It is quite difficult to determine the reference clinical chemistry values in non-clinical toxicity studies in the laboratory rat. There are 3 reasons for this: 1) the blood volume of rats is less than that of humans, and sampling induces considerable stress in rats, 2) the use of human reagents for clinical chemistry in rats sometimes fails to provide the necessary specificity, and 3) when statistically significant differences are evident in the clinical parameters evaluated in toxicity studies, the lack of correlation with histopathological evaluation makes the findings meaningless in many cases. Therefore, a clinical chemistry reference data in rats was established based on the values in untreated or negative control animals (approx. 5,000rats) in toxicity studies which have been conducted since 1994 at 9 member laboratories of the Committee on Animal Clinical Chemistry, Japan Society of Clinical Chemistry. Measurement conditions were confirmed and factors which may affect the determination of standard values were investigated. In this paper, the method of database preparation and the outline are presented, and technical factors including artifacts which may affect the determination of reference values are discussed based on the literature. Subsequently, the standard values of individual parameters are discussed by assigned authors. Also, the relationship between albumin and reagents is reported separately.

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The present study investigated the possibility of providing data interchangeability among laboratories and to establish common reference values for each clinical chemistry parameter in rats. We analyzed the historical control data of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in untreated animals and those used as negative controls in non-clinical toxicity studies. The data were provided by 9 laboratories, which covered a study population of approximately 5,000 rats. The major factors contributing to the effects on ALT and AST were age and mode of measurement. The influence of age on ALT and AST values can be reduced by collecting data from a large number of male and female rats. It is also considered that common reference values can be established by classifying data by each mode of measurement or by standardization of measurement methods. On the basis of the significant differences between serum AST and plasma AST, it is considered necessary to use heparinized blood, which is not affected by the period from blood collection to plasma separation or by the methods of storage. The observed influences on ALP activity were age, sex, fasting/feeding behavior, and measurement mode, with these factors showing the greatest variation in the reference data. Accordingly, it is necessary to classify the data by each of these factors. As for measurement methods, N-methyl-D-glucamine (MEG) is expected to be used as the buffer of ALP measurement methods in rats.

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We researched the factors affecting the historical data regarding total protein, glucose, cholesterol, and urea nitrogen from rats used as negative controls in non-clinical toxicity studies. The data were provided by nine laboratories, which covered a study population of approximately 5,000 rats. As for the difference in the food contents (crude protein, crude fat), it confirmed that it influenced total protein, cholesterol, and urea nitrogen in the blood. Furthermore, as for these influences, it was suggested that the influence increased by long-term breeding. Moreover, it was confirmed that glucose and urea nitrogen decreased by fasting before blood collection. There was a difference in the response of total protein between the standard material and rat specimens at the time of measurement, which influenced the measurement value. Therefore, data regarding the areas of total protein, glucose, cholesterol, and urea nitrogen should be collected with special attention being paid to both the animal's feed content and its fasting/feeding conditions. Furthermore, once the standard rat material is marketed, comprehensive gathering of total protein data is expected.

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We investigated the factors affecting sodium, potassium, and calcium values in rat blood in order to determine the standard clinical chemistry parameters in the non-clinical toxicology field. The data were collected from rats used as negative controls in non-clinical toxicity studies conducted by a laboratories. The data regarding sodium and calcium showed an extremely small coefficient of variation and no evident factor that might affect their measurement was found. It was considered that anesthesia and anticoagulants might influence the values of potassium. It is important to separate serum from blood as soon as possible after coagulation when using serum and to minimize damage to the orbital venous plexus when sampling blood from it.

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[in Japanese]

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To determine the potential use of C-reactive protein (CRP) reagents in dogs, CRP was measured in untreated beagles from an experimental animal facility and in inflammation models. CRP in 80% of the beagles from the experimental animal facility was under 20 mg/l which is clinically said to be the highest normal range in veterinary medicine. CRP in dogs had a sensitive reaction to inflammation and tissue damage. From these results, CRP was judged to be an effective marker for diagnosis of latent infectious diseases and a good parameter for determining normal animals in commercial experimental animals. Because of its sensitive reaction to inflammation and tissue damage, CRP is also a good marker for screening toxicity in non-clinical studies.

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A proposed recommendation method for the determination of total cholesterol (TC) using comparative method for evaluation in serum is described. This proposed method is a full enzymatic cholesterol assay system (CE-CD-UV method) combining cholesterol dehydrogenase (CD, EC number not assigned) from Nocardia species and cholesterol esterase (CE, EC 3. 2. 1. 13) from Pseudomonas species. Cholesterol esters in serum are converted to free cholester-ol by hydrolysis with CE in the first stage of the assay. The free cholesterol is then quantita-tively oxidized by CD to LΔ-4-cholesten-3-one re-sulting in the production of a stoichiometric amount of reduced P-nicotinamide-adenine-di-nucleotide (β-NADH). The P-NADH is measured spectrophotometrically to quantitate total serum cholesterol. The performance of the proposed method is described below. The average withinay and day-to-day precisions expressed as coefficient of variation were 0.37 to 0.78% and 0.42 to 0.49%, respectively. The assay was linear from 0 mg/dl to approximately 720 mg/dl (18.6 mmol/l) total serum cholesterol. Reduced substances (bilirubin, etc.) did not interfere. The correlation between the TC values determined by the project facilities using the proposed method (y) and the values determined by the Abell-Levy-Brodie-Kendall method (χ)(Osaka Medical Center for Cancer & Cardiovascular Disease: a member of U. S. National Cholesterol Reference Method Laboratory Network) was coincidence. The equation was best fit as y= 1.004x+0.450, 7=0.999 and n=12. In addition, an assay for TC using CD coupled with alkaline saponification (the alkaline saponification method) was investigated to confirm that the hydrolysis of CE was complete or not. The results of the proposed method (y) showed a linear correlation with the alkaline saponification method (x). The equation was best fit as y=1.003x-2.849, r=0.993 and n=25. In conclusion, it is recommended that this proposed method is a comparative method for total serum cholesterol determination in serum.

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2-Ethylaminoethanol (EAE) method has widely been used for determination of serum alka-line phosphatase (ALP) activity in Japanese clin-ical laboratory. We have demonstrated that theactivity of ALP changed with levels of both high-molecular mass intestinal alkaline phospha-tase (HIAP) and normal-molecular mass intestin-al alkaline phosphatase (NIAP). We classified the 65 healthy subjects into the two groups of blood group B or O secretors (n=30) and other blood groups (n=35). ALP activity in blood group B or O secretor at fasting and 3 h after fatty meal were 194.2± 54.8 U/I (mean±SD) and 218.3±58.0U/I (mean±SD), respectively, and ALP activity in other blood groups at fast- ing and 3 h after fatty meal were 157.8±40.7U/I (mean±SD) and 162.2±42.5 U/I (mean±SD), respectively. The activity of ALP-(HIAP +NIAP) in blood group B or O secretor at fast-ing and 3 h after fatty meal were approximately the same as those of other blood groups. These results suggest that differences of ALP activity in blood group B or O secretors and other blood groups are closely related to the HIAP and NIAP levels.

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N-3 eicosapentanoic acid is known to have potential antithrombolic and antiatherosclerotic actions. We established a new method using lipoprotein lipase hydrolyzing total lipids to determine the serum levels of the fatty acid fractions. After total lipids (glycerides, phospholipids and cholesterolesters) were hydrolyzed by the lipoprotein lipase for 2 hours at 37°, the released fatty acids were extracted by 1-4'olch reagent and methylesterized by BF3 methanol for 10 minutes at 50°, and injected into gas- chromatography. This new method could maintain the temperature in every step at a level low enough for the fatty acid of become biologically stable. Therefore, the reproducibility both in within-series and between-series of the fatty acid measurement was improved, which was also supported by a relatively low coefficient of variation (2-10% and 3-10%, n=10, respectively). Analytical recoveries of 500 pg fatty acids added to serum were 90.6-104.1%.

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