E.M.S. Schmidt; R.R. Lange; E.G.L. Hoppe; A.C. Paulillo
FCAV/Unesp-Rodovia de Acesso Prof Paulo Donato Castellane, Jaboticabal, SP, Brazil
The Chelidae family is represented by few genera that occur only in Australasia and South America. Most part of this Chelidae species are endowed with a sinuous, long neck that earns this family its common name: snake-necked turtles. South American snake-necked turtles or side-necked turtles (H. tectifera) are semi-aquatic animals that inhabit mainly lentic water bodies, occurring in southern Brazil, Paraguay, Uruguay and Argentina (Locatelli-Diettrich et al., 2001; Fagundes & Bager, 2007; Lange & Schmidt, 2007). Plasma proteins are important complementary constituents in the diagnosis of gastrointestinal, hepatic, renal, or infectious diseases (Lumeij, 2008). Blood proteins play important roles in the maintenance of colloid osmotic pressure, as a rapid substitute for indispensable amino acids, assuring glucose through gluconeogenesis, in transport of minerals and hormones, in build of enzymes, and in the organism immune system (Eckersall, 2008). Some studies have reported that the results of protein estimation by refractometry for domestic mammals, birds and reptiles correlate well with those obtained by the biuret method, although others have reported both high and lower refractometric results compared with biuret results (George, 2001). According to George (2001) and Jacobson (1992) the most marked inconsistencies between refractometry and biuret results have been found for avian and reptile samples, respectively. Andreasen et al. (1989), Lumeij & Bruijne (1985) and Lumeij (2008) suggested that the accuracy of refractometric protein values should be determined for each species, and an effort should be made to limit variables such as lipemia, hemolysis, and icterus that would cause inaccurate results. Thus, the purpose of this study was to evaluate the accuracy of hand-held refractometer in determining plasma protein concentrations in South American snake-necked turtles (H. tectifera) as compared with the standard biuret method.
Material and Methods
Thirty-two adult South American snake-necked turtles were captured, males and females, that survived from an environmental accident in Iguaçu River, at Araucaria, Paraná State, Brazil. Blood samples were collected from the supraoccipital sinus by non-traumatic technique and placed in heparinized collection tubes. The blood was promptly centrifuged after collection and the obtained plasma was immediately removed following centrifugation. All tested plasma samples were clear. Plasma total protein concentrations were determined by the biuret method and with a hand-held refractometer. The biuret test was performed at 25°C, 525 nm absorbance with an automated plasma/serum chemistry analyzer (Celm® SBA200). The refractometry measurements were obtained with an Atago-type non temperature-compensated refractometer and the instrument calibration was checked with distilled water. All readings were made at room temperature (approximately 25°C) by the same person. Protein concentrations (g/dL) are expressed in results as mean (± standard deviation). Statistical differences between the two methods were analyzed by paired t-test and the correlation was tested by Pearson's correlation. Normality of the sample was confirmed by D'Agostino and Pearson test. P value was set in 0.05. The statistical analysis was performed using GraphPad Prism v. 5.00.
In biuret method, plasma protein concentrations (g/dL) were 4.55 (±1.34), while in hand-held refractometer (g/dL) were 4.26 (± 0.89), with no statistical differences observed at paired t-test (t = 1.805, DF = 31, p = 0.081). High positive correlation was observed for plasmatic proteins in biuret and refractometer methods (r = 0.7286, p < 0.0001).
Discussion and Conclusions
Jacobson (1992) found results by the biuret method that was significantly different from values for the same samples determined by refractometry. This author reports that the biuret method appears to be more accurate and should be the preferred method for reptilian blood. According to George (2001), because of known interspecies variation, refractometer use should not be extended to samples from exotic species without correlation to standard protein methods. Thus, the accuracy of refractometric plasma protein values should be determined for each species of reptile. Refractometers are especially useful in a clinical setting, where plasma protein may be conveniently determined from the plasma contained in a centrifuged microhematocrit capillary tube. Refractometers are also especially useful in clinical routine and for experimental purposes, in which plasma or serum protein concentrations may be determined, because no chemical reagents are necessary and results are rapidly obtained. In the present study, refractometer readings for plasma proteins correlated closely with the results of the biuret test and showed no statistical differences. This finding indicates that plasma protein values may be determined accurately for South American snake-necked turtles with an Atago-type hand-held refractometer. Bolten & Bjorndal (1992) also found that refractometry is useful for plasma protein determination in Chelonia mydas, and we suggest that more studies about refractometry for plasma protein determination should be accomplished in other reptile species.
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