University of Nigeria Research Publications

NAMONDO, Lyonga Agnes

Aut

hor

Title

Further Studies On The Properties Of Katsina 1990 (KAT. 90) As An Antisickling

Agent

Facu

lty

Biological Sciences

Dep

artm

ent

Biochemistry

Dat

e

May, 1995

Sign

atur

e

FURTHER STUDIES ON THE PROPE!?TIES OF KATSINA 1 9 9 0

( K A T . ~ ~ ) AS AN A N T I S I C K L I N G AGENT

LYONGA, AGNES NAMONDO PG/MoSc /90 /9654

B .Sc0 ( ~ o n s ) UNIVERSITY OF LAGOS, N I G E R I A "

BEING A DISSERTATION SUBMITTED TO THE DEPARTMENT OF BIOCHEMISTRY I N PARTIAL

FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF A MASTER OF SCIENCE DEGREE I N

MEDICAL BIOCHEMISTRY

UNIVERSITY OF N IGERIA , NSUKKA

CERTIFICATION

Lyonga, Agnes Namondo, a P o s t g r a d u a t e s t u d e n t

i n t h e Depar tment of B i o c h e m i s t r y and w i t h t h e

R e g i s t r a t i o n Number PG/M.Sc/90/9654 has s a t i s f a c -

t o r i l y comple ted t h e r e q u i r e m e n t f o r c o u r s e and

r e s e a r c h work f o r t h e award of t h e d e g r e e of Mas te r

of S c i e n c e (M.Sc.) of t h e U n i v e r s i t y of N i g e r i a i n

Med ica l B i o c h e m i s t r y .

D r . P o N o Uzoegwu S u p e r v i s o r

....................... Head of Depar tment

.................... E x t e r n a l Examiner

iii

DEDICATION

To m y s i s t e r , F r i d a and my b r o t h e r , N j o h ,

ABSTRACT -

The c o n s t i t u e n t s and mode o f a c t i o n of KAT"90

powder were i n v e s t i g a t e d b e c a u s e i t had been o b s e r v e d

t o p o s s e s s b o t h a n t i s i c k l i n g and a n t i g e l l i n g p r o p e r t i e s .

K ~ T , 9 0 ( 1 ) a d m i n i s t e r e d t o p a t i e n t s ( u n d e r 10 y e a r s

o l d ) was p a r t i a l l y s o l u b l e i n d i s t i l l e d w a t e r , m e t h a n o l ,

m e t h a n o l - w a t e r (1:2) s o l v e n t s y s t e m and s o l u b l e i n

d i m e t h y l s u l p h o x i d e . KAT,90(11) a d m i n i s t e r e d t o

p a t i e n t s above 10 y e a r s o l d was p a r t i a l l y s o l u b l e i n

m e t h a n o l , s o l u b l e i n d i s t i l l e d w a t e r , d i m e t h y l s u l p h o -

x i d e and v e r y s o l u b l e i n m e t h a n o l - w a t e r (1 :2) s o l v e n t

s y s t e m , T h i n l a y e r ch roma tography of K A T . ~ ~ ( I I ) w i t h a p p a r e n t l y

m e t h a n o l - c h l o r o f o r m ( 1 : 9 ) s o l v e n t s y s t e m / r e v e a l e d - o n l y

0 b i p h a s i c manner w i t h one m e l t i n g p o i n t a t 146 C

( s i m i l a r t o t h a t of D-g lucose ) and t h e o t h e r above

3 6 0 ' ~ which was t o o h i g h t o be d e t e r m i n e d . Both

b a t c h e s have an a c i d i c pH of 4 and p r o b a b l y have the

same c h e m i c a l g r o u p s a s r e v e a l e d by t h e i r i d e n t i c a l

Chemica l t e s t s on KAT.90 showed t h e p r e s e n c e o f

s t a r c h , s u g a r s , g l y c o s i d e s and a p h e n o l i c compound.

P r o t e i n s , l i p i d s , a l k a l o i d s , s a p o n i n s , t a n n i n s ,

s t e r o l s and t r i t e r p e n e s were n o t d e t e c t e d . The

p r e s e n c e o f a p h e n o l i c compound i s s u g g e s t e d by t h 2

d e t e c t i o n o f C-0 and 0-H c h e m i c a l g r o u p s by t h e I R -

s p e c t r a l a n a l y s i s , t h e a c i d i t y o f t h e compound and

t h e d e t e c t i o n o f an a r o m a t i c r i n g as shown by t h e

a b s o r b a n c e peak i n t h e UV-spectrum. No amide bands

we re seen i n t h e I R - s p e c t r u m o f t h e p r e p a r e d amide

d e r i v a t i v e .

KAT.90 i n c r e a s e d t h e d e l a y t i m e o f g e l a t i o n o f

deoxyhaemog lob in S e s p e c i a l l y a t 4mg drug/ rn l o f

t r e a t m e n t w h i c h gave a . s i g n i f i c a n t i n h i b i t i o n o f

g e l a t i o n ( P < 0.05). F u r t h e r m o r e , t h e v i s c o s i t y o f

o x y g e n a t e d haernog lob in S was r e d u c e d by t h e d rug ,

These a c t i v i t i e s seem t o s u p p o r t t h e a n t i s i c k l i n g

and a n t i g e l l i n g a c t i v i t i e s o f t h e d rug .

Oxygena ted and d e o x y g e n a t e d h a e m o g l o b i n A a n d S

showed i n c r e a s e s i n oxygen o f f i n i t y when t r e a t e d w i t h

KAT.90 (1-3mg d rug /m l ) . T h e r e were s i g n i f i c a n t

i n c r e a s e s i n oxygen a f f i n i t y f o r t h e o x y g e n a t e d

haernog lob in S samples ( ~ < 0 . 0 5 ) , However a d e c r e a s e

i n oxygen a f f i n i t y was o b s e r v e d i n a l l t h e haemog lob in

samples when t r e a t e d w i t h t h e d r u g a t a h i g h e r concen-

t r a t i o n (4mg d r u g / m l ) and above.

ACKNOWLEDGEMENT

I e x p r e s s my p r o f o u n d g r a t i t u d e t o my S u p e r v i s o r ,

D r , P.N. Uzoegwu, f o r h i s c o n t r i b u t i o n s and m a r v e l o u s

s u p e r v i s i o n t o make t h i s r e s e a r c h wo rk a success . I

am a l s o i n d e b t e d t o D r , P o Awach ie f o r h i s g r e a t

a s s i s t a n c e i n t h e c h e m i c a l a s p e c t o f t h e work .

My t h a n k s go t o Dr . C, O k u n j i and D r , B. O k i d e

b o t h o f t h e F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s a n d

D r , M.J. K e l l e h e r o f t h e Depa r tmen t o f P u r e and

I n d u s t r i a l C h e m i s t r y , U n i v e r s i t y o f N i g e r i a , Nsukka

f o r t h e i r i n d i v i d u a l c o n t r i b u t i o n s .

I a p p r e c i a t e t h e encouragement g i v e n t o me by

P r o f . 0. Obidoa, Dr. M O O . Eze and Dr. O.F.C. Nwodo,

w h i c h k e p t me g o i n g , I acknowledge M r . E s s i e t ,

M r . Mba, M r . I t o d o f o r t h e i r t e c h n i c a l a s s i s t a n c e and

a l s o M r . Lawrence Eze f o r h i s h e l p .

My s i n c e r e g r a t i t u d e goes t o M r . G.A. A l e m n j i

whose s a c r i f i c i a l w o r k s made t h e s p e c t r a o b t a i n a b l e .

My s p e c i a l t h a n k s t o M i s s C, Monago and M r . A. Okpe

f o r t h e i r l o v e and c o n c e r n i n t i m e s o f d i f f i c u l t i e s

d u r i n g t h i s p e r i o d .

To my mother , Mrs . S o p h i e Lyonga and my s i s t e r ,

C h r i s t i e Lyonga, I say t h a n k you f o r p l a y i n g my r o l e i n

vii

the family in my absence, I also acknowledge, my

sister, Koko for her challenging kindness in a

period like this.

I express my thanks to my friends, Messrs

Gobi F. Elive, 0, Soludo, P, Mbah, T o Nwankwo and

Mrsc Onunwa.

I wish to express my sincere gratitude to all

my brethren, especially those in GSF who offered

prayers continually on my behalf.

Above all, I praise my prayer-answering God for

the revelations and grace to continue in the midst of

impossibilities encountered during this project work,

LYONGA, AGNES NAMONDO,

viii

TABLE OF CONTENTS

Certification --- --- --- --- --- ii

Abstract --- --- --- --- --- --- iv

CHAPTER ONE: --- --- --- - - INTRODUCT ION 1 1.1 KATSINA 1990 ( K A T . ~ ~ ) DRUG --- 1

1.2 The Haemoglobin Molecule --- --- 2

1.3 Normal and Abnormal Haemoglobins --- 4

1,4 Sickle Cell Anaemia --- --- 5

1.5 Clinical Features and Management of Sickle Cell Anaemia --- --- 8

1.6 The Role of Red Blood Cell Membrane in Sickle Cell Anaemia --- --- 12

1.7 Role of Oxygen Affinity and Haemo- globin Gelation in Sickle Cell Anaemia 17

1.8 Kinetics of Gelation and Polymer Formation --- --- --- --- 23

1.9 Antisickling Agents --- --- 24

1.9.1 Haemoglobin Modifiers --- --- 26

1.9.2 Membrane Modifiers --- --- 30

CHAPTER TWO :

2.1

2.2

2.2.1

2.2.2

2.2.3

2.2.4

2.2.5

2.2.6

2.2.7

Gene Activator --- --- --- Other Antisickling Agents --- Research Rationale and Objectives

MATERIALS AND METHODS --- Collection of Blood Samples

Preparation of Reagents

MayerUs Reagent --- Wagner's Reagent --- Picric Acid (1%) --- Dragendorffus Reagent

Fehling's Reagent

Iodine Vapour --- 0.1M Potassium Phosphate Buffer (pH 7.8) --- --- --- Biuret Reagent --- --- Melting Point Determination

pH Determination --- --- Sol.ubility Tests --- --- Thin Layer Chromatography of KAT.90

Other Tests to Identify the Consti- tuents of KAT.90 --- --- --- Protein --- --- --- ---

2.7.5 G l y c o s i d e s --- --- --- 2.7.6 A l k a l o i d s --- --- --- --- 2.7.7 S a p o n i n s --- --- --- --- 2.7.8 ' P h e n o l i c Compounds --- --- 2.7.9 T a n n i n s --- --- --- --- 2.7.10 S t e r o l s and T r i t e r p e n e s ( S a l k o w s k i u s

T e s t ) --- --- --- --- 2.8 Inf ra-Red S p e c t r o s c o p i c S tudy of

KAT , 90 --- --- --- --- 2.9 U l t r a v i o l e t S p e c t r o s c o p y of KAT.90

2.10 Convers ion of KAT.90 t o an Amide D e r i v a t i v e --- --- ---

2.11 T u r b i d i t y and Oxygen A f f i n i t y S t u d i e s

2.11.1 I s o l a t i o n of Haemoglobin --- --- 2.11.2 Deoxygenat ion o f Haemoglobin --- 2.11.3 T u r b i d i t y S t u d i e s --- --- 2.11.4 Oxygen A f f i n i t y A n a l y s i s --- ---

CHAPTER T H R E E : R E S U L T S --- --- --- 3.1 M e l t i n g P o i n t D e t e r m i n a t i o n --- 3.2 pH D e t e r m i n a t i o n --- --- --- 3.3 S o l u b i l i t y T e s t --- --- --- 3.4 T h i n Layer Chromatography of KAT.90

3.5 O t h e r T e s t s t o I d e n t i f y t h e C o n s t i - t u e n t s of KAT.90 --- --- ---

3.5.1 P r o t e i n --- --- --- ---

CHAPTER FOUR :

4.1

4.2

REFERENCES

APPENDIX I

APPENDIX I1

APPENDIX I11

L i p i d s --- --- --- S t a r c h --- --- --- R e d u c i n g S u g a r s --- --- G l y c o s i d e s --- --- A l k a l o i d s --- --- --- S a p o n i n s --- --- --- P h e n o l i c Compounds --- T a n n i n s --- --- --- S t e r o l s a n d T r i t e r p e n e s --- I n f r a - R e d S p e c t r u m o f KAT.90

U l t r a v i o l e t S p e c t r u m o f KAT.90

R e s u l t s f r o m D e r i v a t i z a t i o n o f

T u r b i d i t y S t u d i e s --- Oxygen A f f i n i t y A n a l y s i s ---

DISCUSSION AND CONCLUSION --- D i s c u s s i o n --- --- --- C o n c l u s i o n --- --- --- S u g g e s t i o n s f o r F u r t h e r S t u d i e s ---

x i i

L I S T OF TABLES -

TABLE PAGE

1 Oxygen A f f i n i t y o f Oxygena ted Haemog lob in Samples --- --- --- --- 64

2 Oxygen A f f i n i t y o f Deoxygena ted Haemog lob in Samples --- --- --- --- 6 5

F I G U R E

1

2

3

4

5

6

7

8

9

10

1 1

12

13

14

15

16

17

18

x i i i

LIST O F FIGURES

PAGE

Haeme ( F e - p r o t ~ p o r ~ h y r i n i x ) --- --- 3

Dimethyl a d i p i m i d a t e --- --- --- 28

C e t i e d i l --- --- --- --- --- 3 1

Desmethyl Ch lo rp romaz ine --- --- --- 3 1

Lawsone (2,04-1,4-naphthoquinone) --- 32

P r o c a i n e h y d r o c h l o r i d e --- --- --- 33

2-OH-methyl b e n z o i c a c i d --- --- --- 3 5

P-hydroxybenzoic a c i d --- --- --- 3 5

V a n i l l i c a c i d --- --- --- --- 3 5

Xant h o x y l o l --- --- --- --- 3 5

P r o p r a n o l o l --- --- --- --- 37

Formula o f t h e r e a c t i o n --- --- --- 61

I n f r a - R e d Spect rum o f P o t a s s i u m Bromide a s t he R e f e r e n c e Spect rum --- --- --- 66

In f ra -Red Spect rum of K ~ T . 9 0 ( 1 ) --- --- 67

In f ra -Red Spect rum of KAT.90(11) --- --- 68

A b s o r p t i o n Spect rum o f K A T . ~ ~ from 200-400nm --- --- --- - - 69

In f ra -Red Spect rum of t h e P r e p a r e d Amide D e r i v a t i v e of KAT.90 --- --- --- 7 0

P l o t of Absorbance a g a i n s t Tempera tu re f o r T r e a t e d and U n t r e a t e d Deoxyhaemoglobin S Samples , a t C o n c e n t r a t i o n Range o f 1-4mg K A T . 9 O / d --- --- --- --- 71

x i v

FIGURE PAGE

19 A b s o r p t i o n Spec t rum o f Oxygena ted Haemog lob in A T r e a t e d w i t h KAT.90 - - 7 2

2 0 A b s o r p t i o n Spec t rum o f Oxygena ted Haemog lob in S T r e a t e d w i t h KAT.90 --- 73

21 P l o t o f Abso rbance a g a i n s t T e m p e r a t u r e f o r T r e a t e d and U n t r e a t e d Oxyhaemog lob in S Samples, a t C o n c e n t r a t i o n Range o f

1-4rng K ~ ~ . 9 0 / m l --- --- --- --- 74

CHAPTER ONE

INTRODUCTION

1 , l KATSINA 1990 ( K A T , ~ ~ ) DRUG

K a t s i n a 1990 ( K A T , ~ ~ ) was a d m i n i s t e r e d t o s i c k l e

c e l l p a t i e n t s by D r , K . Somasunderam, a S r i L a n k a n M e d i c a l

D o c t o r w i t h Canad ian n a t i o n a l i t y . D r , K. Somasunderam

worked a t K a t s i n a Government G e n e r a l H o s p i t a l i n K a t s i n a ,

N i g e r i a , The d r u g whose c o m p o s i t i o n i s n o t known was

a d m i n i s t e r e d t o many s i c k l e c e l l p a t i e n t s i n t h e N o r t h e r n

and E a s t e r n p a r t s o f N i g e r i a , The f a m i l i e s o f t h e p a t i e n t s

who had t a k e n t h e d r u g i n d i c a t e d a marked r e d u c t i o n i n t h e

s e v e r i t y and f r e q u e n c y o f c r i s i s i n t h e p a t i e n t s , KAT.90

was c l a i m e d b y t h e D o c t o r t o have t h e p o t e n t i a l t o change

t h e g e n o t y p e o f s i c k l e c e l l anaemia p a t i e n t s f r o m HbSS t o

HbAS o r HbAA a f t e r t h r e e y e a r s o f t a k i n g t h e d r u g a c c o r d i n g

t o h i s p r e s c r i p t i o n .

KAT.90 was s u p p l i e d i n two b a t c h e s w h i c h i s d e n o t e d

i n t h i s p r o j e c t as KAT.90 ( I ) and KAT.90 (11). KAT.90 ( I )

i s w h i t i s h i n c o l o u r , powdery and t a k e n o r a l l y b y children

u n d e r t h e age o f t e n y e a r s , w h i l e KAT.90 (11) i s d i r t y

w h i t e , powdery and t a k e n o r a l l y b y p e r s o n s above t h e age

o f t e n y e a r s , A s i c k l e c e l l anaemia p a t i e n t i s e x p e c t e d

t o t a k e t h r e e grammes o f KATo90 p e r day f o r 5 days, a t an

2

i n t e r v a l o f f o u r months f o r t h e f i r s t y e a r , t h e n two

t i m e s f o r t h e n e x t y e a r and once f o r t h e t h i r d y e a r .

1,2 THE HAEMOGLOBIN MOLECULE

Haemog lob in i s t h e m a j o r c o n s t i t u e n t o f t h e r e d

b l o o d c e l l s . A h a e m o g l o b i n m o l e c u l e i s composed o f f o u r

s u b u n i t s a r r a n g e d t o f o r m two i d e n t i c a l h a l f m o l e c u l e s .

Each o f t h e f o u r s u b u n i t s i s made up o f two p a r t s : a

p o l y p e p t i d e c h a i n , g l o b i n and an i r o n c o n t a i n i n g

c o n s t i t u e n t c a l l e d haem ( ~ h o m p s o n and Thompson, 1 9 7 3 ) "

The f o u r p o l y p e p t i d e c h a i n s a r e f o l d e d and f i t t e d

t o g e t h e r t o f o r m a r o u g h l y g l o b u l a r m o l e c u l e w i t h a

m o l e c u l a r w e i g h t o f a p p r o x i m a t e l y 68,000 ( I n g r a m , 1959) .

A d u l t haemog lob ins a r e composed o f two a l p h a c h a i n s ,

each w i t h 141 amino a c i d s and two b e t a c h a i n s each w i t h

146 amino a c i d s . Each c h a i n s u r r o u n d s t h e i r o n p o r p h y r i n

p r o s t h e t i c g r o u p c a l l e d haem, w h i c h c o n t a i n s t h e c e n t r a l

i r o n a tom t o w h i c h oxygen can be r e v e r s i b l y bound (Dean

and S c h e c h t e r , 1 9 7 8 ) "

The r e d b l o o d c e l l o f t h e u n b o r n c h i l d c o n t a i n s

c h i e f l y f o e t a l o r F haemog lob in , F o e t a l h a e m o g l o b i n

has t w o a l p h a and two gamma c h a i n s . D u r i n g t h e f i r s t

y e a r o f l i f e , t h e gamma c h a i n s a r e g r a d u a l l y r e p l a c e d b y

a d u l t b e t a h a e m o g l o b i n c h a i n s ( ~ r u o n g -- e t a l . , 1964).

F i g . 1 Haerne ( F e - p r o t o p o r p h y r i n i x )

1 ,3 NORMAL AND ABNORMAL HAEMOGLOB INS

T h e normal and abnormal haemoglobins have t h e same

i r o n atoms and t h e same p o r p h y r i n complexes , They d i f f e r

o n l y i n t h e amino a c i d compos i t ion of t h e p o l y p e p t i d e

c h a i n s . Most of t h e abnormal haemoglobins a r e produced

by m u t a t i o n s i n t h e s t r u c t u r a l genes which d e t e r m i n e t h e

amino a c i d sequence of t h e g l o b i n p o r t i o n of t h e haemo-

g l o b i n molecu le ( ~ h o r n ~ s o n and Thompson, 1973)" I n t h e

amino a c i d s e q u e n c e s of d i f f e r e n t haemoglobin c h a i n s ,

t h e r e a r e c o n s t a n t o r i n v a r i a n t a s w e l l a s s i m i l a r

r e s i d u e s . Any s u b s t i t u t i o n of t h e i n v a r i a n t r e s i d u e s

f o r o t h e r s a s i n abnormal haemoglobins l e a d s t o t h e

a l t e r a t i o n of t h e p r o p e r t i e s o f t h e p r o t e i n ( P e r u t z and

Lehmann, 1968).

A l l t h e known abnormal haemoglobins c o u l d be shown

t o be i n h e r i t e d i n a s i m p l e Mendel ian manner ( Ing ram,

1990). Sometimes two genes f o r d i f f e r e n t abnormal

haemoglobins a f f e c t i n g both a l p h a and b e t a c h a i n s a r e

p r e s e n t i n t h e same i n d i v i d u a l . Abnormal haemoglobins

a r e i d e n t i f i e d and c h a r a c t e r i z e d by t h e i r d i f f e r e n t

e l e c t r o p h o r e t i c m o b i l i t y . The a b i l i t y t o p i n p o i n t t h e

amino a c i d s u b s t i t u t i o n s i n t h e v a r i o u s abnormal haemo-

g l o b i n s l e d t o a c l a s s i f i c a t i o n o f t h e s e m u t a t i o n s , where

t h e a c t u a l amino a c i d s u b s t i t u t i o n had o c c u r r e d , f o r

5

e x a m p l e Hbj3 6Va1?'n6GA~ich t h e s i x t h amino a c i d i n t h e

b e t a h a e m o g l o b i n c h a i n i s v a l i n e ( l n g r a m , 1990),

T h e h a e m o g l o b i n i n t h e r e d b l o o d c e l l p e r f o r m s v i t a l

f u n c t i o n s , c a r r y i n g o x y g e n f rom t h e l u n g s t o t h e t i s s u e s

a n d a l s o f a c i l i t a t i n g t h e t r a n s p o r t o f c a r b o n d i o x i d e

f rom t h e t i s s u e s t o t h e l u n g s , The r e d c e l l s a r e o n e

t h o u s a n d times more numerous t h a n t h e w h i t e c e l l s a n d

c o n s t i t u t e h a l f o f t h e vo lume o f t h e c i r c u l a t i n g b l o o d

( ~ i n t r o b e , 1961) , One t h i r d o f t h e w e i g h t o f t h e r e d

c e l l i s d u e t o i t s h a e m o g l o b i n c o n t e n t , t h e o t h e r two

t h i r d s b e i n g c h i e f l y w a t e r , When t h e h a e m o g l o b i n i s

a b n o r m a l , t h e r e i s a t e n d e n c y f o r i t t o come o u t o f

s o l u t i o n ( H a r r i s , 1963) ,

When b l o o d i s f u l l y o x y g e n a t e d , i t h a s a b r i g h t - r e d

c o l o u r a n d when o x y g e n i s l o s t o r t h e h a e m o g l o b i n i s

r e d u c e d i t h a s a d a r k - r e d c o l o u r . When o x y g e n a t e d

h a e m o g l o b i n i s f u r t h e r o x i d i z e d m e t h a e m o g l o b i n i s

f o r m e d a n d t h i s h a s a c h o c o l a t e brown c o l o u r .

1,4 S I C K L E C E L L ANAEMIA

Haemoglob in S , a m u t a n t o f w h i c h v a l i n e i s s u b -

s t i t u t e d f o r g l u t a m i c a c i d a t p o s i t i o n s i x o f t h e b e t a

c h a i n i l l u s t r a t e s how a s i n g l e amino a c i d s u b s t i t u t i o n

c o u l d a d d a new p r o p e r t y t o a p r o t e i n m o l e c u l e , t h a t i s ,

t h e c a p a c i t y t o po lymer ize . I t s i n h e r i t a n c e p a t t e r n had

a s i g n i f i c a n t impact on g e n e t i c s b e c a u s e i t showed t h a t

t h e c o n c e p t of one gene , one p o l y p e p t i d e c h a i n was

a p p l i c a b l e t o mammals ( Ing ram, 1 9 9 0 ) "

S i c k l e c e l l anaemia i s a g e n e t i c d i s e a s e , t h e

symptons of which r e s u l t from an u l t i m a t e a g g r e g a t i o n

of haemoglobins i n t h e haemoglobin S e r y t h r o c y t e s due

t o lowered oxygen t e n s i o n , r e s u l t i n g i n t h e c h a r a c t e r i s -

t i c d i s t o r t e d s i c k l e d s h a p e s (Adesanya and Sofowora ,

1983) . S i c k l e c e l l anaemia i s a l s o a p o t e n t i a l l y

l e t h a l d i s e a s e w i t h c l i n i c a l m a n i f e s t a t i o n s r e s u l t i n g

from t h e homozygous e x p r e s s i o n of a mutant g l o b i n gene .

The major m a n i f e s t a t i o n s of t h i s d i s e a s e a r e c h r o n i c

h a e m o l y t i c anaemia and v a s o - o c c l u s i v e c r i s i s t h a t c a u s e

s e v e r e p a i n a s w e l l a s long- t e rm and w i d e s p r e a d o r g a n

damage, I n a d d i t i o n t h e r e a r e s y s t e m i c e f f e c t s from

s i c k l e c e l l d i s e a s e such a s i n c r e a s e d s u s c e p t i b i l i t y t o

i n f e c t i o n s and i m p a i r e d growth and development (Konotey-

A h u l u , 1974) .

A pe r son may c a r r y both A - ( n o r m a l ) haemoglobin

and S - ( s i c k l e ) haemoglobin i n which c a s e he i s t e rmed

a " c a r r i e r " ( g e n o t y p e AS) w i t h r e f e r e n c e t o s i c k l e c e l l

d i s e a s e o r he may have a homozygous haemoglobin A -

( g e n o t y p e AA) o r homozygous haemoglobin S - ( g e n o t y p e S S ) ,

A c a r r i e r does no t s u f f e r from t h e c h a r a c t e r i s t i c c r i s i s

i n s i c k l e c e l l d i s e a s e (Sofowora -- e t a l , , 1979) .

The oxygena ted form of haemoglobin S i s s i m i l a r

i n s t r u c t u r e and b e h a v i o u r t o haemoglobin A. However,

when haemoglobin S i s deoxygena ted , i t s p r o p e r t i e s

d i f f e r markedly from t h o s e of deoxygenated haernoglobin

A due t o p o l y m e r i z a t i o n of t h e haemoglobin S m o l e c u l e s

i n t o l o n g f i l a m e n t s which d i s t o r t t h e r e d b lood c e l l s .

A l l i s o n (1957) showed t h a t s i c k l e c e l l oxyhaemoglobin

has t h e same t y p e of v i s c o s i t y b e h a v i o u r a s normal a d u l t

oxyhaemoglobin. According t o Rampling and S i r s (1973)

and Bookchin e t a l , ( 1 9 7 6 ) , t h r e e t y p e s of c e l l s a r e -- found when s i c k l e b lood i s deoxygena ted : normal ,

b i z a r r e ( w r i n k l e d c e l l s u r f a c e ) , and h o l l y l e a f c e l l s

( a t l e a s t one s h a r p - p o i n t e d p r o j e c t i o n ) .

The d i s e a s e has i t s h i g h e s t i n c i d e n c e i n b l a c k

A f r i c a n s and Afro-Americans. I t i s a l s o found i n

M e d i t e r r a n e a n c o u n t r i e s l i k e Greece , I t a l y and I s r a e l

a s w e l l a s i n S a u d i A r a b i a and I n d i a . A f r i c a n s have

s u f f e r e d from t h e d i s e a s e f o r g e n e r a t i o n s and i t has

been t r a c e d back a s f a r a s 1670 i n one f a m i l y b e l o n g i n g

t o t h e Krobo t r i b e i n Ghana ( K o n o t e y - ~ h u l u , 1974)"

The common types o f haernoglobinopathy are i d e n t i f i e d

by t h e genotypes of t h e i n d i v i d u a l s u f f e r e r s . These

i n c l u d e s i c k l e c e l l t r a i t , haemoglobin C t r a i t and

thalassaernia t r a i t . The t h r e e t ypes of s i c k l e c e l l

d i s e a s e a r e : s i c k l e c e l l anaemia, s i c k l e c e l l

haemoglobin C d i s e a s e , and s i c k l e c e l l t h a l a s s a e m i a ,

The l a t t e r i s a combination of s i c k l e c e l l t r a i t and

be t a thalassaernia t r a i t which produces a c l i n i c a l

p i c t u r e s i m i l a r t o , but sometimes l e s s s e v e r e than

s i c k l e c e l l anaemia (Wea the ra l l , 1968).

1 , 5 C L I N I C A L FEATURES AND MANAGEMENT O F SICKLE CELL A N A E M I A

During i n f e c t i o n s r e d c e l l p roduc t ion i n s i c k l e

c e l l anaemia p a t i e n t s may be c u r t a i l e d t o abnormal o r

subnormal l e v e l s ( a p l a s t i c c r i s i s ) and t h e mean r e d

c e l l age suddenly i n c r e a s e s (Diggs, 1965) , I n s i c k l e

c e l l anaemia p a t i e n t s , haernoglobin c o n c e n t r a t i o n i s a s

low a s 7g/IOOml of blood - /Normal: male, 13.5- 18.O9/100rn1;

female, 12.0-16,0g/100ml ( T i l k i a n -- e t a l . , 1979)7. - Some

d a t a showed t h a t t h e mean r a t e of haem ca tabo l i sm i s

approximately s i x t imes t h e normal, b u t v a r i e s from

3 t o 14 t imes i n normal i n d i v i d u a l p a t i e n t s (Coburn,

1963) , I n haernolysis t h e c e l l s a r e d e s t r o y e d e i t h e r

intravascularly or extravascularly releasing t h e contents

of the erythrocytes,

The incidence and severity of the pathogenic

manifestations o f sickle cell anaemia vary from patient

to patient, Ranney (1970) gave an explanation for this

variance in severity observed in different patients.

Clinical and laboratory investigations of patients

whose red blood cells contain haemoglobin S together

with another haemoglobin showed that different haemoglobins

participate to different extents in the formation of

deoxyhaemoglobin gels (Ranney, 1970) . Other genetic

determinants such as glucose-6-phosphate dehydrogenase

deficiency could also contribute to this ( P i o r n e l l i - et -.I a 1

1972) . The foetal haernoglobin level has

been implicated as a mediating factor in the incidence

and severity of the crisis as well (Bertles -- et al., 1970) .

A variety of alpha chain mutations interacting with

haemoglobin S are also thought to serve as factors in

the explanation of the differences in severity from one

patient to another. Dietary and environmental factors

as well as geographic locations may also influence

severity, Although there is a wide spectrum of clinical

severity in this disease for life expectancy, frequency

of crisis, degree o f anaemia and extent of organ invove-

ment, the factors that contribute to this variation have

not as yet been fully elucidated. The only variable

that appears to correlate with protection of patients

from the manifestation of having haemoglobin S is the

presence of normal adult haemoglobin (Lessin and Jensen,

1974). Occasionally there is a sudden massive -- in v i v o

sickling that depletes the b o d y U s essential oxygen hence

the patient runs into a state of crisis which is usually

painful (Konotey-Ahulu, 1974). The clinical features of

cell disorders are best understood in terms of vaso-

occlusive and haernatologic effects. Konotey-Ahulu (1974)

described the vaso-occlusive crisis as infarctive crisis

with pain, while haemolytic crisis occurs as a result of

sudden red blood cell haemolysis.

Vaso-occlusive features of cardiovascular

abnormalities like cardiac enlargement, systolic murmurs

and ventricular gallops are common. Also bone and joint

changes are the results o f ischaemia in the marrow o f

the toes and fingers and eventually other sites. Such

events are quite common in sickle cell anaemia in

Jamaica and elsewhere, where the disease seems to

pursue a more benign course (~erjeant, 1969),

A d u l t s w i t h s i c k l e c e l l anaemia u s u a l l y have

c o m p l a i n t s of p a i n s i n s i d e t h e l o n g bones o r abdomen.

T h o r a c i c , o r l o i n p a i n s a r e a l s o e x p e r i e n c e d d u r i n g

c r i s i s . The p a i n i s i n t e n s e and c o n s t a n t , u s u a l l y

l a s t i n g a b o u t f i v e days , though v e r y v a r i a b l e . C r i s i s

r e c u r a t i r r e g u l a r i n t e r v a l s from a b o u t once i n two

weeks t o once i n a y e a r ( I s a a c s - S o d e y e , 1975).

I n t h e c a s e of h a e m a t o l o g i c a l f e a t u r e s , r e d c e l l

s u r v i v a l i s c h r o n i c a l l y s h o r t , c o m p l i c a t e d by a c u t e

h a e m o l y t i c e p i s o d e s . Haemolys is l e a d s t o j a u n d i c e and

t h e f o r m a t i o n of pigment s t o n e s , Chron ic h a e m a t o l y t i c

t y p e of t h i s anaemia may be a s s o c i a t e d w i t h weakness ,

d i z z i n e s s , y e l l o w d i s c o l o u r a t i o n , j a u n d i c e , v o m i t t i n g

and p a i n i n t h e e p i g a s t r i c and s p l e n i c r e g i o n .

T r e a t m e n t i n s i c k l e c e l l d i s e a s e may be d i r e c t e d

a t an improvement of t h e s t e a d y s t a t e and t h e p r e v e n t i o n

of c r i s i s o r c o m p l i c a t i o n s . The n u t r i t i o n o f t h e i n d i -

v i d u a l w i t h s i c k l e c e l l d i s e a s e differs from t h a t of t h e

a v e r a g e pe r son i n t h a t t h e r e i s a g r e a t e r r e q u i r e m e n t

f o r f o l i c a c i d which i s r e q u i r e d t o s u p p o r t t h e h igh

r a t e o f e r y t h r o p o e i s i s ( L i n d e n t a u m and K l i p s t e i n , 1963) ,

I n t h e p r e s e n c e o f m e g a l o b l a s t o s i s , f o l i c a c i d i s o f

p r o v e n v a l u e (Wa tson -Wi l l i ams , 1965).

D e s p i t e t h e h i g h r a t e o f t u r n o v e r o f i r o n , t h e

i n d i v i d u a l w i t h s i c k l e c e l l d i s e a s e does n o t seem t o

be p r o n e t o t h e deve lopment o f i r o n d e f i c i e n c y (Reyno lds ,

1965).

I n s i c k l e c e l l d i s e a s e , t h e r e i s u s u a l l y a c e r t a i n

d e g r e e o f anaemia w h i c h has been e x p l a i n e d on t h e b a s i s

t h a t t h e s h i f t i n oxygen d i s s o c i a t i o n c u r v e a l l o w s t h e

t i s s u e s an abundance o f oxygen d e s p i t e t h e r e d u c e d l e v e l

o f haemog lob in S ( B e l l i n g h a m and Huehns, 1968) . However,

when t h e haemog lob in l e v e l f a l l s b e l o w 5 grammes i n

s i c k l e c e l l anaemia, b l o o d t r a n s f u s i o n s may be g i v e n ,

Some i n v e s t i g a t o r s remove a s m a l l vo lume of t h e

p a t i e n t o s b l o o d and r e p l a c e i t w i t h n o r m a l b l o o d i n

a programme o f p a r t i a l exchange t r a n s f u s i o n ( A n d e r s o n

e t a l , , 1963), -- 1.6 THE ROLE OF RED BLOOD CELL MEMBRANE

I N S ICKLE CELL ANAEMIA

Much o f what i s known c o n c e r n i n g t h e m o l e c u l a r

s t r u c t u r e o f t h e c e l l membrane has come f r o m t h e s t u d y

o f t h e r e d b l o o d c e l l s o f mammals, These d i s c - s h a p e d

c e l l s t h a t have l o s t t h e i r n u c l e i can be r e a d i l y

empt i ed of t h e r e d c e l l haemoglobin t h e y c a r r y ,

l e a v i n g f o r s t u d y t h e r e d c e l l g h o s t , which i s

m o s t l y membrane ( B e r r i l l , 1966) .

S h a k l a i -- e t a l , (1987) r e p o r t e d t h a t c r o s s l i n k i n g

of i s o l a t e d c y t o s k e l e t a l p r o t e i n s w i t h haemoglobin was

a p o s s i b l e damage i n f l i c t e d t o t h e r e d c e l l membrane,

and t h i s may p r o v i d e a r a t i o n a l e f o r t h e l o s s of

membrane f l e x i b i l i t y , which l e a d s t o c e l l d i s t o r t i o n

i n v a r i o u s h a e m o g l o b i n o p a t h i e s .

The amounts of haem c o n t a i n e d i n normal and

s i c k l e human e r y t h r o c y t e g h o s t membranes were compared

w i t h haem t r u l y a s s o c i a t e d w i t h i n s i d e - o u t membranes

(Kuross -- e t a l . , 1988) , T h e r e were abnormal amounts

of haem t r u l y a s s o c i a t e d with t h e s i c k l e r e d b lood

c e l l membranes which s u g g e s t that i t p a r t i c i p a t e s i n

t h e p a t h o b i o l o g y o f sickle red b lood c e l l s , p a r t i c u l a r l y

i n t h i o l o x i d a t i o n .

Haemoglobin S e x h i b i t e d enhanced b i n d i n g t o r e d

b lood c e l l membranes when compared t o haemoglobin A

(Fung e t a l . , 1 9 8 3 ) , The d i f f e r e n c e between normal - - and s i c k l e haemoglobin p e r s i s t e d a t bo th pH 7 . 4 and low

pH v a l u e s , The c o n c e n t r a t i o n s of haemoglobin a t t h e

s a t u r a t i o n l e v e l were c l o s e t o p h y s i o l o g i c a l concen-

t r a t i o n s , Removal of s p e c t r i n - a c t i n p r o t e i n m o l e c u l e s

from t h e membranes caused l i t t l e change i n t h e i n t e r -

a c t i o n s i n d i c a t i n g t h a t t h e r emain ing membrane p r o t e i n s

p l a y a pr imary r o l e i n haemoglobin membrane i n t e r a c t i o n s .

Haemoglobin e x i s t s i n normal r e d c e l l s a t con-

c e n t r a t i o n s c l o s e t o i t s maximum s o l u b i l i t y . Hence

d e h y d r a t i o n o r changes i n s o l u b i l i t y by m u t a t i o n may

r e s u l t i n t h e p r e c i p i t a t i o n of e x c e s s haemoglobin on

t h e c y t o p l a s m i c membrane s u r f a c e . Indeed membrane

a t t a c h e d haemoglobin i s a common f e a t u r e i n d e h y d r a t e d

and haemoglob inopa th ic red c e l l s , and t h e r e f o r e , may

p l a y a role i n s p e c t r i n haemoglobin c r o s s l i n k i n g by

be ing a s s o c i a t e d w i t h t h e membrane p r i o r t o o x i d a n t

a t t a c k . Mutants w i t h l ower s o l u b i l i t y t h a n haemoglobin

A t e t r a m e r s , l i k e f r e e c h a i n s i n t h a l a s s a e m i a o r

haemoglobin S i n s i c k l e c e l l anaemia, t h e r e f o r e t e n d

t o s e t t l e on t h e membrane, I t i s e s t a b l i s h e d t h a t

non-covalent a s s o c i a t i o n s of c y t o s k e l e t a l p r o t e i n s a r e

c r u c i a l f o r t h e r e d c e l l membrane f l e x i b i l i t y (L iu and

P a l e k , 1980).

The e f f e c t of t h e deoxygena t ion r a t e on t h e

f o r m a t i o n of i r r e v e r s i b l y s i c k l e d c e l l s was o b s e r v e d

t o r e q u i r e calcium ions . The amount of s i c k l e d c e l l s

formed was a l s o found t o be dependent on t h e r a t e of

deoxygenation (Hor iuch i -- e t a l . , 1988).

Eluwa e t a l . (1987) i n v e s t i g a t e d t h e v a r i a t i o n s -.1-

i n t h e r e l a t i v e a c t i v i t i e s o f e r y t h r o c y t e membrane

ATPase w i t h changes i n s e v e r i t y of s i c k l e c e l l anaemia.

The ATPase a c t i v i t i e s were c o r r e l a t e d w i t h t h e v a r i o u s

s t a g e s of s e v e r i t y i n each p a t i e n t a s determined by

c l i n i c a l parameters . The r e s u l t s showed t h a t i n c r e a s e s

i n ATPase a c t i v i t i e s were a s s o c i a t e d w i t h i n c r e a s e s i n

t h e percen tage s e v e r i t y of s i c k l e c e l l anaemia.

S e v e r i t y c o r r e l a t e d i n v e r s e l y w i t h f o e t a l haemoglobin

l e v e l s i n t h e s i c k l e c e l l p a t i e n t s . Also they showed

t h a t t h e a c t i v i t y o f ATPase enzyme was g e n e r a l l y high

i n SS than i n AS and normal AA i n d i v i d u a l s . T h i s may be compensatory f o r t h e s i c k l e r dur ing c r i s i s .

S i c k l e c e l l t r a i t AS c e l l s c o n t a i n about 35 t o

40 per cen t haemoglobin 5 and do not g e n e r a l l y s i c k l e

i n t h e c i r c u l a t i o n ( ~ a l e k , 1977), hence t h e i r membranes

and i o n i c composit ion appear normal. I n h y p e r t o n i c

media, where t h e i r mean c e l l haemoglobin c o n c e n t r a t i o n

(MCHC) i s r a i s e d , AS c e l l s show s i c k l i n g p a t t e r n s and

oxygen e q u i l i b r i a very s i m i l a r t o t h o s e of SS c e l l s i n

i s o t o n i c media (Asakura et a l . , 1977). --

Red c e l l m e t a b o l i c f u n c t i o n s of haemoglobin S

a p p a r e n t l y do n o t c a u s e ma jo r a l t e r a t i o n i n r e d c e l l s

( C l a r k -- e t a l . , 1 9 7 8 ) " The high 2 ,3-d iphospho g l y c e r a t e

(2,3-DPG) l e v e l s i n SS c e l l s r e p r e s e n t a p h y s i o l o g i c a l

r e s p o n s e t o t h e anaemia, I t i s n o t c l e a r why i r r e v e r -

s i b l y s i c k l e d c e l l s have low l e v e l s of 2,3-DPG. T h e r e

a r e a l s o c o n f l i c t i n g r e p o r t s a b o u t whether t h e i r ATP

l e v e l s a r e low o r normal . Damage of s i c k l e c e l l

membrane i s p r e v a l e n t i n t h e i r r e v e r s i b l y s i c k l e

c e l l s ( S e r j e a n t -- e t - a l , , 1 9 6 9 ) , The c e l l s a p p e a r

t o be p r e l y t i c and a r e r a p i d l y c l e a r e d from c i r c u l a t i o n .

These membrane damaged c e l l s t e n d t o c a u s e o b s t r u c t i o n

of b lood v e s s e l s (Hebbel , 1981). Membrane damage a l s o

i s i n v o l v e d i n t h e p a t h o g e n e s i s o f t h e c l i n i c a l

a b n o r m a l i t i e s i n s i c k l e c e l l anaemia ( S e r j e a n t e t a l . , -- 1972).

T h e o x i d a n t - a n t i o x i d a n t b a l a n c e was o b s e r v e d t o

be d i s t u r b e d i n s i c k l e c e l l anaemia e ebb el, 1986).

Fur the rmore t h e development o f e r y t h r o c y t e membrane

d e f e c t i n t h i s d i s e a s e i s r e l a t e d t o e x c e s s i v e a u t o x i -

d a t i o n , T h e s u p p r e s s i o n i n i n c u b a t i o n - i n d u c e d o x i d a t i v e

s t r e s s by a n t i o x i d a n t f r e e r a d i c a l s c a v e n g e r s and an i r o n

( F e ) c h e l a t o r s u g g e s t s t h a t o x i d a t i o n p r o d u c t s o f mernbrane-

bound haemoglobin c o n t r i b u t e towards the p a t h o l o g y o f t h e

d i s e a s e ice-Evans e t a l . , 1986). - --

I t i s i m p o r t a n t t o d e t e c t a p p a r e n t a n t i s i c k l i n g

e f f e c t s which r e s u l t from membrane changes which c o u l d

a f f e c t c e l l s u r v i v a l , T h e r e a r e two l i k e l y p o s s i b i l i t i e s

f o r such a mode of a c t i o n . F i r s t , a decrease i n membrane

f l e x i b i l i t y might p r e v e n t c e l l s from assuming a s i c k l e d

s h a p e d e s p i t e p o l y m e r i z a t i o n o f t h e haemoglobin , Such

a " p i c k l i n g " e f f e c t c o u l d be d e t e c t e d by c e l l f i l t r a t i o n ,

o r by a n o t h e r t e s t o f c e l l d e f o r m a b i l i t y , Secondly,

membrane changes r e s u l t i n g i n a n e t i n c r e a s e i n c e l l

w a t e r c o u l d d e c r e a s e p o l y m e r i z a t i o n by l o w e r i n g t h e

c e l l haemoglobin c o n c e n t r a t i o n ,

1.7 ROLE OF OXYGEN AFFINITY AND HAEMOGLOBLN - GELATION I N SICKLE CELL - A N A E M I A

I n p a t i e n t s w i t h s i c k l e c e l l d i s e a s e , oxygen

e q u i l i b r i u m c u r v e s p r o v i d e a v a l u a b l e g u i d e t o t h e

oxygen d e l i v e r y p o t e n t i a l of the b lood , T h e r e i s now

c o n s i d e r a b l e e v i d e n c e t h a t p o l y m e r i z a t i o n o f haernoglobin

S s u b s t a n t i a l l y l o w e r s i t s o v e r a l l oxygen a f f i n i t y

( J a y a l a k s m i and S e a k i n s , 1976). I t i s t h e r e f o r e

i m p o r t a n t t o u n d e r s t a n d t h e r e l a t i o n s h i p s between

oxygen t e n s i o n , oxygen s a t u r a t i o n and p o l y m e r i z a t i o n

of haemoglobin S. The i n f l u e n c e of haemoglobin deoxyge-

n a t i o n on s i c k l e c e l l anaemia e r y t h r o c y t e s i s p a r t i c u l a r l y

significant in view of its effect on the solubility

of haemoglobin S molecules and the consequent sickling

phenomenon.

A s blood pH is lowered from 7.4 to 7.2, the oxygen

affinity of S S blood falls almost twice as sharply as

that of normal red blood cells (Rosa -- et al., 19791, This

marked increase in the whole blood Bohr effect occurs

in the same pH range over which a small decrease in pH

results in a large increase in the gelling tendency of

deoxyhaemoglobin S (Bookchin -- et al., 1976).

At a pH of 7.4 the oxygen affinity of S S blood

is just moderately lower than normal, A large part of

the difference at this pH can be attributed fo the

increased level of red cell 2,3-diphosphoglycerate, and

would therefore not be corrected much in vitro by counter- - -- acting polymerization. The maximal difference in oxygen

affinity between SS and normal blood cells was found at

a blood pH of about 7,2 and polymerization apparently

accounts for most of this large difference. Therefore

the lower pH range, 7.1 to 7.2 would be most sensitive

for detecting normalization of the oxygen affinity of

S S blood due to a drug" antipolymerization activity

(Rosa e t al., l979) , --

The s l i g h t i n c r e a s e of mean c e l l haemoglobin

c o n c e n t r a t i o n from 35,9grn/dl i n c o n t r o l t o 36,7gm/dl

i n p redeoxygena ted b lood was s u g g e s t e d t o enhance

i n t r a c e l l u l a r p o l y m e r i z a t i o n ( S i n e t and P o c i d a l o , 1981) .

The lower pH o b s e r v e d i n p redeoxygena ted samples might

a l s o promote haemoglobin S p o l y m e r i z a t i o n . F u r t h e r m o r e ,

t h e p e r s i s t e n t i n c r e a s e i n t h e p e r c e n t a g e of s i c k l e c e l l s

a f t e r deoxygena t ion might be due t o t h e m o d i f i c a t i o n of

s e v e r a l v a r i a b l e s i n v o l v i n g a change i n t h e p o l y m e r i z a t i o n

p r o c e s s .

S i c k l i n g and r e v e r s a l o f s i c k l i n g a r e f a s t r e a c t i o n s

( P a d i l l a e t a l . , 1973). T h e f u n c t i o n a l p r o p e r t i e s o f -- -- haemoglobin S i s s t r i k i n g l y d i f f e r e n t , depend ing on

w h e t h e r t h e y a re s t u d i e d i n p u r e d i l u t e s o l u t i o n s ,

i n p u r e c o n c e n t r a t e d s o l u t i o n s , o r u n d e r ' c o n d i t i o n s

p r e v a i l i n g i n t h e e r y t h r o c y t e . I n d i l u t e s o l u t i o n s ,

haemoglobin S e x h i b i t s t h e same f u n c t i o n a l p r o p e r t i e s

a s haemoglobin A ( P e n n e l l y and Noble, 197'8). However

a t low oxygen t e n s i o n , t h e haemoglobin S p o l y m e r i z e s and

hence s t o p s b i n d i n g oxygen ( G i l l e t a l . , 1979). The -- a c t i o n o f s m e a l l o s t e r i c e f f e c t o r s o n t h e haemoglobin

m o l e c u l e a l s o i n t e n s i f i e s b e c a u s e o f t h e two f o l d

i n f l u e n c e of t h e s e e f f e c t o r s , p a r t i c u l a r l y hydrogen

i o n s and 2 , 3 - d i p h o s p h o g l y c e r a t e , on oxygen-haernoglobin

a f f i n i t y and on t h e e x t e n t of p o l y m e r i z a t i o n ( ~ i a n -- e t a l . ,

1971) .

Serum e r y t h r o p o i e t i n l e v e l s i n s i c k l e c e l l anaemia

i n c r e a s e d a t a lower haemoglobin c o n c e n t r a t i o n and a r e

of a lower magni tude t h a n t h o s e of the o t h e r anaemia

(Sherwood -- e t al., 1986), The e r y t h r a p o e t i c r e s p o n s e

i n s i c k l e c e l l anaemia i s p r o b a b l y d e c r e a s e d by t h e low

oxygen a f f i n i t y of s i c k l e c e l l s (Be l l ingham and Huehns,

1968) , and t h e c o r r e s p o n d i n g low h a e m a t o c r i t p r o b a b l y

p r o v i d e s some p r o t e c t i o n a g a i n s t the i n c r e a s e d v i s c o s i t y

of oxy- (Chien -- e t a l . , 1970) , and deoxy s i c k l e b lood .

Both t h e a n t i s i c k l i n g e f f e c t and t h e i n c r e a s e d oxygen

a f f i n i t y t e n d t o r e d u c e t h e anaemia , t h e f o r m e r by

i n c r e a s i n g r e d c e l l l i f e span and t h e l a t t e r by s t i m u l a -

t i n g e r y t h r o p o i e s i s .

The s t u d y on t h e e f f e c t of oxygen s a t u r a t i o n on

t h e i n t r a c e l l u l a r p o l y m e r i z a t i o n o f sickle haernoglobin

r e v e a l e d t h a t t h e amount of polymer formed i n s i c k l e

c e l l e r y t h r o c y t e s a s a r e s u l t o f i n t r a c e l l u l a r g e l a t i o n

i n c r e a s e d m o n o t o n i c a l l y w i t h d e c r e a s i n g oxygen s a t u r a t i o n

(Noguchi e t a l . , 1980) w i t h no a p p a r e n t s i g m o i d c h a r a c t e r -- a s opposed t o t h a t s u g g e s t e d by s t u d i e s of c e l l s i c k l i n g ,

The i n v e s t i g a t o r s t h e r e f o r e s u g g e s t e d t h a t i n t r a c e l l u l a r

g e l a t i o n and c e l l s i c k l i n g may r e p r e s e n t d i f f e r e n t

c e l l u l a r e v e n t s . Human haemoglobin S polymer b i n d s

oxygen n o n c o o p e r a t i v e l y , w i t h an a f f i n i t y t h a t i s

a p p r o x i m a t e l y t h r e e f o l d lower than t h a t o f t e n s e -

s t a t e haemoglobin ( S u n s h i n e -- e t a l , , 1980).

Oxygen t r a n s p o r t e x p e r i m e n t s f a r normal and s i c k l e

e r y t h r o c y t e s showed t h a t t h e r a t e o f oxygen s a t u r a t i o n

d e c r e a s e d more f o r s i c k l e cells t h a n f o r normal c e l l s

( S t a t h o p o u l o s and Hellums, 1 9 8 6 ) . S i c k l e cebj w o u l d

be e x p e c t e d t o have a h i g h e r d i f f u s i o n a l r e s i s t a n c e t o

oxygen t r a n s p o r t t h a n normal c e l l s . However, t h e l o w e r

oxygen a f f i n i t y of t h e s i c k l e c e l l s t e n d t o i n c r e a s e

t h e oxygen d e l i v e r y r a t e , The d i f f e r e n c e i n oxygen

a f f i n i t y t h e r e f o r e , a p p e a r s t o a c c o u n t f o r t h e

d i f f e r e n c e i n oxygen delivery r a t e s between normal and

s i c k l e c e l l s ,

G i l l e t a l . (1978) o b s e r v e d t h a t a f t e r p a r t i a l -- deoxygena t ion , a s h a r p d r o p i n oxygen a f f i n i t y was

a s s o c i a t e d w i t h haemoglobin a g g r e g a t i o n . S ince t h e

polymer phase does n o t b i n d oxygen, t h e a p p a r e n t oxygen

a f f i n i t y of haernoglobin i s s t r o n g l y dependen t on t h e

p o l y m e r i z a t i o n p r o c e s s and c o n s e q u e n t l y on a l l changes

t h a t modify t h e g e l l i n g p r o p e r t i e s of haernoglobin S

( H o f r i c h t e r , 1978) .

Many a n t i s i c k l i n g compounds have been p roposed

which b i n d w i t h haernoglobin S and i n h i b i t g e l a t i o n ,

S i n c e most o f t h e s e compounds r e q u i r e h igh c o n c e n t r a -

t i o n s t o i n h i b i t g e l a t i o n e f f e c t i v e l y , t h e i r use i n c u r s

a d v e r s e s i d e e f f e c t s such t h a t t h e y canno t be used

c l i n i c a l l y o v e r a l o n g t e rm i n s i c k l e c e l l p a t i e n t s

( O h n i s h i e t a l . , 1982) . 7 --

Some of t h e p r o p e r t i e s of t h i s g e l and t h e

c o n d i t i o n s f o r i t s f o r m a t i o n have been found t o depend

on t h e f o l l o w i n g v a r i a b l e s : q u a t e n a r y s t r u c t u r e o f the

haernoglobin, hydrogen i o n c o n c e n t r a t i o n , haernoglobin

c o n c e n t r a t i o n , t e m p e r a t u r e , i o n i c s t r e n g t h , and t h e

p r e s e n c e of o t h e r haemoglobins , I n t h e c a s e of t h e

f i r s t v a r i a b l e , o n l y t h e deoxy form seems t o be

c o m p a t i b l e w i t h t h e po lymer ized s t a t e ( ~ o o k c h i n and

Nagel , 1973) . The g e l a t i o n o f deoxyhaernoglobin S i s

h i g h l y dependent on haernoglobin c o n c e n t r a t i o n . Thus

an a b r u p t phase t r a n s i t i o n will occur as soon a s a

c r i t i c a l haemoglobin c o n c e n t r a t i o n i s r e a c h e d , The

g e l of deoxyhaernoglobin S has a n e g a t i v e t e m p e r a t u r e

c o e f f i c i e n t . T h i s p r o p e r t y i s c h a r a c t e r i s t i c of

hydrophobic i n t e r a c t i o n s , Hence such f o r c e s were

main ly r e s p o n s i b l e f o r t h e s t a b i l i z a t i o n of t h e

polymer ( A l l i s o n , 1957). M a g d o f f - F a i r c h i l d -- e t a l .

(1976) o b s e r v e d t h a t , t h o i n t e r a c t i o n between m o l e c u l e s

of deoxyhaemoglobin S i n t h e g e l i s p r e d o m i n a n t l y hydro-

phobic . The i n c l u s i o n o f deoxyhaemoglobin A i n t h e

polymer i n h i b i t s g e l a t i o n a s shown by t h e i n c r e a s e i n

s u p e r n a t a n t c o n c e n t r a t i o n ( ~ h e e t h a m - e t -- a l . , 1 9 7 9 ) "

T h i s i n h i b i t i o n i s e x p e c t e d from t h e p r e s e n c e of t h e

cha rged g l u t a m a t e r e s i d u e a t be ta -6 i n haernoglobin A

i n p l a c e of t h e hydrophobic v a l i n e r e s i d u e i n huemaglo-

b i n S o

1.8 - KINETICS OF GELATION AND POLYMER FORMATION - S i n c e p o l y m e r i z a t i o n o f haemoglobin S i n t o o r d e r e d

bund les o r f i b e r s i s r e s p o n s i b l e f o r s i c k l i n g i n

e r y t h r o c y t e s , t h e p r e v e n t i o n of the p o l y m e r i z a t i o n

p r o c e s s s h o u l d be accompanied by t h e d i s r u p t i o n of

f o r c e s i n t h e l o c a l i z e d a r e a s o f c o n t a c t between

haemoglobin molecu les . Such d i s r u p t i o n would i n c r e a s e

t h e s o l u b i l i t y of deoxyhaemoglobin S and even s m a l l

changes i n s o l v b i l i t y w i l l g r e a t l y r e t a r d t h e k i n e t i c s

of p o l y m e r f o r m a t i o n ( H o f r i c h t e r -- e t a l , , 1974) .

The gel may be tentatively described as consisting

of two phases in reversible equilibrium: a liquid phase

containing mainly monomeric haemoglobin and a solid

phase containing polymeric haemoglobin in the form of

bundles o f long straight fibers. A t a sufficiently low

concentration o f deoxyhaemoglobin S the formation of

fibers is delayed by many minutes ( S t r y e r , 1981). An

important breakthrough in understanding the rate o f

polymer formation came when a "delay time" before

gelation was definitively measured. Hofrichter et al, -- (1974) gave a report that, there is a delay period

during which no observable polymerization takes place

when gelation is induced by temperature jumps. The

delay time is therefore defined as the interval between

deoxygenation and the onset of gelation. The length of

time before a change in signal occurs depends on the

haemoglobin concentration. The delay time was found to

depend inversely on an extraordinary high power o f the

deoxyhaernoglobin S concentration and also on temperature

(Hofrichter - e t -- al., 1976),

1.9 ANTISICKLING AGENTS

Various investigators have tried to reverse the

sickle shape o f erythrocytes - . in vitro by the incubation

of haemoglobin S b lood c e l l s w i t h d i f f e r e n t chemica l

a g e n t s (Cerami and Manning, 1971). Many of t h e s e

a g e n t s have many d i s a d v a n t a g e s a s p o t e n t i a l t h e r a -

p e u t i c a g e n t s i n t h e manugement of s i c k l e c e l l anaemia .

The i m p o r t a n c e o f s t r u c t u r e i n r e l a t i o n t o t h e r a p e u t i c

e f f e c t i v e n e s s of a n a n t i s i c k l i n g c h e m i c a l a g e n t has

r e c e n t l y been h i g h l i g h t e d ( ~ b r u h a m e t a l . , 1984) . The - -- i n v e s t i g a t o r s p roceeded t o d e s i g n , s y n t h e s i z e and t e s t

a wide r a n g e of p o t e n t i a l a n t i s i c k l i n g a g e n t s . They

were a b l e t o show t h u t c e r t a i n d i s u b s t i t u t e d b e n z o i c

a c i d d e r i v a t i v e s p o s s e s s s i g n i f i c a n t a n t i s i c k l i n g

p r o p e r t i e s . A s i n g l e s e t o f o x y g e n a t i o n curves u s a

f u n c t i o n of haemoglobin c o n c e n t r a t i o n y i e l d s p a r a m e t e r s

which havebeen a p p l i e d t o an e v a l u a t i o n o f a s e r i e s of

a n t i s i c k l i n g a g e n t s which may be grouped i n t o t h r e e

c a t e g o r i e s a c c o r d i n g t o t h e i r mechanisms of a c t i o n

(Benesch e t a l . , 1979): F i r s t l y , a g e n t s which i n c r e a s e -- t h e i n t r i n s i c oxygen a f f i n i t y and t h e r e f o r e f a v o u r t h e

non-polymer iz ing oxy-conformat ion o f haemoglobin S a t a

g i v e n oxygen p r e s s u r e , These w i l l be i n e f f e c t i v e under

s t r i c t l y a n a e r o b i c c o n d i t i o n s , S e c o n d l y , compounds

which d i r e c t l y i n t e r f e r e w i t h t h e p o l y m e r i z a t i o n of

deoxyhaemoglobin S . The e f f e c t of t h e s e i s of c o u r s e

i n d e p e n d e n t o f t h e o x y g e n t e n s i o n . T h i r d l y , a g e n t s

w i t h o u t e f f e c t on h a e m o g l o b i n , T h e s e c o n d c l a s s o f

a n t i s i c k l i n g compounds i n c r e a s e s t h e minimum g e l l i n g

c o n c e n t r a t i o n a n d t h e r e f o r e decreases t h e oxygen

a f f i n i t y o n l y a b o v e t h e end p o i n t ,

A n t i s i c k l i n g a g e n t s h a v e been t e n t a t i v e l y c l a s s i f i e d

i n t o g r o u p s s u c h a s :

1 , 9 . 1 Haemoqlob in M o d i f i e r s

G l y c e r a l d e h y d e h a s b e e n shown t o h a v e s i g n i f i c a n t

a n t i s i c k l i n g a c t i v i t y - i n v i t r o t h r o u g h i t s a b i l i t y t o

m o d i f y h a e m o g l o b i n S (Chang -- e t a l . , 1 9 8 3 ) . T h i s

m o d i f i c a t i o n o c c u r s a s a r e s u l t o f t h e r e a c t i o n b e t w e e n

t h e a l d e h y d e p o r t i o n o f t h e m o l e c u l e a n d some o f t h e

amino g r o u p s o f h a e m o g l o b i n S ( A c h a r y a a n d Manning ,

1 9 8 0 ) . T h e m a j o r e f f e c t o f t h i s r e a c t i o n i s a d e c r e a s e

i n t h e a b i l i t y o f d e o x y h a e m o g l o b i n S t o p o l y m e r i z e .

A c h a r y a e t a l , ( 1 9 8 4 ) o b s e r v e d t h a t t h e m o d i f i c a t i o n o f -- t h e d e o x y h a e m o g l o b i n S h a s a s l i g h t e f f e c t on t h e o x y g e n

a f f i n i t y o f t h e h a e m o g l o b i n a t low c o n c e n t r a t i o n s o f

g l y c e r a l d e h y d e . However , a t h i g h e r c o n c e n t r a t i o n s o f

t h e compound, t h e r e i s a m e a s u r a b l e d e c r e a s e i n t h e

o x y g e n p r e s s u r e ( i n mmHg) a t w h i c h the t e s t b l o o d was

h a l f s a t u r a t e d ( P ) o

50

Human haemoglobin r e a c t e d w i t h 4 - i so th iocyana to-

benzene sulfonamide shows an i n c r e a s e i n oxygen a f f i n i t y

below pH 7,3 and a normal response t o c h l o r i d e i o n s

( C u r r e l l - e t al 19861, The e f f e c t of t h i s a roma t i c

i s o t h i o c y a n a t e on t h e f u n c t i o n a l p r o p e r t i e s of human

haernoglobin could have a p o s s i b l e a p p l i c a t i o n i n

s i c k l e cell anaemia therapy .

The a l k y l a t i n g r e a g e n t s , f o r example a l k y l i o d i d e ,

iodoacetamide and i o d o a c e t a n i l i d e forms a c o v a l e n t bond

w i t h haemoglobin molecule, hence i r r e v e r s i b l y modif ied

t h e s t r u c t u r e (Benesch -- e t a l , , 1979). I o d o a c e t a n i l i d e

r e p r e s e n t s an i n t e r e s t i n g new a n t i s i c k l i n g agen t which

i n c r e a s e s t h e s o l u b i l i t y o f deoxyhaernoglobin S by 30$

wi thou t a major i n f l u e n c e on t h e oxygen a f f i n i t y . T h i s

l a r g e e f f e c t i s undoubtedly a s s o c i a t e d w i t h t h e

i n t r o d u c t i o n o f t h e bulky a roma t i c r e s i d u e t o

haemoglobin S (ROSS a n d Subrarnanian, 1977).

Dimethyl ad ip imida t e (DMA] i s a c r o s s - l i n k i n g

r eagen t (Woterman -- e t a l . , 1975) which i n c r e a s e s t h e

oxygen a f f i n i t y and may a l s o block s u r f a c e s i t e s on t h e

haemoglobin molecule necessary f o r agg rega t ion . Dirnethyl

ad ip imida t e combines c o v a l e n t l y w i t h t h e haemoglobin

molecule, modi f ies i t t o dec rease s i c k l i n g r a t h e r than

s h i f t i n g t h e haemoglobin S oxygenat ion curve ( ~ o t a n o

F i g , 2 Dirnethyl a d i p i m i d a t e

C y a n a t e d i s p l a c e s 2 , 3 - d i p h o s p h o g l y c e r a t e from

t h e b i n d i n g s i t e , t h e r e b y i n c r e a s i n g oxygen a f f i n i t y

( ~ v n n and B r i e h l , 1970) . Ca rbo rny la t ion by c y a n o t e

(Njikam e t aL., 1973) t h e r e f o r e p r e v e n t s g e l a t i o n , a s -- oxygen a f f i n i t y o f haernoglobin S is i n c r e a s e d . A l s o

c y a n a t e combines c o v a l e n t l y w i t h t h e haemoglobin

m o l e c u l e (Votano e t a l , , 19771, R e a g e n t s w i t h t h e -- t h i o l (-SH) g r o u p r e a c t w i t h c y s t e i n e o f b e t a - 9 3 o f

haemoglobin a n d a s a r e s u l t i n c r e a s e t h e oxygen a f f i n i t y

o f haemoglobin ( ~ a r e l -- e t a l . , 1986). The r a t e s o f

p r o d u c t i o n of g l u t a t h i o n y l haemoglobin depend on t h e

s t r u c t u r e o f the t h i o l (-SH) r e a g e n t l i n k e d t o

c y s t e i n e beta-93, Up t o 25% g l u t a t h i o n y l haernoglobin

can be p roduced i n normal and s i c k l e r e d c e l l s because

of t h e h i g h i n t r a c e l l u l a r c o n c e n t r a t i o n o f reduced

g l u t a t h i o n e . T h i s h i g h l e v e l of g l u t a t h i o n y l haemoglobin

i n normal c e l l s i n c r e a s e s t h e oxygen a f f i n i t y by

approximately 35% and dec rease s heme-heme i n t e r a c t i o n s .

I n h i b i t i o n of po lymer iza t ion of deoxyhaemoglobin

S i s a l s o d u e t o a d i r e c t i n h i b i t i o n of i n t e r m o l e c u l a r

c o n t a c t s i n t h e f i b e r s a s demonstrated by t h e i n c r e a s e d

s o l u b i l i t y and i n c r e a s e d de lay t ime of g l u t a t h i o n y l

haemoglobin S (Gare l -- e t a l . , 1986). Homoserine has been

found t o i n h i b i t e r y t h r o c y t e s i c k l i n g ( ~ ~ m e n , 1975).

The a c t i o n of t r a d i t i o n a l l y de r ived pharmacological

l i g a n d s i n t h e i n f l u e n c e o f s i c k l e c e l l anaemia r e s u l t :

i n t h e i r c a p a c i t y t o a f t e r g lob in s t r u c t u r e w h i c h consc

quen t ly i n c r e a s e s t h e a f f i n i t y of haemoglobin t o b ind

oxygen a t t h e l i g a n d b ind ing s i t e . I n N i g e r i a , t h e rot

of Fagara zan thoxylo ides and r e l a t e d s p e c i e s a r e used (

chewing s t i c k f o r c l e a n i n g t h e t e e t h a s well a s f o r t h c

t r e a tmen t of too thache . Some a c t i v e p r i n c i p l e s have

been i s o l a t e d from t h i s r o o t and found t o possess some

a n t i s i c k l i n g p o t e n t i a l ( ~ l u j o b a and Sofowora, 1977).

Furthermore, a f r a c t i o n ob t a ined from t h e water e x t r a c t

of Fagara zan thoxy lo ides , i d e n t i f i e d t o c o n t a i n p h e n o l i c

a c i d s had a n t i m i c r o b i a l a s we l l a s a n t i s i c k l i n g a c t i v i t y

(Odebiyi and Sofowora, 1979) .

1 , 9 , 2 Membrane Modifiers

Sickle red cell membranes exhibit a number of

abnormalities which are most severe in the irreversibly

sickle cells (Lux et al,, 1976) and the sickle cell -- fraction with the shortest svrvival ( ~ e r t l e s and Milner,

1 9 6 8 ) " Because of their extensive membrane alterations,

sickle cells may respond differently to antisickling

agents in vitro t han do the newly formed in vivo. - -- Besides correcting abnormalities, antisickling

agents could themselves alter the membrane, and induce

small changes in function which might have little impor-

tance for normal red cells. They might affect the survival

of sickle cells. For example, a membrane alteration

resulting in a small decrease in cell water could

counterbalance, a partial antisickling effect by raising

the intracellular haemoglobin concentration. If such

an effect took several hours or days to develop fully,

it might be missed during - in vitro tests of antisickling

activity.

Cetiedil, a vasodialator used in treatment of

heart disease was found to inhibit sickling through

what appears to be a membrane linked interaction

(Asakura et al,, 1980), --

Fig . 3 C e t i e d i l

E a r l i e r s t u d i e s i n v i t r o i n d i c a t e d t h a t even - though t h e r e was a d i s c e r n i b l e amount o f r e a c t i o n o f

glycera ldehyde w i t h r ed c e l l membrane p r o t e i n s , t h e r e

was no adve r se e f f e c t on e i t h e r t h e osmot ic f r a g i l i t y

o r t h e f i l t e r a b i l i t y of t h e c e l l s (Nigen and Manning,

1978) o r on t h e a c t i v i t y of r e d c e l l enzymes

(Manning and D r i s o l l , 1979).

The a n t i s i c k l i n g e f f e c t of desme-thyl chlorpromazine

may b e due t o e r y t h r o c y t e membrane s t a b i l i t y which r e s u l t s

i n reduced pe rmeab i l i t y t o water a s i n t h e c a s e of o t h e r

phenoth iaz ine d e r i v a t i v e s (Kwant and Seeman, 19693,

Fig , 4 Desmethyl chlorpromazine

An a n t i s i c k l i n g drug, lawsone, was observed to

p r o t e c t s i c k l e e r y t h r o c y t e a g a i n s t membrane damage by

r e a c t i o n w i t h t r a n s i e n t o x i d a t i v e s p e c i e s (C la rke e t a 1 - * I

1986). A l s o d i f f e r e n t i a l e f f e c t s observed between normal

and s i c k l e c e l l s were a t t r i b u t e d t o t h e d i f f e r e n t membrane

composit ion of i r r e v e r s i b l y s i c k l e d e r y t h r o c y t e s ,

F i g . 5 Lawsone. (2,0H-1,4-naphth0quinone)

Sex hormones, f o r example p roges t e rone and

t e s t o s t e r o n e were r e p o r t e d t o i n h i b i t and r e v e r s e

s i c k l i n g - i n v i t r o by s t i m u l a t i n g ~ a ' + - ~ ~ ~ a s e a c t i v i t y

hence s t a b i l i z i n g the membrane {Olawoye -- e t a l . , 1989).

C a t i o n i c a n a e s t h e t i c s such as p roca ine hydro-

c h l o r i d e p reven t t h e d i scocy te -ech inocyte t rans forma-

t i o n and improve r e d c e l l d e f o r m a b i l i t y i n normal and

s i c k l e r e d c e l l s undergoing ATP d e p l e t i o n (Baker - e t - 0 1 a 1

1974). I t i s , o f i n t e r e s t t h a t t h e maintenance of t h e

b i c o n c a v e s h a p e o f ATP-depleted c e l l s by p r o c a i n e

h y d r o c h l o r i d e i s associated w i t h a marked improvement

i n v i s c o s i t y and f i l t e r a b i l i t y ( P a l e k - e t -*I a 1 1977).

0

Fig . 6 P r o c a i n e h y d r o c h l o r i d e

Z i n c has been shown t o i n h i b i t t h e c a l m o d u l i n

a c t i v a t i o n of ca l c ium ATPase, p h o s p h o d i e s t e r a s e and

a d e n y l c y c l a s e (Brewer, 1980). Low l e v e l s of z i n c

was shown t a e x h i b i t a n t i s i c k l i n g e f f e c t on membrane

{Brewer - e t - m t a1 1977) by i n c r e a s i n g t h e oxygen a f f i n i t y

of s t r i p p e d haernoglobin ( O e l s h l e g a l e t a l . , 1974)"

The ether f r a c t i o n of the aqueous e x t r a c t of a

p l a n t , Zanthoxylurn x a n t h o x y l o i d e s was r e p o r t e d t o

reverse a l r e a d y s i c k l e d r e d b lood c e l l s - i n v i t r o

(E lu joba and Sofowora, 1977). C l i n i c a l and t o x i c o -

l o g i c a l t r i a l s showed t h a t t h i s aqueous e x t r a c t c o u l d

p l a y a useful r o l e i n t h e management of s i c k l e c e l l

d i s e a s e ,

The active principle of Zanthoxylum xanthoxyloides

was later characterized to be benzoic acid derivatives

and their synthetic analogues were prepared (Adesanya

and Sofowora, 1983). The antisickling activity of the

roots has also been confirmed while zanthoxylol isolated

from the same root, as well as a synthetic derivative,

3 , 4 dihydro-2, 2-dimethyl - 2H-1 benzo-pyran-6 butyric acid (DBA), have been shown to have antisickling activity

in vitro (Ekong et al,, 1975), The series of benzoic - acid derivatives reported to have antisickling activity

have been found to have the following chemical characteris-

tics in common: a single benzene ring, a carboxylic acid

grouping as well as an electron rich grouping (~lujoba

and Sofowora, 1977). The active principle being

lipophilic is more likely to act on the cell membrane

since the crude extract was shown to also revert crenated

haernoglobin A erythrocytes to normal configuration

(Sofowora'and Isaacs,'l971), The lipophilicity and

electronegativity were observed to increase the anti-

sickling activity of the active principle (Adesanya

and Sofowora, 1983).

1 . -OH

F i g , 7 2-OH-methyl benzoic acid Fig, 8 P-hydroxybenzoic acid

F i g , 9 Vanillic acid Fig, 10 Xanthoxylol

1,9,3 Gene Activator

Most recently a preliminary study suggests that

butyrate, a widely used flavour enhancer can overcome

the basic cause o f sickle cell disease (Faller, 1993) "

Six patients o f the disease and thalassaemia showed a

dramat ic improvement a f t e r r e c e i v i n g t h e b u t y r a t e

i n j e c t i o n f o r two t o t h r e e weeks. The appa ren t

mechanism of a c t i o n was s p e c u l a t e d t o be t h a t t h e

b u t y r o t e induces t h e p roduc t ion of f o e t a l haemoglobin

by swi tch ing on aga in t h e gene t h a t d i r e c t s i t s

s y n t h e s i s .

1.9.4 Other A n t i s i c k l i n g Agents

Ekeke and Shode ( 1 9 8 5 1 , worked o n t h e seeds of

Cajanus c a j a n and r e p o r t e d t h a t , t h e water and e t h a n o l i c

e x t r a c t s of t h e p l a n t were a b l e t o g i v e s i g n i f i c a n t

i n h i b i t o r y e f f e c t on s i c k l i n g even when sodium meta-

b f s u l p h i t e was p r e s e n t . Iwu -- e t a l . ( 1 9 8 8 ) have a l s o

shown t h a t methanol ic and wate r e x t r a c t s from t h e s eed

o f Cajanus ca j an e x h i b i t e d b e n e f i c i a l e f f e c t on s i c k l e

c e l l d i s e a s e . T h i s i s due t o t h e a b i l i t y of t h e amino-

g l y c a s i d i c compound, cajaminose i s o l a t e d from t h e

rnethanolic e x t r a c t of t h e seed t o i n h i b i t i n v i t r o - sodium metab isu lph i te - induced s i c k l i n g . I n t h e g e l l i n g

0 assay , a t 25 C t h e m i n i m u m g e l l i n g c o n c e n t r a t i o n i n c r e a s e d

f r o m 24.8% f o r t h e u n t r e a t e d deoxyhaemoglobin S t o 32.7%

w i t h 0,1% of t h e aqueous f r a c t i o n of Cajanus ca j an and

t o 42.8% w i t h 0.01% cajaminose. Also i n ' t h i s r e p o r t ,

oxygen a f f i n i t y was cons ide rab ly g r e a t e r than t h a t of

t h e u n t r e a t e d haemoglobins,

D i c h l o r o i s o p r o t e r e n o 1 ( D C I ) and p r o p r a n o l o l were

found t o i n h i b i t s i c k l i n g - i n v i t r o when they were added

t o s i c k l e r e d - c e l l s u s p e n s i o n s p r i o r t o d e o x y g e n a t i o n .

D i c h l o r o i s o p r o t e r e n o 1 was r e p o r t e d t o i n c r e a s e oxygen

a f f i n i t y of bo th normal and s i c k l e r e d c e l l s , The changes

i n t h e d e l a y t i m e of g e l a t i o n of haernoglobin S may be a s

a r e s u l t o f t h e i r e f f e c t s on t h e oxygen a f f i n i t y of

haemoglobin S. If a change i n oxygen a f f i n i t y i s t a k i n g

p l a c e , i t would e x p l a i n why D C I e x h i b i t e d a r e m a r k a b l e

a n t i s i c k l i n g e f f e c t ( O h n i s h i -- e t a l . , 1982) .

o c y

Fig . 1 1 P r o p r a n o l o l

S imple chemica l a g e n t s such as u r e a ( N a l b a r d i a n

e t a l . , 19711, a r o m a t i c amino a c i d s , t h e i r a n a l o g u e s , -- a s we l l a s p e p t i d e s have been shown t o i n h i b i t t h e

g e l l i n g a n d s i c k l i n g t endency o f s i c k l e c e l l haemoglobin

(Noguchi and S c h e c h t e r , 19771, P h e n y l a l a n i n e , an a r o m a t i c

amino a c i d was found t o be t h e most e f f e c t i v e among a

s e r i e s of t e n amino acids i n i n c r e a s i n , g t h e s o l u b i l i t y

o f deoxyhaemoglobin S g e l s (Noguch i and S

Benesch e t a l . (1979) showed haw p h e n y l a l -- mean g e l l i n g c o n c e n t r a t i o n w i t h o u t s i g n i f ~ c a n t l y chang lng

t h e oxygen a f f i n i t y ,

L -asparag ine and L - g l u t a m i n e decreased t h e

carbon monoxide b i n d i n g a f f i n i t y o f haemoglob in S ,

w h i l e no e f f e c t o f t h e s e or any o t h e r amino a c i d s c o u l d

be demons t ra ted w i t h haemoglob in A (~wmen, 1975).

Votano -- e t a l . (1977) examined s e v e r a l o l i g o p e p f i d e s

and found t h a t t h e most e f f e c t i v e i n h i b i t o r s o f g e l a t i o n

were t h e ones wh ich had b o t h a h y d r o p h o b i c r e s i d u e such

as p h e n y l a l a n i n e and a h y d r o p h y l i c r e s i d u e such as

a r g i n i n e o r l y s i n e ,

Based on t h e f a c t t h a t u r i c a c i d i s s t r u c t u r a l l y

c l o s e i n f u n c t i o n a l g roup c h a r a c t e r i s t i c s , n o t o n l y

t o some a r o m a t i c amino a c i d s known t o be a n t i s i c k l i n g

{Noguch i and Schech te r , 1977) b u t a l s o t o some b e n z o i c

a c i d d e r i v a t i v e s (Abraham - e t * I a 1 1984), i t was i n v e s -

t i g a t e d and f o u n d t o possess a n t i s i c k l i n g a c t i v i t y on

s i c k l e c e l l e r y t h r o c y t e s , i n p a r t i c u l a r ' a t h i g h e r u r a t e

c o n c e n t r a t i o n s (Ekeke and Nduka, 1987).

1.10 RESEARCH RATIONALE AND OBJECTIVES

The - i n v i t r o s t u d i e s on I(AT.90, b.y Chidume (1991.) , ,

r e v e a l e d t h a t t h e d r u g c o u l d n o t o n l y p r e v e n t t h e s i c k l i n g

of r e d b lood c e l l s b u t a l s o i n h i b i t t h e g e l a t i o n of

s i c k l e haemoglobin . The r e s u l t s o f t h e s e s t u d i e s a l s o

showed t h a t t h e K A T ~ ~ O r e v e r s e d a l r e a d y s i c k l e d r e d

b lood c e l l s t o t h e i r normal morphology. T h e d r u g was

a l s o o b s e r v e d t o i n t e r f e r w i t h t h e a g g r e g a t i o n o f t h e

s i c k l e haernoglobin. T h e s e r e s u l t s seem t o e x p l a i n t h e

o b s e r v a t i o n t h a t t h e d r u g d e c r e a s e d b o t h t h e f r e q u e n c y

and s e v e r i t y o f c r i s i s i n s i c k l e c e ons who had

t a k e n i t ( ~ z o e g w u , P.N., p e r s o n a l c u ~ ~ ~ t ~ r ~ ~ ~ a t i o n ) .

F u r t h e r s t u d i e s by Chidume (1991) r e v e a l e d t h a t above

t h e d r u g c o n c e n t r a t i o n , o f 3mg/ml o f b l o o d , i t however

seems t o have o x i d i z e d t h e f e r r o u s i o n s of t h e no rma l

a n d s i c k l e haemoglobin S t o i r r e v e r s i b l y f e r r i c s t a t e

t h e r e b y fo rming met-haemoglobin, T h i s d r u g was

t h e r e f o r e r e g a r d e d a s an o x i d a n t a t a h i g h c o n c e n t r a t i o n .

A l l these s t u d i e s were however n o t e x t e n d e d t o t h e

i n v e s t i g a t i o n s on t h e c o n s t i t u e n t s of KAT.90 n o r the

mechanism o f a c t i o n o f t h i s p o t e n t i a l a n t i s i c k l i n g and

a n t i g e l l i n g d rug .

T h i s p r o j e c t i s t h e r e f o r e a imed a t f u r t h e r s t u d i e s

on KAT.90 drug w i t h a view t o d i s c o v e r i t s c o n s t i t u e n t s

a n d p o s s i b l y e l u c i d a t e t h e mechanism o f i t s a n t i s i c k l i n g

and a n t i g e l l i n g a c t i v i t i e s on s i c k l e r e d b l o o d c e l l s and

s i c k l e haemoglobin r e s p e c t i v e l y .

CHAPTER TWO

MATERIALS AND METHODS

2.1 COLLECTION OF BLOOD SAMPLES

Venous b lood samples were c o l l e c t e d from p a t i e n t s

a t t h e Haematology Department , U n i v e r s i t y o f N i g e r i a

Teach ing H o s p i t a l , Enugu. T h e homazygous s i c k l e c e l l

c o n d i t i o n was conf i rmed by e l e c t r o p h o r e s i s . A l l t h e

p a t i e n t s were n o t on t h e KAT.90 drug a t t h e t i m e o f

c o l l e c t i o n . Blood samples from i n d i v i d u a l s w i t h geno-

t y p e o f AA were used a s c o n t r o l s . A l l t h e p a t i e n t s were

between t h e a g e s of 15 and 25 y e a r s .

2.2 PREPARATION OF REAGENTS

2.2.1 Mayeros Reagent

Mercur i c c h l o r i d e (1 .3589) was d i s s o l v e d i n 60ml

o f d i s t i l l e d w a t e r and mixed w i t h 50% (w/v) p o t a s s i u m

i o d i d e s o l u t i o n . T h e m i x t u r e was made u p t o lOOml w i t h

d i s t i l l e d w a t e r .

2 ,2 .2 Wagneros Reagent

C i t r i c a c i d ( 5 g ) and s a l i c y l i c a c i d (0 .2g) were

d i s s o l v e d i n d i s t i l l e d w a t e r and d i l u t e d t o 200m1,

2.2.3 P i c r i c A c i d ( 1 % )

P i c r i c a c i d ( l g ) was d i s s o l v e d i n lOOml of

d i s t i l l e d wa te r .

2 , 2 , 4 D r a g e n d o r f f o s Reagent

T a r t a r i c a c i d ( 1 9 9 g ) , was d i s s o l v e d i n 400ml

o f d i s t i l l e d wa te r . B i s m u t h o x i d e n i t r a t e (8.5g)

was added and shaken f o r one hour. The s o l u t i o n

was then mixed w i t h 200rnl of 40% (w/v) potass ium

i o d i d e s o l u t i o n and shaken v i g o r o u s l y . The mix tu r e

was a l lowed t o s t a n d f o r 24 hours b e f o r e f i l t r a t i o n

was c a r r i e d o u t . The f i l t r a t e which i s t h e s t o c k

s o l u t i o n was s t o r e d a t roam t empe ra tu r e i n brown

b o t t l e s . The spray reagent was p repa red j u s t b e f o r e

use by mixing 10.0ml stock s o l u t i o n w i t h 40.01~11

g l a c i a l a c e t i c a c i d and 50.01~11 d i s t i l l e d wa t e r .

2 , 2 , 5 F e h l i n g u s Reagent (Po tass ium c u p r i c t a r t a r a t e s o l u t i o n )

( a > F e h l i n g U s s o l u t i o n I: Copper s u l p h a t e

(3.464g) was d i s s o l v e d i n 0.51-111 s u l p h u r i c

a c i d and made u p t o 50ml w i t h d i s t i l l e d

wa te r .

( b F e h l i n g " s s o l u t i o n 11: Potass ium t a r t a r a t e

( 17.69) and sodium hydroxide (7. J g ) were

d i s s o l v e d i n enough d i s t i l l e d water t o produce

a f i n a l volume of 50ml.

2 , 2 , 6 I o d i n e Vapour

I o d i n e c r y s t a l s were p l a c e d i n an i o d i n e t a n k

and covered t i g h t l y with t h e a i d of v a s e l i n e a p p l i e d

a t t h e edges of t h e t a n k l i d ,

2 ,2 .7 0,lM Po tas s ium Phospha te B u f f e r (pH 7.8)

P o t a s s i u m d ihydrogen p h o s p h a t e (2.72149) and

d i p o t a s s i u m hydrogen p h o s p h a t e (13.9349) were weighed

o u t and d i s s o l v e d i n 500ml d i s t i l l e d w a t e r and t h e n

made up t o one l i t r e ,

2 , 2 . 8 B i u r e t Reagent .

I n 500rnl d i s t i l l e d w a t e r , were d i s s o l v e d 1.5g

copper s u l p h a t e ( p e n t a h ~ d r a t e ) and 6.09 sodium p o t a s s i u m

t a r t a r a t e . About 300ml of 10% sodium hydroxide s o l u t i o n

was added t o t h e m i x t u r e w i t h c o n s t a n t s t i r r i n g . The

r e s u l t i n g s o l u t i o n was made u p t o one l i t r e w i t h

d i s t i l l e d w a t e r and s t o r e d i n a p l a s t i c b a t t l e ,

2,3 MELTING POINT DETERMINATION

M e l t i n g p o i n t s o f K A T ~ ~ O ( I ) and KAT.?O(II) were

d e t e r m i n e d u s i n g an e l e c t r o t h e r m a l m e l t i n g p o i n t appa-

r a t u s . A s m a l l q u a n t i t y of t h e d rug was p l a c e d i n a

c a p i l l a r y t u b e and mounted o n t h e a p p a r a t u s . The

t e m p e r a t u r e a t which the s o l u t e s t a r t e d d i s s o l v i n g

was obse rved .

2.4 PH DETERMINATION

The pH was determined using a pH paper. The

drug (2mg) was dissolved in 1ml of distilled water.

Blue and red litmus paper were dipped in the solution

and the colour change observed, This colour was

matched with that of the pH paper.

2.5 SOLUBILITY TESTS

Solubility tests were carriea out on the two

batches of the drug to determine the solvents and

solvent systems in'which they are soluble.

Different solvents and solvent systems were used.

Two milli-grammes of the drug and 1.0ml of

the solvent or solvent 'system were mixed at room

temperature and dissolution observed. The solvents

and solvent systems used were: Distilled water,

ethylacetate, methanol, Conc, ammonia, ethanol,

tetra hydrofolate (THF), dioxane, dimethyl sulphoxide

(DMso), formic acid, n-butanol, tween 20, methanol-

water {1:2), ethylacetate-methanol-formic acid (1:1:1),

THF-DMSO-water (1:1:1), dioxane-DMSO-water (1:1:1),

THF-DMSO ( 1 : 1 ; 1 : 2 ) , dioxane-DMSO-water ( 1 : 1 : 1 ) , ethylacetate-methanol-formic acid (3:3:1).

Z 6 T H I N LAYER CHROMATOGRAPHY OF KAT.90

S i l i c a g e l (G.60) was mixed w i t h d i s t i l l e d w a t e r

i n a f l a s k i n the r a t i o of 1:2, The f l a s k was c o r k e d

t i g h t l y and shaken vigorously, The m i x t u r e was t h e n 2 s p r e a d e v e n l y on 20 x 10cm g l a s s p l a t e s , 0,25mm t h i c k

u s i n g a spreader (CAMAG) machine. T h e p l a t e s were o

a l lowed to d r y and t h e n a c t i v a t e d i n an oven a t 110 C

f o r one hour.

T h e s o l u t i o n s o f t h e d rug i n t h e s o l v e n t s used

f o r s o l u b i l i t y t e s t s ( s e c t i o n 2.5) were s p o t t e d on

t h e p l a t e s and a l l o w e d t o d r y . The chroma-tank was

a l l o w e d t o be s a t u r a t e d w i t h t he s p e c i f i e d s o l v e n t

sys tem f o r 30 minu tes b e f o r e s e p a r a t i o n began, Each

s p o t t e d p l a t e was p l a c e d i n t h e t a n k i n such a way

t h a t t h e o r i g i n a l s p o t s were a b o u t 2.cm above t h e

s o l v e n t l e v e l , The s o l v e n t s were a l l o w e d t o r u n

t h rough t h e c o a t e d l a y e r f o r some t ime. , The s o l v e n t

f r o n t was no ted . T h e s e p a r a t e d s p o t s were e i t h e r

viewed under u n t r a v i o l e t lamp o r deve loped i n i o d i n e

vapaur .

The s o l v e n t sys tems used f o r t h e s e p a r a t i o n were

a s f o l l o w s : C h l o r o f o r m - e t h y l a c e t a t e - m e t h a n o l (6 :3 :1) ,

e t h y l a c e t a t e - m e t h a n o l - c o n c , ammonia (4 ,25 :0 .5 :0 ,25) ,

e t h y l a c e t a t e - c h l o r o f o r m (2:8) , m e t h a n o l - e t h y l a c e t a t e -

w a t e r (20:25:5) , ch loroform-methanol ( 9 : 1 ) , w a t e r -

methano l - ch lo ro fo rm (2:13:35;1:5:44),ethylacetate (100%) ,

benzene (100%).

Commerc ia l ly p r e p a r e d s i l i c a g e l - f l u o r e s c e n c e

(silica- gel-^^^^) p l a t e was a l s o used. About 40mg o f

K A T , ~ O ( I I ) was d i s s o l v e d i n lml o f d i s t i l l e d w a t e r ,

0,5m1 of m e t h a n o l was added , T h i s was s p o t t e d on

c h r o m a t o p l a t e s and d e v e l o p e d i n ch lo ro fo rm-methano l

( 9 : l ) s o l v e n t sys t em, I o d i n e v a p o u r was u s e d t o d e t e c t

any s p o t on t h e p l a t e ,

2 , 7 OTHER TESTS TO IDENTIFY T H E CONSTITUENTS OF KAT.90

2 . 7 , l P r o t e i n

B i u r e t r e a g e n t (2ml ) was added t o t he s o l u t i o n

o f t h e d r u g i n a t e s t t u b e and o b s e r v e d f o r v i o l e t o r

p u r p l e c o l o u r .

2.7.2. L i p i d s

The d r u g was rubbed on a f i l t e r p a p e r and checked

f o r any t r a n s l u s c e n t a r e a . F u r t h e r m o r e n i n h y d r i n was

s p r a y e d on some q u a n t i t i e s o f KAT.90 and o b s e r v e d f o r

b l u e a n d p u r p l e c o l o u r s t o i d e n t i f y amino c o n t a i n i n g l i p i d s .

2 . 7 , 3 S t a r c h

( a > A d rop o f i o d i n e s o l u t i o n was added t o 0 ,3ml

of t h e s o l u t i o n of t h e d r u g a n d t h e c o l o u r

change : o b s e r v e d .

( b Two d r o p s of i o d i n e s o l u t i o n were a l s o

added . t o a few grammes of t h e d r u g on

a watch g l a s s ,

2 , 7 , 4 Reduc ing S u g a r s

To 50mg o f t h e t e s t compound i n a t e s t t u b e

was a d d e d 2.0ml o f b o i l i n g d i s t i l l e d w a t e r and 0 .04ml

o f F e h l i n g ' s s o l u t i o n s I and 11. The m i x t u r e was h e a t e d

f o r 15 m i n u t e s i n a w a t e r b a t h .

2 , 7 , 5 G l y c o s i d e s

A s o l u t i o n o f t h e d r u g was f i l t e r e d and 0.61111

of d i l u t e s u l p h u r i c a c i d (0.1M) was added t o t h e

f i l t r a t e . T h i s was b o i l e d f o r 15 m i n u t e s , c o o l e d and

n e u t r a l i z e d w i t h 0 ,6ml o f 20% p o t a s s i u m h y d r o x i d e . Each ( 0 . 2 m l )

o f F e h l i n g ' s s o l u t i o n s 1 and I I / w e r e - added and t h e

m i x t u r e b o i l e d i n a w a t e r b a t h f o r 1 5 m i n u t e s .

2 .7 .6 A l k a l o i d s

The d r u g ( 0 . l g ) was d i s s o l v e d i n m e t h a n o l a l o n e ,

and i n w a t e r - m e t h a n o l (2 :3) s o l v e n t s y s t e m ,

They. were s p o t t e d on c h r o m a t o p l a t e s , r u n i n

w a t e r - m e t h a n o l - c h l o r o f o r m (1 :5 :44 ) s o l v e n t

s y s t e m , and a f t e r a i r d r y i n g , s p r a y e d w i t h

D r a g e n d o r f f u s r e a g e n t and o b s e r v e d f o r brown

s p o t s which would be i n d i c a t i v e o f t h e p r e s e n c e

o f a l k a l o i d s .

( b ) KAT.90 (0 . lmg) was d i s s o l v e d i n 0.5ml of

d i s t i l l e d w a t e r and lml of a s o l u t i o n of

A l u m i n i u m C h l o r i d e ( 1 % ) added. T h i s was

shaken f o r f i v e m i n u t e s and a l l o w e d t o

s e t t l e . A y e l l o w p r e c i p i t a t e would be

i n d i c a t i v e of t h e p r e s e n c e of a l k a l o i d s .

( 4 0.2M s u l p h u r i c a c i d ( I m l ) was poured on

0.08mg drug i n a f l a s k . P i c r i c a c i d

s o l u t i o n ( I rn l ) was added and o b s e r v e d

f o r any ye l low p r e c i p i t a t e , wh ich .wou ld

i n d i c a t e t h e j r e s e n c e of a l k a l o i d s .

( d ) Two d r o p s of M a y e r U s r e a g e n t were added

t o a s o l u t i o n of t h e d rug . A w h i t e

p r e c i p i t a t e would be i n d i c a t i v e of t h e

p r e s e n c e of a l k a l o i d s .

( 4 Two d r o p s of WagnerUs r e a g e n t were added t o

a s o l u t i o n of t h e d rug . A brown p r e c i p i t a t e

would i n d i c a t e t h e p r e s e n c e of a l k a l o i d s .

2.7.7 S a p o n i n s

The drug ( 0 . 5 9 ) was i n t r o d u c e d i n t o a f l a s k

c o n t a i n i n g 2 . 5 ~ 1 1 of d i s t i l l e d w a t e r i n a t e s t t u b e and

shaken v i g o r o u s l y . The s o l u t i o n was o b s e r v e d f o r t h e

p r e s e n c e of f r o t h i n g which would c o n f i r m t h e p r e s e n c e

of s a p o n i n s . :

2,7 .8 P h e n o l i c Compounds

T h e d rug (0 .05g) was d i s s o l v e d i n 0.25ml of

d i s t i l l e d w a t e r i n a t e s t t u b e , A drop of NaOH

s o l u t i o n (pH 8 ) was added. A y e l l o w c o l o u r a t i o n

would i n d i c a t e t h e p r e s e n c e of p h e n o l i c compounds.

2 .7.9 Tann ins

The drug (100mg) was d i s s o l v e d i n 0.5ml of

d i s t i l l e d w a t e r and f i l t e r e d . Two d r o p s of f e r r i c

c h l o r i d e s o l u t i o n ( 0 , l ~ ) were added t o 1.0ml of t h e

f i l t r a t e i n a t e s t t u b e . A g r e e n i s h - b l a c k p r e c i p i t a t e

would i n d i c a t e t h e p r e s e n c e of t a n n i n s .

2.7.10 S t e r o l s and T r i t e r p e n e s : S a l k o w s k i U s t e s t

C o n c e n t r a t e d s u l p h u r i c a c i d was added t o 2ml

of 2% (w/v) s o l u t i o n of KAT.90 s o a s t o form an under an

l a y e r . The p r e s e n c e o f / u n d e r l a y e r , and a r e d c o l o u r a - - t i o n a t t h e i n t e r f a c e would conf i rm t h e p r e s e n c e of

s t e r o l s a n d t r i t e r p e n e s .

2 .8 INFRA-RED SPECTROSCOPIC STUDY OF KAT.90

I n f r a - r e d s p e c t r o s c o p i c s t u d i e s were c a r r i e d o u t

on t h e compound t o d e t e r m i n e some of i t s components.

T h i s d e t e r m i n a t i o n c o u l d r a v e a l t h e f u n c t i o n a l grGups

i n t h e a c t i v e p r i n c i p l e s i n v o l v e d i n t h e a n t i g e l l i n g

and a n t i s i c k l i n g a c t i v i t i e s of t h i s d rug .

K ~ T . 9 0 ( 1 ) and K A T . ~ O ( I I ) were s u b j e c t e d t o i n f r a -

r e d s p e c t r o s c o p y a n a l y s i s u s i n g SP3 Pye-Unicam In f ra -Red

S p e c t r o p h o t o m e t e r ( P y e - ~ n i c a m Ltd . Cambridge, E n g l a n d ) .

Po tass ium bromide was t aken a s t h e r e f e r e n c e spec t rum

f o r a l l t h e i n f r a - r e d s p e c t r a l a n a l y s i s i n t h i s work

( F i g . 1 3 ) .

2 ,9 ULTRAVIOLET SPECTROSCOPY OF KAT .90

The absence of d e f i n e d peaks a t t h e f i n g e r p r i n t

r e g i o n ( F i g . 1 4 and Fig.15) of t h e IR-Spec t ra of KAT,90

n e c e s s i t a t e d an u l t r a v i o l e t s p e c t r o p h o t o m e t r i c test f o r

a r o m a t i c i t y t o conf i rm t h e p r e s e n c e o f pheno l ,

T h r e e milii-grarnrnes o f KAT.90 were d i s s o l v e d i n

3,Ornl (0.03% w/v) of d i s t i l l e d wa te r . F u r t h e r d i l u t i o n

w i t h d i s t i l l e d wa te r was c a r r i e d o u t t o make a f i n a l

c o n c e n t r a t i o n of 0.143rng/rnl. T h e a b s o r b a n c e o f t h e KAT,90

s o l u t i o n was scanned o v e r a wavelength r a n g e o f 200-400nm

a t 5nrn i n t e r v a l . An u n t r a v i o l e t spec t rum was produced by

p l o t t i n g t h e absorbance of t h e drug a g a i n s t t h e c o r r e s -

ponding wavelength . The benzenoid band i n benzene

d e r i v a t i v e s g e n e r a l l y o c c u r s between 250 and 280nm,

2,10 CONVERSION OF KAT.90 TO AN A M I D E D E R I V A T I V E

Although t h e c o n s t i t u e n t s o f KAT.90 a r e n o t known,

i t was d i s c o v e r e d t o be a c i d i c and was o b s e r v e d t o c o n t a i n

some benzene group. S i n c e some compounds c o n t a i n i n g

b e n z o i c a c i d d e r i v a t i v e s were found t o have a n t i -

s i c k l i n g and a n t i g e l l i n g a c t i v i t i e s ( E l u j o b a and

Sofowora, 1977), t h i s expe r imen t was aimed a t

f i n d i n g o u t i f K A T . ~ ~ c o n t a i n s some c a r b o x y l g roup .

A m i x t u r e of KAT,90 ( 0 . 2 g ) and t h i o n y l c h l o r i d e

(2 ,5ml ) was r e f l u x e d on a h o t p l a t e f o r one hour , The

r e a c t i o n m i x t u r e was c o o l e d and e x c e s s t h i o n y l c h l o r i d e

d i s t i l l e d o f f . The s u p p o s e d l y a c i d c h l o r i d e t h u s

o b t a i n e d was t r e a t e d w i t h 4.0ml of a n i l i n e and t h e n

warmed w i t h s t i r r i n g f o r t h r e e h o u r s . The f l a s k was

c o o l e d i n i c e . T h i s r e a c t i o n m i x t u r e was s u c c e s s i v e l y

e x t r a c t e d w i t h 20ml of c h l o r o f o r m and c o n c e n t r a t e d u s i n g

a r o t a r y e v a p o r a t o r . Some q u a n t i t y of t h i s e x t r a c t was

s p o t t e d on c o a t e d t h i n l a y e r chromatography p l a t e s and

t h e components s e p a r a t e d w i t h benzene s o l v e n t sys t em.

A n i l i n e was a l s o s p o t t e d a l o n g w i t h t h e e x t r a c t o f t h e

r e a c t i o n m i x t u r e o b t a i n e d t o i d e n t i f y any e x c e s s a n i l i n e .

Two majo r bands were o b s e r v e d , t h e upper band c o r r e s p o n -

ded w i t h t h e band o f a n i l i n e .

The lower band was s c r a p e d o f f and soaked i n

e t h a n o l o v e r n i g h t , T h e s o l u t i o n was f i l t e r e d w i t h

s u c t i o n p u m p on a b u t c h n e r f u n n e l . More e t h a n o l was

added t o wash t h e g e l i n t h e f u n n e l and t h e f i l t r a t e

c o l l e c t e d , The d e r i v a t i v e was a l l o w e d t o c r y s t a l l i z e 0

a t -4 C from e t h a n o l and t h e n d r i e d i n a d e s s i c a t o r

c o n t a i n i n g anhydrous Calcium C h l o r i d e p e l l e t s , The

m e l t i n g p o i n t of t h e s o l i d d e r i v a t i v e was d e t e r m i n e d

a s o u t l i n e d i n s e c t i o n 2 .3 above. Also t h e d r i e d

samples were a n a l y s e d by i n f r a - r e d s p e c t r o s c o p y .

2 ,11 TURBIDITY AND O X Y G E N A F F I N I T Y STUDIES

2.11.1 I s o l a t i o n o f Haernoalobin

F r e s h b lood samples (5ml ) from p e r s o n s w i t h t h e

geno type o f SS and normal i n d i v i d u a l s w i t h t h e g e n o t y p e

of AA were c o l l e c t e d by v e n i p u n c t u r e i n sample t u b e s

w i t h Img EDTA a s a n t i c o a g u l a n t . The b lood c e l l s were

a l l o w e d some t i m e t o s e t t l e and t h e serum a s p i r a t e d .

The b lood c e l l s were t h e n suspended i n t w i c e t h e i r

volume of normal s a l i n e f o r washing. The b l o o d c e l l

s u s p e n s i o n s were c e n t r i f u g e d a t 3 ,000xg f o r lOmin,

a f t e r which t h e s u p e r n a t a n t was d i s c a r d e d . Washing

was done t h r i c e and t h e s u p e r n a t a n t was a s p i r a t e d a f t e r

each c e n t r i f u g a t i o n . The c e l l s were t h e n l y s e d i n

d i s t i l l e d w a t e r t o r e l e a s e t h e haemoglobin.

The s o l u t i o n was a l l o w e d t o s t a n d f o r f o r t y 0

m i n u t e s 3t 27 C a f t e r which i t was c e n t r i f u g e d a t

6,000xg f o r t h i r t y m i n u t e s , T h e r e s u l t a n t haernoglobin

s u p e r n a t a n t was saved w h i l e t h e g h o s t and u n l y s e d r e d

b lood c e l l s were d i s c a r d e d . The haemoglobin s o l u t i o n

was t h e n p a s s e d th rough a column c o n t a i n i n g sephadex

G.25 t o t r a p t h e l ower m o l e c u l a r we igh t i o n s such a s

i r o n and phosphorus a s w e l l a s some o t h e r low m o l e c u l a r

we igh t m a t e r i a l s . Only t h e s u b s t a n c e w i t h m o l e c u l a r

we igh t above 64 ,000 lJere e l u t e d th rough t h e column u s i n g

0,lM p o t a s s i u m p h o s p h a t e b u f f e r , pH 7.8 a s t h e e l u t i n g

s o l v e n t , I n t h i s e x p e r i m e n t , t h i s e l u t e d haemoglobin

s o l u t i o n was used a s oxygena ted samples .

2.11.2 Deoxygenat ion of Haemoglobin

Normal and s i c k l e haemoglobin samples were

deoxygenated a s d e s c r i b e d by Chidume ( 1 9 9 1 ) . The

oxygena ted haemoglobin samples (0.6ml) were d i l u t e d

w i t h 0.31-111 of p h o s p h a t e b u f f e r , ( p H 7 . 8 ) i n t e s t

t u b e s . The m i x t u r e was cove red w i t h 1.0ml of l i q u i d

p a r a f f i n . Two p e r c e n t (2$, w/v) f r e s h l y p r e p a r e d

sodium m e t a b i s u l p h i t e was added under t h e p a r a f f i n

l a y e r i n each t u b e w i t h t h e h e l p o f a p a s t o r p i p e t t e

and t h e n i n c u b a t e d f o r an hour .

2 , 1 1 , 3 T u r b i d i t y S t u d i e s

T u r b i d i t y s t u d i e s were c a r r i e d o u t a t tempera- 0 0

t u r e s between 283 K and 323 K a c c o r d i n g t o t h e m o d i f i e d

method of ~ d u : ( 1 9 8 6 ) .

I n t h i s e x p e r i m e n t t h e s o l u t i o n o f h a e m o g l o b i n S

u s e d was d e o x y g e n a t c d a s d e s c r i b e d i n s e c t i o n 2,11,2

a t OOC, KAT.90 d r u g s o l u t i o n was a d d e d i n t o t h e t e s t

t u b e s c o n t a i n i n g 0.5ml o f s i c k l e (SS) o x y g e n a t e d a n d

d e o x y g e n a t e d h a e m o g l o b i n s o l u t i o n s i n s u c h a way t h a t

t h e f i n a l c o n c e n t r a t i o n s o f t h e d r u g were l r n g / m l , 2 m g / m l ,

3mg/ml a n d 4mg/ml r e s p e c t i v e l y , O t h e r t u b e s c o n t a i n i n g

o n l y t h e h a e m o g l o b i n s a m p l e s w h i c h were made up w i t h t h e

d i l u t i n g b u f f e r s e r v e d a s c o n t r o l s . A l l t h e t e s t t u b e s

were a r r a n g e d i n d u p l i c a t e . T h e m i x t u r e i n e a c h t u b e

was c o v e r e d w i t h I n 1 o f p a r a f f i n s o l u t i o n t o a v o i d

r e - o x y g e n a t i o n a n d e v a p o r a t i o n , e v e n b e f o r e t h e d r u g

was i n t r o d u c e d i n t o t h e h a e m o g l o b i n samples . T h e 0

s a m p l e s w e r e i n c u b a t e d f o r 40 m i n s a t 0 C. T h e

p a r a f f i n l a y e r was t h e n removed a n d the h a e m o g l o b i n

s o l u t i o n s were u s e d f o r t h e t e s t .

T h e t u b e s c o n t a i n i n g d r u g - t r e a t e d and u n t r e a t e d

h a e m o g l o b i n s a m p l e s were q u i c k l y removed f r o m i c e and

prewarmed t o t h e a p p r o p r i a t e t e m p e r a t u r e i n a w a t e r

b a t h , A s p e c i f i c t ime i n t e r v a l f o r t h e p r o c e s s was

h e l d c o n s t a n t f o r a l l t h e c o n c e n t r a t i o n o f t h e d r u g

a t e a c h t e m p e r a t u r e . A s t o p w a t c h was u s e d i n t h i s

e x p e r i m e n t . A f t e r I m i n , i n t e r v a l s , t h e t r e a t e d , a n d

u n t r e a t e d h a e m o g l o b i n s a m p l e s were t r a n s f e r r e d t o q u a r t z

c u v e t s and t h e abso rbance r e a d a t 700nm w i t h Pye-Unicam

SP6-200 s p e c t r o p h o t o m e t e r , Phospha te b u f f e r , ( p ~ 7,8)

was used as a b l a n k t o z e r o t h e s p e c t r o p h o t o m e t e r . A

g r a p h o f abso rbance a g a i n s t t e m p e r a t u r e was p l o t t e d .

2,11,4 Oxygen A f f i n i t y A n a l y s i s

The e x t e n t t o w h i c h t h e h a e m o g l o b i n t r e a t e d w i t h

t h e d rug , c a p t u r e d and r e t a i n e d oxygen was e s t i m a t e d b y

m e a s u r i n g t h e d i f f e r e n c e s i n t h e r a t i o o f t h e a b s o r b a n c e

a t 540 and 575nm (Ndu, 1 9 8 6 ) " The a b s o r p t i o n due t o 3-f-

g e n e r a t i o n o f Fe a t 630nm was a l s o m o n i t o r e d ,

Oxygena ted and d e o x y g e n a t e d h a e m o g l o b i n s A and S

samples (0 .5ml ) were p l a c e d i n t e s t t u b e s i n d u p l i c a t e .

KAT,90 was t h e n added t o t h e h a e m o g l o b i n s o l u t i o n s w i t h

t h e h e l p o f t h e p a s t o r p i p e t t e , u n d e r t h e p a r a f f i n l a y e r ,

t o make a f i n a l c o n c e n t r a t i o n o f Img/ml, 2mg/ml, 3rng/rnl

a n d 4mg/ml. They were i n c u b a t e d f o r 40min. a t room

t e m p e r a t u r e . The p a r a f f i n was removed a f t e r i n c u b a t i o n

and t h e haemog lob in samples were d i l u t e d w i t h 0.1M

p o t a s s i u m p h o s p h a t e b u f f e r , Haemog lob in samp les w h i c h

were n o t t r e a t e d w i t h t h e d r u g b u t s u b j e c t e d t o t h e same

e x p e r i m e n t a l c o n d i t i o n s were u s e d as c o n t r o l s . The

o p t i c a l d e n s i t i e s o f t h e r e s p e c t i v e samp les a t t h e

s p e c i f i e d w a v e l e n g t h s were d e t e r m i n e d w i t h an u l t r a -

v i o l e t s p e c t r o p h o t o m e t e r ( ~ n i c a m SP-500, S e r i e s 2,

U l t r a v i o l e t and V i s i b l e S p e c t r o p h o t o m e t e r ) , Q u a r t z

c u v e t s were u s e d f o r t h i s d e t e r m i n a t i o n , An a b s o r b a n c e

s p e c t r u m f o r one o f t h e o x y g e n a t e d h a e m o g l o b i n A a n d S

samp les t r e a t e d w i t h 3mg/ml KAT.90 was o b t a i n e d u s i n g

Pye-Unicam SP6-200 s p e c t r o p h o t o m e t e r ( ~ i ~ s . 19 and

20 r e s p e c t i v e l y ) . The r e s u l t s we re o b t a i n e d i n

d u p l i c a t e . The l e v e l o f s i g n i f i c a n c e o b t a i n e d be tween

u n t r e a t e d and t r e a t e d haemog lob in samp les were c a l c u l a t e d

u s i n g s t u d e n t U s t - t e s t ( S t e e l and T o r i e , 1980; A p p e n d i x I ) ,

CHAPTER THREE

RESULTS

3,l MELTING POINT DETERMINATION

Both batches of KAT.90 seem to have biphasic

melting points. Initially some of the drug melted

at 1 4 6 ~ ~ . There was no further melting of the

remaining portions of the drug as the temperature 0

was increased up to 360 C, the upper limit of the

thermometer used for the determination.

The pH of both KAT.90(1) and K~T.90(11) solutions

were determined to be 4. This pH indicates that the

solution of this compound is acidic.

3.3 SOLUBILITY TEST

The solubility test shows that KAT.BO(1) was

slightly soluble in distilled water, methanol,

methanol-water (1:2) and sufficiently soluble in

dimethyl sulphoxide. KAT,90(II) was soluble in

distilled water, dimethyl sulphoxide, and very

soluble in methanol-water ( 1 : 2 ) . Both KAT.90(1) and

(11) were insoluble in every other solvent or solvent

system tested.

3 . 4 T H I N LAYER CHROMATOGRAPHY O F K A T .90

KAT.90 drug c o u l d no t be s e p a r a t e d w i t h most of

t h e s o l v e n t sys tems t r i e d . However, ch loroform-methanol

( 9 : l ) s o l v e n t system a c h i e v e d a s i n g l e p o i n t s e p a r a t i o n

w i t h R f v a l u e of 0 .63 .

3 . 5 O T H E R TESTS TO I D E N T I F Y THE CONSTITUENTS O F KAT.90

3 .5 .1 P r o t e i n

T h e r e was no v i o l e t o r p u r p l e c o l o u r o b s e r v e d ,

i n s t e a d a w h i t e p r e c i p i t a t e was o b s e r v e d . T h i s i n -

d i c a t e d t h a t t h e r e was no p r o t e i n p r e s e n t i n t h e drug

samples .

3 .5 .2 L i p i d s

The absence of t h e t r a n s l u s c e n t a r e a on t h e

f i l t e r paper i n d i c a t e d t h e absence of f a t s and o i l s .

The absence of b l u e and p u r p l e c o l o u r on t h e drug

s p r a y e d w i t h n i n h y d r i n i n d i c a t e d t h e absence of amino

l i p i d s i n t h e sample.

3 , 5 , 3 S t a r c h

When a drop of i o d i n e was a p p l i e d t o t h e d rug

s o l u t i o n t h e r e was a b l u e - b l a c k c o l o u r a t i o n i n d i c a t i v e

o f t h e p r e s e n c e o f s t a r c h . T h i s was conf i rmed by t h e

i n t e n s e b l u e - b l a c k c o l o u r a t i o n o b s e r v e d w h e n i o d i n e

s o l u t i o n was d i r e c t l y a p p l i e d on t h e drug i n a watch '

g l a s s .

3 , 5 , 4 Reducing S u g a r s

B r i c k - r e d p r e c i p i t a t e o b s e r v e d i n d i c a t e d t h e

p r e s e n c e of r e d u c i n g s u g a r s .

3 , 5 . 5 G l v c o s i d e s

B r i c k - r e d p r e c i p i t a t e o b s e r v e d was i n d i c a t i v e

o f t h e p r e s e n c e o f g l y c o s i d e .

3.5.6 A l k a l o i d s

A l l t h e t e s t s c a r r i e d o u t were n e g a t i v e a s t h e r e

were no development of t h e r e s p e c t i v e c o l o u r s a s ment ioned

i n t h e method ( s e c t i o n 2 .7 .6 ) . These r e s u l t s i n d i c a t e

t h a t a l k a l o i d s a r e a b s e n t i n t h e t e s t drug.

3 , 5 , 7 S a u o n i n s

The a b s e n c e o f f r o t h i n g showed t h e a b s e n c e of

s a p o n i n s .

3,5,8 P h e n o l i c Compounds

The y e l l o w i s h c o l o u r a t i o n o b s e r v e d i n t h e t e s t

t u b e i n d i c a t e d t h e p r e s e n c e o f a p h e n o l i c compound,

3,5.9 T a n n i n s

Absence o f g r e e n i s h - b l a c k p r e c i p i t a t e i n t h e

t u b e s i n d i c a t e d t h e absence o f t a n n i n s i n t h e d rug .

3.5,10 S t e r o l s and T r i t e r p e n e s

The absence o f t h e e x p e c t e d u n d e r l a y e r and r e d

c o l o u r a t i o n a t t h e i n t e r f a c e showed t h a t s t e r o l s and

t r i t e r p e n e s w e r e n o t p r e s e n t i n KAT.90.

3.6 INFRA-RED SPECTRUM OF KAT.90

The b r o a d a b s o r p t i o n band o b s e r v e d a t 3500-

3110cm-~ i s p r o b a b l y due t o t h e p r e s e n c e o f h y d r o x y l

g r o u p s (0-H s t r e t c h ) , T h i s g r o u p c o u l d a r i s e due t o

t h e p r e s e n c e o f a l c o h o l s , p h e n o l s o r s u g a r s . The

(c-0) s t r e t c h i n g bands n e a r 1200cm-~ and 1410cm-~

( F i g s . 1 4 and 15) a r e n o t e w o r t h y i n f r a - r e d c h a r a c -

t e r i s t i c s o f p h e n o l s , A l c o h o l s c o u l d a l s o be

p r e s e n t s i n c e t h e i r (C-0) bands a r e i n t h e r e g i o n o f

1200- 1000cm-~ . The a b s o r p t i o n band n e a r 1600cm-~

gave an i d e a o f t h e p r e s e n c e o f an a r o m a t i c (c=c)

s t r e t c h bu t was n o t c l e a r enough s i n c e t h e f i n g e r

p r i n t r e g i o n d i d n o t . g i v e d e f i n e d peaks . However

t h e r e was a broad a b s o r p t i o n band i n t h e r e g i o n

c o v e r i n g 740-600cm-' which c o u l d n o t be, d e f i n e d ,

3 , 7 ULTRAVIOLET SPECTRUM OF KAT.90

The a b s o r p t i o n band o b s e r v e d a t 275nm on t h e

s p e c t r u m i n d i c a t e s t h e p r e s e n c e o f a benzene d e r i v a -

t i v e i n KAT.90 ( F i g . 1 6 ) "

3 , 8 RESULTS FROM D E R I V A T I Z A T I O N OF K A T , 9 0

The lower band on t h e c h r o m a t o p l a t e was c o l o u r e d

p ink when exposed t o a i r and l i g h t . T h i s r e a c t i o n i s

t y p i c a l of a monohydric p h e n o l ,

M e l t i n g p o i n t d e t e r m i n a t i o n of t h e s o l i d d e r i v a - 0

t i v e gave a r e s u l t above 360 C which was t h e maximum

t e m p e r a t u r e on t h e thermometer .

The e x p e c t e d band of t h e amide from t h e i n f r a - r e d

s p e c t r u m was n o t o b s e r v e d a t 1650cm-~ o r 1550cm-I ( ~ i ~ , 17).

T h i s shows t h e a b s e n c e of a c a r b o x y l group i n KAT.90.

However, t h e r e was an a r o m a t i c (C=C s t r e t c h ) n e a r 1500cm-~

whi'ch g i v e s an i d e a o f t h e p r e s e n c e of an a r o m a t i c

compound. The e x p e c t e d fo rmula f o r t h e r e a c t i o n i f t h e

amide of t h e drug was formed would be

9 "% B -c-,+,,,I ,-.- LCI-+ & - c - , a

Carboxy g roup T h i o n y l Acid A n i l i n e Aromatic arnide o f KAT.90 C h l o r i d e C h l o r i d e o f KAT.90

o f K A T . ~ ~

F i g , l 2 Formula of t h e r e a c t i o n

T h e d e t e c t i o n of t h e a r o m a t i c (C=C s t r e t c h ) ,

t h e b road a b s o r p t i o n band i n t h e r e g i o n 3500- - I

3200cm ( i n d i c a t i n g t h e p r e s e n c e of a hydroxyl

compound) and t h e d e t e c t i o n of C-0 a b s o r p t i o n

band ( 1200-1 100cm-') s u g g e s t t h e p r e s e n c e of

a l c o h o l s o r pheno l s a s t h e p r o d u c t o f t h e r e a c t i o n

( F i g , l 7 ) , The (C=H) o u t of p l a n e d e f o r m a t i o n s a t - 1

680cm and 740cm-I ( F i g . 17) show m o n o s u b s t i t u t e d

benzene p a t t e r n . T h i s c o n f i r m s t h e p r e s e n c e o f

pheno l a s t h e p r o d u c t of t h e whole r e a c t i o n ,

3.9 T U R B I D I T Y S T U D I E S

F i g . 18 and F i g . 2 1 show t h e r e s u l t s o f t h e

a v e r a g e a b s o r b a n c e of haemoglobin s a m p l e s w i t h

t h e c o r r e s p o n d i n g t e m p e r a t u r e s f o r d i f f e r e n t

c o n c e n t r a t i o n s of t h e d r u g .

3.10 OXYGEN AFFINITY ANALYSIS

T a b l e s 1 and 2 show t h e r e s u l t s o f t h e

a b s o r b a n c e a t d i f f e r e n t w a v e l e n g t h s and t h e oxygen

a f f i n i t i e s f o r u n t r e a t e d and t r e a t e d haernog lob ins

a t d i f f e r e n t d r u g c o n c e n t r a t i o n s ,

TABLE 1 : OXYGEN A F F I N I T Y OF OXYGENATED HAEMOGLOBIN SAMPLES

C o n c n o f D r u g i n O x y g e n a t e d ABSORBANCE ( A ) AT G I V E N WAVELENGTH

OXYGEN H a e m o g l o b i n S a m p l e s (mg / rn l )

i n nrn A F F I N I T Y

TABLE 2: OXYGEN AFFINITY OF DEOXYGENATED HAEMOGLOBIN SAMPLES

ABSORBANCE ( A ) AT Concn o f D r u g i -n Deoxygena ted GIVEN WAVELENGTH OXYGEN H a e m o g l o b i n Samples (mg/ml) i n nm AFFINITY

540nm 575nm 630nm

HbA = H a e m o g l o b i n A Samples

HbS = H a e m o g l o b i n S Samples

Concn = C o n c e n t r a t i o n

Oxyhaemog lob in a b s o r b s a t 540nm

Deoxyhaemog lob in a b s o r b s a t 575nm

M e t h a e m o g l o b i n a b s o r b s a t 630nm

F i g . 13: INFRA-RED SPECTRUM OF POTASSIUM BROMIDE AS THE REFERENCE SPECTRUM.

F i g o 1 4 : INFRA-RED SPECTRUM OF K A T . 9 0 ( 1 ) .

Fig.15: INFRA-RED SPECTRUM OF KA~.90(11),

d, I I I I

200 1

225 I I I f

250 275 XX) 325 350 375 400 W A V E L E N G T H ( n m )

F i g " l 7 : INFRA-RED SPECTRUM OF THE PREPARED AMIDE D E R I V A T I V E OF KAT .90.

F i g , l B : PLOT OF ABSORBANCE AGAINST TEMPERATURE FOR TREATED AND UNTREATED DEOXYHAEMOGLOBIN S SAMPLES, AT CONCENTRATION RANGE OF 1-4mg K ~ T , 9 0 / m l ,

a-es C o n t r o l

0-8 1. Omg KAT, 9 0 / m l

X-x 2.0mg KAT . 9 0 / m l

8-a' 3.0rng KAT, 90/rnP

B!%-Ff4 4. Orng KAT . 9 0 / m l

0. OZ

0.07

OOE

0.0:

0.04

0-0 3

0.0 2

0.01

C -1 I I I

2 83 293 203 31 3 323 f

T E M P E R A T U R E ( O K 1

F i g . 1 9 : ABSORPTION SPECTRUM OF OXYGENATED HAEMOGLOBIN A TREATED WITH KAT.90 FROM 400-700nrn.

0-0 C o n t r o l

0-0 3.0mg KAT.90 /ml

F i g u 2 0 : ABSORPTION SPECTRUM OF OXYGENATED HAEMOGLOBIN S TREATED WITH KAT.90 FROM 400-700nm.

0-0 C o n t r o l

o-e 3.0mg K A T o 9 0 / r n l

)-

- 3-

b-0. ) i I I I 1 I I I I

443 480 520 5 60 600 --+9-=i

400 640 680 W A V E L E N G T H ( n m )

F i g . 21: PLOT OF ABSORBANCE AGAINST TEMPERATURE FOR TREATED AND UNTREATED OXYHAEMOGLOBIN S SAMPLES, AT C O N C E N T R A T I O N R A N G E OF 1-4mg KAT. 9O/ml,

o C o n t r o l

0-0 l.Omg K~T.90/ml

x-x 2.0mg K ~ ~ . 9 0 / r n l

&-m 3.0rng K ~ ~ . 9 0 / m l

m- 4.0rng KAT .90/ml

-7'1 I I I I 1 283 293 303 313 323

T E M P E R A T U R E ( O K )

CHAPTER FOUR

DISCUSSION AND CONCLUSION

4,1 DISCUSSION

I n t h i s p r o j e c t a t t e m p t s have been made t o i . d e n t , i f y

t h e c o n s t i t u e n t s o f KAT,90, c h a r a c t e r i z e i t f u r t h e r and

i n v e s t i g a t e i t s mode o f a n t i s i c k l i n g and a n t i g e l l i n g

a c t i o n .

The b i p h a s i c m e l t i n g p o i n t s o b s e r v e d i n d i c a t e t h a t

t h e d r u g m i g h t be a m i x t u r e o f compounds whose c o n s t i t u e n t 0

p a r t s have a m e l t i n g p o i n t o f 146 C w h i c h c o r r e s p o n d s t o

t h e m e l t i n g p o i n t o f D - g l u c o s e (~inar;1973) and above 0

360 C, t h e l i m i t o f t h e t e c h n i q u e .

The s l i g h t l y weak a c i d i c n a t u r e o f t h e a g e n t w h i c h

changed c o l o u r when t r e a t e d w i t h a base, a t y p i c a l

r e a c t i o n o f a p h e n o l i c compound ( ~ a r b o r n e , 1973) seems

t o s u g g e s t t h a t a p h e n o l i c compound m i g h t be one o f t h e

c o n s t i t u e n t s o f KAT.90, P h e n o l s a r e weak a c i d s a n d t h e

f o r m a t i o n o f t h e p h e n o x i d e i o n i n b a s i c s o l u t i o n i s

accompan ied b y b a t h o c h r o m i c and h y p e r c h r o m i c s h i f t s o f

c o n s i d e r a b l e m a g n i t u d e ( P h i l l i p s , 1 9 6 4 ) "

7 6

KAT.90 may c o n t a i n a p o l a r c o n s t i t u e n t a s t h e

a g e n t i s more s o l u b l e i n p o l a r s o l v e n t s such a s w a t e r .

The i n c r e a s e i n s o l u b i l i t y of K ~ T , 9 0 ( 1 1 ) i n w a t e r

might be i n t e r p r e t e d t o mean t h a t t h e w a t e r s o l u b l e

c o n s t i t u e n t s of t h e a g e n t may have been more i n

c o n t e n t t h a n i n K A T . ~ O ( I ) "

T h i n l a y e r c h r o m a t o g r a p h i c a n a l y s i s r e v e a l e d

t h a t most of t h e o r g a n i c s o l v e n t s and s o l v e n t s y s t e m s

used c o u l d n o t s e p a r a t e t h e d rug i n t o i t s c o n s t i t u e n t

bands . However, one band was o b t a i n e d w i t h ch lo ro fo rm-

me thano l ( 9 : l ) s o l v e n t sys t em,

O t h e r chemica l t e s t s showed K A T . ~ ~ t o be p o s i t i v e

f o r s t a r c h and s u g c r s . D-glucose c o u l d be one o f t h e

s u g a r s p r e s e n t and might a l s o c o n t r i b u t e t o g i v e t h e

p o s i t i v e r e s u l t of t h e p r e s e n c e of s t a r c h . Ano the r

i m p o r t a n t o b s e r v a t i c n i s t h a t t h e a g e n t was p o s i t i v e

f o r g l y c o s i d e b u t t h e s p e c i f i c g l y c o s i d e c o u l d n o t be

i d e n t i f i e d w i t h i n t h e p r e v a i l i n g c c n d i t i o n s . The

a c e t a l d e r i v a t i v e , g l y c o s i d e , i s formed when t h e

h e m i a c e t a l form of a s u g a r r e a c t s w i t h a m o l e c u l e of

an a l c o h o l o r pheno l ( F i n a r , l 9 7 3 ) , The . p r e s e n c e of

g l y c o s i d e s i n t h e a g e n t s u g g e s t s t h a t a p h e n o l i c g roup

and a sugar moeity a r e p r e s e n t . Th i s f u r t h e r conf i rms

t h e p resence of sugar a s observed i n t h e chemical t e s t s

mentioned above, The a c i d i t y and t h e r e s u l t o f t h e

r e a c t i o n of KAT.90 w i t h sodium hydroxide r u l e s o u t t h e

p re sence c f a l c o h o l s , Glycos ides might be t h e a c t i v e

a n t i g e l l i n g agen t i n KAT,90 s i n c e an aminoglycoside has

been i d e n t i f i e d a s an a c t i v e a n t i g e l l i n g p r i n c i p l e of

t h e e x t r a c t i v e s from t h e seed of Cajanus ca j an (1wu

e t a 1 1988). No f u r t h e r t e s t was c a r r i e d ou t t o - - I

i d e n t i f y t h e aglycone. The two p r e s e n t a t i o n s of KAT.90 have t h e same

chemical c o n s t i t u e n t s a s shown i n F igs . 14 and 15.

I t could be t h a t t h e two ba tches of KAT,90 d i f f e r e d

on ly i n t h e q u a n t i t i e s of s t a r c h and suga r they c o n t a i n .

S o l u b i l i t y t e s t s showed t h a t they c o n t a i n d i f f e r e n t

q u a n t i t i e s of t h e a c t i v e p r i n c i p l e f o r s i c k l e c e l l

p a t i e n t s of d i f f e r e n t ages ,

F u r t h e r con f i rma t ion of t h e p resence of g l y c o s i d e s

i n KAT.90 was observed from t h e i n f r a - r e d spectrum

coupled w i t h t h e u l t r a v i o l e t s p e c t r a l a n a l y s i s of t h e

a g e n t , The a b s o r p t i o n band on t h e u l t r a v i o l e t spectrum

a t 275nm (Dr Michael Ke l l ehe r , D e p t cf P u r e and

I n d u s t r i a l Chemistry, U n i v e r s i t y of N ige r i a , Nsukka,

p e r s o n a l c o m m u n i c a t i o n ) may i n d i c a t e t h e p r e s e n c e o f

s u b s t i t u t e d benzene ( ~ a f f e / and O r c h i n , 1962) i n KAT.90.

Some amino a c i d s , f o r example p h e n y l a l a n i n e w h i c h

i s t h e mos t e f f e c t i v e among a s e r i e s o f t e n amino a c i d s

i n i n c r e a s i n g t h e s o l u b i l i t y o f deoxyhaemog lob in g e l s

p o s s e s s a p h e n y l g r o u p ( N o g u c h i and S c h e c h t e r , 1977) . ,

P h e n y l a l a n i n e was a l s o o b s e r v e d t o a l t e r t h e minimum

g e l l i n g c o n c e n t r a t i o n o f h a e m c g l o b i n S w i t h o u t s i g n i -

f i c a n t l y c h a n g i n g t h e oxygen a f f i n i t y ( N o g u c h i and

S c h e c h t e r , 1977) . However, p r o t e i n was a b s e n t i n

KAT"90. A f r a c t i o n o b t a i n e d f r o m t h e w a t e r e x t r a c t

o f F a g a r a z a n t h o x y l o i d e s was i d e n t i f i e d t o c o n t a i n

p h e n o l i c a c i d s w h i c h had a n t i s i c k l i n g a c t i v i t y

( O d e b i y i a n d So fowora , 1979) .

The amide band was n o t d e t e c t e d i n t h e i n f r a -

r e d s p e c t r u m ( F i g . 1 7 ) i m p l y i n g t h a t t h e a g e n t does

n o t possess a c a r b o x y l g roup , The i n t e n d e d amide

d e r i v a t i v e o f KAT.90 seemed n o t t o have been fo rmed.

Pe rhaps , t h e components o f KAT.90 decomposed d u r i n g

t h e v i g o r o u s r e f l u x i n g i n v o l v e d i n t h e s y n t h e s i s and

p r o b a b l y l o s t t h e s u g a r m o l e c u l e s s i n c e t h e m e l t i n g

p o i n t o f 1 4 6 ' ~ o f KAT.90 was n o t o b s e r v e d i n t h e

d e r i v a t i v e . M o r e o v e r , t h e b r o a d h y d r o x y l b a n d s f o r

s u g a r s were n o t d e t e c t e d on t h e i n f r a - r e d s p e c t r u m

o f t h e i n t e n d e d a m i d e d e r i v a t i v e a s i n t h e c a s e c f

KAT.90. A l s o t h e t h i n l a y e r c h r c r n a t o g r a p h ~ r e s u l t s

o f t h e i n t e n d e d arnide d e r i v a t i v e s u g g e s t s t h e p r e s e n c e

o f m o n o s u b s t i t u t e d p h e n o l , w h i c h t u r n s p i n k on e x p o s u r e

t o l i g h t a n d a i r ( F i n a r , 1 9 7 3 3 , T h e s u s p i c i o n o f

KATU90 u n d e r g o i n g d e c o m p o s i t i o n d u r i n g t h e r e f l u x i n g

p r o c e s s c o u l d a l s o be s u b s t a n t i a t e d by t h e f a c t t h a t

t h e i n f r a - r e d s p e c t r u m o f t h e i n t e n d e d d e r i v a t i v e shows

t h e p r e s e n c e o f p h e n o l .

T h e t u r b i d i t y t e s t c a r r i e d o u t a t t e m p e r a t u r e s 0 0

b e t w e e n 2 8 3 K a n d 3 2 3 K on o x y g e n a t e d a n d d e o x y g e n a t e d g e n e r a l

h a e m o g l o b i n S showed a / d e c r e a s e - i n t u r b i d i t y when

t r e a t e d w i t h KAT.90 d r u g a t c o n c e n t r c t i o n s o f 1-4mg

d r u g / m l a s c o m p a r e d w i t h t h e u n t r e a t e d h a e m o g l o b i n S

s a m p l e s . D e c r e a s e i n v i s c o s i t y i n d i c a t e s a d e c r e a s e

i n t h e f o r m a t i o n o f h a e n i o g l o b i n S f i b e r s ( V o t a n o -- e t a l . ,

1 9 7 7 ) . T h i s f i n d i n g i s i n c o n s o n a n c e w i t h t h e o b s e r v a -

t i o n s t h a t compounds t h a t i n h i b i t t h e f o r m a t i o n o f

hae rnog lob in S f i b e r s a r e u s e d i n s i c k l e c e l l a n a e m i a

t h e r a p y ( S t r y e r , 1 9 8 1 ) a s t h e y c o u l d d e c r e a s e g e l l i n g ,

T h e d e c r e a s e i n t u r b i d i t y by K A T , ~ ~ i s e v i d e n c e d by

t h e l o w a b s o r b a n c e v a l u e o f t h e t r e a t e d h a e r n o g l o b i n

s a m p l e s w h i c h , a c c o r d i n g t o I w u -- e t a l . (1988) s u g g e s t s

t h a t t h e a g e n t p r o l o n g s g e l a t i o n time o f d e o x y h a e ~ n o g l o b i n s

a t v a r i o u s t e r n p e r c t u r e s .

T h e i n h i b i t i o n o f g e l a t i o n o f d e o x y g e n a t e d

hae rnog lob in S was s i g n i f i c a n t a t 3rng d r u g / m l a n d

4mg d r u g / m l t r e a t m e n t ( P < 0 , 0 5 ) . T h i s l a r g e i n h i b i t i o n

o f g e l a t i o n , a c c o r d i n g t o R o s s a n d S u b r a m a n i a n ( 1 9 7 7 ) ,

c o u l d b e a s s o c i a t e d w i t h t h e i n t r o d u c t i o n o f t h e b u l k y

a r o m a t i c r e s i d u e . V i s c o s i t y d e c r e a s e d f o r t h e o x y g e n a t e d

s a m p l e s o f h a e m o g l o b i n S a s t h e c o n c e n t r a t i c n o f t h e d r u g

i n c r e a s e d t o 4mg d r u g / m l . T h e s e r e s u l t s a g r e e w i t h t h e

f i n d i n g s t h a t KAT.90 i s an a n t i g e l l i n g a g e n t (Ch idume,

Compounds t h a t d e c r e a s e t h e c c n c e n t r a t i o n o f

t h e deoxy fo rm o f h a e m o g l o b i n S by i n c r e a s i n g i t s

o x y g e n a f f i n i t y a r e a n t i s i c k l i n g a g e n t s ( S t r y e r , 1 9 8 1 ) . c o n t a i n i n g

KAT,90 c o u l d b e m e n t i o n e d a s / o n e - o f t h e s e compounds

f r o m t h e oxygen a f f i n i t y r e s u l t s .

S a m p l e s o f o x y g e n a t e d h a e m o g l o b i n S t r e a t e d w i t h

1-3mg o f KAT.90 p e r m l showed s i g n i f i c a n t i n c r e a s e s

in oxygen affinity (P<0,05) when compared with the

drug - untreated sickle haemoglobin samples. However

at 4mg drug/ml treatment the oxygen affinity

decreased. This could be due to large amount of

methaemoglobin formation which could not combine

with oxygen since methaemoglobin does not carry

oxygen molecules. This observation is in cgreement

with that of Chidume (1991) who observed that, at

4mg drug/ml concentration of KAT.90, 9% of the

deoxyhaemoglobin was converted to methaemoglobin.

She also observed that 3mg drug/ml concentration of

KAT,90 is the limit concentration at which almost all

the sickled cells seemed to be reversed and prevented

from sickling - in vitro, The result of the oxygen

affinity experiment is supported by this observation

as the maximum oxygen affinity was observed at this

concentration (3mg drug/ml) after which the affinity

decreased drastically.

Oxygen affinity of sickle haemoglobin samples

was higher than that of normal haemoglobin (HbA)

indicating that the drug might have some positive

effect on its graducl capacity to bind oxygen.

The i n c r e a s e i n oxygen a f f i n i t y of t h e

haemoglobin samples , even when t r e a t e d w i t h sodium

m e t a b i s u l p h i t e (more s i c k l e d haemoglobin) was however,

i n s i g n i f i c a n t .

A normal p h y s i o l o g i c a l f u n c t i o n of haemoglobin

i s the r e v e r s i b l e b i n d i n g of dioxygen which can o n l y

o c c u r w i t h t h e haeme i r o n i n t h e r e d u c e d ( f e r r o u s )

s t a t e (Wal l ace -- e t a l , , 1 9 8 2 ) , T h u s i r o n ( 1 1 ) w i t h

t h e l o s s of an e l e c t r o n i s c o n v e r t e d t o t h e o x i d i z e d

i r o n (111) ( f o r m a t i o n of methaemoglobin) which i s n o t

a b l e t o b i n d dioxygen..

A t 630nm methcemoglobin r i s e was d e t e c t e d i n

haemoglobin A and S samples t r e a t e d w i t h KAT.90.

I t s f o r m a t i o n i n c r e a s e d w i t h i n c r e a s e i n d rug concen-

t r a t i o n i n a l l t h e haemcglobin samples t e s t e d . The

d i s c o v e r y of a p h e n o l i c group i n t h e a g e n t c o u l d a l s o

be j u s t i f i e d by i t s a c t i v i t y on oxyhaemoglobin (HbO ) . 2

Pheno l s r e a c t w i t h HbO t o g i v e methaemoglobin, t h e 2 a c t i v e s p e c i e s be ing t h e p h e n o l a t e a n i o n ( w a l l a c e

and C.aughey, 1975). T h e r e a c t i o n of HbO w i t h 2 p h e n o l s t o produce methaemoglobin shows i n v e r s e r a t e

+ dependence upon (H ) , d i r e c t dependence upon t h e

c o n c e n t r a t i o n of oxyhaemoglobin and p h e n o l s , and a r a t e

t h a t c o r r e l a t e s w i t h t h e e l e c t r o n dono r c h a r a c t e r i s -

t i c s o f t h e r e a g e n t ( W a l l a c e and Caughey, 1?75),

O x i d a n t d r u g s w h i c h i n c l u d e such d r u g s as t h e

a n t i m a l a r i a l s o f t h e 8 - a m i n o q u i n o l i n e g roup , c s p i r i n ,

s u l f o n a m i d e s and p h e n y l h y d r a z i n e s a r e a l l a c t i v e one-

e l e c t r o n d o n o r s and can r e a d i l y a c t t o p r o m o t e t h e

two e l e c t r o n r e d u c t i o n o f d i o x y g e n bound t o haemog lob i r i

i n e x a c t l y t h e way d e s c r i b e d f o r p h e n o l s ( w a l l a c e and

Caughey, 1975) .

A l t h o u g h me thaemog lob in i s u s e l e s s a s an oxygen

c a r r i e r , i t a l s o r a i s e s t h e oxygen a f f i n i t y o f t h e

r e m a i n i n g f e r r o h a e m o g l o b i n ( ~ a r l i n ~ and Roughton, 1942) .

T h i s f a c t i s d e m o n s t r a t e d b y t h e oxygen a f f i n i t y r e s u l t s

o f KAT.90. A d r u g c o u l d i n c r e a s e m e t h a e m o g l o b i n l e v e l s

e i t h e r b y a l t e r i n g t h e enzyme sys tems w h i c h n o r m a l l y

r e d u c e i t, o r by d i r e c t e f f e c t s o n h a e m o g l o b i n m o l e c u l e s

( B o o k c h i n -- e t a l . , 1 9 7 9 ) " Methaernog lob in r c d u c t a s e and

o t h e r enzyme sys tems n o r m a l l y r e t u r n m e t h a e m o g l o b i n t o

t h e f u n c t i o n a l ( r e d v c e d ) f o r m a s e x p l a i n e d b y M e t z l e r

( 1 9 7 7 ) . However, excess me thaemog lob in l e v e l s o f more

t h a n 2g/100ml i s t o x i c ( W h i t b y -- e t a l . , 1980) .

4 , 2 CONCLUSION - T h i s s t u d y r e v e a l s t h a t K a t s i n a 1990 (KAT.90)probabl

c o n t a i n s a g l y c o s i d e which c o n s i s t s of a p h e n o l i c

and a s u g a r moei ty . The m e l t i n g p o i n t of 1 4 6 ' ~ c o u l d

be t h a t of D-glucose r e l e a s e d d u r i n g t h e h e a t i n g

p r o c e s s . The a c i d i t y of t h e a g e n t c o u l d a l s o a r i s e

due t o t h e p r e s e n c e of t h e p h e n o l i c g roup . Al though

KAT090(I) and KAT.90(11) d i f f e r i n c o l o u r and e x t e n t

of s o l u b i l i t y , we s u g g e s t t h a t KAT,90(I) and ( 1 1 )

c o n s i s t of t h e same compounds bu t t h e compos i t e p a r t s

a r e i n d i f f e r e n t p r o p o r t i o n s .

KAT.90 i s s u g g e s t e d t o p r o l o n g t h e d e l a y t ime

of g e l a t i o n of deoxygenated haemoglobin S, e s p e c i a l l y

a t a drug c o n c e n t r a t i o n of 4mg drug/ml a t which i t was

o b s e r v e d t o be s i g n i f i c a n t ( P < 0 .05) . Also , d e c r e a s e

i n v i s c o s i t y of oxygena ted haemoglobin S by t h e a g e n t

was e v i d e n t .

KAT.90 was found t o i n c r e a s e oxygen a f f i n i t y

of oxygena ted and deoxygenated haemoglobin A and S

samples . The drug c o n c e n t r a t i o n of 3mg drug/ml gave

t h e most s i g n i f i c a n t i n c r e a s e i n oxygen a f f i n i t y f o r c o n c e n t r a t i o n s of

both haemoglobins ( P C 0 . 0 5 ) . However, at/4mg - drug/ml

and above a d e c r e a s e i n oxygen a f f i n i t y was o b s e r v e d

i n a l l t h e s i c k l e c e l l haemoglobin samples ,

85

T h e r e was a g r a d u a l r i s e of methaemoglobin

p r o d u c t i o n w i t h i n c r e a s i n g c o n c e n t r a t i o n of t h e a g e n t .

T h e p r e s e n c e of t h e p h e n o l a t e a n i o n c o u l d be r e s p o n s i b l e

f o r such a r e a c t i o n a s o b s e r v e d by Wal l ace and Caughey

( 1975).

4 . 3 SUGGESTIONS FOR F U R T H E R STUDIES

I n v i v o e x p e r i m e n t s a r e s u g g e s t e d t o be c a r r i e d - -- o u t w i t h low c o n c e n t r a t i o n s of KAT.90 t o d e t e r m i n e t h e

e x t e n t t o which t h e body ' s d e f e n c e mechanism can l i m i t

t h e c o n c e n t r a t i o n s of methaernoglobin. The i n t e r -

r e l a t i o n s between p o l y m e r i z a t i o n and oxygen a f f i n i t y

a r e r e l e v a n t t o t h e mode of a n t i s i c k l i n g by a d rug .

S t u d i e s on t h e s e a r e a l s o encouraged , F u r t h e r m o r e , t h e

d e t e r m i n a t i o n of t h e e x a c t c o n s t i t u e n t s o f KAT,90 i s

e s s e n t i a l ,

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APPENDIX I

FORMULA FOR THE STUDENTUS t-TEST USED IN THE CALCULATIONS

&Y = Sum of observations

f y2 = Sum of squared observations

= mean of observations 2 ~ ( Y - v ) ~ = $Y - ~ y ) ~ / n where,

2 S(Y - v ) = sum of squared deviations

(SY) 2

= squared sum of observations

n = number of observations

: 2 = $.(Yl - Yl) ---- where

n, - 1 I 2

S1 = Variance of first set of observations.

where ng- 1

2 S2 = Variance of second of

observations,

s(i1-y2 = pooled standard error.

1 t = V 1 - V 2

where, 1

t = calculated t

E f f e c t i v e d e g r e e o f f r eedom ( d f ) =

1 Then, compare c a l c u l a t e d t ( t ) w i t h t a b u l a t e d t

a t e f f e c t i v e d f t o see w h e t h e r t h e d i f f e r e n c e i s

s i g n i f i c a n t .

CALCULATION OF OXYGEN AFFINITY

Abso rbance a t 540 = X

Abso rbance a t 575 = Y

R a t i o = X:Y X - Y

D i f f e r e n c e i n t h e r a t i o = - - X+Y X+Y

APPENDIX I1

MEAN ABSORBANCE OF DEOXYGENATED HAEMOGLOBIN S SAMPLES AT 700nm AT S P E C I F I C T I M E I N T E R V A L S , t ( s e c ) .

- T e m p e r a - ABSORBANCE ( A ) AT G I V E N CONCEN-

T i m e t u r e

T R A T I O N S OF K A T o 9 0 ( m g / r n l ) 0 ,, b e d 0,O 1 ,O 2,O 3,O 4.0

A P P E N D I X I11

MEAN ABSORBANCE O F OXYGENATED HAEMOGLOBIN S SAMPLES AT 7 0 0 n m A T S P E C I F I C T I M E I N T E R V A L S ,

T e m p e r a - ABSORBANCE ( A ) AT G I V E N CONCEN-

T i m e ture

T R A T I O N S OF K A T .9O ( m g / m l )

OK ( s e c ) 0.0 1 .O 2.0 3,O 4.0

m g / m l m d m l m g / m l m g / m l m g / m l