# Presentation: 936991

## Lipidomic Profiling Eicosanoid Changes in CarcinogenesisA. The LIPID MAPS Initiative B. Lipid Classification & Databasing  C. Eicosanoid Standards D. Eicosanoid Lipidomics

- Edward A. Dennis
- Departments of Pharmacology and Chemistry/Biochemistry
- University of California, San Diego

## LIPID MAPS

- _Lipid_ _M_etabolites _A_nd _P_athways _S_trategy
- GENOMICS
- PROTEOMICS
- METABOLOMICS
- “LIPIDOMICS”

## LIPID MAPS

- _Lipid_ _M_etabolites _A_nd _P_athways _S_trategy
- GENOMICS
- PROTEOMICS
- METABOLOMICS
- “LIPIDOMICS”

- 4 base side chains

- 20 amino acid side chains

- nucleic acids, amino acids,
- sugars and fats

- all the fats

- TM

## Lipid Metabolites and Pathways Strategy

- LIPID MAPS GOALS

- To separate and detect all of the lipids in a specific cell and to discover and characterize any novel lipids that may be present.
- To quantitate each of the lipid metabolites present and to quantitate the changes in their levels and location during cellular function.
- To define the biochemical pathways for each lipid and develop lipid maps which define the interaction networks.

## LIPID MAPS

- NIGMS Large Scale Collaborative Grant
- “Glue Grant” [NIH U54 GM 69338]
**	**Mouse Macrophage:	RAW cell line & primary cell
- 	Environmental Agonist:	initially LPS, then oxidized LDL
-  	HPLC/Mass Spectrometer:	identify known & new, quantify
- 	Synthesis/Characterize:	new ones, MS quantitative standards
- 	Bioinformatics:	informatics and lipid networks
- 	Website: 	_[http://www.lipidmaps.org](http://www.lipidmaps.org/)_

##  LIPID MAPS CORES

**Bioinformatics**

**Glycerolipids**

**Prenols/Structural Lipidomics**

**Sterols**

**Fatty Acyls/Eicosanoids**

**Glycerophospholipids**

**Sphingolipids/Gangliosides**

**Lipid Synthesis/Characterization**** **

**Michael VanNieuwenhze - *****UCSD***
**Walter Shaw -***** Avanti Polar Lipids***
**Stephen H. White***** - UC Irvine***

**Shankar Subramaniam - *****UCSD***

**Robert C. Murphy - *****Colorado ***

**Christian Raetz - *****Duke ***

**David W. Russell - *****UTSW***

**     ****Alfred H. Merrill - *****Georgia Tech***

**H. Alex Brown -***** Vanderbilt ***

**Macrophage Biology**

**Christopher Glass - *****UCSD***

**Edward A. Dennis - UCSD**

**LC/Mass Spec**

**Robert C. Murphy - *****Colorado***** ***** ***

** **** **

**PI**
** ****Edward A. Dennis - *****UCSD***

**TM**

## Participating InvestigatorsBoger, Dale  Scripps Research InstituteBrown, Michael  University Texas Southwestern MedicalCantley, Lewis  Harvard Medical SchoolCostello, Catherine  Boston University School of MedicineFalck, Camille  University Texas Southwestern MedicalGoldstein, Joseph  University Texas Southwestern MedicalHannun, Yusuf Medical University of South CarolinaHenson, Peter  National Jewish Medical/Research CenterMarnett, Lawrence  Vanderbilt Institute of Chemical BiologyMaxey, Kirk  Cayman ChemicalsPalsson, Bernhard  University of California, San DiegoPoulter, Dale  University of UtahRoberts, Jack  Vanderbilt University Medical CenterSerhan, Charles  Harvard Medical SchoolShipley, Graham  Boston University School of MedicineSpiegel, Sarah  Virginia Commonwealth UniversityUlevitch, Richard  Scripps Research InstituteVestal, Marvin  Applied BiosystemsVoeker, Dennis  National Jewish Medical/Research CenterYu, Robert  Medical College of GeorgiaNIHChin, Jean  National Insititute of General Medical Sciences

**	****	****	****	****Key Personnel**
**Principal Investigator**
**Dennis, Edward A.  University of California, San Diego**
**Core / Bridge Directors**
**Brown, H. Alex  Vanderbilt University Medical Center**
**Dennis, Edward  University of California, San Diego**
**Glass, Christopher  University of California, San Diego**
**Merrill, Alfred  Georgia Institute of Technology**
**Murphy, Robert  National Jewish Medical/Research Center**
**Raetz, Christian  Duke University Medical Center**
**Russell, David  University Texas Southwestern Medical**
**Shaw, Walter  Avanti Polar Lipids, Inc.**
**Subramaniam, Shankar  University of California, San Diego**
**VanNieuwenhze, Michael  University of California, San Diego**
**White, Stephen H.  University of California, Irvine**
**Witztum, Joseph  University of California, San Diego**
**Advisory Committee**
**FitzGerald, Garret  University of Pennsylvania**
**Henry, Susan  Cornell University**
**Samuelsson, Bengt  Karolinska Institute (Stockholm)**
**Vance, Dennis  University of Alberta (Canada)**
**UCSD Advisory Committee**
**Dixon, Jack  University of California, San Diego**
**Olefsky, Jerry  University of California, San Diego**
**Taylor, Susan  University of California, San Diego**
**Wooley, John  University of California, San Diego**

## LIPID CLASSIFICATION

**TM**

**LIPID CLASSIFICATION**

**Eoin Fahy**

## LIPID MAPS: Key Objectives from a Bioinformatics Perspective

**Define a classification scheme for lipids**
**Hierarchical scheme covering all lipids (eukaryotic and prokaryotic)**
**Logical categories consistent with structure and function**
**Clarify a systematic nomenclature system for lipids**
**Systematic names in compliance  with IUPAC conventions**
**Use of core structures to simplify nomenclature**
**Define stereochemistry and double-bond geometry**
**Establish recommendations for drawing lipid structures**
**Maintain consistency within classes and across classes, where possible**
**Represent chiral centers unambiguously**

## J. Lipid Res. : Classification publicationJournal of Lipid Research, Vol. 46, 839-862, May 2005 

**J. Lipid Res. : Classification publication****Journal of Lipid Research, Vol. 46, 839-862, May 2005**

## Lipid Categories and Examples 

## LIPID MAPS: Compound ID

**LIPID MAPS compound ID**
**LM****GP****01****01****0005**
**Database identifier**
**Database: LM (LIPID MAPS)**
**Category: GP (GP: Glycerophospholipids)**
**Class: 01 (Glycerophosphocholines)**
**Subclass: 01 (Diacylglycerophosphocholines)**
**Unique identifier within subclass: 0005**

**1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine**

## LIPID MAPS: Recommendations for drawing structures Consistent structure representation across classes

**Fatty Acyls (FA)**

**Sterol Lipids**** ****(ST)**

**Glycerophospholipids (GP)**

**Sphingolipids**** ****(SP)**

**Prenol Lipids (PR)**

**Glycerolipids (GL)**

## PubChem (NCBI)LIPID MAPS lipid structures now available on PubChem (http://pubchem.ncbi.nlm.nih.gov)

## KEGG  (Japan) (http://www.genome.ad.jp/brite/)

**Adoption of LIPID MAPS classification scheme by KEGG****Links from Lipid to Protein to Pathway**

## LIPID MAPS Proteome Database 

## LIPIDOMICS COMPONENTS

- Infrastructure

**Lipids**

- Proteins

- Pathways

- Annotations

- Genes

**LIPIDOMICS COMPONENTS**

- Mass Spec
- Lipid
- Standards

##  LIPID  MAPS

** ****LIPID  MAPS**

**Eicosanoid Lipidomics**

**Identify and Quantitate All the Fatty Acids and Fatty Acid Metabolites Produced by Macrophages Employing LC-MS Methodologies**

**TM**

***Rick Harkewicz***

## Arachidonic Acid

**Arachidonic Acid**

**15 LO**

**HETEs**

**15(S)HETE**

**COX**

**Prostaglandins**

**PGE****2**

**14,15-EET**

**P450**

**Epoxyeicosatrienoids**

**5 LO**

**Leukotrienes**

**LTB****4**

## Detection of Eicosanoids

- TM

**Liquid Chromatography – Mass Spectrometry (LC-MS)**

****

***sensitive***

****

***comprehensive***

## Eicosanoid Standard MixtureLC/MS - MRM

_***M***_***ultiple ***_***R***_***eaction ***_***M***_***onitoring***

**HETEs**

**11(S) HETE****(319/167)**

**12(S) HETE****(319/179)**

**8(S) HETE****(319/155)**

**5(S) HETE****(319/115)**

- TM

**(hydroxyeicosatetraenoic acid)**

**15(S) HETE****(319/113)**

## TM

## Example: Eicosanoid Library PGD2 MSMS Fragmentation Spectrum Page

- [M-H]-
- 351.2177

- C20H31O5-

- m/z = 271.2067

- C19H27O -

- m/z = 233.1183

*or alternatively*

- C14H17O3 -

- m/z = 233.1183

- C14H17O3 -

- m/z = 189.1285

- C13H17O -

***Example*****: Eicosanoid Library** **PGD****2 ****MSMS Fragmentation Spectrum Page**

- TM

## Provides:

**Provides:**

- TM

## Library of Lipid Standards

## Eicosanoid Lipidomics   RAW 264.7 macrophagesLPS & Kdo2-Lipid A

- Nuclei – DAPI
- Mitochondria – MitoTracker Red

**Eicosanoid Lipidomics****   RAW 264.7 macrophages****LPS & Kdo****2****-Lipid A**

## ACTIVATE RAW CELLS WITH

**Induction of inflammatory and **
**immune response genes**

**Cytokines (IL-1********, TNF********), **
**Tissue factor, B7, etc.**

**MAP3K**

**(Proteolysis)**

**Macrophage or endothelial**
** ****cell membrane**

**Direct Activation**

**ACTIVATE RAW CELLS WITH**
**      ****LPS and Kdo2-Lipid A**
**	****(Chris Raetz/Walt Shaw)**

## Kdo2-Lipid A

- Kdo2-Lipid A

## TLR-4 Selectivity of Kdo2-Lipid A

**TLR-4 Selectivity of Kdo****2****-Lipid A**
** ****in Mouse Bone Marrow Macrophages**

**R. Ulevitch and C. Fearns**

## Major Eicosanoids Detected in Media of LPS Stimulated RAW Cells  

**[ LPS (100 ng/ml), 18 h ]**

- TM

**RPLC - MRM**

**cis – trans isomers**
**15d-PGD****2**

**cis – tran isomer**
**15d-PGJ****2**

## RAW Cells - LPS Stimulation Time Course [100 ng/ml]

**RAW Cells - LPS Stimulation Time Course ****[100 ng/ml]**

- PlateCells

- 22 h

- Collect Media

- SerumStarve

- LPS

- 1 h

- 0.5 – 24 h

**Time Line:**

## Eicosanoid Release from RAW Cells

**Eicosanoid Release from RAW Cells**

**LPS vs. KDO****2**** Lipid A**

## Dose Response of RAW Cells with KDO2 Lipid A Stimulation [24 h] (secreted eicosanoids)

**PGE****2**
**Rmax****	****5.1 ± 0.4 ng/10****6 ****cells**
**EC****50****	****35 ± 8 ng/ml**

**11******** PGF****2******
**Rmax****	****4.6 ± 0.2 ng/10****6 ****cells**
**EC****50****	****18 ± 2 ng/ml**

**PGD****2**
**Rmax****	****39 ± 1 ng/10****6 ****cells**
**EC****50****	****15 ± 1 ng/ml**

**PGF****2******
**Rmax****	****4.8 ± 0.2 ng/10****6 ****cells**
**EC****50****	****28 ± 4 ng/ml**

**Notes:**
JACLM 1-29

## Major Eicosanoids Detected in Media of LPS Stimulated RAW Cells  

**[ LPS (100 ng/ml), 18 h ]**

**0 h**

**[100 ng/ml]**

**cis****–****trans isomers**
**15d-PGD****2**

**cis****–****trans isomer**
**    ****15d-PGJ****2**

**LC-MS (MRM)**

***Are low-level, ***_***biologically significant***_***, eicosanoids being produced but overlooked ?***

## AA-d8 Supplementation

**AA-d****8**** Supplementation**

**0 h**

**6 h**

## Mass-offset analog products – indicative of AA origin

***Mass-offset analog***** *****products***** – *****indicative of AA origin***

**PGD****2**

**NO AA - d****8**** supplementation**

***CONTROL***

- 351

- 352

- 353

***Full-scan MS***

***all samples*****: LPS (100 ng/ml), 18 h **

## Software For Processing and Comparing Control and AA-d8 Supplemented Full-Scan MS Data Sets

***Efficiently flags analog mass pairs and possible AA origin***

***NO***** AA-d****8**** supplementation (*****Control*****)**

**AA-d****8**** supplementation**

## The search for low-level  AA metabolites

**cis****–****trans isomers**
**15d-PGD****2**

**cis****–****trans isomer**
**    ****15d-PGJ****2**

**The search for **_***low-level ***_** AA metabolites**

## Slide 39

## Eicosanoid Enzymatic and Non-Enzymatic Products in RAW Cells 

**AA**

**PGG****2**

***COX***

**11(R)HETE**

***COX***

**PGH****2**

***(15-hydroxyprostaglandin dehydrogenase)***

***PGE******2 ******9-ketoreductase***

**11****β****-PGF****2****α**

***PGD******2 ******11-ketoreductase***

**13,14-dihydro-15-keto-PGD****2**

***15-PGDH***

**11****β****-13,14-dihydro-15-keto-PGF****2****α**

***15-PGDH***

** ****Dennis Group June, 2004**

**  ****RAY**
**DEEMS**

**        ****RICK**
**  ****HARKEWICZ**

**    ****MATT**
**BUCZYNSKI**

**         ****REBBECA**
** ****BOWERS-GENTRY**

## NIH U54 GM 69338

**26**

## Acknowledgements

**Rick Harkewicz**
-  **Eoin Fahy**
**Dawn Cotter**
**Shankar Subramaniam**
**Jean Chin, NIGMS**
-  [NIH U54 GM 69338]

** ****JLR Manuscript Authors**
** ****van Meer, Gerrit Utrecht**
**Seyama, Yousuke Ochanomizu**
**Shimizu, Takao Tokyo**
**Spener, Fritz Graz**
**Brown, H. Alex  Vanderbilt**
**Dennis, Edward  UCSD**
**Glass, Christopher  UCSD**
**Merrill, Alfred  Georgia Tech**
**Murphy, Robert  Colorado**
**Raetz, Christian  Duke **
**Russell, David  UT Southwestern**
**Shaw, Walter  Avanti**
**Subramaniam, Shankar  UCSD**
**VanNieuwenhze, Michael  UCSD**
**White, Stephen H.  UC, Irvine**
**Witztum, Joseph  UCSD**

## TM

**TM**

## THE LEITMOTIF OF “LIPIDOMICS”

- “Premiums in the shape of sensational discoveries may be hoped for, but cannot be assured even to the greatest genius. But what has to penetrate, relative to this question, more completely into the consciousness of pathologists, is this, that to understand zymoses, to be able to counteract them by rational, as distinguished from empirical or accidentally discovered means, is only possible by the aid of a complete knowledge of the chemical constitution of all the tissues, organs and juices of the body, and of all their possible products.”
-                        Johann Ludwig Wilhelm Thudichum (1829-1901)
-                        A Treatise on the Chemical Constitution of the Brain (1884)
-  Quoted in:
-  [Joseph Needham (1971) The Chemistry of Life, Cambridge University Press, pg. 199]

**Notes:**
“Premiums in the shape of sensational discoveries may be hoped for, but cannot be assured even to the greatest genuis. But what has to penetrate, relative to this question, more completely into the consciousness of pathologists, is this, that to understand zymoses, to be able to conteract them by rational, as distinguished from empirical or accidentally discovered means, is only possible by the aid of a complete knowledge of the chemical constitution of all the tissues, organs and juices of the body, and of all their possible products.