SFRP1 promotor methylation analysis of FTA card touch-prep samples derived from colonic polyps.

Whatman FTA® cards provide the most reliable method for DNA storage and extraction, however, the literature lacks reports on the epigenetic analysis of FTA card-derived tumor DNA. Therefore, this study aimed at demonstrating that punches from colonic adenoma samples preserved on FTA filter cards are suitable for methylation analysis by real-time methylation-specific PCR (MSP). Genomic DNA was isolated from a total of 40 sporadic colorectal adenoma samples stored on FTA cards for a median of 59.60 (range 48-72) months. After bisulfite treatment, deaminated DNA was analyzed by SYBR Green real-time MSP using primers specific for methylated and unmethylated promotor sequences of the secreted frizzled-related protein 1 (SFRP1) gene. Amplifiable DNA could be isolated from all FTA card punches while SFRP1 promotor methylation was present in 34/40 (85.0%) colorectal adenomas. Our results indicate that genomic DNA isolated from colonic tumor samples preserved on FTA cards is suitable for downstream methylation detection methodologies such as MSP even after prolonged storage periods.

Iron status determination in individuals with Helicobacter pylori infection: conventional vs. new laboratory biomarkers.

Background Helicobacter pylori has been associated with iron deficiency (ID). This study is aimed at investigating ID with conventional (ferritin, transferrin saturation [TSAT]) and new biomarkers (soluble transferrin receptor [sTfR], sTfR/log ferritin, reticulocyte hemoglobin content [CHr], hepcidin-25) in patients sub-grouped by the presence or absence of H. pylori infection. Methods In total, 200 consecutive outpatients, who were referred for the H. pylori 13C-urea breath test (13C-UBT), underwent blood testing for ID. Additionally, Thomas-plot (TP)-analyses (sTfR/log ferritin, CHr) were calculated. Results Fifty-three and 147 individuals were found with and without H. pylori infection, respectively. Patients with H. pylori infection showed a higher sTfR concentration (p<0.02) and a higher sTfR/log ferritin ratio (p<0.05). Based on a ferritin <30 µg/L and/or a TSAT <20%, 25/53 (47.2%) patients with H. pylori infection and 63/147 (42.9%) without H. pylori infection showed ID. Based on TP-analyses, 10/53 (18.9%) patients with and 17/147 (11.6%) without H. pylori infection were identified with ID. Completed eradication therapy tended to be associated with functional ID. Conclusions Helicobacter pylori infection was associated with significantly higher plasma sTfR concentrations and sTfR/log ferritin ratios. Patients with H. pylori eradication therapy were more often detected with functional ID compared to patients without eradication therapy, when using the new biomarkers.

Prospective plasma lipid profiling in individuals with and without depression.

BACKGROUND: So far, studies on possible association of plasma lipid levels and depressive disorder are contradictory. This prospective work aimed at assessing a plasma lipid profile in individuals with major depression and healthy controls. METHODS: In total, 94 patients with major depression and 152 healthy controls were included in this prospective study. After an overnight fasting state of 12 h they underwent blood drawing for triglyzerides (TG), total cholesterol, low-density lipoprotein (LDL)- and high-density lipoprotein (HDL)-cholesterol measurements. All participants were evaluated in a clinical interview and filled out the self-rating Beck Depression Inventory (BDI-II) scale to identify depressive symptomatology. RESULTS: Ninety-four patients with major depression showed significantly higher median (interquartile range) plasma TG levels (108.0 [75.8-154.1] vs. 84.0 [63.0-132.2] mg/dL, P = 0.014) and significantly lower HDL-cholesterol levels (55.0 [46.9-123.0] vs. 61.5 [47.4-72.6] mg/dL, P = 0.049) compared to 152 individuals without depression, respectively. Total and LDL-cholesterol concentrations were observed slightly higher in patients with major depression. Significant positive correlation was found between TG, total cholesterol and LDL-cholesterol concentrations and the BDI-II score (p = 0.027, 0.048 and 0.018), and in tendency negative correlation between HDL-cholesterol levels and the BDI-II score (P = 0.091), respectively. CONCLUSIONS: Depressive individuals were found with adverse plasma lipid patterns of higher TG and lower HDL-cholesterol levels compared to healthy controls. On this basis, the authors would suggest the implementation of routine lipid measurements in order to stratify these patients by their cardiovascular risk.

SLCO1B1 c.521T>C Genotyping in the Austrian Population Using 2 Commercial Real-Time Polymerase Chain Reaction Assays: An Implementation Study.

Statin-induced myopathy is reported to be significantly associated with the SCLO1B1 c.521T>C polymorphism. To date, SLCO1B1 c.521T>C epidemiologic data for the Austrian population is still lacking. Therefore, this study aimed at assessing the genotype and allele frequencies of the SLCO1B1 c.521T>C variant in Austria and evaluating the clinical performance of 2 commercial real-time polymerase chain reaction (PCR) assays. Genomic DNA isolated from 181 healthy individuals was analyzed for the SLCO1B1 c.521T>C polymorphism in a comparative manner using the SLCO1B1 c.521T>C RealFastTM Assay and the BioPro SLCO1B1 Genotyping real-time PCR Kit. A total of 10 (5.5%) and 44 (24.3%) out of 181 individuals were SLCO1B1 c.521T>C C/C-homo- and -C/T-heterozygotes, the genotypes indicative of high and increased risk of statin-induced myopathy, respectively. The SLCO1B1 c.521C allele frequency rate was 17.7%. In conclusion, the genetic predisposition of elevated statin-induced myopathy risk in the Austrian population is frequent. Both real-time PCR assays under investigation here are reliable and robust SLCO1B1 c.521T>C genotyping tools in clinical routine.

Performance evaluation of a rapid whole-blood immunoassay for the detection of IgG antibodies against Helicobacter pylori in daily clinical practice.

BACKGROUND: A growing number of rapid Helicobacter pylori antibody tests are commercially available now, however, some of these tests are often used without sufficient evaluation. The aim of this study was to evaluate the performance of a commercially available rapid whole-blood immunoassay (gabControl(®) H. pylori; gabmed GmbH, Köln, Germany), for the qualitative detection of IgG antibodies against H. pylori with the (13)C-urea breath test ((13)C-UBT) serving as a reference method. METHODS: A total of 108 consecutive outpatients, who were referred for (13)C-UBT by general practitioners and specialists, were also tested for H. pylori infection by the gabControl(®) H. pylori immunoassay. The clinical performance of this rapid whole-blood test was evaluated by determining the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) compared to the (13)C-UBT. The agreement between the two tests was calculated using Cohen's Kappa (κ) with 95 % confidence intervals (CI). RESULTS: The agreement between the gabControl(®) H. pylori assay and the (13)C-UBT was 0.62 [95 % confidence intervals (CIs) 0.47-0.76; P < 0.001]. With the (13)C-UBT serving as the non-invasive gold standard method of H. pylori diagnosis, the gabControl(®) H. pylori assay demonstrated a sensitivity and specificity of 91.4 and 76.7 %, respectively, with a PPV of 65.3 % and a NPV of 94.9 %. Seventeen (15.7 %) individuals with a positive H. pylori anamnesis showed a negative (13)C-UBT and were typed positive by the gabControl(®) H. pylori assay. Of these, 13 (76.5 %) and 3 individuals (17.6 %) had completed one and two eradication therapies, respectively. CONCLUSIONS: The gabControl(®) H. pylori immunoassay is a rapid and easy to use first line screening tool for H. pylori IgG antibody detection in daily clinical practice. However, this assay should not be used for confirmation of the successful H. pylori eradication after antibiotic treatment.

Lactase Non-Persistence Genotyping: Comparison of Two Real-Time PCR Assays and Assessment of Concomitant Fructose/Sorbitol Malabsorption Rates.

BACKGROUND: Genetic testing is a standard technique for the diagnosis of primary adult-type hypolactasia, also referred to as lactase non-persistence. The aim of this study was to compare the lactase gene (LCT) C/T-13910 polymorphism genotyping results of two commercially available real-time (RT)-PCR assays in patients referred to our outpatient clinic for primary lactose malabsorption testing. Furthermore, concomitant conditions of fructose/sorbitol malabsorption were assessed. METHODS: Samples obtained from 100 patients were tested in parallel using the LCT T-13910C ToolSet for Light Cycler (Roche, Rotkreuz, Switzerland) and the LCT-13910C>T RealFast Assay (ViennaLab Diagnostics GmbH, Vienna, Austria). Additionally, patients were also screened for the presence of fructose/sorbitol malabsorption by functional hydrogen (H2)/methane (CH4) breath testing (HMBT). Cohen's Kappa (κ) was used to calculate the agreement between the two genotyping methods. The exact Chi-Square test was performed to compare fructose/sorbitol HMBT with LCT genotyping results. RESULTS: Twenty-one (21.0%) patients had a LCT C/C-13910 genotype suggestive of lactase non-persistence, and 79 (79.0%) patients were identified with either a LCT T/C-13910 or T/T-13910 genotype (i.e., lactase persistence). In all genotype groups, concordance between the two RT-PCR assays was 100%. Cohen's κ demonstrated perfect observed agreement (p < 0.001, κ = 1). Fructose and sorbitol malabsorption was observed in 13/100 (13.0%) and 25/100 (25.0%) individuals, respectively. CONCLUSIONS: Both RT-PCR assays are robust and reliable LCT genotyping tools in a routine clinical setting. Concomitant fructose and/or sorbitol malabsorption should be considered in individuals with suspected lactase-non-persistence. However, standardization of clinical interpretation of laboratory HMBT results is required.

Hepcidin-25 vs. conventional clinical biomarkers in the diagnosis of functional iron deficiency.

OBJECTIVES: As hepcidin-25 is considered as a key regulator of human iron homoeostasis, this study aimed to compare this parameter with conventional biomarkers and diagnostic tools of iron deficiency (ID). METHODS: In total, 233 hospitalised adult patients, who underwent routine blood testing for ID, were included. All subjects were investigated for hepcidin-25, reticulocyte haemoglobin content (CHr), soluble transferrin receptor (sTfR)/log ferritin ratio (i.e. Thomas plot), sTfR, ferritin, transferrin saturation (TSAT), C-reactive protein (CRP) and for complete blood cell count. Functional ID was defined as a CHr < 28 pg. Separate logistic regression models were calculated with all potential biomarkers to evaluate and compare the predictive performance with respect to functional ID in patients without (CRP ≤ 0.5 mg/dL) and with (CRP > 0.5 mg/dL) acute-phase reaction, respectively. RESULTS: One hundred seventeen patients with CRP > 0.5 mg/dL showed a distinctly higher hepcidin-25 median value [35.60 (range: 4.27-80.03) ng/mL] as compared to 116 patients with CRP ≤ 0.5 mg/dL [18.55 (range: 3.77-73.01) ng/mL]. With respect to functional ID, sTfR/log ferritin ratio and sTfR were of better positive predictive value (PPV) (sTfR/log ferritin ratio: 58.33% and 70.83%; sTfR: 60.00% and 60.00%) than when compared to hepcidin-25 (PPV: 37.74% and 42.86%) and ferritin (PPV: 27.54% and 46.15%) in both subgroups. CONCLUSIONS: The sTfR/log ferritin ratio, as well as sTfR, were better predictors of functional ID in patients with and without acute-phase reaction as compared to hepcidin-25 and ferritin.

Carbohydrate Malabsorption and Putative Carbohydrate-Specific Small Intestinal Bacterial Overgrowth: Prevalence and Diagnostic Overlap Observed in an Austrian Outpatient Center.

BACKGROUND/AIMS: While lactose malabsorption is a well-investigated condition, few epidemiologic data are available for fructose and sorbitol malabsorption. The aim of this study was to assess the prevalence rates for primary lactose malabsorption, fructose and sorbitol malabsorption, and carbohydrate-specific small intestinal bacterial overgrowth (cs-SIBO) in an Austrian outpatient center. METHODS: In total, 306 adult patients, who were primarily referred with suspected carbohydrate malabsorption by general practitioners to our outpatient clinic, underwent genetic testing (C/T-13910 polymorphism) for primary lactose malabsorption, and a combined hydrogen (H2)/methane (CH4) breath test for fructose (25 g) and sorbitol (12.5 g) malabsorption. Cohen's kappa (κ) was calculated for agreement between positive breath test results and self-reported symptoms during the test. RESULTS: Seventy-eight (25.49%) patients were C/C-13910 homozygotes, indicating primary lactose malabsorption. Thirty-four (11.11%) and 57 (18.63%) patients were classified as fructose and sorbitol malabsorbers. Cohen's κ measuring agreements between positive fructose and sorbitol breath test results and self-reported symptoms during the test were 0.33 and 0.49, respectively. Twenty-nine (9.50%) patients with an early H2/CH4 peak (i.e. within 60 minutes after fructose and/or sorbitol ingestion) were diagnosed with cs-SIBO. CONCLUSION: In Austria, carbohydrate malabsorption is a frequent condition in patients referred by general practitioners to carbohydrate malabsorption testing.

25-hydroxy-Vitamin D status: limitations in comparison and clinical interpretation of serum-levels across different assay methods.

Background: Over the last decade, clinical interest to evaluate human 25-hydroxy-vitamin D (25[OH]D) serum levels has increased exponentially. In the present study, four chemiluminescence immunoassays (CLIA), one radioimmunoassy (RIA), and one high performance liquid chromatography (HPLC) method were compared and also with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in view of 25(OH)D serum level determination. Methods: For the method comparison, blood samples from 133 consecutive patients were prospectively collected. All participants gave written informed consent for their blood samples to be used in this study. They came to the Department of Nuclear Medicine of the Central Hospital Steyr (Austria) for osteodensidometric measurement as part of their preventive medical check-up. Pearson's correlation coefficients, Bland-Altman plots, and paired t-tests were calculated. Assay-specific reference ranges were considered using blood samples from persons with normal parathormone, calcium, and total-protein values (n = 97). Results: The highest correlation was between the HPLC and the LC-MS/MS method (r = 0.96). The lowest correlation was between the cobas Vitamin D3 assay (Roche) and any of the evaluated assays (r = 0.46 - 0.63). Bland-Altman plots revealed a big negative mean bias in three assays (cobas Vitamin D3 assay [Roche]: -22.8; DiaSorin LIAISON [25[OH]D total CLIA [Diasorin]: -18.4; Diasorin 25[OH]D125 I RIA [Diasorin]: -23.8 [nmol/L]) and a much smaller positive mean bias in the other assays (ClinRep complete 25[OH]D2/D3 HPLC kit [Recipe]: 2.7; ADVIA Centaur Vitamin D total assay [Siemens]: 8.2; IDS total vitamin D assay [Immunodiagnostic Systems]: 12.1 [nmol/L]) compared to the LC-MS/MS method. Meanwhile, the manufacturer has withdrawn the cobas Vitamin D3 assay from the market. Conclusions: Poor antibody specificity with cross-reactivity to other vitamin D metabolites, incomplete extraction of the 25(OH)D analyte from the vitamin D-binding protein (DBP), and confounding matrix substances such as lipids could be potential reasons for the unacceptable performance of the cobas Vitamin D3 assay (Roche) and also the significant differences in the 25(OH)D determination between various assays. Standardization and harmonization of 25(OH)D measurements are therefore urgently needed. The widespread introduction of well standardized assays in clinical laboratories is the challenge in the next years. (Clin. Lab. 2014;60:1541-1550. DOI: 10.7754/Clin.Lab.2014.131114)

Diagnosis of anaemia: old things rearranged.

Anaemia is one of the most leading causes of morbidity and mortality, as declared by the World Health Organisation. This syndrome is characterised by low haemoglobin levels and nonspecific clinical symptoms such as weakness, fatigue and dyspnoea. The symptoms are unspecific as the underlying causes are heterogeneous. Thus, good knowledge of the useful biomarkers and their correct assignment is needed to allow rapid and targeted diagnosis.

The use of inflammatory markers as a method for discharging patients post hip or knee arthroplasty.

BACKGROUND: Complications after orthopaedic surgery have a substantial impact on resources, including the financial resources of the health-care system. Early detection of patients developing complications, thus excluding those patients without complications should have a major impact on patients' management. METHODS: We evaluated the prognostic value of blood levels of inflammatory markers (interleukin-6, C-reactive protein, total white blood cell count) for complications after primary hip and knee arthroplasty in a routine setting, and their impact on patient management. First, the three inflammatory markers were evaluated in 68 consecutive patients treated at the Department of Orthopaedics. Laboratory markers were measured on days 2 and 4 after surgery. Second, patient management before and after adapting the routine order for laboratory parameters after arthroplasty were evaluated. RESULTS: We could show that interleukin-6 and the C-reactive protein had an almost equal negative predictive value for the exclusion of postsurgical complications. Nevertheless, interleukin-6 returned below its reference levels almost 2 days earlier compared to the other markers. This made an earlier discharge of patients with presumable complications-less postsurgical process possible. CONCLUSIONS: So, the total number of patients treated as well as the financial reimbursement could be increased and concurrently occupancy days and averaged length of stay were reduced.

Plasma free DNA: Evaluation of temperature-associated storage effects observed for Roche Cell-Free DNA collection tubes.

INTRODUCTION: Standardized pre-analytical blood sample procedures for the analysis of circulating cell-free DNA (ccfDNA) are still not available. Therefore, the present study aimed at evaluating the impact of storage conditions related to different times (24 and 48 h) and temperatures (room temperature (RT) and 4 - 8 °C) on the plasma ccfDNA concentration of blood samples drawn into Cell-Free DNA collection tubes (Roche Diagnostics GmbH, Mannheim, Germany). MATERIALS AND METHODS: Venous blood from 30 healthy individuals was collected into five 8.5 mL Cell-Free DNA Collection Tubes (Roche Diagnostics GmbH) each. Plasma samples were processed at time point of blood collection (tube 1), and after storage under the following conditions: 24 h at RT (tube 2) or 4-8 °C (tube 3), and 48 h at RT (tube 4) or 4 - 8 °C (tube 5). Circulating cell-free DNA concentrations were determined by EvaGreen chemistry-based droplet digital PCR (ddPCR). RESULTS: No statistically significant differences between median (interquartile range) plasma ccfDNA concentrations (ng/mL) at time point of blood collection (3.17 (2.13 - 3.76)) and after storage for 24 h (RT: 3.02 (2.41 - 3.68); 4-8 °C: 3.21 (2.19 - 3.46)) and 48 h (RT: 3.13 (2.10 - 3.76); 4-8 °C: 3.09 (2.19 - 3.50)) were observed (P values from 0.102 - 0.975). CONCLUSIONS: No unwanted release of genomic DNA from white blood cells could be detected in plasma samples after tube storage for 24 and 48 h regardless of storage temperature.

EGFR Mutational Profiling in Non-Small Cell Lung Cancer: The Clinical Performance of a Sensitive Reverse-Hybridization Assay.

In patients with non-small cell lung cancer (NSCLC), epidermal growth factor receptor (EGFR) mutations have been associated with the tumor response to targeted therapy with EGFR tyrosine kinase inhibitors. Although labor intensive and not very sensitive (ie, an analytical sensitivity of 20%), direct sequencing is widely used for mutation detection. This study aimed at evaluating the potential of a test strip-based reverse-hybridization assay (EGFR StripAssay), designed for the simultaneous detection of 16 mutations in exons 18 to 21 of the EGFR gene, to sensitively identify EGFR mutation in DNA from NSCLC tissue samples. Formalin-fixed paraffin-embedded (FFPE) DNA samples from 59 patients with a histologically confirmed primary NSCLC tumor were used to compare the performance of the EGFR StripAssay against that of the Sanger sequencing. The EGFR StripAssay analysis identified 7 (11.8%) of 59 FFPE samples to carry an EGFR mutation, of which 4 (57.1%) and 3 (42.8%) samples were positive for exon 19 and 21 mutations, respectively. Of note, no sample was identified with EGFR exon 18 or 20 mutation. All mutations were confirmed by DNA sequencing. Using 50 ng of template DNA, the EGFR StripAssay demonstrated a detection limit of 1% mutant sequence in a background of normal DNA. The EGFR StripAssay is a fast and robust platform for the sensitive detection of EGFR mutation in FFPE DNA. Therefore, this assay could be considered as an alternative protocol to Sanger sequencing for EGFR mutation testing on limited-quantity samples.

Association between increased plasma levels of homocysteine and depression observed in individuals with primary lactose malabsorption.

BACKGROUND: Current literature proposes associations between homocysteine (HCY), folic acid (FA), vitamin B12 metabolism and depression. However, the exact underlying biological mechanisms remain unclear. This study aimed at evaluating a possible link between primary adult-type lactose malabsorption (PALM), HCY, FA and vitamin B12 metabolism and depressive disorder. METHODS: Plasma levels of HCY, FA and vitamin B12 were determined in 78 patients with PALM and 160 individuals with lactase persistence sub-grouped by the presence or absence of major depression. RESULTS: In 78 patients with PALM, the subgroup of 22 individuals with major depression showed significantly higher median (interquartile range) HCY (10.10 [8.46-12.03] vs. 8.9 [7.54-9.86] µmol/L, p = 0.029) and lower plasma FA levels (5.7 [4.68-9.14] vs. 6.95 [5.24-10.56] µmol/L, p = 0.272) compared to the subgroup of 56 individuals without depression, respectively. No such associations could be observed for those 160 individuals without PALM (i.e., lactase persistence) Plasma HCY levels were positively correlated with depressive symptoms (p = 0.052), and showed negative correlations with FA (p = < 0.001) and vitamin B12 (p = 0.029), respectively. CONCLUSION: Depressed individuals with PALM were found with significantly higher HCY and lower FA levels compared to non-depressed individuals with PALM, however, this association was absent in the subgroup of lactase persistent individuals. These findings suggest an association between increased HCY levels, lactose malabsorption and depression.

Assessment of tryptophan metabolism and signs of depression in individuals with carbohydrate malabsorption.

This prospective cross-sectional study aimed to investigate the potential association between primary-adult lactose malabsorption, fructose malabsorption, tryptophan (TRP) metabolism and the presence of depressive signs. Overall 251 patients, who were referred for lactase gene C/T-13910 polymorphism genotyping and fructose hydrogen/methane breath testing, were included. All participants filled out the Beck Depression Inventory (BDI II). Serum concentrations of tryptophan (TRP), kynurenine (KYN), kynuric acid (KYNA), and TRP competing amino acids (leucine, isoleucine, valine, phenylalanine, tyrosine) were measured by high-pressure liquid-chromatography. Logistic regression analysis was performed with lactose malabsorption, fructose malabsorption and all potential biomarkers of TRP metabolism to assess the effect on signs of depression, defined as a BDI II score > 13. Primary-adult lactose malabsorption and fructose malabsorption was detected in 65 (25.90%) and 65 (25.90%) patients, respectively. Fructose malabsorption was significantly associated with BDI II score, whereas no such relationship was found for lactose malabsorption. Serum levels of TRP and TRP metabolites were no predictors of depression. The authors suggest to conduct further prospective longitudinal studies in order to get further insight of associations between carbohydrate malabsorption, biomarkers and mood disorders.

Comparison of fluorescent polarization immunoassay (FPIA) versus HPLC to measure everolimus blood concentrations in clinical transplantation.

Clinical management of transplant patients depends on therapeutic drug monitoring (TDM) and regulation of immunosuppressive therapy. TDM of whole-blood concentrations is mandatory for everolimus (ERL) dosage individualisation. We compared the new semi-automated immunoassay (Innofluor Certican Assay System, Seradyn Inc) using FPIA technology on Abbott TDxFLx analyzers with established HPLC-UV as reference method. A total of 165 samples were analyzed from 52 transplant patients (40 kidney, 12 heart) receiving ERL or another immunosuppressive agent as part their routine care after organ transplantation. The correlation coefficient was r(2)=0.8229, and the regression equation (95% IC) yielded FPIA=1.111 x (HPLC)+0.378. FPIA compared to HPLC gave a positive bias of 1.19 ng/ml. The FPIA assay so appears to have a diagnostic efficacy comparable to HPLC for assessing the risk of acute rejection in transplant recipients. However, the values of the FPIA were higher than those calculated from HPLC measurements, because of the cross-reactivity of the antibody used in the FPIA assay with the ERL metabolite and/or with sirolimus; this cross-reactivity occurs frequently when transplant patients are switched from sirolimus to ERL.

Reference values for plasma concentrations of asymmetrical dimethylarginine (ADMA) and other arginine metabolites in men after validation of a chromatographic method.

BACKGROUND: Owing to the growing number of reports in the literature on ADMA as a possibly useful marker of endothelial health, its use in the clinical laboratory is of increasing interest. Age dependency and the small, but statistically significant differences between healthy subjects and disease groups are difficult to interpret. Additionally, levels of ADMA in comparable patient groups of different studies vary widely, even when similar methods have been used. METHODS: After analytical evaluation of a chromatographic method according to international guidelines, we analysed asymmetrical (ADMA) and symmetrical dimethyl arginine (SDMA), homo-arginine and arginine in EDTA plasma of 292 healthy males aged 20 to 75 years (y) who had passed strict inclusion/exclusion criteria. For statistical analysis, 4 age groups were formed. Group differences were identified with the non-parametric Kruskal-Wallis test. RESULTS: Calibration curves were linear throughout the selected ranges; the standard deviation for the regression line, recovery, imprecision, and accuracy results were all highly satisfactory. The reference ranges of ADMA for the 4 age groups are presented as age (mean+/-SD of age group, y); number of subjects; median, 2.5th-97.5th percentile: group <35 y: 26.7+/-4.0 y; n=78; 0.58, 0.43-0.69 micromol/L; group 35-49 y: 41.6+/-4.0 y; n=93; 0.59, 0.45-0.73 micromol/L; group 50-65 y: 57.5+/-4.2 y; n=82; 0.61, 0.46-0.78 micromol/L; and group >65 y: 69.6+/-3.3 y; n=39; 0.64, 0.54-0.79 micromol/L. CONCLUSIONS: Only highly precise methods are able to detect small differences between groups. The application of an evaluated method to a well defined group of healthy subjects should provide a basis for comparison of ADMA concentrations in different patient populations of future studies.

Serum diamine oxidase activity is associated with lactose malabsorption phenotypic variation.

OBJECTIVES: Recently, an intermediate lactose intolerance (LI) phenotype based on the lactase gene (LCT) C/T-13910 polymorphism was proposed. However, a multifactorial genesis of LI phenotypic variation including endogenous and exogenous factors cannot be ruled out. Therefore, this study was conducted to investigate a possible association between serum diamine oxidase (DAO) and LI phenotypes in individuals with lactose malabsorption (LM). DESIGN AND METHODS: A total of 121 ambulatory patients with LM were included in this retrospective study. The lactose hydrogen breath test (LHBT) and serum DAO activity measurements were performed on the same day. A thorough anamnesis with respect to gastrointestinal symptoms was carried out at the initial consultation. RESULTS: In total, 44 (36.4%) patients with a serum DAO activity <10U/mL showed higher H2 levels after 60 (mean: 53.7±57.6 vs 34.5±31.7 parts per million [ppm], p=0.116), 90 (mean: 70.3±57.5 vs 52.7±41.4ppm, p=0.184) and 120min (mean: 98.9±72.5 vs 67.9±44.9ppm, p=0.012) during LHBT compared to 77 (63.6%) patients with a serum DAO activity ≥10U/mL. Individuals with a serum DAO activity <10U/mL tended to report gastrointestinal symptoms during the LHBT more often (p=0.091). CONCLUSIONS: Our findings suggest that patients with LM and a serum DAO activity level<10U/mL had higher end-expiratory H2 levels and tended to be more symptomatic during the LHBT compared to LM patients with DAO activity levels ≥10U/mL.