LINOLEIC ACID AND
LEUKOCYTE INFLAMMATORY MARKERS IN
PERIPARTURIENT DAIRY COWS
By
William Raphael

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Comparative Medicine and Integrative Biology – Doctor of Philosophy
2014

ABSTRACT
LINOLEIC ACID AND LEUKOCYTE INFLAMMATORY MARKERS IN
PERIPARTURIENT DAIRY COWS
By
William Raphael
Incidence and severity of dairy cow disease is greatest in the periparturient period. Mobilization
of saturated and unsaturated fatty acids from adipose tissue into plasma is a well-established risk
factor for periparturient disease. Lipid mobilization was associated with changes in peripheral
leukocyte content of polyunsaturated fatty acids and could impact production of the mediators of
inflammatory pathways known as oxylipids. Previous bovine leukocyte studies associated
expression of some proinflammatory markers and enhanced inflammatory responses to endotoxin
with increased disease incidence and severity during the periparturient period. However, the
potential impact of oxylipids on leukocyte inflammatory marker expression during the
periparturient period is unknown. The hypothesis of the Chapter 2 study was that a relationship
exists between the profile of pro- and anti-inflammatory plasma oxylipids and the inflammatory
phenotype of peripheral blood leukocytes during the periparturient period. Correlations were
identified between concentrations of several plasma oxylipids, including some derived from
linoleic acid, and expression of some proinflammatory markers by peripheral blood leukocytes.
Previous reports of increased linoleic acid concentrations within mononuclear leukocytes during
early lactation were confirmed. Human studies showed proinflammatory effects of linoleic acid in
several cell types through oxylipid production. Early lactation monocytes were modeled in vitro
to evaluate effects of increased linoleic acid concentrations on oxylipid production and
inflammatory marker expression. The overall hypothesis of Chapter 3 studies was that increased
monocyte linoleic acid concentrations enhance production of linoleic acid-derived oxylipids,

induce a pro-inflammatory phenotype, and enhance inflammatory responses to endotoxin. Results
showed increased production of several linoleic acid-derived oxylipids in primary bovine and
murine RAW 264.7 monocytes with linoleic acid concentrations similar to early lactation cows.
Increasing linoleic acid concentrations in monocytes did not impact pro- or anti-inflammatory
marker expression and did not influence endotoxin-stimulated inflammatory responses. Future
studies should evaluate the impact of individual oxylipids, produced by monocytes in response to
increased linoleic acid concentration, on inflammatory marker expression.

Copyright by
WILLIAM RAPHAEL
2014

This work is dedicated to improved understanding of dairy cow disease mechanisms by animal
and veterinary scientists.

v

ACKNOWLEDGEMENTS

This work was made possible by important contributions from several people and organizations.
Lorraine M. Sordillo, Professor and Meadow Brook Endowed Chair in Farm Animal Health and
Well-being, graciously provided significant time and effort for academic guidance. Ray Geor,
Chairperson of the Department of Large Animal Clinical Sciences, designated significant
Departmental financial support. Funding was also generously provided by the Matilda R. Wilson
Fund (Detroit, MI), and the Agriculture and Food Research Initiative Competitive Grants Program
of the USDA National Institute for Food and Agriculture (2012-67011-20019). Valuable technical
assistance was provided by Jeffery Gandy, Christine Corl, Dr. Edward Kabara, Sarah Peek, Valerie
E. Ryman, Vengai Mavangira, and staff of the Michigan State University Research Technology
Support Facility Mass Spectrometry Core, especially Drs. Dan Jones and Scott Smith. The love
and support provided by Jennifer, Andrew, Quinton, Oliver, Macarthur, Neil, and Margaret
Raphael are also acknowledged.

vi

TABLE OF CONTENTS

LIST OF TABLES .......................................................................................................................... x
LIST OF FIGURES ....................................................................................................................... xi
KEY TO SYMBOLS AND ABBREVIATIONS ........................................................................ xiii
CHAPTER 1 ................................................................................................................................... 1
DIETARY POLYUNSATURATED FATTY ACIDS AND INFLAMMATION: THE ROLE OF
PHOSPHOLIPID BIOSYNTHESIS ............................................................................................... 1
Abstract ...................................................................................................................................... 2
Introduction ................................................................................................................................ 3
Trends in Dietary Fatty Acid Composition ................................................................................ 4
Dietary Polyunsaturated Fatty Acids and Inflammation ............................................................ 5
Phospholipids of Inflammatory Cells ..................................................................................... 6
Inflammatory Pathways Influenced by Fatty Acid Components of Phospholipids ................ 7
Phospholipid-dependent Biosynthesis of Lipid Mediators ..................................................... 8
Dietary PUFA Influence Inflammation Through Lipid Mediators ....................................... 10
Further Lipid Mediator Research .......................................................................................... 11
Delivery and Utilization of Dietary PUFA for Phospholipid Biosynthesis ............................. 13
Digestion and Absorption of Dietary PUFA ......................................................................... 13
Phospholipid Substrate from Non-dietary PUFA ................................................................. 14
Effects of Disease, Physiological State, and Other Nutrients on Phospholipid Biosynthesis
............................................................................................................................................... 16
Regulation of the Acyl Composition of Phospholipid .......................................................... 19
Conclusions .............................................................................................................................. 20
Acknowledgments .................................................................................................................... 21
Conflicts of Interest .................................................................................................................. 22
CHAPTER 2 ................................................................................................................................. 23
ASSOCIATION BETWEEN POLYUNSATURATED FATTY ACID-DERIVED OXYLIPID
BIOSYNTHESIS AND LEUKOCYTE INFLAMMATORY MARKER EXPRESSION IN
PERIPARTURIENT DAIRY COWS ........................................................................................... 23
Interpretive Summary ............................................................................................................... 24
Abstract .................................................................................................................................... 25
Introduction .............................................................................................................................. 26
Materials and methods.............................................................................................................. 28
Animals ................................................................................................................................. 28
Plasma Oxylipid Quantification ............................................................................................ 29
Oxylipid Extraction .......................................................................................................... 29
Liquid Chromatography/Mass Spectrometry (LC/MS) .................................................... 29
PBMC Fatty Acid Methyl Ester Quantification .................................................................... 30
Lipid Extraction ................................................................................................................ 30
Derivation ......................................................................................................................... 30
vii

Gas Chromatography/Mass Spectrometry (GC/MS) ........................................................ 31
FAME Quantification ....................................................................................................... 32
Leukocyte mRNA Quantification by qPCR.......................................................................... 33
Statistical Evaluation ............................................................................................................ 34
Results ...................................................................................................................................... 34
Animal Production and Health .............................................................................................. 34
Plasma Oxylipid Profile ........................................................................................................ 35
Leukocyte mRNA Expression .............................................................................................. 35
PBMC FAME Content .......................................................................................................... 35
Statistical Relationships ........................................................................................................ 36
Discussion ................................................................................................................................ 36
Acknowledgements .................................................................................................................. 41
CHAPTER 3 ................................................................................................................................. 42
IMPACT OF INCREASED LINOLEIC ACID CONCENTRATION ON INFLAMMATORY
MARKER EXPRESSION IN BOVINE MONOCYTES ............................................................. 42
Abstract .................................................................................................................................... 43
Introduction .............................................................................................................................. 45
Materials and Methods ............................................................................................................. 47
Cell Culture ........................................................................................................................... 47
Flow cytometry ..................................................................................................................... 49
PBMC Fatty Acid Methyl Ester Quantification .................................................................... 49
Lipid Extraction ................................................................................................................ 49
Derivation ......................................................................................................................... 49
Gas Chromatography/Mass Spectrometry (GC/MS) ........................................................ 49
FAME Quantification ....................................................................................................... 50
Plasma Oxylipid Quantification ............................................................................................ 51
Oxylipid Extraction .......................................................................................................... 51
Liquid Chromatography/Mass Spectrometry (LC/MS) .................................................... 51
Monocyte mRNA Quantification by qPCR .......................................................................... 52
Statistical Evaluation ............................................................................................................ 53
Results ...................................................................................................................................... 53
Cell culture ............................................................................................................................ 53
Transcriptional responses to increased cell LA concentration.............................................. 54
LA-derived oxylipid production with increased cell LA concentration ............................... 54
Proinflammatory challenge with increased cell LA concentration ....................................... 55
Discussion ................................................................................................................................ 55
Acknowledgements .................................................................................................................. 59
CHAPTER 4 ................................................................................................................................. 60
CONCLUSIONS........................................................................................................................... 60
Plasma oxylipid profile changes across the periparturient period of dairy cattle .................... 61
Plasma oxylipids are associated with leukocyte transcript ...................................................... 63
Cellular linoleic acid concentration does not impact monocyte inflammatory phenotype ...... 63
Summary of strengths and limitations ...................................................................................... 64

viii

APPENDICES .............................................................................................................................. 67
Appendix 1. Authorization to publish Chapter 2 in Dissertation. ............................................ 68
Appendix 2. Tables................................................................................................................... 69
Appendix 3. Figures. ................................................................................................................ 73
Appendix 4. Supplementary Figures. ....................................................................................... 88
REFERENCES ............................................................................................................................. 95

ix

LIST OF TABLES

Table 1. Gene targets for quantitative PCR in Chapter 2. ............................................................ 69
Table 2. Plasma oxylipids with stable concentration across the periparturient period (mean ± SE,
pg / ul, n = 7). ................................................................................................................................ 70
Table 3. Correlations between plasma oxylipid concentrations and peripheral leukocyte mRNA
expression, adjusted for multiple tests. ......................................................................................... 71
Table 4. Gene targets for quantitative PCR in Chapter 3. ............................................................ 72

x

LIST OF FIGURES

Figure 1. Phospholipids consist of glycerol, usually esterified to a saturated long chain fatty acid
at sn-1 and to an unsaturated long chain fatty acid at sn-2, and to a phosphorylated head group at
sn-3................................................................................................................................................ 73
Figure 2. Fatty acyl chains of phospholipids located within the membranes of cells and
organelles may be enzymatically oxidized into lipid mediators. ...................................................... 74
Figure 3. The utilization of long chain fatty acids for de novo phospholipid biosynthesis
(Kennedy pathway, red arrows and boxes), post-synthetic modification of phospholipids by
reacylation (Lands’ cycle, green arrow), and the metabolism of phospholipids to oxidized lipid
mediators of inflammation (orange arrow and box) in critical cells during inflammatory
responses, such as vascular endothelial cells and mononuclear leukocytes. ................................. 75
Figure 4. Biosynthetic pathway of major phospholipids in vascular endothelial tissue and
mononuclear leukocytes. ................................................................................................................ 76
Figure 5. Plasma concentrations of arachidonic acid-derived oxylipid, 11hydroxyeicosatetraenoic acid (11-HETE) changed across the periparturient period. Reported as
least square mean ± SE (p < 0.05, n = 7). ..................................................................................... 77
Figure 6. Plasma concentration of linoleic acid-derived oxylipid, 9-hydroxyoctadecadienoic acid
(HODE) (A) and 13-HODE (B), changed in the periparturient period. ....................................... 78
Figure 7. Relative (±SE) 15-lipoxygenase (LOX) 1 (A) and 15-hydroxyprostaglandin
dehydrogenase (HPGD) (B) mRNA expression from peripheral leukocytes, calibrated to
expression at 84 d (p < 0.05, n = 7). ............................................................................................. 79
Figure 8. Relative (±SE) IL-12 mRNA expression from peripheral leukocytes, calibrated to
expression at 84 d (p < 0.05, n = 7). ............................................................................................. 80
Figure 9. Linoleic (C18:2 n6, ▲) and arachidonic acid (C20:4 n6, ○) concentrations in
peripheral blood mononuclear cells increased in early lactation relative to prepartum samples,
while eicosapentaenoic (C20:5 n3, ◊) acid concentrations were unchanged. ............................... 81
Figure 10. Linoleic acid methyl ester concentration in peripheral blood mononuclear cells at 10
d postpartum (in vivo, n = 7) versus cells from mid-lactation cows cultured in vitro (n = 6) for 12
h in media supplemented with various concentrations of linoleic acid. ....................................... 82
Figure 11. Transcript expression of tumor necrosis factor (TNF)- in primary bovine monocytes
(A) and IL-1 in murine RAW 264.7 monocytes (B) after 16 h incubation in media
supplemented with various concentrations of linoleic acid (LA). ................................................ 83
Figure 12. Fold changes in primary bovine monocyte culture media concentrations of 9hydroxyoctadecadienoic (HODE, A), 13-HODE (B), 9-oxooctadecadienoic acid (OxoODE, C),
and 13-OxoODE (D). .................................................................................................................... 84
xi

Figure 13. Primary bovine monocyte culture media concentrations of (R)hydroxyoctadecadienoic acid ((R)-hode, Fig. 13A) and (S)-hode (Fig. 13B), and the ratio of (R)hode / (S)-hode (Fig. 13C). ........................................................................................................... 85
Figure 14. Fold changes in murine RAW 264.7 monocyte culture media concentrations of 9hydroxyoctadecadienoic (HODE, A), 13-HODE (B), 9-oxooctadecadienoic acid (OxoODE, C),
and 13-OxoODE (D). .................................................................................................................... 86
Figure 15. Ratio of 9-hydroxyoctadecadienoic (HODE) / 9-oxooctadecadienoic (OxoODE) acids
in lipopolysaccharide stimulated (25 ng / ml) primary monocyte cultures supplemented with
various concentrations of linoleic acid (LA). LA was delivered in ethanol (EtOH) (n = 3, p <
0.05). ............................................................................................................................................. 87
Figure 16. Unidentified compound in lipid extracts of RAW 264.7 monocytes elutes at identical
retention time as pure docosahexaenoic acid methyl ester (C22:6 n3 FAME) standard. ............. 88
Figure 17. Linoleic acid (LA, c18:2 n6) methyl ester abundance in solvent controls (n = 3, open
bars) and RAW 264.7 cell lipid extracts (n = 3, cross hashed bars) spiked with 50 ug LA fatty
acid (Sigma-Aldrich, St. Louis, MO) either at start of experiment or between lipid extraction and
fractionation of lipid types. Other samples were spiked with 50 ug LA methyl ester (SigmaAldrich) at end of experiment. ...................................................................................................... 89
Figure 18. Decreased 15-lipoxygenase1 (15-LOX1) and tumor necrosis factor (TNF)-α mRNA
expression in lipopolysaccharide stimulated primary bovine monocytes associated with increased
concentrations of fatty acid free-bovine serum albumin (BSA). .................................................. 90
Figure 19. Decreased antioxidant potential induced by 2,2′-azobis (2-methylpropionamidine)
dihydrochloride (AAPH, Sigma-Aldrich, St. Louis, MO) in RAW 264.7 monocytes. ................ 91
Figure 20. Induction of 9-hydroxyoctadecadienoic acid (9-HDOE) production by 2,2′-azobis (2methylpropionamidine) dihydrochloride (AAPH, Sigma-Aldrich, St. Louis, MO) in RAW 264.7
monocytes. .................................................................................................................................... 92
Figure 21. Viability is decreased by 2,2′-azobis (2-methylpropionamidine) dihydrochloride
(AAPH, Sigma-Aldrich, St. Louis, MO) in RAW 264.7 monocytes............................................ 93
Figure 22. Linoleic acid does not affect IL-1 (a) or IL-10 (b) transcript induction by 2,2′-azobis
(2-methylpropionamidine) dihydrochloride (AAPH, Sigma-Aldrich, St. Louis, MO) in RAW
264.7 monocytes. .......................................................................................................................... 94

xii

KEY TO SYMBOLS AND ABBREVIATIONS

AA; arachidonic acid
ACSL; acyl CoA synthase (ligase)
AGPAT; acylglycerol-3-phosphoacyltransferase
ALA; α-linolenic acid
AMU; atomic mass units
BHT; butylated hydroxytoluene
CDP; cytidine diphosphate
COX; cyclooxygenase
CTP; cytidine triphosphate
DGAT; diacylglycerol acyltransferase
DHA; docosahexaenoic acid
EPA; eicosapentaenoic acid
FAME; fatty acid methyl esters
GC/MS; gas chromatography/mass spectrometry
GPAT; glycerol-3-phosphoacyltransferase
HETE; hydroxyeicosatetraenoic acids
HODE; hydroxyoctadecadienoic acids
HpDHA; hydroperoxydocosahexaenoic acids
HPETE; hydroperoxyeicosatetraenoic acids
HPGD; 15-hydroxyprostaglandin dehydrogenase
HPODE; hydroperoxyoctadecadienoic acids
LA; linoleic acid
LC/MS; liquid chromatography/mass spectrometry

xiii

LOX; lipoxygenase
LPS; lipopolysaccharide
LT; leukotrienes
MaR1; 7-maresin1
NEFA; non-esterified fatty acids
NOS2; inducible nitric oxide synthase 2
OxoETE; oxoeicosatetraenoic acids
OxoODE; oxooctadecadienoic acids
PBMC; peripheral blood mononuclear cells
PD; protectins
PG; prostaglandins
PPAP; phosphatidic acid phosphatase (lipin)
PPAR-γ; peroxisome proliferator activated receptor-γ
PUFA; polyunsaturated fatty acids
RIN; RNA integrity number
TLR; toll like receptor
TNF; tumor necrosis factor-α
TX; thromboxane

xiv

CHAPTER 1

DIETARY POLYUNSATURATED FATTY ACIDS AND INFLAMMATION: THE ROLE
OF PHOSPHOLIPID BIOSYNTHESIS1
W. Raphael and L. M. Sordillo*
Department of Large Animal Clinical Sciences, College of Veterinary Medicine,
Michigan State University, 736 Wilson Rd., Room D202, East Lansing, MI 48824, USA;
E-mail: raphaelw@msu.edu
*Author to whom correspondence should be addressed; E-Mail: sordillo@msu.edu;
Tel.: +1-517-432-8821; Fax: +1-517-432-8822

1

© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
license (http://creativecommons.org/licenses/by/3.0/).
Published: Int J Mol Sci, 2013, 14, 21167-21188; doi:10.3390/ijms141021167
1

Abstract
The composition of fatty acids in the diets of both human and domestic animal species can regulate
inflammation through the biosynthesis of potent lipid mediators. The substrates for lipid mediator
biosynthesis are derived primarily from membrane phospholipids and reflect dietary fatty acid
intake. Inflammation can be exacerbated with intake of certain dietary fatty acids, such as some
ω-6 polyunsaturated fatty acids (PUFA), and subsequent incorporation into membrane
phospholipids. Inflammation, however, can be resolved with ingestion of other fatty acids, such as
ω-3 PUFA. The influence of dietary PUFA on phospholipid composition is influenced by factors
that control phospholipid biosynthesis within cellular membranes, such as preferential
incorporation of some fatty acids, competition between newly ingested PUFA and fatty acids
released from stores such as adipose, and the impacts of carbohydrate metabolism and
physiological state. The objective of this review is to explain these factors as potential obstacles
to manipulating PUFA composition of tissue phospholipids by specific dietary fatty acids. A better
understanding of the factors that influence how dietary fatty acids can be incorporated into
phospholipids may lead to nutritional intervention strategies that optimize health.
Keywords: diet; phospholipid; fatty acid; lipid mediator; eicosanoid; inflammation; lipoxygenase;
cyclooxygenase

2

Introduction
Dietary fatty acids can control the incidence and severity of inflammation in some diseases of
humans and domestic animals. Current evidence suggests that the dietary ω-6 (n-6) to ω-3 (n-3)
polyunsaturated fatty acid (PUFA) ratio is directly associated with inflammatory-based pathology
in human diseases such as cancer, rheumatoid arthritis, atherosclerosis, and obesity [22, 23, 148].
Studies in production animals used for human food also suggest that dietary intake of certain PUFA
can impact inflammatory responses to common infectious diseases such as mastitis and metritis
[32, 153]. The associations between dietary fatty acids and diseases are partially explained by the
incorporation of dietary n-6 and n-3 PUFA into membrane phospholipids [64]. The PUFA content
of phospholipids influence inflammation through several mechanisms including membrane
fluidity, lipid raft formation, and receptor function[147, 160], but the focus of this review is on the
biosynthesis of potent phospholipid-derived lipid mediators that have pro- or anti-inflammatory
functions. For example, many lipid mediators produced from n-6 PUFA have pro-inflammatory
functions and the overexpression of these lipid mediators is associated with the pathogenesis of
inflammation during disease. Conversely, lipid mediators derived from n-3 PUFA have
inflammation-resolving properties, and partly explain the disease-protective effects that are
sometimes seen with n-3 PUFA ingestion [118]. Inconsistent health benefits seen with n-3 PUFA
supplementation could be due to the regulation of dietary and stored PUFA incorporation into
phospholipids. Digestion, for example, can change the profile of ingested fatty acids by reducing
the number of double carbon bonds [9, 19]. Additionally, fatty acid incorporation into
phospholipids is sometimes selective [173]. Also, phospholipid synthetic pathways are regulated
at multiple levels and are sometimes controlled by other nutrients or metabolites, such as
diacylglycerol and glucose [97, 188]. The objectives of this review are to explain how dietary n-6
3

and n-3 PUFA are incorporated into membrane phospholipids, explain how the subsequent
biosynthesis of lipid mediators can influence inflammatory responses, and describe factors
confounding the influence of dietary PUFA on phospholipid composition. Future studies of these
confounding factors will improve knowledge of phospholipid biosynthesis in humans and
domestic animals and will reveal all health outcomes of n-3 PUFA dietary supplements.
Trends in Dietary Fatty Acid Composition
The consumption of n-3 and n-6 PUFA in Western diets changed considerably over the last several
decades [15]. The n-6 PUFA content of Western human diets increased to some extent because
plants or plant-derivatives, such as corn oil, are popular dietary ingredients [33]. In contrast to
modern Western human diets, Northern (e.g., Greenland) and Eastern (e.g., Southeast Asian) diets
contain more n-3 PUFA because proportionally more dietary fatty acid is derived from fish [8].
Marine plants and fish are rich in n-3 PUFA, relative to terrestrial plant- or animal-derived
foodstuffs that are abundant in Western diets. Hence the dietary n-6 to n-3 PUFA ratio is higher in
modern Western human diets compared to regions where fish remain a significant source of PUFA.
In spite of these differences in PUFA intake between regions, both n-6 and n-3 remain important
dietary ingredients in all human and domestic animals. Linoleic acid (LA, C18:2 n-6) nutritional
deficiency results in disease of multiple systems, including skin [50] and α-linolenic acid (ALA,
C18:3 n-3) deficiency results in neurological impairment [38]. These PUFA cannot be synthesized
de novo but, once absorbed, they can be converted to other PUFA within the same omega
designation [61]. Domestic animals and humans cannot synthesize n-3 PUFA from n-6 because
they lack specific desaturase enzymes, such as that transcribed from fat-1 in lower order eukaryotes
[148]. Therefore, the n-6 to n-3 PUFA ratio in tissue phospholipids is a direct reflection of dietary
4

composition, although the profiles of individual n-6 and n-3 PUFA are also subject to de novo
elongation and desaturation[61].
Like humans, many species of domestic animals have experienced significant dietary change over
the past several decades. This is partly due to industrialization of Western agriculture, which
reduced or eliminated pasture feeding and introduced concentrated-nutrient forages into the diet
of many species, including ruminants. Meat and milk derived from intensively-raised ruminants
may be partly responsible for elevated n-6 to n-3 PUFA ratios in Western human diets [33] as
modern ruminant diets were shown to affect the n-6 to n-3 PUFA ratio of meat [133] and milk
[177] from these animals. As with humans, there is increased disease risk in ruminants fed these
modern diets [42], which can be partly explained by the n-6 to n-3 PUFA ratio in phospholipids
and the effect this has on severity and duration of host inflammatory responses [29].
Dietary Polyunsaturated Fatty Acids and Inflammation
Inflammation is an essential component of the innate immune response to tissue injury. Movement
of serum proteins, lipids, and blood leukocytes into affected tissues eliminate or neutralize the
source of tissue injury then restore normal tissue structure and function. Failure to control the
magnitude and duration of the inflammatory response can cause damage to host tissues and
contribute to pathology, independent of the original insult. The ratio of n-6 to n-3 PUFA in the diet
has long been recognized as an important influence on the pathogenesis of inflammation because
PUFA derivatives can initiate and exacerbate inflammatory responses [22]. This was demonstrated
in several human and animal diseases, including atherosclerosis, sepsis, mastitis, and cancer [32,
89, 148], and involves derivatives of n-6 PUFA. In contrast, human diseases with inflammatorybased pathology were prevented with n-3 PUFA dietary supplementation. Increased fish-derived
5

n-3 PUFA consumption, for example, was shown to lower risk of cardiovascular diseases and
improved survival from myocardial infarction [1, 57, 175]. Evidence also exists that dietary n-3
PUFA dietary supplementation can prevent several forms of cancer [131]. The inflammatory
response of healthy human subjects is also affected by n-3 PUFA ingestion. For example, human
mononuclear leukocytes from subjects supplemented with dietary fish-oil have decreased TNF-
expression relative to subjects consuming control diets [168]. Despite this evidence, there remains
some controversy concerning the health benefits of n-3 PUFA ingestion because not all data were
conclusive. Dietary supplementation with 1 g of n-3 PUFA daily, for example, did not reduce the
rate of adverse cardiovascular events in patients that had or were at risk of type 2 diabetes [17].
Also a recent meta-study found no association between n-3 PUFA intake and incidence of major
cardiovascular disease [129]. A better understanding of how dietary PUFA are integrated into
membrane phospholipids and therefore available for biosynthesis of pro-resolving mediators of
inflammation may explain these equivocal results.

Phospholipids of Inflammatory Cells
The link between dietary PUFA, inflammation, and disease susceptibility is partially due to
changes in the PUFA content of phospholipids in cells involved in the inflammatory response,
such as monocytes, macrophages, and vascular endothelial cells [7, 36]. Phospholipids are
composed of 2 fatty acids esterified to glycerol at sn-1 and sn-2 and a phosphorylated head group
esterified at sn-3 (Figure 1). The n-6 and n-3 PUFA, including arachidonic acid (AA, C20:4 n-6),
LA, eicosapentaenoic acid (EPA, C20:5 n-3) and docosahexaenoic acid (DHA, C22:6 n-3) are
incorporated into the sn-2 position of phospholipids in cellular membranes [173]. Phospholipid
head groups are cytidine-monophosphate, hydroxyl, choline, ethanolamine, serine, or inositols
[173]. Phosphocholine and phosphoethanolamine are concentrated in macrophage plasma
6

membranes [6], are the major phospholipids in human plasma (76% phosphocholine and 17%
phosphoethanolamine) [119], and are also the most abundant phospholipids in human erythrocytes
[43]. As such, phosphocholine and phosphoethanolamine are the largest phospholipid reservoirs
of dietary n-6 and n-3 PUFA in cells and fluids involved in inflammatory responses.
Other less abundant phospholipids can affect inflammatory responses, but independent of lipid
mediator production. For example, diphosphoglycerol (cardiolipin) consists of 2 molecules of
phosphatidic acid esterified to glycerol, is the most abundant phospholipid in the inner
mitochondrial membrane [116], and can compromise cellular respiration [103] and mediate
apoptosis [184] under conditions of oxidative stress. Another example is platelet activating factor
(1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), which directly enhances cytokine and
adhesion molecules expression in vascular endothelium [34]. These are important effects of
phospholipids but the objective here is to explain how major phospholipids in cells and fluids that
are critical in inflammatory processes are influenced by dietary PUFA and regulate inflammatory
processes when PUFAs are converted to lipid mediators of inflammation.

Inflammatory Pathways Influenced by Fatty Acid Components of Phospholipids
The fatty acid composition of membrane phospholipids in macrophages and endothelium can
influence inflammatory responses in several different ways. Physical properties of membranes,
such as fluidity and lipid raft formation, are influenced by specific fatty acids in membrane
phospholipids and modify membrane-generated signaling cascades. For example, murine
macrophage and adipocyte and human vascular endothelial cell culture studies, including some
with transgenic mice, demonstrated that saturated fatty acids can directly activate proinflammatory
signaling pathways through Toll-like receptor (TLR)-mediated mechanisms [80, 91, 162].
Saturated fatty acids increase proinflammatory gene expression in macrophages by inducing the
7

dimerization and recruitment of TLR4-related signaling proteins into lipid rafts in a way that
mimics endotoxin [91, 178]. These responses to saturated fatty acids in phospholipids involve
downstream pro-inflammatory signaling pathways. For example, treatment of murine macrophage
cell lines with palmitic (C16:0) and lauric (C12:0) acids increased phosphorylation of JNK and
ERK, resulting in enhanced expression of proinflammatory cytokines [178]. Interestingly, the
saturated fatty acid-induced increase in phosphorylation of MAPK signaling subunits was
attenuated by increasing the DHA content of cells [178]. This effect may be partially attributable
to the direct action of n-3 PUFA and independent of conversion to lipid mediators [49, 94].
Although saturated fatty acids and PUFA have important direct effects on health, the focus of this
review is on conversion of n-3 and n-6 PUFA, derived from membrane phospholipids, into the
more potent lipid mediators [49].

Phospholipid-dependent Biosynthesis of Lipid Mediators
Several hundred lipid mediators have been identified and they collectively regulate the initiation,
magnitude and duration of inflammatory responses [46]. Eicosanoids are the most widely studied
class of lipid mediators. These are derived from AA and include hydroperoxyeicosatetraenoic acids
(HPETE), prostaglandins (PG), thromboxanes (TX), leukotrienes (LT), and lipoxins. The first step
in eicosanoid biosynthesis occurs when esterified AA is released from membrane phospholipids
by phospholipase enzymes. Intracellular AA may then be metabolized by the cyclooxygenase
(COX), lipoxygenase (LOX), or epoxygenase (e.g. cytochrome P450) enzymatic pathways, which
ultimately determine the class of eicosanoid generated (Figure 2). Two isoforms of COX enzymes,
denoted as COX1 and COX2, are involved in the enzymatic oxidation pathways. Both COX
isoforms first catalyze the oxidation of AA to prostaglandin G2 (PGG2) followed by a peroxidase
reaction that reduces PGG2 to PGH2 [44]. From PGH2, specific downstream PG synthases produce
8

PGE2, PGD2, PGI2, and PGF2α. Alternatively, thromboxane synthases convert PGH2 to TXA2 and
TXB2. Similar to the COX family, there are several isoforms of LOX involved in the enzymatic
oxidation of PUFA. For example, 5-LOX catalyzes the oxidation of AA to 5-HPETE that can be
further metabolized to produce LT. Moreover, both 15-LOX1 and 15-LOX2 oxidize AA to 15HPETE [86]. This can be further metabolized to lipoxin A4 by 5-LOX [140]. The eicosanoid class
of lipid mediators is predominantly proinflammatory, with a small number of exceptions. For
example, lipoxin A4 is pro-resolving [140] and PGE2 has both proinflammatory and resolving
effects [21].
Whereas AA is the common precursor of the eicosanoid class of lipid mediators, there are other
important mediators produced from the enzymatic oxidation of LA, DHA, and EPA. For example,
LA is metabolized by several enzymes including 15-LOX, COX2, and the epoxygenases into
hydroperoxyoctadecadienoic acid (HPODE). This is reduced to hydroxyoctadecadienoic acid
(HODE) which is dehydrogenated to form oxooctadecadienoic acid (OxoODE) [51]. The effects
of LA-derived mediators can be either pro- or anti-inflammatory [5, 106, 111]. Lipid mediators
derived from n-3 PUFA are the most recently discovered [140]. These mediators are synthesized
from DHA by 12-LOX or 15-LOX and are referred to as resolvins, protectins, and maresin. In
contrast to the mediators derived from n-6 PUFA, the function of mediators derived from n-3
PUFA are primarily anti-inflammatory or pro-resolving [140]. COX2 also synthesizes resolvins
from EPA, but only in patients treated with aspirin (Figure 2). Alternatively, these EPA-derived
resolvins can be synthesized by epoxygenases [140].
Enzymatic biosynthesis of lipid mediators is important because enzymes can be overexpressed in
diseases such as atherosclerosis and sepsis [94, 185]. Both n-6 and n-3 PUFA, however, may also
undergo non-enzymatic oxidation with reactive oxygen species, resulting in production of lipid
9

mediators with similar biological effects to those derived from enzymatic oxygenation [182]. For
example, some 15-HPETE and 13-HPODE isomers produced by non-enzymatic oxidation are
identical to those synthesized by enzymatic oxidation [47, 186]. These hydroperoxides are
themselves reactive oxygen species, thus creating positive feedback loops on PUFA oxidation
during an inflammatory response that can exacerbate disease pathogenesis.

Dietary PUFA Influence Inflammation Through Lipid Mediators
There is strong evidence that the dietary n-6 to n-3 PUFA ratio influences the profile of lipid
mediators within tissue [78, 118, 120]. For example, a recent human study demonstrated increased
plasma HODE and OxoODE with increased dietary LA [120]. Dietary supplementation with AA
increased mononuclear leukocyte secretion of pro-inflammatory eicosanoids, including LTB4 [78].
EPA and DHA dietary supplementation were shown to increase lipoxins and E and D series
resolvins in mouse tissue, while also decreasing pro-inflammatory AA-derived eicosanoids [118].
Therefore in vivo production of lipid mediators is regulated by substrate availability. Some of these
studies have confirmed that dietary n-6 to n-3 PUFA ratio does in fact influence lipid mediator
biosynthesis through changes in PUFA composition of tissue phospholipids [63, 118].
Manipulation of the profile of tissue lipid mediators by dietary PUFA is significant because lipid
mediators are known to influence health. For example, increased AA-derived PGE2 biosynthesis
is associated with human colon cancer [176, 193]. There is increased expression of LA-derived
metabolites, 9-HODE and 13-HODE, in atherosclerotic vascular lesions [174]. Specific effects of
eicosanoids on inflammation and disease pathogenesis have been demonstrated using in vivo, in
vitro, and transgenic animal models of disease. For example, DHA-derived resolvin D2 was shown
to ameliorate the inflammatory response in murine models of sepsis [157] and blockade of resolvin
receptors in human polymorphonuclear leukocytes resulted in loss of the anti-inflammatory effects
10

of resolvin D1 [107]. PGE2 receptor deletion reduced pathology in murine models of colon cancer
[176]. Several studies showed that deletion of the 12/15-LOX gene was effective in decreasing
atherosclerosis pathology in mice [35, 130].
Despite cumulative evidence that changes in the profiles of eicosanoids and other lipid mediators
are a major factor contributing to the health effects of PUFA intake, there are several important
studies that are inconclusive. For example, a recent review of 15 human LA dietary trials found no
association between intake and pro-inflammatory markers, including eicosanoids and other lipid
mediators [74]. A recent meta-analysis of the protective effects of n-3 PUFA supplementation
against human cardiovascular diseases failed to identify any significant health benefits [129].
There are several possible reasons why such inconsistency may exist in the literature. For example,
lipid mediator biosynthesis is regulated at multiple levels in addition to supply of PUFA substrate.
These include regulation of enzyme transcript and protein expression and enzyme activity [18, 30,
60, 70, 159, 185, 192]. Also, some n-6 PUFA are substrate for pro-resolving mediators, which can
be displaced by n-3 PUFA supplementation [118]. Additionally, enzymes that synthesize lipid
mediators are selective in their use of substrate [84, 85, 151].

Further Lipid Mediator Research
There are some important questions that remain unanswered in this field, such as “are there
potentially deleterious health effects of decreasing the tissue n-6 to n-3 PUFA ratio?” The
associations between PUFA content of phospholipids and lipid mediators reveal some possible
problems. For example, decreased AA-derived lipoxin-A4 biosynthesis occurs with n-3 PUFA
dietary supplementation [69, 108, 118]. Effects such as this may not always be beneficial for health
as, for example, decreased lipoxin A4 is associated with severe human asthma [93]. The influence
of DHA and EPA dietary supplements on asthmatics is not known. To address this, n-3 dietary
11

trials should quantify all lipid mediator substrates within both diet and phospholipids of immune
and vascular cells, in contrast to reporting the n-6 to n-3 PUFA ratio [65, 125]. Additionally, the
tissue lipid mediator profile should be assessed, in contrast to sporadic mediators in previous
dietary trials [63, 118]. A comprehensive profile, rather than isolated lipid mediators, is important
because the lipid mediator network can function in antagonistic, synergistic, or additive manners.
Having identified interactions among lipid mediators and their substrates, future research should
then investigate the possible causes and interventional targets of such relationships.
Another important question is “whether n-3 PUFA supplementation ameliorates inflammation
during human and animal disease because of increased biosynthesis of resolving lipid mediators
from n-3 PUFA or decreased proinflammatory lipid mediator biosynthesis from n-6 PUFA?” It
was reported that decreasing the tissue n-6 to n-3 PUFA ratio increases the biosynthesis of
resolving type lipid mediators from n-3 PUFA and decreases the biosynthesis of proinflammatory
lipid mediators from n-6 PUFA [69, 108], but comparative health responses between low n-6
PUFA and high n-3 PUFA consumption are not clear. This question could be initially addressed
in carefully designed dietary trials that measure phospholipid PUFA composition and lipid
mediator biosynthesis of tissue.
The final question that this body of literature raises is “under which circumstances should
inflammation be manipulated by dietary n-3 PUFA?” Inflammation is an essential component of
mammalian physiology and exists to facilitate restoration of homeostasis in tissue. Antiinflammatory effects of dietary PUFA may, therefore, have adverse consequences in disease
recovery. This has in fact, been demonstrated with dietary n-3 PUFA supplementation in murine
models of gastrointestinal inflammation [179]. This does not imply that treatment of uncontrolled
inflammation is contraindicated, but rather, that manipulation of lipid mediator biosynthesis likely
12

has multiple outcomes and that some outcomes can be undesirable. Further evidence of this is the
discovery that selective COX2 inhibitors affect vasoactive prostaglandin biosynthesis and increase
risk of thromboembolic disease in humans [25, 54].
Delivery and Utilization of Dietary PUFA for Phospholipid Biosynthesis
A better understanding of critical regulatory steps that impact phospholipid biosynthesis from
dietary PUFA will provide insight into the variable success of dietary manipulation of
inflammatory conditions. For example, though phospholipid biosynthesis sometimes displays a
preference for absorbed PUFA, not all dietary PUFA will be used for this purpose. Also, enzymes
which synthesize phospholipids are influenced by disease and physiological states, such as
reproduction. Additionally, PUFA may arise at sites of phospholipid biosynthesis from body
stores, such as adipose tissue, and displace dietary PUFA as substrate.

Digestion and Absorption of Dietary PUFA
PUFA are ingested in several forms, including non-esterified fatty acid, triacylglycerol, and
phospholipids. Ester bonds of ingested lipids are hydrolyzed by pancreatic lipase to produce
predominantly non-esterified fatty acids and monoacylglycerol [19]. This is the primary digestive
process for monogastric species, such as humans and swine and is not currently known to affect
PUFA supply. However, PUFA from which lipid mediators are derived are very susceptible to
non-enzymatic oxidation. For example, the anti-inflammatory effect of n-3 ingestion is increased
if co-ingested with antioxidants [168], although it is not clear if the antioxidant effect occurs prior
to n-3 absorption, or after, as demonstrated in other models [180]. Multi-gastric species such as
ruminants experience an additional digestive process which, in contrast to lipase hydrolysis, has
been demonstrated to affect dietary PUFA content [9]. This is referred to as biohydrogenation and
13

describes hydrogenation of PUFA by the micro-flora of the fore-stomachs. Biohydrogenation
reduces the efficiency of absorption of some PUFA, particularly those present in the diet at low
concentrations such as EPA and DHA. For example, in vivo goat models of biohydrogenation
demonstrated transfer of only 3.5 to 7.6% of dietary EPA or DHA into milk [81]. Apparently this
is sufficient to induce changes in tissue phospholipid composition, possibly because the quantity
of absorbed n-3 PUFA is large relative to pre-supplementation tissue and milk levels [177].
The absorption process for available PUFA is similar across all domestic animal species and
humans and occurs through the intestinal lymphatic system and the venous side of the vascular
system [19]. In this way, absorbed PUFA are protected from hepatic metabolism in the first pass
through the circulatory system, and immediately available for biosynthesis of phospholipids in
organs and cells critical in determining inflammatory responses, such as bone marrow, peripheral
leukocytes, and vascular tissue [19, 173].

Phospholipid Substrate from Non-dietary PUFA
There are several important sources of non-dietary PUFA that confound the influence of dietary
PUFA on inflammation (Figure 3) [113] . These include PUFA synthesized de novo or stored in
sites such as adipose tissue. The relative efficiencies of n-3 and n-6 PUFA de novo biosynthesis
are controversial. For example, de novo biosynthesis of EPA and DHA from ALA has been
described as modest [82] but there is evidence that humans have greater capacity for this than de
novo biosynthesis of AA from LA [51]. Therefore de novo biosynthesis of some n-3 PUFA cannot
be discounted as having potential health benefits if this results in increased tissue content of DHA
and EPA-derived pro-resolving type lipid mediators.
Adipose-derived PUFA may also be used for phospholipid biosynthesis in inflammatory based
14

cells (Figure 3). This is an abundant substrate for phospholipid synthesis during dietary restriction
or large, chronic expenditures of energy because the rate of lipolysis exceeds that of lipogenesis
and adipose tissue is mobilized [172]. This occurs, for example, in humans and domestic animals
during the periparturient period [31, 76] because of energy expenditure associated with pregnancy
and lactation and a concomitant deficit in calorie intake. During adipose mobilization, the liver
can utilize adipose-derived fatty acid for ketone body biosynthesis, particularly if carbohydrate
ingestion or substrates for gluconeogenesis are restricted relative to the requirements for glucose
[19]. Alternatively, fatty acid in the liver may be stored as triacylglycerol. In other organs, such as
skeletal muscle, dietary and adipose-derived fatty acid can be energy substrates through oxidation, or in functional mammary glands, are excreted as milk lipid. Hence when adiposederived fatty acids are mobilized into circulation, phospholipid biosynthesis must compete against
ketogenesis, -oxidation, and milk-lipid excretion for PUFA substrate. In spite of this competition,
there is in vitro evidence that suggests adipose-derived fatty acids modify the composition of
phospholipids in vascular endothelial tissue and induce inflammation through lipid mediators, and
also evidence that this effect can be reversed with n-3 PUFA supplementation [29, 30]. This is
particularly interesting as there is an exacerbated inflammatory response in vivo in some species
coincident with adipose mobilization [145]. This suggests that decreasing the n-6 to n-3 PUFA
ratio in adipose could potentially improve health during periods of adipose mobilization. Dietary
PUFA are incorporated into adipose triacylglycerol by de novo triacylglycerol biosynthesis and
remodeling through reacylation [73, 169]. The rate of de novo synthesis is 20% of total
triacylglycerol in healthy human adipose tissue after nine-weeks [161]. The kinetics of
triacylglycerol reacylation are not reported but since the half-life of all adipose triacylglycerol is
6-months to 2-years [67], it is reasonable to interpret that reacylation of adipose triacylglycerol

15

occurs over several months or years in vivo. These data suggest that a long-term change in diet is
required to induce change in adipose n-3 PUFA content. Since DHA and EPA are substrate to
several inflammation-resolving lipid mediators, long term ingestion of these, particularly during
periods of triacylglycerol synthesis, could be therapeutic in inflammatory conditions associated
with mobilization of adipose tissue.
Another potential origin of PUFA for phospholipids is recycled use of fatty acyl chains that have
been hydrolyzed from existing phospholipids (Figure 3). Little is known about the role of this pool
of fatty acids in biosynthesis of phospholipids. However, it is unlikely that the plasma membrane,
the largest PUFA reservoir, has a major role in supplying PUFA for de novo phospholipids as the
machinery for this are located in the endoplasmic reticulum and mitochondrion [173].

Effects of Disease, Physiological State, and Other Nutrients on Phospholipid Biosynthesis
De novo biosynthesis of phospholipids occurs in what is commonly referred to as the Kennedy
pathway [79]. A brief description of this pathway is useful to understand regulation of dietary
PUFA incorporation into phospholipids. The first reaction in this pathway combines long-chain
fatty acid with CoA and is catalyzed by acyl CoA synthase (ligase) (ACSL, Figure 4). The fatty
acyl CoA is then esterified to glycerol at sn-1 on the mitochondria by glycerol-3phosphoacyltransferase (GPAT) [173]. The second acyltransferase is 1-acylglycerol-3phosphoacyltransferase (AGPAT). Substrate for AGPAT is transferred from the mitochondria to
the endoplasmic reticulum, and acylation occurs at sn-2 of glycerol [173]. Glycerol esterified to
fatty acid at sn-1 and sn-2 is known as phosphatidic acid. Phosphatidic acid can be metabolized by
phosphatidic acid phosphatase (PPAP or lipin) to synthesize diacylglycerol. This combines with
cytidine diphosphate (CDP)-choline to produce phosphocholine, which may be metabolized to
phosphoethanolamine or phosphoserine. The enzyme cytidine triphosphate (CTP):phosphocholine
16

cytidylyltransferase (CCT) catalyzes CDP-choline biosynthesis, and CDP-choline:1,2diacylglycerol cholinephosphotransferase catalyzes the diacylglycerol reaction with CDP-choline
[173]. Triacylglycerol is the alternative diacylglycerol product, catalyzed by diacylglycerol
acyltransferase (DGAT) [95]. If phosphatidic acid is not utilized for biosynthesis of diacylglycerol,
then it can be utilized by CDP-diacylglycerol synthase for synthesis of phosphoinositol,
phosphoglycerol, or cardiolipin.
Expression of some phospholipid synthesizing enzymes is influenced by pathogen associated
molecular patterns and so is likely influenced by bacterial infection. For example, expression of
ACSL1, 3, and 4 is increased in RAW 264.7 murine macrophages in response to the TLR-4 agonist,
Kdo2 [2], an endotoxin-like molecule involved in sepsis and mastitis [145]. Monocyte expression
of ACSL1 is also increased in mouse models of type 1 diabetes, and is associated with an
inflammatory phenotype and atherosclerosis [75]. Other rodent models indicate that insulin
resistance is associated with AGPAT2 and PPAP1 deficiency [163]. The clinical relevance of these
data is not currently understood but transcriptional effects suggest de novo biosynthesis of some
phospholipids may change during these diseases and therefore affect how dietary PUFA are
incorporated into membrane phospholipids in some patients.
Expression of some phospholipid synthesizing enzymes is also affected by physiological state. For
example, ACSL1 hepatic and mammary tissue expressions are increased in dairy cattle during the
postpartum period, relative to prepartum [13, 98]. GPAT, AGPAT, and PPAP expressions are also
increased after bovine parturition [13, 98, 172]. Activities of some enzymes also change with
physiological state. Activity of CCT, the rate limiting enzyme in phosphocholine biosynthesis, is
regulated by diacylglycerol-induced intracellular translocation to membranes [173]. Translocation
results in 1.8-fold increased hepatic activity in the period immediately following parturition in
17

cows [14]. This may also occur when plasma and hepatic non-esterified fatty acid and di- and
triacylglycerol are increased, such as in obese humans [181]. In fact, human adipose expression of
a CCT gene, Pcyt1a, is positively correlated with adipose mass [142]. These findings indicate that
the capacity for de novo biosynthesis of phospholipids may change by reproductive state and
adipose mass, and warrant further investigation at all levels of enzyme regulation. Identifying
certain physiological states where dietary PUFA is more successful in influencing the PUFA
composition of tissue phospholipids could be used to mitigate disease risk.
Glucose has an important influence on biosynthesis of phospholipids. Glucose availability
positively affects cyclin-dependent kinase cdc28, which up-regulates triacylglycerol lipase-4
activity [97]. This results in stored fatty acids being available to phospholipids when glucose is
abundant and there is a need for new membrane formation and cell proliferation. Loewen (2012)
reports that glucose metabolism also influences biosynthesis of phospholipids through
phosphatidic acid and the repressive transcription factor Opi1 [97]. Specifically, under conditions
of glucose starvation, intracellular pH declines and results in the release of bound Opi1 from
phosphatidic acid. The released Opil translocates to the nucleus resulting in repression of genes
involved in biosynthesis of phospholipids, and thus a conservation of stored fatty acid for more
critical needs such as -oxidation [188]. These data indicate that phospholipid biosynthesis is
affected by glucose homeostasis and suggest that dietary PUFA incorporation into phospholipids
be investigated with consideration of dietary carbohydrate ingredients.
After de novo biosynthesis in the Kennedy pathway, phospholipids are continuously susceptible
to post-synthetic deacylation and reacylation at the sn-1 and sn-2 fatty acyl positions (Figure 3) in
what is referred to as the Lands’ cycle [88]. This allows absorbed PUFA to quickly change the
composition of tissue phospholipids, apparently within several weeks [102]. Manipulation of
18

PUFA in tissue phospholipids by the Lands’ cycle may be particularly successful during bacterial
disease as expression of some acyltransferase enzymes is induced by TLR ligands [144]. Further
investigation of this effect may identify therapeutic applications for dietary PUFA during these
diseases.

Regulation of the Acyl Composition of Phospholipid
Phospholipids have a diverse fatty acid composition. For example Quehenberger et al (2010)
measured 31 fatty-acyl variants of phosphocholine and 38 fatty-acyl variants of
phosphoethanolamine in human plasma [119]. This diversity is attributable to different
combinations of long chain fatty acids at sn-1 and sn-2 of glycerol [119] and is determined by
enzyme preference for specific fatty acid substrate. This is important because enzymes in the
Kennedy pathway will define the milieu of lipid mediators produced in tissue when they show
preference for substrate of lipid mediators. For example, ACSL-3 and -4 show preference for AA
and EPA [28]. AGPAT-1, prefers myristic acid (C14:0), palmitic acid, and LA-CoAs, and
AGPAT-2, prefers AA-CoA [144]. These enzymes are differentially expressed in models of sepsis
and diabetes, and because of their selection of substrate, may partly explain lipid mediator
biosynthesis and inflammation during these diseases [2, 75]. Substrate selection by enzymes
involved in tissue phospholipid biosynthesis has not been evaluated in PUFA dietary studies, but
should be considered when interpreting future studies that examine the influence of dietary PUFA
on lipid mediator biosynthesis.
Some Lands’ cycle enzymes are selective between fatty acids. For example, the conversion of
phosphoethanolamine

to

phosphocholine

by

CDP-ethanolamine:1,2-diacylglycerol

ethanolaminephosphotransferase favors biosynthesis of sn-1 palmitic acid/ sn-2 DHA. This
phospholipid then undergoes rapid reacylation with stearic acid (C18:0) at sn-1, and AA, LA, or
19

DHA at sn-2. In fact, the sn-1 stearic acid/sn-2 AA forms of phosphocholine and
phosphoethanolamine are usually synthesized by replacement of other acyl chains in existing
phospholipids, rather than directly [173]. Therefore the Lands’ cycle can result in rapid and
preferential incorporation of DHA and other PUFA into pre-existing phospholipids. This indicates
that dietary supplementation with some n-3 PUFA could have especially rapid effects on health,
in light of what is known about the effect of dietary n-3 PUFA supplements on inflammation [118].
As mentioned previously, adipose-derived fatty acid can be a major substrate for biosynthesis of
phospholipids in some physiological and disease states. In this context, selectivity of fatty acyl
incorporation into triacylglycerol could affect the composition of the fatty pool available for
phospholipid synthesis during mobilization of adipose. The final step in triacylglycerol synthesis
involves DGAT, which adds fatty acyls to the sn-3 position of diacylglycerol. Unfortunately little
research has been conducted into substrate selectivity of mammalian DGAT, but in plants it
appears that DGAT-2 demonstrates preference for the less abundant, trans isomers of PUFA, and
there is similar DGAT-1 and -2 specificity for palmitic acid, oleic acid (C18:1), LA, and ALA
fatty acyls [95]. In yeast, both DGAT-1 and -2 have broad substrate preferences [170]. In reviews
of these enzymes, Yen et al (2008) suggest DGAT-1 prefers oleic over saturated fatty acyls [183],
while others indicate DGAT-2 is required for essential fatty acyl (i.e. LA and ALA) incorporation
into triacylglycerol [191]. In summary, it appears that much work must be done before the
selectivity of mammalian DGAT, and their influence on PUFA incorporation into adipose tissue,
will be fully understood.
Conclusions
Human and animal research indicate that for dietary n-6 and n-3 PUFA to modify the pathology
20

of disease, the PUFA content of phospholipids in cells involved in the inflammatory response must
reflect the dietary PUFA composition [63, 118] and specific phospholipid-derived PUFA must be
utilized for biosynthesis of pro-inflammatory or pro-resolving lipid mediators [118]. The roles of
lipid mediators in maintenance of health and regulation of inflammation during disease have been
introduced in the literature. Whether lipid mediators are pro-inflammatory or pro-resolving is
partly determined by the specific PUFA from which the lipid mediator is derived. There are trends
in the literature supporting a role for n-3 PUFA in control and prevention of inflammation during
disease but it is also important to recognize that some n-6 PUFA are essential nutrients and produce
pro- and anti-inflammatory lipid mediators. A number of clinical trials with patients supplemented
with fish-oil showed no clear health benefit, and could be explained by factors that affect dietary
PUFA incorporation into phospholipids. In conclusion, future studies of the impact of dietary
PUFA on tissue phospholipid biosynthesis should examine digestive processes, competition for
absorbed PUFA by other physiological processes, supply of PUFA from sources other than diet,
obesity and reproductive state, glucose homeostasis, and selectivity of phospholipid synthesizing
enzymes for fatty acids. Such research will improve knowledge of phospholipid biosynthesis in
humans and domestic animals. Complementary studies that measure inflammatory outcomes and
lipid mediator biosynthesis should reveal the true health benefit or cost of n-3 PUFA dietary
supplements.
Acknowledgments
The authors acknowledge research support from the Agriculture and Food Research Initiative
Competitive Grants Program (2011-67015-30179, 2012-67011-20019) from the USDA National
Institute for Food and Agriculture, and an endowment from the Matilda R. Wilson Fund (Detroit,
MI, USA).
21

Conflicts of Interest
The authors declare no conflict of interest.

22

CHAPTER 2

ASSOCIATION BETWEEN POLYUNSATURATED FATTY ACID-DERIVED
OXYLIPID BIOSYNTHESIS AND LEUKOCYTE INFLAMMATORY MARKER
EXPRESSION IN PERIPARTURIENT DAIRY COWS2

W. Raphael, L. Halbert, G. A. Contreras, and L. M. Sordillo*
Department of Large Animal Clinical Sciences, College of Veterinary Medicine,
Michigan State University, 736 Wilson Rd., Room D202, East Lansing, MI 48824, USA;
*Author to whom correspondence should be addressed; E-Mail: sordillo@msu.edu;
Tel.: +1-517-432-8821; Fax: +1-517-432-8822

2

Used by permission of the Journal of Dairy Science (Appendix 1). Accepted; J. Dairy. Sci. 2014,
97; doi:10.3168/jds.2013-7656.
23

Interpretive Summary
Unregulated inflammatory responses in cows can enhance disease pathogenesis during the
periparturient period. Previous research showed that oxylipids regulate inflammation, but their role
in periparturient inflammatory responses is not known. This study describes changes in both
plasma oxylipids concentrations and their substrates across the periparturient period and correlates
these profiles with inflammatory gene expression in peripheral leukocytes. Future studies will
investigate sources of polyunsaturated fatty acid substrates of plasma oxylipids and roles of
specific plasma oxylipids in inflammatory pathways.

24

Abstract
Peripheral blood mononuclear leukocytes from periparturient cows can have exacerbated
inflammatory responses that contribute to disease incidence and severity. Oxylipids derived from
the oxygenation of polyunsaturated fatty acids (PUFA) can regulate the magnitude and duration
of inflammation. Although PUFA substrate for oxylipid biosynthesis in leukocytes is known to
change across the periparturient period, the plasma oxylipid profile and how this profile relates to
leukocyte inflammatory phenotype is not clear. The objective of this study was to determine if a
relationship exists between the profile of pro- and anti-inflammatory plasma oxylipids and the
inflammatory phenotype of peripheral blood leukocytes during the periparturient period. Seven
multiparous Holsteins were sampled from the prepartum period through peak lactation. Plasma
oxylipids were measured by liquid chromatography-mass spectrometry, peripheral leukocyte
mRNA expression was measured by qPCR, and PUFA content of peripheral blood mononuclear
cells was measured by gas chromatography-mass spectrometry. Concentrations of several
hydroxyl products of linoleic and arachidonic acid changed over time. Linoleic acid and
arachidonic acid concentrations in leukocytes increased during early lactation, suggesting that
substrate availability for hydroxyoctadecadienoic and hydroxyeicosatetraenoic acid biosynthesis
may influence the oxylipid profile. Leukocyte mRNA expression of IL-12B, IL-1B, inducible nitric
oxide synthase 2, and cyclooxygenase 2 were correlated with several plasma oxylipids. These are
the first observations linking leukocyte inflammatory gene responses with shifts in oxylipid
biosynthesis in periparturient dairy cows.
Keywords: inflammation, periparturient, eicosanoid, oxylipid, lipid mediator

25

Introduction
A major factor contributing to decreased productivity and longevity of dairy cows is the incidence
of disease in the first month after parturition [117]. Dysfunctional inflammatory responses during
the periparturient period can contribute to disease incidence and severity [154]. For example,
bovine peripheral blood mononuclear cells (PBMC) have an exacerbated inflammatory response
to lipopolysaccharide in the periparturient period, compared with cells from mid-lactation cows
[152]. Uncontrolled inflammatory responses also were associated with increased severity of
clinical mastitis [145]. Control and prevention of periparturient disease is currently difficult, partly
because it is unclear why dysfunctional inflammatory responses occur around parturition.
Oxylipids are defined as the class of PUFA-derived metabolites that regulate all aspects of
the inflammatory response. The biosynthesis of oxylipids involves a number of complex pathways
that start with the peroxidation of cell membrane-derived PUFA including linoleic (C18:2 n6),
arachidonic (C20:4 n6), eicosapentaenoic (C20:5 n3), and docosahexaenoic acids (C22:6 n3).
Initial peroxidation of these PUFA can occur enzymatically through the cyclooxygenases (COX),
lipoxygenases (LOX), and epoxygenases, but are also mediated by free oxygen radicals. Oxylipids
produced by PUFA peroxidation include 9- and 13- hydroperoxyoctadecadienoic acid (HPODE),
prostaglandin (PG) H2, 5- and 15-hydroperoxyeicosatetraenoic acid (HPETE) and 14- and 17hydroperoxydocosahexaenoic acids (HpDHA) [140, 150]. These are unstable lipid mediators that
are quickly reduced to more stable hydroxyls, such as hydroxyoctadecadienoic acid (HODE) and
hydroxyeicosatetraenoic acid (HETE). The hydroxyl oxylipids can serve as substrates to a vast
number of downstream metabolites, such as oxooctadecadienoic acids (OxoODE) [120],
leukotrienes (LT) [105], thromboxanes (TX), PGD2, PGE2, PGF2, PGI2 [150], 7-maresin1 (MaR1),
and protectins (PD) [140]. Thus, the rate of PUFA oxidation and the extent of metabolism into
26

downstream products determine the relative abundance of oxylipids with different functional
capabilities.
A major determinant of whether oxylipids initiate or resolve inflammation is the PUFA
from which they are derived. Both C20:5 n3 and C22:6 n3-derived oxylipids predominantly
abrogate inflammation. For example, MaR1 is produced from C22:6 n3 and reduces neutrophil
recruitment during peritoneal inflammation [141]. In contrast to the n-3 PUFA-derived oxylipids,
the downstream products of n-6 PUFA peroxides have multiple effects in inflammatory pathways.
Different PG, for example, can stimulate (e.g. PGF2) or abrogate (e.g. PGD2, PGE2) inflammatory
events [138]. The effects of some n-6 derived oxylipids are dependent on species and cell types.
For example, HODE activates adhesion molecule expression on endothelial cells [56], thus
facilitating leukocyte extravasation during inflammation. However, HODE also binds to nuclear
receptors, and abrogates tumor necrosis factor- (TNF- induced inflammatory responses in
human epithelial tissue [5]. Several studies showed that dietary changes in PUFA substrate supply
could effectively change oxylipid profiles [123]. For example, n-3 PUFA dietary supplementation
in humans and rodents was shown to increase tissue phospholipid content of these PUFA and
increase n-3 PUFA-derived oxylipids in tissue [118, 120]. Recent studies in periparturient dairy
cows identified C18:2 n6 as more abundant in several tissues after parturition, including PBMC
[4, 31], but the influence of C18:2 n6 on oxylipid production and leukocyte inflammatory
pathways in cows is not known.
Concentrations and relative potency of individual oxylipids [104, 107] determine the net
effect of the tissue oxylipid profile on inflammatory processes. The abundance of proinflammatory oxylipids relative to pro-resolving oxylipids impacts the chronicity and resolution
of disease by initiating or resolving segments of the inflammatory pathway [140]. Describing the
27

plasma concentrations of oxylipids in periparturient cows, in particular those oxylipids which may
affect circulating immune cells and vascular endothelium, may assist in explaining why
periparturient PBMC may have enhanced inflammatory responses following exposure to bacterial
agonists [152]. The hypothesis of this study was that there is a relationship between the profile of
pro- and anti-inflammatory plasma oxylipids and the inflammatory phenotype of peripheral blood
leukocytes during the periparturient period.
Materials and methods

Animals
This study was conducted in the autumn months of 2012 in a 1500-cow, intensively housed
commercial dairy herd located in Michigan. The herd was fed total mixed rations that contained
corn silage and alfalfa haylage as predominant forages. Seven Holstein cows were randomly
selected from all healthy cows within 8 to 12 d of expected parturition at study commencement.
Median parity for the observed parturition was 3, and ranged from 2 to 4. Blood (50 mL) was
aseptically collected in EDTA vacutainers (Becton, Dickinson and Co., Franklin Lakes, NJ) by
coccygeal venipuncture, and immediately stored on ice. The distribution of sampling time relative
to parturition was 14 d prepartum (range = 6 to 17), immediately after parturition (range = 1 to 3
d postpartum), 10 d postpartum (range = 7 to 10), 28 d postpartum (range = 21 to 32), and 84 d
postpartum (range = 77 to 88). Plasma non-esterified fatty acids (NEFA) were measured in 3
randomly selected cows by an enzymatic colorimetric method (Wako NEFA HR(2), Wako
Chemicals USA, Richmond, VA) conducted at the Diagnostic Center for Population and Animal
Health, Lansing, MI. Michigan State University Animal Care and Use Committee preapproved all
animal use and care.

28

Plasma Oxylipid Quantification
Oxylipid Extraction
Plasma (500 µL) was separated from whole blood prior to PBMC isolation (931 g for 30 min,
4°C). Formic acid (1.7 uL) and antioxidants/reducing agents were added (4 µL of combined 2.4
mg EDTA in 3 mL water, 2.4 mg butylated hydroxytoluene (BHT) 1.5 mL ethanol, 24 mg
triphenylphosphine in 6 mL methanol (Sigma-Aldrich, St. Louis, MO), and 24 mg indomethacin
in 1.5 mL ethanol (Cayman Chemical, Ann Arbor, MI)). Deuterated internal standards (LTB4-d4,
TXB2-d4, PGF2α-d4, PGE2-d4, PGD2-d4, 13(S)-HODE-d4, 6-keto PGF1α-d4, 12(S)-HETE-d8, and 15(S)HETE-d8, Cayman Chemical) were combined in ethanol:water (1:1), to achieve 0.1 ng / uL for each
standard, and 200 uL of this mixture (20 ng of each standard) was added to samples. Methanol
was then added to samples (1 mL, -20°C), and protein precipitated by brief vortexing and freezing
(-20°C for 3 h). Samples were then centrifuged at 18,000 g for 15 min (4°C). The supernatant was
diluted in 9.2 mL water containing 0.1% formic acid (v/v). Oxylipids were extracted by solid phase
methods as previously reported [53], with the following modifications. Extraction columns (StrataX 60 mg, 3 mL, Phenomenex, Torrance, CA) were conditioned (3 mL methanol then 3 mL water),
washed (3 mL 50% methanol (v/v) in high performance liquid chromatography grade water), dried,
and samples eluted in 3 mL methanol:acetonitrile (1:1, v/v). Samples were then vacuum dried,
suspended in 200 uL acetonitrile:water:formic acid (1850:3150:1, v/v/v), and centrifuged at 18,000
g for 30 min (4°C), immediately prior to chromatography.
Liquid Chromatography/Mass Spectrometry (LC/MS)
Oxylipids were measured using LC/MS as we reported previously [53] but with quantification of
8-iso PGF2α, PGE2, PGD2, PGF2α, TxB2, 6-keto PGF1α, resolvin D1 (RD1), and RD2 utilizing an
isocratic mobile phase of acetonitrile:water:0.1% formic acid (35:55:10; v/v/v). 9-HODE, 1329

HODE, 15-oxoeicosatetraenoic acid (OxoETE), 5-OxoETE, 5-HETE, 8-HETE, 11-HETE 12HETE, 15-HETE, 20-HETE, 9-oxoODE, 13-oxoODE, MaR1, PD1, and LTD4 were measured as
previously described [53]. Oxylipids were identified in samples by matching their deprotonated
(i.e., [M-H]-) m/z values and LC retention times with those of pure standards. Oxylipids were
quantified relative to internal standard abundance, and calibration against standard curves.

PBMC Fatty Acid Methyl Ester Quantification
Lipid Extraction
PBMC in 45 mL whole blood were isolated using a Ficoll-Paque Plus gradient (GE Healthcare,
Waukesha, WI), as previously described [31]. Pelletized PBMC were frozen in liquid nitrogen and
stored at -80°C until completion of sample collections. Total lipid was extracted using methanol
and chloroform, as previously reported [16, 55]. Briefly, PBMC pellets were suspended in 1.0 mL
1x PBS, then proteins precipitated by adding 3.0 mL of -20°C methanol (500 mg / L BHT). After
30 min, 1.0 mL 1x PBS, 2.0 mL methanol (500 mg / L BHT), 7.0 uL formic acid, and 7.5 µg C13:0
fatty acid (1 mg / mL in hexane) were added. Samples were then sonicated (30 min, 25°C), and
2.5 mL chloroform added. Samples were then shaken for 16 h (25°C), and a further 2.5 mL of
chloroform, and 2.5 mL 2 % sodium chloride (w/v, in high performance liquid chromatography
grade water) added. Following centrifugation, (2095 g for 15 min, 8°C), chloroform was aspirated
and stored, and extraction repeated with 2.5 mL chloroform and centrifugation (2095 g for 15 min,
8°C). Samples were then dried under vacuum. Contaminant lipids were measured in cell-free
samples, concurrent to PBMC samples.
Derivation
Sample lipids were saponified in 0.5 mL methanol, utilizing 1.0 mL freshly made 0.5 M potassium
hydroxide in methanol, and heat (100° C for 10 min). After cooling to 4° C, derivation of PUFA
30

to methyl esters (FAME) was performed under nitrogen gas, utilizing 3.0 mL fresh 20% v/v
methanolic hydrochloric acid [115], and heat (90° C for 16 h). After cooling to 4° C, acid was
neutralized using 10.0 mL of 6 % potassium carbonate (w/v, in high performance liquid
chromatography grade water). FAMES were then extracted with 3.0, 1.5, and 1.5 mL n-hexanes
by cetrifugation (524 g for 5 min at 8° C). Samples were then vacuum dried, resuspended in 35 uL
n-hexanes, and transferred to a GC microinsert vial. Finally, 7.5 µg C15:0 FAME (5 mg / mL in
hexane) was added, and samples immediately analyzed by gas chromatography/mass
spectrometry.
Gas Chromatography/Mass Spectrometry (GC/MS)
The gas chromatograph was Agilent Technologies model 6890N (Agilent Technologies, Santa
Clara, CA), fitted with a DB-23 column (30 m x 0.25 mm internal diameter x 0.25 um film
thickness, Agilent Technologies). Inlet temperature was 240o C. The method utilized helium gas
at 60.2 kPa psi, a 20:1 split ratio, and a combined flow rate of 25.9 mL / min. Column temperature
was optimized and set at 50o C for 1 min, then increased at 25o C / min to 175o C, then increased
at 4o C / min to 240o C and held constant for 5 min. Total run time was 27.25 min. Injection volume
was 1.0 uL. A pre- and post-injection delay of 1 s was applied.
The mass spectrometer was Agilent Technologies model 5975B inert XLMSD (Agilent
Technologies). Transfer line, source, and quadrupole temperatures were 240o, 230o, and 150oC,
respectively. Detection criteria were 40 to 400 amu, and a 3 min solvent delay was applied. Total
ion chromatograms and mass spectra were generated using Agilent ChemStation software (Agilent
Technologies). A 37-component FAME standard mix was utilized to determine retention times
(Sigma-Aldrich). Analytes were identified with at least 90% similarity between analyte and
reference mass spectra (National Institute of Standards and Technology). Total ion chromatograms
31

were created for FAMES of C13:0, C15:0, C18:2 n6, and C20:4 n6. Extracted ion chromatograms
were created for FAMES of C15:0 (74 atomic mass units [amu]), C20:5 (79 amu) and C22:6 (79
amu) at the appropriate retention times.
GC/MS was validated in several ways. The within-assay and between-assay coefficient of
variation for standard grade C18:2 n6 FAME was calculated to be 5 and 15 %, respectively.
Second, a single cow sample was divided into 3 aliquots and analyzed separately at the beginning,
midway, and completion of chromatography. The coefficient of variation for C18:2 n6 to C15:0
FAMES ratio across these 3 aliquots was 5.4%. Finally, because chromatograms were performed
in 2 batches, variance in chromatogram intensity of the C15:0 FAME internal standard between
batches was assessed by calculating the ratio of intensity in samples to that of calibration standards
for each batch (coefficient of variation = 0.9 %). Batch variance in methylation efficiency was also
evaluated and the coefficient of variation was 10.3 %.
FAME Quantification
Sample internal standards were added prior to extraction (C13:0 fatty acid) and following
derivation (C15:0 FAME). Calibration curves for C18:2 n6, C20:4 n6, C20:5 n3, and C22:6 n3,
and C13:0 FAMEs were created using freshly prepared standard-grade FAMES, analyzed before
and after the samples. Standard dilutions were 0.7 fold in n-hexanes, and concentrations extended
2 dilutions above the maximum sample concentration and below the minimum sample
concentration. Calibration equations were calculated by linear regression of the known standard
concentration against the ratio of analyte to C15:0 FAME (R2 > 0.975). This ratio was calculated
from analyte and C15:0 FAME chromatogram intensities.
Analyte concentration in samples was calculated by comparing the intensity ratio of analyte

32

to C15:0 FAME in samples to that of the linear regression equation from calibration. Analyte
concentrations in samples were corrected for methylation efficiency, by comparing the C13:0
FAME abundance in samples to the expected abundance from C13:0 fatty acid internal standard.
Analyte concentrations also were corrected for FAME abundance in cell free samples.

Leukocyte mRNA Quantification by qPCR
Total RNA was extracted from 3 mL whole blood for quantification of mRNA using the QIAamp
RNA Blood Mini Kit (Qiagen, Valencia, CA) with DNase. cDNA was synthesized using the High
Capacity cDNA reverse transcriptase kit with RNase inhibitor (Applied Biosystems, Foster City,
CA). A single sample was excluded for poor RNA integrity (RIN < 6, Agilent Bioanalyzer).
Primers were derived from the Bos taurus genome (Table 1). The cDNA was amplified using
TaqMan PreAmp Kit (Applied Biosystems). The qPCR was conducted as previously reported [29].
Briefly, PCR was performed in triplicate, using 20 µL reaction mixture per replicate, comprising
11.7 µL TaqMan Gene Expression PCR Master Mix (2x, Applied Biosystems), 1.2 µL (20x)
TaqMan Gene Expression Assay Mix (Applied Biosystems), 5 µL amplified cDNA, and 2.1 µL
nuclease-free water. Pre-designed (20x) TaqMan Gene Expression Assays from Applied
Biosystems for bovine TBP, PGK1, and ACTB were selected as reference genes. Each PCR plate
included a non-template control. Two-step PCR thermal cycling conditions were utilized: stage 1,
50°C for 2 min; stage 2, 95°C for 10 min; stage 3, 95°C for 15 s; stage 4, 60°C for 1 min, with 40
replicates of stages 3 and 4. Quantification was carried out with the relative quantification method
[96]. Briefly, the abundance of target genes is normalized to the average of the reference genes,
and calculated relative to calibrator samples by 2–ΔΔCt, where Ct is the cycle number at which the
fluorescence signal of the product crosses an arbitrary threshold set with exponential phase of the
PCR, and ΔΔCt = (Ct target gene unknown sample – Ct average of endogenous control genes unknown sample) – (Ct
33

target gene calibrator sample

– Ct average of endogenous control genes calibrator sample). Samples collected at 84 d

were the calibrator samples.

Statistical Evaluation
Temporal oxylipid, mRNA (mean ΔCt), and FAME concentrations were evaluated in a mixed
analysis of variance model, with cow as a random source of variance, the dependent variables
repeated across time and within cow, and the model solved for the fixed effect of time. Covariance
structures (compound symmetry, unstructured, or first order autoregressive) were selected
according to Aikake’s information criterion. Differences between least square means were
calculated with Sidak’s adjustment. Pearson correlations were calculated to measure associations
between plasma oxylipid concentrations and leukocyte mRNA expression (23 oxylipids x 8 genes
= 184 correlations), and between PBMC PUFA and plasma oxylipid concentrations (3 PUFA x 23
oxylipids = 69 correlations), and between PBMC PUFA concentrations and leukocyte mRNA
expression (3 PUFA x 8 genes = 24 correlations). Mean Ct was multiplied by -1 prior to
calculation of Pearson coefficients. Significance of correlation was adjusted for false discovery
associated with multiple tests.
Results

Animal Production and Health
All cows remained free from clinical disease during the period of study. Mean (± standard
deviation) 305 d mature equivalent milk production was 12,324 (± 2,518) kg. Mean (± standard
error) plasma NEFA concentration in 3 randomly selected cows was significantly greater at 0 days
(0.70 (± 0.15) mEq / L, P < 0.0136) than -14 days (0.08 (± 0.01) mEq / L) and 10 days (0.43 (±
0.09) mEq / L).
34

Plasma Oxylipid Profile
Plasma oxylipid concentration differed across the periparturient period for 3 of the 23 oxylipids
measured (Figures 5 and 6). Early lactation (10 d) was associated with increased 13-HODE relative
to prepartum. Samples at 84 d had greatest 9-HODE and 11-HETE concentrations. Plasma
concentrations of 9-HODE were approximately 2-fold higher than 13-HODE. Both 9- and 13HODE concentrations were approximately 10-fold higher than 11-HETE. No significant changes
over time were observed in plasma concentrations of 20 oxylipids (Table 2). The limit of detection
for oxylipids was 0.01 ng.

Leukocyte mRNA Expression
The inflammatory phenotype of peripheral leukocytes showed minor changes across the period of
study. The expression of 15-LOX1 and 15-hydroxyprostaglandin dehydrogenase (HPGD) mRNA
in peripheral leukocytes were decreased significantly at 10 d postpartum, relative to prepartum
samples, and also relative to 84 d samples for 15-LOX1 (Figure 7). The expression of IL-12B
mRNA was significantly increased at parturition and 10 d, relative to 28 d samples (Figure 8). No
significant changes were observed in the gene expression of IL-10, inducible nitric oxide synthase
2 (NOS2), IL-1, TNF-, or COX2 during the entire experimental period.

PBMC FAME Content
C18:2 n6 and C20:4 n6 FAMEs were consistently detected in PBMC, across all cows and sample
periods. The concentrations of C18:2 n6 and C20:4 n6 were significantly increased at 10 d relative
to prepartum samples (Figure 9). PBMC C20:5 n3 FAME was not detected in 20 of 35 PBMC
samples. Mean (± standard error) sample mass of C20:5 n3 FAME was 899 (± 132) ng. Minimum
mass of C20:5 n3 FAME standard utilized and detected was 4 ng. C22:6 n3 FAME was detected

35

in one sample therefore no statistical analysis for this FAME was performed. The limit of detection
of C22:6 n3 FAME standard was 0.57 ng. Samples contained a chromatogram peak at the identical
retention time as C22:6 n3 did in standards, but this peak was abundant in 239 amu, was not the
mass spectra of C22:6 n3 nor typical of any methyl ester, and was not identified. C18:2 n6 was the
only FAME of interest detected in cell free control samples (mean ± SE of 5 extraction batches =
3912 ± 860 ng).

Statistical Relationships
There were no significant correlations between C18:2 n6, C20:4 n6, or C20:5 n3 in PBMC and
plasma oxylipids. Among the leukocyte genes assessed, IL-12B, IL-1B, COX2, and NOS2
expression were associated with plasma oxylipids (Table 3). Expression of HPGD was associated
with C20:5 n3 concentrations (r = -0.75, p = 0.0052).
Discussion
This study showed for the first time how concentrations of select pro- and anti-inflammatory
plasma oxylipids change in dairy cows that successfully transitioned from late gestation to early
lactation without clinical illness. Until now, only select bovine oxylipids were studied in
physiological processes, such as luteolysis and maintenance of pregnancy [90, 149], and also in
case-control studies of bovine pathology [126, 136], but little was known about the relationships
between plasma oxylipids and peripheral leukocyte inflammatory responses. Leukocytes are a
primary target for the actions of oxylipids so changes in PBMC inflammatory gene expression in
the absence of clinical disease may be due to the actions of these potent lipid mediators. Results
from this study revealed several notable features of the periparturient oxylipid profile. For
example, hydroxyl forms of C18:2 n6 (13-HODE) was the most abundant plasma oxylipid
36

especially during early lactation. There are several proinflammatory effects of HODE, such as
induction of adhesion molecule expression on human vascular tissue [56]. HODEs also were the
most abundant oxylipids found in human atherosclerotic lesions [60] and therefore have been
investigated as potential contributors to chronic, uncontrolled inflammatory conditions. In
contrast, HODEs can be important activators of peroxisome proliferator activated receptors [104],
which can lead to decreased expression of inflammation-based genes such as inducible nitric oxide
synthase [128]. Thus, the current literature suggests that HODE could be pro- or anti-inflammatory
oxylipids in different situations. The significance of enhanced HODE biosynthesis during the
periparturient period on bovine inflammatory responses is not known. The possibility that HODEs
may contribute to dysfunctional inflammatory reactions in early lactation cows with metabolic or
infectious diseases would be a logical question to ask in future studies.
The enhanced expression of HODE in periparturient cows could be due to several factors
including substrate availability. The substrate for HODE, C18:2 n6, increased in PBMC during
early lactation (Figure 9). These findings are consistent with earlier studies that reported an
increase in plasma and liver C18:2 n6 concentrations of recently calved dairy cows [4, 31]. Both
in vivo and in vitro studies support the contention that increased HODE biosynthesis is at least
partly regulated by substrate availability. For example, increased human dietary C18:2 n6 intake
is associated with increased plasma HODE [120]. Increased HODE biosynthesis was observed in
cultured bovine endothelial cells exposed to a NEFA mixture with high C18:2 n6 content that is
typically found in bovine plasma during the periparturient period [30]. Therefore, increased plasma
HODE may be a reflection of increased tissue, leukocyte, or plasma contents of C18:2 n6. The
tissue and cellular source of plasma HODES measured in this study were not assessed. Oxidation
of C18:2 n6 may occur in leukocytes or other cell types by COX, LOX, epoxygenase, and non37

enzymatic oxidation. Previous studies showed increased COX2 and 15-LOX1 mRNA and COX2
protein expression in bovine vascular endothelial cells in response to NEFA typical of those
present in bovine plasma during the periparturient period [30]. Chemical inhibition of COX and
15-LOX enzymes, however, did not abrogate endothelial HODE biosynthesis from NEFA and
suggests that non-enzymatic oxidation of plasma C18:2 n6 could contribute to endothelial cell
production of plasma HODE [29]. Reduced transcript expression of leukocyte 15-LOX1 in this
study also may suggest that there is a decreased capacity for the enzymatic oxidation of C18:2 n6
in early lactation. Identifying the primary cellular sources of HODE and the mechanism of
oxidation could have future implications of how to not only regulate its biosynthesis, but also
influence the relative contribution to inflammatory responses.
The ratios of HODE to OxoODE reveal changes to C18:2 n-6 metabolism in early lactation.
Mean 13-HODE to 13-OxODE ratios (Figure 6d) indicate lesser abundance of the ketone
derivative than hydroxyl derivative of C18:2 n-6 during the first weeks of lactation (ratio > 1.0)
relative to prepartum, parturition, and 84 days (ratio < 1.0). Moreover, the range of both 13-HODE
to 13-OxODE and 9-HODE to 9-OxoODE ratios (Figure 6c) were greatest in early lactation.
HODE is the reduced product of HPODE, which is more stable and can be converted into OxoODE
by dehydrogenation [120]. The extent of HPODE metabolism is directly associated with antiinflammatory potential [56, 104], therefore increasing HODE to OxoODE ratios may indicate a
change in the net effect of C18:2 n6 derived oxylipids on inflammatory pathways. If future studies
prove the HODE to OxoODE ratio to be influential in bovine inflammatory responses, then the
precise pathways by which HODES are degraded in cows should be investigated as a potential
target of inflammatory disease intervention [62, 187].
Temporal changes in the plasma oxylipid profile can provide some insight as to how bovine
38

leukocytes may respond within that microenvironment. Whereas the HODEs increased during
early lactation, it is important to also note those oxylipids that did not change concentration in this
study. For example, the steady state biosynthesis of certain anti-inflammatory oxylipids could
influence leukocyte phenotype when considered as a key component of the overall oxylipid profile.
In this study, several potent anti-inflammatory oxylipids (PD1, RD1, RD2, and 7-MaR1) did not
significantly change throughout the experimental period in the absence of clinical disease. Several
recent studies documented the significance of these oxylipids in resolving inflammation and
restoring tissue to normal function following infections [138, 140]. The precise role of these antiinflammatory oxylipids on bovine leukocytes during the periparturient period is not known. The
present study does, however, provide baseline information of the overall oxylipid profile in cows
that successfully transition into lactation without succumbing to disease. Plasma oxylipids will
likely differ by health state as oxylipid enzyme expression is influenced by disease [135].
Correlations between plasma oxylipids and peripheral leukocyte mRNA expression were
conducted to assess the potential impact that certain pro-inflammatory and anti-inflammatory
bovine oxylipids may have in the absence of clinical disease. In the present study, no dramatic
changes were observed in several markers used previously to assess leukocyte pro-inflammatory
responses during the periparturient period. This is in contrast to previous studies that documented
significant cytokine gene expression in PBMC obtained from periparturient cows subjected to
metabolic, environmental or microbial challenge [101, 152, 165]. The present study, however,
used healthy animals and the results are consistent with previous reports noting no change in
PBMC pro-inflammatory cytokine mRNA expression in periparturient cows that remain free of
clinical disease [112]. Pearson coefficients did identify several correlations between oxylipids and
PBMC gene expression although no clear conclusion can be drawn from the inconsistent findings
39

(Table 3). Although some oxylipids with known pro-inflammatory functions (PGE2 and LTD4)
were positively correlated with pro-inflammatory gene expression as would be expected, there
were several instances where pro-inflammatory oxylipids (TXB2, 9-HODE, and 13-HODE) were
negatively correlated with the pro-inflammatory markers (Table 3). One explanation is that
cytokine expression and pro-inflammatory oxylipid biosynthesis may form negative feed-back
loops with each other. Expression of epoxygenase enzymes, for example, can be suppressed by
TNF- [10, 11]. Another possibility is that certain oxylipids may be more effective in controlling
leukocyte inflammatory responses than other lipid mediators within the profile. Without
conducting more controlled cause-effect studies, it is not possible to determine which oxylipids
may be the most potent in regulating PBMC gene responses. Based on the finding presented in this
study, however, it is clear that the oxylipid network is complex and the relative impact on leukocyte
responses during the periparturient period will likely be determined by the relative composition of
the oxylipid profile.
In conclusion, this study showed for the first time how concentrations of a select group of
oxylipids change during the periparturient period in healthy animals. The most abundant oxylipids
during early lactation were those derived from C18:2 n6 and C20:4 n6 with significant increases
in their respective hydroxyl products (HODE and 11-HETE) as lactation progressed. Although
plasma oxylipids are likely derived from many cellular sources, an interesting observation in this
study was that the significant increases in HODEs and 11-HETE corresponded to significant
increases in PBMC content of their respective PUFA substrates (C18:2 n6 and C20:4 n6). These
findings suggest that it may be feasible to manipulate oxylipid concentrations during the
periparturient period by altering the dietary content of available PUFA that can be metabolized
through various peroxidation pathways. Future studies that focus on how individual oxylipids may
40

directly impact the pro-inflammatory responses of bovine leukocytes would provide the foundation
for targeted therapies used to optimize innate immune defenses during the periparturient period.
Acknowledgements
The authors acknowledge the resources and staff of the Michigan State University Research
Technology Support Facility Mass Spectrometry Core, especially Dan Jones and Scott Smith. The
authors also acknowledge Jeff Gandy and Edward Kabara (College of Veterinary Medicine,
Michigan State University), Juan Steibel (College of Agriculture and Natural Resources, Michigan
State University), and the owners and staff of the herd for their assistance. This work was funded,
in part, through grants (2011-67015-30179 and 2012-67011-20019) from the Agriculture and Food
Research Initiative Competitive Grants Programs of the USDA National Institute for Food and
Agriculture and by an endowment from the Matilda R. Wilson Fund (Detroit, MI, USA).

41

CHAPTER 3

IMPACT OF INCREASED LINOLEIC ACID CONCENTRATION ON
INFLAMMATORY MARKER EXPRESSION IN BOVINE MONOCYTES

42

Abstract
Incidence and severity of dairy cow disease is greatest in the periparturient period. Disease risk is
increased with mobilization of adipose-derived fatty acids, such as linoleic acid, into plasma.
Tissue and cellular linoleic acid concentrations are increased in early lactation, including within
peripheral monocytes. Expression of proinflammatory markers by monocytes in the periparturient
period was associated with incidence of some diseases. Enhanced monocyte inflammatory
responses to endotoxin in the periparturient period were associated with severity of some diseases.
Metabolism of linoleic acid into inflammatory mediators known as oxylipids impacts the incidence
and severity of human disease but the impacts of increased linoleic acid on bovine monocyte
phenotype and inflammatory responses are unknown. The hypothesis of this research was that
increased bovine monocyte linoleic acid concentration enhances production of linoleic acidderived oxylipids, induces a proinflammatory phenotype, and enhances inflammatory responses
to bacterial endotoxin. Primary bovine monocytes and murine RAW 264.7 monocytes were
cultured in vitro in 25 and 50 uM linoleic acid to emulate in vivo monocyte linoleic acid content
in early lactation. Inflammatory phenotype and endotoxin responses, defined by cytokine transcript
expression and measured by qPCR, were not influenced by monocyte content of linoleic acid.
Oxylipid production was measured in cell culture media by liquid chromatography / mass
spectrometry.

Monocyte

enrichment

with

linoleic

showed

increased

production

of

hydroxyoctadecadienoic and oxooctadecadienoic acids, and no change in arachidonic or n-3 fatty
acid-derived oxylipids. In conclusion, monocyte inflammatory markers were not influenced by
cell linoleic acid concentration. Future studies are required to assess the specific impact of
individual linoleic acid-derived oxylipids on monocyte inflammatory pathways.

43

Keywords: monocyte; linoleic acid; hydroxyoctadecadienoic acid; oxooctadecadienoic acid;
inflammation; bovine; oxylipid

44

Introduction
Incidence and severity of several forms of clinical disease in dairy cows peak in the periparturient
period [117]. The most common diseases are mastitis and metritis and incidence of these peaks
within the first 2 weeks of lactation [37, 59, 114]. Despite previous research into disease prevention
and pathogenesis, periparturient disease continues to have negative impacts on farm economics
[45] and animal welfare [146]. However, previous research has identified important risk factors
that are related to host inflammatory responses.
Epidemiological studies showed that mobilization of adipose-derived lipid into plasma as
non-esterified fatty acids (NEFA) during the periparturient period increased risk of several
diseases, including metritis [24, 77, 137]. Mobilization of adipose-derived lipid is an adaptive
response to increased energy requirements for lactation [32]. Studies have identified potential
mechanisms by which NEFA may influence disease risk. For example, early lactation plasma had
increased concentrations of linoleic acid (LA, C18:2 n6), and this change in plasma fatty acid was
associated with increased LA concentrations in peripheral mononuclear leukocytes (PBMC) [31].
LA is associated with increased incidence and severity of some human diseases with
inflammatory based pathology. For example, the risk of chronic inflammatory vascular conditions,
such as coronary artery disease, was associated with increased dietary LA [121]. Rodent models
of inflammatory bowel disease showed increased disease severity with increasing dietary LA
[171]. In vitro models of human breast cancer demonstrated pro-tumorigenic effects of LA that
involved inflammatory pathways [100]. These associations between LA and inflammatory based
pathology in humans and rodents suggest that increased LA concentration in PBMC from early
lactation cows could influence monocyte inflammatory phenotype and responses.
45

Monocytes are critical cells in innate immune responses during diseases such as mastitis
and metritis. Previous studies identified changes in some monocyte inflammatory responses during
the periparturient period. For example, similar doses of lipopolysaccharide (LPS) elicited greater
tumor necrosis factor (TNF)- expression in monocytes from early lactation cows compared to
monocytes from mid-lactation cows [152]. TNF- influences multiple inflammatory processes,
including fever [40]. Subsequent research in cows demonstrated a greater febrile response with
intramammary Escherichia coli challenge in early lactation relative to mid-lactation [145].
Therefore some clinical signs of gram negative mastitis are more severe in early lactation than
mid-lactation and this difference in disease severity is partly due to an exacerbated monocyte
inflammatory response.
Subsequent research showed that the inflammatory phenotype of monocytes from
periparturient cows was predictive of future disease incidence. For example, monocyte TNF-

expression was greater in the first two weeks of lactation in cows that went on to develop clinical
metritis, than in cows that remained healthy[58]. Therefore in vivo expression of some
proinflammatory cytokines are predictive of future disease in cows. It is not clear why
periparturient monocyte phenotype differs between groups of cows that are more or less likely to
develop clinical disease, or why periparturient monocyte inflammatory responses to LPS differ by
stage of lactation. An objective of these studies was to evaluate the impact of increased cell LA
concentration on monocyte phenotype and inflammatory responses. Previous research of human
diseases suggests that metabolism of LA into oxylipids could be involved with changes in
phenotype and inflammatory responses [71].
Oxylipids are mediators of inflammatory processes derived from some polyunsaturated

46

fatty acids (PUFA). Major substrates are LA, arachidonic (AA, C20:4 n6), eicosapentaenoic (EPA,
C20:5 n3), and docosahexaenoic (DHA, C20:6 n3) acids that are stored within cell membranes
and released during inflammatory responses [108]. While many EPA and DHA-derived oxylipids
have anti-inflammatory effects [156], AA and LA-derived oxylipids are balanced between
proinflammatory and anti-inflammatory effects [123]. For example, the LA-peroxide, 13hydroperoxyoctadecadienoic acid (HPODE), stimulated proinflammatory marker expression in
human vascular smooth muscle cells [48]. Hydroxyoctadecadienoic acid (HODE) stimulated
proinflammatory cytokine production in human mononuclear leukocytes [83] but attenuated
clinical signs of inflammation in a skin disease model [20]. The HODE product,
oxooctadecadienoic acid (OxoODE) reduced TNF- stimulated inflammatory responses in
epithelial cells [5]. The net effect of the milieu of oxylipids on monocyte inflammatory phenotype
and responses depends on the relative concentrations, receptor affinity, and efficacy of each
oxylipid [104, 107]. Production of many oxylipids is regulated by the supply of PUFA substrate
within cells [108, 118]. However the impact of increased LA in bovine monocytes on oxylipid
production is not known. An objective of these studies was to describe the profile of oxylipids
produced by bovine monocytes in response to increased LA. The overall hypothesis of this
research was that increased monocyte LA concentration enhances production of LA-derived
oxylipids, induces a pro-inflammatory phenotype, and enhances inflammatory responses to
bacterial endotoxin.
Materials and Methods

Cell Culture
An in vitro model was developed to emulate PBMC concentration of LA in early lactation. The

47

model utilized PBMC from mid-lactation cows that had lower PBMC concentrations than earlylactation cows (Figure 10). PBMC were cultured in media containing various concentrations of
supplementary LA (6.25, 25, and 50 uM). LA was mixed in ethanol, diluted in media (0.01%), and
filtered (0.22 um). PBMC were utilized immediately after extraction from bovine whole blood by
Ficoll-Paque Plus gradient (GE Healthcare, Waukesha, WI), as previously described [31]. All
animal use and care was preapproved by Michigan State University Animal Care and Use
Committee.
LA stimulated oxylipid biosynthesis and cytokine transcript expression were assessed in
primary bovine monocytes and murine-derived monocyte-like macrophages (RAW 264.7, ATCC,
Manassas VA). Primary monocytes were isolated from PBMC by plate adhesion, as previously
described [110]. PBMC were seeded immediately after isolation from blood, and monocytes used
immediately after isolation from PBMC. Ethanol (0.01%) associated oxylipid biosynthesis and
cytokine transcript expression was assessed to control for non-specific effects of the fatty acid
vehicle. Arachidonic acid (50 uM) was utilized to control for non-specific PUFA effects.
RPMI media was utilized, with 5% (RAW 264.7) or 10% (primary cells) fetal bovine
serum, L-glutamine (Life Technologies, Grand Island NY), and 1X antibiotic/antimycotic (Life
Technologies). Cells were incubated at 37°C with 5% carbon dioxide and the start of experiments
defined as 0 hour or when cells were first exposed to LA. Culture within LA mixtures occurred
from hour 0 to 12 (PBMC), hour 0 to 16 (primary monocytes), and 0 to 13 (RAW 264.7). LPS (25
ng / mL, Sigma-Aldrich, St. Louis MO) was added to primary monocyte media from hour 12 to
16, Kdo2 Lipid A (100 ng / mL, Avanti Polar Lipids, Alabaster AL) to RAW 264.7 cell media
from hour 12 to 13. Viability was measured in PBMC and monocytes by trypan blue exclusion
under light microscopy.
48

Flow cytometry
Enrichment of monocytes from PBMC was assessed by fluorescent antibody staining and flow
cytometry methods as previously described [110]. Murine-derived primary monoclonal antibodies
against bovine CD172a (monocyte/granulocyte, 1:100, v/v), CD14 (monocyte, 1:50, v/v), CD3 (Tcell receptor, 1:100, v/v), and CD18 (integrin beta-2, negative control, 1:100, v/v, VMRD, Pullman
WA) and secondary goat anti-mouse antibody (1:2000, Life Technologies) were utilized. Results
were corrected for non-specific secondary antibody binding.

PBMC Fatty Acid Methyl Ester Quantification
Lipid Extraction
Adherent and non-adherent PBMC were collected by centrifugation of media and gentle plate
scraping, rinsed in 1X PBS, pelletized, then frozen in liquid nitrogen and stored at -80°C until
completion of sample collections. Total lipid was extracted using methanol and chloroform, based
on published methods [16, 55] and used previously by our group [122]. Contaminant LA was
measured in cell-free samples, concurrent to PBMC samples.
Derivation
Sample lipids were saponified using 1.0 mL freshly made 0.5 M potassium hydroxide in methanol,
and heat (100° C for 10 min), as previously reported [122]. After cooling to 4° C, derivation of
PUFA to methyl esters (FAME) was performed under nitrogen gas, utilizing 3.0 mL fresh 20%
v/v methanolic hydrochloric acid [115], and heat (90° C for 16 h). FAMES were then extracted
with 6.0 mL n-hexanes by cetrifugation (524 g for 5 min at 8° C). Samples were then vacuum
dried, resuspended in 35 uL n-hexanes, and transferred to a GC microinsert vial.
Gas Chromatography/Mass Spectrometry (GC/MS)

49

The gas chromatograph was Agilent Technologies model 6890N (Agilent Technologies, Santa
Clara, CA), fitted with a DB-23 column (30 m x 0.25 mm internal diameter x 0.25 um film
thickness, Agilent Technologies). Inlet temperature was 240o C. The method utilized helium gas
at 8.73 psi, a 20:1 split ratio, and a combined flow rate of 25.9 mL / min. Column temperature was
optimized and set at 50o C for 1 min, then increased at 25o C / min to 175o C, then increased at 4o
C / min to 240o C and held constant for 5 min. Total run time was 27.25 min. Injection volume
was 1.0 uL. A pre- and post-injection delay of 1 s was applied.
The mass spectrometer was Agilent Technologies model 5975B inert XLMSD (Agilent
Technologies). Transfer line, source, and quadrupole temperatures were 240o, 230o, and 150oC,
respectively. Detection criteria were 40 to 400 amu, and a 3 min solvent delay was applied. Total
ion chromatograms and mass spectra were generated using Agilent ChemStation software (Agilent
Technologies). A 37-component FAME standard mix was utilized to determine retention times
(Sigma-Aldrich). Analytes were identified with at least 90% similarity between analyte and
reference mass spectra (National Institute of Standards and Technology). Total ion chromatograms
were created for FAMES of C13:0, C15:0, and C18:2 n6. The within-assay and between-assay
coefficient of variation for standard grade C18:2 n6 FAME was calculated to be 5 and 15 %,
respectively.
FAME Quantification
Sample internal standards were added prior to extraction (C13:0 fatty acid) and following
derivation (C15:0 FAME). Calibration curves for C18:2 n6 and C13:0 FAMEs were created using
freshly prepared standard-grade FAMES, analyzed before and after the samples. Standard dilutions
were 0.7 fold in n-hexanes, and concentrations extended 2 dilutions above the maximum sample
concentration and below the minimum sample concentration. Calibration equations were
50

calculated by linear regression of the known standard concentration against the ratio of analyte to
C15:0 FAME (R2 > 0.975). This ratio was calculated from analyte and C15:0 FAME
chromatogram intensities.
Analyte concentration in samples was calculated by comparing the intensity ratio of analyte
to C15:0 FAME in samples to that of the linear regression equation from calibration. Analyte
concentrations in samples were corrected for methylation efficiency and FAME abundance in cell
free sample. Methylation efficiency was calculated by comparing the C13:0 FAME abundance in
samples to the expected abundance from C13:0 fatty acid internal standard. Treatment groups were
normalized by cell count.

Plasma Oxylipid Quantification
Oxylipid Extraction
Solid phase extraction methods were identical to those previously used by our group [122].
Deuterated internal standards were utilized (leukotriene B4-d4, thromboxane B2-d4, prostaglandin
(PG)

F2α-d4,

PGE2-d4,

PGD2-d4,

13(S)-HODE-d4,

6-keto

PGF1α-d4,

12(S)-

hydroxyeicosatetraenoic acid-d8 (12(S)-HETE-d8), and 15(S)-HETE-d8, Cayman Chemical, Ann
Arbor MI).
Liquid Chromatography/Mass Spectrometry (LC/MS)
Oxylipids were measured using LC/MS as previously reported [122]. Chiral HODE isomers were
separated using a Lux 3u Cellulose-1, 150mm x 2 mm column (Phenomenex, Torrance, CA) held
at 35º C. Isocratic elution at 0.225 mL / min lasted 30 m and utilized acetonitrile:methanol:water
(39/3/58, v/v/v), all with 0.1% acetic acid.
Abundance of PGD2, PGE2, PGF2, 6-keto-PGF1, 8-iso-PGF1, thromboxane B2,
51

leukotriene D4, resolvin D1 and resolvin D2, protectin D1, 7-maresin1, 5-, 8-, 11-, 12-, and 15HETE, 9- and 13-HODE, 9- and 13-OxoODE were quantified in culture media. Oxylipids were
identified in samples by matching their deprotonated (i.e., [M-H]-) m/z values and LC retention
times with those of pure standards. Oxylipids were quantified relative to internal standard
abundance, and calibration against standard curves. Treatment groups were normalized by sample
volume, DNA mass (Quant-iT dsDNA broad range, Life Technologies), and protein mass
(Bradford assay).

Monocyte mRNA Quantification by qPCR
Total RNA was extracted from monocytes for quantification of mRNA using the RNeasy Mini Kit
(Qiagen, Valencia, CA) with DNase. cDNA was synthesized using the High Capacity cDNA
reverse transcriptase kit with RNase inhibitor (Applied Biosystems, Foster City, CA). RNA
concentration was measured by Nanodrop (Thermo Fisher Scientific, Waltham, MA) and integrity
by Agilent Bioanalyzer (Santa Clara, CA). Primers were derived from the Bos taurus and Mus
musculus genome (Table 4). Primary monocyte cDNA was amplified using TaqMan PreAmp Kit
(Applied Biosystems). The qPCR was conducted as previously reported [29]. Briefly, PCR was
performed in triplicate, using 20 µL reaction mixture per replicate of primary monocyte cDNA,
comprising 11.7 µL TaqMan Gene Expression PCR Master Mix (2x, Applied Biosystems), 1.2 µL
(20x) TaqMan Gene Expression Assay Mix (Applied Biosystems), 5 µL amplified cDNA, and 2.1
µL nuclease-free water. RAW 264.7 cDNA was not amplified and PCR performed in triplicate
using 10 uL reaction mixture per replicate, comprising 5 µL TaqMan Gene Expression PCR Master
Mix (2x, Applied Biosystems), 0.5 µL (20x) TaqMan Gene Expression Assay Mix (Applied
Biosystems), 0.75 µL cDNA, and 3.75 µL nuclease-free water. Pre-designed (20x) TaqMan Gene
Expression Assays from Applied Biosystems for bovine TBP, PGK1, and ACTB and murine
52

GUSB, PGK1, and TBP were selected as reference genes. Each PCR plate included a non-template
control. Two-step PCR thermal cycling conditions were utilized: stage 1, 50°C for 2 min; stage 2,
95°C for 10 min; stage 3, 95°C for 15 s; stage 4, 60°C for 1 min, with 40 replicates of stages 3 and
4. Quantification was carried out with the relative quantification method [96]. Briefly, the
abundance of target genes is normalized to the average of the reference genes, and calculated
relative to calibrator samples by 2–ΔΔCt, where Ct is the cycle number at which the fluorescence
signal of the product crosses an arbitrary threshold set with exponential phase of the PCR, and
ΔΔCt = (Ct

target gene unknown sample

calibrator sample

– Ct

average of endogenous control genes unknown sample)

– (Ct

target gene

– Ct average of endogenous control genes calibrator sample).

Statistical Evaluation
Oxylipid, mRNA (mean ΔCt), and FAME concentrations were evaluated in a mixed analysis of
variance model, with RAW 264.7 cell clone or primary monocyte donor as random sources of
variance, and the model solved for the fixed effects of cell treatments. Differences between least
square means were calculated with Sidak’s adjustment.
Results

Cell culture
Mean viability of PBMC after Ficoll separation and 12 h culture ranged from 89% (media control
group) to 95% (6.25 and 50 uM LA, n = 3). Mean viability of primary monocytes after enrichment
procedures and 16 h culture ranged from 81% (50 uM AA, 25 ng / mL LPS) to 94 % (media control
group, n = 3). Proportion of total monocytes positive for CD172a, CD14, CD3, and CD18 were
91%, 79%, 3%, and 4% respectively. Culture of PBMC in media with 25 uM or 50 uM
supplementary LA increased cell LA concentrations to levels similar to peak in vivo concentrations
53

[122]. Results of statistical tests were similar for oxylipid samples normalized by DNA, protein,
and volume. Oxylipids concentrations are reported as ng / mL media.

Transcriptional responses to increased cell LA concentration
Transcript expression of TNF-, IL-10, IL-12B, 15-lipoxygenase (LOX) 1, and cyclooxygenase
(COX) 2 in primary monocytes and TNF-, IL10, IL-1, IL-6, 15-LOX1, and COX2 in RAW 264.7
cells were unchanged with increasing cell LA (p > 0.05, n = 3). In contrast, 50 uM AA increased
TNF- expression in primary monocytes and IL-1 in RAW 264.7 cells (p < 0.05, Figure 11).
Ethanol had no effect on transcript expression at the concentration used (0.01 %). Expression of
murine IL-12B mRNA was not detected by the utilized methods.

LA-derived oxylipid production with increased cell LA concentration
Production of 9- and 13-HODE and 9- and 13-OxoODE in primary monocyte culture media
increased with 25 uM and 50 uM LA supplementation (Figure 12). Concentrations of 9-OxoODE
were approximately twice that of 13-OxODE under similar culture conditions, although 9- and 13HODE concentrations did not differ. 9-OxODE was consistently more abundant than 9-HODE
with LA supplementation of 25 uM and 50 uM. Ratios of 9-, 13-, and total HODE / OxoODE did
not change significantly with LA supplementation (p > 0.05). The HODE chiral ratio (R / S)
increased with 6.25 uM and 25 uM LA supplementation (Figure 13). Production of 9- and 13HODE and 9-OxoODE also increased in RAW 264.7 cell culture media containing 50 uM
supplementary LA (Figure 14). Ratios of 9-, 13-, and total HODE / OxoODE did not change
significantly in RAW 264.7 cell culture media with LA supplementation (p > 0.05).
Monocyte production of PGF2a, thromboxane B2, 5-, 11-, and 15-HETE increased with AA
supplementation (p < 0.05, n= 3), but was not affected by LA supplementation or ethanol. Protectin
54

D1, 7-maresin1, and leukotriene D4 production was independent of fatty acid treatment.

Proinflammatory challenge with increased cell LA concentration
Primary monocyte transcript expression of TNF-, IL-10, IL-12B, and COX2 were induced by LPS
(25 ng / mL, p < 0.05, n = 3), but LPS induced expression was not modified by 6.25, 25, or 50 uM
LA, 50 uM AA, or ethanol (p > 0.05, n = 3). Expression of 15-LOX1 did not differ significantly
between LPS, LA, AA, or ethanol cell treatment groups.
LA-derived oxylipid production was not influenced by LPS stimulation of monocytes
enriched with 0 or 25 uM LA, but 9-HODE production increased with LPS stimulation in
monocytes enriched with 50 uM LA and 13-HODE production decreased with LPS stimulation in
monocytes enriched with 6.25 uM LA (p < 0.05, n = 3). HODE and OxoODE production response
to LA was similar between LPS stimulated cell culture media and unstimulated cell culture media.
However, the ratio of 9-HODE / 9-OxoODE in culture media from LPS stimulated monocytes was
significantly less with 25 uM or 50 uM LA supplementation than 0 uM (Figure 15, p < 0.05, n =
3). Influence of fatty acids on production of other oxylipids was similar to unstimulated
monocytes, except production of PGF2a and 5-HETE was not increased by AA in LPS stimulated
monocytes. (p > 0.05, n = 3).
Discussion
Despite previous reviews [27, 113], in vivo [121, 134], and in vitro studies [39, 66, 166, 167, 189]
that suggest increasing concentrations of LA enhanced the risk of some diseases by exacerbating
inflammatory responses, results showed that monocyte LA concentration was not associated with
inflammatory phenotype in either primary bovine monocytes or RAW 264.7 monocyte-like
macrophages and did not impact LPS-stimulated inflammatory responses in primary bovine
55

monocytes. These experiments are the first, to the best of our knowledge, which measure the
impact of cell LA concentration on monocyte inflammatory markers of any type. Previous
ruminant studies utilized primary ovine lymphocytes and reported no impact of LA (0.8 – 100 uM)
on antibody secretion [87]. Some previous monocyte studies focused on conjugated linoleic acid
effects but also utilized single concentrations of LA as a fatty acid treatment control. For instance,
interferon- stimulated RAW 264.7 cells expressed less TNF- transcript and protein when cells
were cultured in 200 uM LA [190]. According to these results (Figure 10), doses of this magnitude
could possibly exceed bovine physiological concentrations. Other studies utilized primary human
monocytes with 30 uM LA and failed to change activity of the critical pro-inflammatory
transcription factor, nuclear factor- [158].
This study defined inflammatory phenotype at the transcript level because previous studies
showed that the selected cytokines, including TNF- and IL-1are transcriptionally regulated
within the time frames of the cell culture experiments reported here [41, 52]. The rationale for
gene selection was to describe a profile of pro- and anti-inflammatory cytokines involved in early
inflammatory responses during several types of periparturient bovine disease [58, 145] or murine
models of fatty acid-induced inflammation [171]. Some cytokines, such as IL-12B, TNF-and
IL-1, were also selected because expression was associated with in vivo oxylipid profiles in
normal and diseased cows and mice [122, 164]. Murine-derived RAW 264.7 monocytes are used
to model fatty acid metabolism in some human diseases, such as sepsis [108], but the impact of
LA concentration on such models was not previously investigated. Hence the results from these
studies enhance understanding of the impact of LA on monocyte inflammatory pathways in
humans, in addition to ruminant species.

56

The second objective was to describe the profile of oxylipids produced by bovine
monocytes in response to increased cell LA concentrations. Production of 9- and 13-HODE and
9- and 13-OxoODE increased with LA concentration in primary bovine and RAW 264.7
monocytes. This suggests that monocyte production of 13-HODE may increase in periparturient
cows as cell LA concentrations increase and could, therefore, contribute to increased plasma 13HODE concentrations that were recently described in early lactation [122]. The magnitude of
production responses to LA differed between bovine and murine cells (Figures 12 and 14), but
similar LA-dose effects were seen across species. The ratio of HODE to OxoODE did not differ
by cell LA concentration in bovine primary or RAW 264.7 monocytes, and also did not change in
vivo during the periparturient period (Figure 6). Production of other oxylipids, such as HETES and
anti-inflammatory ω-3 derived oxylipids, were not influenced by monocyte LA content, and also
did not change in vivo [122]. Therefore, the oxylipid profiles in primary bovine monocyte and
murine monocyte culture media have several similar features to the periparturient plasma oxylipid
profile. However, in vitro production of some oxylipids, such as leukotrienes, may not completely
mimic in vivo production as biosynthesis of some oxylipids is dependent on monocyte-endothelial
cell interaction [105].
The oxylipid profile associated with increasing LA concentration in monocytes was further
defined by measurement of the ratio of (R) to (S)-HODE chiral isomers. Previous research showed
non-enzymatic oxidation of LA produced equal proportions of (R) and (S) isomers [186]. Hence
increased (R) / (S)-HODE ratios associated with increasing primary monocyte LA concentration
(Figure 13) could indicate changes in enzymatic oxidation. Transcript expression of COX2 and
15-LOX1 was not influenced by cell LA concentration (p > 0.05, n = 3), but protein expression
and post translational regulation were not evaluated. These experiments were designed to describe
57

the profile of oxylipids produced in response to increased monocyte availability of LA but were
not designed to investigate the specific pathway by which the metabolites were derived. Therefore,
specific enzyme pathways by which metabolites are produced in these experiments cannot be
determined.
Only production of LA-derived oxylipids changed with increasing cell LA concentration.
HODES and OxoODES both increased, but how either individual oxylipids impact the phenotype
or inflammatory responses of monocytes cannot be determined from these studies. Future studies
should investigate the individual effects of HODES and OxoODES in bovine monocytes, as
previous research suggests they have opposing effects on inflammatory phenotype that could
potentially be counteractive when present in similar concentrations [5, 83]. Future studies could,
for example, utilize purified oxylipids, such as 13-HODE, in primary bovine monocyte culture,
and assess time and dose related effects on inflammatory phenotype.
Oxylipid production in LPS-stimulated monocytes was largely similar to production in
unstimulated monocytes. However, 9-HODE production was greater in LPS-stimulated monocytes
(50 uM LA, p < 0.05, n = 3). Expression of some oxylipid producing enzymes, such as COX2, was
increased by LPS in this and previous studies [72]. COX2 is one of the enzymes that metabolizes
LA to HPODES [68], therefore could be involved with increased 9-HODE production in the LPSstimulated monocytes, although the results of these experiments are not definitive.
In conclusion, the results showed that increasing LA concentration within bovine and
murine monocytes did not impact inflammatory phenotype and did not influence LPS-stimulated
inflammatory responses. Monocyte production of 9- and 13-HODE and 9- and 13-OxoODE
increased with increasing cell LA concentration, as did the ratio of (R)-HODE / (S)-HODE chiral
58

isomers. Future studies are required to assess the specific impact of individual LA-derived
oxylipids on monocyte inflammatory pathways.
Acknowledgements
The author acknowledges the resources and staff of the Michigan State University Research
Technology Support Facility Mass Spectrometry Core, especially Drs. Dan Jones and Scott Smith.
The author also acknowledges Jeff Gandy and Dr. Edward Kabara for their assistance. This work
was funded, in part, through a grant (2012-67011-20019) from the Agriculture and Food Research
Initiative Competitive Grants Programs of the USDA National Institute for Food and Agriculture
and by an endowment from the Matilda R. Wilson Fund (Detroit, MI, USA).

59

CHAPTER 4

CONCLUSIONS

60

Plasma oxylipid profile changes across the periparturient period of dairy cattle
The objective of Chapter 4 is to describe significance of the results and new gaps in knowledge.
Chapter 2 shows that the oxylipid profile of dairy cows is dynamic across the periparturient period.
Previous studies showed that plasma content of non-esterified fatty acids (NEFA) are also dynamic
across the periparturient period [24, 31]. Changes in circulating NEFA could influence the fatty
acid pool available for oxylipid production within plasma, cells, and organs [123]. In fact,
peripheral blood mononuclear cell (PBMC) concentrations of linoleic acid (LA) are increased in
early lactation relative to prepartum concentrations [31]. Changes in polyunsaturated fatty acid
(PUFA) composition of murine monocytes impacts oxylipid production [108]. The results from
Chapter 3 indicate that a consequence of bovine monocyte enrichment with LA is increased
production of 9- and 13-hydroxyoctadecadienoic (HODE) acids. Circulating monocytes may
secrete oxylipids such as HODES in response to increased supply of LA, which could ultimately
affect plasma HODE concentrations. Previous in vitro studies also showed enhanced HODE
production by bovine vascular endothelial cells in response to a mixture of NEFA that mimicked
periparturient plasma [30]. Therefore, monocytes, endothelial cells and possibly other cell
populations should be considered as potential contributors to plasma HODES. Previous human
studies showed that plasma oxylipid substrate was influenced by dietary fatty acid composition
[120]. Although periparturient dairy cows usually experience a diet change at the onset of lactation,
the potential impact of this on plasma fatty acids and oxylipid production has not been researched.
Periparturient changes in plasma oxylipid concentration could be influenced by changes in
oxylipid enzyme expression and activity in various cell types. Previous research showed increased
15-lipoxygenase transcript expression in bovine mammary tissue in early lactation [3].
Cyclooxygenase-2 transcript and protein expression were increased in bovine vascular endothelial
61

cell culture in response to NEFA that mimicked periparturient plasma [30]. Chapter 2 showed
decreased 15-lipoxygenase and 15-hydroxyprostaglandin dehydrogenase transcript expression in
peripheral leukocytes which could impact oxylipid production and secretion into plasma. Although
enzyme activity in mammary tissue, peripheral leukocytes, vascular endothelium, and other sites,
could result in biosynthesis of oxylipids that are secreted into plasma, the relative rates of oxylipid
biosynthesis in each cell type is unclear.
Non-enzymatic peroxidation of plasma NEFA could contribute to some oxylipid changes
in the periparturient period. Previous research showed that early lactation plasma has increased
oxidative capacity [12] and increased concentration of LA [31]. Research in other species showed
that PUFA readily react with free-radicals to produce hydroperoxy-type oxylipids [187].
Therefore, non-enzymatic peroxidation of plasma LA should be investigated as a potential cause
of increased plasma HODE in early lactation. Should non-enzymatic lipid peroxidation occur in
bovine plasma or cells, the influence of antioxidant pharmaceuticals or nutraceuticals on oxylipid
production should be evaluated in periparturient cows.
Previous studies indicate that incidence and severity of disease are greatest in the
periparturient period [117, 145]. Relationships between periparturient disease and the oxylipid
profile are not known. Recent mouse studies showed that the oxylipid profile was associated with
the severity of disease after viral challenge but similar studies have not been conducted in cows
[164]. Bovine case-control or challenge studies could be useful in describing the oxylipid profile
associated with disease. Once the oxylipids that are associated with disease in cows are identified,
oxylipids could be used as markers that predict future disease incidents or trigger early intervention
strategies.

62

Plasma oxylipids are associated with leukocyte transcript
Specific components of the bovine oxylipid profile that potentially influence periparturient
disease incidence and severity are not known. Previous in vitro research showed that some
oxylipids do influence monocyte and endothelial cell inflammatory transcript and protein
expression in humans and cows [56, 83, 155]. However, the effects of specific oxylipids that
changed concentration during the periparturient period, or were associated with leukocyte
transcript expression in Chapter 2, have not been evaluated in cows. Future studies of bovine
monocytes that use controlled in vitro exposure to oxylipids will enhance understanding of the
correlations presented in Chapter 2. Having identified the specific oxylipids that are relevant to
periparturient health, subsequent research should identify how such oxylipids are produced or exert
such effects so that disease intervention strategies can be implemented. Oxylipid effects in some
human and animal diseases are abrogated by inhibitors of oxylipid receptors [92] or steroidal and
non-steroidal anti-inflammatory drugs that affect expression and activity of oxylipid producing
enzymes [109, 139, 143]. Antioxidants are used to reduce free-radical mediated production of
oxylipids [187]. Production of some oxylipids is controlled by increasing or decreasing
polyunsaturated NEFA supply [108, 118, 120]. Omega-3 NEFA supplementation, for example,
increased production of some ω-3 and ω-6 derived anti-inflammatory oxylipids in bovine
endothelial cells [29]. The same study showed that ω-3 fatty acids abrogated inflammatory
responses induced by a NEFA mixture that mimicked periparturient plasma [29]. Which
intervention strategies are utilized in periparturient cows will be determined after the biological
effects and biosynthetic pathways of the periparturient oxylipid profile have been identified.
Cellular linoleic acid concentration does not impact monocyte inflammatory phenotype

63

There are a preponderance of reviews [27, 113], in vivo [121, 134], and in vitro studies [39, 66,
166, 167, 189] that suggest increasing concentrations of LA enhanced the risk of some diseases by
exacerbating inflammatory responses. Peripheral blood mononuclear cells from early lactation
cows were recently reported to have increased LA content relative to cells from prepartum cows
[31]. Peripheral blood mononuclear cells from early lactation cows were shown to have
exacerbated inflammatory responses to lipopolysaccharide (LPS) [152]. Until now, the
relationship between increased LA and LPS-induced inflammatory responses in PBMC was
unknown. Chapter 3 shows that the bovine monocyte inflammatory cytokine response to LPS is
not associated with cellular concentration of LA. Although previous research showed proinflammatory effects of HODES in human monocytes [83], enhanced HODE production in this
study was not associated with changes in cytokine transcript. However, Chapter 3 transcript
expression must be interpreted in the context of all LA-derived metabolites. For example,
oxooctadecadienoic acid (OxoODE) production also increased in response to LA enrichment.
Human and mouse cell culture studies indicate that OxoODEs are ligands of nuclear receptors
including peroxisome proliferator activated receptor (PPAR)- [104]. Activation of PPAR- can
lead to inhibition of nuclear factor– mediated transcript responses, such as those induced by
LPS [26]. Cytokine transcript of bovine monocytes could be regulated by balanced production of
proinflammatory HODEs and anti-inflammatory OxoODEs, but further research is required to
confirm this.
Summary of strengths and limitations
Several features of the experiments are novel and advance knowledge of bovine physiology. For
example, this dissertation is the first report of quantitative assessment of PUFA in ruminant cells

64

by gas chromatography/mass spectrometry. Quantification by these methods could be superior to
qualitative assessment when samples differ in total lipid content. Total lipid in monocytes and
macrophages increases in some pathophysiological states [99], so quantitative assessment of
PUFA is important for monocyte studies, such as those in Chapter 2 and 3. A disadvantage of
quantitative assessment, compared to qualitative assessment, is that not all fatty acids are measured
[108]. Qualitative chromatography, in contrast, requires that all chromatogram peaks are identified
[127]. Disadvantages such as this can be overcome in quantitative assessment by creating
calibration curves for the complete fatty acid profile. However, these calibrations were not required
for Chapter 2 and 3 objectives.
Chapter 2 provides the first report of changes in monocyte AA concentration during the
periparturient period. Arachidonic acid produces oxylipids that were proinflammatory in bovine
endothelial cell studies [155], but oxylipid effects have not been evaluated in bovine monocytes.
Additionally, the impact of bovine monocyte AA concentration on inflammatory phenotype has
not been evaluated. AA was used in Chapter 3 to control for the non-specific effects of PUFA and
results showed that AA directly induced inflammation in monocytes. However, the significance of
these results is unclear because the in vitro AA dose required to mimic in vivo AA concentrations
was not calculated. Subsequent experiments should address potential pro-inflammatory effects of
AA at specific concentrations present in periparturient monocytes.
Chapter 2 also showed, for the first time, simultaneous changes in LA and 13-HODE, and
relationships between 13-HODE and inflammatory phenotype of monocytes from dairy cows that
successfully transitioned into lactation. Subsequent studies were conducted in vitro to identify the
potential impact of monocyte LA concentration on 13-HODE production and inflammatory
phenotype. Results showed that increasing monocyte LA concentrations resulted in increased 1365

HODE production and, therefore, it is likely that monocytes are the source of some plasma 13HODE in vivo.
Linoleic acid did not induce inflammation in bovine monocytes or modify the monocyte
inflammatory response to LPS. These results are conclusive within the context of the experimental
conditions, and how these conditions relate to in vivo conditions impacts the significance of these
results to dairy cow health. Additional in vitro modeling of the complexity of periparturient
physiology would enhance the significance of results. Reactive oxygen metabolites, for example,
are more abundant in plasma after parturition compared to prepartum [12]. Such conditions could
be mimicked in vitro by inducing oxidative stress, and could enhance understanding of LA
peroxidation by non-enzymatic means during the periparturient period. Also, the complexity of
paracrine mediated oxylipid production could be lost in the current in vitro model. Endothelialmonocyte co-culture experiments could enhance understanding of oxylipid biosynthesis in
periparturient dairy cows that is mediated by interactions between these cell types [105].

66

APPENDICES

67

Appendix 1. Authorization to publish Chapter 2 in Dissertation.

68

Appendix 2. Tables.
Table 1. Gene targets for quantitative PCR in Chapter 2.
Gene1

Reference Sequence2

TaqMan Assay ID3

IL-10
IL-12B
NOS2
IL-1
TNF-
15-LOX1
COX2
HPGD
TBP4
PGK14
ACTB4

NM_174088.1
NM_174356.1
NM_001076799.1
NM_174093.1
NM_173966.3
NM_174501.2
NM_174445.2
NM_001034419.2
NM_001075742.1
NM_001034299.1
NM_173979.3

Bt03212724_m1
Bt03213923_m1
Bt03249598_m1
Bt03212741_m1
Bt03259156_m1
Bt03214775_m1
Bt03214492_m1
Bt03226254_m1
Bt03241946_m1
Bt03225857_m1
Bt03279174_g1

NOS2 = inducible nitric oxide synthase 2; TNF-tumor necrosis factor-; 15-LOX1 = 15lipoxygenase1; COX2 = cyclooxygenase2; HPGD = 15-hydroxyprostaglandin dehydrogenase;
TBP = TATA box binding protein; PGK1 = phosphoglycerate kinase ; ACTB = beta actin
2
National Center for Biotechnology Information reference sequence
3
Life Technologies, Grand Island, NY
4
Endogenous controls
1

69

Table 2. Plasma oxylipids with stable concentration across the periparturient period (mean ± SE, pg / uL, n = 7).
Days in Milk
Oxylipid1

-14

0

10

28

84

0.02 ± 0.02
0.09 ± 0.04
0.06 ± 0.04
0.02 ± 0.02
0.06 ± 0.06
5-HETE
0.11
±
0.06
0.09
±
0.06
0.05
±
0.03
0.02
±
0.02
0.06 ± 0.06
8-HETE
0.08 ± 0.06
0.13 ± 0.07
0.06 ± 0.06
0.02 ± 0.02
0.04 ± 0.04
12-HETE
0.15 ± 0.06
0.23 ± 0.05
0.15 ± 0.05
0.23 ± 0.05
0.21 ± 0.05
15-HETE
0.19 ± 0.06
4.15 ± 0.76
2.80 ± 0.46
0.82 ± 0.24
0.33 ± 0.14
20-HETE
0.70 ± 0.32
0.17 ± 0.06
0.51 ± 0.20
0.57 ± 0.23
0.78 ± 0.13
5-OxoETE
0.12 ± 0.06
0.08 ± 0.04
0.06 ± 0.06
0.14 ± 0.03
0.06 ± 0.03
15-OxoETE
0.19 ± 0.04
0.38 ± 0.11
0.29 ± 0.05
0.17 ± 0.06
0.32 ± 0.10
PGD2
0.41 ± 0.19
0.48 ± 0.15
0.36 ± 0.08
0.32 ± 0.09
0.36 ± 0.08
PGE2
32.14 ± 18.86
23.12 ± 11.36
7.85 ± 3.64
12.04 ± 5.58
25.22 ± 15.64
PGF2
0.38 ± 0.05
0.34 ± 0.03
0.38 ± 0.05
0.34 ± 0.05
0.40 ± 0.04
6-keto-PGF1
3.28 ± 1.67
3.01 ± 1.41
1.60 ± 0.98
0.72 ± 0.37
1.07 ± 0.50
8-iso-PGF2
0.13 ± 0.05
0.13 ± 0.07
0.36 ± 0.10
0.46 ± 0.20
0.36 ± 0.15
TXB2
37.73 ± 13.66
54.84 ± 18.86
60.10 ± 20.74
62.55 ± 16.49
119.26 ± 70.29
LTD4
3.58 ± 0.41
6.04 ± 1.53
5.98 ± 1.34
4.95 ± 0.97
7.77 ± 2.03
9-OxoODE
2.44 ± 0.34
4.61 ± 1.07
3.54 ± 0.77
3.39 ± 0.85
5.16 ± 0.97
13-OxoODE
2.10 ± 0.44
2.99 ± 0.59
3.79 ± 0.40
3.87 ± 0.60
11.47 ± 4.91
PD1
2
0.11 ± 0.06
0.06 ± 0.06
0.15 ± 0.06
ND
0.29 ± 0.20
RD1
0.04 ± 0.02
0.15 ± 0.07
0.17 ± 0.05
0.17 ± 0.05
0.17 ± 0.05
RD2
1.43 ± 0.24
1.05 ± 0.20
0.70 ± 0.12
0.98 ± 0.12
1.92 ± 0.73
7-MaR1
1
HETE = hydroxyeicosatetraenoic acid; OxoETE = oxoeicosatetraenoic acid; PG = prostaglandin; TX = thromboxane; LT =
leukotriene; OxoODE = oxooctadecadienoic acid; PD = protectin D; RD = resolvin D; MaR = maresin. 2ND = not detected

70

Table 3. Correlations between plasma oxylipid concentrations and peripheral leukocyte mRNA
expression, adjusted for multiple tests.

Pearson
Oxylipid1
Gene
Coefficient
P
< 0.0001
PGE2
IL-12B
0.575
LTD4
IL-12B
0.553
0.0131
PGD2
IL-12B
0.481
0.0460
9-HODE
IL-12B
-0.524
0.0131
13-HODE
IL-12B
-0.554
0.0131
5-OxoETE
IL-12B
-0.637
<0.0001
PGF2
IL-12B
-0.66
< 0.0001
IL-12B
-0.69
< 0.0001
8-iso-PGF2
9-HODE
NOS2
0.631
< 0.0001
13-HODE
NOS2
0.629
< 0.0001
PGF2
NOS2
0.626
< 0.0001
8-iso-PGF2
NOS2
0.618
< 0.0001
5-OxoETE
NOS2
0.594
< 0.0001
15-HETE
NOS2
0.532
0.0131
TXB2
COX2
-0.565
< 0.0001
TXB2
-0.489
0.0368
IL-1
1
PG = prostaglandin; LT = leukotriene; HODE =
hydroxyoctadecadienoic acid; OxoETE = oxoeicosatetraenoic
acid; HETE = hydroxyeicosatetraenoic acid; TX =
thromboxane

71

Table 4. Gene targets for quantitative PCR in Chapter 3.

Species

Gene1

Reference Sequence2

TaqMan Assay ID3

IL-10
NM_174088.1
Bt03212724_m1
Bovine
IL-12B
NM_174356.1
Bt03213923_m1
Bovine
NM_173966.3
Bt03259156_m1
TNF-
Bovine
15-LOX1
NM_174501.2
Bt03214775_m1
Bovine
COX2
NM_174445.2
Bt03214492_m1
Bovine
4
TBP
NM_001075742.1
Bt03241946_m1
Bovine
4
PGK1
NM_001034299.1
Bt03225857_m1
Bovine
4
ACTB
NM_173979.3
Bt03279174_g1
Bovine
NM_008361.3
Mm01336189_m1
IL-1
Murine
IL-6
NM_031168.1
Mm00446190_m1
Murine
IL-10
NM_010548.2
Mm00439614_m1
Murine
IL-12B
NM_008352.2
Mm00434174_m1
Murine
NM_013693.3
Mm00443258_m1
TNF-
Murine
15-LOX1
NM_009660.3
Mm00507789_m1
Murine
COX2
NM_011198.3
Mm01307329_m1
Murine
4
GUSB
NM_010368.1
Mm00446953_m1
Murine
4
TBP
Mm00446971_m1
Murine
NM_013684.3
4
PGK1
NM_008828.2
Mm00435617_m1
Murine
1
TNF-tumor necrosis factor-; 15-LOX1 = 15-lipoxygenase1;
COX2 = cyclooxygenase2; TBP = TATA box binding protein; PGK1 =
phosphoglycerate kinase ; ACTB = beta actin; GUSB = beta
glucuronidase
2
National Center for Biotechnology Information reference sequence
3
Life Technologies, Grand Island, NY
4
Endogenous controls

72

Appendix 3. Figures.
Figure 1. Phospholipids consist of glycerol, usually esterified to a saturated long chain fatty acid
at sn-1 and to an unsaturated long chain fatty acid at sn-2, and to a phosphorylated head group at sn3.
This example is 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine.

73

Figure 2. Fatty acyl chains of phospholipids located within the membranes of cells and organelles
may be enzymatically oxidized into lipid mediators.
Examples of proinflammatory (orange), resolving (blue), and variable function (green) lipid
mediators are illustrated by fatty acid substrate (yellow) and biosynthetic enzyme. *: aspirin
acetylated; LA: linoleic acid; AA: arachidonic acid; EPA: eicosapentaenoic acid; DHA:
docosahexaenoic acid; COX: cyclooxygenase; Cyt-P450: cytochrome-P450 complex; HEPE:
hydroxyeicosapentaenoic

acid;

HETE:

hydroxyeicosatetraenoic

acid;

HODE:

hydroxyoctadecadienoic acid; LOX: lipoxygenase; LT: leukotriene; LX: lipoxin; MaR: maresin;
PG: prostaglandin; Rv: resolvin; PD: protectin; TX: thromboxane.

74

Figure 3. The utilization of long chain fatty acids for de novo phospholipid biosynthesis (Kennedy
pathway, red arrows and boxes), post-synthetic modification of phospholipids by reacylation
(Lands’ cycle, green arrow), and the metabolism of phospholipids to oxidized lipid mediators of
inflammation (orange arrow and box) in critical cells during inflammatory responses, such as
vascular endothelial cells and mononuclear leukocytes.
Origins of fatty acid substrate addressed in this review are (solid blue arrows and boxes) adipose
triacylglycerol-derived fatty acid1, fatty acid synthesized de novo2, recently absorbed dietary fatty
acid3, and phospholipid-derived fatty acid, cleaved by phospholipases then recycled for
phospholipid biosynthesis4 (broken blue arrow).

75

Figure 4. Biosynthetic pathway of major phospholipids in vascular endothelial tissue and
mononuclear leukocytes.
Metabolites are boxed, enzymes are unboxed. Note that diacylglycerol is a substrate for both
triacylglycerol and phospholipids. Key: ACSL: acyl CoA synthase (ligase); AGPAT: 1acylglycerol-3-phosphoacyltransferase; CDP-choline: cytidine-diphosphate choline; CDS:
cytidine-diphosphate

diacylglycerol

synthase;

CL:

cardiolipin;

CCT:

cytidine-

triphosphate:phosphocholine cytidylyltransferase; DG: diacylglycerol; DGAT: diacylglycerol
acyltransferase;

GPAT:

glycerol-3-phosphoacyltransferase;

PA:

phosphatidic acid;

PC:

phosphocholine; PE: phosphoethanolamine; PG: phosphoglycerol; PI: phosphoinositol; PPAP:
phosphatidic acid phosphatase; PS: phosphoserine; TG: triacylglycerol.

76

Figure 5. Plasma concentrations of arachidonic acid-derived oxylipid, 11-hydroxyeicosatetraenoic
acid (11-HETE) changed across the periparturient period. Reported as least square mean ± SE (p
< 0.05, differences labelled with different letters, n = 7).

77

Figure 6. Plasma concentration of linoleic acid-derived oxylipid, 9-hydroxyoctadecadienoic acid
(HODE) (A) and 13-HODE (B), changed in the periparturient period.
The ratios of 9-HODE to 9-oxooctadecadienoic acid (OxoODE) (C) and 13-HODE to 13-OxoODE
(D) show increased variance in the early lactation period and the ratio is higher for 9-isomers than
13- at similar stages of lactation. The ratio of 13-HODE to 13-OxoODE inverted in early lactation
(D), indicating that mean 13-HODE concentrations exceeded mean 13-OxoODE concentration in
early lactation. Reported as least square mean ± SE, p < 0.05, n = 7.

78

Figure 7. Relative (±SE) 15-lipoxygenase (LOX) 1 (A) and 15-hydroxyprostaglandin
dehydrogenase (HPGD) (B) mRNA expression from peripheral leukocytes, calibrated to
expression at 84 d (p < 0.05, n = 7).

79

Figure 8. Relative (±SE) IL-12B mRNA expression from peripheral leukocytes, calibrated to
expression at 84 d (p < 0.05, n = 7).

80

Figure 9. Linoleic (C18:2 n6, ▲) and arachidonic acid (C20:4 n6, ○) concentrations in peripheral
blood mononuclear cells increased in early lactation relative to prepartum samples, while
eicosapentaenoic (C20:5 n3, ◊) acid concentrations were unchanged.

Reported as least square mean ± SE, p < 0.05, n = 7.

81

Figure 10. Linoleic acid methyl ester concentration in peripheral blood mononuclear cells at 10 d
postpartum (in vivo, n = 7) versus cells from mid-lactation cows cultured in vitro (n = 6) for 12 h
in media supplemented with various concentrations of linoleic acid.
Letters indicate significant (p < 0.05) differences between cells by culture conditions and *
indicates significant (p < 0.05) difference between in vitro and in vivo cell concentrations.

82

Figure 11. Transcript expression of tumor necrosis factor (TNF)- in primary bovine monocytes
(A) and IL-1 in murine RAW 264.7 monocytes (B) after 16 h incubation in media supplemented
with various concentrations of linoleic acid (LA).
The effect of the fatty acid vehicle (ethanol, EtOH) and an alternative omega-6 fatty acid
(arachidonic acid, AA) were also evaluated (p < 0.05, n = 3).

83

Figure 12. Fold changes in primary bovine monocyte culture media concentrations of 9hydroxyoctadecadienoic (HODE, A), 13-HODE (B), 9-oxooctadecadienoic acid (OxoODE, C),
and 13-OxoODE (D).
Reference groups are cells cultured in 0 uM linoleic acid (LA) and no ethanol (EtOH). Increased
cellular concentrations of LA were associated with increased production of 9- and 13-HODE and
9- and 13-OxoODE (n = 3, p < 0.05). The effect of the fatty acid vehicle (EtOH) and an alternative
omega-6 fatty acid (arachidonic acid, AA) were also evaluated.

84

Figure 13. Primary bovine monocyte culture media concentrations of (R)-hydroxyoctadecadienoic
acid ((R)-HODE, Fig. 13A) and (S)-HODE (Fig. 13B), and the ratio of (R)-HODE / (S)-HODE
(Fig. 13C).
Production of (R)- and (S)-HODE isomers, and the (R)-HODE / (S)-HODE ratio all increased with
cellular LA concentration (n = 3, p < 0.05).

85

Figure 14. Fold changes in murine RAW 264.7 monocyte culture media concentrations of 9hydroxyoctadecadienoic (HODE, A), 13-HODE (B), 9-oxooctadecadienoic acid (OxoODE, C),
and 13-OxoODE (D).
Reference groups are cells cultured in 0 uM linoleic acid (LA) and no ethanol (EtOH). Increased
cellular concentrations of LA were associated with increased production of 9- and 13-HODE and
9-OxoODE (n = 3, p < 0.05). The effect of the fatty acid vehicle (EtOH) was also evaluated.

86

Figure 15. Ratio of 9-hydroxyoctadecadienoic (HODE) / 9-oxooctadecadienoic (OxoODE) acids
in lipopolysaccharide stimulated (25 ng / mL) primary monocyte cultures supplemented with
various concentrations of linoleic acid (LA). LA was delivered in ethanol (EtOH) (n = 3, p < 0.05).

87

Appendix 4. Supplementary Figures.
Figure 16. Unidentified compound in lipid extracts of RAW 264.7 monocytes elutes at identical
retention time as pure docosahexaenoic acid methyl ester (C22:6 n3 FAME) standard.
(A). Mass spectra of pure C22:6 n3 FAME standard (Sigma-Aldrich, St. Louis, MO). Gas
chromatograph/mass spectrometry methods are described in Chapter 2.
(B). Gas chromatogram of RAW 264.7 cell lipid extract at retention time of pure standard in (A).
Extractions methods are described in Chapter 1.
(C). Mass spectra of chromatogram peak in (B).

88

Figure 17. Linoleic acid (LA, C18:2 n6) methyl ester abundance in solvent controls (n = 3, open
bars) and RAW 264.7 cell lipid extracts (n = 3, cross hashed bars) spiked with 50 ug LA fatty acid
(Sigma-Aldrich, St. Louis, MO) either at start of experiment or between lipid extraction and
fractionation of lipid types. Other samples were spiked with 50 ug LA methyl ester (SigmaAldrich) at end of experiment.
Lipid extraction and methylation methods are similar to Chapter 2, except these samples were not
saponified prior to methylation. Fractionation utilized 500 mg aminopropyl cartridges (Strata NH2,
Phenomenex, Torrance, CA) and methods by Ruiz et al., (2004) [132]. Abundance is reported as
gas chromatogram peak area.

89

Figure 18. Decreased 15-lipoxygenase1 (15-LOX1) and tumor necrosis factor (TNF)-α mRNA
expression in lipopolysaccharide stimulated primary bovine monocytes associated with increased
concentrations of fatty acid free-bovine serum albumin (BSA).
(A) 15-LOX1 mRNA expression in lipopolysaccharide (LPS)-stimulated cells versus
supplemental BSA concentration. Results reported relative to unstimulated cells (closed
bar). Cells were cultured for 17 hours in 1XRPMI, with 10% fetal bovine serum and 0.1
uM sodium selenite. LPS utilized at 25 or 33 ng / mL media during last 4 hours of culture.
qPCR methods are described in Chapter 2 (n = 2, 0 and 1.67 uM BSA; n = 1, 0.11 to 1.25
uM BSA).
(B) Similar data for TNF-α expression.

90

Figure 19. Decreased antioxidant potential induced by 2,2′-azobis (2-methylpropionamidine)
dihydrochloride (AAPH, Sigma-Aldrich, St. Louis, MO) in RAW 264.7 monocytes.
Cells were cultured in 1XRPMI with 5% fetal bovine serum and 50 uM linoleic acid (LA, C18:2
n6, Sigma-Aldrich, St. Louis, MO, 0.01% ethanol) for 12 hours. AAPH was then added to the
original media or replacement 0 uM LA media then cells were cultured for an additional 18 h.
Kdo2-lipidA (100 ng / mL, Avanti Polar Lipids, Alabaster, AL) was added to media for the last 1
hour of culture (n = 3, p < 0.05). Antioxidant potential was measured as previously reported [124].

91

Figure 20. Induction of 9-hydroxyoctadecadienoic acid (9-HODE) production by 2,2′-azobis (2methylpropionamidine) dihydrochloride (AAPH, Sigma-Aldrich, St. Louis, MO) in RAW 264.7
monocytes.
Cells were cultured in 1XRPMI with 5% fetal bovine serum and 50 uM linoleic acid (LA, C18:2
n6, Sigma-Aldrich, St. Louis, MO, 0.01% ethanol) for 12 hours. AAPH was then added to the
original media or replacement 0 uM LA media then cells were cultured for an additional 18 h.
Kdo2-lipidA (100 ng / mL, Avanti Polar Lipids, Alabaster, AL) was added to media for the last 1
hour of culture (n = 3, p < 0.05). 9-HODE was measured by liquid chromatography/mass
spectrometry, as described in Chapter 2.

92

Figure 21. Viability is decreased by 2,2′-azobis (2-methylpropionamidine) dihydrochloride
(AAPH, Sigma-Aldrich, St. Louis, MO) in RAW 264.7 monocytes.
(A) Cells were cultured in 1XRPMI with 5% fetal bovine serum and 50 uM linoleic acid (LA,
C18:2 n6, Sigma-Aldrich, St. Louis, MO, 0.01% ethanol) for 12 hours. AAPH was then
added to the original media or replacement 0 uM LA media then cells were cultured for an
additional 18 h. Kdo2-lipidA (100 ng / mL, Avanti Polar Lipids, Alabaster, AL) was added
to media for the last 1 hour of culture (n = 3, p < 0.05). Cell viability was measured by the
CellTiter-Glo assay for adenosine triphosphate (Promega, Madison, WI).
(B) Similar data for APPH culture times of 10 to 16 h.

93

Figure 22. Linoleic acid does not affect IL-1 (A) or IL-10 (B) transcript induction by 2,2′-azobis
(2-methylpropionamidine) dihydrochloride (AAPH, Sigma-Aldrich, St. Louis, MO) in RAW
264.7 monocytes.
Cells were cultured in 1XRPMI with 5% fetal bovine serum and supplemented with various
concentrations of linoleic acid (LA, C18:2 n6, Sigma-Aldrich, St. Louis, MO, 0.01% ethanol), 50
uM arachidonic acid (AA, C20:4 n6, Sigma-Aldrich, 0.01% ethanol) or ethanol for 12 hours.
AAPH was then added to the original media and cells were cultured for an additional 12 h. Kdo2lipidA (100 ng / mL, Avanti Polar Lipids, Alabaster, AL) was added to media for the last 1 hour
of culture (n = 3, p < 0.05). qPCR methods are described in Chapter 2.

94

REFERENCES

95

REFERENCES

[1]
Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after
myocardial infarction: results of the GISSI-Prevenzione trial. Gruppo Italiano per lo Studio della
Sopravvivenza nell'Infarto miocardico. Lancet 354:447-455; 1999.
[2]
The LIPID MAPS–Nature Lipidomics Gateway. Last accessed March 1, 2012.
http://www.lipidmaps.org
[3]
Aitken, S. L.; Karcher, E. L.; Rezamand, P.; Gandy, J. C.; VandeHaar, M. J.; Capuco, A.
V.; Sordillo, L. M. Evaluation of antioxidant and proinflammatory gene expression in bovine
mammary tissue during the periparturient period. J Dairy Sci 92:589-598; 2009.
[4]
Akbar, H.; Schmitt, E.; Ballou, M. A.; Corrêa, M. N.; DePeters, E. J.; Loor, J. J. Dietary
lipid during late-pregnancy and early-lactation to manipulate metabolic and inflammatory gene
network expression in dairy cattle liver with a focus on PPARs. Gene Regul Syst Bio 7:103-123;
2013.
[5]
Altmann, R.; Hausmann, M.; Spöttl, T.; Gruber, M.; Bull, A. W.; Menzel, K.; Vogl, D.;
Herfarth, H.; Schölmerich, J.; Falk, W.; Rogler, G. 13-Oxo-ODE is an endogenous ligand for
PPARγ in human colonic epithelial cells. Biochem Pharmacol 74:612-622; 2007.
[6]
Andreyev, A. Y.; Fahy, E.; Guan, Z.; Kelly, S.; Li, X.; McDonald, J. G.; Milne, S.; Myers,
D.; Park, H.; Ryan, A.; Thompson, B. M.; Wang, E.; Zhao, Y.; Brown, H. A.; Merrill, A. H.; Raetz,
C. R. H.; Russell, D. W.; Subramaniam, S.; Dennis, E. A. Subcellular organelle lipidomics in TLR4-activated macrophages. J Lipid Res 51:2785-2797; 2010.
[7]
Arterburn, L. M.; Hall, E. B.; Oken, H. Distribution, interconversion, and dose response of
n-3 fatty acids in humans. Am J Clin Nutr 83:1467S-1476S; 2006.
[8]
Bang, H. O.; Dyerberg, J.; Hjørne, N. The Composition of Food Consumed by Greenland
Eskimos. Acta Med Scand 200:69-73; 1976.
[9]
Bauman, D. E.; Harvatine, K. J.; Lock, A. L. Nutrigenomics, Rumen-Derived Bioactive
Fatty Acids, and the Regulation of Milk Fat Synthesis. Annu Rev Nutr 31:299-319; 2011.
[10] Bell, J. C. Expression and regulation of human cytochrome P450 4F isoforms in tissue
samples and under TNF-alpha challenges. Graduate School of Biomedical Sciences. Houston:
University of Texas; 2011.
96

[11] Bell, J. C.; Strobel, H. W. Regulation of cytochrome P450 4F11 by nuclear transcription
factor-κB. Drug Metab Dispos 40:205-211; 2012.
[12] Bernabucci, U.; Ronchi, B.; Lacetera, N.; Nardone, A. Influence of body condition score
on relationships between metabolic status and oxidative stress in periparturient dairy cows. J Dairy
Sci 88:2017-2026; 2005.
[13] Bionaz, M.; Loor, J. J. ACSL1, AGPAT6, FABP3, LPIN1, and SLC27A6 are the most
abundant isoforms in bovine mammary tissue and their expression is affected by stage of lactation.
J Nutr 138:1019-1024; 2008.
[14] Bladergroen, B. A.; Wensing, T.; Van Golde, L. M. G.; Geelen, M. J. H. Reversible
translocation of CTP:phosphocholine cytidylyltransferase from cytosol to membranes in the adult
bovine liver around parturition. Biochim Biophys Acta - Lipids and Lipid Metabolism 1391:233240; 1998.
[15] Blasbalg, T. L.; Hibbeln, J. R.; Ramsden, C. E.; Majchrzak, S. F.; Rawlings, R. R. Changes
in consumption of omega-3 and omega-6 fatty acids in the United States during the 20th century.
Am J Clin Nutr 93:950-962; 2011.
[16] Bligh, E. G.; Dyer, W. J. A rapid method of total lipid extraction and purification. Can J
Biochem Physiol 37:911-917; 1959.
[17] Bosch, J.; Gerstein, H. C.; Dagenais, G. R.; Diaz, R.; Dyal, L.; Jung, H.; Maggiono, A. P.;
Probstfield, J.; Ramachandran, A.; Riddle, M. C.; Ryden, L. E.; Yusuf, S. n-3 fatty acids and
cardiovascular outcomes in patients with dysglycemia. N Engl J Med 367:309-318; 2012.
[18] Brinckmann, R.; Schnurr, K.; Heydeck, D.; Rosenbach, T.; Kolde, G.; Kühn, H. Membrane
translocation of 15-lipoxygenase in hematopoietic cells is calcium-dependent and activates the
oxygenase activity of the enzyme. Blood 91:64-74; 1998.
[19] Bruss, M. L. Lipids and ketones. In: Kaneko, J. J.; Harvey, J. W.; Bruss, M. L., eds. Clinical
Biochemistry of Domestic Animals. St. Louis: Elsevier; 2008: 81-115.
[20] Buckley, T. L.; Van de Velde, M. J.; Henricks, P. A.; Engels, F.; Nijkamp, F. P. 13hydroxyoctadecadienoic acid attenuates oedema formation induced by leukotriene B4 in vivo in
rabbit skin. Eur J Pharmacol 218:369-372; 1992.

97

[21] Calder, P. C. n–3 Polyunsaturated fatty acids, inflammation, and inflammatory diseases.
Am J Clin Nutr 83:S1505-S1519; 2006.
[22] Calder, P. C. Fatty acids and inflammation: The cutting edge between food and pharma.
Eur J Pharmacol 668:S50-S58; 2011.
[23] Calder, P. C.; Albers, R.; Antoine, J. M.; Blum, S.; Bourdet-Sicard, R.; Ferns, G.; Folkerts,
G.; Friedmann, P.; Frost, G.; Guarner, F. Inflammatory disease processes and interactions with
nutrition. Br J Nutr 101:1-45; 2009.
[24] Cameron, R. E.; Dyk, P. B.; Herdt, T. H.; Kaneene, J. B.; Miller, R.; Bucholtz, H. F.;
Liesman, J. S.; Vandehaar, M. J.; Emery, R. S. Dry cow diet, management, and energy balance as
risk factors for displaced abomasum in high producing dairy herds. J Dairy Sci 81:132-139; 1998.
[25] Cheng, Y.; Austin, S. C.; Rocca, B.; Koller, B. H.; Coffman, T. M.; Grosser, T.; Lawson,
J. A.; FitzGerald, G. A. Role of Prostacyclin in the Cardiovascular Response to Thromboxane A2.
Science 296:539-541; 2002.
[26] Chinetti, G.; Fruchart, J. C.; Staels, B. Peroxisome proliferator-activated receptors
(PPARs): Nuclear receptors at the crossroads between lipid metabolism and inflammation.
Inflamm Res 49:497-505; 2000.
[27] Choque, B.; Catheline, D.; Rioux, V.; Legrand, P. Linoleic acid: Between doubts and
certainties. Biochimie 96:14-21; 2014.
[28] Coleman, R. A.; Lewin, T. M.; Van Horn, C. G.; Gonzalez-Baró, M. R. Do long-chain
acyl-CoA synthetases regulate fatty acid entry into synthetic versus degradative pathways? J Nutr
132:2123-2126; 2002.
[29] Contreras, G.; Mattmiller, S.; Raphael, W.; Gandy, J.; Sordillo, L. Enhanced n-3
phospholipid content reduces inflammatory responses in bovine endothelial cells. J Dairy Sci
95:7137-7150; 2012.
[30] Contreras, G.; Raphael, W.; Mattmiller, S.; Gandy, J.; Sordillo, L. Nonesterified fatty acids
modify inflammatory response and eicosanoid biosynthesis in bovine endothelial cells. J Dairy Sci
95:5011-5023; 2012.

98

[31] Contreras, G. A.; O'Boyle, N. J.; Herdt, T. H.; Sordillo, L. M. Lipomobilization in
periparturient dairy cows influences the composition of plasma nonesterified fatty acids and
leukocyte phospholipid fatty acids. J Dairy Sci 93:2508-2516; 2010.
[32] Contreras, G. A.; Sordillo, L. M. Lipid mobilization and inflammatory responses during
the transition period of dairy cows. Comp Immunol Microbiol Infect Dis 34:281-289; 2011.
[33] Cordain, L.; Eaton, S. B.; Sebastian, A.; Mann, N.; Lindeberg, S.; Watkins, B. A.; O’Keefe,
J. H.; Brand-Miller, J. Origins and evolution of the Western diet: health implications for the 21st
century. Am J Clin Nutr 81:341-354; 2005.
[34] Corl, C.; Gandy, J.; Sordillo, L. Platelet activating factor production and proinflammatory
gene expression in endotoxin-challenged bovine mammary endothelial cells. J Dairy Sci 91:30673078; 2008.
[35] Cyrus, T.; Witztum, J. L.; Rader, D. J.; Tangirala, R.; Fazio, S.; Linton, M.; Funk, C. D.
Disruption of the 12/15-lipoxygenase gene diminishes atherosclerosis in apo E-deficient mice. J
Clin Invest 103:1597-1604; 1999.
[36] de Mello, V. D. F.; Kolehmanien, M.; Schwab, U.; Pulkkinen, L.; Uusitupa, M. Gene
expression of peripheral blood mononuclear cells as a tool in dietary intervention studies: What do
we know so far? Mol Nutr Food Res 56:1160-1172; 2012.
[37] De Vliegher, S.; Fox, L. K.; Piepers, S.; McDougall, S.; Barkema, H. W. Invited review:
Mastitis in dairy heifers: Nature of the disease, potential impact, prevention, and control. J Dairy
Sci 95:1025-1040; 2012.
[38] Delion, S.; Chalon, S.; Hérault, J.; Guilloteau, D.; Besnard, J. C.; Durand, G. Chronic
dietary alpha-linolenic acid deficiency alters dopaminergic and serotoninergic neurotransmission
in rats. J Nutr 124:2466-2476; 1994.
[39] Dichtl, W.; Ares, M. P. S. Linoleic acid-stimulated vascular adhesion molecule-1
expression in endothelial cells depends on nuclear factor-[kappa] B activation* 1. Metabolism
51:327-333; 2002.
[40] Dinarello, C. A. Review: Infection, fever, and exogenous and endogenous pyrogens: some
concepts have changed. J Endotoxin Res 10:201-222; 2004.

99

[41] Dinarello, C. A. A clinical perspective of IL-1β as the gatekeeper of inflammation. Eur J
Immunol 41:1203-1217; 2011.
[42] Douglas, G.; Rehage, J.; Beaulieu, A.; Bahaa, A.; Drackley, J. Prepartum nutrition alters
fatty acid composition in plasma, adipose tissue, and liver lipids of periparturient dairy cows. J
Dairy Sci 90:2941-2959; 2007.
[43] Dowhan, W.; Bogdanov, M.; Mileykovskaya, E. Functional roles of lipids in membranes.
In: Vance, D. E.; Vance, J. E., eds. Biochemistry of Lipids, Lipoproteins and Membranes. St. Louis,
MO, USA: Elsevier; 2008: 1-38.
[44] Dubois, R. N.; Abramson, S. B.; Crofford, L.; Gupta, R. A.; Simon, L. S.; A. Van De Putte,
L. B.; Lipsky, P. E. Cyclooxygenase in biology and disease. FASEB J 12:1063-1073; 1998.
[45] Dubuc, J.; Duffield, T. F.; Leslie, K. E.; Walton, J. S.; LeBlanc, S. J. Effects of postpartum
uterine diseases on milk production and culling in dairy cows. J Dairy Sci 94:1339-1346; 2011.
[46] Dumlao, D. S.; Buczynski, M. W.; Norris, P. C.; Harkewicz, R.; Dennis, E. A. Highthroughput lipidomic analysis of fatty acid derived eicosanoids and N-acylethanolamines. Biochim
Biophys Acta - Molecular and Cell Biology of Lipids 1811:724-736; 2011.
[47] Dussault, P.; Lee, I. Q. The total synthesis of 15 (S)-HPETE (hydroperoxyeicosatetraenoic
acid). J Org Chem 57:1952-1954; 1992.
[48] Dwarakanath, R. S.; Sahar, S.; Reddy, M. A.; Castanotto, D.; Rossi, J. J.; Natarajan, R.
Regulation of monocyte chemoattractant protein-1 by the oxidized lipid, 13hydroperoxyoctadecadienoic acid, in vascular smooth muscle cells via nuclear factor-kappa B
(NF-kB). J Mol Cell Cardiol 36:585-595; 2004.
[49] Edwards, I. J.; Flaherty, J. T. Omega-3 Fatty Acids and PPARg in Cancer. PPAR Res
2008:358052:358051–358052:358014; 2008.
[50] Elias, P. M.; Brown, B. E.; Ziboh, V. A. The permeability barrier in essential fatty acid
deficiency: evidence for a direct role for linoleic acid in barrier function. J Invest Dermatol 74:230233; 1980.
[51] Emken, E. A.; Adlof, R. O.; Gulley, R. M. Dietary linoleic acid influences desaturation and
acylation of deuterium-labeled linoleic and linolenic acids in young adult males. Biochim Biophys
Acta - Lipids and Lipid Metabolism 1213:277-288; 1994.
100

[52] Faour, W. H.; Alaaeddine, N.; Mancini, A.; He, Q. W.; Jovanovic, D.; Di Battista, J. A.
Early Growth Response Factor-1 Mediates Prostaglandin E2-dependent Transcriptional
Suppression of Cytokine-induced Tumor Necrosis Factor-α Gene Expression in Human
Macrophages and Rheumatoid Arthritis-affected Synovial Fibroblasts. J Biol Chem 280:95369546; 2005.
[53] Farney, J. K.; Mamedova, L. K.; Coetzee, J. F.; KuKanich, B.; Sordillo, L. M.; Stoakes, S.
K.; Minton, J. E.; Hollis, L. C.; Bradford, B. J. Anti-inflammatory salicylate treatment alters the
metabolic adaptations to lactation in dairy cattle. Am J Physiol Regul Integr Comp Physiol DOI:
10.1152/ajpregu.00152.2013; 2013.
[54]

Fitzgerald, G. A. Coxibs and cardiovascular disease. N Engl J Med 351:1709-1711; 2004.

[55] Folch, J.; Lees, M.; Sloane-Stanley, G. A simple method for the isolation and purification
of total lipids from animal tissues. J Biol Chem 226:497-509; 1957.
[56] Friedrichs, B.; Toborek, M.; Hennig, B.; Heinevetter, L.; Müller, C.; Brigelius Flohé, R.
13 HPODE and 13 HODE modulate cytokine induced expression of endothelial cell adhesion
molecules differently. Biofactors 9:61-72; 1999.
[57] Galli, C.; Risé, P. Fish Consumption, Omega 3 Fatty Acids and Cardiovascular Disease.
The Science and the Clinical Trials. Nutr Health 20:11-20; 2009.
[58] Galvao, K. N.; Felippe, M. J.; Brittin, S. B.; Sper, R.; Fraga, M.; Galvao, J. S.; Caixeta, L.;
Guard, C. L.; Ricci, A.; Gilbert, R. O. Evaluation of cytokine expression by blood monocytes of
lactating Holstein cows with or without postpartum uterine disease. Theriogenology 77:356-372;
2012.
[59] Galvão, K. N.; Flaminio, M. J. B. F.; Brittin, S. B.; Sper, R.; Fraga, M.; Caixeta, L.; Ricci,
A.; Guard, C. L.; Butler, W. R.; Gilbert, R. O. Association between uterine disease and indicators
of neutrophil and systemic energy status in lactating Holstein cows. J Dairy Sci 93:2926-2937;
2010.
[60] Gertow, K.; Nobili, E.; Folkersen, L.; Newman, J. W.; Pedersen, T. L.; Ekstrand, J.;
Swedenborg, J.; Kuhn, H.; Wheelock, C. E.; Hansson, G. K.; Hedin, U.; Haeggstrom, J. Z.;
Gabrielsen, A. 12- and 15-lipoxygenases in human carotid atherosclerotic lesions: Associations
with cerebrovascular symptoms. Atherosclerosis 215:411-416; 2011.

101

[61] Guillou, H.; Zadravec, D.; Martin, P. G. P.; Jacobsson, A. The key roles of elongases and
desaturases in mammalian fatty acid metabolism: Insights from transgenic mice. Prog Lipid Res
49:186-199; 2010.
[62] Hamberg, M.; Samuelsson, B. Stereochemistry in the formation of 9-hydroxy-10,12octadecadienoic acid and 13-hydroxy-9,11-octadecadienoic acid from linoleic acid by fatty acid
cyclooxygenase. Biochim Biophys Acta - Lipids and Lipid Metabolism 617:545-547; 1980.
[63] Hardardottir, I.; Kinsella, J. E. Tumor necrosis factor production by murine resident
peritoneal macrophages is enhanced by dietary n-3 polyunsaturated fatty acids. Biochim Biophys
Acta 1095:187-195; 1991.
[64] Harris, W. S.; Poston, W. C.; Haddock, C. K. Tissue n−3 and n−6 fatty acids and risk for
coronary heart disease events. Atherosclerosis 193:1-10; 2007.
[65] Healy, D. A.; Wallace, F. A.; Miles, E. A.; Calder, P. C.; Newsholme, P. Effect of low-tomoderate amounts of dietary fish oil on neutrophil lipid composition and function. Lipids 35:763768; 2000.
[66] Hennig, B.; Toborek, M.; Joshi-Barve, S.; Barger, S. W.; Barve, S.; Mattson, M. P.;
McClain, C. J. Linoleic acid activates nuclear transcription factor-kappa B (NF-kappa B) and
induces NF-kappa B-dependent transcription in cultured endothelial cells. Am J Clin Nutr 63:322328; 1996.
[67] Hodson, L.; Skeaff, C. M.; Fielding, B. A. Fatty acid composition of adipose tissue and
blood in humans and its use as a biomarker of dietary intake. Prog Lipid Res 47:348-380; 2008.
[68] Hoffmann, I.; Hamberg, M.; Lindh, R.; Oliw, E. H. Novel insights into cyclooxygenases,
linoleate diol synthases, and lipoxygenases from deuterium kinetic isotope effects and oxidation
of substrate analogs. Biochim Biophys Acta - Molecular and Cell Biology of Lipids 1821:15081517; 2012.
[69] Hung, N. D.; Kim, M. R.; Sok, D. E. Oral administration of 2-docosahexaenoyl
lysophosphatidylcholine displayed anti-inflammatory effects on zymosan A-induced peritonitis.
Inflammation 34:147-160; 2011.
[70] Huo, Y.; Zhao, L.; Hyman, M. C.; Shashkin, P.; Harry, B. L.; Burcin, T.; Forlow, S. B.;
Stark, M. A.; Smith, D. F.; Clarke, S.; Srinivasan, S.; Hedrick, C. C.; Pratico, D.; Witztum, J. L.;
Nadler, J. L.; Funk, C. D.; Ley, K. Critical role of macrophage 12/15-lipoxygenase for
atherosclerosis in apolipoprotein E-deficient mice. Circulation 110:2024-2031; 2004.
102

[71] Hutchins, P. M.; Moore, E. E.; Murphy, R. C. Electrospray MS/MS reveals extensive and
nonspecific oxidation of cholesterol esters in human peripheral vascular lesions. J Lipid Res
52:2070-2083; 2011.
[72] Hwang, D.; Jang, B. C.; Yu, G.; Boudreau, M. Expression of mitogen-inducible
cyclooxygenase induced by lipopolysaccharide: Mediation through both mitogen-activated protein
kinase and nf-kb signaling pathways in macrophages. Biochem Pharmacol 54:87-96; 1997.
[73] Iwasaki, Y.; Yamane, T. Enzymatic synthesis of structured lipids. Adv Biochem Eng
Biotechnol 90:760-760; 2004.
[74] Johnson, G. H.; Fritsche, K. Effect of Dietary Linoleic Acid on Markers of Inflammation
in Healthy Persons: A Systematic Review of Randomized Controlled Trials. J Acad Nutr Diet
112:1029-1041.e1015; 2012.
[75] Kanter, J. E.; Kramer, F.; Barnhart, S.; Averill, M. M.; Vivekanandan-Giri, A.; Vickery,
T.; Li, L. O.; Becker, L.; Yuan, W.; Chait, A.; Braun, K. R.; Potter-Perigo, S.; Sanda, S.; Wight,
T. N.; Pennathur, S.; Serhan, C. N.; Heinecke, J. W.; Coleman, R. A.; Bornfeldt, K. E. Diabetes
promotes an inflammatory macrophage phenotype and atherosclerosis through acyl-CoA
synthetase 1. Proc Natl Acad Sci U S A 109:E715-E724; 2012.
[76] Kaplan, A.; Lee, V. F. Serum lipid levels in infants and mothers at parturition. Clin Chim
Acta 12:258-263; 1965.
[77] Kaufmann, T. B.; Drillich, M.; Tenhagen, B. A.; Heuwieser, W. Correlations between
periparturient serum concentrations of non-esterified fatty acids, beta-hydroxybutyric acid,
bilirubin, and urea and the occurrence of clinical and subclinical postpartum bovine endometritis.
BMC Vet Res 6:47; 2010.
[78] Kelley, D. S.; Taylor, P. C.; Nelson, G. J.; Mackey, B. E. Arachidonic acid supplementation
enhances synthesis of eicosanoids without suppressing immune functions in young healthy men.
Lipids 33:125-130; 1998.
[79] Kennedy, E. P.; Weiss, S. B. The function of cytidine coenzymes in the biosynthesis of
phospholipides. J Biol Chem 222:193-214; 1956.
[80] Kim, F.; Pham, M.; Luttrell, I.; Bannerman, D. D.; Tupper, J.; Thaler, J.; Hawn, T. R.;
Raines, E. W.; Schwartz, M. W. Toll-like receptor-4 mediates vascular inflammation and insulin
resistance in diet-induced obesity. Circ Res 100:1589-1596; 2007.
103

[81] Kitessa, S. M.; Gulati, S. K.; Ashes, J. R.; Fleck, E.; Scott, T. W.; Nichols, P. D. Utilisation
of fish oil in ruminants: II. Transfer of fish oil fatty acids into goats’ milk. Anim Feed Sci Technol
89:201-208; 2001.
[82] Kris-Etherton, P. M.; Harris, W. S.; Appel, L. J. Fish Consumption, Fish Oil, Omega-3
Fatty Acids, and Cardiovascular Disease. Arterioscler Thromb Vasc Biol 23:e20-e30; 2003.
[83] Ku, G.; Thomas, C. E.; Akeson, A. L.; Jackson, R. L. Induction of interleukin 1 beta
expression from human peripheral blood monocyte-derived macrophages by 9hydroxyoctadecadienoic acid. J Biol Chem 267:14183; 1992.
[84] Kuhn, H.; Barnett, J.; Grunberger, D.; Baecker, P.; Chow, J.; Nguyen, B.; BursztynPettegrew, H.; Chan, H.; Sigal, E. Overexpression, purification and characterization of human
recombinant 15-lipoxygenase. Biochim Biophys Acta - Lipids and Lipid Metabolism 1169:80-89;
1993.
[85] Kühn, H.; Belkner, J.; Suzuki, H.; Yamamoto, S. Oxidative modification of human
lipoproteins by lipoxygenases of different positional specificities. J Lipid Res 35:1749; 1994.
[86] Kuhn, H.; Thiele, B. J. The diversity of the lipoxygenase family: Many sequence data but
little information on biological significance. FEBS Lett 449:7-11; 1999.
[87] Lacetera, N.; Franci, O.; Scalia, D.; Bernabucci, U.; Ronchi, B.; Nardone, A. Effects on
functions of ovine blood mononuclear cells for each of several fatty acids at concentrations found
in plasma of healthy and ketotic ewes. Am J Vet Res 63:958-962; 2002.
[88] Lands, W. E. Metabolism of glycerolipides; a comparison of lecithin and triglyceride
synthesis. J Biol Chem 231:883-888; 1958.
[89] Lawrence, T.; Gilroy, D. W. Chronic inflammation: a failure of resolution? Int J Exp Pathol
88:85-94; 2007.
[90] Lee, J.; McCracken, J. A.; Banu, S. K.; Arosh, J. A. Intrauterine inhibition of prostaglandin
transporter protein blocks release of luteolytic PGF2 pulses without suppressing endometrial
expression of estradiol or oxytocin receptor in ruminants. Biol Reprod
10.1095/biolreprod.112.106427; 2013.

104

[91] Lee, J. Y.; Sohn, K. H.; Rhee, S. H.; Hwang, D. Saturated fatty acids, but not unsaturated
fatty acids, induce the expression of cyclooxygenase-2 mediated through Toll-like receptor 4. J
Biol Chem 276:16683-16689; 2001.
[92] Leff, J. A.; Busse, W. W.; Pearlman, D.; Bronsky, E. A.; Kemp, J.; Hendeles, L.; Dockhorn,
R.; Kundu, S.; Zhang, J.; Seidenberg, B. C.; Reiss, T. F. Montelukast, a Leukotriene-Receptor
Antagonist, for the Treatment of Mild Asthma and Exercise-Induced Bronchoconstriction. N Engl
J Med 339:147-152; 1998.
[93] Levy, B. D.; Bonnans, C.; Silverman, E. S.; Palmer, L. J.; Marigowda, G.; Israel, E.
Diminished Lipoxin Biosynthesis in Severe Asthma. Am J Respir Crit Care Med 172:824-830;
2005.
[94] Li, X.; Yu, Y.; Funk, C. D. Cyclooxygenase-2 induction in macrophages is modulated by
docosahexaenoic acid via interactions with free fatty acid receptor 4 (FFA4). FASEB J; 2013.
[95] Liu, Q.; Siloto, R. M. P.; Lehner, R.; Stone, S. J.; Weselake, R. J. Acyl-CoA:diacylglycerol
acyltransferase: Molecular biology, biochemistry and biotechnology. Prog Lipid Res 51:350-377;
2012.
[96] Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using real-time
quantitative PCR and the 2-CT method. Methods 25:402-408; 2001.
[97]

Loewen, C. J. R. Lipids as conductors in the orchestra of life. F1000 Biol Rep 4; 2012.

[98] Loor, J. J.; Dann, H. M.; Everts, R. E.; Oliveira, R.; Green, C. A.; Guretzky, N. A. J.;
Rodriguez-Zas, S. L.; Lewin, H. A.; Drackley, J. K. Temporal gene expression profiling of liver
from periparturient dairy cows reveals complex adaptive mechanisms in hepatic function. Physiol
Genomics 23:217-226; 2005.
[99] McLaren, J. E.; Michael, D. R.; Ashlin, T. G.; Ramji, D. P. Cytokines, macrophage lipid
metabolism and foam cells: Implications for cardiovascular disease therapy. Prog Lipid Res
50:331-347; 2011.
[100] Mouradian, M.; Kikawa, K. D.; Johnson, E. D.; Beck, K. L.; Pardini, R. S. Key roles for
GRB2-associated-binding protein 1, phosphatidylinositol-3-kinase, cyclooxygenase 2,
prostaglandin E2 and transforming growth factor alpha in linoleic acid-induced upregulation of
lung and breast cancer cell growth. Prostaglandins Leukot Essent Fatty Acids 90:105-115; 2014.

105

[101] Moyes, K.; Drackley, J.; Morin, D.; Loor, J. Greater expression of TLR2, TLR4, and IL6
due to negative energy balance is associated with lower expression of HLA-DRA and HLA-A in
bovine blood neutrophils after intramammary mastitis challenge with Streptococcus uberis. Funct
Integr Genomics 10:53-61; 2010.
[102] Mulligan, C. M.; Sparagna, G. C.; Le, C. H.; De Mooy, A. B.; Routh, M. A.; Holmes, M.
G.; Hickson-Bick, D. L.; Zarini, S.; Murphy, R. C.; Xu, F. Y.; Hatch, G. M.; McCune, S. A.;
Moore, R. L.; Chicco, A. J. Dietary linoleate preserves cardiolipin and attenuates mitochondrial
dysfunction in the failing rat heart. Cardiovasc Res 94:460-468; 2012.
[103] Musatov, A.; Robinson, N. C. Susceptibility of mitochondrial electron-transport complexes
to oxidative damage. Focus on cytochrome c oxidase. Free Radic Res 46:1313-1326; 2012.
[104] Nagy, L.; Tontonoz, P.; Alvarez, J. G. A.; Chen, H.; Evans, R. M. Oxidized LDL regulates
macrophage gene expression through ligand activation of PPARγ. Cell 93:229-240; 1998.
[105] Nakamura, M.; Shimizu, T. Leukotriene receptors. Chem Rev 111:6231-6298; 2011.
[106] Natarajan, R.; Reddy, M. A.; Malik, K. U.; Fatima, S.; Khan, B. V. Signaling mechanisms
of
nuclear
factor-κB-mediated
activation
of
inflammatory
genes
by
13hydroperoxyoctadecadienoic acid in cultured vascular smooth muscle cells. Arterioscler Thromb
Vasc Biol 21:1408-1413; 2001.
[107] Norling, L. V.; Dalli, J.; Flower, R. J.; Serhan, C. N.; Perretti, M. Resolvin D1 limits
polymorphonuclear leukocyte recruitment to inflammatory loci: receptor-dependent actions.
Arterioscler. Thromb. Vasc. Biol. 32:1970-1978; 2012.
[108] Norris, P. C.; Dennis, E. A. Omega-3 fatty acids cause dramatic changes in TLR4 and
purinergic eicosanoid signaling. Proc Natl Acad Sci U S A 109:8517-8522; 2012.
[109] O'Banion, M. K.; Winn, V. D.; Young, D. A. cDNA cloning and functional activity of a
glucocorticoid-regulated inflammatory cyclooxygenase. Proc Natl Acad Sci U S A 89:4888; 1992.
[110] O’Boyle, N. J.; Contreras, G. A.; Mattmiller, S. A.; Sordillo, L. M. Changes in glucose
transporter expression in monocytes of periparturient dairy cows. J Dairy Sci 95:5709-5719; 2012.
[111] Ogawa, E.; Owada, Y.; Ikawa, S.; Adachi, Y.; Egawa, T.; Nemoto, K.; Suzuki, K.;
Hishinuma, T.; Kawashima, H.; Kondo, H.; Muto, M.; Aiba, S.; Okuyama, R. Epidermal FABP

106

(FABP5) Regulates Keratinocyte Differentiation by 13(S)-HODE-Mediated Activation of the NFkB Signaling Pathway. J Invest Dermatol 131:604-612; 2011.
[112] Ohtsuka, H.; Watanabe, C.; Kohiruimaki, M.; Ando, T.; Watanabe, D.; Masui, M.;
Hayashi, T.; Abe, R.; Koiwa, M.; Sato, S.; Kawamura, S. Comparison of Two Different Nutritive
Conditions against the Changes in Peripheral Blood Mononuclear Cells of Periparturient Dairy
Cows. J Vet Med Sci 68:1161-1166; 2006.
[113] Okuyama, H.; Kobayashi, T.; Watanabe, S. Dietary fatty acids — The n-6n-3 balance and
chronic elderly diseases. Excess linoleic acid and relative n-3 deficiency syndrome seen in Japan.
Prog Lipid Res 35:409-457; 1996.
[114] Olde Riekerink, R. G. M.; Barkema, H. W.; Kelton, D. F.; Scholl, D. T. Incidence Rate of
Clinical Mastitis on Canadian Dairy Farms. J Dairy Sci 91:1366-1377; 2008.
[115] Palmquist, D.; Jenkins, T. Challenges with fats and fatty acid methods. J Anim Sci 81:32503254; 2003.
[116] Paradies, G.; Petrosillo, G.; Paradies, V.; Reiter, R. J.; Ruggiero, F. M. Melatonin,
cardiolipin and mitochondrial bioenergetics in health and disease. J Pineal Res 48:297-310; 2010.
[117] Pinedo, P.; De Vries, A.; Webb, D. Dynamics of culling risk with disposal codes reported
by Dairy Herd Improvement dairy herds. J Dairy Sci 93:2250-2261; 2010.
[118] Poulsen, R. C.; Gotlinger, K. H.; Serhan, C. N.; Kruger, M. C. Identification of
inflammatory and proresolving lipid mediators in bone marrow and their lipidomic profiles with
ovariectomy and omega-3 intake. Am J Hematol 83:437-445; 2008.
[119] Quehenberger, O.; Armando, A. M.; Brown, A. H.; Milne, S. B.; Myers, D. S.; Merrill, A.
H.; Bandyopadhyay, S.; Jones, K. N.; Kelly, S.; Shaner, R. L. Lipidomics reveals a remarkable
diversity of lipids in human plasma. J Lipid Res 51:3299-3305; 2010.
[120] Ramsden, C. E.; Ringel, A.; Feldstein, A. E.; Taha, A. Y.; MacIntosh, B. A.; Hibbeln, J.
R.; Majchrzak-Hong, S. F.; Faurot, K. R.; Rapoport, S. I.; Cheon, Y.; Chung, Y.-M.; Berk, M.;
Douglas Mann, J. Lowering dietary linoleic acid reduces bioactive oxidized linoleic acid
metabolites in humans. Prostaglandins Leukot Essent Fatty Acids 87:135-141; 2012.
[121] Ramsden, C. E.; Zamora, D.; Leelarthaepin, B.; Majchrzak-Hong, S. F.; Faurot, K. R.;
Suchindran, C. M.; Ringel, A.; Davis, J. M.; Hibbeln, J. R. Use of dietary linoleic acid for
107

secondary prevention of coronary heart disease and death: evaluation of recovered data from the
Sydney Diet Heart Study and updated meta-analysis. BMJ 346; 2013.
[122] Raphael, W.; Halbert, L.; Contreras, G. A.; Sordillo, L. M. Association between
polyunsaturated fatty acid-derived oxylipid biosynthesis and leukocyte inflammatory marker
expression in periparturient dairy cows. J Dairy Sci Accepted; 2014.
[123] Raphael, W.; Sordillo, L. M. Dietary polyunsaturated fatty acids and inflammation: the role
of phospholipid biosynthesis. Int J Mol Sci 14:21167-21188; 2013.
[124] Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant
activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med
26:1231-1237; 1999.
[125] Rees, D.; Miles, E. A.; Banerjee, T.; Wells, S. J.; Roynette, C. E.; Wahle, K. W.; Calder,
P. C. Dose-related effects of eicosapentaenoic acid on innate immune function in healthy humans:
a comparison of young and older men. Am J Clin Nutr 83:331-342; 2006.
[126] Reinhold, P.; Becher, G.; Rothe, M. Evaluation of the measurement of leukotriene B4
concentrations in exhaled condensate as a noninvasive method for assessing mediators of
inflammation in the lungs of calves. Am J Vet Res 61:742-749; 2000.
[127] Rico, J. E.; Allen, M. S.; Lock, A. L. Compared with stearic acid, palmitic acid increased
the yield of milk fat and improved feed efficiency across production level of cows. J Dairy Sci
97:1057-1066; 2014.
[128] Ricote, M.; Li, A. C.; Willson, T. M.; Kelly, C. J.; Glass, C. K. The peroxisome
proliferator-activated receptor- is a negative regulator of macrophage activation. Nature 391:7982; 1998.
[129] Rizos, E. C.; Ntzani, E. E.; Bika, E.; Kostapanos, M. S.; Elisaf, M. S. Association between
omega-3 fatty acid supplementation and risk of major cardiovascular disease events: a systematic
review and meta-analysis. JAMA 308:1024-1033; 2012.
[130] Rong, S.; Cao, Q.; Liu, M.; Seo, J.; Jia, L.; Boudyguina, E.; Gebre, A. K.; Colvin, P. L.;
Smith, T. L.; Murphy, R. C.; Mishra, N.; Parks, J. S. Macrophage 12/15 lipoxygenase expression
increases plasma and hepatic lipid levels and exacerbates atherosclerosis. J Lipid Res 53:686-695;
2012.

108

[131] Rose, D. P.; Connolly, J. M. Omega-3 fatty acids as cancer chemopreventive agents.
Pharmacol Ther 83:217-244; 1999.
[132] Ruiz, J.; Antequera, T.; Andres, A.; Petron, M.; Muriel, E. Improvement of a solid phase
extraction method for analysis of lipid fractions in muscle foods. Anal Chim Acta 520:201-205;
2004.
[133] Rule, D. C.; Broughton, K. S.; Shellito, S. M.; Maiorano, G. Comparison of muscle fatty
acid profiles and cholesterol concentrations of bison, beef cattle, elk, and chicken. J Anim Sci
80:1202-1211; 2002.
[134] Santoro, N.; Caprio, S.; Feldstein, A. E. Oxidized metabolites of linoleic acid as biomarkers
of liver injury in nonalcoholic steatohepatitis. Clin Lipidol; 2013: 411+.
[135] Schmitz, S.; Pfaffl, M. W.; Meyer, H. H. D.; Bruckmaier, R. M. Short-term changes of
mRNA expression of various inflammatory factors and milk proteins in mammary tissue during
LPS-induced mastitis. Domest Anim Endocrinol 26:111-126; 2004.
[136] Seals, R. C.; Matamoros, I.; Lewis, G. S. Relationship between postpartum changes in 13,
14-dihydro-15-keto-PGF2alpha concentrations in Holstein cows and their susceptibility to
endometritis. J Anim Sci 80:1068-1073; 2002.
[137] Seifi, H. A.; LeBlanc, S. J.; Leslie, K. E.; Duffield, T. F. Metabolic predictors of postpartum disease and culling risk in dairy cattle. Vet J 188:216-220; 2011.
[138] Serhan, C. N.; Chiang, N.; Van Dyke, T. E. Resolving inflammation: Dual antiinflammatory and pro-resolution lipid mediators. Nat Rev Immunol 8:349-361; 2008.
[139] Serhan, C. N.; Clish, C. B.; Brannon, J.; Colgan, S. P.; Chiang, N.; Gronert, K. Novel
functional sets of lipid-derived mediators with antiinflammatory actions generated from omega-3
fatty acids via cyclooxygenase 2-nonsteroidal antiinflammatory drugs and transcellular
processing. J Exp Med 192:1197-1204; 2000.
[140] Serhan, C. N.; Petasis, N. A. Resolvins and protectins in inflammation resolution. Chem
Rev 111:5922-5943; 2011.
[141] Serhan, C. N.; Yang, R.; Martinod, K.; Kasuga, K.; Pillai, P. S.; Porter, T. F.; Oh, S. F.;
Spite, M. Maresins: novel macrophage mediators with potent antiinflammatory and proresolving
actions. J Exp Med 206:15-23; 2009.
109

[142] Sharma, N. K.; Langberg, K. A.; Mondal, A. K.; Das, S. K. Phospholipid Biosynthesis
Genes and Susceptibility to Obesity: Analysis of Expression and Polymorphisms. PLoS One
8:e65303; 2013.
[143] Sharma, N. P.; Dong, L.; Yuan, C.; Noon, K. R.; Smith, W. L. Asymmetric Acetylation of
the Cyclooxygenase-2 Homodimer by Aspirin and Its Effects on the Oxygenation of Arachidonic,
Eicosapentaenoic, and Docosahexaenoic Acids. Mol Pharmacol 77:979-986; 2010.
[144] Shindou, H.; Hishikawa, D.; Harayama, T.; Yuki, K.; Shimizu, T. Recent progress on acyl
CoA: lysophospholipid acyltransferase research. J Lipid Res 50:S46-S51; 2009.
[145] Shuster, D.; Lee, E.; Kehrli Jr, M. Bacterial growth, inflammatory cytokine production,
and neutrophil recruitment during coliform mastitis in cows within ten days after calving,
compared with cows at midlactation. Am J Vet Res 57:1569-1575; 1996.
[146] Siivonen, J.; Taponen, S.; Hovinen, M.; Pastell, M.; Lensink, B. J.; Pyörälä, S.; Hänninen,
L. Impact of acute clinical mastitis on cow behaviour. Appl Anim Behav Sci 132:101-106; 2011.
[147] Simons, K.; Toomre, D. Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1:3139; 2000.
[148] Simopoulos, A. P. Evolutionary aspects of diet, the omega-6/omega-3 ratio and genetic
variation: nutritional implications for chronic diseases. Biomed Pharmacother 60:502-507; 2006.
[149] Skarzynski, D. J.; Piotrowska-Tomala, K. K.; Lukasik, K.; Galvão, A.; Farberov, S.;
Zalman, Y.; Meidan, R. Growth and regression in bovine corpora lutea: Regulation by local
survival and death pathways. Reprod Domest Anim 48:25-37; 2013.
[150] Smith, W. L.; Urade, Y.; Jakobsson, P. J. Enzymes of the cyclooxygenase pathways of
prostanoid biosynthesis. Chem Rev 111:5821-5865; 2011.
[151] Soberman, R.; Harper, T.; Betteridge, D.; Lewis, R.; Austen, K. Characterization and
separation of the arachidonic acid 5-lipoxygenase and linoleic acid omega-6 lipoxygenase
(arachidonic acid 15-lipoxygenase) of human polymorphonuclear leukocytes. J Biol Chem
260:4508-4515; 1985.
[152] Sordillo, L.; Pighetti, G.; Davis, M. Enhanced production of bovine tumor necrosis factor[alpha] during the periparturient period. Vet Immunol Immunopathol 49:263-270; 1995.

110

[153] Sordillo, L. M.; Contreras, G. A.; Aitken, S. L. Metabolic factors affecting the
inflammatory response of periparturient dairy cows. Anim Health Res Rev 10:53-63; 2009.
[154] Sordillo, L. M.; Raphael, W. Significance of Metabolic Stress, Lipid Mobilization, and
Inflammation on Transition Cow Disorders. Vet Clin North Am Food Anim Pract 29:267-278;
2013.
[155] Sordillo, L. M.; Streicher, K. L.; Mullarky, I. K.; Gandy, J. C.; Trigona, W.; Corl, C. M.
Selenium inhibits 15-hydroperoxyoctadecadienoic acid-induced intracellular adhesion molecule
expression in aortic endothelial cells. Free Radic Biol Med 44:34-43; 2008.
[156] Spite, M.; Clària, J.; Serhan, Charles N. Resolvins, Specialized Proresolving Lipid
Mediators, and Their Potential Roles in Metabolic Diseases. Cell Metab 19:21-36; 2014.
[157] Spite, M.; Norling, L. V.; Summers, L.; Yang, R.; Cooper, D.; Petasis, N. A.; Flower, R.
J.; Perretti, M.; Serhan, C. N. Resolvin D2 is a potent regulator of leukocytes and controls
microbial sepsis. Nature 461:1287-1291; 2009.
[158] Stachowska, E.; Dolegowska, B.; Dziedziejko, V.; Rybicka, M.; Kaczmarczyk, M.; Bober,
J.; Rac, M.; Machalinski, B.; Chlubek, D. Prostaglandin E2 (PGE2) and thromboxane A2 (TXA2)
synthesis is regulated by conjugated linoleic acids (CLA) in human macrophages. Acta Physiol
Pol 60:77; 2009.
[159] Stachowska, E.; Dziedziejko, V.; Safranow, K.; Jakubowska, K.; Olszewska, M.;
Machaliñski, B.; Chlubek, D. Effect of conjugated linoleic acids on the activity and mRNA
expression of 5-and 15-lipoxygenases in human macrophages. J Agric Food Chem 55:5335-5342;
2007.
[160] Stillwell, W.; Wassall, S. R. Docosahexaenoic acid: membrane properties of a unique fatty
acid. Chem Phys Lipids 126:1-27; 2003.
[161] Strawford, A.; Antelo, F.; Christiansen, M.; Hellerstein, M. K. Adipose tissue triglyceride
turnover, de novo lipogenesis, and cell proliferation in humans measured with 2H2O. Am J Physiol
- Endocrinology And Metabolism 286:E577-E588; 2004.
[162] Suganami, T.; Tanimoto-Koyama, K.; Nishida, J.; Itoh, M.; Yuan, X.; Mizuarai, S.; Kotani,
H.; Yamaoka, S.; Miyake, K.; Aoe, S.; Kamei, Y.; Ogawa, Y. Role of the Toll-like receptor 4/NFkappaB pathway in saturated fatty acid-induced inflammatory changes in the interaction between
adipocytes and macrophages. Arterioscler Thromb Vasc Biol 27:84-91; 2007.
111

[163] Takeuchi, K.; Reue, K. Biochemistry, physiology, and genetics of GPAT, AGPAT, and
lipin enzymes in triglyceride synthesis. Am J Physiol - Endocrinology And Metabolism
296:E1195-E1209; 2009.
[164] Tam, Vincent C.; Quehenberger, O.; Oshansky, Christine M.; Suen, R.; Armando,
Aaron M.; Treuting, Piper M.; Thomas, Paul G.; Dennis, Edward A.; Aderem, A. Lipidomic
Profiling of Influenza Infection Identifies Mediators that Induce and Resolve Inflammation. Cell
154:213-227; 2013.
[165] Tao, S.; Connor, E. E.; Bubolz, J. W.; Thompson, I. M.; do Amaral, B. C.; Hayen, M. J.;
Dahl, G. E. Short communication: Effect of heat stress during the dry period on gene expression
in mammary tissue and peripheral blood mononuclear cells. J Dairy Sci 96:378-383; 2013.
[166] Toborek, M.; Barger, S. W.; Mattson, M. P.; Barve, S.; McClain, C. J.; Hennig, B. Linoleic
acid and TNF-alpha cross-amplify oxidative injury and dysfunction of endothelial cells. J Lipid
Res 37:123-135; 1996.
[167] Toborek, M.; Blanc, E. M.; Kaiser, S.; Mattson, M.; Hennig, B. Linoleic acid potentiates
TNF-mediated oxidative stress, disruption of calcium homeostasis, and apoptosis of cultured
vascular endothelial cells. J Lipid Res 38:2155; 1997.
[168] Trebble, T.; Arden, N. K.; Stroud, M. A.; Wootton, S. A.; Burdge, G. C.; Miles, E. A.;
Ballinger, A. B.; Thompson, R. L.; Calder, P. C. Inhibition of tumour necrosis factor-α and
interleukin 6 production by mononuclear cells following dietary fish-oil supplementation in
healthy men and response to antioxidant co-supplementation. Br J Nutr 90:405-412; 2003.
[169] Triggiani, M.; Oriente, A.; Marone, G. Differential roles for triglyceride and phospholipid
pools of arachidonic acid in human lung macrophages. J Immunol 152:1394-1403; 1994.
[170] Turkish, A.; Sturley, S. L. Regulation of triglyceride metabolism. I. Eukaryotic neutral
lipid synthesis: “Many ways to skin ACAT or a DGAT”. Am J Physiol - Gastrointestinal & Liver
Physiology 292:G953-G957; 2007.
[171] Tyagi, A.; Kumar, U.; Reddy, S.; S. Santosh, V.; B. Mohammed, S.; Ehtesham, N. Z.;
Ibrahim, A. Attenuation of colonic inflammation by partial replacement of dietary linoleic acid
with α-linolenic acid in a rat model of inflammatory bowel disease. Br J Nutr 108:1612-1622;
2012.
[172] van den Top, A. M.; Geelen, M. J. H.; Wensing, T.; Wentink, G. H.; van 'T Klooster, A.
T.; Beynen, A. C. Higher postpartum hepatic triacylglycerol concentrations in dairy cows with free
112

rather than restricted access to feed during the dry period are associated with lower activities of
hepatic glycerolphosphate acyltransferase. J Nutr 126:76-85; 1996.
[173] Vance, D. E.; Vance, J. E. Phospholipid biosynthesis in eukaryotes. In: Vance, D. E.;
Vance, J. E., eds. Biochemistry of Lipids, Lipoproteins and Membranes. St. Louis, MO: Elsevier;
2008: 213-244.
[174] Vangaveti, V.; Baune, B. T.; Kennedy, R. L. Review: Hydroxyoctadecadienoic acids:
novel regulators of macrophage differentiation and atherogenesis. Ther Adv Endocrinol Metab
1:51-60; 2010.
[175] Virtanen, J. K.; Mozaffarian, D.; Chiuve, S. E.; Rimm, E. B. Fish consumption and risk of
major chronic disease in men. Am J Clin Nutr 88:1618-1625; 2008.
[176] Wang, D.; DuBois, R. N. Prostaglandins and cancer. Gut 55:115-122; 2006.
[177] White, S. L.; Bertrand, J. A.; Wade, M. R.; Washburn, S. P.; Green Jr, J. T.; Jenkins, T. C.
Comparison of fatty acid content of milk from Jersey and Holstein cows consuming pasture or a
total mixed ration. J Dairy Sci 84:2295-2301; 2001.
[178] Wong, S. W.; Kwon, M. J.; Choi, A. M.; Kim, H. P.; Nakahira, K.; Hwang, D. H. Fatty
acids modulate Toll-like receptor 4 activation through regulation of receptor dimerization and
recruitment into lipid rafts in a reactive oxygen species-dependent manner. J Biol Chem
284:27384-27392; 2009.
[179] Woodworth, H. L.; McCaskey, S. J.; Duriancik, D. M.; Clinthorne, J. F.; Langohr, I. M.;
Gardner, E. M.; Fenton, J. I. Dietary fish oil alters T lymphocyte cell populations and exacerbates
disease in a mouse model of inflammatory colitis. Cancer Res 70:7960-7969; 2010.
[180] Xiong, A.; Yu, W.; Tiwary, R.; Sanders, B. G.; Kline, K. Distinct roles of different forms
of vitamin E in DHA-induced apoptosis in triple-negative breast cancer cells. Mol Nutr Food Res
56:923-934; 2012.
[181] Xu, A.; Wang, Y.; Xu, J. Y.; Stejskal, D.; Tam, S.; Zhang, J.; Wat, N. M. S.; Wong, W.
K.; Lam, K. S. L. Adipocyte fatty acid–binding protein is a plasma biomarker closely associated
with obesity and metabolic syndrome. Clin Chem 52:405-413; 2006.
[182] Xu, L.; Davis, T. A.; Porter, N. A. Rate Constants for Peroxidation of Polyunsaturated
Fatty Acids and Sterols in Solution and in Liposomes. J Am Chem Soc 131:13037-13044; 2009.
113

[183] Yen, C.-L. E.; Stone, S. J.; Koliwad, S.; Harris, C.; Farese, R. V. Thematic Review Series:
Glycerolipids. DGAT enzymes and triacylglycerol biosynthesis. J Lipid Res 49:2283-2301; 2008.
[184] Yin, H.; Zhu, M. Free radical oxidation of cardiolipin: chemical mechanisms, detection
and implication in apoptosis, mitochondrial dysfunction and human diseases. Free Radic Res
46:959-974; 2012.
[185] Ylä-Herttuala, S.; Rosenfeld, M. E.; Parthasarathy, S.; Glass, C. K.; Sigal, E.; Witztum, J.
L.; Steinberg, D. Colocalization of 15-lipoxygenase mRNA and protein with epitopes of oxidized
low density lipoprotein in macrophage-rich areas of atherosclerotic lesions. Proc Natl Acad Sci U
S A 87:6959-6963; 1990.
[186] Yoshida, Y.; Niki, E. Hydroxyoctadecadienoic acid (HODE) as a marker of linoleic acid
oxidation. In: Aldini, G.; Yeum, K.-J.; Niki, E.; Russell, R. M., eds. Biomarkers for Antioxidant
Defense and Oxidative Damage: Principles and Practical Applications. Ames, IA: Wiley; 2010:
85-97.
[187] Yoshida, Y.; Umeno, A.; Shichiri, M. Lipid peroxidation biomarkers for evaluating
oxidative stress and assessing antioxidant capacity in vivo. J Clin Biochem Nutr 52:9-16; 2013.
[188] Young, B. P.; Shin, J. J. H.; Orij, R.; Chao, J. T.; Li, S. C.; Guan, X. L.; Khong, A.; Jan,
E.; Wenk, M. R.; Prinz, W. A. Phosphatidic acid is a pH biosensor that links membrane biogenesis
to metabolism. Sci Signal 329:1085-1088; 2010.
[189] Young, V. M.; Toborek, M.; Yang, F.; McClain, C. J.; Hennig, B. Effect of linoleic acid
on endothelial cell inflammatory mediators. Metabolism 47:566-572; 1998.
[190] Yu, Y.; Correll, P. H.; Vanden Heuvel, J. P. Conjugated linoleic acid decreases production
of pro-inflammatory products in macrophages: evidence for a PPARγ-dependent mechanism.
Biochim Biophys Acta - Molecular and Cell Biology of Lipids 1581:89-99; 2002.
[191] Zammit, V. A.; Buckett, L. K.; Turnbull, A. V.; Wure, H.; Proven, A. Diacylglycerol
acyltransferases: Potential roles as pharmacological targets. Pharmacol Ther 118:295-302; 2008.
[192] Zou, H.; Yuan, C.; Dong, L.; Sidhu, R. S.; Hong, Y. H.; Kuklev, D. V.; Smith, W. L.
Human cyclooxygenase-1 activity and its responses to COX inhibitors are allosterically regulated
by nonsubstrate fatty acids. J Lipid Res 53:1336-1347; 2012.

114

[193] Zuo, X.; Shureiqi, I. Eicosanoid profiling in colon cancer: Emergence of a pattern.
Prostaglandins Other Lipid Mediat 104-105:139-143; 2013.

115