349
Clinica Chimica Acta, 68 (1976) 349-353 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
SHORT COMMUNICATION CCA 7664
AUTOMATED CONTAINING
MICROANALYSIS OF SULFUR AMINO ACIDS AND THEIR DERIVATIVES
L. BOWIE *, J.C. CRAWHALL
**, N. GOCHMAN, K. JOHNSON and J.A. SCHNEIDER
Laboratory Services, Veterans Administration Hospital, San Diego and Department Pediatrics, University of California, San Diego, La Jolla, Calif. (U.S.A.)
of
(Received September 15, 1975)
Introduction
The introduction of Durrum D-500 amino acid analyzer, (Durrum Instrument Co., Palo Alto, Calif. 94303) has made possible the rapid analysis and automatic quantitation of amino acids on microsamples from physiologic fluids. We have utilized this system for measuring intracellular amino acids in cultured human fibroblasts, and in particular, to measure the intracellular cystine content in fibroblasts obtained from patients with cystinosis, utilizing as little as 10’ cells for the assay procedure. Materials and methods
The procedure for the separation of amino acids from physiological mixtures was carried out essentially as described by Lee [ 11, involving single column, high pressure liquid chromatography. Amino acid preparations of extracts of cultured human fibroblasts from patients with cystinosis were prepared essentially as described by Kroll et al. [ 21. Standard amino acid mixtures (500 pmol/l) used for calibration were obtained from Pierce Chemical Co. (Rockford, Ill. 61105). High sensitivity standards (50 pmol/l and 5 pmol/l) were prepared by diluting the 500 pmol/l standard with 0.2 N lithium citrate buffer (pH 2.20) containing 1% (v/v) thiodiglycol (Pierce Chemical Co.) and 0.1% (v/v) liquefied phenol (Mallinkrodt Chemical Works, St. Louis, MO. 63160) as a preservative. All buffers were prepared as de-
* Correspondence to: Dr. L. Bowie. Laboratory Services. V.A. Hospital, 3350 La Jolla Village Drive, San Diego, Calif. 92161, U.S.A. ** Visiting Associate Professor of Pediatrics. on leave of absence from McGill University Clinic, Royal Victoria Hospital, Montreal, P.Q., Canada.
350
scribed by Lee [l]. Chemical Co.
Ninhydrin
reagent
(“Nin-sol”)
was purchased
from Pierce
Results In this investigation we have defined the characteristics of this form of automated amino acid analysis for a variety of sulfur containing amino acids and their derivatives. The identification of particular amino acids is principally dependent on a comparison of the elution time of the amino acid with a known physiological amino acid mixture. The analyzer at the normal sensitivity is capable of detecting amino acids in the range between 1 and 10 nmol per 20 ~1 loading volume. Fig. 1 shows the analysis of a physiologic mixture of amino acids analyzed at a 100-fold higher sensitivity (10 pmol-100 pmol). All of the sulfur containing amino acids can be analyzed at this sensitivity. At this sensitivity, however, we were not able to carry out the analysis for more basic amino acids due to a large change in baseline after the elution of homocystine. Table I summarizes the relative elution times and peak areas per nmol for various sulfur containing amino acids and their chemical derivatives. CystineNEM is eluted in the form of two peaks resulting from the formation of diastereoisomers as has been reported in other ion exchange chromatography systems [ 31. The advantages of this system have been utilized in studies being carried out
IO
20
30
40
,‘ 52.57mn
70
150 s
80, “‘Wi’ 86-101 mn TIME,
121.l23mm
160 a
170 *
180 a
190 ’
135-141 mn
MINUTES
Fig. 1. Typical chromatogram obtained for the separation of a physiological amino acid standard mixture with a 100 pm01 loading of each amino acid. The chromatogram was terminated when homocystine was eluted and prior to the elution of the more basic amino acids. Baseline shifts after a-aminoadipic acid. valine, and the shift between norleucine and tyrosine and phenylalanine and @-alanine have been partially deleted by a command to the computer to stop recording during those periods of time (5 min at 52 min. 15 min at 86 min, 2 min at 121 min. and 6 min at 135 min). The diagonal arrow indicates a spike at 102 min resulting from the change from the first to second buffer.
351 TABLE
I
ELUTION
CHARACTERISTICS
DERIVATIVES
OF VARIOUS
SULFUR
CONTAINING
____---~-.~.-.-
AMINO
ACIDS
AND
THEIR
_--
Compound
Elution time (mm; s)
DL-Cyst& acid S-Carboxymethyl cysteine DL-Methionine sulfoxide DL-Methionine sulfone Glu~th~one-NEM ** Cysteine-NEM **
5; 26; 32; 31; 27; 47; 60; 104;
49 26 05 05 45 15 15 52
39 41 44 34 29 30 23 30
106: 107; 109; 110:
26 16 34 34
38 311 31021 58 960 33 957
128: 36 173; 41
58 194 32 686
L-Cystine GCysteine-D-penicillamine Mixed disulfide Methionine D-PeniciIlamine disulfide Cystathionine L-Homocysteine-L-cysteine Mixed disulfide L-Homocystine -
Peak area * (per nmol) ..___ ___I_-
-.
.--_-~
_____-
360 261 072 655 531 056 402 192
--
* Peak areas are expressed as a summation of digitized photometric output by the computer. * * Glutathione-NEM and cysteine-NEM refer to the N-ethyimaleimide condensation products thiol groups of these substances (viz. S-(1~thyl-2,5dioxopyrrolidin-3-yl-L-cysteine).
with the
into the factors controlling the cystine content of cultured human fibroblasts obtained from patients with nephropathjc cystinosis. The amino acid chromatogram depicted in Fig. 2 was obtained using a protein precipitated extract of 3 X 10’ fibroblasts. In this chromatogram the cystine peak represents 0.56 nmol. Chromatograms of extracts of normal cells showed no evidence of cystine at comparable instrument sensitivity and cell loading. Further results of these studies will be reported subsequently.
0.2 A
0. I
J
2
Jo
0
IO 20 30 40
50 60 70 80 90 100 110 120 130 TIME,
MINUTES
Fig. 2. Typical analysis of amino acid content of fibroblast cells from a patient with cystinosis using 3 X 10’ cells. Cystinotic cells washed in phosphate buffered saline were disrupted by ultra-sonication and the protein was precipitated with sulfosalicyiic acid. The supernatant solution was adjusted to PH 2 with lithium hydroxide (1 N). The mixture was then lyaphihzed, redissolved in distilled water (0.1 ml), and a 20-&I diquot used for the analysis. The peak height for cysteine (103 min) corresponds to 0.56 mnol.
352
Discussion The various benefits of computer control and readout, the very high sensitivity, fast analysis time, and continuous loading procedures which are a feature of the Durrum D-500 amino acid analyzer open up new possibilities of biological analysis. One field of investigation in which this is of particular value is that of the analysis of free amino acids in cells in tissue culture where only small quantities of amino acid are normally available. In terms of cystine analysis, we estimate that the sensitivity is nearly 100 times greater than that of other amino acid analyzers as normally operated in the high sensitivity mode. This statement is based on our demonstration that 10 pmol of cystine can be measured with a comparable precision to 1 nmol in other analyzers. It should be noted that Stein et al. [4], Roth [ 51, and Benson and Hare [6] have described fluorometric reagents which extend the detection level of conventional analyzers for amino acids and primary amines into the pmol range. However, the sensitivity of these ~uorometri~ systems is approximately half of that which we described for cystine on the Durrum system. This level of sensitivity makes possible the estimation of cystine in cells obtained from heterozygous carriers for cystinosis. The only other analytical technique with comparable sensitivity is that presently being developed using a cystine binding protein isolated from the shock fluid of E. coli [ 7 1. Both methods have their own characteristic advantages. The relevant advantages of the Durrum system in comparison are that data on other amino acids can be obtained simultaneously and a visual record is available to ensure the specificity of the cystine measurement. This system may be especially advantageous in the prenatal detection of cystinotic fetuses [S]. This disease is detected in utero by finding an elevated content of free (acid soluble) cystine in cultured amniotic fluid cells from women at risk for having children with this autosomal recessive disorder. Because it is necessary to establish the diagnosis as soon as possible, and because of the limited sensitivity of the usual methods of amino acid analysis, this diagnosis has usually been accomplished by using a pulse-labelling technique with [35S]cystine. The sensitivity of the system described in this communication, however, is such that it should be possible to detect cystinotic amniotic fluid cells by direct measurement of their cystine content.
This investigation was supported by VA research grant No. MRIS 3181, USPHS grant No. GM 17702 and American Heart Association grant No. 71-981. One of us (J.C.C.) was the recipient of a Schering Travelling Fellowship. J.A.S. is an Established Investigator of the American Heart Association. Methionine sulfoxide and sulfone as well as the various disulfides were a gift from Mr. Stan Kulovich of the Department of Pediatrics, UCSD. References 1 Lee. P.L.Y. (19’74) Biochem. Med. 10. 107 2 Kroll, W.A., Becker, F.L.A. and Schneider, J.A. (1974) Biochem. Med. 10, 368 3 Smyth, D.G., Blumenfeld, 0.0. and Konigsberg, W. (1964) Biochem. J. 91, 589
353 4 Stein. S., Bohlen. P.. Stone, J.. Dairman. W. and Udenfriend, S. (1973) 5 6 7 8
Arch. Biochem. Biophys. 155.
203 Roth, M. (1971) Anal. Chem. 43. 880 Benson, J.R. and Hare. P.E. (1975) Proc. NatI. Acad. Sci. U.S.A. 72,619 Oshima. R.G., Willis. R.C.. Furlong. C.E. and Schneider, J.A. (1974) J. Biol. Chem. 249.6033 Schneider. J.A., Verroust, F.M.. KroII. W.A.. Garvin. A.J.. Horger, III, E.O.. Wong, V.G., Spear. G.S.. Jacobson. C.. PeUett, O.L. and Becker. F.L.A. (1974) New. EngI. J. Med. 290. 878
Clinica Chimica Acta, 68 (1976) 349-353 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
SHORT COMMUNICATION CCA 7664
AUTOMATED CONTAINING
MICROANALYSIS OF SULFUR AMINO ACIDS AND THEIR DERIVATIVES
L. BOWIE *, J.C. CRAWHALL
**, N. GOCHMAN, K. JOHNSON and J.A. SCHNEIDER
Laboratory Services, Veterans Administration Hospital, San Diego and Department Pediatrics, University of California, San Diego, La Jolla, Calif. (U.S.A.)
of
(Received September 15, 1975)
Introduction
The introduction of Durrum D-500 amino acid analyzer, (Durrum Instrument Co., Palo Alto, Calif. 94303) has made possible the rapid analysis and automatic quantitation of amino acids on microsamples from physiologic fluids. We have utilized this system for measuring intracellular amino acids in cultured human fibroblasts, and in particular, to measure the intracellular cystine content in fibroblasts obtained from patients with cystinosis, utilizing as little as 10’ cells for the assay procedure. Materials and methods
The procedure for the separation of amino acids from physiological mixtures was carried out essentially as described by Lee [ 11, involving single column, high pressure liquid chromatography. Amino acid preparations of extracts of cultured human fibroblasts from patients with cystinosis were prepared essentially as described by Kroll et al. [ 21. Standard amino acid mixtures (500 pmol/l) used for calibration were obtained from Pierce Chemical Co. (Rockford, Ill. 61105). High sensitivity standards (50 pmol/l and 5 pmol/l) were prepared by diluting the 500 pmol/l standard with 0.2 N lithium citrate buffer (pH 2.20) containing 1% (v/v) thiodiglycol (Pierce Chemical Co.) and 0.1% (v/v) liquefied phenol (Mallinkrodt Chemical Works, St. Louis, MO. 63160) as a preservative. All buffers were prepared as de-
* Correspondence to: Dr. L. Bowie. Laboratory Services. V.A. Hospital, 3350 La Jolla Village Drive, San Diego, Calif. 92161, U.S.A. ** Visiting Associate Professor of Pediatrics. on leave of absence from McGill University Clinic, Royal Victoria Hospital, Montreal, P.Q., Canada.
350
scribed by Lee [l]. Chemical Co.
Ninhydrin
reagent
(“Nin-sol”)
was purchased
from Pierce
Results In this investigation we have defined the characteristics of this form of automated amino acid analysis for a variety of sulfur containing amino acids and their derivatives. The identification of particular amino acids is principally dependent on a comparison of the elution time of the amino acid with a known physiological amino acid mixture. The analyzer at the normal sensitivity is capable of detecting amino acids in the range between 1 and 10 nmol per 20 ~1 loading volume. Fig. 1 shows the analysis of a physiologic mixture of amino acids analyzed at a 100-fold higher sensitivity (10 pmol-100 pmol). All of the sulfur containing amino acids can be analyzed at this sensitivity. At this sensitivity, however, we were not able to carry out the analysis for more basic amino acids due to a large change in baseline after the elution of homocystine. Table I summarizes the relative elution times and peak areas per nmol for various sulfur containing amino acids and their chemical derivatives. CystineNEM is eluted in the form of two peaks resulting from the formation of diastereoisomers as has been reported in other ion exchange chromatography systems [ 31. The advantages of this system have been utilized in studies being carried out
IO
20
30
40
,‘ 52.57mn
70
150 s
80, “‘Wi’ 86-101 mn TIME,
121.l23mm
160 a
170 *
180 a
190 ’
135-141 mn
MINUTES
Fig. 1. Typical chromatogram obtained for the separation of a physiological amino acid standard mixture with a 100 pm01 loading of each amino acid. The chromatogram was terminated when homocystine was eluted and prior to the elution of the more basic amino acids. Baseline shifts after a-aminoadipic acid. valine, and the shift between norleucine and tyrosine and phenylalanine and @-alanine have been partially deleted by a command to the computer to stop recording during those periods of time (5 min at 52 min. 15 min at 86 min, 2 min at 121 min. and 6 min at 135 min). The diagonal arrow indicates a spike at 102 min resulting from the change from the first to second buffer.
351 TABLE
I
ELUTION
CHARACTERISTICS
DERIVATIVES
OF VARIOUS
SULFUR
CONTAINING
____---~-.~.-.-
AMINO
ACIDS
AND
THEIR
_--
Compound
Elution time (mm; s)
DL-Cyst& acid S-Carboxymethyl cysteine DL-Methionine sulfoxide DL-Methionine sulfone Glu~th~one-NEM ** Cysteine-NEM **
5; 26; 32; 31; 27; 47; 60; 104;
49 26 05 05 45 15 15 52
39 41 44 34 29 30 23 30
106: 107; 109; 110:
26 16 34 34
38 311 31021 58 960 33 957
128: 36 173; 41
58 194 32 686
L-Cystine GCysteine-D-penicillamine Mixed disulfide Methionine D-PeniciIlamine disulfide Cystathionine L-Homocysteine-L-cysteine Mixed disulfide L-Homocystine -
Peak area * (per nmol) ..___ ___I_-
-.
.--_-~
_____-
360 261 072 655 531 056 402 192
--
* Peak areas are expressed as a summation of digitized photometric output by the computer. * * Glutathione-NEM and cysteine-NEM refer to the N-ethyimaleimide condensation products thiol groups of these substances (viz. S-(1~thyl-2,5dioxopyrrolidin-3-yl-L-cysteine).
with the
into the factors controlling the cystine content of cultured human fibroblasts obtained from patients with nephropathjc cystinosis. The amino acid chromatogram depicted in Fig. 2 was obtained using a protein precipitated extract of 3 X 10’ fibroblasts. In this chromatogram the cystine peak represents 0.56 nmol. Chromatograms of extracts of normal cells showed no evidence of cystine at comparable instrument sensitivity and cell loading. Further results of these studies will be reported subsequently.
0.2 A
0. I
J
2
Jo
0
IO 20 30 40
50 60 70 80 90 100 110 120 130 TIME,
MINUTES
Fig. 2. Typical analysis of amino acid content of fibroblast cells from a patient with cystinosis using 3 X 10’ cells. Cystinotic cells washed in phosphate buffered saline were disrupted by ultra-sonication and the protein was precipitated with sulfosalicyiic acid. The supernatant solution was adjusted to PH 2 with lithium hydroxide (1 N). The mixture was then lyaphihzed, redissolved in distilled water (0.1 ml), and a 20-&I diquot used for the analysis. The peak height for cysteine (103 min) corresponds to 0.56 mnol.
352
Discussion The various benefits of computer control and readout, the very high sensitivity, fast analysis time, and continuous loading procedures which are a feature of the Durrum D-500 amino acid analyzer open up new possibilities of biological analysis. One field of investigation in which this is of particular value is that of the analysis of free amino acids in cells in tissue culture where only small quantities of amino acid are normally available. In terms of cystine analysis, we estimate that the sensitivity is nearly 100 times greater than that of other amino acid analyzers as normally operated in the high sensitivity mode. This statement is based on our demonstration that 10 pmol of cystine can be measured with a comparable precision to 1 nmol in other analyzers. It should be noted that Stein et al. [4], Roth [ 51, and Benson and Hare [6] have described fluorometric reagents which extend the detection level of conventional analyzers for amino acids and primary amines into the pmol range. However, the sensitivity of these ~uorometri~ systems is approximately half of that which we described for cystine on the Durrum system. This level of sensitivity makes possible the estimation of cystine in cells obtained from heterozygous carriers for cystinosis. The only other analytical technique with comparable sensitivity is that presently being developed using a cystine binding protein isolated from the shock fluid of E. coli [ 7 1. Both methods have their own characteristic advantages. The relevant advantages of the Durrum system in comparison are that data on other amino acids can be obtained simultaneously and a visual record is available to ensure the specificity of the cystine measurement. This system may be especially advantageous in the prenatal detection of cystinotic fetuses [S]. This disease is detected in utero by finding an elevated content of free (acid soluble) cystine in cultured amniotic fluid cells from women at risk for having children with this autosomal recessive disorder. Because it is necessary to establish the diagnosis as soon as possible, and because of the limited sensitivity of the usual methods of amino acid analysis, this diagnosis has usually been accomplished by using a pulse-labelling technique with [35S]cystine. The sensitivity of the system described in this communication, however, is such that it should be possible to detect cystinotic amniotic fluid cells by direct measurement of their cystine content.
This investigation was supported by VA research grant No. MRIS 3181, USPHS grant No. GM 17702 and American Heart Association grant No. 71-981. One of us (J.C.C.) was the recipient of a Schering Travelling Fellowship. J.A.S. is an Established Investigator of the American Heart Association. Methionine sulfoxide and sulfone as well as the various disulfides were a gift from Mr. Stan Kulovich of the Department of Pediatrics, UCSD. References 1 Lee. P.L.Y. (19’74) Biochem. Med. 10. 107 2 Kroll, W.A., Becker, F.L.A. and Schneider, J.A. (1974) Biochem. Med. 10, 368 3 Smyth, D.G., Blumenfeld, 0.0. and Konigsberg, W. (1964) Biochem. J. 91, 589
353 4 Stein. S., Bohlen. P.. Stone, J.. Dairman. W. and Udenfriend, S. (1973) 5 6 7 8
Arch. Biochem. Biophys. 155.
203 Roth, M. (1971) Anal. Chem. 43. 880 Benson, J.R. and Hare. P.E. (1975) Proc. NatI. Acad. Sci. U.S.A. 72,619 Oshima. R.G., Willis. R.C.. Furlong. C.E. and Schneider, J.A. (1974) J. Biol. Chem. 249.6033 Schneider. J.A., Verroust, F.M.. KroII. W.A.. Garvin. A.J.. Horger, III, E.O.. Wong, V.G., Spear. G.S.. Jacobson. C.. PeUett, O.L. and Becker. F.L.A. (1974) New. EngI. J. Med. 290. 878
