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1 5 FUJIFILM RESEARCH & DEVELOPMENT (No. 56-2011) 111 1 1,2- A&M Steiner v-LIP-P Development of FUJI DRI-CHEM v-LIP-P Slide that Has the High Specificity to Pancreatic LipaseKentaro NAKAMURA*, Shigeki KAGEYAMA*, Hideaki TANAKA**, Kazuya KAWASAKI*, and Kaoru TERASHIMA*AbstractThe lipase of blood is mainly a deviation enzyme from pancreas, and its activity is a highly evaluated index for pancreatitis diagnosis in human medical care.

2 However, the sensitivity and specificity for the pancreatitis of the blood lipase activity is low in the veterinarian medical care for dogs and cats , and making rapid diagnosis has been difficult to conduct. The Pancreatic Lipase Immunoreactivity (PLI), a blood diagnosis method, is the most superior in sensitivity and specificity as a pancreatitis marker for the dogs and cats at present. However, because PLI is not easy and rapid, there is a strong need for the development of an easy and rapid method of pancreatitis screening. FUJI DRI-CHEM (FDC), a dry chemistry method, is a system that enables measurement of various enzymic activities in the blood easily and rapidly, which has widely been used in veterinary care. Now we have developed the FDC v-LIP-P Slide having high specificity to measurable pancreas lipase. The measurement principle we used is the triglyceride method. The specificity for the pancreas lipase has also been improved by prescribing colipase, bile salt and sodium dodecyl benzene sulphonate, which is an anionic detergent.

3 In addition, the reactivity has been improved by prescribing triolein, an emulsified substrate, to secure the area of the oil-water interface for the reaction to the pancreatic lipase. It was confirmed that the correlation of FDC v-LIP-P Slide and PLI was excellent. This shows that this slide is a very effective diagnostic product for the rapid screening of pancreatitis. 2010 12 2 * R&D 258-8577 577 * Pharmaceutical & Healthcare Research Laboratories Research & Development Management Headquaters FUJIFILM Corporation Ushijima, Kaisei-machi, Ashigarakami-gun, Kanagawa 258-8577, Japan ** 250-0193 210 ** Imaging Materials Production Division Kanagawa Factory FUJIFILM Corporation Nakanuma, Minamiashigara, Kanagawa 250-0193, Japan6 v-LIP-P PLI PLI 2 3 DGGR 1.

4 2- 4 FDC 211 2111 211111 FDC Table 1 1,2- 5 6 7 DGGR 4 8 DGGR Table 1 The list of the lipase measurement principles. EK 7 IDEXX 1,2- 5 6 1 DGGR 4 2 LPL 8 1 DGGR 211121 DGGR 1,2-O- -rac- -3- - 6 - - 1,2-O- -rac- - 6 - - 580nm Fig.

5 1 5 DGGR DGGR pH 1 pH pH pH 8 NMR MASS 2 pH pH pH pH 8 FDC pH 8 DGGR FDC pH 8 45 C 2 3 DGGR -( 6 - )- OH ONOCH3 O-O(CH2)3 OOO-+ =580nm OOORRO(CH2)3 OONOCH3O OH OOORRHO(CH2)3 OONOCH3OO-+1,2-O- -rac- -3- -(6 - )- Fig. 1 Reaction scheme of the DGGR RESEARCH & DEVELOPMENT (No. 56-2011) 1 2121 2121115 11kDa 9 10 lid lid Fig.

6 2 11 12 - - DGGR Substrate emalsioncolipas eBil e saltPancreatic lipas eC-terminal domeinN-terminal domein Lid domeinSubstrate emulsioncolipas eBil e saltPancreatic lipas eC-terminal domeinN-terminal domein Lid domeinFig. 2 Schematic view of the reaction of the pancreas 212121 Fig. 3 Lipase, ColipaseTriolein + 2H2O 2-Monoglyceride + 2 Oleic acidMGLP2-Monoglyceride + H2O Glycerol + Oleic acidGK, MgCl2 Glycerol + ATP L- -glycerophosphate+ ADPGPO L- -glycerophosphate+ O2H2O2 + DihydroxyacetonephosphatePODD iarylimidazoleleucodye + H2O2 Blue color dye + 2H2 OLipase, ColipaseTriolein + 2H2O 2-Monoglyceride + 2 Oleic acidMGLP2-Monoglyceride + H2O Glycerol + Oleic acidGK, MgCl2 Glycerol + ATP L- -glycerophosphate+ ADPGPO L- -glycerophosphate+ O2H2O2 + DihydroxyacetonephosphatePODD iarylimidazoleleucodye + H2O2 Blue color dye + 2H2 OLipase, ColipaseTriolein + 2H2O 2-Monoglyceride + 2 Oleic acidMGLP2-Monoglyceride + H2O Glycerol + Oleic acidGK.

7 MgCl2 Glycerol + ATP L- -glycerophosphate+ ADPGPO L- -glycerophosphate+ O2H2O2 + DihydroxyacetonephosphatePODD iarylimidazoleleucodye + H2O2 Blue color dye + 2H2 OFig. 3 Reaction scheme of the FDC v-LIP-P Slide. DGGR r = DGGR II DGGR Fig. 4a Colipase 1 SDBS 0 =46 y = - = (H7180,U/L)FDC v-LIP-P (U/L)r = .Colipase 1 SDBS 1 (n=46)y = - = 015001800 DGGR (H7180,U/L)FDC v-LIP-P (U/L)r = .Fig. 4 Canine sample correlation of FDC v-LIP-P Slide and DGGR method (a. without sodium dodecylbenzene sulphonate (SDBS), b. with SDBS). 13 14 15 r = Fig.

8 4b 1 212121 8 v-LIP-P 200nm 800U/L 40U/L 30 Table 2 Table 2 Reactivity comparison by difference of state of substrate (triolein) in FDC v-LIP-P Slide. ODr/min 100 100 130 2121 FDC v-LIP-P Fig. 5 Sprea ding layer Triolein emulsio n(Triorein, Sodium Dodecylb enzene Sulphonate,Gelatine, Polyv inylp yrrolidone)ColipaseBile acidSodium Dodecylbenzene SulphonateMonoglyceride lip aseCalcium chloride Reagent layer PolymerGlycerol kinase, Magnesium chlorideAdenosine triphosphate disodium saltGlycerol-3-phosphate oxidasePeroxidaseDiarylimidazole leuco dye Transparent support PET filmFig.

9 5 Structure of FDC v-LIP-P Slide. 2 FDC v-LIP-P 2- ATP Mg2+ L- - L- - -3- 650nm OD FDC1v-LIP-P 211 2111 FDC v-LIP-P PLI Spec cPL IDEXX PLI LIPA IDEXX 1,2- 1,2- DGGR Fig. 6 IDEXX N=40 r = 060 080 0 PLI ( ) /L)IDEXX LIP U/L)FD C v-LIP-P (N =40)r= 040 060 080 0020 040 0600800 PLI g/L)FDC v-LIP-P U/L).

10 1, 2- N=24) = 040 060 080 0100012001400020 040 060 080 0 PLI ( ) /L)Wako LIP U/L)DGGR N=40)r = 040 060 080 0 PLI ( ) /L)Roche LIP U/L) ..Fig. 6 Canine sample correlation of PLI and various measurement of the lipase activity (a. FDC v-LIP-P Slide (dry chemistry), b. Diacetinase method (dry chemistry), c. DGGR method (wet chemistry), ,2-Diglyceride method (wet chemistry)).9 FUJIFILM RESEARCH & DEVELOPMENT (No. 56-2011) FDC v-LIP-P PLI r FDC r DGGR r 1,2- r DGGR PLI FDC v-LIP-P PLI 1,2- PLI DGGR FDC v-LIP-P PLI DGGR FDC v-LIP-P Fig.