Rapid short-column chromatography of amino acids

ANALYTICAL

Rapid

20, 299-311 (1967)

BIOCHEMISTRY

Short-Column

A Method

Chromatography

of

Amino

Acids’

for Blood and Urine Specimens in the Diagnosis and Treatment of Metabolic Disease VIVIAN

E. SHIH,Z MARY L. EFRON,3 GERALD L. MECHANIC?

AND

Departments of Neurology and Orthopedic Surgery, Harvard Medical School, and The Joseph P. Kennedy, Jr., Memon’al Laboratories at the Massachusetts General Hospital, Boston, Massachusetts 0.2114 Received February

10, 1967

Recent advances in the methods of ion-exchange chromatography been directed largely toward accelerated analyses and the use of smaller samples (l-5). While the major impetus for this has been the great interest in protein structure, a parallel growth in knowledge of amino acid metabolism as related to clinical disease has produced an increasing demand for rapid analysis of the amino acids in physiological fluids, especially blood and urine. These fluids differ from protein hydrolyzates in that they may contain 60 or more ninhydrin-positive substances, as opposed to the 20 amino acids in most proteins. For this reason, accelerated analyses are often achieved only at the expense of overlapping peaks and loss of resolution (5). It is often unnecessary in the course of treatment of a metabolic disease to know the concentration of each amino acid. Sn investigator or therapist may need to measure only one amino acid, or at most a few amino acids, of interest in the particular clinical situation. He may also need t’o make repeated measurements of one amino acid, e.g., when folhave

lowing tests.

blood

or urine

levels

during

dietary

treatment,

or during

loading

For these reasons, we have attempted to achieve a rapid separation on short columns of the relevant amino acids for diagnosis and moni‘Preliminary Report, see also reference 13. ’ Research Fellow in Neurology, Massachusetts General Hospital, and Harvard Medical School. 3 Assistant in Neurology and Assistant Biochemist, Massachusetts General HORpital; Assistant Professor in Neurology, Harvard Medical School. 4 Research Associate in Biological Chemistry, Harvard Medical School ; Research Fellow

in Orthopedic

Surgery,

Massachusetts

General 299

Hospital.

300

SHIH,

EFRON,

AND

MECHANIC

toring of each presently known clinical disorder of amino acid metabolism in man (6). The separation of tyrosine and phenylalanine in 15 min using the short-column method principle has already been published (7). This report described analogous methods for studying disorders involving other amino acids. MATERIALS

AND

METHODS

The short columns may be attached to any amino acid analyzer. The analyses shown in Figures l-10 were performed by two of us (V.S. and M.E.) on a Technicon amino acid analyzer with a modified ninhydrin system (8, 9). The preparation of the sample and application to the column were as previously described (7-9). All analyses, except for that shown in Figure 11, an accelerated analysis described later, were performed using a 35 cm jacketed column, 0.9 cm i.d., packed with resin (Spherix ~~8-60-0, a generous gift from Phoenix Precision Company) to a 20 cm level. The buffer was pumped at a flow rate of 1.0 ml/min. The compositions of the buffers used were devised as variants of the two buffers (pH 2.875, 0.2 M Na citrate, and pH 4.74, 0.8 M Na citrate (10) used for the complete amino acid analysis on 50 or I.20 cm columns (7, 8, 9). The sodium concentration of the buffers was 0.2 M except as noted. Buffers and water jacket temperatures were varied as described below, depending on the separation desired. BUFFERS

AND

TEMPERATURE

System 1. Hydroxyproline For separation of hydroxyproline on short columns (Fig. l), it was. necessary to lower the temperature to 33” and the pH of the buffer to 2.45. Hydroxyproline is eluted in 60 min. System II. Sarcosine Using a buffer of pH 2.65 and a temperature of 65”, sarcosine is eluted as a separate peak between glutamine and glutamic acid (Fig. 2). The, temperature is important; at 28-37” the emergence of glutamine is delayed so that it is separated from threonine and serine, and overlaps sarcosine. The buffer can be made from the standard pH 2.875 buffer by adding; two drops of concentrated hydrochloric acid to 100 ml buffer.

RAPID

SEPARATION

OF

URINE HYDROXYPROLINEMIC

AMINO

OF

ACIDS

301

A PATIENT

L 20

40

60

80

MINUTES

Fro. 1. System I: separation of hydroxyproline.

System III.

Proline

through

Valine

When the pH 2.875 is used alone, proline and glycine are readily separated from all other amino acids (Fig. 3). Proline is eluted in 60 min in this system, glycine at 70 min, and alanine at 80 min followed by a-aminobutyric acid and valine. Since valine is not eiuted until 105 min, it is more satisfactory to use System VII (see below) for this amino acid. System IV. Cystine and Cysteine-Penicillamine Disulfide Cystinuric patients on penicillamine therapy excrete both cystine and the mixed disulfide in the urine. Both are resolved in a buffer with a pH of approximately 3.0. This buffer is prepared by adding 2-3 mi of pH 4.74 to 150 ml of pH 2.875 buffer. The absolute* pH should be adjusted so that the cystine, which is very sensitive to pH changes, is eluted between valine and methionine. The mixed disulfide is &ted after the cystine

and overlaps

methionine

(Fig.

4). For practical

purposes,

1;

opTICALp

DENglTY

OPTICAL

DENSITY

RAPID

SEPARATION

20

URINE

OF AMINO

303

ACIDS

OF A CYSTINURIC PATIENT ON 0- PENICILLAMINE

10 t

30

40

60

80

120

100

140

MlN”TES

Fm. 4. System IV: separation on penicillamine therapy.

of amino acids in urine from a cystinuric

patient

this is not important, because cystinurics (and normals) rarely excrete more than 10 mg of methionine per day. Homocitrulline does not interfere in this system, in which it overlaps valine. System V. Cystathionine A pH 3.25 buffer was made by mixing the 2.875 and 4.74 buffers in the proportion of 175 to 5 ml. Cystathionine was eluted at 85 min following methionine. Cystine emerges at 55 min, just before valine; URINE OF A PATIENT WITH NEUROBLASTOMA + STANDARD AMINO ACID MIXTURE

20

40

60

80

100

120

MlN”iES

FIG. 5. System V: separation

of methionine

and cystathionine.

304

SHIH,

EF’RON;

AND

MECHANIC

it is not separated from homocitrulline (Fig; 5). When homocitrulline is known not to be present in the sample, this system can also be used to quantitate cystine. Cystathionine is also sensitive to pH. In the next system (pH 3.51))~ it overlaps valine. System VI. Homocitdine This buffer is a mixture of pH 2.875 and 4.74 buffers in the proportion of 150 to 5 ml. The pH is approximately 3.30 and the sodium 2

Cl.5;:.

;

‘_ :’ :, : ,..

..:..:’

.4 CQ z ifI 00 .3-

d 9 Lo

.2 -

:,.. CI.1 -

20 M;tuouTES

FIG. 6. System VI:

separation

60

of homocitdline.

concentration 0.22 M. Homocitrulline is eluted before valine, while cystine overlaps alanine (Fig. 6). The position of homocitrulline is fairly sensitive to pH. At lower pH values, e.g. 3.10, it is moved toward valine. System’VII. Branched-Chain Amino Acids The pH 3.50, 0.24M Na buffer for the separation of the branchedchain amino acids can be made up conveniently by mixing the two standard buffers (pH 2.875 and 4.74) in the proportion 14 to 1. The

RAPID

SEPARATION

0~

AMINO

305

ACIDS

PLASMA (ISCLEUCINE LOADING TEST)

2.0

1

L20 MINUTES

FIG. 7. System VII:

separation

of branched-chain

amino acids suitable

for moni-

toring maple syrup urine disease. amino acids were eluted in the same sequence as with the 2.875 buffer. except for cystine, which is very sensitive to pH and which emerges before valine, being eluted in a mixed peak with glycine and alanine (Fig. 7). Alloisoleucine is well resolved in this system, making the method quite satisfactory for studies of maple syrup urine disease (11). System VIII.

Tyrosine-Phenylalanine

Tyrosine and phenylalanine are resolved using a pH 4.30, 0.4 M Na buffer. This is described completely in a previous publication (7). It is noted that buffer wit,11 sodium concentration of 0.4 M gives the same resolution as that of 0.38M, and that the resolution is satisfactory with any pH between 4.30 and 4.50. System IX.

Methionilre-Homocystine, @Alanine. y-Aminobutyric Acid, and Argininosuccinic Acid

For monitoring homocystinuria, it was necessary to use two buffers. These patients have not only homocystine but also methionine in excess

306

SHIH,

EFRON,

AND

MECHANIC

in the blood and urine. In order to resolve the two amino acids in the same analysis, the pH 3.50, 0.24M Na buffer was used until after the emergence of methionine, followed by the same pH 4.30, 0.4 M Na buffer which is used for the tyrosine-phenylalanine separation. In our system, there is a 20 min lag before the second buffer reaches the column. The time of buffer change may have to be varied from the 55 STANDARD AMINO ACIO MfXTURE 2.0l.O-

0.5: 9 0.4: d 0.3? 8 0.2-

0.1 -

1

20

f

40

I

60

1

I

I

100

I

120

MN&S

FIG. 8. System IX: a short-column and hyperbetaalaninemia.

method suitable

for study of homocystinuria

min that has proved satisfactory on our analyzer, if a different system with a different lag time is used (Fig. 8). In the same figure, the positions of J-alanine, P-aminoisobutyric acid, and y-aminobutyric acid are also shown. Specimens containing argininosuccinic acid are usually boiled in acid before analysis to convert the unstable free argininosuccinic acid to its anhydrides, B and C of Westall (12). Anhydride B falls between leucine and tyrosine, while anhydride C emerges near homocystine (Fig. 9).

RAPID

URINE + 2.0

SEPARATION

OF AMINO

307

ACIDS

OF AN ARGININOSUCCINICACIDURIC STANDARD AMINO ACID MIXTURE

PATIENT

-

l.O-

m al 0.5 c’ 0.4v, 6 0 0.3;: u b8 0.2.

I 0.1 -

I

I

20

40

I

I

60

80

I

I

I

I

IO0

120

MINUTES

FIG.

9. Separation of argininosuccinic acid and its anhydrides

by

System

IS.

System X. The Basic Amino Acids The pH 4.79, 0.4 M Na buffer can be prepared by diluting the pH 4.74 buffer used in the standard long-column analysis 1: 1 with water) and titrating to pH 4.79 with hydrochloric acid. In order to separate histidine and ammonia, the jacket temperature is maintained at 33”. After the emergence of ammonia, the temperature is raised to 65” and the buffer is changed to one of pH 5.8, 0.8 M Na. This is done in order to hasten the emergence of arginine (Fig. 10). It is apparent that there was no marked baseline shift despite the abrupt change in salt concentration. ACCELERBTED

ANALYSIS

The analyses described in Figures l-10 can be further ure 11 is an example; it shows the analysis described requiring only one-third the time. This analysis was performed by one of us (G.M.1 in VG8000 amino acid analyzer. For this analysis. a resin

accelerated. Figin System V but a Phoenix model which gives het-

52

I3

G-

r

c

Leucine

lsoleucme

-

~A,onin;Wcine

2 and

Arginine

Histidine

uffer

Temperature

DENSITY

Change

F====

>

>-=-Ammonia

Lysine

OPTICAL

RAPID

SEPARATION

OF AMINO

AClDS

309

ter resolution was used (Spherix ~~-907-10, which is now commercially available from Phoenix Precision Company, Philadelphia, Pennsylvania). The resin column was packed in a 0.9 cm column to a height of 10 cm. The flow rate was 2.0 ml/min. STANDARD

SOLUTIONS

Cysteic acid is used as a standard for all analyzers. For each buffer system, the constant for each amino acid (i.e., its color value relative to that of cysteic acid) is determined initially. Thereafter, standardization with cysteic acid is performed each day on each column to be used. One milliliter of a solution containing 0.1 pmole/ml cysteic acid is pumped through the column, using the first buffer to be used for analysis of the unknown. After this peak is recorded, the unknown is placed on the column and analyzed. This daily standardization is necessary on the Technicon amino acid analyzer; such frequent standardization is unnecessary on other analyzers in which the entire effluent enters the recording system. REGENERATION

OF

THE

COLUMNS

When the run is completed, the buffer is removed from the top of the resin column, and the space filled with 0.4 M NaOH containing 0.1% EDTA. The equilibrating buffer line is then attached and the buffer pumped for 20 min, after which the column is both regenerated and equilibrated for the next analysis. DISCUSSION

The column methods described in this paper can be adapted for use with any amino acid analyzer. With better resins, such as Spherix xX907-10 (11-14 p) , the analyses can be performed with higher flow rates, e.g., 2.0 ml/mm on an 0.9 X 10 cm column. This permits even more rapid analyses with as little as one-third the time required for the analyses described above. With two columns, one can be regenerated and equilibrated while t’he amino acids are being eluted and quantitated on the other column. Many analyses can, therefore, be performed in one day. The method is ideally suited to automation, and to procedures for accelerated analyses (1, 2). A gradient system with an Autograd can also be used with the short columns by using the same composition of buffers as for the long columns but proportionately less buffer in each chamber. Obviously, one or two buffer systems are preferable for rapid runs because preparation of the Autograd is time consuming and a gradient elution offers no advantage over single or discontinuous two-buffer systems.

310

SHIH,

EFRON,

AND

MECHANIC

In this laboratory, we routinely perform two 14 hr analyses on two 50 X 0.9 cm columns overnight (9). This is used for measurement of samples in which the entire amino acid content is to be quantitated. During the working day, short-column runs are performed on samples in which only one or a few amino acids are to be measured. Three pumps are used during the day, one for regeneration of the long columns, one for regeneration of one short column, and the third for the amino acid analysis on the other short column. In this way, efficient use can be made of the ninhydrin and recording systems throughout the entire day and night. It is expected that this system will prove useful for screening and treatment programs which, although concerned almost exclusively with phenylketonuria at present, are now expanding to include other amino acid disorders. SUMMARY

Methods are described for the rapid analysis of certain individual amino acids (e.g., homocystine) and of groups of amino acids (e.g., the branched-chain amino acids), which are relevant to particular metabolic diseases. All the methods use ion-exchange chromatography on a 20 )( 0.9 cm column. These methods were developed especially for clinical application in the diagnosis and therapy of inborn errors of amino acid metabolism. All analyses are completed in less than 2.5 hr. ACKNOWLEDGMENT This investigation was supported in part by grants from the U. S. Public Health Service (NB-5269, NB-05096 and AM-%375) and by The Joseph P. Kennedy, Jr., Foundation and The John A. Hartford Fund. The authors are grateful to Miss Maryellen McCarthy for expert technical assistance. REFERENCES P. B., Federation Proc. 18, 241 (1959). P. B., in “Technicon Symposium on Automation in Analytical Chemistry, New York, N. Y. (1965),” p. 702. Mediad Inc., New York, 1966. DICKENSON, J. C., RQSENBLUM, H., AND HAMILTON, P. B., Pediatrics 36, 2 (1965). “Spinco DS-248,” Beckman Instruments, Inc. CATRAVAS, G. N., in. “Technicon Symposium on Automation in Analytical Chemistry, New York, N. Y. (1965),” p. 648. Mediad Inc., New York, 1966. EFRON, M. L., New Engl. J. Med. 272, 1053, 1107 (1965). MECHANIC, G., EFRON, M. L., AND SHIH, V. E., Anal. Biochem. 16, 420 (1966). EFRON, M. L., in “Technicon Symposium on Automation in Analytical Chemistry, New York, N. Y. (1965),” p. 637. Mediad Inc., New York, 1966. EFECON, M. L., SHIH, V. E., AND MECHANIC, G., in preparation. HAMILTON, P. B., Anal. Chem. 35, 2055 (1963).

1. HAMILMN, 2. HAMILTON,

3. 4. 5. 6. 7. 8. 9. 10.

RAPID

SEPARATION

OF AMINO

ACIDS

311

11. NORTON, P. M., ROITMAN, E., SNYDERMAN, S. E., .~ND HOLT, L. E., Lancet 1, 26 (1962). 12. ARMSTRONG, M. D., YATES, K. N., AND STEMMERMANN, M. G., Pediatrics 33, 280 (1964). 13. MECHANIC, G. L., SHIH, V. E., AND EFFCON, M. L., in “Technicon Symposium on Automation in Analytical Chemistry, New York, N. Y. (1966).” Mediad Inc., Sew York, in press.