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A study of glyceryl ethers—I. Content of α-glyceryl ethers in marine invertebrates from the sea of Japan and tropical regions of the Pacific Ocean
A STUDY OF GLYCERYL ETHERS-1. CONTENT OF a-GLYCERYL ETHERS IN MARINE INVERTEBRATES FROM THE SEA OF JAPAN AND TROPICAL REGIONS OF THE PACIFIC OCEAN S.
V.
ISAY.’
M.
A. MAI(AR~HLNI
.\>I)
V. E. VASICOVSK’~.’
’ Pacific
lnstitute of Bio-Organic Chcmistry. Far F:ast Sc~encc Ccntrc. USSR Academy of Sciences. Vladl\ostok-27. 1 .S.S.R. ‘Institute of Marine Biology. Far East Sclencc Cents-c. l!.S.S.R. Academg of Sciences. Vladi\ostok-22. I .S.S.R
Abstract 1. A method has been developed for dctermln,rtion 01’ ~-~l>ccrqI ether contcnts In total lipid cxtracts. 2. The method was applied to analyse about 100 \pccica of marine inicrtebratea from thc Sea of Japan and tropical regions of the Pacific. 3. The preliminary data obtained do not allow any dcfinitc conclus~on\ to bc made regarding a relationship between the r-glycerll ether content\ in mar~nc in\crtebratcs and thelr phylogenetic posilion or ecologl
INTRODI
CTION
method of Pande c’t (/1. (1963). The extracts were kcpt in a rel’rlgerator at 1 <‘ and controlled hy thln-layer chromatographq on Glica gel KCK in chloroform methanol
Glyceryl ethers are widely distributed in nature. and their biological role is highly significant (Snyder. 1969a~-c; Lazurievsky. 1966). It is common knowledge that glyceryl ether: are contained in marine organisms in particularly large amounts (Karnovsky & Rapson, 1946: Karnovsky & Brumm. 1955: Kayama PI LI/.. 1971 : Seino et u/., 1971 : Malins & Wekell. 1970: Malins & Varanasi. 1971). However, available evidente is rather fragmentary. Karno\skq & Rapson (1946). who screened glyceryl ether contents in marine animals, devoted much of their attention to fìshcs. and les:, to marine invertebrates. The present paper IS concerned with the distribution of glyceryl ethers in marine invertebrates of different t&&o~y and ecology Suitable techniques of the analysis developed in our laboratories have been used. \lATk:RIAI.S
“Absolute” poration for
AUD
wlth
A 0.02 M 1.5 moles of
contnmmg
perlodate
solution
(0.5 ml).
ZO”,,
hodium hlcarbonate per 1mole of periodic acad. was added in ;I 10 ml graduated tube to a 44”,, aqueous iroprnpanol solution (1 mi) of glyceryl ether. required for oxidation of not morc than 10 /LM periodate. Aftcr Incubatlng the miwture at room temprrature for (0.5 ml) ~vas added. 1 1.5 hl-. ;t 0.05 M rhamnoae solutlon and tilc mlnutea Iatcr a Nash reagent (1 ml) was added. The mlxture was hcuted at 50 C for 15 min. In case’r when thc solutions wcrc turbid. isopropyl alcohol (1 ml) was added. After coolins with running water. distllled water was added till the graduation mark. Following thorough agitation. thc \olution tias photometered in cuvctlcs ( I cm) at 412 nm. To IIICI-cax thc scnsltikitj of the reaction. thc final \olumc can hc decrca$cd to 5 ml. lcaving the amounts of the t-cagcnts thc
ME:THODS
diethyl ethf:r. chimyl alcohol Biochemical Investigations.
water (65:25:4. v \). Spots were detected ammonium tx\ulphate with heating at 1X0 C
4 \\cighcd portkon dissolced in ahsolutc
(California CorU.S.A.), and an
ethereal solution of lithium aluminium hydride, prepared from a commercial preparation in accord with Dillard (1952). were used as reagents. The hydride concentration
of the Iipid extract (20 <5 m_g) was ether (5 IOml). Thcn a lithium alu-
minlum hydride solutmn (double the weighed portion of the lipld extract) wa\ curefully added batchwise. 4fter adding the la\r hydride portion. the reaction mlxturr was hept fnr 30 min at room temperature and thcn r&xcd for 30 mm. Subsequent to cooling the reaction mixture. the cxccs~ of lithium aluminium hydride wah destroycd wlth distilleti mater (tirst b‘ drops). and the resultant CeI uaa thssolvcd \\lth hiu hydrochloric acid. The ether laycr was beparatcd. and the aqucous phase extracted four times Mlth ether. The cxtracts werf combined and c\aporated to minimum volume: the solution was then spread onto a platc 19 r 12 cm) wlth \IIIC:I gel KCK. the plate having heen prewa\hed \vith ether. The system uscd wab ether-conccntratcd ammonium hydroxide (100:1). The Itpid /ones were \ isualued bl
in the solutlon was dctermined by Kreshkov’s method 11961). All other reagents uscd in the work were prepared in accord with our previolls technique (Vaskovsky & lsaq.
Iwí9). The animals used in tne work were collccted in Posiet Bay, Sea of Japan. in July October 1972. and in thc tropical regions of the Pacific in December 1970 and Januarq 1971. Frcshly captured invertebrates were used to preparc total lipid extracts in accord with Folch PI trl. (1957). The amount of lipids in cach extract was dctcrmmed by the 301
As usual. the z-glyceryl ether determination consisted of the following stages: decomposition of lipide: isolation of glyceryl ether fraction: oxidation of fraction with periodate or lead tctracetatc: and yuantification of resultant formaldehyde. In our laboratory. a sensitivc and suitahlc method for determining the formaldehyde that farms durmg oxidation of substances with periodate had previously been developed (Vaskovsky & Isay. 1969). On the basis of this method, we developed a proccdurc for quantitating glyccryl ethers. In recent ycars. reduction wsith lithium aluminium hydride was more frequently used for splitting lipids
A study of glyceryl
tions. After trying several solvents and their mixtures, we chose 440,, aqueous isopropanol, since it gives a transparent reaction solution. which, unlike other solvents. remains transparent when diluted with water. Glyceryl ether fractions are generally eluted from the silica gel with diethyl ether (Wood & Snyder, 1968) or 15”;) diethyl ether in hexane (Snyder, 1969a). Inasmuch as we conducted further analysis in 44% aqueous isopropanol, it appeared more suitable to immediately obtain isoinropanol extracts. The advan-
ethers -~ J
303
tage of aqueous isopropanol was in that under similar experimental conditions it fully washed out the substance. whereas ethers washed out only 507”. Thus, we developed a suitable procedure that made possible a relatively easy analysis of a large number of marine invertebrates. We quantitated the a-glyceryl ether content in 63 species from the Sea of Japan and 39 species from the tropical regions of the Pacific. The results are cited in Tables 1 and 2. The tropical invertebrates are shown only as classes and phyla,
since not all of them were assigned species. There
is u correlation
between
the
to genera
and
taxonomie
pos-
and thcir lipid content and composition. Thus. it was shown that the qualitative and quantitative contents of glycolipids and their monosaccharides are in a certain way associated with the evolutionary leve1 of marine invertebrates (Vaskovsky er td.. 1970). However. the analytical data for a large number of marine invcrtebrates belonging to eight phyla and about 20 classes does not suggest any definitc relationship bctween the contents of r-glyceryl ethers and taxonomy of thc animals. The amount of x-glqceryl ethers found is almost the same for al1 of the classes and types. varying from I to S”,,. exccpt for Anthoroa and certain mollusc species. whose x-glyceryl ether content cxceeds X”,,. This holds truc for both Japan Sea and tropical Pacific invertebratcs. The class Holothurioidea is also distinguished by a high r.-glyceryl ether content. but this applies only to invcrtebrates from the Sea of Japan. Thcre are no other sharp diffcrcnces between the invertebrates from the two regions. The contents of rc-glyceryl ethers appears to bc more dependent on the individual propertics of organisms. Tables 1 and 2 show that the amount of r.-glyccryl ethers even in closely related species is highly \aricd. It is diffìcult to compare our data with thosc of other authors. since \‘crq fcw works are known on glyceryl ethers of marine invertebrates. and therc is virtually no cvidence for the species that we determined. Moreover. cken the available fìgurcs can hardly bc compared with the results of the present paper and with each othcr. since al1 authors ~1st:relatively different values to express thr amounts of r-glyceryl ethers. e.g. unsaponifiablc fractions. neutral Iipids and phospholipids (Taro rt trl.. 1941: Karnovsky & Rapson, 1946; Karnovsky & Brumm, 1955: Rapport & Alonzo, 1960; Seino. 1971). But our results confirm the main conclusion of these works that cc-glyceryl ether is essential for marine lipids. ition
of
marine
provided us with specimens of tropical orgamstns. and to 0. D. Korotchenko for her unstinted aid in preparing lipid extracts from Japan Sea mvertehrates.
invertebrates
Ac~nowIrdyr,,lorts E. Y. Kostetsk) IFar
Our East
heartfelt thanks are dut: to State
University),
who
Dr. kmdl\i
BLANK M. L.. (‘~1~s F. A.. S’rLI’Hl-NS N. & SN’~LXR I-. (1971 l On the analyses of long-chain alkane diols and glycerol ether\ 111hiochemical studies. .I. lip Rr,\. 12, 63X- 640. DII t.b.Rt> c‘. R. (195?l An apparatus for storing and measuring solutions of lithium aluminium hydride. J. C/ICVI. r:tll,<,. 29. I 2‘) 130. Fmc1f J.. LCIS M. & SLOAXI S. G. H. (1957) A simple method lor thc isolution and purifìcation of total lipids from animal tissues. .I. bio/. C/lu/n. 226, 497-509. KARNOVSK~ M. L.. RAPSON W. S. & BLACK M. (1946) South African fish products ~XXIV. The occurrence of a-glyceryl ethers in the unsaponifiahle fractions of natura1 rats. .1. SOC,.C/ICV~ /rr
MAL.ISS D. C. & WI iri~ F.). pp. 297 312. Academie Prees. New York. PANW s. v.. PAKAkI I(. R. & ~‘t‘IKI?ASI!BKAMANIAS T A. (1963) Microdctermlnation of Ilpids and serum total fatty acids. iln~rirr. Hio&w~. 6. 41 5 -413 RAPPORI M. M. & At.oNLo N. F. (1960) The structure of plasmalogens. V Lipids of marine tnvertebrates. ./. bioi. C‘hem. I35, 1953-6. SFINO H.. WAI‘ANABF S.. KAWADA K. & ABE, Y. (1971) Liver oil of marine Crustacea. 1. Stcrols and glycerol ethers in the liber oils of Chionocwl<~,\ ~~pilio and Lirkod~x !III‘virus. YukoqctkL~ 20, XO7~XOh: (‘. 1. 76, 441 3x7 (19711.
A study of glycerql ethers
SNYDER F. (1969a) Ethers-linked lipids in neoplasms of man and animals: methods of measurement and thc occurrence and nature of the alkyl and alk- I -en yl moieMutlicinv rrntl Riokq~. ties. In Adrancrs in E.rperimenfol Vol. 4. pp. 609 ~621. Plenum. New York. SNYDER F. (1969b) The biochemistry of hpids containing ether bonds. In Pwyess in the Chrmistr~ of Ftrts trrd (Edited by HOLMAN R. T.). Vol. 10. pp. Other Lqids 287-335, Pergamon Press, Oxford. SNYDER F. (1969~) The chemistry. phyaical properties. and chromatography of lipids containing ether bonds. In
Proyres,s in Th-Lajvr Chromtrtoy,trpl~~, trnd Rrltrrrtl Methods (Edited by PATAKI G. & NIEDERM I~SER A.). Ann Arbor, Ann Arbor. Michigan. .TAKO M.. MASAKADLI: Y. & YOSHIYC,KI T. (1943) The cryatalline fraction of thc unsaponifiable substances in the fat of 4 srt2ricl.rrollrstcwi and Clrclrmtrr-itr ~~hronhjdmi. J. ~~/w»I. SN. ./
I
W5
THOMPSOPVCr. A. & LI-!- P. (1965) Studies of the r-gllccryl ether liplds occurring in molluscan tissues. Biochlm. biophl,\. Actf~ 98, 151~ 159. VASKOVSKY V. E. & ISA~ S. V. (1969) Quantitativc determmation of formaldehyde liherated with periodate o\idatIon. -Irl[l/vr. Biochcw. 30, 3 3 1. VASKO\SK> V. E.. Ko~T~TsK~ F-. Y.. S\I 1 ~SHI \ \. 1.. Zlir ho\% 1. c;. & SM1KWV.A G. P. (19701 Glycohplds of marine in\ertebrateï. C~ì~np. B~~C/ICHI Pil I~\liJ/. 34. 163 177. Woon R. Hr SN~III K F. (1968) Quantitati\e dctcrmlnatton of alk-l-cnyl and alkvl glyceryl ethers 111ncutral Iipids and phoqholipids. Liptd.5 3, 1% 135. ZIUIUSKI T. & BOROWSKI E. (1966) New spray rcagcnt replac~ng sulfuric acid in thin-layer chïomatogaph> J. (‘/7r.l~~nllriU/.23. 3x0 4x7.
V.
ISAY.’
M.
A. MAI(AR~HLNI
.\>I)
V. E. VASICOVSK’~.’
’ Pacific
lnstitute of Bio-Organic Chcmistry. Far F:ast Sc~encc Ccntrc. USSR Academy of Sciences. Vladl\ostok-27. 1 .S.S.R. ‘Institute of Marine Biology. Far East Sclencc Cents-c. l!.S.S.R. Academg of Sciences. Vladi\ostok-22. I .S.S.R
Abstract 1. A method has been developed for dctermln,rtion 01’ ~-~l>ccrqI ether contcnts In total lipid cxtracts. 2. The method was applied to analyse about 100 \pccica of marine inicrtebratea from thc Sea of Japan and tropical regions of the Pacific. 3. The preliminary data obtained do not allow any dcfinitc conclus~on\ to bc made regarding a relationship between the r-glycerll ether content\ in mar~nc in\crtebratcs and thelr phylogenetic posilion or ecologl
INTRODI
CTION
method of Pande c’t (/1. (1963). The extracts were kcpt in a rel’rlgerator at 1 <‘ and controlled hy thln-layer chromatographq on Glica gel KCK in chloroform methanol
Glyceryl ethers are widely distributed in nature. and their biological role is highly significant (Snyder. 1969a~-c; Lazurievsky. 1966). It is common knowledge that glyceryl ether: are contained in marine organisms in particularly large amounts (Karnovsky & Rapson, 1946: Karnovsky & Brumm. 1955: Kayama PI LI/.. 1971 : Seino et u/., 1971 : Malins & Wekell. 1970: Malins & Varanasi. 1971). However, available evidente is rather fragmentary. Karno\skq & Rapson (1946). who screened glyceryl ether contents in marine animals, devoted much of their attention to fìshcs. and les:, to marine invertebrates. The present paper IS concerned with the distribution of glyceryl ethers in marine invertebrates of different t&&o~y and ecology Suitable techniques of the analysis developed in our laboratories have been used. \lATk:RIAI.S
“Absolute” poration for
AUD
wlth
A 0.02 M 1.5 moles of
contnmmg
perlodate
solution
(0.5 ml).
ZO”,,
hodium hlcarbonate per 1mole of periodic acad. was added in ;I 10 ml graduated tube to a 44”,, aqueous iroprnpanol solution (1 mi) of glyceryl ether. required for oxidation of not morc than 10 /LM periodate. Aftcr Incubatlng the miwture at room temprrature for (0.5 ml) ~vas added. 1 1.5 hl-. ;t 0.05 M rhamnoae solutlon and tilc mlnutea Iatcr a Nash reagent (1 ml) was added. The mlxture was hcuted at 50 C for 15 min. In case’r when thc solutions wcrc turbid. isopropyl alcohol (1 ml) was added. After coolins with running water. distllled water was added till the graduation mark. Following thorough agitation. thc \olution tias photometered in cuvctlcs ( I cm) at 412 nm. To IIICI-cax thc scnsltikitj of the reaction. thc final \olumc can hc decrca$cd to 5 ml. lcaving the amounts of the t-cagcnts thc
ME:THODS
diethyl ethf:r. chimyl alcohol Biochemical Investigations.
water (65:25:4. v \). Spots were detected ammonium tx\ulphate with heating at 1X0 C
4 \\cighcd portkon dissolced in ahsolutc
(California CorU.S.A.), and an
ethereal solution of lithium aluminium hydride, prepared from a commercial preparation in accord with Dillard (1952). were used as reagents. The hydride concentration
of the Iipid extract (20 <5 m_g) was ether (5 IOml). Thcn a lithium alu-
minlum hydride solutmn (double the weighed portion of the lipld extract) wa\ curefully added batchwise. 4fter adding the la\r hydride portion. the reaction mlxturr was hept fnr 30 min at room temperature and thcn r&xcd for 30 mm. Subsequent to cooling the reaction mixture. the cxccs~ of lithium aluminium hydride wah destroycd wlth distilleti mater (tirst b‘ drops). and the resultant CeI uaa thssolvcd \\lth hiu hydrochloric acid. The ether laycr was beparatcd. and the aqucous phase extracted four times Mlth ether. The cxtracts werf combined and c\aporated to minimum volume: the solution was then spread onto a platc 19 r 12 cm) wlth \IIIC:I gel KCK. the plate having heen prewa\hed \vith ether. The system uscd wab ether-conccntratcd ammonium hydroxide (100:1). The Itpid /ones were \ isualued bl
in the solutlon was dctermined by Kreshkov’s method 11961). All other reagents uscd in the work were prepared in accord with our previolls technique (Vaskovsky & lsaq.
Iwí9). The animals used in tne work were collccted in Posiet Bay, Sea of Japan. in July October 1972. and in thc tropical regions of the Pacific in December 1970 and Januarq 1971. Frcshly captured invertebrates were used to preparc total lipid extracts in accord with Folch PI trl. (1957). The amount of lipids in cach extract was dctcrmmed by the 301
As usual. the z-glyceryl ether determination consisted of the following stages: decomposition of lipide: isolation of glyceryl ether fraction: oxidation of fraction with periodate or lead tctracetatc: and yuantification of resultant formaldehyde. In our laboratory. a sensitivc and suitahlc method for determining the formaldehyde that farms durmg oxidation of substances with periodate had previously been developed (Vaskovsky & Isay. 1969). On the basis of this method, we developed a proccdurc for quantitating glyccryl ethers. In recent ycars. reduction wsith lithium aluminium hydride was more frequently used for splitting lipids
A study of glyceryl
tions. After trying several solvents and their mixtures, we chose 440,, aqueous isopropanol, since it gives a transparent reaction solution. which, unlike other solvents. remains transparent when diluted with water. Glyceryl ether fractions are generally eluted from the silica gel with diethyl ether (Wood & Snyder, 1968) or 15”;) diethyl ether in hexane (Snyder, 1969a). Inasmuch as we conducted further analysis in 44% aqueous isopropanol, it appeared more suitable to immediately obtain isoinropanol extracts. The advan-
ethers -~ J
303
tage of aqueous isopropanol was in that under similar experimental conditions it fully washed out the substance. whereas ethers washed out only 507”. Thus, we developed a suitable procedure that made possible a relatively easy analysis of a large number of marine invertebrates. We quantitated the a-glyceryl ether content in 63 species from the Sea of Japan and 39 species from the tropical regions of the Pacific. The results are cited in Tables 1 and 2. The tropical invertebrates are shown only as classes and phyla,
since not all of them were assigned species. There
is u correlation
between
the
to genera
and
taxonomie
pos-
and thcir lipid content and composition. Thus. it was shown that the qualitative and quantitative contents of glycolipids and their monosaccharides are in a certain way associated with the evolutionary leve1 of marine invertebrates (Vaskovsky er td.. 1970). However. the analytical data for a large number of marine invcrtebrates belonging to eight phyla and about 20 classes does not suggest any definitc relationship bctween the contents of r-glyceryl ethers and taxonomy of thc animals. The amount of x-glqceryl ethers found is almost the same for al1 of the classes and types. varying from I to S”,,. exccpt for Anthoroa and certain mollusc species. whose x-glyceryl ether content cxceeds X”,,. This holds truc for both Japan Sea and tropical Pacific invertebratcs. The class Holothurioidea is also distinguished by a high r.-glyceryl ether content. but this applies only to invcrtebrates from the Sea of Japan. Thcre are no other sharp diffcrcnces between the invertebrates from the two regions. The contents of rc-glyceryl ethers appears to bc more dependent on the individual propertics of organisms. Tables 1 and 2 show that the amount of r.-glyccryl ethers even in closely related species is highly \aricd. It is diffìcult to compare our data with thosc of other authors. since \‘crq fcw works are known on glyceryl ethers of marine invertebrates. and therc is virtually no cvidence for the species that we determined. Moreover. cken the available fìgurcs can hardly bc compared with the results of the present paper and with each othcr. since al1 authors ~1st:relatively different values to express thr amounts of r-glyceryl ethers. e.g. unsaponifiablc fractions. neutral Iipids and phospholipids (Taro rt trl.. 1941: Karnovsky & Rapson, 1946; Karnovsky & Brumm, 1955: Rapport & Alonzo, 1960; Seino. 1971). But our results confirm the main conclusion of these works that cc-glyceryl ether is essential for marine lipids. ition
of
marine
provided us with specimens of tropical orgamstns. and to 0. D. Korotchenko for her unstinted aid in preparing lipid extracts from Japan Sea mvertehrates.
invertebrates
Ac~nowIrdyr,,lorts E. Y. Kostetsk) IFar
Our East
heartfelt thanks are dut: to State
University),
who
Dr. kmdl\i
BLANK M. L.. (‘~1~s F. A.. S’rLI’Hl-NS N. & SN’~LXR I-. (1971 l On the analyses of long-chain alkane diols and glycerol ether\ 111hiochemical studies. .I. lip Rr,\. 12, 63X- 640. DII t.b.Rt> c‘. R. (195?l An apparatus for storing and measuring solutions of lithium aluminium hydride. J. C/ICVI. r:tll,<,. 29. I 2‘) 130. Fmc1f J.. LCIS M. & SLOAXI S. G. H. (1957) A simple method lor thc isolution and purifìcation of total lipids from animal tissues. .I. bio/. C/lu/n. 226, 497-509. KARNOVSK~ M. L.. RAPSON W. S. & BLACK M. (1946) South African fish products ~XXIV. The occurrence of a-glyceryl ethers in the unsaponifiahle fractions of natura1 rats. .1. SOC,.C/ICV~ /rr
MAL.ISS D. C. & WI i
A study of glycerql ethers
SNYDER F. (1969a) Ethers-linked lipids in neoplasms of man and animals: methods of measurement and thc occurrence and nature of the alkyl and alk- I -en yl moieMutlicinv rrntl Riokq~. ties. In Adrancrs in E.rperimenfol Vol. 4. pp. 609 ~621. Plenum. New York. SNYDER F. (1969b) The biochemistry of hpids containing ether bonds. In Pwyess in the Chrmistr~ of Ftrts trrd (Edited by HOLMAN R. T.). Vol. 10. pp. Other Lqids 287-335, Pergamon Press, Oxford. SNYDER F. (1969~) The chemistry. phyaical properties. and chromatography of lipids containing ether bonds. In
Proyres,s in Th-Lajvr Chromtrtoy,trpl~~, trnd Rrltrrrtl Methods (Edited by PATAKI G. & NIEDERM I~SER A.). Ann Arbor, Ann Arbor. Michigan. .TAKO M.. MASAKADLI: Y. & YOSHIYC,KI T. (1943) The cryatalline fraction of thc unsaponifiable substances in the fat of 4 srt2ricl.rrollrstcwi and Clrclrmtrr-itr ~~hronhjdmi. J. ~~/w»I. SN. ./
I
W5
THOMPSOPVCr. A. & LI-!- P. (1965) Studies of the r-gllccryl ether liplds occurring in molluscan tissues. Biochlm. biophl,\. Actf~ 98, 151~ 159. VASKOVSKY V. E. & ISA~ S. V. (1969) Quantitativc determmation of formaldehyde liherated with periodate o\idatIon. -Irl[l/vr. Biochcw. 30, 3 3 1. VASKO\SK> V. E.. Ko~T~TsK~ F-. Y.. S\I 1 ~SHI \ \. 1.. Zlir ho\% 1. c;. & SM1KWV.A G. P. (19701 Glycohplds of marine in\ertebrateï. C~ì~np. B~~C/ICHI Pil I~\liJ/. 34. 163 177. Woon R. Hr SN~III K F. (1968) Quantitati\e dctcrmlnatton of alk-l-cnyl and alkvl glyceryl ethers 111ncutral Iipids and phoqholipids. Liptd.5 3, 1% 135. ZIUIUSKI T. & BOROWSKI E. (1966) New spray rcagcnt replac~ng sulfuric acid in thin-layer chïomatogaph> J. (‘/7r.l~~nllriU/.23. 3x0 4x7.