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List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals. Reprints of Papers I and II are published with permission from Elsevier Ltd.

I Garscha, U., Nilsson T., and Oliw, E.H. (2008) Enanti-

omeric separation and analysis of unsaturated hydroperoxy fatty acids by chiral column chromatography-mass spec-trometry. J. Chromatogr. B. 872, 90-98.

II Oliw, E.H., Garscha, U., Nilsson T., and Cristea M. (2006) Payne rearrangement during analysis of epoxyalcohols of linoleic and alpha-linolenic acids by normal phase liquid chromatography with tandem mass spectrometry. Anal. Biochem. 354, 111-126.

III Nilsson, T., Martínez, E., Manresa, A., and Oliw, E.H. LC-MS/MS analysis of 7,10-dihydroxyoctadecenoic acid, its isotopomers, and other 7,10-dihydroxy fatty acids formed by Pseudomonas aeruginosa 42A2. Submitted manuscript.

IV Nilsson, T., Ivanov, I.V., and Oliw, E.H. LC-MS/MS analysis of epoxyalcohols and epoxides of arachidonic acid, and their oxygenation by recombinant CYP4F8 and CYP4F22. Submitted manuscript.

Contents

1 Introduction .........................................................................................11 1.1 Oxylipins ....................................................................................11 1.2 Lipoxygenases ............................................................................12

1.2.1 Nomenclature of lipoxygenases.............................................13 1.2.2 Lipoxygenases in humans......................................................13 1.2.3 Lipoxygenases in human skin................................................15

1.3 The case of ichthyosis ................................................................16 1.4 Cytochrome P450 .......................................................................18

1.4.1 CYP4 Family .........................................................................19 1.4.2 CYP4F8 .................................................................................19 1.4.3 CYP4F22 ...............................................................................20

1.5 Mass Spectrometry .....................................................................21 1.5.1 Electrospray ionization ..........................................................22 1.5.2 Linear quadrupole ion trap.....................................................22 1.5.3 Analysis of fatty acid derivatives by mass spectrometry.......23 1.5.4 Identification of detected ions................................................24

1.5.4.1 Charge-remote or charge-driven fragmentation .........24 1.5.4.2 Uncharacteristic fragmentation...................................25 1.5.4.3 Characteristic fragmentation.......................................27 1.5.4.4 Verification of fragments............................................27

2 Aims.....................................................................................................29

3 Methods ...............................................................................................30 3.1 Preparation of hydroperoxides ...................................................30

3.1.1 Synthesis of hydroperoxides ..................................................30 3.1.2 Purification of hydroperoxides ..............................................30 3.1.3 Separation of hydroperoxides ................................................31

3.2 Preparation of epoxyalcohols .....................................................31 3.2.1 Synthesis of epoxyalcohols....................................................31 3.2.2 Separation of epoxyalcohols ..................................................32

3.3 Preparation of epoxides ..............................................................33 3.4 Preparation of diols ....................................................................33 3.5 Preparation of isotopomers.........................................................34 3.6 Expression system ......................................................................34 3.7 Enzyme assay .............................................................................35

3.8 Analysis of epoxyalcohols in human cornea ..............................35 3.9 Analysis of metabolites ..............................................................36

4 Results and discussion .........................................................................37 4.1 Separation of hydroperoxides (Paper I)......................................37 4.2 MS/MS analysis of hydroperoxides (Papers I and II) ..................38 4.3 Separation of epoxyalcohols (Papers II and IV).........................39 4.4 MS/MS analysis of epoxyalcohols (Papers II and IV) ..................40 4.5 MS/MS analysis of diols (Paper III)...........................................42 4.6 Detection of epoxyalcohols in corneal tissue (Paper IV) ...........44 4.7 Catalytic properties of CYP4F22 (Paper IV) .............................45 4.8 Catalytic properties of CYP4F8 (Paper IV) ...............................47

5 Conclusions .........................................................................................49

6 Future perspectives ..............................................................................50

Populärvetenskaplig sammanfattning ...........................................................51

Acknowledgements.......................................................................................53

References.....................................................................................................55

Abbreviations

18:1n-9 9Z-Octadecenoic acid 18:2n-6 9Z,12Z-Octadecadienoic acid 18:3n-3 9Z,12Z,15Z-Octadecatrienoic acid 20:1n-11 9Z-Eicosenoic acid 7,10-(OH)2-18:0 7,10-Dihydroxyoctadecanoic acid 7,10-(OH)2-18:1 7,10-Dihydroxyoctadecenoic acid 7,10-(OH)2-20:1 7,10-Dihydroxyeicosenoic acid APCI Atmospheric pressure chemical ionization BCD Bietti’s crystalline corneoretinal dystrophy COX Cyclooxygenase CYP Cytochrome P450 EET Epoxyeicosatrienoic acid eLOX3 Epidermal lipoxygenase 3 ER Endoplasmatic reticulum ESI Electrospray ionization GC-MS Gas chromatography-mass spectrometry HEET Hydroxyepoxyeicosatrienoic acid HETE Hydroxyeicosatetraenoic acid HPETE Hydroperoxyeicosatetraenoic acid HPLC High-performance liquid chromatography HPODE Hydroperoxyoctadecadienoic acid HXA3 Hepoxilin A3 HXB3 Hepoxilin B3 KETE Ketoeicosatetraenoic acid LC-MS Liquid chromatography-mass spectrometry LI Lamellar ichthyosis LOX Lipoxygenase LT Leukotriene MALDI Matrix-assisted laser desorption/ionization MS Mass spectrometry MS/MS Tandem mass spectrometry MS3 Triple tandem mass spectrometry NMR Nuclear magnetic resonance NP-HPLC Normal phase-high-performance liquid chromatography PG Prostaglandin RP-HPLC Reverse phase-high-performance liquid chromatography

RT-PCR Reverse transcriptase-polymerase chain reaction SIM Selective Ion Monitoring SNP Single Nucleotide Polymorphism THETE Trihydroxyeicosatrienoic acid TIC Total ion current TLC Thin layer chromatography TXA2 Tromboxane A2

Preface

‘Think you, ‘mid all this mighty sum

Of things for ever speaking, That nothing of itself will come,

But we must still be seeking?

William Wordsworth, 1798

Isaac Newton used the phrase ”hypothesis non fingo”, when debating what caused gravity. Why does gravity exist? Since he did not know, he rational-ized that empirical data had to speak for itself, and speculations were for philosophers. “I feign no hypothesis” as the expression is usually translated, or “shut up and calculate”, as some people likes to put it. In an ironical way, I find the expression perfect to describe the essence of mass spectrometry. Who knows exactly what is going on in there? So many theories. So many different mechanisms. And in the end – large diversities between theories and real world data output. We are back in a world of interpretations.

With this, I don’t want to say that the theories are wrong. They are mostly not. But there are uncertainties. Which mechanism is really applicable for this specific ion? Why does it look like a less energetic favorable mechanism is occurring here? Even so it has to be agreed upon that mass spectrometry is a marvelous analytical tool. Just like Newton’s laws it can tell us much, without answering the question why.

This thesis is about using mass spectrometry for identifying compounds derived from fatty acids. It tries to explain that the fragmentation patterns described are based on scientifically valid mechanisms, and are verified by applicable methodology. But just remember what I said about hypothesis non fingo. The research project behind the thesis is also pragmatic. Mass spec-trometry is utilized to identify substrates and metabolites generated. Finding unique “fingerprint” spectra is one piece in this puzzle. Interpretation of the spectra is another. To be able to explain the mechanism behind the generated ions is a third. The project is also biologically based, and the scope has never been a full validation of methods in use, but instead to improve methods point-blank.

The epigraph above is a verse from the poem Expostulation and Reply, by the English naturalist William Wordsworth. The poem is also about the question “Why?”, but perhaps even more about the question “How?”. Why do we achieve knowledge? How do we achieve knowledge? In accordance with the Romantic Movement the implied answer to the verse is no. As a more scientifically minded person, I would answer the question with a yes. The journey starts here.

Tomas Nilsson Uppsala, October 2009

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1 Introduction

1.1 Oxylipins

As more and more people come aware of, fatty acids are not only energetic nutrients to love or hate, but important multi-functional constituents of the human body. Actually, most living organisms are dependent upon them, since they are both important components in cell membranes etc., but also mediate cell communication, mostly via their metabolites [1]. The essential fatty acids have that in common that they all are polyunsaturated with a long carbon chain of 18-22 carbons. Many of the special properties of these fatty acids are due to the cis-conformation of the double bonds, making the struc-ture flexible. In membranes this feature is utilized to optimize fluidity by changing fatty acid composition, and the open structure facilitate reaction with molecular oxygen [2], to produce biological active metabolites, so called oxylipins. The double bonds are here crucial since few enzymatic systems have the capability to break a C–H bond in a non-allylic system.

Oxylipin is the collective name of enzymatically oxygenated fatty acids. Some use the word only for non-animal compounds, and instead use the word eicosanoids for structures derived from arachidonic acid (eikosi; greek for twenty). In mammals arachidonic acid is the major precursor for these derivatives, but in many other organisms linoleic or linolenic acid have this position. Oxylipins include, inter alia, hydroperoxides, oxo-, epoxy-, keto-, hydroxy-, dihydroxy-, trihydroxy-, and epoxyalcohol compounds. All of them have been found with biological activity, but different oxylipins are formed in different species, and have different importance. There are several enzymatic systems responsible for the oxygenation of fatty acids to oxylip-ins. Hydroperoxides, which are important intermediates for further biotrans-formation, can be formed by lipoxygenases, diol synthases, cyclooxy-genases, and �-dioxygenases. They can also be formed non-enzymatically, a feature utilized further on in this thesis. Hundreds of other oxylipins consti-tutively have their origin from hydroperoxides.

In humans there are three main enzymatic systems responsible for the oxygenation of arachidonic acid to eicosanoids; cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 (CYP)(Figure 1) [3,4]. Among the oxylipins produced by cyclooxygenases one notice the important media-

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tors prostaglandins and thromboxanes, but this thesis will only discuss oxylipins generated by lipoxygenases or cytochrome P450 enzymes.

Figure 1. Metabolites formed from arachidonic acid by the three major pathways.

1.2 Lipoxygenases

The first lipoxygenase was discovered in soybeans during the early 1930s [5]. For long, the theory said that this group of enzymes was only related to plants and lower organisms, and it was not until 1974 the first animal li-poxygenase was disclosed, when human platelets were incubated with ara-chidonic acid, and 12-HPETE was detected [6]. Today, lipoxygenases have been realized as an essential system in most type of living organisms and have been found in, inter alia, mammals, plants, fungi, invertebrates, and bacteria [1,7,8]. Lipoxygenases are iron or manganese containing enzymes, catalyzing a stereoselective oxygenation of compounds with at least one diene with a 1Z,4Z structure [8]. By abstraction of the hydrogen on the car-bon between the double bonds, a free radical is generated (Figure 2). By reaction with molecular oxygen a hydroperoxy compound is formed with the functional group in the +2 or -2 position from the original carbon radical [9,10], or, in one known case, in position zero [11]. Dependent on the selec-tivity of the enzyme, different oxylipins can be formed by this mechanism. From linoleic acid, the most common precursor in e.g. plants, two hydroper-oxy products can be formed; 9-HPETE or 13-HPETE [12]. From arachidonic acid, four lipoxygenase reactions have been reported producing 5-, 8-, 12-, or 15-HPETE in mammals [9]. Only one of these enzymes, 12R-LOX, is known to yield hydroperoxy products with R configuration [13].

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Figure 2. Mechanism for the formation of hydroperoxides by lipoxygenases, via a pentadienyl radical.

1.2.1 Nomenclature of lipoxygenases

The nomenclature of LOX enzymes is logic, but scientifically sometimes confusing. The names simply tell which carbon the enzyme oxygenates. In the case of several isoforms the type is denoted to the tissue where it was first described. There are for example three different types of 12S-LOX; platelet-type; leukocyte-type, and epidermis-type. But the expression profile differs between species, and so does the preferred position of oxygenation. Consequently, leukocyte type 12S-LOX in mouse equals 15S-LOX in hu-mans, and platelet-type 12S-LOX is found in skin in humans, whereas epi-dermis-type 12S-LOX in humans is just a pseudogene [5,14]. Some of the isozymes are denoted with a numeral, most often indicating the order of discovery. Since the list of discovered LOX enzymes never stops increasing, attempts have been made to exchange the old position based nomenclature, with a new. But so far researchers have not been able to present an alterna-tive that has been generally accepted. In this thesis, the names customary used will be practiced.

1.2.2 Lipoxygenases in humans

Enzymatic studies in human tissues almost always suffer from methodologi-cal problems with indirect, instead of direct, measurements. By adding or blocking enzymatic substrates or products, and study a certain effect, con-clusions are drawn on clinical function. But biological systems are seldom so simple, and it is hard to evaluate exactly what is causing the determined ef-fect. As just one example, inhibition of 12-LOX or COX-2 in pancreatic islets increases the secretion of insulin, but this is most likely because of the accumulation of arachidonic acid, and not by the products, per se [1]. It is also hard to use comparative studies from other mammals, due to big differ-

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ences in expression and which oxylipin each homolog is producing. Many investigations have been conducted showing expression of different LOX in different tissues, especially using RT-PCR or detection of metabolites after addition of substrate. Most of them have not been verified by other methods showing an expressed functional protein. Studies on tissue or cell expression are also sometimes criticized for probable contamination of other cell types [15], and catalytic studies cannot separate between different isoforms. With this in mind, follows a short introduction to the six lipoxygenases known in humans; 5-LOX, platelet type 12S-LOX, 12R-LOX, retinocyte-type 15S-LOX, epidermis-type 15-LOX, and epidermal LOX3 (eLOX3).

5-LOX is expressed in different types of leukocytes and mast cells, and is involved in the biosynthesis of leukotrienes (LT) after inflammatory onset [16]. Leukotrienes are implicated in the inflammatory cascade as a chemoat-tractant and chemoactivator. They also contract smooth muscles in the air-ways by ligation of the G-coupled receptor cysLT1 [17]. In resting cells, 5-LOX is located to the cytosol or the nucleus, but after cell activation it is translocated to the nuclear membrane, where it is presented to arachidonic acid by the helping protein FLAP [16]. After the first formation of 5-HPETE, 5-LOX is also able to convert it further to leukotrieneA4 (LTA4), a precursor of other leukotrienes [17].

As already described, the 12-LOX enzymes were the first lipoxygenases to be reported in humans. The biological relevance of the 12S-HPETE is still unclear. It is converted by glutathione peroxidase to the alcohol 12-HETE, and some studies have tried to link this compound to, inter alia, tumours, cardiovascular conditions, or diabetes (e.g. ref [18-20]). But so far the results are non-conclusive. Many of the effects of 12S-LOX could also be attributed to the formation of hepoxilins. The term hepoxilin refers to the epoxyalco-hols of 12S-HPETE, and the name derives from a combination of its struc-ture (Hydroxy EPOXide) and biological function (release of InsuLIN was the first reported effect of the hepoxilins). There are two different types, the A- and B-type, determined by the position of the hydroxyl group. Hepoxilin A3 (HxA3) is the racemic 11S,12S-epoxy-8-hydroxyeicosatrienoic acid, and Hepoxilin B3 (HxB3) is the corresponding 11S,12S-epoxy-10-hydroxy-eicosatrienoic acid. HxA3 has been demonstrated to have several biological impacts, inter alia, as an inflammatory mediator, a potent vasoconstrictor of smooth muscles, control of intracellular calcium release, opening of potas-sium channels, and, as already mentioned, stimulation of insulin release [21-23]. In skin both hepoxilins have been found to increase the vascular perme-ability [24,25]. It seems that HxB3 is the prominent form expressed in this tissue [26], and the level increases in psoriatic lesions [26,27].

Apart from 12S-LOX, humans also express an enzyme producing 12-HPETE of the R-configuration. This property is uncommon but has been reported in some marine organisms, and in rodents [13]. 12R-LOX seems to

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be skin specific, but has also been found in tonsils in lower amounts [13]. The enzyme will be discussed further under section 1.2.3 below.

The general 15S-LOX enzyme in humans is of the retinocyte-type, and is phylogenetically closely related to the murine leukocyte-type 12S-LOX. The expression is well established in airway epithelial cells, eosinofils, mono-cytes and reticulocytes [12,19]. In 1997, another lipoxygenase was cloned from human hair roots, and was demonstrated to also produce 15S-HPETE from arachidonic acid [28]. This second enzyme was named epidermis-type 15S-LOX, but is often simply called 15S-LOX-2. Due to this second en-zyme, it is hard to establish which enzyme is responsible for the reaction in many tissues where a 15-LOX activity has been noticed. Epidermis-type 15S-LOX seems to be expressed in at least skin, prostate, lung, and cornea [28]. Still, no evidence is provided telling that retinocyte-type 15S-LOX could not also be present in these tissues. Since the type of enzyme is not of significance to this thesis, all reactions producing 15S-HPETE will simply be attributed to 15S-LOX.

Biologically 15S-LOX has been related to several functions. In reticulo-cytes (immature red blood cells where this enzyme was first discovered in animals) evidences suggest an involvement of the enzyme in the maturation process [15]. 15S-LOX is strongly induced by interleukin-4 [15], and seems to have significance in inflammation, but the experimental data are so far too contradictive to draw any conclusions, or to even say if the enzyme has a pro-inflammatory or an anti-inflammatory role [15]. In bronchial asthma the levels of 15-HETE is highly increased. 15-HETE has also been shown to be contractive to smooth muscles in vitro, but clinical studies on the effect in asthma have so far not been evident [29]. Other areas under investigation for 15S-LOX impact are cancer and atherosclerosis, but also these areas need further research to be conclusive [29].

The last human lipoxygenase, eLOX3, is essentially not even a lipoxy-genase, but a hydroperoxide isomerase [30]. As the name implies, it was the third type of lipoxygenase found in mammal epidermis. It was first thought to be an inactive enzyme, since it could use neither linoleic acid nor arachi-donic acid as substrate, in contrast to all other LOX enzymes. But in 2003 it was shown that the 12R-LOX product 12R-HPETE was a good substrate [30], converting it to the epoxyalcohol 8R-hydroxy-11R,12R-epoxyeicosa-trienoic acid (8R,11R,12R-HEET). As this lipoxygenase is mostly implicated in skin, it will be more extensively discussed under section 1.2.3.

1.2.3 Lipoxygenases in human skin

In human skin all six lipoxygenases are present, and the major metabolites formed are from 15S-LOX and 12S-LOX [23,24]. Detection of 12-HETE in human epidermis was first made in 1975, in psoriatic lesions, where it was

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found to be greatly increased, in comparison to normal epidermis [31]. Later it has been realized that the main form induced in this condition is the 12R-LOX [32]. The inflammatory response to 12R-HPETE is relatively weak and the theory today is that this increase is linked to keratinocyte hyperprolifera-tion [33].

With increasing evidence, 12R-LOX is more and more related to an in-volvement in the development of the epidermal water permeability barrier. In 2007, a study was published with 12R-LOX-deficient mice [34]. The ho-mozygous neonates soon after birth began to develop a red, shiny appear-ance, and they all died within 3-5 hours. The transepidermal water loss for the homozygous mice were 8 times higher than for wild-type or heteroge-nous mice, supporting the position that 12R-LOX deficiency led to an im-paired water barrier. Staining and fluorescence further confirmed a more water permeable epidermis in 12R-LOX-deficient mice [34]. These findings have also been turned out to have clinical implications in humans. In 2002, a study could show an association between mutations in the genes for both 12R-LOX and eLOX3, and the rare skin disease lamellar ichthyosis [35,36]. This will be discussed under the next section below. Recently, a further study on the deficient mice demonstrated that the character of their skin, much resembled the ichthyosis patients with mutations in the 12R-LOX gene [37].

1.3 The case of ichthyosis

Ichthyosis is a heterogeneous group of disorders to the skin with a variety in severity, etiology, and symptoms. The most common, and also mildest form, is ichthyosis vulgaris, with a prevalence of about 1:250-1000 [38]. The most severe form is the usually fatal harlequin disease. Lamellar ichthyosis (LI) is an intermediate severe form, with a prevalence of about 1:300,000-500,000 [39]. It is a recessive congenital disease characterized by large, dark, some-times plate-like scales, and often hyperkeratosis [38,39]. Irrespective of form, all ichthyosis patients have a dysfunctional permeability barrier of the skin [40].

By several large studies, researchers have been able to link genetic varia-tions to separate types of ichthyosis. About half of the cases of LI have been associated to specific mutation in any of six different genes [35,36]. Two of these genes are the ones for 12R-LOX and eLOX3, making up about 6-12%, and 5% of the cases, respectively [35,41,42]. Noticeably, mutations in any of these genes give the same phenotype of LI, having the researchers conclude that they are both part of a consecutive metabolic pathway. This makes sense, since the 12R-HPETE product of 12R-LOX has been shown to be a good substrate for eLOX3 [30]. One of the other six genes involved is the so

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called ichthyin gene, discovered in 2004 [43,44]. This gene is thought to code for a receptor, probably of the G-protein coupled type. As ligands for this receptor the researchers have proposed the trihydroxy product (THETE) formed from the eLOX3 produced epoxyalcohol, or the �-carboxyl metabo-lite of THETE (Figure 3). This theory was supported by the discovery that another of the mutated genes in LI was the one for CYP4F22 [39]. CYP4 enzymes are known to catalyze �-hydroxylation reactions, as discussed un-der section 1.4.1. So far, no investigation of the catalytic activity of CYP4F22 has been conducted, and this function is hypothetic [33].

Figure 3. The hepoxilin pathway according to the hypothesis. 12R-LOX, eLOX3, and CYP4F22 are all linked to lamellar ichthyosis. The THETE (bottom left) and �-carboxyl THETE (top right) are thought to be ligands for the ichthyin receptor. CYP4F22 is thought to catalyze the �-hydroxylation in the pathway.

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1.4 Cytochrome P450

In contrast to the lipoxygenases, which have dioxygenase activity, the CYPs are monoxygenases. They are membrane bound to the endoplasmatic reticulum (ER), and by electron transfer from NADPH, catalyzed by P450 reductase, CYP enzymes activate molecular oxygen and introduce atomic oxygen to the substrate [45,46]. The foremost reaction catalyzed by this mechanism is hy-droxylation of carbons, but CYPs can also perform, inter alia, dealkylation of amines, hydroxylations of other heteroatoms, and epoxidations. Not until 1981 it was discovered that CYP enzymes take part in arachidonic acid metabolism [47]. Four separate types of CYP reactions occur for eicosanoids [48]; (1) ally-lic oxidation generating six regioisomeric cis-trans conjugated HETEs, (2) bis-allylic oxidation generating three regioisomeric HETEs, (3) hydroxylation of sp3 carbon at the � end, and (4) alkene epoxidation generating 5,6-, 8,9-, 11,12-, or 14,15-EET. The HETEs obtained by bis-allylic oxidation are unsta-ble, transforming to cis-trans conjugated HETEs under acid conditions [49].

When an enzyme was characterized in the 1960s, with a novel chromo-phore at 450 nm in the reduced carbon monoxide spectrum [50], the scien-tists still believed this metabolic system consisted of just one enzyme. Since then, the complexity of this system has become clear. All together over 11,000 CYPs have been reported (August 2009)[51], in most living organ-isms, including mammals, bacteria, viruses, fungi, and plants. Studies on the sequenced human genome have suggested 57 functional CYP genes and 58 pseudogenes [51,52]. This superfamily of cytochrome P450 is further di-vided into families with over 40% amino acid identity, and subfamilies with over 55% amino acid identity [53]. Human isozymes belong to 18 families and 43 subfamilies.

Families 1-3 are usually accounted for catalysis of exogenous substrates, e.g. drugs, pollutants, or other foreign chemicals. About 75% of all drugs are metabolized by CYPs [54], making it a principal system both from a clinical and a pharmaceutical perspective. Families of higher numerative generally have endogenous substrates. They are, for instance, involved in synthesis and catalysis of several eicosanoids, e.g. prostaglandins and thromboxanes, but also steroids, vitamin D3 and cholesterol [55]. It has been apparent that the expression of different CYPs does not vary only between species, but also between individuals and between different tissues in the same individual. Not surprisingly, the liver is the tissue with highest CYP expression, especially in families 1-3. Even so, there are big differences between different isozymes, and each enzyme and each tissue has its own profile [56]. Presently, a lot of research focuses on characterizing the different CYPs, when it comes to ex-pression, gene regulation and substrate specificity. Even so, several so called “orphan” CYP enzymes still have not been proposed with a physiologic func-tion [57,58]. Of these, about half belong to the CYP4 family.

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1.4.1 CYP4 Family

Fatty acid �-hydroxylation was early demonstrated as cytochrome P450- dependent, but it took until 1987 before an enzyme responsible for the �-hydroxylation of lauric acid was cloned from rat liver [59], and recognized to be of a new CYP family [60]. Today the human CYP4 family is consid-ered to be the principal system of hydroxylation of fatty acids at the � end [60]. It consists of six subfamilies; 4A, 4B, 4F, 4V, 4X and 4Z, comprising twelve isozymes [60,61].

Although the major efforts in cytochrome P450 research have been put on families 1-3, due to their importance in metabolism of xenobiotics, the clini-cal influences of family 4 have been realized the last decade. SNP analyses have associated CYP4A11 [62,63], CYP4F2 [52,53] and CYP4V2 [64] with cardiovascular diseases. The mechanisms explaining these clinical data still need to be established, but the proposed theories are all based on the en-zymes properties as �-hydroxylase. CYP4F2 and CYP4F3 are also the main enzymes for �-hydroxylation of leukotriene B4 [65-67], an important media-tor in inflammatory processes. CYP4V2 has been linked to the rare eye dis-ease Bietti’s crystalline corneoretinal dystrophy (BCD), by mutation analysis of patients [68-70]. Recently it was demonstrated to produce the �-hydroxy metabolites of middle chain fatty acids [71], but if any of these are the physiological substrate in cornea is questionable. It has been shown that BCD patients have a defective incorporation of �3 long chain fatty acids [72], but no further investigations of catalytic properties of this enzyme has been presented. Rodent CYP4B1 orthologs have been demonstrated to me-tabolize lauric acid and several drugs, but so far no catalytic studies with recombinant human enzyme have succeeded [73]. Also CYP4F12 is able to metabolize drugs, ebastine [74], and converts arachidonic acid to the �3 metabolite [75], but to a much lower rate than other CYP4 enzymes. CYP4F11, CYP4X1 and CYP4Z1 still lack good substrate candidates [60].

1.4.2 CYP4F8

In 1999, CYP4F8 was first discovered and cloned, in the search for a candi-date for the enzymatic production of 19-hydroxyprostaglandins in human seminal vesicles [76]. These prostanoids are found in large quantities in seminal fluids [77], and are thought to be involved in the fertilization process presumably as immunosuppressors. The next year, recombinant CYP4F8 were demonstrated to convert both PGH2 and PGH2 analogs to the 19-hydroxy metabolites [78]. A correlation between COX-2, prostaglandin H synthase, and CYP4F8 has been found [79], and the theory is that the forma-tion of 19-OH-PGH2 from arachidonic acid by these three enzymes, is

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closely linked in the ER. An interesting feature is the large interindividual variability of the rate of prostaglandin 19-hydroxylation in seminal vesicles. Two distinct groups of slow or rapid metabolizer, have been distinguished [80,81], and may arise from polymorphism of the present gene [80]. So far, three missense SNPs, and one nonsense SNP have been found in the CYP4F8 open reading frame. One of these has been studied, but was not found to alter the production of PGH2 [72]. Apart from seminal vesicles, CYP4F8 is expressed in several tissues, above all in skin [82]. Since 19-OH-PGH has not been detected there, CYP4F8 may have a further function than the one in reproductive tissues. Studies have also shown that CYP4F8 is induced in psoriatic lesions [82]. Catalytically, CYP4F8 forms 18-HETE from arachidonic acid [78], but this compound has not been found in physio-logical fluids, and the significance of this biosynthesis is unclear.

1.4.3 CYP4F22

The enzyme CYP4F22 has so far not been thoroughly investigated. The gene was discovered among several other human CYP enzymes after the comple-tion of the HUGO project [83]. One study of the tissue distribution of mRNA expression saw high expression in human keratinocytes, and in testis [84]. Lower expression was also seen in placenta, skeletal muscles, brain, and kidney. A real-time RT-PCR study conducted in connection to this the-sis, detected levels of CYP4F22 in both retina and cornea (unpublished data). The highest levels of CYP4 enzymes in both these tissues were CYP4V2 and CYP4X1, but CYP4F22 levels were as high as CYP4F8. As described in section 1.3, a mutation analysis has associated CYP4F22 with lamellar ichthyosis [39]. By sequencing the 12 exons and the exon-intron boundaries of the CYP4F22 gene in 21 patients, researchers detected seven different mutations. In a later mutation analysis 8% of the patients with la-mellar ichthyosis had a mutated gene for CYP4F22 [42]. Unfortunately, no catalytic data is available for this enzyme.

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1.5 Mass Spectrometry

Separation of compounds is of upmost importance for both preparative and analytical purposes. Distinctions between the physical properties of the compounds are utilized to detect or collect them separately. Chroma-tographic techniques are based on differences in distribution ratio between a mobile phase and a stationary phase. Mass spectrometry, on the other hand, separates compounds based on their molecular weight and ion charge. Usu-ally it is necessary to combine these two separation procedures in a so called on-line system, where compounds of a crude mixture is first separated chro-matographically, and then directed into a mass spectrometer for further sepa-ration and detection. Another quality of mass spectrometry is the ability to break the isolated ions and detect the fragments. The fragmentation is prede-termined based on properties of the original structure, creating a unique spectrum of ions for each molecule. This spectrum can be used as a specific “fingerprint” to identify the detected compound.

The conception of mass spectrometry is based on the notion that charged particles can be moved in space under the influence of electric or magnetic fields. The basic science behind the technique goes back to the late nine-teenth century, when Eugene Goldstein demonstrated that luminous rays travel in straight lines within a low pressure tube [85]. In 1913, John Joseph Thomson developed a device that could separate the isotopes of neon, and his pupil Francis William Aston a couple of years later improved the ma-chine to the extent that he in 1922 received the Nobel Prize in chemistry, for the invention of the mass spectrometer [85].

All mass spectrometers are generally based on three components: an ions source to produce ions, a selector to filter out all unwanted ions, and a detec-tor. An essential aspect is that the ions have to be in the gaseous phase. This had the consequence that for more then half a century of mass spectrometry the technique was limited to systems where the molecules were already pre-sent in the gas phase, e.g., as detectors in gas chromatography (GC-MS). The compounds available for analysis were also limited to volatile and non-labile substances. All this changed during the 1980s, when several major develop-ments were made, especially in ionization techniques. In 1989, John Bennet Fenn demonstrated that mass spectrometry with so called electrospray ioni-zation (ESI) successfully could be used for analysis of peptides [86]. This achievement, together with other “soft” ionization techniques, e.g., matrix-assisted laser desorption/ionization (MALDI) developed in the same period, converted mass spectrometry to the detection technique of choice, also for non-volatile and thermally unstable substances [87]. These advancements also made mass spectrometry available for other separation methods than GC, above all, liquid chromatography (LC-MS) [88]. Suddenly, mass spec-trometry was no longer a peripheral method, but an alternative for almost all

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compounds, as long as they could be ionized [89]. Fenn received the Nobel Prize in chemistry in 2002 for his achievements, together with the inventor of the MALDI technique Koichi Tanaka [87].

1.5.1 Electrospray ionization

The concept of electrospray ionization was actually developed in the 1960s by Malcolm Dole [90], but Fenn was the first to be able to use the approach to introduce ions into a mass spectrometer. The mechanism of formation of gas phase ions from a solvent is generally well accepted [85,91,92]. Liquid with ionic analytes is forced through a thin capillary, into a closed chamber with atmospheric pressure. A high potential (in this thesis usually 3 kV) is set between the capillary and an opposing electrode at the other end of the chamber. The intense electric field will push the liquid into a conical menis-cus, a so called Taylor cone, at the tip of the capillary [85,92]. Due to elec-trolytes, the conductivity of the liquid will create a polarization within the solvent in the capillary [91]. At the surface of the cone ions of the same charge will accumulate dependent on the potential of the electric field. From the top of the Taylor cone a jet of liquid will be ejected into the chamber. On the surface of this jet “varicose waves” will emerge due to interactions be-tween the viscosity and the surface tension [92]. As the amplitude of these waves increases, the jet will split into a fine spray of droplets. Within the chamber, solvent will evaporate from the surface of the droplets. When the droplet size decreases the Coulomb repulsion of the charges within the drop-lets will increase. At a point, known as the Rayleigh limit, the Coulomb re-pulsion will overcome surface tension, and the droplet will collapse into smaller droplets. Eventually, gas phase ions emerge from the smaller and smaller droplets. The exact mechanism of production of these ions from the droplets is still under discussion [93]. Either ions are formed due to the com-plete evaporation of solvent (charge residue model), or by evaporation of the ions from the very small droplets (ion evaporation model). Evidences sup-port the ion evaporation model for smaller organic ions, while for very large ions such as proteins the charge residue model seems more plausible [91].

1.5.2 Linear quadrupole ion trap

The basic principle of quadrupole ion traps is not more complicated than the fact that ions are mobile in an electric field. It is based on the theory of strong focusing of charge particles, and was adapted to mass spectrometry by the German scientist Wolfgang Paul in the beginning of the 1950s [94]. The trap is built up by four perfectly parallel rods (the LTQ system from Thermo Scientific is described in Ref [95]). By an applied potential over the

23

rods, the trajectory of the ions entering are determined in all three dimen-sions, and the ions will ultimately be trapped within the compartment. By changing the electric fields, ions of specific mass to charge ratios (m/z-values) can be isolated, or be forced to exit the trap. In more details, a DC voltage is applied over the four rods creating an axial field in the y-z plane (vertical and forward). A fluctuating voltage at radio frequency of about 1 MHz produces a radial radio electric field in the y-x plane (vertical and hori-zontal). The linear quadrupole ion trap is sometimes denoted as a 2D ion trap, due to its two-dimensional radio electric field.

As noted, the isolation procedure is based on stable trajectories of the ions. Ions with an unstable trajectory will either hit the rods, or exit the con-tainer through slots in the x-rods [94,96]. This concept is also utilized when the isolated ions are ejected to the detector. The stability of the ions is, in the case of a linear quadrupole ion trap, determined by the voltage of the radio power, and the m/z of the ion. This relationship is denoted as the q-value of the ion [94]. Small ions have a higher q-value then larger ions. The limit where ions are exiting the trap is at a q-value of 0.88 [95]. For isolation, the q-value of the ion of choice is increased to 0.83 by a gradual increase of the voltage of the radio electric field. A variable AC field at 5-500 kHz is set over the two x-rods, making all ions but those with q value of 0.83 leave the trap [95,96]. To finally be able to eject the isolated ions from the trap the AC voltage in combination with the gradually amplified radio electric field make the q-values of the ions to steadily increase. When the q-value of 0.88 is reached, the ion will leave the trap for the detector [95,96]. Instead of being relieved to the detector an isolated ion could also be fragmented within the ion trap. The q value will in this case be retained to a lower number (in this thesis the q value of 0.25 is used), before a more gentle AC voltage is ap-plied. This voltage will not make the ions exit the trap, but will attain frag-mentation of the ions [95].

1.5.3 Analysis of fatty acid derivatives by mass spectrometry

During the last decade several methods for analysis of eicosanoids in bio-logical systems have been published [97-102], usually based on a reverse phase HPLC separation with methanol/water or acetonitril/water as mobile phase, often with 0.1% acetic acid. For the more hydrophilic epoxyalcohols and trihydroxyalcohols a reverse phase approach is usually not sufficient. This necessitates the use of a straight phase system with hexane and 1-5% isopropanol. Brash and coworkers [103,104] have presented the usage of a chiral column system applicable for both reverse and straight phase [89, 90]. On this system they succeeded to separate enantiomers of both HEETs and THETEs by UV analysis during HPLC separation. For identification they used GC or electron capture APCI, still demanding derivatization. Although

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the literature of LC-MS/MS spectra of fatty acid metabolites has expanded rapidly the last 15 years (for an excellent review see ref. [105]), almost all recent research on epoxyalcohols and their ensuing trihydroxyalcohols util-izes gas chromatography [26,27,106-111].

1.5.4 Identification of detected ions

Mass spectrometry has several advantages over other detection methods. Selectivity and sensitivity is, for example, often higher than for both UV spectrophotometry or radioactivity based methods. One of the most impor-tant utilities is the ability to generate structure specific fingerprint spectra. Not all MS instruments have this capability. The selector needs to be able to manipulate the ions before they are directed into the detector. As described under section 1.5.2, the linear quadrupole ion trap can disintegrate an iso-lated ion by applying a voltage within the ion trap. Fragments can then again be isolated by the same procedure as the primary ions. This isolation-fragmentation cycle can then be repeated until the ions are released to the detector. Also spectra of fragments of a fragment could give valuable infor-mation of the structure of the original ion, as demonstrated in section 4.5 and in Paper III. The reason why the spectra are so structure specific is that the fragmentation does not happen in random. Energy is needed to break the covalent bonds within the molecule, and fragmentation mechanisms will therefore follow the rules of thermodynamics. Under the same conditions, the same fragmentation will always occur for the same structure. By know-ing the fragmentation mechanisms, a researcher is able to identify, not only structures where the spectra are already published, but also structures that likely will fragment in a similar way. It is important to remember that the conditions are crucial. Different ionization techniques have different mecha-nisms, and there are several different methods to obtain fragmentation [112]. The most important issue is the extent of energy input to dissociate the ions. For example have magnetic sector instruments high input, but quadrupole ion traps have low input, influencing the mechanisms involved [113]. Within all instruments there are also settings to adjust the scale of disintegration. This thesis handles fragmentation mechanism for polyunsaturated fatty acids and their oxygenated metabolites, as analyzed with a linear quadrupole ion trap selector and electrospray ionization. The following sections will discuss known fragmentation mechanisms for these compounds.

1.5.4.1 Charge-remote or charge-driven fragmentation Initiation is a key issue when discussing fragmentation mechanisms. Some-thing has to start it all, for the dissociation to ever occur. Two concepts are often in focus; charge-remote and charge-driven reactions. Charge-remote mechanisms are distinguished by occurring away from the charged in the

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structure. The additional free electron pair of, e.g., the deprotonated carboxyl group of fatty acids, are therefore not involved. For non-oxygenated satu-rated or unsaturated fatty acids the energy input in the ion trap is usually not sufficient for charge-remote fragmentation [114]. But with an additional functional group both charge driven and charge-remote mechanisms are present, due to a lower activation energy needed adjacent to a heteroatom [113]. The exact mechanism for charge-remote fragmentations is still under debate, and has mostly been studied within selectors with high energy input [115]. They were first reported by Gross and his research group in 1983 [116]. This group has proposed the 1,4-elimination mechanism [117], where two C–H bonds and one C–C bond break almost simultaneously, cleaving the ion in two parts. They argue that this mechanism is the most energeti-cally favorable, but its importance seems limited to non-oxygenated fatty acids, where charge-remote fragmentation is seen along the carbon-chain [115,118]. This theory could also not explain all empirical data, why a radi-cal based theory with a C–C bond cleavage was proposed [119,120]. Claeys and coworkers have proposed another mechanism where a C–H bond cleav-age is the starting point [121-123]. They have demonstrated that some fatty acids are cleaved by a homolytic radical mechanism where hydrogen is ini-tially lost. Although this theory is not conclusively accepted, it has additional support by research on isotopic effects, and in empirical data [113,115]. The theory will be handled more extensively during this thesis, whereas several of the mechanisms are based on it.

Charge-driven reactions, on the other hand, are mechanisms where the charge of the fatty acid triggers the dissociation. The charge is initially on the carboxyl group, but charge-migration to the functional group or the ter-minal allyl is not uncommon, and is an important aspect in the understanding of charge-driven mechanisms. Many of the fragmentation mechanisms in this thesis are charge-driven, and will be discussed in detail below.

1.5.4.2 Uncharacteristic fragmentation In a normal MS/MS spectrum, several of the fragment ions do not include information on the structure of the original fatty acid. These uncharacteristic fragmentation mechanisms are even so interesting. All oxilipins will have a loss of water and carbon dioxide, and some of them will also lose molecular hydrogen. If these certain fragments are noticed in the spectra, it is easier to consider the ion to derive from an oxygenated fatty acid.

Molecular hydrogen is lost by a charge-remote mechanism [124,125]. A functional group seems necessary, since this loss is not evident for non-oxygenated fatty acids. The hydrogen on the carbonyl carbon is abstracted by a homolytic cleavage leaving a carbonyl radical. One additional hydrogen radical is lost adjacent to the carbonyl, creating a double bond (Figure 4A). Water could be lost by several different mechanisms either from the car-boxyl group or from any hydroxyl groups [124]. The most probable mecha-

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nism is by abstraction of hydrogen from the �-carbon. The C-O bond will interact with the arisen radical and form a double bond, via homolytic cleav-age (Figure 4B) [124]. In the case of dehydroxylation of the carboxyl group, an initial charge-migration is necessary. The loss of the carboxyl group is also common. Two different structures of the remaining ion have been iden-tified [124]; one with a terminal vinyl bond, and one with a terminal alkyl group. Both seem to be charge-driven reactions, leading to heterolytic cleav-age between C1 and C2. In the former case, one �-hydrogen and the hydro-gen on a hydroxyl group will also be lost (Figure 4D), and in the later case the hydroxyl hydrogen will be transferred to the alkyl group (Figure 4C).

Figure 4. Common fragmentation mechanisms in MS/MS analysis with electrospray ionization. A, Loss of molecular hydrogen. B, Loss of a water molecule. C, Loss of the carboxyl group. D, Loss of the carboxyl group and molecular hydrogen, forming an allyl. E, �-Cleavage by a charge-remote mechanism. F, �-Cleavage by a charge-driven mechanism.

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1.5.4.3 Characteristic fragmentation A heteroatom will affect the stability of the surrounding structure. Therefore disintegration will occur adjacent to the functional group, also with dissocia-tion techniques with low energy input. The dissociation will lead to frag-ments with information on position of the functional group. The concept of characteristic fragment ions will be in focus for much of this thesis, and mechanisms to explain different fragments will be discussed thoroughly.

The most general cleavage reaction close to the functional group is so called �-cleavage. This reaction could either be charge-remote or charge-driven (Figure 4E-F) [102,113,126,127]. In both cases the aldehyde is formed, but the detection of this fragment is dependent on the site of the charge. In the case of a charge-driven mechanism only the allylic fragment will be detected, and in the case of a charge-remote mechanism, it is deter-mined by where the carboxyl group, or another charged functional group, is situated. Monohydroxy fatty acids are always cleaved adjacent to the hy-droxyl group by �-cleavage, and the position of the hydroxyl group is there-fore easily determined [99].

1.5.4.4 Verification of fragments An MS/MS spectrum of an uncomplicated molecule with intense ions could often be interpreted easily. A plausible mechanism is proposed that is in total agreement with the empirical data. Even so, the result is in many aspects just on paper, if there is no possibility to confirm the findings. The other way around, a spectrum which seems to be insoluble could be illuminated, if just some aspects of the fragmentation could be determined.

There are several different ways to investigate if a certain property of a mechanism is really relevant. The importance of verifying that the assump-tions made are actually true could not be underestimated. Some of the avail-able empirical tests to improve mechanism proposals are described below.

An often utilized method to determine which hydrogen being abstracted is by using deuterium labeled compounds. Since the atomic mass of deuterium is 2 Da instead of 1 Da, it is possible to decide if a certain hydrogen is still on the ion, or have been abstracted to a neutral molecule, e.g. water. By cal-culating how much the m/z value for the ion increases, the number of deute-rium is found. In Paper II, linoleic acid with deuterium on the allylic carbons is used, and in Paper IV, arachidonic acid with deuterium in these positions is used. By this approach it is possible to determine which hydrogen being involved in the cleavage reaction, and also, to some extent, conclude which part of the structure being detected. In Paper III the hydrogens of the hy-droxyl groups are instead exchanged for deuterium. Here it is possible to further investigate if the hydroxyl group is intact or if a charge-migration has occurred. Another strategy, also based on isotopomers, is to replace oxygen with 18O. Either the oxygens of the carboxyl group or of the hydroxyl groups

28

could be exchanged, giving information on the origin of the leaving water molecule, or if the carboxyl group has been lost. This strategy is used in Paper III, where the oxygens in the two hydroxyl groups have been labeled. A strategy to determine the length of the carbon chain of the detected ion is to utilize fatty acid isotopomers with 13C labeled carbons, as performed in Paper I.

In Paper III, also another approach was utilized. To determine which part of the substrate a fragment contained, analog substrates with different length of the carbon chain were analyzed. In this case fatty acids with two more or two less carbons than 18:1n-9 were analyzed. Since the corresponding ions m/z values increased or decreased by 28 Da it was conclude that the present ions originated from the omega-end. This will be discussed further in section 4.5.

One of the best ways to ensure that a present fragment has the expected structure is to isolate this ion, and fragment it once more – an MS3 analysis. These spectra are usually easier to interpret, and consists of less uncharacter-istic ions. This strategy is also utilized in Paper III.

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2 Aims

The present work is part of a research project to characterize some of the orphan CYPs, above all for their catalytic performance. To achieve this goal it was important to develop methods to analyze both fatty acids and oxilipins by LC-MS/MS.

The specific aims of this thesis have been to: • Develop a chiral LC system for enantiomeric separation of fatty acid

hydroperoxides as free acids, optimize parameters for tandem mass spec-trometric analysis of these compounds, and preparative isolation of 12R-HPETE.

• Develop a LC-MS/MS system for analysis and separation of epoxyalco-hols generated from hydroperoxides of linoleic acid or arachidonic acid.

• Study the fragmentation mechanism of fatty acid hydroperoxides, ep-oxyalcohols, 7,10-diols, and keto compounds by analysis of isotopomers by tandem mass spectrometry.

• Investigate whether the LC-MS/MS method could be used for analysis of epoxyalcohols in human tissue.

• Develop a recombinant expression system for the orphan CYP4F22. • Study the catalytic properties of CYP4F22 with arachidonic acid and

epoxyalcohols as substrates. • Study the catalytic properties of CYP4F8 with epoxides and epoxyalco-

hols as substrates.

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3 Methods

3.1 Preparation of hydroperoxides 3.1.1 Synthesis of hydroperoxides Spontaneous peroxidation of unsaturated fatty acids is based on a chain of radical reactions [128-130]. By hydrogen abstraction a lipid radical (L•) is formed, which rapidly reacts with molecular oxygen forming the peroxyl radical (L-OO•). This radical abstracts hydrogen from another fatty acid molecule, preferable in the bis-allylic position. Since this radical is delocal-ized over the pentadienyl structure, several hydroperoxides will be gener-ated, mainly the conjugated isomers. By controlled autoxidation six racemic conjugated hydroperoxide isomers are obtained as main products from ara-chidonic acid; 5-, 8-, 9-, 11-, 12-, and 15-HPETE [128]. In this project (Pa-per IV), arachidonic acid was incubated at 37°C under oxygen. By the addi-tion of �-tocopherol the procedure was directed towards the production of cis-trans-isomers, and a more equal distribution of isomers [131].

An alternative way to produce hydroperoxides, also utilized in this pro-ject, is by photooxidation [129,130]. By radiation from a light source in presence of a photosensitiser, in this case methylene blue, reactive singlet oxygen is formed. By this mechanism 9-, 10-, 12-, and 13-HPODE was pre-pared from linoleic acid (Paper II).

Hydroperoxides were also obtained enzymatically (Papers I, II and IV). 13S-HPODE and 15S-HPETE were prepared by soybean lipoxygenase-1 [132], and 9S-HPODE was prepared by tomato lipoxygenases [133].

3.1.2 Purification of hydroperoxides After autoxidation of arachidonic acid the mixture of hydroperoxides were purified on an open column with silicic acid. The remaining arachidonic acid was eluted in 7% ether in hexane, while the oxygenated fatty acids were eluted in 20% ether in hexane. Hydroperoxides from photooxidation and autoxidation of linoleic acid were purified by extraction on a silica cartridge, eluted with diethyl ether in hexane. Hydroperoxides prepared by soybean lipoxygenase were purified by extraction on a C18 silica cartridge, and eluted in ethyl ace-tate. All hydroperoxides were evaporated to dryness and diluted in ethanol for storage, or in 5% isopropanol in hexane for preparative normal phase HPLC.

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3.1.3 Separation of hydroperoxides In Paper I a Reprosil Chiral-NR column was used to separate the enanti-omers of hydroperoxides of arachidonic acid and linoleic acid. This column has a silica base with a covalently bound chiral aromatic selector. Unfortu-nately, the company does not disclose this selector. By a normal phase sys-tem with hexane, and 1-1.2% isopropanol as alcoholic modifier the hydro-peroxides of autoxidation or photooxidation were separated. For all struc-tures mass spectrometry was utilized for detection. It has previously been shown that hydroperoxides are converted to keto compounds within in the mass spectrometer, and the spectra of the corresponding 5-oxo, 12-oxo, and 15-oxo compounds have been published [134]. Hydroperoxides with conju-gated double bonds were also detected by UV spectrophotometry at 235 nm, before entering the mass spectrometer. For further information on the separa-tion and detection of hydroperoxides with Reprosil Chiral-NR see Paper I.

Investigations with commercial or enzymatically synthesized standards showed that the enantiomers with S-configuration eluted ahead of the enan-tiomers with R-configuration, with 8-HPODE as the only exception (Paper II). Since the purpose was to isolate the 12R-HPETE compound, the column Reprosil Chiral-NRR, with reverse chirality, was utilized in Paper IV. In this case the compounds with R-configuration eluted first, and 12R-HPETE did not coelute with any of the other hydroperoxide products.

3.2 Preparation of epoxyalcohols 3.2.1 Synthesis of epoxyalcohols Epoxyalcohols were prepared from hydroperoxides by treatment with hema-tin [135,136]. Ferric iron (Fe3+) of hematin reacts with the hydroperoxide forming an alkoxyl radical, which is rearranged to the epoxyallylic radical (Figure 5). Also in this molecule the radical is delocalized over the pentadi-enyl structure, and by hydroxylation the epoxyalcohol is formed with a hy-droxyl group in either position 1 or 3. In practice, hydroperoxides were dis-solved in KHPO4 buffer, and 1-10 eq. hematin was added. After 10 minutes the reaction was stopped, and products were extracted on a C18 silica car-tridge, eluted in ethyl acetate, evaporated to dryness, and dissolved in etha-nol for analysis, as described under section 3.2.2.

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Figure 5. Formation of epoxyalcohols from the hydroperoxide by treatment with hematin.

3.2.2 Separation of epoxyalcohols Epoxyalcohols of both arachidonic acid and linoleic acid were separated on a normal phase HPLC system, with a silica column, and a mobile phase of 3% isopropanol in hexane with 0.01% acetic acid. For analytical purposes a 2 mm column was utilized and connected to the mass spectrometer for detec-tion. For preparative purposes a 10 mm column was utilized and the effluent collected in 1 minute fractions for individual analysis of content. Samples of the fractions were injected into the mass spectrometer for analysis. Fractions with epoxyalcohols were identified, evaporated to dryness, and diluted in ethanol for storage. For further information on analysis of epoxyalcohols by mass spectrometry see section 4.4.

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3.3 Preparation of epoxides

Epoxides of arachidonic acid were obtained by treating arachidonic acid with 1.1 eq. m-chloroperoxybenzoic acid. First an attempt to separate the epoxides by normal phase HPLC was executed on a preparative 10 mm sil-ica column eluted at 4 mL/min, with 1% isopropanol in hexane as the mobile phase. The effluent passed a photodiode array detector, and measured at 210 nm, and one minute fractions were collected. 8,9-EET was purified by this strategy, but 11,12-EET and 12,13-EET did not separate. Instead they were separated on a preparative RP-HPLC system, with a C18 column, eluted at 1.2 mL/min with methanol/water/HAc (80/20/0.1) as the mobile phase. 5% of the effluent was analyzed by mass spectrometry via an AccurateTM split. The rest of the effluent was collected in one minute fractions. Fractions con-taining epoxides were evaporated to dryness, and dissolved in ethanol for storage. Mass spectra of all epoxides from arachidonic acid have previously been reported [99], and the isolated compounds were in agreement with these results.

3.4 Preparation of diols

The compounds 7,10-dihydroxyoctadecenoic acid (7,10-(OH)2-18:1) and 7,10-dihydroxyeicosenoic acid (7,10-(OH)2-20:1), were obtained by 7,10-diol synthase from the bacterium Pseudomonas aeruginosa. Bacteria were grown on complete media in the presence of oleic acid (10 g/L) for 18 h at 30°C. Thereafter the bacteria were collected by centrifugation (6,000g, 10 min at 4 °C), suspended in Tris-HCl buffer (50 mM, pH 7.0), sonicated and centrifuged (10,000g, 5 min at 4°C). By diafiltration the supernatant was filtered and concentrated. This extract was incubated with either 18:1n-9 or 20:1n-11 for 1 h at 37°C. Fatty acids and their metabolites were then ex-tracted on a C18 silica cartridge and eluted in ethyl acetate. Metabolites were further purified by TLC with hexane, diethyl ether, and acetic acid (75/25/10) as mobile phase. Bands with products were eluted with ethyl ace-tate, evaporated to dryness, and dissolved in ethanol for analysis, as de-scribed under section 4.5. 7,10-dihydroxyoctadecanoic acid (7,10-(OH)2-18:0) was prepared by hydrogenation of 7,10-(OH)2-18:1, catalyzed by Pd/C.

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3.5 Preparation of isotopomers

Standards for [2H8]15-HEETs were prepared by soybean lipoxygenase as described for non-labeled compounds, but with deuterated arachidonic acid as substrate. Standards for [2H8]12S-HEETs were obtained by 12S-lipoxygenase from human trombocytes [137]. The cytosolic fraction of hu-man trombocytes were prepared by breaking the cells by three cycles of freeze-thawing (-80°C/37°C), followed by ultracentrifugation (100,000g) for 1 h at 4°C. 1.5 mL of this fraction was then incubated with deuterated ara-chidonic acid for 10 minutes at 37°C, in the presence of 1 eq. hematin. Thereafter the sample was extracted with ethyl acetate (2 x 5 mL), and the organic phase was evaporated to dryness, dissolved in ethanol, evaporated under nitrogen and dissolved in 5% isopropanol in hexane for analysis, as described under section 3.2.2. Deuterated standards for epoxyalcohols from linoleic acid were prepared like regular epoxyalcohols as described under section 3.2.1, but with deuterated linoleic acid as substrate.

18O-Labeled 7,10-OH-18:1 was produced by extract of Pseudomonas aeruginosa as described under section 3.4, but under an 18O2 atmosphere. To achieve this, the reaction vessel was repeatedly evacuated, and flushed with nitrogene, before 18O2 was introduced and 18:1n-9 injected. 2H-labeled 7,10-OH-18:1 and 7,10-OH-18:0 were obtained by hydrogen exchange. Directly before infusion into the mass spectrometer the substrate was evaporated to dryness and dissolved in 2H2O/2HOCH3 (1:1).

3.6 Expression system

For this project a yeast system with Saccharomyces cerevisiae was utilized. The advantages of a yeast system are that it can express relatively high amounts of enzymes, it is possible to measure P450 spectra directly from whole cells or microsomes, and it has a fairly low proteolysis [138]. Since yeast is an eukaryote, some post-translation modifications are present, in contrast to e.g., E. coli [138]. The strain of S. cerevisiae in use, named W(R), is specially designed for cytochrome P450 expression, with overexpression of NADPH reductase, and with several selection markers, among them defi-ciency of adenine, tryptophan and uracil [139,140]. This strain has previ-ously been successfully used for expression of CYP4F8 [78], but also sev-eral other mammal CYP enzymes, e.g., CYP4F5 [141], CYP4F6 [141], CYP4F12 [75], and CYP4F21 [142].

The protocol for setting up a recombinant enzyme system based on the W(R) strain is well established [78,139,140]. Essentially it was performed as follows. The full length coding region for CYP4F22 was amplified by PCR,

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along with two linker sequences and three additional adenine residues before the ATG to increase the efficiency [143]. The amplicon was sequenced, and two missense mutations were identified. These were restored by site directed mutagenesis, and confirmed by new sequencing. The open reading frame was then subcloned into the yeast expression vector pYeDP60 [144], be-tween the linker sequences, and under a galactose promoter. The vector was transformed into W(R) (made chemically competent by treatment with lith-ium acetate [145]), and grown on minimal media. Uracil and adenine were used for selection, since they are both expressed by the pYeDP60 vector, but were deficient in the media. To obtain a high density culture the yeast was then grown in complete media with glucose, before a final change to galac-tose as energy source. Both the CYP insert and the NADPH reductase gene have galactose promoters, and were induced under these conditions. Since CYP enzymes are membrane bound in the endoplasmatic reticulum, the mi-crosomal fraction was used for analysis of catalytic properties. Microsomes were obtained by harvesting the cells by centrifugation at 6,800g for 4 min, breakage of the cell walls by shaking with glass beads, differential centrifu-gation (20,000 g for 10 min and 100,000 g for 60 min), resuspension of the microsomal fraction in buffer, and homogenization.

3.7 Enzyme assay

Microsomes of CYP4F8 or CYP4F22 were incubated with the substrate in question for 30 min at 37°C in the presence of NADPH. Samples without NADPH were used as controls in all experiments. The reaction was termi-nated by the addition of 4 volumes of ethanol, and the samples were centri-fuged (15,000g for 10 min at 4°C). Fatty acid contents were extracted on a C18 silica cartridge, eluted in ethyl acetate, evaporated to dryness and dis-solved in ethanol for analysis, as described under section 3.9.

3.8 Analysis of epoxyalcohols in human cornea

To evaluate if epoxyalcohols could be analyzed in human tissue, a human cornea was homogenized in KHPO4 buffer (pH 7.4). Arachidonic acid (100 μM) was added, and the sample was incubated for 30 min in 37°C. The reac-tion was terminated with 4 volumes of ethanol, centrifuged (2,000g in 5 min), and evaporated to a volume of about 1 mL. The sample was extracted on a C18 silica cartridge, eluted in ethyl acetate, and dissolved in ethanol. By NP-HPLC-MS/MS the epoxyalcohols were separated and analyzed, as de-scribed under section 3.2.2.

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3.9 Analysis of metabolites

Hydroxylated products of epoxides, arachidonic acid, and linoleic acid were separated on a reverse phase system on a 2 x 250 mm C18 column. They were eluted in 75-80% methanol with 0.01% acetic acid, and detected by mass spectrometry. Spectra of hydroxyeicosatetraenoic acids have been pub-lished [99], with the exception of the spectra for 16-HETE. This metabolite was identified by a characteristic cleavage (loss of 86 Da) adjacent to the hydroxyl group at the omega end. Also �3-hydroxylated EET metabolites were identified by characteristic loss (loss of 58 Da) from the omega end.

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4 Results and discussion

4.1 Separation of hydroperoxides (Paper I)

Previous research has indicated that some hydroperoxides are not readily detected by ESI-MS/MS [146]. A study was therefore conducted to demon-strate separation and detection of all six hydroperoxides from autoxidation of arachidonic acid [89,113] and the corresponding four hydroperoxides for photooxidation of linoleic acid [129]. The results clearly display the impor-tance of the isolation width within the mass spectrometer (Figure 6). As de-scribed earlier, the ions are separated within the ion trap by a radio fre-quency combined with an AC voltage over the rods. The isolation width setting is a window for isolation of the ions of interest. A narrow window gives less unspecific ions, but a narrow window means a higher voltage has to be applied and the energy within the ion trap increases. If the isolation width is too small the unstable hydroperoxides will disintegrate in the trap and cannot be detected. Figure 6 shows that with an isolation width of 1.5 Da (a common setting used in most of our experiments), neither 15-HPETE nor 5-HPETE could be detected. By increasing the isolation width the signal intensity of all the hydroperoxides also increased. Similar results were found for hydroperoxides from both 18:2n-6 and 18:3n-3, but no increased signal could be seen for two related alcohols. Since no problems have been encoun-tered for other compounds this phenomenon seems to be restricted to the relatively unstable hydroperoxides.

For separation of the cis-trans conjugated enantiomers of hydroperoxides of arachidonic acid, a chiral straight phase system, with a Reprosil Chiral NR column, was utilized. Analyzed by LC-MS3 (m/z 335 � m/z 317) seven peaks were recognized, but in a complex mixture of compounds. By using selective ion monitoring for characteristic ions of each compound, all hy-droperoxides except 9-HPETE were separated (Figure 7). The S configura-tion eluted before the R configuration for all enantiomers except 8-HPETE. These results are in contrast to the results by Schneider et al. for the Chiral-pak-AD column, where almost all tested HPETE eluted with the R-configuration first [103]. 5-HPETE eluted last from the Reprosil Chiral-NR column, well separated. Also the four hydroperoxides from autoxidation of linoleic acid were separated on this column, by utilizing selective ion moni-toring (see Paper I).

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Figure 6. MS/MS analysis of 8-HPODE and 10-HPODE with different isolation widths. Bottom chromatogram shows UV spectrophotometric analysis at 208 nm of the same compounds.

Figure 7. Separation of HPETEs by chiral phase HPLC. Top chroma-togram shows total ion current. Other chromatograms show selec-tive ion monitoring of characteristic ions for each compound.

4.2 MS/MS analysis of hydroperoxides (Papers I and II)

It has been demonstrated that hydroperoxy fatty acids and the corresponding keto compounds are subjected to the same dissociation during MS/MS analysis [134]. One explanation would be that the unstable hydroperoxide structure would convert into the keto derivative within the mass spectrome-ter. The spectra of some of the conjugated keto compounds of arachidonic acid have previously been reported [134]. In Paper I these results are com-plemented by the spectra of 8-, 9-, and 11-ketoeicosatetraenoic acid (KETE). They were compared with the spectra of the corresponding HPETE and were concluded to be identical. Figure 8 shows the spectrum and fragmentation of 9-KETE, to visualize the importance of proton shift. The conjugated double bonds mobilize the protons within the system, and double bond migration is therefore common. The mild acetic conditions also make the keto group subjected to acid catalyzed keto-enol tautomerism. By attracting one hydro-gen from the environment to the keto oxygen, a positive charge is put on the carbonyl carbon. By abstraction of another hydrogen from the �-carbon, with an accompanying double bond formation, the charge is restored. This reac-tion is reversible and in equilibrium, and is of great significance for under-

39

standing the fragmentation pattern of conjugated keto compound. If this aspect is not considered some fragment ions of keto compounds appear to arise through cleavage of carbon-carbon double bonds, something that is not energetically plausible.

In the spectra of 9-KETE the loss of carbon dioxide and water is easily distinguished, in the ions at m/z 299 and m/z 273, as described for uncharac-teristic fragmentation in section 1.5.4.2. The ion at m/z 193 has probably the structure as in Figure 8C, if we compare it with the fragmentations proposed for other KETEs [134]. The corresponding ion is also seen in the spectra of 8-KETE and 11-KETE (see Figure 9 in Paper I), and it has been determined to contain the omega end. Any further mechanism to produce this fragment has even so not been proposed, but it appears to involve some rearrangement mechanism. Rearrangement mechanisms are also required to explain the dissociations occurring further away from the functional group, e.g., the ion at m/z 219.

Figure 8. Fragmentation of 9-KETE. A, Keto-enol tautomerism explains the signals at m/z 193 and m/z 219. B, MS/MS spectrum (m/z 317) of 9-KETE. C, Proposed fragment at m/z 193.

4.3 Separation of epoxyalcohols (Papers II and IV)

Epoxyalcohols from 12- and 15-hydroperoxides of arachidonic acid and 9-, 11-, and 13-hydroperoxides of linoleic acid were produced nonenzymatically by hematin treatment [120,132,133]. They were separated on a normal phase system on a silicic acid column, eluted with 3% isopropanol in hexane.

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Figure 9 shows the separation of the epoxyalcohol products of 15-HPETE, analyzed by MS/MS (m/z 335). Four epoxyalcohols were generated as main products for 15S-HPETE; 11(R or S)-hydroxy-14,15-trans-epoxyeicosa-trienoic acid (peak III and IV) and 13(R or S)-hydroxy-14,15-trans-epoxy-eicosatrienoic acid (peak I and II). The erythro isomer eluted ahead of the threo isomer as judged by commercial standards, and in accordance with the elution order previously reported [147]. The minor peaks compose the ep-oxyalcohols of cis configuration. It is not possible to distinguish between the diastereomers by mass spectrometry, but nothing indicates that these results would not be in accordance with the elution order suggested by Chang et al [147]. Also epoxyalcohols from 12-HPETE and HPODEs separated on this normal phase system. The elution order of the later was as described in the literature for epoxyalcohols of linoleic acid [148] (see paper II).

Figure 9. Separation of epoxyalcohols from 15S-HPETE. Material in peaks I and II was identified as 13,14S,15S-HEET, and material in peaks III and IV as 11,14S,15S-HEET.

4.4 MS/MS analysis of epoxyalcohols (Papers II and IV)

Analysis of epoxyalcohols by LC-MS/MS suffers from difficulties due to complex spectra with a lot of abundant ions (see e.g., the 13,14,15-HEET MS/MS spectrum in Figure 11D). The reason for this seems to be a rear-rangement of the hydroxyl and epoxy groups, a so called epoxide migration or Payne rearrangement [149]. This reaction was first described in 1939 [150], and a mechanism for it was proposed in 1957 [151]. If the hydroxyl group is present as an anion it could attack the adjacent carbon and open the epoxy group (Figure 10). By this nucleophilic attack a new epoxy ring would

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be formed. Usually this mechanism demands a strong base to produce the anion [151], but charge migration within the fatty acid derivatives could leave the hydroxyl group charged. The fragmentation within the ion trap will open the epoxide group. In Paper II the spectra of 11-hydroxy-9,10-epoxy- and 9-hydroxy-10,11-epoxy-octadecenoic acid gave virtually identical ions, so did 13-hydroxy-11,12-epoxy- and 11-hydroxy-12,13-epoxy-octadecenoic acid. This confirms that an epoxide migration do occur within the mass spec-trometer. All spectra contained two characteristic fragments separated by 30 (HCOH), showing that both forms are present in the analysis. Based on the abundance of these two ions in the spectra the epoxide opening seems more selective for one of the structures. In Paper IV also the spectrum of 13,14,15-HEET showed evident signs of Payne rearrangement (the ions at m/z 235 and m/z 205, respectively), but interestingly, no such signs were apparent in the spectrum of 10,11,12-HEET.

Figure 10. Payne rearrangement of the vicinal epoxyalcohols.

The complicity of the spectra could be a disadvantage of the LC-MS/MS method. For certain identification appropriate standards are required, espe-cially since mass spectra of fatty acid epoxyalcohols analyzed by LC-MS/MS have previously not been published. These kinds of rearrangements are not present in GC-MS analysis of trimethylsilyl ether derivatives of ep-oxyalcohols, and all compounds could be verified with this technique. GC-MS is still held as the most powerful method for oxilipin analysis [113,152], but for certain fatty acid metabolites, and with the rearrangement in mind, LC-MS/MS is definitely a simple and robust alternative for analysis of ep-oxyalcohols.

Figure 11 shows MS/MS spectra (m/z 335) of the two forms of epoxyal-cohols produced from 12-HPETE and 15-HPETE, respectively. Uncharacter-istic fragmentation from loss of one or two water molecules (ions at m/z 317 and m/z 299, respectively), loss of carbon dioxide (ion at m/z 273), and loss of both carbon dioxide and water (ion at m/z 255), is easily identified in the spectra. The most abundant ions in most of the spectra are from �-cleavage within the epoxy group, or between the epoxy group and the hydroxyl group. Both these reactions will form the terminal aldehyde.

Investigations of epoxyalcohols are also troublesome due to the unstable nature of these compounds. The allylic epoxyalcohols are easily converted to triols also under mild acidic conditions [153], or the epoxides could be opened by epoxide hydrolase activity within a biological assay [154]. This might be a problem not only in the analysis of compounds, but also in the

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production of material for enzyme assays. Since epoxyalcohols do not in-clude a conjugated system and therefore only exhibits end absorption in the UV spectra, it is difficult to determine the amount of epoxyalcohols avail-able after separation. These difficulties were met by simultaneous MS/MS analysis of trihydroxy metabolites (m/z 351). Such analysis revealed very low conversion of epoxyalcohols. In Paper IV, an enzyme assay of epoxides metabolized by CYP4F8 was conducted, as described in section 3.7, where simultaneous analysis of diols did not show any epoxide opening.

Figure 11. MS/MS spectra (m/z 335) of epoxyalcohols formed by hematin treatment from 12S-HPETE and 15S-HPETE. A, 8,11S,12S-HEET. B, 10,11S,12S-HEET. C, 11,14S,15S-HEET. D, 13,14S,15S-HEET.

4.5 MS/MS analysis of diols (Paper III)

Several studies have been published investigating the fragmentation of fatty acid diols [102,127,155]. For all of them the classical mechanisms with

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cleavage adjacent to the hydroxyl groups are attributable, as described under section 1.5.4.3. Surprisingly, the spectra of 7,10-dihydroxyoctadecenoic acid (7,10-(OH)2-18:1) did not coincide with this pattern. The structure of the compound was verified by both GC-MS and NMR analysis. Even so, the expected fragments at m/z 143 and m/z 199 were not noticed in this spec-trum. An even more interesting finding was the great similarities between this spectrum and the spectrum of the hydrogenated compound 7,10-(OH)2-18:0. The uncharacteristic fragmentation differed by 2 Da representing the extra hydrogens, but the additional ions were more or less identical (Figure 12). This indicated that the dissociation was not influenced by the double bond. Although, it could not explain the complex spectra of these com-pounds.

Figure 12. MS/MS spectra (m/z 313 and m/z 315, respectively) of the diol 7,10-(OH)2-18:1 (A), and 7,10-(OH)2-18:0 (B).

An alternative approach was that the two compounds experienced similar, but not identical, charge-remote or charge-driven mechanisms, producing the same ions. Instead of the mentioned expected ions, the characteristic and structural informative ions seems to be three ions at m/z 141, m/z 155, and m/z 169, identified in both spectra. Studies with fatty acids with prolonged or shortened carbon chains (20 or 16 carbons, respectively) confirmed that these ions contained the omega-end of the fatty acids. As discussed under section 1.5.4.3, the most common fragmentation is towards the carboxyl end, but omega-end fragments have been reported in numerous studies [102,124,155].

MS3 analysis (m/z 313 � m/z 269 and m/z 313 � m/z 251) revealed that for both compounds the ions at m/z 141 and m/z 169 had their origin in the fragment at m/z 269, and the ion at m/z 155 had its origin in the fragment m/z 251 (for data see paper III). Further investigations using 2H or 18O labeled hydroxyl groups advanced into the proposed mechanisms displayed in Figure 13, for the three characteristic fragments. Besides the characteristic ions the mechanisms differs between the two compounds in other aspects.

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The initial loss of water from the molecular ion was in the case of 7,10-(OH)2-18:1 from one of the hydroxyl group, but in 7,10-(OH)2-18:0 the wa-ter molecule instead derived from the carboxyl group, according to investi-gations with 18O labeled isotopomers.

Interestingly, with an additional double bond between carbon 11 and 12, the dissociation returned to normal �-cleavage, with the expected ions at m/z 143 and m/z 199 (see Figure 4 in Paper III). This demonstrates the impact of double bonds in fragmentation mechanisms, even though the double bond between C8 and C9 did not seem to be important.

Figure 13. Fragmentation mechanisms for the formations of the ions at m/z 141 (A), m/z 155 (B), and m/z 169 (C) from 7,10-(OH)2-18:1.

4.6 Detection of epoxyalcohols in corneal tissue (Paper IV)

As with epidermis, the cornea has protective purposes, and fatty acids play an important part in normal functionality. When a human cornea was incu-bated with arachidonic acid for 30 min at 37°C, 15-HEETs were unambigu-ously formed (Figure 14). This demonstrated that the procedure (se section 3.8), with extraction of metabolites on a reverse phase C18 cartridge, fol-lowed by separation on a normal phase system with a silica column, the products eluted in 3% isopropanol in hexane, and finally detected by MS/MS, could be utilized for analysis of epoxyalcohols in human tissues.

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Human cornea has previously been reported to contain 15S-LOX [156,157], which is in agreement with these results.

Figure 14. LC-MS/MS analysis (m/z 335) of epoxyalcohols from human cornea incubated with arachidonic acid. Materials in peaks I and II was identified as 13,14S,15S-HEET, and material in peak III as 11,14S,15S-HEET. Peak denoted with an asterisk labels unrelated compounds.

4.7 Catalytic properties of CYP4F22 (Paper IV)

Incubation of CYP4F22 microsomes and arachidonic acid, in the presence of NADPH, yielded two different metabolites compared to control reactions without NADPH; 16-hydroxy- and 18-hydroxyeicosatetraenoic acid (16-HETE and 18-HETE)(Figure 15). The conversion rate of both metabolites was low, and a Km value was therefore not established. The MS/MS spec-trum of 18-HETE was in accordance with published spectra [155]. The mass spectrum of 16-HETE has previously not been reported, but the metabolite was identified by a characteristic loss of the omega end (loss of 86 Da), ad-jacent to the hydroxyl group, as described for other fatty acids with omega end hydroxylation [99,155]. Unfortunately, the production of 16-HETE was not restricted to the system with the CYP4F22 open reading frame, but could also be detected in control experiments where a vector without insert had been transfected into the yeast cells, and also in untransfected yeast. Several sources of W(R) have been tested multiple times, so contamination seems unlikely. Yeast systems are known for only low levels of endogenous cyto-chrome P450 activity [140], although some CYPs are expressed in S. cere-visiae [142]. The best explanation still, is NADPH dependent enzyme activ-ity within the yeast system itself, even though 16-hydroxylase activity has previously not been reported.

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Control experiments did not show any production of 18-HETE, and this metabolite can not be formed non-enzymatically. This speaks for a true me-tabolism of arachidonic acid by CYP4F22. The production of 18-HETE was even so not according to the hypothesis. In 2006, Fischer’s group reported a linkage between mutations in the gene CYP4F22 and lamellar ichthyosis [84]. Their theory was that CYP4F22 is responsible for the �-hydroxylation of either 8R,11R,12R-HEET, or a subsequent trihydroxy metabolite, in the hepoxilin pathway (Figure 3).

Figure 15. MS/MS analysis (m/z 319) of metabolites formed from arachidonic acid by recombinant CYP4F22.

For arachidonic acid the catalytic performance of CYP4F22 seems to be a �3-hydroxylase instead. This result is surprising also from another aspect. Of the CYP4F family members, only CYP4F8 and CYP4F12 have previ-ously been shown to be �3-hydroxylases. All other studied isozymes me-tabolize the substrates in the terminal �-position. In contrast to these en-zymes, CYP4F8 and CYP4F12 do not have a glutamate in the position 328, but a glycin. This glutamate residue has been shown to be crucial for a cova-lent linkage to the heme group, and affects the preferred site of hydroxyla-tion [143,144]. A site directed mutation study, where glycin of CYP4F8 was mutated to a glutamate, demonstrated a change of hydroxylation site towards the �-end [72]. The glutamate 328 is in a much conserved part of the en-zyme, and this part is found also in the sequence of CYP4F22, with the cor-responding glutamate in position 335. The occurrence of the glutamate resi-due within the conserved element, although shifted seven amino acids, also suggests an �-hydroxylase activity of CYP4F22.

No metabolites from enzyme assays with 8,11R,12R-HEET could be de-tected, supporting that CYP4F22 may not be involved in the hepoxilin path-

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way, as hypothesized. But the theory still holds that the 8,11,12-trihydroxy compound is the substrate. For falsification of the hypothesis this compound also need to be tested. A method for production and separation of THETEs has been presented [90], but with the reservation that the quantities of ob-tained products were limited. By following this protocol, a pilot study was conducted where trihydroxy products were produced from epoxyalcohols and separated, but only in low amounts (data not shown). Until superior methods to produce large quantities of THETEs have been developed, the question of the CYP4F22 position in the etiology of lamellar ichthyosis re-mains unanswered.

4.8 Catalytic properties of CYP4F8 (Paper IV)

In previous studies CYP4F8 has been demonstrated to be the main 19-hydrolase of PGH in human seminal vesicles [78]. It has also, inter alia, been shown to catalyze arachidonic acid to 18-HETE [78], linoleic acid to 16-HODE [78], PGI2 to 19-OH-PGI2 [79], and a stable TXA2 analogue to both �2 and �3 oxygenated products [79]. Apart from seminal vesicles, CYP4F8 is also highly expressed in skin, but the function of the enzyme there has not been established.

Figure 16. MS/MS analysis (m/z 351) of metabolites formed from 10,11R,12R-HEET by recombinant CYP4F8.

To determine if CYP4F8 could be involved in the metabolism of epoxyalco-hols recombinant enzyme was incubated with 8,11R,12R-HEET, 10,11R,12R-HEET, 11,14S,15S-HEET, or 13,14S,15S-HEET. Only for 10,11R,12R-HEET any hydroxylated product could be detected; the 18-hydroxy metabolite, as identifies by a characteristic ion at m/z 293 (loss of

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58 Da) in the MS/MS spectrum (m/z 351)(Figure 16). The related compound HXB3 (10,11S,12S-HEET) has been shown to be increased in psoriasis [26,27], and to affect the permeability of the skin [24,25]. The authors of these reports have also not excluded the possibility that it is actually the 10,11R,12R-HEET, since they assumed the 12S-LOX activity (e.g., [106]). In either case, this could make an interesting linkage between the enzymes 12R-LOX and CYP4F8 in skin.

All three other epoxyalcohols tested were not substrates for CYP4F8 in this study. To evaluate if the position of the epoxy group could influence the metabolic preferences, three epoxides of arachidonic acid were investigated. From this study it was clear that CYP4F8 could metabolize 8,9-EET and 11,12-EET, but not 14,15-EET. These results supported the conclusion that products derived from 15S-HPETE are not substrates for CYP4F8.

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5 Conclusions

The main conclusions of this thesis are as follows. • The Reprosil Chiral NR and Reprosil Chiral NRR columns could be

utilized for stereospecific separation of hydroperoxides. By this ap-proach, 12R-HPETE was prepared from autoxidation of arachidonic acid.

• All hydroperoxides from autoxidation of arachidonic acid or photooxida-

tion of linoleic acid could be identified by MS3 analysis, using an isola-tion width of 5 Da within the ion trap.

• Normal phase HPLC-MS/MS could be used for separation and identifi-

cation of epoxyalcohols derived from either linoleic acid or arachidonic acid.

• The fragmentation mechanisms of fatty acid hydroperoxides, epoxyalco-

hols, 7,10-diols, and keto compounds were deduced by MS/MS analysis, and with aid of isotopomers.

• Specific epoxyalcohols for enzyme assays were prepared by hematin

treatment of 12R-HPETE, and the products were separated on normal phase HPLC.

• The LC-MS/MS method could be utilized for analysis of epoxyalcohols

in tissue, as visualized by human corneal samples. • Recombinant CYP4F22 was expressed in yeast. CYP4F22 converted

arachidonic acid to 18-HETE, but epoxyalcolhols of 12R-HPETE were not metabolized.

• Recombinant CYP4F8 converted 10,11,12-HEET to its �3 metabolite,

which suggests a functional link between 12R-LOX and CYP4F8 in epi-dermis.

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6 Future perspectives

A key point in the continuous work to establish the catalytic function of CYP4F22 is the availability of a robust recombinant enzyme system with high activity. Several research groups have been trying to develop such a system for CYP4F22. So far all experiments in E. coli have been unsuccess-ful [158]. If the low conversion rate of arachidonic acid in the yeast system is due to low enzyme activity, or low substrate affinity is still not known. CYP4V2 was recently expressed in baculovirus transfected into insect cells [71]. If the yeast system expressing CYP4F22 continues to give low activity, an alternative would be to change to an insect cell system instead. The link-age between CYP4F22 and lamellar ichthyosis seems to be well established, and definitely worth continuous efforts. The next step in the hepoxilin path-way is the trihydroxy compounds derived from 12R-HPETE. In low amounts they are easy to synthesize, but to gain a large enough quantity for enzyme assays is more difficult. These are the compounds originally hypothesized to be ligands for the ichthyin receptor, together with the �-carboxyl metabolite [43]. If the epoxide is opened before or after �-hydroxylation, in accordance with Figure 3, is unknown, but the results of this study indicate that the ep-oxyalcohol is not the substrate. The progression of this work would be to establish an analytical method to achieve these trihydroxy products and try them as substrates for CYP4F22.

A more clinical approach under consideration is to start up a study to ana-lyze epoxyalcohols in patients with ichthyosis. Cooperation with the skin clinic at Uppsala academic hospital is already initiated. A first step would be to investigate if the successful process of extracting epoxyalcohols from cornea could be applicable also for skin tissue. Since the clinic already has contact with many patients, such a study would not be unattainable.

As for CYP4F8, the good catalytic activity for both epoxides and the 10,11R,12R-HEET is interesting, but the research is complicated due to the unknown function of these compounds in epidermis. Since a high-activity recombinant system is present, and the function of this isozyme in skin has not been established, further studies of catalytic properties could be moti-vated. Another interesting area is the still unknown cause of interindividual variations in �2-hydroxylation of PGH in human seminal vesicles.

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Populärvetenskaplig sammanfattning

Fettsyror är ofta förknippade med kroppens energiförbrukning, men på sena-re år har alltfler börjat inse att de är viktiga även för många andra funktioner i kroppen. Många av dessa funktioner styrs av metaboliter av fettsyrorna, d.v.s. produkter som antingen är mer, eller mindre aktiva än fettsyran själv. Dessa metaboliter kallas ibland oxilipiner, om de har fler syremolekyler än fettsyran. Hos människa bildas de viktigaste oxilipinerna från fettsyran ara-kidonsyra, medan hos många lägre organismer (växter, svampar m.m.) bildas de från linolsyra. Arakidonsyra är en s.k. omega-6-fettsyra och är framför allt känd för att vara inblandad vid inflammation. Men den ingår även som byggsten i cellernas membran, och dess oxilipiner fungerar som signalämnen när olika funktioner i kroppen ska kommunicera med varandra.

Under de senaste 20 åren har man insett att både personer med psoriasis och personer med fiskfjällssjuka, iktyos, har högre nivåer av vissa av dessa metaboliterna i huden. Framför allt gäller det ämnet 12R-HPETE, som är en hydroperoxid, och 8,11R,12R-HEET, som är en epoxialkohol. Dessa ämnen bildas av enzymerna 12R-LOX respektive eLOX3. Man har i studier kunnat visa att mutationer i generna för båda dessa enzymerna är förknippade med lamellär iktyos, som är en ovanlig, men allvarlig form av fiskfjällssjuka. I en djurmodell med råttor som saknade genen för 12R-LOX, uppvisades hudför-ändringar som påminner om dem hos patienter med lamellär iktyos.

Andra gener har också visat sig vara inblandade, och en av dem är CYP4F22. Detta enzym tillhör en stor familj av enzymer kallad cytokrom P450 (CYP), som metaboliserar både kroppsegna och främmande ämnen. Vilken biologisk funktion som CYP4F22 har är ännu inte klarlagt. Ytterliga-re ett enzym som uttrycks i huden, och som hittills inte kunnat förklaras är det närbesläktade enzymet CYP4F8. Intressant är att både CYP4F22 och CYP4F8 verkar uttryckas mer vid hudsjukdomar som psoriasis. Det verkar som om dessa enzymer är viktiga vid utveckladet av normal hud. Det här forskningsprojektet har haft som mål att undersöka om dessa två enzymer är inblandade i metabolismen av framför allt 8,11R,12R-HEET.

Projektet har framför allt inriktat sig på att ta fram metoder för att kunna producera och isolera de aktuella oxilipinerna. 12R-HPETE bildades från arakidonsyra genom att syrgas tillsattes och reaktionen fick stå i 37°C i 36 h. Med vätskekromatografi, där man utnyttjar att olika ämnen i olika utsträck-ning binder in till antingen ett fast, eller ett flytande ämne, kunde 12R-

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metaboliten separeras från samtliga andra hydroperoxider som också bildas. 8,11R,12R-HEET bildades sedan från 12R-HPETE genom att tillsätta ämnet hematin.

Masspekrometri är en annan analysmetod, där man istället separerar mo-lekyler beroende på dess vikt och dess laddning. Tekniken innebär även att man kan slå sönder, fragmentera, molekylen i mindre bitar och på så sätt erhålla ett specifikt spektrum, ett fingeravtryck, för varje enskild molekyl. På det sättet kan man lätt identifiera olika föreningar, och denna metod har i detta projekt används för att säkerställa att rätt metabolit har framställts. Men då det sedan tidigare inte har funnits några rapporterade spektran av dessa ämnen var det först nödvändigt att ta fram standarder, och klargöra hur dess ämnen fragmenterar.

När metaboliterna framställts tillsattes de till rekombinant CYP4F22. Det-ta är ett enzymsystem där den kodande delen av genen för CYP4F22 har tagits ut ur det mänskliga genomet och istället uttrycks i ett annat system, i det här fallet jästceller. På det sättet kan man undersöka vilken effekt detta enzym har isolerat från andra humana enzymer, som annars skulle kunna påverka. Samma metaboliter undersöktes även med rekombinant CYP4F8. Studierna visade att varken CYP4F8 eller CYP4F22 kunde metabolisera 8,11R,12R-HEET. Däremot kunde CYP4F8 metabolisera en annan epoxial-kohol benämnd 10,11R,12R-HEET. CYP4F22 kunde däremot metabolisera arakidonsyra, vilket visar att enzymet är aktivt.

Det huvudsakliga syftet med denna avhandling var dock att visa att hyd-roperoxider och epoxialkoholer, kan tas fram och analyseras med hjälp av en kombination av vätskekromatografi och masspektrometri. Den visar bland annat att epoxialkoholer har kunnat analyseras från human hornhinna. En fortsättning på det projektet skulle kunna vara att analysera epoxialkoholer hos patienter med lamellär iktyos, för att klarlägga dess betydelse vid denna sjukdom

53

Acknowledgements

This study was carried out at the Department of Pharmaceutical Biosciences, Faculty of Pharmacy at Uppsala university, Sweden.

Many people have contributed to this work, in one way or another. I especially want to thank:

My supervisor Prof. Ernst Oliw, for believing in me, and for your struggle to try to teach me “the laboratory way”. You sure know when to push, and when to pull.

My co-supervisor Dr. Johan Bylund for introducing me to both the project and the life as a graduate student.

Former and present members of the Oliw group: Mirela for your joyfulness; Ulrike for all your knowledge and all the laughs; Fredrik for the never ending history lessons and football chats. I actually like your music, and it’s nice to have someone to yell at, when the frustration runs over; Inga for bringing new spirit and new questions into the lab.

My other co-authors: Eriel Martinez, Dr Angeles Manresa, and Dr Igor Ivanov for the scientific input.

Erica Johansson for your endless patience, and for always helping me out with everything.

Raili Engdahl for all the help in the lab, and for the coffee-breaks during the first years.

Former and present PhD-student colleagues: Alfhild, Anna, Carolina, Hanna, Jenny, Johan, Jonathan, Kristina, Loudin, Martin, Sadia, Sara, Tobias, and Uwe. For all help during the years, but also for all the fun at lunches, parties, conferences, or just at “kurslabb”.

Marie for all the chats, and the support when I needed it the most.

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Magnus Jansson for helping me out whenever my computer went nuts.

And all of you outside of BMC who made this possible with your support:

Andreas, Björn, Erik, Johan, and Leo because I know that you are always there.

Anna, Ann-Sofie, Emma, and Magda for a really good time, and a lot of fun. But also for listening when it wasn’t.

All other people who have made my life as a PhD-student even easier by occupying my spare time: spexare (especially Jocke, Erik and Markus), FDRare, theatre people, and many more… My family: Mamma, Pappa, Therese. For always supporting me. You know I love you.

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