Wolfgang Stremmel

Low vitamin E content in plasma of patients with alcoholic liver disease, hemochromatosis and wilson's disease

The RRR-alpha-tocopherol (vitamin E) content in plasma from 46 patients with liver diseases and 23 healthy controls was determined by high performance liquid chromatography and electrochemical detection. Patients were divided into three groups: alcoholic liver diseases (n = 17; group A), hemochromatosis (n = 17; group B) and Wilsons disease (n = 12; group C). Lipid-standardized alpha-tocopherol levels were determined to neutralize differences due to hyperlipemia. The ratio of serum vitamin E to serum lipids (cholesterol, triglycerides, phospholipids) was highest in healthy controls and in patients in group A with cirrhosis and normal transaminases and bilirubin. Patients in group A with acute or chronic ethanol intoxication and high bilirubin levels had a 37% lower lipid-standardized vitamin E level than controls. Patients in group B with hemochromatosis, showing high serum iron (> 180 micrograms/dl), a low free iron binding capacity (< 8 mumol/l) and high ferritin-levels (< 450 micrograms/l), had a 34% lower vitamin E/lipid ratio than healthy controls. No significant lowering of the vitamin E/lipid ratio was observed in the other patients in group B. A significant decrease (37%) in the vitamin E/lipid ratio was only detectable in patients with Wilsons disease (group C) showing high free serum copper (> 10 micrograms/dl). The data support a role for free radicals in the pathogenesis of active liver diseases.

Epidemiology, Clinical Spectrum and Prognosis of Hemochromatosis

EPIDEMIOLOGY Eleven prospective epidemiological studies from various countries have as yet evaluated the gene prevalence of HLA-linked hemochromatosis. The estimated frequency ranged from 0.027-0.107, the frequency of homozygotes from 0.00074-0.0116, and the frequency of heterozygotes from 0.052-0.191. In a meta-analysis of the eleven surveys the frequency is 0.0016 (106/64758 subjects) for homozygotes which corresponds to a gene frequency of 0.041 and a frequency of heterozygotes of 0.078. Further analyses showed that some of these studies have probably underestimated the prevalence which in reality is probably two- to threefold higher than estimated by the meta-analysis. CLINICAL SPECTRUM In the total group of 251 patients diagnosed with hemochromatosis in the University of Düsseldorf from 1959-1992, abnormality in liver function tests (75%), weakness and lethargy (74%), skin hyperpigmentation (70%), diabetes mellitus (48%), arthralgia (44%), impotence (45% in males), and ECG abnormalities (31%) were the most frequent findings and symptoms at diagnosis. In recent years about 50% of patients were detected without having liver cirrhosis and 20% of patients did not have any symptoms and pathology except iron overload. PROGNOSIS Survival analysis in the 251 patients showed that in the absence of cirrhosis and diabetes iron removal by phlebotomy therapy prevents further tissue damage and guarantees a normal life expectancy. Sex and presence of arthropathy did not predict prognosis. However, patients with massive and long-lasting iron overload had a worse prognosis than patients with less severe iron excess. Iron removal in general ameliorated liver disease, weakness and cardiac abnormalities, and also prevented the progression of endocrine alterations. Therapy, however, did not influence arthropathy which even got worse in several patients. Iron removal also failed to reverse insulin-dependent diabetes. During a mean followup of 13.4 years 69 deaths occurred in the 251 patients. In 19 patients death was due to liver cancer, in 14 due to liver cirrhosis, in 5 due to cardiomyopathy, and in 3 due to diabetes mellitus (all causes significantly more frequent than expected for the general population). The other causes of death were as frequent as expected including extrahepatic malignancies. All liver cancers were seen in cirrhotic livers, but often occurred many years or even decades after complete iron removal. Further strategies have to evaluate the design of screening programs in order to diagnose more patients in the precirrhotic and asymptomatic stage.

Iron uptake by rat duodenal microvillous membrane vesicles: evidence for a carrier mediated transport system.

Abstract. The mechanism of iron translocation from intestinal lumen to portal plasma is poorly understood. To examine these processes, uptake of Fe2+ and Fe3+ by rat duodenal microvillous membrane vesicles prepared by a Ca2+ precipitation procedure was studied. Membrane aliquots were incubated with increasing concentrations of 59FeCl3 in the presence of a one‐thousand‐fold molar excess of citrate or 59FeSO4 with a twenty‐fold molar excess of L‐ascorbic acid. After various time intervals the incubation reaction was stopped by addition of 0·1 mM FeCl3 (4°C), and uptake of 59Fe was determined by a vacuum filtration assay. Initial uptake velocity of 59FeCl3 and 59FeSO4 was determined from the slope of the cumulative uptake curves, which was linear for the first 60 s. Initial uptake rates of both, 59Fe3+ and 59Fe2+ revealed an identical saturable uptake component with a Km of 19–22 nm and a Vmax of 8 pmol min−1 mg protein−1. In addition, transport of Fe2+ revealed a linear unspecific uptake phase, which was predominant at high substrate concentrations. Saturable uptake of Fe2+ and Fe3+ was temperature dependent, and significantly reduced by trypsin pretreatment of the microvillous membrane vesicles, indicating the involvement of a protein in the uptake process. This suggestion was pursued by isolation of an iron binding protein from duodenal brushborder membranes. After solubilization of microvillous plasma membranes with 1% Triton × 100, affinity chromatography of the membrane protein mixture over an iron chelate gel derived from epoxy activated Sepharose and elution with 50 mm EDTA yielded a single 52 000 dalton protein. The protein co‐chromatographed over an Ultro‐Pac TSK G 3000SW HPLC column together with 59FeCl3 and 59FeSO4. It showed no immunologic activity to rabbit antibodies against whole rat serum or rat transferrin. Furthermore, by photoaffinity labelling technique a single iron binding protein with a molecular weight of about 52 000 dalton was identified in microvillous membranes of the rat duodenum. These data are compatible with the hypothesis that intestinal iron absorption is mediated by a specific carrier‐dependent transport system.

Evidence for a novel keratinocyte fatty acid uptake mechanism with preference for linoleic acid: Comparison of oleic and linoleic acid uptake by cultured human keratinocytes, fibroblasts and a human hepatoma cell line

Keratinocytes require the essential fatty acid (FA), linoleic acid (LA), for the synthesis of stratum corneum membrane lipids. A plasma membrane-FA binding protein (PM-FABP), is postulated to mediate cellular FA-uptake in hepatocytes and several other tissues, but the mechanism whereby exogenous FA are taken up by keratinocytes has not been investigated. This study examines the uptake of LA and oleic acid (non-essential) in cultured human keratinocytes, in comparison to dermal fibroblasts and the human hepatoma cell line, HepG2. As previously reported for hepatocytes, FA-uptake in keratinocytes was curvilinear, with an initial (30 s) rapid cellular influx. The initial uptake component was temperature dependent, exhibited saturable kinetics and was significantly inhibited by pretreatment with trypsin. In contrast, fibroblast FA-uptake lacked an initial rapid uptake component, was relatively temperature insensitive, and was not inhibited by trypsin. Keratinocytes differed from both hepatocytes and fibroblasts by more rapid uptake of LA in comparison to oleic acid during the initial influx phase. Moreover, FA-uptake in keratinocytes was not inhibited by preincubation with a anti-rat liver PM-FABP antibody. These data provide evidence for a PM-FA transporter in keratinocytes that is distinct from the hepatic PM-FABP. The apparent preference of the putative keratinocyte FA transporter for LA may function to ensure epidermal capture of sufficient LA for barrier lipid synthesis.

Lack of Association of Helicobacter pylori Seroprevalence and Gastric Cancer in a Population with Low Gastric Cancer Incidence

BACKGROUND Previous studies have suggested that infection with Helicobacter pylori is associated with an increased risk of gastric adenocarcinoma. METHODS We examined the sera of 111 Caucasian patients with histologically confirmed gastric cancer (36 with cancer of the cardia, 70 with cancer of the body or antrum, and 5 with stump carcinomas after Billroth-II procedures) and 111 age-matched controls with colorectal carcinomas for the presence of H. pylori IgG antibodies by enzyme-linked immunoassay. RESULTS The overall prevalence of H. pylori infection was 58.6% (65 of 111) in gastric cancer patients as compared with 50.5% (56 of 111) in matched control subjects (odds ratio, 1.39; 95% confidence interval, 0.82 to 2.36). Carcinomas of the cardia were not linked to H. pylori infection (odds ratio, 1.25; 95% confidence interval, 0.65 to 2.46), nor diffuse or intestinal-type carcinomas (odds ratios, 1.79 and 1.0; 95% confidence intervals, 0.69 to 4.67 and 0.34 to 2.91, respectively). Age, sex, and height of the IgG immune response did not affect risk. CONCLUSIONS In contrast to previous results, these data do not provide evidence that the contribution of H. pylori infection to the carcinogenesis of gastric cancer is of major significance in a population with low gastric cancer rates and with high socioeconomic status.

Identification and characterization of a monoclonal antibody to the membrane fatty acid binding protein

A monoclonal antibody to the rat liver membrane fatty acid binding protein (MFABP) was prepared by immunizing mice with purified MFABP isolated from solubilized rat liver plasma membrane proteins by oleate-agarose affinity chromatography technique. The monoclonal antibody K15/6 identified a single 40 kDa protein in rat liver plasma membranes with pI values of 8.5, 8.8 and 9.0, which is identical to the authentic MFABP, but clearly distinct from rat mitochondrial GOT. The antibody K15/6 selectively inhibited cellular influx as well as membrane binding of fatty acids, but not of cholesterol or vitamin E. The same antibody was used in immunofluorescence, ELISA and Western blot analysis to determine the subcellular and organ distribution pattern of MFABP. The protein was identified in rat liver plasma membranes and mitochondria, but in no other cell compartment. It was detectable in homogenates of rat liver but not in homogenates of other organs. Therefore, the monoclonal antibody K15/6 represents an organ specific antibody to MFABP which reveals inhibitory action on membrane binding/transport of fatty acids.

Pathogenesis of genetic haemochromatosis

Abstract. Genetic haemochromatosis is an autosomal recessive inherited iron overload disease. The genetic defect and the underlying metabolic error are not known. Several observations indicate that the 2–4‐fold increase of iron absorption is due to a regulatory defect of a membrane iron transport system in duodenal mucosal cells. The key pathophysiologic factor may be the increase of gut‐derived non‐transferrin bound iron liganded to low‐molecular mass organic molecules. A putative membrane carrier protein for nontransferrin bound iron was identified and preliminary data suggest its enrichment in plasma membranes of human mucosal cells as well as in liver and other organs which are affected in genetic haemochromatosis. Cellular accumulation of ionic iron leads to peroxidative decomposition of organelle membrane phospholipids with the consequence of cell degeneration and cell death. Impairment of organ function and structural alterations such as cirrhosis of the liver are clinical manifestations.

Cellular uptake of conjugated bilirubin and sulfobromophthalein (BSP) by the human hepatoma cell line Hep G2 is mediated by a membrane BSP/bilirubin binding protein

Cellular influx kinetics of 4-50 microM bilirubin diglucuronide and sulfobromophthalein (BSP) by the human hepatoma cell line Hep G2 was examined at 37 degrees C. In confluent monolayer cultures, cellular influx of increasing concentrations of conjugated bilirubin and BSP revealed similar saturation kinetics with Km values of 9.9 and 12.1 microM, and Vmax values of 0.512 and 0.473 nmol.mg cell protein-1.min-1, respectively. Uptake of [3H]bilirubin diglucuronide was competitively inhibited by unlabeled BSP, and was temperature dependent with maximal cellular influx rates at 37 degrees C. When the confluent monolayer cultures were pretreated with a monospecific antibody to the rat liver BSP/bilirubin binding membrane protein, initial uptake rates of conjugated and unconjugated bilirubin as well as of BSP were significantly inhibited, whereas uptake of oleate was not affected. Furthermore, immunoblot analysis of the homogenate of Hep G2 cells with the same antibody revealed predominant reactivity with a 55 kDa protein. These data suggest that cellular uptake of bilirubin and related cholephilic organic anions by the human hepatoma cell line Hep G2 is mediated by a specific 55 kDa membrane BSP/bilirubin binding protein.

The membrane fatty acid-binding protein is not identical to mitochondrial glutamic oxaloacetic transaminase (mGOT)

For evaluation whether the membrane fatty acid-binding protein is related to mGOT, studies on the structure and function of both purified proteins were performed. Physicochemical characterization revealed that both proteins are different: the membrane fatty acid-binding protein has a molecular weight of 40 kD and a pI of 8.5–9.0, whereas rat mGOT has a molecular weight of 44 kD and a pI of 9.5–10.0. According to this distinct differences, they migrated separately on 2-dimensional electrophoresis. Furthermore, monospecific antibodies against the membrane fatty acid binding protein did not react with rat mGOT. In co-chromatography studies only the membrane fatty acid-binding protein showed affinity for long chain fatty acids, but not mGOT. Moreover, membrane binding studies were performed with the monospecific antibody to the membrane fatty acid binding protein. The inhibitory effect of this antibody on plasma membrane binding of oleate was reversed after preabsorption of the antibody with the membrane fatty acid binding protein, but was not affected after preabsorption with mGOT. These results indicate that the membrane fatty acid binding protein and mGOT are structurally and functionally not related. The data also support the significance of this membrane protein in the plasma membrane binding process of long chain fatty acids.

Antiarrhythmic drugs impair hepatic uptake and secretory function by different mechanisms in the isolated perfused rat liver

In the present study the effect of various antiarrhythmic drugs on hepatic perfusion parameters, uptake capacity of organic anions and biliary secretion using the isolated perfused rat liver was examined. Infusion of verapamil (VP), diltiazem, N-propyl-ajmaline (NPAB), and quinidine at pharmacological doses induced consistently a 1.4-1.6-fold increase in portal pressure accompanied by a approximately 60% decrease in bile flow and a approximately 65% inhibition of biliary taurocholate (TC) excretion. Furthermore, hepatic uptake of oxygen, bromosulphthalein (BSP), and TC was significantly reduced. All these effects were dose-dependent and reversible upon withdrawal of the drugs. Studies of the hepatic circulation using a Trypan blue staining technique demonstrated a patchy perfusion pattern during infusion of the antiarrhythmic drugs as compared to the homogenously stained control organ. The hemodynamic alterations and the impairment of the hepatic initial uptake function could be entirely prevented by concomitant administration of the vasodilator papaverine. Bile flow and biliary TC excretion, however, were still inhibited under these conditions. The present results indicate that antiarrhythmic drugs produce cholestasis in the isolated perfused rat liver independently of their adverse effect on hepatic hemodynamics.