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The influence of acute beta 1-receptor blockade using 50 mg metoprolol or beta 1/beta 2-blockade using 40 mg propranolol resulted in an equipotent reduction of cardiac frequency and systolic blood pressure without influencing diastolic pressure in ten healthy probands. There was no reduction of maximal bicycle ergometric exercise by either beta-receptor blocking agent. Serum glucose levels did not change during metoprolol in comparison to pre-test values. In contrast, propranolol resulted in a significant decrease of glucose levels during maximal exercise and 5 minutes after end of exercise. Plasma lactate was moderately lowered by both beta-receptor blockers after 20 min constant exercising when compared to pre-medication. Both substances reduced the insulin level in a comparable way during the exercise test. Serum triglyceride concentrations did not alter significantly during exercise tests. Serum free fatty acid levels showed a decreasing tendency until maximal exercise; however, there was no significant difference between values obtained with metoprolol or propranolol and drug-free pre-test.

In 5 patients with chronic liver disease (chronic persistent hepatitis, chronic aggressive hepatitis, posthepatitic cirrhosis, alcoholic cirrhosis and primary biliary cirrhosis) various parameters of cholesterol turnover were estimated from the specific activity time curve of plasma cholesterol after an intravenous injection of labelled cholesterol. The mean size of the more rapidly exchangeable pool A (which essentially includes cholesterol of plasma, erythrocytes, bile, liver, intestine and some cholesterol of other organs such as spleen, kidney, and lung) was significantly increased as compared with that of 16 healthy individuals. The mean production rate of cholesterol (synthesis plus absorption), the mean rate constant for cholesterol turnover in pool A (kAA), and the mean fraction of cholesterol of pool A which leaves the system per unit of time (kA) were significantly decreased as compared with the corresponding values of the control group. The mean transfer rates between pool A and the more slowly exchangeable pool B (which includes exchangeable cholesterol in tissues other than pool A) and the mean size of pool B were not significantly altered in the patients with chronic liver disease. SVom these findings and from the interrelationships between the various parameters of cholesterol turnover it is suggested that the increased size of pool A may be the result of the decreased removal of cholesterol from this pool. The plasma cholesterol pool is not related to the size of pool A. In chronic liver disease without cholestasis, the decreased production rate of cholesterol is associated with a low plasma cholesterol concentration.

In eleven patients with chronic renal insufficiency treated by intermittent haemodialysis and in ten normal subjects, hepatic and extrahepatic triglyceride lipase activity of post heparin plasma was selectively measured, utilizing the different sensitivity of both enzymes to inhibition by protamine sulphate. In uraemic patients, hepatic triglyceride lipase activity was significantly decreased and extrahepatic triglyceride lipase activity was normal when compared with the control group. The uraemic subjects showed a moderate hypertri-glyceridaemia; their serum cholesterol level, however, was normal. The high triglyceride concentration was due to an increase of very low density lipoproteins and low density lipoproteins of the density between 1.006 and 1.019g/ml (LDL1) The concentration of low density lipoproteins of the density between 1.019 and 1.063g/ml (LDL2) was decreased. LDL2 were relatively rich in triglycerides when compared with LDL2 from the control group.

Background The Gly972Arg mutation in the IRS-1 gene has been found to be associated with insulin resistance and type II diabetes. A recently published study described an association between the Arg allele and an increased risk for coronary artery disease. In the present study we asked whether the presence of the codon 972 mutation in the IRS-1 gene is associated with higher IMT values of the carotid arteries. Materials and methods To address this question, genotypes of the codon 972 polymorphism were determined in 1018 healthy unrelated individuals aged 40–65 years. Three homozygous carriers of the mutation were excluded for statistical analysis. In all subjects, intima media thickness (IMT) and B-scores of carotid arteries as well as a large number of metabolic parameters were determined. Results Heterozygous carriers of the Arg972 allele exhibited significantly lower IMT and B-score values than noncarriers. Total cholesterol, LDL-cholesterol and serum levels of apolipoprotein B were significantly lower in the carriers. Furthermore, a significant interaction between Gly972Arg-carrier status and mean daytime 24-h systolic blood pressure with regard to IMT could be observed; carriers with a systolic blood pressure above the median had lower IMT values than carriers with a systolic blood pressure equal or below the median. All these effects were more pronounced in females and remained significant after adjustment for sex, age, BMI, systolic blood pressure and serum apolipoprotein B levels. No significant differences between the carriers and the noncarriers could be found for BMI, insulin sensitivity or frequency of type II diabetes. Conclusions The results of our study demonstrate that the presence of the Arg972 allele is associated with lower IMT values of the carotid arteries. This finding is partly explained by lower serum levels of apolipoprotein B in carriers. The protective effect of the Gly972 Arg mutation seems to be stronger in the presence of a higher systolic blood pressure. Our data contradict previous findings suggesting an increased risk for insulin resistance, type II diabetes and atherosclerotic vascular disease in carriers of the mutation.

Growth factors are essential for cellular growth and differentiation in both normal and malignant human breast epithelial cells. In the present study we investigated the effect of epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha) and phorbol myristate acetate (PMA) on chicken ovalbumin upstream promoter-transcription factor (COUP-TF) expression in human breast cancer cells. The orphan receptors COUP-TFI and COUP-TFII are members of the nuclear receptor superfamily. The high degree of evolutionary conservation of these proteins strongly argues for an important biological function. COUP-TF expression was highest in SK-BR3 cells (approximately 130 amol/ micro g total RNA), while the lowest COUP-TF expression was observed in MCF-7 cells (3.5 amol/ micro g total RNA). While treatment of EGF, TGFalpha and PMA induced expression of COUP-TFII, COUP-TFI did not respond to these agents. Oncostatin M (OSM) is known to exert an antiproliferative effect in breast cancer cells. Treatment of MCF-7 cells with OSM resulted in an approximately 90% reduction of COUP-TFII mRNA expression. In SK-BR3 cells, treatment with the MEK inhibitor UO126 resulted in a profound suppression of endogenous COUP-TFII expression. Furthermore, cotreatment with UO126 prevented induction of COUP-TFII expression by EGF in MCF-7 cells. In conclusion, our data provide evidence, for the first time, that mitogenic substances which activate the MAP kinase pathway, can induce COUP-TFII expression. Our results strongly suggest that an active MAP kinase pathway is essential for COUP-TFII expression in human breast cancer cells.

An insertion/deletion polymorphism in the gene coding for the angiotensin-converting enzyme (ACE) is strongly associated with ACE activity. This polymorphism may be a marker for an increased risk for cardiovascular events. Our study examined a possible relationship between the D/I polymorphism and myocardial release of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Ninety-six individuals with normal or impaired left ventricular function were included in the study. ANP and BNP plasma levels were measured at rest and after exposure to physical stress. At rest no association of ACE genotypes with ANP and BNP was found. After exercise homozygotes with the genotype DD had significantly higher ANP plasma levels than homozygotes with the genotype II. In contrast to ANP, BNP levels were not significantly different between genotype groups after exercise. Differences in site of production and mode of release between ANP and BNP might explain this difference. We hypothesize that our result might represent a variability gene effect of the ACE gene locus on endocrine processes in the heart during exposure to physical stress.

The orphan receptor ARP-1/COUP-TFII, a member of the chicken ovalbumin upstream promoter transcription factor (COUP-TF) subfamily of nuclear receptors, strongly represses transcriptional activity of numerous genes, including several apolipoprotein-encoding genes. Recently it has been demonstrated that the mechanism by which COUP-TFs reduce transcriptional activity involves active repression and transrepression. To map the domains of ARP-1/COUP-TFII required for repressor activity, a detailed deletion analysis of the protein was performed. Chimeric proteins in which various segments of the ARP-1/COUP-TFII carboxy terminus were fused to the GAL4 DNA binding domain were used to characterize its active repression domain. The smallest segment confering active repressor activity to a heterologous DNA binding domain was found to comprise residues 210 to 414. This domain encompasses the region of ARP-1/COUP-TFII corresponding to helices 3 to 12 in the recently published crystal structure of other members of the nuclear receptor superfamily. It includes the AF-2 AD core domain formed by helix 12 but not the hinge region, which is essential for interaction with a corepressor in the case of the thyroid hormone and retinoic acid receptor. Attachment of the nuclear localization signal from the simian virus 40 large T antigen (Flu tag) to the amino terminus of ARP-1/COUP-TFII abolished its ability to bind to DNA without affecting its repressor activity. By using a series of Flu-tagged mutants, the domains required for transrepressor activity of the protein were mapped. They include the DNA binding domain and the segment spanning residues 193 to 399. Transcriptional activity induced by liver-enriched transactivators such as hepatocyte nuclear factor 3 (HNF-3), C/EBP, or HNF-4 was repressed by ARP-1/COUP-TFII independent of the presence of its cognate binding site, while basal transcription or transcriptional activity induced by ATF or Sp1 was not perturbed by the protein. In conclusion, our results demonstrate that the domains of ARP-1/COUP-TFII required for active repression and transrepression do not coincide. Moreover, they strongly suggest that transrepression is the predominant mechanism underlying repressor activity of ARP-1/COUP-TFII. This mechanism most likely involves interaction of the protein with one or several transcriptional coactivator proteins which are employed by various liver-enriched transactivators but not by ubiquitous factors such as Sp1 or ATF.

This study examined a possible relationship between genetic variation in the gene coding for the angiotensin converting enzyme (ACE) and increased risk for coronary heart disease (CHD) in an Austrian population. Polymerase chain reaction (PCR) was used to determine the genotypes for an insertion/deletion polymorphism in intron 16 of the ACE gene in 315 patients with CHD and in 149 normal controls. In the control group, the relative allele frequencies of the polymorphism were similar to those of previously published European studies. The genotype distribution among our patients was not significantly different from that among controls. We were not able to show a significant association of the DD genotype with coronary heart disease in subgroups containing patients considered at low coronary risk. There was no association of lipid parameters and ACE genotype. From these data we conclude that, in the Austrian population, the insertion/deletion polymorphism in the ACE gene cannot be used as a marker for coronary risk assessment.

In a patient with primary hyperchylomicronemia as a result of lipoprotein lipase (LPL) deficiency, we sequenced all translated exons and intron-exon boundaries of the LPL gene. We found a C-->A mutation in position -3 at the acceptor splice site of intron 6 which caused aberrant splicing. The major transcript showed a deletion of exons 6 through 9 and amounted to about 3% of the normal transcript of a healthy control individual. In addition to this major transcript, we found trace amounts of both a normally spliced LPL mRNA and a second aberrant transcript devoid of exon 7. On the same allele, we detected in the LPL gene of our patient four polymorphic variations, three of which have not as yet been described. A second patient from an unrelated family, but from the same geographic area, was also found to be homozygous for the same mutation. Of the relatives of the two probands studied, 11 were heterozygous and 5 were unaffected by the mutation. LPL activity in postheparin plasma was near zero in the probands and reduced in 4 of the 10 heterozygotes. A third hyperchylomicronemic patient from the same area was found to be a compound heterozygote who carried on one allele the 3' splice site mutation of intron 6 and on the other one an already described missense mutation resulting in Gly188-->Glu substitution.

In a group of 110 subjects with severe coronary artery disease, two were heterozygous for the apolipoprotein (apo) B arginine3,500----glutamine mutation that characterizes familial defective apo B-100. Both affected subjects were moderately hypercholesterolemic, and their low density lipoproteins (LDLs) were deficient in binding to the LDL receptor. Pedigree analysis of the two probands' families established a correlation between the apo B mutation, defective LDL, and a particular apo B haplotype that was characterized by 10 apo B gene markers. In addition to having one allele carrying the arginine3,500----glutamine mutation, one family member may harbor a second mutant apo B allele that causes its gene product to be present in plasma at a lower than normal level, despite the fact that the affinity of the protein for the LDL receptor appears to be normal. The metabolic basis for the underrepresentation of this second allotype remains to be elucidated.

Several recent reports have examined whether there is a correlation between the presence of some minor alleles of the highly polymorphic apolipoprotein B gene and atherosclerosis and premature heart disease. The present study extends this investigation. A high-resolution method was used to study the allele frequencies of a hypervariable minisatellite region close to the apolipoprotein B gene in 110 patients with severe coronary disease and in 117 normal controls. Alleles containing 38, 44, 46, or 48 hypervariable elements showed an association with coronary heart disease. These alleles were also associated with elevated serum levels of total cholesterol and apolipoprotein B among patients and with elevated serum levels of total triglycerides among controls. The hypervariable region showed strong linkage disequilibrium with a polymorphic EcoRI site in exon 29 and was in linkage equilibrium with a polymorphic MspI site in exon 26. Two patients carried a base change at codon 3500 that results in an arginine-to-glutamine substitution; the base change was linked in both instances to the allele with 48 hypervariable elements.

The role of genetic variation at the 3' end of the apolipoprotein B gene locus in the development of coronary heart disease and the regulation of the serum levels of various lipoproteins was studied by using two common restriction fragment length polymorphisms detected with the enzymes Xba I and EcoR I. A group of 106 male patients with coronary heart disease and 118 matched controls of Austrian origin were investigated. The frequency of the R2 allele of the EcoR I polymorphism at cDNA position 12,669 defined by the absence of the polymorphic EcoR I cutting site was significantly higher among patients than among controls. The controls with the R2 allele had significantly higher levels of total triglycerides, very low density lipoprotein (VLDL) triglycerides, and VLDL cholesterol than did the controls without this allele. Among the patients, the R2 allele was associated with higher serum VLDL apolipoprotein B levels. The chemical composition of VLDL in individuals with different genotypes for the EcoR I polymorphism did not differ significantly. For the Xba I polymorphism at cDNA position 7673, no correlation with coronary risk could be demonstrated. Patients and controls homozygous for the X2 allele characterized by the presence of the polymorphic Xba I cutting site showed a higher total and low density lipoprotein cholesterol level than did subjects with the genotype X1X1 or X1X2. This difference, however, was not statistically significant. These findings indicate that the R2 allele of the EcoR I polymorphism is associated with the occurrence of coronary heart disease and that variation at the 3' end of the apo B gene is involved in the regulation of VLDL metabolism.

Several studies have investigated the lipoprotein phenotype in heterozygous carriers of a defective lipoprotein lipase allele. We studied whether heterozygosity for lipoprotein lipase deficiency also affects glucose metabolism beyond its effect on plasma lipids.To address this question 85 heterozygous carriers of either a missense mutation (Gly188Glu) or a splice site mutation (C--A in position -3 at the acceptor splice site of intron 6) in the LPL gene which both result in a catalytically inactive product were compared with 108 unaffected subjects from the same families.Carriers for one of these mutations had higher fasting insulin levels but only a trend towards increased fasting blood glucose concentrations could be detected. HOMA index values were significantly higher in carriers than in non-carriers. Furthermore, in carriers, a significantly higher BMI and a trend towards higher systolic and diastolic blood pressure were observed. Carriers also had significantly higher fasting triglycerides, lower HDL cholesterol, and lipoprotein lipase particles of smaller size, confirming previous reports. Among carriers, subjects with one rare allele of the SstI polymorphism in the apo CIII gene had significantly higher plasma triglyceride levels than those with two common SstI alleles. This difference could not be observed in non-carriers of a mutant lipoprotein-lipase allele. The mean intima media thickness of the carotid arteries was slightly, but not significantly higher in carriers when compared with non-carriers.This study shows that carrier status of one defective lipoprotein-lipase allele is associated with impaired insulin sensitivity, an atherogenic lipoprotein profile and other characteristics of the metabolic syndrome, which are risk factors for atherosclerotic vascular disease. A higher incidence of atherosclerotic vascular disease, however, could not be firmly established in carriers of this study population.

Obesity is the most common nutritional disorder in Western society. Uncoupling protein-2 (UCP2) is a recently identified member of the mitochondrial transporter superfamily that is expressed in many tissues, including adipose tissue. Like its close relatives UCP1 and UCP3, UCP2 uncouples proton entry in the mitochondrial matrix from ATP synthesis and is therefore a candidate gene for obesity. We show here that a common G/A polymorphism in the UCP2 promoter region is associated with enhanced adipose tissue mRNA expression in vivo and results in increased transcription of a reporter gene in the human adipocyte cell line PAZ-6. In analyzing 340 obese and 256 never-obese middle-aged subjects, we found a modest but significant reduction in obesity prevalence associated with the less-common allele. We confirmed this association in a population-based sample of 791 middle-aged subjects from the same geographic area. Despite its modest effect, but because of its high frequency (approximately 63%), the more-common risk allele conferred a relatively large population-attributable risk accounting for 15% of the obesity in the population studied.

Hepatic involvement is an exceptional presentation of lymphangiomatosis. In this case report we describe a patient who underwent liver transplantation secondary to progressive hepatic involvement, which occurred 2 yr after partial hepatectomy. Within 1 yr after liver transplantation the disease condition deteriorated, with rapid progression of pre-existing skeletal lesions and development of pulmonary disease. We conclude that liver transplantation may be a treatment option for hepatic lymphangiomatosis. In the presence of pre-existing extrahepatic lesions, however, liver transplantation seems to be contraindicated.

OBJECTIVE: HELLP syndrome is a severe form of preeclampsia, characterized by hemolysis (H), elevated liver enzymes (EL), and low platelets (LP), whose pathogenesis is unclear. Autoimmunity is thought to play an important role. After the observation of development of type 1 diabetes in a patient with HELLP syndrome, we assumed a possible disease association based on autoimmune reactions. RESEARCH DESIGN AND METHODS: We examined 70 women with HELLP syndrome for the presence of autoimmune markers and glucose intolerance. Free thyroxine, triiodothyronine, thyroid-stimulating hormone, anti-thyroglobulin antibodies, thyroperoxidase antibodies, thyrotropin receptor antibodies, antinuclear antibodies (ANAs) and anti-DNA, islet cell antibodies, GADA, an oral glucose tolerance test, and HbA1c were determined postpartum. Patients with positive autoimmune markers or glucose intolerance were prospectively followed and repeated testing was performed. There were 60 women with a normal course of pregnancy matched for age, BMI, and number of pregnancies, which served as a control group. RESULTS: From the HELLP patients, 22 (31%) compared with only 6 (10%) control subjects had autoimmune antibodies (P < 0.01). There were 16 HELLP patients (23%) who exhibited only 1 kind of autoantibody (5 ANA, 9 thyroid antibodies, and 2 GADA), whereas in 6 HELLP patients (8.5%) 2 different antibodies were found. In all but 4 patients of the study group, these antibodies disappeared during 3 +/- 1.5 years of follow-up. Glucose intolerance was detected in 22 (31%) of the HELLP patients, 17 of them had impaired glucose tolerance (IGT), and 5 had diabetes, whereas only 4 subjects (6.5%) with IGT at postpartum were found in the control group (P < 0.01). During the follow-up, 2 HELLP patients were still diabetic and another 2 HELLP patients (1 GADA positive) had IGT versus 1 control subject. CONCLUSIONS: Our data give evidence that HELLP syndrome is associated with various autoimmune antibodies and glucose intolerance. Because glucose intolerance and/or autoimmune markers persisted during long-term follow-up in 6 patients with HELLP syndrome versus 1 in the control group, it may become advisable to reexamine patients with HELLP syndrome for detection of diabetes and autoimmune disorders.

Sera of 223 dyspeptic patients with endoscopic findings of nonulcer dyspepsia (72%), gastric ulcer (15%) and duodenal ulcer (13%) were tested for antibodies against Helicobacter pylori with an enzyme immunoassay and an immunoblot technique using lysates of Helicobacter pylori cells as antigen source. One hundred and fifty-one (68%) sera were found to be positive for Helicobacter pylori IgG with both methods; 5% of the positive results in the enzyme immunoassay were false-positive due to cross-reactions mainly of proteins with a molecular mass of 43-66 kDa. Since cross-reactivity not only reduces the diagnostic value of the immunoassay but also complicates evaluation of the immunoblot results, an attempt was made to overcome these problems by using specific purified recombinant proteins instead of the crude cell preparations as antigens. Of the commonly recognised immunogens of Helicobacter pylori, antibodies against a cell surface protein of 26 kDa, the small urease subunit (29 kDa) and the cytotoxin-associated protein (130 kDa) were identified as highly sensitive serological markers for inclusion in a recombinant antigen mixture for Helicobacter pylori screening. Only the cytotoxin-associated protein was confirmed to be an indicator immunogen for ulcerogenic strains. To assess the reliability of recombinant fragments of this protein in serological screening, the reactivity of antibody to purified fragments of the cytotoxin-associated protein was compared with that to the natural protein. A C-terminal recombinant fragment of 58 kDa showed results identical to those obtained with the natural protein and was thus considered to be an appropriate component of an antigen mixture for serological detection of Helicobacter pylori.

Cardiovascular diseases are the major cause of death in European and many other countries. During the last years, our understanding of the risk factors and the possibilities of an effective prevention of coronary heart disease (CHD) has substantially increased. Since patients with established CHD are at a very high risk for a further coronary event, secondary prevention plays a predominant role. Above all, the effectiveness of secondary prevention by lowering LDL cholesterol by statins has been impressively demonstrated by a number of controlled clinical trials. Various international task forces have published recommendations with regard to the cholesterol level indicating intervention and goals of treatment. With the currently available statins these goals can be reached in most cases. Diet is an integral part of overall management. It is important to target also for the intervention of the other risk factors (smoking, physical inactivity, hypertension, diabetes and overweight).

Genetic hemochromatosis (GH) is the most common autosomal-recessive disorder (1 in 300 in populations of Celtic origin). Homozygosity for a C282Y mutation in the hemochromatosis (HFE) gene is the underlying defect in ∼80% of patients with GH, and 3.2–13% of Caucasians are heterozygous for this gene alteration. Because the high frequency of this mutation may result from a selection advantage, the hypothesis was tested that the C282Y mutation confers protection against iron deficiency in young women. To address this question the genotype of codon 282 was determined in a cohort of 468 unrelated female healthcare workers, ages 18–40 years. In all study participants, a complete blood count was obtained, and erythrocyte distribution width, serum iron, transferrin, transferrin saturation, and ferritin were measured. Two individuals were homozygous for the C282Y mutation, 44 were heterozygous, and 416 were homozygous for the wild-type allele. Heterozygous women had significantly higher values for hemoglobin (P = 0.006), serum iron (P = 0.013), and transferrin saturation (P = 0.006) than women homozygous for the wild-type allele. Our data provide evidence for a protective role of the C282Y mutation in the HFE gene against iron deficiency in young women and suggest that a more efficient utilization of nutritional iron may have contributed to the high prevalence of the mutation in Caucasian populations.

Although the association between insulin resistance and hypertriglyceridemia has long been recognized, the question of the causal relationship of these two entities is still a matter of debate. To gain more insight into the relationship between hypertriglyceridemia and insulin resistance, we studied insulin sensitivity in two severely hypertriglyceridemic subjects in whom insulin resistance as a cause for hypertriglyceridemia could be positively ruled out. Rather, lipoprotein lipase deficiency due to a mutation in the lipoprotein lipase gene was identified as the cause. In the two study subjects, whole body glucose utilization was measured during a continuous infusion of somatostatin, glucose and insulin. Mean values of plasma glucose and insulin concentrations at 150, 160, 170 and 180 minutes were used to calculate steady state plasma glucose (SSPG) and steady state plasma insulin (SSPI) concentrations. SSPG of the two hypertriglyceridemic patients was in the range of those reported in the literature for healthy subjects without insulin resistance did not differ from those of two control subjects with normal plasma lipid levels. Therefore, the dyslipidemic state of the two patients, characterized by extreme elevation of triglyceride rich plasma lipoproteins and a severe reduction of HDL cholesterol, was clearly not associated with insulin resistance. From these findings we conclude that hypertriglyceridemia per se is not an obligatory cause for insulin resistance.

Background/Aims: Genetic haemochromatosis is the most common autosomal recessive disorder in Northern European populations. A major histocompatibility complex class I-like gene, HLA-H, has been proposed to be responsible for genetic haemochromatosis. The prevalence of HLA-H gene mutations 282(TGC; Cys/TAC; Tyr) and 63(CAT; His/GAT; Asp) was determined in patients of Austrian origin. Methods: DNA extracted from the blood of 40 Austrian patients and 271 controls was used to amplify HLA-H gene fragments by the polymerase chain reaction method. The base changes responsible for mutations Cys282Tyr and His63Asp alter recognition sites for restriction enzymes Sna B I and Bc1 I, respectively. Digestion products were separated by agarose gel electrophoresis and visualised by ethidium bromide staining. Results: Thirty-one (77.5%) genetic haemochromatosis patients were homozygous for mutation Cys282-Tyr and three compound heterozygous for mutations Cys282Tyr and His63Asp. One patient was homozygous for mutation His63Asp but normal for mutation Cys282Tyr. Four patients were normal at both genetic loci and one patient was heterozygous for mutation His63Asp. One control subject homozygous for mutation Cys282Tyr was found on investigation to fulfil diagnostic criteria for haemochromatosis. Eight control subjects homozygous for mutation His63/Asp showed no biochemical or clinical evidence of haemochromatosis indicating that this variant is not directly responsible for haemochromatosis. Absence of the Cys282Tyr mutation in six genetic haemochromatosis patients with distinct haplotypes indicates mutations within the HLA-H gene or at alternative genetic loci are the cause of genetic haemochromatosis in these patients. Conclusions: The HLA-H Cys282Tyr defect is likely to play a key role in the pathogenesis of haemochromatosis in most patients. Predominance of a single HLA-H gene mutation in haemochromatosis allows presymptomatic screening by genotypic analysis.

In August 1995 a 23-year-old man was admitted to the hospital because of acute anuria. 14 days prior to admission he had consumed five fruit bodies of raw mushrooms of the Cortinarius speciocissimus species. The tentative diagnosis of acute renal failure due to orellanine intoxication was confirmed by the histologic finding of an acute interstitial nephritis in a first renal biopsy one week after onset of anuria. The patient required hemodialysis for the following weeks and months, is now on peritoneal dialysis and is awaiting renal transplantation. Six months after onset of symptoms a second renal biopsy was performed, which revealed increasing interstitial fibrosis. In contrast to the findings of Rapior et al. 1989, orellanine could not be detected in this specimen. The negative toxin test in this second renal biopsy is possibly explained by a wide variability of pharmacokinetics of orellanine.

A sample of 66 healthy, unrelated males with normal blood pressure were studied for a possible association between an insertion/deletion polymorphism in the gene coding for the angiotensin converting enzyme and blood pressure response to physical exercise. No association was found between the polymorphism and systolic blood pressure at rest and during stress. Statistically significant associations between the polymorphism and diastolic blood pressure were observed during exercise and post-stress. At maximal workload, among homozygotes for the deletion, the mean diastolic blood pressure was 93 (+/- 10) mmHg, among homozygotes for the insertion it was 82 (+/- 8) mmHg, among heterozygotes it was 85 (+/- 10) mmHg. The difference was still statistically significant 3 minutes post-stress. The angiotensin converting enzyme polymorphism may be a marker for genetically determined differences in the response of the cardiovascular system to physical stress.

In a patient with primary hyperchylomicronemia as a result of lipoprotein lipase (LPL) deficiency, we sequenced all translated exons and intron-exon boundaries of the LPL gene. We found a C--A mutation in position -3 at the acceptor splice site of intron 6 which caused aberrant splicing. The major transcript showed a deletion of exons 6 through 9 and amounted to about 3% of the normal transcript of a healthy control individual. In addition to this major transcript, we found trace amounts of both a normally spliced LPL mRNA and a second aberrant transcript devoid of exon 7. On the same allele, we detected in the LPL gene of our patient four polymorphic variations, three of which have not as yet been described. A second patient from an unrelated family, but from the same geographic area, was also found to be homozygous for the same mutation. Of the relatives of the two probands studied, 11 were heterozygous and 5 were unaffected by the mutation. LPL activity in postheparin plasma was near zero in the probands and reduced in 4 of the 10 heterozygotes. A third hyperchylomicronemic patient from the same area was found to be a compound heterozygote who carried on one allele the 3' splice site mutation of intron 6 and on the other one an already described missense mutation resulting in Gly188--Glu substitution.

PHYSIOLOGY: Lipoproteins (LP) are generally classified according to their density. Triglycerides are mainly transported in chylomicrons and very low density LP (VLDL), cholesterol is mainly transported in low density LP (LDL) and high density LP (HDL). The metabolism of LP is controlled by their apolipoproteins, by specific receptors, enzymes, and transfer proteins. Triglycerides and cholesterol from the diet are transported in chylomicrons. The triglycerides are rapidly hydrolyzed by LP-lipase to yield chylomicron remnants. The released free fatty acids are used either for storage in adipose tissue or for oxidation in other tissues. Dietary cholesterol is transported in the chylomicron remnants to the liver. Cholesterol and triglyceride are also synthesized in the liver and then secreted into the blood in the form of VLDL. VLDL triglycerides are metabolized by LP-lipase to intermediate density LP (IDL), which are either taken up by the liver or further catabolized to LDL. LDL are bound and taken up by specific receptors (LDL receptors) in the liver and many other tissues. By this pathway, cholesterol is transported from the liver to peripheral tissues. LDL can be modified by oxidation and then taken up by macrophages in the arterial intima resulting in the formation of foam cells, an important step in atherogenesis. HDL play an important role in reverse cholesterol transport (transport of cholesterol back to the liver, the only site of cholesterol excretion).Various mutations in the LP-lipase gene or in the apo C-II gene result in LP-lipase deficiency. Homozygous carriers of the mutated gene show defective metabolism of chylomicrons and VLDL with extreme hypertriglyceridemia, eruptive xanthomas, hepatosplenomegaly and recurrent bouts of acute pancreatitis. Many mutations in the LDL receptor gene have been described as the primary cause of familial hypercholesterolemia due to LDL receptor deficiency. LDL receptor deficiency results in the accumulation of LDL in the plasma and deposition of LDL cholesterol in tendons and skin (xanthomas) and arteries (atheromas). In homozygotes, coronary heart disease begins in childhood. Familial defective apo B-100 is caused by a mutation in codon 3500 of the apo B gene. LDL with the mutated apo B is not recognized by the LDL receptor and LDL accumulates in the blood. Mutant forms of apo E (apo E-2 and others) are not bound to the LDL(B,E)-receptor resulting in accumulation of chylomicrons and VLDL remnants (beta-VLDL) and IDL. For the manifestation of type III hyperlipemia, additional genetic, hormonal or environmental factors are involved. Cholesterol deposition in macrophages of the arterial intima and skin gives rise to atherosclerosis of coronary and peripheral arteries and xanthomas. The pathogenesis of familial combined hyperlipemia, the most frequent form of primary hyperlipemias, is multifactorial and has not been clarified in detail.

Previously, we showed that when a DNA fragment extending from -3067 to -2734 of the human apolipoprotein B (apo-B) gene is inserted immediately upstream of an apo-B promoter segment (-139 to +121), transcription from this promoter is reduced by about 10-fold in cultured colon carcinoma cells (CaCo-2) but not in cultured hepatoma cells (HepG2). We postulated that this reducer operates by a mechanism involving active repression of a transcriptional activator that binds to the segment from -111 to -88 of the apo-B promoter (B. Paulweber and B. Levy-Wilson, J. Biol. Chem. 266:24161-24168 1991). In the current study, the reducer element has been localized to a 24-bp sequence from -2801 to -2778 of the apo-B gene that contains a binding site for the negative regulatory protein ARP-1. Furthermore, we have demonstrated that the transcription factor hepatocyte nuclear factor 3 alpha (HNF-3 alpha) binds to the sequence 5'-TGTTTGCTTTTC-3' from -95 to -106 of the apo-B promoter, to stimulate transcription. Transcriptional activation by HNF-3 is repressed when the reducer sequence is inserted immediately upstream of the HNF-3 binding site, suggesting a mechanism by which the reducer-bound protein blocks the activation promoted by HNF-3. Data from cotransfection experiments in which ARP-1 is overexpressed in the absence of its binding site suggest that ARP-1 interacts either directly or via a mediator protein with proteins recognizing the HNF-3 site and that this interaction is sufficient to repress transcriptional activation by HNF-3. Because transcriptional activation by Sp1 is not affected by the reducer, it is unlikely that the reducer interacts directly with basic components of the transcriptional machinery.

In 16 members of two Austrian families affected by a missense mutation at codon 188 of the lipoprotein lipase (LPL) gene (8 heterozygous and 8 normal subjects), carrier status for the mutation as determined by DNA analysis was related to LPL activity in postheparin plasma, to the magnitude of postprandial lipemia, and to concentration, composition, and size of the major lipoprotein classes of postabsorptive plasma. Carriers exhibited clearly reduced LPL activity, normal fasting triglycerides, but pronounced postprandial lipemia. The carriers' impaired triglyceride tolerance, as evident in the postprandial state of challenge only, was associated with a fasting lipoprotein constellation characterized by (a) enrichment of HDL2 with triglycerides, (b) reduced HDL2-cholesterol, (c) enrichment of VLDL and intermediate density lipoprotein (IDL) with cholesteryl esters, (d) elevated IDL levels, and (e) small-sized LDL. Within any given individual, the degrees of expression of these characteristics were quantitatively and continuously related with each other as well as with the magnitude of lipemia and with LPL activity.

To determine the molecular basis for type I hyperlipoproteinemia in two Austrian families, the lipoprotein lipase (LPL) gene of two patients exhibiting LPL deficiency was analyzed by Southern blotting and by direct genomic sequencing of DNA amplified by polymerase chain reaction (PCR). All exons of the LPL gene except part of the noncoding region of exon 10, all splice donor and acceptor sites, as well as 430 basepairs of the 5′-region including the promotor were sequenced. A homozygous substitution of adenine for guanine in the fifth exon at cDNA position 818 of the LPL gene was found in both patients. Our sequencing strategy largely ruled out a linkage disequilibrium of the identified nucleotide change with another defect potentially causing the clinical phenotype. The base change described abolishes a normally present AvaII restriction site allowing the identification of carriers of the mutant allele by AvaII digestion of PCR fragments of exon 5; three members of the two families were homozygous for this mutation and ten members were heterozygous. The activity of LPL in postheparin plasma was almost completely absent in homozygotes and about half normal in heterozygotes. The loss of activity was related to LPL protein structure. This mutation alters the amino acid sequence at residue 188 from Gly to Glu. The conformational preferences of the protein chain around position 188 were calculated with the use of a knowledge-based computerized method. The most probable conformation is a beta-turn formed by residues 189–192. The mutation seems to destabilize the beta-turn and/or a yet larger domain critical for substrate alignment.

BACKGROUNDThe natural history of hepatitis C virus (HCV) infection is variable and factors determining the course of the illness are unclear. AIMSTo determine the natural course of HCV infection in a well characterised group of patients 18 years after an epidemic outbreak of non-A, non-B hepatitis at a plasmapheresis centre.METHODSBetween 1994 and 1996, 20 of 30 affected individuals were studied. HCV infection was confirmed using second and third generation ELISA test kits. HCV RNA was detected by a polymerase chain reaction (PCR) method and HCV genotyping was performed by analysing amplicons from the conserved 5′-non-translated region generated by nested PCR. Thirty two liver biopsies were carried out in 14 patients.RESULTSHCV antibodies were detected in all subjects. Eighteen patients had abnormal liver enzymes and 17 were HCV RNA positive, all of whom were infected with genotype 1a. Ninety per cent of this cohort showed evidence of chronic HCV infection with 50% having progressive liver disease and 20% cirrhosis 18 years after acute onset of non-A, non-B hepatitis. Considerable variation in disease outcome occurred between individuals and no correlation with clinical features of the acute illness was found.CONCLUSIONSVariability in the consequences of HCV infection in cases infected with the same virus suggests that host factors are important in determining disease outcome. The factors which determine differences in the natural history of the disease still remain to be elucidated.

Cholesteryl ester accumulation in macrophages and foam cell formation is believed to play an important role in atherogenesis. The effect of Lp(a) on the incorporation of [14C]oleate into cholesteryl esters was studied in mouse peritoneal macrophages. In view of the physico-chemical similarities between Lp(a) and LDL, the results were compared with those obtained with LDL. Native Lp(a) and LDL did not stimulate cholesteryl ester formation. Incubation of macrophages with Lp(a)- or LDL-dextran sulfate complexes caused a significant increase in cholesteryl ester formation. A similar effect was observed when Lp(a) or LDL were incubated with macrophages in the presence of antibodies directed against the specific Lp(a) apoprotein or against LpB. Treatment of Lp(a) with acetic anhydride or malondialdehyde (MDA) was followed by precipitation of most of the lipoprotein. Therefore, these modifications were not suitable to study the uptake of modified Lp(a) by macrophages. Studies with acetyl-LDL or MDA-treated LDL caused the well-known stimulation of [14C]oleate incorporation into cholesteryl esters. Thus, the modification of Lp(a) by sulfated polysaccharides or by treatment with antibodies yields similar cholesteryl ester deposition in mouse peritoneal macrophages as observed with modified LDL. This might be one mechanism by which Lp(a) exerts its atherogenicity.

An elevated concentration of lipoprotein (a) [Lp(a)] in the serum has been considered a risk factor for coronary heart disease by various investigators. In the present study, the turnover of Lp(a) was investigated in nine individuals with serum Lp(a) levels ranging from 1 to 68 mg/100 ml. After intravenous injection of radioiodinated Lp(a), the radioactivity time-curve of the serum and the specific activitity time-curves of the isolated Lp(a) and Lp(a) apolipoproteins were measured for 14 d. More than 97% of the label was found in the protein moiety of Lp(a). During the entire study period, the serum radioactivity remained with Lp(a), only insignificant amounts of radioactivity were detectable in other lipoprotein fractions. The serum radioactivity time-curves and the specific activity time-curves of the isolated Lp(a) and Lp(a) apolipoproteins were identical. The kinetic parameters of Lp(a) turnover were calculated in terms of a two-compartment model. 76.5+/-5.1% (mean+/-1 SD) of total Lp(a) was contained in the intravascular space. The biological half-life of Lp(a) was 3.32+/-0.52 d, the fractional catabolic rate (FCR) was 0.306+/-0.054/d, and the rate of synthesis was 5.00+/-3.37 mg/kg/d. A positive correlation was found between serum concentration and synthetic rate of Lp(a) apoprotein. No relationship could be demonstrated between serum level and FCR of Lp(a). The results of this study indicate that Lp(a) is not converted to other serum lipoproteins. From the correlations between serum concentration and kinetic parameters of Lp(a) it is concluded that an elevated Lp(a) level is the consequence of an increased Lp(a) apoprotein synthesis.

1. Lecithin: cholesterol acyltransferase (LCAT) in combination with exchange and transfer proteins is known to alter the composition of all plasma lipoprotein fractions. 2. Human plasma from healthy donors was incubated for 24 h at 37°C in the absence and at 4°C and at 37°C in the presence of the LCAT inhibitors sodium iodoacetate (5 mmol/l), and the low density lipoprotein fractions (LDL) were isolated. 3. LDL isolated from LCAT-active plasma (LDL-a) exhibited pronounced alterations in their surface material: the relative content of phospholipids and of free cholesterol was reduced and the content of tetramethylurea-soluble apolipoproteins was increased. LDL isolated from plasma incubated at 37°C with or without sodium iodoacetate showed significantly increased triglyceride concentrations. 4. The LDL fractions from LCAT-active and LCAT-inactive (LDL-i) incubates were iodinated with 125I and 131I respectively, and their metabolic behaviour was studied in humans. 5. LDL-a was cleared from circulation at a slower rate as compared with LDL-i (t1/2 = 3.17 ± 0.47 vs 2.88 ± 0.45 days). The apparent fractional catabolic rate of LDL-a, calculated according to a two-pool model, was reduced by 22.2 ± 3.1%. Comparing LDL-a with LDL isolated from LCAT-inactive plasma which had been incubated at 37°C, the changes in the metabolic variables were less pronounced. 6. It is concluded that physiological alterations of the chemical compositions, caused by LCAT and exchange/transfer proteins, influence the metabolism of LDL.

The major lipoprotein density classes (chylomicrons-VLDL, LDL, HDL(2) and HDL(3)) were isolated from human plasma in a two-step ultracentrifugal procedure using the Ti-14 zonal rotor. The isolation of the two major high density lipoprotein subclasses (HDL(2) and HDL(3)) was achieved in a 24-hr run using a nonlinear NaBr gradient in the density range of 1.00-1.40. The lipoproteins with a density < 1.063 found in the rotor's center were isolated in a second run of 140 min duration using a continuous linear NaBr gradient in the density range of 1.00-1.30. The isolated lipoproteins were analyzed for chemical composition and for electrophoretic mobility; purity of isolated fractions was checked by immunochemistry. The lipoproteins exhibited flotation rates, chemical compositions, and molecular weights similar to those found with the common sequential procedures in angle-head rotors. The amount of lipoprotein lipids in the bottom fraction of the zonal rotor was comparable to that of the angle-head rotor. The described method yields the main lipoprotein density classes free from albumin in a very short running time; compared with the rate-zonal techniques already in use, this method allows the quantitative separation of an additional lipoprotein density class (HDL(2)) without increasing the running time. Furthermore, this procedure proved to be suitable for isolation of plasma lipoproteins from subjects with various types and varying degrees of hyperlipoproteinemia.

125 I-Labeled autologous very low density lipoprotein (VLDL) was injected intravenously into three lipoprotein (a) positive individuals. One other lipoprotein (a) positive subject received 125 I-labeled VLDL from a lipoprotein (a) negative donor. Specific activity of apolipoprotein B in VLDL, low density lipoprotein (LDL) and lipoprotein (a) was measured for 5 days. In the lipoprotein (a) fraction only traces of radioactivity could be detected, which were caused by contamination with labeled LDL. No precursor-product relationship existed between apolipoprotein B in VLDL or LDL and apolipoprotein B in lipoprotein (a). One lipoprotein (a)-positive individual was kept on a fat-free diet for 4 days to prevent chylomicron formation; no change in the serum level of lipoprotein (a) could be detected under these conditions. The data of this study indicate that lipoprotein (a) is not a metabolic product of VLDL or LDL. Also chylomicrons are not likely to play a role as a precursor for lipoprotein (a). It is concluded that lipoprotein (a) is synthesized as a separate lipoprotein.

Durch die Infusion von Propranolol kommt es beim Menschen zu einer Senkung der Konzentration der freien Fettsauren, zu einer signifikanten Erniedrigung der Umsatzrate der freien Fettsauren und einer signifikanten Erniedrigung der „Fractional turnover rate“ der freien Fettsauren im Plasma. Die Bildung der endogenen Plasmatriglyceride aus freien Fettsauren wird signifikant erniedrigt.

1. Durch konstante Infusion von 9,10-H3-Palmitinsaure und Auftrennung der Hauptlipidfraktionen durch Dunnschichtchromatographie wurden Umsatzraten der freien Fettsauren und der Plasmatriglyceride sowie Plasmatriglycerid-Clearence und Plasmatriglycerid-Umsatzzeit bei Stoffwechselgesunden und Patienten mit essentieller Hyperlipamie untersucht. 2. Bei Patienten mit essentieller Hyperlipamie ist die Einbaurate der freien Fettsauren in Plasmatriglyceride im nuchternen Zustand im Mittel gegenuber Stoffwechselgesunden leicht erhoht. Diese Erhohung wird vermutlich durch die erhohte Konzentration an freien Fettsauren im Plasma dieser Patienten verursacht. Die Triglycerid-Clearence ist bei diesen Patienten aber auf das Funffache erniedrigt und die Triglycerid-Umsatzzeit entsprechend auf das Zehnfache erhoht. 3. Auf Grund dieser Ergebnisse ist eine Storung im Abtransport der Plasmatriglyceride als Ursache des extrem erhohten Triglyceridspiegels bei Patienten mit essentieller Hyperlipamie anzusehen.

An sechs stoffwechselgesunden Personen wurden wahrend einer intravenosen Glucoseinfusion (1 ml einer 50%igen Losung pro Minute, Infusionsdauer 190 min) die Umsatzrate der freien Fettsauren (FFS) des Plasmas und die Einbaurate der Plasma-FFS in Plasmatriglyceride untersucht. Weiter wurde bei denselben Personen an einem der folgenden Tage der Einflus einer intravenosen Infusion von Noradrenalin (0,25/min/kg) auf die genannten Stoffwechselgrosen wahrend der Glucosebelastung untersucht, wobei mit der Verabreichung von Noradrenalin 60 min nach dem Start der Glucoseinfusion begonnen wurde. Der FFS-Umsatz und der Einbau der Plasma-FFS in Plasmatriglyceride wurden durch konstante Infusion von Palmitinsaure-9,10-H3 bestimmt, zur Berechnung der Umsatzgrosen wurde der Zeitraum von 130–190 min nach Beginn der Glucoseinfusion bzw. 70–130 min nach Beginn der Infusion von Noradrenalin herangezogen. Die Ergebnisse wurden mit den entsprechenden Werten einer Kontrollgruppe von elf Personen ohne medikamentose Beeinflussung sowie den Werten von 16 Normalpersonen wahrend einer Noradrenalininfusion in derselben Dosierung, jedoch ohne gleichzeitige Glucosebelastung, verglichen.