AMERICAN JOURNAL OK CLINICAL PATHOLOGY
Vol. 32, No. 5, November. 1959, pp. 465-467
Printed in U.S.A.
DETERMINATIONS OF EXTRACELLULAR FLUID SPACE, (ECF) FROM SMALL
SAMPLES OF TISSUE
A N T H O N Y N O R M A N , A.B., P A U L A. R O N D E L L , P H . D . , AND D . F . B O H R , M.D.
Department of Physiology, University of Michigan, Ann Arbor, Michigan
Increasing interest in the electrolyte
composition of tissue makes it desirable to
have a procedure for in vitro determination
of extracellular fluid space (ECF) suitable
for tissues of small experimental animals
and for biopsy samples. In principle, the
reliability of estimations of concentrations
of intracellular electrolyte is dependent upon
the accuracy and precision of determinations of ECF. In practice, the calculation of
intracellular concentrations is further limited
by insufficient sensitivity of common methods of determining ECF. The following
report deals with the description of (1) the
use of a color reaction sufficiently sensitive
to determine inulin and sucrose spaces of
tissue fragments at least as small as 14 mg.
wet weight,* (2) technics developed for
achieving a useful degree of precision with
this reaction, and discusses (3) the problem
of accuracy of determinations of ECF.
M A T E R I A L S AND
METHODS
Dreywood's anthrone color reaction for
carbohydrates 1 was observed to fulfill the
requirement of high sensitivity. It has been
used for measurement of blood glucose,2
blood and urine amylase,3 plasma and urine
inulin,9 and low concentrations of sucrose.5
It is approximately 10 times as sensitive as
the Seliwanoff reaction8 ordinarily used for
determinations of inulin. As little as 4 gamRccoivcd, March 10, 1959; accepted for publication J u l y 15.
Mr. Norman is Student Research Trainee, D r .
Rondell is Assistant Professor of Physiology, and
D r . Bohr is Professor of Physiology, Universit.y of
Michigan.
* The d a t a in this paper were derived from studies
of samples of tissue approximately 14 nig. in
weight. The procedure has been successfully
applied in this laboratory to samples of tissue of
1 to 2 mg. wet weight, by means of increasing the
concentration of inulin in the loading solution to
3 per cent. This study was supported by a grant
from the Michigan H e a r t Association.
465
mas of sucrose or inulin in 2 ml. of solution
can be measured.
Samples of vessel wall from the carotid
and aorta were obtained from healthy dogs
fed Pard dog meal and tap water ad libitum.
The tissue was removed from anesthetized
animals, kept moistened with a carbohydrate-free Krebs solution, and rapidly
cleaned of loose adventitia. The samples
were immediately cut into small hemicylindrical pieces weighing approximately 14 mg.
and placed in inulin or sucrose Krebs solution at room temperature. One per cent
inulin and 1.7 per cent sucrose were observed
to be suitable concentrations to use with
strips of this size. Isotonicity between the
cells and the bath was obtained by means of
adjusting the sodium chloride content of the
Krebs solution. Fragments were allowed to
equilibrate for approximately 8 hr.; at the
end of this period, the tissue was removed,
blotted, and placed in 5 ml. of distilled
water. The carbohydrate-loaded strips were
permitted to remain in the distilled water
for 12 hr., at which time it was assumed that
all the inulin or sucrose that the muscle
strips had absorbed from the Krebs-carbohydrate bath had become distributed
equally between the tissue and the distilled
water.
Two-milliliter aliquots of the supernatant
fluid were placed in 22 by 175 mm. pyrex
tubes and cooled to 8 ± 2 C. in an ice bath.
The anthrone reagent, consisting of 0.20
per cent anthrone and 1 per cent thiourea
(weight per volume) in 96 per cent sulfuric
acid, was also cooled to this temperature.
The thiourea was present in the reagent to
prevent oxidation of the active enol tautomer of anthrone to the inactive keto
tautomer. 7 Precooling the anthrone reagent
and the unknown solutions reduced the heat
of mixing, which produces an excessive premature color formation. Anthrone reagent
(4 ml.) was added to the 2 ml. of the carbo-
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NORMAN ET AL.
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TIME (HOURS)
FIG. 1. Tissue was obtained from dog aorta just proximal to the bifurcation
of the iliacs. The curves for 1 per cent inulin and 1.7 per cent sucrose were obtained
from the aorta removed from 1 dog; the 5 per cent inulin curve from the aorta of
another. The level plateaus in all loading curves imply complete equilibrium with
ECF.
hydrate solution in the ice bath. After all of
the samples in the run were brought to this
stage, they were immediately heated for
10 min. at 90 ± 2 C , cooled for 5 min. in
an ice bath, and then allowed to come to
room temperature. According to Koehler,4
heating for 10 min. is optimal for maximal
development of color for sucrose. Although
inulin manifests a maximal development of
color after 2 min. of heating, a greater uniformity in the color of duplicate samples,
and only slightly less total color, results
from heating the samples for 10 min. The
resulting turquoise-green solutions were read
in a Klett-Summerson colorimeter at 620 m/aThe mean Klett reading of nonloaded tissue
blanks was subtracted from the readings of
the unknowns.* Carbohydrate concentration
* Calculation of apparent ECF of 20 tissue blanks
yielded a mean ECF of 2.07 per cent wet weight.
This sample blank probably arises from glucose
or other soluble carbohydrates present in the
tissue. A part of sample-to-sample variability
may be owing to variation in the content of glucose. When samples of tissue in the range of 1 to
5 mg. wet weight are used with higher loading concentrations of inulin, the color of tissue blanks
becomes negligible.
was determined from a calibration curve of
standards made from dilutions of the loading solutions.
RESULTS AND DISCUSSION
Rate-of-entry studies were performed with
both inulin and sucrose. Fragments of aorta
tissue were immersed in the carbohydrate
solutions for varying time intervals, but
were otherwise treated as described above.
A plot of the data (Fig. 1) indicates (.1.)
that saturation of the sucrose and inulin
spaces occurs in approximately 2 and 3 hr.,
respectively, and (2) that the sucrose space
is slightly larger than the inulin space.
A semilog plot of the ECF:wet weight
ratio versus time results in a curve that
consists of 2 distinct components, each a
straight line with its own specific slope. This
suggests dual phases in the loading rates of
both sucrose and inulin that are entirely
parallel with those obtained by means of the
in vivo measurements of Nichols and his
associates.6
Examination of ECF determinations of
17 samples from a single carotid indicated
the mean ECF to be 39.19 ± 1.92 per cent
of total wet weight, whereas similar analysis
Nov. 1959
ECF
4G7
DETERMINATIONS
of 10 samples of carotid of another animal
indicated a mean ECF of 37.49 ± 1.99 per
cent. The precision of the analysis is reflected in the standard deviations; i.e.,
approximately ± 5 per cent of the calculated mean value. Furthermore, the average
deviation from the mean value of duplicate
determinations of 14 vessel wall fragments
was 0.69 per cent of the mean value. It
would seem that most of the variability of
multiple samples is caused by an actual
fragment-to-fragment variation in inulin
space or endogenous carbohydrate and not
to uncontrolled variables in the reaction
procedure.
None of the materials routinely used for
ECF approximations has yet been unequivocally demonstrated to penetrate a "true
ECF." Thorough studies of Nichols and his
associates,6 for example, led to the conclusion that the difference between chloride
and inulin or thiosulfate spaces was caused
by slow penetration of connective tissue
water by the larger molecules and that, although chloride spaces are more accurately
"true E C F , " inulin or thiosulfate space
represents the more "active" compartment.
The tissue we have studied contains large
but unquantified amounts of both collagenous and elastic connective tissue, and the
effect of this tissue on the apparent E C F
has not been evaluated.
SUMMARY
A method is described for the in vitro
estimation of extracellular fluid space (ECF)
of unusually small samples of tissue. The
method is suitable for precise measurement
of small changes or differences in any carbohydrate space.
SUMMARIO I N
1NTEULINGUA
Es describite un methodo pro le estimation in vitro del spatio de fluido extracellular
in inusualmente micre specimens de histo.
Le methodo es usabile pro le mesuration precise de micre alterationes o differentias in
non importa qua! spatio de hydrato de
carbon.
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