Effects of alcohol consumption on iron metabolism.

BACKGROUND/OBJECTIVES: Patients with alcohol abuse frequently suffer from malnutrition which may result in insufficient iron distribution and iron overload or deficiency. Iron metabolism can be described by a combination of biochemical soluble transferrin receptor, ferritin, C-reactive protein (CRP), and hematological parameters. Here, vitamin B12 and folic acid state were assessed. Results on iron metabolism in patients with alcohol dependence in comparison with social drinkers are presented. MATERIALS/METHODS: Samples from 101 patients with dependent alcohol consumption were included. The control group comprised 115 social drinkers. Inclusion criteria for patients with chronic regular drinking/social drinkers were positive/negative score of the Alcohol Use Disorders Identification Test (AUDIT), and positive/negative score for alcohol abuse/dependence (DSM-IV criteria). RESULTS: Absolute values for ferritin and sTfR are increased in patients with alcohol dependence with current consumption (ALC) compared with social drinkers. No major differences are observed in the ratio of sTfR/log ferritin in comparison with social drinkers. Hemoglobin concentrations correlated between the two groups. Mean corpuscular volume (MCV) was significantly increased in the ALC collective compared to social drinkers. Eighty patients of the alcohol-dependent group had sufficient iron repletion, 11 had iron overload, 6 are suspicious for functional iron deficiency, and 4 are suspicious for reduced iron supply. No vitamin B12/folate deficiencies are observed in alcohol-dependent patients. CONCLUSIONS AND SCIENTIFIC SIGNIFICANCE: No major abnormalities of iron metabolism are seen in patients with chronic alcohol ingestion besides the well-known macrocytic anemia. Iron overload is relatively frequent and observed in 9% of cases. No differences in vitamin B12 and folate levels were found between individuals with alcohol dependence and social drinkers.

Validity of carbohydrate-deficient transferrin (%CDT), gamma-glutamyltransferase (gamma-GT) and mean corpuscular erythrocyte volume (MCV) as biomarkers for chronic alcohol abuse: a study in patients with alcohol dependence and liver disorders of non-alcoholic and alcoholic origin.

AIM: To test the clinical performance of carbohydrate-deficient transferrin (%CDT), gamma-glutamyltransferase (gamma-GT) and mean corpuscular erythrocyte volume (MCV) as biomarkers for alcoholism with a special focus on patients suffering from liver diseases. DESIGN: Well-characterized collectives of alcohol-dependent patients with current consumption (ALC patients, n = 101), and relevant control groups (115 social drinkers, 46 patients with unspecifically increased gamma-GT, 51 hepatitis patients and 20/31 patients with non-alcohol/alcohol-dependent liver cirrhosis) were included into the study. The Positive Alcohol Use Disorders Test (AUDIT) score, International Classification of Diseases version 10 (ICD-10)/Diagnostic and Statistical Manual version IV (DSM-IV) criteria and blood drawn within 4 days of last drinking were inclusion criteria for subjects with regular heavy drinking. %CDT was determined using an automated assay which recently had been completely modified. FINDINGS: Median AUDIT scores of patients without/with regular heavy drinking were 1-3/27. The following medians/95th percentiles were obtained for %CDT: social drinkers 2.2/3.0, patients with unspecifically increased gamma-GT 2.1/3.0, hepatitis 2.0/4.4, non-alcohol-dependent liver cirrhosis 2.4/4.8, alcohol-dependent liver cirrhosis 3.0/5.9, ALC patients 3.9/14.9. Differences between patients without and with alcohol abuse were highly significant (P < 0.001). No differences in CDT values were found between males and females. There was no correlation between %CDT values, gamma-GT, MCV and the amount of alcohol consumed in ALC patients; 3.0%CDT (95th percentile social drinkers) is proposed as cut-off for the test used (Tina-quant %CDT 2nd-generation). At this cut-off, the sensitivity for ALC patients was 73.3%, whereas gamma-GT/MCV had a sensitivity of 71.3%/64.4%. Multivariate analysis performed at 95% specificity resulted in an improvement of the sensitivity by combining %CDT with gamma-GT (83.2%). A further enhancement of the sensitivity to 88.1% was obtained by combination of %CDT, gamma-GT and MCV. The diagnostic specificity of %CDT calculated at the cut-off of 3% was 93.5% in patients with unspecifically increased gamma-GT, 88.2% in hepatitis patients and 70.0% in patients with non-alcohol-dependent liver cirrhosis. %CDT was more specific in these patient collectives than MCV, and especially more than gamma-GT (specificity in hepatitis 52.9%, and 35.0% in non-alcohol-dependent liver cirrhosis). CONCLUSION: %CDT is of high diagnostic value to support diagnosis of alcohol-use disorders. The specificity of this marker in patient groups with liver disorders is superior to the biomarkers gamma-GT and MCV.

Therapeutic drug monitoring on COBAS INTEGRA 400--evaluation results.

The COBAS INTEGRA 400 (Roche Diagnostics GmbH) is a random access analyzer with a consolidated test menue for routine clinical chemistry, specific proteins, drugs of abuse screening and therapeutic drug monitoring (TDM) and different measuring technologies. It was the aim of the present study to evaluate the suitability of this instrument as dedicated analyzer for TDM. Eight assays based on three different technologies were included: Acetaminophen (enzymatic method), Amikacin/ Phenytoin/ Free Phenytoin/ Lidocaine (fluorescence polarization immunoassays; FPIA), Digitoxin/Digoxin (kinetic interaction of microparticles in solution; KIMS). The study comprised the determination of imprecision according to NCCLS EP-T protocol, method comparison and linearity studies. The assays were compared with the corresponding methods on AxSYM or TDx analyzers (Abbott Laboratories). For Acetaminophen and Amikacin COBAS INTEGRA 700 was used as additional comparison instrument. The results are summarized in a table (table 6). Precision results are well acceptable with within-run CVs < 5% and total CVs < 6% except for Digitoxin and Digoxin which show a somewhat higher imprecision at low concentrations. Results obtained for Acetaminophen and Amikacin on COBAS INTEGRA 400 and 700 show excellent agreement. A good comparability is also found between COBAS INTEGRA 400 and AxSYM or TDx methods with slight systematic deviations for Acetaminophen, Amikacin and Free Phenytoin. The lower correlation coefficient for the digoxin method comparison can be attributed to two discrepant samples. Linearity throughout the range studied which covered > 80% of the measuring range was confirmed for the five assays tested (Digitoxin, Digoxin, Lidocaine, Free Phenytoin, Phenytoin) based on the acceptance criteria of +/- 10% deviation of the measured values from the theoretical values. Based on the analytical performance of the TDM tests studied it can be concluded that the COBAS INTEGRA 400 is very well suited for routine TDM analysis.

Precision and comparability of Abuscreen OnLine assays for drugs of abuse screening in urine on Hitachi 917 with other immunochemical tests and with GC/MS.

Abuscreen OnLine assays for drugs of abuse screening in urine have recently been developed for use on Hitachi 917 analyzers (Roche Diagnostics GmbH). The assays are based on the kinetic interaction of microparticles as measured by changes in light transmission. Drug in a sample inhibits the formation of particle aggregates and diminishes absorbance change increases. It was the goal of this study to evaluate precision and comparability of the new asssys with CEDIA drugs of abuse tests on Hitachi 917 in different laboratories (three European and three US). The assays were calibrated in the nonlinear mode with four to six standards (semiquantitative application). Initial within-run (21 replicates, four labs) and between-day (10 days, two labs) imprecision studies using Abuscreen OnLine tests and commercial negative (0.5 x cut-off) and positive (1.5 x cut-off) controls revealed the following median CVs [withinrun neg./pos. control/between-day neg./pos. control]: amphetamines 1.9/1.3/3.4/2.4, barbiturates 3.0/1.6/3.9/3.1, benzodiazepines 4.7/1.5/6.3/3.0, cocaine metabolite 1.8/0.9/2.4/1.7, methadone 5.4/1.6/5.5/2.2, opiates 5.5/2.8/5.3/2.7, THC 8.9/4.8/21.8/12.1. CVs < 10% were obtained for the THC test using controls with concentrations closer to the cut-off. An identical set of 170 GC/MS analyzed urine samples was distributed to the six laboratories and measured with Abuscreen OnLine tests on Hitachi 917. The median values for each individual sample were calculated and compared with the results obtained on individual Hitachi 917 analyzers by Passing-Bablok regression analysis. A good agreement between the laboratories was found with less than +/- 11% slope deviation and intercepts below 7% of the cut-off except for benzodiazepines (one slope 17%, one slope--26%) and THC (one slope 34%, one slope--18%). The comparability with CEDIA tests was analyzed by concordance plots using randomized routine samples in three laboratories. The following results were obtained in one of the participating laboratories [cut-off ng/mL] (No. of positive/negative/discrepant samples): amphetamines [500] 2/147/0, barbiturates [200] 1/148/0, benzodiazepines [100] 52/91/7, cocaine metabolite [300] 17/129/3, methadone [300] 113/34/2, opiates [300] 31/114/4, THC [50] 66/81/2. GC/MS was performed for clarification of the discrepant results. In summary, Abuscreen OnLine tests on Hitachi 917 give precise results which compare well when analyzed in different laboratories. They can be rated as convenient and flexible methods for drugs of abuse screening in the routine.

Multicenter evaluation of the Elecsys CA 125 II assay.

The technical performance and clinical usefulness of the newly developed Elecsys CA 125 II assay (Boehringer Mannheim) was evaluated in a multicenter study. Imprecision studies were carried out using control sera and human pool sera with CA 125 concentrations from 11 to 1026 U/ml. Within-run CVs between 0.7 to 4.8% (median 1.7%) and between-day CVs between 2.4 to 10.9% (median 5.7%) were found. Method comparison studies with Enzymun-Test CA 125 II carried out in four laboratories yielded slopes between 0.94 to 1.07 and intercepts < 3 U/ml. A good comparability of the Elecsys CA 125 II assay was also found with one MEIA and the Centocor" IRMA. For a second MEIA and a second IRMA the slopes were 1.23 and 1.42, and the corresponding correlation coefficients were 0.987 and 0.977, respectively. The Elecys CA 125 II concentrations are clearly related to the tumor stage of ovarian carcinoma patients. The maximum of diagnostic efficiency of ovarian carcinoma patients compared with patients of benign gynecological diseases is reached at 150 U/ml with a specificity of 93% and a sensitivity of 69%. Follow-up studies of ovarian carcinoma patients reflect the status of the disease and the effect of various therapeutic applications. The technical and clinical evaluation of the Elecsys CA 125 II assay show a superior analytical performance with a broad measuring range up to 5000 U/ml and a short measuring time of 18 minutes.

Analytical and clinical evaluation of an electrochemiluminescence immunoassay for the determination of CA 125.

The CA 125 II assay on the Elecsys(R) 2010 analyzer was evaluated in an international multicenter trial. Imprecision studies yielded within-run CVs of 0.8-3.3% and between-day CVs of 2.4-10.9%; CVs for total imprecision in the manufacturer's laboratory were 2.4-7.8%. The linear range of the assay extended to at least 4500 kilounits/L (three decades). Interference from triglycerides (10.3 mmol/L), bilirubin (850 micromol/L), hemoglobin (1.1 mmol/L), anticoagulants (plasma), and several widely used drugs was undetectable. Method comparisons with five other CA 125 II assays showed good correlation but differences in standardization. A 95th percentile cutoff value of 35 kilounits/L was calculated from values measured in 593 apparently healthy (pre- and postmenopausal) women. In 95% of patients with benign gynecological diseases CA 125 was 190 kilounits/L. A comparison of CA 125 values obtained with the Elecsys test and with other common CA 125 tests in monitored patients being treated for ovarian cancer showed identical patterns. In conclusion, the Elecsys CA 125 II assay is linear over a broad range, yields precise and accurate results, is free from interferences, and compares well with other assays.

Comparability of a new turbidimetric digoxin test with other immunochemical tests and with HPLC--a multicenter evaluation.

A new turbidimetric inhibition immunoassay for digoxin (Tina-quant [a] Digoxin, Boehringer Mannheim) was evaluated in seven laboratories. It can be performed without sample pretreatment with ready-to-use reagents on nondedicated analyzers in combination with routine clinical chemistry. The studies revealed a good analytical performance: lower limit of detection 0.12 microg/L (3 SD from mean of blank); linearity up to 7.5 microg/L; median between-run CVs 8.1% (0.6 microg/L), 2.8% (1.5 microg/L), 1.9% (3 microg/L); mean analytical recovery in control sera 98-102%; slopes from 0.97 to 1.09 and intercepts from -0.28 to 0.10 microg/L in comparison with four immunoassays; and a high resistance to common interferents. The test was more resistant to digoxin-like immunoreactive factor (DLIF) interference than other methods, showing cross-reactivity only in some intensive care patient samples. Among 192 patients in whom DLIF is expected (e.g., pregnant women, patients with renal failure, newborns), 90% of results were < or =0.26 microg/L digoxin. Cortisol showed no cross-reactivity and digoxigenin had a low reactivity. An interlaboratory survey revealed a good comparability of the Tina-quant [a] test with the median of all methods (slope 0.99, intercept -0.06 microg/L). An HPLC method for digoxin based on isocratic separation of samples on an RP-18 column followed by detection by an immunoassay yielded a reasonable comparability with the immunochemical tests with noncritical samples. Divergent results of immunoassays caused by DLIFs or different cross-reactivities with digoxin metabolites or derivatives can be explained by the use of this HPLC method.

Multicentre evaluation of the Boehringer Mannheim/Hitachi 911 Analysis System.

The analytical performance and practicability of the Boehringer Mannheim (BM)/Hitachi 911 analysis system have been assessed in a multicentre evaluation, which involved six laboratories from European countries. Analytes commonly used in classical clinical chemistry were tested in a core programme, which mainly followed the ECCLS guidelines. In addition, a satellite programme covered other analytes, such as proteins, drugs and urine analytes. In total, the study comprised more than 100 000 data items collected over a three-month period. The evaluation was supported with 'Computer Aided Evaluation' (CAEv) and telecommunications.Acceptance criteria for the results were established at the beginning of the study. Nearly all of the analytes met the imprecision limits: within-run imprecision (as CVs) was 2% for enzyme and substrate assays, 1% for ISE methods and 5% for immunoassays; between-day imprecision was 3l% for enzyme and substrate assays, 2% for ISE methods and 10% for immunoassays.No relevant drift effects (systematic deviation >/= 3%) were observed over eight hours. The methods were linear over a wide range. Sample-related and reagent-dependent carry-over can be reduced to a negligible amount by integration of a softwarecontrolled wash-step.Endogenous interferences were found for creatinine (Jaffé method) and uric acid assays (caused by bilirubin), for creatine kinase, creatine kinase MB isoform and gamma-glutamyltransferase (caused by haemoglobin), and for immunoglobulin A (caused by lipaemia)Accuracy was checked by an interlaboratory survey, recovery studies in control materials and method comparison studies. The survey showed that, with the exception of cholesterol and iron in two laboratories, the recovery of analytes did not deviate by more than 5%. Sixty-six of the 77 method comparisons performed met the acceptance criteria. The deviations of the remaining 11 results could be explained by differences in either calibration, application or by the use of different methods.Practicability was assessed using a questionnaire which covered all of the important aspects of an analysis system in the clinical laboratory. Twelve groups of attributes out of 14 were rater higher for the BM/Hitachi 911 than for the present situation in the laboratories concerned. Especially high scores were given for the versatility group.The acceptance criteria for the analytical performance of the BM/Hitachi 911 analysis system were fulfilled in all laboratory segments with few exceptions. The practicability exceeded the requirements in most of the attributes. The results of the study confirmed the usefulness of the system as a consolidated workstation in small- to medium-sized clinical laboratories and in STAT laboratories, or as an instrument for special analytes like proteins and drugs, or for urinalysis in large laboratories.

Results of the multicenter evaluation of the CEDIA Phenobarbital assay.

The CEDIA Phenobarbital assay has been evaluated in twelve clinical laboratories in Europe and U.S.A. on Boehringer Mannheim/Hitachi analysis systems. The evaluation focused on the analysis of imprecision and accuracy. Within-run and between-day coefficients of variations of the new assay were comparable to those of established routine methods. As demonstrated in an interlaboratory survey study with controls and human sera, results obtained in different laboratories showed a good agreement. The CEDIA Phenobarbital assay measured very accurately, as particularly confirmed by comparison with HPLC. It can be recommended as a reliable and practicable test for monitoring of phenobarbital on Boehringer Mannheim/Hitachi analyzers used in routine clinical chemistry.

Endogenous digoxin-like immunoreactive factors (DLIF) as measured by the CEDIA digoxin assay and a fluorescence polarization immunoassay.

The sensitivity of a new homogeneous enzyme immunoassay for the determination of digoxin (CEDIA Digoxin assay) and a fluorescence polarization immunoassay (FPIA) to interference by digoxin-like immunoreactive factors (DLIF) was studied in sera from pregnant women, newborns, patients undergoing hemodialysis and patients with renal insufficiency, but without hemodialysis. None of the patients had been treated with digoxin or digitoxin. Cross-reactivity of DLIF in the CEDIA assay was generally lower than in the FPIA. Data on the distribution DLIF of values and method comparisons showed that sera of the four patient groups reacted in a completely different way in both assays, suggesting that the nature of DLIF in the four groups is not identical. Addition of digoxin to sera of patients not treated with this drug resulted in a reduction of the apparent DLIF concentration in the CEDIA assay and the FPIA. This shows that DLIF interference may be less pronounced in sera of patients undergoing digoxin therapy compared to untreated persons. Although the CEDIA assay is less sensitive to DLIF interference than the FPIA, further efforts are needed to reduce the extent of this interference.

Synthesis of herpes simplex virus proteins and nucleic acids in interferon-treated macrophages.

Mouse macrophages grown from spleen cells were found to be very sensitive to the interferon (IFN) activity against herpes simplex virus type 1 (HSV-1). Therefore we have used these cells to investigate the level at which IFN blocks the replication of HSV-1. IFN treatment resulted in a strong inhibition of the induction of HSV DNA polymerase and other beta proteins. RNA hybridization experiments revealed that the amount of mRNA for the beta protein thymidine kinase was strongly reduced in IFN treated HSV-1 infected cells. Analysis of the effect of IFN on expression of the alpha genes indicated a strong inhibition of alpha protein synthesis. In contrast the synthesis of mRNA of the alpha protein ICP 4 was only moderately inhibited. The results indicate that IFN primarily acts on the translation of HSV alpha proteins.

Inhibition of replication of herpes simplex virus in mouse macrophages by interferons.

The replication of herpes simplex virus (HSV) type 1 in macrophages grown from spleen cells of mouse strains susceptible to HSV infection in vivo was very sensitive to interferon (IFN). Different types of mouse IFN (alpha, beta, gamma) exhibited similar antiviral activities. However, treatment of cells with IFN-gamma in combination with IFN-alpha or IFN-beta resulted in a synergistic inhibition of virus growth. As shown by assaying HSV DNA polymerase, IFN inhibited expression of the beta-genes. Inhibition of enzyme induction correlated well with the reduction of viral yield. Induction of HSV DNA polymerase was delayed by IFN in a dose-dependent manner. These results show that IFN inhibits HSV replication at an early step prior to or during the synthesis of beta-proteins.

Role of interferon in the resistance of C3H/HeJ mice to infection with herpes simplex virus.

C3H/HeJ mice known to be defective in their responses to bacterial lipopolysaccharides, are more resistant to infection with herpes simplex virus (HSV) than the closely related strain C3HeB/FeJ. The increased resistance is reflected in higher early local interferon titers after HSV infection. However, NK cell activation by HSV is not correlated with resistance, since the NK cell response of C3H/HeJ mice was significantly lower than that of the control strain.

[The effect of dietary lipids on microsomal electron transport proteins in the rat]. / Wirkung von Nahrungslipiden auf mikrosomale Elektronentransportproteine der Ratte.

Feeding a fat-free diet for 3 weeks decreases the activity of ascorbate:ferricytochrome b5 oxidoreductase and increases NADH:monodehydroascorbate oxidoreductase in rat liver microsomes. Contrary to Vmax, the apparent Km for monodehydroascorbate of the first enzyme was not depressed. The activity of ascorbate:ferricytochrome b5 oxidoreductase is increased by incubation with microsomal lipid extract, thus showing that the loss of activity results from dietary-induced changes of microsomal fatty acid composition.

Experimental infection of inbred mice with herpes simplex virus. VI. Effect of interferon on in vitro virus replication in macrophages.

Peritoneal exudate cells (PEC) of DBA/2 mice, after 7 days of in vitro preculture and consisting of virtually 100 per cent macrophages, were able to support the replication of Herpes Simplex Virus type 1 strain WAL (HSV). Using a standard medium based on Dulbecco's Modified Eagle Medium (D-MEM), no virus replication was observed in freshly isolated PEC. However a medium based on RPMI 1640 consistently yielded higher virus titres in precultured PEC than the D-MEM medium, and also allowed virus replication in freshly isolated PEC. Macrophages derived from the spleens or the bone marrow, and precultured in the same way as PEC represented a highly pure population and were permissive for infection with HSV. Titres of about 10(6) PFU HSV were observed in PEC 48 hours after infection with 10(3) or 10(6) PFU. However, whereas a complete destruction of the cell monolayer was observed 24 hours after infection with 10(6) PFU, complete cytopathogenicity in PEC infected with 10(3) PFU required at least twice this time. In the latter situation, plaque formation was observed 24 hours after infection. PEC of different strains of mice were compared. Of these, PEC of all mice that are susceptible to HSV infection in vivo replicated HSV to the same degree as PEC of DBA/2 mice, whereas PEC of resistant C57BL/6 and C3H/HeJ mice produced 1000 fold lower titres of viral progeny. Whereas the number of infectious centres were equal in PEC of DBA/2 and C57BL/6 mice, the plaques observed after infection of confluent PEC with a low MOI were considerable smaller in cells from C57BL/6 mice. Furthermore, significantly higher titres of interferon were measured in the supernatants of HSV-infected C57BL/6 macrophages than in those of DBA/2 macrophages, and the former were made fully susceptible by the in vitro addition of an anti-interferon serum.

Reduction of alloxan by microsomal electron transport proteins.

Alloxan behaves as a substrate for NADH:ferricytochrome b5 oxidoreductase (EC 1.6.2.2). The apparent Km for alloxan was 10 mM in liver microsomes and 20 mM with the enzyme prepared by lysosomal digestion. The apparent Km for NADH was the same with microsomes and the isolated enzyme (30 microM). The maximum turnover rate was calculated as 426 moles electrons/min X mole enzyme. Cytochrome b5 was shown to reduce alloxan nonenzymatically.