trends in analytical chemistry, vol. 8, no. 2,1989 67

Thin-layer chromatography of eicosanoids

Elisabeth Granstrijm Stockholm, Sweden

The growing complexity of the eicosanoid field, where a multitude of (structurally) closely related products are formed simultaneously from the same precursor, necessi- tates the use of profiling assay methods in many types of studies. Examples of such studies are the in vitro conversion of exogenous, labeled arachidonic acid by an organ or a tis- sue, and the further metabolism in vivo of a prostaglandin or other eicosanoid. Thin-layer chromatography is one of the most frequently used methods for such studies, generally as a normal-phase system using silica gel as the stationary phase and a non-polar solvent as the mobile phase. Recently, reversed-phase chromatography, which is particularly suit- able for some of the more polar metabolites, has been uti- lized. An improved technique, i.e. two-dimensional thin-lay- er chromatography, with its far higher resolution, provides considerably more information and may also reveal that the results from the conventional one-dimensional technique could be highly misleading.

Introduction The eicosanoids are a very large, heterogeneous

group of structurally and/or biochemically related oxygenated fatty acids (Fig. 1). Several subgroups exist: prostaglandins (PGs), thromboxanes (TXs), leukotrienes (LTs), lipoxins (LXs) and mono-, di- and trihydroxylic fatty acids. They also occur in dif- ferent series, depending on the degree of unsatura- tion: the number of double bonds is shown by an index after the name of the compound. Although

12-HETE 12-HPETE

8 MDA

HO&OOH _

6H TXB2

they were at first identified as a series of CzO fatty acids (hence the designation ‘eicosanoids’), it was later shown that at least some of the eicosanoids have counterparts with other chain lengths’.

In addition to this already complex picture, the further metabolic fates of these compounds give rise to numerous break-down products. Metabolic stud- ies of single compounds, e.g. PGF,,, TXB, etc., have often revealed the formation of twenty or more final degradation products excreted into urine2. In addition, several of the eicosanoids and their metab- olites are chemically unstable and give rise to a num- ber of degradation products, or can occur in several different chemical forms in equilibrium. Thus, the full spectrum of formed products in any given biolog- ical system can be extremely complex.

Research in this field has consequently always been hampered by analytical difficulties. Early ap- proaches to eicosanoid assay were often aimed at measuring single compounds, which required ex- tremely specific analytical methods and/or extensive purification of the samples. Later, when it became increasingly clear how complex this area is, and how different metabolic pathways can interact with each other, the opposite concept gradually gained ground. Today many profiling assays have been de- veloped, where the aim is to get as complete a pic- ture of formed products as possible.

One of the most widely used approaches in the analysis of the full spectrum of eicosanoids formed in a biological system is to use radiolabelled substrate

Arachidmic acid S-HPETE

czlYH 6H

HHT

\

Ye” &OH

PGG2

I 15-HPETE

- Leukotrienes

Hoh - OH HOT HOT Ox”“” 6H

6-K&o PGFld PG12 PGE2 PGD2

Fig. 1. Simplified scheme of some prostaglandins, thromboxanes and leukotrienes formed from arachidonic acid.

01659936/89/$03.00. 0 Elsevier Science Publishers B .V.

68 trends in analytical chemistry, vol. 8, no. 2,1989

and thin-layer chromatography (TLC) of a lipophilic extract of the material. Nearly all assays are of the normal-phase type, using conventional silica gel as the stationary phase, and various medium to non-po- lar solvent mixtures as the mobile phase3.

Limitations in TLC assay There are certain limitations to this analytical ap-

proach. One of the major limitations does not reside in the type of chromatography chosen, but is due to the radiolabel of the precursor fatty acid being near- ly always in the l-position. There are at present three different types of precursor commercially available: a uniformly 14C-labelled arachidonic acid, the [5,6,8,9,11,12,14,15-3H,]-labelled compound, and [l-‘4C]-labelled arachidonic acid. The first of these is very expensive; employing a tritium labelled compound implies difficulties in detection of its products; hence, the [l-‘4C]-labelled precursor is preferred in most studies.

However, since a major fate of the eicosanoids - like other fatty acids - in the body is P-oxidation, the l-label is rapidly lost in many biological systems. Thus, [l-14C]arachidonic acid can only be used in certain simple in vitro systems where P-oxidation is negligible. In other studies, such as in vivo, multiple tritium-labelled precursors are employed. Consider- ing the extremely complex metabolic picture men- tioned above, it is obvious that, in addition to tritium detection, the scientist is facing quite a formidable analytical problem, regardless of the assay em-

Solvent front 1

TLC scan after solvent system I

f dart

ployed. This is particularly the case with TLC, since all the products have to be separated in a space of lo-20 cm, which calls for exceptionally high resolu- tion. Nonetheless, TLC has proved quite adequate for this purpose, if suitably modified.

Comparison between one- and two-dimensional TLC

Fig. 2 shows a profile of PGF,, metabolites in rab- bit plasma, 20 min after the intravenous injection of [9B-3H]PGF,,. The 9P-position is metabolically stable in this species, hence all circulating PGF me- tabolites will be labelled and can be detected with sufficient sensitivity. Two different techniques were employed for this analysis: the conventional one-di- mensional (1D) TLC using a scanner for detection (left panel) and two dimensional (2D) TLC with a combination of two suitable solvent systems, fol- lowed by autoradiography as the detection method (right panel). The conventional method is obviously not satisfactory: resolution is far from sufficient, and the scanty information obtained is misleading, since it suggests that the major compound at this stage is unconverted PGF,, itself. This we know is not the case - the compound does not escape undegraded.

On the other hand, the 2D-TLC technique, with its far higher resolution, displays some 25 clearly separated spots, and distinctly shows that virtually no radioactivity migrates as unconverted PGF,. The greater amount of information obtained by this technique has its price, however: the method is very

Xi-ketodihydro-PGFzd

1

Solvent system I F

-

-

-

-

Fig. 2. Metabolites of [9f3-3H]PGF,, in rabbit blood 20 min after intravenous injection of the compound. Total sample radioactivity, 200 000 dpm. Film, LKB Ultrofilm ‘H. Exposure time, seven weeks. The results of the 20 analysis using autoradiography (right) are compared with a radioscan obtained after ID-TLC (left). Solvent systems: I, ethyl acetate-acetic acid-isooctane (100:10:30). KI, chloro- form-n-butanol-acetic acid (80:20:2) [Reproduced from E. Granstrom, Methods Enzymol., 86 (1982) 493, with permission from Aca- demic Press.]

trends in analytical chemistry, vol. 8, no. 2,1989 69

Fig. 3. Products of [I-t4C]arachidonic acid in a whole homogenate of guinea pig lung. The incubation was interrupted with 25 volumes of methanol after 1 min. Total sample radioactivity, 30 000 dpm. Film, Osray M-3. Exposure time, 7 days. [Reproduced from E. Granstrom, Methods Enzymol., 86 (1982) 493, with permission from Academic Press.]

time-consuming. For detection of such small amounts of tritium (the weakest spots contained only about 50 dpm each), a very sensitive film is required. The most sensitive one on the market at present is the LKB Ultrofilm, which is specifically designed for detection of the weak p-emission of tritium. For ex- ample, it lacks the antiscratch layer of gelatin of other films, which normally absorbs 95% of the ra- diation. Despite the use of this film, the 2D-TLC in Fig. 2 required several weeks exposure. On the other hand, it might be argued that to obtain all the infor- mation the 2D method provides, all one has to do is wait!

Fig. 3 shows a similar comparison between the two TLC techniques. In this experiment, [l-‘4C]arachi- donic acid was incubated with guinea pig lung, which is known to have a high capacity for thromboxane A, biosynthesis. The incubation was treated with a large excess of methanol, which converted the formed thromboxane A, into mono-O-methyl TXB, (two isomers) instead of the regular hydrolysis product, TXB,. The lD-TLC analysis (by scanner) clearly shows this, as well as small amounts of prostaglan- dins. However, the large radioactive peak migrating close to the solvent front would normally be inter- preted simply as unconverted arachidonic acid. It is very common in such experiments to calculate the capacity of a certain tissue for arachidonic acid con- version from the radioactivity of such TLC peaks.

However, after analysis by 2D-TLC, the results will be interpreted quite differently. Fig. 3 (right panel) clearly shows that there are four separate compounds migrating close to the solvent front, only

the topmost of which is unconverted arachidonic acid, and a minor compound at that. The other three, more prominent, compounds most likely are different mono-hydroxylated eicosatetraenoic acids, which are known to be formed in significant amounts by lung tissue.

Structural information The 2D-TLC method can also provide other kinds

of information, such as on the structures of certain compounds. For example, one major thromboxane metabolite in the body is 11-dehydro-TXB, (Fig. 4). Dehydrogenation at C-11 actually converts the thromboxane into a dicarboxylic metabolite, and this compound can occur in two different forms: one retaining the thromboxane ring (a 8-lactone in this case), and an open form (Fig. 4). The two forms can be rapidly converted into each other. This intercon- version is sometimes uncontrollable - it may for ex- ample occur in some TLC systems. Fig. 5 shows a 2D-TLC of the [1-14C]TXB, metabolite profile in rabbit lung. This tissue efficiently converts the com-

OH OH ,,e~COOH

;

OH

Open form and S -lactone form of

11-Dehydro-TXBZ

Fig. 4.11-Dehydro-TXB, in its open and Nactone forms.

trends in analytical chemistry, vol. 8, no. 2,1989

Fig, 5. Formation of [I-14C]11-dehydro-TXBz during incubation of rabbit lung whole homogenate with [l-‘4C]TXBz, as analyzed by 2D-TLC. Solvent systems: I, ethyl acetate-acetic acid-isooc- tane (100:10:15); II, chloroform-n-butanol-acetic acid (75:25:2). Note the occurrence of ll-dehydro-TXB, in two chem- ical forms. [Reproduced from E. Granstrom and M. Kumlin, Adv. Prostaglandin Thromboxane Leukotriene Res., 17B (1987) 587, with permission from Raven Press.]

pound into its 11-dehydrogenated counterpart. The closed lactone form of this metabolite is relatively non-polar and displays an R, value of about 0.7 in both systems; the polar open form migrates consid- erably less. It can be seen that some interconversion between the two forms obviously took place in the first solvent system: the spot corresponding to the closed form displays pronounced tailing. Also the polar open form showed some tendency to conver- sion and appears sickle-shaped, which is very char- acteristic for this particular compound.

Another compound which is notoriously difficult to analyse by chromatography is 6-keto-PGF,,. All compounds with the 6-keto structure can occur in at least two forms: the ‘open’, keto form and a ring- closed, lactol form. However, in TLC this compound frequently occurs as three clearly separated spots3. Various attempts at explaining this behaviour have been made, and certain extraction procedures have 5een identified that either induce or suppress the formation of unknown products3. However, the chromatographic behaviour of 6-keto-PGF,, re- mains unpredictable. The appearance of three spots with RF values characteristically related to each other can, however, be used as a strong indicator of the 6-keto-PGF structure (c$ Fig. 6).

Fig. 6. 2D-TLC profile of [I-r4C]-labelled metabolites of arachi- donic acid in chopped human asthmatic lung challenged with spe- cific antigen. For details of the experiment, see ref. 4. Note 6-keto- PGF,, appearing as three characteristic spots.

Sources of error in TLC In lD-TLC, it is sometimes seen that a major part

of the radioactivity migrates as one or a few bands, although a totally different result was expected. Fig. 3 showed one such case, where the explanation sim- ply was insufficient resolution due to the use of an unsuitable solvent system, and the combination of two solvent systems in 2D-TLC revealed the true picture. 2D-TLC may, however, also reveal anoth- er, more serious cause of misinterpretation of TLC results. Fig. 7 shows an extreme example of this. It is sometimes seen that part of, or almost all, polar compounds in a sample may co-chromatograph with some non-polar constituent in the first run, separat- ing only in the second run. The reason for this phe- nomenon is not completely known: it seems to vary somewhat between batches of TLC plates, but the major reason seems to be the presence of large amounts of lipophilic compounds, such as cholester- ol, triglycerides or long chain fatty acids, in the sam- ple extract. The more polar components probably dissolve in the lipophilic constituents and are never adsorbed to the silica gel. When this phenomenon occurs, the only compounds that are retained to their proper places on the plate are those which were added as internal references (cf. Fig. 7).

Since lD-TLC is one of the most widely used assay methods in this field - it is even used as a quantita- tive method, when relative amounts of formed prod-

‘rends in analytical chemistry, vol. 8, no. 2,1989 71

Fig. 7, Extreme co-migration phenomenon in TLC. A human endometrial biopsy was incubated with [l-‘4C]arachidonic acid and the lipophilic extract analysed by ZD-TLC. Solvent systems: I, hexane-ethyl acetate-acetic acid (25:75:2); II, chloroform-diethyl ether- methanol-acetic acid (45:45:5:2). That the results were not simply caused by decomposition of arachidonic acid was checked by separate analyses of this compound. [Reproduced from E. Granstriim and H. Kindahl, Adv. Prostaglandin Thromboxane Leukotriene Res., 11 (I 983) 173, with permission from Raven Press.]

ucts are calculated - this source of error deserves some attention: otherwise the metabolic profile ob- tained might be a function of what particular refer- ence substances happened to be available that par- ticular day.

Acknowledgment This study was supported by a grant from the

Swedish Medical Research Council (project number 03X-05915).

References 1 E. Oliw, E. Granstrijm and E. iinggbrd, in C. Pace-Asiak and

E. Granstrijm (Editors), Prostaglandins and Related Sub-

stances (New Comprehensive Biochemistry, Vol. S), Else- vier, Amsterdam, 1983, Ch. 1, p. 1.

2 E. Granstriim and M. Kumlin, in C. Benedetto, R. G. McDo- nald-Gibson, S. Nigam and T. F. Slater, Prostaglandins and Related Substances, IRL Press, Oxford, 1987, Ch. 1, p. 1.

3 J. A. Salmon and R. J. Flower, Methods Enzymol., 86 (1982) 477.

4 S.-E. DahlCn, G. Hansson, P. Hedqvist, T. BjGrck, E. Granstrdm and B. DahlCn, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 1712.

Elisabeth Granstriim was born in 1943, Sweden. She received her M.D. in 1972. She joined the Department of Physiological Chem- istry, Karolinska Institutet, Stockholm, Sweden, in 1963, becom- ing Assistant Professor in 1972. In 1987she was appointed Assis- tant Professor at the Reproductive Endocrinology Research Unit, Karolinska Hospital, Stockholm, Sweden.

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