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Automated analysis of common basic amino acids, mono-, di-, and polyamines, phenolicamines, and indoleamines in crude biological samples
ANALYTICAL
BIOCHEMISTRY
91, 264-275 (1978)
Automated Analysis of Common Basic Amino Acids, Mono-, Di-, and Polyamines, Phenolicamines, and lndoleamines in Crude Biological Samples VICTOR R. VILLANUEVA AND RAMESH C. ADLAKHA lnstitut
de Chimie
des Substances
Natwelles.
CNRS.
91 I90 Gif sur Yvette,
France
Received May 1, 1978 A fully automated, fast, and sensitive method for the separation of common basic amino acids and mono-, di-, and poiyamines as well as phenolic- and indoleamines is described. Picomole level determination of hydroxytryptophan, tryptophan, histidine. lysine, ethanol amine, arginine, noradrenaline, diaminopropane, putrescine, histamine, cadaverine, dopamine, hexamethylenediamine, agmatine, tyramine, phenethylamine, serotonin, S,4-dihydroxytryptamine, Smethoxytryptamine, tryptamine, spermidine, and spermine is carried out by ionexchange column chromatography on a single sample in 170 min oftotal analysis. This method is well suited for crude extracts without preliminary purification, thus reducing preparative losses. The reproducibility of the method has been studied and the percentage recovery of the different compounds after column chromatography is reported. Its application to crude samples from different biological sources such as microorganisms, vegetables, platelets, and urine is presented. This method could serve as a powerful tool for the analysis of these amino compounds in which there is currently a considerable interest.
The significance of mono-, di-, and polyamines in biochemical and physiological processes is well known (l-4) and has stimulated the development of a large number of methods for their rapid and sensitive assay. Ion-exchange column chromatography with automated inst~ments is the most favored method for routine analysis of these compounds (5-13). In general, however, most of these methods concern only separations of putrescine, cadaverine, spermidine, spermine, and a few other related compounds. We recently published a chromatographic procedure for the separation of nine of these compounds (putrescine, histamine, cadaverine, spermidine, hexamethylenediamine, agmatine, tyramine, phenethylamine, and spermine) (11,12). We have now improved this method to allow separation of common basic amino acids and have enlarged the list of related amino compounds by including the group of indoleamines. To the best of our knowledge this is the first method described to separate such a large number of naturally occurring mono, di-, and polyamines, phenolicamines, and indoleamines in a single sample at the picomole level, in a short time and applicable to any kind of crude sample 0003-2697/78/0911-0264$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
264
POLYAMINES
ANALYSIS
IN CRUDE BIOLOGICAL
MATERIALS
SAMPLES
265
AND METHODS
Synthetic mixture of amines and related compounds. The amino acids used: Tyrosine’ (Tyr), phenylalanine (Phe), hydroxytryptophan (OH-Trp), tryptophan (Trp), histidine (His), lysine (Lys), arginine (Arg), 1,3diaminopropane (DAP), putrescine (PU), histamine (HA), cadaverine (Cd), spermidine (Sd), hexamethylenediamine (HDA), agmatine (Agm), dopamine (DA), noradrenaline (NA). tyramine (TA), phenethylamine (PA), spermine (Sm), 5-methoxytryptamine (5MT), and tryptamine (T) as hydrochlorides and 5,6-dihydroxytryptamine (5,6-DHT), and S-hydroxytryptamine (5-HT) as creatinine sulfate derivatives were obtained from Sigma Chemical Company, St Louis, Missouri. Ethanolamine (EtA) was from Merck, Darmstadt, Germany and benzylamine (internal standard) from Fluka AG, Buchs, Switzerland. Except for 5,6-dihydroxytryptamine (5 x 1O-5 M) and for the internal standard, benzylamine (1 x 10e5 M), all the amine compounds were made up to a concentration of 5 x 10e6 M in 0.1 M HCL. o-Phthalaldehyde was obtained from Fluka AG, Buchs and all other chemicals used for the preparation of buffers and reagent were obtained as the highest purity grade from Merck. Sample Preparation Escherichia coli B grown to a density of 3 x IO” cells/ml in 5 ml of phosphate-based minimal medium (14) were harvested and extracted for 1 h with 1 ml of 5% TCA in 0.05 M HCL at 4°C. The extract was centrifuged and the supernatant was washed with ether to remove the TCA. Aliquots (25-100 ~1) of the extract was subsequently used for analysis. Tomatoes obtained from the local market were homogenized in 2 vol of 5% TCA in 0.05 M HCl. The homogenates were then centrifuged to remove debris and after ether extraction a lOO-~1 aliquot was used for analysis. Platelets. Blood was obtained from normal adult donors who had not taken medication of any kind in the IO-day period prior to venesection. Twenty-seven milliliters of the blood was mixed immediately with 3 ml of 3.8% trisodium citrate and citrate platelet-rich plasma (C-PRP) was separated from whole blood by centrifugation at 2008 for 20 min at room temperature. To obtain platelet pellet, C-PRP was spun at 3000g for 20 min at 4°C. After elimination of the supernatant, the pellet was washed with NaCl ’ Abbreviations used: Tyr. tyrosine; Phe. phenylalanine; OH-Trp. hydroxytryptophan; Trp, tryptophan; His. histidine; lys, lysine; Arg, arginine: DAP, 1,3-diaminopropane; PU. putrescine; HA. histamine; Cd. cadaverine: Sd, spermidine; HDA, hexamethylenediamine; Agm. agmatine; DA. dopamine; NA. noradrenaline; TA, tyramine; PA. phenethylamine; Sm. spermine; 5MT. 5methoxytryptamine; T, tryptamine; 5,6-DHT. 5,6-dihydroxytryptamine; 5-HT. 5hydroxytryptamine; EtA, ethanol amine; TCA. trichloroacetic acid; C-PRP. citrate platelet-rich plasma; EDTA. ethylenediaminetetraacetic acid.
266
VILLANUEVA
AND
ADLAKHA
at 9%0, centrifuged, and finally suspended in 0.4 ml of the same NaCl solution and counted under a phase microscope. Contamination by red cells and leukocytes was negligible. Amines were extracted from this suspension by addition of 0.6 ml of a 10% perchloric acid solution. After 5 min of homogenization followed by centrifugation at 300g for 5 min, the pellet was again extracted with 0.5 ml of a 5% perchloric acid solution and recentrifuged. The supernatants were pooled and a loo-p1 aliquot was submitted directly to analysis. Urine. One milliliter of urine was treated with 50 ~1 of 80% TCA and centrifuged at 200g for 5 min. One hundred microliters of the supernatant was used directly for analysis. Hydrolysis of samples. All samples were hydrolyzed as described by Gehrke et al. (13), i.e., by addition of an equal volume of concentrated HCl (12 M) and heating at 150°C for 4 h. After hydrolysis the samples were filtered and dried under vacuum. HCl was eliminated by repeated washing with water and subsequent drying. The samples were then dissolved in 0.1 M HCl and loo-p1 aliquots were submitted to analysis. Equipment. An amino acid analyzer (Liquimat-Labotron) equipped with a fluorimeter (Labotron FFM-31, Kontron, Velizy-Villacoublay) using a 50-~1 flow cell was employed. An integrator (ICAP-10, LTT, Sainte-Honorine) was coupled to the fluorimeter for quantification of the amines by the method of internal standard. The recorder (W + W 600Tarkan, Kontron) was set at 100 mV for 100% relative fluorescence. The sensitivity of the recorder could be increased lo-fold further if necessary. Chromatographic
Conditions
Separation of the individual amines was achieved on a 0.45 x 10.5-cm column of DC-4A cation-exchange resin (Durrum Chemical Corp., Palo Alto, Calif.). The composition of the eluting buffers and the elution program were as indicated in Table 1. Buffers were prepared from a freshly doubly distilled/deionized water, brought to the required pH with concentrated HCl, and filtered through a Millipore filter (0.22-pm pore size). After filtration the alcohols were added and the final pH was readjusted. The fluorogenic reagent was prepared by dissolving 800 mg of o-phthalaldehyde in 10 ml of ethanol to which 2 ml of @mercaptoethanol was added. This mixture was then added to 1 liter of 2.5% boric acid solution (brought to pH 10.40 with 45% KOH), containing 900 mg of Brij-35. Buffers were driven at a flow rate of 26 ml/h and reagent at the rate of 25 ml/h. Column back pressure generated during analysis was approximately 25 kg/cm*. Mixing of reagent with column effluent and fluorometric detection were as described by Benson and Hare (15). Two temperatures were used, 66°C during the first 50 min and thereafter
POLYAMINES
ANALYSIS
IN CRUDE BIOLOGICAL TABLE
COMPOSITION
AND
CONDITIONS
SAMPLES
267
I OF
ELUT~NG BUFFERS Buffers
Sodium citrate dihydrate” Sodium chIoride’z Alcohol added Final pH Time (min)
First
Second
Third
0.20 0.30 Ethanol (5.5%) 5.55 It 0.01 37
0.20 2.30 Ethanol (6%) 5.63 t 0.01 40
0.20 2.55 Isopropanol ( 10.5%) 5.73 rt 0.01 93
CLMolarity in Na+.
78°C till the end of the chromatogram. The column was regenerated with 0.2 M NaOH containing 250 mg of EDTAiliter for 10 min and equilibrated by passing the first buffer for 30 min. RESULTS
Figure 1 shows the chromatographic resolution of 22 compounds, consisting of the common basic amino acids, mono-, di-, and polyamines, phenolicamines, and indoleamines. The components of the mixture, the amount of each applied to the column, the retention time, the relative fluorescence peak areas, and the constant values (KC) (benzylamine taken as internal standard) are shown in Table 2. The column, and the elution program employed gave good overall resolution. With the first buffer, the acidic and the neutral amino acids (aspartic acid through phenylalanine included) were eluted in a group. Hydroxytryptophan, tryptophan, histidine, lysine, ethanolamine, ammonia, and arginine were subsequently eluted, in that order, with good resolution. The second buffer eluted and separated well the mono, di-, and polyamines and phenolicamines but not the indoleamines. The elution of indoleamines was achieved by the use of a third buffer of higher ionic strength, containing 10.5% isopropanol, and by raising the column temperature from 66 to 78°C. Complete analysis was effected in 170 min. The presence of alcohol in the buffers is nesessary to obtain a good resolution and sharp peaks. The influence and optimal concentration of four different alcohols (methanol, ethanol, propanol, and isopropanol) in the buffers was studied. Those which gave better resolution, sharpness of peaks, and eluting force were finally adopted. The optimal concentrations for the alcohols were as indicated in Table 1. Isopropanol was chosen for the third buffer as it accelerated well the elution of the indoleamines and no baseline shift was observed. The eluting force of the different alcohols for the indoleamines was in the following order: methanol < ethanol < propanol < isopro-
258
5.MT
OH-Trp Trp His bs EtA NH, A% NA DAP PU HA Cd I.S. DA Sd HDA Agm TA PA 5.6.DHT SED 5-m
Abbreviation
AND A~WRACY
k i2 +
i 0.2 2 0.2 z 0.3 -+ 0.3 1 0.2 IO.2 ;t 0.2 t 0.1 2 0.3 * 0.3 i 0.3 -r 0.2
50.2 51.9 55.0 58.2
63.6 67.2 70.6 73.3 76.0 79.6 84.7 89.8 95.8 101.5 156.1 164.5
0.1 0.2 0.3 0.2
f + If+ I i t It
9.6 12.4 14.7 l&b 23.1 25.9 32.7 45.9
0.1 0.1 0.2 O.? 0.1 0.2 0.2 0.1
Retention time” OtlilI)
-
Ur-.
1148.69 757.69 597.73 1042.05 775.46 1011.18 984.06 1302.09 260.05 1177.69
27,919 25.677
2.60 1.98 I.74 3.25
1743.61 438.71 1196.40 663.21 1788.51 722.14 451.33 745.85
55,632 50.897 18,789 68,405 54.683 58,278 43,723 42,923 44,226 38.364 34,325 32,099 49.%7 29,705 54,743 28.434 4.58 0.93 4.59
1.80
1.55 3,40
3.13 0.86 6.36 0.97 3.27 1.24 1.03 1.74
2166.42 2202.90 1609.8S 2552.09 1574.30
5.57 5‘49 2.81 4.?0 2.70
RSD cw
AND PHENOLK
48.349 33,113
77.871 40.022
1535.01 483.61
1152.43 1267.49 831.9% 939.16
929.80
1844.64
605.21 1712.74 1613.59 781.67 900.67 1132.79 1113.14
1133.72
1590.20 1018.67
i382.49
1676.26
988.12 1356.35 1273.09
SD
2.06 2.11 2.58 2.48 1.42 1.70 i.07 2.35 3.17 I.46
2.21 1.30 1.20
2.23 1.93 4.62 0.80 2.10
2.29 2.77 2.10 3.01 2.18
RSD wd
80.5 72.7 48. I 85.7 61.6 43.2 70.3 71.1 57.8 77.5
72.5 57.3
77.9 96.5 76.6 90.6 67.2 79.7
90.2 81.8 94.6 97.3 91.9
AND INDOLEAMINES
392 429 1163 319 399 375 1.9. 509 494 569 636 681 437 7359 399 768 783 851
401 374
561 444 381
16.99 10.47
12.05 II.98
9.00 1.24 5.69 7.82 1.63 5.20 60.34
7.02 3.22 11.51 6.86 0.68 7.01
9.65 7.45 4.95 t.40 6.67
1.77 0.25 I.04 1.23 0.24 1.19 0.82 3.02 1.56 2.17 1.23
1.79 0.75 0.99 2.15 0.17 1.87
1.72 1.37 1.30 0.35 I.78
standard (IOQO pmol) aad S&DHT (5OlB pmoll. RSD (Yei;)= SDimean x 100.
68.761
54.939 52.756 71.362 37.455 8L.115
60.300 74,947
71.365 52,743 24.529 75,470 81,403 73,133
55,blb 63,483
60,585
43,128 49.041
Mean
Surface area before the column chromatography’
AND POLYAMINES
38.902 40,116 57,314 S4,SLd 58,331
SD
Surface area after the column chromatography”
OF MONO-,
Mean
OF THE ANALYSIS
2
n Average of IO determinatmns of thr mix!ure containing SM) pmol of rach of the products except for the inter&
5-Hydroxytryptophan Tryptophan Histidine Lysine EIh~ttolamine Ammonia Arginine Noradrenaline 1.3.Diaminopropane Futrescine H&%?li%?e Cadawine Benzylamine Dopamine Spermidine Hexa~ethylened~mi~~ Agmatine Tyramirre ~e~thy~mine S.d-Dihydroxytryptamine Spermine .%Hydroxytryptamine Tryptamine S-Methoxytryptamine
Compound
PRECISION
TABLE
270
VILLANUEVAANDADLAKHA
panol. The recovery of the different compounds after column chromatography was studied, and the results are presented in Table 2. Comparison was made between the surface areas of the peaks obtained after the chromatography and those obtained by the direct reaction of the different products with the reagent without passing through the column (this was done by connecting the inlet and outlet of the column together). The lowest recoveries after column chromatography were observed in cases of 5,6-dihydroxytryptamine (43%), agmatine (48%), dopamine (57%), and tryptamine (58%). For all other compounds recoveries varied between 60 and 97%. It must be pointed out that hydroxytryptamine derivatives
I
Pu *c
I
‘I
E.COLl
sd
0
20
40
60 TIME
( min
)
FIG. 2. Chromatogram of a 50-~1 sample of a TCA extract of E. co/i. Besides putrescine and spermidine two peaks with the same retention time as that of histamine and cadaverine which remained after hydrolysis of the sample were also observed. See Table 2 for the identities of the products.
POLYAMINESANALYSISINCRUDE
~~OLOGICALSAMPL~S
L-
& Y)
R I% I 33N33S3101Tlj
3hUVl3M
0
271
272
VILLANUEVA
AND ADLAKHA.
undergo gradual decomposition even at -20°C. Thus when the center of interest is the detection of these compounds, the samples must be analyzed immediately after extraction. Attempts are being made to overcome this difficulty. Quantification was done with the aid of an integrator coupled to the fluorimeter and by employing an internal standard. The integrator was calibrated before with a standard mixture, as indicated in Table 2. Figures 2, 3, 4, and 5 show the chromatograms obtained from crude samples of E. Cali B, tomatoes, platelets, and human urine, respectively. Values obtained for the different polyamines and other amino compounds in the samples analyzed are in agreement with those reported in the literature. E. Cofi B. Putrescine and spermidine were the major compounds present, as reported earlier (5). A peak with the same retention time as that of cadaverine was observed. This peak remained even after hydrolysis of the sample. An increase in the spermidine content was also observed after hydrolysis. Tomatoes. The analysis of the tomato sample was a good test for our
PLATELETS
FIG. 4. Chromatogram of a IOO-~1 sample of a perchloric Table 2 for the identities of the products.
acid extract of ptatelets. See
POLYAMINES
ANALYSIS
IN CRUDE
~ ----
-.-._
-._A
BIOLOGICAL
SAMPLES
273
274
VILLANUEVA
AND
ADLAKHA
method as it contained most of the mono-, di-, and polyamines as well as serotonine and tryptamine. The amounts of these last two indoleamines were of the same order as already reported (16). Platelets. Polyamine and indoleamine content found was in accordance with previous results (17-20). In addition to putrescine, histamine, spermidine, spermine, and serotonine we observed a peak with the same retention time as that of cadaverine which remained even after hydrolysis. Urine. Analysis of free polyamines and total polyamines in human urine was also in good agreement with published results (5,9,13). Phenolic and indoleamines were also present (21-23). DISCUSSION
In the present work we describe a method which separates a wide range of biologically impo~ant amines with an automatic amino acid analyzer without any modification of the analyzer. With this method we were able to analyze in a relatively short time the common basic amino acids and mono-, di-, poly-, phenolic-, and indoleamines in the picomole range in a single crude sample. A three-step change of buffers and two temperatures were employed. Incorporation of alcohols into the buffers dramatically improved the separation and sharpness of peaks. Strongly bound compounds such as tryptamine and 5-methoxytryptamine could also be eluted by using isopropanol without much increase in the ionic strength. The main goal of this work was to develop a method for the direct analysis of biological materials. This was achieved by use of the appropriate ionic strength and concentration of alcohols in each of the three eluting buffers. The first buffer eluted amino acids only and the second and third buffer the amines. The prior purification of samples, which is often time consuming, was not necessary and thereby the risk of losing amino compounds, which could be present in trace amounts was avoided. The retention times of different amino compounds were not altered by our procedure. This was verified by cochromatographing the crude samples with standard mixtures of amino compounds. Compared with the previously described methods, ours has the principal advantage of separating a large number of related amino compounds in a single chromatographic analysis in a relatively short time with high sensitivity (detection limit, picomole level). The use of an integrator and of an internal standard in the samples contributes to an easier and reproducible quantification. The reproducibility of the method is similar to that of automatic analysis of amino acids and is now a routine procedure in our laboratory. REFERENCES 1. Tabor, 2. Cohen,
C., and Tabor. H. (1976) Annu. Rev. Biochem. S. S. (1978) Adv. Polyamine Res. 1, l-10.
45, 285-306.
POLYAMINES
ANALYSIS
IN CRUDE BIOLOGICAL
SAMPLES
275
3. Durie, B. G. M., Salmon, S. S., and Russell, D. H. (1977) Cancer Res. 37, 214-221. 4. Smith, T. A. (1977) in Progress in Phytochemistry (Reinhold, L., Harborne, J. B.. and Swain, T.. eds.), Vol. 4, pp. 27-81, Pergamon Press, New York. 5. Tabor, H., Tabor, C. W., and Irreverre. F. (1973) Anal. Biochem. 55, 457-467. 6. Veening, H., Pitt, W. W., Jr., and Jones, G., Jr., (1974) J. Chromatogr, 90, 129- 139. 7. Marton, L. J., and Lee, P. L. Y. (1975) Clin. Chem. 21, 1721- 1724. 8. Gehrke. C. W., Kuo. K. C.. Zumwalt, R. W.. and Waalkes, T. P. (1974)5. Chromotogr. 89, 231-238. 9. Adler, H.. Margoshes. M.. Snyder, L. R.. and Spitzer, C. (1977) J. Chromafogr. 143, 125- 136. 10. Marton, L. J., Heby, O., Levin, V. A.. Lubich, W. P., Crafts. D. C., and Wilson, C. B. ( 1976) Cuncer Res. 36, 973-977. 11. Villanueva, V. R.. Adlakha, R. C., and Cantera-Soler, A. M. (1977) .I. Chromatogr. 139, 381-385. 12. Villanueva, V. R., Adlakha, R. C.. and Cantera-Soler. A. M. (1978) Phytochemistrv, 17, 1245- 1249. 13. Gehrke, C. H., Kuo, K. C., and Ellis, R. L. (1977) J. Chromatogr. 143, 345-361. 14. Vogel, H. J., and Bonner, D. M. (1956) J. Biol. Chem. 218, 97- 106. 15. Benson, J. R., and Hare, P. E. (1975) Proc. Nat. Acad. Sci. USA 72, 619-622. 16. Undenfriend, S., Lovenberg, W., and Sjoerdsma, A. (1959) Arch. Biochem. Biophys. 85, 487-490. 17. Udenfriend, S., Weissbach, H., and Clark, C. T. (1955) J. Biol. Chem. 215, 337-344. 18. Porter. J. F. (1972) Physiol. Rev. 52, 361-381. 19. Tidball, M. E. (1971) Ann. J. Physiol. 221, 1064-1070. 20. Cooper. K. D., Shukla, J. B., and Rennet?, 0. M. (1976) Clin. Chim. Acta 73, 71-88. 21. Kakimoto, Y., and Armstrong, M. D. (1962) J. Biol. Chem. 237, 208-214. 22. Perry. T. L. (1962) Sciences 136, 879-880. 23. Altman, P. L., and Dittmer. D. S. (1974) in Biology Data Book, 2nd ed., Vol. 3, pp. 1496-1507, Federation of American Societies for Experimental Biology, Bethesda, Md.
BIOCHEMISTRY
91, 264-275 (1978)
Automated Analysis of Common Basic Amino Acids, Mono-, Di-, and Polyamines, Phenolicamines, and lndoleamines in Crude Biological Samples VICTOR R. VILLANUEVA AND RAMESH C. ADLAKHA lnstitut
de Chimie
des Substances
Natwelles.
CNRS.
91 I90 Gif sur Yvette,
France
Received May 1, 1978 A fully automated, fast, and sensitive method for the separation of common basic amino acids and mono-, di-, and poiyamines as well as phenolic- and indoleamines is described. Picomole level determination of hydroxytryptophan, tryptophan, histidine. lysine, ethanol amine, arginine, noradrenaline, diaminopropane, putrescine, histamine, cadaverine, dopamine, hexamethylenediamine, agmatine, tyramine, phenethylamine, serotonin, S,4-dihydroxytryptamine, Smethoxytryptamine, tryptamine, spermidine, and spermine is carried out by ionexchange column chromatography on a single sample in 170 min oftotal analysis. This method is well suited for crude extracts without preliminary purification, thus reducing preparative losses. The reproducibility of the method has been studied and the percentage recovery of the different compounds after column chromatography is reported. Its application to crude samples from different biological sources such as microorganisms, vegetables, platelets, and urine is presented. This method could serve as a powerful tool for the analysis of these amino compounds in which there is currently a considerable interest.
The significance of mono-, di-, and polyamines in biochemical and physiological processes is well known (l-4) and has stimulated the development of a large number of methods for their rapid and sensitive assay. Ion-exchange column chromatography with automated inst~ments is the most favored method for routine analysis of these compounds (5-13). In general, however, most of these methods concern only separations of putrescine, cadaverine, spermidine, spermine, and a few other related compounds. We recently published a chromatographic procedure for the separation of nine of these compounds (putrescine, histamine, cadaverine, spermidine, hexamethylenediamine, agmatine, tyramine, phenethylamine, and spermine) (11,12). We have now improved this method to allow separation of common basic amino acids and have enlarged the list of related amino compounds by including the group of indoleamines. To the best of our knowledge this is the first method described to separate such a large number of naturally occurring mono, di-, and polyamines, phenolicamines, and indoleamines in a single sample at the picomole level, in a short time and applicable to any kind of crude sample 0003-2697/78/0911-0264$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
264
POLYAMINES
ANALYSIS
IN CRUDE BIOLOGICAL
MATERIALS
SAMPLES
265
AND METHODS
Synthetic mixture of amines and related compounds. The amino acids used: Tyrosine’ (Tyr), phenylalanine (Phe), hydroxytryptophan (OH-Trp), tryptophan (Trp), histidine (His), lysine (Lys), arginine (Arg), 1,3diaminopropane (DAP), putrescine (PU), histamine (HA), cadaverine (Cd), spermidine (Sd), hexamethylenediamine (HDA), agmatine (Agm), dopamine (DA), noradrenaline (NA). tyramine (TA), phenethylamine (PA), spermine (Sm), 5-methoxytryptamine (5MT), and tryptamine (T) as hydrochlorides and 5,6-dihydroxytryptamine (5,6-DHT), and S-hydroxytryptamine (5-HT) as creatinine sulfate derivatives were obtained from Sigma Chemical Company, St Louis, Missouri. Ethanolamine (EtA) was from Merck, Darmstadt, Germany and benzylamine (internal standard) from Fluka AG, Buchs, Switzerland. Except for 5,6-dihydroxytryptamine (5 x 1O-5 M) and for the internal standard, benzylamine (1 x 10e5 M), all the amine compounds were made up to a concentration of 5 x 10e6 M in 0.1 M HCL. o-Phthalaldehyde was obtained from Fluka AG, Buchs and all other chemicals used for the preparation of buffers and reagent were obtained as the highest purity grade from Merck. Sample Preparation Escherichia coli B grown to a density of 3 x IO” cells/ml in 5 ml of phosphate-based minimal medium (14) were harvested and extracted for 1 h with 1 ml of 5% TCA in 0.05 M HCL at 4°C. The extract was centrifuged and the supernatant was washed with ether to remove the TCA. Aliquots (25-100 ~1) of the extract was subsequently used for analysis. Tomatoes obtained from the local market were homogenized in 2 vol of 5% TCA in 0.05 M HCl. The homogenates were then centrifuged to remove debris and after ether extraction a lOO-~1 aliquot was used for analysis. Platelets. Blood was obtained from normal adult donors who had not taken medication of any kind in the IO-day period prior to venesection. Twenty-seven milliliters of the blood was mixed immediately with 3 ml of 3.8% trisodium citrate and citrate platelet-rich plasma (C-PRP) was separated from whole blood by centrifugation at 2008 for 20 min at room temperature. To obtain platelet pellet, C-PRP was spun at 3000g for 20 min at 4°C. After elimination of the supernatant, the pellet was washed with NaCl ’ Abbreviations used: Tyr. tyrosine; Phe. phenylalanine; OH-Trp. hydroxytryptophan; Trp, tryptophan; His. histidine; lys, lysine; Arg, arginine: DAP, 1,3-diaminopropane; PU. putrescine; HA. histamine; Cd. cadaverine: Sd, spermidine; HDA, hexamethylenediamine; Agm. agmatine; DA. dopamine; NA. noradrenaline; TA, tyramine; PA. phenethylamine; Sm. spermine; 5MT. 5methoxytryptamine; T, tryptamine; 5,6-DHT. 5,6-dihydroxytryptamine; 5-HT. 5hydroxytryptamine; EtA, ethanol amine; TCA. trichloroacetic acid; C-PRP. citrate platelet-rich plasma; EDTA. ethylenediaminetetraacetic acid.
266
VILLANUEVA
AND
ADLAKHA
at 9%0, centrifuged, and finally suspended in 0.4 ml of the same NaCl solution and counted under a phase microscope. Contamination by red cells and leukocytes was negligible. Amines were extracted from this suspension by addition of 0.6 ml of a 10% perchloric acid solution. After 5 min of homogenization followed by centrifugation at 300g for 5 min, the pellet was again extracted with 0.5 ml of a 5% perchloric acid solution and recentrifuged. The supernatants were pooled and a loo-p1 aliquot was submitted directly to analysis. Urine. One milliliter of urine was treated with 50 ~1 of 80% TCA and centrifuged at 200g for 5 min. One hundred microliters of the supernatant was used directly for analysis. Hydrolysis of samples. All samples were hydrolyzed as described by Gehrke et al. (13), i.e., by addition of an equal volume of concentrated HCl (12 M) and heating at 150°C for 4 h. After hydrolysis the samples were filtered and dried under vacuum. HCl was eliminated by repeated washing with water and subsequent drying. The samples were then dissolved in 0.1 M HCl and loo-p1 aliquots were submitted to analysis. Equipment. An amino acid analyzer (Liquimat-Labotron) equipped with a fluorimeter (Labotron FFM-31, Kontron, Velizy-Villacoublay) using a 50-~1 flow cell was employed. An integrator (ICAP-10, LTT, Sainte-Honorine) was coupled to the fluorimeter for quantification of the amines by the method of internal standard. The recorder (W + W 600Tarkan, Kontron) was set at 100 mV for 100% relative fluorescence. The sensitivity of the recorder could be increased lo-fold further if necessary. Chromatographic
Conditions
Separation of the individual amines was achieved on a 0.45 x 10.5-cm column of DC-4A cation-exchange resin (Durrum Chemical Corp., Palo Alto, Calif.). The composition of the eluting buffers and the elution program were as indicated in Table 1. Buffers were prepared from a freshly doubly distilled/deionized water, brought to the required pH with concentrated HCl, and filtered through a Millipore filter (0.22-pm pore size). After filtration the alcohols were added and the final pH was readjusted. The fluorogenic reagent was prepared by dissolving 800 mg of o-phthalaldehyde in 10 ml of ethanol to which 2 ml of @mercaptoethanol was added. This mixture was then added to 1 liter of 2.5% boric acid solution (brought to pH 10.40 with 45% KOH), containing 900 mg of Brij-35. Buffers were driven at a flow rate of 26 ml/h and reagent at the rate of 25 ml/h. Column back pressure generated during analysis was approximately 25 kg/cm*. Mixing of reagent with column effluent and fluorometric detection were as described by Benson and Hare (15). Two temperatures were used, 66°C during the first 50 min and thereafter
POLYAMINES
ANALYSIS
IN CRUDE BIOLOGICAL TABLE
COMPOSITION
AND
CONDITIONS
SAMPLES
267
I OF
ELUT~NG BUFFERS Buffers
Sodium citrate dihydrate” Sodium chIoride’z Alcohol added Final pH Time (min)
First
Second
Third
0.20 0.30 Ethanol (5.5%) 5.55 It 0.01 37
0.20 2.30 Ethanol (6%) 5.63 t 0.01 40
0.20 2.55 Isopropanol ( 10.5%) 5.73 rt 0.01 93
CLMolarity in Na+.
78°C till the end of the chromatogram. The column was regenerated with 0.2 M NaOH containing 250 mg of EDTAiliter for 10 min and equilibrated by passing the first buffer for 30 min. RESULTS
Figure 1 shows the chromatographic resolution of 22 compounds, consisting of the common basic amino acids, mono-, di-, and polyamines, phenolicamines, and indoleamines. The components of the mixture, the amount of each applied to the column, the retention time, the relative fluorescence peak areas, and the constant values (KC) (benzylamine taken as internal standard) are shown in Table 2. The column, and the elution program employed gave good overall resolution. With the first buffer, the acidic and the neutral amino acids (aspartic acid through phenylalanine included) were eluted in a group. Hydroxytryptophan, tryptophan, histidine, lysine, ethanolamine, ammonia, and arginine were subsequently eluted, in that order, with good resolution. The second buffer eluted and separated well the mono, di-, and polyamines and phenolicamines but not the indoleamines. The elution of indoleamines was achieved by the use of a third buffer of higher ionic strength, containing 10.5% isopropanol, and by raising the column temperature from 66 to 78°C. Complete analysis was effected in 170 min. The presence of alcohol in the buffers is nesessary to obtain a good resolution and sharp peaks. The influence and optimal concentration of four different alcohols (methanol, ethanol, propanol, and isopropanol) in the buffers was studied. Those which gave better resolution, sharpness of peaks, and eluting force were finally adopted. The optimal concentrations for the alcohols were as indicated in Table 1. Isopropanol was chosen for the third buffer as it accelerated well the elution of the indoleamines and no baseline shift was observed. The eluting force of the different alcohols for the indoleamines was in the following order: methanol < ethanol < propanol < isopro-
258
5.MT
OH-Trp Trp His bs EtA NH, A% NA DAP PU HA Cd I.S. DA Sd HDA Agm TA PA 5.6.DHT SED 5-m
Abbreviation
AND A~WRACY
k i2 +
i 0.2 2 0.2 z 0.3 -+ 0.3 1 0.2 IO.2 ;t 0.2 t 0.1 2 0.3 * 0.3 i 0.3 -r 0.2
50.2 51.9 55.0 58.2
63.6 67.2 70.6 73.3 76.0 79.6 84.7 89.8 95.8 101.5 156.1 164.5
0.1 0.2 0.3 0.2
f + If+ I i t It
9.6 12.4 14.7 l&b 23.1 25.9 32.7 45.9
0.1 0.1 0.2 O.? 0.1 0.2 0.2 0.1
Retention time” OtlilI)
-
Ur-.
1148.69 757.69 597.73 1042.05 775.46 1011.18 984.06 1302.09 260.05 1177.69
27,919 25.677
2.60 1.98 I.74 3.25
1743.61 438.71 1196.40 663.21 1788.51 722.14 451.33 745.85
55,632 50.897 18,789 68,405 54.683 58,278 43,723 42,923 44,226 38.364 34,325 32,099 49.%7 29,705 54,743 28.434 4.58 0.93 4.59
1.80
1.55 3,40
3.13 0.86 6.36 0.97 3.27 1.24 1.03 1.74
2166.42 2202.90 1609.8S 2552.09 1574.30
5.57 5‘49 2.81 4.?0 2.70
RSD cw
AND PHENOLK
48.349 33,113
77.871 40.022
1535.01 483.61
1152.43 1267.49 831.9% 939.16
929.80
1844.64
605.21 1712.74 1613.59 781.67 900.67 1132.79 1113.14
1133.72
1590.20 1018.67
i382.49
1676.26
988.12 1356.35 1273.09
SD
2.06 2.11 2.58 2.48 1.42 1.70 i.07 2.35 3.17 I.46
2.21 1.30 1.20
2.23 1.93 4.62 0.80 2.10
2.29 2.77 2.10 3.01 2.18
RSD wd
80.5 72.7 48. I 85.7 61.6 43.2 70.3 71.1 57.8 77.5
72.5 57.3
77.9 96.5 76.6 90.6 67.2 79.7
90.2 81.8 94.6 97.3 91.9
AND INDOLEAMINES
392 429 1163 319 399 375 1.9. 509 494 569 636 681 437 7359 399 768 783 851
401 374
561 444 381
16.99 10.47
12.05 II.98
9.00 1.24 5.69 7.82 1.63 5.20 60.34
7.02 3.22 11.51 6.86 0.68 7.01
9.65 7.45 4.95 t.40 6.67
1.77 0.25 I.04 1.23 0.24 1.19 0.82 3.02 1.56 2.17 1.23
1.79 0.75 0.99 2.15 0.17 1.87
1.72 1.37 1.30 0.35 I.78
standard (IOQO pmol) aad S&DHT (5OlB pmoll. RSD (Yei;)= SDimean x 100.
68.761
54.939 52.756 71.362 37.455 8L.115
60.300 74,947
71.365 52,743 24.529 75,470 81,403 73,133
55,blb 63,483
60,585
43,128 49.041
Mean
Surface area before the column chromatography’
AND POLYAMINES
38.902 40,116 57,314 S4,SLd 58,331
SD
Surface area after the column chromatography”
OF MONO-,
Mean
OF THE ANALYSIS
2
n Average of IO determinatmns of thr mix!ure containing SM) pmol of rach of the products except for the inter&
5-Hydroxytryptophan Tryptophan Histidine Lysine EIh~ttolamine Ammonia Arginine Noradrenaline 1.3.Diaminopropane Futrescine H&%?li%?e Cadawine Benzylamine Dopamine Spermidine Hexa~ethylened~mi~~ Agmatine Tyramirre ~e~thy~mine S.d-Dihydroxytryptamine Spermine .%Hydroxytryptamine Tryptamine S-Methoxytryptamine
Compound
PRECISION
TABLE
270
VILLANUEVAANDADLAKHA
panol. The recovery of the different compounds after column chromatography was studied, and the results are presented in Table 2. Comparison was made between the surface areas of the peaks obtained after the chromatography and those obtained by the direct reaction of the different products with the reagent without passing through the column (this was done by connecting the inlet and outlet of the column together). The lowest recoveries after column chromatography were observed in cases of 5,6-dihydroxytryptamine (43%), agmatine (48%), dopamine (57%), and tryptamine (58%). For all other compounds recoveries varied between 60 and 97%. It must be pointed out that hydroxytryptamine derivatives
I
Pu *c
I
‘I
E.COLl
sd
0
20
40
60 TIME
( min
)
FIG. 2. Chromatogram of a 50-~1 sample of a TCA extract of E. co/i. Besides putrescine and spermidine two peaks with the same retention time as that of histamine and cadaverine which remained after hydrolysis of the sample were also observed. See Table 2 for the identities of the products.
POLYAMINESANALYSISINCRUDE
~~OLOGICALSAMPL~S
L-
& Y)
R I% I 33N33S3101Tlj
3hUVl3M
0
271
272
VILLANUEVA
AND ADLAKHA.
undergo gradual decomposition even at -20°C. Thus when the center of interest is the detection of these compounds, the samples must be analyzed immediately after extraction. Attempts are being made to overcome this difficulty. Quantification was done with the aid of an integrator coupled to the fluorimeter and by employing an internal standard. The integrator was calibrated before with a standard mixture, as indicated in Table 2. Figures 2, 3, 4, and 5 show the chromatograms obtained from crude samples of E. Cali B, tomatoes, platelets, and human urine, respectively. Values obtained for the different polyamines and other amino compounds in the samples analyzed are in agreement with those reported in the literature. E. Cofi B. Putrescine and spermidine were the major compounds present, as reported earlier (5). A peak with the same retention time as that of cadaverine was observed. This peak remained even after hydrolysis of the sample. An increase in the spermidine content was also observed after hydrolysis. Tomatoes. The analysis of the tomato sample was a good test for our
PLATELETS
FIG. 4. Chromatogram of a IOO-~1 sample of a perchloric Table 2 for the identities of the products.
acid extract of ptatelets. See
POLYAMINES
ANALYSIS
IN CRUDE
~ ----
-.-._
-._A
BIOLOGICAL
SAMPLES
273
274
VILLANUEVA
AND
ADLAKHA
method as it contained most of the mono-, di-, and polyamines as well as serotonine and tryptamine. The amounts of these last two indoleamines were of the same order as already reported (16). Platelets. Polyamine and indoleamine content found was in accordance with previous results (17-20). In addition to putrescine, histamine, spermidine, spermine, and serotonine we observed a peak with the same retention time as that of cadaverine which remained even after hydrolysis. Urine. Analysis of free polyamines and total polyamines in human urine was also in good agreement with published results (5,9,13). Phenolic and indoleamines were also present (21-23). DISCUSSION
In the present work we describe a method which separates a wide range of biologically impo~ant amines with an automatic amino acid analyzer without any modification of the analyzer. With this method we were able to analyze in a relatively short time the common basic amino acids and mono-, di-, poly-, phenolic-, and indoleamines in the picomole range in a single crude sample. A three-step change of buffers and two temperatures were employed. Incorporation of alcohols into the buffers dramatically improved the separation and sharpness of peaks. Strongly bound compounds such as tryptamine and 5-methoxytryptamine could also be eluted by using isopropanol without much increase in the ionic strength. The main goal of this work was to develop a method for the direct analysis of biological materials. This was achieved by use of the appropriate ionic strength and concentration of alcohols in each of the three eluting buffers. The first buffer eluted amino acids only and the second and third buffer the amines. The prior purification of samples, which is often time consuming, was not necessary and thereby the risk of losing amino compounds, which could be present in trace amounts was avoided. The retention times of different amino compounds were not altered by our procedure. This was verified by cochromatographing the crude samples with standard mixtures of amino compounds. Compared with the previously described methods, ours has the principal advantage of separating a large number of related amino compounds in a single chromatographic analysis in a relatively short time with high sensitivity (detection limit, picomole level). The use of an integrator and of an internal standard in the samples contributes to an easier and reproducible quantification. The reproducibility of the method is similar to that of automatic analysis of amino acids and is now a routine procedure in our laboratory. REFERENCES 1. Tabor, 2. Cohen,
C., and Tabor. H. (1976) Annu. Rev. Biochem. S. S. (1978) Adv. Polyamine Res. 1, l-10.
45, 285-306.
POLYAMINES
ANALYSIS
IN CRUDE BIOLOGICAL
SAMPLES
275
3. Durie, B. G. M., Salmon, S. S., and Russell, D. H. (1977) Cancer Res. 37, 214-221. 4. Smith, T. A. (1977) in Progress in Phytochemistry (Reinhold, L., Harborne, J. B.. and Swain, T.. eds.), Vol. 4, pp. 27-81, Pergamon Press, New York. 5. Tabor, H., Tabor, C. W., and Irreverre. F. (1973) Anal. Biochem. 55, 457-467. 6. Veening, H., Pitt, W. W., Jr., and Jones, G., Jr., (1974) J. Chromatogr, 90, 129- 139. 7. Marton, L. J., and Lee, P. L. Y. (1975) Clin. Chem. 21, 1721- 1724. 8. Gehrke. C. W., Kuo. K. C.. Zumwalt, R. W.. and Waalkes, T. P. (1974)5. Chromotogr. 89, 231-238. 9. Adler, H.. Margoshes. M.. Snyder, L. R.. and Spitzer, C. (1977) J. Chromafogr. 143, 125- 136. 10. Marton, L. J., Heby, O., Levin, V. A.. Lubich, W. P., Crafts. D. C., and Wilson, C. B. ( 1976) Cuncer Res. 36, 973-977. 11. Villanueva, V. R.. Adlakha, R. C., and Cantera-Soler, A. M. (1977) .I. Chromatogr. 139, 381-385. 12. Villanueva, V. R., Adlakha, R. C.. and Cantera-Soler. A. M. (1978) Phytochemistrv, 17, 1245- 1249. 13. Gehrke, C. H., Kuo, K. C., and Ellis, R. L. (1977) J. Chromatogr. 143, 345-361. 14. Vogel, H. J., and Bonner, D. M. (1956) J. Biol. Chem. 218, 97- 106. 15. Benson, J. R., and Hare, P. E. (1975) Proc. Nat. Acad. Sci. USA 72, 619-622. 16. Undenfriend, S., Lovenberg, W., and Sjoerdsma, A. (1959) Arch. Biochem. Biophys. 85, 487-490. 17. Udenfriend, S., Weissbach, H., and Clark, C. T. (1955) J. Biol. Chem. 215, 337-344. 18. Porter. J. F. (1972) Physiol. Rev. 52, 361-381. 19. Tidball, M. E. (1971) Ann. J. Physiol. 221, 1064-1070. 20. Cooper. K. D., Shukla, J. B., and Rennet?, 0. M. (1976) Clin. Chim. Acta 73, 71-88. 21. Kakimoto, Y., and Armstrong, M. D. (1962) J. Biol. Chem. 237, 208-214. 22. Perry. T. L. (1962) Sciences 136, 879-880. 23. Altman, P. L., and Dittmer. D. S. (1974) in Biology Data Book, 2nd ed., Vol. 3, pp. 1496-1507, Federation of American Societies for Experimental Biology, Bethesda, Md.