NERC-LV-539-32
THE BEHAVIOR OF 131I IN AN ARTIFICIAL RUMEN AND IN
THE SIMULATED FLUIDS OF THE ABOMASUM AND INTESTINE

               Earl L. Whittaker
               Delbtirt S,  Bart*
 National Environmental Research Center-Las Vegas

     U. S.  ENVIRONMENTAL PROTECTION AGENCY
              Lis. ¥gs9 NV  89114

               Published May 1974
     This research performed under a Memorandum
         of Understanding No. AT(26-l}-539
                     for the
          U. S. ATOMIC ENERGY COMMISSION

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                                                   NERC-LV-539-32
THE BEHAVIOR OF 131I IN AN ARTIFICIAL RUMEN AND IN
THE SIMULATED FLUIDS OF THE ABOMASUM AND INTESTINE
                      by
                  Julius Barth
               Earl  L. Whittaker
               Delbert S. Barth
 National  Environmental Research Center-Las  Vegas

     U.  S.  ENVIRONMENTAL PROTECTION AGENCY
              Las Vegas, NV  89114
               Published  May 1974
     This  research performed under a Memorandum
        of Understanding No. AT(26-l)-539
                     for the
          U.  S.  ATOMIC ENERGY COMMISSION

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                            ABSTRACT

                           131
The in vitro conversion of    I-labeled sodium iodide  to  volatile  iodine

was investigated in the artificial  rumen and in simulated abomasal  and
                                                    101
intestinal fluids.  In addition, the association of    I-labeled iodide

with rumen juice sediment, which includes microflora and  feed debris, was

also studied.  The results show that under the conditions reported here,
101                                                    1 01
   I is not volatile.   As much as three percent of the    I  was  shown to

be associated with rumen juice sediment in the artificial rumen.  This

value was reduced to 0.52 percent and 0.038 percent in the simulated

abomasal and intestinal fluids, respectively.
                                 11

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                        ACKNOWLEDGMENTS

The authors thank J. A. Reagan for statistical  analyses of the data
and G. D. Potter and W. W. Sutton for their helpful  suggestions in
the preparation of this manuscript.

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                           INTRODUCTION

Gases of the rumen may be expelled by eructation and subsequently absorbed
by the lungs.  At this laboratory it was observed that    I appears in the
blood and milk of cattle very soon after an oral administration of    I
iodide.  It was therefore postulated that    I could have been converted to
volatile iodine in the rumen and transported into the respiratory system by
eructation where it could be absorbed.  Experiments using the artificial
rumen were designed to test this hypothesis.

Dougherty et aj_. (1965), discussing work of others concerning eructated rumen
gas, stated the following:  A repetition by Dougherty ejt al_. (1962) of an
experiment performed many years ago by Dougherty (1940) very effectively
demonstrated that components of rumen gas which reach the lungs are
absorbed.  Dougherty ejb al_. (1962) and Shipe et^ a\_. (1962) demonstrated
that certain odors carried in eructated gases from the rumen to the lungs
were absorbed, thereby contaminating the mammary blood supply and trans-
mitting off-flavors to the milk.  The same substances tested in these
experiments were also absorbed by the portal blood; however, contamination
of milk was much slower by this route than it was when eructated gases
were allowed to enter the lungs.
                                                       131
This study was designed to assess the possibility that     I  iodide might
be converted to volatile iodine or methyl iodide in rumen juice which
could then be eructated into the respiratory tract.   If  iodide were indeed
                                1

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converted to iodine in the gastrointestinal  tract, it would then be
necessary to account for    I losses by eructation as well  as  recycling
of    I via the respiratory system.  Volatilization of    I iodide from
simulated abomasal and intestinal juice was  included in  this study.   In
                         131
addition, the binding of    I to rumen microflora and other sediment  was
also studied.

Barth and Bruckner (1969  a,b) effectively employed an artificial  rumen
followed by simulated abomasal and intestinal  fluids to  study  the uptake
by binding agents of    Cs, strontium, and other essential  cations in order
to reduce radionuclide transport to milk.  This procedure also provides
a means for investigating the solubility or availability for absorption
of fallout radionuclides in a natural medium,  taken directly from a
rumen-fistulated steer, in the presence of viable rumen  microflora.
                         METHODS AND MATERIALS

The artificial rumen and simulated abomasal  and intestinal  fluid procedure
used is similar to that employed by Barth and Bruckner (1969  a,b) and is
briefly described here with necessary modifications.   Each digestion flask
was prepared by addition of 250 ml of rumen juice to  250 ml of basal
medium in a one-liter Ehrlenmeyer flask containing 3.75 g of powdered
cellulose.  The C02 outlet from each Ehrlenmeyer flask was connected
to an    I scrubber consisting of a 1.5- by 20-cm column of activated
charcoal.  The content of each flask was adjusted to  pH 6.5 with saturated
sodium carbonate solution and dosed with 0.7 to 1.2 yCi of carrier-free
  131
Na   I.  Digestion flasks were incubated for about 21  hours in a water bath
at 39°C, with a continuous stream of COp passing through the contents of each
flask.

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The artificial rumen fluid in each flask was  then converted to simulated
abomasal fluid by addition of 125 ml of simulated abomasal  juice contain-
ing 0.25 g pepsin and by adjusting the pH to  three with 5.0 N HC1.   The
resulting mixture was incubated for three hours.   The C02 flow was  allowed
                                             131
to continue slowly in order to sweep gaseous     I that might be produced
to the  charcoal scrubber.
Following the abomasal digestion phase, the simulated abomasal fluid was
converted to a simulated intestinal fluid.  One-hundred milliliters of
0.1N NaHC03 was added followed by 40 ml of bile.   Fifty milliliters of
an enzyme preparation containing 6.0 g pancreatin, 0.3 g trypsin, and 0.3
g erepsin was added and the pH was adjusted to six with 5.0 N NaOH.  In-
cubation continued for an additional three hours  with a slow rate of (XL
flow.
At the beginning and end of each digestive phase, a 5-ml sample of  rumen
juice was removed and pipetted directly into  50 ml of 0.1N NaOH for
                 131
radioanalysis of    I.  Upon completion of each digestive phase, an ad-
ditional 10-ml sample from each flask was removed and centrifuged for 20
minutes at the maximum speed of a Sen/all Table Model Centrifuge, Type M
(over 5000 rpm).  The supernatant was decanted into 0.1N NaOH for radio-
analysis while the sediment was washed twice  by resuspension in 5 ml of
water and recentrifuged.  All supernatants were combined.  The washed
sediment was analyzed for radioiodine.  Fresh charcoal scrubbers were
                                                           131
used during each digestive phase for collection of gaseous    I.  Gamma
spectrometry was carried out using opposed 5- by 9-inch sodium iodide
crystals associated with a TMC 400-Channel Analyzer.  Digestion flasks
were run in duplicate for three separate trials.

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A preliminary experiment was designed so that gas chromatography could
be used to determine the chemical  form of any volatile  iodine.   This
operation was similar to the procedure described above  except that the
C02 outlet was connected to a small  filter flask which  served as a trap.
This was followed by passing the gas through a 1.5- by  20-cm column of
a mixture of calcium chloride pellets and anhydrous calcium sulfate
which served as a drying agent.  Plastic tubing was used to connect
this column to the first scrubber which consisted of a  "U"  tube containing
4 ml of toluene.  The toluene scrubber was placed in a  Dewar flask contain-
ing a slurry of ground dry ice and isopropyl alcohol.  This was subsequently
attached to a column of activated charcoal which served as  a backup scrubber.
Toluene was used only during the artificial rumen phase, while the charcoal
scrubber was used during all digestion phases.

During the preliminary trial only, one digestion flask  was  used.  Rumen
juice was sampled at the beginning of the phase and at  one, six, and
16-1/2 hours after dosing.  Samples  were collected at the beginning and
                                                              131
end of the abomasal and intestinal phases.  The collection of    I by
the toluene liquid scrubber permitted later separation  of organic and
                  131
inorganic gaseous    I by gas-liquid chromatography and possible
identification of organic and other forms.

                       RESULTS AND DISCUSSION

Results of the preliminary trial (data not shown) indicate practically no
loss of    I from the digestive juices during the incubation periods.
             1 "31
Virtually no    I was recovered in the toluene and charcoal scrubbers or

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in the plastic tubing used.  Chromatographic analysis,  originally
scheduled on the toluene to determine the form of    I  present,  was
not necessary since the toluene contained no radioactivity.

The averages of the results of the three in vitro trials  are  shown  in
Table I.  An analysis of variance indicates that there  was  no significant
difference (p »0.05) between the activity levels in any  of the  nine
fluids sampled.  This indicates little, if any, loss due  to volatility
of    I or binding of    I to sediment.

Radioactivity associated with the sediment was not significantly above
background.  Therefore, although some activity is associated  with the
sediment, it is within the limits that might be expected  from experimental
error.  There was a sharp drop in sediment-associated    I  following
abomasal digestion and almost no    I was associated with sediment  during
the intestinal phase.  This may be due to enzymatic digestion of the
rumen microflora and other sediment present.
           I 01
Absence of    I deposited on the charcoal scrubbers also  demonstrates
little or no    I volatility in these digestive fluids.  Though  not
statistically significant (p »0.05), there appears to  be a slight  drop
in    I remaining in whole juice and supernatant between  the  beginning
and the end of the abomasal phase.  This cannot be accounted  for by
   I association with sediment or deposition on the charcoal  scrubbers
                                  131
and is possibly due to binding of    I to the container during incuba-
tion or centrifugation.

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              131
             TABLE  I

I DISTRIBUTION  DURING  IN VITRO DIGESTION

Whole juice start
Percent of I remaining
Artificial
Rumen %
100.00
102.36*
Abomasal
Phase %
100.00
96.52
Intestinal
Phase %
100.00
98.59
  at end of phase
           101
Percent of    I in supernatant
  at end of phase
           131
Percent of    I in sediment
  at end of phase
Percent of    I deposited
  on charcoal during phase
                   98.16
                    3.13
94.53
 0.521
99.82
 0.038
                    0.00062     0.001038    0.0000
*Value greater than 100$ is due to counting error.

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                           1 "31
These results suggest that    I iodide present in rumen contents is


not volatilized and it is not likely that it would be eructated as


gaseous iodine into the bovine respiratory system where it might be


absorbed through the tissues  of the respiratory tract.

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                            REFERENCES
1.  Barth, J., Bruckner, B.  H., Evaluation of  Clays  as Binding Agents for
    Reduction of Radionuclides in Milk.   Binding  Properties of Natural and
    Hydrogen Form Clays with Strontium and Essential  Cations  in Artificial
    Rumen and in Simulated Abomasal  and Intestinal  Fluids.  J. Agr. Food
    Chem. 17:1340-1343, I969a.

2.  Barth, J., Bruckner, B.  H., Evaluation of  Clays  as Binding Agents for
    Reduction of Radionuclides in Milk.   Binding  Properties of Clays with
    134cs in Artificial Rumen and in Simulated Abomasal and Intestinal
    Fluids, and Uptake of '34cs by Rumen Microflora.  J. Aqr. Food Chem.
    17:1344-1346, 1969b.                                  	

3.  Dougherty, R. W., Physiological  Studies of Induced and Natural Bloat
    in Dairy Cattle.  J. Am. Vet. Med. Assoc.  96_:43-46, 1940.

4.  Dougherty, R. W., Mullenax, C. H, Allison, M.  J., Physiological
    Phenomena Associated with Eructation in Ruminants, in Dougherty, R. W.,
    Allen, R. S., Burroughs, W., Jacobson, N.  L.,  McGilliard, A.  D.,
    Physiology of Digestion in the Ruminant, pp.  159-170, Butterworth,
    Washington, 1965T

5.  Dougherty, R. W., Shipe, W. F.,  Gudnason,  G.  V., Ledford, R.  A.,
    Peterson, R. D. Scarpellino, R., Physiological  Mechanisms Involved
    in Transmitting Flavors and Odor to Milk.   I.   Contribution of
    Eructated Gases to Milk Flavor.   J. Dairy  Sci. _45:472-476, 1962.

6.  Dougherty, R. W., Stewart, W. E., Nold, M. M.,  Lindahl,  I. L.,
    Mullenax, C. H., Leek, B. F., Pulmonary Absorption of Eructated
    Gas in Ruminants.  Am. J. Vet. Res. _23:205-212,  1962.

7.  Shipe, W. F., Ledford, R. A., Peterson, R. D.,  Scanlan,  R. A.,
    Geerken, H. F., Dougherty, R. W., Morgan,  M.  E., Physiological
    Mechanisms Involved in Transmitting Flavors  and Odors to  Milk.
    II.  Transmission of Some Flavor Components  of Silage.  J. Dairy Scj.
    45:477-480, 1962.

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