The oxygenated derivatives of fatty acids, known as oxylipins, are pivotal signaling molecules in animals and terrestrial plants. In animal systems, eicosanoids regulate cell differentiation, immune responses, and homeostasis. In contrast, terrestrial plants use derivatives of C18 and C16 fatty acids as developmental or defense hormones. Marine algae have emerged early in the evolution of eukaryotes as several distinct phyla, independent from the animal and green-plant lineages. The occurrence of oxylipins of the eicosanoid family is well documented in marine red algae, but their biological roles remain an enigma. Here we address the hypothesis that they are involved with the defense mechanisms of the red alga Chondrus crispus. By investigating its association with a green algal endophyte Acrochaete operculata, which becomes invasive in the diploid generation of this red alga, we showed that (1) when challenged by pathogen extracts, the resistant haploid phase of C. crispus produced both C20 and C18 oxylipins, (2) elicitation with pathogen extracts or methyl jasmonate activated the metabolism of C20 and C18 polyunsaturated fatty acids to generate hydroperoxides and cyclopentenones such as prostaglandins and jasmonates, and (3) C20 and C18 hydroperoxides as well as methyl jasmonate did induce shikimate dehydrogenase and Phe ammonialyase activities in C. crispus and conferred an induced resistance to the diploid phase, while inhibitors of fatty acid oxidation reduced the natural resistance of the haploid generation. The dual nature of oxylipin metabolism in this alga suggests that early eukaryotes featured both animal- (eicosanoids) and plant-like (octadecanoids) oxylipins as essential components of innate immunity mechanisms.

This paper provides a comprehensive review of high-performance liquid chromatography (HPLC) methods for fatty acid analysis in various sample matrices. After a brief introduction about the generalities of HPLC, including its modes and applications, the review focuses on various aspects of fatty acid analysis by HPLC, such as sample preparation, mobile phases, stationary phases, detection techniques and the main matrices were fatty acids can be accessed, emphasizing the importance of this analysis in biological samples.

Hypertension is a leading cause of cardiovascular, cerebral, and renal disease morbidity and mortality. Here we show that disruption of the Cyp 4a14 gene causes hypertension, which is, like most human hypertension, more severe in males. Male Cyp 4a14 (−/−) mice show increases in plasma androgens, kidney Cyp 4a12 expression, and the formation of prohypertensive 20-hydroxyarachidonate. Castration normalizes the blood pressure of Cyp 4a14 (−/−) mice and minimizes Cyp 4a12 expression and arachidonate ω-hydroxylation. Androgen replacement restores hypertensive phenotype, Cyp 4a12 expression, and 20-hydroxy-arachidonate formation. We conclude that the androgen-mediated regulation of Cyp 4a arachidonate monooxygenases is an important component of the renal mechanisms that control systemic blood pressures. These results provide direct evidence for a role of Cyp 4a isoforms in cardiovascular physiology, establish Cyp 4a14 (−/−) mice as a monogenic model for the study of cause/effect relationships between blood pressure, sex hormones, and P450 ω-hydroxylases, and suggest the human CYP 4A homologues as candidate genes for the analysis of the genetic and molecular basis of human hypertension.

Background Long-term ethanol consumption in laboratory animals is associated with histological alterations of liver cells and modifications of fatty acid metabolism.

Human liver microsomes and recombinant human P450 have been used as enzyme source in order to better understand the requirement for the optimal rate of omega and (omega-1)-hydroxylations of fatty acids by cytochromes P450 2E1 and 4A, Three parameters were studied: alkyl chain length, presence and configuration of double bond(s) in the alkyl chain, and involvement of carboxylic function in the fatty acid binding inside the access channel of P450 active site. The total rate of metabolite formation decreased when increasing the allyl chain length of saturated fatty acids (from C12 to C16), while no hydroxylated metabolite was detected when liver microsomes were incubated with stearic acid. However, unsaturated fatty acids, such as oleic, elaidic and linoleic acids, were omega and (omega-1)-hydroxylated with an efficiency at least similar to palmitic acid. The (omega-1)/omega, ratio decreased from 2.8 to 1 with lauric, myristic and palmitic acids as substrates, while the reverse was observed for unsaturated C18 fatty acids which are mainly omega-hydroxylated, except for elaidic acid showing a metabolic profile quite similar to those of saturated fatty acids. The double bond configuration did not significantly modify the ability of hydroxylation of fatty acid, while the negatively charged carboxylic group allowed a configuration energetically favourable for omega and (omega-1)-hydroxylation inside the access channel of active site.

The liver microsomal fractions of seven mammalian species including rat, dog, monkey, hamster, mouse, gerbil and humans, catalyzed the hydroxylation of saturated (lauric, myristic and palmitic) and unsaturated (oleic and linoleic) fatty acids to the corresponding ω and (ω-1)-hydroxylated derivatives, while stearic acid was not metabolized. Lauric acid was the most efficiently hydroxylated, and the rank of catalytic activity was lauric>myristic>oleic>palmitic>linoleic. Among the mammalian species studied, mouse and hamster presented the highest level of fatty acid ω and (ω-1)-hydroxylases, while the lowest activity was observed in dog and monkey. In all the animal species, the (ω-1)-hydroxylation of fatty acids correlated significantly with the immunodetectable content of CYP2E1 and the 4-nitrophenol hydroxylation activity, known to be mediated by cytochrome P450 2E1. On the contrary, only the ω-hydroxylation of lauric acid slighly correlated with the level of cytochrome P450 4A, while no significant correlation was found with the ω-hydroxylation of the other fatty acids. Furthermore, chemical and immuno-inhibitions of the hydroxylations of fatty acids led to the conclusion that fatty acid (ω-1)-hydroxylase activity is catalyzed by P450 2E1 in all the mammalian species, while the fatty acid ω-hydroxylase activity may be catalyzed by cytochromes P450 from the 4A family. Therefore, lauric acid (ω-1)-hydroxylation along with 4-nitrophenol hydroxylation can be used as a specific and sensitive method to measure the level of CYP2E1 induction in humans and various animals.

Human liver microsomes and recombinant human P450 have been used as enzyme source in order to better understand the requirement for the optimal rate of v and ( v –1)-hydroxylations of fatty acids by cytochromes P450 2E1 and 4A. Three parameters were studied: alkyl chain length, presence and configuration of double bond(s) in the alkyl chain, and involvement of carboxylic function in the fatty acid binding inside the access channel of P450 active site. The total rate of metabolite formation decreased when increasing the alkyl chain length of saturated fatty acids (from C12 to C16), while no hydroxylated metabolite was detected when liver microsomes were incubated with stearic acid. However, unsaturated fatty acids, such as oleic, elaidic and linoleic acids, were v and ( v –1)-hydroxylated with an efficiency at least similar to palmitic acid. The ( v –1)/ v ratio decreased from 2.8 to 1 with lauric, myristic and palmitic acids as substrates, while the reverse was observed for unsaturated C18 fatty acids which are mainly v -hydroxylated, except for elaidic acid showing a metabolic profile quite similar to those of saturated fatty acids. The double bond configuration did not significantly modify the ability of hydroxylation of fatty acid, while the negatively charged carboxylic group allowed a configuration energetically favourable for v and ( v –1)-hydroxylation inside the access channel of active site. —Adas, F., J.P. Salaün, F. Berthou, D. Picart, B. Simon, and Y. Amet. Requirement for v and ( v – 1)-hydroxylations of fatty acids by human cytochromes P450 2E1 and 4A11. J. Lipid Res. 1999. 40: 1990–1997. Supplementary key words CYP4A11 • CYP2E1 • v and ( v –1)-hydroxylation • cytochrome b5 • human liver microsomes The ubiquitous cytochrome P450 (P450) enzymes comprise a superfamily of monooxygenases present in both eukaryote and procaryote organisms (1). Cytochromes P450 from mammals are involved in the oxidation of a large number of exogenous and endogenous compounds including fatty acids (2). The physiological role of cytochromes P450 catalyzing the hydroxylation of fatty acids remains to be clarified. The CYP4A family appears to be mainly involved in oxidation of fatty acids and derivatives. The physiological role of certain bioactive metabolites generated from eicosanoids by this P450 family is now increasingly documented, and several data show their involvement in various cell functions. In addition, a significant regulatory role of CYP4A induction in the overall balance of fatty acid degradation by b -oxidation system is becoming increasingly evident (3). The peroxisomal b oxidation system is particularly well suited for b -oxidation of fatty acids which are poor substrates for mitochondrial b -oxidation system (i.e., long-chain fatty acids). Unbalance for fatty acid degradation between a very efficient peroxisomal b -oxidation and the mitochondrial system should result in a rapid accumulation of toxic shortand medium-chain free fatty acids (i.e., lauric acid) generated by peroxisomes. Consequently, induction of members of CYP2E and CYP4A families should be associated with a detoxification process needed to reduce accumulation by cells of free fatty acids with the goal to maintain the membrane integrity. Study of the substrate specificity and the structure of metabolites generated by the major catalysts of fatty acid oxidation that belong to the CYP2E and CYP4A families are essential to demonstrate the individual role of P450s in the catabolic process. Several P450 isozymes are effective catalysts of hydroxylation of mediumand long-chain saturated and unsaturated fatty acids, although they show very different substrate selectivity and regiospecificity of the oxygene attack. The ethanol-inducible CYP2E1 isoform catalyzes not only the bioactivation of a large number of lipophilic compounds with low molecular weight, including aromatic and halogenated hydrocarbons, alcohols, ketones and nitrosamines (4–6), but also the hydroxylation of fatty acids. Recently, it was demonstrated that CYP2E1 from rat (7, 8), rabbit (9) and human (8, 10) livers was involved in the microsomal ( v –1)-hydroxyAbbreviations: P450, cytochrome P450 (E.C. 1.14.14.1) or CYP; RPHPLC, reverse phase-high performance liquid chromatography; APCI, atmospheric pressure chemical ionization; LC–MS, liquid chromatography– mass spectrometry; MNNG, 1-methyl-3-nitro-1-nitrosoganidine; PPAR, peroxisome proliferator-activated receptor. 1 To whom correspondence should be addressed. by gest, on S etem er 8, 2017 w w w .j.org D ow nladed fom

The A. thaliana EST database was screened using consensus motifs derived from P450 families CYP52 and CYP4 catalyzing the omega-hydroxylation of fatty acids and alkanes in Candida and in mammals. One EST cDNA fragment was detected in this way and the corresponding full-length cDNA was cloned from a cDNA library of A. thaliana. This cDNA coded the first member of a new plant P450 family and was termed CYP86A1. The deduced peptide sequence showed highest homology with P450s from families 4 and 52. To confirm the catalytic function, CYP86A1 was expressed in a yeast overexpressing its own NADPH-P450 reductase. Efficient expression was evidenced by spectrophotometry, SDS-PAGE and catalytic activity. CYP86A1 was found to catalyze the omega-hydroxylation of saturated and unsaturated fatty acids with chain lengths from C12 to C18 but not of hexadecane. Genomic organization analyzed by Southern blot suggested a single gene encoding CYP86A1 in A. thaliana.

In order to characterize the nature of the active site of cytochrome P450 2E1, the metabolism of various fatty acids with cis/trans geometric configurations has been investigated. A system coupling atmospheric pressure chemical ionization-mass spectrometry detection with HPLC separation was developed as an alternative method for the characterization of hydroxylated metabolites of oleic and elaidic acids in rat and human liver microsomes. Oxidation of oleic and elaidic acids led to the formation of two main metabolites which were identified by LC-MS and GC-MS as omega and (omega-1)-hydroxylated (or 17-OH and 18-OH) fatty acids, on the basis of their pseudo-molecular mass and their fragmentation. The assay was accurate and reproducible, with a detection limit of 25 ng per injection, a linear range from 25 to 1128 ng per injection, no recorded interference, intra-day and inter-day precision with variation coefficients <14%. This LC-MS method was validated with oleic acid by using both radiometric and mass spectrometric detections. A significant correlation was found between the two methods in human (r=0.86 and 0.94 with P<0.05 and 0.01) and rat liver microsomes (r=0.90 and 0.85 with P<0.01 and 0.05) for 17-OH and 18-OH metabolites, respectively. HPLC coupled to mass spectrometry for the analysis of hydroxylated metabolites of elaidic acid offers considerable advantages since the method does not require use of a radioactive molecule, completely separates the two hydroxymetabolites, confirms the identification of each metabolite, and is as sensitive as the radiometric analysis method. This method allowed the comparative study of oleic and elaidic acid hydroxylations by both human and rat liver microsomal preparations.

Arachidonic acid concentrations in liver are decreased in response to ethanol administration. In addition, the oxygenated products of arachidonic acid metabolites could affect the severity of alcoholic liver injury. Selective utilization of arachidonic acid by the cytochrome P-450 system could, in part, account for the decrease in arachidonic acid. To evaluate this pathway further, male Wistar rats were fed different dietary fats: medium chain triglycerides, palm oil, and corn oil or fish oil with either ethanol or isocaloric amounts of dextrose. Histopathology, cytochrome P-4502E1 (CYP2E1) and cytochrome P-4504A (CYP4A), and omega- and (omega-1)-hydroxylation products of lauric and arachidonic acids were evaluated. Ethanol induction of CYP2E1 was related to the concentration of polyunsaturated fatty acids in the diet; induction of CYP4A by ethanol was seen in all groups. The highest levels of 11-hydroxy-lauric acid and 19-hydroxyarachidonic acid (omega-1) were seen in rats fed ethanol with palm oil and corn oil. Highly significant correlations were seen between the (omega-1)-hydroxylation products and CYP2E1 activity. No correlation was seen between the omega-hydroxylation products and CYP2E1 activity. In contrast, the levels of omega-hydroxylation products correlated with CYP4A. The overall results showed a significant increase in (omega-1)-hydroxylation products in rats fed diets containing significant amounts of linoleic acid (i.e., palm oil and corn oil).

In vitro techniques have been used to investigate the nature of microsomal cytochrome P450 involved in the metabolism of oleic acid, a physiological monounsaturated fatty acid. Like lauric acid, which is currently used as a model substrate of fatty acid metabolism, the alkyl chain of oleic acid is hydroxylated on its v and ( v –1) carbons. The identity of these hydroxylated metabolites was ascertained by GC/MS and LC/MS. The v / v –1 ratio of oleic acid metabolites (1.22 6 0.01) was found to be similar to that obtained with lauric acid in rat liver microsomes (1.10 6 0.02), while in human liver microsomes this ratio was 0.75 6 0.5 for lauric acid and 5.2 6 2.6 for oleic acid. After treatment of rats with ethanol or clofibrate, inducers of CYP2E1 and CYP4A, respectively, the hydroxylations of oleic acid were shown to be less inducible than those of lauric acid. Five in vitro approaches were used to identify the P450 isoform(s) responsible for the microsomal ( v –1)-hydroxylation of oleic acid: effect of various inducers in rats, correlation studies between specific P450 catalytic activities in a panel of 25 human liver microsomes, chemical inhibitions, immuno-inhibitions and metabolism by cDNA-expressed human P450 enzymes. From the above results, it can be ascertained that P450 2E1 is the main enzyme involved in the ( v –1)-hydroxylation of oleic acid. Furthermore, the v -hydroxylation of oleic acid was shown to be mainly catalyzed by P450 4A enzymes in human liver microsomes. The turnover number of ( v –1)-hydroxylation of lauric and oleic acids decreased from 7.8 to 1.5 min 2 1 , respectively, suggesting that the dodecane alkyl chain allows optimal binding to the active site of CYP2E1.— Adas, F., F. Berthou, D. Picart, P. Lozac’h, F. Beaugé, and Y. Amet. Involvement of cytochrome P450 2E1 in the ( v –1)-hydroxylation of oleic acid in human and rat liver microsomes. J. Lipid Res. 1998. 39: 1210–1219. Supplementary key words CYP2E1 • lauric acid • v -oxidation • monooxygenase enzymatic activities • xenobiotics • chlorzoxazone • 4-nitrophenol • mass spectrometry Cytochrome P450s (1) are the heme-thiolate proteins of the microsomal mixed function monooxygenase system. They are involved in the metabolism of xenobiotics and endogenous compounds, such as steroids and fatty acids. Many P450 enzymes are characterized by specific substrates that are regiospecifically metabolized. Accordingly, lauric acid has been described as a model substrate for hydroxylations catalyzed by P450 in human liver and kidney microsomes (2, 3). More recently, the P450 involved in these hydroxylations has been reported both in the microsomal fractions of rat (4) and in human (5, 6) livers. In both species, lauric acid was regiospecifically metabolized to form v and ( v –1)-hydroxylated metabolites, and the ratio of these two products varied significantly after starvation, diabetes (7–9), administration of clofibrate and other peroxisome proliferators (10, 11), ethanol or CYP2E1 inducers (4, 12). Fatty acid v -oxidation is a minor pathway that accounts for less than 10% of total liver fatty acid oxidation under normal physiological conditions (13). However, studies performed with mammalian systems suggest that v -hydroxylases could be involved in the first step of fatty acid catabolism (14). Moreover, it was described that starvation or intake of certain dietary fat composition could strongly enhance fatty acid hydroxylation activities (15). Oleic acid is an unsaturated physiological fatty acid present in the free fatty acid fraction and represents approximately 25% of this fraction (16). It is one of the cis unsaturated free fatty acids (with arachidonic acid) which is released from the sn -2 position of phospholipids (17). It plays an important physiological role by activating protein Abbreviations: P450, cytochrome P450 (EC 1.14.14.1) or CYP; 17OH-oleic acid, 17-hydroxyoleic acid or ( v –1)-hydroxyoleic acid; 18OH-oleic acid, 18-hydroxyoleic acid or v -hydroxyoleic acid; PKC, protein kinase C; CHZ, chlorzoxazone; 4-NP, 4-nitrophenol; 17-ODYA, 17-octadecynoic acid; BSTFA, N,O-bis-trimethylsilyl-trifluoroacetamide; TMCS, trimethylchlorosilane; HPLC, high performance liquid chromatography; APCI-LC/MS, atmospheric pressure chemical ionization liquid chromatography/mass spectrometry; GC/EIMS, gas chromatography/electron ionization mass spectrometry. 1 To whom correspondence should be addressed. by gest, on S etem er 0, 2017 w w w .j.org D ow nladed fom

In vitro techniques have been used to investigate the nature of microsomal cytochrome P450 involved in the metabolism of oleic acid, a physiological monounsaturated fatty acid. Like lauric acid, which is cur rently used as a model substrate of fatty acid metabolism, the alkyl chain of oleic acid is hydroxylated on its ω and (ω–1) carbons. The identity of these hydroxylated metabolites was ascertained by GC/MS and LC/MS. The ω/ω–1 ratio of oleic acid metabolites (1.22 ± 0.01) was found to be similar to that obtained with lauric acid in rat liver microsomes (1.10 ± 0.02), while in human liver microsomes this ratio was 0.75 ± 0.5 for lauric acid and 5.2 ± 2.6 for oleic acid. After treatment of rats with ethanol or clofibrate, inducers of CYP2E1 and CYP4A, respectively, the hydroxylations of oleic acid were shown to be less inducible than those of lauric acid. Five in vitro approaches were used to identify the P450 isoform(s) responsible for the microsomal (ω–1)-hydroxylation of oleic acid: effect of various inducers in rats, correlation studies between specific P450 catalytic activities in a panel of 25 human liver microsomes, chemical inhibitions, immuno-inhibitions and metabolism by cDNA-expressed human P450 enzymes. From the above results, it can be ascertained that P450 2E1 is the main enzyme involved in the (ω–1)-hydroxylation of oleic acid. Furthermore, the ω-hydroxylation of oleic acid was shown to be mainly catalyzed by P450 4A enzymes in human liver microsomes. The turnover number of (ω–1)-hydroxylation of lauric and oleic acids decreased from 7.8 to 1.5 min-1, respectively, suggesting that the dodecane alkyl chain allows optimal binding to the active site of CYP2E1.—Adas, F., F. Berthou, D. Picart, P. Lozac'h, F. Beaugé, and Y. Amet. Involvement of cytochrome P450 2E1 in the (ω–1)-hydroxylation of oleic acid in human and rat liver microsomes. J. Lipid Res. 1998. 39: 1210–1219.