A CHEMICAL METHOD FOR THE DETERMINATION OF

FREE CHOLINE IN PLASMA

BY HAROLD D. APPLETON, BERT N. LA DU, JR., BETTY B. LEVY,

J. MURRAY STEELE, AND BERNARD B. BRODIE
(From the Research Service, Third (New York University) Medical Division, Goldwater
Memorial Hospital, New York, New York, and the Laboratory of Chemical
Pharmacology, National Heart Institute, National Institutes of Health,
United States Department of Health, Education, and Welfare,
Bethesda, Maryland)

(Received for publication, July 2, 1953)

In the course of studies on the intermediary metabolism of choline, a

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large number of determinations of free choline in plasma was required.
As none of the methods hitherto described was satisfactory for our purpose,
a sensitive chemical method was developed by which the free choline could
be accurately determined by using small amounts of plasma.
The chemical methods most commonly used for the estimation of choline
involve (a) precipitation with Reinecke salt to yield choline reineckate,
followed by calorimetric measurement of the complex (l-lo), and (6) pre-
cipitation with potassium triiodide to yield choline periodide (choline en-
neaiodide) with subsequent determination of the iodine (11-15). In both
of these procedures the choline derivative must be washed free of excess
reagent before it can be assayed, and small amounts are lost in the washing
of the precipitate. In the determination of mg. quantities of choline this
loss is insignificant, but with small amounts of choline the loss becomes
appreciable. For this reason these procedures are not well suited for the
estimation of free choline in plasma.
It was observed in this laboratory that choline periodide is soluble in
ethylene dichloride and in this solvent exhibits an ultraviolet absorption
spectrum which differs markedly from that of free iodine (Fig. 1). This
permits the measurement of choline periodide in the presence of free iodine
adsorbed on the surface of the precipitated complex and thus eliminates
the need for washing the precipitate. With this principle, a method has
been developed which permits the estimation of as little as 5 y of choline.
This procedure has been applied to the estimation of free choline in plasma.

EXPERIMENTAL

Reagents
n-Butanol saturated with 2 N HCl. n-Butanol, reagent grade, is
shaken with 2 N HCl at room temperature. The butanol should be satu-
rated with the aqueous acid solution the day it is used.
803
804 DETERMINATION OF CHOLINE IN PLASMA

Isobutanol saturated with 2 N HCl. Isobutanol, reagent grade, is

treated in the same way as the n-butanol.
Potassium triiodide reagent. 15.7 gm. of iodine, reagent grade, and 20
gm. of potassium iodide, reagent grade, are dissolved in 100 ml. of water.
The mixture is shaken for 45 minutes on a mechanical shaker to effect
complete solution. The reagent is indefinitely stable at 4”.
0.600 [ I
I I I I I

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bk--
260 300 320 340 360 380 400

WAVELENGTH mp
FIG. 1. Absorption spectra of choline periodide (A) and iodine (B) in ethylene
dichloride. Choline periodide concentration, 16.9 -y per ml. (equivalent to 1.65 y
per ml. of choline). Iodine concentration, 10 y per ml.; cell thickness, 1 cm.

Ethylene dichloride. The solvent obtained from some sources contains

impurities which cause a gradual decomposition of dissolved choline peri-
odide.
Ethylene dichloride obtained from the Eastman Kodak Company (puri-
fied grade) or the Union Carbide and Carbon Corporation (technical grade)
is usually free of interfering substance.
The stability of the choline complex should be tested in each lot of
ethylene dichloride before use.
Choline periodide. The complex was prepared as follows: To 30 mg.
of choline chloride in 5 ml. of water were added 2.5 ml. of potassium tri-
iodide reagent. The mixture was allowed to stand at 4’ for 20 minutes,
centrifuged, and the supernatant fluid removed by aspiration. The pre-
 APPLETON, LA DU, LEVY, STEELE, AND BRODIE 805

cipitate was washed several times with ice-cold water to free it from ad-
sorbed iodine reagent and dried on filter paper over Drierite in a desiccator
at 4”. Weighed amounts of the dry black crystalline precipitate were
analyzed for iodine following sodium fusion, and for nitrogen by the micro-
Kjeldahl procedure.
CSH,~ONIO. Calculated. N 1.12, I 91.8
Found. “ 1.10, “92.0 (Sample 1)
“ 1.10, “91.0 ( “ 2)

The results are in agreement with the theoretical value required for the
compound to contain 9 atoms of iodine, and they confirm the findings of
Stan&k (11). 16.9 mg. of the complex were dissolved in a liter of ethylene

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dichloride to form a reference standard equivalent to 1.65 y of choline per
ml. This solution was found to be stable at refrigerator temperature for
a period of 2 years.
Standard Choline Solution-Choline chloride c.p. is dried over concen-
trated sulfuric acid in vacua. A standard solution is prepared containing
1 mg. of choline per ml. of water. The stock solution is stable at 4’ for
at least 2 weeks. Working standards are prepared by suitable dilution
of the stock solution with water.
Special Apparatus-A specially designed 15 ml. glass-stoppered centri-
fuge tube with a finely tapered tip, and graduated at 0.5, 1.0, 2.0, and 10
ml.,l was used. The finely tapered tip facilitates aspiration of the super-
natant fluid from the choline periodide precipitate.
Extraction of Free Choline from Plasma--Free choline in plasma is sepa-
rated from proteins and phospholipides by extraction into acetone. The
acetone is removed by evaporation and the aqueous residue extracted
with ether to remove neutral fat.
In preliminary experiments, the choline periodide was precipitated from
the ether-extracted aqueous solution. However, counter-current distri-
bution of the apparent choline disclosed the presence of considerable non-
choline material that also reacted with the triiodide reagent to yield an
ethylene dichloride-soluble complex which absorbed light at 365 mp. It
was found that the interfering material could be removed by extractions
of the aqueous choline solution with n-butanol and isobutanol. A small
fraction of the choline is also removed by these extractions but correction
for this loss is made by running choline standards through the same pro-
cedure as plasma.
Procedure-Pipette 3 ml. of plasma into a test-tube and add, with mixing,
12 ml. of acetone (reagent grade). Centrifuge the tube and decant the
supernatant solution into a 50 ml. beaker. Resuspend the precipitate in
10 ml. of 80 per cent acetone and centrifuge. Pool the acetone extracts
1 From E. Machlett and Son, New York.
806 DETERMINATION OF CHOLINE IN PLASMA

and evaporate to approximately 1 ml. at room temperature in a stream of

air. Transfer the sample as completely as possible to the finely tapered
glass-stoppered centrifuge tube. Complete the transfer by using in suc-
cession three 2 ml. portions of acetone and three 2 ml. portions of absolute
ether. Evaporate the solution to about 1 ml. at room temperature in a
stream of air. Shake with 10 ml. of ether, centrifuge, and remove the
ether layer by aspiration with a fine tipped capillary tube. Remove the
ether dissolved in the aqueous phase by carefully heating the tube in a
boiling water bath.
Add 0.17 ml. of 12 N HCl and adjust the volume with water to 1.0 ml.
Immediately2 extract the solution twice with 0.4 ml. of n-butanol and then

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three times with 2 ml. portions of isobutanol. Following each extraction,
centrifuge the tube and, without loss of the aqueous phase, carefully re-
move the organic phase by aspiration with a fine tipped capillary tube.
Remove the dissolved alcohol by evaporation of the aqueous residue to
0.5 ml. in a water bath. This aqueous solution is used for the determina-
tion of choline as described below.

Precipitation and Estimation of Choline As Periodide

The aqueous solution containing free choline is treated with potassium
triiodide reagent to precipitate choline as the periodide. After removal
of the supernatant solution, the periodide complex is dissolved in ethylene
dichloride and determined spectrophotometrically at 365 rnp.
The absorption spectrum of choline periodide shows two peaks, one at
295 rnp and the other at 365 rnp. The 365 mp wave-length was chosen for
the measurement of choline periodide since the interference from iodine
is less at this wave-length (Fig. 1).
Procedure-Cool the centrifuge tube (containing 5 to 40 y of choline in
0.5 ml. of solution) to 4’ and add 0.2 ml. of cold potassium triiodide reagent.
Tap the tube gently to promote mixing but avoid excessive spreading of
the reagent over the upper portion of the tube. Allow the tube to stand
at 4’ for at least 20 minutes and then centrifuge for 10 minutes at 2500
r.p.m. Using a fine tipped capillary tube carefully, aspirate the superna-
tant fluid from the firmly packed precipitate until the centrifuge tube is
essentially dry. Immediat.ely dissolve the choline periodide in a small
2 Small amounts of choline-containing compounds, perhaps residual phospho-
lipides, gradually yield free choline in the acid solution. For this reason, the butanol
and isobutanol extractions which remove these compounds are undertaken with a
minimum of delay after acidification.
3 Choline periodide, which contains 9 atoms of iodine, gradually decomposes at
room temperature to the more stable choline hexaiodide, a black oil. The ultraviolet,
absorption spectra of the two iodine complexes are qualitatively identical, but the
optical density of the choline hcxaiodide is only about two-thirds that of the ennea-
iodide.
 SPPLETON, LA DU, LEVY, STEELE, AND BRODIE 807

amount of ethylene dichloride with a fine tipped stirring rod, and dilute
the solution with additional solvent to a volume of 10 ml. (when the amount
of choline is less than 7 y, dilute to 5 ml.). Transfer about 3 ml. of the solu-
tion to a cuvette and read the optical density of the solution at 365 mp,
with ethylene dichloride for the zero setting. A reagent blank run through
the entire procedure should give a reading of less than 0.030 against ethylene
dichloride (Beckman ultraviolet spectrophotometer).
25 y of choline run through the precipitation procedure give an optical
density of about 0.515. The sensitivity may be doubled by dissolving
the complex in 5 ml. instead of in 10 ml. of ethylene dichloride. When
known amounts of choline are run through the entire plasma procedure,

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the optical densities are about 10 per cent lower, since small amounts of
choline are lost by extraction into butanol and isobutanol. Since the dis-

TABLE I
Recovery of Choline from Aqueous Solution

Choline added No. of recoveries Average recovery Standard deviation

Y gercent gerGent
100 21 99 2.7
50 26 100 2.1
30 12 98 2.4
25 46 97 3.4
20 15 99 2.7
10 16 94 5.0
5 28 94 8.0

tribution of choline between water and these organic solvents varies some-
what from day to day, standards should be run along with each series of
plasma samples.
Recovery of Choline-Various amounts of choline in 0.5 ml. of water were
treated with the triiodide reagent and the choline was assayed as described
above. The optical densities were found to be proportional to concentra-
tion and closely coincided with those obtained by spectrophotometric meas-
urement, in ethylene dichloride solution, of equivalent amounts of choline
periodide (see “Reagents”). Precipitation of the choline as the iodine
complex was essentially complete, but when quantities as small as 5 and
10 y were analyzed, the recovery was somewhat low and variable (Table I).
The reproducibility of the plasma choline method and the recovery of
choline added to plasma were checked as follows: A sample of pooled hu-
man plasma was divided into four portions. The first portion was ana-
lyzed directly for choline, with six replicates of 3.0 ml. each. The other
three portions were analyzed after the addition of 5, 10, and 15 y of choline
808 DETERMINATION OF CHOLINE IN PLASMA

per ml. of plasma. The results indicate that the reproducibility and re-
covery are satisfactory (Table II).
Assay of XpeciJicity-The specificity of the method for the estimation of
free choline in plasma was assayed in the plasma of two human subjects
by the Craig counter-current distribution technique (16). The choline
was isolated from 40 ml. samples of plasma as described in the analytical
method, and the aqueous residue was evaporated to dryness at room tem-
perature in a stream of air. The choline was quantitatively extracted
into two 20 ml. portions of absolute alcohol and the alcohol was evaporated
to dryness at room temperature. This step served to separate the choline

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TABLE II
Recovery of Choline Added to S Ml. of Human Plasma

Choline added Total choline expected* Total choline found kcovery of added choline

y $3‘3 ml. y per ml. y per ml. y fier ml.

5.0 12.3 12.5 5.2
5.0 12.3 12.7 5.4
10.0 17.3 17.8 10.5
10.0 17.3 18.2 10.9
10.0 17.3 17.4 10.1
10.0 17.3 17.1 9.8
15.0 22.3 22.1 14.8
15.0 22.3 21.8 14.5
15.0 22.3 20.7 13.4
15.0 22.3 20.9 13.6

* The endogenous choline value was 7.3 y per ml. (the average of the six values,
7.7,7.7,7.3,7.0,7.2, and 7.1).

from considerable amounts of inorganic salts, which would markedly affect

the counter-current distribution described below. The apparent choline
was subjected to a counter-current distribution involving three transfers.
The distribution was effected in a series of 50 ml. glass-stoppered centri-
fuge tubes between n-butanol (30 volumes) and 2 N HCl (1 volume).
Each solvent was saturated with the other. After the counter-current
distribution, the partition ratio of the choline in each phase was determined
by the analysis of the choline in suitable aliquots evaporated to dryness at
room temperature in a current of air. In Table III is shown the total
amount of apparent choline present in each tube, together with the frac-
tion present in the butanol phase. The ratio of the amount of solute in the
butanol phase to t.he total amount in both phases was constant, and almost
identical with that found for authentic choline measured at the same time
with the same solvents.
 APPLETON, LA DU, LEVY, STEELE, AND BRODIE 809

The specificity of the choline method was further checked by the tech-
nique of comparative partition ratios (17). Choline was isolated from 45
ml. of human plasma as described above for the counter-current distribu-
tion. The partition ratios of the apparent choline from plasma were com-
pared with those of authentic choline in a series of two-phase systems
consisting of 2 N I-ICl and n-butanol containing various concentrations of
petroleum ether. The result,s indicated that both substances possessed the
same solubility characteristics and were, therefore, presumably the same
compound (Table IV).

TABLE III

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Counter-Current Distribution Choline from Plasma of Two Normal
of Apparent
Subjects
The choline in plasma was extracted from about 40 ml. of plasma as described in
the text, and was subjected to a three transfer counter-current distribution in glass-
stoppered test-tubes between 2 N HCl (1 volume) and n-butanol (30 volumes). The
partition ratio of apparent choline in each tube is expressed as the ratio of the amount
of solute in the butanol phase to the total amount in both phases.

Tube No. I y per tube

Subject A

Fraction in buta-
no1 phase*
I
y per tube
Subject B

Fraction in buta-
no1 phase’
___~
1 24 0.60 8 0.58
2 113 0.63 56 0.60
3 183 0.64 94 0.60
4 102 0.63 54 0.60

Total. . . .. 422 212

I i -
*When authentic choline was distributed between the solvents used in this
experiment, the fraction in the butanol phase was 0.62.

The possibility was considered that part of the choline determined as

free choline had been derived during the course of the analytical procedure
from choline-containing precursors, such as phospholipides, phosphoryl-
choline, and glycerylphosphorylcholine.
The effect of phospholipides on the plasma choline level was investigated
as follows: Phospholipides from a sample of plasma were extracted with a
mixture of alcohol-ether according to the procedure of Bloor (18), after
which the solvent was evaporated to dryness and the phospholipides taken
up in petroleum ether. Aliquots of the petroleum ether solution were
evaporated to dryness and various samples of plasma were added to the
residues. Analysis of the plasmas showed that the addition of phospho-
lipides did not affect the free choline levels.
810 DETERMINATION OF CHOLINE IN PLASMA

Phosphorylcholine does not react with potassium triiodide and is not

easily hydrolyzed to free choline. The addition of phosphorylcholine to
plasma was found to have no effect on the estimated free choline level.
Glycerylphosphorylcholine likewise does not react with the triiodide re-
agent, but is readily hydrolyzed to yield choline under the acidic conditions
employed in the butanol and isobutanol extraction steps of the procedure.
However, glycerylphosphorylcholine is stable in neutral solution. In the
analysis of a number of plasma samples, the aqueous phase was buffered
to pH 7 for the butanol and isobutanol extraction steps. This variation
in the procedure gave the same levels of free choline as the regular proce-

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TABLE IV
Distribution of Choline and Apparent Choline between 2 N HCI and Various
Butanol-Petroleum Ether Mixtures
The apparent choline was extracted from human plasma as described in the text,
and taken up into 2 N HCl. Aliquots of this solution and of an authentic choline
solution were distributed between 2 N HCI (1 volume) and various butanol-petroleum
ether mixtures (75 volumes). The fraction of the compound extracted with the
various solvent mixtures is expressed as the ratio of the amount of compound in the
organic phase to total compound.

Per cent petroleum ether in Authentic choline, fraction in Apparent choline from plasma,
butanol phase organic solvent fraction in organic solvent

0 0.75 0.79
1 0.61 0.59
3 0.47 0.46
4 0.37 0.37
5 0.28 0.29
7 0.11 0.10
10 0.00 0.00

dure. The results indicate that glycerylphosphorylcholine in appreciable

amounts is not a normal component of human plasma. Others have also
shown that this choline derivative is not present in plasma (19).
Results
Free choline in plasma was determined in twenty-one adult healthy males
in the fasting state. The samples of blood were treated with oxalate and
centrifuged immediately, and the plasma was analyzed as soon as possible.
The concentration of choline in the plasma averaged 4.4 y per ml. and
ranged from 2.5 to 9.9 y per ml. In only three individuals in this group
were t,he levels above 6.0 y per ml. The concentration of free choline in
the plasma of patients with cirrhosis (six subjects), hypertension (six sub-
jects), diabetes (twelve subjects), and hyperthyroidism (six subjects) was
in the same range as in the normal subjects.
 APPLETON, LA DU, LEVY, STEELE, AND BRODIE 811

The variation in free plasma choline throughout the day was measured
in four of the normal individuals. None of the values, including those
taken 1 to 2 hours after a meal, was appreciably different from the morning
fasting value, indicating that the dietary intake of choline did not affect
the plasma levels.
The variation in free plasma choline over a period of 4 weeks was deter-
mined in thirteen normal male individuals. Generally speaking, the cho-
line levels for a given individual were found to remain relatively constant
from week to week (Table V). The levels in ten other subjects were meas-
ured several months apart and found to be relatively unchanged. This
latter group contained two individuals whose plasma levels were about 10

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y per ml.

TABLE V
Plasma Levels of Free Choline in Normal Individuals Measured at Weekly Intervals
The values are given in micrograms per ml.

Subject 1st wk. 2nd wk. 1 3rd wk. 4th wk. Subject 1st wk. 2nd wk. 3rd wk. 4th wk.
.~

Ro 7.0 6.0 7.6 7.5 La 4.6 5.3 4.8 4.2

MC 4.5 3.7 4.1 4.7 Ch 5.1 5.3 4.5 4.2
Pe 2.6 3.4 3.7 4.4 AP 6.6 6.6 4.8
Ra 3.9 4.2 3.6 Ja 3.2 2.9 3.7 3.1
Ch 3.6 3.8 / i:; 3.2 Vi 3.7 3.3 4.1 3.4
Kr 2.5 3.6 4.7 Ge 3.2 3.2 3.7 3.1
Ca 4.1 2.7 4.1
I 4.3 I
The effect on plasma choline of feeding large amounts of choline was
investigated. Two subjects received orally 5 gm. of choline as the bicar-
bonate salt (in the form of a 14 per cent syrup) and plasma levels were
measured in 30 minutes and at various times thereafter for 12 hours.
Elevation of the plasma choline level was not observed. Two cirrhotic
subjects also received 5 gm. of choline as the bicarbonate salt daily for a
period of 4 weeks. Again the plasma level of choline was not signifi-
cantly affected.
The constancy of the plasma level of free choline in a given individual
suggests that some regulatory mechanism is involved. Further evidence
of this was obtained by measuring the plasma levels of choline of a dog
which received 625 mg. of choline chloride by intravenous infusion over a
period of 3 hours. During the infusion the plasma level of choline rose
progressively from 7 y per ml. to 18 y per ml. 35 minutes after stopping
the infusion the plasma choline had fallen to 10 y per ml., and within 1
hour had returned to the preinject.ion level. The urinary excretion of
choline accounted at most for only a few per cent of the injected dose.
812 DETERMINATION OF CHOLINE IN PLASMA

It was concluded therefore that the major part of the choline had undergone
metabolic transformation.

DISCUSSION

Biological, microbiological, and chemical methods have been used for

the estimation of free choline in plasma. By these methods a wide scatter
of values for free choline in plasma has been reported, as summarized by
Bligh (20).
The specificity of the various chemical and microbiological methods has
not been demonstrated, and indeed the high values usually reported with
these methods suggest that they are unspecific. Methods in which choline

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is bioassayed as acetylcholine possess high sensitivity and perhaps speci-
ficity, but technical difficulties in the assay procedure limit their usefulness.
The present method for plasma appears to be specific, as shown by counter-
current distribution and partition ratio studies. However, application of
the plasma method to urine and homogenized organ tissues has disclosed
the presence of a considerable amount of non-choline material which is
also assayed as choline. Modification of the method will therefore be
necessary before it can be used for the analysis of urine and tissues.
The free choline level in plasma is relatively constant in a given individ-
ual and is not appreciably changed even after the ingestion of large amounts
of choline. Ifi the dog the level of choline is elevated during the intra-
venous infusion of choline, but on discontinuance of the infusion the level
rapidly returns io the preinjection value. Similar results have been re-
ported by Bligh (21). Obviously, therefore, there exists a mechanism in
the body for maintenance of the plasma choline at a constant level. The
results indicate that urinary excretion is only a minor factor in controlling
the plasma level, and it seems probable therefore that the choline level in
plasma is regulated by metabolic processes. The nature of these regula-
tory mechanisms is under investigation.
SUMMARY

A sensitive and specific method for the estimation of free choline in

plasma has been described. The method involves extraction of the choline
into acetone, evaporation of the acetone, and removal of interfering sub-
stances from the aqueous residue by butanol and isobutanol extraction.
Choline is precipitated as the enneaiodide, which is dissolved in ethylene
dichloride and measured spectrophotometrically at 365 rnp. Iodine ad-
sorbed on the periodide precipitate does not interfere in the measurement,
and as little as 5 y of choline can be determined.
The free choline level in plasma of normal male adu1t.s averages about
4.4 y per ml. Analyses made over a period of several months indicat,e
 APPLETON, L.4 DU, LEVY, STEELE, AND BRODIE 813

that each individual maintains a relatively constant plasma level and this
level is not increased after meals or by the oral administration of large
amounts of choline.
Evidence is presented suggesting that the level of free choline in plasma
is controlled by a process involving metabolic transformation.

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 A CHEMICAL METHOD FOR THE
DETERMINATION OF FREE CHOLINE
IN PLASMA
Harold D. Appleton, Bert N. La Du, Jr., Betty
B. Levy, J. Murray Steele and Bernard B.
Brodie
J. Biol. Chem. 1953, 205:803-813.

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