Investigations have been carried out in three separate areas
of research related to epilepsy.I. In the first of these investigations an attempt was made to
clarify a possible role of folic acid derivatives in epilepsy.
This investigation took the form of two self- contained studies
involving dogs.A. The first study concerned the effects of anticonvulsant
treatment on
(a) folate activity in cerebrospinal fluid (c.s.f.).
(b) the concentrations of 5- hydroxyindol- 3- ylacetic
acid (5 -HIAA) and hornovanillic acid (HVA) in c.s.f.Routine sampling of ventricular and cisternal c.s.f.
in the dog was achieved by the implantation of permanent
guide tubes to the lateral ventricle and the cisterna magna.
5 -HIAA and HVA in c.s.f. samples were estimated by fluorescence assay techniques. Folate activity in c.s.f. and
plasma samples was estimated microbiologically with a folatedependent strain of 1. casei.In control studies the c.s.f. folate activity was found
to be between 2 and 7 times higher than the plasma folate
activity. A positive correlation was found between c.s.f.
and plasma folate activities. HVA and 5 -HIAA studies confirmed the concentration gradient which exists for these two acids between the lateral ventricular c.s.f. and the
cisternal c.s.f.Selected anticonvulsant drugs (diphenylhydantoin,
7.5 mg/kg; phenobarbitone, 15 mg/kg; sulthiame 15 mg/kg;
carbamazepine 15 mg /kg) were administered daily to the dogs
for a period of five weeks. During this Period no change
was seen in the folate activity of either c.s.f. or plasma.
The anticonvulsants were also without effect on the concentrations of HVA and 5 -HIAA in c.s.f.These findings are discussed in relation to the folate
deficiency states produced in man by chronic anticonvulsant
therapy.B. The second study in the folate investigations was
concerned with the effect of oral folic acid administration
(a) folate activity in plasma, plasma ultrafiltrate
and c.s.f.
(b) the concentrations of HVA and 5 -HIAA in the c.s.f.During the control period of this study no correlation
was found between c.s.f. and plasma folate activity, such as
was found in the first study. Possible reasons to account
for this discrepancy are discussed. Estimation of folate
activity in plasma and plasma ultrafiltrate confirmed that
much of the folate activity in dog plasma is protein- bound.Folic acid (31 mg/kg) was administered daily to dogs
over a 5 -week period. During this treatment period the
mean plasma folate activity was 10 times higher than normal;
plasma ultrafiltrate folate activity was 5 times higher the
normal; but c.s.f. folate activity was unchanged. These
findings were interpreted as evidence for an increased plasma
level of a folate derivative, probably folic acid itself,
which was more strongly bound to protein than normal plasma
folate derivatives, and was unable to enter the c.s.f. In
two experiments in the rabbit no evidence was found for the
active transport of methyl -tetrahydrofolate (normally the
predominant form of folate in the plasma) into the c.s.f.
HVA and 5 -HIAA concentrations in the c.s.f. were unaltered
during the period of folic acid treatment. It was concluded
that oral folic acid administration is unlikely to have any
significant effect on normal cerebral function.The implications of this study are discussed in relation
to
(a) the role of folic acid derivatives in epilepsy
and mental illness.
(b) the mechanisms responsible for the normal distribution of folate activity between c.s.f. and plasma.II. The second investigation concerned the effects of barbiturate
anaesthetics and anticonvulsant drugs on the c.s.f. potassium
fluxes of the conscious dog.A new technique was developed for "open" perfusion of the
cerebroventricular system of the conscious dog. The dog was
perfused from lateral ventricle to cisterna magna with artificial
r c.s.f. containing inulin, and tracer amounts of 42K. K, total
potassium and inulin assays were performed on the inflow and out - flow fluids. From this data it was possible to calculate, for
each sample of outflow fluid collected, values for both the potassium efflux from, and the potassium influx into, the c.s.f.
After a period of between 65 -100 minutes of perfusion a "steady - state" was reached in which the potassium fluxes, as measured by
this technique, were relatively constant. Drugs actions were
studied by administering the drug during the "steady- state" period
and ascertaining the effect upon the "steady- state" potassium
fluxes.It was shown that -
(1) The barbiturate anaesthetics, sodium pentobarbitone and
o sodium thipentone, in doses sufficient to induce light
anaesthesia, may depress both potassium influx into, and
potassium efflux from c.s.f. Both fluxes were affected
equally on any one occasion. Flux alterations of up to
50% were observed.
(2) Diazepam has a consistent depressant effect on the
potassium fluxes of the c.s.f. This effect was less marked,
but more consistently observed, than in the case of the
barbiturate anaesthetics.
- v -
(3) Diphenylhydantoin has no significant effect on potassium
efflux from the c.s.f. but may have a slight depressant
effect on potassium influx into the c.s.f.
(4) Paraldehyde in a dose sufficient to induce light
anaesthesia slightly increases the c.s.f. potassium fluxes.
These results are discussed in relation to
(a) previously published studies on c.s.f. potassium
fluxes in anaesthetised animals
(b) possible mechanisms of action of these drugs.The effects of the barbiturate anaesthetics and diazepam were
interpreted as being secondary to a decrease in potassium exchange
between brain intracellular and entracellular compartments.It was concluded that the anaesthetic or anticonvulsant
action of a drug is not related to the drug's effect on c.s.f.
potassium fluxes.III. The third investigation concerned the development and preliminary application of a method for the analysis of GABA and
associated amino acids in small areas of tissue from the primary
and secondary epileptic foci of rats with a cobalt- induced epileptogenic lesion.The existing specific enzymic methods for GABA analysis were
examined and found to be either too insensitive or too complex to
act as suitable micro- methods. The standard techniques for amino
acid analysis, involving column separation and colorimetric estimation were also considered to be too insensitive.Reaction of the amino group of amino acids with 1- dimethyl- amnonapthalene-5- sulphonyl chloride (dansyl chloride) produces stable,
highly- fluorescent derivatives which can be readily separated by
thin -layer chromatography.A highly sensitive isotope dilution assay, based on the preparation of dansyl derivatives, was evolved for the estimation of
free amino acids in brain tissue.The tissue was homogenised in perchloric acid and known amounts
of ¹⁴C -amino acid standards were added to an aliquot of the perchlorate extract to act as internal standards for recovery of
amino acid through the method. The extract was then taken to pH
9.3 - 9.8 with potassium carbonate solution, precipitating perchlorate as its insoluble potassium salt. An aliquot of the
alkaline extract was dansylated with an equal volume of H³-dansyl
chloride in acetone. After a 30 minute reaction period the
dansylation mixture was taken to dryness and extracted with acetone:
glacial acetic acid solution (3:2). An aliquot of this extract
was applied to a polyamide micro- chromatography plate which was
then developed in two dimensions. Under u.v. light specific
dansyl -amino acid spots were identified by their characteristic
position on the developed plate. These spots were cut from the
plate and counted for ³H and ¹⁴C activity in a liquid scintillation
counter.The total amount of dansyl-amino acid in a spot was calculated
from its ³H activity. Combining this figure with the ¹⁴C activity
of the spot allowed calculation of the specific activity (w.r.t.
¹⁴C) of the dansyl-amino acid in the spot. Comparison of this
specific activity with the specific activity of the ¹⁴C -amino acid
standard which had been added as a recovery standard gave a measure
of the dilution of exogenous radioactive amino acid by endogenous
non-radioactive amino acid. From this dilution factor it was
possible to calculate the concentration of endogenous amino acid
in the original perchlorate extract of brain tissue. Determination of the weight of protein precipitated from the tissue sample
by the perchloric acid extraction allowed calculation of the amino
acid content of tissue in terms of }moles amino acid per 100 mg
protein.The two dimensional chromatography did not achieve a separation
of dansyl glutamine and dansyl -threonine; as a result glutamine
and threonine were estimated as the sum total of their two
concentrations.The method was applied to the estimation of control values
for GABA, glutamate, glutamine/threonine, glycine, aspartate and
hydroxyproline levels in rat cortex. The values obtained for
GABA, glutamate, glutamine /threonine and glycine were highly reproducible and in good agreement with previously published values.
Aspartate was not satisfactorily estimated. Hyroxyproline was
not detected in rat cortex.The dansylation method was applied to the analysis of brain
tissue from three rats with 11 day-old cobalt-induced epileptogenic
lesions in the frontal cortex. There was evidence that GABA and
glutamate levels were lowered, and glutamine/threonine and glycine
levels raised, in the region of the secondary epileptogenic
( "mirror ") focus. This was the site from which epileptiform spike
discharges were most frequent.The significance of these findings have been discussed along
with general comments on the problems of amino acid studies in
brain. It was concluded that disorders in amino acid metabolism
may be involved in epileptic processes
