1 Introduction

Serum protein measurement is frequently used to assess inflammation resulting from neoplasia, infectious diseases, and other pathophysiological conditions [1]. Serum protein measurement is also used as important marker for the diagnosis of hepatic, renal and gastrointestinal disorders [2].

Different methods for total serum protein are available however biuret and refractometery are considered the most common methods of total protein determination [3]. These methods are frequently available in veterinary clinics [4]. The basic principle of biuret assay is based on the reaction between peptide bonds of protein with copper at alkaline pH resulting in violet colored complex [5]. This assay acts as reference method and usually preferred by analysts. The limitation of this assay is its cost, turnaround time as a result it is difficult to use it out of the laboratory [3].

Measurement of protein using refractometers as compared to biuret is cheaper and requires less turnaround time. The principle of refractometery is based on the measurement of angle of refraction by the sample. The angel of refraction is a contribution of protein and other solutes in the sample. In refractometery interferences by other substances including glucose and cholesterol have been reported [6]. Refractometers are simple portable instruments which can be used to determine protein in wide range of animal’s dogs, cats, cattle’s, sheep’s, goats, etc. [7].

A pre-analytical variable such as transport, storage and improper handling has to be noted during processing samples [8, 9]. Considering the veterinary clinical practice, there is transport of samples from clinics to laboratory; thus it is important to consider optimal management of samples in preanalytic phase as it has probability of causing errors in results [10, 11].

It has been noted that refractometers could be a reliable and easy method for routine screening of serum total protein concentrations. However limited studies by different scholars [12, 13], have addressed the applicability of refractometers in determination of serum total protein in animals including horses [14, 15]. Therefore, the objectives of current study were to compare the protein measurement using refractometery and biuret methods and addressed the applicability of refractometery in horse.

2 Materials and methods

2.1 Study area

This research was conducted at Biochemistry Laboratory of College of Veterinary Medicine and Agriculture, Addis Ababa University, Bishoftu. The Bishoftu town located in the East-Shewa Zone of the Oromia, Ethiopia from April to May 2023.

2.2 Study population

The study population was selected randomly and included 50 male horses of Selale breed, aged 3–8 years, brought to SPANA for various clinical investigations such as parasitic diseases, skin problems, wound and eye cases etc. during the study period. This population is comprised of young male horses with normal body conditions based on their recorded history and their clinical inspection.

2.3 Sample size determination

The sample size was determined as per the principles of evaluation of clinical chemistry methods and approved guideline for method comparison and bias estimation document that 40 to 100 samples are needed to be assayed on both methods on the same day over 8–20 days (preferably within 4 h) [14, 15]. Accordingly the current study utilized 50 samples from horse.

2.4 Sample collection

Ten ml blood sample was collected by asking the owner's permission and using vacutainer tubes without anticoagulant and a sterile needle and holder from the jugular vein. Each sample was clearly labeled with the animal’s identification and date. Samples were brought to the physiology laboratory at CVMA and AAU by using an ice box from SPANA, and they were left to clot at room temperature for 30 min. Serum was separated by centrifugation at 1200 xg 10 min and immediately transferred to Eppendorf safe locks. The serum, which was transferred to Eppendorf safe-locks, was analyzed for total protein using refractometery and biuret.

2.5 Instrumental setup

Spectroscopic analysis was performed using an EMP-168 semi-automated chemistry analyzer. The instrument was calibrated using a calibrator (Autocal) and normal (N) and pathological (P) quality control samples were run for validation before the samples were used for testing. Refractive index was measured using portable clinical refractometer RETK-70 with three scales displaying specific gravity, total serum protein, and refractive index. The measuring range of the refractometer was 0–12.0 g/dl (SI-Unit 0 –120 g/L), and the scale interval was 0.2 g/dl (SI-Unit 2.0 g/L). Before measuring serum proteins, the refractometer calibration was checked with distilled water and all measurements were made at room temperature. A fixed amount of serum 0.3 ml was used in all measurements in duplicate and the mean values was used.

2.6 Total protein using refractometry

A refractometer is primarily used to measure the refractive index of a substance, which describes how light is bent or refracted as it passes through the substance. The refractive index is related to the concentration or composition of a solute in a solution. The principle of a refractometer is based on Snell's law, which states that the angle of light refraction is dependent on the refractive indices of the two media through which it passes. In a refractometer, a serum sample is placed on a prism or a glass plate with a known refractive index. A light source is directed into the serum sample, and the refracted light is measured using a detector. By comparing the angle of refraction with a reference value, the refractive index of the sample can be determined. This refractive index value can then be correlated to concentration or other relevant parameters using calibration curves or established relationships [6].

2.7 Total protein using biuret

The reaction in the biuret test is a colorimetric reaction where the result is indicated by a color change from blue violet. In an alkaline environment, the cupric (Cu+2) ions in the biuret reagent bind to the nitrogen atoms in the peptide bonds of proteins, forming a violet-coloured copper coordination complex. The formation of a purple color indicates the presence of peptide bonds in the sample. The intensity of the color is directly proportional to the concentration of total protein in serum [16].

2.8 Statistical analysis

Statistical analyses were performed using IBM SPSS 20. Normality distribution of the data was tested using the Kolmogorov–Sminorv test prior to statistical analysis. The statistical differences between both methods were analyzed using a paired t-test and the correlation was determined by spearman correlation test. P < 0.05 was considered significant. Bland–Altman plots were created by SPSS statistics to evaluate a bias between the mean differences, and to estimate an agreement interval.

3 Results

The present study evaluates refractometry as an alternative to biuret method for measurement of total protein in horse serum. The data obtained from biuret and refractometery method ranged from 55.8 to 74.4 g/L and 56 to 78 g/L respectively. The overall comparison of result using paired t test were run and no statistical differences were observed between the two methods (Table 1).

Table 1 Mean values ± SD of the serum total protein concentration determined with the biuret and refractometry in horse
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The study examines a bias between the mean differences to estimate an agreement interval using Bland–Altman plots. Bland–Altman plots indicated that the bias for each paired measurement ranged from −7 to 8 g/L and only two results shown biases indicating the analytic error. The error rate was (4/50) 8% (Fig. 1).

Fig. 1
figure 1

Bland–Altman plots of Total protein concentrations in serum specimens of horses (Mean ± SD bias, 0.172 ± 2.977 g/L; 95% limits of agreement, −0.674 to 1.018 g/L)

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The study also examines the correlation between two methods using spearman correlations and revealed that moderate to high correlation between refractometry and biuret assay measurements of TP concentration r = 0.855; P < 0.001) (Fig. 2).

Fig. 2
figure 2

Scatterplots demonstrating the correlation between biuret method and refractometry in horse (n = 50)

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Calculation of Spearman rank correlations revealed a moderate to high correlation between refractometry and biuret assay measurements of TP concentration r = 0.855.

4 Discussion

The present study is to evaluate refractometry as an alternative to biuret method to measure total protein in horse serum. Refractometry has been reported to give both higher and lower values than those obtained with biuret measurement underlining the necessity of reevaluation of refractometers for total protein determination [17]. According to previous studies carried out in canine and feline species, comparison of refractometry and biuret measurement showed significant difference in total protein levels both in serum and plasma sample [18]. Similarly the present study obtained result with no significant difference among the two methods.

The suitability of refractometers for horse serum has been addressed by some scholars [12]. Similarly in sheep where no significant difference was recorded among refractometers and biuret method. However in contrast to our findings the total protein values determined with the refractometer were significantly lower than those measured with the biuret method in cattle and goats [13].

In the present study the two methods had 0.855 correlation so based on Cohen (1988) provided guidelines for the purposes of interpreting the magnitude of a correlation, as well as estimating power, The two method lie between 0.5 and 1.0 so they have strong correlation between refractometery and biuret to measure the total protein.

The analysis of Bland–Altman plots revealed that the biases indicating the analytic and user error were 8% which is analytically insignificant as it was less than 10% the maximum allowable error for total protein determination [19]. Similar to our study the analysis of Bland–Altman pots revealed that the biases indicating less than 10% allowable analytic error in cattle (6%), sheep (0.5%), goats (5.2%) [13].

Generally the findings obtained in this study seem to suggest that refractometry is an alternative method to measures total protein in horse serum.

5 Limitations of the study

The study was unable to conduct comparison studies on large sample size and a wide range of concentrations. Besides the study was limited to conduct on serum collected from male horses due to unavailability of female horses.

6 Conclusion and recommendation

Portable refractometery is reliable, easy to use, characterized by a small volume of sample needed, a quick turnaround time, rapid examination, being inexpensive, and giving good results like biuret. Based on the current study the performance of refractometer was sufficiently accurate for the determination of serum total protein concentrations in horse. However it is recommended that it needs further study to find the effect of other non-protein substances like glucose, cholesterol, urea, sodium, and bilirubin components of total solids in serum and storage condition to see the change in readings of refractomter. Lastly, refractometers can be used as an alternative to biuret method for serum total protein in horse.