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Strain differences in response to dietary cholesterol by JAX rabbits: Correlation with esterase patterns
435
Atherosclerosis, 28 (1977) 435-446 0 Elsevier/North-Holland Scientific
Publishers,
Ltd.
STRAIN DIFFERENCES IN RESPONSE TO DIETARY CHOLESTEROL JAX RABBITS: CORRELATION WITH ESTERASE PATTERNS
L.F.M. VAN ZUTPHEN
BY
and R.R. FOX
Veterinary Faculty, Department of Animal Genetics, University of Utrecht, Utrecht (The Netherlands) and Jackson Laboratory, Bar Harbor, Me. 04609
(U.S.A.)
(Received 23 May, 1977) (Revised, received 29 August, 1977) (Accepted 2 September, 1977)
Summary Six strains of genetically defined JAX rabbits were tested for their serum cholesterol levels (total and free) in response to a 0.5% cholesterol diet. Marked differences in response between the 6 strains were found. IIIVO/J and WH/J are low responding strains, X/J and ACEP/J are intermediate responding strains, and OS/J and AX/J are high responding strains. After 4 weeks of the cholesterol diet the total serum cholesterol level of the high responding AX/J strain was about 5-fold greater than the level of the low responding IIIVO/J strain. The esterified/total (E/T) ratio appeared to be higher in the high responding strains, indicating a synergistic effect in the process of atherosclerosis. The response of the individual rabbits to the cholesterol diet was compared with the patterns of serum and liver esterase zymograms. This comparison indicated a correlation of the dietary cholesterol susceptibility with the presence or absence of the e&erase zones in the anodal, fast moving region of the gel. Key words:
Atherosclerosis
- Cholesterol
- Esterase -Rabbit
Introduction Additive inheritance of serum cholesterol levels has been reported for several mammals, including man [l-8]. The susceptibility to dietary induced hypercholesterolemia is different in various species of mammals and in man [9,10]. Also within a given species differences can occur. These differences have led to the introduction of the terms hyper- and hyporesponders. Influences of genetic factors on the susceptibility to dietary cholesterol are reported for several
436
mammalian species [ll-131. In the squirrel monkey the mechanism for genetic control of hypercholesterolemia seems to depend on the conversion of cholesterol to bile acids [ 141. The physiological basis for the differences in response to dietary cholesterol is still unknown for the other species. Adams et al. [15] found no evidence for a greater excretion of cholesterol in the hyporesponding rabbit. Both Wolman’s disease and cholesterol ester storage disease are believed to be caused by deficiency of acid esterase activity [16,17]. Esterase activity is considered to be a factor in atherosclerosis [ 181. After absorption, most of the dietary cholesterol is being esterified with fatty acids. Studies of Stokke [19] and Redgrave [20] have suggested that hydrolysis of these cholesterol esters by cholesterolesterase can be considered as an important process in the regulation of mammalian cholesterol metabolism. These data emphasize the possible role of the esterases in hypercholesterolemia and atherosclerosis. Differences in esterase zymogram patterns might therefore be correlated with differences in susceptibility to dietary cholesterol and atherosclerosis. A preliminary study of van Zutphen and den Bieman (unpublished) comparing the response to dietary cholesterol with the prealbumin esterase phenotype of the rabbit indicated that such a correlation might exist. The rabbit is frequently used as a model in studies on hypercholesterolemia and atherosclerosis. The value of this animal for these studies has recently been emphasized by Shore and Shore [21]. The availability of inbred strains of rabbits can greatly facilitate these studies, in particular the search for the genetic factors which are involved. Several inbred and partly inbred strains of JAX rabbits [22-241 have recently been characterized for the prealbumin serum esterase phenotypes [25]. Because of this unique availability, we tested several of these strains for dietary cholesterol susceptibility and compared their different susceptibilities with the esterase patterns in both serum and liver. Materials and methods Animals Five young adult healthy male rabbits from each of 6 strains (IIIVO/J, AX/J, OS/J, ACEP/J, WH/J and X/J) from the Jackson Laboratory colony were used [22-251. The inbreeding coefficients for these strains are approximately as follows: IIIVO/J, 0.97; WH/J, 0.72; X/J, 0.88; ACEP/J, 0.92; OS/J, 0.85; AX/J, 0.73. The ages of the rabbits in each strain at the start of the experiment were similar: e.g., IIIVO/J, 122.0 + 4.5; ACEP/J, 119.6 f 5.0; AX/J, 131.8 + 6.6; WH/J, 129.6 f 7.7; OS/J, 136.6 + 10.7; and X/J, 124.6 f 9.0 days. The rabbit room was on a 13-h light-11-h dark cycle. The light period was from 4:30 a.m. to 5:30 p.m. The temperature range in the rabbit room was 13--18°C with a mode value of 15.5”C. Prior to the experiment the rabbits had a lo-day acclimatization period. Feeding Before and during the acclimatization period all rabbits were fed Ralston Purina Rabbit Chow Checkers ad libitum. After acclimatization all rabbits
437
were fed the same food which was fortified with 0.5% pure cholesterol (Sigma) by Purina (5838C6F6 - lot 06995) for 28 days at a rate of 40 g/kg of body weight/day. With exception of the day of bleeding, all feeding was done each day at 8:00 a.m. On the day of bleeding rabbits were fed immediately after bleeding. During the acclimatization period and after bleeding on day 28 the rabbits were fed regular Purina Chow Checkers 20 (5325) also at a rate of 40 g/kg of body weight/day. All rabbits were weighed once a week. By error the feeding on day 12 was omitted. Bleeding All rabbits were bled on day 0, 7,14, 21, 28, 35 and 49, just prior to feeding. Day 0 indicates the first day of the cholesterol diet after the acclimatization period. Bleeding on day 0 preceded the administration of the cholesterol diet. All but terminal bleedings were performed according to Hoppe et al. [26] between 8:00 a.m. and 10:00 a.m. to minimize diurnal variation in cholesterol levels [27] with the order randomized each day. Terminal bleeding on day 49 was performed by cardiac puncture between 8:00 a.m. and 1l:OO a.m. Processing of blood Blood was allowed to clot at room temperature for 30 min, then was refrigerated at 5°C for 30-60 min and centrifuged at 1100 X g. Serum was obtained and aliquoted into two samples. One sample was frozen (-60°C) and the other assayed immediately for total and free cholesterol levels. Au topsy After bleeding on day 49 rabbits were killed by cervical dislocation. Samples of liver, kidney, pancreas and ileum were obtained and stored at -60°C for further study. All animals were necropsied and in particular the great vessels were checked grossly for atherosclerotic plaques. When plaques were observed, samples of the vessels were fixed in Bouin’s fixative, sectioned at 12 pm, and stained with H-E for histological confirmation by Dr. Hans Meier, Senior Staff Scientist, Jackson Laboratory. Determination of es terase phenotypes The esterase patterns of the serum were obtained after electrophoresis according to van Zutphen [28-301. Zymograms for prealbumin esterase loci Est-I, Est-2 and Est-3 and for the P-globulin esterase patterns were determined. The latter are analogous to the patterns of the red cell locus (Es-l). After autopsy of the animals on day 49 part of the liver of each rabbit was homogenized with equal volumes of distilled water, centrifuged at 35,000 X g after freezing and thawing, the esterase zymogram was then determined using the supematant according to the procedures of van Zutphen [28-301. Cholesterol determination Total and free cholesterol was measured in the serum on days 0, 7, 14, 21, 28, 35 and 49 using the enzymatic method described by Roschlau et al. [31]. The free cholesterol is transformed to A4-cholestenone and hydrogen peroxide by cholesteroloxidase. In the presence of catalase hydrogen peroxide oxidizes
438
methanol to formaldehyde which reacts with ammonium ions and acetylacetone to 3,5diacetyl-1,4-dihydrolutidine. The intensity of the yellow color of this product was measured on a Gilford 300 spectrophotometer at 410 nm. For determination of total cholesterol, all cholesterol esters in the serum are hydrolyzed by cholesterol e&erase. All chemicals for total and free cholesterol determinations were obtained as a test combination set from Boehringer, Mannheim. Results The dietary level of 40 g/kg body weight was a maintenance diet but did allow a slight weight gain, e.g., from day 0 to day 28 the average gain per rabbit was 65 g and no change in weight was observed from day 28 to day 49. The data of the individual total and free serum cholesterol levels for each of the bleeding days were summarized by strain, and means and standard errors were calculated. Table 1 gives these mean values + SE for each of the 6 strains. These results indicate marked differences in response to dietary cholesterol between the strains. This may be illustrated by comparing the increase in total cholesterol from day 0 until 28. This increase was about 1100% in the hyporesponding IIIVO/J strain and about 7000% in the hyperresponding AX/J strain. Fig. 1 represents a graphical illustration of the differences between the strains. Twenty-eight days is obviously too short a time for reaching a steadystate level when feeding a 0.5% cholesterol diet. The cholesterol levels in the high responding strains are still rising sharply after 4 weeks of feeding the cholesterol diet. Because we were primarily interested in differences in the initial response of the serum level to dietary cholesterol, the cholesterol diet was stopped at day 28. Fig. 2 demonstrates diagrammatically the individual levels of total and free cholesterol on the final day of the cholesterol diet (day 28). The variation within the strains might be due to the fact that these strains are not yet completely inbred. As can be seen from Fig. 2 the animals with the high total cholesterol levels have relatively lower free cholesterol levels. Consequently, the higher the total cholesterol level is, the higher the esterified/ total (E/T) ratio. A high positive correlation coefficient of 0.95 (y = 0.00016x + 0.36) was calculated for the total cholesterol levels and the E/T ratio at day 28. The increasing E/T ratio is illustrated for each of the strains in Fig. 3. The slightly lower ratios on day 14 may be due to the fact that, by error, the rabbits were not fed on day 12. It is obvious that the strains with the high cholesterol levels have higher E/T ratios than the strains with the lower total cholesterol levels. The total cholesterol levels of the individual rabbits at day 28 were compared with the esterase zymograms of serum and liver. A correlation with the e&erase zones in the anodal, fast moving region of the gel might exist (Table 2). The presence or absence of the most anodal serum esterase zone is genetically determined and is controlled by alleles of the Es&2 locus [30], previously called the As locus of linkage group VI [32]. There are two forms of the anodal serum e&erase zone. One stains very intensively with cr-naphthylacetate as a substrate. This zone has been indicated as F’. The other zone (f’) which has the same RI value, stains rather faint. Either the F’ or the f’ zone was
Total cholesterol
IIIVO/J
=
Totalcholesterol Free cholesterol F/Tratio
TotaI cholesterol Free cholesterol FITratio
Total cholesterol Free cholesterol F/Tratio
Total cholesterol Free cholesterol FITratio
X/J
ACEP/J
OS/J
AX/J
5 5 5
5 5 5
5 5 5
5 5 5
5 5 5
5 5 5
n
2.1 1.3
1.7 1.3
22.0 f 4.1 18.4 fi 4.0 0.84
32.2 r 2.0 22.8 k 0.6 0.71
28.4 f 20.6 f 0.13
36.4 k 13.4 25.8 + 1.4 0.71
12.0 t 1.5 9.2 r 1.3 0.77
28.8 f 22.6 f 0.78
0a
Day
340.4 f 38.0 147.4 t 19.9 0.43
326.4 f 40.4 131.6 f 15.2 0.40
233.6 f 37.1 95.2 f 10.0 0.41
247.6 + 61.6 111.0 f 19.8 0.45
150.6 k 21.2 71.8 + 5.5 0.48
146.6 + 19.5 72.4 + 5.2 0.49
7
31.8 11.1
728.4 i 134.9 304.6 + 41.2 0.42
668.6 + 163.8 292.0 i: 53.0 0.44
308.8 t 31.6 148.6 f 11.1 0.48
301.4 k 73.1 166.6 f 36.1 0.54
21
24.4 11.9
928.2 + 193.4 410.2 f 48.2 0.44
903.4 + 198.3 407.4 ? 62.4 0.45
359.0 f 207.0 f 0.58
319.2 t 58.6 195.8 + 31.6 0.61
28b
922.0 * 155.3 1296.8 k 118.3 1530.6 k 108.7 386.0 k 49.2 492.0 f 36.4 611.6 f 39.1 0.42 0.40 0.38
1011.8 f 150.8 1135.6 + 183.8 1408.8 f 215.6 389.0 + 32.7 431.6 + 53.4 572.4 f. 58.3 0.38 0.38 0.41
429.6 t 112.1 226.0 + 32.1 0.53
632.4 f 154.1 308.6 2 56.4 0.49
241.2 k 145.4 ? 0.59
223.6 + 60.9 130.0 f 33.1 0.58
14
28DAYSOFA0.59bCHOLESTEROLDIET
aDay OleveIsamprecholesteroldietleveIs. Alllevelson aIIdaysarebasedonsemm collectedbefore feedkztbatday. diet stopped on day 28 and normal diet(same butwithoutcholesterol)resumed. b0.5% cholesterolin C Totaland freeserumcholesterolvaluesgiveninmg/100mlserum.
Totalcholesterol Freecholesterol F/Tratio
WI-I/J
Free cbolesterolC F/Tratio
Parameter
Strain
SERUMCHOLESTEROLLEVELSIN6STRAINSOFJAXRABBITSHAVING
TABLE1
8.1 7.2
23.4 19.1
798.6 + 108.5 429.0 + 37.1 0.54
795.8 + 151.8 399.6 f 53.4 0.50
393.6 k 94.3 242.0 r 45.0 0.61
330.4 f 101.2 214.8 k 51.8 0.65
89.4 f 68.6 r 0.17
130.8 + 100.0 f 0.76
35
13.6 10.2
12.4 10.2
71.3 21.6
50.1 18.8
301.2 f 108.8 158.4 f 49.1 0.53
392.2 2 198.4 f 0.51
253.2 f 153.6 f 0.61
141.4 r 18.8 91.0 f 43.3 0.62
49.4 f 36.8 f 0.14
72.4 + 55.8 + 0.77
49
0
14
7
21
DAYS
.28
35
49
Fig. 1. Graphical demonstration of the response in serum total cholesterol levels of 6 strains of JAX rabbits on feeding a 0.5% cholesterol diet for 28 days followed by 21 days of the normal diet. Five rabbits were used Per strain.
ElFREE I
III VO/J
ESTERIFIED
WH/J
X/J
AMP/J
OS/J
STRAIN Fig. 2. Strain differences and individual variation of feeding a 0.5% cholesterol diet.
of serum cholesterol
levels (free and total)
after 28 days
441
_ _ _ _ ACE”/J
0
7
14
21 DAYS
28
35
49
Fig. 3. Esterified/total cholesterol ratio in the serum of rabbits of 6 JAX strains, fed a 0.5% cholesterol diet from day 0 until day 26 followed by 21 days of normal diet.
Fig. 4. Zymogram of rabbit liver esterases. The two phenotypes the anodal zone a. am demonstrated.
A and B. respectively,
with and without
442
present in the rabbits of strains IIIVO/J (phenotypes V and VI), WH/J (phenotypes IIIF’ and VF’) and ACEP/J (phenotype VF’) and in 3 rabbits of strain X/J (phenotype III). All of the other rabbits were missing such a zone (phenotypes II and Vf). Although the rabbits of strain WH/J are classified as IIIF’ or VF’, which means that all these rabbits possessed the most intensive stained anodal F’ zone, we had some problems with phenotyping this strain, because of the fact that in this strain the anodal zone stained somewhat less than F’, but more intense than f’. The genetic basis for the presence or absence of the anodal liver esterase zone has not yet been established. Two different phenotypes A and B were found. An anodal, fast moving zone (a) is present in phenotype A and absent in B (Fig. 4). The intensity of this zone varied but, since we do not know TABLE 2 COMPARISON OF THE SERUM AND LIVER ESTERASE PHENOTYPES OF 30 JAX RABBITS WITH THE TOTAL CHOLESTEROL LEVELS FOLLOWING 28 DAYS OF FEEDING A 0.5% CHOLESTEROL DIET Strain
Serum esterase phenotype
Liver esterase phenotype
Anodal 8enlm esterase
Anodal liver esterme
zone (F’ or f’)
zone (a)
Total chol. (IIxglOO ml)
IIIVO/J
VI VI VI VI VI
B B B B B
+ + + + +
-
228 248 266 306 648
WH/J
vF’ vF’ IIIF’ IIIF’ vF’
B B B A A
+ + + + +
-
303 303 365 409 415
III III III II II
A A A A A
+ + + -
+ + + +a +a
463 601 695 1329 1429
B B B B A
+ + + + +
-
653 676 799 961 1663
-
-
Vf Vf Vf Vf
B A A A A
+ + + +
814 1023 1573 1634 2000
II II II Vf vf
A A A A A
-
+ + + + +
1223 1344 1586 1689 1811
X/J
ACEP/J
;:; $1 vF’
OS/J
AX/J
vf
+ +
+
a The anodial liver esterase zone of these samples were more distinct than normal.
443
whether this is a genetically determined character, all zymograms which showed the anodal zone were indicated as A and those in which the anodal zone was missing as B. Comparing the presence or absence of these fast moving zones of liver and serum with the cholesterol levels at day 28, it was most striking that absence of the anodal zone in the serum and presence of such a zone in the liver are correlated with relatively high serum cholesterol levels, whereas these levels are lower when the anodal zone is present in the serum and absent in the liver (Table 2). Discussion The differences in susceptibility for dietary cholesterol between the strains found in this study are more pronounced than expected, based on the data of Adams et al. [15]. They have shown that the New Zealand White and the Dutch breeds differ in their susceptibility for dietary cholesterol. After 4 weeks of a 0.5% cholesterol diet the male rabbits of the New Zealand White breed had a serum cholesterol level which was about 50% higher than that of the Dutch breed. Our results indicate that after 4 weeks of a similar regimen the male rabbits of the high responding AX/J strain have a serum cholesterol level that is about 5-fold greater than the level of the lowest responding IIIVO/J strain. The availability of high responding and low responding inbred strains of rabbits can be of great value in the research on hypercholesterolemia and atherosclerosis. From Fig. 1 it is obvious that the period of 28 days is too short for reaching the steady-state level. According to Redgrave and West [33] cholesterol levels reach a plateau only after 5-7 weeks of a cholesterol diet. The concentration at that time will be about 50% greater than the concentration at 3 weeks. Their findings could be used for extrapolation of our data as an indication of what the various steady-state levels of total serum cholesterol would have been if the cholesterol diet was continued after 28 days. Fig. 1 shows clearly that we have 2 hyporesponding strains (IIIVO/J and WH/J), 2 intermediate strains (X/J and ACEP/J) and 2 hyperresponding strains (OS/J and AX/J). These differences are not associated with body weight or general activity level. The highest responding AX/J strain and the lowest responding IIIVO/J strain are both large strains, whereas all other strains are small. Also AX/J and IIIVO/J are rather docile, ACEP/J and X/J are very hyperactive and the other 2 strains are intermediate. Blood pressure values for the respective strains [ 341, either systolic or diastolic, appeared not to be correlated with the cholesterol level at day 28. Absorbed cholesterol is found in the serum partly in the free form whereas the rest is esterified with fatty acids. The data of Table 1 shows that the ratio of the free and esterified cholesterol is correlated with the degree of the response to dietary cholesterol. The free cholesterol level is proportionately higher in the hyporesponding IIIVO/J and WH/J rabbits than in the hyperresponding OS/J and AX/J rabbits. These differences in free/total (F/T) ratio are manifested from the beginning of the experiment (with exception of day 0 on which the levels are probably too low for a reliable calculation of this ratio). A high serum cholesterol level is generally accepted as a predisposing factor
444
in the process of atherosclerosis. In particular, the cholesterol esters are more damaging to the arterial wall than the free cholesterol. Thus, the fact that higher cholesterol levels have extra high esterified levels (Figs. 2 and 3) might be an important observation, because it indicates a synergistic effect in the process of atherosclerosis. For example, we examined the major vessels at necropsy for plaque formations and found that 8 rabbits had plaques in the proximal part of the ascending aorta. As might be expected, these plaques were found in rabbits of strain OS/J (3), AX/J (3), X/J (1) and ACEP/J (1). The response to dietary cholesterol is higher in the rabbit than in man. This is probably caused primarily by differences in the cholesterol absorptive capacity which is higher in the rabbit than in man [35-361. The differences in response within species are probably not caused by differences in the rate of absorption [14-l 51. A possible role of the esterases in controlling the cholesterol level is conceivable and is supported by the work of several authors [ 16201. In our opinion it is quite possible that these hydrolytic enzymes are involved in the regulation of the F/T ratio and thus in the response of the F/T ratio to dietary cholesterol. The results of the comparison between the serum and liver esterase patterns and the susceptibility to cholesterol diets are in agreement with this hypothesis. It is not very likely that the striking correlation of the cholesterol level at day 28 with the esterase patterns is just coincidental. The data of Table 2 suggest that, rather than to an additive inheritance, the substantial differences in response are mainly due to a limited number of major genes. It seems that presence of the most anodal serum esterase zone has a decreasing influence on the serum cholesterol level. Presence of the most anodal liver esterase zone seems to have a more or less increasing effect. However, the limited number of animals tested in this study stresses a careful interpretation. Although no conclusion can be drawn yet, the results of the present study support the view that esterases play an important role in the susceptibility to hypercholesterolemia. We are continuing our studies in order to determine the cholesteryl ester hydrolase activity of the different esterase phenotypes. The availability of inbred strains of rabbits with apparent differences in susceptibility to hypercholesterolemia will be of great value for this study. Acknowledgements This research was conducted during the time Dr. van Zutphen was working as an NIH Fogarty Fellow at the Jackson Laboratory. This investigation was supported in part by NIH Grants F05 TW 2438 from the Fogarty International Center, RR 00251 from the Division of Research Resources, and EY 01408 from the National Eye Institute. Institutional funds were also available from the University of Utrecht. The Jackson Laboratory is fully accredited by the American Association for Accrediation of Laboratory Animal Care. References 1 Ymamoto. R.s.. Crittenden. L.B.. Sokoloff. L. and Jay, G.E.. Genetic variations in Plasma lipid content in mice. J. Lipid Res.. 4 (1963) 413413.
445 2 Bmell, J.H., Additive inheritance of serum cholesterol level in mice. Science, 142 (1963) 1664-1666. 3 Eapen, V.J.. Goswami. O.B. and Pillai. S.U., Inheritance of serum cholesterol and its relation to bodyweight in white mice, J. Genet.. 60 (1971) 222-229. 4 Weibust, R.S., Inheritance of plasma cholesterol levels in mice, Genetics, 73 (1973) 303-312. 5 Roberts, D.C.K. and West, C.E., The inheritability of plasma cholesterol concentration in the rabbit, Heredity, 33 (1974) 347-351. 6 Stufflebean, C.E. and Lasley. F.F.. Heredity basis of serum cholesterol level in beef cattle, J. Hered., 60 (1969) 15-16. 7 Schaeffer, L.E.. Adlersberg, G.D. and Steinbeck, A.G.. Heredity, environment and serum cholesterol, Circulation, 17 (1958) 537-542. 8 Pikkaralnen. J.. Takkunen. J. and Kulonen, E., Serum cholesterol in Finnish twins, Amer. J. Hum. Genet., 18 (1966) 115-126. 9 Kritchevsky. D.. Experimental atherosclerosis. In: R. Raoletti (Ed.), Lipid Pharmacology, Academic Press, New York, 1963. pp. 63-130. 10 Clarkson. T.B., Animal models for atherosclerosis, N.C. Med. J.. 32 (1971) 88-98. 11 Clarkson, T.B., L&and, M.B.. Bullock, B.C. and Goodman, H.O., Genetic control of plasma cholesterol, Arch. Path., 92 (1971) 3745. hypercholesterolemia in 12 Imai. Y. and Matsumura, H.. Genetic studies on induced and spontaneous rats, Atherosclerosis. 18 (1973) 59-64. 13 Roberts, D.C.K., West, C.E.. Redgrave, T.G. and Smith, J.B.. Plasma cholesterol concentration in normal and cholesterol fed rabbits. Its variation and herltablllty. Atherosclerosis, 19 (1974) 369380. 14 Lofland. H.B.. Clarkson. T.B., St. Clair, R.W. and Lehner, N.D.M., Studies on the regulation of plasma cholesterol levels in squirrel monkeys of two genotypes, J. Lipid Res., 13 (1972) 39-47. 15 Adams, C.W., Gamsn. E.M. and Feigenbaum. A.S., Breed differences in the response of rabbits to atherogenic diets, Atherosclerosis, 16 (1972) 405411. 16 Lake, B.D. and Patrick. A.D.. Wolman’s disease: deficiency of EGOO-resistant acid esterase activity with storage of lipids in lysomes. J. Pediat.. 76 (1970) 262-266. 17 Sloan, H.R. and Fredrickson. O.S., Enzyme deficiency in cholesterol ester storage disease. J. Clin. Invest., 51 (1972) 1923-1926. 18 Wolman. M., Acid esterase as a factor in atheromatosls. Atherosclerosis, 20 (1974) 217-223. 19 Stokke. K.T., The existence of acid cholesterol e&erase in human liver, Biochim. Biophys. Acta, 270 (1972) 156-166. 20 Redgrave, T.G., Cholesterol feeding alters the metabolism of thoracicduct lymph lipoprotein cholesterol in rabbits but not in rats. Biochem. J.. 136 (1973) 109-113. 21 Shore, B. and Shore, V.. Rabbits as a model for the study of hyperlipoprotelnemia and atherosclerosis. In: Ch.E. Day (Ed.), Atherosclerosis Drug Discovery, Plenum, New York, 1976, PP. 123-141. 22 Diwan. B.A., Fox, R.R. and Meier, H., Strain differences in arylhydrocarbon hydroxylase induction by 3-methylcholanthrene in rabbits, Proc. Sot. EXP. Biol. Med., 149 (1975) 526-529. 23 Fox, R.R., Handbook on Genetically Standardized JAX Rabbits, Jackson Laboratory, Bar Harbor, Me., 1975. 24 Fox. R.R., Weisbroth, S.H., Crary. D.D. and Scher, S.. Accessory spleens in the domestic rabbit cuniculus). I. Frequency, description and genetic factors, Teratology, 13 (1976) 243(Oryctoicgus 252. 25 Fox, R.R. and Zutphen. L.F.M. van, Strain differences in the prealbumin serum esterases of JAX rabbits, J. Hered., 68 (1977) in press. 26 Hoppe, P.C., Lalrd, C.W. and Fox, R.R., A simple technique for bleeding the rabbit ear vein, Lab. Anim. Care, 19 (1969) 524-525. 27 Lalrd, C.W. and Fox, R.R., Diurnal variations in rabbits: biochemical indicators of thyroid function, Life Sci., 9 (1970) 191-202. 28 Zutphen, L.F.M. van. Serum e&erase genetics in rabbits. I. Phenotypic variation of the prealbumln esterase and classification of atroplnesterase and cocaine&erase, Biochem. Genet.. 12 (1974) 309326. 29 Zutphen. L.F.M. van, Serum e&erase genetics in rabbits. II. Genetic amlvsis of the prealbumin ester ase system, including atroplnesterase and cocainesterase polymorphism. Biochem. Genet., 12 (1974) 327-343. 30 Zutphen. L.F.M. van. Bieman, M.G.C.W. den and Bouw. J.. Serum esterase genetics in rabbits. IV. The prealbumin and 6-globulin systems, Biochem. Genet., 15 (1977) in press. 31 Roschlau, P., Bern& E. and Gruber, W., Enzymatische Bestimmung des Gesamt-Cholesterlns in Serum, Z. Klin. Chem. Klin. Biochem., 12 (1974) 403-416. 32 Fox. R.R.. Taxonomy and genetics. In: S.H. Welsbroth. R.E. Flatt and A.L. Kraus (Eds.), The Biology of the Laboratory Rabbit, Academic Press, New York, 1974, pp. l-22. 33 Redgrave, T.G. and West, C.E.. Differential effect of piperarlne on cholesterol metabolism in male and female rabbits, Au& J. Exp. Biol. Med. Sci., 50 (1972) 153-164.
446 34 Fox. R.R., Schlager, G. and Laird. C.W.. Blood oressure in thirteen strains of rabbits, J. Hered.. 60 (1969) 312-314. 35 Cook, R.P., Kliman. A. and Fieser. L.F., The absorption and metabolism of cholesterol and its main companions in the rabbit - with observations on the atherogenic nature of the sterols. Arch. Biothem.. 62 (1954) 439. 36 Briggs, M.W.. Kritchevsky, D.. Coldman. D.. Gofman. J.W.. Jones, H.B., Lindbergen. F.F.. Hyde, G. and Lyon, T.B.. Observations on the fate of ingested cholesterol in man. Circulation, 6 (1952) 359366.
Atherosclerosis, 28 (1977) 435-446 0 Elsevier/North-Holland Scientific
Publishers,
Ltd.
STRAIN DIFFERENCES IN RESPONSE TO DIETARY CHOLESTEROL JAX RABBITS: CORRELATION WITH ESTERASE PATTERNS
L.F.M. VAN ZUTPHEN
BY
and R.R. FOX
Veterinary Faculty, Department of Animal Genetics, University of Utrecht, Utrecht (The Netherlands) and Jackson Laboratory, Bar Harbor, Me. 04609
(U.S.A.)
(Received 23 May, 1977) (Revised, received 29 August, 1977) (Accepted 2 September, 1977)
Summary Six strains of genetically defined JAX rabbits were tested for their serum cholesterol levels (total and free) in response to a 0.5% cholesterol diet. Marked differences in response between the 6 strains were found. IIIVO/J and WH/J are low responding strains, X/J and ACEP/J are intermediate responding strains, and OS/J and AX/J are high responding strains. After 4 weeks of the cholesterol diet the total serum cholesterol level of the high responding AX/J strain was about 5-fold greater than the level of the low responding IIIVO/J strain. The esterified/total (E/T) ratio appeared to be higher in the high responding strains, indicating a synergistic effect in the process of atherosclerosis. The response of the individual rabbits to the cholesterol diet was compared with the patterns of serum and liver esterase zymograms. This comparison indicated a correlation of the dietary cholesterol susceptibility with the presence or absence of the e&erase zones in the anodal, fast moving region of the gel. Key words:
Atherosclerosis
- Cholesterol
- Esterase -Rabbit
Introduction Additive inheritance of serum cholesterol levels has been reported for several mammals, including man [l-8]. The susceptibility to dietary induced hypercholesterolemia is different in various species of mammals and in man [9,10]. Also within a given species differences can occur. These differences have led to the introduction of the terms hyper- and hyporesponders. Influences of genetic factors on the susceptibility to dietary cholesterol are reported for several
436
mammalian species [ll-131. In the squirrel monkey the mechanism for genetic control of hypercholesterolemia seems to depend on the conversion of cholesterol to bile acids [ 141. The physiological basis for the differences in response to dietary cholesterol is still unknown for the other species. Adams et al. [15] found no evidence for a greater excretion of cholesterol in the hyporesponding rabbit. Both Wolman’s disease and cholesterol ester storage disease are believed to be caused by deficiency of acid esterase activity [16,17]. Esterase activity is considered to be a factor in atherosclerosis [ 181. After absorption, most of the dietary cholesterol is being esterified with fatty acids. Studies of Stokke [19] and Redgrave [20] have suggested that hydrolysis of these cholesterol esters by cholesterolesterase can be considered as an important process in the regulation of mammalian cholesterol metabolism. These data emphasize the possible role of the esterases in hypercholesterolemia and atherosclerosis. Differences in esterase zymogram patterns might therefore be correlated with differences in susceptibility to dietary cholesterol and atherosclerosis. A preliminary study of van Zutphen and den Bieman (unpublished) comparing the response to dietary cholesterol with the prealbumin esterase phenotype of the rabbit indicated that such a correlation might exist. The rabbit is frequently used as a model in studies on hypercholesterolemia and atherosclerosis. The value of this animal for these studies has recently been emphasized by Shore and Shore [21]. The availability of inbred strains of rabbits can greatly facilitate these studies, in particular the search for the genetic factors which are involved. Several inbred and partly inbred strains of JAX rabbits [22-241 have recently been characterized for the prealbumin serum esterase phenotypes [25]. Because of this unique availability, we tested several of these strains for dietary cholesterol susceptibility and compared their different susceptibilities with the esterase patterns in both serum and liver. Materials and methods Animals Five young adult healthy male rabbits from each of 6 strains (IIIVO/J, AX/J, OS/J, ACEP/J, WH/J and X/J) from the Jackson Laboratory colony were used [22-251. The inbreeding coefficients for these strains are approximately as follows: IIIVO/J, 0.97; WH/J, 0.72; X/J, 0.88; ACEP/J, 0.92; OS/J, 0.85; AX/J, 0.73. The ages of the rabbits in each strain at the start of the experiment were similar: e.g., IIIVO/J, 122.0 + 4.5; ACEP/J, 119.6 f 5.0; AX/J, 131.8 + 6.6; WH/J, 129.6 f 7.7; OS/J, 136.6 + 10.7; and X/J, 124.6 f 9.0 days. The rabbit room was on a 13-h light-11-h dark cycle. The light period was from 4:30 a.m. to 5:30 p.m. The temperature range in the rabbit room was 13--18°C with a mode value of 15.5”C. Prior to the experiment the rabbits had a lo-day acclimatization period. Feeding Before and during the acclimatization period all rabbits were fed Ralston Purina Rabbit Chow Checkers ad libitum. After acclimatization all rabbits
437
were fed the same food which was fortified with 0.5% pure cholesterol (Sigma) by Purina (5838C6F6 - lot 06995) for 28 days at a rate of 40 g/kg of body weight/day. With exception of the day of bleeding, all feeding was done each day at 8:00 a.m. On the day of bleeding rabbits were fed immediately after bleeding. During the acclimatization period and after bleeding on day 28 the rabbits were fed regular Purina Chow Checkers 20 (5325) also at a rate of 40 g/kg of body weight/day. All rabbits were weighed once a week. By error the feeding on day 12 was omitted. Bleeding All rabbits were bled on day 0, 7,14, 21, 28, 35 and 49, just prior to feeding. Day 0 indicates the first day of the cholesterol diet after the acclimatization period. Bleeding on day 0 preceded the administration of the cholesterol diet. All but terminal bleedings were performed according to Hoppe et al. [26] between 8:00 a.m. and 10:00 a.m. to minimize diurnal variation in cholesterol levels [27] with the order randomized each day. Terminal bleeding on day 49 was performed by cardiac puncture between 8:00 a.m. and 1l:OO a.m. Processing of blood Blood was allowed to clot at room temperature for 30 min, then was refrigerated at 5°C for 30-60 min and centrifuged at 1100 X g. Serum was obtained and aliquoted into two samples. One sample was frozen (-60°C) and the other assayed immediately for total and free cholesterol levels. Au topsy After bleeding on day 49 rabbits were killed by cervical dislocation. Samples of liver, kidney, pancreas and ileum were obtained and stored at -60°C for further study. All animals were necropsied and in particular the great vessels were checked grossly for atherosclerotic plaques. When plaques were observed, samples of the vessels were fixed in Bouin’s fixative, sectioned at 12 pm, and stained with H-E for histological confirmation by Dr. Hans Meier, Senior Staff Scientist, Jackson Laboratory. Determination of es terase phenotypes The esterase patterns of the serum were obtained after electrophoresis according to van Zutphen [28-301. Zymograms for prealbumin esterase loci Est-I, Est-2 and Est-3 and for the P-globulin esterase patterns were determined. The latter are analogous to the patterns of the red cell locus (Es-l). After autopsy of the animals on day 49 part of the liver of each rabbit was homogenized with equal volumes of distilled water, centrifuged at 35,000 X g after freezing and thawing, the esterase zymogram was then determined using the supematant according to the procedures of van Zutphen [28-301. Cholesterol determination Total and free cholesterol was measured in the serum on days 0, 7, 14, 21, 28, 35 and 49 using the enzymatic method described by Roschlau et al. [31]. The free cholesterol is transformed to A4-cholestenone and hydrogen peroxide by cholesteroloxidase. In the presence of catalase hydrogen peroxide oxidizes
438
methanol to formaldehyde which reacts with ammonium ions and acetylacetone to 3,5diacetyl-1,4-dihydrolutidine. The intensity of the yellow color of this product was measured on a Gilford 300 spectrophotometer at 410 nm. For determination of total cholesterol, all cholesterol esters in the serum are hydrolyzed by cholesterol e&erase. All chemicals for total and free cholesterol determinations were obtained as a test combination set from Boehringer, Mannheim. Results The dietary level of 40 g/kg body weight was a maintenance diet but did allow a slight weight gain, e.g., from day 0 to day 28 the average gain per rabbit was 65 g and no change in weight was observed from day 28 to day 49. The data of the individual total and free serum cholesterol levels for each of the bleeding days were summarized by strain, and means and standard errors were calculated. Table 1 gives these mean values + SE for each of the 6 strains. These results indicate marked differences in response to dietary cholesterol between the strains. This may be illustrated by comparing the increase in total cholesterol from day 0 until 28. This increase was about 1100% in the hyporesponding IIIVO/J strain and about 7000% in the hyperresponding AX/J strain. Fig. 1 represents a graphical illustration of the differences between the strains. Twenty-eight days is obviously too short a time for reaching a steadystate level when feeding a 0.5% cholesterol diet. The cholesterol levels in the high responding strains are still rising sharply after 4 weeks of feeding the cholesterol diet. Because we were primarily interested in differences in the initial response of the serum level to dietary cholesterol, the cholesterol diet was stopped at day 28. Fig. 2 demonstrates diagrammatically the individual levels of total and free cholesterol on the final day of the cholesterol diet (day 28). The variation within the strains might be due to the fact that these strains are not yet completely inbred. As can be seen from Fig. 2 the animals with the high total cholesterol levels have relatively lower free cholesterol levels. Consequently, the higher the total cholesterol level is, the higher the esterified/ total (E/T) ratio. A high positive correlation coefficient of 0.95 (y = 0.00016x + 0.36) was calculated for the total cholesterol levels and the E/T ratio at day 28. The increasing E/T ratio is illustrated for each of the strains in Fig. 3. The slightly lower ratios on day 14 may be due to the fact that, by error, the rabbits were not fed on day 12. It is obvious that the strains with the high cholesterol levels have higher E/T ratios than the strains with the lower total cholesterol levels. The total cholesterol levels of the individual rabbits at day 28 were compared with the esterase zymograms of serum and liver. A correlation with the e&erase zones in the anodal, fast moving region of the gel might exist (Table 2). The presence or absence of the most anodal serum esterase zone is genetically determined and is controlled by alleles of the Es&2 locus [30], previously called the As locus of linkage group VI [32]. There are two forms of the anodal serum e&erase zone. One stains very intensively with cr-naphthylacetate as a substrate. This zone has been indicated as F’. The other zone (f’) which has the same RI value, stains rather faint. Either the F’ or the f’ zone was
Total cholesterol
IIIVO/J
=
Totalcholesterol Free cholesterol F/Tratio
TotaI cholesterol Free cholesterol FITratio
Total cholesterol Free cholesterol F/Tratio
Total cholesterol Free cholesterol FITratio
X/J
ACEP/J
OS/J
AX/J
5 5 5
5 5 5
5 5 5
5 5 5
5 5 5
5 5 5
n
2.1 1.3
1.7 1.3
22.0 f 4.1 18.4 fi 4.0 0.84
32.2 r 2.0 22.8 k 0.6 0.71
28.4 f 20.6 f 0.13
36.4 k 13.4 25.8 + 1.4 0.71
12.0 t 1.5 9.2 r 1.3 0.77
28.8 f 22.6 f 0.78
0a
Day
340.4 f 38.0 147.4 t 19.9 0.43
326.4 f 40.4 131.6 f 15.2 0.40
233.6 f 37.1 95.2 f 10.0 0.41
247.6 + 61.6 111.0 f 19.8 0.45
150.6 k 21.2 71.8 + 5.5 0.48
146.6 + 19.5 72.4 + 5.2 0.49
7
31.8 11.1
728.4 i 134.9 304.6 + 41.2 0.42
668.6 + 163.8 292.0 i: 53.0 0.44
308.8 t 31.6 148.6 f 11.1 0.48
301.4 k 73.1 166.6 f 36.1 0.54
21
24.4 11.9
928.2 + 193.4 410.2 f 48.2 0.44
903.4 + 198.3 407.4 ? 62.4 0.45
359.0 f 207.0 f 0.58
319.2 t 58.6 195.8 + 31.6 0.61
28b
922.0 * 155.3 1296.8 k 118.3 1530.6 k 108.7 386.0 k 49.2 492.0 f 36.4 611.6 f 39.1 0.42 0.40 0.38
1011.8 f 150.8 1135.6 + 183.8 1408.8 f 215.6 389.0 + 32.7 431.6 + 53.4 572.4 f. 58.3 0.38 0.38 0.41
429.6 t 112.1 226.0 + 32.1 0.53
632.4 f 154.1 308.6 2 56.4 0.49
241.2 k 145.4 ? 0.59
223.6 + 60.9 130.0 f 33.1 0.58
14
28DAYSOFA0.59bCHOLESTEROLDIET
aDay OleveIsamprecholesteroldietleveIs. Alllevelson aIIdaysarebasedonsemm collectedbefore feedkztbatday. diet stopped on day 28 and normal diet(same butwithoutcholesterol)resumed. b0.5% cholesterolin C Totaland freeserumcholesterolvaluesgiveninmg/100mlserum.
Totalcholesterol Freecholesterol F/Tratio
WI-I/J
Free cbolesterolC F/Tratio
Parameter
Strain
SERUMCHOLESTEROLLEVELSIN6STRAINSOFJAXRABBITSHAVING
TABLE1
8.1 7.2
23.4 19.1
798.6 + 108.5 429.0 + 37.1 0.54
795.8 + 151.8 399.6 f 53.4 0.50
393.6 k 94.3 242.0 r 45.0 0.61
330.4 f 101.2 214.8 k 51.8 0.65
89.4 f 68.6 r 0.17
130.8 + 100.0 f 0.76
35
13.6 10.2
12.4 10.2
71.3 21.6
50.1 18.8
301.2 f 108.8 158.4 f 49.1 0.53
392.2 2 198.4 f 0.51
253.2 f 153.6 f 0.61
141.4 r 18.8 91.0 f 43.3 0.62
49.4 f 36.8 f 0.14
72.4 + 55.8 + 0.77
49
0
14
7
21
DAYS
.28
35
49
Fig. 1. Graphical demonstration of the response in serum total cholesterol levels of 6 strains of JAX rabbits on feeding a 0.5% cholesterol diet for 28 days followed by 21 days of the normal diet. Five rabbits were used Per strain.
ElFREE I
III VO/J
ESTERIFIED
WH/J
X/J
AMP/J
OS/J
STRAIN Fig. 2. Strain differences and individual variation of feeding a 0.5% cholesterol diet.
of serum cholesterol
levels (free and total)
after 28 days
441
_ _ _ _ ACE”/J
0
7
14
21 DAYS
28
35
49
Fig. 3. Esterified/total cholesterol ratio in the serum of rabbits of 6 JAX strains, fed a 0.5% cholesterol diet from day 0 until day 26 followed by 21 days of normal diet.
Fig. 4. Zymogram of rabbit liver esterases. The two phenotypes the anodal zone a. am demonstrated.
A and B. respectively,
with and without
442
present in the rabbits of strains IIIVO/J (phenotypes V and VI), WH/J (phenotypes IIIF’ and VF’) and ACEP/J (phenotype VF’) and in 3 rabbits of strain X/J (phenotype III). All of the other rabbits were missing such a zone (phenotypes II and Vf). Although the rabbits of strain WH/J are classified as IIIF’ or VF’, which means that all these rabbits possessed the most intensive stained anodal F’ zone, we had some problems with phenotyping this strain, because of the fact that in this strain the anodal zone stained somewhat less than F’, but more intense than f’. The genetic basis for the presence or absence of the anodal liver esterase zone has not yet been established. Two different phenotypes A and B were found. An anodal, fast moving zone (a) is present in phenotype A and absent in B (Fig. 4). The intensity of this zone varied but, since we do not know TABLE 2 COMPARISON OF THE SERUM AND LIVER ESTERASE PHENOTYPES OF 30 JAX RABBITS WITH THE TOTAL CHOLESTEROL LEVELS FOLLOWING 28 DAYS OF FEEDING A 0.5% CHOLESTEROL DIET Strain
Serum esterase phenotype
Liver esterase phenotype
Anodal 8enlm esterase
Anodal liver esterme
zone (F’ or f’)
zone (a)
Total chol. (IIxglOO ml)
IIIVO/J
VI VI VI VI VI
B B B B B
+ + + + +
-
228 248 266 306 648
WH/J
vF’ vF’ IIIF’ IIIF’ vF’
B B B A A
+ + + + +
-
303 303 365 409 415
III III III II II
A A A A A
+ + + -
+ + + +a +a
463 601 695 1329 1429
B B B B A
+ + + + +
-
653 676 799 961 1663
-
-
Vf Vf Vf Vf
B A A A A
+ + + +
814 1023 1573 1634 2000
II II II Vf vf
A A A A A
-
+ + + + +
1223 1344 1586 1689 1811
X/J
ACEP/J
;:; $1 vF’
OS/J
AX/J
vf
+ +
+
a The anodial liver esterase zone of these samples were more distinct than normal.
443
whether this is a genetically determined character, all zymograms which showed the anodal zone were indicated as A and those in which the anodal zone was missing as B. Comparing the presence or absence of these fast moving zones of liver and serum with the cholesterol levels at day 28, it was most striking that absence of the anodal zone in the serum and presence of such a zone in the liver are correlated with relatively high serum cholesterol levels, whereas these levels are lower when the anodal zone is present in the serum and absent in the liver (Table 2). Discussion The differences in susceptibility for dietary cholesterol between the strains found in this study are more pronounced than expected, based on the data of Adams et al. [15]. They have shown that the New Zealand White and the Dutch breeds differ in their susceptibility for dietary cholesterol. After 4 weeks of a 0.5% cholesterol diet the male rabbits of the New Zealand White breed had a serum cholesterol level which was about 50% higher than that of the Dutch breed. Our results indicate that after 4 weeks of a similar regimen the male rabbits of the high responding AX/J strain have a serum cholesterol level that is about 5-fold greater than the level of the lowest responding IIIVO/J strain. The availability of high responding and low responding inbred strains of rabbits can be of great value in the research on hypercholesterolemia and atherosclerosis. From Fig. 1 it is obvious that the period of 28 days is too short for reaching the steady-state level. According to Redgrave and West [33] cholesterol levels reach a plateau only after 5-7 weeks of a cholesterol diet. The concentration at that time will be about 50% greater than the concentration at 3 weeks. Their findings could be used for extrapolation of our data as an indication of what the various steady-state levels of total serum cholesterol would have been if the cholesterol diet was continued after 28 days. Fig. 1 shows clearly that we have 2 hyporesponding strains (IIIVO/J and WH/J), 2 intermediate strains (X/J and ACEP/J) and 2 hyperresponding strains (OS/J and AX/J). These differences are not associated with body weight or general activity level. The highest responding AX/J strain and the lowest responding IIIVO/J strain are both large strains, whereas all other strains are small. Also AX/J and IIIVO/J are rather docile, ACEP/J and X/J are very hyperactive and the other 2 strains are intermediate. Blood pressure values for the respective strains [ 341, either systolic or diastolic, appeared not to be correlated with the cholesterol level at day 28. Absorbed cholesterol is found in the serum partly in the free form whereas the rest is esterified with fatty acids. The data of Table 1 shows that the ratio of the free and esterified cholesterol is correlated with the degree of the response to dietary cholesterol. The free cholesterol level is proportionately higher in the hyporesponding IIIVO/J and WH/J rabbits than in the hyperresponding OS/J and AX/J rabbits. These differences in free/total (F/T) ratio are manifested from the beginning of the experiment (with exception of day 0 on which the levels are probably too low for a reliable calculation of this ratio). A high serum cholesterol level is generally accepted as a predisposing factor
444
in the process of atherosclerosis. In particular, the cholesterol esters are more damaging to the arterial wall than the free cholesterol. Thus, the fact that higher cholesterol levels have extra high esterified levels (Figs. 2 and 3) might be an important observation, because it indicates a synergistic effect in the process of atherosclerosis. For example, we examined the major vessels at necropsy for plaque formations and found that 8 rabbits had plaques in the proximal part of the ascending aorta. As might be expected, these plaques were found in rabbits of strain OS/J (3), AX/J (3), X/J (1) and ACEP/J (1). The response to dietary cholesterol is higher in the rabbit than in man. This is probably caused primarily by differences in the cholesterol absorptive capacity which is higher in the rabbit than in man [35-361. The differences in response within species are probably not caused by differences in the rate of absorption [14-l 51. A possible role of the esterases in controlling the cholesterol level is conceivable and is supported by the work of several authors [ 16201. In our opinion it is quite possible that these hydrolytic enzymes are involved in the regulation of the F/T ratio and thus in the response of the F/T ratio to dietary cholesterol. The results of the comparison between the serum and liver esterase patterns and the susceptibility to cholesterol diets are in agreement with this hypothesis. It is not very likely that the striking correlation of the cholesterol level at day 28 with the esterase patterns is just coincidental. The data of Table 2 suggest that, rather than to an additive inheritance, the substantial differences in response are mainly due to a limited number of major genes. It seems that presence of the most anodal serum esterase zone has a decreasing influence on the serum cholesterol level. Presence of the most anodal liver esterase zone seems to have a more or less increasing effect. However, the limited number of animals tested in this study stresses a careful interpretation. Although no conclusion can be drawn yet, the results of the present study support the view that esterases play an important role in the susceptibility to hypercholesterolemia. We are continuing our studies in order to determine the cholesteryl ester hydrolase activity of the different esterase phenotypes. The availability of inbred strains of rabbits with apparent differences in susceptibility to hypercholesterolemia will be of great value for this study. Acknowledgements This research was conducted during the time Dr. van Zutphen was working as an NIH Fogarty Fellow at the Jackson Laboratory. This investigation was supported in part by NIH Grants F05 TW 2438 from the Fogarty International Center, RR 00251 from the Division of Research Resources, and EY 01408 from the National Eye Institute. Institutional funds were also available from the University of Utrecht. The Jackson Laboratory is fully accredited by the American Association for Accrediation of Laboratory Animal Care. References 1 Ymamoto. R.s.. Crittenden. L.B.. Sokoloff. L. and Jay, G.E.. Genetic variations in Plasma lipid content in mice. J. Lipid Res.. 4 (1963) 413413.
445 2 Bmell, J.H., Additive inheritance of serum cholesterol level in mice. Science, 142 (1963) 1664-1666. 3 Eapen, V.J.. Goswami. O.B. and Pillai. S.U., Inheritance of serum cholesterol and its relation to bodyweight in white mice, J. Genet.. 60 (1971) 222-229. 4 Weibust, R.S., Inheritance of plasma cholesterol levels in mice, Genetics, 73 (1973) 303-312. 5 Roberts, D.C.K. and West, C.E., The inheritability of plasma cholesterol concentration in the rabbit, Heredity, 33 (1974) 347-351. 6 Stufflebean, C.E. and Lasley. F.F.. Heredity basis of serum cholesterol level in beef cattle, J. Hered., 60 (1969) 15-16. 7 Schaeffer, L.E.. Adlersberg, G.D. and Steinbeck, A.G.. Heredity, environment and serum cholesterol, Circulation, 17 (1958) 537-542. 8 Pikkaralnen. J.. Takkunen. J. and Kulonen, E., Serum cholesterol in Finnish twins, Amer. J. Hum. Genet., 18 (1966) 115-126. 9 Kritchevsky. D.. Experimental atherosclerosis. In: R. Raoletti (Ed.), Lipid Pharmacology, Academic Press, New York, 1963. pp. 63-130. 10 Clarkson. T.B., Animal models for atherosclerosis, N.C. Med. J.. 32 (1971) 88-98. 11 Clarkson, T.B., L&and, M.B.. Bullock, B.C. and Goodman, H.O., Genetic control of plasma cholesterol, Arch. Path., 92 (1971) 3745. hypercholesterolemia in 12 Imai. Y. and Matsumura, H.. Genetic studies on induced and spontaneous rats, Atherosclerosis. 18 (1973) 59-64. 13 Roberts, D.C.K., West, C.E.. Redgrave, T.G. and Smith, J.B.. Plasma cholesterol concentration in normal and cholesterol fed rabbits. Its variation and herltablllty. Atherosclerosis, 19 (1974) 369380. 14 Lofland. H.B.. Clarkson. T.B., St. Clair, R.W. and Lehner, N.D.M., Studies on the regulation of plasma cholesterol levels in squirrel monkeys of two genotypes, J. Lipid Res., 13 (1972) 39-47. 15 Adams, C.W., Gamsn. E.M. and Feigenbaum. A.S., Breed differences in the response of rabbits to atherogenic diets, Atherosclerosis, 16 (1972) 405411. 16 Lake, B.D. and Patrick. A.D.. Wolman’s disease: deficiency of EGOO-resistant acid esterase activity with storage of lipids in lysomes. J. Pediat.. 76 (1970) 262-266. 17 Sloan, H.R. and Fredrickson. O.S., Enzyme deficiency in cholesterol ester storage disease. J. Clin. Invest., 51 (1972) 1923-1926. 18 Wolman. M., Acid esterase as a factor in atheromatosls. Atherosclerosis, 20 (1974) 217-223. 19 Stokke. K.T., The existence of acid cholesterol e&erase in human liver, Biochim. Biophys. Acta, 270 (1972) 156-166. 20 Redgrave, T.G., Cholesterol feeding alters the metabolism of thoracicduct lymph lipoprotein cholesterol in rabbits but not in rats. Biochem. J.. 136 (1973) 109-113. 21 Shore, B. and Shore, V.. Rabbits as a model for the study of hyperlipoprotelnemia and atherosclerosis. In: Ch.E. Day (Ed.), Atherosclerosis Drug Discovery, Plenum, New York, 1976, PP. 123-141. 22 Diwan. B.A., Fox, R.R. and Meier, H., Strain differences in arylhydrocarbon hydroxylase induction by 3-methylcholanthrene in rabbits, Proc. Sot. EXP. Biol. Med., 149 (1975) 526-529. 23 Fox, R.R., Handbook on Genetically Standardized JAX Rabbits, Jackson Laboratory, Bar Harbor, Me., 1975. 24 Fox. R.R., Weisbroth, S.H., Crary. D.D. and Scher, S.. Accessory spleens in the domestic rabbit cuniculus). I. Frequency, description and genetic factors, Teratology, 13 (1976) 243(Oryctoicgus 252. 25 Fox, R.R. and Zutphen. L.F.M. van, Strain differences in the prealbumin serum esterases of JAX rabbits, J. Hered., 68 (1977) in press. 26 Hoppe, P.C., Lalrd, C.W. and Fox, R.R., A simple technique for bleeding the rabbit ear vein, Lab. Anim. Care, 19 (1969) 524-525. 27 Lalrd, C.W. and Fox, R.R., Diurnal variations in rabbits: biochemical indicators of thyroid function, Life Sci., 9 (1970) 191-202. 28 Zutphen, L.F.M. van. Serum e&erase genetics in rabbits. I. Phenotypic variation of the prealbumln esterase and classification of atroplnesterase and cocaine&erase, Biochem. Genet.. 12 (1974) 309326. 29 Zutphen. L.F.M. van, Serum e&erase genetics in rabbits. II. Genetic amlvsis of the prealbumin ester ase system, including atroplnesterase and cocainesterase polymorphism. Biochem. Genet., 12 (1974) 327-343. 30 Zutphen. L.F.M. van. Bieman, M.G.C.W. den and Bouw. J.. Serum esterase genetics in rabbits. IV. The prealbumin and 6-globulin systems, Biochem. Genet., 15 (1977) in press. 31 Roschlau, P., Bern& E. and Gruber, W., Enzymatische Bestimmung des Gesamt-Cholesterlns in Serum, Z. Klin. Chem. Klin. Biochem., 12 (1974) 403-416. 32 Fox. R.R.. Taxonomy and genetics. In: S.H. Welsbroth. R.E. Flatt and A.L. Kraus (Eds.), The Biology of the Laboratory Rabbit, Academic Press, New York, 1974, pp. l-22. 33 Redgrave, T.G. and West, C.E.. Differential effect of piperarlne on cholesterol metabolism in male and female rabbits, Au& J. Exp. Biol. Med. Sci., 50 (1972) 153-164.
446 34 Fox. R.R., Schlager, G. and Laird. C.W.. Blood oressure in thirteen strains of rabbits, J. Hered.. 60 (1969) 312-314. 35 Cook, R.P., Kliman. A. and Fieser. L.F., The absorption and metabolism of cholesterol and its main companions in the rabbit - with observations on the atherogenic nature of the sterols. Arch. Biothem.. 62 (1954) 439. 36 Briggs, M.W.. Kritchevsky, D.. Coldman. D.. Gofman. J.W.. Jones, H.B., Lindbergen. F.F.. Hyde, G. and Lyon, T.B.. Observations on the fate of ingested cholesterol in man. Circulation, 6 (1952) 359366.