Proinflammatory Extracellular Vesicle-Mediated Signaling Contributes to the Induction of Neuroinflammation in Animal Models of Endotoxemia and Peripheral Surgical Stress.

Peripheral inflammation induced by endotoxemia or surgical stress induces neuroinflammation thereby causing neurological symptoms ranging from sickness behavior to delirium. Thus, proinflammatory signaling must be operative between the periphery and the central nervous system (CNS). In the present study, we tested whether nanometer-sized extracellular vesicles (EVs) that were produced during the peripheral inflammatory process have the capacity to induce neuroinflammation. Conditions of endotoxemia or surgical intervention were simulated in rats by lipopolysaccharide (LPS) injection or partial hepatectomy (HpX). EVs were concentrated from these animals and tested for their proinflammatory action (I) in a microglial cell line and (II) by intracerebroventricular and (III) by intravenous injections into healthy rats. EVs from both conditions induced the secretion of cytokines from the glial cell line. Intracerebroventricular injection of the EVs caused the release of inflammatory cytokines to the cerebrospinal fluid indicating their pro-neuroinflammatory capacity. Finally, proinflammatory EVs were shown to pass the blood-brain barrier and induce neuroinflammation after their intravenous injection. Based on these data, we suggest that EV-associated proinflammatory signaling contributes to the induction of neuroinflammation in endotoxemia and peripheral surgical stress. Preliminary results suggest that peripheral cholinergic signals might be involved in the control of proinflammatory EV-mediated signaling from the periphery to the brain.

Low-sulphate water sample preparation for LSC detection of 35S avoiding sulphate precipitation.

Information about groundwater residence times is essential for evaluating appropriate groundwater abstraction rates and aquifer vulnerabilities and hence for sustainable groundwater management in general. Naturally occurring radionuclides are suitable tools for related investigations. While the applicability of several long-lived radionuclides for the investigation of long-term processes has been demonstrated frequently, residence times of less than one year are only scarcely discussed in the literature. That is due to the rather small number of applicable radionuclides that show adequately short half-lives. A promising approach for investigating sub-yearly residence times applies radioactive sulphur. 35S is continuously produced in the upper atmosphere from where it is transferred with the rain to the groundwater. As soon as the water enters the subsurface its 35S activity concentration decreases with an 87.4 day half-life. This makes 35S suitable for investigating sub-yearly groundwater residence times. However, the low 35S activities in natural waters require sulphate pre-concentration for 35S detection by means of liquid scintillation counting (LSC). That is usually done by sulphate extraction from large water samples with an anion-exchange resin (Amberlite IRA400, Cl-form), elution from the resin with NaCl, and precipitation as BaSO4. Our study aimed at optimizing the standard sample preparation procedure by avoiding the laborious precipitation step. We suggest (i) sulphate extraction using the exchange resin Amberlite IRA67 (OH-form), (ii) elution with ammonium hydroxide, (iii) evaporation of the eluate and (iv) dissolving the resulting dry precipitate in 2 ml H2O. In contrast to the standard approach our method results in a final sample solution of low ionic strength, which allows applying the water miscible scintillation cocktail Hionic-Fluor®. Since Hionic-Fluor accepts only aqueous solutions of low ionic strength the approach is applicable for waters with high 35S/32SO42- ratios, i.e., low total sulphate sample loads (e.g. rainwater).

Improved approach for LSC detection of 35S aiming at its application as tracer for short groundwater residence times.

The knowledge of groundwater residence times in (vulnerable) aquifers is essential for the sustainable management of the associated groundwater resources. A powerful tool for related investigations is the application of naturally occurring radioisotopes as water age indicators. However, due to the limited number of suitable (i.e. omnipresent, short-lived and easily detectable) radionuclides only few studies focus on groundwater ages below one year. A natural radionuclide that does have the potential to cover this time range is 35S (87.4 day half-life). 35S is continually produced in the upper atmosphere and transferred with the rain to the groundwater. Since no natural sources of 35S exist in the subsurface the decrease of the 35S activity concentration in such young groundwater can be used for the determination of its age. Still, 35S activities in precipitation (and hence even more in groundwater) are very low and necessitate appropriate analytical protocols based on liquid scintillation counting (LSC). This turns out to be challenging due to the required large sample volumes and due to potentially high SO42- loads of the samples, both limiting the range of possible applications of 35S as indicator for short groundwater residence times. In the paper we present an improved straightforward LSC based approach for the detection of 35S in natural water samples. We recommend using Insta-Gel Plus as scintillation cocktail for allowing a homogeneous suspension of 35S-containing BaSO4 in the cocktail. The recommended improvements in instrument setting concern the LSC (TriCarb 3170 Tr/SL) counting window, the pulse decay discriminator setting and the delay before burst setting. The settings allow measuring low activity concentrations of 35S, which was previously pre-concentrated from natural water samples, containing SO42- loads of up to 1500 mg with a reasonably high statistical reliability.

[Proteome analysis of undiluted vitreous humor in patients with branch retinal vein occlusion]. / Proteomanalyse unverdünnter Glaskörperflüssigkeit bei Patienten mit einem Venenastverschluss.

BACKGROUND: The pathophysiological mechanisms of macular edema secondary to branch retinal vein occlusion (BRVO) remain unclear. OBJECTIVES: To analyze the protein profile of human vitreous of patients with BRVO and to identify specific dysregulated proteins. MATERIALS AND METHODS: Undiluted vitreous humor samples from patients with treatment naïve BRVO and 15 controls with idiopathic floaters were analyzed in this clinical-experimental study using capillary electrophoresis coupled to a mass spectrometer (CE-MS) and tandem mass spectrometry (MS/MS). Quantitative analysis of the dysregulated proteins was performed with enzyme-linked immunosorbent assay (ELISA). Protein-protein interactions were depicted with the STRING database. RESULTS: A total of 84 proteins were found in the human vitreous samples of 15 patients with BRVO and 15 controls. In all, 14 proteins were significant when comparing the signal intensities of BRVO and control samples. Six significant dysregulated proteins with p < 0.001 were further verified with ELISA. Clusterin, complement factor C3, prostaglandin-H2 D­isomerase and vitronectin were significantly upregulated in the BRVO group and opticin was downregulated. The protein interactions analysis showed associations with inflammatory cascades, matrix changes, mechanisms of cell survival und death. CONCLUSIONS: The results of the study reveal that the proteomic composition of vitreous humor differed significantly between the patients with BRVO and the controls. Whether the identified proteins may serve as potential biomarkers for pathophysiology, diagnostics or therapy should be examine in further studies.

Combining Click Chemistry-Based Proteomics With Dox-Inducible Gene Expression.

Inactivating mutations in single genes can trigger, prevent, promote, or alleviate diseases. Identifying such disease-related genes is a main pillar of medical research. Since proteins play a crucial role in mediating these effects, their impact on the diseased cells' proteome including posttranslational modifications has to be elucidated for a detailed understanding of the role of these genes in the disease process. In complex disorders, like cancer, several genes contribute to the disease process, thereby hampering the assignment of a proteomic change to the corresponding causative gene. To enable comprehensive screening for the impact of inactivation of a gene, e.g., loss of a tumor suppressor in cancer, on the cellular proteome, we present a strategy based on combination of three technologies that is recombinase-mediated cassette exchange, click chemistry, and mass spectrometry. The methodology is exemplified by the analysis of the proteomic changes induced by the loss of a tumor suppressor gene in colorectal cancer cells. To demonstrate the applicability to screen for posttranslational modification changes, we also describe the analysis of protein glycosylation changes caused by the tumor suppressor inactivation. In principle, this strategy can be applied to analyze the effects of any gene of interest on protein expression as well as posttranslational modification by glycosylation. Moreover adaptation of the strategy to an appropriate cell culture model has the potential for application on a broad range of diseases where the disease-promoting mutations have been identified.

Sweet complementarity: the functional pairing of glycans with lectins.

Carbohydrates establish the third alphabet of life. As part of cellular glycoconjugates, the glycans generate a multitude of signals in a minimum of space. The presence of distinct glycotopes and the glycome diversity are mapped by sugar receptors (antibodies and lectins). Endogenous (tissue) lectins can read the sugar-encoded information and translate it into functional aspects of cell sociology. Illustrated by instructive examples, each glycan has its own ligand properties. Lectins with different folds can converge to target the same epitope, while intrafamily diversification enables functional cooperation and antagonism. The emerging evidence for the concept of a network calls for a detailed fingerprinting. Due to the high degree of plasticity and dynamics of the display of genes for lectins the validity of extrapolations between different organisms of the phylogenetic tree yet is inevitably limited.

[Role of energy metabolism in retinal pigment epithelium]. / Rolle des Energiestoffwechsels im retinalen Pigmentepithel.

The universal energy source adenosine triphosphate (ATP)is reduced by approximately 30 % in the retinal pigment epithelium (RPE) of elderly persons. Increased oxidative stress and decreased antioxidative capacity, such as glutathione in aging eyes cause impairment of energy-dependent RPE processes and lead to loss of visual function. We developed a cell culture model of aging RPE using atractyloside to inhibit mitochondrial ATP synthesis and tert-butyl hydroperoxide as oxidant. The ATP levels were reduced by 30 % and oxidative damaged proteins and DNA increased whereas antioxidative glutathione decreased. Autophagy as an internal cellular repair mechanism and phagocytosis of photoreceptors were impaired. Antioxidative and mitochondria-activating Ginkgo biloba extract EGb 761 increased the intracellular ATP level and antioxidative glutathione. This cell culture model seems to be suitable to investigate in vitro the effect of protective substances and their compounds on aging processes in RPE.

Notch1 signaling promotes survival of glioblastoma cells via EGFR-mediated induction of anti-apoptotic Mcl-1.

The Notch1-mediated signaling pathway has a central role in the maintenance of neural stem cells and contributes to growth and progression of glioblastomas, the most frequent malignant brain tumors in adults. Here, we demonstrate that the Notch1 receptor promotes survival of glioblastoma cells by regulation of the anti-apoptotic Mcl-1 protein. Notch1-dependent regulation of Mcl-1 occurs cell type dependent at a transcriptional or post-translational level and is mediated by the induction of epidermal growth factor receptor (EGFR). Inhibition of the Notch1 pathway overcomes apoptosis resistance and sensitizes glioblastoma cells to apoptosis induced by ionizing radiation, the death ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) or the Bcl-2/Bcl-XL inhibitor ABT-737. In conclusion, targeting Notch1 might represent a promising novel strategy in the treatment of glioblastomas.

[Juvenile neuronal ceroid lipofuscinosis. Ophthalmologic findings and differential diagnosis]. / Juvenile neuronale Zeroidlipofuszinose. Ophthalmologische Diagnostik und Differenzialdiagnose.

Neuronal ceroid lipofuscinoses (NCL) are a heterogeneous group of neurodegenerative diseases with mostly autosomal recessive inheritance whose common feature is the intralysosomal accumulation of ceroid lipofuscin. With varying manifestation ages the diseases result in cognitive and motor deterioration, epilepsy, diffuse retinal degeneration, and eventually death. Juvenile ceroid lipofuscinosis (JNCL, CLN3, Batten disease) has the distinctive feature that the ophthalmologic symptoms precede the neurologic symptoms by several years, and thus the ophthalmologist plays a central role in early diagnosis. Important clinical signs of JNCL include bull's eye maculopathy, severely reduced Ganzfeld ERG already at initial presentation, and unusually rapid progression of the functional decline. If JNCL is clinically suspected the diagnosis can be made by means of a standard blood smear and confirmed by genetic detection of the mutation. Although causal therapeutic options are currently only in the developmental stage, early diagnosis by the ophthalmologist is of utmost importance to allow for medical and educational support of the affected child and for adequate counseling of the parents.

[Pathomechanisms for aging of retinal pigment epithelium (RPE) and prophylactic therapy options in regard to AMD]. / Pathomechanismen der Alterung des RPE und prophylaktische Therapieoptionen im Hinblick auf die AMD.

An intact retinal pigment epithelium (RPE) represents an essential condition for the visual process. This post-mitotic RPE monolayer combines different functions such as degradation of photoreceptor outer segments, vitamin A cycle, support of retinal metabolism and maintenance of the outer blood-retina barrier. As a consequence of excessive metabolism, high oxygen levels, exposition to light of short wave length and ensuing radical formation, the RPE is highly dependent on protective systems. In spite of differentiated defence mechanisms, aging processes cause cumulative RPE damage, representing a major component of age-related macular degeneration (AMD), the leading cause of irreversible severe vision loss in people over 50 years old. A better understanding of the underlying pathophysiology will help to develop new prophylactic options which is becoming more and more important with increasing life expectancy.

EEG changes and serum anticholinergic activity measured in patients with delirium in the intensive care unit.

The aim of this study was to examine whether serum anticholinergic activity (SAA) is a reliable indicator of delirium in the ICU, and whether there is a significant correlation between SAA and quantitative electroencephalographic (EEG) data in delirious patients. In a prospective cohort study, we assessed ICU patients diagnosed with delirium (n = 37). EEG measurements and blood analysis including SAA were performed 48 h following ICU admission. The presence of delirium was evaluated using the Confusion Assessment Method for critically ill patients in ICU (CAM-ICU). The SAA level was measured using a competitive radioreceptor binding assay for muscarinergic receptors and quantitative EEG was measured using the CATEEM system. We found that, under comparable conditions, patients in the delirium group showed a higher relative EEG theta power and a reduced alpha power (n = 17) than did the non-delirious patients (n = 20). No difference in measured SAA levels were seen; therefore, there was no correlation between SAA and EEG measurements in delirious patients. We conclude that, in contrast to the EEG, the SAA level cannot be proposed as a tool for diagnosing delirium in ICU patients.

Coeliac disease is associated with impaired expression of acyl-CoA-synthetase 5.

BACKGROUND AND AIMS: Coeliac disease and other disorders of the small intestine are associated with disturbances in mucosal architecture. The most severe injury to tissue architecture is villus atrophy. In coeliac disease, molecules reflecting the state of the villus architecture are not well characterized at present. MATERIALS AND METHODS: Expression of acyl-CoA-synthetase 5 (ACS5) was studied in unaffected human small/large intestinal tissue and in coeliac disease using several methods including molecular techniques, as well as an in situ approach using a novel established monoclonal antibody directed against human ACS5. RESULTS: Strong expression, synthesis, and enzymatic activity of ACS5 were found in normal small intestinal mucosa compared with unaffected colon mucosa. In normal small intestine, ACS5 preferentially located to the epithelium covering villi. In coeliac disease, expression of ACS5 was regularly associated with differentiation of villi. Thus, ACS5 was found in the villus epithelium of the small intestine with coeliac disease of Marsh grades I, II, IIIa, or IIIb respectively. In Marsh grade IIIc coeliac disease lesions, strong expression of ACS5 was detectable neither in the surface epithelium nor in the epithelium lining hyperplastic crypts. CONCLUSION: These data suggest that ACS5 is a very suitable marker molecule for the detection of villus atrophy in the small intestine.

Lipids and lipid peroxidation products in the pathogenesis of age-related macular degeneration.

In people over 50, age-related macular degeneration (ARMD) has become the most common cause for severe visual loss and legal blindness in all industrialized nations. Currently, there is no effective treatment for the majority of patients. To develop new and effective modes of therapy, understanding of the molecular basis of the disease in mandatory. However, the pathogenesis of ARMD is still poorly understood. Several lines of evidence suggest that aging changes of the retinal pigment epithelium (RPE), in particular the accumulation of autofluorescent lipofuscin granules in the lysosomal compartment of postmitotic RPE cells, play a key role in the pathogenesis of the disease. Recent studies indicate that lipidic compounds of lipofuscin, represented by the retinoid A2-E, and protein damage by lipid peroxidation products, in particular malondialdehyde and 4-hydroxynonenal, induce lysosomal dysfunction and lipofuscinogenesis in the RPE. The possible mechanisms underlying this lysosomal dysfunction and the resulting adverse effects on overall RPE function are discussed.

Plasma amisulpride levels in schizophrenia or schizoaffective disorder.

The atypical antipsychotic drug amisulpride is a benzamide with specific antagonistic properties, which target dopamine D(2) and D(3) receptors, preferentially in the limbic system. Amisulpride is readily absorbed from the gastrointestinal tract, distributed to all body systems with little binding to plasma proteins. Elimination occurs mainly through the kidneys as unchanged drug. In contrast, hepatic metabolism is of minor significance and primarily yields two inactive metabolites. Very little is known about the plasma concentrations of amisulpride in patients at varying oral doses or about clinically relevant interactions with co-medication. The aim of the present investigation was to elucidate the factors, which affect amisulpride levels in schizophrenic patients. The plasma amisulpride levels of 85 patients with schizophrenia or schizoaffective disorder (mean age: 34.0+/-11.4 years; 40 women, 45 men) were assessed by high-performance liquid chromatography (HPLC) with fluorometric detection. The average daily dose of amisulpride was 772.3 mg (S.D. 346.7 mg) and the mean amisulpride plasma concentration was 424.4 ng/ml (S.D. 292.8 ng/ml). The interindividual variance of the amisulpride plasma concentration was high; furthermore, the plasma concentration increased linearly with the daily oral dose (r=0.50, p<0.001). Age and gender showed a significant effect on the dose-corrected amisulpride plasma concentrations-older patients and women had higher dose-corrected amisulpride plasma concentrations than younger patients and men. However, cigarette consumption had no effect on the amisulpride plasma concentrations. Regarding co-medication with lithium and/or clozapine, significantly higher amisulpride plasma concentrations were found as compared to monotherapy, whereas other co-medications such as benzodiazepines and various conventional antipsychotics had no effect on the amisulpride plasma concentrations. The results, the possible pathomechanisms and the clinical relevance are discussed. The findings need to be confirmed in larger patient samples and with a wider range of co-medications.

Olanzapine plasma concentration, average daily dose, and interaction with co-medication in schizophrenic patients.

BACKGROUND: Olanzapine, a thienobenzodiazepine, is one of the relatively new atypical antipsychotic drugs. The lowest threshold of effective olanzapine plasma levels in inpatient treatment is assumed to be 9 ng/ml. Very little is known about the plasma concentration in patients at various oral doses of olanzapine or about the clinically relevant interactions with co-medications. METHODS: In 71 schizophrenic patients (age 32.6 +/- 12.1, range 18-63 years; 31 women, 40 men), plasma olanzapine levels were assessed in 377 tests by high-performance liquid chromatography (HPLC) with electrochemical detection. Fifty-six of these plasma levels were assessed while patients were receiving olanzapine as monotherapy; otherwise, the plasma levels were assessed with the patients receiving various co-medications. RESULTS: The mean daily oral dose of olanzapine was 17.5 mg (SD = 7.0, range 5-40 mg), and the mean olanzapine plasma concentration was 54.2 ng/ml (SD 37.8 ng/ml, range 1.2-208 ng/ml). The plasma concentration of olanzapine increased linearly with the daily oral dose (r = 0.64, p < 0.001). A multiple variance analysis considering age and sex as covariables showed a significant difference in the dose-corrected plasma levels of olanzapine among 40 smokers and 31 non-smokers; age and sex did not affect the dose-corrected plasma levels. However, women received a significantly lower daily dose of olanzapine under routine clinical study conditions. No differences could be detected among the dose-corrected plasma concentration of those patients who were taken off olanzapine because they did not respond (n = 14) or because of side effects (n = 5) and those who were discharged while still on olanzapine. Under the co-medication with fluvoxamine, significantly higher dose-corrected olanzapine plasma concentrations were found than with olanzapine monotherapy, whereas significantly lower dose-corrected olanzapine plasma concentrations were detected under lithium and trimipramine co-medication. Under co-medication with amitriptyline, benperidol, carbamazepine, flupentixol, and lorazepam, the dose-corrected olanzapine plasma concentrations were no different than the plasma levels under olanzapine monotherapy. CONCLUSIONS: The relevance of therapeutic drug monitoring is emphasized with respect to the data presented and to the literature. Future studies should examine, in particular, the effects of a wider range of co-medications in a larger patient sample.

Inhibition of the ATP-driven proton pump in RPE lysosomes by the major lipofuscin fluorophore A2-E may contribute to the pathogenesis of age-related macular degeneration.

Lipofuscin accumulation in the retinal pigment epithelium (RPE) is associated with various blinding retinal diseases, including age-related macular degeneration (AMD). The major lipofuscin fluorophor A2-E is thought to play an important pathogenetic role. In previous studies A2-E was shown to severely impair lysosomal function of RPE cells. However, the underlying molecular mechanism remained obscure. Using purified lysosomes from RPE cells we now demonstrate that A2-E is a potent inhibitor of the ATP-driven proton pump located in the lysosomal membrane. Such inhibition of proton transport to the lysosomal lumen results in an increase of the lysosomal pH with subsequent inhibition of lysosomal hydrolases. An essential task of the lysosomal apparatus of postmitotic RPE for normal photoreceptor function is phagocytosis and degradation of membranous discs shed from photoreceptor outer segments (POS) and of biomolecules from autophagy. When the lysosomes of cultured RPE cells were experimentally loaded with A2-E, we observed intracellular accumulation of exogenously added POS with subsequent congestion of the phagocytic process. Moreover, the autophagic sequestration of cytoplasmic material was also markedly reduced after A2-E loading. These data support the hypothesis that A2-E-induced lysosomal dysfunction contributes to the pathogenesis of AMD and other retinal diseases associated with excessive lipofuscin accumulation.

Towards quality assurance in the determination of lysosomal enzymes: a two-centre study.

The definitive diagnosis of lysosomal storage disorders depends on the determination of enzymatic activities in cells, tissues or body fluids. At present, neither an evaluation of the different methods nor an interlaboratory quality assurance scheme is available. We have therefore determined the activities of total hexosaminidase, hexosaminidase A and beta-galactosidase in the same samples (n = 15) at two metabolic centres in Germany. Three different enzymatic methods were employed, two of which were based on leukocytes as enzyme source and one on dried blood spots. The results obtained by the two different methods using leukocytes proved comparable. In contrast, assays with dried blood spots showed poor correlation with results from leukocytes, possibly because enzymatic activity in dried blood is mainly derived from soluble plasma proteins. Nevertheless, accurate detection of a true enzyme deficiency was also possible in dried blood spots. All enzymes were highly stable when mailed frozen (recovery 98-120%). Enzymatic activities in dried blood samples were also stable at room temperature and were not affected even by exposure to elevated temperatures (50 degrees C for 3 h). Dried blood seems to be especially well suited for mailing from distant healthcare facilities, although more accurate results can be expected from leukocytes. In summary, comparability and pitfalls within a lysosomal quality assurance programme were evaluated.

Omeprazole reduces clozapine plasma concentrations. A case report.

A number of interactions of the atypical antipsychotic clozapine with other drugs are well known, some of which can be attributed in part to the pharmacokinetic interactions associated with cytochrome P450 enzymes during drug metabolism. Clozapine is mainly metabolized by the cytochrome P450 isoenzyme 1A2. The proton pump inhibitor omeprazole can induce CYP1A2. We report on two patients with schizoaffective disorder who received omeprazole in addition to clozapine because of gastrointestinal complaints. Before the co-medication with omeprazole was started, the patients had been receiving clozapine for 78 and 41 days and for 40 and 8 days at a stable daily dose of 325 mg (patients 1 and 2, respectively). The co-medication with omeprazole was associated with a reduction in the plasma levels of clozapine of 41.9 % and 44.7 %, respectively, in these patients. The decrease in the plasma concentrations of clozapine in the presence of omeprazole might be due to the induction of the cytochrome P450 isoenzyme CYP1A2. If patients are receiving omeprazole as co-medication, close monitoring of plasma clozapine levels is recommended. If clozapine levels drop, the drug should be adjusted accordingly. If necessary, an alternative to omeprazole should be chosen.

[Detergent-like effects of the lipofuscin retinoid component A2-E in retinal pigment epithelial cells]. / Detergenzienwirkung des Lipofuszin-Retinoidbestandteils A2-E in retinalen Pigmentepithelzellen.

PURPOSE: Several lines of evidence suggest that excessive accumulation of lipofuscin in postmitotic retinal pigment epithelial (RPE) cells with age and in various hereditary retinal diseases, plays a pathogenetic role. The lipofuscin retinoid component A2-E (N-retinylidene-N-retinylethanolamine) inhibits lysosomal degradation. Here we sought to evaluate additional toxic mechanisms of A2-E, whereby possible detergent-like effects on various membranes in human RPE cells were investigated by latency measurements. METHODS: A postnuclear supernatant prepared from cultured human RPE cells was used to isolate intact lysosomes by fractionation of cellular organelles in two sequential gradients. Destabilization of the lysosomal membrane was tested by incubating the purified lysosomal fraction in the presence of A2-E and subsequent measurement of the latency of the lysosomal luminal marker beta-hexosaminidase. In order to compare the effect of A2-E on other cellular membranes, latencies of the specific markers succinate dehydrogenase and UDP-galactosyltransferase were assessed using partially purified mitochondria and microsomes. Intactness of the plasma membrane was tested by including A2-E in the culture medium before leakage of lactate dehydrogenase into the medium was determined. RESULTS: A more than 100-fold purification of the lysosomal fraction was achieved. Except for a minor activity of the mitochondrial marker, no contamination with other cell fractions was observed. Intactness of the purified lysosomes was well preserved during incubation in isotonic media and provided the basis for investigations on a possible detergent-like action of A2-E on lysosomal integrity. At concentrations above 2 microM A2-E, progressive leakage of the lysosomal marker was observed. In comparison leakage of the mitochondrial marker was induced at significantly lower concentrations (1 microM), whereas ER/Golgi membranes and the plasma membrane were relatively insensitive to a detergent effect of the retinoid. CONCLUSIONS: The described practical and fast methodology to obtain highly purified and intact lysosomes from RPE cells, provides a very suitable tool for investigations on compounds affecting the lysosomal structure. The results suggest that A2-E causes disintegration of the lysosomal membrane at relatively low concentrations which may implicate an involvement of such a mechanism in triggering lipofuscin-induced dysfunction of aged RPE in vivo. Secondary to disintegration of the lysosomal membrane, damage to mitochondria might be an additional pathogenic mechanism. Our data provide evidence for surfactant-like properties of A2-E on biomembranes which might be operative in retinal diseases associated with excessive lipofuscin accumulation including age-related macular degeneration.