Vitamin C requirement of human adults: Diet

a  .' _J EXPERIMENTAL DETAILS  57 III. EXPERIMENTAL DETAILS A. Particulars and Management of Volunteers Particulars The details of the ages, weights, heights, occupations and period of participation in the trial of the twenty volunteers are given in Table 28. They all lived in the Sorby Research Institute. Ten of them had participated in the vitamin A trial which preceded the present experiment. Management The general management of the volunteers was along the lines described in the Report of the vitamin A trial (Hume and Krebs, 1949). The volunteers could be relied upon to co-operate in every respect. They assisted in the laboratory work, keeping of records, and the general running of the Institute. They were fully aware of the nature of the experiment and were kept informed of the decisions of the Committee. However, for the first 6 months they did not know to which group they belonged, whether totally deprived of vitamin C, or supplemented with it, with the exception of two, Bartley and Garling, who for reasons arising from the organization of the trial, had to be kept informed. During the later stages, beginning in May 1945, the grouping became obvious from the appearance of the signs of scurvy. B. Diet DIETARY HISTORY OF THE VOLUNTEERS BEFORE THE TRIAL The ten volunteers who had taken part in the vitamin A trial had consumed 50 mg. vitamin C daily throughout the experiment, which finished 2 to 6 months before the present trial (Table 29). In the interval they had an unrestricted diet. No relevant information was available on the dietary history of the other ten volunteers. TABLE 29 History of vitamin C intake, before the beginning of the trial, of the 10 volunteers who took part in the previous experiment with vitamin A (Hume and Krebs, 1949) Name Dates for intake Controlled diet supplemented with 50 mg. vitamin C daily Unrestricted diet Bartley .. Drake  .. Garling 16.8.42 to 10.7.44 23.3.43 to 30.6.44 10.8.42 to 15.6.44 11.7.44 to 30.9.44 1.7.44 to 30.9.44 16.6.44 to 30.9.44 Golding 10.8.42 to  1.6.44 2.6.44 to 30.9.44 Proctor 10.8.42 to 29.7.44 30.7.44 to 30.9.44 Tridgell 13.8.42 to 30.6.44 1.7.44 to 30.9.44 Williams, D. 10.8.42 to  1.4.44 2.4.44 to 30.9.44 Wodeman 10.8.42 to 17.4.44 18.4.44 to 30.9.44 Woodhouse 10.8.42 to 11.5.44 12.5.44 to 30.9.44 Another 10.8.42 to  2.7.44 3.7.44 to 30.9.44 56 Pe r io d on exp er imen t ( day s )  14 9  67  5 81 17 4  L abora tory tec hn ic ia n  La bo ra tory te c hn ic ia n  3 4 1  1 15  5 2  5 2  15 7  Com m er c ia l trave l le r, S. R. I. *  S. R. I.  4 70  1 37  63  5 4  16 3  Civ il se rva n t, S. R. I.  S. R. I.  4 4 4  11 7  5 3  5 61  1 69  Insu ra nc e c ler k, S. R. I.  Pro ba t ion o fficer  474 ,  134  6 1 5 5 1 65 Dra ug htsm an  Dra ug hts ma n  445  14 4  65  5 81 17 4  La n d w or ker  S. R. I.  46 9  1 38  6 2  5 81  17 4  S h ip p ing c ler k  S. R. I.  3 87  153  69  5 101  179  Stu de n t  S. R. I.  47 1  1 1 6  53  5 5  16 5  Var iou s  La bo r a tory tec hn ic ian  2 62  1 24  5 6  5 5$  1 66  Ba ker, S. R. I.  S. R. I.  474  9 1  4 1  5 2  157  Var iou s, S. R. I.  S. R. I.  45 1  14 0  64  5 9  1 7 5  La n d w o r ker  S. R. I.  4 71  13 6  62  5 9  175  C ler k, S. R. I.  S. R. I.  4 76  144  66  5 9  17 5  Ma le nu rse  S. R. I:  379  150  68  5 11  181  Var io u s  S. R. I.  3 9 4  1 4 1  64  5 8  17 3  Lan d w or ke r, S. R. I.  Ma le n urse  227  124  56 5 6 168 Var iou s  Var ious  2 9 8  17 3  79  6 0  18 3  Va r ious, S. R. I.  S. R. I.  47 3  14 6  67  5 11j 18 2  C ler k  C le r k  474  134  6 1  5 9  17 5  Ar c hitec t 's c ler k, s tu de n t  Stu de n t  3 09 Ag e a t s tar t o f  We ig ht a t Nam e  Sex  exp er im en t  s tar t o f  He ig ht exp er ime n t (y ea rs )  (m on t hs ) ( lb. )  ( kg. ) ( ft. in. )  (cm. ) N M N M N N N N .~ -~ M ~n N N N M N N M N N wca00xx,  P. P , 4C/2 Q x  h 3333¢  1; ; i. A  59 58  VITAMIN C REQUIREMENT OF HUMAN ADULTS EXPERIMENTAL DETAILS DIET DURING THE PRELIMINARY PERIOD Base line data for fifteen of the volunteers were collected for a preliminary period of 6 weeks, during which they consumed an ordinary diet containing about 60 mg. vitamin C daily from natural sources. Apples were used to adjust the vitamin C intake to that level. The other five volunteers joined the team a few weeks later and they were given, for the preliminary period, the basal diet with a supplement of 70 mg. vitamin C daily. Details are given in Table 30. TABLE 30 Duration of the preliminary period, and the periods of administration and amounts of vitamin C supplements received by the volunteers Name Period on preliminary diet* (days) Vitamin C supplement (mg. daily) Period on supplement Dates No. of days Bartley 42 70 13.11.44 to  7. 9.45 299 Drake 42 0 13.11.44 to  3. 7.45 233 10 4. 7.45 to  4.11.45 124 20 5.11.45 to  3.  1.46 60 510 4.  1.46 to 14.  1.46 it Garling 42 70 13.11.44 to  3.10.45 325 50 4.10.45 to 10.12.45 68 630 11. 12.45 to 19.12.45 9 Golding 42 10 13.11.44 to  8.  1.46 422 570 9.  1.46 to 18.  1.46 10 Hill  .. 42 70 13.11.44 to  7.10.45 329 50 8.10.45 to 10.12.45 64 630 11.12.45 to 19.12.45 10 Hudson 42 0 13.11.44 to 30. 7.45 260 10 31. 7.45 to 18.11.45 111 20 19.11.45 to  3.  1.46 46 670 4.  1.46 to 13.  1.46 10 Jackson 14 0 11.12.44 to 17.12.44 7 10 18.12.44 to 27. 5.45 161 0 28. 5.45 to  7. 8.45 71 5 8. 8.45 to 10.12.45 125 630 11.12.45 to 19.12.45 9 Milburn 42 0 13.11.44 to 20. 7.45 250 6,000 21. 7.45 1 100 22. 7.45 to 31. 8.45 41 Uncontrolled intake 1. 9.45 to  3.10.45 33 20 4.10.45 to  3. 1.46 92 670 4. 1.46 to 15. 1.46 12 Parry 10 10 18.12.44 to 26. 8.45 252 TABLE 30 (continued) Period on preliminary diet* (days) Vitamin C Period on supplement supplement (mg. daily) Dates No. of days 42 10 560 13.11.44 to  8.  1.46 9.  1.46 to 18. 1.46 422 10 21 0 10 20 420 13.11.44 to 26. 7.45 27. 7.45 to 18.11.45 19. 11.45 to  3. 1.46 4. 1.46 to 16.  1.46 256 115 46 13 42 0 13.11.44 to 26. 7.45 256 10 27. 7.45 to 18.11.45 115 20 19.11.45 to  3.  1.46 46 660 4. 1.46 to 15.  1.46 12 42 0 13.11.44 to  4. 8.45 265 10 5. 8.45 to  8. 1.46 157 620 9.  1.46 to 20.  1.46 12 11 10 18.12.44 to 27. 5.45 161 0 28. 5.45 to  4. 6.45 8 10 5. 6.45 to  1. 7.45 27 0 2. 7.45 to  7. 8.45 37 5 8. 8.45 to 10.12.45 125 670 11.12.45 to 20.12.45 10 20 0 11.12.44 to 17.12.44 7 10 18.12.44 to 27. 5.45 161 0 28. 5.45 to  7. 8.45 71 5 8. 8.45 to 10.12.45 125 670 11.12.45 to 19.12.45 10 42 0 13.11.44 to 16. 5.45 185 500 17. 5.45 to ? 42 0 13.11.44 to  4. 6.45 204 20 5. 6.45 to 26. 7.45 52 42 0 13.11.44 to 24. 6.45 224 10 25. 6.45 to  3.10.45 101 20 4.10.45 to  3. 1.46 92 830 4. 1.46 to 17. 1.46 14 42 10 13.11.44 to  8.  1.46 422 640 9. 1.46 to 18. 1.46 10 42 0 13.11.44 to  6. 8.45 267 * A mixed diet containing about 50 to 70 mg vitamin C daily, natural or synthetic. BASAL DIET Permitted Foods The choice of the ingredients was determined by the availability of foods in Britain at the time of the trial and by the amount of the vitamin C which they Name Proctor Sanderson .. Tridgell 3 i S Way .. Whinfield Williams, D. Williams, H. Wodeman Woodhouse .. Another Robinson 60  VITAMIN C REQUIREMENT OF HUMAN ADULTS contained. The number of food items which are almost free from vitamin C is rather small, and in order to avoid monotony means were devised to reduce the vitamin C content of milk, potatoes and vegetables to so low a level that the inclusion of these foods in the basal diet would be possible. A number of items whose vitamin C content was known to be variable were analysed before they were included in the diet. Dehydrated meat, dehydrated potatoes, dehydrated carrots, plum jam, synthetic lemon curd and specially treated milk were obtained in bulk by courtesy of the Ministry of Food and commercial firms. This simplified the control of the diet and the check on the vitamin C content. A list of the foods which were permitted is given in Table 31. TABLE 31 Experimental basal diet deficient in vitamin C: foods of low vitamin C content from which selection was permitted Bread  Milk, processed, fresh, whole Flour  dehydrated Rice  Eggs, fresh or dried Pearl barley  Butter and margarine Macaroni  Cheese Semolina Oatmeal  Sultanas Cornflour  Raisins Breakfast foods (wheat flakes etc.)  Prunes Soya flour  Dates Meat, special dried pork and beef,  Figs canned  Sauces, various, tested for absence of Oxo  vitamin C Bovril Marmite  Sugar Bacon  Lard Coffee Fish, herring, fresh  Cocoa cod, frozen  Tea haddock, kipper, smoked  Mustard paste  Salt tinned, not in tomato sauce  Custard powder Potatoes, dehydrated  Baking powder Peas and beans, dried  Curry powder Carrots, dehydrated  specially cooked  Peanut butter Peanuts Lemon curd, synthetic  Mineral waters Plum jam, yellow  Vinegar Plum jam, greengage  Pepper Toffee  Cashew nuts Golden syrup  Volatile oil of mint 1 made by steam Chocolate, plain, non-fortified  Volatile oil of onion)  distillation Special Treatment of Foods to reduce Vitamin C Content Potatoes. The dehydrated potato strips, as received, were put in a large pan with about 12 volumes of cold water, which was brought to the boil and allowed to stand without further heating for about 1 2 hours. The supernatant liquid was EXPERIMENTAL DETAILS  61 U  pp z  b N o° h R ibo flav in (µ g. / 1 00 g. ) IIIIIIIIII~III ~  m 8 Cd  ~ ~~ Iillllllll0 0 V itam in C con ten t a fter trea tmen t by : fo rmo l me t ho d (-g. / 1 00 g• ) I  I o 0 0 0 0 0 l  l  l o 0 0 l  l dy e me t ho d (mg. / 100 g. ) 00 Vl ti h 00  d' N 0~ 00 C~ Tre a tmen t 3 0  b Ri b  Z R'i Yb  ~b 0 0 0 0 0 0 0 0 0 0  0 0 ZZZ ZZZZ ZZZ.<  ZZ Vitam in C con ten t be for e tr ea tme n t (mg• / 10 0 g• ) I~ IIIIIIIIIII  II 0 f~-i ~  • ,ti w o y C~  N ° R,  U b V N  a  •o 0 O  ?;  b  0  o  >  w w ~ 3  0 • b  aa3 1 2  0 o , o  M U C cC  Cp Q  UA ~4 3:  ~a  u 63 62  VITAMIN C REQUIREMENT OF HUMAN ADULTS poured off and replaced by an equal quantity of cold water. The mixture was again poured off and the potatoes were mashed hot by means of a wire masher. Pan and contents were kept hot for half an hour before serving. During the early part of the experiment a sample was analysed for vitamin C before consumption was allowed, but detectable quantities of vitamin C (more than 0.02 mg. per 100 g.) were never found, so the daily examination of the potatoes was, after some months, replaced by occasional checks. Carrots. The procedure was the same as that described above except for the omission of mashing; the whole cooked strips were kept hot for half an hour. Samples were tested as for the potatoes. Dried peas and beans. Vitamin C can be formed during germination, so the dry pulses were plunged into a large quantity of boiling water to kill the seeds. They were then left to soak overnight. The following morning the supernatant liquid was poured away and the pulses were cooked in plenty of water with the addition of sodium bicarbonate. Milk. After various preliminary experiments on the destruction of vitamin C in fresh milk the following method was adopted. Copper sulphate was added to fresh milk to produce a final concentration of 1 part copper in a million parts of milk. The milk thus treated was heated to 70° C. and aerated for half an hour, which reduced the vitamin C content of the milk to below 0.04 mg. per 100 ml. The milk was treated in bulk in this manner by United Dairies, Ltd. and spray dried. The dried product contained no measurable amount of vitamin C. During the early part of the trial, when there was a break in the delivery of the bulk supply, aeration was carried out for a short time in the laboratory. Vitamin Content of Certain Foods used in the Basal Diet To supplement the information available in food tables a number of items were specially tested for vitamin C, vitamin B 1i riboflavin and nicotinic acid. The results are given in Table 32. The data were used in computing the vitamin content of the diet. SUPPLEMENTS In order to ensure a satisfactory intake of vitamins A and D, each volunteer was given 8 "Adexolin" capsules weekly, supplying about 5,000 I.U. vitamin A and 1,000 I.U. vitamin D daily. On February 5, 1945 it was decided to give all volunteers 50 mg. anhydrous ferrous sulphate daily, so that iron deficiency could be excluded as a cause of any anaemia which might develop. One volunteer, Whinfield, later dropped the iron supplement because he was liable to gastric pain after taking it. ADEQUACY OF THE DIET At three different stages of the experiment the amounts of food consumed by the volunteers were measured for a period of a week. From the data, which are given in Table 33, the average food intake per head daily was calculated (Table 34). It will be seen that the values of the different nutrients are near to those of the recommended allowances of the National Research Council (1948) of the United States. EXPERIMENTAL DETAILS TABLE 33 Gross amounts of foods entering kitchen for consumption in the basal diet of the volunteers in three sample weeks of 1944-5 Amount of food in one week: Food for 17 people (November 1944) for 20 people (March 1945) for ( June-July p le 945) (lb.) (kg.) (lb.) (kg.) (lb.) (kg.) Sugar 13 5.99 16 7.35 17 7.82 Cheese 14 6.45 16 7.35 15 6.90 Tea 3 1.37 2~ 1.15 21 1.15 Margarine 8 3.7 71r 3.45 7 3.22 Bread 70 32.2 68 31.3 70 32.2 Lard .. 6 2.6 7 3.22 41 2.07 Wheat flakes 1 0.46 - - 4j 2.07 Dried egg 3'j 1.72 4 1 84 - - Barley flakes 6 2.6 7 3.22 11 0.69 Haricot beans 4~ 2.07 6 2.60 - - Rice  ..  .. 11 0.69 - - - - Sultanas 41 2.07 - - 6 2.6 Prunes 1 0.46 4 1.84 - - Dates 2 0.92 - - 1 0.69 Figs  .. 1 0.23 - - 1 0.69 Tinned salmon 3 0.34 - - - - Spam, tinned meat .. 41 2.07 21 1.15 2} 1.03 Flour 12 5.52 11 5.0 10 4.6 Frozen cod .. 14 6.45 - - - - Bacon 5 2.3 5 2.3 31 1.72 Dehydrated meat  .. 41 2.07 7(?) 3.22 81 3-91 Dehydrated carrots 21 1.03 21 1.03 1i 0.69 Dehydrated potatoes 21 9.66 20 9.2 12; 5.87 Chicken and ham roll 3 1.37 - - - Butter 21 1.15 21 1.15 2lj 1.15 Dried milk 10 4.6 17j 8.05 161 7.46 Lemon curd 7 3.22 9 4.15 51 2.53 Jam  .. 4t 1.95 4j 2.07 41j 2.07 Raisins - - 9 4.15 - - Salt beef - - 5 2.3 - - Biscuits - - 2 0.92 4 1.84 Fish paste  .. - - 1 0.23 0.34 Peas  .. - - 4 1.84 4j 2.07 Syrup - - 6j 2.99 - - Eggs, shell - - 2j 1.15 - - Mackerel, tinned - - 3 1.38 - - Oxo  .. - - 1 0.23 - - Salt pork - - - - 4j 2.07 Smoked haddock  .. - - - - 7 3.22 Kippers - - - - 4 1.84 Herrings - - - - 4 1.84 Suet  .. - - - - 1= 0.23 Tinned beans - - - - 9 4.15 Semolina - - - l 0.46 Macaroni - - - - 1 0.46 Cocoa - - - - 0.11 TABLE 34 Average daily nutritive value per head, calculated from Food Tables,* of the basal diet (edible portion) consumed by the volunteers in three sample weeks (see Table 33), compared with the standards of requirements set up by the National Research Council (1948) of the United States and by the League of Nations Health Organisation (1938) (The supplements of vitamin A and of iron are not included in the computations.) Nutrient Average daily nutritive value calculated for food intake in: Requirements of moderately active man weighing 70 kg. 1st sample week November 1944 (average weight of the 17  volunteers = 57.6 kg.) 2nd sample week March 1945 (average weight of the 20 volunteers = 61.4 kg.) 3rd sample week June-July 1945 (average weight of the 18 volunteers = 61.6 kg.) Standards of National Research Council (1948) Standards of League. of Nations Health Organisation (1938) Calories Protein (g.) "Available"  carbohydrate  (as starch, g.) Fat (g.) Calcium (g.) Iron (mg.) .. Vitamin B i (mg.)  .. Riboflavin (mg.)  .. Nicotinic acid (mg.) Vitamin A potencyt(LU.) 2,980 96 371 120 1.2 17 1.2 1.8 13.6 5,510 2,910 105 362 122 1.3 17 1.1 2.0 10.3 5,520 2,870 99 355 121 1.3 17 1.1 1.9 13.1 4,130 3,000 70 — — 1.0 12 1.5 1.8 15 5,000 3,000 70 — 80-125 0.75 10 0.9 — — 2,000-4,000 * For the special dehydrated foods the values were taken from Table 32; otherwise War Memorandum No. 14 (Medical Research Council, 1945) was used, and for nicotinic acid, the Food Composition Tables for the Commonwealth of Australia Council for Scientific and Industrial Research (Marston and Dawbarn, 1944). t Calculated by adding together the values for preformed vitamin A and for one third of the carotene value. Preformed vitamin A contributed 2,290-2,680 I.U.; total carotene 5,500-8,770 I.U. bdrnW  ItjbW4a ndnWo ~ . yy d C7  C CD ~ m d (nnp w w G  a w oQ Q B Q w a a y 5 F: ° D. o o 'O C9 N  C]. (D' C G  (D  N n Q•  to  N  O 0  = o~  ao-  ` D c 0°o o. d aay B B  F 5  v  ~~ p  m  co a rn w  c °°n  O D W C " n Sv n a 0 CD  la , P• G y  P• : a  N  P• N w N r°  w o rL ti ao  CD  0 a t:j  B  w  w y ov  p A Q.  n G a.  a b 0 a  ti  N w w o N o w JP to  O  rn O A OO  N NPA O N ~1 O oo A O A A I I I of I of i of ~I I I of I I I I W CD ~  N 0 0 O O C i ? 00 U CT N W O O w A v N i A v i i v N i .P to .1 O~ to O v~ J O o0 A v, in CL C O tl0 a `a :, Cq p z ) w n su o o I u n o my V ITA MIN C RE QUIREM ENT OF HUMAN A D ULTS S 'I I V. La Q 'I V. L N a w i ll i l dX a 't 66  VITAMIN C REQUIREMENT OF HUMAN ADULTS In the vitamin A trial good agreement was found between the food values calculated and the food values directly determined by analysis (Hume and Krebs, 1949, Table 36), and it was, therefore, thought unnecessary to analyse the food directly. The adequacy of the diet was confirmed by the fact that the volunteers who acted as controls by consuming the basal diet supplemented with 70 mg. vitamin C daily remained in normal health throughout the trial. Further samples of the food intake were collected from two volunteers, one, Wodeman, a consistently large eater, the other, Drake, a consistently small eater, when they were showing severe signs of depletion. The data are given in Tables 35 and 36; they show that the average food consumption was unchanged when frank scurvy had appeared. TABLE 36 Average daily nutritive value per head of the diet set out in Table 35 (The food values were calculated from the Tables for the edible portion (Medical Research Council, 1945). For the special dehydrated foods, the values for vitamin B i , riboflavin and nicotinic acid were obtained by direct analysis. The carotene content of the diet was divided by 3 to give the total vitamin A potency.) Calculated nutrient value: Wodeman  Drake Iron (mg.) Calcium (g.) Protein (g.) Fat (g.) Carbohydrate (g.) Calories  3,362  2,594  421  261  129  124  132  118  29.3  23.3  1-2  1.0 Vitamin B i (mg.)  12  0.9 Riboflavin (mg.)  2.0  1.6 Nicotinic acid (mg.) . .  13 , 5  11.6 Vitamin A potency (I.U.)  4,041  3,617 VITAMIN C POTENCY OF THE DIET None of the foodstuffs consumed by the volunteers, with the exception of jam, contained measurable amounts of vitamin C (Table 32). The jam was not analysed by the formol method and its true vitamin C content is therefore uncertain. The figure obtained on the assumption that the whole indophenol titre of the jam represented vitamin C is thus the maximum vitamin C content of the jam. The average weekly consumption of jam per head was 110 g. On the basis of an average figure of 1.3 mg. vitamin C per 100 g., the weekly intake of vitamin C from this jam was 1.43 mg., and the daily intake 0.20 mg. Other items, though they contained no measurable amounts of vitamin C may have contained traces. With the limits of accuracy of the analyses in mind, it may be safely assumed that the basal diet contained daily less than 1.0 mg. vitamin C and most probably even less than 0.5 mg. EXPERIMENTAL DETAILS  67 VITAMIN C SUPPLEMENTS Vitamin C supplements were given in the form of tablets containing 5 or 10 mg. ascorbic acid. It was desired that the volunteers should not know to which group they belonged so each day all were given 7 tablets identical in size, appearance and taste, some containing vitamin C and others being dummies. The latter consisted of 5 mg. tartaric acid, 94.5 mg. lactose, 22 mg. starch, 3 mg. sugar, 5 mg. talc and 0.5 mg. stearic acid. C. Biochemical Methods ESTIMATION OF VITAMIN C Blood Collection of Samples Between 7.0 a.m. and 8.0 a.m., 25 ml. of blood were drawn from the cubital vein of the fasting subject. Sodium oxalate was added as the anticoagulant. Six ml. of the bloodwere set aside for estimation of vitamin C, and the remainder was immediately centrifuged to separate the plasma, the white cell layer and the red cell layer. Determination of the vitamin C content of the plasma and white cells was begun as soon as possible. The determinations on whole blood were done at Oxford on the following day. During the saturation tests additional specimens of blood and plasma were despatched to Oxford on the morning of collection, and determination of the vitamin C content by the method of Roe and Kuether was done in the evening. Plasma: Procedure of the Oxford Team Estimations of vitamin C in the plasma were made independently by the Oxford team and the Sheffield team. Two methods were used by the Oxford team. Titration with 2:6-dichlorophenolindophenol. Three ml. of plasma were slowly added to 12 ml. of metaphosphoric acid solution freshly prepared; the product was well mixed, centrifuged, and filtered in the dark through a 9 cm. Whatman No. 42 filter paper. Duplicate samples of 5 ml. of the filtrate were then titrated with the dye, freshly dissolved and standardized against pure vitamin C, and of such a strength that 100 ml. was equivalent to 2 mg. vitamin C. The titrations were made with a Conway microburette, and the end point was taken as a faint pink colour persisting for 30 seconds. The reading was corrected for a blank value. The figures reported are the averages of those duplicate estimations which agreed within 0.04 mg. per 100 ml. plasma. Method of Roe and Kuether (1943). In this procedure vitamin C is converted into dehydroascorbic acid by treatment with charcoal; the dehydroascorbic acid is treated with 2:4-dinitrophenylhydrazine and the product, probably a 2:4dinitrophenylosazone, is estimated colorimetrically in sulphuric acid solution. The method estimates the sum of ascorbic acid and dehydroascorbic acid. Five ml. of the plasma were added drop by drop to 15 ml. trichloroacetic acid and shaken well. After it had stood for 5 minutes, the mixture was centrifuged; about 0.75 g. of washed "Norite" was added to it, and it was again shaken, and left to stand. The mixture was centrifuged and filtered through a 9 cm. Whatman No. 42 filter paper. Duplicate samples of 4 ml. of the filtrate were placed in two Evelyn colorimeter tubes, and 2 drops of 10 per cent thiourea solution in alcohol were added to each. To one of the tubes 1 ml. of 2 per cent 2:4-dinitrophenylhydrazine in 9N H,SO 4 was added, and the tube was incubated for 3 hours at Nutrient EXPERIMENTAL DETAILS  69 TABLE 37 Effect of the delay caused by transit on the apparent vitamin C content of samples of urine sent from Sheffield to Oxford as estimated by titration with 2:6-dichlorophenol indophenol Apparent vitamin C content of 24-hour specimen of urine: estimated in Sheffield at end of 24-hour period of collection (rng.) 17.8 192 27.0 28.8 36.4 35.4 43.5 72.5 90.0 194 estimated in Oxford 10 to 24 hours after end of collection (MB.) 15.8 17.3 25.0 25.8 32.4 34.4 43.5 71.0 85.8 176 68  VITAMIN C REQUIREMENT OF HUMAN ADULTS 37° C. The tube was then cooled in ice water, and 5 ml. of 85 per cent sulphuric acid were added slowly, the temperature being kept low. Similar amounts of dinitrophenylhydrazine solution and sulphuric acid were then added to the other tube. After 30 minutes the optical density of the incubated tube was determined by means of an Evelyn photo-electric colorimeter, the tube not incubated providing the blank. Recovery experiments indicated that the results were accurate to within 0.05 mg. per 100 ml. plasma. Results obtained from the same plasma by the two methods are given in Table 42 (p. 122). Plasma: Procedure of the Sheffield Team The method used was a modification of the dye titration procedure. Four ml. of oxalated plasma, 2 ml. of water and 4 ml. of 20 per cent metaphosphoric acid, freshly prepared, were thoroughly mixed and filtered through a 7 cm. filter paper. A little more than 5 ml. of filtrate was obtained, and duplicate determinations were made on two 2.5 ml. samples. One hundred ml. of the dye solution was equivalent to 1 mg. of ascorbic acid, hence the volume in ml. of the dye solution required was numerically identical with the concentration of vitamin C in mg. in 100 ml. of the plasma. The method requires twice the amount of plasma usually used but the accuracy is greater, duplicates agreeing to within 0.03 mg. per cent. Whole Blood The estimations of vitamin C in whole blood were made by the Oxford team, who used the method of Roe and Kuether. White Cells The estimations of vitamin C in white cells were made by the Oxford team who used the method of Butler and Cushman (1940). After the blood had been centrifuged for 15 minutes at 15,000 r.p.m. and the plasma had been removed, the bully layer was carefully transferred by means of a wide-bore Pasteur pipette to a tube similar to a Wintrobe haematocrit tube, having internally a length of 110 mm. and a diameter of 4 mm. It was then centrifuged for 30 minutes at 4,000 r.p.m. The white cells were thus packed into a clearly defined column which, after the supernatant plasma had been carefully removed, could be taken up with a pipette, transferred to a small centrifuge tube and weighed. To disintegrate them, 0.5 ml. of distilled water and 1.5 ml. of 10 per cent metaphosphoric acid were mixed with the cells. The mixture was centrifuged for 10 minutes at 2,500 r.p.m., and the whole of the clear supernatant fluid was used for determining the vitamin C content by titration with dichlorophenolindophenol. The content was expressed in mg. per 100 g. white cells. Urine Collection of Samples Urine was collected for 24-hour periods beginning at 8 a.m. It was placed in dark-coloured bottles containing 40 g. of metaphosphoric acid, sufficient to give a final concentration of about 2 per cent. Analyses were made independently at Oxford and Cambridge, the specimens reaching their destination 10 to 24 hours after the end of the collecting period. The extent of the loss of reducing substances during transit can be gauged from the figures in Table 37. For the saturation tests members of the Oxford and Cambridge teams were stationed at Sheffield and determinations by the dye titration methods were made independently. Methods. Three methods were used: (1) Titration with 2:6-dichlorophenolindophenol (Harris, Ray and Ward, 1933). The method is widely used for clinical purposes but is known to be rather unspecific and to include a variety of reducing substances in the estimation. (2) Titration with 2:6-dichlorophenolindophenol in the presence of formaldehyde. The procedure, an elaboration of Lugg's (1942, a, b) method, is described in Appendix A. (3) The colorimetric method of Roe and Kuether (1943) with 2:4-dinitrophenylhydrazine. Methods (2) and (3) are known to be more specific than method (1). Food The method described by Mapson (1943) was used. It is based on titration with 2:6-dichlorophenolindophenol at different acidities in the presence of formaldehyde. ESTIMATION OF B VITAMINS Vitamin B l was estimated by the method of Harris and Wang (1941), nicotinic acid and nicotinamide by the method of Kodicek (1940), and riboflavin by the method of Wang and Kodicek (1943) (see Table 32). ESTIMATION OF BLOOD CONSTITUENTS OTHER THAN VITAMIN C Constituents of blood other than vitamin C were estimated by the Oxford team. Plasma Proteins The micro-Kjeldahl method was used to determine total protein, albumin and fibrinogen nitrogen. Globulin was calculated by difference. The sodium sulphate method (Howe, 1921) was used to separate albumin and globulin. For the digestion the quantities used were: