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The determination of choline in human semen by the enzymic method
79
Forensic Science International, 17 (1981) 79 - 84 @ Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
THE DETERMINATION ENZYMIC METHOD
TAKEHIKO TAKATORI Department
of Legal
OF CHOLINE
IN HUMAN SEMEN BY THE
and SEIBEI TOM11
Medicine,
Hokkaido
University
School
of Medicine,
Sapporo
060
(Japan) TOSHINOBU TANAKA Department of Obstetrics Sapporo 060 (Japan)
(Received December 1,1979;
and
Gynecology,
Hokkaido
University
School
of Medicine,
accepted September 3, 1980)
Summary Studies are reported on the specific enzymic microdetermination of free choline in human semen. The concentration of choline in normal semen was found to be 0.9 1.4 mg/ml. The minimum detectable concentration of choline using the method described was 1.5 yg. Normal levels of choline in vaginal fluid, saliva, serum and urine could not be detected by this procedure.
Introduction For the detection of the presence of semen in vaginal contents and seminal stains in the medicolegal field, the demonstration of the presence of some of the enzymes found in semen, such as acid phosphatase [ 1, 21, y-glutamyl transpeptidase [3, 41 or diamine oxidase [5, 61, has been widely investigated as a preliminary examination of semen. However, these enzymic activities are frequently reduced under various circumstances. Therefore, the determination of the presence of some organic compounds found in semen which remain unaffected by various chemical conditions would be expected to be a better marker for presumptive evidence of semen than the presence of seminal enzymes. Although the Florence test [ 7,8] for choline is not sufficiently sensitive to identify a trace amount of semen, it has been used as a preliminary test for semen, indicating that the seminal fluid contains a certain amount of choline [9] . Since choline is more stable under various chemical conditions than seminal enzyme activities, its determination as a means of demonstrating the presence of semen would appear to be advantageous in the medicolegal field. In this paper we attempt to establish a simple method for the microdetermination of free choline in semen.
80
Materials and methods Samples Vaginal fluid samples from women who had not had sexual intercourse for at least two weeks were collected in the Department of Obstetrics and Gynecology ‘of Hokkaido University Hospital. Normal semen, serum, saliva and urine were collected from healthy individuals. All specimens were kept at -20 “C until assayed. Procedure for determination of choline The principle of the assay procedure is illustrated in Fig. 1. The free choline in the sample is first oxidized by choline oxidase to produce hydrogen peroxide and betaine. The produced hydrogen peroxide, in the presence of peroxidase as a catalyst, oxidizes and condenses phenol and 4-aminoantipyrine to generate a red quinone stoichiometrically. The red quinone is then determined by measuring the absorbance at 500 nm using a Hitachi Model 200-20 spectrophotometer. The standard assay procedure was carried out as follows. The incubation mixture consisted of 4 ml of 0.1 M Tris-HCl buffer (pH 7.4) containing 5 units of choline oxidase [EC 1 .1.99.1] ,50 units of peroxidase [EC 1.11.1.71, 400 pmol of phenol and 40 pmol of 4-aminoantipyrine. After addition of either 20 ~1 of the standard solution (choline chloride solution) or of the sample (in the case of semen 10 ~1 of sample were used), the mixture was incubated at 37 “C for 20 min. The red quinone product was then measured at 500 nm. The colour was stable for up to 60 min after incubation. Chemicals Choline oxidase from alcaligenes (10 units/mg of protein) and peroxidase from horseradish (250 purpurogallin units/mg) were purchased from Toyobo Co., Japan. 4:Aminoantipyrine was obtained from Tokyo Kasei Co., Tokyo, Japan, and all other reagents were from Wako Pure Chemical Ind., Osaka, Japan. (CH,),-N-CH,-CH,-OH
+
20~
+
H,O
O’H choline
COD
)
2H,02
$
I
betaine
Phenol 4-AA
POD
red
quinone
$
4H20
Fig. 1. The principle of the assay method of free choline. COD, POD and 4-AA represent choline oiidase, peroxidase and 4-aminoantipyrine, respectively.
81
Results The effect of different buffer solutions on the activities of the choline oxidase-peroxidase mixture was tested with four different buffers (pH 7.4). Table 1 shows that 0.1 M Tris-HCl buffer gave the maximum activites of the coupled enzymes. For the standard assay system, therefore, 0.1 M TrisHCl buffer was used. The effect of changing the pH of the 0.1 M Tris-HCl buffer on the activities of the choline oxidase-peroxidase mixture showed that the optimum pH was between 7.2 and 7.5 as illustrated in Fig. 2. Thus, pH 7.4 was adopted for the standard assay system. When the concentration of the standard choline chloride solution was varied as shown in Fig. 3, there was a linear relationship between the concentration of the choline chloride TABLE 1 The effect of different buffers on the activity of the choline oxidase-peroxidase in the assay system
mixture
The incubation was carried out using the standard assay system described except that the buffer was changed and 10 pg of choline were added to the incubation mixture. Buffers (all at pH 7.4)
Absorbance at 500 nm
0.1 0.1 0.1 0.2
0.452 0.386 0.526 0.511
M Sodium phosphate M Borate M Tris-HCl M Tris-HCl
/
1.2 06
i
1.0
0.0 . 0.4
d d
d
0.4
0.2 0
0.6
v’
;P 0.2 I 0 5
6
7
6
9
PH
Fig. 2. The effect of pH on the red quinone production
01
0
6
12 Choline
10
20
c@pl
at 500 nm by choline oxidase
and peroxidase. The incubation conditions were the same as the standard assay procedure except that the pH of the 0.1 M Tris-HCl buffer was changed. Fig. 3. Calibration curve for the production of red quinone (absorbance at 500 nm) from choline chloride.
82
and the red quinone product. Five units of choline oxidase and 50 units of peroxidase were enough to convert 20 pg of choline (0.165 pmol of choline chloride) to the corresponding red quinone products. Using this procedure a level of free choline as low as 1.5 pg in the assay system could be detected. The concentrations of free choline in various biological fluids determined by this method are summarized in Table 2. The level of free choline in normal semen was 0.9 - 1.4 mg/ml, but no choline could be detected in normal vaginal fluid, saliva, serum or urine by the procedure used here. In order to test the effect of contaminants contained in biological fluids on the activities of the choline oxidase-peroxidase mixture, different volumes of the biological fluids were added to a constant volume of semen (10 ~1) in the assay system, but there was little interference in the enzyme system (Table 3).
TABLE 2 The concentration
of free choline in biological fluids
Biological fluids
No. of determinations
mg free choline per ml
Semen Vaginal fluid Saliva Serum Urine
12 7 10 7 10
1.15 i 0.236* N.D.** N.D. N.D. N.D.
*Mean f S.D. **Not detected. TABLE
3
The effect of contaminants containing in biological fluids on the activity of the choline oxidase-peroxidase mixture in the assay system The incubation was carried out using the standard assay system described except that semen (10 ~1) alone or with other biological fluids was added and the total volume was made up to 4.11 ml with buffer. Biological fluids
Absorbance at 500 nm Addition of biological fluids (~1)
Semen Semen Semen Semen Semen
20
40
100
0.656 0.660 0.653 0.660
0.667 0.659 0.697 0.666
0.672 0.670 0.690 0.636
0.665 + + + +
vaginal fluid saliva serum urine
83
Discussion In this paper we describe a method for the measurement of a small amount of free choline in biological fluids using an enzymic method containing choline oxidase and peroxidase coupled to phenol and 4-aminoantipyrine. This method is very specific for choline, and no interference by contaminants contained in biological fluids used as samples has been demonstrated (Table 3). Bound compounds of choline in seminal plasma such as phosphorylcholine, glycerophosphorylcholine or phosphatidylcholine are not detected by this procedure. If a biological fluid contains a slight amount of serum together with ester compounds of choline such as acetylcholine and benzoylcholine, pseudocholinesterase [EC 3.1 .1.8] present in the serum hydrolyzes the esters of choline to free choline. However, the samples contaminated with serum will only occur very rarely. Phosphorylcholine, which is secreted into the seminal plasma mostly from seminal vesicles, breaks down rapidly to free choline and inorganic phosphate after ejaculation by the action of acid phosphatase in the seminal plasma [ 9 - 111. The inorganic phosphate liberated binds to spermine to form spermine phosphate which is measured by Barberio’s reaction [8] . Glycerophosphorylcholine, which is also one of the compounds containing choline precursor in seminal fluid and secreted from epididymis, is not hydrolyzed by acid phosphatase [lo] . Therefore, the origin of free choline in seminal fluid appears to be mainly due to hydrolysis of phosphorylcholine. Normal semen contained a considerable amount of free choline; the level was found to be 0.9 - 1.4 mg/ml of semen. Two microlitres of seminal fluid were enough to determine the content of free choline, and as little as 1.5 pg of choline could be measured by this method. The free choline levels in other biological fluids were not detectable by this procedure. The activity of pseudocholinesterase in the seminal fluid was not determined. These results indicate that even though a slight amount of semen is contaminated with other biological fluids, the free choline from the semen can be quantitatively determined. Although Appleton et al. [12] reported a chemical determination of free choline in plasma by using a triiodide reagent to precipitate the periodide complex of free choline, their procedure was very tedious and the sensitivity was far lower than that of our present method. The enzymic method reported here is a more useful tool for presumptive evidence of seminal fluid together with a microscopical examination of spermatozoa, for example, from vaginal contents in cases of sexual offence.
References 1 S.
S.
Kind, The acid phosphatase test. In A. S. Curry (ed.), Methods of Forensic Vol. 3, Interscience, New York, 1964, pp. 267 - 287. 2 E. C. Adams and B. G. Wraxall, Phosphatase in body fluids: The differentiation of semen and vaginal secretion. Forensic Sci., 3 (1974) 57 - 62.
Science,
84 3 S. B. Rosalki and J. A. Rowe, Gamma-glutamyl-transpeptidase activity of human seminal fluid. Lance& i (1973) 323 - 324. 4 K. Nakanishi, Studies on y-glutamyl transpeptidase of semen from the medico-legal aspects. Part I. r-Glutamyl transpeptidase in human seminal fluid. Jpn. J. Leg. Med., 30 (1976) 281 - 287. 5 H. Tabor, Amine oxidase. B. Diamine oxidase (histamine) from hog kidney. In C. Kaplan (ed.), Methods in Enzymology, Vol. II, Academic Press, New York, 1955, pp. 394 - 396. 6 0. Suzuki, M. Oya, Y. Katsumata, S. Yada and K. Higashide, A medico-legal study on diamine oxidase in human semen. Jpn. J. Leg. Med., 33 (1979) 1 - 6. 7 J. Beeman, Notes on the identification of seminal stains by means of the Florence reaction. Arch. Pathol., 34 (1942) 932 - 933. 8 C. J. Polson and D. J. Gee, The Essentials of Forensic Medicine, Pergamon Press, Oxford, 1973, pp. 507 - 512. 9 T. Mann and C. Lutwak-Mann, Secretory function of male accessory organs of reproduction in mammals. Physiol. Reu., 31 (1951) 27 _ 55. 10 R. M. C. Dawson, T. Mann and I. G. White, Glycerophosphorylcholine and phosphorylcholine in semen, and their relation to choline. Biochem. J., 65 (1957) 627 - 634. 11 T. Mann, Biochemistry of semen. In D. W. Hamilton and R. 0. Greep (eds.), Handbook of Physiology, Section 7, Vol. V, American Physiological Society, Washington, DC, 1975, pp. 461 - 471. 12 H. D. Appleton, B. N. LaDu, Jr., B. B. Levy, J. M. Steele and B. B. Brodie, A chemical method for the determination of free choline in plasma. J. Biol. Chem., 205 (1953) 803 - 813.
Forensic Science International, 17 (1981) 79 - 84 @ Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
THE DETERMINATION ENZYMIC METHOD
TAKEHIKO TAKATORI Department
of Legal
OF CHOLINE
IN HUMAN SEMEN BY THE
and SEIBEI TOM11
Medicine,
Hokkaido
University
School
of Medicine,
Sapporo
060
(Japan) TOSHINOBU TANAKA Department of Obstetrics Sapporo 060 (Japan)
(Received December 1,1979;
and
Gynecology,
Hokkaido
University
School
of Medicine,
accepted September 3, 1980)
Summary Studies are reported on the specific enzymic microdetermination of free choline in human semen. The concentration of choline in normal semen was found to be 0.9 1.4 mg/ml. The minimum detectable concentration of choline using the method described was 1.5 yg. Normal levels of choline in vaginal fluid, saliva, serum and urine could not be detected by this procedure.
Introduction For the detection of the presence of semen in vaginal contents and seminal stains in the medicolegal field, the demonstration of the presence of some of the enzymes found in semen, such as acid phosphatase [ 1, 21, y-glutamyl transpeptidase [3, 41 or diamine oxidase [5, 61, has been widely investigated as a preliminary examination of semen. However, these enzymic activities are frequently reduced under various circumstances. Therefore, the determination of the presence of some organic compounds found in semen which remain unaffected by various chemical conditions would be expected to be a better marker for presumptive evidence of semen than the presence of seminal enzymes. Although the Florence test [ 7,8] for choline is not sufficiently sensitive to identify a trace amount of semen, it has been used as a preliminary test for semen, indicating that the seminal fluid contains a certain amount of choline [9] . Since choline is more stable under various chemical conditions than seminal enzyme activities, its determination as a means of demonstrating the presence of semen would appear to be advantageous in the medicolegal field. In this paper we attempt to establish a simple method for the microdetermination of free choline in semen.
80
Materials and methods Samples Vaginal fluid samples from women who had not had sexual intercourse for at least two weeks were collected in the Department of Obstetrics and Gynecology ‘of Hokkaido University Hospital. Normal semen, serum, saliva and urine were collected from healthy individuals. All specimens were kept at -20 “C until assayed. Procedure for determination of choline The principle of the assay procedure is illustrated in Fig. 1. The free choline in the sample is first oxidized by choline oxidase to produce hydrogen peroxide and betaine. The produced hydrogen peroxide, in the presence of peroxidase as a catalyst, oxidizes and condenses phenol and 4-aminoantipyrine to generate a red quinone stoichiometrically. The red quinone is then determined by measuring the absorbance at 500 nm using a Hitachi Model 200-20 spectrophotometer. The standard assay procedure was carried out as follows. The incubation mixture consisted of 4 ml of 0.1 M Tris-HCl buffer (pH 7.4) containing 5 units of choline oxidase [EC 1 .1.99.1] ,50 units of peroxidase [EC 1.11.1.71, 400 pmol of phenol and 40 pmol of 4-aminoantipyrine. After addition of either 20 ~1 of the standard solution (choline chloride solution) or of the sample (in the case of semen 10 ~1 of sample were used), the mixture was incubated at 37 “C for 20 min. The red quinone product was then measured at 500 nm. The colour was stable for up to 60 min after incubation. Chemicals Choline oxidase from alcaligenes (10 units/mg of protein) and peroxidase from horseradish (250 purpurogallin units/mg) were purchased from Toyobo Co., Japan. 4:Aminoantipyrine was obtained from Tokyo Kasei Co., Tokyo, Japan, and all other reagents were from Wako Pure Chemical Ind., Osaka, Japan. (CH,),-N-CH,-CH,-OH
+
20~
+
H,O
O’H choline
COD
)
2H,02
$
I
betaine
Phenol 4-AA
POD
red
quinone
$
4H20
Fig. 1. The principle of the assay method of free choline. COD, POD and 4-AA represent choline oiidase, peroxidase and 4-aminoantipyrine, respectively.
81
Results The effect of different buffer solutions on the activities of the choline oxidase-peroxidase mixture was tested with four different buffers (pH 7.4). Table 1 shows that 0.1 M Tris-HCl buffer gave the maximum activites of the coupled enzymes. For the standard assay system, therefore, 0.1 M TrisHCl buffer was used. The effect of changing the pH of the 0.1 M Tris-HCl buffer on the activities of the choline oxidase-peroxidase mixture showed that the optimum pH was between 7.2 and 7.5 as illustrated in Fig. 2. Thus, pH 7.4 was adopted for the standard assay system. When the concentration of the standard choline chloride solution was varied as shown in Fig. 3, there was a linear relationship between the concentration of the choline chloride TABLE 1 The effect of different buffers on the activity of the choline oxidase-peroxidase in the assay system
mixture
The incubation was carried out using the standard assay system described except that the buffer was changed and 10 pg of choline were added to the incubation mixture. Buffers (all at pH 7.4)
Absorbance at 500 nm
0.1 0.1 0.1 0.2
0.452 0.386 0.526 0.511
M Sodium phosphate M Borate M Tris-HCl M Tris-HCl
/
1.2 06
i
1.0
0.0 . 0.4
d d
d
0.4
0.2 0
0.6
v’
;P 0.2 I 0 5
6
7
6
9
PH
Fig. 2. The effect of pH on the red quinone production
01
0
6
12 Choline
10
20
c@pl
at 500 nm by choline oxidase
and peroxidase. The incubation conditions were the same as the standard assay procedure except that the pH of the 0.1 M Tris-HCl buffer was changed. Fig. 3. Calibration curve for the production of red quinone (absorbance at 500 nm) from choline chloride.
82
and the red quinone product. Five units of choline oxidase and 50 units of peroxidase were enough to convert 20 pg of choline (0.165 pmol of choline chloride) to the corresponding red quinone products. Using this procedure a level of free choline as low as 1.5 pg in the assay system could be detected. The concentrations of free choline in various biological fluids determined by this method are summarized in Table 2. The level of free choline in normal semen was 0.9 - 1.4 mg/ml, but no choline could be detected in normal vaginal fluid, saliva, serum or urine by the procedure used here. In order to test the effect of contaminants contained in biological fluids on the activities of the choline oxidase-peroxidase mixture, different volumes of the biological fluids were added to a constant volume of semen (10 ~1) in the assay system, but there was little interference in the enzyme system (Table 3).
TABLE 2 The concentration
of free choline in biological fluids
Biological fluids
No. of determinations
mg free choline per ml
Semen Vaginal fluid Saliva Serum Urine
12 7 10 7 10
1.15 i 0.236* N.D.** N.D. N.D. N.D.
*Mean f S.D. **Not detected. TABLE
3
The effect of contaminants containing in biological fluids on the activity of the choline oxidase-peroxidase mixture in the assay system The incubation was carried out using the standard assay system described except that semen (10 ~1) alone or with other biological fluids was added and the total volume was made up to 4.11 ml with buffer. Biological fluids
Absorbance at 500 nm Addition of biological fluids (~1)
Semen Semen Semen Semen Semen
20
40
100
0.656 0.660 0.653 0.660
0.667 0.659 0.697 0.666
0.672 0.670 0.690 0.636
0.665 + + + +
vaginal fluid saliva serum urine
83
Discussion In this paper we describe a method for the measurement of a small amount of free choline in biological fluids using an enzymic method containing choline oxidase and peroxidase coupled to phenol and 4-aminoantipyrine. This method is very specific for choline, and no interference by contaminants contained in biological fluids used as samples has been demonstrated (Table 3). Bound compounds of choline in seminal plasma such as phosphorylcholine, glycerophosphorylcholine or phosphatidylcholine are not detected by this procedure. If a biological fluid contains a slight amount of serum together with ester compounds of choline such as acetylcholine and benzoylcholine, pseudocholinesterase [EC 3.1 .1.8] present in the serum hydrolyzes the esters of choline to free choline. However, the samples contaminated with serum will only occur very rarely. Phosphorylcholine, which is secreted into the seminal plasma mostly from seminal vesicles, breaks down rapidly to free choline and inorganic phosphate after ejaculation by the action of acid phosphatase in the seminal plasma [ 9 - 111. The inorganic phosphate liberated binds to spermine to form spermine phosphate which is measured by Barberio’s reaction [8] . Glycerophosphorylcholine, which is also one of the compounds containing choline precursor in seminal fluid and secreted from epididymis, is not hydrolyzed by acid phosphatase [lo] . Therefore, the origin of free choline in seminal fluid appears to be mainly due to hydrolysis of phosphorylcholine. Normal semen contained a considerable amount of free choline; the level was found to be 0.9 - 1.4 mg/ml of semen. Two microlitres of seminal fluid were enough to determine the content of free choline, and as little as 1.5 pg of choline could be measured by this method. The free choline levels in other biological fluids were not detectable by this procedure. The activity of pseudocholinesterase in the seminal fluid was not determined. These results indicate that even though a slight amount of semen is contaminated with other biological fluids, the free choline from the semen can be quantitatively determined. Although Appleton et al. [12] reported a chemical determination of free choline in plasma by using a triiodide reagent to precipitate the periodide complex of free choline, their procedure was very tedious and the sensitivity was far lower than that of our present method. The enzymic method reported here is a more useful tool for presumptive evidence of seminal fluid together with a microscopical examination of spermatozoa, for example, from vaginal contents in cases of sexual offence.
References 1 S.
S.
Kind, The acid phosphatase test. In A. S. Curry (ed.), Methods of Forensic Vol. 3, Interscience, New York, 1964, pp. 267 - 287. 2 E. C. Adams and B. G. Wraxall, Phosphatase in body fluids: The differentiation of semen and vaginal secretion. Forensic Sci., 3 (1974) 57 - 62.
Science,
84 3 S. B. Rosalki and J. A. Rowe, Gamma-glutamyl-transpeptidase activity of human seminal fluid. Lance& i (1973) 323 - 324. 4 K. Nakanishi, Studies on y-glutamyl transpeptidase of semen from the medico-legal aspects. Part I. r-Glutamyl transpeptidase in human seminal fluid. Jpn. J. Leg. Med., 30 (1976) 281 - 287. 5 H. Tabor, Amine oxidase. B. Diamine oxidase (histamine) from hog kidney. In C. Kaplan (ed.), Methods in Enzymology, Vol. II, Academic Press, New York, 1955, pp. 394 - 396. 6 0. Suzuki, M. Oya, Y. Katsumata, S. Yada and K. Higashide, A medico-legal study on diamine oxidase in human semen. Jpn. J. Leg. Med., 33 (1979) 1 - 6. 7 J. Beeman, Notes on the identification of seminal stains by means of the Florence reaction. Arch. Pathol., 34 (1942) 932 - 933. 8 C. J. Polson and D. J. Gee, The Essentials of Forensic Medicine, Pergamon Press, Oxford, 1973, pp. 507 - 512. 9 T. Mann and C. Lutwak-Mann, Secretory function of male accessory organs of reproduction in mammals. Physiol. Reu., 31 (1951) 27 _ 55. 10 R. M. C. Dawson, T. Mann and I. G. White, Glycerophosphorylcholine and phosphorylcholine in semen, and their relation to choline. Biochem. J., 65 (1957) 627 - 634. 11 T. Mann, Biochemistry of semen. In D. W. Hamilton and R. 0. Greep (eds.), Handbook of Physiology, Section 7, Vol. V, American Physiological Society, Washington, DC, 1975, pp. 461 - 471. 12 H. D. Appleton, B. N. LaDu, Jr., B. B. Levy, J. M. Steele and B. B. Brodie, A chemical method for the determination of free choline in plasma. J. Biol. Chem., 205 (1953) 803 - 813.