Article Figures & Data
Figures
- Fig. 1.
Western blot analysis of human CES1 and CES2 in pediatric hepatic microsomal samples. (A) Western blot with anti-CES1 antibody; lane 1, molecular weight standards as shown (kDa); lanes 2–6, purified recombinant CES2 standards (6.5, 12, 25, 50, and 100 ng); lanes 7–18, microsomal hepatic protein samples (10 µg each). (B) Western blot with anti-CES1 antibody; lanes 1–12, microsomal hepatic protein samples (10 µg each); lanes 13–17, purified recombinant CES1 standards (12.5, 25, 50, 100, 150 ng); lane 18, molecular weight standards as shown (kDa). (C) Western blot with anti-CES2 antibody; lane 1, molecular weight standards as shown (kDa); lanes 2–6, purified recombinant CES2 standards (6.5, 12, 25, 50, and 100 ng); lanes 7–18, microsomal hepatic protein samples (10 µg each). (D) Western blot with anti-CES2 antibody; lanes 1–12, microsomal hepatic protein samples (10 µg each); lanes 13–17, purified recombinant CES1 standards; lane 18, molecular weight standards as shown. Microsomal samples from differing age groups were loaded in a nonordered fashion.
- Fig. 2.
The relationship between human CES1 and age in postmortem microsomal and cytosolic liver samples. (A) Overall relationship with microsomal CES1 in samples from donors aged birth to 18 years (n = 161). The two added vertical lines represent the two nodes, aged 3 weeks (left line) and 6 years (right line), selected by classification regression tree analysis as indicative of appropriate age groupings. The 3-week age classification (solid line) was confirmed on Kruskal-Wallis statistical testing, whereas the 6-year grouping (indicated by a dotted line) was not. (B) Relationship between microsomal CES1 and age in liver samples from the subset of subjects younger than 1 year (n = 102). The added solid vertical line represents the 3-week time point selected by classification trees and confirmed by statistical testing as appropriate age stratification. (C) Relationship between cytosolic CES1 and age in postmortem cytosolic liver samples from birth to 18 years (n = 162). The vertical lines represent the two nodes selected by classification trees as indicative of appropriate age groupings: 3 weeks (left line) and 6 years (right line). Both were confirmed as statistically significant using Kruskal-Wallis testing. (D) Relationship between cytosolic CES1 and age in postmortem cytosolic liver samples from a subset of subjects younger than 1 year (n = 101). The vertical line represents the time point selected by classification trees as an appropriate age grouping (3 weeks).
- Fig. 3.
Summary of microsomal (white boxes) and cytosolic (gray boxes) human CES1 developmental expression pattern. CES1-specific content as a function of age was grouped using classification tree analysis to minimize differences within while maximizing differences between age brackets. The resulting data are shown as box and whisker plots in which the horizontal bar represents median CES1 content, boxes the upper and lower quartiles, and vertical bars the 5th to 95th percentiles. Outliers, defined as having specific contents outside 1.5 times the 25th to 75th percentiles, are shown as open circles but were excluded from the analyses except for determining absolute ranges in expression. The youngest age group differed significantly from the other two for both matrices (P < 0.001, each comparison; Kruskal-Wallis testing), whereas the middle age group was modestly significantly different from the older group in CES1 cytosolic content (P = 0.05) but did not differ in CES1 microsomal content (P = 0.13).
- Fig. 4.
The relationship between human CES2 and age in postmortem microsomal and cytosolic liver samples. (A) The relationship between microsomal CES2 and age in postmortem liver samples from donors aged birth to 18 years (n = 161). The vertical lines represent the two nodes selected by classification trees as indicative of appropriate age groupings: 3 weeks (left line) and 312 weeks or 6 years (right line). Both were confirmed as statistically significant using Kruskal-Wallis testing. (B) The relationship between microsomal CES2 and age in postmortem liver samples from the subset of subjects younger than 1 year (n = 102). The vertical line represents the time point selected by classification trees as an appropriate age grouping (3 weeks). (C) Relationship between cytosolic CES2 and age from postmortem liver samples (n = 162). The two vertical lines at 3 weeks (left) and 6 years (right) of age represent the nodes selected by classification trees. The 3-week age classification (solid line) was confirmed on Kruskal-Wallis statistical testing, whereas the 6-year grouping (indicated by a dotted line) was not. (D) Relationship between cytosolic CES2 and age in postmortem liver samples from a subset of subjects younger than 1 year (n = 101). The vertical line at 3 weeks represents the time point selected by classification trees and statistically confirmed (Kruskal-Wallis) as an appropriate age grouping.
- Fig. 5.
Summary of microsomal (white boxes) and cytosolic (gray boxes) human CES2 developmental expression pattern. CES2-specific content as a function of age was grouped using classification regression trees to minimize differences within age groups while maximizing differences between age brackets. The resulting data are shown as box and whisker plots in which the horizontal bar represents median CES2 content, boxes are the upper and lower quartiles, and vertical bars the 5th to 95th percentiles. Outliers, defined as having specific contents outside 1.5 times the 25th to 75th percentiles, are shown as open circles but were excluded from the analyses except for reporting absolute expression ranges. The youngest age group differed significantly from the other two for both matrices (***P < 0.001, each comparison, Kruskal-Wallis testing), whereas the middle age group was significantly different from the older group in CES1 microsomal content (P ≤ 0.001) but did not differ in CES1 cytosolic content (P = 0.26).
Tables
- TABLE 1
Tissue sample donor demographics
To ensure good age representation and adequate power, target sample sizes for birth to 30 days, greater than 30 days to 1 year, greater than 1–5 years, greater than 5–10 years, and greater than 10–18 years age brackets were developed based on data from the existing literature on drug and toxicant metabolizing enzyme ontogeny. Samples sizes were sufficient to provide at least 80% power to detect a 1 S.D. change in enzyme specific content between age brackets assuming α = 0.05.
Variable Median Range Age at death (mo) 3.7 0–212 Postmortem interval (h) 17 1–41 No. % of Total Hepatic tissue samples 165 100 Sex Male 104 63 Female 58 35 Unknown 3 2 Ethnicity/race Northern European white 79 48 African American 62 38 Hispanic 16 10 Asian 2 1 Native American 1 <1 Unknown 5 3 - TABLE 2
Immunodetected carboxylesterase (CES) content from postmortem fractionated hepatic samples
Data are given in median pmol/mg protein (interquartile range). Each enzyme is compared across the three donor race/ethnicity groups.
Enzyme African American (n = 62) White (n = 79) Hispanic (n = 16) Overall P Valuea Microsomal CES 1 14.99* (9.62–18.51) 17.95 (13.60–20.67) 10.86** (4.33–20.72) 0.01 Cytosolic CES1 13.62* (5.51–16.91) 15.46 (0.35–19.58) 11.94 (0–23.21) 0.155 Microsomal CES2 2.79 (2.05–3.98) 2.99 (2.20–3.93) 1.79*** (1.52–1.98) 0.001 Cytosolic CES2 1.83 (1.22–2.23) 1.69 (1.13–2.07) 1.51 (0–2.12) 0.23 ↵a The overall P value was derived from Kruskal-Wallis comparison across the three groups, whereas the asterisk P value designations within the table represent two-way comparisons using the Mann-Whitney U test.
↵* P ≤ 0.05 compared with whites; **P ≤ 0.01 compared with the other two groups; ***P ≤ 0.001 compared with the other two groups
In this issue
Human Hepatic CES1 and 2 Postnatal Ontogeny
