State Enterprise “L. I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety”, Ministry of Health of Ukraine, Kyiv, Ukraine
ABSTRACT. Objective. Creation of historical control of certain serum biochemistry parameters of Wistar Hannover rats.
Materials and methods. The data were obtained from 1,180 Wistar Hannover rats of the control groups that were kept in conventional vivarium during the period from 2013 to 2018 in the toxicological studies conducted as per GLP. Eight serum biochemistry parameters: alkaline phosphatase, aspartate transaminase, alanine transaminase, total protein, urea, cholesterol, triglycerides, glucose were determined in rats. Biochemistry parameters were determined using open type biochemical analyser Vitalab Flexor E (The Netherlands) with reagents manufactured by Global Scientific (USA) and BioSystems (Spain).
Results. The obtained data were analysed by age and gender of biological models. The average values and standard deviations for each parameter were determined during the creation of historical control. Minimal and maximal values and normal ranges were established for each parameter. Changes in historical control were reviewed by year.
Conclusion. Created historical control can be used for an objective interpretation of obtained serum biochemistry parameters in toxicological studies as well as for assessment and monitoring of physiological conditions of laboratory rats.
Keywords: serum, biochemistry parameters, historical control, Wistar Hannover rats.
Introduction. Blood biochemistry is a method of laboratory diagnostics that allows evaluating many internal organs functioning. Blood biochemistry involves the determination of a number of parameters that reflect the status of protein, carbohydrate, lipid and mineral metabolism, as well as the activity of some key serum enzymes. At present, toxicological and pharmacological studies are difficult to imagine without biochemical test. Biochemical parameters are very important for assessing the effect of the study drug on the organs and tissues, target organ, the correctness of the formed experimental condition.
In toxicological studies, sporadic results that do not depend on the effect of the study substance, but may be misinterpreted as the substance toxicity are common. Setting the reference values of health parameters for experimental biological models allows planning experiment correctly and interpret its results objectively.
Following the analysis of various literature sources, it can be observed that published reference values of blood biochemical parameters of the most common biological model — laboratory rats differ significantly depending on the line, age, husbandry and feeding of animals. The problem of establishing the reference values of biological parameters of the condition of laboratory animals has been highlighted since the last century in the paper edited by Prof. I. M. Trakhtenberh Aspects of normal ranges in toxicology and in other publications [1 – 5]. Therefore, the question of creating historical control of the condition of the body of certain species of laboratory animals is still relevant.
Objective. Creation of historical control of certain serum biochemistry parameters of Wistar Hannover rats based on the results of toxicological studies during 2013–2018 and analysis of the obtained data by age and years.
Materials and methods. The data were obtained during subchronic oral toxicity studies of chemical substances from control Wistar Hannover rats during 2013–2018. The study was performed at the Laboratory of Experimental Toxicology and Mutagenesis (LETAM) of the State Enterprise “L. I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety” of the MoH of Ukraine as per GLP requirements. Animals were handled according to the guidelines “Guide for the care and use of laboratory animals”, National Academy Press, USA, 1996 [6], and according to Directive 2010/63/EU of the European Parliament [7].
All animals were received from SPF breeding nursery for small laboratory animals at the State Enterprise ‘L. I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety’ of the MoH of Ukraine and transferred to the conventional barrier-type vivarium with further acclimatization. Blood sampling for biochemical studies was performed under fasting conditions at the same time. Animal blood samples were collected from the femoral vein after the previous analgesia. The obtained blood was centrifuged at 1,500 rpm for 10 minutes. Biochemical parameters of the obtained serum were investigated on an automated open type biochemical analyser Vitalab Flexor E (The Netherlands) with a set of reagents manufactured by Global Scientific (USA) and BioSystems (Spain). Eight biochemical parameters of rat blood serum were determined: alkaline phosphatase, aspartate transaminase, alanine transaminase, total protein, urea, cholesterol, triglycerides, glucose. Analytical measurements were carried out in accordance with standardised methods on an automated device, which allowed minimizing errors and to eliminate the impact of the human factor with mandatory two-level quality control (normal and abnormal values). The data obtained were statistically processed using the Microsoft Excel software package. Mean (M), standard deviation (SD), and minimum (Min) and maximum (Max) values were calculated. Reference values are provided as (M ± SD) [8].
Results and discussion. The Wistar Hannover rats, selected as the biological model for the study, is one of the most common lines of laboratory rats for biological studies. This line was received by Scientific Products Farm Ltd., (predecessor of Charles River United Kingdom [CR.UK]) from the Wistar Institute in 1947. In 1975, the stock was transferred from CRUK to CRL breeding nursery, USA. The core of the current colony was obtained in 1975 by rederivation via caesarean section. The breeding core of the Wistar Hannover line kept at the SPF breeding nursery for small laboratory animals of the State Enterprise ‘L. I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety’ of the MoH of Ukraine during 2013–2014 was received in 2010 from Charles River Laboratories (France) SPF breeding nursery for laboratory animals. In the second half of 2014, the breeding core was replaced, a new core was received from Envigo RMS B.V. (The Netherlands) breeding nursery (ex-Harlan Sparque Dawley Inc.).
In 2013–2018 study, blood samples from a total of 1,180 Wistar Hannover rats were used, out of which 580 were females and 600 males. All experimental animals were free from pathogenic microflora, had no signs of morbidity, injury, and physical disability. During the study, animals were kept at the conventional vivarium at the Laboratory of Experimental Toxicology and Mutagenesis (LETAM) of the State Enterprise “L. I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety” of the MoH of Ukraine with a stable environmental parameters (air temperature: 19–25оС; humidity: 30–70%; lightning: artificial, 12 hours of light and 12 hours of darkness). All animals were on a standard feed for small rodents from Altromin (Germany) and decontaminated filtered water. In all animals, samples of venous blood were taken in the morning, within 10:00 am to 12:00 pm under fasting conditions. The biochemical test of the blood of Wistar Hannover rats included blood serum parameters the most commonly used in the studies: parameters of enzymatic activity (alkaline phosphatase, aspartate transaminase [AST], alanine transaminase [ALT]), total protein, urea, cholesterol, triglycerides, glucose.
The results of the conducted study of the biochemical parameters of rat serum from 2013 to 2018, taking into account the age and gender of animals, are shown in Table 1, and taking in to account the years of study — in Tables 2 and 3.
Table 1
Results of the historical control of the serum biochemical parameters of Wistar Hannover rats (Laboratory of Experimental Toxicology and Mutagenesis [LETAM]) 
As can be seen from Table 1, biochemical parameters are predominantly relatively stable in different age groups. There is some trend of increasing aspartate transaminase within acceptable limits, as well as some gender and age differences at the level of total protein and alkaline phosphatase, which is also reported in the literature [9, 10]. Parameters of lipid metabolism, urea and glucose remain virtually unchanged during ontogeny, which can also be confirmed by data from other studies [11–14].
Data in Tables 2 and 3 indicate relative stability in biochemical parameters of rat blood serum during 2013–2018.
Table 2
Results of the study of serum biochemical parameters of male Wistar Hannover rats by years, LETAM
Table 3
Results of the study of serum biochemical parameters of female Wistar Hannover rats by years, LETAM
A slightly increased (P > 0.05) alkaline phosphatase, alanine and aspartate transaminase activity were observed in 2013 and 2014 compared to the rest of the study period, with increased serum enzymatic activity more clearly manifested in male rats.
Comparing the obtained data with the normal physiological parameters of serum of laboratory rats of different lines provided in the literature [12–14], it can be seen that the obtained data generally do not go beyond the normal physiological fluctuations. An exception may be the somewhat increased (P > 0.05) enzymatic activity (in particular aspartate transaminase) in 2013 and 2014 compared to other years of the study, which may be explained by the characteristics of origin of the breeding core in those years.
Conclusion. During 2013–2018, the Laboratory of Experimental Toxicology and Mutagenesis (LETAM) of the State Enterprise “L. I. Medved’s Research Center of Preventive Toxicology” of the MoH of Ukraine have created the historical control of eight most common serum parameters used in toxicological studies. The level of gender and age dependence of the studied serum parameters was determined. Changes in the study parameters were established by years. Created historical control can be used for assessment and monitoring of physiological conditions of laboratory rats, it will help to perform an objective interpretation of obtained serum biochemistry parameters in toxicological studies of chemical substances and to improve the significance of data for justification of hygienic parameters.
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Надійшла до редакції 18.09.2019 р.