Lipids of Bovine Thyroid P.G. SATYASWAROOP, Radiation Medicine Centre, Medical Division, Bhabha Atomic Research Centre, Tara Memorial Hospital, Parel, Bombay-12, I ndia

ABSTRACT

The lipid composit ion of the freshly slaughtered bovine thyroid tissue has been investigated. The phospholipid pat- terns of microsomal and mitochondrial fractions obtained from homogenates of bovine ~hyroids have also been deter- mined. They resemble the phospholipid composit ion of the corresponding subcel- lular fractions from other tissues. The fat ty acid composit ion of the various phospholipid species of these subcellular components have also been estimated by gas liquid chromatography. These analy- ses reveal that the fat ty acids are not particularly characteristic of the subcel- lular organelle but tend to be character- istic of the lipid species. There is a high percentage of nervonic acid (C24:1) in all the subcellular phospholipid species ex- amined.

Phospholipid patterns of sheep and dog thyroids have been studied (1,2) and phospho- lipids have been postulated as the possible mediators of thyroidal iodide transport (3,4). The unsaturated fat ty acids of phospholipids are considered to be the probable site of iodinat ion (4). For example, nervonic acid (24:1) present at the /3-position of the human thyroidal phosphat idyl choline has been impli- cated in the role of binding and carriage of iodide ion (5). The enhanced 32p incorporat ion into phospholipids of thyrotropin-st imulated thyroids has been well documented (2,5,7) and the mitochondria and microsomes of these stimulated thyroids are the subcellular organ- elles which exhibit increased turnover of phos- pholipids (8,9). However, there are no reports on the subcellular phospholipid distribution in the thyroid tissue. The present studies were undertaken to provide information on the proport ions of individual phospholipids of the bovine thyroidal mitochondrial and microsomal fractions. The fat ty acid composit ion of the individual phospholipid species of these organ- elles have been determined by gas liquid chro- matography (GLC).

MATERIALS AND METHODS

All solvents employed for extract ion and

chromatography were Analar grade and were redistilled before use. Kieselgel H was obtained from E. Merck, Darmstadt, Germany. Standard GLC pure fat ty acid methyl esters were from Applied Science Lab., State College, Pa. and were donated by Fred Snyder, Oak Ridge Laboratories.

Bovine thyroids from freshly slaughtered animals were transported to the laboratory frozen in ice. After removal of adhering fat ty material and connective tissue, the tissue was sliced, washed in saline, blot ted with filter paper, weighed and taken for lipid extraction. Each analysis contained thyroid slices pooled from at least five different thyroids.

The thyroid slices were homogenized in CHC13/MeOH (2:1 v/v) and the total lipids were prepared by the procedure of Folch et al. (10).

Thin Layer Chromatography

All thin layer separations were carried out in glass tanks containing chromatography paper as wicks to facilitate solvent equilibration. Thin layers of Kieselget H (1 hr activation) were used to separate the phospholipid classes using the s o l v e n t system chloroform-methanol-acetic acid-water (50:25:8 :4 v/v). Lipids resolved by thin layer chromatography (TLC) were sprayed with concentrated H2SO 4 and charred. Phos- pholipids were also visualized by exposure of the TLC plates to iodine vapor. Phospholipid zones were scraped and quantitatively trans- ferred to test tubes, and the lipids were extracted from the silica gel for phosphorus estimation employing three batches of the same solvent system used for the development of the chromatograms. Total lipid P and individual phospholipid P were determined by Bartlett 's method (I I) . Cholesterol a n d cholesterol esters were estimated by the method of AbeU et al. (12).

Gas Liquid Chromatography

Methyl esters of the fat ty acids of the individual phospholipids were prepared by re- fluxing the Kieselgel scrapings of phospholipid zones for 5 hr with H2SO 4 (1%) in dry me thano l

Panchromatograph (Model 12103, W.G. Pye and Co., U.K.) equipped with a 90Sr ionizing detector was used in this study.

A 152 cm x 30 mm o.d. pyrex glass column

661

662 P.G. SATYASWAROOP

THYROID TISSUE

Homogenized in chilled 0.25 M sucrose filtered through gauze

FILTERED HOMOGENATE

[ RESIDUE

(unbroken cells and nuclei-discarded)

1000 g for 10 min

[ RESIDUE

Crude Mitochondria

washed with xl0,000g 0.25 M sucrose for 20 min

I RESIDUE

Washed Mitochondria

1 SUPERNATANT

(Discard)

RE ~IDUE Crude mlcrosomes

washed with

0.25 M S U C r O S e x 105,000 g for

60 rain

I SUPERNATANT I

I xlO,O00 g 20 rain

I SUPERNATANT II

1 I RESI DUE SUPE RNATANT

Washed Microsomes

Scheme 1. Procedure for subcellular fraetionation of bovine thyroidal homogenate.

x105,000 g for 60 rain

I SUPERNATANT

packed with 15% EGS on 80-100 mesh chromo- sorb W was used for all analysis. Temperature of the column was 178-184 C, temperature of the detector, 225 C; argon flow, 60 ml/min.

Fatty acids were identified by: (a) compar- ison of relative retention times with standard compounds and with those given in literature, (b) analysis of GLC results before and after hydrogenation to fix the identity of unsatu- rated fatty acids, and (c) by plotting retention times relative to methyl stearate against chain length and determining the chain length of unknown peaks from the curve.

Quantitation of fatty acids was done by the triangulation method and results were ex- pressed as per cent of the total fatty acids obtained by GLC.

Sub-Cellular Fractionation

The thyroid slices were homogenized with four volumes of chilled 0.25 ml sucrose solu- tion and subcellutar fractions were prepared by the differential centrifugation of the homoge- nate. The entire procedure for separation of the subcellular organeUes was carded out at 4 C. The subcellular fractionation procedures era-

ployed were essentially the same as those of Kogl and van Deenen (8) and that of DeGroot and Carvalho (15). They are schematically shown in Scheme 1.

RESULTS

The lipid composition of the bovine thyroids is the following. The total llpids, five samples, form 1.8 -+ 0.47/100 g of the wet weight of the fresh tissue (the value preceded by + is the standard deviation); lipid phosphorus, four sam- ples, forms 19.1 + 4.1 pg/mg lipid; total cholesterol, five samples, forms 120.0 + 15.0 pg/mg lipid; free cholesterol, five samples, forms 102.0 + 9.6 pg/mg lipid; cholesterol ester, five samples, forms 18.0 + 4.4 pg/mg lipid (cholesterol ester values were obtained by subtracting free cholesterol from total choles- terol).

The distribution of phospholipids in the whole thyroid tissue, and the tliyroidal, mito- chondrial and microsomal fractions are given in Table I. Phosphatidyl choline (PC) (42%) is the predominant phospholipid followed by phos- phatidyl ethanolamine (PE) (26%), sphingomy-

LIPIDS, VOL. 6, NO. 9

LIPIDS OF BOVINE THYROID

TABLE 1

Phospholipid Patterns in Bovine Thyroid and Its Subcellular Distribution a

663

Mitochondrial Microsomal Phospholipid species Whole thyroid, % fraction, % fraction, %

Lysolecithin 3.0 3.5 2.0 (1.5-4.0) (3.0-4.0) (1.5-3.0)

Sphingomyelin 18.0 14.0 16.0 (15.5-19.5) (12.0-16.0) (15.0-18.0)

Phosphatidyl choline 42.0 36.0 46.0 (41.0-44.0) (34.0-39.0) (45.0-47.0)

Phosphatidyl serine + 11.0 12.0 12.0 Phosphatidyl inositol ( 10.5-11.6) ( 10.5-14.0) ( 11.0-14.0) Phosphatidyl ethanolamine 26.0 34.5 28.0

(24.0-28.5) (34.0-35.0) (26.0-31.0)

aThe results are the percentage of individual phospholipid P as determined from TLC. Each value represents the mean of three triplicates. The value given in parentheses are the ranges. Cardiolipin and phosphatidic acid which move with the solvent front were not estimated.

elin (18%), phospha t idy l serine (PS), and phos- pha t idy l inosi tol (PI) (11%) and lysoleci thin (3%) in the whole t hy ro id tissue lipids. Phos- phol ipids cons t i tu te the major p r o p o r t i o n of the lipids of b o t h subcellular f ract ions, mi to- chondr ia and microsomes , analyzed. Total l ipid P in the mi tochondr i a and microsomes were 28.5 /ag P /mg lipid and 31.5 /~g/mg lipid, respectively, More PE appears to be in mi to- chondr ia (34.5%) than in microsomes (28.0%); the p r e d o m i n a n t phospho l ip id of the micro- somes is PC (46%). In mi tochondr i a , PC (36%) and PE (34.5%) toge ther cons t i tu te more than 70% o f the phosphol ip ids . Cardiol ipin, shown to be localized in mi tochondr i a of all t issues

examined , has no t been measured in our studies as they travel along the solvent f ron t wi th phospha t id ic acid (PA) and o the r lipids.

The fa t ty acid profi le of the individual phosphol ip ids of mi tochondr ia l and microsomal f rac t ions analyzed ext~bi t variat ion among themselves (Tables II and III). The same phos- phol ip id species, w h e t h e r localized in the mi to- chondr ia l or microsomal f ract ions , show simi- larity in the i r f a t t y acid con ten t . However , individual phosphol ip ids , when compared wi th- in each f rac t ion , show striking variat ion in their p ropor t ions of fa t ty acids. Thus, PC in b o t h f ract ions show the highest percentage of pal- mitic acid (37.0% and 40.0%) fo l lowed by oleic

TABLE II

Fatty Acid Composition of Individual Phospholipid8 of Bovine Thyroidal Mitochondrial Fractions a

Phosphatidyl inositoi +

Fatty Phosphatidyl Phosphatidyl phosphatidyl acid choline ethanolamine Sphingomyelin serine

C12:0 1.77 9.85 5.00 9.63 C14:0 3.03 11.13 4.37 11.65 C14:i 1.91 16.16 6.39 4.35 C16:0 40.02 17.50 17.75 27.83 C18:0 16.85 13.38 8.59 9.07 C18:1 17.44 15.15 5.91 10.46 C18:2 2.90 3.27 2.57 0.69 C18:3 . . . . 3.01 14.00 C20:0 . . . . . . . . . . . C20:? 7.93 3.16 3.83 1.12 C22:0 0.76 1.46 15.68 8.69 C22:1 4.11 3.56 3.46 1.70 C22:? - - 2.07 15.92 12.21 C24:0 0.13 0.77 1.65 --- C24:1 3.97 2.92 19.90 --- C22:5 - - 3.94 . . . . . . C22:6 --- 2.51 1.89 - -

aResults are expressed as area per cent of total fatty acids as determined by GLC. Results given are the arithmetic mean of three individual samples. Experimental details in text.

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664 P.G. SATYASWAROOP

TABLE III

Fatty Acid Composition of Individual Phospholipids of Bovine Thyroidal Mierosomal Fractions a

Phosphatidyl inos i to l +

Fatty Phosphatidyl Phosphatidyl phosphatidyl acid choline ethanolamine serine Sphingomyelin

C12:0 1.12 5.75 9.14 5.32 C14:0 1.63 6.58 7.95 8.42 C14:1 1.33 16.15 4.08 19.39 C16:0 37.55 17.17 27.19 20.33 C18:0 15.22 12.15 12.10 6.96 C18:1 24.36 22.42 10.22 5.86 C18:2 6.72 3.68 1.70 1.93 C18:3 0.30 1.52 3.02 1.63 C20:0 0.66 1.78 0.67 -- C20:? 3.80 2.70 1.16 1.19 C22:0 3.76 4.10 5.50 12.20 C20:4 -- 9.39 10.16 -- C22:? 1.18 5.10 10.63 9.08 C24:0 . . . . . . 3.46 C24:1 4.90 4.56 4.03 6.00 C22: 5 -- 1.07 -- -- C22:6 -- 1.20 -- --

aResults are expressed as area per cent of total fatty acids as determined by GLC. Results are arithmetic mean of three individual samples. Experimental details given in text.

acid (24.0% and 27.0%). The o ther phospha- tides, PE, sphingomyel in , PS and PI contain a very high percentage (about 20% to 30%) of fa t ty acids wi th chain lengths shorter than 16:0. All the phosphat ide species contain a significant p ropor t ion of nervonic acid (24:1) . Mitochondr ia l sphingomyel in contains as much as 19% of this fa t ty acid. Considerable amounts of 22 :0 is also present in this phosphol ipid .

DISCUSSION

The only available reports on phosphol ipid dis t r ibut ion in thyroids are those of Freinkel (1) and Scot t et al. (2). Freinkel has shown in sheep thyroids that the alkali stable phosphorus comprised 20% of the to ta l lipid phosphorus. The present data obta ined by the direct deter- ruination of phosphorus of bovine thyroidal phosphokipids f rom the Kiesetgel plates are in good agreement . Scot t et al. (2) have es t imated the amounts o f plasmalogen, alkyl e ther lipids and PA, apart f rom the o ther major phospha- tides in two dog thyroids. The methods em- p loyed in the present s tudy do no t resolve these phosphatides.

The general thyroidal lipid classes o f bo th the whole tissue and the subcelhilar fract ions show no striking differences when compared with the lipid classes o f various o ther tissues. The dis t r ibut ion of phospholipids of the thy- roidal mi tochondr ia l and the microsomal frac- t ions show qui te a distinctive pat tern, however , their f a t ty acid profiles do not reveal any

significant organelle specifici ty. In mi tochon- dria, PE and PC are present in equal amount s ; in microsomes, PC is almost 1.5 t imes that of PE. Similar phospholipid pat terns in subcellular organelles of various tissues have been repor ted ( t3 ) .

The fat ty acid pat terns in the individual phospholipids of mi tochondr ia and microsomes show a lack of differential subcellular distribu- t ion. That they are no t part icularly characteris- t ic o f the organelle but they tend to be characterist ic o f the lipid species has been demonst ra ted by many workers. Villki and Jaakonmaki (5) have repor ted a significant percentage of nervonic acid (24 :1) in human PC at the r -pos i t ion and have demonst ra ted its impor tance in complexing iodide. They have postulated an iodide ion carrier role for this species of phosphol ipid. The fa t ty acid analysis of thyroidal neutral lipids and phospholipids or the fa t ty acid pat terns of the subcellular organ- elles studied do no t show the presence of this fa t ty acid species perhaps due to its di lut ion to low concent ra t ions when these componen t s are analyzed. However , the individual phosphol ipid fa t ty acids of the mi tochondr ia and microsomes showed a significant percentage of this compo- nent. Mitochondria l sphingomyel ins showed a particularly high (19%) con ten t of this fa t ty acid. If the presence of this fa t ty acid in the phosphol ipid molecule is a prerequisi te for iodide binding, then all the phosphol ipid spe- cies might be expec ted to complex iodide and sphingomyel in should exhibi t this iodide-

LIPIDS, VOL. 6, NO. 9

LIPIDS OF BOVINE THYROID 665

complex ing p h e n o m e n o n in higher p ropor t ions . However , sph ingomyel in is a lmost inert in this regard (4,14). The results of Nagashima and Suzuki (4) and those of Shah and S h o w n k e e n (14) f rom this labora tory show the presence of an u n k n o w n frac t ion wi th the highest iodide- complex ing capaci ty. GLC analysis of this u n k n o w n frac t ion f rom sheep thyro ids conta in no traceable quant i t ies of nervonic acid (un- publ ished data).

ACKNOWLEDGMENT

D.H. Shah gave helpful suggestions.

REFERENCES

1. Freinkel, N. Biochem. J. 68:327 (1958). 2. Scott, T.W., S.M. Jay and N. Freinkel, Endocrin-

ology 79:591 (1966). 3. Villki, P., Arch. Biochem. Biophys. 97:425

(1962). 4. Nagasbima, N., and M. Suzuki, Gunma J. Med.

Sci. 4:291 (1965). 5. Villki, P., and O. Jaakonmaki, Endocrinology

78:453 (1966). 6. Morton, M.E., and J.R. Schwartz, Science

117:103 (1953). 7. Freinkel, N., Endocrinology 61:448 (1957). 8. Kogl, F., and L.L.M. Van Deenen, Acta Endocr.

36:9 (1961). 9. Kerkof, P.R., and J.R. Tata, Biochem. J. 112:'729

(1969). 10. Folch, J., M. Lees and G.A. Sloane-Stanley, J.

Biol. Chem. 226:497 (1957). 11. Bartlett, G.R., Ibid. 234:466 (1959). 12. Abell, L.L., B.B. Levy, B.B. Brodie and F.E.

Kendall, Ibid. 195:357 (1952). 13. Bartley, W., in "Metabolism and Physiological

Significance of Lipids," Edited by R.M.C. Dawson and D.N. Rhodes, John Wiley and Sons Ltd., New York, 1964, p. 369.

14. Shah, D.H., and R.C. Shownkeen, Symposium on the Chemistry and Metabolism of Lipids, New Delhi, 1969, p. 32.

15. DeGroot, L.J., and E. Carvalho, J. Biol. Chem. 235:1390 (1960).

[ Revised manuscr ip t received April 2, 197 t ]

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