THE

VITAMIN

B

COMPLEX.

BY

H. M.

Sinclair, M.A., B.Sc., B.M., B.Ch.,

Department of Biochemistry, University of Oxford Fellow and Tutor, Magdalen College, Oxford.

;

Introduction.

Although scurvy, beriberi, rickets and pellagra have been known for centuries, the discovery of diseases due

vitamin

to

beginning made

of this

deficiencies

century.

dates

from the

only

It is true that Lind

admirable observations

(1753)

scurvy ; that Stark in 1769 twice induced the disease in himself by some

careful dietetic

died)

;

isolate

experiments (as

a

on

result of which he

and that Michael made excellent the

antiscorbutic

principle

attempts

early

in

to

the

Lunin

(1881) clearly eighteenth century. Further, of that small unknown substances quantities recognized essential to were life, and Pekelharing (1905) demonstrated the existence of diseases due to

dietary

first Hopkins (1912) realized the full significance of vitamins. Since then their number has increased with such rapidity that it is a year or two since the discovery of vitamin T was claimed ; vitamin B has been split into some dozen

deficiency.

173

Yet it

was

who

Dr. H. M. Sinclair

174

possible factors, and about two new papers on B and C are published each day of the year. Much of this work will not stand

vitamins

critical

analysis, hope to be fully conversant with the scientific work being done in this field. It is the object of this paper briefly to summarize recent work, done mainly upon animals, on the vitamin B complex, and as far as possible to indicate applications of this work. a

and it is obvious that the clinician cannot

The term used

for

"

water-soluble vitamin B

the

antineuritic

vitamin

"

was

originally by

discovered

the last twelve years it has been shown that this factor is multiple. Vitamin B2

Eijkman

in 1897.

distinguished pellagra and was

was

Vitamins all

During

in 1926

B3, B4, B5

water-soluble and

are

as a

factor which

prevented

heat-stable than vitamin Bj. and B6 were later recognized ;

more

present in fresh yeast, and all

necessary for the nutrition of pigeons or rats. Confusion then arose. Bats fed on a diet deficient in

are

vitamin B2 failed to grow and and this was believed to be When the fluorescent

developed a dermatitis, analogous to pellagra. compounds called flavins came

prominence in 1933, it was (riboflavin) could be isolated

into

vitamin

B2

and

would

adequately provided

found that

one

of these

preparations of promote growth in rats from

with vitamins

B1? B4 and B6.

But pure riboflavin was found to have no effect upon this dermatitis of rats or upon human pellagra ; it was obvious, therefore, that vitamin B2 must consist of more

than

one

factor which and

so

factor.

prevented

In 1935 it

was

rat dermatitis

shown that the was

vitamin

B6,

for the rat vitamin B, consisted of riboflavin

The Vitamin B Complex

175

and vitamin B6. Neither of these components, either alone or in combination, cured human pellagra, and "

"

therefore the original vitamin B2 (which, of course, had never been isolated pure) consisted of at least three factors

riboflavin, vitamin B6, and the P-P (pellagra-preventive) factor, now believed to be nicotinic acid or a derivative (see p. 185). These factors are all adsorbed on fuller's earth, but there is a fourth :

factor necessary for the which is not adsorbed "

filtrate

growth

(and called These

factor").

the vitamin B2 complex. Vitamins B3, B4 and

B5

four

are

of rats and chicks on

this account the

factors not

make

up

known to have

any clinical significance, and other alleged constituents of the vitamin B complex (such as factor W, choline, vitamin

H, and the

"

anti-gizzard-erosion

factor for

chicks" described by Bird et al. (1936)) need not detain us from the more detailed consideration of vitamin

Bx and the vitamin B2 complex. Vitamin

Bx.

Clinical Considerations. More than that beriberi

fifty was

years ago Baron Takaki deduced of dietary origin, and, by adding

vegetables to the diet of Japanese sailors and by partly replacing the customary rice by wheat and barley, greatly reduced the incidence of the disease in the Japanese navy. Despite the mass of work which has been published upon the clinical manifestations of deficiency of vitamin Bx, this is still a frequent cause of illness and death in such countries as China, Japan, Brazil, the Philippine more

meat and

Islands and

some

parts of the United States.

Gross

Dr. H. M. Sinclair

176

deficiency is undoubtedly and subclinical states

rare

in this

country, but mild

probably fairly common. In man symptoms of deficiency begin and progress somewhat as follows. First there is loss of appetite, with "

lack

heaviness

of the calf

are

and muscular weakness energy, of the legs ; this is followed by tenderness

of "

muscles, and

"

burning

"

of the soles of the

feet with loss of vibration-sense in the

parsesthesise in the legs become weakness increases and is intermittent

more

marked, the

perhaps accompanied by patella

and the Achilles and

claudication,

reflexes become weak and later absent.

spreads

The

legs.

to the extensors of the

The weakness

toes, then of the foot

and next to the extensors and flexors of the

leg.

Toe-

and

foot-drop are therefore found. At the same time there is hyperesthesia followed by anaesthesia advancing up the leg. Atrophy of the muscles and skin occurs. The upper extremity is usually affected later. The central nervous system is rarely involved, but the memory is usually defective. Some authors consider that both Korsakov's psychosis and the toxic amblyopia of alcohol- and tobacco-addicts are due to deficiency of vitamin So far

dyspnoea,

as "

cardiovascular

palpitations

"

symptoms

and

are

tachycardia

concerned, are

found.

Later the heart dilates.

normal and the

pulse

The arterial pressure is usually pressure slightly increased. The

circulation rate is increased because the small vessels

probable extreme.

are

cause

dilated

of the

Death

;

peripheral

and this dilatation is the

oedema, which may be mild

finally

occurs

autopsy very few pathological changes usually found except the dilatation of the heart. At

or

from heart failure. are

In

The Vitamin B Complex

chronic

there is marked

cases

myelin sheaths of peripheral in the

of the

periphery

degeneration this

nerves ;

long

177

nerves

of the

commences

and

gradually

extends towards the cell. Two

may

factors, acting either singly

be

responsible

vitaminosis

Bx

for

the

or

in

conjunction,

occurrence

of

hypo-

:

(i) Inadequate ingestion of the vitamin (ii) Failure to assimilate the vitamin.

;

Each of these will be considered in turn.

1.

Inadequate Ingestion.

Cowgill1 (1934)

has shown that the

the total metabolism variables

determining

Beriberi

is

because

their

females.

the

among young adult males metabolism is higher than that of

commonest

The breast-fed infant is next in order of

susceptibility. and among the

paratively

or

body weight and calories are two important requirement of vitamin

rare.

The incidence among women is elderly and the aged beriberi is Since the metabolism is raised

less, com-

during

pregnancy, and since it has been shown that lactating animals require three to five times more vitamin Bx than normal become more

ones, it is not

vitamin Bx

Strauss and McDonald

surprising that women susceptible during pregnancy and lactation. Several workers have suggested that the polyneuritis of pregnancy is due to deficiency of only

the

;

to beriberi

but

polyneuritis

gravidarum in doses of yeast.

of

also

the

not

hyperemesis by generous recently reported relief

their

many Authors have

(1933) cured

cases

Dr. H. M. Sinclair

178

of this condition

administration of vitamin

by

and have advised its pregnancy labour and

has

it

regular

been

even

afterwards for

Bx,

in pregnancy. In used for inducing

use

the

increasing

period

of

lactation!

factor, apart from pregnancy, which

other

Any

increases metabolism will increase the to

of vitamin

deficiency

reports of beriberi following prolonged

numerous

fevers,

susceptibility

The literature contains

Bx.

and this association has led

clinicians to

some

adopt the view that beriberi is an infection and not a dietary deficiency. Polyneuritis occasionally accompanies influenza, typhoid fever, leprosy, rabies, smallpox, and chronic pulmonary tuberculosis ; vitamin Bx is stated to be effective in post-influenzal neuritis and in the neuritis of leprosy. One might expect to find symptoms of deficiency occasionally in cases of hyperthyroidism (although such patients usually take a liberal

diet)

Means

has

such

;

cases

have been

recorded, and

Weiss's

that

recently quoted vitamin B deficiency patients with hyperthyroidism and cardiac symptoms." "

.

.

.

opinion explain why

some may dilatation cardiac have

Actual deficient intake in otherwise normal is

undoubtedly

rare

in this

Those

country.

people people

who, for financial reasons, are on low diets, have low caloric intakes, and therefore (as Cowgill has shown)

they

tend not to

reason

that

anorexia "

a

It is for that in

cases

of

nervosa.

Alcoholic

due to to

develop polyneuritis. polyneuritis is very rare "

polyneuritis

is

now

deficiency of vitamin Bx and "

neurotoxic"

effect

of

believed to be not

alcohol.

primarily Goodhart

179

The Vitamin B Complex and Jolliffe2

(1938) have finally proved this.

subjects of this form of polyneuritis take

a

The

high

caloric diet in which most of the energy is supplied by alcohol ; as no form of spirits contains any

B1} these individuals suffer

vitamin

a

deficient intake may be exacerbated

deficiency. This by the presence

of

achlorhydria or hypochlorhydria in about 80 per of patients with the disease (Minot) since hypoacidity is alleged to prevent the assimilation of cent,

the vitamin. Other forms of

polyneuritis

have been claimed to

be due to

deficiency of vitamin Bx. Vorhaus, Williams and Waterman (1935) treated one hundred unselected cases

of

and localized neuritis of diverse

multiple

aetiology with vitamin Bx ; it was claimed that only eight failed to show improvement, forty-four being cured and forty-eight improved. Toxic forms due to arsenic, lead, thallium, mercury, and nicotine have all been claimed to be

Bj. regard

to

improved by

claims

Such

treatment with vitamin

extravagant, especially in heavy metals such as lead, which produce a seem

polyneuritis clinically different from that caused by deficiency of vitamin B^ Good effects of vitamin therapy have been reported by several authors in various forms of neuritis (localized, acute, postdiphtheritic, etc.) ; improvement has been claimed in sciatica and claims

are

true

polyneuritis and

deficiency

and fanciful. of

nutritional and

of diabetes

treated

in disseminated sclerosis.

extravagant

said is that causes

even

below).

vitamin

All that

These can

be

Bx undoubtedly "

"

alcoholic polyneuritis, possibly polyneuritis of pregnancy (gastrogenous polyneuritis will be

180

Dr. H. M. Sinclair 2.

(ii)

Failure to assimilate vitamin B

This may be due to destruction in the

x.

(i) failure to absorb the vitamin gut, or possibly (iii) failure to

phosphorylate it. Many cases of deficiency of vitamin Bi have achlorhydria, although this is not caused by the vitamin deficiency. It has been suggested by many workers that cases of pernicious anaemia might be partially deficient, and that subacute combined degeneration is due to chronic deficiency of vitamin Analyses of blood which I have done during the last two years lend

no

to this view.

support

have found low values in

some

cases

hypochromic anaemia, which is, of caused

by

nutritional

deficiency

failure to absorb the iron

of

However, I of

idiopathic

course, sometimes

iron, sometimes by

achlorhydria. In idiopathic hypochromic anaemia we probably have the same two causes tending to produce low vitamin Bx in the blood

as

we

owing

have in the

to

case

of

"

alcoholic

"

polyneuritis, namely deficient intake and deficient absorption. But in the cases with anaemia, unlike the others, the intake is not markedly deficient and so the blood values do not fall so low. Possible deficiency of vitamin Bx, probably through failure of absorption, has been observed in other disorders of the

gastrocanal, such as after gastro-enterostomy, entero-enterostomy, gastrectomy, in ulcerative colitis intestinal

and in chronic

bacillary dysentery. ingest or absorb the vitamin, Apart deficiency is possible through its destruction in the gastro-intestinal canal. A number of cases of polyneuritis secondary to pyloric stenosis have been from failure to

The Vitamin B Complex

described,

181

Bx blood values in

and I have found low

three such cases.3 It seems probable that prolonged fermentation in the dilated stomach in an alkaline medium

destruction of the

causes

vitamin, particularly

since it is known to be unstable in presence of alkali. Indeed, it is possible that deficiency in the other cases of

mentioned

achlorhydria

actual

destruction

failure

to

however,

dary course

to

absorb

may be due vitamin rather than

the

should

It

it.

that

be

to to

mentioned,

case4 of

polyneuritis seconhad hyperchlorhydria. Of may be lost through vomiting, described the neuritis preceded

Ungley's pyloric stenosis

the vitamin

but in several the

of

above

cases

vomiting. Chemistry

and Mode

Action

of

Vitamin

of

Bx.

The chemical work done in various laboratories

led

finally

to the

synthesis

of vitamin

Bj by Williams

and Cline in contains

a

1936, and also by others. The molecule pyrimidine and a thiazole ring (Fig. 1). ch3 n

=

c

c-nh2.hci

II"/

h3c-c II N

c?ch2?n / \ II

CH

CI

=

och2.ch?oh

C ?S

H Fig. Fio. Vitamin B

x

1.

hydrochloride.

The way in which the vitamin acts in the body has been worked out mainly by Peters (see e.g. Peters,& 1936). Using pigeons and rats, he has shown that

when vitamin Bx is deficient, lactic acids accumulate in the

body,

because

and

they

are

pyruvic formed

Dr. H. M. Sinclair

182 from

carbohydrate

but cannot be further broken down.

Avitaminous brain tissue in vitro is unable to oxidize

pyruvic acid, added.

are

unaffected vitamin

but does

so

when minute amounts of Bx

Normal tissue oxidizes

by

additional

Bx.

pyruvic

It therefore

seems

is necessary for the oxidation of

Bx

acid in the

body

that

pyruvic

:

+0 Glucose

acid and is

^lactic acid

+0

^pyruvic

acid

^C02 +H20 t

vitamin Bi This work

on

lower animals is confirmed

study of patients with beriberi.

Japanese

showed that lactic acid accumulated in the this

disease, and

more

have shown that the

by

workers

body

Piatt and Lu6

recently

the in

(1937)

pyruvic acid. change particularly in the brain, and this goes far towards explaining the symptoms of beriberi. Pigeons which are moribund (blind and opisthotonic) from acute deficiency of vitamin Bx recover completely within half an hour of the injection of the pure vitamin. The change producing these symptoms must obviously be biochemical, for no structural lesions can be demonstrated by ordinary histological methods of staining. It seems that the biochemical change in the nerve cells is sufficient to explain the peripheral neuritis of beriberi, This biochemical

same

is true of

occurs

because if the metabolism of the the

will be affected

nerve

cell is disturbed

It is not distally surprising, therefore, that the early symptoms occur as a result of changes at the distal end of the long nerves. The fact that the respiration of the auricle is also neurone

at first.

183

The Vitamin B Complex

lowered in deficiency of vitamin Bx helps

to

explain

the cardiac manifestations.

readily available for therapeutic use, either orally or parenterally. It is certain that there is no danger of overdosage or cumulative toxic effects, since a therapeutic dose of 5 mg. intravenously is about 1/7,200th of the toxic Pure

vitamin

Bx is

now

dose. Intravenous administration of as much as 100 mg. is without untoward effects. It would advisable to administer the vitamin by injection if there is doubt about its efficacy by mouth ; for

seem

instance, Badger and Patrick found little effect on the neuritis of leprosy after oral administration, but good results after intramuscular

injection. Treatment should or intravenous injection

be started with intramuscular

of from 20 to 50 mg. daily. may be given orally, or the 10 mg. of

daily

until the

Later the

same

dose

injections decreased

patient

is

completely

to

relieved

symptoms. Vitamin B2 Complex. It has

been stated that vitamin B2 is now to consist of at least four factors : riboflavin,

known

already

vitamin B6, filtrate factor and nicotinic acid.

Riboflavin, which has been synthesized and has the formula shown in figure 2, is necessary for growth in various

animals

and

certain

disorders

in

alopecia,

cataract*

probably rats

and

(such even

It

prevents conjunctivitis, pediculosis), and

man. as

* It has recently been claimed that cataract is due to deficiency ?f vitamin B1? since in human lenses with cataract the amount of this

vitamin

is very low and the amount of

pyruvic

acid is much increased. 1ST

Vol. LV.

No. 209.

Dr. H. M. Sinclair

184

far, it has no human significance. It is present in yeast, milk and liver, and is destroyed by light; it will be further mentioned on dermatitis in

So

turkeys.

p. 187. H

CH2-(HCOH)3-CH2OH

CNN

H3C -C h3c-c

/ \ / \/ \ C

C

C

C

nh

\ / \ / \ C

N

C:0

/ C 0

H Fig. 2.

Riboflavin.

Vitamin B6 has recently been crystallized, but its formula is not yet known. In rats it prevents a "

"

symmetrical dermatitis which is called rat acrodynia because it is alleged to resemble pink disease in children. It may be mentioned that pink disease is There is believed by many to be a deficiency disease. as

yet little

it

seems

evidence in favour of this view, and probable that the primary cause

or no

rather

more

will prove to be a virus. This very interesting disease cannot be further discussed here ; it may be remarked,

however, that in the

aetiology, preparation. convenient 30 g.

easily

daily

our

present

state of

ignorance

it would be wise to administer Dried

source

is

a

powdered

brewers'

of the vitamin B

reasonable dose for

administered

stirred

into

milk,

about

yeast

yeast is

complex an

a

;

adult. or

in

a

about It is warm

water with salt.

Pellagra *

An American

in America bus.

is very

recently

rarely recognized

colleague saw a

in this

country,*

with considerable experience of pellagra with pellagrous dermatitis in a London

tramp

185

The Vitamin B Complex still

although

common

in

The

parts of America.

some

symptoms may be summarized as weakness and lassitude, anorexia, diarrhoea, sore ulcerated mouth and ulcerated atrophic tongue of a fiery red, mental and

changes

consist of

a

seborrhoea over

lesions

dermatitis

about

the

on

the

genitalia,

the face and

neck, and hyperkeratoses bony prominences (particularly the elbow). The on

of the

discovery of the P-P factor summarized. Warburg and von Euler

story be

of

surfaces,

exposed

characteristic

very

These latter

cutaneous lesions.

typical

nicotinic

acid

amide

is

important

may briefly showed that

the

active group in two led to its use in other This

coenzymes. and late last year Elvehjem and others showed fields, that it cured " black-tongue "?a disease of dogs to

analogous

immediately in

cases

of

pellagra. Several groups of workers tried it and certain related compounds and there

pellagra,

the disease

seems no

doubt that

completely cured by the oral administration of nicotinic acid (Fig. 3) or its amide ; coramine, which is a related compound, is also effective (Spies et al., 1938).7 It is interesting to note that the peripheral neuritis of pellagra is not relieved by nicotinic acid, but is cured by vitamin Pellagra is occasionally seen in this country accompanying ulcerative colitis, and recently nicotinic acid

has

acid

is

Spies

is

be

can

The

been tried in this latter condition.

cheap,

and

total

of

the

dose

recommended

500

by by

mg. a day, given The dose is carefully mg. stated because it must be emphasized that, unlike a

mouth in doses of 50

vitamin

large

B1?

nicotinic

amounts

acid

producing

is

definitely

flushing,

burning

toxic, and

Dr. H. M. Sinclair

186

itching

skin, and increased motility of the

of the

stomach. COOH N Fig. 3. Nicotinic acid.

Enzymes, Coenzymes and Phosphorylation. There is

striking

a

certain

connection between

members of the vitamin B

complex (and

one

or

two

An vitamins) coenzymes enzymes. enzyme is an organic catalyst which is a protein ; a other

and

coenzyme is

a

or

non-protein organic compound

which is

fairly specific and associated with an enzyme in nature. Enzymes are believed to consist of an active (" prosthetic ") group, which is often unstable, joined to or part of a protein. If the prosthetic group is stable and becomes detached from its carrier, it may easily be mistaken for a coenzyme. It is probable that

nothing

than the active

part composition of some coenzymes is known ; two which are important in cell respiration and fermentation contain nicotinic acid amide (see p. 185) combined through phosphate molecules with adenosine (a nucleoside). It has recently been shown cocarboxylase by Lohmann that the coenzyme the of breakdown (which catalyses pyruvic acid) is nothing else than vitamin Bx combined with two

some

coenzymes of the enzymes.

are

more

The

"

phosphate

molecules.

riboflavin acts in the and

a

factor which

"

Further, it is believed that

body was

combined with shown

phosphate ; by Warburg to be

The Vitamin B Complex

187

important in cell respiration (and is sometimes called the "yellow enzyme") consists of riboflavin joined through phosphate to protein. Hence we have at least three members of the vitamin B complex (Bx, riboflavin and

body

with

nicotinic acid

phosphate

amide) combined in the

to form coenzymes

or

(if protein

be

attached) enzymes which are important in cell respiration or metabolism. By analogy with riboflavin it is tempting to believe that vitamin Bx diphosphate is not

merely the coenzyme which is necessary for the degradation of pyruvic acid, but is the active part of the enzyme which catalyses this degradation. In support of this hypothesis, I have recently found that neither vitamin Bi nor the diphosphate exists free in blood, but that the vitamin is combined probably with

protein.

Verzar8 has stressed the

tion in the

importance

He has shown that

of

phosphorylafatty acids and

body. carbohydrates are absorbed from the intestine an increased rate by being combined with phosphate

certain at

in the cells of the intestinal to

For this process riboflavin phosphate is

mucosa.

the

presence of necessary, and for riboflavin to be phosphorylated the hormone of the adrenal cortex is necessary. He occur

maintains that adrenalectomized animals show

decrease in the

"

"

are not

tion

absorbed from the intestine unless

can

occur, he

suggests

that

large

in their livers, and fed with riboflavin

yellow enzyme be caused to grow by being phosphate. Further, since he believes that

can

a

cases

fatty acids

phosphorylaof

idiopathic

sprue and coeliac disease should be treated with the adrenal cortical hormone or with riboflavin phosphate. This work, however, is still in

steatorrhea,

Dr. H. M. Sinclair

188 the

possibility should be borne in mind that symptoms of deficiency of vitamin Bx (or of other factors) may arise, not through actual deficiency of the vitamin, but through failure of it to be phosphorylated (and possibly combined with protein). Vitamin Bx is phosphorylated in the liver (Ochoa and Peters) and possibly also in the intestine (Tauber). Apparent B1 deficiency occurs occasionally in

But the

experimental stage.

of coeliac disease and sprue, and has been

cases

described in liver disease.

Anaemia and the vitamin B

complex.

A relation between the vitamin B various forms of anaemia has been

complex and repeatedly claimed

since

(1932) stated that

"

1929.

vitamin

Castle and Strauss "

closely related substance was the extrinsic factor in pernicious ansemia, but this was later discarded. More recently it has been shown that pigeons on a synthetic diet develop an ansemia which is cured by yeast but not by vitamins Bx or B6 or B2

riboflavin.

Clutterbuck,

consider

that

complex

cures

been

or a

stated

a

Evans

and

factor related to

nutritional ansemia in that

the

filtrate

Wills

(1937) the vitamin B2

monkeys

factor

is

;

it has

concerned.

Gyorgy has produced aplastic ansemia with symptoms of hsemorrhagic diathesis in rats by dietary means, and cured it with nicotinic acid. Others have produced a severe microcytic hypochromic ansemia in puppies by a

diet deficient in vitamin

difficult to correlate at

possible

These results are B6. present, but they indicate a

relation between

vitamin B

complex

a

factor

or

factors in the

and normal blood formation.

189

The Vitamin B Complex

A short time ago vitamins were thought to have a narrow clinical application, being useful only in such

classical

diseases

pellagra.

swung too far in the direction and there is a danger of diseases

opposite

Now the

not of nutritional

preparations there

are

neuritis

rickets, scurvy, beriberi and

as

pendulum has origin being

to the exclusion of

treated with vitamin

appropriate therapy

;

syphilis, diabetic being diagnosed as

instances of cardiovascular and

deficiency

other

conditions

Bx and treated with the pure

of vitamin

vitamin. The rapid advance of laboratory work in this field makes the careful clinical investigation of each

case

ignorance ease

even

important and emphasizes

more

of nutritional diseases in

The relative

man.

with which several of such diseases

our

can

be treated

with pure preparations is unfortunately apt to overshadow the important problem of preventing nutritional

disorders

that everyone

by ensuring

can

consume

a

well-balanced diet. Summary. The various factors which compose the vitamin B complex are mentioned. 1.

2.

The clinical

aspects of deficiency of vitamin Bx,

and factors which may cause that deficiency, are described. The vitamin has been synthetized, and it is concerned in the

body

with the oxidation of

pyruvic

acid. The four known factors which constitute the vitamin B2 complex (riboflavin, vitamin B6, filtrate 3.

factor and nicotinic acid) are briefly described. The symptoms and nature of pellagra are mentioned.

The Vitamin B Complex

190 4.

There is

a

connection between certain

striking

members of the vitamin B

complex and coenzymes or importance of phosphorylation in the

enzymes. The body is stressed. 5.

A relation between

complex

deficiency

of the vitamin B

and anaemia is indicated. REFERENCES.

1

Cowgill,

2

Goodhart, R., and Jolliffe, N., Jour. Amer. Med. Assoc., 1938,

3

Laurent, L. P. E., and Sinclair, H. M., Lancet, 1938, i. 1045.

4

Ungley,

5

Petters, R. A., Idem., 1936, i. 1161.

6

Piatt, B. S., and Lu, G. D., Quart. Jour. Med., 1936,

G. R., The Vitamin B

Requirement of

Man.

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