Lipoprotein (a) levels in those with high molecular weight apo (a) isoforms may remain low in a significant proportion of patients with end-stage renal disease

Darren S. Parsons1, David A. Reaveley2, Darrell V. Pavitt2, Madhukar Misra1 and Edwina A. Brown1

1 Department of Renal Medicine and 2 Department of Clinical Chemistry, Faculty of Medicine, Imperial College School of Science, Technology and Medicine, Charing Cross Hospital, London, UK

Correspondence and offprint requests to: Dr D. S. Parsons, Department of Renal Medicine, Charing Cross Hospital, Fulham Palace Road, London W6 8RF, UK. Email: dspcx{at}yahoo.com



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Studies have reported an increase in median Lipoprotein (Lp) (a) in patients with high molecular weight (HMW) apolipoprotein (apo) (a) isoforms and renal impairment. Some studies identify Lp (a) levels as a risk factor for vascular disease in renal failure whilst others have demonstrated an association with apo (a) isoform type and vascular disease.

Methods. A total of 239 patients at end-stage renal failure (ESRF) were studied prior to the initiation of dialysis. Blood was taken for Lp (a) levels and apo (a) isoforms. Clinical vascular disease (CVD) was assessed on the basis of clinical history and Rose questionnaire. The control group for Lp (a) levels consisted of 228 healthy volunteers.

Results. Despite a higher median Lp (a) level in those with HMW isoforms, 30% of patients had Lp (a) levels <10 mg/dl. Overall, 49% patients were identified as having CVD. Diabetes, smoking history and Lp (a) levels were significantly associated with CVD in logistic regression analysis, although when patients with low molecular weight (LMW) and HMW isoforms were analysed separately, Lp (a) levels were not significantly associated with CVD in those with LMW isoforms. The rates of CVD in those with HMW isoform and low Lp (a) levels were significantly lower than those with HMW isoforms and elevated Lp (a) levels, 34 vs 57% (P < 0.01).

Conclusions. Although median Lp (a) levels in those patients at ESRF with HMW isoforms are higher than controls, in a third of such patients Lp (a) levels remain relatively low. These patients have lower rates of CVD than those with high levels of Lp (a).

Keywords: atherosclerosis; lipoprotein (a); renal failure



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Atherosclerotic complications such as coronary artery disease (CAD), peripheral vascular disease (PVD) and cerebrovascular disease (CeVD) are a common cause of morbidity and mortality in renal failure. Prospective studies have shown cardiovascular mortality in dialysis patients may be increased as much as 10 times that of general population and more than 40 times in diabetic patients with end-stage renal failure (ESRF) [1]. This excess of atherosclerosis may be partly explained by the higher prevalence of traditional risk factors in patients on dialysis and with renal failure, such as diabetes, hypertension and dyslipidaemia, but newer vascular risk factors such as lipoprotein (Lp) (a) may also be important.

Lp (a) is an LDL-like particle, which has a highly polymorphic apolipoprotein (apo) (a) glycoprotein bound to the apoB moiety of LDL. There is considerable variation in the genetic size polymorphisms of apo (a) with over 30 different isoforms [2]. These different isoforms can be grouped into low (LMW) and high molecular weight (HMW) isoforms according to the number of kringle IV repeats in the apo (a) molecule. In healthy subjects Lp (a) levels are mainly dependent on the isoform type, with those with LMW isoforms having high levels and those with HMW isoforms having lower levels. Lp (a) levels are not significantly affected by age or sex, diabetes, dietary cholesterol or HMG-CoA-reductase inhibitors.

A number of studies have reported elevated Lp (a) levels in patients with advanced renal failure [3,4] or in patients established on dialysis [59]. Some studies have reported elevation in both HMW and LMW isoforms in patients with renal failure with a relative smaller increase in those with LMW isoforms [3,10]; however, this finding has not been confirmed in other studies with an increase in Lp (a) level only being demonstrated in patients with HMW isoforms [7,8,11].

Results from case control or prospective studies of Lp (a) levels as a risk factor for vascular disease in dialysis patients do not show consistent results. Several case control studies have shown a positive association between Lp (a) levels and vascular disease [6,12,13]. Similarly, results from prospective studies are conflicting with some studies demonstrating Lp (a) levels as an independent risk factor for vascular events in dialysis patients [9,14] and others showing no association [15]. Conflicting results may be in part related to small sample sizes, different end-points and failure to measure apo (a) isoforms. In some studies the isoform type has been shown to be more predictive of vascular disease, with the presence of LMW isoforms being an independent risk factor for vascular disease rather than the Lp (a) level [7,16].

Although it has been established that median Lp (a) levels are increased in those with HMW isoforms and advanced renal failure, it was our impression that appreciable absolute change in Lp (a) does not occur in all patients with HMW isoforms and a significant number develop either a very small change or one that is not detectable. This observation has not been examined previously.

The aim of this study was to determine the proportion of patients at ESRF with HMW isoforms without high Lp (a) levels and to examine the rates of CVD in such patients compared with those with elevated Lp (a) levels.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
A total of 239 patients [age (mean ± SD) 63.5 ± 14.3, range 30–88 years] with renal impairment (mean ± SD plasma creatinine 570 ± 231 mmol/l) were studied prior to starting renal replacement therapy. The cause of renal impairment was diabetes mellitus 28%, hypertensive nephropathy 13%, chronic glomerulonephritis 13%, adult polycystic kidney disease 8%, reno-vascular 6%, unknown 18% and miscellaneous in 14%. Exclusions included patients <30 years old, those with chronic liver disease or taking steroids, those who had had an acute vascular event in the previous 6 months or with evidence of an acute infective or inflammatory response. Patients who were of Afro-Caribbean origin were also excluded, as such individuals have significantly higher Lp (a) levels compared with Caucasians and the relevance of Lp (a) as a risk factor for vascular disease is less clear in this group.

Our local ethics committee approved the protocol and all patients gave written informed consent.

Following an overnight fast, venous blood was taken for Lp (a) levels and apo (a) isoforms, plasma creatinine and fasting lipids (total cholesterol, HDL-cholesterol, LDL-cholesterol and triglycerides). Urinary protein excretion was determined from a 24-h urine collection.

Serum for Lp (a) was stored at -70°C and Lp (a) levels were measured using an enzyme-linked immunoabsorbent sandwich assay (TintElize, Biopool AB, Umea, Sweden). Apo (a) isoforms were determined by SDS–PAGE using the method described previously [5]. Two control sera each with HMW and LMW apo (a) isoform was included in each batch of isoforms run. The number of kringle IV repeat units in these control isoforms were accurately known and were used to determine the number of kringle IV repeats in the patient samples. The control isoforms were calibrated against four sera each containing two isoforms whose number of kringle IV repeats had been accurately determined. These four sera were a gift from Dr Brian Knight (MRC Lipoprotein Team, Hammersmith Hospital, London, UK). Because of the large number of different isoforms we divided apo (a) phenotypes into two groups according to the molecular weight of the apo (a) isoforms as has been done in previous studies [7,16]. Those individuals who had only isoforms with more than 22 kringle IV repeats were classified as the HMW group and those with at least one isoform with less than 22 kringle IV repeats were classified as LMW. The control population for Lp (a) levels consisted of 228 healthy volunteers (50% male), aged <65 years (mean age 55.1 ± 15.1 years).

Vascular disease was assessed on the basis of clinical history, notes review and standardized Rose questionnaire. A positive diagnosis of CAD was made on the basis of one or more of the criteria: a positive diagnosis of angina made on the Rose questionnaire [17], positive exercise tolerance test, thallium scan or angiography, or history of coronary artery bypass surgery or myocardial infarction (with compatible ECG changes). A positive diagnosis of PVD was made on the basis of one or more of the following criteria: diagnosis of intermittent claudication on the Rose questionnaire, positive Doppler studies or angiography or history of angioplasty/bypass surgery or amputation for PVD. Similarly, CeVD was considered present on the basis of criteria: history of ischaemic stroke or transient ischaemic event (confirmed on CT scan) or previous carotid endartectomy. Patients were classified as having clinical vascular disease (CVD+) if they fulfilled criteria for CAD, PVD or CeVD. Assessment of the presence of CVD was made blinded to laboratory results. Smoking history was also recorded and patients were considered smokers if they were currently smoking or had ceased within the last 12 months.

Median Lp (a) was calculated for Lp (a) in those with HMW and LMW isoforms. Lp (a) levels were compared with controls using the Mann–Whitney test as Lp (a) levels were not normally distributed. Correlation between Lp (a) levels in those with HMW isoforms and other variables was tested using Spearman’s rank correlation for non-normally distributed variables. In those with HMW isoform, a comparison was made between those with Lp (a) levels <10 mg/dl and those >10 mg/dl. This level of 10 mg/dl was chosen as this was the median level of Lp (a) in control subjects with HMW isoforms. Univariate analysis was performed using unpaired t-test, Mann–Whitney or Pearson’s {chi}2 test.

Patients were grouped in to CVD+ and CVD- and compared in univariate analysis by unpaired t-test for normally distributed variables or by Mann–Whitney test for non-normally distributed variables. Categorical variables were compared by Pearson’s {chi}2 test. Logistic regression analysis was performed to identify variables independently associated with CVD+. Patients were analysed as a whole and also by isoform type.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
There was a significantly higher median Lp (a) in patients with HMW and LMW isoforms compared with controls (Table 1). There was no significant difference in the frequency of LMW apo (a) isoform between patients and the controls. Patients with HMW isoforms had a median Lp (a) of 27.5 compared with a control value of 10.5 mg/dl, P-value = 0.0001. There was a smaller relative increase in Lp (a) levels in those with LMW isoforms, median (range) Lp (a) of 62.0 (1.6–163.7) vs 42.6 (3.4–88.7) mg/dl, P-value = 0.01.


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Table 1. Lp (a) levels median (range) in patients and controls

 
Despite the higher median Lp (a) level in patients with HMW isoforms, a considerable proportion (29%) had Lp (a) levels below the median of the control group (10.5 mg/dl). Figure 1 shows the distribution of Lp (a) in patients and controls in those with HMW isoforms. Lp (a) levels in those with HMW isoforms were not significantly associated with serum albumin or proteinuria. No significant differences were seen in Lp (a) levels between those with diabetes or those taking HMG-CoA-reductase inhibitors.



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Fig. 1. Comparison of Lp (a) levels in those with HMW apo (a) isoforms in patients compared with controls.

 
Overall, 49% patients were identified as having CVD, with higher rates of vascular disease in those with diabetes mellitus compared with non-diabetic patients, 62 vs 43%, P = 0.01. The frequency of CAD was higher in patients with diabetes compared to non-diabetics, 44 vs 31%, P = 0.05 and similarly the rates of PAD were also higher in patients with diabetes, 35 vs 19%, P = 0.01. No significant difference was seen in the frequency of CeVD between diabetic and non-diabetic patients.

Univariate analysis (see Table 2) demonstrated a significant association between CVD and age (P-value = 0.0001), smoking (P-value = 0.02), diabetes mellitus (P-value =0.01) and Lp (a) levels (P-value = 0.02). No association was seen between CVD and other fasting lipid parameters, body mass index, plasma creatinine, serum albumin, calcium or phosphate levels, haemoglobin, 24-h protein excretion or apo (a) isoform type. Similarly male sex and HMG-CoA-reductase inhibitor use were not significantly associated with vascular disease.


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Table 2. Univariate comparisons of patients with clinical vascular disease characteristics (CVD+) compared with patients without vascular disease (CVD–)

 
Logistic regression analysis identified age, diabetes mellitus, smoking and Lp (a) level as independent risk factors for CVD (see Table 3). Subgroup analysis by isoform type showed that in those with HMW isoforms, age, diabetes, smoking and Lp (a) level remained independent predictors of CVD, but in those with LMW isoforms no association was seen between Lp (a) level and CVD.


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Table 3. Logistic regression analyses to predict clinical vascular disease in patients with ESRF

 
The rates of CVD in those patients with HMW isoforms and Lp (a) levels <10 mg/dl were significantly lower than compared with those with HMW isoforms and elevated Lp (a) levels, 34 vs 54%, P-value <0.01; however, these patients did not differ in terms of other cardiovascular risk factors (see Table 4).


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Table 4. Univariate comparison of patients with HMW isoforms and Lp (a) levels >10 mg/dl compared with those with Lp (a) levels <10 mg/dl

 


   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Although a higher median Lp (a) in those with HMW apo (a) isoforms at ESRF was seen, confirming the findings of previous studies in patients with advanced renal failure or on dialysis [3,7,8,11], a significant proportion of such patients in the current study did not have high Lp (a) levels. Figure 1 shows that the distribution of Lp (a) levels in those controls with HMW isoforms is not simply shifted to the left with the development of advanced renal failure. It would seem likely that there is relatively greater increase in Lp (a) in some individuals who develop renal impairment than in others. Nearly a third of patients with HMW isoforms and advanced renal failure had Lp (a) levels below the median of the control group suggesting that although elevation of Lp (a) can occur with declining renal function, Lp (a) levels in those with HMW isoforms may remain relatively low. We were unable to identify any factor that differentiated those with HMW isoforms and elevated levels from those in whom Lp (a) levels were not elevated. Diabetic status, HMG-CoA-reductase use and plasma albumin were not significantly different between those with HMW isoforms and elevated Lp (a) and those with levels <10 mg/dl. Similarly, although elevation of Lp (a) levels in all isoform types has been described in patients with nephrotic syndrome [18] and levels of Lp (a) have been correlated with proteinuria in some studies of patients with renal impairment [3], although not in others [11], the degree of proteinuria in the current study did not correlate with Lp (a) levels in those with HMW isoforms.

We demonstrated a lower frequency of LMW apo (a) isoforms than in some previous studies. In the 228 controls the frequency of the LMW apo (a) isoforms was 17%. No significant difference was seen in the frequency of LMW isoforms between patients at ESRF and controls. Some previous studies in different populations have reported a frequency of LMW isoforms of 20–35% [11,16]. However, the lower frequency of LMW isoforms in the current study is similar to the frequency we have previously seen in populations in West London [12]. Apo (a) isoforms were determined using SDS–PAGE electrophoresis, which in our hands provides similar resolution to SDS agarose gel electrophoresis, which has been used in some previous studies used. SDS–PAGE electrophoresis has been used to identify over 28 individual apo (a) bands [19].

In the current study Lp (a) levels were increased in both those with HMW and LMW isoforms. We demonstrated significant elevation in both isoforms types, although the relative change in those with LMW isoforms was much smaller than in those with HMW isoforms. A number of studies have reported elevation in both isoform groups with a relative smaller increase in those with LMW isoforms [3,10]. However, this finding has not been confirmed by other studies in which elevation of Lp (a) levels was only seen in those with HMW isoforms [7,8,11]. It may be that these differences in results are related to study size. The number of patients with LMW isoforms in the current study was small, which limits any conclusions concerning the effect of renal failure on Lp (a) levels in those with LMW isoforms.

The mechanism of elevation of Lp (a) in patients with renal impairment is undetermined. In nephrotic syndrome the elevation of Lp (a) levels seen may be secondary to generalized increased hepatic protein synthesis [20]. In patients without significant proteinuria this mechanism does not account for the elevation of Lp (a) levels. It is possible that the kidney has a metabolic role in the breakdown of Lp (a), a theory which is supported by the arterio-venous differences in Lp (a) concentrations that have been described by some [21] and by the findings of apo (a) fragments in the urine [22], the excretion of which is reduced in renal impairment [23]. Our data demonstrate an association between Lp (a) levels and CVD in those with HMW isoforms. The same association was not seen in those with LMW isoforms although the number of patients with LMW isoforms was small.

An association has been described previously between vascular disease and isoform type, with the LMW apo (a) isoform associated with vascular disease in patients with advanced renal failure [7,13,16]. No such association was seen in the current study but this should be interpreted with caution given the relatively small number of patients with LMW isoforms.

In the current study, those patients with HMW isoforms and low levels of Lp (a) had significantly less CVD than those with elevated levels despite a lack of significant differences in other cardiovascular risk factors, suggesting that those individuals with HMW isoforms in whom Lp (a) levels remain relatively low may to some extent be protected from vascular disease. It is unknown what additional factors other than renal function might influence the change in Lp (a) levels in those with HMW isoforms. In the current study neither the degree of proteinuria or diabetic status could explain why some patients with HMW isoforms exhibit elevated Lp (a) levels and in others Lp (a) remain unchanged. It is likely that an additional genetic or environmental factor influences the change in Lp (a) in patients with HMW isoforms. If the determinants of the increase in Lp (a) level in those with HMW isoforms could be ascertained this might lead to additional therapeutic options for prevention of vascular disease in patients with renal impairment.

In conclusion, although higher median Lp (a) levels in patients with HMW isoforms and advanced renal failure have been previously reported, the observation that in a considerable proportion of patients little or no absolute change is seen in Lp (a) levels has not been reported. In 30% of such patients we have shown that little absolute increase in Lp (a) levels occurs. Although the size of the current study limits firm conclusions these patients without greatly elevated Lp (a) levels had less CVD. This is an area which requires further study.

Conflict of interest statement. None declared.



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 24. 8.02
Accepted in revised form: 4. 4.03





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