The passage of ascorbic acid across the membrane of the human erythrocyte

The Passage of Ascorbic Acid Across the Membrane Human Erythrocyte

of the

Ralph Golden’ and Frederick Sargent, II’ From the Army Medical Nutrition Laboratory, Chicago, Illinois23 3

Received August, 15, 1951

Cellular ascorbic acid behaves differently in viva than in vitro. In the intact organism the vitamin can be shown to move slowly across the membrane of the leukocyte and the erythrocyte (l-4). Under in vitro conditions, however, it, has only been possible to demonstrate that ascorbic acid will move into the leukocyte (5, G, 7). Whether or not ascorbic acid will move out of the cellular elements under these conditions has not been studied. The transfer of ascorbic acid across the cell membrane may be influenced by insulin. Sherry and Ralli (8) reported t’hat, both in vivo and in vitro, insulin caused a transfer of ascorbic acid into the leukocyte but not into the erythr0cyt.e. These observations suggest that ascorbic acid will much more readily move into and out of the leukocyte than the erythrocyte. In either case, however, the vitamin seems to move into the cellular elements more readily than it moves out. Since these facts are not consistent with free diffusion, the suggestion has been made (9, 10) that ascorbic acid is bound to a nondiffusible constituent of the cell. Eidel’mann and Gordon (11) claim to have demonstrated that adrenaline causes release of bound ascorbic acid from the cellular elements. Whether or not, ascorbigen (12, 13) is the nondiffusible form of ascorbic acid in question is not known. Matusis (14) and Sargent and Golden (15) failed to find ascorbigen in plasma. In the present, investigation, a study was made 1 Present address: Department of Physiology, University of Illinois, Urbana, Illinois. 2 An installation under the jurisdiction of the Surgeon General, U. S. Army. 3 The opinions expressed in this paper are those of the authors and do not necessarily represent the official views of any governmental agency. 138

ASCORBIC

ACID

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of the transfer of ascorbic acid across the membrane of the erythrocyte and of the ascorbigen content of whole blood. METHODS

In Vitro Equilibratiml The plasma was separated from the erythrocytes of heparinized venous blood by centrifugation. The erythrocytes were washed” two to three times with 2 vol. of cold Ringer-Locke’s solution and resuspended in approximately equal volumes of plasma or Ringer-Locke’s solution. After thorough mixing, the suspension was divided into three parts: one for determining the initial concentrations of ascorbic acid, one maintained 4°C. as a control,’ and one transferred to a modified Barcroft tonometera and equilibrated7 for 1 hr. in a water bath maintained at 37°C. After equilibration, the ascorbic acid concentrations of the control and equilibrated suspensions were determined. .411the equilibrations were conducted in an atmosphere of room air.

CO2 Experiments Eight experiments were conducted in w-hich heparinized venous blood was equilibrated under a wide range of pC0, levels. In these tests the standard Barcroft tonometer was employed. The equilibration time was 20 min. in a water bath maintained at 37°C. The specimens of blood were divided into three portions: one maintained at 4°C. as a control, one introduced into a tonometer with a low $02, and one introduced into a tonometer with a high pCOn. After equilibration, the blood samples were removed from the tonometers under mineral oil. The cells and plasma were separated hy cent,rifugation under oil.

Three hcslthy young males participated: J. R., age 3X; F. S., age 30; and R. G., age 26. After pre1iminar.v determinations of the ascorbic acid concentration of erythrocytes and plasma, these men were given a loading test of 500 mg. of ascorbic acid orally. Daily thereafter these subjects took, in addition to their 4 Washing the erythrocytes did not appreciably alter their ascorbic acid content. In 16 experiments the ascorbic acid concentration of washed cells averaged 100.3 f 12.4% of that of unwashed cells. 6 According to Heinemann and Hald (5) there is no transfer of ascorbic acid across the cell membrane at 7°C. 6 Some of the equilibration experiments were conducted with the standard Barcroft tonometer. The modified tonometer w-as made for these studied by Mr. George Kokinis of the Chicago Scientific Glass Works. The tonometer was essentially a miniature of the standard vessel. It had a lo-ml. capacity and a groundglass stopper in place of the conventional rubber stopper. 7 A special equilibration apparatus for rotating the toqometers in the water bath was designed by the authors and built by Mr. George E. Wendt of Chicago, Illinois.

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customary diets, 500 mg. ascorbic acid in divided doses for 25 days and then 1000 mg. for 5 days. Following this period of “saturation,” the subjects began to consume a diet low in ascorbic acid. On the 18th day of the latter regimen, an oral loading test was conducted, using 1000 mg. of ascorbic acid. The subjects then continued the low ascorbic acid diets for an additional 23 days. On the 21st dav the loading test was repeated.

Acid Hydrolysis Acid hydrolysis was carried ciates (12, 13, 16-19).

out according

to the method

of Guha and asso-

ilscorbic Acid Analysis In the experiments with COs and in the acid hydrolyses, ascorbic acid was assayed by the method of Roe and Kuether (20). The majority of the analyses made during equilibration experiments were carried out by means of a modification of the micro method of Bessey et al. (21). The reagents were prepared and used exactly as described. The quantities were changed as follows: One ml. of sample was added to 4 ml. of 5y0 trichloroacetic acid. To 1 ml. of the centrifugate, 0.25 ml. of the 2,4-dinitrophenylhydrazine reagent was added. After a 3-hr. incubation at 37”C., 1.25 ml. of cold 65% sulfuric acid was added drop by drop. The erythrocyte ascorbic acid concentration was calculated as described by Sargent (10). RESULTS

AND DISCUSSION

In Vitro Equilibration

(Table I)

No evidence was obtained that ascorbic acid either passed into or out of the erythrocyte when these cells were suspended in media containing 0.00-4.60 mg./lOO ml. of the vitamin. If ascorbic acid could cross the membrane of the erythrocyte by free diffusion, appreciable changes in the concentration of ascorbic acid should have occurred with 1 hr. at 37°C. (15). Furthermore, the presence of adrenaline and insulin did not cause any significant gain or loss of erythrocyte ascorbic acid.8 These latter findings fail to confirm the claim of Eidel’mann and Gordon (11) but do confirm the report of Sherry and Ralli (8). CO2 Experiments Johnson et al. (23) have shown that large changes in the pcoz produced significant shifts in cellular chloride and lactate. The ionic transfer was 8 Preliminary experiments indicated that when the ascorbic acid concentration ranged between 4.70 and 8.28 mg./lOO ml., the erythrocyte ascorbic acid concentration increased 15(t2OOo/o in the presence of insulin (1 unit/ml.) both at 4” and 37°C. NO such transfer occurred in the absence of insulin. Inconsistent results were obtained with adrenalin (2 rg./ml.). The explanation of the phenomenon awaits further study.

ASCORBIC

141

.4CID

in agreement with the Donnan theory of membrane equilibrium. In the present series of experiments, the behavior of ascorbic acid was studied under similar conditions. The low p C02values studied ranged from 8 to 22 mm. Hg; the high pCo, values from 159 to 218 mm. Hg. In none of the experiments did a consistent and significant change occur either in the

--__

TABLE I Summary of in Vitro Equilibration

Conditions of experiment

Ascorbic acid in ““gSp&C mg./lOO ml.

in Ringer-Locke

I ~

~~__~Original

--~ mg./100

Solution

Erythrocyte ascorbic acid= --.-~1 hr.. 4T. 1 hr., 37°C.

ml.

mg./lOO

ml.

mg./100

ml.

-

A. No ascorbic acid

added: Ringer-Locke solution (R. L.) R. L. plus insulin (I unit/ml.) R. L. plus adrenalin (2 w/ml.) R. Ascorbic acid added: Ringer-Locke solution (R. L.) It. L. plus insulin (1 unit/ml.) R. 1,. plus adrenalin

O.oo-O.25 (12)b 1.37 f

0.53 1.13 f

0.451.10 f

0.63

0.00.10

(6)

1.48 f

0.461.40 f

0.101.26 f

0.32

0.00-O. 13 (4)

11.21 f

0.33 1.22 f

0.41 1.19 f

0.17

1 1 1.05-4.55 (10) 1.18 f

0.21 1.27 f

0.481.13 f

0.48

3.054.60

(9)

1.56 f

0.421.40 f

0.481.50 f

0.53

2.75-3.88 (8)

1.46 f

0.01 1.43 f

0.041.40 f

0.07

(2 xf4lml.) (1Mean f standard deviation. by t test (22). b Number of experiments.

None of the means were significantly

different

ascorbic acid concentration of the erythrocytes or plasma or in the ratio of cellular ascorbic acid to plasma ascorbic acid (Table 11). Therefore, the ratio of cellular to plasma ascorbic acid is not conditioned by the Donnan equilibrium. In Vivo Equilibration Prior to saturation, plasma and erythrocyte ascorbic acid concentra1.98 and 1.69 mg./lOO ml. for subject J. R., 1.26 and 1.13 for subject F. S., and 1.10 and 1.18 for subject R. G., respectively. During the Sl-day period of saturation the plasma and erythrocyte concentrations of ascorbic acid averaged 1.98 and 1.73 mg./lOO ml. for

tions averaged

142

RALPH

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subject J. R., 1.40 and 1.29 for subject F. S., and 1.84 and 1.71 for subject R. G., respectively. These results indicate that with relatively small increases in the plasma concent,ration of ascorbic arid, the erythrocyte will eventually show a proportional increase. Most, of the increase in eryt’hrocyte ascorbic acid which did occur t)ook place within the 3-hr. period following the load-test (Fig. 2). Within 3-4 days, F. S. and R. G. had come into equilibrium and no appreciable changes occurred thereafter. Doubling the daily intake of ascorbic acid during the last 5 days caused no additional increase. Alfter the period of satura,t,ion, the subjects began the low ascorbic acid regimen (Fig. 1). The fact, that the erythrocyte ascorbic acid cow

l’ypical

Experiment

on Equilibration

Conditions of experiment

pco,

of Blood at Different .-~-

Ascorbic acid ~~-.__~Plasma ; Erythrocyte CAP)

mm. Hg.

Conho Low PCO? High PCO~

i !

15 218

pcot Levels

(AC)

~ mg./lOO ml.

I

;$j

~

;I;

’

1.1s

~

1.33

rng.jlOO

~

AC -&

ml.

::;; 1.13

centration decreased much more slowly thaIi that of the plasma argues against free diffusion being the mechanism of t’ransfer of aworbic acid between the red blood cell and the plasma. In Fig. 2 the results of the second loading t’est conducted while the subjects were “unsaturated” are compared with loading test performed while the subjects were on their customary diets. When “normal” all three subjects showed large and sustained increases in plasma ascorbic acid. The erythrocytes, on the other hand, showed no (J. R.) or small increases (F. 8. and R. G.). The increases occurred during the first hour. With further elevation of the plasma levels, no more ascorbic acid was t,aken up by t,he erythrocyte. These data then suggest that., in ZQ’ZV, ascorbic acid may be transferred int,o the erythrocyte t,o a limit,ed extent within 1 hr.-- a finding which contrast,s with t,he in vitro st,udies (Table I). Loading tests performed when the subjects were “unsaturated” likewise indicated that a transfer of ascorbic acid into the erythrocyte okcurred. The plasma levels underwent large and sustained increases fol

ISCORBIC

9CID

143

FIG. 1. Comparative rates of decrease of erythrocyte and plasma ascorbic acid concentrations in three healthy male subjects during first 18 days of low ascorbic acid regimen.

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RALPH

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SARGENT,

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a 3-hr. period. During the first hour none of the subjects showed significant changes in the erythrocyte ascorbic acid concentrations. During the second hour the erythrocyte levels of subjects J. R. and F. S. increased appreciably. The increases were sustained up to 48 hr. in spite of falling plasma levels. Subject R. G.‘s erythrocytes failed t,o take up any ascorbic acid for 24 hr. COh%?ARATIvE CHANGES IN ERYTHROCYTE AND PLASMA ASCORBIC AC4D Dl,t?lffi LOADING TESTS: SUBJECTS NORMAL AND UNSATURATED WITH RESPECT TO ASCORBIC ACID suwJ.R.

SUBJECT F.S

SlJiWZT

R.G

FIG. 2. Comparative changes in erythrocyte and plasma ascorbic acid concentrations during loading tests. Before beginning the low ascorbic acid regimen (“normal”), the three subjects were given 500 mg. of ascorbic acid orally. After becoming “unsaturated” by subsisting on a low vitamin regimen, the three subjects were given 1000 mg. of ascorbic acid orally.

The in vivo studies, therefore, bring out clearly the marked difference between the rate of transfer of ascorbic acid into and out of the erythrocyte. That ascorbic acid more readily enters the erythrocyte than it leaves this cell bespeaks chemical combination with an intracelmlar nondiffusible substance. This substance readily accepts ascorbic acid molecules but only slowly gives them up. Acid Hydrolysis The bound form of ascorbic acid described by Saha, Majumdar, and Guha (12, 13) suggested itself as the compound involved in the above

AKCOHBIC

ACID

145

reaction. In eleven experiments, the heated hydrolyzate contained less ascorbic acid than the unheated hydrolyzate. The loss of ascorbic acid ranged from 16 to 67%,, averaging 40.4yC. Since ascorbigen is identified by an increase in the ascorbic acid content of the heated acid hydrolyzate, the results fail to confirm the findings of Guha and his associates. Furthermore, since this compound is not present in plasma (14, 15), the data suggest that ascorbigen is probably not the hypothetical intracellular binding substance. SUMMARY

1. Under controlled conditions in vitro, when the ascorbic acid concentration outside washed erythrocytes ranged between 0 and 4.60 mg./lOO ml., there was no evidence that ascorbic acid either moved out of or into the cell. 2. Wide variations in pcoz and the presence or absence of adrenaline or insulin did not alter, within this range of concentrations, t,he t’ransfer of ascorbic acid across the cell membrane. 3. In vivo, ascorbic acid moved slowly across the cell membrane. Ascorbic acid passed into the erythrocyte more rapidly than it passed out. 4. The presence of ascorbigen (bound ascorbic acid) in laked whole blood was not confirmed. REFERENCES 1. BUTLER, A. M., AND CUSHMAN, M., J. Clin. Invest. 19, 459 (1940). 2. CRANDON, J. H., LUND, C. C., AND DILL, D. B., New England J. Med. 223. 353 (1939). 3. MEDICAL RESEARCH COUNCIL, VITAMIN C SUBCOMMITTEE OF ACCESSORY FOOD FACTORS COMMITTEE, Lancet 264, 853 (1948). 4. SOSSAI, A., hll. sot. ital. biol. sper. 16, 738 (1941) [C. A. 40, 6588 (1946)j. 5. HEINEMANS, M., AND HALD, P. M., J. Clin. Invest. 19. 469 (1940). 6. HEINEMANN, M., J. Clin. Znvest. 20, 467 (1941). 7. BORSOOK, H., DAVENPORT, H. W., JEFFREYS, C. E. P., AND WARNER, R. C., J. Biol. Chem. 117, 237 (1937). 8. SHERRY, S., AND RALLI, E. F., J. Clin. Znvest. 27, 217 (1948). 9. HEINEIVIANN, M., J. Clin. Invest. 20, 39 (1941). 10. SARGENT, F., J. Biol. Chem. 1’71, 471 (1947). 11. EIDEL’MANN, M. M., AND GORDON, F. Y., Biokhimiya 14, 58 (1949). 12. SAHA, K. C., MAJUMDAR, A. C., AND GUHA, B. C., Ann. Riocheni. Exptl. Med. 1, 135 (1941). 13. SAHA, K. C., MAJUMDAR, A. C., AND GWA, B. C., dnn. Biochem. Exptl. Med. (India) 1, 139 (1941).

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II

MATUSIS, I. I., Bull. Eksptl. Biol. Med. 20, 66 (1945) [C. A. 40, 7321 (1946)]. SARGENT, F., II, AND GOLDEN, R., J. Biol. Chem. 188, 773 (1951). PAL, J. C., AND GIJHA, B. C., J. 1mLm Chew Sot. 16, 481 (1939). SEN-GUPTA, P. N., AND GUHA, B. C., J. In&m Chew Sot. 16, 496 (1939). SEN-GUPTA, I’. N., ANI) GUHA, B. C., J. Indian Chm. Sot. 16. 549 (1939). GHOSH, B., AXU GUHA, B. C., J. Indian Chem. Sot. 16, 505 (1939). ROE, J. H., AND KUETHER: C. A.? J. Riol. Chern. 147, 399 (1943). BESSEY, 0. A., LOWRY, 0. II., AND BROW, 31. ,J., J. %oZ. Chew 168, 1!)7 (1947). 22. RIDER, P. R., An Introduction to Modem Statistical Methods. John Wiley h Sons, Inc., N. Y., 1939. 23. JOHNSON, R. E., EDWARDS, H. T., DILL, D. B., AND WILSON, J. W., J. Biol. Chem. 167, 461 (1945,.