THE ENZYMATIC DEAMINATION OF AMPHETAMINE
(BENZEDRINE)

By JULIUS AXELROD

(From the Laboratory of Chemical Pharmacology; National Heart Institute, National
Institutes. of Health, Public Health Service, United States Depariment of
Health, Education, and Welfare, Bethesda, Maryland)

Reprinted from Tun Jovnwa. or Brotoaica, Cammretry
Val, 214, No. 2, June, 1955

Made in United States af America
Reprinted from Tur JourNAL oF BioLogicaL CHEMISTRY
Vol, 214, No. 2, June, 1955
Made in United States of America

THE ENZYMATIC DEAMINATION OF AMPHETAMINE
(BENZEDRINE)*

By JULIUS AXELROD

(From the Laboratory of Chemical Pharmacology, National Heart Institute, National
Institutes of Health, Public Health Service, United States Department of
Health, Education, and Welfare, Bethesda, Maryland)

(Received for publication, November 23, 1954)

A previous study on the metabolism of amphetamine ( 1-pheny1-2-amino-
propane), widely used as a stimulant of the central nervous system, showed
that its major route of biotransformation in the rat and dog involved hy-
droxylation of the aromatic ring. In the rabbit, however, the compound
was not hydroxylated, but was found to be transformed in another man-
ner (2),

The present report describes an enzyme system in the rabbit which
deaminates amphetamine to yield phenylacetone. It will be shown that
the enzyme system is localized in the microsomal fraction of the cell and
requires oxygen and reduced triphosphopyridine nucleotide. Furthermore,
these studies indicate that there are inhibitory factors present in the liver
of the dog, rat, and guinea pig which may explain, in part, the inability of
these species to deaminate amphetamine.

Materials—t- and d-amphetamine sulfate was obtained through the cour-
tesy of the Smith, Kline and French Laboratories. [-Norephedrine hydro-
chloride was kindly supplied by K. H. Beyer of Sharpe and Dohme. Tri-
phosphopyridine nucleotide (TPN) and diphosphopyridine nucleotide
(DPN) 80 per cent purity, glucose-6-phosphate dehydrogenase, and glu-
cose-6-phosphate were obtained from the Sigma Chemical Company. Re-
duced triphosphopyridine nucleotide (TPNH) was prepared by the proce-
dure of Kaplan et al. (3).

Methods—Amphetamine, ephedrine, norephedrine, and methylampheta-
mine were determined by the methyl orange reaction of Brodie and Uden-
friend (4) with benzene as the extractant, as previously described (2, 5).
An essentially similar procedure was used for the estimation of 1-phenyl-1-
aminopropane, 1-phenyl-3-aminobutane, phenylethylamine, isoamylamine,
2-aminoheptane, and benzylamine with chloroform as the extractant.
Tyramine was estimated according to Udenfriend and Cooper (6) and p-

* A preliminary report of this work has appeared (1). The material in this in-
vestigation will appear in a thesis in partial fulfilment of the requirements for the
degree of Doctor of Philosophy in the Department of Pharmacology, Graduate
Council, George Washington University.

753
754 ENZYMATIC DEAMINATION OF AMPHETAMINE

hydroxyamphetamine according to Axelrod (2). Ammonia was estimated
by the Conway microdiffusion procedure (7).

Phenylacetone was determined by its reaction with 2,4-dinitrophenyl-
hydrazine as follows: An aliquot of biological material was transferred to a
40 ml. glass-stoppered centrifuge tube containing 0.5 ml. of 1 n NaOH
and 20 ml. of carbon tetrachloride (reagent grade) and shaken for 20 min-
utes. The tube was centrifuged and the aqueous phase removed by as-
piration. The carbon tetrachloride extract was washed with 5 ml. of
water to remove “blank” material which reacted with dinitrophenylhy-
drazine. 15 mil. of the carbon tetrachloride phase were transferred to a
40 ml. glass-stoppered centrifuge tube containing 5 ml. of 0.1 per cent
2,4-dinitrophenylhydrazine in 2 Nn HCl! and shaken for 2 hours. The
aqueous phase was removed by aspiration and the organic phase washed
three times with 5 ml. portions of 2 Nn HCl to remove unchanged dinitro-
phenylhydrazine. 6 ml. of the carbon tetrachloride extract containing
the dinitrophenylhydrazone derivative of phenylacetone were transferred
to a cuvette containing | ml. of 10 per cent KOH in ethanol, and the optical
density was read in a spectrophotometer at 430 mp. A blank consisting of
biological material carried through the procedure was set at 100 per cent
transmission. Standards were prepared by adding a known amount of
phenylacetone to biological material and proceeding as above. Phenyl-
acetone added to biological materials in amounts from 0.5 to 5 wmoles are
recovered with adequate precision 90 -+ 6 per cent.

Preparation of Tissue Samples—The preparation of all tissue samples
was carried out at 0-3°.

Male albino rabbits were stunned and exsanguinated. The livers were
immediately removed and homogenized with 2 volumes of 0.1 M phosphate
buffer, pH 7, with a Potter-Elvehjem type of homogenizer. The homog-
enates were centrifuged at 9000 X g for 10 minutes to remove unbroken
cells, nuclei, and mitochondria, and the resulting supernatant fraction was
dialyzed against 0.01 m phosphate buffer, pH 7.0, for 20 hours. The super-
natant fraction could be stored for at least 2 weeks at —10° without loss
of activity.

The particulate fractions of liver were prepared by differential centrifu-
gation of 12 per cent homogenates in 0.25 m sucrose (8). The nuclear
fraction was sedimented by centrifugation at 600 X g for 10 minutes; the
mitochondria at 9000 X g for 10 minutes; microsomes were separated from
the soluble supernatant fraction by centrifugation at 78,000 x g for 45
minutes. The microsomes were washed once with 0.25 m sucrose and re-
centrifuged. Particulate fractions were resuspended in a 0.1 m phosphate
buffer, pH 7.0, to a concentration 3 times that of the original homogenate.
The particulate and soluble supernatant fractions were dialyzed against
0.01 m phosphate buffer, pH 7.0, for 20 hours.
J. AXELROD 755

Measurement of Enzyme Activity—A typical incubation mixture was pre-
pared as follows: In a 20 ml. beaker were placed 0.3 ml. of enzyme prep-
aration (equivalent to 100 mg. of liver), 5 wmoles of nicotinamide, 0.1
umole of TPN, 5 wmoles of MgCh, 0.6 umole of Lamphetamine, 0.5 ml. of
phosphate buffer pH 7.4 (0.2 m), and water to make a final volume of 4
ml, The mixture was incubated in a Dubnoff metabolic shaking apparatus
for 2 hours at 37° in air, At the end of the incubation period an aliquot of
the reaction mixture was immediately transferred to a 60 ml. glass-stop-
pered bottle containing 0.5 ml. of 1 n NaOH and assayed for amphetamine.
Enzyme activity was expressed as micromoles of amphetamine metab-
olized.

After the incubation the residual amphetamine was identified by com-
paring its distribution ratios between buffers of various pH values and
benzene with those of authentic amphetamine. The results indicated that
the apparent amphetamine measured after incubation had the same solu-
bility characteristics as authentic amphetamine and that the compounds
were presumably the same.

Results

Tissue Distribution of Enzyme Activity—l-Amphetamine (0.6 ymole) was
incubated with 100 mg. of minced liver, lung, diaphragm, muscle, kidney,
and brain of the rabbit under the conditions described under “Methods.”
The liver metabolized 0.35 umole of l-amphetamine in 2 hours, while the
other tissues were unable to metabolize the drug.

Properties of Enzyme System—An undialyzed supernatant fraction of
rabbit liver failed to metabolize l-amphetamine in appreciable amounts
without the addition of nicotinamide and either TPN or DPN. Nicotin-
amide presumably served to protect the pyridine nucleotides against
enzymatic destruction (9). The effect of the two pyridine nucleotides
was then examined in a supernatant fraction of rabbit liver dialyzed for
20 hours. It was found that the activity of the dialyzed supernatant
fraction was restored after the addition of TPN, while DPN was inactive
(Fig. 1). Mg** stimulated activity about 10 per cent. Negligible activity
was observed under anaerobic conditions, but incubation in air was as
effective as incubation in oxygen. In phosphate buffers the maximal en-
zyme activity occurred at pH 7.4, and the activity was sharply reduced
below and above this pH.

Rate of Metabolism—The rate of metabolism of amphetamine by the
rabbit supernatant fraction is shown in Fig. 2. About 50 per cent of the
drug was metabolized in 3 hours, with no further metabolism after this
time. To examine the lability of the enzyme, the rabbit liver supernatant
fraction was preincubated at 37° for 3 hours. On the addition of Lam-
phetamine, no metabolism of the drug took place.
756 ENZYMATIC DEAMINATION OF AMPHETAMINE

The relationship between substrate concentration and substrate dis-
appearance is shown in Table I. At concentrations of amphetamine vary-
ing from 0.6 to 2.4 wmoles a constant amount of the drug was metabolized.

Intracellular Localization of Enzyme System—Nuclear, mitochondrial,
microsomal, and soluble supernatant fractions of the rabbit liver were sep-
arated by differential centrifugation (8) and assayed for their capacity to
metabolize l-amphetamine. Negligible enzyme activity was observed in
each cellular fraction (Table IT). However, when the microsomal and
soluble supernatant fractions were combined, the drug was metabolized

 

 

0.3

TPN

0.2 0.2

    

 

 

 

 

 

pM AMPHETAMINE METABOLIZED
BM AMPHETAMINE METABOLIZED

0! 7 O.bK
DPN
0 l ! 1 L t ! t ! !
0.02 0.06 G.10 0 { 2 3 4
#M TPN OR DPN ADDED TIME IN HOURS
Fia. 1 Fig. 2

Fie. 1. Requirement for TPN. 0.3 ml. of dialyzed rabbit liver supernatant frac-
tion was incubated for 2 hours at 37° with 0.6 umole of /-amphetamine, varying
amounts of TPN or DPN, and cofactors described under ‘‘Methods.”’

Fic. 2. Rate of metabolism of /-amphetamine. Six beakers, each containing 0.3
ml. of rabbit liver supernatant fraction, were incubated at 37° with 0.6 umole of
l-amphetamine and cofactors described under ‘‘Methods.”’

almost as effectively as by the whole homogenate. From these observa-
tions it was evident that factors present in both the microsomes and the
soluble supernatant fraction were required to carry out the metabolism of
amphetamine.

Requirement for TPNH—The possible réle of the soluble supernatant
fraction in the metabolism of amphetamine was examined by adding a
number of substrates normally present in this fraction to the microsomes.
It was found that by replacing the soluble supernatant fluid with TPN and
either glucose-6-phosphate or isocitric acid, unwashed microsomes metab-
olized amphetamine; washed microsomes in the presence of these factors
were unable to metabolize the drug. These observations suggested that
TPN-dependent dehydrogenases associated with the unwashed micro-
somes were involved in the enzymatic conversion of amphetamine. In
J. AXELROD 757

the presence of glucose-6-phosphate, glucose-6-phosphate dehydrogenase,
and TPN, washed microsomes metabolized amphetamine (Table III).
These results suggested that reduced TPN was the actual cofactor and. that

TaBLe I
Relationship between Substrate Concentration and Substrate Disappearance

0.3 ml. of rabbit liver supernatant fraction was incubated at 37° for 2 hours with
various amounts of /-amphetamine and cofactors described under “Methods.”

 

 

 

i-Amphetamine added !-Amphetamine metabolized
pmoles umole
0.12 0.12
0.30 0.20
0.60 0.25
1.20 0.24
2.40 0.22
Tase II

Intracellular Distribution of Amphetamine Deamination Activity

Various intracellular fractions were prepared from rabbit liver by differential
centrifugation as described under ‘‘Methods.”’ Aliquots of the various fractions
equivalent to that contained in 100 mg. of whole rabbit liver were incubated at 37°
for 2 hours with 0.6 umole of /-amphetamine and cofactors described under ‘‘Meth-
ods.”’

 

 

Intracellular fraction l-Amphetamine metabolized

umole
Whole homogenate. .......... 000.00 0.27
Nuclei*¥. 00. e nent e ees 0.06
Mitochondria... 0... 0... e ees 0.00
Microsomes. ... 2.6... cnn ent een ne ene 0.03
Soluble supernatant. .........0...0 0000s cece eee eee eee 0.00
Nuclei* and soluble supernatant...................00008. 0.08
Mitochondria and soluble supernatant................... 0.02
Microsomes and soluble supernatant..................... 0.20

 

* This fraction contained unbroken cells and red blood cells as well as nuclei.

TPN-dependent dehydrogenases in the soluble fraction of the cell served
to generate this cofactor. To confirm this, the effect of chemically pre-
pared TPNH on the metabolism of amphetamine by microsomes was ex-
amined. Reduced TPN was found to be as effective as the supernatant
fraction in promoting the metabolism of amphetamine by the washed
microsomal fraction of rabbit liver (Table III). DPNH could not replace
TPNH. From these results, it appears that the supernatant fraction
758 ENZYMATIC DEAMINATION OF AMPHETAMINE

served to maintain a reservoir of reduced TPN by catalyzing the oxidation
of glucose-6-phosphate and other substrates and that the deamination of
amphetamine was mediated by a TPNH-dependent enzyme located in the
microsomal fraction of the rabbit liver.

Amphetamine Disappearance and Phenylacetone Formation—Supernatant
fluid obtained from 20 gm. of rabbit liver was incubated at 37° for 1 hour
with 100 ymoles of nicotinamide, 10 umoles of TPN, 100 umoles of MgClo,
120 pmoles of l-amphetamine, 10 ml. of phosphate buffer, pH 7 (0.2 m),
and examined for the presence of phenylacetone, the deaminated metabo-
lite of amphetamine. Apparent phenylacetone formed in the enzymatic

Tass IIT
Requirement for Reduced TPN

0.3 ml. of liver microsomal! fraction, 0.6 umole of l-amphetamine, 5 umoles of
MgCh, 5 umoles of nicotinamide, 0.5 ml. of 0.2 m phosphate buffer, pH 7.4, added
cofactors, and water to make a final volume of 4 ml. were incubated at 37° for 2 hours.

 

 

Cofactors added i-Amphetamine metabolized

pmole

TPN 1 wmole*. 2... tenes 0.01

‘© 0.1 pmole, glucose-6-phosphate 5 wmoles............ 0.03
« a1“ a 5 “ — glucose-6-

phosphate dehydrogenase 1 mg.............0.......... | 0.25

TPNH 1 umole*.... 20. cee 0.26

DPNH 1 6 ooo eects 0.03
Soluble supernatant from 100 mg. of rabbit liver + TPN

OL pmole... 2. eee ee 0.26

 

* These cofactors were added in five divided portions over a period of 90 minutes.

deamination of amphetamine was extracted from the incubation mixture
with carbon tetrachloride and treated with 2 ,4-dinitrophenylhydrazine as
described under ‘“Methods.”’ The absorption spectra of the hydrazone of
the enzymatically formed phenylacetone and that of the hydrazone of an
authentic sample of phenylacetone were identical. Further evidence for
the identity of the hydrazone of phenylacetone was obtained by ascending
paper chromatography with water, tbutanol, and n-butanol (6:6:5) as
the solvent system. The apparent and authentic hydrazones of phenyl-
acetone were found to have the same FR» (0.70).

The quantity of phenylacetone in the enzymatic deamination of Lam-
phetamine was equivalent to only about one-half that of the /-ampheta-
mine metabolized (Table IV). However, phenylacetone incubated with
rabbit microsomes and TPNH was also metabolized. On incubation of
0.5, 1, and 2 ymoles of phenylacetone with rabbit liver microsomes, about
J. AXELROD 759

the same percentage of the phenylacetone disappeared. It was possible,
therefore, to make an approximate correction for the metabolism of phenyl-
acetone formed in the enzymatic deamination of l-amphetamine. The re-

TaBLe IV
Enzymatic Deamination of l-Amphetamine to Phenylacetone

Each beaker containing 1.5 ml. of rabbit liver microsomal fraction, 25 ymoles of
MgCh, 25 umoles of nicotinamide, 2 ml. of phosphate buffer, pH 7.4 (0.2 m), added
cofactors, substrates, and water to make a final volume of 10 ml. was incubated at
37° for 2 hours. Experiments 1 and 1A contained 3 ymoles of TPNH added in five
divided portions over a period of 90 minutes. Experiments 2 and 2A contained 10
nmoles of glucose-6-phosphate, 1 mg. of glucose-6-phosphate dehydrogenase, and
0.3 pmole of TPN.

 

 

a Calculated
Experi- Amphetamine] Phenylace- eny!- amount 0
ment No. Substrate added metabolized | tone found m acetone ed pheny|-
formed
moles umole per cent umoles
1 l-Amphetamine 4 umoles 1.26 0.63 1.21
1A Phenylacetone 1.0 umole 48
2 i-Amphetamine 4 pmoles 1.10 0.66 1.02
2A Phenylacetone 1.0 umole 35

 

 

 

 

 

Z 190 T t T T T |
= [~ 8:

2 HEATED RAT
° 160/- MICROSOMES 7
W130; 4
Ke

+ 100

ul

a

b 70h 4
ws A. _|
Qo 40r @ UNTREATED RAT
ti MICROSOMES
2 19 ! 1 Lol

 

0 2 4 6 8 10

RATIO OF RAT MICROSOMES TO
RABBIT MICROSOMES

Fig. 3. Inhibition and activation of amphetamine-deaminating activity of rab-
bit liver microsomes by rat liver microsomes. A, each beaker contained 0.6 umole
of l-amphetamine, 0.1 pmole of TPN, 5 ymoles of MgCh, 5 wmoles of nicotinamide,
0.5 ml. of phosphate buffer, pH 7.4 (0.2 M), microsomal fraction and soluble super-
natant fraction obtained from 100 mg. of rabbit liver, and varying amounts of micro-
somes obtained from rat liver. The reaction mixture was incubated at 37° for 2
hours. 8, same as A, but rat microsomes were preheated for 2 minutes in a boiling
water bath.
760 ENZYMATIC DEAMINATION OF AMPHETAMINE

sults shown in Table IV indicate that about 1 mole of phenylacetone was
formed for each mole of amphetamine metabolized.

From 50 to 100 per cent of the theoretical amount of ammonia was
found to be liberated in the enzymatic deamination of amphetamine, but,

TaBLe V
Substrate Specificity
0.3 ml. of rabbit liver supernatant fraction was incubated at 37° for 2 hours with
0.6 pmole of substrate and cofactors as described under “Methods.’’? The reaction

mixture was examined for the amount of substrate remaining. In the absence of
TPN none of the substrates were metabolized.

 

 

 

Substrate Relative activity
per cent
l-Amphetamine.......0.....0 000000 e cece ees 100
d-Amphetamine...........0.0 00002 c eee eee 27
l-p-Hydroxyamphetamine................ 000.000 ee seers 14
l-Ephedrine........0 200.0002 cee 150
d-Ephedrine......0.0.0.0 00000 cece eect eens il
l-Norephedrine......... 00.0000. 2 eee eee 70
di-Methylamphetamine................0. 00000 e eee eee 77
dl-\-Phenyl-l-aminopropane..........-.....-...-25.0005- 40
dl-1-Pheny]-3-aminobutane..........-..-..-0-00-02 00000 70
Phenylethylamine*.............0. 0560600000 e eee eee eee 30
dl-a-Phenylethylamine*.. 0.0.00... 000.0 c eee eee 27
Tyramine*... 0.000. 0e enee 0
Benzylamine*.....................008 cence eee nee ee 0
2-Aminoheptane..........00.0 00.0600 c eet eee 20
Isoamylamine*.........0.0 00000 e eee eee eee 0

 

* The supernatant fluid was preincubated at 37° for 10 minutes with 10-* m iso-
propylisonicotinylhydrazine before the addition of substrates metabolized by mono-
amine oxidase. At this concentration isopropylisonicotinylhydrazine completely
inhibits monoamine oxidase activity (10) without effecting amphetamine-deaminat-
ing activity.

owing to the relatively large amounts of endogenous ammonia also formed
by the enzyme preparations, an accurate balance study could not be made.

Enzyme Activity in Various Species—The activity of the amphetarhine-
deaminating enzyme was examined in dog, guinea pig, and rat liver super-
natant fraction. Only small amounts of enzyme activity were found in
these species compared to the rabbit. The possibility was entertained
that the low degree of deamination activity resulted from inhibitory factors
in microsomes. This was examined by measuring the effect of microsomes
of dog, guinea pig, and rat liver on the enzyme activity of rabbit micro-
somes. The activity of the rabbit liver microsomes was markedly de-
J. AXELROD 761

pressed in the presence of the microsomes of these species. On the other
hand, the rabbit microsomal enzyme was not inhibited by the soluble
supernatant fractions of dog, guinea pig, or rat liver. These observations
indicated that an inhibitory factor was present in the microsomes of dog,
guinea pig, and rat liver. Fig. 3, A shows the inhibitory action of rat
microsomes on the amphetamine-deaminating activity of rabbit liver
microsomes. When the microsomal fraction of rat liver was heated at
100° for 2 minutes prior to its addition to the rabbit enzyme, a marked
stimulation of enzyme activity was observed (Fig. 3, B). These results
indicate the presence of a heat-stable activating factor which is masked
by a heat-labile inhibitory factor. Preheated microsomes of dog, guinea
pig, and rabbit liver also possessed stimulatory activity.

Substrate Specificity—The metabolism of a number of amines by rabbit
liver supernatant fraction is recorded in Table V. Substrates having a
phenylpropylamine or phenylbutylamine structure were extensively me-
tabolized and the enzyme preparation showed relative specificity for the
levo isomers. None of the amines were metabolized in the absence of
TPN. It appeared that phenolic-substituted amines, phenylethylamines,
and aliphatic amines were metabolized slightly or not at all. It was pos-
sible that the arylamines could also be metabolized by hydroxylation of
the aromatic nucleus. However, it seemed unlikely that the phenyliso-
propylamines were hydroxylated in the rabbit, since it was demonstrated
that this species could not hydroxylate these amines in vivo (2, 5). It
seemed likely that the rabbit liver microsomal enzyme system metabolized
the amphetamine analogues by deamination.

DISCUSSION

On the basis of the studies described in this paper, the over-all reaction
for the enzymatic deamination of amphetamine is represented in the ac-
companying scheme. Amphetamine is deaminated to phenylacetone and
ammonia in the presence of reduced TPN and oxygen by an enzyme sys-
tem which is localized in the microsomal fraction of the rabbit liver. A
reservoir of reduced coenzyme is maintained by TPN-dependent dehydro-
genases and their oxidizable substrates present in the soluble supernatant
fraction.

H; H Fiz

C—C— CH; C—-C— CH; + NH;

 

NH, TPNH oO
762 ENZYMATIC DEAMINATION OF AMPHETAMINE

The enzyme which deaminates amphetamine differs from other deami-
nating enzymes such as monoamine oxidase, D-amino acid oxidase, L-amino
acid oxidase, and glutamic acid dehydrogenase with respect to its sub-
strate specificity, cellular localization, and cofactor requirements.

The réle of reduced TPN in an enzyme system catalyzing the oxidative
deamination of an amine is not understood. Reduced TPN could con-
ceivably act by the generation of hydrogen peroxide through the transfer
of its hydrogen by an intermediate electron transport system to molecular
oxygen. Preliminary studies indicate that hydrogen peroxide generated
from D-amino acid oxidase was unable to replace TPNH in the deamina-
tion of amphetamine by rabbit liver microsomes. The activating action
of heated rat microsomes suggests the requirement for other factors be-
sides TPNH. The nature of the additional cofactor is unknown.

Previous studies have shown considerable species differences in the me-
tabolism of amphetamine in vivo (2). The dog and the rat transformed
amphetamine mainly by hydroxylation, while the rabbit metabolized the
drug, presumably by deamination. The species differences in the metab-
olism of amphetamine may be explained in part by the presence of inhibi-
tory factors in the dog and rat. Inhibitory factors in these species may
act by blocking the deamination of amphetamine so that the drug is
excreted unchanged or metabolized by alternative pathways involving
hydroxylation.

It is becoming increasingly evident that enzymes in liver microsomes
which have a specific requirement for reduced TPN and oxygen are of
major importance in the detoxification of many drugs and foreign organic
compounds. Such enzyme systems have been found to carry out a variety
of reactions such as dealkylation of alkylamines (11, 12), side chain oxida-
tion of barbiturates,! cleavage of aromatic ethers,? and hydroxylation of
aromatic compounds (13). Another property common to these enzyme
systems is their inhibition by 6-diethylaminoethyl diphenylpropylacetate
(14, 15).

SUMMARY

An enzyme system in rabbit liver microsomes catalyzes the deamipation
of amphetamine to yield phenylacetone and ammonia. The enzyme sys-
tem requires reduced triphosphopyridine nucleotide and oxygen. The
TPN-dependent dehydrogenases in the soluble supernatant fraction of the
liver serve to maintain a reservoir of reduced triphosphopyridine nucleo-
tide.

Species differences in the metabolism of amphetamine may be explained,
in part, by the presence of inhibitory factors in the microsomes of the dog

1 Cooper, J. R., and Brodie, B. B., unpublished findings.
2 Axelrod, J., unpublished work.
J. AXELROD 763

and rat liver. A heat-stable factor which can stimulate the enzymatic
deamination of amphetamine is also present in the microsomes.

Ww be

1m oe

oO

11.
12,

13.
14.

15.

BIBLIOGRAPHY

. Axelrod, J., J. Pharmacol. and Exp. Therap., 110, 2 (1954).
. Axelrod, J., J. Pharmacol. and Exp. Therap., 110, 315 (1954).
. Kaplan, N. O., Colowick, 8. P., and Neufeld, E. F., J. Biol. Chem., 195, 107

(1952).

. Brodie, B. B., and Udenfriend, 8., J. Biol. Chem., 158, 705 (1945).

. Axelrod, J., J. Pharmacol. and Exp. Therap., 109, 62 (1953).

. Udenfriend, 8., and Cooper, J. R., J. Biol. Chem., 196, 227 (1952).

. Conway, E. J., Micro-diffusion analysis and volumetric error, London, 2nd edi-

tion (1947).

. Schneider, W. C., J. Biol. Chem., 176, 259 (1948).
. Mann, J. J. G., and Quastel, J. H., Biochem. J., 36, 502 (1941).
. Zeller, E. A., Barsky, J., Fouts, J. R., Kircheimer, W. F., and Van Orden, L. S.,

Experientia, 8, 349 (1952).

Axelrod, J., Federation Proc., 18, 332 (1954).

La Du, B. N., Gaudette, L., Trousof, N., and Brodie, B. B., J. Biol. Chem., 214,
741 (1955).

Mitoma, C., and Udenfriend, 8., J. Pharmacol. and Exp. Therap., in press.

Axelrod, J., Reichenthal, J., and Brodie, B. B., J. Pharmacol. and Exp. Therap.,
112, 49 (1954).

Cooper, J. R., Axelrod, J., and Brodie, B. B., J. Pharmacol. and Exp. Therap.,
112, 55 (1954).