DETERMINATION OF NICOTINIC ACID IN BIOLOGICAL MATERIALS BY MEANS OF PHOTOELECTRIC

COLORIMETRY *

BY DANIEL MELNICK? AND HENRY FIELD, JR.

(From the Department of Internal Medicine, Medical School, University of Michigan, Ann Arbor)

(Received for publication, February 12, 1940)

The pyridine nucleus reacts with cyanogen bromide and an aromatic amine to give a yellow compound which can be esti- mated calorimetrically. This reaction has been made the basis for the chemical determination of nicotinic acid (l-9). In the present study the same reaction has been used for the analysis of biological materials but the errors, due to inadequate extraction or to adsorption, are avoided by direct acid hydrolysis of the test substance, followed by preferential charcoal adsorption for the decolorization of the hydrolysate. For the measurement of the final yellow color resulting from the reaction between nicotinic acid and reagents, a photoelectric calorimeter’ is used, and in the application of the test to biological materials a new proce- dure is described for making the blank corrections and for evalu- ating the effects of interfering substances which modify the sensi- tivity of the reaction.

Nicotinamide2 rather than nicotinic acid was used in the re-

* The expenses of this investigation were defrayed by grants from The Upjohn Company, Kalamazoo, and from the Horace H. Rackham School of Graduate Studies, University of Michigan.

t Upjohn Fellow in Clinical Research, 1937-40. 1 Obtained from the Rubicon Company, Philadelphia, Pennsylvania.

The design and principle of operation of this instrument are described by Evelyn (10). For a more complete description, including the significance of the terms used in the present study, the reader is referred to “Evelyn photoelectric calorimeter, Notes on operation” (1939), Rubicon Company, Philadelphia.

* The crystalline nicotinamide was kindly furnished by General Bio- chemicals, Inc., Cleveland, Ohio. The compound melted at 126”. A

1

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2 Determination of Nicotinic Acid

covery experiments. The amide reacts also with the reagents to give the same yellow color but of an intensity corresponding to only one-half of that obtained with an equivalent amount of the acid. Under the conditions of our test the amide is completely hydrolyzed to give the theoretical amount of free acid. This quantitative conversion of amide to acid in tests with the com- pound added to biological materials is adequate proof that any nicotinamide-containing coenzymes present are also hydrolyzed to yield free nicotinic acid. Von Euler and associates (3) have shown that the union of the pyridine ring to carbohydrate in codehydrogenase I and II is much more labile to acid hydrolysis than the acid amide group. The present method for the determi- nation of nicotinic acid has been used in the analyses of yeast, liver, rice polish, and wheat germ preparations and of urine, milk, saliva, plasma, and whole blood.

EXPERIMENTAL

Reagents for Chemical Reaction- Cyanogen bromide. Water saturated with bromine at 5-10”

is just decolorized in the cold by the addition of a 10 per cent KCN solution. From 70 to 75 cc. of the KCN solution are used in the titration of 500 cc. of the bromine water.

Absolute ethyl alcohol. This reagent is generally free from pyridine compounds.

Aniline solution. Redistilled aniline is dissolved in absolute ethyl alcohol to make a 4.0 per cent solution.

Standard nicotinic acid3 solution. This contains 100 micro- grams per cc. of absolute ethyl alcohol.

Results Obtained in Tests with Pure Solutions of Nicotinic Acid- The procedure used in carrying out the reaction is that reported by Shaw and MacDonald (2). To the nicotinic acid in 3 cc. of solution (2 parts of water to 1 part of ethyl alcohol) 6 cc. of the cyanogen bromide reagent are added from a burette. This is

stock solution was made by dissolving 496 mg. of nicotinamide in 500 cc. of absolute ethyl alcohol. This solution is equivalent on the basis of comparative molecular weights of the solutes to one containing 1 mg. of nicotinic acid per cc.

*Obtained from Eastman Kodak Company, Rochester, New York. After recrystallization from hot water, the compound melted at 231”.

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D. Melnick and H. Field, Jr. 3

followed immediately with the addition of 1 cc. of the aniline solution. The- solutions are stirred after the addition of each reagent. The maximal yellow color4 is then read in the Evelyn photoelectric calorimeter, with Filter 420.

With quantities of nicotinic acid ranging from 3 to 50 micro- grams the ratio of photometric density (L value) to the concen- tration of the test substance was constant to within fl per cent. There was a significant positive deviation from the constant K value when smaller amounts of nicotinic acid were tested, but because of the excellent reproducibility of the results accurate values may still be obtained in the very low range by applying an appropriate correction factor.

Xpecijicity of Reaction for Nicotinic Acid-This subject has been more extensively investigated by others (l-6, 9). Their studies indicate that the reaction is specific not for nicotinic acid alone but for the pyridine ring, provided the nitrogen is trivalent. However, the reaction appears to be even more specific than this inasmuch as vitamin Bs, which also contains the pyridine ring with trivalent nitrogen (ll), fails to give a positive color test.s

Reproducibility and Stability of Reagents--In order to study this problem adequately more than 100 tests with twenty-five different cyanogen bromide reagents and six different aniline solu- tions were carried out. Representative data are presented in Table I. The K values obtained with the cyanogen bromide reagents were not constant but varied from 0.215 to 0.259. How- ever, with each of the particular reagents used the K value was constant in tests conducted with varying quantities of nicotinic acid. This finding indicates that no previously determined reference curve can be used for this determination but that along with each series of tests a K value should also be obtained. We have preferred in analyses of biological materials to determine for each test solution its own particular K value. To an aliquot of the test solution 10 micrograms of nicotinic acid are added and the difference in photometric density yields the proper K value to be used in the calculations. This is necessary since other

4 This color reaches a maximum in from 3 to 5 minutes and remains constant for at least the next 5 minutes.

6 The crystalline vitamin Ba was kindly furnished by Merck and Com- pany, Inc., Rahway, New Jersey.

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4 Determination of Nicotinic Acid

substances in solution may either inhibit the reaction (e.g., excess of free acid or free alkali) or make it more sensitive (e.g., sodium chloride).

Examination of Table I also reveals that the reagents, when properly stored, are suitable for use for a period of at least 5

TABLE I

Reproducibility and Stability of Reagents Used in Chemical Determination of Nicotinic Acid

Fleagents Age of reagents

CNBr

-

Blank :enter set- ting, gal- Rxnometer reading

Nicotinic cid tested K valuet

CNBr CsHsNHn BHSNHZ

k!s his “C. 7

I I 0 0 5 81’ 5 10 20

II 0 75 5 821 10 III 0 171 5 1362$ 10

II III 0 0 5 802 5 10 20

III 31 31 5 7S2 10 II 150 55 5 781 10

III 31 31 25-30 792 5 10 20

II 150 55 25-30 781 10

* The aniline solutions were stored at ioom temperature throughout. t This value corresponds to the photometric density obtained in each

test when expressed in terms of 10 micrograms of nicotinic acid. $ When the galvanometer in this case was adjusted to 100 with the blank

tube, the center setting was not on the galvanometer scale. The recorded value was obtained by adjusting the galvanometer to 50 with the blank tube and multiplying the resulting center setting by 2.

0.262 0.256 0.258 0.258 0.258 0.213 0.216 0.215 0.218 0.206 0.178 0.179 0.175 0.072

-

months. This is in contrast to all methods previously published, which advocate the use of freshly prepared reagents. The aniline solutions were stored at room temperature in glass-stoppered, brown glass bottles. The center settings obtained with these solutions and the freshly prepared cyanogen bromide reagent indicate that there is a marked increase in the color of the former

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D. Melnick and H. Field, Jr. 5

with increasing age, However, this change in the aniline reagent merely alters the center setting without affecting the accuracy of the results. The cyanogen bromide is stored also in a glass- stoppered bottle but in the refrigerator at about 5”. When kept at room temperature the reagent gradually deteriorates with a concomitant decrease in the experimentally determined K values.

Despite the fact that nicotinic acid is relatively a very stable compound, we have repeatedly observed a rather rapid decrease in its concentration when dilute aqueous solutions (10 micrograms per cc.) are allowed to stand at room temperature. Because the factor responsible for this is bacterial contamination, all standard nicotinic acid solutions are made up in absolute alcohol.

Use of Preferential Charcoal Adsorption for Decolorization of Nicotinic Acid SoZutions-Analyses of biological materials for nicotinic acid have been complicated by the presence in test solutions of pigments which mask the color produced by the reaction and because of their intensity make impossible colori- metric measurements. Charcoal adsorbs nicotinic acid quan- titatively from pure aqueous solutions and to an appreciable extent from biological solutions. For this reason charcoal ad- sorption has been abandoned and other procedures, though much more tedious, adopted for the preparation of colorless solutions (l-9). However, by making use of preferential charcoal adsorp- tion we have been successful in the decolorization of solutions with no concomitant loss of nicotinic acid.

The validity of this new procedure is indicated by tests with pure solutions of nicotinic acid of known concentration. Variable amounts of charcoal6 were added to aqueous, strongly acidic, alcoholic, and strongly acidic alcoholic solutions of nicotinic’ acid. The mixtures were shaken and filtered’ at room temperature. The filtrates were adjusted* in the cold to pH 7 and tests conducted

6 Darco, a vegetable charcoal, obtained from The Coleman and Bell Company, Inc., Norwood, Ohio.

r Whitall Tatum filter paper, manufactured by the Armstrong Cork Company, Lancaster, Pennsylvania, was used.

8 Concentrated NaOH (18 N) was used to neutralize the acid filtrates with phenolphthalein as an inside indicator. For the final adjustment 1 N

NaOH was added with litmus paper as an outside indicator. The phenol- phthalein does not influence the reaction and at the pH of the test is in its colorless form.

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6 Determination of Nicotinic Acid

with 3 cc. samples, containing 2 parts of water to 1 part of alcohol and equivalent to one-tenth of the original solutions. The results, presented in Table II, indicate that quantities of charcoal up to 200 mg. may be added to 25 cc. of the acidic alcoholic solution with no loss of nicotinic acid, Losses from simple acidic or alcoholic solutions are large but the coupling of the two mediums

TABLE II Injluence of Solvent upon Adsorptj ion of Nicotinic Acid by Charcoal

Solvent for nieotinic acid* Charmalt added iicotinic acid adsorbed

15 cc. Hz0

15 “ 4 N HCI

15 “ Ha0 + 10 cc. CzHhOH

15 “ 4 N Hcl + 10 cc. CeH60H

nag. per cent 100 100 200 100 300 100 500 100 loo 85 200 92 300 92 500 95 100 67 200 84 300 88 500 93 100 3 200 0 300 17 500 30

* In each case 100 micrograms of nicotinic acid were added; for the calorimetric tests 10 per cent aliquots were used.

t Darco, a vegetable charcoal, obtained from The Coleman and Bell Company, Norwood, Ohio.

is followed by a synergistic action in the prevention of charcoal adsorption of nicotinic acid.

Procedure for Evaluating Residual Color in Test Solution and for Converting Photometric Density into Absolute Units of Nicotinic Acid -For the decolorization of biological solutions the technique of preferential charcoal adsorption is used. No attempt ‘is made to obtain complete decolorization of the alcoholic hydrolysates inasmuch as correction can easily be made for the amount of residual color. The procedure described by Bandier (5) for

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D. Melnick and H. Field, Jr. 7

making this correction involves the addition of all reagents except the base to an aliquot of the test solution. However, since cyanogen bromide reacts to an appreciable extent with nicotinic acid despite the absence of the base to give the same color, false results are obtained. We have found in tests with pure solutions of nicotinic acid ranging from 3 to 50 micrograms that the yellow color developed without the aniline is equivalent to from 33 to 20 per cent respectively of that obtained when the base is added. It is therefore necessary to carry out an independent blank deter- mination for the residual color subsequent to decolorization in addition to that obtained with the reagents.

The procedure used for making these two independent blank corrections and its validity are presented in Table III. The tests were conducted on 3 cc. aliquots of three 30 cc. nicotinic acid solutions, containing in each case 100 micrograms of the acid in 2 parts of water to 1 part of ethyl alcohol. The solutions were colored to varying degrees by thymol blue indicator. The initial photometric densities were calculated from the galvanom- eter readings of the samples diluted with a buffer solutions to simulate test conditions. The same Filter 420 was used but the center setting was that obtained with a blank tube containing only the pure solvents. Subsequent to the chemical reactions the maximal photometric densities were measured, the center setting obtained with the blank on the reagents being used. By sub- tracting from the total photometric density that corresponding to the color initially present in the test solution prior to the addi- tion of the reagents, the photometric density resulting from the chemical reaction alone was obtained. For the conversion of this value into micrograms of nicotinic acid use was made of the K value corresponding to the increase in photometric density when 10 micrograms of nicotinic acid in 0.1 cc. of absolute ethyl alcohol were added to another 3 cc. aliquot of the test solution. The

8 This solution is composed of 1960 cc. of water, 10 cc. of H,POd (85 per cent), 30 cc. of 15 per cent NaOH, and 333 cc. of absolute ethyl alcohol. With phenolphthalein as the indicator, 7 cc. of the solution have a ti- tratable acidity equivalent to 10 cc. of 0.05 N NaOH; its pH is 2.4. These values correspond to the titratable acidity and pH of the pooled reagents, 6 cc. of CNBr + 1 cc. of CeHhNHz, estimated at from 5 to 10 minutes after being mixed at room temperature.

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TABL

E III

00

De

term

inat

ion

of N

icotin

ic Ac

id

in

Colo

red

Test

Sol

utio

ns;

Expe

rimen

tal

Conf

irmat

ion

of V

alid

ity

of P

TOce

dUTe

Us

ed

in

Mak

ing

“Dou

ble

Blan

k”*

Cowe

ctio

n

Solu-

tio

n NO.

I II III IV V VI

VII

VIII IX

X XI

XII

- I De

scrip

tion

2 cc

. Hz0

+

1 cc

. C2

H60H

+

7 cc

. bu

ffers

Sa

me

as I

+

sligh

t am

ount

of

pig

men

tll “

I‘ I‘

+ co

nsid

erab

le

pigm

entjl

‘I “

“ +

larg

e am

ount

of

pig

men

t11

2 cc

. Hz0

+

1 cc

. CzH

sOH

+ 6

cc. C

NBr

+ 1

co.

C&NH

z 10

y n

icotin

ic ac

id

test

ed

in p

rese

nce

of p

igm

ent

as in

II

Sam

e as

VI

-I- i

ncre

men

t of

10

y ni

cotin

ic ac

id

10 y

nico

tinic

acid

te

sted

in

pre

senc

e of

pig

men

t as

in

III

Sam

e as

VIII

f

incr

emen

t of

10

y ni

cotin

ic ac

id

10 y

nico

tinic

acid

te

sted

in

pre

senc

e of

pig

men

t as

in

IV

Sam

e as

X

+ in

crem

ent

of 1

0 y

nico

tinic

acid

0 y

nico

tinic

acid

te

sted

in

pr

esen

ce

of p

igm

ent

as in

IV

kite

r 3e

tting

70

70

70

70

801

801

801

801

80’

801

801

801

Of

ampk

, .

_

100 921

71’

491

100 54’

321

42

243

291

171

491

Photo

metr

ic de

n&f

of sa

mple

0.00

0 0.

035

0.14

7 0.

308

0.00

0

0.26

2

0.49

1 (K

=

0.22

9)T

0.37

7

0.60

6 (K

=

0.22

9)T

0.53

4

0.76

3 (K

=

0.22

9)8

0.30

8

Calcu

lation

s, nic

otinic

ac

id rec

overe

d

0.26

2 -

0.03

5 %

x

10 =

0.

229

9.9

z Z’

s C.

0.37

7 -

0.14

7 3

x 10

=

0.22

9 10

.0

;;’

b 5.

0.53

4 -

0.30

8 i=

0.22

9 x

10 =

9.

9

0.30

8 -

0.30

8 0.

229

x 10

=

0.0

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* Th

e re

ason

fo

r co

nduc

ting

the

two

blan

k te

sts,

on

e on

the

re

agen

ts

and

the

othe

r on

the

in

itial

colo

r of

th

e so

lu-

tion,

in

depe

nden

tly

is d

iscus

sed

in

the

text

. f

Thes

e va

lues

ar

e co

rrect

ed

for

the

sligh

t de

viatio

ns

from

tru

e lin

earit

y of

the

rela

tion

betw

een

curre

nt

and

de-

flect

ion.

$

This

is

anal

ogou

s to

op

tical

de

nsity

as

mea

sure

d on

a

spec

troph

otom

eter

an

d co

rresp

onds

to

th

e qu

antit

y (2

-

log10

of

the

galva

nom

eter

re

adin

g).

0 A

14 p

er c

ent

alco

holic

so

lutio

n wi

th

the

sam

e pH

an

d tit

rata

ble

acid

ity

as 6

cc.

of

the

cyan

ogen

br

omid

e re

agen

t +

1 cc

. of

the

anilin

e so

lutio

n.

In t

he p

hoto

elec

tric

calo

rimet

er

it gi

ves

the

sam

e va

lue

as d

istille

d wa

ter.

Its c

ompo

si-

tion

is d

escr

ibed

in

fo

ot-n

ote

9.

11 Th

ymol

blu

e in

dica

tor

was

used

to

im

part

colo

r to

th

e te

st

solu

tions

. 7

This

K v

alue

, ca

lcula

ted

by d

iffer

ence

, co

rresp

onds

to

the

in

crea

se

in p

hoto

met

ric

dens

ity

when

th

e te

st

is c

arrie

d ou

t wi

th

10 m

icrog

ram

s of

nico

tinic

acid

ad

ded

to a

n al

iquo

t of

the

test

so

lutio

n.

W

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Determination of Nicotinic Acid

validity of these procedures is indicated by the theoretical values obtained with all three nicotinic acid solutions (Table III).

Determination of Nicotinic Acid in Biological Materials-The test material, containing from 10 to 400 micrograms of nicotinic acid, is added to a test-tube graduated at the 10 and 15 cc. marks. 5 cc. of concentrated hydrochloric acid (specific gravity of about 1.18) are added and this is followed by distilled water until the total volume measures 15 cc. The acidity is approximately 4 N. The test-tube is immersed in a boiling water bath and the hydrolysis allowed to proceed for a period of from 30 to 40 minutes with occasional stirring. The sample is cooled to room temperature and the volume restored to the original 15 cc. 10 cc. of absolute ethyl alcohol are added and the solution (or suspension) trans- ferred to a 150 cc. Erlenmeyer flask. Exactly 200 mg. of char- coal6 are added, and the mixture shaken and filtered’ at room temperature. An aliquot of the filtrate, 8.33 cc., is pipetted into the graduated test-tube, 1 drop of phenolphthalein added, and the solution neutralized8 in the cold to pH 7. The final volume is brought to the 10 cc. mark.

3 cc. aliquots of the test solution, already containing alcohol in the proper ratio of 1 part to 2 parts of water and equivalent to one-tenth of the original sample, are used for the tests. To the first sample 7 cc. of an alcoholic buffer solution9 are added. The photometric density of the color remaining after the charcoal decolorization is then estimated according to the procedure described in the preceding section of this paper. A second 3 cc. aliquot of the test solution is used for the chemical reaction and the maximal total photometric density measured. By difference that resulting from the chemical reaction alone is obtained. This value is converted into micrograms of nicotinic acid by use of the K value obtained when 10 micrograms of the acid are added to a third 3 cc. aliquot of the test solution. The validity of these procedures with examples to illustrate the method of calculation has been presented in Table III.

Representative data obtained in the course of numerous analyses of food sources, rich in the vitamin B complex, and of other materials of biological interest are given in Table IV. The theo- retical recoveries of added nicotinamide as nicotinic acid from the hydrolysates support the validity of the procedures used.

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D. Melnick and H. Field, Jr.

In Table IV there is included a column to show what may be considered optimal amounts of the test materials to be used for the analysis. Test solutions, such as saliva and urine, are first concentrated over a steam bath and then brought to volume with the concentrated hydrochloric acid and wash water.‘O In analyses of material, such as milk, plasma, and ‘blood, the concentrated acid is first pipetted into the graduated test-tube. 5 drops of caprylic alcohol are added in order to reduce subsequent foaming. While the acid is being stirred mechanically, the sample is slowly added. The mixture, with the same stirring rod, is then immersed in the boiling water bath. During the first 15 minutes of the hydrolysis the samples are not disturbed. By that time they become sufficiently liquid for adequate stirring.

Alkaline hydrolysis (NaOH) of the test material has been employed by Bandier (4, 5) with good recoveries of added nic- otinamide. Such a procedure yields a lighter colored solution than acid hydrolysis. Our own experience with alkaline hydroly- sis (2.5 to 9 N), especially of urine, has also shown that added nicotinamide is completely coverted to the free acid. However, the yield of “nicotinic acid” from urine samples subjected to such alkaline hydrolysis is far in excess of that obtained by hydrolysis with hydrochloric acid of equivalent normality and increases progressively with increasing concentration of the alkali used. The acid hydrolysis yields constant values despite varying con- centrations of the hydrochloric acid used. In all of these cases theoretical recoveries of added nicotinamide are obtained. Fur- thermore, a normal intake of some materials, devoid of anti- pellagra activity, has been found to augment up to 700 per cent the yield of the substances in urine reacting like nicotinic acid when alkaline hydrolysis (9 N) is employed. Inasmuch as the intake of these substances does not appreciably increase the yield of nicotinic acid obtained by acid hydrolysis, we have preferred to use acid rather than alkali for the hydrolysis of the test mate-

lo The usual 30 to 40 minute acid hydrolysis is satisfactory for the conversion of all nicotinamide in normal urine to the free acid. Prolonged hydrolysis (5 hours duration) does not increase the yield of nicotinic acid. However, a 5 hour hydrolysis is essential when urine is collected from subjects receiving test doses of extra nicotinic acid. Only under these conditions will the voided nicotinuric acid be completely hydrolyzed.

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s;E

-

A B C

D

E F G

H I

TABL

E IV

;

Dete

rmin

atio

n of

Nico

tinic

Acid

in

Bi

olog

ical

Mat

erial

s an

d Va

lidity

of

Pro

cedu

res

Used

Desc

riptio

n of

prep

arat

ion

Live

r ex

tract

po

wder

20%

al

coho

lic

liver

ex

tract

Dehy

drat

ed

liver

an

d ye

ast

conc

entra

te

Drie

d ye

ast

powd

er

Yeas

t ex

tract

pa

ste

Swee

tene

d,

conc

entra

ted

aque

- ou

s ex

tract

of

yea

st

Yeas

t ex

tract

po

wder

Aque

ous

conc

entra

te

of

rice

polis

h ex

tract

W

heat

ge

rm

powd

er

Qua

ntity

use

d in

ana

lysi

s

mg.

100

100

200

200

160

160

200

200

100

100

200

200 50

50

25

25

300

300

I 8

-

Nico

- tin

- m

ide

bdde

d’

Y 0

200 0

200 0

200 0

200 0

200 0

200 0

200 0

200 0

200

- I R

$+;

aliq

uot

of te

st

SOlW

0

tion,

t ph

oto-

m

etric

E

dens

ity -

K va

lue:

lbts

ina

with

PU

P3

solu

tion

K va

lp$

‘btwt

Ed

test

3o

lutio

n

0.04

7 0.

244

0.25

0 0.

054

0.24

4 0.

229

0.04

3 0.

244

0.23

6 0.

037

0.24

4 0.

220

0.10

5 0.

240

0.24

5 0.

153

0.24

0 0.

258

0.01

8 0.

240

0.26

0 0.

025

0.24

0 0.

243

0.01

4 0.

237

0.23

5 0.

013

0.23

7 0.

217

0.07

6 0.

237

0.24

8 0.

047

0.23

7 0.

231

0.00

8 0.

229

0.23

0 0.

008

0.22

9 0.

223

0.01

1 0.

240

0.23

4 0.

013

0.24

0 0.

226

0.06

7 0.

233

0.25

8 0.

090

0.23

3 0.

245

Nico

tinic

acid

foun

d

Y

115

(1.1

5 m

g.

per

gm.)

317

151

(0.7

6 m

g.

per

pm.)

367

194

(1.2

1 m

g.

per

gm.)

392 85

(0.

43 m

g.

per

gm.)

287

155

(1.5

5 m

g.

per

gm.)

357 84 (

0.42

mg.

pe

r gm

.) 28

3 12

6 (2

.52

mg.

pe

r gm

.) 32

0 71 (

2.84

mg.

pe

r gm

.) 26

9 45

(0.1

5 m

g.

per

gm.)

248

101

E g*

108

o

99

; K 10

1 s I-.

s

101

g;’

100

FF

E 97

99

102

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cc.

J Hu

man

sa

liva

30

0 0.

014

0.23

8 0.

241

I 17

(0.

07 m

g.

%)

30

100

0.01

8 0.

238

0.24

6 10

2 85

11

K (I

urin

e (2

4 hr

. sa

mpl

e =

28 (

a hr

.) 0

0.15

3 0.

233

0.23

8 77

(3

.7

mg.

pe

r 24

hrs

.) 13

40 c

c.)

28

(3

“) 10

0 0.

132

0.23

3 0.

244

176

99

L Co

w’s

milk

y 10

0

0.17

6 0.

244

0.27

7 45

(0

.45

mg.

%

) 10

20

0 0.

177

0.24

4 0.

256

245

100

M

Hum

an

plas

maf

f 10

0

0.12

6 0.

254

0.27

4 13

(0

.13

mg.

%

) 10

10

0 0.

115

0.25

4 0.

272

116

103

N

‘I ox

alat

ed

bloo

d**

10

0 0.

182

0.24

8 0.

247

72

(0.7

2 m

g.

%)

10

100

0.20

6 0.

248

0.23

3 17

0 98

-

- -

* Ex

pres

sed

in t

erm

s of

nico

tinic

acid

. Th

e am

ide

was

adde

d as

suc

h bu

t re

cove

red

as th

e fre

e ac

id.

t Th

is re

pres

ents

th

e am

ount

of

pig

men

t re

mai

ning

in

a

one-

tent

h al

iquo

t of

the

te

st

solu

tion

subs

eque

nt

to

char

coal

de

colo

rizat

ion.

Su

ch

an

aliq

uot

is u

sed

for

the

chem

ical

test

. Th

e te

rm

phot

omet

ric

dens

ity

is a

nalo

gous

to

op

tical

de

nsity

as

mea

sure

d on

a s

pect

roph

otom

eter

an

d co

rresp

onds

to

the

qua

ntity

(2

-

log,

, of

the

galva

nom

eter

re

adin

g).

$ Th

is va

lue

corre

spon

ds

to

the

phot

omet

ric

dens

ity

resu

lting

from

th

e ch

emica

l re

actio

n be

twee

n 10

micr

ogra

ms

of

nico

tinic

acid

in

pur

e so

lutio

n an

d th

e re

agen

ts.

g Th

is K

valu

e,

calcu

late

d by

diff

eren

ce,

corre

spon

ds

to t

he

incr

ease

in

ph

otom

etric

de

nsity

wh

en

the

test

is

carri

ed

out

with

10

micr

ogra

ms

of n

icotin

ic ac

id

adde

d to

ano

ther

on

e-te

nth

aliq

uot

of th

e te

st s

olut

ion.

jl

Cons

isten

t low

re

cove

ries

of 8

5 pe

r ce

nt

of a

dded

ni

cotin

amid

e ar

e ob

tain

ed

in t

ests

with

sa

liva.

Th

ese

low

valu

es

are

not

due

to

any

dest

ruct

ion

of n

icotin

amid

e by

sa

liva

or

to

adso

rptio

n of

nico

tinic

acid

du

ring

char

coal

de

colo

rizat

ion.

Be

caus

e th

e lo

ss i

s no

t an

abs

olut

e on

e bu

t a

cons

tant

fra

ctio

n of

the

nico

tinam

ide

adde

d,

the

foun

d va

lue

of m

g.

per

cent

of

nico

tinic

acid

in

sal

iva

has

been

co

rrect

ed

for

this

lo

st

fract

ion.

7

One

20

0 m

g.

char

coal

ad

ditio

n is

em

ploy

ed

for

the

deco

loriz

atio

n of

eac

h of

the

te

st

solu

tions

, Sa

mpl

es

A to

K.

In

Sam

ples

L

and

M a

n ad

ditio

nal

treat

men

t wi

th

100

mg.

of c

harc

oal

is c

arrie

d ou

t to

obt

ain

satis

fact

ory

deco

lorie

atio

n.

** F

or

the

deco

loriz

atio

n of

th

e al

coho

lic

hydr

olys

ate

of w

hole

blood

th

ree

char

coal

ad

ditio

ns

of 3

00 m

g. a

re e

mpl

oyed

. 40

mg.

of p

otas

sium

ox

alat

e po

wder

ar

e us

ed a

s th

e an

ticoa

gula

nt

for

10 c

c. o

f bl

ood.

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14 Determination of Nicotinic Acid

rials. A more detailed and extended report of these findings will be the subject of a subsequent paper.

In the fifth column of Table IV, the amount of color remaining in an aliquot of each of the test samples subsequent to charcoal decolorization and neutralization is indicated. The specimens from Samples A to K contained very little color. In the case of Samples L to N the decolorization was not satisfactory when the usual single addition of 200 mg. of charcoal was used. Additional charcoal treatments were essential. In the analysis of materials such as milk and plasma the initial charcoal adsorbate is removed by centrifugation and an additional 100 mg. quantity of charcoal added to the decanted solution. The mixture is then filtered. With whole blood three additions of 300 mg. of charcoal are employed to decolorize the alcoholic hydrolysates. Because substances in biological solutions inhibit adsorption of nicotinic acid, amounts of charcoal can be used for the decolorization of such solutions in excess of the maximum found in tests with pure acidic alcoholic solutions of nicotinic acid (see Table II). The excellent recoveries of the nicotinamide added to Samples L to N support the validity of this new procedure of stepwise preferential charcoal adsorption for the decolorization of solutions with no loss of nicotinic acid.

In Table IV comparison is made between the K values obtained in tests with 10 micrograms of nicotinic acid in pure solutions and those calculated by difference when the 10 microgram quantities of nicotinic acid were added to aliquots of the test solutions. The discrepancies between these two series of K values are real and do not represent errors in the analyses. The variations in these values among the different test solutions are due to the presence of other substances in varying concentration in these solutions which tend to modify the sensitivity of the reaction. However, in any given case the K value is constant over the range of nicotinic acid concentration determined by the present method. This was indicated by additional tests with graded am0unt.s of nicotinic acid (from 1 to 50 micrograms) added to the same test solution.

Von Euler and associates (3) have pointed out that the color produced by the reaction of the reagents with nicotinamide is fully 4 times as great as that obtained with the dihydropyridine remaining after acid hydrolysis of the reduced codehydrogenases.

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D. Melnick and H. Field, Jr. 15

They state that these compounds are completely oxidized by atmospheric oxygen during simple aqueous extraction and that solutions of the reduced codehydrogenases are obtained only by alkaline extraction. The yield of nicotinic acid from the test materials of this study was not increased by preliminary aeration11 in the presence of hydrogen peroxide at pH 7 and at 37” for 30 minutes.

SUMMARY

Nicotinic acid reacts with cyanogen bromide to give a yellow color, the intensity of which is increased 3- to 5-fold by the addition of aniline. The ratio of maximal photometric density, measured by the Evelyn photoelectric calorimeter, to the quantity of the acid (from 3 to 50 micrograms) is a constant. With application of the proper correction factor as little as 1 microgram of nicotinic acid may be determined. The reagents, when properly stored, are suitable for use for a period of at least 5 months. The chemical reaction has been used in the analyses of yeast, liver, rice polish, and wheat germ preparations and of urine, milk, saliva, plasma, and whole blood. The test material is subjected to direct acid hydrolysis, followed by preferential charcoal adsorption for the decolorization of the solution with no concomitant loss of nicotinic acid. A new procedure is described for the estimation of the photometric density due solely to the reaction between nicotinic acid and reagents and for the conversion of this value into absolute units of the acid. The necessity for using acid in preference to alkaline hydrolysis is indicated. Oxidation prior to hydrolysis does not appear to be essential.

Addendum-While this paper was in press, the report by Harris and Raymond (13) appeared, advocating the addition of aniline to the blank test. The base was found to react directly with substances in the hydroly- sates to give colors indistinguishable from that obtained in tests for nico- tinic acid. We had also found this to be true. However, our more extensive studies indicated conclusively that in the presence of cyanogen bromide

l1 In the case of the whole blood samples complete hemolysis was ef- fected in one series by simple dilution with water, in the other by the addition of saponin. The reason for this preliminary treatment is the fact that the nicotinamide-containing coeneymes of the blood are entirely in the cells (12).

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Determination of Nicotinic Acid

these interfering side reactions do not occur. The details of these studies are now in press. Accordingly, we omitted aniline from the blank test and used the procedure described in this paper for evaluating residual color in the hydrolysate.

BIBLIOGRAPHY

1. Swaminathan, M., Indian J. Med. Research, 26, 427 (1938). 2. Shaw, G. E., and MacDonald, C. A., Quart. J. Pharm. and Pharmacol.,

11,380 (1938). 3. von Euler, H., Schlenk, F., Heiwinkel, H., and Hogberg, B., 2. physiol.

Chem., 266, 208 (1938). 4. Bandier, E., and Hald, J., Biochem. J., 33, 264 (1939). 5. Bandier, E., Biochem. J., 33, 1130, 1787 (1939). 6. Askeliif, E., and Holmberg, C., Svensk Farm. Tidskr., 43, 301, 321

(1939); Chem. Abst., 33, 8229 (1939). 7. Pearson, P. B., J. Biol. Chem., 129, 491 (1939). 8. Ritsert, K., K&in. Voch., 18, 934 (1939). 9. Kringstad, H., and Naess, T., 2. physio2. Chem., 260, 108 (1939).

10. Evelyn, K. A., J. Biol. Chem., 116, 63 (1936). 11. Stiller, E. T., Keresztesy, J. C., and Stevens, J. R., J. Am. Chem. SO:.,

61, 1237 (1939). 12. Kohn, H. I., and Bernheim, F., J. CZin. Znv., 18, 585 (1939). 13. Harris, L. J., and Raymond, W. D., Biochem. J., 33, 2037 (1939).

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Daniel Melnick and Henry Field, Jr.COLORIMETRY

MEANS OF PHOTOELECTRICIN BIOLOGICAL MATERIALS BY

DETERMINATION OF NICOTINIC ACID

1940, 134:1-16.J. Biol. Chem. 

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