22 (1975) 629-636
Atherosclerosis,
629
0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Short Communication
CRYSTALS
IN
C. W. M. ADAMS
ATHEROSCLEROTIC
AND
LESIONS:
REAL
OR
ARTEFACT?
0. B. BAYLISS
Department of Pathology, Guy’s Hospital London SE1 9RT (Great Britain)
Medical
School,
St. Thomas’ Street,
University
of London,
(Received May 13th, 1975) (Revised, received June 30th, 1975) (Accepted June 30th, 1975)
SUMMARY
Atherosclerotic lesions were obtained cholesterol-fed rabbits. They were maintained were prepared
from man during surgery and from at about 37 “C during handling. Smears
on glass slides and these were examined
microscopically
at 37 “C.
Solid rhomboidal or thick needle-like crystals were present at 37 “C but increased in numbers or in size or both on cooling. Staining studies and measurement of melting point (133-153 “C) suggested that such crystals are composed largely of free cholesterol or a related sterol. Liquid crystals exhibiting conic focal (Maltese cross) anisotropism were present at 37 “C and did not appreciably increase in either size or numbers on cooling. Staining studies and their resistance to digitonin suggested that these Maltese cross crystals are largely esterified cholesterol. Thin needle-like crystals arranged like feathers or in rosettes were seen in smears of adipose tissue and were attributed to triglycerides.
Key words:
Atherosclerosis - Crystals - EsteriJied cholesterol - Free cholesterol Triglycerides
INTRODUCTION
It is not clear whether
crystals
are present
in various
The authors are grateful to the British Heart Foundation
atherosclerotic
for partial support.
lesions
630
SHORT
COMMUNICATION
during life. Some workers have held that the typical crystals in frozen sections or clefts in paraffin-embedded tissues represent precipitation of cholesterol from solution in other lipids brought about by cooling after deathl. The chemical nature of the various
types of crystal in atherosclerotic
lesions is also not clear: some may be com-
posed of free cholesterol, mixed free and esterified cholesterol, lipid mixtures or even esterified cholesterol alonee,s,“. The purpose
of this paper is to distinguish
cholesterol-phospho-
some types of crystalline
structures
seen in atherosclerotic lesions and, by examining and photographing them at 37°C to determine whether they were likely to have been present in the lesion during life. METHODS
AND
MATERIALS
Lipid preparations Samples of fibrofatty plaques were taken from the thoracic aorta of 4 rabbits who had been fed cholesterol for 12 weeks and then allowed a normal diet for 6 months. Atheromatous plaques were obtained from the thoracic aorta of 2 rabbits fed cholesterol for 12 weeks and then killed. The diet was the customary 1% powdered cholesterol rubbed into SGl pellets previously “dressed” with sunflower seed oil. Ascending thoracic human aorta was obtained at surgery from 2 cases undergoing aortic valve replacement. This tissue showed occasional fatty streaks (man of 29 yr) or small fibrofatty Donald
plaques
Ross, Surgeon,
(man of 53 yr); it was made available
by kindness
of Mr.
Guy’s Hospital.
The tissues were maintained at or near 37°C by the following manoeuvres. Rabbits were killed with nembutal anaesthesia and then dissected in a hot room maintained at 37-38°C. Human tissue was placed directly at surgery into a glass container previously warmed to about 37°C. The glass container was then placed in the investigator’s axilla and transported back to the hot room with this thermal protection. The investigators’ axillary temperature varied between 35.5 and 37.o”C, but this would seem to represent or be consistent with normal diurnal variation in bodily temperature. Atheromatous and atherosclerotic lesions were scraped with a warm scalpel and then thinly smeared on glass slides at 37°C; the preparation was protected with a warm coverslip.
The preparations
were examined
and photographed
by polarized
light or by the specimen’s refractivity using a Leitz Orthomat photomicroscope; the apparatus had been equilibrated at 37°C in the hot room for 24 h before use. Melting points of some crystals were determined on a solid brass 12-V heated stage (up to 360°C) fitted to a solid brass Leitz monocular microscope (Professor G. A. D. Hazelwood). Smears were extracted with the solvents or stained by the histochemical methods specified in Table 1. Solubility was established under the microscope by observing the lipid smear after a drop of solvent had been allowed by capillary attraction to run between the slide and coverslip. Free cholesterol in the smears was converted to its digitonide by exposure for 3 h at 20°C to an 0.5 % solution of digitonin in 40 % ethanol.
631
SHORT COMMUNICATION TABLE
1
Crystal shape Birefringence Melting point Oil Red 05sF (triglycerides, cholesterol esters) Perchloric acid naphthoquinone5,” (free and ester cholesterol) Bromine Sudan black6z7 (free and ester cholesterol, triglycerides, phospholipids) Acetone Chloroform-methanol (2:1, v/v) Water N-HCL
rhomboidal anisotropic 133-153°C
Maltese cross conic focal anisotropic ?
feathery* isotropic 15-22°C
I
i
+
+
sol. sol. insol. insol.
sol. sol. insol. insol.
sol. sol. insol. insol.
* On cooling to 4°C; liquid at room temperature.
RESULTS
Direct smears of lipids obtained from both progressive and regressive atherosclerotic rabbit aortas, maintained at 37-38°C showed that most lipid in these tissues was present as liquid crystals that exhibited ‘Maltese cross’ birefringence (Figs. 1, 2, conic focal anisotropism). However, a number of smaller or larger rhomboidal true crystals were also seen (Fig. 3); such crystals were more abundant in the deeper scrapings from rabbit atheromatous lesions. Both rhomboidal and liquid crystals were also recovered 29 yr and moderate
from the two specimens of human in the man of 53 yr (Fig. 4).
Cooling
(-
to room temperature
aorta;
22°C) or 4°C caused
scanty
in the man of
no increase
in droplets
with Maltese cross birefringence, but often increased the number of rhomboidal crystals, sometimes to a marked degree (Figs. 5 and 6). Feathery crystals appeared on cooling, both in the smears of atheroma lipids and in smears of whole aortic wall (Fig. 7). On testing on the hot-stage
microscope,
the feathery
crystals melted between
15 and 22°C whereas the rhomboidal crystals largely melted between 133 and 153°C. The staining and solubility features of these various crystals is shown in Table 1. COMMENT
The results show that true crystals are present in atheromatous and atherosclerotic lesions maintained outside the body at 37°C. Such crystals often increase in size and numbers on cooling. This suggests that a proportion of true crystals seen in material treated in the conventional way are derived by precipitation on cooling the atheromatous lipid deposits, either as new crystals or by enlargement of existing crystals. The melting point and histochemical results with these crystals suggest that they are mainly free cholesterol, possibly admixed with a related sterol.
632
Fig. 1. Liquid crystals showing Maltese cross anisotropism. atheroma, polarized light, photographed at 37”C, x 310.
Fig. 2. Same view as Fig. 1 in ordinary light.
SHORT COMMUNICATION
Smear fromrabbit
aorta with progressive
SHORT COMMUNICATION
633
Fig. 3. Rhomboidal light, 37”C, x 310.
crystals in smear from regressive atherosclerosis
in rabbit aorta. Polarized
Fig. 4. Rhomboidal 37”C, x 310.
crystals in smears from ascending aorta from man of 53 yr. Polarized light,
634
SHORT COMMUNICATION
B Fig. 5. Rhomboidal crystals in smear of regressive rabbit atheroma, polarized light. A: at 37°C; B: similar field but after cooling to 4 “C. Note increase in number of rhomboidal crystals and further crystals forming on the surface of the rhomboidal crystals, polarized light, x 310.
SHORT COMMUNICATION
635
Fig. 6. Left, scanty Maltese cross crystals in smear of progressive rabbit atheroma, polarized light, 37°C. Right, same field but after cooling to 4°C; note newly-formed rhomboidal crystals, where there were previously none, some forming over surface of Maltese cross crystals, polarized light, x 3 IO.
Fig. 7. Feathery crystals in smear of whole rabbit aorta; these appeared on cooling to 4°C but melted between 15 and 22”C, x 310.
636
SHORT COMMUNICATION
Maltese cross birefringence would seem to be due to cholesterol esters, as sugby Insull and co-workers394 and, seemingly, by Learyl. Thus, these liquid crystals do not change their form after treatment with digitonin and remain soluble in acetone: their non-reactivity with digitonin is against a compositions of free gested
cholesterol
mixed with phospholipid.
Feathery are prominent of triglycerides.
crystals are entirely
an artefact
of cooling below 15°C. As such crystals
in smears of adipose tissue (see alsoi), It must
be pointed
out, however,
they would seem to be composed that these crystals
in smears
of
atheroma lipids may only represent contamination by adventitial adipose tissue. A conclusion from this work is that a proportion of crystals of free cholesterol exist in vivo. Such crystals being in a physically inaccessible state - hydrophobic, not dispersed, largely extracellular and presumably dissociated from lipoprotein would constitute an important part of the slowly miscible8 or relatively inert9 pool of cholesterol
in the atheromatous
lesion.
REFERENCES 1 LEARY,T., Crystalline ester cholesterol and atherosclerosis, Arch. Path., 47 (1949) 1. 2 WELLER,R. O., Cytochemistry of lipids in atherosclerosis, J. Path. Bact., 94 (1967) 171. 3 LANG, P. D. AND INSULL, JR., W., Lipid droplets in atherosclerotic fatty streaks of human aorta, J. Clin. Invest., 49 (1970) 1479. 4 HATA, Y., HOWER, J. AND INSULL, JR., W., Cholesteryl ester-rich inclusions from human aortic fatty streak and fibrous plaque lesions of atherosclerosis, Part 1 (Crystalline properties, size and internal structure), Amer. J. Path., 75 (1974) 423. 5 ADAMS, C. W. M., Lipid histochemistry. In: D. KRITCHEVSKYAND R. PAOLETTI(Eds.), Advances in Lipid Research, Vol. 7, Academic Press, New York, 1969, p. 1. 6 ADAMS, C. W. M. AND BAYLISS,0. B., Lipid histochemistry. In: D. GLICK AND R. ROSENBAUM (Eds.), Techniques of Biochemical and Biophysical Morphology, Vol. 2, John Wiley, Baltimore, Md., 1975, p. 99. 7 BAYLISS,0. B. AND ADAMS, C. W. M., Bromine-Sudan black (BSB). A general stain for tissue lipids including free cholesterol, Histochem. J., 4 (1972) 505. 8 LOFLAND,H. B. AND CLARKSON,T. B., The bi-directional transfer of cholesterol in normal aorta, fatty streaks and atheromatous plaques, Proc. Sot. Exp. Biol. Med., 133 (1970) 1. 9 ADAMS, C. W. M., Tissue changes and lipid entry in developing atheroma. In: Atherogenesis: Initiating Factors (CIBA Foundation Symposium NS No. 12), Excerpta Medica, Amsterdam, 1973, p. 5.
Atherosclerosis,
629
0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Short Communication
CRYSTALS
IN
C. W. M. ADAMS
ATHEROSCLEROTIC
AND
LESIONS:
REAL
OR
ARTEFACT?
0. B. BAYLISS
Department of Pathology, Guy’s Hospital London SE1 9RT (Great Britain)
Medical
School,
St. Thomas’ Street,
University
of London,
(Received May 13th, 1975) (Revised, received June 30th, 1975) (Accepted June 30th, 1975)
SUMMARY
Atherosclerotic lesions were obtained cholesterol-fed rabbits. They were maintained were prepared
from man during surgery and from at about 37 “C during handling. Smears
on glass slides and these were examined
microscopically
at 37 “C.
Solid rhomboidal or thick needle-like crystals were present at 37 “C but increased in numbers or in size or both on cooling. Staining studies and measurement of melting point (133-153 “C) suggested that such crystals are composed largely of free cholesterol or a related sterol. Liquid crystals exhibiting conic focal (Maltese cross) anisotropism were present at 37 “C and did not appreciably increase in either size or numbers on cooling. Staining studies and their resistance to digitonin suggested that these Maltese cross crystals are largely esterified cholesterol. Thin needle-like crystals arranged like feathers or in rosettes were seen in smears of adipose tissue and were attributed to triglycerides.
Key words:
Atherosclerosis - Crystals - EsteriJied cholesterol - Free cholesterol Triglycerides
INTRODUCTION
It is not clear whether
crystals
are present
in various
The authors are grateful to the British Heart Foundation
atherosclerotic
for partial support.
lesions
630
SHORT
COMMUNICATION
during life. Some workers have held that the typical crystals in frozen sections or clefts in paraffin-embedded tissues represent precipitation of cholesterol from solution in other lipids brought about by cooling after deathl. The chemical nature of the various
types of crystal in atherosclerotic
lesions is also not clear: some may be com-
posed of free cholesterol, mixed free and esterified cholesterol, lipid mixtures or even esterified cholesterol alonee,s,“. The purpose
of this paper is to distinguish
cholesterol-phospho-
some types of crystalline
structures
seen in atherosclerotic lesions and, by examining and photographing them at 37°C to determine whether they were likely to have been present in the lesion during life. METHODS
AND
MATERIALS
Lipid preparations Samples of fibrofatty plaques were taken from the thoracic aorta of 4 rabbits who had been fed cholesterol for 12 weeks and then allowed a normal diet for 6 months. Atheromatous plaques were obtained from the thoracic aorta of 2 rabbits fed cholesterol for 12 weeks and then killed. The diet was the customary 1% powdered cholesterol rubbed into SGl pellets previously “dressed” with sunflower seed oil. Ascending thoracic human aorta was obtained at surgery from 2 cases undergoing aortic valve replacement. This tissue showed occasional fatty streaks (man of 29 yr) or small fibrofatty Donald
plaques
Ross, Surgeon,
(man of 53 yr); it was made available
by kindness
of Mr.
Guy’s Hospital.
The tissues were maintained at or near 37°C by the following manoeuvres. Rabbits were killed with nembutal anaesthesia and then dissected in a hot room maintained at 37-38°C. Human tissue was placed directly at surgery into a glass container previously warmed to about 37°C. The glass container was then placed in the investigator’s axilla and transported back to the hot room with this thermal protection. The investigators’ axillary temperature varied between 35.5 and 37.o”C, but this would seem to represent or be consistent with normal diurnal variation in bodily temperature. Atheromatous and atherosclerotic lesions were scraped with a warm scalpel and then thinly smeared on glass slides at 37°C; the preparation was protected with a warm coverslip.
The preparations
were examined
and photographed
by polarized
light or by the specimen’s refractivity using a Leitz Orthomat photomicroscope; the apparatus had been equilibrated at 37°C in the hot room for 24 h before use. Melting points of some crystals were determined on a solid brass 12-V heated stage (up to 360°C) fitted to a solid brass Leitz monocular microscope (Professor G. A. D. Hazelwood). Smears were extracted with the solvents or stained by the histochemical methods specified in Table 1. Solubility was established under the microscope by observing the lipid smear after a drop of solvent had been allowed by capillary attraction to run between the slide and coverslip. Free cholesterol in the smears was converted to its digitonide by exposure for 3 h at 20°C to an 0.5 % solution of digitonin in 40 % ethanol.
631
SHORT COMMUNICATION TABLE
1
Crystal shape Birefringence Melting point Oil Red 05sF (triglycerides, cholesterol esters) Perchloric acid naphthoquinone5,” (free and ester cholesterol) Bromine Sudan black6z7 (free and ester cholesterol, triglycerides, phospholipids) Acetone Chloroform-methanol (2:1, v/v) Water N-HCL
rhomboidal anisotropic 133-153°C
Maltese cross conic focal anisotropic ?
feathery* isotropic 15-22°C
I
i
+
+
sol. sol. insol. insol.
sol. sol. insol. insol.
sol. sol. insol. insol.
* On cooling to 4°C; liquid at room temperature.
RESULTS
Direct smears of lipids obtained from both progressive and regressive atherosclerotic rabbit aortas, maintained at 37-38°C showed that most lipid in these tissues was present as liquid crystals that exhibited ‘Maltese cross’ birefringence (Figs. 1, 2, conic focal anisotropism). However, a number of smaller or larger rhomboidal true crystals were also seen (Fig. 3); such crystals were more abundant in the deeper scrapings from rabbit atheromatous lesions. Both rhomboidal and liquid crystals were also recovered 29 yr and moderate
from the two specimens of human in the man of 53 yr (Fig. 4).
Cooling
(-
to room temperature
aorta;
22°C) or 4°C caused
scanty
in the man of
no increase
in droplets
with Maltese cross birefringence, but often increased the number of rhomboidal crystals, sometimes to a marked degree (Figs. 5 and 6). Feathery crystals appeared on cooling, both in the smears of atheroma lipids and in smears of whole aortic wall (Fig. 7). On testing on the hot-stage
microscope,
the feathery
crystals melted between
15 and 22°C whereas the rhomboidal crystals largely melted between 133 and 153°C. The staining and solubility features of these various crystals is shown in Table 1. COMMENT
The results show that true crystals are present in atheromatous and atherosclerotic lesions maintained outside the body at 37°C. Such crystals often increase in size and numbers on cooling. This suggests that a proportion of true crystals seen in material treated in the conventional way are derived by precipitation on cooling the atheromatous lipid deposits, either as new crystals or by enlargement of existing crystals. The melting point and histochemical results with these crystals suggest that they are mainly free cholesterol, possibly admixed with a related sterol.
632
Fig. 1. Liquid crystals showing Maltese cross anisotropism. atheroma, polarized light, photographed at 37”C, x 310.
Fig. 2. Same view as Fig. 1 in ordinary light.
SHORT COMMUNICATION
Smear fromrabbit
aorta with progressive
SHORT COMMUNICATION
633
Fig. 3. Rhomboidal light, 37”C, x 310.
crystals in smear from regressive atherosclerosis
in rabbit aorta. Polarized
Fig. 4. Rhomboidal 37”C, x 310.
crystals in smears from ascending aorta from man of 53 yr. Polarized light,
634
SHORT COMMUNICATION
B Fig. 5. Rhomboidal crystals in smear of regressive rabbit atheroma, polarized light. A: at 37°C; B: similar field but after cooling to 4 “C. Note increase in number of rhomboidal crystals and further crystals forming on the surface of the rhomboidal crystals, polarized light, x 310.
SHORT COMMUNICATION
635
Fig. 6. Left, scanty Maltese cross crystals in smear of progressive rabbit atheroma, polarized light, 37°C. Right, same field but after cooling to 4°C; note newly-formed rhomboidal crystals, where there were previously none, some forming over surface of Maltese cross crystals, polarized light, x 3 IO.
Fig. 7. Feathery crystals in smear of whole rabbit aorta; these appeared on cooling to 4°C but melted between 15 and 22”C, x 310.
636
SHORT COMMUNICATION
Maltese cross birefringence would seem to be due to cholesterol esters, as sugby Insull and co-workers394 and, seemingly, by Learyl. Thus, these liquid crystals do not change their form after treatment with digitonin and remain soluble in acetone: their non-reactivity with digitonin is against a compositions of free gested
cholesterol
mixed with phospholipid.
Feathery are prominent of triglycerides.
crystals are entirely
an artefact
of cooling below 15°C. As such crystals
in smears of adipose tissue (see alsoi), It must
be pointed
out, however,
they would seem to be composed that these crystals
in smears
of
atheroma lipids may only represent contamination by adventitial adipose tissue. A conclusion from this work is that a proportion of crystals of free cholesterol exist in vivo. Such crystals being in a physically inaccessible state - hydrophobic, not dispersed, largely extracellular and presumably dissociated from lipoprotein would constitute an important part of the slowly miscible8 or relatively inert9 pool of cholesterol
in the atheromatous
lesion.
REFERENCES 1 LEARY,T., Crystalline ester cholesterol and atherosclerosis, Arch. Path., 47 (1949) 1. 2 WELLER,R. O., Cytochemistry of lipids in atherosclerosis, J. Path. Bact., 94 (1967) 171. 3 LANG, P. D. AND INSULL, JR., W., Lipid droplets in atherosclerotic fatty streaks of human aorta, J. Clin. Invest., 49 (1970) 1479. 4 HATA, Y., HOWER, J. AND INSULL, JR., W., Cholesteryl ester-rich inclusions from human aortic fatty streak and fibrous plaque lesions of atherosclerosis, Part 1 (Crystalline properties, size and internal structure), Amer. J. Path., 75 (1974) 423. 5 ADAMS, C. W. M., Lipid histochemistry. In: D. KRITCHEVSKYAND R. PAOLETTI(Eds.), Advances in Lipid Research, Vol. 7, Academic Press, New York, 1969, p. 1. 6 ADAMS, C. W. M. AND BAYLISS,0. B., Lipid histochemistry. In: D. GLICK AND R. ROSENBAUM (Eds.), Techniques of Biochemical and Biophysical Morphology, Vol. 2, John Wiley, Baltimore, Md., 1975, p. 99. 7 BAYLISS,0. B. AND ADAMS, C. W. M., Bromine-Sudan black (BSB). A general stain for tissue lipids including free cholesterol, Histochem. J., 4 (1972) 505. 8 LOFLAND,H. B. AND CLARKSON,T. B., The bi-directional transfer of cholesterol in normal aorta, fatty streaks and atheromatous plaques, Proc. Sot. Exp. Biol. Med., 133 (1970) 1. 9 ADAMS, C. W. M., Tissue changes and lipid entry in developing atheroma. In: Atherogenesis: Initiating Factors (CIBA Foundation Symposium NS No. 12), Excerpta Medica, Amsterdam, 1973, p. 5.
