Correction of peripheral blood mononuclear cell cytosolic protein for hemoglobin contamination

Pharmacodynamic (PD) analysis requires accurate and precise quantification of enzyme activity targeted by anticancer agents in surrogate cells like peripheral blood mononuclear cells (PBMCs). Enzyme activity is normally reported per mass unit of protein input. However, high and fluctuating hemoglobi...

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Bibliographic Details
Published in:Analytical and bioanalytical chemistry 2013-03, Vol.405 (7), p.2391-2395
Main Authors: Pluim, Dick, Jacobs, Bart A. W., Krähenbühl, Martin D., Ruijter, Anna E. M., Beijnen, Jos H., Schellens, Jan H. M.
Format: Article
Language:English
Subjects:
Analytical Chemistry
Anticancer properties
Antimitotic agents
Antineoplastic agents
Biochemistry
Blood
Blood cells
Cancer
Characterization and Evaluation of Materials
Chemistry
Chemistry and Materials Science
Cold
Contamination
Cytosol - chemistry
Cytosol - enzymology
Dehydrogenases
Dihydrouracil Dehydrogenase (NADP) - analysis
Dosage and administration
Enzymatic activity
Enzyme activity
Enzymes
Female
Fluctuation
Food Science
Health aspects
Hemoglobin
Hemoglobins - analysis
Humans
Identification and classification
Laboratory Medicine
Leukocytes, Mononuclear - chemistry
Leukocytes, Mononuclear - enzymology
Male
Mathematical analysis
Methods
Monitoring/Environmental Analysis
Pharmacodynamics
Pharmacokinetics
Physiological aspects
Proteins
Proteins - analysis
Spectrophotometry
Spectrophotometry - methods
Technical Note
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Description
Summary:Pharmacodynamic (PD) analysis requires accurate and precise quantification of enzyme activity targeted by anticancer agents in surrogate cells like peripheral blood mononuclear cells (PBMCs). Enzyme activity is normally reported per mass unit of protein input. However, high and fluctuating hemoglobin (Hb) contamination strongly influences the protein content of PBMC cytosolic lysate. We present the development and validation of a spectrophotometrical Hb quantification method to correct for this contamination. The applicability of Hb correction was demonstrated by determination of the dihydropyrimidine dehydrogenase enzyme activity in PBMC cytosolic lysates.
ISSN:1618-2642
1618-2650
DOI:10.1007/s00216-012-6614-2