Proc. Nati. Acad. Sci. USA Vol. 76, No. 12, pp. 6652-6655, December 1979

Medical Sciences

Polyamine-pyridoxal Schiff bases in urine (excretion/metabolism/vitamin B-6)

ROSEMARIE AIGNER-HELD*, ROBERT A. CAMPBELLt, AND G. DOYLE DAVES, JR.* *Department of Chemistry and Biochemical Sciences, Oregon Graduate Center, Beaverton, Oregon 97005; and tPediatric Renal-Metabolic Laboratory, University of Oregon Health Sciences Center, Portland, Oregon 97201

Communicated by Karl Folkers, September 28, 1979

ABSTRACT Schiff bases of the diamines 1,3-diaminopropane, putrescine, and cadaverine and the polyamines spermidine and spermine with pyridoxal or pyridoxal phosphate occur in human urine, as shown by gas chromatographic/mass spectrometric selected ion-monitoring techniques. By use of synthetic standards, procedures were devised for conversion of the Schiff bases to stable derivatives amenable to gas chromatospectrometric analysis. These procedures involve Fraphic/mass borohydride reduction of the C=N double bond, hydrolytic removal of the phosphate group, chromatographic separation from the bulk of urinary constituents, and trifluoroacetylation of polar functional groups. The levels of the polyamine-pyridoxal Schiff bases were estimated to be in the range of pmol/ml of urine.

N (CH2)4 NH2

N (CH2)3 NH2

II CH

HO H3C

CH

OH

CH2-0-P-O NO

HO

H 3C

N

The presence in human urine of Schiff bases of the diamines 1,3-diaminopropane, putrescine, and cadaverine and the polyamines spermidine and spermine with pyridoxal or pyridoxal phosphate (Fig. 1) has been established by gas chromatographic/mass spectrometric selected ion-monitoring techniques. Pyridoxal phosphate undergoes Schiff base formation with a variety of endogenous amines (1). Indeed, the principal storage forms of pyridoxal phosphate are as Schiff base conjugates with various proteins (1-6). To our knowledge, the occurrence of polyamine-pyridoxal Schiff bases in body fluids has not been recognized previously although these conjugates are known synthetically (7, 8).

HO

f

H3C

N

CH

OH

CH2 -O-P-O OH

OH

$CH2-O-P-O

HO H3C N

OH

Spermine-

Cadaverine-

N (CH2)3NH (CH2)4 NH2

N (CH2)4NH (CH2)3NH2

CH

CH

OH

CH2-O-P0O

HO

-13C

OH

N(CH2)3NH (CH2)4NH (CH2)3 NH2

(CH2)5NH2

CH

CCH2-0-P-O N

Putrescine-

1 ,3-Diaminopropane-

N

OH

N

1 -N-Spermidine-

O

HO H3C

OH

CH2-0-P10 CK N

OH

8-N-Spermidine-

FIG. 1. Polyamine-pyridoxal phosphate Schiff bases.

EXPERIMENTAL Materials and Instrumentation. All chemicals used were reagent grade; glassware was rinsed in 5 M HC1 prior to use. Gas chromatography/mass spectrometry (gc/ms) experiments were carried out with a DuPont 21-491B mass spectrometer interfaced with a Varian 2700 gas chromatograph. This system is equipped with a four-channel DuPont MSID accessory for selected ion monitoring (SIM) analysis. Outputs from the gas chromatograph flame ionization detector and the selected ion monitors were recorded on a Gould Brush 260 six-channel recorder. The 1 m X 2 mm glass column was packed with 3% Dexsil 300 on Chrom W AW DMCS 80/100 mesh (Varian Aerograph). The column was treated with DMCS prior to packing. The column temperature was programmed from 100 to 400°C at 20°C/min. Urine Samples. Morning urine samples of three healthy young adults were obtained. All received at least the recommended daily allowance of vitamin B-6 for 3 days prior to urine collection. The collected urine (400-500 ml) was frozen either immediately or after reduction with sodium borohydride. Aliquots of 10-100 ml of urine were analyzed. Preparation of Polyamine-Pyridoxal Phosphate Schiff Bases. To 1 mmol of the appropriate polyamine or polyamine

hydrochloride in water was added 0.8 mmol of pyridoxal phosphate. The pH of the solution was adjusted to 8 with aqueous sodium bicarbonate and the completion of the reaction was verified by ultraviolet spectroscopy (7, 8). Reduction. For analysis of the Schiff bases, samples were stabilized by reduction with sodium borohydride. The yellow solutions of Schiff bases produced as described above were reduced by adding NaBH4 until discoloration was complete. For reduction of Schiff bases present in urine samples, NaBH4 (-100 mg/20 ml of urine) was added slowly and the mixture was stirred until foaming ceased. In an alternative procedure, a mixture of 0.5 g of platinum oxide and 100 ml of urine was shaken for 4 hr under 2 atm of hydrogen pressure (1 atm = 1.013 X 105 pascals). The catalyst was then removed by filtration.

Hydrolysis. The phosphate group was removed from the reduced polyamine-pyridoxal Schiff base by acid hydrolysis at pH 4.1 or with the sample 0.5 M in HC1 (9, 10). Samples were heated under reflux in a nitrogen atmosphere for a minimum of 12 hr. In initial experiments using synthesized materials, completion of phosphate hydrolysis was monitored by measuring the formation of heteropolyacids spectroscopically at 650 nm (11). Alternatively, samples were hydrolyzed enzymatically with

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Abbreviations: gc/ms, gas chromatography/mass spectrometry; SIM, selected ion monitoring; m/e, mass-to-charge ratio. 6652

Medical Sciences:

Proc. Natl. Acad. Sci. USA 76 (1979)

Aigner-Held et al.

6653

CF3

1001

0

C- 679CF3 --1-----|

154

IH F3CCN-CH2 -CH2-CH2-LCH2-N-CH2-CH2-CH2-N-,CO6

-636

4).

CH2

I F3C-C-0

679 H3C N"

4)

-

0

-622

0

0

I CHZ21I O-C-CF3

345

566

663-322

776

679

[ml*

276

0

566

4).

a:

663

498

LI

622

I1

80

I

I

6361 6I 6 111 I

700

800

1001

Ill

5 500

I

600

400

i1 1i

300

200

100

679 345

H3Cl

154

663

10 .O-

C

- 50-

776

276

[Ml+,

566

650

.O-

a:

636

I

I I -~~~~~~ .

a

I

I.

800

700

1622 -

1,iI

I

I

I

l

Li

II *, ,, I . ....,, ., .. s I II.I. m

,

600

.

500

400

.

iIiI

III , 300

Is

li

.

I II 11 200

m 100

mie

FIG. 2. Mass spectra of the reduced trifluoroacetylated spermidine-pyridoxal Schiff base isomers. For assignment of isomer structures, Fig. 3 and Discussion. m/e, Mass-to-charge ratio.

see

acid phosphatase (12). The enzyme (2 mg) was added to a solution of urine or synthesized sample that had been buffered with acetic acid/KOH at pH 5.10. The acid phosphatase-treated samples were then incubated at 37 + 0.50C for 60-90 min with stirring.

Column Chromatography. After reduction and hydrolysis, solution was concentrated at 450C under reduced pressure to t2 ml, neutralized (if necessary) with sodium bicarbonate, and introduced onto a dry-packed silica gel column (1.5 X 9 cm). The column was then washed with 100 ml each of methanol, methanol/water, 1:1 (vol/vol), and water. The polyamine-pyridoxal conjugate was then eluted with 100 ml of 30 mM HC1. a

Trifluoroacetylation. The HCI eluate obtained upon chromatography was evaporated to dryness under reduced pressure. To the resulting residue, 5 ml of trifluoroacetic anhydride was added and the reaction vessel was sealed and placed in an ultrasonic bath for 30-60 min. The unreacted trifluoroacetic anhydride was removed with a stream of nitrogen; the residue was dissolved in 200 Al of dichloromethane for gc/ms analysis.

RESULTS Polyamine-Pyridoxal Schiff Bases. The preparation of Schiff bases (imines) of polyamines and pyridoxal or pyridoxal phosphate has been reported (7, 8). Because the Schiff bases are hydrolytically unstable and involatile, chemical stabilization and derivatization was required to permit gc/ms analysis. Stable, volatile derivatives of the Schiff bases were prepared by a three-step procedure involving sequential reduction of the C- N double bond with sodium borohydride, removal of the phosphate group (if present) either enzymatically or by acid hydrolysis, and trifluoroacetylation of polar functional groups.

The gas chromatographic retention times, the ions chosen for use in SIM analysis, and the ion abundance ratios used for recognition are included in Table 1. Two pyridoxal Schiff base derivatives for the unsymmetrical polyamine spermidine were observed by SIM analysis. Comparison of the mass spectra of the two spermidine-pyridoxal conjugate trifluoroacetyl derivatives (Fig. 2) reveals few differences. However, structures were assigned to the isomeric

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Medical Sciences: Aigner-Held et al.

Proc. Natt. Acad. Sci. USA 76 (1979)

Table 1. Chromatographic retention temperatures for reduced polyamine-pyridoxal Schiff base trifluoroacetyl derivatives and identification of ions used in gc/ms SIM experiments Polyamine-pyridoxal Retention Ion monitored Ion m/e conjugate temp., 0C I2/I1 (I3/I2)* Assignment 1,3-Diaminopropane 248 1 M+609 6 2 496 (M-CF3CO2)+ Putrescine 255 1 M+623 7

Cadaverine

250

2 1

510 637

(M-CF3CO2)+ M+-

305

2 1

524 776

(M-CF3CO2)+ W+

2

663

(M-CF3CO2)+

3 1

679 816

(M-CF3CO)+ (M-CF3CO2)+

2

832

(M-CF3CO)+

3

Spermidine

2

(1.5) Spermine

365

0.5 * Monitored intensities of the respective ion currents.

conjugates by comparison of the relative abundances of ions at m/e 622 and m/e 636 measured accurately by SIM analysis in the spectrum of each. The origins of these ions for each isomer and the results of the SIM analysis that permit assignment of structures are shown in Fig. 3. Analysis of Urine Samples. Analysis of urine samples from healthy individuals by procedures developed by using authentic polyamine-pyridoxal phosphate Schiff bases prepared synthetically resulted in identification of the six polyamine-pyridoxal Schiff bases (for which structures are shown in Fig. 1) as urinary constituents. Fig. 4 contains the selected ion recordings of the analysis of a sample prepared from a urine specimen which, together with retention values and ion intensity data (Table 1) obtained with reference compounds, establishes unequivocally the presence of the polyamine-pyridoxal Schiff bases. The procedure developed for preparation and isolation of polyamine-pyridoxal Schiff bases from urine incorporates the derivatization steps together with a silica gel column chromatographic step that allows the urinary conjugates, after reduction and removal of phosphate esters, to be separated from the

bulk of urinary constituents. The procedure uses borohydride reduction of the C=N double bond and results in raising the pH of a urine sample from ;5.5 to -8. The possibility that the urine conjugates are artifacts formed during the reduction process (polyamine-pyridoxal phosphate Schiff bases form more rapidly at higher pH) was ruled out when catalytic reduction of a urine sample over platinum also permitted identification of urinary Schiff bases. As presently developed, the procedure does not distinguish between phosphorylated and nonphosphorylated pyridoxal species. Similarly, Schiff base conjugates of pyridoxal or pyridoxal phosphate and acetylated or other modified forms of polyamines might occur in urine and be converted to the corresponding free Schiff bases upon acid hydrolysis. Although the present study is qualitative, an estimation of the urinary levels of polyamine-pyridoxal Schiff bases was made. Using a SIM response curve for the derivative prepared from the putrescine-pyridoxal phosphate conjugate, we estimate that the concentration of each of the six urinary Schiff bases is in the pmol/ml range. Preparation of Schiff base standards by using deuterated polyamines (13, 14) will permit accurate determination of levels. ,CF3 C=O

0

I

II

F3CCNHCH2CH2CH2,CH2N

Channel A

6361

m/e

m/e 622 = 167 m/e 636

0

L- 622

o..CF3

C=O

1 F3CCNHCH2CH2CH2N

Channel B

L 636

I

m/e 622 = 0.56

| 6221

m/e

I

°C

I

m/e 636

320 305290 FIG. 3. Selected ion recordings for the two fragment ions m/e 622 and m/e 636 in the mass spectra of isomeric spermidine-pyridoxal conjugates. Indicated are retention temperatures, the monitored ions, and the ion intensity ratios. Upper, 1-N-spermidine derivative; Lower, 8-N-spermidine

derivative.

Medical Sciences:

Aigner-Held et al.

Proc. Natl. Acad. Sci. USA 76 (1979)

v Cadaverine

Spermidine v

r

663l

1

679

524

M/e

637

rm/e

Channel B

1

V Spermine

Diaminopropone

Channel A

II

816

1

-496

1

609 rm/e y Putrescine

mT/e

Channel B

1 832 Spermidine I Channel A

m

663

1 510

rm/e

Channel B

7776 I,,,, II,11,,1

400

Appreciation is expressed to the National Cancer Institute (CA 16328) and the M. J. Murdock Charitable Trust for financial support.

Channel A

I

6655

350

1 623

I,,,I

300

r,/e

1°C , ,

250

200

FIG. 4. Two-channel SIM analysis of six reduced and trifluoroacetylated polyamine-pyridoxal Schiff bases of a sample prepared from human urine. The column emergence temperatures of these compounds, the points at which changes were made in the ions monitored, and the assignment of ions to SIM runs (I, II, or III) and to ion-monitoring channels (A or B) are indicated. In runs I and III, a third channel was monitoring perfluorokerosene reference ions mie 505 and m/e 655; in run II, mle 493 and mle 743. See Experimental for chromatographic conditions and Table 1 for identification of the specific ions selected for monitoring.

DISCUSSION It is increasingly recognized that conjugated as well as free polyamines occur in body fluids. Thus, we have recently shown that 1-N-acetylspermidine occurs in serum (15) and both 1-Nand 8-N-acetylspermidines and acetylated forms of 1,3-diaminopropane, putrescine, and cadaverine occur in relatively large amounts in urine. (16, 17). In addition, peptides incorporating polyamines have been reported as occurring in both urine and serum (18-21).

The occurrence of polyamine-pyridoxal Schiff base conjugates in tissues or body fluids other than urine has not been established. Still, recognition that polyamine-pyridoxal Schiff base conjugates occur in urine is noteworthy. A number of pathological states, including uremia and cancer, are accompanied by increased polyamine levels in body fluids (21-24) and by vitamin B-6 insufficiency (25, 26). The possibility that the dynamics of transport and physiological actions of the polyamines and pyridoxal (pyridoxal phosphate) in body fluids are linked is an intriguing one and is consistent with the occurrence of polyamine-pyridoxal Schiff bases in urine.

1. Snell, E. E., Braunstein, A. E., Severin, E. E. & Torchinsky, M. Yu. (1968) Pyridoxal Catalysis: Enzymes and Model Systems (Wiley, New York). 2. Snell, E. E. (1971) Vitam. Horm. (N.Y.) 28,265-290. 3. Davis, C. & Metzler, D. E. (1973) in The Enzymes, ed. Boyer, P. D. (Academic, New York), 3rd Ed., Vol. 7, pp. 33-74. 4. Bosron, W. F., Veitch, R. L., Lumeng, L. & Li, T. K. (1978) J. Biol. Chem. 253, 1488-1492. 5. Lumeng, L., Ryan, M. P. & Li, T. K. (1978) J. Nutr. 108, 545553. 6. Nandi, D. L. (1978) Arch. Biochem. Biophys. 188,266-271. 7. O'Leary, M. H. (1971) Biochim. Biophys. Acta 242,484-492. 8. Heller, J. S., Canellakis, E. S., Bussolotti, D. L. & Coward, J. K. (1975) Biochim. Biophys. Acta 403, 197-207. 9. Hudson, R. F. (1965) Structure and Mechanisms in Organic Phosphorus Chemistry (Academic, New York), pp. 271-272. 10. Tabor, H., Tabor, C. W. & Irreverri, F. (1973) Anal. Biochem.

55,457-467. 11. Halmann, M. (1972) Analytical Chemistry of Phosphorus Compounds (Wiley-Interscience, New York), p. 51. 12. Takahashi, Y. & Matsuda, M. (1975) Jikeikai Med. J. 22, 1319. 13. Smith, R. G. & Daves, G. D., Jr. (1977) Biomed. Mass Spectrom. 4, 146-151. 14. Bartos, F., Bartos, D., Grettie, D. P., Campbell, R. A., Marton, L. J., Smith, R. G. & Daves, G. D., Jr. (1977) Biochem. Biophys.

Res. Commun. 175,915-919. 15. Smith, R. G., Bartos, D., Bartos, F., Grettie, D. P., Frick, W., Campbell, R. A. & Daves, G. D., Jr. (1978) Biomed. Mass Spectrom. 5, 515-517. 16. Abdel-Monem, M. M., Ohno, K., Newton, N. E. & Weeks, C. E. (1978) in Advances in Polyamine Research, eds.Campbell, R. A., Morris, D. R., Bartos, D., Daves, G. D., Jr. & Bartos, F. (Raven, New York), Vol. 2, pp. 37-49. 17. Tsuji, M., Nakajima, T. & Sano, I. (1975) Clin. Chim. Acta 52, 161-167. 18. Rosenblum, M. G. & Russell, D. H. (1977) Cancer Res. 37,4751. 19. Rosenblum, M. G., Durie, B. G. M., Beckerman, R. C., Taussig, L. M. & Russell, D. H. (1978) Science 200, 1496-1497. 20. Seale, T. W., Chan, W. Y., Shukla, J. B. & Rennert, 0. M. (1978) Pediat. Res. 12,512. 21. Russell, D. H. & Durie, B. G. M. (1978) Progress in Cancer Research and Therapy (Raven, New York), Vol. 8, pp. 135-136. 22. Campbell, R. A., Talwalker, Y. B., Bartos, D., Bartos, F., Musgrave, J., Harner, M., Puri, H., Grettie, D. P., Dolney, A. M. & Loggan, B. (1978) in Advances in Polyamines Research, eds. Campbell, R. A., Morris, D. R., Bartos, D., Daves, G. D., Jr. & Bartos, F.(Raven, New York), Vol. 2, pp. 319-343. 23. Nishioka, K. & Romsdahl, M. M. (1974) Clin. Chim. Acta 57, 155-161. 24. Russell, D. H., Levy, C. C., Schimpff, S. C. & Hawk, I. A. (1971) Cancer Res. 31, 1555-1558. 25. Dobbelstein, H., Korner, W. F., Mempel, W., Grosse-Wilde, H. & Edel, H. H. (1974) Kidney Int. 5, 233. 26. Wachstein, M., Kellner, J. D. & Ortiz, J. M. (1960) Proc. Soc. Exp.

Biol. Med. 105,563-566.

Polyamine-pyridoxal Schiff bases in urine.

Proc. Nati. Acad. Sci. USA Vol. 76, No. 12, pp. 6652-6655, December 1979 Medical Sciences Polyamine-pyridoxal Schiff bases in urine (excretion/metab...
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