Comparison of Absolute Expression and Turnover Number of COX-1 and COX-2 in Human and Rodent Cells and Tissues

Figures & data

Figure 1 Typical chromatograms for LC-MS method specificity and selectivity. The retention times of representative peptides for rat COXs and human COXs were at 4.9, 2.6, 5.8 and 6.5 min, respectively (A–D). For Raw264.7 cells with LPS treatment, signal was only detected in rodent COX-2 MRM transition while it was below the detection limit in rodent COX-1 transition (E and F). No interference was found in human COX-1 and COX-2 transition (G and H). For EOMA cells with LPS treatment, signal was detected in rodent COXs’ transitions but not interfered in human COXs’ transitions (I–L). Similarly, the signal was only detected in human COXs’ transitions for the human recombinant enzymes samples and no background interference was found in rodent COXs’ transitions (M–T).

Figure 2 LC-MS method validation results in the heatmap. The newly developed LC-MS method was fully validated following the bioanalytical method validation guidelines for pharmaceutical industry released by USFDA. All inter- and intra-day accuracy and precision, recovery and standard addition accuracy were within acceptable range. Error rate = Accuracy - 100%.

Table 1 COX-1, COX-2 and PGE₂ Levels in Different Matrices.

Biological Samples	Source	LPS	Amount of COX-1 (pmol/mg)	Amount of COX-2 (pmol/mg)	Concentration of PGE2 (ng/mL)
Human COX-1 enzyme	Human	—	356.35 ± 25.99	<0.17	—
Human COX-2 enzyme		—	<0.17	3037.68 ± 469.80	—
SCC Patient Specimen #1	Human	—	0.656	<0.056	33.72
SCC Patient Specimen #2		—	1.011	<0.056	25.96
SCC Patient Specimen #3		—	0.736	<0.056	32.06
Human SI microsomes	Human	—	<0.056	<0.056	—
Human colon microsomes		—	<0.056	<0.056	—
SCC9 cells	Human	No	<0.083	<0.083	1.03 ± 0.08
SCC9 cells		Yes	<0.083	0.090 ± 0.009	5.48 ± 0.21
Caco-2	Human	No	<0.083	<0.083	<0.050
Caco-2		Yes	<0.083	<0.083	<0.050
Pirc polyps microsomes	Rat	—	0.84 ± 0.12	0.091 ± 0.075	—
Pirc colon microsomes		—	0.48 ± 0.22	0.023 ± 0.001	—
F344 colon microsomes		—	0.41	<0.014	—
Raw264.7 cells	Mouse	No	<0.042	<0.042	<0.050
Raw264.7 cells		Yes	<0.042	50.89 ± 1.23	12.50 ± 0.61
EOMA cells	Mouse	No	<0.042	4.15 ± 0.04	6.30 ± 0.40
EOMA cells		Yes	0.58 ± 0.10	10.91 ± 0.63	14.30 ± 0.17

Notes: In cell lysate, LLOQ of rodent and human COXs was 0.042pmol/mg and 0.083pmol/mg, respectively. In tissue microsome, LLOQ of rat and human COXs was 0.014pmol/mg and 0.056pmol/mg, respectively. In recombinant enzyme, LLOQ of human COXs was 0.17pmol/mg. LLOQ of PGE₂ in cell culture medium was 0.050 ng/mL.

Table 2 Relative Turnover Numbers of COX-1 and COX-2 Enzymes.

Biological Samples	Treatment	Turnover Number (ng·h^−1·pmol^−1)
Human COX-2	–	162.26 ± 3.21
Human COX-1	–	11.19 ± 1.01
Mouse COX-2	LPS 8h	48.57 ± 2.03
Mouse COX-2	LPS18h	30.77 ± 3.96

Figure 3 The correlation between PGE₂ levels and COX-2 levels from Raw264.7 cells with or without LPS induction and celecoxib inhibition. (A) PGE₂ production and COX-2 expression levels in Raw264.7 cells in the absence or presence of LPS for 8h, 18h and 24h. (B) PGE₂ production and COX-2 expression levels in the presence of celecoxib at different concentrations (0, 0.1, 1, and 10 µM). (C) Western blot results of Raw264.7 cells with LPS stimulation at indicated time (8h, 18h and 24h). (D) WB statistical results for relative COX-2 expression level in Raw264.7 cells with LPS treatment.