Download Enzymatic Hydrolysis of Pilocarpine

Enzymatic hydrolysis of pilocarpine
Philip P. Ellis, Kathrine Littlejohn, and Richard A. Deitrich
Enzymatic hydrolysis of pilocarpine by rabbit and human serum, ocular tissues, and liver was
demonstrated. Within each species the enzyme concentration in ocidar tissues appeared highest
in uveal and retinal tissues and lowest in the lens. The Michaelis-Menten constants (Km) for
the pilocarpine inactivating enzyme was 6.04 ± 2.52 x 10-* M. for rabbit serum and 4.63 ± 2.02
x 10~s M. for human serum.
Key words: pilocarpine, enzymatic hydrolysis, enzyme kinetics, rabbit serum, human serum,
enzyme concentration, rabbit ocular tissues, human ocular tissues.
E
lyzing enzyme in rabbit and human serum
were determined. The level of this enzyme
in rabbit and human ocular tissues and
liver also was measured.
Inzymatic hydrolysis of pilocarpine has
been demonstrated in the serum of rats,
rabbits, and humans and in the secondary
aqueous humor of rabbits and humans.1-2
Evidence has been presented that the enzyme, apparently an esterase, is probably
cation dependent. Enzyme activity is lost
by heating or with the addition of various
chelating agents: EDTA (ethylene diaminetetraacetate), trien, penicillamine,
neomycin sulfate, kanamycin sulfate, and
ethambutol hydrochloride. Unpublished
observations in our laboratory have established that the enzyme responsible for pilocarpine hydrolysis is not cholinesterase.3
In the present study, Michaelis-Menten
(Km) kinetics of the pilocarpine hydro-
Methods
PUocarpine determination. Quantitative determination of pilocarpine hydrochloride followed the
colorimetric technique of Hestrin as modified by
Schonberg and Ellis.2 This reaction requires the
presence of an ester bond; thus residual pilocarpine
is measured. Each sample was run in duplicate
and compared to a reagent blank consisting of a
comparable tissue or serum preparation without
substrate.
Time periods began with the addition of the
pilocarpine to serum or tissue homogenates previously brought to the incubation temperature.
The reaction was stopped with the addition of 2
ml. of alkaline hydroxylamine. With high substrate concentrations, the test solutions were diluted at the end of the incubation period and 1 ml.
of the resulting solution was used for analysis.
Klett units were converted to micrograms of
pilocarpine from a standard curve constructed
periodically using various quantities of the pilocarpine solution.
Serum preparation. Blood was collected from
normal adult New Zealand albino rabbits by
cardiac puncture. Samples were allowed to clot
at room temperature, refrigerated several hours,
From the Division of Ophthalmology and Department of Pharmacology, University of Colorado
Medical Center, Denver.
Supported by an unrestricted grant from Research
to Prevent Blindness, Inc.
Manuscript submitted April 24, 1972; manuscript
accepted June 12, 1972.
Reprint requests: Dr. Philip P. Ellis, 4200 E. Ninth
Ave., Denver, Colo. 80220.
747
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Investigative Ophthalmology
September 1972
748 Ellis, Littlejohn, and Deitrich
centrifuged five minutes at 400g, and the serum
decanted. Human blood was drawn by venipuncture from blood-bank donors and volunteers (ages
ranged from 18 to 75 years) and the serum prepared as above. All serum samples were either
iced or stored at -32° C. and thawed at room
temperature just prior to use.
Preparation of rabbit tissues. Rabbit tissues
were obtained from New Zealand albino rabbits
weighing 2.5 to 3 kilograms. Animals were injected intravenously with 1,000 units of sodium
heparin, killed by cervical dislocation, and the
head immediately perfused through the carotid
arteries with cold 0.25 M sucrose. Each eye was
enucleated to include extraocular muscle, rinsed
in cold sucrose, and dissected.
Following removal of the extraocular muscles,
an incision 3 mm. posterior to the limbus freed the
anterior portion of the globe from which the iris,
lens, and cornea were removed. The intact posterior sclera, choroid, and retina were immersed
in iced sucrose, tissues bisected and the retina
and choroid layers carefully scraped from the
sclera until only a small section of attachment remained. Gentle shaking released the retina and
the choroid was scraped free. Excess sucrose was
removed with filter paper.
A small piece of liver was removed immediately
following enucleation, rinsed in cold sucrose and
blotted on filter paper.
In another group of animals the technique of
collection and storage of eyes was similar to that
for human eye bank specimens. Animals were
killed by cervical dislocation, the eyelids were
closed, and enucleation performed three hours
after death. The eyes were placed in a standard
glass human eye bank container which had approximately 1 ml. of a solution containing 1.75 mg.
of neomycin, 0.025 mg. of gramicidin, and 5,000
units of polymyxin B per milliliter (Neosporin)
in the bottom. The eyes were refrigerated at 4°
C. for two to five days prior to dissection.
All tissues were placed in preweighed homogenizer tubes. Lens capsule-cortex, extraocular muscle and liver were used from individual rabbits,
other similar tissues from two animals were
grouped, and homogenates prepared with cold
0.25 M sucrose. The cornea was prepared as a 5
per cent homogenate and all other tissues as 10
per cent homogenates.
Preparation of human tissues. Human eyes were
obtained from the Colorado Eye Bank and dissected within five days of enucleation. The dissection was performed on an ice-filled Petri dish.
Similar tissues from each pair of eyes were
grouped in preweighed homogenizing tubes and
10 per cent homogenates prepared with cold
sucrose. Human liver samples were obtained at
autopsy, four to 22 hours after death, and prepared as above.
Michaelis-Menten constants (Km). Pilocarpine
HC1 at final concentrations of 2 x 10"4, 2.9 x
10-*, 5 x 10-4, 2 x 10-3, and 4 x 10-3 M was
mixed with 0.2 ml. aliquots of nine rabbit serum
samples, incubated for two minutes at 37° C ,
and the residual pilocarpine determined.
Pilocarpine HC1 at final concentrations of 0.5
x lO-3, 1 x 10-3, 2 x 10-3, 4 x IQ-3 ) a n ( i Q.Q X
10-3 M w a s m ixed with 0.5 ml. aliquots of seven
human serum samples, incubated for 50 minutes
at 37° C, and the residual pilocarpine determined. Although the incubation time was necessarily long, because of low activity, between 31
and 65 per cent of the substrate disappeared during this time for the 1 x 10~3 M concentration.
Linear Lineweaver-Burk plots were obtained under
these conditions, indicating that the Km determination is probably valid.
Enzyme levels in rabbit and human serum.
Pilocarpine 6.04 x 10-3 M (10 x Km) was mixed
with 0.2 ml. aliquots of 15 rabbit serum samples,
incubated for 15 minutes at 37° C , and the residual pilocarpine determined.
Pilocarpine 4.1 x 10~2 M (10 x Km) was mixed
with 0.8 ml. aliquots of 48 human serum samples
and incubated for seven hours at 42° C. The test
solution was diluted tenfold with distilled water
just prior to pilocarpine analysis.
Tissue water content. Individual tissues were
placed in preweighed 1 to 10 ml. pyrex beakers,
wet weight noted, and dried at 101° C. until dry
weights were unchanged with additional time.
Enzyme levels in rabbit and human tissues.
K,t, values were not determined for tissues. Preliminary tests indicated that enzyme levels were
lower than in sera. Pilocarpine levels were used
equal to those for determination of enzyme levels
in sera; this insured that the substrate was in excess and therefore not rate limiting.
Pilocarpine 6.04 x 10~3 M was mixed with 0.5
ml. of each rabbit tissue homogenate and incubated for five hours at 37° C. The test solution
was diluted by half with distilled water prior to
pilocarpine analysis.
Pilocarpine 4.1 x 10"2 M was mixed with 0.5
ml. of each human tissue homogenate and incubated for seven hours at 42° C. The test solution
was diluted as for human serum prior to pilocarpine
analysis.
Results
All results are given as the mean ± the
standard deviation.
Michaelis-Menten constants. For both
normal rabbit and human serum, the data
were plotted in the double reciprocal manner of Lineweaver and Burk.4 The mean
Km for duplicate determinations of 9 rabbit
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Volume 11
Number 9
Enzymatic hydrolysis of pilocarpine 749
Table I. Water content of rabbit and human tissues
Human
Water
(%)
Serum
Cornea
Iris-ciliary body
Lens capsule cortex
Chorokl
Retina
Optic nerve
Extraocular muscle
Liver
Rabbit
Water
(%)
Solid
(%)
91.40
85.39
91.08
73.05
90.21
91.42
78.56
8.60
14.61
8.92
26.95
9.79
8.58
21.44
(5)'
(6)
(5)
(6)
(6)
(6)
77.47
22.52
(8)
(5)
Solid
(%)
92.14
75.15
84.64
68.07
84.37
83.55
7.86
24.85
15.36
31.93
15.63
16.45
(5)
(5)
(5)
(5)
(5)
(5)
78.17
74.05
21.83
25.95
(5)
(5)
"Number of tissue srjecimens given in parentheses.
serum samples was 6.04 ± 2.52 x 10~4 M.
The mean value for triplicate determinations of 9 human samples was 4.63 ± 2.02 x
10"3 M. A representative Lineweaver-Burk
plot is shown in Fig. 1.
Tissue water content. The water content
of the tissues used appears in Table I.
Values are the arithmetic mean with the
number of observations given in parentheses.
Serum enzyme levels. The incubation of
6.04 x 10-3 M pilocarpine (1.480 mg. total
in a final volume of 1 ml.) with 0.2 ml.
rabbit serum resulted in the hydrolysis of
0.698 ± 0.114 mg. of pilocarpine in 15 minutes.
The incubation of 4.1 x 10~2 M pilocarpine (10.045 mg.) with 0.8 ml. of human serum (final total volume was 1 ml.)
resulted in the hydrolysis of 4.798 ± 0.777
mg. of pilocarpine in 7 hours.
Tissue enzyme levels. Fresh rabbit tissue
homogenates incubated with 6.04 x 10~3 M
pilocaqDine hydrolyzed substrate ranging
from 0.100 ± 0.069 mg. for cornea to 0.181
± 0.138 mg. for iris-ciliary body. Tissues
processed two to five days after death were
very similar in their activity to corresponding fresh tissues and in no instance was the
difference significant at a confidence level
of 0.05. Values are based on the 1.4 mg. of
pilocarpine recovered in controls which
contained no enzyme.
To compare the activity of all sera and
tissues tested, results were calculated on
the basis of milligrams of pilocarpine hy-
2CH
15n
O
X
>
10-
5-
5
10
1/B1X10
15
20
2
Fig. 1. Representative Lineweaver-Burk plot. Km
= 4.67 x 10~3 M. S = substrate concentration
moles per liter. V = Mg substrate hydrolyzed in
50 minutes.
drolyzed per gram dry weight of tissue per
hour of incubation. (Table II)
Discussion
This study indicates that a pilocarpine
hydroly:dng enzyme is present in ocular
tissues, liver, and serum of both rabbits
and humans. The enzyme would most
properly be called a lactonase since the
ester bond hydrolyzed is a lactone. Quantitative comparisons of tissue-enzyme levels
between the two species cannot be made
since the rabbit eyes were perfused prior
to tissue preparation for enzyme analysis
while the human eyes were not perfused.
However, this cannot account for the higher
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Investigative Ophthalmology
September 1972
750 Ellis, Littlejohn, and Deitrich
Table II. Milligrams of pilocarpine inactivated per gram dry weight tissue
per hour
Tissues stored 2 to 5 days
Fresh tissues
Rabbit:
Serum
Cornea
Iris-ciliary body
Lens capsule + cortex
Retina
Choroid
Extraocular muscle
Liver
174.376
3.564
4.738
1.624
2.729
3.415
1.748
2.155
±
±
±
±
±
±
±
±
28.723
2.860
3.536
1.111
2.345
3.203
1.486
1.259
(15)
(7)
(8)
(16)
(8)
(8)
(15)
(14)
Human:
Serum
Cornea
Iris-ciliary body
Lens capsule + cortex
Retina
Choroid
Optic nerve
Liver
9.681
11.155
31.741
8.978
33.072
30.909
10.309
11.288
±
±
±
±
±
±
±
±
1.568
14.960
16.394
4.331
23.325
12.754
11.422
6.883
(48)
(8)
(8)
(5)
(9)
(9)
(7)
(7)
3.559
1.627
3.337
4.387
±
±
±
±
1.489
0.471
1.361
2.708
(5)
(10)
(5)
(4)
Values are the arithmetic mean; ± values are the standard deviation. Number of tissue specimens are given in parentheses.
Rabbit tissues with exception of liver, perfused with cold 0.25 M sucrose. Human tissues not perfused.
enzyme levels in human tissue since human serum contains so little enzyme. Serum
enzyme levels are substantially higher in
the rabbit than in the human. Within each
species the enzyme concentration in ocular
tissues seems to be highest in uveal and
retinal tissues and least in the lens. In
the rabbit the concentration of the enzyme
in the cornea was high in comparison to
the enzyme concentration in other tissues
whereas the relative concentration of enzyme in extraocular muscle was low. In
the human the concentration of the enzyme
in the cornea and optic nerve was low in
comparison to enzyme concentration in
other tissues.
Like other esterases, the pilocarpine
hydrolyzing enzyme appears to be quite
stable. There was little difference in the
hydrolysis of pilocarpine by fresh and refrigerated ocular tissues of the rabbit.
The Km value for the pilocarpine hydrolyzing enzyme was 6.04 ± 2.52 x 10~4 M
for rabbit serum, and 4.63 ± 2.02 x 10"3 M
for human serum. Km values cannot be used
to compare enzyme concentrations quantitatively. Actually, an enzyme with a lower
Km value is more efficient at low concentrations of pilocarpine even if the Vinilx (the
velocity of the reaction at saturating concentrations of substrate) is less than that
for another enzyme with a higher Km value.
The significance of the pilocarpine hydrolyzing enzyme is unknown. It is generally held that pilocarpine is largely excreted by the kidneys and enzymatic inactivation of pilocarpine is not believed
to be a major method for removal of the
drug. Furthermore, it should be pointed
out that the possible pharmacologic activity of the products of enzymatic hydrolysis of pilocarpine is unknown.
Studies are in progress in our laboratories
to examine the concentration of the enzyme
in serum of glaucoma patients including
those treated with pilocarpine. Conceivably, patients with higher enzyme levels
might be less affected by pilocarpine therapy than those patients with low concentrations of the enzyme. Also under investigation is the possible value of inactivators of the pilocarpine hydrolyzing enzyme to increase the ocular hypotensive
action of pilocarpine.
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Volume 11
Number 9
REFERENCES
1. Lavallee, W. F., and Rosenkrantz, H.: Evidence for pilocarpine transformation by serum,
Biochem. Pharm. 15: 206, 1966.
2. Schonberg, S. S., and Ellis, P. P.: Pilocarpine
inactivation, Arch. Ophthalmol. 82: 351, 1969.
Enzymatic hydrolysis of pilocarpine 751
3. Allen, A. W., and Ellis, P. P.. Unpublished
observations.
4. Lineweaver, H., and Burk, D.: The determination of enzyme dissociation constants, J.
Am. Chem. Soc. 56: 658, 1934.
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