A STUDY OF THE ANTIMONY TRICHLORIDE COLOR REACTION FOR VITAMIN A.

BY EARL R. NORRIS ANJI ANNA E. CHURCH.

(From the Division of Biochemistry, University of Washington, Seattle.)

(Received for publication, August 20, 1929.)

In 1926 Carr and Price (1) pointed out that a blue color, which did not fade perceptibly in 3 minutes, was produced when a chloroform solution of antimony trichloride was added to a potent cod liver oil, and suggested that the blue color observed was a direct measure of vitamin A activity. Other investigators have examined this test and it is generally considered the most reliable color test so far suggested. Recently there has been considerable controversy as to the reliability and specific nature of the test; therefore a critical study of the antimony trichloride color test has been undertaken in this laboratory.

If the blue color produced by antimony trichloride with a potent oil is a measure of the vitamin content of the oil, a correlation should exist between the color test and the biological test of the oil. The blue color produced should also be proportional to the active substance present and consequently to the amount of any one oil used.

In comparing the calorimetric and biological assay of an oil Rosenheim (2) suggested the use of 20 mg. of cod liver oil as the standard amount, because this amount would restore normal growth to rats on a diet other- wise free of vitamin A. This amount of oil they found to equal 10 Lovi- bond standard blue units when mixed with 1 cc. of reagent and measured in a layer of 10mm. Wokes and Willimott (3) have shown with three potent cod liver oils the effect of varying amounts of oil with a given amount of reagent. At higher concentrations they found that the blue color pro- duced was not proportional to the amount of oil used and the flattening of the curves was not the same for different oils. They state that the curves cease to be linear functions above 2 to 3 per cent and therefore amounts of oil taken for the test should give readings not higher than 15 to 20 Lovi- bond blue units. Norris and Danielson (4) have shown that such con- centrations cannot be used as a basis for quantitative comparison of the

477

by guest on April 12, 2019

http://ww

w.jbc.org/

Dow

nloaded from

478 Color Reaction for Vitamin A

factor causing the blue color. They found that at no concentration of a potent oil was the color produced a linear function of the amount of oil used and therefore not proportional to the amount of vitamin A present. A reading of 10 to 20 blue units cannot be compared directly with another oil, but if a sufficiently low value be selected the function approaches a straight line and a comparison can be made which is in close agreement with values determined by biological assay.

Wokes and Willimott (3, 5) state that the reaction consists of a color change of blue through yellow to red; and have shown that the inte’nsity of the observed color varies with the strength of the reagent, the tempera- ture, and the time. The reaction is accelerated when the vitamin is rendered unstable by irradiation and aeration (change by irradiation may be due to ozone); olive oil and oleic acid appear to exert some inhibitory effect upon the application of the color tests; pure olive oil and oleic acid give no blue color and the inhibitory effect may be due to the presence of some organic peroxides which would destroy vitamin A.

In the present work a study was made of the color reaction of the non-saponifiable substances of cod liver oil with a chloroform solution of antimony trichloride, and the effect of various factors upon the color developed.

EXPERIMENTAL.

To obtain the unsaponifiable portion of cod liver oil two methods were used. The first method is similar to that probosed by Takahashi and Kawakami (6). 100 gm. of cod liver oil are added to 200 cc. of 20 per cent alcoholic potassium hydroxide and re- fluxed on a water bath for 30 minutes. After cooling, 200 cc. of water are added and 150 cc. of petroleum ether (b.p. 30-50”). The mixture is shaken and after it has separated into two layers the water layer is again extracted with petroleum ether. This is repeated twice more and the petroleum ether extractions are col- lected and washed with water until there is no further tendency to form an emulsion. The petroleum ether extract is concentrated by distilling under partial pressure in an atmosphere of carbon dioxide.

The second method is that of Marcus (7). 6.5 gm. of potassium hydroxide are dissolved in 6.25 cc. of water. The solution while warm is added to 25 cc. of cod liver oil. The mixture is stirred and 0.25 cc. of alcohol added. The mass stiffens while the tem- perature rises. After cooling, 2.25 cc. of water are added and then the mass is extracted several times with ethylene dichloride. The

by guest on April 12, 2019

http://ww

w.jbc.org/

Dow

nloaded from

E. R. Norris and A. E. Church 479

second method is much simpler and quicker. At the present time we have not been able to extract the vitamin quantitatively by either method.

To use the extract for color assay either the petroleum ether extract or the ethylene dichloride extract is evaporated almost to dryness under partial pressure in an atmosphere of carbon dioxide and the residue is taken up in chloroform.

The antimony trichloride reagent is prepared by dissolving 30 gm. of C.P. Baker’s Analyzed antimony trichloride, which is washed in anhydrous chloroform, in 100 cc. of anhydrous chloro- form. The solution is stored in the refrigerator and when used is brought to equilibrium at the temperature of an ice and water bath.

The color tests were carried out according to the technique out- lined by Norris and Danielson (4) ; 0.3 cc. of the chloroform extract or oil is pipetted by means of a serological pipette into a half ingh cell, and 3 cc. of the antimony trichloride reagent cooled to 2-4”,

. added m a rapid stream from a pipette. A reading of the color developed is taken by means of a Lovibond tintometer 30 seconds after beginning the addition of antimony trichloride. The period is timed by a stop-watch. The results given in Tables I to V are averages of three or more separate readings. In the tables B, Y, and R refer to Lovibond standards, blue series 1180, yellow series 510, and red series 200 respectively. In order to show the relation- ship between the color developed by different concentrations an extract was diluted with chloroform. In each case the original ex- tract is referred to as 100 per cent by volume and the more dilute as 75 per cent, 50 per cent, etc., with regard to the proportion of the original extract and chloroform added.

Table I and Fig. 1 show the results of four distinct extractions of the unsaponifiable portion of cod liver oil, and also the results of using varying amounts of a typical cod liver oil (Squibb). The oil was diluted with chloroform in the same manner as the unsaponi- fiable extract.

Extracts 3 and 4 were kept in an ice box and samples tested at the end of 2 and 4 months. The values differed by less than 0.2 of a blue unit from the original readings. This would indicate that the blue-producing substance is stable in petroleum ether in the cold and dark.

From Table I and Fig. 1 it will be observed that the extracts of

by guest on April 12, 2019

http://ww

w.jbc.org/

Dow

nloaded from

TABL

E I.

Color

Pr

oduc

ed

by

Cod

Liver

O

il an

d Ex

tracts

oj

Un

sapo

nijia

ble

Porti

on

of

Cod

Liver

O

il wi

th

Antim

ony

Trich

lorid

e.

Extra

ct

ml

per

cent

. 50

pe

r ce

nt.

25 p

er

cent

. 12

.5

per

cent

. 6.

25

per

cent

.

NO.

Subs

tanc

e.

B Y

R

B Y

R

BYRB

YRBY

R ~~

--~--~

-- -_

_---

1 Ex

tract

of

unsa

pon-

6.

6 2.

8 3.

1 1.

3 1.

5 0.

6 0.

8 0.

4 ifia

ble

porti

on

(Squ

ibb).

2 Sa

me

(Pat

ch).

7.3

3.2

3.7

1.5

2.0

0.6

1.1

0.6

3 “

( ‘(

). 14

.1

5.8

7.3

1.8

3.6

1.4

1.9

0.8

0.9

0.3

4 “

(Squ

ibb).

8.8

3.7

4.4

1.7

2.2

0.9

1.1

0.5

5 Co

d liv

er

oil

14.0

8.

8 28

.8

14.0

11

.8

13.0

1.

8 1.

8 9.

7 2.

1 1.

4 5.

6 0.

8 0.

8 (S

quibb

). --

B,

Y,

and

R de

note

Lo

vibon

d sta

ndar

ds,

blue

serie

s 11

80,

yello

w se

ries

510,

an

d re

d se

ries

200

resp

ectiv

ely.

The

orig

inal

ex

tract

is r

eferre

d to

as

10

0 pe

r ce

nt

by

volum

e,

and

the

mor

e di

lute

as

50

pe

r ce

nt,

etc.

by guest on April 12, 2019http://www.jbc.org/Downloaded from

E. R. Norris and A. E. Church

the unsaponifiable portion of cod liver oil produce no red colora- tion and that the blue color of the extracts is a linear function of the percentage concentration, while the curve produced with cod liver oil is not a linear function. Work is now being carried on in this laboratory to correlate the biological rat unit with the Lovibond blue unit.

In preparing the above extracts of unsaponifiable portion of cod liver oil, petroleum ether and ethylene dichloride solutions were

~ONCENTPATION ( mmm )

FIG. 1. Intensity of color plotted against the concentration of solution used in the test. Curves 1, 2, 3, and 4 indicate chloroform solutions of the unsaponifiable portion of cod liver oil; Curve 5, cod liver oil (Squibb).

evaporated almost to dryness, the residue being taken up in chloro- form. This would give a solution containing traces of the other two solvents. In order to determine whether the small amounts of the above solvents left in the chloroform solution would have any effect upon the intensity of color produced, solutions were pre- pared containing varying amounts of these and other solvents and color tests made in the usual manner.

by guest on April 12, 2019

http://ww

w.jbc.org/

Dow

nloaded from

482 Color Reaction for Vitamin A

In preparing the solutions 1 cc. of a chloroform extract was pi- petted into each of four tubes, 0, 1,2, and 3 cc. of the solvent to be studied added to successive tubes, and each tube diluted to 4 cc. with chloroform; this gives a chloroform solution containing 0, 25, 50, and 75 per cent by volume of the second solvent. U.S.P.

reagents were used and also reagents dried over calcium chloride and anhydrous sodium sulfate. The results given in Table II would indicate that traces of moisture have little or no effect upon the development of color. No solvent was found in which a deeper

TABLE II.

Effect of Solvents upon Color Produced by Antimony Trichloride upon Eztract of Unsaponifiable Portion of Cod Liver Oil.

Results are expressed in Lovibond units.

Solvent.

Petroleum ether ..................... “ ether-dried CM&. .......

Absolute alcohol ................... 95 percent “ ................... Acetone-dried CaCl~. ................ Acetone ............................ Benzene dried over NatSOr ......... CCL “ “ “ .......... Cc&. ............................... Ether-dried NaGSO ................. C&-dried NazSOd. ................... csz. ............................... Ethylene dichloride redistilled ......

0 25 50 75 per cent. per cent. per cent. per cent.

~~ ____- BYBYBYBY

----__---

3.6 1.5 3.6 1.5 3.5 1.7 3.3 1.4 3.6 1.2 3.6 1.2 3.5 1.3 3.2 1.3 3.6 1.2 3.2 1.5 2.5 1.4 1.1 0.8 3.6 1.2 1.1 1.1 3.6 1.2 3.3 1.4 2.7 1.4 2.1 1.4 3.6 1.2 2.1 1.4 3.7 1.5 3.7 1.4 3.5 1.3 3.3 1.3 3.7 1.5 3.7 1.3 3.5 1.3 3.3 1.3 3.7 1.5 3.3 1.3 3.7 1.5 2.8 1.3 1.2 1.3 3.7 1.5 3.4 1.3 3.5 1.3 3.3 1.3 3.7 1.5 3.3 1.3 3.3 1.3 3.3 1.0 3.3 1.0 3.3 1.0

blue was produced ‘than in chloroform. Petroleum ether and ethylene dichloride had no effect up to 25 per cent by volume, consequently the traces remaining in the extracts previously used would have no influence upon the color. Alcohol, ether, and ace- tone of the solvents tried decreased the color most markedly, but in no case was there any development of red coloration.

Since the curve for cod liver oil differs so radically from that for the purified unsaponifiable fraction of the same oil, and at the same time the divergence of the concentration curve from a linear function varies with different cod liver oils, it is evident that

by guest on April 12, 2019

http://ww

w.jbc.org/

Dow

nloaded from

E. R. Norris and A. E. Church

some chemical compound common to all oils but varying in amount in different oils, and which is removed by saponification and extraction, must have an inhibitory effect upon the observed blue color. Oils differ in free fatty acids, oxidation products, and impurities, depending upon the process of preparation and purification.

The effect of various saturated and unsaturated free fatty acids upon the intensity of the blue color produced by the action of the unsaponifiable portion of cod liver oil and chloroform solution of antimony trichloride was measured by setting up series of tubes, each containing an equal amount of a chloroform extract of the unsaponifiable portion of cod liver oil, and adding to successive tubes varying amounts of chloroform solutions of fatty acids such that the final concentrations of fatty acids should be 0, 7.5, 12.5, 25,50, and 75 per cent when each tube was diluted with chloroform to the same volume. If the fatty acid has any appreciable effect upon the color produced, the measured color value will vary from the standard or 0 per cent. The results given in Table III show that the saturated fatty acids have no effect upon the color ob- served. It is more difficult to obtain pure unsaturated fatty acids; of those used aa-hexenic, crotonic, and cinnamic acids showed only a slight inhibiting effect on the development of a blue color, while U.S.P. oleic acid showed a very marked decrease in blue with increasing concentration and the rapid development of a red color- ation. The saturated and other unsaturated fatty acids used showed absolutely no red coloration with the exception of a very light color, 0.4 red units, with 50 per cent crotonic acid.

The oleic acid used in Table III was of U.S.P. grade and of yellow color; as the hexenic acid had very little effect upon the color pro- duced it was probably not the unsaturated fatty acid itself but some impurity or oxidation product which caused the red color. The purest oleic acid obtainable on the market (free from linoleic acid) had a slight straw color and gave a very marked red with antimony trichloride. Effort was made to purify oleic acid both by its lead salt and by fractionally distilling several times under reduced pressure in an atmosphere of carbon dioxide. A colorless product was obtained which gave only a very slight pink color when first mixed with antimony trichloride reagent, but developed into a deep red. Bubbling oxygen through oleic acid for 2 hours at room

by guest on April 12, 2019

http://ww

w.jbc.org/

Dow

nloaded from

TABL

E III

.

Effe

ct

of

Vario

us

Fatty

Ac

ids

upon

Co

lor

Prod

uced

by

An

timon

y Tr

ichlo

ride

with

Ex

tract

of

Unsa

poni

jiabl

e Po

rtion

of

Cod

Liv

er

Oil.

Resu

lts

are

expr

esse

d in

Lo

vibon

d un

its.

9

Ex%

?t

1 2 3 4 5 6 7 8 9 10

11

- _-

-

Fatty

ac

id.

Prop

ionic.

Bu

tyric,

b.

p.

160-

165”

. Iso

butyr

ic,

b.p.

15

5-16

0’.

Laur

ie.

Myr

istic.

Pa

lmitic

. St

earic

. Aa

-Hex

enic.

Cr

oton

ic.

Cinn

amic.

O

leic

(u.8

.p.).

B 4.7

7.7

7.7

7.5

7.5

7.5

7.5

7.5

11.5

11

.5

5.3

0 Y 1.8

3.2

3.2

3.3

3.3

3.3

3.3

3.1

4.8

4.8

2.1

__

R

Per

cent

of fa

tty

acid

in fin

al m

ixtur

e. T

- B - 7.

i

LO.

LO.!

2.1

-

7.5

Y R

-- 2.7

4.0

4.7

1.6

O.!

12.5

B Y

R ---

25.0

B Y

R ---

4.3

1.8

50.0

B Y

R ---

4.3

1.8

7.6

3.0

7.5

2.8

7.6

3.2

7.5

2.9

7.5

2.9

7.6

3.2

7.5

3.0

7.1

2.4

6.8

2.4

6.6

2.4

7.5

3.6

5.5

2.4

3.3

1.4

0.4

0.8

4.8

4.1

0.2

7.8

1O.c

75.0

if B

Y R

0 ---

c.

4.0

1.8

E 7.

6 3.

0 7.

4 2.

9 $ 5 s+

9 b-

. 6.

3 2.

4

. .

. .

” ,.

_^

^ .

by guest on April 12, 2019http://www.jbc.org/Downloaded from

E. R. Norris and A. E. Church 485

temperature had no effect upon the color produced when mixed with a solution of the unsaponifiable fraction of cod liver oil and added to antimony trichloride reagent.

Saturated oils as coconut oil, even when strongly rancid, give no color with antimony trichloride reagent, while unsaturated oils, as olive, peanut, and linseed, give varying degrees of deep red but no blue color. The effect seems not to be due to oxidation to a single hydroxy group, as ricinolein (castor oil) gives only a very slight pink. The results produced by an oil as olive oil when mixed in varying proportions with a fixed amount of the unsaponifiable portion of cod liver oil, Table IV, might be explained on the basis

TABLE IV.

Effect of Olive Oil upon Color Produced by Antimony Trichloride with Extract of Unsaponifiable Portion of Cod Liver Oil.

Results are expressed in Lovibond units.

Olive oil.

per cent

0.0 3.12 6.25

12.5 25.0 75.0

-

i

B

7.5 3.2 5.7 1.9 5.5 2.1 5.2 2.0 4.7 2.0 0.3 3.7 1.8 0.3

Color produced.

Y I R

Decrease of blue.

per cent

0.0 24.0 26.7 30.6 37.3 50.6

of the free fatty acid present. The olive oil used had the fol\owing constants: iodine number (Hanus) 80.7 and acid number 0.799.

Oleic acid and unsaturated oils show a very marked inhibiting effect upon the blue color when measured according to the tech- nique of Norris and Danielson, at 30 seconds after mixing the solu- tions. However immediately upon mixing a solution of the unsa- ponifiable portion of cod liver oil containing oleic acid or unsatu- rated oils with antimony trichloride reagent, a blue flash lasting in the higher concentrations of oleic acid only a very short period of time was observed, the blue color fading very rapidly. This would indicate that the oleic acid or an impurity in the acid did not destroy the blue-producing substance nor absolutely inhibit the formation of a blue color but accelerated the rate of fading of the

by guest on April 12, 2019

http://ww

w.jbc.org/

Dow

nloaded from

486 Color Reaction for Vitamin A

blue. Table V gives the results of tests showing the effect of oleic acid (Eimer and Amend, c.P., free of linoleic) upon the rate of fad- ing of the blue color and the development of a red coloration. The rate of fading of the blue is greatly increased by increasing amounts of oleic acid used, so that readings taken at 30 seconds vary g,reatly in the measured intensity of blue although the amount of blue- producing substance in each case is known to be the same. Oleic

TABLE V.

E$ect of Oleic Acid upon Rate of Color Development of Extract of Unsaponi- $able Portion of Cod Liver Oil with Antimony Trichloride Reagent.

Results are expressed in Lovibond units.

T- Time.

. - see.

5 10 20 30

min.

1 2 3 4 5 6 7 8 9

0.0 T 6.25 I 12.5 I 25.0 1 50.0

B Y R ---

8.5 2.6 7.9 2.8 7.4 2.1 5.7 2.7 1.13.43 8.4 2.6 7.2 2.2 4.5 2.3 2.5 8.3 2.6 6.6 2.2 0.3 4.9 1.8 0.6 4.4 2.8 3.62.94

8.1 2.6 7.7 2.4 6.7 2.1 6.1 1.8 5.1 1.4 4.3 1.1 3.8 1.1 0.1 3.7 1.1 0.3 3.6 1.1 0.4

5.7 2.2 0.9 4.5 2.0 1.9 3.7 2.4 4.72.74 4.5 2.2 1.8 3.1 2.0 2.9 2.9 2.4 5.82.74 4.1 2.2 2.2 3.2 1.8 3.3 2.7 2.5 5.6 3.7 2.0 2.6 2.7 1.8 3.5 2.4 2.7 6.1 3.3 1.9 2.6 2.4. 1.8 3.5 2.3 2.7 6.3 2.5 1.7 2.6 2.3 1.8 3.6 2.0 2.7 6.4 2.8 1.7 2.6 2.3 1.8 3.7 2.2 2.9 6.5 2.61 1.71 2.61 ) / j 2.11 2.91 6.61 1

Per cent oleic acid.

R

3.7 4.1

7.2

10.3 12.0

acid without the extract of the UnsaponifIable portion of cod liver oil shows a development of red color reaching a maximum with each concentration of acid similar to the red values obtained when oleic acid is added to the extract of unsaponifiable substance of cod liver oil. Therefore it would seem that the color change for vitamin A, if the color be due to the vitamin, is not one from blue through yellow to red as suggested by Wokes and Willimott but, the substance producing the “blue” color produces a greenish blue with a ratio of blue to yellow of approximately 1:0.4 when meas-

by guest on April 12, 2019

http://ww

w.jbc.org/

Dow

nloaded from

E. R. Norris and A. E. Church 487

ured in Lovibond units, and the color fades out to a colorless solu- tion. The red coloration is produced by some other substance present in oleic acid and unsaturated oils.

In attempting to determine the vitamin A by the intensity of the blue col,or produced by cod liver oil with a chloroform solution

0 10 20 30 40 50 60 70 .so go 100 CONCENTRATION ( PERCENT )

FIG. 2. The effect of oleic acid upon the intensity of color produced plotted against the concentration of the solution used in the test. Curve 1 indicates a chloroform solution of the unsaponifiable portion of cod liver oil; Curve 2, the same containing 6.25 per cent oleic acid; Curve 3, the same containing 12.5 per cent obic acid; Curve 4, the same containing 25.0 per cent oleic acid; Curve 5, the same containing 50.0 per cent oleic acid.

of antimony trichloride, two variables must be considered, the first the amount of vitamin A or blue-producing substance and the second that which increases the rate of fading of the blue and probably also causes the red coloration. The results obtained with known but varying amounts of these two factors are given in Fig. 2. Tubes were set up in three series such that each tube of a

by guest on April 12, 2019

http://ww

w.jbc.org/

Dow

nloaded from

Color Reaction for Vitamin A

given series would contain the same amount of the unsaponifiable extract of cod liver oil producing a clear greenish blue and succes- sive tubes of the series to have a final concentration of 0.0, 6.25, 12.5, 25.0, and 50.0 per cent oleic acid. The amount of unsaponi- fiable portion of cod liver oil in the first series is designated as 100 per cent, the second series contained two-thirds of the first, and the third series one-third of the first. Color tests were made according to the technique of Norris and Danielson, readings being taken at 30 seconds. Fig. 2 gives the data (the values for yellow and red being omitted) calculated to represent the effect obtained with five oils each having exactly the same amount of vitamin A but differ- ing from one another in the amount of oleic acid or red-producing substance present in the initial oil, the initial oils each being diluted to two-thirds and one-third by volume with chloroform, and color tests made upon the resulting solutions. As the amount of oleic acid increases, the divergence of the curve from a linear function becomes greater; 100 per cent of the initial oils known to contain the same amount of vitamin A is seen to give color values varying from 2.9 to 9.6 blue units. At the lower color values as the oils become more dilute the interference of the red-producing sub- stance becomes less marked; the curves approach more closely a linear function so that the color observed for a definite known vita- min content checks more closely in the several oils. Wfth 12.5 per cent oleic acid the color for the oil (4.9) is approximately 50 per cent of that with no oleic acid; when diluted to one-third with chloroform, the observed value is 3.2 blue units, within 10 per cent of that without oleic acid (3.5). Therefore with the ratio of the interfering substance to vitamin A present to the extent that would be represented by 12.5 per cent of the oleic acid used, if the compar- ison were made below a color value of 3.0 blue units the results would vary by fess than 10 per cent; if the comparison were made above 4 blue units the error would be as great as 50 per cent. These results substantiate the findings of Norris and Danielson that at sufficiently low color values the curves for oils approach linear functions and a comparison can be made between the colarimetric and feeding experiments. However the color tests must be very carefully controlled as to time, temperature, and concentration of the reagent. The tests are best made in a con- stant temperature room; if tests be made on a single oil at various

by guest on April 12, 2019

http://ww

w.jbc.org/

Dow

nloaded from

E. R. Norris and A. E. Church

times during the course of an experiment extending through differ- ent seasons so that the room temperature will vary by several degrees, observed values at later dates may be greater or less than the initial observed value depending upon whether the room tem- perature be cooler or warmer.

SUMMARY.

1. Vitamin A, or the substance producing the “blue” color with antimony trichloride reagent, produces a greenish blue which fades to a colorless solution; the ratio of blue to yellow is approxi- mately 1.0 to 0.4 Lovibond unit.

2. The blue color produced by an extract of the unsaponifiable portion of cod liver oil with a chloroform solution of antimony trichloride is a linear function of the percentage concentration of the extract, and no red coloration devefops on standing.

3. Traces of petroleum ether and ethy!ene dichloride have no effect upon the intensity of the color produced.

4. Saturated fatty acids and oils have no effect upon the color produced by the unsaponifiable portion of cod liver oil with anti- mony trichloride reagent.

5. Oleic acid and unsaturated oils accelerate the rate of fading of the blue color.

6. Deviation of the observed blue color produced with varying amounts of cod liver oil from a linear function is due to an in- creased rate of fading of the blue color.

7. Quantitative comparison of the color values between differ- ent oils or between calorimetric and feeding experiments can only be made at a sufficiently low value so that the dilution curve approaches a linear function, or be made on the unsaponifiable portion.

BIBLIOGRAPHY.

1. Carr, F. H., and Price, E. A., Biochem. J., 20, 497 (1926). 2. Rosenheim, O., Biochem. J., 21, 1329 (1927). 3. Wokes, F., and Willimott, S. G., Analyst, 62, 515 (1927); Biochem. J.,

21, 419 (1927). 4. Norris, E. R., and Danielson, I. S., J. Biol. Chem., 83, 469 (1929). 5. Wokes, F., and Willimott, S. G., Lancet, 2, 8 (1927); Pharm. J., 118,217

(1927). Willimott, S. G., and Wokes, F., Pharm. J., 118, 752 (1927). 6. Takahashi, K., and Kawakami, K., J. Chem. Sot. Japan, 44, 590 (1923). 7. Marcus, J. K., J. Biol. Chem., 80,9 (1928).

by guest on April 12, 2019

http://ww

w.jbc.org/

Dow

nloaded from

Earl R. Norris and Anna E. ChurchFOR VITAMIN A

TRICHLORIDE COLOR REACTION A STUDY OF THE ANTIMONY

1930, 85:477-489.J. Biol. Chem. 

  http://www.jbc.org/content/85/2/477.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

alerts to choose from all of JBC's e-mailClick here

  ml#ref-list-1

http://www.jbc.org/content/85/2/477.citation.full.htaccessed free atThis article cites 0 references, 0 of which can be

by guest on April 12, 2019

http://ww

w.jbc.org/

Dow

nloaded from