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Lipids in the haemolymph of waxmoth larvae during starvation
7. Ins. Physiol., 1965, Vol. 11, pp. 11 to 20. Pergamon Press Ltd.
Printed in Great Britain
LIPIDS IN THE HAEMOLYMPH OF WAXMOTH DURING STARVATION PAULINA Department
WLODAWER
of Biochemistry,
Nencki
and ALICJA
Institute
(Received
WISNIEWSKA
of Experimental
11 May
LARVAE
Biology,
Warsaw,
Poland
1964)
Abstract-The
composition of lipids in the haemolymph of waxmoth larvae was Total lipids which represented investigated during feeding and starvation. about 2.4 per cent of the haemolymph contained about 22 per cent phospholipids, 15 per cent sterols (including cholesterol and a ‘fast acting’ sterol of unknown nature), 8 per cent unesterified fatty acids, and a high proportion of neutral glycerides. The main components of the phospholipid fraction appeared to be phosphatidylcholine, which comprised about 60 per cent of the total lipid phosphorus, and phosphatidylethanolamine (about 20 per cent). Sphingomyelin was not detected. Neither the concentration nor the composition of the lipids in the haemolymph was found to be markedly influenced by starvation; in particular, the phospholipid content was remarkably stable. No change in the proportions of choline and ethanolamine containing phospholipids could be observed. The loss of the lipid constituents from the haemolymph during starvation roughly However, paralleled the loss of body weight and of the amount of haemolymph. the relative loss of total lipids and phospholipids in the ‘residual larval body’, which consisted chiefly of the fat body, muscles, and integument, greatly exceeded the relative loss of body weight. Part of these lipids might contribute in maintaining the stable lipid level in the haemolymph.
INTRODUCTION
IT
HAS been shown recently in this laboratory (LENARTOWICZ and NIEMIERKO, 1964) that the contents of some organic phosphorus esters present in the haemolymph of waxmoth larvae undergo considerable changes during prolonged starvation. This is particularly true as regards phosphorylcholine and phosphorylethanolamine. During starvation of the larvae phosphorylethanolamine disappears from the haemolymph to a much greater extent than phosphorylcholine. Consequently, the phosphorylcholine to phosphorylethanolamine ratio rises from about O-7 in the fed larvae to approximately 3 in the 4 weeks starved insects. The reasons for the marked differences in the behaviour between the two phosphorus esters are poorly understood as yet. As both phosphorylcholine and phosphorylethanolamine are components of corresponding phospholipid molecules, the possibility was not to be excluded that these results might reflect some differences in the utilization of phosphatidylcholine (lecithin) and phosphatidylethanolamine, respectively, by the starved larvae. 11
12
PAULINAWLODAWER ANDALICJAWISNIEWSKA
The purpose of the present study was to follow the fate of lecithin and phosphatidylethanolamine in the haemolymph of waxmoth larvae during starvation and to gain information on the other lipid fractions of insect blood during feeding and fasting. MATERIAL
AND METHODS
Fully grown waxmoth larvae (Galleria mellonella L.) weighing 170-200 mg each were investigated. The insects were bred on beecomb and kept at constant temperature of 30°C. The larvae to be starved were ligated behind the head in order to prevent metamorphosis (NIEMIERKO and WOJTCZAK, 1950). Haemolymph was collected as described by LENARTOWICZet al. (1964). From the haemolymph pooled from about 100 larvae, lipids were extracted with 25 vol of chloroformmethanol (2 : 1 v/v), the solvent was evaporated under reduced pressure and the residue was re-extracted with petroleum ether-chloroform (2 : 1 v/v). The extract was taken to dryness and the amount of total lipids was determined gravimetrically. Sterols were determined by the method of SEARCY et al. (1960). Lipid phosphorus was estimated according to FISKE and SUBBAROW (1925) in samples digested with nitric acid, and the amount of phospholipids was calculated by multiplying the phosphorus value by 25. The composition of the phospholipid fraction was checked qualitatively by thin-layer chromatography (WAGNER et al., 1961), and the percentage contribution of the main components was evaluated quantitatively by chromatography on silicic acid impregnated filter paper, according to the method of MARINETTI (1962). The chromatograms were run in diisobutylketone-acetic acid-water (40 : 25 : 5) ; phospholipids were detected by staining with rhodamine 6G and viewed in ultraviolet light. Spots corresponding to individual phospholipids were excised and eluted with 1 N HCl in methanol. The eluates were evaporated, digested in 60% perchloric acid, and phosphorus was determined according to BARTLETT (1959). Chromatography of total lipids was performed on Whatman No. 3 filter paper impregnated with silicic acid, according to DOBIAQOVAand MICHALEC (private communication). The developing solvent was 10% ethyl ether in petroleum ether. Lipid spots were detected with rhodamine 6G or rhodamine B and viewed in U.V. while wet. RESULTS The concentrations of total lipids, sterols, and phospholipids in the haemolymph of the fed and of the starved larvae are shown in Table 1. As may be seen, total lipids represented about 2.4 per cent of the larval blood, and this concentration did not change markedly during 28 days of starvation. The differences found in various periods of starvation do not seem to be statistically significant. Phospholipid fraction comprised 0.52 per cent of the haemolymph and practically no changes in concentration were observed during the whole period of starvation. Total sterols made up 0.35 per cent in the blood of the fed larvae and 0.27 per cent in those starved for 7 days. Further starvation did not affect the sterol concentration significantly.
LIPIDS IN THE
HABMOLYMPH
OFWAXMOTH
LARVAE
DURING
13
STARVATION
An attempt was made to examine the nature of the sterols. As mentioned in Methods, sterols were determined calorimetrically by the method of SEARCY et al. (1960), and the determinations were referred to a cholesterol standard curve. The colour produced by the haemolymph sterols was the same as given by authentic cholesterol. On paper chromatograms the haemolymph sterols gave one spot closely corresponding to cholesterol used as reference compound. With the ferric chloride reagent of ZAK et al. (1954), wh’rch may serve to differentiate between TABLE~-LIPIDSINTHEHAEMOLYMPHOFWAXMOTHLARVAEDURINGFEEDINGANDSTARVATION (MEAN
Period of starvation (days) 0 7 18 28
VALUES
Total lipids in the haemolymph (%) 2.4 2.0 2.2 2.5
+ + f +
0.3 (5) 0.2 (5) 0.1 (4) 0.07 (5)
AND
STANDARD
DEVIATION
Sterols (expressed as cholesterol*)
Phospholipids In haemolymph (%) 0.52 0.53 0.51 0.53
& 0.02 kO.02 f0.02 + 0.03
(5) (5) (3) (4)
OF THE MEAN)
In lipids (%)
In haemolymph (%)
In lipids (%)
22 27 23 21
0.35 + O-03 (6) 0.2710.03 (5) 0.28 & 0.03 (6) 0.25 k 0.02 (5)
15 14 13 10
The figures in parentheses indicate the number of determinations. * Determinations referred to a cholesterol standard curve.
various sterols, the same characteristic coloured rings and the same final purple colour as given by authentic cholesterol were observed. With the modified Schoenheimer-Sperry reagent (MOORE and BAUMANN, 1952) the haemolymph sterols developed the blue-green colour much faster than authentic cholesterol; but instead of fading after several minutes, like that of the typical ‘fast acting’ sterols, the colour due to the haemolymph sterols markedly increased with time. According to MOORE and BAUMANN (1952), this type of colour reaction is the same as that observed when suitable mixtures of cholesterol and a ‘fast acting’ sterol (like 7-dehydrocholesterol) were treated with the reagent. On the basis of these observations, it seems that the sterols of the waxmoth haemolymph consist of both cholesterol and some other, ‘fast acting’, sterol. Paper chromatography of the total lipids revealed (Fig. 2) that in addition to phospholipids and sterols large amounts of triglycerides and appreciable amounts of diglycerides were always present in the haemolymph. However, the particular glycerides were not determined quantitatively. It has been found in another study (WLODAWER and BARA&SKA, unpublished) that the unesterified fatty acids (FFA) in the haemolymph represent about 8 per cent of the total lipids. Thus the composition of the lipids in the haemolymph appeared to be as follows: phospholipids, 22%; total sterols, 15%; FFA, 8%; tri- and diglycerides, 55% (obtained by subtraction of the sum of phospholipid, sterol, and FFA percentages from 100).
14
PAULINA WLODAWER AND ALICJA WI~NIEWSKA
Both thin-layer and paper chromatography revealed that the major components of the phospholipid fraction were lecithin and phosphatidylethanolamine, other constituents (as lysolecithin and a fast-moving phospholipid with an R., similar to that of cardiolipins) being present only in small amounts. No sphmgomyelin could be found. It is seen in Table 2 that lecithin represented approximately 58 per cent and phosphatidylethanolamine about 20 per cent of the total phospholipids. The amounts of the minor phospholipid constituents were too small to be determined by the method used and were not examined further. Determinations of lecithin and phosphatidylethanolamine in the blood of starved larvae indicated that no changes in the proportions of these two major phospholipids took place during prolonged starvation (Table 2). TABLE 2-THE
CONTENTS OF PHOSPHATIDYLCHOLINE AND PHOSPHATIDYLETHANOLAMINE IN THE PHOSPHOLIPIDS OF WAXMOTH BLOOD
Period of starvation
Phosphatidylcholine
Phosphatidylethanolamine
(days)
(%>
(%I
0
7
18 28
60.0’ 55.8 -58.0 58.0 56.0’ 59.2 57.0 52.0’ 56.0 54.0 56.6’ z57.3 58.0
It has previously been shown by LENARTOWICZ and NIEMIERKO (1964) that the percentage contribution of each particular organ to the body weight of waxmoth larvae was not markedly influenced by starvation. Thus, during the whole period of starvation (up to 4 weeks) haemolymph comprised about 28 per cent of the larval body. On the basis of this observation, the loss of blood lipids during starvation could be calculated and expressed as percentage of the initial amount present in the blood of the fed larvae. Fig. 1 shows that nearly half the initial, amount of both total lipids and phospholipids disappeared from the blood during 28 days of starvation. At the same time, the larvae had lost about 45 per cent of their body weight and therefore as much of haemolymph. The loss of lipids was, therefore, proportional to the loss of haemolymph and their concentration remained unchanged. As both the concentration and the composition of lipids in the haemolymph were found to be rather stable, it seemed appropriate to examine the behaviour of lipids in some other larval tissues. Analyses were therefore performed on the
LIPIDS IN THE HAEMOLYMPH
OF WAXMOTH LARVAE DURING STARVATION
60 %
0
7
I8
28
FIG. 1. Body weight and lipids in the haemolymph during starvation. Ordinate : per cent of the initial value. Abscissa: period of starvation in days. Black circlesbody weight; squares-total lipids; triangles-phospholipids.
0
00
006
a-FIG. 2. Tracing of a chromatogram of the haemolymph lipids. (A) Reference compounds: lecithin, dipahnitin, cholesterol, and tripalmitin. (B) Lipids in the haemolymph of fed larvae (20 pg). (C) L ipi ds in the haemolymph of 7 days fasted larvae (40 pg). (1) phospholipids, (2) diglycerides, (3) free cholesterol, (4) triglycerides (and cholesterol esters ?).
15
16
PAULINAWLODAWERANDALICJA WISNIEWSRA
tissues which remained after removal of the haemolymph, intestine, and the silk glands from the larvae. The so-called ‘residual larval body’ consisted chiefly of fat body, muscles, and integument. Lipids were extracted and analysed as TARLE 3-TOTAL
LIPIDS AND PHOSPHOLIPIDSIN THE ‘RESIDUALLARVALBODY’ (SEE TEXT) (MEANVALUESOF THREEEXPERIMENTS) Phospholipids
Period of starvation (days)
Total lipids in tissues (%)
In tissues (%)
In lipids (%)
0 7 18 28
38.0 26.4 20.0 19.0
1.9 0.9 0.7 0.7
5-O 3.4 3.5 3.7
(%)
Phosphatidylethanolamine in phospholipids (%)
60 62 57 59
21 22 21 22
Lecithin in phospholipids
L I o1
IO
FIG. 3. Body weight and lipids in the ‘residual larval body’ during starvation. Ordinate: per cent of the initial amount. Abscissa: period of starvation in days. Black circles-body weight; squares-total lipids; triangles-phospholipids.
described for the haemolymph. The results are presented in Table 3. A marked decrease in the total lipid content could be observed after the first 7 days of starvation, followed by a further decline during the subsequent 10 days, and by only a slight change in the remaining period. The content of lipids in the ‘residual
LIPIDS
IN
THE
HAEMOLYMPH
OF WAXMOTH
LARVAE
DURING
STARVATION
17
larval body’ fell from 38 per cent in the fed larvae to 26 per cent after 7 days and to 20 per cent after 18 days of starvation. The analysed tissues contained only a small amount of phospholipids, part of which disappeared during fasting. The phospholipid content decreased from l-9 per cent to O-9 per cent after 7 days fasting and to O-7 per cent after 18 days. It could be calculated (Fig. 3) that 46 per cent of total lipids and about 60 per cent of phospholipids disappeared from the ‘residual larval body’ during the first 7 days of starvation and 73 per cent and 80 per cent, respectively, during the whole period of 28 days. The composition of phospholipids in these tissues strongly resembled that in the haemolymph, lecithin being the main constituent, phosphatidylethanolamine comprising about 20 per cent of the total lipid phosphorus, and sphingomyelin being absent. DISCUSSION
Very little is known about the amount and composition of the lipids in insect blood. One per cent of lipids has been found in the haemolymph of Phormia larvae (HOPF, 1940), O-85 per cent in that of Deilephila euphorbiae pupae (HELLER,1932), 0.32 per cent in Pro&&a larva (BUCK, 1953), and 0.45 per cent in the blood of the bee larva (BISHOPet al., 1925). The lipid content in the haemolymph of waxmoth larvae (2.4 per cent) far exceeds the above values. However, in view of the highly insufficient data available, it would be unwise to attribute the high level of lipids in waxmoth blood to the unique way of feeding of larvae on the beecomb, which contains about 50 per cent of wax. It should be mentioned, nevertheless, that the fully grown waxmoth larvae are particularly rich in lipids, which comprise about 25 per cent of the body weight. As the lipids could be completely extracted from the haemolymph only if polar solvents like ethanol or methanol were used (unpublished data), there are good reasons to assume that they occur, partly at least, in waxmoth blood not in the free form but bound to some proteins as in the blood of vertebrates. Not only the total amount but also the composition of the lipids in waxmoth haemolymph differs markedly from the composition of lipids in vertebrate blood. Large amounts of neutral glycerides occur both in the fed and in the starved insects. The phospholipid content in the haemolymph was found to be twice as high as in human serum (PHILLIPS, 1958). According to PHILLIPS (1958) and ETIENNEand POLONOV~KI(1960), th e main phospholipid components of human serum are lecithin (more than 60 per cent of the total lipid phosphorus) and sphingomyelin (about 20 per cent), phosphatidylethanolamine and lysolecithin being present only in small amounts. There are only a few investigations concerned with insect phospholipids. HOPF (1940) found 2.5 mg phospholipids per 1 ml of haemolymph of CaZZiphoralarvae and l-5 mg/l ml in those of Phormia regina. The phospholipid fraction was not further fractionated. According to CHOJNACKI and KORZYBSKI (1962) the phospholipids of the moth, Arctia caja, contain 47 per cent lecithin, 24 per cent phosSmall amounts of phatidylethanolamine, and 10.7 per cent sphingomyelin.
18
PAULINA WLODAWER ANDALICJAWI~NIEWSKA
phosphatidylserine (2 per cent) and of inositolphosphatides (1.8 per cent) have also been found. BIEBERst al. (1961) investigated the phospholipid patterns in various developmental stages of the blowfly, Phormia regina, and found the major constituents to be similar in the egg, larva, and adult and to consist chiefly of ethanolamine- and choline-phospholipids (60 and 25 per cent respectively). Sphingomyelin was not detected. Similarly, according to CRONE and BRIDGES (1963), the major components of the phospholipid fraction of the housefly, Musca domestica, are phosphatidylethanolamine (65 per cent) and lecithin (17 per cent), and no sphingomyelin could be found. On the contrary, comparatively high amounts of lecithin were found by the same authors in two species of cockroach (Periplaneta americana, 44 per cent, and Blattella germanica, 53 per cent, of the total lipid phosphorus). As in the cockroach, but unlike the blowfly and housefly, the waxmoth phospholipids contain much more lecithin than phosphatidylethanolamine (58 and 20 per cent respectively), the common feature of the phospholipid patterns in the flies and waxmoth being the absence of sphingomyelin. The failure to detect any sphingomyelin in at least several species of insects would represent a notable departure from the phospholipid pattern in vertebrates. The relatively large amount of sterols found in the haemolymph of waxmoth larvae seems to be of interest, if one takes into account that the only source of lipids for these insects is the beeswax. Although, according to GIL~V~OUR (1961), no sterol has been found in insect wax, the lipids obtained by exhaustive extraction of beecomb contained compounds which gave a faint but distinct colour reactian with the ferrous sulphate reagent of SEAFEYet al. (1960). These substances were present in small but measurable amounts and comprised less than 3 per cent of the wax. They might be derived from plant pollen and bee exuviae and excreta present in the beecomb. In all probability these compounds differ from cholesterol and related sterols, as they do not produce any colour reaction with the modified Schoenheimer-Sperry reagent (MOORE and BAUMANN, 1952) nor with the ferric chloride reagent of ZAK et al. (1954). On the contrary, the sterols present in the waxmoth blood, when treated with the ferric chloride reagent, gave rise to the same coloured rings and produced the same final purple colour as did authentic cholesterol. The type of colour formation with the modified SchoenheimerSperry reagent clearly pointed to the presence of two different sterols, one being cholesterol (or a compound so closely related to it that it could not be distinguished from it by the methods used), the second-a ‘fast acting’ sterol of unknown nature, which is likely to be 7-dehydrocholesterol (CASIDA et al., 1957). Mixtures of ‘slow’ and ‘fast’ sterols have been found by some other authors in various insects. CASIDA et al. (1957) found cholesterol (‘slow acting’) and a second, ‘fast acting’, sterol generally distributed among the organ systems of Periplaneta americana. BECK and KAPAIXA (1957) were able to determine in the larvae of Tribolium confusum two sterols, one of which was chemically and chromatographically indistinguishable from cholesterol and one a ‘fast’ sterol indistinguishUnusual sterols different from cholesterol able from 7-dehydrocholesterol. were found by AGARWALet al. (1961) in houseflies.
LIPIDS IN THE HAEMOLYMPH OF WAXMOTHLARVAEDURINGSTARVATION
19
It seems to be well established that insects, unlike the higher animals, require a dietary source of sterols (LIPKE and FRAENKEL,1956; BECKand KAPADIA, 1957; CLARK and BLOCH, 1959) and, according to LIPKE and FRAENKEL(1956), ‘no exception has been found to this rule’. The nutritional requirement for sterols is indicative of an inability to synthesize the steroid nucleus. However, it has been demonstrated by numerous authors (FRAENKELand BLEWETT, 1943; BECK and KAPADIA, 1957; CASIDAet al., 1957; BERGMANNet al., 1959; CLARK and BLOCH, 1959a; AGARWALet al., 1961) that various insects are able to convert some dietary sterols including ‘phytosterols’ into typical ‘zoosterols’, like cholesterol or compounds related to it. It is therefore reasonable to suppose that Galleria larvae are able to utilize sterols present: in the beecomb and to transform them into the sterols of their own body. Determinations performed on starved waxmoth larvae indicated that prolonged starvation affected neither the relative lipid content nor the lipid composition of the haemolymph. Similarly, no changes in the proportions of the major phospholipid fractions could be observed. There is no evidence for selective utilization of any phospholipid component during starvation, either in the haemolymph or in the other tissues analysed. It seems reasonable to suppose that some mechanisms are active in the insect which may regulate the concentration of lipids in the haemolymph. Large amounts of lipids have been found to disappear from the ‘residual larval body’ during starvation of the larvae. These lipids may be derived chiefly from the fat body (which comprises the greatest part of the ‘residual larval body’), and either utilized directly by this tissue or released into the haemolymph and transported to other sites of utilization. The failure to find any significant increase in the concentration of lipids in the haemolymph during starvation indicates that the utilization of the lipids in the tissues may correspond closely to the rate of their release. Acknowledgement-The and helpful criticism.
authors wish to thank Professor W. NIEMIERKOfor his interest REFERENCES
AGARWALH. C., CASIDAJ. E., and BECK S. D. (1961) An unusual sterol from houseflies. J. Ins. Physiol. 7, 32-45. BARTLETTG. R. (1959) Phosphorus assay in column chromatography. 3. biol. Chem. 234, 466-468. BECK S. D. and KAPADIAG. G. (1957) Insect nutrition and metabolism of sterols. Science 126, 258-259. BERGMANNE. D. and LEVINSON2. H. (1954) Steroid requirements of housefly larvae. Nature, Land. 143, 211-212. BERGMANNE. D., RABINOVITZM., and LEVINSON2. H. (1959) The synthesis and biological availability of some lower homologs of cholesterol. J. Amer. Chem. Sot. 81, 1239-1243. BIEBER L. L., HODGSONE., CHELDELINV. H., BROOKESV. J., and NEWBLIRGHR. W. (1961) Phospholipid patterns in the blowfly, Phormia regina (Heigen). J. biol. Chem. 236, 2.590-259s. BISHOP G. H., BRIGGSA. P., and RONZONIE. (1925) Body fluids of the honey bee larva. II. Chemical constituents of the blood and their osmotic effects. 3’. biol. Chem. 66, 77-88.
20
PAULINA WLODAWERAND ALICJA WI~NIEWSKA
BUCK J. B. (1953) Physical properties and chemical composition of insect blood. In Insect Physiology (Ed. by ROEDERK. D.), p. 188. John Wiley, New York. CASIDA J. E., BECK S. D., and COLE M. J. (1957) Sterol metabolism in the American cockroach. 3. biol. Chem. 224, 365-371. CHOJNACKIT. and KORZYBSKIT. (1962) Biosynthesis of phospholipids in insects. The incorporation of (““P) orthophosphate into phospholipids of Arc& c&u moths. Acta biochim. Polon. 9, 95-110. CLARK A. J. and BLOCH K. (1959) The absence of sterol synthesis in insects. J. biol. Chem. 234, 2578-2582. CLARK A. J. and BLOCH K. (1959a) Conversion of ergosterol to 22-dehydrocholesterol in Blattella germanica. J. biol. Chem. 234, 2589-2594. CRONEH. D. and BRIDGESR. G. (1963) The phospholipids of the housefly, Musca domestica. Biochem. J. 89, 11-21. ETIENNE J. and POLONOVSKIJ. (1960) Evolution des lipides au tours de l’incubation du &urn-III. Etude des phospholipides. Bull. Sot. chim. Biol. 42, 857-866. FISKE C. H. and SUBBAROWK. (1925) The calorimetric determination of phosphorus. J. biol. Chem. 66, 375-400. FRAENKELG. and BLEWETTM. (1943) The sterol requirements of several insects. Bi0chem.J. 37, 692-695. GILMOUR D. (1961) Biochemistry of Insects, p. 212. Academic Press, New York. WELLERJ. (1932) Uber den Anteil der Hiimolymphe und StofIwechsel der Schmetterlingspuppen. Biochem. Z. 255,205-221. HOPF H. S. (1940) The physiological action of abnormally high temperatures on poikilothermic animals. 3. Some changes occurring in the phosphorus distribution of the haemolymph of insects under the influence of abnormally high temperature, Biochem. J. 34, 1396-1403. LENARTOWICZE. and NIEMIERKOS. (1964) Phosphorylethanolamine and phosphorylcholine in the haemolymph of larvae of Galleria mellonella L. during starvation. J. Ins. Physiol. 10, 831-837. LENARTOWICZE., RUDZISZB., and NIEMIERKO S. (1964) Distribution of nonhydrolysable phosphorus compounds in the body of Galleria mellonella L. larvae. J. Ins. Physiol. 10, 89-96. LIPKE H. and FRAENKELG. (1956) Insect nutrition. Annu. Rev. Ent. 1, 17-44. MARINETTI G. V. (1962) Chromatographic separation, identification, and analysis of phosphatides. J. Lipid Res. 3, I-20. MOORE P. R. and BAUMANN C. A. (1952) Skin sterols. 1. Calorimetric determination of cholesterol and other sterols in skin. J. biol. Chem. 195, 615-621. NIEMIERKO W. and WOJTCZAK L. (1950) Oxygen consumption by the waxmoth larvae during starvation. Acta biol. Exp. 15, 79-90. PHILLIPS G. B. (1958) The isolation and quantitation of the principal phospholipid components of human serum using chromatography on silicic acid. Biochim. biophys. Acta 29, 59+602. SEARCYR. L., BERGQUISTL. M. and JUNG R. C. (1960) Rapid ultramicro estimation of serum total cholesterol. J. Lipid Res. 1, 349-351. WAGNER H., HOERNAMMERL., and WOLF P. (1961) D iinnschichtchromatographie von Phosphatiden und Glykolipiden. Biochem. Z. 334, 175-184. ZAK. B., Moss N., BOYLE A. J., and ZLATKIS A. (1954) Reactions of certain unsaturated steroids with acid iron reagent. Anal. Chem. 26, 776-777.
Printed in Great Britain
LIPIDS IN THE HAEMOLYMPH OF WAXMOTH DURING STARVATION PAULINA Department
WLODAWER
of Biochemistry,
Nencki
and ALICJA
Institute
(Received
WISNIEWSKA
of Experimental
11 May
LARVAE
Biology,
Warsaw,
Poland
1964)
Abstract-The
composition of lipids in the haemolymph of waxmoth larvae was Total lipids which represented investigated during feeding and starvation. about 2.4 per cent of the haemolymph contained about 22 per cent phospholipids, 15 per cent sterols (including cholesterol and a ‘fast acting’ sterol of unknown nature), 8 per cent unesterified fatty acids, and a high proportion of neutral glycerides. The main components of the phospholipid fraction appeared to be phosphatidylcholine, which comprised about 60 per cent of the total lipid phosphorus, and phosphatidylethanolamine (about 20 per cent). Sphingomyelin was not detected. Neither the concentration nor the composition of the lipids in the haemolymph was found to be markedly influenced by starvation; in particular, the phospholipid content was remarkably stable. No change in the proportions of choline and ethanolamine containing phospholipids could be observed. The loss of the lipid constituents from the haemolymph during starvation roughly However, paralleled the loss of body weight and of the amount of haemolymph. the relative loss of total lipids and phospholipids in the ‘residual larval body’, which consisted chiefly of the fat body, muscles, and integument, greatly exceeded the relative loss of body weight. Part of these lipids might contribute in maintaining the stable lipid level in the haemolymph.
INTRODUCTION
IT
HAS been shown recently in this laboratory (LENARTOWICZ and NIEMIERKO, 1964) that the contents of some organic phosphorus esters present in the haemolymph of waxmoth larvae undergo considerable changes during prolonged starvation. This is particularly true as regards phosphorylcholine and phosphorylethanolamine. During starvation of the larvae phosphorylethanolamine disappears from the haemolymph to a much greater extent than phosphorylcholine. Consequently, the phosphorylcholine to phosphorylethanolamine ratio rises from about O-7 in the fed larvae to approximately 3 in the 4 weeks starved insects. The reasons for the marked differences in the behaviour between the two phosphorus esters are poorly understood as yet. As both phosphorylcholine and phosphorylethanolamine are components of corresponding phospholipid molecules, the possibility was not to be excluded that these results might reflect some differences in the utilization of phosphatidylcholine (lecithin) and phosphatidylethanolamine, respectively, by the starved larvae. 11
12
PAULINAWLODAWER ANDALICJAWISNIEWSKA
The purpose of the present study was to follow the fate of lecithin and phosphatidylethanolamine in the haemolymph of waxmoth larvae during starvation and to gain information on the other lipid fractions of insect blood during feeding and fasting. MATERIAL
AND METHODS
Fully grown waxmoth larvae (Galleria mellonella L.) weighing 170-200 mg each were investigated. The insects were bred on beecomb and kept at constant temperature of 30°C. The larvae to be starved were ligated behind the head in order to prevent metamorphosis (NIEMIERKO and WOJTCZAK, 1950). Haemolymph was collected as described by LENARTOWICZet al. (1964). From the haemolymph pooled from about 100 larvae, lipids were extracted with 25 vol of chloroformmethanol (2 : 1 v/v), the solvent was evaporated under reduced pressure and the residue was re-extracted with petroleum ether-chloroform (2 : 1 v/v). The extract was taken to dryness and the amount of total lipids was determined gravimetrically. Sterols were determined by the method of SEARCY et al. (1960). Lipid phosphorus was estimated according to FISKE and SUBBAROW (1925) in samples digested with nitric acid, and the amount of phospholipids was calculated by multiplying the phosphorus value by 25. The composition of the phospholipid fraction was checked qualitatively by thin-layer chromatography (WAGNER et al., 1961), and the percentage contribution of the main components was evaluated quantitatively by chromatography on silicic acid impregnated filter paper, according to the method of MARINETTI (1962). The chromatograms were run in diisobutylketone-acetic acid-water (40 : 25 : 5) ; phospholipids were detected by staining with rhodamine 6G and viewed in ultraviolet light. Spots corresponding to individual phospholipids were excised and eluted with 1 N HCl in methanol. The eluates were evaporated, digested in 60% perchloric acid, and phosphorus was determined according to BARTLETT (1959). Chromatography of total lipids was performed on Whatman No. 3 filter paper impregnated with silicic acid, according to DOBIAQOVAand MICHALEC (private communication). The developing solvent was 10% ethyl ether in petroleum ether. Lipid spots were detected with rhodamine 6G or rhodamine B and viewed in U.V. while wet. RESULTS The concentrations of total lipids, sterols, and phospholipids in the haemolymph of the fed and of the starved larvae are shown in Table 1. As may be seen, total lipids represented about 2.4 per cent of the larval blood, and this concentration did not change markedly during 28 days of starvation. The differences found in various periods of starvation do not seem to be statistically significant. Phospholipid fraction comprised 0.52 per cent of the haemolymph and practically no changes in concentration were observed during the whole period of starvation. Total sterols made up 0.35 per cent in the blood of the fed larvae and 0.27 per cent in those starved for 7 days. Further starvation did not affect the sterol concentration significantly.
LIPIDS IN THE
HABMOLYMPH
OFWAXMOTH
LARVAE
DURING
13
STARVATION
An attempt was made to examine the nature of the sterols. As mentioned in Methods, sterols were determined calorimetrically by the method of SEARCY et al. (1960), and the determinations were referred to a cholesterol standard curve. The colour produced by the haemolymph sterols was the same as given by authentic cholesterol. On paper chromatograms the haemolymph sterols gave one spot closely corresponding to cholesterol used as reference compound. With the ferric chloride reagent of ZAK et al. (1954), wh’rch may serve to differentiate between TABLE~-LIPIDSINTHEHAEMOLYMPHOFWAXMOTHLARVAEDURINGFEEDINGANDSTARVATION (MEAN
Period of starvation (days) 0 7 18 28
VALUES
Total lipids in the haemolymph (%) 2.4 2.0 2.2 2.5
+ + f +
0.3 (5) 0.2 (5) 0.1 (4) 0.07 (5)
AND
STANDARD
DEVIATION
Sterols (expressed as cholesterol*)
Phospholipids In haemolymph (%) 0.52 0.53 0.51 0.53
& 0.02 kO.02 f0.02 + 0.03
(5) (5) (3) (4)
OF THE MEAN)
In lipids (%)
In haemolymph (%)
In lipids (%)
22 27 23 21
0.35 + O-03 (6) 0.2710.03 (5) 0.28 & 0.03 (6) 0.25 k 0.02 (5)
15 14 13 10
The figures in parentheses indicate the number of determinations. * Determinations referred to a cholesterol standard curve.
various sterols, the same characteristic coloured rings and the same final purple colour as given by authentic cholesterol were observed. With the modified Schoenheimer-Sperry reagent (MOORE and BAUMANN, 1952) the haemolymph sterols developed the blue-green colour much faster than authentic cholesterol; but instead of fading after several minutes, like that of the typical ‘fast acting’ sterols, the colour due to the haemolymph sterols markedly increased with time. According to MOORE and BAUMANN (1952), this type of colour reaction is the same as that observed when suitable mixtures of cholesterol and a ‘fast acting’ sterol (like 7-dehydrocholesterol) were treated with the reagent. On the basis of these observations, it seems that the sterols of the waxmoth haemolymph consist of both cholesterol and some other, ‘fast acting’, sterol. Paper chromatography of the total lipids revealed (Fig. 2) that in addition to phospholipids and sterols large amounts of triglycerides and appreciable amounts of diglycerides were always present in the haemolymph. However, the particular glycerides were not determined quantitatively. It has been found in another study (WLODAWER and BARA&SKA, unpublished) that the unesterified fatty acids (FFA) in the haemolymph represent about 8 per cent of the total lipids. Thus the composition of the lipids in the haemolymph appeared to be as follows: phospholipids, 22%; total sterols, 15%; FFA, 8%; tri- and diglycerides, 55% (obtained by subtraction of the sum of phospholipid, sterol, and FFA percentages from 100).
14
PAULINA WLODAWER AND ALICJA WI~NIEWSKA
Both thin-layer and paper chromatography revealed that the major components of the phospholipid fraction were lecithin and phosphatidylethanolamine, other constituents (as lysolecithin and a fast-moving phospholipid with an R., similar to that of cardiolipins) being present only in small amounts. No sphmgomyelin could be found. It is seen in Table 2 that lecithin represented approximately 58 per cent and phosphatidylethanolamine about 20 per cent of the total phospholipids. The amounts of the minor phospholipid constituents were too small to be determined by the method used and were not examined further. Determinations of lecithin and phosphatidylethanolamine in the blood of starved larvae indicated that no changes in the proportions of these two major phospholipids took place during prolonged starvation (Table 2). TABLE 2-THE
CONTENTS OF PHOSPHATIDYLCHOLINE AND PHOSPHATIDYLETHANOLAMINE IN THE PHOSPHOLIPIDS OF WAXMOTH BLOOD
Period of starvation
Phosphatidylcholine
Phosphatidylethanolamine
(days)
(%>
(%I
0
7
18 28
60.0’ 55.8 -58.0 58.0 56.0’ 59.2 57.0 52.0’ 56.0 54.0 56.6’ z57.3 58.0
It has previously been shown by LENARTOWICZ and NIEMIERKO (1964) that the percentage contribution of each particular organ to the body weight of waxmoth larvae was not markedly influenced by starvation. Thus, during the whole period of starvation (up to 4 weeks) haemolymph comprised about 28 per cent of the larval body. On the basis of this observation, the loss of blood lipids during starvation could be calculated and expressed as percentage of the initial amount present in the blood of the fed larvae. Fig. 1 shows that nearly half the initial, amount of both total lipids and phospholipids disappeared from the blood during 28 days of starvation. At the same time, the larvae had lost about 45 per cent of their body weight and therefore as much of haemolymph. The loss of lipids was, therefore, proportional to the loss of haemolymph and their concentration remained unchanged. As both the concentration and the composition of lipids in the haemolymph were found to be rather stable, it seemed appropriate to examine the behaviour of lipids in some other larval tissues. Analyses were therefore performed on the
LIPIDS IN THE HAEMOLYMPH
OF WAXMOTH LARVAE DURING STARVATION
60 %
0
7
I8
28
FIG. 1. Body weight and lipids in the haemolymph during starvation. Ordinate : per cent of the initial value. Abscissa: period of starvation in days. Black circlesbody weight; squares-total lipids; triangles-phospholipids.
0
00
006
a-FIG. 2. Tracing of a chromatogram of the haemolymph lipids. (A) Reference compounds: lecithin, dipahnitin, cholesterol, and tripalmitin. (B) Lipids in the haemolymph of fed larvae (20 pg). (C) L ipi ds in the haemolymph of 7 days fasted larvae (40 pg). (1) phospholipids, (2) diglycerides, (3) free cholesterol, (4) triglycerides (and cholesterol esters ?).
15
16
PAULINAWLODAWERANDALICJA WISNIEWSRA
tissues which remained after removal of the haemolymph, intestine, and the silk glands from the larvae. The so-called ‘residual larval body’ consisted chiefly of fat body, muscles, and integument. Lipids were extracted and analysed as TARLE 3-TOTAL
LIPIDS AND PHOSPHOLIPIDSIN THE ‘RESIDUALLARVALBODY’ (SEE TEXT) (MEANVALUESOF THREEEXPERIMENTS) Phospholipids
Period of starvation (days)
Total lipids in tissues (%)
In tissues (%)
In lipids (%)
0 7 18 28
38.0 26.4 20.0 19.0
1.9 0.9 0.7 0.7
5-O 3.4 3.5 3.7
(%)
Phosphatidylethanolamine in phospholipids (%)
60 62 57 59
21 22 21 22
Lecithin in phospholipids
L I o1
IO
FIG. 3. Body weight and lipids in the ‘residual larval body’ during starvation. Ordinate: per cent of the initial amount. Abscissa: period of starvation in days. Black circles-body weight; squares-total lipids; triangles-phospholipids.
described for the haemolymph. The results are presented in Table 3. A marked decrease in the total lipid content could be observed after the first 7 days of starvation, followed by a further decline during the subsequent 10 days, and by only a slight change in the remaining period. The content of lipids in the ‘residual
LIPIDS
IN
THE
HAEMOLYMPH
OF WAXMOTH
LARVAE
DURING
STARVATION
17
larval body’ fell from 38 per cent in the fed larvae to 26 per cent after 7 days and to 20 per cent after 18 days of starvation. The analysed tissues contained only a small amount of phospholipids, part of which disappeared during fasting. The phospholipid content decreased from l-9 per cent to O-9 per cent after 7 days fasting and to O-7 per cent after 18 days. It could be calculated (Fig. 3) that 46 per cent of total lipids and about 60 per cent of phospholipids disappeared from the ‘residual larval body’ during the first 7 days of starvation and 73 per cent and 80 per cent, respectively, during the whole period of 28 days. The composition of phospholipids in these tissues strongly resembled that in the haemolymph, lecithin being the main constituent, phosphatidylethanolamine comprising about 20 per cent of the total lipid phosphorus, and sphingomyelin being absent. DISCUSSION
Very little is known about the amount and composition of the lipids in insect blood. One per cent of lipids has been found in the haemolymph of Phormia larvae (HOPF, 1940), O-85 per cent in that of Deilephila euphorbiae pupae (HELLER,1932), 0.32 per cent in Pro&&a larva (BUCK, 1953), and 0.45 per cent in the blood of the bee larva (BISHOPet al., 1925). The lipid content in the haemolymph of waxmoth larvae (2.4 per cent) far exceeds the above values. However, in view of the highly insufficient data available, it would be unwise to attribute the high level of lipids in waxmoth blood to the unique way of feeding of larvae on the beecomb, which contains about 50 per cent of wax. It should be mentioned, nevertheless, that the fully grown waxmoth larvae are particularly rich in lipids, which comprise about 25 per cent of the body weight. As the lipids could be completely extracted from the haemolymph only if polar solvents like ethanol or methanol were used (unpublished data), there are good reasons to assume that they occur, partly at least, in waxmoth blood not in the free form but bound to some proteins as in the blood of vertebrates. Not only the total amount but also the composition of the lipids in waxmoth haemolymph differs markedly from the composition of lipids in vertebrate blood. Large amounts of neutral glycerides occur both in the fed and in the starved insects. The phospholipid content in the haemolymph was found to be twice as high as in human serum (PHILLIPS, 1958). According to PHILLIPS (1958) and ETIENNEand POLONOV~KI(1960), th e main phospholipid components of human serum are lecithin (more than 60 per cent of the total lipid phosphorus) and sphingomyelin (about 20 per cent), phosphatidylethanolamine and lysolecithin being present only in small amounts. There are only a few investigations concerned with insect phospholipids. HOPF (1940) found 2.5 mg phospholipids per 1 ml of haemolymph of CaZZiphoralarvae and l-5 mg/l ml in those of Phormia regina. The phospholipid fraction was not further fractionated. According to CHOJNACKI and KORZYBSKI (1962) the phospholipids of the moth, Arctia caja, contain 47 per cent lecithin, 24 per cent phosSmall amounts of phatidylethanolamine, and 10.7 per cent sphingomyelin.
18
PAULINA WLODAWER ANDALICJAWI~NIEWSKA
phosphatidylserine (2 per cent) and of inositolphosphatides (1.8 per cent) have also been found. BIEBERst al. (1961) investigated the phospholipid patterns in various developmental stages of the blowfly, Phormia regina, and found the major constituents to be similar in the egg, larva, and adult and to consist chiefly of ethanolamine- and choline-phospholipids (60 and 25 per cent respectively). Sphingomyelin was not detected. Similarly, according to CRONE and BRIDGES (1963), the major components of the phospholipid fraction of the housefly, Musca domestica, are phosphatidylethanolamine (65 per cent) and lecithin (17 per cent), and no sphingomyelin could be found. On the contrary, comparatively high amounts of lecithin were found by the same authors in two species of cockroach (Periplaneta americana, 44 per cent, and Blattella germanica, 53 per cent, of the total lipid phosphorus). As in the cockroach, but unlike the blowfly and housefly, the waxmoth phospholipids contain much more lecithin than phosphatidylethanolamine (58 and 20 per cent respectively), the common feature of the phospholipid patterns in the flies and waxmoth being the absence of sphingomyelin. The failure to detect any sphingomyelin in at least several species of insects would represent a notable departure from the phospholipid pattern in vertebrates. The relatively large amount of sterols found in the haemolymph of waxmoth larvae seems to be of interest, if one takes into account that the only source of lipids for these insects is the beeswax. Although, according to GIL~V~OUR (1961), no sterol has been found in insect wax, the lipids obtained by exhaustive extraction of beecomb contained compounds which gave a faint but distinct colour reactian with the ferrous sulphate reagent of SEAFEYet al. (1960). These substances were present in small but measurable amounts and comprised less than 3 per cent of the wax. They might be derived from plant pollen and bee exuviae and excreta present in the beecomb. In all probability these compounds differ from cholesterol and related sterols, as they do not produce any colour reaction with the modified Schoenheimer-Sperry reagent (MOORE and BAUMANN, 1952) nor with the ferric chloride reagent of ZAK et al. (1954). On the contrary, the sterols present in the waxmoth blood, when treated with the ferric chloride reagent, gave rise to the same coloured rings and produced the same final purple colour as did authentic cholesterol. The type of colour formation with the modified SchoenheimerSperry reagent clearly pointed to the presence of two different sterols, one being cholesterol (or a compound so closely related to it that it could not be distinguished from it by the methods used), the second-a ‘fast acting’ sterol of unknown nature, which is likely to be 7-dehydrocholesterol (CASIDA et al., 1957). Mixtures of ‘slow’ and ‘fast’ sterols have been found by some other authors in various insects. CASIDA et al. (1957) found cholesterol (‘slow acting’) and a second, ‘fast acting’, sterol generally distributed among the organ systems of Periplaneta americana. BECK and KAPAIXA (1957) were able to determine in the larvae of Tribolium confusum two sterols, one of which was chemically and chromatographically indistinguishable from cholesterol and one a ‘fast’ sterol indistinguishUnusual sterols different from cholesterol able from 7-dehydrocholesterol. were found by AGARWALet al. (1961) in houseflies.
LIPIDS IN THE HAEMOLYMPH OF WAXMOTHLARVAEDURINGSTARVATION
19
It seems to be well established that insects, unlike the higher animals, require a dietary source of sterols (LIPKE and FRAENKEL,1956; BECKand KAPADIA, 1957; CLARK and BLOCH, 1959) and, according to LIPKE and FRAENKEL(1956), ‘no exception has been found to this rule’. The nutritional requirement for sterols is indicative of an inability to synthesize the steroid nucleus. However, it has been demonstrated by numerous authors (FRAENKELand BLEWETT, 1943; BECK and KAPADIA, 1957; CASIDAet al., 1957; BERGMANNet al., 1959; CLARK and BLOCH, 1959a; AGARWALet al., 1961) that various insects are able to convert some dietary sterols including ‘phytosterols’ into typical ‘zoosterols’, like cholesterol or compounds related to it. It is therefore reasonable to suppose that Galleria larvae are able to utilize sterols present: in the beecomb and to transform them into the sterols of their own body. Determinations performed on starved waxmoth larvae indicated that prolonged starvation affected neither the relative lipid content nor the lipid composition of the haemolymph. Similarly, no changes in the proportions of the major phospholipid fractions could be observed. There is no evidence for selective utilization of any phospholipid component during starvation, either in the haemolymph or in the other tissues analysed. It seems reasonable to suppose that some mechanisms are active in the insect which may regulate the concentration of lipids in the haemolymph. Large amounts of lipids have been found to disappear from the ‘residual larval body’ during starvation of the larvae. These lipids may be derived chiefly from the fat body (which comprises the greatest part of the ‘residual larval body’), and either utilized directly by this tissue or released into the haemolymph and transported to other sites of utilization. The failure to find any significant increase in the concentration of lipids in the haemolymph during starvation indicates that the utilization of the lipids in the tissues may correspond closely to the rate of their release. Acknowledgement-The and helpful criticism.
authors wish to thank Professor W. NIEMIERKOfor his interest REFERENCES
AGARWALH. C., CASIDAJ. E., and BECK S. D. (1961) An unusual sterol from houseflies. J. Ins. Physiol. 7, 32-45. BARTLETTG. R. (1959) Phosphorus assay in column chromatography. 3. biol. Chem. 234, 466-468. BECK S. D. and KAPADIAG. G. (1957) Insect nutrition and metabolism of sterols. Science 126, 258-259. BERGMANNE. D. and LEVINSON2. H. (1954) Steroid requirements of housefly larvae. Nature, Land. 143, 211-212. BERGMANNE. D., RABINOVITZM., and LEVINSON2. H. (1959) The synthesis and biological availability of some lower homologs of cholesterol. J. Amer. Chem. Sot. 81, 1239-1243. BIEBER L. L., HODGSONE., CHELDELINV. H., BROOKESV. J., and NEWBLIRGHR. W. (1961) Phospholipid patterns in the blowfly, Phormia regina (Heigen). J. biol. Chem. 236, 2.590-259s. BISHOP G. H., BRIGGSA. P., and RONZONIE. (1925) Body fluids of the honey bee larva. II. Chemical constituents of the blood and their osmotic effects. 3’. biol. Chem. 66, 77-88.
20
PAULINA WLODAWERAND ALICJA WI~NIEWSKA
BUCK J. B. (1953) Physical properties and chemical composition of insect blood. In Insect Physiology (Ed. by ROEDERK. D.), p. 188. John Wiley, New York. CASIDA J. E., BECK S. D., and COLE M. J. (1957) Sterol metabolism in the American cockroach. 3. biol. Chem. 224, 365-371. CHOJNACKIT. and KORZYBSKIT. (1962) Biosynthesis of phospholipids in insects. The incorporation of (““P) orthophosphate into phospholipids of Arc& c&u moths. Acta biochim. Polon. 9, 95-110. CLARK A. J. and BLOCH K. (1959) The absence of sterol synthesis in insects. J. biol. Chem. 234, 2578-2582. CLARK A. J. and BLOCH K. (1959a) Conversion of ergosterol to 22-dehydrocholesterol in Blattella germanica. J. biol. Chem. 234, 2589-2594. CRONEH. D. and BRIDGESR. G. (1963) The phospholipids of the housefly, Musca domestica. Biochem. J. 89, 11-21. ETIENNE J. and POLONOVSKIJ. (1960) Evolution des lipides au tours de l’incubation du &urn-III. Etude des phospholipides. Bull. Sot. chim. Biol. 42, 857-866. FISKE C. H. and SUBBAROWK. (1925) The calorimetric determination of phosphorus. J. biol. Chem. 66, 375-400. FRAENKELG. and BLEWETTM. (1943) The sterol requirements of several insects. Bi0chem.J. 37, 692-695. GILMOUR D. (1961) Biochemistry of Insects, p. 212. Academic Press, New York. WELLERJ. (1932) Uber den Anteil der Hiimolymphe und StofIwechsel der Schmetterlingspuppen. Biochem. Z. 255,205-221. HOPF H. S. (1940) The physiological action of abnormally high temperatures on poikilothermic animals. 3. Some changes occurring in the phosphorus distribution of the haemolymph of insects under the influence of abnormally high temperature, Biochem. J. 34, 1396-1403. LENARTOWICZE. and NIEMIERKOS. (1964) Phosphorylethanolamine and phosphorylcholine in the haemolymph of larvae of Galleria mellonella L. during starvation. J. Ins. Physiol. 10, 831-837. LENARTOWICZE., RUDZISZB., and NIEMIERKO S. (1964) Distribution of nonhydrolysable phosphorus compounds in the body of Galleria mellonella L. larvae. J. Ins. Physiol. 10, 89-96. LIPKE H. and FRAENKELG. (1956) Insect nutrition. Annu. Rev. Ent. 1, 17-44. MARINETTI G. V. (1962) Chromatographic separation, identification, and analysis of phosphatides. J. Lipid Res. 3, I-20. MOORE P. R. and BAUMANN C. A. (1952) Skin sterols. 1. Calorimetric determination of cholesterol and other sterols in skin. J. biol. Chem. 195, 615-621. NIEMIERKO W. and WOJTCZAK L. (1950) Oxygen consumption by the waxmoth larvae during starvation. Acta biol. Exp. 15, 79-90. PHILLIPS G. B. (1958) The isolation and quantitation of the principal phospholipid components of human serum using chromatography on silicic acid. Biochim. biophys. Acta 29, 59+602. SEARCYR. L., BERGQUISTL. M. and JUNG R. C. (1960) Rapid ultramicro estimation of serum total cholesterol. J. Lipid Res. 1, 349-351. WAGNER H., HOERNAMMERL., and WOLF P. (1961) D iinnschichtchromatographie von Phosphatiden und Glykolipiden. Biochem. Z. 334, 175-184. ZAK. B., Moss N., BOYLE A. J., and ZLATKIS A. (1954) Reactions of certain unsaturated steroids with acid iron reagent. Anal. Chem. 26, 776-777.