الفهرس 
Material and methodsOnly 14 pages are availabe for public view
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Abstract
Fractionation of N. nigricoJJis, N. nivea, N. meJanoJeuca
and N. haJe venoms using ion-exchange chromatography on Amberlite
CG-50 and ammonium bicarbonate elution gradient, yielded, 13, 12,
11 & 7 different fractions respectively. There were 2, 4, 4 & 2
lethal fractions for N. nigricoJJis, N. nivea,
N. haJe venoms.
N. nigricoJJis venom caused heamorrhagic patched at the site
N. meJanoJeuca &
of inj ection, lungs and stomach; this activity was only shown
with some of the other venoms fractions when injected
doses.
in higher
The i.m LD”,., in mice were 0.235::,0.042, 0.45::,0.073,
1.75::,0.154 & 3.0::,0.269 mg/kg for N. melanoleucd, N. n i v e e , N.
haJe and N. nigricoJJis venoms respectively. The dose mortality
curves for the 4 venoms were nearly parellel, revealing a possible
similarity in the causes of death.
The 4 NaJa venoms studied showed similar effects on the isolated
guinea-pig heart and atrium. The venoms caused marked negative
inotropic and chronotropic effects followed by contracture
which terminated in complete cardiac arrest. The effects were not
reversed by extensive washing and not blocked by either
hexamethonium, or atropine excluding the possibil ity of a
cholinergic component in their action. Calcium chloride, however,
completely antagonised the venom-induced contracture and cardiac
depression. It was concluded that binding of the cardiotoxins in
the venoms is inhibited by increased Ca++.
The most common electrocardiographic effects of the venoms
were, bradycardia, AV-block, ischemia and inferior wall in farction.
The bradycardia is probably due to the direct depressant
effect of the venoms as was evidenced from the results on the
isolated preparations • Various types of AV-block were produced.
.First degree heart block in the early phases and second degree
heart block in the later phases of envenomation. This indicated
interference by the venoms of the conducting system at different
levels in the AV-bundle. Myocardial ischemia was evidenced from
depression of ST-segment particularly in I & aVL. This was supported
by the decrease in perfusion rate of coronary flow in the
isolated hearts. Inferior wall infarction was evidenced from the
elevation of ST segment in II, III & aVF in all venoms except N.
nivea where elevation the ST segment occured in III & Vl. In addition,
the T wave was inverted in different leads including I ,
II, aVL, Vl & V5. With the exception of N. nivea venom, the three
other venoms induced a tall peaked and slender T wave in all
leads indicating the presence of hyperkalemia. It is worth mentioning
here that N. nivea venom was the least potent in causing
electrocardiographic changes and in decreasing coronary flow. On
a weight bases, N. melanoleuca venom was the most potent in causing
the electrocardiographic effects.
Incubating venoms with their specific antivenoms completely
prevented venoms-induced cardiac
fects.
and electrocardiographic ef-
In the present study, some of the NaJa venoms caused a rise
or fall in the arterial blood pressure of the anaesthetized cat.
This may be due to release of medullary
tamine and or acetylcholine.
catecholamines,
I
his-
The action of Naja venoms on the nerve muscle pr parations
was mainly confined to the cholinergic neuroeffector tr nsmission
sites. The venoms bind strongly and specifically to the cholinergic
motor end plate and the blocking effect was not reversed by
washing. The Naja venoms block the transmission mainly by a postjunctional
mechanism. This was evidenced from the protection from
the blocking effect of the venoms by the competitive neuromuscular
blocking drugs, d-tubocurarine or gallamine. The protection
was related to the bulk of the drug, its better fit to the receptors
and the number of onium groups as better protection was
achieved with gallamine than with d-tubocurarine. The complete
protection from the block by succinylcholine was unexpected. This
effect is probably due to binding of succinylcholine to the
receptors and because .of its aliphatic chain residue, the drug is
expected to possess a very flexible molecule that can fit in the
cholinergic receptor better than the rather rigid mplecules of dtubocurarine
or gallamine. The block caused by the venoms was
mainly of the non-depolarizing type as was shown from the absence
of any contraction of the skeletal muscle preparations used on
addition of the venoms, the flaccid paralysis of the conscious
chicks injected with the venoms and that the intensity of
blockade was greater at higher frequencies of nerve stimulation
or following application of tetanus. Neostigmine caused a transient
reversal of the venom-induced blocking activity in the
phrenic nerve-hemidiaphragm and partial recovery of the response
to acetylcholine in rectus abdominis muscle. Tetraethlyammonium
did not antagonise the blocking action of the venoms on the
phrenic nerve-hemidiaphragm but restored a partial recovery after
venoms in the rectus abdomins preparation. The effect of both
drugs is probably caused by increasing the concentration of
acetylcholine which in turn antagonizes the competitive neuromuscular
blocking action of the venoms. These effects refer to a
possible presynaptic mechanism in the ac tion of the venoms.
neuromuscular
Choline did not antagonise the venom-induced
blockade in both the phrenic nerve-hemidiaphragm and rectus abdominis.
This suggests that NaJa venoms either do not act on
choline transport mechanism or that any action at this site is
none-competitive in nature. Naja venoms act partly through
depolarization as well maintained tetanus was obtained during
transmission failure. In this respect N. nigricollis venom caused
spastic paralysis in the conscious chick and contracture of the
phrenic nerve-hemidiaphragm. Also, both N. nigricollis and N. haJe
venoms caused contracture of the chick biventer cervicis muscle
probably due to their contents of cardiotoxins. The contracture
might also be due to the release of acetylcholine.
Lowering the Ca++ concentration in the Krebs solution enchanced
the blocking effect induced by the NaJa venoms on the
phrenic nerve-hemidiaphragm. The effects of N. nigricollis and N.
haJe venoms were particularly affected in this ~espect. On the
contrary, lowering the magnisum ion concentration caused a par-
tial protection from the venoms effect. The effect was particularly
significant in case of N. nigricollis and N. nivea
venoms. This indicates that venoms studied may interfer with the
release mechanism.
The most lethal fractions were those possessing neuromuscular
blocking effect; their blocking effect was prevented by
pretreatment of the muscle preparatons with d-tubocurarine or
gallamine denoting their a-neurotoxin nature. They are FII and FV
of N. haje, FII of N. nigricollis,
FVI of N. melanoleuca venoms.
Incubating the venoms with their spicific antivenoms,
FIV and FVII of N. nivea and
completely
prevented their neuromuscular blocking effect.
The 4 Naja venoms caused stimulation of the isolated guineapig
ileum, an effect which was subject to tachyphylaxis. The action
may be due to the release of either acetylcholine or histamine.
In higher concentrations all venoms decreased the size of
the twitches of the field stimulated guinea-pig ileum and the
response to subsequent doses of acetylcholine. Pretreatment with
morphine did not influence the inhibition. This indicates that
the venoms in’the doses used are either more potent than morphine
in inhibiting the release of acetylcholine, or they inhibit the
release of the transmitter by a mechanism different of that of
morphine. Only N. nigricollis venom antagonised the effect of
histamine on the isolated guinea-pig ileum. The antagonism is
likely due to the stimulation of the sympathatic nerve endings.
Naja venoms stimulated rat uterus in both de Jalon and
ringer locke solutions, an effect which was subject to
tachyphylaxis. The stimulant effect was not blocked by
hexamethonium, atropine, cyproheptadine or indomethacin excluding
the possibility of being mediated via
choline, serotonine or prostaglandins.
the release of acetyl-
The effect of N. nivea
venom on the spontaneously contracting rat uterus was an exception
since it was blocked by either cyproheptadine or indomethacin.
Also the effect of N. melanoleuca venom on the uterus
in de jalon solution was exception since it was blocked by indomethacin.
Meclofenamic acid completetly antagonised the spasmogenic
effects of all NaJa venoms studied which suggests that
NaJa venoms act through release of kinins or slow reacting substanc
(SRSA). All NaJa venoms attenuated the response of rat
uterus to serotonin. This may be attributed to an a-adrenergic
blocking effect.
NaJa venoms reduced the size of the twitches of the field
stimulated rat vas deference. They also attenuated the response
to the exogenously added norepinephrine. Pretreatment with indomethacin,
antagonised the inhibitory effect of N. melanoleuca
venom. This indicates that the action of the venom be due to
release of prostaglandines. In the reserpine treated rat vas
deferens, the inhibitory effect of NaJa venoms was pronounced and
led to complete blockade of twitch activity. This effect is attributed
to the a-adrenergic blocking effect of the venoms.
Plotting of blood radioactivity of the labelled NaJa venoms
or their labelled toxins mixed with non-labelled venom vs time
revealed a triexponential behaviour characteristic of a three
compartment open-model. The values for the distribution halflives
(t~ n) associated with the rapidly equlibrating compartment
ranged from 3 to 5 minutes. These values reflect the very rapid
uptake of the venoms and their toxins by the shallow compartment.
On the other hand, the distribution half lives (t~ a) for the
slowly equlibrating compartment, ranged from 22 to 47 minutes,
thus indicating a much slower uptake. The overall elimination
half lives (t~ S) ranged between 15 and 29 hours. N.
had the shortest elimination half life (15 hours) among
haJe toxin
all the
venoms and toxins studied. The graphical estimation of the equilibrium
distribution ratios of the venoms and toxins concentrations
in the shallow compartment to the central compartment
(blood) are 0.5, 0.9; 0.8, 0.9; 1.6,1.0 and 1.7,1.0 for N.
melanolecua, N. nigricollis, N. nivea and N. haJe venoms and
their toxins respectively. This reveals a high affinity to the
shallow compartment in case of N. nivea and N. haJe venoms and
approximatly equal distribution of toxins in the shallow and
central compartments. The ratios of venoms and toxins concentration
in the deep compartment to the central compartment (blood)
are 3.7, 4.0; 3.3, 3.4; 3.0, 1.7 and 1.3,2.7 for N. nigricollis,
N. melanoleuca, N. nivea and N. haJe venoms and their toxins
respectively. These values reflect the much higher affinity of
the venoms and toxins to the deep compartment than to either the
central or the shallow compartments. N. haje venom and N. nivea
toxin showed the least affinity to the deep compartment compared
to all venoms and toxins studied.
The peak tissue levels in the
shallow compartment was reached within 20-25 minutes following
injection. However, most effects started 30-60 minutes. This indicates
that the possible site of action of venoms or toxins are
probably not located in the shallow compartment and would refer
to the deep tissue compartment as the probable site of effect, as
the peak tissue level in the deep compartment was reached 100-200
minutes following venom or toxin injection.
The highest radioactivity was found in the thyroid gland
followed by kidney, liver, lung and heart. Also very high
radioactivity was found in the urine,
bile and stomach contents.
The very high activity found in the stomach contents would refer
to the stomach as a possible route for elimination of the venoms.
Also the high radioactivity found in the urine would refer to the
kidney as the principal route for excretion and the bile as the
next major route for elimination. All venoms and toxins showed a
very high radioactivity in the thyroid gland except N. haje venom
and its toxin which showed much lower values.
This may be due to
inhibition of the concentration mechanism for iodide by the
thyroid tissue or to the possibility of their low metabolism.
All venoms caused significant decrease in the gastric volume
and free and total acidity together with a significant increase
in the peptic activity. The ulceration and hemorrhagic patches
observed follOWing venom injection are probably due to the increased
peptic activity in all venoms and
decrease of mucin.
the significant
Hyperimmunizing rabbits against N. nigricollis, N.
mel.anoleuca, N. nivea and N. haje venoms or a mixture of the 4
venoms over a period of 4-8 months, yielded specific and
polyvalent antivenoms of high potency. Using the immunodiffusion
experiments, precipitin bands were obtained with venoms or venom
fractions in concentrations as low as 10 mg/ml and using serum
dilutions up to 1: 8. Also each antivenom showed definite
and the
precipitin bands with its own venom and its fractions,
other venoms and their fractions. This indicates the presence of
common antigens in the venoms. The antivenoms and the immunoglobulins
separated were highly effective in neutralizing all
the pharmacological effects induced by venoms. They also
protected the mice against lethal doses equivalent to 5-50 times
the LD~(:I.
Except N. nivea vemom, the other three venoms casued hyperhypourecemia,
hyper-uremia, hypophosphatimia
hypocalcemia. The hyperglycemia could not be due
and
to
The
glycogenolysis since the rabbits were fasted for 17 hours.
gluconeogenesis appeared to come mainly from lipolysis since urea
level was not altered at times where blood glucose level was
elevated. N. nivea venom on the other hand caused hypoglycemia,
hyperurecemia, hypocalcemia and hypophosphatemia; serum urea and
creatinine were not altered. This indicated that gluconeogenesis
occurs mainly from lipolysis and not from amino acids. This would
render the glucose generation lesser than in the case of the
other three venoms. Also no muscular damage Dccured wiht N. nivea
venom reflecting normal glucose utilization which coupled with
decreased generation would lead to hypoglycemia.
The decrease in serum uric acid caused by the 3 venoms is
likely due to inhibition of uric acid synthesis. On the other
hand the increased uric acid in case of N. nivea may be due to
some components in the venom which may interfer with uric acid
secretion in the renal tubules.
The effect of the 4 NaJa venoms on serum sodium, potassium,
calcium and phosphorous were nearly similar and reflects a picture
of inhibion of the adrenal and parathyroid glands. The effects
of the 4 NaJa venoms on total proteins, albumin and
globulins are more or less similar and consisted of significant
decrease possibly secondray to the haemorrhages induced in the
lungs, stomach and intestine. Albumin loss is likely to be of
renal origin.
The different NaJa venoms caused a gradual increase in serum
urea which was apparent only 12-24 hours. This increase was not
associated with any significant increase. in serum creatinine or
uric acid except with N. nive~ venom which caused an increase in
uric acid. This indicates that the effect of NaJa venoms in the
doses used on the kidney function is minimal.
The NaJa venoms, with the exception of that of N. nivea,
caused a significant increase in LDH, AST, ALT and CK activities
reflecting a picture that correlates very well with the inferior
wall infarction revealed in the electrocardiographic studies. The
effect on CK activity reflects the action of the venoms on both
myocardial and skeletal muscles. On the contrary, N. nivea venom
decreased serum LDH, AST, ALT and CK activity, although the venom
induced inferior wall infarction was evidenced from the results
of the electrocardiographic studies. It is possible that N. nivea
venom inhibited the activity of enzymes after their release by
some components or released factors.
All
OnIy N.
venoms seemed to increase ALT and decrease ALK.Phos.
nigricollis venoms caused significant increased in
bilirubin level which is likely due to the hemolytic effect of
the venom. The effect of the venoms on the liver function appeared
to be non-significant.
All venoms increased serum chloride level. This may be due
to metabolic acidosis and decreased chloride shift. An indirect
evidence comes from the decreased volume and acidity of the
gastric juice following venom injection.