95

ESTIMATION OF ECDYSTERONE

FROM SULFURIC ACID INDUCED FLUORESCENCE

Michael W. Gilgan and Maurice E. Zinckl

Fisheries Research Board of Canada Halifax Laboratory

Halifax, Nova Scotia

Received : 3127172

ABSTRACT

The sulfuric acid induction of ecdysterone (2P,3P,l&u,20R,22R, 2.5-hexahydroxy-SP-cholest-7-en-6-one) fluorescence was examined under various conditions of acid concentrations, acid diluents, reaction duration and reaction temperature. With appropriate conditions ecdysterone standards could be estimated in amounts from ca. 5 ng to in excess of 10 Pg. The similarity of the fluorescence spectra and yields of some other ecdysteroids suggested that the method is applicable to other compounds of this type.

INTRODUCTION

Work on arthropod molt hormones has been encumbered by an almost

total reliance on bioassays utilizing various insects or their larvae

(e.g., 2). A chemical method for ecdysteroid analysis as a substitute

for insect bioassay3 would be desirable. The most promising substitute

appeared to be acid-induced fluorescence, investigated briefly by

T. Takemoto et al __. (3) if high sensitivity could be achieved.

Sulfuric acid-induced fluorescence is an established method for

the precise, if not always accurate, estimation of small amounts of

steroids. Hence optimal conditions for procedures such as glassware

cleansing and acid dilution have already been defined. It remained

96 STEROIDS

to determine the combination of variables which would give sufficient

sensitivity to ecdysterone with reasonable specificity (low acid

concentration). A principal requirement for estimation of ecdysterone

in arthropod extracts was a sensitivity to submicrogram quantities of

the steroids, comparable to that of the housefly bioassay (2). A much

lower assay sensitivity would require starting-samples excessively

large for most animal research.

MATERIALS

Sulfuric acid

Baker and Adamson, reagent grade, or Matheson, Coleman and Bell, fluorescent grade acids were found to be adequate. Other products examined exhibited too high a background fluorescence to be useful.

Solvents

All solvents used for cleansing glassware or preparing standard solutions were reagent grade, redistilled. The alcohols used as sulfuric acid diluents were refluxed with NaOH pellets and then distilled twice. Distilled water was redistilled in all-glass apparatus.

Glassware

The "cuvettes" used were Kimax 0.6 x 5 cm culture tubes selected for minimum variation in light path with a fluorescent sulfuric acid- ethanol mixture at maximum fluorometer sensitivity. The Kimax tubes used had the advantage of thinner walls which permitted more solution to be illuminated by the incident beam and a lower background due to fluorescence or light scattering in the glass.

All glassware was cleaned by the procedure suggested by Udenfriend (4).

Standards

Inokosterone (2P,3P,14a,20R,22R,26-hexahydroxy-5P-cholest-7- en-bone) and ecdysterone (20-hydroxyecdysterone, crustecdysone, P-ecdysone) were purchased from Mann Laboratories and recrystallized before use. The other ecdysteroids were gifts:- a-ecdysone (2P,3P, ll+a,22R,25-pentahydroxy-5S-cholest-7-en-bone) from Dr. R.I. Dorfman, insect 20-hydroxyecdysone from Dr. J.N. Kaplanis and ponasterone A

July 1972 STEROIDS 97

(2P,3P,14u,20R,22R-pentahydroxy-5P-cholest-7-en-bone) from Dr. K. Nakanishi, and were used as supplied.

The concentration of standard ecdysterone solutions was determined from optical density at 243 nm (extinction coefficient, 1.24x 104 (5)). Chromatographic examination has never shown any contamination of the recrystallized ecdysterone. However, since the present studies were completed, inokosterone has always been found to contain an ecdysterone contaminant (lo%, as estimated visually in UV light).

Instrumentation

Spectra were determined with quartz micro-cuvettes in an automatic scanning and recording Farrand spectrofluorometer. Routine fluorometry was done with the Kimax culture tubes in a Turner Model 110 with the micro-adaptor kit. The most suitable filter combination for the Turner fluorometer was 7-60 and 47B + 2A for the excitation and fluorescence beams respectively.

GENERAL PROCEDURES

Preparation of diluted acid solutions

An appropriate volume of diluting solvent was cooled in an ice bath by rapid stirring with a magnetic stirrer. The required amount of sulfuric acid was then added dropwise from a small burette with continuous cooling and rapid stirring. The reagent was normally prepared fresh, but on occasions was stored at 6°C for 24 to 48 hr, .and allowed to come to room temperature before use.

Assay procedure

The most convenient procedure used was as follows: Samples to be assayed, normally triplicates, were pipetted into small test tubes with precleaned Drummond micropipettes, the tubes covered immediately with Parafilm and the solvent evaporated with a stream of nitrogen. The needle-jets of the N-evaporator (Organomation Assoc., Shrewsbury, Mass.) were covered with cleaned Pasteur pipettes sealed to the butt of the needle with gum rubber tubing. The pipette-covered needles were inserted through the parafilm to direct the gas stream onto the solution. To prevent cross-contamination of samples the pipette needle covers were discarded after use.

The appropriate diluted sulfuric acid solution (1.0 ml) was added to each sample from a Repipette (Labindustries, Berkeley, Calif.) and the solution immediately mixed vigorously with a vortex mixer. The vigorous mixing tended to trap air in the more viscous acid solutions, hence, if initial fluorescence values were of concern, the solutions were briefly centrifuged. A sample of each reaction mixture was then

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transferred to the micro culture tube cuvettes with a washed, disposable Pasteur pipette and allowed to stand at room temperature until fluorescence was determined. All tubes containing reagents or samples were kept covered with Parafilm caps at all times. When heating was necessary the reaction mixtures were incubated in the original reaction tubes.

All fluorometric estimations were done at room temperature and with minimum exposure to the heat and intense light beam of the instrument.

EXPERIMENTAL METHODS AND RESULTS

Wavelenpth maxima

Approximately one microgram each of alpha-ecdysone, ponasterone A, inokosterone and ecdysterone (from three sources) were reacted for at least 60 min at room temperature with 50% sulfuric acid/ethanol. All spectra were quite similar. The different ecdysteroids produced only slight variations in the maxima, Table 1, which were not considered very significant since the wavelength accuracy of the Farrand instrument was low. Samples of ecdysterone from three sources, commercial, insect (from Dr. Kaplanis), and Yew tree (isolated in this laboratory, essentially according to Imai et al. (6)), gave the same spectrum and -- showed similar fluorescence yields. One would assume that all these ecdysteroids could be analysed by the same methods and achieve similar sensitivities.

Table 1

Fluorescence (F) and excitation (E) maxima of the fluorophores produced from ca. 1 Vg of ecdysteroids by sulfuric acid/ethanol.

Insect 20-hydroxyecdysone 375 431 Yew ecdysterone 374 431 Commercial ecdysterone 375 425 a-ecdysone 378 - 380 425 - 427 Ponasterone A 377 - 380 425 - 427 Inokosterone 375 431

Reactions with one microgram or more of ecdysterone

An examination of the rates of fluorescence development of five pg of ecdysterone in three concentrations of sulfuric acid (Fig.la)

July 1972 STEROIDS 99

showed that the reaction was largely complete in 60% and 70% sulfuric acid/ethanol after 30 min at room temperature. Only minor increases in fluorescence were found after 60 min. Lower sulfuric acid concentrations yielded slower fluorescence development. An acid concentration of 60% was therefore chosen to demonstrate the effect of diluent on the fluorescence production,

0 cTnA*Ol. OILULIIT A YCTHAUOL OILUEWT

Fig. 1. The rate of fluorescence development at room temperature from quadruplicate ecdysterone samples (5.0 pg), (a) in 50, 60 and 70% sulfuric acid/ethanol (b) in 60% sulfuric acid diluted with water, methanol and ethanol. All values were determined at minimum instrument sensitivity. Variations indicated are mean + S.E.

A comparison of the polarity of the diluting solvents, water, methanol and ethanol, with the fluorescence yield of ecdysterone (Fig.1 (b)), shows that reduced diluent polarity increases fluorescent yield. However, a further reduction in polarity through the use of isopropanol did not improve the fluorescence yield and, in addition, resulted in a higher fluorescence background. Mixtures of n-butanol and H2SO4 were not sufficiently stable to allow determinations.

Experiments similar to that shown in Figure l(a) for ethanol were also performed to determine the effect of acid concentration with water and methanol as diluents. In all cases, the fluorescence intensity was lower for equivalent acid concentrations in water and methanol than in ethanol. The higher polarity of diluent resulted in lower fluorescence yields apparently due to slower reaction rates (Fig.1 (b)). Both alcohols permitted higher yields than water. The methanolic mixtures continued to increase significantly in fluorescence intensity for the duration of the observations while the ethanolic mixture more nearly stabilized at its higher level. Unfortunately, although ethanol mixtures produced the highest sensitivity, they also showed much greater variation as indicated in Figure l(b). At 60 min in 60% sulfuric acid

100 STEROIDS 2O:l

the percent standard error, (S.E./Mean) x 100, was 1.22 for methanol and 2.01 for ethanol. The difference in precision was even greater in 50% sulfuric acid, errors were 0.55% for methanol and 4.6% for ethanol. This has been found to be typical even though the error difference was greater than expected from the observed difference in fluorscence yields.

Of note also, 50% sulfuric acid/ethanol gave the greatest fluorescence yield on long standing (180 min, Fig.l(a)). Unfortunately, the 50% sulfuric acid/methanol samples did not show any tendency to stabilize at the higher levels. The fluorescence was reasonably stable on standing at room temperature at the higher acid concentrations.

Further investigation with amounts of ecdysterone greater than 5 pg showed a more pronounced upward drift in fluorescence intensity with time at all three sulfuric acid concentrations. Only 70% sulfuric acid/ethanol could be considered to give sufficiently stable fluorescence yield for ca. 10 Pg amounts of ecdysterone. Therefore, 70% sulfuric acid/ethanol was used to produce a standard curve for ecdysterone in amounts from 1 to 10 pg (Fig.2 (a)). The 10 kg samples were nearly off-scale at minimum instrument sensitivity.

Reactions with less than one microgram of ecdysterone

Subsequently attempts were made to improve the fluorescence analysis to accommodate the measurement of sub-microgram quantities of ecdysterone. Two principal avenues were followed: attempts to optimize the above conditions for sub-microgram amounts of material, and attempts to increase the fluorescence yield by heating the reactions at a moderate temperature prior to fluorescence determination.

The result of the former attempts was the adoption of the present methods for purifying the solvents, cleaning glassware

3. and

preparation of the reagent (described in the General Procedure Not surprisingly, variations in the glassware cleaning methods were found to have a negligible effect on the reproducibility of the assay since these methods are standard for fluorescence analyses. The purification of the solvents and selection of source acid samples (described above) both permitted a considerable reduction in the fluorescence blank. Careful preparation of the reagent as described allowed much more reproducible individual estimates and standard curves (Fig.2 (b)).

July 1772 STEROIDS 101

ECDYSTERONE (ng)

Fig. 2. Ecdysterone fluorescence standard curves: (a) for one to ten micrograms reacted with 70% sulfuric

acid/ethanol, (b) for 50 to 200 nanograms reacted in 50% sulfuric

acid/ethanol on two different days. .A11 reactions were conducted at room temperature. Values indicated were the means of duplicates (a) or triplicates (b) + S.E.

When the acid induced fluorescence method was applied to sub- microgram quantities of ecdysterone it was found that factors apparently significant to larger quantities were not as important. The effect of diluent and acid concentration were both much reduced. Also, the rate of production of maximum fluorescence was much greater and the resultant fluorescence intensity more stable with time. Therefore, lower acid concentration and a shorter reaction time was permissible. A reaction time of 30 min and an acid concentration of 50% (lowered to reduce blank) in ethanol was therefore adopted. Standard curves determined on two consecutive days under the above conditions are shown in Figure 2 (b). Where additional specificity might be required (generally assumed to increase with lower acid concentrations), it would be possible to determine like amounts of ecdysterone with an even lower sulfuric acid concentration and with little loss of sensitivity or time.

Heating the reaction mixtures increased sensitivity but should have decreased specificity, and hence increased noise in samples other than pure standards. An example of the effect of heating on the fluorescence yield is shown in Figure 3 (a). All samples were cooled at least ten minutes on the bench before the fluorescence was determined. Hence all samples had reacted with 60% sulfuric acid for 45 minutes total before the fluorescence was determined. Blanks were low (7 units) and essentially unaffected by the heating.

102 STEROIDS 2O:l

j=j , 4 1 0: 0 IS SO

HEATING TIME (Min.) AT 5OC

b

Fig. 3. The effect of heating (SOY) on fluorescence development in 605% sulfuric acid. (a) rate of reaction of 500 ng ecdysterone (means of

triplicate values corrected for blank), (b) standard curve for 5 to 37.5 ng ecdysterone heated

for 20 min (means of triplicates corrected for blank).

The curve shown in Fig,3 (a) suggests that the fluorescence yield continued to increase on heating even after 30 min. The effect may have been partially due to solvent loss, although the tubes were capped to minimize this. Most of the increase would appear to have been achieved in about 20 minutes. To utilize the increase in sensitivity permitted, but to minimize the potential variation in results due to variable solvent losses, a heating time interval of twenty minutes was selected for an examination of the effect of heating on a high sensitivity standard curve (Fig.3 (b).

Little or no change in fluorescence occurred in these samples after the heating period for a total elapsed time of up to 180 min. For example, the 37.5 ng standard changed from a fluorescence value of 38.6 to 37.6 (both corrected for blank) at the total elapsed times of 35 and 180 mintes. The principal difficulty encountered in such high sensitivity standard curves was inadequate blank correction which could give erroneous low value estimations. The curve was suitably linear. The percent standard error on triplicate samples ranged from 9.4 for 25 ng to 2.0 for 37.5 ng. The overall sensitivity was approximately twice that achieved without heating.

July 1972 STEROIDS 103

DISCUSSION

The behavior of the fluorescence development reaction appears

to be to a fair degree concentration dependent. Therefore, we have

included sufficient data to allow a future investigator to judge the

most appropriate conditions for his analysis.

The fluorescence analysis procedure has had only limited use.

Samples of yew-tree extracts, which contain large amounts of ecdysterone

(6), were purified by silicic acid column chromatography followed by

thin-layer chromatography. The appropriate purified samples were

assayed for ecdysterone by housefly bioassay (2) and fluorescence.

Within the limitations of the housefly bioassay the results were

comparable. The over-all method was not considered very useful, however,

due to poor recovery (f+O-50%) from the thin-layer plates. Hence the

suitability of the method for routine analyses, particularly of the

trace amounts of ecdysteroids normally found in animals, remains to be

determined.

The housefly bioassay of Kaplanis et a1.(2), while superior to --

many bioassays, suffers from high variation ($ SE = 13 at 61% full

response with 75 animals (2)) and, with the large number of test

animals required, is not extremely sensitive (150 ng for one adequate

test (2)).

High blank values would limit the fluorescence procedure to

amounts of purified ecdysterone in excess of 50 ng. However, even at

such relatively low sensitivities the fluorescence method achieves a

sensitivity similar to that of the insect assay. The principal

disadvantages of the fluorescence method are its lack of specificity

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which dictates that all glassware must be extremely clean and that the

method must be used in concert with adequate chromatography. The

principal advantage of the fluorescence assay is that it is faster

and less variable than the bioassay and that it avoids the continuous

maintenance of an insect colony.

REFERENCES

1. Present address: Fisheries Research Board of Canada, Marine Ecology Laboratory, P.O. Box 1006, Dartmouth, Nova Scotia, Canada.

2. Kaplanis, J.N., Tabor, L.A., Thompson, M.J., Robbins, W.E. and Sortino, T.J., STEROIDS 8, 625 (1966).

3. Takemoto, T., Ogawa, S., Morita, M., Nishimoto, N., Dome, K. and Morishima, K., YAKUGAKU ZASSHI 88, 39 (1968).

4. Udenfriend, S., FLUORESCENCE ASSAY IN BIOLOGY AND MEDICINE, Academic Press, N.Y. (1962).

5. Hocks, P. and Wiechert, R., TETRAHEDRON LETTERS, 2989 (1966).

6. Imai, S., Fujioka, S., Nakanishi, K., Koreeda, M. and T. Kurokawa, STEROIDS I-0, 557 (1967).