Pergamon Press

Life Sciences, Vol . 25, pp . 157-164 Printed in the U.S .A .

HARMAN IN HUMAN PLATELETS T .G . Bidder, D .W . Shoemaker, A .G . Boettger, M. Evens and J .T . Cummins Department of Psychiatry, University of California, Los Angeles, and VA Medical Center, 16111 Plummer Street, Sepulveda, CA 91343 and Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA 91103 (Received in final form May 21, 1979) SUMMARY The ß-carbolines present is human platelets have been extracted with diethyl ether, isolated by liquid column chromatography and thin-layer chromatography (TLC), and identified by ultraviolet fluorimetry, gas liquid chromatography (GC) and mass spectrometry (MS) . Harman (1-methylß-carboline) was the only ß-carboline unequivocally identified in platelet samples with these techniques . Since barman is thought to be biosynthesized by the condensation of tryptamine and acetaldehyde, its formation may be of importance in the metabolism and the pharmacological-toxicological actions of alcohol. Recent attempts to understand the pharmacological and toxicological actions of ethyl alcohol have increasingly focused on the properties of its mayor metabolite, acetaldehyde (1,2) . Oae of acetaldehyde's actions is of particular interest since it cosetitutes a possible mechanism whereby catecholaminergic and/or serotonergic processes could be modified as a result of the ingestion of alcoholic beverages . This action involves the condensation of acetaldehyde with catecholamines to form isoquinolines and with indole-ethylamines to form ß-carboliaes (3,4) . Since these condensation reactions occur in vitro under conditions of pH and temperature which prevail in living tissues(5), studies have been conducted to determine whether, in fact, isoquinolines and ß-carbolines are pres ent is animal (6,7) and human (8,9,10) tissues. We have an interest in possible individual differences in the metabolism of ethanol as constituting one of the factors determining single or multiple abnormal human response to alcohol subsumed under the clinical label of the alcoholisma (11) . Consequently, we have been examining one aspect of the metabolism of ethanol - the formation of ß-carbolinee - in an accessible human tissue, the platelet . The present communication records our findings that barman (1-methgl-ß-carboline) exista is the human platelet . Methode and Materials Human platelet concentrates were prepared from the blood of anonymous Red Cross donors by differential centrifugation (10) and were used within 72 hours after venesection . Tha platelet-rich plasma from 5 donors was freed of erythro cgtea and leukocytes by repeated centrifugation for one-minute periods at 1,500 x g at room temperature. Platelets were isolated by centrifugation of the 0024-3205/79/020157-0802 .00/0 Copyright (c) 1979 Pergamon Press Ltd

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platelet-rich plasma at 1,750 x g for 20 minutes . The platelet pellet was gently dispersed in washing medium (150 mM NaCl, 1 mM EDTA, 10 mM Trie :HCl buffer, pH - 7 .5) and recentrifuged at 1,750 x g for 20 minutes . After three such washings, the platelet suspension was determined to be free of contaminating leukocytes sad erythrocytes by phase-contrast microscopy . ß-carboliaea were extracted from the platelet pellet by homogenization in saturated borate buffer, pH 9 .5, followed by 10-minute periods of vortexing with two volumes of peroxide-free diethyl ether . Three such diethyl ether extractions were pooled, their volume reduced to 1-2 ml, and the concentrated extract was subjected to liquid column chromatography on silica gel . After waehiag the column with diethyl ether (5 ml) and n-heptane (5 ml), elution was carried out with 4 .35 N acetic acid . The ß-carboline content and concentration of the column eluates were ascertained by fluorimetry employing an Aminco-Bowman Fluorimeter . The ß-carboline-containing eluate fractions were lyophilized and the residue was taken up in a small volume of methanol for preparative TLC on silica gel glees plates with n-butanol(4) :glacial acetic acid(1) :water (1) and/or chloroform (9) : methanol(1) . The platelet and reference ß-carboline spots were visualized by fluorescence with ultraviolet light (ChromataVu, Ultraviolet Products, Inc .) aad identified by their characteristic hues and ~ values . The ß-carboline spots were scraped off the plates aad extracted with methanol . The methanolic extracts were taken down to dryness with nitrogen and dissolved in a known (5-10 ul) volume of 0 .1 methanolic formic acid for GC and GC-MS . These analytical procedures were carried out without derivatization . GC analyses were performed with a Varian Model 2700 gas chromatograph Coiled 6-foot glass columns, 2 .0 equipped with a flame ionization detector . mm i .d ., were packed with 3X OV-17 on Gaschrom Q (100/120 mesh) and operated isothermally at 246°C. Dry helium, passed through a gas purifier, was used as the carrier gas at an initial pressure of 60 psi and a final flow rate of 37 ml/minute . The inlet temperature was 275°C and the detector temperature wse 300°C . Combined GC-MS was performed with a Victoreen Model 4000 gas chromatograph coupled to a Hitachi-Perkin-Eimer RMS-4 single focusing mass spectrometer operated with an ionization energy of 70 electron-volts . The Victoreen gas chromatograph employed as identical column and was operated in the manner described above for the Varian instrument . The glass transfer line coupling the gas chromatograph to the mass spectrometer was heated to 300°C . Perfluorokerosene was used as a reference standard for calibration of the mass spectrometer . Harman aad other reference ß-carbolines were purchased from Aldrich Chemical Company, Milwaukee, Wisconsin. The barman gave a single spot on the TLC (a-butanol :acetic acid water) and the single peak which it produced oa GC gave a molecular ion value of 182 (formula weight of barman e 182) and an ion fragment at mesa 154 (loss of NCH2 from the pyridine ring) when it was introduced into the mesa spectrometer . Inorganic and organic chemicals were purchased from commercial sources in Silica gel for column chromatography (0 .2-0 .5 mm maximal obtainable purity . Q6 Quanta/Gram Silica gel particle size) was obtained from Merck-Darmstadt . glass chromatographic plates (0 .25 mm thick) were purchased from Quantum Industries, New Jersey . OV-17 (3X) on Gaschrom Q (100-120 mesh) and other gas chromatographic materials were obtained from Applied Science Laboratories,

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State College, Pennsylvania . Diethyl ether was freed of peroxides with acidic ferrous sulfate and subsequent distillation . N-heptane was re~istilled before use. Results When the concentrated diethyl ether extract of borate-buffered (pH 9 .5) platelet homogenate was absorbed on a silica gel column, the constituent ßcarboliaes, se measured by ultraviolet fluorimetry, were eluted in the first 2 .0 ml of 4 .35 N acetic acid emerging from the column . The platelet fluorochrome in the slants shows the characteristic barman emission peak at 430 nm with an incident or excitation wavelength of 300 nm (Table 1) . Emission peaks characteristic of other ß-carbolines were not evident when the incident light wave-length was varied between 300-370 nm . TABLE I Characteristics of Platelet Fluorochrome

Compound

Primary Fluorescent PeaksB

TLC Rf Valuesb

Excitation

BAW

Emission

CM

GC Retention Timeç (minutes)

Platelet fluorochrome

300 nm

430 nm

0 .59

0 .51

2 .50

1-methyl-ßcarboline (Harman)

300 nm

430 nm

0 .59

0 .51

2 .50

_a - Ultraviolet fluorimetric emission spectrum for the platelet sample was run on the first (2 .0 ml) fraction with 4 .35 N acetic acid elution from the silica gel column . The reference ß-carboline's spectrum was run on a solution of the compound in 4.35 N acetic acid . _b - The TLC Rf values were determined for n-butanol (4) :glacial acetic acid (1) :water (1) ~ BAW or for chloroform (9) :methanol (1) = CM . c - Retention times were measured from the time of injection .

Lyophilization of ß-carboline-containing coli~n eluatea was followed by resolubilization in methanol and thin-layer chromatography on silica gel glass plates together with reference ß-carboliaea . Long wave-length (366 nm) ultraviolet visualization of the chromatogram after development revealed the platelet fluorochrome to exist as a single spot which had a hue and Rf value identical to that of barman in two solvent systems (Table 1) . When the platelet fluorochrome spot and the reference ß-carboline spots were scraped off the TLC plates, extracted with methanol and subjected to GC ., the platelet sample showed a prominent peak with a retention time ident ical to that of the barman TLC sample and of a standard solution of barman (Table 1, Figure 1) . This peak was absent from a methanolic extract of a control area of the TLC plate . The ß-carboline GC effluent peak for the platelet sample and for the barman standard were sampled in the coupled mass spectrometer (Figure 2) . Mass spectrum relative inteasity~/e bar graphs derived from these records are shown is Figure 3 . The mesa spectrum bar graph for a reference sample

Vol . 25, No . 2, 1979

Harman in üuman Platelets

Platd~t TLC

FIG . 1 GC patterns of TLC spots . Left to right : fluorescent spot from platelet sample with same Rf as reference barman spot ; TLC barman spot ; barman standard (not from TLC) ; extract of control area of TLC plate . PLATELET ~ee-~ N

M`

na

C !0-

0

_ay LMm

0

~-/ f

~oo

ma

aso

M/E

M PLATELET

182 1

HARMAN

M`

n~

~oo-

c

a

,o-

rM_ 9 ~I+

s

n~

~-,' F

FIG . 2 Mass spectrum (GC-MS) of barman standard (upper record) and platelet sample . M/e of 182 indicated by arrows .

l oio

nôo

aé0

M~E

FIG . 3

Mass spectrum relative intensityM/e bar graphs for GC-MS of platelet sample and barman standard .

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Harman in Human Platelets

HARMAN PROBE

16 1

+ M iss

~oo-

so-

~M-ss~ ~a~

0 ô oc

MIE FIG . 4 Mana spectrum relative intensity-M/e bar graph for barman standard introduced directly into mass spectrometer via probe . of barman introduced directly into the mass spectrometer via a probe is shown in Figure 4 . These mass spectra show the platelet sample and the barman samples to have identical peaks at 182 (the molecular ion for barman) and at 154 . The latter fragment arises from the loss of H2CN (mass ~ 28) from the ß-carboline nucleus (Figure 4) . Discussion Two platelet samples, each comprised of 5 Red Cross donors, show the same characteristics as an authentic sample of 1-methyl-ß-carboline (barman) . The platelet and barman samples have the same fluorescent spectra and the single spots which each sample shows on thin-layer chromatography have identical hues and Rf values . The platelet sample has a prominent GC peak with the same retention time ae the peak obtained with the barman sample . Finally, the mass spectra of the two samples show identical peaks at 182, the molecular ion for barman, and at 154, an ion fragment arising from the loss of H2CN (mass ~ 28) from the 2-3 position of the barman molecule's pyridine ring . (Fig . 4) . These findings establish the presence of lrmethyl-ß-carboline (barman) as a constituent of the human platelet . In view of the ease with which oxidation of dihydro- and tetrahydro-ß-carbolines occurs upon exposure to light and atmospheric oxygen (9), it is possible that unsaturated compounds such ae 3,4-dihydroharman and 1,2,3,4- tatrahydroharman have been converted to barman during the extractive, ieolative and analytical procedures employed in these studies .

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Speculations about the mechanisms whereby ß-carbolinea are biosynthesized invoke a Mahnich-type (Pictet-Spengler) condensation of an indole-ethylamine and an aldehyde as the terminal step (13,14) (Fig . 5) : R~

R2CH0

Ri=OH, OCHS,

Ri

R2=

H

H,

CH 3

FIG . 5 Mahnich-type (Pictet-Spengler) condensation reaction The fact that condensations of this type occur under conditions of pH and temperature similar to those existing in living tissues has lent support to this mechanism. Because of the availability in human body tissues of a variety of endogeaoua indole-ethylamines (e .g ., tryptamine, 5-hydroxytryptamiae) and aldehydes (e .g ., formaldehyde, acetaldehyde), it is probable that a variety of ß carbolinea exist in platelets and other structures . ß-carbolinea have been isolated from the cataractous lens of older humane (9) . In a recent abstract report, tetrahydronorharmane and 6-hydroxyharmane are stated to be present in the platelets and urine of human subjects (10) . The presence of barman and other ß-carbolinea in the human platelet raises questions about their origin(s) . It is possible that these compounds are biosynthesized in the platelet from indole-ethylamihe and aldehyde pre curaora which are present in this organelle (15) . In view of the capacity of platelets to take up ß-carbolinea (16), it is also possible that ß-carbolinea synthesized by other tissues or absorbed from external sources e.g ., from inhalation of cigarette smoke (17) will be taken up by platelets . The fact that barman is a major ß-carboline constituent of the human platelet ie of spatial interest since the aldehydic component of this parThe small concentrations of acetaldehyde ticular compound is acetaldehyde . derived from normal metabolism and the larger amounts generated from alcohol beverage ingestion are probably available for ß-carboline biosynthesis . Their It ie likely that ß-carbolinea have biological significance . synthesis from pharmacologically-active indole-ethylamines and aldehydes can be conceived of ae a mechanism for modifying the actions of these constituent molecules . And the actions of ß-carbolinea reported to occur in animals and These include increases in man lend support to their biological importance . whole-brain concentrations of 5-hydroxytryptamine (18), inhibition of membrane tranalocatidn processes (19,20,21,22), inhibition of monoamine oxidases (23) and cardiovascular actions (24) . The central nervous system effects of the ßcarbolinea include tremorogenic and convulsive actions as well as changes in body temperature (25) . In the human, naturallyroccurring ß-carbolinea (e .g ., the harmala alkaloids) have long been known £or their potent psychotomimetic effects (26) . Finally, as intriguing effect of extremely small doses of a ß-carboline has been reported in rate : intraventricular injection evokes a marked and prolonged increase in voluntary ethanol ingestion (27) .

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Harman in Human Platelets

16 3

In view of these actions and their possible relationship to the pharmacological and toxicological actions of ethanol, the rate of formation of ßStudies of ß carboliaes and their tissue concentrations are of interest . carboline concentrations in platelets from alcoholic and non-alcoholic aubjecta are currently being pursuAd in this laboratory . Acknowledgments The authors wish to express their appreciation to Dr . Carroll Spurliag, Ms . Marcia Breakin and Ms . S.J . Ruoff of the Los Angeles Chapter, American National Red Cross, for supplying us with human platelet samples . The skilled technical assistance of M.J . Holleabeck, R.N ., sad P . Shamblin is gratefully acknowledged . These studies were supported by funds from the Kimberly-Clark Corporation and from the Veterans Administration sad by Grant RR00922, NIH Research Resources Branch and Contract NAS7-100, Technical Utilization Office, NASA . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 .

R.G . RAHWAN, Life Sciences 15 617-633 (1975) . R.G . RAHWAN, Toxicol . Appl .Pharmacol. 34 3-27 (1975) . M. SHAMMA, _The Isoquinoline Alkaloids, Âcademic Press, New York (1972) . R.H .F . MANSRE, _The Alkaloide : Chemistry _and Physiology (Volume VIII : The Carboline Alkaloids), Eds . R.H .F . MANSRE and H.L . HOLMES, Chapter 3, pp 47-52, Academic Press, New York (1965) . W.M . McISAAC, Biothun. Biophys . Acta _52 607-609 (1961) . H. ROMMELSPACHER and B . GREINER, Naunyn-Schmiedebergs Arch . Pharm. _302 R60 (1978) . G. COHEN, Adv . in Exptl . Med . sad Biol . _35 33-44 (1973) . M. SANDLER, S .B . CARTER, R.R . HUNTER and G.M . STERN, Nature _241 439-443 (1973) . J. DILLON, A. SPECTOR and R . NARANISHI, Nature _259 422-423 (1976) . H. HONECKER and H. COPER, Nauayn-Schmiedebergs Arch . Pharmacol . _302 R63 (1978) . G. EDWARDS, M.M . GROSS, M. KELLER, J . MOSER and R . ROOM (Eds), AlcoholRelated Disabilities (World Health Organization Offset Publication No . 32) pp 45-46, World Health Organization Geneva (1977) . AMERICAN ASSOCIATION OF BLOOD BANKS Technical Manual , Chapter 3 : Components, pp 43-44 (Platelet Concentrates) American Association of Blood Banks Washington, D.C . P. LADURON sad J . LEYSEN, Biochem. Pharmacol. 24 929-932 (1975) . H. ROSENGARTEN, G . MELLER sad A. FRIEDHOFF, Biochem . Pharmacol . _24 1759-1762 (1975) . S .M . STAHL, Arch . Gen . Paychiat . _34 509-516 (1977) . M.M . AIRARSINEN, T-T HUANG, B .T . HO sad D. TAYLOR, Acta Pharmacol . et Toxicol . 41 (Suppl . 4) 39 (1977) . I . SCHMELTZ and D . HOFFMAN, Chem . Revs . _77 295-311 (1977) . L.J . POIRIER, P. SINGH and R. BOUCHER, Caned . J. Physiol . Pharmacol . _46 585-589 (1968) . L. TUOMISTO sad J . TUOMISTO, Naunyn-Schmiedebergs Arch . Pharmacol . _279 371-380 (1973) . H.O . GREEN and T.A . SLOTRIN, Mol . Pharmacol . _9 748-755 (1973) . F.V . SEPULVEDA and J .W .L . ROBINSON, Naunyn-Schmiedebergs Arch . Pharm . _291 201-212 (1975) . M. CANESSA, E . JAIMOVICH and M. de la FUENTE, J. Membr . Biol . _13 263-282 (1973) . N.S . BUCRHOLTZ and W.O . BOGGAN, Biochem. Pharmacol . 26 1991-1996 (1977) .

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T .A . SLOTKIN and V . DiSTEFANO, Proc . Soc . Exptl . Biol . Med . _133 663-664 (1970) . B .T . H0, Current Developments in Psychopharmacology 4 153-177 (1977) . C . NARANJO, Ethnopharmacological Search _for Psychoactive Drugs , Eds . D.H . EFRON, B . HOLMSTEDT and N.S . KLINE, pp . 385-392 U .S . Public Health Washington, D .C . (1967) . Service, R.D . MYERS and M.M . OBLINGER, Drug and Alcohol Dependence _2 469-483 (1977) .

Harman in human platelets.

Pergamon Press Life Sciences, Vol . 25, pp . 157-164 Printed in the U.S .A . HARMAN IN HUMAN PLATELETS T .G . Bidder, D .W . Shoemaker, A .G . Boett...
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