Vitamin в6: Pyridoxine, Pyridoxal and Pyridoxamine T . U R B A Ń S K I , D .Sc .

Department of Chemistry, Institute of Technology, Warsaw-

V I T A M I N B 6 , also known as pyridoxine, or adermin, is a derivative of pyridine and in that respect is similar to nicotinic acid and nicotinamide, which also belong to the complex mixture originally called vitamin в.

According to our present knowledge, vitamin в 6 is a group of three substances : an alcohol, pyridoxine, an aldehyde, pyridoxal and an amine, pyridoxamine. A l l three are derivatives of /У-hydroxypyridinc, and thus there is a profound similarity between the vitamin в 0 group and nicotinic acid and its derivatives : in all of them the /^-position in the pyridine ring is substituted by a group strongly influencing the character of the molecule—the phenolic group in vitamin в в and the carboxylic group in nicotinic acid.

The principal component of vitamin B C , pyridoxine, has the structure a-lnethyl-3-hydrOxy-4.5-di(hydroxymethyl) pyridine, / .

H O

C H ,

C H . O H

• \ C H . O H

1 4

This was elucidated in 1939 by experiments described below, but the first mention of the r e l a t i o n s h i p b e t w e e n /)'-hydroxypyridinc and vitamin в was given by R . R . WILLIAMS 1

in 1921 i.e. long before vita­min B E was isolated.

A few years later J . GOLDBEROER and R . D . L i L L i E - found certain types of dermatitis in rats fed on diet free from vitamin в , (lactoflavin). The dermatitis was produced by an unknown factor present in vitamin в 2 , since the rats d id not recover on administration of pure vitamin в г .

Then S. J . О Н П Л К Е 3 isolated a new crystalline substance from rice bran. H e found the cmpyrical formula of the hydrochloride of this product to be C e H j , 0 , N . H C l . It was certainly pyridoxine, although Ohdakc d id not recognize the vitamin character of the substance.

The name ' vitamin в 0 ' appeared in 1934, bestowed by P. G Y O R G Y 1 who isolated this substance from yeast, rice bran and liver. H e found it to bc an antipellagra factor, which can accompany vitamin в 2 in yeast, liver and other ' vitamin в ' sources. The same author, together with T . W . BlRCH 5 , found vitamin B 0 to bc a nitrogen base which forms a hydrochloride.

In a series of papers 6 - 1 0 published in 1938 improved methods of isolating vitamin в 6 were described. Further experiments showed that vitamin B, is present in animal or vegetable tissue in the form of a complex with proteins or carbo­hydrates which is subject to cleavage by the action of enzymes or heat. The richest sources of vitamin в 6

are yeast and rice bran. Various seeds including corn also contain a considerable quantity, while lesser amounts occur in molasses and liver. M i l k , egg yolk, spinach and green vegetables are poor sources of vitamin в с .

C H E M I C A L P R O P E R T I E S A N D S T R U C T U R E OF P Y R I D O X I N E

The free base pyridoxine has the empirical formula C 8 H u 0 3 N . It is a white crystalline substance with m. pt i6o°C. It is soluble in water and most organic solvents, and forms several salts e.g. the hydrochloride (m. pt 204~2о6°С with decomposition). R . K U H N and his co-workers1 1 determined its structure in an extremely skilful way within a very short time. The main facts which led to their conclusions were :

/ The substance contains three hydroxyl groups, but only one of them can be methylated with diazomethane ; thus the substance has one phenolic and two alcoholic groups.

s If pyridoxine methylated with diazomethane was subjected to heating with lead tetracetate, no oxidation occurred. This indicates that the alcoholic groups are not attached to the neighbouring carbon atoms since lead tetracetate is a reagent which oxidizes oc-glycols.

3 Pyridoxine methylated with diazomethane can be oxidized with cold potassium permanganate. One of the alcoholic groups is oxidized to a carboxylic group which readily forms a lactone with the second alcoholic group. As only y'- and sometimes (5-lactones can be formed in this way, the alcoholic groups can only occupy the 1.4 or 1.5 positions.

4 The ultraviolet absorption spectrum shows a great similarity between pyridoxine and /}-hydroxy-pyridine. Both pyridoxine and /5-hydroxypyridine give a cherry red colour with ferric chloride, which indicates the phenolic character of their hydroxyl groups : a- and y-hydroxypyridine are not phenolic in character and do not become coloured with ferric chloride.

Research 2 - 1 1 т. U R B A Ń S K I : Vitamin в6 : Pyridoxine, Pyridoxal and Pyridoxamine

5 If pyridoxine methylated with diazomethane is oxidized with hot potassium permanganate in alkaline medium a tribasic acid is formed which gives a brown colour with ferrous sulphate. This colour reaction is characteristic of pyridinic acids, which contain a carboxylic group in a-position. O n heating the acid looses a molecule of water and a molecule of carbon dioxide, forming the anhydride of a dibasic acid. The acid produced from this anhydride does not give the colour reaction with ferrous sulphate.

These facts, together with the analytical data, brought K u h n to the following conclusions :

a the three carboxyl groups are formed by the oxidation of two primary alcoholic and one methyl group ;

b the carboxyl group, which is eliminated as C 0 2

on heating, occupies the a-position ;

с the two remaining carboxyl groups are attached to the neighbouring carbons. O n this basis K u h n gives the structure of the anhydride / / and three possible structures of pyridoxine, la, lb and Ic :

HO,

CHJ

HO,

C H . . O H / N \ C H , O H

NT (la)

C H . O H CH.OH

со—о CHJOJ^NCO

4 N X

(Щ C H 3

но , / \ сн .он t N J c H 2 O H

(Ic)

/

(lb)

6 K u h n and co-workers synthesized the anhydride which possesses the structure / / . It proved to be identical with that obtained from the methyl ether of pyridoxine.

7 Careful oxidation of the methyl ether of. pyridoxine with barium permanganate led to a dibasic acid with an untouched methyl group. This acid does not give any colour with ferrous sulphate. It has therefore two possible structures, Ilia and 111b :•

C O O H

C H : , 0 C O O H

C H ! N J (Ilia)

сн,о C O O H

C O O H I I 4 N (Hlb)

C H ,

Consequently the structure Ic for pyridoxine should be rejected.

8 K u h n and his co-workers synthesized a dibasic acid Ilia. It proved to be identical with the acid formed from the methyl ether of pyridoxine, so formula lb was therefore rejected.

Independently the structure of pyridoxine was also established by a team of workers in the U . S . A .

Thus the acid Ilia was obtained1- by oxidizing the methyl ether of pyridoxine IV. This acid was subsequently synthesized1 3 by a different method. Both syntheses of the acid Ilia were important in providing a basis for the preparation of pyridoxine, which both groups of investigators achieved shortly afterwards.

S Y N T H E S I S O F P Y R I D O X I N E

R . K u h n and his co-workers1 4 synthesized pyridoxine by starting from the acid Ilia and transforming thé' carboxyl groups according to the scheme :

- C O O H -* - C O N H . -+ - C N -* - C H . N H , -* - C H . O H .

Thus the acid Ilia gave the methyl ether of

pyridoxine IV :

C O O H C H . O H

C H . O

C H I ix N (Ilia)

C O O H сн3о,х ^ C K O H

C H I I (IV)

which was demcthylated to give the hydrobromidc : CH.Br C H 2 O H

HBr H o / ^ C H . B r H,о Н О / ^ С К О Н (IV)

(66%) C H , .+! N

H В Г (V)

C H 1 +1

/

H В l '­

A n identical synthesis was also described by A . ICHIBA and K . M i e n i 1 5 .

The method of synthesizing pyridoxine developed by S . Л . HA R R I S and K . F O L K E R S 1 0 gave the best practical results from the viewpoints of yield and cost of production. They started from ethoxy-acetylacetonc VI and cyanacetamide VII which with piperidine in alcoholic solution underwent ring closure and formed a pyridine derivative VIII :

C H ,

CH.,

- co

C H . O C . H ; I

C O

+ C H 2 — C N piperidive

yield 8,".

(VI)

/ N H ,

(VII)

c = o CH.,

C H . O C H j

C N

= 0 N H

(VIII)

5 0 8

т. U R B A Ń S K I : Vi tamin в в : Pyridoxine, Pyridoxal and Pyridoxamine Research 2 - 1 1

C H . , O C , H 5

J С Н . О С Н - ,

(VIII) NO.,, v C N

H « ( P T >. N H ,

yield 32%

C N

C O O C H 3

H O / N C O O C H ,

C O O C H 3

HOf ^ N c o o c H ,

C H J N )=оуШвГ5"'°сн1 )=o N H N H

CH

(IX) (A') I

C H 2 C , H 5

[XVII)

CH. N

(XVIII) Substance VIII was nitrated to IX and then reduced by platinum black to the amine X. Substance X, which is an a-pyridone derivative, can be transformed by phosphorus pcntachloridc and phosphorus oxy-chloridc into an oc-chloropyridinc derivative, because a-pyridone reacts tautomerically as a-hydroxy-pyridine :

{ J=o < ' V ' O H

N H N

a-pyridone a-hydroxypyridine

Thus product X can be transformed into A ' / :

С Н 2 О С , Н 5 C H 2 O C 2 H ,

PCI. + POCI, NHj/NcN H,(pt-Pd) N H / N C H . N H , ,

yieldi6-5% C H , I N N J C I yUUjn-5% C H , I N K

The product XVIII can be converted into pyridoxine in the conventional way by stepwise transformation of - C O O C H 3 into - C H j O H . This synthesis suggested the possible functioning of a-alanine as a natural precursor of pyridoxine and it is interesting to note that E . E . S N E L L 1 9 found alanine capable of replacing vitamin в 6 for the growth of Lactobacillus casei and Streptococcus faecalis R .

P Y R I D O X A L A N D P Y R I D O X A M I N E ( P S E U D O P Y R I D O X I N E )

It was found in 1938-39 that vitamin в с is a growth-promoting agent for yeast and bacteria. A biological method for the assay of pyridoxine was then

C H 2 O H СН..ОН HCI â N H / ^ C H j N H j C I HOMO H O ^ ^ C H . O H

(XI) (XII)

yield7(sy)>„ C H J 4 | 4 J

(XIII)

yield 45-4°,'oCV\

(la)

This was reduced by hydrogen on platinum and palladium with the simultaneous removal of chlorine, giving the amine XII. Hydrolysis with hydrochloric acid at i 7 5 - i 8 o ° C yielded the aminoalcohol XIII which on diazotization finally gave pyridoxine. A n almost identical method was adopted by Merck A . G . in Germany for the commercial synthesis of pyridoxine 1 7 .

Recently A . C O H E N 1 8 proposed a new method of preparing pyridoxine by starting from a-alanine derivatives : N-benzyl-a-alaninc ester XIV and a-hydroxymethylenesuccinic ester XV were cyclized to a 3-kcto-i .2.3.4-tetrahydropyridine derivative XVI :

СООС..Н,

I СН3--СН + \

N H

СООСН3

сн2 • \

С.СООСН.,

0 =

CH.

C O O C H 3

соосн.,

Ć H , C , H , H O Ć H (XIV) (XV)

I C H X t H 5

(XVI)

The product was dehydrogenated to XVII and after hydrogenolysis of the N-benzyl group the substance XVIII was obtained.

developed2 0. Experiments on the growth of bacteria on a medium containing pyridoxine led to some important conclusions. E . E . S N E L L , В. M . GUIRARD and R . J . W I L L I A M S 2 1 found that Strep lactis R would grow on such a medium many times faster than could be accounted for on the basis of actual pyridoxine content. The explanation is that pyridoxine was converted into a more highly active metabolite prior to utilization by the bacterial organism. This hypothetical metabolite was called pseudopyridoxine.

In his later experiments E . E . S N E L L 2 2 found that media containing pyridoxine could be activated by simple heating in an autoclave. He advanced a suggestion that pseudopyridoxine was formed by the interaction of pyridoxine and aminoacids present in the mixtures. He developed2 3 his experiments further and heated pyridoxine a with ammonia in an autoclave b with manganese dioxide.

In both instances he obtained products much more active than pyridoxine. In the experiments a they were 140 times more active than pyridoxine, and in b 50 times. He therefore suggested that animation and partial oxidation are possible reactions which can transform pyridoxine into pseudo­pyridoxine. Pseudopyridoxine is therefore a mixture

5 0 9

Research 2 - 1 1 т . U R B A Ń S K I : V i t a m i n в 6 : Pyr idox ine , P y r i d o x a l and P y r i d o x a m i n e

of two substances, the product of oxidation, which Snell called pyridoxal, and the product of amination, which he called pyridoxamine. The following reaction scheme represents this transformation :

[O] R . C H 2 O H

Ptridoxine

NH: i

R . C H O <lt R . C H Pyridoxal

[H] 5t R . C H . N H . , N H

Pyridoxamine

Snell's metabolic experiments with rats and human beings also led to the conclusion that pyridoxine is partially converted by animal organisms into pseudopyridoxinc.

C H . N H ,

н о / Ч с н . . о н

C H N ' (XIX)

Independently R . K U H N ! 1 found that patients cured with pyridoxine excreted in their urine a new substance much more potent than pyridoxine, which possesses the structure XIX.

No details were given of how he arrived at this conclusion.

The structures of pyridoxal and pyridoxamine were established by S. A . HARRIS , D . H E Y L and K . F O L K E R S 2 5 by synthesizing these two products and their isomers. They started from substance XX,

of the alcohol Xlla. homologous with substance XII, the ethyl ether of the same alcohol. Diazotization of XX gave the product XXI. O n action of thionyl chloride the alcoholic group was replaced by chlorine

XXII which was then substituted by the amino group XXIII ; finally the ether group was hydrolyscd with hydrochloric acid at i70- i8o ° C and product XXIV was obtained. There was no doubt about the structure of this compound, which on bio-assay proved to be non-identical with pyridoxamine and did not possess its activity.

the methyl ether

Ć H , O H N H 2 / \ c H , N H . .

C H : i 4 N J [Xlla)

The product possessed the properties of pyridoxamine, as determined from its action on the growth of Lact casei and Strep lactis R .

Harris, Heyl and Folkers also prepared pyridoxal XXVaXià transformed it into pyridoxamine, providing evidence that the same 4-hydroxymcthyl group was transformed into an aldehyde and methylamino group respectively. Pyridoxal was prepared by a careful oxidation of pyridoxine in an alkaline medium :

C H , O H

H O / N C H . O H

CH : L

H O KMnO,

~ ^ C H ,

C H O C H . . O H NH..OH

N (XXV)

C H = N O H

H O / N C H . , O H

с н з ч J (XXVI)

C H . N H .

[H] Н О / >СН,ОН

C H I I T N N (XIX)

The aldehyde XXV was transformed into the oxime and this was catalytically reduced to pyridoxamine.

H O

C H ,

СН..ОН , C H O

4 N {XXVII)

Further, E .

Final confirmation of the struc­ture of pyridoxal was obtained when the same authors synthesized the isomeric aldehyde XXVII, which does not possess the growth promoting activity of pyridoxal.

E . S N E L L 2 0 found that all three members of the vitamin в 0 group occur in natural tissues. In various papers E . E . SNELL and his co-worker2 7

showed that the three compounds pyridoxine, pyridoxal and pyridoxamine each behave very differently towards different organisms.

Thus they found pyridoxal has 5,000-8,000 times and pyridoxamine 6,000-9,000 times the activity of

N H 2

C H

C H , O C H ; 1

C H . . N H ,

(XX)

С Н . О С К ,

н о / ^ с н - р н

СНз\|ч| J (XXI)

SOCI H O

C H ,

C H 2 O C H : l

СН..С1 H O

СН..ОСН,

'NcH-.NH,

N '

(XXII)

C H J V N

СН..ОН

H O ^ C H . N H ,

(+ 2НС1) {170-180")

C H

(XXIII i (XXIV)

Acting on substance XXI with ammonia at 140°C in methyl alcohol, they obtained the substance XIX identical with pyridoxamine :

C H , O C H ; 1

H Ó / \ C H , O H

C H N (XXI)

H O

C H ,

C H . N H , / X j C H . O H

(XIX)

pyridoxine as a growth factor for Strep lactis R . For other organisms, such as Lact casei, pyridoxal was also found to be highly active, but pyridoxine and pyridoxamine almost inactive. O n the contrary, for Saccharomyces carlsbcrgensis all the compounds have approximately equal activities. It is assumed that the variation in activity is due to differences in the ease with which the various organisms convert pyridoxine into pyridoxamine and pyridoxal.

т. U R B A Ń S K I : Vi tamin в с : Pyridoxine, Pyridoxal and Pyridoxamine Research 2 — 11

The original presence or formation of the members of vitamin в„ groups renders microbiological assay for vitamin B g invalid if the growth of unsuitable organisms is examined. For that reason it is preferable to use organisms which respond equally to all three members of the group e.g. Sacch carlsbergcnsis and Neurosppra sitophila.

P H Y S I O L O G I C A L A C T I O N OF V I T A M I N B 6

None of the members of the vitamin B E group produces a definite pharmacological action and this is the main reason why the part played by this vitamin was obscure. The earliest experiments showed a connection between vitamin в 6 and certain skin disorders, so typical for most в group vitamins. This was confirmed by later experiments. A special type of dermatitis, acrodinia, occurring on peripheral parts of the bodies of animals is due to lack of vitamin в 6 . It was also suggested that vitamin в с

is an agent in the metabolism of unsaturated fatty acids2". Experiments with rats gave evidence of the importance of pyridoxine in the process of transformation of proteins into fats2 9.

More recently a relation between the vitamin в 0

deficiency and anaemia has been clearly shown by several authors. S. L E P K O V S K Y 3 0 found that anaemia produced by the lack of pyridoxine does not respond to iron and copper, but prompt improvement follows administration of pyridoxine. M . M . WINTROBF. et alii31 showed that pigs fed on a diet supplemented with vitamins A and D and all known vitamins в except B E develop a severe microcytic anaemia frequently accompanied by epileptic con­vulsions. Administration of pyridoxine produced rapid regeneration of blood.

It is known that kynurenine, an amino acid, and xanthurenic acid, a quinoline derivative, can be excreted by animals suffering from nutritional disorders. It has also been shown that both sub­stances are products of incomplete metabolism of tryptophane. S. LEPKOVSKY et alii3- established the exact dietary deficiency necessary to produce the excretion of xanfhurenic acid. They found that

pyridoxine deficient rats excreted in their urine a complex of xanthurenic acid and iron. This excretion of iron might be one of the factors causing anaemia. O n the basis of this and some later work the role of pyridoxine was established as a factor producing metabolism of tryptophane and par­ticularly of kynurenine and xanthurenic acid. This property of pyridoxine was later elucidated in the course of work on the coenzyme action of phos-phorylated pyridoxal quoted 4 0 below.

The discovery that vitamin B g is closely related to certain enzymes was the most important part of the research on its biological action. The first observation was related to the role of vitamin в 6

in the transformation of certain amino acids to amines i.e. of their decarboxylation by decarboxylase. Thus W . D . B E L L A M Y and I. C . G U N S A L U S 3 3 found that addition of pyridoxine and nicotinic acid or pyridoxal stimulated production of tyrosine decar­boxylase. Then J . BADDILEY and E . F. G A L E 3 4

and, almost simultaneously, W . W . UMBREIT and I. C . G U N S A L U S 3 5 discovered that phosphorylated pyridoxal can act as co-decarboxylase. In addition, others3 0 prepared phosphorylated pyridoxal with 40 to 50 per cent coenzyme activity from pyridoxal and phosphorus oxychloride. The exact position of the phosphoric acid group in this coenzyme is not yet known.

A further discovery was the function of pyridoxine derivatives as agents playing important parts in biological transaminations. Previously E . E . S N E L L 3 7

had suggested that the possible conversion of pyridoxal into pyridoxamine may be explained by a trans­amination reaction. In a number of papers he showed that a relation existed between members of vitamin B E group and the process of transamination. Then H . C . LICHSTEIN, I. C . GUNSALUS and W . W . U M B R E I T 3 3 found that pyridoxal phosphate functioned as the coenzyme of the transaminase of certain acids.

The process of transamination shown below was suggested by F. SCHLENK and A . F I S C H E R 3 9 .

Another function of pyridoxal phosphate was found by W . W . UMBREIT, W . A . WOOD and I. C . GUNSALUS 4 0 . They proved it to be the coenzyme

C O + N H , I I

C O O H C H , J

H O |

C H 1 V N '

C H . O H

protein

fyndoxamine-protein complex

C= 1

N

H O O C C H . , !

H O ,

R I

C H — N

II H O O C C H

H O O C

C H — N H . , -|- C H O I

C H . . O H НО? X | C H , O H H<Y ( C H . O H

r u \protein •'^ N ^

pyridoxal-protein complex

Research 2 - 11 j . п. B U R G O Y N E : Inflammability of Gases

for tryptophanase which catalyses the breakdown of tryptophane to indole, pyruvic acid and ammonia.

The complex behaviour of the vitamin в 6 group gives no clear indication of its use in therapy. They may be useful remedies against some types of anaemia and some skin disorders, including such symptoms of pellagra which arc not relieved by the usual treatment with vitamin в , , в 2 and nicotinic acid. It is certain that pyridoxine is an invaluable remedy against certain nervous disorders accom­panied by insomnia and irritability, and also against vomiting in pregnancy. It is of value in treating muscular dystrophy, paralysis agitans and chorea.

R E F E R E N C E S 1 WILLIAMS, R. R. Ind. Eng. Chem. 13 (1921) 1107 2 GOLDBERGER, J . and LiLLiE, R. D. U.S. Publ. Health

Service 41 (1926) 1025 3 OHDAKE, S. J . Bull. agr. chem. Soc. Japan 8 (1932) 111 4 GYÔRGY, P. Nature, Land. 133 (1934) 498

— Biochem. J. 29 (1935) 74 1 . 76°> 7»7 5 BIRCH, T . W . and GYÓRGY, P. ibid 30 (1936) 304 6 K U H N , R. and WENDT, G . Ber. 71 (1938) 780, 1118 7 LEPKOVSKY, S. Science 87(1938) 169 8 KERESZTESY, J . C . and STEVENS, J . R. Proc. Soc. exper.

biol. Med. 3 8 ( 1 9 3 8 ) 6 4 * ICHIBA, Л. and MICHI, K . Inst. phys. chem. Res., Tokyo

34 (1938) 623 1 0 GYORGY, P. J. Amer. chem. Soc. 60 (1938) 983 " K U H N , R. and WENDT, G . Ber. 71 (1938) 780,

1118, 1534 — — ibid 72 (1939) 305 — ANDERSAG, H., WESTPHAL, K . and WENDT, G .

ibid 72 (1939) 309 — WENDT,, G . and WESTPHAL, К . ibid 72

(> 939) 310 1 1 STILLER, E. J., KERESZTESY, J . C. and STEVENS, J . R.

J. Amer. chem. Soc. 61 (1939) 1237 1 : 1 HARRIS, S. A . , STILLER, E. J . and FOLKERS, K . ibid Gi

(1939) 1242 1 1 K U H N , R., WESTPHAL, К., WENDT, G . and WESTPHAL,О.

.Xaturwiss. 27 (1939) 469 1 0 ICHIBA, A . and Mieni, K . Inst. phys. chem. Res., Tokyo

35 (1938) 73 — — ibid 36 (1939) i , 173

L C HARRIS, S. A . and FOLKERS, K . J. Amer. chem. Soc. 61

(>939) 1245. ЗЗ07 1 7 B.I.O.S. Report Mo. 766, 159 1 8 COHEN, A . Xlth intern. Congr. Chem. London, 1947

Abstr. 247/3 1 0 SNELL, E . E. J. biol. Chem. 158 (1945) 497 2 0 ATKIN, L . , SCHULTZ, A . S. and FREY, G . N . J. Amer,

chem. Soc. 61 (1939) 1931 — — — and WILLIAMS, W. L. Ind. Eng. Chem.

Anal. 15 (1943) 141 2 1 SNELL, E. E. , GUIRARD, B. M . and WILLIAMS, R . J . J .

biol. Chem. 143 (1942) 519 2 2 — Proc. Soc. exper. biol. Med. 51 (1942) 356 2 3 — J. Amer. chem. Soc. 66 (1944) 2082 2 ł C.I.O.S. Report XXIV—13 2 6 HARRIS, S. A . , H E Y L , D. and FOLKERS, K . J. Amer,

chem. Soc. 66 (1944) 2088 2 0 SNELL, E . E.- J. biol. Chem. 157 (1945) 491 2 7 e.g. — and RANNEFELD, A . N . ibid 157(1945)475 2 8 BIRCH, T . W. ibid 124 (1938) 775 2 9 MCHENRY , E . W. and GAVIN, G . ibid 138(1941)471 3 0 LEPKOVSKY, S. ibid 149 (194З) ' 9 5

— ibid 155 (1944) 299 3 1 WiNTROBE, M . M . , SAMTER, M . and Lisco, H . Bull.

John, Hopkins Hosp. 64 (1939) 399 — FOLLIS, R. H., MILLER, M . H., STEIN, H . J.,

ALEAYAGO, R., HUMPHREYS, S., SUKSTA, A . and CART-WRIGHT, G . E . ibid 72 (1943) i CARTWRIGHT, G . E. , WINTROBE, M . M . and HUM­PHREYS, S. J. biol. Chem. 153 (1944) 171

3 2 LEPKOVSKY, S., ROBOZ, E . and HAAGEN-SMIT, A . J . ibid 149 (194З) "95

REID, D. F., LEPKOVSKY, S., BONNER, D. and TATUM, E. L . ibid 155 (1944) 2 9 9

3 3 BELLAMY, W. D. and GUNSALUS, I. C. J. Bad. 48 (1944) 191

— — J . biol. Chem. 155 (1944) 357 1 1 BADDILEY, J . and GALE , E. F. Mature, Land. 155

(1945) 727 3 5 UMBREIT, W. W. and GUNSALUS, I. C. J. biol. Chem.

159 (1945) 333 3 6 H E Y L , D., HARRIS, S. A . and FOLKERS, K . iwtli Meet.

Amer. chem. Soc. 1946 35 в 3 7 SNELL, E. E . J. biol. Chem. 154 (1944) 313 3 8 LICHSTEIN, H . C , GUNSALUS, L C , UMBREIT, W. \V.

ibid 161 (1945) 311' 3 9 SCHI.ENK, F. and FISCHER, A . Arch. Biol. 12 (1947) 69 4 1 1 UMBREIT, W. W., WOOD, W. A . and GUNSALUS, I. C.

J. biol. Chem. 165 (1946) 731 — — — ibid "169 (1947) 631

Inflammability of Gases J . H . B U R G O Y N E , D . S c , F . R . I . C .

Imperial College of Science and Technology, London

L I M I T S OF I N F L A M M A B I L I T Y A T A T M O S P H E R I C T E M P E R A T U R E

I T has long been known that gases capable of flame propagation in air can only be inflamed between certain limits of concentration. A moment's rcllection shows the theoretical necessity of such

limits. Thus, while a mixture containing the fuel gas and oxygen in stoichiometric proportions may be expected to propagate flame mere readily than others because it has a maximum calorific value, dilution with either constituent may be expected to reduce the calorific value such that eventually the tern-