The vitamins. II. The water-soluble vitamins

of the blood of avitaminous pigeons is probably due The VITAMINS* entirely to p p v i c acid. Cardiac damage: hypertrophy of the right heart;

11. THE WATER-SOL UBLE VITAHINs decompensation. The cardiac involvement in vitamin

VICTOR E. LEVINE BI deficiency is not amenable to treatment with digi- talis, but is very favorably influenced by vitamin B,

Creighton University School of Medicine, Omaha, Nebraska therapy. VITAMIN B1

SYNONYMS: Water-Soluble B1; Anti-neuritic Vita- min; Anti-beriberi Vitamin; AppetiteStimulating Vi- tamin.

Vitamin B1 has been obtained in crystalline form. According to R. R. Williams (1935) the formula for vitamin B1 indicates the presence in the molecule of a pyrimidine base and of a thiazole derivative.

I I I C-CCH2:CHzOH

HC /I c-N<CHS.HCI /I N-CGHs

vitamin B1 hydrochloride (Williams, 19361.

BIOLOGIC FUNCTIONS

Promotes growth. Essential to normal nuclear metabolism. Stimulates appetite. Essential for the maintenance of the gastrointestinal

tract. Essential for normal reproduction and lactation. Concerned in the intermediate metabolism of carbo-

hydrate. Essential for the normal functioning of the nervous

system. Increases resistance to infection.

PATHOLOGICAL FINDINGS AS A RESULT OF DEFICIENCY

Impaired growth, loss of weight, emaciation. Nuclear degeneration. Loss of vigor, muscular weakness, fatigue. Impairment of digestive processes: hss of appetite,

idammation of the tongue, gastrointestinal stasis, decreased motility of the stomach, lowered gastric acidity, intestinal atony, constipation, ulceration, colitis.

Lowered energy metabolism: fall in basal metabolism, subnormal temperature, lowered blood pressure, slowed pulse rate (bradycardia).

Disturbances in the metabolism of carbohydrate: lowered glucose tolerance, hyperglycemia with termi- nal hypoglycemia, increase in the lactic acid content, and decrease in the oxygen uptake of brain tissue.

Thompson and Johnson (1935) found abnormally large units of bisulfite-binding substances in the blood of B1 avitaminous pigeons and rats, an axcumulation independent of the accompanying starvation. This accumulation is specifically related to vitamin Bt deficiency. The increase in bisulfite-binding capacity

'Continued from the August issue, pp. 35752.

~ n ~ ~ l v e m e n t of the nervous system: loss of co- ordinating power of the muscles, leading to paralysis of the limbs. Little atrophy of the central nervous system, the paralytic symptoms being due mainly to impaired functional activity of the nerve cells rather than to their degeneration. Convulsions; vomiting. In birds: opisthotonos, emprosthotonos, convulsive seiz- ures, cartwheel turning.

Sterility in the male due to atrophy of the testes with shrunken tubules and absence of spermatozoa.

Sterility in the female due to cessation of cestrus cycle as the result of failure of ovulation.

Diminished resistance to infection, especially in the gastrointestinal system and in other organs as a result of the passage of bacteria through an impaired alimentary tract.

Fat has a sparing action on vitamin B1. Fat spares vitamin B1 by decreasing the amount dissipated in the metabolism of the rat (Evans and Lepkovsky, 1935).

Specific disease produced as a result of deficiency: beriberi.

CHEMICAL STABILITY

Destroyed on heating, the extent of the destruction depending upon temperature and hydrogen-ion concen- tration. The higher the temperature or the less acid the medium the greater is the destruction.

Sherman and Grose (1923)'aetermined the effect of heat on vitamin B complex (which may be interpreted to hold good for vitamin B1) of tomato juice a t its natural acidity (pH 4.3). At 100°C., for one hour the inactivation was 20 per cent.; a t llO°C., 33 per cent., a t 120°C., 45 per cent., and a t 130°C,, 58 per cent.

Sherman and Burton (1926) reported the effect of heating vitamin B1 in tomato juice for one hour a t 100°C. under different hydrogen-ion concentrations. At pH 5.4, the inactivation was 10 per cent., a t pH 7.9, 30 to 40 per cent.; a t pH 9.2, 60 to 70 per cent.; a t pH 10.9,90 to 100 per cent. -

The destruction of vitamin BI is not due to oxidation, since.the same results are obtained whether the heating is done in loosely covered flasks or under very strictly anaerobic conditions.

ARTIFICIAL SOURCES AND CONCENTRATES

Marmite (67). T o ~ l i n . Filtrate from mercuric sulfate precipitated in the

preparation of torulii (vitamin Bl concentrates from baker's yeast) by the Kinnersley and Peter's method (1927).

Aqueous extract of baker's yeast is treated with neutral lead acetate (1). The precipitate (1) is dis-

carded. The filtrate (1) is treated with baryta water and the precipitate is again discarded (2). The filtrate (2) is treated with sulfuric acid and the precipitated barium sulfate (3) discarded. The filtrate (3) is treated with mercuric sulfate and the precipitate re- served for the preparation of vitamin B4.

The last filtrate is made up to pH 7.0 and treated with norite charcoal and the filtrate is again treated with norite charcoal. The charcoal collected on the filter paper is extracted with 0.1N HC1. The filtrate is discarded. The norite is now extracted with 50 per cent. alcohol and filtered. The alcoholic extract known as torulin is rich in vitamin BI.

Cereals: wheat germ (63); middlings (40); bran (20) ; malt extract (10) ; whole-grain cereals (10).

Vegetables: pulses (13); parsnips (7); artichokes (5) ; cabbage (5) ; leeks (5) ; tomatoes (5) ; potatoes (5).

Hanning (1934) has determined the number of Chase and Sherman units of vitamin B1 in one-ounce samples of the following foodstuffs: canned strained tomatoes (20-24), canned strained peas (7-8), canned strained green beans (4.7), canned strained carrots (4.3), canned strained beets (3.3), and canned strained spinach (2.5).

Fruits: orange juice (6). Seedless Thompson grapes, according to Morgan,

Kimmel, Field, and Nichols (1935), contain .20 to 25 Chase and Sherman units of vitamin B1.

Morgan, Hunt, and Squier (1935) have reported that dried California (French) prune flesh contains 22.7 to 28.4 Chase and Sherman units of vitamin BI per ounce. This quantity is approximately one-tenth the amount found in wheat germ and nearly one-half of the vitamin B1 content of whole wheat.

Fresh figs-Kadota and Adriatic varieties--each contain 10 units of Chase and Sherman units of vitamin B1 per ounce. Unsulfured dried Adriatics retain 61 per cent. and the dried sulfured Kadota and Adriatic varieties retain 37 per cent. or less of their vitamin B1 content. (Morgan, Field, Kimmelf and Nichols, 1935.)

Douglas, Halloway, Williams, and Garrison (1934) have found that one ounce of fresh Bosc pear contains 4.7 Chase and Sherman units of vitamin B1.

Dairy products: dried skim milk (10); egg yolk (7). Meats andfLsh: kidney (8) ; liver (7). Oysters contain 42.5 Chase and Sherman units per

ounce (Whipple, 1935). No appreciable destruction of vitamin B1 occurs during the cooking of oysters.

Miscellaneous: dried brewer's yeast (100); nuts (2040); roes (7).

BIOLOGIC TEST

The biologic test for vitamin Bl is carried out on animals kept on a vitamin B1-free diet. Any substance administered is said to have vitamin BI potency when -

* The figures in parentheses, uoless otherwise stated, indicate the relative vitamin B1 potency according to PLIMMER, RAY- MOND, AND LOWDES (1933), taking dried brewer's yeast as the standard with a potency of 100.

i t induces the resumption of growth, the return of the appetite to normal, and the disappearance of the neuro- genic symptoms characteristic of the polyneuritic syndrome due to lack of vitamin BI.

CLINICAL TEST

(1) Vestibular function test for vitamin BI deficiency. The vestibular test consists of a t least three successive observations on the duration of nystagmus, the direction of rotation being reversed after each observation. The durations of nystagmus following clockwise rotations are averaged, and similarly the durations following counter-clockwise rotations. The mean of these two averages is taken as the result of the test. Vita- min B,-deficient animals show an increase in the duration of nystagmus. The increase in duration makes its appearance before any other neurologic symptom.

(2) The catatorulin test, which indicates the power of vitamin B1 to restore a specific in vitro respiration in avitaminous-B1 tissue (Passmore, Peters, and Sinclair, 1933; Kinnersley, O'Brien, and Peters, 1935).

CHEMICAL TEST

Formaldehyde-Azo Test (Kinnersley and Peters, 1934). Diazotized sulfanilic acid is added to a reagent containing an aqueous solution of sodium bicarbonate and sodium hydroxide. After one minute a drop of 40 per cent. formaldehyde and the vitamin solution of an acidity greater than pH 4.0 are added. A pink color develops which slowly fades to yellow. The limit of sensitivity is 1.5 to 2 . 0 ~ of crystallime vitamin B1. Reasonably accurate estimates of color can be made with solution containing 5 to 107 crystalline vitamin B1. :. . .

SPECTROSCOPIC TEST

Crystalline vitamin B1 in acid alcohol shows selec- tive absorption with a maximum at 245-247 mp (Holi- day, 1935).

SYNONYMS: WaterSoluble Bz; Vitamin G; Anti-pellagra Vitamin; Anti-dermatitis Vitamin.

BIOLOGIC FUNCTIONS

Essential for proper functioning of the gastrointestinal tract.

Essential for normal nutrition and maintenance of the cutaneous system.

Involved in normal hematopoiesis. Essential for normal maintenance of the visual appa-

ratus.

PATHOLOGICAL PINDINGS AS A RESULT OF DEFICIENCY

Cessation of growth and cachexia. Disturbances in the alimentary system: stomatitis,

soreness, and inflammation of tongue, gastric hypo- acidity or anacidity, enteritis, marked diarrhea, putre- faction as indicated by the large amounts of indican in the urine.

Disturbances in the cutaneous system: in the human, being, symmetrical lesions on face, feet, back of neck, scrotum, showing erythema, thickening, and ulceration.

Disturbances in the nervous system due to organic lesions in the spinal cord and in the central nervous system, which, in the human being, may lead to de- mentia.

Disturbances in the hematopoietic system: severe anemia responding to specific treatment with vitamin Bz.

Disturbances in the visual apparatus: in the experimental animal, soreness of the eyes and abnormal secre- tion, with a tendency to sticky lids and frequent ruh- bing and occasional scratching of the eyelids by the animal. The fur may fall out around the eyes, leaving bald and inflamed lids. Formation of cataract.

Specific disease produced as a result of deficiency: pellagra.

CHEMICAL STABILITY

More stable to heat and alkalies than vitamin BI. Vitamin B, is relatively thermo-labile, whereas vitamin B, is relatively thermo-stable.

Dried yeast heated in an autoclave for 1.5 hours a t 120°C. loses anti-neuritic potency, but retains prac- tically all of the anti-pellagrous component.

Heating dried yeast a t 105°C. for two weeks does not affect its vitamin Bz potency. Heating dried yeast at 150°C. for the same length of time brings abont complete loss of potency.

Vitamin Bz is relatively heat-stable in acid solution, but is eventually destroyed in alkaline medium, the rate of destruction increasing rapidly with increasing alka- linity.

Vitamin B2 is comparatively stable to prolonged heating when the reaction mixture is acid (pH 5.0 to 3.0), retaining half of its potency after 4 hours a t 122 to 124°C.

Heating for 1 hour a t 120°C. a t pH 7.2-8.7 an aqueous extract of yeast or marmite or a watery liver extract results in a loss of 50 per cent. of the priginal vitamin Bz potency.

Vitamin Bz (at pH 8.0 to pH 10.0) loses 30 per cent. of its potency in 10 days a t room temperature, 50 per cent. in 2 hours, a t 122'C., 75 to 100 per cent. in 4 to 6 hours a t 122'C.


Dried brewer's yeast autoclaved for 1.5 hours a t 12O0C.

Liver extract used in the treatment of pernicious anemia.

Cereals: wheat germ. Cereals are rich in vitamin B1, but relatively poor in vitamin Be.

Vegetables: turnip tops (85) ; beet tops (71) ; carrot tops (57); peas, dried (28-35); spinach, winter (28); -

The figures in parentheses represent vitamin BI values in Baurquin units per ounce, following the system adopted by H. C. Sherman.

water cress (28) ; cabbage (14-28) ; carrots (14-21) ; lettuce (14-21) ; beets (14) ; cauliflower (14) ; pigeon peas (14) ; turnips (7-14) ; potatoes (6-8) ; onions (3-4).

Hanning (1934) has reported the number of Bourquin and Sherman units in one-ounce samples of the following foodstuffs: canned strained spinach (7.5-11.8), canned strained peas (7.5-8.6), canned strained tomatoes (6.7), canned strained green beans (6.0), canned strained beets (3.0), and canned strained carrots (2.5).

Fruits: ripe plantain (56.7) ; pears (14) ; bananas (10) ; oranges (6-18) ; apples (6-7) ; watermelons (3).

Douglas, Halloway, Williams, and Garrison have found one ounce of fresh Bosc pear to contain 6.1 Bour- quin and Sherman units of vitamin Bz.

Morgan, Hunt, and Squier (1935) bave determined the vitamin Bz content of dried California (French) prunes. The dried prune flesh contains 75 Bourquin and Sherman units per ounce. This quantity is larger than has been reported for any other fruit, and is com- parable with the value found for wheat germ, and is one-half to one-fourth that of dried brewers' yeast and liver.

Vitamin BI is present in figs to the extent of 8.6 to 14.2 Bourquin and Sherman units per ounce (Morgan, Field, Kimmel, and Nichols, 1935).

Dairy products: milk, malted, dry (71); milk (11- 21); egg yolk (65); egg white (2142); egg, whole (28). Milk is comparatively poor in vitamin BI but relatively rich in vitamin Bz.

Meats andfish: beef liver (227-284) ; veal liver (242) ; beef kidney (227); veal kidney (227); beef heart (85); veal, round, lean (42); beef, round, lean (26-31); beef, average fat (23-26); bacon, average'fat (8-14).

Miscellaneous: brewer's yeast, dried (213425).

YITAMIN Be AND TEIE FLAVEiS

The flavins are organic compounds which yield bright yellow-green fluorescence in solution?. The isolation and composition of the flavins bave been investigated by a number of workers-Warburg and Cbristien (1933), Kuhn, Gyorgy, and Wagner-Jauregg (1933), Ellinger and Koschara (1934), Karra, Salomon, and Schopp (1934), Stern (1934), and Itter, Orent, and Mc- Collum (1935). The flavins isolated from egg-white (ovoflavin), from whey (lactoflavin), from liver (hepatoflavin), and from yeast are very similar if not identi- cal. The formula attributed to the flavins is C11Hzo- N4Os.

Withii the last two years attempts have been made by means of animal experimentation to identify the flavins with vitamin B2. Gyorgy, Kuhn, and Wagner- Jauregg (1933), employing the Bourquin-Sherman diet supplemented with vitamin B1 and with vitamin B4, reported the interesting fact that 5 micrograms of flavin was required by a rat per day to increase its weight by 40 grams in 4 weeks. Karrer and von Euler (1933) prepared a flavin from liver which gave a good response to growth on a vitamin B2-deficient ration. Kuhn (1933) and Booher (1933) described a flavin from milk

with very great vitamin Bz potency. Booher's compound displayed vitamin BZ activity in a dose as low as one milligram per rat per day.

Elvehjem and Koehn (1935) have just recently dis- proved the identity of the flavins with vitamin Bz. They claim that vitamin Bz is a chemical entity separate and distinct from flavins. They succeeded in preparing a vitamin Bt concentrate from liver extract by adsorp- tion with fuller's earth. The material removed by the fuller's earth carried the hepatoflavin, but i t was de- void of vitamin Bz potency. The filtrate, however, possessed vitamin Bz potency.

Elvehjem and Koehn departed from the usual procedure. They did not employ growth as the biologic indicator for the presence of vitamin Bz. They used the chick as the experimental animal and the appearance of the cutaneous symptoms of pellagra as the criterion for vitamin B2 deficiency. The animals re- ceiving the basal ration plus the flavins (free from vitamin Bz) showed more severe symptoms of pellagra than those on the basal ration alone.

VITAMIN B, BIOLOGIC FUNCTION.

Promotes growth in the pigeon.

CHEMICAL STABILITY

Exceedingly thermo-labile, since it is destrbyed by a temperature as low as 60°C.

NATURAL SOURCES

Cereals: whole wheat; wheat germ. Miscellaneous: yeast.

VITAMIN Ba BIOLOGIC FUNCTIONS

Promotes growth in rats. Essential in neuromuscular,equilibrium. Required for the production of litters in the rat.

C

PATHOLOGICAL F I N D I . AS A RESULT OF DEFICIENCY

The pathological findings are best given by describing the course of vitamin B4 deficiency. Rats are raised to adult size on a basal diet plus vitamins B1, Bz, and B4. They are then deprived of vitamins Bl and B4 until polyneuritis sets in (Reader, 1930).

Feeding vitamin B1 a t this stage cures the convulsions and the paralysis in a few hours. The weight now ceases to fall and is maintained on a level.

Although relieved of the symptoms of polyneuritis the animals still show general muscular weakness, swollen, red paws, a tendency to sit in a hunched po- sition, and a tendency to walk with a rolliig gait. The fur appears yellowed and greased, and nasal hemorrhage may occur.

Addition a t this point of a food substance or concentrate containing vitamin B4 leads to an increase in weight and to greater bodily activity, and the gait becomes normal in about a week.

If no vitamin Bp is administered, the rat becomes worse and dies within 10 to 14 days.

From the above picture we may be led to believe that beriberi is probably B1 + B4 deficiency and that pellagra is probably Bz + B4 deficiency.

CHEMICAL STABILITY

Stable in acid medium, but loses potency when kept in storage in aqueous solution even a t O°C.

In contrast to vitamin Bz, vitamin Ba is thermo-labile in alkaline medium. Yeast heated a t pH 9.4 for 5 hours a t 15 pounds pressure loses its vitamin B4 potency, whereas under the same conditions the vitamin B2 present still shows biologic activity.

The stabilities of vitamins B, Ba (G) and B4 have very recently been investigated by Keenan, mine, Elvehjem, and Hart (1935). They observed that vitamin B1 in yeast, liver, and in a natural grain ration was destroyed completely by autoclaving, and inactivated to a great extent in yeast and liver by heating in the fresh state a t 100°C. for 24 hours. Vitamin Bz (G) was inactivated in the foodstuffs tested by a dry heat treatment a t 120°C. maintained for a period of 24 hours. Vitamin B1 is relatively stable to this ther- mal treatment. Under such conditions vitamin Bz (G) does not conform to the conception that i t is the more heat-stable factor.

Vitamin B4 is similar in heat stability to vitamin BI. The similarity in heat stability of vitamins BI and B4 in yeast and in liver raises the question as to whether some of the abnormalities which have been ascribed to the syndrome of polyneuritis may not be due in part a t least to a deficiency in vi.tamin B4.

Barnes, O'Brien, and. eider (1933) have isolated from yeast an unknown base, CaH4N4HCl.'/aHpO, which has the biologic properties of vitamin B4, and which consists of colorless, rod-like crystals, subliming a t 27OoC., soluble in water, insoluble in absolute alcohol, ether, or acetone, and quiokly inactivated by alkali. Tschesche (1933) confirmed the findings of Barnes, O'Brien, and Reader and identified the compound as adenine.

Since it has been recently shown that pure adenine has no potency, i t is reasonable to state that vitamin B4 accompanies this compound as an impurity.


The mercuric sulfate precipitate from the preparation of torulin is used for the preparation of vitamin B4 concentrate (Reader, 1934).

Vitamin B4 concentrate may also be prepared by ad- sorption upon fuller's earth (Seidell) following a lead acetate precipitation (Guha, 1931).

NATURAL SOURCES

Cereals: whole wheat; wheat germ. Vegetables: lettuce; spinach; alfalfa. Dairy products: milk. Meats and fish: liver; ox muscle. Miscellaneous: yeast.

VITAMIN C SYNONYMS: Water-Soluble C; Anti-scurvy Vita-

min; Anti-scorbutic Vitamin; Ascorbic Acid; Ascor- binic Acid; Cevitamic Acid.

Formula for Vitamin C (Ascorbic Acid) (Karru. 1933).

HOC-OH I

CHSOHCHOH-CH I

SYNTHESIS OF ASCORBIC ACID IN THE LABORATORY

Reichstein in Ziirich has succeeded in preparing ascorbic acid by synthesis from 1-xylose.

CHO CH=N.NHGHa CHO

I I

COOH

I CHOH

I

I CHOH Hydrolysk +

I CHOH

I CHOH

I CHOH

I co co I saponification I CHOH C CHOH

I I ~ H O H CHOH

I I &H~OH CHZOH e P Enolization r 0 I! - H a

COH --f COH I I

CHOH I CH

I A

I CHOH

I CHzOH

I CHOH

I A%arbie Acid

CHZOH

SYNTHESIS OF ASCORBIC ACID IN THE BODY

The mechanism involving the synthesis of ascorbic acid in the body has been suggested by Mosonyi (1934). This investigator observed that the reducing ability (ascorbic acid content) of the adrenals of the rat increased to the same degree after the administration of

Henze's ketol (3-hydroxyhexane-1,5-dione) as after the administration of ascorbic acid. The ketol is probably an intermediate product.

The synthesis of ascorbic acid may follow the chemical pattern given below:

CHs CHa

do COH I I

HCOH II

COH 1 Enoliratian fHsO

CHa __f CH ---+ I I II

I I C H z CHI 3-Hydroxyh-eraeral,5diooe

CHI oxidation COOH c h I + COH

I COH

I COH I

/I COH

II COH

I/ 0

HdoH HdoH c H

HdoH HcoH I I HCOH

I Oxidation CH, + L o H

I CHSOH

Ascorbic Acid

BIOLOGIC FUNCTIONS

Required for the formation and maintenance of in- tercellular cement substance.

Required for normal bone and tooth formation and maintenance.

Involved in normal hematopoiesis. Increases resistance to infection.

PATHOLOGICAL FINDINGS AS A RESVLT OF DEFICIENCY . . Loss in weight, fatigue, loss of appetite. Derangement in bone and teeth: decalcification, de-

struction of odontoblasts, decay of teeth, loosening and shedding of teeth due to swelling and hemorrhage in gums; fragility of bones, spontaneous fracture.

Hemorrhage as a result of loss of infercellular cement substance with increased fragdity of the capillaries in mucous membranes, skm, gums, joints, limbs, bone marrow, muscle and other tissues.

1)cgeneration of the skeletal musdes accompanied by foci of fibrinoid degeneration and hyperplastic connec- tive tissue.

Rheumatic pain and swelling in joints and limbs. Pathologic changes in the joints characterized by

hyaline fibrinous material in joint spaces, usually ad- hering to or replacing the synovia, and by areas of hemorrhage and eosinophilic hyaline material in the subsynovial, capsular, and periarticular tissues.

Lowered resistance to infection. The pathologic similarities between rheumatic fever

and experimental scurvy combined with infection have been strikingly demonstrated by Rinehart, Connor, and Mettier (1934).

Anemia, not curable by the administration of massive doses of iron salts or liver extract, but specifically by the administration of vitamin C.

Speci6c disease produced as a result of deficiency: scurvy.

CHEMICAL STABILITY

More stable in acid than in alkaline medium. Spraying of orange trees with lead acetate causes

reduction in the acidity of the juice of the orange, a decrease in sucrose with a corresponding increase in in- vert sugar (Gray and Ryan, 1921; Juritz, 1925; Haw- kms and Barger, 1927). The vitamin C content of oranges from trees sprayed with lead arsenate is con- siderably lower than that of oranges from unsprayed trees of the same variety and same degree of maturity (Nelson and Mottern, 1932).

Destroyed by oxidation or on heating in the presence of air. Heat alone in the absence of air or oxygen does not destroy the potency of vitamin C.

By removing dissolved oxygen in liquid and solid foods by vacuum exhaust and by operating in atmos- pheres free from oxygen, canning of foods maybecarried out with retention of anti-scorbutic potency.

A specific oxidase, ascorbic acid oxidase, has been reported by Tauber and Kleiner (1935) to oxidize ascorbic acid. The enzyme requires the presence of atmos- pheric oxygen, whicli acts as a hydrogen acceptor. The oxidized ascorbic acid has no reducing properties. It may, however, be transformed into its original state by treatment with hydrogen sulfide.


Ascorbic acid (hexuronic acid) prepared according to the methods of Szeut-Gyorgyi (1928), Svirbely and Szent-Gyorgyi (1932), and King and Waugh (1932).

Cereals: corn, fresh (5); corn, canned (3). Vitamin C is lacking in dry mature seeds (cereals, legumes), but develops in the process of germination.

Vegetables: peppers, raw (25); spinach, raw (25); cabbage, raw (20); parsley Q5); peas, raw (15); tomatoes, raw (15); canned (15); peas, canned (10); spinach, canned (10); cabbage, sauerkraut (5); turnips (4); beets (3); carrots (3); celery (3); escarole (3) ; onions, white (3) ; potatoes, white (3) ; potatoes, sweet (3) ; egg plant (2) ; lettuce (2).

Fruits: grapefruit (15); lemons (15); oranges (15); tangerines (15); strawberries (10); bananas (5); ' peaches, raw and canned (5) ; pineapple (5) ; apples (5) ; grapes (2).

Manville, McMinis, and Chuinard (1934) reported the vitamin C units oer ounce of the followine varieties - of apples: Yellow Newton (B), Delicious (3), Rome Beauty (3), Stayman (3), and Winesap (3). Mauville and Chuinard (1934) also reported the vitamin C content of a few varieties of pears (after a short storage period): D'Anjou (4), Winter Nelis (3), Bosc (2.5).

The figures in parentheses represent units of vitamin C per ounce, following the system of Rice and Munxll, and adopted by H. C. Sherman.

After prolonged storage the figures obtained were as follows: D'Anjou (2) and Winter Nelis (3).

Dairy Products: milk (raw). Meals: liver.

QUANTITATIVE CHEMICAL DETERMINATION OF ASCORBIC

ACID

At present the vitamins are assayed in terms of biologic units or in terms of chemical units (International Units). As ow knowledge relating to the chemical composition, isolation, synthesis, and. chemical identi- fication increases, the unit system is bound to give way to the method usually followed in quantitative pro- cedures, and the results of analysis will be given in absolute terms, in percentage, or in milligrams or grams per definite weight. A beginning in this direction has already been made with reference to ascorbic acid, which may be determined by a purely chemical procedure (reduction of 2,6-dichlorophenolindophenol).

Birch, Harris, and Ray (1933), using the chemical procedure mentioned, give the following figures for the ascorbic acid conteut of a variety of foods:

Prui1r Lemon juice orange juice Grapefrvit juice Pineapple jviee Banana Apples:

Bramley's seedlings cortex peel

Newton Winoor cortex pee1

Blenhein orange cortex peel : 7

Bdward VIl . . mrter peel

COX'S orant? Pippin cortex peel

Vcmloblelcr

Rhubarb potato

Suprarend cortex, or Live.. or Milk, mw'a -

Becker (1935) determined the ascorbic-acid conteut of vitapric, a jam-like substance produced from the juice of ripened paprika fruits, having a spicy and some- what sweetish taste and a deep red color, and used as a jam or a coloring agent in foods. It is extremely rich in ascorbic acid (0.45 per cent. or 4.5 mg. per gram.)

D. Gleria (1934) reported the following figures for milligrams of ascorbic acid per gram:

Vacloblcr which is markedly lowered in scurvy. Owing to lack Spinach Bow 1.16 ~~~~t~ flour 1.09 of cement substance the capillaries become very fragile. T O ~ ~ ~ O chew= 1.05 On the application of positive or negative pressure, Tomato juice 0.48 ~ d e d tomatoes 0.14 petechial hemorrhages make their appearance (Leede, Fresh spinach 0 08 Onion 0.04

1911; Hess, 1914; Stephan, 1900; ( h e l l , 1928; Gothliu, 1931; Dalldorf, 1931; Dalldorf and Russell,

Ghosh and Guha (1935) reported on the ascorbic 1935). acid content of Indian foodstuffs:

P d Mg. p ~ r z r o m CAEMICAL TESTS cunvn 1.04 M~~~~ (lsngra -ietg) 0.69 (1) Ascorbic acid in aqueous solution reacts with Lichi . 0.48 L~~~~ 0.19 benzoylhydraziue to form a compound which crystal- Orange 0.18 lizes in yellow clumps of needles melting. a t 192-194'C.

(zj L nm by the e the incis, anti-scorl stained i uuiforml! fied pred, blasts, aI C is com! thin, and is calcifil continuoc into the of scurv) observed the body. index ma animal t method 1 and by E

(3) Th' of the inc placed or food con1

The te - * The I

Organizati* logic unit 1

BIOLOGIC TEST*

(1) The test is carried out with guinea pigs that have developed the characteristic symptoms of scurvy on a basal diet lacking in vitamin C. The addition to the basal diet of a foodstuff or food concentrate containing this vitamin brings about a cure of the deficiency disease.

,-\ ?.., e test is carried out according to Hojer (1926) xamination of a cross-section of the roots of ors of the guinea pig. On a fully protected butic diet, a section of the root of the incisor vith hematoxylin and eosin shows a broad y stained pink layer of dentin, a white uncalci- entin, a layer of long, slender, parallel odonto- id a pulp of star-shaped cells. When vitamin pletely lacking in the diet, the dentin is rather the predentin is stained very dark because i t

:d. The odontoblasts no longer exist as a 1s layer, but seem to be on a disordered march hyperemic pulp. The changes characteristic r with reference to dental pathology may be in the teeth earlier than in any other part of

The histological test, using the incisor as the rterial, may be made 10 to 14 days after the as been placed on a scorbutic diet. The as been found satisfactory by Goetsch (1928) tddy (1929). e test consists in measuring the rate d growth ,isor teeth of the guinea pig when the animal is 1 a basal diet plus the particular foodstuff or :entrate that is being assayed for vitamin C.

CLINICAL TEST

st consists in measuring capillary resistance,

(R. ~ischer, 1935). - -

(2) Ascorbic acid in aqueous solution, when boiled with hydrochloric acid, decomposes with the evolution of carbon dioxide and with the formation of furfural. The furfural may be identified by the color reactions obtained with anilme acetate, phloroglucin, or orcinol.

(3) A slightly alkalime solution of ascorbic acid is treated with ferrous sulfate. In contact with air the reaction forms a beautiful, dark violet color (Szent- Gydrgi, 1934). The violet color is due to the formation of an oxide of the primary ferro-vitamin complex. It may be reduced to a leuko compound by a small amount of hydrosulfite, and the color may be restored by shaking with air.

(4) Ascorbic acid has powerful reducing properties. -It reduces in the cold sodium selenite in acid medium (Leviue and Rosenthal, 1934), selenious acid and gold chloride (Emmerie, 1934), methylene blue (Martini and Bonsignore, 1934), dichlorophenoliudophenol (TeU- mans, Hirsch, and Hirsch, 1932), phosphomolybdic acid complex (Bezssonoff, 1923). silicomolybdic acid (Levine, Smazal, and Booher, 1935), and ferricyanide (Tauber and Kleiner, 1935).

Quantitative methods for the determination of vitamin C have been devised on the basis of its ability to reduce methylene blue (Martini and Bonsignore, 1934), to reduce 2,6-dichlorophenoliudophenol (Till- mans, Hirsch, and Hirsch, 1932), and toieduce ferricyanide with the subsequent formation of Prussian blue and its colorimetric estimation (Tauber and Kleiner, 1935).

SPECTROSCOPIC TEST - Solutions of ascorbic acid in acetate butfer (pH 5) con- - - . ~

nternational Conference (League of Nations. Health taining 0.001 N KCN show a band a t 265 mp (Robert- m) ha adopted 0.05 mg. of L-ascorbic acid a the KO- Son, 1934). The acidity and the cyanide are required 'or vitamin C . to stabilize the ascorbic acid.

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The vitamins. II. The water-soluble vitamins