C-reactive protein (CRP) measurement in canine serum following experimentally-induced acute gastric mucosal injury Kouji Otabe', Tsuneo Ito', Tetsuro Sugimoto' & Shizuo Yamamoto2 lSafety Assessment Laboratory, Chugai Pharmaceutical Co., Ltd, 1-135 Komakado, Gotemba-shi Shizuoka 412-8513 and 2Department of Immunology, College of Environmental and Health Sciences, Azabu University, Sagamihara-shi, Kanagawa 229, Japan

Summary To establish the diagnostic significance of canine C-reactive protein (CRP)in gastrointestinal

disorders, the serum canine CRP concentration was measured in dogs with experimentallyinduced acute gastric mucosal injury. Gastric injury was induced in one male and one female beagle by a single dose oral administration of acetylsalicylic acid (200 mg/kg body weight I or indomethacin (60 mg/kg body weight), or sodium chloride (1000 mg/kg body weight). CRP was measured prior to dose, and I, 3, 7, and 14 days after the administration of the drugs, together with the total leucocyte counts and serum iron. Changes in the serum CRP in dogs with gastric injury were similar for the three test compounds, and reflected by the endoscopic findings. CRP values increased from 87 to 390 mg/l within 1 to 3 days after the compound administration but returned nearly to the pre-

dose levels within 14 days. Endoscopy revealed haemorrhagic erosion of the gastric mucosa in all dogs one day after dosing, with no evidence of the erosions observed after 7 days in many of the dogs. Changes of the total leucocyte and serum iron also occurred following gastric injury, but these changes were not as marked as those observed for CRP. The results of this study suggest that serum CRP level may be a useful indicator of a gastrointestinal mucosal injury in dogs. Keywords Dog; C-reactive protein; gastrointestinal indomethacin; sodium chloride

C-reactive protein is one of the acute phase reactant proteins, which reflect the severity of acute inflammation and tissue damages, and it is also found in humans (Okamura et al. 1990, Hodgson 19941. Canine CRP has been used as a possible marker of inflammation in some experimental models (Ganrot 1973, Laurent & Panayi 1980, Caspi et al. 1987, Ndung'u et al. 1991, Yamamoto et al. 1993), although additional results must still be obtained. Correspondence

to: Kouji Otabe

Accepted 21 March 2000

©

mucosal injury; acetylsalicylic acid;

Our previous studies showed that there are no marked intra individual variations in dogs, and little variation occurred during either 24 h or 28-day periods (Otabe et al. 1998). In this study, we used a canine model of acute injury of the gastric mucosa by using a single oral dose administration of one of the following: acetylsalicylic acid, indomethacin, and sodium chloride; we monitored clinical symptoms, endoscopic findings, serum CRP, total leucocyte counts and serum iron concentration. Laboratory Animals Ltd. Laboratory Animals (2000) 34, 434-438

Serum CRP in dogs with gastric mucosal injury

Materials and methods Animals and husbandry Three male and three female beagle dogs aged one year (body weight 7.5-11.9 kg) were used in the study. The dogs were obtained from CSK Research Park Inc. (Nagano, Japan) and individually housed in metal cages (W700x 0900 x H 1090mm) in an animal room adjusted to a temperature of 23±2°C, humidity of 55± 10%, illuminated for 12 h (06:00-18:00 hI, and air changes 14-16 times/ h. They were given about 250 g/animal/ day of a solid stock food (CD-5, Clea Japan, Inc., Tokyol and were allowed to drink water ad libitum. The animals used in this experiment were treated in accordance with the guide for the care and use of laboratory animals by the Chugai Pharmaceutical. Co., Ltd. Preparation of experimental model of acute gastric mucosal injury After the dogs were fasted for 12 h, acetylsalicylic acid (200 mg/kg body weight), indomethacin (60 mg/kg body weightl, or sodium chloride (1 g/kg body weightl packed in gelatin capsules were administered orally once to one animal. All chemicals were purchased from Wako Pure Chemical Industries Ltd. [Osaka, Japan). ' Observation

and examinations

Clinical observations Clinical signs for each animal were recorded daily from one day before to 14 days after dosing. Endoscopic findings The interior of the stomach was examined using an endoscope [CV-l, Olympus Optical Co., Ltd., Tokyo, Japan) under pentobarbital anaesthesia (Nembutal®, Dainippon Pharmaceutical Co., Ltd., 25 mg/kg body weight) after a 12 h fast on the day before and 1, 3, and 7 days after the administration. Blood sampling Blood was sampled from the cephalic vein on the day before and I, 3, 7, and 14 days after

435

the dosing, always after an overnight fast. Blood samples were collected into anticoagulant (dipotassium ethylenediamine tetra-acetic acid, EDTA-2K)for totalleucocyte count analysis. Serum samples were separated by centrifugation (1870 g, 4°C, 10 min) and stored at - 80°C until the analysis for serum CRP and iron. Serum CRP Canine CRP was assayed in serum using the sandwich method of enzyme-linked immunosorbent assay (ELISAlas previously described (Yamamoto et al. 1992). The microplate (Nunc, Roskilde, Denmark) was coated with the goat anti-dog CRP IgG antibody. After adsorption, specific binding was blocked with 1% bovine serum albumin. After washings with phosphate buffered saline containing 0.1% of Tween 20, multiply diluted, 444 Ilg/ml of CRP and the test serum were added to each well. The plates were washed again, then incubated using the horseradish peroxidase (Sigma type VI, RZ 3.0, Sigma Chemicals Co.llabelled with anti-dog CRP IgG, and substrate (ABTS®,Boehringer Mannheim GmbH, Germany), containing 0.03% of hydrogen peroxide. The absorbance was then measured at 405 nm.

Blood leucocyte counts and serum iron Total leucocyte counts was measured with automatic cell counter (K2000,TOA Medical Electronics Co., Ltd., Kobe, Japan). Serum iron was measured with an automatic analyzer (7170, Hitachi, Ltd., Tokyo, Japan)using a reagent kit (Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan) based on the ferrozine colour reaction. Results Clinical observations The observations after dosing with three compounds are shown in Table 1. Endoscopic findings Haemorrhagic erosions were observed in both dogs with all three compounds 1 day Laboratory

Animals

(2000) 34

436 Table 1

Otabe et al. Clinical observations following

Days after dosing

dosing with acetylsalicylic acid, indomethacin

and sodium chloride

Acetylsalicylic acid (200 mg/kg)

Indomethacin (60 mg/kg)

1 Male

3 Male

4 Female

S Male

6 Female

+c

+a,c

+b

+a,c

2 Female

Sodium chloride (1 g/kg)

Pre

1-3 4-7 8-14 a = vomit; b = blood vomit; c = haemafecia

administration with acetylsalicylic acid and sodium chloride. However no clear change was observed in the total leucocyte counts and serum iron levels after dosing with indomethacin.

after dosing, and persisted for 3 to 7 days (Table 2). Serum CRP, totalleucocyte serum iron

counts and

The serum eRP concentrations, totalleucocyte counts and serum iron levels in individual dogs treated with each compound are shown in Table 3. CRP changes occurred in both male and female dogs with three compounds. CRP values increased to values ranging from 87.3 to 390.6 mg/l within 1 to 3 days after dosing and then returned nearly to the pre-dose levels after 7 or 14 days. The peak level was lO-fold the pre-dosing level in the male and 22-fold in the female dogs dosed with acetylsalicylic acid, lO-fold in the male and 45-fold in the female dogs dosed with indomethacin, 5-fold in the male and 18-fold in the female dogs dosed with sodium chloride. The total leucocyte counts was increased in 1 or 3 days after dosing with acetylsalicylic acid and sodium chloride. Serum iron changes was observed in 1 and 3 days after the

Discussion C-reactive protein, an acute phase reactant protein, has no specificity for disease, but increases rapidly in the presence of inflammation or tissue destruction (Gewurz et a1. 1982). Therefore, evaluation of its relative changes could be useful for the early diagnosis and monitoring of diseases, for the confirmation of therapeutic effects, and secondary diagnostic method (Okamura et a1. 1990, Hodgson 1994). Serum CRP concentration showed similar changes in dogs with acute gastric mucosal injury induced with three different compounds. These changes in the serum CRP reflected the development and disappearance of the gastric mucosal lesions observed by endoscopy; and correlated with the gastric mucosal injuries.

Table 2 Endoscopic findings of acute gastric mucosal lesions following indomethacin and sodium chloride Acetylsalicylic acid (200 mg/kg)

dosing with

Indomethacin (60 mg/kg)

acetylsalicylic

Sodium chloride (1 g/kg)

2 Female

3 Male

4 Female

S Male

6 Female

+a

++a

+a

+a

+a

+b

+a

+a

+a

+a

Days after dosing

1 Male

Pre 1 3 7

+a

Rating: -: normal. +: haemorrhagic erosion (single), ++: haemorrhagic erosion (few areas) a = pyloric region of the stomach; b = fundic region of the stomach Laboratory Animals (2000) 34

+b

acid,

Serum CRP in dogs with gastric mucosal injury Table 3 Serum CRP, total leucocyte indomethacin and sodium chloride

Days after dosing Pre

3

7

14

counts

Variable Serum CRP (mg/I) Total leucocyte counts Serum iron (fl mol/I) Serum CRP (mg/I) Total leucocyte counts Serum iron (fl mol/I) Serum CRP (mg/I) Total leucocyte counts Serum iron (fl mol/I) Serum CRP (mg/I) Total leucocyte counts Serum iron (fl mol/I) Serum CRP (mg/l) Total leucocyte counts Serum iron (fl mol/I)

(109/1)

(109/1)

(109/1)

(109/1)

(109/1)

437 and

serum

iron

following

dosing

with

acetylsalicylic

acid,

Acetylsalicylic acid (200 mg/kg)

Indomethacin (60 mg/kg)

Sodium chloride (1 g/kg)

1 Male

2 Female

3 Male

4 Female

5 Male

6 Female

11.2 10.8 30 76.5 18.9 11 111.4 14.3 49 51.3 14.6 35 17.5 12.5 24

17.6 10.5 21 390.6 19.5 7 143.2 11.8 55 41.2 11.5 31 11.7 11.0 18

8.9 12.5 19 12.2 12.4 29 87.3 13.8 17 14.2 11.7 30 3.2 13.3 16

3.2 13.2 23 13.8 13.6 42 144.7 14.2 13 37.3 13.0 23 9.5 13.7 18

23.7 8.2 32 131.3 13.2 25 34.7 9.1 42 12.8 8.5 21 39.8 9.6 28

7.2 10.0 29 131.8 12.8 23 37.9 13.6 44 13.2 8.2 25 5.1 8.4 18

In veterinary medicine, in which endoscopic examination is still an uncommon procedure, the diagnosis of gastrointestinal disorders in dogs can be dependent on findings of blood in excrement or vomitus, and the value of these examinations is limited. The reliability of the diagnosis and follow-up of gastrointestinal disorders could be improved by using serum CRP measurements, which showed changes in association with endoscopic Bndings of the development and disappearance of gastric mucosal lesions. In this study, CRP level increased in several times with the development of gastric mucosal injuries. CRP showed sensitive changes in the indomethacin-treated animals, in which no marked changes were observed for the total leucocyte counts and serum iron. Serum CRP concentration and leucocyte count in dogs have been compared previously in surgical trauma where changes of CRP concentration were detected later than alterations of blood cell counts (Burton et al. 1994).

It was noted that clinical examinations failed to show changes in dogs with indomethacin: this may be due to destruction by the mechanism of the gastric mucosa. Acetylsalicylic acid and sodium chloride directly

injure the gastric mucosa, but indomethacin does not (Hoshino et al. 1993). Serum CRP concentration in this study from six dogs before dosing ranged from 3.223.7 mg/lj these values were similar to our previous study of the 10 normal dogs (Otabe et al. 1998). The slight increase of CRP in 14 days after dosing with sodium chloride in male dogs, was considered to be a transient response. In this study the diagnostic value of the canine CRP changes in drug-induced gastrointestinal injuries has been demonstrated. References Burton SA, Honor DJ, Mackenzie AL, Eckersall PD, Markham RJ, Horney BS 119941C-reactive protein concentration in dogs with inflammatory leukograms. American Journal of Veterinary Research 55, 613-18 Caspi D, Snel FWJJ, Batt RM, Bennett D, Rutteman GR, Hartman EG, Baltz ML, Gruys E, Pepys MB (1987) C-reactive protein in dogs. American Journal of Veterinary Research 48, 919-21 Gamot K 119731Plasma protein response in experimental inflammation in the dog. Research in Experimental Medicine 161, 251-61 Gewurz H, Mold C, Siegel J, Fiedel B 11982) C-reactive protein and the acute phase response. Advances in Internal Medicine 27, 345-72 Laboratory Animals (2000) 34

438

Hodgson HJF (1994) Why measure C reactive protein? Gastroenterology 35, 5-7 Hoshino T, Endo T, Kusakari K, Ishida M (19931 Gastric mucosal injury by transendoscopic compressed air delivery of powdered NaCl, KCl or N5AIDs in dogs. Digestive Endoscopy 5, 373-7 Laurent MR, Panayi G5 (1980) Biochemical parameters in the assessment of anti-inflammatory drugs-a review. Agents and Actions, Supplement 7,310-17 Ndung'u JM, Eckersall PD, Jennings FW 119911 Elevation of the concentration of acute phase proteins in dogs infected with Trypanosoma brucei. Acta Tropica 49, 77-85 Okamura JM, Miyagi JM, Terada K, Hokama Y 119901 Potential clinical applications of C-reactive pro-

Laboratory Animals (2000) 34

Otabe et a/.

tein. TournaI of Clinical Laboratory Analysis 4, 231-5 Otabe K, 5ugimoto T, Jinbo T, Honda M, Kitao 5, Hayashi 5, 5himizu M, Yamamoto 5 119981Physiologicallevels of C-reactive protein in normal canine sera. Veterinary Research Communications 22, 77-85 Yamamoto 5, Tagata K, Nagahata H, Ishikawa Y, Morimatsu M, Naiki M (19921 Isolation of canine C-reactive protein and characterization of its properties. Veterinary Immunology and Immunopathology 30, 329-39 Yamamoto 5, 5hida T, Miyaji 5, 5antsuka H, Fujise H, Mukawa K, Furukawa E, Nagae T, Naiki M 119931 Changes in serum C-reactive protein levels in dogs with various disorders and surgical traumas. Veterinary Research Communications 17, 85-93