CHAPTER III ORAL CONTRACEPTIVES AND LIPID...

Part I

Part II

CHAPTER III

ORAL CONTRACEPTIVES AND LIPID METABOLISM

Effect of administration of high dose oral contra

ceptive on the metabolism of lipid

Comparative study of the effects of oral contra

ceptives (OCs) containing high & low dose of

estrogen on the metabolism of lipid

Part III Comparative study of the effects of estrogen &

progestin, components of OCs on the metabolism of

lipid

Part IV Effect of low and high dose oral contraceptives on

the metabolism of lipid in rats fed high fat high

cholesterol diet

60

Part I

Effect of administration of high dose oral contraceptive

on the metabolism of lipid

The available reports on the effect of oral

contraceptives (OCs) on serum lipids & lipoprotein

metabolism have been reviewed in the introduction. Depending

on the estrogen content of the OCs, increase in plasma

triglycerides, cholesterol, very low density lipoprotein and

157-162low density lipoprotein have been reported • Very few

studies seems to be carried out, however, on the

mechanism(s)

apart from

involved by which hyperlipidemia is

a report by Letterie et al. 163

produced,

that OC

administration containing 0.1 mg menstranol and 1 mg of

ethynodiol diacetate enhances the activity of hepatic HMG-

CoA reductase, which catalyses the rate limiting step in

cholesterol biosynthesis. Result of another investigation

shows that rate of in vitro incorporation of hepatic

triglycerides increases in rats administered OC containing

164high concentration of estrogen •

61

The changes induced by OCs in lipid profile are

important to consider in view of well established influence

of decreased HDL and increased LDL and total cholesterol

concentrations as primary risk factors of ischemic heart

disease. Therefore, the current investigation which is part

of series of studies was carried out on the effect of Ovulen

on the metabolism of lipids using rats as experimental

animals. Ovulen, which contains 0.1 mg of menstranol & 1 mg

of ethynodiol diacetate was administered to rats for

different period of 3, 6 & 12 cycles and the results are

being reported in this part.

Section I

Effect of administration of Ovulen on the concentration of

cholesterol, triglycerides and phospholipids

Materials and Methods

Female albino rats (Sprague-Dawley strain, average

weight 150g) were divided into four groups of 12 rats each.

Group 1. Control rats.

62

*Group 2. Rats administered Ovulen 3 cycles (21 days).

Group 3. Rats administered Ovulen for six cycles (42 days).

Group 4. Rats administered Ovulen for 12 cycles (84 days).

Rats of all groups were fed diet which contained

the following composition: (g/100g)-dextrose-69, casein

(Vitamin and fat free)-18, groundnut oil-8, salt mixture-4

and the Vitamin" mixture-l. Vitamin mixture used had the

following composition: (per 100g diet)-retinyl palmitate-

1000 IU, ergocalciferol-ISO IU, a-tocopherol-12mg,

menadione-0.3mg, thiamine-l.Omg, riboflavin-l.Omg, pyrido-

xine -0.6mg, niacin-lO.Omg, calcium pantothenate-S.Omg,

inositol 20.0mg, choline chloride-300.0mg, folic acid-0.4mg,

vitamin B12-3.0pg, P-aminobenzoic acid-S.Omg, biotin-

20.0ug, made upto 19 with corn starch. The salt mixture

contained the following (g/kg); sodium chloride-10S.00,

potassium chloride-120.00, potassium dihydrogen phosphate-

310.00, calcium phosphate-149.00, calcium carbonate-2l0.00,

Ferrous phosphate-14.70, manganese sulphate (anhydrous)-

0.20, potassium aluminium sulphate-0.09, copper sulphate-

0.39, sodium fluoride-0.S7, potassium iodide-O.OS, magnesium

sulphate (anhydrous)-90.00, ZnC1 2 and CoC1 2 .6H 20 were also

added to the diet at a concentration of lS.O and O.lS mg/kg

* Ovulen is an oral pill which containestrogen (menstranol) and 1 mg of(ethynodiol diacetate) per tablet.

100 ug ofsynthetic

syntheticprogestin

63

diet respectively. All the chemicals used for the salt

mixture were of analytical grade. Since the diet consumption

in the rats of experimental groups was lower, the rats of

group 1 were also given the same quantity of diet as

consumed by rats of groups 2, 3 and 4 (9.2+ 1.18g) to

maintain caloric intake similar in all groups. The rats in

each groups were housed individually in polypropylene cages

with wire mesh floor in room maintained at 2So C. Water was

provided ad libitum. Each rat of all experimental groups was

administered lOpg of synthetic estrogen, menstranol and

lOOug of synthetic progestin, ethynodiol diacetate. The oral

contraceptive formulation was dissolved in O.lml of

propylene glycol and given orally by a tube. The same

quantity of propylene glycol was administered orally to the

control rats. The duration of the experiment for groups 2, 3

and 4 were 3 (21 days), 6 (42 days) and 12 (84 days) cycles

respectively. At the end of each period, the rats were

deprived of food overnight, stunned by a blow at the back of

the neck and killed by decapitation. Blood and tissues were

removed to ice cold containers for estimations of

cholesterol, phospholipids and triglycerides. Details of the

procedure for the estimation of cholesterol, phospholipids

and triglycerides are given in chapter II.

64

Results

The diet consumption was similar in rats of all

groups (9.2+ 1.18g) but the rats of the experimental group

showed higher gain in weight {31.5± 1.5g for control rats

and 35.6+ 1.2 for the experimental group~.

a. Concentration of cholesterol in the serum, liver, aorta

and heart

Results are given in Table 1

It' values are given in Table lao

Concentration of cholesterol in serum,

liver and aorta were significantly higher in groups

and 4 when compared with group 1.

heart,

2, 3

b. Concentration of triglycerides in serum and tissues


It' values are given Table 2a

Rats administered estrogen-progestin formulation

(Ovulen) showed significant increase in concentration of

triglycerides in serum, Liver,aorta, adipose and heart when

compared to the control rats.

65

c. Concentration of phospholipids in serum & tissues

Results are given in Table 3.

It I values are given in Table 3a.

Concentration of phospholipid in serum and heart

in group 2 did not change in comparison with group 1,

whereas its levels increased significantly in serum, liver,

heart & aorta of all Ovulen administered groups when

compared with group 1.

Section II

Activity of HMG-CoA reductase and incorporation

of (l,2l4c-acetate) into liver cholesterol

The activity of HMG-CoA reductase in liver and

incorporation of (1,2 l4c)-acetate into liver cholesterol

were studied in rats administered Ovulen for different

duration and the results are discussed in this section.



Group 1. Control rats

Group 2. Rats administered Ovulen for 3 cycles

66

Table 1 Concentration of Cholesterol in serum and tissues

Cholesterol (mg/lOO gm tissue) (mg/lOO ml serum)

Tissue Group 1 Group 2 Group 3 Group 4

Liver 328.15+8.2 439.15+13.6 a 494.7+15.26 a 529.40+17.5a

176.15+4.4 286.44+8.02a a 322.84+11.50aHeart 291.6+8.9

Aorta 188.35+5.7 423.80+12.6 a 468.0+15.2a 523.80+17.5a

75.88+2.2 b a aSerum 86.47+2.8 89.6+3.00 93.04+3.4

Values are mean + SEM for 6 rats.

Groups 2, 3 & 4 are compared with group 1.

a - p < O.Gl, b - 0.01 < p < 0.05.

Table la It I values for Table 1

Tissue

Liver

Heart

Aorta

Serum

1 and 2

7.0

12.1

17.0

3.0

It' values between groups

1 and 3

9.6

11.6

17.2

3.7

1 and 4

10.4

12.0

18.7

4.2

67

Table 2 Concentration of Triglycerides in serum and tissues

(mg/100 gm wet tissue) (mg/l00 ml serum)


Liver 545.10+13.6 649.65+19.4a 710.4+17.76a 985.60+24.6 a

56.20+1.4 67.30+1.7 a a 97.90+2.93aHeart 80.4+ 2.12

Aorta 447.90+11.2 490.40+12.26b 527.0+18.1a 785.00+22.6a

7.55+0.19 a a 10.68+0.30aSerum 9.39+0.22 10.4+0.26

Adipose 6.96+0.17 10.14+0.25a 12.42+0.34a 17.74+0.54a

(gm/l00gmwet tissue)

Footnotes same as in Table 1.

Table 2a It I values for Table 2

't' values between groups

Tissue

Liver

Heart

Aorta

Serum

Adipose

1 and 2

4.4

5.0

2.7

6.3

10.6

1 and 3

7.4

9.5

3.7

8.9

14.4

1 and 4

15.7

12.9

13.4

8.8

19.0

68

Table 3 Concentration of phospholipids in serum and tissues

(mg/IOO gm wet tissues, mg/IOO ml serum)


-----------------------------------------------------------------

Liver 2532.00+88.3 3942.2+101a 4070.16+ll5.05a 4508.7+132.5 a

Heart 1831.35+60.8 1989.0+74.7 2333.90+86.3 a 3469.7+90.7a

525.38+13.1 627.7+17 a a 734.7+20.9aAorta 647.58+19.1

122.80+3.0 131.6+4.6 147.45+4.9a aSerum 163.8+6.0



Tissue

Liver

Heart

Aorta

Serum

1 and 2

10.5

1.6

4.8

1.6


1 and 3

10.6

4.8

5.3

4.3

1 and 4

12.4

15.0

8.5

6.1

body weight l50g) were divided into four groups of 12 rats each

69



The composition of the diet and its consumption,

dose of oral pill, and duration of experiment and all other

experimental details are the same as in section I.

Activity of HMG-CoA reductase was determined as

described by Rao and Ramakrishnan by determining the ratio

of HMG-CoA to mevalonate. The details of the procedure are

given in chapter II. Details of the procedure used for the

14incorporation of C-acetate into liver cholesterol are also

given in chapter II.

Results

a. Activity of HMG-CoA reductase in liver


It' values are given in Table 4a.

The activity of the enzyme in rats administered

Ovulen was significantly higher in the liver when compared

to the control rats.

70

b. Incorporation of (1,2l4c-acetate) into the cholesterol

in liver



In vivo incorporation of (1,2 l4C) acetate into

cholesterol of liver was significantly higher in rats

treated with Ovulen.

section III

concentration of hepatic bile acids and activity

of lipogenic enzymes in liver

In previous sections, the effect of Ovulen, an

oral contraceptive on the concentration of lipids in the

tissues and hepatic cholesterogenesis as measured by the

activity of HMG-CoA reductase and incorporation of labelled

acetate into hepatic cholesterol were discussed.

In this section, the effect of administration of

OC on the concentration of hepatic bile acids and activity

of lipogenic enzymes have been studied and the results

obtained are discussed.

Table 4

71

Activity of HMG-CoA Reductase and in vivo incorporation

of (l,2l4

c-acetate) into hepatic cholesterol

Group

1

2

3

4

Activity of hepaticHMG-CoA reductase*

Liver

3.00+0.075

a2.51+0.06

2.35+0.045a

a2.25+0.04

Incorporation of (14Cacetate) into hepaticcholesterol c/M/g tissue

Liver

1022+25.6

1414+35.3a

1865+49.68a

2002+50.0 a


* Ratio of HMG-CoA/Mevalonate, lower ratio indicates high enzyme

activity.

Table 4a 't' values for Table 4

HMG-CoA reductase ratio ofHMG-CoA toMevalonate

Rate ofincorporation

1 and 2

5.1

9.0

It' values between groups1 and 3

7.5

15.1

1 and 4

8.9

72


The experimental procedure used were exactly the

same as described in previous sections.


body weight l50g) were divided into 4 groups of 12 rats

each.


Group 2. Rats treated with Ovulen for 3 cycles.

Group 3. Rats treated with Ovulen for 6 cycles.

Group 4. Rats treated with Ovulen for 12 cycles

The composition & consumption of the diet,

duration of the experiment, dose of oral pill, and all other

experimental details were the Same as described in the

previous sections. At the end of each experimental period

the rats were sacrificed and the liver was collected in ice

cold containers for estimation of hepatic bile acids and

activity of L-malate & glucose-6-phosphate dehydrogenase.

Details of the procedures used for the extraction and

estimation of hepatic bile acids and the enzymes are given

in chapter II.

73

Results

a. Concentration of hepatic bile acids



The concentration of hepatic bile acid was

significantly lower in the rats administered ovulen when

compared to control rats.

b. Activity of Glucose-6-phosphate dehydrogenase and L

Malate dehydrogenase


It' values are given Table 6a.

Activity of the both enzymes in rats administered

Ovulen showed significant increase when compared to the

control rats.

74

Table 5 Concentration of bile acids in liver

(mg/l00 g tissue)

Group

1

2

3

4

Hepatic bile acids

45.7+1.5

a35.5+1.1

a30.6+0.77

a13.6+0.34


Table 5a It' values for Table 5


Tissue

Liver

1 and 2

5.5

1 and 3

6.1

1 and 4

20.9

Table 6

Group

75

Activity of lipogenic enzymes

Glucose 6 phosphate

*dehydrogenase

Malate

dehydrogenase:lt

1 103+2.6 1108.8+27.7

2 135+3.0a 1174.4+29.0

3 141+3.5a 1200.5+31.2b

4 154+4.6a 1344.7+37.5a

Footnotes same as in Table 1.* Unit = amount of enzyme causes initial change of OD of

l/min.:It Unit = amount of enzyme increases OD of O.Ol/min.



G-6-phosphate

L malate

1 and 2

8.0

1.6

1 and 3

8.7

2.2

.1 and 4

9.7

5.0

76

Section IV

Activity of lipoprotein lipase in heart and adipose tissue,

activity of LCAT and concentration of cholesterol

in lipoprotein fractions



body weight 150g) were divided into four groups of 12 rats

each:



Group 3. Rats administered Ovulen for 6 cycles.

Group 4. Rats administered Ovulen for 12 cycles.

The details of the procedures used such

77

adipose tissue was estimated according to the Procedure of

Krauss et al. Details of the procedure is given in chapter

II. Procedure for the estimation of cholesterol in

lipoprotein fractions and activity of plasma LCAT are also

given in chapter II. Protein was estimated in the enzyme

extracts after TCA precipitation, by the method of Lowry et

al. as described in Chapter II.

Results

a. Activity of lipoprotein lipase in heart and adipose

tissues


It' value are given in Table 7a.

The enzyme activity in heart and adipose tissues

showed a significant decrease in animals treated with Ovulen

when compared to the control group.

b. Activity of plasma lecithin cholesterol acyl transferase

(LCAT)



Rats administered Ovulen showed

decrease in the enzyme activity when compared

rats. No data is available for group 4.

significant

to control

Table 7

78

Lipoprotein lipase activity (u mol glycero1/h/g

protein)

Group Adipose Heart

Group 1 129.3+3.2 33.7+1.0

Group 2 a 34.1+1.2106.2+3.0

3 a aGroup 95.6+4.9 27.1+0.95

4 a aGroup 89.1+2.5 25.5+0.7




Tissue

Adipose

Heart

1 and 2

5.3

0.3

1 and 3

5.8

4.8

1 and 4

10.0

6.7

Table 8

79

Activity of lecithin cholesterol acyl transferase

(LeAT)

Activity of LCAT expressed as % increase in ratio of ester

cholesterol to free cholesterol during incubation

Group 1

30.5+0.8

Group 2

a20.2+7.0

Group 3

l5.5+0.55a

Footnotes same as in Table 1


It I values between groups

1 & 2

9.7

1 & 3

15.5

significant

in all the

80

Thus, rats administered Ovulen, an estrogen

progestin formulation showed decrease in activity of

lipoprotein lipase of the extra hepatic tissue. Activity of

plasma LCAT was also decreased in all experimental groups.

c. Concentration of cholesterol in BDL, LDL and VLDL

fractions


It' values are given in Table 9a

Rat administered Ovulen showed

increase in the concentration of cholesterol

experimental groups in the LDL and VLDL fractions as

compared to control rats but HDL cholesterol showed a

considerable decrease in groups 2, 3 & 4 when compared with

control group.

Section V

Release of lipoprotein into circulation

In the previous sections, the effect of

administration of Ovulen, an oral contraceptive on some

aspects of cholesterol metabolism was discussed. In this

section, the effect of administration of Ovulen on the

81

Table 9 Concentration of cholesterol in HOL, LOL and VLOL

fractions (mg/lOO ml serum)

Group VLDL LDL HDL

Group 1 5.57+0.14 9.63+0.24 58.2+1.7

Group 2 15.00+0.45a 24.39+0.62a a47.8+1.39

Group 3 17.52+0.56a 24.59+9.68a a47.1+1.50

4 19.72+0.70a a aGroup 27.5+0.85 45.67+1.2




Tissue

VLDL

LDL

HDL

1 and 2

20.0

22.4

4.7

1 and 3

20.6

20.8

4.9

1 and 4

20.0

20.3

5.9

82

release of lipoproteins into the circulation was studied and

the results are discussed.


same as

The experimental procedure used was

in the previous experiments. Female

exactly the

albino rats

(Sprague-Dawley strain, average body weight 150g) were

divided into four groups of 12 rats in each:

Group 1.

Group 2.

Group 3.

Group 4.

Control rats

Experimental rats fed Ovulen for 3 cycles.

Experimental rats fed Ovu1en for 6 cycles.

Experimental rats fed Ovu1en for 12 cycles.

Diet composition and consumption was the same as

in previous sections. Dose of oral pill administered to all

groups and duration were exactly the same as in previous

sections. At the end of each experimental period, release of

lipoproteins into circulation was studied using Triton WR

1339. Details of the procedure used are given in chapter II.

Estimation of cholesterol in the serum was carried

out as described in chapter II.

83

Results

Release of lipoproteins into circulation


It I values are given in Table lOa

All groups of Ovulen treated rats showed

significant increase in release of lipoproteins into

circulation-

84

Table 10 Release of lipoproteins in to the circulation

(Lipoprotein cholesterol mg/100ml)

Group

1

2

3


Release of lipoproteininto the circulation% increase in cholesterol

(Triton vs Saline)

75.7+2.2

a130.1+3.7

a145.5+4.5

Table lOa It I values for Table 10

It I values between groups1 and 2 1 and 3

12.6 14.0

85

Part II

Comparative study of the effects of oral contraceptives

(OCs) containing high & low dose of estrogen

on the metabolism of lipid

Hyperlipidemic effect of high dose oral

contraceptives (Ovulen) was studied in detail in the

previous part. Epidemiological studies have attributed

changes in lipids & lipoprotein profile leading to

development of CVD in women using DCs to the

, 1 'II 165 destrogen content In ora pl s Mea

dominance of

et al. has

demonstrated that reduction in concentration of estrogen &

progestin of DCs is accompanied by lower incidence of the

ischemic heart disease166 . These studies have resulted in

formulation of several low dose DCs and they have been

summarised in Table III in the introduction. Triphasic oral

contraceptives which contain low concentration of estrogen

and combined with high potency of progestin such as

Levonergestrel (LNG) & Desogestral (DSG) have been

extensively investigated in recent years. Results of these

studies, although appear to be in contradictory, have been

86

reviewed in the introduction. Depending on the dose and

potency of progestin component, increase or no alteration in

the levels of total cholesterol, LDL & HDL cholesterol and

167-179plasma triglyceride have been reported ~ Studies in

animal model with preparation containing EE30, LNG150, EE30,

LNG250 indicate that plasma lipids practically

180unchanged .

remain

Thus, it appears that very few detailed studies

have been conducted with preparations containing low doses

of estrogen in combination with low potency progestins such

as norethindrone acetate or ethynodiol acetate. Few reports

available in this regard are suggestive that these type of

theundertook&

OCs exert minimal alteration on the concentration of serum

1 , . f . 45,48,72lpoproteln ractlons . Welipids

present comparative study on the effects ofOCs containing

low and high concentration of estrogen with roughly same

potency and content of progestins on the metabolism of

lipids. Low dose OC chosen for the study was N-Mala which is

a very popular OC by virtue of its distribution through

various Government agencies throughout India. Ovulen & N-

Mala were administered in rats for a period of 6 cycles.

87

Section 1

Effect of administration of Ovulen and N-Mala

on concentration of cholesterol, triglycerides

and phospholipids

The effect of administration of Ovulen and N-Mala,

a high and low dose oral contraceptives (OC) on concen-

tration of cholesterol, triglycerides & phospholipids in

serum and tissues were studied.

Materials and methods


body weight 150 g) were divided into 3 groups at 12 rats

each.

Group 1

Group 2

Group 3

Control group

Ovulen group

*N-Mala group

The details of the composition of diet is same as

in part I. The consumption of diet was adjusted to be the

* N-Mala is manufactured by BUPHARMAParle (East), Bombay for Ministry ofWelfare, New Delhi, contains 30 pg ofmg norethisterone acetate.

LABORATORIES, VileHealth and Familyethylestradiol & 1

88

same for all groups. Rats of Ovulen group were administered

10 ug of estrogen and 100 ug of progestin, where as rats of

N-Mala group were administered 3 pg of estrogen and 100 pg

of progestin. Duration of experiment was six cycles (42

days). At the end of the experimental period, the animals

were deprived food for overnight & sacrificed and Serum and

tissues were collected in ice-cold containers for various

estimations. For histopathological studies, aorta of N-Mala

group was removed and fixed in 10% buffered formalin

solution. Procedure for estimation of cholesterol,

triglycerides and phospholipids are given in chapter II.

Procedure for histopathological examination also given in

chapter II.

Results

The diet consumption was similar in rats of all

groups (lO.2+1.5g) but the rats of the Ovulen group showed

higher weight gain, whereas N-Mala group did not show

significant alteration in weight gain (33.1+1.7g for control

group and 37.8~1.2g & 34.1+1.4g for Ovulen & N-Mala groups

respectively).

89

a) Concentration of cholesterol in serum and tissues

Results are given in Fig.ll. It I values are given

in Table lla.

Concentration of cholesterol in plasma and tissue

was increased in Ovulen administered rats when compared with

control group. Cholesterol level in plasma and heart of N

Mala group did not alter, however, significant increase in

cholesterol concentration in liver and aorta of the same

group was observed in comparison with the control group.

b) Concentration of triglycerides in serum and tissues

Results are given in Table 12. It I values are

given in table 12a.

Plasma triglyceride of N-Mala group did not change

when compared to the group 1. However, concentration of

triglyceride in all tissues of N-Mala group and as well as

the Ovulen group increased significantly in comparison with

control rats.

c) Concentration of phospholipids in plasma and tissues of

ovulen and N-Mala treated rats

Results are given in Fig.13. It I values are given

in Table 13a.

90

Fig. 11- Concentration of cholesterolin serum and tissues

(mg/l00 9 wet tissue, mg/l00 ml serum)

500.----------------------------,

400·'

800 -

200

100

oSerum Liver Aorta Heart

1:::::::::::1 Group 1

• Significant. Group 2 & 3 comparod witt)Group 1. Values are mean + SEM lor 6 ratsa. p <0.01; b. 0.01 <p <0,05

_ Group 2 ffi::ill Group 8

Table lla 't' values for Fig.ll


Tissue

Serum

Liver

Aorta

Heart

1&2

4.0

5.9

15.5

14.4

1&3

0.2

3.8

12.5

0.8

91

Table 12 Concentration of trig1ycerides in serum and tissues

(mg/100m1) ( •••. mg/100g wet tissue •••• )

Group Serum Aorta Heart Liver Adipose

(g/100g)

9.2+0.25a 567.9+14.9a 145.4+3.5a 815.15+22.0a

6.95+0.16 525.7+12.5a 72.59+2.0a 710.3+18.0a

1

2

3

7.3+0.18 437.9+11.2 56.48+1.5 560.1+14.0 6.96+0.17

a9.42+0.27

a8.1+0.21

Footnotes same as in Fig.ll.



Tissue 1&2 1&3

------------------------------

Serum 6.4 1.5

Aorta 6.9 5.2

Heart 23.4 6.4

Liver 9.8 6.4

Adipose 7.7 4.2

92

Fig. 13 - Concentration of phospholipidsin serum and tissues

(mg/l00 g wet tissue, mg/l00 ml serum)

Thousands4....------------------------------,

*3 .

2 .

1

*Serum Aorla Hearl Liver

_ Group" \;:::::::::::1 Group 2 _ Group 3

Footnotes same as in Fig, 11

_._-_....._---------_.._----_._._---==============:!I

Table 13a It I values for Fig.13


Tissue

Serum

Aorta

Heart

Liver

1&2

5.1

6.8

6.7

6.7

1&3

5.8

4.5

4.8

5.5

93

Phospholipids concentration were markedly increa

sed in plasma and tissues of both Ovulen and N-Mala treated

groups when compared with group 1.

d) Histopathological examinations

As shown in (Fig.2, Plate 1), significant lipid

deposition in aorta of N-Mala group in comparison with

normal aorta showing uniform thin intima (Fig.l, Plate 1).

Section II

Activity of HMG-CoA reductase and in vivo incorporation

of (l,2l4C) acetate into hepatic cholesterol

vivo

The activity of HMG-CoA reductase in liver and

incorporation of [1,2 14C]-acetate into liver

in

was

studied in rats administered oral contraceptives of Ovulen

and N-Mala. Results are being discussed.



body weight 150 g) were divided into 3 groups of 12 rats

each.

Group I

Group 2

Group 3

94

Control rats

Experimental rats administered Ovulen

Experimental rats administered N-Mala

The composition of diet, dose of oral pill

administered and route of administration, duration of the

experiment and all other experimental details are exactly

the same as in Section I.

Activity of HMG-CoA reductase was determined as

described by Rao and Ramakrishnan by determining the ratio

of HMG-CoA to Mevalonate. The details of the procedure are

given in Chapter II. Details of the procedure used for the

incorporation of l4C-acetate into liver cholesterol are also

given in Chapter II.

Results

a) Activity of HMG-CoA reductase in liver and intestine

Results are given in Fig.14. It' values are given

in Table 14a.

Activity ofHMG-CoA increased significantly in

liver of both experimental groups when compared with control

rats. However, increase in the case of Ovulen group was much

more than the N-Mala group.

95

b) In vivo incorporation of [1,2 l4C] acetate into liver

Results are given in Fig.15. 't' values are given

in Table 15a.

In vivo incorporation of [1,2 14C] acetate into

cholesterol in liver increased significantly in rats

administered Ovulen and N-Mala. Rate of incorporation in

Ovulen group was considerably high in comparison with N-Mala

group.

Section III

Concentration of hepatic bile acids and activity of

lipogenic enzymes in rats administered ovulen and N-Mala

In the above sections, the effect of N-Mala and

ovulen, low and high dose oral contraceptives on the

concentration of lipids in serum & tissues and hepatic

cholesterogenesis as measured by the activity of HMG-CoA

reductase and in vivo incorporation of labelled acetate into

hepatic cholesterol were discussed.

In this section, the effect of administration of

oral contraceptives on the concentration of hepatic bile

acids and activity of L-malate & glucose-6-phosphate

dehydro-genase were studied.

96

Fig. 14 -Activity of HMG-CoA reductase+ in Liver

4 r------------------------------,

3 ~ .

. ------- *. -----------* ..2 . _ _ _ _._ _ _ .._._ __.._ -

OL.-----'----------'---------....L- -.l

Group 1

+Ratlo of HMG-CoA/mevalonate,lawer ratioIndiCflt8fl higher enzyme activity.Footnotes same as in Fig. 11.

Group '2

- Liver

Group 3



Tissue

Liver

1&2

6.5

1&3

5

97

Fig. 16 -Invivo incorporation of [1,214

CJ acetateinto liver cholesterol

2500.---------------------------,

2000 _ .

1500

*...........~ _ _ _ _ ..

1000 f- _ ..-..- _ _ .

500

O'------'-----------'----------'-----~

Group 1

Oounts/min/g tissue.Footnotes same as In Fig.11,

Group 2

~ liver

Group 3

Table ISa 't' values for Fig.IS


Liver

1&2

17.3

1&3

2.7

98



body weight 150 g) were divided into 3 groups of 12 rats in

each

Group 1

Group 2

Group 3

Control rats



The details of diet composition dose of oral pill

administered, route of administration and other experimental

details are exactly the same as in Section I of part II.

Rats of experimental groups at end of 6 cycles were deprived

food for overnight and sacrificed. Liver was collected to

ice-cold container for estimation of bile acid and

determination of the activities of LM & G6PDH. The procedure

for extraction and estimation hepatic bile acids are given

in Chapter II. Procedure for determination of the activities

of LM & G6PDH are also given in chapter II. Protein was

estimated in the enzyme extracts after TCA precipitation, by

method of Lowry et al., as described in chapter II.

99

Results

a) Concentration of hepatic acids in Ovulen and N-Mala

administered rats

Results are given in Fig.l6. It' values are given

in Table l6a.

Concentration of bile acids in liver of both

Ovulen and N-Mala groups significantly decreased when

compared with control group. In this case also decrease in

concentration of hepatic bile acids was much high compared

with N-Mala group.

b) The activity of lipogenic enzymes in rats administered

Ovulen and N-Mala

Results are given in Fig.l? It' values are given

in Table 17a.

The activity of glucose-6-phosphate & L-malate

increased significantly in both experimental groups when

compared with the control rats.

Section IV

Activity of LPL in tissues, activity of plasma LCAT &

cholesterol concentration in lipoprotein fractions

100

FIg. 18 - Concentration of hepatic bile acids(mg/100 g wet tissue)

60,.---------------------------,

50 .

40 ~..........._ __ ~~:................~--_._.-

30 , ~ : _ : ..

20 f- ..

10 f- ..

o'- .....J1'- ---'1 ---'-1 ---'

Group 1 Group 2 Group 3

- Liver


Table l6a 't' values for Fig.l6


Tissue

Liver

1&2

10.8

1&3

8.0

*Flg.17- Activity of glucose-6-phosphate andL-malate * dehydrogenase

G cQ 7063

G6PDH

L -malate

o 500 1000 1500

Footnote same as in Fig, 11.

_ Group1 k::<] Group 2 _ Group 3

* Unit = amount of enzyme causes initial change of OD of l/min.*Unit = amount of enzyme increases OD of O.Ol/min.



Tissue

G-6-P

L-malate

1&2

9.1

4.3

1&3

8.4

3.5

102

The effect of administration of Ovulen and N-Mala,

high and low dose OCs on activity of lipoprotein lipase in

heart and adipose tissue, cholesterol levels in HDL, LDL &

VLDL and activity of plasma LCAT on female rats were studied

and the results are being discussed.



body weight 150 g) were divided into 3 groups of 12 rats in

each

Group 1

Group 2

Group 3

Control group

Ovulen group

N-Mala group

The details of diet composition, dose of oral

contraceptives administered to the both experimental groups,

route of administration, duration of experiment and other

experimental details are exactly the same as in Section I of

this part. Experimental rats of both groups at the end of 6

cycles were sacrificed and the heart and adipose tissues

were collected into the ice-cold containers for determina

tion of lipoprotein lipase activity. Plasma was collected to

103

the heparinsed ice-cold container for determining the

activity of plasma LCAT. Blood also was collected for

estimation of cholesterol levels in lipoprotein fractions.

Experimental procedure for determination of lipoproteins

lipase activity, concentration of cholesterol in the

lipoprotein fractions and that of LCAT are given in Chapter

II.

Results

a) Activity of lipoprotein lipase in heart and adipose

tissues

Results are given in Fig.18. 't' values are

in Table l8a.

The activity of lipoprotein lipase in

administered N-Mala showed a significant increase in

and showed no alteration in adipose tissue, but the

activity in both the tissues viz., heart and

decreased significantly in rats administered Ovulen.

given

rats

heart

enzyme

adipose

b) Activity plasma LCAT


in Table 19a.

The activity of plasma LCAT in rats administered

and VLDL

104

ovulen decreased significantly, where as the activity of

LCAT enzyme in plasma of N-Mala group did not alter

significantly when compared with control group.

c) Concentration of cholesterol in IIDL, LDL

fractions


in Table 20a.

Concentration of cholesterol in LDL in rats

treated with N-Mala did rise significantly, whereas in HDL

and VLDL cholesterol did not show any significant change

when compared with control group. In rats administered

Ovulen, HDL-cholesterol did fall significantly and the

cholesterol level in LDL and VLDL-cholesterol increased

significantly when compared with the control rats.

Section V

Release of lipoprotein into circulation

In previous sections, the effect of administration

of Ovulen and N-Mala on some aspects of cholesterol

metabolism was discussed. in this section we intended to

study the effect of administration of Ovulen and N-Mala on

105

Fig. 18- Activity of lipopr .otein lipase in heartand adipose

(u mole glycerol IIberated/h/g/proteln)

140.-----------------------------,

120

100

80

60 " " " " """".." " .

*4 0 1- - : __ -.... . _ .

*20 = .o L..- .....JIL..- ----'I ---LI~__---'


Heart -l- Adipose

Footnotes same as in Fig. 11

Table 18a It' values for Fig.18


Tissue

Heart

Adipose

1&2

10.9

8.8

1&3

5.1

1.3

106

Flg.19- Activity of LeATActivity of LCAT expressed % Increase In

.ratio of EO to FO during Incubation

40 ---..---,....-----------------------------,

......'O~•

....... *

./.........•. ..L. " .

20 1-............................................................................................................................................ . _ - - - - ..-•....

10 1--....................••.._ - - •..- .................................•........•........•......................._ -.-..-..•-...•.•....-- - ••.....•...--

oL- ..1.--1 -'1'-- -'--1 ---'


Activity of LCAT

Footnote same as In Fig. 11

Table 19a 't' values for Fig.19


1&2 1&3------------------------------

9.9 1.7

107

Fig.20- Concentration of cholesterol in HDL,LDL and VLDL fractions

(mg/100 ml serum)

80 ,-------------------------------,

HDL VLDL LDL

_ Group 1 1::::::::::::::1 Group 2 _ Group 3


Table 20a t values for Fig.20


HDL

VLDL

LDL

1&2

4.5

12.80

13.8

1&3

1.0

1.7

7.0

108

the release of lipoprotein into circulation and the results

obtained are being discussed.


The experimental procedure adopted in this study

was exactly the same as in previous sections. Female albino

rats (Sprague-Dawley strain, average body weight 150 g) were

divided into three groups of 12 rats each.

Group 1

Group 2

Group 3

Control rats



The diet composition~ dose of oral pill, route of

administration and other experimental details are same as in

the previous sections. At the end of 6 cycles, release of

lipoprotein into circulation was studied using Triton WR

1339. Details of the procedure used for the estimation of

lipoprotein release is given in Chapter II. The estimation

of cholesterol in serum was carried out as described in

chapter II.

estrogen

circulation

N-Mala,

109

Results

Results are given in Fig.2l. It I values are

in Table 21a.

Rats administered ovulen and

contraceptives containing high and low

increased release of lipoprotein into

compared with the control group;

given

oral

showed

when

110

Flg.21- Release of lipoprotein into circulation(percent dillerenCfl Iriton vS saline)

(mg/100 ml serum)

120.-------------------------~

*100

80 _ , M _ ••••••••••••

eo , " ·..·._·.· M.· ·••·••.....

4 0 _ ..

2 0 _ _ _ _ - ..

Group 3Group 20'-----......1---------....1---------...1.-----,----'

Group 1

Triton vs saline


Table 2la It I values for Fig.2l


1&2 1&3

7.58 1.8

111

PART III

Comparative study of the effects of estrogen & progestin,

components of OCs on the metabolism of lipid

Hypertriglyceridemia has been suggested as an

independent risk factor for coronary heart disease and

exogenous estrogen has been shown to elevate plasma

triacylglycerol levels in men & womenl05 ,106. It is clear

from other studies that higher concentrations of estrogen

can induce marked increase in VLDL:HDL ratio and result in

hypertriglyceridemia18l . Analysis of the levels of the major

apolipoproteins and their turnover rates led to the

conclusion

increases

that estrogen treatment (0.1 mg/day)

94in the synthesis of apo Band apo Al . A

induced

variety

of in vivo studies indicate that estrogens influence several

hepatic function in human beings, ranging from alterations

in drug metabolism to modification of the rate of secretion

of plasma protein182 . In vivo studies with estrogen based on

measuring rates of clearance of inject radioactively

labelled lipoproteins, it was found that hormone primarily

affected the rates of synthesis of apolipoproteins rather

94than rates of clearance . However, in contrast, a similar

112

study carried out in postmenopausal women indicated that

hormone was only affecting apolipoprotein turnover183 •

Further study indicate liver of estrogen treated chicks

synthesise more triglycerides from pr~cursors such as

acetate or palmitate than those of control birds l07 ,112. On

the contrary, available reports are suggestive that most of

the effects produced by estrogen administration are

antagonised by progestins, on account of their antiestogenic

or androgenic properties. Progestins inhibits hepatic

production of triglycerides VLDL and apolipoprotein Al,

stimulates the activity of hepatic triglyceride lipase and

decrease the lecithin content of phospholipidsl1 4 ,115.

However, despite numerous studies performed with

estrogen & progestin, it seems very few comparative

investigations have been conducted with estrogen and

progestin in the same dose as they are present in OCs. The

present study, therefore, was carried out to evaluate

metabolic impact of estrogen & progestin administered in the

same concentration as they are in Ovulen & N-Mala, oral

contraceptives studied in part II.

113

Section I

Effect of oral administration of estrogen and progestin

on the concentration of cholesterol, triglycerides

and phospholipids



body weight 150 g) were divided into four groups of 12 rats

each.

Group 1

Group 2

Group 3

Group 4

Control rats

Progestin* administered rats

Low dose estrogen administered rats

High dose estrogen administered rats

The composition of diet used was the same as in

the previous sections. Diet consumption was adjusted to be

the same for the rats of all the groups. Rats of group 2,

were orally administered 100 pg of synthetic progestin,

norethesterone acetate and rats of group 3 & 4, were orally

* Regesterone is an oral progestin pill which contains 5 mgof norethesterone acetate per tablet.

of

cycles

were

estrogen

formalin

estimation

are given

progestin

114

administered 3 pg & 10 pg of synthetic estrogen

estradiol respectively by a tube for a period of 6

(42 days). At the end of experimental period rats

sacrificed and tissues and serum collected to the ice-cold

containers for various estimations. Aorta of low dose

group was dissected entire and fixed in 10%

for histopathological studies. Procedure for

of cholesterol, triglycerides and phospholipids

in Chapter II. Procedure for histopathological

examination is also given in chapter II.

Results

There was no significant changes in weight gains

in rats of all groups, except in high dose estrogen group

which exhibited slightly higher weight gain.

a) Concentration of cholesterol in tissues and serum

Results are given in Fig.22.


Concentration of cholesterol in

administered rats showed significant decrease

heart and serum, whereas no change was observed

tissue when compared with control group. In

in aorta,

in liver

low dose

115

estrogen group, concentration of cholesterol in liver and

aorta increased significantly, where as considerable

decrease was noted in heart and no changes was observed in

serum. On the other hand, rats of group 4 exhibited high

levels of cholesterol in serum, liver, aorta and heart when

compared with control rats.

b) Concentration of triglycerides in tissues and serum



Triglycerides levels in progestin administered

rats was decreased significantly in aorta and heart tissues

but increased considerably in liver while it did not change

in plasma when compared with control group. Concentration of

triglycerides increased significantly in serum, heart, liver

and aorta of both groups 3 & 4 in comparison with control

group. However, incrase in concentration of triglycerides in

group 4 was much higher than in group 3.

c) Concentration of phospholipids in tissues and plasma



Phospholipids concentration decreased considerably

in serum, heart and aorta, while it did not change

116

Flg.22-Concentration of cholesterol inserum and tissues

(mg/100 g wet tissue, mg/100 ml serum)

500.-------------

*

*

*

*

* !Emr=' mil!

......... .., .

....t ............., •• t ••

*

Serum. Liver Aorta Hearto

100

200

400

300

_ Group 1 [;:1 Group 2 _ Group 3 E:;:~U Group 4

Values are mean + SEM for 6 ratsGroups 2, 3 and 4 compared with Group 1a; P.O.Ol b; 0.01 <p <0.05



1 & 2 1 & 3 1 & 4

Serum

Liver

Aorta

6.2

0.9

6.3

0.5

4.6

8.6

5.0

7.9

13.9

Heart 6.0 1.5 8.7

117

Table 23 Concentration of triglycerides in serum & tissues

Serum Liver Aorta Heart

Group (mg/100m1) (mg/100 mg wet tissue)

1 8.9+0.29 560.2+16.8 445.1+13.4 56.9+1.8

2 8.64+0.26 650.5+20.5a a a189.9+6.6 46.2+1.4

3 a 668.8+21.2b 499.2+15.0b a10.5+0.34 67.9+2.2. a

766.0+23.7 a 996.2+29.9 a a4 11.5+0.4 114.8+3.5

Footnotes same as in Fig.22.

Table 23a It I values for table 23


Serum

Liver

Aorta

Heart

1 & 2

0.7

3.4

17.1

4.7

1 & 3

3.7

4.0

2.7

3.8

1 & 4

5.4

7.0

16.8

14.7

118

Fig.24- Concentration of phospholipidsin serum and tissues

(mg/l00 g wet tissue, mg/100 ml serum)

Thousands4.------------------------------,

*

1 .

3 .

*

HeartAortaLiver

**~TIill_

Serumo

2 .

._ Group 1 k«1 Group 2 _ Group 3 EiuIill Group 4

Footnote Berne as In Fig. 22



1 & 2 1 & 3 1 & 4

Serum 3.5 2.2 5.1

Liver 1.1 0.76 7.5

Aorta 5.1 1.5 12.4

Heart 3.9 0.8 7.3

119

considerably in liver of progestin group in comparison with

control rats. Low concentration of estrogen group showed no

considerable alteration in concentration of phospholipids in

liver, aorta but considerably enhanced in plasma and

decreased significantly in heart when compared with control

rats. Levels of phospholipids increased in serum & tissues

in rats of group 4 in comparison with group I.

d) Histopathological examination of aorta in low dose

estrogen group

As shown in Plate 1 - Fig.3, aorta of low dose

estrogen group contains Sudanophilic (black) droplets in the

intima in comparison with Fig.l of the same plate which

shows a uniform thin intima.

Section II

Activity of HMG-CoA reductase and in vivo incorporation

at [1,2 14C]-acetate into hepatic cholesterol

Effect of administration of 10 & 3 ug of estrogen

and 100 ug of progestin on the activity of HMG-CoA reductase

and in vivo incorporation of [1,21 4C]-acetate into

cholesterol in liver were studied and results are discussed

divided into

Group 1

Group 2

Group 3

Group 4

120

in this section.

Female albino rats (Sprague-Dawley strain) were

f~u~ !groups of 12 rats in each.

Control group

progestin administered rats

Low dose estrogen administered rats

High dose estrogen administered rats

The diet composition used, dose of estrogen and

progestin administered, duration of experiment and all other

experimental details are exactly same in as previous

section. At the end of experimental period of 6-cycles, rats

were sacrificed, tissues were collected into ice cold

containers for determination of the activity of HMG-CoA

reductase, details of which are given in Chapter II.

Experimental procedure for in vivo incorporation of

[l,2 l4c]-acetate into hepatic cholesterol is also given in

Chapter II.

Results

a) Activity of HMG-CoA reductase



121

Activity of HMG-CoA reductase did not change in

liver of progestin administered rats in comparison with

control group. However, the activity of enzyme increased

significantly in liver of estrogen administered groups when

compared to control rats.

b) In vivo incorporation of [1,2 14c ]-acetate into

cholesterol in liver


't' values are given in Table 26a.

Rate of in vivo incorporation of (1,2 l4c )-acetate

into liver cholesterol in rats of group 2, did not alter

considerably when compared with rats of group 1. However,

rats of group 3 & 4, showed significant increase in rate

( 1 2 l4C) t' t" l' h l·t 1, -ace ate lncorpora lon lnto lver c 0 es ero

of

in

comparison with control group, increase in the case of

Ovulen group was significantly high.

Section III

Concentration of hepatic bile acid, activity of

glucose-6-phosphate dehydrogenase & L-Malate

In this section we intended to study the influence

122

Flg.26- Actvlty of hepatic HMG-CoA reductase"

4.-----------------------------,

3 _ . . , _ _ _ - _ - .

**...

2 _ _ - _ _ _ .

Group 4Group 3

o '------'-1 ....J1'- .L.- -'-__--'

Group 1 Group 2

- Liver

# Retio of HII4G..CoA/mevelonete. lov.errallo Indicete8 higher enzyme eotlyityFootnote 88ma 83 In Fig. 22


It I values between groups'

1 & 2 1 & 3 1 & 4

Liver 0.85 5.5 7.8

Group 4

123

FIg.26-lnvivo incorporation of [1,2'" cI acetateinto hepatic cholesterol

ICOllnl6/mlnllto/g IIB6110)

2000r---------------------------,

*1500 f-- _ _ : __ _ _ _.- -_ _ _ _ _.... ~-~ ..

*

1000 _ _._ __ _ _._ - _.._ __ _ _ .

500 f- , _ _ _ .

0'---__......1 .1.--1 ..1.-1 --'-__-----'


Liver

Footnotes same as In Fig. 22



1 & 2

1.2

1 & 3

5.5

1 & 4

9.2

124

of components of OC viz., estrogen and progestin on the

concentration of hepatic bile acids and activities of

glucose-6-phosphate dehydrogenase and L-malate.


Female albino rats (Sprague-Dawley strain) average


each.

Group 1

Group 2

Group 3

Group 4

Control group

Progestin administered group

Low dose estrogen administered group

High dose estrogen administered group

L

The composition of diet used, dose of estrogen and

progestin, duration of experimental and all other experi

mental details were same as in section I of this part.

Animals at the end of experimental period were sacrificed

and the tissues were collected into ice-cold containers for

estimation of bile acids and activities of enzymes. Details

of procedure for extraction and estimation of bile acids are

given in Chapter II. Procedure for determination of the

activlty of glucose-6-phosphate and L-malate are also given

in Chapter II.

125

Results

a) Concentration of bile acids in liver



Concentration of bile acids in liver of progestin

group did not change significantly when compared with

control group, whereas in estrogen groups~ concentration of

hepatic bile acids decreased significantly in comparison

with control group.

b) Activity of glucose-6-phosphate and L-malate dehydrogenase



The activity of glucose-6-phosphate and L-malate

decreased considerably in progestin administered group,

whereas the activities of both the enzymes were increased

significantly in both estrogen groups when compared with

control group.

Section IV

Activity of lipoprotein lipase in heart and adipose tissues,

LCAT in plasma and concentration of cholesterol in

lipoprotein fractions

126

Flg.27-Concentration of hepatic bile acids(mg/l00 g weI tissue)

40 ,..-----------------------------,

30 ,... ::.::40: .

*2 0 .. .

*---

10 f- _ - - - - ..- _ - - .

o L..-__--'1'-- --L.1 --I.1 --'1'--_--'

Group 1 Group 2 Group 3 Group 4

--Footnotes same as In Fig. 22


Liver


Liver

1 & 2

1.3

1 & 3

7.6

1 & 4

9.6

G6PDH

L-malate

127

Flg.28- Activity of glucose-a-phosphate ~

and L-malate ++ dehydrogenase

···--·---·--T--- --------i'-----y--! I I

! i! !

i. j I

!i

o 200 400 600 800 .1000 1200 1400

~~ Group 1 [ZIJ Group 2 _ Oroup 8 U::::ill Group 4

Footnotes same as In Flg.22+ Unit·Amount of enzyme causes Initial change 01 0.0 01 1/mln.++Unit-Amount of enzyme Increases In 00 01 O.01/min.



G6PDH

LNDH

1 & 2

5.1

3.4

1 & 3

2.5

2.5

1 & 4

5.8

5.0

128

Effect of oral administration of estrogen and

progestin on the activity of lipoprotein lipase (LPL) in

heart and adipose tissues, activity of plasma LCAT and

cholesterol levels in lipoprotein fractions were studied in

this section.




each.

Group 1

Group 2

Group 3

Group 4

Control group

Progestin group

Low dose estrogen group

High dose estrogen group

1he composition of diet, dose of estrogen and

progestin, duration of experiment and all other experimental

details were exactly same as in the previous sections. Rats

at the end of experimental period were sacrificed and

tissues such as heart and adipose were collected to ice-cold

containers for the determination of enzyme activity. Blood

also collected to ice-cold heparinsed tube for estimation of

129

the activity of LCAT. Details of procedure used for

determinig the activities of LPL, plasma LCAT and

L-...

cholesterol levels in VLDL, LDL and HDL are given in Chapter

II. Procedure for the estimation of cholesterol levels in

HOL, LDL and VLDL are also given in chapter II. Protein in

the enzyme extract was determined after TCA precipitation by

the method of Lowry et ale also given in chapter II.

Results

a) Activity of lipoprotein lipase in heart and adipose



Activity of lipoprotein lipase in progestin group

did not change significantly in heart and adipose tissues

when compared with control group: In low dose estrogen group

activity of LPL enhanced considerably in heart but did not

change significantly in adipose tissue in comparison with

control group. On the other hand, activity of LPL decreased

significantly in both tissues of group 4 when compared with

control rats.

b) Activity of plasma LCAT

Results are given in Fig.3D.

130


Activity of LCAT did not change significantly in

group 2 & 3 in comparison with control group. However,

enzyme activity decreased significantly in group 4 when

compared with control rats.

c) Concentration of cholesterol in VLDL, LDL and HDL



Concentration of cholesterol in VLDL & LDL

decreased significantly in progestin group, whereas in HDL

fraction no significant alteration was observed in

comparison with control group. On the other hand, in group 3

the concentration of cholesterol did not change in VLDL and

HDL cholesterol but increased considerably in LDL

cholesterol. Whereas in group 4, increased levels of LDL and

VLDL cholesterol but decreased concentration in HDL

cholesterol were observed, when compared with control group.

Section V

Release of lipoprotein into the circulation

In this. section, effects of estrogen and progestin

were studied on the release of lipoproteins into the

circulation.

Heart

Adipose

131

Flg.29- Activity of Iiooprotein lipase(u mole glycerol Ilborated/hr Ig protein)

jjj~jjj~!j~~!1~jjjl~~!~j~jjjj~jl~j~jjjlj~j~~l~jjjjljl1jjl1~~1!1~jjl~jlljln---+-----~I.UI.I.UI. UI. I. .. I.,~.I. ..LLI.&..u I. I...~L.Ll.U" u u,~u. ••

*

o 60 100 160

_ Group 1 Cd Group 2 ~ Group 3 Ulilill Group 4

Footnote same as In Fig. 22



Heart

Adipose

1 & 2

1.8

1.7

1 & 3

4.7

0.3

1 & 4

21.6

8.7

132

Flg.30-Activity of lecithin acyl cholesteroltransferase (LeAl)

40~--------------------------,

30 - ....•_._. =----.... :.....= _ -.- .

*20 _ _ > _ _ _ _ - _ - -_ .

10 1- , - _ _.- __ - _ _ _.._ _ _ _.._._._ .

OL..----l.-------..L.----__--!.. -l.-__---J

Group 1 Group 2 Group 3 Group 4

AOtlvlty 01 lQAT expressed aa '" IncreeseIn r!ltlo of EO to FO during inoul>6tion.Footnotes same 8S In Fig. 22.

---- Activity of LCAT



1 & 2

1.0

1 & 3

1.7

1 & 4

5.4

133

IF""'--~===---~~=======================;]

Flg.31- Concentration of cholesterol in HOL,VLOL and LOL

(rng/100 ml serum)

VLDL

LDL

HDL

o 20 40 60

_ Group 1 1::::::::::::::1 Group 2 _ Group 3 1:::::::1 Group 4


Table 3la 't I values for Fig.3l


VLDL

LDL

HDL

1 & 2

14.2

22.2

0.9

1 & 3

1.7

7.4

0.4

1 & 4

20.6

20.3

4.0

134




in each. Grouping of rats, composition & consumption of

diet, dose of estrogen & progestin and all other

experimental details were exactly same as in previous

sections. Release of lipoproteins were studied at the end of

experimental period (6 cycles), using Triton WR 1339.

Details of the procedure used for the estimation of

lipoproteins are given in Chapter II. Estimation of

cholesterol in serum was carried out as per procedure

described in Chapter II.

Results



In rats of group 2, no significant release of

lipoprotein was observed in comparison with control group.

However, in the estrogen administered groups, release of

lipoproteins into the circulation were considerably high

when compared with control rats.

135

==

Flg.32- Release of lipoprotein into circulation(% Increase In cholesterol

Inlo circulation)

200,----------------------------,

150 *.....................................................- , _••••................

*100 .. .

...

50 .

Group 4Group 3Group 2

oL-__......l' --'- -L- ---I__--'

Group 1

---- Triton vs saline

Footnotes same as In Fig. 22



1 & 2 1 & 3 1 & 4

1.6 8.6 14.9

136

Part IV

Effect of low and high dose oral contraceptives

on the metabolism of lipid in rats fed

high fat high cholesterol diet

The results reported in the previous parts

indicate that OCs containing high and low concentration of

estrogen causes changes in plasma and aorta similar to those

produced in atherosclerosis. These results have been

obtained in rats fed with low fat, low cholesterol diet. It

is well known that a diet containing a high level of fat and

cholesterol by itself contribute towards atherosclerosis in

most species. Report of a study by Claes-Herik & co-workers

in this regard concludes that livers from estrogen-treated

rabbits when perfused under identical conditions as normal

livers and with the same lipoproteins, the uptake of

cholesterol rich VLDL was increased by 76% compared with 21%

for normal VLDL184 . Studies performed by an experienced

Adams et al. 185 u$ing cynomdgus monkey 38% fatgroup, on

diet for 7 months pre treatment period and for the 2 years

~hat the study animals received vaginal rings that contained

Levonorgesterol and estradiol, indicated that the extent of

137

coronary atherosclerosis was much severe in the experimental

186group. On the other hand, Rampratap et al. found that in

rabbits on 2% cholesterol containing diet and treated with

estrogen, no significant alteration in plasma & aorta were

observed to be similar to that of atherosclerosis. Apart

from these studies, it appears that not much work has been

carried out in this aspect.

Therefore, in view of the results obtained from

previous parts, it was considered desirable to study whether

the atherogenic effect of OCs would be enhanced when a high

fat cholesterol diet is also fed.

Section I

Effect of low and high dose oral contraceptives on

the concentration of cholesterol, triglycerides

and phospholipids.



body weight 150 g) were divided into three groups of 12 rats

in each.

138

Group 1 - Control group

Group 2 - Ovulen group

Group 3 - N-Mala group

Rats fed the high fat cholesterol diet used in

this study was an atherogenic diet commonly used in this

laboratory and it contained 2% cholesterol and 15% coconut

oil. The salt mixture and vitamin mixture used had the same

composition as given in part I. The diet consumption was

adjusted to be same (11.2~1.2g) for groups I, 2 and 3. The

rats of group 2, were administered 10 pg of synthetic

estrogen and 100 pg of progestin (Ovulen) and rats of group

3, were administered 3 pg of synthetic estrogen and 100 pg

of progestin (N-Mala), orally by a tube. Duration of

experiment was for three months. At the end of experimental

period rats were deprived of food for overnight and

sacrificed. Then tissues and serum were collected into ice-

cold containers for various estimations. Aorta of all

groups were dissected entire and fixed in 10% formalin for

histopathological examination. The details of the procedures

for estimation of cholesterol, triglycerides, phospholipids

and histopathological examinations are given in chapter II.

139

Results

The diet consumption which was adjusted to be the

same in rats of all groups, however, Ovulen treated rats

exhibited considerable increase in weight gain.

of

heart

N-Mala

a) Concentration of cholesterol in serum and tissues



The levels of cholesterol enhanced significantly

whenin serum, aorta, heart and liver in rats given Ovulen

compared with control group. The concentration

cholesterol increased markedly in aorta, liver and

while it was not significantly altered in serum of

administered group in comparison with rats of group 1.

b) Concentration of triglycerides in serum and tissues



The levels of triglycerides in serum, liver, heart

and aorta of Ovulen administered rats increased

significantly in comparison with control group. On the

other hand, the concentration of triglycerides in serum,

liver and aorta of N-Mala group increased considerably but

140

Table 33 Concentration of cholesterol in serum and tissues

Group Serum

(mg/l00ml) (

Aorta Liver

mg/l00 g wet tissue

Heart

. . . .)

1

2

3

125.2+3.7

a172.5+5.7

131.5+4.2

369.1+11.0

840.6+26.9a

683.6+21.8a

1751.2+52.5

3687.5+120.1a

2272.1+81.7a

225.1+7.3

a320.5+9.8

a288.1+8.6

Groups 2 and 3 compared with group 1.

a, p < 0.01 b, 0.01 < P < 0.05

Table 33a •t' values for above Table 33

It I between Groups

Serum

Aorta

Liver

Heart

1 & 2

7.0

16.3

14.8

6.3

1 & 3

1.1

12.9

5.3

3.9

141

Table 34 Concentration of triglycerides in serum and

tissues.

mg/100 g wet tissue

Group Serum

(mg/100m1) (.

Liver Heart Aorta

. )

1

2

3

14.5+0.43

a22.2+0.58

18.7+0.49a

950.6+28.7

1510.8+47.9a

1182.8+38.5a

68.2+2.1

a88.9+3.2

73.0+2.4

1350.1+40.7

3036.6+91.0a

3066.5+95.1a


Table 34a 't' values for above Table 34

't' between Groups

Serum

Liver

Heart

Aorta

1 & 2

10.0

10.2

5.0

16.5

1 & 3

6.3

4.7

1.5

16.2

142

did not change in heart of the same group when compared with

group 1.

c) Concentration of phospholipids in serum and tissues


"t" values are given in Table 35a.

In group 2, concentration of phospholipids

increased significantly in liver, aorta and heart in

comparison with group 1. In group 3, the levels of

phospholipids remained unchanged in heart but increased

significantly in liver and aorta when compared with group 1.

d) Histopathological examination of aorta

Aort.a of rats fed h.igh fat diet & treated with "

Mala showing diffuse intimal deposition for Sudanophilic

droplets (Plate 1, Fig.5). Aorta of Ovulen treated group on

high fat diet showing thickening of the intima and extensive

deposition of Sudanophilic droplets (Plate 1, Fig.G).

Section II

Effect of low and high dose oral contraceptives on the

activity of BMG-COA reductase, liPOgenic enzymes and on

the concentration of bile acids in rats on high fat

cholesterol diet

143

Table 35. Concentration of phospholipids in tissues

Group

1

2

3

Liver

(. . .

4020.1+120.0

6836.6+218.7a

4931.7+162.6a

Aorta

mg/100 9 wet tissue

1890.6+56.7

6835.0+218.7a

5443.3+179.6a

Heart

. . . . .)

1750.1+52.5

3314.4+90.7a

1875.8+64.0


Table 35a •t· va1ues for above Table 35

It I between Groups

Liver

Aorta

Heart

1 & 2

11 .. 3

21 .. 9

13.2

1 & 3

4.5

18.9

1 .. 5

144



body weight 150 9) were divided into three groups of 12 rats

in each.

Group 1

Group 2

Group 3

Control group

OVulen administered group

N-Mala administered group

The composition of diet used was the same as in

section I. The dose of Ovulen and N-Mala given to rats of

group 2 & J, duration of experiment and all other

experimental details were exactly same as in section I. At

the ell1lru of experimental period, rats were sacrificed and

tissues ~ere re~oved quickly to the ice-cold containers for

determination of the activity of HMG-CoA reductase, hepatic

L-malate and g11llcose-6-phosphate dehydrogenase & esti~tion

of fulepattic bi.le acids. JDletails of procedures for

determinattion of H:lIMIG-CoA reductase, lipogemic enzymmes and

hepatic bile acids are given i.n chapter II. Protein im the

enzyme extract was deterDined after TeA precipitation by the

method of~ et al. also given in Chapter II.

145

Results

a) The activity of HMG-CoA reductase in liver



The activity of HMG-CoA reductase increased

significantly in rats of both experimental groups in

comparison with the control group.

b The activity of lipogenic enzymes

Results ar~ given in Table 37.

'tl values are given in Table 37a.

The activity glucose-6-phosphate & L-malate

dehydrogenase increased significantly in rats administered

Ovulen in comparison with control group. Activity of the

enzymes also considerably enhanced in N-mala group when

compared with control group 1. However, enzymes activity in

the case of Ovulen found to be much more in comparison with

N-Mala group.

c Concentration of hepatic bile acids



The concentration of bile acid increased

significantly in both experimental" groups when compared with

control group.

146

Table 36 The activity of HMG-CoA reductase* in liver

Group

1

2

3

Liver

3.6+0.1

2.5+0.08a

a2.8+0.09

* Ratio of HMG-CoA/Mevalonate, lower ratio indicates higher

enzyme activity.


Table 36a It' values for Table 36


Liver

1 and 2

7.6

1 and 3

5.3

147

Table 37 Activity of lipogenic enzymes

Group

1

2

3

Glucose-6-phosphate

*dehydrogenase

80.1+2.9

a120.2+3.5

a97.8+3.1

L-malate

dehydrogenase#

820.1+29.7

1114.9+39.5a

912.0+35.5

Footnotes same as in Table 33.* Unit = amount of enzyme causes initial change of 00 of

l/min.# Unit = amount of enzyme increases 00 of O.Ol/min.



G-6-PDH

L-ma1ate

1 and 2

7.4

6.0

1 and 3

4.2

2.0

148

Table 38 Concentration of hepatic bile acids

Group

1

2

3


Liver

(mg/lOOg Issue)

35.5+1.0

a44.7+1.4

a56.2+1.9

Table 38a •t' va1ues for Table 38


Liver

1 and 2

5.3

1 and 3

9.7

149

Section III

Effect of low and high dose oral contraceptives on

the activity of lipoprotein lipase, plasma LCAT

and concentration of cholesterol in HDL & VLDL+LDL

fractions in rats on high fat cholesterol diet

In previous section effect of low and high dose

oral contraceptives on the activity of HMG-CoA reductase,

and concentration of hepatic bile acids were discussed. The

present section deals with the effect of these OCs on the

activities of Lipoprotein Lipase, Plasma LCAT and

concentration of cholesterol in lipoprotein fractions. The

results obtained are discussed in this section.


Female albino rate (Sprague-Dawley strain, average

body weight l50g) were divided into 3 groups of 12 rats in

each.

Group 1

Group 2

Group 3

- Control group

- Ovulen administered group

- N. Mala administered group

150

The composition of diet, dose of Ovulen and N

Mala administered to group 2 & 3 respectively, duration of

experiment and all other experimental details were exactly

same as in section I. The rats at the end of experimental

period were sacrificed as described in previous sections and

the tissues were removed to the ice-cold containers for the

determination of lipoprotein lipase activity. Blood was

collected in heparinsed tube and the plasma was separated at

4°C for determination of the activities of LCAT. Protein in

the enzyme extract was determined after TCA precipitation by

the method of lowry et al. Procedure for determination of

the activities of LPL and plasma LCAT are given in chapter

II, Details of the procedure for the estimation of the

levels' of cholesterol in HDL and LDL + VLDL are also given

in chapter II.

Results

a The activity of lipoprotein lipase in heart and adipose

tissues


It' Values are given in Table 39a

151

The activity of lipoprotein lipase enzyme

decreased significantly in heart and adipose tissues of

ovulen treated group in comparison with control group.

Significant decrease was also observed in the activity of

enzyme in N. Mala treated groups when compared with group 1.

b Activity of plasma LCAT


It' Values are given in Table 40a.

The activity of plasma LCAT decreased considerably

in both experimental groups in comparison with control

group.

c Concentration of cholesterol in BDL, and LDL + VLDL


It' Values are given in Table 4la.

The levels of cholesterol did not alter

considerably in HDL fraction but increased significantly in

LDL+VLDL, in rats administered Ovulen in comparison with

control group. On the other hand, concentration of

cholesterol in HDL & LDL+VLDL did not alter considerably in

group 3 when compared with group 1.

152

Table 39 Activity of lipoprotein lipase in heart and adipose

(p mole glycerol liberated/h/g/protein>

Group

1

2

3

Heart

19.2+0.6

a14.5+0.4

a16.1+0.5

Adipose

112.5+3.8

a92.9+3.0

a95.6+3.2


Table 39a 't' Values for Table 39

It' Values between groups

Heart

Adipose

1 and 2

6.5

4.0

1 and 3

4.0

3.4

153

Table 40 Activity of LCAT in plasma

Activity of LCAT expressed as % increase in ratio of ester

choesterol to free cholesterol during incubation

Group 1

22.5+0.7

Group 2

a15.1+0.5

Group 3

a17.5+0.6


Table 40a It I Values for Table 40

't' Values between groups

1 and 2

8.6

1 and 3

5.4

154

Table 41 Concentration of Cholesterol in HDL and LDL+VLDL

Group HDL

(. . .LDL + VLDL

mg/100 m1 ...)

------------------------------------------------------------

1

2

3

37.4+1.1

40.6+1.5

36.2+1.3

80.6+3.5

a131.4+5.9

90.2+4.8


Table 4la It I Values for Table 41

It' Values between groups

HDL

VLDL + LDL

1 and 2

1.7

7.5

1 and 3

0.7

1.6

Legend to the figures

1. Normal aorta showing uniform thin intima and

spaced parallel elastic fibres

2. Rat aorta after administration of low dose

equally

x 694

oral

contraceptive (N-Mala) showing Sudanophilic (black)

droplets in the intima x 708

3. Aorta of rat after low concentration of estrogen

administration. Lipid deposition is seen in the intima

x 689

4. Aorta of rat on high fat diet showing normal intima and

elastic bundles low power x 693

5. Aorta of rat

contraceptive

the diffuse

fed high fat diet and given oral

containing low dose of estrogen showing

intimal deposition for Sudanophilic

droplets x 788

6. Aorta of rat fed high fat diet supplemented with oral

cont7aceptive containing high doses of estrogen

(Ovulen) showing thickening of intima and extensiye

deposition of Sudanophilic droplets x 1062

J.55

Discussion

The serious side. effects of oral contraceptives

(Des) include an increase in the incidence of circulatory

disorders, cardiovascular disease (CVD) and the occurrence

recognised that

of certain metabolic changes such as

. . 187-189 F 't h bllpoprote1n • or years]. as een

altered 1ipids/

there

and

is a relationship between estrogen content of an

certain CVD such as thromboembo1ism190 ,19l,

OC

the

mechanism of this relationship is not totally understood,

although, epidemiological studies indicate a link between

coronary heart disease and altered lipid/lipoprotein levels,

in particular decreased HDL-cho1esterol192 .

In the present study two oral contraceptives

Ovulen and N-Mala which contain high and low concentration

of estrogen respectively but the same dose of progestin were

studied regarding their effects on lipid metabolism. The

components of these oral contraceptives viz., estrogen and

progestin were also studied separately in the same dose as

they are present in the combination.

Results now obtained on administration of Ovulen

for varied period of 3, 6 & 12 cycles clearly demonstrates

progressive increase in lipid profiles both in serum and

tissues with prolonged duration of treatment with OC. This

156

observation is consistent with previous reports indicating

that prolonged use of OCs increases the risk of coronary

d ' 71,193 I . d f 1heart lsease . n our comparatlve stu y 0 Ovu en & N-

Mala administered to experimental rats for duration of 6

cycles, Ovulen clearly induced higher levels of cholesterol,

triglycerides & phospholipids in serum, liver, heart and

aorta. On the other hand, N-Mala practically had no impact

upon plasma lipids but increased cholesterol, triglycerides

&phospholipids slightly but significantly in liver & aorta.

Elevated levels of lipids in serum caused by Ovulen,

165 194compares well with previous reports ' and particularly

with Larsson-Cohn et al. 195 whose study indicates that

estrogen dominance in OCs preparation causes elevated levels

of plasma triglycerides and also with result of

hypertriglyceridemia reported in fasted and fed rats on

d ' , . f h' h d f b k K' 164a m1nlstratlon 0 19 ose 0 OC Y Ka -Joong 1m •

Elevated levels of cholesterol, triglycerides &

64phospholipids have been reported by Harvengt et ale due to

estrogen dominance of OC preparation. Bottiger et al. 36 has

attributed the development of CVD in OCs user to the over

dose of estrogen in certain preparation and this aspect has

been reviewed recently by Sitruk-Ware et al. 160 . Results

reports ofobtained with N-Mala are in agreement with

several workers 72 ,196,197 and notably by Arora et al.l98

157

whose study with N-Mala preparation on women for a period of

six months concludes that it has no impact on plasma

lipids/lipoprotein levels. No data seems to be available on

effect of OCs in tissue lipid levels. Histopathological

studies conducted in aorta of N-Mala group (Plate 1, Fig.2)

showed significant lipid accumulation. These results

indicate that the deleterious effects of Ovulen on lipid

metabolism may probably be due to high concentration of

estrogen present in these combinations, while taking into

consideration that progestin used in both preparation are

approximately equipotent.

Results obtained on administration of estrogen &

progestin in the same dose as they are present in Ovulen &

N-Mala indicate that high concentration of estrogen (10 pg)

caused higher levels of cholesterol, triglycerides &

phospholipids in serum, liver, heart & aorta. Low dose of

estrogen (3 pg) generally exerted no significant alteration

on lipid levels except for slight but considerable rise in

triglycerides concentration in serum & heart and cholesterol

level in aorta & liver. In contrast, progestin treated group

showed either decrease in concentration of cholesterol,

triglycerides & phospholipid or did not produce any

significant alteration with exception of considerable rise

in the levels of liver triglycerides. These observations are

158

in agreement with the reports of previous studies.

Administration of high dose of estrogen reported to cause

hypertriglyceridemia in rats while progestin treatment did

1 1 ,' d t t' 164 S' '1 1not a ter lpl concen ra lon • lml ar y, estrogen

treatment has also been reported to elevate plasma

t . 1 'd 86,174,199ng ycerl es and the development of hyperlipi-

demia has been reported in avian species by administration

107 108of estrogen ' • Studies of hens treated with high

concentration of estrogen have demonstrated that the hormone

increases the hepatic production of VLDL112,113 . High

concentration of estrogen (25 mg/Kg body weight) admini-

stered to chicks resulted in higher levels of triglycerides

in liver. 200

tissue reported by Park et ale Tkocz & co-

workers have found that norethisterone aceate administration

did not influence plasma lipid levels 20l . It has also been

reported that androgenic progestins (levonorgstrel, LNG)

~re effectively counter the estrogenic effect

., 57 194 202 203nonandrogenlc progestlns (desogestrol) ., , , •

than

Metabolic behaviour shown by the experimental rats

fed high fat diet and administered both Ovulen & N-Mala was

more or less similar to the results of these combinations in

rats on normal laboratory diet. The concentration of

cholesterol, triglycerides and phospholipids in serum,

heart, liver & aorta was much higher in rats treated Ovulen

r

159

than those administered N-Mala. Similar pattern has been

observed in histopathological studies conducted in aorta of

N-Mala & Ovulen groups. As shown in (Plate 1, Figs.5 & 6)

lipid accumulation in latter being much higher than the

former. In this regard, results of a study by Adams et

al. 185 using Cynomogus monkey on 38% fat diet indicated that

extent of coronary atherosclerosis found to be more severe

in animals received vaginal rings that contained

levonogesterol and estradiol. Luigi et ale has concluded

that rabbits on atherogenic diet and administered with high

dose of the synthetic progestogen northesterone exhibited a

more marked reduction of atherosclerosis than control

rabbi ts fed with same d ' t204le . Further study in this

connection also reveals that in female Cynomogus macaques

fed a moderately atherogenic diet and treated with OCs

containing EE, norgestrol and EE, EDD, the concentration of

205plasma HDL-C decreased in experimental groups

In the case of Ovulen and high concentration of

estrogen significant elevation in serum cholesterol is

manifested by increase in LDL and VLDL-cholesterol while

HDL-cholesterol showed decrease. The progestin administered

rats showed decrease in LDL and VLDL cholesterol and no

significant alteration in HDL-C. In this regard, several

workers 79 ,8l,206,207,208 have reported similar observation

160

of decreased HDL-C & increased LDL-C as a result of high

dose OC. Elevated levels of LDL-cholesterol have also been

reported with formulations preparation containing androgenic

. 162,209 "kk 1 187progest1.n . T1. anen et a. has further asserted

(l) that contraceptive estrogens elevate plasma LDL and

triglycerides levels (2) that androgenic progestins derived

from 19,nortestosterone reduce plasma VLDL and triglyceride

concentrations, whereas progesterone derived progestins have

little effect, and (3) that in combination pr~parations, the

relative potencies of estrogen & progestin determine the net

effect on plasma VLDL and triglyceride levels.

The results now obtained with Ovulen and high

concentration of estrogen are quite relevant in the light of

changes observed in atheromatous rats. The increase in serum

and aortic lipids and that of LDL cholesterol and decrease

in HOL cholesterol are observed in atheromatous rats2l0-2l2.

Similar results are now observed in the case of Ovulen and

high dose of estrogen, clearly indicating that admini

stration of Ovulen and high concentration of estrogen may be

accompanied by the risk of development of atherosclerosis.

The concentration of cholesterol in serum and

tissues is determined by the balance between the rate of

synthesis and degradation to bile acids. Hepatic

cholestrogenesis and degradation to bile acids have been

161

examined. Increased hepatic cholesterognesis in rats fed

laboratory diet and treated with both preparations and high

concentration of estrogen separately was observed as evident

from increased activity of HMG-CoA reductase and increased

rate of incorporation of labelled [1,2 14C] acetate into

liver cholesterol. Elevated hepatic cholerogenesis was

further noted in rats fed high fat diet and given Ovulen and

N-Mala due to increased activity of HMG-CoA reductase, an

enzyme which catalyses the rate limiting step in cholesterol

biosynthesis and its activity generally correlates with

tissue cholesterognesis. The increased activity of HMG-CoA

reductase observed in rats treated with OC is

. h .. 1 . 1 d b L . 1 163Wlt Slml ar anlma stu y y etterle et a.

consistent

Increased

activity of this enzyme with estrogen administration is also

in agreement with results reported by Carlson et al. 213

Increased rate of incorporation in the case of Ovulen and N-

Mala and high dose of estrogen are similar with previous

studies that employed a similar animal models which have

shown increased rate of in vitro incorporation of Hepatic

triglycerides in animals exposed to high concentration of

1 . .. . h . 164estrogen a one or ln comblnatl0n Wlt progestln

Additional investigation of a variety of· animal species

treated with higher doses of estrogens, reported similar in

vitro results l07 ,108,111,112.

162

On the other hand, rats administered progestin

neither influenced the activity of HMG-CoA nor significantly

altered the rate of incorporation which indicate no

considerable alteration in cholesterogenesis. This is also

. l' . h . 163in carre atlon Wlt a prevlous report

The increased HMG-CoA reductase activity found in

the present study are suggestive of a failure of LDL-

receptor, mediated catabolism and the feed back mechanisms

necessary for HMG-CoA reductase suppression. The increased

enzyme activity noted is similar to that in familial

hypercholesterolemia.

In rats fed high fat cholesterol diet and given

oral contraceptives the activity of HMG-CoA reductase is

increased even though in the control rats, the activity of

this enzyme is decreased when compared to normal rats fed

usual laboratory diet, the decrease being due to the

suppression of this enzyme activity by dietary cholesterol.

The increase in the activity HMG-CoA reductase in rats given

high fat cholesterol diet and administered oral

contraceptives may probably be due to the fact that the OCs

by themselves cause increase in the activity of this enzyme

and this increase may counteract the inhibitory effect of

dietary cholesterol.

Activities of hepatic malic enzyme and glucose-6-

163

phosphate dehydragenase were monitored and their activities

increased in Ovulen administered rats for varied duration,

and in rats treated with high concentration of estrogen

whereas their activity were not altered significantly with

treatment of N-Mala low dose estrogen and progestin. Simon-

eho et 214a1. has reported estrogen induced activity of

malic enzyme and G6PDH in chicks. The increase of both malic

enzyme and G6PDH activity appears to be consistant with the

stimulation of hepatic acety1-eoA reductase and fatty acids

215 216synthetase by estrogen ' .

The degradation of hepatic cholesterol to bile

acids is also decreased in rats administered high dose of

estrogen and the combinations (Ovu1en and N-Ma1a) as

evident from the decreased concentration of hepatic bile

acids. On the other hand, rats administered Progestin showed

no significant alteration in the concentration of hepatic

bile acids. These results indicate that increase in the case

of hepatic cholesterol in rats administered estrogen and the

combination (Ovu1en) may be due to increased hepatic

cholestrogenesis and decreased hepatic degradation to bile

acids. Absence of significant alteration in hepatic bile

acids concentration and that of cholesterol in Progestin

administered rats may be due to the lack of significant

effect on hepatic cholesterogenesis and the degradation of

164

cholesterol to bile acids. In high fat cholesterol diet fed

rats given oral contraceptive, the concentration of hepatic

bile acids showed significant increase. The increase in the

concentration of hepatic cholesterol in this group may

probably be due to the fact that the increase in the rate

of its synthesis more than offsets the increase in the rate

of its degradation to bile acids.

The concentration of lipoproteins in the

circulation is regulated by the balance between the release

of lipoproteins into circulation and the activity of

lipoprotein lipase, an enzyme responsible for uptake of

these triglyceride rich lipoproteins (chylomicrons and VLDL)

from extra hepatic tissues. Increased release of

lipoproteins into circulation were found in rats

administered Ovulen, N-Mala and high dose of estrogen,

whereas in low dose estrogen and progestin treated groups no

considerable alterations were observed. The activity of

lipoprotein lipase (LPL) in rats administered high

concentration of estrogen and Ovulen decreased which may

indicate decreased clearance of triglyceride rich

lipoprotein in the circulation since the enzyme is involved

in the uptake of triglyceride rich lipoproteins by the extra

hepatic tissues. The increase in the concentration of

triglycerides in the serum in these animals may, therefore,

165

be due to the decreased activity of this enzyme. In

'progestin administered rats the concentration of serum

triglycerides remained unchanged. This may probably be due

to the fact that the activity of LPL is not not altered

significantly. On the other hand, there is increase in

enzyme activity in rats administered N-Mala, even though the

concentration of serum triglycerides is not significantly

altered. In the latter case the release of lipoprotein into

the circulation is increased, and the lack of significant

alteration is due to the balance between the increased

clearance and increased release. These findings are in

agreement with the results reported by Berr et al. 99 that

oral contraceptives has been shown to enhance chylomicron

clearance from the circulation. It has also been reported

that estrogen administration depresses LPL activity in

female rats 217 Similar observation has been reported by.Gluek et ale 218 Progestin treatment has been reported to.induce hepatic lipase activity219 Kunsi et ale 114 has.further demonstrated that the two progestins studied namely

LNG and DSG had a different effect on hepatic lipase

activities. It is stimulated by LNG than by DSG.

The activity of plasma LCAT was decreased in rats

given high dose estrogen and Ovulen while it was not

significantly altered in animals given N-Mala. The activity

166

of LCAT has been reported to be

'd220 Th' b 'sterol • lS enzyme rlngs

decreased with anobolic

about esterification of

cholesterol on the surface of HDL. High density lip9proteins

and plasma LCAT are believed to be involved in transport of

cholesterol from the extra hepatic tissues to the liver for

its catabolism. The decrease in the activity of this enzyme

correlates with higher concentration of cholesterol in the

aorta in rats administered Ovulen high concentration of

estrogen and in the animals administered N-Mala.

Thus, the results obtained indicate that the

higher concentration of estrogen in the Ovulen may be

responsible for the adverse effect on lipid metabolism on

women using this oral contraceptive. N-Mala with low

estrogen appears to be safer. Progestin has generally been

reported to have an antiatherogenic action and this effect

may probably counteract the atherogenic effect of lower

concentration of estrogen present in N-Mala but not

sufficiently high to counteract the higher atherogenic

effect of the higher concentration of estrogen present in

the Ovulen.

Thus, the serious risk of cardiovascular

complication developed due to use of OCs can be reduced by

adequate reduction in estrogen content to 30-35 pg and in

adaptation with low potency progestins such as NET or EDD.

CHAPTER III ORAL CONTRACEPTIVES AND LIPID...

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