CYP450 Metabolites Analysis Service

Arachidonic  acid (AA) is a widely distributed polyunsaturated fatty acid once liberated  from phospholipids by phospholipases. AA can undergo either monooxygenation or  epoxidation by enzymes in the cytochrome P450 (CYP450) family. CYP-AA  metabolites include 19- and 20-hydroxyeicosatetraenoic acids (HETEs),  epoxyeicosatrienoic acids (EETs) and diHETEs. These metabolites have different  biological functions based on sites of production- the endothelium of vessels  in many organs, the lung, the tubular and corneal epithelium, the liver. The  likely targets are either enzymes (Na+-K+-ATPase) or ion channels (calcium activated  potassium channels). The involvement of conventional receptor physiology in the  tissue responses to CYP-AA products remains an open question. Several isoforms  of CYP families that metabolize AA have been identified and cloned.

Recombinant  CYP4A1, 4A2 and 4A3 enzymes, and possibly 4A8, have been reported to produce  20-HETE when incubated with AA. CYP4A2 and 4A3 mRNA have been shown in small  arterioles of the rat kidney cat brain, skeletal muscle, and liver. Human liver  and kidney express mRNA for the CYP4A11 and CYP4F2 isoforms; however, CYP4F2  now appears to be the isoform primarily responsible for the formation of  20-HETE in the human kidney, rat glomeruli, proximal tubule and thick ascending  limb of the loop of Henle (TALH) of rats, and in both the airways and  vasculature of the lungs of rabbits. Microsomes prepared from all these tissues  produce 20-HETE when incubated with AA in the presence of adequate oxygen  tension (PO2 > 6650 Pa) and reduced nicotinamide adenine dinucleotide  phosphate (NADP).

The  CYP2C family is involved in the production of EETs. Specific examples of this  family include a CYP2C11 enzyme present in astrocytes in the brain, CYP2C8 and  2C9 in human liver and endothelial cells, 2C2 in rabbit kidney, CYP2C29, 2C38  and 2C39 in the liver, kidney and brain of mice and CYP2C23 in the kidney of  rats. Also CYP1A, 2B and 2J catalyze the formation of EETs in various tissues.  The formation in various tissues is highly dependent on experimental  conditions.

CYP-AA  metabolites, 19- and 20-hydroxyeicosatetraenoic acids (HETEs),  epoxyeicosatrienoic acids (EETs) and diHETEs have different biological  properties based on sites of production and can be stored in various tissue  lipids and released in response to hormonal stimuli. 20-HETE is a vasoconstrictor,  causing blockade of Ca++-activated K+ (KCa) channels. EETs, produced by the  vascular endothelium, are potent dilators. EETs hyperpolarize VSM cells by  activating KCa channels. Several investigators have proposed that one or more  EETs may serve as endothelial-derived hyperpolarizing factors (EDHF).

Currently, a reliable and  reproducible method using highly  sensitive LC-MS/MS platform for the  identification and quantification of diverse  CYP450 metabolites in different sample types has been established by the  scientists at Creative Proteomics, which can satisfy the needs of academic and  industrial study in your lab.

Platform

• LC-MS/MS

Summary
 Identification and quantification ofCYP450  metabolites (eicosanoids) by mixed organic solvent  extractionin plasma or tissue. CYP450 metabolites are extracted and  concentrated using solid phase extraction. The eluant is dried and re-suspended  for LC-MS separation and measured using MRM methods.

Sample  Requirement

• Normal Volume: 200 uL plasma, 20 mg tissue, 1e7 cells
• Minimal  Volume: 50uL,  5 mg tissue, 6 e6 cells

Report

• A full report including all raw data, MS/MS instrument parameters and step-by-step calculations will be  provided (Excel and PDF formats).
• Analytes are reported as uM or ug/mg (tissue), and CV's are  generally<10%.

Ordering Procedure:

*If your organization requires signing  of a confidentiality agreement, please contact us by email.

Staffed  by experienced biological scientists, Creative Proteomics can provide a  wide range of services ranging from the sample preparation to the lipid  extraction, characterization, identification and quantification. We promise accurate and reliable  analysis, in shorter duration of time! You are welcome to discuss your project with us.