Hypoalbuminemia increases lysophosphatidylcholine in low-density lipoprotein of normocholesterolemic subjects

Kidney International, Vol. 55 (1999), pp. 1005–1010

Hypoalbuminemia increases lysophosphatidylcholine in low-density lipoprotein of normocholesterolemic subjects THI DANH VUONG, ERIC S.G. STROES, NEL WILLEKES-KOOLSCHIJN, TON J. RABELINK, HEIN A. KOOMANS, and JAAP A. JOLES Department of Nephrology and Hypertension, Utrecht University, Utrecht, The Netherlands

Hypoalbuminemia increases lysophosphatidylcholine in lowdensity lipoprotein of normocholesterolemic subjects. Background. A phospholipid, lysophosphatidylcholine (LPC), is the major determinant of the atherosclerotic properties of oxidized low-density lipoprotein (LDL). Under normal circumstances most LPC is bound to albumin. We hypothesized that lipoprotein LPC concentrations are increased in hypoalbuminemic patients with the nephrotic syndrome, irrespective of their lipid levels. To test this hypothesis, we selected nephrotic and control subjects with matched LDL cholesterol levels. Methods. Lipoproteins and the albumin-rich lipoproteindeficient fractions were separated by ultracentrifugation and their phospholipid composition was analyzed by thin-layer chromatography. Results. Nephrotic subjects (albumin 23 6 2 g/liter and LDL cholesterol 3.1 6 0.2 mmol/liter) had a LDL LPC concentration that was increased (P , 0.05) to 66 6 7 vs. 35 6 6 mmol/ liter in matched controls (albumin 42 6 5 g/liter and LDL cholesterol 3.1 6 0.2 mmol/liter). LPC in very low-density lipoprotein plus intermediate-density lipoprotein (VLDL 1 IDL) in these subjects was also increased to 33 6 7 vs. 9 6 2 mmol/liter in controls (P , 0.05). Conversely, LPC was decreased to 19 6 4 mmol/liter in the albumin-containing fraction of these hypoalbuminemic patients, as compared to 46 6 10 mmol/liter in the controls (P , 0.05). LPC was also low (14 6 4 mmol/liter) in the albumin-containing fraction of hypoalbuminemic, hypocholesterolemic patients with nonrenal diseases. In hyperlipidemic nephrotic subjects (albumin 21 6 2 g/liter and LDL cholesterol 5.7 6 0.5 mmol/liter) the LPC levels in LDL and VLDL 1 IDL were further increased, to 95 6 20 and 56 6 23 mmol/liter, respectively (P , 0.05). Conclusion. These findings suggest that in the presence of hypoalbuminemia in combination with proteinuria, LPC shifts from albumin to VLDL, IDL and LDL. This effect is independent of hyperlipidemia. Increased LPC in lipoproteins may be an important factor in the disproportionate increase in cardiovascular disease in nephrotic patients with hypoalbuminemia.

Key words: hypoalbuminemia, atherosclerosis, nephrotic syndrome, proteinuria, hyperlipidemia. Received for publication February 17, 1998 and in revised form September 22, 1998 Accepted for publication September 22, 1998

 1999 by the International Society of Nephrology

Elevated plasma levels of low-density lipoprotein (LDL) have been associated with the development of atherosclerosis [1]. The interphase between these conditions is considered to be oxidation of LDL in the artery wall [2]. During this process of oxidation phosphatidylcholine is extensively hydrolyzed to lysophosphatidylcholine (LPC) by a phospholipase A2 called plateletactivating factor acetylhydrolase that exists in plasma largely in association with LDL [3–5]. LPC is present in a concentration of about 500 nmol/mg protein in oxidized LDL, whereas in native LDL derived from healthy subjects concentrations of only 25 nmol/mg protein have been found [6, 7]. Lysophosphatidylcholine has been proposed as an important determinant of the atherosclerotic properties of oxidized LDL [8], although it should be noted that many other chemical changes are induced by oxidation which also contribute to the biological effects of oxidized LDL [8, 9]. Impairment of endothelial reactivity, caused by oxidized LDL in vitro, was abolished by LPC depletion [10]. Other effects of LPC on the endothelium include superoxide production [11–13], activation of protein kinase C [11, 14, 15], and increased expression of leukocyte adhesion molecules [7]. Some increases in LDL LPC content have been identified in patients with dyslipidemia due to diabetes [16, 17], or familial hypercholesterolemia [18]. However, whether increased LPC levels can occur in circulating LDL in normolipidemic patients and whether this is associated with detrimental effects is unknown. Approximately 10% of total human plasma phospholipid consists of LPC [17, 19–21]. About 80% of this LPC [20] is tightly bound to albumin [19, 22]. However, on a molar basis the capacity and affinity for LPC is much higher for LDL than for albumin [23]. Indeed, studies with both nephrotic and analbuminemic rats with dyslipidemia demonstrated that most of the LPC normally bound to albumin was shifted to lipoproteins [24]. If increased LPC levels in circulating LDL have adverse effects, insight into the separate effects of hyperlipidemia and hypoalbuminemia on the distribution of LPC in lipo-

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Vuong et al: LDL lysophosphatidylcholine in hypoalbuminemia Table 1. Clinical characteristics NS-H

Sex (M/F) Age years Total protein g/liter Albumin g/liter Cholesterol mmol /liter Phosphoipids mmol /liter Triglycerides mmol /liter Creatinine lmol /liter Proteinuria g/day

7/1 42 (25–57) 47.6 6 2.9a 20.7 6 1.9b 10.05 6 1.64a 4.28 6 0.56b 3.6 6 1.0a 78.5 6 9.5 6 (2–7)

NS-N

CON

NRD

SNA

6/1 46 (24–57) 55.1 6 1.4a 23.2 6 1.5b 5.46 6 0.58 2.74 6 0.20 2.1 6 0.5 114.7 6 23.0 8 (1–16)

6/0 32 (25–50) 66.0 6 2.8 42.1 6 4.7 4.95 6 0.27 2.52 6 0.09 1.5 6 0.3 81.7 6 7.6 ND

4/4 52 (17–79) 52.3 6 3.3a 24.2 6 1.1b 3.18 6 0.30a 2.28 6 0.20 1.8 6 0.4 75.1 6 13.0 ND

4/2 41 (22–60) 55.7 6 3.3a 29.1 6 1.0b 6.68 6 0.89 3.17 6 0.39 1.7 6 0.3 110.7 6 16.2 7 (2–10)

Data are means 6 sem, except for age and proteinuria which are presented as median and range. Abbreviations are: NS-H, hyperlipidemic nephrotic syndrome; NS-N, normolipidemic nephrotic syndrome; CON, control subjects; NRD, non-renal diseases; SNA, sub-normal albumin; ND, not determined. a P , 0.05 vs. control b P , 0.01 vs. control

proteins will have important implications for therapeutic strategies. In hyperlipidemic patients with the nephrotic syndrome we found increased LPC in LDL, in comparison to controls with normal lipid and albumin levels [21]. However, it is not clear whether this was due to hyperlipidemia or hypoalbuminemia. To test the hypothesis that lipoprotein LPC concentrations are increased in hypoalbuminemic patients irrespective of their lipid levels, we utilized larger groups of subjects to select nephrotic and control subjects with matched total plasma phospholipid and LDL cholesterol levels. Furthermore, we also studied patients with low albumin as well as low cholesterol levels due to nonrenal diseases. METHODS Subjects Lipoprotein composition was measured in five groups. Three groups were formed from a cohort of 21 hypoalbuminemic patients with renal disease: a group of patients with hyperlipidemic nephrotic syndrome (NS-H, N 5 8), a group with normolipidemic nephrotic syndrome (NS-N, N 5 7), and a group with subnormal albumin (SNA, N 5 6). We also studied patients with nonrenal diseases (NRD, N 5 8), and healthy controls (CON, N 5 6). The inclusion criteria were: for the NS patients, plasma albumin , 26 g/liter; NRD patients, plasma albumin , 30 g/liter; SNA patients, plasma albumin 26–35 g/liter; and CON, plasma albumin . 35 g/liter and total phospholipids and LDL cholesterol matched to the NS-N. The latter were selected from a large group of healthy controls. Clinical characteristics are summarized in Table 1. The histologic diagnosis of patients with NS-H was membranous glomerulophathy (N 5 4), and minimal lesions (N 5 4). Of the NS-N patients five had membranous glomerulopathy, one chronic transplantation nephropathy, and one had minimal lesions. Of the NRD patients one had short bowel syndrome, one pancreas necrosis, one liver cirrhosis and pancreatitis, one

Crohn’s disease with protein depletion, one acute respiratory distress syndrome in association with multiple myeloma, one neuromuscular disease, one hemihepatectomy after an isolated metastasis, and one sepsis with perforated diverticulitis. Of the SNA patients, one had membranous glomerulopathy, one focal segmental glomerulosclerosis, one minimal change lesion, one mesangioproliferative glomerulonephritis, and two IgA-nephritis. Protocol Blood was collected, after an overnight fast, in chilled K-EDTA coated tubes and immediately centrifuged at 48C for 10 minutes at 1000 3 g. Lipoproteins were isolated from fresh plasma. The study protocol was approved by the Utrecht University Hospital Ethics Committee for study in human beings. Patients and subjects gave written informed consent after explanation of the protocol. Laboratory parameters Albumin, creatinine, cholesterol, phospholipid, and triglyceride determination. Plasma albumin was determined by immuno-electrophoresis. Standard dye-binding methods tend to overestimate albumin concentrations during hypoalbuminemia because of aspecific binding to globulins [25]. Plasma creatinine was determined colorimetrically. Cholesterol, triglyceride (Boehringer GmbH, Mannheim, Germany) and phospholipid (bioMe´rieux, Marcy-l’Etoile, France) concentrations were assayed enzymatically. Lipoprotein isolation by density-gradient ultracentrifugation. Plasma lipoproteins and the albumin-rich lipoprotein-deficient fractions were separated by densitygradient ultracentrifugation into four fractions: very lowdensity lipoprotein (VLDL) plus intermediate-density lipoprotein (IDL), density (d) , 1.019 g/ml; low-density lipoprotein (LDL), d 1.019 to 1.063; high-density lipoprotein, HDL, d 1.063 to 1.21 g/ml and lipoprotein-deficient plasma (LDP) with a d . 1.21 g/ml) [24].

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Vuong et al: LDL lysophosphatidylcholine in hypoalbuminemia Table 2. Lipoprotein cholesterol (mmol/liter)

N VLDL 1 IDL LDL HDL LDP

NS-H

NS-N

CON

NRD

SNA

8 2.41 6 0.85a 5.72 6 0.49b 1.61 6 0.18 0.07 6 0.03

7 1.23 6 0.27a 3.09 6 0.23 1.06 6 0.11 0.05 6 0.01

6 0.45 6 0.06 3.11 6 0.18 1.22 6 0.15 0.03 6 0.01

8 0.54 6 0.11 1.80 6 0.24b 0.76 6 0.13 0.06 6 0.01

6 1.09 6 0.24 3.88 6 0.50 1.53 6 0.26 0.04 6 0.01

Data are mean 6 sem. Groups as in Table 1. Abbreviations are: TOT, total cholesterol; VLDL 1 IDL, a combination of very low-density and intermediatedensity lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein; LDP, lipoprotein-deficient plasma. a P , 0.05 vs control b P , 0.01 vs control

Phospholipid composition of lipoproteins and the albumin-rich LDP fraction analyzed by thin-layer chromatography. Phospholipids were extracted in a stepwise fashion. First with a mixture of methanol (2.53 3 fraction volume) and chloroform (1.27 3 fraction volume), then with chloroform and water (both 1.27 3 fraction volume). The chloroform fraction was then evaporated, and the residue dissolved in 2 ml chloroform:methanol (2:1, vol:vol). Phosphorus content was determined with a modified Bartlett procedure [26]. Phospholipid species were separated by thin-layer chromatography. An aliquot containing 300 nmol phosphorus was evaporated, and the residue dissolved in 100 ml chloroform:methanol (2:1, vol:vol) and spotted onto the plate. Separation of phospholipid species was achieved using a solvent composed of chloroform:methanol:acetic acid:water (100:50:16:4, vol:vol). Phospholipids were visualized with iodine, scraped from the plate, and phosphorus content of the various species [lysophosphatidylcholine (LPC), sphingomyelin, phosphatidylcholine and phosphatidylethanolamine] in each lipoprotein was determined with the modified Bartlett procedure [26]. Statistics Results are expressed as means 6 sem. One-way ANOVA was used to evaluate the statistical significance between values obtained in different groups. If variance ratios reached statistical significance, the difference between the means were analyzed with the posthoc tests versus controls (Dunnett) for P , 0.05 and P , 0.01. If data were not normally distributed, logarithmic transformation was applied. RESULTS Characteristics of the subjects Clinical characteristics of the different groups are shown in Table 1. No statistically significant differences were observed with respect to gender, age and plasma creatinine levels. Patients with nephrotic syndrome (NS-H and NS-N), subnormal albumin (SNA), and nonrenal disease (NRD) had statistically lower levels of total proteins compared to the controls (P , 0.05). Albumin

Table 3. Lipoprotein lysophosphatidylcholine (lmol/liter) NS-H

NS-N

N 8 7 TOTAL 364 6 83 277 6 29 VLDL 1 IDL 56 6 23a 33 6 7a LDL 95 6 20a 66 6 7a HDL 92 6 9 89 6 3 19 6 4a LDP 22 6 5a

CON

NRD

SNA

6 247 6 49 962 35 6 6 98 6 16 46 6 10

8 200 6 17 11 6 3 27 6 7 62 6 10 14 6 4a

6 380 6 121 38 6 12a 82 6 18a 123 6 14 36 6 8

Data are means 6 sem. Abbreviations are in Table 2. a P , 0.05 vs. control

levels were markedly decreased in all groups (P , 0.01). Cholesterol, phospholipids and triglycerides were all increased in the NS-H group and cholesterol was decreased in NRD group. The median level of proteinuria was similar in the three groups with renal disease (NS-H, NS-N and SNA). Lipoprotein cholesterol VLDL 1 IDL-cholesterol level was significantly higher than controls in both the NS-H and NS-N groups (P , 0.05; Table 2). However, the NS-H group exhibited significantly higher LDL-cholesterol levels (P , 0.01), while cholesterol levels were significantly decreased in the NRD group (P , 0.05). No significant differences were found in the other two groups (NS-N and SNA) in comparison to control subjects. Lipoprotein lysophosphatidylcholine Lysophosphatidylcholine (LPC) is a phospholipid which is derived from PC [3, 28, 29]. As can be seen in Table 3, VLDL 1 IDL-LPC level was increased in the NS-H, NS-N and SNA groups (P , 0.05). The LDLLPC level were also significantly higher than CON in these groups (P , 0.05). In the NRD group, where LDL cholesterol levels were low, LDL-LPC levels were not increased. There were no significant differences in HDLLPC levels. Conversely, LPC levels were significantly decreased in the albumin-rich LDP fraction in the NS-H, NS-N, and NRD groups (P , 0.05). Significant increases

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Vuong et al: LDL lysophosphatidylcholine in hypoalbuminemia

Fig. 1. Lysophosphatidylcholine/cholesterol ratios in low-density lipoprotein of hypoalbuminemic patients. Data are means 6 sem. Abbreviations are: NS-H, hyperlipidemic nephrotic syndrome; NS-N, normolipidemic nephrotic syndrome; CON, control subjects; NRD, nonrenal diseases; SNA, subnormal albumin. *P , 0.05 vs. control.

in LDL LPC/cholesterol ratios were observed in the NS-N and SNA groups (Fig. 1). DISCUSSION In hypoalbuminemic patients with proteinuria, with or without hyperlipidemia, LPC was increased in VLDL 1 IDL and LDL. Conversely, in all hypoalbuminemic patients LPC was decreased in lipoprotein-deficient plasma, the albumin-containing fraction. These findings suggest that in the presence of hypoalbuminemia and either normolipidemia or hyperlipidemia, LPC shifts from albumin to VLDL, IDL and LDL. Increased LPC levels in LDL are commonly associated with oxidation in vitro. The LPC level can be increased 6- to 30-fold depending on the degree of oxidation [6, 7, 10, 24, 29, 30]. Previously, it has been found that in circulating LDL, LPC levels are much less variable [23, 29], which probably reflects the effectiveness of antioxidative defense mechanisms in the circulation. Some increases in LDL LPC content have been identified in patients with dyslipidemia due to diabetes [16, 17], or familial hypercholesterolemia [18]. These increases may be due to a prolonged half-life [31] or increased oxidative stress [32]. However, the increase in LPC content was only 40% [10, 33], consistently less than the nearly twofold increase that we found in LDL of normolipidemic, hypoalbuminemic subjects in the present study, in whom lipoprotein metabolism was probably not grossly abnormal. Moreover, in the presence of hyperlipidemia, the increase in LPC in LDL was nearly threefold in the hypoalbuminemic nephrotic patients. However, per mmol cholesterol LDL LPC levels were not significantly increased in these patients. Whether this implies that LDL particle size was larger in the hyperlipidemic than in the normolipidemic nephrotic patients, and that there were relatively more LDL particles in the normolipidemic patients is not certain. Nevertheless, the total load

of LPC in LDL is increased in most nephrotic subjects, even when hypoalbuminemia is relatively mild (SNA group). Thus, hypoalbuminemia in the presence of normolipidemia or hyperlipidemia appears to have a specific effect on lipoprotein LPC content. This effect is not restricted to LDL, but also occurs in VLDL and IDL. It is not clear why this does not take place in HDL, but the resistance of HDL to the sequestration of LPC may enable this particle to exert its anti-atherosclerotic effect [34–36]. In human plasma, albumin is the most abundant protein with a normal concentration of approximately 40 g/ liter [37, 38]. It is highly soluble and binds a variety of exogenous (metals, drugs) and endogenous substances (nonesterified fatty acids, bilirubin, amino acids and LPC) [20, 38]. Previously it has been shown that hypoalbuminemia of the nephrotic syndrome is accompanied by an increase in the nonesterified fatty acid content of lipoproteins [39, 40]. Presently, we have documented such an effect for the LPC content of lipoproteins. LPC is formed out of PC by phospholipase A2 [4, 5, 27–29, 41] or as a product of the lecithin:cholesterol acyl transferase reaction (LCAT) [42]. Phospholipase A2 is either associated with lipoproteins [3–5] or secreted by platelets [43]. LCAT activity is enhanced in the nephrotic syndrome [24, 44]. Both phospholipase A2 and LCAT transfer fatty acids from the sn-2 position of phosphatidylcholine [5, 30, 41, 42] resulting in the formation of LPC. Irrespective of its route of formation, LPC is always transferable to albumin [45]. Previously, albumin has been identified as an antioxidant [46–49]. Whether this is due to binding of transition metals [38, 48, 49], LPC, or both is unclear. Irrespective of the exact nature of its antioxidant effect, it is well known that incubation of oxidized lipoproteins with albumin can reverse their deleterious effects on endothelial function [6, 10, 50, 51]. Hence, it follows that in vivo albumin also has such an effect. Presumably this function is proportionally reduced under hypoalbuminemic circumstances. In hypoalbuminemia, increased LPC levels in circulating LDL will probably cause effects that resemble those caused by oxidized LDL, albeit to a lesser degree. Whether the increased LPC levels in VLDL and IDL also have deleterious effects on endothelial function is not clear, but evidence is accumulating that triglyceriderich particles [34, 52] and their cholesterol-rich remnants, primarily IDL [34], have pro-atherosclerotic effects. Recent data from the MARS study underscore the potential relevance of IDL for progression of atherosclerotic disease [53]. Indeed, we recently found a correlation between the reduction of IDL cholesterol and the level of improvement of endothelial function in patients with combined hyperlipidemia [54]. Thus, LPC accumulation in VLDL and IDL may also not be innocuous. Albumin-binding leads to a decrease in the transfer

Vuong et al: LDL lysophosphatidylcholine in hypoalbuminemia

of LPC from oxidized LDL to other compartments such as the endothelial or red cell membrane. It has been shown that albumin can reduce the uptake of LPC by cultured endothelial cells [27]. The consequence is less LPC-induced suppression of endothelium-dependent arterial tone [10]. Thus, it is not inconceivable that during hypoalbuminemia, endothelial function will be disturbed due to a quantitative increase in the amount of membrane-bound LPC. It is also well-known that there is rapid exchange of LPC between red cell membranes and either albumin [55] or lipoproteins [56]. Indeed, we found that red cells in analbuminemic rats showed a marked increase in LPC content with a concomitant reduction in deformability. These changes could be normalized by the addition of albumin [57]. Most probably this effect will also be present in hypoalbuminemic humans, and may contribute to the increased risk of atherosclerosis present in hypoalbuminemic patients with the nephrotic syndrome [58]. Even in hypolipidemic subjects with NRD, hypoalbuminemia may have resulted in a shift of LPC from albumin to nonlipoprotein compartments. In conclusion, hypoalbuminemia due to proteinuria causes decreased binding of LPC by albumin. This results in increased LPC levels in both LDL and VLDL 1 IDL. Increased LPC in lipoproteins and possibly also in cell membranes may be an important factor in the disproportionate increase in cardiovascular disease in nephrotic patients with hypoalbuminemia.

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ACKNOWLEDGMENTS This research was supported by the Dutch Kidney Foundation, grant number C96.1607. Portions of this work appear in abstract form (J Am Soc Nephrol 8:71A, 1997). Reprint requests to Jaap A. Joles, D.V.M., Ph.D., Department of Nephrology and Hypertension (FO3.226), Utrecht University Hospital, PO Box 85500, 3508 GA Utrecht, The Netherlands. E-mail: [email protected]

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