a ·-o-·· - tdl
TRANSCRIPT
BILE PIGMENT METABOLISM DURING ANURAN METAMORPHOSIS
by
Kenneth D. Cole, B.S.
A DISSERTATION
IN
MEDICAL BIOCHEMISTRY
Submitted to the Graduate Faculty of Texas Tech University School of Medicine at Lubbock
in Partial Fulfillment of the Requirements for
the Degree of
DOCTOR OF PHILOSOPHY
Approved
I ' r ' ._. f
,/ ( v-""""" -
7\ I l, -----
A /l
·-\J· ·-o-·· ... -... r '5 ~ v
Accepted
x
0 ~ean ct} the G~aduat e School
December, 1981
ACKNOWLEDGEMENTS
I wish to thank Dr. G.H. Little for the opportunity to work
in his laboratory and for his expert guidance. Appreciation is
expressed to the members of my committee for helpful criticisms
and discussions. Expert technical assistance was provided by
Jim Mason, Martha Benson, Janice Brady, and Harvey Olney. The
patience and encouragement of my wife and parents has allowed
me to complete these studies.
ii
TABLE OF CONTENTS
ACKNOWLEDGMENTS . • • • • . . • • • • . . . . . . . . . . . . LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . LIST OF FIGURES • • . . . • • • • • • • • • . . . . . • • • •
I. INTRODUCTION • • • • • • • • . . . . . • • • • • • •
II. HIGH PERFORMANCE LIQUID CHROMOTOGRAPHY OF BILE PIGMENTS • • • • • • • • • •
III. BILE PIGMENT METABOLISM IN ADULT AMPHIBIANS • • • •
IV. BILE PIGMENT METABOLISM DURING ANURAN METAMORPHOSIS • • • • • • • • • • . • • • • • • • •
v. BILE 6-GLUCURONIDASE . . • • • • • • • . . . . • • •
LIST OF REFERENCES • • • • • • • • • • • • • • • • • • •
iii
11
1V
v
1
17
52
79
115
128
tIST OF TABLES
Table Page
3.1 Comparative Concentrations of Bile Pigments • • • • • . • • • . . . . . . . . . . . . . .
3.2
4.1
4.2
Comparative Activity of Hepatic Bilirubin UDP-Glucuronyl Transferase
Ratio of the Bile Concentrations of Bilirubin IXa to Bilirubin IXy • • •
• • • • • • • • •
. . . . . . . . . Concentrations of Bile Bilirubin IXa and Biliverdin IXa during Spontaneous Metamorphosis . • • . . . . . . . . . . .
4.3 Plasma Bilirubin Concentrations of T3 Injected Tadpoles and Control Tadpoles • . • • • • . • • . • • • . . . . . . . . . .
4.4 Plasma Bilirubin Concentrations of Phenylhydrazine-Immersed and Control Tad poles . . . . . . . . . . . . . . . . . .
5.1 Organisms Detected in Adult Bile • •
5.2 pH of Bile in Spontaneous Metamorphosis of Bullfrog Tadpoles
. . . . . • • •
. . . .
and Adult Bullfrogs ••••••••..•••••
lV
62
68
85
87
101
108
124
126
Figure
1.1
1.2
1.3
1.4
2.1
2.2
2.3
2.4
2.5
LIST OF FIGURES
Oxidative Cleavage of Heme to its Respective Bilirubin Isomers • • • • . . . . . . . . . Thin Layer Chromotography System of Heirwegh et. al. (1974) ••..•.• • • . . • • . . . Involuted Structure of Bilirubin IXa • • • • • •
Serum Transport, Intracellular Metabolism, and Excretion of Bilirubin IXa •••••• . . . . . . HPLC Seperation of p-Iodoaniline Azodipyrroles • • • • • . • • . • . . . . . . . . . . . The Base Catalyzed Transesterification of the Conjugated Azodipyrroles of Dog Bile . . . . . . . The Enzymatic Hydrolysis of the Conjugated Azodipyrroles of Dog Bile by Bacterial 8-Glucuronidase • • • • . . . . • . . • . .
The Enzymatic Hydrolysis of the Conjugated Azodipyrroles of Dog Bile by Almond
. . . . . .
8-Glucosidase •.•.•.•.••.••... . . . . . Enzymatic Synthesis of Bilirubin Conjugates • •
2.6 Enzymatic Hydrolysis of Conjugated Peaks 3 and 4 to Reveal their Respective Endovinyl, Exovinyl Isomers . . . . . . . . . . . . . . . . .
2.7 Azodipyrroles Resulting from the Coupling of the Isomers of Bilirubin IX with Diazotized p-Iodoanaline ..••...•.....••.
2.8 HPLC Separation of the Azodipyrroles of Bilirubin IXa, Bilirubin IXS, Bilirubin IXY and Bilirubin Ixc. • • • . • . • • • • . . . • . . . • . • .
2.9 HPLC Separation of the Bilirubin Azodipyrroles. . 2. 10 HPLC Separations of the Isomers of Biliverdin IX
v
. . .
. • .
. . .
Page
2
4
6
10
28
31
33
35
36
39
41
42
45
47
Figure
2.11
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
The Simultaneous Absorbance Monitoring of a Synthetic Mixture of Bilirubin Azodipyrroles (Peaks 7 and 8) and Biliverdin IX (l~) at 658 run Run at 0.5 AUFS and 5465 nm Run at 0.2 AUFS •• . . . . . . . HPLC of p-Iodoaniline Coupled A.) Mouse Bile; B.) Bullfrog Bile; C.) Bullfrog Serum
HPLC Tracing of Bullfrog Bile Diazo Coupled with p-Iodoanaline • • • • . • . • • • • • • . . . . . Simultaneous HPLC Monitoring of Biliverdin IXa and the Azodipyrroles of Bilirubin IXa ••••
Standard Curve of the Azodipyrroles of Bilirubin IXa •.•••••••••••. . . .
. . . .
• • . . Standard Curve for Biliverdin Ixa •. . . . HPLC Tracing (Absorbance at 546 nm) of an Assay Bilirubin UDP-Glucuronyl Transferase using Bullfrog Liver Homogenate ...•• . . . . . . Dependence of Bilirubin Glucuronide Synthesis on a Volume of Homogenate added to the Standard Bilirubin UDP-Glucuronyl Transferase Assay ••• . . . The Effect of pH on the Activity of Bullfrog Liver UDP-Glucuronyl Transferase • • • . • • • . • • • • • .
3.9 Hydrolysis of Bilirubin Glucuronide by Bullfrog
3 .10
3.11
4.1
4.2
Bile • • • . . . . . . . . . . . . . . • • •
Hydrolysis of the Glucuronic Acid Conjugates of the Azodipyrroles of Bilirubin IX by Frog Bile.
The In Vivo Glucuronidation of Bilirubin IXa by Bullfrogs . • • • • • • . • • • • • . • • •
Bile Concentrations of Bilirubin IXa During Spontaneous Metamorphosis of Bullfrog Tadpoles
. . .
. . .
. . .
and in Adult Bullfrogs ..•.......•.. • ..
Bile Concentrations of Bilirubin IXy During Spontaneous Metamorphosis of Bullfrog Tadpoles and in Adult Bullfrogs ....•..• · • · • · • · ·
vi
Page
49
59
60
61
64
65
66
69
70
72
73
74
83
84
Figure
4.3
4.4
4.5
4.6
4.7
4.8
4.9
4.10
4.11
4.12
Plasma Bilirubin Concentrations During Spontaneous Metamorphosis of Bullfrog Tadpoles and Serum Bilirubin Concentrations in Adult Bullfrogs .••••••••.••• • • • • •
Hepatic Activity of Bilirubin UDP-Glucuronyl Transferase (UDPGT) During Spontaneous Metamorphosis of Bullfrog Tadpoles and in Adult Bullfrogs ••••••••.•••••• • • • •
pH Dependence of UDP-Glcuronyl Transferase in Liver Homogenates of an Adult Bullfrog (A) and a Stage X Tadpole (e) . . . . . . . . . . . . . . . . Detection of Bilirubin Glucuronide in the Bile of Froglets Injected with Bilirubin and Saccharic Acid Lactone • • . • • • • • • • • • • • • • • . • • •
Base Catalyzed Transesterifiction in Methanol of Ester Azodipyrroles Conjugates ••••••• . . . . Detection of Bilirubin Glucuronide in the Bile of Stage X Tadpoles Injected with Bilirubin and Saccharic Acid Lactone ••••••••••••..
Bile Concentrations of Bilirubin IXa in the Biles of T3 injected Tadpoles (&) and Fasted Control Tadpoles (e) . . . . . . . . . . . . . . . . .
Bile Concentration of Bilirubin IXy in the Biles of T3 Injected Tadpoles (&) and Fasted Control
. .
. .
Tadpoles (e) . . . . . . . . • . . . . . . . . . • • •
Bile Concentrations of A.) Bilirubin IXS and B.) Bilirubin IXo of T3 Injected (&) and Control (e) Tadpoles ••••.••••••••
Specific Activity of Bilirubin UDP-Glucuronyl Transferase in the Livers of T3 Injected Tadpoles (&) and Fasted Control Tadpoles (e) .
. . . .
4.13 Hematocrit Values for Phenylhydrazine-
Page
88
89
91
92
93
96
97
98
100
102
Immersed Tadpoles (&) and Control Tadpoles (e) . • • . 103
4.14 Bile Concentrations of Bilirubin IXa in Phenylhydrazine-Immersed Tadpoles (&) and Control Tadpoles (e) • . . . • . • • . • . . . . • • . 104
vii
Figure
4.15
4.16
5.1
Concentrations of Bile Bilirubin IXy in the Bile of Phenylhydrazine-Immersed Tadpoles (!) and Fasted Control Tadpoles (•) ••••
Bile Concentrations of Biliverdin IXa in Phenylhydrazine-Immersed Tadpoles (!)
• • • • • •
and Control Tadpoles (e) ....... . • • • • • • • •
pH Dependence of S-Glucuronidase Acitivity in Liver Homogenate of an Adult Bullfrog (e) and Bile Pooled from Several Adult Bullfrogs (A) •• . . . . . . . . . .
5.2 Saccharic Acid Lactone Inhibition
5.3
5.4
of S-Glucuronidase in Liver Homogenate of an Adult Bullfrog (e) and Bile Pooled from Several Adult Bullfrogs (A) ••••••••••••
Cellulose Acetate Electrophoresis of A.) Bullfrog Liver Homogenate and B.) Bullfrog Bile •••.•..•
. . . . . . . . . .
. . . . . . . . . . Biliary S-Glucuronidase Activity During Spontaneous Metamorphosis and in Adult Bullfrogs ••••. . . . . . . . . . . . .
viii
Page
106
107
120
121
122
125
Historical Perspective
CHAPTER I
INTRODUCTION
Bilirubin was first shown to be the breakdown product of heme when
Fischer and Plieninger (1942) showed that bilirubin consisted of four
pyrrole rings joined by three carbon bridges. They showed that the
sequence of the side chains of the pyrrole rings are identical to those
of protoporphyrin IX and that bilirubin could be formed from protopor
phyrin IX by the oxidative removal of the alpha (a) methylene bridge.
This form of bilirubin was called bilirubin IXa (Fig. 1.1), because
of its obvious derivation from protoporphyrin IX.
The use of Ehrlich's diazo reaction by Van den Berg (1916) allowed
for the easy and sensitive assay of bilirubin by converting it to its
more stable diazo derivatives. Diazo coupling of bilirubin leads to
cleavage at the central methylene bridge with the formation of two
isomeric dipyrrolic azopigments. It became clear that there were at
least two types of bilirubin formed in man. The serum of patients with
hemolytic disease required the addition of alcohol to give a positive
reaction. The requirement of alcohol for the reaction resulted in this
type of biliribin being called indirect bilirubin. Normal bile or the
serum from patients with obstructive jaundice did not require the presence
of an alcohol and this type of bilirubin was called direct ·bilirubin.
The use of reverse phase partition chromatography revealed that
direct bilirubin contained two components termed I and II (Cole et al.,
1954). Conversion of indirect bilirubin to its stable azodipyrrole
yielded two molecules of what was called azopigment A. Pigment II,
when diazo coupled, yielded two molecules of a more polar azopigment
called azopigment B. Pigment I, when diazo coupled, yielded equal amounts
of azopigment A and azopigment B. Purification of azopigment B and its
characterization revealed that it was a glucuronide ester of azopigment
A (Billing et al., 1957; Schmid, 1956). It thus appeared that bilirubin
was conjugated with glucuronic acid and excreted into the bile mainly
1
Bilirubin IX d Bilirubin IX ex
(V)
(M) CH~ II
d rs ()( cl ~~ __ c 011
C H3-
1
___._ : J----.- CH 3 (M)
N-Fe-N
HO-c-c-c -'---rl N:, "'-=="- C=CH2 (V) HEME H2 H1 H
Protoporphyrin ( IX)
Bilirubin IX ~
c--( ",=c cs H- .d
CH2 . CH2 I
o=c I
OH
( P)
CH3 (M)
MP P MM VM V
~t1=cDcDc=U~ 0 N H N Hz N H N 0
H H H H
Bilirubin IX .8
Fig. 1.1. Oxidative Cleavage of Heme to its Respective Bilirubin Isomers.
2
as the diglucuronide although it was later shown that the two isomeric
monoconjugates do occur in bile (Jansen and Billing, 1971).
3
It was not until the introduction of the high resolution thin layer
chromatography system (TLC) of Heirwegh et al., (1970) which was able to
separate the bilirubin azodipyrroles that bile was shown to be a much
more complex mixture than suspected (Fig. 1.2). Heirwegh and his
associates diazo couple bile pigments with the diazonium salts of ethyl
anthranilate or p-iodoaniline. Diazotized ethyl anthranilate reacts only
with conjugated bilirubin under suitable conditions while diazotized
p-iodoaniline will react quantitatively with total bilirubin (Heirwegh
et al., 1974). From normal dog bile twenty tetrapyrrolic diazo-positive
bile pigments were resolved (Heirwegh et al., (1975). The tetrapyrroles
were identified following conversion to their corresponding azodipyrroles.
Recent Developments in Bile Pigment Structure
Heirwegh and his associates have characterized the azodipyrroles
based on their migration on TLC (Fig. 1.2). The most polar, or the
delta (o) spot has been shown to consist of the two isomeric dipyrrolic
azopigments derived from bilirubin IXa 1-0-acyl glucuronide (Compernolle)
et al., 1978; Blanckaert et al., 1978). The majority of the beta (B)
and gannna (y) TLC azopigments have been shown to be derived from bili
rubin IXa 2, 3 and 4-0-acyl glucuronide (Compernolle et al., 1978;
Blanckaert et al., 1978). These pigments will spontaneously form from
aqueous solutions of bilirubin 1-0-acyl glucuronide by the sequential
migration at alkaline pH (Blanckaert et al., 1978). Increased amounts
of the B and y azopigments are found in bile pigments of patients with
biliary stasis (Blanckaert et al., 1978).
The ao spot has been shown to be a mixture of the two isomeric
dipyrrolic azopigments derived from bilirubin IXa (Jansen and Stoll,
1971; Compernolle et al., 1970). These two isomers differ only in the
orientation of the methyl and vinyl side chains. Azopigment spot a 2
------------------------ F I a.r + etF
W/W/ffd/J ~ ~
I a,z
V////Z//hi °'3
I {j
{jx
I f'
~ _____ ...., _______________________ s
Fig. 1.2. Thin Layer Chromatography System of Heirwegh et al. (1974)-. Azopigments of dog bile characterized by their migration. They are designated into groups by Greek letters. Single azopigments spots may contain more than one species. Darker shading indicated higher concentrations in dog bile. S indicates sample application and F indicates solvent front.
4
has been shown to be a mixture of the two dipyrrolic isomers of bili
rubin IXa esterified with xylose (Fevery et al., 197la). The a spot 3
has been shown to correspond to the glucose ester conjugate of the
isomeric dipyrrole azopigments of bilirubin IXa (Fevery et al., 197la;
Gordon et al., 1974). Fevery et al., (1977), studied the bile of animals
and found glucuronic acid conjugates of bilirubin IXa appear to be
quantitatively the most abundant, with glucose and xylose conjugates of
bilirubin IXa found in low high concentrations d~pending upon the
species studied. In most animals monoconjugates seem to dominate, al
though in the rat, dog, and human, bilirubin diglucuronide is the major
bile pigment (Fevery et al., 1977; Gordon et al., 1976; Blanckaert et
al., 1980). Trace amounts of bilirubin IXB, IXy and IXo have also
5
been observed in the biles of animals in an apparently unconjugated form
(Heirwegh et al., 1977; Blanckaert et al., 1977b; O'Carra and Colleran
1970).
Structure of Bilirubin IXa
A question that comes to mind when the structure of bilirubin IXa
is examined is why the organism needs to go to the metabolic expense
to conjugate bilirubin with a carbohydrate before it is excreted.
Bilirubin IXa contains two propionic acid side chains that,would be
expected to ionize at physiological pH and aid in the solubility of the
molecule. The actual solubility of bilirubin in aqueous solutions is
exceedingly small but it is readily soluble in organic solvents such as
chloroform.
Fog and Jellum (1963) first proposed that bilirubin IXa had an
involuted conformation in which the propronic acid side chains were
hydrogen bonded to pyrrole ring nitrogens and thus their ionization would
be supressed (Fig. 1.3). This involuted structure has been shown by
x-ray diffraction to be the actual structure for crystalline bilirubin
IXa (Bonnett et al., 1976). If this involuted structure of bilirubin
IXa is the configuration it assumes in aqueous solutions it would ex
plain certain properties of bilirubin. For bilirubin to undergo the
Van den Berg reaction it would require an alcohol to disrupt the
6
Fig. 1.3. Involuted Structure of Bilirubin IXa.
7
hydrogen bonding and open the molecule for attack by a diazonium salt.
Esterif ication of the propionic acid side chains with carbohydrate
molecules would presumably disrupt the involuted structure and would
result in a more soluble molecule. Biliverdin IXa with its central
methylene bridge in a non-reduced state would be more hindered and not
able to form the involuted structure. Biliverdin IXa is much more sol
uble in aqueous solutions and apparently does not require conjugation for
its efficient excretion (Barrowman et al., 1976).
Bilirubin has four possible geometric isomers, the two methylene
bridges in the tetrapyrrole molecule can exist in a cis or trans form.
X-ray crystallographic experiments (Bonnett et al., 1976) have shown
that bilirubin exists in the more thermodynamically stable trans, trans
isomer (also known as the ZZ form). It is the ZZ form that allows the
involuted hydrogen bonded structure to occur {Figure 1.3). Presumably
the cis, cis form (also known as the E-E form) or the cis, trans (E-Z)
or the trans, cis (Z-E) would not be able to assume the hydrogen bonded
form. It has not been directly shown that this isomerization (transfor
mation from Z to E) can occur with bilirubin but studies with other
organic models suggest that such an isomerization is light catalyzed
(Falk and Grubmayr, 1977).
Although the structures of the non-a isomers of bilirubin have not
been investigated by X-ray diffraction, they presumably do not form such
hydrogen bonded structures. Blanckaert et al., (1977a) have investigated
the biliary excretion of bilirubin IXa, IXS, IXy and IXo in normal
rats and the bilirubin UDP-glucuronyl transf erase genetically deficient
Gunn rat. After loading the rats with 14c-labeled bilirubin isomers
the normal rat quickly excreted all four isomers, IXa in a predominantly
conjugated state and the non-a isomers in varying amounts of conjugated
and unconjugated forms. Gunn rats were severely deficient in the biliary
excretion of bilirubin IXa but were able to excrete efficiently the
non-a isomers in an unconjugated form. These experiments indicate that
conjugation with glucuronic acid has been developed as a mechanism to
rid the body of bilirubin IXa.
Formation of Bilirubin
A microsomal enzyme (heme oxygenase) utilizing molecular oxygen
and NADPH is capable of converting heme to biliverdin (Tenhunen et al.,
1969). This enzyme is most active with free heme or heme solubilized
with albumin. Heme oxygenase apparently produces only the a isomer of
biliverdin (Tenhunen et al., 1969). In mammals a cytoplasmic NADPH
requiring enzyme, biliverdin reductase, is present in excess and reduces
any biliverdin IXa to bilirubin IXa (Tenhunen et al., 1970). Bili
verdin reductase also appears to be specific for the IXa isomer of
biliverdin (Colleran and O'Carra, 1977).
The formation of biliverdin from heme has also been proposed to
occur through what is called a coupled oxidation reaction. A coupled
oxidation is so called because it requires oxygen and a reducing agent
(usually ascorbic acid}. Hemoglobin under these conditions will produce
small amounts of biliverdin IXa and also significant amounts of IXS
(O'Carra and Colleran, 1969). Complexing hemoglobin with haptoglobin
increases the preference for the a isomer (O'Carra and Colleran, 1969).
In this type of mechanism the heme protein appears to direct its own
destruction but the importance of this mechanism in the organism is
unclear.
It is now believed that hemoglobin in the blood and hemoglobin in
intact senescent erythrocytes are catabolized by two different cell
populations (Bissel, 1975). The catabolism of intact erythrocytes is
believed to be accomplished by the reticuloendothelial system (spleen,
bone marrow, and liver sinusoidal cells) (Bissel et al., 1972a; Bissel
et al., 1972b). Hemoglobin in the blood bound or not bound to hapto
globin is catabolized by liver parenchyrnal cells (Bissel et al., 1972b;
Hershko et al., 1972).
8
There are many other heme proteins in the cell in addition to hemo
globin. The cytochromes of the endoplasmic reticulum and mitochondria,
some enzymes, and myoglobin are examples. By labeling heme with 14c
glycine it was found that in addition to a large peak of radioactivity
occuring in the bile appearing at the time of the half life of red cells,
an early peak of radioactivity was observed (Robinson, 1968). Studies
9
with isolated perfused livers showed that the liver was the source of
the early labeled peak (Robinson et al., 1965). The kinetics of the
early labeled peak indicate the presence of two or more components
(Schmid et al., 1966). The kinetics of the early phase of the early
labeled peak has a half life of one hour and does not correspond to the
turnover of any known hemeprotein in the liver (Marver and Schmid, 1972).
The later kinetics of the early labeled peak appear to be linked to the
turnover of cytochrome P-450 (Bissel, 1975).
Transport of Bilirubin
Bilirubin binds to serum albumin at a high affinity site in a
ratio of 1:1 (Ostrow and Schmid, 1963). This is believed to be the
major mechanism in which this insoluble compound is transported in
the body. It is possible for bilirubin to be displaced from serum
albumin by fatty acids and other compunds resulting in high levels of
"free" bilirubin (Odell, 1966). High levels of serum bilirubin are
apparently harmless to adults but high bilirubin concentrations in the
serum of newborn human are responsible for neural damage (kernicteus).
The most important determinant in the development of neural damage is
the fraction of bilirubin not bound to serum albumin (Maisels, 1972).
In the liver cell bilirubin binding proteins termed Y and Z exist
(Levi et al., 1969). Y protein (also called ligandin) appear to be
more specific for bilirubin. The exact role of Y and Z in bilirubin
transport and metabolism have not yet been fully clarified, however,
albumin appears to have a greater affinity for bilirubin than Y protein
in vivo (Fleischner and Arias, 1976).
Conjugation of Bilirubin by the Liver
The conjugation of bilirubin with glucuronic acid is accomplished
by bilirubin UDP-glucuronyl transf erase. This enzyme that is tightly
bound to the microsomal fraction of liver homogenates and requires UDP
glucuronic acid as a second substrate (Schmid et al., 1957; Heirwegh
et al., 1972). In most assays currently in use for bilirubin UDP-glucur
onyl transferase only bilirubin monoglucuronide is formed. The exact
BLOOD -- -
Bilirubin Albumin Complex
J
~
Free Bilirubin
~
Pool
HEPAT OCYTE MICROSOMES
81lirubin + UDPGA UDPG T..._ 81lirubin G/ucuronide
) .,I
-- - ---- ----- - - - - - - - - - - - - - - --
Bilirubin '" Ligondin
Complex
I
'
UOPGA NAOH+H+
J
UDPGDH
UOPG NAO+
CYTOSOL
BILE
... Bilirubln Glucuronide
Fig. 1.4. Serum Transport, Intracellular Metabolism, and Excretion of Bilirubin IXa. Abbreviations used are UDP-glucuronic acid (UDPGA), UDP-glucose (UDP glucose), UDP-glucuronyl transferase (UDPGT), nicotinamide adenine dinucleotide (NAD+), and nicotinamide adenine dinucleotide reduced form (NADH).
1---1 0
11
mechanism for the formation of bilirubin diglucuronide is still contro
versial. Jansen et al., (1977) have presented evidence that the con
version of bilirubin monoglucuronide to bilirubin diglucuronide may
occur at the plasma membrane of liver cells. They propose that bili
rubin diglucuronide formation results from the transfer of a glucuronyl
group from one monoglucuronide to a second monoglucuronide molecule to
form one molecule of bilirubin diglucuronide and one molecule of un-
conj ugated bilirubin. Whether one enzyme catalyzes the formation of both
bilirubin mono and diglucuronide or seperate enzymes exist, will ulti
mately be answered by purification and characterization of the respective
enzyme{s).
Bilirubin in Development
Neonatal hyperbilirubinemia is a connnon occurrence in humans. The
increase in serum bilirubin levels of newborns is due apparently to an
increased catabolism of heme and a deficiency in the capacity of the
bilirubin excretory pathway (Lester et al., 1963; Bernstein et al.,
1969; Schenker et al., 1964). Elimination of bilirubin produced by the
fetus is accomplished by transfer of bilirubin IXa across the placenta
(Lester et al., 1963; Bernstein et al., 1969; Palma et al., 1977). At
birth, with the pacental route of excretion removed, the fetus must
handle its own bilirubin excretion and until its excretory system has
developed, neonatal hyperbilirubinemia will be the result.
The f orrnation of bilirubin glucuronide in vitro by livers of human
(Lathe and Walker, 1958), and monkey fetuses (Lucey et al., 1963) has
been shown to be nearly absent. Recently a study was performed using
high perf orrnance liquid chromatography (HPLC) to measure low levels of
bilirubin UDP-glucuronyl transf erase in human fetal and newborn livers
was performed (Kawade and Onishi, 1981). In the livers of young
fetuses levels of bilirubin UDP-glucuronyl transf erase corresponding to
about 0.1% of the adult levels were detected. Between 30 and 40 weeks
(term) of development, activity increased from 0.1% to 1.0% of the adult
levels. After birth, levels increased rapidly and soon reached adult
levels (Kawade and Onishi, 1981). Fetal livers are also low in the intra
cellular anion binding protein ligandin (Levi et al., 1970). Assuming
that ligandin plays an important role in the transport of bilirubin
this deficiency could result in impared ability to excrete bilirubin.
The first bile pigment detected in the bile of human fetuses is
bilirubin rxs occuring between 14 and 15 weeks of development
(Blumenthal et al., 1980). The next bile pigment to be detected is
bilirubin IXa, followed by monoconjugates of bilirubin IXa with
12
glucose and xylose. Between 20 and 23 weeks of development monocon
jugates of bilirubin IXa with glucuronic acid are detected. These mono
conjugates increase to become the major pigment in newborns (Blumenthal
et al., 1980).
Concentrations of UDP-glucuronic acid can also affect the conjuga
tion of bilirubin with glucuronic acid. UDP-glucose dehydrogenase,
the enzyme responsible for the production of UDP-glucuronic acid from
UDP-glucose is found in lowered levels in fetal livers (Fyffe and
Dutton, 1975; Brown and Zueler, 1958). In the fetal pig, levels of
UDP-glucose are high but UDP-glucuronic acid levels are low (Zhivkov
et al., 1975). It might be expected that if UDP-glucose levels are
high, UDP-glucosyl transferase activities would compensate for deficient
UDP-glucuronyl transferase activity. Fevery et al., (1977b) found in the
fetal rat livers xylosyl-, glucosyl-, and glucuronyl transferase activi
ties toward bilirubin were equally low in activity. The f~nding that
glucose and xylose conjugates appear before glucuronic acid conjugates
of bilirubin IXa. in the human fetus indicates that xylosyl- and
glucosyl transf erase activities appear before the bilirubin glucuronyl
transferase activities.
Using fetal rats it has been shown that there are at least two
separate types of UDP-glucuronyl transf erases with activity towards
different substrates (Wilhart et al., 1978). These two activities are
different with respect to when they reach adult levels, their induci
bility by glucocorticoids, their stimulation by xenobiotics and their
subcellular location (Wishart et al., 1978). According to Wishart et
al., (1978), the first type of activity develops in the late fetal
period and its apparent substrates include 4-nitrophenol, 1-naphthol~
and serotonin. The second type of activity develops in the neonatal
13
period and includes bilirubin, morphine, testosterone, and other
hormones as substrates. Activities of the late fetal group rise from
low levels to adult levels several days before birth, while the neonatal
group of activities do not start to rise to adult levels after birth
(Wishart et al., 1978).
The UDP-glucuronyl transf erases of the late fetal group can be
prematurely increased by exposure to glucocorticoid hormones in utero
(Wishart and Dutton, 1977) or in organ culture (Catz and Yaffe, 1968).
The increase is dependent upon protein synthesis and probably represents
the synthesis of new enzyme (Wishart and Dutton, 1977). In rats at
the time of increase of the late fetal group of enzyme activities, fetal
glucocorticoid levels are rising (Wishart et al., 1977). It thus appears
that glucocorticoids are responsible for the in vivo induction of the
late fetal UDP glucuronyl transferases.
Using adult rats Brock et al., (1977) found that with 3-methylcho
lanthrene stimulation of the glucuronidation activities toward 4-nitro
phenol and 1-naphthol was greater than stimulation of activities toward
bilirubin, chloramphenicol and morphine. Phenobarbital had an inverse
effect, increasing the levels of glucuronidation activity toward bili
rubin, chloramphenicol and morphine over 4-nitrophenol and 1-naphthol
glucuronidation activities.
In adult rats the late fetal glucuronyl transferase activities tend
to predominate in microsomal fractions derived from rough endoplasmic
reticulum and the neonatal glucuronyl transf erases are concentrated in
the microsomal fraction corresponding to smooth endoplasmic reticulum
(Wishart et al., 1977). This observation is important in light of the
fact that rough endoplasmic reticulum is the dominant form of endo
plasmic reticulum in the fetus and that after birth the smooth endo
plasmic reticulum appears (Greengard, 1974). The ability of phenobarbi
tal to increase the rate of bilirubin glucuronide formation is apparently
dependent upon the appearance of smooth endoplasmic reticultnn in develop
ment (Chedid and Nair, 1974).
The next section will detail an easily manipulated animal which
appears to be well suited for elucidating the signals responsible for the
development of the bilirubin excretory pathway.
Amphibian Metamorphosis as a Model for Mannnalian Development
During its metamorphosis the tadpole undergoes a transition from
a free swirmning aquatic form to a predominantly terrestrial adult form.
The mammalian fetus undergoes a similar transition from the aquatic
life in the womb to a terrestrial existance outside the womb. There
exists many biochemical events in the development of the late fetus
that are paralleled in amphibian metamorphosis (Greengard, 1969).
14
It has been shown that during metamorphosis a shift from excretion
of ammonia to urea takes place (Ashley et al., 1968). Along with this
change are corresponding increases in the urea cycle enzymes in the
livers of tadpoles undergoing metamorphosis (Cohen, 1970). The urea
cycle enzymes make their appearance late in development of the marmnalian
fetus (Kennan and Cohen, 1959).
Since the early discovery by Grudernatsch (1912) that feeding
tadpoles thyroid tissue causes their precocious metamorphosis, much
effort has been devoted toward using amphibian metamorphosis as a model
for thyroid hormone action. Injection of premetamorphic tadpoles with
thyroxine has been shown to cause increases in the urea cycle enzymes
to levels corresponding to those of spontaneous metamorphosis (Adamson
and Ingbar, 1967). Greengard (1969) has shown that a single injection
of thyroxine will accelerate the prenatal rise in the levels of the
urea cycle enzyme, arginase.
During metamorphosis there is an accumulation of glycogen in the
liver of tadpoles that is depleted at the end of metamorphosis (Bilewicz,
1938). A similar pattern of glycogen accumulation is seen in the liver
of fetal rats that is rapidly depleted after birth (Dawkins, 1963). The
enzyme responsible for releasing glucose after the initial breakdown of
glycogen, glucose-6-phosphatase increases in spontaneous metamorphosis
and after thyroxine treatment in tadpoles (Frienden and Mathews, 1958).
Glucose-6-phosphatase rises in late fetal development and also after
birth in fetal guinea pigs (Nemeth, 1954) and fetal rats (Dawkins, 1963).
15
There is a large increase in serum albumin levels during the meta
morphosis of the tadpole (Herner and Freiden, 1960; Feldhoff, 1971).
Ledford and Freiden (1973) found that esposure to triiodothyronine (T3
)
increased the levels of serum albumin synthesis in premetamorphic tad
poles. Albumin synthesis is also high during the late fetal development
of the rat also (Yeah and Morgan, 1974).
From these examples of parallel biochemical differentiation in the
fetal mannnalian liver and the tadpole liver it is clear that enough
similar events are taking place to justify the use of amphibian meta
morphosis as a model to study the development of the bilirubin excretory
pathway. It is in this light that we have studied the tadpole as an
experimental model for bilirubin excretion.
Bile Pigments in Amphibians
Bile pigment metabolism has not been thoroughly investigated in
amphibians. Lester and Schmid (1961) studied the bile of several
representative adult amphibians and the tadpoles of the bullfrog, Rana
catesbiana. They were able to detect bilirubin and frequently biliverdin
in the bile of the adults examined. Analysis by paper chromatography
indicated the bilirubin in the bile to be unconjugated. Bilirubin and
biliverdin were undetectable or present in trace amounts in the bile of
the tadpoles examined. The serum of both adults and tadpoles could not
be shown to contain bilirubin. Lester and Schmid also measured UDP
glucuronyl transferase activities using 0-aminophenol as a substrate.
They found significant levels of glucuronyl transferase in the livers of
the predominantly terrestrial adults (Rana catesbiana and Rana pipens)
but activity was not demonstrable in the livers of Rana catesbiana
tadpoles or predominantly aquatic adults (Xenopus laevis and Necturus
maculosus).
Maickel et al., (1958) have reported that adult frogs and toads
were able to conjugate phenols with glucuronic acid. Ozon and Brewer
(1966) found that microsomes of the urodele (Pleurodeles waltlii :1ichah)
were capable of forming glucuronide conjugates of estrone, estradiol-178
and estradiol-17a in the presence of UDP-glucuronic acid. They also
16
were able to detect estrone 3-glucuronide, estradiol-176 and estra
diol-17a in the presence of UDP-glucuronic acid. They also were able
to detect estrone 3-glucuronide, estradiol-176 3-monoglucuronide, and
estradiol-17a 3-monoglucuronide in the water of animals soaked in
estrone after forty hours, indicating in vivo glucuronidation had taken
place. No studies concerning bilirubin UDP-glucuronyl transf erase in
amphibians have been reported.
Studies with the liver anion binding proteins Y and Z have been
conducted with amphibians (Levine et al., 1971). In the tadpoles of
Rana catesbiana before hind limb emergence, Y and Z were present in
trace amounts or at undetectable levels. After hind limb emergence but
before complete tail resorption Z protein became demonstratable. The
adult terrestrial amphibians had demonstratable amounts of both Z and Y
forms (Levine et al., 1971).
One report indicates that the livers of amphibians do not possess
NADPH dependent biliverdin reductase activity and to excrete only un
conjugated biliverdin (Colleran and O'Carra, 1977). This report is
in conflict with the data of Lester and Schmid, in which they could
consistantly find bilirubin in the biles of amphibians and could not
always demonstrate the presence of biliverdin.
CHAPTER II
HIGH PERFORMANCE LIQUID CHROMOTOGRAPHY OF BILE PIGMENTS
Introduction
This chapter describes the development of a sensitive and quanti
tative method for the separation of bilirubin azodipyrroles and the
isomers of biliverdin. The separations of the azodipyrroles are
based on the initial high performance liquid chromatography (HPLC)
system of Little (1979). This HPLC system has been modified to improve
the resolution of the azodipyrroles. This report also includes methods
for the identification of the conjugated azodipyrroles.
In the original HPLC separation of the azodipyrroles of dog bile,
the identity of all of the compounds separated was not known. Sub
sequent studies described here have led to the identification of these
compounds. The separated compounds have been identified through four
lines of evidence: 1.) transesterfication; 2.) comparison with thin
later chromatography (TLC) of isolated azodipyrroles; 3.) digestion by
specific hydrolytic enzymes; and 4.) their enzymatic synthesis. Also, we
have applied these techniques for the separation of azodipyrroles
derived from the non-a isomers of bilirubin. We have also developed
methods for the separation of the isomers of biliverdin, since bili
verdin has been implied to be present in the bile of amphibians
(Lester and Schmid, 1961).
Recently Onishi et al., (1980) have published a HPLC system for
the separation of the azodipyrroles derived from dog bile. Their
separation is effected by a gradient solvent system whereas our system
relies on a simpler isocratic method.
Experimental
Chemicals
Reagent grade solvents were obtained from Matheson, Coleman and
Bell (Cincinnati, Ohio, U.S.A.) or Fisher Scientific (Pittsburg,
Pennsylvania, U.S.A.) and were filtered through an 0.45 µm fluoropore
filter (Millipore) before use. Human serum albumin (fraction V),
hemin (type 1), bacterial S-glucuronidase (type 1), S-glucuronidase
(bovine liver type B-1), S-glucosidase (almonds), uridine
17
5'-diphosphoglucuronic acid (ammonium salt), uridine 5'-diphospho
glucose (sodium salt), uridine 5'-diphosphoxylose (sodium salt),
D-saccharic acid 1,4-lactone, and D-gluconic acid lactone (grade IX)
were obtained from Sigma Chemical Company (St. Louis, Missouri,
U.S.A.). Biliverdin IXa {dihydrochloride) and bilirubin IXa were
obtained from Porphyrin Products (Logan, Utah, U.S.A.). Para-iodo
aniline was obtained from Fisher Scientific and was recrystallized
from hexane and stored at -8o0 c. Tetrabutyl annnonium hydroxide (40%
solution) was obtained from Aldrich Chemical Company. Tetraheptyl
ammonium chloride and ethyl anthranilate (Eastman) were obtained from
Fisher Scientific Company.
Apparatus
Al~ separations described employed an ALC 200 series liquid
chromatography apparatus (Waters Associates, Milford, Massachusetts,
U.S.A.) equipped with model U6K injectors and model 440 absorbance
detectors monitoring at a single fixed wavelength of 365 nm, 546 nm
or 658 nm. All separations were performed on reversed phase columns
(4.6 x 150 mm) obtained from various sources prepacked with LiChrosorb
5 RPS (5µm).
HPLC Operating Conditions
Separations were performed at ambient temperature with a flow
rate of 1.0 ml/min (column inlet pressure 500-1000 p.s.i.). The
solvent system used was a combination of acetonitrile, ethyl acetate,
methanol, a dilute aqueous buffer and a counter ion(s). The systems
are flexible to meet the needs of the separation by varying the water
content, pH and counter ion.
18
The mobile phase core was formulated by mixing 30 ml of acetonitrile,
33 ml of ethyl acetate, 45 ml of methanol, and 50 ml of water. The
appropriate counter ion was added and pH adjusted with phosphoric acid
or acetic acid (to establish a weak buffer). The solvent was then taken
to its final volume and the pH was checked and readjusted if necessary.
The following solvents have proved useful in our HPLC separations:
Solvent A contained the mobile phase core with lOmM tetrabutyl
annnonium hydroxide (TBAH) adjusted to a final pH of 6.1
with 4.4N phosphoric acid to a total volume of 200 ml.
Solvent B contained the mobile phase core with lOmM TBAH
adjusted to a final pH of 6.1 with 4.4N phosphoric acid
and a total volume of 155 ml.
Solvent C contained the mobile phase core with 10 mM TBAH
adjusted to a final pH of 4.25 with a solution of 2.2N
phosphoric and 2.2N acetic acid and a total volume of
200 ml.
Solvent D contained the mobile phase core with 10 mM TBAH
adjusted to a final pH of 4.0 with a solution of 2.2N
phosphoric and 2.2N acetic acid and a total volume of
240 ml.
Solvent E contained the mobile phase core with 4.8 mM TBAH
and 5.2 mM tetraheptyl ammonium chloride (THAC) adjusted
to a final pH of 6.1 with 4.4N phosphoric acid to a total
volume of 350 ml.
Preparation of Samples
19
Bile samples were collected from the gall bladders of anesthetized
dogs via a hypodermic needle. The bile was stabilized by dilution with
one volume of pH 4.0 citrate phosphate buffer (Compernolle et al.,
1978) and stored at -so0 c protected from light. For some of the initial
experiments dog bile was diazo coupled with diazotized ethyl anthranilate.
Since diazotized p-iodoaniline reacts quantitatively with unconjugated
bilirubin, it was used exclusively in the later experiments. Dog bile
which contains little if any unconjugated bilirubin, when diazo coupled
with either diazotized ethyl anthranilate or diazotized p-iodoaniline
gives virtually identical results. The resulting azodipyrroles have
very similar chromatographic properties (Heirwegh et al., 1974).
All operations with bile pigments were carried out in subdued light.
Diazo coupling of bile pigments with diazotized ethyl anthranilate was
accomplished as described by Heirwegh et al., (1974). The ethyl
anthranilate diazo reagent is prepared by suspending 0.1 ml of ethyl anthranilate (6.76 M) in 10 ml of 0.15 N HCl and mixing with 0.3 ml
of sodium nitrite (5 mg/ml prepared fresh before each use) and allowed
to stand at room temperature for 5 min. To this solution 0.1 ml of
ammonium sulfamate (10 mg/ml) was added. The diazo reagent was used
after 3 min. Samples were diazo coupled by mixing with one volume of
20
0.4 M glycine/RC! buffer pH 2.7 and one volume of diazo reagent. Samples
were allowed to remain at room temperature for 30 min. At the end of
this time, one volume of ascorbic acid solution (10 mg/ml prepared fresh
immediately before use) was added and the azopigments were extracted
with 3 volumes of 2-pentanone. The tubes were centrifuged and small
aliquots of the organic layer were evaporated to dryness with N2
at 30°c.
Diazo coupling of bile pigments with p-iodoaniline was also accom
plished as described by Heirwegh et al., (1974). The following solutions
for the reaction were prepared:
A. Accelerator: acetone-ethanol (1:1) containing 15 ml of 2,6-di
tertbutyl-p-cresol per ml of solution.
B. NaN02 (100 ml/ml) prepared weekly and stored in an amber
bottle at 4°C.
C. 2 M p-toluene sulfonic acid
D. 1.5 M ammonium sulfamate
E. Termination solution: ascorbic acid (10 mg/ml) prepared
immediately before use.
F. Extraction solvent: n-butyl acetate
The diazo reagent was prepared by mixing 0.15 ml of solution B with 4 ml
of solution C and adding 2 ml of the resulting solution to 21 mg of
p-iodoaniline dissolved in 1 ml of glacial acetic acid. This solution
was allowed to remain at room temperature for 2 min. Five ml of water
was added, the solution was mixed and 0.1 ml of solution D was added.
The solution was mixed again and placed in ice for 5 min.
The aqueous bile sample (typically 0.5 ml) was placed in a screw cap
centrifuge tube and placed on crushed ice. Four volumes of the accel
erator solution (solution A) was added along with 1 volume of the diazo
reagent. The tubes were mixed and allowed to sit in ice for 60 min. At
the end of 60 min. 6 volumes of the termination solution (solution E) was
21
added, the tube was mixed and 4 volumes of the extraction solvent added.
The tubes were placed on a mechanical invertor for 15 min. and then
centrifuged at half speed (1470 x g) in a clinical centrifuge
(International Clinical Centrifuge Model CL) for 20 min. Aliquots were
then removed from the organic phase and placed in plastic centrifuge
tubes (1.5 ml) and the organic solvent removed by blowing a gentle
stream of N2 into the tubes while they were maintained at 30°c. The
dried diazo pigments were stored at -8o0 c protected from light. Immed
iately before HPLC, the samples were dissolved in small amounts of meth
anol-ethyl acetate (1:1). Typically 10µ1 was injected.
Transesterif ication of the Conjugated Azodipyrroles of Bilirubin
The dried azodipyrroles obtained from dog bile were dissolved in
methanol. The azodipyrroles were mixed with an equal volume of NaOH
(10 mg/ml in methanol) or methanol (for control) for 60 sec. The reac
tion was terminated by 10 volumes of 0.4 M glycine/RC! pH 2.7 buffer.
The azodipyrroles were extracted with 2 volumes of chloroform. An
aliquot of the chloroform phase was removed and evaporated to dryness
with N2 at 30°c.
Analysis of Azodipyrroles by TLC
Bilirubin IXa was diazo coupled with either p-iodoaniline or
ethyl anthranilate and the dried azopigments was dissolved in methanol
and spotted on precoated silica gel plates (DC-Kiselgel, E. Merck,
Darmstadt, G.F.R.). The plates were repeatedly developed in the dark
with chloroform-ethyl acetate (1:1) to resolve the exovinyl and endo
vinyl azodipyrroles derived from bilirubin IXa (Compernolle et al.,
1970). On TLC the faster migrating spot corresponds to the endovinyl
isomer and the slower moving spot corresponds to the exovinyl isomer.
The isolated spots were scraped from the plates, eluted with methanol,
centrifuged for 5 min. and rechromatographed by HPLC.
The glucuronic acid, glucose and xylose conjugated azodipyrroles
of bilirubin IXa were isolated by TLC. The dried azopigment organic
extract obtained from dog bile was dissolved in methanol and spotted
on silica gel plates. The plates were developed in the dark with
chloroform/methanol/water (65:25:3). The isolated azopigments were
identified on the basis of their Rf values (Heirwegh et al., 1974).
Identification of the Endovinyl and Exovinyl Azodipyrrole Conjugates of Bilirubin
22
The methanol eluates of the conjugated azodipyrroles obtained from
dog bile diazo coupled with ethyl anthranilate were subjected to HPLC
with solvent A and the resolved endovinyl and exovinyl isomers isolated
by collecting the effluent innnediately after it passed through the
flow cell of the detector. The isolated fractions were added to 0.5 ml
of 0.05 M acetate buffer pH 5.0 and a gentle stream of N2
was bubbled
through the fractions for ten minutes to remove the organic solvents.
To the isolated glucuronic acid and xylose conjugated azodipyrroles
0.2 ml of bovine S-glucuronidase (8 mg/ml) in 0.05 M acetate buffer
ph 5.0 was added. To the isolated glucose confugated azodipyrroles
0.2 ml of almond S-glucosidase (16 mg/ml) was added. The fractions were
capped and incubated at 37°C for 30 min. At the end of 30 min. the tubes
were placed on ice and 0.5 ml of 2.0 M glycine/RC! pH 2.0 buffer was
added and the samples were extracted with 1.0 ml of pentanone. The
samples were centrifuged for 10 min. and 0.8 ml of the organic phase was 0 placed in a tube and evaporated to dryness with N
2 at 30 C. The dried -
azopigments were dissolved in methanol and subjected to HPLC (solvent A)
along with the azodipyrroles of bilirubin rxa.
Enzymatic Hydrolysis of the Conjugated Azodipyrroles of Dog Bile
Six aliquots (50µ1) of the organic extract of dog bile diazo
coupled with p-iodoaniline were evaporated to dryness with N2 at 30°c.
The dried diazo pigments were redissolved in 20µ1 of Me2 so2 . The tubes
were incubated with either bacterial B-glucuronidase or S-glucosidase
(almonds) at 37°c for 1 hour. The tubes were prepared as follows:
Tube 1: contained 0.39 ml of 0.06 M phosphate buffer pH 7.0
and 0.1 ml of S-glucuronidase (1 mg/ml dissolved in phosphate
buffer).
Tube 2: contained 0.29 ml of phosphate buffer, 0.1 ml of
8-glucuronidase and 0.1 ml of saccharic acid lactone (20
mg/ml neutralized in phosphate buffer).
Tube 3: contained 0.49 ml of phosphate buffer.
Tube 4: contained 0.39 ml of 0.05 M acetate buffer pH 5.0
and 0.1 ml of S-glucosidase (12 mg/ml dissolved in acetate
buffer).
Tube 5: contained 0.29 ml of acetate buffer, 0.1 ml of
B-glucosidase, and 0.1 ml of gluconic acid lactone (30
mg/ml adjusted to pH 5.0 in acetate buffer).
Tube 6: contained 0.49 ml of acetate buffer.
At the end of the incubation period the tubes were placed on ice.
To each of the tubes 0.2 ml of 2.0 M glycine/HCl buffer pH 2.0 was
23
added. The azopigments were then extracted with 0.5 ml of butyl acetate.
The tubes were centrifuged (2575 x g) for 10 min. and 0.25 ml of the 0 organic phase was evaporated to dryness with N
2 at 30 C. The dried
extracts were redissolved in methanol/ethyl acetate (1:1) and subjected
to HPLC.
Enzymatic Synthesis of Bilirubin IX Conjugates
The in vitro formation of the conjugates of bilirubin was based on
the bilirubin UDP-glucuronyl transf erase assay conditions of Heirwegh
et al., (1972) and the UDP-glycosyl and UDP-xylosyl transferase assay of
Fevery et al., (1972). These assay conditions were combined with the
HPLC systems outlined in this chapter to synthetize the conjugates of
bilirubin. Rat liver served as a source of conjugating enzymes.
Male Spraque-Dawley rats were sacrificed and bled. The livers were
removed, blotted and weighed. Livers were finely minced with a razor
blade and diluted with three volumes of ice cold 0.25 M sucrose. The
livers were homogenized with 6 passes at 1000 rpm in a Potter Elvehjem
24
homogenizer. Digitonin (12 mg/ml) was solubilized in sucrose by placing
the solution into a boiling water bath for a few minutes. Equal volumes
of liver homogenate and digitonin solution were mixed and left on ice
for 30 min.
The bilirubin substrate solution was prepared by dissolving 2.5 mg
of bilirubin in 0.2 ml of 0.05 N NaOH and diluting to 10 ml with human
serum albumin (24 mg/ml, dialyzed overnight against 1 mM EDTA pH 7.4).
The incubation mixture was prepared by adding the following to screw
cap round bottom tubes:
1. 0.1 ml of the digitonin activated liver homogenate
2. 0.1 ml of the bilirubin-albumin solution
3. 0.1 ml of 0.4 M HEPES pH 7.8, 25 mM Mg Cl2
buffer
4. 0.03 ml of the appropriate UDP sugar (33.3 mg UDP
glucuronic acid/ml; 36.3 mg UDP-xylose/ml; or 80 mg UDP
glucose/ml).
Control tubes were prepared by omitting bilirubin and the UDP sugars
for the incubation period. The tubes were placed in a 30°c shaking water
bath for 20 min. At the end of the incubation period the tubes were
removed and placed on ice. Bilirubin and the appropriate UDP sugar were
added to the blank tubes. The samples were then diazo coupled with
diazotized p-iodoaniline as previously described and extracted with
1.0 ml of butyl acetate. An aliquot (0.5 ml) of the organic phase was 0
evaporated to dryness with N2 at 30 C.
Preparation of the Isomers of Biliverdin IX and Bilirubin IX Azodipyrroles
Biliverdin dimethyl esters were synthesized by the coupled oxidation
of heroin as described by Bonnet and McDonagh (1973). The procedure used
is as follows:
1. 25 mg of heroin in 400 ml of aqueous pyridine (3:1) was bubbled
with o2
for 20 min. at 37°c.
2. The o2
source was withdrawn from the solution to just above its
surface. The solution was rapidly stirred and 300 mg of ascorbic
acid in 5 ml of distilled water was added and timed for 3 min.
3. This solution was rapidly extracted into 100 ml of ice cold
chloroform (flushed with N2).
4. The aqueous phase was reextracted with 30 ml of ice cold
chloroform (N2
flushed).
5. The combined chloroform extract was washed with 100 ml of ice
cold distilled water (N2
flushed).
25
6. The chloroform phase was separated and placed in a flask under
N2 and approximately 5 g of anhydrous sodium sulfate is added.
7. The chloroform solution was filtered through chloroform
moistened filter paper, and concentrated to a moist green residue 0 in a rotary evaporator at 43 C.
8. The residue was dissolved in 1.5 ml of chloroform and added to
7.5 ml petroleum ether in a glass screw capped conical centrifuge
tube. The tube was centrifuged (2575 x g).
9. The precipitate was collected and resuspended in petroleum ether
and recentrifuged. Again it was resuspended in petroleum ether
and recentrifuged.
10. The precipate was dissolved in 6.25 ml of methanol (N2
flushed)
and 0.5 ml of 2 N NaOH is added and shaken for 1 min.
11. 6.25 ml of BF3
(boron trifluoride) in methanol was added and the
mixture was refluxed under N2
for 15 min.
12. The mixture was then removed from reflux and left overnight
under N2
•
13. The mixture was then diluted with 40 ml of distilled water
(N2
flush) and extracted with 50 ml of chloroform.
14. The chloroform extract was washed twice with 30 ml of distilled
water (N2
flushed), separated and dried with anhydrous Naso4
under
N2
for ten min.
15. The chloroform extract was then filtered through chloroform
moistened filter paper and concentrated to almost dryness in a
rotatary evaporator.
16. The concentrate was dissolved in 5 ml of chloroform/benzene
(1:9) and placed on a column of deactivated alumina (S g) equili
brated with chloroform/benzene (1:9).
17. A blue green band was eluted from the alumina column with
chloroform/benzene (9:1).
18. The fraction was concentrated and dissolved in chloroform.
19. The biliverdin dimethyl esters were lightly streaked on
silica gel plates {previously activated for 2 hours at l00°c).
20. The plates were loaded into a TLC chamber and flushed with
N2 for 15 min. before development with chloroform/acetone (19:1)
until the solvent front reached the top border of the plate.
26
21. The biliverdin dimethyl esters were scraped into conical centri
fuge tubes, eluted with methanol and centrifuged (2575 x g) for
10 min.
The order of migration from the origin on the TLC plates was bili
verdin IX6 dimethyl ester, biliverdin IXy dimethyl ester, biliverdin
IX a dimethyl ester, and biliverdin IX S dimethyl ester. At this point
the isolated biliverdin dimethyl esters may be subjected to alkaline
hydrolysis to form the biliverdin isomers or subjected to reduction,
hydrolysis and diazo coupling to form the bilirubin azodipyrroles
(Blanckaert et al., 1976).
The biliverdin isomers were prepared as follows:
1. 2.0 ml of the methanol eluate from the silica gel fractions was
added to 1 ml of 1 N NaOH in a round bottom screw cap tube, flushed
with N2
and placed in a 37°C water bath for 30 min.
2. The solution was neutralized with glacial acetic acid (3-4
drops) and 5.0 ml of 0.4 M glycine/HCl pH 2.7 buffer was added.
3. 2.0 ml of chloroform was added, the tubes were shaken and
centrifuged (2575 x g) for 10 min.
4. Aliquots of the chloroform extract were dried at 30°C with
N2
and stored at -80°c until use.
The bilirubin azodipyrroles were synthesized as follows:
1. 1.5 ml of the methanol eluate of the silica gel was mixed
with NaBH4
{10-20 mg) for 30 sec. or until the solution just
turned yellow.
2. The reaction was terminated by placing in ice and adding 4.0
ml of 0.4 M glycine/HCl pH 2.7 buffer containing ascorbic acid
(10 mg/ml).
3. The bilirubin dimethyl esters were extracted in 1.0 ml of
chloroform and the chloroform phase was placed into a centrifuge
tube and washed three times with ice cold N flushed distilled 2
water.
4. The chloroform phase was removed and evaporated to dryness
by a gentle stream of NZ.
5. The concentrated bilirubin dimethyl esters were resuspended
in Z.O ml of methanol and 1 ml of 1 N NaOH containing 20 mg of
ascorbic acid and a trace of EDTA is added.
6. The tubes were flushed with NZ and placed in a 37°c water
bath for 30 min.
7. The tubes were then neutralized with acetic acid and 4.0 ml
of 0.4 M glycine/HCl pH 2.7 buffer was added.
8. The tubes were extracted with 2.0 ml chloroform, the phases
separated and reextrated with 1.0 ml chloroform.
9. The pooled chloroform phases are concentrated to dryness with 0
N2
at 30 C.
10. The isolated bilirubin isomers were resuspended in ethanol/
acetone (1:1) and diazo coupled with p-iodoaniline as previously
described.
Results
Dog bile, when diazo coupled with either p-iodoaniline or ethyl
anthranilate, yields 8 major peaks in our HPLC systems (Fig. 2.1.A).
27
It has been shown by mass spectroscopy (Compermolle et al., 1970) that
upon diazo coupling, bilirubin IXa yields two azodipyrrole molecules that
differ only in the order of the vinyl and methyl side chains (the endo
vinyl and exovinyl isomers). It is possible to separate a mixture of the
endovinyl and exovinyl isomers (Fig. 2.7. for structure) by repeated TLC
developments. When the azodipyrroles are isolated by TLC and subjected
to HPLC, it is revealed that the endovinyl azodipyrrole isomer preceeds
the exovinyl isomer in our systems (Fig. 2.1. B, C and D). By comparison
with these standards two of the peaks of dog bile (Fig. 2.1.i\) were shown
28
Fig. 2.1. HPLC Separation of p-Iodoaniline Azodipyrroles. HPLC operating conditions are solvent A and as described in text. Absorbance tracings were monitored at 546 nm. A.) and E) diazo coupled dog bile; B.) bilirubin IXa azodipyrroles; C.) endovinyl azodipyrrole isomer; D.) exovinyl azodipyrrole isomer; F.) delta TLC spot; G.) a3 TLC spot; H.) a 2 TLC spot.
,_
A
B
c
D
I 2
7
8
0 I I I 20 TIME (MIN.)
E
F
G
H
29
2
2
I
1
3 4 -
56
0 I I I 20 TIME (MIN.)
30
to be chromatographically identical to the azodipyrroles obtained from
bilirubin IXa. These unconjugated azodipyrrole molecules are probably
derived from monoconjugates of bilirubin IXa since they are also
present in dog bile diazo coupled with ethyl anthranilate which has been
shown to react almost exclusively with conjugated bilirubin.
Transesterification of Conjugated Azodipyrroles
Base catalyzed transesterification in methanol of conjugated azodi
pyrroles has been used as a quick diagnostic test for the presence of
ester conjugates (Heirwegh et al., 1975). Under the conditions of this
reaction azodipyrrole conjugates are quantitatively converted to the
methyl ester azodipyrroles. Using our standard HPLC system (solvent A)
the hydrophobic methyl ester azodipyrroles are retained by the column for
prolonged periods. The use of solvent system B, which has a low content
of water, allows the resolution of the methyl ester azodipyrroles in a
reasonable length of time. Fig. 2.2 illustrates the transesterification
reaction performed on dog bile. Fig. 2.2.A illustrates normal dog bile
(NaOH ommitted from the reaction) run on solvent B. The individual
azodipyrrole peaks 1-6 are not well resolved but are distinguishable.
Fig 2.2.B illustrates dog bile incubated for 60 sec in alkaline methanol.
The conjugated azodipyrroles (peaks 1-6) are quantitatively converted to
the more hydrophobic methyl ester azodipyrroles (peaks 1 Mand 2 M). The
unconjugated azodipyrroles (peaks 7 and 8) remain unchanged (fig. 2.2.A
and B). These results indicate that peaks 1-6 are ester conjugated
azodipyrroles of bilirubin IXa.
Identification of HPLC Peaks by Comparison with Standards Prepared by TLC
Dog bile has been shown to contain significant amounts of bilirubin
IXa conjugated with glucuronic acid, glucose, and xylose (Heirwegh
et al., 1970; Fevery et al., 1971). Heirwegh and his associates have
chemically characterized these conjugates after their conversion to the
azodipyrroles. Using the TLC systems developed by Heirwegh and his
associates (Fig. 1.2), standards of azodipyrrole conjugates of glucuronic
acid (o spot), glucose (a3
spot) and xylose (a2
spot) were prepared. The
31
A
1M2.M
B
0 I t I 5 TI ME (MIN.)
Fig. 2.2. The Base Catalyzed Transesterification of the Conjugated Azodipyrroles of Dog Bile. Transesterification reaction was performed as described in text. HPLC operating conditions are solvent B and as described in text. Absorbance tracing at 546 nm are A.) control experiment (NaOH omitted) of diazocoupled dog bile; B.) base catalyzed transesterification of diazo coupled dog bile.
32
results of this experiment are shown in Fig. 2.1. The glucuronic acid
conjugates of the azodipyrroles of bilirubin IXa were identical to
peaks 1 and 2 of dog bile (Fig. 2.1.F). The glucose conjugates were
chromatographicallyidentical to peaks 3 and 4 of dog bile (Fig. 2.1.G).
The xylose conjugates were chromotographically identical to peaks 5
and 6 of dog bile (Fig. 2.1.H).
Susceptibility of Peaks to Enzymatic Hydrolysis
To confirm the identity of these HPLC peaks, we examined their
susceptibility to enzymatic hydrolysis. Fig. 2.3 illustrates the effect
of a bacterial B-glucuronidase on the peaks from dog bile. The
B-glucuronidase preparation almost completely hydrolyzed peaks 1 and 2.
The hydrolysis was completely inhibited by 21 mM saccharic acid lactone,
a specific inhibitor of S-glucuronidase. Fig. 2.4 illustrates the effect
of a S-glucosidase preparation on the peaks from dog bile. The
B-glucosidase almost completely hydrolyzed peaks 3 and 4, and hydrolyzed
peaks 5 and 6 approximately 50%. Peaks 1 and 2 were hydrolyzed to a minor
extent by this preparation. The addition of 31 rnM gluconic acid lactone
to the incubation mixture inhibited the hydrolysis of peaks 3, 4, 5 and 6.
The gluconic acid lactone had no effect on the hydrolysis of peaks 1 and
2.
Enzymatic Synthesis of HPLC Peaks
Rat liver contains large amounts of bilirubin UDP-glucuronyl trans
f erase activity and is also a good source of bilirubin UDP-glucosyl and
UDP-xylosyl transferase activities. Fig. 2.5 illustrates that when rat
liver is incubated with bilirubin and UDP-glucuronic acid, two peaks
are found in the diazo coupled incubation mixture that are chromatog
raphically identical to peaks 1 and 2 found in diazo coupled dog bile.
When rat liver is incubated with bilirubin and UDP-glucose, peaks are
found that are chromatographically identical to peaks 3 and 4 which were
found in dog bile. The peaks obtained from the incubation mixture con
taining bilirubin and UDP-xylose are chromatographically identical to
peaks 5 and 6 found in dog bile.
33
Fig. 2.3. The Enzymatic Hydrolysis of the Conjugated Azodipyrroles of Dog Bile by Bacterial 8-Glucuronidase. Experimental conditions are as described in text. HPLC operating conditions are solvent A and as described in text. Absorbance tracings at 546 nm are A.) diazotized p-iodoaniline-coupled dog bile incubated in phosphate buffer; B.) diazo coupled dog bile incubated with 8-glucuronidase in phosphate buffer; C.) diazo coupled dog bile incubated with B-glucuronidase and saccharic acid lactone in phosphate buffer.
A
B
c
2 4 3
2
5 6
7
8
7 8
0 I I I 20 TIME (MIN.)
A 5 6
12 7
8 B
c 7 5 6 8
0 I I I 20 TIME(MIN.)
Fig. 2.4. The Enzymatic Hydrolysis of the Conjugated Azodipyrroles of Dog Bile by Almond S-Glucosidase. Experimental conditions are as described in text. HPLC operating conditions are solvent A and as
35
described in text. Absorbance tracings at 546 nm are A.) p-Iodoaniline coupled dog bile incubated in acetate buffer; B.) Diazo coupled dog bile incubated with S-glucosidase in acetate buffer; C.) diazo coupled dog bile incubated with S-glucosidase and gluconic acid lactone in acetate buffer.
36
Fig. 2.5. Enzymatic Synthesis of Bilirubin Conjugates. HPLC operating conditions are solvent A and as described in text. Incubations with rat liver homogenate and post incubation diazo coupling with p-iodoaniline was accomplished as described in text. Absorbance tracings at 546 nm are A.) dog bile diazo coupled with diazotized p-iodoaniline; B.) incubation containing bilirubin and UDP-glucuronic acid, tracing run at 0.5 absorbance units full scale (AUFS); C.) incubation with bilirubin and UDP-glucose, tracing run at 0.05 AUFS; D.) incubation with bilirubin and UDP-xylose, tracing run at 0.05 AUFS; E.) incubation blank, bilirubin and UDPsugar added after incubation period, tracing run at 0.05 AUFS and 0.5 AUFS at arrow.
37 7
8
A E
5 6 7 8
I I I 20 TIME (MIN.)
8 2
3
c 4
6
D
0 I I I TIME(MIN.)
20
Identification of the Endovinyl and Exovinyl Identity of Peaks 1-6 of Dog Bile
38
Peaks 1-6 of dog bile were isolated by collecting the effluent of
the HPLC flow cell during the individual peak elution. Reinjection of
the fractions thus isolated, revealed a single specie. Fig. 2.6 illus
trates an experiment that shows the endovinyl, exovinyl identity of peaks
3 and 4. The isolated peaks were enzymatically hydrolyzed. The experi
ment in Fig. 2.6 is representative of the results obtained from experi
ments using peaks 1-6. As can be seen from this experiment peaks 1-6
retain the same endovinyl, exovinyl order of elution as the unconjugated
azodipyrroles of bilirubin (peaks 7 and 8).
HPLC of Non-a Azodipyrroles and Occurance in Dog Bile
When diazo coupled dog bile is run with solvent B, small amounts of
hydrophobic azopigments that are retained longer than the methyl ester
azodipyrroles of bilirubin are seen. It has been shown that small
amounts of the non-a isomers of bilirubin occur in the bile of animals
(Petryka, 1966; O'Carra and Colleran, 1970; Blanckaert et al., 1977;
Heirwegh et al., 1977). When bilirubin !Xo and bilirubin IXS are diazo
coupled, they give rise to an extremely polar azodipyrrole (Sx Fig, 2.7)
and two extremely non-polar azodipyrroles (aF' and aF respectively,
Fig. 2.7). Bilirubin IXy, when diazo coupled, yields two'azodipyrroles
of intermediate polarity (Fig. 2.7). Fig. 2.8.A is a chromatograph of- a
synthetic mixture of the azodipyrroles resulting the diazo coupling of
bilirubin !Xa, IXS, IXy and IXo. Small amounts of nonpolar azo
dipyrroles derived from IXS (Fig. 2.8.B) and from IXo (Fig. 2.8.B)are
seen in dog bile. In the dog bile of Fig. 2.8, aF made up 0.62% and
aF' made up 0.12% of the total diazo positive material. The azodipyrroles
aF and aF' only make up half of the parent tetrapyrrole molecule. Bili
rubin IXS made up 1.2% and bilirubin IXo made up 0.42% of the total diazo
positive material in this dog bile sample.
The hydrophilic azodipyrrole Sx is not resolved from the IXa
conjugates in the normal solvent system (solvent A) well enough to allow
39
Fig. 2.6. Enzymatic Hydrolysis of Conjugated Peaks 3 and 4 to Reveal their Respective Endovinyl, Exovinyl Isomers. Tracings are absorbance monitoring at 546 nm. HPLC conditions are solvent A and as described in text. A.) Dog bile coupled with diazotized ethyl anthranilate, fractions were collected during the intervals indicated by the bars, and hydrolyzed as described in text. B.) Results of the enzymatic hydrolysis of peak 3, showing the formation of the endovinyl azodipyrrole isomer. C.) Results of the enzymatic hydrolysis of peak 4, showing the formation of the exovinyl azodipyrrole isomer.
4
3
A
B 3
c 8 4
O I I I I 30
TIME(MIN.)
41
HO OH • I
9=0 CH2 CH2 CH2 CH2 II t 1 II
O=C
~ rJr 1,H· rrT·J Bilirubin IXO<--:P + o'' N ''c N N=N N=N N c'' N ~o
Endovi~yl H H Jr ~ H ~xo~inyl (O<o) V V (CXo)
I I
Bilirubin IX B
I I
Fig. 2.7. Azodipyrroles Resulting from the Coupling of the Isomers of Bilirubin IX with Diazotized p-Iodoaniline. Symbols in parenthesis are TLC nomenclature.
42
Fig. 2.8. HPLC Separation of the Azodipyrroles of Bilirubin IXa, Bilirubin IXS, Bilirubin IXy, and Bilirubin IXo. Samples were diazo coupled with diazotized p-iodoaniline as described in text. HPLC operating conditions are solvent B and as described in text. Absorbance tracings at 546 nm are A.) Synthetic mixture of azodipyrroles from the isomers of bilirubin IX. Peak 7 and 8 are derived from bilirubin IXa. Peaks 9 and 10 are derived from bilirubin IXy. Bilirubin IXS when diazo coupled yields peaks 12 (aF) and 0 (Bx); B.) Diazo coupled dog bile demonstrating small amounts of azopigments Corresponding to the azodipyrroles derived from bilirubin IXS (peak 12) and bilirubin IXo (peak 11).
43
0
B
78 12
0 ---------------- 2 0
TIME (Ml N.)
44
for its detection. Lowering the pH of the standard solvent (solvent A)
to pH 4.25 resolves Bx from the conjugate peaks and sharpens it (Fig.
2.9). At this pH the glucuronic acid peaks are slowed so that the
exovinyl glucuronic acid azodipyrrole appears between the two glucose
azodipyrroles. As seen in Fig. 2.9, small amounts of a peak chromatogra
phically identical to Bx is seen in dog bile.
HPLC Separation of the Isomers of Biliverdin
Also shown in Fig. 2.9 is the chromatographic behavior of the tetra
pyrrole biliverdin IXa. Biliverdin IXa elutes from the column before
the glucuronic acid conjugates of bilirubin IXa. By lowering the pH
to 4.0 and increasing the water content (solvent D)~ all four of the
isomeric biliverdin isomers can be separated. Fig. 2.10 shows the HPLC
separation of biliverdin IXa, IXB, IXy and IXo. The TLC separation of
biliverdin IXa dimethyl ester and biliverdin IXy dimethyl ester is
usually poor. In the preparation shown in Fig. 2.10, biliverdin IXa and
IXy were obtained as a single fraction (Fig. 2.10.D). Identification
was provided by the availability of commercially pure IXa (Fig, 2.10.B).
In other preparations, it was possible to isolate IXa and IXy but never
in a form completely free of the other isomer. Fig. 2.11 illustrates
the application of solvent system E to separate biliverdin IXa from the
azodipyrroles of bilirubin IXa. This solvent system selectively slows
the migration of biliverdin to allow for its more complete separation from
chromagenic components in bile that interfer with its detection in bile
when the normal solvent system is used (solvent A). As illustrated in
Fig. 2.11 biliverdin has about 3.4 times more absorbance at 658 nm than
it does at 546 nm. The azodipyrroles absorb very little at 658 nm but
absorb very strong'ly at 546 nm.
Discussion
This chapter outlines the separation and identification of the
azodipyrroles of dog bile and the azodipyrroles derived from bilirubin
IXa, IXB, IXy and IXo. Separation methods for the isomers of bili
verdin are also presented. Azodipyrroles are used in this study because
45
Fig. 2.9. HPLC Separation of the Bilirubin Azodipyrroles. HPLC operating conditions are solvent C and as described in text. Absorbance tracings at 546 nm are A.) p-iodoaniline diazo coupled dog bile; B.) o azopigment spot; C.) a azopigment spot; D.) a 2 azopigment spot; E.) Bx azodipyrrole; ~.) diazo coupled dog bile with added biliverdin IXa (l'), Bx azodipyrrole (0) and bilirubin IXy azodipyrroles (9 and 10); G.) biliverdin IXa; H.) azodipyrroles of bilirubin IXy.
.._
46
A F
9 10
2
B
G
3
4
H~ - ~ 0 I 30 ~1--- 60
c
TIME(MIN.) 5
D 6
E 0
0 I 30 6 0 TIME (MIN.)
47
Fig. 2.10. HPLC Separations of the Isomers of Biliverdin IX. HPLC operating conditions are solvent D and as described in text. Absorbance monitoring at 365 nm are A.) synthetic mixture of the isomers of biliverdin IX; B.) biliverdin IXa obtained from commercial sources; C.) biliverdin IXc; D.) mixture of biliverdin IXa and biliverdin IXy; E.) biliverdin IXS. Order of elution is biliverdin IXa, IXc, IXS, and lXy.
48
A
8
c
D
E
0 t I I 20 TIME (MI NJ
49
A (
7
8
B
0 I I 20
TIME (MIN.)
Fig. 2.11. The Simultaneous Absorbance Monitoring of a Synthetic Mixture of Bilirubin Azodipyrroles (peaks 7 and 8) and Biliverdin IXa (1') at 658 run Run at 0.5 AUFS (A) and 546 nm Run at 0.2 AUFS (B). HPLC operating conditions are solvent E and as described in text.
50
they are more stable than their parent tetrapyrrole molecules. The
conversion of bilirubin isomers to diazodipyrroles allows for their more
efficient extraction into organic solvent which aids in subsequent manip
ulation. Another reason for employing azodipyrrole derivatives is that
these compounds are well characterized both as to their chemical and
thin layer chromatographic properties. Although some information about
the parent tetrapyrrole molecule is lost, (i.e. the ability to distinguish
monoconjugates from diconjugates) the advantages in using azodipyrroles
for most purposes offsets any disadvantages.
The HPLC systems in this chapter are able to resolve the endovinyl
and exovinyl azodipyrroles of bilirubin IXa and its conjugates. Separa
tion of the endovinyl and exovinyl azodipyrroles of bilirubin IXa by
TLC is possible only after multiple developments. Separations of the
endovinyl and exovinyl azodipyrrole isomers of the conjugates of bilirubin
IXa by TLC has not yet been published. The standard solvent allows the
rapid separation and easy quantitation of the eight major azodipyrroles
found in dog bile. The HPLC solvent systems in the chapter are flexible,
in that the amount of water and the pH of the solvent can be varied to
suit the needs of a particular separation. The resolution of the more
hydrophilic azodipyrroles is increased by increasing the water content
and/or lowering the pH. The resolution (and decreased analysis time) of
the more hydrophobic azodipyrroles can be improved by lowering the water
content and/or raising the pH.
The identity the HPLC peaks 1-6 found in dog bile have been confirmed
by four criteria. The first criterion is that the peaks are readily con
verted to methyl esters azodipyrroles. This result indicates the HPLC
peaks are ester conjugates. The second criterion used is the suscepti
bility of the peaks enzymatic hydrolysis. The hydrolysis of peaks 1
and 2 were inhibited by the specific S-glucuronidase inhibitor, saccharic
acid lactone (Levvy and Conchie, 1966). The hydrolysis of peaks 3 and 4
by S-glucosidase was inhibited by the specific B-glucosidase inhibitor
(Conchie et al., 1967). The B-glucosidase preparation resulted in peaks
5 and 6 being hydrolyzed approximately 50%. Peaks 5 and 6 are in much
lower concentration compared to peaks 3 and 4 so this activity is
51
relatively minor. The hydrolysis of peaks 5 and 6 was also inhibited by
gluconic acid lactone. Fevery et al., (1971) also found that a
S-glucosidase preparation (Emulsin-almonds) hydrolyzed the glucose and
xylose azodipyrroles. They found that gluconic acid lactone inhibited
both hydrolytic activities. The third criterion used in the identifica
tion of peaks 1-6 is their comparison to fractions isolated by TLC systems
that have been extensively chemically characterized (Compernolle et al.,
1970; Heirwegh et al., 1970; Jansen and Stoll et al., 1971; Fevery et al.,
1971; Gordon et al., 1974). The fourth criterion used is the enzymatic
synthesis of peaks 1-6. Based on the evidence presented the identity
of the HPLC peaks 1-6 of dog bile can be assigned:
Peak 1 = endovinyl azodipyrrole S-glucuronide
Peak 2 = exovinyl azodipyrrole S-glucuronide
Peak 3 = endovinyl azodipyrrole B-glucoside
Peak 4 = exovinyl azodipyrrole S-glucoside
Peak 5 = endovinyl azodipyrrole S-xyloside
Peak 6 = exovinyl azodipyrrole S-xyloside
Separation methods for biliverdin are also presented in this chapter.
The biliverdin isomers are separated as the tetrapyrrole molecules. The
methods presented here allow for the sensitive and selective measurement
of biliverdin that represents a significant advance over the existing
methodology.
CHAPTER III
BILE PIGMENT METABOLISM IN ADULT AMPHIBIANS
Introduction
There are few reports in the literature that have examined the bile
pigment metabolism of amphibians. Lester and Schmid (1961) consistently
found unconjugated bilirubin in the bile of adult amphibians. They also
found significant levels of o-aminophenol UDP-glucuronyl transf erase
activities in adult amphibian livers. Based on the finding of unconju
gated bilirubin in the bile, they speculated that the glucuronyl trans
f erase of frog liver had not "adapted" itself for the conjugation of
bilirubin. If it is true that amphibians do not conjugate bilirubin,
they obviously must have a mechanism for the excretion of bilirubin in
an unconjugated form. The excretion of significant quantities of bili
rubin in an unconjugated form is a feat apparently not possible in
mammals.
In conflict with the view that amphibians excrete bilirubin in an
unconjugated form is the view of Colleran and O'Carra (1977) who stated
that amphibians excrete unconjugated biliverdin and do not produce
bilirubin. Their statements are apparently based on the absence of
detectable reduced pyridine nucleotide dependent biliverdin reductase
activity in the livers of amphibians and the presence of biliverdin in
the bile.
Due to the lack of consistent data in the literature, we have ex- -
amined the bile pigments of adult amphibians, asking these basic questions:
1. What is the major pigment(s) of adult amphibian bile?
2. What is the major bile pigment(s) of adult amphibian serum?
3. Does amphibian liver posses bilirubin UDP-glucuronyl trans
ferase activity?
4. Whether amphibians excrete bilirubin in an unconjugated or
conjugated form?
To facilitate comparison of amphibians and mammals, we used a represen
tative mammal (laboratory mouse) and a representative anuran amphibian
(bullfrog) in these studies.
52
53
Experimental
Animals
Adult male mice (C57BL/5J) were obtained from Jackson Labs (Bar
Harbor, ME., U.S.A.). Adult bullfrogs (Rana catesbiana) were obtained
from Riverside Biological Center (Somerset, WI., U.S.A.). The bullfrogs
were maintained in separate cages and were fed a tadpole once a week.
Chemicals
Saccharic acid 1-4 lactone was obtained from Sigma Chemical Co.,
(St. Louis, MO., U.S.A.) and dissolved in water, neutralized with SM
NaOH and lyophilyzed. Tricane (ethyl m-amino benzoate) was obtained from
Sigma Chemical Co.
Amphibian Saline
In lL of distilled H20 the following was added: NaCl (4.3 g), KCl
(0.3 g) and Cacl2
(O.l g).
Protein Determination
Protein concentration of the crude liver homogenates (diluted 1:100
with lN NaOH and stored at -40°C) was determined with the Bio Rad protein
assay (Bio-Rad Laboratories, Richmond CA., U.S.A.). Bovine serum albumin
(fraction V) served as a protein standard. Diluted homogenates were
placed in a 45°c water bath for approximately 15 min to completely
solubilize the protein and the protein assay was perf orrned according to
instructions supplied with the reagent.
Collection of Biological Samples
Adult bullfrogs were anesthetized by an intraperitoneal injection
of tricane in the amount of 0.002 ml/g of body weight. The heart was
surgically exposed and blood collected by cutting the conus arteriosus
and allowing the blood to flow into a conical centrifuge tube. Serum
was obtained by allowing the blood to clot at room temperature for
15 min. The sample was then centrifuged for 15 min (2575 x g) in an
International Clinical Centrifuge (model CL) and aliquots of the super
natant were removed. The abdominal cavity was then surgically exposed
54
and the bile collected by aspiration into a hypodermic needle. The bile
was inunediately stored on ice and protected from light. The liver was
then excised and placed in a beaker with ice-cold 0.25 M sucrose.
Preparation of Sample for HPLC or TLC
Bile and serum samples were reacted with diazoitzed p-iodoaniline
as described in Chapter II. Briefly this was accomplished as follows:
bile samples were diluted approxiamtely 5 to 10-fold to a total volume
of 0.5 ml and added to 2.0 ml accelerator solution and 0.5 ml of diazo
reagent. Serum samples were diazo coupled undiluted in the same manner.
Standard bilirubin solutions were prepared by dissolving 2.5 mg of
bilirubin in 0.2 ml of 0.05 N NaOH and diluting to 10 ml with human serum
albumin (24 mg/ml dialyzed against lmM EDTA, pH 7.4 overnight). A bili
verdin standard solution was prepared by dissolving 2.46 mg of biliverdin
in 2.0 ml of methanol. A bilirubin-biliverdin standard was prepared by
placing 0.05 ml of the bilirubin standard solution and 0.050 ml of the
biliverdin standard solution in a tube with 0.400 ml of distilled water.
Three combined standards were prepared and treated along with the bile
and serum samples. All samples were allowed to remain in ice for one
hour and then 3.0 ml of ascorbic acid solution (10 mg/ml) was added along
with 2.0 ml of butyl acetate. The samples were mixed on a mechanical
inverter for 15 min and centrifuged (1470xg) in a clinical ·centrifuge
for 20 min. An aliquot (0.5 ml) of the organic phase was evaporated to
dryness with a gentle stream of N2
at 30°c.
HPLC and TLC Analysis of Bile Pigments
HPLC and TLC analysis of bile pigments was accomplished as described
in Chapter II. The first set of biles and serums was subjected to HPLC
solvent system A to test for the presence of bilirubin conjugates.
Samples were also screened by TLC (developed with chloroform, methanol
H20; 65:25:3). Diazo pigments were identified by their characteristic
retention time or Rf values when compared to standards prepared from dog
bile or the synthetic azodipyrroles of bilirubin isomers prepared as
described in Chapter II. Initially bile samples were divided, one half
was diazotized and one half was used as a diazo blank prepared by omitting
55
the NaN02 from the diazo reagent. In this way peaks on HPLC were con
firmed to be actually diazo positive. A second set of bile samples was
subjected to HPLC with solvent system E to identify and quantitate
biliverdin IXa. For these samples a detector with the capacity for the
simultaneous monitoring of absorbances at 546 nm and 658 nm was utilized.
Assay of Bilirubin UDP-Glucuronyl Transferase
Livers are blotted, weighed and finely minced with a razor blade.
The minced livers are placed in a glass-teflon homogenizer and diluted
with 3 volumes of ice-cold 0.25M sucrose. The livers were homogenized
by 6 passes of a motor driven pestle at 1000 rpm. In some assays the
effects of digitonin was tested by solubilizing digitonin in sucrose
by placing solutions into a boiling water bath for a few min. Equal
volumes of digitonin and liver homogenate were mixed and placed on ice
for 30 min before assay.
Assay incubation mixtures were prepared by adding the following to
round bottom screw cap tubes:
1. 0.1 ml of 0.4 M HEPES-25 mM MgC12
buffer (pH 7.15 for the
bullfrog and pH 7.8 for the mouse).
2. O.l ml of a bilirubin-albumin solution prepared by dissolving
2.5 mg of bilirubin in 0.2 ml of 0.05 N NaOH and diluting to
10 ml with human serum albumin (24 mg/ml dialyzed overnight
against 1 mM EDTA pH 7.4).
3. 0.03 ml of UDP glucuronic acid (33.3 mg/ml). In some experi
ments UDP glucose (83.3 mg/ml) or UDP xylose (36.3 mg/ml) was
substituted.
4. 0.03 ml of saccharic acid lactone (128.4 mg/ml)
5. O.l ml of liver homogenate to begin the reaction.
Blank tubes were prepared by omitting the UDP glucuronic acid and bili
rubin solutions. All tubes were mixed and placed in a shaking water bath
at 30°c for 20 min. At the end of the incubation period the tubes were
removed and placed in ice. Bilirubin standards were prenared by placing
36 µl of the bilirubin-albumin solution and 0.324 ml of water in a screw
cap tube. To all tubes 0.36 ml p-iodoaniline diazo reagent was added.
At the end of 1 hr, 2.15 ml of ascorbic acid solution (10 mg/ml) and
56
1.0 ml of butyl acetate were added to all tubes. The tubes were mixed on
a mechanical invertor for 15 min, then centrifuged for 20 min (1470 x g).
An aliquot (0.5 ml) of the organic phase was removed and concentrated
to dryness with a gentle stream of N2
at 30°c. The dried azopigments 0
were stored at -80 C protected from light until HPLC (solvent system A).
Quantitation of Azodipyrroles and Biliverdin IXa
Azodipyrroles were identified by their characteristic retention
times on HPLC when compared with azodipyrrole standards prepared from
dog bile and synthetic azodipyrroles prepared as described in Chapter II.
Biliverdin IXa was identified by its retention time and its characteristic
absorbances at 658 and 546 nm. Azodipyrroles or biliverdin were calcu
lated by the following formulas after correction for the blank.
X moles in standard moles of azodipyrrole = area of azodipyrroles average of standard
moles of biliverdin IXa = peak height X moles in standard average peak height of standard
Moles of bilirbuin IXa and bilirubin IXy were obtained by adding the moles
of the two corresponding azodipyrroles obtained from the respective
parent tetrapyrroles. Moles of bilirubin IXS and bilirubin IXo were
obtained by multiplying the area of azopigment, aF or a'F, respectively,
by 2. Areas are determined by triangulation. Concentration of bile
pigments were determined by dividing the moles present in the original
sample by the original volume of the sample.
Hydrolysis of Bilirubin Glucuronide of Dog Bile by Frog Bile
Frog bile (2.0 ml pooled from several individuals) was applied to a
Sephadex G-50-150 column (15 x 400 nnn) equilibrated with O.lM sodium
phosphate pH 7.0. Fractions (100 drops) were collected and assayed for
S-glucuronidase activity (Chapter V). The fractions with maximal activity
were pooled (total volume 10.0 ml) and used as an enzyme source.
An aliquot of the pooled fractions was placed in a boiling water
bath for 15 min to serve as a boiled control. In a screw cap tube, 1.5 ml
57
of the pooled fractions was added to 0.04 ml of dog bile (previously
diluted with one voltnne of citrate/phosphate buffer as described in
Chapter II) and a 0.25 ml aliquot is taken for a time zero sample. To
another screw cap tube 1.5 ml of the boiled control preparation was
added to 0.04 ml of dog bile and a 0.25 ml aliquot was taken for a time
zero sample. Time zero samples were placed on ice and the incubation
tubes were placed in a 30°c heating block. Aliquots (0.25 ml) were
taken at 15 min intervals and placed on ice. All aliquots were then
diazo coupled with p-iodoaniline as previously described.
In Vivo Bilirubin Glucuronide Demonstration
Adult frogs were injected intraperitoneally with a solution of
saccharic acid lactone (333 mg/ml) at a dose of 0.002 ml per gram of body
weight. One and one-half hours later the animals were restrained and a
small incision made in the abdomen and the gallbladder exposed. A
hypodermic needle was inserted in the gallbladder and the bile was re
moved. The gallbladder was then flushed with 0.5 ml of amphibian saline
and completely evacuated. A solution of 200 mM saccharic acid lactone in
amphibian saline was injected into the gallbladder and approximately
0.15 ml was left in the gallbladder. The incision in the abdomen was
sutured with ethicon suture. The animals were then injected intraperi
toneally with a solution of bilirubin (2.5 mg/ml dissolved in Me2so2) in
the amount of 0.002 ml/g of body weight.
Four hours later the animals were restrained and the incision was
reopened and the gallbladder was flushed with 0.5 ml of amphibian
saline. The gallbladder was completely evacuated and the bile sample
was placed on ice protected from light. The gallbladder was reinjected
with approximately 0.15 ml of 200 mM saccharic acid lactone-amphibian
saline solution. The incision was then resutured. The next day (26.5 hr
after initial bile sample) the animals were restrained, the incision
reopened and the gallbladder was flushed with 0.5 ml of amphibian saline.
The gallbaldder was evacuated and the bile sample placed in ice protected
from light. The bile samples were then diazo coupled using p-iodoaniline
as previously described.
58
Results
Bile Pigments of Adult Bullfrog Bile
When adult bullfrog bile was subjected to diazo coupling using
p-iodoaniline, the major azopigments obtained were chromatographically
identical to the azodipyrroles obtained from bilirubin IXa (Fig. 3.1).
Also present were azopigments chromatographically identical to azodi
pyrroles obtained from bilirubin IXy. Bilirubin IXy was present at
levels about 18% of the levels of bilirubin IXa (Table 3.1). When diazo
coupled bullfrog bile was run on HPLC solvent system B, non-polar
azopigments corresponding to aF' and aF (derived from bilirubin IXo and
bilirubin IXS, respectively) were occasionally seen in some individuals
(Fig. 3.2). For those individuals that posessed detectable levels of
bilirubin IXS the results are sumarized in Table 3.1. A diazopigment
was often seen in the bile of bullfrogs that resolves later than either
aF' and aF {Fig. 3.2). This nonpolar azopigment was not an ester
conjugate as shown by its inability hydrolyze in alkaline methanol.
Frog bile when subjected to transesterification in alkaline methanol
consistantly failed to yield detectable amounts of ester conjugates.
Frog bile was not found to contain detectable amounts of azodipyrrole
conjugates of glucuronic acid, glucose, or xylose when compared to
standards prepared from dog bile when chromatographed in our HPLC systems
or by the TLC systems of Heirwegh et al., (1974).
A green bile pigment with the same Rf (0.71) value as biliverdin
IXa was seen when frog bile is subjected to TLC with chloroform, methanol
and water (65:25:3). The bile pigment, after elution from silica gel had
an absorption spectrum very similar to that of biliverdin IXa {absorption
maxima at 660 nm and 375 nm). When bullfrog bile was chromatographed on
solvent system E, a non-diazo positive pigment was seen with the same
elution time as biliverdin IXa (Fig. 3.3). This bile pigment in bull
frogs had the same characteristic absorption ratio at 658 nm and 546 nm
as authenic biliverdin IXa (average ratio about 3.4). When a second set
of adult bullfrog biles was analyzed using this method to identify
biliverdin IXa, the bile levels of biliverdin IXa, were about 49% of the
bile levels of bilirubin IXa (Table 3.1).
• l•
' 't"' . . , '
n ' .
. '
59
A 12
8
c
o I I I 20 Tl ME(MIN.)
Fig. 3.1. HPLC of p-Iodoaniline Coupled A.) Mouse Bile; B.) Bullfrog Bile; C.) Bullfrog Serum. Tracings represent absorbance at 546 nm. Peaks 1 and 2 are chromatographically identical to the glucuronic acid conjugates of the azodipyrroles of bilirubin rxa. Peaks 3 and -4 are identical to the azodipyrroles of bilirubin IXa and peaks 5 and 6 are identical to the azodipyrroles obtained from bilirubin IXy. HPLC was accomplished with solvent system A and as described in text.
12
3 4
TIME(MIN.) 25
Fig. 3.2. HPLC Tracing of Bullfrog Bile Diazo Coupled with p-Iodoaniline. Tracing represents absorbance at 546 run. Peaks
60
1 and 2 are identical to the azodipyrroles obtained from bilirubin IXa. Peaks 3 and 4 are identical to the azodipyrroles of bilirubin !Xy. Peak 5 is chromatographically identical to the hydrophobic azodipyrrole (aF') derived from bilirubin IXo. Peak 6 is chromatographically identical to the hydrophobic azodipyrrole (aF) derived from bilirubin IXo. Peak 7 is an azopigment of unknown identity. HPLC was accomplished with solvent system B and as described in text.
61
A
L---'L 658nm AT 0.5 AUFS 2
3
546nm AT 0.2 AUFS 8
658nm AT 0.02 AUFS
2 3
4 5 546nm AT 0.05 AUFS
0 I I I I 20
TIME (MIN.)
Fig. 3.3. Simultaneous HPLC Monitoring of Biliverdin IXa and the Azodipyrroles of Bilirubin IXa. A.) synthetic mixture of biliverdin IXa (1) and the azodipyrroles of bilirubin IXa (2 and 3); B.) bullfrog bile diazo coupled with p-iodoaniline. Peak 1 in B.) is chromatographically identical to biliverdin IXa in A.). Peaks 2 and 3 are chromatographically identical to the azodipyrroles of bilirubin IXa. Peaks 4 and 5 are identical to the azodipyrroles derived from bilirubin IXy. HPLC was accomplished with solvent system E and as described in text.
Species
Bullfrog
Bile Set 1 Bile Set 2 Serum Set 1 Ser_um Set 2
House
Bile Serum
Table 3.1
Comparative Concentrations of Bile Pigments
Bilirubin XIa* Bilirubin XIci* Bilirubin IXy* Bilirubin X!O* Glucuronide
50 ± 18 (12) 62 ± 24 (9)
0 • 2 6 ± 0 • 04 ( 9 ) 0. 19 ± 0. 03 (18)
94 ± 13 (8) 1.46 ± 0.21 (IO)
N. D. 9 ± 2 (8) trace 11 ± 4 (9)
102 ± 10 ( 8)
Bilirubin IXl3*
1.3 ± 0.4 (5)
*Concentrations are expresed in lo-6 H ± 1 standard error of the mean (S.E.H.) and individuals (N). N.D. = not detectable.
Bi 1 iverdin IX a*
30 ± 10 (9)
< 0.1
O'\ N
63
Bile Pigments of Bullfrog Serum
Diazo pigments corresponding to the azodipyrroles of bilirubin IXa
were re~dily detectable in the serum of adult bullfrogs (Table 3.1).
When adult bullfrog serum was analyzed using HPLC solvent system E and
simultaneously monitoring at 658 run and 546 nm, biliverdin IXa could
not be demonstrated to occur in the serum samples examined. Biliverdin
IXa, if present, would occur at levels less than the sensitivity of the
method (less than 0.1 µM). If serum biliverdin IXa was present at all
it would occur at levels less than 50% of the serum bilirubin IXa levels.
Quantitation of Bile and Serum Bile Pigments
The results of typical standard curve determination for bilirubin
IXa and biliverdin IXa are shown in Fig. 3.4 and 3.5. Standards were
prepared. by adding varying amounts of bilirubin and biliverdin to tubes.
The standards were diazo coupled, extracted and subjected to HPLC. The
area of a single standard was then plotted versus the amount of bile
pigment added. For the bile and serums analyzed, triplicate standards
within the range of the standard curve were prepared and the mean area
used in the calculations.
Bullfrog Liver Bilirubin UDP-Glucuronyl Transferase
Adult bullfrog livers were examined for bilirubin UDP-glucuronyl
transf erase activity and significant levels were found to be present
(Fig. 3.6; Table 3.2). From preliminary experiments, it was found that
the greatest, most consistant activity was found in crude liver homo
genates. Fig. 3.7 illustrates that bilirubin glucuronide synthesis was
linear over the liver homogenate concentrations used.
Although bilirubin glucuronide synthesis was linear for over one
hour under the assay conditions, a standard incubation period of 20 min
was chosen to facilitate comparison with the developmental stages (Chapter
IV). Digitonin when added to an equal volume of liver homogenate at
concentrations of 1, 6, 12, and 18 mg/ml failed to increase bilirubin
UDP-glucuronyl transferase activities and at higher levels inhibited the
activity. When assayed under our conditions the enzyme had a pH optimum
of 6.85 (Fig. 3.8). Doubling the amount of UDP-glucuronic acid did not
64
10000
9000
8000
7000
6000
((j'5000
~ ~4000 <{ w ~3000
2000
1000
O..,._ ____ __,_ ______ _._ ____ ~L-------'-------"-------1.----
0 10 20 30 40 50 - 60
BILIRUBIN (J0-9 MOLES)
Fig. 3.4. Standard Curve of the Azodipyrroles of Bilirubin IXa. Varying amounts of bilirubin were diazo coupled, extracted, dried, and subjected to HPLC with solvent system A at 546 nm and as described in text. Areas were measured by triangulation and normalized to the area the peaks would occupy at 0.02 AUFS.
1000
800
~
5600 r-I <.!)
w I ~400 <! LU 0...
200
65
0------------'---------'----------'----------lL---------J..-----0 100 150 200
BILIVERDIN (10-9MOLES) 250
Fig. 3.5. Standard Curve for Biliverdin IXa. Varying amounts of biliverdin were diazo coupled, extracted, dried, and subjected to HPLC with solvent system A at 546 nm and as described in text. Peak heights were measured and normalized to the height the peaks would be when measured at 0.02 AUFS.
66
Fig. 3.6. HPLC Tracing (absorbance at 546 nm) of an Assay of Bilirubin UDP-glucuronyl Transferase using Bullfrog Liver Homogenate. A.) Liver homogenate was incubated with bilirubin IXa, UDP-glucuronic acid, HEPES buffer and saccharic acid lactone for 20 min at 30°C. The sample was then diazo coupled with p-iodoaniline as described in text. B.) Liver homogenate incubated with HEPES buffer and saccharic acid lactone for 20 min at 30°C. Bilirubin and UDP-glucuronic acid were added after the incubation period and sample was diazo coupled with p-iodoaniline as described in text. HPLC was accomplished with solvent system A and as described in text. The samples were run at 0.02 AUFS then changed to 0.5 AUFS at the arrow. Peaks 1 and 2 are chromatographically identical to the gluduronic acid conjugated of the azodipyrroles of bilirubin IXa.
A 2
B
0 --~--~K.J---- 15 TIME(MIN.)
Table 3.2
Comparative Activity of Hepatic Bilirubin UDP-Glucuronyl Transferase
68
Species Liver Bilirubin UDP-Glucuronyl Transferase*
Bullfrog Set 1 Set 2
Mouse
0.51 + 0.04 (11) 0.47 + 0.06 (9)
2.25 + 0.17 (8)
*activity expressed in micromoles of bilirubin glucuronide synthesized per hour per gram liver protein at 30 degrees C; ± 1 S.E.M. (N).
~ ~
cso.3 E c '-'
-ca ::) ~ -..... -ca 0 ~--~--...... ---'"--~.L--~-'-~---~--~------
0 0.05 0.1 0. 15 0.2 HOMOGENATE (ml)
Fig. 3.7. Dependence of Bilirubin Glucuronide Synthesis on a Volume of Homogenate Added to the Standard Bilirubin UDP-Glucuronyl Transferase Assay. Bilirubin glucuronide was measured in the incubation mixture after 20 min at 30°C.
69
(/) w _J 0 ~
cro '-J
L1.J 0 z 0.4 0 0::: :::> u0.3 => _J (.!)
z0.2 CD :::> 0::: _J
CD
0.l
6.5 7.0
pH
7.5
Fig. 3.8. The Effect of pH on the Activity of Bullfrog Liver UDP-glucuronyl Transferase. Final pH of incubation mixture was measured by means of a micro pH probe. Bilirubin glucuronide was measured in the standard incubation mixture after 20 min at 3ooc.
70
71
increase activity, indicating saturating conditions for this substrate in
the standard assay. The inclusion of of lmM UDP-N-acetyl glucosamine did
not increase bilirubin UDP-glucuronyl transferase in the standard assay.
No apparent conjugate synthesis was detected when liver homogenates were
incubated with UDP-glucose or UDP-xylose.
Hydrolysis of Dog Bile by Frog Bile
Frog bile is capable of hydrolyzing bilirubin glucuronide (Fig. 3.9).
Dog bile was used as a source of bilirubin glucuronide. At these con
centrations of frog bile hydrolytic activity (0.12µmoles of 4-methyl
umbelliferyl S-D-glucuronide per ml per hr; Chapter V) and bilirubin
glucuronide (6.247 µM) hydrolysis was virtually complete in 45 min. The
decrease in bilirubin glucuronide conjugates and increase in unconjugated
bilirubin for this experiment are summarized in Fig. 3.10. At these
concentrations the reaction does not appear to be linear for long. Using
the initial rate, 1.0 ml of frog bile is capable of hydrolyzing 13.6
nmoles of bilirubin glucuronide per hour. Boiling the enzyme preparation
for 15 min completely abolished the hydrolytic activity (Fig. 3.9).
Boiling the enzyme preparation also degraded residual unconjugated bili
rubin in the enzyme source resulting in lower levels of initial unconju
gated bilirubin in the boiled control (Fig. 3.9 and 3.10). As can be
seen from Fig. 3.9 hydrolysis of the glucose and xylose conjugates of
bilirubin was insignificant indicating that frog bile does not contain
significant amounts of hydrolytic activity toward these substrates.
In Vivo Bilirubin Glucuronide Synthesis by Bullfrogs
Shown in Fig. 3.11 is an experiment in which an adult bullfrog was
injected with saccharic acid lactone and bilirubin. Significant amounts
of azopigments chromatographically identical to the glucuronic acid
conjugates of bilirubin IXa were detected by TLC and HPLC. In this ex
periment, two adults were used. Four hours after injection of bili
rubin, 0.8 nmoles and 2.4 nmoles of glucuronic acid were found in their
biles. The glucuronic acid conjugated were present at levels of 24.3%
and 12.2% respectively of the levels of total (conjugated and unconjugated)
A
8
2 4 3
7
7
72
8
8
Fig. 3.9. Hydrolysis of Bilirubin Glucuronide by Bullfrog Bile. Dog bile served as a source of bilirubin glucuronide. Dog bile was mixed with frog bile and aliquots removed at various times and diazo-coupled with p-iodoaniline as described in text. Samples were run on HPLC solvent system A and as described in text. Tracings are absorbance monitoring at 546 run. A.) dog bile mixed with boiled frog bile at time zero. B.) dog bile and boiled frog bile incubated for 45 min. C.) dog bile and frog bile at time zero. D.) dog bile and frog bile incubated 45 min. As shown in Chapter 11 peaks 1 and 2 are the glucuronic acid conjugated azodipyrroles of bilirubin IXa. Peaks 3 and 4 are the glucose conjugates. Peaks 5 and 6 are the xylose conjugates. Peaks 7 and 8 are the azodipyrroles of bilirubin IXa.
73
11
9
"""' L 8 c..9 'o '-.I 7 z 0
6 ~ <( e:::: ~ 5 z w u 4 z 0 u
3
2
0 15 30 45 TIME (MIN)
Fig. 3.10. Hydrolysis of the Glucuronic Acid Conjugates of the Azodipyrroles of Bilirubin IXa by Frog Bile. This is the graphic representation of the experiment in Fig. 3.9. The open symbols are the change in concentration of the glucuronic acid conjugated azodipyrroles (o) and the unconjugated azodipyrroles (6) when dog bile is incubated with boiled frog bile. The closed symbols represent the change in concentration of the glucuronic acid conjugated azodipyrroles ( •) and the unconjugated azodipyrroles (.) when do~ bile is incubated with frog bile.
A
B I 2
0 I I I ·20 TIME(MIN.)
74
Fig. 3.11. The In Vivo Glucuronidation of Bilirubin IXa by Bullfrogs. Adult Bullfrogs had an injection of saccharic acid lactone. Their gallbladders were then surgically emptied and a small amount of saccharic acid lactone left in their gallbladder. Animals were injected with bilirubin IXa and the bile surgically removed after four hours. A.) Represents the initial bile sample from an adult bullfrog. B.) Represents the bile sample from the same animal four hours after the injection of bilirubin. Peaks 1 and 2 are chromatographically identical to the glucuronic acid conjugates of the azodipyrroles of bilirubin I~a. Peaks 3 and 4 are chromatographically identical to the azodipyrroles of bilirubin IXa.
75
bilirubin IXa in their biles. The two adults had their gallbladders
emptied again 26.5 hours after the initial bilirubin injection. One
adult had negligible levels of glucuronic acid conjugates and the second
adult had 3.5 runoles of glucuronic acid present at levels of 20.2% of
the total bilirubin IXa in their bile.
Discussion
These results demonstrate that the major bile pigment in the bile
and serum of adult bullfrogs is bilirubin IXa. Biliverdin IXa is also
present in the bile. Bilirubin IXa and biliverdin IXa pigments made up
the bulk of the bile pigments of bullfrogs. Bilirubin IXy surprisingly
is present in significant amounts. Bilirubin IXS and bilirubin IXo are
present in trace concentrations. These results indicate that the bulk of
heme catabolism in the bullfrog is directed toward cleavage at the a
methylene bridge. Cleavage at the y methylene bridge also represents a
significant pathway of heme catabolism in the bullfrog. These results
also indicate that a mechanism for the reduction of biliverdin to
bilirubin occurs in vivo. During preliminary experiments with liver
homogenates, considerable biliverdin reductase was seen that was not
dependent upon added NADPH or NADH. Whether trace levels of biliverdin
reductase are present or biliverdin reduction is accomplished by an
alternate mechanism will require additional experiments to distinguish
between the two. The existance of both biliverdin and bilirubin in the
bile of the bullfrog might indicate the existance of two pools of heme
catabolism, one resulting in biliverdin and the other in bilirubin.
Another possible explanation for the presence of bilirubin and biliverdin
in bile would be that during heme catabolism the biliverdin reducing
system could exist at a low level so that all of the biliverdin would not
be reduced to bilirubin and could be excreted unaltered as biliverdin.
These results show that bilirubin IXa is the major bile pigment in
bile and the major bile pigment of serum. This indicates that in the
adult bullfrog bilirubin IXa is the major end product of heme metabolism.
As can be seen from Table 3.1 the amount of bilirubin in the bile of adult
frogs is considerably variable. This may be due to variations in the
time elapsed in bile sampling and the last discharge of the bile. Frogs
76
are able to go long intervals without eating. Also it has been shown that
the gallbladders of bullfrogs have considerable concentrative powers
(Hober and Titajew, 1929). The adult bullfrogs used in this study were
eating regularly and appeared healthy.
As can be seen from Table 3.1, the serum bilirubin levels of the
bullfrog are present at about 15% of those of the mouse. The low levels
of serum bilirubin in the bullfrogs are not surprising, considering the
low metabolic rate of amphibians and their long lived red cells (Altland
and Bruce, 1962). The bile levels of bilirubin are comparable in the
bullfrog and mouse. The main difference in the bile of the two species
is the absence of conjugates in the frog. As implied in the argument in
the preceeding paragraph the levels of bilirubin in the bile of bullfrogs
could be elevated due to a degree of cholestasis and concentrative powers
of the bullfrog gallbladder.
When bilirubin UDP-glucuronyl transf erase from the mouse and the
bullfrog are both assayed at 30°c, the bullfrog liver had activity
corresponding to about 22% of the levels of mouse liver activity. When
assayed at 37°C the mouse bilirubin UDP-glucuronyl transferase activity
approximately doubled. If it is possible to relate the rates of in vitro
glucuronidation to the situation in vivo frog liver would have approxi
mately 10% of the activity that the mouse possesses. Thus, it appears
that the bullfrog possesses sufficient liver bilirubin UDP-glucuronyl
transf erase to conjugate bilirubin.
The most striking difference between the two species is their quali
tative patterns of bile pigments. The mouse bile contains approximately
equal amounts of glucuronide conjugates of bilirubin azodipyrroles and
unconjugated azodipyrroles. This pattern is consistant with measurements
by Fevery et al., (1977a) thatmouse bile contains 100% monoconjugates.
A consistant feature of bullfrog bile is the presence of large
amounts of unconjugated bilirubin and the absence of conjugated biliru
bin. We have shown that frog liver contains significant amounts of bili
rubin UDP-glucuronyl transferase. There are two possibilities to explain
the presense of unconjugated bilirubin in frog bile. The first possi
bility is that bilirubin is secreted in an unconjugated form. The
second possibility is that bilirubin is excreted into the bile in a
77
conjugated form and the conjugated bilirubin is hydrolyzed in the bile.
To test the ability of frog bile to hydrolyze bilirubin conjugates,
frog bile was incubated with dog bile. Frog bile contained large amounts
of S-glucuronide hydrolytic activity. If the assumption is made that the
bilirubin in frog bile was 100% monoconjugated with glucuronic acid when
it was excreted, the hydrolytic activity of the frog bile shown in
Fig. 3. 9 would completely hydrolyze the bilirubin in approximately one
hour using the initial rate of reaction and the average value for bili
rubin bile concentration. This experiment illustrates the difficulty
in detecting endogeneous produced bilirubin glucuronide when basal
bilirubin excretion is small and large amounts of S-glucuronide hydrolytic
activity exist in the bile.
We have established that frog livers contain bilirubin UDP-glucuronyl
transferase and their bile contains hydrolytic activity. The question
remains whether they make bilirubin glucuronide in vivo. Preliminary
experiments with bilirubin injections to increase the basal excretion rate
did not result in detectable amounts of bilirubin glucuronide in the bile.
To test the possibility that S-glucuronidase was responsible for the bile
hydrolytic activity, frogs were injected with the specific inhibitor,
saccharic acid lactone. When bullfrogs were injected with bilirubin and
saccharic acid lactone, detectable amounts of bilirubin glucuronide were ~
found in their biles (Fig. 3.11). This experiment indicates that frogs
possess mechanisms for the serum transport, hepatic uptake, hepatic con
jugation with glucuronic acid, and hepatic excretion of bilirubin.
The injection of bilirubin and saccharic acid lactone resulted in
levels of bilirubin glucuronide of about 22% of the total bilirubin IXa
present in the bile. If this bilirubin was excreted as monoconjugated
bilirubin, it would indicate that about 44% of the bilirubin excreted
into the bile was in a conjugated form. Failure to see higher percentages
of conjugate? bilirubin is probably due to two factors. The initial
draining of the gallbladder may not have removed all of the unconjugated
bilirubin, and it probably did not remove any unconjugated bilirubin
higher up in the biliary system. Also saccharic acid lactone must enter
the bile in sufficient quantities to inhibit the powerful hydrolytic
78
activity. Any residual activity will result in significant hydrolysis.
Akamatsu et al., (1961) achieved an approximately 60% inhibition of the
liver type 8-glucuronidase 30 min after the oral administration of
saccharic acid lactone at the rate of 400 mg per kg of body weight in
mice. They found that the inhibition almost completely disappeared in
several hours. The critical factors in inhibiting biliary S-glucuronidase
is getting sufficient quantities of saccharic acid lactone in the bile
and maintaining these levels long enough to measure in vivo glucuron
idation of bilirubin. The results of Akamatsu et al., indicate that for
the liver, the inhibition occurs rapidly and that saccharic acid lactone
is cleared rapidly. Injection of adult bullfrogs with large amounts of
saccharic acid lactone failed to yield any detectable bilirubin glucuron
ide in their bile overnight. Trace levels of S-glucuronidase activity
can probably efficiently hydrolyze the basal production of bilirubin
glucuronide.
In sunnnary the data in this chapter indicates that in the healthy
adult bullfrog, bilirubin IXa is the major pigment of bile and of serum.
It also indicates that adult bullfrogs contain low but significant and
adequate amounts of bilirubin UDP-glucuronyl transf erase. The ex
periments presented in this chapter also demonstrate that bullfrogs
secrete bilirubin conjugated with glucuronic acid that can be detected ,,
in the bile if the biliary S-glucuronidase is inhibited. The in vivo
experiment did not demonstrate that all of the bilirubin was excreted in
a conjugated form but considering the limitation of the S-glucuronidase
inhibitor (saccharic acid lactone), it is likely that the adult bullfrog
has the same mechanism for bilirubin IXa excretion that has been shown
in other animals.
CHAPTER IV
BILE PIGMENT METABOLISM DURING ANURAN METAMORPHOSIS
Introduction
Hepatic bilirubin UDP-glucuronyl transferase has been shown to be
the rate-limiting enzyme in the bilirubin excretory pathway in the new
born rhesus monkey (Gartner et al., 1977). Recently Kwade and Onishi
(1981) have shown that bilirubin UDP-glucuronyl transferase is present
in the livers of human fetuses at activities corresponding to approxi
mately 0.1% of the mature activity. A slow increase in activity
occurred in utero in the late developing fetus and a much more rapid
increase in activity was seen in newborns. There is some evidence that
tadpoles are deficient in the conjugation of foreign phenols with glu
curonic acid. Lester and Schmid (1961) failed to detect a-amino phenol
glucuronyl transf erase activity in the livers of Rana catesbiana tad
poles. They were able to detect significant transferase activity in the
livers of terrestrial adult amphibians. Maickel et al., (1959) stated
that tadpoles of Rana pipens lacked both the UDP-glucose dehydrogenase
and glucuronyl transferase activity for foreign phenols.
Brodie and associates have developed a hypothesis that the ability
to metabolize lipid soluble organic compounds has develope~ along with
the evolution of animals from an aquatic existance to a land living
existance (Jondorf et al., 1958; Jondorf, 1979). According to this
theory fish and aquatic amphibia lack the enzymes responsible for the
oxidation and conjugation of many foreign drugs. Terrestrial amphibia
and "higher" vertebrates possess the enzymes for detoxification. Pre
sumably tadpoles of terrestrial frogs and toads develop the enzymes
during metamorphosis. Tadpoles and aquatic adult amphibians are
thought to be able to eliminate lipid-soluble exogenous compounds by
dialysis through the lipoidal membranes of the gills and skin. Pre
sumably bilirubin should fall into the class of lipophilic compounds
that tadpoles should be able to "dialyze" away through their gills.
Therefore tadpoles should not require the glucuronyl transferase system
necessary for biliary excretion of bilirubin. During metamorphosis
79
80
tadpoles resorb their gills and undergo other adaptations for terrestrial
existence. If an analogy between amphibian and mammalian development
exists, adaptations in bile pigment metabolism, including increased
activity of bilirubin UDP-glucuronyl transferase activity, would be
expected to occur. The stimuli for development of UDP-glucuronyl
transferase activity in mammals are not unknown; but induction by the
increased bilirubin load resulting from fetal hemoglobin replacement has
been proposed.
Herner and Freiden (1961) have shown that there is a replacement of
larval type hemoglobins by adult type hemoglobins during spontaneous and
thyroid hormone-induced metamorphosis of Rana catesbiana. The transition
from the larval type hemoglobins to adult type hemoglobins results in
the entire larval hemoglobin population being scavenged and catabolized
(Moss and Ingram, 1968a; Moss and Ingram, 1968b). This should result
in increased heme catabolism and increasing bilirubin concentrations;
which, by analogy to mammalian development, might be expected to result
in increased UDP-glucuronyl transferase activity.
To establish whether amphibian metamorphosis is a suitable model
to study the development of the mammalian bilirubin excretory pathway
the following parameters were measured during spontaneous metamorphosis.
1. Bile pigment pattern
2. Plasma bilirubin concentrations
3. Hepatic bilirubin UDP-glucuronyl transferase activity
Changes in the first parameter should reflect changes in the enzymatic
activity of the bilirubin excretory pathway (i.e. presence of conjugates).
Changes in the second parameter should reflect the rate of heme
catabolism and the efficiency of excretion. Changes in the third para
meter should reflect the metabolic needs of the animal for conjugation
of bilirubin. If amphibian metamorphosis is a suitable model for the
development of the mammalian bilirubin excretory pathway, increases in
the third parameter should be of a similar magnitude as that seen in
mammalian development.
To examine the possible effects of thyroid hormones in the develop-
ment of the bilirubin excretory pathway, parallel experiments will be
81
done using thyroid hormones to induce precocious metamorphosis in tad
poles. In addition, phenylhydrazine-induced hemolysis will be used to
see if the hemolysis results in increased plasma and bile bilirubin
levels. lIIllllersion of tadpoles in solutions of pheynlhydrazine will
induce a rapid and almost complete anemia (Flores and Freiden, 1968).
Hepatic bilirubin UDP-glucuronyl transf erase activity will be examined
to see what effect the expected increased bilirubin load will have on
its activity.
Experimental
Chemicals
Phenylhydrazine hydrochloride and 3, 3', 5-triiodo-L-thyronine
(free acid) were obtained from Sigma Chemical Co. (St. Louis, Missouri,
U.S.A.).
Animals
Rana catesbiana tadpoles were obtained from Howe Brothers Minnow
Farm (Atlanta, Texas, U.S.A.). Animals were maintained in an aquarium
in the laboratory and fed ad libitum, a diet consisting of trout chow,
agar and gelatin formed into cakes.
Thyroid Hormone Injection of Tadpoles
Premetamorphic tadpoles (stage X-XII) were injected w~th triio
dothyronine (T3
) intraperitoneally through the tail at the rate of
3 x 10-lO moles/gm body weight. This was accomplished by dissolving
1.95 mg of T3
(free acid) in 1 ml of 1% NaOH and diluting to 10 ml.
Tadpoles were then injected with 1 µl of this solution per gm body
weight. Control animals were injected with a solution of 0.1% NaOH
at the rate of 1 µl per gm body weight. Control and T3 treated tad
poles were maintained separately in plastic tubs and not allowed
access to food.
Phenylhydrazine Immersion of Tadpoles
Premetamorphic tadpoles (stage X-XII) were immersed in a solution
of phenylhydrazine (1.75 x 10-SM in tapwater pH 7.5; 6 animals per
liter of solution). The water was changed after 1 day and every other
day thereafter. Control and phenylhydrazine treated tadpoles were
maintained separately in plastic tubs and not allowed access to food.
Diazo Coupling of Samples
Bile (diluted 5-10 fold) and serum samples were diazo coupled to
p-iodoaniline as described in Chapter II.
HPLC and TLC Analysis of Samples
82
Bile and serum samples were analyzed by HPLC and TLC as described
in Chapter II.
UDP-glucuronyl Transf erase Assay
Bilirubin UDP-glucuronyl transf erase assays were performed on
tadpole livers as described in Chapter III.
In Vivo Bilirubin Glucuronide Synthesis
Animals were injected intraperitoneally with a solution of bili
rubin (3.0 mg dissolved in 0.2 ml of O.OSN NaOH and diluted with 2.5 ml
of human serum albumin 24 mg/ml) at the rate of 0.02 ml per gram body
weight. Animals were also injected intraperitoneally with a solution
of saccharic acid lactone (176 mg/ml). Four hours later the animals
were anesthetized by irrrrnersion in 0.1% tricane and their bile collected
as previously described.
Statistical Analysis
Control and treated animals were statistically compared, by means
of the Independent Student's t test.
Results
Spontaneous Metamorphosis
Bile Pigm£nts
Figure 4.1 shows the bile concentrations of bilirubin IXa during
spontaneous metamorphosis of Rana catesbiana. Low concentrations
of bilirubin IXa were found in the bile of premetamorphic tadpoles.
Bile concentrations of bilirubin IXa begin to rise at stage XX and
reach maximum concentrations in froglets. Figure 4.2 shows the bile
concentrations of bilirubin IXy during spontaneous metamorphosis.
Bile bilirubin IXy roughly parallels bilirubin IX~ concentrations
although the degree of increase is not as large (Fig. 4.2, Table 4.1).
•(5356) •(5243) • (3305) • (3103)
2800 • 2600
" ~2400 b • 02200 •• ~2000 .. -=:1soo •
•• • ai::
~1600 • •• • LI.I • ~l400 • o. • • • (.)1200 ••
•• • 1000 • • •• 800 • 600 • • • • 400
200 .. ' • • 0 • , ...
x XVIII xx XXll FROG LET ADULT STAGE
Fig. 4.1. Bile Concentrations of Bilirubin IXa During Spontaneous Metamorphosis of Bullfrog Tadpoles and in Adult Bullfrogs.
83
• •
• (44) • (79) 40 •
35
30
" ::E c.o
I
S:2s '1J
z 0 -~20 ai:: .... z • LI.I c.> zlS • 0 c.>
• • 10
• •• • • • • .. • • 5 .. • •• • .: • ., • • ~ I 0
x XVIII xx
• •
• •
• • • • • •
• I
XXll STAGE
•
•
• •• •
• • •• • • • •• • • • ••
• •• • • • • ••
FROG LET ADULT
Fig. 4.2. Bile Concentrations of Bilirubin IXy During Spontaneous Metamorphosis of Bullfrog Tadpoles and in Adult Bullfrogs.
84
Table 4.1
Ratio of the Bile Concentrations of Bilirubin IXa to Bilirubin IXy
Stage in Spontaneous Metamorphosis
x
XVIII
xx
XXII
Froglet
Adult
Ratio
4.4
6.5
33.9
47.4
114.0
5.5
Day after T3 Injection Ratio Controls Ratio Injected
0
2
4
7
9
11
Days after Phenylhydrazine Immersion
0
1
2
3
4
3.4
2.0
3.1
6.3
10.3
21.0
Ratio Controls
3.2
8.7
4.7
3.3
2.6
4.4
5.3
2.1
4.5
7.2
Ratio Immersed
9.1
7.1
15. 9
24.4
36.1
85
86
Biles were screened for the presence of azodipyrroles derived from bili
rubin IXS IXo on HPLC solvent system E. Small amounts of azodipyrrole
aF (derived from IXS) and azodipyrrole aF' (derived from IXo) were seen
in biles of stage X animals. An experiment using the pooled bile of 2
individuals was done. The stage X bile samples had concentrations of
bilirubin IXS of 0.93, 0.20, 0.38, 0.48, 0.82 µMand 2 samples that
were below the limits of detection. Bilirubin IXo could sometimes be
detected but as concentrations too low to be accurately quantitated.
Biliverdin IXa was measured in the bile of another group of tad
poles by using HPLC solvent system E and simultaneously monitoring
the absorbance at 658 run and 546 run. The results are contained in
Table 4.2. Biliverdin IXa bile concentrations rise in parallel with
bilirubin IXa concentrations. The data presented in Table 4.2 indicates
that biliverdin IXa is not the major bile pigment during spontaneous
metamorphosis.
Transesterification reactions performed on biles of stage X, XX,
XXII tadpoles, and froglets yielded no detectable amounts of ester
conjugates. Biles screened on HPLC solvent system A did not contain
detectable concentrations of azopigments corresponding to the glucuronic
acid, glucose, or xylose conjugated azodipyrroles prepared from dog
bile. Biles screened for the presence of conjugates on TLC (Heirwegh
et al., 1974) failed to yield detectable azopigments corresponding to
the conjugated azodipyrroles prepared from dog bile.
Figure 4.3 illustrates plasma concentrations of bilirubin IXa
measured during spontaneous metamorphosis. Plasma levels rise from
low concentrations to maximal concentrations at metamorphic climax
(stage XXII to froglet). Adult serum concentrations of bilirubin were
found to be approximately 2-fold higher than premetamorphic concen
trations.
Bilirubin UDP-glucuronyl Transf erase
Hepatic bilirubin UDP-glucuronyl transferase activity was present
in all stages of metamorphosis examined. Figure 4.4 illustrates the
specific activity of hepatic bilirubin UDP-glucuronyl transferase
measured during spontaneous metamorphosis. Variations in individuals
Table 4.2
Concentrations of Bile Bilirubin IXa and Biliverdin IXa during Spontaneous Metamorphosis
Stage of Metamorphosis Bilirubin IXa* Biliverdin IXa*
x 7.5 + 2. 9 ( 18) 12. 1 + 4.4 (18)
XVIII 139 + 4 7 (9) 36 + 12 (9)
xx 630 + 220 (7) 320 + 140 ( 7)
XXII 3600 + 1100 (4) 3600 + 1400 (4)
Frog let 880 + 210 (7) 190 + 30 (7)
-6 *values are 10 M ± 1 S.E.M. (N).
87
"" 0.5 -&
(0 I
C> ..... '-' 0.4 -z Q -=c 0.3 lo-
A:: .... z LI.I
~0.2 -Q c.>
0.1 T(lO) -..1.
x
,(13) ~
XVIII
- ... (8)
--
xx STAGE
--(10)
_ ._
XIII
88
--(15)
--
-~Cl 6)
--
FROGLET ADULT
Fig. 4.3. Plasma Bilirubin Concentrations During Spontaneous Metamorphosis of Bullfrog Tadpoles and Serum Bilirubin Concentrations in Adult Bullfrogs. Bars represent mean values and T-lines are± 1 S.E.M. (N).
" Cl) .... -z
0.5 ..
0.4 ..
3o.3 ... .... " A. Q
::;:) 0.2 ...
0.1.
----
--
-~(20)
----(12)
. ... --(7)
(8)
----(6) . ._
O ... ._ •. _. __ __. ........ .._.._ __ ...... •...-__ __.__.._...._ __ .,,_ •. ....r..----1....1•_.i.....
I XVIII XX XXll FROGLET ADULT STAGE
89
Fig. 4.4. Hepatic Activity of Bilirubin UDP-Glucuronyl Transferase (UDPGT) During Spontaneous Metamorphosis of Bullfrog Tadpoles and in Adult Bullfrogs. Units are defined as µmoles of bilirubin glucuronide synthesized per hr per gm of protein at 30°C. Bars represent mean values and T-lines ± 1 S.E.M. (N).
90
and error associated with the assay make detection of small differences
between stages difficult. From the data, it is clear that a dramatic
increase in hepatic UDP-glucuronyl transf erase does not occur during
spontaneous metamorphosis. Activity of bilirubin UDP-glucuronyl
transferase appears to be depressed at stage XXII. The in vitro pro
duction of bilirubin glucuronide by stage XXII liver homogenates was
only linear for 20-30 min, whereas production by other stages was
linear for at least 60 min. A 20 min incubation was employed in all
assays, since it falls in the linear range for all stages. Fig. 4.5
is a pH dependence curve for bilirubin UDP-glucuronyl transf erase
using an adult bullfrog liver and a stage X tadpole liver. The hepatic
UDP-glucuronyl transf erase activities in adult and stage X tadpole
showed essentially the same pH dependence.
Digitonin treatment at concentrations of 12 mg/ml and 1 mg/ml
failed to significantly increase bilirubin glucuronide formation in
livers of stage X, XX, XXII tadpoles or in froglets. Doubling the
concentration of UDP-glucuronic acid did not increase the synthesis of
bilirubin glucuronide in stage X, XX, XXII tadpoles or in froglet
liver homogenates. This result indicates that the concentration of
UDP-glucuronic acid is at saturating concentrations in the standard
assay for those stages examined.
In Vivo Glucuronidation of Bilirubin
Froglets, when injected intraperitoneally with bilirubin and high
concentrations of saccharic acid lactone, had detectable amounts of
bile bilirubin glucuronide conjugates after 4 hours (Fig. 4.6).
Bilirubin glucuronide was identified on the basis of the chromatographic
behavior of the azodipyrroles on HPLC and TLC systems. The presence
of ester conjugates in the bile of bilirubin-saccharic acid lactone
injected froglets was confirmed by transesterification reactions.
Fig. 4.7 shows the results of a transesterification reaction performed
on the bile of a froglet injected with bilirubin and saccharic acid
lactone. The bile of froglets injected only with bilirubin contained
only low or undetectable amounts of bilirubin glucuronide as judged
by comparison of their biles with glucuronide conjugates prepared from
'QP.4 -0 E c .,.,., ~0.3 -z 0 D: = c.>0.2 = .... c.:J
z iii 0.1 = D: -.... -m 0
6.5 7.0
pH
91
7.5
Fig. 4.5. pH Dependence of UDP-Glucuronyl Transferase in Liver Homogenates of an Adult Bullfrog (A) and a Stage X Tadpole ( •). Bilirubin glucuronide was measured in the incubation mixture after 20 min at JQOC using the assay conditions described in the text. The pH of the incubation mixture was measured using micro pH probe.
1 92
2
A
1
i 2
B
0 20 TIME (MIN.)
Fig. 4.6. Detection of Bilirubin Glucuronide in the Bile of Froglets Injected with Bilirubin and Saccharic Acid Lactone. Tracings are absorbance monitoring at 546 nm. HPLC was accomplished using solvent system A and as described in text. A.) p-iodoaniline-coupled dog bile showing the presence of azodipyrroles corresponding to the glucuronic acid conjugated azodipyrroles prepared dog bile. The sample was run at 0.05 AUFS and at 1.0 AUFS at arrow. B.) glucuronic acid conjugated azodipyrroles from dog bile.
93
Fig. 4.7. Base Catalyzed Transesterification in Methanol of Ester Azodipyrroles Conjugates. Transesterification reactions were performed as described in text. Absorbance monitorings at 546 nm were accomplished using HPLC solvent system B and as described in text. A.) Control (NaOH omitted) transesterification of diazo coupled dog bile. B.) Transesterified diazo coupled dog bile showing the formation of the hydrophobic methyl ester azodipyrroles (peaks lM and 2M). C.) Control sample transesterification reaction of diazo coupled bile obtained 4 hours after a froglett was injected with bilirubin and saccharic acid lactone. D.) Transesterification reaction of diazo coupled bile obtained 4 hours after a froglett was injected with bilirubin and saccharic acid lactone, showing formation of methyl ester azodipyrroles (111 and 2M). Tracings in C and D were run at 0.05 AUFS after arrow.
A B
c D
1
2M
1M
2M
9
95
dog bile and transesterification reactions. In a typical experiment 3
froglets injected with bilirubin and saccharic acid lactone had
biliary bilirubin glucuronide levels of 48.7, 51.3 and 6.5 µM which
made up 2.2, 3.4, and 0.5% respectively of the total bilirubin IXa
in their bile. In the same experiment 3 froglets were injected with
bilirubin only. Two of these tadpoles had undetectable concentrations
of bilirubin glucuronide; the third had a bilirubin glucuronide
concentration of 10.9 µM which was 0.7% of the total bilirubin IXa
present in the bile.
Stage X tadpoles when injected with bilirubin and saccharic acid
lactone also produced amounts of bilirubin glucuronide detectable 4
hours later (Fig. 4.8). Bilirubin glucuronide was identified on the
basis of the chromatographic behavior of the azodipyrroles on HPLC
(solvent system A) and TLC when compared to glucuronide conjugated
azodipyrroles prepared from dog bile. The presence of ester conjugates
in the bile of bilirubin-saccharic acid lactone injected tadpoles was
also confirmed by transesterification. Stage X tadpoles injected with
bilirubin alone also contained bilirubin glucuronide in their biles
after 4 hours. In a typical experiment 3 tadpoles injected with
bilirubin and saccharic acid lactone contained 10.0, 28.7, and 60.9
µM bilirubin glucuronide in their biles. This amount of bilirubin
glucuronide represents 11.9, 16.5 and 3.11% respectively of the total
bilirubin IXa in their bile. Three stage X tadpoles injected with
only bilirubin had 7.9, 36.2 and 14.4 µM bilirubin glucuronide in their
biles. This amount of bilirubin glucuronide represents 4.3, 21.2 and
13.4% respectively of the total bilirubin Xa in their bile.
Thyroid Hormone-Induced Metamorphosis
Bile Pigments
Fig. 4.9 illustrates the bile concentrations of bilirubin IXa after
triiodothyronine (T3
) injection of premetarnorphic stage tadpoles
(X-XII). The bile concentrations of bilirubin IXa are approximately 2-
fold higher in T3
treated animals than in fasted control tadpoles.
Concentrations of bile bilirubin IXy are approximately 6-f old higher
in T3
treated tadpoles (Fig. 4.10). The differences in the means of
96 1
A 2
B 1
2
0 25 TIME (MIN.)
Fig. 4.8. Detection of Bilirubin Glucuronide in the Bile of Stage X Tadpoles Injected with Bilirubin and Saccharic Acid Lactone. Tracings are absorbance monitored at 546 nm. HPLC operating conditions are solvent A and as described in text. A.) Bile of stage X tadpole containing azodipyrroles chromatographically identical to the glucuronic acid conjugated azodipyrriles prepared from dog bile. B.) Glucuronic acid conjugated azodipyrroles prepared form dog bile.
300
"250 :E co
I
0 .... '-" 200 • z Q t -.... cc Cl:: 150 .... z e6A .... • u z • =100 • u • .,
• • •• •• • 50 • eM
• •• • I 0 ,
0 2 4 7 9 11 TIME (DAYS)
Fig. 4.9. Bile Concentrations of Bilirubin IXa in the Biles of T3 Injected Tadpoles <•) and Fasted Control Tadpoles ( • ) •
97
35
" ~30 <O 0 ..... '1J t 2 25 0 -.... c e:20 z LI.I u z ~ 15
• • 10 •
• s • 5 •• • .. ~
• • • .. • 0 .. 0 2 4 7 9
Tl ME (DAYS)
Fig. 4.10. Bile Concentrations of Bilirubin IXy in the Biles of T3 Injected Tadpoles (A) and Fasted Control Tadpoles ( •).
98
• • L •
-' • • 11
bilirubin IXa and bilirubin IXy from their respective control values
were found to be significantly different at day 11 (p<0.05 level).
Biliverdin IXa was screened for in the biles of 3 control tadpoles
and 3 T3-injected tadpoles at days 0, 2, 4, 7, 9 and 11 using HPLC
solvent system E and simultaneously monitoring absorbance at 658 and
99
546 run. In the tadpoles examined biliverdin IXa was generally below
detectable levels and did not appear to increase significantly in either
group.
Fig. 4.11 contains the bile concentrations of bilirubin IXS and
IXo in the bile of T3-injected and control tadpoles. Bilirubin IXS and
IXo concentrations are normally at low or undetectable concentrations
in the bile of stage X tadpoles. It can be seen from the data presented
in Fig. 4.11 that bilirubin IXB and IXo rise to concentrations signi
ficantly higher than controls. Table 4.3 contains the serum bilirubin
IXa concentrations of control and T3-treated tadpoles. Concentrations
of plasma bilirubin are higher in T3-treated tadpoles than control
concentrations 7 days after T3
injection.
Bilirubin UDP-glucuronyl Transf erase Activity
Fig. 4.12 contains the activities of bilirubin UDP-glucuronyl
transferase in control and T3-treated tadpoles. Enzymatic activities
of T3-treated livers are approximately twice as high as th9se of control
tadpole livers, 7 days after r3
injection. The differences in the means
of T -treated and control hepatic UDP-glucuronyl transf erase activities 3
at day 7, and day 9 was judged to be significant at the p<0.05 level.
The hepatic bilirubin UDP-glucuronyl transferase of T3-treated tadpoles
appears to decline by day 11.
Phenylhydrazine-Induced Hemolysis
Bile Pigments
Hematocrits of phenylhydrazine immersed and control tadpoles are
shown in Fig. 4.13. Hematocrits of phenylhydrazine immersed tadpoles
rapidly decline to low levels. Fig. 4.14 contains bile bilirubin IX,
concentrations in control and phenylhydrazine treated tadpoles. Bile
concentrations of bilirubin IXa increase approximately 12-fold in
100
8
" & :E
co A I
Q 6 ....
'-'
z & Q & • - 4 ... •• c .:.:: ... z LI.I 2 A & c.> • z & • • Q ... A c.> & •
I A 0
0 2 4 7 9 11 TIME (DAYS)
" ~3 &
I
Q .... t '-'
z 2 Q
& & -... • c & .:.:: & ... 1 A z • LI.I t • •& c.> • z • & Q A & c.> 0
0 2 4 7 9 11 TIME (DAYS)
Fig. 4.11. Bile Concentrations of A.) Bilirubin IXS and B.) Bilirubin IXo of T3 Injected (A) and Control (•) Tadpoles.
Table 4.3
Plasma Bilirubin Concentrations of T3 Injected Tadpoles and Control Tadpoles
Days after Injection Bilirubin IXa*
Controls T)-Injected
0 0.073 + 0.004 (3) 0.092 + 0.011
2 0.059 + 0.004 (3) 0.075 + 0.028
4 0 .140 + 0.038 (3) 0.178 + 0.025
7 0.146 + 0.016 (3) 0.223 + 0.026
9 0.154 + 0.018 (3) 0.207 + 0.048
11 0.174 ± 0.055 (3) 0.461 + 0.164
*Concentration in 10-6 M + 1 S.E.M. (N).
101
(3)
(3)
(3)
(3)
(3)
(3)
102
1.0
0.9
0.8 A A
0.7 •• en o.& •A ... I t - A ~ 0.5 M A
"-' t A .t ~ 0.4 •t
·1 ... • A. I• •• Q ,. • ~ 0.3 .. ! • • ..
• • I A ,_ I 0.2 • • ' • • t • ... 0.1 •
0
0 2 4 7 9 11 TIME (DAYS)
Fig. 4.12. Specific Activity of Bilirubin UDP-Glucuronyl Transferase in the Livers of T
3 Injected Tadpoles ( •) and Fasted Control Tadpoles
(.).
103
30
(&JI <S>J C&>I <&>I <6)I 20
1(6) .... -~ u 0
ti10 &
B&> .... :z:
0 I<&> 6) I(&)
0 1 2 3 4 TIME (DAYS)
Fig. 4.13. Hematocrit Values for Phenylhydrazine-Irrnnersed Tadpoles (A) and Control Tadpoles (• ). T-lines represent 1 S.E.M. (N).
'"'300 • co I
0 ..... ...,,_,
z200 0 -.... c ai:: .... z 1.a.1100 c.> z 0 (.)
0
(6)
6)
0
104
(6)
1(6) (6)
IC6) <&>I (6) (6):i
1 2 3 4 TIME (DAYS)
Fig. 4.14. Bile Concentrations of Bilirubin IXa in PhenylhydrazineImmersed Tadpoles (A) and Control Tadpoles ( • ) . T-lines represent 1 S • E • M . ( N) •
105
phenylhydrazine treated tadpoles over control concentrations. Fig. 4.15
reveals that bile concentrations of bilirubin IXy do not appear to differ
significantly in phenylhydrazine treated and fasted control tadpoles.
Concentrations of bile biliverdin IXa increase significantly in phenyl
hydrozine treated tadpoles over control tadpoles at days 2, 3 and 4
(Fig. 4.16). Table 4.4 contains the plasma levels of bilirubin IXa of
phenylhydrazine-treated and control tadpoles. By day 4 phenylhydrazine
treated tadpoles had a significantly higher serum bilirubin concentration
than control animals (p<0.05).
UDP-glucuronyl Transferase
The livers of control and phenylhydrazine treated tadpoles were
assayed for bilirubin UDP-glucuronyl transf erase activity at days 0,
2, and 4. The hepatic activity of bilirubin UDP-glucuronyl transferase
did not significantly change in control tadpoles. Day 0 phenylhydrazine
tadpoles liver contained approximately the same activity. Day 2 and day
4 phenylhydrazine treated tadpole livers did not contain detectable
amounts of bilirubin UDP-glucuronyl transferase activity.
Discussion
As can be seen in Fig. 4.1 bile concentrations of bilirubin IXa
rise dramatically during spontaneous metamorphosis. This rise is
most likely due to increased heme catabolism from the transition from
larval to adult hemoglobin. Interpretation of bile pigment concentra-
tions are complicated by several factors, which probably contribute
to the considerable variability observed. Variations in the frequency
of emptying of the gall bladder and the rate of bile flow could explain
some of the variability in bile samples seen. The amphibian gall
bladder is capable of considerable concentrative action and a bile
sample could reflect bile pigment production ranging from hours to
weeks. During spontaneous metamorphosis, tadpoles cease to eat at the
stage of forelimb emergence (stage XX). The fasting of animals while
they are undergoing metamorphic climax probably results in some degree
of bile stasis (cholestasis). The control stage X tadpoles showed
increased bile concentrations of bilirubin during the course of the
experiment. Whether the increase due to increased cholestasis or
106
20 •
" :E CD
I 15 e ..... '-J • z •• • 0 -I-
10 c ai:: I- • • z ..... A u z • • •• h 0 5 •• • • t. u ... •• • A •
• t •t • e£
• -:, • • • 0 •
0 1 2 3 4 TIME (DAYS)
Fig. 4.15. Concentrations of Bile Bilirubin IXy in the Bile of Phenylhydrazine-Immersed Tadpoles (A) and Fasted Control Tadpoles ( •-).
107
1600
"" ~1400 I
c 01200 (6)
~1000 -.... c 800 ai:: .... z LI.I
600 u z 6) 0 u 400 I<6)
200
O....u~~~~i'_6_~_(~6~)..._ __ ~(6~)SJ-__ ~(6~)AA-__ 0 1 2 3 4
TIME (DAYS)
Fig. 4.16 Bile Concentration of Biliverdin IXa in PhenylhydrazineImmersed Tadpoles (A) and Control Tadpoles ( •). T-lines represent 1 S.E.M. (N).
Table 4.4
Plasma Bilirubin Concentrations of PhenylhydrazineImmersed and Control Tadpoles
Days after Immersion Bilirubin IXa*
108
Controls Phenylhydrazine-Immersed
0 0.128 + 0.011 (6) 0.115 ± 0.011 (6)
1 0.136 + 0.018 (6) 0. 121 + 0.0176 (6)
2 0.098 + 0.016 (6) 0.141 + 0.030 (6)
3 0.112 + 0.016 (4) 0.156 + 0.024 (6)
4 0.118 + 0.007 (6) o. 187 + 0.020 (6)
*Concentration . -6 + 1 S.E.M. (N) • 1n 10 M value
109
increased production of bilirubin due to fasting is at present not clear.
Another possible source of the variation seen in the bile samples is
uncertainty in staging. Some of the stages have a definite morphological
event that occurs (i.e. forelimb emergence at stage XX). Froglett is
a loosely applied term that means a tadpole that has completed tail
resorption but has not reached sexual maturity. Variations in length
of time since a Froglett has completed metamorphosis could contribute
to the variations observed.
Bile bilirubin IXy concentrations also rise during spontaneous
metamorphosis but the magnitude of the increase of IXy is not as large
as the increase in bilirubin IXa (Table 4.1). Bilirubin IXS and IXo in
bile appear not to increase significantly during spontaneous metamorpho
sis. The non-a isomers of bilirubin are very liable (Blanckaert et al.,
1976) and their stability in bile, if significant cholestasis occurs,
is not presently known. If significant cholestasis is present, the
concentrations of thenon-aisomers may be artificially low.
Plasma concentrations of bilirubin IXa increase approximately 4-fold
during spontaneous metamorphosis from premetamorphic stages. Plasma
bilirubin concentrations decline from metamorphic climax stages to adult
values that are approximately double the premetamorphic concentrations.
The increased plasma bilirubin values are probably due to increased heme
turnover during the transition from larval to adult type hemoglobins.
The loss of gills may also cause increased plasma concentrations of
bilirubin during metamorphosis and in the adult because of decreased
excretion of bilirubin via this route. Increased amounts of enteric re
absorption of unconjugated bilirubin is partially responsible for the
increased serum concentrations of bilirubin in human newborns (Poland
and Odell, 1971). At metamorphic climax tadpole bile can contain up to
5 mM unconjugated bilirubin. Although these mechanisms have not yet
been demonstrated in tadpoles, reabsorption of unconjugated bilirubin
from the gallbladder and enteric reabsorption could also cause increased
plasma bilirubin concentrations during spontaneous metamorphosis.
Bilirubin UDP-glucuronyl transf erase is a tightly bound membrane
enzyme. Treatment of liver homogenates or microsomes with digitonin
generally results in increased enzymatic activity, the magnitude of the
110
increase apparently being species specific. A wide variety of detergents
and enzyme treatment has been used to "activate" glucuronyl transferase
activity toward 4-nitrophenol (Vessey and Zakim, 1971; Zakim and Vessey,
1976). Two hypotheses have been put forth to explain the increase in
glucuronyl transferase activity (activation) by detergent and enzymatic
digestion. One hypothesis is that glucuronyl transf erase is constrained
by its lipid environment and treatments that modify its lipid environment
can increase the activity. The second hypothesis is the compartmentation
of glucuronyl transferase in the microsomal membrane results in limited
access to its substrates. A "permease" protein is postulated to control
transport of UDP-glucuronic acid to the enzyme (Berry and Hallinan,
1976). Activation of glucuronyl transferase by UDP-N-acetyl glucuosamine
is considered evidence for the second hypothesis (Berry and Hallinan,
1976; Berry, 1978).
Treatment of tadpole liver homogenates with digitonin failed to in
crease bilirubin UDP-glucuronyl transferase activity. The addition of
UDP-N-acetyl glucuosamine also failed to increase enzymatic activity.
Fevery et al., (1977a) found that digitonin increased the activity of
rat liver homogenates approximately 13 times over control activity. They
found that guinea pig liver homogenate had activity increased less than
2 times control values. This obvious species dependence of digitonin
activation could possibly be due to differences in the lipid composition
of the liver microsomal membranes.
The assay of bilirubin UDP-glucuronyl transf erase used in this study
is subject to assay to assay variation. The HPLC assay developed in this
study is more sensitive and selective than the spectrophometric assay of
Heirwegh et al., (1972). The spectrophotometric assay is dependent upon
the assumption that diazotized ethyl anthranilate only reacts with bili
rubin glucuronide in the incubation mixture. The HPLC assay has the
advantage that smaller amounts of bilirubin glucuronide can be detected
and identification of bilirubin glucuronide azodipyrroles being formed
is more unequivocal than the spectrophometric assay. The HPLC assay
suffers in that sample preparation is more complex and tedious. The
HPLC assay is dependent upon the separation of the conjugated
111
azodipyrrole from chromogenic pigments in the liver. Error is introduced
during the integration of the sharp bilirubin glucuronide azodipyrroles
by triangulation. A bilirubin glucuronide standard is not yet commer
cially available. With the limitations of the assay in mind, it can be
stated that bilirubin UDP-glucuronyl transf erase activity does not
dramatically increase (less than two-fold) during spontaneous meta
morphosis. Individual variations in tadpoles also contribute to the
variations seen in the assay. The lack of a genetically defined bull
frog strain undoubtedly contributes to this variation seen in individ
uals.
The absoluate increases in bile and serum concentrations in T 3
treatment are not as large as those in spontaneous metamorphosis. The
lower bile levels could reflect less cholestasis in T3
treatment
compared to spontaneous metamorphosis. Biliverdin IXa was not seen to
increase significantly during T3
treatment. If biliverdin is the result
of oxidation of unconjugated bilirubin during cholestasis, the result
might indicate less cholestasis. T3
treatment was terminated after
11 days. No treated tadpoles live longer than 11 days. After 11 days
of T3
treatment tail resorption was not complete. Highest serum and
bile concentrations of bilirubin were observed in froglets, in which
tail regression was complete.
The physiological significance of the non-a isomers of bilirubin is
not yet clear. Heme oxygenase from mammalian sources appears to direct
cleavage exclusively at the a methylene bridge of heme (Tenhunen, et al.,
1969). It is possible that other heme oxygenases present in lower
amounts catalyze cleavage at the non-a methylene bridges. In vitro
it is possible for heme proteins to direct their own catabolism by a
coupled oxidation producing significant amounts of the non-a isomers
(O'Carra and Calleran, 1969). Whether heme proteins direct their own
destruction in vivo producing non-a isomers of bilirubin is not yet
known. The occurrence of bilirubin IXa in nature is relatively rare.
The occurrance of significant amounts of bilirubin IXa in the bile of
bullfrog tadpoles is an interesting aspect of comnar~tive biochemistry.
The increase of bilirubin IXB and IXo in the bile of T3-treated tadpoles
is an interesting observation, the significance of which is not yet
known.
112
T3 treatment of tadpoles resulted in a 2-fold increase in the
activity of hepatic bilirubin UDP-glucuronyl transferase. Whether the
increase in enzymatic activity of T3-treated tadpoles represents an
activation of existing enzyme or represents the induction (new protein
synthesis) of more enzyme is uncertain.
Phenylhydrazine had the unexpected effect of completely inhibiting
bilirubin UDP-glucuronyl transferase activity in the livers of treated
animals. Whether phenylhydrazine has a direct effect on the enzyme or
its presence interferes with the assay is not presently known.
The results of the phenylhydrazine experiments are complicated by
the fact that phenylhydrazine is probably not entirely specific for the
destruction of red cells. Beutler (1969) has ascribed the action of
"oxidant" hemolytic drugs to inhibiting enzymes that maintain gluta
thione in a reduced form or which synthesize glutathione. Phenylhy
drazine hemoglobin complex cannot be reduced as methemoglobin (Jaffe
and Neuman, 1968). The damaged red cell is removed by the reticulo
endothelial cells (London, 1961; Jandl, 1960; Card et al., 1968). The
large increase in biliverdin IXa seen in the bile of phenylhydrazine
treated tadpoles could be due to inhibition of the biliverdin reductase ,_
system. The main conclusion that can be drawn from the phenylhydrazine
experiments is that hemolysis induced by phenylhydrazine results in
large amounts of the a isomers of bilirubin and biliverdin being
excreted into the bile.
The conjugation of bilirubin with glucuronic acid could be
demonstrated in both premetamorphic stage X tadpoles and froglets under
conditions of bilirubin loading. The highest percentage of bilirubin
glucuronide formed in the bile of a stage X animal under these con
ditions was 36% of the total bilirubinIXa (conjugated and unconjugated).
If this bilirubin were excreted as monconjugates, this would mean
that 72% of the bilirubin was excreted in a conjugated form. This value
could be an underestimate due to two things. The bile already contains
unconjugated bilirubin before the injection. Failure of sufficient
saccharic acid lactone reaching the bile to completely inhibit the
113
S-glucuronidase activity would also result in increased unconjugated
bilirubin. From the experiments done with stage X tadpoles injection
of saccharic acid lactone did not appear necessary to measure detectable
amounts of bilirubin glucuronide after the injection of a bilirubin
bolus. This is probably due to the generally low hydrolytic activity
of premetamorphic tadpole bile which will be discussed in Chapter V.
The detection of bilirubin glucuronide in the bile of froglets was
dependent upon an injection of saccharic acid lactone. The amount of
bilirubin glucuronide was also a much smaller percentage of the total
bilirubin IXa in the bile. These observations are probably due to the
generally higher hydrolytic activity of froglet bile (Chapter V) and
the much higher levels of unconjugated bilirubin in the bile of froglets.
Presented in this chapter is the unexpected result that premeta
morphic tadpoles possess a fully functional biliary excretion mechanism
for bilirubin. Also conclusively demonstrated is the presence of
bilirubin in the serum and bile of premetamorphic tadpoles. The findings
indicate that in premetamorphic tadpoles, the following steps are
functional:
1. heme oxygenase(s)
2. biliverdin reductase system(s)
3. serum transport of bilirubin
4. hepatic uptake of bilirubin
5. UDP-glucose dehydrogenase enzyme for the production of
UDP-glucuronic acid
6. bilirubin UDP-glucuronyl transferase
7. biliary excretion of conjugated bilirubin
In summary the results presented in this chapter indicate that the
development of the bilirubin excretory pathway in amphibian metamorphosis
is not completely analogous to primate development. The premetamorphic
tadpole appears to be competent for the biliary excretion of bilirubin.
Spontaneous and thyroid hormone-induced metamorphosis results in in
creased heme catabolism to bilirubin as judged by increased bile and
plasma bilirubin concentrations. The activity of hepatic bilirubin
UDP-glucuronyl transf erase appears to increase during spontaneous and
114
T3-induced metamorphosis but the increase is not of a similar magnitude
as the increase in activity seen in primate fetal development, which
may be as 100-fold.
CHAPTER V
BILE B-GLUCURONIDASE
Many alcohols, phenols, and carboxylic acids are combined with
D-glucuronic acid to form B-D-glucosiduonic acids. The enzyme that
catalyzes the hydrolysis of B-D-glucosiduronic acids is commonly called
B-glucuronidase. B-glucuronidase is widely found in plant and animal
kingdoms. In rat kidney liver, cartilage, and preputial gland,
B-glucuonidase has been detected in the lysosome and the endoplasmic
reticulum (Fishman et al., 1967).
The occurrence of the enzyme in bacteria is sporadic. S-glucuron
idase in bacteria may be adaptive or constitutive, extracellular or
intracellular (Levvy and Marsh, 1959). S-glucuronidase in Escherichia
coli is secreted extracellularly and its concentrations increase greatly
when cultured in the presence of menthol glucuronide (Buehler, et al.,
1951). B-glucuronidase activity has been detected in species of
Streptococcus, Staphylococcus, some Corynebacterium and sheep rumen
microorganisms (Levvy and Marsch, 1959). Hawksworth et al., (1971), in
a study on the effect of intestinal bacteria on the hydrolysis of
glycosidic bonds, found that the greatest amounts of S-glucuronidase
were produced by E. coli, and intermediate amounts were produced by
Nagler-positive Clostridia and Bacteroides bacteria. ,_
Larusso and Fowler (1979) measured the activities of three lysosomal
glycosidases (B-glucuronidase, B-galactosidase, and N-acetyl-S-gluco
samidase) in bile collected from rats with biliary fistula. They found
that biliary excretion of the three enzymes varied considerably during
a 24 hour period. Although the total activity varied, the three hydrol
ases were excreted in a parallel (coordinate) manner. The influence of
pH on the activity of both liver and bile enzymes was measured. They
found an acid pH optimum for each enzyme and the curves were not
different for the liver or bile enzyme. They proposed that bulk dis
charge of hepatocyte lysosomes as the most likely mechanism for the
biliary excretion of the lysosomal enzymes.
B-glucuronidase in bile is believed to contribute to the formation
of bilirubin pigment gallstones. S-glucuronidase, if active in the
115
116
bile, will deconjugate bilirubin glucuronide producing unconjugated
bilirubin. The bilirubin found in pigment gallstones is unconjugated
(Trotman et al., 1974). Maki (1966) has proposed that G-glucuronidase
present in the bile from bacterial infection hydrolyzes bilirubin
glucuronide. The resulting bilirubin combines with calcium to form an
insoluble calcium bilirubinate salt. The pigment present in pigment
gallstones has been shown to be calcium bilirubinate (Toyoda, 1966).
Sato et al., (1964) examined the bile of patients with either calcium
bilirubinate or cholesterol gallstones. They characterized the
S-gluconidase activity of the bile of both groups by its pH optimum.
The pH optimum of the bile from patients with calcium bilirubinate stones
was from 5.8 to 7.3. The pH optimum of the bile from patients with
cholesterol stones was from 3.8 to 6.2. E. coli was found to be present
in the bile of those patients with pigment gallstones. Enzyme obtained
from E. coli cultured from the bile of patients with pigment stones had
a pH optimum around neutrality, similar to the pH optimum of the bile
of patients with pigment stones. The pH dependence curve of the
S-glucuronidase activity of bile obtained from patients with cholesterol
stones had a similiar shape and optimal pH (acidic) as the enzyme ob
tained from serum and liver enzymes.
Boonyapisit et al., (1978) measured the rate of hydrolysis of
conjugated bilirubin to unconjugated bilirubin in bile of human patients
with cholesterol and pigment gallstones. They found that only 3 out of
24 bile samples from patients with cholesterol stones showed significant
hydrolysis. In the bile of patients with pigment stones, 10 out of 19
showed significant hydrolysis. They cultured 15 bile samples for the
presence of bacteria. Two of the bile samples grew E. coli but there
was no relationship between the presence of bacteria and the ability of
the bile to hydrolyze conjugated bilirubin to unconjugated bilirubin.
Recently Ho et al., (1979) have described a biliary 8-glucuronidase
in rats that they believe is secreted into the bile by the liver or the
biliary epithelium. They collected bile from rats with permanent bile
dust fistula. They found that activity of bile samples was extremely 0
variable. The enzyme had a pH optimum of 6.0 when assayed at 56 C
using phenolpthalein 8-D-glucuronide as a substrate.
117
The experiments undertaken in this chapter were done to investigate
the S-glucuronidase activity observed in bullfrog bile. Indirect
evidence for B-glucuronidase activity in bullfrog bile was obtained
from the bilirubin loading experiments. Detectable amounts of bili
rubin glucuronide in the bile of bullfrogs were only found when large
amounts of saccharic acid lactone, a specific inhibitor of B-glucuron
idase, were injected into bullfrogs. To establish the possible source
of the hydrolytic activity, we have attempted to characterize the bile
enzyme and screen the bile for bacteria that could be the source of
the enzyme.
Experimental
Chemicals
Phenolphthalein 8-D-glucuronic acid (sodium salt from rabbit
urine), 4-methylumbelliferone (practical grade), 4-methylumbelliferyl
S-D-glucuronide, and p-nitrophenyl B-D-glucuronide were obtained from
Sigma Chem. Co. (St. Louis, MO., U.S.A.). Brain heart infusion and
nutrient broth were obtained from BBL (Cockeyville, MA., U.S.A.).
Blood agar plates were obtained from REMEL (Lenex, KA., U.S.A.).
Fluorometric Assay of B-Glucuronidase
The following were added to 3.0 ml fluorescence cuvette: 0.5 ml of
0.2 M phosphate pH 7.0 buffer or 0.2 M acetate ph 4.75 buffer, 25 µl of
4-methyl-umbelliferyl S-D-glucuronide (0.54 mg/ml), diluted bile and
water to a total volume of 2.0 ml. The increase in fluorescence was
measured with a total fluorescence accessory on a Beckman Acta M VI.
The excitation wavelength was 340 run (slit width 7 nnn). The assay was
performed at room temperature. The instrument was calibrated with a
solution of 10-6 M 4-methylumbelliferone. The standard was prepared by
dissolving 176.2 mg of 4-methylumbelliferone into 100 ml of methanol
and performing serial dilutions with 0.05 M phosphate pH 7.0 buffer or
0.05 M acetate buffer pH 4.75. Standards were stored at -40°C until use.
Bile was routinely diluted 10-fold. Ten µl was used for assay.
Spectrophotometric Assay of B-Glucuronidase
The spectrophotometric assay of B-glucuronidase, using
118
phenolphthalein B-glucuonide as a substrate, was based on the assay of
Levvey and Conchie (1966). To a test tube the following was added:
0.75 ml buffer, 0.3 ml of phenophthalein S-glucuronide (5.62 mg/ml)
diluted enzyme source, and water to a total volume of 3.0 ml. The
solution was mixed. A 0.8 ml aliquot was removed and placed on ice.
The test tube was then placed in a 30°c water bath. Aliquots were re
moved at timed intervals and placed on ice. At the end of the incubation
period, the aliquots were mixed with 0.4 ml of 0.4 M glycine/NaOH pH 10.7
buff er and the absorbance read at 540 nm. The change in absorbance was
obtained by subtracting the time zero reading from the incubated mix
ture reading. The buffers used was 0.2 M phosphate pH 7.0 buffer (for
bile) or 0.2 M acetate pH 4.75 buffer (for liver). For the pH profiles
a buffer consisting of 0.07 M citrate, 0.07 M phosphate and 0.07 M
glycine adjusted to the desired pH with 10 N NaOH was used. The final
pH of the incubation mixture was measured using a micro pH probe.
Electrophoresis
Electrophoresis was accomplished using a Beckman Microzone electro
phoresis system with cellulose acetate membranes and B-2 barbital buffer
(250V~30 min). B-glucuronidase activity was detected by placing the
cellulose acetate membrane face down upon a petri dish containing agar
and 4-methylumbellif eryl S-D-glucuronide and incubating for approximately
30 min at 37°c. Activity was detected by irradiating t 1.1f; membrane with
an ultraviolet lamp and observing the strongly fluorescing bands. The
petri dishes were prepared by mixing 250 ml of 0.1 M phosphate buffer
pH 6.5 with 3.75 g of agar and 42 mg of EDTA and heating to boiling. The
solution was mixed and 25 mg of 4-methylumbelliferyl B-D-glucuronide
added and poured into petri dishes.
Collection of Biological Materials
Adult bullfrogs were anesthetized by an intraperitoneal injection
of tricane at the dose 0.002 ml/g of body weight. The gallbladder was
surgically exposed, swabed with 95% ethanol, and bile was drawn into
sterile hypodermic syringe. The liver was removed, blotted, weighed and
placed in ice-cold distilled water. The liver was minced with a razor
h~_;1rl,, and homogenized with a polytron homogenizer (for 30 sec at setting
119
#5). The liver was appropriately diluted with distilled water.
Bacteriology of Bullfrog Bile
Initial isolation and culturing of bacteria from bullfrog bile was
accompished using standard microbiological techniques. Organisms in
adult bullfrog whole bile were identified by Dr. D. H. Lewis of Texas
A&M University College of Veterinary Medicine.
Results
Characterization
Fig. 5.1 contains the pH profile obtained from bullfrog liver and
bile S-glucuronidases using phenolphthalein S-D-glucuronide as a sub
strate. The liver enzyme had a pH optimum of approximately 4.7. The
bile activity had a broad pH optimum of between 7.0 and 7.5.
Fig. 5.2 indicates the effect of saccharic acid lactone on the
hydrolysis of phenophthalein S-D-glucuronidase. When assayed under these
conditions, saccharic acid lactone inhibits hydrolysis by the liver
enzyme much more efficiently than the bile enzyme.
When subjected to electrophoresis the bile enzyme migrated more
rapidly than the liver enzyme (Fig. 5.3). S-glucuronidase activity with
the same electrophoretic mobility as the bile activities was not detect
able in liver homogenates. When frog bile was subjected to gel filtra
tion column chromatography, with either sephadex G-150 or sephacryl
S-300, the S-glucuronidase activity always chromatographed with blue 6 dextran (2xl0 daltons). The inclusion of 0.5 M NaCl in the elution
buffer did not change the chromatographic properties and resulted in loss
of most of the activity.
Attempts to Demonstrate Bacterial Origin of the Biliary S-Glucuronidase
The bile of adult bullfrogs consistantly indicated the presence of
considerable numbers of bacteria when plated on blood agar plates.
Isolated bacterial colonies or whole bile were cultured in suspensions
of nutrient broth or brain-heart infusion broth. Cultures were periodi
cally sampled for up to 1 month and assayed for C-glucuronidase activity
using the fluorometric assay. Cultures containing 10 mM phenolphthalein
B-D-glucuronide in either nutrient broth or brain-heart infusion broth
100
>....
90
80
70
60
(350 0 .... ~40 ~
30
20
10
o ...... __ .._ __ ..._ __ .._. __ ...____.m.;;::._.~----'-3 4 5 6
pH 7 8 9 '10
Fig. 5.1. pH Dependence of S-Glucuronidase Activity in Liver Homogenate of an Adult Bullfrog ( •) and Bile Pooled from Several Adult Bullfrogs (~). Assay was performed at 30°C using phenolphthalein S-D-glucuronide as described in text.
120
100
80
~ 60 -.... -!! 40 ::c z -~ 20
121
0.001 0.01 0.1 1 10 100 CONCENTRATION (mM)
Fig. 5.2. Saccharic Acid Lactone Inhibition of B-Glucuronidase in Liver Homogenate of an Adult Bullfrog (•). and Bile Pooled from Several Adult Bullfrogs (~). Results of two seperate determinations were averaged for each curve. Assay of B-glucuronidase was performed at 30°c using phenolphthalein B-D-glucuronide as described in text.
122
0 I
A
(+)
B ) I
Fig. 5.3 Cellulose Acetate Electrophoresis of A.) Bullfrog Liver Homogenate and B.) Bullfrog Bile. Electrophoresis was accomplished as described in text. S-glucuronidase activity was visualized as described in text. 0 is the site of sample application.
123
were also periodically monitored for S-glucuronidase activity. Tests
for the presence of S-glucuronidase activity in cultures grown in the
presence or absence of glucuronide substrates were negative. The results
obtained from bacteriological analysis of adult bullfrog are summarized
in Table 5.1.
S-glucuronidase Activity during Spontaneous Metamorphosis.
Fig. 5.4 illustrates S-glucuronidase activity measured during spon
taneous metamorphosis of bullfrog tadpoles and in adult bullfrogs
measured at pH 7.0 using the fluorometric assay. Considerable variabiJ
ity is observed in the biliary activity. An additional group of 21
stage X tadpoles had their biles screened for S-glucuronidase activity
at pH 4.75 and 7.0 using the fluorometric assay. Of the 21 individuals
screened, 11 had greater activity at pH 7.0, 6 had activity higher at
pH 4.75 and 4 gave no detectable activity at either pH. The results of
pH measurements of bullfrog tadpole bile are sunnnarized in table 5.2.
Discussion
The results contained in this chapter indicate that bullfrog bile
contains a S-glucuronidase whose characteristics differ greatly from
the liver type S-glucuronidase. The pH optimum for the bile S-glucuroni
dase is higher than the published pH optimum of 6.2 for E. coli using
phenolphthalein S-D-glucuronide (Buehler et al., 1951). The published
value for the K. for saccharic acid lactone using the hydrolysis of 1
phenolphthalein S-D-glucuronide by rat preputial gland S-glucuronidase
is 0.11 x l0-6M (Levvy et al., 1958). The published value for the K. i
for saccharic acid lactone using the hydrolysis of phenolphthalein
S-D-glucuronic acid by S-glucuronidase from sheep-rumen microorganisms
is 19 x 10-6M (Marsch, 1955). This indicates that saccharic acid lactone
is a much less effective inhibitor of the bacterial enzyme compared to
the mannnalian enzyme. Due to the failure to obtain bacterially produced
B-glucuronidase in culture it is impossible at this time to state that
bacteria are sources of the enzyme. It is possible that the biliary
B-glucuronidase is excreted by the liver or the gallbladder into the
bile. If the enzyme is rapidly excreted it is likely that the liver or
gallbladder would not contain large enough amounts of the enzyme to
Table 5.1
Organisms Detected in Adult Bile
Organism
Corynebacterium pyogenes
Alcaligenes aquamarinus
Achromobacter pinnatum
Brevibacterium maris
Pseudomonas piscidida
Unknown (still working on)
Number of Individuals
2
1
1
6
1
1
124
125
6.5
6.0 • 5.5 •
"50 en ~
z4.5 = '-' 4.0 I LI.I
~3.5 Q -z 3.0 • 0 • ai::: = 2.5 • ' (.) • • = ~2.0 • I
~1.5 • • • I • • 1.0 • •
I • • ~ 0.5 • ... 0 • •:\..
x XVIII xx XXll FROG LET ADULT STAGE
Fig. 5. 4. Biliary S-Glucuronidase Activity During Spontaneous ~1etamorphosis and in Adult Bullfrogs. Assay was performed at room temperature by measuring the hydrolysis of 4-methyl unbellif erone-8-D-glucuronide. Units are defined as 1 µmol of substrate hydrolyzed per hr per ml of bile.
Table 5.2
pH of Bile in Spontaneous Metamorphosis of Bullfrog Tadpoles and Adult Bullfrogs
Stage pH of Bile*
x 6.6 ± o. 1 (8)
XVIII 6.6 ± o. 3 (7)
xx 6.3 ± o. 1 (11)
XXII 6.1 ± 0 .1 (13)
Froglet 6.1 ± 0. 1 (7)
Adult 6.3 ± 0. 2 (4)
*pH units ± 1 S.E.M. (N)
126
127
detect by these techniques.
The values obtained for S-glucuonidase activity measured at pH 7.0
show considerable variability (Fig. 5.4). Differences in bile flow and
the degree of cholestasis could possibly account for some of the vari
ability observed. It is also possible that bacterial colonization of
the gallbladder has occurred in some animals and not others. This could
explain the very high activity observed in some individuals and in
detectable activity in other individuals of the same stage. Multiple
bile samples from a single animal would help to clarify if only some
animals are producers of the enzyme or, if bile levels are variable
within an individual.
From the survey of stage X tadpole biles, it appears that it is
possible that low levels of the liver type S-glucuronidase (pH optimum
5.0) occur in the bile of animals that have no apparent activity at
pH 7.0. Liver type B-glucuronidase would still have considerable activ
ity at the pH of the bile (Table 5.2 and Fig. 5.1). This activity could
explain the lack of conjugates in stage X tadpoles that have no apparent
activity at pH 7.0. It is difficult at the present time to explain the
lack of glucuronide conjugates in the bile of tadpoles that do not have
any apparent B-glucuronidase activity at either pH, unless the enzyme is
transient or hydrolysis occurred higher in the biliary tree. It is
likely that trace activities of S-glucuronidase activity are sufficient
to hydrolyze the low endogeneous bilirubin glucuronide production if
indeed bilirubin is excreted in a conjugated form by the tadpole.
REFERENCES
Adamson, L. F., and Ingbar, S. H. (1967) Endo. 81, 1362.
Akamatsu, S., Kiyomoto, A., Harigaya, S., and Ohsima, S. (1961) Nature 191, 1298.
Ashley, H., Katti, P., and Frieden, E. (1968) Dev. Biol. 17, 293.
Atland, P. D., and Brace, K. C. (1962) Am. J. Physiol. 203, 1182.
Barrowman, J. A., Bonnett, R., and Bray, P. J. (1976) Biochim. Biophys. Acta 444, 333.
Bernstein, R. B., Novy, M. J., Piasecki, G. J., Lester, R., and Jackson, B. T. (1969) J. Clin. Invest. 48, 1678.
Berry, C. S. (1978) in Conjugation Reactions in Drug Biotransformation p. 233, Elsevier/North Holland Biomedical Press. Amsterdam.
Berry, C. S., and Hallinan, T. (1976) Biochem. Soc. Trans. 4, 650.
Beutler, E. (1969) Phann. Rev. 21, 73.
Bilewicz, S. (1938) Biochem. z~ 297, 379.
Billing, B. H., Cole, P. G., and Lathe, G. H. (1957) Biochem. J. 65, 774.
Bissel, D. M. (1975) Gastro. 69, 519.
Bissel, D. M., Hammaker, L. E., and Schmid, R. (1972a) Blood 40, 812.
Bissel, D. M., Hammaker, L. E., and Schmid, R. (1972b) J. Cell Biol. 54' 107.
Blanckaert, N. (1980) Biochem. J. 185, 115.
Blanckaert, N., Heirwegh, K. P. M., and Compernolle, T. (1976) Biochem. J. 155, 405.
Blanckaert, N., Heirwegh, K. P. M. and Zaman, Z. (1977a) Biochem. J. 164, 229.
Blanckaert, N., Fevery J., Heirwegh, K. P. M., and Compernolle, F. (1977b) Biochem. J. 164, 237.
128
129
Blanckaert, N., Compernolle, F., Leroy, P., Van Routte, R. Fevery, J., and Heirwegh, K. P. M. (1978) Biochem. J. 171, 203.
Blumenthal, S. G., Stucker, T., Rasmussen, R. D., Ikeda, R. M., Rubner, B. H., Bergstrom, D. E., and Hanson, F. W. (1980) Biochem. J. 186, 693.
Bonnett, R., and McDonagh, A. F. (1973) J. Chem. Soc. (Perkin) 881.
Bonnett, R., Davies, J.E., and Hursthours, M. B. (1976) Nature 262, 326.
Boonyapisit, S. T., Trotman, B. W., and Ostrow, J. D. (1978) Gastro. 74, 70.
Brock, K. W., von Clausbruch, U.C., Josting, D., and Ottenwalder, H. (1977) Biochem. Pharm. 26, 1097.
Brown, A. K., and Zuelzer, W.W. (1958) J. Clin. Invest. 37, 332.
Buehler, H. J., Katzman, P. A., and Daisy, E. A. (1951) Proc. Soc. Exptl. Biol. Med. 76, 672.
Card, R. T., Paulson, E. J., and Valberg, L. S. (1968) Am. J. Physiol. 214, 45.
Catz, C., and Yaffe, S. (1968) Ped. Res.~, 361.
Chedid, A., and Nair, V. (1974) Dev. Biol. 39, 49.
Cohen, P. P. (1970) Science 168, 533.
Cole, P. G., Lathe, G. H., and Billing, B. H. (1954) Biochem. J. 57, 514.
Colleran, E., and O'Carra, P. (1977) in Chemistry and Physiology of Bile Pigments p. 69 U.S. Dept. of Health, Education, and Welfare, N.I.H.
Compernolle, F., Jansen, T. H., and Heirwegh, K. P. M. (1970) Biochem. J. 120, 891.
Compernolle, F., Van Rees, G.D. Blanckaert, N., and Heirwegh, K. P. M. (1978) Biochem. J. 171, 185.
Conchie, J., Gelman, A. L., and Levvy, G. A. (1967) Biochem. J. 103, 609.
Dawkins, M. J. R. (1963) Quart. J. Exptl. Physiol. 48, 265.
130
Falk, H., and Grubmayr, K. (1977) Partial struktur von Gallenpigmenten Montash Chem. 108, 625.
Feldhoff, R. C. (1971) Comp. Biochem. Physiol. 40B, 733.
Fevery, J., Van Hees, G. P., Leroy, P., Compernolle, F., and Heirwegh, K. P. M. (1971) Biochem J. 125, 803.
Fevery, J., Van De Vijver, M., Michiels, R., and Heirwegh, K. P. M. (1977a) Biochem. J. 164, 737.
Fevery, J., De Wolf-Peeters, C., De Vos, R., Desmet, V., and Heirwegh, K. P. M. (1977b) Biol. Neonate 32, 336.
Fisher, H., and Plienger, H. (1942) Hoppe-Seylers Zeitschrift Fur Physiologische Chemie 274, 234.
Fishman, W. H., Goldman, S. S., and De Lellis, R. (1967) Nature 213, 457.
Fleischner, G., and Arias, I. M. (1976) Progress 1n Liver Disease 2_, 172.
Flores, G., and Frieden, E. (1968) Science 159, 101.
Fog, J., and Jellum, E. (1963) Nature 198, 88.
Frieden, E., and Mathews, H. (1958) Arch. Biochem. Biophy. 73, 107.
Fyffe, J., and Dutton, G. J. (1975) Biochim. Biophys. Acta 411, 41.
Gartner, L. M., Lee, K., Vaisman, S., Lane, D., and Zarafu, I. (1977) J. Ped. 90, 513.
Gordon, E •. R., Daudon, M., Coresky, C. A., Chan, T., and Perlin, A. S. (1974) Biochem. J. 143, 97.
Gordon, E. R., Goresky, C. A., Chang, T., and Perlin, A. S. (1976) Biochem. J. 155, 477.
Greengard, O. (1969) Adv. Enz. Reg. z, 283.
Greengard, O. (1974) in Perinatal Pharmacology: Problems and Priorities p. 15. Raven Press, N. Y.
Gudernatsch, J. F. (1912) Arch. Entwicklungs Mech. Organismen 35, 457.
Hawksworth, G., Drasar, B. S., and Hill, M. J. (1971) J. Med Micro. 4' 451.
Heirwegh, K. P. M., Van Rees, G. P., Leroy, P., Van Roy, F. P., and Jansen, F. H. (1970) Biochem. J. 120, 877.
131
Heirwegh, K. P. M., Van De Vijver, M., and Fevery, J. (1972) Biochem J. 12 9' 605.
Heirwegh, K. P. M., Fevery, J., Meuwissen, J. A. T. P., and DeGroote, J. (1974) in Methods of Biochemical Analysis 22, p. 205.
Heirwegh, K. P. M., Fevery, J Mi.chi.els R Van Rees G P and . ' ., ' .. , Compernolle, F. (1975) Biochem. J. 145, 18S.
Heirwegh, K. P. M., Blanckaert, N. Compernolle, F. Fevery, J., and Ziaman, z. (1977) Biochem. Soc. Trans. ~' 316.
Herner, A. E., and Frieden, E. (1961) Arch. Biochem. Biophys, 9S, 25.
Hershko, C., Cook, J. D., and Finch, C. A. (1972) J. Lab. Clin. Med. 80' 624.
Ho, K. J., Ho, L. H., and Kruger, O. R. (1979) J. Lab. Clin. Med. 93, 916.
Hober, R. ' and Titajew, A. (1929) Arch. ges. Physiol. 223, 180.
Jaffe, E. R.' and Neumann, G. (1968) Ann, N. Y. Acad. Sci. lSl, 795.
Jandl, J. H. (1960) J. Lab. Clin. Med. SS, 663.
Jansen, F. H.' and Billing, B. H. (1971) Biochem. J. 12S' 917. ,_
Jansen, F. H.' and Stoll, M. s. (1971) Biochem. J. 12S, S8S.
Jansen, P. L. M.' Chowdhury, J. R. , Fischberg, F. B. ' and Arias, I. M. (1977) J. Biol. Chem. 2S2, 2710.
Jondorf, W. R. (1979) Trends in Biochem. Sci. 4, 141.
Jondorf, W. R., Maickel, R. P. Brodie, B. B. (19S9) Fed. Proc. 18, 407.
Kawade, N., and Onishi, S. (1981) Biochem J. 196, 2S7.
Kennan, A. L., and Cohen, P. P. (19S9) Dev. Biol. l, 511.
La Russo, N. F., and Fowler, S. (1979) J. Clin. Invest. 64, 948.
Lathe, G. H., and Walker, M. (1958) Biochem. J. 70, 705.
Ledord, B. E., and Frieden, E. (1973) Dev. Biol. 30, 187.
Lester, R., and Schmid, R. (1961) Nature 190, 452.
Lester, R., Behrman, R. E. , and Lucey, J. F. ( 1963) Ped. 32, 416.
Levi, A. J., Gatmaitan, z. , and Arias, I. M. (1969) J. Cl in Invest. 48, 2156.
Levi, A. J., Gatmaitan, z., and Arias, I. M. (1970) N. Eng. J. Med. 283, 1136.
132
Levine, R. I., Reyes, H., Levi, A. J., Gatmaitan, z., and Arias, I. M. (1971) Nature New Biol. 231, 277.
Levvy, G. A., and Marsch, C. A. (1959) 1n Advances in Carbohydate Chemistry Vol. 14 p. 381, Acad. Press, New York.
Levvy, G. A., and Conchie, J. (1966) in Methods in Enzymology Vol. VIII p. 571, Acad. Press, New York.
Levvy, G. A., McAllan, A., and Marsh, C. A. (1958) Biochem. J. 69, 22.
Little, G. H. (1979) J. Chrom, 163, 81.
London, I. M. (1960) N. Y. Acad. Med. 36, 79.
Lucey, J. F., Behrman, R. E., and Warshaw, A. L. (1963) Am. J. Dis. Child. 106, 305.
Maickel, R. P., Jondorf, W. R., and Brodie, B. B. (1958) Fed. Proc. Q, 390.
Maickel R. P., Jondorf, W. R. and Brodie, B. B. (1959) Fed. Proc. ~' 418.
Maisels, M. J. (1972) Ped Clin. North Am. 19, 477.
Maki, T. (1966) Ann. Surg. 164, 90.
Maniatis, G. M., and Ingram, V. M. (1972) Dev. Biol. 27, 580.
Marsch, c. A. (1955) Biochem. J. 59, 3 75.
Marver, H. s. , and Schmid, R. (1972) in Metabolic Basis of Inherited Disease 3rd ed. P• 1087, McGraw Hill Co., New York.
Moss, M. ' and Ingram, v. M. (1968a) J. Mol. Biol. 32, 481.
Moss, M., and Ingram, v. M. (1968b) J. Mol. Biol. 32, 493.
Nemeth, A. M. (1954) J. Biol. Chem. 208, 733.
133
O'Carra, p. , and Colleran, E. (1969) FEBS Lett. 2_, 295.
O' Carra, p. , and Colleran, E. ( 19 70) J. Chrom. 50, 458.
Odel 1, G. B. (1966) J. Ped. 68, 164.
Onishi, S., Itoh, S., Kawade, N., Isobe, K., and Sugiyama, S. (1980) J. Chrom. 182, 105.
Ostrow, J. D. and Schmid, R. (1963) J. Clin. Invest. 42, 1286.
Ozon, R., and Breuer, H. (1966) Gen. Comp. Endo, 6, 295.
Palma, L., McDonagh, A. F., and Schmid R. (1977) Gastro. 73, 1238.
Petryka, Z. J. (1966) Proc. Soc. Biol. Med. 123, 464.
Poland, R. L., and Odell, G. B. (1971) N. Eng. J. Med. 284, 2.
Robinson, S. H. (1968) N. Eng. J. Med. 279, 143.
Robinson, S. H., Owen, C. A., and Flock, E. V. (1965) Blood 26 823.
Sato, T., Matsashiro, T., Oikawa, T. and Sato, H. (1964). Tohoku J. Exp. Med. 84, 154.
Schenker, S., Dawber, N. H., and Schmid, R. (1964) J. Clin. Invest. 43, 32.
Schmid, R. (1956) Science 124, 76.
Schmid, R., Hammaker, L., and Axelrod, J. (1957) Arch. Biochem. Biophys. 70, 285.
Schmid, R., Marver, H. S., and Hammaker, L. (1966) Biochem. Biophys. Res. Comm. 24, 319.
Taylor, A. C., and Kollros, J. J. (1946) Anat. Rec. 94, 413.
Tenhunen, R., Marver, H. S., and Schmid, R. (1969) J. Biol. Chem. 244, 6388.
Tenhunen, R., Ross, M. E., Marver, H. S., and Schmid, R. (1970) B iochem. 2_, 2 98.
Toyoda, M. (1966) Tohuku J. Exp. Med. 90, 303.
Trotman, B. W., Ostrow, J. D., and Soloway, J. D. (1974) Dig. Dis. _!2., 5 85.
van den Berg, H. A., and Mueller, P. (1916) Biochem. Z. 77, 90.
Vessey, D. A., and Zakim, D. (1971) J. Biol. Chem. 246, 4649.
Wishart, G. J., and Dutton, G. J. (1977) Biochem J. 168, 507.
Wishart, G. J., Goheer, M.A., Leakey, J.E. A., and Dutton, G. J. (1977) Biochem. J. 166, 249.
134
Wishart, G. J., Campbell, M. T., and Dutton, G. J. (1978) in Conjugation Reaction in Drug Biotransformation p. 179, Elsevier, North-Holland, Amsterdam and New York.
Yoeh, G. C. T., and Morgan, E. H. (1974) Biochem. J. 144, 215.
Zakim, D., and Vessey, D. A. (1976) in Enzymes of Biological Membranes p. 443, Plenum, New York.
Zhivkov, V., Tosheva, R., and Zhivkova, Y. (1975) Comp. Biochem. Physiol. 51B, 421.
