Serum alpha-fetoprotein Values in Dogs with Various Hepatic Diseases

Takatsugu YAMADA,1,2), Megumi FUJITA1), Satoshi KITAO3), Yoshinori ASHIDA1), Kazuya NISHIZONO1), Ryo TSUCHIYA1), Takuo SHIDA4), and Kousaku KOBAYASHI1) 1)Laboratory of Veterinary Internal Medicine and 4)Laboratory of Veterinary Radiology, School of Veterinary Medicine; 2)Research Institute of Bioscience, Azabu University, 1Ð17Ð71 Fuchinobe, Sagamihara, Kanagawa, 229Ð8501 and 3)Doubutsu Medical Center, 1Ð6Ð45 Nakahozumi, Ibaraki-shi, Osaka 567Ð0034, Japan

(Received 2 December 1997/Accepted 10 February 1999)

ABSTRACT. Serum alpha-fetoprotein (AFP) values were measured in hepatic diseased dogs with or without tumor and non-hepatic tumor bearing dogs by a sandwich ELISA using anti-dog AFP antiserum. Serum AFP values were less than 70 ng/ml in clinically healthy dogs. The values in dogs with hepatocellular carcinoma were higher than 1,400 ng/ml in 7 of 9 dogs, wherever those in two dogs with cholangiocarcinoma were in the normal range. Serum AFP values in hepatic diseased dogs without tumor were also high, however, the values were below 500 ng/ml in 90% of the dogs. In non-hepatic tumor dogs, serum AFP values were less than 500 ng/ml in 76% of the dogs. In the surgically removal cases with hepatocellular carcinoma, serum AFP values rapidly decreased. These results suggested that the sandwich ELISA using anti-dog AFP antiserum was an available method for diagnosis of hepatocellular carcinoma in dogs.—KEY WORDS: alpha-fetoprotein, canine, ELISA, tumor. J. Vet. Med. Sci. 61(6): 657Ð659, 1999

Serum alpha-fetoprotein (AFP) values have been used with hepatocellular carcinoma were also monitored AFP for the diagnosis of hepatocellular carcinoma [4, 5Ð6, 14] values after surgical removal of masses. and also for the understanding pathophysiological states in Serum: Sera were collected by the usual methods, and hepatic failure [8Ð9] in man. The values in dogs measured kept below －20°C until assay. by enzyme-linked immunosorbent assay (ELISA) or radio Preparation of AFP and antibodies: Canine AFP and anti- immuno-assay (RIA) using anti-human AFP antibody were dog AFP antiserum were prepared according to the also reported [7, 9, 15], as the incidence of hepatocellular previously described method [17]. carcinoma has been a concomitant increase in dogs with ELISA for canine AFP: The protein concentration of age advanced. However, these reports included some purified AFP was measured by the Coomassie protein assay discrepancies with the low AFP values in dogs with methods (Pierce, U.S.A.) using bovine serum albumin (BSA) hepatocellular carcinoma, suggesting low sensitivity of these as a standard. The ELISA for canine AFP was developed methods for the measurement of canine AFP. It has been by the previously reported avidin-biotin sandwich method widely accepted that the human AFP molecule is similar to with minor modifications [17]. Briefly, the immunoglobulin that of other mammal species [10, 12], however, some AFP G (IgG) fraction of anti-dog AFP antiserum from rabbits from animals shows low affinity compared to that from was coated on 96-well microplates (flat-bottom) at 5 µg per man [13]. The AFP values measured by ELISA using well. The wells were blocked with PBS containing 0.5% monoclonal anti-human AFP antibody were different from BSA, and washed with PBS containing 0.05% Tween 20 that by RIA using polyclonal anti-human AFP antibody [1]. (TPBS) by an ELISA plate washer (Immunowash; BioRad, The diagnosis of hepatocellular carcinoma in dogs is usually USA). Serum samples were diluted with TPBS at 1:2, 1:10, difficult due to the absence of remarkable clinical sign, and and 1:50. A standard solution of canine AFP was also specific parameters in the early stage of the disease, since diluted at concentrations of 0.25 ng/ml to 600 ng/ml in no reliable serum AFP values were able to measure by the TPBS. The IgG fraction of anti-dog AFP antiserum was high sensitive and specific method for canine AFP. This labeled with N-hydroxysuccimidobiotins (Sigma, U.S.A.) as paper deals with the serum AFP values in hepatic disease shown by Coligan et al. [3]. The peroxidase-conjugated dogs with or without tumor and also non-hepatic tumor streptavidin was purchased from Dako (Denmark) and the bearing dogs by a sandwich ELISA for canine AFP. chromogen 2',2' azino-bis (3-ethylbenzthiazolin-6-sulfonic acid) from Sigma (U.S.A.). Absorption of the samples and MATERIALS AND METHODS standards were measured at 405 nm. Serum AFP was measured in less than 3 weeks after storage, because storage Animals: Twenty clinically healthy dogs (10 male and 10 time would affect AFP values [7]. female, from 2 to 11 year-old) kept in our laboratory and total of 86 dogs (2 to 16 year-old) referred to the Animal RESULTS Hospital of Azabu University, Ibaraki Doubutsu Medical Center and some private animal hospitals asked for Serum AFP values: Serum AFP values in clinically examination of serum AFP values were used. Five dogs healthy dogs were less than 70 ng/ml. Serum AFP values 658 T. YAMADA ET AL.

Table 1. Serum AFP values in dogs with various hepatic diseases

Group No. Means ± SD (ng/ml) Range

Hepatic diseases With tumor Hepatocellular carcinoma 9 2860 ± 3910 20Ð12300 Cholangiocarcinoma 2 75 ± 12 66Ð84 Without tumor Hepatitis, Colangitis 21 310 ± 426 18Ð1700 Non-hepatic tumora) 54 366 ± 555 13Ð2990

a) Lymphosarcoma, Mammary tumor, Melanoma, Squamous-cell carcinoma, Mas- tocytoma, Stomach cancer.

Fig. 2. Changes of serum AFP values in dogs with Fig. 1. Serum AFP values in 20 clinically healthy dogs and hepatocellular carcinoma after surgically removal. ●—●: diseased dogs, included 9 hepatocellular carcinoma, 2 case 1, a 12-year-old spayed female beagle, detected cholangiocarcinoma, 21 hepatic disease without tumor, and 54 metastasis and died after one year; ○…○: case 2, a 10-years- non-hepatic tumor (lymphosarcoma, mammary tumor, old, mixed-breed, castrated male survived during observation; melanoma, squamous-cell carcinoma, mastocytoma, stomach ▼—▼: case 3, 12-years-old Maltese female, detected no cancer). The descriptive statistic test box plot graphed the metastasis and died after one month by non tumor reason; ▽ percentiles and mean of column data. The ends of the boxes …▽: case 4, 11-years-old spayed female mongrel, had defined the 25th and 75th percentile, with a line at the mean metastasis and vascular invasion, and died after one week; (thick bar) and error bars defining the 10th and 90th ■—■: patient 5, 14-years-old spayed female mongrel, percentiles. Each data plots were also shown by computer detected no metastasis, and survived during observation. software “Sigma Plot”. in hepatocellular carcinoma dogs were remarkably high, DISCUSSION except in the one case (20 ng/ml), being more than 1,400 ng/ml in 7 out of 9 dogs (Table 1) (Fig. 1), whereas those in In man, serum AFP values remarkably increased in cases two dogs with cholangiocarcinoma were in normal range. of hepatocellular carcinoma, hepatoblastoma, and yolk sack Serum AFP values in hepatic diseased dogs without tumors tumor, and also moderately or mildly increased in acute or were 18 to 1,700 ng/ml, however, 90% of the dogs showed chronic hepatitis [4, 6, 14]. In most of clinically healthy under 500 ng/ml. Non-hepatic tumor bearing dogs showed dogs, AFP values were under 70 ng/ml, and were similar variable serum AFP values, and 92% of the dogs showed levels to those in our previous report [17]. Lowseth et al. under 1,000 ng/ml. [7] reported that serum AFP values measured by an ELISA Changes in serum AFP values after removing for human AFP, were approximately 220 ng/ml in neonatal hepatocellular carcinoma masses: The serum AFP levels in dogs and were 30Ð60 ng/ml in adult dogs. However, our all dogs with hepatocellular carcinomas decreased rapidly previous report [17] demonstrated that serum AFP values in after removal of the confined tumor masses. The dogs that newborn puppy were approximately 14 mg/ml with mean, AFP values maintained low levels had not a relapse the and the values were 2 × 105 fold higher levels than those in tumor, however, one case (case 1) that the AFP values adult dogs. These results suggested that anti-human AFP gradually increased and recurred the tumor (Fig. 2). antibody used in the ELISA had a low sensitivity against canine AFP and induced false-positive results. ALPHA-FETOPROTEIN IN HEPATIC DISEASED DOGS 659

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