CHAPTER I

STUDIES ON LIPID METABOLISM - EFFECT OF ORAL

CONTRACEPTIVES ON THE METABOLISM OF LIPIDS

Introduction

This thesis incorporates the results of studies on

the effect of oral contraceptives (Des) on lipid metabolism

particularly that of cholesterol, using rats as experimental

animals. Many reports are available which indicate that the

use of oral contraceptives in women may lead to

hyperlipidemia which is one of the major risk factor in

atherosclerosis. It is proposed to review briefly the

salient aspects of the available information in

respect.

"this

It is recognized that hyperlipidemia is a major

risk factor for cardiovascular disease l ,2. Since the lipids

in the serum circulate as lipoproteins, recent emphasis has

been on the changes in serum lipoproteins in cardiovascular

disease. A brief mention of the changes in serum lipoprotein

and its impact on cardiovascular disease is made before

reviewing the relationship between the use of oral contra­

ceptives and incidence of coronary heart diseases in women.

2

The serum lipoproteins represents water soluble

macromolecules of lipids with specific proteins termed

apolipoproteins or apoproteins, held together by

noncovalent, primarily hydrophobic forces. The lipoproteins

serve as carries of lipids in the plasma. The main function

of these lipoproteins is to transport hydrophobic lipids of

dietary or endogenase origin within the hydrophilic

environment of the plasma to the tissues which utilize the

constituent fatty acids or cholesterol.

Lipoproteins appear as spherical particles with

water insoluble lipids at the centre or 3core . The

hydrophobic triacylglycerols and cholesteryl esters occupy

the central core and the more hydrophobic lipids

particularly cholesterol and phospholipids form a surface

interfacial monolayer. Specific amphipathic helical regions

identified within the structures of the apolipoproteins4

apparently permit lipid binding and orientation of the

apoprotein and the phospholipid within the surface mono

layer5 .

Human plasma lipoproteins are commonly divided

into five major classes based on the density at which they

float on ultracentrifugation. chylomicrons are the least

dense and largest lipoproteins, synthesized by the intestine

to transport dietary triglycerides and cholesterol from the

3

site of absorption in the intestinal epithelium. They reach

circulation via the lymphatic system. Very low density

lipoproteins (VLDL), the next largest particles, are

triacylglycerol rich and are mostly of hepatic origin. They

transport triglycerides and cholesterol from the liver for

redistribution to various tissues. Intermediate density

lipoproteins (IDL) are cholesterol-enriched lipoproteins,

smaller than LDL and represent the intermediate stages in

the conversion of VLDL to low density lipoproteins. Low

density lipoprotein (LDL) are among the most significant

with respect to arteriosclerosis and represent the end

product of VLDL catabolism. LDL are the major cholesterol

transporting lipoproteins. High density lipoproteins (HDL),

the smallest of lipoproteins appear to arise from various

sources including the liver and intestine. In addition, HDL

or HDL precursors appear to be produced within the plasma

compartment during lipolytic processing of chylomicrons. The

HDL family members are relatively rich in protein and they

contain approximately one-half protein and one-half lipid,

by weight. The HDL are sub-divided into 2 categories, HDL2

and HDL 3 .

In addition, lipoprotein (a)6 LP(a), has also been

identified in virtually all individuals, except

abetalipoproteinemic subjects. Lp (a) contains apo B (62%),

•

4

albumin, and the unique Lp (a) immunological determinant,

apo (a) (20%)7, which is rich in carbohydrates. Apo Band

apo (a) are linked to each other by one or more disulphide

bonds. Lp (a) is cholesterol rich and because of the

presence of apo B on its surface, Lp (a) resembles LDL with

regard to their plasma clearance rates and cellular receptor

. t' 8lnterac lon .

The average chemical composition, size, molecular

weight and electrophoretic migration of lipoproteins are

given in table (1).

The apo1ipoproteins

The apolipoproteins of various lipoproteins have

1 . d 9 f h 1 1 . hbeen recent y reVlewe . A summary 0 t e mo ecu ar welg ts,

major biosynthetic sites, plasma distributions and

concentrations, and ascribed functions of the major

apolipoproteins are presented in Table (2). The molecular

weights of the apoproteins vary from over 500,000 for apo B

to less than 9000 for apo A-II and the C apoproteins. The

biosynthetic sites are predominantly the liver and the

intestine. The amino acid sequences of human apo A-I,

A-II, C-I, C-II, C-III, and E are known. Defined

amphipathic alpha helical regions have been identified in

Table I Physical properties and chemical composition of normal human plasma lipoproteins

MWxl06 Per cent compositionDiameter Density(nm) (g/ml)

Fraction Electro­phoreticmobility

Chylo- Remains at 75-1200 0.93 AOO 2.0 90.0 "\ .0 3.0 £\.0 ~

microns origin

VLDL Pre- 30-80 0.93-1.006 10-80 8.0 55.0 7.0 12.0 18.0lipoproteins

IDL Slow pre- 25-35 1.006-1.019 5-10 17 .8 29.5 5.8 22.7 24.2 Nllipoproteins U1

LDL - 18-25 1.019-1.063 2.3 20.5 5. 1 8.5 42.3 23.6 Nllipoproteins

Lp{a) Pre- 23-24 1.08-1. 10 5.4 24.1 1.9 9. 1 38.2 14.6 12.0lipoproteins

HDL2 - 9-12 1.063-1. 125 0.36 46.1 2.5 3.6 18.8 28.1· 1.0lipoproteins

HDL3 - 5-9 1. 125- 1.210 0.18 61.0 1.9 1.9 14.2 20.0 1.0lipoproteins

~---------------------------------------------------------------------------------------------------- ~

'J~

~

Table II Characteristics of plasma apolipoproteins in normal humans

Apolipo­protein

Plasmaconcen­tration(mg/dL)

MW Majorbiosyntheticsite (s)

Plasmadistribution

Ascribedfunction

(s )

---------------------------------------------------------;--------------------A-I 130 28,016 Intestine, HDL 3 , nascent CM LCAT activation;

liver receptor binding

VLDL, LDL Receptor binding;particle formation

CM and CM remnants Particle formation

A- II

A-IV

8-100 }}

8-48 }

C-I

C-II

C- II I

oE- II }E-III }E-IV }

40

15

80

6

3

12

10

5

17,414 Intestine,liver

44,465 Intestine

264,000 Liver

550,000 Intestine

6,630 Liver

8,900 Liver

8,800 Liver

22,000 ?

34,145 Liver

HDL 3 , nascent eM

HDL, nascent CM

CM, VLDL, HDL

CM, VLDL, HDL

CM, VLDL, HDL

HDL 2 ,HDL 3

CM, VLDL, HDL 2

Hepatic lipaseactivator

LCAT activation

LCAT activator;LPL activator?LPL activator

Inhibition of LPLand prematureremnant clearance

Unknown

Receptor binding;LCAT activation

C'\

------------------------------------------------------------------------------* eM - Chylomicrons

7

apo A-I, A-II, C-I, C-II, C-III,and E which are believed to

be responsible for lipid-binding properties of the

apoproteins. Manyapoprotein like apo A-IV.B, C-III, D and

E are glycoproteins whil~ others like apo A-I, A-II, C-I and

C-II do not contain any carbohydrate.

Lipoprotein and Cardiovascular disease (CVD)

Several studies have substantiated an association

between lipoprotein fractions and coronary artery disease

(CAD). It is not intended to review this aspect since there

are several recent review in this regard. Current research

data, however, indicate that the apolipoproteins A-I and B

could be very sensitive and specific markers for

characterization of individuals with heart . 10 11dlsease ' .

Apo B been positively correlated with incidence of coronary

ailing disease and apo AI is believed to show a negative

correlation. Recently, the apo A-I/apo B ratio has been

recognized to be a very good discriminator between patients

with ischemic heart disease and controls12 .

Oral contraceptives, Lipids and Cardiovascular disease

The explosive increase in population particularly

8

in third world countries has become a great concern to the

epidemiologists, demographers, obstetricians and economists

around the globe. Frantic efforts are being made to check

population growth and the oral contraceptives have been

found to be an important device for family planning.

Combined pill

Since the first clinical demonstration of

usefulness of an oral combination of estrogen-progestin by

Pincus (1955), various commercial preparations have been in

use. These preparations contain ethinylestradiol or its 3­

methyl ether (mestranol) as the estrogen, and a progestin

either belonging to 10-northestosterone group or derived

from progesterone. The "original pill" which entered into

the market in 1960 contained 100-200 meg of synthetic

estrogen and 10 mg of progestin. Since then, a number of

improvement have been made to reduce the undesirable side

effects of the pill by reducing the content of both the

estrogen and progestin. At the present time, the

formulations of the combined pill contain below 100 meg of

estrogen and 0.5 to 1 mg of a progestin.

9

Progestin only pill (POP)

This is commonly referred to as "minipill" or

"micropill". It contains only progestine, which is given in

small doses throughout the cycle. The commonly used

progestines are norethisterone and desogestrel. The

progestin-only pills never gained widespread use because of

poor cycle control and an increased pregnancy rate13 .

However, they have a definite place in modern-day

,,-

contraception. They could be prescribed to older women for

whom the combined pill is contraindicated because of

cardiovascular risks. They may also be considered in young

. h . k f fl' 14women Wlt rlS actors or neop aSla •

Once-a-month (long-acting) pill

Experiments with once-a-month oral pill in which

quinestrol, a long-acting estrogen is given in combination

with a short-acting progestine, have been disappointing15 •

The pregnancy rate is too high to be acceptable. In

addition, bleeding tends to be irregular.

10

Male pill

The search for a male contraceptive began in 1950.

Research is following 4 main lines of approach: (a)

preventing spermatogenesis, (b) interfering with sperm

storage and maturation, (c) preventing sperm transport in

the vas, and (d) affecting constituents of the seminal

fluid. Most of the research is concentrated on interference

with spermatogenesis. An ideal male contraceptive would

decrease sperm count while leaving testosterone at .normal

levels. But hormones that suppress sperm production tend to

lower testosterone and affect potency and libido.

A male pill made of gossypol-a has been effective

in producing azoospermia or severe oligospermia, but shows a

narrow margin between effective toxic doses. At present it

does not seem that gossypol will ever be widely used as a

1 . 16rna e contraceptlve

Mode of action of oral pills

The mechanism of action of the combined oral pill

is to prevent the release of the ovum from the ovary. This

is achieved by blocking the pituitary secretion of

gonadotropin that is necessary for ovulation to occur.

11

Progestin-only preparations render the cervical mucus thick

and scanty and thereby inhibit sperm penetration. Progestins

also inhibit tubal motility and delay the transport of the

d f th t ·· 17sperm an 0 e ovum to the u erlne cavlty .

Risks and benefits

Oral contraceptives were first marketed in 1960,

and their use increased steadily until 1973 when they were

used by an estimated 36% of married women of reproductive

age in United States17 • Oral contraceptive use declined

because of concerns among the women about adverse effects of

the pill. A report indicates that only 20% of married women

and 29% of all women of reproductive age were using oral

contraceptives in United States in 198218 .

Once again the use of oral contraceptives

increased from 1982 onwards. A nation-wide survey in 1987 at

United States revealed that, of the 57.4 million women in

reproductive (15 to 44 years) age group, about 16 million

(30%) were not exposed to unwanted 19pregnancy .

Sterilization and oral contraceptives were the most popular

methods.

However # despite popularity of oral contraceptives

the concern of women for their adverse side effects

12

continues. A report of survey conducted in 1985 at United

States indicates that 75% of the women surveyed believed

that oral pills caused serious health problems; one third

thought they caused cancer, and a similar number were

concerned about serious cardiovascular effects such as blood

1 k d d ' 1 'f ,20cots, stro e, an myocar la ln arctlon .

The three major concerns are the possible,

neoplastic, reproductive, and metabolic effects. It has been

shown that oral contraceptive actually reduce the risk of

developing two lethal cancers: Cancer of the uterine

endometrium and cancer of the ovary. There is no evidence

that oral contraceptive use definitely increases the risk of

Preinvasive cervical neoplasia can usually be

developing

21cancer

any other cancer except possibly cervical

diagnosed and treated before it becomes malignant. The only

adverse effect on future reproduction is a delay in the time

to conception after stopping the medication that occurs in

22some oral pill users, and this effect is temporary .

The type of oral contraceptives formulation,

details of their components and their proprietary names have

been summarized in Table III. The most commonly used oral

pill in India, appears to be N-Mala which is being

distributed through family planning wing of the Government

of India. A recent survey23 indicates that OCs containing

high dose of estrogen are being prescribed by the Doctors.

13

Table III proprietary names of formulations (EE, ethynylo­estradiol; ME, mestranol; LNG, levonorgestrel;DSG, desogestrel; NET, norethisterone; EDD,ethynodiol 1 diacetate; LYN, lynestrenol)

Formulation

EE30, LNG150

EE30, LNG250EE50, LNG250

EE30,40,30 LNG50,75, 125 (Triphasicformulation)

EE30, DSG150

EE35, NET400EE35, NET500EE30, NET 1000EE55, NET1000

ME50, NET1000

EE30, EDD2000EE50, EDD1000ME100,EDD1000

EE50, LYN1000

Proprietary Name

Microgynon 30, Microvlar, Ovranette,Lo - ovralEugynon 30, Ovran 30Eugynon 50, Ovran*, Ovral*, Stedvil*

Logynon, Trinordiol, Triquilar

Marvelon

Ovcon 35Brevicon, Brevinor, Modicon, OvysmenN-MalaBrevicon-l, Brevinor-l, Normin orthoNovum 1/35, Ovysmen 1/35Noriny-l, Ortho-Novum 1/50 ortest 21Minovler

Conova 30, Demulen 30Demulen, Ovulen 1/50Ovulen 1 mg

Lyndiol 1 mg, Ovostat

* denotes that formulation alsoinactive dextronorgestre1. Nobetween NET and NET acetate.

contains biologicallydistinction is made

14

Oral contraceptive and cardiovascular disease (CVD)

Of the major adverse side-effects of OC use the

most serious, particularly in terms of the number of ,women

that might be affected, is CVD, especially coronary heart

d ' 24lsease . In the Royal College of General Practioners

Study25 the 40% excess mortality in women using OCs was due

to almost entirely to cardiovascular disease. In respect of

this condition, epidemiological studies have identified a

number of 'risk factors' which can be used to predict the

possibility of the development of CVD in groups of subjects.

The primary risk factors are considered to be hypertension,

smoking and hyperlipidaemia with overweight, lack of

exercise, diabetes mellitus, severe electrocardiographic

abnormalities, personality type and previous myocardial

infarctions as secondary risk factors.

Authoritative reports have established that oral

contraception increases the risk of atherosclerotic vascular

disease (ASVD) and thromboembolism leading to various

ischemic

ischemic

vascular disease like coronary heart disease and

. 25-34cerebrovascular dlsease . During the late

1960's and early 1970's a large number of epidemiological

studies showed an increased risk, approximately fourfold, of

developing venous thromboembolism, particularly idiopathic

15

deep vein thrombosis, in women using DCs. These studies have

been 35summarised by Vessey . The risk appears to correlate

with dose of estrogen but not of the progestin. Reduction of

the estrogen dose to 50 pg or less was associated with a

reduction in the number or thromboembolic incidents 36 and it

might be expected that the now widespread use of DCs

containing only 30 pg estrogen would further reduce the

incidence. However, the Royal College of General

Practitioner's study suggested that this association with

estrogen dose applied only to superficial vein thrombosis

and that in this type the progesterone dose may also be

important. The effect of the DC on the structure of the

vessel walls and venous return and on the coagulation and

fibrinolytic systems, particularly the latter (since the

development of venous thrombosis seem to be more common in

women with a defective vessel-wall fibrinolytic system), is

probably also important in the development of thrombo-

embolism. The risk of death from venous thromboembolism in

association with DC use is reported to be very small (about

1 per 290,000 women years of follow up).

Myocardial infarction is uncommon in young women

and factors influencing this condition in women under 50

years of age have been studied in a number of reports in

recent past, notably by Rosenberg et al. 3? and reviewed by

16

Johanssan et al. 38 . These factors include cholesterol levels

above 250 mg/100 ml or HDL-cholesterol below 40 mg/100 ml,

hypertension, diabetes, family history and blood group A.

The risk increases with age and is multiplied by OC use.

Although

age, the

the relative risk does not change very much with

30number of incident does ; the incidence of

myocardial infarction per 100,000 women years in the age

groups 30 to 39 years and 40 to 44 years was 4 and 22 in

non-users of OCs and 11 and 39 in OC users. In women who

smoked the attributable risk, but not the relative risk, was

markedly increased; thus for women aged 40 to 44 years the

incidence for those who smoked less than 15 cigarettes per

day was 12 and 47 per 100,000 woman years in controls and OC

users respectively.

Thus it appears to be evident that the association

between the use of OC and the risk of myocardial infraction

appears to be greater in women who have pre-existing

coronary atherosclerosis and other risk factors, such as

hypertension, changes in lipid and lipoprotein levels and a

decrease in glucose tolerance. The risk may also be related

to the dose of estrogen and progestine.

A number of reports also indicate a relationship

between cerebrovascular accidents and OC use. This was

substantiated by the first large-scale . " 38lnvestlgatl0n , a

17

case control study which suggested that thrombotic stroke

was four times, and haemorrhagic stroke twice, as common in

OC users 25as in controls. The study suggests a relative

risk of about 3 for both the incidence and mortality of

cerebrovascular disease in OC users compared to controls and

this same relative risk appears to apply also to women who

have discontinued use of OCs. As in the case of myocardial

infarction, smoking is an important risk factor, as is also

hypertension; the dose of estrogen and progestine in OC is

also important and part of their involvement may relate to

the changes in lipid levels.

OC users

A small rise in blood pressure is observed in many

and it decreases after discontinuation39 ,40. In

most women the rise in blood pressure with OC use is small

but it has been reported to be a progressive rise with

continued use so that it becomes significant enough to

affect cardiovascular risk. The relationship between OCs and

cardiovascular disease has been considered in more detail in

t25,35,41,42some repor s .

Numerous studies carried out by various workers

have established a relationship between OC use and the

levels . 43 44of lipids and lipoproteins ln the serum ' . These

changes caused by OCs depending on dose, potency of estrogen

& progestin and duration of treatment. It is proposed tc

of

18

review briefly the available reports on the changes in lipid

and lipoprotein metabolism with the use of DC in human

volunteers. The particulars of various DCs used in several

studies have already been summarised in Table III.

Effect of use of DCs for 2 to 4 months on plasma lipids and

lipoproteins

Powell et aI, Taggart et al have reported elevated

levels of serum total cholesterol with use of EE30 LNG 150

. 1 45,46. . 1 1 hId 1 1ln human vo unteers . Slml ar y,t e e evate eve s

triglycerides have been observed by Larsson-Chon et aI,

Kraus 1 . 1 . k 1 47 - 49 ,50et ~, SamSloe et a & Bergln et a • The

concentration of LDL-C found to be increased as reported by

Harvengt et a1 51 & Helenius et a152

but the HDL-C levels

decreased with use of same Oc 53 . On the contrary, Rossner et

a1 54 & Newton et a1 55 have asserted that the concentration

of total serum cholesterol remains unaltered by this

indicates

combination. Similar findings have been reported on the

levels of triglycerides by Shouby et a1 56 , Briggs et a1 57 &

Newton et a1 55 and also on the concentration of LDL & HDL­

cholestero146 ,58,59. A report of study48 in this regard also

that the levels of HDL 2-C decreased while the

concentration of HDL 3-C remains unchanged. Further infor-

19

mation available reveals that the levels of apoprotein B is

enhanced whereas the concentration of apoprotein A-I did not

alter with use of EE30 LNG150.

Results of studies with the use of EE30 LNG250

indicate that it increases the levels of triglycerides while

it has no influence on total serum cholesterol but decreases

the HDL-Cholesterol as reported by Larsson-Cohn, et al 47 •

From a report by Vermeulen. et al 60 it appears

that use of EE50 LNG150 does not alter the levels of total

serum cholesterol, triglycerides, LDL-Cholesterol and HDL-

Cholesterol.

Investigations conducted with use of EE50 LNG250

shows that it enhances levels of total serum cholesterol,

triglycerides, LDL-C but decrease the concentration of HDL-

Elevated levels of serum triglycerides has also been

reported by Nash 62et al. • However, some workers have

observed no change in the concentration of cholesterol,

triglycerides, LDL-Cholesterol and HDL-Cholesterol with use

f h ' fl' 60,62o t lS ormu atlon .

Triphasic OCs which generally contain low dose of

estrogen appears to be extensively studied by many workers

in recent years. There are no data to suggest that use of

these OCs enhances the levels of total serum cholesterol and

LDL-Cholesterol. However, there are reports which suggest

20

elevated levels of triglycerides as reported by Bertolini et

al.63

, and decreased concentration of HDL-Cholestero1 52 ,64.

On the other hand, there are many reports which indicate

that use of triphasic formulations in human volunteers

stero1 55 ,59,64,65 ,

have no influence upon the concentration

, 1 'd 67,68 LDLtrlg ycerl es ,

of

and

chole-

HDL-

cholesteroI 63 ,66,68. It has also been reported by Bertolini

et al. 63 that the concentration of HDL 3 & HDL 2 does not

alter. Further data in this regard suggest that the levels

of apolipoprotein Band apolipoprotein A-I remains

58unchanged , but on the contrary, some workers have reported

elevated levels of apolipoprotein A-I and apolipoprotein

B5l ,55,68.

There are no reports to show that use of EE30,DSG

150 increases the concentration of total serum cholesterol,

but many investigators have found that it enhances

considerably the levels of triglycerides as reported

62 , 50 5~ 63 65Nash , Berglnk et al. , Newton et al. & other '

by

and

&

be

by

to

reported

et al. 64

appearsconcentrationThe

concentration of HDL-cholesterol as

1 69 . 1 49 t~. , SamSl0e et~. , Horveng

the

Bergink et

th 56,63o ers .

also

enhanced while the levels of HDL 2-C remains unchanged 63 ,67.

The available reports in this regard also suggest that the

concentration of apoprotein B remains unaltered50 ,68 , but

21

. 50 55 68the levels of apolipoprotein A-I lncreases ' , .

Use of EE35 NET500 appears to elevate the

concentration of serum total cholesterol as reported by

Rossuer et al. 70 , Burkman et al. 71 triglycerides by Pasauale

et al. 72 . It reduces the concentration of HDL-c57 • However,

there are few reports which are suggestive that EE35 NET 500

formulation has no influence upon the levels of total serum

57 72. . 70 70-72cholesterol ' , trlg1ycerldes , LDL-cholesterol and

HDL-cholestero1 61 ,70.

Investigation with EE35, NET 1000 reveals that it

enhances the concentration of total serum cholesterol and

triglycerides as reported by Briggs et al. 57 and Burkman et

al. 71 but decreases the concentration HDL-cholesterol as

reported 70by Rossner et ale 61& Briggs et ale . In this

regard, there are also reports which state this drug has

no impact on the concentration of cholesterol & trigly­

cerides70 ,72, LDL-cholestero1 71 ,72 and HDL-Cholestero1 72 •

Result of a study70 with EE50, NET 1000 prepara-

tion indicate that it enhances the levels of serum total

cholesterol,. LDL-cholesterol but decreases the concen­

tration of the HDL-Cholesterol. Burkman et al. 71 has also

reported elevated levels of total serum cholesterol and

triglycerides. However, Pasquale et al. 72 in his study has

observed that EE50, NET 1000 does not alter the levels at

22

total cholesterol, triglycerides, LDL-Cholesterol and HDL-

Cholesterol.

45Powell et al. has reported that use of EE50, EDD

1000 has no impact on the levels of total cholesterol and

HDL-cholesterol but increases the concentration of

triglycerides and LDL-Cholesterol. His study with ME 100,

EDD 1000 reveals, that it increases the levels of total

serum cholesterol, triglycerides, LDL-Cholesterol, but does

not alter the concentration of the HDL-Cholesterol. In this

connection, 73McConathy et al. has observed that use of ME

100, EDD 1000, increases the concentration of triglycerides

but it has no influence on the levels of total serum

cholesterol, LDL-C and HDL-C. Only information available of

the effect of EE50, LYN 1000 indicates that it decreases the

concentration of HDL-Cholesterol as reported by Briggs et

al. 6l • The ratio of apo A-I/apo B seems to be decreased with

use of EE30 LNG 150 & triphasic ocs5l . On the other hand,

Bergink et al has reported that ratio of apo A-I/apo B

increases with use of OCs containing EE30 DSG150 50 .

Effect of use of OCs for 6 or more months on plasma lipids

and lipoproteins

Larson-Cohn et al. 47 & Bertolini et al. 74 have

23

reported the elevated levels of serum cholesterol with use

of OC containing EE30 LNG 150 for period of six months.

Similar increase in the concentration of triglycerides have

76 77also been noted by Wynn et ale & Mendoza et ale . The

52concentration of LDL-C also reported to be enhanced but it

decreases the levels of HDL-C as indicated by Harvengt et

ale 51 Ho et ale 53 Havekes et ale 78& th 52,58,74 On the, , o er •--

contrary, reports by Newton et ale 55Ahren et ale 58

- , ,. 1 69 th 53,59,75,76 . hBerglnk et a. & 0 ers are suggestlve t at the

concentration of serum cholesterol does not alter by this

combination. Similar observation on the levels of trigly-

. 57 58cerldes ' , LDL_C 53 ,75 and on the HDL_c 69 ,77 have been

reported. Further information in this regard indicate that

the concentration of HDL 2-C appears to be decreased while

it has no impact on the levels of HDL 3-C but enhances

the levels of apolipoprotein B and apolipoprotein

A_I 49 ,51,57,58.

Use of EE30 LNG 250 enhances the concentration of

total serum cholesterol while triglycerides levels remains

reported

78report also

unchanged but decreases the HDL-C concentration as

47 76by Larsson-Cohn et al~ & Wynn et ale • A

indicates that levels of apolipoproteins are increased with

the use of this preparation.

Results of investigations with use of EE 50, LNG

24

250 reveals that it increases the concentration of plasma

cholesterol as reported by Briggs et a1 57 , Mendoza et al. 77

& others74 ,78,79, triglycerides and LDL-cholesterol observed

19 74by Wahl et ale , Bertolini et ale but decreases the HDL-C

concentration57 ,74,78-81.

As far as triphasic OC preparations are concerned

no data are available to suggest that they enhance the

levels of plasma total cholesterol, triglyceride and LDL­

64cholesterol. However, Harvengt et ale and Hanne Refn et

al. 82 have reported that the use of triphasic formulation

decreases the concentration of HDL-cholesterol. On the other

hand, many workers have observed that these combinations

does not alter the levels of the total serum cholesterol,

, l' 'd d h 1 60,67,83, 'Itrlg ycerl es an HDL-c 0 esterol . Slml ar findings

in this regard have been reported by others on total serum

47 51 65 82 , ,65 66cholesterol ' , , , trlglycerldes' on LDL-chole-

stero1 82 and HDL-cholestero1 52 ,56,8l. It has further been

reported that triphasic preparations have no influence on

th 1 1 f HDL C67 I' ,83 d I' t 'e eve s 0 2-' apo lpoproteln an apo lpopro eln

A_I 51 ,55. Result of some studies, however, indicate elevated

1 1 f 1 ' " , h ' OC 51, 55eve s 0 apo lpoprotelns wlth use of trlp aS1C s •

The concentration of total plasma cholesterol and

LDL-cholesterol seems to be unaffected with use of EE 30,

DSG 150 while the concentration of HDL-cholesterol increased

triglycerides

as reported by

19lesias-Cortit

25

Bergink et al. 69 , Schweppe--59et ale . The levels of

et al. 84 and

increased Significantly with use of EE30 DSG150 as reported

by Harvengt et al. 64 Ho et al. 53 and others 47 ,57. On the

other hand, results of some investigations indicate that use

of this combination does not alter the levels of total serum

cholestero1 55 ,56,66,67,83 triglycerides 65 ,66,83,85, LDL-

cholestero183 ,84, and HDL-cholestero1 66 ,67,83. There are

some reports which are suggestive that use of this

combinations decreases apolipoprotein B83 levels and

, th . l' 'A 55,83lncreases e concentratl0n at apo lpoproteln -I •

71Ronald et ale has reported that use of EE35,

NET500 increases the concentration of plasma cholesterol and

triglycerides. The concentration of LDL and HDL-cholesterol

appears to be unaffected with use of the same OC as reported

81by Bradley et ale .

Results of a study indicates that the concen-

tration of Plasma cholesterol and triglycerides have

increased and LDL-cholesterol level unchanged with use EE35,

NET 100071 . Similar observation have been made by others on

57 64, . 57 71 86cholesterol ' , trlg1ycerldes ' and LDL-cholesterol •

Use of EE50, NETIOOO enhance the levels of

total cholestero1 71 ,79 triglycerides 78 ,79 and

plasma

has no

influence on the concentration LDL-cholestero171 and HDL-

26

78 83cholesterol ' . Similarly the levels of HDL 3-C and. 61

cholesterol remain unchanged while apolipoprotein

increases with use of same OC.

HDL ~2

A_I 78

Result of investigation carried out by Briggs et

al. 57 indicate that the concentration of cholesterol and

triglycerides increases while the levels of HDL-cholesterol

decreases with use of preparation containing EE30, EDD2000.

The oral contraceptive containing EE 50, EDD 1000 increase

th . f h 1 1 71 ,79 '1 'd 79 de concentratl0n 0 c 0 estero , trlg ycerl es an

LDL cholestero1 69 ,7l. While Wahl et al. 79 has reported that

the use of OC containing MEIOO, EDDlOOO enhances the levels

of plasma cholesterol, triglycerides, LDL-cholesterol but

decreases the concentration of HDL-cholesterol. Only

information seems to be available on EE50, LNYlOOO indicates

that it enhances the levels of HDL-C cholestero1 78 .

In view of well established certain abnormalities

caused by oral pills some of which are recognized to be risk

factors for development of CVD87 - 94 , recent studies have

considered to some extent, the effects of estrogen and

progestin, components of OCs on the lipids and lipoprotein

metabolism. Briefly stating that estrogen component may

increase total cholesterol, phospholipids, VLDL, LDL

cholesterol and apolipoprotein A-I and B95 - 97 , enhances the

synthesis of hepatic B:E and E receptors which are important

27

for the clearance of intermediate density lipoprotein, LDL

d h 1 . 98-100 . .. h . . 1 . dan c y omlcron remnants, lnhlblt epatlc trlg ycerl e

lipase, which degrades the triglycerides and phospholipids

of HDL _c lOl and increase the lecithin content of2

phospholipidsl0 2 ,103. The concentration of triglycerides in

the VLDL and HDL has been found to increase 1.5 to 2.5 fold

with the use of oral contraceptives containing higher

estrogen concentration87 . Serum HDL-concentrations has been

reported to increase with increasing dose of estrogen and

d . th' . d f . 104ecrease Wl lncreaslng ose 0 progestln Estrogen

administration also enhances the levels of triacylglycerol

in 105 106men and women ' . The development of hyperlipidemia

has been reported in avian species by administration of

107 108estrogen' The estrogen induced hyperlipidemia in

chicks is characterized by a marked elevation in the levels

of plasma triglycerides and, to a less extent, cholesterol

and phospholipidl07-110. Studies of hens treated with

estrogen have demonstrated that the hormone increases the

. l' . 111-113hepatic production of very low denslty lpoproteln •

By contrast most of the effects of estrogen are

antagonized by progestins used in oral pills. Progestins

inhibit the hepatic production of triglycerides, very low­

density lipoprotein, and apolipoprotein Al , stimulate the

activity of hepatic triglyceride lipase, and decrease the

28

lecithin content of phOSPholipidsl0 2 ,114,115. Comparison of

the OCs studied demonstrates that metabolic effects are

related not only to estrogen but also the dose ·of

progestinl16 . However, potency of progestin must be taken

into account in calculation of its dose. A review by Laneta

J fl ' 117 ,. d' h. Dor 1nger on progest1ns 1n 1cate t at norethindrone,

norethindrone acetate and ~thynodiol diacetate are roughly

equivalent in potency while norgestrol is roughly five to

ten times and levonorgestrol ten to 20 times as potent. The

relative potency of various progestins used in oral pills

have been summarized in Table IV.

Table IV Summary of relative progestin potency

Norethindrone

Norethindrone acetate

Ethynodiol Diacetate

Norgestrel

Levonorgestrol

1

1

1

5-10

10-20

It is

29

known that in atherosclerosis, the

metabolism of glycosaminoglycans (GAG) and glycoproteins

(GP) is also deranged in addition to that of lipidsl18-120.

Decreased concentration of sulphated GAG has been reported

in the aorta of atheromatous rats, while an increase in many

121-126carbohydrate components in GP has been reported . But

no systematic investigations seems to have been carried out

on the effect of oral contraceptives on the metabolism of

these complex macromolecular components.

There are reports that lipid peroxidation leading

to formation of oxidised LDL takes place in

atherosclerosis1 27 . This modified LDL is taken up rapidly by

scavenger receptors on macrophages which eventually leads to

deposition of cholesterol in the cells. Although the use of

DCs have been reported to increase the risk of

atherosclerosis disease, no reports seems to be available on

the level of lipid peroxidation in women using DC.

Thus, results of numerous studies reported on the

effect of DCs on lipid and lipoprotein metabolism found to

be in disagreement. Increased, decreased and no changes have

been reported. The mechanism(s) involved for causing these

changes remain largely unknown and it appears that not much

work has been conducted in this aspect. Above review also

indicates that our knowledge of the effects of DCs on lipid

30

metabolism rests mainly upon their effect on serum levels of

certain lipids. There seems to be very little information on

OCs containing low & high dose of estrogen when it is

combined with low potency progestin. Very few reports of

previous investigations are also available regarding OCs

influence on the levels of lipid peroxide, GAG and GP

metabolism. Therefore, we undertook to carry out detailed

studies by selecting Ovulen & N-Mala, oral contraceptive

agents containing high and low dose of estrogen respectively

and investigations conducted in this regard are as follows:

1. Effect of administration of Ovulen for different

duration on rats fed cholesterol free and cholesterol

containing diet, on concentration of cholesterol,

phospholipids and triglycerides in serum and tissues.

Concentration of cholesterol in HDL, LDL and VLDL

fractions of the serum. Release of the lipoproteins

into the circulation, activity of lipoprotein lipase in

the extra hepatic tissues, activity of HMG-CoA

reductase and in vivo incorporation of 141,2 C-acetate

into Liver Cholesterol, concentration of bile acids in

the Liver and intestine, activity of lipogenic enzymes

and histopathological studies of the aorta.

2. Comparative study of the effect of administration of N-

Mala and Ovulen, oral contraceptives agents on the

31

metabolism of lipids. The parameters as mentioned above

were investigated.

3. Effect of administration of estrogen and progestin

separately (Components of oral contraceptive agents) on

lipid metabolism with parameters as mentioned above.

4. Effect of administration of N-Mala on lipid peroxide

metabolism viz. Concentration of malondialdehyde,

hydroperoxides, conjugated dienes and free fatty acids

in tissues, activity of superoxide dismutase & catalase

in tissues, concentration of glutathione in liver and

ceruloplasmin in the serum.

5. Effect of administration of Ovulen on the metabolism of

glycosaminoglycans and glycoproteins concentration of

total glycosaminoglycans in tissues, concentration of

total hexose, fucose and sialic acid in tissue

glycoprotein.

The results of these investigations are discussed

in this thesis.