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Spontaneous coronary lesions in howler monkeys (Alouatta caraya)
Journal of A therosclerosis Research Elsevier Publishing Company, Amsterdam - - Printed in The Netherlands
SPONTANEOUS CORONARY LESIONS IN HOWLER MONKEYS (ALOUATTA CARAYA)*
M. R. MALINOW AND C. A. STORVICK Department of Cardiovascular Physiology, Oregon Regional Primate Research Center, Beaverton; University of Oregon Medical School, Portland; and Department of Foods and Nutrition, School of Home Economics, Oregon State University, Corvallis, Ore. (U.S.A.) (Received September 21st, 1967)
SUMMARY The m a i n c o r o n a r y arteries were s t u d i e d in free-ranging howler m o n k e y s (Alouatta caraya). Values for p l a s m a cholesterol a n d blood p y r i d o x i n e levels were d e t e r m i n e d . The c o r o n a r y arteries showed in n u m e r o u s instances, s e g m e n t a l lesions consisting of s u b i n t i m a l cell proliferation, a c c u m u l a t i o n of acid m u c o p o l y s a c c h a r i d e , a n d d e g e n e r a t i v e changes in t h e i n t e r n a l elastic lamella. The c o r o n a r y plaques were c o m m o n in older m o n k e y s b u t r e l a t i v e l y rare in the y o u n g e r animals. The occurrence of c o r o n a r y lesions bore no relation to sex nor to the presence or absence of p r e g n a n c y . No correlation b e t w e e n the lesions a n d h y p e r c h o l e s t e r o l e m i a or low p y r i d o x i n e b l o o d levels could be established.
INTRODUCTION The occurrence of atherosclerotic lesions in a free-ranging p o p u l a t i o n of howler m o n k e y s was r e p o r t e d previously; i n t i m a l plaques showing fibrosis a n d / o r fat deposits were o b s e r v e d in t h e aorta, the m a i n vessels arising from t h e arch, a n d the iliac arteries 1. The s t u d y of the c o r o n a r y arteries at t h a t t i m e was l i m i t e d to a few animals selected from a m o n g those showing the most p r o m i n e n t aortic lesions. F i n d i n g s i n d i c a t e d the presence of c o r o n a r y lesions. However, v e r y y o u n g animals were n o t i n c l u d e d in the s t u d y so no progression of t h e c o r o n a r y changes w i t h age could be recorded. F u r t h e r m o r e , no b l o o d d e t e r m i n a t i o n s were p e r f o r m e d since the a n i m a l s were s t u d i e d after death. I n order to s u p p l y the d a t a t h a t were n e e d e d to c o m p a r e these findings in Alouatta caraya w i t h those in o t h e r species, we felt t h a t a more system a t i c e x a m i n a t i o n of t h e c o r o n a r y tree was necessary. To o b t a i n b l o o d s a m p l e s for cholesterol a n d v i t a m i n B6 d e t e r m i n a t i o n s , we t o o k a d v a n t a g e of s i m u l t a n e o u s studies t h a t were being p e r f o r m e d on certain a n i m a l s to test liver function 2.
Publication No. 303 of the Oregon Regional Primate Research Center. Aided by Operating Grant FR-OO-163 of the U.S. Public Health Service and the John C. Higgins Foundation (Argentina). J. Atheroscler. Res., 1968, 8:421-431
M. R. MALINOW, C. A. STORVICK
422 TABLE 1 DENTAL
GRADING
IN SEXUALLY
MATURE
HOWL~ER MONKEYS*
Grade
Criteria
I
Upper first molars with well-defined buccal cusps. Attrition has exposed lingual cusp dentin which is dark and discolored. The buccal cusps of teeth other than the maxillary first molars are not worn, with the occasional exception of the premolars Upper first molar cusps not well-defined. Attrition has worn them down to the external cingulum, but has not obliterated the cingulum or the mesostyle (dentin exposed and dark on all molars and premolars; wear on all premolar and molar cusps as well as canines and incisors) Upper first molar worn to or below buccal cingulum (wear on premolars, canines, and incisors very marked so that cusps are obliterated and darkened dentin is apparent buccally as well as lingually) Upper first molar may be worn below buccal cingulum and with only a slight or no lingual rim of enamel left (upper premolars are "pegs" worn to the pulp cavities and incisors are almost without enamel rims, or, may also be "pegs")
II
III IV
* Based on permanent dentition, with upper third molar fully erupted and in occlusion. Reproduced from MALINOWet al. 4. Dr. M. R. Zingeser, University of Oregon Dental School, provided valuable assistance in formulation of criteria. MATERIALS AND METHODS Two series of free-ranging howler m o n k e y s (Alouatta caraya) were studied. The first series consisted of 314 a n i m a l s o b t a i n e d in Bella Vista, Corrientes, A r g e n t i n a , in A u g u s t 1964 (to be called Series 1964). The m e t h o d of collection a n d classification into different groups has been described1, ~-5 a n d will only be s u m m a r i z e d here. The a n i m a l s were shot a n d b r o u g h t to the field l a b o r a t o r y within a few hours after death. Some h u n t e r s spared adult females carrying y o u n g babies, otherwise the sample was n o t biased a n d represents b o t h sexes a n d p r o b a b l y all ages. A b o d y weight of 4.0 kg appeared to correspond to sexual m a t u r a t i o n in b o t h sexes a n d was associated with a definite increase in testicular mass for males a n d the smallest weight for early p r e g n a n c y in females. P r e p u b e r a l animals were divided into two equal intervals below a n d above 2.0 kg. A n i m a l s above 4.0 kg h a d p e r m a n e n t d e n t i t i o n , classified on the basis of wear from criteria listed in T a b l e 1. The first upper molar was found to reflect grinding wear most i n t e n s i v e l y a n d was used to grade the a n i m a l s i n t o four (I-IV) d e n t a l a t t r i t i o n categories. Five "age" groups were thus defined in order of increasing age: p r e p u b e r a l 1 a n d 2 a n d p o s t p u b e r a l 1, 2, a n d 3: the p o s t p u b e r a l correspond to d e n t a l a t t r i t i o n I a n d II, with I I I a n d IV combined, respectively. The second series of a n i m a l s consisted of 52 howler m o n k e y s o b t a i n e d in the same location in J u n e a n d J u l y of 1967 (Series 1967). The m o n k e y s were t r a p p e d alive, given water a n d food, a n d studied w i t h i n 24-72 h. These a n i m a l s were classified as p r e p u b e r a l or postpuberal according to b o d y weight, the presence of p e r m a n e n t d e n t i t i o n , a n d the d e v e l o p m e n t of secondary sexual characteristics. U n d e r S e r n y l a n | * Sernylan| phencyclidine [1-(1 phenylcyclohexyl) piperidine hydrochloride] was generously provided by Parke Davis and Comp., Detroit, Mich. j . Atherosder. Res., 1968, 8:42t-431
SPONTANEOUS CORONARY LESIONS IN HOWLER MONKEYS
423
or pentobarbital anesthesia, blood was withdrawn from the femoral artery; the heart and aorta were excised and fixed in buffered 10 % formaldehyde. The coronary arteries were studied as follows: in 28 of the animals from Series 1964 and in 50 of Series 1967, blocks were obtained from the left and right a - v groove near the aorta, as well as from the anterior and posterior interventricular groove near their upper origin. These blocks included the left and right main coronary artery and the main anterior and posterior descending coronary branches. The blocks were embedded in paraffin and stained with hematoxylin-eosin; additional sections were studied with yon Kossa's technique for calcium 6, Gomori's for ironT, Rhinehart and Abul-Haj's method for acid mucopolysaccharidess and Gomori's method for elastic fibers 9. When indicated, slides were studied with polarized light or by fluorescence technique. Part of the blocks were also stained using Oil-red 0 in frozen sections without paraffin embedding. Five to 10 sections were reviewed per block with each technique. Sixty-five animals from Series 1964 were chosen "blindly" to include in each "age" group 5 males and 5 females. The postpuberal animals included also an equal number of pregnant females. Representative blocks from ttle free wall of the upper portion of the left ventricle or from the upper region of both ventricles, in the case of small animals, were embedded in paraffin and sections were stained with hematoxylineosin, as well as by the Rinehart and Abul-Haj's method for acid mucopolysaccharides and by the Gomori method for elastic fibers. Four sections were reviewed with each method, but only arteries with a diameter larger than 0.25 mm have been included in this part of the study. In 2 monkeys, no such arteries were seen in the available sections, and consequently, the animals were deleted from the present series. Blood was collected with potassium oxalate, protected from light, and frozen immediately on dry ice for later vitamin B6 determination with S. carlsbergensis 1~ Another sample was obtained with KzEDTA (dipotassium ethylene diamine tetraacetate) as anticoagulant, centrifuged immediately, and the plasma frozen for later cholesterol determination 11 on an alcohol-acetone extract. All samples were kept on dry ice and the analyses were carried out in the United States. One determination of vitamin B6 was deleted from this report because a large clot had formed in that sample. Observations were conducted without access to information on the age or sex of the animals. The distribution, according to sex, for these 2 series of monkeys studied and reported here is shown in Table 2. RESULTS
Normal arteries The main coronary arteries had a diameter of 1.0-1.5 mm and a wall thickness of 50-100 #. They showed a thick, continuous wavy internal elastic lamella (IEL) and generally, very thin elastic fibers condensed somewhat loosely into an external elastic lamella (EEL). The endothelial cells lay directly on the I E L and no subintimal material could be detected with light microscopy. The media consisted of 5-10 rows of j . Atheroscler. Res., 1968, 8:421-431
424
M. R. MALINOW, C. A. STORVICK
TABLE 2 NUMBER
OF F R E E - R A N G I N G
1964 (314") 1967 (52)
HOWLER MONKEYS
STUDIED
Main coronary arteries
~/Iyocardial block
male
male
female
female
A
B
A
B
A
B
A
B
6 7
9 15
4 7
9 21
10
15
10
30
* Total population sampled. A, prepuberal; B, postpuberal.
c i r c u l a r l y a r r a n g e d s m o o t h m u s c l e cells, w h i c h was g r a d u a l l y t r a n s f o r m e d i n t o a pred o m i n a n t l y fibrous a d v e n t i t i a . A s m a l l a m o u n t of a c i d m u c o p o l y s a c c h a r i d e (AMP) w a s p r e s e n t a r o u n d t h e i n t e r n a l elastic l a m e l l a a n d also in t h e a d v e n t i t i a w h e r e it was i n t e r s p e r s e d w i t h t h i c k c o l l a g e n o u s b u n d l e s . A s i m i l a r d e s c r i p t i o n applies to t h e s m a l l e r c o r o n a r y b r a n c h e s , b u t no E E L was seen.
Fig. 1. Concentric intimal thickening. Coronary artery of a postpuberal 3 male howler monkey. Arrow shows a disruption of the internal elastic lamella. Gomori's stain for elastic fibers; • 150. j . Athernscler. Res., 1968, 8:421 431
SPONTANEOUS CORONARY LESIONS IN HOWLER MONKEYS
425
Fig. 2. Coronary artery showing subintimal proliferation; disrupted internal elastic lamella marked with arrow. Vacuoles m a y represent fat deposition. N o n p r e g n a n t , postpuberal 1 female howler monkey. H e m a t o x y l i n - e o s i n ; • 400.
Abnormal arteries (Figs. I through 4) Arteries departing from the above description were classified as abnormal; intimal "cushions" near points of bifurcation were not considered. Although in many instances abundant clear vacuoles were present in the media of the coronary arteries, they were not taken into consideration. The significance of this finding is debatable, some authors ascribing it to postmortem changes 12. Duplication of the internal elastic lamella (IE L ). In very few instances, the predominant abnormality was a duplication of the I E L in a segment of the arterial section. A small number of ceils were present between both elastic lamellae and AMP also accumulated in the same region.* "Early" intimal plaques. In a few animals, a small accumulation of interstitial material was observed between the endothelium and the internal elastic lamella. In addition, several "spindle" cells and an increased amount of AMP were found here. The I E L was disrupted and clumps of AMP were also present in the media. "Full-grown" intimal plaques. In most of the postpuberal animals, as well as in 2 older juvenile females, extensive intimal plaques, 10-30 # thick, were observed.
* In this and the forthcoming paragraphs, accumulation of the AMP was estimated s e m i q u a n t i t a tively b y visual comparison with sections of normal arteries.
J. Atheroscler. Res., 1968, 8:421-4131
426
M. R. MALINOW, C. A. STORVICK
Several rows of " s p i n d l e " cells were seen b e t w e e n t h e e n d o t h e l i u m a n d t h e I E L ; no foam cells could be identified with c e r t a i n t y . I n t e r s t i t i a l m a t e r i a l , some of which consisted of AMP, was a b u n d a n t in this a r e a a n d clear vacuoles were seen in a few i n s t a n c e s b e t w e e n cells. I t was also c o m m o n to observe a region t h a t r e a c t e d p o s i t i v e l y to iron in the deeper portions of the i n t i m a l plaques. Below the plaque, t h e I E L was d i s r u p t e d a n d occasionally a b s e n t or d u p l i c a t e d . I n some monkeys, lysis of the elastic fibers led to the f o r m a t i o n of a meshwork, in the interstices of which cellular e l e m e n t s as well as c l u m p s of A M P were seen. I n certain cases, t h e elastic l a m e l l a was s t a i n e d red with R i n e h a r t a n d A b u l - H a j ' s m e t h o d which p r o b a b l y i n d i c a t e d the presence of collagen material. I n some instances, t h e m e d i a showed toss of o r i e n t a t i o n of the muscle cells as well as an increased a m o u n t of A M P a n d of collagen. Large plaques e n c r o a c h e d on the lumen, b u t no i m p o r t a n t o b s t r u c t i o n was recorded in our material. L i p i d m a t e r i a l s t a i n a b l e w i t h Oil-red O was n o t o b s e r v e d in t h e i n t i m a or m e d i a of t h e arteries reviewed.
Elastic basophilia. I n 2 of the Series 1967 p o s t p u b e r a l males which also e x h i b i t e d i n t i m a l plaques, the I E L was i n t e n s e l y basophilic in small segments of the circumference of the m a i n c o r o n a r y a r t e r y . T h e m e m b r a n e was s t r a i g h t e n e d a n d d i d not show d a r k color in slides s t a i n e d only with eosin. T h e changes were o b s e r v e d s e g m e n t ally also, in o t h e r vessels of the s a m e animals, e.g., in e x t e r n a l elastic lamellae of the aorta, a n d of one brachiocephalic t r u n k a n d in small portions of elastic lamellae s i t u a t e d n e a r t h e lumen in the aorta. The basophilic s e g m e n t s were n o t b i r e f r i n g e n t u n d e r p o l a r i z e d light, nor d i d t h e y e x h i b i t fluorescence, b u t d i d stain w i t h von
Fig. 3. Coronary artery with intimal thickening in a postpuberal 3 male howler monkey. Hematoxylin-eosin; • 150.
J. Alhe~'oscler. Res., 1968, 8:421-431
427
SPONTANEOUS CORONARY LESIONS IN HOWLER MONKEYS
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49-8
M.R. MALINOW, C. A. STORVICK
Fig. 4. Coronary plaque in a postpuberal 1 male howler monkey. Arrow: internal elastic lamina. ttematoxylin-eosin; • 450. Kossa's t e c h n i q u e a n d iron-positive areas were present near the modified elastic fibers. Distribution of the s u b i n t i m a l lesions
T h e incidence of coronary a b n o r m a l i t i e s is shown in Table 3. Most of the prep u b e r a l a n i m a l s were normal. D u p l i c a t i o n of I E L or v e r y small s u b i n t i m a l thickenings were observable in only a few of them. B y contrast, slight i n t i n m l thickenings or extensive s u b i n t i m a l plaques were v e r y c o m m o n in the p o s t p u b e r a l animals. I n practically every instance, such changes were observed in the right coronary artery, whereas, in only half the cases could anomalies be detected in the left coronary a r t e r y or in its a n t e r i o r descending branch. The lesions seemed to be somewhat less c o m m o n in the posterior descending b r a n c h . No sex-related differences were observed in the incidence of lesions. T a b l e 4 shows the changes observed in the coronary arteries of the m o n k e y s of TABLE 5 CHOLESTEROL AND PVRIDOXINE LEVELS IN F R E E - R A N G I N G HOWLER MONKEYS (MEAN Z~
Prepuberal Postpuberal Males Females
Plasma cholesterol (rag/I O0 ml)
Blood vitamin B~ (ttg/1 O0 ml) *
101 96 94 100
4.6 7.1 6.4 7.1
~ • -•
7 5 6 6
(6)** (16) (9) (13)
SEM)
(3) =k 0.4 (16) • 0.6 (10) -b 0.6 (9)
* For the determination of blood vitamin B6, we are indebted to Eva M. Benson, Margaret A. Edwards, and Jean M. Peters, Nutrition Research Laboratory, School of Home Economics, Oregon State University. ** Number of animals. j . Atheroscler. Res., 1968, 8:421-431
SPONTANEOUS CORONARYLESIONS IN HOWLER MONKEYS
429
Series 1964, where a more precise breakdown could be made as to relative "age", sex, and the presence or absence of pregnancy. The arteries studied in this series were the larger, generally subepicardial branches found in the myocardial blocks. Most of the prepuberal animals were normal. One female showed elastic duplication, 2 of them showed "early" intimal plaques, while 2 other females had more advanced coronary lesions. "Full-grown" intimal plaques were very common among the adult monkeys, lesions being found in practically all of the "older" animals. Testing for the significance of these differences was performed with the Z 2 method. The animals were considered positive when any of the lesions described under Abnormal arteries were found. Although pre- and postpuberal animals differed significantly, no differences attributable to sex or to the presence or absence of pregnancy could be detected.
Blood findings (see Table 5) Plasma cholesterol was around 100 rag/100 ml. No differences attributable to puberty or to sex were present. The levels of vitamin Be were between 6 and 7 #g/100 ml and were similar in all groups. The lower levels reported in the only 3 prepuberal animals are due to the inclusion of a single value of 1.74/zg/100 ml, the lowest one observed in the whole series. DISCUSSION The occurrence of "spontaneous" coronary lesions has been established in a number of nonhuman primates including Cebus, Saimiri, Callithrix, Papio, Macaca, Cercoeebus, Hylobates, Pan, Gorilla, and Pongo (see bibliography in ref. 13). The lesions described here are similar to those found in the coronary arteries of other monkeys (cf., for instance ref. 14) and conform in general to the "preatheromatous phase of atherosclerosis" as defined b y SCOTT et al. 15. However, based on strictly morphologic grounds, it cannot be stated whether the same process is at work in humans and in howlers. Lipid deposits, previously observed in the coronary arteries of two howler monkeys 1, could not be detected in the present series. In our previous report 1, we studied the coronary arteries of only a small number of adult monkeys selected from among those with the most extensive aortic involvement. Obviously the span of age for that study was limited and no progression of the coronary lesions with body maturation was observed. In the present study the inclusion of younger animals made it possible to show that the incidence of intimal plaques increases with "age". However, it must be realized that age estimation can only be approximate in free-ranging monkeys, hence only a rough comparison can be obtained by using different parameters1, 3-5. The curious "mineralization" of the internal elastic lamella observed in the coronary arteries of 2 monkeys was probably not related to intimal fibrosis since it was also found in unrelated places, such as in the external elastic lamellae of the innominate trunk. I t probably represents some generalized disturbance, the nature of which cannot be decided now.
j. $theroscler. Res., 1968, 8:421-431
430
M . R . MALINOW, C. A. STORVICK
In contrast to our findings in the aorta 1, the occurrence of the fibrotic coronary plaques bears no relation to sex. Similar observations have been reported in freeranging baboons 16 as well as in Aves, where the incidence of coronary atherosclerosis has been the same in roosters and hens 17. Furthermore, and again in contrast to the results obtained in the aorta, gestation does not modify the incidence of the presently described coronary lesions as already observed b y McGILL et al. in baboons 16. Apparently a similar situation m a y exist among humans as seen in a detailed study performed b y SCOTTet al. on the coronary arteries of American and African individuals where no sex-related differences were found in the extent of preatheromatous lesions 15. The findings in howler monkeys which are exclusively vegetarian animals is with low plasma cholesterol levels, seem then to agree with the hypothesis that coronary atherogenesis comprises at least two relatively independent processes: one consists mainly of intimal fibrosis, the other shows mainly lipid infiltration. The evidence reported here as well as elsewhere 17 indicates that primary intimal fibrosis is not related to a high intake of cholesterol, that it m a y occur in the presence of low blood cholesterol levels, and that it bears no relation to sex. These lesions appear to be different from atheromatosis which seems to be influenced b y several factors including high cholesterol intake and female sex hormones (see bibliography in ref. 19). The etiology of the coronary lesions in howler monkeys is not known. The morphology of the intimal plaques is somewhat similar to that described in pyridoxinedeficient M a c a c a mulatta 2~ In rhesus monkeys, however, such an avitaminosis also induces dental caries and f a t t y liver infiltrations leading to cirrhosis 21. No dental caries were observed in this population of Alouatta caraya 22 and the hepatic changes 23 appear to be different from those mentioned by RINEHART AND GREENBERG21. The free-ranging monkeys reported here showed an apparently adequate state of nutrition as judged b y the abundant subcutaneous and retroperitoneal fat tissue even when studied in winter time when, presumably, the food intake m a y be at its lowest 3. Furthermore, the blood values of vitamin B6 were within the levels considered normal in the human 24. The occurrence of pyridoxine deficiency seems unlikely, and other factors are probably responsible for the arterial lesions observed. ACKNOWLEDGEMENTS
We gratefully acknowledge the criticism of Dr. Sefton R. Wellings, Professor and Chairman, Department of Pathology, University of Oregon Medical School, Portland, Ore., and the helpful suggestions of Dr. Wilbur P. McNulty, Chairman, Department of Pathology, Oregon Regional Primate Research Center, Beaverton, Ore. REFERENCES 1 MALINOW, M. R. AND C. A. MARUFFO, N a t u r a l l y - o c c u r r i n g atherosclerosis in h o w l e r m o n k e y s (Alouatta caraya), J. Atheroscler. Res., 1966, 6: 368. KATZ, S., C. E. CORNELIUS AND M. R. MALINOW, u n p u b l i s h e d o b s e r v a t i o n s . 3 MALINOW, M. R., I n t r o d u c t i o n . In: M. R. MALINOW (Ed.), Biology of the Howler Monhey (Alou-
J. Atheroscler. Res., 1968, 8 : 4 2 1 - 4 3 1
SPONTANEOUS CORONARY LESIONS IN HOWLER MONKEYS
431
atta caraya), K a r g e r , Basel, 1967. 4 MALINOW, M. R., W. R. STAHL, C. A. MARUFFO, BETTY L. POPE AND 1~. DEPAOLI, G r o w t h in t h e howler m o n k e y (Alouatta caraya), Primates, 1966, 7: 433. 5 MALINOW, M. R. AND ANN CORCORAN, G r o w t h of t h e lens in howler m o n k e y s (Alouatta carava), J. Mammal., 1966, 47: 58. 6 VON KOSSA, J., l~ber die im O r g a n i s m u s kflnstlich e r z e u g b a r e n V e r k a l k u n g e n , Beitr. path. Anat., 1901, 29: 163. 7 Manual of Histologic and Special Technics, A r m e d Forces I n s t i t u t e of P a t h o l o g y , W a s h i n g t o n , D.C., 1957, p. 149. 8 RINEHART, J. F. AND S. K. ABuL-HAJ, An i m p r o v e d m e t h o d for histological d e m o n s t r a t i o n of acid m u c o p o l y s a c e h a r i d e s in tissues, A . M . A . Arch. Path., 1951, 52: 189. 9 GOMORI, G., A l d e h y d e fuchsin: a n e w stain for elastic tissues, Amer. J. clin. Path., 1950, 20: 655. 10 STORVICK, C. A., E. i . BENSON, M. A. EDWARDS AND M. J. WOODRING, C h e m i c a l a n d microbiological d e t e r m i n a t i o n of v i t a m i n BG, Meth. biochem. Anal., 1964, 12: 183. 11 ZLATKIS, A., B. ZAK AND A. J. BOYLE, New m e t h o d for direct d e t e r m i n a t i o n of s e r u m cholesterol, J. Lab. clin. Med., 1953, 41: 486. 12 LINDSAY, S. AND I. L. CHAIKOFF, C o r o n a r y arterial sclerosis of birds; c o m p a r i s o n of s p o n t a n e o u s a n d arterial lesions, Arch. Path., 1950, 49: 434. 13 MALINOW, M. R., Atherosclerosis in s u b h u m a n p r i m a t e s , Folia primat., 1965, 3: 277. 14 GILBERT, C. AND J. GILLMAN, S t r u c t u r a l modifications in t h e c o r o n a r y a r t e r y of t h e b a b o o n (Papio ursinus) w i t h special reference to a g e a n d endocrine s t a t u s , S. Aft. J. reed. Sci., 1960, 25 : 59. 15 SCOTT, R. F., R. A. FLORENTIN, A. S. DAOUD, E. S. MORRISON, R. M. JONES AND M. S. R. HUTT, C o r o n a r y arteries of children a n d y o u n g adults. A c o m p a r i s o n of lipids a n d a n a t o m i c f e a t u r e s in New Y o r k e r s a n d E a s t Africans, Exp. Mol. Path., 1966, 5: 12. l0 McGILL, H. C., JR., J. P. STRONG, R. L. HOLMAN AND N. T. WERTHESSEN, Arterial lesions in t h e K e n y a b a b o o n , Circulat. Res., 1960, 8: 670. 17 MAL1NOW, M. R., B. LEMA AND S. J. KAPLAN, S p o n t a n e o u s c o r o n a r y atherosclerosis in birds, J. Atheroscler. Res., 1963, 3: 321. 18 HILL, W. C. O., Primates, Comparative Anatomy and Taxonomy, Vol. 5 (Cebidae), Part B, U n i v e r s i t y Press, E d i n b u r g h , 1962. 19 MALINOW, M. R., H o r m o n e s a n d atherosclerosis, Advanc. Pharmacol., 1963, 2: 211. 2o RINEHART, J. F., AND L. D. GREENBERG, Arteriosclerotic lesions in p y r i d o x i n e - d e f i c i e n t mon-keys, Amer. J. Path., 1949, 25: 481. 21 RINEHART, J. V. AND L. D. GREENBERG, V i t a m i n BG deficiency in t h e r h e s u s m o n k e y Amer. J. clin. Nutr., 1956, 4: 318. 22 HALL, W. B., H. E. GRUPE AND C. K. CLAYCOMB, T h e p e r i d o n t i u m a n d p e r i o d o n t a l p a t h o l o g y , In: M. R. MALINOW (Ed.), Biology of the Howler Monkey (Alouatta caraya), Karger, Basel, 1967. 23 MARUFFO, C. A., M. R. MALINOW, S. KATZ AND J. R. DEPAOLI, P i g m e n t a r y liver disease in howler m o n k e y s , Amer. ar. Path., 1966, 49: 445. 24 KARLIN, R., O n t h e V i t a m i n B6 c o n t e n t of h u m a n whole blood. V a r i a t i o n s of t h e B6 level in m a t e r n a l a n d fetal blood d u r i n g labor, C. R. Soe. Biol. (Paris), 1962, 156: 858.
J. Atheroscler. Res., 1968, 8 : 4 2 1 - 4 3 1
SPONTANEOUS CORONARY LESIONS IN HOWLER MONKEYS (ALOUATTA CARAYA)*
M. R. MALINOW AND C. A. STORVICK Department of Cardiovascular Physiology, Oregon Regional Primate Research Center, Beaverton; University of Oregon Medical School, Portland; and Department of Foods and Nutrition, School of Home Economics, Oregon State University, Corvallis, Ore. (U.S.A.) (Received September 21st, 1967)
SUMMARY The m a i n c o r o n a r y arteries were s t u d i e d in free-ranging howler m o n k e y s (Alouatta caraya). Values for p l a s m a cholesterol a n d blood p y r i d o x i n e levels were d e t e r m i n e d . The c o r o n a r y arteries showed in n u m e r o u s instances, s e g m e n t a l lesions consisting of s u b i n t i m a l cell proliferation, a c c u m u l a t i o n of acid m u c o p o l y s a c c h a r i d e , a n d d e g e n e r a t i v e changes in t h e i n t e r n a l elastic lamella. The c o r o n a r y plaques were c o m m o n in older m o n k e y s b u t r e l a t i v e l y rare in the y o u n g e r animals. The occurrence of c o r o n a r y lesions bore no relation to sex nor to the presence or absence of p r e g n a n c y . No correlation b e t w e e n the lesions a n d h y p e r c h o l e s t e r o l e m i a or low p y r i d o x i n e b l o o d levels could be established.
INTRODUCTION The occurrence of atherosclerotic lesions in a free-ranging p o p u l a t i o n of howler m o n k e y s was r e p o r t e d previously; i n t i m a l plaques showing fibrosis a n d / o r fat deposits were o b s e r v e d in t h e aorta, the m a i n vessels arising from t h e arch, a n d the iliac arteries 1. The s t u d y of the c o r o n a r y arteries at t h a t t i m e was l i m i t e d to a few animals selected from a m o n g those showing the most p r o m i n e n t aortic lesions. F i n d i n g s i n d i c a t e d the presence of c o r o n a r y lesions. However, v e r y y o u n g animals were n o t i n c l u d e d in the s t u d y so no progression of t h e c o r o n a r y changes w i t h age could be recorded. F u r t h e r m o r e , no b l o o d d e t e r m i n a t i o n s were p e r f o r m e d since the a n i m a l s were s t u d i e d after death. I n order to s u p p l y the d a t a t h a t were n e e d e d to c o m p a r e these findings in Alouatta caraya w i t h those in o t h e r species, we felt t h a t a more system a t i c e x a m i n a t i o n of t h e c o r o n a r y tree was necessary. To o b t a i n b l o o d s a m p l e s for cholesterol a n d v i t a m i n B6 d e t e r m i n a t i o n s , we t o o k a d v a n t a g e of s i m u l t a n e o u s studies t h a t were being p e r f o r m e d on certain a n i m a l s to test liver function 2.
Publication No. 303 of the Oregon Regional Primate Research Center. Aided by Operating Grant FR-OO-163 of the U.S. Public Health Service and the John C. Higgins Foundation (Argentina). J. Atheroscler. Res., 1968, 8:421-431
M. R. MALINOW, C. A. STORVICK
422 TABLE 1 DENTAL
GRADING
IN SEXUALLY
MATURE
HOWL~ER MONKEYS*
Grade
Criteria
I
Upper first molars with well-defined buccal cusps. Attrition has exposed lingual cusp dentin which is dark and discolored. The buccal cusps of teeth other than the maxillary first molars are not worn, with the occasional exception of the premolars Upper first molar cusps not well-defined. Attrition has worn them down to the external cingulum, but has not obliterated the cingulum or the mesostyle (dentin exposed and dark on all molars and premolars; wear on all premolar and molar cusps as well as canines and incisors) Upper first molar worn to or below buccal cingulum (wear on premolars, canines, and incisors very marked so that cusps are obliterated and darkened dentin is apparent buccally as well as lingually) Upper first molar may be worn below buccal cingulum and with only a slight or no lingual rim of enamel left (upper premolars are "pegs" worn to the pulp cavities and incisors are almost without enamel rims, or, may also be "pegs")
II
III IV
* Based on permanent dentition, with upper third molar fully erupted and in occlusion. Reproduced from MALINOWet al. 4. Dr. M. R. Zingeser, University of Oregon Dental School, provided valuable assistance in formulation of criteria. MATERIALS AND METHODS Two series of free-ranging howler m o n k e y s (Alouatta caraya) were studied. The first series consisted of 314 a n i m a l s o b t a i n e d in Bella Vista, Corrientes, A r g e n t i n a , in A u g u s t 1964 (to be called Series 1964). The m e t h o d of collection a n d classification into different groups has been described1, ~-5 a n d will only be s u m m a r i z e d here. The a n i m a l s were shot a n d b r o u g h t to the field l a b o r a t o r y within a few hours after death. Some h u n t e r s spared adult females carrying y o u n g babies, otherwise the sample was n o t biased a n d represents b o t h sexes a n d p r o b a b l y all ages. A b o d y weight of 4.0 kg appeared to correspond to sexual m a t u r a t i o n in b o t h sexes a n d was associated with a definite increase in testicular mass for males a n d the smallest weight for early p r e g n a n c y in females. P r e p u b e r a l animals were divided into two equal intervals below a n d above 2.0 kg. A n i m a l s above 4.0 kg h a d p e r m a n e n t d e n t i t i o n , classified on the basis of wear from criteria listed in T a b l e 1. The first upper molar was found to reflect grinding wear most i n t e n s i v e l y a n d was used to grade the a n i m a l s i n t o four (I-IV) d e n t a l a t t r i t i o n categories. Five "age" groups were thus defined in order of increasing age: p r e p u b e r a l 1 a n d 2 a n d p o s t p u b e r a l 1, 2, a n d 3: the p o s t p u b e r a l correspond to d e n t a l a t t r i t i o n I a n d II, with I I I a n d IV combined, respectively. The second series of a n i m a l s consisted of 52 howler m o n k e y s o b t a i n e d in the same location in J u n e a n d J u l y of 1967 (Series 1967). The m o n k e y s were t r a p p e d alive, given water a n d food, a n d studied w i t h i n 24-72 h. These a n i m a l s were classified as p r e p u b e r a l or postpuberal according to b o d y weight, the presence of p e r m a n e n t d e n t i t i o n , a n d the d e v e l o p m e n t of secondary sexual characteristics. U n d e r S e r n y l a n | * Sernylan| phencyclidine [1-(1 phenylcyclohexyl) piperidine hydrochloride] was generously provided by Parke Davis and Comp., Detroit, Mich. j . Atherosder. Res., 1968, 8:42t-431
SPONTANEOUS CORONARY LESIONS IN HOWLER MONKEYS
423
or pentobarbital anesthesia, blood was withdrawn from the femoral artery; the heart and aorta were excised and fixed in buffered 10 % formaldehyde. The coronary arteries were studied as follows: in 28 of the animals from Series 1964 and in 50 of Series 1967, blocks were obtained from the left and right a - v groove near the aorta, as well as from the anterior and posterior interventricular groove near their upper origin. These blocks included the left and right main coronary artery and the main anterior and posterior descending coronary branches. The blocks were embedded in paraffin and stained with hematoxylin-eosin; additional sections were studied with yon Kossa's technique for calcium 6, Gomori's for ironT, Rhinehart and Abul-Haj's method for acid mucopolysaccharidess and Gomori's method for elastic fibers 9. When indicated, slides were studied with polarized light or by fluorescence technique. Part of the blocks were also stained using Oil-red 0 in frozen sections without paraffin embedding. Five to 10 sections were reviewed per block with each technique. Sixty-five animals from Series 1964 were chosen "blindly" to include in each "age" group 5 males and 5 females. The postpuberal animals included also an equal number of pregnant females. Representative blocks from ttle free wall of the upper portion of the left ventricle or from the upper region of both ventricles, in the case of small animals, were embedded in paraffin and sections were stained with hematoxylineosin, as well as by the Rinehart and Abul-Haj's method for acid mucopolysaccharides and by the Gomori method for elastic fibers. Four sections were reviewed with each method, but only arteries with a diameter larger than 0.25 mm have been included in this part of the study. In 2 monkeys, no such arteries were seen in the available sections, and consequently, the animals were deleted from the present series. Blood was collected with potassium oxalate, protected from light, and frozen immediately on dry ice for later vitamin B6 determination with S. carlsbergensis 1~ Another sample was obtained with KzEDTA (dipotassium ethylene diamine tetraacetate) as anticoagulant, centrifuged immediately, and the plasma frozen for later cholesterol determination 11 on an alcohol-acetone extract. All samples were kept on dry ice and the analyses were carried out in the United States. One determination of vitamin B6 was deleted from this report because a large clot had formed in that sample. Observations were conducted without access to information on the age or sex of the animals. The distribution, according to sex, for these 2 series of monkeys studied and reported here is shown in Table 2. RESULTS
Normal arteries The main coronary arteries had a diameter of 1.0-1.5 mm and a wall thickness of 50-100 #. They showed a thick, continuous wavy internal elastic lamella (IEL) and generally, very thin elastic fibers condensed somewhat loosely into an external elastic lamella (EEL). The endothelial cells lay directly on the I E L and no subintimal material could be detected with light microscopy. The media consisted of 5-10 rows of j . Atheroscler. Res., 1968, 8:421-431
424
M. R. MALINOW, C. A. STORVICK
TABLE 2 NUMBER
OF F R E E - R A N G I N G
1964 (314") 1967 (52)
HOWLER MONKEYS
STUDIED
Main coronary arteries
~/Iyocardial block
male
male
female
female
A
B
A
B
A
B
A
B
6 7
9 15
4 7
9 21
10
15
10
30
* Total population sampled. A, prepuberal; B, postpuberal.
c i r c u l a r l y a r r a n g e d s m o o t h m u s c l e cells, w h i c h was g r a d u a l l y t r a n s f o r m e d i n t o a pred o m i n a n t l y fibrous a d v e n t i t i a . A s m a l l a m o u n t of a c i d m u c o p o l y s a c c h a r i d e (AMP) w a s p r e s e n t a r o u n d t h e i n t e r n a l elastic l a m e l l a a n d also in t h e a d v e n t i t i a w h e r e it was i n t e r s p e r s e d w i t h t h i c k c o l l a g e n o u s b u n d l e s . A s i m i l a r d e s c r i p t i o n applies to t h e s m a l l e r c o r o n a r y b r a n c h e s , b u t no E E L was seen.
Fig. 1. Concentric intimal thickening. Coronary artery of a postpuberal 3 male howler monkey. Arrow shows a disruption of the internal elastic lamella. Gomori's stain for elastic fibers; • 150. j . Athernscler. Res., 1968, 8:421 431
SPONTANEOUS CORONARY LESIONS IN HOWLER MONKEYS
425
Fig. 2. Coronary artery showing subintimal proliferation; disrupted internal elastic lamella marked with arrow. Vacuoles m a y represent fat deposition. N o n p r e g n a n t , postpuberal 1 female howler monkey. H e m a t o x y l i n - e o s i n ; • 400.
Abnormal arteries (Figs. I through 4) Arteries departing from the above description were classified as abnormal; intimal "cushions" near points of bifurcation were not considered. Although in many instances abundant clear vacuoles were present in the media of the coronary arteries, they were not taken into consideration. The significance of this finding is debatable, some authors ascribing it to postmortem changes 12. Duplication of the internal elastic lamella (IE L ). In very few instances, the predominant abnormality was a duplication of the I E L in a segment of the arterial section. A small number of ceils were present between both elastic lamellae and AMP also accumulated in the same region.* "Early" intimal plaques. In a few animals, a small accumulation of interstitial material was observed between the endothelium and the internal elastic lamella. In addition, several "spindle" cells and an increased amount of AMP were found here. The I E L was disrupted and clumps of AMP were also present in the media. "Full-grown" intimal plaques. In most of the postpuberal animals, as well as in 2 older juvenile females, extensive intimal plaques, 10-30 # thick, were observed.
* In this and the forthcoming paragraphs, accumulation of the AMP was estimated s e m i q u a n t i t a tively b y visual comparison with sections of normal arteries.
J. Atheroscler. Res., 1968, 8:421-4131
426
M. R. MALINOW, C. A. STORVICK
Several rows of " s p i n d l e " cells were seen b e t w e e n t h e e n d o t h e l i u m a n d t h e I E L ; no foam cells could be identified with c e r t a i n t y . I n t e r s t i t i a l m a t e r i a l , some of which consisted of AMP, was a b u n d a n t in this a r e a a n d clear vacuoles were seen in a few i n s t a n c e s b e t w e e n cells. I t was also c o m m o n to observe a region t h a t r e a c t e d p o s i t i v e l y to iron in the deeper portions of the i n t i m a l plaques. Below the plaque, t h e I E L was d i s r u p t e d a n d occasionally a b s e n t or d u p l i c a t e d . I n some monkeys, lysis of the elastic fibers led to the f o r m a t i o n of a meshwork, in the interstices of which cellular e l e m e n t s as well as c l u m p s of A M P were seen. I n certain cases, t h e elastic l a m e l l a was s t a i n e d red with R i n e h a r t a n d A b u l - H a j ' s m e t h o d which p r o b a b l y i n d i c a t e d the presence of collagen material. I n some instances, t h e m e d i a showed toss of o r i e n t a t i o n of the muscle cells as well as an increased a m o u n t of A M P a n d of collagen. Large plaques e n c r o a c h e d on the lumen, b u t no i m p o r t a n t o b s t r u c t i o n was recorded in our material. L i p i d m a t e r i a l s t a i n a b l e w i t h Oil-red O was n o t o b s e r v e d in t h e i n t i m a or m e d i a of t h e arteries reviewed.
Elastic basophilia. I n 2 of the Series 1967 p o s t p u b e r a l males which also e x h i b i t e d i n t i m a l plaques, the I E L was i n t e n s e l y basophilic in small segments of the circumference of the m a i n c o r o n a r y a r t e r y . T h e m e m b r a n e was s t r a i g h t e n e d a n d d i d not show d a r k color in slides s t a i n e d only with eosin. T h e changes were o b s e r v e d s e g m e n t ally also, in o t h e r vessels of the s a m e animals, e.g., in e x t e r n a l elastic lamellae of the aorta, a n d of one brachiocephalic t r u n k a n d in small portions of elastic lamellae s i t u a t e d n e a r t h e lumen in the aorta. The basophilic s e g m e n t s were n o t b i r e f r i n g e n t u n d e r p o l a r i z e d light, nor d i d t h e y e x h i b i t fluorescence, b u t d i d stain w i t h von
Fig. 3. Coronary artery with intimal thickening in a postpuberal 3 male howler monkey. Hematoxylin-eosin; • 150.
J. Alhe~'oscler. Res., 1968, 8:421-431
427
SPONTANEOUS CORONARY LESIONS IN HOWLER MONKEYS
-5
o
<
tr Z 0
b 0 O 0 0)
N
Z O m O
z M Z o
N
,-I
O
o Z N
Z
e O Z
v
Z
O 2; <
b~ Z
<
m
m O Z m
Z O
O
O
-~;
O
z
m
~ o ~
a ~ 0 0 0
.:%
j . Atheroscler. Res., 1968, 8 : 4 2 1 - 4 3 1
49-8
M.R. MALINOW, C. A. STORVICK
Fig. 4. Coronary plaque in a postpuberal 1 male howler monkey. Arrow: internal elastic lamina. ttematoxylin-eosin; • 450. Kossa's t e c h n i q u e a n d iron-positive areas were present near the modified elastic fibers. Distribution of the s u b i n t i m a l lesions
T h e incidence of coronary a b n o r m a l i t i e s is shown in Table 3. Most of the prep u b e r a l a n i m a l s were normal. D u p l i c a t i o n of I E L or v e r y small s u b i n t i m a l thickenings were observable in only a few of them. B y contrast, slight i n t i n m l thickenings or extensive s u b i n t i m a l plaques were v e r y c o m m o n in the p o s t p u b e r a l animals. I n practically every instance, such changes were observed in the right coronary artery, whereas, in only half the cases could anomalies be detected in the left coronary a r t e r y or in its a n t e r i o r descending branch. The lesions seemed to be somewhat less c o m m o n in the posterior descending b r a n c h . No sex-related differences were observed in the incidence of lesions. T a b l e 4 shows the changes observed in the coronary arteries of the m o n k e y s of TABLE 5 CHOLESTEROL AND PVRIDOXINE LEVELS IN F R E E - R A N G I N G HOWLER MONKEYS (MEAN Z~
Prepuberal Postpuberal Males Females
Plasma cholesterol (rag/I O0 ml)
Blood vitamin B~ (ttg/1 O0 ml) *
101 96 94 100
4.6 7.1 6.4 7.1
~ • -•
7 5 6 6
(6)** (16) (9) (13)
SEM)
(3) =k 0.4 (16) • 0.6 (10) -b 0.6 (9)
* For the determination of blood vitamin B6, we are indebted to Eva M. Benson, Margaret A. Edwards, and Jean M. Peters, Nutrition Research Laboratory, School of Home Economics, Oregon State University. ** Number of animals. j . Atheroscler. Res., 1968, 8:421-431
SPONTANEOUS CORONARYLESIONS IN HOWLER MONKEYS
429
Series 1964, where a more precise breakdown could be made as to relative "age", sex, and the presence or absence of pregnancy. The arteries studied in this series were the larger, generally subepicardial branches found in the myocardial blocks. Most of the prepuberal animals were normal. One female showed elastic duplication, 2 of them showed "early" intimal plaques, while 2 other females had more advanced coronary lesions. "Full-grown" intimal plaques were very common among the adult monkeys, lesions being found in practically all of the "older" animals. Testing for the significance of these differences was performed with the Z 2 method. The animals were considered positive when any of the lesions described under Abnormal arteries were found. Although pre- and postpuberal animals differed significantly, no differences attributable to sex or to the presence or absence of pregnancy could be detected.
Blood findings (see Table 5) Plasma cholesterol was around 100 rag/100 ml. No differences attributable to puberty or to sex were present. The levels of vitamin Be were between 6 and 7 #g/100 ml and were similar in all groups. The lower levels reported in the only 3 prepuberal animals are due to the inclusion of a single value of 1.74/zg/100 ml, the lowest one observed in the whole series. DISCUSSION The occurrence of "spontaneous" coronary lesions has been established in a number of nonhuman primates including Cebus, Saimiri, Callithrix, Papio, Macaca, Cercoeebus, Hylobates, Pan, Gorilla, and Pongo (see bibliography in ref. 13). The lesions described here are similar to those found in the coronary arteries of other monkeys (cf., for instance ref. 14) and conform in general to the "preatheromatous phase of atherosclerosis" as defined b y SCOTT et al. 15. However, based on strictly morphologic grounds, it cannot be stated whether the same process is at work in humans and in howlers. Lipid deposits, previously observed in the coronary arteries of two howler monkeys 1, could not be detected in the present series. In our previous report 1, we studied the coronary arteries of only a small number of adult monkeys selected from among those with the most extensive aortic involvement. Obviously the span of age for that study was limited and no progression of the coronary lesions with body maturation was observed. In the present study the inclusion of younger animals made it possible to show that the incidence of intimal plaques increases with "age". However, it must be realized that age estimation can only be approximate in free-ranging monkeys, hence only a rough comparison can be obtained by using different parameters1, 3-5. The curious "mineralization" of the internal elastic lamella observed in the coronary arteries of 2 monkeys was probably not related to intimal fibrosis since it was also found in unrelated places, such as in the external elastic lamellae of the innominate trunk. I t probably represents some generalized disturbance, the nature of which cannot be decided now.
j. $theroscler. Res., 1968, 8:421-431
430
M . R . MALINOW, C. A. STORVICK
In contrast to our findings in the aorta 1, the occurrence of the fibrotic coronary plaques bears no relation to sex. Similar observations have been reported in freeranging baboons 16 as well as in Aves, where the incidence of coronary atherosclerosis has been the same in roosters and hens 17. Furthermore, and again in contrast to the results obtained in the aorta, gestation does not modify the incidence of the presently described coronary lesions as already observed b y McGILL et al. in baboons 16. Apparently a similar situation m a y exist among humans as seen in a detailed study performed b y SCOTTet al. on the coronary arteries of American and African individuals where no sex-related differences were found in the extent of preatheromatous lesions 15. The findings in howler monkeys which are exclusively vegetarian animals is with low plasma cholesterol levels, seem then to agree with the hypothesis that coronary atherogenesis comprises at least two relatively independent processes: one consists mainly of intimal fibrosis, the other shows mainly lipid infiltration. The evidence reported here as well as elsewhere 17 indicates that primary intimal fibrosis is not related to a high intake of cholesterol, that it m a y occur in the presence of low blood cholesterol levels, and that it bears no relation to sex. These lesions appear to be different from atheromatosis which seems to be influenced b y several factors including high cholesterol intake and female sex hormones (see bibliography in ref. 19). The etiology of the coronary lesions in howler monkeys is not known. The morphology of the intimal plaques is somewhat similar to that described in pyridoxinedeficient M a c a c a mulatta 2~ In rhesus monkeys, however, such an avitaminosis also induces dental caries and f a t t y liver infiltrations leading to cirrhosis 21. No dental caries were observed in this population of Alouatta caraya 22 and the hepatic changes 23 appear to be different from those mentioned by RINEHART AND GREENBERG21. The free-ranging monkeys reported here showed an apparently adequate state of nutrition as judged b y the abundant subcutaneous and retroperitoneal fat tissue even when studied in winter time when, presumably, the food intake m a y be at its lowest 3. Furthermore, the blood values of vitamin B6 were within the levels considered normal in the human 24. The occurrence of pyridoxine deficiency seems unlikely, and other factors are probably responsible for the arterial lesions observed. ACKNOWLEDGEMENTS
We gratefully acknowledge the criticism of Dr. Sefton R. Wellings, Professor and Chairman, Department of Pathology, University of Oregon Medical School, Portland, Ore., and the helpful suggestions of Dr. Wilbur P. McNulty, Chairman, Department of Pathology, Oregon Regional Primate Research Center, Beaverton, Ore. REFERENCES 1 MALINOW, M. R. AND C. A. MARUFFO, N a t u r a l l y - o c c u r r i n g atherosclerosis in h o w l e r m o n k e y s (Alouatta caraya), J. Atheroscler. Res., 1966, 6: 368. KATZ, S., C. E. CORNELIUS AND M. R. MALINOW, u n p u b l i s h e d o b s e r v a t i o n s . 3 MALINOW, M. R., I n t r o d u c t i o n . In: M. R. MALINOW (Ed.), Biology of the Howler Monhey (Alou-
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atta caraya), K a r g e r , Basel, 1967. 4 MALINOW, M. R., W. R. STAHL, C. A. MARUFFO, BETTY L. POPE AND 1~. DEPAOLI, G r o w t h in t h e howler m o n k e y (Alouatta caraya), Primates, 1966, 7: 433. 5 MALINOW, M. R. AND ANN CORCORAN, G r o w t h of t h e lens in howler m o n k e y s (Alouatta carava), J. Mammal., 1966, 47: 58. 6 VON KOSSA, J., l~ber die im O r g a n i s m u s kflnstlich e r z e u g b a r e n V e r k a l k u n g e n , Beitr. path. Anat., 1901, 29: 163. 7 Manual of Histologic and Special Technics, A r m e d Forces I n s t i t u t e of P a t h o l o g y , W a s h i n g t o n , D.C., 1957, p. 149. 8 RINEHART, J. F. AND S. K. ABuL-HAJ, An i m p r o v e d m e t h o d for histological d e m o n s t r a t i o n of acid m u c o p o l y s a c e h a r i d e s in tissues, A . M . A . Arch. Path., 1951, 52: 189. 9 GOMORI, G., A l d e h y d e fuchsin: a n e w stain for elastic tissues, Amer. J. clin. Path., 1950, 20: 655. 10 STORVICK, C. A., E. i . BENSON, M. A. EDWARDS AND M. J. WOODRING, C h e m i c a l a n d microbiological d e t e r m i n a t i o n of v i t a m i n BG, Meth. biochem. Anal., 1964, 12: 183. 11 ZLATKIS, A., B. ZAK AND A. J. BOYLE, New m e t h o d for direct d e t e r m i n a t i o n of s e r u m cholesterol, J. Lab. clin. Med., 1953, 41: 486. 12 LINDSAY, S. AND I. L. CHAIKOFF, C o r o n a r y arterial sclerosis of birds; c o m p a r i s o n of s p o n t a n e o u s a n d arterial lesions, Arch. Path., 1950, 49: 434. 13 MALINOW, M. R., Atherosclerosis in s u b h u m a n p r i m a t e s , Folia primat., 1965, 3: 277. 14 GILBERT, C. AND J. GILLMAN, S t r u c t u r a l modifications in t h e c o r o n a r y a r t e r y of t h e b a b o o n (Papio ursinus) w i t h special reference to a g e a n d endocrine s t a t u s , S. Aft. J. reed. Sci., 1960, 25 : 59. 15 SCOTT, R. F., R. A. FLORENTIN, A. S. DAOUD, E. S. MORRISON, R. M. JONES AND M. S. R. HUTT, C o r o n a r y arteries of children a n d y o u n g adults. A c o m p a r i s o n of lipids a n d a n a t o m i c f e a t u r e s in New Y o r k e r s a n d E a s t Africans, Exp. Mol. Path., 1966, 5: 12. l0 McGILL, H. C., JR., J. P. STRONG, R. L. HOLMAN AND N. T. WERTHESSEN, Arterial lesions in t h e K e n y a b a b o o n , Circulat. Res., 1960, 8: 670. 17 MAL1NOW, M. R., B. LEMA AND S. J. KAPLAN, S p o n t a n e o u s c o r o n a r y atherosclerosis in birds, J. Atheroscler. Res., 1963, 3: 321. 18 HILL, W. C. O., Primates, Comparative Anatomy and Taxonomy, Vol. 5 (Cebidae), Part B, U n i v e r s i t y Press, E d i n b u r g h , 1962. 19 MALINOW, M. R., H o r m o n e s a n d atherosclerosis, Advanc. Pharmacol., 1963, 2: 211. 2o RINEHART, J. F., AND L. D. GREENBERG, Arteriosclerotic lesions in p y r i d o x i n e - d e f i c i e n t mon-keys, Amer. J. Path., 1949, 25: 481. 21 RINEHART, J. V. AND L. D. GREENBERG, V i t a m i n BG deficiency in t h e r h e s u s m o n k e y Amer. J. clin. Nutr., 1956, 4: 318. 22 HALL, W. B., H. E. GRUPE AND C. K. CLAYCOMB, T h e p e r i d o n t i u m a n d p e r i o d o n t a l p a t h o l o g y , In: M. R. MALINOW (Ed.), Biology of the Howler Monkey (Alouatta caraya), Karger, Basel, 1967. 23 MARUFFO, C. A., M. R. MALINOW, S. KATZ AND J. R. DEPAOLI, P i g m e n t a r y liver disease in howler m o n k e y s , Amer. ar. Path., 1966, 49: 445. 24 KARLIN, R., O n t h e V i t a m i n B6 c o n t e n t of h u m a n whole blood. V a r i a t i o n s of t h e B6 level in m a t e r n a l a n d fetal blood d u r i n g labor, C. R. Soe. Biol. (Paris), 1962, 156: 858.
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