8-Hydroxy-2'-deoxyguanosine as a marker of oxidative DNA damage related to occupational and environmental exposures.

Oxidative DNA damage is considered to play an important role in pathophysiological processes, ageing and cancer. So far major interest has been on measuring 8-hydroxy-2'-deoxyguanosine (8-OHdG), the preferred methods relying on HPLC or GC-mass spectrometry. The high biological relevance of 8-OHdG is due to its ability to induce G-->T transversions, which are among the most frequent somatic mutations found in human cancers. Effects of workplace exposures on the level of white blood cell 8-OHdG or urinary 8-OHdG have been reported with controversial results. Exposures examined include asbestos, azo-dyes, benzene, fine particulate matter (PM(2.5)), glassworks, polycyclic aromatic hydrocarbons (PAHs), rubber manufacturing, silica, metals, styrene, toluene and xylenes. The available data indicate that there is still a lack of well established dose-response relations between occupational or environmental exposures and the induction of 8-OHdG. Smoking has been most consistently identified as a confounder for 8-OHdG, but various occupational studies did not reveal higher levels of 8-OHdG in smokers. Despite the conflicting results, the reported studies show promise for 8-OHdG as a biomarker of oxidative stress associated with chemical exposure. However, there are critical aspects related to the analytical challenge, artifactual production of 8-OHdG, inter- and intra-individual variation, confounding factors and inter-laboratory differences, implying that further work is needed to reach a consensus on the background level of 8-OHdG.

Prolonged asystole provoked by head-up tilt testing.

We describe a patient with a history of neurocardiogenic syncopes who had a positive headup tilt test that resulted in an lasting asystole lasting 34 seconds. However, the previously carried out Schellong test with a 30-min phase of standing showed a normal result. The patient showed typical orthostatic symptoms while tilted at the angle of 75 degrees. Shortly before asystole occurred, heart rate variability showed high frequency bands, indicating vagal stimulation. The pathophysiology of neurocardiogenic syncope (NCS) in context with heart rate variability is discussed. This patient was successfully treated with propranolol. This case shows the utility of a provocative head-up tilt test in establishing the diagnosis of NCS. If the Schellong test is normal, still further examination by tilt-table test is indispensable.

Lead exposure levels and duration of exposure absence predict neurobehavioral performance.

OBJECTIVES: We investigated cognitive functions in currently lead exposed and formerly lead exposed men. The aim of the study was to verify the hypothesis that lead induced cognitive deficits are reversible. METHODS: Forty-seven lead exposed subjects with a mean blood level (PbB) of 30.8 microg/100 ml and 48 formerly exposed aged-matched subjects (PbB: 5.5 microg/100 ml) with the same socio-economic background were investigated. Both groups were matched on verbal intelligence. Cognitive functions were assessed by using the Modified Wisconsin Card Sorting Test, Block Design Test, Visual Recognition Test, Simple Reaction Time, Choice Reaction and Digit Symbol Substitution. Lead exposure was assessed by using current and cumulative measures. The sample was divided according to their exposure duration versus exposure absence into four groups for subsequent ANOVA models analyses. RESULTS: Currently exposed men performed significantly worse in the Wisconsin Test and Block Design Test. No differences were found in the Visual Recognition Test, Simple Reaction Time, Choice Reaction and Digit Symbol Substitution. Splitting the sample according to their exposure duration versus exposure absence resulted in better cognitive performance of subjects with shorter exposure duration and longer exposure absence in the Block Design Test and the Wisconsin Test. CONCLUSIONS: Our results provide further evidence that cognitive deficits induced by occupationally lead are most likely reversible.

Reproductive toxicology in occupational settings: an update.

OBJECTIVE: This article mainly attempts to review the recent human literature on the adverse effects of occupational factors on fertility, developmental effects and genetic changes in the germ line, which lead to genetic malformations or to genetic disease. The secondary study aim is to answer whether occupational exposures are quantitative momentously for 15% involuntarily childless couples, 10-20% spontaneous abortions and 3% birth defects. METHODS: A literature survey was conducted for publications on these subjects focusing on the latest publications. PubMed (Medline. 2005) was used for this literature search. RESULTS: Publication bias and a large amount of confounding factors, which have to be controlled, make the design of human fertility studies difficult. Epidemiologic studies using time to conception techniques have been useful in identifying substances and exposure scenarios with proven toxic effect on fertility. The collected studies suggest that the exposure to the following substances or occupational settings may affect fertility function: lead, organic mercury compounds, manganese, carbon disulfide, 2-bromopropane and dibromochloropropane, welding, professional driving and working with heat. Concerning developmental effects even for methyl mercury, which was in group A of the German MAK list, to date no reliable evidence of the damaging effect on the human fetus under actual work conditions has been obtained. It is also difficult to classify substances according to their mutagenic potential for the germ cell, since no direct evidence of an association between exposure against a physical or chemical pollutant and the occurrence of a hereditary disorder has been found yet. CONCLUSION: In conclusion there are only a few substances which may affect reproductive function in the workplace without a doubt. The decreasing fertility of women in Western countries can be explained by the increasing female reproduction age, rather than by occupational exposures. Also the rates for spontaneous abortions and birth defects cannot be explained by industrial exposures at the workplace.

Chromosomal damage in human diploid fibroblasts by intermittent exposure to extremely low-frequency electromagnetic fields.

Environmental exposure to extremely low-frequency electromagnetic fields (ELF-EMFs) has been implicated in the development of cancer in humans. An important basis for assessing a potential cancer risk due to ELF-EMF exposure is knowledge of biological effects on human cells at the chromosomal level. Therefore, we investigated in the present study the effect of intermittent ELF electromagnetic fields (50 Hz, sinusoidal, 5'field-on/10'field-off, 2-24 h, 1 mT) on the induction of micronuclei (MN) and chromosomal aberrations in cultured human fibroblasts. ELF-EMF radiation resulted in a time-dependent increase of micronuclei, which became significant after 10 h of intermittent exposure at a flux density of 1 mT. After approximately 15 h a constant level of micronuclei of about three times the basal level was reached. In addition, chromosomal aberrations were increased up to 10-fold above basal levels. Our data strongly indicate a clastogenic potential of intermittent low-frequency electromagnetic fields, which may lead to considerable chromosomal damage in dividing cells.

Functional adrenergic receptor polymorphisms and idiopathic orthostatic intolerance.

OBJECTIVES: Idiopathic orthostatic intolerance (IOI) is a common disorder that is characterized by chronic orthostatic symptoms and substantial increases in heart rate and plasma norepinephrine concentrations that are disproportionately high while standing. Several features of the syndrome, including the tachycardia, tremulousness, and exaggerated norepinephrine have been considered potentially due to hypoactive or hyperactive states of adrenergic receptors of the sympathetic nervous system. The aim of this study was therefore to ascertain whether genotypes at eight polymorphic loci within five relevant adrenergic receptor genes (alpha2A, alpha2B, alpha2C, beta1 and beta2) influence the risk for IOI. METHODS: We studied 80 young men in military service (20 patients with IOI and 60 age-matched controls). All participants underwent a tilt table test including monitoring of blood pressure, heart rate and plasma catecholamines, in the supine position and during 30 min of standing. Genotyping at the eight loci (alpha2ALys251, alpha2BDel301-303, alpha2CDel322-325, beta1Gly49, beta1Arg389, beta2Arg16, beta2Glu27, beta2Ile164) was performed in all participants. Chi-square tests of independence were used to test for associations between IOI and genotype. In addition, an association of the polymorphisms with haemodynamic variables (heart rate, supine and upright blood pressure) was ascertained using one-way variance analysis. RESULTS: For the beta1Gly49 polymorphism we found a decrease in the risk of IOI among persons who were homozygous (odds ratio, 0.88; 95% confidence interval, 0.81-0.97). In addition, we found an association between beta1Gly49 and decreased heart rate in the upright position, regardless of IOI diagnosis. There were no associations with the other studied polymorphisms and IOI. CONCLUSIONS: Our current results suggest that the beta1Gly49 polymorphism is protective for IOI. This is likely one of several common genetic loci that may represent modifiers of IOI phenotypes.

[Health problems due to night shift work and jetlag]. / Gesundheitliche Probleme bei Nacht- und Schichtarbeit sowie beim Jetlag.

Technical demands, economic considerations and allocation of services around the clock bring about, that night work constantly represents a portion of 12-15% in Germany. Work against the biologic clock increases the risks for accidents and may produce health risks as sleeping or gastro-intestinal disorders, depression, cardio-vascular diseases, overweight, and a disturbed sexual activity and fertility. Adaptation to an altered day/night rhythm during night shift work takes more than a week, and even then the time shift is rarely complete. In contrast the duration of time shift is much shorter during a jetlag and is mostly completed after 2-3 days. Therefore, much less health risk is to be expected from jetlag as compared to night shift work. About 15% of all healthy adults are insufficiently adaptable to night shift work. These individuals carry a particularly high health risk, if regularly participating in night shift work.

Diagnostic management of orthostatic intolerance in the workplace.

OBJECTIVE: Orthostatic intolerance (OI) is a syndrome that is characterised by headache, concentration difficulties, palpitation of the heart, dizziness associated with postural tachycardia and plasma norepinephrine concentrations that are disproportionately high when the sufferer is in the upright posture. In contrast to other forms of orthostatic dysregulation - orthostatic hypotension (OH) and postural orthostatic tachycardia syndrome (POTS) - OI, hitherto, could be diagnosed only by a tilt table examination, with high expenditure. In this paper we examine the reliability and validity of a questionnaire as a screening instrument for OI. METHODS: We studied 138 young men (mean age 21.6 years) who were undergoing military service. After a medical check and filling in the questionnaire, the participants underwent a tilt table test including monitoring of blood pressure, heart rate and plasma catecholamines, in the supine position and during 30 min of standing. The questionnaire consisted of ten items registering presence and frequency of typical OI symptoms. RESULTS: Probands (104) showed normal tilt table test results. OI was diagnosed in 14 probands, OH in 6 and POTS in 14. The OI participants scored significantly higher in the questionnaire than the healthy subjects did: the mean score of the OI group was 22.6, the healthy participants had a mean score of 3.9. Participants with POTS had a mean score of 13.5 and subjects with OH had a mean score of 17.0. Reliability analysis showed a Cronbach's alpha of 0.888. Validity analysis showed that 93.5% of the probands with any kind of orthostatic dysregulation can be detected. CONCLUSIONS: We were able to establish a short questionnaire as a reliable and valid screening instrument for OI. Usage of this questionnaire can simplify enormously the diagnostic management of patients with suspected OI.

Urinary excretion of 8-hydroxy-2'-deoxyguanosine measured by high-performance liquid chromatography with electrochemical detection.

There is good evidence that oxidative DNA damage permanently occurs in living cells. The oxidative DNA damage product 8-hydroxy-2'-deoxyguanosine (8-OHdG) is one of the predominant forms of radical-induced lesions to DNA, and has therefore been widely used as a biomarker for oxidative stress, either in cellular DNA or as DNA repair product in urine. In this paper we describe the use of a high-performance liquid chromatographic procedure with electrochemical detection for the measurement of urinary 8-OHdG. Our study has addressed the questions (i) of baseline urinary levels of 8-OHdG in spot urine and 24-h urine, (ii) of inter- and intra-individual variation of this biomarker, and (iii) of confounding factors for the excretion of 8-OHdG. No significant difference between the mean group levels of 8-OHdG/creatinine in spot urine (2.03+/-1.21 micromol/mol, n=148) and in 24-h urine (1.86+/-1.09 micromol/mol, n=67) was observed. However, when only 24-h urine was used for analysis, 8-OHdG was found to be statistically significantly higher in smokers. By multiple linear regression analysis, urinary creatinine was identified as the only predictor of 8-OHdG/24 h (r(p)=0.33, P=0.007). High intra-individual coefficients of variation of 8-OHdG/24 h were observed in two healthy subjects over a period of 10 consecutive days (37 and 57%, respectively), indicating that the intra-individual fluctuation of urinary 8-OHdG has so far been underestimated. Therefore, we suggest that single values of 8-OHdG should be considered with caution, in particular in small study groups and when spot urine is used.

Health consequences of an intravenous injection of metallic mercury.

BACKGROUND: Mercury poisoning presents a variety of clinical pictures depending on chemical structure, the route of exposure, amount absorbed and individual factors. Thus, an injection of metallic mercury can be considered relatively harmless in contrast to inhalation of mercury vapor. Injection of elemental mercury is rare, and a total of only 78 cases have been reported in the literature over the period 1923-2000. CASE REPORT: We report a suicide attempt by intravenous injection of approximately 8 g metallic mercury. By X-ray examination widespread multiple mercury shadows were visible in the whole lung and also in the subcutaneous region of the cubital fossa, the small pelvis and the right hypogastrium. Mercury excretion after treatment with 2,3-dimercaptopropane-1-sulfonate (DMPS) was significantly higher than in occupationally exposed workers. CLINICAL SYMPTOMS: The patient showed symptoms typical of acute mercury intoxication, including gastroenteritis, ulceromembranous colitis and stomatitis mercuralis. No biochemical abnormalities in hepatic or renal function occurred, despite the persistence of metallic densities in the body. The patient's lung function was normal. The patient transitionally developed erethismus and tremor mercuralis. After 1 month of DMPS treatment, the mercury levels in blood were still high and the tremor was persistent. Three years after the suicide attempt the surgical removal of residual mercury in the left fossa cubitalis was performed. The extirpation of residual mercury was successful in cutting the mercury levels to almost half. After the operation the patient showed no symptoms of chronic mercury intoxication. CONCLUSIONS: Since only 1 mg of mercury per day could be removed with DMPS treatment, it can be calculated, that it would take about 8,000 daily treatments to remove a total of 8 g solely by DMPS. Although DMPS itself does not dissolve the metallic deposits, it may considerably reduce the blood level of mercury and may therefore mitigate clinical symptoms, albeit transitorily. We therefore recommend that in cases of symptomatic metallic mercury injections, where the mercury cannot be removed by surgery, the patient's condition should be managed by repeated long-term DMPS treatment in order to control blood mercury levels.

Neurobehavioral effects of vanadium.

It was our aim to study whether chronic exposure to vanadium reduces cognitive abilities. We investigated effects on attention, visuospatial and visuomotor functioning, reaction time, short-term memory, and prefrontal functioning. Forty-nine vanadium exposed subjects with a mean vanadium level in urine (VanU) of 14.4 micro/L and 49 controls (VanU: 0.8 microg/L) with the same socioeconomic background were investigated. Neuropsychological tests were done using a modified Wisconsin Card Sorting Test (WCST), Block Design Test (BDT), Visual Recognition Test (VRT), Simple Reaction Time (SRT), Choice Reaction (CR), Digit Symbol Substitution (DSS), and Digit Span (DS). Exposure was assessed by using the vanadium level in urine and serum. While there were significant differences in BDT and DSS, no differences were found in WCST, SRT, CR, and DS. Significant correlations existed between the vanadium levels in urine and serum and the cognitive deficits. Vanadium concentrations around 14.2 microg/L in urine reduce neurobehavioral abilities, particularly visuospatial abilities and attention.

Longitudinal study of urinary 8-hydroxy-2'-deoxyguanosine excretion in healthy adults.

Numerous studies have investigated the urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a biomarker for the assessment of oxidative DNA damage in humans. In this study, we performed six consecutive series of measurement of urinary levels of 8-OHdG in 68 healthy probands, in order to provide information on the intra- and inter-individual variability of 8-OHdG and to estimate the influence of smoking, age, sex, body weight and body mass index (BMI) on the excretion of 8-OHdG. The intra-individual coefficient of variation (CV) of urinary 8-OHdG/24 h ranged from 0.18 to 1.06 (mean CV = 0.48). Women excreted significantly lower amounts of 8-OHdG/24 h than men, but the difference lost its significance when the body weight or urinary creatinine were used as covariates. By multiple linear regression analysis significant correlations between the mean individual levels of 8-OHdG/24 h excretion and urinary creatinine (rp = 0.61), and cotinine (rp = 0.27) have been observed, whereas no statistically significant effect of age, body weight and BMI was found. The 8-OHdG/creatinine ratio was found to be significantly increased in 23 smokers (1.95 +/- 0.40 mumol/mol) opposed to 45 non-smoking probands (1.62 +/- 0.50 mumol/mol), which is in good agreement with previously published data. No effect of passive smoking on the excretion of 8-OHdG was found. From our data we conclude that the intra-individual variability of urinary 8-OHdG excretion has been underestimated so far, indicating that values of 8-OHdG measured by single spot monitoring are not representative for individual base levels.

[Chronic anemia and abdominal pain as a sequela of lead poisoning]. / Chronische Anämie und abdominelle Schmerzen als Folge einer Bleiintoxikation.

HISTORY AND ADMISSION FINDINGS: 3 years ago, a 63-year-old man underwent laparotomia due to clinical signs of acute abdominal pain. The anemia was treated with erythrocyte concentrates. Several weeks before the current admission to our outpatient department, the patient was again hospitalized because of acute abdominal pain and anemia. The patient was then transferred to the University clinic for further medical tests. INVESTIGATIONS: In addition to a normocytic anemia, a secondary porphyria was found. Further medical tests demonstrated lead poisoning. DIAGNOSIS, TREATMENT AND COURSE: Initially, the cause of the lead poisoning remained unclear because the patient had reported neither an occuppational nor an environmental exposure to lead. Finally, a prescription-prepared ointment was evaluated, which the patient had applied to his feet and lips over the past 3 years. CONCLUSION: Lead poisoning can be found outside the realm of occupational exposure; therefore, the knowledge of lead poisoning is still important.

Long-term monitoring of sister chromatid exchanges and micronucleus frequencies in pharmacy personnel occupationally exposed to cytostatic drugs.

OBJECTIVES: Many antineoplastic drugs were found to have carcinogenic, mutagenic and teratogenic potential. The aim of this study was to carry out cytogenetic and internal dose monitoring of hospital pharmacy personnel regularly involved in the preparation of cytostatic agents, in order to test possible cytostatics-induced genotoxic effects due to occupational exposure under routine working conditions, and in cases of accidental contamination. METHODS: Platinum in whole blood and anthracyclines in plasma were measured to assess internal exposure to cytostatics. The level of cytogenetic damage was determined in peripheral blood lymphocytes with the micronucleus test and the sister chromatid exchange assay. Five series of monitoring were performed over a period of 2 years. RESULTS: No significant differences in the mean frequencies of sister chromatid exchanges (SCE) and micronuclei (MN) were found between occupationally exposed probands and controls (9.9 +/- 1.4 vs 10.1 +/- 1.2 SCEs/cell and 21.2 +/- 7.2 vs 23.3 +/- 7.5 MN/2000 binucleated (BN) cells, n = 16). Significant elevations of SCE or MN were detected in seven out of 12 cases of accidental contamination at the workplace, whereas no increase in platinum in blood and anthracyclines in plasma was observed in these probands. Two cases of non-reported contamination were identified by measurement of epirubicin in plasma. Smoking was found to increase the SCE significantly. No correlation between individual SCE scores and MN scores was observed. CONCLUSIONS: Our findings support a transient increase in SCE or MN after relevant exposure to cytostatic drugs in cases of accidental contamination. The lack of significant differences in SCE and MN between hospital pharmacy personnel and unexposed controls, points to high standards of safety at the corresponding workplaces.

8-Hydroxydeoxyguanosine in leukocyte DNA and urine of quartz-exposed workers and patients with silicosis.

OBJECTIVE: To examine radical-induced DNA damage and its elimination in workers exposed to quartz and in patients with silicosis, and to assess the relationship of these effects to lung function. METHODS: Blood and spontaneous urine samples were obtained from active, quartz-exposed workers without silicosis (n = 63), and from retired workers with silicosis (n = 42). Levels of 8-hydroxydeoxyguanosine (8-OHdG) were determined in peripheral blood leukocyte DNA and urine, by the use of high-performance liquid chromatography coupled with ultra violet- (UV) and electrochemical detection. RESULTS: No significant differences in the mean levels of 8-OHdG in leukocyte DNA and of urinary excretion of 8-OHdG were found between silicosis patients and quartz-exposed healthy workers. However, in the group of silicosis patients with increased oxidative DNA damage the urinary excretion of 8-OHdG was lower than in the corresponding group of active workers without silicosis. In the case of silicosis, urinary 8-OHdG correlated positively, and 8-OHdG in DNA correlated negatively, with forced expiratory volume in one second (FEV1) and forced vital capacity (FVC). Healthy workers with a personally estimated high dust exposure in the workplace showed higher levels of 8-OHdG in DNA than did workers with moderate dust exposure. No association of 8-OHdG formation and/or elimination with duration of employment, field of activity, smoking or age was found. CONCLUSION: Our findings suggest that a less effective repair of 8-OHdG is associated with a higher degree of pulmonary airway obstruction in patients with silicosis.

Radiation synovectomy using 165Dy ferric-hydroxide and oxidative DNA damage in patients with different types of arthritis.

UNLABELLED: Radiation synovectomy is an effective treatment for chronic synovitis refractory to pharmacological treatment in patients with rheumatoid or seronegative arthritis. Concerns persist about possible radiation-induced cytogenetic damage after radiation synovectomy leading to recommendations to use this technique only in the elderly. Micronucleus (MN) frequency in lymphocytes and urinary excretion of 8-hydroxy-2'-deoxyguanosine (8OHdG) as an indicator of cellular oxidative DNA base damage are biomarkers of radiation-induced cytogenetic damage. The course of both biomarkers was studied in patients with different types of chronic synovitis undergoing radiation synovectomy with very short-lived 165Dy-ferric-hydroxide (DFH). METHODS: Radiation synovectomy of the knee was performed in 13 men and 12 women (mean age, 44+/-15 y) using a mean activity of 9.48+/-1.65 GBq 165Dy-DFH in 27 consecutive treatments. MN frequency in lymphocytes and urinary excretion of 8OHdG, measured by high-performance liquid chromatography, were assessed before and 4 (MN only) and 20 h after radiation synovectomy. RESULTS: Urinary excretion of 8OHdG in patients (in micromol/mol creatinine; pretreatment mean, 3.1+/-3.4; median, 2.27) was not significantly different from that in healthy volunteers (mean, 2.0+/-1.2; median, 1.87) and not altered by radiation synovectomy (post-treatment mean, 2.5+/-1.5; median, 2.04, NS). An increase in 8OHdG levels after radiation synovectomy of more than 1 SD was found in only 1 patient, who experienced leakage to the lymph nodes but who already had elevated urinary 8OHdG levels before treatment. The frequency of MN/500 binucleated cells (BNCs) was slightly lower in patients (pretreatment mean, 4.3+/-2.6; median, 4.25) than in healthy volunteers (mean, 5.4+/-2.3; median, 5.3) and did not significantly change after therapy, either (4-h post-treatment mean, 3.9+/-2.1, median, 3.8; 20-h post-treatment mean, 4.1+/-2, median 3.8 MN/500 BNC). In 22 of 27 treatments, no leakage to nontarget organs could be monitored, whereas leakage to the local lymph nodes and the liver was detected after 5 treatments. CONCLUSION: Radiation synovectomy using 165Dy-DFH causes no significant radiation burden to most patients as indicated by the absence of adverse changes in levels of biomarkers of cytogenetic damage and a low incidence of leakage. These data suggest that the risk of malignancy may not be elevated.

Association of impaired phosphatidylinositol 3-kinase activity in GLUT1-containing vesicles with malinsertion of glucose transporters into the plasma membrane of fibroblasts from a patient with severe insulin resistance and clinical features of Werner syndrome.

The purpose of this study was to examine the molecular mechanism responsible for the defective insulin-stimulated glucose transport in cultured fibroblasts from a patient (VH) with clinical features of Werner syndrome and severe insulin resistance. Thus, in cells derived from VH, the subcellular distribution, structure, functional activity, as well as plasma membrane insertion of GLUT1 glucose transporters were analyzed. Furthermore, the insulin signal transduction pathway leading to activation of phosphatidylinositol (PI) 3-kinase as well as components of GLUT1-containing membrane vesicles were characterized. In fibroblasts derived from VH, GLUT1 glucose transporters were overexpressed by 8-fold in plasma membranes (PM) and by 5-fold in high density microsomes, respectively. Exofacial photolabeling revealed that only 14% of the overexpressed PM-GLUT1 transporters were properly inserted into the plasma membrane. The complementary DNA structure of the patient's insulin receptor and the GLUT1 glucose transporter, the intrinsic activity of plasma membrane glucose transporters, the tyrosine phosphorylation, as well as the protein expression of insulin receptor substrate-1/2 and p85 alpha/beta- and p110 alpha/beta-subunits of PI 3-kinase were normal. However, insulin-stimulated association of the p85 subunit of PI 3-kinase was defective in fibroblasts derived from VH compared to those from controls, and this defect was associated with a reduced IRS-1-dependent activation of PI 3-kinase by 50.2% and 63.6% after incubation for 5 and 10 min with 100 nmol/L insulin, respectively. Furthermore, immunodetection of small GTP-binding Rab proteins in subcellular membrane fractions indicated a decreased expression of Rab4 in total cellular homogenates as well as in high density microsomes by 70% and 58%, respectively. After preparation of GLUT1-containing vesicles, Rab4 was not detected to be a component of these vesicles. Analysis of the PI 3-kinase in GLUT1-containing membrane vesicles revealed insulin-dependent targeting of the p85 subunit to the vesicles immunoadsorbed from VH and control fibroblasts. Importantly, the association of the p85 subunit as well as the p85-immunoprecipitable PI 3-kinase activity were markedly reduced in GLUT1-vesicles derived from the patient. In conclusion, impaired PI 3-kinase activity in GLUT1-containing membrane vesicles derived from fibroblasts of VH is associated with a defective docking and/or fusion process of glucose transporters with the plasma membrane and thus might contribute to the molecular defect causing insulin resistance in this patient.