Identification of disease markers in human cerebrospinal fluid using lipidomic and proteomic methods
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Disease Markers 22 (2006) 39–64 39
IOS Press
Identification of disease markers in human
cerebrospinal fluid using lipidomic and
proteomic methods
Alfred N. Fonteha,∗, Robert J. Harringtona , Andreas F. Huhmer b , Roger G. Biringerb ,
James N. Riggins a and Michael G. Harringtona
a
Molecular Neurology Program, Huntington Medical Research Institutes, Pasadena, CA 91101, USA
b
Thermo, San Jose, CA 95134, USA
Abstract. Lipids comprise the bulk of the dry mass of the brain. In addition to providing structural integrity to membranes,
insulation to cells and acting as a source of energy, lipids can be rapidly converted to mediators of inflammation or to signaling
molecules that control molecular and cellular events in the brain. The advent of soft ionization procedures such as electrospray
ionization (ESI) and atmospheric pressure chemical ionization (APCI) have made it possible for compositional studies of the
diverse lipid structures that are present in brain. These include phospholipids, ceramides, sphingomyelin, cerebrosides, cholesterol
and their oxidized derivatives. Lipid analyses have delineated metabolic defects in disease conditions including mental retardation,
Parkinson’s Disease (PD), schizophrenia, Alzheimer’s Disease (AD), depression, brain development, and ischemic stroke. In
this review, we examine the structure of the major lipid classes in the brain, describe methods used for their characterization,
and evaluate their role in neurological diseases. The potential utility of characterizing lipid markers in the brain, with specific
emphasis on disease mechanisms, will be discussed. Additionally, we describe several proteomic strategies for characterizing
lipid-metabolizing proteins in human cerebrospinal fluid (CSF). These proteins may be potential therapeutic targets since they
transport lipids required for neuronal growth or convert lipids into molecules that control brain physiology. Combining lipidomics
and proteomics will enhance existing knowledge of disease pathology and increase the likelihood of discovering specific markers
and biochemical mechanisms of brain diseases.
Keywords: Lipidomics, phospholipidomics, sphingolipidomics, cholesterol, proteomics, mass spectrometry, electrospray ioniza-
tion, phospholipases, enzymes, lipoproteins, cytochrome P450, acetylhydrolases, fatty acids, eicosanoids, secretion, ion channels,
receptors, inflammation, oxidation, cerebrospinal fluid, brain, neurological diseases
Abbreviations used:
AA, arachidonic acid
Apo, apolipoprotein
APCI, atmospheric pressure chemical ionization
BBB, Blood brain barrier
CDP, cystidine diphosphate
CSF, cerebrospinal fluid
COX, cyclooxygenase
CYP, cytochrome P
ESI, electrospray ionization
ESI EPA, eicosapentaenoic acid
∗ Corresponding author. Tel.: +1 626 795 4343; E-mail: afonteh@
hmri.org.
ISSN 0278-0240/06/$17.00 © 2006 – IOS Press and the authors. All rights reserved
40 A.N. Fonteh et al. / Identification of disease markers in human cerebrospinal fluid
(EPA) DHA, docosahexaenoic acid
(DHA) HETEs
HDL, low density lipoprotein
IP, inositol phosphate
LC-MS2 , liquid chromatography tandem mass spectrometry
LDL, low density lipoprotein LO, lipoxygenase
LT, leukotriene
PA, phosphatidic acid
PAF, platelet-activating factor
PAFA, platelet-activating factor acetylhydrolase
PC, phosphatidylcholine
PE, phosphatidylethanolamine
PhosGl, phosphatidylglycerol
PG, prostaglandin
PI, phosphatidylinositol
PIP, phosphatidylinositol phosphate
PL, phospholipase
PLA2 , phospholipase A2
PLC, phospholipase C
PLD, phospholipase D
PS, phosphatidylserine
PUFA, polyunsaturated fatty acid
SRM, selected reaction monitoring
pAD, probable Alzheimer’s disease
PD, Parkinson’s disease
SRM, selected reaction monitoring
Glossary
Lipidome- All known lipids, includes phospholipids, fatty acids and cholesterol. The study of structure, biosynthesis and function of all
lipids is lipidomics.
Phospholipidome- All known phospholipid classes, subclasses and molecular species. The study of structure, cellular distribution, biosyn-
thesis and function of phospholipids is phospholipidomics.
Sphingolipidome- All known sphingolipid classes and molecular species. The study of structure, cellular distribution, biosynthesis and
function of sphingolipids is sphingolipidomics.
1. What are lipids and why are they important in carry a myriad of biological functions. For example,
brain function? arachidonic acid (20:4, n-6) can be released and sub-
sequently converted to eicosanoids by cycloxygenases
Lipids are organic compounds with long chain hy- (COX), epoxygenases, lipoxygenase (LO) in combina-
drocarbon molecules that are soluble in organic sol- tion with prostaglandin or leukotriene synthases [6,160,
vents but not soluble in water. Lipids are derived from 180,190,195]. Eicosanoids act on specific receptors or
living organisms; some examples of lipids include long ion channels to influence physiological processes such
chain hydrocarbons, alcohols, aldehydes, fatty acids, as sleep and pain [94,95]. Likewise, several neuros-
their derivatives (glycerides, wax esters, phospholipids, teroids derived from cholesterol have been shown to
glycolipids, sulfolipids, and fatty acid esters), fat solu- have important physiologic functions in the brain [10,
ble vitamins (A, D, E and K), carotenoids and sterols. 99,116,170,213]. In addition to providing signaling
Lipids are usually subdivided into neutral or polar lipids molecules, lipids are the building blocks of cell mem-
and are now classified into eight categories based on branes that confer structure and insulation to nerve cells
hydrophobic and hydrophilic composition [68]. About and are a major reservoir of stored energy. With these
half of the dry weight of the brain is made of lipids. important functions, changes in their amounts or de-
Lipids are important in many brain functions includ- fects in lipid metabolic pathways can have a signifi-
ing membrane composition, signal transduction, and cant impact on brain function. Therefore, an accurate
biological messenger functions [18,39,40,61,67,69,88, measure of lipid concentrations in the central nervous
206,221]. Thus, changes in the concentrations of brain system is needed for understanding their role in the
lipids may reflect physiopathologic processes. pathology of diseases.
One class of lipids proposed to be important in brain Lipids encompass a range of structurally dissimi-
function is the polyunsaturated fatty acids (PUFAs). lar molecules consisting of several isomers that are
PUFAs are released from phospholipids by lipases to difficult to isolate. Lipids do not easily ionize and
A.N. Fonteh et al. / Identification of disease markers in human cerebrospinal fluid 41
upon collision-induced dissociation, fragment into ions or 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine. PAF
that can not be useful fingerprints for distinguishing is a mediator of hypersensitivity, acute inflammation,
the thousands of molecular species found in cells. anaphylactic shock, platelet aggregation and serotonin
No single ionization method can be used for all lipid release [31,37,149,152]. Bazan et al. have proposed
classes. Moreover, differently charged headgroups in that PAF is important in brain plasticity [12].
lipids make some lipids easy to ionize in the positive In most phospholipids, the fatty acid at the sn-1 po-
mode while others are better measured in the negative sition is palmitic (16:0), stearic (18:0) or oleic acid
mode. However, recent advances in mass spectrome- (18:1) while either a saturated, unsaturated or a polyun-
try have made it possible to use electrospray ionization saturated fatty acids (PUFAs) can be found at the sn-2
(ESI) [90,105,129,133] with negative or positive ions position of the glycerol backbone. In certain classes
under atmospheric pressure conditions to measure sev- and subclasses found in cells or tissues, PUFAs are
eral molecular species of lipids. Combined with liquid the major fatty acids at the sn-2 position. This dis-
or gas chromatography, hundreds of lipid molecular tribution gives rise to subclasses of lipids that are tar-
species can now be identified. geted for the release of PUFAs and the generation
of specific lipid mediators and signaling molecules.
An example is 1-alkyl-2-arachidonoyl-sn-glycero-3-
2. Structures of lipids detected in human brain phosphocholine, the precursor of platelet activating fac-
and CSF and their biosynthetic pathways tor and leukotrienes [77,122]. The incorporation of
PUFAs into phospholipid subclasses is highly chore-
2.1. Phospholipidome ographed such that most PUFAs are initially incor-
porated into 1,2-diacyl phospholipids subclasses be-
The phospholipidome consists of the major polar fore they are remodeled into the ether-linked phos-
lipid class found in mammalian cells and the study of pholipids classes by coenzyme A (CoA)-dependent
their structure, biosynthesis and catabolism is hence- or CoA-independent transacylase activities [73,84,126,
forth referred to as phospholipidomics. Structurally, 165,167].
phospholipids are composed of a glycerol backbone to Phospholipids are not only variable in their head
which is esterified a fatty acid at the sn-1 and sn-2 car- groups, fatty acids and bond profiles on the glyc-
bon and a phosphor-headgroup moiety at the sn-3 posi- erol backbone, but they are asymmetrically distributed
tion. When the headgroup is choline, ethanolamine or within lipid bilayers of cells. This molecular diver-
serine, the phospholipids are known as phosphatidyl- sity results in hundreds of molecular species in a given
choline (PC), phosphatidylethanolamine (PE) or phos- cell and theoretically thousands of species in mam-
phatidylserine, respectively (Fig. 1A). Phosphatidyli- mals. Even more intriguing is the fact that different or-
nositol (PI) and phosphatidylglycerol (PhosGl) are ganelles may be enriched with specific lipid classes or
formed when inositol and glycerol are the headgroups, subclasses. These distributions are unique in establish-
respectively (Fig. 1A). Phosphatidic acid (PA) and ing the functions of different organelles by influencing
diphosphatidylglycerol (cardiolipin) are other impor- membrane fluidity or generating signaling molecules
tant phospholipids classes with structures depicted on in specific sites when cells are stimulated.
Fig. 1. Phospholipids are synthesized when cystidine
Phospholipids are further divided into subclasses diphosphate (CDP)-activated polar headgroups are at-
based on the type of linkage of fatty acids at the sn-1 tached to PA (1,2-diacyl-sn-glycerol-3-phosphate) or
position. In ester lipids, fatty acids are attached via when CDP-activated diacylglycerol (DAG) is attached
1-acyl bonds while for ether lipids or plasmalogens, to polar headgroups. For example, PC or lecithin
fatty acids at the sn-1 position are linked via 1-alkyl- is synthesized when choline is phosphorylated by
or 1-alk-1-enyl- bonds (Fig. 1B). 1-Acyl-, 1-alkyl or cholinephosphotransferase [106] and then coupled to
1-alk-1-enyl- subclasses are predominant in PC and PE CDP prior to attachment to PA. Cholinephosphotrans-
while diacyl-linked subclasses comprise most of PI, PS ferase catalyzes the final step in the synthesis of
and PG found in cells. Ethanolamine plasmalogens are PC via the Kennedy pathway by transferring phos-
abundant in myelin sheath and changes in their compo- phocholine from CDP-choline to DAG [98]. For
sition are proposed for several diseases including AD. PE biosynthesis, ethanolaminephosphotransferase cat-
Choline plasmalogen is a precursor of the potent biolog- alyzes a similar transfer of ethanolamine with CDP-
ical mediator known as platelet-activating factor (PAF) ethanolamine as the intermediate. PC can also be ob-
42 A.N. Fonteh et al. / Identification of disease markers in human cerebrospinal fluid
(A)
O O
H2C O_C_R1
sn1
_ _ 1
H2C O C R
O O
sn2
R2_ C_O CH R2_ C_O CH
O O
sn3
H2C O P O_CH2 CH2 N+(CH3)3 H2C O P O_CH2 CH2 N+ H3
O- O-
PC (Phosphatidylcholine or 1,2-Diacyl-sn- PE (Phosphatidylethanolamine or 1,2-Diacyl-sn-
glycero-3-phosphorylcholine or lecithin) glycero-3-phosphorylethanolamine)
O O
_ _ 1
H2C O C R H2C O_C_R1
O O
R2_ C_O CH R2_ C_O CH
O O
H2C O P O OH
H2C O P O_CH2 CH(+NH3)COO- OH
O- O- HO OH
HO
PS (Phosphatidylserine or 1,2-Diacyl-sn- PI (Phosphatidylinositol or 1,2-Diacyl-sn-
glycero-3-phosphorylserine) glycero-3-phosphorylinositol)
O
_ _ 1
H2C O C R O O O
_ _ 1
O H2C O C R CH2OH _ _ 1
H2C O C R CH2O P O CH2
O O
R2_ C_O CH O O-
R2_ C_O CH CHOH R2_ C_O CH CHOH CH O_C_R3
O
H2C O P OH O O CH2 O_C_R4
O- H2C O P O CH2 H2C O P O CH2 O
O- O-
PA (Phosphatidic acid or PG (Phosphatidylglycerol
Cardiolipin (diphosphatidylglycerol)
1,2-Diacyl-sn-glycero-3 or 1,2-Diacyl-sn-glycero-3-
-phosphate) phosphoryl-1 -sn-glycerol)
(B)
H2C O_R1 H2C O_CH=CH_R
O O
R2_ C_O CH R2_ C_O CH
O O
H2C O P O_CH2 CH2 N+(CH3)3 H2C O P O_CH2 CH2 N+(CH3)3
O- O-
1-Alkyl-2-acyl-sn-glycero-3 1-Alk-1-enyl-2-acyl-sn-glycero-3
-phosphorylcholine -phosphorylcholine or plasmalogen
Fig. 1. Structure of phospholipids- Ester-linked phospholipids (A) and ether-linked phospholipids (B). Stereospecific numbering (sn) system
of nomenclature for the glycerol backbone is indicated while R1 and R2 denote fatty acyl moieties. The classification of phospholipids into
subclasses (1-acyl-, 1-alkyl- or 1-alk-1-enyl-) is shown by the fatty acyl-bond at the sn-1 position of glycerol.
tained when PS is decarboxylated to PE followed by N- differentiation and synaptic vesicle formation [59,177].
methylation of PE by S-adenosylmethionine-dependent The phospholipidome undergoes dynamic remodel-
methyl transferase [216]. Base exchange reactions ing of fatty acids and phospho-headgroups under rest-
when ethanolamine is exchanged for serine in PE re- ing conditions and this remodeling process is enhanced
sults in PS biosynthesis. Similar to PC biosynthesis, when cells are stimulated and decreased when cells
PI is formed when 1,2 DAG is activated by CDP fol- are undergoing apoptosis [79,84]. Examples of en-
lowed by condensation with myo-inositol. PI can un- zymes that catabolize phospholipids and the products
dergo a series of phosphorylations catalyzed by various they generate are listed on Table 1. The major en-
PI-kinases to form phosphopolyinositides. PIP 2 is an zymes that modify phospholipids include phospholi-
example of phosphorylated PI that has been well char- pases (PLA1 , PLA2 , PLC, PLD), acyl transferases and
acterized and been shown to be a signal for cell growth, PI specific kinases. Phospholipases hydrolyze ester
A.N. Fonteh et al. / Identification of disease markers in human cerebrospinal fluid 43
PLA1 2.2. Sphingolipidome
O The sphingolipidome is a subset of the phospho-
sn1
_ _ 1
lipidome consisting of sphingomyelin and glycosphin-
H2C O C R golipids (cerebrosides, sulfatides, globosides and gan-
O gliosides). Sphingolipids are composed of a polar head-
sn2
R C_O CH
2_ group and two non-polar tails (Fig. 3). A long chain
O amino alcohol known as sphingosine is linked via an
sn3 amine and a long chain fatty acid is attached to carbon-
H2C O P O_CH2 CH2 N+(CH3)3 2 to yield ceramide. Sphingolipids are components
PLA2 O- of membranes found mainly in myelin sheaths. The
major route of sphingolipid formation is the transfer
PLC PLD of phosphorylcholine from PC to ceramide by sphin-
gomyelin synthase (Fig. 3). Sphingomyelins are im-
Fig. 2. Sites of action of phospholipases on phosphatidylcholine- portant in nerve cell membranes where very long chain
The major groups of phospholipases include PLA1 , PLA2 , PLC and
PLD. Products of these enzyme activities and their relevance to the
saturated and monounsaturated fatty acids are the main
brain are shown on Table 1. N-acylated molecules at carbon-2 of sphingosine [96,
142,188].
The action of sphingomyelinase on sphingomyelin
bonds shown on Fig. 2. Many isoforms of PLA 2 that
forms choline and ceramide. Ceramide can be fur-
differ in structure, substrate specificity, requirements ther broken-down to sphingosine by ceramidase or can
for calcium ions, mode of activation and cellular local- be converted to glucosylcerebroside by glycosyl ce-
ization have been cloned and described in various mam- ramide synthase [179,184] (Fig. 3). Sphingosine phos-
malian cells [123]. Several PLA 2 isoforms have been phate and related molecules have recently been shown
characterized in the brain [73,204]. One PLA 2 isoform to regulate apoptosis [128,153]. Although these novel
(cPLA2 ) co-localizes with reactive astrocytes and glial mediators have not been characterized in the brain,
cells and is involved in neurodegeneration [47]. Var- their presence may account for brain diseases that are
ious PLC isoforms are involved in the generation of known to result from defects in sphingolipid biosyn-
DAG and IP3 in the rat and human brain [183,192]. thesis. Thus, one may postulate that changes in sphin-
Two isoforms of PLD (PLD 1 and PLD2 ) generate sig- gosine phosphate levels in the neurodegenerative brain
naling molecules in the brain and involved in the gen- may be early indicators/biomarkers of brain atrophy.
eration of choline required for acetylcholine biosynthe- In summary, the phospholipidome provides struc-
sis [230]. PLD isoforms are implicated in neural out- ture, is the precursor of signaling molecules and plays
growth and hormonal/stress signaling [219,220,236]. an important role in the formation of vesicles re-
Lysophospholipids generated by the action of PLA 2 can quired for neurotransmitter release and the transport of
accept acyl groups from other phospholipids in a reac- metabolites. These important functions make it neces-
tion catalyzed by lysolecithin-lecithin-acyltransferase sary to study the composition of brain phospholipidome
because early changes are possible events in brain dis-
(LLAT) [203]. Other CoA-dependent and -independent
eases.
remodeling of PUFAs are responsible for the buildup
of PUFAs in ether-linked phospholipids [82,121,165].
2.3. Cholesterol and hormones
Lecithin cholesterol acyltransferase (LCAT), a protein
with both lipase and transferase activity transfers fatty The brain is a very rich source of cholesterol and is
acids from lecithin to cholesterol [1,110]. LCAT is estimated to represent 25% of total cholesterol in hu-
associated with lipoproteins that are involved in the mans [60]. Although it is known that a major portion
transport of lipids from the liver to organs and vice of this cholesterol is localized in myelin sheaths, lit-
versa. Although not recognized as the major path- tle is known about the metabolic pathways that control
way by which cholesterol is excreted from the brain, this vast reservoir of cholesterol. Brain cholesterol is
lipoprotein-mediated transport is important in neural mainly produced by de novo synthesis (Fig. 4) and is
outgrowth and may be the major transcellular transport segregated from plasma cholesterol by the blood brain
mechanism within the brain. barrier (BBB). Homeostatic control of brain cholesterol
44 A.N. Fonteh et al. / Identification of disease markers in human cerebrospinal fluid
Table 1
Some enzymes that modify phospholipids, their products and functions in CNS
Enzymes Lipid substrates and products Importance of products
PLA2 Hydrolyzes the ester bond at the sn-2 position of phos- If the FFA is a PUFA, it may be a substrate of COX, CYP
pholipids (Fig. 2) to release free fatty acids (FFA) and or LO. PLA2 activity is implicated in endocytosis, fusion
lysophospholipids. For example, PC is hydrolyzed to and membrane structure and asymmetry, neurodegenerative
LPC and FFA or PA hydrolyzed to LPA and FFA. diseases and autistic disorders [20,73,204,208].
PLC Hydrolyses phospholipids to DAG and phospho- Important in vesicle priming and synaptic vesicle dock-
headgroup moiety, e.g. PI(4,5)P2 to DAG and ing [52,59]. DAG activates PKC and/or PKA while IP3
I(1,4,5)P3 [21] induces intracellular calcium release [22].
PLD Releases PA and free headgroup from phospholipids Implicated in synaptic vesicle fusion. PA promotes exo-
(Fig. 2). An example is the release of choline from cytosis, phagocytosis, membrane trafficking and cytoskele-
PC. tal structure [48,51,62,147,219,220]. Headgroup such as
choline can be used for neurotransmitter biosynthesis.
Lysophospholipase Glycerophosphate and free fatty acids [218]. Im- Important in signal transduction [174,210,218].
plicated in ether lipid formation by brain micro-
somes [229] and anandamide biosynthesis [205]
PI kinase or PIP Phosphorylate PI to form PI(4)P and PI(4)P to Required for synaptic vesicle formation, fusion, trafficking
kinases and PITP PI(4,5)P2 . PITP is involved in membrane remodeling. and exocytosis [49,50]
involves biosynthesis and cytochrome P450 (CYP)- the AD brain [33]. Several other brain diseases are as-
mediated excretion with 24S-hydroxycholestrol as the sociated with metabolism, transport, recycling, excre-
major product [24,28]. Cholesterol serves several im- tion and degradation of cholesterol [2,24,64,120,135,
portant functions in the brain. First, cholesterol helps 143,161,163,178,202,227].
maintain brain structure and is important in controlling Levels of cholesterol in the brain are controlled by
lipid fluidity and the transport or permeability of ions the rate of biosynthesis, catabolism, transport and ex-
and metabolites. Second, cholesterol provides the nec- cretion. Two major pathways for cholesterol biosyn-
essary insulation to neurons that allows efficient prop- thesis are known in mammals (Fig. 4). Both path-
agation of an action potential. Third, cholesterol is ways require acetyl-CoA derived from glucose oxida-
needed for the growth and development of neurons. tion or from fatty acid metabolism (Fig. 4) [85,87,112,
Fourth, cholesterol is the precursor for the synthesis 113]. Conversion of HMG-CoA to mevolonic acid by
of steroid hormones that are important in controlling HMG-CoA reductase is the rate-limiting step for both
physiologic processes such as stress, plasticity, and de- pathways in many cells including glial cells. In one
pression [11]. Fifth, the cholesterol biosynthetic path- pathway, 7-dehydrocholesterolis an intermediate while
way generates molecules (isoprenenyl-pyrophosphate, the alternate route involves 7-dehydrodesmosterol as
geranyl-pyrophosphate, farnesyl-pyrophosphate) used an intermediate (Fig. 4). Several enzymes involved in
for the modification of proteins and RNA and for the cholesterol synthesis have been characterized in vari-
biosynthesis of ubiquinone, dolichol and Heme A [65, ous brain cells. The differential expression of these en-
66,196]. Modification of G-proteins is critical for zymes in brain cells suggests that there is transcellular
their function. Thus cholesterol biosynthesis may in- metabolism or highly specialized functions of choles-
directly influence receptor mediated physiologic pro- terol products by brain cells or specific brain regions.
cesses controlled by G-protein coupled receptors. Fi- For example, astrocytes synthesize two to three times
nally, the cholesterol composition affects the activity more cholesterol than neurons and fibroblasts [169].
of transmembrane proteins and receptors. For exam- Oligodendrocytes that are normally involved in myeli-
ple, gamma amino butyric acid (GABA) transport re- nation have higher capacity to synthesize cholesterol,
quires cholesterol and lipid rafts/coated pits that are im- underscoring the need and importance of cholesterol
plicated in endocytosis and protein remodeling are all for myelination [212].
eriched with cholesterol [138,193,200,209,227]. These Cholesterol synthesis and distribution may play a
functions of cholesterol underscore its importance in critical role in the onset and progression of degener-
brain function. For example, 24S-hydroxycholesterol, ative brain disorders. A study showing that choles-
the major excretion product and has been shown to in- terol synthesis is higher in developing neurons but is
crease in CSF of AD subjects [24,60]. Moreover, CYP decreased with aging has several implications for ag-
enzymes that oxidize cholesterol are differentially ex- ing and neurodegenerative diseases [227]. While total
pressed in brain cells and around amyloid plaques in cholesterol may not change, the distribution of choles-
A.N. Fonteh et al. / Identification of disease markers in human cerebrospinal fluid 45
CDP-choline
+
Ceramide
Sphingomyelin
synthase
CDP
Sphingosyl Sphingosyl- Ceramide
phosphocholine phosphocholine Phosphocholine
+ transferase +
acyltransferase
Fatty acyl CoA Phosphatidylcholine
Sphingomyelin
Sphingomyelinase
(acid-, neutral or
Zn-cativated)
choline
Ceramidase
Sphingosine Ceramide Ceramide
Ceramide kinase phosphate
synthase Ceramide
Sphingosine
kinase Glucosyl
Glucosyl Ceramide
Ceramidase Synthase
Sphingosine-1-
Phosphate
Glucosylcerebroside
Fig. 3. Metabolism of sphingomyelin and ceramide.
terol favors amyloid peptide accumulation upon ag- mated half-life of 5 years in the brain, enhanced degra-
ing. In terms of biomarker discovery, it may be impor- dation or CYP-mediated catabolism of cholesterol will
tant to examine the biosynthetic pathway in individuals significantly alter its levels [28,33,136,224].
with a neurodegenerative disease to determine whether Lipoprotein-mediated uptake and reverse transport
cholesterol biosynthesis is slower than in normal sub- is proposed to play a small role in the removal of
jects. Alternatively, a compromised BBB may disrupt cholesterol from the brain. However, several groups
the tight control of brain cholesterol levels. Finally, have characterized many cholesterol-binding proteins
enhanced catabolism via CYP-dependent mechanisms in the CSF and brain [2,53,131,217,226]. If not di-
or by auto-oxidation may generate oxidative products rectly involved in cholesterol transport out of the brain,
that cross the BBB easily and are excreted from the these proteins may be crucial in transporting cholesterol
brain. Any decrease in cholesterol level may result within the brain from sites of synthesis to sites needed
in enhanced proteolytic digestion of membrane bound for neuronal growth or for the synthesis of neuro-
proteins by exposing them to proteases or secretases in hormones. There may also be cross-talk between 24S-
the case of amyloidosis (AD) [27,60]. Other ramifi- hydroxycholesterol and lipoprotein-dependent trans-
cations may include enhanced oxidation or increased port since 24S-hydroxycholesterol has been shown to
inflammation, leading to neuronal cell death. enhance ApoA1 dependent efflux of cholesterol from
Since cholesterol is independently controlled in the cultured cells. Several lipoproteins synthesized in the
brain, it is unlikely to be strongly influenced by in- brain have been detected in CSF by 2D gels [80].
hibitors of biosynthesis if these do not cross the BBB These include ApoA1, ApoD, ApoJ, and ApoE. Ex-
or by dietary manipulation, since the plasma pool is pression of one ApoE allele (ApoE4) increases the risk
not interchangeable with the brain pool. Given an esti- of late onset AD [32,41,124,127,134,238]. ApoE pro-
46 A.N. Fonteh et al. / Identification of disease markers in human cerebrospinal fluid
Glucose Fatty acid
2-Acetyl-CoA
Acetoacetyl-CoA thiolase
Acetoacetyl-CoA
HMG-CoA synthase
HMG-CoA
HMG-CoA reductase
(rat e limiting enz yme)
Mevalonic acid
Mevolonate k inase
5-phosphomevalonate
phosphomevalonate k inase
5-pyrophosphate mevalonate
Phosphomevalonat e decarboxylase
Isopentenyl-pyrophosphate (IPP) Isopentenyl tRNAs
IPP isomerase
Dimethylallyl-pyrophosphate
Farnesyl- PP transferase
Geranyl-pyrophosphate
Farnesyl- PP transferase
Farnesyl-pyrophosphate Dolichol
Squalene synt hase
Squalene Heme A
Prenylated Squalene epoxidase
proteins
Squalene 2,3-epoxide Ubiquinone
Oxidosqualene cyclase
Lanosterol
3 -Hydoxy- 5-sterol Dehydrocholesterol-
-24 reductase 7-reductase
Desmosterol 7-dehydro-cholesterol
20
18
17 24 25
12
19
C 14
D 15
1 9
A B
3 5
6
HO
HO Cholesterol esters of
Cholesterol
fatty acids. Sulfates,
Myelin/cell membrane glucuronides
24-hydroxy cholesterol
Lipoproteins Neurosteroids Glycosides
Steroid hormones
Fig. 4. Metabolism of cholesterol- Cholesterol is synthesized by a multi-enzyme pathway starting with 2 molecules of acetyl-CoA. Hy-
droxyl-methylglutaryl-CoA reductase is the rate limiting enzyme of this pathway. Several intermediates of cholesterol biosynthesis are substrates
for molecules such as dolichol, Heme A and ubiquinone. Some intermediates are also used for post-translational modification of proteins. Once
formed in the brain, cholesterol is used for nerve cell growth, for the formation of steroids and glucosides. CYP activity converts cholesterol to
hydroxycholesterol that easily crosses the BBB for excretion. Cholesterol is also converted to sulfate or glucuronides dereivatives.
duced in high amounts by astrocytes is important in LDL particles that are the suggested route of cholesterol
cholesterol transport in the brain and is a major com- transport from the liver to organs and vise versa. Scav-
ponent of cholesterol-containing lipoproteins found in enger receptors (SR-B1) or other lipoprotein recep-
CSF. In vitro studies show that ApoE3 is more efficient tors may be responsible for cholesterol removal from
in cholesterol transport and delivery to neurons than the brain. LDL receptor (LDLR) is expressed in the
ApoE4. In addition, ApoE3 from astrocytes stimu- brain [17]. Seven members of the LDLR family have
lates neuronal outgrowth more than ApoE4 expressing been characterized. Typical features of the LDLR in-
cells [156]. Lipoproteins are integral parts of HDL or clude a ligand binding domain, an EGF repeat, a trans-
A.N. Fonteh et al. / Identification of disease markers in human cerebrospinal fluid 47
4
A 6
Cholesterol 1) Sulfate
5 2) Unknown
50000 RT: 15.23
AA: 743045 3) Linoleneate
40000
AH: 38641 4) Linoleate
5) Oleate
3 6) Palmitate
30000 7) unknown
7 8) Stearate
20000 2 9) Arachidonate
1 RT: 28.62
RT: 13.21 RT: 23.98 AA: 57022
AH: 5833
Intensity
10000 AA: 17434 AA: 38448
AH: 3950 AH: 3644 8
9
0 RT: 15.15
RT: 30.42
AA: 223279
AA: 308641
B AH: 15390
AH: 14854
14000
[2H8]-Cholesterol [2H8]-Cholesteryl
octanoate
12000
10000
8000
6000
4000
2000
0
0 5 10 15 20 25 30 35 40 45
Time (min)
Fig. 5. LC-APCI tandem MS of cholesterol in CSF- Cholesterol and esters in CSF from 79 year old female- Positive ion mass spectra obtained
by APCI/MS2 for cholesterol and its derivatives in CSF (A) and SRM of internal standards (B). Peaks for cholesterol sulfate, free cholesterol and
fatty acid esters of cholesterol are numbered from 1–9 (A). Deuterated internal standards are shown on Fig. 5(B).
membrane segment and a cytosplasmic tail containing port of cholesterol from areas of synthesis to other parts
a NPxY motif that controls endocytosis and interac- of the brain where it may be needed for hormone syn-
tion with the phosphotyrosine binding-containing pro- thesis or neuronal growth. Alternatively, cholesterol
teins [17,158,166,185]. ApoE is a common ligand of and its esters may exist in lipoprotein bound particles
the LDLR family. ApoE binds to LDLR and initiates a that are needed for LDLR binding. Studies showing
signaling pathway that promotes cell survival (Akt).
LCAT, cholesterol esters and various constituents of
In addition to lipoproteins, several ATP-binding
lipoproteins in CSF suggest that this mode of choles-
cassette (ABC) transporters assist in the shuttling of
cholesterol from glial cells to neurons [217]. Prelim- terol transport may be important within the brain for
inary results from our laboratory combining 2D-LC neuronal function. These studies underscore the im-
with a linear ion trap mass spectrometer have revealed portance of cholesterol in growth and neurodevelop-
several isoforms of ABC proteins in CSF (unpublished ment. Considerable levels of esterified cholesterol in
data). Studies to determine whether their expression human CSF may reflect a preferred mode of extracellu-
or isoform profiles change in brain diseases or whether lar transport since most cholesterol within brain cells is
their expression is linked to cholesterol are underway free. It remains to be determined whether levels of free
in our laboratory. or esterified cholesterol are altered in CSF from sub-
Cholesterol metabolism is associated with several
jects with neurological diseases compared to normal
diseases including neurodegeneration, hypercholes-
subjects. Moreover, it would be of interest to determine
terolemia, AD, multiple sclerosis, and Niemann Pick
disease type C. CNS cholesterol is mainly unmodified; whether profiles of cholesterol, CYP, lipoproteins, re-
it is not conjugated to fatty acids,or modified by sulfates ceptors and ATP-binding cassette proteins can form a
and glucuronides. Our studies show several cholesterol multiplex biomarker panel for the detection of neuro-
molecular species in CSF including fatty acid esters logical pathologies linked to cholesterol metabolism in
and sulfates (Fig. 5). CSF may be a medium of trans- the brain.
48 A.N. Fonteh et al. / Identification of disease markers in human cerebrospinal fluid
PLA2
PLA2
PLA2, acyltrasnferase
+ PLD
PLA2
COOH
COOH
Docosahexaenoic Acid (DHA) 22:6 Eicosapentaenoic Acid (EPA) 20:5
O
COOH
5-LO (EC 1.13.1134) N
5-HP ETE OH
Arachidonoyl ethanolomide
Arachidonic Acid (AA) 20:4 Auto
Docosa trienes Re solvins OH
-ox id O
31) a tio OH
5-LO
1 3.1 1 n
O
(EC 1.13.1134) 1.
O (E C 15-LO
P4
50 CH3
(Ep
1 2 -L (EC 1.13.1133 ) PGHS ox Arachidonoyl glycerol
yg Isoprostanes
(EC 1.14.99.1 ) en
as
e) HNE, MDH
LTA4 12-HP ETE FAAH
PGG2
EC EETs
1 .1 15-HP ETE HETEs
1 .1 AA + ethanolamine or
.7
EC 3.3.2.6 PGHS glycerol
(EC 1.14.99.1 )
LT
5-HETE
C4
TXBS 11-Dehydro-TX B2
TXA2
-
TXB2
S
PGH 2 (EC 5.3.99.5)
(E
C
LTB4
4.
41
.2
Lipoxin PGIS
)0
(EC 5.3.99.4) PGDS EC 1.1.1.188
(EC 5.3.99.2) PGD 2 11-epi-P GF2
EC 1.14.1330
LTC4 PGI 2
20-OH-LTB4 PGES
EC 2.3.2.2 (EC 5.3.99.3)
12-Keto-LTB 4
PGF2
PGJ2
20-COOH-LTB4 LTD4
EC 1.1.1.184
EC 2.3.2- EC 1.1.1.189 EC 1.1.1.196
6-Keto-PGF 1
LTE4
15-Keto-P GF2
PGE2
EC 2.3.2-
LTF4 6-Keto-PGE1
Products of PUFA oxygenation and endocannabinoids act on specific receptors
(G-protein coupled) to control physiologic processes such as sleep,
pain, secretion or inflammation.
Fig. 6. Oxygenation of PUFAs and endocannabinoid biosynthesis- PUFAs are released from the lipid bilayer by the action of PLA2 enzymes.
Free PUFAs such as AA, DHA or EPA are metabolized by major oxygenases pathways numbered 1–4. 1) Lipoxygenases (5-LO, 12-LO or 15-LO)
generate hydroxyperoxyeicosatetraenoic acid (HPETE) that are subsequently converted to leukotrienes (5-LO) or lipoxins (15-LO). 2) PUFAs can
also be metabolized via the Prostaglandin H synthase (PGHS) pathway to form PGH2 . Terminal synthases convert PGH2 to prostanoids. PGIS,
PGES, PGDS and TXBS are the major terminal synthases responsible for PGI2 , PGE2 , PGD2 and TXB2 biosynthesis. 3) Cytochrome P450
monooxygenases (P450) convert PUFAs by hydroxylation to hydroxyeicosatetraenoic acids (HETEs), by allylic oxidation to generate isomers
of HETEs and by olefin bond epoxidation to generate regioisomers of epoxyeicosatetraenoic acids (EETs). 4) Auto-oxidation of PUFAs can
generate isoprostanes or neuroprostanes and oxidized lipid products such as malonaldeylde and 4-hydoxynonenal (HNE). 5) DHA and EPA can
serve as substrates of these oxygenases to for docosatrienes and resolvins, respectively. 6) A combination of acyltransferase activity in concert
with PLD is proposed for the biosynthesis of the endocannabinoid, arachidonoyl ethanolamide (AEA). AEA levels in the brain may be controlled
in part by fatty acid amide hydrolase (FAAH) activity. Overall, products from these pathways are implicated in processes such as sleep, pain,
secretion and inflammation.
2.4. Fatty acids, eicosanoids and endocannabinoids where n denotes the total number of carbon atoms and
the numbers denote the presence of a double bond 6
Compared to other tissues, the brain is highly en- or 3 carbon atoms from the terminal omega (ω) carbon
riched with PUFAs . The major classes of PUFAs in atom. The major PUFA species are arachidonic acid
the human brain belong to the n − 6 or n − 3 classes (AA, 20:4, n-6), eicosapentaenoic acid (EPA, 20:5, n-A.N. Fonteh et al. / Identification of disease markers in human cerebrospinal fluid 49
TXs PGs LTs HETEs FAs NL:
100 3.26E5
TIC F: MS
29.71 CSF_1050_
90 15-deoxy-PGJ 2 1
80
Relative Abundance
29.43
29.99
70 34.46
60
50
40 29.32
40.42
30
20 14.15 14.87
13.59 18.76 36.95
3.78 40.97
4.12 21.38 28.03 43.86
10 5.12
9.25
47.60
0 0 10 20 30 40 50
Time (min)
Fig. 7. LC-ESI tandem MS of eicosanoids, isoprostanes and fatty acids in CSF- After the addition of 2 ng deuterated internal standards to
400 ul CSF, eicosanoids and free fatty acids were extracted using ethyl acetate. Samples were reconstituted in water/methanol (70:30) containing
0.01% acetic acid. LC-negative ion ESI-MS2 was performed with SRM of parent and product ions for thromboxanes (TX), prostanoids (PTs),
leukotrienes (LT), isoprostanes and fatty acids (PUFAs).
3) and docosahexaenoic acid (DHA, 22:6, n-3) [146]. still awaits discovery [115,140]. PUFA levels may be
Mammals cannot synthesize AA, EPA and DHA be- influenced by uptake from the diet, biosynthesis and
cause they lack the desaturase enzyme required to in- release/catabolism within the brain.
troduce a double bond at the n-6 and n-3 position [125]. Phospholipases release PUFAs from membrane
Therefore, the major precursors of PUFAs must be pro- phospholipids. Released PUFAs are converted to
vided by the diet and are thus referred to as essential bioactive lipids or other signaling molecules (Fig. 6).
fatty acids (EFAs). Linoleic acid (18:2, n-6) and α- Bazan has recently reviewed the significance of PU-
linolenic acid (18:3, n-3) are the major plant oil-derived FAs in synaptic lipid signaling [14]. Likewise, the im-
EFAs. EFAs are important in brain development and portance of phospholipases in releasing PUFAs from
function [54,125,186,214]. Once ingested, EFAs are phospholipids and their potential role in brain func-
subjected to elongation and desaturation to form sev- tion has been reviewed [73]. However, the isoforms
eral PUFAs including AA and DHA [199]. Consider- of phospholipases and the source and mechanism by
able evidence suggest that PUFAs diffuse through the which PUFAs may be mobilized for eicosanoid for-
lipid bilayer and FA transporters have also been im- mation are not yet defined in CSF. Given the pres-
plicated in their uptake [173,176]. Upon internaliza- ence of several oxygenases and terminal synthases that
tion, PUFAs are converted to CoA-derivatives by acyl- can convert PUFAs into eicosanoids, resolvins and do-
CoA synthetases (ACS), which require ATP. PUFA- cosanoids/neuroprostanes [139,186] (Fig. 6), it is im-
CoAs are utilized for the synthesis of phospholipids, portant to determine the PUFA composition of brain
triacylglycerides or can be utilized for energy gener- lipids and the ancillary pathways that control their avail-
ation via mitochondrial β-oxidation [38,130,145,157]. ability to neurons. Using LC-negative ion ESI tandem
PUFA-CoAs also function as signaling molecules [38, MS, we have detected many eicosanoids, isoprostanes
93]. PUFA levels and metabolism have been implicated and fatty acids in human CSF (Fig. 7). Changes in the
in several neurological processes. These include neural amounts of these molecules in CSF may not only be
outgrowth, neurodegeneration, depression, membrane an indication of physiologic process in the brain, but
activity of receptors and the sodium pump, synaptic could suggest pathologic conditions.
lipid signaling and plasticity [29,86,115,140,142,194, In addition to eicosanoids, another AA-derived class
214,232]. Although PUFAs are thought to be involved of molecules implicated in pain reduction, motor regu-
in diseases ranging from AD, PD, stroke, and anxi- lation, learning, memory, reward and appetite has been
ety, the mechanisms that would account for their roles recently described [57,100,215]. These molecules are50 A.N. Fonteh et al. / Identification of disease markers in human cerebrospinal fluid
A B
RT: 0.00 - 68.16 RT: 0.00 - 68.16
31.27 NL: RT: 31.30 NL: 1.30E8
100 1.39E8 100 TIC F: + c sid=-30.00 Full pr
Relative Abundance
Relative Abundance
TIC F: MS 184.14@-10.00[ 400.00-950.00]
80 80 MS ICIS
PL_CSF_100
6_Parent_NL PL_CSF_1006_Parent_NL_320
60 60 6
_3206
32.76
40 30.15 40
33.13 45.06 RT: 16.77
20 6.51 6.88 20 RT: 45.09
27.17
59.96 61.45 RT: 6.34
0 0
0 10 20 30 40 50 60 0 20 40 60
Time (min) Time (min)
C D
464.44 760.88
100 100
Relative Abundance
Relative Abundance
80 80
60 60
485.52
847.74
40 550.60 40
640.45 867.38
725.32 20 734.70
20 838.08 810.68
891.14 706.69 834.88 876.82 913.79
0 0
400 500 600 700 800 900 650 700 750 800 850 900 950
m/z m/z
E 703.64
F 526.68
100 731.74 100
Relative Abundance
Relative Abundance
80 80
60 60
40 813.64 40
20 759.64 20
675.94
819.30 867.15 946.48 451.40 485.44 522.51 530.84 585.44 635.64
0 0
650 700 750 800 850 900 950 400 450 500 550 600 650
m/z m/z
Fig. 8. LC-ESI tandem MS of phospholipids in human CSF- 10 ng phospholipids standards were added to 200 µl CSF and total lipids were
extracted using chloroform and methanol. Samples were reconstituted in chloroform/methanol (2:1) and LC-positive ion ESI-MS2 was performed
with parent ion monitoring for choline containing phospholipids or neutral ion loss for PE, PI, PG and PS. Figure 8(A) shows the TIC obtained
for all phospholipids classes while Fig. 8(B) shows a chromatograph of choline-containing molecular species. Figure 8(C) shows spectra of
sphingosylphosphocholine/phosphocholine species eluting at 1–20 min, PC at 20–35 min (Fig. 8D), sphingomyelin at 35–45 min (Fig. 8E) and
lysophosphatidylcholine/PAF at 45–65 min (Fig. 8F). Over 450 choline-containing molecular species are identified in CSF.
known as endogenous cannabinoid ligands or endo- clarified endocannabinoid signaling. Upon release by
cannabinoids. The first endocannabinoid was initially postsynaptic neurons, endocannabinoids diffuse back
identified as an ethanol amide derivative of AA and to the presynapatic neurons where they act on CB1 re-
termed arachidonamide or anandamide (Fig. 6). A sec- ceptors to inhibit release of neurotransmitters such as
ond endocannabinoid also contains AA that is attached GABA and glutamate [3]. Interestingly, recent stud-
to glycerol (2-arachidonoyl glyceryl ester or 2AG). ies show that CB1 receptors are coupled to another
Endocannabinoids are synthesized and released from lipid second messenger, ceramide [3]. Also a pro-
neurons upon stimulation [215]. Inactivation of anan- posed pathway for endocannabinoid-induced apoptosis
damide and 2-AG is accomplished by rapid uptake via involves ceramide. Structural similarities between 2-
a membrane transporter (AMT) followed by intracel- AG and lysophosphatidic acid (LPA) also suggest pos-
lular enzymatic degradation by fatty acid amide hydro- sible cross-talk between these lipid-signaling ligands
lase (FAAH) [100,215]. Endocannabinoids bind to the and their receptors. Measurement of these signaling
CB1 receptor. Similar to the CB1 receptor, the distribu- molecules in CSF and correlation of any changes in
tion of AMT and FAAH are high in the hippocampus, their levels with enzyme activity or protein levels will
cerebellum and cerebral cortex. Recent studies have represent disease markers and provide tangible path-A.N. Fonteh et al. / Identification of disease markers in human cerebrospinal fluid 51
ways that can be influenced by therapy. 4. Proteomic strategies for the identification of
lipid-metabolizing proteins in CSF
While lipidomic approaches reveal lipid composi-
3. Methods for measuring lipids in CSF tion, protein expression and the putative biosynthetic
pathways in CSF that account for specific changes in
lipid levels or distribution can best be understood if the
Early methods for analyzing the lipidome depended
levels and activity of metabolizing enzymes and trans-
on organic extraction coupled with thin layer chro- port proteins are determined. For example, if the level
matography (TLC) or high performance liquid chro- of PAF is found to change in CSF for a specific disease,
matography (HPLC). Further structural identification various possibilities may account for this change. An
of lipid classes and molecular species were possible increase in PLA2 activity resulting in the generation of
only after extensive digestion of lipids, derivatization lysophosphatidylcholine (LPC) and subsequent acety-
and further chromatography (Table 2). This laborious lation of LPC by acetyltransferase can result in an in-
strategy employed over many decades revealed sev- crease in PAF. Likewise, a decrease in PAF acetylhy-
eral metabolic pathways. However, low sensitivity and drolase activity may result in a build up in PAF levels.
the labor-intensive process limited extensive profiling Alternatively, an increase in PAF acetylhydrolase activ-
of lipid molecular species. Advances in mass spec- ity will decrease PAF levels. Similar arguments can be
trometry have made it possible to detect thousands of applied to most lipid signaling molecules (eicosanoids,
lipid molecular species. Initial advances in lipid anal- DAG, IP3 etc). One way of understanding why there is
yses were made by Murphy and colleagues using fast a change in the lipidome is to examine all the proteins
atomic bombardment [44,117]. The advent of soft in the biosynthetic and catabolic pathways associated
with specific lipid molecules as outlined on Fig. 9. In
ionization processes such as ESI-MS 2 [108,171,175]
addition to the use of systematic animal knockout mod-
or APCI-MS2 [35] have revolutionized metabolic pro-
els, enzyme analyses or use of proteins chips, 2D gel
filing of lipids. Not only are these modern instru-
electrophoresis and shotgun sequencing of CSF pro-
ments sensitive, but enzyme digestion and derivatiza- teins are the most valuable approaches for interrogat-
tion procedures are not needed for most lipids. By ing changes in lipid-metabolizing proteins. In 2D gel
combining LC with MS, many more molecular species electrophoresis, CSF proteins are identified by their
of lipids can be measured. For example, Fig. 8(A) isolelectric point (pI) and by their size. A 2D map is
shows the total ion current (TIC) obtained from CSF then obtained by staining gels with fluorescent dyes
phospholipids monitored using LC tandem MS with or with silver stains [92,101]. Densitometric profiles
parent ion monitoring of PC or neutral ion loss for and normalized total densities are obtained for relative
PE, PI, PS and PhosGl. Figure 8(B) shows the TIC quantification. Proteins on 2D gels are identified by
of choline-containing phospholipids species. Spectra extraction and MS sequencing, immunoblotting, or by
of choline-containing molecular species correspond- reference to published 2D database (SWISS-2DPAGE,
ing to sphingosylphosphocholine/phosphocholine, PC, http://www.expasy.org/ch2d/). While very laborious,
sphingomyelin and PAF/LPC are shown in Figs 8(C), 2D gels are useful because they resolve posttranslation-
8(D), 8(E) and 8(F), respectively. Over 450 different ally modified isoforms of proteins [101].
molecular species were identified in human CSF. Sim- In shotgun sequencing, CSF proteins are reduced,
denatured and alkylated prior to enzyme digestion, of-
ilar to PC, hundreds of PE, PI, PS and PhosGl molec-
ten with trypsin. Peptide fragments are subsequently
ular species were identified in CSF (data not shown).
resolved using capillary columns and detected using
Other studies use 2D mass spectrometry with either an ion trap mass spectrometer. Sequences are ob-
parent ion monitoring, neutral ion loss of specific lipid tained using several software packages such as Se-
fragments or multiple reaction monitoring (MRM) to quest/Bioworks and multidimensional protein identifi-
identify hundreds of lipid species [102,207] (Table 2). cation technology [225]. Improvements in mass spec-
Information from these studies is important in obtain- trometric methods and the availability of human se-
ing structure, composition of lipids in cells, turnover of quence databases makes it possible to analyze complex
lipids and characterization of lipid synthesis/transport mixtures of peptides in CSF by liquid chromatography
and degradation pathways. coupled to tandem mass spectrometry. The handling of52 A.N. Fonteh et al. / Identification of disease markers in human cerebrospinal fluid
LIPIDS
Uptake or biosynthesis Binding and transport Catabolism
(anabolism)
Lipases, synthases,
Apolipoprotein, lipid oxidases, peroxidases,
Composition of lipid binding proteins, dismutases, cytochrome
molecular species albumin, lipocalin P450
Growth and
Membrane Cell Inflammation Oxidation
regeneration
structure
of nerve cells Signaling
(receptors, ion
channels)
Protein processing & Apoptosis of
vesicle trafficking nerve cells
Pathophysiological changes in the brain characteristic of specific diseases.
Fig. 9. An overview of lipid metabolism and its pathological significance- A comprehensive analysis of CSF lipids composition by LC tandem
MS will reveal how lipids get into the brain, their usage once in the brain and physiologic processes that they influence. Lipid compositional
data are complemented by measures of proteins that metabolize lipids in the CSF. This approach will result in a better understanding of the
pathophysiology of brain diseases.
data using automated data processing with improved al- teins may be classified by their functions in biosynthe-
gorithms has increased confidence in the identification sis, transport or catabolism/degradation of lipids. Our
of proteins [42,63]. studies show nine different prostaglandin D synthase
Once both the identities and the levels of CSF pro- (PGDS) isoforms, 4 apolipoprotein (ApoA1) isoforms,
teins have been determined, apparent differences in the and 9 apoliprotein J (Apo J) isoforms in CSF [80].
proteomes of normal and diseased individuals can be These initial studies are being expanded using 2D-
used to explain the mechanisms by which changes in capillary LC in combination with high resolution, high
the lipidome occur in disease. Mechanistic changes sensitivity linear ion trap mass spectrometry. With this
could be reflected in observed differences in protein approach, we have been able to increase the dynamic
abundance (COX, 5-LO, PLA 2 , etc.), or variance in range more than 7 fold such that less abundant pro-
the levels of protein activation (e.g., phosphorylation of teins not visualized on 2D gels are discovered with high
cPLA2 ). This combinatorial strategy will be useful in statistical confidence [104].
assessing confidence for novel lipid or protein biomark- Lipid metabolizing proteins that we have found in
ers and allow for direct comparison between variable CSF include enzymes with a variety of functions such
levels attributable to chance, diet, genetic variability as: ligases, synthases, transferases, lipid binding pro-
and environment and changes related to the onset and teins, ABC cassette proteins, all major lipoprotein iso-
progression of disease. Furthermore, this strategy will forms, lipases, COX, LO, CYP450 enzymes, kinases
prove to be clinically useful in helping to develop inter- for choline, ethanolamine, inositol and PI, and recep-
vention strategies involving drug inhibitor discovery as
tors for steroids and prostaglandins (unpublished data).
well as in the rational design of diet/lifestyle changes
Bazan and colleagues, and others have extensively
necessary for disease prevention.
reviewed roles of COX in brain physiology [13,16].
4.1. Some examples of lipid metabolizing proteins of Other studies have shown close association of cPLA 2
interest in CSF to amyloid plaques in AD brain and the importance
of phospholipases in the brain has been reviewed [47,
Several 2D gel electrophoresis studies have revealed 70,72,73,201]. The importance of apolipoproteins in
lipid-metabolizing proteins in human CSF. These pro- lipid transport in the brain has been suggested based onA.N. Fonteh et al. / Identification of disease markers in human cerebrospinal fluid 53
Table 2
Methods for profiling lipids classes
LIPIDS Lipidomic procedures
Phospholipids 1) Normal phase HPLC or TLC followed by PLC digestion and derivatization of diglycerides.
([76] and references therein)
2) LC-ESI-MS2 [102,207]
3) ESI-MS2 [91,171,198,235]
4) NMR [25,26]
Ceramides, 1) LC-ESI-MS2 [34]
sphingomyelin. 2) ESI-MS2 [91]
Cholesterol and 1) LC-ESI-MS2 [91,132,172]
steroid hormones 2) GC/MS [187]
Fatty acids and eicosanoids 1) Saponification, derivatization and GC or GC/MS [7,89]
2) LC-ESI-MS2 [83]
3) LC-APCI-MS2 [83]
4) NMR [107,228]
Triacylglycerides 1) ESI-MS2 [91]
2) APCI-MS [36]
cell studies showing increased secretion of ApoE and diminished by hydrolysis of the acetyl moiety or the
cholesterol by astrocytes over-expressing ApoE [156]. short-chain oxidized esters.
Close association of ApoE4 alleles to late onset AD PAFA hydrolysis of the sn-2 ester bond of platelet-
and epidemiological studies showing a link between activating factor (PAF) and PAF-like oxidized phospho-
high cholesterol to AD has given more credence to a lipid species release an acetate or oxidized moiety and
link between ApoE and cholesterol transport [137,150, lysophosphatidylcholine, thus attenuating the bioactiv-
156]. However, the functions of other CSF lipopro- ity of PAF by reducing its levels. Several PAFA iso-
teins are not known. Given that ApoA1 interacts with forms (1 secreted and 4 intracellular) have been cloned
the ABC cassette transporter and ATP phospholipid or characterized based on substrate specificity, cellu-
binding proteins to transport cholesterol in cells [223, lar localization and structure. Isoform1b forms a G-
237], it is likely that a similar mechanism takes place protein-like complex consisting of two catalytic sub-
in the brain. While of great importance to brain lipid units (a1 and a2) and a regulatory subunit (b). Another
metabolism, these can not be fully addressed because well-characterized isoform of PAFA consists of a sin-
of the limited knowledge in the living brain. We will gle polypeptide and is homologous to plasma PAFA.
concentrate on our discovery of groups of enzymes that Isoform II has anti-oxidant properties and a catalytic
exemplify synthesis and degradation of bioactive lipid triad of amino acids characteristic of most esterases,
molecules. Specifically, we will review platelet ac- including the G motif found in most serine esterases
tivating factor acetylhydrolase and CYP enzyme iso- and lipases [4,114].
forms that are important in cholesterol, neurosteroids Deficiency in plasma PAFA is associated with stroke,
and PUFA metabolism. asthma, myocardial infarction, brain hemorrhage and
non-familial cardiomyopathy [4,114]. Animal studies
4.2. Platelet-activating factor acetylhydrolase (PAFA) and preclinical studies show that recombinant plasma
PAFA can prevent inflammation and thus has the poten-
At the site of inflammation, several cells are recruited tial of controlling human inflammatory diseases [211,
to counteract tissue damage. Cells are recruited to 222]. As much as two thirds of PAFA activity is asso-
these sites by mediators such as platelet-activating fac- ciated with LDL and the remainder is found in HDL
tor (PAF). PAF is a phospholipid that is a potent me- particles. Expression of PAFA is regulated by bacterial
diator of inflammation. PAF is formed by sequential liposacharides (LPS), cytokines, PAF and the lyso-PAF
action of PLA2 on PC to generate LPC that is subse- concentration. PAFA levels are strongly correlated to
quently acetylated at the sn-2 position by acetyltrans- LDL cholesterol and an increase in PAFA has been
ferase. PAF-like molecules can also be formed by ox- shown in essential hypertension, vascular disease, is-
idative fragmentation of PUFAs at the sn-2 position chemic stroke, diabetes mellitus, rheumatoid and non-
of PC [46,77,97]. PAF receptor antagonists, throm- rheumatoid arthritis [8,159,211,231]. In other diseases
boxane B2 (TxB2 ) and leukotrienes C 4 (LTC4 ) reduce such as asthma, Crohn’s disease, sepsis, acute myocar-
PAF activity [69]. The biological properties of PAF are dial infarction, multiple organ failure, juvenile rheuma-54 A.N. Fonteh et al. / Identification of disease markers in human cerebrospinal fluid
toid arthritis and systemic lupus erythematosis, PAFA eral neurosteroids that are known to influence its func-
has been shown to decrease. Given the proposed role of tion. Steroids are implicated in behavior, mental ill-
PAF in plasticity and the role of inflammation in brain ness, activation of the immune system, fatigue, de-
diseases, PAFA levels may change with diseases and pression, some forms of epilepsy and dementia [131].
control of PAFA activity may influence neurological The role of CYP family enzymes in PUFA metabolism,
pathologies. steroid biosynthesis and neurotransmitter modification
underlines their importance in brain function.
4.3. CYP450 and brain function
Cytochrome P450 (CYP) are phase 1 enzymes in- 5. Lipid-related mechanisms in neurological
volved in oxidative activation /deactivation of com- diseases
pounds or toxins [233]. CYPs belong to four major
families (CYP1, CYP2, CYP3 and CYP4) that are fur- The brain is composed of cells (neurons, astrocytes,
ther subdivided into subfamily members. The liver is a glial cells, oligodendrocytes, etc) that communicate via
very rich source of CYP [233]. However, whole brain chemical or electrical signals in response to stimula-
CYP activity is approximately 1% activity of the liver. tion. These cells are highly shielded to prevent fluctu-
Specific brain regions or cell types may have higher ations in ion or chemical concentrations and are sepa-
expression of CYP than others. For example, the major rated from the rest of the body by a blood brain barrier.
CYP families are localized at the BBB, choroid plexus The cell membranes of cells are critical in maintaining
and posterior pituitary [148,168,197,233]. CYP1 fam- ion and chemical balance in the brain. Therefore, dras-
ily proteins are often expressed in basal ganglia and tic changes in lipid composition may have significant
cerebellum of human brain. CYP2, especially the D6 pathological ramifications (Fig. 9).
subfamily has been mapped to the BBB of human brain,
are expressed mainly in arachnoid, choroid plexus and 5.1. Structure and distribution
vascular areas as well as in neuronal cells. CYP3 family
enzymes are mainly localized in pituitary cells and are Cell membranes are composed of complex lipids (di-
probably involved in the regulation of growth hormone. acyl glycerophospholipids, sphingomyelin, ether phos-
Higher levels of CYP are associated with oxida- pholipids, plasmalogens and cholesterol) and contain
tive stress. Drugs, xenobiotics and endogenous com- ion channels as well as several receptors for neuro-
pounds are known to be substrates of CYP. Endogenous transmitters, neuropeptides or neurohormones. The
substrates include neurotransmitters such as dopamine, complexity of lipids is reflected in important ways.
neurosteroids and PUFAs. PUFAs are modified by First, lipids are composed of different classes and sub-
CYP1A, 2A, 2C, 2D, 2E, 2J and 4A [23,103,131]. classes (e.g., PC, PE and plasmalogen). Second, each
Arachidonic acid is converted to epoxygenase metabo- class is further differentiated by the fatty acid content
lites (14,15, 11,12, 8,9, 5,6-epoxyeicosatetraenoic acid, that make up thousands of molecular species. Third,
EETs) or ω-terminal hydroxylase metabolites (20, 19, some complex lipids such as glycerophosphatidylinos-
18, 17, 16 hydroxyeicosatetraenoic acids, HETES) itols are modified by phosphorylation of the inositol
and lipoxygenase-like metabolites (15-, 12-, 9-, 8-, 5 base at the 3, 4 or 5 position to generate PIP, PIP 2
HETES) [45,131,234]. In the brain, EETs are pro- or PIP3 derivatives. Fourth, complex lipids are asym-
duced by astrocytes close to cerebral microvesicles and metrically distributed within cell membranes and lipid
are likely involved in control of cerebral blood flow. composition varies within organelles of the same cell.
The pituitary and hypothalamus both produce HETEs For example, lipid rafts have been shown to be rich
that stimulate neuropeptide formation. HETEs are also in PI-anchored proteins and cholesterol while PUFAs
potent vasoactive agents that modulate normal brain seem to initially accumulate in the nuclear membrane
function and are altered in cerebrovascular pathologies. before being remodeled to organelles [155,227]. Fifth,
CYPs are also involved in cholesterol and steroid the difference in distribution is not limited to specific
biosynthesis. Steroid hormones influence brain growth cells but also to specific regions of the brain. Sixth,
and development [131,144,182,191]. Steroids pro- these complex lipids are subject to constant remodel-
duced by the adrenal glands and the gonads readily ing of fatty acyl groups and are rapidly degraded by
cross the BBB. In addition to steroids synthesized out- phospholipases (A 2 , C or D) to generate several lipid
side the CNS, the brain is the site of synthesis of sev- derivatives [14]. This complexity coupled with the factYou can also read
