Aldehyde oxidase activity in donor-matched fresh and cryopreserved human hepatocytes and assessment of variability in 75 donors.
Studies were conducted to evaluate the impact of time and cryopreservation on aldehyde oxidase (AO) activity in human hepatocytes isolated from 10 donor livers, using O(6)-benzylguanine as a probe substrate. In addition, variability in activity was assessed using cryopreserved hepatocytes from 75 donors. Substantial donor-dependent loss in AO activity within 24 hours after isolation of hepatocytes was observed (average loss of 42%, range 15%-81%). Meanwhile, AO activity in cryopreserved hepatocytes more closely represented the activity observed in fresh hepatocytes that were incubated immediately after isolation for the same donors (within 81% of fresh, range 48%-100%). Activity of AO in cryopreserved hepatocytes from 75 donors varied by at least 17-fold (≤ 5.4 to 90 ml/minute per kilogram of body weight), with 63% of the donors having higher activity than a pooled 19-donor lot (34.2 ml/minute per kilogram). Comparison of demographics such as gender, body mass index, age, and ethnicity showed no statistically significant correlations with activity. Evaluation of medical histories revealed that three of the five donors with no measurable activity had immediate histories of extensive alcohol abuse. Meanwhile, two single nucleotide polymorphisms (SNPs) for AOX1 (rs3731772 and rs55754655) were detected in our donor pool and showed allelic frequencies similar to those reported from other cohort studies. However, these SNPs did not correlate with a statistically significant difference in intrinsic clearance compared with wild-type donors. With a general lack of clarity about what causes highly variable AO activity, prescreening donors for AO activity and creating a custom high-activity pooled lot of cryopreserved hepatocytes are advised to minimize underpredictions of clearance.
Boehringer-Ingelheim Pharmaceuticals Inc. Medicinal Chemistry, Drug Discovery Support (DMPK), Ridgefield, Connecticut (J.M.H., Y.S.Y.), BioreclamationIVT, Baltimore, Maryland (C.B., S.H., T.M.).
J Matthew
JM
Hutzler
Young-Sun
YS
Yang
Caitlin
C
Brown
Scott
S
Heyward
Timothy
T
Moeller
D000328:Adult; D000368:Aged; D042931:Aldehyde Oxidase / Q000235:genetics* / Q000378:metabolism*; D002648:Child; D002675:Child, Preschool; D015925:Cryopreservation / Q000379:methods; D004789:Enzyme Activation / Q000502:physiology; D005260:Female; D022781:Hepatocytes / Q000201:enzymology*; D006801:Humans; D008297:Male; D008657:Metabolic Clearance Rate / Q000502:physiology; D008875:Middle Aged; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D014019:Tissue Donors
D042931:Aldehyde Oxidase