July 1, 1939
"In a study from the 1930s-early 1940s, they experimented with doses of vitamin C to determine minimum requirement to avoid scurvy. The researchers supplemented 10mg/day. In their trials, they found that that the 10mg amount was sufficient not only to prevent scurvy but also to reverse scurvy. A few of the participants were given reduced doses, after 160 days with only 10 mg a day, three volunteers were left on less, which averaged 3.2, 3.2, and 4.5 mg vitamin C daily, and even that was enough to prevent scurvy."
Medical experiments carried out in Sheffield on conscientious objectors to military service during the 1939-45 war
The Sheffield Experiment on the Vitamin C Requirement of Human Adults*
BY H. A. KREBS, Medical Research Council Unit for Research in Cell Metabolism, Department of Biochemistry, University of Shefield
In 1938 the League of Nations Technical Commission on Nutrition estimated the daily vitamin C requirement of human adults at 30 mg (League of Nations Health Organization: Technical Commission on Nutrition, 1938). In 1943 the (U.S.A.) National Research Council Committee on Food and Nutrition recommended an allowance of 75 mg (National Research Council: Food and Nutrition Board, 1943). Some authorities, e.g. Zilva (1941, 1944) put the daily requirements much below 30 mg. These were the main data on which dietary planning had to be based during the war years. The divergences in the estimates arose from the different standards used in assessing the requirement. The high values were based on studies of the saturation of the body with vitamin C, whereas the lower values were inferred from observations on the prevention and cure of the clinical manifestations of scurvy. The differences were also an indication of the lack of accurate information, and in 1944 Professor R. A. Peters suggested to the Medical Research Council that it would be worth while obtaining additional information on the vitamin C requirements of human adults by a trial on human volunteers who had offered themselves for this kind of investigation at the Sorby Research Institute at Sheffield. Professor Peters’s suggestion was accepted and the Vitamin C Subcommittee of the Medical Research Council under the chairmanship of Professor Peters was entrusted with the task of designing and conducting the trial. This was carried out at Sheffield from October 1944 to February 1946. The main plan was to induce scurvy by a vitamin C-deficient diet and to establish the minimum dose of the vitamin that cures scurvy. A subsidiary aim was to study, the clinical signs and symptoms of scurvy and to correlate them with laboratory findings. Owing to the limited number of volunteers, the scope and design of the trial were bound to be restricted. Nineteen men and one woman, aged 21-34, volunteered for the experiment. They lived a normal life without strenuous physical work. Their basal diet was designed to be as low as possible in vitamin C but complete in every other respect. It was sufficiently varied to be acceptable. From chemical analyses it was calculated that on the average a volunteer obtained not more than I mg vitamin C daily from the diet. To obtain base-line data the trial began with a preliminary period, in most cases of 6 weeks, on a complete diet including about 70 mg vitamin C daily. At the end of the period all the volunteers were given the basal deficient diet and divided into three groups, ten having no supplements, seven 10 mg of vitamin C daily, and three 70 mg vitamin C daily. The group receiving 70 mg was intended to serve as a positive control and the group receiving 10 mg as a prophylactic test. The volunteers did not know to which group they belonged, nor did the physicians responsible for the clinical investigation. All the volunteers were daily given seven supplementary tablets of identical taste and appearance, some containing vitamin C, the others being dummies. Investigations, made on the volunteers at regular intervals, included general clinical examinations, chemical analyses of blood and urine, haematological examinations, capillary-fragility tests, capillaroscopy, radiography, electrocardiography, studies of fatigue, and studies of experimental wounds.
Development of signs of deficiency The clinical examinations, by inspection and physical methods, revealed no definite changes during the first 17 weeks of deprivation. The first changes which retrospectively were recognized as significant were enlargement and keratosis of the hair follicles in one volunteer, particularly on the outer aspect of the upper arm. After 21 weeks six of the ten deprived volunteers had developed follicular changes, and after 26 weeks all had done so. The main areas affected were the upper arms, backs, buttocks, back of thighs, calves and shins. In all of them except one the enlarged hair follicles eventually became haemorrhagic. Examination with the skin microscope showed that the initial change was the plugging of follicles by horny material in which the hair was coiled or looped. A few weeks later the enlarged follicles turned red. Under the microscope this redness presented itself as congestion and proliferation of the blood-vessels round the hair follicles. The colour gradually deepened and within another week or two the enlarged hair follicles became haemorrhagic, the red colour turning dark purple and no longer disappearing on compression; at this stage many red cells could be seen outside the vessels. After 26 weeks of deprivation, six of the ten volunteers, and 9 weeks later nine of the ten, had numerous haemorrhagic follicles. In general it was on the legs that the follicles showed the greatest tendency to become haemorrhagic. No subjective sensations accompanied the appearance of the haemorrhages. As the development of enlarged and haemorrhagic hair follicles progressed, five of the ten deprived volunteers showed a very pronounced exacerbation of the acne present in a mild form at the start of the experiment. The papules became more numerous after about 22 weeks; they increased in size and later became bright red and eventually haemorrhagic. The other five deprived volunteers who had no acne at the start remained free throughout the experiment. Other changes generally noted during the period of deprivation were in the gums. The earliest signs were tiny haemorrhages in the tips of the interdental papillae, and swelling, seen first after 26 weeks of deprivation. About 10 weeks later, nine of the ten deprived volunteers had developed abnormalities of the gums, gross in two cases, less advanced but definite in five other cases, and slight in the remaining two cases. Another striking observation, in agreement with the old accounts of scurvy, was made after 30 weeks in six of the ten deprived volunteers. It concerned the behaviour of the scars of experimental wounds that had been made to study wound healing. Scars of wounds made earlier during the trial, whose healing had proceeded normally, became red and livid. New wounds made at the stage of pronounced haemorrhagic scurvy showed a reduced tendency to heal. Some important abnormalities were observed in single cases. One man developed effusions into both knee joints and ecchymoses of the leg during the 30th week of deprivation after a long walk. Another was taken ill 4 weeks later, 19 h after heavy physical exercise. He had severe pain in the lower sternal region, and became dyspnoeic and cyanosed. The pulse was rapid and the blood pressure low. The clinical picture was that of an acute cardiac emergency. He was immediately admitted to hospital and dosed with vitamin C. The lower sternal pain, which at first had increased in intensity, passed off after 9 h. The electrocardiogram showed high ST levels in leads I and 11. A radiogram of the chest showed no abnormality. Eighteen days later another deprived volunteer complained of a sudden constrictive pain in the chest. Physical examination revealed a systolic murmur which had not been heard before, and the electrocardiogram showed a partial heart-block, the P-R interval being 0.32 sec. Before the experiment the electrocardiogram had been normal with a P-R interval of 0.20 sec. It was thought necessary to treat this volunteer immediately with large doses of vitamin C. The chest pain and the systolic murmur disappeared within 24 h, but during the following months the P-R interval showed variable periods between 0.13 and 0.32 sec. depending on posture, breathing, administration of drugs, and other factors. The pathological process underlying this cardiac emergency is bound to remain uncertain. The older records of scurvy contain many references to sudden death. Lind (1757, p. 137) writes: ‘Persons that appear to be but slightly scorbutic, are apt to be suddenly and unexpectedly seized with some of its worse symptoms. Their dropping down dead upon an exertion of their strength, or change of air, is not easily foretold’. As these incidents occur at a stage when multiple skin petechiae are the main clinical manifestation of scurvy, and when general fitness still appears to be fairly good, it is not improbable that a haemorrhage at a critical point of the conducting system of the heart forms the basis of the cardiac syndrome. Negative findings during the period of deprivation included no significant change in body-weight, no increased incidence of infection, and no change in the appearance of the capillaries of the nail bed and of the conjunctivae. Dark adaptation, haemoglobin concentration, red-cell count, total and differential leucocyte counts, platelet count, and bleeding time remained normal. There were no complaints of general pains or weakness. The capillary-resistance tests of Hess (1920, p. 212) and of Gothlin (1931) showed no consistent trends throughout the period of deprivation. To sum up, the clinical course of the development of scurvy was fairly uniform in the ten volunteers: no clinical signs for about 17 weeks; the first sign, after 17-21 weeks, was hyperkeratosis of the hair follicles; after 26-34 weeks perifollicular haemorrhages occurred and after 30-38 weeks swelling and haemorrhages of the gums. Exacerbation of acne, not apparently hitherto recognized as a sign of scurvy, began after 22 weeks. Like all the other single clinical signs of scurvy, neither hyperkeratosis nor congestion of the hair follicles is a specific sign, and the occurrence of gradual development of either of them in a person does not necessarily indicate lack of vitamin C. They occur in many people ‘saturated’ with vitamin C. Deficiency in this vitamin is only one of a variety of causes that can evoke them. In the Sheffield experiment the appearance and disappearance of the skin changes reflected the intake of vitamin C, and this proved beyond doubt that they were the early stages of the typical haemorrhagic spots of scurvy. It is noteworthy that vitamin A deficiency, in a similar trial, did not lead to hyperkeratosis (Hume & Krebs, 1949). The gum lesions appeared always after the skin lesions. Though this may not always be true of scurvy, it might nevertheless be a useful diagnostic pointer in deciding on the cause of gum lesions of doubtful origin. Many signs listed as scorbutic in the classical description of the disease, e.g. pallor, dryness of the skin, anaemia, and night-blindness, were not observed. It is probable that classical scurvy was often a multiple deficiency.
Effects of vitamin C supplements
In the seven volunteers receiving a supplement of 10 mg vitamin C daily no abnormalities were noted during the first 160 days of the experimental period. It was then decided that four of the volunteers should continue with the 10 mg supplement and three of them be deprived of it, the object being to ascertain whether signs of deficiency would develop quickly on withdrawal of the supplement. Three of the four volunteers who received 10 mg continued for another 264 days, and one abandoned the experiment after another 92 days. No abnormalities were recorded. The second group of three volunteers had no supplement for 71 days, broken in one case by a 26-day period on a supplement of 10 mg. Again no definite signs of deficiency developed. To sum up, a prophylactic dose of 10 mg vitamin C daily gave complete protection from clinical scurvy for periods up to 424 days. The three volunteers who served as positive controls for 300, 326 and 331 days and received 70 mg vitamin C daily also showed no abnormalities. Curative efJects of various doses of vitamin C Seven deprived volunteers, all showing unequivocal signs of scurvy-multiple skin haemorrhages and gum lesions-were available for dosing tests. In choosing the dose the intention was to give the smallest dose likely to produce a cure within a reasonable time but to aim too low rather than too high, since the dose could be increased later if necessary. A daily dose of 10 mg was chosen and given to six of the seven volunteers. The seventh received 20 mg because this volunteer was not available for long. The response to the dose of 10 mg followed the same pattern in all six cases. Within a week haemorrhages into the perifollicular region ceased, and within I or 2 weeks the older haemorrhages began to lose their dark purple colour and gradually faded. Within a month the hair in most of the follicles uncoiled, lifting out the plug. The dilation and congestion of the capillaries round the hair follicles disappeared, and within 7-9 weeks the skin appeared normal except for a slight brown pigmentation at the site of the former haemorrhages. The liability to haemorrhage in the wound tissue and the failure of the wounds to heal disappeared as the follicular eruptions regressed. The wound haemorrhages disappeared within 2 months, the original blue and purple colour gradually giving way to a pure red, pink, and finally pale brown, and changes in the appearance of the wounds indicated improved healing. The acneiform papules likewise regressed to the pre-experimental state, though in most cases somewhat more slowly than the other skin signs. The initial state was regained within 10-18 weeks. The gum lesions did not respond as promptly to dosing as the follicular skin lesions. When improvement began, the first sign was a change from livid blue to bright red, followed by the normal pink. Slowly the swelling decreased and the consistency of the gums improved, restoration being complete within 10-14 weeks. In brief, a dose of 10 mg daily was followed in all cases by a gradual disappearance of the clinical manifestations of scurvy.
Vitamin C requirement The main facts relevant to the assessment of the requirement are as follows : (I) A supplement of 10 mg cured clinical scurvy in all six cases examined. (2) A supplement of 10 mg protected seven volunteers throughout the period of observation, which, €or three of them, extended to 424 days. (3) When a 10 mg supplement was withdrawn from three volunteers after 160 days and was followed by a period of 195 days during which the intake varied slightly, with an average for the three of 3.2, 3.2, and 4.5 mg vitamin C daily, no definite clinical signs of scurvy appeared. These facts suggest that in the group under test the ‘minimum protective dose’ of vitamin C, as measured by the criteria of the presence of scurvy, was in the region of, perhaps somewhat below, 10 mg daily.