Lipoprotein(a)

Evidence for increased synthesis of lipoprotein(a) in the nephrotic syndrome

MG De Sain-Van Der Velden, DJ Reijngoud, GA Kaysen, MM Gadellaa, H Voorbij, F Stellaard, HA Koomans and TJ Rabelink
Department of Nephrology and Hypertension, University Hospital Utrecht, The Netherlands.

In patients with the nephrotic syndrome, markedly increased levels of lipoprotein(a) (Lp(a)) concentration have been frequently reported, and it has been suggested that this may contribute to the increased cardiovascular risk in these patients. The mechanism, however, is not clear. In the present study, in vivo fractional synthesis rate of Lp(a) was measured using incorporation of the stable isotope 13C valine. Under steady-state conditions, fractional synthesis rate equals fractional catabolic rate (FCR). FCR of Lp(a) was estimated in five patients with the nephrotic syndrome and compared with five control subjects. The mean plasma Lp(a) concentration in the patients (1749+/- 612 mg/L) was higher than in control subjects (553+/-96 mg/L). Two patients were heterozygous for apolipoprotein(a) (range, 19 to 30 kringle IV domains), whereas all control subjects were each homozygous with regard to apolipoprotein(a) phenotype (range, 18 to 28 kringle IV domains). The FCR of Lp(a) was comparable between control subjects (0.072+/-0.032 pools/d) and patients (0.064+/-0.029 pools/d) despite the wide variance in plasma concentration. This suggests that differences in Lp(a) levels are caused by differences in synthesis rate. Indeed, the absolute synthetic rate of Lp(a) correlated directly with plasma Lp(a) concentration (P < 0.0001) in all subjects. The present results demonstrate that increased synthesis, rather than decreased catabolism, causes elevated plasma Lp(a) concentrations in the nephrotic syndrome.

Decreases in apolipoprotein(a) after renal transplantation: implications for lipoprotein(a) metabolism

IW Black and DE Wilcken
Department of Cardiovascular Medicine, Prince Henry Hospital, University of New South Wales, Sydney, Australia.

Serum concentrations of apolipoprotein(a) [apo(a)], the unique glycoprotein of lipoprotein(a), are increased in patients with end- stage renal failure. We prospectively studied serum apo(a) and other lipoproteins in 20 consecutive patients, ages 46 +/- 11 years, before and for six months after successful renal transplantation. All patients received cyclosporine, and no patient was treated for hyperlipidemia. The mean creatinine clearance increased from 7.5 mL/min before transplant surgery to 40.9 mL/min six months afterwards (P less than 0.001). Apo(a) decreased from a median of 403 units/L before transplantation to 184 units/L at one week (P less than 0.001) and was 170 units/L (P less than 0.001) at six months. For the assay used, 1 unit of apo(a) is equivalent to 1 mg of lipoprotein(a). In contrast, from baseline to six months, increases were found for low-density lipoprotein (LDL) cholesterol (P = 0.03), high-density lipoprotein cholesterol (P = 0.06), apo B (P = 0.07), and apo A-I (P = 0.01). The decrease in apo(a) in individual patients was significantly correlated with the increase in creatinine clearance (r = -0.48, P less than 0.001). The single patient who developed nephrotic syndrome after renal transplantation had marked increases in apo(a) (693-1595 units/L), apo B, and LDL cholesterol, which paralleled the degree of proteinuria. These findings suggest that abnormal renal function affects the regulation of lipoprotein(a) metabolism.

Lipoprotein(a) Reduction by Ascorbate
Matthias Rath M.D. and Linus Pauling Ph.D. (1992)

Journal of Orthomolecular Medicine 7: 81-82.

Introduction

Lipoprotein(a) [Lp(a)] is associated with an increased risk of atherogenesis and thrombogenesis. Recently it was proposed that Lp(a) is a surrogate for ascorbate.1 This proposal suggested a role of ascorbate in the regulation of Lp(a) synthesis: namely, that increased intake of ascorbate, a strong natural reducing agent, would lower Lp(a) plasma levels. N-Acetylcysteine (NAC) was then also proposed to lower Lp(a) plasma levels and was reported to do this to a variable degree.2,3 The effect of ascorbate in lowering Lp(a) plasma levels was studied in a clinical pilot study with the results reported here.
Patients, Materials and Methods

Eleven outpatients with coronary heart disease and elevated Lp(a) levels consented to participate in this study. The patients received 9 grams of ascorbic acid (Bronson Pharmaceuticals, La Canada, California) per day for a period of 14 weeks. Plasma Lp(a) levels were determined at the beginning and at the end of the study. Lp(a) plasma levels were determined by a sandwich ELISA method with monoclonal capture antibodies against apo(a) and monoclonal peroxidase-labeled antibodies against the apoB-100 portion of the Lp(a) molecule. 4 The antibodies were a gift from Dr. J. C. Fruchart, Lille, France.

Results

In this study ascorbate was found to lower Lp(a) plasma levels on average by 27% with a median value also of 27% (Table 1). Two of the 11 patients showed no decrease of Lp(a) during this time period. Lp(a) in the same plasma samples was also measured with immunological assays using monoclonal antibodies against the apo(a) portion of the Lp(a) molecule for both, capturing and revealing (radioimmunoassay[RIA], Pharmacia Diagnostics; anti-apo(a) sandwich ELISA). Changes in Lp(a) plasma concentrations were measured for RIA mean +2%, median -7.5 % and for ELISA mean -4%, median -12%. The mean values for vitamin C plasma levels were 48.6 uM at the beginning and 94.4 uM at the end of the study.


Table 1.

Discussion

Two factors may account for the differences between the assay including an antibody against apoB and the assays using exclusively anti-apo(a) antibodies. One factor could be the variation in epitopes of the apo(a) molecule as a result of the variation of the molecular size determined by the genetic isoforms. This factor was largely excluded in this study by determining the apo(a) isoforms by means of SDS PAGE and subsequent immunoblotting with anti apo(a) antibodies.

The second possible factor accounting for these differences is the effect of reducing agents on the intramolecular disulfide bonds of the apo(a) molecule. This factor is discussed here in more detail. Apo(a) has been proposed to function as a proteinthiol1 and the disulfide bonds of the repetitive plasminogen kringle IV structure are known to have different dissociation constants. Elevated plasma concentrations of reducing agents such as ascorbate or NAC could alter the epitope constellation of the apo(a) molecule in vivo by reducing some of the many disulfide bonds to sulfhydryl groups. Underthis condition, assays using only anti-apo(a) antibodies could give falsely positive results, dependent on the specific epitopes they recognize in the repetitive kringle structures of the apo(a) molecule.

In contrast, an assay measuring the apoB portion of the Lp(a) molecule should provide more reliable results since apoB contains less disulfide bonds and in addition has a constant molecular size. This conclusion could also explain the fact that the only two studies reporting a lowering of Lp(a) plasma levels with reducing agents included assays using anti-apoB antibodies for detection (2, and this paper). In contrast, assays exclusively based on antibodies against apo(a) gave variable results in the presence of reducing agents. 2,3

From in vitro studies with NAC it was recently concluded that supraphysiological concentrations of reducing agents above 1 mM decrease the immunoreactivity for Lp(a). 5 The extrapolation of these results to the in vivo situation must, of course, be handled with care. The highest molar concentration of ascorbate measured in the study reported here was 154 uM, a level that does not decrease the immunoreactivity of apo(a) or Lp(a). The effect of physiological levels of ascorbate on the reduction of disulfide bonds of the apo(a) molecule as well as the possible immunological implications need further investigation

The results of the clinical study reported here, namely that dietary ascorbate supplementation reduces Lp(a) plasma levels, was supported by in vitro studies in our laboratory with human liver cells (HepG2, data not shown). In metabolic studies using S35 methionine increasing concentrations of ascorbate in the cell culture medium decreased the amount of Lp(a) secreted by these cells. Ascorbate concentrations up to 2.25 mM did not reveal any dissociation of apo(a) from apoB. It is, therefore, concluded that the effect of ascorbate on Lp(a) plasma levels is the result of a decreased rate of synthesis of Lp(a) particles in the liver.

In conclusion, ascorbate is a physiological reducing agent involved in the metabolic regulation of Lp(a) synthesis. Dietary supplementation of ascorbate, as an adjunct to conventional therapy, should contribute to reducing elevated Lp(a) plasma levels and the risk of cardiovascular disease. Prolonged supplementation of ascorbate may be required to achieve these effects.
References

1. Rath M, Pauling L. Hypothesis: Lipoprotein(a) is a surrogate for ascorbate. Proceedings of the National Academy of Sciences USA 1990; 87: 6204-6207.

2. Gavish D, Breslow J. Lipoprotein(a) reduction by N-acetylcysteine. Lancet 1991; 337:203-204.

3. Stalenhoef AFH, Kroon A, Demacker PNM. N-acetylcysteine and lipoprotein. Lancet 1991; 337: 491.

4. Vu Dac N, Mezdour H, Parra HJ, Luc G, Luyeye I, Fruchart JC. A selective bi-site immunoenzymatic procedure for human Lp(a) lipoprotein quantification using monclonal antibodies against apo(a) and apoB. Journal of Lipid Research 1989; 30: 1437-1443.

5. Scanu AM, Pfaffinger D, Fless GM, Makino K, Eisenbart J, Hinman J. Attenuation of immunologic reactivity of lipoprotein(a) by thiols and cysteine-containing compounds. Arteriosclerosis and Thrombosis 1992; 12: 424-429

Metabolism of Apo(a) and ApoB100 of Lipoprotein(a) in Women: Effect of Postmenopausal Estrogen Replacement
Wanfang Su, Hannia Campos, Helena Judge, Brian W. Walsh and Frank M. Sacks

Department of Nutrition (W.S., H.C., H.J., F.M.S.), Harvard School of Public Health, and the Departments of Medicine (F.M.S.), and Obstetrics and Gynecology (B.W.W.), Brigham and Women’s Hospital, and Harvard Medical School, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Frank M. Sacks, M.D., Department of Nutrition, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115. E-mail: fsacks@hsph.harvard.edu.

The metabolism in plasma of apo(a) and apoB100, the major protein components of lipoprotein(a) [Lp(a)], and the mechanism by which estrogen lowers Lp(a) concentration are both not well understood. Estrogen or placebo were administered to 12 postmenopausal women in a double-blind cross-over design; and after each treatment, apo(a) and apoB100 in Lp(a) were endogenously labeled by iv trideuterated leucine. After estrogen treatment, mean Lp(a) concentration decreased during estrogen, from 25 mg/dL, by 20% (P < 0.01); and the mean production rate of apo(a) decreased, from 0.31 nmol/kg·day, by 34% (P = 0.046). In contrast, the mean fractional catabolic rates of apo(a) were similar, 0.36 vs. 0.31/day (P = 0.23). In 6 women, the kinetics of apo(a) and apoB100, the two major proteins of Lp(a), were studied during estrogen and placebo periods. During both periods, the rate of appearance of tracer was similar in Lp(a)-apo(a) and Lp(a)-apoB100, as were the resulting metabolic rates and the changes during estrogen treatment. In conclusion, the findings are more compatible with intracellular synthesis of Lp(a) from nascent apo(a) and apoB100 than extracellular assembly from plasma low-density lipoproteins. Reduced flux into plasma of Lp(a), an atherogenic lipoprotein, could contribute to the lower cardiovascular disease rates in women receiving estrogen replacement therapy.

Lipoprotein(a), plasmin modulation, and atherogenesis.
Harpel PC, Hermann A, Zhang X, Ostfeld I, Borth W.

Department of Medicine, Mount Sinai Medical Center, New York, NY 10029, USA.

Lipoprotein(a) [Lp(a)] is an atherogenic lipoprotein however the mechanisms by which Lp(a) promote the atherosclerotic process are not clear. The apolipoprotein(a) portion of Lp(a) shares partial homology with plasminogen, a finding that has stimulated numerous studies. Lp(a) binds to fibrin and the affinity between fibrin surfaces and Lp(a) appears to be related to the state of oxidation of the lipoprotein particle. Lp(a) also effects fibrin-dependent plasminogen activation. Recent findings suggest that dependent plasminogen activation. Recent findings suggest that depending upon the in vitro conditions, Lp(a) either promotes or inhibits plasmin formation. Lp(a) also inhibits cell-surface dependent plasmin generation that is associated with an inhibition of transforming growth factor-beta (TGF-beta) production in cell coculture systems. Lp(a) stimulates smooth muscle cell migration and proliferation as a secondary response to this decrease in TGF-beta concentration. Studies in transgenic mice containing the human apolipoprotein(a) gene, document that both plasmin and TGF-beta formation in the media of the aorta is markedly decreased in the presence of apo(a). Thus the atherogenicity of Lp(a) may be mediated, in part, through its modulation of plasmin and TGF-beta production in the blood vessel wall.

Lipoprotein (a) inhibits the generation of transforming growth factor beta: an endogenous inhibitor of smooth muscle cell migration.
Kojima S, Harpel PC, Rifkin DB.

Department of Cell Biology, New York University Medical School, New York 10016.

Conditioned medium (CM) derived from co-cultures of bovine aortic endothelial cells (BAECs) and bovine smooth muscle cells (BSMCs) contains transforming growth factor-beta (TGF-beta) formed via a plasmin-dependent activation of latent TGF-beta (LTGF beta), which occurs in heterotypic but not in homotypic cultures (Sato, Y., and D. B. Rifkin. 1989. J. Cell Biol. 107: 1199-1205). The TGF-beta formed is able to block the migration of BSMCs or BAECs. We have found that the simultaneous addition to heterotypic culture medium of plasminogen and the atherogenic lipoprotein, lipoprotein (a) (Lp(a)), which contains plasminogen-like kringles, inhibits the activation of LTGF-beta in a dose-dependent manner. The inclusion of LDL in the culture medium did not show such an effect. Control experiments indicated that Lp(a) does not interfere with the basal level of cell migration, the activity of exogenous added TGF-beta, the release of LTGF-beta from cells, the activation of LTGF-beta either by plasmin or by transient acidification, or the activity of plasminogen activator. The addition of Lp(a) to the culture medium decreased the amount of plasmin found in BAECs/BSMCs cultures. Similar results were obtained using CM derived from cocultures of human umbilical vein endothelial cells and human foreskin fibroblasts. These results suggest that Lp(a) can inhibit the activation of LTGF-beta by competing with the binding of plasminogen to cell or matrix surfaces. Therefore, high plasma levels of Lp(a) might enhance smooth muscle cell migration by decreasing the levels of the migration inhibitor TGF-beta thus contributing to generation of the atheromatous lesions.