediately after bleeding for analyses of gene expression (, , , , , , , , , ), skin histological characteristics (inflammation, erosion or ulceration, and necrosis), and enzyme activity (alkaline phosphatase and adenosine triphosphatase). The number of lesions was counted on each carcass, and the color was assessed visually by a pictorial chart and instrumentally through a spectrophotometer. Delta values (Δ) were calculated as the difference between the value of the lesion and the value of the control for all measures, except for the histological analysis. Results indicated that visual color observation was not sufficiently accurate to discriminate lesions by time of infliction ( > 0.10), while the spectrophotometer ΔL* and Δa* values variation allowed the identification of < 7 h or > 25 h old lesions ( < 0.05). Similarly, the expression of , , , , and genes was higher ( < 0.05) in < 7 h old lesions, while gene expression was higher ( < 0.05) in < 25 h old lesions. As for the histological analysis, the severity of inflammation was correlated with the age of the lesion (lower score in < 7 h old lesions and higher score in > 25 h old lesions; < 0.05). To conclude, the spectrophotometric color assessment of the carcass lesions at slaughter appears to be a reliable method to discriminate between fresh and older lesions on the carcass at the abattoir.

Detection and measurement of alpha-amylase in canine saliva and changes after an experimentally induced sympathetic activation.

BACKGROUND: Salivary alpha-amylase (sAA) is considered a biomarker of sympathetic activation in humans, but there is controversy regarding the existence of sAA in dogs. The hypothesis of this study was that sAA exists in dogs and it could change in situations of sympathetic stimulation. Therefore, the aims of this study were: 1) to demonstrate the presence of alpha-amylase in saliva of dogs by Western-Blot, 2) to validate an spectrophotometric method for the measurement of sAA activity and 3) to evaluate the possible changes in sAA activity after the induction of an ejaculation in dogs which is known to produce a sympathetic activation. RESULTS: Western-Blot demonstrated a band in dog saliva specimens between 60 kDa and 50 kDa, similar to purified sAA. The spectrophotometric assay validated showed an adequate inter- and intra-assay precision, and a high correlation coefficient (r = 0.999) in the linearity under dilution study. sAA median activity significantly increased just after ejaculation compared with just before the ejaculation (2.06-fold, P = 0.005). CONCLUSIONS: This study demonstrated the existence of alpha-amylase in saliva of dogs and that this enzyme can be measured by a spectrophotometric assay. In addition, results showed that sAA increase after a sympathetic activation and could be potentially used as non-invasive biomarker of sympathetic activity in this species.

Measurement of Creatine kinase and Aspartate aminotransferase in saliva of dogs: a pilot study.

BACKGROUND: Muscle enzymes in saliva have been reported to be possible markers of heart and muscle damage in humans. The aim of this study was to assess if Creatine kinase (CK) and Aspartate aminotransferase (AST) activities could be measured in canine saliva, and to evaluate their possible changes in situations of muscle damage. RESULTS: The spectrophotometric assays for CK and AST measurement in saliva of dogs showed intra- and inter-assay imprecision lower than 1 and 16% and coefficients of correlation close to 1 in linearity under dilution tests. Healthy dogs showed activities in saliva of CK between 27 and 121 U/L and AST between 46 and 144 U/L, whereas in saliva of dogs with muscle damage CK ranged between 132 and 3862 U/L and AST between 154 and 4340 U/L. Positive moderate correlations were found between saliva and serum activities of the two enzymes (CK, r = 0.579; P = 0.001; AST, r = 0.674; P = 0.001). CONCLUSIONS: CK and AST activities can be measured in canine saliva with commercially available spectrophotometric assays. In addition these enzymes show higher values in saliva of dogs with muscle damage and their values are moderately correlated with those of serum.

The effect of urea on refractometric total protein measurement in dogs and cats with azotemia.

BACKGROUND: While protein is the predominant solute measured in plasma or serum by a refractometer, nonprotein substances also contribute to the angle of refraction. There is debate in the current literature regarding which nonprotein substances cause factitiously high refractometric total protein measurements, as compared to the biuret assay. OBJECTIVES: The purpose of the study was to determine if the blood of azotemic animals, specifically with increased blood urea concentration, will have significantly higher refractometric total protein concentrations compared to the total protein concentrations measured by biuret assay. METHODS: A prospective case series was conducted by collecting data from azotemic (n = 26) and nonazotemic (n = 34) dogs and cats. In addition, an in vitro study was performed where urea was added to an enhanced electrolyte solution at increasing concentrations, and total protein was assessed by both the refractometer and spectrophotometer. Statistical analysis was performed to determine the effect of urea. RESULTS: The refractometric total protein measurement showed a positive bias when compared to the biuret protein measurement in both groups, but the bias was higher in the azotemic group vs the nonazotemic group. The mean difference in total protein measurements of the nonazotemic group (0.59 g/dL) was significantly less (P < .01) than the mean difference of the azotemic group (0.95 g/dL). The in vitro experiment revealed a positive bias with a proportional error. CONCLUSIONS: This study demonstrated that increasing concentrations of urea significantly increased the total protein concentration measured by the refractometer as compared to the biuret assay, both in vivo and in vitro.

Comparison of 3 options for choosing control limits in biochemistry testing.

BACKGROUND: The purpose of statistical quality control (QC) is to provide peace of mind with regard to the production of results that are suitable for analytically sound clinical interpretation and making reliable decisions about patient diagnosis, monitoring, and prognosis. OBJECTIVES: In this study, we compared 3 options for choosing control limits for biochemistry testing. They focus on the probability of error detection (Ped) and probability of false rejection (Pfr) achievable for a veterinary biochemical analyzer using the following 3 combinations: the quality control material (QCM) manufacturer's acceptable ranges; a standard 12s rule customized for the instrument's observed performance; and candidate rules selected for the instrument's observed performance using a computerized program (EZrules). METHODS: For assessing customized QC, we used mean, SD, CV, bias, total error, and sigma metrics calculated from 3 months of control measurements on a laboratory biochemical analyzer, for 24 commonly used analytes, on 2 QCM levels. RESULTS: Given the desirable combination of high Ped (> 90%) and low Pfr (≤ 5%), the candidate rules selected by the computerized program-related EZrules provided the best performance combinations. CONCLUSIONS: The present work shows acceptable QC performance basing the QC on customization of the acceptable ranges of results from the achievable performance of an individual instrument. The QC performance is maximized by the application of candidate rules based on customized ranges obtained from a computerized QC tool, providing the ability to achieve the highest Ped and acceptably low Pfr values.