•
CONFIDENT/AL
L-HISTIDINE
AMMONIA-LYASE IMMOBILIZED BY
MICROINCAPSULATION
WITHIN
ARTIrICIAL
CILLS: ENZYME XINITICS, STABILITY, AND IN
VITRO
SIMULATION or HISTIDINE
DEPLETION
rOR BISTIDINEMIA
by
A
thesis
Rajesh
submitted
KHANNA
to the
Faculty
Graduate Studies Bnd Research in
fulfil1ment
of
of
partial
the requirements of
the
degree of Master of Science.
Department of Physiology
McGill University
Montreal
September 1989
"
(
('1
, 1 l
.
.,..;.
'-"
\...
©
TABLI OF CONTINTS
ACKNOWLIDGIMINTS ................................... .
1
ARS TRACT """""""""""""""" . """"""" . """""""""""""""""""
2
RESUME """",,""""""""""""""""""",,",,"""""""""""""""""""
3
5
1. INTRODUCTION
(,
Inborn Krrors of Netabolis • . . . . . . . . . . . . . . . . . . . . . . . . .
6
Ristidine.ia
7
" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " "
Treat.ent of RistidiDe.ia """""""""""""""""""""""""""
12
Inzy.e Replace.ent Therapy ......................... .
14
I ••obilized IDz~e. """""""""""""""""""""""""""""""""
15
Artificial Cella """"""""""""""""""""""""""""""""""""
16
Bi.tidase """""""""""""""""""""""""""""",,""""""""""""
21
Ai.s of the Present Reaearcb Project . ....... ........
25
Bibliography .. """"""""""""""""""""""""""""""""""""
26
'1
"
II. MITRODS - APPINDIX A
38
Basic Principle of Preparation of Artificial Cella
39
Reagents Required for Preparation of Microcapsules
40
Procedure for Preparation of Artificial Cella. ......
43
References """"""""""""""""",,""""""""""""""""""""""""
44
(
•
III. METHODS - APPENDIX B
Ristidase Solution Assay ..............•.....•.•.....
46
Microencapsulated Histidase Assay...... ....... ......
50
References ..................... ,.................................
56
IV. CHARACTERIZATION or L- BISTIDINE
AMMONIA-LYASE IMMOBILIZED DY
MICROENCAPSULATION IN ARTlrICIAL
CELLS: PREPARATION, KINETICS,
STABILITY, AND IN VITRO DEPLETIOU
or HISTIDINE
58
ABSTRACT ............................................
60
INTRODUCTION ......................................................................
61
MATERIALS AND METHODS ...............................
62
Preparation of Artificial Cells .. ........ ......
62
Kinetic Analysis of Microencapsulated ......... .
Histidase
63
Kinetic
of Histidase in Solution ......
64
Storage Stability Analysis of Histidase .. ......
Solution and Microencapsulated Histidase
Stored at 4°C and 37°C
64
Histidase Encapsulated within Artificial
Cells: In Vitro Depletion of L- Histidine
65
~nalysis
RESULTS AND DISCUSSION ..............................
66
Kinetic Behavior of Microencapsulated .... ......
Ristidase
66
Storage Stability Behavior of
Microencapsulated Ristidase
69
Microencapsulated Ristidase: Effectiveness
for Depletion of L- Histidine In Vitro
74
.........................................................
77
ACKNOWLEDGEMENTS ................................... .
78
RErlRENCES .................................................................. .
79
GENERAL DISCUSSION
ACKNOWLKDGKMBNTS
(
1 aJII very grateful ta Dr.
T.M.S.
Chal.lg M.D. Ph.D.,
Director of
the Artificial Cells and Organs Research
Centre, for allowing me the opportunity ta carry out my
gradua te
st udies
in
hi s
lab.
His
interes t,
encouragement,
and
support were very helpful in
aIl
stages of this research project.
1 also wi sh
to express rny
grat i t ude ta Mr.
Col in
J,ister,
Chief Technician of the Artificial Cells
and
Organs
Research Centre for his
hel~
during this
research project.
His assistance in mastering certain
technical procedures, the Perkin-Elmer La~bda 4B UV/VIS
spectrophotometer,
and preparation of m:,' seminar
are
much appreciated.
1 am pleased ta have had the opportunity to work in the
same center as Dr.
Rajender Sipehia.
His
friendship,
encouragemeut,
and good humor was of great value ta me
over
the course of this
research.
His
help
in
proofreading this thesis was very valuable.
1 am grateful to Emma Resurrecion and Pina
their friendship.
(
S~rrini
for
During the
period of this research l
have had
the
opportunity to make acquaintances with fellow
~raduate
and summer students at the Artificial Cells and Organs
Research Centre and in the Department of Physio~ogy. To
them
1 exp~ess special thanks for making my ~tudies
intellectually and culturally rewarding.
1 must extend a note of thanks to Riccardo Garberi
and
Alison J.
McTavish
for
their friendship,
and
for
allowing me
to process part of this thesis
on their
computer.
1 must also extend special thanks to Daniel Duguay
and
Normand Rousseau for providing the French
translation
of the abstract of this thesis.
Finally 1 wish to acknowledge the moral support from my
family
for
being there in easy times and
periods
of
stress. To my brother Rick and my sister Madhu for your
inquisitiveness which provided motivation for me 1 owe
you much thanks. My father and my mother were always
there with their support, encouragement, and patience.
(
'"
This
research work was supported by grant MT9100
from
the Medical
Research Council and
the MEEST Virage
Centre of Excellence grant
to Dr.
T.M.S. Chang.
Financial
support
from the
FONDS FCAR
through a
graduate fellowship
they
awarded to me
i8 greatly
appreciated.
1
.
ABSTRACT
L-histidine
within
ammonia-lyase (histidase) was encapsulated
cellulose
kinetic
nitrate artificial
parameters were
evaluated.
histidase
had an apparent activity
50%
the
of
activity
of
cells.
and
its
Microencapsulated
of
histidase
approximately
in
solution.
Encapsulation did not alter the KM of histidase. The KM
of
histidase
solution and the
KMapparent
encapsulated histidase were both 20mM.
histidase
resulted in increased stability of enzymatic
histidase
activity
micro-
Encapsulation of
at storage temperatures of 4°C and
activity
37°C
of
solution reached 50' of
after
microencapsulated
days
9.5
histidase
of
37°C.
its
original
while
storage.
reached
the
At
same
level
after 15 days. At 4°C histidase solution had 63' of its
original
activity
after
21 days
encapsulated histidase had 95'.
of
storage.
while
In vitro experiments to
evaluate the feasibility of microencapsulated histidase
for possible experimental therapy in histidinemia
carried
effectiveness
histidine.
loaded
..
of
v~lume
Three different
A
evaluated
encapsulated histidase in
artificial
tested.
experiments
These
out.
cells
ratio
of
depletion
after
histidine
depletion
to substrate
1:100
120 hours.
after
ratios of
allowed
histidase
hours.
AI: 25
allowed 40' histidine depletion after 24 hours .
2
were
histidine
A 1:50 ratio allowed
72
the
depleting
solution
25'
were
35%
rat.io
RESUME
(
(histidase) L-histidine a ~té mise
L'ammonia-lyase
capsules
en
dans des cellules artificielles de nitrate de
ce Il u 1 ose e.t ses paraml't res k'lne"'t'1ques on t
L'hiatidase
mise en micro-capsules a eu
,e"t""
e eva 1 ues.
une
activité
apparente d'environ 50% de l'activitl de l'histidase en
La mise en capsules n'a pas
solution.
le KM de
l'histidase
qui comme le KMapparent de l'histidase
~n
capsules
~t:
de
a
l'histidase
stabilité
a
après
20mM.
La
mise
en
,-
pour consequence
une
l'activit: enzymatique aux
de
d'histldase
de
eu
d'entreposage
(
affect~
4°C
de
atteint
son
9.5 jours d'entreposage,
plus
activité'"
solution
originelle
tandis que l'histidase
capsules atte~nt le m~me niveau apr~s 15
en
la
dWhistidase a atteint
solution
grande
températures
la
et 37°C.
50% de
capsules
jours.
63%
dt~
,
A
son
activité originelle après 21 jours d'entreposage tandis
que
l'histidase
en
capsules
a atteint
activité dans les m~mes conditions.
,
,
experlences
in
95%
cie
son
On a effectué
vitro pour évaluer la
faisabilité
des
de
l'histidase en capsules <lui pourrait ~tre utilisée dans
une therapie expérimentale pour traiter l'histidinemie.
Ces
en
, ,
experlences
on t
capsules
diff'rentes
'
1 ue1 l a capaC1't e1 d e
eva
~ réduire l'histidine.
quantit{s
l'histidase
On a testé
d'histidase dans
les
trois
cellules
artificielles. Un taux de 1: 100 a permis de r&duire 25%
(
"
d'histidine après 120 heures.
3
Un taux de 1:50 a permis
de réduire 35% d'histidine apr~s 72 heures.
1:25
a
permis
de réduire 40%
heures .
.-.
-
......
4
d'histidine
Un taux de
,
apres
24
1. INTRODUCTION
(
5
INBORN BRRORS of MKTABOLISM
The
~oncept
modern
started
from
(Stanbury
are
of inborn
the studies of
et al.,
inherited
1978).
errors
Garrod
of
on
metabclism
a1captonuria
Inborn errors of metabolism
genetic disorders,
where
proteins
~re
enzymes involved in metabolic pathways
or
deficient or
defective.
In
their
account
monograph Stanbury et al.
of
(1978)
the various documented
inborn
offer
an
errors
of
metabolism. Geuetic defects manifest themselves in Many
forms.
If the defect affects an enzyme that cata1yzes a
metabolic
reactlon,
then at the site of the rnetabolic
sub~trate
block there will be an accumulation of
defective
".
l_
The
reaction and reduc2d formatiun
elevated
accumulated
1evels of subatrate May
of
he
of lhe
product.
taxic.
substrate at the metabolic block May
The
also
result in overproduction of a1ternate metabo1ites.
The consequences of inborn errors of rnetabolism
on
the
depend
toxicity of the accumulated substrate and
necessity
of
the absent
pro~uct.
The
expression
the
of
inborn errors of metabolism can range from asymptomatic
and nonconsequential to lethal.
for
Treatment
intake
of
precursors,
Metabolites
enzyme
deficiencies
accumulated
and
supplying
filubstrate
the
include
derived from the product.
substrale
and
absent
product
or
Histidinemia is
an example of an inborn error of metabolism.
6
limiting
BISTIDINEMIA
(
Hi.slidinemia,
firat
showing
chloride
with
~961
described in
sisters
(Ghadimi et
positive
test were
levels
metabolism,
was
1961) .
Two
al.,
results
with
ferric
t.he
misdiagnosed as
in~tially
phenylkelonuria,
elevated
hi~tidine
a disorder of
patients
but amino acid analysis
showed
of histidine in the blood and
urine.
The enzymatic defect in the histidine metabolic pathway
waa
with
elucidated through
histidinemia
glutamic
acid
studies.
biochemic~l
could metabolize urocanic
but
not
histidine,
contalned
elevated
leve)s
(Auerbach
et
1961,
al,
of
and
imidazole
1962) .
Ammonia-lyase
histidine
(histidase).
acid
their
urine
fos t ered
This
t- he
L-T,istidine
Histidase
deamillates
DlrecT proof
to produce urocanic acid.
to
metabolites
suspicion that the enzymatic defecl was in
(
P~tients
shown by demonstrating a lack of histidase activity
was
in
the stratum corneum from skin biopsies of histidinewics
( LaD u etaI., 1962; Z an non i an d t. a Du,
1963).
The
histidine
metabolic
in
pathw~ys
which
can
participate are shown in Figure 1. The dominant pathway
of
histidine
produce
glutamic
acid.
trensaminated
with
pyruvic
This
acid.
(
degradation
acid.
involves
Histidine
pyruvate
i~
to
deamination
can
produce
also
to
be
imidazole
converted to imidazole
lactic
Imidazole pyruvic acid lS responsible for giving
positive
results
(Auerbach et al.,
with Phenistix and
1962,
7
ferric
chloride
Ghadimi et ai., 1962). Normal
,
Figure 1:
Pathways of histidine
Rosenberg and Scriver, 1980)
metabo1ism
(from
'.
8
(
<»
ProtelO synthuis
t~Oigelltlde synthesis (c.11110Slne: homoc.1moslneJ H
)
HC~C-CHZCH~OOH
(Histidine)
I~ ~I
NH
Na). . <!lNH
~~.7
/H
HC=C CH?CHZNHZ
1
1
N
NH
..
ru
HC=C--c~C-COOH
*
H!5!!di15!:
~
1 1H
N
;
!
~
Urocanase
O:aC.=C-CH,CH.,COOH
\ : / (Histamine)
H
1
N
/
~~/
Imlduale .lcetlC
aeld
1
NH
1
1
N
NH
_ ..
~c/
HC=C CH, C-COOH
\
NH
'\0 /'
t
8
H
(lmldazoleoyruv,c
aCldJ
\
Imldazale l.letlc
Clmlduale :roolonlc 3c.dl
H
HOOC-C-CH,CH,COOH
1
HN
NH
~ /
CH
(Fo/mlmlnoglutam,c
.1Clol (FIG LUI
F,~,I ~leHCH ,CH,
- THFA
~
/'"
Glulamlc ~
.. ::a
Deficient in hbtiùinemia.
(
.
rHiA
"OOH
~H
CH
11
o
<Fannviglut;unic 3CIO)
individuals have negligible levels of i.idazole pyruvic
acid
in
their urine,
while in hist id inemic
1eveIs are elevated 30 fold (Ghadimi,
1974).
can
form
also
This
undergo decarboxylation to
pathway
convers ion
BeaU,
The
is of minor
in
significance
the normal ind i v idual
patients
Histidine
histamine.
in
histidine
(Van Ars de Il
1960).
current
biochellical parameters used
to
diagnose
histidinemia include elevated plasma histidine,
histidine
excretion,
imidazole
pyruvic
imidazole
lactic
Ghadimi,
........
and
acid,
8cid
Ito
1981:
urinary
metabolites
imidazole acetic
(Thomas
and
such
as
acid,
and
Howell,
1981).
et al.,
urinary
1973;
Urocanic acid in
sweat is negligible (La Du, 1963; Yokoya et al., 1983) •
Formiminoglutamic
acid (FIGLU)
in urine is present
very low levels compared to normal individuals
et
1982) .
al. ,
determination
of
Confirmat ion
histidase
is
measured in the stratum corneum (Kihara et
1978).
La Du,
is
ascertained
parameters,
loads
(Matsuda
made
activity.
at
through
Histidase
al.,
is
1968 j
The status of histidinemic heterozygotes
by determining the
and record i ng the i r
(Hague and Holton,
1971;
above
biochemical
responses to hi s tid i ne
Kuroda et al.,
1979,
1980). Metabolism of histamine is increased (Imamura et
al.,
1984). As research in histidinemia has continued,
variant
cases from the classical histidinemic
have been noted (Kuroda et al.
9
1985).
profile
During the infancy of reseorch in histidinemia,
regarded
as
inborn error cf
The
occurence.
genet ic
an
from
countries
of
rare
advent of mass screening programs
disorders
Result.s
lIetabol ism
it was
in
mass
neonates
screening
changed
programs
In U. S. A.
are now known.
this
for
view.
in
various
the frequency
histidinemio is 1 pel' 14000 births (Ghadimi,
1981).
Aus tria the frequency is 1: 17000 (Tha1a.mer, 1972).
frequency
higher
of
the frequency of other inborn
births
inherited
and
(
The
much
errors
of
(Tada
et
al.,
1982).
os an autosoMal recessive
Scriver,
1980) ,
but
cases
Ristidinemia
trait
are
is
(Rosenberg
decribed
where
inheritance oppears to be autosomal dominant (Kuroda et
al.,
The
1985).
clinical
various
and
majority
disorders
speech,
inc1uded
and
1964,
Gordon,
1981;
other
(Ga1jaard,
1980).
The
reported
La Du et al.,
1967;
Carr
and
Fensom,
1972;
1985) .
et
al
with
(1985)
10
histidinemia
found
very
Ho1ton et
Rolton,
1968;
Ghadimi,
1974,
Further
supported the notion of speech
association
in
abnorma1ities
1963;
and
ear1y
the
defects
behavioral
Neville et al.,
Benson
in
retardation,
subt1e
1962;
1970;
Pieniazek
are
mi1d
Waisman,
description
in
disp1ay
histidinemia
(Ghadimi et al,
al. ,
histidinemics
controversia1
of
literature
(
In
Manifestation of histidinemio occurs 1 pel'
metabolism.
8000
histidinemia occurence in Japan is
than
of
(La
10w
clinica1
impairment
Du
1978) .
1evels
of
histidiase
activity
sta.mering.
in
did
not
histidinemia
reports
probability
of
his tid inemia
(Popkin
consistent
physiological
display
patients
any
clinical
1980). A statistical analysis
abnormalities (Galjaard,
of
with
Cases are a1so documented where
histidinemia
with
chi1dren
shows that there
speech
disorders
et al.,
is
occuring
1974) .
histidinemia
1ed
deBree,
An
1979).
some
1974)
editorial,
results
receive
retardation
tests.
questioned
any
as
to
in
1974
need
for
patients
Patients with histidinemia who
did
treatment
any
shown
by
did
not
manifest
standardized
intelligence
The correlat ion between hist idinemia and speech
maternaI
histidinemia
disorder
in
reports
their
did
not
children.
Tada' s analys i s of
reveal
any
MaternaI
serious
histidinemia
also show conflicting results (Neville et al.,
Lyon et aL, 1974; Whitman et aL, 1977).
hist idinemic mouse (Bu 1 field et al.,
al.,
balance
1977;
and
In 1982 Tada et al reported
defects was not eval ua ted in depth.
1971;
the
a
(WadllaD
of their five year fo11ow up of
with histidinemia.
not
of
investigators
editoria1 pub1ished
treatment of histidinemia.
the
with
The 1ack
question the clinica1 severity of histidinemia
(Lancet
40'
correlation between clinical disorders
biochemical
and
a
1973,
1977).
maternaI
1974;
In the
Kacser
histidinemia
et
produces
defect syndromes in the infant (Kacser et al.,
Burns
and Kacser,
Il
1987).
Raisova
(1986)
has
suggested
(
that there may be two types of histidinemia,
one malignant which produces phenotypic disorders,
the
other benign with no serious side effects.
Histidinemia is often compared to
phenylk~tonuria.
80th
inborn errors of amino acid metabolism involve a defect
in
the first step of their primary metabolic
Much
attention is therefore paid to the
pathway.
question
why
phenylketonuria produces severe clinical defects, while
histidinemia is much less conspicous.
The elevation in
histidine levels in histidineaia (4-10 times normal) is
less
than
the
elevation in phenylalanine
phenylketonuria (20-30 times) (Ghadimi,
levels
in
It May
1974).
be that the subsidiary pathway in histidine metabolism,
tranaaination,
is
more effecient than the
pathways
in
phenylalanine
clearance
of
histidine is also higher
phenylalanine
serotonin
(Tada
et
metabolism.
al. ,
subsidiary
The
than
1982).
renal
that
of
Furthermore
metabolism is not affected in
histidinemia,
unlike in phenylketonuria (Douay and Kamoun, 1980).
TR.ATMIMT of BISTIDIHIMIA
His t idinemic
patients,
abnormal i t i es,
al1eviate
usually
lower
have
their
who
been
c1inica1
have
shown
subjected
condition.
centered on dietary substrate
the
majority
"
hist idine
derived
of
from
histidine.
the reports of
levels
dropped
12
ft
dietary
by
varying
therapy
to
Treatllen t
has
restriction
to
levels of elevated histidine and
Metabolites
(
to
phenotypic
In
imidazole
the
therapy,
amounts,
vast
plasma
but
histidine
restriction
improvellent
(Waisman,
Gatfie1d et al.,
reduced
did not result in any
1967;
1969;
Cain and
growth and development (Ga1jaard,
a
histidinemic
placed
on
infant
with
concentration
1968;
1975).
incompatible
1980).
myoclonic
a histidine restriction
histidine
Holton,
Duffner and Cohen,
intake of histidine may be
clinical
with
In one report
seizures
di et .
fell to 50% of
The
His
its
was
plasma
original
leve1, and his clinical symptolls subsided (Zachary Dyme
et
al,
1983). The
why
explain
authors of this report
substrate
successfu1.
Substrate
patients
under
is
restriction
restriction
scrutiny
at
cou1d
therapy
in
present
not
was
histidinemic
because
of
question being examined is the prudence
of
severa1 unres01ved questions.
The
first
histidine
restriction.
histidine
is
growth
and
Gatewood,
Baker,
an
It
is weIl
essential
nutrition
dietary
in 10wer
that
requirement
ma_mals
Klein and Halver,
1954j
established
1971 ;
for
(Nasset
and
Easter
and
The requirement of humans for histidine
1977).
is unclear at the present time (Wisek, 1984). Two views
exist
on this subject.
The first
is that histidine is
an essentia1 amino acid for the human body.
of
histidine
decreased
resu1t
serum
in
a1bumin,
histidine,
and
reduced
Swendseid,
1975,
1979).
13
negative
plasma
nitrogen
histidine,
balance,
muscle
(Kop~le
and
other view is that
the
erythropoiesis
The
Deprivation
human
(
body has a limited capacity for
the
endogenous
synthesis of histidine, because of incorporation of lsN
into
the
free
imidazole ring of histidine under
intake conditions (Wixom
Thus
histidine
status
is at present
(Wisek,
1984).
It
infants (Snyderman et al.,
and
histidine
Anderson,
accorded
1979).
semiessential
is requi:-ed for growth
in
1963; Snyderman, 1979), but
its requireaent May decrease with age.
The
other
ares of controversy is
is
treatment
real1y necessary (Popkin
Wadman and deBree,
manifest
1979).
(
asymptomatic
preventive
et
al,
Sorne histidinemic
clinical abnormalities,
clincially normal (La Du,
an
whether
patients
whlle others
appear
The logic of placing
1978).
histidine.ic
1974;
infant
on
histidine
restriction as a preventive treatment is controversial.
ENZYME REPLACEMENT TBERAPY
As
mentioned
in the previous
in
cases
his t idinemi a
where therapy has been a t tempted,
centered
dietary
on
potential
errors
therapy.
one
in
avenue
Enzyme
The
of
approach
restriction.
available
enzyme
pathway.
~nzyme
Excess
i t has
Atlother
inborn
replacement
substrate
is
to produce the lacking product.
replacement therapy is potentially
fl~9!~!E~~!
for
of
functional enzyme replaces the defective
the metabolic
histidinemia,
Previous
substrate
of metabolism treatment is
converted by the
(
section,
because
histidase
possible in
from
is commercia11y avai1able.
uses of enzymes in therapy include their uses
14
ft
in
the
treatment
of
cancer,
digestive
inflammation, clotting disorders,
disorders,
and drug intoxication
(Holcenberg, 1982; Poznansky, 1984). However widespread
application of enzymes in therapy is 1imited because of
certain
difficulties
(Chang,
1977a,
1984).
encountered
1988;
with
Holcenberg,
enzyme
1982;
usage
Poznansky,
Enzymes in solution are rapidly inactivated
vivo
d~e
the
biological lifespan of free enzymes is short,
to proteolysis,
repeated
removal,
enzyme administration
enzymes are antigenic in vivo.
hypersensitivity
and
administration,
the
enzyme.
resulting
in
and excretion. Thus
ia
required.
and
Foreign
They elicit immunogenic
reactions
with
continued
in reduced effectiveness of
Targeting the free enzyme to the site
of
substrate for action con also be a problem.
IMMOBILIZED ENZYMES
The
difficulties associated with enzyme usage
avoided
circumvent
Immobilized
These
first
problems associated with the
enzymes
entrapment,
be
enzymes
free
enzyme.
are classified into four
groups.
are (1) adsorption,
matrix
Immobilized
by immobilizing enzymes.
can
and
linkage,
(3)
microencapsu1ation.
The
(2) covalent
(4)
three classes of immobilized
enzymes
emphasize
the
microenvironment of the enzyme and are predominant
in
industrial,
applications
diagnostic,
(Chang,
1977a).
and
public
Microencapsulation
emphasizes the intracellular environment in which
15
health
most
r------------------- -
enzymes
(
and
1977a).
proteins
are located in
Microencapsulated
nature
enzymes
(Chang,
are
employed
predominantly in therapeutic applications.
ARTIfICIAL CRLLS
The cell is the fundimental unit of biological systems.
Examination
of the cell shows that it is an
enclosure
of biological materials within a semipermeable membrane
made
of
lipid
bilayer.
microencapsulate
In
1957
Chang
heaoglobin
membrane (Chang, 1965,
is
p~oteins
a fluidized mosaic of embedded
an
where
to
ultrathin
contents
are
within a semipermeable ultrathin membrane and
seperated from the external surroundings.
(~
a
Thus the artificial cell
a form of microencapsulation,
enclosed
attempted
within
1972).
in
monograph
Chang in his
on artificial cells (1972) stated
artificial
that
celI should not be considered as an
the
actual
physical entity able to perform aIl cellular functions.
Rather
it
is a concept involving the
artificial
possible
structures
replacement
cell functions
A
by
of
dimensions
or supplementation of
(Chang,
of
for
deficient
1972).
large variety of artificial cells have been prepared
using
different
membrane
microcapsule membrane.
membranes
prepared
(
cellular
preparation
include
derived
by
from
different
16
for
the
Artificial cells with ultrathin
synthetic
polymers
methodologies.
interfacial
emulsification (Chang,
compositions
coacervation
1964,
These
can
be
methods
following
1965, 1972, 1977a, 1977b,
1984,
1985,
1987b;
Chan'! et al., 1966a), i.nterfacial
polymerization followint emulsification
1965,
1972,
al. ,
1966a),
(Chang,
1966b,
and
1977a, 1977b, 1984,
1964,
1985, 1987b; Chang et
multicompartmental
membrane
systems
1965, 1972), secondary emulsions (Chang,
1972,
Li,
1977a,
1972).
addi tion ta membranes
po1ymers,
artificial
cel1
bi
0
logica 1
membranes
cross-linked protein (Chang,
Chang
1965,
1977b) and liquid membranes (May
In
synthetic
1977b;
(Chang,
et al.,
and
1966a),
prepared
using
1965, 1972,
1977a,
1ipids
(Mue11er
Rudin, 1968), lipid-protein (Chang, 1969a, 1972,
--
1977b,
Rosenthal
1982),
and Chang,
1 iposomes
al.,
1981,
1971),
and
biodegradable po1ymers (Chang, 1976b).
The
contents
artificial
include
that
cells
successfully
have
can
1965,
1972,
multienzyme
Chang,
enzymes
al.,
1984,
diverse.
(Chang,
within
Materiels
1965,
]9668,
1987a), cell extracts
(Chang,
1987a;
1964,
Yuan
and
Chang,
systems with cofactor recycling
1975,
1987,
microbial
quite
encapsulated
microencapsu1ated within artificial cella
single
and Chang,
been
be
19€8a, 1971a, 1972, 1984,
--
et
and
1977a,
Ch~ng,
1980; Yu and
(Gregoriadis
fro.
b iodegradab 1 e
have been
1964,
made
1988),
(Chang,
1966;
ce1ls (Chang,
1976;
Yu and Chang,
biological
1982;
cella
1986),
(Campbell
Gu
and
including
1965; Chang et al., 1965a; Mosbacl. et
Ergan et al.,
1965,
1972,
17
1984 J
1984,
1987) and
1988;
mammal ian
Chang et al.,
1966a;
Wong and Chang,
1964,
1965,
1971b,
1988), proteins {Chaug,
1984,
1972;
1987a},
1979),
(Chang,
antibodies, vaccines, hormones
1977a, 1977b, Ashkar et al., 1980), and
detoxification (Chang,
magnetic
Kato et al.,
materials
labelled
radioisotope
dntigens,
1972,
1966b,
(~hang,
materials
1986,
1972),
(Chang,
1976b,
adsorbents for
1969b, 1972, 1975, 1976a, 1984,
;988; Wolfe and Chang, 1987).
Characterization
of
depending
the
upon
artificial
ultrathin membrane,
Angstroms
radius
(Chang,
of
the
material
cell~
has
shown
used
to
prepare
its thickness varies from
1965,
1972).
that
200-500
The equivalent
semipermeable membrane has
pore
also
been
determined
and has a value of 17 Angstroms (Chang
Poznansky,
1968b;
Chang,
artificial cella.
and
1972). The pore radius size
cao he adjusted by modifying the preparation
of
the
procedure
This equivalent pore radius size
restricts passage of proteins like Hh and antibodies in
and out of the artificia1 cell.
the
molecular
artificial
he
cut
off
for
cell is 5000 daltons.
prepared
altering
weight
With this pore
in
a wide range of
parameters
in
the
entry
radius
into
the
Artificial ce11s can
diameters
again
preparation
by
procedure
(Chang,
1965, 1972). ArtificiGl cells have an enormous
surface
aree
te
volume
relationship.
Tt
has
ca1culated that 1.0 ml of artificial cells of
20
um
offered
has
by
ft
larger surface area (2 meter 2
the
conventional
18
artificial
)
be~n
diameter
than
kidne~
that
(1
meter 2
(Chang,
)
1966b, 1972). Because artificial cells
have a large surface area to volume relationship,
permits rapid equilibration
the artificial cell.
c~lls
can
this
of permeant Molecules into
Like red blood cells,
artificial
undergo crenation or swelling depending
the medium in which they are suspended
(Chang,
on
1966a,
1972) .
The
schematic representation of how
function
is
membrane
illustrated in Figure
of
the
microcapsule
artificial
2.
is
The
cells
ultrathin
impermeable
ta
Macromolecules such as proteins and antibodies. However
small
molecular
weight
Molecules
ultrathin
membrane into and out of
Thus
microencapsulated
the
and proteolytic enzymes.
molecular
size
substrates
converts
the
enzyme is
antibodies
microcapsule,
can
cross
the
microcapsule.
isolated
It acts on
diffusing
into
them to products,
which
from
small
the
then
diffuse out of the microcapsule.
The
experimental therapies in which
have
been employed are various.
cells
as
a
red
b100d
extensively investigated.
cons idered
for
blood
cell
1972,
1984).
polymerie adducts,
cella
The use of artificial
substitute
has
been
Some of the strategies being
substitutes
encapsulated within artificial cells,
soluble
artificial
include
Hb
Hb conjugated to
and polymerized Rb (Chang,
1980, 1984, 19878, 1988; Keipert and Chang,
1983,
Artificial cells containing enzymes have
been
19
(
meter 2
J966b, 1972). Because actificial cells
(Chang,
)
have a large surface area to volume relationship,
permits rapid equilibration
the artificial cell.
cells
th~
can
this
of permeant Molecules into
Like red blood cell",
artificial
undergo crenation or swe11ing depending
medium in whieh they are suspended
(Chang,
on
1966a,
1972).
The
schematic representation of how
function
illustrated in Figure
is
of
membrane
the
microcapsule
artificial
2.
is
The
cells
u1trathin
lmpermeable
to
macromo1ecu1es such as proteins and antibodies. However
small
(
molecular
weight
Molecules
ultrathin
~embrane
Thus
microencapsulated
the
into and out of
and proteolytic enzymes.
molecular
size
substrates
eonverts
the
enzyme is
antibodies
microcapsule,
ean
cross
the
microcapsule.
isolated
It acts on
diffusing
into
them to products,
which
from
smali
the
then
diffuse out of the microcapsule.
The
experimental therapies in which
have
been employed are various.
cells
as
a
red
blood
extensive1y investigated.
considered
for
blood
cell
polymerie adducts,
substitute
has
1984) .
substitutes
include
Rb
Hb conjugated to
and polymerized Rb (Chang,
Artificial cells containing enzymes have
19
been
Sorne of the strategies being
1972, 1980, 1984, 1987a, 1988; Keipert and Chang,
(
cells
The use of artifieial
encapsulated within artificial cells,
soluble
artificial
1983,
been
Figure 2:
Schematic representation of the
cell (from Chang, 1972)
20
artificial
(
FIGURE 2
M/CROENCAPSULATED
ENZYME
membrane wlth !ele~l'le çerrr..eabirty
ct
';::,=====
SUeSiRATE
====:::=:è 0
À
F~CLJC7
8
~.
for
used
{
enzyme
replacement
deficiencies (Chang,
1987a,
1988),
1972,
notable
in
hereditary
enzyme
1973a, 1977a, 1977b, 1984,
examples
of
this
include
acatalase.ia (Chang and Poznansky, 1968a; Poznansky and
Chang,
1974),
1984,
and phenylketonuria (Bourget and Chang,
1985,
1986).
microencapsu1ated
asparagine
1971a,
Asaparaginase
has
within artificial cells for
been
use
in
dependent tu.or suppression therapy (Chang,
1974).
Artificial
ce11s containing enzymes auch as urease and
tyrosinase
have
1973b;
been
1966b,
Siuchong and Chang,
used
1969a,
as assists in organ
failure
(Chang,
1970,1972, 1977a, 1984, 1987a; Shu and
Chang, 1980, 1981; Wolfe and Chang, 1987). The research
for
the present thesis is on the microencapsulation of
hi s t idase.
BISTIDASB
The
enzyme
found
L-Histidine Ammonia-Lyase
in a variety of bacteria,
(Hanson
and
Havir,
1972).
(histidase)
plants,
and
Histidase
reaction
1953;
has
mammals
deaminates
histidine to produce urocanic acid and ammonia
and Tabor,
is
(Mehler
Meh1er, 1957). The mechanism of this
been investigated and the properties
of
histidase characterized (Peterkofsky, 1962; Peterkofsky
and Mehler,
from
~!~Y~QmQq~~ f!YQr~!ç~~~
been
(
1963; Givot et al., 1969, 1970). Histidase
been
200000
(Williams
and
shùwn
to have a molecular
210000
and
has been purified and
daltons
Hiroms,
21
by
1967)
weight
sucrose
and
has
between
gradients
sedimentation
equilibriuDi
dissociated
(Rechler,
into
in
Havir,
1972).
Thiols
reduced form
The
activity is reported
and
enzyme
such
as
can
weight
DTT,
1955;
glutalhione
confined
to
lnhib i tors
to
and
maximal
(Tabor
1962 i Rechler, 1969 i Rechler
Histidase substrate specificity is
L-histidine (Rechler
of hist idase
are
Hanson
to he between 9.0 and 9.5
a~d
cysteine (Peterkofsky
urocanate
1969;
pH at which histidase has
Peterkofsky,
1971).
Tabor,
(Rechler,
be
35000
histidase activity by a110wing the enzyme
remain
et al,
The
subunits of mo1ecu1ar
1969).
maximize
1969) .
(RechIer,
and
Tabor,
1971).
i nc 1 ude
EDTA,
g1 yc ine,
and
Mehler,
1963). Histidine and
physiological inducers of histidase
in
Pseudomonas (Lessie and Neidhardt, 1967). Regulation of
histidase is accomplished by catabolite repression
sequential
feedback inhibition (Lessie and
and
Neidhardt,
1967; Hug et al., 1968).
The
assay
for histidase is a
(Tabor et al., 1955;
of
the
acid
reveals
minimal
-
that
peak
absorbance
Tabor,
urocanic
at
277
nm,
acid
while
above 240 nm
1955;
Rechler
activity is conveniently
recording
the
concommitant
increase
22
and
urocanic
a
maximal
has
histidine
(Mehler
and Tabor,
histidase
production
one
1971; Klee, 1971). Analysis
absorption spectra of histidine
absorbance
1953;
La Du,
spectrophotometric
and
Tabor,
1971).
monitered
of urocanic acid
in absorbance at
shows
277
Thus
through
and
the
nm.
The
aeeepted
(
value of the molar absorption eoeffeeient
in
aeid
urocanic
1 i terature
1969; Reeh 1 er and Tabor,
Histidaae
is
18800
(Barman,
1971).
in mammals can be found in the 1iver and
stratum corneum of the epidermis (Spo1ter et
the
Hanson
1963;
and
oî
Havir,
1972;
Scott,
in
al. ,
1981).
The
metabolism of histidine in the two organs is different.
In
the
1iver histidine is ultimately
glutamic
aeid,
formic aeid,
into
and ammonia through
deamination pathway (Greenberg,
histidase eonverts histidine
eonverted
the
1961). In the epidermis
into urocanic
acid,
but
the other enzymes of the deamination pathway are absent
(Baden and Pathak,
(
1967;
Scott,
1981). Urocanic acid
therefore accumu1ates in the epidermis.
of
The high levels
urocanic acid in the skin has led to the suggestion
that
urocan,c
provide
acts as a
natural
1967;
irradiation
urocanate
of
levels
However
Yamamoto
humans
et
al.,
is associated
Different
mammals
because
liver
and
properties
with
increased
and
negligib1e
but they do not
isozymes of histidase )Day
epidermis
Strych,
to sunlight radiation
characterization
reveals
of
and
Sunlight
histidinemic patients have
hypersensitivity
1978).
(Baden
to
1974).
in the epidermis (Hais
levels of urocanic acid in their skin,
show
sunscreen
protection against UV irradiation
Pathak,
1967).
acid
(La
exist
histidase
differences
of histidase from these two sources
in
Du,
in
from
the
(Baden
and Gavioli, 1974). Histidase of histidinemic mice do es
23
not
immunotitrate
prepared
anti-histidase
ant ibody
against histidase of normal mice
1982).
al. •
against
protein
The
main inducer of histidase is a
intake,
catabolism,
(Wright
but
glucagon,
hormones
et
high
associated
with
cortisol also induce the enzyme
(Lee and Harper, 1971).
There
are
sorne
immobilization
immobilized
activity
reports
of
in
the
hislidase.
literature
Yamamoto et
of
the
(1974)
histidase
a polacrylamide gel lattice to allow
continous production of urocanic acid.
(1977)
al
microbial cells possessing high
in
on
Jack and
immobil ized microb ial cells for the
urocanic
acid
by
covalent
for
Zajic
product ion
linkage
to
carboxymethylcellulose supports. The histidase activity
of
these
immobilized microbial cells stayed
constant
over a period of 16 days of cont inous
ope rat ion.
Wood
and
histidase
froID
Whateley
(1982)
immobilized
by
microcapsules
They
microencapsulation
prepared by interfacial
reported
microencaps ulated
that
the
enzyme
is
histidase in solution.
24
in
polymerization.
activity
40% of the
of
act i vit Y
the
of
AIMS of the PRISENT RISEARCR PROJECT
c
The
present
research project involves:
(1)
Analyzing
the possibility
of
microencapsulating
derived from ~!~Y~QmQU~! f!YQr~!~~U!
histidase
within
artificial cells.
(2) Analysis of
to
assess
enzyme kinetics and storage
stability
the effects of microencapsulation on
these
parameters.
(3)
possibility of
The
for
histidase
histidinemic
experiments
the
state
employing
depletion
was
in vitro.
of
evaluated
microencapsulated
histidine
with
in
the
pre 1 iminary
Histidine depletion experiments
were performed in vitro in a stirred-batch reactor with
different volume ratios of microencapsulated
to substrate solution.
which
treated
feasibility
detailed
These were in vitro experiments
histidine
as
a
single
pool.
study can be used for the design
IIUIt
icompartllen tal
vivo studies.
25
histidase
of
in vitro analys is and
This
more
in
BIBLIOGRAPHY
....
Ashkar
F.S., Buehler R.J., Chan T.
and Hourani M.
Radioimmunoassay
of free thyroxine
with
prebound anti T4 microcapsuIes, J. Nucl. Med., ~Q, 956-
(1980) ,
960
Auerbach
V.H.,
DiGeorge
A.M.,
Baldridge
R. C. ,
Tourtellote
C.D.,
and
Brigham
M.P.
(1961),
Histidinemia: a deficiency in liv~r histidase resul ting
in the urinary excretion of histidine and of imidazole
pyruvic acid, Clin. Res., ~, 334-337
Auerbach
V.H.,
DiGeorge
A.M.,
Baldridge
R.C.,
Tourtellote C.D., and Drigham M.P. (1962) Histidinemia:
a deficiency in histidase resulting in
the urinary
excretion
of histidine and of imidazole pyruvic acid,
J. Pediatr., §Q, 487-497
Baden H.P.
and Pathak M.A.
(1967), Metabolism and
function of urocanic acid in skin, J. Invest. Dermatol.
4~,
11-17
Baden H.P. and Gavioli L. (1974), Histidase activity in
rat liver and epidermis, J. Invest. Dermatol., §~, 479481
-
Barman T.E.
(ed)
(1969),
Springer-VerIag, 799-800
Enzyme
Handbook
Vol
II,
Denson P.F. and Fensom A.H. (1985), Genetic Biochemical
------in Oxford Monographs on Medical Genetics, No.
12, Oxford University Press, 252-255
-----------
~!~~rg~~!
Bourget
L.
and Chang T.M.S.
(1984),
Artificial cell
microencapsulated phenylalanine ammonia-Iyase,
Appl.
Biochem. Biotech., lQ, 57-59
Bourget 1..
and Chang T.M.S.
(1985),
Phenylalanine
ammonia-Iyase
immobilized in semipermeable
microcapsules for enzyme replacement
in phenylketonuria,
FEBS Letters, l~Q, 5-8
Bourget L.
and Chang T.M.S.
(1986),
Phenylalanine
ammonia-lyase immobilized
in microcapsules for the
depletion of phenylalanine in plasma in pheny1ketonuric
rat model, Biochim. Diophys. Acta, ~~~, 432-438
Bulfield G. and Kacser H. (1974), Histidinemia in mouse
and man, Arch. Dis. Child., 1~, 545-552
.
...
Burns J.E. and Kacser H. (1987), Genetic effects on the
susceptibility to histidine induced teratogenesis in
the mouse, Genet. Res., ~q, 147-153
26
Cain A.R.R.
and Holton J.B.
(1968), Histidinemia!
child and his family, Arch. Dis. Chi1d., ~~, 62-68
a
Campbell J.
and Chang T.M.S.
(1975),
Enzymatic
recycl ing
of coenzymes by amuI tienzyme
system
immobilized
within
semipermeable
microcapsules,
8iochim. 8iophys. Acta, ~~Z, 101-109
Campbell J.
and Chang T.M.S. (1976), Recycling of NAD+
(free and
iJIIJllobilized) within semipermeable aqueous
microcapsules containing a multienzyme system, 8iochem.
8iophys. Res. Commun., §~, 562-569
Chang 'l'.M. S.
(1964),
Science, !1§, 524-525
Semipermeable
microcapsules,
Chang T.M.S.
(1965), Semipermeable aqueous microcapsules,
Ph.D.
Thesis,
McGi11 University, Montreal,
Canada
Chang T.M.S., Macintosh F.C., and Mason F.G. (1966a),
Semipermeable aqueous JIIicrocapsules: I. Preparation and
properties,
Cano
J. Physiol. Pharmac01., 44, 115-128
-#
l
Chang T.M.S.
(1966b), Semiperlleab1e aqueous microcapsules
("artificial cells"):
with emphasis
on
experiments
in an extracorporeal shunt system,
Trans.
Amer. Soc. Artif. Intern. Organs, 19, 13-19
Chang T.M.S.
and Poznansky M.J. (1968a), Semipermeable
microcapsu1es
containing
catalase
for
enzyme
replacement in acatalasemic mice, Nature, gl~, 242-245
Chang T.M.S.
and Poznansky M.J. (1968b), Semipermeable
aqueous
microcapsules
(artificial
cells):
V.
Per-JIleability characteristics, J.
Biomed. Mater. Res.,
~, 187-199
Chang T.M.S.
(1969a), Clinical
potentia1of
techno1ogy, Science Tools, l§i~l, 33-39
enzyme
Chang T.M.S.
(1969b),
Removal
of endogenous and
exogenous toxins by microencapsulated adsorbent,
Cano
J. Phyaiol. Pharmacol., ~1, 1043-1045
Chang T.M.S.
and
Loa S.K.
(1970),
urease
and ammonia adsorbents
in
Physio10gist, l~, 70
Urea removal by
the
intestine,
Chang T.M.S.
(1971a), In vivo effects of semipermeable
microcapsu1es
containing L-asparaginase on
6C3HED
lymphosarcoma, Nature, ~~~, 117-118
27
;-
Chang T.M.S.
(1971b),
Stabilization of enzymes
by
microencapsulation with a concentrated protein solution
or by microencapsulat i on followed by cross-l i nking wi th
glutaraldehyde,
Biochem. Biophys.
Res.
Commun., 1~,
1531-1536
Chang
T.M.S.
(1972),
~!:!:!f!~!!!!
Q~!!~,
Thomas Publisher, Spr i ngfield, Il lino is
Charles
C.
Chang
T.M.S.
(1973a),
Immobilization of enzymes,
adsorbents,
or both within semipermeable micro~apsules
(artifi.cial
cells)
for clinical
and
experillental
treatment of metabolite related disorders in D. Bergslla
(ed)
~!!~~!!:!~
Tg~!:!!e~ f~!!: QQ!!g~!üt.~l
!!!~~~~~~
(Birth
Defects: Original Article Series, Vol IX), Williams and
Wilkins Co., 66-76
Chang T.M.S. (l973b), L-asparaginase immobilized withJn
sellipermeab le
.. icrocapsules:
In vit ro and in v i VI)
stability, En?yme, 14, 95-104
Chang
T. M. S.
(1975) ,
Mi croencapsulated
adsorbent
helloper+"'.4s:on
for uremia,
intoxication,
and hepatic
failure, K!dney lot., 7, S387-S392
Chang T.M.S.
(1976a),
Microcapsule artificial kidney:
including updated preparative procedures and properties
Kidney, Int., lQ, S218-S224
Chang
T.M.S.
(l976b),
Biodegradable semipermeable
lIicrocapsules contain ing enzymes,
horllones,
vaccines,
and other biologicals, J. Bioeng., l, 25-32
Chang T.M.S.
(ed) (1977a),
~j9m~~i~~1 6~~li~~!19~~
l~~g~ilj~~~ ~~~~~~! ~~g frg!~jD!
Vol l,
Plenum
9f
Press,
New York
Chang T. M. S.
(ed) (l977b),
!!!QI!~ç!!ç:~! ~eel!ç:!!t.!.Q!!~
!m~Q~!!!~~g ~!!~~m~! ~!!g ~rQt.~!!!~ Vo)
II,
Qf
Plenum Press,
New York
Chang T.M.S. (1980), Artificial red b100d cells, Trans.
Amer. Soc. Art if. In tern. Organs, ~§, 354-357
Chang T.M.S.
(1984),
Artificial cells in Medicine and
biotechnology, Appl. Biochem. Biotech., 1Q, 5-24
Chang T.M.S. (1985), Artificial cells with regenerating
multienzyme systems, Meth. Enzymol., l!~, 195-203
-
Chang T.M.S.
(1987a), Applications of artificial cells
in medicine and bjotechno1ogy,
Int.
J.
Biomat.
Art.
Cells Art. Organs, 1.2, 1-20
28
(
Chang T.M.S.
(1987b),
Recycling of NAD(P) by multienzyme systema
immobilized by microencapsulation in
artificial cells, Meth. Enzymol., !~~, 67-82
Chang
T.M.S.
(1988),
Medical
applications
of
and
cells,
Meth.
immobil i zed protei ns,
enzymes,
Enzymol., 1~1, 444-457
Douay O.
and Kamoun P.P.
(1980),
Serotonin in
experimental histidinemia, Neurochem. Res., Q, 897-903
Duffner P.K.
and Cohen M.E.
(1975),
Infantile spasms
associated with histidinemia, Neurology, g§, 195-197
Raster R.A.
and Baker P.H. (1977), Nitrogen metabolism
tissue carnosine concentration and blood chemistry of
gravid swine fed graded levels of histidine,
J. Nutr.,
H!7., 120-125
Ergan F., Thomas D., and Chang T.M.S. (1984), Selection
and microencapsulation of a NADH-oxidizing bacterium
regeneration,
Appl.
Biochem.
and
its
use for
NAD
Biotech., lQ, 61-71
Ergan F.,
Thomas D.,
and Chang T.M.S.
(1987),
I •• obilization of
the "NADH oxidase" function of the
bacterium Leuconostoc mesenteroides in matrix ~Apported
microcapsules for
continuous cofactor
regeneration,
Ann. New York Acad. Sei., §Q!, 372-376
(1980), g~!!~t!~ M~i~QQ!!~ !!!~~~~~~, ~~!:!~
Qi~g!!Q~i~ ~!!~ ~!:~!!~i~! !!!~!f!i~, Elsevier/North Holland
Gal jaard H.
Biomedical Press, 406-414
Gatfield P.O.,
Knight R.M.,
Deveruz M.,
and Pozonyi
(1969), Histidinemia: Report of four new cases in
one family and the effect of low histidine diets,
Cano
Med. Assoc. J., !Q!, 465-469
J.P.
Ghadimi H.,
Partington M.W.,
and Hunter A.
(1961), A
familial disturbance of histidine metabolism, New Engl.
J. Med., ~§§, 221-224
Ghadimi H.,
Partington M.W.,
and Hunter A.
(1962),
lnborn error of histidine metabolism,
Pediatrics,
~~,
714-728
Ghadimi H. and Partington M.W. (1967), Salient features
of histidinemia, Amer. J. Dis. Child., 11~, 83-87
Ghadimi H.
(1974),
Histidinemia:
emerging clinical
picture,
in W.L.
Nyhan (ed),
H~!:!i~Q!~ Qi~Q!:g~!:! Qf
(
!~1~~ !~ig M~!~~~li~~l
~~g g~n~!l~
f~!!~!n~ ~f gllni~~l E~~!~~~i~~
~~!i~!lgn,
John Wiley and Sons, 265-292
29
ft
Ghadimi
H.
(1981),
Histidinemia:
behavior, Amer. J. Dis. Chi Id., 1~§,
biochemistry
210-211
and
Givot I.L., Sm\th T.A., and Abeles R.H. (1969), Studies
on the mechanism of action and the structure of
the
e1ectrophi1ic
center of histidine
ammonia-lyase,
J.
Biol. Chem., ~11, 6341-6353
Givot 1. J..
and Abeles R. H. (1970), Mammalian histidine
ammonia-lyase - in vivo inactivation and presence of an
electrophil1c center at the act1ve site,
J.
Biol.
Chem., ~~§, 3271-3273
Gordon N. (1970), De1ayed speech and histidinemia, Dev.
Med. Child. Neurol., 1~, 104-106
Greenberg D. M.
(1961) , Carbon catabolism of amino
aeids,
in D.M.
Greenberg
(ed),
M~.!~~.Q1i~
f~.!h~~.Y!
Vol II, Academic Press, 133-143
Gregcriadis G.,
Leathwood P.O., and Ryman B.E. (1971),
Enzyme entrapment in liposomes, FEBS Letters, 1~, 95-99
-
and Chang T.M.S. (1987), Conversion of ammonia
Gu K.F.
or urea into essential amino acids
(L-leucine,
Lvaline,
L-isoleucine)
using multienzyme systems
and
NADH-Dextran jmmobi1ized in artificiel cells,
Int.
J.
Biomat. Art. Cells Art. Organs, 1§, 297-304
Gu K.F.
and Chang
ketoglutarate
into
ammonium source using
NAD+
immobilized
artificial cells in a
363-368
T.M.S.
(1988), Conversion of
L-glutamic aCld with urea
as
multienzyme systems and Dextranby
microencapsulation
within
bioreactor, Biotech. Bioeng., ~~,
Hague R.V.
and Holton J.B.
(1971),
An
intravenous
histidine load test for the deteetion of heterozygotes
for histidinemia, Clin. Chim. Acta, ~~, 462-464
Hais r.M.
and Strych A.
(1967), Increase in urocanate
concentration
in human epidermis following insolaticn,
Experentia, ~~, 231-232
Hanson
K.R.
and Havir
E.A.
(1972),
elimination of ammonia,
in P.D. Boyer (~d)
Vol VII, 3rd ed., Academie Press, 75-166
Enzymatic
1h~ ~~~.Y~~!
Holcenberg J. S.
(1982),
Enzyme therapy:
problems and
solutions, Ann. Rev. Biochem. 1 §1, 795-812
-....
Holton J.B.,
Lewis
F.J.W.,
Biochemical
investigation of
Path., 11, 671-675
30
and Moore G.R.
(1964),
histidinemia,
J.
Clin.
(
Hug D.H.,
Roth O., and Hunter J. (1968), Regulation of
histidine catabolism by succinate in Pseudomonas putida
J. Bact., ~§, 396-402
lmamura 1., Watanabe T.,
Hase Y., Sakamoto Y., Fukada
Y.,
Yamamoto H.,
Tsuruhara T.,
and Wada H.
(1984),
Histamine metabolism in patients with histidinemia:
determination of urinary leve1s of histamine,
Nmethylhistamine,
imidazole
acetic
acid and
its
conjugHtes, J. Biochem., ~§, 1925-1929
Ito F.,
Aoki K.,
and Eto Y.
(1981),
Histidinemia:
biochemlcal parameters for diagnosis,
Amer.
J.
Dis.
Chi Id. , 1~§, 227-229
Jack T.R.
and
Zajic J.E.
(1977),
The enzymatic
conversion of
L-histidine to urocanic acid by whole
cells of Micrococcus luteus immobilized on carbodimide
activated carboxymethylcellulose, Biotech. Bioeng., 1~,
631-648
Kacser H.,
Bulf'ie1d G.,
and Wallace M.E.
Histidinemic mutant in the mouse, Nature, ~11,
Kacser H.
and Bulfield G.
Path. Bull., ~, 3-4
(
(1973),
77-79
(1977), Histidinemia, Comp.
Kacser H.,
Mya Mya K. 1
Duncker M. 1
Wright
A.F"
Bulfield G., Mclaren A. 1 and Lyon M. F. (1977), MaternaI
histidine
metabolism and its
effects
on
foetal
development in the Mouse, Nature, g§§, 262-266
Keipert P.
and Chang T.M.S. (1983), In vivo assessment
of pyridoxylated cross-linked
polyhemoglobin
as an
artificial red cell substitute in rats,
Trans.
Amer.
Soc. Artif. lntern. Organs, ~~, 329-333
Keipert P.
and Chang T.M.S. (1984), Preparation and in
vitro characteristics of a blood substitute based on
pyridoxylated polyhemoglobin,
Appl. Biochem. Biotech.,
lQ, 133-141
Kihara H.
Boggs D.E.,
Lassi la E. L.,
and Wright S.W.
(1968),
Histidinemia: studies on histidase activity in
the stratum corneum , Biochem. Med., ~, 243-250
j
Klee C.B. (1971) 1 Histidine ammonia-lyase (Pseudomonas)
Meth. Enzymo1., !L{~l, 69-73
Klein R.G.
and Halver J.E.
(1971),
Nutrition of
salmanoid
fishes:
arginine and histidine requirements
of chinook and colio salmon, J. Nutr., !QQr 1105-1110
(
31
i
L
Kopple J.O.
and Swendseid M.E.
(1975)1
Evjdence that
histidine is an essential amino acid in normal and
chronically uremie man, J. Clin. Invest., ~~, 881-891
Kopple J.O.,
Figueroa W.G., and Swendseid M.R. (1979),
Histidine,
an essential amino acid ln adult humans, in
R.
Kluthe and N.
Katz (eds) Mi~!igi~~= ~~!~~Qli~m,
glj~iE~l
~~P~E!~,
1h~r~p~~!iE
Q~~,
George Thieme
Publishers, 8-18
Kuroda V., rto M., Ogawa T., Takeda E., Toshima K., and
Miyao M.
(1979),
A new sensitive method for assay of
histidase in human skin and detection of heterozygotes
for histidinemia, Clin. Chim. Acta, ~§, 139-144
Kuroda V.,
Ogawa T.,
lto M.,
Watanabe T., Takeda E.,
Toshima K.,
and Miyao M.
(1980), Relationship between
skin histidase activity and blood histidine response to
histidine intake in patients with histidinemia, J.
Pediatr., ~1, 269-272
Kuroda V.,
Watanabe T., Ito M., Takeda E., Toshima K.,
and
Miyao
M.
(1985),
Genetic heterogenity
of
histidinemia detected by screening newborn infants in
Japan, Jpn. J. Human Genet., ~Q, 287-295
Howell R.R., Jacoby G.A., Seegmiller J.E.,
La Du B.N.,
and Zannoni V.G.
(1962),
The enzymatic defect in
histidinemia,
Biochem.
Biophys. Res. Commun., 7, 398402
La Du B.N.,
Howell R.R., Jacoby G.A., Seegmiller J.E.,
Sober E.K.,
Zannoni V.G., Canby J.P., and Ziegler L.K.
(1963), Clinical and biochemical studies on two cases
of histidinemia, Pediatrics, ~Q, 216-227
La Du B.N.
(1967), Histidinemia: current status, Amer.
J. Dis. Child., !!~, 88-92
La Du S.N.
(1971),
Histidine ammonia-lyase
(human
stratum corneum), Meth. Enzymol., 17i~1, 891-894
La Du B.N.
(1978), Histidinemia in J.B. Stanbury, J.B.
Wyngaarden and D.S.
Friedrickson (eda),
Th~ M~~~Q2!!~
~~~i~ QI l~h~ri!~g ~i!~~!~ 4th ed.,
McGraw-Hill,
317327
Lancet editorial (1974),
to treat?, Lancet, 719
Histidinemia:to treat or not
Lee S.C. and Harper A.E. (1971), Responses of mammalien
histidine catabolic enzymes to dietary and hormonal
treatments, Biochim. Biophys. Acta, g11, 135-145
32
(
Lessie T.G. and Neidhardt F.C.
(1967),
Formation and
operation
of the histidine degrading pathway
in
Pseudomonas aeruginosa, J. Bact., ~~, 1800-1810
Lyon I.C.T., Gardner H.J.M.,
and Veale A.M.O. (1974),
MaternaI histidinemia, Arch. Dis. Child. 4~, 581-583
Matsuda 1., Matsuo K.,
Endo F., Uehara 1., Nagata N.,
Jinno V., Chikazawa S., Miyakita T., and Miura H.
and
urine
(1982),
Skin
histidase
activity
with
formiminog1utamic
acid
(FIGLU)
in patients
histidinemia found by screening newborn infants, Cl in.
Chim. Acta, !!~, 319-328
May S. W.
and Li N.N.
(1972),
The immobi1ization of
urease
us ing 1 iqu id-s urfactant lIemb ranes,
B iochem.
Biophys. Res. Commun., 1L, 1179-1185
Mehler
A.H.
and Tabor H.
(1953),
Deamination
of
histidine to form urocanic acid in the liver,
J. Biol.
Che •. , ~Q!, 775-784
Mehler A.H.
(1957),
Academie Press, 332-339
!!ltrQgyç!:.üm
iQ
Mosbach K. and Mosbdch H. (1966), Entrapment of enzymes
and microorganisms in synthetic crosslinked polymers
and their applications in column techniques, Acta Chem.
Scand., gQ, 2807-2810
Muel1er P. and Rudin D.O.
(1968),
Resting and action
potentials in experimental bimolecular lipid membranes,
J. Theor. Biol., !~, 222-258
Nasset E. S. and Gatewood V. H. (1954), Nitrogen balance
and hemoglobin of adult rats fed amino acid diets
low
in L- and D-histidine, J. Nutr., Q~, 163-176
Neville B.G.R.,
Harris R.F.,
Stern D.J. and Stern J.
(1971),
MaternaI histidinemia,
Arch. Dis. Child., 1~,
119-121
Neville B.G.R., Bentovim A., Clay ton B.E., and Shepherd
J.
(1972), Histidinemia- study of
relation between
clinical and biological findings in 7 subjects, Arch.
Dis. Child., .17, 190-200
Peterkofsky
A.
(1962),
histidase:
amino-enzyme
reactions, J. Biol. Chem.,
(.
Mechanism of action
of
formation
and
partial
~~L, 787-795
Peterkofsky A.
and Mehler L. N.
(1963),
Inhibition of
Pseudomonas hist idase,
evidence for a metal
cofactor,
Biochim. Biophys. Acta, L~, 159-162
33
ft
-
Pieniazek D.,
Stecko E.,
Kubalaska J., Krassowska A.,
and Zychowicz K.
(1985), Metabolism of histidine in
children with speech abnormalities- histidase actlvity
in stratum corneum, Acta Med. Pol., ~§, 27-36
Popkin J.S.,
Clow C.L., Grove J.,
and Scriver C.R.
(1974) ,
Is herediatry histidinemia harllful?,
Lancet,
721-722
Poznansky M.J.
and Chang T.M.S.
(1974) Comparison of
the enzyme kineties and immunologieal
properties of
catalase immobilized by mieroencapsulation and catalase
in free solu t i on for
enzyme rep lacement,
B ioeh im.
Biophys. Acta, ~~1, 103-115
Poznansky M. J .
(1984) ,
Enzyme-albumi n polymerH: new
approaches to
the use of enzymes in Medicine, App.
Biochem. Biotech., !q, 41-56
Rai SO'1a V.
and Hyanek J.
(1986) , Speech d isorders
assoeiated with histidinemia and other
hereditary
disorders of amino acid metabolism, Folia Phoniat., ~~,
43-48
Rechler M. M.
( 1969) ,
The pur i ficat ion and characterization of L-Histidine aamonia-lyase (Pseudomonas), J.
Biol. Chem., ~11t 551-559
Rechler M.M.
and Tabor H.
(1971), Histidine ammonialyase (Pseudomonas), Meth. Enzymol., 11i~2, 63-69
Rosenberg L.R.
and Scriver C.R.
(1980),
Disorders of
amino acid metabolism, in P.K. Bondy and L.E. Rosenberg
(eds),
M~!~~Qli~
ÇQ~!~Ql
~~g Di~~~!~
8th ed, W.8.
Saunders Co., 651-655
Rosenthal A.M.
and Chang T.M.S.
(1980), Incorporation
of
lipid
and Na+-K+-ATPase
into
membranes
of
semipermeable microcapsules, J. Memb. Sei., Q, 329-338
Scott I.H.
(1981),
Factors control1ing the expressed
activity of histidine ammonia-lyase in
the epidermis
and
the resulting accumulation of urocanic aeid,
Bioehem. J., !~4, 829-838
Shu
C.D.
and Chang
T.M.S.
(1980),
Tyrosinase
immobilized within artificial cells for detoxifieation
in liver failure,
1. Preparation and in vitro studies,
lnt. J. Artif. Organs, ~, 287-291
Shu
C.D.
and Chang
T.M.S.
(1981),
Tyrosinase
immobilized within artificial cells for
detoxifieation
in liver failure,
II.
In vivo studies in fulminant
hepatic failure rats, Int. J. Artif. Organs, 1, 82-84
34
(
Siuchong R. and Chang T.M.S. (1974), In vivo effects of
intraperitoneally injected L-asparaginase solution and
L-asparaginase immobilized within semipermeable nylon
microcapsules with emphasis on blood L-asparaginase,
body L-asparaginase,
and plasma asparagine levels,
Enzyme, l~, 218-239
Snyderman S.E., Boyer A.,
Raitman E., Holt L.R., and
Prose Jr.,
P.H.
(1963),
The histidine requirement of
the infant, Pediatrics, ~!, 786-801
Snyderman
S.E.
(1979),
The histidine requirement of
Katz (eds),
Hi!!igi!!~=
Ih~r~p~~!j~ y!~, George
infant~,
in R. Kluthe and N.
M~!~~~lj!~,
glj~is~l !!p~s!~,
Thieme Publishers, 2-7
Spolter P.O.
and Oaldridge R.C.
(1963), Metabolism of
histidine- on the assay of enzymes in the rat liver, J.
Biol. Chem., ~~~, 2071-2074
Stanbury J.O., Wyngaarden J.B., and Friedrickson D.S.
(1978),
Inherited variation and metabolic abnormality,
in J.B. Stanbury, J.B. Wyngaarden,and O.S. Friedrickson
(eds), lb~ M~!~QQliS ~~!i! ~f l~~~ri!~~ ~i~~~!~ 4th ed.
McGraw-Hill, 2-33
«
Tabor
H.
(1955),
Degradation of histidine,
in
McElroy and B. Glass (eds),
§Ymp~~j~~ Q~ !~i~Q
M~!~~Qlj!m, John Hopkins Press, 373-390
Tabor
H.
and Mehler A.H.
(1955),
urocanase, Meth. Enzymol., ~, 228-231
Hist.idase
W.D.
!sig
and
Tada K., Ta teda H., Arashima S., Sakai K., Ki tagawa T.,
Aoki K., Suwa S.,
Kawamura M., Oura T., Takesada M.,
Kuroda Y.,
Yamashita F.,
Matsuda 1., and Narusa H.
(1982),
Intellectual development
in patients with
untreated histidinemia, J. Pediatr., l~l, 562-563
Thalammar O.
(1972), Austria newborn screening program
for inborn errora of metabolism, Abstract from meeting
of European Society of Pedia tric Research,
Heid1eberg,
West. Germany
Thomas G. H.
l~!!!
fQ!
and Howell R. R.
(1973),
g~~~!j~ M~!~QQljs ~j!~~!~§,
~~!~~~~H!
~~[~~!!!.!!g
Yearbook Medical
Publishers, 62-66
Van Arsdell P.P. and BealI G.N. (1960), The metabolism
and functions of histamine, Arch. lnt. Med., 1~§, 714
.(
Wadman S.K.
and deBree P.K.
(1979),
Histidinemia,
symptom or di sease?,
in R.
KI uthe and N.
Katz (eds),
Hi!!igj!!~= M~!~~Qli§~,
y!~,
Çlini~~l
!§p~~!!,
George Thieme Publishers, 43-51
35
1h~!~p~y!i~
--
--------------------------
Waisman
H.A.
(1967),
Variation
in clinical and
laboratory findings in histidinemia,
Amer.
J.
Dis.
Child., !!~, 93-94
Whitman R.D., Maher B.A., and Abeles R. (1977), Deficit
in discrimination and Maze learning resulting from
maternaI histidinemia in rats,
J.
Abnorm. Psych., ~§,
659-661
Williams V.R.
and Hiroms J.M. (1967), Reversibility of
the "Irreversible" histidine ammonia-lyase
reaction,
Biochim. Biophys. Acta, !~~, 214-216
Wisek W. J.
(1984) , An update of concepts of essent i al
amino acids, Ann. Rev. Nutr., 1, 137-155
Wixom R.L. and Anderson H.L. (1979), Histidine- 1imited
synthesis in normal adult man, in R. Kluthe and N. Katz
(eds),
Hl!!igi~~= M~!~~~li!~,
gli~l~~l
~!P~E!!,
!h~r~p~~!19 y!~, George Thieme Publishers, 19-33
Wolfe E.A.
and Chang T.M.S.
(1987),
Orally ingested
microencapsulated urease and an adsorbent,
zirconium
phosphate to remove urea in kidney failure,
Int.
J.
Art. Organs, lQ, 43-48
....
Wong H.
and Chang T.M.S.
(1986), Bioartificial li ver:
imp1anted artificial
ce1ls microencapsu1ated living
hepatocytes increase survival of liver failure rats,
Int. J. Artif. Organs, ~, 335-336
Wong H.
and Chang T.M.S.
(1988),
Viability and
regeneration of artificial cells microencapsulated rat
hepatocytes xenograft
transp lant in Mice,
Int.
J.
Biomat. Art. Cells Art. Organs, !§, 731-741
Wood D.A. and Whateley T. (1982), A study of enzyme and
protein microencapsulation- snme factors affecting the
low apparent
enzymic activity yields,
J.
Pharm.
Pharmacol., ~1, 552-557
Wright
A. F.,
Bulfield G.,
Arfin S. M.
and Kacser H.
(1982),
Comparison of the properties of histidine
ammonia-Iyase
in normal and histidinemic mutant mice,
Biochem. Genet., ~Q, 245-262
Yamamoto K.,
Sato T.,
Tosa T., and Chibata 1. (1974),
Continuous production of urocanic acid by immobilized
Achromobacter
liquidum cells,
Biotech. Bioeng.,
!§,
1601-1610
-
Yokoya S., Tokuhiro E., Suwa S., and Maesaka H. (1983),
Measurement of the skin urocanic acid content in normal
J. Pediatr., !1Q, 330and histidinemic infants, Eur.
332
36
('
Yu Y.T. and Chang T.M.S.
(1981),
Ultrathin lipidpolymer membrane microcapsules containing mu1tienzymes,
cofactors,
and
substrates for
multistep
enzyme
reactions, FEBS Letters, 1~§, 94-96
Yu Y.T. and Chang T.M.S.
(1982),
Multienzymes and
cofactors immobilized within lipid polyamide membrane
microcapsu1es for sequential substrate
conversion,
Enzyme Eng., §, 163-164
Yuan Z.Y.
and Chang T.M.S.
(1986), Rat microsomes and
cytoso1 immobilized by microencapsu1ation in artificial
cells, Int. J. Artif. Organs, ~, 63-68
Zachary Dyme 1., Horowitz S.J.,
Bacchus B., and Kerr
O.S.
(1983),
Histidinemia- a case of resolution of
myoclonic seizures after treatment with a low histidine
diet, Amer. J. Dis. Child, 1~1, 256-258
Zannoni V. G.
and La Du B. N.
(1963) , Determinat ion of
histidine deaminase in human stratum corneum and its
absence in histidinemia, Biochem. J., ~~, 160-163
(~
(
37
..
II. METBODS - APPINDIX A
38
APPENDIX A
(
~~~ig ~t!ngi2!~
Artificial
Qf
cells
~t~2~t~~iQn
~t~!f!gi§! Q~!!!:
are made according to the
updated
Both control artificial
method of Chang (1987).
and
Qf
cells
histidase loaded artificial cells are prepared
by
the same procedure.
There are a veriety of Methodologies available for
preparation
depend
of artificial cells.
on
the
artificial cells.
to
of
the
and on the type of polymer
to make artificial cells
polymerization
methodologies
preparation
make the artificial cell membrane.
methods
(
principle
These
and
In
include
interfacial
this
of
employed
The more common
interfacial
precipitation.
Interfacial precipitation is also known as
coacervation.
the
interfacial
research work artificial
cell
membranes
were made of the polymer
cellulose
nitrate
prepal'ed
from
Cellulose
nitrate
artificial
Collodion
cells
precipitation
are
U.S.P.
prepared
procedure.
by
the
The principle
interfacial
of
preparing
artificial cells by this method using cellulose nitrate
as
the
membrane had been outlined previously
(Chang.
1972) and is as follows:
(1) Two phases.
one aqueous containing the contents to
be encapsulated,
the other an immiscible organic phase.
are emulsi fied.
The resulting emulsification
produces
aqueous microdroplets in an organic phase.
(2)
The material which will make up
membrane.
the
in this case cellulose nitrate.
39
microcapsule
is added to
r
r,
(
1t
the emulsion. The membrane material precipitates at the
interface
of
each
microcapsule.
The
aqueous
microdroplet
to
form
microcapsules are now suspended
a
in
the organic phase.
(3) Using a surfactant, Tween 20,
washed
an
the microcapsules are
and then transferred from the organic phase
aqueous
one.
The microcapsules are stored
in
to
an
aqueous medium for later use.
(1) Remoglobin solution :
(2X
dialyzed,
Crystallized,
Chemical
Co.,
Missouri,
of
O.lM
Tris
mIs
buffer
8.75.
pH
desired
pR
15 grams of bovine hemoglobin
lyophilized
from
U.S.A.) was dissolved in 100
(Tris-hydroxymethylaminomethane)
The Tris buffer was brought
with
Sigma
IN
HCI.
Dissolution
of
to
the
Rb
is
accomplished by dissolving the Hb solution in a
shaker
for
it
dissolved,
paper
2
hrs
at 4°C.
After
Rb
has
is filtered through Whatman #42
to remove further impurities.
requires
the
rotary
at least 5 hrs,
been
filter
This step usually
and is carried out
at
4°C.
After the Rb solution has been completely filtered, the
concentration
of Rb is rechecked.
Hb concentration is
determined by the cyanomethmoglobin method.
concentration
12 %
dissolved
a
Rb solution of at Ieast 10%.
in
l
11-
The preparation of stable microcapsules
this
Rb
40
i
final
of the Hb solution is usually about
( gm s / d 1) .
requires
The
solution
at
Histidase is
the
desired
concentration for encapsulation.
(2)
Cellulose
nitrate
solution
Collodion
U.S.P.
(Mallinckrodt Inc.) is left to evaporate on a stainless
steel tray. The tray had been previously treated with a
Zerostat
anti-static
nitrate
sheets
remove
The
gun.
are
resulting
cellulose
dried for 24 hrs in a
aIl ether and alcohol.
The
beaker
cellulose
to
nitrate
solution is prepared as follows. 4 gms of the cellulose
nitrate
mIs
52.5
sheet is placed in an Rrlenmeyer
of absolute ethanol (Commercial Alcohols Ltd.) and
mIs of anhydrous ether (BDH Chemicals) are
into the flask.
solution
(
17.5
flask.
After
stirred
the
another
second.
ether
seperatory
is
and
cellulose
funnel.
the
nitrate.
dissolved,
30 mIs of ant.ydrous ether is added
anhydrous
rate,
to dissolve the
cellulose nitrate is completely
covered
foil
The flask is foil stoppered,
added
t hrolLgh
This
a
amount
of
drop
per
added at the rate of 1
It is important that the ether is added at this
because
otherwise
precipitate. The final
nitrate
the cellulose
concentration
solution should be 4gms/dl.
cellulose
nitrate
of the cellulose
The
solution
of
nitrate is placed in either a dark bottle or
an aluminum foil covered bottie for atorage.
regarding
May
preparation
AlI
and usage of cellulose
work
nitrate
solution is done in a weIl ventilated fumehood.
(3)
(
Water
prepared
Saturated Ether
H20 saturated
ether
by mixing 500 mIs of anhydrous diethyl
is
ether
(BDH Chemicals) with 100 mIs of double deionized H20 in
41
-
•
l
---------------
-
a
glass
1000
stoppered
contents
ml
seperatory
funnel.
of the funnel are shaken for 15 minutes
intermittent
release of gas.
bottom phase which consists of water is discarded.
The
to
phase which consists of water saturated
collected
The
are allowed
H20 and
The
top
ether,
with
seperate.
H20
saturated
The two phases,
The
and
water
ether
stored in a dark bottle for later
saturated
ether solution
should
is
use.
be
used
within a week.
(4)
n-Butyl
Benzoat3
n-Butyl
benzoate
(Aldrich
Chemical Co.) is used as supplied by the supplier.
(5)
50' Tween 20 solution:
prepared by
Co.)
50% Tween 20 solution
diluting 500 mIs of Tween 20 (Atlas Powder
500 mIs of double deionized H20.
with
Tween 20 solution is used as an emulsifier to
artificial
is
This
50'
transfer
cells from the organic phase to the aqueous
phase.
(6)
l ' Tween 20 solution:
prepared
100%
The 1% Tween 20 solution is
in O.lM Tris-HCl buffer (pH 9.2).
Tween
20
buffer (pH 9.2).
wes diluted in 990 mls
of
10 mIs
O.lM
The 1% Tween 20 solution 1s used
of
Tris
for
washing the artificial cells to ensure that aIl organic
membrane
is
is also used as a storage medium for
the
residue
present
removed.
It
on
the
microcapsule
microcapsules to prevent aggregation.
42
~~i~!!~Q
(
fQ[
erQ~~g~[~
Ç~!!!:
The
standard procedure for
taken
from
(1987).
making artificial cells is
updated version
published
Chang
The following is a description of how a
batch
150 ml beaker containing a 4 cm stirring
To a
units
histidase (BC 4.3.1.3) (Sigma Chemical
of
Mis s II.) uri ,
H20 saturated ether containing 1% Span 85
emulsifying
a
agent.
speed
equi val ent
(v/v)
is
Span 85 (Atlas Powder Co.) is
an
The
contents
of 5.
setting
500-550
to
cellulose
nitrate
continued
for
are
emulsified
by
This
rpms.
stirring
speed
25
of
Then
mIs
solution is added and
another 60
seconds.
stirring
These
steps
is
the
is
are
carried out at ambient room temperature.
The beaker is
covered
and placed at
with a watchglass and parafilm,
for 45 minutes.
4°C
as
sett le
cells
precipitates
solution
During this time
the
around
cellulose
each
is then decsnted.
is
step
stirred
is
artificial
minutes.
the
artificial
nitrate
microdroplet.
The
and the
for 30 sec st a speed setting of
done
cells
at
ambient
polymer
organic
25 mIs of n-butyl benzoate
containing l ' Span 85 (v/v) is added,
(
\
,,
Co.,
stirring for 5 sec on the Fisher Jumbo Magnetic Stirrer
(
\
A further 25 mIs
U • S . A. ) / ml o f Hb i sad d e d .
added to the beaker.
at
~
2.5 mIs of the Hemoglobin solution containing 300
bar,
of
d[t!f!~!~!
by
is made.
the
Qf
ih~ f~~e~[~i!Qll
room
mixture
5.
This
temperature.
The
are placed uncovered at 4°C
for
30
During this period the but yI benzoate acts as
43
"
,
.
a
plasticizer on the cellulose nitrate
membrane.
The
but yI benzoate is aspirated off the microcapsules.
The
artificial
cells are stirred with 25 mIs of 50%
Tween
20 at a speed setting of 7 (1500-1700 rpms) for 30 sec.
Stirring is slowed to speed 5,
added
to the mixture.
and 25 mIs of water
is
Stirring is allowed to continue
for another 30 sec. The mixture of artificial cells and
Tween 20 is placed in centrifuge tubes and
in
IEC universal
the
the
sediment
aspirated off.
of
mIs
1%
centrifuge at 500-600
microcapsules.
The
rpms
supernatant
removed.
This
This
is
centrifuged
9.2
vitro
is repeateC: 7
the
times.
The
for use in studies of enzyme kinetics
histidine
storage
for
artificial
is
and
artificial cells are stored at 4°C in O.lM Tris
pH
to
The artificial cells are washed with 50
Tween 20.
supernatant
centrifuged
depletion
stability
studies.
~tudies
For
buffer
and
long
histidase
in
term
loaded
cells are stored in 1% Tween 20 - O.lM Tris
buffer pH 9.2.
Chang T.M.S.
(1972), ~!:t!f!~i~! Q~!!~,
Thomas Publisher, Springfield, Illinois
Chang T. M. S.
Charles
(1987), Meth. Enzymol., !;!§, 67-82
44
C.
(
III.
MITHODS - APPINDIX B
(
45
APPBNDIX B
Hi!!ig~~~ §Qly!iQ~ ~!§~y
The
enzyme
purchased
L-Histidine ammonia-lyase (histidase)
from
the
Sigma
Chemical
Co.
was
(Missouri,
U.S.A.). Histidase (EC 4.3.1.3) received from Sigma was
e~~~gQ~Qrr~~
derived from
f!~Qr~~~~rr~.
It was
supplied
as a partial1y purified soluble preparation.
activity
units
specified
per
deaminate
1
nanomole of
The
mg
activity.
500-800
by Sigma was stated to be
protein.
nanomole
ul~canic
supplier
Histidase
One unit
of
histidase
of L- histidine
to
will
prod ce
1
acid per minute at pH 9.0 at 25°C.
provided this
Before
definition
attempting
any
of
histidase
encapsulation
of
histidase, we checked histidase activity by assaying it
with the protocol set by the supplier.
Tabor
(1955) set forth the principle of the
assay.
Histidase
urocanic acid.
by
has
deaminates
histidine
to
produce
Thus histidase activity can be
assayed
moniteriag urocanic acid production.
an
(Tabor,
absorbance
1955).
absorbance
urocanic
change
in
1971).
The
peak at a
Histidine
above 240
peaks
acid
determined
histidase
production
by
Urocanic acid
wavelength
spectra
nm
from
of
277
does not show
(Tabor,
1955).
any
Thus
can
be
moniterillg spectrophotometrically
the
absorbance at 277 nm
rate
of
histidine
nm
(Rechler
and
urocanlc acid production
Tabor,
is
an
evaluation of histidase activity. The rate of change of
absorbance
at
277
nm is
46
converted
into
amount
of
(
urocanic
acid
produced by using the molar
coeffecient of urocanic acid at 277 nm.
value
of
Rech 1er
this coeffecient
and
histidase
Tabor.
in
(Barman.
activity
in
UV/VIS Spectrophotometer.
histidase
assay
microencapsulated
1969;
experiments
solution or histidase loaded
spectrophotometrlcally
the
The literature
18800
In a 11
1971) .
histidase
cells.
is
absorption
wi th
artificial
was
assayed
the perkin Elmer Lambda
4B
One important difference
in
for
histidase
histidase
is
the
solution
and
following.
For
histidase solution assays absorbance recordings at
nm
were
carried
out
continuously.
The
277
assay
of
histidase solution was carried out in a cuvette reactor
(
placed
in
regulated
cuvette
histidase
out
at
the spectrophotometer
assays
chamber.
with
For
a
temperature
microencapsulated
absorbance measurements were carried
intervals.
The
assay
of
microencapsulated
histidase was carried out in a Lab-Line
Environ
Shaker with temperature regulation features.
time
for
At preset
intervals an aliquot of supernatant was
absorbance
readings
at 277 nm
in
Orbit
the
obtained
spectro-
photometer.
This
where
appendix
recorded
histidase solution was assayed in
photometer.
(
includes sample sets of
The
contents
of the assay
solution
wer~
represent
their final concentration.
the
following.
47
the
The
of
spectrohistidase
values
ImM
data
denoted
MgC12.
2mM
reduced glutathione pH 9.2 (GSH),
500uM L-histidine pH
9.2, O.IM Tris buffer pH 9.2, and 200 uL histidase (400
units/ml).
The
final total volume was
containers
for
the
cuvettes
histidase
(Montreal, Quebec).
The
first
This
reaction
printout
increase
reaction
histidine.
mIs.
were
cm lighl path) from Scie-Bec
(1
Inc.
linear
3.0
data chart (Figure
contained both
quartz
Electronics
shows
BI)
in absorbance at wavelength
mixture
The
a
277
nm.
histidase
and
The data printout shows the results'of this
experiment. The histidase reactions were carried out in
triplicate,
form.
the
and the results are presented in overlayed
The vertical axis denotes the
bottom
point
denoting
initiation of the reaction.
the
absorbance,
absorbance
with
before
The top point denotes
the
absorbance
at the end of the time period during
absorbance
was monitered.
The horizontal axis denotes
period
which
the
time
during
the
which
react ion
was
monitered.
The information at the bottom describes the
length
the lag period.
of
In this period
commencement
of the histidase reaction,
measurements
are
variables
is
measured,
graph
data
reaction
and
The
and
rates
calculation
absorbance
calculated
numbers in the upper righl part
individual
denote
spectrophotometer.
cuvettes.
The
These
the
no absorbance
represent the intervals at which
respectively.
the
taken.
after
cells
cells contained the
of
of
the
reactor
The histidase reaction rates were calculated
48
r!,~[~
~!~ Data recording of histidase solution assay.
Absorbance 277 nm is plotted as a function of time.
Histidase and histidine are included in the assay.
49
LAIIE:DH 4E: l'wu'"rD 001 ~~7 ülHI SLT
111[':
F:SP ~: FHCi
1 13 Hi 10 00 Il!II'CELL ::
r:
............
,,~_.
..-::::::::::::
. ='~....""~
.................
..•.
_.--_..--_.-_.--_.- .-.-..
.........-...-
l
..-_.
.-_.-
l:~::.:::::::::::::=::=:-------·--....~ ;;.--:~:'
t:'
- -.-.- .-.- - -. -~---
.......-.....
----
H
~
L:-<!;:
..
(l'I)
Ct
:
\.11.1
EII(:: 10 :JO [1,..7,..:
ü
~I:I
M
.
:
"
,) 1:
::
.:'
{
using
The
the Kinetics software provided by
Perkin-Elmer.
Kinetics software calculates the rate of change of
absorbance.
reaction
These
rates are converted into
using
velocities
the
histidase
molar
absorption
coeffecient of urocanic acid.
The second printout (Figure 82) show the results of the
control
histidine
(Figure
substrate
studies where histidase was not added
substrate
83)
is
solution.
another
The
control
to
third
study
printout
where
is not included in the
histidine
containing histidase.
the
the
reaction
The important point worth noting
is that in both control studies there is no increase in
absorbance at 277 nm.
(
A preliminary
effectiveness
assessment was made
of
of
the
potential
aicroencapsulated histidase for
the
depletion of histidine from the extracellular fluid
histidinemia.
histidase
reactor.
Histidine depletion by microencapsulated
was
studied
Various
volume
in vitro in
ratios of
a
stirred-batch
histidase
artificiel cells to total substrate solution
were
tested.
concentration
(
in
In
of
these
in
histidine
vitro
was
400uM.
level of
for
is
This
level
(vol/vol)
experiments,
corresponds to the elevated plasma
histidinemia.
10aded
used
This
the
level
histidine
as
the
extracellular fluid concentration for histidine.
The
three volu.e ratios of histidase loaded artificial
50
[i,y~~ ~~~ Data recording of histidase solution
assay.
Histidase is Dot included in the reaction.
Absorbance
277 nm is p10tted as a function of time.
)
51
(
ri
Li1tl2DH H fil- U ,ID 00 1 2~~ Oiltl ';Li
1 ilDE
;: ': F : FH C i l (1 TC ï 10 0Ü t1 ~ ;1 CEL L 3
'l'Et
H
.
H
,",
1
(
",
"
rL
/0 000
H
1(1
(
ft
(1(;'
)
~igYr~ ~~~ Data recording of histidase solution
asssy.
Histidine
is not included in the resction.
Absorbance
277 nm is plotted as a function of tiae.
)
52
(
(
FIGURE B3
(
ft
-
~
.. .
r. : i
000
~
1 11 [: E
- .-
r NI.
t1
1
H
- r
.-
1
C
OÜÜ
10 '.11.1
H
1,)
~
.' .)'(1
LHG=
~)
O\) E:1C = 1(1 (t Ü
O~T,.=
~
0 .
"'.
r' l
_ '
•
:
~)
~)
\)'
:1 ~:
{
cells
to total substrate solution
were
1:100.
1:50.
1:25. Controls consisted of artificial cells containing
no histjdase.
show
the
ratios
Figures 84.
results
85, and 86 of this appendix
obtained with
respectively.
these
three
The initial histidine
volume
level
of
6.21 mg/dl ls equal to 400uM.
The
results of histidase loaded artificial cells at
volume
ratio of 1:100 are presented
ls clear
in Figure 84.
It
that with this ratio histidine conversion
by
microencapsulated
After
noticeable.
(
histidase
ls
and
both low
slow.
24 hours the drop in histidine levels is
required
low~r
a
Wlth
for
volume
this
ratio
histidase loaded
histidine
levels
120
hours
are
cells
artificial
by 25% from
hardly
to
original
their
value.
The
results of histidase loaded artificial cells in
volume
ratio of 1:50 are presented in
doubling
the
amount
of
microcapsules
Figure
in
85.
histidine
depletion
is also
doubled.
the rate
With
volume ratio histidine levels have fallen by a
after approximately 48 hours.
By
substrate
solution to achieve the volume ratio of 1:50.
of
a
this
quarter
After 72 hours histidase
loaded artificial cells had lowered histidine levels to
65% of their original value.
The
final volume ratio of histidase loaded
cells
(
results
to
substrate
obtained
solution
tested
was
artificial
1:25.
with this ratio are shown in
53
The
Figure
)
~!gy~~
~1~
In vitro depletion
of
L-histidine:
Artificial cells in
a volume ratio of 1:100
to
substrate solution.
Rate of decrease of histidine
concentration (mg/dl) is plotted as a function of time.
54
l
FIGURE B4
l'
L
r.,
-!_____ .-
6.0 ~_~~~i:_====Q-2
-!
Q----------- 2 - - - - - 2 - - - - - 2
...
--
...
5.0
C
"C
Cl
E
-----!------
-.
!
4.0
w
z
0
lCI}
3.0
.....
.....
.!J
.......
hlstldase loaded artificial cells
-.-
control artificial cells
-0-
2.0
.
~.
1.0
1
O~I-----------r--------~----------~--------~----------~
120
48
96
24
72
o
TIME (hours)
6.0
--
l~
5.0 . .
____-!-------2-2
!------- .
2------Q
...
"0
------
2----__
Cl
E
4.0
LU
C
fCfJ
.!J
3.0
histld:J.se loaded artificial cells
-.-
contrai artificial cells
-0-
2.0
(
1.0 -
O~I-----------r----------.---------~----------~----------~-
a
24
48
72
TIME (hours)
(
96
120
~)
)
E!lnH::~
!!Q~
In
vitro
depletion
of
l,-histidine:
Artificial cells in a volume ratio of 1:50 to substrate
solution.
Rate of decrease of histidine concentration
(mg/dl) is plotted as a function of Ume.
)
55
(
(
FIGURE B5
(
6.0
-
5.0
~
.....
Cl
E
-
4.0
C
1C/J
3.0
~
~~
2 - - - - - - - - -_ _ _ __
2
!--------- ---------~
2
•...
UJ
Z
-
control artificial cells
J:
......
1
...J
histidase loaded artificial cells
--e----0--
2.0
....
~
1.0
a ;-----~----~----r---~~--~----~----~----,_----~
24
40
48
5ô
64
72
16
32
o
8
TIME (heurs)
B6.
(
After
24
hours lIicroencapsulated
histidase
had
lowered histidine levels by 40'. This volume ratio gave
the Most satisfactory results.
study
The histidine depletion
for this volume ratio was followed for a
of 24 hours.
period
As will be discussed in the next section,
these results represent in vitro simulation of rate
depletion
of
Histidine
in the body is distributed in extracellular
and
histidine
intracellular
concentrations.
required
if
A
the
from
fluid
extracellular
of
compartments
multicompartmental
purpose
is
to
at
different
model
study
fI u id.
will
total
be
body
depletion of histidine.
(
Bar.an T. E.
(ed)
(1969) ,
Springer-Verlag, 799-800
Rechler
l.7.L~l,
M.M.
63-69
and Tabor
H.
~n!~~~
(1971),
H~m~~QQk
Meth.
Vol
II,
Enzymol.,
Tabor H.
(1955),
Degradation of Histidine in w.n.
McElroy
and B.
Glass (eds). §X~p~~iy~ ~E !~iE~ !~ig
~~!~~~li!~. John Hopkins Press. 373-390
(
56
.
)
~
}
Eigy[~
~~L
In vitro depletion
of
L-histidine:
Artificial cells in a volume ratio of 1:25 to substrate
solution.
Rate of decrease of histidine concentration
(mg/dl) ls plotted as a function of time.
57
(~
FIGURE B6
(
....
......
6.0
-
k.l~"2""2"'2-2""2
.......a
""a
.L. ....a
2
-'-..........
5.0
"C
.......
0)
E
......
-
4.0
-•
LlJ
~
c
-
1-
3.0
en
l
...
~
",..
1
-l
histidase loaded artificial ce!ls
•
control artificial cells
0
2.0
1.0
O~----~-----r----~-----.-----.-----.---
a
4
8
12
T1ME (hours)
16
20
24
(
IV. CHARACTIRIZATION
IMMOBILIZID
(
CILLS:
or
L-HISTIDINI
AMMONIA-LYASI
BY MICROINCAPSULATION IN
PRIPARATION,
ARTlrlCIAL
IINITICS, STABILITY, AND IN
VITRO DIPLETION or HISTIDINE
(Manuscript Submitted for Publication)
{
58
-CBARACTIRIZATION or L-BISTIDINI ANNONIALYASI
IN
IMMOBILIZID BY MICROINCAPSULATION
ARTlrlCIAL
IINITICS,
CILLS:
STABILITY,
PIlIPARATION,
AND
IN
VITRO
BIPLITION or HISTIDINI
......
Rajesh KHANNA and
T.M.S. CHANG
& Organs Research Centre
Artificial Cells
Faculty of Medicine
McGill University
3655 Drummond Street
Montreal, Quebec, CANADA, H3G IY6
59
ABSTRACT
(
L-histidine
within
ammonia-lyase (histidase) was encapsulated
cellulose
kinetic
parameters were
the
of
50~
activity
of
and
its
Microencapsulated
evaluated.
had an apparent activity
histidase
ce~ls,
nitrate artificial
of
histidase
approximately
in
solution.
Encapsulation did not alter the KM of histidase. The KM
of
histidase
solution and the
KMapparent
of
micro-
encapsulated histidase were both 20mM. Encapsulation of
histidase
activity
37°C
resulted in increased stability of enzymatic
at storage temperatures of 4°C and
histidase
activity
(
after
microencapsulated
after 15 days.
original
50~
solution reached
days
9.5
histidase
of
of
37°C.
its
original
storage,
reached
the
At
while
same
level
At 4°C histidase solution had 63% of its
activity
after
21 days
95~.
encapsu1ated histidase had
of
storage,
while
In vitro experiments to
evaluate the feasibi1ity of microencapsulated histidase
for possible experimental therapy in histidinemia
carried
out.
effectiveness
histidine.
loaded
of
A
the
Three different volume ratios of
histidase
cells
ratio
of
after
histidine
depletion
40~
evaluated
depleting
depletion
allowed
experiments
encapsulated histidase in
artificia1
tested.
(
These
were
to substrate
1:100
120 hours.
after
allowed
solution
25%
histidine
A 1:50 ratio allowed
72
hours.
A
1:25
histidine depletion after 24 hours.
60
were
35%
ratio
._---------------------------------------------------~
INTRODUCTION
The
enzymatic defect in the inborn error of metabolism
histidinemia,
lies in the enzyme L-histidine
lyase
(Ee 4.3.1.3) (1).
step
of
the
ammonia-
Histidase catalyzes the rirst
deamination
pathway
of
histidine
metabolism. It deaminates histidine to produce urocanic
acid.
In
Histidine
histidinemia histidine accumulates in
metabol ism
and
transamination
becomes
shi fted
decarboxylation
vivo.
towards
with
increased
production of imidazole Metabolites (2,3) and histamine
(4)
respectively.
patients
can
defects,
subt le
The
manifest
symptoms
that
clinically
mental
histidinemic
include
retardation,
speech
other
and
behavioral abnormalities (2,3,5,6).
Treatment
of
histidinemia has centered
restriction therapy.
on
substrate
The histidinemic patient
reduces
his intake of histidine to the minimal required levels.
In
most
of the reported cases
of
histidine
dietary
therapy
(2,3,7,8),
reduced,
however there was no clin i ca 1 improvement.
a
few
cases
blood
of histidine
L-histidine
dietary
levels
therapy
were
In
clinical
improvement in histidinemics was noted (9).
An
alternative
errors
type
approach for the treatment
of metabolism is enzyme
of
defective
therapy
one.
replacement.
the functional enzyme
Certain
problems
implementation of enzyme therapy (10).
61
of
inborn
In
replaces
arise
this
the
with
A novel solution
to
(
these problems involves the immobilization
desired
cells
of
enzyme by microencapsulation within artificial
(11).
Microencapsulation of the enzyme prevents
it from participating in immunological reactions
~nzymes
Microencapsulated
in
the
experimental
therapy
acatalsemia
(12,13),
asparagine
dependent
have been used
for inborn
(10).
successfully
errors
such
phenylketonuria (14,15),
substrate
tumors
as
for L-
(16,17),
for
liver failure (18), and for kidney failure (19).
In
this
report
encapsulated
histidase
the
results
histidase
derived
from
are
of
studies
presented.
regarding
Commercial
f~~~4~~~~~~ flY~r~~s~E!
can be
successfully microencapsulated within cellulose nitrate
artificial
cells.
microencapsulated
The in vitro storage
histidase is assessed,
with the stabillty of the free enzyme.
vitro
stability
depletion
studies
of
of
and compared
Preliminary
L-histidine
in
with
microencapsulated histidase are reported.
MATBRIALS AND MITRODS
fr~F~r~!i~~
Qf
Artificial
cells
~r!ifisi~l Ç~11!:
were
made according to the
method
of Chang,
described elsewhere (20).
loaded
and control artificial cells were
(
purified
contained
Hb.
Histidase
prepared
the same procedure. Control artificial cells
only
updated
contained
Histidase loaded artificial
histidase (EC 4.3.1.3) (Sigma Chemical
Missouri, U.S.A.).
62
.
by
cells
Co.,
......
Histidase
loaded
artifical
concentration
of 300
bovine
The
Hb.
histidase
is
a
cells
activity
assay
The
for
modification of Wood
Montreal,
a
starting
of histidase/ml of purified
~nits
procedure (21). Quartz cuvettes
Inc.,
had
encapsulated
and
Whateley's
(Scie-Bec
Electronics
Quebec) were used as assay
contents of the assay were lmM MgC12,
reactors.
2mM reduced
glutathione (GSH) pH 9.2 (Sigma Chemical Co., Missouri,
U.S.A.),
cells,
0.1 ml of packed histidase loaded
O.lM
Chemica1
Tris
Co. ,
buffer pH 9.2.
Missouri,
artificial
L-histidine
(Sigma
U.S.A.) concentrations
varied from ImM to 10mM for kinetic analysis.
were
The total
volume of the reactor was 3.0 mIs.
The assay for histidase activity involves measurlng the
increase
in absorbance at wavelength 277 nm (22).
The
molar absorption coeffecient of urocanic acid at 277 nm
is reported in the literature as 18800 (23,24).
Cuvette
reactors
histidine
loss
24°C,
minus
are sealed with cuvette caps to prevent
of
placing
containing the assay contents
volume.
the
Histidase activity is
reactors at ambient room
for 30 min.
any
maximized
by
temperature
of
Histidine is added to the reactors.
Cuvettes are recapped and resealed.
The absorbance
at
wavelength 277 nm was determined using the Perkin Elmer
Lambda 4B UV/VIS spectropholometer. Reactors are placed
in a Lab-Line Environ Orbit Shaker.
The
shaking
speed is 80 rpm.
63
The temperature ls
At
preset
time
(
intervals
reactors
absorbance
returned
are removed from the
readings
to
the
are recorded.
AlI
shaker.
The
kinetic
shaker,
and
cuvettes
are
analysis
eucapsu]ated hiptidase was done in triplicate.
were
done
in the same
except
manner,
of
Controls
thet
control
artificial cells containing no histidase were used.
Tests
was
for
leakage of histidase from artificial
also
enalysis
carried out as follows.
ls
eentrifuged
the
eompleted,
After
assey
the
cells
kinetic
suspension
was
in an IEC Universal model centrifuge.
The
supernatant is collected and assayed in the same manner
as histidase in free solution.
(
Histidase
in
solution
spectrophotometer.
is
assayed
directly
in
Reaction rates are determined
the
with
the Perkjn-Elmer Kinetics software. The concentration of
the
contents
in
this
assay are the
same
as
mentioned above for the encapsulated histidase
The
total volume of the assay is 3.0 mIs.
anelysis
of
triplicate.
histidese
in
solution
was
those
assays.
AlI kinetic
donc
in
Controls consisted of both reaetions where
either histldase or histldine were left out.
The artificial cells were stored in 1% Tween 20
Tris
(
buffer
pH 9.2.
At different
storage
- O.IM
intervals
microencapsulated histidase activity was determined.
AlI
storage
stability analysis
64
•
of
microencapsulated
histidase
and
histidase
solution
was
done
in
triplicate.
Hislidase
loaded
concentration
artificial
(plis
had
R
starling
of 250 units of histidase/al of Hb.
The
feasibility of microencapsulated histidase waB assessed
by evaluating its efficacy in depleting histidine in an
in vitro stirred-batch reactor.
ratios
of
microcapsules to
ratios
were
Three different volume
substrate
1:100,
solution
tested.
The
1:50,
studies
were done at physiological pH and
in
constant volume vessel reRctors.
of
L-histidine used was 400uM.
histidine
The concentration
of
is the level seen in histidinemic
reaction temperature was 37°C.
was
BO
rpm.
returned
277
At preset time intervals
to
nm was
the
recorded.
reactors
to
Controls
histidine
The
keep
consisted of artificial cells
65
speed
were
and absorbance at
aliquots
the
ratio
were
of
Ali in vitro
analysis was done in
histidase.
palients.
aliquots
microcapsules to total solution constant.
depletion
plasma
The shaking
from the reaction suspension,
wavelength
These
temperature
This level
The
taken
1:25.
were
triplicate.
containing
no
RISULTS AND DISCUSSION
(
Figures
analysis
obtained
1
Figure
with
microencapsulated
shows the results
artificial
is
(
histidase.
histidase
loaded
in
Figure 1 (from ImM to
the
6mM),
histidase
Urocanic acid production,
by the rise in absorbance at wavelength 277
linear over the time period of the assay.
kinetic
data
compares
the
histidase
with that of histidase
results
show
that
activity.
This
as
nm,
Figure 2
microencapsulated
of
in
solution.
These
microencapsulation
of
histidase
the apparent Vmax
of
histidase
in a drop in
results
of
For the range of substrate concentrations
reactian rate is linear.
shown
of the
kinetic
cells assay with varying concentrations
L-histidine.
shown
and 3 show the results of the
2,
l,
decrease
the
in
Vmax
of
r
J
1
microencapsulated enzymes has been attributed mainly to
diffusion restrictions regarding the substrate into the
microcapsule. It is postulaled that the membrane of the
artificial
cell
serves as a diffusion barrier to
the
substrate (11,16). The results in Figure 2 suggest that
microencapsulated
histidase
has
approximately
50%
apparent Vmax activity, of the activity of histidase in
solution.
Figure
experiments
cells
(
kinetic
was
2
also
shows the
where histidase leakage
assessed.
The
experiments
done
results
from
varying
the
artificial
results show that
with
of
for
aIl
L-histidine
i
j
66
•
}
cells.
[!gYr~
!~ Assay of histidase loaded artificial
Rate of conversion of histidine is plotted as changes
in
Absorbance at 277 nm.
Each reaction mixture
contained
0.1 mIs of histidase loaded artificial
cells.
4 different substrate concentrations
were
studied.
67
(
(
FIGURE l
{
ft
......
2.8
L-histidine
L-histidine
L-histidine
L-histidine
2.4
Êc
"-
"-
'"
w
:1mM
:2mM
:4mM
:6mM
-.-.-.-
- â -
2.0
1.6
()
Z
oC(
1.2
cc
ct
o
(IJ
al
0.8
oC(
0.4
o
o
30
60
90
120
TIME (minutes)
150
180
(
2.8
l
ri /
2.4
Êc:
(
1'1'-
-z
(\j
-1/.
•
2.0
1.6
;'/
w
Ü
«
co
i----
0.4
o
o
--.-
--.-
--A-
_______ •
.----
1 ______ 1
1
1
1
1
1
30
60
90
120
150
TIME (minutes)
(
--e-
1/
;; /
0.8
: 1 mM
:2mM
:4mM
:6mM
/1
I#
li./
1.2
Cl:
aen
co
«
L-his tidine
L-histidine
iL-histidine
L-hislidine
180
.....
FIGURE 2
(
(
FIGURE 2
•
18.0
16.0
14.0
oQ)
c_
as :1
o c
0·-
.-
~
E
î
12.0
::1 ....
_"'0
o
rn
Q)
(J
:1
Q)"'O
-
...."
--
o 0..
E Q.
0"'0
c ._
as (J
c «1
AcroenC3 P SUI3ted histidase - e -
10.0
-...
l
8.0
/1
>
(.)
0
...J
w
histidase solution
microencapsulated histidase
supernatant
6.0
!
>
/î/
4.0
2/!
2.0
!
0
4
i
0
2
4
6
8
L- HISTIDINE (mM)
10
-0-4-
concentrations,
(
leakage of histidase
the
shows
3
Figure
no
took
Vmax for both
histidase
20mM.
cells
apparent
loaded
value
of
of
variety
a
the
made
have
enzymes
observation (13,14).
Both had a
on
reports
Previous
microencapsulated
be
The KM of
are reported in Table 1.
are the same.
KM
The
solution and KMapparent of histidase
artificial
of
plots
Lineweaver-Burk
encapsulated histidase and histidase solution.
and
place.
same
The Vmax data (Table 1) shows the
Vmax of histidase loaded artificial cells
50%
of
the Vmax of histidase
in
solution.
to
This
observation is consistent with previously reported work
microcapsules
(
(21) .
The
was
histidase
which
in
prepared by
yield
microencapsulation
of
was
microencapsulated
interfacial
reported
polymerization
upon
activity
histidase
in
40%
to be
of
free
histidase.
Figure
4
displays the storage stability
microencapsulated
profiles
of
histidase and histidase solution
at
two different storage temperatures,
stability
profile
of
histidase
4°C and 37°C.
stored
demonstrates that encapsulation of histidase
the
stability
Histidase
of
histidase
activity
loaded
at
incresses
storage.
solution reaches 50% of its original enzymic
activity after 9.5 days of storage at
{
upon
The
artificial
cells reach 50% of
69
37°C.
their
Histidase
original
)
[igYr~
~L
Lineweaver-Burk plot of
histidase and histidase in solution.
70
microencapsulated
(
(
FIGURE 3
.
0.8
0.7
microencapsulated - e histidase
histidasa solution
-
r
0 -
0.6
)-.
0.5
-f-
(J
"',
0
..J
W
0.4
~'
>
.....
,...
0.3
/./
0.2
r
• 6
0.1
0/
o~
-1
KM 0
200
400
600
1IL-HISTIDINE
800
1000
TABLI 1: IINBTIC PARAWITIRS
KM
(mM)
or BISTIDASI
Vmax
(nano.ols of urocanic
acid produced/minute)
Microencapsulated
histidase
20
33
Histidase in
solution
20
67
71
l
.....
lU
...
r!g~[~ 1~ Storage stability profile of histidase stored
at 4°C and 37°C.
The histidase activity retained is
presented as a
percentage of the histidase enzymic
activity at time zero,
and is plotted as a function of
tilDe.
72
TIME (days)
(
FIGURE 4
•
TIME (days)
enzymic Dctivity af'ter 15 days of storage at 37° C. Even
(
after
da ys of storage at
36
artificial
maintain
cells
original
enzy.'lÎc
histidase
st 0
encapsulation
more
activity.
at
rt~d
histidaae
37° C,
than
of
25%
also
demonstrates
stabilizes histi.lase activity.
days
of storage at ,qoC hist.idase solution has
its
original
histidase
still
Howe"er
95%
m~intains
~f
after 21 days of stora"e at 4°C.
days.
After
days
77
of
of
that
After 21
63%
of
microencapsulated
ita original actjvity
The storage stability
of histidase stawed at 4°C was followed
atudy
their
The sttt!Jil i ty profile
4° C
activity.
loaded
storage
for
77
microencapsulated
histidase still maintaine more than 50% of its original
(
enzymic
activity.
Hiatil\ase
shows 40% of its original
of
increased
after
storage
(25).
~ctivity.
sta~ility
microencapsulation
investigators
for
solution after
have
a var'iety of
days
77
Theae observations
of
enzymic
been
noted
activity
by
encapsulated
other
enzymes
This incredse in stability is attributed to
the
following. Enzymes encapsulated within artificial cella
are
stabilized
concentration.
containing
stable
(
the
of
high
this
For
enzymes
concentration
functions
by
are
Hb
intracellular
reason
made
(JO
to
gœs/IOO
of Hb were thus two foId,
microcapsules,
and
second
stability of encapsulated histidase.
73
protein
artificial
coutain
mIs)
high
a
(26) •
first to
to
cells
The
prepare
increase
the
The
first
two
set of
studies
of
microencapsulated
histidase showed that histidase retained activity
microencapsulation.
also
Microencapsulhted
more stable than histidase
these
to
histidase
solution.
ln
potential
depletion
vitro
effectlveness
of
were
microencapsulat.ed
histidase
for
Therefore
carried
.,as
regard
histidase
of histidine in histidinemia.
experiments
was
However
studies did not provide lnformation with
the
upon
out
used to
'in
where
deplete
L-
histidine in a stirred-batch reactor. The concentration
of
histidine in these experiments was
considered
,
..
histidine
to
in
individuals
around
the
plasma
the
is
In
4-10
found
in
minimaJ
for
mean
100uM.
histidine
levels
be
elevated
histidinemla.
levels of blood
histidinemir
times higher
normal
This
400~M.
level
In
of
normal
histidlne
individuals
than
the
individuals
19
nre
blood
histidine
Three
(2,3).
different
volume ratios of histidase loaded artificial
cells
total
to
tested.
the
These were 1:100,
results
ratios.
The
se'd
After
ratio
solution
1:50,
(vol/vol)
with these three
different
volume
6.21mgjdl
is
Histidase loaded artificial cells at a
of 1: 100 showed the
following
120 hours microencapsulated histidase
histidine
were
1:25. Figure 5 shows
initial histidine level of
equal to 400uM.
volume
substrate
results.
decreased
levels to about 75% of their original value.
74
(
The
fi st
two
set of
of
studies
microencapsulated
hlstidase showed that hlstidase retained activity
Microencapsulated
microencapsulation.
also
more stable than histidase in
tbese
potential
depletion
solution.
effectiveness
of
were
microencapsulated
histidase
histidine
to
in
individuals
around
the
plasma
the
18
In
4-10
found
in
minimal
for
.ean
100uM.
h~stidine
levels
he
deplete
levels of blood
normal
different
volume ratios of
cells
total
to
tested.
the
ratios.
The
seen
After
ratio
In
than
the
histidas~
solution
L-
is
of
normal
are
blood
histidine
(2,3).
Three
loalcd artificial
(vol/vol)
were
1:25. Figure 5 shows
with these three
different
voluae
6.21mg/dl
is
Histidase loaded artificial cells at a
of 1:100 showed the
following
120 hours microencapsulated histidase
histidine
level
individuals
individuals
1:50,
This
histidine
initial histidine level of
equal to 400uM.
volume
suhstrate
These were 1:100,
results
histidinemia.
times higher
in
The concentration
elevated
histidinemic
for
where
histidine in these experiments was 400uM.
con8idered
regard
out
used to
histidine in a stirred-batch reactor.
{
However
Therefore
carried
was
was
histidase
of histidine in histidinemia.
experiments
vitro
of
histidase
studies did nol provide information with
the
to
upon
re8ults.
decreased
levels to about 75% of their original value.
74
~~
-
-
---------.
)
ri'Yr~ Q~ In vitro dep1et~on of L-histidine: Artificial
ce1ls in the volume ratio of 1/100,
1/50,
1/25 were
used.
Rate of decrease
of histidine
concentration
(mg/dl) is p10tted as 8 function of time.
)
75
(
(
FIGURE 5
(
-
6.0
lI~.".
,.i·~-----..!'2!~J: -----------= ~.----- ----:r----------ï
---.:-..;. 5"':~-"::-'::"~-----
1______
1
1
• 5 :) -;
l
\"
~
~
-
......
...
....
.....ë
......
~
4.0
1
LW
Q
1(/')
-
1
...1
~1
1
3°1
2.0
1.0
"!
t,;. - - - - - - -
4_4
-
- ____
! ____
'-o.
.1.---""'"
'---
hlstidase loaded arllflclal cells
•
t
control arllflcl;'}1 ccII,:
substrate solution
substrate solution
1/100
-4-
1/100
----- .:. -----
1/50
- t -
1/50
---- 0 .----
1/25
--e-
1/25
---- 0 ----
O~-----------~----------~-----------~----------~----------_.__
o
24
48
72
96
120
TIME (hours)
The
volume ratio of 1:100 was used as a sim14]ation
~dministered
the amounl of microcapsules
would
ratio
This
situatlon.
total
body
water.
This
to
simplification
of the situation in vivo.
compartlllents
experimental desIgn
requir~s
in vivo.
drop
in histidine level.
levels
The
a
of
though this is not
1:50
in
was
cells
at
levels
a
would
used
lowered
as
a
hl~cidine
1:25
expected as the
These
p~ol
artificial
faster.
Of
the
cells
rate
in
of histidine
of
results
substrate
of
artificial
lowered
dilution
hours.
simulation
Histidase loaded
volume ratIo of
be
decreases,
the
artificial
histidine
to 60% of their original value after 24
loaded
the
1:50
extracellular -"ater.
within that compa rtmen t.
As
Hlstidase loaded
histidine as a single
treat
This
of h;stidine
consider~tion
volume ratio of
microcapsules
also
ln the body,
to 65% of their original value after 72
ratIo
a
Wllh rontrol ffllcrocapsules there was nu
case
at
of
is
compartmentalized.
l'1re
as a single pool of substrate,
cèlls
c1inical
ratio
th~
mic.rocapsules
fluid
(
be
in a
of
hours.
of
histidase
substrate
solution
depleti~n
becomes
the three different volume ratios
testerl,
ratio of 1:25 gave the Most satisfactory
results.
The histidine levels fell &y 40% over a 24 hour period.
This
ratio
artificial
(
is
equivalent to doubling the
cells used in the volume ratio of 1:50
of
for
depleting substrate in the extracellular compartment.
76
l
volume
GENERAL DISCUSSION
.'
In this repcrt the following evidence is presented.
ammonia-lyase (EC 4.3.1.3)
histidine
(histidase)
was
microenca~sulated
Lfrom
successfully
withln artificial cells.
of
histidase solution and apparent KM of microencapsulated
hist.idase
enzyme.
hisfidase
and
Whate]ey for histidase
microcapsulp5
storage
upon
free
This result i8 in agreement with those reported
Wood
nylo~
(21) .
encapsulat~d
At both 4°C and
stability of histidase activity
microencapsulation.
being
Histidase
in an enclosed environment.
pcesence
the
Microencapsulated
an apparent Vmax of approximately 50% of the
has
by
are both 20mM.
is
histidine
Preliminary
depletion
out.
feasibility
possible
These
sLab111zed
It is also
ln
studies of
il!
vitro experiments assessed
of histidase loaded artificial
experimental therapy in
cells
histidinemia.
vitro
were
the
for
These
of experiments evaluated three different volume
ratios
of
in
artificial
cells
suhstrate
solution
effeciency for histidine.
.'
the
series
in
suhstrate
solution.
Microencapsulated histidase in a volume ratio of
-
by
within
with encapsulated histidase
i~
the
increased
of a high concentration of hemoglobin
microcapsules.
carried
LS
37°C
in
in
did not show
high
1:100
conversion
Microencapsulated
histidase
a volume ratio of 1:50 in substrate solution showed
better
histidine conversion
77
effec1ency .
However
the
duration
(
is
required to lower histidine levels by a third
long.
The volume ratio of 1 part histidase
loaded
microcapsules in 25 parts substrate solution showed 40'
conversion in 24 hours.
histidine
effective
that
histidase
show
histidine
with
of
depletion
microencapsulated
These resuits
requires using
a
ratio of 1:25 of microcapsules in substrate
voluae
solution.
This single pool model is an oversimplification
situation
existing
distributed
in
differences
in vivo.
different
In
vivo,
fluid
histidine
is
with
compartments,
intracellular
in
of the
extracellular
and
concentrations. The present in vitro simulation is more
applicable
depletion
in
to the assessment of the
effectiveness
of histidine by mieroeneapsulated
extracellular fluid.
of
histidase
The next step is to carry out
studies in a histidinemie animal model.
A
number
These
of models of the histidinemic
histidinemie models inelude
histidinemie
experimentally
mutants
in
the
state
naturally
mouse
exist.
oecuring
(27,28),
indueed histidinemie states in
and
animaIs
(29,30).
ACI.OWL.DGIMI.~S
The
grant support to TMSC from MRC (MT9l00) and
Virage
Centre
acknowledged.
(
graduate
of
Excellence
Support
fellowship
78
are
from the FONDS FCAR
to
acknowledged.
grant
RI
is
also
MESST
gratefully
through
a
gratefully
RIFIRINCIS
(1)
La Du B.N., Howell R.R.,
Jacoby A.,
Seegmiller
J.B.,
and Zannoni V.G. (1962). Bioche •.
Biophys.
Res. Commun., L, 398-402
(2)
Ghadimi H. (1974), Histidinemia: Emerging Clinical
Picture in W.L. Nyhan (ed.) Heritable Disorders of
!~in~ !~i4 M~!~~~li!~l
f~!!~i~!--~f---g!j~j~~!
E~P!~!!i~D ~n~ g~n~!i~ Y~!l!!i~D,
2nd ed., John
Wi1ey and Sons Inc., 265-292
(3)
La Du B.N.
(1978), Histidinemia in J.B. Stanbury,
J.B.
Wyngaarden, O.S.
Fredrickson (eds.) !h~
M~!!~Ql1~ »!!1! Qi lnh~!1!~4 ~1!~!!~,
4th ed.,
McGraw-Hill Inc., 317-327
(4)
Imamura L. t Watanabe T., Hase V., Sakamoto V.,
Fukada V., Yamamoto H., Tsuruhara T., and Wada H.
(1984), J. Biochem., ~Q, 1925-1929
(5)
Gordon N.
104-105
(6,
Ghadimi H.
211
(7)
Gatfield P.D.,
Pozsonyi J.P.
465-469
(8)
Duffner P.K. and Cohen M.R. (1975), Neurology,
195-197
(9)
Zachary Dyme 1.,
Horowitz S.J.,
Bacchus B.,
and
Kerr O.S. (1983), Am. J. Dis. Child., l~l, 256-258
(1970),
Dev.
Med.
Chi1d Neurol.,
!~§.,
210-
Devereux M.,
Knights R.M.,
Med. Ass. J.,
(1969),
Canad.
lQl,
(1981),
Am. J. Dis. Child.,
!~1,
(10) Chang T.M.S. (1988), Meth. Rnzymol.,
(11) Chang T.M.S. (1964), Science,
(12) Chang T.M.S.
~!~,
!~,
l!§,
and Poznanaky M.J.
and
~§.,
444-457
524-525
(1968),
Nature,
242-245
(13) Poznansky M.J.
Biophys. Acta,
(14) Bourget L.
l~Q, 5-8
and Chang T.M.S.
103-115
(1974), Biochill.
~~~,
and Chang T. M. S.
(1985) ,
(15) Bourget L. and Chang T.M.S.
Biophys. Acta, ~~~, 432-438
(16) Chang T.M.S. (1973), Bnzyme,
FBBS Let t. ,
(1986),
l~i~l,
8iochill.
95-104
79
...
{
(17) Siu Chong E.
218-239
(18) Shu C.D.
!~,
(1974), Enzyme,
(1981), Int. J. Artif.
and Chang T.M.S.
~,
Organs,
and Chang T.M.S.
82-84
(19) Chang T.M.S. (1969), Science Tools,
!§(~l,
(20) Chang T.M.S. (1987), Meth. Enzymol.,
(21) Wood
D.A. and
Pharmacol., ~1,
1~§,
(1982),
Whateley T.L.
33-39
68-82
J.
Pharll.
552-557
(22) Tabor H.
(1955), Degradation of Histidine in W.D.
McElroy and 8.
Glass (eds.),
§~~~~~iy~ Q~
~min~
~E1Q M~!~Q~11~~, John Hopkins Press, 373-390
(ed.) (1969), ~!!~~!!~ H!!!!~1!QQ! Vol II,
Springer-Verlag, 799-800
(23) Barman T.IL
l
1
(24) Rechler M.M.
11.0U 63-69
(
(25) Chang T.M.S.
(1977), Encapsulation of Enzymes,
Cell
Contents,
Cells,
Vaccines,
Ant igens,
Anliserum,
Cofactors,
Hormones. and Proteins in
T.M.S.
Chang (ed.), ~i~~~giE~l ~ppliE~!!Q~ g!
lm!QQili~~g
~~~y~~~
~~g frQ!~i~! Vol
l,
Plenum
Press, 69-90
(26) Chang
COllmun.
T.M.S.
(1971).
1531-1536
Nature,
Biochem.
Diophys.
Res.
,~~.
(27) Kacser H.,
l,
(1971), Meth. Enzymol.,
and Tabor H.
Bulfield G.,
~~1,
(28) Kacser H.
Bull..~..c~.2,
and Wallace M. E.
(1973),
77-79
and Bul field G.
3-4
(1977), Compar. Path.
(29) Lee S.C. and Wang M.L. (1975). Nutr. Metabol .• 18.
79-88
(30) Brand
L.M.
and Harper A.R.
Biophys. Acta, ~11, 294-306
(
80
l
.
(976) ,
Diochim.