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Method for the histochemical identification of choline-containing phospholipids
109
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eleetrophoretic precipitation b a n d (Fig. 1C) as well as the considerable decrease of the fl-lipoprotein fraction (Fig. 1A, B) showed t h a t the interaction of the aortic MPS with serum fl-lipoproteins resulted in a significantly higher m o b i l i t y of the latter. The mobility of all o t h e r serum protein fractions r e m a i n e d u n c h a n g e d , indicating t h a t only the fl-lipoproteins form complexes with the aortic MPS. B o t h the gel electrophoretic a n d immunoelectrophoretic p a t t e r n of a s u p e r n a t a n t after the precipitation of s e r u m fl-lipoproteins with aortic MPS showed (Fig. 2) virtually complete r e m o v a l of fl-lipoproteins under our e x p e r i m e n t a l conditions. The results of our present investigations give further evidences for the specific ability of binding fl-lipoproteins - - u n d e r certain experimental conditions - - of the aortic MPS. To t h r o w more light on the pathogenesis of atherosclerosis it is necessary to obtain more informations u n d e r a wide v a r i e t y of conditions a b o u t the functional properties of the vascular MPS.
REFERENCES 1 S. GER6, J. GERGELY, T. Dt~V/~NYI, L. JAKAB, J. SZEKELY AND S. VIR2{G, Nature, 187 (1960) 152. 2 S. GER6, J. GERGELY, T. DI~Va~NYI, L. JAKAB, j. SZ~KELY AND S. gIRtinG, .]. Atheroscler. Res., 1 (1961) 67. a j. S. AMENTAAND L. L. WATERS, Yale dr. Biol. Med., 33 (1960) 112. 4 M. BIHARI-VARGA,J. GERGELYAND S. GER6, Orv. Hetilap, 104 (1963) 1401. 5 T. D~VI~NYI, M. BIHARI-VARGA, L. JAKAB AND J. GERGELY, Kisdrl. Orvostud., 15 (1963) 326.
6 7 8 9 lO 11
M. DYRBYE AND J. E. KIRK, J. Gerontol., 12 (1957) 20. Z. STARY, F. BURSA, O. I~ALEOGLU AND M. BILEN, Bull. Fac. 2~ldd. fstanbul, 13 (1950) 243. C. RIMINGTON, Biochem. J., 34 (1940) 931. W. AYALA, L. V. MOOR AND E. L. HESS, J. Clin. Invest., 37 (1951) 81. Z. DISCHE, J. Biol. Chem., 167 (1947) 189. j . j . SCHEIDEGGER, Intern. Arch. Allergy, Appl. Immunol., 7 (1955) 103.
j . Atheroscler. Res., 4 (1964) 106-109
METHOD FOR THE HISTOCHEMICAL IDENTIFICATION
OF
CHOLINE-CONTAINING PHOSPHOLIPIDS C. J. F. B(DTTCHER AND E. BOELSMA-VAN HOUTE The Gaubius Institute, Leyden University, Leyden (The Netherlands)
(Received June 10th, 1963)
INTRODUCTION
I n t h e histochemical s t u d y of t h e localization of different lipids in various stages of atherosclerosis of the arterial wall, specific staining m e t h o d s were needed. S t a r t i n g from the colorimetric m i c r o - d e t e r m i n a t i o n of free and b o u n d choline b y B(~TTCHER J. Atheroscler. Res., 4 (1964) 109-112
110
C.J.F.
BOTTCHER, E. BOELSMA-VAN HOUTE
et al. 1, which is based on the colorimetric estimation of quaternary a m m o n i u m bases according to SAss et al. 2, a method was developed for the histochemical localization of choline-containing phospholipids, t h a t is to say of lecithins, lyso-lecithins and sphingomyelins together. MATERIAL AND METHODS
H u m a n aortic tissue, obtained from autopsy, was cut into 10/z thick sections on a Pearse t y p e cryostat microtome. Sections were applied to quartz slides and fixed in 4 % formaldehyde to which 1 % of calcium chloride was added. To protect lipids against dissolving in the lipid solvents used, sections were rinsed in t a p water and treated with a 10 % cobalt chloride solution in water for 24 h at room t e m p e r a t u r e ; then after another very thorough rinse in t a p w a t e r t h e y were placed in a solution of 1 % sodium periodate in water during 1 h at 37°C. Slides were rinsed once more in tap w a t e r and sections were dehydrated in graded alcohols, ending with two washings in absolute ethanol. Ethanol was carefully r e m o v e d from the sections b y toluene and thereafter slides were transferred into acetic anhydride/toluene (1 : 1 v/v). The colour was developed in a 2½ % solution of cis-aconitic anhydride in acetic anhydride/ toluene (2 : 3 v/v), prepared 24 h earlier; the reaction was carried out in quartz staining vessels for ½-1 h at room temperature. Excess reagent was r e m o v e d b y rinsing in acetic anhydride/toluene ( 1 : 1 v/v), sections were cleared in toluene, m o u n t e d and covered b y a normal glass coverslip. As the colonr is not stable, sections had to be examined within a few hours. RESULTS
Results are shown in Figs. 1-6. Lecithins, lysolecithins and sphingomyelins formed violet coloured complexes with the cis-aconitic anhydride. DISCUSSION
Method Several steps of tile staining method need further explanation, for instance the use of quartz slides and quartz staining vessels. This proved to be necessary because normal glassware is contaminated with LEWIS bases which interfere with complex formation and cause a dark brown b a c k g r o u n d colour in the sections. Once the coloured complex between the q u a t e r n a r y b o u n d nitrogen and the cis-aconitic anhydride has been developed and the excess of colour reagent removed, a normal glass coverslip can be used. Fixation of lipids was carried out through oxidation a n d polymerization b y means of cobalt and periodate successively and not b y dichromate, which is used as a m o r d a n t in BAKER'S acid haematein test for phospholipids s. The former m e t h o d was chosen because the use of dichromate gives rise to insoluble precipitates of polymerized J. Atheroscler. Res., 4 (1964) 109-112
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111
Fig. 1. D e t a i l of a f i b r o u s p l a q u e ; h u m a n aorta, a t h e r o s c l e r o s i s s t a g e I I I . Violet coloured c o m p l e x e s of c h o l i n e - c o n t a i n i n g p h o s p h o l i p i d s w i t h cis-aconitic a n h y d r i d e . A c o n s i d e r a b l e a m o u n t of t h e c o l o u r e d c o m p l e x c a n be s e e n e x t r a - c e l l u l a r l y . × 400. Fig. 2. C h o l e s t e r o l clefts w i t h i n a fibrous p l a q u e ; h u m a n a o r t a , a t h e r o s c l e r o s i s s t a g e I I I . Clefts are s u r r o u n d e d b y c h o l i n e - c o n t a i n i n g p h o s p h o l i p i d s . E x t r a c e l l u l a r lecithins, l y s o l e c i t h i n s a n d / o r s p h i n g o m y e l i n s a p p e a r to be p r e s e n t in s m a l l c o n c e n t r a t i o n . × 400. Fig. 3. C h o l i n e - c o n t a i n i n g p h o s p h o l i p i d s in t h e cell n u c l e i a n d in e x t r a c e l l u l a r r e g i o n s of a f i b r o u s p l a q u e of h u m a n a o r t a , a t h e r o s c l e r o s i s s t a g e I I I . T h e c h o l e s t e r o l clefts a r e s u r r o u n d e d b y lipids t h a t s t a i n p o s i t i v e l y w i t h cis-aconitic a n h y d r i d e . X 400. Fig. 4. M e d i a of y o u n g u n d i s e a s e d h u m a n aorta. S m o o t h m u s c l e ceils a n d elastic l a m e U a e p o s i t i v e t o cis-aconitic a n h y d r i d e . S m a l l a m o u n t s of violet c o l o u r e d c o m p l e x e s b e t w e e n elastic l a m e l l a e . × 400. Fig. 5. M e d i a of h u m a n a o r t a , a t h e r o s c l e r o s i s s t a g e I. S t a i n e d w i t h cis-aconitic a n h y d r i d e . A l a r g e a m o u n t of p o s i t i v e l y s t a i n e d p h o s p h o l i p i d s c a n be s e e n in t h e i n t e r l a m e l l a r spaces. T h e e l a s t i c l a m e l l a e are also p o s i t i v e . × 400. Fig. 6. I n t i m a a n d m e d i a of y o u n g u n d i s e a s e d h u m a n a o r t a . C h o l i n e - c o n t a i n i n g p h o s p h o l i p i d s in t h e e n d o t h e l i a l l i n i n g , in t h e i n t e r n a l elastic m e m b r a n e a n d t h e elastic l a m e l l a e of t h e m e d i a , a n d in t h e cell nuclei. T h e i n t i m a c o n t a i n s h a r d l y a n y p o s i t i v e l y s t a i n e d m a t e r i a l . × 400.
J. Atheroscler. Res., 4 (1964) 1 0 9 - 1 1 2
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c h r o m i c h y d r o x i d e 4. These p r e c i p i t a t e s also p r e v e n t c o m p l e x f o r m a t i o n . F o r t h e s a m e reason t h e c o b a l t chloride has to be w a s h e d o u t v e r y t h o r o u g h l y before a p p l i c a t i o n of t h e slides to p e r i o d a t e . P o s s i b l y t h e c o b a l t s a l t can be a d d e d to t h e f i x a t i v e as well (compare McMANuS5). T h e n e x t d e h y d r a t i o n of t h e tissue sections h a s to b e d o n e w i t h care, as r e m a i n i n g w a t e r r e a c t s w i t h t h e cis-aconitic a n h y d r i d e t o give c i s - a c o n i t i c acid; e t h a n o l also d e s t r o y s t h e cis-aconitic a n h y d r i d e , so t h a t i t s r e p l a c e m e n t b y t o l u e n e a n d acetic a n h y d r i d e / t o l u e n e h a s to be c o m p l e t e as well. Results
As SASS 2 h a s p o i n t e d out, t h e cis-aconitic a n h y d r i d e r e a c t s w i t h b o t h q u a t e r n a r y a n d t e r t i a r y b o u n d nitrogen. T h e m e t h o d is, h o w e v e r , of p o o r s e n s i t i v i t y for t e r t i a r y amines, so t h a t t h e p o s i t i v e r e a c t i o n of t h e elastic l a m e l l a e of the h u m a n a o r t a is m o s t l i k e l y due to t h e i r c o n t e n t of c h o l i n e - c o n t a i n i n g p h o s p h o l i p i d s a n d n o t to histidine w i t h its t e r t i a r y b o u n d n i t r o g e n , t h a t occurs in s m a l l a m o u n t s in elastin 6. L i p i d s in elastic l a m e l l a e of t h e h u m a n a o r t a h a v e b e e n r e p o r t e d , a m o n g s t o t h e r s b y ADAMS7, ADAMS AND TUQUAN 8 a n d LANSING9. T h e p o s i t i v e r e a c t i o n of t h e elastic l a m e l l a e is in g o o d a c c o r d a n c e w i t h ADAMS' o b s e r v a t i o n 1° t h a t s p h i n g o m y e l i n is t h e c h a r a c t e r istic l i p i d of t h e a r t e r i a l elastica. I t is t h e m o r e p r o b a b l e t h a t choline is t h e p o s i t i v e r e a c t i n g c o m p o n e n t as collagen c o n t a i n s t e n t i m e s m o r e h i s t i d i n e t h a n elastin does 6 a n d collagen shows no p o s i t i v e r e a c t i o n a t all. A n o t h e r a r g u m e n t in f a v o u r of t h e p h o s p h o l i p i d c o n t e n t of t h e elastic l a m e l l a e is t h e decrease in cis-aconitic a n h y d r i d e p o s i t i v i t y of e l a s t i n a f t e r e x t r a c t i o n w i t h m e t h a n o l / c h l o r o f o r m (1 : 2 v/v). T h e s a m e h o l d s for t h e p o s i t i v e r e a c t i o n of cell nuclei: nucleic acids c o n t a i n t e r t i a r y b o u n d n i t r o g e n , b u t t h e p o s i t i v e r e a c t i o n is a b s e n t a f t e r l i p i d ext r a c t i o n so t h a t t h e p o s i t i v e r e a c t i o n m u s t be a t t r i b u t e d t o a n i n c r e a s e d c o n t e n t of choline-containing phospholipids. ACKNOWLEDGEMENT T h e a u t h o r s are g r a t e f u l for t h e v a l u a b l e s u g g e s t i o n s g i v e n b y Drs. C. PRIES. REFERENCES
a C. J. F. BOTTCHER, C. PRIES AND C. M. VAN GENT, Rec. Tray. Chim., 80 (1961) 1169. 2 S. SAss, J. J. KAUFMAN,A. A. CARDENASAND J. J. MARTIN, Anal. Chem., 30 (1958) 529." 3 j . 1~. BAKER, Quart. J. Microscop. Sci., 87 (1946) 441. 4 F. J. ~v[. HESLINGA, De Bichromaatbeitsing en de Kleurbaarheid van Gebeitst Lipoid Onderzocht met Behulp van Reflectometrie, Thesis, Leiden, 1957, p. 29. 5 j. F. A. MCMANUS, J. Pathol. Bacteriol., 58 (1946) 93. 6 A. I. LANSING, in A. I. LANSING (Ed.), The Arterial Wall, Williams and Wilkins Comp., Baltimore, 1959, p. 140. 7 C. W. M. ADAMS, Lancet, i (1959) 1075. 8 C. W. M. ADAMSAND 2q'. A. TUQUAN, J. Pathol. Bacteriol., 82 (1961) 131. 9 A. I. LANSING,T. B. ROSENTHAL, M. ALEX AND E. W. DAMPSEY, Anat. Record, 114 (1952) 555. 10 C. W. M. ADAMSAND O. B. BAYLISS, J. Pathol. Bacteriol., 85 (1963) 113. j . Atheroscler. Res., 4 (1964) 109-112
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eleetrophoretic precipitation b a n d (Fig. 1C) as well as the considerable decrease of the fl-lipoprotein fraction (Fig. 1A, B) showed t h a t the interaction of the aortic MPS with serum fl-lipoproteins resulted in a significantly higher m o b i l i t y of the latter. The mobility of all o t h e r serum protein fractions r e m a i n e d u n c h a n g e d , indicating t h a t only the fl-lipoproteins form complexes with the aortic MPS. B o t h the gel electrophoretic a n d immunoelectrophoretic p a t t e r n of a s u p e r n a t a n t after the precipitation of s e r u m fl-lipoproteins with aortic MPS showed (Fig. 2) virtually complete r e m o v a l of fl-lipoproteins under our e x p e r i m e n t a l conditions. The results of our present investigations give further evidences for the specific ability of binding fl-lipoproteins - - u n d e r certain experimental conditions - - of the aortic MPS. To t h r o w more light on the pathogenesis of atherosclerosis it is necessary to obtain more informations u n d e r a wide v a r i e t y of conditions a b o u t the functional properties of the vascular MPS.
REFERENCES 1 S. GER6, J. GERGELY, T. Dt~V/~NYI, L. JAKAB, J. SZEKELY AND S. VIR2{G, Nature, 187 (1960) 152. 2 S. GER6, J. GERGELY, T. DI~Va~NYI, L. JAKAB, j. SZ~KELY AND S. gIRtinG, .]. Atheroscler. Res., 1 (1961) 67. a j. S. AMENTAAND L. L. WATERS, Yale dr. Biol. Med., 33 (1960) 112. 4 M. BIHARI-VARGA,J. GERGELYAND S. GER6, Orv. Hetilap, 104 (1963) 1401. 5 T. D~VI~NYI, M. BIHARI-VARGA, L. JAKAB AND J. GERGELY, Kisdrl. Orvostud., 15 (1963) 326.
6 7 8 9 lO 11
M. DYRBYE AND J. E. KIRK, J. Gerontol., 12 (1957) 20. Z. STARY, F. BURSA, O. I~ALEOGLU AND M. BILEN, Bull. Fac. 2~ldd. fstanbul, 13 (1950) 243. C. RIMINGTON, Biochem. J., 34 (1940) 931. W. AYALA, L. V. MOOR AND E. L. HESS, J. Clin. Invest., 37 (1951) 81. Z. DISCHE, J. Biol. Chem., 167 (1947) 189. j . j . SCHEIDEGGER, Intern. Arch. Allergy, Appl. Immunol., 7 (1955) 103.
j . Atheroscler. Res., 4 (1964) 106-109
METHOD FOR THE HISTOCHEMICAL IDENTIFICATION
OF
CHOLINE-CONTAINING PHOSPHOLIPIDS C. J. F. B(DTTCHER AND E. BOELSMA-VAN HOUTE The Gaubius Institute, Leyden University, Leyden (The Netherlands)
(Received June 10th, 1963)
INTRODUCTION
I n t h e histochemical s t u d y of t h e localization of different lipids in various stages of atherosclerosis of the arterial wall, specific staining m e t h o d s were needed. S t a r t i n g from the colorimetric m i c r o - d e t e r m i n a t i o n of free and b o u n d choline b y B(~TTCHER J. Atheroscler. Res., 4 (1964) 109-112
110
C.J.F.
BOTTCHER, E. BOELSMA-VAN HOUTE
et al. 1, which is based on the colorimetric estimation of quaternary a m m o n i u m bases according to SAss et al. 2, a method was developed for the histochemical localization of choline-containing phospholipids, t h a t is to say of lecithins, lyso-lecithins and sphingomyelins together. MATERIAL AND METHODS
H u m a n aortic tissue, obtained from autopsy, was cut into 10/z thick sections on a Pearse t y p e cryostat microtome. Sections were applied to quartz slides and fixed in 4 % formaldehyde to which 1 % of calcium chloride was added. To protect lipids against dissolving in the lipid solvents used, sections were rinsed in t a p water and treated with a 10 % cobalt chloride solution in water for 24 h at room t e m p e r a t u r e ; then after another very thorough rinse in t a p w a t e r t h e y were placed in a solution of 1 % sodium periodate in water during 1 h at 37°C. Slides were rinsed once more in tap w a t e r and sections were dehydrated in graded alcohols, ending with two washings in absolute ethanol. Ethanol was carefully r e m o v e d from the sections b y toluene and thereafter slides were transferred into acetic anhydride/toluene (1 : 1 v/v). The colour was developed in a 2½ % solution of cis-aconitic anhydride in acetic anhydride/ toluene (2 : 3 v/v), prepared 24 h earlier; the reaction was carried out in quartz staining vessels for ½-1 h at room temperature. Excess reagent was r e m o v e d b y rinsing in acetic anhydride/toluene ( 1 : 1 v/v), sections were cleared in toluene, m o u n t e d and covered b y a normal glass coverslip. As the colonr is not stable, sections had to be examined within a few hours. RESULTS
Results are shown in Figs. 1-6. Lecithins, lysolecithins and sphingomyelins formed violet coloured complexes with the cis-aconitic anhydride. DISCUSSION
Method Several steps of tile staining method need further explanation, for instance the use of quartz slides and quartz staining vessels. This proved to be necessary because normal glassware is contaminated with LEWIS bases which interfere with complex formation and cause a dark brown b a c k g r o u n d colour in the sections. Once the coloured complex between the q u a t e r n a r y b o u n d nitrogen and the cis-aconitic anhydride has been developed and the excess of colour reagent removed, a normal glass coverslip can be used. Fixation of lipids was carried out through oxidation a n d polymerization b y means of cobalt and periodate successively and not b y dichromate, which is used as a m o r d a n t in BAKER'S acid haematein test for phospholipids s. The former m e t h o d was chosen because the use of dichromate gives rise to insoluble precipitates of polymerized J. Atheroscler. Res., 4 (1964) 109-112
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111
Fig. 1. D e t a i l of a f i b r o u s p l a q u e ; h u m a n aorta, a t h e r o s c l e r o s i s s t a g e I I I . Violet coloured c o m p l e x e s of c h o l i n e - c o n t a i n i n g p h o s p h o l i p i d s w i t h cis-aconitic a n h y d r i d e . A c o n s i d e r a b l e a m o u n t of t h e c o l o u r e d c o m p l e x c a n be s e e n e x t r a - c e l l u l a r l y . × 400. Fig. 2. C h o l e s t e r o l clefts w i t h i n a fibrous p l a q u e ; h u m a n a o r t a , a t h e r o s c l e r o s i s s t a g e I I I . Clefts are s u r r o u n d e d b y c h o l i n e - c o n t a i n i n g p h o s p h o l i p i d s . E x t r a c e l l u l a r lecithins, l y s o l e c i t h i n s a n d / o r s p h i n g o m y e l i n s a p p e a r to be p r e s e n t in s m a l l c o n c e n t r a t i o n . × 400. Fig. 3. C h o l i n e - c o n t a i n i n g p h o s p h o l i p i d s in t h e cell n u c l e i a n d in e x t r a c e l l u l a r r e g i o n s of a f i b r o u s p l a q u e of h u m a n a o r t a , a t h e r o s c l e r o s i s s t a g e I I I . T h e c h o l e s t e r o l clefts a r e s u r r o u n d e d b y lipids t h a t s t a i n p o s i t i v e l y w i t h cis-aconitic a n h y d r i d e . X 400. Fig. 4. M e d i a of y o u n g u n d i s e a s e d h u m a n aorta. S m o o t h m u s c l e ceils a n d elastic l a m e U a e p o s i t i v e t o cis-aconitic a n h y d r i d e . S m a l l a m o u n t s of violet c o l o u r e d c o m p l e x e s b e t w e e n elastic l a m e l l a e . × 400. Fig. 5. M e d i a of h u m a n a o r t a , a t h e r o s c l e r o s i s s t a g e I. S t a i n e d w i t h cis-aconitic a n h y d r i d e . A l a r g e a m o u n t of p o s i t i v e l y s t a i n e d p h o s p h o l i p i d s c a n be s e e n in t h e i n t e r l a m e l l a r spaces. T h e e l a s t i c l a m e l l a e are also p o s i t i v e . × 400. Fig. 6. I n t i m a a n d m e d i a of y o u n g u n d i s e a s e d h u m a n a o r t a . C h o l i n e - c o n t a i n i n g p h o s p h o l i p i d s in t h e e n d o t h e l i a l l i n i n g , in t h e i n t e r n a l elastic m e m b r a n e a n d t h e elastic l a m e l l a e of t h e m e d i a , a n d in t h e cell nuclei. T h e i n t i m a c o n t a i n s h a r d l y a n y p o s i t i v e l y s t a i n e d m a t e r i a l . × 400.
J. Atheroscler. Res., 4 (1964) 1 0 9 - 1 1 2
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BOTTCHER, E. BOELSMA-VAN H O U T E
c h r o m i c h y d r o x i d e 4. These p r e c i p i t a t e s also p r e v e n t c o m p l e x f o r m a t i o n . F o r t h e s a m e reason t h e c o b a l t chloride has to be w a s h e d o u t v e r y t h o r o u g h l y before a p p l i c a t i o n of t h e slides to p e r i o d a t e . P o s s i b l y t h e c o b a l t s a l t can be a d d e d to t h e f i x a t i v e as well (compare McMANuS5). T h e n e x t d e h y d r a t i o n of t h e tissue sections h a s to b e d o n e w i t h care, as r e m a i n i n g w a t e r r e a c t s w i t h t h e cis-aconitic a n h y d r i d e t o give c i s - a c o n i t i c acid; e t h a n o l also d e s t r o y s t h e cis-aconitic a n h y d r i d e , so t h a t i t s r e p l a c e m e n t b y t o l u e n e a n d acetic a n h y d r i d e / t o l u e n e h a s to be c o m p l e t e as well. Results
As SASS 2 h a s p o i n t e d out, t h e cis-aconitic a n h y d r i d e r e a c t s w i t h b o t h q u a t e r n a r y a n d t e r t i a r y b o u n d nitrogen. T h e m e t h o d is, h o w e v e r , of p o o r s e n s i t i v i t y for t e r t i a r y amines, so t h a t t h e p o s i t i v e r e a c t i o n of t h e elastic l a m e l l a e of the h u m a n a o r t a is m o s t l i k e l y due to t h e i r c o n t e n t of c h o l i n e - c o n t a i n i n g p h o s p h o l i p i d s a n d n o t to histidine w i t h its t e r t i a r y b o u n d n i t r o g e n , t h a t occurs in s m a l l a m o u n t s in elastin 6. L i p i d s in elastic l a m e l l a e of t h e h u m a n a o r t a h a v e b e e n r e p o r t e d , a m o n g s t o t h e r s b y ADAMS7, ADAMS AND TUQUAN 8 a n d LANSING9. T h e p o s i t i v e r e a c t i o n of t h e elastic l a m e l l a e is in g o o d a c c o r d a n c e w i t h ADAMS' o b s e r v a t i o n 1° t h a t s p h i n g o m y e l i n is t h e c h a r a c t e r istic l i p i d of t h e a r t e r i a l elastica. I t is t h e m o r e p r o b a b l e t h a t choline is t h e p o s i t i v e r e a c t i n g c o m p o n e n t as collagen c o n t a i n s t e n t i m e s m o r e h i s t i d i n e t h a n elastin does 6 a n d collagen shows no p o s i t i v e r e a c t i o n a t all. A n o t h e r a r g u m e n t in f a v o u r of t h e p h o s p h o l i p i d c o n t e n t of t h e elastic l a m e l l a e is t h e decrease in cis-aconitic a n h y d r i d e p o s i t i v i t y of e l a s t i n a f t e r e x t r a c t i o n w i t h m e t h a n o l / c h l o r o f o r m (1 : 2 v/v). T h e s a m e h o l d s for t h e p o s i t i v e r e a c t i o n of cell nuclei: nucleic acids c o n t a i n t e r t i a r y b o u n d n i t r o g e n , b u t t h e p o s i t i v e r e a c t i o n is a b s e n t a f t e r l i p i d ext r a c t i o n so t h a t t h e p o s i t i v e r e a c t i o n m u s t be a t t r i b u t e d t o a n i n c r e a s e d c o n t e n t of choline-containing phospholipids. ACKNOWLEDGEMENT T h e a u t h o r s are g r a t e f u l for t h e v a l u a b l e s u g g e s t i o n s g i v e n b y Drs. C. PRIES. REFERENCES
a C. J. F. BOTTCHER, C. PRIES AND C. M. VAN GENT, Rec. Tray. Chim., 80 (1961) 1169. 2 S. SAss, J. J. KAUFMAN,A. A. CARDENASAND J. J. MARTIN, Anal. Chem., 30 (1958) 529." 3 j . 1~. BAKER, Quart. J. Microscop. Sci., 87 (1946) 441. 4 F. J. ~v[. HESLINGA, De Bichromaatbeitsing en de Kleurbaarheid van Gebeitst Lipoid Onderzocht met Behulp van Reflectometrie, Thesis, Leiden, 1957, p. 29. 5 j. F. A. MCMANUS, J. Pathol. Bacteriol., 58 (1946) 93. 6 A. I. LANSING, in A. I. LANSING (Ed.), The Arterial Wall, Williams and Wilkins Comp., Baltimore, 1959, p. 140. 7 C. W. M. ADAMS, Lancet, i (1959) 1075. 8 C. W. M. ADAMSAND 2q'. A. TUQUAN, J. Pathol. Bacteriol., 82 (1961) 131. 9 A. I. LANSING,T. B. ROSENTHAL, M. ALEX AND E. W. DAMPSEY, Anat. Record, 114 (1952) 555. 10 C. W. M. ADAMSAND O. B. BAYLISS, J. Pathol. Bacteriol., 85 (1963) 113. j . Atheroscler. Res., 4 (1964) 109-112