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CONFIDENTIAL
Author : Eldon D . Nielson MRR-B, 1969, No . 2
Biological Research Division

To : Dr . Murray Senkus February 24, 1969

Director of Research

Re : MONTHLY RESEARCH REPORT Period Covered :

Biological Research Division January 16 through
1969, No . 2 February 14, 1969

No . of Pages : 6

A . SMOKING PHYSIOLOGY

I . Fate of Smoke Constituents in Animals

Dotriacontane-16-17-C14 was administered to a rabbit as a vapor aerosol ;

the animal was sacrificed at 120 minutes and the distribution of C14 in the
individual tissues determined . Nearly 100% of the C14 activity was found in
the lung tissue with trace amounts detected in the trachea and stomach contents .
Presumedly, the small amount of C14 activity in the stomach could originate
from the lungs by means of the mucus transport system and subsequent swallowing
of the mucus material . Only negligible amounts of activity were detected in
the blood and urine . Thus, this compound unlike all the others we have investi-
gated shows no tendency to be taken up after administration to the lungs . It
remains to be ascertained as to whether the C14 activity in the lungs is intra-
cellular or merely deposited on the alveolar surface, also, whether it-undergoes
any metabolic changes .

II . Development of Biochemical Methods

a . Saponification of Triglycerides or Phospholipids and Methylation

__ of the Released FattyAcids ___________________

The present method used for saponification and methylation of the

triglycerides and phospholipids using dimethoxypropane and H2SO4 often
yields a polymeric material and some trace components which interfere
with the subsequent gas chromatographic analysis of the methyl esters
of the fatty acids . Alternate methods have been investigated and an
improved method based on saponification and methylation with methanolic-
HC1 is being evaluated . Thus far, the application of this method to
standard tripalmitin, crude lecithin, and crude triolein indicates that
it will meet our requirements .

b . Quantitative Analysis of Phosphorous in Phospholipids Separated

__by Thin-Layer ChromatogrAphy __________^______

In order to determine the amount of phospholipids present in individual

fractions, it is essential that a method for determination of phosphorous
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in these compounds be developed . Preliminary investigations indicate

that it will be possible to measure the amount of phosphorous present
in the various phospholipids classes present in lung, liver, and kidney
by a modification of the Fiske-Subbarow Method, which is applied to the
phospholipids separated by TLC without elution from the silicic acid .

III . Rabbits

Smoke exposure in the group of rabbits is now in its eighth month .

Progress has been uneventful . Blood serum analysis and EKG's are apparently
still within normal range .

At the present time, a microbial screen to detect Bordetello sQ . in the

rabbits has been initiated . Bordetello sp . has been implicated as a causative
agent in a number of chronic pulmonary diseases in laboratory animals . Elimi-
nation of rabbits carrying this microorganism prior to smoke exposure may help
in the evaluation of tissue sections .

IV . Mice

An experiment to determine the effect of smoke exposure (30 days) on

clearance of C14 from various organs of mice has been completed, and tissue
samples are being analyzed by the Biochemistry Section .

B . BIOLOGICAL TESTING

I . Abscisic Acid

Free samples of abscisic acid were sent to thirteen qualified research

workers .

II . Primary Screenin&

a . CNS

Of the twenty-seven compounds received and screened this month, two

showed activity . SFF100A scored significantly as an anti-depressant but
only at the highest dose level . ST19A showed significant behavior
depressant activity at 50 mg/kg, I .P . ; but toxicity was noted at 100 and
400 mg/kg, I .P .

b . Anti-convulsant

Regular screening of all compounds for anti-convulsant activity has

been incorporated into our program . We are using the chemical analeptic
pentylenetetrazol, and also supra maximal electro shock as the routine
inducers of convulsions in mice . SAA123A has shown significant activity
in both the pentylenetetrazol and the electro shock assays . Further work
will be done with this compound .

c ._ Cardiovascular Screening_

Of the sixteen compounds evaluated, two, SMM11A and ST17A, have

shown repeated hypotensive activity in the guinea pig . These two
compounds will be tested further in cats .
 3

The time-dose response of four standard hypotensive agents are

now being run to determine the efficiency of this assay in guinea pigs .

d ._ Metabolic Screen

Twenty-six compounds were run and one, SAA124A, will be retested '
for hypocholesteremic activity . SMM19G produced a hypoglycemic activity
in two of five rats equivalent to that produced by Tolbutamide . This
effect of SMM18G will be rechecked and tested further .

III . Secondary Evaluation

SB43A was administered I .P . to a group of rats for five days . There was
no apparent hypocholesteremic activity, however, the animals lost considerable
weight at this dose of 300 mg/kg . This route of administration of SB43A will
be tested further at lower doses .

C . STARCH BIOCHEMISTRY

I . Total Saccharification

The evaluation of the enzymatic activity of the a-amylases is still in

progress .

II . Biochemical Analysis

The unfermentable sugars (sugars other than glucose, fructose and mannose)
in enzymatic and alkali isomerized GF syrups were determined by yeast fermenta-
tion and vapor-phase chromatography . The alkali GF syrup, before fermentation,
contained 65 .7% glucose, 32 .6% ketoses and 1 .2% G2 sugars . After the yeast
fermentation, this syrup contained from 3 to 4% (DWB) unfermented monosaccharides
which are not normally detected since they are eluted with the TMS-D-fructose
and TMS-a-D glucose peaks . The characterization of these sugars is presently
under investigation . The enzymatic isomerized syrups contained no unfermentable
monosaccharides .

The alkali-converted syrup also contained approximately 0 .6% isomaltose,

0 .6% maltulose (4-0-a-D-glucopyranosyl-D-fructose) and 1 to 4% mannose . No
mannose was observed in the isomerase converted syrups .

III . Glucose Isomerase

Constitutive mutants obtained from Arthrobacter RJR 2453-Y yielded higher

enzymatic activities than the mutants from RJR 2453-2 . The Yc mutants when
grown in 2% glucose produce as much as two-thirds of the isomerase activity
produced by the wild type cells grown in 2% xylose . Further testing of these
mutants under various growth conditions is in progress .

Shake-flask studies have demonstrated that the xylose content of the

isomerase medium can be reduced to 1% by the addition of 2% ethanol and 1%
sorbitol . The enzymatic activity in these cases was 660 yU/ml . after 56
hours . Initial experiments have also shown that the xylose can be completely
eliminated by adding 1% ethanol and 0 .5 to 1 .0% sorbitol, and glucose isomerase
activities of approximately 500 uU/ml . were observed . These results will be
further substantiated by 30-gallon fermentation studies .
 4

A continuous diffusion method was developed for the isomerization of

glucose to fructose . The apparatus consists of 25 stacked chambers each
separated by polyvinyl chloride membranes . A 30% glucose syrup was continu-
ously pumped through the apparatus at a flow rate of 30 ml . per hour .
Diffusion through the membrane did not appear to be a rate limiting step
since a 34% conversion rate was maintained during the run . Variations in
flow rate, temperature, cell concentration, etc ., are under investigation .

IV . Glucoamylase

The growth characteristics and enzymatic activity of the two Rhizopus

cultures are presently being evaluated . There are no significant results
to report at this time .

V . Microbial Transformation of a-Ionone

Many microbial cultures including stock cultures of Rhizopus and

Aspergillus are being screened to determine their ability to form 4-keto-l-
hydroxy-a-ionone from a- or B-ionones . Known standards of the various inter-
mediates in the chemical conversion have been obtained from Dr . Donald Roberts,
and procedures for rapid analysis of these intermediates and final products
are under investigation .

D . FOOD MICROBIOLOGY

Examination of 54 samples of food products currently under development

involving 332 laboratory tests was completed . Coagulase-positive Staphylococci
were found in Flavored Bits, Chicken Log, and in the chopped celery, red peppers,
and frozen fried eggs used in the preparation of chicken fried rice . But they
were not present in the finished chicken fried rice product .

The food pilot plant was surveyed for microbiological cleanliness . High
total counts of yeasts, molds, coliform bacteria, and coagulase-positive bac-
teria were encountered . It was therefore recommended that all equipment be
sanitized according to a prescribed 5-step procedure and that the last two
steps be repeated just prior to use of the equipment .

E . MISCELLANEOUS

I . Control of Fungal, Bacterial and Insect Damage in Flue-Cured

Tobacco During AginR

Twenty hogsheads of grades 44X and 7X tobaccos of the 1966 crop were
packed in polyethylene-lined hogsheads . Twenty of the hogsheads were opened
and examined after storage for 2 years in sheds where mold and beetle damage
have been known to occur . None of the hogsheads contained tobacco damaged
by these agents during the two-year storage period . This appeared to contra-
dict previous laboratory observations indicating the ability of beetles to
bore through polyethylene film . However, under natural storage conditions,
beetles failed to enter the protected hogsheads .
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The tobaccos were examined by Mr . William Scales of the Leaf Processing

Department, Mr . J . A . Glenn, Jr ., and Mr . W . L . Gibbs of the Leaf Department,
who declared that the tobaccos had aged fully and properly and were typical
for the grades in question . The results suggested that such liners could be
used advantageously for that purpose . Cost of the film was estimated to be
about $ .60 per hogshead .

II . Tobacco Beetle Control

a ._ Phostoxin as a FumiRant

Further experiments have been carried out to study the relationship

between cigarette beetle mortality and Phostoxin dosage-exposure time-
temperature . Three dosage levels .were studied from time intervals of
eight to ninety-six hours and at three different temperatures ; 40°,
450, and 50° C . At these low temperatures, complete mortality was not
attained until an exposure of forty-eight hours or longer . In most
cases where complete kill was not attained, there was more that 95%
mortality . The beetles that were not killed were in most cases newly-
hatched adults . These results suggest that there may be certain stages
in the insecth development where it is much more resistant to the
phosphine . Further work will have to be conducted to determine if some
of the stages are indeed much more resistant .

b ._ Fate of Phosphine Taken up by Tobacco

Previously, it was reported that small amounts of P32 were bound to

tobacco after exposure to P32 phosphine . Trace amounts of P32 were
transferred into the gaseous phase and particulate phase of the main-
stream smoke of the cigarette . By studying the transfer of the P32 into
the above fractions after two days, two weeks, and five weeks of storage,
evidence has been obtained that indicates the P32 activity in the gas
phase decreases steadily and will approach 0 after six months storage .
This could be explained by assuming that one of the first reaction
products of phosphine in tobacco, which liberates phosphine on burning,
undergoes subsequent change during the stora e period, perhaps a slow
oxidation to phosphate ion . The amount of P12 in the particulate phase,
however, appears to remain constant even after five weeks storage . The
material present in the particulate phase is therefore presumed to be
some phosphorous compound or compounds other than phosphine .

Whole grains of corn and wheat take up a significant amount of P32

activity after exposure to P32 phosphine . As in tobacco, the P32 activity
is retained after aeration and heating . This is of interest since phos-
phine has been used for the fumigation of grains for several years in
the United States .
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F . MANPOWER DISTRIBUTION FOR FEBRUARY, 1969

14%

135 12%

130 8%

007 6%

003 5%

131 5%

013 1%

903 1%

1%

652 1%

132 1%

930 1%

614 1%

eeL , 1 / G, ~~~ .,
Eldon D . Nielson

Distribution :

Dr . Murray Senkus Mr . M . R . Haxton

Dr . K . J . Mysels Mr . L . A . Willson, Jr .
Dr . P . C . Markunas Dr . Herbert J . Bluhm
Dr . E . D . Nielson Mr . E . C . Ritchell
Dr . C . E . Teague, Jr . Dr . . R . U . Lemieux
Mr . J . L . Keaton
Library (2)
Dr . R . E . Farrar Submitted : February 24, 1969
Mr . E . H . Harwood
Completed : February 24, 1969
Dr . S . 0 . Jones
From manuscript :vbl
Mr . N . G . Fox
Mr . E . J . Monohan
Mr . William H . Stevenson, III Approved :
M. S.
r^ .~.q 3 1969