Influence of short-term storage on electrophoretic profile of bovine serum proteins

Abstract

Storage represents a basic step of the pre-analytical phase for the accuracy of screening tests both in human and veterinary diagnostic laboratories. This study aimed to evaluate the effect of short-term storage on electrophoretic profile of serum proteins in dairy cows. Blood was collected from 18 clinically healthy cows and serum was divided into four aliquots. The first aliquot was immediately analysed (time 0). The second and the third aliquots were stored at +4°C for 24 and 48 h, respectively. The fourth aliquot was frozen at −20° C for 48 hours. A statistical analysis was performed on the obtained data. Albumin and α-globulins relative concentration and A/G ratio showed significant changes after 48 h storage. The results obtained in the current study showed that short-term storage influenced electrophoretic pattern of bovine serum proteins and the A/G ratio. Therefore, the refrigeration of bovine sera for 24 h at the most should be suggested to practitioners in order to obtain reliable serum protein concentrations as useful screening test in veterinary diagnostic laboratory.

Keywords:

• protein fractions
• electrophoresis
• storage
• bovine serum

Introduction

The electrophoretic technique is the current standard method to evaluate serum protein fractions. Reference values for bovine serum protein profiles are available in different conditions (Piccione et al. 2009, 2011; Alberghina et al. 2011). In bovine practice, the need to carry samples from the stable to the laboratory makes the pre-analytical phase the major source of variability in laboratory testing. Fresh serum should be recommended for electrophoretic analyses because protein degradation during storage may cause alterations in serum protein fractions (Wijnen & Van Dieijen-Visser 1996). Correct sample storage is extremely important in order to evaluate serum albumin and globulin levels. Factors that affect albumin levels could change percentage fraction and consequently albumin/globulin (A/G) ratio. In bovine serum albumin fraction decrease after 2, 7 and 21 days freezing (Tothova et al. 2010). The aim of the present study was to determine the stability of electrophoretic profile of bovine serum proteins after short-term storage: at +4°C for 24 h and 48 h, and at −20°C for 48 h. This could be useful for pre-analytic phase in order to include refrigeration and freezing of samples in bovine practice.

Materials and methods

Subjects and management

The experiment was carried out in a farm situated in Ragusa, Sicily (Italy 36°55’ N; 14°43’ E), on 18 multiparous healthy Holstein Friesian cows in good nutritional condition (6 years old, mean body weight 660±45 kg) during dry period. All housing and care conditions were conformed to the standards recommended by the Guide for the Care and Use of Laboratory Animals and Directive 86/609 CEE. Their health status was evaluated based on rectal temperature, heart rate, respiratory heart, appetite, faecal consistency and hematochemical and haematological profiles. All animals were free from internal and external parasites and resulted negative to the coprologic examination (flotation method) carried out before starting the experimental period. No treatments were administered before the start of the experiment.

Collection of blood samples

Blood samples were taken by jugular venipuncture into 10 ml vacutainer tubes (Terumo Corporation, Tokyo, Japan) and then centrifuged. The obtained sera were transferred with a plastic pipette into graduate Eppendorf microtubes in order to fix the amount of dead space. Serum samples were neither lipemic nor hemolysed. Four Eppendorf tubes were prepared for each sample. Later, serum samples were carried to diagnostic laboratory to assess electrophoretic fractions.

Samples treatment for electrophoresis

The first aliquot was analysed within 3 h after collection, the second one was refrigerated at +4°C for 24 h, the third one was refrigerated at +4°C for 48 h and the last one was frozen at −20°C for 48 h. The values obtained within 3 h were considered the initial concentrations (Time 0). A normal bovin serum was used as control in 12 replicated position. Before the analysis, the stored samples were thawed at room temperature (18–20 °C). The protein fractions were performed using an automated system (Selvet 24, SELEO Enginering, Naples, Italy) according to a procedure described in detail elsewhere (Piccione et al. 2011). For each sample, 25 µl of serum were applied to numbered sample wells. Each holder accommodate up to 24 samples. Running conditions were 125 V for 30 min. After electrophoresis, acetate cellulose films were fixed simultaneously using an automated system, stained in red stain acid solution for 3 min and then dried at 37°C. For protein fractionation, the midpoint of the elecropherogram, which lies between the α₂ and the β₁- peaks, was identified, following the recommendation of Kaneko et al. (1997). Relative migration distances for each protein fraction about the albumin fraction were used to standardise serum protein fraction identification. The major protein fractions were divided from cathode to anode as albumin, α₁-, α₂-, β₁-, β₂-, γ-globulins, respectively. All samples were assayed by the same operator each time. The relative concentrations of the protein fractions were determined as the percentage of the optical absorbance.

Statistical analysis

One-way repeated measures analysis of variance (ANOVA) was used to determine significant differences of time on refrigerated samples. Bonferroni multiple comparison test was applied for post hoc comparison. The Wilcoxon signed-rank test was performed to compare results obtained at time 0 and after 48 h of freezing. P values <0.05 were considered statistically significant. Data were analysed using the software Statistica 8 (Statsoft Inc., Oklahoma USA).

Results

The coefficient of variation of serum control was less than 3.8% for albumin, 10% for α₁, 4% for α₂, 8% for β₁, 10% for β₂ and 4% for γ-globulins. Table 1 shows the effect of refrigeration after 24 h and 48 h on relative concentration of serum protein fractions and A/G ratio. ANOVA showed the following results: albumin (F_(2,34)=9.91, P<0.001), α₁-globulins (F_(2,34)=13.47, P<0.001) and A/G ratio (F_(2,34)= 7.5, P<0.01). Table 2 shows the effect of 48 h freezing on serum protein fractions and A/G ratio. Albumin, α₁- and α₂-globulin fractions and A/G ratio decrease significantly (P<0.01).

Table 1. The effect of refrigeration (+4°C) for 24 h and 48 h on relative concentrations (mean ± SD) of serum protein fractions and A/G ratio in 18 healthy cows.

	Experimental conditions
Analytes	Time 0	After 24 h at +4°C	After 48 h at +4°C
Albumin (%)	43.7±5.13	42.5±4.58^a	40.1±3.93^c
α1-globulin (%)	6.05±1.14	7.46±1.44^c	7.01±1.14^b
α2-globulin (%)	3.66±0.82	4.18±1.28	3.71±0.69^d
β1-globulin (%)	9.34±0.99	9.41±1.66	9.36±1.31
β2-globulin (%)	10.25±1.84	10.76±1.79	10.30±1.48
γ-globulin (%)	27.0±4.74	26.7±3.87	27.9±3.66
A/G ratio	0.79±0.19	0.75±0.15	0.68±0.18^a

Bonferroni multiple comparison test: vs. time 0 ^aP<0.05; ^bP<0.01; ^cP<0.001; vs previous ^dP <0.05.

Table 2. Comparison of relative concentrations (mean±S.D.) before and after 48 h of freezing storage of serum protein fractions (percentage in parenthesis) and A/G ratio in18 healthy cows.

	Time of analysis
Fraction	Time 0	After 48 h at −20°C	P
Albumin (%)	43.7±5.13	40.4±4.65	0.003
α1-Globulin (%)	6.05±1.14	6.68±0.95	0.005
α2-Globulin (%)	3.66±0.82	4.20±1.14	0.005
β1-Globulin (%)	9.36±0.99	9.86±1.36	0.097
β2-Globulin (%)	10.25±1.84	11.06±1.60	0.058
γ-globulin (%)	26.9±4.74	27.8±3.81	0.097
A/G ratio	0.79±0.19	0.69±0.14	0.003

P, significance of Wilcoxon Test.

Discussion

The obtained results showed the influence of sample storage on bovine serum protein analysis. Investigating the effect of storage on serum analytical results is very important in veterinary medicine, as samples have to be carried out from the farm to the laboratory. However, little is known about the effect of storage time and temperature for bovine samples as an important pre-analytical factor (Tothova et al. 2010). The results obtained in the present study showed that within 24 h storage at +4°C no change occurred in serum protein fraction's relative concentration and A/G ratio. Instead, +4°C and −20°C storage for 48 h determined significant changes in bovine serum protein fraction areas because of disproportions among them. Storage conditions may alter the percentage area of protein fractions differently. This might be due to different effects of temperature and time on the degradation of molecular configuration of albumin with respect to other globulins. Almost all plasma proteins, except albumin, are glycoproteins (Kaneko et al. 1997). Bovine serum albumin has several molecular forms and exists as a monomer and higher aggregate states, the monomer is stable for 5 months at +8°C, whereas dimer and tetramer dissociated into smaller forms within few hours (Atmeh et al. 2007). The influence of short-term storage on electrophoretic distribution of globulins fraction in mammalian species of veterinary interest is still almost unknown. In conclusion, the results of this study showed that short-term storage has statistically significant effect on serum protein fractions and A/G ratio. We hope these results contribute to assess storage conditions, reducing pre-analytical variation and preventing misleading results. We recommend analysing bovine sera for electrophoresis within 24 h at refrigerate temperature. Reliable information about pre-analytical variation helps bovine practitioners to handle and store bovine serum samples appropriately, as a good quality sample is essential for a correct evaluation.

Acknowledgements

This study was supported by MIUR-PRIN 2008 funds – University of Messina.