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Lipid composition of erythrocytes in various mammalian species
BIOCHIMICA
BBA
ET BIOPHYSICA
ACTA
221
55366
LIPID
COMPOSITION
OF ERYTHROCYTES
IN VARIOUS
MAMMALIAN
SPECIES
GARY
J. NELSON
Bio-Medical Research Division, Lierermore, Calif. (U..S.A .) (Received
February
(Revised
manuscript
Lawrence Radiaiion
Laboratory,
University
of California,
13th, 1967) received April zsth,
1967)
SUMMARY
Lipid distribution in the erythrocyte was investigated in several common mammals, including rat, rabbit, pig, dog, horse, sheep, cow, goat, cat and guinea pig. Lipids were extracted from fresh, thoroughly washed, whole packed cells. The lipids were purified by Sephadex column chromato~aphy and separated by thin-layer chromato~aphy. Infrared spectrophotometry was also used to identify components. The distribution of cholesterol, glycolipids, and the phospholipids is reported. Cholesterol was approx. 26% of total lipid in all species while phospholipids ranged between 50 and 70%. The glycolipids were considerably more variable, accounting for 5.3% of the lipids in the rabbit erythrocyte and 23.5% of the lipids in the horse erythrocyte. The glycolipid fraction was not analyzed in detail. The phospholipids were separated into the various classes, but neither vinyl nor glyceryl ether compounds were separated from diacyl derivatives. The phospholipids common to all species were phosphatidic acid, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidy1 inositol, and sphingomyelin. There are, however, marked species variations in the relative abundance of these erythrocyte phospholipids. No lecithin was detected in sheep, cow, or goat. However, an as yet unidentified phospholipid was detected in these species.
INTRODUCTION
The procedures now available allow accurate estimation and quantitation of most lipids in any organ or subcellular particle l - 3. Yet despite the current interest in the lipid composition of the erythrocyte, accurate analytical data on lipid composition is fragmentary at best for most mammalian species. Earlier investigators often grouped several types of lipids when they reported their results+’ or used analytical techniques that failed to resolve all components%*. Human erythrocytes, perhaps because of their chnical interest, have been studied most frequently and with most precision: WAYS AND HANAHANIO, WAYS, REED AND HANAHAN’~, VAN GASTEI, et uZ.la, Bdochim. Biophys.
Acta,
I++ (1967) 221-232
222
G.
J. NELSON
HILL, KUKSIS AND BEVERIDGE~~~‘~,and FARQ!UHAR~~have reported detailed studies on the distribution of the erythrocyte phospholipids. The latest techniques have not heretofore been used, however, to clarify the situation in other mammals. Data now in the literature indicate marked species differences in the distribution of phospholipids, particularly lecithin, in the mammalian erythrocyte. TURNER in some pioneering studieslapr7 reported that lecithin was absent in ruminant erythrocytes, but abundant in most other species including humans. DAWSON, HEMINGTON AND LINDSEY’*, DE GIER AND VAN DEENEN~, and HANAHAN, WATTS AND PAPPAJOHN~ confirmed these results, but in most of these reports, including TURNER’S original work, many of the lipids present were either not resolved or not reported. Because accurate analytical data on erythrocyte lipids are pertinent to theories of the structure and function of their membrane, a series of common mammalian species was investigated in an attempt to elucidate their erythrocyte lipid composition using accurate analytical methods. This report gives the results for total lipid, cholesterol, glycolipid, and phospholipid of the erythrocytes of several domestic mammals, both herbivores and carnivores. The animals investigated were rat, rabbit, pig, dog, horse, sheep, cow, goat ,cat, and guinea pig. MATERIALSAND METHODS All solvents used in this work were redistilled from glass stills and deoxygenated by bubbling N, through them before use. Silica Gel HR, 20 cm x 20 cm borosilicate glass plates, thin-layer chromatographic plate spreader, and spreading template were obtained from Brinkman Instruments, Westbury, New York. MgSiO, was obtained from Allegheny Chemical, Butler, New Jersey. Sephadex was purchased from Pharmacia Fine Chemicals, New York. Standard phospholipids were obtained from General Biochemicals, Chagrin Falls, Ohio, and Applied Science Laboratories, State College, Pennsylvania; they included phosphatidic acid, phosphatidyl ethanolamine, phosphatidyl serine, lecithin, lysolecithin, and sphingomyelin. Phosphatidyl inositol was a gift of Dr. C. BALLOU. Infrared spectra were determined in a sealed liquid cell with an NaCl window, or, alternatively, from a pellet prepared from the solid sample mixed with KBr (infrared grade, Harshaw Chemical Company, Cleveland, Ohio), and were recorded on a Perkin-Elmer Model 521 double-beam infrared-grating spectrophotometer. Sampling
and extraction
All animals in this investigation were maintained at this laboratory using standard animal husbandry methods; except the horse and pig, samples from which were obtained from local slaughter houses using only freshly killed animals. Only sexually mature animals in good health were selected for sampling. The guinea-pig, rat and rabbit samples were pooled from 6, 3 and 2 animals, respectively. All other samples were drawn from a single animal. Blood was drawn by venipuncture into sterile plastic blood bags as reported earlier’@ for the large animals in this series, or directly into polypropylene centrifuge tubes for the small animals. Heparin, 500 USP units per IOO ml blood, was the anticoagulant. The blood was cooled immediately to o0 in an ice bath and kept at that temperature throughout processing and until subsequent analysis. Biochim.
Biophys.
Acta,
144 (1967)
221-232
LIPID COMPOSITIONOF MAMMALIANERYTROCYTES
223
Erythrocytes were separated from plasma by centrifugation at 1570xg for 20 min in a refrigerated centrifuge. The plasma was removed by aspiration, and the packed cells were washed 3 or 4 times with an equal volume of 310 mosM phosphate buffer (pH 7.4). After each washing, the upper phase, and the top 2 or 3 mm of packed cells, was removed by aspiration. This procedure ensured almost complete removal of other formed elements of blood from the final erythrocyte preparation. Smears of the packed red cells stained with Giemsa stain yielded less than oneleucocyteper 104 erythrocyte in all cases. The cells were extracted immediately after the final centrifugation. Extraction
procedure
The packed cells were transferred to the extraction flask with a volumetric pipette of appropriate size, usually 20 ml. The cells were added slowly to cold methanol that was continuously stirred by a magnetic mixer. After the cells were introduced, the flask was brought to volume with chloroform and vigorous stirring was continued for 5 min. The volumes of methanol and chloroform were selected to yield a final chloroform-to-methanol ratio of Z/I (v/v) and a solvent-to-packed-cell ratio of 50/r (v/v). The solution was then filtered using a fast, pre-washed filter paper (Whatman 41H, or Schleicher and Schuell Sharkskin) into a round-bottomed boiling flask. The solvent was removed by rotary evaporation at low temperation (~15”) and a reduced pressure of N,. Before the flask became dry, the residue was transferred to a small volumetric flask (2 to 5 ml) with chloroform-methanol (rg : I, v/v). The crude lipid extract was then chromatographed on a Sephadex column to separate gangliosides and remove non-lipid contaminants. Re-extraction of the insoluble matter with chloroform-methanol (2 : I, v/v) yielded less than 0.2% of the lipid obtained from the initial extraction. Digestion of the residue with aqueous KOH (ref. 19) indicated that no more than 0.05% total fatty acid in the cells remained after the solvent extraction. Sephadex
column chromatography The procedure used was essentially that of SIAKOTOS AND ROUSERS with only slight modifications. The Sephadex (G-25, coarse) was washed and the column was prepared as described2, except that the pre-washing of the packed column was simplified by going through the normal elution scheme once after packing the column and before applying the sample. Columns (2.5 cm x IO cm) were fitted with Teflon needle-valve stopcocks allowing easy adjustment of the flow rate. Three fractions were collected. Fraction I, 170 ml of chloroform-methanol (19: I, v/v) satd. with water, contained all the neutral lipids, phospholipids and glycolipids other than gangliosides. Fraction II, 350 ml of 5 parts chloroform-methanol (19:1, v/v) and I part acetic acid satd. with water, followed by 170 ml of 5 parts chloroform-methanol (9: I, v/v) and I part acetic acid satd. with water, contained the gangliosides. Fraction III, 350 ml of methanol-water (I: I, v/v) contained the non-lipid impurities in the initial extract. Fractions were collected at 20’. The first fraction was collected at a flow rate of I to 2 ml/min and the following fractions at 3 to 4 ml/min. A column was allowed to stand in methanol-water (I : I, v/v) for at least 48 h before reuse, and 500 ml of chloroform-methanol (19:1, v/v) Biochim.
Biophys.
Acta,
144 (1967) 221--2p
224
G. J. NELSON
satd. with water was passed through the column immediately before the sample was applied. The solvent was removed from Fraction I as described above for the initial red cell extract, without allowing the flask to become dry before transferring the sample to a glass-stoppered graduated cylinder with 5 ml chloroform. At this time an aliquot was weighed to determine the total amount of lipid in this fraction. The remainder was stored as the solution at --IO' until further analyses could be performed. Fraction II was handled similarly except that it was transferred to a tared 8-ml screw-cap vial with chloroform-methanol (19: I, v/v) and the solvent was evaporated by passing a stream of N, over it. The sample was then placed in desiccator under vacuum for at least 2 h, weighed, and stored dry at -IO' until further analysis. Fraction III was taken to dryness on the rotary evaporator, transferred to a tared S-ml screw-cap vial with methanol-water (4:1, v/v) and then handled like Fraction II, except that further analysis was not usually performed. Analytical procedures for lipids in Fractions
I and II
Cholesterol was analyzed in all samples, using gas-liquid chromatography, thinlayer chromatography, and infrared spectrophotometry. The details and accuracy of the procedures have been reported elsewherezO. The phospholipids were separated by two-dimensional thin-layer chromatography using methods developed by ROUSER and co-workers1~21with thin-layer chromatography plates spread with Silica Gel HR mixed with 10% MgSiO, by wt. Layer thickness was 0.25 mm. Plates were activated for 20 min at IZOO,then cooled in air for 30 min before the sample was applied. Samples and standards were spotted on the thin-layer chromatography plates from Lang-Levy micropipes of appropriate volumes, usually IO ~1. Approx. 500 pg of phospholipid was applied to the thin-layer chromatography plate. Samples were run in both pairs of solvent system developed by ROUSERand co-workers1~21; the results in the two systems were the same within experimental error which was determined by running triplicate determinations on horse and sheep extracts. One pair of solvents consisted of chloroform-methanol-ammonia (65 : 35 : 5, by vol.) followed by chloroform-acetone-methanol-acetic acid-water (5 : 2 : I :I :0.5, by vol.) in the second dimension. The second pair consisted of chloroform-methanol-water, (65 : 35 : 4, by vol.) followed by butanol-acetic acid-water (40: 20 :20, by vol.) in the second dimension. Spots were visualized with a char reagent (0.6 g potassium dichromate per IOO ml of 55% by wt. H2S0JZ2, which was sprayed on the thin-layer chromatography plate after the developing solvents had evaporated completely. The plate was charred by heating for 20 min at 180’ in a forced-draft oven. The plates were photographed as previously described18 and also viewed under ultraviolet light to detect trace components. Phospholipids were analyzed on the charred plates by phosphorus analysis by a1.The spots were scraped off the the method of ROUSER,SIAKOTOSAND FLEISCHER plate with a razor blade into 5-ml glass-stoppered tubes to which the color reagents were added directly without removing the absorbant. The total sample was also spotted in the opposite corner of the plate so that total recovery could be estimated. Color development was done by the methods previously describedlo. Biochint.
Biophys.
Acta.
I++ (1967)
221-232
LIPID COMPOSITION OF MAMMALIAN
ERYTROCYTES
225
Additional plates spotted and developed in an identical manner were sprayed with ninhydrin or Dragendorff’s reagent to locate amino- and choline-containing lipids respectively. Glycolipids were located with cr-naphthol spray. Still other plates were sprayed with water and the spots outlined while the plates were wet. They were then dried, the spots were scraped into sintered-glass funnels, and the lipids were eluted with an appropriate solvent. Lipids recovered from the thin-layer chromatography plates were repurified by passing through small Sephadex columns. The solvent was removed by a stream of N, and the infrared spectra of the lipids were obtained as described above. Fraction II containing the gangliosides was analyzed for phosphorus and cholesterol and its infrared spectrum was recorded; further characterization of the gangliosides was not attempted at this time. The phosphorus content of Fraction II averaged0.05%; no cholesterol was detected in this fraction in any sample. Other glycolipids in Fraction I were estimated by subtracting the amounts of phospholipid and cholesterol found in this fraction from the total weight, but detailed analyses were not attempted. RESULTS
The analyses reported here were performed on blood drawn from one animal from each of the species studied except when it was necessary to pool samples from several animals, as noted above. Previous studies on sheep’@ and unpublished work on cows carried out in this laboratory, indicated that erythrocyte lipid distribution does not vary significantly among members of the same species. Hence, analyzing the erythrocyte lipids from a single animal, randomly selected, in each species should yield a representative analysis of the erythrocyte lipid for that species. The results in Tables I and II are data from single experiments for each species. Table III presents the results of a triplicate analysis of horse erythrocyte phospholipids run by both thin-layer chromatography procedures. The results show the excellent reproducibility of the method. Data in Table IV are averages of the phosphorus determinations of the Sephadex F-I fraction run in both thin-layer chromatography systems for all species in this investigation. Table I presents the results from the Sephadex column chromatography of the chloroform-methanol extracts. Recoveries of phosphorus from the Sephadex columns were close to or over 100% in all cases. Most of the phosphorus was recovered in Fraction I except for a trace of water-soluble phosphorus recovered in Fraction III that was probably inorganic material. Recoveries well over 100% (dog, goat and guinea pig) do not represent errors of the method but rather indicate the presence of material insoluble in chloroform-methanol (rg: I, v/v) and later solubilized with either acid solvents or methanol-water (I : I, v/v). Material from the initial extracts was transferred quantitatively to Sephadex columns of possible; this usually included a fair amount of particulate (nonlipid) material. The low recoveries (rabbit, cat) may have resulted from failure to transfer all of the extracted material to the Sephadex column. Since this insoluble material is not lipoidal, the accuracy of the succeeding analytical procedures was unaffected. The difficulties in quantitating the recoveries from the Sephadex columns result primarily from the nonlipid material carried in the initial extract. To avoid decompoBiochim.
Biophys.
Acta,
144 (1967) 221-232
G. J, NELSOIi
226 TABLE
I
RESULTS OF SEPHADEX COLUMN CHROMATOGRAPHY FROM VARIOUS MAMMALIAN SPECIES
OB CHLOROFORM-METHANOL
The solution used was chloroform-mcthanoi Species
(2: 1, v/v) ~Hemato- Volume wit* packed cells
Sex
---__ Rat, Sprague-Dawley Rabbit, Rev.7 Zealand white Pig, Poland-China Dog, beagle
M M M M
.~_ 56 42 54 53
Horse, western quarter F Sheep, Hampshire F Cow, Holstein F Goat, African pigmy M Cat, domestic short hair F Guinea pig M -* Determined at 1570 x g for 20 min
extracted (ml) ___._ 7.0
52 50 so 40 35 40
EXTRACT
OF
ERYTHROCYTES
._- .__-_ Weight of chromatographic Hecovevy fractions -..-I_-._ ..III Eg) (mg) (?I) img) _ -_--_ _ _---_._ ..~_ 2.3 9.0 101 34.0
Total wt. of extract (mg) 45.6
16.0 20.0 20.0
92.6 100.2 120.1
IO.1 25.0 27.4 15.2 16.8
69.9 143.4 142.8 97.5 123.7
14.0
79.2
83.9
70.7
2.9
2.j
13.8
101.6
13.8
+a.1
136
42.3
11.4 8.6 6.7
19.8 23.5 ‘24.2
105
5.3
LT.6
113.0
114.9 88. I 9.24 78.4 ~~~
9.0 1.7 -.
13.2
r3.7 10.7 - .. ..--
9.5
100
IO1 102
124 93 122 ~___. .-
at 0’.
sition and loss of lipid the samples were not dried or filtered before Sephadex chromatography, hence it is difficult to obtain an accurate weight for the initial extracts. However, when Fractions I, II and III from the Sephadex column were recombined for samples from sheep, horse, and cat and rechromatographed, the distributions obtained in the new fractions were within 2%, respectively, of the original values in each case. Table 11 lists total lipid (Fraction I plus Fraction II from the Sephadex chromatography}, and also percentages of cholesterol, glycolipid, and phospholjpid in the erythrocyte of each species examined. Total lipid in mg/ml is relatively constant for all species. Conversely, total lipid per cell is highly variable among the species, reflecting a similarly wide range in the mean corpuscular volume of the different species erythrocyteP. Cholesterol averages about 26% of the total lipid and shows little species variation. Glycolipids show more species variation than either cholesterol or total phosTABLE
II
LIPID DISTRIBUTION -__.-_Species
IN ERYTHROCYTES --
Total lipid* (mglml
FROM
Total lipid* (g/cell) **
Packed cells)
Rat Rabbit Pig Dog Horse Sheep COW Goat
Cat Guinea pig
5.08
4.57 4.33 5.76 5.37 4.91 4.44 6.14 6.04 5.72
MAMMALIAN
Per cent of total lipid* ___. Gljdipid Neutral
SPECIES ---__l_.-
..___-..___ Ganglioside
-_--
Phospholipid
lipid 3*rg*10-'3
24-7
4.15 *IO-=
28.9
2.52.w'3
26.8 24.7 24.5 26.5
4.84*10-I3 2.58.10-13
1.62 -IO-~~ 2.58.10-1” 1.23 *IO-= 3.45’ 10-13 4.41. x0-13
* Lipid of Fraction
** Calculated
VARIOUS -
10.9 8.0 2.5 2.2
27.5 26.2
17.9 3.1 15.2
26.8 27.0
I plus Fraction II. using mean corpuscular volumes23.
Biochim. Biophys. Acta, 144 (19G7) 221-232
2.0 0.8 IO.1
_.~
6.3 4.5 3.3 II.8
15.5 7.8 55 5-7 8.8 2.2
67.0 65.8 59.8 52.6 >z.a 63.2 64.8 50.2 61.3
55.6
LIPID COMPOSITION OF MAMMALIAN ERYTROCYTES
227
pholipids. Ganglioside varies from 2 to 16% of the total lipid, and the other glycolipids from I to 18%. There is no apparent correlation between the amounts of ganglioside and the other glycolipids. The gangliosides appear to be a rather complex mixture. The other glycolipids may be ceramide polyhexosides, or perhaps globosides?. These glycolipids were not characterized further, but their extremely polar thin-layer chromatography migration indicates that they may be di- or trihexoses, TABLE
III
REPRODUCIBILITY ANALYSIS
IN
BOTH
OF
THIN-LAYER
THIN-LAYER
CHROMATOGRAPHY CHROMATOGRAPHY
METHOD SOLVENT
FOR
SYSTEMS
PHOSPHOLIPID
PHOSPHORUS
FOR HORSE ERYTHROCYTE
EXTRACT, SEPHADEX FRACTION I Values are expressed in weight percent of total phospholipid Phospholipid
Phosphatidic acid Phosphatidyl ethanolamine* * * Phosphatidyl serine Phosphatidyl inositol Phosphatidyl choline Sphingomyelin Lysophosphatidyl choline
phosphorus. System 2**
SystemI* a
b
0.32 24.19 18.03 0.20 42.69 13.05 1.63
0.15 24.65 18.21 0.17 42.10 12.92 1.72
c
b
a
c
0.28
0.29
O.ZI
23.55 17.74 0.34 41.97 13.51 1.84
25.05 17.86 0.38 43.11 13.67 1.95
24.30 17.92 0.41 42.82 14.03 1.49
0.19 24.38 18.27 0.70 42.59 13.81 1.54
* 1. CHCl,-methanol-NH, (65 : 35 : 5, by vol.) ; 2. CHCl,-acetone-methanol-H,0 (5 : 2 : I : 0.5, by vol.). ** I. CHCl,-methanol-H,0 (65 : 35 : 4, by vol.) ; 2. butanol-acetic acid-H,0 (40: 20: 20, by vol.). *** Includes vinyl or glyceryl ethers if present.
and perhaps higher polymers. Cow and sheep erythrocytes contain practically none of these compounds, whereas those of the goat, which belongs to the same family, bovidae, contain large quantities. Fig. I presents the infrared spectra of phospholipids isolated from the erythrocytes of several species. The spectra were identified by comparison to spectra obtained on commercial reference compounds. The commercial standards were checked for purity by thin-layer chromatography; they were used for infrared comparisons only if they appeared chromatographically homogeneous in both z-dimensional thin-layer chromatography systems. The spectra of the ethanolamine phospholipids from all species show evidence of vinyl or glyceryl ether compounds as indicated by the reduced intensity of the carbonyl absorption at 1740 cm-l, and a lower carbonyl to phosphate ester absorption, 1740 cm-l to 1230 cm-l, ratio. The phospholipid class composition for each species is given in Table IV. No polyglycerol phospholipid (cardiolipin) was detected in any species. This result supports the current hypothesis that this particular compound is associated exclusively with mitochondriaa6. Sulphatide, cerebroside, and lysophospholipid (except for small amounts of lysolecithin) were also absent. Fig. 2 shows representative thin-layer chromatography separations of the erythrocyte lipids of dog, cat, horse and rat. The results obtained here show striking species variations in the choline phosphatides similar to those reported previously by other workers4-7~r~~18.However, absolutely no lecithin was detected in the erythrocytes of any of the true ruminants (cow, sheep, goat), and sphingomyelin accounted for about 50% of the total phospholipids. In contrast, the erythrocytes of the horse (a pseudoruminant, order perissoBiochim. Biophys, Acta, 144 (1967) 221-232
G. J. NELSON
228
WAVELENGTH 2.5 100
3 1
I
I
/,,I,
2000
1600
QL) 7
8
9 IO 12
60
4000
3000
FREQUENCY
1200
600
(Cdl
Fig. r. Infrared spectra of phospholipids isolated from thin-layer chromatogram as described in the text. Curve A, phosphatidyl inositol from cat erythrocytes. Curve B, phosphatidyl serine from goat erythrocytes. Curve C, sphingomyelin from cow erythrocytes. Curve D, lecithin from dog erythrocytes. Curve E, unidentified phospholipid from sheep erythrocytes. Curve F, phosphatidyl ethanolamine (probably contains glyceryl ether analogues) from goat erythrocytes. Curves A, C, and E were obtained on solid samples in KBr pellets (5.0 mm diameter, 50 mg KBr). Curve B is asolutionspectruminCC1,. 50 mg/ml, in a 0.1.ml path, NaCl cell. Curve D and F are solution spectra in CS,, 50 mg/ml in a o.r-mm path, NaCl cell. TABLE
IV
PHOSPHOLIPID
DISTRIBUTION*
IN
Phospholipid
ERYTHROCYTES _____
FROM
VARIOUS
MAMMALIAN
SPECIES
Sp&?S
Rabbit
Rat
Pig
Dog
Horse
Sheep
Cow
Goat
Cat
Phosphatidic acid Phosphatidyl ethanolamine** Phosphatidyl serine Phosphatidyl inositol Phosphatidyl choline Sphingomyelin Lysophosphatidyl choline Unidentified
co.3
1.6
21.5
31.9
IO.8
12.2
3.5 47.5 12.8 3.8
1.6 33.9 19.0 co.3
Guinea
Pig
_______ <0.3
0.5
29.7
22.4
17.8 1.8 23.3 26.5 0.9
15.4 2.2 46.9 IO.8 I.8
to.3
24.3
18.0 <0.3 42.4 ‘3.5 1.7
ico.3
26.2
I4.I 2.9 51.0 4.8
<0.3
co.3
0.8
4.2
zg.1
27.9
22.2
24.6
19.3 3.7 40.2
20.8 4.6 45.9 0.8
13.2 7.4 30.5 26.1 <0.3 -
16.8 2.4 41.r II.1
<0.3 -
1.7 -_-. * Data are average phosphorus determinations obtained using duplicate with two pairs of thin-layer chromatography solvents for each sample ; presented as weight percent phosphorus of the total phospholipid phosphorus recovered for each phospholipid. ** Includes ethanolamine vinyl or glyceryl ether, if present. Biochim.
Biophys.
Acta,
144 (1967) 221-232
LIPID COMPOSITION OF MAMMALIAN
ERYTROCYTES
229
Fig. 2. Thin-layer chromatograms of dog, horse, cat, and pig erythrocytes Iipid after purification by Sephadex column chromato~aphy. Differences in various lipids, particularly the glycolipids, are readily apparent. The solvent used was chloroform-methanol-ammonia (65 : 34 : 4, by vol.)in the first &me&on followed by chloroform-acetone-methanol-acetic acid-water (5 :z :I :0.5, by vol.). Abbreviations are: Ch, cholesterol; H, heme pigments; PA, phosphatidic acid; PE, phosphatidyl ethanolamine; PC, lecithin; PS, phosphatidyl serine; PI, phosphatidyl inositol; Sp, sphingomyelin; GL, glycolipid ; 0, origin. ~uc~yla) are high in lecithin and rather low in sphingomyelin, and their total phospholipid dist~bution is more similar to that of the dog or rat than to that of the true ruminant. Sphingomyelin is quite low in certain other species such as the guinea pig and dog, but it is never zero as is the lecithin of the true ruminant. The erythrocytes of true ruminants appear to contain a phospholipid, as yet unidentified, not found in the erythrocytes of the other species, all of which contain lecithin. The amount is largest in the sheep (4.8%) and lowest in the goat (0.8%). The compound gives a negative test for free amino groups and for choline, and judging from its thin-layer chromatography migration it is probably acidic, not a zwitterion and may be phosphatidyl glycerol. Fig. 3 shows a series of thin-layer chromatography plates on which this compound is distinguished from a lecithin standard in sheep erythrocytes. Only traces of lysolecithin were detected in these species, except for the rat; even in the rat the observed value could be artifactual or a misinterpretation of the character of the thin-layer chromatography spot, since the amount was too small to be analyzed in detail. No trace of lysolecithin could be detected in the sheep, cow or goat, consistent with the lack of lecithin in the erythrocytes of these species. Biochiwt. Biophys. Acta, 144 (x967) 221-232
G. J. NELSOW
a lecithin standard, 5 pg; B, sheep erythrocyte lipid extract after Sephadex column chromatography, 500 pg; C, lecithin standard and the sheep erythrocyte lipid spotted on the same plate, in the same amounts as on plates A and B. This figure shows that lecithin is absent from sheep erythrocytes and that the spot labelled UK is not lecithin. Developing solvents were the same as listed in Fig. a. Spots were visualized by charring. Abbreviations are defined in the caption to Fig. 2. UK designates unidentified phospholipid.
Phosphatidyl ethanolamine or its vinyl or glyceryl ether analogue is the major non-choline phospholipid present in all of the species analyzed here. Phosphatidyl ethanolamine varied between a low of 21.5% in the rat and a high of 31.9% in the rabbit. Phosphatidyl serine varied over a slightly greater range, from 110/o in the rat to 21% in the goat. Phosphatidic acid and phosphatidyl inositol were the only other phospholipids detected; together they constituted less than 5% of the total phospholipids in most cases. The quantitative values reported in Table IV are subject to experimental errors of -& 5% for the major components present in amounts greater than 5% of the total phospholipids, while minor components can be in error as much as & 207/,, determined by thin-layer chromatography analyses performed in triplicate on horse and sheep samples using both thin-layer chromatography systems. See Table III for an example of the reproducibility of the phosphorus determinations. UISCUSSION
The results of DE GIER AND VAN DEENEN~ for the choline phospholipids in sheep, cow, pig, rabbit and rat are essentially in agreement with these results. Sheep and cow erythrocytes were high in sphingomyelin and low in lecithin, while those of the rat, rabbit, and pig were higher in lecithin than in sphingomyelin. A major discrepancy between this work and theirs was in the values for serine phosphatides. The highest values reported by DE GIER AND VAN DEENEN were only 7% for the bovine erythrocyte; much lower values were found in the other species. The average value for all species in this work was about 15%. The values reported by DAWSON, HEMINGTON AND LINDSAY~~ and CONDREA et aLzs for phosphatidyl serine were higher than those of DE GIER AND VAN DEENEN, although lower than those reported here. In his original reports, TURNER*~F~~ indicated that no lecithin existed in ruminants, but later investigators usually found small but measurable amounts4-7*26. The present is report in agreement with TURNER’S original finding that no detectable lecithin (detection limits 0.27~) occurs in cow, sheep or goat erythrocytes. Some of the past confusion in the literature may well have arisen from failure to remove all of the serum lipids from the packed-cell preparation. In all of these Riochiv~.Rio&~.
Acta,
144 (1967)
221-232
LIPID COMPOSITIONOF MAMMALIAN ERYTROCYTES
231
species the serum contains lecithin, which is, in fact, the major phospholipid of the soluble lipoprotein without exception (G. J. NELSON, paper in preparation). In previous work on the composition of neutral lipid of the erythrocyteao, it was concluded that cholesterol accounted for more than 99% of the neutral lipid in the erythrocyte membrane, and reports indicating otherwise were probably the results of failure to remove serum lipoproteins or leukocytes from the erythrocyte preparation. Hence, it may be that previous reports4-’ of lecithin in sheep, cow and goats actually reflected the phospholipid composition of the serum lipoprotein. Another source of error that might have influenced the results of previous investigations is the autoxidation of phosphatidyl ethanolamine to an as yet unidentified compound (not, however, lysophosphatidyl ethanolamine). The conversion occurs rapidly in the presence of oxygen and may well be catalyzed by heme pigments in the lipid extract. This substance can be confused with lecithin in one-dimensional thin-layer chromatography or silicic-acid column chromatography. DODGE AND PHILLIPS~’ have recently studied this problem in human erythrocyte lipid extracts in some detail. Little earlier quantitative information is available on the amounts of ganglioside and other glycolipids in the erythrocytes
of various species. This work, however, con-
firms previous results which indicated that the glycolipid content of the erythrocytes of various species is highly variable28-30. Attempts to correlate known physiological differences between the erythrocytes of various species with the lipid composition of their erythrocytes has not proven particularly successful. JACOBS, GLASSMAN AND PARPART~~ reported data on the permeability of the erythrocytes of several species, and noted striking differences in the rate of hemolysis and in permeability to various non-electrolytes. VAN DEENEN et aZ.32 using the data of JACOBS, GLASSMAN AND PARPART 31attempted to correlate permeability of the erythrocyte with its lipid composition and suggested that increased permeability was related to increasing lecithin content of the erythrocyte. Recently DE GIER, VAN DEENEN AND VAN SENDEN~~were able to distinguish several animal species by the rates at which their erythrocyte hemolyzed in 0.3 M glycerol, but these authors concluded that no significant correlation could be drawn along these lines although there was a trend suggesting that erythrocytes
containing large amounts
of lecithin were often more permeable than those containing only small amounts. TURNER and co-workers18~17suggested that lysis by snake venom is related to phospholipid composition of the erythrocyte, particularly the lecithin content. However, CONDREA et aLz6, who discussed this phenomenon in detail, noted that although some ruminant erythrocytes were indeed resistant to lysis, the camel erythrocyte, which is high in lecithin, was also resistant. These authors concluded that venominduced lysis is related primarily to the proteins of the erythrocyte membrane, not to the lipids. The observations suggest that the key to membrane function reside in the protein-lipid interaction and that lipid-lipid or lipid-environmental interactions have less physiological significance. KORN has in a recent review34summarized criticism of the current molecular theories of membrane structure. Current work on mitochondria2593smay also indicate a need for drastic revision of the simple bilayer leaflet theory. Lipids and proteins are certainly integral parts of all biological membranes now known. Thus it is reasonable that the key to the structure and function of memBiochim. Biophys. Acta, 144 (1967)
221-232
232
6. J. NELSON
brane is in the interaction of these substances, particularly since studies of either alone have failed to provide an acceptable model for a biological membrane.
The technical assistance of ROBERT BOOTH is gratefully acknowledged. The author is also indebted to Dr. GECXXX ROUSEIR for many useful discussions pertaining to thin-layer rneth~o~~ and for stjrnn~a~~~~my interest in this area. This work was performed under the auspieces of the U.S. Atomic Energy &omm~ss~on. REFERENCES I G. ROUSER,C.GALLI,E. LIEBER,M. 1;.BLANK AND 0.S. P~r~9r~,J.-~?~.OibCltemdsts'Soc., 41 (1964)836. 2 A. N. SIAICOTOS AND G, ROUSER, 1, Am. CM Cb~&s’ Sot., *!A(1965)gr3. 3 M. A. WELW AWD f.C. DITT~~ER,BiOC~~~~StYy, 5 (1g66) 340$‘ 4 .D.J. HANAHAK, R. x. WATTS AXD D. PjtPPAJOHN,J. Lip&Be&, I {i9f.@42r. 3 J.DE GIE~ AND L. L. M. VAN DEENEN, Biuchim. Bioph~s. AC&, 49 (1961)286. 6 1.DE GIBR AND L. L. M. VAN DEENEN,B~o&z~~.Biofihys. Acta, 84 (1964)294.
BBA
ET BIOPHYSICA
ACTA
221
55366
LIPID
COMPOSITION
OF ERYTHROCYTES
IN VARIOUS
MAMMALIAN
SPECIES
GARY
J. NELSON
Bio-Medical Research Division, Lierermore, Calif. (U..S.A .) (Received
February
(Revised
manuscript
Lawrence Radiaiion
Laboratory,
University
of California,
13th, 1967) received April zsth,
1967)
SUMMARY
Lipid distribution in the erythrocyte was investigated in several common mammals, including rat, rabbit, pig, dog, horse, sheep, cow, goat, cat and guinea pig. Lipids were extracted from fresh, thoroughly washed, whole packed cells. The lipids were purified by Sephadex column chromato~aphy and separated by thin-layer chromato~aphy. Infrared spectrophotometry was also used to identify components. The distribution of cholesterol, glycolipids, and the phospholipids is reported. Cholesterol was approx. 26% of total lipid in all species while phospholipids ranged between 50 and 70%. The glycolipids were considerably more variable, accounting for 5.3% of the lipids in the rabbit erythrocyte and 23.5% of the lipids in the horse erythrocyte. The glycolipid fraction was not analyzed in detail. The phospholipids were separated into the various classes, but neither vinyl nor glyceryl ether compounds were separated from diacyl derivatives. The phospholipids common to all species were phosphatidic acid, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidy1 inositol, and sphingomyelin. There are, however, marked species variations in the relative abundance of these erythrocyte phospholipids. No lecithin was detected in sheep, cow, or goat. However, an as yet unidentified phospholipid was detected in these species.
INTRODUCTION
The procedures now available allow accurate estimation and quantitation of most lipids in any organ or subcellular particle l - 3. Yet despite the current interest in the lipid composition of the erythrocyte, accurate analytical data on lipid composition is fragmentary at best for most mammalian species. Earlier investigators often grouped several types of lipids when they reported their results+’ or used analytical techniques that failed to resolve all components%*. Human erythrocytes, perhaps because of their chnical interest, have been studied most frequently and with most precision: WAYS AND HANAHANIO, WAYS, REED AND HANAHAN’~, VAN GASTEI, et uZ.la, Bdochim. Biophys.
Acta,
I++ (1967) 221-232
222
G.
J. NELSON
HILL, KUKSIS AND BEVERIDGE~~~‘~,and FARQ!UHAR~~have reported detailed studies on the distribution of the erythrocyte phospholipids. The latest techniques have not heretofore been used, however, to clarify the situation in other mammals. Data now in the literature indicate marked species differences in the distribution of phospholipids, particularly lecithin, in the mammalian erythrocyte. TURNER in some pioneering studieslapr7 reported that lecithin was absent in ruminant erythrocytes, but abundant in most other species including humans. DAWSON, HEMINGTON AND LINDSEY’*, DE GIER AND VAN DEENEN~, and HANAHAN, WATTS AND PAPPAJOHN~ confirmed these results, but in most of these reports, including TURNER’S original work, many of the lipids present were either not resolved or not reported. Because accurate analytical data on erythrocyte lipids are pertinent to theories of the structure and function of their membrane, a series of common mammalian species was investigated in an attempt to elucidate their erythrocyte lipid composition using accurate analytical methods. This report gives the results for total lipid, cholesterol, glycolipid, and phospholipid of the erythrocytes of several domestic mammals, both herbivores and carnivores. The animals investigated were rat, rabbit, pig, dog, horse, sheep, cow, goat ,cat, and guinea pig. MATERIALSAND METHODS All solvents used in this work were redistilled from glass stills and deoxygenated by bubbling N, through them before use. Silica Gel HR, 20 cm x 20 cm borosilicate glass plates, thin-layer chromatographic plate spreader, and spreading template were obtained from Brinkman Instruments, Westbury, New York. MgSiO, was obtained from Allegheny Chemical, Butler, New Jersey. Sephadex was purchased from Pharmacia Fine Chemicals, New York. Standard phospholipids were obtained from General Biochemicals, Chagrin Falls, Ohio, and Applied Science Laboratories, State College, Pennsylvania; they included phosphatidic acid, phosphatidyl ethanolamine, phosphatidyl serine, lecithin, lysolecithin, and sphingomyelin. Phosphatidyl inositol was a gift of Dr. C. BALLOU. Infrared spectra were determined in a sealed liquid cell with an NaCl window, or, alternatively, from a pellet prepared from the solid sample mixed with KBr (infrared grade, Harshaw Chemical Company, Cleveland, Ohio), and were recorded on a Perkin-Elmer Model 521 double-beam infrared-grating spectrophotometer. Sampling
and extraction
All animals in this investigation were maintained at this laboratory using standard animal husbandry methods; except the horse and pig, samples from which were obtained from local slaughter houses using only freshly killed animals. Only sexually mature animals in good health were selected for sampling. The guinea-pig, rat and rabbit samples were pooled from 6, 3 and 2 animals, respectively. All other samples were drawn from a single animal. Blood was drawn by venipuncture into sterile plastic blood bags as reported earlier’@ for the large animals in this series, or directly into polypropylene centrifuge tubes for the small animals. Heparin, 500 USP units per IOO ml blood, was the anticoagulant. The blood was cooled immediately to o0 in an ice bath and kept at that temperature throughout processing and until subsequent analysis. Biochim.
Biophys.
Acta,
144 (1967)
221-232
LIPID COMPOSITIONOF MAMMALIANERYTROCYTES
223
Erythrocytes were separated from plasma by centrifugation at 1570xg for 20 min in a refrigerated centrifuge. The plasma was removed by aspiration, and the packed cells were washed 3 or 4 times with an equal volume of 310 mosM phosphate buffer (pH 7.4). After each washing, the upper phase, and the top 2 or 3 mm of packed cells, was removed by aspiration. This procedure ensured almost complete removal of other formed elements of blood from the final erythrocyte preparation. Smears of the packed red cells stained with Giemsa stain yielded less than oneleucocyteper 104 erythrocyte in all cases. The cells were extracted immediately after the final centrifugation. Extraction
procedure
The packed cells were transferred to the extraction flask with a volumetric pipette of appropriate size, usually 20 ml. The cells were added slowly to cold methanol that was continuously stirred by a magnetic mixer. After the cells were introduced, the flask was brought to volume with chloroform and vigorous stirring was continued for 5 min. The volumes of methanol and chloroform were selected to yield a final chloroform-to-methanol ratio of Z/I (v/v) and a solvent-to-packed-cell ratio of 50/r (v/v). The solution was then filtered using a fast, pre-washed filter paper (Whatman 41H, or Schleicher and Schuell Sharkskin) into a round-bottomed boiling flask. The solvent was removed by rotary evaporation at low temperation (~15”) and a reduced pressure of N,. Before the flask became dry, the residue was transferred to a small volumetric flask (2 to 5 ml) with chloroform-methanol (rg : I, v/v). The crude lipid extract was then chromatographed on a Sephadex column to separate gangliosides and remove non-lipid contaminants. Re-extraction of the insoluble matter with chloroform-methanol (2 : I, v/v) yielded less than 0.2% of the lipid obtained from the initial extraction. Digestion of the residue with aqueous KOH (ref. 19) indicated that no more than 0.05% total fatty acid in the cells remained after the solvent extraction. Sephadex
column chromatography The procedure used was essentially that of SIAKOTOS AND ROUSERS with only slight modifications. The Sephadex (G-25, coarse) was washed and the column was prepared as described2, except that the pre-washing of the packed column was simplified by going through the normal elution scheme once after packing the column and before applying the sample. Columns (2.5 cm x IO cm) were fitted with Teflon needle-valve stopcocks allowing easy adjustment of the flow rate. Three fractions were collected. Fraction I, 170 ml of chloroform-methanol (19: I, v/v) satd. with water, contained all the neutral lipids, phospholipids and glycolipids other than gangliosides. Fraction II, 350 ml of 5 parts chloroform-methanol (19:1, v/v) and I part acetic acid satd. with water, followed by 170 ml of 5 parts chloroform-methanol (9: I, v/v) and I part acetic acid satd. with water, contained the gangliosides. Fraction III, 350 ml of methanol-water (I: I, v/v) contained the non-lipid impurities in the initial extract. Fractions were collected at 20’. The first fraction was collected at a flow rate of I to 2 ml/min and the following fractions at 3 to 4 ml/min. A column was allowed to stand in methanol-water (I : I, v/v) for at least 48 h before reuse, and 500 ml of chloroform-methanol (19:1, v/v) Biochim.
Biophys.
Acta,
144 (1967) 221--2p
224
G. J. NELSON
satd. with water was passed through the column immediately before the sample was applied. The solvent was removed from Fraction I as described above for the initial red cell extract, without allowing the flask to become dry before transferring the sample to a glass-stoppered graduated cylinder with 5 ml chloroform. At this time an aliquot was weighed to determine the total amount of lipid in this fraction. The remainder was stored as the solution at --IO' until further analyses could be performed. Fraction II was handled similarly except that it was transferred to a tared 8-ml screw-cap vial with chloroform-methanol (19: I, v/v) and the solvent was evaporated by passing a stream of N, over it. The sample was then placed in desiccator under vacuum for at least 2 h, weighed, and stored dry at -IO' until further analysis. Fraction III was taken to dryness on the rotary evaporator, transferred to a tared S-ml screw-cap vial with methanol-water (4:1, v/v) and then handled like Fraction II, except that further analysis was not usually performed. Analytical procedures for lipids in Fractions
I and II
Cholesterol was analyzed in all samples, using gas-liquid chromatography, thinlayer chromatography, and infrared spectrophotometry. The details and accuracy of the procedures have been reported elsewherezO. The phospholipids were separated by two-dimensional thin-layer chromatography using methods developed by ROUSER and co-workers1~21with thin-layer chromatography plates spread with Silica Gel HR mixed with 10% MgSiO, by wt. Layer thickness was 0.25 mm. Plates were activated for 20 min at IZOO,then cooled in air for 30 min before the sample was applied. Samples and standards were spotted on the thin-layer chromatography plates from Lang-Levy micropipes of appropriate volumes, usually IO ~1. Approx. 500 pg of phospholipid was applied to the thin-layer chromatography plate. Samples were run in both pairs of solvent system developed by ROUSERand co-workers1~21; the results in the two systems were the same within experimental error which was determined by running triplicate determinations on horse and sheep extracts. One pair of solvents consisted of chloroform-methanol-ammonia (65 : 35 : 5, by vol.) followed by chloroform-acetone-methanol-acetic acid-water (5 : 2 : I :I :0.5, by vol.) in the second dimension. The second pair consisted of chloroform-methanol-water, (65 : 35 : 4, by vol.) followed by butanol-acetic acid-water (40: 20 :20, by vol.) in the second dimension. Spots were visualized with a char reagent (0.6 g potassium dichromate per IOO ml of 55% by wt. H2S0JZ2, which was sprayed on the thin-layer chromatography plate after the developing solvents had evaporated completely. The plate was charred by heating for 20 min at 180’ in a forced-draft oven. The plates were photographed as previously described18 and also viewed under ultraviolet light to detect trace components. Phospholipids were analyzed on the charred plates by phosphorus analysis by a1.The spots were scraped off the the method of ROUSER,SIAKOTOSAND FLEISCHER plate with a razor blade into 5-ml glass-stoppered tubes to which the color reagents were added directly without removing the absorbant. The total sample was also spotted in the opposite corner of the plate so that total recovery could be estimated. Color development was done by the methods previously describedlo. Biochint.
Biophys.
Acta.
I++ (1967)
221-232
LIPID COMPOSITION OF MAMMALIAN
ERYTROCYTES
225
Additional plates spotted and developed in an identical manner were sprayed with ninhydrin or Dragendorff’s reagent to locate amino- and choline-containing lipids respectively. Glycolipids were located with cr-naphthol spray. Still other plates were sprayed with water and the spots outlined while the plates were wet. They were then dried, the spots were scraped into sintered-glass funnels, and the lipids were eluted with an appropriate solvent. Lipids recovered from the thin-layer chromatography plates were repurified by passing through small Sephadex columns. The solvent was removed by a stream of N, and the infrared spectra of the lipids were obtained as described above. Fraction II containing the gangliosides was analyzed for phosphorus and cholesterol and its infrared spectrum was recorded; further characterization of the gangliosides was not attempted at this time. The phosphorus content of Fraction II averaged0.05%; no cholesterol was detected in this fraction in any sample. Other glycolipids in Fraction I were estimated by subtracting the amounts of phospholipid and cholesterol found in this fraction from the total weight, but detailed analyses were not attempted. RESULTS
The analyses reported here were performed on blood drawn from one animal from each of the species studied except when it was necessary to pool samples from several animals, as noted above. Previous studies on sheep’@ and unpublished work on cows carried out in this laboratory, indicated that erythrocyte lipid distribution does not vary significantly among members of the same species. Hence, analyzing the erythrocyte lipids from a single animal, randomly selected, in each species should yield a representative analysis of the erythrocyte lipid for that species. The results in Tables I and II are data from single experiments for each species. Table III presents the results of a triplicate analysis of horse erythrocyte phospholipids run by both thin-layer chromatography procedures. The results show the excellent reproducibility of the method. Data in Table IV are averages of the phosphorus determinations of the Sephadex F-I fraction run in both thin-layer chromatography systems for all species in this investigation. Table I presents the results from the Sephadex column chromatography of the chloroform-methanol extracts. Recoveries of phosphorus from the Sephadex columns were close to or over 100% in all cases. Most of the phosphorus was recovered in Fraction I except for a trace of water-soluble phosphorus recovered in Fraction III that was probably inorganic material. Recoveries well over 100% (dog, goat and guinea pig) do not represent errors of the method but rather indicate the presence of material insoluble in chloroform-methanol (rg: I, v/v) and later solubilized with either acid solvents or methanol-water (I : I, v/v). Material from the initial extracts was transferred quantitatively to Sephadex columns of possible; this usually included a fair amount of particulate (nonlipid) material. The low recoveries (rabbit, cat) may have resulted from failure to transfer all of the extracted material to the Sephadex column. Since this insoluble material is not lipoidal, the accuracy of the succeeding analytical procedures was unaffected. The difficulties in quantitating the recoveries from the Sephadex columns result primarily from the nonlipid material carried in the initial extract. To avoid decompoBiochim.
Biophys.
Acta,
144 (1967) 221-232
G. J, NELSOIi
226 TABLE
I
RESULTS OF SEPHADEX COLUMN CHROMATOGRAPHY FROM VARIOUS MAMMALIAN SPECIES
OB CHLOROFORM-METHANOL
The solution used was chloroform-mcthanoi Species
(2: 1, v/v) ~Hemato- Volume wit* packed cells
Sex
---__ Rat, Sprague-Dawley Rabbit, Rev.7 Zealand white Pig, Poland-China Dog, beagle
M M M M
.~_ 56 42 54 53
Horse, western quarter F Sheep, Hampshire F Cow, Holstein F Goat, African pigmy M Cat, domestic short hair F Guinea pig M -* Determined at 1570 x g for 20 min
extracted (ml) ___._ 7.0
52 50 so 40 35 40
EXTRACT
OF
ERYTHROCYTES
._- .__-_ Weight of chromatographic Hecovevy fractions -..-I_-._ ..III Eg) (mg) (?I) img) _ -_--_ _ _---_._ ..~_ 2.3 9.0 101 34.0
Total wt. of extract (mg) 45.6
16.0 20.0 20.0
92.6 100.2 120.1
IO.1 25.0 27.4 15.2 16.8
69.9 143.4 142.8 97.5 123.7
14.0
79.2
83.9
70.7
2.9
2.j
13.8
101.6
13.8
+a.1
136
42.3
11.4 8.6 6.7
19.8 23.5 ‘24.2
105
5.3
LT.6
113.0
114.9 88. I 9.24 78.4 ~~~
9.0 1.7 -.
13.2
r3.7 10.7 - .. ..--
9.5
100
IO1 102
124 93 122 ~___. .-
at 0’.
sition and loss of lipid the samples were not dried or filtered before Sephadex chromatography, hence it is difficult to obtain an accurate weight for the initial extracts. However, when Fractions I, II and III from the Sephadex column were recombined for samples from sheep, horse, and cat and rechromatographed, the distributions obtained in the new fractions were within 2%, respectively, of the original values in each case. Table 11 lists total lipid (Fraction I plus Fraction II from the Sephadex chromatography}, and also percentages of cholesterol, glycolipid, and phospholjpid in the erythrocyte of each species examined. Total lipid in mg/ml is relatively constant for all species. Conversely, total lipid per cell is highly variable among the species, reflecting a similarly wide range in the mean corpuscular volume of the different species erythrocyteP. Cholesterol averages about 26% of the total lipid and shows little species variation. Glycolipids show more species variation than either cholesterol or total phosTABLE
II
LIPID DISTRIBUTION -__.-_Species
IN ERYTHROCYTES --
Total lipid* (mglml
FROM
Total lipid* (g/cell) **
Packed cells)
Rat Rabbit Pig Dog Horse Sheep COW Goat
Cat Guinea pig
5.08
4.57 4.33 5.76 5.37 4.91 4.44 6.14 6.04 5.72
MAMMALIAN
Per cent of total lipid* ___. Gljdipid Neutral
SPECIES ---__l_.-
..___-..___ Ganglioside
-_--
Phospholipid
lipid 3*rg*10-'3
24-7
4.15 *IO-=
28.9
2.52.w'3
26.8 24.7 24.5 26.5
4.84*10-I3 2.58.10-13
1.62 -IO-~~ 2.58.10-1” 1.23 *IO-= 3.45’ 10-13 4.41. x0-13
* Lipid of Fraction
** Calculated
VARIOUS -
10.9 8.0 2.5 2.2
27.5 26.2
17.9 3.1 15.2
26.8 27.0
I plus Fraction II. using mean corpuscular volumes23.
Biochim. Biophys. Acta, 144 (19G7) 221-232
2.0 0.8 IO.1
_.~
6.3 4.5 3.3 II.8
15.5 7.8 55 5-7 8.8 2.2
67.0 65.8 59.8 52.6 >z.a 63.2 64.8 50.2 61.3
55.6
LIPID COMPOSITION OF MAMMALIAN ERYTROCYTES
227
pholipids. Ganglioside varies from 2 to 16% of the total lipid, and the other glycolipids from I to 18%. There is no apparent correlation between the amounts of ganglioside and the other glycolipids. The gangliosides appear to be a rather complex mixture. The other glycolipids may be ceramide polyhexosides, or perhaps globosides?. These glycolipids were not characterized further, but their extremely polar thin-layer chromatography migration indicates that they may be di- or trihexoses, TABLE
III
REPRODUCIBILITY ANALYSIS
IN
BOTH
OF
THIN-LAYER
THIN-LAYER
CHROMATOGRAPHY CHROMATOGRAPHY
METHOD SOLVENT
FOR
SYSTEMS
PHOSPHOLIPID
PHOSPHORUS
FOR HORSE ERYTHROCYTE
EXTRACT, SEPHADEX FRACTION I Values are expressed in weight percent of total phospholipid Phospholipid
Phosphatidic acid Phosphatidyl ethanolamine* * * Phosphatidyl serine Phosphatidyl inositol Phosphatidyl choline Sphingomyelin Lysophosphatidyl choline
phosphorus. System 2**
SystemI* a
b
0.32 24.19 18.03 0.20 42.69 13.05 1.63
0.15 24.65 18.21 0.17 42.10 12.92 1.72
c
b
a
c
0.28
0.29
O.ZI
23.55 17.74 0.34 41.97 13.51 1.84
25.05 17.86 0.38 43.11 13.67 1.95
24.30 17.92 0.41 42.82 14.03 1.49
0.19 24.38 18.27 0.70 42.59 13.81 1.54
* 1. CHCl,-methanol-NH, (65 : 35 : 5, by vol.) ; 2. CHCl,-acetone-methanol-H,0 (5 : 2 : I : 0.5, by vol.). ** I. CHCl,-methanol-H,0 (65 : 35 : 4, by vol.) ; 2. butanol-acetic acid-H,0 (40: 20: 20, by vol.). *** Includes vinyl or glyceryl ethers if present.
and perhaps higher polymers. Cow and sheep erythrocytes contain practically none of these compounds, whereas those of the goat, which belongs to the same family, bovidae, contain large quantities. Fig. I presents the infrared spectra of phospholipids isolated from the erythrocytes of several species. The spectra were identified by comparison to spectra obtained on commercial reference compounds. The commercial standards were checked for purity by thin-layer chromatography; they were used for infrared comparisons only if they appeared chromatographically homogeneous in both z-dimensional thin-layer chromatography systems. The spectra of the ethanolamine phospholipids from all species show evidence of vinyl or glyceryl ether compounds as indicated by the reduced intensity of the carbonyl absorption at 1740 cm-l, and a lower carbonyl to phosphate ester absorption, 1740 cm-l to 1230 cm-l, ratio. The phospholipid class composition for each species is given in Table IV. No polyglycerol phospholipid (cardiolipin) was detected in any species. This result supports the current hypothesis that this particular compound is associated exclusively with mitochondriaa6. Sulphatide, cerebroside, and lysophospholipid (except for small amounts of lysolecithin) were also absent. Fig. 2 shows representative thin-layer chromatography separations of the erythrocyte lipids of dog, cat, horse and rat. The results obtained here show striking species variations in the choline phosphatides similar to those reported previously by other workers4-7~r~~18.However, absolutely no lecithin was detected in the erythrocytes of any of the true ruminants (cow, sheep, goat), and sphingomyelin accounted for about 50% of the total phospholipids. In contrast, the erythrocytes of the horse (a pseudoruminant, order perissoBiochim. Biophys, Acta, 144 (1967) 221-232
G. J. NELSON
228
WAVELENGTH 2.5 100
3 1
I
I
/,,I,
2000
1600
QL) 7
8
9 IO 12
60
4000
3000
FREQUENCY
1200
600
(Cdl
Fig. r. Infrared spectra of phospholipids isolated from thin-layer chromatogram as described in the text. Curve A, phosphatidyl inositol from cat erythrocytes. Curve B, phosphatidyl serine from goat erythrocytes. Curve C, sphingomyelin from cow erythrocytes. Curve D, lecithin from dog erythrocytes. Curve E, unidentified phospholipid from sheep erythrocytes. Curve F, phosphatidyl ethanolamine (probably contains glyceryl ether analogues) from goat erythrocytes. Curves A, C, and E were obtained on solid samples in KBr pellets (5.0 mm diameter, 50 mg KBr). Curve B is asolutionspectruminCC1,. 50 mg/ml, in a 0.1.ml path, NaCl cell. Curve D and F are solution spectra in CS,, 50 mg/ml in a o.r-mm path, NaCl cell. TABLE
IV
PHOSPHOLIPID
DISTRIBUTION*
IN
Phospholipid
ERYTHROCYTES _____
FROM
VARIOUS
MAMMALIAN
SPECIES
Sp&?S
Rabbit
Rat
Pig
Dog
Horse
Sheep
Cow
Goat
Cat
Phosphatidic acid Phosphatidyl ethanolamine** Phosphatidyl serine Phosphatidyl inositol Phosphatidyl choline Sphingomyelin Lysophosphatidyl choline Unidentified
co.3
1.6
21.5
31.9
IO.8
12.2
3.5 47.5 12.8 3.8
1.6 33.9 19.0 co.3
Guinea
Pig
_______ <0.3
0.5
29.7
22.4
17.8 1.8 23.3 26.5 0.9
15.4 2.2 46.9 IO.8 I.8
to.3
24.3
18.0 <0.3 42.4 ‘3.5 1.7
ico.3
26.2
I4.I 2.9 51.0 4.8
<0.3
co.3
0.8
4.2
zg.1
27.9
22.2
24.6
19.3 3.7 40.2
20.8 4.6 45.9 0.8
13.2 7.4 30.5 26.1 <0.3 -
16.8 2.4 41.r II.1
<0.3 -
1.7 -_-. * Data are average phosphorus determinations obtained using duplicate with two pairs of thin-layer chromatography solvents for each sample ; presented as weight percent phosphorus of the total phospholipid phosphorus recovered for each phospholipid. ** Includes ethanolamine vinyl or glyceryl ether, if present. Biochim.
Biophys.
Acta,
144 (1967) 221-232
LIPID COMPOSITION OF MAMMALIAN
ERYTROCYTES
229
Fig. 2. Thin-layer chromatograms of dog, horse, cat, and pig erythrocytes Iipid after purification by Sephadex column chromato~aphy. Differences in various lipids, particularly the glycolipids, are readily apparent. The solvent used was chloroform-methanol-ammonia (65 : 34 : 4, by vol.)in the first &me&on followed by chloroform-acetone-methanol-acetic acid-water (5 :z :I :0.5, by vol.). Abbreviations are: Ch, cholesterol; H, heme pigments; PA, phosphatidic acid; PE, phosphatidyl ethanolamine; PC, lecithin; PS, phosphatidyl serine; PI, phosphatidyl inositol; Sp, sphingomyelin; GL, glycolipid ; 0, origin. ~uc~yla) are high in lecithin and rather low in sphingomyelin, and their total phospholipid dist~bution is more similar to that of the dog or rat than to that of the true ruminant. Sphingomyelin is quite low in certain other species such as the guinea pig and dog, but it is never zero as is the lecithin of the true ruminant. The erythrocytes of true ruminants appear to contain a phospholipid, as yet unidentified, not found in the erythrocytes of the other species, all of which contain lecithin. The amount is largest in the sheep (4.8%) and lowest in the goat (0.8%). The compound gives a negative test for free amino groups and for choline, and judging from its thin-layer chromatography migration it is probably acidic, not a zwitterion and may be phosphatidyl glycerol. Fig. 3 shows a series of thin-layer chromatography plates on which this compound is distinguished from a lecithin standard in sheep erythrocytes. Only traces of lysolecithin were detected in these species, except for the rat; even in the rat the observed value could be artifactual or a misinterpretation of the character of the thin-layer chromatography spot, since the amount was too small to be analyzed in detail. No trace of lysolecithin could be detected in the sheep, cow or goat, consistent with the lack of lecithin in the erythrocytes of these species. Biochiwt. Biophys. Acta, 144 (x967) 221-232
G. J. NELSOW
a lecithin standard, 5 pg; B, sheep erythrocyte lipid extract after Sephadex column chromatography, 500 pg; C, lecithin standard and the sheep erythrocyte lipid spotted on the same plate, in the same amounts as on plates A and B. This figure shows that lecithin is absent from sheep erythrocytes and that the spot labelled UK is not lecithin. Developing solvents were the same as listed in Fig. a. Spots were visualized by charring. Abbreviations are defined in the caption to Fig. 2. UK designates unidentified phospholipid.
Phosphatidyl ethanolamine or its vinyl or glyceryl ether analogue is the major non-choline phospholipid present in all of the species analyzed here. Phosphatidyl ethanolamine varied between a low of 21.5% in the rat and a high of 31.9% in the rabbit. Phosphatidyl serine varied over a slightly greater range, from 110/o in the rat to 21% in the goat. Phosphatidic acid and phosphatidyl inositol were the only other phospholipids detected; together they constituted less than 5% of the total phospholipids in most cases. The quantitative values reported in Table IV are subject to experimental errors of -& 5% for the major components present in amounts greater than 5% of the total phospholipids, while minor components can be in error as much as & 207/,, determined by thin-layer chromatography analyses performed in triplicate on horse and sheep samples using both thin-layer chromatography systems. See Table III for an example of the reproducibility of the phosphorus determinations. UISCUSSION
The results of DE GIER AND VAN DEENEN~ for the choline phospholipids in sheep, cow, pig, rabbit and rat are essentially in agreement with these results. Sheep and cow erythrocytes were high in sphingomyelin and low in lecithin, while those of the rat, rabbit, and pig were higher in lecithin than in sphingomyelin. A major discrepancy between this work and theirs was in the values for serine phosphatides. The highest values reported by DE GIER AND VAN DEENEN were only 7% for the bovine erythrocyte; much lower values were found in the other species. The average value for all species in this work was about 15%. The values reported by DAWSON, HEMINGTON AND LINDSAY~~ and CONDREA et aLzs for phosphatidyl serine were higher than those of DE GIER AND VAN DEENEN, although lower than those reported here. In his original reports, TURNER*~F~~ indicated that no lecithin existed in ruminants, but later investigators usually found small but measurable amounts4-7*26. The present is report in agreement with TURNER’S original finding that no detectable lecithin (detection limits 0.27~) occurs in cow, sheep or goat erythrocytes. Some of the past confusion in the literature may well have arisen from failure to remove all of the serum lipids from the packed-cell preparation. In all of these Riochiv~.Rio&~.
Acta,
144 (1967)
221-232
LIPID COMPOSITIONOF MAMMALIAN ERYTROCYTES
231
species the serum contains lecithin, which is, in fact, the major phospholipid of the soluble lipoprotein without exception (G. J. NELSON, paper in preparation). In previous work on the composition of neutral lipid of the erythrocyteao, it was concluded that cholesterol accounted for more than 99% of the neutral lipid in the erythrocyte membrane, and reports indicating otherwise were probably the results of failure to remove serum lipoproteins or leukocytes from the erythrocyte preparation. Hence, it may be that previous reports4-’ of lecithin in sheep, cow and goats actually reflected the phospholipid composition of the serum lipoprotein. Another source of error that might have influenced the results of previous investigations is the autoxidation of phosphatidyl ethanolamine to an as yet unidentified compound (not, however, lysophosphatidyl ethanolamine). The conversion occurs rapidly in the presence of oxygen and may well be catalyzed by heme pigments in the lipid extract. This substance can be confused with lecithin in one-dimensional thin-layer chromatography or silicic-acid column chromatography. DODGE AND PHILLIPS~’ have recently studied this problem in human erythrocyte lipid extracts in some detail. Little earlier quantitative information is available on the amounts of ganglioside and other glycolipids in the erythrocytes
of various species. This work, however, con-
firms previous results which indicated that the glycolipid content of the erythrocytes of various species is highly variable28-30. Attempts to correlate known physiological differences between the erythrocytes of various species with the lipid composition of their erythrocytes has not proven particularly successful. JACOBS, GLASSMAN AND PARPART~~ reported data on the permeability of the erythrocytes of several species, and noted striking differences in the rate of hemolysis and in permeability to various non-electrolytes. VAN DEENEN et aZ.32 using the data of JACOBS, GLASSMAN AND PARPART 31attempted to correlate permeability of the erythrocyte with its lipid composition and suggested that increased permeability was related to increasing lecithin content of the erythrocyte. Recently DE GIER, VAN DEENEN AND VAN SENDEN~~were able to distinguish several animal species by the rates at which their erythrocyte hemolyzed in 0.3 M glycerol, but these authors concluded that no significant correlation could be drawn along these lines although there was a trend suggesting that erythrocytes
containing large amounts
of lecithin were often more permeable than those containing only small amounts. TURNER and co-workers18~17suggested that lysis by snake venom is related to phospholipid composition of the erythrocyte, particularly the lecithin content. However, CONDREA et aLz6, who discussed this phenomenon in detail, noted that although some ruminant erythrocytes were indeed resistant to lysis, the camel erythrocyte, which is high in lecithin, was also resistant. These authors concluded that venominduced lysis is related primarily to the proteins of the erythrocyte membrane, not to the lipids. The observations suggest that the key to membrane function reside in the protein-lipid interaction and that lipid-lipid or lipid-environmental interactions have less physiological significance. KORN has in a recent review34summarized criticism of the current molecular theories of membrane structure. Current work on mitochondria2593smay also indicate a need for drastic revision of the simple bilayer leaflet theory. Lipids and proteins are certainly integral parts of all biological membranes now known. Thus it is reasonable that the key to the structure and function of memBiochim. Biophys. Acta, 144 (1967)
221-232
232
6. J. NELSON
brane is in the interaction of these substances, particularly since studies of either alone have failed to provide an acceptable model for a biological membrane.
The technical assistance of ROBERT BOOTH is gratefully acknowledged. The author is also indebted to Dr. GECXXX ROUSEIR for many useful discussions pertaining to thin-layer rneth~o~~ and for stjrnn~a~~~~my interest in this area. This work was performed under the auspieces of the U.S. Atomic Energy &omm~ss~on. REFERENCES I G. ROUSER,C.GALLI,E. LIEBER,M. 1;.BLANK AND 0.S. P~r~9r~,J.-~?~.OibCltemdsts'Soc., 41 (1964)836. 2 A. N. SIAICOTOS AND G, ROUSER, 1, Am. CM Cb~&s’ Sot., *!A(1965)gr3. 3 M. A. WELW AWD f.C. DITT~~ER,BiOC~~~~StYy, 5 (1g66) 340$‘ 4 .D.J. HANAHAK, R. x. WATTS AXD D. PjtPPAJOHN,J. Lip&Be&, I {i9f.@42r. 3 J.DE GIE~ AND L. L. M. VAN DEENEN, Biuchim. Bioph~s. AC&, 49 (1961)286. 6 1.DE GIBR AND L. L. M. VAN DEENEN,B~o&z~~.Biofihys. Acta, 84 (1964)294.