Arnold von Eckardstein

Prospective assessment of risk factors for recurrent stroke during childhood-a 5-year follow-up study

BACKGROUNDnRisk factors for arterial stroke in children include congenital heart malformations, vasculopathies, infectious diseases, collagen tissue diseases, and metabolic disorders. Results of previous case-control studies have shown an association between ischaemic stroke and hereditary prothrombotic risk factors: factor V G1691A and factor II G20210A mutations, raised lipoprotein (a), and deficiencies in antithrombin, protein C, and protein S. The relevance of these factors to a second ischaemic stroke event is not known.nnnMETHODSnWe assessed the risk of a second arterial ischaemic stroke associated with these prothrombotic risk factors, with underlying diseases or stroke comorbidities, and with stroke subtypes (cardiac, vascular, infectious, idiopathic). 167 boys and 134 girls aged between 6 months and 18 years of age (median 7 years) with a first episode of ischaemic stroke were followed-up prospectively for a median of 44 months (range 20-56).nnnFINDINGSnRecurrent ischaemic stroke was diagnosed in 20 of 301 children who survived (6.6%) at a median of 5 months (range 1.5-36) after first stroke onset. The relative risk of having a second stroke was significantly increased in patients with raised lipoprotein (a) (relative risk 4.4, 95% CI 1.9-10.5) and in children with familial protein C deficiency (3.5, 1.1-10.9). Additionally, survival analysis showed that a first ischaemic stroke of vascular origin was significantly associated with having a second stroke (odds ratio 3.9, 95% CI 1.4-10.6).nnnINTERPRETATIONnRaised lipoprotein (a), protein C deficiency, and stroke of vascular origin are risk factors for recurrent arterial ischaemic stroke in childhood.

Overexpression and Accumulation of Apolipoprotein E as a Cause of Hypertriglyceridemia

The molecular mechanisms of hypertriglyceridemia (HTG), a common lipid metabolic disorder in humans, often of genetic origin, are not well understood. In studying the effect of apolipoprotein (apo) E on the metabolism of triglyceride-rich lipoproteins, we found that expressing high plasma levels of human apoE3 in transgenic mice lacking endogenous mouse apoE caused HTG. These transgenic animals had 3-fold higher plasma triglyceride levels, higher very low density lipoproteins (VLDL), and lower high density lipoproteins than did nontransgenics. Removing one or both low density lipoprotein receptor alleles in the apoE3-overexpressing mice caused severe HTG (8–11-fold over nontransgenics) and increased VLDL and decreased low and high density lipoproteins, and apoE3-enriched VLDL were markedly depleted in apoC-II. At least two mechanisms could explain HTG associated with apoE3 overexpression: stimulated VLDL triglyceride production and impaired VLDL lipolysis. The apoE3 mice with HTG had a 50% increase in hepatic VLDL triglyceride production. Furthermore, overexpression of apoE (E2, E3, or E4) in cultured hepatocytes (McA-RH7777 cells) correlated positively with secretion of VLDL into the medium. However, apoE3 overexpression–associated HTG was only partially explained by VLDL overproduction, as lipoprotein lipase–mediated VLDL lipolysis was also decreased 20–86% depending on apoE3 levels, most likely by displacing or masking apoC-II on the particles. In human subjects, HTG correlated positively with increased VLDL triglyceride and plasma and VLDL apoE levels. However, plasma and VLDL apoE correlated negatively with VLDL apoC-II levels and lipoprotein lipase-mediated VLDL lipolysis. Thus, optimal expression of apoE is crucial for normal metabolism of triglyceride-rich lipoproteins, and overexpression and/or accumulation of apoE may contribute to HTG by stimulating VLDL triglyceride production and by impairing VLDL lipolysis. The apoE3-overexpressing mice will be useful for studying the pathophysiology of this disorder.

Iron metabolism and the risk of Restless Legs Syndrome in an elderly general population – The MEMO-Study

Background: Low iron and ferritin blood levels have been observed in patients with Restless Legs Syndrome (RLS) with an inverse relation between symptom severity and ferritin level. All reports are based on single cases or case series of hospitalized patients or those from outpatient clinics. No data from population studies are available. Methods: Cross-sectional study examining the associations between 5 measures of iron metabolism and RLS in an elderly general population in southern Germany. All 365 participants, aged 65 to 83 years, were examined neurologically and interviewed using standardized questions addressing the four minimal criteria for RLS. Iron, ferritin, transferrin, soluble transferrin receptor and C-Reactive Protein were analysed with standard laboratory methods. Results: The prevalence of Restless Legs Syndrome in this population was 9.8 %. Odds Ratios associated with Restless Legs were significantly increased in the fourth quintile of iron (OR 3.08 95 % CI 1.02–9.29) and transferrin saturation (OR 5.68 95 % CI 1.18–27.26) compared with the third (middle) quintile. Increases in the first (lowest) quintile of both measures were not or borderline significant. No associations with ferritin and soluble transferrin receptor were found. Conclusions: No evidence was found that iron or ferritin deficiency are a major cause of RLS in this population study. The results support the hypothesis that changes in the complex regulation of iron metabolism contribute to the occurence of RLS.

The plasminogen activator inhibitor (PAI)‐1 promoter 4G/4G genotype is not associated with ischemic stroke in a population of German children

Abstract: Objectives: To investigate the relationship between an insertion/deletion (4G/5G) polymorphism of the plasminogen activator inhibitor (PAI)‐1 gene and childhood patients with a past history of ischemic stroke. Methods: The PAI‐1 4G/4G genotype and the coinheritance with lipoprotein (Lp) (a) levels, the factor V (FV) G1691A mutation, the prothrombin (PT) G20210A variant, and the methylenetetrahydrofolate reductase (MTHFR) T677T genotype were studied in 198 Caucasian children with stroke and 951 controls (same age, sex and ethnical distribution). In a randomly selected subgroup of patients/controls (n=60) PAI‐1 activities have been investigated. Results: The distribution of the 4G/5G genotypes was no different in childhood stroke patients and controls, with a 4G allele frequency of 55.8% in patients compared with 53.8% in control subjects (P=0.49). The 4G/4G genotype compared with the remaining genotypes was present in 43 cases and 167 (17.6% vs. 21.7%; OR/CI: 1.30/0.89–1.98; P=0.3). PAI‐1 activity was significantly elevated (P<0.001) in the patient group. Conclusions: Data presented here suggest that the 4G/4G genotype is not a major risk factor in the aetiology of childhood ischemic stroke.

Structural and functional properties of natural and chemical variants of apolipoprotein A-I

Four isoforms of human apolipoprotein A-I (apo A-I): the normal allele product and the corresponding Lys-107 deletion mutant, and apo A-I with sulfoxidized Met-112 and Met-148 residues and the corresponding reduced form, were investigated in their lipid binding properties, structures, and abilities to activate lecithin-cholesterol acyltransferase. All apo A-I isoforms reacted completely with palmitoyloleoylphosphatidylcholine to give reconstituted high density lipoprotein (rHDL) particles with diameters of 96 A. These particles reacted with low density lipoprotein (LDL) and lecithin-cholesterol acyltransferase (LCAT) equally well, except that the Lys-107 deletion mutant was resistant to structural rearrangements in the presence of LDL. The spectral measurements revealed only minor structural differences among the free apo A-I forms or among their rHDL products, but showed a decreased stability of the Lys-107 deletion mutant and the isoform with reduced Met towards denaturation by guanidine hydrochloride. The results demonstrate that these specific alterations of the apo A-I sequence, which change the helix orientation and hydrophobic moment in one or two putative lipid binding regions, are not sufficient to disrupt the overall properties of the apo A-I complexes with lipid nor to impair significantly their ability to activate LCAT.

Associations Between Homocysteine and Coagulation Factors — A Cross-Sectional Study in Two Populations of Central Europe

Plasma homocysteine has been associated with vascular disease and mortality. Experimental studies and studies on patients with vascular disease have indicated a thrombogenic potential of raised homocysteine levels. Studies on community samples are rare. We investigated the associations between homocysteine levels and selected coagulation factors in population-based random samples of 187 men from Pardubice (Czech Republic) and 147 men from Augsburg (Germany), aged 45 to 64 years. Czech men had higher mean levels of plasma homocysteine (10.3 vs. 8.9 micromol/l, P<.001) and of fibrinogen, von Willebrand factor (vWF), prothrombin fragment 1+2 (F 1+2) and D-Dimer (each P<.05). Plasma homocysteine was positively correlated with fibrinogen (r=.34) and vWF (r=.23, each P<.001) only in Czechs, and with D-Dimer in both Czechs and Germans (r=.26 and.21, respectively). Formal testing for interaction regarding the intercountry differences in the relationship with homocysteine revealed significance only for fibrinogen (P<.01). In multivariate analyses, the association of homocysteine with D-Dimer remained statistically significant after adjustment for indicators of chronic inflammation and fibrinogen. No significant correlation was found with Factor VII (F VII) activity or F 1+2. Homocysteine levels were also unrelated to traditional risk factors. In conclusion, in these cross-sectional studies we found moderate to strong associations between homocysteine and components of the endogenous hemostatic and fibrinolytic systems. The associations were slightly different between Czech and German men. These findings may help to better understand the role of homocysteine in atherothrombotic diseases.

Effects of Genotype and Diet on Cholesterol Efflux into Plasma and Lipoproteins of Normal, Apolipoprotein A-I-, and Apolipoprotein E-Deficient Mice

We investigated the contribution of apoE to cholesterol efflux into plasmas of normal, apoA-I-, and apoE-deficient mice, which were fed with chow- and cholesterol-rich diets. Plasmas of normal and apoA-I-deficient mice contain apoE in pre-beta-migrating VLDL as well as in HDL-like lipoproteins, which have either electrophoretic alpha- or gamma-mobilities. The latter particle resembled gamma-LpE in human plasma also by its mobility on nondenaturing two-dimensional electrophoresis. No apoE-containing lipoproteins were found in plasmas of apoE-deficient mice. When apoA-I- and apoE-deficient mice received both chow- and fat-rich diets, their plasmas released significantly less 3H-cholesterol from radiolabeled fibroblasts than did plasma of normal mice. Removal of apoE from plasmas of normal and apoA-I-deficient mice by anti-apoE immunoaffinity chromatography decreased their cholesterol efflux capacities (per 1 minute/per 1 hour) by 26%/40% (P = 0.0092/0.0007) and 30%/26% (P = 0.0092/0.0003), respectively. Net cholesterol efflux from fibroblasts into apoA-I-deficient plasma was 45% lower compared with plasma of normal mice. Incubation of fibroblasts with apoE-deficient plasma caused net influx of cholesterol. Prior addition of human apoE to or removal of apoB-containing lipoproteins from apoE-deficient plasma restored its ability to cause net cholesterol efflux to 50% of normal plasma. Some of the differences between cholesterol efflux into normal and apoE-deficient plasmas were attributable to the failure of apoE-deficient plasmas to take up cell-derived 3H-cholesterol into gamma-LpE. Compared with normal plasma, both apoA-I-deficient and apoE-deficient plasmas were significantly decreased in their activity to esterify cell-derived 3H-cholesterol. Anti-apoE chromatography decreased significantly cholesterol esterification in normal plasma and apoA-I-deficient plasma but not in apoE-deficient plasma. Taken together, the data provide evidence that apoE is an important contributor to reverse cholesterol transport, partially because of initial uptake of cell-derived cholesterol by gamma-LpE and partially because of the contribution of apoE-containing lipoproteins to esterification of cholesterol in plasma.

Polymorphic site study at codon 347 of apolipoprotein A-IV in a Japanese population

In a population of Japanese subjects, we surveyed codon 347 of the apolipoprotein (apo) A-IV gene and found that the frequency of a rare allele at this point was extremely low compared to that in western populations. Only one of 850 unrelated samples showed mutation at the enzyme recognition site by agarose gel electrophoresis. However, direct sequencing of the coding region revealed that it did not result from the ACT (Thr) to TCT (Ser) mutation which has been reported in western countries, but from an ACT to ACG (Thr) mutation, which does not affect the primary structure of apo A-IV. Two additional family members showed the same point mutation at codon 347.

HDL and Reverse Transport of Cholesterol: Insights from Mutants

Several epidemiological and clinical studies revealed an inverse correlation between low plasma concentrations of high density lipoprotein (HDL) cholesterol as well as its major protein component apolipoprotein A–I (apo A–I) and the risk of myocardial infarction (reviewed in 1). Family and twin studies suggested partial heredity of low HDL-cholesterol levels and have put the influence of genes at 35 to 50% (2,3). Frequently, familial HDL cholesterol- deficiency was paralleled with a family history of premature coronary heart disease (CHD) (4,5).

Relationship of HDL Cholesterol to Incidence of Atherosclerotic Coronary Heart Disease: The Procam Experience

The incidence of atherosclerotic coronary heart disease (CHD) was assessed in 4559 male partecipants of the Prospective Cardiovascular Munster (PROCAM) study, aged 40 to 64 years, over — 6 year follow-up period. In this time, 186 study partecipants developed atherosclerotic CHD (134 definite nonfatal myocardial infarctions and 52 definite atherosclerotic CHD deaths including 21 sudden cardiac deaths and 31 fatal myocardial infarctions). Univariate analysis revealed — significant association between the incidence of atherosclerotic CHD, and high density lipoprotein (HDL) cholesterol (p<0.001), which remained after adjustment for other risk factors.