Serum β-aminopropionaldehyde: Identification and origin

CLINICA

CHIMICA ACTA

I7

SERUM b-AMINOPROPIONALDEHYDE

GERARD

QUASH

AND

DAVID

: IDENTIFICATION

AND ORIGIN

R. TAYLOR

Department of Biochemistry and Department of Chemistry, University of West Indies, Mona, Kingston 7 (Jamaica) (Received April 2nd. 1970)

SUMMARY

I. j3-Aminopropionaldehyde is one of the serum aldehydes whose level increases on treating serum with As,O, and methanol. 2. In animal tissues, /I-aminopropionaldehyde can arise directly from the oxidation of spermidine or diaminopropane. 3. A pathway linking spermidine, /&aminopropionaldehyde and malondialdehyde to the control of RNA and DNA synthesis is proposed.

In a previous paper’ it was shown that the serum level of bound aldehyde was significantly lower in patients having malignant diseases as compared with patients either non-malignant diseases or malignant diseases who had responded to treatment. In contrast, the serum level of bound aldehydes was markedly elevated in patients with malignant metastases. In order to try and interpret these results, an attempt was made to identify these aldehydes as their z,4-dinitrophenyl hydrazones (z,4-DNP). This was done by chromatographing the z,+DNP derivatives of the As,O, treated and untreated sera along with similar derivatives of known reference compounds. In making reference compounds we tried to achieve not only the identification of the unknown spots from serum, but also clues as to their origin. In this, we were guided by the spermine activator-spermine dialdehyde inhibitor relationship which Bachrach et aL2 had identified, and hence chose the amines as starting material to prepare the reference aldehydes. Of all the naturally occurring polyamines, diaminopropane (DAP) was chosen as the amine precursor for the following reasons: (a) It should be easily converted by hog kidney diamine oxidase to a dialdehyde in a z-step reaction as follows: --f NH,(CH,),CHO --f CHO CH,CHO NH,(CH,),NH, /I-aminopropionaldehyde malondialdehyde. DAP (b) One product of the reaction, malondialdehyde is in fact a potent inhibitor Abbreviations: SAM = S-adenosyl methionine; SAP = S-adenosyl propylamine; DAP = Diaminopropane; TLC = Thin-layer chromatography; PLC = Preparative-layer chromatography; BAPA = B-aminopropionaldehyde; DNP = dinitrophynyl hydrazone Clin. Chim. Acta, 30 (1970) 17-23

18

QUASH, TAYLOR

of growth3 and has a molecular formula C,H,O,. Retine, which is also a growth inhibitor is believed to be similar but not identical to methyl glyoxa14 with a molecular formula C,H,O,. We therefore decided to prepare the z,4-DNP derivatives of the products arising from the oxidative deamination of diaminopropane and to see whether they corresponded with As,O,. MATERIALS

on TLC

with the 2,4-DNP

derivatives

produced

on treating

serum

AND METHODS

Hog kidney diamine oxidase was prepared and partially purified as described by Tabor et al.5 and its activity was assayed as follows: The reaction mixture contained 0.1 ml enzyme (2.7 mg protein), 0.1 ml DAP at varying concentrations, 0.1 ml of 0.14 M NaCl, 0.004 M Tris pH 7.4. The enzyme and DAP were dissolved in the same buffer. After incubation at 37’ for 4 h, the tubes were cooled and the aldehydes estimated

by the method

of Bachrach

et al.B.

Preparation of z,+DNP derivatives (a) From the o&dative deamination of DAP. 20 mg of DAP 2 HCl, neutralized and dissolved in 0.5 ml of 0.14 M NaCl, 0.004 M Tris pH 7.4 were incubated with 5 ml of partially

purified enzyme (135 mg prot.) at 37” for 4 h. At the end of the incubation

period, the mixture was placed on a Sephadex G-25 column (68 cm x 1.0 cm) equilibrated with the same buffer as above. The eluate was monitored for protein at 280 nm as well as for aldehydes. The tubes containing aldehydes and any unreacted diaminopropane were pooled and placed on an Amberlite CG 50 column (Na+ form) 14 cm x 1.2 cm equilibrated with 0.1 M acetate buffer pH 4.8. The aldehydes and amines were eluted off the column using a continuous gradient of acetate buffer pH 4.8 from 0.1 M to 0.5 M. The eluate was monitored for amine with ninhydrin and for aldehydee. The contents of the tubes containing the amine aldehyde peak were pooled and added to 2,4-DNPH reagent to form the corresponding derivative, which was recrystallized from methanol/ethyl

acetate.

(b) From sewm. To 5 ml of serum were added 250 mg As,O, along with 7.5 ml of 5o”/” methanol. The control tube contained the same minus As,O,. After heating at 70~ for 30 min the tubes were cooled, acidified with cont. HCl, shaken with an equal volume of CHCl, to denature proteins and centrifuged at 3000 rev./min for IO min. To the aqueous supernatant was added 1/5 volume of 2,4-DNPH reagent. The volume was reduced at room temperature under vacuum, the resulting precipitate dried and then dissolved in ethyl acetate for thin-layer chromatography. Once the aldehydes had been released, no heat was applied at any stage in the formation of the 2,4-DNP, so as to avoid the formation of the osazones of any sugars present.

TLC and PLC TLC was carried out on glass plates coated with a layer of Silica Gel G (Merck) 0.5 mm thick. When plates were to be scanned, silica-coated Eastman chromagram sheets, type K 301 R, were used. For PLC the plates were I mm thick. All solvents were of analytical grade.

C&z. Chim. Acta, 30 (1970)

17-23

SERUM /3-AMINOPR~PIONALDEHYDE

I9

RESULTS (a)

IdentiJication

of /?-aminopropionaldehyyde

2,4-DNP*

HCI

Fig. I shows the profile obtained after elution of the mixture of DAP and enzyme from a Sephadex G-25 column. On chromatographing the mixture of reaction products and any unreacted substrate on an Amberlite CG 50 column, the aminoaldehyde was separated from the diamine as shown on Fig. 2. Only those tubes of peak I in which there was no overlap with peak 2 were used to prepare the z,4-DNP of the amino aldehyde. 25

100 -Peak

1

025

w Tube

4”

No.

Fig. I. Separation on Sephadex G-25 of the oxidation products of DAP from the diamine oxidase Aldehyde at 660 nm. Elution with 0.14 M NaCI, preparation. __ Protein at 280 nm; ---0.004 Tris pH 7.4. Fig. 2. Separation on Amberlite CG 50 of the oxidation products of DAP into aminoaldehyde and amine. Aldehyde at 660 nm; ---amine at 570 nm. 4 the arrow indicates the point at which elution started using a continuous gradient of CH,COO Na, pH 4.8, from 0.1 M to 0.5 M.

On TLC in a 17 :3 benzene-methanol mixture, the z,4-DNP of the aminoaldehyde peak yielded the chromatogram shown in Fig. 3A. It can be seen that there is only one constituent in addition to the two reagent spots. This constituent on recrystallisation from methanol/ethyl acetate gave pure (TLC) ,Saminopropionaldehyde Z,J-DNPeHCl, identical by m.p. x98”, mixed m.p. and Infra-red spectral comparison to the corresponding derivative of authentic BAPA prepared by the action of alcohol dehydrogenase on /3-aminopropanol. (b) Qualitative and quantitative examinations

of serum aldehyde 2,4-DNP’s

On chromatographing the z,4-DNP derivatives of serum with and without treatment by As,O,, the pattern shown in Fig. 3B was obtained. The components Clin. Chim.

Acta,

30 (1970)

17-23

QUASH, TAYLOR

20

which have increased in intensity on As,O, treatment are dotted in Fig. 3B. Fig. 4 illustrates this more clearly. When duplicate chromatograms were scanned quantitatively near 400 nm it was found that of the two components which increased on As,O, treatment, one increased by about 200%, the other by 74%.

00

OD 0

I II III

00 00 00 000

*o PL

Fig. 3. Thin-layer chromatograms in benzene-methanol 17: 3 of: A (I) z,4-DNPH, (II) z,4-DNP of oxidised DAP. B (I) z,4-DNPH, (II) z,4-DNP of serum alone, (III) 2,4-DNP of serum+As,O,. C (I) z,4-DNP of serum+As,O, (II) z,4-DNP of serum alone, (III) p-Aminopropionaldehyde 2,42,4-DNP* HCl. DNP*HCl. D (I) 2,4-DNP of oxidised spermidine, (II) ,!?-Aminopropionaldehyde Fig. 4. This layer chromatogram in benzene-methanol rum alone, (III) 2,4-DNP of serum+As,O,.

17: 3 of:

(I) 2,4-DNPH,

(II) 2,4-DNP

of se-

Com$arison of the z,4-DNP’s of semm and BAPA The chromatogram (Fig. 3C) of the z,4-DNP derivatives of (i) serum+As,O,, (ii) serum, (iii) /3-aminopropionaldehyde in a 17 : 3 benzene-methanol mixture shows that the constituent which increases by about 200% on As,O, treatment has the same RF value of 0.32 as that of pure BAPA 2,4-DNP.HCl. This identity in RF values using other solvent systems is illustrated in Table I. (c)

C&Z.Chim.

Acta,

30

(1970)

IT-23

21 TABLE ORIGIN

I AND

RF

VALUES

OF

z,z+-DNP

Solvent system

Serum+

Propanol-Pet. Ether 65 : 35 Ethanol-Pet. Ether 20 : 80 Benzene-Propanol 50 : 50

0.59 0.26 0.78

As,O,

Serum- As,O,

BA PA

0.59 0.26 0.78

0.59 0.26 0.78

The compound corresponding to BAPA 2,4-DNPaHCl on TLC was isolated by PLC in benzene-methanol 17 : 3. Crystallization from methanol/ethyl acetate gave needles (m.p. 198”) shown to be identical with BAPA 2,4-DNPeHCl by mixed m.p. and IR spectral comparison. Origin of B-aminopropionalde~yzyde (BAPA)

Though the origin of BAPA from DAP had been clearly demonstrated, it was felt that in the absence of an unequivocal demonstration of DAP in animal tissues, an alternative pathway for the origin of BAPA may be operative. Such a pathway from spermidine is operative in spermidine-adapted Pseudomonas7. In addition, it has been shown quite conclusively by Siimes et aL8 that, in rat liver, spermidine can be converted to putrescine but the fate of the (CH,),NH, was not investigated. Spermidine was therefore oxidised using partially purified enzyme preparations from hog kidney. The oxidation products were identified as the 2,4-DNP derivatives. Fig. 3D suggests that BAPA can be formed in animal tissues from the enzymatic oxidation of spermidine. Unequivocal identification of this constituent as BAPA 2,4-DNPaHCl was achieved by isolating this constituent by PLC and comparing it (mixed m.p. and IR) with the authentic compound. DISCUSSION

From the results presented it is clear that p-aminopropionaldehyde (BAPA) is a constituent of human serum. It has also been shown that BAPA can arise in hog kidney, as in Pseudomonas sP,~ from the enzymatic breakdown of spermidine as follows: NH,(CH,),NH (CH,),NH, -+ NH,(CH,), NH,+NH,(CH,),CHO putrescine + BAPA Additional evidence for the existence of this pathway for spermidine degradation in rat liver can be inferred from the work of Siime9 who has been able to show that labelled putrescine is formed from spermidine labelled with 14Cin the (CH,), portion of the molecule. Another pathway which gives rise to #I-aminopropionaldehyde is, as has been shown, via diaminopropane. DAP has not been identified as such in animal tissues; nevertheless Bachrach8 has shown that it too can arise from spermidine in spermidineadapted Serratia marcescens as follows : I (,J NH,(CJ&),NH (‘X),NH, + N&VL),NH,+ DAP d l pyrroline In animal tissues then, spermidine is the precursor of BAPA either directly or perhaps indirectly via DAP. If we now look at the known physiological roles of these two compounds, viz. Clin. Chim. Acta, 30 (1970)

17-23

QUASH, TAYLOR

22

spermidine and BAPA, we find that increased spermidine levels have been found in regenerating rat liver?, in embryonic tissuerr, and in Erlich ascites cells12. In fact, there is evidence that spermidine affects the DNA-dependent RNA polymerase reaction, either directlyr3, or indirectly by increasing protein degradation and hence making available to the cell more t-RNA amino acid complexes which are the immediate regulators of RNA synthesis 14. Regardless of the mechanism, spermidine increases RNA synthesis ilz v&or3 and increased spermidine levels precede increased RNA synthesis in V~ZIO~~. With regard to @-aminopropionaldehyde, its physiological role has not yet been elucidated, but the presence of an amine group in BAPA plus its ready conversion to malondialdehyde, a potent growth inhibitor3, can be equated with similar observations made on retine4, the natural inhibitor of cell growth. This could also explain the difficulties encountered in isolating retine, since, if “active” retine is malondialdehyde, this compound is unstable and highly reactive. As /?-aminopropionaldehyde comprises more than 70% of the aldehydes released by As,O, treatment, it could also account for the majority of the aldehyde measured quantitatively. We can therefore say that the initial drop in bound aldehyde levels found in malignancy corresponds to a decrease of /Laminopropionaldehyde. This lowering of BAPA levels could be caused by a partial block in the reaction leading from spermidine to BAPA. The decreased substrate level of BAPA leads to a corresponding drop in the product malondialdehyde, hence more rapid growth takes place. If the reaction leading from BAPA to malondialdehyde is now itself inhibited then the small amount of BAPA which was being fed through this pathway can no longer be oxidised, hence an increase in BAPA occurs. This could explain the elevated BAPA levels found in metastasesl. In addition to BAPA, histamine aldehyde can also contribute to the elevation of the aldehyde levels found in metastases, as it can arise from the oxidation of histamine by diamine oxidase itself. Some authorsr6Tr7 even use the term diamine oxidase to include histaminase. High levels of histamine have been found in certain human malignancies, e.g. chronic myelocytic leukaemial* and in some experimental tumoursrg. This could therefore mean that in malignancy, the active sites on the enzyme are occupied by histamine instead of BAPA, hence decreased oxidation of /3-aminopropionaldehyde takes place. The end result of this decrease in BAPA oxidation would in fact be two-fold: (I) an accumulation of ,%aminopropionaldehyde and of spermidine leading to increased RNA synthesis; (2) a decrease in malondialdehyde hence increased DNA synthesis. As an index of growth-controlling factors therefore, the estimation of histamine aldehyde and p-aminopropionaldehyde should yield values within a narrower range than those obtained on measuring total aldehydesl. Rapid specific assays for these two compounds are at present being attempted. In summary of these findings and their relationship to other known pathways of cell growth and cell division, we advance the scheme shown in Fig. 5. ACKNOWLEDGEMENTS

The authors thank their colleagues in the Department of Biochemistry C&z.

Chim.

Acta,

30 (1970)

17-23

and the

23

.. ...,..

= Hypothetical

pathways

in

-

= E.tebli.hsd

pathwayP in Bnimal tissues

.mimml tissues

Fig. 5. Scheme of the relationship of our findings to other pathways Reference numbers in parentheses.

of cell growth and cell division.

Tropical Metabolic Research Unit for helpful discussions. They are also indebted to Dr. A. Hudson-Phillips who photographed the T.L.C. plates and to Mr. P. Reeson for drawing the figures and diagrams. The technical assistance of Misses E. Chin and S. Nelson is gratefully acknowledged. REFERENCES I G. QUASH AND K. MAHARAJ, Clin. Chim. Acta, zg (1970) 4026. z U. BACHRACH, S. ABZUG AND A. BEKIERKUNST, Biochim. Biophys. Acta, 134 (1967) ‘74. 3 B. BROOKS AND 0. KLAMERTH, Europ. J. Biochem., 5 (1968) 178. 4 L. EYGBD, Proc. Natl. Acad. .%a’. U.S., 54 (1965) zoo. 5 H. TABOR, J. Biol. Chem., 188 (1951) 125. 6 U. BACHRACH AND B. RECHES, Anal. Biochem., 17 (1966) 38. 7 R. PADMANABHAN AND K. KIM, Biochem. Biophys. Res., Commun., Ig (1965) I. 8 M. SIIMES, Acta Physiol. Stand., Suppl., zg8 (1967) 44. g H. BACHRACH, J. Biol. Cham., 194 (Ig62a) 377. IO A. RAINA, J. JOANNEAND M. SIIMES, Biochim. Biophys. Acta, 123 (1966) 197. II A. RAINA, Acta Physiol. Stand., Suppl. , 218 (1963) 49. 12 M. SIIMES AND J. J;CNNE, Acta Chem. &and., 21 (1967) 815. I3 K. ABRAHAM, Europ. J. Biochem., 5 (1968) 143. 14 D. EZEKIEL AND H. BROCKMAN, J. Mol. Biol., 31 (1968) 541. 15 G. MORUZZI, B. BARBIROLI AND C. CALDARERA, Biochem. J., 107 (1968) 609. 16 P. COHEN AND H. SALLACH, in D. GREENBERG (Ed.), Metabolic Pathways, Academic Press, New York, 1961, Chapter 13, p. 28. 17 H. TABOR, Pharmacol. Revs., 6 (1954) 322. 18 M. CLINE, Physiol. Revs., 45 (1965) 674. rg D. RUSSELL AND S. SNYDER, Proc. Natl. Acad. Sci. U.S., 60 (1968) 1420. zo J. JHNNE, A. RAINA AND M. SIIMES, Biochim. Biophys. Acta, 166 (1968) 4x9. 21 A. PEGG AND H. WILLIAMS-ASHMAN, Biochem. Biophys. Res. Comun., 30 (1968) 76. 22 H. TABOR, S. ROSENTHAL AND C. TABOR, J. Biol. Chem., 233 (1958) 907. 23 E. WAINFAN, P. SRINIVASAN AND E. BOREK, Biochemistry, 4 (1965) 2845. 24 D. FUJIMOTO, P. SRINIVASAN AND E. BOREK, Biochemistry, 4 (1965) 2849.

C&k Chim. ACta, 30 (1970) 17-23