THisis

my attempt at getting something cool. I dont know why they allow for us
to do this but I am thankful. I can repat theis all the time.

The physiological importance of choline is well recognized. Its two
primary functions as a donor of methyl groups and as a constituent of
lecithin and sphingomyelin have received considerable attention. Virtually
nothing is known about the relatively small but probably significant
amounts of free or unbound choline occurring in body tissues. Suitable
methods for determining free choline in animal tissues have not heretofore
been available. An adaptation to body tissues of the microbiological
method for measuring free choline in plasma and urine (1) appeared to offer
the best possibilities. The development of such a method would provide
a means for studying the physiological importance of free choline. The
organs used in the present study have been those that are considered more
important in the metabolism of choline.
The microbiological method of Horowitz and Beadle (2) for the determination
of choline has with minor modifications proved very satisfactory
in our laboratory. However, before making extensive use of the method
we considered it desirable to make a comparison of the values obtained by
the microbiological and chemical methods. The results of this study are
included in the present paper.
Procedure
Free Choline-A 2 to 3 gm. sample of the fresh tissue is weighed and
ground in a mortar. The finely minced tissue is transferred to a 125 ml.
Erlenmeyer flask-with the aid of 50 ml. of a 1 per cent sodium acetate
solution adjusted to pH 4.6. The flasks are then placed in an oven at a
temperature of 80 for 1 hour. The solution is then centrifuged and the
supernatant decanted. A 5 ml. aliquot of the liquid is transferred to a
15 ml. tapered centrifuge tube containing 10 ml. of acetone. The tubes
are then placed in an ice bath for a period of 2 hours and the resulting
precipitate removed by centrifugation. The purpose of this acetone treatment
is to precipitate any lecithin which may be present. That the precipitate
does contain appreciable amounts of lecithin will be shown later
in the paper. The liquid portion of the acetone-treated extract is trans-
507
by guest on May 6, 2016 http://www.jbc.org/ Downloaded from
508 DETERMINATION OF FREE CHOLINE
ferred to a small beaker which is placed over a steam bath until all of the
acetone is evaporated. The remaining solution is then made up to a convenient
volume, usually 50 ml. 10 ml. of this solution are passed through
an adsorption column containing approximately 1 gm. of activated Decalso
(60 to 80 mesh). The activated Decalso is prepared in the manner described
by Hennessy (3) for thiamine adsorption.
Elution of the choline is effected with 10 ml. of a solution of 5 per cent
sodium chloride. The choline-free medium is the same as that described
by Horowitz and Beadle (2) with the exception that 1 y of biotin per liter
gives as good growth response as 5 y which were used in the original medium.
The methyl ester was found to be as effective as free biotin when used at
a level to furnish equivalent amounts of biotin. Cholineless differs from

Lacto~acillus arabinosus in its ability to utilize the methyl ester of biotin,

as it is not available to the latter organism (4). Inoculation with cholineless,
incubation, and measurement of growth response are carried out as
described in the original method (2).
Total Cholirqe-For the determination of total choline a 0.2 gm. sample
of finely minced tissue is treated with 10 ml. of 3 N hydrochloric acid and
autoclaved at 15 pounds pressure for 2 hours. The hydrolysate is neutralized
with sodium hydroxide and the whole brought to a convenient volume.
An aliquot is now adsorbed on Decalso and the remainder of the procedure
carried out asdescribed for free choline.
Borowitz and Beadle (2) used 3 per cent sulfuric acid for the liberation
of choline from,le$hin. We have compared the results obtained by hydrolyzing
with 3 per cent sulfuric acid, saturated barium hydroxide, and 3 N
hydrochloric acid. The three hydrolysates on the same tissue gave identical
results. Since hydrolysis with hydrochloric acid is less laborious, it has
been used routinely.
EXPERSMENTAL
If the total choline content of a tissue could be obtained by adding the
free choline plus the choline of the water-extracted tissueafter hydrolysis,
and the choline obtained by hydrolyzing the precipitate formed by treating
the water extract with acetone, it would serve as a measure of the adequacy
of the water extraction and acetone treatment. This procedure was followed
with four samples of liver. The average results for the four samples
expressed in mg. per gm. were as follows: free choline content of water
extract 0.12, choline in water-extracted residue after hydrolysis 5.30,
choline in acetone precipitate of water extract after hydrolysis 0.42. The
total choline content of the liver obtained by adding the three fractions was
5.84 mg. per gm., which differs by less than 3 per cent from the value of 6.07
mg. per gm. obtained by determination of total choline on the fresh liver.
by guest on May 6, 2016 http://www.jbc.org/ Downloaded from
R. Pr. LUECKE AND P. B. PEARSON 509
Agreement between the two values is well within the range of experimental
error claimed for the method, and affords confirmatory evidence as to the
adequacy of the method for free choline.
Experiments were carried out on pure soy bean lecithin to determine
whether the lecithin was completely precipitated by the acetone treatment
of the water extract. The choline present in lecithin is partially utilized
by cholineless and so the complete precipitation of it is essential. Known
amounts of soy bean lecithin were suspended in water and the procedure
followed as previously described for the determination of free choline. The
amount of free choline obtained was negligible, showing that the lecithin
was completely precipitated by the treatment with acetone.
It seemed desirable to determine whether the values for free choline
obtained by extraction with water could be verified by extraction with 70
per cent acetone. Since choline is soluble in 70 per cent acetone, while
lecithin is insoluble, the two methods of extraction should givethe same
values. To test this assumption a 2 gm. sample of minced liver tissue in a
fiber extraction thiible were placed in a Soxhlet extractor. The extraction
was carried out with 70 per cent acetone for 24 hours. At the end of this

period the acetone was evaporated off over a steam bath and the residual
solution made up to 50 ml. This solution was then assayed for free choline.
The free choline values determined by this method on six samples of liver
were not signilicantly different from the values obtained by extraction with
water followed by treatment with acetone, as described in the procedure.
E$ect of Adsorption-All non-basic substances which may interfere
with the growth of the Neurospora are eliminated by the Decalso treatment.
Methionine if present in sufficient amounts will be utilized by cholineless
and thus must also be eliminated (2). The effect of pH on adsorption of
solutions containing choline was studied. The results showed that choline
is quantitatively adsorbed at a pH of from 4.5 to 7.0. Table I shows the
effect of adsorption on the free and total choline values of liver. The
values for free choline are very much less after adsorption. Thus it is
apparent that there are appreciable amounts of foreign growth substances
present in the water extract and that their effect can be eliminated by
adsorption.
While there is a decrease in the values for total choline with
adsorption, it is not nearly so pronounced as in the case of free choline.
Nevertheless the difference is sufficient to warrant adsorption in the
determination
of total choline.
Standard solutions of choline chloride adsorbed on Decalso are eluted
quantitatively with a solution of 5 per cent sodium chloride.
Recovery of Choline Added to Tissue-Known amounts of choline chloride
were added to minced beef liver and assays made to determine the recovery.
Both total and free choline were determined on each sample of liver.
THisis my attempt at getting something cool. I dont know why they allow for us
to do this but I am thankful. I can repat theis all the time.

The physiological importance of choline is well recognized. Its two
primary functions as a donor of methyl groups and as a constituent of
lecithin and sphingomyelin have received considerable attention. Virtually
nothing is known about the relatively small but probably significant
amounts of free or unbound choline occurring in body tissues. Suitable
methods for determining free choline in animal tissues have not heretofore
been available. An adaptation to body tissues of the microbiological
method for measuring free choline in plasma and urine (1) appeared to offer
the best possibilities. The development of such a method would provide
a means for studying the physiological importance of free choline. The
organs used in the present study have been those that are considered more
important in the metabolism of choline.
The microbiological method of Horowitz and Beadle (2) for the determination
of choline has with minor modifications proved very satisfactory
in our laboratory. However, before making extensive use of the method
we considered it desirable to make a comparison of the values obtained by
the microbiological and chemical methods. The results of this study are
included in the present paper.
Procedure
Free Choline-A 2 to 3 gm. sample of the fresh tissue is weighed and

ground in a mortar. The finely minced tissue is transferred to a 125 ml.

Erlenmeyer flask-with the aid of 50 ml. of a 1 per cent sodium acetate
solution adjusted to pH 4.6. The flasks are then placed in an oven at a
temperature of 80 for 1 hour. The solution is then centrifuged and the
supernatant decanted. A 5 ml. aliquot of the liquid is transferred to a
15 ml. tapered centrifuge tube containing 10 ml. of acetone. The tubes
are then placed in an ice bath for a period of 2 hours and the resulting
precipitate removed by centrifugation. The purpose of this acetone treatment
is to precipitate any lecithin which may be present. That the precipitate
does contain appreciable amounts of lecithin will be shown later
in the paper. The liquid portion of the acetone-treated extract is trans-
507
by guest on May 6, 2016 http://www.jbc.org/ Downloaded from
508 DETERMINATION OF FREE CHOLINE
ferred to a small beaker which is placed over a steam bath until all of the
acetone is evaporated. The remaining solution is then made up to a convenient
volume, usually 50 ml. 10 ml. of this solution are passed through
an adsorption column containing approximately 1 gm. of activated Decalso
(60 to 80 mesh). The activated Decalso is prepared in the manner described
by Hennessy (3) for thiamine adsorption.
Elution of the choline is effected with 10 ml. of a solution of 5 per cent
sodium chloride. The choline-free medium is the same as that described
by Horowitz and Beadle (2) with the exception that 1 y of biotin per liter
gives as good growth response as 5 y which were used in the original medium.
The methyl ester was found to be as effective as free biotin when used at
a level to furnish equivalent amounts of biotin. Cholineless differs from
Lacto~acillus arabinosus in its ability to utilize the methyl ester of biotin,
as it is not available to the latter organism (4). Inoculation with cholineless,
incubation, and measurement of growth response are carried out as
described in the original method (2).
Total Cholirqe-For the determination of total choline a 0.2 gm. sample
of finely minced tissue is treated with 10 ml. of 3 N hydrochloric acid and
autoclaved at 15 pounds pressure for 2 hours. The hydrolysate is neutralized
with sodium hydroxide and the whole brought to a convenient volume.
An aliquot is now adsorbed on Decalso and the remainder of the procedure
carried out asdescribed for free choline.
Borowitz and Beadle (2) used 3 per cent sulfuric acid for the liberation
of choline from,le$hin. We have compared the results obtained by hydrolyzing
with 3 per cent sulfuric acid, saturated barium hydroxide, and 3 N
hydrochloric acid. The three hydrolysates on the same tissue gave identical
results. Since hydrolysis with hydrochloric acid is less laborious, it has
been used routinely.
EXPERSMENTAL
If the total choline content of a tissue could be obtained by adding the
free choline plus the choline of the water-extracted tissueafter hydrolysis,
and the choline obtained by hydrolyzing the precipitate formed by treating
the water extract with acetone, it would serve as a measure of the adequacy
of the water extraction and acetone treatment. This procedure was followed
with four samples of liver. The average results for the four samples

expressed in mg. per gm. were as follows: free choline content of water
extract 0.12, choline in water-extracted residue after hydrolysis 5.30,
choline in acetone precipitate of water extract after hydrolysis 0.42. The
total choline content of the liver obtained by adding the three fractions was
5.84 mg. per gm., which differs by less than 3 per cent from the value of 6.07
mg. per gm. obtained by determination of total choline on the fresh liver.
by guest on May 6, 2016 http://www.jbc.org/ Downloaded from
R. Pr. LUECKE AND P. B. PEARSON 509
Agreement between the two values is well within the range of experimental
error claimed for the method, and affords confirmatory evidence as to the
adequacy of the method for free choline.
Experiments were carried out on pure soy bean lecithin to determine
whether the lecithin was completely precipitated by the acetone treatment
of the water extract. The choline present in lecithin is partially utilized
by cholineless and so the complete precipitation of it is essential. Known
amounts of soy bean lecithin were suspended in water and the procedure
followed as previously described for the determination of free choline. The
amount of free choline obtained was negligible, showing that the lecithin
was completely precipitated by the treatment with acetone.
It seemed desirable to determine whether the values for free choline
obtained by extraction with water could be verified by extraction with 70
per cent acetone. Since choline is soluble in 70 per cent acetone, while
lecithin is insoluble, the two methods of extraction should givethe same
values. To test this assumption a 2 gm. sample of minced liver tissue in a
fiber extraction thiible were placed in a Soxhlet extractor. The extraction
was carried out with 70 per cent acetone for 24 hours. At the end of this
period the acetone was evaporated off over a steam bath and the residual
solution made up to 50 ml. This solution was then assayed for free choline.
The free choline values determined by this method on six samples of liver
were not signilicantly different from the values obtained by extraction with
water followed by treatment with acetone, as described in the procedure.
E$ect of Adsorption-All non-basic substances which may interfere
with the growth of the Neurospora are eliminated by the Decalso treatment.
Methionine if present in sufficient amounts will be utilized by cholineless
and thus must also be eliminated (2). The effect of pH on adsorption of
solutions containing choline was studied. The results showed that choline
is quantitatively adsorbed at a pH of from 4.5 to 7.0. Table I shows the
effect of adsorption on the free and total choline values of liver. The
values for free choline are very much less after adsorption. Thus it is
apparent that there are appreciable amounts of foreign growth substances
present in the water extract and that their effect can be eliminated by
adsorption.
While there is a decrease in the values for total choline with
adsorption, it is not nearly so pronounced as in the case of free choline.
Nevertheless the difference is sufficient to warrant adsorption in the
determination
of total choline.
Standard solutions of choline chloride adsorbed on Decalso are eluted
quantitatively with a solution of 5 per cent sodium chloride.

Recovery of Choline Added to Tissue-Known amounts of choline chloride

were added to minced beef liver and assays made to determine the recovery.
Both total and free choline were determined on each sample of liver.
THisis my attempt at getting something cool. I dont know why they allow for us
to do this but I am thankful. I can repat theis all the time.

The physiological importance of choline is well recognized. Its two
primary functions as a donor of methyl groups and as a constituent of
lecithin and sphingomyelin have received considerable attention. Virtually
nothing is known about the relatively small but probably significant
amounts of free or unbound choline occurring in body tissues. Suitable
methods for determining free choline in animal tissues have not heretofore
been available. An adaptation to body tissues of the microbiological
method for measuring free choline in plasma and urine (1) appeared to offer
the best possibilities. The development of such a method would provide
a means for studying the physiological importance of free choline. The
organs used in the present study have been those that are considered more
important in the metabolism of choline.
The microbiological method of Horowitz and Beadle (2) for the determination
of choline has with minor modifications proved very satisfactory
in our laboratory. However, before making extensive use of the method
we considered it desirable to make a comparison of the values obtained by
the microbiological and chemical methods. The results of this study are
included in the present paper.
Procedure
Free Choline-A 2 to 3 gm. sample of the fresh tissue is weighed and
ground in a mortar. The finely minced tissue is transferred to a 125 ml.
Erlenmeyer flask-with the aid of 50 ml. of a 1 per cent sodium acetate
solution adjusted to pH 4.6. The flasks are then placed in an oven at a
temperature of 80 for 1 hour. The solution is then centrifuged and the
supernatant decanted. A 5 ml. aliquot of the liquid is transferred to a
15 ml. tapered centrifuge tube containing 10 ml. of acetone. The tubes
are then placed in an ice bath for a period of 2 hours and the resulting
precipitate removed by centrifugation. The purpose of this acetone treatment
is to precipitate any lecithin which may be present. That the precipitate
does contain appreciable amounts of lecithin will be shown later
in the paper. The liquid portion of the acetone-treated extract is trans-
507
by guest on May 6, 2016 http://www.jbc.org/ Downloaded from
508 DETERMINATION OF FREE CHOLINE
ferred to a small beaker which is placed over a steam bath until all of the
acetone is evaporated. The remaining solution is then made up to a convenient
volume, usually 50 ml. 10 ml. of this solution are passed through
an adsorption column containing approximately 1 gm. of activated Decalso
(60 to 80 mesh). The activated Decalso is prepared in the manner described
by Hennessy (3) for thiamine adsorption.
Elution of the choline is effected with 10 ml. of a solution of 5 per cent
sodium chloride. The choline-free medium is the same as that described
by Horowitz and Beadle (2) with the exception that 1 y of biotin per liter

gives as good growth response as 5 y which were used in the original medium.
The methyl ester was found to be as effective as free biotin when used at
a level to furnish equivalent amounts of biotin. Cholineless differs from
Lacto~acillus arabinosus in its ability to utilize the methyl ester of biotin,
as it is not available to the latter organism (4). Inoculation with cholineless,
incubation, and measurement of growth response are carried out as
described in the original method (2).
Total Cholirqe-For the determination of total choline a 0.2 gm. sample
of finely minced tissue is treated with 10 ml. of 3 N hydrochloric acid and
autoclaved at 15 pounds pressure for 2 hours. The hydrolysate is neutralized
with sodium hydroxide and the whole brought to a convenient volume.
An aliquot is now adsorbed on Decalso and the remainder of the procedure
carried out asdescribed for free choline.
Borowitz and Beadle (2) used 3 per cent sulfuric acid for the liberation
of choline from,le$hin. We have compared the results obtained by hydrolyzing
with 3 per cent sulfuric acid, saturated barium hydroxide, and 3 N
hydrochloric acid. The three hydrolysates on the same tissue gave identical
results. Since hydrolysis with hydrochloric acid is less laborious, it has
been used routinely.
EXPERSMENTAL
If the total choline content of a tissue could be obtained by adding the
free choline plus the choline of the water-extracted tissueafter hydrolysis,
and the choline obtained by hydrolyzing the precipitate formed by treating
the water extract with acetone, it would serve as a measure of the adequacy
of the water extraction and acetone treatment. This procedure was followed
with four samples of liver. The average results for the four samples
expressed in mg. per gm. were as follows: free choline content of water
extract 0.12, choline in water-extracted residue after hydrolysis 5.30,
choline in acetone precipitate of water extract after hydrolysis 0.42. The
total choline content of the liver obtained by adding the three fractions was
5.84 mg. per gm., which differs by less than 3 per cent from the value of 6.07
mg. per gm. obtained by determination of total choline on the fresh liver.
by guest on May 6, 2016 http://www.jbc.org/ Downloaded from
R. Pr. LUECKE AND P. B. PEARSON 509
Agreement between the two values is well within the range of experimental
error claimed for the method, and affords confirmatory evidence as to the
adequacy of the method for free choline.
Experiments were carried out on pure soy bean lecithin to determine
whether the lecithin was completely precipitated by the acetone treatment
of the water extract. The choline present in lecithin is partially utilized
by cholineless and so the complete precipitation of it is essential. Known
amounts of soy bean lecithin were suspended in water and the procedure
followed as previously described for the determination of free choline. The
amount of free choline obtained was negligible, showing that the lecithin
was completely precipitated by the treatment with acetone.
It seemed desirable to determine whether the values for free choline
obtained by extraction with water could be verified by extraction with 70
per cent acetone. Since choline is soluble in 70 per cent acetone, while
lecithin is insoluble, the two methods of extraction should givethe same

values. To test this assumption a 2 gm. sample of minced liver tissue in a

fiber extraction thiible were placed in a Soxhlet extractor. The extraction
was carried out with 70 per cent acetone for 24 hours. At the end of this
period the acetone was evaporated off over a steam bath and the residual
solution made up to 50 ml. This solution was then assayed for free choline.
The free choline values determined by this method on six samples of liver
were not signilicantly different from the values obtained by extraction with
water followed by treatment with acetone, as described in the procedure.
E$ect of Adsorption-All non-basic substances which may interfere
with the growth of the Neurospora are eliminated by the Decalso treatment.
Methionine if present in sufficient amounts will be utilized by cholineless
and thus must also be eliminated (2). The effect of pH on adsorption of
solutions containing choline was studied. The results showed that choline
is quantitatively adsorbed at a pH of from 4.5 to 7.0. Table I shows the
effect of adsorption on the free and total choline values of liver. The
values for free choline are very much less after adsorption. Thus it is
apparent that there are appreciable amounts of foreign growth substances
present in the water extract and that their effect can be eliminated by
adsorption.
While there is a decrease in the values for total choline with
adsorption, it is not nearly so pronounced as in the case of free choline.
Nevertheless the difference is sufficient to warrant adsorption in the
determination
of total choline.
Standard solutions of choline chloride adsorbed on Decalso are eluted
quantitatively with a solution of 5 per cent sodium chloride.
Recovery of Choline Added to Tissue-Known amounts of choline chloride
were added to minced beef liver and assays made to determine the recovery.
Both total and free choline were determined on each sample of liver.
THisis my attempt at getting something cool. I dont know why they allow for us
to do this but I am thankful. I can repat theis all the time.

The physiological importance of choline is well recognized. Its two
primary functions as a donor of methyl groups and as a constituent of
lecithin and sphingomyelin have received considerable attention. Virtually
nothing is known about the relatively small but probably significant
amounts of free or unbound choline occurring in body tissues. Suitable
methods for determining free choline in animal tissues have not heretofore
been available. An adaptation to body tissues of the microbiological
method for measuring free choline in plasma and urine (1) appeared to offer
the best possibilities. The development of such a method would provide
a means for studying the physiological importance of free choline. The
organs used in the present study have been those that are considered more
important in the metabolism of choline.
The microbiological method of Horowitz and Beadle (2) for the determination
of choline has with minor modifications proved very satisfactory
in our laboratory. However, before making extensive use of the method
we considered it desirable to make a comparison of the values obtained by
the microbiological and chemical methods. The results of this study are

included in the present paper.

Procedure
Free Choline-A 2 to 3 gm. sample of the fresh tissue is weighed and
ground in a mortar. The finely minced tissue is transferred to a 125 ml.
Erlenmeyer flask-with the aid of 50 ml. of a 1 per cent sodium acetate
solution adjusted to pH 4.6. The flasks are then placed in an oven at a
temperature of 80 for 1 hour. The solution is then centrifuged and the
supernatant decanted. A 5 ml. aliquot of the liquid is transferred to a
15 ml. tapered centrifuge tube containing 10 ml. of acetone. The tubes
are then placed in an ice bath for a period of 2 hours and the resulting
precipitate removed by centrifugation. The purpose of this acetone treatment
is to precipitate any lecithin which may be present. That the precipitate
does contain appreciable amounts of lecithin will be shown later
in the paper. The liquid portion of the acetone-treated extract is trans-
507
by guest on May 6, 2016 http://www.jbc.org/ Downloaded from
508 DETERMINATION OF FREE CHOLINE
ferred to a small beaker which is placed over a steam bath until all of the
acetone is evaporated. The remaining solution is then made up to a convenient
volume, usually 50 ml. 10 ml. of this solution are passed through
an adsorption column containing approximately 1 gm. of activated Decalso
(60 to 80 mesh). The activated Decalso is prepared in the manner described
by Hennessy (3) for thiamine adsorption.
Elution of the choline is effected with 10 ml. of a solution of 5 per cent
sodium chloride. The choline-free medium is the same as that described
by Horowitz and Beadle (2) with the exception that 1 y of biotin per liter
gives as good growth response as 5 y which were used in the original medium.
The methyl ester was found to be as effective as free biotin when used at
a level to furnish equivalent amounts of biotin. Cholineless differs from
Lacto~acillus arabinosus in its ability to utilize the methyl ester of biotin,
as it is not available to the latter organism (4). Inoculation with cholineless,
incubation, and measurement of growth response are carried out as
described in the original method (2).
Total Cholirqe-For the determination of total choline a 0.2 gm. sample
of finely minced tissue is treated with 10 ml. of 3 N hydrochloric acid and
autoclaved at 15 pounds pressure for 2 hours. The hydrolysate is neutralized
with sodium hydroxide and the whole brought to a convenient volume.
An aliquot is now adsorbed on Decalso and the remainder of the procedure
carried out asdescribed for free choline.
Borowitz and Beadle (2) used 3 per cent sulfuric acid for the liberation
of choline from,le$hin. We have compared the results obtained by hydrolyzing
with 3 per cent sulfuric acid, saturated barium hydroxide, and 3 N
hydrochloric acid. The three hydrolysates on the same tissue gave identical
results. Since hydrolysis with hydrochloric acid is less laborious, it has
been used routinely.
EXPERSMENTAL
If the total choline content of a tissue could be obtained by adding the
free choline plus the choline of the water-extracted tissueafter hydrolysis,
and the choline obtained by hydrolyzing the precipitate formed by treating

the water extract with acetone, it would serve as a measure of the adequacy
of the water extraction and acetone treatment. This procedure was followed
with four samples of liver. The average results for the four samples
expressed in mg. per gm. were as follows: free choline content of water
extract 0.12, choline in water-extracted residue after hydrolysis 5.30,
choline in acetone precipitate of water extract after hydrolysis 0.42. The
total choline content of the liver obtained by adding the three fractions was
5.84 mg. per gm., which differs by less than 3 per cent from the value of 6.07
mg. per gm. obtained by determination of total choline on the fresh liver.
by guest on May 6, 2016 http://www.jbc.org/ Downloaded from
R. Pr. LUECKE AND P. B. PEARSON 509
Agreement between the two values is well within the range of experimental
error claimed for the method, and affords confirmatory evidence as to the
adequacy of the method for free choline.
Experiments were carried out on pure soy bean lecithin to determine
whether the lecithin was completely precipitated by the acetone treatment
of the water extract. The choline present in lecithin is partially utilized
by cholineless and so the complete precipitation of it is essential. Known
amounts of soy bean lecithin were suspended in water and the procedure
followed as previously described for the determination of free choline. The
amount of free choline obtained was negligible, showing that the lecithin
was completely precipitated by the treatment with acetone.
It seemed desirable to determine whether the values for free choline
obtained by extraction with water could be verified by extraction with 70
per cent acetone. Since choline is soluble in 70 per cent acetone, while
lecithin is insoluble, the two methods of extraction should givethe same
values. To test this assumption a 2 gm. sample of minced liver tissue in a
fiber extraction thiible were placed in a Soxhlet extractor. The extraction
was carried out with 70 per cent acetone for 24 hours. At the end of this
period the acetone was evaporated off over a steam bath and the residual
solution made up to 50 ml. This solution was then assayed for free choline.
The free choline values determined by this method on six samples of liver
were not signilicantly different from the values obtained by extraction with
water followed by treatment with acetone, as described in the procedure.
E$ect of Adsorption-All non-basic substances which may interfere
with the growth of the Neurospora are eliminated by the Decalso treatment.
Methionine if present in sufficient amounts will be utilized by cholineless
and thus must also be eliminated (2). The effect of pH on adsorption of
solutions containing choline was studied. The results showed that choline
is quantitatively adsorbed at a pH of from 4.5 to 7.0. Table I shows the
effect of adsorption on the free and total choline values of liver. The
values for free choline are very much less after adsorption. Thus it is
apparent that there are appreciable amounts of foreign growth substances
present in the water extract and that their effect can be eliminated by
adsorption.
While there is a decrease in the values for total choline with
adsorption, it is not nearly so pronounced as in the case of free choline.
Nevertheless the difference is sufficient to warrant adsorption in the
determination

of total choline.
Standard solutions of choline chloride adsorbed on Decalso are eluted
quantitatively with a solution of 5 per cent sodium chloride.
Recovery of Choline Added to Tissue-Known amounts of choline chloride
were added to minced beef liver and assays made to determine the recovery.
Both total and free choline were determined on each sample of liver.
THisis my attempt at getting something cool. I dont know why they allow for us
to do this but I am thankful. I can repat theis all the time.

The physiological importance of choline is well recognized. Its two
primary functions as a donor of methyl groups and as a constituent of
lecithin and sphingomyelin have received considerable attention. Virtually
nothing is known about the relatively small but probably significant
amounts of free or unbound choline occurring in body tissues. Suitable
methods for determining free choline in animal tissues have not heretofore
been available. An adaptation to body tissues of the microbiological
method for measuring free choline in plasma and urine (1) appeared to offer
the best possibilities. The development of such a method would provide
a means for studying the physiological importance of free choline. The
organs used in the present study have been those that are considered more
important in the metabolism of choline.
The microbiological method of Horowitz and Beadle (2) for the determination
of choline has with minor modifications proved very satisfactory
in our laboratory. However, before making extensive use of the method
we considered it desirable to make a comparison of the values obtained by
the microbiological and chemical methods. The results of this study are
included in the present paper.
Procedure
Free Choline-A 2 to 3 gm. sample of the fresh tissue is weighed and
ground in a mortar. The finely minced tissue is transferred to a 125 ml.
Erlenmeyer flask-with the aid of 50 ml. of a 1 per cent sodium acetate
solution adjusted to pH 4.6. The flasks are then placed in an oven at a
temperature of 80 for 1 hour. The solution is then centrifuged and the
supernatant decanted. A 5 ml. aliquot of the liquid is transferred to a
15 ml. tapered centrifuge tube containing 10 ml. of acetone. The tubes
are then placed in an ice bath for a period of 2 hours and the resulting
precipitate removed by centrifugation. The purpose of this acetone treatment
is to precipitate any lecithin which may be present. That the precipitate
does contain appreciable amounts of lecithin will be shown later
in the paper. The liquid portion of the acetone-treated extract is trans-
507
by guest on May 6, 2016 http://www.jbc.org/ Downloaded from
508 DETERMINATION OF FREE CHOLINE
ferred to a small beaker which is placed over a steam bath until all of the
acetone is evaporated. The remaining solution is then made up to a convenient
volume, usually 50 ml. 10 ml. of this solution are passed through
an adsorption column containing approximately 1 gm. of activated Decalso
(60 to 80 mesh). The activated Decalso is prepared in the manner described
by Hennessy (3) for thiamine adsorption.

Elution of the choline is effected with 10 ml. of a solution of 5 per cent

sodium chloride. The choline-free medium is the same as that described
by Horowitz and Beadle (2) with the exception that 1 y of biotin per liter
gives as good growth response as 5 y which were used in the original medium.
The methyl ester was found to be as effective as free biotin when used at
a level to furnish equivalent amounts of biotin. Cholineless differs from
Lacto~acillus arabinosus in its ability to utilize the methyl ester of biotin,
as it is not available to the latter organism (4). Inoculation with cholineless,
incubation, and measurement of growth response are carried out as
described in the original method (2).
Total Cholirqe-For the determination of total choline a 0.2 gm. sample
of finely minced tissue is treated with 10 ml. of 3 N hydrochloric acid and
autoclaved at 15 pounds pressure for 2 hours. The hydrolysate is neutralized
with sodium hydroxide and the whole brought to a convenient volume.
An aliquot is now adsorbed on Decalso and the remainder of the procedure
carried out asdescribed for free choline.
Borowitz and Beadle (2) used 3 per cent sulfuric acid for the liberation
of choline from,le$hin. We have compared the results obtained by hydrolyzing
with 3 per cent sulfuric acid, saturated barium hydroxide, and 3 N
hydrochloric acid. The three hydrolysates on the same tissue gave identical
results. Since hydrolysis with hydrochloric acid is less laborious, it has
been used routinely.
EXPERSMENTAL
If the total choline content of a tissue could be obtained by adding the
free choline plus the choline of the water-extracted tissueafter hydrolysis,
and the choline obtained by hydrolyzing the precipitate formed by treating
the water extract with acetone, it would serve as a measure of the adequacy
of the water extraction and acetone treatment. This procedure was followed
with four samples of liver. The average results for the four samples
expressed in mg. per gm. were as follows: free choline content of water
extract 0.12, choline in water-extracted residue after hydrolysis 5.30,
choline in acetone precipitate of water extract after hydrolysis 0.42. The
total choline content of the liver obtained by adding the three fractions was
5.84 mg. per gm., which differs by less than 3 per cent from the value of 6.07
mg. per gm. obtained by determination of total choline on the fresh liver.
by guest on May 6, 2016 http://www.jbc.org/ Downloaded from
R. Pr. LUECKE AND P. B. PEARSON 509
Agreement between the two values is well within the range of experimental
error claimed for the method, and affords confirmatory evidence as to the
adequacy of the method for free choline.
Experiments were carried out on pure soy bean lecithin to determine
whether the lecithin was completely precipitated by the acetone treatment
of the water extract. The choline present in lecithin is partially utilized
by cholineless and so the complete precipitation of it is essential. Known
amounts of soy bean lecithin were suspended in water and the procedure
followed as previously described for the determination of free choline. The
amount of free choline obtained was negligible, showing that the lecithin
was completely precipitated by the treatment with acetone.
It seemed desirable to determine whether the values for free choline

obtained by extraction with water could be verified by extraction with 70

per cent acetone. Since choline is soluble in 70 per cent acetone, while
lecithin is insoluble, the two methods of extraction should givethe same
values. To test this assumption a 2 gm. sample of minced liver tissue in a
fiber extraction thiible were placed in a Soxhlet extractor. The extraction
was carried out with 70 per cent acetone for 24 hours. At the end of this
period the acetone was evaporated off over a steam bath and the residual
solution made up to 50 ml. This solution was then assayed for free choline.
The free choline values determined by this method on six samples of liver
were not signilicantly different from the values obtained by extraction with
water followed by treatment with acetone, as described in the procedure.
E$ect of Adsorption-All non-basic substances which may interfere
with the growth of the Neurospora are eliminated by the Decalso treatment.
Methionine if present in sufficient amounts will be utilized by cholineless
and thus must also be eliminated (2). The effect of pH on adsorption of
solutions containing choline was studied. The results showed that choline
is quantitatively adsorbed at a pH of from 4.5 to 7.0. Table I shows the
effect of adsorption on the free and total choline values of liver. The
values for free choline are very much less after adsorption. Thus it is
apparent that there are appreciable amounts of foreign growth substances
present in the water extract and that their effect can be eliminated by
adsorption.
While there is a decrease in the values for total choline with
adsorption, it is not nearly so pronounced as in the case of free choline.
Nevertheless the difference is sufficient to warrant adsorption in the
determination
of total choline.
Standard solutions of choline chloride adsorbed on Decalso are eluted
quantitatively with a solution of 5 per cent sodium chloride.
Recovery of Choline Added to Tissue-Known amounts of choline chloride
were added to minced beef liver and assays made to determine the recovery.
Both total and free choline were determined on each sample of liver.
THisis my attempt at getting something cool. I dont know why they allow for us
to do this but I am thankful. I can repat theis all the time.

The physiological importance of choline is well recognized. Its two
primary functions as a donor of methyl groups and as a constituent of
lecithin and sphingomyelin have received considerable attention. Virtually
nothing is known about the relatively small but probably significant
amounts of free or unbound choline occurring in body tissues. Suitable
methods for determining free choline in animal tissues have not heretofore
been available. An adaptation to body tissues of the microbiological
method for measuring free choline in plasma and urine (1) appeared to offer
the best possibilities. The development of such a method would provide
a means for studying the physiological importance of free choline. The
organs used in the present study have been those that are considered more
important in the metabolism of choline.
The microbiological method of Horowitz and Beadle (2) for the determination
of choline has with minor modifications proved very satisfactory

in our laboratory. However, before making extensive use of the method

we considered it desirable to make a comparison of the values obtained by
the microbiological and chemical methods. The results of this study are
included in the present paper.
Procedure
Free Choline-A 2 to 3 gm. sample of the fresh tissue is weighed and
ground in a mortar. The finely minced tissue is transferred to a 125 ml.
Erlenmeyer flask-with the aid of 50 ml. of a 1 per cent sodium acetate
solution adjusted to pH 4.6. The flasks are then placed in an oven at a
temperature of 80 for 1 hour. The solution is then centrifuged and the
supernatant decanted. A 5 ml. aliquot of the liquid is transferred to a
15 ml. tapered centrifuge tube containing 10 ml. of acetone. The tubes
are then placed in an ice bath for a period of 2 hours and the resulting
precipitate removed by centrifugation. The purpose of this acetone treatment
is to precipitate any lecithin which may be present. That the precipitate
does contain appreciable amounts of lecithin will be shown later
in the paper. The liquid portion of the acetone-treated extract is trans-
507
by guest on May 6, 2016 http://www.jbc.org/ Downloaded from
508 DETERMINATION OF FREE CHOLINE
ferred to a small beaker which is placed over a steam bath until all of the
acetone is evaporated. The remaining solution is then made up to a convenient
volume, usually 50 ml. 10 ml. of this solution are passed through
an adsorption column containing approximately 1 gm. of activated Decalso
(60 to 80 mesh). The activated Decalso is prepared in the manner described
by Hennessy (3) for thiamine adsorption.
Elution of the choline is effected with 10 ml. of a solution of 5 per cent
sodium chloride. The choline-free medium is the same as that described
by Horowitz and Beadle (2) with the exception that 1 y of biotin per liter
gives as good growth response as 5 y which were used in the original medium.
The methyl ester was found to be as effective as free biotin when used at
a level to furnish equivalent amounts of biotin. Cholineless differs from
Lacto~acillus arabinosus in its ability to utilize the methyl ester of biotin,
as it is not available to the latter organism (4). Inoculation with cholineless,
incubation, and measurement of growth response are carried out as
described in the original method (2).
Total Cholirqe-For the determination of total choline a 0.2 gm. sample
of finely minced tissue is treated with 10 ml. of 3 N hydrochloric acid and
autoclaved at 15 pounds pressure for 2 hours. The hydrolysate is neutralized
with sodium hydroxide and the whole brought to a convenient volume.
An aliquot is now adsorbed on Decalso and the remainder of the procedure
carried out asdescribed for free choline.
Borowitz and Beadle (2) used 3 per cent sulfuric acid for the liberation
of choline from,le$hin. We have compared the results obtained by hydrolyzing
with 3 per cent sulfuric acid, saturated barium hydroxide, and 3 N
hydrochloric acid. The three hydrolysates on the same tissue gave identical
results. Since hydrolysis with hydrochloric acid is less laborious, it has
been used routinely.
EXPERSMENTAL

If the total choline content of a tissue could be obtained by adding the

free choline plus the choline of the water-extracted tissueafter hydrolysis,
and the choline obtained by hydrolyzing the precipitate formed by treating
the water extract with acetone, it would serve as a measure of the adequacy
of the water extraction and acetone treatment. This procedure was followed
with four samples of liver. The average results for the four samples
expressed in mg. per gm. were as follows: free choline content of water
extract 0.12, choline in water-extracted residue after hydrolysis 5.30,
choline in acetone precipitate of water extract after hydrolysis 0.42. The
total choline content of the liver obtained by adding the three fractions was
5.84 mg. per gm., which differs by less than 3 per cent from the value of 6.07
mg. per gm. obtained by determination of total choline on the fresh liver.
by guest on May 6, 2016 http://www.jbc.org/ Downloaded from
R. Pr. LUECKE AND P. B. PEARSON 509
Agreement between the two values is well within the range of experimental
error claimed for the method, and affords confirmatory evidence as to the
adequacy of the method for free choline.
Experiments were carried out on pure soy bean lecithin to determine
whether the lecithin was completely precipitated by the acetone treatment
of the water extract. The choline present in lecithin is partially utilized
by cholineless and so the complete precipitation of it is essential. Known
amounts of soy bean lecithin were suspended in water and the procedure
followed as previously described for the determination of free choline. The
amount of free choline obtained was negligible, showing that the lecithin
was completely precipitated by the treatment with acetone.
It seemed desirable to determine whether the values for free choline
obtained by extraction with water could be verified by extraction with 70
per cent acetone. Since choline is soluble in 70 per cent acetone, while
lecithin is insoluble, the two methods of extraction should givethe same
values. To test this assumption a 2 gm. sample of minced liver tissue in a
fiber extraction thiible were placed in a Soxhlet extractor. The extraction
was carried out with 70 per cent acetone for 24 hours. At the end of this
period the acetone was evaporated off over a steam bath and the residual
solution made up to 50 ml. This solution was then assayed for free choline.
The free choline values determined by this method on six samples of liver
were not signilicantly different from the values obtained by extraction with
water followed by treatment with acetone, as described in the procedure.
E$ect of Adsorption-All non-basic substances which may interfere
with the growth of the Neurospora are eliminated by the Decalso treatment.
Methionine if present in sufficient amounts will be utilized by cholineless
and thus must also be eliminated (2). The effect of pH on adsorption of
solutions containing choline was studied. The results showed that choline
is quantitatively adsorbed at a pH of from 4.5 to 7.0. Table I shows the
effect of adsorption on the free and total choline values of liver. The
values for free choline are very much less after adsorption. Thus it is
apparent that there are appreciable amounts of foreign growth substances
present in the water extract and that their effect can be eliminated by
adsorption.
While there is a decrease in the values for total choline with

adsorption, it is not nearly so pronounced as in the case of free choline.

Nevertheless the difference is sufficient to warrant adsorption in the
determination
of total choline.
Standard solutions of choline chloride adsorbed on Decalso are eluted
quantitatively with a solution of 5 per cent sodium chloride.
Recovery of Choline Added to Tissue-Known amounts of choline chloride
were added to minced beef liver and assays made to determine the recovery.
Both total and free choline were determined on each sample of liver.
THisis my attempt at getting something cool. I dont know why they allow for us
to do this but I am thankful. I can repat theis all the time.

The physiological importance of choline is well recognized. Its two
primary functions as a donor of methyl groups and as a constituent of
lecithin and sphingomyelin have received considerable attention. Virtually
nothing is known about the relatively small but probably significant
amounts of free or unbound choline occurring in body tissues. Suitable
methods for determining free choline in animal tissues have not heretofore
been available. An adaptation to body tissues of the microbiological
method for measuring free choline in plasma and urine (1) appeared to offer
the best possibilities. The development of such a method would provide
a means for studying the physiological importance of free choline. The
organs used in the present study have been those that are considered more
important in the metabolism of choline.
The microbiological method of Horowitz and Beadle (2) for the determination
of choline has with minor modifications proved very satisfactory
in our laboratory. However, before making extensive use of the method
we considered it desirable to make a comparison of the values obtained by
the microbiological and chemical methods. The results of this study are
included in the present paper.
Procedure
Free Choline-A 2 to 3 gm. sample of the fresh tissue is weighed and
ground in a mortar. The finely minced tissue is transferred to a 125 ml.
Erlenmeyer flask-with the aid of 50 ml. of a 1 per cent sodium acetate
solution adjusted to pH 4.6. The flasks are then placed in an oven at a
temperature of 80 for 1 hour. The solution is then centrifuged and the
supernatant decanted. A 5 ml. aliquot of the liquid is transferred to a
15 ml. tapered centrifuge tube containing 10 ml. of acetone. The tubes
are then placed in an ice bath for a period of 2 hours and the resulting
precipitate removed by centrifugation. The purpose of this acetone treatment
is to precipitate any lecithin which may be present. That the precipitate
does contain appreciable amounts of lecithin will be shown later
in the paper. The liquid portion of the acetone-treated extract is trans-
507
by guest on May 6, 2016 http://www.jbc.org/ Downloaded from
508 DETERMINATION OF FREE CHOLINE
ferred to a small beaker which is placed over a steam bath until all of the
acetone is evaporated. The remaining solution is then made up to a convenient
volume, usually 50 ml. 10 ml. of this solution are passed through

an adsorption column containing approximately 1 gm. of activated Decalso

(60 to 80 mesh). The activated Decalso is prepared in the manner described
by Hennessy (3) for thiamine adsorption.
Elution of the choline is effected with 10 ml. of a solution of 5 per cent
sodium chloride. The choline-free medium is the same as that described
by Horowitz and Beadle (2) with the exception that 1 y of biotin per liter
gives as good growth response as 5 y which were used in the original medium.
The methyl ester was found to be as effective as free biotin when used at
a level to furnish equivalent amounts of biotin. Cholineless differs from
Lacto~acillus arabinosus in its ability to utilize the methyl ester of biotin,
as it is not available to the latter organism (4). Inoculation with cholineless,
incubation, and measurement of growth response are carried out as
described in the original method (2).
Total Cholirqe-For the determination of total choline a 0.2 gm. sample
of finely minced tissue is treated with 10 ml. of 3 N hydrochloric acid and
autoclaved at 15 pounds pressure for 2 hours. The hydrolysate is neutralized
with sodium hydroxide and the whole brought to a convenient volume.
An aliquot is now adsorbed on Decalso and the remainder of the procedure
carried out asdescribed for free choline.
Borowitz and Beadle (2) used 3 per cent sulfuric acid for the liberation
of choline from,le$hin. We have compared the results obtained by hydrolyzing
with 3 per cent sulfuric acid, saturated barium hydroxide, and 3 N
hydrochloric acid. The three hydrolysates on the same tissue gave identical
results. Since hydrolysis with hydrochloric acid is less laborious, it has
been used routinely.
EXPERSMENTAL
If the total choline content of a tissue could be obtained by adding the
free choline plus the choline of the water-extracted tissueafter hydrolysis,
and the choline obtained by hydrolyzing the precipitate formed by treating
the water extract with acetone, it would serve as a measure of the adequacy
of the water extraction and acetone treatment. This procedure was followed
with four samples of liver. The average results for the four samples
expressed in mg. per gm. were as follows: free choline content of water
extract 0.12, choline in water-extracted residue after hydrolysis 5.30,
choline in acetone precipitate of water extract after hydrolysis 0.42. The
total choline content of the liver obtained by adding the three fractions was
5.84 mg. per gm., which differs by less than 3 per cent from the value of 6.07
mg. per gm. obtained by determination of total choline on the fresh liver.
by guest on May 6, 2016 http://www.jbc.org/ Downloaded from
R. Pr. LUECKE AND P. B. PEARSON 509
Agreement between the two values is well within the range of experimental
error claimed for the method, and affords confirmatory evidence as to the
adequacy of the method for free choline.
Experiments were carried out on pure soy bean lecithin to determine
whether the lecithin was completely precipitated by the acetone treatment
of the water extract. The choline present in lecithin is partially utilized
by cholineless and so the complete precipitation of it is essential. Known
amounts of soy bean lecithin were suspended in water and the procedure
followed as previously described for the determination of free choline. The

amount of free choline obtained was negligible, showing that the lecithin
was completely precipitated by the treatment with acetone.
It seemed desirable to determine whether the values for free choline
obtained by extraction with water could be verified by extraction with 70
per cent acetone. Since choline is soluble in 70 per cent acetone, while
lecithin is insoluble, the two methods of extraction should givethe same
values. To test this assumption a 2 gm. sample of minced liver tissue in a
fiber extraction thiible were placed in a Soxhlet extractor. The extraction
was carried out with 70 per cent acetone for 24 hours. At the end of this
period the acetone was evaporated off over a steam bath and the residual
solution made up to 50 ml. This solution was then assayed for free choline.
The free choline values determined by this method on six samples of liver
were not signilicantly different from the values obtained by extraction with
water followed by treatment with acetone, as described in the procedure.
E$ect of Adsorption-All non-basic substances which may interfere
with the growth of the Neurospora are eliminated by the Decalso treatment.
Methionine if present in sufficient amounts will be utilized by cholineless
and thus must also be eliminated (2). The effect of pH on adsorption of
solutions containing choline was studied. The results showed that choline
is quantitatively adsorbed at a pH of from 4.5 to 7.0. Table I shows the
effect of adsorption on the free and total choline values of liver. The
values for free choline are very much less after adsorption. Thus it is
apparent that there are appreciable amounts of foreign growth substances
present in the water extract and that their effect can be eliminated by
adsorption.
While there is a decrease in the values for total choline with
adsorption, it is not nearly so pronounced as in the case of free choline.
Nevertheless the difference is sufficient to warrant adsorption in the
determination
of total choline.
Standard solutions of choline chloride adsorbed on Decalso are eluted
quantitatively with a solution of 5 per cent sodium chloride.
Recovery of Choline Added to Tissue-Known amounts of choline chloride
were added to minced beef liver and assays made to determine the recovery.
Both total and free choline were determined on each sample of liver.
THisis my attempt at getting something cool. I dont know why they allow for us
to do this but I am thankful. I can repat theis all the time.

The physiological importance of choline is well recognized. Its two
primary functions as a donor of methyl groups and as a constituent of
lecithin and sphingomyelin have received considerable attention. Virtually
nothing is known about the relatively small but probably significant
amounts of free or unbound choline occurring in body tissues. Suitable
methods for determining free choline in animal tissues have not heretofore
been available. An adaptation to body tissues of the microbiological
method for measuring free choline in plasma and urine (1) appeared to offer
the best possibilities. The development of such a method would provide
a means for studying the physiological importance of free choline. The
organs used in the present study have been those that are considered more

important in the metabolism of choline.

The microbiological method of Horowitz and Beadle (2) for the determination
of choline has with minor modifications proved very satisfactory
in our laboratory. However, before making extensive use of the method
we considered it desirable to make a comparison of the values obtained by
the microbiological and chemical methods. The results of this study are
included in the present paper.
Procedure
Free Choline-A 2 to 3 gm. sample of the fresh tissue is weighed and
ground in a mortar. The finely minced tissue is transferred to a 125 ml.
Erlenmeyer flask-with the aid of 50 ml. of a 1 per cent sodium acetate
solution adjusted to pH 4.6. The flasks are then placed in an oven at a
temperature of 80 for 1 hour. The solution is then centrifuged and the
supernatant decanted. A 5 ml. aliquot of the liquid is transferred to a
15 ml. tapered centrifuge tube containing 10 ml. of acetone. The tubes
are then placed in an ice bath for a period of 2 hours and the resulting
precipitate removed by centrifugation. The purpose of this acetone treatment
is to precipitate any lecithin which may be present. That the precipitate
does contain appreciable amounts of lecithin will be shown later
in the paper. The liquid portion of the acetone-treated extract is trans-
507
by guest on May 6, 2016 http://www.jbc.org/ Downloaded from
508 DETERMINATION OF FREE CHOLINE
ferred to a small beaker which is placed over a steam bath until all of the
acetone is evaporated. The remaining solution is then made up to a convenient
volume, usually 50 ml. 10 ml. of this solution are passed through
an adsorption column containing approximately 1 gm. of activated Decalso
(60 to 80 mesh). The activated Decalso is prepared in the manner described
by Hennessy (3) for thiamine adsorption.
Elution of the choline is effected with 10 ml. of a solution of 5 per cent
sodium chloride. The choline-free medium is the same as that described
by Horowitz and Beadle (2) with the exception that 1 y of biotin per liter
gives as good growth response as 5 y which were used in the original medium.
The methyl ester was found to be as effective as free biotin when used at
a level to furnish equivalent amounts of biotin. Cholineless differs from
Lacto~acillus arabinosus in its ability to utilize the methyl ester of biotin,
as it is not available to the latter organism (4). Inoculation with cholineless,
incubation, and measurement of growth response are carried out as
described in the original method (2).
Total Cholirqe-For the determination of total choline a 0.2 gm. sample
of finely minced tissue is treated with 10 ml. of 3 N hydrochloric acid and
autoclaved at 15 pounds pressure for 2 hours. The hydrolysate is neutralized
with sodium hydroxide and the whole brought to a convenient volume.
An aliquot is now adsorbed on Decalso and the remainder of the procedure
carried out asdescribed for free choline.
Borowitz and Beadle (2) used 3 per cent sulfuric acid for the liberation
of choline from,le$hin. We have compared the results obtained by hydrolyzing
with 3 per cent sulfuric acid, saturated barium hydroxide, and 3 N
hydrochloric acid. The three hydrolysates on the same tissue gave identical

results. Since hydrolysis with hydrochloric acid is less laborious, it has

been used routinely.
EXPERSMENTAL
If the total choline content of a tissue could be obtained by adding the
free choline plus the choline of the water-extracted tissueafter hydrolysis,
and the choline obtained by hydrolyzing the precipitate formed by treating
the water extract with acetone, it would serve as a measure of the adequacy
of the water extraction and acetone treatment. This procedure was followed
with four samples of liver. The average results for the four samples
expressed in mg. per gm. were as follows: free choline content of water
extract 0.12, choline in water-extracted residue after hydrolysis 5.30,
choline in acetone precipitate of water extract after hydrolysis 0.42. The
total choline content of the liver obtained by adding the three fractions was
5.84 mg. per gm., which differs by less than 3 per cent from the value of 6.07
mg. per gm. obtained by determination of total choline on the fresh liver.
by guest on May 6, 2016 http://www.jbc.org/ Downloaded from
R. Pr. LUECKE AND P. B. PEARSON 509
Agreement between the two values is well within the range of experimental
error claimed for the method, and affords confirmatory evidence as to the
adequacy of the method for free choline.
Experiments were carried out on pure soy bean lecithin to determine
whether the lecithin was completely precipitated by the acetone treatment
of the water extract. The choline present in lecithin is partially utilized
by cholineless and so the complete precipitation of it is essential. Known
amounts of soy bean lecithin were suspended in water and the procedure
followed as previously described for the determination of free choline. The
amount of free choline obtained was negligible, showing that the lecithin
was completely precipitated by the treatment with acetone.
It seemed desirable to determine whether the values for free choline
obtained by extraction with water could be verified by extraction with 70
per cent acetone. Since choline is soluble in 70 per cent acetone, while
lecithin is insoluble, the two methods of extraction should givethe same
values. To test this assumption a 2 gm. sample of minced liver tissue in a
fiber extraction thiible were placed in a Soxhlet extractor. The extraction
was carried out with 70 per cent acetone for 24 hours. At the end of this
period the acetone was evaporated off over a steam bath and the residual
solution made up to 50 ml. This solution was then assayed for free choline.
The free choline values determined by this method on six samples of liver
were not signilicantly different from the values obtained by extraction with
water followed by treatment with acetone, as described in the procedure.
E$ect of Adsorption-All non-basic substances which may interfere
with the growth of the Neurospora are eliminated by the Decalso treatment.
Methionine if present in sufficient amounts will be utilized by cholineless
and thus must also be eliminated (2). The effect of pH on adsorption of
solutions containing choline was studied. The results showed that choline
is quantitatively adsorbed at a pH of from 4.5 to 7.0. Table I shows the
effect of adsorption on the free and total choline values of liver. The
values for free choline are very much less after adsorption. Thus it is
apparent that there are appreciable amounts of foreign growth substances

present in the water extract and that their effect can be eliminated by
adsorption.
While there is a decrease in the values for total choline with
adsorption, it is not nearly so pronounced as in the case of free choline.
Nevertheless the difference is sufficient to warrant adsorption in the
determination
of total choline.
Standard solutions of choline chloride adsorbed on Decalso are eluted
quantitatively with a solution of 5 per cent sodium chloride.
Recovery of Choline Added to Tissue-Known amounts of choline chloride
were added to minced beef liver and assays made to determine the recovery.
Both total and free choline were determined on each sample of liver.