IDENYIFICATION AND QUANTITATION OF
TRYPTOPHAN MET ABOLITES IN URINE OF NORMAL
AND ENDOTOXIN - POISONED MICE

Dissertation for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY -
KATHERINE MARIE MORRIS
1973

LIBRARY

Michigan S
University

 

.‘v. .

‘ kilns is to-eertify that the

‘ ( ‘

thesis entitled

IDENTIFICATION AND QUANTITATION OF
TRYPTOPHAN METABOLITES IN URINE
OF NORMAL AND ENDOTOXIN-
POISONED MICE

presented by

Katherine Marie Morris

has been accepted towards fulfillment
of the requirements for

Ph . D . degree in Microbiology and
Public Health

 

 

Wm

MajcMarofessor

Date 8;”1/73

07639

 

ABSTRACT

IDENTIFICATION AND QUANTITATION OF
TRYPTOPHAN METABOLITES IN URINE
OF NORMAL AND ENDOTOXIN-
POISONED MICE

BY

Katherine Marie Morris

Twenty tryptOphan metabolites from urine of normal
and endotoxin-poisoned mice given D,L-tryptophan (benzene
ring-14C) with or without tryptophan load have been sepa-
rated by thin layer and DEAE-cellulose chromatography and
tentatively identified by fluorescence and color reactions
and mass spectral analyses. Of these, eleven are meta-
bolities of the kynurenine pathway and nine are meta-
bolites of the serotonin pathway. Injection of 5-hydroxy-
tryptamine—3'-14C instead of labeled tryptophan revealed
four additional serotonin metabolites, three of which could
be identified. Eleven of the major kynurenine and sero-
tonin pathway metabolites were quantitated. Significantly
less tryptophan and its metabolites were excreted by en-
dotoxin-poisoned mice. The decreased excretion of radio-
activity was accompanied by significant decreases in the
amount of kynurenine metabolites and increases in the sero-

tonin metabolites excreted. After one hour in normal mice

—~
I
/ I

E: 75¢3ar“ Katherine Marie Morris

with load 43.4% of the metabolites are from the kynuren-
ine pathway and 8.4% from the serotonin pathway. By con-
trast, in endotoxin-poisoned mice, 30.4% are from the
kynurenine pathway and 17.5% from the serotonin pathway.
Similar results are seen in mice given only the labeled
amino acid. These results strongly suggest that a shift
in tryptophan metabolism occurs in_yizg in endotoxin-
poisoned animals with resultant increases in the production
of serotonin pathway metabolites and decreases in kynuren-
ine pathway metabolites. The implications of these data
with respect to the hyper-reactivity of endotoxin—poisoned

mice to tryptOphan are discussed.

IDENTIFICATION AND QUANTITATION OF
TRYPTOPHAN METABOLITES IN URINE
OF NORMAL AND ENDOTOXIN-

POISONED MICE

BY

Katherine Marie Morris

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health

1973

ACKNOWLEDGMENTS

I wish to thank my major professor, Dr. Robert
J. Moon, for encouragement during the period of my research
and for providing me with an appreciation of complex bio-
logical systems.

I would especially like to thank Dr. David Bing for
his assistance throughout my graduate studies, Dr. Harold C.
Miller for his assistance in obtaining a postdoctoral posi-
tion, and Dr. Walter Esselman for his guidance and assist—
ance and help in doing the gas chromatography-mass spectrometry.

I would also like to thank Dr. Mark Bieber and Mr.
Jack Harten of the Mass Spectrometry Laboratory at Michigan
State under the direction of Dr. Charles C. Sweeley for their
help with the mass spectral analyses.

The assistance of Mr. Daniel P. Roman, Jr., in draw-
ing the figures and Ms. Susan Schmiege, and Ms. Betty Tavella
in providing continual support are all greatly appreciated.

My father and mother, Mr. and Mrs. Charles G. Morris
have provided me with a continual source of moral and finan-
cial support and I am extremely grateful to them for their

invaluable efforts and love.

ii

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . .

LIST OF FIGURES O O O O O O O O O O O O 0

INTRODUCTION . . . . . . . . . . . . . .

LITERATURE REVIEW . . . . . . . . . . . .

Part I. Tryptophan Metabolism . . . . . . .
Introduction . . . . . . . . . . . . .
The Kynurenine Pathway . . . . . . . . .
The Serotonin Pathway . . . . . . . . . .
Minor Pathways . . . . . . . .
Tryptophan Metabolites Found in Urine . .
The Influence of Tryptophan and Its Metabolites

on Enzyme Levels . . . . . . . . . .

Part II. Endotoxin . . . . . . . . . . .
Introduction . . . . . . . . . . . .
Biochemistry . . . . . . . . . . . . .
Localization and Detoxification . . . . . .
Molecular Aspects of Biological Activity . .

The Effects of Endotoxin on Tryptophan Metabolism.
Part III. Methods of Separation and Identification
of Tryptophan Metabolites . . . . . . . .
Paper Chromatography . . . . . . . . . .
Thin Layer Chromatography . . . . . . . .
Column Chromatography . . . . . . . . .
Gas Chromatography . . . . . . . . . . .

MATERIALS AND METHODS . . . . . . . . . . .

Chemicals and RadioisotOpes . . . . . . . .
Mice C O O O O O O O O O O O O O O O
Endotoxin . . . . . . . . . . .

Collection and Quantitation of Total Radioactivity
in Urine . . . . . . . . . . . . . .

iii

Page
vi

viii

U'l

wr—uommm

P4P

22

24
25
28
29
32

36
36
37
38
39

41
41
42
42

43

Page

Collection of Urine for Identification of

Tryptophan Metabolites . . . . . . . . . 44
Thin Layer Chromatography . . . . . . . . . 45
Autoradiography . . . . . . . . . . . . 47
Spray Reagents . . . . . . . . . . . . . 48
Column Chromatography . . . . . . 49
Preparation of Methyl Esters for Gas Chromatography-

Mass Spectrometry . . . . . . . . . . 51
Preparation of Trimethylsilyl (TMSi) Ethers or

Esters for Gas Chromatography-Mass Spectrometry . 52
Gas Chromatography-Mass Spectrometry . . . . . 53
Quantitation of Urinary Tryptophan Metabolites . . 53
Statistical Methods . . . . . . . . . . . 54

RESULTS . . . . . . . . . . . . . . . . 56
Rf Values, Fluorescence, and Color Reactions of

Standard Tryptophan Metabolites . . . . . . 56
DEAE-Cellulose Chromatography of Standard Tryptophan

Metabolites . . . . . . . . . 56

Gas Chromatography-Mass Spectrometry of ME-TMSi and
TMSi Derivatives of Standard Tryptophan
Metabolites . . . . . . . . . 59
Thin Layer and DEAE- Cellulose Chromatography of
Tryptophan Metabolites in Urine of Normal and
Endotoxin-Poisoned Mice . . . . . . 68
Gas Chromatography-Mass Spectrometry of ME- TMSi and
TMSi Derivatives of Tryptophan and Serotonin
Metabolites from Urine of Normal and Endotoxin-
Poisoned Mice . . . . . . . . . . . . 75
Tentative Identification of Kynurenine Pathway
Metabolites Isolated from Urine of Normal and
Endotoxin- Poisoned Mice Given D,L-Tryptophan
(Benzene Ring-l C) . . . . . . . . . 80
Tentative Identification of Serotonin Pathway
Metabolites Isolated from Urine of Normal and
Endotoxin- Poisoned Mice given 5- -Hydroxytryptamine-
3'-14C or D, L- -Tryptophan (Benzene Ring-l
Quantity of Radioactivity Recovered from Urine of
Normal and Endotoxin-Poisoned Mice Given D,L-
Tryptophan (Benzene Ring-14C) With and Without
Load . . . . . . . . . . . . . . . 90
Quantitation of Tryptophan Metabolites in Urine
of Normal and Endotoxin-Poisoned Mice Given D,
L-Tryptophan (Benzene Ring-14C) With and Without
Loan . . . . . . . . . . . . . . . 93

87

iv

Quantitative Enumeration of Individual Metabolites
of the Kynurenine and Serotonin Pathways in
Normal and Endotoxin-Poisoned Mice Given D,L-
Tryptophan (Benzene Ring-14) . . . . . . .

DISCUSSION 0 O O O O O O O O O O O O 0
LI ST OF REFERENCES 0 O O O O O O O O O O

APPENDICES O I O O O O O O O O O O O O

Page

96
103

112
124

\1

Cl?

‘C’
o

LIST OF TABLES

Table ' Page
1. Characteristic Endotoxic Reactions . . . . . 31

2. Rf Values of Standard Tryptophan Metabolites in
Two Solvent Systems . . . . . . . . . 57

3. Characteristics of Standard Tryptophan Meta-
bolites with Respect to Fluorescence, Color
Reactions with van Urk's Reagent, and
Fluorescence and Color Reactions with
Prochazka's Reagent . . . . . . . . . 58

4. Elution Pattern of Standard Tryptophan Meta-
bolites from a DEAE-Cellulose (Formate Form)
COlumn O O O O O O O O O O O O O 60

5. Tentative Identification and Characterization
of Kynurenine Pathway Metabolites in Urine of
Normal and Endotoxin-Poisoned Mice Given D,
L—TryptOphan (Benzene Ring-14C) . . . . . 85

6. Tentative Identification, Rf Values in Two
Solvent Systems, and Elution Pattern from
DEAE-Cellulose of Serotonin Pathway Metabolites
in Urine of Normal and Endotoxin-Poisoned Mice
Given D,L—Tryptophan (Benzene Ring-14C). . . 88

7. Quantity of Radioactivity Recovered from Urine of
Normal and Endotoxin-Poisoned Mice Given D,L—
Tryptophan (Benzene Ring-14C) Without Load. . 9O

8. Quantity of Radioactivity Recovered from Urine
of Normal and Endotoxin-Poisoned Mice Given
D,L-TryptOphan (Benzene Ring-14C) With Load . 92

9. Relative Percentage of Kynurenine or Serotonin
Pathway Metabolites in Urine of Normal and
Endotoxin-Poisoned Mice Given D,L-Tryptophan
(Benzene Ring-14) With Load . . . . . . 94

vi

Table Page

10. Relative Percentage of Kynuenine or Serotonin
Pathway Metabolites in Urine of Normal and
Endotoxin-Poisoned Mice Given D,L—Tryptophan
(Benzene Ring-14C) Without Load . . . . . 95

11. Relative Percentage of Individual Kynurenine
and Serotonin Pathway Metabolites in Urine of
Normal and Endotoxin-Poisoned Mice Given D,L-
Tryptophan (Benzene Ring-14C) After One Hour . 97

12. Relative Percentage of Individual Kynurenine
and Serotonin Pathway Metabolites in Urine of
Normal and Endotoxin-Poisoned Mice Given D,L-
Tryptophan (Benzene Ring-14C) After Two Hours. 99

13. Relative Percentage of Individual Kynurenine
and Serotonin Pathway Metabolites in Urine
of Normal and Endotoxin-Poisoned Mice Given
D,L—Tryptophan (Benzene Ring-14C) After
Three Hours . . . . . . . . . . . . 100

14. Relative Percentage of Individual Kynurenine
and Serotonin Pathway Metabolites in Urine
of Normal and Endotoxin-Poisoned Mice Given
D,L-Tryptophan (Benzene Ring-14C) After
Six Hours . . . . . . . . . . . . 102

A1. Rf Values and Color Reactions of "Simple"
Indole Derivatives (145). . . . . . . . 126

A2. Rf Values and Detection of TryptoPhan Meta-
bolites Utilizing 4-Dimethylaminobenzalde-
hyde Reagent (119). . . . . . . . . . 127

A3. Rf Values of Serotonin Metabolites and Their
Variations . . . . . . . . . . . . 128

vii

Figure

1.

10.

LIST OF FIGURES

The Kynurenine Pathway . . . . . . . . .
The Serotonin Pathway . . . . . . . . .
The Tryptamine Pathway . . . . . . . . .

The Proposed Structure of the Lipopolysaccharide
of Salmonella tryphimurium . . . . . . .

 

Mass Spectra Obtained from the ME-TMSi Deriva-
tives of Kynurenine and 5-Hydroxytryptamine
Demonstrating the Similarities in Spectra . .

Mass Spectra Obtained from o-Aminophenol,
Anthranilic Acid, Nicotinic Acid, Nico-
tinamide, and N-Methylnicotinamide Prepared
as ME—TMSi Derivatives But Due to Structure
or Insolubility Forming Only TMSi Ethers or
Esters . . . . . . . . . . . . .

Mass Spectra Obtained from the TMSi Derivatives
of Kynurenic Acid, Anthranilic Acid, N-
Methylnicotinamide and 5-Hydroxyindoleacetic
ACid O O O O O O O O O O O O O C

A Representative Autoradiogram of a Two Dimen-
sional Thin Layer Development of a Urine
Sample from an Individual Mouse Given D,L-
Tryptophan (Benzene Ring-14C) . . . . . .

A Representative Autoradiogram of a Two Dimen-
sional Thin Layer Development of a Urine
Sample from an Individual Mouse Given 5-
Hydroxytryptamine-3'-14C. . . . . . . .

Radioactive Profile of a Pooled Urine Sample from
Ten Normal or Endotoxin-Poisoned Mice Given
D,L-TryptOphan (Benzene Ring-14C) Chromato-
graphed on a DEAE-Cellulose (Formate Form)
Column . . . . . . . . . . . . .

viii

Page

10
12

27

62

64

66

69

71

73

Figure

11.

12.

13.

14.

B1.

BZ.

B3.

B4.

B5.

Radioactive Profile of Pooled Urine Sample
from Ten Normal or Endotoxin- oisoned Mice
Given S-Hydroxytryptamine-B'l C Without Load
Chromatographed on a DEAR-Cellulose Column
(Formate Form) . . . . . . . . . . .

Radioactive Profile of a Pooled Urine Sample
from Ten Normal or Endotoxin-Poisoned Mice
Given 5-Hydroxytryptamine-3'14C With Load
Chromatographed on a DEAE-Cellulose Column
(Formate Form) . . . . . . . . . . .

Mass Spectra Obtained from TMSi-S-Hydroxy-
tryptamine and Background . . . . . . .

Mass Spectra of TMSi-5-Hydroxyindoleacetic
Acid and TMSi-S-Hydroxytryptophol . . . .

Mass Spectra Obtained from the ME-TMSi Deri-
vatives of 3-Hydroxykynurenine, Kynurenic
Acid, Xanthurenic Acid, and 5-Hydroxy-
tryptophol Demonstrating the Similarities
in Spectra Obtained . . . . . . . . .

Major Patterns Obtained from Mass Spectra of
Unknown Tryptophan and Serotonin Metabolites
from Urine of Mice Given D,L-Tryptophan
(Benzene Ring-14C) or 5-Hydroxytryptamine-
3'-14C Prepared as the TM81 Derivatives . .

Mass Spectra Obtained from ME-TMSi Derivatives
of Kynurenine-O-Sulfate and N—Acetylkynurenine

Mass Spectra Obtained from ME-TMSi Derivatives
of 5-Hydroxytryptamine and 5-Hydroxyin-
doleacetic Acid . . . . . . . . . .

Mass Spectra Obtained from ME—TMSi Derivatives
of 5-Hydroxyindoleacetic Acid-O-Glucuronide,
S-Hydroxytryptophol-O-Glucuronide, 5-
Hydroxytryptamine-O—Glucuronide, and 5-
Hydroxyindoleacetic Acid—O—Sulfate . . . .

ix

Page

76

78

81

83

130

132

134

136

138

INTRODUCTION

Endotoxin-poisoned mice frequently die in convul-
sions within 8 hours after a delayed but not concurrent
injection of tryptOphan load (1 mg/gm body weight). Death
occurs 24 to 36 hours sooner than in mice given endotoxin
alone (11,102,104). While the immediate reasons for the
increased hyper-reactivity of endotoxin-poisoned mice to
tryptophan have not been established, such responses may
be due to the increased production in give of potentially
toxic tryptOphan metabolites such as serotonin. Supporting
this latter hypothesis is the fact that pretreatment of
endotoxin-poisoned mice with cyproheptadine, an anti-
serotonin drug, protects against the enchanced lethality
of a delayed injection of tryptophan (104). Further,
cyproheptadine protects against serotonin induced hypo-
thermia and the tryptophan induced hypothermia in endotoxin-
poisoned mice housed at 150C.(103).

Depression of the activity of the adaptive liver
enzyme tryptophan oxygenase has been the primary enzymatic
lesion in tryptophan metabolism described to date in endo-
toxemia (1,11,104). While it is known that tryptophan oxygen-

ase is depressed, no definitive evidence exists as to whether

there is a correlation between the depressed enzyme activ-

ity as monitored in vitro and altered tryptophan metabolism

 

in vivo.

 

Funneling of tryptophan from one pathway to another
due to changes in tryptophan oxygenase activity has been
suggested by Curzon and Green (30) who showed that increased
tryptophan oxygenase activity in stessed rats correlated_.
with decreased brain serotonin. Further, Schmike (138)
and Sourkes (150) have independently shown that B-methyl-
tryptophan-mediated increases in tryptophan oxygenase corre-
lated with decreases in blood levels of tryptophan and
subsequent decreases in brain serotonin and 5-hydroxyindole—
acetic acid levels as well.

In an effort to provide more quantitative data on
whether funneling of tryptophan into the serotonin pathway
occurs in endotoxin-poisoned animals, the primary objective
of the present study has been to determine whether or not
there are significant increases in serotonin pathway meta-
bolites in the urine of endotoxin-poisoned compared to
normal mice. Studies have been done in animals without
and with tryptophan load, the former to monitor tryptophan
metabolism at dose levels well below quantities which pro-
duce hyper—reactivity and the latter to compare these data
with changes in the mice given a potentially lethal dose
of the amino acid. Monitoring of urinary tryptophan meta-

bolites as a method of estimating in vivo enzyme activity

has been used previously (3,144) and it is believed that
the accurate quantitative monitoring of urinary tryptOphan
metabolites should lead to an accurate description of the
ig_zizg catabolism of this amino acid.

To accomplish our objective certain specific goals
had to be reached. Techniques had to be devised which
would not only allow us to study the wide variety and di-
verse nature of tryptophan metabolites but would also be
sensitive enough to detect the minute quantities of trypto-
phan metabolites excreted in a single mouse urine sample.
Thin layer chromatography (including fluorescence and color
reactions), column chromatography, and gas chromatography-
mass spectrometry were chosen as methods for this study.
Initially Rf values, fluorescence, color reactions, and
mass spectral data were established for standard trypt0phan
metabolites. The use of radioactive D,L—tryptophan (ben-
zene ring-14C) and 5-hydroxytryptamine-3'-14C provided
markers in the tryptophan metabolites excreted in urine.
From autoradiographs of thin layer plates, individual meta-
bolites could be estimated, Rf values measured, and when
concentrations of metabolites were great enough, fluor-
escence and color reactions determined. Elution profiles
from DEAE-cellulose chromatography and gas chromatographic-
mass spectral analyses aided in identification of unknown

tryptophan metabolites scraped and eluted from thin layer

chromatographic plates. From a combination of these data
tentative identification of the unknown urinary tryptophan
metabolites could be made. To quantitate the individual
metabolites, the location of isotope on the thin layer
chromatographic plate was determined by autoradiography

and individual spots were scraped and counted by liquid
scintillation spectrometry. By comparing the relative quan-
tities of isotope found in the products of the serotonin and
kynurenine pathways, one could then determine whether there
was a shift in metabolism from the kynurenine pathway in
endotoxin-poisoned mice and evaluate the effect of a tryp-

tophan load on the excretion of these metabolites.

LITERATURE REVIEW

PART I

TRYPTOPHAN METABOLISM

Introduction

 

Metabolism of tryptophan by mammalian systems can
proceed through several pathways, yielding numerically more
metabolites than any other amino acid. The major pathways
primarily lead either to the synthesis of nicotinamide
adenine dinucleotide (NAD) and glutaryl CoA, hereafter re-
ferred to as the kynurenine pathway, or to the synthesis
of 5-hydroxytryptamine (serotonin), hereafter referred to
as the serotonin pathway. A third pathway produces trypta-
mine and metabolites similar in structure to those of the
serotonin pathway. The enzymes of this latter pathway are
found both in intestinal flora and mammalian systems. Sev-
eral minor branch pathways are also found throughout the
primary pathways. A comprehensive review of tryptophan
metabolism has been compiled by Meister (99). These path-
ways for tryptophan metabolism are presumably regulated

not only by substrate availability (43,45,102) but also

by adaptive enzyme activity (44,45,83) and can be dis-
rupted by effectors such as endotoxin (61,102). The pur-
pose of the first two parts of the present review is to
familiarize the reader with tryptophan metabolism and endo-
toxin, and to present the relationship between these two
diverse subjects. The third part of the review will summar-
ize some of the techniques utilized to isolate and identify

tryptOphan metabolites.

The Kynurenine Pathway

The initial reaction in the kynurenine pathway is
cleavage of the indole nucleus of tryptophan between C2
and C3 with the addition of molecular oxygen to form
formylkynurenine (Figure l). Tryptophan oxygenase (L-
tryptophan:oxygen oxidoreductase, EC 1.13.1.12), is the
adaptive liver enzyme which catalyzes this reaction. Hema-
tin is a cofactor for tryptophan oxygenase (25,42). The
activity of this enzyme can be increased by its substrate
tryptophan (103,104), glucocorticoids (12,43,62,83,86),
and a variety of substrate analogues (104,150). A decrease
in the activity of this enzyme can be caused by numerous in-
hibitors (102,146) including endotixin (61,104).

Formylkynurenine is converted to kynurenine by the
enzyme kynurenine formylase found in the mammalian liver
(84). Kynurenine is metabolized primarily to 3-hydroxy-

xykynurenine by kynurenine-3-hydroxylase and requires NADPH

0 NH,
H I
T“: C—CHf-CH—COOH
| CHf—CH—COOH ,
N — CH
H H
H o
Tryptophan N-Formylkynurenine
0 NH COOH ""2
2
II I + CH3—CH—COOH
C-CHz-CH—COOH / N“;
G Anthrmflic acid Alamn' e
NH2
Kynurenine \ OH
\ \
——>
/
COOH COOH
(I? T“: Kynurenic acid Quinaldic acid
C—CH2 —CH —COOH
OH
NH
2 \ \ \
OH
_—_.’ /
3-Hydroxykynurenine COOH N COOH
OH OH
Xanthurenic acid 8-Hydroxyquinaldic acid
COOH
COOH COOH
HOOC “”2 N coon
o-Aminophenol 3-Hydroxymthrmflic a-Amino—fl- Quinolinic acid
acid carboxymuconic-A-
seminldehyde pRpp
-—CO,
1‘00: Pi
/
9.1 a m / .
HO HOOC NH: *—— = H +
CC C OH COOH HOOC N 2 \T
a-Ketoadipic a-Aminomuconic a-Aminomuconic— RP
' ' - midld h d
acid acrd A se e y e Niacin ribo-
1 nucleotide
I
I
l
l
‘1
Glutaryl
CoA

Figure l.-—The Kynurenine Pathway.

and molecular oxygen. The cleavage of alanine from both
kynurenine and 3-hydroxykynurenine yielding anthranilic
acid and 3-hydroxyanthranilic acid respectively, is cata-
lyzed by the enzyme kynureninase, a pyridoxal phosphate
dependent enzyme. Similarly, both kynurenine and 3-hydroxy-
kynurenine may be converted to their keto acids by
kynurenine transaminase (160). The keto acids undergo
spontaneous ring closure to form kynurenic acid and
xanthurenic acid which may then be converted to quinaldic
acid and 8-hydroxyquinaldic acid.

3-Hydroxyanthranilic acid may also be converted
to the end product o-aminophenol or to a-amino-B-carboxy-
mucconic-Z-semialdehyde (2—acroleyl—3—aminofumaric acid).
The latter reaction is catalyzed by the enzyme 3-hydroxy—
anthranilic acid oxidase which is found in the liver and
kidney (77,112). The semialdehyde is the branch point
between the pathways which lead to either NAD or glutaryl
CoA. 'Spontaneous ring closure produces quinolinic acid
which is converted in one step to quinolinic acid ribo-
nucleotide. This compound is decarboxylated to nicotinic
acid ribonucleotide. It condenses with ATP and is aminated
with glutamine to synthesize NAD. Nicotinic acid is not
a substrate for these reaction (79).

Glutaryl CoA is synthesized by the decarboxylation

of a-amino-B-carboxymuconic-6-semialdehyde by the liver

enzyme picolinic carboxylase to yield d-aminomuconic-d-seim-
aldehyde. This compound is oxidized to a-aminomuconic acid
(y-oxalocrotonic acid) by a-hydroxymuconic-Z—semialdehyde
dehydrogenase and required NAD. The acid is reductively
deaminated to a-ketoadipic acid in an NADH requiring re-
action. a-Ketoadipic acid is oxidatively decarboxylated

to glutaryl CoA by the d-ketoglutarate dehydrogenase com-

plex.

The Serotonin Pathway

 

In the serotonin pathway, tryptophan is hydroxylated
by tryptophan—S-hydroxylase to form 5-hydroxytryptophan
(Figure 2). This reaction is the rate limiting step in the
biosynthesis of 5-hydroxytryptamine (serotonin) (58).
Tryptophan may also be hydroxylated in the 5 position by
phenylalanine hydroxylase (127). These enzymes are found
in liver (158), intestinal mucosa cells, and kidney (119),
but the specific tryptOphan-S-hydroxylase is found in high-
est concentration in the pineal body (82,90,162). Sero-
tonin (5HT) is formed from 5-hydroxytryptophan (5HTP) by
the substrate Specific enzyme, 5HTP decarboxylase (49).

This enzyme is found in the mammalian kidney, with lesser
amounts present in the liver (60) and pineal body (13,58,
93). Nerve tissue, sympathetic ganglia, and adrenal medulla

also contain substantial 5HTP-decarboxylase activity (49).

10

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Aromatic amino acid decarboxylase has a very low affinity
for tryptOphan and does not play a major role in its decarbox-
ylation (99).

Serotonin may be catabolized to S-hydroxytryptophal
(5-hydr0xyindoleacetaldehyde) by monoamine oxidase. This
compound is oxidized to S-hydroxyindoleacetic acid (SHIAA)
by aldehyde dehydrogenase. Monoamine oxidase can also form
the alcohol, 5-hydroxytrypt0phol, from 5HT (99). Two impor-
tant amines found in the brain, bufetonin and melatonin,
are synthesized by N-methylation of N'-methylserotonin and
N'-acetylserotonin, respectively. Most of the serotonin
metabolites may also exist as the 5-methoxy, O-sulfate,

and O-glucuronide conjugates (87).

Minor Pathways

 

In the third pathway, tryptophan is decarboxylated
to tryptamine by the liver enzyme, aromatic amino acid de-
carboxylase (Figure 3). This enzyme has a very low affinity
for tryptophan. Tryptamine, which has no specific role in
mammals, may then be oxidized by monoamine oxidase to the
aldehyde which is acted upon by aldehyde oxidase to form
indoleacetic acid. The major route for the synthesis of
indoleacetic acid, though, is by transamination of trypto-
phan to produce indolepyruvic acid which is oxidatively
decarboxylated to yield the acid. Enzymes present in bac-

teria and yeast also catalyze these same reactions (99).

12

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13

Many of the tryptophan metabolites synthesized
within the major pathways can take alternate metabolic
routes. In the mouse kynurenine can also be hydroxylated
in the 5 position and then decarboxylated to S-hydroxy-
kynuramine (104). The other hydroxykynurenine, 3-hydroxy—
kynurenine, can be converted to 4,8-dihydroxyquinolinic
acid, instead of xanthurenic acid, by mouse liver homogen-
ates. This occurs by the decarboxylation of 3-hydroxy-
kynurenine and cyclization to form 3-hydroxykynuramine (94).

Many bacteria and yeast also contain the enzyme
tryptophan 2,3-dioxygenase but the metabolic pathways are
not the same as in mammals. The microorganisms produce
kynurenine which is degraded to either anthranilic acid,
3-hydroxykynurenine, or kynurenic acid. Both anthranilic
acid and kynurenic acid are end products in mammals but
are the starting point for tryptOphan metabolism in bac-
teria. The end products in bacteria and yeast are nicotinic
acid, succinic acid,and acetic acid, and a-ketoglutarate,
oxaloacetate, and ammonia (99). Anthranilic acid may be

utilized in the synthesis of tryptophan in bacteria (99).

Tryptophan Metabolites Found in Urine
The variety of the kynurenine metabolites isolated
from urine is almost equal to the variety of techniques
and pathological conditions utilized to obtain them. There

are fewer studies on urinary kynurenine metabolites from

14

normal subjects than abnormal. However, in one study,
Benassi et_gl, (8) identified kynurenine, 3-hydroxy-
kynurenine, kynurenic acid, xanthurenic acid, xanthurenic
acid-8-methyl ether, anthranilic acid, 3-hydroxyanthranilic
acid, and 8-methy1-oxyanthranilic acid from normal human
urine.

The effects of a loading dose of tryptophan on
tryptophan metabolism have been studied in normal and patho—
logical conditions. Hankes and coworkers (65,66,67) studied
the effects on tryptophan metabolism of loading doses of
tryptophan and other kynurenine metabolites utilizing D,
L-tryptophan-Z—Cl4 as a tracer. They monitored kynurenic
acid, xanthurenic acid, 3-hydroxykynurenine, kynurenine,
anthranilic acid, quinolinic acid, nicotinic acid, and N-
methylnicotinamide. Only xanthurenic and kynurenic acids
were reported isotopically. In all three studies they
measured total radioactivity found in urine and carbon
dioxide. These values are not as valid as possible be—
cause they utilized tryptOphan labeled on the side chain.
The label is cleaved from tryptophan as alanine in the
formation of both anthranilic acid and 3-hydroxyanthranilic
acid. These are both early events in the kynurenine path-
way. Therefore, they are more accurately monitoring ala—
nine metabolism rather than tryptophan metabolism. An

interesting observation to come from one of their studies

15

(67) was that a loading dose of 3-hydroxyanthrani1ic acid
caused a decrease in CO2 respired and urinary output of
quinolinic acid. TryptOphan oxygenase was not blocked be-
cause the quantity of kynurenine, xanthurenic acid, and
kynurenic acid excreted remained the same. They hypothe-
sized that 3-hydroxyanthranilic acid oxidase may have been
blocked due to excess substrate.

Many of the enzymes of the kynurenine pathway re-
quire pyridoxyl phosphate. This fact has been utilized
to study tryptophan metabolism in both humans and rats with
a Vitamin B6 deficiency. Yess gt_al. (170) found that
there was an increase of kynurenine, 3-hydroxykynurenine,
xanthurenic acid, and kynurenic acid in the urine of
pyridoxine deficient humans. 3-Hydroxykynurenine was
found to be the most sensitive indicator of Vitamin B6 de-
pletion. In 1952, Dalgliesch (32) did an extensive study
on the relationship between pyridoxin and tryptophan meta-
bolism in rats. Unlike the previous authors (170) who
utilized a fluorometric assay for determining the trypto-
phan metabolites, Dalgliesch separated the individual
urinary tryptOphan metabolites by paper chromatography.
Utilizing fluorescence and different spray reagents, he
determined that 3-hydroxykynurenine was also excreted as

its glucuronide, sulfate, and acetyl conjugates. The

acetyl derivative of kynurenine was also present. These

16

results indicate that the animal cannot metabolize these
compounds and is therefore detoxifying them so that they
may be excreted. Rothstein and Greenberg (134) found vari-
ations in the conjugates of xanthurenic acid excreted in
pyridoxine deficient animals. Rats excreted xanthurenic
acid alone and as the mono- and diglucuronides with a
serine attached in an amide linkage. Rabbits, however, ex-
creted one O-sulfate conjugate linked to serine.

Musajo et_al. (106) found that normal human urine
contains only very slight amounts of kynurenic and xanthur-
enic acids. In patients with neOplastic diseases, urinary
kynurenine pathway metabolites are not altered. On the con-
trary, patients with hemoblastic diseases show an increase
in kynurenine, 3-hydroxykynurenine, a-N—acetylkynurenine,
a-N-acetyl-3-hyroxykynurenine, and 3-hydroxykynurenine-O—
glucuronide. In another type of cancer, bilharzial bladder
cancer, Khalfallah and Abul-Fadl (80) found that urinary
excretion of 3-hydroxyanthranilic acid was increased about
eight times, anthranilic acid about six times, 5-hydroxy-
indoleacetic acid about four times and kynurenic acid about
two times. Kynurenic and xanthurenic acid excretion de-
creased about 50%. These results indicate a general increase
in activity in the major route of both pathways of trypt0phan
metabolism. Along similar lines Watanabe and coworkers (162,

163) have found that 3-hydroxyanthranilic acid and

l7

3-hydroxykynurenine are carcinogenic and cause bladder can-
cer. The O-sulfate and O-glucuronide conjugates of these
compounds may be cleaved in the kidney to produce the toxic
metabolites. Recently, Gailani gt_al. (50) observed in-
creased kynurenine and 3-hydroxykynurenine levels in pre-
and post-operative patients with bladder cancer.

Enzyme regulation by drugs, hormones, and substrate
can be determined by monitoring variations in urinary excre-
tion of tryptophan metabolites. For example, to determine if
kynurenine kydroxylase required a reduced pteridine cofactor,
an aromatic pteridine which would inhibit the reaction if
the cofactor was necessary, was given to rats. Quantitative
changes in kynurenine and 3-hydroxykynurenine were measured.
It was demonstrated that the aromatic pteridine caused an
enhancement of tryptOphan oxygenase activity and a concomi-
tant rise in kynurenine excretion but 3-hydroxykynurenine
could not even be detected in the urine of treated animals.
These results showed that the enzyme did require the reduced
pteridine for activity. In another study concerned with
enzyme levels, Altman and Greengard (3) showed that the in-
crease in the in_yit52 activity of tryptophan oxygenase
caused by either hydroxortisone or tryptOphan administration
could be correlated with increased urinary kynurenine excre-
tion. Shaw and Feigin (144) found that, under the stress

of a bacterial infection, glucocorticoids elevated tryptophan

18

oxygenase and urinary excretion of kynurenic acid and xanth-
urenic acid were increased. During endotoxic shock, however,
tryptophan oxygenase was decreased as were the levels of
kynurenic acid and xanthurenic acid excreted in the urine.

The metabolism of metabolites within the kynurenine
pathway has been studied too. Kaihara and Price (78) demon-
strated that two thirds of an oral dose of kynurenic and
xanthurenic acids was dehydroxylated by the rabbit. After
subcutaneous injection, only 2-10% of the quinoline deriva-
tives were dehydroxylated. They were unable to determine
if dehydrogenation of kynurenic and xanthurenic acids occur-
red in the tissues or in the gut by the bacterial flora.
Either oral or subcutaneous administration of quinaldic acid
resulted in over 90% recovery of unchanged quinaldic acid
in the urine.

Serotonin may be degraded or conjugated to a wide
variety of compounds, many of which can be isolated from
urine. The major catabolic pathway for serotonin is through
oxidative deamination by monoamine oxidase to form 5-hydroxy-
indoleacetaldehyde (5-hydroxytryptophal) 27,98). This com-
pound may be further oxidized by the enzyme aldehyde dehydro-
genase to yield 5-hydroxyindoleacetic acid (SHIAA), the major
excretory product of serotonin in humans and many other ani-
mals (5,68). In dogs, rats, and humans, which are carnis

vores, SHIAA concentration in urine ranges from 1.5 to 4.0

19

ug/ml (108). However, herbivores, such as mice, guinea pigs,
rabbits, and horses, excrete only small amounts (less than

0.3 ug/ml) of SHIAA in urine (42,108). Instead of producing
the acid, herbivores form the alcohol, 5-hydroxytryptophol
(5HTOH). Evidence suggests that 5HTOH may enter pigment forma-
tion (108).

Kveder, Iskrie, and Keglevic (87) identified 5HTOH
in human urine. When serotonin is administered to humans,
5HTOH and its conjugates account for two percent of the sero-
tonin injected. They also identified the glucuronide of
5HTOH in the urine of rats and showed that 60-70% of 5HTOH
was being oxidized to SHIAA or conjugated.

In a very detailed and complete study on serotonin
metabolism in normal and cirrhotic rats, Pentikainen, Mekki,
and Mustala (121) identified 5HT and its glucuronide, 5-hydroxy-
indoleacetaldehyde—O—sulfate, and the O-sulfate and O-
glucuronide derivates of 5HTOH and SHIAA. They found that in
normal rats given l4C-SHT intravenously, SHIAA was initially
the predominant metabolite but its relative amount steadily
decreased with time while 5HT-O-glucuronide continually in-
creased. If these rats were cirrhotic, they produced signifi-
cantly more 5-HT-O-glucuronide and SHIAA-O-glucuronide than
other metabolites throughout the whole experiment.

N-Acetylserotonin has been described as a major meta-

bolite by some workers (98) but further investigation has

20

shown that although it is present in urine it is not a major
metabolite (87). Kveder gE_§l. (64) found that the compound
identified as N-acetylserotonin by McIsaac and Page (98) was
composed primarily of the glucuronide of 5HTOH. The two com-
pounds had similar chromatographic proPerties under the
conditions utilized by McIsaac and Page (98).

Delvigs et_al. (36) studied the metabolic fate of
5-methoxytryptophol, which is localized in the pineal gland.
This compound is rapidly metabolized; with an average of 91%
of the administered activity appearing in urine within 24
hours. As much as 65% of the activity in one hour and 92%
in 2 hours was found in the urine of some animals. The
major metabolite excreted was 5-methoxyindoleacetic acid
(SMIAA) representing 93% of the administered 5—methoxy-
tryptophol.

Formation of conjugates through the 5-hydroxyl
group is yet another route of metabolism for serotonin and
its products. This is a common method of detoxification
for these compounds. Liver homogenates form serotonin-O-
sulfate (20, 133) and this compound has been isolated from
urine, especially when monoamine exidase is inhibited (20).
Chadwick (20) also found the O-sulfate derivatives of 5HIAA,
in urine. The O-sulfate derivatives of 5—hydroxyindole-
acetaldehyde, 5HIAA, and 5HTOH have also been described (121,

122). The O-glucuronide derivatives are very common, with

21

serotonin-O-glucuronide being a major urinary metabolite in
rats and humans (2,58,97,98,121,122,165). 5-Hydroxy—
indoleacetic acid and 5-hydroxytryptophol form O-glucuronide
conjugates (97, 98,121,122) and, in addition, 5HIAA may also
combine with glycine to form 5-hydroxyindoleaceturic acid
(97,98).

Other products of serotonin found in urine are the.
N-methyl and N-acetyl derivatives. Although N-methylation
is uncommon in mammals, Bumpus and Page (15) identified trace
amounts of N-methylserotonin in human urine. Trace amounts
of N-acetylserotonin have also been identified in urine (68,
98). Generally, this compound is O-methylated in the pineal
body to form the pigment melatonin (82) but it may also be
catabolized to 5HIAA (87).

Many of the 5-hydroxyindoles were first isolated
from urine of carcinoid patients. A carcinoid is an argen-
taffin cell tumor with the primary lesion usually appearing
in the ileum but lesions also may be found in the stomach
and pancreas (119). Although it has been shown that the
metabolic pathways of tryptophan do not vary between car-
cinoid and normal tissues, approximately 60% of orally in-
gested tryptophan proceeds to 5-hydroxyindoles in carcinoid
patients as compared to approximately one percent in normal
individuals (119). The tumors have been characterized as

producing excess amounts of serotonin with the subsequent

22

excretion of increased quantities of 5HIAA in the urine (41,
98). Feldstein (47) has reported results contradictory to
this thesis.
The Influence ofTryptophan and its
Metabolites on Enzyme Levels

TryptOphan increases the activity of tryptophan 2,
3-dioxygenase by converting the inactive apoenzyme to the
active holoenzyme. Substrate activation is accomplished by
the binding of tryptOphan at both the allosteric and cataly-
tic sites (14). This binding also exerts a stabilizing ef-
fect on tryptophan oxygenase and prevents degradation both
in_zi£gg (26,43,108) and in vivo (138,139,140,l46).

Recent evidence suggests that in addition to inhibiting de-
gradation, tryptophan also stimulates mRNA synthesis by
stimulating adrenocortical hormonal secretion (146).

Unlike tryptophan which elevates tryptophan oxygen-
ase levels for 8-12 hours, a—methyltryptOphan elevates the
enzyme for at least a week. This compound binds to the en-
zyme and stabilizes it but cannot be metabolized. Due to the
increased metabolism of available tryptophan, thus limiting
substrate availability, there is decreased protein synthesis
(138, 150). Along similar lines it has been shown that
tryptOphan plays a special role in the regulation of poly-
ribosome aggregation and protein synthesis in the liver (147).

Brain 5HT nad 5HIAA are also decreased after administration

23

of a-methyltryptophan. This deficiency is due to decreased
blood levels of tryptophan and thus less substrate availa-
bility. Although tryptophan oxygenase cannot metabolize
a-methyltryptophan, tryptophan hydroxylase and S-hydroxy-
tryptophan decarboxylase can, and forms a-methyl-S-hydroxy-
tryptophan and o-methyl—S-hydroxytryptamine. However, mono-
amine oxidase cannot convert the a-methylamine to the acid
(120).

Other tryptophan metabolites also effect the activ-
ity of tryptOphan oxygenase. Cho-Chung and Pitot (24) demon-
strated that the highly active form of tryptophan oxygenase
is sensitive to inhibition by its end product NADPH. Kinetic
analysis of the inhibition indicates that it is a type of
allosteric inhibition. In a somewhat contradictory report,
Powanda and Wannemacher (123) reported that NAD synthesis
from tryptophan was regulated by the substrate availability
rather than variations in tryptophan oxygenase activity.

They found that allopurinol given simultaneously with trypto-
phan decreased tryptophan oxygenase activity but did not
reduce the increase in NAD concentration. Hydrocortisone

and a-methyltryptophan, which increase tryptophan oxygenase
activity, did not increase NAD levels.

Tagliamonte and co—workers (155) have stated that
the concentration of tryptOphan in the brain is a more im—

portant control mechanism than the concentrations of

 

 

I."

24

tryptophan hydroxylase. Another interpretation of their
data might suggest that the rate of serotonin synthesis could
control the concentration of tryptophan in the brain.

Foster gt_al. (48) described a paradoxical effect
of tryptophan on the gluconeogenic enzyme phosphopyruvate
carboxylase (phOSphoenolpyruvate carboxykinase - PEPCK).
Exogenously administered tryptophan enhanced PEPCK activity
in Xitrg_but depressed it in the intact rat (48,159). Quin-
olinate, a catabolite of the kynurenine pathway, appears to
be the metabolite primarily responsible for the in_xizg in-
hibition of the enzyme. The mechanism of inhibition is not
known but may be partially due to the metal chelator proper-
ties of quinolinate (150). Other metabolites of tryptophan,
such as xanthurenic acid, reduce PEPCK activity to some ex-

tent (150, 159).
PART I I
ENDOTOXIN

Introduction

 

Classically, the term "endotoxin" was introduced
because of the View that this material was an internal con-
stituent of gram—negative bacteria and that disruption of
cells was essential for its release. It is now known that,
not only is endotoxin concentrated at the cell surface, but

that intact young cells release substantial amounts of

endotoxin into the medium (29) making the term endotoxin a
misnomer. The primary chemical composition of endotoxin
makes it a lipopolysaccharide, suggesting the name lipopoly-
saccharide toxin, but this name does not cover the toxic
extracts from heptoseless mutants which do not appear to
contain any true polysaccharide. Milner, Rudbach, and Ribi
(101) suggest the term endotoxic phospholipopolysaccharide
and give the following somewhat detailed definition.

Endotoxic phospholipopolysaccharides (endotoxins)
are found principally at or near the cell surfaces of
gram-negative bacteria. As extracted by common pro-
cedures, they are macromolecular aggregates of sub-
units, united in various forms by hydrophobic bonds,
incorporating both the major somatic antigens of the
bacteria and a large number of toxic and other host—
reactive properties that are described collectively
as "endotoxic." All are stable to boiling in neutral
water. The probability of correctly identifying a
substance as an endotoxin increases rapidly with the
number of typical host responses that are demonstrated.
Of particular value in this regard are the production,
in suitable animals, of characteristic biphasic fever,
lethal shock after a latent period, the Sanarelli-
Shwartzman reactions, hemorrhagic necrosis of trans-
plantable tumors, leukopenia followed by leukocytosis,
enhancement of the antigenicity of proteins, and non-
specific resistance to infection or to damage by ir-
radiation. Dasic to many of these pathOphysiological
effects of endotoxin is an injury to the cardiovascu-
lar system, by means not yet fully understood, which
alters or disrupts normal function.

From this definition one can succinctly see the diversity

Of manifestations elicited by this molecule.

Biochemistry

 

Biochemically, endotoxin is composed of three re-

gions: the O-specific chain, the basal core polysaccharide,

26

and lipid A (Figure 4). The lipopolysaccharide may be ex-
tracted from the cell by a number of methods, the most
common being the phenol water method (166,167) and the aqu-
eous ether method (128,129,130).

Over thirty aldoses have been identified in O-anti-
gens of gram-negative bacteria. Another important sugar, 3-
deoxy-D-mannooctulosonic acid (KDO), has been regularly
isolated from lipopolysaccharides. This sugar forms a
stable glycosidic linkage with heptose. Most lip0polysac-
charides contain five or more sugar constituents. Glucosa-
mine, heptose, KDO, glactose, and glucose are, however, the
only ones commonly encountered and represent the units of
the core polysaccharide (region II, Figure 4). The exact
structures and linkages of the sugars have been elaborately
worked out by the combination of gas chromatography with
mass spectrometry [for a review, see Luderitz eE_al. (91)].

Lipid A is a complex structure containing glycosidi-
cally linked glucosamine units. The hydroxyl groups of
glucosamine are substituted by long-chain fatty acids, such
as lauric, myristic, palmitic, and B-hydroxymyristic acids.
B-Hydroxymyristic acid is also bound to the amino groups of
the glucosamine units. KDO is linked directly to lipid A

(91).

 

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28

Localization and Detoxification
To gain a clearer understanding of where endotoxin
localizes in the host, studies have been undertaken utiliz-

ing 14C, 32P, 51Cr, and 131I labeled endotoxins. Noyes et al.

(117) found that 14C-labeled endotoxin persists in the blood
and localized in the liver. It appears to be sequestered in
phagocytes of the reticuloendothelial system. Willerson

et al. (169) found that intravenously administered 14C-la-

beled Salmonella enteritidis endotoxin was recovered predomi-

 

nantly from the nuclear and mitochondrial fractions prepared

from livers and spleens of mice and rats. Mice intravenously

inoculated with endotoxin labeled with 51Cr (18), 32

or 1311 (144) retained a major portion of the endotoxin in

P (76) r

the liver over several hours to several days. Immediately
after intravenous injection Carey (18) found endotoxin in the
buffy coat of blood samples. By immunofluorescence, Ruben-
stein gt_al. (137) detected endotoxin in patchy distribution
throughout the walls of the peripheral vascular system. Golub,
Groschel, and Nowotny (56) found that detoxified endotoxin is
not taken up by the liver and spleen and remains in circula-
tion for a relatively long time.

Although there is some controversy, both humoral and
cell-mediated mechanisms of the host are probably inseparably
involved in the detoxification of endotoxin. PrOposing a hum-
oral mechanism for detoxification, several authors (63,141,142,

148) have presented evidence for two serum alpha globulins

29

which have the nonspecific, carboxylic type of esterase activ-
ity. The first is a heat—stable alpha lipoprotein which leads
to degradation or disaggregation of the large molecules of
endotoxin. The exposed toxic particles are then degraded by
a heat-labile alpha globulin. The reactions are inhibited
by physiological concentrations of serum Ca++ but Skarnes (148)
found a gradual drop in Ca++ levels with time in post-endotoxin
serum which coincided with an increase in the enzyme levels
and detoxification.

Rutenberg (137) found that peritoneal macrophages

combined with immune serum detoxified E. coli 011 endotoxin

 

more effectively than macrophages in normal serum. Chedid

51Cr—labeled endotoxin

and coworkers (21,22) found that toxic
was present in plasma six hours after injection and smooth
strains of salmonellae are not removed as rapidly as rough
strains. Opsinizing antibodies directed toward the R anti-
gen, which must be exposed, may be necessary for detoxifica-
tion to occur. They also observed that only non-toxic
degraded endotoxin can pass the kidney and occur in urine.
Rowley (135) suggests that the decisive event in the destruc-
tion of bacteria is phagocytosis and its efficiency is depend-
ent upon the presence of specific antibody which may be free
or bound to the cell.

Molecular Aspects of
Biological Activity

 

 

In the introduction to Bacterial Endotoxins, Ivan

 

Bennett, Jr., sums up the expansive variety of biological

30

effects of endotoxin with the statement: "an investigator

in almost any biological field is likely to obtain a 'posi-
tive' result if he tried endotoxin in the experimental system
he is using" (10). A summary of the major biological effects
of endotoxin is presented in Table 1.

It is assumed that the elicitation of the diverse
endotoxin reactions do not involve the entire lipopoly-
saccharide molecule but are localized in certain active
sites. These active sites are formed by specific steric
arrangements of certain functional groups of the structure
(116). The polysaccharide moiety is responsible for O-anti-
genic specificity and has never been shown to be involved in
the toxic manifestations. Glycolipids extracted from rough
mutants, and thus lacking the polysaccharide moiety, are
fully toxic. Lipid A moieties, some of which are composed
of only KDO and lipid A, appear to contain the active sites
and are responsible for the potent endotoxic activity (68,100,
156). When KDO is removed from the glycolipid by mild acid
hydrolysis, the free Lipid A has a lower activity. However,
this appears to be due to its insolubility in water as glyco-
lipids treated with agents that chemically modify the KDO
(130) or free Lipid A combined with water soluble bovine
serum albumin (51) are fully toxic. These results suggest
that the polysaccharide portion and the KDO of the glycolipid
act as water—solubilizing carriers for the insoluble Lipid A

which represents the biologically active center of the molecule.

 

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31

Table l.--Characteristic Endotoxic Reactions (116).

 

Pyrogenicity

Release of endogenous pyrogen
Immunogenicity

Adjuvant effect and inhibition of antibody production
Effect on properdin or natural antibody levels
LeukOpenia and leukocytosis

Protection against irradiation

Effect on RES

Development of tolerance

Enhancement of nonspecific resistance
Mobilization of interferon

Changes in blood clotting

Metabolic changes

Endocrinological changes

Release and sensitization to histamine
Vascular effects

Sanarelli-Shwartzman phenomenon
Cytotoxicity

Abortion

Tumor-necrotizing effect

Interaction with complement

Shock and lethality

 

The Effects of Endotoxin on
Tryptophan Metabolism

 

 

Tryptophan oxygenase is depressed in endotoxin-
poisoned mice (1,11,12,104). This enzyme catalyzes the
first reaction in the pathway leading to the formation of
pyridine nucleotides. Its depression implies a block in
the biosynthesis of these compounds, the consequence of
which could be biologically significant in endotoxin-poison-
ing. This concept is consistent with evidence that adminis-
tration of the end products NAD or nicotinamide concurrent
with endotoxin protects mice from endotoxin lethality (11).

The adrenocortical hormone, cortisone, is known to
elevate tryptophan oxygenase (44,45,83). When cortisone is
given concurrently with endotoxin, mice are protected
against endotoxin lethality (12,52,86,104) and their level
of total oxidized pyridine nucleotides is maintained (12).
The protective role of cortisone and its elevation of trypto-
phan oxygenase are dependent upon the time of administration
of cortisone relative to the administration of endotoxin
(11,12). If 5 mg of cortisone is given more than one hour
after 1 LD50 of endotoxin, it fails to maintain the trypto-
phan oxygenase activity at control levels and to protect
mice from endotoxin lethality. Corresponding to these re-
sults, Shaw and Fegin (143) found that children having gram-
negative infections had increased kynurenic and xanthurenic

acid excretion and postulated that this was due to stimulated

33

adrenocortical function whereas those suffering from severe
endotoxic shock had decreased excretion of the acids, possi-
bly due to inhibited tryptOphan oxygenase activity.
Tryptophan elevated tryptophan oxygenase activity
in normal mice (43,45,104). When tryptophan is given con-

currently with 1 LD of endotoxin, it fails to maintain

50
tryptophan oxygenase levels and does not protect against
lethality (11,104).

Elevation of tryptophan oxygenase activity with
a—methyltryptophan does not decrease, nor does depression
with 5-hydroxytryptophan increase, protection from endotoxin
lethality (11,104). These results imply that tryptophan
oxygenase per se is not directly related to survival of en-
dotoxin-poisoned mice.

When endotoxin-poisoned mice are given 20 mg of L-
tryptophan at a time when tryptophan oxygenase is depressed,
they frequently die in convulsions within 8 hours. Death
occurs 24 to 36 hours sooner than in mice given endotoxin
alone (11,102,104). This hyper-reactivity to tryptophan
may be due to the altered enzymatic and metabolic homeo-
stasis in endotoxin-poisoned animals which results in the
disruption of the normal balance of tryptophan metabolites.

Prior treatment of endotoxin-poisoned mice with
cyproheptadine, an antiserotonin drug, protected mice from

the convulsive death (104). This result suggests that

34

tryptophan is funneled in excess into serotonin production.
Further indications of the excess conversion of tryptophan
to serotonin synthesis are observed in the protection afforded
by cyproheptadine against hypothermia produced in mice kept
at 150C and given serotonin alone, or against hypothermia pro-
duced by tryptophan in endotoxin-poisoned mice (103).

If serotonin is important in the altered response
of endotoxin-poisoned mice to tryptophan, injection of sero-
tonin might be expected to cause enhancement of toxicity.
Conflicting data on this subject have been obtained. Gordon
and Lipton (57) demonstrated that a subcutaneous injection
of either 0.8 or 1.6 milligrams of serotonin per killogram
of body weight reduced mortality in mice given 24 or 32 mg/kg
of endotoxin intraperitoneally 30 minutes after serotonin.
Des Prez gt_al. (37) also demonstrated that the precursor of
serotonin, 5—hydroxytryptophan, and l-benzyl-2,5-dimethyl-
serotonin (BAS), an antimetabolite of serotonin, protected
mice from the toxicity of endotoxin. It is believed that
protection from endotoxin-poisoning by serotonin may be medi-
ated by the adrenal corticoids, since serotonin administra-
tion increases ACTH output. In the same group of experiments
Des Prez (37) also found that when the monoamine oxidase in-
hibitory Baphenylisopropyl hydrazine (PIH) was injected prior
to endotoxin, it made mice more susceptible to endotoxin.

Lasker (88) found similar results. Contrary to these reports

35

Susman eE_al. (7) and Davis et_al,(35) found that monoamine
oxidase inhibitors afforded protection to endotoxin. David
utilized PIH and l—(2-(benzylcarbamyl) ether)-2-isonicotinoyl-
hydrazine (nialamide), which inhibits both monoamine oxidases,
and N,N-dimethyl-2-phenylcyclopr0pylcyclopropylamine hydro-
chloride (S.K.F. 556), which is specific for monoamine oxi-
dases. The probable reason for the different results using
the same inhibitor (PIH) was that Des Prez used a larger dose
(40mg/kg) as compared to 10 mg/kg used by Davis. Davis also
found varying results with dose variations.

In addition to increased serotonin synthesis, there
is also an increase in serotonin released from platelets
after endotoxin administration (38,39). Recently, Nomura
and coworkers (113,114,115) isolated and purified a pro-
thrombin-like protein from plasma which causes the release of
5-hydroxytryptamine from platelets. This factor appears to
induce a change in the permeability of the platelet membrane,
thereby facilitating the entry of calcium ions into plate-
lets which in turn release 5HT from the storage granule.

The implications of funneling of tryptOphan from one
pathway to another are demonstrated by the work of Curzon and
Green (30). When tryptophan oxygenase was increased in
stressed rats, there was a decrease in brain serotonin due
to the flow of tryptophan into the kynurenine pathway. Along
similar lines, Schmike (140) and Sourkes (150) have independ-

ently shown that elevation of tryptophan oxygenase by

36

administration of u-methyltryptophan to rats causes a de-
crease in blood levels of tryptOphan with a consequent de-
crease in brain serotonin and 5HIAA levels. These results
suggest that although tryptophan oxygenase itself probably
does not influence survival of mice given endotoxin, the
funneling of tryptophan into other pathways and the produc-
tion of potentially toxic matabolites may be a factor in
convulsive death observed in endotoxin-poisoned mice given

a loading dose of tryptophan.

PART III

METHODS FOR SEPARATION AND IDENTIFICATION

OF TRYPTOPHAN METABOLITES

Paper Chromatography

 

Classically, one dimensional ascending paper chroma-
tography has been the method of choice for the separation of
urinary tryptophan metabolites. The solvent system most
widely utilized to separate metabolites of the kynurenine
pathway is composed of butanol, acetic acid, and water in
various combinations (54,55,80,106,133,134). For better reso-
lution of the metabolites Dalgliesch (33) utilized descending
paper chromatography with butanol, acetic acid, and water to
separate a wide variety of tryptophan metabolites. Of the
several other solvent systems utilized, methanol, butanol,

benzene, and water in the proportion 2:1:l:1 was preferred (134).

37

A wide variety of solvent systems have also been
used to separate the serotonin metabolites. Butanol, acetic
acid, and water (4:1:5) and several other organic solvent
systems have been utilized to separate 5HT, 5HIAA, 5HTOH,
and a few other serotonin metabolites (87,97,98). Pentikai-
nen and coworkers (121,122) used another combination of the
butanol system along with butanol, pyridine, and water
(1:1:1) to isolate nine different 5HT metabolites. Rodnight
(132) separated urinary indoles by column chromatography and
then used paper chromatographic separation in a variety of
solvents for identification. Urinary indoles extracted under
alkaline and acidic conditions have also been separated by
two dimensional chromatography with isopropanol, ammonium
hydroxide, and water (8:1:1) followed by butanol, acetic

acid and water (4:1:5) (151).

Thin Layer Chromatography

 

 

During the last decade thin layer chromatography
has become an excellent analytical tool for the rapid sepa-
ration and detection of minute amounts of biogenic amines
and other tryptophan metabolites. Diamenstein and Ehrhart
(40) found that either methyl acetate, iSOpropyl alcohol,
and ammonium hydroxide, 45:35:20, or chloroform, methanol,
and acetic acid, 90:5:5, were good solvent systems to sepa—
rate a variety of biologically relevant tryptophan metabolites

by one or two dimensional thin layer chromatography. Cotte

 

()

U)

g£_§1. (28) and Stahl and Kaldeway (153) used the methyl
acetate, isopropanol, and ammonium hydroxide solvent system
but used chloroform and acetic acid (95:5) for the second di-
rection runs. They found this method was a rapid, sensitive
way of detecting metabolites of the kynurenine and serotonin
pathways.

Aures et_al. (6) developed a microanalysis of tissue
amines utilizing a wide variety of solvent systems for thin
layer chromatography and then o-phthaldehyde as a spray rea-
gent for the detection of the compounds. Hill gE_§l. (69)
also used fluorescent techniques after development on thin
layer chromatOgraphy with chloroform, methanol, acetic acid
solvent system (75:20:5) and removal from the gel to detect

xanthurenic acid, kynurenic acid, and kynurenine.

Column Chromatography

Separation of tryptophan metabolites by column
chromatography presents an interesting problem. The meta-
bolites are all similar in molecular weight and are either
similar in molecular weight and are either similar in chemi-
cal nature such as many of the serotonin metabolites or
diverse as are some of the kynurenine metabolites.

Burtis and Warren (16) utilized a high pressure anion
exchange chromatographic system to separate more than 100 UV-
absorbing urinary metabolites. Of these, nine were tentatively

identified as tryptOphan metabolites. Due to some technical

39

problems this same group of workers (17) prefractionated the
human urine samples on Sephadex G—10 and thus simplified the
anion-exchange chromatograms. Nishino et_§l. (111) also

used ion exchange chromatography and separated 5HTP, 5HT, and
5HIAA on Dowex l (acetate form). Chen and Gholson (23) re-
cently reported the fractionation of a variety of tryptophan

metabolites on DEAR—cellulose (amine and formate forms).

Gas Chromatography

 

The use of gas chromatography to separate and iden-
tify tryptophan metabolites as well as other biologically im-
portant amine and acids has been hampered by the relative
insolubility of the kynurenine metabolites in the solvents
used for derivatization and by the formation of multiple
derivatives. Problems have also encompassed partial or com-
plete decomposition of the derivatives, trailing of peaks
and incomplete separation of closely related substances.
However, several studies have been reported. Urinary acids
contain several tryptophan metabolites which have been iso-
lated by various techniques (34,71,73,74,75). Dalgliesch
§E_31. (34) prepared the methyl esters, trimethylsilyl esters
and ethers, and their combinations of many acids of both the
kynurenine and serotonin pathways found in urine and sepa-
rated them on a 6 ft. x 3.5 mm glass U-shaped column contain-

ing F-60 coated on Gas Chrom P, 80/100 mesh. The Hornings

40

and their coworkers (72,73,74,75) also studied a wide vari-
ety of urinary metabolites and the different methods of deri-
vatizing and chromatographing them. Maruyama and Takemori
(96) described a method for separating 5—hydroxyindoleacetic
acid and 5-hydroxytryptamine from biological samples and
chtomatographing them on 3% OV-l7. Altschuler and Gold (4)
Chromatographed the metabolites of the kynurenine pathway on
2% OV-l7 using N,O—bis—trimethylsilylacetamide (BSA) as a

silylating agent.

 

 

 

k

U)

MATERIALS AND METHODS

Chemicals and Radioisotopes

 

N-Acetylserotonin, 5-hydroxyindoleacetic acid, 5-
hydroxytryptamine creatinine sulfate, indole-3-acetic acid,
kynurenic acid, D,L-kynurenine, N-methylnicotinamide, nico-
tinamide, nicotinic acid, quinolinic acid, and xanthurenic
acid were purchased from Nutritional Biochemicals Co. (Cleve-
land, OH). Bufetonin, 3-hydroxy-D,L-kynurenine, D,L-5—
hydroxytryptophan, L-5-hydroxytrypt0phan, indole, and trypta-
mine were purchased from Sigma Chemical Co. (St. Louis, MO).
o-Aminophenol, anthranilic acid, 3-hydroxyanthranilic acid,
and tryptophan were purchased from Aldrich Chemical Co.,
Inc., (Milwaukee, WI). 5-hydroxytryptophol, 5-methoxyindole-
acetic acid, 5—methoxyserotonin, and 5-methoxytryptophol were
purchased from Regis Chemical Co. (Chicago, IL). Quinaldic
acid was purchased from Eastman Organic Chemicals (Rochester,
NY).

D,L—Tryptophan (benzene ring-14C), specific activity
95 mCi/mM, and 5—hydroxytryptamine-3'-14C creatinine sulfate,
specific activity 55 mCi/mM, were purchased in 50 uCi ali-
quots from Amersham/Serle (Des Plaines, IL). Each was diluted
to 0.5 uCi/ml (1.1 x 106 dpm/ml) in 100 ml of sterile non-

pyrogenic physiological saline (Baxter Laboratories, Morton

41

 

f—J

42

Grove, IL). The radioactive tryptophan and serotonin were
frozen in dry ice, lyophilized, and stored at -700C to re-
duce degradation. They were reconstituted to original VOlume
in deionized distilled water (to maintain the prOper salt
concentration) just prior to use. For tryptophan load, stud-
ies, 20 mg of unlabeled L-tryptOphan was dissolved per milli-
liter of the radioactive solution just prior to injection.
All injections of the radioactive tryptophan and serotonin
with and without load were given intraperitoneally to the

mice at 0900.

Eighteen to 20 gram female CF—l mice (Carworth Farms,
Portage, MI) were used throughout the study. They were
housed six per cage with wood chips as bedding. Purina Lab-
oratory chow (Ralston Purina Co., St. Louis, MO) and water

were provided ad libitum. All mice were starved for 17 hours

 

prior to tryptophan injection (seven hours prior to endotoxin).

Endotoxin

 

Heat killed cells of Salmonella typhimurium, strain

 

SR—ll, served as the source of endotoxin in all experiments.
Cultures were grown for 18 hours in brain heart infusion

broth to a concentration of approximately 109 cells per milli-
liter and havested by continuous flow centrifugation in a
Sorvall RC—2B refrigerated centrifuge. Following centrifu-
gation, the cells were washed twice with isotonic non-

pyrogenic saline (Baxter Laboratories, Morton Grove, IL) and

 

I“, “a

.‘A

“:b

in

o“

m2.

PxV

NI»
~\,~

43

finally resuspended in saline to approximately ten times

their original concentration. Cells were heat-killed by
exposure to 1150C for six minutes. Lack of growth on sub-
culture served as proof of sterility. The LD50 of this prepa-
ration for mice was determined according to the method of

Reed and Muench (126). The endotoxin was diluted with non-

pyrogenic physiological saline so that 1 LD (approximately

50
4 x 109 heat-killed organisms) was contained in 0.5 m1.
Endotoxin was administered to mice intraperitoneally 10
hours prior to administration of radioactive tryptophan or
serotonin (at 2300).

Collection and Quantitation of Total
Radioactivity in Urine

 

 

To determine the total amount of radioactivity ex-
creted in urine, each mouse was placed in a 500 ml Erlenmeyer
flask which had a 9.0 cm Whatman #1 filter paper disc stapled
to the underside of a circular piece of aluminum screen.

Upon completion of the experiment, the filter paper was re-
moved from the screen, dried, cut into six pieces and placed
in scintillation vials containing 20 ml of a Scintillation
cocktail consisting of 4 g of PPO (2,5—diphenyloxazole) and
50 mg of POPOP (1,4—bis[2—(5-phenyloxazolyl)]benzene) in 1
liter of toluene. To determine counts per minute (cpm), each
sample was counted for 10 minutes in a Packard 2420 liquid

scintillation spectrometer and the background counts

-!>
sh

subtracted. A counting efficiency of 62% for filter paper
samples was used for conversion of cpm to disintegrations
per minute (dpm).

Collection of Urine for Identification

of Tryptophan Metabolites

Immediately after mice were injected with the iso-
tope they were placed in a metabolism cage with a screen
bottom which allowed the urine to be collected in a test
tube. At the end of the experiment each mouse was removed
from the cage and killed by cervical dislocation. Any urine
excretedafl:this time onto a metal tray was collected with a
micropipette and added to the tube. If the urine was cloudy
or contained solid material, it was centrifuged for 5 minutes
at 4000 rpm in a Phillips-Drucker Model 708 combination cen—
trifuge (Astoria, OR). Depending on the volume of urine ex—
creted, duplicate samples of 5, 10, or 20 ul were spotted on
strips of filter paper and counted by liquid scintillation
spectrometry in a Packard Model 2420 Tri—Carb spectrometer.

The urine sample was treated in one of several ways.
Generally, 40 to 60 ul of urine from an individual mouse were
spotted directly onto thin layer plates for two dimensional
thin layer chromatography. For DEAE-cellulose chromatography,
urine from 10 mice was pooled, the volume measured, and dupli-
cate 5 ul samples spotted on filter paper for liquid scintilla-

tion spectrometry (to determine the cpm/ul and the total cpm/

 

Ii

 

u.

m.‘

L)

ro-

‘1“

‘1

45

sample). The remaining urine was frozen in dry ice and kept

at -700C until it was loaded onto the column.

Thin Layer Chromatography

Ascending two dimensional thin layer chromato-
graphy was done in multiplate developing chambers (E. Merck
Ag, Brinkmann Instruments, Inc., Westbury, NY) lined on two
sides with paper towels. The solvent system for the first
development was composed of methyl acetate, isopropyl alco-
hol, and ammonium hydroxide in the proportion of 45:35:20.
Methyl acetate was prepared by refluxing molar quantities of
methanol (303 ml) and glacial acetic acid (598 ml) and 12 ml
of concentrated sulfuric acid for 30 minutes. The addition
of slight excess of acetic acid and distillation of the re-
sulting methyl acetate from the reaction mixture facilitated
the formation of the product. Further removal of any water
was accomplished by two additions of 12 grams of silicic acid.
A second distillation at 57.1OC yielded methyl acetate. Iso-
propyl alcohol and ammonium hydroxide were purchased commer-
cially.

The second solvent system was composed on n—butyl
alcohol, glacial acetic acid, and deionized distilled water
in the prOportion of 12:3:5. n—Butanol was redistilled and
Linde Type 3A (1/16") molecular sieves (Matheson, Coleman,
and Bell, Norwood, OH) added to the storage container. One

hundred and fifty milliliters of the two solvent systems were

46

prepared fresh daily and added to individual tanks from which
the solvents of the previous day had been just removed. The
tanks were covered tightly using vacuum grease on the lid and
allowed to equilibrate for at least one hour. This procedure
has been shown to give optimum tank saturation and resolution
(89).

The two solvent systems were chosen after testing
several combinations of different solvents. Opienska-Blauth
(118) reported instances where tryptophan metabolites were
decomposed in preparation for chromatOgraphy and in develop-
ment, but Lipton et_al,(89) and Benassi et_§l. (9) have re-
ported that with proper procedures degradation was eliminated.
This was the experience in this laboratory.

Precoated 20 cm x 20 cm Silica Gel F-254 glass
plates, purchased from E. Merck Ag., Brinkmann Instruments,
Inc., Westbury, NY, were used in all experiments. Prelimi-
nary results showed that heat activation of the plates was
not necessary. Plates were marked lightly with a soft lead
pencil 2 cm and 17 cm from the bottom and on one side to give
a two dimensional run of 15 cm x 15 cm. Samples were spotted
with "microcaps" lambda pipettes (A. H. Thomas Co., Phila—
delphia, PA) as one spot on the 2 cm point at the left side
of the plate. A warm air drier was employed between drop ap-
plication to facilitate drying and to insure a small sample

spot. DevelOping time usually required 3 to 3 1/2 hours for

47

the first run. After the plates were removed from the cham-
ber, they were allowed to dry thoroughly to remove all traces
of solvents and then turned 900 and placed in the second solv-
ent system which required 6 to 6 l/2 hours for development.
After removal from the second solvent, the plates were again
dried thoroughly. All Operations were performed in a hood.
One dimensional ascending thin layer chromatography
utilizing the two solvents previously discussed was done on
reference standards and eluates from the DEAE-cellulose
column. For these runs plates were marked lightly with a
soft lead pencil 2 cm and 17 cm from the bottom. Samples
were spotted on the 2 cm line at least 1.75 cm from the edge.
One dimensional runs of 5 to 20 ug of each standard compound
were made at least ten times in each solvent system. One
milligram of each reference standard was dissolved in either
distilled water, ethanol, or pyridine and 5 to 20 ul spotted
and develOped. Each compound was also dissolved in normal
mouse urine and Chromatographed. Fluorescent compounds were
circled lightly with a pencil while viewed under "long wave-
length" (356 A) ultraviolet light in a Chromato-Vue Cabinet
Model CC—20 (A. H. Thomas, Philadelphia, PA). Rf—values were
calculated at the distance from the origin to the center of

the spot divided by the distance the solvent traveled.

Autoradiography

 

All manipulations were performed in a dark room

using a safety light. Thin layer chromatographic plates

48

were placed in an 8" x 10" (20.3 cm x 25.4 cm) x-ray expos-
ure holder (Eastman Kodak Co., Rochester, NY). One 5" x 7"
Kodak No-Screen-Medical x—ray film (tinted estar safety base)
was placed over the plate near the bottom and another half
piece (2 1/2" x 7") was placed above this to cover the whole
plate. Spots of radioactive material had been put on the
edges of the plate and their location circled with a pencil
to determine the exact position of the film on the plate.
After 5 to 7 days exposure at room temperature, the film was
developed for 5 minutes at 200C or until spots were observed
on the film in Kodak liquid x-ray developer and then fixed in
Kodak liquid x-ray fixer for 8 minutes or until clearing of
the film occurred. After this, the film was rinsed in water
for 10 minutes to remove the fixer, and then dried. The two
pieces of film were taped together. A dark spot denoted the

presence of a radioactive compound on the thin layer plate.

Spray Reagents

 

Prochazka's formaldehyde-HCl reagent for detecting
indole derivatives was prepared just prior to use by mixing
10 ml of formaldehyde (about 35%), 10 ml of pure HCl, 25%,
and 20 ml of ethanol.

The reagent was sprayed onto the thin layer plates,
which were then heated to 1000C for 5 minutes producing yellow,

orange, and greenish fluorescent colors (152).

49

Van Urk's reagent (152), 4-dimethylaminobenzaldehyde,
was a second reagent used for detection of indole derivatives.
One gram of 4-dimethylaminobenzaldehyde was dissolved in 50
ml of HCl and then 50 ml of ethanol was added. The thin layer
plates were heated to 600C for 5 minutes and then sprayed ex-
haustively until they became transparent. After the plates
were dried in air, fluorescent and visible colors of the com-
pounds were recorded.

Ninhydrin (152) was used to detect primary and second-
ary amines. It was prepared by dissolving 0.3 g of ninhydrin
in 100 ml of n-butanol and mixing this with 3 m1 of glacial
acetic acid. This solution was sprayed onto the thin layer
plates and heated to 600C for 30 minutes or 110°C for 10
minutes.

1,3-Dihydroxynaphthalene (Naphthoresorcinol)-trich-
loroacetic acid (152) was used to detect glucuronic acid resi-
dues present in conjugates. This reagent was prepared by dis-
solving 0.2 g of naphthoresorcinol in 100 ml of ethanol and
mixing it with an equal volume of an aqueons 20% solution of
trichloroacetic acid. The plates were sprayed and heated to

70-800C for 10 to 15 minutes in a humid atmosphere (water

bath).

Column Chromatography

 

DEAE-cellulose, Cellex—D (Bio-Rad Laboratories,

Rockville Centre, NY), was prepared by a slight modification

50

of the method of Chen and Gholson (23). Twenty grams of

resin were added with stirring to 400 ml of 0.5 N HCl in a

1 liter beaker and stirred for 10 minutes. After sitting

for 30 minutes, the supernatant was decanted or filtered
through a Buchner funnel. The resin was washed extensively

(8 to 10 times; 2.5 to 3.0 l) with deionized distilled water
until the supernatant was pH 4 with pH paper. All water used
was deionized and distilled. The resin was poured into a 2
liter beaker containing approximately 1 liter of 0.5 N NaOH

(5 times the volume of swollen exchanger), stirred gently for
10 minutes and allowed to sit for 30 minutes. The supernatant
was decanted or filtered and the resin again washed with water
(approximately 2 liters) until the pH was 10 with pH paper.

One liter of l M sodium formate was added to the
resin, allowed to sit for 1 hour and filtered. Another liter
of 1 M sodium formate was added and after 45 minutes, filtered,
and washed 4 times with water to remove the sodium. The
resin should be almost white at this point.

The resin was the put into an aspirator bottle and
stirred. A Chromaflex 1.2 cm x 30 cm column (Kontes Glass
Co., Franklin Park, IL) was filled with 0.001 M triethylamine-
formate buffer and the column stopcock was opened. Nitrogen
pressure (7 lb/sq in) was applied to the top of the mixing
chamber forcing the resin into the column as the buffer flowed

out. The column was packed to a height of 10 cm. It was

51

placed in a 40C room and equilibrated with 0.001 M triethyla-
mine-formate pH 4.0. Standard solutions or urine samples

were loaded carefully onto the column and 80 ml eluted in 2 ml
samples with 0.001 M triethylamine-formate. A gradient was
then set up by placing 250 ml of 0.001 M triethylamine-formate
buffer (pH 4.0) in a cylindrical mixing chamber that was con-
nected by tubing to a reservoir of equal dimensions containing
250 ml of 0.1 M triethylamine-formate buffer (pH 4.0). Frac-
tions of 4 ml were collected. All samples were frozen in dry
ice, stored at -700C if necessary, and lyophilized in a Refrig-
eration for Science lyophilizer (Island Park, NY). They were
reconstitut to 0.1 ml with deionized distilled water. Five
microliters from each tube was spotted on filter paper and
counted by liquid scintillation spectrometry. The remaining
sample was used for thin layer chromatography or gas chroma-
tography-mass spectrometry.

Preparation of Methyl Esters for Gas
Chromatographnyass Spectrometry

 

 

Initially ethereal diazomethane made from N-nitro-
N-nitroso-N-methylguanidine (NMNG) was used for methylation;
but due to the insolubility in both ether and methanol, plus
the hazardous nature of diazomethane and NMNG, methyl esters
were prepared using 2,2-dimethoxypropane. One dram vials with
teflon lined screw caps containing 100 ug of a reference

standard, lyophilized or dissolved in 100 pl of methanol, or

52

an unknown urine sample were used throughout derivatization.
' Unknowns were isolated by drawing a vacuum through the small
end of a Pasteur pipette containing a small piece of glass
wool and removing the gel from the thin layer plate where a
spot was observed on an autoradiograph. Working in a hood
100 ul of an aqueous HCl (36%) solution and then 2 ml of 2,
2-dimethoxypropane (Fisher Chemical Co., Detroit, MI) were
added directly to the reference sample or run through the
Pasteur pipette with the unknown and mixed slightly. Darken-
ing of the solution occurred with sitting. After 19 hours
the sample was removed from the vial with a Pasteur pipette
and placed in a 5 or 10 m1 pear-shaped flask (Ace Glassware,
Vineland, NJ). The solution was evaporated to dryness in a
rotary evaporator over a 500C water bath to insure removal
of the HCl. The sample was reconstituted in 50 ul of pyridine,
transferred to a pyridine rinsed one dram vial, and recapped
tightly for storage at 40C.

Preparation of Trimethylsilyl (TMSi) Ethers

or Esters for Gas Chromatography-Mass
Spectrometry

 

Twenty five to 50 microliters of Regisil® (bis-
(trimethylsilyl)trifluoroacetamide) (Regis Chemical Co.,
Chicago, IL) was added to the vial containing the methyl
esters or to unknown or standard compounds (prepared as stated
in the previous section) containing 25 ul of pyridine, re-

capped tightly, and allowed to sit at room temperature or

53

heated at 84°C for 10 minutes. The first is preferred as
heating of the samples may cause degradation of the deriva-
tives. The samples should be kept dry as TMSi esters and
ethers hydrolyze easily. The pyridine in the samples did not
interfere with derivative formation or gas chromatographic-
mass spectral analyses. Two microliters of the methyl ester-
trimethylsilyl ethers (ME-TMSi) or trimethylsilyl (TMSi) esters
and ethers of the standards and variable amounts of the un-
knowns were injected directly into the gas chromatograph-mass

spectrometer.

Gas Chromatography-Mass Spectrometry

 

Mass spectrometry was performed at 100, 130, 150, or
2000C on a combined gas-chromatograph-mass spectrometer LKB
9000 equipped with a column (6' x 3 mm) of either 3% SE-30
or 2.5% SP-2401 on Supelcoport 100/120 (Supelco Inc., Belle-
fort, PA). The runs were made with helium as the carrier gas,
an ionizing electron energy of 70 ev, the molecular separator
at 2300C and the ion source at 2900C. The spectra were recorded

as bar graphs by means of an on-line computer system (154).

Quantitation of Urinary
Tryptophan Metabolites

After thin layer plates were developed and autora-
diographs made, the Rf values of the radioactive spots were
measured and the spots carefully scraped from the silica gel

layer with a small spatula, collected on the spatula by means

54

of a small paint brush and placed in scintillation vials.

The sample was crushed to a fine powder with a spatula and

10 m1 of toluene, PPO, and POPOP scintillation fluid was
added to 5 ml of ethyleneglycolmonomethylether:ethanolamine
solution in the vial. The samples were counted in a Packard
Model 2420 Tri-Carb liquid scintillation spectrometer and dpm
were determined by the calculated efficiency of 62% for these
samples. The percent of total dpm recovered from the urine
was calculated for each spot on the chromatogram. 'Several
plates, with the spots observed by autoradiography removed,
were sectioned into 150, 1 cm squares, scraped, and counted
to determine the quantity of residual radioactivity on the
plate. No more than 1% of the original isotope remained on

the plate.

Statistical Methods

 

Since the absolute quantities of individual meta-
bolites varied extensively among the plates as a direct
consequence of the amount of radioactivity spotted, all data
were calculated as a function of a percentage of the total
radioactivity recovered. The amount of total radioactivity
recovered was consistently over 99%. The statistical dif-
ferences between individual metabolitesvnnxadetermined by ap-
plying the White Rank Test (168) to the individual percentage
calculated. For statistical comparison of the total kynurenine

or serotonin pathway metabolites excreted, the relative

55

percentages of total pathway metabolites were obtained by
adding the percentages of individual metabolites from each

given pathway of a single mouse.

RESULTS

Rf Values, Fluorescence, and Color
Reactions of Standard
Tryptophan Metabolites

 

Tryptophan and 24 of its metabolites were each dis-
solved in an appropriate solvent (either distilled water,
ethanol, or pyridine) to a concentration of 1 mg/ml. Com-
pounds were also dissolved in mouse urine. Five, 10, or 20
micrograms of each compound were spotted on thin layer plates.
The plates were develOped in either the butanol, acetic acid,
and water solvent system or the methyl acetate, isopropanol,
ammonium hydroxide solvent system. Rf values were calculated
and are reported in Table 2. The fluorescence and color re-
actions with Prochazka's formaldehyde-HCl reagent and van
Urk's 4-dimethylaminobenzaldehyde reagent are presented in
Table 3. No significant change in Rf values or color reac-
tions were observed after refrigeration of the standard solu-
tions at 40C for 24 hours or after dissolving them in urine.

Similar results of other investigators are shown in Appendix A,

Tables Al, A2, and A3.

DEAE-Cellulose Chromatography of
Standard Tryptophan Metabolites

One hundred micrograms of each of 22 standard trypto-

phan metabolites were dissolved in 0.001 M Triethylamine-formate

56

TABLE 2.--Rf values of standard tryptophan metabolites in two

solvent systems.

 

E

 

METABOLITE SOLVENT Rf1 sz
Tryptophan H20 0.273 0.45
5-Hydroxytrypt0phan H20 0.21 0.37
5-Hydroxytryptamine H20 0.63 0.47
5-Hydroxytryptophol H20 0.84 0.75
5-Hydroxyindoleacetic Acid EtOH 0.27 0.73
N-Acetylserotonin H20 0.82 0.69
S-Methoxytryptamine H20 0.66 0.50
5—Methoxytryptophol H20 0.87 0.73
S-Methoxyindoleacetic Acid EtOH 0.28 0.75
Bufetonin H20 0.81 0.33
Tryptamine H20 0.77 0.58
Indoleacetic Acid EtOH 0.30 0.75
Indole EtOH 0.94 0.79
Kynurenine Pyr 0.27' 0.38
3-Hydroxykynurenine Pyr 0.23 0.43
Anthranilic Acid EtOH 0.39 0.77
3-Hydroxyanthranilic Acid Pyr 0.22 0.84
Kynurenic Acid Pyr 0.37 0.43
Xanthurenic Acid Pyr 0.43 0.47
Quinaldic Acid Pyr 0.46 0.55
Quinolinic Acid EtOH 0.03 0.03
o-Aminophenol EtOH 0.80 0.67
Nicotinamide EtOH 0.76 0.58
N-Methylnicotinamide EtOH 0.85 0.54
Nicotinic Acid EtOH 0.37 0.47

 

l - Methyl Acetate, Isopropyl Alcohol, Ammonium Hydroxide

(45:35:20)

2 - Butyl Alcohol, Acetic Acid, Water (12:3:5)

3 - All values are averages of at least 25 individual runs

with deviations no greater than $0.05 cm.

58

TABLE 3.--Characteristics of standard tryptophan metabolites with respect
to fluorescence, color reactions with van Urk's reagent, and
fluorescence and color reactions with Prochazka's reagent.

 

 

 

METABOLITE FLUORESCENCEl VAN URK'S PROCHAZKA'S
VISIBLE FLUORS.

Tryptophan - b 92 b g+gr y y
5-Hydroxytrypt0phan - y t b g - 9 br b gr br
S-Hydroxytryptamine t br b g - br dk y t
5-Hydroxytryptophol br ro dk b t ro t o t ro
5-Hydroxyindoleacetic Acid pi t br b y be br
N-Acetylserotonin - t b gr 9 br dk y
5-Methoxytryptamine - y t b t br t br
5-Methoxytryptophol - y t b t br t br
5-Methoxyindoleacetic Acid - t b t br t br
Bufetonin - t b g+b y br y br
Tryptamine - y t b g y br y o
Indoleacetic Acid t b y br y
Indole ro b g ro v
Kynurenine b y o y pi y
3-Hydroxykynurenine ro o y o dk 0 br 0 ru
Anthranilic Acid p b y pale b br be y
3-Hydroxyanthranilic Acid br v b o y dk y b o y
Kynurenic Acid p with gr - - p
Xanthurenic Acid y - - -
Quinaldic Acid - - - -
Quinolinic Acid - br - - -
o-Aminophenol y o ru dk 0 br dk ro br
Nicotinamide - - - -
N-Methylnicotinamide - - - -
Nicotinic Acid - y - - -

 

1

All characteristics represent data collected from

runs.

at least 25 individual

2Abbreviations: b=blue, be=beige, br=brown, dk=dark, g=grey, gr=green,

o=orange,

v=violet, y=yellow, -=colorless.

=purple, pi=pink, ro=rose, ru=rust, t=tan,

59

buffer (1 mg/ml) and Chromatographed on a DEAE-cellulose
column. After 1y0philization and reconstitution, each frac-
tion was run on one dimensional thin layer chromatography in
both solvent systems and Rf values calculated. These values
combined with fluorescence and color reactions were used to
determine in what fraction a given compound was eluted (Table
4). The neutral and basic compounds were recovered first
while the acids appeared in later fractions.

Gas Chromatography-Mass Spectrometry of

ME-TMSi and TMSi Derivatives of
Standard TryptOphan Metabolites

 

 

 

Gas chromatography-mass spectrometry proved helpful
in characterization of tryptophan metabolites in this study.
Most of the ME-TMSi derivatives of the tryptophan metabolites-
gave multiple peaks with similar retension times on gas chroma-
tographic analysis. The indole derivatives (tryptophan, 5-
hydroxytryptophan, 5-hydroxytryptamine, N-acetylserotonin, 5-
methoxytryptamine, 5-hydroxytryptophol, 5-methoxytryptophol,
5-hydroxyindoleacetic acid, 5-methoxyindoleacetic acid, trypta-
mine, and indole acetic acid) and the kynurenine metabolites
(kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid,
xanthurenic acid, kynurenic acid, quinolinic acid, and quin-
aldic acid) also gave almost identical mass spectra. A major
peak with mass 131, whose structure was not elucidated, was
consistently observed in analysis of these compounds. Several

other peaks with masses of 285, 270, 257, or 258, 217, 185,

 

 

 

60

TABLE 4.--Elution pattern of standard tryptOphan metabolites
from a DEAE-cellulose (formate form) column.

 

 

METABOLITE DEAE-ELUANT
S-Methoxytryptophol 5 - 8a
o-Aminophenol 5 - 12
Tryptamine 5 - 10
5-Hydroxytryptamine 5 - 12
Bufetonin 5 - 12
Kynurenine 5 - 12
Tryptophan 8 - l6
5-Hydroxytryptophan 8 - l6
Nicotinamide 9 - 12
N-Acetylserotonin 9 - l7
5-Hydroxytryptophol 10 - 12
N-Methylnicotinamide l7 - 21
Anthranilic Acid 19 — 46
3-Hydroxykynurenine 40 - 55
3-Hydroxyanthranilic Acid 43 - 61
Indoleacetic Acid 48 - 70
5-Hydroxyindoleacetic Acid 40 — 89
Quinolinic Acid 51 - 130
5-Methoxyindoleacetic Acid 62 - 68
Kynurenic Acid 81'- 105
Xanthurenic Acid 110 - 130
Quinaldic Acid 110 - 130

 

aAverage of two individual DEAE-
cellulose column runs.

61

and 170 were also regularly observed. Although these spectra
were similar, small variations occurred which allowed general
conclusions about basic structure to be drawn. For example,
only kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic
acid, and xanthurenic acid contained a peak at mass 105, and
only xanthurenic acid had a major peak at 240. The spectra

of the ME-TMSi derivatives obtained from kynurenine and 5-
hydroxytryptamine are shown in Figure 5. Those obtained from
the ME—TMSi derivatives of 3-hydroxykynurenine, kynurenic acid,
and xanthurenic acid, and 5-hydroxytryptophol are shown in
Appendix B, Figure Bl. Spectra of the terminal metabolites

of the kynurenine patheay: i.e., o-aminophenol, anthranilic
acid, nicotininc acid, nicotinamide, and N-methylnicotinamide
showed that methyl esters of these compounds were not formed
and only silylation occurred. These compounds either did not
have a carboxylic acid group or were insoluble in the reagents.
These spectra are shown in Figure 6.

Preparation of TMSi derivatives was simple and rapid
but care has to be taken in manipulation of the derivatized
samples as they hydrolyzed easily to give multiple gas chroma-
tography peaks. The spectra obtained from these derivatized
standards generally gave a molecular ion and predictable break-
down ions. Spectra of the TMSi derivatives obtained from
kynurenic acid, anthranilic acid, N-methylnicotinamide, and

S-hydroxyindoleacetic acid are shown in Figure 7.

 

 

 

 

62

Figure 5.—-Mass Spectra Obtained from the ME—TMSI
Derivatives of Kynurenine and 5-Hydroxytryptamine
Demonstrating the Similarities in Spectra.

 

 

63

 

 

 

 

 

 

 

 

 

 

 

 

 

oasesm: smn 13 KYNURENINE
100 — —_ ~ a —— , 7
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100 T11

80“ X4 L-

so~ I-

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50 100 150 200 250 300 350 400

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64

Figure 6.--Mass Spectra Obtained from o—Aminophenol,
Anthranilic Acid, Nicotinic Acid, Nicotinamide,
and N-Methylnicotinamide Prepared as ME-TMSi
Derivatives But Due to Structure or Insolubility
Forming Only the TMSi Ethers or Esters.

 

 

65

05173KM1 SCAN 11 TMSi-o-AMNOPHENOL

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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50 100 150 200 250. 300 350 400

66

Figure 7.--Mass Spectra Obtained from the TMSi Derivatives
of Kynurenic Acid, Anthranilic Acid, N-Methyl-
nicotinamide and S-Hydroxyindoleacetic Acid.

 

 

44. ._A_..——— _._’—-—-—

 

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68

Thin Layer and DEAF-Cellulose Chromatography
of TryptOphan Metabolites in Urine of
Normal and Endotoxin—Poisoned Mice

 

Autoradiograms of two dimensional thin layer plates
revealed between 8 and 25 different tryptophan metabolites
in the urine of normal and endotoxin-poisoned mice given D,
L-tryptophan (benzene ring-14C) with and without a tryptophan
load. A representative autoradiogram is shown in Figure 8.
When normal and endotoxin-poisoned mice were given S-hydroxy-

14C with and without load, eight to 12 meta-

tryptamine-3'-
bolites were isolated. A representative autoradiogram from

a two dimensional develOpment of urine from these mice is
shown in Figure 9.

Pooled urine from ten normal or endotoxin-poisoned
mice given labeled tryptOphan with or without load was chro-
matographed on DEAF-cellulose. Samples were lyOphilized and
reconstituted in 100 pl of deionized distilled water. Five
microliters of each sample were spotted on filter paper and
counted by liquid scintillation spectrometry. Radioactive
profiles obtained from normal and endotoxin-poisoned mice
given labeled tryptophan without load are shown in Figure 10.
Profiles obtained from mice with load were essentially the
same. To determine the metabolites recovered, individual
fractions containing more than 100 cpm were spotted on two
different thin layer plates, developed in the butanol, acetic
acid, and water and in the methyl acetate, isopropanol, and

ammonium hydroxide solvent systems, autoradiographed, and Rf

Values determined.

69

Figure 8.--A Representative Autoradiogram of a Two Dimen-
sional Thin Layer Development of a Urine Sample
from an Individual Mouse Given D,L-Tryptophan
(Benzene Ring-14C).

 

 

 

 

7O

REPRESENTATIVE AUTORADIOGRAN OF URINE
FROM NICE GIVEN D,L-TRYPTOPHAN(BENZENE RING-14c)

 

71

Figure 9.--A Representative Autoradiogram of a Two Dimen-
sional Thin Layer Development of a Urine Sample
froT4an Individual Mouse Given 5-Hydroxytryptamine-
3'- C.

 

RI1

 

REPRESENTATIVE AUTORADIOGRAN OF URINE
FROM NICE GIVEN 5-NVDR0XVTRVPTANINE-3'-14c

............
.........

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73

Figure lO.—-Radioactive Profiles of a Pooled Urine Sample
from Ten Normal or Endotoxin-Poisoned Mice
Given D,L-Tryptophan (Benzene Ring—14C)
Chromatographed on a DEAF—Cellulose (Formate
Form) Column.

 

CPA! x 10'3

CPI! x 10'3

14

12

1O

74

DEAE-cELLuosE GNRGNATOGRARIIV or URINE FROM NICE
GIVEN D,L-TRYPTOPHAN(BENZENE RING-c")

 

I

 

 

 

 

 

 

 

NORMAL

I WITHOUT LOAD
' I
+—
t ENDOTOXIN

L WITHOUT LOAD

#-

‘F 5’

 

I l l l I l

 

 

20 4O 60 80 100 120 140 160
FRACTION NUMBER

75

The same procedures were followed for the urine of

normal and endotoxin-poisoned animals given 5-hydroxytryptamine

-3'-14C with and without load. The radioactive profiles from

normal and endotoxin-poisoned mice without load are shown in

Figure 11 and with load in Figure 12.

Gas Chromatography-Mass Spectrometry of
ME-TMSi and TMSi Derivatives of
Tryptophan and Serotonin
Metabolites from Urine of
Normal and Endotoxin-

Poisoned Mice

 

 

 

 

 

 

Mass spectra of unconjugated urinary tryptophan or
serotonin metabolites reisolated from thin layer plates gave
similar results to the ME-TMSi derivatives of known standards.
Metabolites tentatively identified as conjugates had strong
79 and 128,129 peaks but no mass 131 peak. Several of the
serotonin metabolites also thought to be conjugates had the
79, and 128, 129 mass spectral pattern.

The spectra of the TMSi derivatives of the unknown
urinary tryptophan and serotonin metabolites gave two major
patterns and did not resemble the spectra of the standards.
One group had major peaks at 137, 159, 185, 193, 215, 237,
257, and 331 and the other at 129, 157, 185, 259, 329, 343,
and 396 (Appendix B, Figure B2).

On analysis of the spectra obtained from the compound
tentatively identified as 5—hydroxytryptamine, the mass peaks
of the TMSi derivatives of 5-hydroxytryptamine: 393 = M + 1,

291 = (M + 1) - 102, 276 = (M - 116) - 43, and 219 = (M-116) - 43,

76

Figure ll.--Radioactive Profiles of a Pooled Urine Sample
from Ten Normal or Endotoxin-Poisoned Mice
Given S-Hydroxytryptamine—3'14C Without Load
Chromatographed on a DEAR-Cellulose (Formate

Form) Column.

 

 

7 7
DEAE-CELLULOSE CHROMATOGRAPHY OF URINE PROM MICE
GIVEN s-IIVOROXVTRVPTAMINE-a'-c“

CPM “0'3

 

1O

3O '-
25 I"
20 '-
15

10-

 

 

NORMAL
WITHOUT LOAD

EN DOTOXIN
WITHOUT LOAD

0PM x 10‘3

 

 

 

 

 

 

 

 

1 l

 

1
20 4O 60 BO 100 120 140 160
FRACTION NUMBER

78

Figure 12.--Radioactive Profiles of a Pooled Urine Sample
from Ten Normal or Endotoxin- oisoned Mice
Given 5-Hydroxytryptamine-3'l C With Load

Chromatographed on a DEAF—Cellulose Column
(Formate Form).

 

 

CPM x 10'3

CPM x 10'3

79

DEAE-CELLULOSE CHROMATOGRAPHY OF URINE FROM MICE
GIVEN 5-HVOROXVTRVPTAMINE-3'-c'4

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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WITH LOAD
l I I J l
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FRACTION NUMBER

 

80

were present but in smaller proportions than other peaks found
in most of these spectra (Figure 13, top spectra). The mass
peaks identified as belonging to serotonin did not appear in
a spectra from the same run which did contain the other peaks
observed in the spectra with 5-hydroxytryptamine (Figure 13,
bottom spectra). Assuming this was a problem in concentration,
urine from normal mice given 5-hydroxytryptamine-3'14C was
spiked with 100 pg of 5HIAA and 5HTOH and Chromatographed on
a DEAE-cellulose column. The effluents from several fractions
were lyophilized and TMSi derivatives formed. The spectra
of the sample were screened and of them all, two serotonin
metabolites were observed. They were tentatively identified
as 5-hydroxyindoleacetic acid with one TMSi group and 5-
hydroxytryptophol with two TMSi groups (Figure 14).

Tentative Identification of Kynurenine

Pathway Metabolites Isolated from Urine

of Normal and Endotoxin-POisoned Mice

Given D,L-Tryptophan
(Benzene Ring-l4C)

 

 

 

 

 

Eleven kynurenine metabolites were selected for iden-
tification. The data obtained from thin layer and DEAE-cellu-
lose chromatography, fluorescence and color reactions along
with their tentative identification is contained in Table 5.
Kynurenine, 3-hydroxykynurenine, kynurenic acid, and xanthur-
enic acid were tentatively identified by comparison of fluor-
escence, color reactions, mass spectra, and Rf values with

known authenic standards.

81

Figure 13.--Mass Spectra Obtained from TMSi-S—Hydroxy-
tryptamine and Background.

 

 

:‘4

82

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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83

Figure l4.--Mass Spectra of TMSi-S-Hydroxyindoleacetic
Acid and TMSi-S-Hydroxytryptophol.

 

 

 

 

 

84

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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86

The tentative identification of kynurenine-O—sulfate
was based on its Rf value in the butanol, acetic acid, and
water solvent system (32,106), its fluorescence and color
reactions. It also gave a yellow color when sprayed with
naphthoresorcinol indicating a conjugate. The mass spectrum
of the ME-TMSi derivative gave major peaks at 79, 128, and
129 indicative of conjugation (Appendix B, Figure B3).

The other kynurenine metabolite was less easily iden-

tified. The fluorescence and color reactions indicated an

Ianconjugated, non-indole. The high Rf values in both solvent

ssystems indicated it could contain an alcohol function or an
aicetyl group. The standard compounds with Rf values in the
:range of this unknown were o-aminophenol, N-methylnicotinamide,

.S-hydroxytryptophol, and indole. 5—Hydroxytryptophol and in-

ciole were eliminated as they gave positive (blue and rose tan)

Ireactions with van Urk's reagent. 5-Hydroxytryptophol was

c>ften isolated from the same sample several times. The mass

sspectra of this unknown (Appendix B, Figure B3) and negative re-

aiction with naphthoresorcinol excluded a conjugate. Based on

tlhis information plus that of other workers (8,32,106) this
compound was identified as N—acetylkynurenine.

Five other kynurenine metabolites were also periodi-
<3Eilly observed but they appeared in concentrations too low

13C) give reliable results with the spray reagents and thus re-

EPIRDducible identification. Anthranilic and 3-hydroxyanthranilic

87

acid were observed and were tentatively identified by com-
parison to reference standards. Quinolinic acid, N-methyl-
nicotinamide, and o-aminophenol also appeared sporadically.
Quinolinic acid, due to its low Rf values, merged with the
residual radioactivity at the origin and hence was not always
easy to identify. Tryptophan was also regularly identified
and is included with these metabolites for identification.
Tentative Identification of Serotonin
Pathway Metabolites Isolated from
Urine of Normal and Endotoxin-
Poisoned MiEe Given 5-
Hydroxytryptamine-
3r-14C or D,L-Tryp—
tophan (Benzene
Ring-14C).

To aid in identification of serotonin metabolites in
IJrine from mice given the labeled tryptophan, normal and endo-
‘toxin-poisoned mice were injected with 5-hydroxytryptamine-
:3'-14C creatinine sulfate with and without a tryptophan load.
CPwo dimensional thin layer chromatography and column chroma-
1:ography revealed ten different serotonin metabolites of which
11ine could be tentatively identified (Table 6). With this in-
ffiarmation as a guideline, six serotonin metabolites could be
Inegularly identified in the urine of mice given D,L-tryptophan
(kbenzene ring-14C) with two other appearing sporadically.

5—Hydroxytryptophol, 5—hydroxyindoleacetic acid, and

£5--.hydroxytryptamine were tentatively identified by comparing

'tlleix'characteristic Rf values, fluorescence, color reactions,

88

TABLE 6.——Tentative Identification, Rf Values in Two Solvent Systems,
and Elution Pattern from DEAR-cellulose of Serotonin Pathway
Metabolites in Urine of Normal and Endotoxin-Poisoned Mice
Given D,L-Tryptophan (Benzene Ring-14C).

 

 

NO . TENTATIVE IDENTI F ICATION Rf l Rf 2 DEAE-ELUATE
5 5-Hydroxytryptamine 0.673 0.45 5 - 16
10 S-Hydroxyindoleacetic Acid 0.25 0.76 60 - 85
3 5-Hydroxytryptophol 0.88 0.78 5 - 8
218 5HT~O—Glucuronide 0.08 0.45 8 - 16
2C) 5HTOH-O-Glucuronide 0.01 0.26 80 - 95
119 5HIAA-O-Glucuronide 0.02 0.30 103 - 150
:22 SHIAA-O-Sulfate 0.07 0.18 82 - 150
2L6 '5HTOH-O-Sulfate 0.13 0.31 8 - l6
2L4 5HT-O-Sulfate 0.25 0.33 5 - 12
3.7 ? 0.02 0.53 43 - 67

 

urine samples .

raciiograph.

2Butyl Alcohol, Acetic Acid, Water (12:3:5).

l . .
Methyl Acetate, Isopropyl Alcohol, Ammonium HydrOXide

(45:35:20).

3Average Rf values calculated from at least 25 individual mouse

4 . .
Corresponds tx) metabolite number on the representative auto-

89

and mass spectra (Appendix B, Figure B4) to known standards.
S-Hydroxytryptamine was only observed in the urine of mice

given labeled 5-hydroxytryptamine.
Six metabolites were tentatively identified by compar-
ing their Rf values to those obtained by Pentikainen (122),

Tyce (157) and Dalgliesch (32). They were identified as 5-

hydroxytryptamine-O-glucuronide, 5-hydroxytryptophol-O-
glucuronide, 5-hydroxyindoleacetic acid-O-glucuronide, 5-
Jhydroxytryptamine-O-sulfate, S-hydroxytryptophol-O-sulfate,
aand S-hydroxyindoleacetic acid-O-sulfate.

The compounds tentatively identified as the glucuron-
:ides of 5—hydroxytryptamine, 5-hydroxytryptophol, and S-hydroxy-
:Lndoleacetic acid were analyzed by gas chromatography-mass
espectrometry and found to give the 79, 128 and 129 mass peaks
c>f conjugates instead of the major 131 peak (Appendix B, Figure
I35). 5-Hydroxytryptamine-O-glucuronide was present in concen-

t:rations high enough to give a positive naphthoresorcinol re-

action .
The sulfate conjugates of the three major metabolites

were tentatively identified by comparison of their Rf values

vvjsth those reported by other workers (32,122,157). The mass

Spectra (Appendix B, Figure B4) gave the characteristic mass

Peaks for conjugates.

90

Quantity of Radioactivitngecovered from
Urine of Normal and Endotoxin-Poisoned
Mice Giyen D,L-Tryptophan (Benzene
Ring-14C) With and Without Load.
Within one hour after intraperitoneal injection of
labeled tryptophan without load, normal mice excreted 21.6%

of the label in their urine (Table 7). The amount gradually

increased to a total of 29.2% after 6 hours. Endotoxin-
poisoned animals excreted only 6.0% of the label in the first
.hour. At 6 hours they had excreted 17.0% of the radioactivity

.in their urine. All decreases were statistically significant

(p<0.01). The dpm/ul did not vary significantly between the
Ilormal and endotoxin-poisoned mice without load at any time
15eriod except at three hours.

The variations in total urinary excretion observed
kaetween normal and endotoxin-poisoned mice with load were
ssignificant at one (p<0.001) but not at six hours (Table 8).

The presence of tryptophan load did not statistically
Eilter the total quantity of label excreted in either normal
c>r endotoxin-poisoned mice (Table 8 vs Table 7) but dpm/ul
Ciid vary at one point where statistically less label (p<0.01)

lJabel was excreted by normal mice without load than with load

(Crable 8 vs Table 7, 1 hour).

91

TABLE 7.--Quantity of Radioactivity Recovered from Urine of
Normal and Endotoxin-Poisoned Mice Given D,L-Trypto-
phan (Benzene Rine-14C) Without Load.

 

 

 

TIME NORMALa ENDOTOXIN
(HR) TOTAL DPM DPM/111 TOTAL DPM DPM/111
(%) + S.D (%) : S.D.
1 238,990** 661.5 66,412 675.1
(21.6) i 296.2 (6.0) 1 298.6
2 261,105** 493.7 108,487 738.4
(23.7) i 140.5 (9.9) : 362.0
3 298,807** 351.1* 132,650 530.6
(27.2) i 5.0 (12.1) i 97.9
6 321,243** 486.2 187,505 579.0
(29.2) 1 152.6 (17.0) 1 137.6
aAll values represent averages of data from at least
5 mice.

*
p<0.05

**p<0.01

 

TABI.

TIME
(HR)

5m

92

TABLE 8.--Quantity of Radioactivity Recovered from Urine of
Normal and Endotoxin-Poisoned Mice Given D,LdTrypto-
phan (Benzene Ring-14C) With Load.

 

 

 

TIME NORMALa ENDOTOXIN
(HR) TOTAL DPM DPM/pl TOTAL DPM DPM/Ill
(%) : S.D. (%) : S.D.
1 250,695*** 168l.5** 104,464 665.8
(22.8) : 415.8 (9.5) : 240.0
2 310,442** 598.6 153,707 574.8
(28.2) 1 107.0 (14.0) 1 149.4
3 313,440*** 473.5 168,071 370.8
(28.5) : 158.3 (15.3) :_89.3
6 276,566 453.0 210,219 711.5
(25.1) i,60-2 (19.1) : 271.8
aAll values represent averages of data from at least
5 mice.
*p<0.05
**p<0.0l

***p<0.001

 

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93

Quantitation of Tryptophan Metabolites in
Urine of Normal and Endotoxin-Poisoned
Mice Given D,L-Tryptophan (Benzene
Ring—14C) With and Without Load

To determine if quantitative variations occurred among
the distribution of tryptophan metabolites among the major
pathways in yiyg in normal and endotoxin—poisoned mice with
and without tryptophan load, individual radioactive metabolites
were scraped from two dimensional thin layer plates and the

quantity of isotope determined by liquid scintillation spec-

trometry. Preliminary studies showed that scraping all radio-

active spots observed on autoradiograms removed 99% of the

radioactivity from the thin layer plate. Therefore, the total

.radioactivity recovered (assumed to be 100%) per urine sample

Ivas calculated for each metabolite. The per cent radioactiv-

jtty recovered as metabolites of either the kynurenine or the
serotonin pathways is summarized in Table 9 with load and
Table 10 without load.

By one hour after tryptophan excretion of kynurenine

parthway metabolites was significantly (p<0.01) decreased in

endotoxin-poisoned mice compared to normal mice (13.0% with

load or 9.1% without load). Throughout the six hour time

perjxod, kynurenine pathway excretion was significantly reduced
(p<C).Ol) except at 3 and 6 hours without load and 6 hours with
loati. The overall percentage of metabolites accounted for as

kynurenine metabolites also decreased with time in all groups

94

TABLE 9.-—Relative Percentage of Kynurenine or Serotonin
Pathway Metabolites in Urine of Normal and
Endotoxin-Poisoned Mice Given D,L-Tryptophan
(Benzene Ring—14C) With Load.

 

 

 

TIME KYNURENINE SEROTONIN
(HR) NORMALa ENDOTOXIN NORMAL ENDOTOXIN
1 43.4 i 2.5** 30.4 i 3.4 8.4 i 1.0** 17.5 i 4.1
2 38.3 i 3.9** 32.1 i 3.2 13.2 i 4.5 20.7 :_1.5
.3 40.8 i 8.0** 29.5 i 3.6 10.1 i 2.6** 24.2 i 3.2
es 31.4 i 5.7 24.6 i 6.8 9.5 i 1.3** 19.3 i 0.6
**p<0.01

aAll values represent averages of data from at least
5 mice.

95

TABLE 10.—-Relative Percentage of Kynurenine or Serotonin

Pathway Metabolites in Urine of Normal and
Endotoxin-Poisoned Mice Given D,L-Tryptophan
(Benzene Rine-14C) Without Load.

 

 

 

TIME KYNURENINE SEROTONIN
(HR) NORMALa ENDOTOXIN NORMAL ENDOTOXIN
1 45.2 i 6.7** 36.1 i 3.3 13.2 i 1.9** 17.9 i 2.7
2 45.7 i 4.3** 33.1 i 7.2 16.1 i 2.8* 21.1 i 4.7
3 41.0 i 8.5 34.0 i 2.3 15.8 i 1.4** 24.0 :_5.4
6 35.3 i 11.3 24.2 i 5.4 14.0 i 0.7** 20.7 i 3.2
*p<0.05
**p<0.01

5.mice.

aAll values represent averages of data from at least

96

of mice. No significant differences in excretion patterns
between animals with and without tryptophan load were observed.
Excretion of serotonin metabolites was significantly
increased in endotoxin-poisoned animals at all periods tested
except at two hours with load. By one hour endotoxin-
poisoned mice with load excreted 9.1% (p<0.01) more serotonin
metabolites than normal animals. After three hours endotoxin-
poisoned animals with load had excreted 14.1% (p<0.01) more
serotonin metabolites than normal animals and after six hours
serotonin pathway metabolites still predominated (p<0.01) with
load. Without load endotoxin-poisoned mice excreted 6.7%
(p<0.01) more serotonin metabolites than normal mice. The in-
creased excretion of serotonin pathway metabolites is also
significant at 2 (p<0.05), 3 (p<0.01), and 6(p<0.01) hours.
Quantitative Enumeration of Individual
Metabolites of the Kynurenine and
Serotonin Pathways in Normal and

Endotoxin-Poisoned Mice Given D,
L-Tryptophan (Benzene Ring-l4C)

 

 

 

 

 

The quantitative variations in individual metabolites
of the kynurenine and serotonin pathways between normal and
endotoxin-poisoned mice with and without a tryptOphan load
at one hour are shown in Table 11. At one hour with load
only kynurenic acid was significantly reduced (p<0.05) in
endotoxin-poisoned compared with normal animals. Without load
in normal mice kynurenine (p<0.05) and N-acetylkynurenine

(p<0.05) were significantly reduced. Endotoxin-poisoned mice

97

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98

with load excreted a significant amount more S-hydroxyindole-
acetic acid than did normal mice (p<0.05).

At two hours kynurenic acid was significantly reduced
(p<0.05) between normal and endotoxin-poisoned mice with load
and kynurenine was again significantly reduced (p<0.01) with-
out load (Table 12). N-Acetylkynurenine was significantly
increased in endotoxin-poisoned mice without load (p<0.05).
Interestingly, 5-hydroxytryptamine-O-glucuronide was signifi-
cantly reduced in endotoxin-poisoned mice with load. The glu-
curonide conjugates of 5-hydroxytryptophol and S-hydroxyindole-
acetic acid, however, were increased significantly (p<0.05)
in endotoxin-poisoned mice without load.

After three hours no statistically significant differ-
ences in the kynurenine metabolites between normal and endo-
toxin-poisoned mice with or without load were detected (Table
13). However, among the serotonin metabolites, 5-hydroxy-
indoleacetic acid was significantly increased (p<0.05) in
endotoxin-poisoned mice both with and without load. 5-
Hydroxytngptophol—and 5-hydroxyindoleacetic acid-glucuronides
were also increased significantly (p<0.05) in endotoxin-
poisoned mice with load.

At six hours kynurenine and kynurenine-O-sulfate were
reduced (p<0.01 and p<0.05, respectively) in endotoxin-poisoned
mice with load and xanthurenic acid and 3-hydroxykynurenine
in the poisoned animals without load. Once again S-hydroxy-

indoleacetic acid was increased significantly (p<0.05) in

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101

endotoxin-poisoned animals with load as was S-hydroxytryptamine-
O-glucuronide (p<0.01) (Table 14).
In mice given tryptophan load between 26 and 35% of
the radioactivity was excreted as tryptophan at all time
points tested. In mice not given load, approximately 17% of

the label was excreted as tryptophan (Tables 11,12,13,l4).’

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DISCUSSION

In determining which tryptophan metabolites were
present in urine of normal and endotoxin-poisoned mice, it
was necessary to consider not only the 50 or more known
metabolites of tryptOphan but also the numerous tryptophan
metabolites which have been recognized but not identified
(119,152). This problem is further complicated by the fact
that there are over 200 "common" acids in urine. Of these
only one (5-hydroxyindoleacetic acid) or sometimes two
(5-hydroxyindoleacetic acid and indoleacetic acid) of the 18
known acid metabolites of tryptOphan appear in quantities
large enough to be included in the "commonfi urinary acids
(16,17,34). Hence, tryptOphan labeled with 14C in the benzene
ring provided an invaluable tool for the accomplishment of our
primary objective. Due to the position of the label, it was
retained in the metabolites of the serotonin and tryptamine
pathways and all but the terminal reactions of the kynurenine

pathway. In addition, 5—hydroxytryptamine-3'-l4

C provided a
useful tool, particularly in identifying the serotonin path—
way metabolites. The various isotopes allowed us to identify

and quantify specific metabolites even though they were pres-

ent in only nanogram amounts.

103

104

A major emphasis in this project has been placed on
devising techniques which would establish distinctive charac-
teristics for each individual metabolite or group of meta-
bolites in the kynurenine and serotonin pathways and thereby
aid in identification. Thin layer chromatography proved a
simple, rapid and reliable method for the separation of trypto-
phan metabolites. Although Rf values of compounds obtained
from a given biological sample may vary slightly from indi-
vidual standards or other biological samples, their relative
position usually remains constant as does their fluorescence
and color reactions. Throughout the experiments great care
had to be exercised in handling the radioactive tracers and
urine samples since many tryptophan metabolites are readily
degraded.

Elution patterns from DEAE-Cellulose columns are
characteristic (23). Being able to separate groups of
tryptophan metabolites based on ionic charge, provided a
means of categorizing the metabolites into subgroups which
could then be more easily identified on thin layer chroma-
tography.

Gas chromatography combined with mass spectrometry
also proved useful in confirming the identity of certain un-
known tryptophan and serotonin metabolites. Several papers
have been published on the derivatization of the serotonin

metabolites for gas chromatography-mass spectrometry (19,34,

105

75,96,110). Such information is less available on the kynur-
enine metabolites (4,111). Multiple peaks on gas chromatog-
raphy have been described for kynurenine metabolites deriva-
tized with trimethylsilyl groups. The serotonin pathway meta-
bolites readily formed TMSi derivatives as did a few of the
kynurenine metabolites. These conjugates hydrolyze within a
short time and therefore must be analyzed by gas chromatography
or gas chromatography-mass spectrometry within 24 hours. Spec-
tra of several TMSi derivatives of authenic compounds were ob-
tained (Figures 6,7,13,14). Preliminary studies showed con-
siderable discrepancies between the tentative identification
of unknown compounds and reference mass spectra. Further in-
vestigation showed that this discrepancy was not due to the
method but rather to the concentration of the unknown meta-
bolites available for study. When urine from normal mice
given 5-hydroxytryptamine-3'14C was spiked with 5-hydroxy-
tryptophol and 5-hydroxyindoleacetic acid, the spectra re-
sembled the reference standards (Figure 14). The unlabeled
material co-migrated with the label on thin layer plates.

The ME-TMSi derivatives are more stable than the TMSi
derivatives. The most common method for the preparation of
methyl esters (diazomethane) is not applicable to many of the
tryptophan metabolites because of the insolubility of these
compounds in methanol and ether; hence, 2,2-dimethoxypropane
‘Was used for methylation. Following preparation of the ME-

'TMSi derivatives, multiple gas chromatography peaks were

106

obtained and mass spectral analysis demonstrated that the
majority of the authenic as well as unknown tryptophan meta-
bolites formed similar ions with a major peak at 131 (Figure
5 and Appendix B, Figure Bl). Although the structure of this
compound has not been elucidated, the mass matches that of a
quinone type structure commonly produced in the oxidation of
these indoles (30,95). The kynurenine metabolites which
formed the 131 peak in the gas chromatograph had structures
which could form the quinone-like structure upon rearrange-
ment.

Several conclusions drawn from the spectra of un-
knowns were particularly valuable in identification. For
example, the compounds tentatively identified as conjugates
gave different major ion peaks at 79, 128, and 129 (Appen-
dix B, Figure BS) than their parent compounds (Appendix B,
Figure Bl). Further, the spectra showed that the major
metabolites of urine from mice given labeled tryptophan were
the unconjugated metabolites from either the initial reactions
of the kynurenine pathway (kynurenine, 3-hydroxykynurenine,
kynurenic acid, xanthurenic acid, and 3-hydroxyanthranilic
acid) or the primary serotonin pathway (5-hydroxytryptamine,
5-hydroxytryptophol, and 5-hydroxyindoleacetic acid). The
interpretation of spectra coincided with the results from Rf
values, fluorescence, color reactions, and DEAE-cellulose
chromatography elution patterns. The metabolites of the

kynurenine pathway (nicotinamide, nicotinic acid,

107

N-methylnicotinamide, o-aminophenol, and anthranilic acid),
which formed only TMSi esters and not methyl esters, were not
major metabolites. Although anthranilic acid, N-methylnico-
tinamide, and o—aminophenol were observed in mouse urine,
their concentrations were well below those where gas chroma-
tography-mass spectrometry would be feasible.

Eleven kynurenine metabolites were isolated from
urine of normal and endotoxin-poisoned mice given 0.5 “Ci of
D,L-tryptophan (benzene ring-14C) with and without tryptophan
load. Kynurenine, 3-hydroxykynurenine, kynurenic acid,

xanthurenic acid, kynurenine-O-sulfate, and N-acetyl—

kynurenine were the major kynurenine pathway metabolites.
Anthranilic acid, 3-hydroxyanthranilic acid, N-methylnicotina-
mide, o—aminophenol, and quinolinic acid also were tentatively
identified but they appeared infrequently.

Six serotonin metabolites were isolated from urine of
mice given the labeled tryptophan. They were tentatively
identified as 5-hydroxyindoleacetic acid, 5-hydroxytryptophol,
5—hydroxytryptamine-O-glucuronide, and 5-hydroxyindoleacetic
acid-O-sulfate. When mice were given 5—hydroxytryptamine-3'
—14C, four additional metabolites were observed. These were
5-hydroxytryptamine, 5-hydroxytrypt0phol-O-sulfate, 5—hydroxy-
tryptamine-O-sulfate, and an unidentified metabolite.

When endotoxin—poisoned mice were given benzene ring-

labeled tryptophan without load, they excreted significantly

108

less tryptophan and its metabolites than normal mice through-
out the six hour time period (Table 7). Tryptophan load did
not significantly alter the quantity of label appearing in
urine, except after one hour in normal mice despite the fact
that 20,000 times as much tryptophan was administered (Table
8). Interestingly, the amount of label per unit volume was
similar in both normal and endotoxin—poisoned mice with and
without load except again at 1 hour in normal mice (Tables 7
and 8). These data suggest a passive rather than active
mechanism for the filtration of tryptophan metabolites. The
decrease observed in urine of endotoxin-poisoned mice is
probably due to the decreased blood flow to the kidneys due
to vascular shock (70,107).

The decreased excretion of radioactivity in endotoxin-
poisoned mice was accompanied by significant decreases in the
amount of kynurenine metabolites and increases in serotonin
metabolites excreted (Table 9 and 10). These results suggest
a shift in metabolism from the kynurenine to the serotonin
pathway in endotoxin-poisoned mice. These results may be due
to the well-established depression of tryptophan oxygenase in
endotoxin-poisoned mice (1,11,104) or to the pooling of blood
in the intestine, a major site of serotonin synthesis (70,119).
Further studies, perhaps using isolated perfused organ systems
rather than whole animals, should aid considerably in determin—

ing whether metabolic or physiologic alterations are ultimately

109

responsible for the observed in yiyg alterations in trypto-
phan metabolism in poisoned mice.

Analysis of individual metabolites showed that there
were significant decreases in all of the kynurenine meta-
bolites evaluated (kynurenine, 3—hydroxykynurenine, kynurenic
acid, xanthurenic acid, and kynurenine-O-sulfate) except N-
acetylkynurenine. These data do not suggest the location of
any specific enzymatic lesions along the kynurenine pathway.
The increases in N—acetylkynurenine in endotoxin-poisoned
mice cannot be explained at present. Although tryptophan
oxygenase levels are depressed in endotoxin-poisoned mice,
the enzyme is not completely inhibited. It is possible,
though only suggested until enzyme levels are measured, that
there may be a depression of an enzyme, possible kynurenine
hydroxylase, so that metabolite flow beyond that point is
sharply curtailed. Such an occurrence could account for the
increase in the production of N-acetylkynurenine. Such a
hypothesis would also anticipate an increase in both anthra-
nilic acid and kynurenic acid. The former was not concentrated
enough to monitor reliably, and the latter was not increased.

Endotoxin-poisoned mice consistently excreted signifi-
cantly more 5-hydroxyindoleacetic acid than normal mice. At
two, three, and six hours, the glucuronide conjugates of the
serotonin metabolites were also significantly increased in

endotoxin-poisoned animals. Glucuronides are known to be

110

detoxification products in mammals (2,58,98,121). The data
show that there was a significant shift in the flow of trypto-
phan from the kynurenine to the serotonin pathway. 5-Hydroxy-
indoleacetic acid, which has been used as an indicator for
metabolic changes in several diseases (41,98,119), seemed to
be a valid marker for endotoxemia as well.

5—Hydroxytryptophol appeared as only a minor meta-
bolite in our studies. This data is in direct conflict with
a report by Nakai (108) suggesting that 5-hydroxytryptophol
and not 5-hydroxyindoleacetic acid was the major serotonin
metabolite of herbivores. While 5—hydroxyindoleacetic acid
was the major metabolite isolated, the glucuronide of 5-
hydroxytryptOphol was also present in significant amounts.
This may have been the metabolite observed by Nakai (108).
Pentikainen (121) also reported that although 5-hydroxy-
tryptophol is a metabolite of serotonin, it is not excreted
in appreciable quantities in urine of rats.

Although hyper-reactivity to endotoxin occurrs only
with a tryptophan load, no differences were noted in the
quantity of radioactivity excreted in individual metabolites
or pathways among normal or endotoxin—poisoned mice with and
without load. It must be remembered however, that there was
20,000 times more unlabeled tryptophan given to mice with
load so that the per cent of radioactivity represents a much
higher concentration of metabolites both excreted and remain-

ing in vivo.

111

The high excretion rate of unmetabolized tryptophan
(Tables ll-l4) was not expected” In animals given tryptophan
load, some tryptophan may be excreted but greater than 30%
seems excessive. In animals given the isotope alone, 17% of
the label was excreted as tryptophan after 6 hours. It is
possible that the D-isomer of the label was being selectively
excreted. Although mice have the enzymes to convert D-
tryptOphan to L-tryptOphan (64), there may have been no need
to do so, especially in experiments with load. This problem
should not alter the interpretation of our data on pathway
flow since evaluation of the relative distribution of label
among pathways only took into account the amount of metabolite
which entered the pathway. If the D-isomer is being selec-
tively excreted however, such an occurrence could lead to
slight misinterpretations on the significance of data describ-
ing the distribution of labeled tryptophan among the organs
and tissues of normal and endotoxin-poisoned mice (Moon et_
a1., unpublished data). Clarification of this issue must

await further investigation.

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APPENDICES

124

APPENDIX A

125

126

TABLE A1.--Rf Values and Color Reactions of "Simple" Indole

Derivatives (145).

 

 

* Color with Color with Fluorescence

Compound Rf van Urk's Prochazka's in Prochazka
Reagent Reagent Reagent
Indole 0.84 dark red to pale green green
violet
Indole-3- 0.86 pink orange with 2,4-dinitro-
aldehyde phenyl hydrazine reagent
Indole-3- 0.86 reddish yellow with 2,4-dinitro-
acetalde- brown phenyl hydrazine reagent
hyde
Indole-3- 0.31 blue, tinge yellow yellow with
acetic of violet green border
acid
5HIAA 0.19 blue to pale yellow deep violet
violet to beige
Tryptamine 0.77 blue-green yellow yellow with
blue border
Serotonin 0.65 grey yellow brown
D,L- 0.23 blue-green yellow yellow with
Tryptophan blue border
D,L-SHTP 0.14 blue-grey pale yellow yellow
to beige
Anthranilic 0.33 becomes pale beige blue, turning
Acid intensive brown
yellow

 

*
Rf Values in methyl acetate, isopropyl alcohol, and
ammonium hydroxide, 45:35:20.

127

TABLE A2.--Rf Values and Detection of Tryptophan Metabolites
Utilizing 4-Dimethy1aminobenzaldehyde Reagent (119).

 

Detection with
p-Dimethylaminobenzaldehyde

 

 

Substance Rf Reagent
Fluorescence Color

Tryptophan 0.25 - Violet

Indole 0.90 Blue Violet

Indican 0.61 Brown Brown

D,L-Kynurenine 0.32 Green-blue Yellow-brown

3-Hydroxy- 0.16 Yellow green Orange

Kynurenine

Kynurenic 0.45 Green after -

Acid 12 hours

Xanthurenic 0.45 Grey -

Acid

Anthranilic 0.45. Light blue Yellow

Acid

3-Hydroxy- 0.31 Light blue Yellow

Anthranilic

Acid

 

*Rf Values in methyl acetate, isopropyl alcohol, and
ammonium hydroxide, 45:35:20.

 

.‘

 

128

TABLE A3.--Rf Values of Serotonin Metabolites and Their
Variations (121).

 

Compound Rfa

 

S-Hydroxytryptamine Glucuronide 0.13
. (0.11 - 0.16)

5-Hydroxyindoleaceturic Acid Sulfate 0.15

5-Hydroxyindoleacetic Acid Glucuronide 0.28
(0.23 - 0.32)

5-Hydroxytryptophol Glucuronide 0.28
(0.23 - 0.33)

S-Hydroxyindoleacetic Acid Sulfate 0.35

S-Hydroxytryptamine 0.37
(0.35 - 0.44)

5-Hydroxyindoleacetic Acid 0.75

5-Hydroxytrypt0phol 0.78
(0.66 - 0.88)

5-Hydroxytrypt0phol Sulfate 0.27
‘ (0.24 - 0.32)

 

 

aDeveloped in butanol, acetic acid, and water,
12:3:5.

.‘

 

 

APPENDIX B

130

Figure B1.--Mass Spectra Obtained from the ME-TMSi Deriva-
tives of 3-Hydroxykynurenine, Kynurenic Acid,
Xanthurenic Acid, and 5—Hydroxytryptophol,
Demonstrating the Similarities in Spectra
Obtained.

 

 

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Figure B2.--Major Patterns Obtained from Mass Spectra of
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(Benzene Ring-14C) or 5-Hydroxytryptamine-3'14C
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135

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