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Determination of 3-mercaptolactic acid by amino acid analyzer after aminoethylation
ANALYTICAL
BIOCHEMISTRY
145, 120-123 (1985)
Determination of 3-Mercaptolactic Acid by Amino Acid Analyzer after Aminoethylation’ BERNARD~PENSA,MARACOSTA, Istituto
ANDDORIANOCAVALLINI
di Chimica Biologica, Universitri di Roma “La Sapienza” e Centro lo Studio della Biologia Molecolare de1 CNR, Rome, Italy
per
Received April 2, 1984 A quantitative determination of 3-mercaptolactic acid was performed after its conversion into Saminoethylmercaptolactic acid by reacting with excess of 2-bromoethylamine. 9 aminoethylmercaptolactic acid was quantitated by an amino acid analyzer. Other thiols were shown not to interfere with the determination of 3-mercaptolactic acid. The sensitivity of the method was at the nanomoles level. The application of the method to the determination of 3-mercaptolactic acid in human urine is also reported. 0 198s Academic PI=, IIIC. KEY WORDS: general chromatography; amino acid metabolism; 3-mercaptolactic acid; 3mercaptolactate-cysteine disulfide; 2-bromoethylamine.
3-Mercaptolactic acid (MLA)’ is a normal constituent of human urine and it has been found as mixed disulfide with cysteine (1). Transamination of cysteine to 3-mercaptopyruvic acid has been reported (2). Reduction of 3-mercaptopyruvic to 3-mercaptolactic acid by lactate dehydrogenase has also been described (3,4). Therefore the biosynthesis of MLA is thought to follow this route. An increased excretion of MLA has been seen in some pathological conditions (5,6). Hence the utility of devising a method to quantitate 3-mercaptolactate. One such sensitive gas-chromatographic method for normal and pathological urines has been developed by Hannestad (Hannestad and Sorb0 (7)). Recently the method has been used to determine 3-mercaptolactate in the urine of newborn infants (8). This paper describes a simple alternative method for quantitation of MLA after its conversion into an aminoethylated derivative
using 2-bromoethylamine as reagent. The resultant ninhydrin-positive compound is then accurately quantitated by an amino acid analyzer. The procedure has been applied to determine 3-mercaptolactate in normal human urine. MATERIALS
Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.
METHODS
3-Mercaptolactic acid was synthesized according to Costa et al. (9). Glutathione (reduced form), 2-bromoethylamine hydrobromide, and dithiothreitol were from Sigma (St. Louis, MO.). 3-Mercaptopyruvic acid (sodium salt) was from USBC (Cleveland, Ohio). All other materials were Merck reagent grade. Fresh solutions of MLA were titrated according to Ellman (11). Thiopropyl-Sepharose 6B was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden) in its mixed disuhide form with 2-thiopyridine. This was easily converted into its free-thiol form by removing the 2-thiopyridyl protecting groups, treating it with 1% (w/v) dithiothreitol solution. The resultant fully reduced gel contained 35 cLmo1 of thiol groups per milliliter swollen gel. Organomercurial agarose (A&Gel 501) was
’ This work was partially supported by a grant from the Italian Minister0 della Pubblica Istruzione. 2 Abbreviations used: MLA, 3-mercaptolactic acid, AEML, S-aminoethylmercaptolactic acid, GSH, glutathione (reduced form); DTT, dithiothreitol.
0003-2697185 $3.00
AND
120
DETERMINATION
OF 3-MERCAPTOLACTIC
obtained ready for use from Bio-Rad (Richmond, Calif.). The reaction with 2-bromoethylamine was carried out in a TTT2 Titrator equipped with an ABU 12 Radiometer automatic burette. Automatic amino acid analyses were performed using an Optica Aminolyzer (Milane, Italy) with a 40-cm column at 45°C and a single 0.6 M lithium citrate buffer at pH 4.5 (flow rate, 103 ml/h). S-Aminoethylmercaptolactic acid (AEML) was eluted 38 min after sample loading and produced a single well-resolved peak. The AEML content was calculated from peak area using a standard solution reference containing 10 pmol/ ml of authentic AEML synthesized according to Costa et al. (9). Creatinine was detected employing the Jaffe reaction. RESULTS
AND
DISCUSSION
Different quantities (0.0 1- 1 pmol/ml) of MLA were reacted with a constant amount of 2-bromoethylamine (240 pmol/ml) in the presence of 5 mM EDTA and under a N2 stream to avoid thiol oxidation. The reaction mixture, in a final volume of 5 ml, was brought to pH 10 with 2 N NaOH and kept constant thereafter by the automatic addition of 2 N NaOH. Most of the reaction occurs in the first half hour of incubation. However, to be sure that absolutely all MLA converts
FIG. 1. Extent of reaction of 3-mercaptolactic acid (10m4 (MLA) with 2-bromoethylamine (0.24 as a function of pH. For all pHs tested, the reaction was stopped after 3 h.
M),
M)
121
ACID o
lo-
oa-” 0
1 02
1 04
1
1 06
’ 06 pmok?*MLA/rnl
1 10
FIG.
2. Amounts of Saminoethylmercaptolactate (AEML) obtained vs 3-mercaptolactic acid (MLA). 2Bromoethylamine and pH were kept constant at 240 ~mol/ml and 10, respectively, for 3 h.
to AEML even at the lowest concentration used, the reaction is left to run its full course for 3 h. At the end of the reaction the absence of -SH groups and S-S bonds was tested with Ellman’s reagent before and after reduction with NaBH,. The pH of the mixture was then lowered to 5 by coned HCl and appropriate aliquots were applied on the amino acid analyzer column. The extent of the reaction was seen to depend on pH (Fig. 1). This is due to the ionization of the MLA thiol group. In any case, from pH 10 to 12, the amount of AEML obtained was seen to reach a maximum within 3 h. The AEML appeared stable at alkaline pHs-not even after standing over night at pH 12 was it seen to have decreased significantly. As shown in Fig. 2, 3-mercaptolactate is completely converted into its aminoethylated derivative within a concentration range of 10m3 to 10m5 M, provided the 2-bromoethylamine and pH were kept constant at 240 pmol/ml and 10, respectively. In order to test whether other thiol compounds might interfere with our determination, the reaction of MLA with 2-bromoethylamine was performed in the presence of cysteine, mercaptopyruvic acid, mercaptoacetic acid, and glutathione. GSH, mercaptopyruvic acid, and mercaptoacetic acid were added to the reaction mixture at the same concentration as that of MLA ( 10m4M). Cysteine was added at a concentration of 10m3
122
PENSA, TABLE
COSTA,
I
RECOVERYOF MLA ADDED TO URINE SAMPLES Endogenous concentration (nmol/ml)
Final concentration of added MLA @Wml)
16.5 15.8 16.1
1.oo 0.50 0.10
16.8 17.2
0.03
0.01
Recovery @) 100.2* 104.8 97.6 98.7 99.2
* Mean of triplicate determinations.
because its concentration in UtitX iS much higher than that of other thiols. 2-Bromoethylamine was kept constant. These compounds in the reaction mixture did not appear to affect the quantitative determination of MLA. The elution times of the various S-aminoethyl derivatives assayed were: Saminoethylpyruvic acid, 23.5 min; S-aminoethylglutathione, 34.5 min; S-AEML, 38 min, and S-aminoethylmercaptoacetic acid, 44.3 min. S-aminoethylcysteine (or thialysine) is a basic amino acid which does not elute with the buffer used in our procedure. The procedure described above was also used to quantitate MLA in normal human urine as follows: 5 ml of fresh urine samples was added with 0.2 ml 0.1 M EDTA. The pH was brought to 10 with 5 M NH@H and the volume made up to 6 ml with water. The mixture was centrifuged at 3000 rpm for 5 min. The supernatant was mixed with 2 ml of a thiopropylsepharose suspension containing 25 pmol of -SH groups per milliliter and then stirred for 60 min at room temperature. This enabled complete release of the 3-mercaptolactic acid from the mixed disulfide with cysteine as described by Hannestad (7). The sample was then acidified to pH 4.5 with 4 N CHJCOOH and centrifuged. The supernatant was applied to a 0.8 X 6-cm column of Affi-Gel 501 (organomercurial agarose) equilibrated to pH 8 with 50 mM phosphate buffer. The column, M
AND
CAVALLINI
washed with 5 ml of water, was eluted with 5 ml 2 X 10m2DTTM. The eluate, containing free 3-mercaptolactic acid, was reacted with 2-bromoethylamine as described above. After 3 h the reaction was stopped by lowering the pH to 5 with coned HCl. Appropriate aliquots were then run through the amino acid analyzer column and the amount of S-aminoethylmercaptolactic acid calculated from the peak area using a standard solution of authenitic 4aminoethylmercaptolactic acid as reference. To test our procedure in urinary assays, various amounts of authentic 3-mercaptolactic acid were added to 5 ml of urine specimen and quantitative determinations were carried out as described. Recoveries are reported in Table 1. Urinary MLA levels from nine healthy subjects are listed in Table 2. Both male and female values are the means of four different subjects on a free diet and are related to creatinine elimination. The values obtained are in satisfactory agreement with those reported by Hannestad. The MLA content in urine of one female fed on L-cystine, 1 g/day for 1 week, was also determined. In this case nearly twice as much 3-mercaptolactic acid was seen to be eliminated. This result agrees with the previously reported findings of Ubuka et al. (10). In conclusion, the procedure proposed TABLE
2
VALUESOF~-MERCAFTOLACTICACIDEXCRETION IN HUMAN URINE
Subjects Male” Female b Female fed on L-cystine’
nmol MLA/pmol
creatinine
2.11 f 0.46 1.64 f 0.66 3.05
DMean of assaysin four healthy males f SD. ’ Mean of assaysin four healthy females f SD. c Mean of two determinations on different samples from the same subject.
DETERMINATION
OF 3-MERCAPTOLACTIC
herein appears to be simple and suitable for the quantitative determination of MLA at nanomole levels in normal human urine. The method does not call for any complex manipulation of urine samples before determination and recoveries are quantitative. Other thiol compounds normally present in urine do not appear to interfere with the determination, and the method is suitable for clinical investigations. REFERENCES 1. Ubuka, T., Kobayashi, K., Yao, K., Kodama, H., Fujii, K., Hirayama, K., Kuwaki, T., and Mizuhara, S. ( 1968) Biochim. Biophys. Acfa 158, 493495. 2. Ubuka, T., Umemura, S., Ishimoto, Y., and Shimomma, M. (1977) Ph.ysiol. Chem. Phys. 9, 9196.
ACID
123
3. Kun, E. (1957) Biochim. Biophys. Acta 25, 135137. 4. Pensa, B., Costa, M., Colosimo, A., and Cavallini, D. (1982) Mol. Cell. Biochem. 44, 107- 112. 5. Crawhall, J. C., Parker, R.. Sneddon, W., Young, E. P., Ampola, M. G., Efron, M. L., and Bixby, E. M. (1968) Science (Washington, D. C.) 160, 4 19-420. 6. Niederwieser, A., Giliberti, P., and Baerlocher, K. (1973) Clin. Chim. Acta 43, 405-416. 7. Hannestad, U., and Sorbo, B. (1979) Clin. Chim. Acta 95, 189-200. 8. Finnstrom. O., Lundqvist, P., Martensson, J., and Sorbo, B. (1983) Metabolism 32, 732-735. 9. Costa, M., Pensa, B., Iavarone, C., and Cavallini. D. ( 1983) Prep. Biochem. 12, 4 17-427. 10. Ubuka, T., Akagi, R., Yuasa, S., Masuoka. N., Kinuta, M., Yao, K.. and Hosaki, Y. (1982) International Symposium on Sulfur Amino Acids and 5th Annual Meeting of Japanese Research Society on Sulfur Amino Acids (Tokyo), Poster I-8, p. 52. 1 I. Ellman. G. L. (1959) Arch. Biochem. Biophqls, 82, 70-77.
BIOCHEMISTRY
145, 120-123 (1985)
Determination of 3-Mercaptolactic Acid by Amino Acid Analyzer after Aminoethylation’ BERNARD~PENSA,MARACOSTA, Istituto
ANDDORIANOCAVALLINI
di Chimica Biologica, Universitri di Roma “La Sapienza” e Centro lo Studio della Biologia Molecolare de1 CNR, Rome, Italy
per
Received April 2, 1984 A quantitative determination of 3-mercaptolactic acid was performed after its conversion into Saminoethylmercaptolactic acid by reacting with excess of 2-bromoethylamine. 9 aminoethylmercaptolactic acid was quantitated by an amino acid analyzer. Other thiols were shown not to interfere with the determination of 3-mercaptolactic acid. The sensitivity of the method was at the nanomoles level. The application of the method to the determination of 3-mercaptolactic acid in human urine is also reported. 0 198s Academic PI=, IIIC. KEY WORDS: general chromatography; amino acid metabolism; 3-mercaptolactic acid; 3mercaptolactate-cysteine disulfide; 2-bromoethylamine.
3-Mercaptolactic acid (MLA)’ is a normal constituent of human urine and it has been found as mixed disulfide with cysteine (1). Transamination of cysteine to 3-mercaptopyruvic acid has been reported (2). Reduction of 3-mercaptopyruvic to 3-mercaptolactic acid by lactate dehydrogenase has also been described (3,4). Therefore the biosynthesis of MLA is thought to follow this route. An increased excretion of MLA has been seen in some pathological conditions (5,6). Hence the utility of devising a method to quantitate 3-mercaptolactate. One such sensitive gas-chromatographic method for normal and pathological urines has been developed by Hannestad (Hannestad and Sorb0 (7)). Recently the method has been used to determine 3-mercaptolactate in the urine of newborn infants (8). This paper describes a simple alternative method for quantitation of MLA after its conversion into an aminoethylated derivative
using 2-bromoethylamine as reagent. The resultant ninhydrin-positive compound is then accurately quantitated by an amino acid analyzer. The procedure has been applied to determine 3-mercaptolactate in normal human urine. MATERIALS
Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.
METHODS
3-Mercaptolactic acid was synthesized according to Costa et al. (9). Glutathione (reduced form), 2-bromoethylamine hydrobromide, and dithiothreitol were from Sigma (St. Louis, MO.). 3-Mercaptopyruvic acid (sodium salt) was from USBC (Cleveland, Ohio). All other materials were Merck reagent grade. Fresh solutions of MLA were titrated according to Ellman (11). Thiopropyl-Sepharose 6B was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden) in its mixed disuhide form with 2-thiopyridine. This was easily converted into its free-thiol form by removing the 2-thiopyridyl protecting groups, treating it with 1% (w/v) dithiothreitol solution. The resultant fully reduced gel contained 35 cLmo1 of thiol groups per milliliter swollen gel. Organomercurial agarose (A&Gel 501) was
’ This work was partially supported by a grant from the Italian Minister0 della Pubblica Istruzione. 2 Abbreviations used: MLA, 3-mercaptolactic acid, AEML, S-aminoethylmercaptolactic acid, GSH, glutathione (reduced form); DTT, dithiothreitol.
0003-2697185 $3.00
AND
120
DETERMINATION
OF 3-MERCAPTOLACTIC
obtained ready for use from Bio-Rad (Richmond, Calif.). The reaction with 2-bromoethylamine was carried out in a TTT2 Titrator equipped with an ABU 12 Radiometer automatic burette. Automatic amino acid analyses were performed using an Optica Aminolyzer (Milane, Italy) with a 40-cm column at 45°C and a single 0.6 M lithium citrate buffer at pH 4.5 (flow rate, 103 ml/h). S-Aminoethylmercaptolactic acid (AEML) was eluted 38 min after sample loading and produced a single well-resolved peak. The AEML content was calculated from peak area using a standard solution reference containing 10 pmol/ ml of authentic AEML synthesized according to Costa et al. (9). Creatinine was detected employing the Jaffe reaction. RESULTS
AND
DISCUSSION
Different quantities (0.0 1- 1 pmol/ml) of MLA were reacted with a constant amount of 2-bromoethylamine (240 pmol/ml) in the presence of 5 mM EDTA and under a N2 stream to avoid thiol oxidation. The reaction mixture, in a final volume of 5 ml, was brought to pH 10 with 2 N NaOH and kept constant thereafter by the automatic addition of 2 N NaOH. Most of the reaction occurs in the first half hour of incubation. However, to be sure that absolutely all MLA converts
FIG. 1. Extent of reaction of 3-mercaptolactic acid (10m4 (MLA) with 2-bromoethylamine (0.24 as a function of pH. For all pHs tested, the reaction was stopped after 3 h.
M),
M)
121
ACID o
lo-
oa-” 0
1 02
1 04
1
1 06
’ 06 pmok?*MLA/rnl
1 10
FIG.
2. Amounts of Saminoethylmercaptolactate (AEML) obtained vs 3-mercaptolactic acid (MLA). 2Bromoethylamine and pH were kept constant at 240 ~mol/ml and 10, respectively, for 3 h.
to AEML even at the lowest concentration used, the reaction is left to run its full course for 3 h. At the end of the reaction the absence of -SH groups and S-S bonds was tested with Ellman’s reagent before and after reduction with NaBH,. The pH of the mixture was then lowered to 5 by coned HCl and appropriate aliquots were applied on the amino acid analyzer column. The extent of the reaction was seen to depend on pH (Fig. 1). This is due to the ionization of the MLA thiol group. In any case, from pH 10 to 12, the amount of AEML obtained was seen to reach a maximum within 3 h. The AEML appeared stable at alkaline pHs-not even after standing over night at pH 12 was it seen to have decreased significantly. As shown in Fig. 2, 3-mercaptolactate is completely converted into its aminoethylated derivative within a concentration range of 10m3 to 10m5 M, provided the 2-bromoethylamine and pH were kept constant at 240 pmol/ml and 10, respectively. In order to test whether other thiol compounds might interfere with our determination, the reaction of MLA with 2-bromoethylamine was performed in the presence of cysteine, mercaptopyruvic acid, mercaptoacetic acid, and glutathione. GSH, mercaptopyruvic acid, and mercaptoacetic acid were added to the reaction mixture at the same concentration as that of MLA ( 10m4M). Cysteine was added at a concentration of 10m3
122
PENSA, TABLE
COSTA,
I
RECOVERYOF MLA ADDED TO URINE SAMPLES Endogenous concentration (nmol/ml)
Final concentration of added MLA @Wml)
16.5 15.8 16.1
1.oo 0.50 0.10
16.8 17.2
0.03
0.01
Recovery @) 100.2* 104.8 97.6 98.7 99.2
* Mean of triplicate determinations.
because its concentration in UtitX iS much higher than that of other thiols. 2-Bromoethylamine was kept constant. These compounds in the reaction mixture did not appear to affect the quantitative determination of MLA. The elution times of the various S-aminoethyl derivatives assayed were: Saminoethylpyruvic acid, 23.5 min; S-aminoethylglutathione, 34.5 min; S-AEML, 38 min, and S-aminoethylmercaptoacetic acid, 44.3 min. S-aminoethylcysteine (or thialysine) is a basic amino acid which does not elute with the buffer used in our procedure. The procedure described above was also used to quantitate MLA in normal human urine as follows: 5 ml of fresh urine samples was added with 0.2 ml 0.1 M EDTA. The pH was brought to 10 with 5 M NH@H and the volume made up to 6 ml with water. The mixture was centrifuged at 3000 rpm for 5 min. The supernatant was mixed with 2 ml of a thiopropylsepharose suspension containing 25 pmol of -SH groups per milliliter and then stirred for 60 min at room temperature. This enabled complete release of the 3-mercaptolactic acid from the mixed disulfide with cysteine as described by Hannestad (7). The sample was then acidified to pH 4.5 with 4 N CHJCOOH and centrifuged. The supernatant was applied to a 0.8 X 6-cm column of Affi-Gel 501 (organomercurial agarose) equilibrated to pH 8 with 50 mM phosphate buffer. The column, M
AND
CAVALLINI
washed with 5 ml of water, was eluted with 5 ml 2 X 10m2DTTM. The eluate, containing free 3-mercaptolactic acid, was reacted with 2-bromoethylamine as described above. After 3 h the reaction was stopped by lowering the pH to 5 with coned HCl. Appropriate aliquots were then run through the amino acid analyzer column and the amount of S-aminoethylmercaptolactic acid calculated from the peak area using a standard solution of authenitic 4aminoethylmercaptolactic acid as reference. To test our procedure in urinary assays, various amounts of authentic 3-mercaptolactic acid were added to 5 ml of urine specimen and quantitative determinations were carried out as described. Recoveries are reported in Table 1. Urinary MLA levels from nine healthy subjects are listed in Table 2. Both male and female values are the means of four different subjects on a free diet and are related to creatinine elimination. The values obtained are in satisfactory agreement with those reported by Hannestad. The MLA content in urine of one female fed on L-cystine, 1 g/day for 1 week, was also determined. In this case nearly twice as much 3-mercaptolactic acid was seen to be eliminated. This result agrees with the previously reported findings of Ubuka et al. (10). In conclusion, the procedure proposed TABLE
2
VALUESOF~-MERCAFTOLACTICACIDEXCRETION IN HUMAN URINE
Subjects Male” Female b Female fed on L-cystine’
nmol MLA/pmol
creatinine
2.11 f 0.46 1.64 f 0.66 3.05
DMean of assaysin four healthy males f SD. ’ Mean of assaysin four healthy females f SD. c Mean of two determinations on different samples from the same subject.
DETERMINATION
OF 3-MERCAPTOLACTIC
herein appears to be simple and suitable for the quantitative determination of MLA at nanomole levels in normal human urine. The method does not call for any complex manipulation of urine samples before determination and recoveries are quantitative. Other thiol compounds normally present in urine do not appear to interfere with the determination, and the method is suitable for clinical investigations. REFERENCES 1. Ubuka, T., Kobayashi, K., Yao, K., Kodama, H., Fujii, K., Hirayama, K., Kuwaki, T., and Mizuhara, S. ( 1968) Biochim. Biophys. Acfa 158, 493495. 2. Ubuka, T., Umemura, S., Ishimoto, Y., and Shimomma, M. (1977) Ph.ysiol. Chem. Phys. 9, 9196.
ACID
123
3. Kun, E. (1957) Biochim. Biophys. Acta 25, 135137. 4. Pensa, B., Costa, M., Colosimo, A., and Cavallini, D. (1982) Mol. Cell. Biochem. 44, 107- 112. 5. Crawhall, J. C., Parker, R.. Sneddon, W., Young, E. P., Ampola, M. G., Efron, M. L., and Bixby, E. M. (1968) Science (Washington, D. C.) 160, 4 19-420. 6. Niederwieser, A., Giliberti, P., and Baerlocher, K. (1973) Clin. Chim. Acta 43, 405-416. 7. Hannestad, U., and Sorbo, B. (1979) Clin. Chim. Acta 95, 189-200. 8. Finnstrom. O., Lundqvist, P., Martensson, J., and Sorbo, B. (1983) Metabolism 32, 732-735. 9. Costa, M., Pensa, B., Iavarone, C., and Cavallini. D. ( 1983) Prep. Biochem. 12, 4 17-427. 10. Ubuka, T., Akagi, R., Yuasa, S., Masuoka. N., Kinuta, M., Yao, K.. and Hosaki, Y. (1982) International Symposium on Sulfur Amino Acids and 5th Annual Meeting of Japanese Research Society on Sulfur Amino Acids (Tokyo), Poster I-8, p. 52. 1 I. Ellman. G. L. (1959) Arch. Biochem. Biophqls, 82, 70-77.