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This is to certify that the

thesis entitled

Determination of the Prostagiandin E Metaboiite (PGE-M)
By Gas Chromatography-Mass Spectrometry and Excretion
of PGE-M By Women Consuming a Gariic Extract

presented by

Ching-Hsun Chiou

has been accepted towards fulfillment
of the requirements for

Masters Science

degree in

 

 

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Date j ” 52 3 _ Q»);

0-7539 MS U is an Affirmative Action/Equal Opportunity Institution

 

PLACE IN RETURN BOX to remove thte checkout from your record.
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MSU Ie An Afflnnettve Action/Ewe! Opportunity Inetttulon

 

 

 

 

 

 

 

 

 

 

 

 

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DUE JLI

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DETERMINATION OF TEE PROSTAGLANDIN E METABOLITE
(PGE—M) BY GAS CHROMATOGRAPHY-MASS SPECTROMETRY
AND EXCRETION OP PGE-M BY WOMEN CONSUMING A
GARLIC EXTRACT

BY

CHING-HSUN CHIOU

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1995

AIMSTTUHBT'

DETERMINATION OF THE PROSTAGLANDIN E METABOLITE
(PGE-M) B! GAS CHROMATOGRAPHY-MASS SPECTROMETRY
AND EXCRETION OF PGE-M B! WOMEN CONSUMING A
GARLIC EXTRACT

BY

CHING-HSUN CHIOU

A study was conducted to develop a method to quantitate
urinary excretion of PCB-M, an indicator of whole body
turnover of prostaglandin El and 32. PGE-M excretion in one
individual taking aspirin and eight women consuming a garlic
extract (GE) were also examined. PGE-M and [ZH7J—PGE-M
(internal standard) were co-extracted from 20 ml of urine,
methylated, partially purified, methoximated, and
trimethylsilated. PGE-M was analyzed by gas chromatography-
mass spectrometry using the selected ion monitoring mode at
m/z 365 and m/z 372. Aspirin but not GE consumption decreased

PGE-M excretion. There was no difference in PGE-M excretion

before (42.7 f 9.5 ug/24 hr) and after three months of garlic
consumption (35.9 t 4.7 ug/24 hr) (P = 0.29). It is concluded
that the GE did not contain sufficient prostaglandin

inhibitors to reduce PGE-M excretion.

ACKNOWLEDGMENTS

I would like to sincerely thank my major professor, Dr.
Maurice R. Bennink, for his support and guidance throughout
this study. Appreciation is also expressed to Dr. Dale R.
Romsos and Dr. wanda E. Chenoweth for serving on my guidance
committee.

Thanks to Dr. J. T. watson and everyone who have
assisted me in the mass-spectrometry facility room in the
Biochemistry department.

I also appreciate my parents, family and friends who
always stand by me and give me their support and

encouragement.

iii

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

INTRODUCTION

REVIEW OF LITERATURE

A.

BIOLOGY OF EICOSANOIDS

A.1.

A.2.

A.3.

AC4.

Precursors of Eicosanoids

Regulation of the Eicosanoid Precursors
Release

Biosynthesis of Eicosanoids

Physiological function of Eicosanoids

THE E SERIES OF PROSTAGLANDINS (PGE)

3.1.
3.2.

BOB.

3.4.
B.5.

Biosynthesis of PGE
Catabolism of PGE

Quantitative Analysis of PGE-M by Gas
Chromatography-Mass Spectrometry (GO-MS)

Biological Function OF PGE

PGE and Tumorgenesis

REGULATION OF PROSTAGLANDIN METABOLISM

C01.

CCZO

Stimulators of Prostaglandin Synthesis

Inhibitors of Prostaglandin Synthesis

PHARMACOLOGICAL EFFECT OF GARLIC

D01.

D.2.

Introduction

The Chemistry of Garlic

iv

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11

14
15
15
16
16
17
18
18
18

D.3. Garlic Inhibits Arachidonic Acid
Metabolism

D.4. Garlic and Carcinogenesis
E. JUSTIFICATION
F. OBJECTIVE
G. HYPOTHESIS
H. NULL HYPOTHESIS
MATERIALS AND METHODS
EXPERIMENT DESIGN
SUBJECTS
GARLIC SUPPLEMENTATION
URINE SAMPLES
REAGENTS AND MATERIALS
EQUIPMENT
METHODS
GLASSWARE PREPARATION
PGE-M EXTRACTION FROM URINE
DERIVATIVE FORMATION
Methylester formation (ME)
Methoximation (M0)
Trimethylsilation (TMS)
QUANTITATIVE ANALYSIS OF PGE-M BY GC-MS
STATISTICAL METHODS
RESULTS AND DISCUSSION
SUMMARY AND CONCLUSIONS
RECOMMENDATION FOR FUTURE RESEARCH

BIBLIOGRAPHY

20
23
25
25
26
26
27
27
27
27
28
28
30
30
30
32

34
34
34
35
36
37
54
56
58

Table

Table

Table

Table

Table

LIST OF TABLES

Organosulfur compounds in garlic tested
in vitro and in vivo assays for possible
anticarcinogenic activity

Garlic supplementation and urine
collection protocol

Twenty-four hour urinary PGE-M excretion
before and after aspirin treatment

Twenty-four hour urinary PGE-M excretion

(ug/24 hr) for the human subjects before

and after three months consumption of an
aqueous-ethanol garlic extract

Organosulfur compounds in Kyolic"
concentrate and garlic

vi

21

29

50

51

52

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

10.

11.

LIST OF FIGURES

Arachidonate cascade

Biosynthetic pathway for prostanoid
formation. The cyclooxygenase activity
is inhibited by non-steroid anti-
inflammatory drugs (NSAIDs)

Sequence of reactions involved in the
degradation of PGEz and formation of
the major urinary metabolite (PGE-M)

Possible cancer-preventive foods and

ingredients

Organosulfur compounds transformation

in garlic

Schematic diagram of a Gas
chromatography-Mass spectrometry

Scheme for the analysis of urinary

PGE-M

Chemical structures of PGE-M, [237]-
PGE-M and their ME-MO-TMS derivatives

Total ion current (TIC) chromatogram.of

ME-MO-TMS [2H7]-PGE-M

Scans of the mass spectrum of the most

intense peaks in the TIC chromatogram of

ME-MO-TMS [ZH7J-PGE-M at the retention
time of 10.241min (a) and 10.132 min

(b)

Scans of the mass spectrum of the most

intense peaks in the TIC chromatogram of

ME-MO-TMS [2371-PGE-H at the retention
time of 9.838min (a) and 9.701 min

(1))

vii

7

10

13

19

22

31

33

38

39

41

42

Figure

Figure

Figure

Figure

Figure

12.

13.

14.

15.

16.

Scans the mass spectrum of the intense

peaks in the TIC chromatogram of ME-MO-
TMS [ZH7J-PGE—M at the retention time of

7.544min (a) and 7.289 min (b)

Analyte fragmentation occurres within
the mass chamber

From the TIC profile of ME-MO-TMS
[2H7J-PGE-M, the m/z 372 ion was
monitored to create a new mass
chromatogram, which identified the
retention time of this standard
compound

The relationship between amount of
[2H7]-PGE-M injected (0.1 ng - 200 ng)
and the GC-MS detector response

SIM chromatograms of an urine sample

viii

43

44

45

47
48

INTRODUCTION

Cancer has become a major cause of death all over the
world during the last several decades. The incidence of
several kinds of cancer, such as colon, breast, ovary,
prostate and endometrial, has been related to diet as shown
by both epidemiological and laboratory studies (weisburger
and Rivenson, 1993). For example, colon cancer, the third
most common malignant cancer in the United States, was found
to be associated with high intakes of fat and animal protein,
and with low intakes of fiber. People who consume high fat
diets also have higher incidence of breast, pancreatic and
colorectal cancers.

Carcinogens can be formed during food preparation.
Examples are polycyclic aromatic hydrocarbons and heterocylic
aromatic amines - compounds that can initiate mammary and
colon cancer in experimental animals. Conversely, many
possible cancer preventive components have been found in the
diet, particularly in foods from plant sources. Foods with
chemopreventive potential include garlic, soybean, licorice,
green tea, umbelliferous vegetables, and citrus fruits
(Caragay, 1992).

In fact, a variety of anti-carcinogens and potential

carcinogens co-exist in our diet, and these naturally

2
occurring substances in foods can affect cancer initiation,
promotion, and progression by different mechanisms. The
dietary anti-carcinogens suppress carcinogenesis development
by inducing biotransforming enzymes that block initiation and
reduce carcinogenicity, or by inhibiting tumor promotion
(Davis, 1989).

Oxidative damage, prostaglandins and related eicosanoids
play an important role in the tumor promotion stage of
carcinogenesis. The promotion process can be blocked by
compounds such as phenolics, salicylates, flavonoids,
polyacetylenes, and sulfides. Retinols and tocopherols found
in vegetables and animal foods, and organosulfates found in
garlic and onion, also inhibit tumor promotion (Hayes et al.,
1987; Wargovich et al., 1987; Sparnins et al., 1988).

The Experimental Foods Program.from.the National Cancer
Institute (NCI) identified over forty foods with cancer
preventive activity, and garlic was considered the food with
the most anti-cancer potential. The ancient pharmacological
and health promoting attributes of garlic have been known for
thousands of years. In addition, modern science suggests that
several of the organosulfur compounds in garlic are
biologically active and elicit a number of physiological
functions, including inhibition of prostaglandin biosynthesis
and inhibition of cancer (Davis, 1989; Lau et al., 1993).

The objective of this study was to develop a method to
determine the prostaglandin E metabolite in urine and to

determine if a garlic extract would suppress prostaglandin E

3
production in humans. The quantitative measurement of
prostaglandin E metabolite (PGE-M) excretion in urine, which

is an indicator of whole body biosynthesis of prostaglandin

E1 and E2, was used to assess the effect of the garlic

extract on prostaglandin biosynthesis.

REVIEW OF LITERATURE

A. BIOLOGY OF EICOSANOIDS

A.1. Precursors of eicosanoids
Prostaglandins, leukotrienes, and related hydroxy fatty
acids are members of the eicosanoid family that accounts for

a variety of significant physiological functions in humans.

Dihomo-gamma-linolenate (20:5m6), arachidonic acid (AA,
20:4w6), and eicosapentaenolate (20:5w3) are immediate
precursors for eicosanoid biosynthesis. Of these, AA is the
main unsaturated fatty acid substrate used for eicosanoid
synthesis in most mammalian cells.

AA is obtained either directly from the diet, mainly
from animal products, or AA is synthesized from linoleic
acid, via desaturation and elongation reactions. Once AA is
synthesized, it is incorporated into cell membrane
phospholipids. AA is esterified primarily to the 8-position
of phosphatidylinositol, phosphatidylcholine, phosphatidyl-
ethanolamine or phosphotidylserine.

Since only unesterified polyunsaturated precursors can
enter the eicosanoid formation cascade (Struyck et al.,1966),
the release of non-esterified arachidonic acid (NEAA) is an
important point for regulation of eicosanoid biosynthesis

4

5
(Smith, 1989). AA release from membranes is relatively quick

and is typically accompanied by turnover of inositol
containing lipids (Lapetina et al., 1978). Deesterification
by phospholipase A2 (PLAz), phosphololipase C (PLC) and
diacylglyceral lipase occurs in response to particular
hormone stimuli in specific cells.

Phospholipase A2 is the key enzyme which catalyzes the
breakage of the 8—bond to release AA from phospholipids; thus
stimulation of PLA2 plays a pivotal role in AA release. The
mechanism of AA release is not the same as for the release of
fatty acids from triglycerides in adipose tissue. Fatty acids
release from adipose tissue is regulated by hormone sensitive
lipase (Hagenfeldt et al., 1975a). The release of AA from
phospholipids is increased by stress (Deby-Dupont et
al.,1983) but is not affected by exercise (Hagenfeldt and
wahzen, 1975b). Plasma non-esterified fatty acids other than
AA and from adipose tissue are increased by both stress and

exercise.

A.2. Regulation of the Eicosanoid Precursors Release
The biochemical details involved in the deesterification
process have not been well resolved. Many types of hormones,
autocoids, growth factors and tumor promoters elicit AA
release by receptor mediated action. Alternately, mechanical
stress on the cell such as shear forces acting on vascular
endothelial cells can produce NEAA. Hormones such as
angiotensin II (Nolan et al., 1981), bradykinin (Hong and

Levine, 1976) and catecholamines (Norcia and Evans, 1962) are

6
stimulators of PLA2, Glucocorticoids (Di Rosa and Persico,

1979; Flower and Blackwell, 1979), and lipomodulin protein
(Hirata, 1982) inhibit PLAz activity. Cellular calcium.and
cyclic AMP (cAMP) regulate AA release also. For example, cAMP
can inhibit the release of eicosanoid precursors from
membrane phospholipids by decreasing phospholipase activity
(Minkes et al., 1977). Ca2+ stimulates phospholipase activity
and inhibits adenylcyclase while cAMP decreases cytosolic

Ca2+ concentration (Gemsa et al., 1979; Lin et al., 1981).

A.3. Biosynthesis of Eicosanoids

Once deesterified from phospholipids, free AA can be
oxidized and further metabolized to a variety of biologically
active C20 eicosanoid products (Figure 1). The formation of
eicosanoids is species specific and tissue specific as well
(Sun et al., 1977). In mammalian cells, almost all cells
except red blood cells (RBC) can produce prostaglandins and
related compounds in response to a particular stimuli (Voet,
1990). Depending on the specific cells, there are three
different pathways for AA oxygenation- the cyclic pathway
leads to the formation of prostaglandins and thromboxanes
(prostanoids), the lipoxygenase pathway leads to the
synthesis of leukotrienes, mono-, di-, and tri— hydroxy
acids, and the epoxygenase pathway to the biosynthesis of
epoxides. Details for the biosynthesis of prostanoids will be
discussed in more detail in section B.1.

Eicosanoids are formed in response to various stimuli to

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generate a variety of general biological functions or

specific physiological responses resembling steroid hormone
activity. Similar to hormones, extremely low concentrations
of eicosanoids are sufficient to elicit physiological
effects. However, unlike hormones, the eicosanoids are not
transported through the blood stream to target sites; rather,
they are unstable substances utilized rapidly inside the cell
where they are produced. In other words, the eicosanoids act
as local hormones that are synthesized in the cell and
function within the same environment (Ferreia and Vane,

1967).

A.4. Physiological Function of Eicosanoids

Since eicosanoids are synthesized in most tissues, their
biological activities are broadly distributed in most organs,
i.e., cardiovascular, renal, reproductive, respiratory,
digestive, and neurotropic systems. Besides, eicosanoids are
also involved in platelet aggregation, pain and fever
production, inflammatory response, induction of blood
clotting, reproduction control, and sleep/wake cycle
regulation, etc. (VOet, 1990). The targets and functional
activities of eicosanoids may be associated with their
structural characteristics, i.e., their hydrophilic or
lipophilic properties (Euler, 1963). In some cases, different
eicosanoid families are antagonist to each other. For
example, in platelets, thromboxane A2 (TxAz) acts as a
vasoconstrictor and stimulator of platelet aggregation,

whereas prostaglandin 12 (P612) is a vasodilator and an

9
inhibitor of platelet aggregation. In normal conditions,

homeostatic mechanism.maintain prostanoid balance in the

cardiovascular system.
B. THE E SERIES 01" PROSTAGLANDINS (POE)

B.1. Biosynthesis of PGE

Among eicosanoids, prostaglandin Ez (PGEz) is
characterized as the most common and most potent mammalian
prostaglandin synthesized in the cyclic pathway of
arachidonic acid metabolism (Figure 2). The first step of
arachidonic acid oxygenation is mediated by prostaglandin
synthetase which has two enzyme activities - cyclooxygenase
activity involved in PGGz formation by adding two:molecules
of oxygen to AA and hydroperoxidase activity which adds two
electrons to reduce PGGz to yield PGH2.

The cyclooxygenase enzyme activity is specifically
inhibited by non-steroid anti-inflammatory drugs (NSAIDs)
such as aspirin, indomethacin, ibuprofen, phenylbutazone,
etc. (vane et al., 1971; Flower, 1974; Gryglewski, 1975).
Many NSAIDs are therapeutically effective in relieving pain,
fever and general inflammatory response by inhibition of
prostanoid biosynthesis. NSAIDs are commonly utilized as
prostanoid biosynthesis inhibitors in laboratory studies.

Since cyclooxygenase is ubiquitous in human nucleated
cells, prostanoids (prostaglandins and thromboxanes) are
generally produced in most cells with the exception of red

blood cells. As mentioned earlier, prostaglandins act like

10

STIMPLUS

 

 

 

F
I Cell Membrane
\

Activation of , .
Phospholipases\./ PhosPhOllpldS

Arachidonic Acid
2 o 2 «sam-

cyclooxygenase
PGG2
2 6' i Hydroperoxidase

PGH-PGD Prostacyclin

isornerase PGH 2 synthase

Thromboxane PGI
PGH-PGE PGFZ synthase 2

isomerase re ductase
GS

   
 

TXA
PGE

(1

 

 

 

Figure 2. Biosynthetic pathway for prostanoid formation. The
cyclooxygenase activity is inhibited by non-steroid anti-
inflammatory drugs (NSAIDs).

(Adapted from Smith, 1989)

 

11
local hormones. Thus, formation of specific prostaglandins

depends upon tissue need and presence of enzymes for specific
prostanoid synthesis.

Focusing on PGE2, prostaglandin endoperoxidase E
isomerase (PGH2/PGE2 isomerase) is responsible for converting
PGH2 to PGEz. This enzyme was extensively purified from sheep
seminal vesicle microsomes and found to be a glutathione

dependent enzyme (Ogino et al., 1977).

8.2. Catabolisn of PG

Normally, prostanoids are efficiently catabolized in
tissues where they are formed. However, the lung play an
important role in PG uptake and catabolism.as well. The lungs
serve to "filter" biologically active substances from the
blood stream to ensure that these compounds are at optimum
concentrations in the arterial circulation. If excess
prostaglandins are synthesized in peripheral tissues, the
lung inactives and degradates prostanoids. This process is
very efficient since 80-95% of PGE2 and PGan is catabolized
in lung of guinea pig, rat, rabbit, cat, dog, and human
(Piper et al., 1970).

The initial step in PG catabolism.by lung is PG uptake
from extracellular space into the cytoplasm where PG
degrading enzymes are located (Bito et al., 1977). PGs are
transported across the plasma membrane by a carrier mechanism
since PGs do not freely pass across biological membranes. All
PGs compete for a single carrier mechanism (Eling and Ally,

1981).

12
The enzyme responsible for classical prostaglandin

catabolism is 15-hydroxy prostaglandin dehydrogenase (15-
PGDH) (step 1 in Figure 3). Two types of 15-PGDH have been
identified (Nakano, 1969). Type I (lS-PGDH), NAD+ dependent
enzyme, is present in most tissues including lung, spleen,
parts of gastro-intestinal tract, kidney and heart. Type II
(15-PGDH) enzyme, which needs NADP+ as a cofactor is found in
kidney, brain, and erythrocytes. Among PGs, PGE2 is the best
substrate for both types of lS-PGDH.

After the P63 are oxidized to 15-keto PGs by 15-PGDH,
the next enzyme involved in catabolism is prostaglandin
reductase (PGR; step 2 in Figure 3). PGR is an NADH or NADPH
dependent enzyme, which reduces the Cl3-C14 double bond to
yield 13, 14-dihydro-15-oxo-prostaglandin derivatives, the
major metabolites found in plasma and lung effluents. The 13,
l4-dihydro-15-oxo-PGE2 has very little biological activity
and a 8 min half life in blood circulation in man (Hamberg
and Samuelsson, 1971). In comparison, PGEz has a 30 second
half life. Further metabolism is mediated by B orIw-oxidizing
enzymes (step 3 and 4, Figure 3) in liver, kidney, and
intestine (Sammuelson et al., 1971). These enzymes are not
specific for prostaglandins, they act on many fatty acids.
The final metabolite, 11a-hydroxy-9,15,-dioxo-2,3,4,5,20-
pentanor-l9-carboxyprostanoic acid (PGE-M), is excreted in
the urine (Hamberg and Samuelsson, 1971; Ferretti et al.,
1987).

The level of urinary PGE-M is a better indicator of

13

(3) B-oxidation

 

, h
“0 4 I k ‘.
I I I OH ‘\ \\
I \ w
i -
(2) PGR I (4) Hydroxylation &
' Oxidation
( 1 ) 1 S-PGDH
PGEZ
o
coon
. coon
H6 0
PG E-M

1 1a-hydroxy-9, 15 ,-dioxo-2,3 ,4,5 ,20-
pentanor-19-carboxyprostanoic acid

Figure 3. Sequence of reactions involved in the degradation of
PGE2 and formation of the major urinary metabolite
(PCB-M). 15-PDGH: lS-hydroxy-dehydrogenase, PGR:
Prostaglandin reductase.

14
whole body PGE turnover than PGE concentrations in plasma or

urine. Considering that white blood cells, platelets,

kidney, and prostate cells produce PGE, measuring PGE in

blood or urine does not accurately reflect whole body

turnover for prostaglandins. Assessment of urinary PGE-M

could be a valuable indicator for nutrition and clinical

modulation studies that are designed to modulate PGE turnover

(Ferretti et al., 1988; Ferretti et al., 1989; Schweer et al,

1990).

8.3. Quantitative analysis of PGE-M by Gas
Chromatography-Mass Spectrometry' (GC-MS)

GC-MS is a powerful microanalytical technique that
combines two sample analyzers - a gas chromatograph that
separates analytes, and a mass spectrometer that causes the
analytes to decompose into fragments which can then be used
for sample identification (Watson, 1985). Various strategies
are available to optimize analyte identification and
quantification. For example, in the total ion current (TIC)
chromatography mode, the mass analyzer records the abundance
of all ions generated by analyte degradation. TIC provides an
overall ion performance of the sample. The selected ion
monitoring (SIM) mode records only specific mass-to-charge
(m/z) values that have been selected because they
characterize the analyte of choice. The SIM technique
features high selectivity and high sensitivity and is ideal
for quantitative assays of biological samples (Seulter and

Watson, 1990).

15
8.4. Biological Function of PGE

Prostaglandin E1 (PGE1) and Prostaglandin E2 (PGEz) are
the major PGs of the E series and they are found in
relatively high concentrations in the renal system, coronary
tissues, and immune cells. Thus, the physiological activities
of PGE are closely associated with renal function, blood
pressure regulation, and immune response modulation. Not only
PGEz, but all the prostaglandins and leukotrienes, conduct
their physiological operation through the G protein-linked
receptors. The role of PGEz in water reabsorption in renal
collecting tubules represents a typical model of eicosanoid
action (Smith, 1989). Immuno-regulation is an important
normal function of PGE5 also. However, excessive
prostaglandin production is considered to promote

carcinogenesis.

8.5. PGE and Tumorgenesis

More and more studies have shown that PGs may play a
role in tumorgenesis, especially in tumor promotion. However,
discerning the role of PGE in tumor promotion is very
complicated since PGE is widely distributed throughout the
body and often PG synthesis is under tissue specific
regulation. Besides, low or high doses of PGs may exhibit
different or even opposite effects on tumor regulation; both
tumor inhibition and tumor promotion effects have been
suggested. For example, in many tumor cell lines, PG
synthesis is persistently raised, particularly the E series

PGs (Karmali et al., 1979; Bennett et al., 1979). In vivio it

16
is not known if the increase of PGs are produced by tumor

cells or by host-derived macrophages (Evans, 1973; Svennevig
and Svarr, 1979).

Phorbol esters, such as TPA, are potent tumor promoters.
TPA leads to the activation of PLA2 and enhances PG
synthesis; the tumor promoting effect of TPA may be in part

due to increased PG synthesis.

C . REGULATION OF PROSTAGLANDIN METABOLISM

C.1. Stimulators of Prostaglandin Synthesis

Under normal circumstances, the prostaglandins are
maintained at specific levels for their tissue specific
physiological functions; however, in many occasions, cells
may over-produce PGs and related eicosanoids and cause
improper physiological performance. Stimulators of PG
synthesis include hypersensitivity mediators such as
cytokines and lymphokines (Rankin et al., 1982), platelet
activating factor (Voelkel et al., 1982), ultraviolet light
(Morris et al., 1978), calcium.ionophores (Jakschik et al.,
1977), etc. In addition, as mentioned earlier, PG synthesis
in many tumor cell lines is significantly increased;
particularly the E series of PCs (Karmali et al., 1979;
Bennett et al., 1979). Thus, compounds that can affect
eicosanoid metabolism are important modulating reagents when
the body over-produces these physiologically active

eicosanoids.

l7
C.2. Inhibitors of prostaglandin synthesis

Drugs used to inhibit prostaglandin synthesis affect
different targets in the eicosanoid biosynthetic pathway.
Steroid hormones act up stream of AA.metabolism.by inducing
synthesis of proteins that inhibit PLA2. Consequently,
steroid hormones reduce the availability of PG precursors by
inhibiting AA release from phospholipids (Flower and
Blackwell, 1979). In addition, cyclooxygenase activity can be
inhibited by various drugs. These include the nonsteroid
anti-inflammatory drugs (NSAIDs) (Vane, 1971), the eicosa-
5,8,11,14 tetraenoic acids which act as analogs of AA (Ahern
and Douing, 1970), antioxidants (Gwebo, 1978; Bauman et al.,
1980), some phenolic compounds (Baumann et al., 1979), and
compounds in garlic and onion (Vanderhoek et al., 1980).
These substances can down regulate excessive production of
arachidonic acid metabolites and thereby modulate the
physiological abnormalities due to over-production of
prostanoids.

From a nutrition point of view, it is valuable to
identify dietary compounds that promote health and that may
have pharmacological activity. Garlic possess these
advantages and has been utilized for these purposes for
thousands of years. The Experimental Foods Program of the
National Cancer Institute (NCI) points out that over forty
foods with cancer preventive potential have been identified.
These food include garlic, cabbage, licorice, soybeans,

ginger, and umbelliferae; garlic is considered to have the

18
greatest anticancer potential (Caragay, 1992) (Figure 4). The

anti-cancer properties of garlic is associated with
inhibition of prostaglandin synthesis (Srivastava and Tyagi,
1993; Ali et al., 1993). Additional supportive studies will

be discussed in section D.

D. PHARMACOLOGICAL EFFECT OF GARLIC

0.1. Introduction

For thousands of years, garlic (Allium sativum) has been
used as a herbal medicine for a variety of ailments including
headache, heart problems, animal bites, intestinal parasites,
etc. (Block, 1985). Recent studies demonstrate that compounds
in garlic produce remarkable biological actions that may

correlate with the pharmacological effects noted in folklore.

13.2. The Chemistry of Garlic

In 1944, Cavallito et a1. (1944) produced an ethyl
alcohol extract of garlic that had anti-bacteria and anti—
fungal properties (Cavallito et al., 1944; Appleton and
Tansey, 1975; Barone and Tansey, 1977). Later studies have
shown that dietary garlic oil reduced the severity of
atherosclerosis in rabbits (Bordia et al., 1977) and serum.
cholesterol in rat (Sodimu, 1984). Several researchers
reported that various garlic extracts inhibit human platelet
aggregation by a variety of mechanisms (Bordia, 1978;
Vanderhoek et al., 1980; Aptiz-Castro et al., 1983; Mohammad,

1986; Mayeux et al., 1988; Shalinsky et al., 1989).

 

19

    
     

Garlic

Cabbage
Soybeans Ginger

Umbelliferae
(carrots, celery, parsnips)

Increase importance

        
       
    
   
  
 

 

Onion Tea Tumeric

Citrus (orange, lemon, grapefruit)
Whole wheat Flax Brown Rice
Solanacae (tomato, eggplant, peppers)

 

Cruciferous
(broccoli, cauliflower, brussels sprouts)
Oats Mints Oregano Cucumber

Rosemary Sage Potato Thyme Chives
Cantaloupe Basil Tarragon Barley Berries

 

Figure 4. Possible cancer-preventive foods and ingredients.

(Adapted from Caragay, 1992)

20
Many kinds of organosulfur compounds have been

identified in garlic; some of these compounds account for the
potent biological activities attributed to garlic (Block,
1985; Dorant, 1993) (Table 1). In fresh garlic, the majority
of the organosulfur compounds exist as y-Glutamyl-S-
allylcysteine (GSAC) (Figure 5). GSAC can be proteolytically
hydrolyzed to produce alliin during storage of the garlic
cloves. When a clove is crushed or cut, the enzyme allinase
is activated and it converts alliin to allicin, the source of
garlic odor. Allicin is responsible for much of the anti-
bacterial and anti-fungal properties of garlic. Allicin is
not stable and it self-condenses to form another potent
compound, ajoene. Ajoene was found to be an effective anti—
thrombotic agent that prevents platelet aggregation (Block et
al., 1986). Other compounds such as diallylsulfides, allixin
and even crude garlic extracts inhibit aflatoxin Bl induced

mutagenesis (Yamasaki et al., 1991).

D.3. Garlic inhibit Arachidonic Acid Metabolis-

The anti-thrombotic effect of garlic was associated with
arachidonate metabolism. It has been found that the 5-
lipoxygenase and prostaglandin synthetase activity were
strongly inhibited by ajoene (wagner et al., 1987). An anti-
platelet aggregation event was triggered by ajoene possibily
by the alteration of AA metabolism (Srivastava and Tyagi,
1983). Chemically synthesized allicin also showed an
inhibitory effect on platelet aggregation and neutrophil

enzyme release, but no direct effect on cyclooxygenase,

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y -Glutamyl-S-allycysteine (fresh garlic)
+ Proteolysis

Alliin (after storage)
( allyl-cysteine-sulfoxide)

* Allinase

Allicin —> anti-bacterial & anti-fungal

+ Self-condensation

Ajoene -—> anti-thrombotic agent

Figure 5. Organosulfur compound transformation in garlic.

23
thromboxane synthase, or prostacyclin synthase from.a number

of different cell sources (Mayeux et al., 1988). A.later
study confirmed that allicin did not alter cyclooxygenase
activity, but showed a dose dependent reduction of P632
formation, suggesting that allicin may act on the glutathione
dependent PGEz/PGEz isomerase, the enzyme responsible for
PGEz biosynthesis (Shalinsky et al., 1989). The prostaglandin
synthesis in the ovine ureter was also inhibited by an

aqueous extracts of garlic (Ali et al., 1993).

13.4. Garlic and Carcinogenesis

Cancer mortality has increased remarkably all over the
world during the last decades. Thus, many therapeutic anti-
carcinogenic agents have been developed. It is notable that
anti-cancer compounds are also found in the diet; and garlic
is one dietary source of anti-carcinogens.

Many epidemiological and laboratory studies have
demonstrated that garlic has anti-carcinogenic activity
(Dorant et al., 1993). In an area of China, increasing
consumption of allium vegetables including garlic,
sinificantly reduced gastric cancer development (You et al.,
1989). Ajoene, a natural sulfur-containing compound in
garlic, was tested for cytostatic activity towards primary
cultures, a permanent cell line, and a tumorgenic cell line.
Ajoene was most cytostatic to the tumor cells and least
cytostatic to the primary cells. This suggested that tumor
cells are more senstive to ajoene than non-tumor cells

(Scharfenberg et al., 1990). Diallyl disulfide, an

24
organosulfur metabolite found in garlic, suppressed

dimethylhydrazine (DMH)-induced colon cancer and nitrosa
methylbenzylamine (NMBA)-induced oesophageal cancer in
animals (Sumdyoshi and wargovich, 1989).

Basically, carcinogenesis involves initiation,
promotion, and progression stages. In order to prevent or
inhibit cancer, dietary anti-carcinogens should affect one or
more of the three broad stages of carcinogenesis. First,
carcinogenicity can be reduced by the biotransforming enzymes
such as cytochrome p450 and mixed function oxidases that can
be induced by anti-carcinogens. Second, initiation may be
blocked by preventing covalent binding of cancer causing
substances to the DNA. Furthermore, tumor promotion may be
inhibited by many compounds including retinol, tocopherol,
and organosulfates. Garlic and onion, rich sources of
organosulfates, are considered as inhibitors of tumor
promotion (Davis, 1989).

In a liver tumor model initiated by 1,2-
dimethylhydrazine (OMB) and promoted by orotic acid, diallyl-
dilsulfide (OAS), isolated from garlic, inhibited the
promotion process (Hayes et al., 1987). In a mouse skin tumor
model, which was initiated by 7,12-dimethylbenz[a]anthracene
(DMBA) and promoted by phorbol-myristate-acetate (PMA), onion
and garlic extracts elicited a dose-dependent inhibition on
EMA promotion (Belman, 1983). PMA stimulates prostaglandin
synthesis also (Bresnick et al., 1979; Ashendel et al.,

1979).

25
Lau et al. (1993) suggested several possible cancer

protective mechanism for compounds in garlic. The host immune
response may be modulated by garlic to act against tumor
growth. In addition, compounds in garlic could directly
inhibit tumor cell metabolism or inhibit the initiation
and/or promotion process of carcinogenesis. Since
prostaglandins and related eicosanoids play an important role
in the tumor promotion stage of carcinogenesis, the anti-
carcinogenic activity of organosulfur compounds may be
attributed to inhibition of tumor promotion by inhibiting

prostaglandin biosynthesis.

B . JUSTIFICATIOI

Several studies have indicated that the organosulfur
compounds in garlic inhibit biosynthesis of PGE. PGE is a
chemoattractant to macrophages which release 3202 that in
turn causes oxidative damage and cancer promotion.
Macrophages also secrete growth factors and proteases, two
critical biomolecules required during tumor promotion and
progression. Thus, overproduction of PGE may be a critical
step in the overall promotion and progression stages of

carcinogenesis.

I" . OBJECTIVE

The objective of this study was to develope a method to
determine the prostaglandin E metabolite in urine and to
determine if a garlic extract would suppress prostaglandin E

production in humans.

26
G . HYPOTBESIS

The main hypothesis of this study is that an
aqueous-ethanol garlic extract reduces PGE biosynthesis and

urinary PGE-M excretion in women.

[1. NULL HYPOTBBSIS

The null hypothesis to be tested in this study is that
consumption of an aqueous-ethanol garlic extract by women
will not cause a difference in PGE biosynthesis and urinary

PGE-M excretion.

MATERIALS AND METHODS

SZPSRMMAL DESIGN. The overall experimental design was
specified by the National Cancer Institute (NCI). Eight women
consumed garlic for three months in addition to their regular
daily diet. The efficacy of garlic in suppressing
prostaglandin E synthesis was assessed by measuring the
prostaglandin E metabolite (PGE-M) excretion before and after
three months of garlic supplementation. This study was
approved by the Michigan State University Committee on
Research Involving Human Subjects (IRB# 90—480).

SUBJECTS. Eight female subjects, 21 to 41 years of age,
were selected from.among the staff at the Life Sciences
building at Michigan State University. All were healthy with
no evidence of any disease based on history, physical
examination, cell blood count, urinalysis, and biochemical
profile. All subjects signed an informed consent form.prior
to participation in the study.

GARLIC SUPPLMNTATION. The garlic supplement
(I(yolic"l concentrate) used in this study was an aqueous-
ethanol garlic extract provided by the NCI. The organosulfur
composition of the supplement was determined by Dr. L.D.
Lawson at the Plant Bioactives Research Institute

(Springville, Utah). The analysis was not available until
27

28
after the feeding study was completed. Ten ml of the Kyolic'

concentrate was taken at breakfast every day for three months
in orange or vegetable (V8) juice (Campbell Co., Camden, NJ).

URINE SAMPLES. Twenty-four hour urinary excretion was
collected six times as shown in Table 2. Total volume of each
collection was measured, and then stored at -20°C. After
samples were thawed and thoroughly mixed, a 20 ml aliquot was
acidified to pH 3.5 for PGE-M analysis. Only the zero time
and the three month specimens were analyzed due to the
difficulty in measuring PGE-M.

Additional twenty-four hour urine collections were made
for three different days from a 28 yr female to establish
methodology. Also, urine samples were collected from one
individual (male, 49 years of age) before and after taking
aspirin. Aspirin was used as a "positive control treatment”.
This individual did not take any vitamin supplements or
:medicine for at least three days prior to the urine
collection. A baseline (no aspirin) 24 hr urine collection
and two 24 hr urine collections during aspirin treatment were
made. Urine collections during aspirin treatment started 6
hours after the first aspirin treatment (650 mg, 4
times/day). Each 24 hr urine sample was collected in
silanized glass bottles, and kept at 4°C during the

collection period.

REAGENTS AND MATERIALS. The internal standard, seven
deuterium labeled PGE-M ([2371—PGE-M) , was chemically

synthesized as described by Meese (1992). A 50 ng/ul stock

29

Table 2. Garlic supplemention and urine collection protocol.

 

STUDY PERIOD

 

Urine collection -1(wk) 0(mo) 1(mo) 2(mo) 3(mo) 4(mo)

Supplement Start --------------------- End

 

30
solution was prepared in absolute ethanol. All organic

solvents were either chromatography grade or redistilled
before use. Glassware silanizing reagent,
dimethyldichlorosilane (DMCS), was from Aldrich (Milwaukee,
WI). Fifty percent formic acid was prepared from 80%
concentrated formic acid with deionized water. The Pierce
nox®, and BSTEA - 1%TMCS reagents were used for methoximine
and trimethylsilate derivatiation respectively. 013 Sep-Pak
(500 mg) and silica Sep-Pak (500 mg) solid phase extraction
columns were purchased from.Waters Associates (Milford, MA).
One ml cone shaped vials with teflon lined caps were

purchased from Kimble (Vineland, NJ).

EQUIPMENT. Gas chromatography- mass spectrometry (GC-
MS) was done with a Hewlett Packard 5890 Gas Chromatography
and a 597GB Mass Selective Detector (MSD) (Figure 6). A DB"l -
5ms capillary column (15 M x 0.25 mm I.D, 0.25 un film
thickness) was used to separate the analytes. The GC oven was
temperature programmed from 190°C to 250°C at 5°C/min, from

250°C to 310°C at 30°C/min, and held at 310°C for l min.

IEETTNIDS!

GLASSHARB PREPARATION. A11 analytical operations were
done in silanized glassware. Glassware was soaked with 10%
Dimethyldichlorosilane (DMCS) for 5-10 min. Excess DMCS was
removed by rinsing with toluene, drying, soaking in methanol

for 5-10 min, rinsing with excess fresh methanol, rinsing

31

Ion Source

      
 

 

 

 

Mass Analyzer

 

 

 

to
vacuum

 

 

l

vacuum

vacuum

Figure 6. Schematic diagram of a Gas chromatography-Mass
spectrom try.

(Adapted from Watson, 1985)

32
‘with toluene and drying before use.

PGE-M Extraction from Urine. The overall procedure
for PGE-M extraction from urine was as described by Ferretti,
et al., (1983) with slight modification (Figure 7). ,
Twenty ml of urine was acidified to pH 3.5 by adding 50%
formic acid. Deuterium labeled PGE-M internal standard (200
ng) was added to the acidified urine and mixed with 20 ml of
conditioned XAD-Z resin in a 50 ml silanized glass beaker at
4°C for 1 hr. After filtration with No.50 filter paper under
suction, the resin was rinsed with 500 ml deionized water,
and then 100 ml of petroleum ether. The organic materials of
interest were eluted with a total of 120 ml absolute ethanol
in sequential 30 md rinses. The extract was rotoevaporated at
room temperature, then the residue was transferred in a small
amount of ethanol into a 20 m1 silanized glass tube and the
ethanol was evaporated under N2 gas at 38°C. The ethanolic
extract residue was methylated as described below. The
methylated residue was dissolved in 0.5 ml methanol, 9.5 ml
of HzO/MeOH (85:10, v/v) was added and the mixture was passed
through a conditioned octadecylsilyl (ODS) silica (Sep-Pak
C13 cartridge). The C13 cartridge was rinsed sequentially
with 20 ml deionized water, 10 ml of petroleum.ether, and 5
ml of methyl-formate/petroleum ether (5:95, v/v). PGE-M,
still bound to the C18 column, was eluted with 5 ml of
methyl-formate/petroleum.ether (25:75, v/v) and collected in

a silanized screw capped tube. After solvent evaporation

under nitrogen, the residue was dissolved in 100 pl of ethyl

33

20 ml Urine + 200 ng of [2H7]-PGE-M
1
mix with XAD-Z resin
1
elute PGE-M with 100% Ethanol

l
Methylate

1
clean up with 013 Sep-Pak

1
clean up with Silica Sep-Pak

l
Methoximate (MO)

1
Trimethylsilate (TMS)
l
GC-MS
Selected Ion Monitoring (SIM)

 

Figure 7. Scheme for the analysis of urinary PGE-M.

34
acetate/2,2,4-trimethy1 pentane (2:1, v/v), and

chromatographed over a 3 ml silica gel column. The PGE-M
methyl ester was collected in the third to eighth ml of
eluent (ethyl acetate/2,2,4-trimethyl pentane (2:1, v/v)).
After evaporation of solvent under nitrogen, methoximine and

trimethylsilyl ethers were prepared as described below.

lufltFMATIVE'FKHUImTION'

.Mbthylester formation (ME). Methanol (0.5 ml) and
2 ml of ethereal diazomethane solution were added to the
dried ethanol eluate and vortexed thoroughly. After reacting
for 10-15 min at room temperature, the solvent was evaporated
under a nitrogen stream.

Mothoxination (MO). Methoximines were prepared by
adding 125 pl of MOX® reagent to the dried residue eluted
from the silica gel Sep-Pak, vortexing thoroughly and heating
for 3 hours at 60°C. The pyridine was evaporated, 0.5 ml of
320 was added, and the methoximated derivatives were
extracted three times with 0.5 ml benzene. The benzene
extracts were combined for further derivatization.

Trimethylsilation (TMS). The dried residue from the
benzene extract of methoximated compounds was treated with
100 pl of the BSTFA - 1%TMCS reagent. After incubation at
80°C for 1 hr, the pyridine was evaporated and the residue
was redissolved in 10 ul of ethyl acetate. The sample was

then ready for GC-MS analysis.

Since the endogenous PGE-M and the [2871-PGE-M were

processed through the entire assay together, the efficiencies

35
of isolation, purification, and derivitization and the extent

of sample loss should be comparable for both the endogenous

PGE—M and the internal standard PGE-M. The [2H71-PGE-M was
utilized to identify the retention time of the endogenous
PGE-M.and to estimate the quantity of endogenous PGE-M as
described in the section below. The chemical and physical
characteristics for the endogenous PGE-M and the [287]-PGE-M
standard are identical except for the mass difference of
seven that comes from the deuterium atoms in the internal

standard.

QUANTITATIVE ANALYSIS OF PGE-N BI GC-HS

PGE-M was quantitated by GC-MS. Two ions, m/z 365 and
m/z 372, were determined in the Selected Ion Monitoring (SIM)
mode. Endogenous PGE-M was identified at m/z 365 according to
the retention time of the internal standard PGE-M on the mass
chromatogram. Quantification of endogenous PGE-M was

calculated by the equation:

:Endogenous PGE-M PEAK AREA.endogenous pGEEM (m/z365)

 

 

[237l-PGE-M PEAK AREA [237]-PGE-M (m/z372)

Since PGE-M slowly degenerates during storage,
correction for decay of PGE-M during urine storage was done
as described by Seyberth el al., (1976) . PGE-M has a half

life of 318 days; the data were corrected by the equation:

36
ln C = ln Co — 0.002180t where C = estimated PGE-M

concentration, Co = adjusted PGE-M concentration, and

t = days of storage before PGE-M analysis.

STATISTICAL METHODS
GC-MS detector response for PGE-M injection was tested
by linear regression analysis. Garlic treatment effect on

urinary PGE—M excretion was evaluated by the paired t-test.

RESULTS AND DISCUSSION

In this study, the measurement of urinary PGE-M was done
by SIM since SIM can distinguish the [2371-PGE-M from the
endogenous PGE-M and because of the high sensitivity and
selectivity of SIM GC-MS. Prior to analysis by GC-MS, both the
endogenous and internal standard PGE-M had to be extracted,
concentrated and partially purified from the complex mixture
of biological compounds in urine specimens. Sample clean up
was absolutely essential to minimize interference from ions
derived from the overwhelming amounts of other urinary
compounds relative to PGE-M. In addition, in order to
increase thermal stability and volatility and to decrease
polarity, PGE-M had to be derivatized for GC analysis. PGEeM,
the lla-hydroxy-9,15,-dioxo-2,3,4,5,20-pentanor-19-
carboxyprostanoic acid, with its one hydroxyl, two keto, and
two carboxyl groups, was derivatized to the trimethylsilyl
ether, o-methyloxime, and methyl ester (ME-MO-TMS) derivative
which could then be analyzed by GC-MS (Figure 8). Figure 9
shows the TIC chromatogram for the ME-MO-TMS derivative of
[2317-PGE-M under the GC conditions that were described in
the Materials and Methods section.

The molecular ion (M+), or the intact ionized molecule

of the ME—MO-TMS [ZH]7-PGE-M, was determined at a m/z of 493.

37

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Figure 9. Total ion current (TIC) chromatogram of ME-MO-TMS
[2H7]-PGE-M.

 

40
The base peak, the most intense peak in the mass spectrum, of

the ME-MO-TMS [2H17-PGE-M was determined at m/z 372 which
represents the [M+ minus (0CH3) minus (CH3)3 minus SiOH)] ion
([M+-0CH3-(CH3)3-Si0H]). Scanning the mass spectrum of each
peak in Figure 9 showed that the base peak was the
predominant ion detected for peaks with retention times of
10.241 min (Figure 10a), 10.132 min (Figure 10b), 9.838 min
(Figure 11a), and 9.701 min (Figure 11b). The peaks at 7.544
min (Figure 12a) and 7.289 min (Figure 12b) had a different
pattern of mass to charge ions. These peaks were found in
"reagent blanks" and most likely resulted from the
derivatization reagents.

The highest mass shown on the mass spectrum.in Figure 10
and Figure 11 was m/z 462 instead of the molecular ion (M+)
of m/z 493. Frequently, the M+ is not found in a mass
spectrum because all of the analyte is fragmented within the
mass chamber and none of the parent compound with a mass of
m/z 493 remains. The -0CH3 radical was released from the
oximine moiety of the M+ and this [M-31]+ fragment was then
detected at m/z 462 (Figure 13). However, the predominant
fragment was [M+ - ~ocn3 - °(CH3)3 SiOH], ([M-121]"'), which
was detected at m/z 372. Figure 14 shows the mass
chromatogram when the GC-MS was operated in the SIM mode and
the m/z 372 was monitored. Figure 14 is consistent with the
TIC for the [2H71-PGE-M derivative for retention times 9.6-
10.5 min as shown in Figure 9. Four peaks were produced by

the standard since each keto group produced a syn- and an

41

 

a. Scan 419 (18.24lm1n) \
1 372
3 (2155‘
l
v 2 95?
U .1
c 1
N
“U .
c ?3
:1 1
a ./
1 055*

 

 

  

100 200 308 400
Mass/Charge

b. Scan 414119.132 min)
2.2554; \
2.955% 372
1.955%
1.651

 

 

1.4551 73
1.255l/ -
.055?
.054?

a .
6.0E4‘; / l \
4 .

Rbundance

 

 

    

100 200 300 400
Misc/Charge

 

 

 

Figure 10. Scans of the mass spectrum of the most intense peaks in
the TIC chromatogram of ME-MO-‘I‘MS [2H7]-PGE-M at
the retention time of 10. 241min (a) and 10.132 min
(b). The m/z 372 ion is the base peak shown in both
mass spectrums.

442

 

 

 

 

 

 

 

 

a. Scan 399 (9.999 min} \
9.054% 372
3.054%
7.054%
5.054?
3s 05 i 73
c. 4‘.
S 1///
§4.GE41
g' :
3.0546 259
1 /// 462
x 340
10000 \\\ j? I /
G Li I ,1 l - .
100 200 300 400
Mass/Charge
Scan 392 (9.70] min)
b. \
372
4.054‘
/73
3.054-
O
o C
S
t
$2.054-
1E ‘21 462
259
193 / \
/ 357
10000 25
[L I II \I
. , II 1‘ I11 ,
100 200 300 400
Mass/Charge

 

 

 

Figure 11. Scans of the mass spectrum of the most intense peaks in
the TIC chromatogram of ME—MO-TMS [2H71-PGE-M at .
the retention time of 9.838min (a) and 9.701 min (b).
The m/z 372 ion is the base peak shown in both mass
spunflh1rnis.

43

 

 

 

 

 

2L Scan 281 (7.544 min)
. 250
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100 208 300 400
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191

179

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1.05s€ \ -

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180 200
Hess/Chagggi

 

 

 

 

 

 

 

 

 

Figure 12. Scans the mass spectrum of the intense peaks in the
TIC chromatogram of ME—MO-TMS [21171-901514 at. the
retention time of 7.544min (a) and 7.289 min (b).
Compared with the previous mass spectrums in Figure 10

and Figure l 1, different spectrum patterns are produced,
and the m/z 372 ion is not present.

44

 

 

 

 

 

 

 

 

 

[M-121]+4—. +
(m/z 372) [M31] "
(m/z 462) [Mr

(m/z 493)

 

 

Figure 13. Analyte fragmentation occurrs within the mass
chamber with release of OCH3 and ~(CH3)3SiOH
radicals from the molecular ion (M+, m/z 493). If only
OCH 3 is released, the [M-31]+ fragment is formed and
the m/z 462 ion is produced; if both OCH3 and

-(CH 3) 3 SiOl-l are released, then the [M-121]+ fragment
is formed and the m/z 372 ion is produced.

45

 

 

 

 

 

 

 

  

 

 

 

 

Ion 372.00 amu.
4.0ES‘ ‘
3.0ES- ._
. m
:53
m ._
o .
5 2 055-
‘o. ' .
S
E E
‘ a;
1.055- 2
‘ (D
. a;
'9 ' 1 F ' ‘1 I ' ' ' ff ' - v v 1 w v
9.8 9.8 10.0 10.2 10.4 10.8
Time (m1n.)

 

 

Figure 14 . From the TIC profile of ME-MO-TMS [Mu-poem, the
m/z 372 ion was monitored to create a new mass
chromatogram, which identified the retention time of

this standard compound.

46
anti- methoxime isomer; therefore, four isomers were formed

from the two keto groups on PGE-M.

Various amounts of ME-MO-TMS [2H7]-PGE-M were injected
into the GC-MS and the abundance of the m/z 372 ion was
recorded. A linear relationship ( Y = 161839 + 289758 (r=
0.992)) was observed when 0.1, 0.5, 1.0, 5.0, 10.0, 100.0,
and 200.0 mg of PGE-M were injected. The regression equation
and the 95% confidence intervals are shown in Figure 15.
These data demonstrate that the abundance of the m/z 372 ion
was proportional to the amount of PGE-M injected into GC-MS.

Figure 16 shows a typical chromatograph for
quantification of endogenous PGE-M. Two hundred ng of [2H7]-
PGE-M was added to 20 ml of urine (from a female, 28 years of
age) and the PGE-M was extracted as described in the
Materials and Methods section. The abundance of two ions, m/z
365 and m/z 372 which correspond to the [M+-(-0CH3+
~(CH3)3Si0H)] fragments for endogenous PGE-M and [2H7]-PGE-M
respectively, were monitored in the SIM mode. The four
isomers of endogenous PGE-M and of [2H7]-PGE-M are noted on
the chromatogram.

The urinary PGE-M excretion from this subject was
estimated as 315.9 1 33.1 ng/20 ml (mean 1 SEM, three 24 hr
samples) which was similar to that reported (395 1 22 (SE)
ng/ZO ml) by Ferretti, et al.(1983). The 24 hr urinary PGE—M
excretion was calculated as 18.2 t 1.9 ug/24 hr (mean 3 SEM,
three 24 hr samples).

Aspirin and NSAIDs are potent prostaglandin biosynthesis

Detector Response

47

 

 

 

 

40000000 I
x 1
y = 161839 x 4» 289758 ( r = 0.992) .’
35000000- x
3m— 1” 30°.
25000000- ."
20000000- x'
’V
1m - ”l o
I,’ Regression Linc
10000000 - x” - - - o - - - Upper 95% CI
”4' o m95%Cl
50000001 /
47'
o 50 100 150 200 250
ng of PGE-M injection

Figure 15. The relationship between amount of [2H7J-PGE-M
injected (0.1 ng - 200 ng) and the GC—MS detector

response.

48

 

 

[on 365.00 All).

 

 

 

2 055 l 1 2.055
1.555 1 ’4 1.555
s n t:

1.055 3 3, g : .1: n ‘3 g 1.055
n c5 c1 ‘ a: " a:

5.054 c; a; a, °' °' a. a 5.054
W

@J /\1”\/‘ :0

Ion 372.00 am.

1

N
S
n
M
‘—

m
A
m
(I)

l
a
S
a;
s 054 v/\_

8.881

 

 

 

11.1; 9.0 9' 9.4 9.6 _s.0

.0 9.2
Time (Mm)

 

 

a.
b
Figure 16.

SIM chromatograms of an urine sample. The mass
chromatogram of endogenous PGE-M is shown at

m/z 365(a) and the [2H71-PGE-M internal standard is
shown at m/z 372 (b). The endogenous PGE-M peaks
(panel a) relative to the internal standard peaks
(panel b) are identified by the arrow symbols (1).
The peaks identified by (1) are isomers.

49
inhibitors (vane et al., 1971; Flower, 1974). Therefore, the

excretion of PGE-M before and after aspirin (acetyl salicylic
acid) treatment was determined in a 49 year old male to serve
as a ”positive control treatment"; the results are shown in
Table 3. Aspirin treatment (4 x 650 mg aspirin/day) reduced
urinary PGE-M production to 19% to 47% of baseline value.
Seyberth (1976) reported that aspirin treatment (4 x 650 mg
aspirin per day caused a 45% to 70% decrease in PGE-M
excretion in eight 19-30 yrs males.

The effect of garlic treatment on the PGE-M excretion in
eight women is summarized in Table 4. The mean baseline
(before treatment) PGE-M value is 42.7 i 9.5 (mean 1 SEM,
N28) and the PGE-M excretion after three month of garlic
treatment is 35.9 i 4.7 (mean 1 SEM, N28). PGE-M excretion
during baseline and after garlic treatment were not
significantly different as determined by the paired t test
(P = 0.29). The aqueous-ethanol garlic extract did not
decrease the PGE-M excretion as expected. There may be
several reasons why the garlic extract did not inhibit
prostaglandin synthesis. First, the organosulfur compounds
that are in the aqueous-ethanol garlic extract (Table 5) may
not be active inhibitors of prostaglandin biosynthesis.
Allicin and ajoene are the most potent inhibitors of
prostaglandin synthesis (Block, 1985). However, the
concentration of these two compounds in the Kyolic‘
concentrate are very low (Table 5). The amounts of S-Allyl-

mercapto-cysteine, S-trans-l-propenyl-cysteine and

50

Table 3. Twenty-four hour urinary PGE-M excretion before and
after aspirin treatment ( 4 x 650 mg/day ).

 

PGE-M ( ug/ 24 hr)21 Percentage (96)

 

Baseline 44.1 :1: 2.0 100 %
Aspirin Day 1 8.2 :1: 1.2 19 96
Aspirin Day 2 20.7 :1: 1.6 47 %

 

a Values are mean :1: CV.

51

Table 4 . Twenty-four hour urinary PGE-M excretion ( ug/ 24 hr) for
eight women before and after three months consumption
of an aqueous-ethenol garlic extract.

 

 

 

PGE-M (118/24 hr)a
Subject Baseline Garlic Treatment
No. 1 35.8 42.8
No. 2 38.5 30.2
No. 3 72.4 35.0
No. 4 12.1 56.6
No. 5 89.7 24.5
No. 6 21.6 46.9
No. 7 50.8 37.0
No. 8 20.3 13.9
mean 3: SEM 42.7 :t 9.5 35.9 :1: 4.7

 

3- Analyzed in duplicate.

52

 

 

Table 5. Organosulfur compounds in Kyolic” concentrate and

garlic. 3

(micrograms/ gram)
Compound Kyolicn‘ b Crushed C Fresh dried 9
Garlic Garlic
(fresh wt.) (dry Wt.)

Alliin (allyl- 610 400 27,400
cysteine-sulfoxide)
Allicin < 2 3900 < 2
y-Glutamyl-S- 200 4900 8300
allylcysteine
S-Methyl-cysteine 270 40 1 1 0
S-Allyl 185 < 20 < 20
mercaptocysteine
Methionine 95 23 45
Ajoene < 1 < 1 < 5
y-Glutamyl-S-trans- 92 4600 8600
1 ~propenylcysteine
S-Allylcysteine 1165 < 20 1080
S—Trans-l- 530 < 20 310
propenylcysteine

 

3 Analysis was done by Dr. L.D. Lawson (Bioactives Research Institute,
Springville, Utah).

The garlic supplement used in this study.

Garlic cloves (68% moisture content) homogenized in water.
Powder of typical store-purchased garlic.

Allyl sulfide, Diallyl sulfide, Diallyl disulfide, Diallyl trisulfide,
methyl allylsulfide, methyl allyl disulfide and methyl allyl
trisulfide were present in minimal amounts (less than 10 pg/ m1
concentrate).

*Qfic‘

53
Squlylcysteine are high in the Kyolic' concentrate. However,

it is not known if they inhibit PG synthesis.

In fresh garlic, a major organosulfur compound is y-
Glutamyl-S-allylcysteine (GSAC). During storage, GSAC is
proteolytically hydrolyzed to alliin (Figure 5). Neither GSAC
nor alliin is active until chemically transformed to allicin
as described previously. It is assumed that GSAC and alliin
can be converted to allicin during digestion. So, in the case
of fresh and freeze dried garlic, there are large amounts of
allicin and precursors (fresh garlic) or allicin precursors
(freeze dried). In the Kyolic"l concentrate used in this
study, allicin and its precursors are minimal compared to the
fresh and freeze dried garlic. Therefore, the effectiveness
of Kyolic‘ concentrate to inhibit PG synthesis could be very
limited because of insufficient inhibitors.

Moreover, during three months of garlic treatment, there
may be compensation and up regulation of prostaglandin
biosynthesis in response to continued consumption of PG
inhibitors. Thus, a small inhibition of PG synthesis by long
term treatment with garlic extract may not be detected.

The Kyolic'l concentrate was the choice of the NCI, since
it contains S-allylcysteine, trace amounts of diallyl
disulfide, and diallyl trisulfide - chemicals that inhibit
tumorgenesis (Dorant, 1993). However, those allyl-containing
compounds may not be associated with inhibition of PG

synthesis.

SUMMARY AND CONCLUSIONS

Garlic (Allium sativum) contains organosulfur compounds
that inhibit the biosynthesis of prostaglandin E (PGE). PGEs
are associated with inflammatory response and the tumor
promotion stage of carcinogenesis. Therefore, this study was
conducted to determine if an aqueous-ethanol extract of
garlic (Kyolicfll concentrate) could inhibit prostaglandin E
metabolism.

Eight females (21 to 41 years of age) consumed 10 ml of
Kyolic‘ concentrate for three months. Twenty-four hour urine

collections were made before and after three months of garlic

consumption to measure the excretion of the Ila-hydroxy-
9,15,-dioxo-2,3,4,5,20-pentanor-l9-carboxyprostanoic acid
(PGE-M) since PGE-M reflects whole body turn over of
prostaglandin El and Ez. There was no difference in PGE-M
excretion before (42.7 t 9.5 ug/24 hr)(mean r SEM, N= 8) and
after three months of garlic consumption (35.9 t 4.7 ug/24
hr)(mean t SEM, N=8) (P = 0.29). These data show that
consuming 10 ml of Kyolic‘ concentrate daily for three months
did not result in a significant decrease in PGE-M excretion
in women.

The aged aqueous-ethanol garlic extract (Kyolic‘) has
relatively low concentrations of prostaglandin synthesis

54

55
inhibitors (allicin and its precursors) compared to fresh or

freeze-dried garlic. The low intake of allicin and precursors
is the most likely explanation for the lack of treatment

effect in this study.

RECOMMENDATIONS FOR FUTURE RESEARCH

For future research, if the objective is to inhibit PG
synthesis, a product rich in allicin and/or ajoene should be
selected.

Another consideration should be that, the concentration
of urinary PGE-M in a normal individual is extremely low --
in the ng per ml level. Therefore, inhibition of PGE
metabolism.in normal subjects will be very difficult to
detect. Thus, for future studies, the subjects could be
carefully selected to have inflammatory disorders, e.g.
allergy, asthma, rheumatoid arthritis or cystic fibrosis -
patients with these conditions that have elevated
prostaglandins production. The effect of garlic treatment on
PGE-M excretion could then be examined more efficiently and
the results applied to pharmaceutical situations.

In vitro studies would be valuable additions to human
studies. For example, several immunocytes, such as
macrophages and neutrophils, synthesize significant amounts
of prostaglandins. These cells can be isolated from humans or
animals and treated with garlic extracts. The production of
PGs with or without garlic treatment could be determined.
Similar assays could be conducted with the cell lines that

synthesize elevated levels of PGs. In addition, cytotoxic

56

57
effects from various doses of garlic extracts could be also

examined.

3181. I OGRAPHY

BIBLIOGRAPHY

Ahern, D.G. and Downing D.I. Inhibition of prostaglandin
biosynthesis by eicosa-5,8,11,14—tetrayonic acid.
Biochemica. et biophysica. acta. 210:456-461,1970.

Ali, M., Angelo-Khattar, M., Farid, A., Hassan, R.A.H. and
Thulesius, O. Aqueous extracts of garlic (Allium
sativum) inhibit prostaglandin synthesis in the ovine
ureter. Prostagl.Leukotr.Essent. 49:855-859, 1993.

Appleton, J.A. and Tansey, M.R. Inhibition of growth of
zoopathogentic fungi by garlic extract. Mycologia
67:882-885, 1975.

Aptiz-Castro, R., Cabrera, S., Cruz, H.R., Ledezma, E. and
Jain, M.K. Effects of garlic extract and of three pure
components isolated from it on human platelet
aggregation, arachidonic metabolism, release reaction
and platelet ultrastructure. Thromb. Res. 32:155-169,
1983.

Ashendel, C.L. and Boutwell, R.K. Prostaglandin E and F
levels in mouse epidermis are increased by tumor
promoting phorbol esters. Biochem. Biophys. Res. Commun.
90:623-627, 1979.

Barone, F.E. and Tansey, M.R. Isolation, purification,
identification, synthesis, and kinetic of activity of
the anti-candidal component of Allium sativum, and a
hypothesis for its mode of action. Mycologia 69:793-825,
1977.

Baumann, J., van Bruchhausen, F. and WUrm, G. Flavanoids and
related compounds as inhibitors of arachidonic acid
peroxidation. Prostaglandins 20:627-639, 1980.

Baumann, J., VOn Bruchhausen, F. and WUrm, G. A.structure-
activity study on the influence of phenolic compounds
and bioflavanoids on renal prostaglandin synthetase.
Naunyn-Schmiedeberg's Arch. Pharm. 307:73-78,1979.

Belman, S. Onion and garlic oils inhibit tumor promotion.
Carcinogenesis 4:1063-1065, 1983.

58

59

Bennett, A., Boughton, J., Leaper, D.J. and Stamford, I.F.
Cancer growth, response to treatment and survival time
in mice: beneficial effect of prostaglandin synthesis
inhibitor flurbiprofen. Prostaglandins 17: 179, 1979.

Bito, L.z., Baroody, R.A., and Reitz, M.E. Dependence of
pulmonary prostaglandin metabolism.on carrier mediated
transport processes. Am. J. Physiol. 253:273-285, 1977.

Block, E. The chemistry of garlic and onions. Sci. Am.
285:114-119, 1985.

Block, E., Ahmad, S., Catalfamo, J.L., Jain, M.K. and Apitz-
Castro, R. Antithrombotic organosulfur compound from
garlic:structural, mechanistic, and synthetic studies.
J. Am. Chem. Soc. 108:7045-7055, 1986.

Bordia, A., Verma, S.K., vyas, A.K., Khabya, B.L., Rathore,
A.S., Bhu, N., and Bedi, B.K. Effect of essential oil of
onion and garlic on experimental atherosclerosis in
rabbits. Atherosclerosis 26:379-386, 1977.

Bordia, A. Effect of garlic on human platelet aggregation in
vitro. Atherosclerosis 30:355-360, 1978.

Caragay, A.B. Cancer-preventive foods and ingridents. Food

Cavallito, C.J. and Bailey, J.H. Allicin, the anti-bacterial
principle of.Allium.Sativum. 1. Isolation, physical
properties and antibacterial action. J. Am. Chem. Soc.
66:1952-1954, 1944.

Davis, D.L. Natural anticarcinogens, carcinogens, and
changing patterns in cancer: some speculation. Environ.
Res. 50:322-340, 1989.

Di Rosa, M. and Persico, P. Mechanism.of inhibition of
prostaglandin biosynthesis by hydrocortisone in rat
leukocytes. Br. J. Pharmacol. 66:161-163, 1979.

Deby-Dupont, G., Ducarne, E., De Landsheere, C., Ancion,
J.C., Noel, F.X., Radoux, L. and Deby, C. Intense rise
of unesterified arachidonic plasma levels in stressed
humans. Biomed. Pharmacother. 37:386-391, 1983.

Dorant, E. van der Brandt, P.A., Goldbohm, R.A., Hermus,
R.J.J. and Sturmans, F. Garlic and its siginificance for
the prevention of cancer in humans: a critical View.

Br. J. Cancer 67:424-429, 1993.

60

Eling, T.E., and Ally, A.I. Pulmonary biosynthesis and
metabolism of prostaglandins and realted substances.
Bull. Europ. Pysiopathlo. Respir. 17:611-625, 1981.

von Euler, U.S. 0n the specific vasodilating and plain muscle
stimulating substances from accessory genital glands in
man and certain animals (prostaglandin and
vesiglandins). J. Physiol. 88:213-237, 1963.

Evan, R. Macrophages and tumour bearing host. Br. J. Cancer
28 (suppl 1):19-21, 1973.

Ferreira, S.H. and Vane, J.R. Mode of action of anti-
inflammatory agents which are prostaglandin inhibitors.
In Anti-inflammatory drugs-Handbook of Exp. Pharmacol.,
eds J. R. Vane & Ferreira, Berlin: Springer-Verlag, p. 348-
349,1978.

Ferretti, A. Flanagan, V.P. and Roman, J.M. Quantitative
analysis of 11a-hydroxy-9,15,-dioxo-2,3,4,5,20-pentanor-
19-carboxyprostanoic acid, the major urinary metabolite

of E prostaglandin in man. Anal. Biochem. 128:351-358,
1983.

Ferretti, A., Flanagan, V.P. and Reeve, V.B. Stable isotope
dilution assay for prostaglandin E metabolite: 24-hour
urinary output in healthy male subjects. Anal. Biochem.
167:174-180, 1987.

Ferretti, A., Flanagan, V.P. and Reeve, V.B. Occurrence of
prostaglandin E3 in human urine as a result of marine
oil ingestion: gas chromatographic-mass spectromteric
evidence. Biochimica. et Biophysia. Acta. 959:262-268,
1988.

Ferretti, A., Judd, J.T., Taylor, P.R., Schatzkin, A. and
Brown, C. Modulating influence of dietary lipid intake
on the prostaglandin system in adult men. Lipids 24:419-
422, 1989.

Ferretti, A., Judd, J.T. and Ballard-Barbash, R. Effect of
fish oil supplementation on the excretion of the major
metabolite of prostaglandin E in healthy male subjects.
Lipids 26:500-503, 1991.

Flower, R.J. Drugs which inhibit prostaglandin biosynthesis.
Pharm. Rev. 26:33-67, 1974.

Flower, R.J. and Blackwell, G.J. Anti-inflammatory steroids
induce biosynthesis of phospholipase A2 inhibitor which
prevents prostaglandin generation. Nature 278:456-459,
1979.

61

Gemsa, D., Seitz, M., Kramer, W., Grimm, W., Till, G., and
Resch, K. Ionophore A 23187 raises cyclic AMP levels in
macrophages by stimulating Prostaglandin E formation.
Exp. Cell Res. 118:55-62, 1979.

Gryglewski, R. J. Prostaglandins and prostaglandin synthesis
inhibitors in etiology and treatment of inflammation.
In: Matilla, M. J. (ed) Clinical pharmacology, vol 5.
Oxford, Pergamon Press, p.157, 1975.

Gwebo, E.T. Anti-oxidants and the inhibition of lipoxygenase
and cyclo-oxygenase enzyme systems. Ph.D. Thesis, Ohio
State Univ. 1978.

Hirata, F. Lipomodulin; a possible mediator of glucocorticoid
action. In: Vth International Conference on Prostagl.
Abstr. Book, Florence, p.122, 1982.

Hagenfeldt, L., Eagenfeldt, K., and Wennmalm, A. Turnover of
plasma—free arachidonic and oleic acids in men and
women. Horm. Metab. Res. 7:467—471, 1975a.

Hagenfeldt, L. and wahzen, J. Turnover of plasma-free
arachidonic and oleic acids in resting and exercising
human subjects. Metabolism.24:799-806, 1975b.

Hamberg, M. and Samuelsson, B. On the metabolism of
prostaglandin E1 and E2 in man, J. Biol. Chem. 246:6713-
6721, 1971.

Hayes, M.A., Rushmore, T.B. and Goldberg, M.T. Inhibition of
heptocarcinogenic responses to 1,2-dimethylhydrazine by
diallyl sulfide, a component of garlic oil.
Carcinogenesis 8:1155-1157, 1987.

Hong, S.L., and Levine, L. Stimulation of prostaglandin
synthesis by bradykinin and thrombin and their
mechanisms of action on MES-5 fibroblasts. J. Biol.
Chem. 251:5814-5817, 1976.

Jakschik, B.A., Falkenhein, S. and Parker, C.W. Precursor
role of arachidonic acid in the release of SRS. Proc.
Natl. Acad. Sci. USA 74:4577-4591, 1977.

Karmali, R.A., Borrobin, D.F., Menezes, J. and Patel, P. The
relationship between concentrations of prostaglandin

.A1, E1, E2 and Flu and rates of cell proliferation.
Pharmacol. Res. Commun. 11:69, 1979.

Lapetina, E.G., Schmitges, C.J., Chandrabose,K. and
Cuatrocasas, P. Regulation of phospholipase activity in
platelets. Adv. Prostagl. Thromb. Res. 3:127-135, 1978.

62

Lim, L.M., Hunt, N.H., Evans, T. and weidemann, M.J. Rapid
changes in the activities of the enzymes of cyclic AMP
metabolism after addition of ionophore A 23187 to
macrophages. Biochem. Biophys. Res. Commun. 103:745-750,
1981.

Mayeux, P.R., Agrawal, K.C., Tou, J.S.H. and King, E.T. The
pharmacological effects of allicin, a constituent of
garlic oil. Agents Actions 25:182-189, 1988.

Mohammad, S.F. and woodward, S.C. Characterization of a
potent inhibitor of platelet aggregation and release
reaction isolated from Allium sativum (garlic). Thrombo.
Res. 44:793-806, 1986.

Meese, C.O. Novel syntheses of 2,3,4,5-tetranor- and 6-oxo-
prostaglandins. Liebigs Ann. Chem. 1217-1223, 1992.

Minkes, M., Standford, N., Chi, N.Y., Roth, G.J., Needleman,
P. and Majerus, P.W. Cyclic adenosine 3',5'-
monophosphate inhibits the availability of arachidonate
to prostaglandin synthetase in human platelet
suspensions. J. Clin. Invest. 59:449-454, 1977.

Morris, H.R., Taylor, G.W., Piper, P.J., Sirloins, P. and
Tippins. J.R.: SRS-A: purification and characterization.
FEBS Lett. 87:203-206, 1978.

Nakano, J. and McCurdy, J.R. Cardiovascular effects of
prostaglandin E1. Journal of Pharmacol. Exp. Ther. 156:
538-547, 1969.

Nolan, R.D., Dusting, G.J. and Martin, T.J. Phospholipase
inhibition and the mechanism of angiotensin-induced
prostacyclin release from rat mesenteric vasculature.
Biochem. Pharmacol. 30:2121-2125, 1981.

Norcia, L.N., and Evans, J.D. Composition of rabbit plasma
ree fatty acids. Fed. Proceed. 21:290b, 1962.

Ogino, N., Miamoto, Y., Yamamoto, S. and Hayaishi, O.
Prostaglandin endoperoxide E isomerase from bovine
vascular gland microsomes, a glutathione requiring
enzyme. J. Biol. Chem. 252:890-895,1977.

Piper, P.J., Vane, J.R. and wyllie, J. In activation of
prostaglandins by the lungs. Nature 225:600-604, 1970.

Rankin, J.A., Hitchock, B., Merril, N., Bach, M.K., Brashler,
J.R. and Askenase, P.W. IgE dependent release of LTC4
from alveolar macrophage. Nature 297:329-335, 1982.

63

Samuelsson, B., Granstrom, B., Greon, K. and Hamburg, M.
Metabolism of prostaglandin. Ann. NY Acad. Sci. 180:
138-163, 1971.

Samuelsson, B. Endogenous synthesis of prostaglandins in
guinea pig and man: Effects of inhibitors, in
prostaglandin Synthetase Inhibitors Ed by H. J.
Robinson and Y.R. Vane,) Raven Press, New York, p.99,
1974.

Scharfenberg, K., wagner, R. and wagner, K.G. The cytotoxic
effect of ajoene, a natural product from garlic,
investigated with different cell lines Cancer Lett.
53:103-108, 1990.

Schweer, H., Meese, C.O. and Seyberth, H.W. Determination of
11a-hydroxy-9,15,-dioxo-2,3,4,5,20-pentanor-l9-
carboxyprostanoic acid by gas chromatography/negative

ion chemical ionization triple-stage quadrapole mass
spectrometry. Anal. Chem. 189:54-58, 1990.

Seyberth, H.W., Sweetman, B.J., Frolich, J.C., and Oates,
J.A. Quantification of the major urinary metabolite of
the E prostaglandins by mass spectrometry: evaluation of
the method's application to clinical studies.
Prostaglandins 11:381-389, 1976.

Shalisky, D.R., McNamara, D.B. and Agrawal, K.C. Inhibition
of GSH—dependent PGH2 isomerase in mammary
adenocarcinoma cells by allicin. Prostaglandins 37:135-
148, 1989.

Smith, W.L. The eicosanoids and their biochemical mechanisms
of action. Biochem. J. 259:312-324, 1989.

Sodimu, 0., Joseph, P.K. and Augusti, K.T.I. Certain
biochemical effects of garlic oil on rate maintained on
a high fat-high cholesterol diet. Experientia 40:78-80,
1984.

Struyck, C.B., Beerthuis, R-K. and‘Van Dorp, D.A. Specificity
in the enzymatic conversion of polysaturated fatty acids
into prostaglandins.Prostaglandins,Stockholm. In:Nobel
Symposium II, 51-56, 1966.

Suelter, C.H. and watson, J.T. Biomedical applications of
mass spectrometry. Volume 34, Wiley, New York, 1990.

Sumiyoshi, H. and Wargovich, M.J. Effect of organosulfur
compounds on 1.2~dimethy1-hydrazine-induced nuclear
toxicity and GST activity in mouse colon. Proc. Am.
Assoc. Cancer Res. 30:181, 1989.

64

Sun, F.F., Chapman, J.P., and McGuire, J.C. Metabolism of
prostaglandin endoperxide in animal tissues.
Prostaglandins 14:1055-1074, 1977.

Svennevig, J.L. and Svaar, H. Content and distribution of
macrophages and lymphocytes in solid malignant human
tumors. Int. J. Cancer 24:754,1979.

Vanderhoek, J.Y., Makheja, A.N and Bailey, J.M. Inhibition of
fatty acid oxygenases by onion and garlic oil. Evidence
for the mechanism by which these oil inhibit platelet
aggregation. Biochem. Pharmacol. 29:3169-3173,1980.

Vane, J.R. Inhibition of prostaglandin synthetase as a
mechanism of action for aspirin-like drugs. Nature New
Biol. 231:232-235, 1971.

voelkel, N.F., WOrthen, S. and Reevesd, J.T. Non-
immunological production of leukotrienes induced by PAF.
Science 218:286-303, 1982.

voet, D. and Voet, J.G. Biochemistry. Wiley, New York, p.658-
665, 1990.

wagner, H., Wiere, M. and Fessler, B. Effects of garlic
constituents on arachidonate metabolism. Planta medica
53:305-306,1987.

Watson, J .T. Introduction to mass spectrometry. Raven Press,
New York, p.307-331,1984.

Yamasaki, H., Mufson, R.A. and Weinstein, I.B. Phorbol ester

induced prostaglandin synthesis and (3H)-TPA metabolism
by TPA-sensitive and TPA-resistant Friend
erythroleukemia cells. Biochem. Biophys. Res. Commun.
89:1018,1979.

Yamasaki, T., Teel, R.W., and Lau, B.H.S. Effect of allixin,
a phytoalexin produced by garlic, on mutagenesis, DNA-
binding and metabolism of aflatoxin B1. Cancer Lett.

59:89-94, 1991.

You, W.C., Blot, W.J., Chang, Y.S., Ershow, A., Yang, z.T.,
An, 0., Henderson, B.E., Fraumeni, J.F. and wang, T.G.
Allium vegetables and reduced risk of stomach cancer. J.
Natl. Cancer. Inst. 81:162-164, 1989.

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