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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:905.)
© 1996 American Heart Association, Inc.

Articles

Urinary Excretion of Apo(a) Fragments

Role in Apo(a) Catabolism

Karam M. Kostner; Gerald Maurer; Kurt Huber; Thomas Stefenelli; Hans Dieplinger; Ernst Steyrer; Gert M. Kostner

the Second Department of Medicine (K.M.K., G.M., K.H., T.S.), Division of Cardiology, University Hospital of Vienna; the Institute of Medical Biology and Human Genetics (H.D.), University of Innsbruck; and the Institute of Medical Biochemistry (E.S., G.M.K.), University of Graz, Austria.

Correspondence to Dr Karam Kostner, AKH Wien, Department of Cardiology, Währingergürtel 18-20, A-1090 Vienna, Austria.

Abstract

The biosynthesis and assembly of lipoprotein(a) [Lp(a)], a marker for atherosclerotic disease, appears to be well understood. However, information is lacking concerning the mode and site of Lp(a) catabolism. Apo(a) is reported to be excreted into the urine. To study the effect of this pathway on the overall catabolism of Lp(a), urinary apo(a) was characterized by immunoblotting. More than 10 distinct apo(a) bands with molecular masses between 30 and 160 kD were observed. Apo(a) fragments were not complexed to apoB. In more than 30 individuals the size of apo(a) bands was comparable irrespective of their apo(a) phenotype, although marked differences in the relative intensities of the bands were observed. Eight batches of 24-hour urine collections collected from one proband at 2-week intervals exhibited a significant correlation between creatinine and apo(a) concentrations as measured by DELFIA (r=.93; P<.01). In 193 healthy volunteers a highly significant correlation was found between urinary apo(a) concentrations normalized to creatinine levels and plasma Lp(a) values (=0.659; P<.0001). Of the total plasma apo(a), 0.073%, ie, 121 µg apo(a), was excreted in the form of apo(a) fragments in 24-hour urine samples from 12 healthy volunteers. We conclude that the catabolism of Lp(a) via excretion of apo(a) fragments accounts for <1% of the daily Lp(a) catabolism.

Key Words: kidney • urinary excretion • kringle IV

Increased plasma Lp(a) concentrations are strongly associated with premature coronary heart disease and stroke.^{1 2 3 4 5 6} Lp(a) consists of an LDL-like core particle and apo(a) linked together by a disulfide bridge.⁷ Apo(a) shares structural features with plasminogen, ie, it consists of several copies of kringle IV–like repeats and one copy of kringle V in addition to a protease domain.⁸ Each apo(a) subunit contains nine unique kringle IVs that differ from each other by only a few amino acids in addition to a variable number of identical "repetitive" kringle IVs. More than 40 genetic isoforms of apo(a) have been reported, differing in the number of repetitive kringle IVs.⁹ The plasma Lp(a) concentration among individuals varies by a factor of 1000 (<1 to >200 mg/dL). Most notably, some individuals have undetectable amounts of Lp(a).¹⁰ An inverse relationship exists between the number of kringle IV repeats and the plasma concentration of Lp(a),^{11 12} and the degree of inheritance of plasma Lp(a) concentrations is >90%.¹³

Despite intensive investigations by various research groups, little is known about the possible physiological functions of Lp(a). Earlier metabolic studies in humans have suggested that Lp(a) is not an immediate product of VLDL.¹⁴ Our investigation¹⁵ of Lp(a) turnover in 20 normolipemic and hyperlipemic volunteers showed that plasma Lp(a) levels are highly correlated with the rate of biosynthesis, but no significant correlation was found with the fractional catabolic rate. Although much knowledge has accumulated concerning the site of apo(a) synthesis and the mode of Lp(a) assembly,¹⁶ little is known about the catabolism of Lp(a). As an apoB-containing particle, Lp(a) binds weakly to the LDL receptor in vitro, yet in vivo LDL receptor–mediated catabolism seems to play a minor role in the removal of Lp(a).¹⁷

Oida et al¹⁸ report that apo(a) fragments are present in the urine and that their excretion decreases in parallel with the glomerular filtration rate. In the present study we pursued this issue further and addressed the following questions: How does the structure of urinary apo(a) relate to plasma Lp(a)? Are urinary apo(a) levels correlated with plasma Lp(a)? Does the excretion of apo(a) from urine account for plasma Lp(a) catabolism?

Methods

Studied Individuals and Samples
Volunteers were men and women aged 20 to 65 years. All individuals were apparently healthy and did not take any medication other than occasional pain relievers. Some of the women took contraceptives. Blood samples were taken from the antecubital vein after the subjects had fasted overnight, allowed to clot for 30 minutes at room temperature, and centrifuged, and the sera were frozen at -20°C. In previous experiments we ascertained that a single freezing does not affect the assay results. Urine was collected either in the morning only or during a 24-hour period commencing in the early morning and was kept frozen at -20°C until used. Apo(a) in plasma and urine was quantified within one run. For investigating the apo(a) fragments, 1 L urine was concentrated 100- to 200-fold by pressure dialysis and immediately processed further.

Experimental Procedures
Immunoquantification of Lp(a) and Apo(a)
Analyses were performed by using a sandwich DELFIA essentially as recommended by the manufacturer (LKB-Pharmacia).^{10 19} Briefly, a rabbit affinity-purified POAB that had been produced in our laboratory^{10 19} and passed over an affinity column loaded with plasminogen was used to coat 96-well Costar plates. The purified antibody was free of any detectable cross-reactivity against plasminogen, plasmin, or other plasma constituents as tested by immunoblot analysis. Nonspecific binding sites were blocked with 250 µL 0.5% (wt/vol) bovine serum albumin for 30 minutes. Aliquots (200 µL) of the samples were added to the wells and incubated for 2 hours at 20°C. After three successive washing steps with 50 mmol/L Tris-HCl, pH 7.7, the same POAB against apo(a) as above but labeled with europium was added to the wells and further incubated for 2 hours at 20°C. Excess antibody was removed by two further washing steps with 50 mmol/L Tris-HCl, pH 7.7. Enhancement solution (200 µL; Pharmacia) was added, and fluorescence was determined in a DELFIA reader after 15 minutes. For the determination of total apo(a), an Eu-labeled POAB [anti-apo(a)] from rabbit was used (a:a DELFIA). In some cases, the kringle IV–specific anti-apo(a) MAB 1A2 (see below) was used as the detection antibody. Apo(a):apoB complexes were measured by using Eu-labeled antibody against apoB from rabbit as detection antibody¹⁰ (a:B DELFIA). For measuring total apoB, a B:B DELFIA was designed.^{10 18}

Reference Material
Standard curves were produced by using the Lp(a) reference standard from Immuno Diagnostika (lot #2900/170) in each experiment separately. This standard was calibrated in our laboratory by using freshly purified Lp(a) as well as recombinant apo(a) with 18 kringle IV repeats expressed in COS-7 cells²⁰ as primary standard as follows. Lp(a) was purified from a donor with a single apo(a) isoform (22 kringle IV repeats) by stepwise ultracentrifugation at increasing densities followed by column chromatography.²¹ The purity of the material was checked by using SDS-PAGE and immunoelectrophoresis. The concentration of this primary Lp(a) standard was calculated after enzymatic analysis of triglycerides, free and esterified cholesterol, and phospholipids; proteins were analyzed by amino acid analysis. The primary standard for apo(a) containing 18 kringle IV repeats was purified from the incubation medium of transfected COS-7 cells²⁰ by using Lys-Sepharose affinity chromatography.²² Its concentration was also determined by amino acid analysis. The primary standards were solubilized at various concentrations in phosphate-buffered saline containing 6% human serum albumin and measured by using the a:a and a:B DELFIA. The standard curves obtained for Lp(a) and apo(a) were used to calibrate the immuno–reference standard. Of the Lp(a) mass in the immuno–reference standard, 15.1% consisted of apo(a). We assayed primary standards, the immuno–reference standard, and serum at several dilutions by DELFIA and found good parallelism of the curves upon dilution of samples. The assay was linear between 1 and 100 ng of apo(a) per well; the within-run coefficient of variation was <3%. Plasma samples were diluted 1500- to 3000-fold, and urine samples were diluted between 10- and 50-fold.

PAGE and Immunoblotting
Electrophoresis was performed in 3.5% to 13% polyacrylamide gradient gels containing 0.1% SDS with the Mini-protean electrophoresis system from BioRad. Samples were mixed with SDS (final concentration, 10 g/L) in the absence or presence of dithiothreitol and heated for 5 minutes at 100°C. SDS–agarose gel electrophoresis was performed,¹⁰ after which proteins were transferred to a nitrocellulose membrane by electroblotting overnight at 4°C in 10 mmol/L Tris-HCl and 40 mmol/L glycine buffer, pH 7.4.¹⁰ The membrane was blocked for 60 minutes in 5% (wt/vol) powdered skim milk, incubated for 3 hours with the affinity-purified rabbit anti-apo(a) antiserum described above^{10 19 23} (diluted 1:1000), washed extensively in Tris-buffered saline (pH 7.4) containing 0.05% (wt/vol) Tween 20, and incubated for 2 hours with horseradish peroxidase–labeled protein A. After further washing as described above, the membrane was incubated with an enhanced-chemiluminescent Western blotting detection reagent (Amersham) for 1 to 2 minutes and subjected to autoradiography according to the manufacturer's instructions (Amersham). In some cases MAB 1A2 or LM1 was used for apo(a) detection. MAB 1A2 is specific for kringle IV and recognizes a linear epitope on the repetitive kringle IVs as well as on "unique" kringle IV types 2, 3, 4, 6, and 7.²⁴ MAB 1A2 does not react with plasminogen. MAB LM1 is a peptide antibody directed against an oligopeptide of kringle V in apo(a).²⁵ The sensitivity of the immunoblotting method was at least 5 ng.

Phenotyping and determination of the number of kringle IV repeats were performed.²⁶ In brief, the migration of apo(a) bands relative to apoB-100 was measured by using 3 µL of plasma and compared with that of an immuno-isoform standard that consisted of a mixture of five distinct apo(a) isoforms. The apo(a) bands were characterized with regard to the corresponding genotypes by pulsed-field gel electrophoresis.²⁶

Neuraminidase Treatment and Determination of Creatinine
Aliquots (1 mL) of concentrated urine samples were incubated for 6 hours at 37°C in the presence of 1 U neuraminidase from Clostridium perfingens (Sigma) and investigated immediately by using SDS-PAGE.

Creatinine was measured by using the Jaffé method with commercial assay kits from Boehringer Mannheim.

All other chemicals were reagent grade from E Merck if not otherwise stated.

Statistical Analyses
Statistical analyses were performed by using the SPSS program for Macintosh.

Results

For validation of the Lp(a) assay for urine, four urine samples were spiked with various amounts of Lp(a) or recombinant apo(a); in all cases yields ranged from >95% to <107% (Table 1). In addition, apo(a) was measured directly in fresh urine samples from five volunteers with different creatinine and apo(a) concentrations and in urine concentrated 200-fold by pressure dialysis (Table 2). The concentrated urine was diluted for the DELFIA assay in the same way as plasma. The values obtained for diluted and concentrated urine agreed within 10%. The cause for the lower apo(a) levels in the concentrated urine is thought to be due to losses during dialysis. From these results we concluded that our measured apo(a) concentrations were not biased by matrix effects.

View this table: [in this window] [in a new window]   Table 1. Validation of the DELFIA Assay for Apo(A) in Urine: Effect of Spiking With Lp(a) or Apo(a)

View this table: [in this window] [in a new window]   Table 2. Validation of the Apo(a) DELFIA Assay in Urine: Effect of Concentration by Pressure Dialysis

Twenty-four–hour urine samples from 12 normolipemic individuals with plasma Lp(a) levels ranging from 3 to 150 mg/dL were collected and immediately frozen at -20°C. Phenotyping of apo(a) isoforms in plasma revealed that most of the volunteers were heterozygous, with numbers of kringle IV copies ranging from 14 to 40 (Table 3). The apo(a) concentrations in the 24-hour urine of the 12 probands assayed by our a:a DELFIA ranged from 3.7 to 30.4 µg/dL. To characterize the nature of apo(a) secreted into urine, the samples from these single donors were concentrated by pressure dialysis and subjected to SDS-PAGE followed by immunoblotting using a POAB against apo(a). The banding pattern of five of these concentrated urine samples (probands J.G., S.H., G.G., W.R., and P.P. from Table 3) are displayed in Fig 1A. Individual urine samples from all 12 subjects described in Table 3 as well as 20 additional ones (data not shown) exhibited qualitatively very similar patterns irrespective of their phenotype. There were, however, differences in the relative intensities of the different apo(a) bands. Apparent molecular masses of the apo(a) bands ranged from 50 to 160 kD; weak bands, not visible on the blot in Fig 1 within the molecular mass range of 30 to 50 kD, were observed in highly concentrated urine. The apo(a) pattern was identical whether SDS electrophoresis was performed in the presence or absence of 1% mercaptoethanol (data not shown), indicating that the bands did not consist of smaller fragments held together by disulfide bonds.

View this table: [in this window] [in a new window]   Table 3. Characteristics of Plasma and Urine Apo(a) of 12 Probands

View larger version (34K): [in this window] [in a new window]   Figure 1. Urine was concentrated 100-fold by pressure dialysis; 10-20 µL of each sample was subjected to SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. Bands were visualized by chemiluminescence as described in "Methods." Autoradiography was for 1-3 minutes. The electrophoretic migration of molecular weight markers is shown. A, Detection of apo(a) fragments in urine from five subjects (initials indicated at top of lanes) from Table 2. Immunoblotting was performed with a rabbit anti-apo(a) POAB IgG. B, Urine from five volunteers was pooled (lanes 1-3) and processed as indicated above. Plasma (2 µL) from A.I. (Table 1) was electrophoresed in 3.75% SDS gels (lane 4). Immunoblotting was performed with the following antibodies: lane 1, rabbit POAB IgG; lane 2, kringle IV–specific MAB 1A2; and lanes 3 and 4, kringle V–specific MAB LM1. C, Pooled urine was incubated for 6 hours at 37°C in the presence (+) or absence (-) of neuraminidase (NANA) as described in "Methods." Visualization of bands was performed by using a rabbit anti-apo(a) POAB IgG. St d indicates low-molecular-weight protein standard stained with Fastgreen.

To exclude the possibility that apo(a) fragmentation takes place after sampling by the action of proteases, fresh urine from three volunteers was immediately lyophilized. Aliquots of urine samples from the same volunteers were stored for 2 or 3 days at room temperature followed by pressure dialysis. There was absolutely no difference in the individual apo(a) patterns in immunoblots (data not shown).

To address the question of whether urine apo(a) might be complexed to apoB, 10 randomly selected urine samples from donors with Lp(a) levels between 35 and 142 mg/dL were subjected to our a:B DELFIA; the measured signals were not higher than that of the blank. To determine whether apoB was present in urine at all, total apoB was further measured in the 10 urine samples by a B:B DELFIA with a sensitivity of 0.1 µg/dL. Under these conditions, none of the 10 normal urine samples tested were positive. In support of this, apoB was not detectable in urine samples by using immunoblot analysis. Thus, from these results we excluded the possibility that urinary apo(a) was complexed to apoB.

To further pursue the identity of these bands, pooled concentrated urine was investigated by immunoblotting with MABs specific for kringle IV (1A2) or kringle V (LM1) (Fig 1B). 1A2 gave a pattern in immunoblots identical to that obtained with the POAB. With LM1, a distinct pattern of three major bands with apparent molecular masses of 43, 39, and 35 kD was observed. It should be noted that the exposure time for autoradiography of lane 3 in Fig 1B was 10 times longer than that for lanes 1 and 2.

In subsequent experiments, concentrated pooled urine was incubated with neuraminidase and subjected to SDS-PAGE (Fig 1C). Neuraminidase treatment led to a shift of the banding pattern toward lower molecular weights, which led us to conclude that the apo(a) fragments in urine were glycosylated.

We then determined the correlation of urinary with plasma apo(a) values. First, the consistency of apo(a) secretion into urine in each patient was monitored over 4 months. One volunteer (A.I. in Table 3) collected his 24-hour urine samples in 2-week intervals (Fig 2). A highly significant correlation (r=.93, P<.01) between his urine apo(a) concentrations and urinary creatinine levels was observed. The urinary apo(a) level from these eight samples (normalized to 100 mg/dL creatinine) was 21.5±0.5 µg/dL, which suggests that normal individuals have a rather constant secretion rate of apo(a) relative to creatinine.

View larger version (11K): [in this window] [in a new window]   Figure 2. Line graph shows correlation of apo(a) in urine with urinary creatinine. Twenty-four–hour urine samples were collected from one individual (A.I.) eight times at 2-week intervals.

In subsequent experiments, the urine apo(a) values of 193 healthy volunteers were immunoquantified by using the a:a POAB-DELFIA, normalized to 100 mg/dL creatinine, and correlated with fasting plasma apo(a) values. Median values and 25th and 75th percentiles, respectively, for plasma apo(a) were 2.15, 0.88, and 4.72 mg/dL; the corresponding urine apo(a) values were 5.1, 2.84, and 8.70 µg/dL. Plasma and urine apo(a) values correlated significantly (=0.659, P<.0001) by the Spearman rank correlation test (Fig 3).

View larger version (18K): [in this window] [in a new window]   Figure 3. Correlation of apo(a) values in urine with plasma apo(a). Morning urine and fasting plasma from 193 apparently healthy male and female volunteers aged 20-65 years were collected and assayed by DELFIA as described in "Methods." Urinary apo(a) values were normalized to 100 mg/dL creatinine.

Of particular interest was the question of whether individuals with almost undetectable amounts of plasma Lp(a) secrete any apo(a) at all into their urine. For this purpose we selected five probands with plasma apo(a) values far below 1 mg/dL. These apo(a) values were measured by using the a:a DELFIA standardized especially for a very low concentration range. All these probands secreted apo(a) into the urine (Table 4); their urinary apo(a) levels were among the lowest observed in the group of 193 volunteers but definitely higher than those calculated from the regression line derived from Fig 3. This regression line also demonstrates that at low plasma apo(a) concentrations, the apo(a) excretion into urine is proportionately high. The physiological basis for this observation is unknown. The five urine samples from Table 4 also exhibited apo(a) fragments as assessed by immunoblotting that were comparable in size to the other 30 probands studied (results not shown).

View this table: [in this window] [in a new window]   Table 4. Apo(a) Values of Probands With Very Low Plasma Lp(a) Concentrations

The major organ of plasma apo(a) catabolism is unknown. To investigate the significance of apo(a) excreted into urine for the total catabolism of Lp(a), 24-hour urine samples from 12 healthy volunteers (11 men and 1 woman) were collected, and their total content of apo(a) fragments was quantified (Table 3). The median amount of apo(a) fragments excreted per day was 121.0 µg. From plasma apo(a) concentration combined with plasma volume (assuming that 4% of the body weight represents plasma), we calculated a median total apo(a) pool in the 12 individuals of 97.2 mg. From these values we further calculated that during 24 hours a median of 0.073% of plasma apo(a) was excreted into the urine in the form of apo(a) fragments.

Discussion

To understand the pathophysiology of increased plasma concentrations of Lp(a), a detailed knowledge of its metabolism is essential. The fundamental steps of Lp(a) biosynthesis and assembly are reasonably well characterized. Recent work by several research groups strongly suggests an extracellular assembly of Lp(a), ie, LDL complexes with a preformed mature apo(a) glycoprotein.^{16 20 23 27 28 29} In contrast to Lp(a) biosynthesis, information on the site and mode of Lp(a) catabolism is lacking. As an apoB-100–containing lipoprotein, one may anticipate that Lp(a) is catabolized in a way similar to LDL, ie, by the apoB/E-receptor. Although Lp(a) binding to the apoB/E-receptor has been demonstrated in vitro,³⁰ the in vivo situation is less clear. Lipid-lowering drugs, which strongly affect plasma LDL levels by inducing apoB/E-receptor expression in the liver, are hardly effective for reducing Lp(a).¹⁷ On the other hand, Lp(a) in homozygous familial hypercholesterolemia patients is catabolized at a faster rate than LDL,^{15 31} which suggests some specific mechanisms for Lp(a) removal.

The report of Oida et al¹⁸ stimulated our present investigation. We were interested in learning to what extent the kidney might be involved in specific Lp(a) excretion. To answer this question, it was important to characterize the apo(a)-containing fractions in human urine. Pooled urine from probands with various apo(a) phenotypes was investigated. The results of these experiments led to the following picture: apo(a) fragments secreted into the urine are free of apoB and exhibit apparent molecular masses ranging from 50 to 160 kD. Smaller fragments with <50 kD are also present at much lower concentrations. Neuraminidase treatment, which removes the terminal sugar from glycoproteins, shifts the electrophoretic migration of all observed apo(a) bands toward lower molecular masses, indicating that apo(a) fragments in urine are glycosylated. On the basis of the amino acid sequence one may calculate the following molecular masses: kringle IV, 12.5 kD; kringle V, 9.7 kD; and protease domain, 27.9 kD. Assuming 28% glycosylation, a value that has been proposed for plasma apo(a),⁷ the apparent molecular masses upon SDS electrophoresis of the above fragments are 16.0, 12.4, and 35.7 kD, respectively. Thus, the bands reacting with MAB 1A2 in Fig 1B may represent apo(a) fragments with 3 to 10 kringle IVs. Of particular interest was the observation that the major fragments of urinary apo(a) reacted with the POAB against apo(a) and the kringle IV–specific MAB 1A2 yet failed to show a reaction with the kringle V–specific MAB. In addition, there were three apo(a) fragments observed in urine that reacted with the kringle V–specific MOAB with apparent molecular masses of 43, 39, and 35 kD, respectively. From these molecular masses we consider that the two smaller fragments may represent kringle V plus parts of the protease domain; the larger fragment may contain an additional kringle IV or part of one.

Furthermore, we demonstrated that the sizes of apo(a) fragments in individual urine samples from donors with various apo(a) phenotypes were very similar and not affected by reducing agents. This size distribution, independent of apo(a) isoforms, is of particular interest and is certainly subject to further investigation.

In speculating about the possible origin of apo(a) degradation products and their urinary secretion mechanism, it is tempting to assume that specific proteases cleave apo(a) at distinct positions somewhere between the kringle IV repeats. Since reducing agents do not change the banding pattern, a cleavage within kringles is less likely. With respect to the question of origin of apo(a) fragments, it would be important to know whether similar fragments are also present in plasma. We addressed this question by investigating concentrated plasma with immunoblot analysis. Despite the presence of some degradation products, we could not detect any apo(a) pattern resembling that of urine.

The molecular weight range up to 160 kD certainly calls for an active transport mechanism into the urine since the fragments with the high molecular masses, at least, would be too large to undergo the physiological filtration process by the kidney. Since similar bands were detected in urine samples from individuals with differently sized apo(a) isoforms, such an active transport mechanism might be size-selective. On the other hand, an active transport mechanism is not supported by the observed high correlation between urinary apo(a) values and creatinine. A possible explanation for this might be that plasma Lp(a) is degraded at a constant rate. Furthermore, the plasma concentration of Lp(a) in healthy persons is very stable over time. Accordingly, the urinary excretion of apo(a) fragments during 24 hours is constant and thus correlates with the creatinine catabolism.

We also addressed the question of the significance of the urinary loss of apo(a) for the overall Lp(a) catabolism. The total amount of apo(a) secreted into the urine of 12 healthy volunteers (median, 121 µg/d) was considerably higher in persons with high plasma Lp(a) concentrations than those with low concentrations. From the plasma pool (assuming that 4% of the body weight represents blood plasma) and the apo(a) mass in plasma we calculated that a median value of 0.073% of plasma Lp(a) is catabolized via this pathway.

Turnover studies in humans show a fractional catabolic rate for plasma Lp(a) of 31±6% per day.^{14 31 32 33} From the present results we calculated that <1% of the catabolized fraction of apo(a) is excreted in the form of apo(a) fragments into urine. Several studies report increased plasma Lp(a) levels in patients suffering from kidney diseases.³⁴ In fact, plasma Lp(a) levels are three to five times higher in these patients but return to normal after kidney transplantation.³⁵ In animal studies, we have also found that the relative amount of Lp(a) retained in the kidney in relation to LDL is higher than in most other organs.³⁶

Our studies at this time do not allow us to draw any conclusion on the mode of Lp(a) catabolism by the kidney on a molecular basis. It would even be conceivable that the role of the kidney is restricted to scavenging for the breakdown products of apo(a), which may be generated elsewhere in the body. We also suggest the possibility that only free apo(a), which makes up 3% of total apo(a) mass³⁷ and possibly bypasses the assembly with LDL, is processed and secreted into urine. Finally, we have to consider the possibility that the apo(a) fragments present in the urine might be biosynthesized as such, eg, by alternative splicing.

With respect to the physiological significance of apo(a) fragments in the urine, we are thus limited to speculation. It is, however, intriguing to note that distinct fragments of plasminogen, the protein with the highest homology to apo(a), are also secreted into urine. This 38-kD plasminogen fragment, termed angiostatin, consists of an N-terminal portion containing kringles I through III^{38 39} and suppresses metastases of Lewis lung carcinoma. The possible role of urinary apo(a) fragments with regard to angiostatin-like actions is of particular interest and deserves further investigation.

Selected Abbreviations and Acronyms

DELFIA = dissociation-enhanced lanthanide fluoroimmunoassay Lp(a) = lipoprotein(a) MAB = monoclonal antibody PAGE = polyacrylamide gel electrophoresis POAB = polyclonal antibody SDS = sodium dodecyl sulfate

Acknowledgments

This work was supported by grants from the Austrian Research Foundation (grants S 7104 and SFB 702 to Dr G.M. Kostner and P 10090 to Dr Dieplinger) and the Austrian National Bank (ÖNB) (grant 5134 to Dr Steyrer). The technical assistance of Margit Stultschnig, Anton Ibovnik, and Harald Grillhofer is appreciated.

Received November 16, 1995; revision received March 7, 1996; References

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