1 Department of Internal Medicine V, Department of Diabetes, Metabolism and Rheumatology, Wilhelminenspital, 2 Third Department of Medicine, Division of Nephrology and 3 Second Department of Medicine, Division of Cardiology, University of Vienna, Vienna, Austria and 4 Department of Clinical Chemistry, University of the Saarland, Homburg, Germany
Correspondence and offprint requests to: Dr Karam Kostner, AKH Wien, Department of Cardiology, Waehringerguertel 1820, A-1090 Vienna, Austria. Email: karamkostner{at}hotmail.com
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Abstract |
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Methods. Plasma Lp(a), non-LDL-bound apo(a) and urinary apo(a) fragments were measured in 55 kidney disease patients (28 males and 27 females) and matched controls.
Results. Plasma Lp(a) and non-LDL-bound apo(a) were increased in patients, whereas urinary apo(a) was decreased, especially in patients with a creatinine clearance < 70 ml/min. There was a significant correlation between plasma Lp(a) and non-LDL-bound apo(a) in patients and controls.
Conclusion. We conclude that decreased urinary apo(a) excretion could be one possible mechanism of increased plasma Lp(a) and non-LDL-bound apo(a) in patients with decreased kidney function.
Keywords: lipoprotein (a); non-LDL-bound apo(a); renal function; urinary apo(a)
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Introduction |
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There are several indications that the kidney plays an important role in Lp(a) metabolism. Various publications have appeared with the unanimous finding that patients with uraemia and nephrotic syndrome have a 3- to 5-fold increase in their plasma Lp(a). Patients with nephrotic syndrome, for example, exhibit excessively high plasma Lp(a) concentrations, which can be reduced by antiproteinuric therapies [2]. Patients with end-stage renal disease (ESRD) treated with haemodialysis are also found to have elevated Lp(a) levels, and these are even higher in patients treated with continuous ambulatory peritoneal dialysis. Interestingly, Lp(a) is present in the form of different pools. Most studies have measured low-density lipoprotein (LDL)-bound Lp(a), which consists of apolipoprotein B-100 and apo(a) which are covalently bound by a disulfide bridge, and appears in plasma in up to 30 high or low molecular weight isoforms.
In 1987, Gries et al. [3] reported the existence of non-LDL-bound apo(a) in human serum. We and others have published findings that apo(a) immunoreactivity is found in urine in the form of apo(a) fragments [4,5]. We previously reported a highly significant correlation between these urinary apo(a) fragments and plasma Lp(a) levels in healthy volunteers and patients with coronary artery disease [6]. Herrmann et al. [7] also reported increased concentrations of non-LDL-bound apo(a) in patients with coronary artery disease. The same authors also found a strong elevation of non-LDL-bound apo(a) in patients with ESRD and in patients with nephrotic syndrome [8].
Recently, Kronenberg et al. [9] showed an arteriovenous difference in Lp(a) of 10% and concluded that substantial amounts of this atherogenic lipoprotein are removed by the kidney. The same authors could also show that plasma concentrations of non-LDL-bound apo(a) are increased in patients with ESRD [10]. However, the relationship between non-LDL-bound apo(a), plasma Lp(a) and urinary apo(a) fragments still remains unclear.
Therefore, in the present study, we determined the relationship between plasma Lp(a), non-LDL-bound apo(a) and urinary apo(a) fragments in kidney disease patients and compared them with a matched control group.
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Subjects and methods |
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CrCl was measured directly.
Immunoquantitation of Lp(a) and apo(a)
Analyses were carried out by a sandwich DELFIA as recommended by the manufacturer, (LKB-Pharmacia) and described in detail previously [4,6]. Briefly, polyclonal affinity-purified antibody from rabbit produced in our own laboratory [4,6] was passed over an affinity column loaded with plasminogen and was used to coat 96-well Costar plates. The purified antibody was free of any detectable cross-reactivity against plasminogen, plasmin or elastase as tested by immunoblot analysis, but had a slight cross-reactivity with catalase. For control experiments, we used a monoclonal antibody and obtained identical results. Non-specific binding sites were blocked with 250 µl of 0.5% (w/v) bovine serum albumin for 30 min. Aliquots (200 µl) of the samples were added to the wells and incubated for 2 h at 20°C. After three successive washing steps with 50 mM TrisHCl pH 7.7, the same polyclonal antibody against apo(a) as above, labelled with Europium (Eu), was added to the wells and incubated further for 2 h at 20°C. Excess antibody was removed by two further washing steps with 50 mM TrisHCl pH 7.7. A 200 µl aliquot of enhancement solution (Pharmacia, Uppsala, Sweden) was added, and fluorescence was determined after 15 min in a DELFIA reader. For the determination of total apo(a), Eu-labelled polyclonal (POAB) anti-apo(a) from rabbit was used (= a:a DELFIA). Plasma samples were diluted 3000-fold and urine samples were diluted between 10- and 50-fold. The assay was linear between 1 and 100 ng of apo(a) per well; the within-run coefficient of variation was < 3%.
Measurement of non-LDL-bound apo(a)
The enzyme-linked ligand sorbent assay (ELLSA) for fapo(a) (Immuno GmbH, Heidelberg, Germany) captured fapo(a) by a specific peptide fixed on the microwells which carried the amino acid sequence of a non-covalent apo(a)-binding site on apo B (ligand peptide, 20).
Immobilized fapo(a) was detected utilizing horseradish peroxidase-labelled polyclonal apo(a) [7,8]. Cross-reactions with Lp(a)/apo B-containing particles and plasminogen were excluded. The ligand peptide was also coupled to Sepharose and used in affinity chromatography to separate fapo(a) from whole serum, which was demonstrated by immunoblot analysis. Affinity chromatography-purified fapo(a) was free of apo B and did not cross-react with plasminogen. Levels of free apo(a) determined with the ELLSA method correlated well with those measured by electroimmunodiffusion, an agarose gel electrophoresis which precipitates apo B-containing particles [Lp(a) and LDL] and free apo(a) separately during one electrophoretic run.
Determination of other plasma lipids and albumin
Total cholesterol, high-density lipoprotein (HDL) cholesterol and triglycerides were determined with commercially available kits from Roche Diagnostics, Mannheim. LDL cholesterol was calculated using the Friedewald equation. Creatinine was measured by the Jaffee method using commercial assay kits from Boehringer Mannheim. All chemicals were reagent grade obtained from E. Merck, Darmstadt if not stated otherwise.
Statistical analyses
The analysis of our data was performed with the Statistical Package for Social Sciences (SPSS/Mac+). For serum lipids, mean values ± SD were calculated and analysed by a one-way analysis of variance (ANOVA). A Students t-test was applied to assess significant differences of continuous variables among groups. Because of the abnormal distribution of Lp(a), non-LDL-bound apo(a) and urine apo(a), non-parametric tests were carried out and medians instead of means are given. Comparison of serum Lp(a), non-LDL-bound apo(a) and urine apo(a) values among groups was performed by the Wilcoxon test or by ANOVA after logarithmic transformation of values. Correlations of serum apo(a) and urine apo(a) values were performed by the Spearman rank correlation test.
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Results |
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Discussion |
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Lp(a) seems to consist of three pools: (i) the majority seems to circulate in the form of intact apo B (LDL) containing Lp(a), which consists of up to 30 isoforms; (ii) non-LDL-bound or free apo(a); and (iii) apo(a) fragments which appear in urine and plasma. Not very much is known about this non-LDL-bound apo(a), especially since very few assays exist for its measurement. Most of this non-LDL-bound apo(a) seems to consist of apo(a) fragments, identical to those fragments that can be found in urine [4]. Several histopathology studies have shown that atherosclerotic lesions are selectively enriched in non-LDL-bound apo(a). About 50% of the apo(a) extracted from atherosclerotic lesions is not associated with lipids [13].
It could also be shown that the ratio of plasma concentration of apo B-100 to apo(a) in venous graft lesions is only 3:1, whereas it is 9:1 in plasma [14]. Furthermore, individuals with little to no Lp(a) measurable in plasma have detectable apo(a) in arterial lesions [15]. Lastly, Hoff et al. [16] have identified a series of apo(a)-immunoreactive fragments in the > 1.21 g/ml density fraction of human atherosclerotic lesions that were similar in size to those we have characterized.
From the results of the above-mentioned studies, one could speculate that free apo(a) and apo(a) fragments are more atherogenic than apo B-bound apo(a). This would explain why Lp(a) turns out to be a risk factor for coronary artery disease only in some studies. The kidney could play an important role in the metabolism of non-LDL-bound apo(a) by excreting it in the form of apo(a) fragments. To understand better the relationship of these apo(a) fragments, that can be detected in urine and non-LDL-bound apo(a) in plasma in kidney disease patients, we performed the present study. To our knowledge, this is the first study that looks at the relationship between Lp(a), non-LDL-bound apo(a) and urinary apo(a) fragments in patients with kidney disease.
The main finding of the present study was that there exists a positive correlation between non-LDL-bound apo(a), plasma Lp(a) and urinary apo(a) fragments in kidney disease patients and controls. In addition, levels of not only Lp(a), but also non-LDL-bound apo(a) were significantly higher in kidney disease patients with a CrCl < 70 ml/min as compared with controls and kidney disease patients with a normal CrCl. We could also reproduce earlier findings from our group that urinary excretion of apo(a) fragments is significantly decreased in kidney disease patients with a CrCl of < 70 ml/min.
The limitations of our study are the relatively small number of kidney disease patients and the marked heterogeneity of our patient population. We conclude that both Lp(a) and non-LDL-bound apo(a) are elevated in patients with kidney disease as compared with healthy controls. In addition, a significant correlation exists between both Lp(a) and non-LDL-bound apo(a) in patients and controls. Since urinary apo(a) fragments are significantly decreased in kidney disease patients, especially in patients with a CrCl of < 70 ml/min, a decreased ability of the kidney to metabolize Lp(a) could be responsible for the increase of Lp(a) and non-LDL-bound apo(a).
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References |
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