BIOCHIMICA ET BIOPHYSICA ACTA BBA 55629
TISSUES OF MATURE
Ophthalmic Biochemistry Houston, Texas (U.S.A.)
Section, Department of Ofihthalmology, Baylor College of Medicine,
(Received May z?th,1969)
The first detailed analysis of mature bovine and rabbit lens phospholipids is reported. I. The phospholipid class composition of both species is similar. Sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine are the major components, being present in near equal molar concentrations in each species. Lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol were a!so identified. 2. Analysis of the fatty acids of the three major phospholipids, plus phosphatidylserine, revealed that each phospholipid class has its own characteristic composition. The same lipid classes from the two animals are similar, but not identical, in fatty acid concentrations. 3. No polyunsaturated fatty acids were found in any of the lipid classes of either species. 4. Mature bovine and rabbit lenticular phosphatidylethanolamine contains 35 and 29 mole% of ethanolamine plasmalogens, respectively. Plasmalogenic aldehydes from both species are made up primarily of analogues of palmitic, stearic, and oleic acids, oleic being the most abundant in each.
Although lipids were identified as components of ocular tissues over a century ago’, only recently have advances in methodology permitted the accurate chemical characterization of the small amounts of lipids normally found as a complex mixture in these tissues. Consequently, only a few reports2-6 have appeared. FELDMAN et aL3 used two-dimensional thin-layer chromatography to quantify the phospholipids of human lens and showed that the major component was sphingomyelin. They also demonstrated that the human lens contains relatively large amounts of glycolipids2 and gangliosides?. BROEKHUYSE~~~~BROEKHUYSE AND VEERKAMP~ quantitated the phospholipids of rat and calf lens and showed that the Biochim. Biophys. Acta, 187 (1969) 345-353
calf lens could incorporate radioactive phosphate into phospholipidss. FELDMAN~ has recently published a critical review of the lipids of the human lens. We have undertaken detailed analysis of the lipids of various ocular tissues by methods developed in this laboratory8 and by ROUSER et al.g. This paper is the first of a series and describes the phospholipids of mature rabbit and bovine lens. EXPERIMENTAL Tissues
Lenses from mature rabbits were obtained from Pel-Freeze Co., Rogers, Ark. They had been frozen immediately after removal from the animal and were shipped packed in dry ice. Mature bovine eyes were purchased at a local slaughter house. The entire bovine eye was kept in ice (no longer than 4 h) until the various ocular tissues were dissected and frozen. Our laboratory has previously shown that no structural alterations occur in lenticular lipids treated under these conditionslo. Lipid extraction and column chromatography
Rabbit lenses were divided into three groups of 25 lenses each, and bovine lenses were divided into three groups of 20 lenses each, Each group was homogenized in deionized water, lyophilized, and a dry weight was obtained. The dried material was extracted twice with 2 : I, I : I and I : 2 volumetric ratios of chloroform-methanol (200 ml per extraction). The combined extracts were taken to near dryness in a flash evaporator under N, without water partitioning. Small amounts of water (from the solvents) were removed by addition of chloroform and repeated evaporation. The extract was dissolved in 25 ml of chloroform-methanol (19: I, by vol., saturated with water) and applied to a 2.5 cmx30 cm Sephadex G-25 column. The elution scheme of ROUSER et aL9 was followed and total lipids (except gangliosides) were eluted with 750 ml of the solvent in which they were applied. This fraction was taken to near dryness and dissolved in 25 ml of chloroform. An aliquot was removed to determine the weight of the total lipid. 20 ml of the total lipid fraction were applied to a I cm x IO cm column packed with 200-325 mesh Unisil (Clarkson Chemical Co., Williamsport, Pa.). Neutral lipids were eluted with IOO ml of chloroform, glycolipids with 300 ml of acetone and phospholipids with IOO ml of methanol 9. The fractions were taken to near dryness and dissolved in 25 ml of chloroform; the phospholipid fraction was qualitatively examined by thin-layer chromatography. Each of the spots that charred gave a that the methanol eluate contained only positive phosphorus test”, indicating phospholipids. No positive phosphorus tests were obtained after chromatography of the neutral lipid and glycolipid fractions. Thin-layer
Phospholipids were separated on thin layers of Silica Gel HR (Brinkmann Instruments, Westbury, N.Y.), spread 0.5 mm thick on 20 cmx20 cm glass plates. A two-dimensional system was used: direction I, chloroform-methanol-glacial acetic acid-o.9°/0 saline (100: 50: 16: 8, by vol.) ; direction 2, chloroform-methanol-glacial acetic acid-0.9% saline (IOO : 15 : 16 : 4, by vol.). Phospholipids were qualitatively identified after spraying with the reagent of Biochim.
187 (1969) 345-353
LIPIDS OF BOVINE AND RABBIT LENS
DITTMER AND LESTER”. Lipids containing free amines were identified by spraying with a 0.1% n-butanolic solution of ninhydrin. All plates used for quantification of phosphorus were sprayed lightly with 55y0 H,SO, containing 0.6% Na,Cr,O, (ref. 12) and charred at 200’ for 30 min. Lipids to be used for methyl ester analysis were visualized under ultraviolet light after lightly spraying with a o.z~/~ methanolic solution of z’,7’-dichlorofluorescein. Phospholipid phosphorus was determined after two-dimensional thin-layer chromatography by the method of ROUSER et al.‘%. The plasmalogen content of rabbit and bovine phosphatidylethanolamine was thin-layer chromatographic determined by the “separation-reaction-separation” plate was developed technique of SCHMID AND MANGOLD14. Briefly, the thin-layer in the first direction, dried with a stream of N,, exposed to HCl fumes for 5 min, Phosphorus again dried with N,, and finally developed in the second direction. analysis was performed on the phosphatidylethanolamine and the lysophosphatidylethanolamine resulting from the acid hydrolysis of the plasmalogen. No lysophoslane. Plasmalogenic phatidylcholine was observed in the phosphatidylcholine aldehydes were obtained for gas chromatography by applying the procedure of SCHMID AND MANGOLD’* to a sample of total lipid spotted across the bottom of a thin-layer plate. Development was in hexane-ether (80: 20, by vol.). Gas-lip&d
Methyl esters and aldehydes from the phospholipids were analyzed on a Barber-Colman 5000 series 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 Gas Chrom P (Applied Science Laboratories, State College, Pa.) was used. Inlet and detector temperatures were maintained at 250 and 310”, respectively. The oven temperature was 185”, and N, was the carrier gas. The flow rate and oven temperature were adjusted to give base line separation between methyl stearate and methyl oleate in the least amount of time. Miscellaneous
Methyl esters of the fatty acids of phosphatidylseline, phosphatidylethanolamine, and phosphatidylcholine were prepared by heating the phosphclipids at 80” for I h in a 4% H,SO, in anhydrous methanol solution. The amide-linked fatty acids of sphingomyelin were methylated by heating at 80” for 24 h in a 0.5 M anhydrous methanolic HCl solution. Hydrogenation was carried out by bubbling H, through 1.5 ml of a hexaneethanol (2: I, by vol.) solution of methyl esters containing I-Z mg cf platinumoxide. The reaction was completed when the catalyst darkened and settled to the bottom of the test tube (2-3 min). RESULTS AND DISCUSSION Data in Table I show some of the characteristics of bovine and rabbit lenses. Bovine lens contains 66% water and the rabbit lens 5476, The lipid content of the lenses of each species is quite low and is expressed in the table both as percent wet and dry weight. Two-dimensional thin-layer chromatographic plates of bovine and rabbit lens Riochim.
187 (1969) 345-353
R. E. ANDERSON
PROPERTIESOF BOVINE AND RABBIT Lens
Watelf ( Yo)
460 (389-596) * ‘955 (1950-1960)***
56.9 (55.8-57.9)** 66.6 (66.o-67.o)t
0.42 (0.40-0.45) ** 0.25 (o.rg-0.2g)t
0.97 (0.93-1.04) ** 0.84 (0.64-0.98)t
* Average of individual weights of IO lenses, range given in parentheses. ** Average of three groups of 25 lenses each. * * * Average individual lens weights from three groups of 20 lenses each.
7 Average of three groups of 20 lenses each.
are shown in Figs.
IA and IB, respectively.
The spots identified
phosphatidylserine, lysophosphatidylethanolamine, and phosphatidylethanolamine gave positive ninhydrin tests. The separation between phosphatidylserine and phosphatidylinositol was demonstrated using authentic standards (Supelco, Bellefonte, Pa.), compounds
and it was because of the good separation we found between these two that we chose the present solvent system. Other phospholipids were
identified by co-chromatography with standards. Qualitative identity of each phospholipid was made only after each sample had been subjected to two-dimensional thin-layer
in at least four different
(The reader is
Fig. I. Two-dimensional thin-layer chromatograms of phospholipids from mature bovine (A) and mature rabbit (B) lens. Solvent systems: direction I, chloroform-methanol-glacial acetic acid0.9% saline (1oo:50: 16: 8, by vol.) ; direction 2, chloroform-methanol-glacial acetic acid-0.9% saline (100: 15 : 16: 4. by vol.). The spots were produced by spraying with 55% H,SOI containing 0.6% Na,Cr,O, (ref. 12) and charring at 200’ for 30 min. Qualitative identification of the spots is described in the text. Abbreviations: OR, origin; SPH, sphingomyelin; PC, phosphatidylcholine; LPE, lysophosphatidylethanolamine ; PI, phosphatidylinositol; PS, phosphatidylserine ; PE, phosphatidylethanolamine ; SF, solvent front. Xi, X, and X, are unknown phosphorus-positive compounds.
to a recent review by ROUSER et al. 9 for a detailed description of these systems). It should be pointed out that the separations we achieved by two-dimensional thin-layer chromatography affords at best a fractionation of “families” of phospholipids. It is impossible under the described conditions to separate these families on the basis of minor differences of polarity, such as an ether VeYsUsan ester bond or a P-O-C 8eYsUs a P-C bond. The initial development in the acidic solvents caused no damage to the lipids since no decomposition products were observed in any of the vertical lanes. We believe the lysophosphatidylethanolamine is net an artifact. Freshly extracted total lipids from bovine lens (after Sephadex column chromatography and before silicic
187 (1969) 345-353
LIPIDS OF BOVINE AND RABBIT LENS acid chromatography)
and the ninhydrin-positive,
349 to two-dimensional
was detected. In addition, R. M. BROEKHUYSE (personal communication) has also observed this lipid in the human lens. The phospholipid composition of rabbit and bovine lens is given in Table II and is expressed as mole% phosphorus. Recovery of the lipid phosphorus frcm the thin-layer plates was quantitative. The major lipids in each animal are sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine. In addition, lysophosphatidylTABLE
and X, are unknown phosphorus-positive
Mole y. P
Trace * * *
0.2 + 0.2 0.9 Jo0.3 0.4 f 0.2
Sphingomyelin Lysophosphatidylethanolamine Phosphatidylinositol Phosphatidylserine X, X, Phosphatidylcholine Phosphatidylethanolamine Solvent front ~_ * Mean & S.D. for duplicate ** Mean & S.D. for duplicate *** 0.1% or less.
Rabbit * *
22.3 3.1 1.6 8.2 I.2 0.3 26.0 33.6 I.3
Trace * * * 32.2 + 0.9 3.1 * 9.6 I.3 d-10.2 12.3 _i 1.4
5 0.6 + 0.8 _k 0.3 & I.0 * 0.7 & 0.2 +~ 0.7 * 1.6 +
22.6 + 1.4 27.9 i 0.7 9.4 i 0.3
analyses of three tissue samples. analyses of two tissue samples.
were detected. The bovine lens contains three unknown phospholipids, only one of which is found in the rabbit. These constitute less than 3% of the phospholipid phosphorus in the bovine and only traces in the rabbit. The lipids in the solvent front are most probably phosphatidic acid, diphosphatidylglycerol, and phosphatidylglycerol, which were identified in calf lens by BROEKHUYSE~ and BROEKHUYSE AND VEERKAMP~. Our results for the bovine lens agree for the most part with the phospholipid composition reported for calf lens 5+. The high levels of sphingomyelin consistent with earlier observations in this laboratory3,15*r6.
BROEKHUYSE~ reported high levels (50%) of plasmalogens in the phosphatidglethanolamine of calf lens. Our analysis of mature bovine and mature rabbit-lenticular phospholipids also shows that only phosphatidylethanolamine contains plasmalogens; the bovine contains 35% and the rabbit 29%, expressed as mole percentage of phosphatidylethanolamine. The composition of the aldehydes resulting from acid hydrolysis of the phosphatidylethanolamine plasmalogens is shown in Table III. The identities of these aldehydes were established as follows: (I) preparative thinlayer chromatography using known aldehydes in the reference lanes, (2) comparison of gas chromatographic elution times with aldehydes of known structure and (3) reduction of the aldehydes to alcohols with LiAIH4, production of the acetate derivatives, and gas chromatography of the alcohol acetates, as described by SNYDER Biochim.
R. E. ANDERSON et al.
COMPOSITIONOF ALDEHYDES ETHANOLAMIKE
Values are expressed as wt.%. Aldehydes 12:o 14:0 15:o
Trace * Trace* Trace * 26.5 0.7 Trace *
Trace * Trace* 23.2 I.9 0.3 0.9
16:o 16:1 17:o 17:1
58.5 0.6 1.3
AND BLANK”. The composition
of the original
and of the alcohol
acetates was identical. As can be seen in the table, only palmitic, stearic and oleic acid analogues are found. This is similar to the plasmalogenic aldehydes reported for the lipids of normal tissuesls, tumor tissueslg and cells grown in tissue culturezo. Fatty acid compositions of the major phospholipids of bovine and rabbit lens are given in Table IV. Peak identities were established on the following bases: (I) plots of log elution time veYsU.s carbon numbers, (2) co-chromatography with compounds of known structures, (3) relative elution times given in the literature21 and (4) peak areas before and after catalytic hydrogenation. The phosphatidyl cholines from both species are similar.
acid is the
major fatty acid (60-64%), acids f o 11owed by oleic acid (19-20~/~). The unsaturated make up less than 25% of the total fatty acids. No significant quantities of acids with retention times greater than oleic acid were observed. Sphingomyelin, on the other hand, contains large quantities
carbon chain lengths greater than eighteen. Nervonic acid is the major long-chain acid, amounting to 11.7% in the rabbit lens and 30.994 in the bovine lens. Sphingomyelin is the only lens phospholipid that contains nervonic acid, which has been shown to be a major constituent in bovine lens gangliosidesz2 and human-brain sphingomyelinza. fatty
acids. Oleic acid is the major
acid in the phosphatidylethanolamine
species. Both species contain relatively low levels of palmitic acid (S-13%), and neither contain any polyunsaturated acids. Although the phosphatidylethanolamine from the two species is made up in part by ethanolamine plasmalogens, very low concentrations of dimethyl acetals were observed in the chromatograms of the methyl esters. These compounds were reported by DODGE AND PHILLIPS~~ to elute on EGSS-X columns just prior to their methyl ester analogue. For example, 16:o dimethyl acetal would elute as 15 : I, 18 : o dimethyl acetal as 17 : I, and 18 : I dimethyl acetal as 18:o. Evidently the conditions we used to prepare the methyl esters destroyed these fragile materials. The composition of the phosphatidylserine resembles that of the phosphatidylBiochim.
‘; G W
* Average of two groups of tissues.
Trace* * * _-
Trace* * * -
Trace * * *
22 : I
3.4 20.3 -
Trace * * *
3.8 Trace * * *
Values are expressed as wt.%. _______~__ Acid Rabbit*
Trace* * *
Trace * * *
I.9 Trace* * *
0.5 Trace * * *
_______ PhosphatidylPhosphatidylethanolamike serine
- _.__ ** Average of three groups of tissues.
7.8 Trace * * * -
Trace * * * -
Trace * * *
3.0 Trace * * +
Trace * * f 64.0
Bovine* * __Phosphatidylcholine _
Trace * * *
Trace * * * 22.3
2.4 Trace * * * -
Trace * * *
Trace * * *
3.3 Trace * * *
R. E. ANDERSON
ethanolamine in that oleic is the major acid. The levels of palmitic acid in both species are relatively low when compared to that in the sphingomyelin and phosphatidylcholine. Stearic acid is higher in phosphatidylserine of both species than in other lipid class. As was observed for the other lipid classes, phosphatidylserine contains no polyunsaturated acids. The fatty acid compositions given in Table IV agree for the most part with those reported earlierIs. Any differences may be attributany
ed to the lack of purity of the phospholipid raphy in the earlier study. It is readily apparent
from the data
by column chromatog-
the lipids of mature
rabbit lens are quite similar. This is borne out in the phospholipid class compositions, the fatty acid ccmpositions of individual phospholipids and the composition of the aldehydes laboratory have
derived from the ethanolamine plasmalogens. Recent studies in our on the phospholipids of the retina of these two species indicate they
positions2”. The most striking feature of lenticular phospholipids is the absence of polyunsaturated fatty acids, especially linoleic acid. This acid is essential, and its absence from the diet of most monogastric mammals leads to various characteristic deficiency symptoms. While some species are more susceptible to essential fatty-acid deficiencies than others, eicosatrienoic
all try to compensate for the lack of linoleic acid by synthesizing acid26. However, lenticular phospholipids contain none of the latter
acid and, therefore, the absence of polyunsaturated fatty acids in this tissue must be considered “normal”. We suggest that the absence of linoleic acid is indicative that all of the fatty acids found in the lens are synthesized in situ. This is not an unreasonable hypothesis when one considers that the lens contains no vascular system
and derives all its nutrients
from the aqueous humor.
of Mrs. Lutrell
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-04277 from the National Institute of Neurological Diseases and Stroke
I J. J, BERZELIUS, Lehrbuch dev Chemie, Arnold, Dresden, 1825, p. 525. 2 G. L. FELDMAN, L. S. FELDMAN AND G. ROUSER,]. Am. OilChemists’ Sot., 42 (1965) 742. 3 G. L. FELDMAN, L. S. FELDMAN AND G. R~u~~~,Lipids, I (1966) 161. 4 G. L. FELDMAN, L. S. FELDMAN AND G. ROUSER, Lipids, I (1966) 21. ; R. M. BROEKHUYSE, Biochim. Biophys. Acta, 152 (1968) 307. 6 R. M. BROEKHUYSE AND J. H. VEERKAMP, Biochim. Biophys. Acta, 152 (1968) 316. 7 G. L. FELDMAN, Surv. Ophthalmol., 12 (1967) 207. 8 G. L. FELDMAN AND G. ROUSER, J. Am. Oil Chemists’ Sot., 42 (1965) 290. 9 G. ROUSER, G. KRITCHEVSKY AND A. YAMAMOTO,~~ G. V. MARINETTI, LipidChromatographic Analysis, Vol. I, Marcel Dekker, New York, 1967, p. 99. IO G. L. FELDMAN AND L. S. FELDMAN, Invest. Ophthalmol., 4 (1965) 162. II J. D. DITTMER AND R. L. LESTER, J. Lipid Res., 5 (1964) 126. 12 G. ROUSER, C. GALLI, E. LIEBER, M. L. BLANK AND 0. S. PRIVETT, J. Am. Oil Chemists’ Sot., 4’
187 (1969) 345-353
LIPIDS OF BOVINE AND RABBIT
13 G. ROUSER. A. N. SIAKOTOS AND S. FLEISCHER, Lipids, I (1966) 85. Acia, 125 (1966) 182. I4 H. H. SCH~ID AND H. K. MANGOLD, Biochim. Biobhys. I.5 G. L. FELDMAN, T. W. CULP, L. S. FELDMAN, C. K. GRANTHAM AND H. T. JONSSON, Jr., Invest. Opthalmol., 3 (1964) 194. 16 G. L. FELDMAN. in M. U. DARDENNE AND T. NORDMANN, Biochemistry _ of the Eye, Karger, Basel-New York, 1968, p. 348. 130 (1969) IOI. 17 F. SNYDER AND M. L. BLANK, Arch. Biochem. Biophys., 131 (1969) 478. 18 R. WOOD AND F. SNYDER, Arch. Biochem. Bioplzys., 131 (1969) 495. I9 R. WOOD AND R. D. HARLOW, Arch. Biochem. Biophys., Acta, 176 20 R. E. ANDERSON, R. B. CUMMIXG, M. WALTEN AND F. SNYDER, Biochim. Biophys.
(1969) 491. 21 R. G. ACKMAN AND R. D. BURGHER, J. Am. Ozl Chemists’ Sec., 42 (1965) 38. 22 A. S. WINDELER, Ph.D. Thesis, Baylor College of Medicine, Houston, Texas, 1969. 23 J. S. O’BRIEN AND E. L. SAMPSON, J. Lipid Res., 6 (1965) 545. 24 1. T. DODGE AND G. B. PHILLIPS, I. Lipid Res., 8 (1967) 667. _ 25 in. E. ANDERSON, to be published.Proc., zo (1961) 952. 26 J. F. MEAD, Federation Biochim.