Lipids of ocular tissues

Lipids of ocular tissues

367 BIOCHIMICA ET BIOPHYSICA ACTA BBA 55680 LIPIDS OF OCULAR TISSUES II. THE PHOSPHOLIPIDS OF MATURE BOVINE AND RABBIT WHOLE RETINA ROBERT E...

467KB Sizes 0 Downloads 68 Views

367

BIOCHIMICA ET BIOPHYSICA ACTA BBA 55680

LIPIDS

OF OCULAR TISSUES

II. THE PHOSPHOLIPIDS

OF MATURE BOVINE AND RABBIT

WHOLE

RETINA

ROBERT

E.ANDERSON,LUTRELL

S.FELDMANANDGERALD

L.FELDMAN

Ophthalmic Biochemistry Section, Institute of Ophthalmology, and Departments and Biochemistry, Baylor College of Medicine, Houston, Texas 77025 (U.S.A.)

ofOphthalmology

(Received November roth,1969)

SUMMARY

The phospholipid class composition and the fatty acid composition of the individual phospholipids of mature bovine and rabbit whole retina were determined. Both species have identical phospholipid class compositions; phosphatidylcholine and phosphatidylethanolamine are the most abundant. Moreover, the same phospholipid classes of each species have near identical fatty acid compositions. The major acids of phosphatidylethanolamine and phosphatidylserine are 18 : o and 22 : 6; phosphatidylcholine contains mostly saturated and monoenic acids, 16: o being the major component. The phosphatidyl inositols are characterized by high levels of 1810 and 2014.

INTRODUCTION

The retina is a rich source of lipid material, mostly phospholipidsl-s. In 1934 KRAUSE' reported that the major phospholipids of bovine retina are phosphatidylcholine and phosphatidylethanolamine. In recent times, EICHBERG AND HESS showed that these two lipids are also the major lipid components of frog retina and retina outer segments. ADAMS% and FLEISCHER AND MCCONNELL~ reported the qualitative analysis of the phospholipids of bovine retina outer segments, but quantitative data were only recently supplied by POINCELOT AND ZULL'. Calf retina phospholipids were quantified by BROEKHUYSE~, and the fatty acid composition of ox retina total lipids was reported by BARTLEY et aLB. To our knowledge, no detailed report of the phospholipids of mature bovine and rabbit whole retina has appeared. This paper describes the phospholipid class composition of whole retina from these two species and the fatty acid composition of their major phospholipids. Biochim. Biophys. Acta, 202 (1970) 367-373

R. E. ANDERSON tJt al.

368 METHODS

Tissues Mature bovine eyes were obtained at a local slaughter house. They were placed on ice immediately after enucleation and dissected within 4 h. Mature rabbit retinas were obtained from Pel-Freeze Co., Rogers, Ark., and shipped frozen in dry ice. No attempt was made to protect either group of retinas from light. Bovine and rabbit retinas were divided into three groups of approx. 50 retina each. These six groups were treated separately throughout the entire study. All analyses made on any one group were in duplicate, except as noted. Extraction and chromatography of retina lipids The extraction and isolation procedures for ocular lipids were described in detail in an earlier publicationlO. Briefly, the tissues were homogenized in distilled water and lyophilized. The dried residues were extracted twice each with 2oo-ml portions of chloroform and methanol (2: I and I :2, v/v), and the extract chromatographed on a Sephadex G-25 column I1. A total lipid fraction was obtained, which was further chromatographed on a column of Unisil (ref. II) (Clarkson Chemical Co., Williamsport, Pa.), resulting in a preparation of phospholipids devoid of any neutral or glycolipids. Phospholipids were fractionated by two dimensional thin-layer chromatography on Silica Gel HR (Brinkmann Instruments, Westbury, N.Y.). The solvent for the first direction was chloroform-methanol-glacial acetic acid-0.9% saline (100: 50 : 16 : 8, by vol.) ; the same solvents in the volumetric ratio of IOO: 15 : 16:4 were used for development in the second direction*. Phospholipids were identified on the plates after spraying with the reagent of DITTMER AND LESTER’~. Lipids containing free amines were detected after spraying with ninhydrin; phosphatidylcholine was detected with Dragendorf’s reagent. Inositol phospholipids were identified by preparing the trimethylsilyl ether of an acid hydrolysate of the lipid, and identifying hexa(trimethylsilyl) inositol by gas-liquid chromatography. The identity of each phospholipid was made only a,fter each sample had been subjected to (a) the color tests just mentioned, (b) two dimensional thin-layer chromatography in a number of solvent systems and (c) co-chromatography with authentic compounds. Gas-liquid chromatography of methyl esters was performed on a BarberColman series 5000 gas chromatograph equipped with a hydrogen flame ionization detector. A 6 ft x 4 mm (internal diameter) glass U-column packed with 15% EGSS-X on 100/120 mesh Gas Chrom P (Applied Science Laboratories, State College, Pa.) was used for all analyses. Inlet and detector temperatures were maintained at 250’ and 310”, respectively. The column was kept at 185” and the carrier gas was N,. The flow rate and oven temperature were adjusted so that base line sepa.ration was obtained between methyl stearate and methyl oleate when both peaks were contained within the IO inch width of the chart paper. Detector responses were monitored on a CRSIIO electronic integrator (Infotronics, Houston, Texas). Miscellaneous procedures Phospholipid phosphorus

was quantified

by the procedure

of ROUSER, SIAKOTOS

* Photographs of lens phospholipids separated by these systems were given in the first paper of this seriesl0. B&him.

Biophys.

.4&a, 202 (1970)

367-373

369

LIPIDS OF BOVINE AND RABBIT RETINA

Methyl esters of the phospholipid fatty acids were prepared with boron trifluoride-methanol according to MORRISON AND SMITH’*. The “separation-

AND FLEISCHER~~. reaction-separation”

procedure

ROCK+

was used to determine

method

to be quantitative.

Catalytic

hydrogenation

1.5 ml of hexane-ethanol platinum settled

of SCHMID AND MANGOLD’s the plasmalogen

content.

was carried out by bubbling

(2: I,

hydrogen

by

HOR-

gas through

v/v) solution of methyl esters containing

oxide. The reaction was deemed complete

to the bottom

as modified

HORROCKS~~ has shown this

when the catalyst

I-Z

mg of

darkened

and

of the test tube.

RESULTS

Phospholipid class composition The phospholipid class compositions of bovine and rabbit retina are given in Table I. Each value is the average of 6 determinations (duplicate analyses of 3 difTABLE

I

PHOSPHOLIPID

CLASS

COMPOSITION

OF

MATURE

ROVINE

AND

RABBIT

WHOLE

RETINA

Bovine and rabbit retina values are the average of duplicate phosphorus analyses performed after two-dimensional thin-layer chromatography of three different samples from each species (total of six analyses). The values are expressed as mole “/A P*S.D. Rabbit

Lipid Origin Lysophosphatidylcholine Sphingomyelin Phosphatidylcholine Phosphatidylinositol Phosphatidylserine Phosphatidylethanolamine Plasmalogen phosphatidylethanolamine Solvent front Unknown Recovery

ferent samples)

and is expressed

can be seen that the phospholipids

Bovine 0.0

0.0

2.6 &

4.4 43.9 4.3 7.4 24.9 + I.5 9.8 * I.5

* * * i

0.7

0.2

f

0.2

0.7 2.7 0.6 2.0

2.1

*

0.0

43.2 It 0.9 5.6 & 0.8 30.6 & 0.1 3.5 f 9.1

34.7 zt I.9

10.0

*

I.1

34.1

f

2.0

4.8 + 0.8

I.3 + 0.7 1.6 & 0.3 98.5 zt 3.3

0.1

+

98.8 I

as mole percent phosphorus

plus or minus

from the two species are identical.

0.1

3.5

S.D.

It

Phosphatidyl-

accounting for 43% of the total lipid phosphorus. The other major component is phosphatidylethanolamine (34%), which is made up in part by ethanolamine plasmalogens; no choline plasmalogens were found. In addition, lysophosphatidylcholine, sphingomyelin, phosphatidylinositol, and phosphatidylserine were identified. The unknown lipid is in the region of lysophosphatidylserine and lysophosphatidylinositol. The lipids in the solvent front are most probably a mixture of phosphatidic acid, identified in frog retina by EICHBERG AND HESS~, and choline is the most abundant,

diphosphatidylglycerol, class compositions in the table.

We

phatidylcholine contribute

identified

in calf retina by BROEKHUYSE*. The phospholipid

of both the bovine and rabbit retina are not as simple as depicted consistently

observe

and sphingomyelin

several small spots in the region of lysophos-

when we overload

little to the total phosphorus,

our plates.

but do magnify

These unknowns

the complexness

of retina

lipids. B&him.

Biophys.

Acta,

202

(1970)

367-373

370

R. E. ANDERSON

Bovtne

Retina

Phosphotidyl

Swine

Methyl

Esters

et RI.

-50

-‘lo

- 30 :

-20

g LL

-to

% e s!

-0 50 After

g P LL

Hydrogenation 40 30 I

Fig. I. Gas chromatogram of the methyl esters of bovine retina phosphatidylserine Conditions of the separation are given in the text.

fatty acids.

Fatty acid composition

Fig. I depicts a typical gas chromatogram of retina phospholipid methyl esters before and after catalytic hydrogenation. We routinely run major acids off-scale in order that the minor components may be quantified. This poses no problem since our electrometer (Infotronics) amplifies linearly and the electronic integrator accurately integrates these peaks regardless of whether they are totally contained on the chart or not. This particular chromatogram is of methyl esters of the fatty acids of bovine retina phosphatidylserine. Peak identities were made on the following bases : (I) plots of log elution time verszcscarbon number; (2) co-chromatography with compounds of known structure; (3) relative elution times given in the literature”; (4) peak areas before and after catalytic hydrogenation. The fatty acid compositions of the individual phospholipids of bovine and rabbit retina are given in Table II. Values for phosphatidylcholine and phosphatidylethanolamine represent the average of duplicate analyses of 3 different samples. Phosphatidylserine, phosphatidylinositol, and lysophosphatidylcholine were each pooled from duplicate thin-layer plates in order to have enough material for individual analysis of each of the 6 groups of retinas. The data given in Table II are graphically demonstrated for the major acids in Fig. 2. It is readily apparent that each of the phospholipid classes has its own characteristic fatty acid composition, and that the same phospholipid classes from both species have near identical compositions. The phosphatidylcholines contain higher levels of palmitic acid (16:o) and lower levels of polyunsaturates than any of the other lipids. Phosphatidylserine and phosphatidylethanolamine are similar, each containing high levels of stearic acid (18 : o) and docosahexenoic acid (22 : 6). Perhaps Biochinz. Biofihys.

Acta,

202

(1970)

367-373

II

ACID

COMPOSITION

OF INDIVIDUAL

BOVINE

0.2

1.1

&

0.2

1.6

31.6 f

Trace

18.3 f

17:1

18:o

%

-

24:X

24:Y

1.3

-

0.4

5

10.7 *

22:6

-

4.5 f

2.0 * 0.2

1.1

22:5

9.8 k 0.4

29.5 f

8.5 f

3.4 f

0.4 f

0.3

0.2

0.4 f

22x4

4.1 f

0.7 0.2

5.1 f 1.0 f

20~4

Trace

Trace

Trace

Trace

20:3

0.1

0.1

b “, P

0.6 f

Trace

0.3 f

Trace

0.7 +

7.7 f

f

0.3 f

p

-

0.5 & 0.1

Trace

20:o

3

b 20:x F;. 20:2 %

1.7 & 0.2

1.0 & 0.2

1.5

1812

15.8 i

0.7 & 0.1

19.0 * 0.5

2.0

0.2

0.5 f

0.5 f

0.0

*

f

&

17:o

1.9 f

10.4

0.2

0.2

1.9 * 0.2

1.1

0.2

16:1

42.3 *

0.6 f

41.0 + 0.8

0.2

0.2

16:o

+

0.5 +

15:o

2.3

2.0

0.8

0.9

0.2

0.1

0.6

3.1

1.5

0.2

0.1

1.1

0.2

0.2 f

&

f

24.8 f

5.8 f

3.6 f

9.6 f

Trace

Trace

0.8 f

0.8 f -

11.9 f

4.1 f 27.2 f

Trace

0.3 f

8.8

2.3

0.2

Rabbit

Phosphatidylethanolamine

Bovine

Rabbit

18.5 & 1.0

Y

WHOLE

Phosphatidylcholine

18:1

zs.T

RABBIT

Bovine

1q:o

Acid

tU i;. f

:

AND

RETINA

PHOSPHOLIPIDS

3.3

1.4

0.5

0.7

0.2

0.2

1.3

2.3 1.1

0.3

1.0

1.7

0.2

0.7

0.2

f

1.8

1.8

I.5

I.2 f

0.1

4.3 + I.0

24.4

4.5 f

4.2 i

3.5 I!= 0.6

0.6 f

Trace -

Trace

2.3 dz 0.7

15.5 * 0.7

33.7 & 2.8

Trace

Trace

0.3 It 0.3

5.8 f

Trace

Trace

Bovine

Phosphatidylsevine

1.0

0.2

19.5 zt 3.0 -

5.3 k 1.6

3.2 + 0.6

5.7 + 1.1

Trace

Trace

0.2 f

Trace

0.7 * 0.4

43.4 f 4.8 14.8 += 0.1

Trace

Trace

1.3 *

5.5 * 2.8

Trace

Trace

Rabbit

f

f

*

*

-+

+

+

1.1

0.1

0.1

0.0

0.1

0.4

0.5

0.2

0.2

0.5

2.6 & 0.4

0.6 * 0.6

0.8 & 0.3

42.0 f

0.5 f

0.8

0.1

Trace

1.3

7.4

36.2

Trace

0.4

0.2

7.3 i

Trace

Trace

Bovine

f

+

1.9

0.2

-

0.4

4.9

2.2 & 0.6

0.5 i

33.4 i Trace

Trace

Trace

Trace

Trace

0.6 k 0.1

7.6 & 1.0

40.6 + 2.8

0.9 j, 0.5

1.8 + 0.3

12.0

Trace

0.2

Rabbit

Phosphatidylinositol

i_ +

6.8 f

1.4

1.5

-

39.1 5 2.9 -

7.2 i

Trace

-

-

1.3

1.9

0.2

0.0

2.3

0.2

0.2

3.2 + 0.4

13.3 f

11.7 f

Trace

0.2 f

2.6 f

14.4 i

0.2

I.2

Lysophosphatidylcholine Rabbit

Values are expressed as weight yO & S.D. The phosphatidylcholine and phosphatidylethanolamine values are the average of duplicate analyses of three different samples from each species (total of six analyses). The phosphatidylserine, phosphatidylinositol and lysophosphatidylcholine values are the average of single analyses of three different samples from each species. Trace means less than 0.1~/“.

FATTY

TABLE

R. E.

372 50

PC

PE

50

r 40

ANDERSON et d.

i

BOVINE RABBIT

30 20 10 16’0

18.0

18:l

20:4

22:6

4. 0 IIxlL-d 16:0 18:0 18:l 20.4 22:6 50

50 r

r

30

%

20 10

PI

30 20 10

0 16:O 18:O

18:l

M:4

22:6

40 0 IdILk_ 16.0 18:0 18:l ZQ.4 22:6

Fig. 2. Bar graph comparing the major fatty acids of bovine and rabbit retina phospholipids. Abbreviations : PC, phosphatidylcholine ; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol.

the most striking example of the similarity between species is shown by the phosphatidylinositols. The major acids are 18: o and arachidonic (20:4), and the levels of the latter acid are much higher in the phosphatidylinositols classes.

than in any of the other lipid

DISCUSSION The retina contains no novel phospholipids,

in confirmation

of other reports

(refs. 1-4, 7, 8). The percentages we report are similar to those of BROEKHUYSE~ for calf retina and POINCELOT AND ZULL’ for bovine retina outer segments. We found no retinal-phosphatidylethanolamine*

in the whole retina of either species, as was

observed by ADAMS~ and POINCELOT et al. l8 in bovine retina outer segments, but not by POINCELOT AND ZULL’ in a more recent report. Preliminary thin-layer chromatography of bovine retina outer segment phospholipids has also failed to disclose the presence of this lipid. However, our extractions were performed in the light and any retinal-phosphatidylethanolamine may have been destroyed. The phospholipid composition we found for our single preparation of bovine retina outer segments was identical to that for the whole retina. This is in agreement with EICHBERG AND HESS who found that the phospholipid composition of whole retina and retina outer segments from frogs was nearly identical. BARTLEY et al.8 examined the fatty acids of ox retina total lipids and reported that polyunsaturated fatty acids comprised 31% of the total. They identified 20:4 and 22 : 5 or 22 : 6 as the major polyunsaturates. Our results confirm their qualitative identifications and show that 22:6 is the major polyunsaturated acid in all lipid classes except phosphatidylinositol. Our data also show that the whole retina from mature bovine and rabbit is composed of similar phospholipids in identical proportions. Preliminary investigationslQ of mature swine, sheep, dog and human retina have revealed that their phospholipid * Identified by POINCELOT et al.18 as a Schiff base between phosphatidylethanolamine visual pigment chromophore retinal (formerly called retinene). Biochim.

Biofihys. Acta,

202

(1970)

367-373

and the

LIPIDS OF BOVINE AND RABBIT RETINA

373

class compositions are very similar to those reported here. Since the retina is a heterogeneous tissue, we have undertaken the analysis of various retina components, beginning with the rod outer segments. Such a study will give valuable information about the photoreceptors of different species, and compliment the recent work of (rhodopsin) isolated from rat, frog, HELLER~~, who showed that visual pigment-5oo and bovine retina outer segments are of comparable molecular weights (27000) and differ only slightly in amino acid compositions. ACKNOWLEDGEMENTS The technical assistance of Miss MAUREEN B. MAUDE is greatly appreciated. This research was supported by grants from the Moody Foundation, Galveston, Texas, the Robert A. Welch Foundation, Houston, Texas, and U.S. Public Health Service Grant NB-oqz77 from the National Institute of NeurologicalDiseases and Stroke, Bethesda, Md. REFERENCES I A. C. KRAUSE, in The Biochemistry ofthe Eye, Johns Hopkins, Baltimore, Maryland, 1934. p. 6.1. 2 J. EICHBERG AND H. H. HESS, Experientia, 32 (1967) 993. 3 R. ADAMS, J. Lipid Res., 8 (1967) 245. 4 S. FLEISCHER AND D. G. MCCONNELL, Nature, 212 (1966) 1366. 5 F. D. COLLINS, R. M. LOVE AND R. A. MORTON, Bib&m. ,I., 51 (1952) 669. 6 N. I. KRINSKY, A. M. A. Arch. Ophthalmol., 60 (1958) 688. R. P. POINCELOT AND J. E. ZULL, Vision Res., 9 (1969) 647. 87 R. M. BROEKHUYSE, Biochim. Biophys. Acta, 152 (1968) 316. 9 W. BARTLEY, R. VAN HEYNINGEN, B. M. NOTTON AND A. RENSHAW, Biochem. J., 85 (1962) 332. R. E. ANDERSON, M. B. MAUDE AND G. L. FELDMAN, Biochim. Biophys. Acta, 187 (1969) 345. 10 G. ROUSER, G. KRITCHEVSKY AND A. YAMAMOTO, in G. V. MARINETTI, Lipid Chromatographic II Amzlysis, Vol. I, Marcel Dekker, New York, 1967, p. 99. 12 J. D. DITTMER AND R. L. LESTER, J. Lipid Res., 5 (1964) 126. I3 G. ROUSER, A. SIAKOTOS AND S. FLEISCHER, Lipids, I (1966) 85. I4 W. R. MORRISON AND L. M. SMITH, ,I. Lipid Res., 5 (1964) 600. I5 H. H. 0. SCHMID AND H. K. MANGOLD, Biochim. Biophys. Acta, 125 (1966) 182. 16 L. HORROCKS, J. Lipid Res., Q (1968) 469. 17 R. G. ACKMAN AND R. D. BURGHER, J. Am. Oil Chemists’ Sot., 42 (1965) 38. 18 R. P. POINCELOT, P. G. MILLAR, R. I,. KIMBELL AND E. W. ABRAHAMSON, Nature, 221 (1~69) 256. 19 R. E. ANDERSON, to be published. 20 J. HELLER, Biochemistry, 8 (1969) 675.

Biochim. Biophys. Acta, 202 (1970) 367-373