Transcapillary Exchange Rates of Deuterium Oxide
and Thiocyanate in the Forearm of Man*”?

EDWARD D. FREIS, THOMAS F. HIGGINS anp HAROLD J.
MOROWITZ. From the Cardiovascular Research Laboratory, George-
town University Hospital, Department of Medicine, Georgetown Uni-
versity School of Medicine, the Veterans Administration Hospital,
Washington, D. C., and the Heat and Power Division of the National
Bureau of Standards

HE RATE AT WHICH DIFFUSIBLE, nongaseous substances transfer into and out of
the circulation is not well understood. Disappearance slopes of injected labeled ma-
terials represent the integrated effect of filtration and reabsorption and therefore
indicate the net rather than the absolute transfer of such substances out of the blood
stream. Hence, in order to measure the absolute transcapillary movement of a dif-
fusible substance it is essential that the rate of transfer of this material be determined
in the early phases of the initial circulation prior to any significant return of filtered
material from the interstitial fluid to the blood.

This can be accomplished by utilizing a technique of multiple sampling from
the effluent vessel at intervals of seconds rather than'minutes. In addition as pointed
out by Pappenheimer and his associates (1) since the method depends on concentra-
tion measurements, the dilution of the labeled substance by the blood itself also must
be known. This may be accomplished by mixing the labeled, diffusible material with a
second nondiffusible substance prior to injection. Then the concentration of the
nondiffusible substance in the recovered samples will indicate the dilution of both
substances by the blood itself. A similar method has been devised by Chinard (2)
and by Freinkel, Schreiner and Athens (3).

METHODS

The dye T-1824 (Evan’s blue) was used as the nondiffusible tracer since it has
the property of rapid mixing and binds to the relatively impermeable plasma pro-
tems. Deuterium‘ oxide and sodium thiocyanate were the permeable tracer sub-
stances. Twenty per cent ‘salt free’ human serum albumin was added to this mixture
in order to obtain a solution with an osmotic pressure within the physiologic range.
The usual procedure was to mix, under sterile precautions, 5 ml. of deuterium oxide,

Received for publication September 12, 1952.

‘Presented in part at the Scientific Sessions of the American Heart Association, April ro,
1952.

* Supported in part by research grants from the National Heart Institute, U. S. Public Health
Service, the Squibb Institute for Medical Research, New Brunswick, N. J. and Irwin, Neisler and
Company, Decatur, Ill.

Sponsored (in part) by the VA and published with the approval of the Chief Medical Direc-
tor. The statements and conclusions published by the authors are a result of their own study and
do not necessarily reflect the opinion or policy of the Veterans Administration.

* The monochromater used for determining the concentrations of deuterium was constructed
by the Research Department of Leeds and Northrup Company, and was loaned on a field trial basis
to the Heat and Power Division of the National Bureau of Standards.

526
March 1953 TRANSCAPILLARY EXCHANGE RATES 527

9.5 ml. of 5 per cent T-1824, 1.0 ml. of 20 per cent sodium thiocyanate and 1.5 ml.
of 20 per cent human serum albumin.

After withdrawing a sample of this mixture for analysis for T-1824, deuterium
and thiocyanate concentrations, the remainder was taken up in a syringe and injected
at an even rate into the branchial artery over a 2- to 6-second period. The technique
of injection and of sampling which was similar to that described in another communi-
cation (4) involves injection into the brachial artery followed by rapid sampling from
an antecubital vein. In all except one experiment the circulation to the hand was
excluded by inflating a pneumatic cuff wrapped around the wrist to pressures well
above the subject’s systolic blood pressure.

The concentrations of T-1824 and thiocyanate were determined according to
methods previously described (5). The concentration of deuterium oxide was de-
termined by a new spectrophotometric method which is based on the emission
spectra of atomic hydrogen and deuterium in a high frequency electrodeless dis-
charge (6, 7). Using this method it is possible to measure concentrations of deuterium
as low as 1 part in 6500 with an accuracy of +5 per cent with correspondingly greater
accuracy at the higher concentrations measured in these experiments.

Typical T-1824 transit curves through the vessels of the forearm have been
described previously (4). Following the appearance of the dye there is a rapid rise to
peak concentrations followed by a more gradual decline. The concentrations of
deuterium oxide and of thiocyanate were measured at several points along this
curve, but particularly at the early period prior to or at peak concentrations of the
dye.

The estimation of the percentage of the permeable substances which pass out
of the blood is calculated from the formula:

t me
= So X 100 (z)

where L is the percentage loss of the labeled substance (x) from the circulating blood,
Cé is the concentration of x which would be present if none had left the circulation
and C™ is the concentration of « experimentally determined in the sample. The
calculation Cé is based on the premise that no significant amount of T-1824 is lost
from the circulation in a single passage through the forearm capillaries. The concen-
tration of T-1824, therefore, is an index of the volume of blood in which the injected
materials have been diluted. In addition, the relative concentrations of dye and the
permeable substances in the injected mixture are known. Correction must be made,
however, for the passage of diffusible substances into the water of the red cells. In
the present studies it has been assumed that complete equilibrium has occurred
between the water of the red cells and the water of the plasma in each sample.

The above factors are accounted for as follows: consider a venous blood sample
of volume V. This volume represents the sum of two contributing volumes, w the
volume contribution due to the injected solution and y the volume contribution of
the blood. If C, is the concentration of T-1824 in the injecta, the concentration in
the sample under consideration, C¢ is given by

we,
w+ pyW,

where » is the plasma fraction of the sample and W? is the water fraction of plasma.
Thus, the denominator is the éofal fluid volume available to the T-1824, while the

Ce = (2)
528 FREIS, HIGGINS AND MOROWITZ Volume 5

numerator is the amount of T-1824 in this volume. If there were no loss of permeable
tracer substance from the blood, that is no diffusion, the expected concentration,
Ci, would be

we,
wt pyW, + WV,

where C, is the concentration of the labeled substance x in the injected mixture, ¢
is the cell fraction (by volume) of the sample and W’, is the water fraction of cells.
The denominator, therefore, is the éofai fluid volume in the blood available to the
tracer substance including the fluid volume of the red cells.

Solving equations 2 and 3 the following expression is found for C!

t

))

C.Cn
We (c on
pWp e— €

Of these quantities of the right hand side of the equation, Cz, C7”, C., ¢ and p are
measured for each experiment, while W, and W, are considered as constants of blood

Cz = (4)

 

a+

 

W,  .90
can be calculated directly from equation 1.

W.  .6 . .
( ee n). After determining CZ the percentage loss (L) of the material

RESULTS

The transcapillary exchange of deuterium oxide was determined in the forearm
vascular bed in four normal subjects and two hypertensive patients (table 1 and fig.
1). The mean value for the maximum observed loss of deuterium oxide from the
circulation in the six subjects was 94 per cent, S.D. 2.6, No significant differences
were observed between the normal and the hypertensive subjects.

The permeability of thiocyanate was determined simultaneously in five of the
six subjects. The maximum observed loss of thiocyanate from the circulation was
much less than that of deuterium oxide, the mean value for thiocyanate being 48
per cent, S.D. 7.4.

Since it seemed possible that the high local concentrations of thiocyanate used
in these studies might alter normal capillary permeability, sodium chloride in physi-
ologic concentration was substituted for thiocyanate in one experiment (subject J. J.,
table r). In this experiment the rate of loss of deuterium oxide did not differ sig-
nificantly from the others in which thiocyanate was injected.

The maximum percentage loss of both deuterium oxide and thiocyanate from
the circulation occurred early in the circulation of T-1824. At this time there also
was a considerable decline in hematocrit values. In later samples the apparent loss
of both substances diminished probably because of some return of filtered material
to the circulation. Figure 1 illustrates a typical experiment in which the percentage
loss of deuterium oxide decreased from 95 to 79.7 per cent and of thiocyanate from
55.2 to 20.8 per cent. The hematocrit fell from a control value of 39 to 32 at the
peak of the dye concentration curve but returned gradually to 37 in the final samples.

Because of the possibility that such marked and rapid shifts in hematocrit
might influence the exchange rates, the circulation to the hand was not excluded in
subject C, G. (table 1), and the volume of injected materials was made as small as
could be permitted without sacrificing accuracy of determination. In this subject
March 1953 TRANSCAPILLARY EXCHANGE RATES 529

the hematocrit did not fall at any time during the collection period; yet the percentage
losses of deuterium oxide and thiocyanate were, if anything, slightly higher than
those found in the other experiments.

TABLE 1. PERCENTAGE LOSS OF DEUTERIUM OXIDE AND THIOCYANATE ION FROM THE CAPILLARIES
OF THE FOREARM IN NORMAL AND HYPERTENSIVE SUBJECTS. T-1824 CONCENTRATIONS AND
HEMATOCRIT VALUES ALSO ARE GIVEN

ESTIMATED LOSS Lesrnwaren Loss!

 

 

 

 

 

 

 

TIME FROM INJ., | T-1824 CONC., IN . ;

SUBJECT AND DIAGNOSIS SEC. SAMPLE, MG. % OTOXIDE, OF meee NANE: HEMATOCRIT
F.O. ° 39
Normal 6 3.0 95.0 47.6 36

8 8.6 94.6 55-2 31

12 10.2 92.6 34.8 31

16 6.9 88.6 41.3 32

22 5.0 85.1 49.3 34

32 3.2 79-7 20.8 37

M.A. ° 4
Normal 12 2.5 87.3 56.0 38
16 12.0 95.2 53-3 32

28 5.8 84.2 29.6 36

40 2.8 72.5 22.2 39

C.G. ° 50
Normal 14 2.7 95-3 41.0 50
18 5.1 96.4 46.4 51

22 4.2 96.8 47-1 50

32 2.6 87.2 40.0 51

TLE, ° 43
Hypertension 14 1.6 86.7 34.8 42
20 4-7 90.9 46.2 38

30 3.6 88.6 49.1 42

40 2.5 80.5 48.3 43

J.J. ° 42
Normal 14 3.5 95-8 7 38
18 5.4 94-4 38

24 3.4 90.0 39

32 1.3 76.6 41

M.C. ° 37
Hypertension 10 6.2 g1.6 34-1 32
I4 8.6 88.8 35.6 30

16 8.4 go.5 37-8 | 30

22 5.1 86.4 33-1 33

| 34 1.9 65.5 16.9 | 35

 

 

* Sodium chloride replaced thiocyanate in this experiment.

DiIscussION

The validity of the method depends primarily on the assumption that T-1824
is not lost from the blood plasma in any significant amounts during a single circula-
tion. This assumption seems to be valid (8, 9). It is worth noting that if the capil-
laries were permeable to T-1824 the calculated loss of deuterium oxide would be
less than the actual loss. Hence, it is not possible to ascribe the high rate of loss of
deuterium oxide to leakage of T-1824.

Complete mixing of T-1824 with the circulating blood does not seem to be as
important in this determination as in the measurement of total blood volume or
530 FREIS, HIGGINS AND MOROWITZ Volume 5

cardiac output. The dye is well mixed with the diffusible labeled substances prior
to injection. Hence, if complete mixing of T-1824 with blood does not occur, the
same should hold for the diffusible substances (except for differences in diffusion
rates of the various substances).

It is possible that the deuterium may exchange with hydrogen atoms in the
plasma proteins so that they are diluted into a larger volume than the plasma water.
However, addition of deuterium oxide to plasma or blood in vilro indicated that
errors due to such exchanges were negligible.

It is evident that the present method indicates only the loss of permeable sub-
stances from the circulating blood and does not differentiate between passage through

 

ESTIMATED LOSS OF DEUTERIUM OXIDE FROM CIRCULATING BLOOD —- PER CENT

I |
We MANA OA

so}
i
ESTIMATED LOSS OF THIOCYANATE ION FROM CIRCULATING BLOOD-PER CENT
TTT

Lone

PLASMA CONCENTRATION OF T~1824 IN RECOVERED SAMPLES- MG. PER CENT

 

 

 

 

 

 

60r
40r

20r

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

80F
60F

 

 

i]

 

Aor
205

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4 4 1 . 2. 2 2 2 m 1 4 2 Zz a 1

2 4 6 8 10 \2 14 16 1a 20 22 24 26 28 30 32

 

 

 

Fig. 1. PERCENTAGE Loss of deuterium oxide and thiocyanate from the blood during a single
circulation through the capillaries of the forearm. The T-1824 transit curve is shown below. Ordi-
nates are percentages; abscéssae, seconds.

the capillary membrane and penetration into the cells of the blood vessel walls.
Finally, permeability may vary in vascular beds other than those in the forearm so
that results obtained in this area of the circulation are not transferable to other areas.

The blood-extravascular fluid exchange was almost complete in a single circula-
tion. It does not seem likely that hydrostatic pressure alone could accomplish such
an extensive movement of fluid. Further, if water were lost in the arteriolar end of
the capillary by hydrostatic pressure and regained in the venous end by osmotic
pressure as has been supposed (10) the capillary hematocrit would of necessity rise
to unphysiological heights. The magnitude of the water exchanges found in these
experiments makes it seem possible that the transcapillary movement of water is
March 1953 TRANSCAPILLARY EXCHANGE RATES 531

primarily a process of simple diffusion, whereas the forces of hydrostatic and osmotic
pressure are more concerned with the adjustment of the final equilibrium. Flexner
and his associates using a slightly different technique also concluded that diffusion
rather than filtration is the predominant process in the exchange of water, sodium
and chloride across the capillary wall (11).

It is of interest that the descending limb of the curve representing the percentage
loss of deuterium oxide was not steep, indicating that the heavy water was not re-
turning to the blood either rapidly or in large amounts. This suggests that the greater
part of the filtered water immediately enters cells, or moves away in the interstitial
fluid or becomes bound; and that the greater part of the water entering the blood in
that period has not been filtered recently through the capillaries. Such observations
suggest an extensive migration of water in the extravascular tissues.

The percentage loss of thiocyanate ion from the circulating blood was less than
that of water. The lower diffusion rate of thiocyanate may be accounted for by the
fact that significant amounts of thiocyanate bind to plasma proteins particularly in
the albumin fraction (12) and, therefore, cannot penetrate the capillary wall. In
addition the larger thiocyanate molecule does not diffuse as rapidly as deuterium
oxide.

The descending limb of the curve representing the percentage loss of thiocyanate
(fig. 1) usually was steeper than the similar curve for deuterium oxide indicating that
thiocyanate returns more rapidly to the blood. This difference in reabsorption rates
is not surprising, however, since thiocyanate does not penetrate most cells (13) and
therefore remains in the interstitial fluid where it is available for more rapid equilibra-
tion with the circulating blood.

When the volume perfused was smail in comparison to the volume injected the
hematocrit fell indicating a considerable disturbance in this physiologic equilibrium
of the blood. However, despite a massive water exchange this new equilibrium was
maintained in the diluted segment of blood moving through the forearm capillaries.
The fluid content of the blood, therefore, does not seem to be adjusted immediately
by physical forces operating in capillary beds such as those in the forearm.

SUMMARY AND CONCLUSIONS

A method is described for estimating the exchange rates of labeled filterable
substances through capillary walls. The method was applied in studying the trans-
capillary exchange rates of deuterium oxide and thiocyanate ion, and indicated that
approximately 95 per cent of the heavy water and 50 per cent of the thiocyanate ion
transferred out of the blood in a single circulation through the forearm. The major
portion of the deuterium oxide which passed through the capillary walls of the fore-
arm did not return rapidly to the circulation.

The rapidity and magnitude of the water exchange across capillary walls ob-
served in these experiments suggest that simple diffusion rather than hydrostatic
and osmotic pressures is the major influence governing such fluid transfer.

The authors wish to thank Miss Jean Pietras, Miss Barbarba Allison, Mr. Victor Landi and
Mr. Larry C. Gaskins for valuable technical assistance.

REFERENCES

1, PapPENHEIMER, J. R., E. M. RENKIN AND L. M. Borrerp. Am. J. Physiol. 167: 13, 1951.
2. Cuinarp, F. P. Bull. Johns Hopkins Hosp. 88: 489, 1951.
3. FREINKEL, N., G. E. SCHREINER AND J. W. ATHENS. J. Clin. Investigation 31: 629, 1952.
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Frers, E. D. anp R. H. Smitawicx. Am. J. M. Sc. 214: 363, 1947.

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Morowti7z, H. J. anp H. P. Broa. Analy. Chemi. In press.

. GREGERSEN, M. 1. AND R. A. Rawson. Am. J. Physiol. 138: 698, 1943.
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& Wilkins, 1945, p. 25.

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