396 OCCURRENCE OF FREE AMINO ACIDS — VERTEBRATES 
FREE AMINO ACIDS IN THE BLOOD OF MAN AND ANIMALS 
IV. EFFECTS OF METHYL(BIS)§-CHLOROETHYLAMINE 
(NITROGEN MUSTARD), 4-[f-BIS(2-CHLOROETHYLAMINOPHENYL)] 
BUTYRIC ACID (CHLORAMBUCIL) AND PHENYLHYDRAZINE 
GEORGE ROUSER, ARTHUR J. SAMUELS, KEIJI KINUGASA*, BOHDAN JELINEK 
AND DOROTHY HELLER 
Departments of Biochemistry and Medicine, City of Hope Medical Center, 
Duarte, Calif. (U.S.A.) 
This report presents the results of studies on the effects of nitrogen mustard and 
chlorambucil on the free amino acid levels of blood of patients with chronic lymphatic 
leukemia, and the effects of nitrogen mustard and phenylhydrazine on the blood and 
tissue free amino acids of rabbits. 
GENERAL METHODS OF STUDY 
The general methods for the separation of the blood constituents, extraction, and 
chromatographic examination are described in part I. Two carefully controlled studies 
were carried out in two different patients to determine the effects of nitrogen mustard 
and chlorambucil. The first patient (G. Cap. 4) had been followed for several months 
prior to treatment and was then given 0.7 mg of nitrogen mustard intravenously on 
4 consecutive days and the plasma and cell levels of free amino acids were followed 
for 3 months. 
A patient with chronic lymphatic leukemia (W. Ric. 3) was studied for the effects 
of chlorambucil after the oral administration of 2 mg/day for 1 week (total dose 
14 mg). The free amino acids of plasma and blood cells were then followed for about 
2 months. 
The effects of nitrogen mustard on blood and tissue free amino acid levels of rabbits 
was studied with male and female New Zealand white rabbits weighing from 2.4— 
2.9 kg. Several control studies were necessary. One control animal was subjected to 
venipuncture at frequent intervals over a one day period. The results of this study 
are presented in part I of this series. The effects of fasting over the longest period 
of time (about 33 h) that any mustard-treated animals went without food was studied 
in a second animal. Two additional animals were fed from 12 to 4 PM while blood 
samples were drawn at 10 AM. These animals were followed for 4 days in this fashion 
to serve as controls for mustard-treated animals that were examined over the same 
period of time. The controls also serve to establish the reproducibility of the plasma 
and erythrocyte free amino acid levels during the production of a blood loss anemia 
resulting from the withdrawal of 4-5 ml blood samples. 
Nitrogen mustard was administered intravenously to 7 animals. 4 rabbits were 
given 2.5 mg/kg of mustard and 3 animals were given 1 mg/kg. All animals were 
* Present address: Prof. OkINAKA’s Clinic School of Medicine, University of Tokyo, (Japan). 
References p. 447/448 
