CHAPTER IV
ANTITUMOR PROPERTY OF L-ASPARAGINASE ISOLATED
FROM THE AEROMONAS
ANTITUMOR PROPERTY OF L-ASPARAGINASE
ISOLATED FROM AEROMONAS
Various experimental models for testing the antitumor
property of different compounds have been suggested by
Connors and ~oe[210]. The compounds may be tested in
tumor bearing animals, against microbial systems or cell
cultures. The antitumor property of L-asparaginase is
well established and is therapeutically used in human
leukaemia. L-asparaginase is cytotoxic to a large number
of tumor cell lines which require an exogenous supply of
L-asparagine. Table IV.1. gives a list of some of the
tumor systems in which different L-asparaginase
preparations have been tested for their antitumor
property. The antitumor property of L-asparaginases
derived from various sources tested against many tumor
systems viz., 6C3HED Gardner lymphosarcoma, Yoshida
ascites sarcoma, Walker 256 ascites sarcoma, Ehrlich
lymphoma, L-5178 murine lymphoma, ascites fibrosarcoma,
Dalton's lymphoma etc. differ in their antitumor
properties.
6C HED-OG tui4or cells
Tosa et al.[66] From Proteus vulqarisr
6C3HED tumor Edman Peter and L-asparaginase encapsulated Inguar Sjoholm[l31] in polyacrylamide microspheres
6C3HED tumor Alpar et a1.[95] L-asparaginase encapsulated in erythrocytes
6C3HED-JL tumor Boyd & Philips[65] From Serratia marcescens
6C3HED lymphoma Distasio et a1.[681 From Vibrio succinoqenes
Walker 256 De Angeli et a1. [ 4 8 1 From Aspergillus terreus ascites sarcoma
Human ovary carcinoma, Pekhov et a1.[212] Fisher lympholeukaemia and Burkitt's lymphoma
From Pseudomonas boreopolis
Human Jurkat T cell Takase Kozo et a1.[281 From E. coli line
Human acute lympho- Mandelli Franco et a1.[1091 Combination therapy with blastic leukaemia L-asparaginase, Idarubicin,
Vincristine and Methotrexate
Ascites fibrosarcoma and Dalton's lymphoma
Acute lymphatic leukaemia
Spontaneous lymphoma in dogs
Malignant lymphoma in dogs
L-5178 Y murine tumor
Lymphoma in dogs
Non Hodgkins lymphoma in dogs
Childhood non T cell acute lymphoblastic leukaemia
Human hepatopoietic cell lines
Raha et a1.[561
i3elasco et a1.[81
Yoshimoto Takayuki et a1.[118]
Mac Ewen et a1.[119]
Sur Pratima et a1.[213]
Jeglum et a1.[214]
Teska et a1.[130]
Sallan et a1.[2151
Koishi Toshioki et a1.[216]
From Cylindrocarpon obstusisporum
Combination therapy with L-asparaginase, Daunomycin, Vincristine, Prednisone, Tenipsoid and Cytosine ara-C
Polyethylene glycol modified L-asparaginase
Polyethylene glycol modified L-asparaginase
From E. coli
Combination therapy with Vincristine, L-asparaginase and Doxorubicin
Polyethylene glycol modified L-asparayinase
IV. A. SOLID TUMOR REDUCTION STUDIES
In the present study, the antitumor property of
L-asparaginase from the estuarine Aeromonas was studied
using Ehrlich ascites lymphosarcoma system in mice.
Eventhough there was a promise of it being effective
against tumors, as suggested in the previous section,
direct evidences for the same are obtained only through
this study.
MATERIALS AND METHODS
Experimental animals
Inbred Balb/c mice of 7 weeks age, weighing 14-17 y
were used for the study. The mice were obtained from the
stock inbred colony which was maintained by mating of
brothers and sisters. The mice were maintained on dry
pellets (Gold mohur rat feed, Hindustan Lever Ltd.) and
tap water.
Tumor cell lines and their maintenance
Ehrlich ascites tumor cell line, kindly provided by
.Amala Cancer Research Institute. Trissur, was used. The
tumor cell line was maintained by serial intraperitoneal
(IP) transplantation in mice. Full grown tumor cells were
aspirated from the mouse peritoneum, washed thrice with
0.9% saline, and suspended in saline. About 1 x lo5 cells
were injected intraperitoneally into a new healthy mouse.
All operations were done aseptically. Solid tumor growth
was obtained from the ascites cells by injecting 5 x 10 4
cells subcutaneously under an area of shaved skin on the
flank.
Asparaginase preparations
(a) Aeromonas L-asparaginase
Growing of cells, preparation of crude cell free
extract and purification were done as described previously
(Chapter 111). The enzyme preparation obtained after
hydroxyapatite chromatography and lyophilization was used
for the studies, after dissolving in 0.9% saline.
(b) -- E. coli L-a.sparaginase
The commercially available -- E. coli L-asparaginase,
manufactured by Kyowa Hakko Kogyo Co. Ltd.. Japan,
marketed by Aiochem India Ltd. under the trade name
'Leunase' was used to compare the antineoplastic activity
of Aeromonas L-asparaginase. The lyophilized enzyme
preparation was dissolved in 0.9% saline.
Determination of antineoplastic activity of L-asparaginase
Three groups of mice consisting of 6 mice each
(average weight 15.4 g) were inoculated with 5 x 10 4
Ehrlich ascites tumor cells subcutaneously. Seven days
after tumor implantation, L-asparaginase was administrated
through the same route. Group I1 was injected with
Leunase, Group I11 received Aeromonas L-asparaginase and
Group I was maintained as the control. L-asparaginase
was injected into the subcutaneous tumor mass in a single
dose and in three intermittent doses with an interval of
24 hrs between each administration. Controls received
same quantity of sterile saline. The diameter of the
subcutaneous mass (average of two measurements at right
angles) was measured on the 7th day and 12th day.
Antitumor activity is indicated by diminution of tumor
size and is expressed as mean change in tumor size.
Enzyme therapy
The antitumor activity of Reromonas L-asparaginase
preparation was compared with that of Leunase using
Ehrlich ascites tumor in mice. Mice weighing about 14
to 17 g were divided into 3 groups, each consisting of
6 animals. All animals were inoculated intraperitoneally
5 with 1 x 10 tumor cells. 24 hrs after tumor implantation,
the mice were divided in to 3 groups and L-asparaginase
was administered through the same route in a single dose
103
and in three intermittent doses with an interval of 24 hrs
between each administration. Controls received the same
amount of sterile saline. Group I (control) received
sterile normal saline. Group I1 received Leunase and
Group I11 received Aeromonas L-asparaginase preparation in
saline. Several dose levels of the test agents were used
and the survival times of the treated groups were compared
with survival times of controls. Since there was an
inverse relationship between survival time and number of
viable cells injected, the percentage increase in life
span of treated groups was compared with that of control
groups as a direct measure of tumor cell kill as suggested
by Connors and Jones[217].
RESULTS AND DISCUSSION
The antitumor property of L-asparaqinase obtained
from estuarine Aeromonas was compared with that of the
commercially available Leunase and the results are given
in Table IV.2.
When 5 units of Aeromonas L-asparaginase was injected
subcutaneously, there was a reduction in the growth rate
of tumor, when compared to controls. 10 units of enzyme
could bring down the tumor size. 50 units of
L-asparaginase injected into the subcutaneous tumor mass
caused almost complete regression of the tumor mass.
Table IV.2
Comparison of antineoplastic activity of Leunase and Aeromonas L-asparaginase
Dose of Mean change in tumor size (nun)* L-asparaginase (I(,) ....................................
Aeromonas L-asparaginase Leunase Control
5 units
10 units
25 units
50 units -8.1 -8.4
3 intermittent doses -8.4 -8.5 of 10 units each
* Diameter of the tumor mass
6 mice in each group. Injections were done 7 days after tumor implantation and the effect was measured on the 12th day.
Better results were obtained when 3 intermittent doses of
10 units were injected at an interval of 24 hrs between
successive injections. Such decrease in tumor mass was
also obtained with E. coli L-asparaginase at the same dose -- levels. Average reduction in tumor mass was more with
increase in the dose of L-asparaginase. Complete
disappearance of the subcutaneous tumor mass without any
tissue necrosis was obtained with both AerQmonas and
E. coli L-asparaginase,injected in 3 doses of 10 units -- each. This clearly establishes the antineoplastic effect
of L-asparaginase prepared from estuarine Aeromonas, and
is equally comparable with that of Leunase. This shows
that L-asparaginase prepared from estuarine Aeromonas
has cytotoxic effect on Ehrlich ascites tumor but not on
normal cells. Aeromonas L-asparaginase preparation
has the same antineoplastic effect as that of E. coli
L-asparaginase on subcutaneous solid tumors, which is
evident from the photographs appended in Figures IV.l.
and IV. 2.
The effect 0f.Aeromonas L-asparaginase therapy on
Ehrlich ascites sarcoma bearing mice is shown in
Table IV. 3. Duration of the experiment was 60 days
after the tumor implantation. It can be seen from
Table IV.3. that all the animals which received 75 units
of Aeromonas L-asparaginase intraperitoneally in 3
intermittent doses, survived. The average survival period
of the animals were more, when less quantity of the enzyme
protein was injected at a time. The average survival
period could be increased by injecting the same amount of
the enzyme in intermittent doses rather than as a single
dose. There was a reduction in the survival period when
large quantities of the enzyme preparation was
administered intraperitoneally. Even 100 units of the
enzyme from Aeromonas did not protect the tumor bearing
mice when yiven in a single dose. However administration
of Aeromonas L-asparaginase of any dose would increase the
average survival period of the Ehrlich ascites tumor
bearing mice, as evident from the percentage of survivors.
106
Mice with subcutaneous Ehrlich ascites tumor. Photographs were taken 12 days after tumor implantation. Arrow mark indicates the site of tumor implantation.
Figure:I~.l(a). Front view of the tumor growth.
Figure:IV.l(b). Side view of the tumor growth.
Mice treated with Aeromonas L-asparaginase and Leunase. Enzymes were injected in 3 intermittent doses of 10 units each at an interval of 24 hrs, starting from the 7th day onwards. Photographs were taken on the 12th day of tumor implantation. The arrow mark shows the site of tumor implantation.
Figure:IV.2(a). Mouse treated with Aeromonas L-asparaginase.
Figure:IV-Z(b). Mouse treated with Leunase.
Table IV.3
Effect of L-asparaginase isolated from Aeromonas on Ehrlich ascites tumor bearing mice
Dose of NO. of 'Cures1/ Percent- Day of L-aspara- in jec- mice age of Day of sacrifice ginase (IU) tions treated* survivors** death of tumor
free mice
Control 1 0/6 0 14,16,18, - (not treated) 19,22,22
10 1 2/6 100 34,39,43,56 60 (2 mice)
2 5 1 4/6 100 38,54 60 (4 mice)
5 0 1 3/6 100 45,51,56 60 (3 mice)
1 1/6 100 38,47,48, 60 (1 mice) 53,58
3 intermittent 3 6/6 100 - 60 (6 mice) doses of 25 units each
+ Mice that survived uptothe 60th day of experiment
* * Survivors at the time of death of controls. L-asparayinase was administered intraperitoneally in a single dose, 24 hrs after the tumor implantation.
A comparison of the efficiency of L-asparaginases
from E. coli (Leunase) and estuarine Aeromonas, in the
treatment of Ehrlich ascites tumor in mice is given in
Table IV.4.
Table IV.4
Effect of E. coli and Aeromonas L-asparaginases on the growth -- of Ehrlich ascites tumor in mice
Dose of E.coli L-asparaginase Aeromonas L-asparaginase L-aspara- Average Inorease Average Increase ginase (IU) survival in life survival in life
time span ( % I time span ( % ) (days) (days)
Control 18.5 - 18.5 - (not treated)
2 5 61.5 Cures 65.2 Cures
50 65.7 Cures 60.7 Cures
100 62.8 Cures 51.5 278.4
3 intermittent 88.6 Cures 84.8 Cures doses of 25 units each
'Cures' are those which survive upto the end of the experiment (60 days)
Both E. coli and Aeromonas L-asparaginases were -- curative at a dose level of 25 units. The average life
span was slightly higher in mice treated with E. coli -- L-asparaginase than in mice treated with Aeromonas
L-asparaginase. This may be due to the presence of some
other proteins or may be due to the presence of other
components present in the Aeromonas L-asparaginase
preparation since the enzyme was purified only partially.
Gaffar showed that the reduction in the survival period
when large doses were injected could be due to the
toxicity of the enzyme preparation[501. Small doses of
L-asparaginase from Aeromonas were curative whereas high
doses did not show the same trend which may be due to the
reasons mentioned above. Similar reduction in survival
period with increasing doses of Vibrio L-asparaginase in
Yoshida ascites tumor bearing rats was observed by
Selvakumar[57].
Better results were obtained with E, and
Aeromonas L-asparayinases, when these were injected in
intermittent doses. Similar trend was noticed with
L-asparaginase from Vibrio by Selvakumar[S71. The
results clearly establish antineoplastic property of the
L-asparaginase, on Ehrlich ascites tumor, purified from
estuarine Aeromonas and is comparable with that of -- E. coli
L-asparaginase (Leunase). This is more evident from the
photographs appended in Figures IV.3. - IV-5.
Figure:IV.3. A normal mouse without tumor.
Mouse bearing ~hrlfch ascites tumor. Photograph was taken 15 days after the intraperitoneal implantation of tumor.
Tumor bearing mice treated with Aeromonas L-asparaginase and Leunase. 1n jectiozs were done in 3 intermittent doses of 25 units at an interval of 48 hrs. Tumor cells and L-asparaginases were injected intraperitoneally, Photographs were taken 15 days after tumor implantation,
m
Figure:IV.5(a). Mouse treated with Aeromonas L-asparaginase.
- - Figure:IV.5(b), Mouse treated with Leunase.
L-asparaginases from different sources were found
to vary greatly with respect to their antitumor
properties. The enzyme from E. coli[15,82,196], Serratia
marcescens[ 65,2181, Achromobacteraceae[77], Erwinia
aroideae[61], Erwinia carotovora[219], Cylindrocarpon
obstusisporum[561, Acinetobacter[91], Pseudomonas[l971,
Citrobacter[ZOS] and Asperqillus te$reus[48] are known to
suppress tumor growth. L-asparaginases from other
microbial sources viz., Bacillus coagulans[53] and
Saccharomyces cerevisiae[41] were, however, not
inhibitory to tumor growth. The present study shows that
L-asparaginase from estuarine Aeromonas is very effective
in inhibiting the growth of Ehrlich ascites tumor in mice.
All the animals survived when 75 units were injected in
three equal doses. The successful regimen of intermittent
doses of L-asparaginase has already been reported[57] and
the reduction in the survival period of the animals when
large doses were administered could be due to the toxicity
of the enzyme preparation as suggested by Gaffar[501.
More than one form of L-asparaginases have been
reported in - E. - coli[80,82,83,87,196], Mycobacterium
tuberculosis 837 Ra[204] and Citrobacter freundiiC671.
Of the two different forms of L-asparayinase reported in
E. coli and M. tuberculosis- H37 Ra and of the three forms -- - present in - C. freundii, only one each has been found to be
active against tumors. The different L-asparaginases
differ in pH optima, heat inactivation, substrate
affinity and effect of inhibitors[57,82]. However, the
L-asparaginase isolated from estuarine Aeromonas was found
to be antineoplastic.
Factors that might affect the antitumor property of
L-asparaginase include pH optima, Km value and serum half
life of the enzyme in the host[55,57]. Good activity at
the physiological pH, stability at elevated temperature,
high substrate affinity (low Km value) etc. would explain
Aeromonas L-asparaginase as an antitumor drug.
IV. B. BIOCHEMICAL STUDIES
L-asparaginase is an antitumor agent which is
effective in inducing remissions in patients with acute
leukaemia. This enzyme catalyses the hydrolysis of
L-asparagine which can not be synthesized by the sensitive
tumor cells. Normal cells, however, are able to
synthesize L-asparagine from aspartic acid and ammonia
with help of asparagine synthetase. This metabolic
difference between L-asparaginase sensitive tumor cells
and normal cells explains the minimal host toxicity
observed initially.
Nevertheless, this enzyme has other actions that
cannot be ascribed solely to intracellular asparagine
depletion[l60]. Although normal cells do not appear to
have an absolute requirement for exogenous L-asparagine,
the administration of L-asparaginase has been associated
with multisystemic toxic effects involving liver[152,160],
pancreas[161,1661, brain[156,158] and kidneys[156,161].
On L-asparaginase administration, abnormalities in
clotting process[141,143,148] and in liver functions[l56,
2201, pancreatitis[162,164], central nervous system
disturbances[l58,161] and renal failure[156,161] have been
reported. A reduction in plasma levels of hepatic
proteins such as lipoproteins[l56,1611, albumin[156,161],
thyroxine binding globulin[23,160], plasminogen[l44],
antithrombin-I11 [221] and fibrinogen[25, 2211 were also
reported. Elevation of serum enzymes[l61], serum
urea[l61] and serum ylucose[166,170] and reduction of
serum lipids[l611 were also reported. Bosman and Kessel
reported a decreased glycoprotein biosynthesis in murine
lymphoma cells[222].
In this section some of the changes on the metabolism
of carbohydrates, lipids, proteins, and ylycoproteins
have been discussed. Some serum enzymes were also
studied as tumor markers for monitoring therapy with
L-asparaginase. The antineoplastic activity of the
Aeromonas L-asparaginase has been compared with that of
commercially available Leunase from -- E. coli which is
largely used very effectively in the treatment of
leukaemia.
MATERIALS AND METHODS
The maintenance and intraperitoneal implantation of
Ehrlich ascites tumor in Balb/c mice were done as
described in Section A. The enzyme preparation
and mode of injections were explained in detail in
Chapters 111 and Section A of this chapter. Mice (50-60
days old) of average weight 15 5 1.5 y were divided into
7 groups, consisting of 6 mice in each group and were
maintained as follows.
Group 1 - Normal mice maintained with normal laboratory feed.
Group 2 - Control group. Ehrlich agcites tumor implanted mice. About 1 x 10 tumor cells were injected into the peritoneal cavity. These mice were not treated and were sacrificed on the 8th day of tumor implantation.
Group 3 - Tumor implanted mice treated with Leunase. 24 hrs after tumor implantation, Leunase was administered through the same route in 3 intermittent doses of 25 units, at an interval of 48 hrs and were sacrificed on the 8th day of tumor implantation.
Group 4 - Tumor implanted mice, treated with Aeromonas L-asparaginase. Injections were done as described earlier, and sacrificed on the 8th day of tumor implantation.
Group 5 - Control mice bearing tumor, that were not treated. Sacrificed on the 15th day of tumor implantation.
Group 6 - Tumor implanted mice treated with Leunase as in Group 3. Sacrificed on the 15th day of tumor implantation.
Group 7 - Tumor implanted mice, treated with Aeromonas L-asparaginase preparation as in Group 4. Sacrificed on the 15th day. of tumor implantation.
The weight of the animal was noted before and after
the experiment.
Blood from mice was collected, after the stipulated
time by cutting the jugular vein with a sharp blade.
0.1 ml blood was removed immediately to 0.9 ml of
0.18 M TCA and was used for the estimation of glucose.
The remaining blood was allowed to clot and the serum was
collected by centrifugation at 1,500 rpm for 10'. This
serum was immediately used for the assay of various serum
enzymes. The liver and kidney were removed to ice cold
containers,after washinywith saline to remove the blood.
500 mg each of kidney and liver were homogenated with 5 ml
distilled water in a Remi homogenizer to get a 10%
homogenate. This homogenate and serum were used for
estimating lipids, total proteins and glycoproteins. The
following estimations were done.
I. Carbohydrate Metabolism
Estimation of blood glucose
Blood glucose was estimated by the 0-Toluidine method
according to the procedure of John D. Bauer[223].
Reagents
a) 0-Toluidine reagent: 6% 0-Toluidine (EM) in glacial
acetic acid containing 150 mg% thiourea.
b) TCA: 3 g TCA (SRL) in 100 ml distilled water.
C) Glucose standard: 100 mg glucose ( A R ) in 100 ml
saturated benzoic acid solution.
Procedure
0.1 ml of the whole blood was added to 0.9 ml of TCA
solution. Mixed well, allowed to stand for a few minutes
and centrifuged at 1,500 rpm for 5'. 0.5ml of the
supernatant was transferred to another test tube. 0.5 ml
TCA was treafed as blank. For standard, different volumes
of 100 mg% glucose were taken and the volumes made up to - 0.5 ml with TCA. 3.5 ml of 0-Toluidine reagent was added
to all the tubes. Heated in a boiling waterbath for 12'.
Cooled and the absorbance was read against blank at
630 nm.
11. Lipid Metabolism
1. Extraction of serum and tissues for lipid estimation
a) Extraction of serum
Serum was extracted according to the procedure of
Folch et a1.[2241.
'n' ml of the serum sample was added drop wise to 5 n
volume of methanol in a stoppered tube. Then 5 n volume
of chloroform was added and mixed. This mixture was
incubated at 5 5 O ~ for 15'. At the end, another 5 n volume
of chloroform was added so that the proportion of
chloroform to methanol was 2:1 (v/v). After filtration
and washing, the residue was washed with chloroform :
methanol (2:l) at least 3 times, and the combined filtrate
was washed with 0.7% potassium chloride solution. (20% of
the total volume of the extract). The aqueous upper ptase
was removed with a Pasteur pipette and the lower layer was
washed each time with 5 ml of chloroform : methanol t 0.7%
potassium chloride solution (3:48:47 v/v). The washed
lower layer of chloroform was evaporated to dryness and
the residue redissolved in known volume of chloroform.
Aliquots were used for the estimation of lipids.
b) Extraction of tissues for lipid estimation
The tissue was homogenized as mentioned before and
extracted with chloroform : methanol (2:l) and processed
as described for serum.
2. Estimation of cholesterol
Total cholesterol was estimated by the method of
Abe11[225].
Reagents
a) Potassium hydroxide (AR), 33%
b) Absolute ethanol
c) Ethanolic potassium hydroxide: 6 ml of 33%
potassium hydroxide in water was added to 94 ml of
absolute ethanol.
d) Petroleum ether (AR) ( 60°c-80°c)
e) Colour reagent: 20 ml of acetic anhydride (AR)
was cooled in ice. One ml of con. sulphuric acid
(AR) was added to this. It was again cooled for
10' and 10 ml of glacial acetic acid (AR) was
added and allowed to attain room temperature.
Procedure
An aliquot of the lipid extract was pipetted out into
a glass stoppered centrifuge tube and was allowed to
evaporate to dryness. 5 ml of ethanolic KOH was added,
stoppered and was shaken well. It was then warmed in a
water bath at 37-41°c for 5 5 ' . After cooling to room
temperature, 10 ml of petroleum ether (60-80°c) was
added and mixed. 5 ml of water was then added to this
and was shaken vigorously for 1'. It was then centrifuged
at a low speed for 5'. 4 ml of petroleum ether layer
was pipetted out into a test tube and evaporated to
dryness at 60°c. A standard was also treated in the same
manner. 6 ml of colour reagent was added to each tube
and kept at 2 5 O ~ after thorough shaking. 6.0 ml of colour
reagent was taken as blank. After 30-35' the optical
density was read at 620 nm.
3. Estimation of triglycerides
Triglycerides were estimated by the method of Van
Handel and Zilversmit[2261 with the modification that
florisil was used to remove phospholipids.
Reagents
a) Chloroform (AR)
b) Florisil (EM)
C) Ethanolic potassium hydroxide (0.4%): 2 g of
potassium hydroxide was dissolved in 100 ml ethanol.
This was then diluted 5 times with ethanol.
d) Sulphuric acid, 0.2 N
e) Sodium metaperiodate (SRL), 0.05 M
f) Sodium arsenite (AR), 0.5 M
g) Chromotropic acid: 2 g of chromotropic acid (SRL)
was dissolved in 200 ml distilled water. 600 ml of
con. sulphuric acid was added slowly to 300 ml of
distilled water which was already chilled in ice.
This chilled and diluted acid was then added to the
chromotropic acid solution (0.05 mg/ml).
Procedure
2 g florisil was taken in a glass stoppered tube
and 3 ml of chloroform was added. An aliquot of the
extract was layered or the top of florisil and mixed.
Chloroform was then added to this to a total of 10 ml.
It was then stoppered and was shaken intermittently for
about 10'. After filtration, one ml was pipetted out into
each of the 3 tubes. One ml of the working standard was
similarly pipetted out into each of the 3 tubes. The
solvent was evaporated at 60-70°c. Then 0.5 ml of ethanol
was added to the third tube. The tubes were closed and
kept at 60-70°c for 15'. To each tube, 0.5 ml of 0.2 N
sulphuric acid was added and then placed in a gently
boiling water bath for about 15' to remove alcohol. These
were then cooled to room temperature, 0.1 ml sodium
metaperiodate was added to each tube and kept for 10'.
0.1 ml sodium arsenite solution was added. An yellow
colour of iodine appeared and vanished within a few
minutes. To each tube, 5 ml of chromotropic acid was
then added and mixed. The tubes were closed and heated in
a boiling water bath for 30'. These were then cooled and
the absorbance was read at 570 nm.
4. Estimation of phospholipids
~hospholipids were estimated by the method of
Zilversmit and Davis[227].
Reagents
a) Sulphuric acid ( AR) , 5 N
b) Ammoniummolybdate (AR), 2.5%
c) ANSA: 0.2 g of 1-amino 2-naphthol 4-sulfonic acid
(EM) was mixed with 1.2 g of sodium bisulphite and
1.2 g of sodium sulphite. 0.25 g was taken from this
mixture and dissolved in 10 ml of water.
Procedure
An aliquot of the extract was pipetted out into a
Kjeldahl flask and evaporated to dryness. One ml of 5 N
sulphuric acid was added and digested in a digestion rack
till it became light brown. It was then-cooled to room
temperature. One or two drops of 2 N nitric acid were
added and digested again till it became colourless. The
Kjeldahl flask was cooled, one ml of water was added and
heated in a boiling water bath for 5 ' . One ml of 2.5%
ammonium molybdate and 0.1 ml of ANSA were added and the
volume was made up to 10 ml with distilled water. The
absorbance was measured at 660 nm within 10'.
111. Protein Metabolism
1. Estimation of protein
The protein content of the serum and tissues were
estimated by the method of Lowry et a1.[190].
2. Estimation of blood urea
Blood urea was estimated by the diacetyl monoxime
method according to the procedure of Natelson et a1.[228].
Reagents
a) Sodium tungstate (SRL), 10%
b) Sulphuric acid (AR), 2/3 N
C) Diacetyl monoxime (AR), 2% solution in 2% acetic acid
d) Sulphuric acid - Phosphoric acid reagent: 150 ml of
85% phosphoric acid (AR) and 50 ml con. sulphuric
acid mixed with 140 ml distilled water.
Procedure
0.1 ml of the blood was mixed with 3.3 ml water and
0.3 ml 10% tungstate and 0.3 ml 2/3 N sulphuric acid, were
added. Mixed well and centrifuged. To one ml of the
supernatant solution added one ml of water, 0.4 ml
diacetyl monoxime and 1.6 ml of sulphuric acid-phosphoric
acid reagent, placed in a boiling water bath for 3 0 ' ,
cooled and the absorbance was read against a water blank
at 480 nm. At the same time, colour was developed
from one ml of the standard urea containing 0.025 mg urea
per ml.
IV. Glycoprotein Metabolism
1. Extraction of glycoproteins from the tissues
a) Preparation of dry defatted tissue
Acetone dry powder was prepared by keeping the minced
0 tissue or serum in acetone at 0 C for 72 hrs. The acetone
was changed every 24 hrs. The tissue was then extracted
with ether : acetone (3:1, v/v) at 3 7 O ~ for 1 hr followed
by ether for 1 hr. The defatted tissue was then dried
in vaccum to constant weight.
b) Papain digestion
The dry defatted tissue was digested with papain
(crystalline papain (SRL), one third the dry weight of the
tissue) for 7 2 hrs at 6 5 O ~ in 0.2 M acetate buffer
(pH 7.0) containing 2 mg cysteine hydrochloride/ml.
Fresh papain was added every 24 hrs. The digest was then
cooled to room temperature and 4-5 volumes of ethanol was
added at OOC and kept at this temperature for 24 hrs.
It was centrifuged at 5,000 rpm for 5' and the supernatant
was evaporated to dryness in the cold in vaccum. The
residue was dissolved in a known volume of water and
aliquots were used for the analysis of carbohydrate
components. The procedure used was similar to that
described by Wayh et a1.[2291, except that ethanol was
used instead of TCA to deproteinize the digest, since TCA
keeps the tissue glycogen and ylycosaminoglycans in
solution.
2 Estimation of total hexose
Total hexose was estimated by phenol-sulphuric acid
method of Dubois et a1.[230].
Reagents
a) Phenol ( AR) , 5%
h) Con. sulphuric acid
Procedure
An aliquot of the test solution was pipetted out into
a test tube and made upto one ml. One ml of 5% phenol was
added to this and mixed. Appropriate standards
(10-70p g of galactose) and blank were treated
similarly. 5 ml con. sulphuric acid was added to each
tube directly to produce good mixing and heat
distribution. After lo', the tubes were shaken and placed
in a water bath at 25-30°c for 20'. The absorbance was
measured at 490 nm.
3. Estimation of fucose
Fucose was estimated by the method of Dische and
Shettles[231].
Reagents
a) Sulphuric acid reagent: 6 volumes of con. sulphuric
acid and one volume of distilled water were mixed in
the cold and stored in refrigerator.
b) Cysteine reagent: A 3% aqueous cysteine hydrochloride
(SRL) solution was made just prior to use.
Procedure
Samples and standards containing 2-20 k g of fucose
in one ml of water were taken in 18 x 150 mm Pyrex tubes.
A water blank was also prepared. The'tubes were cooled in
ice bath. 4.5 ml of cold sulphuric acid reagent was added
to each tube and shaken vigorously. The tubes were then
brought to room temperature by placing them in a water
bath at 20-22Oc for a few minutes. These were then closed
and placed in a vigorously boiling water bath for exactly
3' and cooled to room temperature. 0.1 ml of cysteine
reagent was adezd to each tube with immediate mixing.
A control tube was also taken for each test and blank
which contained 4.6 ml of sulphuric acid reagent and no
cysteine hydrochloride. The absorbance was read after
1-2 hrs at 396 nm and 427 nm. The absorbance due to
methyl pentose in a given sample was determined by
subtracting OD396 - OD427 of the sample analysed without
cysteine from the OD396 - OD427 of the sample enslysed
with cysteine.
4. Estimation of sialic acid
Sialic acid was estimated by the thiobarbituric acid
method of Warren[232].
Reagents
a) Sodium metaperiodate (SRL), 0.2 Y in 9 M phosphoric
acid.
b) Sodium arsenite (AR), 10% in 0.5 M sodium sulphate
(AR) solution.
C) 2-thiobarbituric acid (EM), 0.6% in 0.5 M sodium
sulphate.
d) Cyclohexanone (SRL).
Procedure
Samples were first hydrolysed in 0.1 N sulphuric acid
at 8 0 O ~ for 1 hr. The samples and standards should
contain 2-18 /' g of sialic aoid dissolved in 0.2 ml water.
It was not necessary to neutralize the acid from the
sulphuric acid hydrolysis. 0.2 ml distilled water was
taken as blank. 0.1 ml of periodate solution was added to
each tube and mixed. It was allowed to stand at room
temperature for 20' and one ml of sodium arsenite was then
added to this. The tubes were shaken until the yellowish
brown colour had disappeared. 3 ml of 2-thiobarbituric
acid solution was added to each tube and the contents
mixed. The tubes were closed, heated in a vigorously
boiling water bath for 15' and then cooled in water for
15'. 5 ml of cyclohexanone was added to each tube and
shaken vigorously in order to extract the red colour into
the organic phase. The tubes were centrifuged for about
3' at 1000 rpm. The clear upper layer was transferred and
the absorbance was read at 549 nm.
V. Assay of Serum Enzymes
1. Estimation of serum lactate dehydrogenase
The serum LDH level was determined by the method of
King[233].
Reagents
a) Glycine buffer, 0.1 M: 7.505 g glycine (SRL) and
5.85 g sodium chloride (AR) in one litre distilled
water.
b) Buffered substrate: Added 125 ml glycine buffer and
75 ml 0.1 N sodium hydroxide to 5 ml of 75% sodium
lactate (EM) solution.
C) NAD: 10 mg NAD (Sigma) in 2 ml distilled water.
Kept at OOC.
d) 2,4 dinitrophenylhydrazine reagent: 20 mg DNPH (EM)
in 100 ml hot 1 N hydrochloric acid.
e) sodium hydroxide, 0.4 N
Procedure
Pipetted out one ml of buffered substrate and 20 /' 1
of serum into each of two tubes. Added 0.2 ml of
distilled water into the blank tube and placed both tubes
in a water bath at 37O~. Allowed to reach the temperature
of the bath. Then to the other tube (test), added 0.2 ml
NAD solution and shaken to mix. Exactly 15' after adding
the NAD, one ml of DNPH solution was added to each, shaken
to mix and left in the water bath for 20'. Then removed
from the bath and to each tube added 10 ml of 0.4 N
sodium hydroxide and the absorbance was read at
440 nm within 5'. Sodium pyruvate solution containing
one micromole of pyruvate per ml was used as standard.
2. Estimation of serum transaminases
Serum levels of alanine and aspartate transaminases
were determined by the method of Mohun and CookL2341.
Reagents
a) Potassium phosphate buffer, 0.075 M (pH 7.5)
b) Buffered substrates:
(i) Aspartate transaminase - 300 mg L-aspartic
acid (Sigma) and 50 mg alpha ketoglutaric acid
(Sigma) in 100 ml phosphate buffer and the pH
was adjusted to 7.5 with sodium hydroxide.
(ii) Alanine transaminase - 5 y DL-alanine (Sigma)
and 20 mg alpha ketoglutaric acid (Sigma) in
100 ml phosphate buffer and the pH was adjusted
to 7.5 with sodium hydroxide.
C) Aniline - Citrate reagent: 50 y citric acid (SRL) in
50 ml of distilled water and to this, an equal volume
of redistilled aniline (EM) was added.
d) Dinitrophenylhydrazine reagent: Dissolved 200 mg of
2,4 dinitrophenylhydrazine (EM) in 85 ml of con.
hydrochloric acid and made up to one litre with
distilled water.
e) Sodium hydroxide, 0.4 N
Procedure
Pipetted out one ml of substrate into two tubes and
placed in a water bath at 3 7 O ~ for a few minutes. To one,
added 0.2 ml serum and shaken gently to mix. Exactly an
hour later in the case of aspartate transaminase and after
30' in the case of alanine transaminase, with the tubes
still in the bath, added 2 drops of aniline-citrate
reagent to both, and 0.2 ml serum to the control tgbe and
left undisturbed for 20'. Then to both the tubes added
one ml of the dinitrophenylhydrazine reagent and left for
another 20'. Then removed from the bath and added 10 ml
0.4 N sodium hydroxide and the absorbance was read at
520 nm after 10'. A solution of sodium pyruvate
containing 10 mg pyruvate per ml was used as standard.
3) Serum phosphatases
Serum phosphatases were assayed following the
procedure o f King and Jagatheesan[235] using
4-aminoantipyrine.
(i) Alkaline phosphatase
Reagents
a) Disodium phenyl phosphate (EM), 0.01 M in distilled
water
b) Sodium carbonate - bicarbonate buffer - 0.1 M: 3.18 g anhydrous sodium carbonate and 1.68 g sodium
bicarbonate dissolved in 500 ml distilled water.
c) Buffered substrate for use: Prepared by mixing equal
volumes of reagent a & b.
d) Sodium hydroxide, 0.5 N
e) Sodium bicarbonate, 0.5 M
f) 4-aminoantipyrine (Sigma), 0.6% in distilled water
g ) Potassium ferricyanide, 2.4% in distilled water
Procedure
2 ml of the buffered substrate was incubated at 3 7 O ~
for a few minutes in a water bath. Then 0.1 ml serum was
added and incubated again for exactly 15'. Removed from
the bath and added 0.8 ml of 0.5 N sodium hydroxide and
1.2 ml of 0.5 M sodium bicarbonate. Similarly another
tube was kept to which serum was added only after adding
sodium bicarbonate. This tube was used as the blank. To
both the tubes added one ml aminoantipyrine reagent and
one ml of potassium ferricyanide solution. For the
standard, 1.1 ml of the buffer and one ml of phenol
solution containing 0.01 my of phenol, and for the
standard blank 1.1 ml buffer and one ml water were taken.
To both tubes, added sodium hydroxide, bicarbonate,
aminoantipyrine and ferricyanide as above. The absorbance
was read at 520 nm.
(ii) Total acid phosphatase
Reagent
a) Citric acid - sodium citrate buffer, 0.1 M (pH 4 . 9 )
All the other reagents were same as for alkaline
phosphatase.
Procedure
The procedure was the same as used for alkaline
~hosphatase except that the citric acid - citrate buffer
was used for preparing the buffered substrate and
incubation was for 1 hr. For developing the colour with
aminoantipyrine, one ml of sodium hydroxide and one ml of
bicarbonate were added.
(iii) Tartarate labile acid phosphatase
The procedure was exactly the same as above, except
that in addition to the test and blank tubes, a third tube
containing 2 ml of buffered substrate, one drop of one
molar L - ( + ) tartarate and one ml serum was also incubated
and the rest of the procedure was exactly the same as for
the total acid phosphatase. The tartarate labile acid
phosphatase was calculated by subtracting this value from
the total acid phosphatase level.
RESULTS AND DISCUSSION
I. Gain in Weight
Gain in weight during tumor growth and L-asparaginase
treatment in mice was noted. The results are given in
Table IV.5. and the 't' values are given in Table IV.S(a).
Table IV.5.
Gain in weight
Table IV.S(a).
* t ' values
Groups Weight (g)
't' between 't' values groups
Average of 6 values in each group + SEM. Group 2 & 5 have been compared with Group 1. Group 3 & 4 have been compared with Group 2. Group 6 & 7 have been compared with Group 5.
b - p between 0.01 and 0.05
Increase in weight was observed in cancer groups when
compared to the normals. It can be seen that the increase
in body weight was proportional to the tumor growth but
Group 5 showed a decrease when compared to Group 2.
Raha et al. suggested that an increase in body weight can
be taken as a measure of tumor burden[56]. The slight
decrease in weight in Group 5 when compared to Group 2
might be due to the regression of tumor. Similar
observation was reported by Raha et a1.[561. I= will. be
quite logic to conclude that this increase in body weight
during the early stage of tumor growth will be due to
increased cell proliferation and slight decrease during
advanced stage of tumor will be due to the development of
non healthy physiology and regression of tumor.
L-asparaginase treated groups showed a decrease in
body weight. Decrease became more marked with increase in
time. Such a decrease in body weight upon L-asparaginase
therapy has been reported by many workers[154,157,1611.
11. Carbohydrate Metabolism
Glucose levels in blood
Blood glucose levels were studied in mice bearing
tumor and in mice treated with L-asparaginase. The
results are given in Table IV.6. and the 't' values are
given in Table IV.6(a).
Tumor cells have high rate of ylycolysis required for
their rapid proliferation[l83]. This high rate of
glycolysis reduced the blood glucose level as observed in
Groups 2 and 5. This decrease in blood glucose level
became more marked with increase of tumor burden, showing
a higher decline in Group 5.
Table IV.6.
~lood glucose levels in tumor bearing and L-asparaginase
treated mice
Table IV.6(a)
' t ' values
Groups Blood glucose 't' between ' t' values level groups
1 147.30 - + 8.05 1 & 2 0.42
5 124.66 - + 8.59 5 & 6 2.22
6 158.33 2 12.53 5 & 7 2.74
7 164.20 - + 11.54~
Values are expressed as my glucose/100 ml blood
Average of 6 values in each group + SEM - Group 2 & 5 have been compared with Group 1. Group 3 & 4 have been compared with Group 2. Group 6 & 7 have been compared with Groug 5.
b - p between 0.01 and 0.05
~dministration of both L-asparaginases caused an
increase in the blood glucose level. This increase was
more marked immediately after L-asparaginase injection and
a partial reversibility was observed few days after the
ceassation of therapy. Hypoinsulinemic hyperglycemia
responding to insulin that shows a partial recovery upon
ceassation of L-asparaginase therapy was observed by many
workers[161,168,170,171,236]. Eventhough many mechanisms
have been suggested including decreased synthesis of
insulin[l641, impairment of insulin release by pancreatic
islets[1661r decreased binding of insulin to the cell
receptors[l681 and post receptor mechanisms[l69], the
exact mechanism for hypoinsulinism and hyperglycemia
associated with L-asparaginase therapy is not established
so far.
111. Lipid Metabolism
1 ) Cholesterol
Cholesterol levels in serum and tissues (liver and
kidney) in untreated and treated groups were compared with
the normal groups. The results are given in Table IV.7.
and the 't' values are given in Table IV.7(a).
Table IV.7.
Serum and tissue cholesterol levels in tumor bearing and L-asparaginase treated mice
Tissues Groups Serum ..................................
Liver Kidney
Values are expressed as my cholestero1/100 ml serum or 100 g wet tissue
Average of 6 values in each group 2 SEM
Group 2 & 5 have been compared with Group 1. Group 3 & 4 have been compared with Group 2. Group 6 & 7 have been compared with Group 5.
a - p less than 0.01 b - p between 0.01 and 0.05
Table IV.7.(a): 't' values
't' between ' t ' values groups ........................................
Serum Liver Kidney
2) Triglycerides
Triglyceride levels in serum and tissues (liver and
kidney) of mice bearing tumor and those treated with
L-asparaginase were studied and these were compared with
those of normal mice. The results are given in
Table IV.8. and the 't' values are given in Table IV.8(a).
Table IV.8.
Triglyceride levels in serum and tissues of tumor bearing and L-asparaginase treated mice
Tissues Groups Serum ..............................
Liver Kidney
Values are expressed as mg glycero1/100 ml serum or 100 g wet tissue
Average of 6 values in each group 2 SEM
Group 2 & 5 have been compared with Group 1. Group 3 & 4 have been compared with Group 2. Group 6 & 7 have been compared with Group 5.
a - p less than 0.01 b - p between 0.01 and 0.05
Table 8(a): ' t ' values
't' between ' t ' values groups .........................................
serum Liver Kidney
3) Phospholipids
The phospholipid levels in serum and tissues (liver
and kidney) of tumor bearing and L-asparayinase treated
mice were studied and compared with those of normal mice.
The results are given in Table IV.9. and the 't' values
are given in Table IV.g(a).
Table IV.9.
Serum and tissue phospholipid levels in tumor bearing and L-asparaginase treated mice
Tissues Croups Serum .................................
Liver Kidney
Values are expressed as mg phospholipids/lOO ml serum or 100 g wet tissue
Averaye of 6 values in each group 2 SEM
Group 2 & 5 have been compared with Group 1. Group 3 & 4 have been compared with Group 2. Group 6 & 7 have been compared with Group 5.
a - p less than 0.01 b - p between 0.01 and 0.05
Table IV.g(a): 't' values
't' between ' t ' values groups .........................................
Serum Liver Kidney
Cholesterol levels in serum, liver and kidney were
found to be decreased significantly with the progression
of tumor growth.
Highly elevated cholesterol level was observed in
liver during the administration of L-asparaginase, but no
significant change was observed in serum and kidney.
Significant elevation of cholesterol levels in serum and
in kidney were observed after the ceassation of therapy.
This increase was pronounced in groups treated Q i t h
Aeromonas L-asparaginase. L-asparaginase administration
resulted in concomitant increase of liver cholesterol
levels. Groups treated with Aeromonas L-asparaginase
showed a partial reversion to near normal level.
A highly significant elevation of the triglyceride
level in serum was found duriny the early stages of tumor
growth and no alterations in triglyceride levels were
found in liver and kidney. Very significant elevation of
serum and liver triglyceride levels was found during the
later stages of tumor growth but no alterations were
observed in kidney.
Administration of L-asparaginase did not cause any
immediate alteration in the triglyceride level of serum.
A slight increase was observed in liver and kidney. A few
days after the ceassation of therapy, a significant
reduction in triglyceride level in serum was observed. In
groups treated with Aeromonas L-asparaqinase, serum
triglyceride values returned to near normal.
Serum phospholipid level increased significantly
during the early stage of tumor growth and phospholigid
level in kidney was found to be decreased. The serum
phospholipid level remains elevated at a later stage of
tumor growth, whereas kidney and liver phospholipid levels
returned to near normal at a later stage of tumor growth.
Phospholipid level in serum was found to be elevated
after L-asparaginase administration. Ceassation of
therapy resulted in a significant decrease in groups
treated with Aeromonas L-asparaginase and no significant
decrease was observed in groups treated with Leunase.
Phospholipid level in liver was not altered significantly
upon the administration of either -- E. coli or Aeromonas
L-asparayinase. The phospholipid level in kidney
showed no decrease immediately after L-asparaginase
administration but a few days after the ceassation of
therapy, the level decreased significantly. This
decrease was more marked in yroups treated with -- E. coli
L-asparaginase.
The lipid profile in patients with malignancies was
studied by Kritchevsky et al. and reported that
cholesterol level in patients with non localized
maligancies dropped below the normal leve1[237]. This is
in agreement with our present findings, here also the
cholesterol level showed a significant decrease with the
progression of the tumor growth. These alterations might
be useful as differential markers with the more mature
leukaemia having more complex glycolipids as suggested by
William et a1.[238]. He found that the composition of
neutral glycosphingolipids in acute leukaemic cells
differs significantly from that found in normal or chronic
leukaemic cells.
Only a few reports are available on the effect of
L-asparaginase on lipid metabolism. Low serum cholesterol
has been reported and has been shown to be related to
decrease in the serum alpha, pre-beta and beta
lipoproteins[l56,161]. Hypocholesterolemia associated with
L-asparaginase therapy may be due to decreased synthesis
of proteins required for the transport of
cholesterol[l72]. Reports available suggests that there is
no clear relationship between tumor growth and
triglyceride pattern. However, in the present study we
observed a significant elevation of serum triglyceride
level. The increased triglyceride levels observed in
serum and liver during tumor growth might be due to
decreased rate of catabolism of triglycerides or may be
due to increased biosynthesis. These increased levels may
be due to the decreased production of enzymes concerned
with the catabolism of triglycerides. Since lipoprotein
lipase, triglyceride lipase and other enzymes of
triglyceride/lipoprotein metabolism could not be studied,
a definite conclusion could not be drawn. At a later
stage of therapy, the triglyceride levels decreased with
Aeromonas L-asparaginase and the value returned to near
normal level. A decrease in the triglyceride level in
the serum during therapy was an important observation
and was observed only with Aeromonas L-asparaginase
treated groups, when compared to the Leunase. This
reduces the secondary effect of tumor in inducing
artherosclerosis, since this is an important risk factor
in artherosclerosis. A significant increase in
phospholipid level was observed in fatty liver. The
elevated levels of phospholipids and triglycerides in
liver and kidney in the present study may be due to fat
infiltration, as suggested by many workers[161,172,173].
Rnalysis of the neutral glycolipid content of the
normal and leukaemic blood cells showed great differences
in their distribution [239].~ypocholesterolemia observed
in both L-asparaginase treated and non treated tumor
bearing mice in the present study is in agreement with
that suggested by Kritchevsky et a1.[237]. Decreased
cholesterol level may be due to the growth of tumor
burden[237] rather than by the direct action of
L-asparaginase. Histological examination of the liver
showed fat infiltration in man during the first two weeks
of treatment which subsides upon ceassation of
therapy[17,156,161]. Yoon Kang Mook et al. observed fat
infiltration and numerous irregular sized lipid droplets
in the hepatic parenchymal cells of mice treated with
L-asparaginase[l73]. In the present study we also
observed such an elevated amount of triglycerides in liver
and kidney.
IV. Protein Metabolism
1) Total protein
Changes in the protein content of serum, liver and
kidney were estimated during tumor growth and treatment
with L-asparaginase. The results are given in Table IV.10
and the 't' values are given in Table IV.lO(a).
Table IV.lO.
Serum and tissue protein levels in tumor bearing and L-asparaginase treated mice
Tissues Groups Serum ...............................
Liver Kidney
Values are expressed as y protein/100 ml serum or 100 g wet tissue
Average of 6 values in each group 2 SEM
Group 2 & 5 have been compared with Group 1. Group 3 & 4 have been compared with Group 2. Group 6 & 7 have been compared with Group 5.
a - p less than 0.01 b - p between 0.01 and 0.05
Table IV.lO(a): It' values
't' between ' t' values groups ........................................
Serum Liver Kidney
2) Blood urea
The blood urea levels in tumor beariny and
L-asparaginase treated groups were determined and
these were compared with the controls. The results
are giveninTable IV.11 and the 't' values are given in
Table IV.ll(a).
Table IV.11.
Blood urea levels in tumor bearing and L-asparaginase treated mice
Table IV.ll(a)
' t' values
Groups Blood urea 't' between groups 't' values
Values expressed as mg urea/100 ml serum
Average of 6 values in each group + SEM Group 2 & 5 have been compared with Group 1. Group 3 & 4 have been compared with Group 2. Group 6 & 7 have been compared with Group 5.
a - p less than 0.01 b - p between 0.01 and 0.05
During the early phase of tumor growth there was a
significant reduction in the protein content of the serum
and kidney but no significant change was observed in the
protein content of liver. As tumor growth proceeded
(Groap 5) the difference in protein content became less
significant and there was little difference in the amount
of protein between the normal and the 15 days old tumor
bearing mice.
In all the cases, administration of L-asparaginase
caused a very significant reduction in the protein content
of the serum, liver and kidney. This reduction was more
marked in the proteins of serum. There was no appreciable
difference between the groups treated with Leunase and
Aeromonas L-asparaginase. Reduction in serum protein
level persisted for long time as observed in Group 6
and 7, but protein levels of kidney and liver returned to
normal levels to some extent. In all the groups the
variations in protein content were more marked in serum
than in liver or kidney.
Blood urea level was found to be increased with
increase of tumor burden and treatment with L-asparayinase
brought this value to near normal level. Significant
increase in urea content of the blood was observed 8 days
after tumor i~nplantation and naximum increase was observed
after 15 days of tumor implantation. After the ceassation
of L-asparaginase therapy the blood urea was found to be
equal to that of the normal group.
Numerous reports are available on the reduction of
protein synthesis upon L-asparaginase administration.
This drug causes partial inhibition of synthesis of
fibrinogen[25,221,2401r thyroxine binding globulin[160,
241,2421, sex hormone binding globulin[23], coagulation
factors[141,144-148,2431, lipoproteins[l561, albumin[l56,
1611. plasminogen[l481 and other proteins[156,220].
It has been shown that the proteins synthesized in the
presence of L-asparaginase do not have an abnormal rate
of catabolism and the reduction in protein content upon
L-asparaginase therapy may be due to the direct inhibition
of protein synthesis by L-asparaginase, by reducing the
level of available L-asparagine[21,24,25]. A reduction
in protein synthesis in rat hepatocytes was observed by
Villa et al. when the liver tissue was cultured with
L-asparaginase[241. They observed that protein
synthesis is inhibited when the glutamine level reaches a
critical leve1[241. This may be due to the presence of
L-glutaminase activity in the L-asparaginase preparation.
Ollenschlaeger et al. supported this view and suggested
that L-glutaminase activity is responsible for the reduced
hepatic synthesis of proteins[l52]. L-asparayinase causes
a rapid decrease in the serum and cellular level of
glutamine124.244-2461. Bartalena et al. have suggested
that different proteins have different thresholds to the
action of this drugLl601. L-asparaginase produced a dose
and time dependent inhibition of the synthesis of
proteins. Apart from the inhibition of protein synthesis,
L-asparaginase also caused degradation of newly
synthesized. proteins[l60].
Some of the patients treated with L-asparaginase
showed an elevated level of blood ammonia[l7] and some
showed renal failure with oliguria[161,2471. In the
present study there was no increase in blood urea level
in groups treated with L-asparaginase, contrary to that
suggested by Whitecar et a1.[1611. He observed an
elevated level of blood urea on administration of
L-asparayinase. According to him the elevated blood urea
level might he due to increased production of ammonia from
L-asparagine by L-asparaginase. One cannot stick on to
the above explanations since normal kidney would eliminate
the excess amount of urea by glomerular filtration. The
increased blood urea level in tumor bearing mice might be
due to the damage or dysfunction of kidney caused by
tumor growth. A decrease in blood urea level during
L-asparaginase therapy in the present study indicates that
L-asparaginase therapy might have corrected the kidney
damage or dysfunction caused by the tumor.
V. Glycoprotein Metabolism
1) Protein bound hexose
Protein bound hexose levels in serum, liver and
kidney were estimated in tumor bearing and L-asparaginase
treated mice. The results are given in Table IV.12 and
the 't' values are given in Table IV.lZ(a).
Table IV.12.
Protein bound hexose levels in serum and tissues of tumor bearing and L-asparaginase treated mice
Tissues Groups Serum ...............................
Liver Kidney
Values are expressed as mg hexose/g dry defatted tissue
Average of 6 values in each group - + SEM
Group 2 8 ' 5 have been compared with Group 1. Group 3 8 4 have been compared with Group 2. Group 6 8 7 have been compared with Group 5.
a - 2 less than 0.01 b - p kzueen 0.01 and 0.05
Table IV.l2(a) : 't' values
't' between ' t ' values groups ............................................
Serum Liver Kidney
2) protein bound fucose
Protein bound fucose levels in serum, liver and
kidney of tumor bearing and L-asparaginase treated
mice were studied. The results of the study are given
in Table IV.13 and the 't' values are given in
Table ZV.i3(a).
Table IV.13.
Protein bound fucose levels in serum and tissues of tumor bearing and L-asparaginase treated mice
Tissues Groups Serum ...............................
Liver Kidney
Values are expressed as mg fucose/g dry defatted tissue
Average of 6 values in each group -+ SEM Group 2 & 5 have been compared with Group 1. Group 3 & 4 have been compared with Group 2. Group 6 & 7 have been compared with Group 5.
a - p less than 0.01 b - p between 0.01 and 0.05
Table IV.l3(a): 't' values
't' between ' t ' values groups ...........................................
Serum Liver Kidney
3) Protein bound sialic acid
Protein bound sialic acid levels of serum, liver and
kidney were studied in tumor bearing and L-asparaginase
treated mice. The results of the study are given
in Table IV.14. and the 't' values are given in
Table IV.l4(a).
Table IV.14.
Protein bound sialic acid levels in serum and tissues of tumor bearing and L-asparaginase treated mice
Groups Tissues
Serum .............................. ~iver Kidney
Values are expressed as mg sialic acid/g dry defatted tissue
Average of 6 values in each group + SEM Group 2 & 5 have been compared with Group 1. Group 3 & 4 have been compared with Group 2. Group 6 & 7 have been compared with Group 5.
a - p less than 0.01 b - p between 0.01 and 0.05
Table IV.l4(a): 't' values
't' between ' t' values groups __-__________-_____-_--_----_------------
Serum Liver Kidney
Serum protein bound hexose level was found to be
increased significantly during tumor growth and this
increase became more significant during the later phase.
It has been found that there was a significant reduction
in the amount of protein bound hexose in kidney during
tumor growth. This reduction was pronounced in the
later phase of tumor growth than in the early phase.
There was no significant alteration in the amount of
protein bound hexose in liver during the early phase of
tumor growth. However, at a later phase of tumor growth a
reduction in the protein bound hexose level in liver was
observed.
~dministration of L-asparaginase caused a significant
reduction in the serum protein bound hexose level and the
values reached near normal levels. On the other hand,
L-asparaginase caused a significant increase in the
protein bound hexose level in kidney, concomitantly with
L-asparaginase administration and reverted to near normal
level after ceassation of therapy. There was no
significant alteration in the protein bound hexose level
in liver upon L-asparaginase administration. The same
pattern was observed with both enzyme preparations.
Protein bound fucose level in serum increased with
tumor growth and was highly significant during the later
phase of tumor growth. The protein bound fucose level in
kidney was reduced significantly during the later phase,
though no considerable change was observed during the
early stage. No significant alteration was found in
protein bound fucose in liver during the early phase of
tumor growth. However, there occured a reduction in the
protein bound fucose level of liver at a later stage of
tumor growth.
No significant alteration in the level of serum
protein bound fucose was obtained immediately after
L-asparaginase therapy. However, administration of
L-asparaginase preparations caused a significant reduction
in the serum fucose level during the later stage.
The protein bound fucose levels of kidney and liver . were not significantly altered on L-asparaginase
administration.
Protein bound sialic acid in serum was found to be
increased during the early stages of tumor growth, but no
alterations were found in liver and kidney. Significant
elevation of protein bound sialic acid level in serum was
observed during the later phase of tumor growth and a
significant reduction was observed in liver and kidney.
The decrease was more pronounced in kidney than in liver.
There were no siynificant alterations in the levels
of protein bound sialic acid in serum and tissues,
immediately after administration of L-asparaginase from
V. coli and Aeromonas. -- A significant reduction in the
level of serum protein bound sialic acid was observed at a
later staye of L-asparaginase therapy. Moreover, an
elevation of the protein bound sialic acid level was found
in kidney on administration of L-asparaginase preparation
from Aeromonas at a later stage. Administration of E. coli -- L-asparaginase produced no significant change. Protein
bound sialic acid level in liver was found to be unaltered
with the administration of L-asparaginases from both the
sources.
Numerous reports on the glycoprotein pattern of
tumor cells are available[248-2511. Comparison of
glycoprotein pattern of normal and cancerous cells would
give valuable information about the molecular changes that
occur in different malignancies[249,252].
Several types of neoplastic transformations are
accompanied by alterations in the composition of cell
glycoproteins which are the major structural components of
the cell surface. One such alteration observed is in the
level of sialic acid on the cell surface. Changes in the
sialic acid level of patients sera reflect growth
process of benign and malignant characters[l77,178].
In monitoring patients with pre therapeutically elevated
sialic acid content in the serum, drooping values
indicate successful therapeutic intervention, and an
increase of post therapeutic values, demonstrate recurrent
process[l77]. Precise determination of the sialic acid
provides information that correlates with clinical status
of cancer patients and thus may be useful as a monitor of
clinical course and response to treatment. The plasma
sialic acid level correlates well with the progression and
regression of diseaseLl771. In our study also similar
results were obtained. However, assay of plasma sialic
acid is not sensitive enough to be used for screening, but
can be used as a prognostic determinant in a variety of
neoplastic conditions as suggested by Dwivedi et a1.[179].
Glycoproteins containing sialic acid are proved to be
responsib1.e for the elevated sialic acid level in the
serum instead of lipid bound sialic acid[l80].
Vellenga et al. reported a reduction of histidine
rich glycoproteins and plasminogen on L-asparaginase
therapy[1481. -- E. coli L-asparaginase is reported to
decrease the glycoprotein biosynthesis in murine
lymphomas[222]. -- E. coli L-asparaginase does not have
proteolytic or glycolytic activities, eventhough it alters
the receptor glycoproteins of the cell surface[l74].
~sparagine is found in many glycoproteins where a
linkage exists between the beta amide of asparagine and
N-acetyl glucosamine residue of the oligosaccharide
chains. Removal of asparagine by L-asparaginase may lead
to decreased glycosylation of proteins resulting in
decreased biosynthesis of glycoproteins in tissues. The
increased level of glycoproteins in the serum may be
derived from the solubilized cell membranes. The exact
mechanism by which L-asparaginase causes alterations in
the glycoprotein pattern is not clear, althouyh it does
not seem to be caused by the proteolytic and glycolytic
activity of the enzyme preparation. Perhaps it may be due
to the generalized effect on protein and glycoprotein
biosynthesis.
VI. Serum Enzymes as Tumor Markers
1) Serum lactate dehydrogenase
Lactate dehydrogenase activity in the serum of tumor
bearing and L-asparaginase treated mice were estimated.
The results are given in Table IV.15. and the 't' values
are given in Table IV.lS(a).
Table IV.15 Table IV.15 (a)
Lactate dehydrogenase activity in serum of tumor bearing and L-asparaginase treated mice
' t ' values
Groups Lactate dehy- 't' between 't' values drogenase groups activity
Values are expressed as IU/100 ml serum
Average of 6 values in each group - + SEM
Group 2 & 5 have been compared with Group 1. Group 3 & 4 have been compared with Group 2. Group 6 & 7 have been compared with Group 5.
a - p less than 0.01
2) Serum transaminases
The serum transaminases activity were detected in
tumor bearing mice and mice treated with L-asparaginase.
The results are given in Table IV.16 and the 't' values
are given in Table IV.l6(a).
Table IV.16.
Serum transaminase levels in tumor bearing and L-asparaginase treated mice
Groups Alanine Aspartate transaminase transaminase activity activity
Values are expressed as IU/100 ml serum
Average of 6 values in each group + SEM Group 2 & 5 have been compared with Group 1. Group 3 & 4 have been compared with Group 2. Group 6 & 7 have been compared with Group 5.
a - p less than 0.01 b - p between 0.01 and 0.05
Table IV.l6(a) : 't' values
't' between ' t ' values groups ..................................
Alanine Aspartate transaminase transaminase
- -
3) Serum phosphatases
Phosphatase activities in the serum of tumor beariny
and L-asparaginase treated mice were studied. The results
are given in Table IV.17 and the 't' values are given in
Table IV.l7(a).
Table IV.17.
serum phosphatase levels in tumor bearing and L-asparaginase treated mice
Total acid Tartarate Alkaline Groups phosphatase labile acid phosphatase
phosphatase
Values are expressed as King Amstrong units/100 ml serum
Average of 6 values in each group + SEM - Group 2 & 5 have been compared with Group 1. Group 3 & 4 have been compared with Group 2. Group 6 & 7 have been compared with Group 5 .
a - p less than 0.01 b - p between 0.01 and 0 . 0 5
Table 1~.17(a): 't' values
't' between ' t ' values groups _____________-_____--------_---_------------
Total acid Tartarate Alkaline phosphatase labile acid phosphatase
phosphatase
There was a significant rise in the serum level of
lactate dehydrogenase with tumor growth and there occured
a significant partial reversion in the level of LDH during
therapy with both L-asparaginases. Few days after the
ceassation of therapy, this reversion became less
significant. Vasanti et al. suggested that serum LDH
level can be used as a prognostic index of tumor burden,
response to therapy and in predicting relapse[l83]. Our
studies have shown that serum LDH level can be used as a
diagnostic index of tumor burden but cannot be used for
monitoring therapy.
There was no significant change in alanine
transaminase level in the serum with tumor growth. Very
significant increase in serum was observed immediately
after L-asparaginase treatment. This increased alanine
transaminase activity remained during later stage in the
case of animals treated with E. coli L-asparaginase. But
in those treated with Aeromonas L-asparaginase, the value
returned to near normal level. Eventhough serum aspartate
transaminase level did not show any significant change
during the early phase of tumor growth, there occured a
very significant rise in the serum aspartate transaminase
level during the later phase of tumor growth. There was a
rise in the serum level of aspartate transaminase
immediately after L-asparayinase administration and a
partial decline was observed after a period of time. Many
workers suggested that elevated serum level of
transaminases manifest alterations in the liver function
caused by nutritional deficiency that reverted to normal
after ceassation of therapy[154,156,157]. The Aeromonas
L-asparaginase treated groups showed a comparatively low
rise in serum alanine transaminase and reverted
immediately. From these observations it can be concluded
that Aeromonas L-asparayinase is less hepatotoxic than
Leunase.
Total acid phosphatase in serum was found to be
increased significantly during the early phase of tumor
growth and showed a partial reversion towards the later
phase. The same trend was observed with tartarate labile
acid phosphatase. In fact the changes in total acid
phosphatase are almost fully due to changes of tartarate
labile fractions. Rdministration of L-asparaginase caused
a significant reduction in the total as well as tartarate
labile serum acid phosphatase levels in tumor bearing mice
immediately after therapy. Mercer suggested that the
prostatic acid phosphatase level in serum increases with
tumor burden and decreases with regression so that it can
he used as a tumor marker to support diagnosis of cancer
and to assist the monitoring of therapy[l84]. In the
present study, it was found that the acid phosphatase
level did not show a direct correlation with tumor burden.
Eventhough acid phosphatase level has been reported as a
tumor marker by many workers[175,176,182,184] the present
study does not agree to it.
Serum alkaline phosphatase showed a significant
increase with tumor growth (Table IV.17). There was
a significant rise in the serum level of alkaline
phosphatase immediately after L-asparayinase
administration and at a later phase, no significant
difference was found between the treated and non treated
groups. Capizzi et al. and Whitecar et al. have reported
an elevation of serum alkaline phosphatase level upon
L-asparaginase therapy and this may be due to hepatic
dysfunction[l54,161]. Bates and Mercer have suggested
that serum alkaline phosphatase level can be used to
diagnose cancer and to monitor cancer therapy[175,184].
Works in .our laboratory prove that alkaline phosphatase
level could not he used to monitor cancer therapy.
From the above results it can be concluded that
L-asparaginase isolated and purified from Aeromonas is
reported for the first time from our laboratory and is
found to be very effective in the treatment of leukaemia.
The drug is superior to Leunase in some respects and can
he recommended to replace the commercially used drug by
our preparation. Since the cultural conditions and
purifications are standardized, it would be possible to
produce the drug industrially for therapeutic purpose.
CHAPTER V
S U M M A R Y
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