Guarnierí G, Panetta G, Toigo G (eds): Metabolic and Nutritional Abnormalities in Kidney Disease. Contrib Nephrol. Basel, Karger, 1992, vol 98, pp 11-19

Apolipoprotein B-Containing Lipoprotein Particles in Progressive Renal Insufficiency Per-OlaAttmana, Marcelo Tavellab, Carolyn Knight-Gibsonb, Ola Samuelsson a, Petar Alaupovicb a Department of Nephrology, University of Göteborg, b Lipoprotein and Atherosclerosis Research Program,

Sweden, and

Abnormal lipid transport is one of the characteristic metabolic features of chronic renal failure (CRF). Although the dyslipoproteinemia of CRF may be manifested as a mild to moderate hypertriglyceridemia with or without increased cholesterol levels, its frequently encountered latent state may not be recognized by changes in plasma lipid concentrations [1]. Studies on lipoprotein density classes have indicated that subtle inverse changes in the levels of very low (VLDL) and high density lipoproteins (HDL) coupled with insignificant changes in the levels of low density lipoproteins (LDL) may be one of the main reasons for the masked dyslipoproteinemia in a substantial number of patients with CRF who have only been characterized by measurement of lipid profile [2]. However, the apolipoprotein profiling has proved to be a very useful means for characterizing dyslipoproteinemia in patients with renal insufficiency [3, 4]. These studies have shown that, regardless of triglyceride concentration, increased levels of ApoC-III are the most characteristic feature of this dyslipoproteinemic state detectable already during the earlier stages of renal insufficiency [4, 5]. A shift in the distribution of ApoC-III from HDL to VLDL was interpreted as an additional evidence for an altered relationship between VLDL and HDL particles in patients with CRF [3, 5]. Recent studies from this and other laboratories have shown that the chemical and metabolic heterogeneity of major lipoprotein density classes is due to the presence of distinct lipoprotein particles characterized by specific apolipoprotein composition rather than nonspecific physical properties [6]. Because apolipoproteins are the determinants of structural integ-

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Oklahoma Medical Research Foundation, Oklahoma City, Okla., USA

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rity and functional specificity of lipoprotein particles, we have proposed that they be used as markers for identifying and characterizing plasma lipoproteins. Accordingly, there are two major classes of lipoproteins, one of which is characterized by ApiA and the other by ApoB as their major apolipoprotein constituents. By applying this conceptual approach to the characterization of ApoB-containing lipoproteins, we have established that this group of lipoproteins consists of cholesterol-rich lipoprotein Β (LP-B) and triglyceride-rich lipoprotein B:C-I:C-II:C-III (LP-B:C) and lipoprotein B:C-L•C-II:C-IIL•E (LP-B:C:E) [6] as the major lipoprotein families. The purpose of this study was to characterize the dyslipoproteinemia of progressive renal insufficiency on the basis of distinct ApoB-containing lipoprotein families and to correlate their concentration profiles with the progression and vascular complications of this disease. Experimental Procedures Patients Thirteen patients (8 men, 5 women) were selected for this study from a larger population of patients with chronic renal insufficiency described earlier [5]. Their mean age was 55 ± 13 years. Seven patients were in the conservatively treated predialytic stage with mild to moderate uremic symptoms. The mean glomerular filtration rate (GFR) was 6.9 ± 2.8 ml/min/1.73 m2 body surface area and the mean body mass index (BMI) was 29.2 ± 5.6 kg/m2. Six patients were on a regular hemodialysis treatment for 2-117 months and had a mean predialysis serum creatinine value of 931 ± 280 µmol/1, a serum urea value of 25.0 ± 7.3 mmol/1 and a mean BIΙ of 23.3 ± 6.5 kg/m2. Nine patients were treated with (3-blocking agents for hypertension. None of the patients had diabetes mellitus and none was on immunosuppressive treatment. Four patients had clinically manifested vascular disease (myocardial infarction 3, peripheral arterial disease 1). Fifteen healthy normolipidemic subjects (7 men, 8 women, age 47 ± 12 years) recruited as described earlier [5] served as controls.

Fractionation of ApoB-Containing Lipoprotein Families Separation of LP-B, LP-B:C and LP-B:C:E particles was performed by sequential immunoprecipitation of VLDL, IDL and LDL with polyclonal antisera to ApoE and ApoC-III according to a previously described procedure [8]. The concentrations of these three ApoB-containing lipoprotein families were expressed in terms of their ApoB con-

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Isolation of Lipoprotein Density Classes Blood samples were obtained from patients and normal controls after an overnight fast of 12 h. Blood was drawn into tubes that contained EDTA, and the plasma samples were collected by low-speed centrifugation. The VLDL (d < 1.006 g/ml), intermediate density lipoproteins (IDL, d 1.006-1.019 g/ml) and LDL (d 1.019-1.063 g/ml) were isolated by sequential ultracentrifugation as previously described [7].

Lipoprotein Particles in Renal Failure

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tents (mg/dl plasma). The recovery of ApoB ranged between 60 and 95% of plasma ApoB levels; all ApoB concentrations were corrected to that of plasma ApoB. Lipid and Apolipoprotein Analyses Total cholesterol and triglyceride were determined by enzymic procedures [3] in the plasma and by a gas chromatographic method [9] in lipoprotein density classes. The HDL-C was estimated as previously described [5]. Apolipoproteins A-I, A-II, Β, C-II, C-III and E were measured by previously described electroimmunoassays [3, 5]. The preparation and characterization of antigens and polyclonal antisera were previously reported [7].

Results and Discussion Mean triglyceride levels were increased in both patient groups (table 1). In the present series only 5/13 patients (39%) had plasma triglyceride values lower than 150 mg/dl (1.7 mmol/1). There was no significant increase in the total cholesterol and no significant decrease in the mean HDL cholesterol as is often described in patients with CRF [2, 3]. However, 5 patients had HDL cholesterol values < 40 mg/dl (1.04 mmol/1). The apolipoprotein profile was characterized by a significant increase in the concentration of ApoC-III in both patient groups. All but 1 patient had

Table 1. Plasma lipid and apolipoprotein profiles of patients with CRF before and during dialysis Subjects

Lipids, mmol/1

Apolipoproteins, mg/dl

choiesterol

triglycelides

HDL cholesterol

A-I

A-II

Β

C-II

C-III

E

CRF before dialysis (n = 7)

5.93 (1.76)

2.36* (1.48)

1.19 (0.49)

86** (26)

44** (9)

143* (68)

4.1 (1.4)

19.3** (7.3)

7.9 (1.7)

CRF hemodialysis (n = 6)

6.27 (2.22)

2.27* (1.40)

1.40 (0.44)

120* (30)

60 (23)

116 (49)

4.6 (1.2)

21.6** (4.2)

14.4* (4.8)

Control (n = 15)

5.23 (1.09)

0.90 (0.43)

1.35 (0.36)

162 (33)

68 (14)

94 (261)

3.3 (1.1)

8.9 (2.1)

9.7 (2.3)

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Mean ± (SD). Significance of difference vs. controls: * p < 0.05; ** p < 0.01.

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plasma ApoC-III levels higher than 1 SD of controls (11.0 mg/dl). The mean concentrations of ApoB were moderately increased in the predialytic patients and to a lesser extent in hemodialysis patients who, in contrast to predialysis patients, had a significant elevation of ApoE levels. These variations in the apolipoprotein profile of patients before and during dialysis already reported from our laboratory [5] are in agreement with the reports by others regarding the increased ΑροΕ concentrations in hemodialysis patients [ 10]. There is no readily available explanation for this variation in ΑροΕ levels between the two uremic patient groups. In view of the importance ascribed to ΑροΕ for recognition and uptake of metabolized lipoproteins [1 1 ], this finding could reflect a partial amelioration of lipoprotein abnormalities during dialysis. However, because the underlying mechanism(s) resulting in this change has not been clarified, it is also possible that the dialysis procedure may not be the only factor responsible for this difference. Predialytic patients had a more pronounced reduction in the levels of ApoA-I and ΑροΑ-II than patients during dialysis, indicating that de-

Table 2. Lipid and apolipoprotein mass in density classes of patients with CRF before and during dialysis Subjects

Lípids, mg/dl

Apolipoproteins, mg/dl

Total, mg/dl

VLDL+IDL LDL

VLDL+IDL LDL

VLDL+IDL LDL

CRF before dialysis n

174.9** (118.5) 7

153.2 (46.1) 7

46.2*** (28.7) 7

84.2 (23.5) 7

221.1** (146.7) 7

225.4 (58.2) 7

CRF hemodialysis n

142.6* (92.3) 5

149.0 (85.3) 5

37.8*** (22.7) 5

81.6 (24.2) 5

180.4** (114.9) 5

230.6 (108.2) 5

Control

60.2 (42.3) 15

115.1 (42.7) 13

11.9 (5.9) 15

63.9 (24.4) 13

72.1 (45.9) 15

182.7 (61.9) 13

n

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Mean ± (SD). Lipid mass is the sum of triglyceride and cholesterol concentrations. Apolipoprotein mass is the sum of ApoB, ApoC-I, ApoC-II, ApoC-III and ΑροΕ in VLDL + IDL and of ΑροΑ-I, ΑροΑ-II, ApoB, ApoC-I, ApoC-II, ApoC-III and ΑροΕ in LDL. Significance of difference vs. controls: * p < 0.05; ** p < 0.01; *** p < 0.001.

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Lipoprotein Particles in Renal Failure

creased concentrations of ApoA-containing lipoproteins is a characteristic feature of advanced renal failure particularly in patients before dialysis [4, 5]. Lower levels of ApoA-I and Α-II may also be found during earlier stages of renal insufficiency [4, 5]. Table 2 shows the distribution and concentrations of lipids (triglyceride and cholesterol) and apolipoproteins (A-I, Α-II, Β, C-I, C-II, C-III and E) in VLDL and LDL of uremic patients in predialytic and dialytic states. There was a three-fold increase in the lipid and apolipoprotein mass of VLDL + IDL, but no change in the corresponding mass of LDL. These changes were less pronounced in patients during dialysis than in patients before dialysis. The increased lipid mass was due to increases in both triglyceride and cholesterol contents. The elevated apolipoprotein mass resulted from increased concentrations of apolipoproteins Β, C and Ε in VLDL + IDL with a proportionally greater increase in ΑρoC-III than ApoE. These findings have confirmed and expanded previous results indicating that renal insufficiency is characterized by a predominant accumulation of ApoB-containing lipoproteins of very low densities enriched not only in lipid constituents but also in ApoC peptides and ΑροΕ [1, 2, 10, 12]. To explore the chemical nature of ApoB-containing lipoproteins accumulating in plasma of patients with CRF, the corresponding VLDL, IDL and LDL were fractionated by sequential immunoprecipitation with antisera to ΑροΕ and ApoC-III. The total amount of ApoB-containing lipoproteins, expressed in terms of ApoB concentrations, was higher in patients before and during dialysis than in normal controls (table 3). However, this Table 3. Concentrations of ApoB-containing lipoprotein particles in plasma of patients with CRF before and during hemodialysis Subjects

LP-B, mg/dl

LP-B:C, mg/dl LP-B:C:E, mg/dl

CRF before dialysis (n = 7)

84.5 (28.0)

31.2** (19.7)

21.9 (12.8)

CRF hemodialysis (n = 6)

73.5 (37.7)

9.9 (6.6)

31.9* (34.6)

Controls (n = 15)

74.4 (19.2)

6.6 (5.4)

12.8 (8.7)

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Values are mean ± (SD) expressed in terms of ApoB concentrations. Significance of difference vs. controls: * p < 0.05; ** p < 0.001.

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increase was mainly due to elevated concentrations of triglyceride-rich LPB:C and LP-B:C:E with no significant change in the levels of cholesterolrich LP-B particles. Patients on hemodialysis differed from patients before dialysis by having lower concentrations of LP-B:C and higher concentrations of LP-B:C:E particles. The identification of LP-B, LP-B:C and LP-B:C:E particles in VLDL, IDL and LDL clearly shows the polydisperse character of all three major ApoB-containing lipoprotein families. Although in normolipidemic subjects the cholesterol-rich LP-B is present mainly in LDL, in uremic patients more than 20% of LP-B particles are detected in VLDL and IDL. In normolipidemic subjects, partially delipidized triglyceride-rich lipoproteins LP-B:C and LP-B:C:E (LP-Be) are also detectable mainly in the low density range. However, in patients with renal insufficiency, these lipoprotein families retain their triglyceride complement to a greater extent than in normolipidemic subjects. Consequently, a greater proportion of LP-BO particles is present in VLDL and IDL of uremic patients than normal controls. It is quite possible that increased concentrations of cholesterol-rich LP-B particles may be the main reason for the already reported increased cholesterol ester content of VLDL in patients with CRF [2]. Conversely, the incompletely delipidized LP-BU particles may be the main contributors to the increased triglyceride content of LDL in these patients [2]. Results of kinetic and metabolic studies suggest that an abnormal transport and catabolism of triglyceride-rich lipoproteins may be the major underlying cause for dyslipoproteinemia in CRF [ 1 ]. The efficiency of catabolic processes is reflected, among others, in the measurement of ApoCIII ratio [ 13]; the higher the ratio of ApoC-III in heparin supernate/ApoCIII in heparin precipitate, the higher the efficiency of these processes. In both normolipidemic and hyperlipidemic subjects, there is a close relationship between the ApoC-III ratio and lipoprotein lipase (LPL) activity [13]. The ApoC-III ratio was markedly reduced in both predialytic and hemodialysis patients (0.32 ± 0.54 and 0.38 ± 0.24 vs. 1.95 ± 1.08). This finding emphasized the significance of decreased degradation and removal of triglyceride-rich ApoB-containing lipoproteins as the most probable cause for the accumulation of LP-B:C and LP-B:C:E particles in CRF. The reduced catabolism may result from decreased activities of l ροlytic enzymes and/or impaired recognition and uptake of lipoprotein particles by receptors. The increased ratio of ApoC-III/ ΑροΕ reflects most probably the increased concentration of LP-B:C particles which may be less readily recognized and cleared by the B:E receptors [14] and, thus,

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Attman/Tavella/Knight-Gibson/Samuelsson/Alaupovic

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more susceptible to alternative pathways for their removal such as the nonreceptor-mediated uptake by macrophages. The clinical significance of the altered lipoprotein profile in CRF is not fully elucidated. The abnormal profile of lipoprotein particles may be related not only to atherogenesis and development of vascular disease but also to further progression of glomerulosclerosis and renal insufficiency [ 15, 16]. In contrast to the ApoA- containing lipoproteins, the ApoB-contaíning lipoproteins have been considered as potentially atherogenic. However, the relative atherogenicity of discrete ApoB- containing lipoproteins has not yet been established. In the present study, 4 patients with vascular disease had higher concentrations of LP-B:C and LP-B:C:E than patients without symptoms of a major vascular disease (table 4). There was no difference in LP-B concentrations. This observation corresponds with our previous reports indicating that CRF patients with vascular disease have higher levels of triglycerides, ApoB and ApoC-III than patients without vascular disease [3, 5]. Increased levels of triglyceride-rich ApoB- containing lipoproteins were associated with a more rapid progression of coronary artery disease in nonrenalpatients in the CLASstudy [17]. It has also been reported that CRF patients with increased plasma levels of triglycerides and ApoB have a more rapid progression of renal insufficiency than patients with lower plasma ApoB and triglyceride concentrations [18]. In summary, results of this preliminary study have shown that the dyslipoproteinemia in patients with renal insufficiency before and during dialysis is characterized by significantly increased levels of triglyceriderich LP-B:C and LP-B:C:E but normal or only slightly increased levels of

Table 4. Plasma profile of ApoB-containing lipoprotein particles in patients with CRF with and without vascular disease Patients

LP-B mg/dl

LP-B:C mg/dl

LP-B:C:E mg/dl

LP-B/LP-BU

Vascular disease (n=4)

76.1 (42.5)

34.6 (21.9)

42.9 (41.6)

1.07 (0.44)

No vascular disease (n = 8)

78.4 (30.0)

16.6 (14.4)

19.9 (20.0)

2.59 (1.43)

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Values are mean ± (SD) expressed in terms of ApoB concentrations.

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cholesterol-rich LP-B particles. The clinical significance of increased levels of LP-B:C and/or LP-B:C:E particles in the development of atherosclerotic vascular disease and glomerulosclerosis remains to be established in future studies. Acknowledgements This work was supported by the Swedish Medical Research Council, Henning och Johan Throne-Holsts stiftelse fdr vetenskaplig forskning, John och Brit Wennerströms forskningsstiftelse fór njursjukdomar, University of Göteborg, Svenska Läkaresällskapet, and by a grant (HR 1-005) from the Oklahoma Center for the Advancement of Science and Technology, State of Oklahoma.

1 Attman PO, Alaupovic P: Lipid abnormalities in chronic renal insufficiency. Kidney Int 1990;39(suρρ131):S16—S23. 2 Norbeck HE, Carlson LA: The uremic dyslipoproteinemia: Its characteristics and relations to clinical factors. Acta Med Scand 1981;209:489-503. 3 Attman PO, Alaupovic P, Gustafson A: Serum apolipoprotein profile of patients with chronic renal failure. Kidney Int 1987;32:368-375. 4 Griitzmacher P, März W, Peschke B, Gross W, Schoeppe N: Lipoproteins and apolipoproteins during the progression of chronic renal disease. Nephron 1988;50:103111. 5 Attman PO, Alaupovic P: Lipid and apolipoprotein profiles of uremic dyslipoproteinemia. The relation to renal function and dialysis. Nephron 1991;57:401-410. 6 Alaupovic P: Apolipoprotein composition as the basis for classifying plasma l ρoproteins. Characterization of ApoA- and ApoB-containing lipoprotein families. Prog Lipid Res 1991;30:105-138. 7 Alaupovic P, Lee DM, McConathy WJ: Studies on the composition and structure of plasma lipoproteins. Distribution of lipoprotein families in major density classes of normal human plasma lipoproteins. Biochim Biophys Acta 1972;260:689-707. 8 Alaupovic P, Tavella M, Fesmire J: Separation and identification of ApoB-containing lipoprotein particles in normolipidemic subjects and patients with hyperlipoproteinemias. Adv Exp Med Bul 1987;210:7-14. 9 Kuksis A, Myher JJ, Marai L, Geher L: Determination of plasma lipid profiles by automated gas chromatography and computerized data analysis. J Chromatogr Sci 1975;13:423-430. 10 Nestel PJ, Fidge NH, Tan ΜΗ: Increased lipoprotein-remnant formation in chronic renal failure. N Engl J Med 1982;307:329-333. 11 Mahley RW: Apolipoprotein E: Cholesterol transport protein with expanding role in cell biology. Science 1988;240:622-630. 12 Attman PO, Alaupovic P: Abnormalities of lipoprotein composition in renal insufficiency. Prog Lipid Res 1991;30:275-279.

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References

Lipoprotein Particles in Renal Failure

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Per-Ola Attman, MD, PhD, University of Göteborg, Department of Nephrology, Sahlgrenska Sjukhuset, S-413 45 Göteborg (Sweden)

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13 Alaupovic P: The biochemical and clinical significance of the interrelationship between very low density and high density lipoproteins. Can J Biochem 1981;59: 565-579. 14 Agnani G, Bard JM, Candelier L, Delattre S, Fruchart JC, Clavey V: Interaction of LpB, LpB:E, LpB:C-III and LpB:C-III:E lipoproteins with the low density lipoprotein receptor of HeLa cells. Arterioscler Thromb 1991;11:1021-1029. 15 Moorhead JF, Chan MK, El Nahas M, Varghese Z: Lipid nephrotoxicity in chronic progressive glomerular and tubulo-interstitial disease. Lancet 1982;i:1309-1311. 16 Keane WF, Mulcahy WS, Kasiske BL, Kim Y, l'Donnel MP: Hyperlipidemia and progressive renal disease. Kidney Int 1991;39(suppl 31):S41—S48. 17 Blankenhorn DH, Alaupovic P, Wickham MS, Chin HP, Azen SP: Prediction of angíographic change in native human coronary arteries and aorto-coronary bypass grafts. Circulation 1 990;81:470-476. 18 Attman PO, Samuelsson O, Alaupovic P: ApoB-containing lipoproteins and progression of renal insufficiency (abstract). VI Int Cong Nutrition and Metabolism in Renal Disease, Harrogate, 1991.

Apolipoprotein B-containing lipoprotein particles in progressive renal insufficiency.

Guarnierí G, Panetta G, Toigo G (eds): Metabolic and Nutritional Abnormalities in Kidney Disease. Contrib Nephrol. Basel, Karger, 1992, vol 98, pp 11-...
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