J. Biol. Chem.-1958-Hellman-923-30.pdf

8/14/2019 J. Biol. Chem.-1958-Hellman-923-30.pdf

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METABOLISM OF L-ASCORBIC ACID-l-Cl4 IN MANBY LEON HELLMAN AND J. J. BURNS

(From the Division of Biophy sics, Sloan-Kettering Institute for Cancer Research,Memorial Center, Research Service, Third (New York University) MedicalDivision, Goldwater Memorial Hospital, New York, New York, and the

Laboratory of Chemical Pharmacology, National Heart Institute,National Institutes of Health, Bethesda, Maryland)Received for publication, September 19, 1957)

n-Ascorbic acid labeled with Cl4 has been utilized in studying the me-tabolism of this vitamin in rats (1, 2) and guinea pigs (3, 4). In bothspecies here is considerable conversion of carboxyl-labeled n-ascorbic acidto CO2 and significant urinary excretion of labeled oxalate. The impor-tant nutritional role of n-ascorbic acid made it desirable to study themetabolism of the labeled vitamin in man with special attention to its rateof disappearance from the body, relative importance of its various routes ofexcretion, and nature of its metabolic products. The results obtained inthis investigation, when compared to those reported in similar studies inguinea pigs, indicate sharp differences in n-ascorbic acid metabolism inthe two species.l

EXPERIMENTALn-Ascorbic acid-1-C4, having a specific activity of 1.0 PC. per mg., wassynthesized by a previously published procedure (6).The labeled n-ascorbic acid was administered intravenously to threepatients in the James Ewing Hospital, Memorial Center. In Table I ispresented information on the physical status of the patients,2 the dose oflabeled compound, and the amounts of L-ascorbic acid in their plasma andurine as determined by the method of Roe and Kuether (7).At various intervals after injection of the labeled n-ascorbic acid, samplesof respiratory COZ were collected by having the subjects breathe for 1minute into a solution of COz-free 0.5 N NaOH (8). These samples werekept protected from atmospheric COZ at all times. For isolation of CY402,excess Bach was added after each sample was warmed to about 70.The BaCOt formed was centrifuged and then washed with water andmethanol. A slurry of BaC03 in methanol was applied to a weighed1 A preliminary publication of this work has been presented 5).2 The patients were free from acute symptoms of their disease and were normalin respect to liver and kidney function. Throughout the course of the experimentsthey received the usual hospital diet.

923


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924 ME TAB OLIS M OF L-ASCORBIC ACID-l-Cl4planchet which was reweighed after evaporation of the methanol and storedin a desiccator until counted.Urine samples were refrigerated, the volume was recorded, and 0.2 ml.taken for counting. To obtain a suitable thickness of material for counting,0.2 ml. of a 5 per cent dextrose solution was added to the planchets con-taining the urine samples. Urine samples were collected in bottles con-taining 25 ml. of 5 N HCl or 300 ml. of 10 per cent oxalic acid. Stoolsamples were homogenized and an aliquot was placed on a weighed planchet.All samples were applied to the planchet together with lens paper to aidin achieving an equal distribution of radioactive material; they were driedand stored in a desiccator. Radioactivity was measured in a windowless

TABLE IClin ical Data and L-Ascorbic Acid Levels

Subject

AA-l. .....AA-2. .....AA-3 ......

Age Sex Weight Diagnosis

3s.433670

I kg. IM. 65.0 Multiple lipomatosis 59.1 sclerosisF. 75.4 Recurrent breast can-

cer with metastase s

L-Ascorbic Level oLc;iscorbicacid-104administered* Urine Plasma

23.9 14.0 0.3533.8 11.0 0.5437.2 20.0 1.0

* The labeled compound was dissolved in 50 ml. of saline and the resulting solu-tion was administered intravenously over a period of 5 minutes.

t Value s determined on the day before administration of labeled compound.

gas flow counter. The methods used to determine the amount of Cl4in urine present as L-ascorbic acid, dehydro-L-ascorbic acid, 2, S-diketo-n-gulonic acid, and oxalic acid were identical to those described previously3,4).

ResultsThe cumulative excretion of Cl4 in urine and feces of the three subjects

is presented in Table II. During a 10 day period an average of 42 per centof the administered dose was found in urine and about 1.0 per cent in thefeces. No Cl4 was detected, however, in the respiratory COZ; i.e., less than5.0 per cent of dose during a 10 day period.

The half life of L-ascorbic acid was estimated by plotting the per centof the administered dose which remained in the body each day as a logarith-mic function against time Fig. 1). Since only a small fraction of theinjected Cl4 was found in COz and feces, the amount remaining in the body


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L. HELLMAN AND J . J. BURNS 925was calculated by subtracting the activity excreted in urine from theadministered dose. The value for the half life ranged from 13 to 20 days,with an average of 16 days.

TABLE IICumulative Excretion of Radioactivity in Urine and Feces by Sub jects Receiving

L-Ascorbic Acid-l-P*

Time

days0.250.501.02.03.04.05.06.07.08.09.0

10.011.012.013.014.015.016.017.018.019.020.021.022.023.024.025.026.027.0

Subject AA-l

er cent10.412.314.618.120.423.726.330.334.638.342.045.948.052.054.957.159.7

FKWfier cent

0.0830.1070.1510.2170.2820.3570.4170.4890.570

0.6510.704

Subject AA-2-I

UrineCT cent10.211.614.018.321.125.429.532.135.938.141.444.7

FW3$53 cent

0.1020.368

0.6320.8001.07

* Value s for amounts of Cl4 expressed as per cent of dose.

Subject AA-3

Urineper ce+d

4.786.468.97

12.315.418.220.525.127.930.432.636.338.542.043.545.546.947.749.150.451.452.653.854.857.758.859.460.160.7

The fraction of total Cl4 excreted in urine as n-ascorbic acid, diketo-n-gulonic acid, and oxalic acid is given in Table III. About 20 per cent oftotal urinary Cl4 was present as n-ascorbic acid, about the same amountas diketo-n-gulonic acid and less than 2 per cent as dehydro-n-ascorbic


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926 METABOLISM OF L-ASCORBIC ACID-l-Cl4acid. An average of 44 per cent of the total radiocarbon excreted in urinewas recovered as oxalate.The body pool of L-ascorbic acid was estimated from the excretion of Cl4during the 3rd day after the administration of labeled n-ascorbic acid

1 ii h50 -z D

L+ 40-0 af .--* AA-2u w AA-3

8 30 0 5 IO 15DAYSFIG. 1. Per cent of dose remaining in body plotted against time (days). Per cent

of dose remaining in body ca lculated by subtracting amount of Cl4 excreted in urineby each subje ct during various time intervals from dose of Cl4 administered.

TABLE IIIExcretion of L-Ascorbic Acid and Its Metabolites in Urine after Administration

of L-Ascorbic Acid-i-Cl4 to Human Sub jects

Subject Day after dose

AA -l.. . . . . . . . . .AA-2. . . .I . . . . .AA-Z..6

I

...............................................................................

I- Per cent total urinary 04 found as-_- t-Ascorbic acid

Diketo-z-gulonicacid

12 1216 1612 1815 1623 1623 1524 12

Oxalic acid

35585563472429

(Table IV). The specific activity of urinary n-ascorbic acid was obtainedby dividing the amount of Cl4 present as L-ascorbic acid in urine by theamount of L-ascorbic acid as determined chemically (7). This value forthe specific activity expressed as per cent of dose per mg. when dividedinto per cent of dose remaining in the body at the end of the 3rd daygave an average body pool of L-ascorbic acid of 22 mg. per kilo.Since no comparable data were available on the body pool of L-ascorbicacid in guinea pigs, it was estimated as follows: Four guinea pigs each


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L. HELLMAN AND J . J. BURNS 927received 1.0 mg. of n-ascorbic acid-l-Cl4 by intraperitoneal injection andthe Cl4 in respiratory COz and urine was determined over a 24 hour periodTable V) .a The animals were then killed and the specific activity of

TABLE IVBody Pool of L-Ascorbic Acid in Human Subjects*

Subject

m g . per 24 hrs. per cent Of doseper mg. nzg. per kg.AA-I. 12 0.56 0.047 80 26AA-2. 13 0.83 0.064 79 21AA-3 ..... 20 1.2 0.060 85 19

* Value for body pool calcula ted from excretion data obtained on 3rd day afteradministration of labeled n-ascorbic acid. The weights of patients are given inTable I.

t The se values includ e total for n-ascorbic acid, dehydro-n-ascorbic acid, anddiketo-n-gu lonic ac id as determined by Roe and Kuether (7).

$ Th is value includes Cl4 present in n-ascorbic acid and diketo-n-gulonic acid.TABLE V

Body Pool of L-Ascorbic Acid in Guinea Pigs *

Weigh t of guineaPigPer cent dose at end of 24 hrs. in Sp ecific activity of BodyI-ascorbic acid poolCO8 Urine Animalt

@.308278300290

15.622.415.632.2

7.97.28.75.0

76.570.475.762.8

per ceGLpw4.226.623.654.57

m g. per kg.59396847

* Guinea pigs used in this experiment were maintained on a vitamin C-free dietsupplemented daily with 6 mg. oral doses of L-ascorbic acid for at least 1 week be-fore the experiment.

t Per cent of dose remaining in anima l calcula ted by subtracting fraction of dosein respiratory CO2 and urine from total ad ministe red.n-ascorbic acid was determined on a pooled sample of liver, kidneys, testes,adrenals, and spleen from each animal.3 The body pool was estimated bydividing the specific activity, expressed as per cent of dose per mg. of

8 The methods used in these experiments for collec tion of urine and respiratoryCot and determination of spe cific activity of tissue of n-ascorbic acid were the sameas those used previously (9).


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928 MET ABOLISM OF L-ASCORBIC ACID-1-C4L-ascorbic acid, into the per cent of dose remaining in the animal. Thiscalculation is based on the previous finding that essentially al l the Cl4 inthe guinea pig 24 hours after administration of n-ascorbic acid-l-04 ispresent as n-ascorbic acid 10). An average body pool of n-ascorbic acidin the guinea pig of 54 mg. per kilo was obtained.

DISCUSSIONThe results of this study show that the half life of n-ascorbic acid averages

16 days in the three human subjects studied. In contrast, the half lifeof n-ascorbic acid in guinea pigs averages only 4 days 4). This differenceis presumably the reason why man requires a much longer period of timeon a vitamin C-free diet to develop scurvy than the guinea pig. For in-

TABLE VIBody Pool and Turnover Rates of L-Ascorbic Acid in Man, Guinea Pig, and Rat

Species

Man, subject AA-l. AA-2. AA-3.Guinea pig.Rat?.

Half life Body poo l Turnover time Turnover ratedays mg . @r k g . &YS m g. per kg. per day13 26 19 1.415 21 22 1.020 19 29 0.664.0* 54 5.8 9.32.9 107 4.1 26

* Previously published data for half life in guinea pigs 4).t Previously published values for rats 1).

stance, guinea pigs on such a diet develop scurvy within about 21 daysalthough man requires 3 to 4 months to show symptoms of the disease.The values obtained in this study for the half life and body pool ofn-ascorbic acid make it possible to estimate the turnover rate of n-ascorbicacid in man (Table VI). For comparison, average data are also given forthe body pool and turnover rate of n-ascorbic acid in the guinea pig andrat. In man, about 1 O mg. per kilo per day is required to maintain a bodypool of 20 mg. per kilo, in the guinea pig an average of 9.3 mg. per kiloper day are required to maintain a body pool of 54 mg. per kilo, and in therat, a species which synthesizes n-ascorbic acid, 26 mg. per kilo per dayare needed to maintain a body pool of 107 mg. per kilo. The results of thisstudy indicate that a smaller dose of n-ascorbic acid, when compared onan equivalent weight basis, is required to maintain the body pool of thevitamin in man than in the guinea pig. This probably explains why thedaily requirements of the vitamin are less for man than for the guinea pig.For instance, about 10 mg. per kilo of n-ascorbic acid are needed daily in


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L. HELLMAN AND J . J. BURNS 929the diet of guinea pigs to prevent signs of vitamin C deficiency comparedto only about 1 mg. per kilo or less for man 11).

The major pathway of metabolism of n-ascorbic acid in guinea pigsinvolves oxidation of its carboxyl carbon to COz. For instance, during a10 day period after administration of L-ascorbic acid-l-C4 to guinea pigs,about 70 per cent of the dose is found in COz and about 20 per cent inurine 3). Additional studies in which uniformly labeled L-ascorbic acidwas employed show that decarboxylation is presumably the first step inthe complete oxidation of carbon chain to CO2 4). In man, on the otherhand, there is practically no oxidation of the carboxyl carbon of L-ascorbicacid to CO2


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930 METABOLISM OF L-ASCORBIC ACID-l-Cl4BIBLIOGRAPHY

1. Burns, J. J., Mosbach, E. H., and Schulenberg, S., J. Biol. Chem., 207,679 1954).2. Curtin, C. OH., and King, C. G., J. Biol. Chem., 216, 539 1955).3. Burns, J. J., Burch, H. B., and King, C. G., J. Biol. Chem., 191,501 (1951).4. Burns, J. J., Dayton, P. G., and Schulenberg, S., J. Biol. Chem., 218, 15 (1956).5. Hellman, L., and Burns, J. J., Federation Proc., 14, 225(1955).6. Salomon, L. L., Burns, J. J., and King, C. G., J. Am. Chem. Sot., 74,516l 1952).7. Roe, J. H., and Kuether, C. A., J. Biol. Chem., 147,399(1943).8. Hellman, L., Bradlow, H. L., Adesman, J., Fukushima, D. K., Kulp, J. L., andGallagher, T. F., J. Clin. Invest., 33, 1106 1954).9. Burns, J. J., and Evans, C., J. Biol. Chem., 223, 897 1956).10. Salomon, L. L., Dissertation, Columbia University 1952).

11. Sebrell, W. H., Jr., and Harris, R. S., The vitamins, New York, 1, 389(1954).12. Cox, E. G., Hirst, E. L., and Reynolds, R. J. W., Nature, 130,888 1932).13. Herbert, R. W., Hirst, E. L., Percival, E. G. V., Reynolds, R. J. W., and Smith,F., J. Chem. SOL, 1270 1933).14. Jeghers, H., and Murphy, R., New England J. Med., 233, 208 1945).15. Lamden, M. P., and Chrystowski, G. A., Proc. Sot. Exp. BioZ . and Med., 86, 1901954).

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J. Biol. Chem.-1958-Hellman-923-30.pdf