The lipid composition of normal rat thymus

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 55077

117

THE LIPID COMPOSITION OF NORMAL RAT THYMUS

DAVID ABRAMSON AND MELVIN BLECHER

Department of Biochemistry, Schools of Medicine and Dentistry, Georgetown University, Washington, D. C. (U.S.A.)

(Received June I&h, 1964)

SUMMARY

Thymic lipids (representing 2.6% of tissue wet weight) from two strains of normal, adult, white rats have been subjected to a variety of chromatographic techniques and chemical analyses leading to the complete separation and quantification of lipid components. Neutral lipids and phospholipids were separated on silicic acid by a batch procedure; neutral lipids represented from 66 to 80% of total lipids. Individual neutral lipids were separated and isolated using column chromatography on Florisil ; neutral lipids were mainly triglycerides (8~-85~~), with cholesterol (6-10%)

and cholesterol esters (3-4%) representing significant contributions to the total; smaller amounts of lower glycerides and free fatty acids were also present. Individual phospholipids were separated and isolated by two-dimensional thin-layer chromatography on Silica Gel G; 8 phospholipids were identified with phosphatidyl choline (49-50% of total), phosphatidyl ethanolamine (Is%), phosphatidyl inositol (IZ%),

sphingomyelin (IO-II~/~), and phosphatidyl serine (5-7%) representing the main components; small amounts of lysolecithin, phosphatidic acid and cardiolipin were also present.

The fatty acid composition of the isolated lipid fractions was determined by quantitative gas-liquid chromatography. Among the neutral lipids, unsaturated fatty acids predominated (48-80O/~ of total acids) ; among unsaturated fatty acids, oleic and linoleic represented the largest proportion, although in certain of the lower glycerides hexadecatrienoic acid represented 35 to 52% of total fatty acids; of the saturated acids, palmitic, stearic and arachidic acids constituted the majority.

Unsaturated fatty acids predominated in most, but not all, of the phospholipids; this relationship depended on the strain of rat. Although oleic, linoleic and linolenic acids comprised a large portion of the unsaturated acids, large amounts of C,, to C, polyunsaturated acids were also found in all phospholipids. Palmitic, stearic, arachi- die and behenic acids represented the bulk of the saturated acids in phospholipids; however, large amounts of 26 : o were also found in a number of phospholipids.

INTRODUCTION

Studies ire vitro with suspensions of rat thymic lymphocytes have revealed a significant and continuing endogenous respiration in the absence of exogenous sub-

Biochim. Biophys. Acta, g8 (1965) 117-127

118 z). ARRAMSON, M. BLECHER

strate, with a respiratory quotient indicative of lipid oxidation ; this oxidation was inhibited by adrenal steroids l. The synthesis of lipids from carbohydrate precursors is also an active process in this tissue 8. As part of our investigations of the endocrino- logical control of lipid metabolism in rat thymic lymphocytes, it was considered necessary to obtain quantitative data on the lipid composition of this tissue; this information is not available in the literature. This paper reports the separation, isolation and quantitative analysis of the lipid components of thymus glands taken from two strains of albino rats.

EXPERIMENTAL

Materials Solvents were either Chromatoquality (Matheson, Coleman and Bell) or were

purified before use by conventional methods. Silicic acid (M~~ckrodt, Ioo mesh) was washed with water, freed of “fines” by repeated suspension in methanol and decantation, then activated at 11s~ for 24 h. Florisil (Floridon, Inc., Tallahassee, Fla.), 6o-100 mesh, was dried at 115’ for 4 h, then equilibrated with water (7%, v/w) for 16 h with shaking. Silica Gel G (E. Merck, A.G., Darmstadt) wasusedwithout further treatment.

Reference lipids were obtained from the following sources: fatty acids, fatty esters, and standard mixtures of fatty esters from Applied Science Laboratories, State College, Pa., and Endocrinology Study Section, National Heart Institute; mono-, di- and triglycerides from the Hormel Foundation, Austin, Minn., and Dr. F. H. Mattson, The Proctor and Gamble Co., Cincinnati, Ohio; cholesterol and cholesterol esters from the Hormel Foundation; phospholipids from sources previously acknowledged3; and Dr. E. Kern, National Heart Institute, kindly provided mixtures of homologous saturated and unsaturated fatty esters, isolated and purified from natural sources.

The purity of neutral lipids was checked by thin-layer chromatography on Silica Gel G (250~p-thick layers) using as solvent systems: 50% methanol in diethyl ether, 50% light petroleum in diethyl ether, and 30% diethyl ether in light petroleum; only single spots were detectable for each reference lipid. The purity of free fatty acid and fatty ester reference compounds was stated to be greater than 99% by the suppliers; this was verified by gas-liquid chromatography as described below. Puri- fication of reference phospholipids was performed by preparative thin-layer chromatography as described previously a.

For storage purpose, all lipid-cont~n~g solutions were maintained at 4” or -zoo and, wherever practical, kept in an anaerobic atmosphere.

Analytical methods Cholesterol, free and ester, was determined essentially according to HANEL

and DAMS. Lipid phosphorous was determined by MARINETTI’S modification& of BARTLETT’S procedure 6. Ester group analysis was performed by a minor m~fication of the method of RAPPORT and ALONZO?. Methods for detection and analysis of phospholipids have been described previouslya.8.

Biochinz. Biophys. Acta, 98 (1965) 1x7-127

LIPID COMPOSITION OF RAT THYMUS 119

Thymus sources Thymus tissue was obtained from two strains of male, albino rats (IO-II

weeks) : Expts. RT-I, Blue Spruce strain, from Blue Spruce Farms, Altamont, N. Y., fed Old Guilford Rat Diet; Expts. RT-z, Holtzman strain. Holtzman Rat Co., Madison, Wise., fed Purina Laboratory Chow. Thymi were removed to cold 0.9% NaCl following sacrifice of rats by decapitation; tissue work-up was begun without delay.

Extraction of &ids from tissue Approx. 5 g of minced thymus tissue pooled from IO animals, were homoge-

nized in 20 vol. of chloroform-methanol, 2 : I (v/v) in a Teflon-glass tissue homogeni- zer. After filtration of the homogenate through a cylindrical, sintered-glass funnel (fine porosity), the residue was re-extracted within the funnel with the original volume of fresh chloroform-methanol (2: I, v/v) using mechanical stirring. Pooled lipid extracts were washed with 0.2 vol. of 0.73% NaCl (ref. 9), and the lower chloroform phase evaporated to dryness in vaczco (rotary evaporator at room temperature). The residue was extracted with 50 ml of chloroform-methanol (2: I, v/v) and the extract clarified by centrifugation (1000 xg for 3 min) ; insoluble material was re- extracted with IO ml of chloroform-methanol (2:1, v/v). The pooled extracts were evaporated to dryness in a rotary evaporator, and the crude lipid residue dissolved in IO ml of light petroleum.

Separation of neutral lipids from phospholipids 6 g of activated silicic acid, in a cylindrical, sintered-glass funnel (fine poro-

sity), were washed with 40 ml of light petroleum. The crude lipid mixture, in IO ml of light petroleum, was added to the adsorbent, and the mixture stirred mechanically for IO min. Following isolation of the light petroleum extract by vacuum filtration, the silicic acid was washed 3 times with successive 6o-ml portions of diethyl ether. The combined filtrates, which contained the neutral lipid fraction, were taken to dryness in vacua, and the residue dissolved in IO ml of n-hexane for further chromatography.

The silicic acid was then extracted 3 times with 6o-ml portions of anhydrous methanol; the extracts were isolated by vacuum filtration. The pooled filtrates, which contained all of the phospholipids, were taken to dryness in vacua, and the residue dissolved in IO ml of chloroform-methanol (2: I, v/v).

Recovery of lipids from this batch separation was, on a weight basis, essentially complete, as seen from the data of Table I.

Column chromatography of neutral lipids 12 g of hydrated Florisil, suspended in H-hexane, were packed by gravity in

a glass, jacketed column as described by CARROLL lo; the dimensions of the packed adsorbent were 12 mmx 140 mm, and the column was maintained at o-4’. The adsorbent was washed with 40 ml of n-hexane, and the flow rate adjusted to about 1.3 ml per min.

A portion of the neutral lipid fraction (cf. Table III for amounts) was filtered into the Florisil, and elution begun according to the pattern described in Table II (ref. IO). Eluate fractions (7-8 ml), collected automatically with a refrigerated frac-

Biochim. Biophys. Ada, 98 (1965) 117-127

120 D. ABRAMSON, M. BLECHER

tion collector (Buchler Instruments, Inc., Fort Lee, Xew Jersey), were maintained at o-4” during collection. Lipids were detected according to LANDS AND DEAN 11 by evaporating 5 ,ul of each fraction on a ferrotype plate; no overlap between lipid fractions was noted using this technique.

Eluate fractions containing each lipid (see Table II) were pooled, evaporated to dryness in vucuo, and dissolved in chloroform-methanol (2:1, v/v) for further analyses. Purity of each lipid fraction was determined by unidimensional thin-layer chromatography on Silica Gel G (250-p layers) using 3 solvent systems: 50% abs. methanol in diethyl ether, 50% light petroleum in diethyl ether, and 10% diethyl ether in

TABLE I

DISTRIBUTION OF NEUTRAL LIPIDS AND PHOSPHOLIPIDS IS RAT THYMUS -- ._.~ -_ -____ _-

RT-r RI’-2

Tissue wet weight (g) --_

5.176 4.817 Total lipid, weight (mg), and

per cent of tissue wet weight Neutral lipids, weight (mg), and

per cent of recovered lipids Phospholipids, weight (mg), and

per cent of recovered lipids Total lipid recovered, per cent of initial weight

~. - .-

133.9 125.3 2.59 2.60

79.9 65.9 61.5 53.2 50.3 58.2 38.5 46.8 97.2 99.0

TABLE II

FRACTIONATION OF NEUTRAL LIPIDS ON FLORISIL

Order of elution

Eluting solvent

.- n-Hexane

5 y0 Diethyl ether in n-hexane 15 o/0 Diethyl ether in n-hexane 25 o’0 Diethyl ether in n-hexane 50 y0 Diethyl ether in n-hexane

2 o/0 Methanol in diethyl ether 4 y0 Acetic acid in diethyl ether

Volume of solvent (ml)

20

50

2 60

75 75

Collection Lipid tube numbers* eluted

I-5 Hydrocarbons 6-10 Cholesterol esters

I I-20 Triglycerides 2x-29 Cholesterol 3~38 Diglycerides 39-46 Monoglycerides

47-55 Free fatty acids

* From 7 to 8 ml of eluate were collected in each tube.

light petroleum ; each lipid migrated as a single spot (detected by iodine vapor or by hy- droxylamine-ferrichloride spray*) with the same RF as reference compounds. The amount of lipid in each fraction was determined both gravimetrically and by specific spectrophotometric analysis (see Analytical methods above). Fatty acid moieties of each iraction were converted to their methyl esters for gas-liquid chromatography as described below.

Thin-layer chromatogra#hy of j&os$holipids

The crude phospholipid mixtures were subjected to two-dimensional thin- layer chromatography on Silica Gel G for the complete separation and quantification of individual phospholipids by techniques described previously a.

Biochim. Biophys. Acfa, 98 (1~65) 117-127


Analysis of fatty acids by gas-liquid chmmatogra~hy Following extraction of separated phcspholipids from chromatoplatesa, ali-

quots (0.02-1.30 holes lipid P) were added to test tubes. After removing the solvent under a stream of nitrogen, LO ml of transesterification reagent (1.9% sulfuric acid-3.6% dimethoxypropane in abs. methanol) was added, the tubes sealed (Teflon- lined screw caps), and the mixtures heated at 65” for 4 h while blowing a stream of cool air across the necks of the tubes. Reaction mixtures were treated with I ml of water, then extracted with 4 successive 24 portions of n-hexane. The pooled hexane extracts were evaporated to dryness under a stream of nitrogen, and the fatty ester residues were dissolved in sufficient n-heptane (usually 0.05-o.30 ml) to make a 0.5% solution.

For neutral lipids the procedure was the same except that samples containing 0.03-7.0 mg of lipid were used, the transesterification reagent was 2.2% sulfuric acid-3.6% dimethoxypropane in abs. methanol, and the heating period was reduced to 2 h.

Gas-liquid chromatography of fatty esters Mixtures of fatty esters were separated and quantified in a Jarrell-Ash Model

700 argon ionization gas chromatograph (using strontium go) equipped with a Disc Integrator. The column was an 8 ft by 4 mm (internal diameter) glass column packed with 15% diethyleneglycol succinate on Anakrom ABS, 80190 mesh (Analabs, Ham- den, Conn.). Argon inlet pressure was 26 lb/in2 (150 ml per min flow rate measured at the exit port). Temperature programming was discontinuous: 150’ through emergence of pahnitoleate, 175” through emergence of docosanoate, and 185” thereafter. Fatty esters were identified by their retention times relative to that of palmitate, the elution patterns having been determined previously with reference fatty esters (see Materials above). Satisfactory linearity of detector response was demonstrated by checking with National Heart Institute fatty ester standard mixtures A, B, C and D.

RESULTS AND DISCUSSION

Table I presents the distribution of neutral lipids and phospholipids in lipid extracts of rat thymic tissue following separation of the two classes of lipids by a batch procedure using silicic acid. In thymi from both strains of rats, lipid represented about 2.6% of tissue wet weight. Although no data for lymphoid tissue is available for comparison, the present results can be contrasted with 6% lipid in mouse liver”. 1% in human erythrocytes I*, 6% and 19% in gray and white matter, respectively, of brain I’, 24% in adrenals lV@, and 62% in adipose tissue”. The distributions of the two classes of lipids in rat thymus observed in the present study (Table I) are similar to those reported for erythrocytes”; in contrast, however, neutral lipids represent over 90% of total lipids in adrenals %I*, less than 50% of total lipids in mouse liver We,

and about 22% of total lipids in rat liver’*. In Table III are presented data on the distribution of individual lipids in the

neutral lipid fraction of rat thymus. Individual components were isolated using column chromatography on Florisil as described in EXPERIMENTAL; the technique employed yielded individual neutral lipids uncontaminated with neighboring fractions, as revealed by thin-layer chromatography of pooled eluates in three solvent systems. Recove-

Biochim. Biophyhys. Acta, $3 (1965) 117-123


ries of neutral lipids following Florisil chromatography were essentially complete (Table III), amounting to 95100% of the lipid placed on the columns. The relative amounts of hydrocarbons, cholesterol esters and triglycerides were very similar in thymi from both strains of rats; however, thymi from Blue Spruce animals (RT-I) contained much more free cholesterol and free fatty acids, and much less of the lower glycerides, than did thymi from Holtzman rats (RT-2). Weights of each type of

TABLE III

NEUTRAL LIPID COMPOSITION OF RAT THYMES

In RT-I and RT-z, Florisil columns were charged with 79.90 mg and 52.56 mg, respectively, of the crude neutral lipid mixtures; recoveries were 100.2 y0 and 95.2 %. respectively, for RT-I and RT-z, baaed upon gravimetric analyses. Analytical weights were calculated from cholesterol and ester analyses. Theoretical molecular weights were calculated on the basis of fatty acid compositions determined for each neutral lipid by gas-liquid chromatography.

Lipid fraction RT-r

Weighf, Weight, Fraction of gravimeiric analytical total

recovered*

(mgl Img) (%I

Hydrocarbons 0.60 - 0.73 Cholesterol esters 2.74 2.83 3.35 Triglycerides 67.02 70.83 82.32 Cholesterol 8.27 8.57 10.14 Diglycerides 0.74 0.67 0.90 Monoglycerides 0.00 0.03 0.04 Free fatty acids 2.14 - 2.62

Totals 81.50 82.93

l Based upon gravimetric analyses.

RT-a

Weight, Weight, Fraction of gratimelric analytical total

recovered*

(mg) (mg) (%ul

0.60 - 1.20 1.81 I.90 3.64

42.34 42.17 85.25 2.92 3.00 5.87 1.08 1.10 2.17 0.57 0.60 1.14

0.34 - 0.68

49.66 48.77 .--

TABLE IV

PHOSPHOLIPID COMPOSITION OR RAT THYMUS

In RT-I and RT-2 the chromatoplates were charged with 0.612 and 0.801 rmole of lipid P, respectively; recoveries of lipid P were 0.576 pmole (94.1 %) and 0.797 /Imole (95.0 %), respectively, for RT-I and RT-2.

Lipid jracfion

Phosphatidyl choline Phosphatidyl ethanolamine Phosphatidyl ihositol Sphingomyelia Phosphatidyl serine Lysophosphatidyl choline Phosphatidic acid Cardiolipin

Per cent of total P recovered

RI-x RT-z

48.61 50.43 18.75 19.44 11.80 12.04 10.76 9.41 6.59 5.25 I.38 0.87 I .04 I .50 1.04 I .oo

neutral lipid were essentially the same when determined gravimetrically or when calculated from analytical data; analytical data could not be obtained for the hydro- carbon fractions, and chemical analyses of the free fatty acid fraction were not performed. In rat thymus, ttiglycerides represented the major neutral lipid (82-85% of total neutral lipids), while free and esterified cholesterol represented 1+14O/~ of the total; free fatty acids were relatively minor components (0.7-2.6%), as were the lower glycerides (~-3%). Although similar distributions have been reported for mouse liver lS,

Biochim. &@hys. Acta, g8 (1965) 1X7-127


in mouse ascites cells, GRAY reports m triglyceride as 56% of total neutral lipids, 1%

cholesterol ester, 22% free cholesterol, 17% monoglyceride, 4% diglyceride, and less than 1% hydrocarbons; contrasting data for adrenal glandsl~ shows, as would be anticipated, the greatest percentage for free and ester cholesterol (61%), with triglyceride amounting to only 31%, diglyceride 3%, hydrocarbons 0.6%, and only traces of free fatty acids.

Table IV presents data on the relative distributions of individual phospholipids of rat thymus. As demonstrated by the authors elsewherea, the two-dimensional thin-layer chromatography technique employed in these studies permits complete

TABLE V

THE FATTY ACID DISTRIBUTION IN RAT THYMUS NEUTRAL LIPIDS

For fatty acids standard shorthand designations are used. Relative retention times are given in Table VI. The symbol T stands for trace amounts; blank spaces indicate no detectable quantities.

Fatty arid

Methyl esters as percentage of total methyl esters

TYi- Di- Mcmo- glycerides glyctides glycm’des

RT-r RT-2 RT-I RT-2 RT-r RT-2

Free fatty Cholesterol acids esters

RT-I RT-a RT-I RT-a

8:o IO : 0

II :o II : I

12 :o 12 : I

13 :o 13 : I 14 : 0 14 : I 15 : 0 15 : I 15 :2 16 : o 16 : I 16 : 2

16 : 3 16 : 4 18 : 0 18 : I 18 : 2

18 : 3 19 : 0 20 : 0 20 : I

20 : 2

20 : 3

20 : 4 22 : 0 22 : 1 22 : 2

22 : 3

22 : 4 22 : 5

22 : 6 24 : 0 24 : I 24 : 2

24 : 3

: 0.29 4.34

T T 0.34

T T T 0.17 T T T T 1.07 5.2I

T T T 0.26 T T

I.29 I .48 T T

17.85 18.63 12.01 29.37

I.65 3.og T 1.48 T 0.73 T

0.65 52.49

3.01 ‘5.47 31.10 x9.22 4;75 19.87

0.94 0.57 6.43

I.79 0.12

0.22

0.28 0.75 0.13

1.07 T T T T

0.53 0.52 0.55

0.10 0.21

0.7’

3.88 13.65 12.56 31.16 11.64 22.25

T 0.59 T T T

6-9

T

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T

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T 3.96 T I.75 3.37 T 0.88

0.88

1.61 IX.05 22.10

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29.10 13.94 22.91

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6.85 1.48

0.59

2.32

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1.78

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1.81 1.78

Biochim. Biophyr Acta, ~8 (x965) .I 17-127

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T

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separation and essentially quantitative recovery of all phospholipids, with the sole exception of phosphatidyl serine which is recoverable in only 85% yield from chromatoplates by this technique. Significant differences between thymi from the two strains of rats employed are evident only in the phosphatidyl choline and sphingomyelin fractions. In both strains, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol and sphingomyelin, in descending order, constituted the bulk of the phospholipid fractions. The relative distributions of phospholipids observed in the present studies with rat thymus, and those reported for a variety of animal tissues, e.g. mouse ascites cellsmpal, mouse liver18, mouse adipose tissuel’, and dog adrenalslb, are all remarkably similar; in human erythrocytes, however, phospha- tidy1 choline represents a’smaller, and sphingomyelins and the cephalins a larger, proportion of total phospholipids than in the other tissues noted aboveIS.

In Table V are presented the relative distributions of fatty acids in the individual neutral lipids as determined by quantitative gas-liquid chromatography. Among the neutral lipids, and for both strains of rats, unsaturated fatty acids predominated (48-80% of total fatty acids). Among unsaturated acids, 18 : I and 18 : 2 predominated, although in the mono- and diglycerides of RT-I 16:3 represented 35% and 52%,

respectively, of total fatty acids. Although there are several exceptions, in the main the present results with rat thymus are similar qualitatively and quantitatively to findings of others with a number of rat 10g22 and mouse 12~1’ tissues.

Table VI gives the relative fatty acid distributions in the eight phospholipids isolated from rat thymus, as determined by quantitative gas-liquid chromatography following separation of the phospholipids by two-dimensional thin-layer chromatography. The retention times of each fatty ester derivative, relative to that of palmitate, are also recorded; it should be emphasized that these ratios were obtained during three-stage, discontinuous temperature programming. In RT-I the saturated acids predominated in all phospholipids except, phosphatidyl serine and sphingomyelin. In RT-z unsaturated acids predominated in all phospholipids except phosphatidyl choline and phosphatidyl inositol. Among all unsaturated acids, 18: I, 18 : 2, 18 : 3,22 : 1;22 : 2 and 24: 3 represented the major portion. Among all saturated acids, 16:o and 18 : o predominated with the exceptions of phosphatidyl serine, in which 22:o represented over one-third of total fatty acids, and of lysolecithin, in which 26:o represented over one-third of total fatty acids. What is probably the most significant difference between the present results with the fatty acids from rat- thymus phospholipids and results of others with a variety of other rat tissues18yag is the presence in thymus phospholipids of large amounts of the higher (C,,-C,,) unsaturated fatty acids; in phospholipids from other rat tissues significantly large amounts only of 20 : 4 have been reported.

ACKNOWLEDGEMENT

This work was supported in part by grants AM-06208 and AM-05475 from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health.

Biochim. Biophys. Actu, g8 (1965) 117-127

LIPID COMPOSITION OF RAT TWYMUS 127

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4 H. K. f%ANEL AND Ei. DAM, l#Gt~ Chcm. SCtWd, 9 (1955) 677. 5 G.V. MABINETTI,_/. LipidRes., 3(1962) I. 6 G. R. &%RTLETT, J. Bid. C%em., 234 (1959) 466. 7 M. L. EZAPPORT AND N. A~onrzo, J. Biol. Chem., 217 (Igs5) 193, 8 W. D. SIUDMORE AND C. ENTENMAI, J. G&d Res., 3 (rg62) 47~ g J. FOLCH, bf. b?.Es AND G. H. SLOANE-STANLEY% j. Bid. C&em., 226 (Ig57j497.

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The lipid composition of normal rat thymus

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