＼ｋｚｋｘｏｔｇｘ｡
Ｙｉｏｋｔｉｋ
Ｐ４＆ ＼ｋｚ４＆ Ｙｉｏ４＆．８６７７／２＆７８．７／２＆ ＆ ８７３８；ＪＵＯ＠＆ ７６４：７：８５ｐ｜ｙ４８６７７４７８４７４８７
Ｘｋｉｋｏ｜ｋｊ＠＆ ７９＆ Ｇｖｘ４＆ ８６７６２＆ Ｇｉｉｋｖｚｋｊ＠＆ ６７＆ Ｐ｛ｒ４＆ ８６７６
Ｕｘｏｍｏｔｇｒ＆Ｇｘｚｏｉｒｋ
*Corresponding author
Tel: +64-7-8348800-7554; Fax: +61-3-97312000 E-mail: Fernanda.Costa@wintec.ac.nz
Ｊｋｚｋｘｓｏｔｇｚｏｕｔ＆ｕｌ＆ｇｔｍｏｕｚｋｔｙｏｔ＆Ｏ３ｉｕｔ｜ｋｘｚｏｔｍ＆ｋｔ｢｡ｓｋ＆ｇｉｚｏ｜ｏｚ｡＆ｏｔ＆ｋｗ｛ｏｔｋ＆
ｈｒｕｕｊ＠＆ｒｇｉｑ＆ｕｌ＆ｇｍｘｋｋｓｋｔｚ＆ｈｋｚ｝ｋｋｔ＆ｓｋｚｎｕｊｙ＆ｕｌ＆ｇｔｇｒ｡ｙｏｙ
Ｓｇｘｏｇ＆Ｌｋｘｔｇｔｊｇ＆ｊｋ＆Ｓ４＆Ｉｕｙｚｇ７２０２＆Ｇｊｘｏｇｔｇ＆Ｑ４＆Ｉｇｘｓｕｔｇ９２＆Ｓｇｘｉｏｕ＆Ｌ４＆Ｓ４＆Ｇｒ｜ｋｙ９２＆Ｚｏｓｕｚｎ｡＆Ｓ４＆Ｘ｡ｇｔ８２＆Ｎｋｒｋｔ＆Ｓ４＆ Ｊｇ｜ｏｋｙ７２＆Ｍｇｘｘ｡＆Ｇ４＆Ｇｔｊｋｘｙｕｔ７２＆Ｘｕｔ＆Ｌ４＆Ｙｒｕｉｕｓｈｋ７
1
Faculty of Veterinary Science, and 2BIO 21 Institute, The University of Melbourne, Victoria 3010, Australia
3
Universidade Federal de São Paulo, São Paulo 04023-900, Brazil
＆＆Ｇｔｍｏｕｚｋｔｙｏｔ３Ｏ＆ｉｕｔ｜ｋｘｚｏｔｍ＆ｋｔ｢｡ｓｋ＆．ＧＩＫ／＆ｏｙ＆ｇ＆ｑｋ｡＆ｘｋｍ｛ｒｇｚｕｘ＆ ｕｌ＆ ｈｒｕｕｊ＆ ｖｘｋｙｙ｛ｘｋ２＆ ｋｒｋｉｚｘｕｒ｡ｚｋｙ＆ ｇｔｊ＆ ｌｒ｛ｏｊ＆ ｎｕｓｋｕｙｚｇｙｏｙ＆ ｚｎｘｕ｛ｍｎ＆ ｉｕｔ｜ｋｘｙｏｕｔ＆ ｕｌ＆ ｇｔｍｏｕｚｋｔｙｏｔ＆ Ｏ＆ ｏｔｚｕ＆ ｇｔｍｏｕｚｋｔｙｏｔ＆ ＯＯ４＆ Ｘｋｉｋｔｚｒ｡２＆ｇ＆ｍｋｔｋｚｏｉ＆ｖｕｒ｡ｓｕｘｖｎｏｙｓ＆ｕｌ＆ｚｎｋ＆ＧＩＫ＆ｍｋｔｋ２＆｝ｎｏｉｎ＆ ｇｉｉｕ｛ｔｚｙ＆ ｌｕｘ＆ ：＝＋＆ ｕｌ＆ ｚｎｋ＆ ｜ｇｘｏｇｚｏｕｔ＆ ｕｌ＆ ＧＩＫ＆ ｇｉｚｏ｜ｏｚ｡＆ ｏｔ＆ ｈｒｕｕｊ２＆ ｎｇｙ＆ ｈｋｋｔ＆ ｇｊ｜ｕｉｇｚｋｊ＆ ｇｙ＆ ｇ＆ ｈｏｕｓｇｘｑｋｘ＆ ｕｌ＆ ｇｚｎｒｋｚｏｉ＆ ｇｖｚｏｚ｛ｊｋ４＆Ｊｏｌｌｋｘｋｔｚ＆ｓｋｚｎｕｊｙ＆ｕｌ＆ｇｔｇｒ｡ｙｏｙ＆ｇｔｊ＆ｊｋｚｋｘｓｏｔｇｚｏｕｔ＆ ｕｌ＆ ＧＩＫ＆ ｇｉｚｏ｜ｏｚ｡＆ ｏｔ＆ ｖｒｇｙｓｇ＆ ｎｇ｜ｋ＆ ｈｋｋｔ＆ ｛ｙｋｊ＆ ｏｔ＆ ｎ｛ｓｇｔ＆ ｇｔｊ＆ ｋｗ｛ｏｔｋ＆ｘｋｙｋｇｘｉｎ＆｝ｏｚｎｕ｛ｚ＆ｇ＆ｉｕｔｙｋｔｙ｛ｙ＆ｕｌ＆ｇ＆ ｍｕｒｊ＆ｙｚｇｔｊｇｘｊ ＆ ｓｋｚｎｕｊ４＆ Ｊｏｌｌｋｘｋｔｚ＆ ｓｋｚｎｕｊｙ＆ ｎｇ｜ｋ＆ ｕｌｚｋｔ＆ ｈｋｋｔ＆ ｛ｙｋｊ＆ ｏｔｚｋｘｉｎｇｔｍｋｇｈｒ｡＆ ｕｘ＆ ｉｏｚｋｊ＆ ｇｙ＆ ｈｋｏｔｍ＆ ｉｕｓｖｇｘｇｈｒｋ＆ ｏｔ＆ ｚｎｋ＆ ｋ～ｏｙｚｏｔｍ＆ｒｏｚｋｘｇｚ｛ｘｋＡ＆ｎｕ｝ｋ｜ｋｘ２＆ｚｎｋ＆ｇｉｚ｛ｇｒ＆ｇｍｘｋｋｓｋｔｚ＆ｈｋｚ｝ｋｋｔ＆ ｇｙｙｇ｡ｙ＆ｎｇｙ＆ｔｕｚ＆ｈｋｋｔ＆ｏｔ｜ｋｙｚｏｍｇｚｋｊ４＆Ｚｎｋｘｋｌｕｘｋ２＆ｏｔ＆ｚｎｏｙ＆ｙｚ｛ｊ｡２＆ ｝ｋ＆ ｋ｜ｇｒ｛ｇｚｋｊ＆ ｚｎｋ＆ ｒｋ｜ｋｒ＆ ｕｌ＆ ｇｍｘｋｋｓｋｔｚ＆ ｈｋｚ｝ｋｋｔ＆ ｚｎｘｋｋ＆ ｊｏｌｌｋｘｋｔｚ＆ ｇｙｙｇ｡ｙ＆ ｛ｙｏｔｍ＆ ｋｗ｛ｏｔｋ＆ ｖｒｇｙｓｇ＆ ｕｈｚｇｏｔｋｊ＆ ｌｘｕｓ＆ ８？＆ ｎｕｘｙｋｙ４＆Ｚ｝ｕ＆ｙｖｋｉｚｘｕｖｎｕｚｕｓｋｚｘｏｉ＆ｇｙｙｇ｡ｙ＆｛ｙｏｔｍ＆Ｌ｛ｘ｡ｒｇｉｘ｡ｒｕ｡ｒ３＆ ｖｎｋｔ｡ｒｇｒｇｔ｡ｒ３ｍｒ｡ｉ｡ｒ３ｍｒ｡ｉｏｔｋ＆ｇｙ＆ｙ｛ｈｙｚｘｇｚｋ＆ｇｔｊ＆ｕｔｋ＆ｌｒ｛ｕｘｏｓｋｚｘｏｉ＆ ｇｙｙｇ｡＆ ｛ｚｏｒｏ｢ｏｔｍ＆ ｕ３ｇｓｏｔｕｈｋｔ｢ｕｏｉ＆ ｇｉｏｊ３ＬＸＱ３．Ｊｔｖ／Ｖ３ＵＮ＆ ｝ｋｘｋ＆ｋｓｖｒｕ｡ｋｊ４＆Ｚｎｋ＆ｘｋｙ｛ｒｚｙ＆ｘｋ｜ｋｇｒｋｊ＆ｚｎｇｚ＆ｚｎｋ＆ｓｋｇｙ｛ｘｋｓｋｔｚｙ＆ ｌｘｕｓ＆ｚｎｋ＆ｊｏｌｌｋｘｋｔｚ＆ｇｙｙｇ｡ｙ＆｝ｋｘｋ＆ｔｕｚ＆ｏｔ＆ｇｍｘｋｋｓｋｔｚ２＆ｏｔｊｏｉｇｚｏｔｍ＆ ｚｎｇｚ＆ ｚｎｋ＆ ｓｋｚｎｕｊｙ＆ ｙｎｕ｛ｒｊ＆ ｔｕｚ＆ ｈｋ＆ ｛ｙｋｊ＆ ｏｔｚｋｘｉｎｇｔｍｋｇｈｒ｡＆ ｌｕｘ＆ ｓｋｇｙ｛ｘｋｓｋｔｚ＆ ｕｌ＆ ｋｗ｛ｏｔｋ＆ ＧＩＫ＆ ｇｉｚｏ｜ｏｚ｡４＆ Ｘｇｚｎｋｘ２＆ ｇ＆ ｙｏｔｍｒｋ＆ ｓｋｚｎｕｊ＆ｕｌ＆ｇｔｇｒ｡ｙｏｙ＆ｙｎｕ｛ｒｊ＆ｈｋ＆ｇｊｕｖｚｋｊ＆ｚｕ＆ｇｉｎｏｋ｜ｋ＆ｉｕｓｖｇｘｇｈｒｋ＆ ｘｋｙ｛ｒｚｙ＆ ｇｔｊ＆ ｉｘｏｚｏｉｇｒ＆ ｇｖｖｘｇｏｙｇｒ＆ ｕｌ＆ ｚｎｋ＆ ｒｏｚｋｘｇｚ｛ｘｋ＆ ｏｙ＆ ｔｋｋｊｋｊ＆ ｝ｎｋｔ＆ ｇｚｚｋｓｖｚｏｔｍ＆ ｚｕ＆ ｉｕｓｖｇｘｋ＆ ｘｋｙ｛ｒｚｙ＆ ｕｈｚｇｏｔｋｊ＆ ｌｘｕｓ＆ ｊｏｌｌｋｘｋｔｚ＆ｇｙｙｇ｡ｙ４
Ｑｋ｡｝ｕｘｊｙ＠ angiotensin I-converting enzyme, assay agreement, biochemistry, equine, methodology
Ｏｔｚｘｕｊ｛ｉｚｏｕｔ＆
Angiotensin I-converting enzyme (ACE) is responsible
The population studied comprised 29 horses, of which 26 (90%) were Thoroughbreds, two (7%) were mixed breeds and one (3%) was a Standardbred. The ages of the horses ranged from 3 to 16 years and there were 16 males (13 geldings and 3 colts) and 13 females. Blood samples (10 mL per horse) were collected between 7 AM and 10 AM by simple venipuncture from one of the jugular veins into heparinized vacuum tubes. The blood was immediately placed and then kept on ice and promptly centrifuged at 3,400 × g for 10 min in a refrigerated centrifuge (GS15R; Beckman, Australia) at 4oC. The resulting plasma was harvested and transferred with sterile disposable plastic pipettes in aliquots into labeled microtubes (Eppendorf, Australia). The sample aliquots were snap-frozen in liquid nitrogen and stored in a −70oC freezer until analysis. Each aliquot was used once, after a single cycle of thawing. Sample aliquots were not thawed and refrozen. From collection to freezing, the process took a maximum of 2 h.
ＧＩＫ＆Ｏｔｌｏｔｏｚ｡＆．ＧＩＫＯｔ／＆ｓｋｚｎｕｊ＆．ｓｋｚｎｕｊ＆７／
This method utilized kinetic spectrophotometry and a commercially available assay kit, ACE Infinity (Thermo- Fisher, USA), developed for ACE measurement in both plasma and serum. This assay is based on the chemical reaction between ACE and the substrate furylacryloyl- phenylalanyl-glycyl-glycine (FAPGG), which causes a decrease in absorbance at 340 nm. The method employs a ready-to-use calibrator and substrate reagents and uses 30 µL of plasma and 300 µL of substrate for each sample analyzed. The linearity of the assay is between 1 and 120 U/L and the sensitivity is described by the manufacturer as 0.084ΔmA/min per U/L. Calibration methodology for the spectrophotometer is provided by the kit manufacturer.
Ｌ｛ｘ｡ｒｇｉｘ｡ｒｕ｡ｒ３ｖｎｋｔ｡ｒｇｒｇｔ｡ｒ３ｍｒ｡ｉ｡ｒ３ｍｒ｡ｉｏｔｋ＆ ．ＬＧＶＭＭ／＆ ｓｋｚｎｕｊ＆．ｓｋｚｎｕｊ＆８／
This method is also a kinetic spectrophotometric method based on the chemical reaction between ACE and FAPGG (F7131; Sigma-Aldrich, Australia). The method utilizes raw chemicals instead of a pre-assembled assay kit. Samples must be run with both blanks and substrate
Fig. 1. Bland and Altman plot showing the limits of agreement (two outer lines), 95% confidence interval of the mean difference (two lines on either side of the mean) and the mean difference between the furylacryloyl-phenylalanyl-glycyl-glycine (FAPGG) method and the angiotensin I-converting enzyme Infinity (ACEIn) method. n = 29.
Fig. 3. Bland and Altman plot showing the limits of agreement (two outer lines), 95% confidence interval of the mean difference (two lines on either side of the mean) and the mean difference between the o-aminobenzoic acid-FRK(Dnp)P-OH (ABZ) method and angiotensin I-converting enzyme Infinity (ACEIn) method. n = 29.
Fig. 2. Bland and Altman plot showing the limits of agreement (two outer lines), 95% confidence interval of the mean difference (two lines on either side of the mean) and the mean difference between the furylacryloyl-phenylalanyl-glycyl-glycine (FAPGG) method and the o-aminobenzoic acid-FRK(Dnp)P-OH (ABZ) method. n = 29.
Ｘｋｙ｛ｒｚｙ
The mean ± SD of 29 samples was 69.0 ± 16.5 U/L for the ACEIn method, 89.8 ± 19.5 U/L for FAPGG method and 85.1 ± 24.8 U/L for the ABZ method. The CV for each method was 4.3% for the ACEIn method, 2.8% for the FAPGG method and 13.6% for the ABZ method. The respective CVw and CVb values were 2.7% and 10.1% for the ACEIn method, 2.5% and 14.5% for the FAPGG method and 9.5% and 14.8% for the ABZ method. Complete ACE activity inhibition was achieved in the ABZ method by adding captopril to the samples before
analysis. Ninety eight percent of the ACE activity was inhibited by EDTA in the ACEIn method, while 97.7% was inhibited in the FAPGG method. The results (Figs. 1-3) showed that measurements of ACE acquired by different methods had poor agreement. For the FAPGG method and ACEIn method, the lower limit of agreement was −8.4 (95%CI −18.2 to 1.4 U/L) and the upper limit of agreement was 49.9 (95%CI 40.2 to 59.7 U/L). For the FAPGG method and ABZ method, the lower limit of agreement was −28.8 (95%CI −40.1 to −17.6 U/L) and the upper limit of agreement was 38.3 (95%CI 27.0 to 49.6 U/L). Finally, for the ABZ method and the ACEIn method, the lower limit of agreement was −26.3 (95%CI −40.6 to −12.1 U/L) and the upper limit of agreement was 58.4 (95%CI 44.2 to 72.6 U/L). The correlation between the difference and average was 0.23 (p = 0.24) for the FAPGG and ACEIn methods, −0.33 (p = 0.08) for the FAPGG and ABZ methods, and 0.44 (p = 0.02) for the ABZ and ACEIn methods. Removal of the observation with the greatest difference between the ABZ method and ACEIn method (difference of 67.6) resulted in the correlation between the difference and average being 0.34 (p = 0.08). Coefficients of determination were 0.45 between the ACEIn method and FAPGG method, 0.26 between the ABZ method and ACEIn method and 0.53 between the ABZ method and FAPGG method. None of the non-linear quadratic coefficients were significant (p values ＞ 0.75).
Ｊｏｙｉ｛ｙｙｏｕｔ
plasma; therefore, these methods should not be used interchangeably. Variation in the mean value of ACE observed with different methods is likely to come from the fact that different protocols (spectrophotometric and fluorimetric) and different substrates (ACEIn and FAPGG vs. ABZ) were used. Nevertheless, a degree of comparability between methods was expected. Judicious analysis of literature utilizing ACE results obtained from one method but utilizing ACE reference values obtained from a second method is necessary. Previous studies on how to determine method agreement for clinical measures have been published and we have followed those guidelines [6,18]. We propose that comparison between results for equine ACE activity obtained from different methods should not be attempted, since this may be misleading. In conclusion, the results of this study demonstrate that different assays have poor agreement and should not be used interchangeably. Accordingly, linear regression should not be used to establish a transformation equation to compare results from different methods, even for clinical purposes. In terms of the precision of each method, the FAPGG method and ACEIn method showed the lowest analytical CV and within-run variation, while the ACEIn method had the lowest between-run coefficient of variation. Overall, the results presented here indicate that it is critical to adhere to a single method of analysis to achieve comparability of samples.
Ｇｉｑｔｕ｝ｒｋｊｍｓｋｔｚｙ
This research was funded by a Joint Collaborative Grant from The University of Melbourne (Australia) and UNIFESP (Brazil), and by The Pathology Section of The Faculty of Veterinary Science at The University of Melbourne.
Ｘｋｌｋｘｋｔｉｋｙ
７４ Ｇｒ｜ｋｙ＆ ＳＬ２＆ Ｇｘｇ｛ｐｕ＆ ＳＩ２＆ Ｐ｛ｒｏｇｔｕ＆ ＳＧ２＆ Ｕｒｏ｜ｋｏｘｇ＆ ＫＳ２＆ Ｑｘｏｋｍｋｘ＆ ＰＫ２＆ Ｉｇｙｇｘｏｔｏ＆ ＪＫ２＆ Ｐ｛ｒｏｇｔｕ＆ Ｒ２＆ Ｉｇｘｓｕｔｇ＆ ＧＱ４＆Ｇ＆ ｉｕｔｚｏｔ｛ｕ｛ｙ＆ｌｒ｛ｕｘｋｙｉｋｔｚ＆ｇｙｙｇ｡＆ｌｕｘ＆ｚｎｋ＆ｊｋｚｋｘｓｏｔｇｚｏｕｔ＆ｕｌ＆ｖｒｇｙｓｇ＆
ｖｒｇｉｋｈｕ３ｉｕｔｚｘｕｒｒｋｊ２＆ ｒｕｔｍ３ｚｋｘｓ＆ ｉｒｏｔｏｉｇｒ＆ ｚｘｏｇｒ４＆ Ｐ＆ ＼ｋｚ＆ Ｉｇｘｊｏｕｒ＆ ７？？？２＆７２＆＝３７＞４
；４ Ｈｋｔｋｚｋｇ｛＆ Ｈ２＆ Ｈｇ｛ｊｏｔ＆ Ｈ２＆ Ｓｕｘｍｇｔｚ＆ Ｍ２＆ Ｍｏｈｕ｛ｊｋｇ｛＆ Ｐ２＆ Ｈｇ｛ｓｇｔｔ＆ ＬＩ４＆Ｇ｛ｚｕｓｇｚｋｊ＆ ｑｏｔｋｚｏｉ＆ ｇｙｙｇ｡＆ ｕｌ＆ ｇｔｍｏｕｚｋｔｙｏｔ３＆ ｉｕｔ｜ｋｘｚｏｔｍ＆ｋｔ｢｡ｓｋ＆ｏｔ＆ｙｋｘ｛ｓ４＆Ｉｒｏｔ＆Ｉｎｋｓ＆７？＞＜２＆９８２＆＞＞：３＞＞＜４ ＜４ Ｈｒｇｔｊ＆ＰＳ２＆Ｇｒｚｓｇｔ＆ＪＭ４＆Ｓｋｇｙ｛ｘｏｔｍ＆ｇｍｘｋｋｓｋｔｚ＆ｏｔ＆ｓｋｚｎｕｊ＆
ｉｕｓｖｇｘｏｙｕｔ＆ｙｚ｛ｊｏｋｙ４＆Ｙｚｇｚ＆Ｓｋｚｎｕｊｙ＆Ｓｋｊ＆Ｘｋｙ＆７？？？２＆＞２＆７９；３＆ ７＜６４
＝４ Ｈｘｇｓ｛ｉｉｏ＆Ｓ２＆Ｓｏｇｔｕ＆Ｇ２＆Ｗ｛ｇｙｙｏｔｚｏ＆Ｒ２＆Ｓｇｉｉｇｘｏ＆Ｋ２＆Ｓ｛ｘｘｏ＆Ｕ２＆ Ｇｓｏｉｏ＆Ｊ４＆Ｖｘｋｙｋｔｉｋ＆ｇｔｊ＆ｉｕｓｖｇｘｏｙｕｔ＆ｕｌ＆Ｇｔｍｏｕｚｋｔｙｏｔ＆ｉｕｔ｜ｋｘｚｏｔｍ＆ ｋｔ｢｡ｓｋ＆ｏｔ＆ｉｕｓｓｋｘｉｏｇｒ＆ｉｋｒｒ＆ｉ｛ｒｚ｛ｘｋ＆ｙｋｘｇ４＆Ｈｏｕｉｎｋｓ＆Ｓｕｒ＆Ｈｏｕｒ＆ Ｏｔｚ＆７？？？２＆：＝２＆７６＝３７７；４
＞４ Ｉｇｘｓｕｔｇ＆ ＧＱ２＆ Ｙｉｎ｝ｇｍｋｘ＆ ＹＲ２＆ Ｐ｛ｒｏｇｔｕ＆ ＳＧ２＆ Ｐ｛ｒｏｇｔｕ＆ Ｒ２＆ Ｙｚ｛ｘｘｕｉｑ＆ＫＪ４＆Ｇ＆ｉｕｔｚｏｔ｛ｕ｛ｙ＆ｌｒ｛ｕｘｋｙｉｋｔｉｋ＆ｘｋｙｕｔｇｔｉｋ＆ｋｔｋｘｍ｡＆ ｚｘｇｔｙｌｋｘ＆ｇｔｍｏｕｚｋｔｙｏｔ＆Ｏ３ｉｕｔ｜ｋｘｚｏｔｍ＆ｋｔ｢｡ｓｋ＆ｇｙｙｇ｡４＆Ｔｇｚ＆Ｖｘｕｚｕｉ＆ ８６６＜２＆７２＆７？＝７３７？＝＜４
？４ Ｉｕｕｓｋｘ＆ＸＶＩ２＆Ｌｕｘｎｋｇｊ＆ＧＰ２＆Ｈｇｚｎｋ＆ＧＶ２＆Ｎｋｇｊ＆ＳＰ４＆Ｖｒｇｙｓｇ＆ ｇｔｍｏｕｚｋｔｙｏｔ３ｉｕｔ｜ｋｘｚｏｔｍ＆ ｋｔ｢｡ｓｋ＆ ．ＧＩＫ／＆ ｉｕｔｉｋｔｚｘｇｚｏｕｔ＆ ｏｔ＆ Ｚｎｕｘｕ｛ｍｎｈｘｋｊ＆ｘｇｉｋｎｕｘｙｋｙ４＆Ｋｗ｛ｏｔｋ＆＼ｋｚ＆Ｐ＆８６６９２＆９；２＆？＜３？＞４ ７６４ Ｉｕｙｚｇ＆ ＳＬＳ４＆Ｇｔｍｏｕｚｋｔｙｏｔ３ｉｕｔ｜ｋｘｚｏｔｍ＆ ｋｔ｢｡ｓｋ＆ ．ＧＩＫ／＆ ｏｔ＆
ｎｕｘｙｋｙ＆ｇｔｊ＆ｏｚｙ＆ｘｋｒｇｚｏｕｔｙｎｏｖ＆ｚｕ＆ｖｋｘｌｕｘｓｇｔｉｋ＆ｇｔｊ＆ｌｏｚｔｋｙｙ４＆Ｖｎ４Ｊ＆ ｊｏｙｙｋｘｚｇｚｏｕｔ４＆ Ｚｎｋ＆ ［ｔｏ｜ｋｘｙｏｚ｡＆ ｕｌ＆ Ｓｋｒｈｕ｛ｘｔｋ２＆ Ｓｋｒｈｕ｛ｘｔｋ２＆ ８６７６４
７７４ Ｉ｛ｙｎｓｇｔ＆Ｊ］２＆Ｉｎｋ｛ｔｍ＆ＮＹ４＆Ｙｖｋｉｚｘｕｖｎｕｚｕｓｋｚｘｏｉ＆ｇｙｙｇ｡＆ｇｔｊ＆ ｖｘｕｖｋｘｚｏｋｙ＆ ｕｌ＆ ｚｎｋ＆ ｇｔｍｏｕｚｋｔｙｏｔ３ｉｕｔ｜ｋｘｚｏｔｍ＆ ｋｔ｢｡ｓｋ＆ ｕｌ＆ ｘｇｈｈｏｚ＆ ｒ｛ｔｍ４＆Ｈｏｕｉｎｋｓ＆Ｖｎｇｘｓｇｉｕｒ＆７？＝７２＆８６２＆７＜９＝３７＜：＞４
７８４ Ｋｒｒｏｙ＆ Ｔ４＆Ｓｕｒｋｉ｛ｒｇｘ＆ ｍｋｔｋｚｏｉ＆ ｉｎｇｘｇｉｚｋｘｏ｢ｇｚｏｕｔ＆ ｕｌ＆ ｚｎｋ＆ ｋｗ｛ｏｔｋ＆ ｇｔｍｏｕｚｋｔｙｏｔ３ｉｕｔ｜ｋｘｚｏｔｍ＆ｋｔ｢｡ｓｋ＆ｍｋｔｋ４＆Ｏｔ＠＆Ｌｇｉ｛ｒｚ｡＆ｕｌ＆＼ｋｚｋｘｏｔｇｘ｡＆ Ｙｉｏｋｔｉｋ４＆ｖｖ４＆９９＞２＆Ｚｎｋ＆［ｔｏ｜ｋｘｙｏｚ｡＆ｕｌ＆Ｙ｡ｊｔｋ｡２＆Ｙ｡ｊｔｋ｡２＆８６６；４ ７９４ Ｌｕｍｇｘｚ｡＆ ＿２＆ Ｌｘｇｙｋｘ＆ ＩＭ２＆ Ｈｘｕ｝ｔｏｔｍ＆ ＳＩＱ４＆Ｏｔｚｘｇ３＆ ｇｔｊ＆
ｏｔｚｋｘ３ｏｔｊｏ｜ｏｊ｛ｇｒ＆ ｜ｇｘｏｇｚｏｕｔ＆ ｕｌ＆ ｙｋｘ｛ｓ＆ ｇｔｍｏｕｚｋｔｙｏｔ＆ ｉｕｔ｜ｋｘｚｏｔｍ＆ ｋｔ｢｡ｓｋ＠＆ｉｒｏｔｏｉｇｒ＆ｏｓｖｒｏｉｇｚｏｕｔｙ４＆Ｇｔｔ＆Ｉｒｏｔ＆Ｈｏｕｉｎｋｓ＆７？＞？２＆８＜２＆ ８６７３８６８４
７：４ Ｌｕｘｎｋｇｊ＆ＧＰ２＆Ｍｏｒｒｋｙｖｏｋ＆ＩＫ２＆Ｌｕ｝ｊｋｔ＆ＧＲ４＆Ｘｕｒｋ＆ｕｌ＆ｉｕｘｚｏｙｕｒ＆ｏｔ＆ ｚｎｋ＆ｕｔｚｕｍｋｔｏｉ＆ｉｕｔｚｘｕｒ＆ｕｌ＆ｖ｛ｒｓｕｔｇｘ｡＆ｇｔｊ＆ｘｋｔｇｒ＆ｇｔｍｏｕｚｋｔｙｏｔ３＆ ｉｕｔ｜ｋｘｚｏｔｍ＆ｋｔ｢｡ｓｋ＆ｏｔ＆ｌｋｚｇｒ＆ｙｎｋｋｖ＆ｔｋｇｘ＆ｚｋｘｓ４＆Ｐ＆Ｖｎ｡ｙｏｕｒ＆８６６６２＆
；８＜２＆：６？３：７＜４
７；４ Ｍｇｔｐｇｓ＆ ＼Ｑ２＆ Ｋ｜ｇｔｙ＆ ＺＰ４＆ Ｋｗ｛ｏｔｋ＆ ｋｔｊｕｓｋｚｘｏｇｒ＆ ｌｏｈｘｕｙｏｙ＆ ｉｕｘｘｋｒｇｚｋｙ＆｝ｏｚｎ＆７７β３ＮＹＪ８２＆ＺＭＬ３β７＆ｇｔｊ＆ＧＩＫ＆ｇｉｚｏ｜ｏｚｏｋｙ４＆Ｓｕｒ＆ Ｉｋｒｒ＆Ｋｔｊｕｉｘｏｔｕｒ＆８６６＜２＆８：＞２＆７６：３７６＞４
７＝４ Ｎ｛ｘｙｚ＆ ＶＲ２＆ Ｒｕ｜ｋｒｒ３Ｙｓｏｚｎ＆ ＩＰ４＆Ｕｖｚｏｓｏ｢ｋｊ＆ ｇｙｙｇ｡＆ ｌｕｘ＆ ｙｋｘ｛ｓ＆ ｇｔｍｏｕｚｋｔｙｏｔ３ｉｕｔ｜ｋｘｚｏｔｍ＆ ｋｔ｢｡ｓｋ＆ ｇｉｚｏ｜ｏｚ｡４＆ Ｉｒｏｔ＆ Ｉｎｋｓ＆ ７？＞７２＆
８＝２＆８６：＞３８６；８４
７＞４ Ｓｇｔｚｎｇ＆Ｙ２＆Ｘｕｏ｢ｋｔ＆ＳＬ２＆Ｌｒｋｏｙｎｋｘ＆ＲＧ２＆Ｚｎｏｙｚｋｊ＆Ｘ２＆Ｌｕｙｙ＆Ｐ４＆
Ｉｕｓｖｇｘｏｔｍ＆ ｓｋｚｎｕｊｙ＆ ｕｌ＆ ｉｒｏｔｏｉｇｒ＆ ｓｋｇｙ｛ｘｋｓｋｔｚ＠＆ ｘｋｖｕｘｚｏｔｍ＆ ｙｚｇｔｊｇｘｊｙ＆ ｌｕｘ＆ Ｈｒｇｔｊ＆ ｇｔｊ＆ Ｇｒｚｓｇｔ＆ ｇｔｇｒ｡ｙｏｙ４＆ Ｇｔｋｙｚｎ＆ Ｇｔｇｒｍ＆ ８６６６２＆？６２＆；？９３＜６８４
７？４ Ｓｕｔｚｍｕｓｋｘ｡＆Ｎ２＆Ｉｒｇｘｑｙｕｔ＆Ｖ２＆Ｈｇｘｔｇｘｊ＆Ｓ２＆Ｈｋｒｒ＆Ｐ２＆Ｈｘ｡ｔｋｙ＆ Ｇ２＆ Ｊｕｒｒｋｘ｡＆ Ｉ２＆ Ｎｇｐｔｇｒ＆ Ｐ２＆ Ｎｋｓｏｔｍ｝ｇ｡＆ Ｎ２＆ Ｓｋｘｉｋｘ＆ Ｊ２＆ Ｐｇｘｓｇｔ＆Ｖ２＆Ｓｇｘｙｎｇｒｒ＆Ｘ２＆Ｖｘｇｙｇｊ＆Ｑ２＆Ｘｇ｡ｙｕｔ＆Ｓ２＆Ｙｇｋｋｊ＆Ｔ２＆ Ｚｇｒｓ｛ｊ＆Ｖ２＆Ｚｎｕｓｇｙ＆Ｒ２＆Ｐ｛ｈｈ＆Ｓ２＆］ｕｘｒｊ＆Ｓ２＆Ｎ｛ｓｖｎｘｏｋｙ＆Ｙ４＆
Ｇｔｍｏｕｚｋｔｙｏｔ３ｉｕｔ｜ｋｘｚｏｔｍ３ｋｔ｢｡ｓｋ＆ ｍｋｔｋ＆ ｏｔｙｋｘｚｏｕｔ５ｊｋｒｋｚｏｕｔ＆ ｖｕｒ｡ｓｕｘｖｎｏｙｓ＆ ｇｔｊ＆ ｘｋｙｖｕｔｙｋ＆ ｚｕ＆ ｖｎ｡ｙｏｉｇｒ＆ ｚｘｇｏｔｏｔｍ４＆ Ｒｇｔｉｋｚ＆ ７？？？２＆９；９２＆；：７３；：；４
８６４ Ｓ｛ｏｘ＆］］＆ＯＯＯ２＆Ｙｇｓｙ＆ＸＧ２＆Ｎ｛ｈｈｋｒｒ＆ＰＧＫ２＆Ｎｏｔｉｎｉｒｏｌｌ＆Ｑ］２＆ Ｍｇｊｇ｝ｙｑｏ＆ Ｐ４＆Ｋｌｌｋｉｚｙ＆ ｕｌ＆ ｋｔｇｒｇｖｘｏｒｇｚ＆ ｕｔ＆ ｉｇｘｊｏｕｘｋｙｖｏｘｇｚｕｘ｡２＆ ｎｋｓｕｊ｡ｔｇｓｏｉ２＆ ｇｔｊ＆ ｎｋｓｇｚｕｒｕｍｏｉ＆ ｜ｇｘｏｇｈｒｋｙ＆ ｏｔ＆ ｋ～ｋｘｉｏｙｏｔｍ＆ ｎｕｘｙｋｙ４＆Ｇｓ＆Ｐ＆＼ｋｚ＆Ｘｋｙ＆８６６７２＆＜８２＆７６６＞３７６７９４
８７４ Ｓ｡ｋｘｙｕｔ＆ Ｙ２＆ Ｎｋｓｏｔｍ｝ｇ｡＆ Ｎ２＆ Ｈ｛ｊｍｋｚ＆ Ｘ２＆ Ｓｇｘｚｏｔ＆ Ｐ２＆ Ｎ｛ｓｖｎｘｏｋｙ＆ Ｙ２＆ Ｓｕｔｚｍｕｓｋｘ｡＆ Ｎ４＆ Ｎ｛ｓｇｔ＆ ｇｔｍｏｕｚｋｔｙｏｔ＆ Ｏ３ｉｕｔ｜ｋｘｚｏｔｍ＆ ｋｔ｢｡ｓｋ＆ ｍｋｔｋ＆ ｇｔｊ＆ ｋｔｊ｛ｘｇｔｉｋ＆ ｖｋｘｌｕｘｓｇｔｉｋ４＆ Ｐ＆ Ｇｖｖｒ＆Ｖｎ｡ｙｏｕｒ＆７？？？２＆＞＝２＆７９７９３７９７＜４
８８４ Ｔｕｉｋｔｚｏ＆ ＳＸ２＆ Ｙｏｓｉｎｕｔ＆ Ｙ２＆ Ｉｏ｢ｋｑ＆ ＲＰ４＆Ｇｔｇｒ｡ｙｏｙ＆ ｕｌ＆ ｚｎｋ＆ ｘｋｔｏｔ３ｇｔｍｏｕｚｋｔｙｏｔ＆ ｙ｡ｙｚｋｓ＆ ｊ｛ｘｏｔｍ＆ ｌｇｙｚｏｔｍ＆ ｏｔ＆ ｇｊ｛ｒｚ＆ ｓｇｒｋ＆ ｘｇｈｈｏｚｙ４＆Ｖｘｕｉ＆Ｙｕｉ＆Ｋ～ｖ＆Ｈｏｕｒ＆Ｓｋｊ＆７？＝；２＆７；６２＆７：８３７：＝４ ８９４ Ｕ－Ｉｕｔｔｕｘ＆ＹＰ２＆Ｌｕ｝ｊｋｔ＆ＧＲ２＆Ｎｕｒｊｙｚｕｉｑ＆Ｔ２＆Ｍｏ｛ｙｙｇｔｏ＆ＪＧ２＆
Ｌｕｘｎｋｇｊ＆ ＧＰ４＆Ｊｋ｜ｋｒｕｖｓｋｔｚｇｒ＆ ｉｎｇｔｍｋｙ＆ ｏｔ＆ ｖ｛ｒｓｕｔｇｘ｡＆ ｇｔｊ＆ ｘｋｔｇｒ＆ｇｔｍｏｕｚｋｔｙｏｔ３ｉｕｔ｜ｋｘｚｏｔｍ＆ｋｔ｢｡ｓｋ＆ｉｕｔｉｋｔｚｘｇｚｏｕｔ＆ｏｔ＆ｌｋｚｇｒ＆
ｇｔｊ＆ｔｋｕｔｇｚｇｒ＆ｎｕｘｙｋｙ４＆Ｘｋｖｘｕｊ＆Ｌｋｘｚｏｒ＆Ｊｋ｜＆８６６８２＆７：２＆：７９３：７＝４ ８：４ Ｘｇｏｓｈｇｉｎ＆ ＹＰ２＆ Ｚｎｕｓｇｙ＆ ＧＲ４＆ Ｘｋｔｏｔ＆ ｇｔｊ＆ ｇｔｍｏｕｚｋｔｙｏｔ＆
ｉｕｔ｜ｋｘｚｏｔｍ＆ｋｔ｢｡ｓｋ＆ｉｕｔｉｋｔｚｘｇｚｏｕｔｙ＆ｏｔ＆ｚｎｋ＆ｌｋｚｇｒ＆ｇｔｊ＆ｔｋｕｔｇｚｇｒ＆ ｍ｛ｏｔｋｇ３ｖｏｍ４＆Ｐ＆Ｖｎ｡ｙｏｕｒ＆７？？６２＆：８９２＆：：７３：；７４
８；４ Ｘｏｍｇｚ＆Ｈ２＆Ｎ｛ｈｋｘｚ＆Ｉ２＆Ｇｒｎｋｔｉ３Ｍｋｒｇｙ＆Ｌ２＆Ｉｇｓｈｏｋｔ＆Ｌ２＆Ｉｕｘ｜ｕｒ＆ Ｖ２＆Ｙｕ｛ｈｘｏｋｘ＆Ｌ４＆Ｇｔ＆ｏｔｙｋｘｚｏｕｔ５ｊｋｒｋｚｏｕｔ＆ｖｕｒ｡ｓｕｘｖｎｏｙｓ＆ｏｔ＆ｚｎｋ＆ ｇｔｍｏｕｚｋｔｙｏｔ＆Ｏ３ｉｕｔ｜ｋｘｚｏｔｍ＆ｋｔ｢｡ｓｋ＆ｍｋｔｋ＆ｇｉｉｕ｛ｔｚｏｔｍ＆ｌｕｘ＆ｎｇｒｌ＆ ｚｎｋ＆｜ｇｘｏｇｔｉｋ＆ｕｌ＆ｙｋｘ｛ｓ＆ｋｔ｢｡ｓｋ＆ｒｋ｜ｋｒｙ４＆Ｐ＆Ｉｒｏｔ＆Ｏｔ｜ｋｙｚ＆７？？６２＆＞＜２＆ ７９：９３７９：＜４
８＜４ Ｘｕｔｉｇ３Ｚｋｙｚｕｔｏ＆ Ｙ４＆ Ｊｏｘｋｉｚ＆ ｙｖｋｉｚｘｕｖｎｕｚｕｓｋｚｘｏｉ＆ ｇｙｙｇ｡＆ ｌｕｘ＆ Ｇｔｍｏｕｚｋｔｙｏｔ３ｉｕｔ｜ｋｘｚｏｔｍ＆ｋｔ｢｡ｓｋ＆ｏｔ＆ｙｋｘ｛ｓ４＆Ｉｒｏｔ＆Ｉｎｋｓ＆７？＞９２＆
８？２＆７６？９３７６？＜４
８＝４ Ｙｇｈｇｚｏｔｏ＆ ＸＧ２＆ Ｈｋｘｙｇｔｋｚｚｏ＆ ＶＧ２＆ Ｌｇｘｏｇｙ＆ ＹＲ２＆ Ｐ｛ｒｏｇｔｕ＆ Ｒ２＆ Ｐ｛ｒｏｇｔｕ＆ ＳＧ２＆ Ｉｇｙｇｘｏｔｏ＆ ＪＫ２＆ Ｉｇｘｓｕｔｇ＆ ＧＱ２＆ Ｖｇｏ｜ｇ＆ ＧＩＳ２＆ Ｖｋｙｗ｛ｋｘｕ＆ ＰＨ４＆Ｊｋｚｋｘｓｏｔｇｚｏｕｔ＆ ｕｌ＆ ｇｔｍｏｕｚｋｔｙｏｔ＆ Ｏ３ｉｕｔ｜ｋｘｚｏｔｍ＆ ｋｔ｢｡ｓｋ＆ｇｉｚｏ｜ｏｚ｡＆ｏｔ＆ｉｋｒｒ＆ｉ｛ｒｚ｛ｘｋ＆｛ｙｏｔｍ＆ｌｒ｛ｕｘｋｙｉｋｔｉｋ＆ｘｋｙｕｔｇｔｉｋ＆ ｋｔｋｘｍ｡＆ｚｘｇｔｙｌｋｘ＆ｖｋｖｚｏｊｋｙ４＆Ｇｔｇｒ＆Ｈｏｕｉｎｋｓ＆８６６＝２＆９＜９２＆８；；３８＜８４ ８＞４ Ｙｉｎ｝ｋｏｙｌ｛ｘｚｎ＆ Ｎ２＆ Ｙｉｎｏöｈｋｘｍ３Ｙｉｎｏｋｍｔｏｚ｢＆ Ｙ４＆ Ｇｙｙｇ｡＆ ｇｔｊ＆ ｈｏｕｉｎｋｓｏｉｇｒ＆ ｉｎｇｘｇｉｚｋｘｏ｢ｇｚｏｕｔ＆ ｕｌ＆ ｇｔｍｏｕｚｋｔｙｏｔ３Ｏ３ｉｕｔ｜ｋｘｚｏｔｍ＆ ｋｔ｢｡ｓｋ＆ｏｔ＆ｉｋｘｋｈｘｕｙｖｏｔｇｒ＆ｌｒ｛ｏｊ４＆Ｋｔ｢｡ｓｋ＆７？＞：２＆９８２＆７８３７？４ ８？４ Ｚｏｒｒｓｇｔ＆ ＲＭ２＆ Ｓｕｕｘｋ＆ ＰＴ４＆Ｙｋｘ｛ｓ＆ ｇｔｍｏｕｚｋｔｙｏｔ＆ ｉｕｔ｜ｋｘｚｏｔｍ＆
ｋｔ｢｡ｓｋ＆ ｇｉｚｏ｜ｏｚ｡＆ ｇｔｊ＆ ｘｋｙｖｕｔｙｋ＆ ｚｕ＆ ｇｔｍｏｕｚｋｔｙｏｔ＆ Ｏ＆ ｏｔ＆ ｎｕｘｙｋｙ４＆ Ｋｗ｛ｏｔｋ＆＼ｋｚ＆Ｐ＆７？＞？２＆８７＆．Ｙ｛ｖｖｒ＆＝／２＆ｙ＞６３＞９４
