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BLOOD GLUCOSE LEVELS
IN
CATTLE IN RESPONSE TO DIFFERENT FORMULATIONS OFBETAHETHASOi�E
A THESIS PRESENTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF PHILOSOPHY IN VE TERINARY SCIENCE
AT MASSEY UNIVERSITY
BY
NI NI MAW
B,v.s.
AUGUST
1981
(
THIS THEf> l �
.i �P��if
ENTS 2 5% OF THE
ASSESSMENT OF THE CANDIDATE
)
i.
ABSTRACT
The hyperg l y c aemi c r e s p on s e in adu l t c ows to the s ub c u t aneou s i n j e c t ion o f b e t ame thas one a t 0 . 04mg / kg b o dy we i gh t was
s tu d i ed u s ing four d i f ferent f o rmu l a t ions o f t h e drug . Th e s e were Be t s op a r t and B e t s o l an , aqueous su s pens i o n s o f b e t ame tha s on e o f lOmg /ml and 2mg /ml r e s p e c t ive l y and Smg /ml and 2mg /ml p r epara t i on s produ c e d by d i lu t ing B e t s opar t w i th wa t e r imme d i a t e l y pr ior t o inj e c t i on . Four t r e a tmen t group s and one c on t r o l group each o f four c ows were b l e d d ai l y f o r 1 2 days w i th the be t ame th a s one b e ing g iven on day 4 . A l l t r i a l b l oo d s amp l e s we re c o l l e c t e d u s ing flu o r i de-oxa l a t e a s an t i c o agu l an t wh i ch wa s sh own t o r e s u l t i n l ower p l a sma g l u c o s e l eve l s than in EDTA p l a sma o r s e rum .
D a i ly p l a sma g l u c o s e c onc e n t r a t i o n s we re de t e rmined u s �ng 3 a n a ly t i c a l me tho d s . The a l ka l ine f e r r i cyan i d e me thod , a s an t i c i p a t e d , gave h i gher g l uc o s e l ev e l s than the g lu c o s e o x i da s e me tho d , and unexpe c t e d l y , t h e oxygen e l e c t r o d e me thod a l s o over e s t ima t ed t h e g l uc o s e c oncen t r a t i on .
D e s p i t e d i f fe r en c e s in the ana l y t i c a l me tho d s i t wa s sh own t h a t the l Omg /ml Be t s opar t and the two 2mg /ml prepara t i ons ( B e t s o l an and d i lu t ed Be t s opar t ) gave s imi l a r p e ak p l a sma g l uc o s e c o n c en t r a t i on s wh i l e the Smg /ml d i l u t e d Be t s op a r t g ave a s i gn i f i c an t l y h i gh e r p e ak . Th e l ongev i ty o f the hyperglyc aemia wa s grea t e s t in the l Omg /ml and Smg /ml
t r e a t e d group s and le s s in the two 2mg /ml group s .
ACKNOWLEDGEf1ENTS
I am d e ep l y indeb t e d t o my superv � s o r Mr J . V . P au l i f o r h i s e n thu s i a s t i c i n t e r e s t , encouragemen t , gui d an c e and g r e a t h e l p through ou t th i s s tudy . I wou l d l ike to exp re s s my
s p ec i a l thank s t o Dr R . M . G r e enway , Depar tmen t o f Chemi s t ry , B iochemi s t ry and B i ophy s i c s , f o r h i s superv i s i on and
va luab l e advi c e throughou t th i s work .
I owe my s in c e r e thank s t o Pro f e s s o r R . E . Mun fo r d , D e p a r tme n t o f Phys i o l o gy a n d Ana tomy , f o r h i s a dv i c e and h e l p w i th the s ta t i s t i c a l ana l y s i s .
My thank s a r e a l s o due to Pro f e s s o r E . D . F i e l d en , D e an o f the Facu l ty o f V e t e r inary S c i e n c e and G l axo Labora t o r i e s ( N . Z . ) L t d f o r the i r in t er e s t and f inanc i a l a s s i s tance w i th th i s exper imen t .
I wou l d l ik e t o thank Dr McKenz i e , Depar tmen t o f Da i ry Hu s b andry , f o r the u s e o f the g l u c o s e ana l y s e r ins t rumen t ; M i s s G . B o r r i e and Mr s J . Rumb a l f o r the i r c o-o p e ra t i on
and techn i c a l h e lp ; and Mr S . McDi a rmid for h i s advi c e in pharma c o l o gy . I am a l s o gra t e fu l t o the Gove rnmen t o f the S o c i a l i s t Repub l i c o f th e Un i on o f Burma f o r the gran t o f fe r e d und e r the Wo r l d B ank l o an pro gramme .
F ina l ly my a p pr e c i a t ion � s due t o my devo t e d Oo and paren t s at h ome for the i r enc ouragemen t and l ove .
AB S T RACT
ACKNOWLEDGEMENTS TABLE OF CONTENTS L I S T OF TABLE S L I S T OF FIGURE S
CHAPTER 1: INTRODUCT I ON
TABLE OF CONTENTS
CHAPTER 2: L ITERATURE REV IEW 2 . 1 Subj e c t Revi ew
iii .
2 . l. l 2 . l. 2
Chemi s t ry o f c o r t ic o s t e r o i d s
Phys io l o g i c a l e f f e c t s and me chan i sm 2 .l. 3
2 . 2 2 . 2 . l 2 . 2 . 2 2 . 2 . 3
o f ac t i on o f g l u c o c or t i c o i d s App l i c a t i on o f g l u c o c or t i c o i d s 1n
ve t e r inary med i c ine
Me thod Rev i ew
F e r r i cyan i d e reduc t ion me thod G l uc o s e o x i d a s e enzyrna t i c me thod Oxy gen e l ec t r o d e me thod
CHAPTER 3: MATERIAL S AND METHOD S
P a ge
i ii
111 V
vii l
2 2 2
3 13
16 16 17 20
22 3 . 1 Organ i s a t ion o f the Tr i a l 22
3 . 1 . 1 Group ing o f th e c ows 22
3 . 1 . 2 B l ee d ing o f the c ows 22 3 . 1 . 3 Admin i s t ra t i on o f the drug 22 3 . 2 C o l l e c t i on and p r e s erva t i on o f the
s amp l e s 24
3 . 3 Ana l y s i s o f the s amp l e s 24 3 . 3 . 1 Alka l ine f e r r i c y an i de r e du c t ion me t h o d 24 3 . 3 . 1 . 1 Rea gen t s 26 3 . 3 . 1 . 2 C o l o r ime t e r adj u s tmen t 26 3 . 3 . 1 . 3 D a t a p r o c e s s ing : F er r i cy an i d e
me th o d 26
3 . 3 . 2 G l uc o s e oxi d a s e enzyme me thod 27
iv .
TABLE OF CONTENTS CONTINUED
P a ge
CHAPTER 3: CONTINUED
3 . 3 . 2 . 1 Re a gen t s 27
3 . 3 . 2 . 2 Co l o r ime t e r a d j u s tmen t 29 3 . 3 . 2 . 3 Da t a Pro c e s s i ng : G luco s e ox i d a s e
me thod 29
3 . 3 . 3 Oxy gen e l e c t rode me thod 29
3 . 3 . 4 P r e c i s i on c on t r o l 30
3 . 4 S t a t i s t i c a l meth o d s 35
CHAPTER 4: RE S ULTS 37
37 4 . 1
4 . 2
4 . 3
4 . 4 4 . 5
I n t r o du c t ion
The e f f e c t o f an t i c o a gu l an t s on p l asma g l u c o s e l eve l s
The e f f e c t o f t ime between b l o o d co l l e c t i on and t h e s epara t ion o f p l a sma o r s e rum f r om the
ery throc y t e s on p l a sma and serum glu c o s e l eve l s
B e t ame tha s one e xper imen t
C omp ar i s ons o f ana l y t i c a l me tho d s
CHAPTER 5: D I S C U S S ION
APPENDIX I
APPENDIX I I REFE RENCES
5 . 1 5 . 2 5 . 3
5 . 4 5 . 5 5 . 6
I n t r o duc t ion
E f fec t of an t i c o a gu l an t s
E f fec t o f t ime be tween b l eed ing and c en t r i fuga t ion
Ana l y t i c a l me tho d s
Th e betame th a s ome t r i a l Con c l u s i on
37
4 0 40 58
62 62 62
64 64 65 69
v .
LIST OF TABLES
Table Page
1 Betamethasone trial protoc o l showing the groupings of the cows by body we ight and breed , the amount o f drug admin i s tered and the composition of the
in j ec ted dose . 2 3
2 The e f fect o f anticoagulants on p lasma glucose leve l s
3 The e f fect of fluoride-oxalate on aqueous glucose s o lutions and se rum
4 E f fect of adding fluor ide -oxal ate to who le blood and EDTA pla sma samp le s
5 E f fect of sodium f luor ide and potas s ium oxalate on p lasma gluco se leve ls when added to whole b lood and p lasma containing EDTA
6 E f fect o f sodium fluoride and pota s s ium oxa late on serum glucose leve l s when added to whole b lood and se rum
7 Mean p la sma glucose leve l s o f trial groups
3 8
3 8
3 9
4 1
4 1
( Ferricyanide method) 4 5
8 Mean plasma glucose leve l s o f trial group s
( Glucose oxidase method ) 4 6
9 Mean plasma glucose leve ls o f trial group s
( Oxygen e le ctrode metho d ) 4 7
1 0 Analysi s o f variance o f log gluco se concentrations for each of the 5 treatment groups 5 2
vi .
LIS T OF TABLES CONTINUED
Tabl e Page
1 1 Compar ison o f s lope s and ( 9 5 % confidence
interva l ) of the regre s s ion l ine s of the five treatment groups
1 2 Compari son o f the intercepts and ( 9 5 % confidence interval ) o f the regre s s ion lines at day 0
( i . e . day 5 of the trial ) for the 5 treatment groups
1 3 Comparison o f three analytical methods by the
5 3
5 4
t-te s t using the precis ion control s e rum data 5 9
1 4 Comparison o f the 3 analytical method by the paired t-te s t us ing the Betamethasone trial
data from days 1 to 4 6 0
1 5 Analysis o f variance ( within cows ) o f the d i f ference s between me thods u s ing the
Betamethasone trial data from days 6 to 1 2 6 1
LIST OF FIGURES
F i gure
1 The pregnane nucleus
2 Cor t i sone
3 Cor tiso l ( hydrocortisone )
4 P rednisone
5 Prednisolone
6 Triamcinolone 7 Betamethasone 8 Corticos te rone
9 The general actions o f glucocorticoids on carbohydrate , lipid and protein metabo l i sm
1 0 ( a ) Acute and ( b ) chronic e f fect o f
glucocorticoids o n carbohydrate metabo l i sm . The arrow end ing in a bar � ) indicate s an inhib i tory e ffect . Unblocked arrows ind icate s t imulation
1 1 The actions o f glucocorticoids and insulin on carbohydrate metabo l i sm
1 2 The ce l lular mechani sm o f action o f glucocortico ids on the i r target t i s sue s .
1 3 Chemlab-analyse r module s and man i fo ld s for Alkal ine ferricyanide reduction method for plasma glucose
vii .
P age
4 4 4 4 5
5
5
5
7
8
1 0
1 1
2 5
v i i i .
L I S T OF F IGURES CONTINUED
Figure P age
1 4 Chemlab-analyser modules and manifold for glucose oxidase -peroxidase me thod for p l a sma gluco se
1 5
1 6
The sensor probe and the membrane o f Y S I Mode l 2 7 Analyser
De te rmination of glucose concentration in contro l serum ( Fe rricyanide method )
1 7 Determinations o f Gluco se concentration in control s erum ( Gl uco se Oxidase Method )
1 8 Determinations o f Gluco se concentration in control serum ( Oxygen Electrode Method )
1 9 The e f fect o f time between blood col l ection and centrifugation on the s tab i l i ty of plasma and serum gluco s e concentrations
method )
( Fe rricyanide
20 The e f fect o f time between b lood col l ection and centrifugation on the stabil i ty of plasma and serum glucos e concentrations ( Gl ucose Oxidase Me thod )
2 1 The e f fect o f t ime between b lood co l lection and centrifugation on the s tabi l i ty of p la sma and serum glucos e concentrations ( Oxygen
Electrode Me thod )
2 2 Mean p lasma glucose leve l s o f tria l cows ( Ferricyan ide Me thod )
2 8
3 1
3 2
3 3
3 4
4 2
4 3
4 4
4 8
L I S T OF FIGURES CONTINUED F igure
2 3
2 4
Mean p lasma glucose leve l s of the trial cows ( Gl ucose Oxidase Method )
Mean p lasma g lucose leve ls of trial cows ( Oxygen Electrode Method )
2 5 S tatis tically derived regre s s ion l ines o f the four betametha sone treatment groups and control group f rom day 5 to 12 ( Ferricyanide
ix .
P age
4 9
50
Method ) 55
2 6 S tati s tically derived regress ion l ines of the four betamethasone treatment groups and control group from day 5 to 12 ( Gl ucose
Oxidase Method) 5 6
2 7 S tat i s tically derived regre s s ion l ine o f the four betame tha some treatment groups and
control group from day 5 to 12 ( Oxygen E lec trode Method )
2 8 The theoretical relationship between b lood steroid concentration and the rate of
g lucogene s i s
2 9 The theoretical relationship between the t ime course of blood s teroid concentrations and the var ious concentrat ions of the in j ected s teroid dos e .
57
6 8
6 8
