Henne A. Kleinveld

Oxidation of lipoprotein(a) and low density lipoprotein containing density gradient ultracentrifugation fractions

Increased plasma concentrations of lipoprotein(a) (Lp(a)) are associated with an increased risk for atherosclerotic cardiovascular disease. It is thought that the atherogenicity of Lp(a) is mediated both through its LDL-like properties and its plasminogen-like properties. In this study we have investigated the LDL-like atherogenic properties of Lp(a) by comparing the susceptibility to in vitro oxidation of Lp(a) and LDL isolated from the same subject. The subjects studied varied widely in plasma Lp(a) concentration (331-1829 mg/l) and Lp(a) phenotype (from B to S4). Lipoproteins are notoriously unstable in vitro, consequently differences in in vitro handling could influence oxidizability. Therefore, the isolation and handling of Lp(a) and LDL were performed in an identical fashion. Lp(a) and LDL containing fractions were obtained by density gradient ultracentrifugation. Separate fractions containing various amounts of Lp(a) and LDL, quantitated by measuring both Lp(a) and apo B-100, were subsequently oxidized on equimolar apo B-100 basis. Despite large differences in the Lp(a)/apo B-100 ratio of the various fractions (ranging from 5.3 +/- 1.7 to 0.2 +/- 0.1) they showed quite similar oxidation characteristics. The most dense Lp(a) containing fractions showed an aberrant susceptibility to oxidation. Subsequent gel filtration and reconstitution experiments showed that this was due to protein (i.e., albumin) contamination. Removal of excess protein revealed an oxidation pattern similar to that of LDL. It is concluded that the susceptibility of Lp(a) to lipid-peroxidation is similar to that of LDL when isolated simultaneously and in the same way from the same subject. Thus, lipid-peroxidation of Lp(a) is not influenced by the presence of its distinguishing apolipoprotein(a).

The rebound of lipoproteins after LDL-apheresis. Effects on chemical composition and LDL-oxidizability.

The changes in low density lipoprotein (LDL) composition and oxidizability after LDL-apheresis (LA) using dextran sulfate cellulose columns were evaluated in 12 hypercholesterolemic men (mean+/-S.D. total cholesterol (TC) 9.7+/-1.8 mmol/l). After 10-20 months on biweekly LA combined with simvastatin 40 mg per day immediate pre-apheresis levels of TC, LDL-cholesterol, and apolipoprotein B were decreased to 5.3+/-1.3 mmol/l, 3.3+/-1.2 mmol/l, and 1.6+/-0.4 g/l, respectively, whereas apheresis induced mean acute reductions of 61, 78, and 76%, respectively. Measurements of copper-induced LDL-oxidizability in vitro showed an increased resistance against oxidation after LA until day 3 post-treatment: lag time (min) (day 0 (before LA) versus day 1 (post-LA)) 112+/-27 versus 130+/-26 (P=0.001), maximal rate of diene production (nmol/min per mg LDL) 11.1+/-2.7 versus 9.1+/-2.1 (P=0.001), and time to maximal diene production (min) 186+/-39 versus 209+/-35 (P=0. 001). Analysis of the chemical composition of LDL revealed a 25% (P<0.001) reduced content of cholesteryl esters and a decrease of the cholesterol to protein ratio of 1.20+/-0.25 to 0.70+/-0.22 (P<0. 001) through the 3rd day post-LA. Linoleic acid and arachidonic acid content of LDL decreased 11 and 18%, respectively, at the expense of palmitic acid. Vitamin E levels (mg/l) were significantly lowered due to reduction of the lipoprotein pool by apheresis; however, vitamin E content of LDL did not change in the days after apheresis when expressed per g protein or per micromol linoleic acid. The changes in fatty acid pattern were strongly associated with changes in LDL-oxidizability indices (P</=0.01). Thus, LA effectively decreased LDL pool size, inducing the presence of less buoyant lipoproteins, which were less susceptible to in vitro oxidation. This was not explained by changes in vitamin E levels, but by short-term changes in the fatty acids composition.

Lipoprotein lipase—enhanced binding of lipoprotein(a) [Lp(a)] to heparan sulfate is improved by apolipoprotein E (apoE) saturation: Secretion-capture process of ApoE is a possible route for the catabolism of Lp(a)☆☆☆

Recently, it has been recognized that cell-bound heparan sulfate (HS) proteoglycans (HSPG) are able to bind and subsequently initiate degradation of lipoproteins. Two mediators of lipoprotein catabolism, both with HS binding capacity, lipoprotein lipase (LPL) and apolipoprotein E (apoE), are involved in this process. This mechanism is known as the secretion-capture process of apoE. Lipoprotein(a) [Lp(a)] was shown to have a strong binding capacity to cell-associated HSPG. This binding capacity was increased by LPL addition. We investigated the effects of recombinant apoE (r-apoE) enrichment of Lp(a) on the binding to HS. Lp(a), isolated by ultracentrifugation and gel filtration, was incubated with r-apoE and reisolated by ultracentrifugation, resulting in r-apoE-enriched Lp(a). ApoE-enriched Lp(a) and control Lp(a) were coated to microtiter plates. The capacity to bind biotin-conjugated HS (b-HS) in the presence or absence of inactivated bovine LPL was studied. R-apoE-enriched Lp(a) showed increased b-HS binding capacity versus control Lp(a). Addition of LPL resulted in an increased b-HS binding capacity of both control and r-apoE-enriched Lp(a). To investigate whether binding of Lp(a) to endothelial cell HSPG occurred in vivo, 39 volunteers were injected with heparin (50 U/kg) and plasma lipid and Lp(a) levels were determined before and 20 minutes after heparin injection. No significant increase in plasma Lp(a) concentrations was found. The results showed that Lp(a) can be enriched with apoE and that this resulted in increased LPL-enhanced binding to HSPG. From the in vitro studies, it can be concluded that the secretion-capture process of apoE is a possible catabolic route for Lp(a). However, whether this also occurs in vivo remains to be confirmed.

Modification of lipoprotein(a) by oxidation or desialylation influences its ability to compete with plasminogen for binding to the extracellular matrix

Lipoprotein (a) [Lp(a)], and to a lesser extent low-density lipoprotein (LDL), have been shown to compete with plasminogen for binding to the extracellular matrix (ECM). Evidence exists that modification of lipoproteins alters their atherogenic properties. Therefore in the present study the effect of modifying Lp(a) and LDL by copper-induced in vitro oxidation on their ability to compete with plasminogen for binding to the ECM was studied. Oxidation of Lp(a) resulted in increased competitiveness for plasminogen binding. This effect was dependent on the Lp(a) concentration used, as well as the extent of oxidation. In the highest Lp(a) concentration used (100 nmol/l apo B100), inhibition of plasminogen binding was further increased with almost 30% compared with native Lp(a). In contrast, oxidation of LDL resulted in an additional inhibition of plasminogen binding of about 10% at all concentrations used. In separate experiments Lp(a) and LDL were modified by neuraminidase treatment. After desialylation a strong tendency for better competitiveness of Lp(a) was observed. Desialylation of LDL had no effect on its ability to compete with plasminogen for binding to the ECM. Modification of the additional and distinguishing apolipoprotein [i.e. apo(a)] in Lp(a) by oxidation and desialylation most likely explains the difference in behaviour of Lp(a) and LDL. It is concluded that modification by oxidation, and to a lesser extent desialylation, increases the anti-fibrinolytic potential of Lp(a).

Rapid and easy procedure for the determination of immunoglobulin class and light chain type of anti-lactate dehydrogenase antibodies in macro-lactate dehydrogenase.

Aim of the studyTo analyze the referral pattern, indications and results of esophageal manometry in our setting. MethodsIn this retrospective study, manometry records of all consecutive patients who underwent esophageal manometry from January 2013 to June 2015 were analyzed. The results were interpreted as per Chicago classification v.3.0. The data was entered in Microsoft excel sheet and analyzed using necessary tests. Results-A total of 220 patients with a mean age of 45 years and male to female ratio of 7:4 formed the study group. The indications for manometric evaluation includeddysphagia (87,39.54%), gastroesophageal reflux(76,34.54%) , non-cardiac chest pain(25,11.36%), dyspepsia(20,9.09%) and prior to fundoplication surgery(12,5.45%). , 39.1% patients could be diagnosed with a definitive motility disorder while 29% had ineffective motility or frequently failed peristalsis. 36% patients had a normal study. ConclusionsEsophageal manometry in our setting is done mainly for dysphagia and reflux symptoms. The main referrals are from fellow gastroenterologists and surgeons. In 39% of cases, a definite diagnosis is possible. Achalsiacardia is the most common cause of motor dysphagia.

Method-dependent increase in lipoprotein(a) in insulin-dependent diabetes mellitus during pregnancy

The current prevalent view is that plasma lipoprotein(a) [Lp(a)] concentrations are under strong genetic control. Most dietary and drug interventions seem to have little or no effect on plasma Lp(a) levels. However, evidence for a possible regulatory rol e of hormones is accumulating, for instance, fluctuations of Lp(a) levels during pregnancy have been reported. Also, in insulin-dependent diabetes mellitus (IDDM) patients, elevated Lp(a) levels have been reported. In the present longitudinal study, plasma lipid concentrations, including Lp(a), were determined in IDDM women before pregnancy, during pregnancy, and 3 months postpartum. In our study population, Lp(a) concentration was not significantly correlated with either hemoglobin A1c (HbA1c) levels of apolipoprotein(a) [apo(a)] phenotype. Changes in other lipid parameters observed during pregnancy in our IDDM population were similar to those reported during normal pregnancy. Lp(a) concentrations were quantified using two different immunochemical methods that possess different sensitivities and specificities: an immunoradiometric assay (IRMA) using two different anti-apo(a) antibodies, and an enzyme-linked immunosorbent assay (ELISA) using an anti-apo(a) and an anti-apo B antibody. Median prepregnancy Lp(a) concentrations were 118 mg/L (range, 15 to 672) as determined with the IRMA and 107 mg/L (range, 21 to 451) as determined with the ELISA. Women with IDDM showed, in general, no significant change in Lp(a) concentration during pregnancy when it was assayed with the IRMA, although a tendency to increased values was observed. When Lp(a) concentrations were determined with the ELISA, a strong and significant increase in Lp(a) from weeks 17 to 24 of pregnancy onward was found. The latter results confirm the prevalent view that during pregnancy Lp(a) levels are increased. However, the present results and those of others and Lp(a) in normal pregnancy strongly emphasize the importance of method selection when determining Lp(a) concentrations.