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Serum and biliary lipid pattern in rabbits feeding a diet enriched with unsaturated fatty acids
University Institute of Clinical Medicine, Universita Cattolica del Sacro Cuore, Rome, Italy
Serum and biliary lipid pattern in rabbits feeding a diet enriched with unsaturated fatty acids By A. V. GRECO and G. MIN GRONE With 5 figures
Address for correspondence: Prof. Dr. ALDo V. GRECO, Istituto di Clinica Medica, Universita Cattolica del Sacro Cuore, Largo Gemelli 8,1-00168 Roma, Italia
Keywords: lipid, serum; lipid, biliary; lipoperoxidation; atherosclerosis; cholesterol esters; triglycerides; phospholipids; glutathione peroxidase; superoxide dismutase; catalase
Summary Adult male New Zealand white rabbits were fed for 3 months a stock diet supplemented with 6% (w/w) soybean oil heated at 240°C for 60 min. After the first month of treatment a significant increase in total lipid content of serum was observed mainly due to the cholesterol ester fraction. Simultaneously, grossly induced atherosclerosis and marked liver damage were histologically and clinically demonstrated. Lipid peroxide values, performed by thiobarbituric acid test in lipid extracts from liver, aorta and bile showed a significant increase as compared to controls. Lipoperoxidation rate increased with the duration of feeding. Parallel to this there was a marked reduction in the activities of glutathione peroxidase, superoxide dismutase and catalase in liver and aorta, all enzymes involved in the mechanism of detoxification of lipid peroxides. The results are in agreement with the hypothesis that lipid peroxidation can playa significant role in the pathogenesis of atherosclerosis.
Introduction Cholesterol feeding of laboratory animals, mainly rabbits, has been regarded as a reliable method for inducing vascular changes which resemble human atheromatosis (WANG et al. 1957). KRITCHEVSKY etal. (1961, 1965, 1967) found that when an oil was added to cholesterol, formation of atheromata became more marked than in the absence of oil, especially if the oil was first heated at 200°C for 15 min before added to the diet. It was suggested that the effect might be related to the increase in fatty acids that occurs by heating the oil. On the other hand, atheromatosis quite similar to that produced by cholesterol feeding, can be induced in rabbits by semisynthetic diet which contained saturated triacylglycerol as the only source offat, i.e., 8,42% hydrogenated coconut oil or 8.42% glyceril-trilaurate (WIGAND 1959; STORMBY and WIGAND 1963). These studies agreed with the hypothesis that diet rich in polyunsaturated fatty acids depresses serum cholesterol levels in hypercholesterolemic patients, so reducing the risks of coronary heart disease (KANNEL et al. 1961; OLIVER and BOYD 1961). The mechanism whereby polysaturated fatty acids in the diet lower plasma lipid levels is not clear. In many individuals the change from saturated to polyunsaturated fat enhances fecal excretion of cholesterol and its catabolites, i.e. bile acids, and thus creates a more negative cholesterol
2*
Exp. Pathol. 40 (1990) 1
19
balance (CONNOR etal. 1969; GRUNDY 1975; MOORE etat. 1968; NESTEL eta1.1975) with a concomitant lowering of plasma cholesterol levels. On the other hand, diets rich in polyunsaturated fatty acids can lead to enhanced formation of cholesterol gallstones or to be a stimulus to carcinogenesis, perhaps in part due to the accumulation of peroxides (STURDEVANT et al. 1973; PEARCE and DAYTON 1971; EDERER etal. 1971). Lipoperoxidation may play an important role in the pathogenesis of atherosclerosis, not only in experimental animals. In fact, LOEPER et al. (1983) demonstrated that plasma of patients with arterial injury exhibited a significant increase in malondialdehyde, indicating a peroxidation of unsaturated fatty acids. In the present paper, rabbits were fed for 3 months a stock diet supplemented with 6 % soybean oil heated at 240°C for 60 min (KRITCHEVSKY modified diet). Experiments were performed in order to investigate: 1) the rate of atherosclerotic lesions and the toxicity of this diet; 2) the effects of unsaturated free fatty acids feeding on lipid peroxidation of liver, bile serum and aorta, and on liver and aortic levels of glutathione peroxide, catalase and superoxide dismutase; 3) the effects of unsaturated fatty acids feeding on lipid composition of both serum and bile, including bile acid analysis.
Materials and Methods Animals and diet
Fifteen adult male New Zealand white rabbits, 3 months old and weighing approximately 1,600 g, were alloted to 3 groups of 5 and fed a stock diet supplemented with 6 % free fatty acids (FFA) from saponified soybean oil. Percentage composition of added unsaturated free fatty acids was: 0.98% C 16:0,2.5% C 18:0,28.1 % C 18:1,62.9% C 18:2,2.6% C 18:3 andO.7% others. Since the stock diet contained 4.1 % (w/w) fats (9% cholesterol, 69% trig1ycerides, 2% sterol esters, 8% free fatty acids and 12% polar lipids) the total fat content of the new diet was 10.1 %. Fifteen male New Zealand white rabbits of the same age and weight, separated into 3 groups, were used as controls and fed the stock diet without heated soybean oil supplement. Since data regarding this control class were similar, we grouped and expressed them as mean ± SD of 15 determinations. Lipid analysis
Extraction: 1 ml of both serum and bile, and 0.5 g of both homogenized fresh liver and aortic tissues, acidified at pH 2.5-3.0 with 0.1 N HC1, were extracted 3 times with 10 m1 of CHC1 3 -CH30H (2: 1, v/v). Pooled extracts were dried over anhydrous NaZS04 and lipids recovered by evaporation of solvents under nitrogen stream. Percentage composition of lipid extracts was performed by thin-layer chromatography (TLC) according to PASSI etal. (1981). Fatty acid analysis
Fatty acids from phospholipids (PL), triglycerides (TG), cholesterol esters (CE) and free fatty acids (FFA) fractions on thin-layer plate were analyzed by reversed phase high-performance liquid chromatography (RP-HPLC) according to the method described by PASSI etal. (1981). Analysis of cholesterol
Cholesterol as trimethyl-silyloxy derivative was analyzed by gas-liquid chromatography (GLC) as proposed by PASSI etal. (1981).
20
Exp. Pathol. 40 (1990) 1
Analysis of bile salts Individual conjugated bile salts were measured in gallbladder bile according to MINGRONE etal. (1980).
Lipid peroxidation The determination of the rate of lipid peroxidation was indirectly evaluated by thiobarbituric acid test (TBA test) on lipid extracts from serum, bile, liver and aorta according to WILLS (1966).
Enzymes Catalase, superoxide dismutase and glutathione peroxidase in both liver or aortic supernatant fractions were assayed according to TSAI and KELLEY (1978).
Morphologic techniques (light microscopy) The organs were placed in a solution of 10% formalin and fixed overnight. The tissues were dehydrated and embedded in paraffin in routine manners. Sections were stained with hematoxylin-eosin. 3000
..,
...::t
2000
o
--------IlI--------_Il I----_____
w ~
1000
o
o
30
eo
~
eo
DAYS Fig. 1. Body weight diagram of rabbits fed either basal . - - . or unsaturated fatty acids supplemented diet 0- - -D. The values are expressed as mean ± SD.
Results The body weight diagram of rabbits feeding unsaturated fatty acid (UFA) enriched diet or normal diet (controls) is represented in fig. 1. This figure shows a highly significant decrease of body weight in treated animals as compared to controls. Starting from the first month of treatment atheromatous lesions were found (fig. 2). In rabbits killed after 30 d the livers were enlarged, abnormally pale and firmer than normal. The histological examination revealed a fat deposition in the reticulo-endothelial system (Kupffer cells) occurring in combination with a moderately coarse or fine degeneration of the cells of liver parenchyma, particularly in the peripheral parts of the lobules (fig. 3). The liver surface in the second group of animals killed at day 60 was dark-yellow and hardly to be cut. After 90 d of treatment 3 of 5 rabbits died, indicating a high toxicity of UFA enriched diet. The autopsy showed in all cases the presence of relevant volumes of ascitic fluid Exp. Pathol. 40 (1990) 1
21
Fig. 2. Atheromatous lesions characterized by the presence of numerous foamy cells in the intima with disruption of the internal elastic lamina. Hematoxylin eosin X 100 (original magnification).
Fig.3. Fatty degeneration of the hepatocytes is more marked in the centrolobular area of liver parenchyma (from rabbits killed after 30 days of treatment) . Hematoxylin eosin x 40 (original magnification) . 22
Exp. Pathol. 40 (1990) I
Table 1. Lipid composition (%) of bile and serum of rabbits fed basal diet or unsaturated fatty acids enriched diet for 30, 60 and 90 days. Polar lipids contain phospholipids, bile acids, glycolipids and other fractions with that the solvent system used to remain at the origin of thinlayer plates. Serum
Polar lipids
Cholesterol
Controls 30 days 60 days 90 days
63.69 20.50 14.37 13.97
± 4.05 ± 3.16± 2.52-· ± 2.62-·
19.39 13.75 15.70 14.97
± 2.77 ± 2.67± 2.70 ± 1.48-
3.00 1.94 2.19 2.27
± 0.95 ± 0.61 ± 0.72 ± 0.75
5.66 5.16 9.84 15.34
Bile Controls 30 days 60 days 90 days
71.44 68.06 69.03 68.30
± 4.40 ± 3.90 ± 2.99 ± 2.79
16.03 17.89 16.67 16.36
± 2.73 ± 1.49 ± 1.38 ± 1.41
2.44 3.52 3.67 3.96
± 0.85 ± 0.62 ± 0.70 ± 0.60
tr 1.81 2.35 2.53
Free fatty acids
Triglycerides
Chloesterol esters
± 1.66 tr ± 1.12 50.65 ± 7.54 ± 1.28-· 57.27 ± 3.01 ± 2.14-·*52.64 ± 2.34
± 0.47 ± 0.34 ± 0.50
tr tr tr tr
The values are expressed as mean ± SD. Statistical significance (Student's t-test, 0.05 < P >0.001): - treated animals vs controls; • 60 days and 90 days vs 30 days; * 90 days vs 60 days.
(10-15 ml) and pronounced liver changes, with a microscopical aspect quite similar to that found in the 2nd group. Table 1 shows the lipid composition (%) of serum and bile of rabbits fed basal diet or UFA enriched diet for 1, 2 and 3 months. The serum polar lipids, which are a heterogeneous class and principally represented by phospholipids, significantly decrease with time in the animals fed a UFA enriched diet reaching to a percentage value of 13.97 ± 2.62% at 90 days vs. 63.69 ± 4.05% found in the controls. Simultaneously, a relevant increase of cholesterol esters, up to a maximum of about 5 times over basal values at 90 d, and of triglycerides, about 3 times over basal values, is observed during treatment. On the other hand, the percentage composition of biliary lipids remained essentially unchanged during the entire period of study. Figs. 4a and 4b show the levels of total fatty acids present in triglycerides, free fatty acids, phospholipids and cholesterol ester fractions from lipid extract of bile and serum of rabbits fed normal or UFA enriched diet. Fatty acids represent about 42 % , 94 % and 72 % of CE, TG and PL fractions. These lipid classes as well as free cholesterol (fig. 5) increase significantly in bile and mainly in serum of treated rabbits as compared to controls. The amounts of individual fatty acids present in the above mentioned fractions both in serum and bile are reported in tables 2 and 3. The pattern of fatty acids (C 18:2, C 18: 1, C 16:0 and C 18:0) ofPL, CE, TG and FFA of both bile and serum is the same either in treated or control rabbits. However, the percentage of unsaturated fatty acids increases progressively during the treatment with the enriched diet and, at the third month, is quite similar to that present in diet. The gas chromatographic analysis of fatty acids present in serum lipids faithfully reflects the percentage lipid composition of individual lipid classes investigated by TLC. In fact, the highest amounts of unsaturated fatty acids are found in cholesterol ester fraction reaching the maximum value at 90d. On the other hand, all biliary lipids simultaneously increased during the period of study. Table 4 reports liver, aortic, biliary and serum lipid peroxidation and enzyme activities (catalase, superoxide dis mutase and glutathione peroxidase) ofliver and aorta in both controls and animals treated for 1, 2 and 3 months. The significant increase of the liperoxidation rate with time in the tissues and fluids examined is coupled with a decrease in the activity of the enzymes involved in the detoxification of lipid peroxides. Exp. Pathol. 40 (1990) 1
23
~
~
I
."
~
~
62.85 ± tr tr tr
64.10 ± 1l.66· ... tr tr tr
60 days Phospholipids Free fatty acids Triglycerides Cholesterol esters
90 days Phospholipids Free fatty acids Triglycerides Cholesterol esters
Controls Phospholipids Free fatty acids Trigl ycerides Cholesterol esters
3l.03 ± tr tr tr
30 days Phospholipids Free fatty acids Triglycerides Cholesterol esters
228.46 3l.86 49.07 24.45
± ± ± ±
Palmitic acid
82.53 5.99 5.54 6.42
5.85°'"
tr tr tr tr 7.74·
Linolenic acid
Controls Phospholipids Free fatty acids Trigl ycerides Cholesterol esters
animals.
6.64
3.49
270.31 15.16 5.65 13.46
± 30.63 ± 2.28 ± 0.96 ± 4.38
Stearic acid
166.71 ± 11.99.... 0 tr tr tr
116.08 ± 13.32· ... tr tr tr
76.03 ± tr tr tr
20.16 ± tr tr tr
Arachidonic acid
3896.33 1669.17 10174.53 31056.99
2615.48 1446.93 5012.02 34333.94
2585.31 729.73 1102.42 13368.59
841.61 67.84 58.64 56.07 70.87 8.29 7.99 17.04
1546.59 137.90 155.70 125.09
Totals
± ± ± ±
128.63 14.31 14.14 27.24
± 320.56.... 0 ± 377.94.... 0 ± 719.29.... 0 ± 4675.89.... 0
± 247.52· ... ± 269.86· ... ± 160.86· ... ± 5623.53· ...
± 363.79· ± 148.03· ± 106.35· ± 2379.55·
± ± ± ±
Linoleic acid
18l.35· 38.50· 95.20· 330.09·
22.28 6.00 4.89 7.55
± 129.58· ± 6l.l4· ... ± 507.58· ... ± 2949.97· ...
± ± ± ±
± ± ± ±
2.10 1.68 1.84 2.32
i1s
± ± ± ±
0.09 0.28 0.11 0.24
2168.64 ± 348.73.... 0 265.39 ± 7l.82· ... 0 6513.81 ± 601.84.... 0 20488.24 ± 3582.15· ...
128l.58 369.31 3883.70 19523.40
1088.63 125.49 714.89 6024.37
185.92 2l.15 42.34 3l.1O
Oleic acid
Table 2. Content of individual fatty acids present in phospholipids, free fatty acids, triglycerides and cholesterol esters in serum of controls and treated
VI
N
~
~
i
'"tI
~
600.51 399.91 1452.06 9524.27
786.86 429.85 1566.91 8853.01
60 days Phospholipids Free fatty acids Triglycerides Cholesterol esters
90 days Phospholipids Free fatty acids Triglycerides Cholesterol esters
± 47.25-. 0 ± 102.99-· ± 63.18-· ± 2136.61-·
± 26.59± 139.11-· ± 112.69-· ± 1586.00-·
± 66.75± 24.13± 30.48± 643.53-
639.04 227.27 205.82 2106.04
393.99 222.89 176.21 3359.98
353.18 121.26 137.25 1503.84
± ± ± ±
± ± ± ±
± ± ± ±
49.05-. 0 80.22-· 22.68-· 439.24-. 0
44.1645.7114.21-· 460.47-·
41.1837.5510.88479.67-
7721.81 2608.09 18461.18 62504.30
5069.83 2419.07 10524.20 66739.61
4666.52 1119.22 2359.30 24944.30
± ± ± ±
± ± ± ±
0
4.40 3.01 9.16 4.87
4.11 3.00 5.49 4.16
4.15 3.02 3.36 3.56
± ± ± ±
± ± ± ±
± ± ± ±
0.190.720.86-. 0 1.22-·
0.500.350.62-· 0.30-
0.220.640.420.58-
90 days vs 60 days. Values are
455.44-. 0 572.86-· 259.49-. 0 6418.24-·
343.99-· 472.13-· 335.59-· 9347.21-·
± 506.62± 239.09± 99.23± 2990.88-
Statistical significance (Student's t-test, 0.05 < P>O.OOI): - treated animals vs controls; • 60 and 90 days vs 30 days; mean ± SD. The values are expressed as f.lg/rnl.
549.09 162.71 404.73 4047.48
30 days Phospholipids Free fatty acids Triglycerides Cholesterol esters
-8
'C 'C
~
~£.
?
><
tTl
IV 0'-
115.52 tr tr
120.95 tr tr
60 days Phospholipids Free fatty acids Triglycerides
90 days Phospholipids Free fatty acids Triglycerides
± 57.19-·
± 20.93-·
± 5.74
± 5.69
228.74 37.82 tr
564.06 72.56 52.92
Controls Phospholipids Free fatty acids Triglycerides
30 days Phospholipids Free fatty acids Triglycerides
± 84.65± 6.89± 9.94-
± 53.56 ± 6.26
Palmitic acid
43.70 tr tr
41.61 tr tr
30 days Phospholipids Free fatty acids Triglycerides
Controls Phospholipids Free fatty acids Triglycerides
Linolenic acid
± 22.45-·
± 26.51-·
± 9.61
± 10.64
110.14 23.46 tr
73.36 15.70 tr
± 17.02± 4.38-
± 11.63 ± 3.71
Stearic acid
205.80 tr tr
205.09 tr tr
74.69 tr tr
83.50 tr tr
Arachidonic acid
3332.25 555.16 225.27
1227.85 162.06
Totals
2065.56 672.72 319.47
2835.53 519.74 283.45
1943.03 405.33 137.22
tr
335.90 65.46
± 152.82± 23.56± 16.50-
± 132.78 ± 16.62
± 161.06eG ± 13.90-. 0 ± 28.84-·
± 189.19-· ± 35.26-· ± 23.57-·
± 139.17± 56.70± 11.72-
± 34.84 ± 3.70
Linoleic acid
4.01 4.77 3.34
3.13 1.76
Q/s
± 62.52-. 0 ± 18.43-· ± 6.32-·
± 90.45-· ± 10.05-· ± 5.26-·
± 65.25± 5.72 ± 5.85-
± 33.05 ± 8.02
± 0.79 ± 0.39± 0.67-
± 0.79 ± 0.31
842.43 128.75 94.01
920.28 132.31 66.15
594.61 53.81 35.13
464.72 44.88 tr
Oleic acid
Table 3. Content of individual fatty acids present in phospholipids, free fatty acids, triglycerides and cholesterol esters in bile of controls and treated animals.
~
!::1
~
~
~ ~
~
'?
530.45 ± 57.72eG 104.75 ± 14.08· .... 119.44 ± 11.24..... 0
90 days Phospholipids Free fatty acids Triglycerides 155.93 ± 93.78.0 65.87 ± 4.57· .... tr
191.14 ± 15.47· 44.11 ± 5.35· .... tr
0
3921.15 ± 142.29..... 0 947.10 ± 45.54..... 0 532.93 ± 29.27..... 0
4982.96 ± 385.09· .... 780.11 ± 51.96· .... 438.90 ± 28.16· ....
Statistical significance (Student's t-test, 0.05
O.OOl):· treated animals vs controls; .... 60 and 90 days vs 30 days; mean ± SO. The values are expressed as Ilg/rnl.
715.37 ± 73.03· .... 83.95 ± 8.77· 89.29 ± 8.44· ....
60 days Phospholipids Free fatty acids Triglycerides
90 days vs 60 days. Values are
4.72 ± 0.30· 4.86 ± 0.38· 3.48 ± 0.46·
4.49 ± 0.09· 5.10 ± 0.33· 3.94 ± 0.53·
:0 '>0 9
<:>
...
0
,....
::r
f!!.
"0
::;
><
tTl
bi
56.78 13.03 45.93 0.62
Lipoperoxidation Liver Aorta Bile Serum
± ± ± ± 5.60 3.60 2.27 0.10
± 3.64
± 0.06 ± 0.23
± 34.25 ± 0.35
92.58 17.61 78.84 0.69
30.00
0.78 0.64
195.90 2.86
30 days
± ± ± ± 1.89· 1.20· 2.08· 0.03
± 4.88·
± 0.05· ± 0.05·
± 46.36 ± 0.21·
128.95 23.45 147.12 0.60
27.55
0.66 0.56
156.42 1.80
60 days
± ± ± ±
8.39· ... 1.44· ... 3.99· ... 0.02'"
± 2.50·
± 0.06· ... ± 0.04· ...
± 22.62· ± 0.12· ...
420.95 38.96 345.84 0.73
13.49
0.46 0.30
83.46 1.31
90 days
± ± ± ±
7.49· ... 2.15· ... 8.02· ... 0.01· ...
* * * *
± 3.67· ... *
± 0.08· ... * ± 0.03· ... *
± 19.18· ... * ± 0.26· ... *
Glutathione peroxidase activity is expressed a nmoles NADPH oxidized/g tissue/min; superoxide dismutase: inhibition of the reduction of nitroblue tetrazolin by superoxide anion by 100 III of tissue supernatant; catalase: nmoles H 2 0 2 utilized/g tissue/min. Lipoperoxidation rate, found by TBA test, is expressed as Ilg malondialdehyde/g tissue/ml fluid.
40.66
0.95 0.93
222.57 3.52
Controls
Catalase Liver, per g liver
Superoxide dis mutase Liver Aorta
Glutathione peroxidase Liver, per g liver Aorta, per g aorta
for 30, 60 and 90 days.
Table 4. Liver, bile and serum lipid peroxidation and liver and aortic enzyme activities in rabbits fed basal diet or unsaturated fatty acids enriched diet
\0
N
~
to
~
~
~
$
GCA
± ± ± ±
0.45 0.03· 0.03· ... 0.02· ... *
1.95 0.70 0.23 0.23
± ± ± ± 0.18 0.03· 0.03· ... 0.01· ...
GCDCA 28.92 23.16 18.49 11.41
± ± ± ±
GDCA 0.53 0.19· 0.32· ... 0.23· ... *
34.37 24.79 19.46 12.24
Glyco
± ± ± ± 1.13 4.45 0.23· 2.26 0.35· ... 1.79 0.25· ... *1.70
± ± ± ±
Tauro 0.46 0.42· 0.03· 0.03·
38.82 27.05 21.25 13.94
Total
± ± ± ±
1.52 7.76 0.46· 11.22 0.36· ... 10.90 0.26· ... * 7.21
± ± ± ±
0.60 1.79 0.33· 0.17'"
G/T Ratio
TDCA = taurodeoxycholic acid; GCA = glycocholic acid; GCDCA = glycochenodeoxycholic acid; GDCA = glycodeoxycholic acid; glyco = total glycocholic acids; tauro = total taurocholic acids; total = total bile acids; G/T ratio = glyco/tauro ratio. For symbols indicating the statistical significance see table 1 test. Values are mean ± SD. The values are expressed as mg/mI.
Controls 4.45 ± 0.46 3.49 30 days 2.26 ± 0.42· 0.93 60 days 1.79 ± 0.05· 0.75 90 days 1.70 ± 0.03· 0.60
TDCA
Table 5. Tauro- and glyco-conjugated bile acids in gallbladder bile samples of controls and treated animals.
-E
10~
C)
:l..
PL
TG
FFA
E
PL
FFA
TG
CE
Fig. 480 and 4b. Levels ([!g/ml) of total fatty acids content in PL, FFA, TG and CE of serum (fig. 4 a) and bile (fig. 4 b) of rabbits fed basal diet ~ or unsaturated fatty acids enriched diet at 30 days IDI, 60 days D, and 90 days Iill. The values are expressed as mean ± SD.
Table 5 reports the levels (mg/ml) of conjugated bile acids found in gallbladder biles of treated rabbits and controls. There is an evident decrease of each conjugated bile acid during the treatment.
Discussion Probably, unsaturated free fatty acids present in the diet were extremely toxic for rabbits as demonstrated by the decrease of body weight of treated animals as compared to controls and death of 60% of animals already after the 3rd month of treatment. This toxic action could be related to in vivo lipoperoxidation. In fact, MATSUO (1954) reported that when the ethyl esters of highly unsaturated fatty acids freshly obtained from cuttle fish oil were heated at 100°C (peroxide value 30
Exp. Pathol. 40 (1990) 1
20
..........................i ........ ··· .. ······· .. ·· ..
~
O~__~-~ --~-~-~ --~-~ --~-~-~-~-~ --_-_ --_-_ -~ ~
30
60
90
DAYS
Fig. 5. Amounts {mg/ml) of free cholesterol (mean ± SD) present in serum of controls .- - - - -. and treated animals *--* and in bile of controls [:., - - - [:., and treated animals
...- ....
240 and iodine value 245.5 of the products), these became fatal for the rat, causing abrupt decrease of body weight, which led to death within 7 d. Fatty acid (in part as free fatty acids) supplement plus 4.1 % lipid present in the stock diet represent an overload for liver and the other tissues capable of metabolizing lipids. In addition, glucidic uptake from diet is probably relatively insufficient to provide an adequate amount of alpha-glycerophosphate for the biosynthesis of triglycerides, which should be stocked in the adipose tissue. The increase in the total lipid content of serum of rabbits fed enriched diet is mainly due to the cholesterol ester fraction, the amount of which , since the first month of treatment, reached levels much higher than those reported in the literature (TSAI and KELLEY 1978). Simultaneously with the increase of CE in serum, a grossly induced atherosclerosis and a marked hepatic damage with ascites formation appearing at the 3rd month were histologically and clinically demonstrated. Normally, the high concentrations of serum CE are attributed to cholesterol feeding, which enhances hepatic fatty acid synthesis (TSAI and KELLY 1978). In our case it would appear that the large quantity of FFA induces an increase of the hepatic biosynthesis of cholesterol and cholesterol-esterase probably to lower the toxic effects of free fatty acids. In fact, the uncoupling effect of FFA on oxidative phosphorylation at the level of mitochondria is well known (PRESSMAN and LARDY 1956; LEHNINGER and REMMERT 1959; HULSMANN etal. 1960). It becomes stronger with increasing chain length and is particularly powerful in the case of unsaturated fatty acids (such as those added to the diet). On the other hand, WIGAND (1959) found a hypercholesterolemia in rabbits fed a diet supplemented with saturated triglycerides as only lipid source. The damage of both liver and endothelium of the arterial wall was reflected in a high rate of lipoperoxidation. There was, in fact, a significant increase of lipoperoxide values, performed by TBA test in lipid extracts of liver, aorta and bile of rabbits fed unsaturated FFA supplemented diet as compared to controls. Lipoperoxidation increased with the duration of feeding. Parallely, there was a marked reduction in the activities of hepatic and aortic glutathion peroxidase, superoxide dismutase and catalase, all enzymes involved in the detoxification process of lipid peroxides. These results are in favour of the hypothesis that lipid peroxidation plays an important role in the pathogenesis of atherosclerosis. Similar data were obtained by TSAI and KELLEY (1978) in rats and guinea pigs fed a diet containing 1 % or 1.5 % cholesterol plus 10% or 15 % soy oil, but not in rabbits fed 1 % CH plus 2 % soy oil. Probably, the different percentage of lipids contained in the diets might account for the conflicting results obtained by these authors. In addition, it must be considered that rabbits are a species known to be more susceptible to atherosclerosis than other species when fed a CH supplemented diet. Exp. Pathol. 40 (1990) 1
31
Our results are in agreement with some data reported in literature, i.e., mainly, that atherogenesis is enhanced (HARMAN 1962) by substances capable of irritating the arterial wall. A source of irritating compounds (HARMAN 1957) could be the reaction of molecular oxygen with polyunsaturated substances present in serum and arterial wall lipids. About 30% of these substances is represented by fatty acids, mainly if esterified, in the lipids of both serum (PATIL and MAGAR 1960) and atherosclerotic plaques (BOTTCHER etal. 1960). Indirect evidence (HARMAN and PIETTE 1966) that lipoperoxidation occurs in serum has been produced by means of electron spin resonance spectroscopy. On the other hand, the presence of ceroid (HARTROFT and PORTA 1965) in the arterial intima represents the indirect evidence of reactions involving molecular oxygen and organic compounds, mainly polyunsaturated. Also pertinent is the observation that the 37 % rise in the incidence of cardiovascular disease during the past 50 years in the USA was accompanied by a 7 % increase in the consumption of polyunsaturated fats (SWELL etal. 1960). Hepatic damage had repercussion on the fairly good levels of TBA test reacting substances in bile. Nevertheless, there is no evidence of gallbladder epithelial damage, visible by O.M. Probably, the TBA test evidences aldehyde byproducts of lipoperoxidation which took place in the liver. During treatment there was an evident decrease of conjugated bile salts in bile, reflecting reduced liver catabolism. However, gallstones were not found, probably because of the increased levels of PL (see table 3), which solubilize the apolar lipids in bile.
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1363-1375. EDERER, F., LEREN, P., TURPENINEN, 0.: Cancer among men on cholesterol-lowering diets. Lancet 1971; 2:
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Exp. Pathol. 1990; 40: 33-34 Gustav Fischer Verlag lena
Book Review
Fortschritte der Osteologie in Diagnostik und Therapie Genetische Knochenerkrankungen, Primire Knochentumoren, Prothetik Osteologia 3 edited by F. H. W. Heuck and E. KECK. 461 pages, with 217 figures, DM 98,-, ISBN: 3-540-50190-8. Springer-Verlag, Berlin-Heidelberg-New York-London-Paris-Tokyo 1989. This volume contains the lectures and articles given at the 3rd session of the "Gennan Society of Osteology", held in Stuttgart 7 -10 October, 1987. The main topics are: genetic diseases of the skeleton, primary bone tumors, news of prosthetic research. .\
Exp. Patho!. 40 (1990) 1
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