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 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

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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

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-

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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 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.

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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 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.

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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, 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

100

50 30 25 20 10 5

21 26 12 46 15 16 28

ger cent ger Gent 99 2.7

100 2.1 98 2.4 97 3.4 99 2.7 94 5.0 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

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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

TABLE II

Recovery of Choline Added to S Ml. of Human Plasma

Total choline expected* Choline added

y $3‘3 ml.

5.0 5.0

10.0 10.0 10.0 10.0 15.0 15.0 15.0 15.0

y per ml.

12.3 12.3 17.3 17.3 17.3 17.3 22.3 22.3 22.3 22.3

Total choline found

y per ml.

12.5 12.7 17.8 18.2 17.4 17.1 22.1 21.8 20.7 20.9

kcovery of added choline

y fier ml.

5.2 5.4

10.5 10.9 10.1 9.8

14.8 14.5 13.4 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.

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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

Counter-Current Distribution of Apparent Choline from Plasma of Two Normal 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.

Fraction in buta- no1 phase’

Subject A Subject B I I y per tube Fraction in buta-

no1 phase* y per tube

24 0.60 8 113 0.63 56 183 0.64 94 102 0.63 54

___~

0.58 0.60 0.60 0.60

Tube No.

1 2 3 4

Total. . . . . 422 I i

212 -

*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.

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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-

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 butanol phase

Authentic choline, fraction in organic solvent

Apparent choline from plasma, 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.

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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 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

Ro MC Pe Ra Ch Kr Ca

1st wk. 2nd wk.

.~

7.0 6.0 4.5 3.7 2.6 3.4 3.9 4.2 3.6 3.8 2.5 3.6 4.1 2.7

1 3rd wk.

7.6 4.1 3.7

/ i:;

4.1

4th wk. Subject

7.5 La 4.7 Ch

4.4 AP

3.6 Ja 3.2 Vi 4.7 Ge

I 4.3 I

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

4.6 5.1 6.6 3.2 3.7 3.2

5.3 5.3

2.9 3.3 3.2

4.8 4.2 4.5 4.2 6.6 4.8 3.7 3.1 4.1 3.4 3.7 3.1

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.

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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 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

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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.

BIBLIOGRAPHY

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(1942). 5. Engel, R. W., J. Biol. Chem., 144,701 (1942). 6. Shaw, F. H., Biochem. J., 32, 1002 (1938). 7. Lovern, J. A., Chem. and Ind., 707 (1950). 8. Trusov, V. I., Biokhimiya, 16,495 (1950). 9. Glick, D., J. BioZ. Chem., 168, 643 (1944).

10. Marenzi, A. D., and Cardini, C. E., J. BioZ. Chem., 147,363 (1943). 11. Stanhk, V., 2. physiol. Chem., 46, 280 (1905). 12. Sharpe, J. S., Biochem. J., 17,41 (1923). 13. Roman, W., Biochem. Z., 219, 218 (1930). 14. Erickson, B. N., Avrin, I., Teague, D. M., and Williams, H. H., J. BioZ. Chem.,

136, 671 (1940). 15. Reifer, I., New Zealand J. SC. and Technol., 22 B, 111 (1941). 16. Craig, L. C., Golumbic, C., Mighton, H., and Titus, E., J. BioZ. Chem., 161, 321

(1945). 17. Brodie, B. B., and Udenfriend, S., J. BioZ. Chem., 158, 705 (1945). 18. Bloor, W. R., J. BioZ. Chem., ‘77, 53 (1928). 19. Kahane, E., and Levy, J., Arch. SC. physiol., 2, 281 (1948). 20. Bligh, J., J. Physiol., 117, 234 (1952). 21. Bligh, J., J. PhysioZ., 120, 53 (1953).

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BrodieB. Levy, J. Murray Steele and Bernard B.

Harold D. Appleton, Bert N. La Du, Jr., BettyIN PLASMA

DETERMINATION OF FREE CHOLINE A CHEMICAL METHOD FOR THE

1953, 205:803-813.J. Biol. Chem. 

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