Armored 
Medical Research Laboratory 
Fort Knox, Kentucky 
Final Report 
On 
PROJECT NO. 18 - INVESTIGATION OF THE EFFECTS OF ACTIVITY AND 
ENVIRONMENT ON ATABRINE THERAPY 
Project No. 18 
23 December J9h3 ARMORED METICAL RESEARCH LABORATORY 
Fort Knox, Kentucky 
Project No. 18 
khlol GNQML 
23 December 19h3 
1. PROJECT: No. 18 - Final Report on. Investigation of the 
Effects oi* Activity and Environment on Atabrine Therapy. 
a. Authority - Reference 3rd Indorsement, Headquarters Army 
Ground Forces, Army War College, Washington, D. C., file 720 (12 July 
GNMED, Bated 31 July 19U3. Subject: Investigation of Atabrine 
Blood Levels at Armored Medical Research Laboratory, Fort Knox, 
Kentucky. 
b. Purposes - 
(1) To determine with each of two racommended suppressive 
regimens administered to troops during active training: 
(a) The time required to develop equilibrium plasma 
atabrine levels; (b) the magnitude of the level in re- 
lation to dosage and (c) the variability in levels 
among individuals on the same regimen. 
(2) To determine the effect of high initial doses in short- 
ening she time required to reach equilibrium levels. 
(3) To deter.nine the effects of a simulated Jungle climate 
upon the plasma atabrine levels. 
(U) To study the rate of reduction of plasma atabrine 
levels after discontinuing administration of the drug. 
(5, To study the relationship between the plasma atabrine 
levels obtained with therapeutic doses and those 
established previously with suppressive regimens. 
2, DISCUSSION: 
a. Two hundred and fifty men were employed who were sub- 
divided for purposes of study, as follows: Two groups of 100 men each 
(from the Armored Replacement Training Center) were given OJ* and 0.6 
gm atabrine per week respectively, a third group of on 0.6 gm/wk 
was rotated through the Laboratory hot room arid a fourth group of 20 
men was used for special studies. 
b. Details of allocation, conditions, dosage regimens and 
sampling schedules and results are given in the appendices. 
1 c. The analytical method employed was that described by 
Brodie and Shannon - Malaria Report No, 9> NRG. 
d. Experience with the drug, the men, and the chemical method 
has led to certain conclusions. In addition, one arrives at inferences 
with respect to the application of this experience to the continued in- 
vestigation of this problem. These points of view are summarized and 
from them the recommendations have evolved. 
e. A table of contents follows this section. 
3* CONCLUSIONS: 
a. Plasma atabrine levels obtained with two constant regimens. 
(1) The group plasma level at any time on a given regimen 
is a function of the daily dose, the pre-existing 
level and the time interval since the last dose. 
(2) The group mean level attained on a given regimen rises 
progressively for from four to eight weeks and finally 
reaches an equilibrium level which remains substantially 
constant thereafter, 
(3) The group mean equilibrium level is directly proportion- 
al to the average daily dose. With dosages of 0.6 
grn/wk, a mean plasma level of 1? micrograms/L is 
reached— and with 0.4 gni/wk, a level of 12 micro- 
grams/L. ' 
(4) Individual plasma concentrations obtained with a given 
regimen differ widely. In a group exhibiting a mean 
equilibrium level of 12 micrograms/L, individual con- 
centrations varied from 5 to 85 micrograms/L. 
b. Plasma atabrine levels with regimens instituted after high 
initial doses. 
(1) The time required to reach the equilibrium level 
characteristic of any regimen can be greatly reduced 
by administration of larger doses prior to initiation 
of the maintenance regimePo 
(2) The final equilibrium level attained with the subsequent 
maintenance dose is not altered by initial priming doses 
of short duration. 
c. Effects of simulated jungle climate. 
(1) The group equilibrium level achieved with a given dosage 
regimen in a hot humid environment is the same as that 
obtained at normal temperatures. (2) Rate of acclimatization and performance of men in a 
hot humid environment are not affected adversely by 
suppressive therapy. 
do Rate of decrease of group mean plasma level when adminis- 
tration of the drug is discontinuedo When the drug is no longer taken 
the concentration of plasma atabrine falls, the level at any time there- 
after being a function of the pre-existing level and tne time interval 
since discontinuing dosage. The level existing at any time is reduced 
by approximately 1C$ in the ensuing 24 hours. 
e. Level obtained with therapeutic dosage. 
(1) Administration of therapeutic doses for one vreek to men 
at equilibrium with suppressive regimens results in a 
degree of rise of plasma level which is a function of the 
added quantity administered and the pre-existing level. 
(2) Men who are characterized by low plasma levels while 
on suppressive dosage regimens will in general have 
relatively low levels when on standard therapeutic 
. regimens, 
fo Analytical method. 
(1) The precision of the method as employed is within 
•f2 micrograras/L. 
(2) Larger errors which affect ail determinations in a 
single day may occur. 
(3) The relative precision of the method is greatest at the 
higher levels. For plasma levels below 10 micrograms/L 
the method must be used witn extreme care. 
g. Toxicity of atabrine. No unequivocal toxic reactions occurred 
in any of the 250 men studied for three months. 
4* RSC0MM5NDATI0NS: 
a. That the following studies be expedited: 
(1) Field investigations in hyperendemic areas to determine 
the minimum plasma atabrine level required for the 
suppression of malaria. 
(2) Further investigation, in the individual, of the value 
of the plasma atabrine level and other indices, as 
measures of antimalarial protection. 
(3) Laboratory studies to improve the chemical method and 
equipment. 
(4) A controlled field study of the toxicity of atabrine. b0 The minimum suppressive plasma atabrine level has not yet 
been established. Breakthrougns have occurred with wedkly dosage of 
0.4 gm. In the present study no toxic reactions occurred with tills or 
larger suppressive doses. Pending results of the suggested studies, 
therefore, it is recommended that the dosage regimen for troops entering 
hyperendemic theaters be established as follows: 
(1) 0.1 gm atabrine twice daily after meals for 1 week, 
to be given not later than the week immediately preceding 
exposure. 
(2) 0.1 gm. atabrine daily thereafter so long as exposed. 
Submitted by: 
Armored Medical Research Laboratory Staff 
Norton A. Nelson, Captain, SnC 
William F. Ashe, Major, M.C. 
Frederick So Brackett, Major, SnC 
Ludwig W. Eichna, Captain, M.C» 
William B. Bean, Captain, M.Ge 
Office of The Surgeon General Staff 
Aims C. McGuinness, Major, M.C0 
Morris Rosenfeld, 1st Lt., M.Co 
Robert 0. Gould, 1st Lt., SnC 
Leonard I). Rosenman, 1st Lt., M.C0 
Maurice Wince, 1st Lt., M,C0 
Edward D. Conner, 1st Lt., M.C» 
Under the general supervision of Dr. James A. Shannon. 
APPROVED 
WILLAItD MACULE 
Colonel, Medical Corps 
Commanding 
4 TABLE OF CONTENTS 
APPENDIX A 
SU’UARY OF RESULTS 
APPENDIX B 
METHODS AND RESULTS OF MATHEMATICAL TREATKffiNT OF DATA 
Section lo Statistical Analysis of Individual Variability 
Section II. Prediction of Group Plas:oa Levels 
APPENDIX C 
PROCEDURES AND RESULTS OF INDIVIDUAL STUDIES 
Section I» Collection, Preparation and Analysis of Plasma 
Section II„ Plasma Atabrine Levels Established in Two Lar&© Groups 
Under Different Dosage Regimens (ARTC Group) 
Section III, Effects of a Simulated Jungle Climate Upon Plasma Levels 
Section IV. Special Studies 
1. Absorption Following a Single Dose and Effect of 
High Initial Doses 
2. Rates of Buildup and Dieaway 
3. Levels Attained with Therapeutic Doses 
Section V. Factors Related to Variability in Individual Plasma Levels 
1. Excretion and Degradation 
2, Variation in Protein Binding 
Section VI„ Effects of Atabrine on Man 
APPENDIX D 
IMPLICATIONS OF THE STUDY AS APPLIED TO FUTURE INVESTIGATIONS 
APPENDIX e 
TABULATION of RAW DATA APPENDIX A 
SUMMARY OF RESULTS 
lo Administration of atabrine under a given dosage regimen 
results in a progressive increase in plasma concentration until an 
equilibrium level is reached which continues thereafter, with little 
variation, so long as the dosage schedule is maintained. After dis- 
continuing the drug, the plasma level decreases rapidly at first and 
then more and more slowly. There are marked variations in plasma levels 
among the individuals of a group subjected to the same regimen, but 
with few exceptions they follow a pattern similar to that exhibited by 
the group as a whole. This characteristic pattern - the progressive 
buildup, the equilibrium and the dieaway - appears to be fixed by a 
simple law which makes it possible to predict the course which the mean 
concentration of a group will follow when subjected to a given dosage 
regimen. The extent of the individual departure from this mean value 
is also predictable. These and other important behavior character- 
istics exhibited by a group of men are considered below. The procedure 
employed and more detailed considerations of the results are presented 
in subsequent sections. 
2, Buildup of plasma atabrine concentration. 
Although different dosage regimens, in which the administration 
is intermittent, exhibit characteristic detailed differences in pattern, 
the regular repetition of dosages each suceeding week, results in a 
general rising course which is susceptible to simple description. The 
plasma concentration increases rapidly during the first week and then 
more and more slowly as equilibrium is approached. At the end of the 
first week approximately one-half of the final equilibrium level is 
attained; at the end of the second week, three-fourths of the equilibrium 
level; at the end of the third week, 87$; at the end of the fourth week, 
9k% and so on; each week halving the remaining difference so that by 
the end of the sixth week no perceptible difference from equilibrium is 
observed. 
3. Equilibrium level attained in relation to dosage regimen. 
At equilibrium the average concentration resulting from ad- 
ministration of a specific amount of drug each week depends upon the 
total weekly dose. Representative values for a group are found to be 
directly proportional to the dose. Thus for the two regimens, 0.4 gm/wk 
and 0.6 gm/wk, the group mean equilibrium levels attained were 12 and 
17 micrograms/L, respectively. In view of this direct proportionality, 
it is a simple matter to calculate, within the general limits of the 
study, the predicted equilibrium plasma level for other dosage regimens. 
The relationship is: 30 micrograms/L for 1 gm weekly dose, (Total) 
1 4. Effect of large initial doses upon the time required to reach 
maintenance levels. 
As was pointed out in par. 2 above, from 4 to 6 weeks are re- 
quired on a regular weekly regimen to attain the resulting equilibrium. 
However, a practical expedient immediately suggests itself wnereby a de- 
sired maintenance level can be obtained within a few days. Since half- 
equilibrium values are reached witn any regimen in 1 week, if double 
the maintenance dosage is given in the first week the level reached 
at the end of this tine will be same as the equilibrium level of the 
maintenance dose. This avoids from 3-5 weeks delay. By administration 
of 3 times tne maintenance dose initially, the required level will be 
reached after four daily doses; with 4 times the maintenance dose, in 
3 days, etc, 
5° Decrease of plasma atabrine concentration after discontinuing 
the drug. 
When the drug is discontinued after the establishment of an 
equilibrium state the plasma atabrine concentration decreases in a 
manner very similar to the initial buildup. Thus, ICXo of the level is 
lost each day and at the end of a week the level will have dropped to 
half the equilibrium value, to one-fourth in two weeks, one-eighth in 
three weeks and so on, each week halving the remaining concentration. 
m 
6. Effect of climatic environment upon equilibrium level. 
An important phase of the present study was to determine the 
effect, if any, of a hot, humid environment upon the course of buildup, 
and upon the equilibrium level attained with a given regimen as compared 
with the equilibrium level reached with the same dosage schedule in a 
temperate climate. The results, as indicated in the table below, show 
that climate had no influence upon the equilibrium plasma atabrine level. 
TABLE 1 
EQUILIBRIUM PLASMA ATABRINE LEVELS ATTAINED UNDER DIFFERENT 
CLIMATIC EXPOSURES MTTH SAMS DOSAGE SCHEDULE - . 
0,6 GM PEP WEEK 
Exposure 
No. 
of 
Results 
No, 
Men 
Group MeariQ Concentration 
7th Through 11th Weeks 
Standard 
Geometric 
Deviation 
Outside, A.R.T.C, 
853 
B5 
17.2 | 1.04 
1.44 
7 wks jungle, 4 wks 
outside Group A 
205 
15 
19.9 ? 1.17 
- 
1.41 
7 wks outside, 4 wks 
jungle Group 3 
205 
15 
19.9 ? 1.08 
♦ 
1.32 It was also found, in this phase of the study, that neither the adminis- 
tration of atabrine concurrent with initial exposure to a hot, humid 
environment, nor the establishment of a suppressive atabrine level 
prior to such exposure, in any way affected the work capacity of the 
subjects in the heat nor their rate of acclimatization, 
7. Plasma levels attained with the therapeutic dosage schedule. 
The increase of plasma concentration with the institution 
of a therapeutic regimen after the establishment of equilibrium under 
a suppressive dosage schedule can be described by the same expressions 
that were derived from the behavior of the drug at the lower dosage 
rates. The concentration approaches a new equilibrium level which is 
directly proportional to the dosage and the manner of increase is the 
same as before. It was found possible to predict the plasma level 
attained with therapeutic dosages in the same manner and with equal 
success as was the case with the concentrations under the suppressive 
regimens, when due account was taken of the transients. 
d. Individual variability. 
The difference between plasma levels in different individuals 
may be great. This variability follows a regular pattern and is 
consistent with the type of statistical analysis (geometric) to which 
the data have been subjected. Description of the statistical procedure 
will be given in the next section. A possible biochemical explanation 
of the wide differences between individual results is offered. It is 
believed that variation in protein binding is the main cause of the 
wide ranges in values. The atabrine concentration in erythrocytes 
varied independently of plasma atabrine level and was more nearly 
proportional to dosage experience. It is inferred from this that a 
variation in partition coefficient rather than differences in total 
quantities involved may be responsible for the variance. Evidence on 
this was not conclusive. 
3 APPENDIX B 
1-BSTHODS AKD RESULTS OF MATHEMATICAL TREATMENT OF DATA 
SECTION I - Statistical Analysis of Individual Variability. 
1. Distribution of individual plasma levels. 
Individuals on the same dosage regimen differ markedly in 
their plasma concentrations• For example, a group of 85 men on a 
0,4 gra/wk regimen were distributed with respect to their plasma levels 
as shown in Chart 1. In order to describe the pattern of variability 
statistical treatment of the data has been employed. It is immediately 
evident from the unsymmetrical pattern shown in Chart 1 that the 
variability does not follow the normal arithmetic probability distribu- 
tion. The arithmetic mean and standard deviation, which apply to the 
normal probability curve, cannot be eiaployed, therefore, to describe 
the distribution of individual plasma atabrine levels. When the varia- 
tions in atabrine concentrations are expressed logarithinically, however, 
the distribution is approximately symmetrical, and is found to approach 
closely a logarithmic probability distribution, as shown in chart 2. 
The normality of the logarithmic distribution is demonstrated by the 
fact that the cumulative frequency curve plotted on logarithmic proba- 
bility paper yields an approximately straignt line over most of the 
range, as seen in Chart 3» This linearity test was applied to every 
set of group data and satisfactory straight-line plots were obtained 
in all cases. It should be pointed out, that a few exceptional indi- 
viduals exhibited abnormally high atabrine levels for extended periods. 
The number is not sufficient, hov/ever, to affect appreciably the group 
values. 
2, Significance of geometric distribution. 
The representative value for a group which is characterized 
by a logarithmic probability distribution is the geometric mean (anti- 
log of the arithmetic mean of the logarithms of the plasma atabrine 
values in a group of data) instead of the customary arithmetic mean, 
and the dispersion of the data is measured by the standard geometric 
deviation rather than by the standard deviation of the arithmetic values. 
The concept of dispersion as measured by the standard geometric de- 
viation (anti-log of the standard arithmetic deviation of the logarithmic 
plasma levels about the logarithmic mean) differs basically from the 
common conception of linear dispersion, measured by the arithmetic 
standard deviation. In the case of a normal arithmetic distribution, 
probabilities ere equal, that deviations of i 1.0 standard deviation 
about the mean will occur0 With a log-probability distribution, on the 
other hand, probabilities are equal, that deviations obtained by multiplying 
or dividing by (1.0) one standard geometric deviation, will occur. Thus, in 
1 CHART - I 
FREQUENCY DISTRIBUTION OF PLASMA ATABRINE 
LEVELS IN CO. B SECTION I. 
7 TH THRU II TH WEEK 
FREQUENCY 
PLASMA ATABRINE IN MICROGRAMS PER LITER 
(DISTRIBUTION BY EQUAL ARITHMETIC CLASS INTERVALS) 
CHART -1 CHART-2 
FREQUENCY DISTRIBUTION OF PLASMA ATABRINE LEVELS 
IN CO. B, SECTION I, 7TH THRU NTH WEEK 
LOGARITHMIC SCALE 
FREQUENCY 
PLASMA ATABRINE IN MICROGRAMS/LITER 
(DISTRIBUTION BY EQUAL LOGARITHMIC CLASS INTERVALS) 
CHART—2 PLASMA CONCENTRATION (LOG SCALE) 
CUMULATIVE PERCENTAGE DISTRIBUTION OF PLASMA ATABRINE LEVELS 
CO- B, SECTION I, 7TH THRU 11TH WEEK 
PERCENT I STATED PLASMA CONCENTRATION 
PROBABILITY SCALE 
CHART-3 
MEANg = 12.5 MICROGRAM/LITER 
SD=-fi= 146 
CHART-3 terms of a normal logarithmic probability curve, the relative significance 
of high and low values becomes quite different from their meaning when 
viewed in arithmetic relation to the mean. Under the geometric concept, 
a level of 80 micrograms/L in a group of data having a mean level of 
20 micrograms/L has the same probability of occurrence as a level of 5 
micrograas (80 . 20 _ 4), whereas, in a normal arithmetic probability 
20 ~ 5 ~ 
distribution, with a mean of 20, values of 5 would occur with much greater 
frequency. It is not suggested that the geometric distribution is an 
invariable characteristic of plasma atabrine levels of a group. It was, 
however, repeatedly observed in this study and provides a method for 
describing variability which is more representative than is the common 
arithmetic method. The standard geometric deviation was found to be 
quite constant for all sets of data, exhibiting relatively little variation 
from week to week. Its average value was 1.45* from which one may predict 
(within the limits of the study) the range and distribution of plasma 
concentrations among a group of men subjected to a given suppressive 
regimen, as in the following table: 
TABLE 2 
PREDICTED DISTRIBUTION OF INDIVIDUAL PLASMA LEVELS 
IN 
MEN OK A GIVEN REGIMEN 
(Presented in a cumulative frequency distribution of the indivi- 
dual plasma levels which arc expressed as ratios 
of the Mean$ of the group) 
Individual Levels 
(Factor by which Meaiv is to 
be multiplied^ 
— 
Percentage of Men Having Stated 
Level or Less 
0,4 x MeanQ 
1— - ■ J 1 1—u- 1— f 
1 
0.5 
3 
0.6 
8 
0.7 
17 
0.8 
27 
1.0 
50 
lo2 
70 
loU 
82 
1.6 
90 
2.0 
97 
2.5 
99 APPENDIX B 
SECTION II - Prediction of Group Plasma Atabrine Levels0 
lo Discussion, 
a0 It is the purpose of this section to describe the simple 
expressions by means of which representative group values can be pre- 
dicted and to present the derivation of these expressions for the 
observed data0 
b. The plasma value which is representative of a group has 
been shown in the previous section to be the geometric mean (MeariQ) of 
the individual plasma atabrine concentration,. This section will be de- 
voted entirely to the consideration of such representative group values„ 
2, Concept of transients and underlying levels„ 
Following the administration of atabrine the concentration of 
the drug in the plasma exhibits rapid changes associated with the 
entrance and redistribution of the drug0 After twenty four hours, how- 
ever, these initial disturbances, have practically disappeared and the 
then observed values can be very simply correlated0 For the purpose of 
this discussion, the initial disturbances will be spoken of as 
transients and the term underlying level will be applied to all values 
found more than 24 hours after administration of any dose. The under- 
lying level may be thought of as existing at all times but concealed by 
the presence of the transients during the first day following administra- 
tion<> The first part of the analysis will deal with the correlation of 
data on underlying levels and the second part with the evaluation of 
the transients® Knowledge of the transients becomes particularly 
necessary during a therapeutic regimen where the doses are administered 
at intervals of less than a day® 
3o Simple mechanism correlating underlying levels0 
a0 The most important fact which emerges from a study of the 
data on underlying levels is that all the values observedcan be computed, 
within experimental error, from two constants. This implies a simple 
mechanism which may be expressed as follows: 
b0 The net change in underlying plasma level is the result of - 
(1) A gain in concentration directly proportional to the 
dose - 23o2 micrograms/L for 1 gm doseQ 
(2) A loss in concentration proportional to the existing concentration and time interval - 10,2 of concentra- 
tion per day, 
4. Computation of underlying level for a daily dose regimen. 
a. In order to determine the pre-dose level on any day from 
the pre-dose level of the previous day, the procedure is as follows: 
(1) To the pre-aose value of the first day add the con- 
tribution of the new dose - 23.2 times dose in gm, 
(2) From this new value subtract lO'T of the new value, 
thus giving the pre-dose level for the second day 
in adcrograms/L, 
b. By means of this procedure, curves have ueen built up day 
by day for each of the grouns. 
c. In Charts 4 and 5> the observed values for the groups are 
shown by the dotted lines, for comparison with the computed values, 
solid lines. In Chart 6 the values are shown for a group (Jungle) 
which was given 1.2 gm in the first week and 0o6 gm thereafter. 
Calculated values are indicated by the solid curve and the observed 
by the dotted curve. 
d. Tables 3* 4, 5 and give the computations for all curves. 
Calculations for the therapeutic levels will be considered later. 
Summary of the more detailed step-by-step calculations for the groups 
is given in Chart 7. 
e. This procedure may be expressed algebraically - 
Lf = L + AD - K (L + AD) 
where L = grouo plasma concentration Just before a 
dose, 
L1 = same for succeeding day, 
D = dose in gm. 
A - gain per gm of dose = 23*2 micrograms/l/gra, 
K = percent of level lost per day = 10,1. 
Then L’ = (l-K) (L + AD) = 0.9 (L + 23.20) 
(D may be zero on dsys of no uose) 
The procedure of adding the gain immediately at ths time of administra- 
tion has been adopted arbitrarily for simplicity and to partially offset 
Tables will be found at the end of this section. COMPARISON OF THEORETICAL AND EXPERIMENTAL PLASMA ATABRINE LEVELS 
0.4 CM. PER WEEK 
PLASMA ATABRINE LEVEL IN MICROGRAMS PER LITER 
EXPERIMENTAL CURVE BASED ON THE RESULTS FOR 85 MEN 
CHART -4 
DAYS 
WEEKS 
THEORETICAL VALUES 
EXPERIMENTAL VALUES 
THERAPEUTIC 
DOSAGE 
CHART —4 CHART —5 
COMPARISON OF THEORETICAL AND EXPERIMENTAL MEANG PLASMA 
ATABRINE LEVELS ON A DOSAGE SCHEDULE OF 0.60 GRAMS PER WEEK 
WEEKS 
PLASMA ATABRINE IN MICROGRAMS PER LITER 
DEFINITIVE THERAPY 
BEGINS (40-41 MEN) 
DIE AWAY BEGINS 
(43 -45 MEN) 
DAYS 
THEORETICAL VALUES 
EXPERIMENTAL VALUES 
NUMBER OF MEN 84 - IOO 
CHART-5 PLASMA ATABRINE LEVEL 
MICROGRAMS PER LITER 
COMPARISON OF THEORETICAL AND EXPERIMENTAL PLASMA ATABRINE LEVELS 
1.2 GM. THE FIRST WEEK AND 0.6 GM. EVERY SUBSEQUENT WEEK 
EXPERIMENTAL CURVE BASED ON THE RESULTS FOR 30 MEN 
CHART - 6 
WEEKS 
DAYS 
theoretical values 
EXPERIMENTAL VALUES 
ABERRANT VALUES 
DOSAGE IN GRAMS 
CHART-6 PLASMA ATABRINE IN MICROGRAMS PER LITER 
JUNGLE GROUPS 
THEORETICAL PLASMA ATABRINE LEVELS OF ALL 
MAJOR GROUPS FOR ENTIRE EXPERIMENT 
CHART-7 
DAYS 
WEEKS 
NOTE EFFECT OF 
DOSAGE SCHEDULE 
CHANGES 
CHART-7 the greater loss arising from transient elevation of levels 
5» Consequences of the simple mechanism^ 
a0 Certain facts in regard to the course which a group will 
follow arise from the two relations postulated for our simple mechan- 
isms (par„ 3» (l) and (2))c 
b0 Rise in atabrine levels If atabrine is administered in 
equal daily doses, underlying level rises by diminishing increments to 
an equilibrium value, while loss per day becomes larger and larger, 
until at equilibrium the loss just equals the dally dose contribution 
The rise is logarithmic, approaching the equilibrium value asymptotically0 
It may be expressed by the equation: 
l s (1 - e-Kt) 
Where L r level at time t0 
= equilibrium level (t =CO)» 
t - time after first dose0(days) 
K = decay constant in l/days s O.L 
e r Naperian base of logarithms0 
c0 This rise may be described as follows: tan percent of the 
remaining rise to equilibrium takes place in each day so that: 50$ of 
the rise takes place in one week, 75$ by the end of the second week, 
87$ by the end of the third, etc0 By the end of the sixth week 98$ 
has taken place and only 2$ of the total rise remains0 This is within 
experimental error0 
do Equilibrium levelo The equilibrium value (L]_) is propor- 
tional to the dose® Thus, for equilibrium (gain = loss); 
AD = K (L1 4 AD) 
4 = ad (1 , 1) 
Since, K = 0.1 
- 9 AD 
Where A - rise per gm of dosec 
D s dose in gm. From paragraph 3-(l) above, A - 23.2 micrograas/L/gm dose. Hence, 
(in micrograms/L) s 209 x D (in gms.) 
This direct proportionality between the equilibrium concentration and 
daily dose is of the greatest practical importance since it provides 
the means for predicting the maintenance level achieved by any dosage 
regimen. Thus, for example, for 0.15 gffl daily the underlying level 
will be: 
209 x 0*15 = 31 o 3 micrograms/L. 
While strictly true only for a regular daily regimen, the average 
equilibrium level can be determined for an irregular regimen, repeated 
weekly, with sufficient accuracy by - 
In - 209 x D» : 30 I D' 
7 
Where D1 is the weekly dose in gm. 
Hence, for a weekly dose of 0,6 gm the underlying level will be 30 x 
0,6 s IS micrograms/L. 
e. Decay of level after termination of dosage. After cessation 
of atabrine administration the level will fall logarithmically: 
-Kt 
L - 
Where L = level at time t« 
L0 s level at time t - 0. 
t - time in days after L0. 
K - decay constant in i/days - 0,1, 
e — Naperian base of logarithms, 
f. This decay is very similar to the rise since: - Ten percent 
of the level is lost each day so that; 50% will be lost after one week; 
75% after two weeks, etc. At the end of six weeks only 2% of the equili- 
brium level will remain. ga The general course - rise, equilibrium and decay - is 
illustrated in Chart 8 for a daily dose of 0o05 gm0 
h, Mathematical derivation for continuous administration,. 
Provided there is approximately continuous administration of the drug, 
these three relationships may be derived mathematically from the simple 
statement in paragraph 3 as follows - 
dL = AD dt - KL dt 
or dL - dt 
AD - KL ” 
by - 1 log (AD - KL) = t - log C 
K 1 
Where dL s change in concentration in time increment, 
dt„ 
= constant of integratione 
A, D, L and K as above. 
. 1 log = t 
K e C 
-Kt 
AD - KL - e 
' C 
t _ AD C .-Kt 
L = T - IT e 
At t rCO, L r = IjS. 
At t = 0, L - 0, so, £ = ~ and C « AD 
K M 
Hencep L = L (1 - e“Kt) = AD (1 - e~Kt) 
and L * D or 232 D 
1 Ool LIMITING LEVEL1 
-DOSAGE PERIOD A 
THEORETICAL CURVES 
CHART - 8 
- NO DOSE PERIOD B 
CHART - 8 The actual value - 209 instead of 232, results from the discontinuous 
administration of the drug and the procedure developed to deal with it, 
in contrast to the continuous adninistration assumed in the foregoing 
analysis. 
io In a period of no dosage, D = 0, so from above differential 
equation: 
dL = - KL dt 
dL - - Kdt 
L 
by integration. Log L = - Kt 4 log C 
L * C e-Kt 
At t s 0, L* Lq, hence 
L B L0e"Kt 
Where L0 is the value at the beginning of decay or termina- 
tion of dosage. This equation is trie expression for dieaway given in 
paragraph 
60 Evaluation of constants from experimental data. The values 
of the constants for the two relations of the simple mechanism were 
obtained in the following manner; 
ac Determination of K0 It is evident that K, the decay 
constant, can be obtained from the experimental data, both from the 
rising portion of the curve and also from the decay. Since none of 
the regimens followed, comprised equal dally doses but rather a weekly 
repetition of dosage pattern, only those plasma concentrations obtained 
at the same time each week could be employed for determination of K. 
The value of K was obtained by adjustment to give the hkst fit on . 
the rising portion of the curves drawn through these corresponding 
points. The resulting values may be then compared with the observed 
decay, See Chart 9, where the points are the actual observed values 
and the line has the slope K. CHART - 9 
EXPONENTIAL NATURE OF THE DISAPPEARANCE 
, OF ATABRINE FROM THE PLASMA 
WHEN DOSAGE IS DISCONTINUED 
PLASMA ATABRINE MICROGRAMS PER LITER 
DIE AWAY POINTS PLOTTED ON SEMI 
LOGARITHMIC SCALE. SLOPE OF LINES 
BASED ON PREDICTED RATES OF 
DISAPPEARANCE 
COMPANY C, SECTION 2 o (0.6 CM GROUP) 
COMPANY C, SECTION I • (0.4 GM GROUP ) 
CHART - 9 b. Determination of A. Knowing the values of K and the 
equilibrium level, I-, for a given dosage rate, D, the value of A, 
(rise per 1 gm dose) .may then be obtained by the relation already 
given in paragraph 3-d: 
Lx r AD (| - 1) or 
A= L1 
dH-TT 
K 
Values of the equilibrium level were obtained for each set of 
group data and the average daily dose, D, determined (e.g0, for the 
Go6 gn/wk groups D = 0*6 gnu 
7 
Co The best value for tine equilibrium levels of all groups 
was found to be: 
- 209 D micrograms/L, 
from which we obtain A = 209 - 23o2 raicrograma/L« 
9 
7. Evaluation of Transientso While the data on changes in plasma 
concentration which immediately follow the administration of atabrine 
are meager, a tentative basis for computation is suggested which has 
satisfactorily predicted the response obtained from therapeutic regimens. 
In a considerable number of cases two maxima occur, one at about 2 
hours after dose and another at about 8 hours after dose (see Chart 10), 
In other cases no bimodal form is noticeable„ However, it is a common 
observation that bimodality may be lost sight of, if the time spacing 
is somewhat variable* The fact that it does occur frequently is strong 
evidence that a dual mechanism is present. We shall therefore assume 
that two distinct transients occur but are so closely spaced that they 
may merge at times. Our concept is as follows: 
a* A first transient (T-,, Charts 11 and 12) begins immediately 
after adroinistration, rises to a maximum in 2 hours and falls to a 
negligable value at the end of 8 hours* 
b. A second transient (T2, Charts 11 and 12) follows more 
gradually after administration, rises to a maximum in 8 hours and then 
decays, losing a half value in each succeeding four-hour interval* 
Thus, it has practically disappeared 24 hours after dosage. CHART- 10 
POST ABSORPTION CURVES OF PLASMA ATABRINE CONCENTRATION 
FOLLOWING 0.2 GM DOSE 
AFTER 16TH DOSE OF 0.2 GM 
{8 MEN ) 
PLASMA ATABRINE-MICROGRAMS / LITER 
AFTER 0.2 GM IN IOTH WEEK 
OF C-1,0-2 REGIMES (7-MEN) 
AFTER 4TH DOSE OF 0.2 GM. 
{4 MEN? 
AFTER 1ST DOSE OF 0.2 GM 
(8 MEN) 
HOURS AFTER ORAL DOSE OF 0.2 GM ATABRINE 
CHART— 10 CHART-11 
TRANSIENTS FOLLOWING SINGLE DOSE 0.2 GM. 
CALCULATED 
AFTER I6TH DOSE 
POINTS OBSERVED 
PLASMA ATABRINE - MICROGRAMS PER LITER 
CALCULATED 
AFTER 4th DOSE 
-CALCULATED 
AFTER Ist DOSE 
HOURS AFTER DOSE OF 0.2 GM 
CHART-11 CHART-12 
TRANSIENTS FOLLOWING SINGLE DOSE 03 GM. 
CALCULATED CURVES—OBSERVED POINTS 
AFTER 4TH DOSE 
PLASMA ATABRINE—MICROGRAMS PER LITER 
HOURS AFTER DOSE OF 0.3 GM. 
CHART-12 c. The contribution of these two transients is additive and 
superimposed on the underlying level which undergoes a gradual change 
from the value of one day to that of the next, 
d. The magnitudes of the peaks of these two transients were 
found to be approximately as follows; 
(1) The two hour transient is proportional to the dose 
and equals 50 micrograms/L/gm dose. 
(2) The 8 hour transient varies from a small peak value 
at the beginning of treatment to a maximum peak 
value when the subject has attained equilibrium on 
that particular dosage regimen. This greatest value 
is found to oe ICO micrograms/gm dose. The increase 
in peak magnitude in the second transient is similar 
to the logarithmic rise of the underlying level. It 
may be roughly expressed by the expression; 
T2 r 100 (l-e~Kt) D 
Where K - 0,1 as before, 
t = day of dose, 
8. Application of theory to therapeutic regimens. 
The underlying level for a therapeutic.regimen may be computed 
in the manner already described. However the doses are administered so 
frequently that a continuous curve may be drawn by consideration of 
the exponential form. Superimposed upon this underlying curve the con- 
tributions of each of both transients from each dose may be determined 
graphically as in Chart 13, 
9. Physiological implications. While the preceding correlation 
of the observed data has been presented on a purely empirical basis, 
possible implications as to physiological mechanism are of some interest. 
a. The first transient. The fact that the magnitude depends 
only on the particular dose causing it and not on the stage of the 
treatment or the underlying level suggests a direct effect of absorption. 
The relatively short time required to reach maximum (2 hours) and the 
brief overall duration is compatible with this interpretation. 
bo The second transient. The marked variation in peak MICR06RAM/LITER 
END OF SUPPRESSIVE 
REGIMEN 
THEORETICAL RESPONSE TO THERAPEUTIC REGIMEN 
TIME IN DAYS (EACH DAY BEGINS AT 0800 HRS.) 
CHART-13 
CALCULATED CONCENTRATION 
TRANSIENTS 
UNDERLYING LEVEL 
OBSERVED CONCENTRATIONS 
CHART-13 magnitude of this transient is suggestive of a contribution arising 
after passage thru a barrier in which partial removal has taken place0 
This barrier at first passes only a small fraction of what later 
passes when saturation of the barrier has taken place0 The greater 
time between administration of dose and maximum of the transient* is 
again suggestive of passage through a barrier0 The nature of the 
progressive rise in magnitude of this transient suggests that the 
saturation of the barrier follows a parallel course to the general 
saturation of the systenu The time of decay of this transient is 
suggestive of a redistribution rather than elimination from the body 
which follows a much slower coursee The liver has been suggested as 
this barriero 
Co The underlying level may be thought of as the concentra- 
tion in the blood plasma maintained by the buffering of the systemically 
stored atabrine0 It is to be expected that the values relating to this 
systemic storage would present the simplest picture0 
11 r> •y 
Jii I 
of 
EX PER 
date 
Pf.O-DOSE 
LEV EL 
THEOii’T 
DOSE 
mg - 
0 
All' 
21 
0 
50 
1 
28 
1.04 
50 
2 
29 
1.98 
0 
5 
30 
1.78 
100 
4 
31 
3.79 
100 
Seiu 
3.30 
50 
6 
ti 
6.00 
50 
7 
3 
6.44 
50 
a 
4 
6.94 
50 
9 
3 
7.29 
0..^ 
10 
6 
6.56 
100 
11 
7 
7.99 
100 
12 
B 
9.28 
5Q 
13 
9 
9.40 
50 
14 
10 
9.50 
30 
13 
11 
9.39 
50 
16 
12 
9.63 
0 
17 
13 
8.71 
100 ! 
IB 
14 
9.93 
100 I 
19 
13 
11.02 
50 i 
20 
16 
10.96 
50 
21 
17 
10.87 
1 
50 
22 
16 
10.83 
23 
19 
10.79 
0 I 
24 
20 
9.71 
100 ! 
25 
21 
10.83 
10;; i 
26 
22 
11.84 
50 
,27 
23 
11.70 
50 
28 
24 
11.57 
30 
29 
25 
11.46 
50 
30 
26 
11.36 
0 
—21 . 
,27 
10.22 
100 
DAu 
of 
I'XFE} 
DATE 
L 
Ju 1 V uL 
Tli OH. T 
DOSE 
mg 
32 
28 
11.29 
100 
33 
29 
12.25 
50 
34 
30 
12.07 
50 
35 
0ci* 
11.90 
50 
36 
11.75 
50 
37 
11.. 2 
0 
38 
4 
10.46 
100 
.. 39 
5 
11.50 
10c 
40 
6 
12.44 
50 
41 
7 
12.24 
50 
42 
8 
12.06 
50 
4? 
9 
u.90 
50 
44 
10 
11.75 
0 
45 
n 
10.57 
100 
46 
12 
11.60 
100 
47 
__13_ 
12.53 
50 
43 
14 
12.32 
50 , 
49 
15 
12.13 
• 
50 
50 
16 
11.96 
50 
51 
17 
11.81 
0 
52 
18 
10.63 
100 
53 
19 
11.65 
100 
54^ 
20 
. _12,5Z. .. 
50 
55 
21 
50 
56 
22 
12.1? 
_ _10 
57 
23 
12.00 
50 
53 
24 
II.84 
0 
59 
25 
10.66 
100 
60 
26 
11.68 
100 
61 
22_ 
12.60 
50 
62 
28 
12.38 
50 
63 
29 
12.19 
50 
r-;XP- R. 
DATS 
nr-dose 
V VEL 
THEOFST 
DOSS 
mg 
64 _ 
12.01 
50 
65 
31 
11.85 
50 
66 
l.ov. 
1 
11^71 
100 
. 67 
2 
12.63 
50 
68 
3 
12.41 
50 
69 
4 
12'. 21 
50 
7- 
5 
12.03 
50 
71 
6 
11.87 
50 
72 
7 
13.73 
50 
73 
8 
11.60 
loo 
74 
9 
12.53 
50 
76 
10 
12.32 
50 
. 
THJOH TICAL PLASUA ATABEIKE 
LhVLLS OL SUPPRESSIV“' DOSAGE 
40C my per week 
COD PALY 0 SECTION 1. 
Dosage Constant - 2,32 oicrograms/liter/ 100 mg, dose 
Die-Away Constant - 0.10. 
Underlined Levels occur on days on which men were bled. 
TABLE 3 I PAY 
r 
PRE-DOS* 
1 of 
1 DATE 
LTVEL 
DOSE 
EXP1R 
> 
TH EGRET 
m£. 
0 
0 
100 
1 
28 
2.09 
200 
2 
29 
6.06 
0 
o 
J> 
30 
5.65 
100 
lx 
31 
6.99 
100 
5_ 
OcJ. 
8.33 
c 
o 
6 
2 
9.63 
0 
7 
3 
B.67 
100 
e 
6 
9.89 
200 
9 
5 
13.07 
0 
10 
6 
XI.76 
100 
1 T 
X-L 
7 
12.67 
100 
12 
3 
13.69 
100 
13 
9 
16.23 
0 
10 
12.81 
100 
15 
11 
13.62 
200 
16 
12 
16.66 
0 
17 
13 
16.80 
100 
18 
16 
15.61 
100 
19 
15 
15.96 
100 
20 
16 
16.15 
0 1 
21 
1? 
16.81 
100 
22 
18 
15.62 
200 
23 
19 
IS. 06 
0 
26 
20 
16.26 
100 1 
25 
21 
16.72 
100 
26 
09 
17.16 
100 
27 
23 
17.52 
0 
2B 
26 
15.77 
100 I 
29 
25 
16.28 
200 
30 
26 
18.32 
0 
31- 
27 
16.91 
1 
100 i 
-HT- 
of 
| DATE 
rare 
LEVEL 
THLORET. 
DOSE 
mg. 
32 
28 
17.34 
100 
-13 
29 
17.69 
100 
. 34 
30 
18.01 
0 
3? 
OcJ,. 
16.21 
100 
36 
2 
16.68 
200 
37 
3 
19.19 
0 
38 
. . 4 
17.27 
100 
39 
3 
17.63 
100 
40 
6 
17.95 
100 
41 
7 
18.24 
0 
42 
3 
16.42 
100 
43 
— 
9 
16.87 
200 
44 
10 1 
IV.36 
0 
43 
ii 
17.42 
100 
46 
12 
17.77 
100 
. 47 
13 
18.09 
100 
48 
14 
18.37 
0 
49 
15 
16.11, 
100 
30 
16 
16.96 
200 
31 
1? 
19.44 
0 
52 
18 
17.50 
100 
33 
19 
17.82 
100 
34 
20 
18.13 
.100 
33 
21 
18.40 
0 
56 
22 
16.56 
IOC 
57 
23 
16.99 
200 
58 
24 
19.47 
0 
39 
25 
17.52 
100 
60 
26 
17.85 
100 
61 
27 
18.15 
100 
62 
2.8 
18.42 
0 
63 . 
29 
16.58 
100 
"DAY""" 
of 
EXPER 
DATE 
► 
PRE-DOSE 
LEVEL |DOSE 
THEORET' ra* 
6 4 
30 
r 
17.01 
100 
65 
31 
17.40 
100 
66 
i,OV , 
1 
17.35 
100 
62 
2 
.10.15 
100 
68 
3 
18.42 
100 
69 
4 
18.6 i. 
0 
70 
5 
16.80 
71 
6 
17.21 
1(X) 
72 
7 
1 
17.56 
inn 
78 
8 
17.89 
inn _ 
74 
9 
18.19 
100 
75 
10 
18,46 
100 
76 
11 
18.70 
0 
77 
12 
16,32. 
100 
T: DOLTICAL PLASMA AT A 31 IKE 
LEVELS 01' rUPPRTSOIVE DOSAGE 
600 mg, per week 
COUP ALT B SECTION* 2. 
Dosage Constant - 2,32 micrograms/liter/lOO rag. dose 
Die-Away Constant - 0.10. 
Underlined Levels occur on days on which nen were bled. 
TABLE L ■ PAY 
— 
of 
DATF 
LFVEL 
DOSE 
EXPi-JR 
• _ , . _ 
TH OHL'T 
im 
0 
\u^. 
0 
200 
1 
10 
4.13 
200 
2 
11 
7.94 
200 
3,... 
12 
11.32 
200 
; L 
13 
14.36 
200 
\ 5 
14 
17.10 
~1J i*J " 
200 
! 
6 
15 
19.57 
0 
7 
16 
17.61 
100 
1 B 
17 
17.93 
100 
L 9 
18 
18.22 
100 . 
rH 
19 
18.49 
100 . . . 
] 
. 11 
20 
18.73 
100 . 
i 12 
21 
18.9^ 
100- 
13 
22 
19.13 
0 
u 
23 
17.22 
0 
rH 
24 
17.59 
100 
. 16 
25 
17.22. 
1? 
26 
13.22 
100 
13 
27 
18.49 
100 
19 
28 
18.73 
100 
20 
29 
18.94 
0 
21 
30 
17.05 
100 
22 
31 
17.43 
100 
23 
Sept. 
17.78 
100 
24 
2 
18.09 
100 
25 
3 
IB. 37 
100 
26 
- Jl 
18.62 
27 
5 
18.85 
0 
28 
6 
16,96 
100 
29 
7 
17.35 
100 
30 
8 
17.71 
100 
31 
9 
18.03 
100 
DAY 
of 
i .XPEfi 
> ’ mr' 
L’iiib 
♦ 
PR?-DOSE 
LEVEL 
TH ,C: T 
DOSE 
nur. 
32 
10 
18,31 
100 
33 
11 
13.57 
100 
34 
12 
18.30 
0 
35 
13 
16.91 
100 
. 36 
14 
17.31 
100 
37 
15 
16.67 
100 
_ 3a 
16 
17.99 
100 
. 39 
17 
13.28 
100 
40 
IB 
18,53 
100 
41 
19 
18.76 
0 
L2. 
20 
16.33 
100 
- 4 .3 
21 
17-23 
TOO 
44 
22 
17,64 
100 
45 
23 
17.96 
100 
46 
24 
18.25 
100 
47 
25 
13.51 
100 
48 
26. 
18.15 
0 
49 
27 
16.37 
100 
. 5u 
23 
17.24 
100 
51 
29 
17.63 
100 
52 
30 
17.95 
100 
53 
Oct^ 
18.24 
100 
34 
2 
18.50 
100 
55 
3 
18.74 
0 
56 
4 
16.37 
100 
57 
5 
17.27 
IOC 
6 
17.63 
- IOC 
59 
7 
19.95 
IOC 
60 
ft 
13.24 
IOC 
61 
9 
18.50 
IOC 
62 
10 
18.74 
0 
63 
11 
16.87 
IOC 
JAY 
of 
JXPER 
DATE 
.'R'-OOSE 
LEVEL 
TH JRET 
JOSE 
mg 
64 
12 
17.27 
100 
65 
13 
17.63 
100 
66 
14 
* 17.95 
100 
67 
15 
18.24 
100 
68 
16 
18.50 
100 
69 
17 
18.74 
0 
70 
18 
16.8? 
100 
71 
19 
17.27 
100 
. 72 
20 
17.63 
100 
77 
21 
17.95 
100 
74 
22 
18.24 
100 
- 75 
23 
18.50 
100 
76 
24 
13.74 
0 
77 . 
25 
16.87 
0 . 
73 
26 
15.00 
0 
.... 79 
27 
13.50 
0 
BO 
28 
12.15 
0 
SI 
29 
10.93 
0 
82 
30 
9.84 
0 
83 
31 
8.36 
c 
84 
Rov 
7.?7 
0 
35 
2 
7.71 
0 
86 
3 
6.45 
0 
8? 
4 
5.80 
0 
88 
5 
5.22 
0 
89 
6 
4.70 
0 
90 
7 
4.2,3 
0 - 
91 
8 
3.81 
100 
. 92 
9 
5.52 
100 
93 
10 
7.06 
100 
. 94 
11 
8,44 
100 
95 
12 
9.68 
100 
... 96 
13 
10.80 
100 
97_ 
_14J 
11.81 
THEORETICAL PUS’A LFV XS OF JUNGLL CROUDS 
CORBIN D A An.) B GROUPS 
1200 nig, dosage in first 6 days, 600 ng. tier week tiiereafter. 
Dosage Constant - 2.32 micrograms per liter per 100 mg. nose, 
Die-Away Constant- 0.10. 
Underlined Levels occur on days on which men were bled. 
TABU' 5 COMPANY 
C SECTION 1 
COMPANY 
C SECTI01 
¥ 2 
DAY 
OF 
EXPSR 
DATS 
PRE-DOSE 
LEVEL 
THEOHET. 
DOSE 
(mg.) 
1 
DAY 
OF 
EXPER 
DATE 
PRE-DOSE 
LEVEL 
THEORET. 
DOSE 
(mg.) 
71 
Nov^ 
1] .87 
■ 
50 
• 
71 
NoVy 
17-21 
100 
72 
8 
11-73 
5o 
72 
8 
17.56 
100 
73 
9 
11.60 
100 
73 
9 
17.69 
100 
7h 
10 
12.53 
50 
7U 
10 
18.19 
IOC 
75 
n 
12o32 
5o 
75 
11 
18.1*6 
100 
76 
12 
12.13 
0 
76 
12 
18.70 
0 
77 
13 
10,92 
0 
77 
13 
16.83 
0 
78 
11* 
9.91 
0 
78 
Hi 
15.15 
0 
79 
15 
8.92 
0 
79 
15 
13.61* 
0 
80 
16 
8.03 
0 
80 
16 
12.28 
0 
81 
17 
7-23 
0 
8i 
17 
' 11.05 
0 
82 
18 
6.51 
0 
82 
18 
9.95 
0 
63 
19 
5o86 
0 
63 
19 
8.96 
0 
81* 
20 
5.26 
0 
81* 
20 
6.06 
0 
PLASMA ATABRINE LEVELS 
THEORETICAL VALUES FOR DIEAWAI AFTER SUPPRESSIVE THERAPY 
Dosage Constant - 2*32 micrograms/liter/lOO mg. dose. 
Dieaway Constant - 0,10 
Underlined Levels occur on day on which men were bled. 
TABLE 6 APPENDIX C 
PROCEDURES AND RESULTS OF INDIVIDUAL STUDIES 
SECTION I - Collection, Preparation and Analysis of Plasma0 
lo Principles of analysis„ 
aQ The procedure used for the analysis of plasma for atabrine 
was that developed by Brodie and Shannon (Malaria Report No0 9* N0R.Co)c 
Briefly* the method is based on measurement of the intensity of 
fluorescence* which is proportional to the concentration of atabrine 
in a suitable medium,. Preliminary removal of interfering fluorescent 
materials is accomplished in two steps; (l) by extraction of the organic 
base, atabrine* from alkalinized plasma by shaking with ethylene di- 
chloride* (2) transfer of the atabrine to concentrated lactic acid, 
again by shaking* from the ethylene dichloride„ The fluorescence is 
read directly in the lactic acid solution with a photoelectric fluoro- 
meter0 Minor modifications were made to facilitate handling large 
numbers of specimens0 
bo Atabrine is present in the leukocytes in very much higher 
concentration than in plasma0 Since leukocytes begin to disintegrate 
shortly after blood is drawn* it is imperative to separate the plasma 
soon after drawing the blood in order to avoid contamination of the 
plasma by leukocyte atabrine0 It is necessary to free the plasma of 
all intact leukocytes by adequate centrifugation*, These conditions 
can be fulfilled only when the blood stands less than 15 minutes be- 
fore centrifugationc The possibility of accidental contamination by 
leukocytes is minimized by the removal of plasma after 15 minutes of 
centrifugation after which the plasma is spun an additional hour0 
Considerations of speed in handling the blood as well as the desira- 
bility of reducing interference with military training to a minimum* 
led to the final procedure for obtaining and processing the blood 
samples„ 
20 Collection of samples0 
ac The bleeding schedules were arranged to collect 20 samples 
every 10 minutes (30 ml each)* This number made for efficient use of 
the centrifuge equipment; each group of 20 specimens was treated as a 
unit, no additional samples being collected until these had been placed 
in the centrifuge» In this way flexibility of schedules was secured 
which permitted minor delays without endangering a large number of 
specimenso The ten-minute deadline insured ample leeway for meeting 
the maximum intervals of 15 minutes between withdrawal of blood and 
start of centrifugingo 
1 b0 Three bleeding stations were required to secure the 20 
specimens in this time0 This required a maximum of 7 bleedings at 
each station in 10 minutes. This rate was easily managed by the team 
of assistant and bleeder manning each station,, Generally the 20 
samples were collected within 7 minutes; a complete section of 100 
men was handled in 50 minutes. The blood was drawn in a 30 ml syringe, 
containing 6 drops of saturated potassium oxalate® 
3® Preparation of samples® 
ae The 6 or 7 samples collected at each bleeding station 
during one bleeding period were delivered to the preparation room in 
the small rack used to hold the tubes during the bleedingo The tubes 
were here transferred to the centrifuge for the first centrifugation 
(15 minutes running time, 3 minutes for deceleration). Thereupon the 
specimen tubes were removed and placed in a rack already half filled 
with numbered conical centrifuge tubes® The plasma was carefully 
aspirated with a clean dry 10 ml syringe fitted with a 3“inch No® 19 
gauge needle and transferred into the conical tube. The schedule 
permitted 15 minutes for the transfer by 2 men of the 20 specimens 
in each run. At the completion of the first separation the plasmas 
were returned for the final 1-hour centrifugation® 
b® When the centrifugation was finished a measured portion 
of the plasma was withdrawn from each conical tube without disturbing 
the sediment, and placed in a correspondingly numbered 60 ml bottle in 
a rack. The final aspiration was accomplished with a syringe pipette 
(Chart 14) calibrated to deliver 10 ml at full extension® The pipette 
was also graduated at full ml intervals down to 6 ml for use when less 
than the full amount of plasma was available® The syringe used to 
transfer plasma was rinsed 3 times with saline after each use® At the 
completion of the transfer a check was made of the plasma samples 
against the bleeding forms and the numbers were transferred to the 
analysis form® The samples (in dust-proof boxes) were held in a 
refrigerator at 40°F for analysis the next day® 
Uo Treatment of samples. 
a® The plasma samples in the 60 ml glass-stoppered bottles 
were delivered to the laboratory in dust-proof boxes and rechecked 
against the analysis forms® 
b. All samples for one day (up to 150 specimens and 25 
controls and standards) was carried through as a unit® The use of 
numbered glassware for the various steps through which the material 
passed and the use of racks which had uniform spacing expedited the 
handling of the large numbers of specimens® 
c® Each specimen and control recovery(on blood-bank plasma) CHART -14 
SYRINGE PIPETTE 
-SOLDERED AFTER 
CALIBRATION 
DIMENSIONS A - B 
10 ML '.1 41 
30 ML ..l| 4 | 
INSERT TO ADAPT 250 ML TRUNNION 
CUPS TO CARRY 5 TEST TUBES 
4 - 
8 
55 COPPER TUBING 35'l.D. 
BRASS TUBING || "|. D. 
CHART - 14 passed through the following steps, in order: 
(1) 3 ml of 0o2il is added to each bottle from 
an automatic 50 ml burette, 
(2) 30 ml of ethylene dichloride is added to each bottle 
from a calibrated syringe pipette, 
(3) 3 racks, containing 10 samples each, are shaken for 
10 minutes in a mechanical shaker (240 cycles per 
minute)0 
(4) The contents of each bottle are then poured into a 
40 ml heavy duty pyrex centrifuge tube supported in 
a racko 
(5) The tubes are centrifuged in a 16-place head, for 10 
minutes at 2000 rpm, 
(6) The supernatant plasma is aspirated and discarded by 
means of a drawn glass tube connected to a water pump, 
{7) A 20 ml aliquot of ethylene dichloride is drawn off 
with a calibrated syringe pipette. The syringe is 
rinsed with ethylene dichloride three times between 
samples, 
i (8) The aliquots are transferred to a second set of 
numbered 60 ml pyrex bottles, 
(9) 11 ml of diluted lactic acid parts purified 
commercial lactic acid to 1 part distilled water) is 
added to each sample from a calibrated syringe pipette 
(10) The bottles are shaken again in groups of thirty for 
10 minutes, 
(11) After the samples are poured into numbered 
cuvettes (preselected 20 x 150 ram pyrex test tubes) 
in a wire reck, 
(12) The cuvettes are centrifuged for 10 minutes at 2000 
rpm in a 16-place head, 
(13) The cuvettes are returned to the wire racks and 
placed in a water bath ready for reading in the 
fluorometer, 
5, Measurement of fluorescence, 
a, A Colman fluorometer (Model 12) was modified to permit the measurement of fluorescence to be made on the upper part of the cuvette. 
By this means it was possible to read the fluorescence of the lactic 
acid solution stratified above the ethylene dichloride, thus 
eliminating transfer to another tube, 
bo Standard Pyrex test tubes (20 x 150 mm) were selected 
for cuvettes on the basis of uniformity of size and freedom from optical 
defects*, A standard atabrine solution was read in each tube and only 
those tubes which gave readings within ± 1% were selected, 
Co The fluorometer was warmed for one hour before any measure- 
ments were made, thus increasing stability of the instrumento The 
electrical zero of the instrument was set with the shutter closedo The 
sensitivity was then set near its maximum, using a lactic acid atabrine 
standard equivalent to 100 micrograms of atabrine per liter (0,667 
micrograms of atabrine base in 11 ml of diluted lactic acid). The 
sensitivity was checked by measuring the fluorescence of a standard 
every $ or 10 readings, and readjusting to the original setting, if 
necessaryc If the change was greater than 2 scale divisions, the tubes 
read in the meantime were retested, 
do The solutions and standards were kept in the same water 
bath for at least half an hour before reading to insure uniformity of 
temperature of all solutions0 Readings were made as rapidly as possible 
to avoid temperature changes of the solutions while in the instrument,* 
60 Calibration and calculation of atabrine concentration0 
a0 The primary standard used contained B5o0 mg of atabrine 
dihydrochloride dihydrate (corresponding to 66,6? mg of atabrine base) 
in a 1 liter of a M/15 phosphate buffer (pH 7*8). This solution was 
kept in the refrigerator and small samples withdrawn as neededc 
be A working standard was prepared daily by diluting 5 of 
the primary standard with 100 ml of phosphate buffer (pH ?08) and 
distilled water to 500 mlo The final concentration of atabrine base 
was 667. microgr&ms/Lo Intermediate standards varying from zero 
atabrine up to the concentration of the working standard were prepared 
by appropriate dilution of the working standard. The concentration 
of buffer was kept uniform in all dilutions, 
Co The final standards used for the daily calibrations were 
prepared by shaking 1 ml of the diluted standard with 10 ml of lactic 
acid and 20 ml of ethylene dichloride, These were transferred to 
cuvettes and centrifuged in the same manner as described for the blood 
sampleso 
The temperature coefficient of atabrine fluorescence in lactic acid 
was found to be approximately linear in the range from 10° to 50°Co It 
amounted to about lo5 microgram/C° for a concentration equivalent to 
100 microgram/L of plasma0 do Permanent standards were made up in lactic acid essentially 
as described above for daily standards except that no ethylene dichloride 
was added* Cuvettes were filled with this solution and sealedo These 
standards were read every day to check the daily standards* Observa- 
tions over a period of three weeks showed no appreciable change in the 
permanent standards0 
e. Control recoveries were prepared by transferring lo0 ml 
of each atabrine dilution to each of two 60 ml bottles containing 10 
ml of human plasma which had been preserved in the frozen state* These 
solutions were then analyzed in the usual way* 
fc The fluorometer readings were converted into plasma 
atabrine concentrations by means of a calibration curve which was 
determined for each daycs readings* Values for both the standards 
and the recoveries were plotted and straight lines were fitted visually* 
During moat of the period of study 6 standards were used (in duplicate) 
corresponding to plasma atabrine concentrations of 0, 12* 5$ 25* 50* 
66, and 100 micrograms per liter and 6 recovery standards (in duplicate) 
on blood-bank plasma corresponding to 0, 8*3, 16*7* 33°3, UUoU and 660? 
micrograms per liter* 
g* Hie recovery calibration curve ordinarily corresponded to 
a recovery of 95% or better over most of the range* However* the zero 
atabrine plasma recovery value was generally slightly higher than the 
zero atabrine standard value* resulting in a crossing of the recovery 
and standard curves near the origin* 
7o Washing of apparatus« All glassware was washed in hot 
calgonite solution, rinsed with hot tap water, then cold distilled 
water and dried in a drying oven. 
8. Discussion of the method and the limits of its accuracy* 
a* It is believed that the management of the bleeding and 
the plasma preparation were sufficiently controlled to meet all the 
requirements of the present study. Accidental contamination by 
leukocytes or extraneous fluorescent materials presented no problem. 
Out of several thousand analyses performed, there were no more than 
8 or 10 instances in which contamination was demonstrated, A decided 
advantage is gained, of course, by the uniformity of handling which 
obtains when analyses are being carried out on a large scale* 
b. Most of the plasma atabrine levels encountered were at 
the lower limits of suitability of the technic, as used. The limiting 
factors in the procedure are; (l) extraneous nonfluorescent light - 
this gave deflections equivalent to 8 to 12 micrograms per liter of 
atabrine; (2) extraneous fluorescence (irreducible blank in reagents) - 
amounting to another 8 to 12 micrograms per liter; (3) instrumental 
limitations, imposed by the sensitivity (always used at maximum 
sensitivity) of the instrument, stability of the instrument, stability of the light source, and the residual inequality of the cuvettes0 
The net results of these factors was such that the reproducibility 
of results in the range up to 30 micrograms was generally within 
jt 2 microgramso The absolute value of the analytical results was 
generally reliable enough to keep the overall precision within this 
range of 2 micrograms, though, in a few runs the calibration pro- 
cedure undoubtedly led to considerably greater absolute errorsa 
c. The convention used in converting fluorometer readings 
to atabrine concentrations consisted in drawing the best straight 
line through the points fixed by the control recoveries on plasma„ 
This procedure implicitly assumes that all plasma samples were sub- 
ject to the same factors that influenced the control samples and 
that the control plasma recoveries in their significant character- 
istics were identical to the plasma samples that were analyzedo On a 
few runs the recoveries were considerably less than the usual 95%° 
In several of these cases it is believed that this procedure of 
assigning values based on the recoveries led to a correct readjust- 
ment of the analytical values; in several others, however, assignment 
of value on the basis of a low recovery line led to results which 
were inconsistent with the previous and subsequent behavior of the 
plasma levels of the men0 In these cases the difficulty was not 
identified but the plasma used for the recoveries must remain suspect. 
In general, of course, the absolute values assigned in a comparative 
procedure such as used here can never be better than the values found 
for the comparison standards (in this case the control recoveries); 
hence, the inherent reproducibility of the procedure influences the 
absolute values and may lead to a systematic error in all determina- 
tions,, An additional hazard which may lead to a small systematic 
error at low levels consists in unrecognized nonlinearity between 
galvanometer deflection and atabrine plasma level, the best line 
drawn through either recoveries or standards generally passed below 
the zero atabrine points Presumably this resulted from nonlinear 
response of the instrument. 
d0 These pitfalls indicate the necessity of careful control 
of the procedure if useful results are to be secured at the lower 
plasma levels«, 
e a Certain fairly simple steps might permit more efficient 
and more reliable work in the very low ranges (up to 10 or 15 micro- 
grams) of plasma atabrine0 These are, (l) concentration of the same 
amount of atabrine into a smaller final volume (requiring modifica- 
tion of the fluorometer)« This would lessen the difficulties due to 
low sensitivity of the instrument, the scattered light, and the blank 
fluorescence, (2) Improve the fluorometer as to stability and 
sensitivity, (3) Careful selection of filters to minimize the 
amount of exciting light passed to the photo-cello (U) If possible, 
find a suitable substitute for lactic acid that can be freed more 
easily from fluorescent materials0 9. Reagentso 
a# Lactic acid - Commercial lactic acid was tested by- 
shaking with ethylene dichloride of known purity (as regards fluorescent 
materials) and reading in the fluorometer* If the blank value thus 
obtained was within one scale division of that given by purified (see 
below) lactic acid no purification was done„ (l scale division » 1,5 
microgram per liter atabrine)0 Some lots of lactic acid have given 
slightly lower readings before purification than after, so it is re- 
garded as desirable to omit purification unless there is definite 
evidence that it is necessary* 
(1) Four-liter lots of lactic acid were shaken in a 
Pyrex glass stoppered bottle with about 4 grams of bone charcoal 
(Norit A Pfanstiehl) and allowed to stand overnight0 The mixture was 
then treated with about lo5 grams of Filter-Cel analytical grade 
(Johns Manville Co0) and filtered with suction through a 10 inch 
Buchner funnel, using two sheets of filter paper (Whatman #2) 0 The 
filtrate in general gave a low blank, but since it usually gave a 
positive Tyndall effect it was refiltered through a 3-1/4 inch 
Pyrex sintred glass funnel, fine porosity0 The filtrate thus obtained 
gave a negative or inappreciable Tyndall effect and a low blank0 It 
was stored in Pyrex glass stoppered bottles and used without further 
purification0 
b„ Diluted lactic acid - 180 c,c0 of distilled water is 
placed in 2 liter volumetric flask and made up to 2 liters with lactic 
acid0 
Co Ethylene dichloride0 In part of the work the ethylene 
dichloride was purified with Norit0 Approximately 4 grams of Norit 
was added to ethylene dichloride in 4 liter bottles shaken, allowed 
to stand Several hours, and filtered twice through filter paper0 
Extreme care must be taken to remove the last trace of charcoal,, 
do Disodium phosphate solution,, A 0o2 molar solution of 
Na^O^o 
effl Phosphate buffer solution was prepared by making a mixture 
of 9 parts of k/7o5 molar and 1 part of M/?o5 molar H^PO^o 
fo. Stock standard - A sample of pure atabrine dihydrochloride 
dihydrate obtained from Drs James A0 Shannon*s laboratory was usedc On 
drying over sulfuric acid in a vacuum dessicator at room temperature 
the material lost 6% in weight, corresponding to B5% of the theoretical 
amount for 2 molecules of water of crystallization, and it also changed 
somewhat in color from canary yellow to orange yellow, 85oO mg of the 
undried material was dissolved in water, 500 ml of M/7o5 buffer, pH?c8 
(e,above) added and diluted to 1 liter with distilled water and stored 
in the refrigerator„ 10. Personnel used, 
a, Bleeding station at the Replacement Training Center. 
(1) Bleeders: - 3 officers. 
(2) Supervisor: 1 officer. 
(3) Assistant Bleeders: 2 (civilians) cleaned equipment 
in spare time and 1 (enlisted man) helped separate 
plasma at end of bleeding. 
(4) Plasma Preparation: 2 (enlisted men) sharpened and 
sterilized the needles, and arranged the equipment for 
the bleeding when not actually handling the samples. 
b. Chemical Laboratory. 
(1) Technicians: (4 enlisted men) 3 of these worked on 
the actual analysis; one purified reagents, etc. 
(2) Supervisor: 1 officer. 
(3) Cleaning and preparation of glassware: 2 civilians, 
11. Reliability of certain results. 
Analytical difficulties with the specimens taken on the follow- 
ing dates gave basis for questioning the reliability of the values 
obtained: Jungle Groups, 9-11-43 (Ho + 3 only) 9-13-43, 9-20-43, 
10-27-435 Co. B, Section-1, 10-15-43, 10-18-43; Co. B, Section 2, 
10-27-43; Co. C, Section 2, 9-18-43. APPENDIX G 
SECTION II - Plasma Atabrine Levels Established in Two large Groups 
Under Different Dosage Regimens. 
1. Procedure. 
a. Subjects. One hundred (100) soldiers were selected from 
Company B and one hundred (100) from Company C of the Fifth Battalion, 
Armored Replacement Training Center. These men had recently been in- 
ducted into the army and were in their first week of basic training. 
Selection was on the basis of platoons rather than on an individual 
volunteer basis for two reasons: First, taere would be less inter- 
ference with training; and second, a random sample would be obtained. 
Men were lost from the experiment from time to time as a result of un- 
avoidable transfer, etc. Consequently, at the end of the study 84 of 
the original 100 men remained in each group (168 in all). The subjects 
were white men from all sections of the United States, approximately 
one half being between IB and 20 years of age, one quarter between 20 
and 25 and one Quarter between 25 and 37. This distribution agreed 
closely with that of the Array as a whole. The average weight for the 
entire group at the beginning of the study was 157 pounds and at the 
end of the study 160 pounds. 
b. Environment. The experiment was carried out during the 
twelve weeks between 26 August and 13 November, 1943* August was dry 
and very hot. October was cooler with comparatively little rainfall. 
November was cold ana damp, (See Chart 15) 
c. Activity. A brief outline of the training program of the 
men during the experimental period follows; 
eek 
Activity 
1-2 
Orientation, School of Soldier. Chiefly indoor 
lectures and demonstrations. 
3-4-5 
Use and firing of rifle, carbine and tommy.gun. 
Chiefly outside duty on ranges. 
6 
Preliminary work on Cal, 30 G. Preparation for 
infiltration and village fighting courses. 
7 
Firing Cal. 30 MG on ranges. 
8 
Chiefly outside review of training. 
9-10-11 
Driving light and medium tanks 6n ranges„ 
12 
Firing 76 aim howitzer, 76 mm tank guns and 81 mm 
.mortar. TEMPERATURE IN °F 
DAILY AVERAGE AND TOTAL RANGE 
CLEAR 
CLOUDY 
HAZE OR FOG 
RAIN AND 
OR SNOW 
RELATIVE HUMIDITY - % 
DAILY AVERAGE AND TOTAL RANGE 
WEATHER DATA FOR THE MONTHS OF THE EXPERIMENT 
CHART- 15 
DAILY PRECIPITATION 
IN INCHES 
CHART-15 In addition to the above, the men had 3 hours of dismounted drill and 
3 hours -or physical training each week; a four-hour march each week lur- 
ing weeks five through seven; arid a twenty-five mile march during week 
eight. 
d. Glotiling. Food and Shelter. 
(1) The men wore standard fatigue uniforms consisting of 
coveralls, helmet liners, canvas le, gings and leather 
shoes. 
(2) Diet consisted of regulation army garrison rations. 
(3) Men lived in a standard army oarracks throughout the 
exper iment. 
e. Dosage of Atabrine, 
(1) Atabrine was administered at the noon mess by one of 
tne officers assigned to the project. In the twelfth 
week when Company 3 was placet on full therapeutic 
doses, it was administered at each meal. On entering 
the mess hall each nan >icK.ed up a cup of water, took 
a tablet, and after swallowing the tablet ana ariracing 
-water, called his name to the officer who checked the 
men as they passed. A similar procedure was followed 
in the field. In the fourth week it was discovered 
that one subject had been able to expectorate the 
drug despite all precautions. Thereafter at intervals 
the oral cavities were examined after administration 
of the drug and as an added precaution six of the men 
having the lowest plasma levels were given the drug 
in the solution for a period of 2 weeks. No change 
in plasma levels of these men occurred. 
(2) The dosage and bleeding schedules for both companies 
for the entire experiment are given in Table ?. They 
were designed wo cause no interference with the regular 
basic training program. :ach group of 100 men was sub- 
divided into two sections of fifty men each.' One 
section from each group received 0,4 gm of atabrine a 
week, ana the second section, 0.6 gm per week. It 
was originally planned to follow the two dosage regimens 
recommended by the Surgeon General's Office.*' But to 
meet these schedules would have necessitated keeping 
the men on the ost every Sunday for three months which, 
for reasons of morale, was felt to oe in dvisablej more- 
over, the- limitation in number of samples that could be 
analyzed in one day reouirea rearrangement of schedules. 
To trust the men to take any uose except umer uirect 
* SGO Circ. Letter r: 1335 August 1743 ODSACtE A.I) SA ’’LL'S SOI; 1UL.S V;X C;V /AT 3 B AH ./ C, FIFTH SAT, A' r' * , A. .T.C. 
Doses in ; llligrams * 
..is:s os•: trough line 
SUN. 
. 
rVJT‘ O 
X ' Hj « 
• 
THUN. 
FRI. 
SAT. 
Co. B. 
h2 & f 2+5 
D 
Section 1 
Mo Drug 
100 * 
100 
50 
50 
50 
50 
H2& H2+5 
Hx 
Co. 9. 
Section 2 
No Drug 
100 
100 
100 
No Drug 
100 
200 
r* hi 
UO. u • 
H2 & H2+5 
Hi I 
Section 1 
Mo Drug 
50 
100 
100 
■ 
50 
50 
50 
1 2 H2+5 
H 
1 
CO « C a 
Section 2 
No Drug 
100 
100 
-t ry \ 
100 
No Drug 
200 
NNcKS T 
CM AMD ;^LhViN 
Co. 3. 
H2 
ii 
Section 1 
1 
50 
100 
50 
■ 50 
50 
50 
50 
Co. B. 
H 
p 
Hi 
Section 2 
100 
100 
100 
100 
No Drug 
100 
100 
Co • C • 
H2 
H 
Section 1 
i 
50 
50 
100 
50 
50 
50 
50 
Co. C 
"2 
H1 
Section 2 
100 
100 
100 
100 
100 
No Drug 
100 
V.NDK T NLVi. 
Co. B 
Ha 
Hb 
He 
Both Sections 
500 
300 
300 
300 
300 
300 
loo 
Co. C. 
Kx 
H 
H 
Both Sections 
.7 
No Drug 
No Drug 
No Drug 
No Drug 
No Drug 
No Drug 
No Drug 
Hi and Ho * 1130 hrs. 
ii0 4 5 — —-—- 1630 hrr . 
' ajbjO—— — Samples During therapeutic 
Dosage 
*tx,y,2—■— Samples for Dleeway 
table 7 observation by an officer was also considered in- 
advisable. Dosage schedules for the first nine 
weeks were therefore slightly altered from the stan- 
dard SGO regimens, as indicated by Table 7. The 
dosage and bleeding schedules of the two sub-groups 
receiving 0.1 grams per week ana the two sub-groups 
receiving 0.6 grams per week were so designed that 
there would be a minimum of difference in pattern of 
dosage between the pairs of groups receiving identical 
total quantities weekly. 
(3) Beginning with the 10th week the men were kept on the 
Post continuously for the remaining three weeks and 
the schedules revised (Taole 7) to fit the SGO 
schedules. 
(4) At the beginning of week 12, Company B was put on 
full therapeutic doses of atabrine as described in 
SGO letter No. 153. Both sections of this company 
were given 0.5 gm atabrine on Sunday, 14 November; 
(0,1 gm at breakfast, 0.2 gm at lunch, and 0.2 gra 
at supper) 0.3 gm daily for the next five days (0.1 
gm at breakfast, lunch and supper) and 0.1 gm at 
breakfast on the seventh day. Blood samples were 
taken before the noon meal on trie 4th, 5th and 6th 
days (18, 19, and 20 November). 
(5) After the last dose for week 11, atabrine was discon- 
tinued in Company C in order to determine the rate 
of decline of the olasma atabrine level, 
f. Observations. A brief medical history which included 
age, state of birth, history of superessive or definitive malaria 
therapy, history of Jaundice, and general statement as to previous 
health, was obtained on each man, A daily check was made during the 
study of all visits to the dispensary by experimental subjects and by 
the other soldiers from both companies. Records were kept of all com- 
plaints except those obviously having no possible connection with 
administration of atabrine; e.g. trauma and upper respiratory infections. 
All members of the experimental group hospitalized for any reason were 
followed curing hospitalization and atabrine dosage was continued un- 
less contraindicated. Biood samples were taken regularly on certain 
men who were hospitalized for minor injuries. Direct questioning as 
to possible gastroenteric tract symptons was scrupulously avoided. 
For the sake of morale, however, the medical officers in cnarge of 
the study were more than ordinarily receptive to the minor complaints 
of the men, 
g. Blooc Sampling," Blood samples (30 ml) were taken at 1130 
hrs,, prior to noon mess, throughout except when special samples were 
* For full details concerning management of blooc samples see Section I drawn at 1630 hrs. in order to determine peak concentrations follovdng 
individual doses of atabrine. The first sample of each week was designated 
as Ht, the second sample H2, ard the sample taken for peak concentration 
H2+5• (See Appendix D, Par. 2) 
h. Presentation of Data. 
(1) Tabulation of raw data. Complete records of the 
plasma atabrine levels for all subjects and for all 
bleedings, listed by weeks and time of bleeding are 
tabulated by Company and Section in Appendix £. 
Levels obtained on therapeutic dosages and the die- 
away concentrations obtained during the twelfth week 
as well as those obtained with the suppressive dosages 
are included. From this complete tabulation one can 
study closely the behavior of each experimental sub- 
ject throughout the experiment, 
(2) The geometric mean levels and standard geometric 
deviations for each group of subjects have been de- 
termined for every set of blood samples. These 
parameters were obtained both graphically from log- 
probability plots of the data and by direct computation 
and the two were in close agreementJtip.ess than + 1 micro- 
grams), In Tables 8, 9, IQ Hand 12," the statistical 
parameters for all the data are summarized, including 
the geometric mean (MIANq) and the standard error of 
the mean (expressed as the 68/S range of the means,) 
and the dispersion of the individual plasma atabrine 
concentrations about the geometric mean (in terms of 
the standard geometric deviation and the 6B% range 
of dispersion) of the individual levels. The arithmetic 
mean plasma atabrine level is also given for each set 
of data in these Tables0 
2. Results. 
a. Time required to reach equilibrium. The characteristic 
behavior of the group mean plasma levels may be seen from inspection of 
Charts 16 and 17 where it will be noted that trie time required to reach 
equilibrium level was in general the same - without regard to magnitude 
of dose,'- Variability' with respect to this secular trend was wide. 
* The apparently irregular rhythmic behavior of certain groups is the 
result of a differing interval between dosage and sampling. The erratic 
results which occurred in the week of 10-15, Chart 16 and 10-22, Chart 17 
were the result of aberrations in the chemical method which affected all 
samples on that day. 
-** These tables will be found at the end of this section0 MEAN6 PLASMA ATABRINE LEVELS OF TWO GROUPS OF MEN RECEIVING 0.4 GM./WEEK 
MICROGRAMS PER LITER 
MICROGRAMS PER LITER 
H +5 MEANG 
MEAN X SD 
MEAN -r SC 
MEAN G 
CO. B 
SECT. I 
CO. c 
SECT. 1 
68 % RANGE 
CHART-16 
WEEKS 
WEEKS 
o 
5 
m 
DATE 
CHANGE IN DOSAGE 
SCHEDULE (SEE TABLE 
CHANGE IN DOSAGE 
SCHEDULE (SEE TABLE 
DOSAGE IN GMS. 
DOSAGE IN GMS. 
CHART-16 MEAN. PLASMA ATABRINE LEVELS OF TWO GROUPS OF MEN RECEIVING 0.6 GM./WEEK 
MICROGRAMS PER LITER 
MICROGRAMS PER LITER 
- MEAN X SD 
- MEAN -r SD 
•MEAN G 
H +5 MEAN. 
CO. C 
SECT. 2 
CO. B 
SECT. 2 
% RANGE 
CHART-17 
DATE 
WEEKS 
DATE 
CHANGE IN DOSAGE 
SCHEDULE (SEE TABLE 
CHANGE in dosage 
SCHEDULE (SEE TABLE) ' 
DOSAGE IN GMS. 
$ 
m 
m 
Tv 
CO 
DOSAGE IN GMS. 
CHART-17 as will be seen later* Approximately 8£, for example, reachea a level 
in two weeks from which they did not subsequently depart significantly. 
Fourteen per cent appeared to have reached equilibrium levels by the 
4th week, whereas 9m jnay not be considered to have stabilised at ail 
since their highest plasma levels occurrea in from the 8th to 11th weeks. 
Vihen, therefore, one speaks of equilibriian stabilization with the dosage 
it is to be recognized that this is a term of convenience ana must remain 
at tiift most a concept which is applied in this discussion to the mean 
plasma atabrine level of a group and not the level for an individual within 
the group. In keeping with the initial statement, in this section, during 
me second ana third weeks the rates of increase in plasma atabrine were 
quite constant; accordingly, individuals who stabilized in the earlier 
weeks, in general dia so at levels which were lower than were those of in- 
dividuals who reached their equilibrium levels in the sixth or seventh 
weeks. Special mention is made of the behavior of these low men at this 
time for tv;o reasons: first, this portion of the group will merit special 
consideration in later discussion of variability in equilibrium levels and 
second, to point out that the time to reacn equilibrium is determined not 
by great difTerences .in the rate at which a given plasma level is approached, 
but rather tiiat with the rate constant, it is the plasma level characteristic 
of the individual which determines the so-callea time of ecuilibriwii. 
b, Magnitude of Group Equilibrium Level. The group mean plasma 
levels at equilibrium were found to be directly proportional to the dosages 
(Chart 18); on a regimen of 0,4 gm/uk the mean level was 12,2 micrograms per 
liter; with 0.6 gm/wk, a mean of 17o2 wicrograms per liter was attained.. 
These values are the meann of all and K2 values of weeks 7 to 11 in- 
clusive. Alterations in time relationship between dosage and sampling may 
change group values. However, when the dosage pattern was changed from 
that regularly followed to that recommended by the EGO, no differences in 
mean plasma levels were noted, c.f. Charts 16, 17, 18. 
c. Variability in Individual Equilibrium Levels. The dispersion 
of individual values about the mean as represented by the standard deviation 
of the data is shown graphically in Charts 16 and 17. The values included 
by a range of 1 sigma on either side of the mean are also listed in the 
tables. Froia the above and the standard error of the means, the stability 
of the means becomes apparent. 
Hot evident from inspection of either the charts or tables 
is the wide variance of certain individuals wnose plasma levels fall outsiae 
the 68f> range and who are characterized by either consistent low values 
or by high values which resulted from sudden departure from previous levels 
and which then, declined somewhat, but in general, remained elevated above 
the remainder of the group. In view of the small size of the universe 
of study the validity of special consideration of the small numbers of 
subjects who lie at the extremes may be questioned since the sample was 
insufficient to make certain that one universe only was under study. One 
assumes, nonetheless, that with a sufficiently large soriole the discontinuity 
would be eliminated. The characteristics of the group at the extremes are 
worthy of discussion, however since they illustrate types of behavior which 
may be of great practical importance. CHART-18 
COMPARISON OF MEANG WEEKLY PLASMA ATABRINE LEVELS 
IN TWO GROUPS OF MEN ON DIFFERENT DOSAGE SCHEDULES 
(H, VALUES) 
WEEKS OF EXPERIMENT 
PLASMA ATABRINE CONCENTRATION 
MICROGRAMS/ LITER 
CHANGE IN DOSAGE 
SCHEDULE { SEE TABLE 7 ) 
EQUILIBRIUM LEVELS 
_ MAINTAINED BY EACH 
'group during the 
7-11 WEEKS 
MEANg WEEKLY LEVEL ON GROUP RECEIVING 0.4 GM /WEEK (84 “100 MEN) 
MEANg WEEKLY LEVEL ON GROUP RECEIVING 0.6 GM /WEEK (84-100 MEN) 
CHART-18 Approximately 7% of men showed unusually low levels as 
compared with the levels of the remainder of the group in both the 0o4 gm 
and 0o6 gm groups. As mentioned above these men reached ecuilibrium 
earlier and maintained consistent low levels, tills being apparently a 
characteristic of individual response to the drug. Thst the low values were 
not the result of failure to take the drug or to lack of solution of the 
tablet in the gut was established by the aaministration of atabrine in 
solution. This resulted in no change in plasma values. Another 7% of the 
subjects in both the 0,4 and 0,6 gm groups exhibited tae phenomenon of 
breakaway with more or less suauen departures from previous plasma levels 
to exceptionally high ones. This change to high levels occurred at various 
times between trie 3rd to 8th week, there being no regularity in its appear- 
ance. After achieving the new high level the behavior varied. In the 
main the breakaway was followed by a graaual decline to a new level signifi- 
cantly higher than either the level from which the subject had previously 
departed, or the level for the group. These sporadic increases in plasma 
level were not associated with clinical evidence of toxicity nor was there 
associated evidence of liver damage. (See Appendix C, Section V) 
Typical curves of plasma atabrine levels illustrating the 
performance of men with unusually high or low levels as compared with the 
usual response are presented in Chart 19. A possible explanation of these 
widely variant levels is given in Appendix G, Section V„ 
d. Significance of variability. The wide dispersion of values 
about the mean becomes a matter of considerable practical importance in 
any consideration of the extent of protection afforded a population by any 
standard dosage regimen. From the point of view of characterizing the 
population as a whole the mean arid standard deviation are useful tools<, 
From the point of view of management of troops in the field, however, it 
is the status of the percentiles of the group with the lowest plasma 
levels that most concerns us since we may properly assume, other things 
being equal, that tnis fraction of the group has the least protection and 
will manifest the highest malaria attack rate, ,ith this in mind we 
examine the population in the different weeks with respect to the per- 
centages of the total which exceed or fail to achieve any arbitrarily 
selected pi:sma level„ In Charts 2C and 21 the distribution of concentration 
in each week is plotted as a single curve. These are smoothed probability 
curves obtained from the calculated geometric means and standard geometric 
deviations of the data.* They represent the most probable prediction curves 
which emerge from the present stuay. Applying the level of 10 micrograms, 
for example, to these charts one finds (Chart2C) that at equilibrium (weeks 
7-11) 37$ of the group receiving 0.4 gm/wk were still below this level 
whereas only 7$ of the 0.6 gm/wk group (Chart21) were below the level when 
stabilized. It is also apparent, even in the 0.6 gm group, that the maximum 
* The curve for week 4 (Fig. 21) is incongruous and has been omitted. Data 
upon which it was derived are unreliable because abnormally high results 
were obtained from all analyse e from the clay of sampling0 CHART- 19 
DIFFERENCES IN 
PLASMA ATABRINE LEVELS OF INDIVIDUALS RECEIVING 0.6 CM./WEEK 
HIGH TYPE OF 
RESPONSE 
PLASMA ATABRINE IN MICROGRAMS PER LITER 
TYPICAL TYPE 
OF RESPONSE 
LOW TYPE OF RESPONSE 
MEN RECEIVED SUPPRESSIVE DOSAGE DURING WEEKS 1-11; THERAPEUTIC DOSAGE DURING WEEK 12 
MEN RECEIVED SUPPRESSIVE DOSAGE DURING WEEKS Ml; NO DRUG DURING WEEK 12 
CHART-19 PLASMA ATABRINE IN MICROGRAMS PER LITER 
CUMULATIVE FREQUENCIES OF MINIMUM WEEKLY PLASMA ATABRINE LEVELS OF GROUPS OF MEN 
NUMBER OF MEN 84 - IOO 
CURVES DERIVED FROM THE 
meang AND STANDARD 
DEVIATION 
(A RTC. COMPANIES BBC) RECEIVING 0.4 GM / WEEK 
CHART - 20 
NUMBERS DENOTE 
WEEK 
CHART-20 PLASMA ATABRINE IN MICROGRAMS PER LITER 
CUMULATIVE FREQUENCIES OF MINIMUM WEEKLY PLASMA ATABRINE LEVELS OF GROUPS OF MEN 
(A RT. C. COMPANIES B 8. C) RECEIVING 0.6 GM. / WEEK 
NUMBER OF MEN 84*100 
CURVES DERIVED FROM THE 
MEANG AND STANDARD 
DEVIATION 
CHART —21 
PERCENT 
NUMBERS DENOTE 
WEEK 
CHART-21 level for the group Is not achieved until the 7th week of administration. 
Another significant fact which emerges from comparison of Charts 20 and 21 
is that the lowest 5% of men who received 0.6 gro/wk had higher plasma 
levels at the eno of the 2na week than aid the lowest 5% of men on 0.4 
gm/v.k when the latter group were at eouilibrium from the 7th to 11th weeks * IT 
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TABLE 12 APPENDIX C 
SECTION III - Effects of a Simulated Jungle Climate Upon Plasma Atabrine 
Levels, 
1. Procedureo 
a* Subjects. 
(1) Thirty (30) enlisted volunteers served as experimental 
subjects. Fifteen (15) were 19 years old, nine (9) 
20 years, two (2) 21 years and one each 22, 2h, 31 
years. They came originally from all parts of the 
United States and had been in the Array or $ months. 
Their military service included reception center, basic 
and battle training. The men were of representative 
body types, and in varying states of physical fitness. 
After three weeks of preliminary training and testing 
they were divided into two groups of 15 men each. 
These groups, hereafter called A and 3, were similar 
in respect to age, bodily configuration, weight, 
section of the country from which the men came, physical 
fitness, cardiovascular and thermal response to a 
standard work procedure. This similarity existed for 
both the group averages and the distributions within 
the groups (e.g, equal numbers of small and tall men in 
each group), 
(2) Throughout the study both groups were handled in exactly 
the same manner in all respects except in climatic 
exposure. One group initially resided in the simulated 
jungle climate, the other in the Fort Knox climate, 
b. Environment. 
(1) In the hot room a hot humid (jangle) climate was simu- 
lated. During the day (OCCO to 1700 hours) the ary 
bulb temperature averaged 90°F with extremes of B9°F to 
93o5°F. The relative humidity was usually 92$ to 95$ 
with extremes of 100$ and BS$. During the night (1800 
hours to 0630 hours) the dry bulb temperature was ?B°F 
to 87°F (usually o/+°F) and the relative humidity 75$ to 
85$. It required one hour to change from day to night 
climate. Ho radiant heat was supplied. Air movement 
was turbulent but not greater than that made by the 
moving men. The men lived continuously in this climate 
except for a 10 minute clean-up in the morning and 
evening. (2) outside environment varied from a hot dry summer 
period to cool fall weather* This climate is indicated 
in Chart 22, showing dry bulb temperature (°F) and 
relative humidity as the'average values for all of the 
readings at 15 minute intervals during each work period 
throughout the week. The hot humid climate is similarly 
plotted on the same chart. 
c. Activity and Work* 
Both groups followed a standard daily work procedure 
consisting of walking five "work-periods" a day at 
2-1/2 miles in 47 minutes (approximately standard army 
pace) carrying a 20 pound pack. Between successive 
work periods there were 13 minutes of rest during which 
observations were made on the men. Two successive work 
periods in the morning and three in the afternoon ( a 
total walk of 12-1/2 miles) constituted a standard work- 
day, Five work periods daily on the first five days of 
the week, three periods on Saturday and none on Sunday 
constituted the standard work-week. One hour periods of 
organised athletics, calisthenics or close order drill 
were at times substituted for work periods in the outside 
group. The energy expenditure during this substituted 
activity was comparable to that of a work period as 
Judged by the responses of the heart rate, rectal temper- 
ature and sweating rate* 
d. Clothing, Food. Water, Sleep. 
During the work periods all men wore the regulation 
fatigue coverall, cotton shorts, socks and shoes* The 
food consisted of standard garrison rations and was 
salted to individual taste. Extra salt was not added* 
During work, water salted to a final concentration of 
0*l£ was permitted as desired. This was the only water 
permitted in the hot room at any time* The group out- 
side drank salted water during the work periods, fresh 
water at other times. Approximately B-9 hours were 
permitted for sleep, 
e. Dose of Atabrine. 
Both groups began taking atabrine on 9 August 1943> the 
same dosage and procedure being followed in each group. 
During six days of the first weak each man received 0*1 
gm with his morning and evening meals, to give a total 
of 1.2 gms/wk* During the following 11 weeks each man 
received 0*6 gm/wk, 0,1 ga daily for six days with the 
noon meal Monday through Friday, with breakfast on Sat- DRY BULB AIR TEMPERATURE , ° F 
RELATIVE HUMIDITY , PERCENT 
HOT ROOM 
OUTSIDE 
CLIMATIC CONDITIONS DURING WORK HOURS 
WEEK OF EXPERIMENT 
CHART- 22 
CHART-22 urday0 No drug was given on Sunday., Rigid precautions 
were taken to insure the swallowing of the tablets. 
f. Rotation of Exposure. 
After 2 weeks of preliminary training for all subjects 
group A entered the jungle climate while group B 
remained outside in the summer climate. On the same day 
(9 August 1943) both groups began suppressive atabrine 
therapy (See Pare e). The men in group A were permitted 
to leave the hot room from 1300 hours Saturday until 2200 
hours Sunday. After seven weeks of exposure in the hot 
room group A was moved outside and group B entered the 
jungle climate. For the next four weeks group B remained 
continuously in this environment; the B men were not 
permitted to leave on Saturday afternoon and Sunday as 
had group A. 
g. Observations. 
(1) Before and after each work period the following 
observations were made; (a) general appearance and 
symptoms, (b) heart rate and blood pressure in both 
erect and supine positions, (c) rectal temperature. 
The water intake ana urine output and the weight (sweat) 
loss, within 10 grams, were determined. 
(2) The 24 hour water intake of the jungle group was measured 
daily and the 24 hour urine output and specific gravity 
of 10 men of each group (A and B). The urinary chloride 
excretion per 24 hours and the plasma chloride concen- 
• tration, the plasma protein and the hemoglobin were 
determined at regular intervals on 10 subjects in each 
group before and during their exposure to heat. 
h. Blood Samplingo 
Thirty (30) ml of blood were withdrawn for each plasma 
atabrine determination. Three samples were taken each 
week (a) - 1115 Monday, before the first atabrine 
dose of the week, approximately 54 hours after the pre- 
ceding dose; (b) H2 - 0630 Saturday before the last 
atabrine dose of the week, IB hours after the preceding 
dose; (c) H2 4 5 - 1130 Saturday, 5 hours after the last 
atabrine dose of the week. 
2, ResultSo 
a. The work performance and rate of acclimatization in the hot 
jungle environment of the two groups of men are compared in Chart 2> The 
two groups differed in that the men in group A received atabrine regularly 
from their first day in the heat whereas group 3 had already received the Chart- 23 
CHANGES IN HEART RATE, RECTAL TEMPERATURE AND BLOOD PRESSURE 
DURING ACCLIMATIZATION TO WORK IN JUNGLE HEAT 
HEART RATE 
(PER MINUTE) 
RECTAL TEMPERATURE 
(°F ) 
BLOOD PRESSURE 
(MM HG.) 
DAY 
cloudy sunny 
ENVIRONMENT 
WORK PERIODS 
- GOOL- 
■ JUNGLE HEAT 
GROUP A {is men) 
GROUP B (15 men) 
NOATABRINE GROUP (e men) 
CHART - 23 drug for seven weeks before exposure to tne jungle environment. For further 
comparison, the performance of a third group who received no atabrine is also 
shown on the Chart. Two iiuportant characteristics of the experiences of these 
three groups must be mentioned. First, the work performances on the first day 
and thereafter and the progress of acclimatization among the subjects did not 
differ in any significant way from that observed in previously reported studies 
of the effect of moist heat upon the performance of young men,* Second, there 
was no essential difference with respect to work performance or rate of accli- 
matization between the three groups. It is evident, therefore, that the pro- 
gressive accumulation of atabrine in the body concurrent with heat exposure, 
or continuing to take atabrine after an equilibrium level has been established 
in the cool do not in any way affect the physiological reactions of young men 
exposed to moist heat. With or without the drug the rate of acclimatization 
and ability to work in a jungle climate are the sane. 
b0 The plasma atabrine levels attained by groups A and B, who re- 
ceived the drug according to the sane dosage schedules out under different en- 
vironmental conditions are given in Tables 13 and 14** and compared in Chart 
24. ilo difference in the rate of increase or of the final equilibrium levels 
will be observed between the two groups, nor was there any significant change 
in the equilibrium level in either groups when the subjects were shifted from 
one environment to the other. In this connection it should be noted that the 
outside exposure of group A, following their jungle exposure represented a 
much greater climatic shift than was experienced by group B who received their 
outside exposure first, during the late summer. Thus, the fact that group A 
showed no measurable alteration in plasma atabrine level is more significant 
than the fact that the level for group B did not change. The increased sweat- 
ing rate experienced in the jungle environment had no effect upon the plasma 
atabrine level as shown in Chart 25. The equilibrium levels reached (7th 
through.11th weeks) by the two groups and the levels reached by the larger 
ARTC group who received the same weekly maintenance dosage did not differ 
significantly from the level of 18 micrograras/L predicted by the dosage schedule, 
the deviations being approximately'equivalent to one standard error. 
Exposure 
No, 
Results 
No. 
Men 
Equilibrium atabrine level 
on 0.6 gra dosage per week 
and dispersion of levels 
(7th through 11th weeks) 
MeanQ 
S. Eo of MeanQ 
Std. Geo. Deviation 
Outside, ARTC 
croup 
B5B 
*5 
17.2 
1.04 
1.44 
7 wks jungle 4 
wks outside. 
Group A 
205 
15 
19o9 
1.17 
1,41 
7 wks outside, 
4 wks jungle. 
Group B 
205 
15 
19.9 
i.oa 
1 
H 
• 
VO 
N' 
* Reference A1SRL Report on Project Wo. 2 (2-7, 11, 13, 15, 17, 19) dated IB 
October 1943* 
** These tables mil be found at the end of this section. PLASMA ATABRINE CONCENTRATION 
MICROGRAMS / LITER 
MEANG PLASMA ATABRINE CONCENTRATIONSJUNGLE GROUPS 
GROUP B OUTSIDE 
GROUP A IN JUNGLE ENVIRONMENT 
WEEKS 
CHART- 24 
DATE 
GROUP A 
GROUP B 
ABERRANT VALUES 
JUNGLE ENVIRONMENT 
GROUP A OUTSIDE 
GROUP B IN 
OUTSIDE 
GROUP B 
OUTSIDE 
GROUP A 
DOSAGE IN GMS. 
CHART- 2 4 SWEAT LOSS DURING WORK 
GRAMS PER 5 WORK PERIODS 
PLASMA ATABRINE 
CONCENTRATION 
MICROGRAMS / LITER 
-GROUP B 7 — WEEK, OUTSIDE 
HIGH RATE OF SWEAT LOSS IN JUNGLE DOES NOT AFFECT PLASMA 
ATABRINE CONCENTRATION 
DAY OF WEEK 
CHART- 25 
-GROUP B WEEK, IN JUNGLE 
DAY OF WEEK 
CHART - £5 Tho.mean levels for the three groups are approximately the same as is 
the degree of individual variability as measured by the standard 
deviations. Both jungle groups had their low and high men and others 
exhibiting breakaways. It is concluded, therefore, that the hot, humid 
environment has no effect upon the process of absorption and storage 
of atabrine and that the required dosage schedule for effective suppres- 
sive therapy is not influenced by the climatic conditions studied H-* 
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p- 
5£A% AND CHARACTERISTICS 
OF WEEKLY PLASMA 
ATABHINE LEVELS 
FOR 
JUNGLE GROUP B 
0o6 gm/wfc 
BLEEDING Hg 
TABLE 14 APPENDIX C 
SECTION IV - Special Studies 
1. Absorption of atabrine following a single dose and effect of 
"booster" doses upon time to reach equilibrium, 
a. Procedure, 
(1) Subjects. In order to obtain further information on 
the behavior of atabrine, with respect to its rate of 
absorption and removal from plasma in relation to dosage, 
certain special studies were carried out employing an 
additional group of 14 healthy, young (18 to 20 years) 
volunteers. These special studies began in mid-summer 
and continued into mid-fall (for environment see Chart 15, 
Section II and Chart 22, Section III. Throughout the 
entire period these men engaged in regular company duty, 
(2) Grouping by dosage schedule - These subjects were divided 
into three groups for study of absorption under different 
dosage schedules. Groups C-l and C-2, of four men each, 
were put under the same basic schedule of single daily 
doses of 0,2 gra for 17 out of the first 19 and 20 days 
for the two groups respectively, and 0,1 gms, 6 days a 
week, for the remainder of the study (10 weeks for C-l 
and 9 weeks for C-2), These and other minor differences 
were considered not sufficiently great to prevent com- 
bining the results obtained for these two groups. Dosage 
schedule for group C-3 consisted in single daily doses of 
0.3 grams for six days during the first week, and daily 
doses of 0,2 grams for six days during the second weekj 
for the next seven weeks the daily maintenance dose of 
0,1 gram, six days per week, was administered. Thus, 
after higher dosage schedules during the first three weeks, 
all three groups were subjected to the same maintenance 
schedule for the remainder of the study. 
(3) Blood sampling - Blood samples (30 ml) for determination 
of absorption curves were taken before the drug was given 
and 2, 4, 6, 8, 12, 24, and at times 48 hours after the 
dose. In addition, samples were obtained regularly three 
times weekly in accordance with the schedule already 
described (H1, H2, H2 + 5). 
b. Results. 
(1) Absorption, The rise and fall of plasma atabrine concen- 
tration immediately following the administration of a 
single dose is shown in Charts 26 and 27 (data in Table 15)* 
* Tables will be found at end of Section. CHART- 10 
POST ABSORPTION CURVES OF PLASMA ATABRINE CONCENTRATION 
FOLLOWING 0.2 GM DOSE 
AFTER 16TH DOSE OF 0.2 GM 
(8 MEN ) 
PLASMA ATABRINE-MICROGRAMS / LITER 
AFTER 0.2 GM IN IOTH WEEK 
OF C-I.C-2 REGIMES (7-MEN) 
AFTER 4TH DOSE OF 0.2 GM 
{ 4 MEN) 
HOURS AFTER ORAL DOSE OF 0.2 GM, ATABRINE 
CHART— CHART- 27 
POST ABSORPTION CURVE OF PLASMA ATABR1NE 
FOLLOWING 4TH DAILY DOSE OF 0.3 GM. 
PLASMA ATABRINE-MICROGRAMS / LITER 
6 MEN 
HOURS AFTER ORAL DOSE OF 0.3 GM. 
CHART- 27 for subjects who had previously received varying amounts 
of the drug, from none up to a sufficient quantity to have 
established equilibrium. The absorption curves have the 
same general shape, although the absolute plasma levels 
resulting from a given dose vary, depending upon the 
previous atabrine dosage. Absorption from the gastro- 
intestinal tract is rapid, A major portion of the rise in 
concentration takes place within 2 hours and the peak level 
is reached within 8 hours. Thereafter there is a decline 
in concentration. In 24 hours the level is reduced to a 
point which is above the pre-dose level, the net gain in 
concentration for a given dose depending upon status of 
the subjects with respect to previous Intake of the drug. 
If no dose is given after one day, the concentration 
continues to decline. With a single dose of 0,3 gm (Chart 2?) 
except for the greater increase in level induced by the 
larger dose, the results were similar to those following 
the 0,2 dose. Some of the curves are bimodal in shape, 
with two peak values which are believed to characterize the 
mechanism of absorption, A more extensive consideration of 
these curves, from the standpoint of their significance 
with respect to the buildup of suppressive and therapeutic 
atabrine levels is given in Appendix B. 
(2) Effect of initial booster dose. It was shown that the 
mean equilibrium plasma atabrine level is directly pro- 
portional to the dosage; and that the time required to 
reach an equilibrium State is independent of dosage 
provided the weekly dosage schedule remains constant. 
Furthermore, it has been demonstrated that the underlying 
plasma level at any time when expressed as a percentage 
of the final equilibrium level, is the same regardless of 
dosage. It follows directly from these statements that 
the time required to reach a desired plasma level can be 
shortened if, at the outset, the drug is administered at 
a dosage rate higher than that required to maintain the 
desired level in equilibrium. An example will make this 
clear. The rate of rise in plasma level in any week is 
approximately 50$ of the difference between the level 
already established and the final equilibrium level. Thus 
at dosages of 0,6 and 1,2 gm/wk the predicted plasma levels 
at the end of the first and subsequent weeks will be: 
Week 
Plasma Concentration 
0o6 gm/wk 
1*2 gm/wk 
1 
9.0 
18.0 
2 
13.5 
27.0 
3 
15.7 
31.5 
4 
16.9 
33.7 
5 
17.4 
34.8 
Equilibrium 
18*0 
36.0 At the higher dosage rate of 1.2 gm/wk the plasma level 
at the end of the first week is as high, according to 
this theoretical relationship, as is trie equilibrium 
level ultimately attained by the 0.6 gm dosage. If, 
, therefore, the dosage auring the first week only is 
doubled as compared with the subsequent maintenance 
dosage, the plasma concentration will be raised to the 
desired level within this period rather than in 5 weeks 
or more, which is required without the initial booster 
dose0 This conclusion is partially borne out by the 
experimental evidence as shown in Chart 28, in which are 
compared the plasma atabrine levels in relation to time 
for the two groups (C-l arid 2, C-3) on the same mainten- 
ance dosage rate but with differing dosage schedules 
during the first two weeks. For comparison, the curve 
of mean plasma level for the AliTG group on the constant 
0,6 gra/wk schedule is shorn. Also plotted on the chart 
is the curve for the jungle group, which received an 
initial booster dose of 1,2 gra/wk for the first week. 
In the C-l and 2 and C-3 groups, plasma concentrations 
in excess of the ultimate maintenance level were reached 
within a week. It will be noted that the time required 
to reach the actual equilibrium state was not changed 
but it was approached by a declining concentration curve 
from higher levels in contrast to the progressively 
rising curves for the group receiving no booster dose. 
The jungle group, receiving the double dose for the first 
week only, did not follow the theoretical prediction 
exactly, showing a decline during tne second week and 
close agreement with the ARTC group thereafter. It is 
to be noted that the equilibrium levels finally attained 
on the maintenance regimen of 0,6 grn/wk did not differ 
significantly among the four groups. The practical 
significance of the initial high dosage schedule is 
great since the time required to reach an effective 
suppressive level may be an important factor in the 
planning of troop movements into malarious areas. The 
regular inclusion of the initial booster dose in the 
schedule of administration of atabrine is therefore 
recommended, 
2, Rates of buildup and dieaway of plasma atabrine level. 
a. Procedure. At the end of the eleventh week both jungle groups 
A and B were allowed to work out-of-doors and administration of the drug 
vras discontinued in order to study the rate of fall of plasma atabrine 
level (dieaway). The subsequent rise of concentration on restoration of DAILY DOSE 
GRAMS OF ATABRINE 
PLASMA ATABRINE- MICROGRAMS/LITERS 
(MEANG OF SAMPLE TAKEN 20'24 HRS. AFTER LAST DOSE) 
PLASMA ATABRINE LEVELS OBTAINED WITH DIFFERENT PRIMING DOSES 
MAINTENANCE DOSAGE, 0.6 GM. / WEEK 
1. COS. B ac, SECT. 2, IOO MEN 
COB DOSE SOLID BARS 
CO.C DOSE OPEN BARS 
2. GROUPS Aae, 30 MEN 
CHART- 28 
DAYS 
3. GROUPS C ft C9,8 MEN 
4 GROUPS 0,, 6 MEN 
CHART-28 sud ressive atabrine therapy (buildup) was also determined. Half of these 
men were without drug for one week and the other half for two weeks. In 
oraer to insure comparability of the two uieaway curves, groups A and B 
were rearranged into two new groups (X and I) which were comparable with 
rsspect to: duration of previous heat exposure, Lime since removal from 
the jungle environment, weight of men, plasma ataurine level over the four 
week period before dieaway, and atabrine levels after six days without drug. 
Atabrine suppressive therapy v/as resumed in group X after an interval of 
one week and in roup Y after two weeks without dru? (See Chart 29). The 
means and dispersion of the data are recorded in Table 16 and the crude 
data will be found in Appendix K0 To obtain additional dieaway data on a 
large group, all of Company C, ARTC group (84) men) were similarly studied. 
Company G, Section 1 (G„4 gri/vk) took their last dose on November 12, 1913; 
Company G, Section 2 (0,6 gm/wk) took their last dose on November 11, 1943; 
both were bled thereafter on November 13, 15, 17 arid 20. Chart 30 compares 
the dieaway curves on the two dosage regimens, Ko buildup studies were 
made on this group, (data in Table l6a) 
b. Results. The mechanisms which delermine the rate of increase 
of plasma level under a constant dosage schedule and tire rate of decrease 
from an equilibrium level after aiscontinning the drug are corrsiaered theo- 
retically in Appendix B and the values predicted from the theory are com- 
pared with the observed concentrations. The rate of dieaway amounts to 
approximately IQ.a per day resulting in a drop of about one-half in a weekQ 
Restoration of the dosage regimen results in Increasing plasma levels which 
approach the equilibrium level at the same percentage rate as obtains with 
the original administration of the drug, namely, 5Qt per week of the 
difference in plasma level at the begiruing of the week ana the equilibrium 
level (See Chart 29). 
3. Plasma levels obtained with therapeutic doses. 
a. Procedure. At the beginning of the 12th week the two sections 
of Company B, ARTC Group, after having developed equilibrium levels on their 
respective dosage schedules of 0.4 ana 0,6 gm/v/k, were placed upon a modified 
therapeutic regimen, as follows: 
1st day - 0,5 gm (0.1 gm at breakfast and C.2 gin at lunch and supper) 
Wext 5 keys - 0,3 gm (0.1 gm at breakfast, lunch and supper) 
?th dap' - 0,1 gm at oreakfast 
Bloou samples were taken before the noon meal on the 4th, 5th and 6th days. 
b. Results. 
• (3.) The increases in plasma levels, as shown in Chart 31, 
for the two sections at the end of the period were approxi- 
mately the same (above tneir previously established equi- 
librium levels, 30.h arid 30,5 micrograms/L respectively), 
These changes in concentration with one institution of 
the new dosage regimen ivere found to follow tie save basic 
law of buildup as irad previous 1/ determined the increase 
toward equilibrium on the lower suppressive regimen and CHART - 29 
INFLUENCE OF INTERRUPTION OF DOSAGE ON 
PLASMA ATABRINE LEVELS 
(0.6 6M. / WEEK SCHEDULE, 30 MEN) 
WEEKS 
PLASMA ATABRINE CONCENTATION 
MICROGRAMS / LITER 
GROUP X 
RESUMED DOSE 
DRUG STOPPED 
GROUP Y 
RESUMED DOSE 
DOSAGE IN GMS. 
GROUP X (MEANg OF 15 MEN ) 
GROUP Y (MEANg OF 14 MEN ) 
CHART- 29 PLASMA ATABRINE IN MICROGRAMS PER LITER 
NO. OF MEN 32 “ 39 
CO. c 
SECT. I 
DIE AWAY CURVE FQR GROUP THAT RECEIVED 
0.4 GRAMS PER WEEK FOR ELEVEN WEEKS 
MEANg PLASMA ATABRINE LEVELS OF TWO GROUPS OF MEN 
FOR ONE WEEK AFTER DISCONTINUING SUPPRESSIVE THERAPY 
DATE 
WEEKS 
CHART-30 
meang 
MEAN X SD) 
MEAN ~ SD) 68 °/o RANGE 
DOSAGE IN GMS. 
DIE AWAY CURVE FOR GROUP THAT RECEIVED 
0.6 GRAMS PER WEEK FOR ELEVEN WEEKS 
CO. c 
SECT 2 
DATE 
NO, OF MEN 43'45 
DOSAGE IN GMS. 
CHART- 30 CHART - 31 
MEANG PLASMA ATABRINE LEVELS OF TWO GROUPS OF MEN 
RECEIVING THERAPEUTIC DOSAGE FOR ONE WEEK 
THERAPEUTIC DOSAGE AFTER RECEIVING 
0.4 GRAMS PER WEEK FOR ELEVEN WEEKS 
THERAPEUTIC DOSAGE AFTER RECEIVING 
0,6 GRAMS PER WEEK FOR ELEVEN WEEKS 
WEEK 
WEEK 
CO. B 
SECT. 1 
CO. B 
SECT. 2 
cl 
LU 
_J 
CL 
LU 
CL 
CO 
< 
CL 
C0 
O 
CL 
0 
-Z. 
LU 
z 
DL 
CD 
1 
< 
CO 
< 
CL 
PLASMA ATABRINE IN MICROGRAMS PER LITER 
DOSAGE IN GMS. 
DATE 
DATE 
NUMBER OF MEN 39-42 
NUMBER OF MEN 40“4I 
MEANG 
MEAN X SDl 
MEAN t- SDj 
68 % RANGE 
CHART — 31 tnere was close agreement between uhe levels predicted 
by the theoretical equation ana the observed values0 
(2) An important objective of this phase of the study was 
to determine whether or not the men with persisting low 
and high plasma levels while on suppressive therapy 
would continue in the same relation to the group mean 
uncier the subsequent therapeutic regimens. The correlations 
are shown in Charts 32 and 33» Chart 32 shows the dis- 
tribution with respect to the equilibrium levels attained 
under the two suppressive regimens am the subsequent 
distribution of these low, central and high men after 
receiving therapeutic doses as outl ined above, ilo great 
shift in distribution will be noted, the majority of the 
subjects remaining in their original categories under the 
therapeutic regimen. The correlation is shown in another 
way in Chart 33 where therapeutic level is plotted against 
suppressive level. A definite relationship is evident 
but the correlation is not high. One cannot, therefore, 
oredict with any c the relative therapeutic 
level for an individual in a group from his relative 
suppressive level except in broad categoriesa NUMBER OF MEN 
PLASMA ATABRINE IN MICROGRAMS PER LITER 
LOW LEVELS ON SUPPRESSIVE THERAPY 25% 
CENTRAL LEVELS ON SUPPRESSIVE THERAPY 50% 
HIGHER LEVELS ON SUPPRESSIVE THERAPY 25% 
(42 MEN) 
0.6 GRAMS PER WEEK 2.1 GRAMS PER WEEK 
RELATIONSHIP BETWEEN LEVEL ATTAINED ON SUPPRESSIVE THERAPY 
AND LEVEL REACHED ON THERAPEUTIC DOSES 
COMPANY B 
SECTION n 
CHART - 32 
PLASMA ATABRINE IN MICROGRAMS PER LITER 
( 39 MEN ) 
0.4 GRAMS PER WEEK 2.1 GRAMS PER WEI 
COMPANY B 
SECTION I 
CHART-32 CHART - 33 
RELATIONSHIP BETWEEN PLASMA ATABRINE LEVEL ATTAINED ON SUPPRESSIVE 
THERAPY AND LEVEL REACHED ON THERAPEUTIC DOSES 
COMPANY B , SECTION I 
(39 MEN) 
0.4 GRAMS PER WEEK 
CORRELATION COEFFICIENT 0.648 
STANDARD DEVIATION ± 0.009 
SUPPRESSIVE LEVEL 
THERAPEUTIC LEVEL 
COMPANY B , SECTION 2 
(42 MEN } 
0.6 GRAMS PER WEEK 
CORRELATION COEFFICIENT 0.412 
STANDARD DEVIATION ± 0.136 
SUPPRESSIVE LEVEL 
THERAPEUTIC LEVEL 
BOTH GROUPS RECEIVED 2.1 GRAMS PER WEEK 
ON THE THERAPEUTIC REGIMEN 
CHART- 33  
Date 
, 
■ ■ T— 
ft /?! 
Cl i 
Day of Experiment 
i I 
L 
( 
Honrs After Drug 
_2 
_ .4 J 
-_4_! 
_JB. ' 
. I2__ 
_Zk 1 
1:42 
4 
6 
8 
12 
24 
Jones 
9 
7 
5 
5 
4 
4 ! 
7 
12 
16 
16 
14 
14 
Gervais 
- 
8 
8 
7 
9 1 
4 1 
7 
il 
22 
231 
20 
15 
Peterson 
- 
10 
n 
7 
9 
5 1 
5 
13 i 
2? 
25 
25 
25 
Hudson 
— 
4 
8 
4 
-. 
5 
- 
3 1 
J 
3 
8 
16 
13 
15 
L 
14 
17 1 
i 
14 
MEANq 
7.3 
804 
5.6 
6.7 
3-9 
« 
?•> 
18.4 
18.0 
19.1] 
18.9 
18.0, 
Date 
tb/2Z~ 
C2 
Day of Experiment 
_ 0 
L 1 1 
?, 
L . — 
Honrs A/ter Drug 
0 
2" 
4 
6 
8 
12 
24 
20-0 
2 
4 
6 
8* 
12 
24 
Clifford 
8 
10 
5 
5 
4 
2 
8 
18 
17 
18 
18 
13 
Hedburg 
- 
8 
10 
7 
5 
4 
3 
10 
22 
19 
18 
21 
22 
18 
Brown 
- 
11 
6 
5 
5 
3 
3 
10 
18 
18 
i 19 
18 
19 
21 
Mills 
7 
5 
4 
2 
3 
2 
4 
12 
10 
10 
10 
12 
8 
MEANq 
8.4 
7.4 
5.1 
4.0 
3.5 . 
2.5 
7.5 
17.1 
15.5 
15.8 
15.6 
17.3 
MEANq (Cl &1C21 
i 
7-,9 
7.9 
5.4 
5.2 
3.7 
3.3 
8.4 
17.7 
16.7 
17.3 
17.6 
17.7 
14.3 
Date 
2/16A- 
L. 
W43 
9/18 
9/18 
9/20 
9/25 
9/25 
9/27 
10/2 
C3 
Day of Experiment 
3 
4 
5 
5 
7 
12 
12 
14 
u 19. 
Hours After Drug 
20-0 
2 
... 4 
6 
8 
12 
24 
20 
5 
53 
20 
5 
SX 
20 
Dunlap 
19 
34 
36 
27 
27 
30 
22 
27 
49 
21 
24 
34 
23 
Blanchard 
18 
2? 
31 
25 
26 
27 
20 
30 
59 
22 
31 
37 
24 
18 
Flores 
30 
45 
49 
43 
44 
44 
37 
46 
100 
44 
43 
50 
35 
- 
Cook 
26 
35 
44 
39 
37 
42 
32 
35 
81 
40 
41 
41 
30 
24 
Holly 
26 
49 
45 
36 
36 
31 
33 
3B 
54 
26 
33 
37 
33 
23 
Hastings 
30 
51 
51 
45 
47 
44 
42 
44 
106 
47 
37 
56 
30 
20 
MEANq 
24*3_ 
39.2 
42.Q 
36.0 
71.6 
31.6 
14*2/ 
-4L«iL 
.-28.8 
21.1 
The men in'Tiroiip~UZ”fecelvec 
by m; 
.sTaTce 
‘0ol gm of atabrine sftortly afteFthe' o' Hour BleeHTHgT" 
Correction of the total dosage was made by a corresponding reduction the following day0 
PLASMA ATABRrNP (Micrograms per Liter) 
GROUP C 
See sheet lx for explanation 
ot dosage schedules* 
TABLE 15 
Snee* 1 1 Date 
i 9/2 . 
' ' “ 
! 
9/3 
9/L 
9/13 
9/18 ! 9/18 
Q /^O 
9/25 
Day of Experiment 
. 
17 
18 
27 
L 32, 
32 
34 
39 
32_ 
Cl 
Hours After Drug 
20-0 
2 
4 
6 
8 
. 12 
24 
L 48 
L„33 
20 
L 2LL 
Jones 
35 
40 
42 
43 
39 
42 
34 
! 19 
f 46 
41 
56 
38 
! 42 
48 
Gervais 
44 
51 
50 
59 
62 
54 
45 
29 
i 32 
27 
36 
20 
27 
32 
Peterson 
49 
61 
56 
66 
79 
61 
53 
31 
29 
26 
32 
20 
27 
26 
Hudson 
34 
46 
39 
48 
39 
49 
33 
27 
28 
24 
31 
25 
26 
32 
MEANq 
UUQ- 
49.4 
46.7 
53.3 
52.2 
51,0 
40,4 
26,1 
33,1 
2818 
27.6 
24,8 
29*9 
33,6 
i 
Date 
„2/2_ 
9/10 
9/11 
9/20 
.5/25 
2/25. 
2/27 
10/2 
10/2 
C2 
Day of Experiment 
.17 . 
18 
19 
28 
33 
33 
35 
40 
40 
Hours After Drug 
2Q-Q 
h 2 
4 . 
6., 
8 
12 
24 
48 
— 
■ 53. 
... 2CL. 
L J5 
53 
5 
Clifford 
26 
42 
26 
37 
42 
38 
32 
25 
28 
31 
I 28 
26 
25 
28 
Hedburg 
35 
46 
47 
45 
47 
41 
36 
28 
20 
22 
26 
20 
20 
25 
Brown 
33 
55 
58 
54 
77 
53 
37 
27 
26 
28 
32 
24 
21 
29 
Mills 
16 
29 
26 
24 
25 
28 
12 
18 
14 
15 
16 
14 
— 
17 
HEANq 
-26.3 
41.9 
36.9 
38.3 
44.1 
39-0 
26.7 
24*1 
21.3 
23.,1 
24.7 
20,4 
21.9 
24.2 
MEAN& (Cl & C2) 
45.5 
41.6 
45,2 
48.0 
44j.6 
32.9 
25.1 
26.5 
25.8 
30.5n 
22.5 
[26.41 
28.6 
Date 
10/2 
10/4 
10/9 
10/9 
10/11 
10/16 
10/16 
10/18 
10/23 
10/23 
10/25 
10/30 
10/30 
11/1 
Day of Experiment 
19 
21 
26 
. 26 
28 
- 33 
33 
35 
40 
42 
47 
47 
49 
Hours After Drug 
5 
53 
. _20___ 
5 
53 
20 
5 
53 
20 
5 
„33 
20 
5 
53 
Dunlap 
21 
17 
14 
18 
16 
15 
24 
15 
19 
19 
16 
13 
17 
14 
Blanchard 
20 
- 
16 
18 
14 
15 
21 
12 
16 
17 
12 
13 
19 
12 
Flores 
35 
16 
24 
33 
- 
27 
38 
25 
27 
30 
21 
2? 
- 
30 
Cook 
31 
20 
25 
31 
26 
29 
37 
22 
29 
30 
24 
20 
30 
23 
Holly 
25 
18 
21 
24 
21 
25 
31 
- 
32 
28 
24 
21 
34 
25 
Hastings 
33 
22 
20 
21 
19 
I 
_ .... _i 
30 
32 
\ 
26 
32 
28 
22 
19 
28 
22 
~ M" -T 
MEANfi 
26.9 | 
18.5 
19,3 
23,5 
18.8 
22.6 
29.8 j 
25.0 
24.7 
19.3 
18.2 | 
24.7 
2o.b 
PLASMA ATAHRINE (Micrograms per Liter 
GROUP C 
See Sheet I4 for explanation 
if dosage schedules,. 
TABLE 15 
Sheet 2 Cl 
Date 
19/27 , 
10/2 
’lo/frl'loAlf' 10/16 
10/16 
lJLQAS 
10/21 
10/23) 
10/25, 
Day of Experiment 
41 
1 46 . 
.... 46__h 
48 
.53 
53 
55 
60 
60 
62 
61 
67 1 
69 
Hours Ax ter Drug 
3 
L 20 
. 5 
53 
20 
5 
53 
20 
_ _5 
53 
20 
5 
31 
Jones 
30 
34 
29 
27 
30 
23 
25 
31 
18 
28 
29 
1 23 
Gervai s 
24 
20 
25 
22 
35 
35 
33 
30 
35 
22 
29 
33 
25 
Foterscn 
20 
- 
25 
20 
2? 
23 
25 
24 
38 
26 
29 
34 
28 
Hudson 
26 
21 
26 
20 
29 
30 
20 
25 
26 
16 
23 
25 
20 
MEANq 
..22JL 
23-3. 
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30.7 
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Si 
0 i 
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10/18 
10/23 
10/21 
10/2? 
rio7ic 
C2 
Day of Experiment 
42. 
47 
47 
49 
54 
54 
56 
61 
61 
63 
68 
Hours After Drug 
. JlL_ 
__J2£L_ 
i_j 
53 
20 
5 
53 
20 
|_ 3 
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20 
Clifford 
27 
27 
20 
29 
16 
- 
Hedburg 
17 
25 
22 
18 
20 
32 
20 
22 
23 
23 
17 
Brown 
16 
23 
27 
20 
22 
26 
16 
29 
38 
25 
19 
Mills 
14 
13 
15 
13 
12 
16 
9 
14 
16 
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11 
MEANq 
15.6 
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22.1 
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19.8 
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14.7 
20.8 
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(Ci & C2) 
12jlQ. 
22.1 
24,6 
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24.1 
27.5 
19.5 
23.6 
28.4 
19-7 j 21.2 
Date 
11/6 
n/6 
11/8 
11/13 
11/15 
C3 
Day of Experiment 
54 
54 
56 
61 
63 
* 
Hours A fter Drug 
20 
.. 3 
53 
20 
53 
Dunlap 
15 
IB 
15 
32 
14 
Blanchard 
17 
15 
14 
- 
13 
Flores 
25 
32 
22 
32 
17 
Cook 
23 
25 
21 
18 
19 
Holly 
25 
34 
23 
32 
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Hastings 
20 
2B 
19 
19 
18 
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PLASMA ATABRTNE (1'icrograms per Liter) 
GROUP C 
See sheet U for explanation of 
dos age s chedules0 
TABLE 15 
Sheet 3  
Date 
110/301 
10/3(1 
i 
10/31 
110/30 
11/1 
11/6 
11/8 
11/13 
11/15 
Cl 
Day of Experiment 
Ik 
74 
7? 
74 
76 
81 
83 
88 
90 . 
Hours After Drug 
20-0 
2 
4 
6 
$ 
12 
24 
20 
54 
20 
53 
_ 20 
53 . 
Jones 
16 
30 
24 
29 
26 
16 
19 
18 
19 
18 
18 
Gervais 
24 
34 
35 
27 
31 
31 
33 
24 
28 
29 
29 
24 
26 
Peterson 
22 
39 
35 
29 
33 
41 
36 
22 
29 
36 
32 
31 
11 
Hudson 
17 
— 
— 
21 
25 
25 
22 
17 
20 
23 
18 
18 
19 
MEAKq 
19.5 
34.1 
35.0 
25.1 
29.4 
30.7 
29.7 
19.5 
23.6 
25.6 
23.7 
22.2 
17o7 
Date 
10/30 
10/301 
10/31 
11/1 
n/6 
n/6 
11/8 
liA- 
11 A1 
C2 
Day of fexperiipent 
68 
68 
69 
70 
75 
75 
77 
82 
84 
riours After Drug 
20^0. 
2 
4 
6 
8 
12 
24 
54 
20 
5 
53 
20 
53 
Clifford 
Hedburg 
17 
28 
31 
22 
26 
28 
29 
19 
25 
23 
19 
22 
23 
Brown 
19 
34 
- 
26 
31 
25 
31 
23 
30 
31 
23 
21 
22 
Mills 
ll 
20 
19 
14 
18 
16 
16 
13 
15 
18 
14 
13 
12 
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24.3 
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24,4 
22.4 
24.3 
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22,4 
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18,3 
18,2 
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(Cl & C2) 
17.5 
30.2 
29.2 
22.8 
27.1 
26.8 
26.9 
- 
20.7 
21.1 
22.1 
21.2 
Groups Cl and C2t- Followed for 91 and 8£ days. The men in these groups received 17 priming doses 
of 0.2 gm per day from day 0 to day 18 (Cl) and day 0 to day 19 (C2)0 Thereafter, each man received 
6 doses of 0,1 gm each weeks Post absorption curves were followed after the 1st, 4th and 16th doses of 
0o2 gm; on 10-30 (day 74 and day 68) a post absorption curve was run after a dose of 0o2 gmQ 
Group C3:~ Followed for 64 days. Received 6 priming doses of 0o3 gm per day during the 1st week| 
6 doses of 0o2 gm per day during the 2nd weeko Thereafter, each man received 6 doses of 0,1 gm each 
week. The post absorption curve was followed after the 4th dose of 0<>3 gm. 
PLASMA ATABRINE (Micrograms per Liter) 
See sheet 4 for explanation 
of dosage schedules. 
TABLE 15 
Sheet 4 MEANq PLASMA ATABRINE LEVELS OF MEN IN THE JUNGLE GROUP - REARRANGED FOR STUDY OF DIEAWAY AND RECOVERY 
WEEK 
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16.1 
ALL BLEEDINGS GROUP X 
TABLE 16 
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TABLE 16 12 
Diov 
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12.8-22.5 
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!• GROUP OF MSN (COMPANY C) RECEIVING NO DRUG AFTER 11 WEEKS OF SUPPRESSIVE DOSAGE 
2. GROUP OF MEN (COMPANY B) RECEIVING THERAPEUTIC DOSAGE AFTER 11 WEEKS OF SUPPRESSIVE DOSAGE 
Company C Section 2 
Drug Discontinued 
Company C Section 1 
MEANq AND CHARACTERISTICS OF 
PLASMA ATABRINE LEVELS 
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Therapeutic Dosage 
Company B Section 1 
TABLE 16a APPENDIX C 
SECTION V - Factors Related to Variability in Individual Plasma Levels0 
lo Excretion and Degradation0 
a® Discussion: 
(1) It became apparent early in the study that individual 
plasma atabrine levels may differ markedly from the 
group mean concentration* and that the levels attained 
were* to a large extent* characteristic of the in- 
dividuals o Thus* certain men maintained high levels 
throughout the period of the study* whereas others 
had levels consistently below the group average., Con- 
sideration of the factors which regulate the plasma 
level led to several lines of inquiry which* it was 
hoped, might make clear the basic cause of these 
differenceso 
(2) Atabrine entering the portal circulation during the 
initial absorption period is very rapidly removed from 
the blood stream® The final level which results from 
a given dosage schedule is a balance between the total 
rate of removal and the rate of entry of the drug0 
From a long-term point of view* the rate of entry 
following single doses is not significant provided 
the same total quantity is absorbed0 No investiga- 
tion of the completeness of absorption has been made 
but it is not believed that it could be responsible 
for the large differences found in equilibrium plasma 
levelso Previous investigations reported by Shannon 
on the fecal excretion of the drug in animals tends 
to bear out this beliefo The second factor which de- 
termines the balance* the rate of removal of the drug* 
is the resultant of several individual rates of removal 
by different avenues0 Among these are (a) extraction 
of atabrine from the blood stream into the tissues 
where it accumulates in high concentrations* (b) 
degradation or destruction* and (c) urinary excretion0 
The plasma level of atabrine measured 24 or 48 hours 
after the last dose of the drug is determined by* and 
reflects the tissue concentration with which it is in 
equilibrium® Constancy of the plasma atabrine level 
over a period of time with a uniform rate of intake* 
therefore, indicates that the tissue concentration 
has become stabilized and that the rate of removal 
(sum of items ‘b and c above) has become equal to the 
rate of intake® Thus* an unusually high plasma level might result from (a) decreased rate of degradation; 
(b) a decreased rate of excretion or (c) a combination 
of bothc Atabrine destruction is believed to occur 
largely in the liver, The possibility arises* there- 
fore, that some impairment of liver function might 
be present in men with high plasma levels. Moreover, 
measurement of excretion rates should indicate whether 
variation in rate of loss into the urine contributes 
significant!y to the differences in plasma level0 
b, Procedure: Liver function tests were applied as follows 
to men whose plasma atabrine levels had remained with some consistency 
in either the upper or lower ranges of their group: bromsulfalein ex- 
cretion, prothrombin time, plasma fibrin concentration, and icterus 
index, Brief descriptions follow: 
(1) Bromsulfalein excretion, 5 mg/kilo were injected 
intravenously in 1 minute , Blood samples were taken 
6, 15 and 30 minutes after the start of injection. 
Heparin was used as an anticoagulant, 0,5 nil of 
plasma were diluted with 2 ml of an M/15 phosphate 
buffer (6 parts of Ha2HP0, to 4 parts of ) and 
read in a spectrophotometer at a wave lengtfi ox 590 
mmUo After the addition of 0o06 ml of 3 NaOH a 
second reading was made at the same wavelength, A 
concentration of 10 mg bromsulfalein/100 ml plasma 
was considered 100/6, The half excretion time was 
estimated graphically, using semilogarithmic paper, 
(2) Prothrombin time. The procedure of Page (J, La, & 
Clin, Med, Page 26: 1366, *41)» using viper venom as 
a thromboplastic agent was employed. Estimations 
were made at several dilutions of the plasma in saline 
in the hope of revealing any differences which might 
have been concealed as a result of the undiluted 
prothrombin time falling on the asymptotic portion 
of the time-dilution curve, 
(3) Icterus index. The acetone procedure of Newburgh 
(Jo Lab, Clin, Med,, 22: 1192 (1937)) was used, 
(4) Plasma fibrin. Plasma was collected as for prothrombin 
time (l ml of M/lO potassium Oxalate for each 9 ml of 
blood), 1 ml plasma was diluted with 4 ml of saline, 
1 ml of 0,025 molar CaCl2 was ac*ded, the fluids mixed 
and allowed to stand 2 hours at room temperature. The 
clot was separated and washed in the same tube by 
centrifugation, once with 10 ml saline and twice with 
the same amounts of distilled water. The fibrin was 
finally transferred to a flask and the nitrogen 
determined by micro-Kjeldahl, The factor 6,25 was 
used to convert to protein. c• Results. 
(1) The results of the liver function tests are to be 
found in Table (17)* and are plotted in Charts 
(3U and 35). There is no necessity for detailed 
discussion. JSach of-the tests indicates that toere 
is no correlation between plasma level of atabrine 
and the hepatic fuiictions measured. The tests all 
yielded results within the normal ranges. Owirg to 
tne insensitivity of liver function tests these 
findings are not necessarily conclusive in eliminating 
liver impairment as one factor responsible for the 
high plasma levels. There is the possibility that 
normal efficiency with respect to the functions 
measured may have existed along with a reduced 
ability to degrade atabrine. One may however, on 
the basis of these findings, eliminate gross dif- 
ferences in liver efficiency as a significant factor, 
(2) Similarly, the results of the urine studies (Table 18 
and Chart 36) failed to show differences in urinary 
excretion that would explain the differences in 
plasma concentrations. Both of the men with high 
plasma levels excreted more atabrine in the urine 
than aid itte law men. The possibility that trie hi$i 
plasma levels are elevated because of decreased loss 
in the urine is thus eliminated. 
2, Variation in Protein Binding. 
a. Discussion; There remains for consideration another in- 
dependent factor which may explain variations in individual plasma 
levels, namely,, differences in the amount of bouno or non-active 
atabrine in the plasma with little or no variation in the free or 
active fraction of the drug. Since the latter constitutes only 10 to 
20 per cent of the total, it is evident that the overall level is largely 
determined by the amount of bound atabrine. Both tne concentration and 
total amount of atabrine in plasma are very small comparer to that 
stored in the rest of the body. One may anticipate that after the 
transient changes in concentration incident to movement of the atabrine 
into the tissues, the free atabrine concentration found in the olasma 
will be determined by the resultant balance of the concentrations in 
the various tissues, determined in turn by the respective partition, 
coefficients between these tissues and the olasma water arc! extra- 
cellular fluid. From this It follows that wnereas the concentration 
of free atabrine will be related to the net tissue concentration, the 
wbolc olasma concentration will be only an indirect measure of tissue 
* Tableswill be found at end of section. SECONDS 
PLASMA ATABRINE 
MICROGRAMS PER LITER 
PROTHROMBIN TIME 
UNDILUTED 
RELATIONSHIP OF PLASMA PROTHROMBIN TIME TO PLASMA ATABRINE LEVEL 
SECONDS 
PLASMA ATABRINE 
MICROGRAMS PER LITER 
PROTHROMBIN TIME 
DILUTED 2 TIMES 
CHART - 34 
SECONDS 
PLASMA ATABRINE 
MICROGRAMS PER LITER 
PROTHROMBIN TIME 
DILUTED 4 TIMES 
CHART — 34 CHART - 35 
RELATIONSHIP OF PLASMA FIBRIN CONCENTRATION AND 
BROMSULFALEIN EXCRETION TO PLASMA ATABRINE LEVEL 
PLASMA FIBRIN 
GRAMS PER 100 ML. 
PLASMA ATABRINE 
MICROGRAMS PER LITER 
BR0MSULFALE1N 
BROMSULFALEIN 
PERCENT RETAINED AT 30 MINUTES 
HALF EXCRETION TIME 
MINUTES 
PLASMA ATABRINE 
M1CR0GRAMS PER LiTER 
PLASMA ATABRINE 
MICROGRAMS PER LITER 
CHART - 35 URINE ATABRINE 
MILLGRAMS/DAY 
PLASMA ATABRINE 
MICROGRAMS / LITER 
PAUL 
24 HOUR EXCRETION OF ATABRINE 
IN RELATION TO PLASMA ATABRINE LEVEL 
HEMP 
/ 
CHART- 36 
GOLDMAN 
JOHNSON, O.B 
DOSAGE IN GMS 
CHART 36 concentration, reliable only so far as the extent of protein binding 
in plasma is predictable., It was not feasible to examine this 
possibility directly, but from incidentally accumulated data the 
following questions are partially answered: 
(1} Does the atabrine level of the cellular elements 
of the blood bear a linear relation to the plasma 
level in both high and low men? 
(2) Is the relationship between free atabrine and whole 
plasma atabrine more, or less, constant than the 
relationship between free atabrine and cellular 
atabrine? 
bo Procedure: Cellular atabrine measurements. In most cases 
atabrine was estimated from measurements of whole blood concentration, 
and per cent of plasma in the blcodc These values were then corrected 
to a standard (7500) leukocyte count. An example will illustrate the 
method. When the whole blood atabrine was found to be 96 micrograms/L, 
plasma from the same specimen gave 28 micrograms/L. Since the plasma 
constituted $8% of the whole blood, there was 0.58 x 28 « 16 micrograms 
in the plasma, and 96 - 16 s 80 micrograms atabrine in the cells from 
1 liter of blood. If we assume that atabrine is present in the 
erythrocytes in twice the concentration it is in plasma* then the 
erythrocyte concentration is 2 x 28 - 56 micrograms/L, The whole blood 
contained 1*2% erythrocytes, whence 0.42 x 56 - 24 micrograms in the 
erythrocytes of the cell phase. Thus, there was 80 - 24 - 56 micro- 
grams in the white cells from 1 liter of blood. The leukocyte count 
was found to be 8600. Thus for a count of 7500, the atabrine would 
have been 7500 x 56 » 49 micrograms in the leukocytes from 1 liter of 
blood. This, plus the estimated content of the erythrocytes of 24 
micrograms which is now returned to the total, gives the corrected 
cellular atabrine value of 73 micrograms which corresponds to the 
atabrine content of the cells from 1 liter of blood corrected to a 
standard leukocyte count of 7500, The symbol Ac? is used to designate 
this quantity in Table 20. The assumed value for the erythrocyte con- 
centration is uncritical in fixing the final value; if it has been 
assumed that there was no atabrine in the erythrocytes the corrected 
value would have been 70 micrograms instead of 73# while for twice the 
assumed erythrocyte concentration the corrected value would have been 
* Although this is not a valid assumption, it is acceptable here 
owing to the fact that the absolute value employed makes little 
difference in the correction (see further discussion in the text). 
The factor of 2 probably does represent a reasonable median value. 76o This arises from the fact that the chief contribution to the 
cellular atabrine is that portion contained in the white cells0 In 
a few experiments effort was made to analyze the several phases in as 
pure a fora as possible0 The leukocyte phase was isolated reasonably 
free from aped cells by the following technic: The whole blood was 
centrifuged for 10 minutes at 1000 rpm, the supernatant plasma was 
aspirated into a clean dry syringe down to the buffy layer0 The 
buffy layer was then aspirated into another syringe, transferred to 
a Kahn tube, diluted with plasma to a volume of approximately 3 ml, 
mixed and recentrifuged at 800 to 1000 rpm for 1 minute * The super- 
natant of the last centrifugation gave a suspension of white cells in 
plasma two to three times more concentrated than in the original 
bloodo The suspension was relatively free from erythrocytes0 In 
our hands this procedure for isolation of the leukocytes frequently 
led to a serious error, owing, perhaps to a preferential sorting or 
selection of the leukocytes according to size or type during the 
double centrifugation0 It offered no advantage over an estimation 
of the cellular atabrine content based upon the levels in plasma, 
whole blood, and red cells as outlined above0 Centrifugation of the 
packed red cells for 1/2 to 1 hour at 2000 rpm yielded a phase in 
which plasma was present to only a small percent and white cell counts 
were regularly below 50/mm^o Comparison of a series of whole blood 
values reached by direct analysis with values secured by combination 
of the results on the separate phases is made in Table 190 
Co Results: 
(l) Non-proportionality of cellular and plasma atabrine 
valueso t 
(a) The findings are collected in Table 2O0 In 
Chart 37 the corrected values for cellular 
atabrine and the ratios of plasma to cellular 
levels are plotted against the corresponding 
plasma atabrine concentrations. As previously 
mentioned, the men studied were selected from 
the extreme ranges of their respective groups4 
Chart 37 shows that there is a fairly definite 
correlation between the two measures of atabrine 
concentration, but not a directly proportional 
relationship such as would result if the partition 
coefficient which governs the distribution was 
the same in all men. The extent of the departures 
from a direct relation has been indicated by 
plotting the ratio of plasma atabrine to cellular 
atabrine against plasma atabrinee The ratio is 
not constant but varies with plasma concentra- . 
tion from 1 for low plasma levels to 1/3 for 
10 
high levels; that is to say, for a four-fold MICROGRAMS / LITER OF BLOOD (FOR _WBC =_7500/CU MM ) 
CELLULAR ATABRINE 
PLASMA ATABRINE IN MICROGRAMS PER LITER 
PROPORTIONALITY BETWEEN PLASMA ATABRINE 
AND CELLULAR ATABRINE 
0.6 GM. GROUP 
CHART - 37 
PLASMA ATABRINE ~ CELLULAR ATABRINE 
PLASMA ATABRINE IN MICROGRAMS PER LITER 
CHART -37 range in plasma concentration the cellular con- 
centration is only doubled. The same general 
pattern is exhibited by the relation between 
erythrocyte and plasma concentrations for a 
smaller series of men in Chart 38* On the 
other hand, leukocyte and erythrocyte concentra- 
tions appear (Chart 39) to bear a constant 
relation to each other for all concentrations, 
which is consistent with an equilibrium governed 
by a uniform distribution coefficient for all 
men. 
(b) Failure of the cellular atabrine concentration 
to increase in direct proportion to plasma con- 
centration might be explained on the grounds of 
saturation of the cell phase at low plasma 
levels, making further increase impossible. 
This explanation was eliminated, however, by an 
experiment in which erythrocytes were equilibrated 
with a solution of atabrine in Ringer*s solution 
of such concentration as to yield a normal 
erythrocyte concentration and a similar solution 
in which the atabrine was about 9 times as con- 
centrated. Approximately equal distributions 
were found, however, at both levels (see Table 21) 
indicating that the original erythrocyte concen- 
trations were far below saturation levels. 
(2) Lesser dispersion of cellular atabrine values. The 
geometric means and the standard deviations of two 
separate sets of parallel cellular and plasma atabrine 
levels are given in Table 22 for the two groups of 
men on dosage schedules of 0.4 gm/wk and 0.6 gm/wk 
respectively. A comparison of these statistical 
values reveals two significant facts: first, the 
standard deviations of the values for cellular 
atabrine levels are far smaller than for the plasma 
values (and incidentally are not much higher than 
the standard deviations of the plasma values of the 
large group from which these extremes were drawn) 
and second, while the mean plasma values for these 
extreme cases bear no relationship to the dosage 
received, the mean values of the cellular atabrine 
levels differ roughly in direct proportion to the 
dosage rates (0.4 and 0.6 gm/wk, respectively). 
These differences are illustrated in a somewhat 
different manner in Chart 40. Here are plotted on 
the same time scale, plasma and cellular atabrine 
values for eight men, four from the 0.4 gm/wk group RED CELL ATABRINE 
MICROGRAMS PER LITER 
PLASMA ATABRINE 
MICROGRAMS PER LITER 
PROPORTIONALITY BETWEEN PLASMA ATABRINE 
AND RED CELL ATABRINE 
CHART - 38 
PLASMA ATABRINE-r 
RED CELL ATABRINE 
PLASMA ATABRINE 
MICROGRAMS PER LITER 
CHART-38 CALCULATED WHITE CELL ATABRINE 
MICROGRAMS PER LITER 
RED CELL ATABRINE 
MICROGRAMS PER LITER 
PROPORTIONALITY BETWEEN WHITE CELL AND RED CELL ATABRINE 
CHART - 39 
RED CELL ATABRINE 
WHITE CELL ATABRINE 
RED CELL ATABRINE 
MICROGRAMS PER LITER 
CHART - 39 TABLE 21 
ATABRINE PARTITION 
Between Phosphate Ringer’s and Erythrocyte* 
37.5°, pH 7.25 
. • n 
• 
i 
Final Ratio firythroc:/te Phase 
, Supernatant 
Plasma 
Supernatant Cone, ± 
Name 
Atabrine 
Approx, 2e5 
Microgram®/L 
Approx, 23 
Micrograrcs/L 
tfeiss 
14 
10 
12 
Ostrowitz 
17 
10 
11 
Canada 
5 
S 
(u) 
Vandertie 
5v 
—— _ 
10 
13 
TABLE 21 TABLE 22 
COMPARISON OF MEANS AND RELATIVE DISPERSIONS 
OF PLASMA ATABRINE LEVELS AND CELLULAR ATABRINE LEVELS 
Dosage 0. 
(B-l 
.4 gm/week 
t C—1} 
Oosage 0,6 mg/week 
(B-2 & C-2) 
• 
plasma 
Aiabrine 
Ap 
Cellular 
Atabrine 
. Ac» 
Plasma 
Atabrine 
Ap 
Cellular 
Atabrine 
Ac» 
Date 
10*2 
9, 30 
10-- 
29, 30 
Number of Men 
11 
n 
13 
13 
MeaiiQ . 
17.6 
65.7 
16.2 
86.5 
1.96 
1.31 
1.95 
1*40 
Date 
11- 
•8, 9 
11- 
-5# 6 
Number of Men 
11 
11 
12 
12 
Mean^ 
16.6 
68.2 
20,3 
93.1 
J 
1*79 
' 1*33 
- ... 
1.79 
1.35 
Ap -micrograras per liter 
Ac*-’micrograms in the cells from 1 liter of blood for a white count of 7500 
TABLE 22 PLASMA ATABRINE 
MICROGRAMS PER LITER 
PLASMA ATABRINE LEVELS 
WEEK OF EXPERIMENT 
COMPARISON OF PLASMA AND CELLULAR ATABRINE LEVELS 
IN EIGHT SUBJECTS 
0 6 6M ATABRINE / WEEK 
0.4 GM ATABRINE / WEEK 
DATE 
CHART - 40 
CELLULAR ATABRINE 
MICROGRAMS / LITER OF BLOOD (FOR _WBC OF 7500 CU MM) 
DOSEAGE IN GM 
CELLULAR ATABRINE LEVELS 
WEEK OF EXPERIMENT 
0.6 GM ATABRINE / WEEK 
0.4 GM ATABRINE / WEEK 
DATE 
DOSEAGE IN GM 
CHART - 40 and four from the 0,6 gra/wk group. It can be seen 
that in the case of the cellular atabrine levels, in 
contrast to plasma concentrations, definite groupings 
of the men according to dosage is achievedo Moreover, 
a marked reduction in the difference between the high 
and the low men is apparent„ 
(3) Equilibration experiment. Limitations of time made 
it impossible to carry the investigation to the logical 
point of actually estimating the concentration of 
free atabrine in the plasma. This probably can be 
done with sufficient accuracy for the purpose by the 
procedure described by Brodie and Shannon, It in- 
volves equilibration of erythrocytes with a sufficient 
series of atabrine-containing, protein-free, media to 
permit determination of the partition coefficient of 
that particular lot of erythrocytes. Some of this 
lot of erythrocytes are then equilibrated with the 
plasma in question, separated and analyzed. Then, 
knowing the partition coefficient of the erythro- 
cytes, the free atabrine concentration in plasma can 
be estimated, A similar but incomplete experiment 
was carried out during this study in which red cells 
from 2 high and 2 low men were equilibrated with 2 
different concentrations of atabrine in a buffered 
Ringer9s solution. At the end of one hour at 37o5°C 
the cells were separated by centrifugation and the 
two phases from each equilibration were analyzed. 
The results are assembled in Table 21, There was 
insufficient material to permit an adequate analysis 
of the original concentration in the erythrocytes so 
that only very limited information can be drawn from 
this experiment. It is apparent, however* that (1) 
saturation was not reached, (2) the partition co- 
efficients are substantially the same in the different 
cells. If the latter is true, we are then justified 
in giving even more weight to the greater uniformity 
of cellular atabrine, 
d. Summary; 
(l) The suggestion is very strong that variation in pro- 
tein binding is the main cause for the large differences 
in plasma levels found in the groups studied. This 
conclusion rests on three points arising out of the 
present studies; (a) The concentration of atabrine 
in the cellular phase of the blood does not vary 
among individuals in direct proportion to the plasma 
atabrine concentration. This lack of proportionality between the two does not/ in the case of a high 
plasma level* result from saturation of the cells 
with atabrine0 It does* however* indicate that 
variation in one or more partition coefficients (plasma 
water to plasma protein* or plasma water to cells) 
does occuTo (b) The concentrations of atabrine in 
the cellular elements of the blood show not only less 
relative dispersion than the plasma atabrine concen- 
trations but are* in the case of individuals with 
extremely low or high plasma levels* more closely 
related to previous dosage than the plasma values0 
This point plus (a) tends to incriminate protein 
binding as a factor more variable than the "plasma 
water to cells** partition coefficient, since* until 
disproved, we must assume that men who have the same 
dosage history will have the same tissue concentra- 
tion of atabrine, (c) One incomplete experiment 
suggested that partition between free atabrine and 
erythrocytes was reasonably uniform in 2 high and 2 
low men, and independent suggestion that cellular 
atabrine may in many instances be a more reliable 
guide to the concentration of free atabrine in plasma 
than is the whole plasma concentration,, 
(2) The information presented here is inconclusive as to 
the probable constancy or uncertainty of the free 
atabrine to protein partition. Sufficient doubt, 
however, is thrown on its previously assumed con- 
stancy to warrant a thorough investigation of this 
variation in an adequate sample of normal men. In 
this way it may be possible to evaluate more com- 
pletely the limitations of the level of atabrine in 
whole plasma as an index of the levels of atabrine 
in other tissues - whether in those of a parasite 
or of the hosto TABLE 17 
RESULTS OF SEVERAL INDICES OP LIVER FUNCTION 
ON SUBJECTS WITH DIFFERENT LEVELS OP 
FLASEA ATABRINE 
. NAME 
-r -n r-r - ! * 
date 
GROUP 
Days on Therapy 
21 
Grams Atabrine 
Taken 
' . 
t 
a) a> 
•Si? 
rs w 
■£ s 
U 
d &o 
i g 
cj o 
r~\ *H 
fU 2g 
Prothrombin Time 
Seconds 
At Dilution 
Bromsulfalein 
• 
e 
« 
3 
lL 
*3 v c 
, § §| 
Half Excretion 
Time 
Minutes 
£ 8 
6 
0 
i 
4 
0 
1 «JC 
cj a) 
aj +> 
Cw 
Canada 
10-14 
CoQ 
C, 
S-l 
43 
2,7 
7: 
18,8 
25.0 
40,6 
1,2 
4.5 
0.32 
5 
Leskewsky 
10-18 
Co, 
B, 
S-2 
53 
4.5 
9 
17.4 
25.8 
36.1 
3o3 
6,0 
0.37 
5 
Vandertie 
10-12 
Co, 
c, 
S-2 
46 
3.9 
11 
, 17.4 
24.5 
33,0 
3.0 
6,7 
0.30 
5 
Pelczar 
10-13 
Co, 
B, 
S-l 
48 
2,7 
13 
18.1 
24 o 9 
37.9 
3.0 
5.5 
0,31 
5 
Paul 
9-18 
A 
hi 
4*1 
2k- 
LIZA 
26.7 
L40JL- 
-3A—. 
,-2A ... 
. —. 
Cox 
9-29 
A 
52 
5.0 
U 
17,2 
24.2 
41.5 
8,7 
8,4 
— 
Hemp 
9-30 
. 
B 
53 
5.1 
16 
20,4 
29.2 
43.7 
0,1 
3.2 
Littleton 
9-29 
A 
52 
5.0 
17 
18,6 
27.8 
39.6 
3.0 
6,2 
- 
— 
Harry 
9-29 
A 
52 
5.0 
25 
12,4 
19.2 
37.5 
2,1 
5.0 
«» 
Pachuoki 
9-30 
B 
-53..-J 
i.5.1 1 
13,6 
25,9 
43.0 
2,8 
5o8 
OlGara 
10-12 
Co, 
C, 
S-2 
46 
3.9 
26 
14.4 
21,1 
32.7 
2,4 
5.4 
0.36 
5 
Perry 
9-30 
B 
53 
5.1 
28r 
18,5 
27.2 
45.8 
4,4 
7.0 
Belleaky 
10-13 
Co, 
B, 
S-l 
48 
2,7 
34 
17.8 
22,0 
34 o 5 
5o2 
7.0 
0.29 
5 
Krawiecki 
10-15 
Co. 
B, 
S-2 
$3 
4.5 
34 
17,2 
25.4 
39.6 
3.5 
5.9 
0,3&. 
5 
Sink 
9-15 
A 
hi 
4,1 
35 
21*5 
25.6: 
46,8 
5.3 
6,6 
Berka 
10-12 
Co, 
c. 
S-2 
46 
3.9 
35 
16.6 
20,0 
30,4 
1.9 
5.3 
0,46 
5 
Smith 
10-14 
Co, 
C, 
S-l 
48 
2,7 
42 
19.3 
25.3 
40.6 
3.0 
6,0 
0,29 
5 
Gordon 
10-13 
Co. 
B, 
S-l 
48 
2o7 
47 
16.7 
27.8 
44.7 
• 6.3 
7.7 
0,29 
5 
Allison 
10-18 
Co, 
B, 
S-2 
53 
4.5 
50 
17o3 
26.1 
44o8 
0,5 
3.5 
0,36 
5 
Johnson, 0,B 
;VL; 
9-18 
A 
41 
4,1 
56 
20,2 
23o7 
42,0 
5.2 
6.8 
— 
* Average 
of 4 values bracke 
ting c 
iate noted H2’s) 
TABLE 17 TABLE 18 
Excretion of Atabrine 
in the Urine 
Name 
Plasma 
Atabrine 
Micrograms/L 
Date 
Urine 
Atabrine 
mg/L 
Urine 
Vol 
ml,/day 
Urine 
Atabrine 
mg/day 
Paul 
11 
10-21 
2.0 
1700 
3.4 
9 
10-22 
1.66 
1900 
3.2 
14 
10-23 
2.46 
1730 
4.3 
13 
10-25 
1.18 
2105 
2.5 
- 
10-26 
1.26 
3430 
4.4 
13 
10-27 
2.43 
1470 
3.6 
Average 
12.0 
10-28 
0.90 
2720 
2.4 
3*4 
Hemp 
15 
10-21 
1.22 
2450 
3*0 
13 
10-22 
2.26 
I960 
4.4 
15 
10-23 
1.08 
3390 
3.7 
17 
10-24 
1.24 
3410 
4.2 
20 
10-25 
0.53 
4400 
2.5 
- 
10-26 
1.22 
2810 
3.4 
17 
10-27 
0.68 
3790 
2.6 
Average 
16.2 
10-28 
1.24 
2370 
2.9 
3.3 
Golman 
25 
10-21 
5.2 
1000 
5.2 
28 
10-22 
3.96 
1420 
5.6 
32 
10-23 
3.44 
1720 
i 5.9 
37 
10-24 
2.22 
2360 
5*2 
33 
10-25 
1.84 
2080 
3.3 
- 
10-26 
2.22 
2530 
5.6 
Average 
33 
32.2 
10-27 
3.14 
2110 
6.6 
5.4 
Johnson,O.B. 
35 
10-21 
3*50 
1320 
4.6 
32 
10-22 
2.76 
1200 
3»3 
42 ' 
10-23 
2.06 
2060 
4o2 
35 
10-25 
2.53 
1730 
4.6 
- 
10-26 
2.72 
1450 
3o9 
37 
10-2? 
3.04 
1120 
3o4 
Average 
36.2 
10-28 
1.53 
1300 
201 
3.7 
Line after 10-23 indicates discontinuance of drug* 
TABLE 18 TABLE 19 
Whole Blood 
Atabrine 
Micrograms/Liter 
Prom Analysis 
of separate phases 
" From Analysis 
bf,whole blood 
232 
155 
U~\ 
to 
1—1 
195 
156 
155 
129 
90 
93 
80 
72 
70 
126 
90 
' 107 
100 
TABLE 19 TABLE 20 
(Sheet 1) 
PLASMA AND CELLULAR ATABRINE VALDES 
ON SELECTED HIGH AND IX*? MEN 
GROUP * 
& 
SECTION 
... ■ 
NAME " 
DATS 
Cw** 
Fc 
Ap 
Awb 
Ac 
Ac® 
i£ 
Ac1 
B-2 
Lhota 
10-29 
10,0 
41.0 
37 
164 
142 
114 
.32 
B-2 
Allison 
ft 
7.5 
42,0 
40 
98 
75 
75 
.53 
McMichael 
H 
. 8.6 
36.0 
31 
152 
132 
118 
.26 
1W2 
Krawiecki 
H 
9.0 
.32JL.. 
27 
r-lUL 
122.. 
.,113 
-24, 
C-2 
V&lente 
10-30 
10,0 
38.5 
32 
162 
142 
112 
.29 
C-2 
Weiss 
m : 
8.9 
34.0 
25 
152 
136 
117 
.21 
G-2 
Berka 
n 
$.5 
3P.5 
19 
100 
.87 
- 99 
.19 
B-2 
Elmore 
10-29 
7.8 
43.0 
7 
70 
66 
62 
.11 
B-2 
Farris 
n 'ii 
7.0 
45.0 
9 
78 
>73 
78 
«12 
B—2 
Leskiowsky 
n 
9.8 
33.0. 
9 
70 
‘ fA 
53 
.17 
C-2 
Q^jora 
10-30 
5.1 
39.5 
a 
52 
47 
66 
.12 
C-2 | 
Skrabis 
It 
10,0 
39.5 
9 
102 
97 
75 
.12 
C-2 
Vandertie 
It 
7.2 
7 
62 
.11 
• 
B—1 
Bellesky 
10-29 
10,5 
41.0 
30 
98 
80 
64 
.47 
.B-l . _ 
Gordon 
it 
3.4 
19 
99_ 
92 
-42 
0-1 
Ostroirits 
10-30 
7.2 
40,0 
30 
84 
66 
68 
.44 
c-l •* 1 
Smith * 
| 
8,6 
42,0 
28 
96 
80 
73 
.38 
C-l ■ 
Hiee 
* 
7n7 
19.0 
20 
86 
74 
72 
-28 
B-l 
Hildner 
10-29 
12,1 
39.0 
10 
82 
76 
50 
o 
fO 
O 
B-l / 
Swigert 
« 
1001 
46 o0 
7 
70 
66 
51 
• 14 
B-l 
Xirk 
R 
6-8 
41-5 
36 
31 . 
34 
.24 
€i 
Miller 
10-30 
4.05 
39.0 
7 
50 
46 
81 
.09 
it 
C-l 
Woods 
tl 
5.0 
39.0 
8 
60 
55 
80 
.10 
C-l 
Canada 
r ■ 
5.9 
38.0 
; 7 
52 
48 
60 t? 
.12 
* Men 1 
n B-l and C-l received 0.4 
gm Atabrine per week, those in B-2 
! and C-2 received 0*6 gm per week 
. 
** Cw - leukocyte count in whole blood. 
thousands/ cu. mm. 
Vc - Bed cell volume, per cent 
Ap - Plasma Atabrine, mic rograma/liter 
f 
Awb - Whole blood Atabrine 
, mic rograms/lit ©r 
At - Red cell Atabrine, micrograms/liier 
- 
Ac - 
Cellular Atabrine, microgr&me in the cella from 1 liter 
of blood 
Ac1 - 
Cellular Atabrine, micrograaa in the 
cella from 1 liter of blood 
for a white count of 7500/cu# 
ism. 
■ V ‘ '* : 
At* *• leukocyte Atabrine, micrograms in the leukocytes from 1 liter of 
blood for a 
count of 7500/cu. 
mm. 
TABLE 20 TABLE 20 
(Sheet 2) 
PLASMA AND CELLULAR ATABRINE VALUES 
ON SELECTED HIGH AND L0» MSN 
GROUP 
& 
’SECTION 
NAME 
DATE 
Cx 
Vc 
AP 
Awb 
Ac 
Ac1 
An 
Ac’ 
B-2 
Lhota 
11-5 
8.9 
- 
39 
172 
148 
130 
.30 
B-2 
Allison 
if 
5.4 
- 
34 
102 
82 
103 
.33 
13-2 
McMichael 
n 
8,4 
- 
26 
182 
166 
151 
.17 
3-2 
Krawiecki 
ft 
8.7 
- 
25 
-.152 
137 
121 
.21 
C-2 
Valente 
11-6 
11.0 
39.4 
27 
134 
118 
8? 
.31 
C-2 
ti 6i S S 
« 
9.6 
34.6 
25 
146 
130 
105 
.24 
C-2 
Berka 
n 
8.8 
34.8 
20 
110 
97 
85 
.24 
B-2 
Elmore 
11-5 
6.4 
14 
90 
82 
94 
.15 
B- 2 . 
Leskov? sky 
u 
9.9 
- 
10 
70 
64 
50 
,20 
C-2 
Ogborn 
11-6 
6.7 
39 o 7 
9 
66 
61 
68 
.13 
C-2 
Skrebis 
rt 
7.0 
42.0 
9 
82 
77 
82 
,11 
C-2 
Vendertie 
it 
8.4 
40 ol 
6 
74 
70 
63 
.10 
B-l 
Gordon 
11-8 
9.3 
38,0 
35 
140 
118 
100 
.35 
B-l 
Dark 
n 
8.5 
39.9 
24 
158 
144 
129 
.19 
B-l 
Fehrle 
n 
- ■ 
- 
- 
- 
- 
- 
- 
C-l 
Ostrowitz 
11-9 
8.3 
40.0 
28 
82 
65 
62 
.45 
C-l 
Smith 
it 
11.0 
40 o 2 
25 
112 
97 
74 
.34 
.c-i 
_J4ec 
« 
5.8 
38.5 
20 
88 
75 
9r3 
.22 
B-l 
Hildner 
11-8 
12.6 
43.1 
9 
94 
89 
56 
.16 
B-l 
Swigert 
t< 
8.1 
42.9 
8 
76 
71 
66 
.12 
B-l 
Kirk 
it 
10.7 
41.9 
7 
. 50 
46 
34 
.21 
C—1 
Miller 
11-9 
9.0 
41.4 
10 
62 
58 
50 
.20 
C-l 
Woods 
it 
8.3 
42.1 
9 
56 
51 
47 
.19 
B-l 
Canada 
it 
8.4 
38.9 
8 
54 
49 
44 
.18 
See sheet 1 for legend 
TABLE 20 TABLE 20 
(Sheet 3) 
PLASMA AW CELLULAR ATABRINE VALUES 
ON SELECTED HIGH AND LOW MEN 
GROUP 
& 
STATION J 
NAME 
DATE 
Oti 
Vc 
Ap 
Awb 
At 
Ac 
Ac* 
Ap 
Ac1 
Ap 
Ar 
V 
C-2 
Ialente 
11-13 
9.7 
41.0 
29 
155 
40 
138 " 
110 
.26 
.73 
94 
C-2 
.eiss 
« 
12.4 
37.5 
30 
195 
52 
176 
114 
.26 
.58 
95 
C-2 
)gborn 
ii 
6.8 
41.0 
15 
80 
32 
71 
79 
.19 
.47 
66 
C-2 
Zandertie 
n 
8.4 
40.5 . 
8 
100 
48 
95 
87 
.09 
.17 
68 
- 0-1 
Sstrowitz 
ti 
11.6 
41.0 
34 
155 
44 
135 
94 
.36 
.77 
76 
C-l 
Smith 
it 
9.4 
40.0 
22 
90 
56 
77 
66 
.33 
.39 
44 
C-l 
.filler 
it 
10.6 
42.0 
10 
70 
24 
66 
50 
.20 
.42 
40 
C—1 
Canada 
ti 
12.7 
41.0 
9 
90 
20 
8? 
54 
.17 
.45 
46 
C-2 
/alente 
M 
M 
1 
o 
7.4 
39.7 
20 
6? 
- 
55 
56 
.36 
- 
- 
C-2 
eiss 
H 
12.0 
37oO 
19 
57 
- 
45 
33 
.58 
- 
- 
C-2 
Igborn 
H 
8.6 
39.9 
9 
50 
- 
45 
40 . 
.23 
- 
- 
C-2 
Zandertie 
tl 
9.7 
38.2 
6 
‘ 30 
- 
46 
57 
.16 
- 
— 
C-l 
)strowitz 
ft 
8.9 
33.0 
20 
57 
- 
44 
39 
.51 
- 
C-l 
Smith 
!! 
9.9 
39.9 
18 
57 
- 
46 
38 
.47 
- 
- 
C-l 
filler 
11.0 
42.0 
5 
33 
- 
30 
22 
.23 
- 
- 
C-l 
Canada 
M 
6.7 
40.1 
4 
27 
- 
25 
28 
.14 
- 
- 
See Sheet 1 for legend 
TABLE 20 APPLNDII C 
SdCTICN VI - Effects of Atabrine on .! an0 
1. Toxicity. 
a. There were no unequivocal toxic reactions to atabrine in 
any of tne 250 men on any of the suppressive dosage regimens. The health 
and well-being of all the men was excellent throughout. The A.H.T.C. 
group made an average gain in weight of 3 pounds during the experimental 
period and the group exposed to hot humid (jungle) climate manifested 
no disturbances that could be referred to the drug. In fact, the tran- 
sient gastroenteric-tract disturbances commonly seen during the first 
days of exposure to heat v;ere less marked in this experimental group 
than in others reviously studied in the hot room. For the most part, 
the drug was given at meals; in instances however, it was taken between 
meals during periods of work in the sun. 
b. All visits to the dispensary by men from both comoanies were' 
carefully checked. In no instances could the complaints be attributed 
to atabrine nor was the frequency of visits by members of the experimental 
group greater than that of the men in the same units not taking the drug. 
c. One case of probable toxic reaction from atabrine in thera- 
peutic doses was encountered. The subject (Shakleford) had an epilepti- 
form seizure on the 5th day of therapeutic dosage. Investigation revealed 
no evidence of previous seizures ana there had been no disturbances while 
the subject was on suppressive therapy. On the day following the seizure 
the subject’s plasma atabrine level was 108 micrograms, the highest 
attained by any of the group on the therapeutic regimen. Forty eight 
hours after the seizure the miasma level nad fallen 6? micrograms, the 
administration of the drug having been continued throughout. No further 
incident occurred. 
d. In view of the frequent reports of atabrine toxicity the 
failure to encounter such during the present study is of interest. This 
experimental group differed from men in combat theatres essentially in 
that the experimental group was not exposed to the psychological stresses 
of approaching combat. One may properly question therefore, if the drug, 
when administered in accordance with the schedule recommended, can be 
considered solely responsible .or the reported complaints. .Whether the 
drug plus emotional stress will account for the.manifestations or whether 
symptoms due to stress and other factors are being attributed to the ata- 
brine remains to be established, 
2. Discoloration. The characteristic yellow discolor alien from 
the drug began to be noticeable at the end of trie first month and in- 
creased progressively thereafter. The intensity of color did not correlate 
with plasma levels. 
34 APPENDIX D 
IMPLICATIONS OF THIS STUDY AS APPLIED TO FUTURE INVESTIGATIONS 
lo Field studies for determination of suppressive plasma level. It 
is generally agreed that one of the first points to be investigated in the 
field is the plasma level of atabrine which is necessary to suppress 
symptoms. For the preliminary organisation and conduct of such an epidemi- 
ological investigation, the relationships established in the present study 
are directly applicable. 
a0 Selection of dosage regimens. The dosage required to develop 
any group mean equilibrium level that is desired can be obtained, as follows: 
Divide the value of the desired equilibrium level (in micrograms/L) oy 209 
to get tne required daily dose in gratis. Example: Desired level * 20 
micrograms/L; daily dose required = 20/209 ~ 0.1 gm/day. If it is desired 
to establish this level rapidly, administer double the daily maintenance 
dose, or 0.2 gm/day, for the first week. 
be Prediction of range and percentage distribution of individual 
equilibrium levels on a given regiuen. The probable range of equilibrium 
levels attained by the individuals in a group on a selected regimen and 
the percentage of the group having concentrations below stated levels can 
be determined from Chert 41. Example: ?*hat percentage out of a group on 
a regimen of 0.1 gm/day will.have plasma atabrine levels = 10 micrograms/L? 
Since the group mean equilibrium c 0,1 x 209 = 21 micrograms/L, the level 
in question - Meanpr x 0.5. According to Chart 41, three (3) percent of 
the group will have plasma levels equal to or less than 10 micrograms/L. 
Similarly, 17 percent will develop plasma concentrations not greater than 
UeariQ x 0o7 or 14 micrograms/L0 
Co Determination of dosage regimen to produce equilibrium plasma 
levels above a desired minimum level in any given percentage of the popu- 
lation P In order to insure that a given percentage of the population will 
have plasma atabrine concentrations above a stated minimum level, the re- 
quired dosage regimen is calculated as follows: 
Divide the desired minimum by the ratio to the mean for the per- 
centage in question as obtained from the log-probability line in Chart 41. 
The result is tne required group mean plasma levelc This value divided by 
209 gives the necessary daily dose in grams. Example: What daily dosage 
is required to maintain plasma atabrine levels equal to or above 10 micro- 
grams /L for all but one man in 100 (99%)? Referring to Chart 41, the 
minimum level which occurs 99 out of ICC times is found to be 0.A2 x Meanq; 
the group mean equilibrium level, therefore is 10 - 23*8 micrograms/L 
Go 42 
and the required daily dose is 23,8/209 r 0,12 gm/day. Similarly for all 
but one man in 1000 (99.9%) to be above 10 micrograms/L, the mean equilibrium 
level v;ill be 10 - 3I°2 micrograms/L and the required dosage is 32/209 
0.32 
- Co 16 gm/day. 
1 PLASMA LEVEL IN TERMS OF MEAN 
PREDICTED DISTRIBUTION OF PLASMA ATABRINE LEVELS 
STATED RATIO~TO~MEANG EQUILIBRIUM LEVEL 
CHART-41 
68% RANGE 
STANDARD GEOMETRIC 
DEVIATION5 1.45 
CHART-41 d. Blood sampling for control. Any scheme for field study must 
be predicated upon the knowledge that the drug is being taken. In the 
absence of complete supervision of administration, some type of analytical 
control procedure is required„ Since this will entail blood sampling and 
access to the troops, it follows that the program should be limited in 
scope to that necessary to establish whether or not the dosage is being 
maintained., The accuracy with which it is desired to establish control will 
largely determine the size of the sample to be obtained. Assume, for ex- 
ample, that the limits within which the mean of any sample of size M should 
fall are group J lo0oSoSo (68$.range of mean). The standard eiror 
of the mean depends upon the size of the sample: 
Assume, as an example, a group mean plasma level of 16 micrograms/L and 
standard geometric deviation of 1.45 to represent the universe and that 
samples of 9 men are taken for examination. 
and 68$ of the means of representative* samples of 9 each should fall within 
the limits of ? 1.13 or 14 to 18 micrograms. If a probability of occurrence 
of once in 1000 times is used as the limiting criterion, then the sample 
means, to be considered representative, must fail within 16 x (S<,S.)3 - 
16 x 1.45 or 11 to 23 micrograms (a range of x (SdiioP includes 99 = 9$ of 
the population)c If the mean of a given sample falls below the selected 
lower limits, it may be concluded that all numbers of the group are not 
receiving atabrine regularly according rto the established regimen. 
20 Analytical method. 
a. Further work on method is desirable. The procedure as used 
was satisfactory in our hands, since, with few exceptions, the results were 
consistent. Nonetheless, it must be remembered that the procedure was 
carried out under the continual supervision of two investigstiers with ex- 
tensive analytical and research experience. Unless workers of equivalent 
training and experience will be available for the ensuing studies, sufficient 
work should be done with the method to make it an entirely reliable tool in 
the hands of technicians. With respect to field investigations in the tropics, 
tine fluorometer now in use is very sensitive to atmospheric conditions. This 
instrument should be improved for use in hot, humid environments and/or a 
visual instrument developed in order that the vagaries of the photoelectric 
element may be eliminated. b. It is evident from considerations of the transients (see 
Appendix B - II) that the choice of a bleeding $ hours after dosage is 
unfortunate since it lies just between the 2-hour and B-hour transients 
where the time variation in the first transient will greatly irfluence 
'the magnitude of the plasma level observed. A better choice would be 
B hours after dosage when the second transient has reached its peak 
value and the first has largely disappeared. 
3. Partition of atabrine in blood. The possibility has been presented 
that the whole plasma atabrine level in tlie individual may in some instances 
be an unreliable measure of the free atabrine concentration in the plasma. 
The evidence for this is inconclusive. Ylhen a study is set up to investigate 
the correlation between olasma level and malarial protection it is obviously 
necessary to have available a reliable index of effective atabrine con- 
centration. It is recommended therefore that further investigation be 
immediately instituted to establish the reliability of whole plasma con- 
centration as well as other measures of free atabrine concentration in the 
plasma. 
4. Toxicity of Atabrine. The short span of this study and whe 
absence of reactions provide no basis for comment with respect to long- 
time toxicity. In the considerations of a future controlled study of acute 
toxicity the entirely negative observations here reported are of significance. 
The freedom from disturbances may be attributed, in part, to the assurance 
given the men that the drug would not disturb then and the absence of other 
psychological factors (fear, etc.) likely to cause gastroenteric tract dis- 
turbances, 
5. Qualifications. It is to be recognized that the foregoing inferences 
and implications may be strictly applied only to populations having 
characteristics like those of the experimental group ana under states of 
health and conditions of environment within the ranges described for this 
stuay. 
3 APPENDIX E 
TABULATION OF RAW DATA 
1. Included in this appendix are the crude data* of all groups 
for the entire experiment in the following order: 
a. A.R.T.C, Co. B Sect. 1 0.4 gm/wk 
b. A.R.T.C, Co. C Sect. 1 0.4 gm/wk 
c. A.R.T.C, Co. B Sect. 2 0.6 gm/wk 
d. A.R.T.C. Co. C Sect. 2 0.6 gm/wk 
o. Jungle Group A 0o6 gm/wk 
f. Jungle Group B 0o6 gm/wk 
g. Group X Dieaway 
hc Group Y Dieaway 
Rearranged from Jungle Groups A and B 
* All Means,-. and standard geometric deviations were determined graphically. APPENDIX E 
The tabulated raw data is not included in this copy of 
the reporto It may be obtained upon request addressed to 
Adjutant, Armored Medical Research Laboratory, Fort Knox, 
Kentuckyo