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Pantethine reduces plasma cholesterol and the severity of arterial lesions in experimental hypercholesterolemic rabbits
Atherosclerosis, 53 (1984) 255-264 Elsevier Scientific Publishers Ireland,
255 Ltd.
ATH 03541
Pantethine Reduces Plasma Cholesterol and the Severity of Arterial Lesions in Experimental Hypercholesterolemic Rabbits P. Carrara
‘, L. Matturri *, M. Galbussera ‘, M.R. Lovati ‘, G. Franceschini ’ and C.R. Sirtori ’
’ Institute of Pharmacology and Pharmacognosy, Center E. Grossi Paoietti and Chemotherapy Chair, University of Milan and ‘Institute of Morbid Anatomy, University of Milan 20129 Milan (Italy)
Summary
Pantethine (P), a coenzyme A precursor, was administered to cholesterol-fed rabbits (0.5% cholesterol diet + 1% pantethine) for 90 days. At the end of treatment, plasma total cholesterol levels were reduced 64.7% and the HDL/total cholesterol ratio increased in P-treated animals; a significant rise of the apo A-I/A-II ratio was detected in HDL. VLDL lipid and protein levels were, on the other hand, reduced by P. The cholesterol-ester content of both liver and aortic tissues was not significantly affected by P. Although the total aortic area with evident plaques was reduced only 18.2%, the microscopical examination of sections from the major vessels of P-treated animals, showed a reduction in the severity of lesions, both in the aorta and in the coronary arteries. These findings suggest that P, in addition to significantly lowering plasma cholesterol levels in rabbits on an experimental diet, may modify lipid deposition in major arteries, possibly by affecting lipoprotein composition and/or exerting an arterial protective effect. Key words: Apoprotein A-I - Atherosclerosis
- Cholesterol feeding - Natural hypo-
lipidemic agents - Pantethine
Presented in part at the 8th International Symposium on Drugs Affecting Lipid Metabolism, Philadelphia, PA, U.S.A., July 1983. Supported in part by the Consiglio Nazionale delle Ricerche of Italy (P.F. Medicina Preventiva, Subproject ATS N. 70 011 13.83). Send reprint requests to: Dr. Maria Rosa Lovati, Institute of Pharmacology and Pharmacognosy, University of Milano, Via A. Del Sarto 21, 20129’Mihq Italy.
0021-9150/84/$03.00
0 1984 Elsevier Scientific
Publishers
Ireland,
Ltd.
256
Introduction Pantethine (P), the disulphate form of pantetheine, D( + )-N-pantothenylaminoethanethiol, precursor of coenzyme A [l], has shown significant hypocholesterolemic properties in man, and positive effects on high density lipoprotein (HDL) cholesterol levels have been described [2]. Animal studies have reported a reduction of plasma cholesterol and an increased arterial cholesterol ester hydrolase in rats on high-fat regimens [3], as well as’stimulated fatty acid oxidation in muscle and liver in experimental diabetes [4]. In rabbits on a mildly hypercholesterolemic regimen, P effectively prevented the cholesterol rise in the P-migrating very low density lipoproteins (VLDL) and in low density lipoproteins (LDL); the percent levels of apoprotein A-I in the HDL, subfraction was increased by treatment [5]. This and other studies have, however, not taken into consideration the possible beneficial effect of the agent on atherosclerosis development. It was the object of this study to investigate, in addition to plasma lipid and lipoprotein changes in rabbits on a moderately cholesterol enriched regimen, the pattern of arterial lesions resulting from dietary treatment, and the effect of P on these. Materials and Methods
Animals and diets Male New Zealand rabbits, weighing 2.0-2.5 kg (Charles River, Calco, Italy) were randomly divided into 3 groups of 10 animals. The 3 groups were treated for 90 days, respectively with: (A) standard diet (Charles River diet for rabbits); (B) 0.5% cholesterol diet (HC) and (C) 0.5% cholesterol diet + 1% pantethine (HC + P). In all 3 dietary regimens, rabbits received 125 g of diet per day; during regimens B and C, cholesterol was given in the morning mixed with 25 g of normal diet, the remaining 100 g of diet (mixed with P in group C) being given after complete consumption of the cholesterol ration.
Biochemical methods Animals were kept fasted 24 h before each blood drawing. Blood, taken from the central ear artery, was collected in EDTA Na, (1 mg/ml) containing tubes. Plasma total cholesterol and triglyceride (TG) levels were determined by enzyme methods [6,7]. Plasma lipoproteins were separated by preparative ultracentrifugation [8], with a 50 Ti rotor in a Beckman Spinco L5-50 ultracentrifuge. The VLDL fraction (inclusive of IDL) was separated at d < 1.019 g/ml, LDL at d = 1.019-1.063 g/ml and HDL at d = 1.063-1.210 g/ml. Lipoprotein fractions were extensively dialyzed against 0.01 M Tris buffer, pH 7.4, containing 0.15 M NaCl and 1% EDTA Na, for 24 h. Cholesterol and TG concentrations were determined enzymatically [7,9], and proteins [lo] after SDS treatment of the lipoprotein preparations [ll]; phospholipids (PL) were measured by a calorimetric method [12]. Liver lipids were frozen and later homogenized in 0.9% saline with a Polytron homogenizer. Lipids were then extracted from the homogenate with chloroform/
257
methanol (2 : 1, v/v, 20 ml/g tissue) [13]. Aliquots of the lipid extracts were spotted on Silica Gel 60 F254 (Merck, Darmstadt, F.R.G.) and separated with hexane/ diethyl ether/acetic acid (70 : 30 : 1, v/v/v), followed by elution of the bands. Free and ester cholesterol were determined [14]; the bands corresponding to TG and PL, after drying the chloroform extract under N,, were dissolved in isopropanol, the lipid contents being assayed by calorimetric methods [12,15]. Aortas, immediately after removal from the animals, were stained with Tissue Blue (Hubbord Ind., Leonidas, MI, U.S.A.), diluted in water, and photographed, to determine the extent of atherosclerotic lesions from coloured slides [16]. They were then cut longitudinally, half of each aorta being used for histology, the other half for biochemical determinations. These latter halves were rapidly frozen in liquid nitrogen, the resulting powder weighed, and homogenized in 0.9% saline, similarly to the liver. Determination of the protein content of aortas [lo] was carried out after denaturation in 1 N NaOH at room temperature for 24 h and at 55 ‘C for 30 min. Aliquots of HDL from all animals were delipidated for isoelectric focusing (IEF) separation of isoproteins [17]; gels were focussed on a flat bed, between pH 4 and 6, and isoprotein distribution determined by scanning (Seroskope, Elvi, Milan, Italy). Histological
evaluation
Sections from the aorta (thoracic and abdominal segment) and liver were taken. Moreover, heart fragments were prepared, taking care to draw segments comprehensive of the myocardium, the epicardium and the initial tract of the descending branch of the left coronary. Tissues, fixed in 10% buffered formalin, were paraffin embedded. Sections (5-6 pm thick) were then stained by the common histological techniques (hematoxylin-eosin, Weigert for elastic fibers, Azan Mallory), and also by the Alcian blue technique (pH 1 and 2.5) and by PAS. Presence of lipids was evaluated by Sudan red staining of cryostat sections [18]. Results Plasma lipid-lipoprotein
findings
Cholesterol treatment (groups B and C) was associated with a rapid rise of cholesterolemia (Fig. 1). Both treatment groups showed significantly higher plasma cholesterol levels, compared to controls, already after 10 days of diet administration. On the other hand, after 24 days of treatment, there was a significantly lower cholesterolemia in P-treated animals compared to the HC group (P -C0.01). This difference remained statistically highly significant up to the end of treatment. HDL-C levels were elevated by both dietary treatments, being slightly lower in the HC + P group; however, at the end of treatment, the HDL/total cholesterol ratio was significantly higher in this latter group (P < 0.01) (Fig. 1). Plasma TG levels were only moderately modified in the 3 groups during treatment. At the end, the HC group had significantly higher levels vs controls, whereas lower levels were detected in the HC + P group, not significantly different from either of the other two (group A: 68.7 + 24.5 mg/dl; group B: 150.6 f 36.4, P < 0.05 vs A; group C: 91.3 + 14.7).
258 TABLE
1
PLASMA LEVELS OF TOTAL CHOLESTEROL AND COMPOSITION TEINS AT THE END OF THE DIETARY TREATMENTS
OF PLASMA
LIPOPRO-
Values are mean + SEM for 10 animals. Standard Total cholesterol
HC regimen
diet
1050.0*15.3
39.8k3.6
HC + pantethine 370.0,11.6
**
**+
‘K,of total VLDL (mg/dl) Total chol TG PL Protein
6.0 + 2.4 24.6 + 1.3 22.7k2.5 7.3 * 3.0
(9.9) (40.6) (37.4) (12.0)
718.2 i 10.9 ** 54.9i11.3 * 288.7 k 78.4 ** 131.2531.4
(60.2)
LDL (mg/dl) Total chol TG PL Protein
8.4+0.3 7.4* 1.2 28.7+ 1.8 5.8+0.3
(16.7) (14.7) (57.1) (11.5)
263.6i 17.9 ** 19.8iO.5 ** 136.0*5.9 ** 87.3k6.5 **
(51.1)
HDL (mg/dl) Total chol TG PL Protein
21.4 12.2 31.2 17.1
(26.1) (14.9) (38.1) (20.9)
61.2 f 17.4 ** 11.6k1.3 58.6+4.7 100.4+ 8.0 **
(26.4)
+ + + k
2.2 1.2 3.5 4.6
(4.6) (24.2) (11.0)
(4.0) (27.4) (17.6)
(5.0) (25.3) (43.3)
222.6 + 5.6 **+ 12.1 f 1.8 65.5 + 14.5 + 35.9k6.4 *+
(66.3)
100.1 + 19.2 ** + 4.3 * 1.7 + 50.5 + 11.7 ** + 33.1,6.1 **+
(53.2)
45.3* 8.8 * 41.2+ 92.6 +
11.2 ** 1.1 7.7 8.6 **
(3.6) (19.5) (10.7)
(2.3) (26.8) (17.6)
(24.1) (4.7) (21.9) (49.3)
* P < 0.05 vs controls. **P i 0.01 vs controls. **+ P
HC
HC+P
N ”
30
60
davr
90
Fig. 1. Plasma cholesterol levels in rabbits on a standard diet (N), and given cholesterol without (HC) and with pantethine (HC + P). Plasma total cholesterol levels were significantly reduced in the HC + P group after 65 days of treatment (*P -z 0.05) and more so after 90 days ( **P i 0.01). At the end of treatment a significant rise of the HDL/totaI cholesterol ratio was evident in the HC + P group.
259
Plasma lipoprotein composition was evaluated at the end of the treatments (Table 1). VLDL were characterized by a marked increase of cholesterol content in the cholesterol-fed animals, significantly reduced in the HC + P group. A reduction of the phospholipid and protein content (about 5% lower compared to HC animals) could also be detected in these animals. Similarly, the experimental diets significantly elevated LDL lipids and proteins. The reduction in LDL cholesterol, TG, PL and protein content by P was statistically significant. The percentage composition of lipoproteins showed the expected reduction of TG content in VLDL; the cholesterol enrichment in LDL occurred at the expense of PL, and only an increased protein content could be detected in HDL. P-treatment did not appear to markedly modify the composition of any of the lipoprotein fractions. HDL proteins were separated by an analytical IEF procedure [17]. Absolute amounts of apoproteins and of the A-I isoproteins changed little, with the exception of a statistically significant reduction of apo A-II and C levels. The percentage composition of HDL apoproteins, however, was dramatically modified by P administration. Both the percentage of total A-I and of its major isoprotein (apo A-I,) were significantly raised after P treatment. Conversely, the apo A-II percentage content was reduced 19.7%, also highly significant. The percent content of apo C changed moderately and no variations were noted in the other apoproteins (Table 2, Fig. 2). Tissue lipidr
Liver lipid composition (Table 3) was characterized, in both treatment groups, by a marked increase of total and esterified cholesterol content. Neither of these changes was modified by concomitant P treatment. TABLE
2
LEVELS AND PERCENTAGE DISTRIBUTION ISOPROTEINS FROM HYPERCHOLESTEROLEMIC COMITANT PANTETHINE TREATMENT
OF THE MAJOR HDL APOPROTEINS ANIMALS, WITH AND WITHOUT
Distribution data are taken from the densitometric scans of the isoelectric focusing relative chromogenicity (see Fig. 2). Values are means f SEM from 10 animals. Cholesterol
Cholesterol
Apo E Apo A-I tot. Apo A-I, Apo A-I a Apo A-II Apo C tot. Others
** ***
gels, correcting
+ pantethine
mg/dI
%
mg/dI
I
10.8 f 0.3 59.6k0.6 43.0*0.5 16.4*0.2 6.lkO.2 15.7+0.9 8.2kO.7
10.8kO.4 59.3kO.8 43.0 + 0.8 16.4kO.3 6.1 f 0.2 15.6 f 1.0 8.2kO.8
10.4 f 0.9 61.6kl.3 45.8 f 1.0 15.7*0.9 4.5*0.1+ 10.5*1.3+ 5.6 f 0.4
11.2kO.6 66.5rtl.l 49.5kO.8 17.OkO.8 4.9*0.1 11.4*1.0 6.1 k 0.6
* P < 0.05 vs cholesterol-treated P -c0.01 vs cholesterol-treated P -c0.005 vs cholesterol-treated + P < 0.05 vs cholesterol-treated
group. group. group. group.
*** *** ** *
AND CON-
for the
260 TABLE
3
LIVER LIPID LEVELS IN RABBITS ON A STANDARD CHOLESTEROL+ PANTETHINE (HC + P) REGIMENS
DIET,
CHOLESTEROL
(HC)
AND
Values are means f SEM from 10 animals mg/g
of fresh tissue
Total cholesterol Free cholesterol Est. cholesterol Free/est. chol. Triglycerides Phospholipids ** P < 0.01 vs standard
Standard diet
HC
3.5 f 0.2 2.1 f 0.1 0.9 f 0.1 2.3 k 0.2 4.5 If:0.2 0.8+0.1
13.5f0.7 4.7kO.2 7.3kO.6 0.6 + 0.1 3.8f0.2 1.0*0.1
HC+P
** ** ** **
14.2 f 0.8 5.8kO.4 8.OkO.5 0.7kO.l 4.1* 0.2 0.9*0.1
** ** ** **
diet.
A similar accumulation of cholesterol esters was noted in the aortas of diet treated animals. P treatment did not seem to modify aortic cholesterol esters (Table 4A). Histological and histochemical observations
A visual estimate of the total area of aortic lesions (lack of uptake of Tissue Blue), was suggestive of a significant reduction in the area of lesions of P-treated animals (- 18.2% compared to HC; P < 0.05) (Table 4B). In the HC treated animals, severe arterial lesions were detected both in the aorta and the anterior descending branch of the left coronary branch, being characterized by lipid deposition both intra-cellularly and in the extra-cellular space. Lipid accumulation was recognizable in the intima, but occasional intra-cellular lipid depositions could be observed in the outer
Fig. 2. Isoelectric focusing pattern of HDL apoproteins from the HC and HC + P groups. A markedly increased A-I, is clearly evident (see Table 2 for the quantitative
and isoproteins at the end of treatment in rabbits density of the protein band corresponding to apo data).
TABLE AORTIC
4A LIPID
Values are mg/g
COMPOSITION of protein,
expressed
Total cholesterol (W est. chol.) Triglycerides
AORTIC
ON THE DIFFERENT
TREATMENT
REGIMENS
as mean f SEM from 10 animals.
Standard diet
HC
3.9*0.4
18.9kO.9 (61.5) 0.2*0.1
(29) 0.1*0.1
** P < 0.01 vs standard
TABLE
IN RABBITS
HC+P
**
18.9* 1.2 ** (56.2) 0.2*0.1
diet.
48 LESIONS
IN RABBITS
ON THE DIFFERENT
Values are means f SEM from 10 animals;
X of aortic surface with visible lesions ** P c 0.01 vs standard vsHC. + P -co.05
assessment
TREATMENT
REGIMENS
by visual method.
Standard diet
HC
8.6*1.2
33.2kl.l
HC+P
**
27.1k1.2
**+
diet;
Fig. 3. Sections of abdominal aorta taken at the corresponding sites from cholesterol fed (HC) and cholesterol+pantethine (HC+ P)-treated rabbits. A:HC. Intense lipid deposition through the whole intimal thickness. Occasional lipid deposits may be observed in the media. Sudan Red, x 300; B: HC + P. The lipid deposition in the abdominal aorta is considerably reduced compared to the previous figure. Sudan Red, x300; C: HC. Myo-intimal thickening of severe degree, associated with lipid deposits and thickening. fragmented internal elastic membrane. Weigert stain, x 250; D: HC + P. Initial myo-intimal The internal elastic membrane is well preserved. Weigert stain, X 250.
262
section of the media (Fig. 3A). A significant increase of the intimal smooth muscle cell population and Alcian positive material at pH 2.5 was also recognizable. In the HC + P treated animals, lipid deposition was reduced (Fig. 3B) and, by specific methods for elastic fibers, in the aortas from HC + P rabbits, there appeared to be a significant preservation of the integrity of the basal elastic lamina, compared to HC animals (Figs. 3C and 3D). HC and HC + P rabbits did not differ in term of cell population and glycosaminoglycan deposition, as assessed from visual examination, these being present to a similar degree in both animal groups. Sections from the coronary arteries examined also showed a reduced severity of lesions in the HC + P group. In the liver, cholesterol feeding was associated with a microvacuolar steatosis in the peripheral areas of the lobule. Some reduction, of a minor degree, could be observed in the P-treated animals. Finally, no significant lesions could be observed in the myocardium. Discussion The cholesterol-lowering activity of P in an animal model resistant to treatment with standard hypolipidemic drugs [19], has been recently reported [5], and is confirmed in the present study. In addition, the reduction of the total cholesterol content of plasma and isolated lipoproteins was accompanied by an amelioration of atherosclerotic lesions. The major biochemical findings in our study are alike those reported by Tomikawa et al. [5], who followed a similar protocol for a shorter time (1 month). We also showed a reduction of the &nigrating VLDL in plasma and a relative, as well as absolute, increase of HDL cholesterolemia in treated animals. By analytical IEF, both total apo A-I and the major isoprotein (apo A-I,) were markedly increased in P-treated animals. The apo A-I, content, in a previous study [20], was significantly correlated with the cholesterol content of HDL. In contrast to Tomikawa et al. [5], we also detected a relative reduction of the apo A-II content in HDL. This finding, i.e. a higher A-I/A-II ratio in HDL, is generally consistent with an increased HDLJHDL, ratio in plasma, in humans and in other animal models [21]. The lack of demonstration of this type of distribution in the HDL subfractions of cholesterolfed rabbits, may only indicate that particles with a higher content of the activator for lecithin cholesterol acyltransferase [22] are probably increased by P treatment. We did not observe any marked changes in the TG levels in drug-treated animals, similarly to what was reported by Tomikawa et al. [5]. Findings of this type are unlikely to be obtained in this model, where triglyceridernia is generally minimally elevated. On the other hand, the results of a concomitant multicenter clinical study, indicating that changes in LDL cholesterol levels (increases or decreases) are a function of pre-treatment LDL cholesterolemia [23], suggest that P treatment may influence the breakdown of TG-rich lipoproteins. An apparent reduction of the severity of atherosclerotic lesions in rabbits receiving cholesterol with P, was present, in spite of the lack of difference in the aortic and liver cholesterol content between HC and HC + P treated rabbits. This rules out the
263
hypothesis that a reduction of atherosclerotic lesions may only be a consequence of lowered cholesterolemia in this model [24]. There also appeared to be a reduction in the extent of lesions in the coronary tree of P-treated animals. The mechanism whereby rabbits seem to be protected from the atherogenic effect of hyperlipidemia may be possibly related to the increased HDL cholesterolemia, in particular, with a raised content of the LCAT activator [22] and/or to the marked reduction in the plasma levels of atherogenic VLDL [25]. Another recently suggested mechanism may be related to a protective effect of P on tissue injury, exerted, e.g., by freezing or by immunological techniques (Yoshida Y., personal communication). Preliminary apoprotein compositional studies of VLDL failed to show any significant changes in, particularly, the content of apoprotein E. Differently, therefore, from an agent like metformin, which also markedly reduces atherosclerosis in rabbits [26], but does not affect plasma cholesterol, while exerting major structural changes in VLDL, P only increases the relative percentage of HDL and possibly also of HDL,. Acknowledgements
Prof. M. Riva, Drs. C. Donati and L. Zanardi (Maggioni S.p.A., Milan) are gratefully acknowledged for the supply of pantethine and for constant help and encouragement in these studies. References 1 Abiko, Y., Metabolism of &enzyme A. In: D.M. Greenberg (Ed.), Metabolic Pathways, Vol. 7, Academic Press, New York, NY, 1975, pp. l-25. 2 Avogaro, P., Bittolo Bon, G. and Fusello, M., Effect of pantethine on lipids, lipoproteins and apolipoproteins in man, Curr. Ther. Res., 33 (1983) 488. 3 Shinomiya, M., Matsuoka, N., Shirai, K., Nobuhiro, M., Sasaki, N., Murano, S., Saito, Y. and Kumagai, A., Effect of pantethine on cholesterol ester metabolism in rat arterial wall, Atherosclerosis, 36 (1980) 75. 4 Kameda, K. and Abiko, Y., Stimulation of fatty acid metabolism by pantethine. In: D. Cavallini et al. (Eds.), Natural Sulfur Compounds - Novel Biochemical and Structural Aspects, Plenum Press, New York,.NY, 1980, pp. 443-452. 5 Tomikawa, M., Nakayasu, T., Twara, K., Kaneda, K.Y. and Abiko, Y., Effect of pantethine on lipoprotein profile and HDL subfractions in experimental hypercholesterolemic rabbits, Atherosclerosis, 41 (1982) 267. 6 Allain, C.C., Poon, L.S., Chan, C.S.G., Richmond, W. and Fu, P.C., Enzymatic determination of total serum cholesterol, Clin. Chem., 20 (1974) 470. 7 Bucolo, G. and David, H., Quantitative determination of serum triglycerides by use of enzymes, Clin. Chem., 19 (1973) 476. 8 Havel, R.J., Eder, H.A. and Bragdon, J.H., Distribution and composition of ultracentrifugally separated lipoproteins in human serum, J. Clin. Invest., 34 (1955) 1345. 9 R&&au, P., Bemt, E. and Grtiber, W., Enzymatische Bestimmung Gesamtcholesterins in Serum, 2. Klin. Chem. Khn. B&hem., 12 (1974) 226. 10 Lowry, O.H., Rosebrough, H.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265. 11 Helenius, A. and Simons, K., Removal of lipids from human plasma low-density lipoproteins by detergents, Biochemistry, 10 (1971) 2542.
264 12 Rouser, G., Yamamoto, A. and Fleischer, S., Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots, Lipids, 5 (1970) 494. 13 Folch, P.J., Lees, M. and Sloane Stanley, C.H., A single method for isolation and purification of total lipids from animal tissue, J. Biol. Chem., 226 (1957) 497. 14 Zlatkis, A., Zak, B. and Boyle, A.J., A new method for the direct determination of serum cholesterol, J. Lab. Clin. Med., 41 (1953) 486. 15 Gottfried, S.P. and Rosenberg B., Improved manual spectrophotometric procedure for determination of serum triglycerides, Clin. Chem., 19 (1973) 1977. 16 Albert, E.N., Kassira, W.N., Muesing, R. and Vahouny, G.V., Pyridinolcarbamate and experimental atherosclerosis, Atherosclerosis, 31 (1978) 205. 17 Gidez, L.I., Swaney, J.B. and Mumane, S., Analysis of rat serum apolipoproteins by isoelectric focusing, Part 1 (Studies on the middle molecular weight subunits), J. Lipid Res., 18 (1977) 59. 18 Coutard, M. and Osborne-Pellegrin, M.J., Spontaneous lesions in the rat caudal artery, Atherosclerosis, 44 (1982) 245. 19 Brattsand, R., The effect of niceritrol (pentaerythritoltetranicotinate) and clofibrate upon hyperlipemia and atherosclerosis induced in rabbits by cholesterol-free semisynthetic diets, Atherosclerosis, 20 (1974) 453. 20 Franceschini, G., Sirtori, M., Gianfranceschi, G. and Sirtori, C.R., Relation between HDL apoproteins and AI isoproteins in subjects with the AI-Milan0 abnormality, Metabolism, 30 (1981) 502. 21 Kostner, G.M., Patsch, J.R., Sailer, S., Braunsteiner, H. and Holasek, A., Polypeptide distribution of the main lipoprotein density classes separated from human plasma by rate zonal ultracentrifugation, Europ. J. B&hem., 45 (1974) 611. 22 Albers, J.J., Cabana, V.G. and Stahl, Y.D.B., Purification and characterization of human plasma lecithin-cholesterol acyltransferase, Biochemistry, 15 (1976) 1084. 23 Gaddi, A., Descovich, G.C., Noseda, G., Fragiacomo, C., Colombo, L., Craveri, A., Montanari, G. and Sirtori, C.R., Controlled evaluation of pantethine, a natural hypolipidemic compound, in patients with different forms of hyperlipoproteinemia, Atherosclerosis, 50 (1984) 73. 24 Brattsand, R., Petersen, H. and Lundholm, L., Action of niceritrol (pentaerythritoltetranicotinate) on lipid accumulation in aortas of cholesterol-fed rabbits independent of contemporary reduction of serum lipids, Atherosclerosis, 20 (1974) 469. 25 Mahley, R.W., Atherogenic hyperlipoproteinemia - The cellular and molecular biology of plasma lipoproteins altered by dietary fat and cholesterol, Med. Clin. N. Amer., 66 (1982) 375. 26 Sirtori, C.R., Catapano, A., Ghiselli, G.C., Innocenti, A.L. and Rodriguez, J., Metformin - an antiatherosclerotic agent modifying very low density lipoproteins in rabbits, Atherosclerosis, 26 (1977) 79.
255 Ltd.
ATH 03541
Pantethine Reduces Plasma Cholesterol and the Severity of Arterial Lesions in Experimental Hypercholesterolemic Rabbits P. Carrara
‘, L. Matturri *, M. Galbussera ‘, M.R. Lovati ‘, G. Franceschini ’ and C.R. Sirtori ’
’ Institute of Pharmacology and Pharmacognosy, Center E. Grossi Paoietti and Chemotherapy Chair, University of Milan and ‘Institute of Morbid Anatomy, University of Milan 20129 Milan (Italy)
Summary
Pantethine (P), a coenzyme A precursor, was administered to cholesterol-fed rabbits (0.5% cholesterol diet + 1% pantethine) for 90 days. At the end of treatment, plasma total cholesterol levels were reduced 64.7% and the HDL/total cholesterol ratio increased in P-treated animals; a significant rise of the apo A-I/A-II ratio was detected in HDL. VLDL lipid and protein levels were, on the other hand, reduced by P. The cholesterol-ester content of both liver and aortic tissues was not significantly affected by P. Although the total aortic area with evident plaques was reduced only 18.2%, the microscopical examination of sections from the major vessels of P-treated animals, showed a reduction in the severity of lesions, both in the aorta and in the coronary arteries. These findings suggest that P, in addition to significantly lowering plasma cholesterol levels in rabbits on an experimental diet, may modify lipid deposition in major arteries, possibly by affecting lipoprotein composition and/or exerting an arterial protective effect. Key words: Apoprotein A-I - Atherosclerosis
- Cholesterol feeding - Natural hypo-
lipidemic agents - Pantethine
Presented in part at the 8th International Symposium on Drugs Affecting Lipid Metabolism, Philadelphia, PA, U.S.A., July 1983. Supported in part by the Consiglio Nazionale delle Ricerche of Italy (P.F. Medicina Preventiva, Subproject ATS N. 70 011 13.83). Send reprint requests to: Dr. Maria Rosa Lovati, Institute of Pharmacology and Pharmacognosy, University of Milano, Via A. Del Sarto 21, 20129’Mihq Italy.
0021-9150/84/$03.00
0 1984 Elsevier Scientific
Publishers
Ireland,
Ltd.
256
Introduction Pantethine (P), the disulphate form of pantetheine, D( + )-N-pantothenylaminoethanethiol, precursor of coenzyme A [l], has shown significant hypocholesterolemic properties in man, and positive effects on high density lipoprotein (HDL) cholesterol levels have been described [2]. Animal studies have reported a reduction of plasma cholesterol and an increased arterial cholesterol ester hydrolase in rats on high-fat regimens [3], as well as’stimulated fatty acid oxidation in muscle and liver in experimental diabetes [4]. In rabbits on a mildly hypercholesterolemic regimen, P effectively prevented the cholesterol rise in the P-migrating very low density lipoproteins (VLDL) and in low density lipoproteins (LDL); the percent levels of apoprotein A-I in the HDL, subfraction was increased by treatment [5]. This and other studies have, however, not taken into consideration the possible beneficial effect of the agent on atherosclerosis development. It was the object of this study to investigate, in addition to plasma lipid and lipoprotein changes in rabbits on a moderately cholesterol enriched regimen, the pattern of arterial lesions resulting from dietary treatment, and the effect of P on these. Materials and Methods
Animals and diets Male New Zealand rabbits, weighing 2.0-2.5 kg (Charles River, Calco, Italy) were randomly divided into 3 groups of 10 animals. The 3 groups were treated for 90 days, respectively with: (A) standard diet (Charles River diet for rabbits); (B) 0.5% cholesterol diet (HC) and (C) 0.5% cholesterol diet + 1% pantethine (HC + P). In all 3 dietary regimens, rabbits received 125 g of diet per day; during regimens B and C, cholesterol was given in the morning mixed with 25 g of normal diet, the remaining 100 g of diet (mixed with P in group C) being given after complete consumption of the cholesterol ration.
Biochemical methods Animals were kept fasted 24 h before each blood drawing. Blood, taken from the central ear artery, was collected in EDTA Na, (1 mg/ml) containing tubes. Plasma total cholesterol and triglyceride (TG) levels were determined by enzyme methods [6,7]. Plasma lipoproteins were separated by preparative ultracentrifugation [8], with a 50 Ti rotor in a Beckman Spinco L5-50 ultracentrifuge. The VLDL fraction (inclusive of IDL) was separated at d < 1.019 g/ml, LDL at d = 1.019-1.063 g/ml and HDL at d = 1.063-1.210 g/ml. Lipoprotein fractions were extensively dialyzed against 0.01 M Tris buffer, pH 7.4, containing 0.15 M NaCl and 1% EDTA Na, for 24 h. Cholesterol and TG concentrations were determined enzymatically [7,9], and proteins [lo] after SDS treatment of the lipoprotein preparations [ll]; phospholipids (PL) were measured by a calorimetric method [12]. Liver lipids were frozen and later homogenized in 0.9% saline with a Polytron homogenizer. Lipids were then extracted from the homogenate with chloroform/
257
methanol (2 : 1, v/v, 20 ml/g tissue) [13]. Aliquots of the lipid extracts were spotted on Silica Gel 60 F254 (Merck, Darmstadt, F.R.G.) and separated with hexane/ diethyl ether/acetic acid (70 : 30 : 1, v/v/v), followed by elution of the bands. Free and ester cholesterol were determined [14]; the bands corresponding to TG and PL, after drying the chloroform extract under N,, were dissolved in isopropanol, the lipid contents being assayed by calorimetric methods [12,15]. Aortas, immediately after removal from the animals, were stained with Tissue Blue (Hubbord Ind., Leonidas, MI, U.S.A.), diluted in water, and photographed, to determine the extent of atherosclerotic lesions from coloured slides [16]. They were then cut longitudinally, half of each aorta being used for histology, the other half for biochemical determinations. These latter halves were rapidly frozen in liquid nitrogen, the resulting powder weighed, and homogenized in 0.9% saline, similarly to the liver. Determination of the protein content of aortas [lo] was carried out after denaturation in 1 N NaOH at room temperature for 24 h and at 55 ‘C for 30 min. Aliquots of HDL from all animals were delipidated for isoelectric focusing (IEF) separation of isoproteins [17]; gels were focussed on a flat bed, between pH 4 and 6, and isoprotein distribution determined by scanning (Seroskope, Elvi, Milan, Italy). Histological
evaluation
Sections from the aorta (thoracic and abdominal segment) and liver were taken. Moreover, heart fragments were prepared, taking care to draw segments comprehensive of the myocardium, the epicardium and the initial tract of the descending branch of the left coronary. Tissues, fixed in 10% buffered formalin, were paraffin embedded. Sections (5-6 pm thick) were then stained by the common histological techniques (hematoxylin-eosin, Weigert for elastic fibers, Azan Mallory), and also by the Alcian blue technique (pH 1 and 2.5) and by PAS. Presence of lipids was evaluated by Sudan red staining of cryostat sections [18]. Results Plasma lipid-lipoprotein
findings
Cholesterol treatment (groups B and C) was associated with a rapid rise of cholesterolemia (Fig. 1). Both treatment groups showed significantly higher plasma cholesterol levels, compared to controls, already after 10 days of diet administration. On the other hand, after 24 days of treatment, there was a significantly lower cholesterolemia in P-treated animals compared to the HC group (P -C0.01). This difference remained statistically highly significant up to the end of treatment. HDL-C levels were elevated by both dietary treatments, being slightly lower in the HC + P group; however, at the end of treatment, the HDL/total cholesterol ratio was significantly higher in this latter group (P < 0.01) (Fig. 1). Plasma TG levels were only moderately modified in the 3 groups during treatment. At the end, the HC group had significantly higher levels vs controls, whereas lower levels were detected in the HC + P group, not significantly different from either of the other two (group A: 68.7 + 24.5 mg/dl; group B: 150.6 f 36.4, P < 0.05 vs A; group C: 91.3 + 14.7).
258 TABLE
1
PLASMA LEVELS OF TOTAL CHOLESTEROL AND COMPOSITION TEINS AT THE END OF THE DIETARY TREATMENTS
OF PLASMA
LIPOPRO-
Values are mean + SEM for 10 animals. Standard Total cholesterol
HC regimen
diet
1050.0*15.3
39.8k3.6
HC + pantethine 370.0,11.6
**
**+
‘K,of total VLDL (mg/dl) Total chol TG PL Protein
6.0 + 2.4 24.6 + 1.3 22.7k2.5 7.3 * 3.0
(9.9) (40.6) (37.4) (12.0)
718.2 i 10.9 ** 54.9i11.3 * 288.7 k 78.4 ** 131.2531.4
(60.2)
LDL (mg/dl) Total chol TG PL Protein
8.4+0.3 7.4* 1.2 28.7+ 1.8 5.8+0.3
(16.7) (14.7) (57.1) (11.5)
263.6i 17.9 ** 19.8iO.5 ** 136.0*5.9 ** 87.3k6.5 **
(51.1)
HDL (mg/dl) Total chol TG PL Protein
21.4 12.2 31.2 17.1
(26.1) (14.9) (38.1) (20.9)
61.2 f 17.4 ** 11.6k1.3 58.6+4.7 100.4+ 8.0 **
(26.4)
+ + + k
2.2 1.2 3.5 4.6
(4.6) (24.2) (11.0)
(4.0) (27.4) (17.6)
(5.0) (25.3) (43.3)
222.6 + 5.6 **+ 12.1 f 1.8 65.5 + 14.5 + 35.9k6.4 *+
(66.3)
100.1 + 19.2 ** + 4.3 * 1.7 + 50.5 + 11.7 ** + 33.1,6.1 **+
(53.2)
45.3* 8.8 * 41.2+ 92.6 +
11.2 ** 1.1 7.7 8.6 **
(3.6) (19.5) (10.7)
(2.3) (26.8) (17.6)
(24.1) (4.7) (21.9) (49.3)
* P < 0.05 vs controls. **P i 0.01 vs controls. **+ P
HC
HC+P
N ”
30
60
davr
90
Fig. 1. Plasma cholesterol levels in rabbits on a standard diet (N), and given cholesterol without (HC) and with pantethine (HC + P). Plasma total cholesterol levels were significantly reduced in the HC + P group after 65 days of treatment (*P -z 0.05) and more so after 90 days ( **P i 0.01). At the end of treatment a significant rise of the HDL/totaI cholesterol ratio was evident in the HC + P group.
259
Plasma lipoprotein composition was evaluated at the end of the treatments (Table 1). VLDL were characterized by a marked increase of cholesterol content in the cholesterol-fed animals, significantly reduced in the HC + P group. A reduction of the phospholipid and protein content (about 5% lower compared to HC animals) could also be detected in these animals. Similarly, the experimental diets significantly elevated LDL lipids and proteins. The reduction in LDL cholesterol, TG, PL and protein content by P was statistically significant. The percentage composition of lipoproteins showed the expected reduction of TG content in VLDL; the cholesterol enrichment in LDL occurred at the expense of PL, and only an increased protein content could be detected in HDL. P-treatment did not appear to markedly modify the composition of any of the lipoprotein fractions. HDL proteins were separated by an analytical IEF procedure [17]. Absolute amounts of apoproteins and of the A-I isoproteins changed little, with the exception of a statistically significant reduction of apo A-II and C levels. The percentage composition of HDL apoproteins, however, was dramatically modified by P administration. Both the percentage of total A-I and of its major isoprotein (apo A-I,) were significantly raised after P treatment. Conversely, the apo A-II percentage content was reduced 19.7%, also highly significant. The percent content of apo C changed moderately and no variations were noted in the other apoproteins (Table 2, Fig. 2). Tissue lipidr
Liver lipid composition (Table 3) was characterized, in both treatment groups, by a marked increase of total and esterified cholesterol content. Neither of these changes was modified by concomitant P treatment. TABLE
2
LEVELS AND PERCENTAGE DISTRIBUTION ISOPROTEINS FROM HYPERCHOLESTEROLEMIC COMITANT PANTETHINE TREATMENT
OF THE MAJOR HDL APOPROTEINS ANIMALS, WITH AND WITHOUT
Distribution data are taken from the densitometric scans of the isoelectric focusing relative chromogenicity (see Fig. 2). Values are means f SEM from 10 animals. Cholesterol
Cholesterol
Apo E Apo A-I tot. Apo A-I, Apo A-I a Apo A-II Apo C tot. Others
** ***
gels, correcting
+ pantethine
mg/dI
%
mg/dI
I
10.8 f 0.3 59.6k0.6 43.0*0.5 16.4*0.2 6.lkO.2 15.7+0.9 8.2kO.7
10.8kO.4 59.3kO.8 43.0 + 0.8 16.4kO.3 6.1 f 0.2 15.6 f 1.0 8.2kO.8
10.4 f 0.9 61.6kl.3 45.8 f 1.0 15.7*0.9 4.5*0.1+ 10.5*1.3+ 5.6 f 0.4
11.2kO.6 66.5rtl.l 49.5kO.8 17.OkO.8 4.9*0.1 11.4*1.0 6.1 k 0.6
* P < 0.05 vs cholesterol-treated P -c0.01 vs cholesterol-treated P -c0.005 vs cholesterol-treated + P < 0.05 vs cholesterol-treated
group. group. group. group.
*** *** ** *
AND CON-
for the
260 TABLE
3
LIVER LIPID LEVELS IN RABBITS ON A STANDARD CHOLESTEROL+ PANTETHINE (HC + P) REGIMENS
DIET,
CHOLESTEROL
(HC)
AND
Values are means f SEM from 10 animals mg/g
of fresh tissue
Total cholesterol Free cholesterol Est. cholesterol Free/est. chol. Triglycerides Phospholipids ** P < 0.01 vs standard
Standard diet
HC
3.5 f 0.2 2.1 f 0.1 0.9 f 0.1 2.3 k 0.2 4.5 If:0.2 0.8+0.1
13.5f0.7 4.7kO.2 7.3kO.6 0.6 + 0.1 3.8f0.2 1.0*0.1
HC+P
** ** ** **
14.2 f 0.8 5.8kO.4 8.OkO.5 0.7kO.l 4.1* 0.2 0.9*0.1
** ** ** **
diet.
A similar accumulation of cholesterol esters was noted in the aortas of diet treated animals. P treatment did not seem to modify aortic cholesterol esters (Table 4A). Histological and histochemical observations
A visual estimate of the total area of aortic lesions (lack of uptake of Tissue Blue), was suggestive of a significant reduction in the area of lesions of P-treated animals (- 18.2% compared to HC; P < 0.05) (Table 4B). In the HC treated animals, severe arterial lesions were detected both in the aorta and the anterior descending branch of the left coronary branch, being characterized by lipid deposition both intra-cellularly and in the extra-cellular space. Lipid accumulation was recognizable in the intima, but occasional intra-cellular lipid depositions could be observed in the outer
Fig. 2. Isoelectric focusing pattern of HDL apoproteins from the HC and HC + P groups. A markedly increased A-I, is clearly evident (see Table 2 for the quantitative
and isoproteins at the end of treatment in rabbits density of the protein band corresponding to apo data).
TABLE AORTIC
4A LIPID
Values are mg/g
COMPOSITION of protein,
expressed
Total cholesterol (W est. chol.) Triglycerides
AORTIC
ON THE DIFFERENT
TREATMENT
REGIMENS
as mean f SEM from 10 animals.
Standard diet
HC
3.9*0.4
18.9kO.9 (61.5) 0.2*0.1
(29) 0.1*0.1
** P < 0.01 vs standard
TABLE
IN RABBITS
HC+P
**
18.9* 1.2 ** (56.2) 0.2*0.1
diet.
48 LESIONS
IN RABBITS
ON THE DIFFERENT
Values are means f SEM from 10 animals;
X of aortic surface with visible lesions ** P c 0.01 vs standard vsHC. + P -co.05
assessment
TREATMENT
REGIMENS
by visual method.
Standard diet
HC
8.6*1.2
33.2kl.l
HC+P
**
27.1k1.2
**+
diet;
Fig. 3. Sections of abdominal aorta taken at the corresponding sites from cholesterol fed (HC) and cholesterol+pantethine (HC+ P)-treated rabbits. A:HC. Intense lipid deposition through the whole intimal thickness. Occasional lipid deposits may be observed in the media. Sudan Red, x 300; B: HC + P. The lipid deposition in the abdominal aorta is considerably reduced compared to the previous figure. Sudan Red, x300; C: HC. Myo-intimal thickening of severe degree, associated with lipid deposits and thickening. fragmented internal elastic membrane. Weigert stain, x 250; D: HC + P. Initial myo-intimal The internal elastic membrane is well preserved. Weigert stain, X 250.
262
section of the media (Fig. 3A). A significant increase of the intimal smooth muscle cell population and Alcian positive material at pH 2.5 was also recognizable. In the HC + P treated animals, lipid deposition was reduced (Fig. 3B) and, by specific methods for elastic fibers, in the aortas from HC + P rabbits, there appeared to be a significant preservation of the integrity of the basal elastic lamina, compared to HC animals (Figs. 3C and 3D). HC and HC + P rabbits did not differ in term of cell population and glycosaminoglycan deposition, as assessed from visual examination, these being present to a similar degree in both animal groups. Sections from the coronary arteries examined also showed a reduced severity of lesions in the HC + P group. In the liver, cholesterol feeding was associated with a microvacuolar steatosis in the peripheral areas of the lobule. Some reduction, of a minor degree, could be observed in the P-treated animals. Finally, no significant lesions could be observed in the myocardium. Discussion The cholesterol-lowering activity of P in an animal model resistant to treatment with standard hypolipidemic drugs [19], has been recently reported [5], and is confirmed in the present study. In addition, the reduction of the total cholesterol content of plasma and isolated lipoproteins was accompanied by an amelioration of atherosclerotic lesions. The major biochemical findings in our study are alike those reported by Tomikawa et al. [5], who followed a similar protocol for a shorter time (1 month). We also showed a reduction of the &nigrating VLDL in plasma and a relative, as well as absolute, increase of HDL cholesterolemia in treated animals. By analytical IEF, both total apo A-I and the major isoprotein (apo A-I,) were markedly increased in P-treated animals. The apo A-I, content, in a previous study [20], was significantly correlated with the cholesterol content of HDL. In contrast to Tomikawa et al. [5], we also detected a relative reduction of the apo A-II content in HDL. This finding, i.e. a higher A-I/A-II ratio in HDL, is generally consistent with an increased HDLJHDL, ratio in plasma, in humans and in other animal models [21]. The lack of demonstration of this type of distribution in the HDL subfractions of cholesterolfed rabbits, may only indicate that particles with a higher content of the activator for lecithin cholesterol acyltransferase [22] are probably increased by P treatment. We did not observe any marked changes in the TG levels in drug-treated animals, similarly to what was reported by Tomikawa et al. [5]. Findings of this type are unlikely to be obtained in this model, where triglyceridernia is generally minimally elevated. On the other hand, the results of a concomitant multicenter clinical study, indicating that changes in LDL cholesterol levels (increases or decreases) are a function of pre-treatment LDL cholesterolemia [23], suggest that P treatment may influence the breakdown of TG-rich lipoproteins. An apparent reduction of the severity of atherosclerotic lesions in rabbits receiving cholesterol with P, was present, in spite of the lack of difference in the aortic and liver cholesterol content between HC and HC + P treated rabbits. This rules out the
263
hypothesis that a reduction of atherosclerotic lesions may only be a consequence of lowered cholesterolemia in this model [24]. There also appeared to be a reduction in the extent of lesions in the coronary tree of P-treated animals. The mechanism whereby rabbits seem to be protected from the atherogenic effect of hyperlipidemia may be possibly related to the increased HDL cholesterolemia, in particular, with a raised content of the LCAT activator [22] and/or to the marked reduction in the plasma levels of atherogenic VLDL [25]. Another recently suggested mechanism may be related to a protective effect of P on tissue injury, exerted, e.g., by freezing or by immunological techniques (Yoshida Y., personal communication). Preliminary apoprotein compositional studies of VLDL failed to show any significant changes in, particularly, the content of apoprotein E. Differently, therefore, from an agent like metformin, which also markedly reduces atherosclerosis in rabbits [26], but does not affect plasma cholesterol, while exerting major structural changes in VLDL, P only increases the relative percentage of HDL and possibly also of HDL,. Acknowledgements
Prof. M. Riva, Drs. C. Donati and L. Zanardi (Maggioni S.p.A., Milan) are gratefully acknowledged for the supply of pantethine and for constant help and encouragement in these studies. References 1 Abiko, Y., Metabolism of &enzyme A. In: D.M. Greenberg (Ed.), Metabolic Pathways, Vol. 7, Academic Press, New York, NY, 1975, pp. l-25. 2 Avogaro, P., Bittolo Bon, G. and Fusello, M., Effect of pantethine on lipids, lipoproteins and apolipoproteins in man, Curr. Ther. Res., 33 (1983) 488. 3 Shinomiya, M., Matsuoka, N., Shirai, K., Nobuhiro, M., Sasaki, N., Murano, S., Saito, Y. and Kumagai, A., Effect of pantethine on cholesterol ester metabolism in rat arterial wall, Atherosclerosis, 36 (1980) 75. 4 Kameda, K. and Abiko, Y., Stimulation of fatty acid metabolism by pantethine. In: D. Cavallini et al. (Eds.), Natural Sulfur Compounds - Novel Biochemical and Structural Aspects, Plenum Press, New York,.NY, 1980, pp. 443-452. 5 Tomikawa, M., Nakayasu, T., Twara, K., Kaneda, K.Y. and Abiko, Y., Effect of pantethine on lipoprotein profile and HDL subfractions in experimental hypercholesterolemic rabbits, Atherosclerosis, 41 (1982) 267. 6 Allain, C.C., Poon, L.S., Chan, C.S.G., Richmond, W. and Fu, P.C., Enzymatic determination of total serum cholesterol, Clin. Chem., 20 (1974) 470. 7 Bucolo, G. and David, H., Quantitative determination of serum triglycerides by use of enzymes, Clin. Chem., 19 (1973) 476. 8 Havel, R.J., Eder, H.A. and Bragdon, J.H., Distribution and composition of ultracentrifugally separated lipoproteins in human serum, J. Clin. Invest., 34 (1955) 1345. 9 R&&au, P., Bemt, E. and Grtiber, W., Enzymatische Bestimmung Gesamtcholesterins in Serum, 2. Klin. Chem. Khn. B&hem., 12 (1974) 226. 10 Lowry, O.H., Rosebrough, H.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265. 11 Helenius, A. and Simons, K., Removal of lipids from human plasma low-density lipoproteins by detergents, Biochemistry, 10 (1971) 2542.
264 12 Rouser, G., Yamamoto, A. and Fleischer, S., Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots, Lipids, 5 (1970) 494. 13 Folch, P.J., Lees, M. and Sloane Stanley, C.H., A single method for isolation and purification of total lipids from animal tissue, J. Biol. Chem., 226 (1957) 497. 14 Zlatkis, A., Zak, B. and Boyle, A.J., A new method for the direct determination of serum cholesterol, J. Lab. Clin. Med., 41 (1953) 486. 15 Gottfried, S.P. and Rosenberg B., Improved manual spectrophotometric procedure for determination of serum triglycerides, Clin. Chem., 19 (1973) 1977. 16 Albert, E.N., Kassira, W.N., Muesing, R. and Vahouny, G.V., Pyridinolcarbamate and experimental atherosclerosis, Atherosclerosis, 31 (1978) 205. 17 Gidez, L.I., Swaney, J.B. and Mumane, S., Analysis of rat serum apolipoproteins by isoelectric focusing, Part 1 (Studies on the middle molecular weight subunits), J. Lipid Res., 18 (1977) 59. 18 Coutard, M. and Osborne-Pellegrin, M.J., Spontaneous lesions in the rat caudal artery, Atherosclerosis, 44 (1982) 245. 19 Brattsand, R., The effect of niceritrol (pentaerythritoltetranicotinate) and clofibrate upon hyperlipemia and atherosclerosis induced in rabbits by cholesterol-free semisynthetic diets, Atherosclerosis, 20 (1974) 453. 20 Franceschini, G., Sirtori, M., Gianfranceschi, G. and Sirtori, C.R., Relation between HDL apoproteins and AI isoproteins in subjects with the AI-Milan0 abnormality, Metabolism, 30 (1981) 502. 21 Kostner, G.M., Patsch, J.R., Sailer, S., Braunsteiner, H. and Holasek, A., Polypeptide distribution of the main lipoprotein density classes separated from human plasma by rate zonal ultracentrifugation, Europ. J. B&hem., 45 (1974) 611. 22 Albers, J.J., Cabana, V.G. and Stahl, Y.D.B., Purification and characterization of human plasma lecithin-cholesterol acyltransferase, Biochemistry, 15 (1976) 1084. 23 Gaddi, A., Descovich, G.C., Noseda, G., Fragiacomo, C., Colombo, L., Craveri, A., Montanari, G. and Sirtori, C.R., Controlled evaluation of pantethine, a natural hypolipidemic compound, in patients with different forms of hyperlipoproteinemia, Atherosclerosis, 50 (1984) 73. 24 Brattsand, R., Petersen, H. and Lundholm, L., Action of niceritrol (pentaerythritoltetranicotinate) on lipid accumulation in aortas of cholesterol-fed rabbits independent of contemporary reduction of serum lipids, Atherosclerosis, 20 (1974) 469. 25 Mahley, R.W., Atherogenic hyperlipoproteinemia - The cellular and molecular biology of plasma lipoproteins altered by dietary fat and cholesterol, Med. Clin. N. Amer., 66 (1982) 375. 26 Sirtori, C.R., Catapano, A., Ghiselli, G.C., Innocenti, A.L. and Rodriguez, J., Metformin - an antiatherosclerotic agent modifying very low density lipoproteins in rabbits, Atherosclerosis, 26 (1977) 79.