692 
PHARMACOLOGY 
MATERIALS AND METHODS 
Experimental Procedures 
The experimental subjects comprised ran- 
domly selected groups of healthy animals of 
both sexes from each of six domesticated mam- 
malian species and chickens. A similar dose 
(0.66 mg/kg, calculated as free base) of d^-am- 
phetamine sulphate (Amfetasul, Pitman-Moore, 
Indianapolis, Ind.) was administered intrave- 
nously to each subject. Venous blood samples 
were collected in tubes, containing dipotassium 
etylenediamine tetraacetate, at fixed time inter- 
vals. Plasma was separated by centrifugation 
and stored at —10° pending analysis. The rate 
of elimination and extent of distribution of am- 
phetamine were studied in Shetland-cross pon- 
ies'', Toggenburg goats^°, Yorkshire swine^, New 
Zealand White rabbits*, mongrel dogs", Ameri- 
can Short-haired cats*' and Leghorn chickens^. 
Experiments were performed to determine quan- 
titatively the unchanged amphetamine, p-hy- 
droxyamphetamine and its conjugates in cumu- 
lative urine samples. A urine collection period 
which exceeded five half-lives of the drug in 
each species was employed. The volume and pH 
of the urine were measured. 
The amounts of unchanged amphetamine ex- 
creted in urine and bile of dogs and swine were 
measured. The influence of nephrectomy upon 
the rate of elimination and extent of distribu- 
tion of amphetamine was studied in nephrec- 
tomized dogs. Under pentobarbital anaesthesia 
eight randomly selected mongrel dogs were bi- 
laterally nephrectomized. The BUN values 
(mean ± S.E.) in five dogs 24 hours after ne- 
phrectomy were 88 ± 6.1 mg/100 ml (normal, 
10-30 mg/100 ml) . Amphetamine was adminis- 
tered 42-48 hours after surgical removal of the 
kidneys. 
The extent of plasma protein binding was de- 
termined in vivo by measuring the ampheta- 
mine concentrations in cerebrospinal fluid and 
plasma samples collected simultaneously after 
distribution was complete. The experimental 
subjects were seven healthy and eight nephrec- 
tomized dogs. 
Blood, free of drug, was collected in tubes 
containing dipotassium EDTA from donor ani- 
mals of the several species used in this study. 
Plasma was separated by centrifugation. The 
total plasma protein concentration was meas- 
ured by refractometry, the plasma was then 
stored at —10° until used in determining the 
extent of amphetamine protein binding (in 
vitro). 
Analytical Techniques 
The amphetamine concentration in the bio- 
logical fluids was measured by a sensitive and 
specific gas chromatographic method.^^ Am- 
phetamine was extracted from alkaline biologic 
fluid into cyclohexane. The trichloroacetamide 
derivative was prepared with trichloroacetyl i 
chloride in the organic phase. This derivative | 
was chromatographed on 3 % OV-1 and detected | 
by electron capture (Beckman Gas Chroma^ j 
tograph. Model GC-5). The concentration of j 
amphetamine in biological fluids of medicated 
animals was estimated by comparing sample | 
peak heights with standard peak heights on the i 
chromatogram. The standard solutions were ana- i 
lyzed in the same manner as the samples to I 
compensate for losses occurring in the proce- j 
dure. 
The concentration of p-hydroxyamphetamine i 
in urine was determined by the colorimetric i 
method of Axelrod.^ The glucuronide and sul- ^ 
phate conjugates of p-hydroxyamphetamine ' 
were assayed as the latter following incubation I 
of urine with appropriate enzyme in a suitable j 
buffer. Glucuronide conjugate: to 3 ml 0.2 M y 
acetate buffer (pH 4.6) was added 3 ml urine | 
and 50 mg /3-glucuronidase (beef liver, salt > 
free, 70,000 to 100,000 units per 10 g; Nutri- i 
tional Biochemicals Corporation, Cleveland, 5 
Ohio). Sulphate conjugate: to 3 ml 0.2 M ace- [ 
tate buffer (pH 5.0) was added 3 ml urine and 1 ; 
unit sulfatase (Sulfatase, Type III: from Lim- i 
pets; Sigma Chemical Company, St. Louis, 
Mo.). The mixtures were incubated for one I 
hour at 37° in a Dubnoff metabolic shaking 
incubator. Following this period the total p-hy- 
droxyamphetamine concentration was deter- ! 
mined in 2.5 ml of the incubated mixture. The i 
concentration of conjugate was estimated by 1 
difference between total and unconjugated hy- f' 
droxyamphetamine concentrations. j 
The equilibrium dialysis technique using 
^H-cZ-amphetamine sulphate (New England 
