- Email: [email protected]
Peripheral white blood cell and differential counts is cholesterol-fed rabbits
Journal of A therosclerosis Research
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
P E R I P H E R A L W H I T E BLOOD CELL AND D I F F E R E N T I A L COUNTS IN CHOLESTEROL-FED RABBITS
S. C. SPRARAGEN Radioisotope Service, V.A. Hospital, and Department of Radiology, State University of N.Y., Downstate Medical Center, Brooklyn, N.Y. (U.S.A.)
(Received January 29th, 1968)
SUMMARY The leukocyte response was studied in adult, female New Zealand white rabbits having supplementary cholesterol of either 250, 500 or 1000 mg per day added to their diets. Although hypercholesterolemia occurred in all test animals b y the end of the first week of feeding, no effect upon either the total white blood cell counts or differential counts was detected under the conditions of this experiment. Total white blood cell and differential counts in New Zealand white female rabbits fed a commonly employed commercial diet is presented to augment normal values cited in the literature.
INTRODUCTION This study of the leukocytic response to cholesterol feeding is the first of several experiments performed in rabbits to investigate the origin of the foam cell found in atheromatous vascular lesions. Work previously reported 1 established that foam cells found in rabbit atheromata were capable of incorporating tritium labeled thymidine (ESHITh), a specific precursor of DNA (ref. 2). This evidence for DNA synthesis challenged the postulate t h a t foam cells are solely degenerative in nature. I t also suggested means whereby the precursor of the foam cell might be identified b y using a modification of the described 1 [3H~Th labeling technique. Currently, two theories for the origin of the foam cell are receiving primary consideration. T h e y are the smooth muscle ce113-5 and the circulating mononuclear cell 6-s hypotheses. Because of our above reported experience and BOND'S et al. 9 demonstration of multipotential cells in the peripheral blood, we decided to first test the blood borne hypothesis. Since the circulating mononuclear cells were to be germane J. Atheroscler. Res., 1968, 8:777-785
778
S.C. SPRARAGEN
to this study, it was deemed appropriate to start b y examining the influence of an atherogenic diet on the peripheral white blood cell population. ALTSCHUL AND MARTIN10 reported an initial lymphocytosis followed b y a general leukocytosis in rabbits fed cholesterol. Our experiment was designed to elaborate upon their findings and to ascertain whether an observed effect was dependent upon the dose of cholesterol employed. Consequent to this study an appreciable volume of data pertaining to the total white blood cell and differential counts of control animals was collected. Inasmuch as references to these entities in the literature are sparse, this data will also be reported. MATERIALS AND METHODS
Experimental design T w e n t y New Zealand white temale rabbits, approximately 1 year of age, weighing 3-5 kg, were caged separately in a temperature-controlled, air conditioned room. To obtain a uniform distribution of animals, baseline plasma cholesterol levels, white blood cell counts, differential counts and body weights were obtained before assigning the animals to a particular group. In this manner, four animals were assigned to each of fi separate groups. Then at random, 2 groups (8 animals) were selected to serve as controls and 1 group (4 animals) was selected for each of the 3 test groups. The quantity of the daily cholesterol supplement served to distinguish the test groups. Diets The control animals were offered 150 g of Purina Rabbit Chow per day. The test diets differed only in that 250 mg, S00 mg, or 1000 mg of cholesterol (U.S.P.) were admixed with the last 50 g of chow added to the feeding trough. The cholesterol diets were prepared in the usual manner; i.e., sprinkling a cholesterol-ether solution of known concentration over evenly spread chow pellets and then allowing the ether to evaporate. Water was permitted ad libitum. Daily food intake along with weekly weights were recorded.
White cell counts White cell counts and the differential counts were performed either once or twice weekly for 4.5 weeks prior to, and then for 6 weeks following the initiation of cholesterol feeding. To help eliminate diurnal variations, determinations were made at the same time of day. The ears were kept closely shaven so t h a t blood could be obtained b y pricking the marginal ear vein. To minimize ear trauma, alternate ears were used for each succeeding determination. Blood for the white cell studies was secured 3 days prior to drawing blood for cholesterol analyses to avoid a possible leukocytosis resulting from a 4 ml blood loss. On test days, food was withheld until the procedures were completed. Except when breakage occurred, the same pipettes and counting chambers were used for a particular animal. Two chambers were used to obtain an average white blood cell count. ]. Atherosclev. Res., 1968, 8:777-785
LEUKOCYTE RESPONSE IN CHOLESTEROL-FED
RABBITS
779
Differential white cell counts Peripheral blood smears for the differential counts were prepared using the coverslip technique. The preparations were stained with Wright's stain and mounted on regular microscope slides. A minimum of 200 cells, 100 from each slip, were recorded for each differential white cell count. Only fields showing a uniform distribution of cells were counted. Care was taken not to include nucleated red blood cells as part of the lymphocyte count. Cholesterol analysis Plasma cholesterol determinations using MANN'S method 11 were performed at weekly intervals for 3 weeks preceding the cholesterol trial, and then at weekly or biweekly intervals for the remainder of the experiment. Blood was drawn alternately from the left and right central ear arteries. All analyses were performed in duplicate. RESULTS
Survival and diet tolerance Early in the trial, 3 animals, 1 from each of the test groups, developed infections which conceivably could have influenced their white cell and differential counts. These animals were dropped from the study. This left 3 animals for s t u d y in each test group along with the 8 control animals. The diets were well tolerated b y the survivors as judged b y their daily food consumption and the fact that all such animals either gained or maintained their weight during the course of the experiment. White blood cell counts The average white blood cell counts for each group are presented along with their respective standard deviationsin Table 1. Values listed for the white blood counts of the test groups represent the average white blood cell count for the 3 animals in the group. Each control value is the average white blood cell count of the 8 control animals. Also presented is the mean white blood cell count of the control animals for the entire period of observation. All values have been rounded off to the nearest 100 white blood cells. The data suggests t h a t the cholesterol diets produced a leukocytosis in all trial groups and that the rate of response is dependent upon the dose of cholesterol employed. The fi00 mg and 1000 mg groups attained an optimal average white blood cell count b y the 24th and 17th day of cholesterol feeding respectively, while the 250 mg group did not appear to show a response until the end of the trial. A glance at Table 1, however, reveals that there is actually no significant difference between the values obtained for the test groups. Similarly there is no statistical difference between any of the test groups and the trial mean, 7900 • 2100, established for the control group. As would be expected, there is greater variability in the average white blood cell counts of the test groups than in the control group due to the smaller number of animals involved. J. Atheroscler. Res., 1968, 8:777-785
780
S.C. SPRARAGEN
TABLE 1 %'HITE
BLOOD
CELL
COUNTS
AND
STANDARD
DEVIATIONS,
PRIOR
TO AND
DURING
CHOLESTEROL
FEEDING
D a y s on trial
D a y s on diet
0 3 10
----
7.6 4- 1.9 7.5 4- 1.1 8.7 + 2.3
17 32 35 38 42
--3 6 10 13
7.8 7.4 7.8 7.5 8.0 7.2
17 24
7.0 4- 1.4 8.5 4- 3.4
45 49 56
59 27 63 31 66 34 70 38 73 41 77 45 Trial mean control animals:
White blood cell counts • ]O3[mm 3 control *L
7.6 8.3 8.0 8.0 9.0 8.2
4- 1.5 4- 2.3 4- 1.7 4- 1.3 -4- 2.2 -4- 1.6 4- 2.7 4- 2.3 4- 2.1 4- 2.1 4- 3.5 4- 1.6
group 1 b 8.1 4- 1.3 8.0 4- 1.8
7.3 8.8 6.6 7.2 7.9 8.4 8.2
4- 3.2 4- 0.9 4- 3.0 4- 0.3 4- 0.9 4- 2.0 -4- 2.6
8.2 4- 1.1 9.0 4- 1.4 8.9 4- 1.0
8.7 8.6 10.0 10.6 11.8
44444-
0.7 2.2 2.0 1.5 1.0
group I I e
group I I I a
5.3 4- 1.1 5.7 4- 1.8 6.0 4- 1.6
7.3 5.4 6.2 6.1 7.8 7.8
4- 3.1 4- 2.5 -4- 2.6 4- 2.4 4- 2.5 4- 1.8
10.3 4- 6.3 l l . O 4- 5.6 11.0 4- 5.0
9.2 8.4 9.2 8.8 8.8
4- 3.6 4- 2.8 4- 3.7 4- 3.3 4- 3.0
8.2 -4- 1.7 7.2 4- 2.0 8.8 4- 2.5
8.1 9.4 9.8 10.2 10.5
4- 3.4 -4- 4.0 4- 2.6 -4- 2.5 4- 0.8 10.0 4- 2.7 11.6 4- 3.3 10.5 4- 2.0 10.3 4- 1.7
8.9 9.3 10.2 10.6 8.8
44444-
1.5 1.5 1.2 1.9 2.3
7.9 4- 2.1
Averages of determinations made on 8 control animals. b Averages of determinations made on 3 animals fed 250 mg cholesterol daily. e Averages of determinations made on 3 animals fed 500 mg cholesterol daily. a Averages of determinations made on 3 animals fed 1000 mg cholesterol daily.
Differential counts
I n performing the differential counts, the large lymphocytes, small l y m p h o c y t e s a n d m o n o c y t o i d or t r a n s i t i o n a l cells were separately t a b u l a t e d . T h e d i a m e t e r of p r o p i n q u a n t red blood cells served to distinguish the large l y m p h o c y t e from the small l y m p h o c y t e . Monocytes, distinguishable in peripheral blood smears of the h u m a n , could n o t be readily identified as such in the r a b b i t . There are, however, r e l a t i v e l y large m o n o n u c l e a r cells h a v i n g a basophillic c y t o p l a s m a n d a large, sometimes convoluted, nucleus which resemble the monocyte. Unlike the monocyte, cytoplasmic g r a n u l a t i o n is i n f r e q u e n t a n d the nuclear c h r o m a t i n of this cell is b o t h lacey a n d c l u m p e d i n appearance. Such cells were t e r m e d t r a n s i t i o n a l cells after the n o m e n clature of SCARBOROUGH12. E x a m i n a t i o n of the d a t a failed to reveal a detectable influence of the cholesterol diet u p o n a n y c o m p o n e n t of the differential count. Accordingly, for simplicity a n d c l a r i t y of presentation, the counts of the i n d i v i d u a l t y p e s of m o n o n u c l e a r cells were t o t a l e d for each a n i m a l a n d t h e n averaged for each group of animals. These pooled values are presented in T a b l e 2. Referring to this table, it is seen t h a t m o n o n u c l e a r cells comprise a p p r o x i m a t e l y 60 ~o of the circulating white blood cells in the r a b b i t . F r o m the data, it is readily a p p a r e n t t h a t s u p p l e m e n t a l cholesterol feedings of u p to J . Atherosder. Res., 1968, 8:777-785
LEUKOCYTE RESPONSE IN CHOLESTEROL-FED RABBITS
781
TABLE2 MONONUCLEAR
WHITE
BLOOD CELLS IN PERIPHERAL
BLOOD,
P R I O R TO A N D D U R I N G
CHOLESTEROL
FEEDING
Days on trial
Days on diet
0 -3 -10 -17 -32 -35 3 38 6 42 10 45 13 49 17 56 24 59 27 63 31 66 34 70 38 73 41 77 45 Trial mean control animals:
Percent mononuclear cells • S.D. control a
group I b
group IIc
group I I I a
61 :z 70466i 63 -464• 55 458 457 462 :k 57455 • 58 458 -456 + 61 455 • 59 4-
10 12 14 13 14 10 7 13 14 10 12 12 7 14 14 11 13
49 • 15 54 -4- 15 61 4- 14 61 + 5 67 4- 15 57 4- 15 63:j: 8 56 4- 6 55 4- 12 54-4- 18 52 4- 6 47 4- 17 57 -4- 17 52 -4- 15 56 4- 10 50 • 9 51 • 7
58 :i: 63 463470 • 74464 :k 66 465 467 • 62467 473 464 469-74 459 • 63 4-
49 4- 5 5 4 4 - 15 684- 9 51 4- 20 5 5 4 - 16 52 4- 10 41 4- 20 51 :J:: 6 61 4- 8 55 4- 16 70 4- 8 62 :t: 10 62 4- 14 68 4- 22 53 4- 13 66 -4- 6 63 4- 13
604-
4
12 13 15 4 13 8 10 2 10 15 9 12 24 8 6 13 23
a Averages of determinations made on 8 control animals. b Averages of determinations made on 3 animals fed 250 mg cholesterol daily. c Averages of determinations made on 3 animals fed 500 mg cholesterol daily. d Averages of determinations made on 3 animals fed 1000 mg cholesterol daily.
1000 m g / d a y d i d n o t a p p r e c i a b l y a l t e r t h e d i f f e r e n t i a l c o u n t . T h e d a t a d o e s n o t w a r r a n t a more sophisticated analysis to establish this point. Cholesterol a n a l y s i s
The average plasma cholesterol values for the control and 3 experimental groups a r e c h a r t e d a l o n g w i t h t h e i r s t a n d a r d d e v i a t i o n s i n T a b l e 3. A l l t e s t g r o u p s d e v e l o p e d a s t a t i s t i c a l l y s i g n i f i c a n t h y p e r c h o l e s t e r o l e m i a b y t h e e n d of t h e f i r s t w e e k of c h o l e s t e r o l f e e d i n g . T h e P v a l u e s f o r g r o u p s I, I I a n d I I I a t t h i s t i m e a r e less t h a n 0.02, 0.01 a n d 0.01 r e s p e c t i v e l y . A P < 0.01 w a s e s t a b l i s h e d b y all t e s t g r o u p s b y t h e 1 4 t h d a y of t h e t r i a l p e r i o d . T h o u g h dependent,
the plasma cholesterol values appear to be dose
statistical analyses failed to reveal a significant difference between the
test groups. Control w h i t e blood cell a n d d i f f e r e n t i a l counts
D u r i n g t h e 11 w e e k c o u r s e of t h i s e x p e r i m e n t
196 w h i t e b l o o d cell c o u n t s a n d
associated differential counts were performed on blood drawn from animals fed only regular Purina Rabbit Chow. The precholesterol feeding period provided data from 20 a n i m a l s w h e r e a s t h e s e o b s e r v a t i o n s w e r e l i m i t e d t o t h e 8 c o n t r o l a n i m a l s d u r i n g J. Atheroscler. Re s . 1968, 8:777-785
S. C. SPRARAGEN
782
tile trial period. These data were compiled and are presented in Table 3 to serve as a supplement to the literature's limited reference to these entities. DISCUSSION No correlation was found to exist between the cholesterol levels and the total white blood cell or differential counts of any of the test groups. This lack of correlation does not appear to be attributable to the levels of supplemental cholesterol used, as all test animals promptly developed a significant hypercholesterolemia which in each instance exceeded 1000 mg per cent b y the end of the experiment. These results are in contrast with those observed by ALTSCHUL10. The amount of cholesterol fed in ALTSCHUL'Sexperiments can only be surmised, but as judged from the listed ingredients the levels of fed cholesterol were likely to have been much larger than those used in this investigation. Nevertheless, the degree and time of onset of the cholesterolemic response in the two studies are comparable. This raises the possibility that the leukocytosis observed by ALTSCHUL AND MARTIN may have resulted from reasons other than dietary cholesterol. I t m a y have been of value had there been more animals in our test groups, but it is unlikely that greater numbers would have altered the results. Supporting this contention is the fact that when the white cell count data of all cholesterol fed animals were pooled and compared with the control data, there was still no statistically significant differences between the treated animals and the control group. Comparing the average white blood cell count, 9200 ~ 3600, of all 9 test animals for the period of cholesterol feeding with the trial average of 7900 =E 2100, for the 8 controls yields a
TABLE 3 PLASMA CHOLESTEROL FEEDING
LEVELS
AND STANDARD
DEVIATIONS,
PRIOR
TO AND
DURING
CHOLESTEROL
D a y s of trial
D a y s on diet
P l a s m a cholesterol level (mg[ l O0 m l ) control a
group I b
group I I e
group I I I a
6 13 20 39 46
---7 14
60
28
74
42
94 4- 34 87 4- 43 834-48 95 4- 32 844-34 784-24 81 4- 26
99 4- 26 96 4- 34 79 4- 14 321 4- 19 559 :=l= 39 9 6 2 4 - 112 1235 4- 424
74 4- 29 6 9 4 - 23 59 4- 13 422 4- 157 824 4- 314 10694-327 1438 4- 37
130 4- 120 1 2 1 4 - 96 103 4- 77 733 4- 415 1421 4- 604 16384-428 1983 4- 197
Values represent averages of determinations made on 8 animals fed regular Purina Rabbit Chow. b Values represent averages of determinations made on 3 animals fed a daily 250 mg cholesterol supplement. e Values represent averages of determinations made on 3 animals fed a daily 500 mg cholesterol supplement. a Values represent averages of determinations made on 3 animals fed a daily 1000 mg cholesterol supplement. j. Atherosd~'. Res., 1968, 8:777-785
LEUKOCYTE
RESPONSE
IN
CHOLESTEROL-FED
783
RABBITS
P value far in excess of 0.5. Pooling the data from the cholesterol-fed animals for this comparison is felt to be reasonable as, again, no significant difference was found to exist between the total white blood cell counts, the differential counts or the cholesterol levels for any of the test groups. The vagaries of the white blood cells, though poorly understood, have long been recognized. Unfortunately they include an aggregate of variables capable of introducing serious error into the results of an experiment designed to study the behavior of these cells. Probably the most important variable is the normally occurring, periodic fluctuation in the number of peripherally circulating white blood cells first reported b y SABIN et al.la. The cyclical changes they described were found to be highly individual and on the average less than 24 hours in duration. Later, REIFENSTEIN et al. 14, found that this circadian r h y t h m can be as short as 1 h. They also report t h a t changes in the white blood cell count m a y range from 10 to 100 % of the total count with an average fluctuation of approximately 3500 white blood cells/ram 8. Though aware of these findings, practical considerations prevented encompassing this information in the design of this experiment. Like ALTSCHULAND MARTIN10, we simply limited the white blood cell determinations to the same time of day. SIMON et al. 15 have reported the presence of cells believed to be lipid laden macrophages in the peripheral blood of cholesterol fed rats, and that a positive correlation exists between the number of these macrophages and the degree of cholesterolemia. They did not, however, provide information pertaining to the corresponding total white blood cell counts or to the relative number of the other circulating white cells. Peripherally circulating lipid laden leukocytes were not looked for in this investigation. Unfortunately, PORTMAN'S AND STARE'S16 comprehensive report on the physiological aspects of feeding cholesterol to experimental animals did not include consideration of the hematological system. The control values listed in Table 4 for the peripheral white blood cell count TABLE
4
AVERAGE PERIPHERAL WHITE BLOOD CELL AND DIFFERENTIAL COUNTS WITH STANDARD DEVIATIONS AND RANGES FOR ADULT, FEMALE NEW ZEALAND WHITE RABBITS FED REGULAR PURINA RABBIT CHOW &
Count (cellslmmZ)
Range (cells/ram3)
77004-2100
3300 - 15,600
Differential cell type
Counts 4- S.D. ( % )
Range ( % )
Lymphocytes Transitional (monocytoid) Polymorphonuclear Bands Eosinophils Basophfls
53 46 433 43 41 44 4-
14 3 13 2
28-81 2-15 9-64 0-10
1
0- 6
3
0-20
Total white blood cell
a See
text. j . Athevoscler. Res., 1968, 8:777-785
784
S.C. SPRARAGEN
a n d t h e ranges for b o t h t h e w h i t e cell a n d differential counts are in good a g r e e m e n t w i t h those r e p o r t e d b y SCARBOROUGH12, ALBRITTON 17 an d CASEY et al. is. O u r finding of a g r e a t e r av er a g e n u m b e r of l y m p h o c y t e s t h a n p o l y m o r p h o n u c l e a r cells in t h e p e r i p h e r a l wh i t e blood cell p o p u l a t io n , on t h e o t h e r hand, is in c o n t r a s t to t h ei r findings. This result m a y be p a r t i a l l y e x p l a i n e d p e r h a p s b y our s e p a r a t e caging of t he a n i m a l s a n d r e m o v a l of a n i m a l s showing signs of infection from t h e a n i m a l ward. Also w o r t h y of consideration is t h e fact t h a t ALBRITTON'S figures represents averages of pooled d a t a published b y m a n y a u t h o r s o v e r several years, while CASEu
data
r e l a t i v e to t h e New Z e a l a n d W h i t e r a b b i t s te m s from 4, 4 .S - m o n t h - o l d males. T h o u g h female r a b b i t s were used in this e x p e r i m e n t , it is unlikely t h a t t h e o b s e r v e d n u m b e r of l y m p h o c y t e s can be a t t r i b u t e d to t h e sex of t h e animals. Th e r e l a t i v e l y a d v a n c e d age of our r a b b i t s , 1 y e a r or older, h o w e v e r , m a y c o n s t i t u t e an i m p o r t a n t factor. ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d in p a r t b y an i n s t i t u t i o n a l grant, N. 7200, from t h e M e h a r r y Medical College; a g r a n t from t h e Middle Tennessee H e a r t Association, an d b y G r a n t HE-08758, from the N a t i o n a l H e a r t I n s t i t u t e , Public H e a l t h Service. T h e a u t h o r wishes to t h a n k Mrs. H. H a m e l for her t ech n i cal assistance.
REFERENCES 1 SPRARAGEN, S. C., V. P. BOND AND L. K. DAHL, Role of hyperplasia in vascular lesions of
cholesterol-fed rabbits studied with thymidine-H s autoradiography, Circulation Res.,1962, 11: 329. '2 FRIEDKIN, M., D. TILSON AND D. ROBERTS, Studies of desoxyribonucleic acid biosynthesis in embryonic tissue with thymidine-C la, J. biol. Chem., 1956, 220: 627. 3 LUGINBUHL, H., J. E. T. JONES AND D. K. DETWEILER, The morphology of spontaneous arteriosclerotic lesions in the dog. In: J. C. ROBERTS, JR. AND R. STRAUS(Eds.), Comparative Atherosclerosis, Harper and Row, New York, 1965, p. 161. 4 BALIS, J. V., M. D. HAUSTAND R. n. MORE. Electron-microscopic studies in human atherosclerosis, cellular elements in aortic fatty streaks, Exp. molec. Path., 1964, 3: 511. 5 PARKER, F., An electron microscopic study of experimental atherosclerosis, Amer. J. Path., 1960, 36: 16. DUFF, G. L., G. C. McMILLAN AND A. C. RITCHIE, The morphology of early atherosclerotic lesions of the aorta demonstrated by the surface technique in rabbit fed cholesterol. Amer. J. Path., 1957, 33: 845. 7 GONZALEZ,I. E. AND R. H. FURMAN,Histochemistry of spontaneous and experimental arterial lesions. In: J. C. ROBERTS, JR. AND R. STRAUS(Eds.), Comparative Atherosclerosis, Harper and Row, New York, 1965, p. 329. s ROBERTSON,A. L., JR., Metabolism and ultrastructure of the arterial wall in atherosclerosis, Cleveland Clin. Quart., 1965, 32: 99. 9 BOND, V. P., E. P. CRONKITE, T. M. FLEIDNER AND P. SCHORK, Deoxyribonucleic acid synthesizing cells in peripheral blood of normal human beings, Science, 1958, 128: 202. 10 ALTSCHUL, R. AND M. E. MARTIN, Experimental cholesterol arteriosclerosis, Part 3 (The reaction of the white blood cells), Arch. Path., 1951, 51: 617. 11 MANN, C-. V., A method for measurement of cholesterol in blood serum, Clin. Chem., 1961, 7: 275. as SCARBOROUGn,R. A., The blood picture of normal laboratory animals, Yale J. Biol. Med., 1930-1, 3: 63. 13 SABIN,F. R., R. S. CVNNINCHAM,C. A. DOANAND J. A. KINDWALL, The normal rhythm of the white blood cells, Bull. Johns Hopk. Hosp., 1925, 37: 14.
j . Atheroscler. Res., 1968, 8:777-785
LEUKOCYTE
R E S P O N S E IN C H O L E S T E R O L - F E D RABBITS
785
14 REIFENSTEIN, G. H., J. H. FERGUSON AND H. G. WEISKOTTEN, Hourly leukocyte variations of normal rabbits, Amer. J. Path., 1941, 17: 219. 15 SIMON, R. C., W. J. S. STme AND R. M. O'NEAL, The circulating lipophage and experimental atherosclerosis, J. A theroscler. Res., 1961, 1: 395. 16 PORTMAN,O. W. AND F. J. STARE, Dietary regulation of serum cholesterol levels, Physiol. Rev., 1959, 39: 407. 17 ALBRITTON, E. C., In: Standard Values in Blood, Saunders, Philadelphia and London, 1952, p. 53. 18 CASEY, A. E., P. D. ROSAnN, C. K. Hu AND L. PEARCE, The hemocytological constitution of adult male rabbits from fifteen standard breeds, J. exp. Med., 1936, 64: 453.
J. Atheroscler. Res., 1968, 8: 777-78S
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
P E R I P H E R A L W H I T E BLOOD CELL AND D I F F E R E N T I A L COUNTS IN CHOLESTEROL-FED RABBITS
S. C. SPRARAGEN Radioisotope Service, V.A. Hospital, and Department of Radiology, State University of N.Y., Downstate Medical Center, Brooklyn, N.Y. (U.S.A.)
(Received January 29th, 1968)
SUMMARY The leukocyte response was studied in adult, female New Zealand white rabbits having supplementary cholesterol of either 250, 500 or 1000 mg per day added to their diets. Although hypercholesterolemia occurred in all test animals b y the end of the first week of feeding, no effect upon either the total white blood cell counts or differential counts was detected under the conditions of this experiment. Total white blood cell and differential counts in New Zealand white female rabbits fed a commonly employed commercial diet is presented to augment normal values cited in the literature.
INTRODUCTION This study of the leukocytic response to cholesterol feeding is the first of several experiments performed in rabbits to investigate the origin of the foam cell found in atheromatous vascular lesions. Work previously reported 1 established that foam cells found in rabbit atheromata were capable of incorporating tritium labeled thymidine (ESHITh), a specific precursor of DNA (ref. 2). This evidence for DNA synthesis challenged the postulate t h a t foam cells are solely degenerative in nature. I t also suggested means whereby the precursor of the foam cell might be identified b y using a modification of the described 1 [3H~Th labeling technique. Currently, two theories for the origin of the foam cell are receiving primary consideration. T h e y are the smooth muscle ce113-5 and the circulating mononuclear cell 6-s hypotheses. Because of our above reported experience and BOND'S et al. 9 demonstration of multipotential cells in the peripheral blood, we decided to first test the blood borne hypothesis. Since the circulating mononuclear cells were to be germane J. Atheroscler. Res., 1968, 8:777-785
778
S.C. SPRARAGEN
to this study, it was deemed appropriate to start b y examining the influence of an atherogenic diet on the peripheral white blood cell population. ALTSCHUL AND MARTIN10 reported an initial lymphocytosis followed b y a general leukocytosis in rabbits fed cholesterol. Our experiment was designed to elaborate upon their findings and to ascertain whether an observed effect was dependent upon the dose of cholesterol employed. Consequent to this study an appreciable volume of data pertaining to the total white blood cell and differential counts of control animals was collected. Inasmuch as references to these entities in the literature are sparse, this data will also be reported. MATERIALS AND METHODS
Experimental design T w e n t y New Zealand white temale rabbits, approximately 1 year of age, weighing 3-5 kg, were caged separately in a temperature-controlled, air conditioned room. To obtain a uniform distribution of animals, baseline plasma cholesterol levels, white blood cell counts, differential counts and body weights were obtained before assigning the animals to a particular group. In this manner, four animals were assigned to each of fi separate groups. Then at random, 2 groups (8 animals) were selected to serve as controls and 1 group (4 animals) was selected for each of the 3 test groups. The quantity of the daily cholesterol supplement served to distinguish the test groups. Diets The control animals were offered 150 g of Purina Rabbit Chow per day. The test diets differed only in that 250 mg, S00 mg, or 1000 mg of cholesterol (U.S.P.) were admixed with the last 50 g of chow added to the feeding trough. The cholesterol diets were prepared in the usual manner; i.e., sprinkling a cholesterol-ether solution of known concentration over evenly spread chow pellets and then allowing the ether to evaporate. Water was permitted ad libitum. Daily food intake along with weekly weights were recorded.
White cell counts White cell counts and the differential counts were performed either once or twice weekly for 4.5 weeks prior to, and then for 6 weeks following the initiation of cholesterol feeding. To help eliminate diurnal variations, determinations were made at the same time of day. The ears were kept closely shaven so t h a t blood could be obtained b y pricking the marginal ear vein. To minimize ear trauma, alternate ears were used for each succeeding determination. Blood for the white cell studies was secured 3 days prior to drawing blood for cholesterol analyses to avoid a possible leukocytosis resulting from a 4 ml blood loss. On test days, food was withheld until the procedures were completed. Except when breakage occurred, the same pipettes and counting chambers were used for a particular animal. Two chambers were used to obtain an average white blood cell count. ]. Atherosclev. Res., 1968, 8:777-785
LEUKOCYTE RESPONSE IN CHOLESTEROL-FED
RABBITS
779
Differential white cell counts Peripheral blood smears for the differential counts were prepared using the coverslip technique. The preparations were stained with Wright's stain and mounted on regular microscope slides. A minimum of 200 cells, 100 from each slip, were recorded for each differential white cell count. Only fields showing a uniform distribution of cells were counted. Care was taken not to include nucleated red blood cells as part of the lymphocyte count. Cholesterol analysis Plasma cholesterol determinations using MANN'S method 11 were performed at weekly intervals for 3 weeks preceding the cholesterol trial, and then at weekly or biweekly intervals for the remainder of the experiment. Blood was drawn alternately from the left and right central ear arteries. All analyses were performed in duplicate. RESULTS
Survival and diet tolerance Early in the trial, 3 animals, 1 from each of the test groups, developed infections which conceivably could have influenced their white cell and differential counts. These animals were dropped from the study. This left 3 animals for s t u d y in each test group along with the 8 control animals. The diets were well tolerated b y the survivors as judged b y their daily food consumption and the fact that all such animals either gained or maintained their weight during the course of the experiment. White blood cell counts The average white blood cell counts for each group are presented along with their respective standard deviationsin Table 1. Values listed for the white blood counts of the test groups represent the average white blood cell count for the 3 animals in the group. Each control value is the average white blood cell count of the 8 control animals. Also presented is the mean white blood cell count of the control animals for the entire period of observation. All values have been rounded off to the nearest 100 white blood cells. The data suggests t h a t the cholesterol diets produced a leukocytosis in all trial groups and that the rate of response is dependent upon the dose of cholesterol employed. The fi00 mg and 1000 mg groups attained an optimal average white blood cell count b y the 24th and 17th day of cholesterol feeding respectively, while the 250 mg group did not appear to show a response until the end of the trial. A glance at Table 1, however, reveals that there is actually no significant difference between the values obtained for the test groups. Similarly there is no statistical difference between any of the test groups and the trial mean, 7900 • 2100, established for the control group. As would be expected, there is greater variability in the average white blood cell counts of the test groups than in the control group due to the smaller number of animals involved. J. Atheroscler. Res., 1968, 8:777-785
780
S.C. SPRARAGEN
TABLE 1 %'HITE
BLOOD
CELL
COUNTS
AND
STANDARD
DEVIATIONS,
PRIOR
TO AND
DURING
CHOLESTEROL
FEEDING
D a y s on trial
D a y s on diet
0 3 10
----
7.6 4- 1.9 7.5 4- 1.1 8.7 + 2.3
17 32 35 38 42
--3 6 10 13
7.8 7.4 7.8 7.5 8.0 7.2
17 24
7.0 4- 1.4 8.5 4- 3.4
45 49 56
59 27 63 31 66 34 70 38 73 41 77 45 Trial mean control animals:
White blood cell counts • ]O3[mm 3 control *L
7.6 8.3 8.0 8.0 9.0 8.2
4- 1.5 4- 2.3 4- 1.7 4- 1.3 -4- 2.2 -4- 1.6 4- 2.7 4- 2.3 4- 2.1 4- 2.1 4- 3.5 4- 1.6
group 1 b 8.1 4- 1.3 8.0 4- 1.8
7.3 8.8 6.6 7.2 7.9 8.4 8.2
4- 3.2 4- 0.9 4- 3.0 4- 0.3 4- 0.9 4- 2.0 -4- 2.6
8.2 4- 1.1 9.0 4- 1.4 8.9 4- 1.0
8.7 8.6 10.0 10.6 11.8
44444-
0.7 2.2 2.0 1.5 1.0
group I I e
group I I I a
5.3 4- 1.1 5.7 4- 1.8 6.0 4- 1.6
7.3 5.4 6.2 6.1 7.8 7.8
4- 3.1 4- 2.5 -4- 2.6 4- 2.4 4- 2.5 4- 1.8
10.3 4- 6.3 l l . O 4- 5.6 11.0 4- 5.0
9.2 8.4 9.2 8.8 8.8
4- 3.6 4- 2.8 4- 3.7 4- 3.3 4- 3.0
8.2 -4- 1.7 7.2 4- 2.0 8.8 4- 2.5
8.1 9.4 9.8 10.2 10.5
4- 3.4 -4- 4.0 4- 2.6 -4- 2.5 4- 0.8 10.0 4- 2.7 11.6 4- 3.3 10.5 4- 2.0 10.3 4- 1.7
8.9 9.3 10.2 10.6 8.8
44444-
1.5 1.5 1.2 1.9 2.3
7.9 4- 2.1
Averages of determinations made on 8 control animals. b Averages of determinations made on 3 animals fed 250 mg cholesterol daily. e Averages of determinations made on 3 animals fed 500 mg cholesterol daily. a Averages of determinations made on 3 animals fed 1000 mg cholesterol daily.
Differential counts
I n performing the differential counts, the large lymphocytes, small l y m p h o c y t e s a n d m o n o c y t o i d or t r a n s i t i o n a l cells were separately t a b u l a t e d . T h e d i a m e t e r of p r o p i n q u a n t red blood cells served to distinguish the large l y m p h o c y t e from the small l y m p h o c y t e . Monocytes, distinguishable in peripheral blood smears of the h u m a n , could n o t be readily identified as such in the r a b b i t . There are, however, r e l a t i v e l y large m o n o n u c l e a r cells h a v i n g a basophillic c y t o p l a s m a n d a large, sometimes convoluted, nucleus which resemble the monocyte. Unlike the monocyte, cytoplasmic g r a n u l a t i o n is i n f r e q u e n t a n d the nuclear c h r o m a t i n of this cell is b o t h lacey a n d c l u m p e d i n appearance. Such cells were t e r m e d t r a n s i t i o n a l cells after the n o m e n clature of SCARBOROUGH12. E x a m i n a t i o n of the d a t a failed to reveal a detectable influence of the cholesterol diet u p o n a n y c o m p o n e n t of the differential count. Accordingly, for simplicity a n d c l a r i t y of presentation, the counts of the i n d i v i d u a l t y p e s of m o n o n u c l e a r cells were t o t a l e d for each a n i m a l a n d t h e n averaged for each group of animals. These pooled values are presented in T a b l e 2. Referring to this table, it is seen t h a t m o n o n u c l e a r cells comprise a p p r o x i m a t e l y 60 ~o of the circulating white blood cells in the r a b b i t . F r o m the data, it is readily a p p a r e n t t h a t s u p p l e m e n t a l cholesterol feedings of u p to J . Atherosder. Res., 1968, 8:777-785
LEUKOCYTE RESPONSE IN CHOLESTEROL-FED RABBITS
781
TABLE2 MONONUCLEAR
WHITE
BLOOD CELLS IN PERIPHERAL
BLOOD,
P R I O R TO A N D D U R I N G
CHOLESTEROL
FEEDING
Days on trial
Days on diet
0 -3 -10 -17 -32 -35 3 38 6 42 10 45 13 49 17 56 24 59 27 63 31 66 34 70 38 73 41 77 45 Trial mean control animals:
Percent mononuclear cells • S.D. control a
group I b
group IIc
group I I I a
61 :z 70466i 63 -464• 55 458 457 462 :k 57455 • 58 458 -456 + 61 455 • 59 4-
10 12 14 13 14 10 7 13 14 10 12 12 7 14 14 11 13
49 • 15 54 -4- 15 61 4- 14 61 + 5 67 4- 15 57 4- 15 63:j: 8 56 4- 6 55 4- 12 54-4- 18 52 4- 6 47 4- 17 57 -4- 17 52 -4- 15 56 4- 10 50 • 9 51 • 7
58 :i: 63 463470 • 74464 :k 66 465 467 • 62467 473 464 469-74 459 • 63 4-
49 4- 5 5 4 4 - 15 684- 9 51 4- 20 5 5 4 - 16 52 4- 10 41 4- 20 51 :J:: 6 61 4- 8 55 4- 16 70 4- 8 62 :t: 10 62 4- 14 68 4- 22 53 4- 13 66 -4- 6 63 4- 13
604-
4
12 13 15 4 13 8 10 2 10 15 9 12 24 8 6 13 23
a Averages of determinations made on 8 control animals. b Averages of determinations made on 3 animals fed 250 mg cholesterol daily. c Averages of determinations made on 3 animals fed 500 mg cholesterol daily. d Averages of determinations made on 3 animals fed 1000 mg cholesterol daily.
1000 m g / d a y d i d n o t a p p r e c i a b l y a l t e r t h e d i f f e r e n t i a l c o u n t . T h e d a t a d o e s n o t w a r r a n t a more sophisticated analysis to establish this point. Cholesterol a n a l y s i s
The average plasma cholesterol values for the control and 3 experimental groups a r e c h a r t e d a l o n g w i t h t h e i r s t a n d a r d d e v i a t i o n s i n T a b l e 3. A l l t e s t g r o u p s d e v e l o p e d a s t a t i s t i c a l l y s i g n i f i c a n t h y p e r c h o l e s t e r o l e m i a b y t h e e n d of t h e f i r s t w e e k of c h o l e s t e r o l f e e d i n g . T h e P v a l u e s f o r g r o u p s I, I I a n d I I I a t t h i s t i m e a r e less t h a n 0.02, 0.01 a n d 0.01 r e s p e c t i v e l y . A P < 0.01 w a s e s t a b l i s h e d b y all t e s t g r o u p s b y t h e 1 4 t h d a y of t h e t r i a l p e r i o d . T h o u g h dependent,
the plasma cholesterol values appear to be dose
statistical analyses failed to reveal a significant difference between the
test groups. Control w h i t e blood cell a n d d i f f e r e n t i a l counts
D u r i n g t h e 11 w e e k c o u r s e of t h i s e x p e r i m e n t
196 w h i t e b l o o d cell c o u n t s a n d
associated differential counts were performed on blood drawn from animals fed only regular Purina Rabbit Chow. The precholesterol feeding period provided data from 20 a n i m a l s w h e r e a s t h e s e o b s e r v a t i o n s w e r e l i m i t e d t o t h e 8 c o n t r o l a n i m a l s d u r i n g J. Atheroscler. Re s . 1968, 8:777-785
S. C. SPRARAGEN
782
tile trial period. These data were compiled and are presented in Table 3 to serve as a supplement to the literature's limited reference to these entities. DISCUSSION No correlation was found to exist between the cholesterol levels and the total white blood cell or differential counts of any of the test groups. This lack of correlation does not appear to be attributable to the levels of supplemental cholesterol used, as all test animals promptly developed a significant hypercholesterolemia which in each instance exceeded 1000 mg per cent b y the end of the experiment. These results are in contrast with those observed by ALTSCHUL10. The amount of cholesterol fed in ALTSCHUL'Sexperiments can only be surmised, but as judged from the listed ingredients the levels of fed cholesterol were likely to have been much larger than those used in this investigation. Nevertheless, the degree and time of onset of the cholesterolemic response in the two studies are comparable. This raises the possibility that the leukocytosis observed by ALTSCHUL AND MARTIN may have resulted from reasons other than dietary cholesterol. I t m a y have been of value had there been more animals in our test groups, but it is unlikely that greater numbers would have altered the results. Supporting this contention is the fact that when the white cell count data of all cholesterol fed animals were pooled and compared with the control data, there was still no statistically significant differences between the treated animals and the control group. Comparing the average white blood cell count, 9200 ~ 3600, of all 9 test animals for the period of cholesterol feeding with the trial average of 7900 =E 2100, for the 8 controls yields a
TABLE 3 PLASMA CHOLESTEROL FEEDING
LEVELS
AND STANDARD
DEVIATIONS,
PRIOR
TO AND
DURING
CHOLESTEROL
D a y s of trial
D a y s on diet
P l a s m a cholesterol level (mg[ l O0 m l ) control a
group I b
group I I e
group I I I a
6 13 20 39 46
---7 14
60
28
74
42
94 4- 34 87 4- 43 834-48 95 4- 32 844-34 784-24 81 4- 26
99 4- 26 96 4- 34 79 4- 14 321 4- 19 559 :=l= 39 9 6 2 4 - 112 1235 4- 424
74 4- 29 6 9 4 - 23 59 4- 13 422 4- 157 824 4- 314 10694-327 1438 4- 37
130 4- 120 1 2 1 4 - 96 103 4- 77 733 4- 415 1421 4- 604 16384-428 1983 4- 197
Values represent averages of determinations made on 8 animals fed regular Purina Rabbit Chow. b Values represent averages of determinations made on 3 animals fed a daily 250 mg cholesterol supplement. e Values represent averages of determinations made on 3 animals fed a daily 500 mg cholesterol supplement. a Values represent averages of determinations made on 3 animals fed a daily 1000 mg cholesterol supplement. j. Atherosd~'. Res., 1968, 8:777-785
LEUKOCYTE
RESPONSE
IN
CHOLESTEROL-FED
783
RABBITS
P value far in excess of 0.5. Pooling the data from the cholesterol-fed animals for this comparison is felt to be reasonable as, again, no significant difference was found to exist between the total white blood cell counts, the differential counts or the cholesterol levels for any of the test groups. The vagaries of the white blood cells, though poorly understood, have long been recognized. Unfortunately they include an aggregate of variables capable of introducing serious error into the results of an experiment designed to study the behavior of these cells. Probably the most important variable is the normally occurring, periodic fluctuation in the number of peripherally circulating white blood cells first reported b y SABIN et al.la. The cyclical changes they described were found to be highly individual and on the average less than 24 hours in duration. Later, REIFENSTEIN et al. 14, found that this circadian r h y t h m can be as short as 1 h. They also report t h a t changes in the white blood cell count m a y range from 10 to 100 % of the total count with an average fluctuation of approximately 3500 white blood cells/ram 8. Though aware of these findings, practical considerations prevented encompassing this information in the design of this experiment. Like ALTSCHULAND MARTIN10, we simply limited the white blood cell determinations to the same time of day. SIMON et al. 15 have reported the presence of cells believed to be lipid laden macrophages in the peripheral blood of cholesterol fed rats, and that a positive correlation exists between the number of these macrophages and the degree of cholesterolemia. They did not, however, provide information pertaining to the corresponding total white blood cell counts or to the relative number of the other circulating white cells. Peripherally circulating lipid laden leukocytes were not looked for in this investigation. Unfortunately, PORTMAN'S AND STARE'S16 comprehensive report on the physiological aspects of feeding cholesterol to experimental animals did not include consideration of the hematological system. The control values listed in Table 4 for the peripheral white blood cell count TABLE
4
AVERAGE PERIPHERAL WHITE BLOOD CELL AND DIFFERENTIAL COUNTS WITH STANDARD DEVIATIONS AND RANGES FOR ADULT, FEMALE NEW ZEALAND WHITE RABBITS FED REGULAR PURINA RABBIT CHOW &
Count (cellslmmZ)
Range (cells/ram3)
77004-2100
3300 - 15,600
Differential cell type
Counts 4- S.D. ( % )
Range ( % )
Lymphocytes Transitional (monocytoid) Polymorphonuclear Bands Eosinophils Basophfls
53 46 433 43 41 44 4-
14 3 13 2
28-81 2-15 9-64 0-10
1
0- 6
3
0-20
Total white blood cell
a See
text. j . Athevoscler. Res., 1968, 8:777-785
784
S.C. SPRARAGEN
a n d t h e ranges for b o t h t h e w h i t e cell a n d differential counts are in good a g r e e m e n t w i t h those r e p o r t e d b y SCARBOROUGH12, ALBRITTON 17 an d CASEY et al. is. O u r finding of a g r e a t e r av er a g e n u m b e r of l y m p h o c y t e s t h a n p o l y m o r p h o n u c l e a r cells in t h e p e r i p h e r a l wh i t e blood cell p o p u l a t io n , on t h e o t h e r hand, is in c o n t r a s t to t h ei r findings. This result m a y be p a r t i a l l y e x p l a i n e d p e r h a p s b y our s e p a r a t e caging of t he a n i m a l s a n d r e m o v a l of a n i m a l s showing signs of infection from t h e a n i m a l ward. Also w o r t h y of consideration is t h e fact t h a t ALBRITTON'S figures represents averages of pooled d a t a published b y m a n y a u t h o r s o v e r several years, while CASEu
data
r e l a t i v e to t h e New Z e a l a n d W h i t e r a b b i t s te m s from 4, 4 .S - m o n t h - o l d males. T h o u g h female r a b b i t s were used in this e x p e r i m e n t , it is unlikely t h a t t h e o b s e r v e d n u m b e r of l y m p h o c y t e s can be a t t r i b u t e d to t h e sex of t h e animals. Th e r e l a t i v e l y a d v a n c e d age of our r a b b i t s , 1 y e a r or older, h o w e v e r , m a y c o n s t i t u t e an i m p o r t a n t factor. ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d in p a r t b y an i n s t i t u t i o n a l grant, N. 7200, from t h e M e h a r r y Medical College; a g r a n t from t h e Middle Tennessee H e a r t Association, an d b y G r a n t HE-08758, from the N a t i o n a l H e a r t I n s t i t u t e , Public H e a l t h Service. T h e a u t h o r wishes to t h a n k Mrs. H. H a m e l for her t ech n i cal assistance.
REFERENCES 1 SPRARAGEN, S. C., V. P. BOND AND L. K. DAHL, Role of hyperplasia in vascular lesions of
cholesterol-fed rabbits studied with thymidine-H s autoradiography, Circulation Res.,1962, 11: 329. '2 FRIEDKIN, M., D. TILSON AND D. ROBERTS, Studies of desoxyribonucleic acid biosynthesis in embryonic tissue with thymidine-C la, J. biol. Chem., 1956, 220: 627. 3 LUGINBUHL, H., J. E. T. JONES AND D. K. DETWEILER, The morphology of spontaneous arteriosclerotic lesions in the dog. In: J. C. ROBERTS, JR. AND R. STRAUS(Eds.), Comparative Atherosclerosis, Harper and Row, New York, 1965, p. 161. 4 BALIS, J. V., M. D. HAUSTAND R. n. MORE. Electron-microscopic studies in human atherosclerosis, cellular elements in aortic fatty streaks, Exp. molec. Path., 1964, 3: 511. 5 PARKER, F., An electron microscopic study of experimental atherosclerosis, Amer. J. Path., 1960, 36: 16. DUFF, G. L., G. C. McMILLAN AND A. C. RITCHIE, The morphology of early atherosclerotic lesions of the aorta demonstrated by the surface technique in rabbit fed cholesterol. Amer. J. Path., 1957, 33: 845. 7 GONZALEZ,I. E. AND R. H. FURMAN,Histochemistry of spontaneous and experimental arterial lesions. In: J. C. ROBERTS, JR. AND R. STRAUS(Eds.), Comparative Atherosclerosis, Harper and Row, New York, 1965, p. 329. s ROBERTSON,A. L., JR., Metabolism and ultrastructure of the arterial wall in atherosclerosis, Cleveland Clin. Quart., 1965, 32: 99. 9 BOND, V. P., E. P. CRONKITE, T. M. FLEIDNER AND P. SCHORK, Deoxyribonucleic acid synthesizing cells in peripheral blood of normal human beings, Science, 1958, 128: 202. 10 ALTSCHUL, R. AND M. E. MARTIN, Experimental cholesterol arteriosclerosis, Part 3 (The reaction of the white blood cells), Arch. Path., 1951, 51: 617. 11 MANN, C-. V., A method for measurement of cholesterol in blood serum, Clin. Chem., 1961, 7: 275. as SCARBOROUGn,R. A., The blood picture of normal laboratory animals, Yale J. Biol. Med., 1930-1, 3: 63. 13 SABIN,F. R., R. S. CVNNINCHAM,C. A. DOANAND J. A. KINDWALL, The normal rhythm of the white blood cells, Bull. Johns Hopk. Hosp., 1925, 37: 14.
j . Atheroscler. Res., 1968, 8:777-785
LEUKOCYTE
R E S P O N S E IN C H O L E S T E R O L - F E D RABBITS
785
14 REIFENSTEIN, G. H., J. H. FERGUSON AND H. G. WEISKOTTEN, Hourly leukocyte variations of normal rabbits, Amer. J. Path., 1941, 17: 219. 15 SIMON, R. C., W. J. S. STme AND R. M. O'NEAL, The circulating lipophage and experimental atherosclerosis, J. A theroscler. Res., 1961, 1: 395. 16 PORTMAN,O. W. AND F. J. STARE, Dietary regulation of serum cholesterol levels, Physiol. Rev., 1959, 39: 407. 17 ALBRITTON, E. C., In: Standard Values in Blood, Saunders, Philadelphia and London, 1952, p. 53. 18 CASEY, A. E., P. D. ROSAnN, C. K. Hu AND L. PEARCE, The hemocytological constitution of adult male rabbits from fifteen standard breeds, J. exp. Med., 1936, 64: 453.
J. Atheroscler. Res., 1968, 8: 777-78S