Vitamin C economy in the human subject

Heeptial (-arer? NUMBER — BULLETIN of the OHNS HOPKINS HOSPITAL THE PUBLICATION OF THE MEDICAL SCHOOL AND HOSPITAL SUPPORTED BY THE DE LAMAR FUND OF THE JOHNS HOPKINS UNIVERSITY November, 1944 The Interrelationships of Glomerular Filtration Rate (Mannitol Clearance), Extracellular Fluid Volume, Surface Area of the Body, and Plasma Concentration of Mannitol. Evuior V. Newman, JAMES Borp.tey, III Anp JANE WINTERNITZ Atypical Pneumonia: A Diagnostic Problem in the Tropics. CapTaiIn Puiuip A. TUMULTY .... Vitamin C Economy in the Human Subject. Mice. Prisoan, Lr. (M.C.) USNR anp EvuGene L. Lozner, Lr. Compr. (M.C.) USNR.. A Note on the Minimum Requirements of Man for Vitamin C and Certain other Vitamins. Vicror A. Najjar, L. Emmett Ho rt, Jr., AnD Harriet M. Royston : The Effect of Intravenous Injection of Epinephrin and Angiotonin Before and After the Production of Neurogenic Hypertension. CAroLINE BepeLL THomas AnD Ross L. McLEAN ....... PUBLISHED MONTHLY THE JOHNS HOPKINS PRESS, Baltimore-18, Maryland Made in Untied States of America Coprricut, 1944, sy Tae Jonns Hopkins Press ogy UnAtypical an His- | (Virus) al Pneury Tract ysicians, College it Pub- . Pneuttacosis -rimary 7: 378, VITAMIN C ECONOMY IN THE HUMAN SUBJECT" MICHEL PIJOAN, Lr. (M.C.) USNR anp EUGENE L. LOZNER, Lr. Compr. (M.C.) USNR Submitted for publication October 20, 1944 The object of this study was to ascertain (1) the minimal daily requirement of ascorbic acid necessary to protect against scurvy, and (2) the period of protection afforded by saturation with the vitamin. There is considerable disagreement among the various authors in the field of nutrition as to the amount of vitamin C man requires to maintain health. A high intake of the vitamin is advised by those who use the saturation test based on urinary excretion as the index of ascorbic acid economy, while those who believe a lower intake to be adequate generally base their recommendation upon blood tissue values supported by clinical observations. The entire subject has been recently reviewed (1). It is the purpose of this inquiry to evaluate, as briefly as possible, these two views and to supply additional data which may help to clarify misunderstandings. To understand the problem of vitamin C economy, it is well to keep in mind two fundamental considerations, which are: (1) That the only known anatomic lesion of vitamin C deficiency is the scorbutic process (2, 3, 4). Therefore, ascorbic acid, whether synthetic or derived from food, serves but two known purposes, the prevention of scurvy and the treatment for scurvy. (It is possible that vitamin C is active in promoting the intermediary metabolism of aromatic amino acids in premature infants (5) and other animals (6), but unequivocal evidence for this function is lacking.) (2) That the evaluation of ascorbic acid deficiency in any given individual from an assay of his diet is untrustworthy. Not until a diet can be demonstrated to produce a steady linear decline in the vitamin C content of the white cell-platelet layer (or some index derived from 1 This article has been released for publication by the Division of Publications of the Bureau of Medicine and Surgery of the United States Navy. The opinions or assertions contained herein are the private ones of the writers and are not to be construed as official or reflecting views of the Navy Department or the Naval Service at large. 303 304 MICHEL PIJOAN AND EUGENE L. LOZNER whole blood), or to produce scurvy, can one call it deficient. The ability to maintain a fixed plasma level of vitamin C, no matter how small, indicates, for that individual a positive ascorbic acid balance. The problem, then, is to ascertain how much ascorbic acid is needed to prevent scurvy and how much is required for reserves on an average basis. The amounts required by normal individuals vary somewhat and such variations cannot be explained purely on the basis of surface area. The subject of collagen and reticulum defect in scurvy has been reviewed elsewhere (1, 7, 8). EXPERIMENTAL PROCEDURE A separate study, previously abstracted (1), was conducted on an individual who, following saturation, consumed no ascorbic acid until such time as his plasma vitamin value was zero and the white cellplatelet layer began to decrease. He then received small amounts of the vitamin from biological sources. At first the diet consisted of rice, certain cheeses, eggs, bran, and synthetic vitamins with the exception of vitamin C. The foods used in the experiment were assayed by the method of Bessey (9). After seventy days on the vitamin C free diet, the subject was given 5 mg. of the vitamin as present in orange juice. The white cell-platelet layer vitamin value continued to drop. At the end of fifty days, the vitamin intake was increased to 10 mg. daily and this was accompanied by a slight continued decrease in the blood tissue values. This continued for eighty days when the intake was increased to 18-25 mg. daily which resulted in an initial increase and a subsequent maintenance of the blood tissue levels which lasted from the seventh to the twenty-second month (fig. 1). At no time during the entire twenty-two months did the subject experience any objective or subjective clinical symptoms. During the twenty-first month an experimental incision, 4 cm. long and 1 cm. deep, was made in the left subscapular area. It was closed with five interrupted sutures. No infection occurred and the wound healed with good tensile strength in six days. The sutures were then removed and a biopsy performed (figs. 2and3). In contrast with other studies (13), there appeared to be sufficient collagen and reticulum formation and there was no evidence of weak areas in the tissue structure. Thus it would appear that a diet containing 25 mg. of vitamin C daily The how re. eded rage what rface been n an until cellts of rice, tion ' the liet, lice. the and ssue ased bsethe the e or ong »sed und hen ther forure. aily VITAMIN C ECONOMY IN HUMAN SUBJECT 305 (fig. 4) or possibly less, was sufficient to protect against scurvy and insure wound healing. Six subjects, volunteers, were placed under hospital conditions.’ Their tissues were first saturated by large intakes of ascorbic acid. They were then placed on a diet of eggs, crackers, well cooked meat (free of vitamin C), rice, macaroni, certain cheeses, debittered yeast -_ 3 Oo S ~ 4 _ at 41. 5 |° o < o € - a 283 & FE $85 BF 2 $23 | F ce 4°13 £€ EH e ; zB § E sé co 3 kt iihifealielierbiinatinalindicnlen ef | ee 1 fNone—p5mg 10mg +18 - 25 mg ‘ 3 MONTHS 123 45 6 7 6 9 1 tH 12 13 14 15 16 I7 16 19 20 2 22 Fic. 1. DETERMINATION OF APPROXIMATE MINIMAL VITAMIN C INTAKE REQUIRED ro MAINTAIN FrxED WBC-PLATELET Ascorbic AcID LEVELS Plasma and white cell-platelet vitamin C values in a 35 year old male whose dietary intake of the vitamin did not exceed 25 mg. daily for a period of twentytwo months. and an adequate intake of all vitamins (in preparations) with the exception of ascorbic acid. Tea, coffee, and sugar were allowed. A variety of dishes were prepared from the above by adding numerous seasoning adjuvants. Every five days vitamin C assays were made (10, 11) on blood tissues and plasma of subjects after a twelve hour fast. ? Carried out in part by the U. S. Indian Service Nutrition Laboratory, Univer sity of New Mexico, Albuquerque, New Mexico. 306 MICHEL PIJOAN AND EUGENE L. LOZNER ) Zz al Z. = = = — ~% a a < oan — ie a £ mG ~ = wf) = vn ¥ _ Md —_ 2 » 2 fz] ~ oS &@ SY ee ow ee -~—sS = @s Se 3 & Secs au Ze weas tan = aS ~~ = oS a , Hicu ) MAGNIFICATION OF HEA 2 Note abundant newly formed fibroblasts with extensiv MonrTus OF A Diet, THE VITAMIN C CONTENT OF WHICH D 3. (Ricnutr) SAME As FIGURE The section shows complete healing by firs Fic. . (Lerr) Biopsy SPECIMEN, Low PoWER 2 “IG. phe section shows Compiete heanne DY rst intention Fic. Note abundant newly formed fibroblasts with extensive collagen between them , HicH MAGNIFICATION 2 (RicHtT) SAME AS FIGURE > 3. VITAMIN C ECONOMY IN HUMAN SUBJECT 307 CASE HISTORIES (1) J. K. (aged 26, male) weighing 142 pounds was admitted to the hospital with the complaint of muscle pain in the region of the middle third of his SATURATION WITH ASCORBIC ACID; URINARY, SPILL OF EXCESS VITAMIN. ae ASCORBIC ACID RESERVES DEVELOP; SAT- | awe URATION MAY TAKE PLACE. VARIATIONS IN AL LOWANG MIN * |HII PLASMA LEVELS AND URINARY SPILL OF VITAMIN. INTAKE INTAKES MG. ASCORBIC ACID DAILY YANO SCURVY DEVELOPS ON MINIMUM PROTECTIVE INTAKE. PLASMA LEVELS OF VITAMIN DROP OR BEY), COME ZERO; FIXED WHITE-CELL-PLATELET CONTENT # OF VITAMIN. NO URINARY SPILL OF VITAMIN. GRADUAL FALL OF WHITE-CELL-PLATELET VITAMIN VALUE. EVENTUAL SCURVY. Fic. 4. Vrramin C Economy in NorMAL SUBJECTS Chart showing schematic representation of various vitamin C intakes and laboratory findings. Since no striking laboratory or pathological changes occur between the so-called needless excess and the protective minimum, no absolute figure can be given as a recommended allowance. Any intake on a daily basis within this range appears to be reasonable. right femur. His antecedent history revealed a compound comminuted fracture of the femur which occurred two years before his present admission. The subject was of a child-like disposition who behaved well but had a great fear of a recurrence of the fracture. He was admitted for physical therapy, reassurance and observation. His past history was unrelated to the present injury 308 MICHEL PIJOAN AND EUGENE L. LOZNER and was essentially non-contributory. The dietary background was fairly good except for an occasional bout of acute alcoholism. The physical examination revealed a well developed male in no apparent physical distress, and revealed no abnormalities whatsoever. The roentgenogram showed excellent union of the fractured shafts of the femur. Objective physical and laboratory measurements were: pulse rate 82, blood pressure 118/84 mm. Hg, hemoglobin 14.4 gm., erythrocytes 4,000,000/cu. mm., white blood cells 6,200/cu.mm. Ascorbic acid study on admission: plasma 0.3 mg./100 ml., white blood cell-platelet value 28 mg./100 gm. A diagnosis of anxiety neurosis was made. He was given 300 mg. of ascorbic acid orally daily for five days at the end of which time more than 75 per cent of his intake was excreted in the urine over a period of twenty-four hours. It was then considered that his tissues were saturated with vitamin C and he was placed on the ascorbic acid-free diet. (2) M. W. (aged 42, male) weighing 190 pounds was admitted to the hospital with treated trachoma but with persistent blindness due to a well cicatrized macroscopic pannus. His past history is non-contributory except for an appendectomy performed eight years previous to admission. Since he was to be kept in the hospital for opthalmologic observations and since he didn’t particularly want to leave he willingly volunteered for these studies. The physical examination was essentially non-contributory other than the presence of an old trachomatous opthalmia with a heavy macroscopic pannus. Objective physical and laboratory measurements were: pulse rate 68, blood pressure 108/88 mm. Hg, hemoglobin 15.2 gm., erythrocytes 4,510,000/cu. mm., white cells 7,800/cu.mm. Ascorbic acid: plasma 0.6 mg./100 ml., white blood cell-platelet value 29 mg./100 gm. This subject then received 300 mg. of ascorbic acid daily, on the sixth day he eliminated through the twenty-four hour urine 84 per cent of his ascorbic acid intake. It was considered that his tissues were saturated with ascorbic acid and he was placed on the vitamin C free diet. (3) T. L. (aged 28, male) weighing 205 pounds was admitted to the hospital with a painful Dupuytrens contracture of the palmar fascia of the right hand. This was the result of local injury and infection seven years prior to the present admission. He was admitted for physical therapy and possible surgical intervention. The past history was non-contributory. The physical examination was not remarkable except for the finding of the contracture and the presence of external hemorrhoids. Objective physical and laboratory measurements were: pulse rate 80, blood pressure 120/80 mm. Hg, hemoglobin 15.4 gm., erythrocytes 4,630,000/ cu. mm., white blood cells 7,220/cu.mm. Ascorbic acid: plasma 0.52 mg./100 ml., white blood cell-platelet value 30 mg./100 gm. He was placed on 400 mg. of accorbic acid orally in divided doses daily for five days at the end of which time 90 per cent of the acid ingested was excreted during twenty-four hours in the urine. He was then placed on the vitamin C free diet. (4) C. L. (aged 64, male) weighing 210 pounds was admitted to the hospital because of bronchiectasis and ureteral stricture. No reliable history could be obtained that would indicate the possibility of other systemic disease. The subject - good on reed no f the ments ythrody on 0 gm. c acid ntake 1 conmn the spital 1acro- ‘tomy > hosleave ssenalmia usurelasma - then h the dered ‘amin spital This dmisition. ot reernal : rate ,000/ ) ml., of acne 90 irine. pital e obVITAMIN C ECONOMY IN HUMAN SUBJECT 309 was rather anxious to remain in the hospital and enthusiastically volunteered for the observations. He had apparently had symptoms of bronchiectasis for fifteen years; the ureteral stricture had been causing inconvenience for two years. The patient complained occasionally of early morning headache. The subject was a well developed male whose physical findings were dependent on the two entities mentioned along with hypertension (arteriosclerotic-cardiovascular syndrome). Objective physical and laboratory findings were: Emphysematous chest with numerous coarse rhonchi at both bases and a cough productive of thick semipurulent sputum, the heart sounds were forceful in character and regular, retina showed considerable A-V nicking but no hemorrhages; pulse rate 74, blood pressure 164/92 mm. Hg hemoglobin 12.8 gm., erythrocytes 3,810,000/cu. mm., white blood cells 9,400/cu. mm., urine had a persistent trace of albumin with many pus cells but no casts, plasma ascorbic acid was 0.24 mg./100 ml. plasma, and the white blood cell-platelet content was 25 mg./100 gm. He was given 400 mg. of ascorbic acid daily in divided doses and on the fourth day excreted 80 per cent of his vitamin intake. He was then placed on the vitamin C free diet. (5) A. M. (aged 26, female) weighing 138 pounds was receiving antiluetic treatment for primary syphilis. She was anxious to remain in the hospital since her domestic life was far from satisfactory and willingly volunteered for the observations. Her present illness dated to six months before admission and was discovered discovered through serological and precipitation-flocculation tests. The past history was irrelevant to the present study, except for one small vulvar condyloma there were no remarkable findings on physical examination. Objective physical and laboratory findings were: pulse rate 80, blood pressure 106/72 mm. Hg, hemoglobin 14.6 gm., erythrocytes 4,450,000/cu. mm., white blood cells 5,800/cu. mm., ascorbic acid in plasma 0.22 mg./100 gm. She was given 400 mg. of ascorbic acid daily and on the fifth day excreted 87 per cent of the ingested vitamin. She was then placed on the vitamin C free regime. The subject continued on her diet for one hundred and forty-nine days without any stigmata of scurvy and desired to leave at that time. She was then re-saturated with vitamin C and discharged from the hospital. (6) C. A. (aged 26, female) weighing 118 pounds was admitted to the hospital for study and physical therapy for post traumatic peripheral pain due to malunion of the lower third of the tibia, and myositis. This illness was dependent on an accident received one year prior to her admission, the fracture having been treated by inexperienced friends. The patient expressed a willingness to remain in the hospital; she was an intelligent person who fully understood the nature of the study and volunteered as a subject. Her past history was non-contributory. The objective physical and laboratory findings were: blood pressure 112/72 mm. Hg, hemoglobin 14.4 gm., erythrocytes 4,420,000/cu. mm., white blood cells 6,200/cu. mm., plasma ascorbic acid 0.57 mg./100 ml., white blood cell-platelet value 24 mg./100 gm. The subject received 350 mg. ascorbic acid daily, in divided doses, for five days, at which time she excreted 77 per cent of the vitamin in the urine. She was then placed on the vitamin C free regime. 310 MICHEL PIJOAN AND EUGENE L. LOZNER Petechial and perifollicular hemorrhages occurred in all subjects. except No. 5, at the end of about 5 months and all were given large doses of vitamin C anda highly nutritious diet. Recovery occurred in seven to ten days. RESULTS The results obtained in the study of the six subjects on a vitamin C free diet following saturation showed there were no clinical findings, either subjective or objective, until two weeks previous to the physical signs of scurvy. The findings noted during this period were easy fatigue, lassitude, and irritability. The degree of these symptoms varied with the individual. Shortly thereafter, scurvy set in and the above mentioned symptoms became more severe. The first physical findings noted were petechial and perifollicular hemorrhages; in one case there were tender swollen gums that bled easily. It was felt that these results demonstrated that a period of from five to six months was required to deplete the individual following saturation; the first four months can be considered a period of protection (table 1). It can be deduced that if saturation should take place every four months it would offer protection against scurvy. This would require approximately 2 gm. of the vitamin in divided doses during the period of saturation or represent approximately 8,000 mg. per annum, the equivalent of about 22 mg. per day, a figure very near to that found to be the minimum protective daily dose. Obviously these comparisons cannot be precise but they are suggestive and close enough to warrant such consideration. Our findings further indicate the value of the blood tissue content to be a reliable index of the ascorbic acid status (12, 13). The fact that gingivitis failed to make its appearance except in one case would militate against the view that gingivitis is an early symptom of scurvy. DISCUSSION The problem of gum lesions and scurvy is one that has received considerable attention and deserves comment. Recently in a separate study (1) the subject has been reviewed. The added information gained indicates the need for further study but it is highly questionable as to whether the gingival lesion is a scorbutic one on the basis of saturation tests and plasma levels (14). According to Hess (2) absolute TABLE 1 Certain Findings Related to Scurvy and Gingivitis in 6 Adult Subjects on a Scorbutic Regi | A. M., FEMALE—26 | C. A., FEMALE—26 Cc. L., MALE—64 T. L., MALE—28 uw. W.. MALE—42 | +s watrte~—74