Neurochemical Research, Vol. 10, No. 11, 1985, pp. 1499-1509

CHANGES IN A X O N A L TRANSPORT OF PHOSPHOLIPIDS IN THE REGENERATING GOLDFISH OPTIC SYSTEM* M. SBASCHNIG-AGLER 1, R. W. LEDEEN 1, R. M. ALPE~T 2, AND B.

GRAFSTEIN 2

~Departments of Neurology and Biochemistry Albert Einstein College of Medicine, Bronx and 2Department of Physiology Cornell University Medical College New York

Accepted April 10, 1985

Changes in axonally transported phospholipids of regenerating goldfish optic nerve were studied by intraocular injection of [2-3H]glycerol 9 days and 16 days after nerve crush at 30~ The four major glycerophospholipids all showed substantial increases in transported radioactivity above non-regenerating controls at both time points, these being maximal (15- to 35-fold) in the optic nerve-tract at 9 days and about half as great at 16 days. In the contralateral optic tectum transported label increased 6- to 13-fold at 9 days and 10- to 25-fold at 16 days in the various glycerophospholipids. While all glycerophospholipids showed absolute increases in both tissues, PS and PI increased relatively more, especially in the tectum. The regeneration-associated increases in transported label of all glycerophospholipids were larger than those previously demonstrated for gangliosides and glycoproteins in the same system.

INTRODUCTION Regeneration of the goldfish optic nerve is accompanied by dramatic changes in the metabolism of the retinal ganglion cells, including large * Special Issue dedicated to Dr. Eugene Kreps. Address correspondence to: Dr. R. W. Ledeen, Albert Einstein College of Medicine, Neurology Dept., F-140, 1300 Morris Park Avenue, Bronx, NY 10461.

1499 0364-3190/85/1100-1499504.50/0 9 1985 Plenum Publishing Corporation

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SBASCHNIG-AGLER ET AL.

increases in the synthesis and axonal transport of proteins, glycoproteins and lipids (1-4). We have recently shown (4) that there is a particularly marked increase in axonal transport of gangliosides: radiolabeling of axonally-transported gangliosides in regenerating nerves was found to increase 8-fold over non-regenerating control nerves, approximately double the increase seen for sialoglycoproteins. The same study also revealed a significant increase in transport radiolabeling of membrane glycerophospholipids, although individual components of this group were not examined. In the present study we have focused on the four major glycerophospholipids of nervous tissue and the changes in transport labeling which they undergo during optic nerve regeneration. EXPERIMENTAL PROCEDURE The source and preliminary treatment of the goldfish, including acclimation to 30~ were the same as previously described (4). At either 9 days or 16 days after crushing the right optic nerve, 25.4 ~Ci of [2-3H]glycerol (2 Ci/mmol; New England Nuclear, Boston, MA) in 4 microliters of water was injected into the vitreous humor of the right eye. The same amount was injected into the right eye in a corresponding group of control (unoperated) fish. Forty eight hours later 12-14 fish per group were sacrificed by decapitation and the optic nerves and optic tecta were dissected, pooled and frozen. The nerve segments were 3-5 mm in length and included a portion beyond the chiasm (optic tract); a 1 mm segment from immediately behind the eye was discarded to eliminate labeling through extra-axonal diffusion. Corresponding samples from the uninjected pathway (left nerve-tract, right tectum) were also collected. Each of the pooled tissue samples was placed in a small glass tissue grinder and homogenized with 1 ml chloroform (C)--methanol (M) 2:1; a little BHT was added as antioxidant. The mixture was centrifuged at 1100 g for 15 min, the supernatant removed to a graduated tube and the residue rehomogenized and centrifuged as above. The combined supernatants were mixed well with 0.2 vol physiological saline and the resulting upper phase removed. The lower phase was re-extracted twice with saline--M 1 : 1, once with M - - w a t e r (W) 1 : 1 followed by rinsing away of residual upper phase with a little of the latter solvent. These washings effectively removed all water-soluble radioactivity. Enough M was then added to the lower phase to make it homogeneous and aliquots were taken for counting and phosphorus determination (5, 6). For TLC, aliquots containing 5-12 tag phosphorus and 700-15,000 DPM were applied to silica gel 60 plates (Merck, VWR Scientific Inc, South Plainfield, NJ 07080). Duplicate plates were developed in two solvent systems: (a) C-M-acetic acid-formic acid-W 35:15:6:2:1 (7); (b) C-M-acetic acid- 10% aq. NaHSO3 50:20:6: 1.5 (8). System (a) had some overlap between PC and PI while system (b) had overlap between PS and PI (Figure 1). P1 was calculated by difference based on clear separation of PS in system (a). PE and sulfatide overlapped in both systems but the latter would not be labeled by [2-3H]glycerol. Bands were visualized with I2, scraped into scintillation vials and sonicated with 3 ml W. This was shaken well with 10 ml of hydrofluor scintillation medium (National Diagnostics, Somerville, NJ 08876) to form a gel and counted with a Packard Tri-Carb 300 Liquid Scintillation System. Protein levels in the delipidated residues were determined by solubilizing in 2 ml of 1 N NaOH for 2-3 days followed by application of the Lees and Paxman procedure (9).

AXONAL TRANSPORT OF PHOSPHOLIPIDS

A

1501

B

FtG 1. Thin-layer chromatograms of lipids separated by two systems: (B) C-M-acetic acidformic acid-W 35:15:6:2:1 as described in (7).; (A) C-M-Acetic acid-10% aq. NaHSO3 50 :20 :6: 1:5 as described in (8). Lane 1, standards; 2, optic nerve-tract lipids; 3, optic tectum lipids. The cerebroside standard was synthetic glucosylceramide. Abbreviations, in addition to those given: Chol, cholesterol; Cer, cerebrosides; Sulf, sulfatides; SM, sphingomyelin. Chromatograms were developed at 4~ and sprayed with cupric acetate-phosphoric acid (33). Note overlap of PI and PS in system (A) and PI and PC in (B). PE and Sulf overlap in both systems but this did not pose a problem because of the few counts in Sulf.

Transported radioactivities were calculated as the difference between specific radioactivities of the substance or mixture isolated from the injected and uninjected pathways. This is the standard means of correcting for background labeling that results from precursor leaking into the blood stream and entering the tissue via the circulation (10).

RESULTS N i n e d a y s after n e r v e c r u s h , w h e n the r e g e n e r a t i n g optic a x o n s are b e g i n n i n g to e n t e r the optic t e c t u m , t r a n s p o r t e d r a d i o a c t i v i t y of total lipid f r o m the r e g e n e r a t i n g optic n e r v e - t r a c t was e l e v a t e d a p p r o x i m a t e l y 20fold a b o v e that in the c o r r e s p o n d i n g s e g m e n t s of c o n t r o l n e r v e s ( F i g u r e 2). B y 16 d a y s , w h e n the a x o n s w o u l d h a v e i n v a d e d the e n t i r e r e c t u m , the a m o u n t o f t r a n s p o r t e d label in the n e r v e - t r a c t was still 6 - 7 fold a b o v e

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SBASCHNIG-AGLER ET AL.

TOTAL LIPIDS (Mainly Glyce rophospholipids)

100

~t I

o X r

NERVE (R-L)

~

o 0')

E

5C

IX.

t~

TECTUM (L-R)

1G

nC

i

9 16 C DAYS POST CRUSH

9

16

FIG. 2. Transported radioactivity of total lipids labeled with [2-3H]Glycerol (mainly glycerophospholipids) in regenerating goldfish optic system. Precursor was injected into the right eyes of several goldfish and the tissues collected 48 hr later (fast transport). Lipids were extracted with C-M, Folch-partitioned, and the washed lower phase counted. Transported radioactivity was calculated as the difference in DPM between total lipids from the injected and uninjected pathways. Error bars represent SEM. Each experiment involved pooling of 12-14 fish; the number of separate experiments were 3 for control and 2 for the others.

AXONAL TRANSPORT OF PHOSPHOLIPIDS

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TABLE I CHANGES lN RADIOLABELINGOF AXONALLYTRANSPORTEDGLYCEROPHOSPHOLIPIDS RELATIVETO CONTROLSFOLLOWINGOPTIC NERVE CRUSH Regeneration/Control Optic Nerve-Tract Optic Tectum

Total Lipids PC PE PS PI

9d

16d

9d

16d

21 27 15 34 16

6.6 5.5 4.1 8.0 7.2

8.2 6.1 6.0 14 14

11 12 11 21 24

Ratios were calculated for transported radioactivities (per mg protein) in comparable samples isolated from regenerating and control tissues. Absolute values can be obtained from Figures 2 and 3. n = 3 for control and 2 for regenerating tissues.

control. These results are roughly comparable to those obtained previously (4) with [~4C]glycerol as precursor although less background labeling occurs with [2-3H]glycerol (see below). In both studies the bulk of radioactivity resided in glycerophospholipids due to limited scrambling or reutilization of label in the nerve-tract (11, 12). Radioactivity transported to the optic tectum at 9 days in the regenerating animals was 8-fold higher than in the control animals, and at 16 days it was l 1-fold higher than controls (Figure 2). The increase during this interval, observed both in this study and a previous study using [14C]glycerol (4), in contrast to the decrease seen in the same period in the regenerating nerve-tract, presumably reflects the increasing volume of regenerating axons occupying the tectum. The changes for total lipid represented the sum of changes in the four individual glycerophospholipids, which behaved somewhat differently from one another (Table I and Figure 3). In the optic nerve-tract, the increases seen at 9 days varied from 16- to 35-fold and were larger for PC and PS than for the others; at 16 days the range of increase was 7- to 16-fold. In the optic tectum, the increases were 6- to 13-fold at 9 days and 10- to 25-fold at 16 days with PS and PI showing the most pronounced elevation at both time points. The relative behavior of individual phospholipids is also represented in the percentage distribution of radiolabel (Table II). Comparisons of specific radioactivities of lipids obtained from the injected and uninjected sides of the visual pathway (Table III) give an indication of the relative contributions from axonal transport and local in-

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TABLE II PERCENTAGE DISTRIBUTION OF RADIOLABEL AMONG AXONALLY TRANSPORTED GLYCEROPHOSPHOLIPIDS

Control % of total

9D % of total

16D % change

% of total

% change

Optic Nerve-Tract PC PE PS PI

49,4 38,2 7,4 5.0

59.1 25.9 11.3 3.6

+ 20 -32 + 54 - 28

50.6 30,9 11.5 6.9

+ 2.4 - 19 + 56 + 38

61.4 25.1 7.1 6.4

- 3.2 - 15 +69 + 113

Optic Tectu m PC PE PS PI

63.4 29.4 4.2 3.0

58.4 26.4 9.0 6.3

- 7.9 - 10 + 114 + 110

Data represent % of 3H counts present in each phospholipid obtained from TLC separation, relative to total 3H counts recovered from the plate. Each value is an average of 2-3 determinations (cf. Figure 2 for variation).

corporation. The latter is presumed due to uptake of label from the circulation or CSF by the glial cells in the nerve-tract and tectum as well as the post-synaptic neurons of the rectum. The ratios for glycerophospholipids in regenerating nerve were all quite high, for both nerve-tract and rectum, indicating that relatively little of the phospholipid label was derived from the circulation or CSF. The ratios in control (non-regenerating) animals were much lower though still significantly above unity, even for the optic rectum. This contrasts with the previous study employing [14C]glycerol in which ratios for control tecta were very low (11.5), pointing to a major contribution from local incorporation. Such comparative data indicate that while glycerol is readily absorbed from the circulation and reutilized for glycerophospholipid synthesis, the 3H-label at the 2-position is largely eliminated in the process.

DISCUSSION Previous studies defining the anatomical parameters of regeneration for the goldfish optic nerve revealed that at 20~ the axons begin to elongate

AXONAL TRANSPORT OF PHOSPHOLIPIDS

NERVE(R-L)

50

1505

TECTUM(L-R)

12.5-

10

4()

7

7.5

0

x 30 ._c

E

:~ 2O Q_

C3

~j~

10

2.5 u

u

_uoLu

-uouJ

CONTROL

g

0 CONTROL DAYS POST CRUSH 16

g

16

FTO. 3. Transported radioactivity of individual glycerophospholipids labeled with [23H]Glycerol in regenerating goldfish optic nerve. Washed lower phase lipids (Figure 2) were separated by TLC (Figure 1) and scraped zones counted. For other particulars see legend to Figure 2. Bar assignments indicated on controls apply (in same sequence) to the two regeneration experiments.

at about 4 days following crush (13) and proceed at a rate of about 0 . 3 0.5 m m per day (13-15). The regenerating axons begin to enter the contralateral optic tectum, their principal site of synaptic termination, at about 12-18 days following crush (13, 15, 16). At 30~ the t e m p e r a t u r e e m p l o y e d in the present study, regeneration proceeds about twice as fast as at 20~ so that some innervation of the rectum is seen as early 7 - 8 days after optic nerve crush and by 12-15 days innervation of the whole tectum has o c c u r r e d (17, 18). A further advantage of the higher temperature is the increased incorporation of label into axonaily transported constituents.

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TABLE

III

RADIOACTIVITY RATIOS FOR SUBSTANCES ISOLATED FROM THE TWO SIDES OF THE VISUAL PATHWAY

Optic Nerve-Tract (R/L) Substance Total lipids PC PE PS PI

Control 22 19 30 19 25

• • • • •

2 6 9 5 8

9D 226 317 339 280 101

• 43 • 84 • 87 _+ 101 +_ 21

16D 129 124 154 135 87

• • • • •

49 63 65 59 41

Optic Tectum (L/R) Total lipids PC PE PS PI

7.4 8.7 8.6 5.4 4.1

• 2.2 • 2.5 - 2.6 • 1.4 • 1.2

51 66 58 46 46

• 4 • 22 • 12 • 8 • 13

100 _+ 10 108 89 91 83

_+ • _+ •

20 11 15 38

Ratios were calculated for transported radioactivities in comparable samples isolated from the injected and uninjected components of the same group of fish. Each experiment involved pooling of 12-14 fish; the number of separate experiments were 3 for control and 2 for the others. Values are mean • SEM. Data were derived from same experiment as Figures 2 and 3.

Phospholipids were previously shown to undergo fast axonal transport in the normal goldfish optic system (19, 20). The present study has demonstrated very substantial increases in the transport labeling of phospholipids entering the optic nerve-tract during regeneration, particularly at 9 days of post-crush when this tissue would have contained a large proportion of newly-regenerated axons. Significant though less pronounced increases were seen in the contralateral optic tectum which receives the growing axons. These results confirm in general terms our previous study (4) employing [14C]glycerol, although [2-3H]glycerol has proved superior to the latter precursor in giving less background labeling. These results also point to a general parallel in the behavior of glycerophospholipids and gangliosides during optic nerve regeneration although the increases in the glycerophospholipids are more pronounced. We have now focused on individual species and revealed that the four principal glycerophospholipids of nervous tissue all show major increases in transport labeling during regeneration. Variations were seen in the magnitude of this increase, depending on the phospholipid, the tissue of origin, and the post-crush period. Thus when the results are expressed as %

AXONAL TRANSPORT OF PHOSPHOLIPIDS

1507

distribution of radiolabel (Table II), the proportion of PC increases by about 20% in the nerve-tract at 9 days accompanied by a corresponding fall in PE. This rather modest rise in the relative contribution of PC does not accord with peripheral nerve studies suggesting that this lipid is preferentially assembled into the regenerating fibers (21-23). The relative labeling of PS rose significantly (50-100%) in both nerve-tract and tectum and P1 appeared to behave similarly, at least in the rectum. It was previously suggested (24) that an increase in the turnover of the latter lipid may be a requisite event in neuritic outgrowth. Moreover, it is possible that the large changes in this lipid which are seen to persist during the period of innervation of the rectum may be related to the important role this lipid is thought to play in nerve conduction and synaptic function (25). Increased transport of lipids and certain proteins presumably reflects augmented demand for membrane components during sprouting and axonal outgrowth. These membrane components are all conveyed by fast axonal transport (26), although they do not all increase to the same extent during regeneration. Thus for goldfish optic nerve, glycoproteins showed rather modest increases in transport labeling as compared to gangliosides and glycerophospholipids (4). This apparent bias in favor of lipid during the early stages of regeneration would seem consistent with earlier morphological evidence (27) for a relative paucity of protein in the growing neurite and growth cone, a proposal now finding confirmation through biochemical analysis of isolated CNS growth cone membranes (28). It should be pointed out that the changes observed in goldfish optic nerve may not be characteristic of all regenerating systems, as suggested by the modest increase of gangliosides observed in regenerating sensory neurons of rat sciatic nerve (29). Studies of the kind described here do not permit direct measurement of quantities of material transported, although it is generally assumed that an increase in labelling of the transported material corresponds to an increase in the amount being transported. This is usually a valid assumption, unless changes have occurred in the cell body that substantially alter the specific radioactivity of the incorporated precursor or result in enhanced turnover. Although both kinds of change may occur to some extent in regenerating goldfish retinal ganglion cells (30~ 31), the increased abundance of at least some transported proteins in regenerating axons (32) as well as the accompanying increase in the size of the cell bodies from which they originate (1), indicates a net increase in synthesis and axonal transport of proteins during regeneration. In the case of lipids, which continue to enter the optic axons for some time after their synthesis is complete, due to delayed release from the cell body (19), their rate of

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SBASCHN1G-AGLER ET AL.

a c c u m u l a t i o n in t h e o p t i c p a t h w a y is l i k e l y a l s o to b e f u r t h e r i n c r e a s e d d u e t o t h e a c c e l e r a t e d v e l o c i t y o f f a s t a x o n a l t r a n s p o r t w h i c h is c h a r a c t e r i s t i c o f r e g e n e r a t i n g g o l d f i s h o p t i c a x o n s (2, 14).

ACKNOWLEDGMENTS This study was supported by PHS grants NS 03356, NS 04834, NS 09015, and NS 16181.

REFERENCES

1. MURRAY, M., and GRAFSTEIN,B. 1969. Changes in the morphology and amino acid incorporation of regenerating goldfish optic neurons. Exp. Neurol. 23:544-223. 2. McQUARRIE, I. G., and GRAFSTEIN,B. 1982. Protein synthesis and fast axonal transport in regenerating goldfish retinal ganglion cells. Brain Res. 235:213-560. 3. HEACOCK,A. M., and AGRANOFF,B. W. 1976. Enhanced labeling of a retinal protein during regeneration of optic nerve in goldfish. Proc. Nat. Acad. Sci. USA 73:828-832. 4. SBASCHNIG-AOLER,M., LEDEEN, R. W., GRAFSTEIN,B., and AL~'ERTR. M. 1984. Ganglioside changes in the regenerating goldfish optic system: Comparison with glycoproreins and phospholipids. J. Neurosci. Res. 12:221-232. 5. MARINETTI,G. V., ERBLAND,J., and STOTZ, E. I959. The quantitative analysis of plasmalogens by paper chromatography. Biochim. Biophys. Acta. 31:251-252. 6. NORTON,W. T., and AuTmIO, L. A. 1966. The lipid composition of purified bovine brain myelin. J. Neurochem. 13:213-222. 7. MACALA, L. J., Yu, R. K., and AND0, S. 1983. Analysis of brain lipids by high performance thin-layer chromatography and densitometry. J. Lipid Res. 24:1243-1250. 8. SBASCHNIG-AGLE~,M., and PULLARKAT,R. 1985. Lysophosphatidylserine-dependent incorporation of Acyl CoA into phospholipids in rat brain microsomes. Neurochem. Int. 7:295-300. 9. LEES, M. B., and PAXMAN,S. t972. Modification of the Lowry procedure for the analysis of proteolipid protein. 47:184-192. 10. McEWEN B. S., and GRAFSTEIN,B. 1968. Fast and slow components in axonal transport of protein. J. Cell Biol. 38:494-508. 11. BENJAMINS,J A. andMCKHANN,a . 1973. [2-3H]Glycerol as a precursor of phospholipids 'in rat brain: Evidence for lack of recycling. J. Neurochem. 20:1111-1120. 12. MmLER, S. L., BENJAMINS,J. A., and MORELL, P. 1977. Metabolism of glycerophospholipids of myelin and microsomes in rat brain. Re-utilization of precursors. J. Biol. Chem. 252:4025-4037. 13. McQuARRIE, I. G., and GRAFSTEIN,B. 1981. Effect of a conditioning lesion on optic nerve regeneration in goldfish. Brain Res. 216:253-264. 14. GRAFSTEIN, B., and MURRAY, M. 1969. Transport of protein in goldfish optic nerve during regeneration. Exp. Neurol. 25:494-508. 15. EDWARDS,D. L., ALPERT,R. M., and GRAFSTEIN,B. 1981. Recovery of vision in regeneration of goldfish optic axons: Enchancement of axonal outgrowth by a conditioning lesion. Exp. Neurol. 72:672-686. 16. HEACOCK,A. M., and AGRANOFF,B. W. 1982. Protein synthesis and transport in the regenerating goldfish visual system. Neurochem. Res. 7:771-788.

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17. SPRINGER,A. D., and AGRANOFF,B. W. 1977. Effect of temperature on rate of goldfish

18. 19. 20.

21. 22.

23.

24. 25. 26. 27.

28.

29.

30.

31.

32.

33.

optic nerve regeneration: A radioautographic and behavioral study. Brain Res. 128:405415. SPRINGER, A. D. 1980. Aberrant regeneration in goldfish after crushing one optic nerve. Brain Res. 199:214-218. GRAFSTEIN, B., MILLER, J. A., LEDEEN, R. W., HALEY, J. and SPECHT, S. C. 1975. Axonal transport of phospholipid in goldfish optic system, Exp. Neurol. 46:261-281. CURRIE, J. R., GRAFSTEIN, B., WHITNALL, M. t-I., and ALPERT, R. 1978. Axonal transport of lipid in goldfish optic axons. Neurochem. Res. 3:479-492. DZIEGIELEWSKA,K. M., EVANS,C. A. N., and SAUNDERS, N. R. 1980. Rapid effect of nerve injury upon axonal transport of phospholipids. J. Physiol. (Lond). 304:83-98. ALBERGHINA, M., MOSCHELLA, F., VIOLA, M., BRANCATI, V., MICALI, G., and Gluv FRIDA, A. M. 1983. Changes in rapid transport of phospholipids in the rat sciatic nerve during axonal regeneration. J. Neurochem. 40:32-38. ALBERGI-UNA,M., VIOLA, M., and GIUFFRIDA,A. M. 1983. Rapid axonal transport of glycerophospholipids in regenerating hypoglossal nerve of the rabbit. J. Neurochem. 40:25-31. TRAYNOR,A. E. 1984. The relationship between neurite extension and phospholipid metabolism in PCI2 cells. Devel. Brain Res. 14:205-210. HAWTHORNE,J. N. 1983. Polyphosphoinositide metabolism in excitable membranes. Biosci. Rep. 3:887-904. GRAFSTEIN, B., and FORMAN, D. S. 1980. Intracellular transport in neurons. Physiol. Rev. 60:1167-1283. SMALL, R. K., and PFENNINGER, K. 1984. Components of the plasma membrane of growing axons. I. Size and distribution of intramembrane particles. J. Cell Biol. 98:14221433. LEDEEN, R. W., SBASCHNIG-AGLER, M., and PFENNINGER, K. 1985. Gangliosides and other lipids of CNS neuronal growth cone membranes. Trans. Am. Soc. Neurochem. 16:346. AQUtNO,D. A., LEDEEN, R. W., and BISBY, M. A. 1985. Bidirectional axonal transport ofgangliosides and neutral glycospingolipids in sensory axons of rat sciatic nerve. Trans. Am. Soc. Neurochem. 16:83. WmTNALL, M. H., and GRAFSTEIN, B. 1981. The relationship between extracellular amino acids and protein synthesis is altered during axonal regeneration. Brain Res. 220: 362-366. WHITNALL, M. H., and GRAFSTEIN,B. 1982. Changes in perikaryal organeltes during axonal regeneration in goldfish retinal ganglion cells: an anlysis of protein synthesis and routing. Brain Res. 272:49-56. PERRY, G. W., BURMEISTER, D. W., and GRAFSTEIN,B. 1985. Changes in protein content of goldfish optic nerve during degeneration and regeneration following nerve crush. J. Neurochem. 44:1142-1151. FEWSTER, M. E., BURNS, B. J., and MEAD, J. F. 1969. Quantitative densitometric thinlayer chromatography of lipids using copper acetate reagent. J. C hromatog 43:120-126.