Journal of Biomedical Research 11(1) : 19-26 (2010) Hematological Evaluation for Quality Control of Minipig as a Non-rodent Laboratory Animal Hyun-Gu Kang 1 *, Sang-Koo Lee 2 1 College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Korea 2 College of Medicine, Hanyang University, Seoul 133-791, Korea (Received Dec 10, 2009 / Revised Feb 23, 2010 / Accepted Feb 26, 2010) ABSTRACT: Pigs have increasingly become useful models for toxicological and pharmacological studies because of the similarities of their anatomical morphology and physiology to those of humans. This study was conducted to evaluate of the quality control level through hematological features of minipig. Thus, we analyzed whether there are differences in hematological parameters between male and female minipigs. There were no significant differences in the levels of erythrocytes, hematocrit, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), leukocytes (WBC), and serum electrolytes between male and female minipigs. But, leukocytes were higher than reference range in both sexes (26.46±2.06 vs 11.6±5.4 for male; 23.58±3.36 vs 10.0±2.0 for female). In differential leukocyte counts, all leukocyte parameters were not significantly different between both sexes. But, neutrophils were higher than reference range in both sexes (60.65±13.92 vs 33.1±18.9 for male; 46.84±12.80 vs 15.4±5.6 for female). In serum biochemical analysis, all biochemical parameters were not significantly different between male and female minipigs. However, aspartate aminotransferase (AST) and alkaline phosphatase (ALT) were higher than reference range, while alanine aminotransferase (ALP) was lower than reference range. In urine analysis, ph of male and female were 8.3±1.0 and 6.4±1.6, showing higher value in male minipig. These data indicate that quality control of minipig must take precedence in order to toxicological and pharmacological studies. Keywords: Hematology, Quality control, Minipig *Corresponding author: Hyun-Gu Kang College of Veterinary Medicine, Chungbuk National University, 410 Sungbong-ro, Heungduk-gu, Cheongju 361-763, Korea Tel: +82-43-261-3359 Fax: +82-43-261-3224 E-mail: kang6467@cbnu.ac.kr
20 Hyun-Gu Kang and Sang-Koo Lee 서론본래돼지는육용가축으로육성된잡식동물이나생리해부학적소견이사람과유사한점 [1, 2] 과근년동물복지에대한높은관심과함께실험동물로서도크게주목받고있다 [3, 4]. 돼지의해부 생리학적구조및생리현상이인간과유사하고그간연구되어왔던개나원숭이와는달리가축화된지수천년이경과하는동안에인간으로전염되는질병이발견되지않은점등이실험동물로써돼지의이용을확대시키고있다 [5]. 돼지의실험동물로서이용분야는다양하다. 또한돼지혹은미니돼지는개나원숭이와달리가축으로분류되기때문에동물복지와관련한이슈에서자유로울뿐아니라돼지를이용한동물실험은그다지윤리적으로제약받지않는것을들수있다 [3, 4]. 돼지를이용한연구분야는다양한생명과학분야즉, 영양학, 순환기, 안과및치과분야, 이종장기이식등이있으며 [5-9], 독성실험분야는일반독성및생식독성에많이이용되고있다 [6, 10]. 또한약물의동태시험, 약효 약리시험등에널리이용되고있으며 [11, 12] 생식독성을수행하는데필요한모든기술적인면들이돼지를이용한시험에서도가능할뿐아니라성성숙, 임신, 산자수등의기본적인데이터들도생식독성시험에적합한동물로판명되고있다 [10]. 미니돼지가독성학분야에사용되기시작한것은 1980년대중반부터이다. 미니돼지가독성분야에본격적으로사용되게된원인은약물의대사및흡수경로가사람과가장유사하다고알려지면서부터이다 [1, 2, 13]. 이상과같이돼지는많은연구분야에사용되고있으나설치류에서제공되는실험동물로서의품질관리는아직이루어지고있지않다. 돼지가실험동물로서사용되기위해서는반드시선행되어야할것이품질관리이다 [14]. 실험동물은살아있는시약이라할만큼품질관리가잘이루어져야결과의신뢰성및무분별한동물의희생을막을수가있다. 현재국내외적으로미니돼지의이용성이증가하면서사육관리및품질관리에대한연구가많이 이루어지고있다 [13, 15-18]. 하지만국내에서는다양한분야에미니돼지가이용되고있음에도불구하고품질관리에대한보고는미미한실정이다. 따라서본연구에서는국내에서독성실험및약물동력학실험에의사용이증가하고있는미니돼지의혈액및혈액생화학적분석을실시하여미니돼지에대한품질관리의지표를제공하고자하였다. 재료및방법실험동물미니돼지 (minipig) 는경기도평택소재 PWG Genetics Korea 사에서구입하였으며미니돼지의연령은 4.0~6.0개월령, 체중은 12~16 kg이었다. 미니돼지는암수각 2마리를시험에사용하였다. 이들미니돼지는 PWG Genetics Korea사실험동물실의스테인리스로만든미니돼지전용케이지에각각 1마리씩수용하였으며, 사료는자체적으로제조한미니돼지용사료를 300 g/day로공급하였으며음수는상수도수를자유로이급여하였다. 미니돼지를전용케이지에수용한후약 1주일간의검역및순화기간을거친다음시험에사용하였다. 사육실환경은온도 22±2 C, 상대습도 50±10% 를유지하였고환기횟수는 12~18회 /hr ( 전외기공조방식 ) 였으며조명시간및명암주기는 12시간점등, 12시간소등하였다. 혈액학및혈액생화학적검사미니돼지는시료채취 16시간이상절식을시킨후경정맥에서약 6 ml의혈액을채취한후, 그중 3 ml를 EDTA가함유된 CBC bottle에넣어혈구자동측정기 (T-540 Coulter Counter, Coulter Electronics Inc., Hialeah, FL, USA) 및 ADVIA120 Hematology system (Bayer Healthcare LLC, Tarrytown, NY, USA) 로혈액학적검사를실시하였으며, 백혈구백분율은혈액도말표본을제작하여광학현미경하에서산정하였다. 혈액학적검사는 white blood
Hematological evaluation of minipig 21 cell (WBC) count, differential leukocyte count, red blood cell (RBC) count, hemoglobin concentration, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), reticulocyte 및혈소판수를측정하였다. 혈액학적검사와동일한시간에채취한혈액은 3,000 rpm에서 10분간원심분리한후혈청을분리하여혈청자동분석기 (Shimadzu CL-7200, Shimadzu Co., Kyoto, Japan) 와전해질분석기 (Ciba-Corning 644 Na/K/Cl analyzer, Ciba-corning, Medfield, MA, USA) 를이용하여다음의항목을측정하였다 ; alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TB), total cholesterol (TC), triglycerides, phospholipids, total proteins (TP), albumin, albumin/globulin (A/G) ratio, glucose, blood urea nitrogen (BUN), creatinine, creatine phosphokinase (CPK), calcium 및 inorganic phosphorous (IP). 혈액응고검사 Prothrombin time (PT) 및 activated partial thromboplastin time (APTT) 은남은혈액과 3.2% sodium citrate를 9:1의비율로섞어 3,000 rpm에서 10분간원심분리하여얻은혈장으로측정하였다. 요검사요분석기기 (UrotronRL, Germany) 를이용하여요비중, ph, protein, ketone, 백혈구수를측정하였다. 통계분석측정된자료는 Microsoft사의 Excel 프로그램으로통계처리하였다. 암수간측정치의비교는 Student s t-test를이용하여검정수준 95% 에서분석하였다. 결과국내에서생산된미니돼지의혈액학적검사를실시한결과는 Table 1 및 2와같다. 혈액학적검사결과국내에서생산된미니돼지에서암수모두백혈구수치가높게나타났으며 (Table 1), 백혈구감별계산에서는중성호성백혈구가 60.65±13.92% 로높은비율을차지하였다 (Table 2). 또한혈소판의수치는낮게관찰되었으며, 나머지혈액학적검사항목에서는유의할만한차이가인정되지않았다. 한편암컷과수컷에따른혈액학적검사결과에대한차이점은인정되지않았다. 미니돼지의혈액화학치검사결과혈당, 단백질, AST, ALT가높게나타났고 BUN, ALT, K은약간높게나타났으며총콜레스테롤, 빌리루빈, 크레아티닌은낮게나타났다. 또한혈액화학치검사에서암수에따른차이는인정할수는없었지만개체별에따른차이가심했다 (Table 3). 혈액응고시험에서는암수에따른차이가인정되지않았으며정상치의범위에있었다 (Table 4). 미니돼지의요검사를실시한결과요의 ph는수컷이 8.3±1.0으로알칼리성을나타내었으며, 암컷이 6.4±1.6으로약산성을나타내었다. 또한요중단백질은수컷이 6.3±12.5, 암컷이 30.0±67.1로암컷에서상당히높은요중단백질이검출되었다. 기타요비중과백혈구, 케톤체는암수에따른차이가관찰되지않았다 (Table 5). 고찰미니돼지는비교적용이하게사람에서의 PK 특성및독성을정확하게예측할수있을뿐아니라개개의약물별로사람과가장유사한대사패턴을나타내는최적의동물모델이라고하였다 [7, 12]. 또한미니돼지는생리 해부학적으로사람과가장비슷한동물로이종장기이식과같은생명공학에그활용성이날로증가하고있다 [7, 19]. 돼지에서생리학적지표를확립하는것은실험실
22 Hyun-Gu Kang and Sang-Koo Lee Table 1. Hematological values of male and female minipigs Sex Items Minipig Reference range Male Female Leucocytes (10 3 /mm 3 ) 26.46 ± 2.06 11.6 ± 5.4 No. of erythrocyte (10 6 /mm 3 ) 8.50 ± 0.31 8.9 ± 1.7 Hemoglobin (g/dl) 13.26 ± 0.60 13.7 ± 2.8 PCV (%) 44.40 ± 2.63 41.5 ± 10.7 Platelet (10 3 /mm 3 ) 440.14 ± 118.14 529.2 ± 198.8 MCV (fl) 52.18 ± 1.42 48.4 ± 3.9 MCH (pg) 15.60 ± 0.32 15.5 ± 1.5 MCHC (g/dl) 29.88 ± 0.72 31.9 ± 0.8 Leucocytes (10 3 /mm 3 ) 23.58 ± 3.36 10 ± 2.0 No. of erythrocyte (10 6 /mm 3 ) 8.59 ± 0.22 8.8 ± 0.7 Hemoglobin (g/dl) 13.52 ± 0.65 12.7 ± 1.0 PCV (%) 45.18 ± 1.73 40.7 ± 2.9 Platelet (10 3 /mm 3 ) 398.18 ± 43.04 466.2 ± 157.3 MCV (fl) 52.42 ± 1.71 46.2 ± 4.5 MCH (pg) 15.72 ± 0.66 14.5 ± 1.7 MCHC (g/dl) 29.94 ± 0.45 31.4 ± 1.0 PCV, packed cell volume; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration. Table 2. Differential leukocyte count of male and female minipigs Sex Items Minipig Reference range Neutrophils (%) 60.65 ± 13.92 33.1 ± 18.9 Eosinophils (%) 0.40 ± 0.54 1.6 ± 0.9 Male Basophils (%) 0 0.5 ± 0.7 Lymphocytes (%) 35.20 ± 14.64 58.6 ± 20.7 Monocytes (%) 3.30 ± 1.09 2.7 ± 1.4 Neutrophils (%) 46.84 ± 12.80 15.4 ± 5.6 Eosinophils (%) 0.76 ± 0.49 2.7 ± 1.3 Female Basophils (%) 0 0.5 ± 0.4 Lymphocytes (%) 48.28 ± 12.82 72.1 ± 19.8 Monocytes (%) 3.86 ± 2.59 2.6 ± 1.1 검사결과의해석에있어서중요한기준이된다 [20, 21]. 혈액학적및혈액생화학적항목은식이, 성, 사 육환경을포함한환경적및생리학적인자에의하여 영향을받는다 [22, 23].
Hematological evaluation of minipig 23 Table 3. Serum biochemical values of male and female minipigs Sex Items Minipig Reference range Male Female Total protein (g/dl) 7.46 ± 0.29 5.7 ± 0.3 Albumin (g/dl) 3.44 ± 0.15 3.5 ± 0.3 Total bilirubin (mg/dl) 0.09 ± 0.01 0.2 ± 0.1 Blood urea nitrogen (mg/dl) 8.44 ± 1.42 7.6 ± 1.8 Total cholesterol (mg/dl) 71.15 ± 7.97 76.0 ± 15.9 Creatinine (mg/dl) 0.61 ± 0.20 1.3 ± 0.2 Total globulin (mg/dl) 21.85 ± 4.81 Albumin/globulin ratio 0.93 ± 0.15 1.6 ± 0.2 Glucose (g/dl) 7.20 ± 5.06 2.2 ± 0.2 ALP (IU/l) 114.16 ± 25.59 258.5 ± 240 AST (IU/l) 181.05 ± 72.25 30.4 ± 5.7 ALT (IU/l) 62.40 ± 6.34 58.0 ± 11.0 Ca 2+ (mg/dl) 9.75 ± 0.22 10.7 ± 0.4 K + (mmol/l) 8.97 ± 0.43 5.1 ± 0.8 Cl - (mmol/l) 101.48 ± 2.16 102.4 ± 2.4 Total protein (g/dl) 7.83 ± 0.16 5.8 ± 0.3 Albumin (g/dl) 4.37 ± 2.42 3.5 ± 0.3 Total bilirubin (mg/dl) 0.08 ± 0.02 0.2 ± 0.1 Blood urea nitrogen (mg/dl) 8.25 ± 0.76 8.0 ± 1.2 Total cholesterol (mg/dl) 101.44 ± 4.12 92.4 ± 14.1 Creatinine (mg/dl) 0.46 ± 0.13 1.4 ± 0.1 Total globulin (mg/dl) 36.76 ± 8.18 Albumin/globulin ratio 0.71 ± 0.09 1.6 ± 0.2 Glucose (g/dl) 7.35 ± 2.13 2.3 ± 0.2 ALP (IU/l) 140.62 ± 50.54 306.6 ± 144.5 AST (IU/l) 197.89 ± 115.16 35.2 ± 17.8 ALT (IU/l) 65.15 ± 15.90 54.5 ± 23.3 Ca 2+ (mg/dl) 9.71 ± 0.19 10.8 ± 0.3 K + (mmol/l) 10.15 ± 0.85 5.2 ± 0.5 Cl - (mmol/l) 100.23 ± 0.55 101.7 ± 2.6 ALT, Alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase. 백혈구는염증성질환을반영하는혈액학적지표로서일반적으로사용된다. 본연구에서백혈구의수치는 26.46±2.06 (10 3 /mm 3 ) 로서높은수치를나타내었다. 백혈구수치가높은이유는부검을실 시하지않아원인을판정하기는곤란하지만 Lee 등 [14] 의보고에서처럼돼지는호흡기질환에민감한동물로서정상적인상태를나타낸돼지에서호흡기질환이존재하고있었다고하여아마도본연구에서
24 Hyun-Gu Kang and Sang-Koo Lee Table 4. Prothrombin time and partial thromboplastin time of male and female minipigs Sex Items Minipig Male Female Prothrombin time (sec) 14.5 ± 1.0 Activated partial thromboplastin time (sec) 19.3 ± 5.3 Prothrombin time (sec) 15.1 ± 0.8 Activated partial thromboplastin time (sec) 17.1 ± 4.7 Table 5. Urine analysis values of male and female minipigs Sex Items Minipig Male Female Specific gravity 1.021 ± 0.007 ph 8.3 ± 1.0 Leukocytes (Leuko/µl) 0.0 ± 0.0 * Protein (mg/dl) 6.3 ± 12.5 Ketone (mmol/l) 0.0 ± 0.0 Specific gravity 1.022 ± 0.014 ph 6.4 ± 1.6 Leukocytes (Leuko/µl) 5.0 ± 11.2 Protein (mg/dl) 30.0 ± 67.1 Ketone (mmol/l) 0.0 ± 0.0 * The value "0" means negative in this table. 도병원성세균의감염으로인한것으로생각된다. 또한백혈구감별계산에서중성호성백혈구의수치 가증가되어있는것 (Table 2) 으로보아감염성원인 을뒷받침하고있다. 혈청화학치중에서 ALT 와 AST 는간의질병을 검사하기위한항목이다 [24]. 본연구에서 AST 가 상당히높게나타났으며, ALT 는중등도로높게나 타났다. 이는간성질환이존재하는것으로해석할 수있지만 ALT 가정상범위의혈액학적지표를나타 내어간성질환은배제되었다. 또한 AST 는약물의 처치나골격근질환이존재할때에높게나타난다고 하였으나 creatinine 이정상범위에있는것으로보아 골격근의질환또한배제되었다. 따라서 AST가높게나타난원인을판정하기가곤란하지만 Friendship 등 [25] 의보고에서와같이돼지에서는 AST에대한정상범위가 36~272로넓다고하였으며, Lee 등 [17] 은 AST가일령이증가하면서높게측정되었다고하였으며이에대한원인을추정할수없다고하였다. 본연구결과에서도정상범위있어정상으로간주할수있지만이에대한더많은연구가필요할것으로생각된다. 한편혈액응고검사는출혈환자나술전검사에서기초적인평가에사용되는검사이다 [24]. 하지만지금까지혈액응고검사에대한보고는이루어지고있지않다. 본연구에서 prothrombin time과 activated partial thromboplastin time을측정한결과암컷과수컷에서유의적인차이를인정할수없었으며그범위는 prothrombin time이 14.5~15.1초, activated partial thromboplastin time은 17.1~19.3초이었다. 지금까지미니돼지에서요분석에대한보고는미미한실정이다. 본연구에서요비중은는암컷과수컷에서유의한차이를관찰할수없었다. 하지만암컷의 ph는 6.4±1.6, 수컷의 ph는 8.3으로암컷의 ph는정상의범위에있었으나수컷의 ph는알칼리성을나타내었다. 일반적으로요의 ph는식이에의해영향을받는데본연구에서암컷과수컷모두에동일한사료를급여한것으로보아식이성영향은아닌것으로생각된다. 또한 ph가증가하는원인으로는대사성또는호흡성알칼리증이있다. 본연구에서수컷에서 ph가높은것은이들의원인으로추정할수는있지만정확한원인에대해서는더많은연구와자료가필요할것으로생각된다. 본연구에서실시한요검사항목중수컷에서는백혈구가검출되지않았으나암컷에서는검출되었다. 또한암컷과수컷에서요중단백질이검출되었다. 정상적인상태에서의요에서는혈구및단백질이거의검출되지않아신장기능의이상으로판단할수있으나이의정확한원인을추정하기가곤란하였으며이에대한더많은연구가필요하다고생각된다. 또한이결과는 Lee 등 [17] 의결과와유사하였다.
Hematological evaluation of minipig 25 이상의결과는국내에서생산되어약리 독성분 야에사용되고있는미니돼지의품질관리에문제가 있음을나타내고있다. 따라서실험동물로서미니돼 지를약리 독성시험에사용하고자한다면좋은건강 상태가확보되어야하며 [14, 26, 27], 결과의정확성 에대한신뢰성이인정될것이다. 특히돼지는호흡 기감염에민감하기때문에사육환경및사육관리 에대한품질관리가이루어지는것이시급하다할 수있다. 참고문헌 1. Namara PJ. Interspecies scaling in phamacokinetics. In: Welling PG, Tse FLS, Dighe SV (eds.). Pharmaceutical bioequivalence. New York: Marcel Dekker; 1991. p. 267-300. 2. Anzenbacher P, Soucek P, Anzenbacherova E, Gut I, Hruby K, Svoboda Z, Kvetina J. Presence and activity of cytochrome p450 isoforms in minipig liver microsomes. Comparison with human liver samples. Drug Metab Disp 1998;26:56-59. 3. Gad SC. Pigs and ferrets as models in toxicology and biological safety assessment. Int J Toxicology 2000;19:149-168. 4. Ellegaard Newsletter (August 2006). 2006:vol 25. 5. Logan JS. Prospects for xenotransplantation. Curr Opin Immunol 2000;12(5):563-568. 6. Bertschinger DR, Beknazar E, Simonutti M, Safran AB, Sahel JA, Rosolen SG, Picaud S, Salzmann J. A review of in vivo animal studies in retinal prosthesis research. Graefes Arch Clin Exp Ophthalmol 2008;246(11):1505-1517. 7. MacKenzie DA, Hullett DA, Sollinger HW. Xenogeneic transplantation of porcine islets: an overview. Transplantation 2003;76(6):887-891. 8. Bustad LK, McClellan RO. Miniature swine: development, management, and utilization. Lab Anim Care 1968;18(2):280-287. 9. Shimizu A, Yamada K. Pathology of renal xenograft rejection in pig to non-human primate transplantation. Clin Transplant 2006;15:46-52. 10. Barrow PC. Reproductive toxicity testing for pharmaceuticals under ICH. Reprod Toxicol 2009;28(2):172-179. 11. Skaanild MT. Porcine cytochrome P450 and metabolism. Curr Pharm Des 2006;12:1421-1427. 12. Zuber R, Anzenbacherová E, Anzenbacher P. Cytochromes P450 and experimental models of drug metabolism. J Cell Mol Med 2002;6(2):189-198. 13. Lehmmann H. The minipig in general toxicity. Scand J Lab Anim Sci 1998;25:59-62. 14. Lee SK, Kweon YS, Solis CD, Rho SJ, Kim TH. Single and 28-day repeated dose toxicity studies of coumarin in the minipig. Lab Anim Res 2008;24(3):277-286. 15. Khan M. Minipig: Advantage and disadvantages as a model in toxicity testing. J Am Coll Toxicil 1984;3:337-342. 16. Ikeda G, Friendman L, Hattan DG. The pig as a model in regulatory toxicity testing. Scand J Lab Anim Sci 1998;25(supp l):99-105. 17. Lee MY, Lee SH, Park BK, Yoo HS, Yoon HJ, Kim DY, Park SH, Han HJ. Comparative analysis of various physilolgical and biochemical values between Yucatan micropigs and conventional pigs. Lab Anim Res 2006;22(3):193-201. 18. Svendsen O. The minipig in toxicology. Exp Toxicol Pathol 2006;57(5-6):335-339. 19. Evans RW, Orians CE, Ascher NL. The potential supply of organ donors: an assessment of the efficiency of organ procurent efforts in the United States. JAMA 1992;67:239-246. 20. Lumsden JH, Mullen K. On establishing reference values. Can J Comp Med 1978;42:293-301. 21. Lumsden JH, Mullen K, Rowe R. Hematology and biochemistry reference values for female holstein cattle. Can J Comp Med 1980;44:24-30. 22. Tumbleson ME, Scholl E. Hematology and clinical
26 Hyun-Gu Kang and Sang-Koo Lee chemistry. In: Leman AD (ed.). Diseases of swine. Ames: Iowa State University Press; 1981. p. 27-40. 23. Wilson S, Norton P, Haverson L, Leigh J, Bailey M. Development of the palatine tonsil in conventional and germ-free piglet. Dev Comp Immunol 2005;29(11):977-987. 24. Meyer D, Coles E, Rich L. Veterinary laboratory medicine. 1st ed. Philadelphia: WB Saunders; 1992. 25. Friendship RM, Lumsden JH, McMillan I, Wilson MR. Hematology and biochemistry reference values for Ontario swine. Can J Comp Med 1984;48(4):390-393. 26. Brown RF, Rice P. The Göttingen minipig in pharmacology and toxicology. Pharmacol Toxicol 1997;80:3-4. 27. Jacoby RO, Lindsey JR. Health care for research animals is essential and affordable. FASEB J 1997;11:609-614.