CHAPTER IV ANTITUMOR PROPERTY OF L-ASPARAGINASE ISOLATED...

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




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



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




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.


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


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


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.


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



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


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



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

CHAPTER IV ANTITUMOR PROPERTY OF L-ASPARAGINASE ISOLATED...

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