Experimental electron microscopic study of the sequential stages of in vitro formation of ceroid

Experimental electron microscopic study of the sequential stages of in vitro formation of ceroid

EXPERIMENTAL AND Experimental MOLECULAR PATHOLOGY 2, 219-233 Electron Microscopic Study of the Sequential Stages of in Vitro Formation of Ceroid...

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EXPERIMENTAL

AND

Experimental

MOLECULAR

PATHOLOGY

2, 219-233

Electron Microscopic Study of the Sequential Stages of in Vitro Formation of Ceroidl EDUARDO

Department

(1963)

of Pathology,

Albany

A. PORTA

Medical

College

Received

July

of Union

University,

Albany,

New

York

5, 1962

INTRODUCTION Ceroid-like pigment, a lipochrome first described in nutritional cirrhosis in rats (Lillie et aZ., 1941)) is also encountered in a wide variety of experimental conditions in animals and in different human tissues in casesof nutritional or metabolic disorders. Ceroid deposits have also been demonstrated within recent mural thrombi in rats fed infarction-producing diets and are a common component of the arteriosclerotic plaques of human aortas and coronary arteries (Pappenheimer and Victor, 1946; Burt, 1952; Hartroft, 1953; Hartroft and Thomas, 1957; Fritz, 1961). The pigment is readily produced in animals by feeding diets deficient in vitamin E and containing unsaturated fatty acids. Ceroid has been produced in vitro by several investigators (Endicott, 1944; Hartroft, 1951; Casselman, 1951) by incubating mixtures of unsaturated fats and organic tissues. Histochemical studies have provided evidence that ceroid might be a product of auto-oxidation of unsaturated lipids. The staining differences between the various forms of lipochromes are apparently a function of the length of time that they have been deposited in the tissue. The electron microscopic configuration of ceroid and similar pigments found in the cells of animals and human tissueshas been describedby Hess (1955), Bondareff (1957), Gadrat et al. (1960)) and Essner and Novikoff (1960). The purpose of this experiment was to study the ultrastructural sequential changes of ceroid formation in vitro, when fresh blood cells were mixed with fats of various degreesof unsaturation. MATERIALS

AND METHODS

Saline washed cells were prepared from the heparinized blood of Wistar albino rats. One part of these cells was mixed with nine parts each of cod liver oil, butter oil, and hydrogenated coconut oil (See Table I for fatty acid composition). Eight aliquots were removed from each of these three mixtures to provide four duplicate samplesfor incubation at 37°C for 6, 8, 10, and 15 days. The residues of altered blood cells were washed six times in iso-propyl alcohol and twice in xylene. Portions of the residueswere embeddedin paraffin and smearson albuminized slideswere also prepared. Paraffin sections and smearswere stained with oil red 0, iron stain, ZiehlNeelsen, and periodic acid-Schiff (PAS). Unstained sections were utilized for fluor1 This Institute,

study was supported Bethesda, Maryland.

by

U. S. Public

Health

219

Grant

H-7155

from

the

National

Heart

220

EDUARDO

A. PORTA

T.1BLE FATTY

Fatty

ACID COMPOSITION”

OF BUTTER

acid

Caproic Caprylic Capric Laurie Myristic Palmitic Stearic Arachidic Lauroleic Myristoleic Palmitoleic Oleic Linoleic Arachidonic Clupanodonic 0 Moles per cent. h Taken from the chart prepared r Unsaturated fatty acids.

OIL,

Butter

I

COD LIVER

oil

OIL,

AND HYDROGENATED

Cod liver

1.4 1.5 2.7 3.1 12.2 25.2 9.3 1.3 0.4” 1.6C 4.oc 29.5” 3.6C

COCONUT

Hydrogenated coconut oil

oil

0.2 8.0 7.0 48.2 17.3 8.8 2.0

5.8 8.4 C.6 -

-

0.26 2c.oc 29.1” -

-

6.0 2.5

-

25.4<: 9.G

by E. F. Drew

and Co., Inc., New

OILY

York

10, New

York.

escent microscopy. To another portion of the residue, after the extraction of soluble fat, five parts of Dalton’s osmic fixative were added. This mixture was centrifuged and the fixative decanted. The sediment was washed with saline and transferred to Petri dishes filled with plain agar which was allowed to jell. Small blocks were cut, dehydrated, and embedded in methacrylate in the usual way. Finally, ultrathin sections were photographed with an RCA electron microscope, Model EMU-3F. RESULTS Light microscopy Sections and smears from the residues of the mixtures containing cod liver oil, incubated for 6 days and stained with oil red 0, showed altered blood cells and a sudanophilic granular material deposited in and between the cells. A few of these coarse granules were found inside the altered white blood cells, while the still recognizable erythrocytes were heavily loaded with a more finely granular sudanophilic material. Granules of different sizes, scattered between cells, were frequently attached to membranes of fragmented cells. This newly formed granular material was irregularly stained by the oil red 0 and PAS stains (Fig. 1) but exhibited a more uniform affmity for acid-fast stain. They also displayed a brilliant yellowish-white autofluorescence of uniform character (Fig. 3). A few small iron-positive granules were seen in some of the sections. After 6 days’ incubation, the residues from the mixtures FIG. 1. Photomicrograph of a section from the residue of a cod liver oil-saline washed blood cell mixture, incubated for 6 days at 37°C. Note the deteriorated leukocyte with coarse sudanophilic granules, some occupying the interior and others attached to the membranes. The deteriorated erythrocytes are filled with more finely granular sudanophilic material. Oil red 0 stain. x 250. FIG. 2. Section from a blood cell-cod liver oil mixture after 15 days incubation. The deteriorated erythrocytes are heavily loaded with sudanophilic granular material. The leukocyte also shows some sudanophilic material in its interior and at the periphery. Oil red 0 stain. X 1000.

ELECTRON

MICROSCOPY

OF CEROID

221

222

EDUARDO

A. PORTA

containing butter oil showed very few morphologic and histochemical differences from those of cod liver oil, but mixtures containing hydrogenated coconut oil were quite different after the same period of incubation. The latter failed to show any granules or material with the tinctorial affinities of the other mixtures and no autofluorescence was observed under ultraviolet light. After 8, 10, and 15 days’ incubation, residues from the different mixtures showed essentially the same findings as observed at 6 days (Fig. 2). It was evident, however, that in the butter and in the cod liver oil mixtures the amount of ceroid had increased in direct relation with the incubation time. With longer incubation, ceroid

FIG. 3. The residues of a blood cell-cod liver oil mixture after 6 days granules of ceroid displaying autofluorescence which was yellowish-white microscope. Unstained paraffin section mounted in glycerine. X 450.

incubation under the

shows the fluorescent

was more sudanophilic and exhibited variable degrees of fluorescence. Most of the pigment granules were definitely yellowish-bronze, although some had the yellowishwhite appearance more commonly seen with shorter incubation times. The light microscopic characteristics are summarized in Table IT. TABLE HISTOCHEMICAL

Mixture: Days: Oil red 0 Periodic acidSchiff Ziehl-Neelsen Autofluorescence PearI’s iron stain

Cod liver 6

8

II OF in vitro

PROPERTIES

oil 10

FORMED

Butter 15

6

8

CEROID

Hydrogenated coconut oil

oil 10

15

8

6

10

15

++ ++

++ ++

+++ ++

+++ ++

+ +

++ ++

++ ++

+++ ++

-

-

-

-

+ ++ -t

++ ++ -

++ ff-t -

++ +++ -

++ + -

++ ++ -

++ ++ -

++ +++ -

-

-

-

-

ELECTRON

MICROSCOPY

OF CEROID

223

Electron microscopy At 6 days, residues from the cod liver oil mixtures showed numerous drops of a dense osmiophilic material inside the altered red blood cells (Fig. 4). These drops of ceroid were contained in round or oblong cavities without any detectable peripheral membranes. This pattern suggested that ceroid, an insoluble fat, had been mixed with other soluble fats which had been extracted by the solvents employed in the preparation. That these drops were not merely unextracted fat was precluded by the thoroughness of the extraction procedure and the fact that no drops were seen in the hydrogenated coconut oil mixture which was similarly treated. In the altered white blood cells the configuration of ceroid was rather similar to that in erythrocytes (Fig. 6). In addition, some of this granular material showed a tendency to coat the surface of the white blood cells. Most of the white blood cells containing ceroid still had swollen but recognizable mitochondria. The granular pigment did not show any relationship with these degenerated organelles. After 6 days’ incubation, residues from butter oil mixtures showed similar granules or drops (Fig. S), although they were fewer than those found in the cod liver oil mixtures. With longer incubation, the amount of ceroid found in cod liver oil and butter oil mixtures progressively increased and the configuration changed, so that in the white blood cells the pigment was found generally as amorphous, and occasionally as crystal-like, osmiophilic material (Figs. 7 and 8). Ceroid in the red blood cells accumulated in the form of big coarse masses of uneven osmiophilic density (Fig. 9). Similar coarse granules in very small amounts were also found outside the cells but may have been extruded from cells to this location during manipulation of the mixtures. No ceroid was observed in the sections from hydrogenated coconut oil mixtures during any of the periods studied (Fig. 10). White and red blood cells, showed better preservation than those of cod liver oil and butter oil mixtures. DISCUSSION Since the first description of ceroid by Lillie et al. (1941), this pigment has been found in almost every tissue of experimental animals and man (Gyorgy, 1944; Popper et al., 1944; Endicott and Lillie, 1944; Mason and Emmel, 1944; Victor and Pappenheimer, 1945; Moore and Wang, 1947; Elftman et al., 1949; Radice and Herraiz, 1949; Pierangeli et al., 1949; Burt, 1952; Hartroft, 1953; Blanc et al., 1958). Ceroid has been described as a granular hyaline substance appearing as rounded or oval up to 20 p in diameter (Endicott and Lillie, 1944; Dam and Mason, 1945 ; Glavind et al., 1949; Hartroft, 1953). Under the conditions of this study, ceroid formed in vitro also appeared in a granular form. The number, size, and general configuration of the granules, as seen by light microscopy, apparently depends on the cellular elements in which they are formed, on the precursor fatty acids employed, and on the incubation time. From previous electron microscopic studies (Hess, 1955; Bondareff, 1957; Gadrat et al., 1960; Essner and Novikoff, 1960), it seems that ceroid accumulates in the cytoplasmic organelles catled Iysosomes as rounded, dense, and sometimes heterogeneous granules varying from 0.2 to several microns in diameter. Electron microscopically, the granules of the in vitro formed pigment in

224

FIG. 4. incubated detectable Unstained.

EDUARDO

A. PORTA

Electron micrograph of a section from the residue of a blood cell-cod liver oil mixture for 6 days. This portion of an altered erythrocyte shows several cavities without any peripheral membrane and containing ceroid in the farm of dense osmiophiIic drops. X 16,000.

ELECTRON

FIG. 5. Portion incubation. Drops similar configurations

MICROSCOPY

OF

CEROID

225

of matrix of an erythrocyte from a blood cell-butter oil mixture after 10 days of ceroid (Ce) contained in cavities without membranes are seen displaying to those in the cod liver oil mixtures. Unstained. x 32,000.

226

EDUARDO

A.

PORTA

FIG. 6. Eiectron micrograph of a portion of an altered leukocyte from the residues cell-cod liver oil mixture after 6 days incubation. Numerous drops of ceroid with osmiophilic density are contained in ill-dehned cavities. Unstained. x 16,000.

of blood moderate

ELECTRON

MICROSCOPY

OF CEROID

FIG. 7. Configuration of ceroid in a deteriorated leukocyte. oil mixture after 8 days incubation. Note the crystal-like cytoplasm of the cell and the accumu!ation of ceroid (thick adjacent leukocyte. Unstained. X 8000.

227

Section from a blood cell-cod liver appearance of ceroid (Ce) in the arrows) coating a membrane of an

228

EDUARDO

A. PORTA

FIG. 8. Electron micrograph showing the configuration of ceroid in a deteriorated leukocyte. Section from a blood cell-cod liver oil mixture after 15 days’ incubation. Note the solid and crystal-like appearance of the pigment. Unstained. X 16,000.

ELECTRON

MICROSCOPY

OF

CEROID

PIG. 9. Configuration of ceroid in a deteriorated erythrocyte mixture after 15 days incubation. Big coarse masses of uneven ill-defined cavities. Unstained. X 22,400.

229

from a blood cell-cod liver oil osmiophilic density are seen in

230

FIG.

oil after

EDUARDO

A. PORTA

10. A portion oi a deteriorated leukocyte from a mixture containing hydrogenated 10 days’ incubation. No ceroid was seen in these sections. Unstained. x 11,200.

coconut

ELECTRON

MICROSCOPY

OF CEROID

231

this study also varied in size and shape, but any relationship to cytoplasmic organelles could not clearly be established, since the alteration of the cells precluded any precise identification. However, in the early stages of the experiment, when some altered mitochondria could still be identified, it was evident that ceroid had no relation to them. On occasions, the pigment showed a tendency to accumulate in the inner surface of the cytoplasmic membrane as irregular osmiophilic masses. No lysosomes were recognized in the altered blood cells. Ceroid, when excited by ultraviolet light, displays an autofluorescence variously described as yellow-green (Dam and Granados, 1945 ; Mason et al., 1946), yellowishwhite (Lillie et al., 1941), orange (Mason et al., 1946), yellow or bronze (Gyorgy, 1944), and brown (Lee, 1950). We have found that the fluorescence of this pigment in vitro varied with incubation time: the yellowish-white of early stages gradually turns to a bronze color. In previous experiments with ceroid accumulation in the liver of rats, autofluorescence was still visible in the unstained sections 3 years after its formation (Porta and Hartroft, 1962). The sudanophilia of ceroid first described by Liliie et al. (1941) has been repeatedly confirmed by other investigators (Mason and Emmel, 1945; Elftman et al., 1949; Casselman, 1951; Hartroft, 1953). Hartroft (1953) has found that its affinity for the fat stain oil red 0 varies and has designated an intensively stained pigment “hemoceroid” and a weakly stained pigment “hyaloceroid.” He also suggested that the sudanophilic intensity varies directly with time. The present experiment confirmed the variability of most of the tinctorial affinities of ceroid. The positive reaction of ceroid with PAS is another property commonly but not consistently observed (Edwards and White, 1941; Dam and Granados, 1945; Casselman, 1951; Terry et al., 1954). The in vitro material in this study showed a rather uniform and practically invariable affinity for this reagent and for acid-fast stain whenever cod liver oil or butter oil was used in the incubation mixtures. Some authors consider ceroid a product of disordered fat metabolism (Edwards and Dalton, 1942; Radice and Herraiz, 1949; Burt, 1952) or as a product of the breakdown of necrotic tissues (Stowell and Lee, 1950). It is readily produced in vitro by mixing unsaturated fats, fatty acids and their esters with blood and other tissues (Endicott, 1944; Hartroft, 1951; Casselman, 1951). Hartroft (1951) produced the pigment in vivo by crushing the fatty mesenteric tissue of the rat and Hass (1938) by the injection of unsaturated natural oils, fatty acids, and esters into the subcutaneous tissue of the guinea pig. Ceroid can also be produced in mice and rats by feeding these species with diets containing some amounts of unsaturated fatty acids. Under these dietary conditions, the pigment is readily detected within one week in the cells of the reticula-endothelial system and appears before any other change can be noted (Porta and Hartroft, 1962). Vitamin E, a strong biological antioxidant, can prevent in vitro ceroid formation but cannot accelerate its removal (Ansanelli and Lane, 1957). Endicott (1944) and de Olivera (1949) believe that the pigment is a polymer of unstable peroxides of unsaturated fatty acids. Different chemical structures have been suggested by others (Hass, 1939; Edwards and Dalton, 1942; Thompson et al., 1960; Lee, 1950; Pearse, 1960). That a certain amount of unsaturated fatty acids is necessary for the in vitro formation of ceroid appeared evident under the condi-

232

EDUARDO

A, PORTA

tions of this experiment. The differences found in the pigment from cod liver oil and butter mixtures appears related to the different proportion of unsaturated fatty acids in the composition of these fats. SUMMARY The results of this study have indicated that a certain amount of unsaturated fatty acids is necessary to the in vitro formation of ceroid, as evidenced by its absence in the hydrogenated coconut oil mixture. The differences in the pigment found in the cod liver oil and in the butter mixtures probably was related to the different unsaturated fatty acid content of these fats. REFERENCES ANSANELLI, V., JR., and LANE, N. (1957). Lipochrome (ceroid) pigment of the small intestine. Ann. Surg. 146, 117-123. BLANC, W. A., REID, J., and ANDERSON, D. H. (1958). A vitaminosis E in cystic fibrosis of the pancreas. Pediatrics 22, 494-W. BONDAREFF, W. (1957). Genesis of intracellular pigment in the spinal ganglia of senile rats. An electron microscope study. /. Gerontol. 12, 364-369. BURT, R. (1952). Incidence of acid-fast pigment (ceroid) in aortic atherosclerosis. Am. J. Clin. Pathol. 22, 135-139. CASSELMAN, W. G. B. (1951). The in vitro preparation and histochemical properties of substances resembling ceroid. J. Exptl. Med. 94, 549-562. DAM, H., and GRANADOS, H. (1945). Peroxidation of body fat in vitamin E deficiency. Acta. Physiol. &and. 10, 162-171. DAM, H., and MASON, K. E. (1945). Vitamin E and brownish discoloration of adipose tissue in rats fed diets high in cod liver oil. Federation Proc. 4, 153. DE OLIVEIR.~, J. D. (1949). Ceroid substance and its meaning. Ann. N.Y. Acad. Sci. 52, 125. EDWARDS, J. E., and WHITE, J. (1941). Pathologic changes with special reference to pigmentation and classification of hepatic tumors in rats fed p-dimethy:aminoazobenzene (butter yellow). J. Natl. Cancer Inst. 2, 157-183. EDWARDS, J. E., and DALTON, A. J. (1942). Induction of cirrhosis of the liver and of hepatomas with carbon tetrachloride. J. Natl. Cancer Inst. 3, 19-41. ELFTMAN, H., KAUNITZ, H., and SLANETZ, C. A. (1949). Histochemistry of uterine pigment in vitamin E-deficient rats. Ann. N.Y. Acad. Sci. 52, 72-79. ENDICOTT, K. M. (1944). Similarity of the acid-fast pigment, ceroid, and oxidized unsaturated fat. Srch. Pathol. 37, 49-53. ENDXOTT, K. M., and L~LIE, R. D. (1944). Ceroid, the pigment of dietary cirrhosis of ratsits characteristics and differentiation from hemofuchsin. Am. J. Pathol. 20, 149-153. ESSNER, E., and NOVIKOFF, A. B. (1960). Human hepatocellular pgiments and lysosomes. J. Ultrastructure Research 3, 374-391. FRITZ, K. E. (1961). A Study of Ceroid in the Human Aorta and Its Relation to Arteriosclerosis. M.A. Thesis, Albany Medical College of Union University, Albany, New York. GADRAT, J., PLANEL, H., GUILHEM, A., and IZARD, J. (1960). Contribution de la microscopic electronique a l’etude des lipopigments du foie et du cortex surrenal. Pathol. Biol. Semaine Hop. 8, 697-708. GLAVIND, J., GRANADOS, H., and HARTMAN, S. (1949). A histochemical method for the demonstration of fat peroxides. Experientia 5, 84-85. GYORGY, P. (1944). Experimental hepatic injury. Am. J. Clin. Path&. 14, 67-88. HARTROFT, W. S. (1951). In vitro and in vivo production of a ceroid-like substance from red blood cells and certain lipids. Science 113, 673-674. HARTROFT, W. S. (1953). Hemoceroid and hyaloceroid in atheromas. J. Gerontol. 8, 158-166. HARTROFT, W. S., and THOMAS, W. A. (1957). Pathological lesions related to disturbances of fat and cholesterol in man. J. Am. Med. Assoc. 164, 1899-1905. HASS, G. M. (1938). Tissue reactions to natural oils and fractions thereof. Arch. Pathol. 26, 956-965.

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G. M. (1939). Membrane formation at lipoid-aqueous interfaces in tissues. II. A correlaof the morphologic and chemical aspects. drch. Pathol. 28, 177-198. HESS, A. (1955). The fine structure of young and old spinal ganglia. Anat. Rec. 123, 399-424. LEE, C. S. (1950). Histochemical studies of the ceroid pigment of rats and mice and its relation to necrosis. J. Natl. Cancer. Inst. 11, 399-349. LILLIE, R. D., DAFT, F. S., and SEBRELL, W. H., JR. (1941). Cirrhosis of the liver in rats on a deficient diet and effects of alcohol. Public Health Rep. (U.S.) 56. MASSON, K. E., and EMMEL, A. F. (1944). Pigmentation of the sex glands in vitamin E-deficient rats. Yale J. Biol. Med. 17, 189-206. MASSON, K. E., and EMILZEL, A. F. (1945). Vitamin E and muscle pigment in the rat. Anat. Rec. 92, 33-59. MASSON, K. E., DAM, H., and GRANADOS, H. (1946). Histological changes in adipose tissue of rats fed a vitamin E-deficient diet high in cod liver oil. Anat. Rec. 94, 265-286. MOORE, T., and WANG, Y. L. (1943). The fluorescence of the tissues in avitaminosis E. Biochem. J. 37, i. PADPENHEIMER, A. M., and VICTOR, J. (1946). Ceroid pigment in human tissues. Am. J. Pathol. 22, 395-41.3. PIERANGELI, E., RADICE, J. C., and HERRAIZ, M. L. (1949). Deposits of fluorescent pigment in atrophic testes of vitamin E-deficient rats. Ann. N.Y. Acad. Sci. 52, 129-131. POPPER, H., GYORGY, P., and GOLDBLATT, H. (1944). Fluorescent material (ceroid) in experimental nutritional cirrhosis. Arch. Pathol. 37, 161-168. PORTA, E. A., and HARTROFT, W. S. (1963). Early deposition of ceroid in Kiipffer cells of mice fed hepatic necrogenic diets. Can. Med. Assoc. J. (in press). RADICE, J. C., and HERRXZ, M. L. (1949). Fluorescent pigment in the uteri of vitamin K-deficient rats. Ann. N.Y. Acad. Sci. 52, 126-128. STOWELL, R. E., and LEE, C. S. (1950). Histochemical studies of mouse liver after single feeding of carbon tetrachloride. Arch. Pathol. 50, 519-537. TERRY, R. D., SPERRY, W. M., and BRODOFF, B. (1954). .4dult lipoidosis resembling NiemannPick’s disease. Am. J. Pathol. 30, 263-285. THOMPSON, S. M., THOMASSEN, R. W., YOST, D. H., and WEIGAND, R. G. (1960). A study on pigment deposited by intravenous fat emulsions. J. Nutr. 71, 37-44. VICTOR, J., and PAPPENHEIMER, A. M. (1945). The influence of choline, cystine, and of a-tocopherol upon the occurrence of ceroid pigment in dietary cirrhosis of rats. J. Erptt. Med. 32, 373-383.

HASS,

tion