Simulation of Electromagnetic Heating of Cryopreserved SAMPLES
THE CONSTRUCTION OF A REPRESENTATIVE HUMAN CRYOPRESERVED HEPATOCYTE POOL FOR METABOLISM STUDY...
1
THE CONSTRUCTION OF A REPRESENTATIVE HUMAN CRYOPRESERVED HEPATOCYTE POOL FOR METABOLISM STUDY Zhihong Zhang O’Brien* , Troy Bremer, Kevin Holme and Yong Hee Lee* 1 In Vitro System, Lion Bioscience, San Diego, CA 92121 Due to the extensive inter-individual difference and polymorphic distribution of liver enzymes, it is necessary to construct a representative human cryopreserved hepatocyte (HCH) pool for metabolism study. To obtain a representative HCH pool, enzyme activity database of total 51 donors (In Vitro Technology (IVT)) was analyzed using statistical tools. Due to the non-normal distribution of all isozymes from vendor database, a median value, its 99% confidence interval (CI) and confidence intervals of 67% typical population for each isozyme were defined. To select a representative pool and minimize the pool-to-pool variation, the following criteria will be applied: 1) at least 80% of individual isozyme activity values must be within their corresponding acceptable activity range; 2) no more than 30-35% of each enzyme activity value/X donors should be outside the acceptable activity range; and 3) mean activity for each enzyme /pool should fall into 99% CI of its corresponding median value. By doing so, it is expected to result in approximate one- to three-fold (except 4-fold for CYP1A2 and 6-fold for CYP2C19) variations of isozyme activities among pools. Thirty 6-donor pools were constructed based on the selection criteria described above, and four of them were tested for isozyme activities using 11 functional probe substrates. Our results showed that: 1) good correlation (0.8slope1.3 and R 2 0.8) is achieved between the mean of each isozyme activity per pool and median values of each enzyme in the database; 2) enzyme activity changing trends of the virtual HCH pools constructed through IVT database are compatible with our actual results; and 3) one- to three-fold isozyme activity differences are observed among pools. ABSTRACT INTRODUCTION METHODS RESULTS CONCLUSION Hepatocyte has been recognized as one of the most powerful in vitro tools in metabolism and toxicity studies. The advent of cryopreservation technologies opened a new field for using hepatocyte more conveniently and wisely, especially for human hepatocyte. Cryopreservation techniqe circumvents the huge waste of freshly isolated human hepatocytes due to the lack of appropriate storage, and provides a tremendous opportunity to minimize the inter-individual differences and polymorphic distribution of liver enzymes. To maintain the consistency of liver enzyme activities and reduce the inter-indivadual enzyme differences in hepatocyte system used in each study, it is better to construct a uniformed hepatocyte pool. However, it is impossible to have a same generic pool for each indivadual study. Then, what would be the reasonable way for the hepatocyte pool construction? Up to now, there hasn’t been any strong interest shown in how to construct the best pool although almost everyone realizes the importance of doing so. Recently, our lab started with a total of 51 human cryopreserved hepatocyte donor database published by In Vitro Technology (IVT). To initiate the investigation, the enzyme activity database of 51 donors was analyzed by using statistical tools. Based on the statistical results, hepatocyte pool selection criteria was established. Thirty-four 6-donor pools were constructed according to the selection criteria and four of them were actually tested in our lab for isozyme activities using 11 functional marker compounds. This study provides a basis for obtaining a representative human cryopreserved hepatocyte (HCH) pool to reduce the chance of causing enzyme activity variation during each study. Table 1. IVT 51-donor enzyme activity database Conventional pool construction for liver fractions Conventional pool construction for liver fractions • Human microsome and S9: 10 to 15 donors as an optimum pool • Human cryopreserved hepatocyte: Not addressed yet Advantages for construction of a representative human cryopreserved hepatocyte Advantages for construction of a representative human cryopreserved hepatocyte pool pool • Reduce the interindividual variations in human liver enzymes • Make experimental results more comparible between studies or labs • Provide experimental data more representation of a “generic human” Study Design: (1) Statistical analysis of IVT 51-donor database to establish the pool selection criteria: • Determine type of distribution of 51-donors for each isozyme • Define median value and its 99% confidence interval (CI) for each isozyme • Define confidence intervals of 67% typical population for each isozyme (2) Construction of thirty-four HCH pools based on the pool selection criteria (3) Actual pool comparisons by measuring isozyme activities using 11 functional markers Table 2. Eleven functional marker compounds Substrate ID Substrate C onc.#1 ( M) a M etabolites M onitored Detection Phenacetine (PH EN ) 250 Acetaminophen LC/M S/M S C oum arin (C O UM) 100 7-Hydroxycoum arin LC/U V Tolbutam ide (TO LB) 500 OH-Tolbutam ide LC/M S/M S (S)-M ephenytoin (M EPH) 250 OH-m ehpenytoin LC/M S/M S D extrom ethorphan (D EX) 50 D extrorphan LC/M S/M S C hlorzoxazone (C ZX) 250 O H -C hlorzoxazone LC/M S/M S M idazolam (M ID) 10 1’-and 4’-O H M idazolam LC/M S/M S 7-H ydroxycoum arin (7-H C ) 250 7-Hydroxycoum arin glucuronide & 7- hydroxycoum arin sulfate LC/U V M ethylum belliferone (M UF) 500 Methylum belliferone glucuronide & methylumbelliferone sulfate LC/U V Trans-4-phenyl-3-butene-2- one (TPBO ) 500 Trans-4-phenyl-3-butene- 2-one glutathione conjugate LC/M S/M S 7-Ethoxycoum arin (7-EC ) 75 7-Hydroxycoum arin 7-Hydroxycoum arin glucuronide 7-Hydroxycoum arin sulfate LC/U V a This saturated concentration (>> Km )w as used forthe m easurem entofm etabolite form ation rates. (1) Statistical analysis of IVT 51-donor database to establish the pool selection criteria: Table 3. Median confidence range and acceptable activity range of indivadual isozyme Substrate: COUM DEX ECOD 7-H CG 7-HCS M EPH TEST TO LB PHEN CZX Enzym e: 2A6 2D 6 2E1 GT ST 2C 19 3A4 2C 9 1A2 2E1 M edian confidence range: 30-60 12-27 25-41 50-117 8-20 3-18 49-113 13-34 8-33 23-36 A cceptable activity range: 21-76 9-33 11-43 8-184 5-27 1-31 25-147 10-46 4-43 20-47 Donor selection criteria: • At least 80% of individual isozyme activity values must be within their corresponding acceptable range. • No more than 30-35% of each enzyme activity value/X donors should be outside the acceptable activity range. • Mean activity for each isozyme/pool should fall into its corresponding median confidence range. • The donor selection criteria based on the statistical analysis successfully provides a basis for obtaining a representative human cryopreserved hepatocyte (HCH) pool. (2) Construction of thirty-four HCH pools based on the pool selection criteria: M edian vs. Pool#1 y = 1.0955x R 2 = 0.8451 0 20 40 60 80 100 120 0 20 40 60 80 Median M edian vs. P ool#2 y = 1.0741x R 2 = 0.9568 0 20 40 60 80 100 0 50 100 Median M edian vs. Pool#3 y= 1.0236x R 2 = 0.8183 0 20 40 60 80 100 0 20 40 60 80 Median M edian vs. P ool#4 y= 1.113x R 2 = 0.977 0 20 40 60 80 100 0 20 40 60 80 Median M edian vs. Pool#5 y = 0.9512x R 2 = 0.9269 0 20 40 60 80 100 0 50 100 Median M edian vs. Pool#6 y = 1.157x R 2 = 0.9589 0 20 40 60 80 100 0 20 40 60 80 Median M edian vs. Pool#7 y = 1.1278x R 2 = 0.9026 0 20 40 60 80 100 0 20 40 60 80 Median M edian vs. P ool#8 y = 0.9709x R 2 = 0.9767 0 20 40 60 80 100 0 20 40 60 80 Median M edian vs. Pool#9 y= 1.1132x R 2 = 0.9589 0 20 40 60 80 100 0 20 40 60 80 Median M edian vs. P ool#10 y= 1.0977x R 2 = 0.9529 0 20 40 60 80 100 0 20 40 60 80 Median M edian vs. P ool#11 y = 0.9571x R 2 = 0.9656 0 20 40 60 80 100 0 20 40 60 80 Median M edian vs. Pool#12 y = 1.0995x R 2 = 0.928 0 20 40 60 80 100 0 20 40 60 80 Median M edian vs. P ool#13 y = 1.0028x R 2 = 0.9853 0 20 40 60 80 100 0 20 40 60 80 Median M edian vs. Pool#14 y= 1.0745x R 2 = 0.9551 0 20 40 60 80 100 0 50 100 Median M edian vs. Pool#15 y= 1.0309x R 2 = 0.948 0 20 40 60 80 100 0 20 40 60 80 Median M edian vs. P ool#16 y= 1.094x R 2 = 0.9616 0 20 40 60 80 100 0 20 40 60 80 Median M edian vs. P ool#17 y = 1.1506x R 2 = 0.9247 0 20 40 60 80 100 0 20 40 60 80 Median M edian vs. P ool#18 y = 1.2552x R 2 = 0.9595 0 20 40 60 80 100 0 20 40 60 80 Median M edian vs. P ool#19 y= 1.1318x R 2 = 0.9698 0 20 40 60 80 100 0 20 40 60 80 Median M edian vs. Pool#20 y= 1.2041x R 2 = 0.9427 0 20 40 60 80 100 0 20 40 60 80 Median M edian vs. Pool#21 y= 0.9706x R 2 = 0.9428 0 20 40 60 80 100 0 20 40 60 80 Median M edian vs. P ool#22 y= 0.944x R 2 = 0.9236 0 20 40 60 80 100 0 20 40 60 80 Median M edian vs. Pool#23 y= 1.0345x R 2 = 0.9331 0 20 40 60 80 100 0 50 100 Median M edian vs. Pool#24 y= 0.9118x R 2 = 0.9722 0 20 40 60 80 100 0 20 40 60 80 Median M edian vs. Pool#25 y = 1.0338x R 2 = 0.9 0 20 40 60 80 100 0 50 100 Median M edian vs. Pool#26 y= 1.0745x R 2 = 0.8868 0 20 40 60 80 100 0 50 100 Median M edian vs. Pool#31(Lion M irrorPool#1) y = 1.2382x R 2 = 0.9322 0 20 40 60 80 100 0 20 40 60 80 Median M edian vs. Pool#32 (Lion M irrorPool#2) y= 1.1918x R 2 = 0.9596 0 20 40 60 80 100 0 20 40 60 80 Median M edian vs. Pool#33 (Lion M irrorPool#3) y= 1.0798x R 2 = 0.9443 0 10 20 30 40 50 60 70 80 90 100 0 50 100 Median M edian vs. Pool#34 (Lion M irrorPool#4) y= 0.7867x R 2 = 0.9013 0 20 40 60 80 100 0 20 40 60 80 Median (3) Actual pool comparisons by measuring isozyme activities using 11 functional markers: Mirror#1vs.Mirror#2 y = 1.0151x R 2 = 0.9869 0 200 400 600 800 1000 0 500 1000 E nzyme activity (pm ole/min/E06 cells) in M irror#1 M irror#1vs. M irror#3 y = 0.4497x R 2 = 0.9383 0 100 200 300 400 500 0 500 1000 E nzym e activity (pmole/m in/E 06 cells) in M irror#1 M irror#1vs.Mirror#4 y = 0.8036x R 2 = 0.9836 0 200 400 600 800 1000 0 500 1000 E nzyme activity(pm ole/min/E 06 cells) in M irror#1 Lot# V iability COUM DEX ECOD 7-HC G 7-HCS M EPH TEST TOLB PHEN CZX G ender 2A6 2D6 2E1 GT ST 2C19 3A4 2C9 1A2 2E1 51 65 55 9 17 47 7 1 115 10 6 23 F 70 74 36 4 22 88 33 12 55 32 43 40 M 83 59 29 24 42 190 25 21 74 39 33 30 M 86 74 51 23 10 50 9 1 38 13 0 18 F 87 71 34 36 43 81 35 3 56 50 4 64 F 89 77 25 21 8 23 6 1 11 13 0 29 F 90 74 30 25 7 13 0 2 19 15 0 16 F 91 73 13 29 66 44 10 12 252 36 27 36 F 94 67 41 12 37 24 4 21 126 40 19 87 F 98 72 75 5 24 57 12 9 45 25 12 32 F 99 86 21 15 7 4 0 1 60 10 0 22 F 102 81 42 40 27 8 13 1 7 6 6 37 M 104 81 63 21 44 247 25 2 58 37 8 20 M 105 67 59 15 24 38 14 1 29 27 12 27 M 107 81 60 28 24 21 0 16 234 41 7 48 F 110 77 45 24 35 23 4 6 206 11 54 36 F 111 71 4 10 9 2 3 3 147 2 19 19 F 113 60 15 47 34 16 0 4 151 51 25 74 F 114 75 39 23 21 10 5 5 59 TBD 3 45 F 117 85 42 16 21 60 25 16 113 45 8 25 F 118 65 81 34 56 208 29 26 208 51 39 13 F 122 70 26 30 29 80 5 1 42 23 4 25 M 129 90 4 24 27 67 18 1 16 33 10 51 F 133 82 67 16 24 129 19 27 96 11 24 32 F ACU 81 53 8 25 74 18 8 80 16 4 23 F CPN 81 28 6 40 100 13 2 168 14 37 21 M DRL 84 25 7 27 100 10 4 43 31 37 31 M EFA 69 9 9 35 47 5 18 66 12 47 67 M ENR 73 69 28 27 124 31 107 77 36 38 32 M EQB 88 14 11 18 65 9 31 49 14 21 35 M ETR 88 30 1 34 13 13 7 13 13 37 69 F EVY 80 2 20 23 218 77 33 24 25 17 38 M GAL 64 52 7 27 59 6 3 94 13 57 23 F GIU 84 87 15 29 55 13 171 86 28 20 33 M GNG 70 57 17 33 54 8 5 95 22 16 22 F GUY 92 65 12 11 73 12 20 90 13 5 8 M HRK 63 69 11 55 124 12 23 136 34 64 22 F IEM 88 34 17 23 129 34 72 48 23 19 34 M KM I 74 32 11 29 192 18 6 69 9 14 18 M KPT 83 9 12 32 248 30 55 65 26 56 32 F KRM 78 126 36 55 83 17 103 98 46 74 44 F KTG 94 135 40 86 111 25 12 60 32 74 66 M M AN 97 31 12 35 119 32 3 40 14 6 34 M MQF 91 79 17 29 10 12 2 85 5 7 46 F M YO 94 40 24 9 24 0 0 12 7 0 11 F NQT 85 76 12 39 80 23 2 151 14 20 34 M OCF 82 91 16 26 98 17 52 147 33 40 32 F OQD 94 80 13 39 233 26 10 135 11 14 28 M PFM 87 21 1 11 67 10 3 116 8 15 33 F RKB 63 42 16 16 100 8 8 36 17 20 19 F RNG 91 119 14 97 298 27 177 207 34 71 41 F Mirror#1vs.M irror#2 y = 0.9466x R 2 = 0.9348 0 40 80 120 0 40 80 120 E nzym e activity in m irror#1 (pm ole/m in/m illion cells) M irror#1vs.M irror#4 y = 0.8481x R 2 = 0.858 0 40 80 120 0 40 80 120 Enzym e activity in m irror #1(pm ole/min/m illion cells) M irror#1vs.M irror#3 y = 0.6156x R 2 = 0.7738 0 40 80 120 0 40 80 120 E nzym e activity in m irror#1 (pm ole/m in/m illion cells) • Good correlation (0.8slope1.3 and R 2 0.8) is achieved between the mean of each isozyme activity per pool and median values of each enzyme in the database. • One- to three-fold isozyme activity differences are observed among pools. • Enzyme activity changing trends of the virtual HCH pools constructed through IVT database are compatible with our actal results. Enzyme activity values are based on IVT database Enzyme activity values are based on Lion actual experimental data
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Transcript of THE CONSTRUCTION OF A REPRESENTATIVE HUMAN CRYOPRESERVED HEPATOCYTE POOL FOR METABOLISM STUDY...
THE CONSTRUCTION OF A REPRESENTATIVE HUMAN CRYOPRESERVED HEPATOCYTE POOL FOR METABOLISM STUDYZhihong Zhang O’Brien*, Troy Bremer, Kevin Holme and Yong Hee Lee*1
In Vitro System, Lion Bioscience, San Diego, CA 92121
THE CONSTRUCTION OF A REPRESENTATIVE HUMAN CRYOPRESERVED HEPATOCYTE POOL FOR METABOLISM STUDYZhihong Zhang O’Brien*, Troy Bremer, Kevin Holme and Yong Hee Lee*1
In Vitro System, Lion Bioscience, San Diego, CA 92121
Due to the extensive inter-individual difference and polymorphic distribution of liver enzymes, it is necessary to construct a representative human cryopreserved hepatocyte (HCH) pool for metabolism study. To obtain a representative HCH pool, enzyme activity database of total 51 donors (In Vitro Technology (IVT)) was analyzed using statistical tools. Due to the non-normal distribution of all isozymes from vendor database, a median value, its 99% confidence interval (CI) and confidence intervals of 67% typical population for each isozyme were defined. To select a representative pool and minimize the pool-to-pool variation, the following criteria will be applied: 1) at least 80% of individual isozyme activity values must be within their corresponding acceptable activity range; 2) no more than 30-35% of each enzyme activity value/X donors should be outside the acceptable activity range; and 3) mean activity for each enzyme /pool should fall into 99% CI of its corresponding median value. By doing so, it is expected to result in approximate one- to three-fold (except 4-fold for CYP1A2 and 6-fold for CYP2C19) variations of isozyme activities among pools. Thirty 6-donor pools were constructed based on the selection criteria described above, and four of them were tested for isozyme activities using 11 functional probe substrates. Our results showed that: 1) good correlation (0.8slope1.3 and R20.8) is achieved between the mean of each isozyme activity per pool and median values of each enzyme in the database; 2) enzyme activity changing trends of the virtual HCH pools constructed through IVT database are compatible with our actual results; and 3) one- to three-fold isozyme activity differences are observed among pools.
ABSTRACTABSTRACT
INTRODUCTIONINTRODUCTION
METHODSMETHODS
RESULTSRESULTS
CONCLUSIONCONCLUSION
Hepatocyte has been recognized as one of the most powerful in vitro tools in metabolism and toxicity studies. The advent of cryopreservation technologies opened a new field for using hepatocyte more conveniently and wisely, especially for human hepatocyte. Cryopreservation techniqe circumvents the huge waste of freshly isolated human hepatocytes due to the lack of appropriate storage, and provides a tremendous opportunity to minimize the inter-individual differences and polymorphic distribution of liver enzymes. To maintain the consistency of liver enzyme activities and reduce the inter-indivadual enzyme differences in hepatocyte system used in each study, it is better to construct a uniformed hepatocyte pool. However, it is impossible to have a same generic pool for each indivadual study. Then, what would be the reasonable way for the hepatocyte pool construction? Up to now, there hasn’t been any strong interest shown in how to construct the best pool although almost everyone realizes the importance of doing so. Recently, our lab started with a total of 51 human cryopreserved hepatocyte donor database published by In Vitro Technology (IVT). To initiate the investigation, the enzyme activity database of 51 donors was analyzed by using statistical tools. Based on the statistical results, hepatocyte pool selection criteria was established. Thirty-four 6-donor pools were constructed according to the selection criteria and four of them were actually tested in our lab for isozyme activities using 11 functional marker compounds. This study provides a basis for obtaining a representative human cryopreserved hepatocyte (HCH) pool to reduce the chance of causing enzyme activity variation during each study.
Table 1. IVT 51-donor enzyme activity database
Conventional pool construction for liver fractionsConventional pool construction for liver fractions
• Human microsome and S9: 10 to 15 donors as an optimum pool• Human cryopreserved hepatocyte: Not addressed yet
Advantages for construction of a representative human cryopreserved hepatocyte poolAdvantages for construction of a representative human cryopreserved hepatocyte pool
• Reduce the interindividual variations in human liver enzymes • Make experimental results more comparible between studies or labs• Provide experimental data more representation of a “generic human”
Study Design:
(1) Statistical analysis of IVT 51-donor database to establish the pool selection criteria:• Determine type of distribution of 51-donors for each isozyme• Define median value and its 99% confidence interval (CI) for each isozyme• Define confidence intervals of 67% typical population for each isozyme
(2) Construction of thirty-four HCH pools based on the pool selection criteria
(3) Actual pool comparisons by measuring isozyme activities using 11 functional markers
Table 2. Eleven functional marker compounds
Substrate ID Substrate Conc. #1(M)a
Metabolites Monitored Detection
Phenacetine (PHEN) 250 Acetaminophen LC/MS/MSCoumarin (COUM) 100 7-Hydroxycoumarin LC/UV
Tolbutamide (TOLB) 500 OH-Tolbutamide LC/MS/MS(S)-Mephenytoin (MEPH) 250 OH-mehpenytoin LC/MS/MSDextromethorphan (DEX) 50 Dextrorphan LC/MS/MS
Chlorzoxazone (CZX) 250 OH-Chlorzoxazone LC/MS/MSMidazolam (MID) 10 1’- and 4’-OH Midazolam LC/MS/MS
7-Hydroxycoumarin (7-HC) 250 7-Hydroxycoumaringlucuronide & 7-
hydroxycoumarin sulfate
LC/UV
Methylumbelliferone (MUF) 500 Methylumbelliferoneglucuronide &
methylumbelliferonesulfate
LC/UV
Trans-4-phenyl-3-butene-2-one (TPBO)
500 Trans-4-phenyl-3-butene-2-one glutathione
conjugate
LC/MS/MS
7-Ethoxycoumarin (7-EC) 75 7-Hydroxycoumarin7-Hydroxycoumarin
glucuronide7-Hydroxycoumarin
sulfate
LC/UV
a This saturated concentration (>> Km) was used for the measurement of metabolite formation rates.
(1) Statistical analysis of IVT 51-donor database to establish the pool selection criteria:
Table 3. Median confidence range and acceptable activity range of indivadual isozyme
Substrate: COUM DEX ECOD 7-HCG 7-HCS MEPH TEST TOLB PHEN CZX
Enzyme: 2A6 2D6 2E1 GT ST 2C19 3A4 2C9 1A2 2E1
Medianconfidence
range:
30-60 12-27 25-41 50-117 8-20 3-18 49-113 13-34 8-33 23-36
Acceptableactivity range:
21-76 9-33 11-43 8-184 5-27 1-31 25-147 10-46 4-43 20-47
Donor selection criteria:• At least 80% of individual isozyme activity values must be within their corresponding acceptable range.• No more than 30-35% of each enzyme activity value/X donors should be outside the acceptable activity range.• Mean activity for each isozyme/pool should fall into its corresponding median confidence range.
• The donor selection criteria based on the statistical analysis successfully provides a basis for obtaining a representative human cryopreserved hepatocyte (HCH) pool.
(2) Construction of thirty-four HCH pools based on the pool selection criteria:
Median vs. Pool#1
y = 1.0955xR2 = 0.8451
0
20
40
60
80
100
120
0 20 40 60 80
Median
Median vs. Pool#2
y = 1.0741x
R2 = 0.9568
0
20
40
60
80
100
0 50 100
Median
Median vs. Pool#3
y = 1.0236xR2 = 0.8183
0
20
40
60
80
100
0 20 40 60 80
Median
Median vs. Pool#4
y = 1.113xR2 = 0.977
0
20
40
60
80
100
0 20 40 60 80
Median
Median vs. Pool#5
y = 0.9512x
R2 = 0.9269
0
20
40
60
80
100
0 50 100
Median
Median vs. Pool#6
y = 1.157xR2 = 0.9589
0
20
40
60
80
100
0 20 40 60 80
Median
Median vs. Pool#7
y = 1.1278xR2 = 0.9026
0
20
40
60
80
100
0 20 40 60 80
Median
Median vs. Pool#8
y = 0.9709xR2 = 0.9767
0
20
40
60
80
100
0 20 40 60 80
Median
Median vs. Pool#9
y = 1.1132xR2 = 0.9589
0
20
40
60
80
100
0 20 40 60 80
Median
Median vs. Pool#10
y = 1.0977xR2 = 0.9529
0
20
40
60
80
100
0 20 40 60 80
Median
Median vs. Pool#11
y = 0.9571xR2 = 0.9656
0
20
40
60
80
100
0 20 40 60 80
Median
Median vs. Pool#12
y = 1.0995xR2 = 0.928
0
20
40
60
80
100
0 20 40 60 80
Median
Median vs. Pool#13
y = 1.0028xR2 = 0.9853
0
20
40
60
80
100
0 20 40 60 80
Median
Median vs. Pool#14
y = 1.0745xR2 = 0.9551
0
20
40
60
80
100
0 50 100
Median
Median vs. Pool#15
y = 1.0309xR2 = 0.948
0
20
40
60
80
100
0 20 40 60 80
Median
Median vs. Pool#16
y = 1.094xR2 = 0.9616
0
20
40
60
80
100
0 20 40 60 80
Median
Median vs. Pool#17
y = 1.1506xR2 = 0.9247
0
20
40
60
80
100
0 20 40 60 80
Median
Median vs. Pool#18
y = 1.2552xR2 = 0.9595
0
20
40
60
80
100
0 20 40 60 80
Median
Median vs. Pool#19
y = 1.1318xR2 = 0.9698
0
20
40
60
80
100
0 20 40 60 80
Median
Median vs. Pool#20
y = 1.2041xR2 = 0.9427
0
20
40
60
80
100
0 20 40 60 80
Median
Median vs. Pool#21
y = 0.9706xR2 = 0.9428
0
20
40
60
80
100
0 20 40 60 80
Median
Median vs. Pool#22
y = 0.944xR2 = 0.9236
0
20
40
60
80
100
0 20 40 60 80
Median
Median vs. Pool#23
y = 1.0345xR2 = 0.9331
0
20
40
60
80
100
0 50 100
Median
Median vs. Pool#24
y = 0.9118xR2 = 0.9722
0
20
40
60
80
100
0 20 40 60 80
Median
Median vs. Pool#25
y = 1.0338xR2 = 0.9
0
20
40
60
80
100
0 50 100
Median
Median vs. Pool#26
y = 1.0745xR2 = 0.8868
0
20
40
60
80
100
0 50 100
Median
Median vs. Pool#31 (Lion Mirror Pool#1)
y = 1.2382xR2 = 0.9322
0
20
40
60
80
100
0 20 40 60 80
Median
Median vs. Pool#32 (Lion Mirror Pool#2)
y = 1.1918xR2 = 0.9596
0
20
40
60
80
100
0 20 40 60 80
Median
Median vs. Pool#33 (Lion Mirror Pool#3)
y = 1.0798xR2 = 0.9443
0102030405060708090
100
0 50 100
Median
Median vs. Pool#34 (Lion Mirror Pool#4)
y = 0.7867xR2 = 0.9013
0
20
40
60
80
100
0 20 40 60 80
Median
(3) Actual pool comparisons by measuring isozyme activities using 11 functional markers:
Mirror#1 vs. Mirror#2
y = 1.0151xR2 = 0.9869
0
200
400
600
800
1000
0 500 1000
Enzyme activity (pmole/min/E06 cells) in Mirror#1
Mirror#1 vs. Mirror#3
y = 0.4497xR2 = 0.9383
0
100
200
300
400
500
0 500 1000
Enzyme activity (pmole/min/E06 cells) in Mirror#1
Mirror#1 vs. Mirror#4
y = 0.8036x
R2 = 0.9836
0
200
400
600
800
1000
0 500 1000
Enzyme activity (pmole/min/E06 cells) in Mirror#1
Lot # Viability COUM DEX ECOD 7-HCG 7-HCS MEPH TEST TOLB PHEN CZX Gender2A6 2D6 2E1 GT ST 2C19 3A4 2C9 1A2 2E1
51 65 55 9 17 47 7 1 115 10 6 23 F70 74 36 4 22 88 33 12 55 32 43 40 M83 59 29 24 42 190 25 21 74 39 33 30 M86 74 51 23 10 50 9 1 38 13 0 18 F87 71 34 36 43 81 35 3 56 50 4 64 F89 77 25 21 8 23 6 1 11 13 0 29 F90 74 30 25 7 13 0 2 19 15 0 16 F91 73 13 29 66 44 10 12 252 36 27 36 F94 67 41 12 37 24 4 21 126 40 19 87 F98 72 75 5 24 57 12 9 45 25 12 32 F99 86 21 15 7 4 0 1 60 10 0 22 F102 81 42 40 27 8 13 1 7 6 6 37 M104 81 63 21 44 247 25 2 58 37 8 20 M105 67 59 15 24 38 14 1 29 27 12 27 M107 81 60 28 24 21 0 16 234 41 7 48 F110 77 45 24 35 23 4 6 206 11 54 36 F111 71 4 10 9 2 3 3 147 2 19 19 F113 60 15 47 34 16 0 4 151 51 25 74 F114 75 39 23 21 10 5 5 59 TBD 3 45 F117 85 42 16 21 60 25 16 113 45 8 25 F118 65 81 34 56 208 29 26 208 51 39 13 F122 70 26 30 29 80 5 1 42 23 4 25 M129 90 4 24 27 67 18 1 16 33 10 51 F133 82 67 16 24 129 19 27 96 11 24 32 F
ACU 81 53 8 25 74 18 8 80 16 4 23 FCPN 81 28 6 40 100 13 2 168 14 37 21 MDRL 84 25 7 27 100 10 4 43 31 37 31 MEFA 69 9 9 35 47 5 18 66 12 47 67 MENR 73 69 28 27 124 31 107 77 36 38 32 MEQB 88 14 11 18 65 9 31 49 14 21 35 METR 88 30 1 34 13 13 7 13 13 37 69 FEVY 80 2 20 23 218 77 33 24 25 17 38 MGAL 64 52 7 27 59 6 3 94 13 57 23 FGIU 84 87 15 29 55 13 171 86 28 20 33 MGNG 70 57 17 33 54 8 5 95 22 16 22 FGUY 92 65 12 11 73 12 20 90 13 5 8 MHRK 63 69 11 55 124 12 23 136 34 64 22 FIEM 88 34 17 23 129 34 72 48 23 19 34 MKMI 74 32 11 29 192 18 6 69 9 14 18 MKPT 83 9 12 32 248 30 55 65 26 56 32 FKRM 78 126 36 55 83 17 103 98 46 74 44 FKTG 94 135 40 86 111 25 12 60 32 74 66 MMAN 97 31 12 35 119 32 3 40 14 6 34 MMQF 91 79 17 29 10 12 2 85 5 7 46 FMYO 94 40 24 9 24 0 0 12 7 0 11 FNQT 85 76 12 39 80 23 2 151 14 20 34 MOCF 82 91 16 26 98 17 52 147 33 40 32 FOQD 94 80 13 39 233 26 10 135 11 14 28 MPFM 87 21 1 11 67 10 3 116 8 15 33 FRKB 63 42 16 16 100 8 8 36 17 20 19 FRNG 91 119 14 97 298 27 177 207 34 71 41 F
Mirror#1 vs. Mirror #2
y = 0.9466xR2 = 0.9348
0
40
80
120
0 40 80 120
Enzyme activity in mirror #1 (pmole/min/million cells)
Mirror #1 vs. Mirror #4
y = 0.8481x
R2 = 0.858
0
40
80
120
0 40 80 120
Enzyme activity in mirror #1 (pmole/min/million cells)
Mirror #1 vs. Mirror #3
y = 0.6156xR2 = 0.7738
0
40
80
120
0 40 80 120
Enzyme activity in mirror #1 (pmole/min/million cells)
• Good correlation (0.8slope1.3 and R20.8) is achieved between the mean of each isozyme activity per pool and median values of each enzyme in the database.
• One- to three-fold isozyme activity differences are observed among pools.
• Enzyme activity changing trends of the virtual HCH pools constructed through IVT database are compatible with our actal results.
Enzyme activity values are based on IVT database
Enzyme activity values are based on Lion actual experimental data
