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ZusammenfassungDieser Übersichtsartikel bietet einen Überblick über klinisch sinnvolle Anwendungsgebiete einer Next-Generation-Sequencing-basierten (NGS) Multi-Genpanel-Teststrategie in den Bereichen Onkologie, hereditärer Tumorsyndrome und Hämatologie. Bei soliden Tumoren (z.B. Lungenkarzinom, Kolonrektalkarzinom) trägt die Detektion somatischer Mutationen nicht nur zu einer besseren diagnostischen, sondern auch therapeutischen Stratifizierung der Betroffenen bei. Die zunehmende genetische Komplexität hereditärer Tumorsyndrome (z.B. Brust- und Ovarialkarzinom, Lynchsyndrom/Polypose) erfordert in betroffenen Familien eine Multi-Genpanel-Analyse von Keimbahnmutationen. Ein weiteres sinnvolles Indikationsgebiet einer Multi-Genpanel-Diagnostik und Prognoseabschätzung sind akute und chronische myeloische Erkrankungen. Die Kriterien der WHO-Klassifikation und des „European LeukemiaNet“-Prognosesystems der akuten myeloischen Leukämie können nur durch eine Multi-Genpanel-Teststrategie erfüllt werden.

BACKGROUD: Trimethylamine oxide (TMAO) represents a gut metabolite and it's physiological concentration is proposed to be influenced by the abundance of precursor trimethylamine (TMA)-producing microbiota in the gut. OBJECTIVE: Lifestyle factors, such as diet and exercise, modify the microbiome, but their long-term impact on plasma TMAO and TMA production in the gut is poorly understood. METHODS: Fecal microbiome composition was analyzed and correlated with TMAO serum concentrations in female Sprague-Dawley rats that received either a healthy normal or a high-fat diet (HFD) for 10 months. Half of the animals performed regular running exercise, the other half served as sedentary controls. RESULTS: HFD consumption induced an alteration of the fecal microbiome with increased alpha diversity on ASV levels. More importantly, HFD reduced the abundance of genera containing potential TMA producers, such as certain members of Clostridia and Lachnospiraceae. In line with this observation, serum TMAO concentrations were found to be lower with HFD and to correlate with the abundance of these genera. Regular exercise also induced changes in microbiome beta diversity but had no effect on serum TMAO in female rats. CONCLUSION: A diet containing high amounts of dietary fat reduced serum TMAO, probably through a reduced intestinal abundance of TMA-producing bacteria. In contrast, regular exercise altered beta diversity of the microbiome composition, but without significant effects on serum TMAO concentrations.

ZusammenfassungNext-Generation-Sequencing ist ein modernes diagnostisches Hochdurchsatz-Verfahren (Multi-Gen-Analysen), durch dessen Einsatz sowohl hereditäre Krebserkrankungen (Tumordispositionssyndrome, Keimbahndiagnostik) als auch somatische Alterationen in Tumoren besser abgeklärt werden können. Der breitere Einsatz dieser Technologie im medizinischen Alltag zeigt das tatsächliche Ausmaß der interindividuellen genetischen Variabilität. Wichtige Bedeutung hat dieses Verfahren für die Untersuchung von heterogenen genetischen Erkrankungen (z. B. Tumorerkrankungen, neurodegenerativen und -muskulären Erkrankungen) erlangt. Weitere Indikationsgebiete stellen die Pharmakogenetik sowie die nicht invasive Pränataldiagnostik dar. Es ist zu erwarten, dass dieses diagnostische Mittel eine breite klinische Anwendung finden wird. Mit der rasanten Zunahme und Komplexität genetischer Dateninformationen nimmt die richtige Interpretation und Übermittlung der Befunde in der humangenetischen Beratung (Keimbahndiagnostik) einen hohen Stellenwert ein. Die genetische Beratung muss entsprechend neu ausgerichtet und adaptiert werden.

Abstract Different foods, especially mushrooms, are a valuable source of vitamin D2. However, published concentrations in mushrooms show large variabilities. One reason for this is certainly the high biological variability caused by growth conditions, and another could also be found in the analytical methodology. Therefore, this study aimed to develop a sensitive and highly selective two-dimensional liquid chromatography mass spectrometry (LC–MS/MS) method for vitamin D2 analysis in mushrooms. After validation, the method was applied to four different mushroom species. The developed method with a one-step extraction procedure showed a limit of detection of 0.01 µg vitamin D2/g dry mass (DM), a limit of quantification of 0.05 µg vitamin D2/g DM, and recovery rates between 87.6 and 94.8%. The total run time including the re-equilibration of the columns for the next injection was 7.5 min. After adding increased concentrations of pure substance to Pleurotus ostreatus, Lentinula edodes, and brown and white button mushrooms the standard addition plot showed excellent correlation coefficients (R2) of > 0.9994. Mean vitamin D2 concentrations were observed at 0.122 ± 0.007, 0.074 ± 0.005, 0.099 ± 0.007, and 0.073 ± 0.005 µg/g DM. The coefficient of variation (CV) was between 5.1 and 7.6%. This well-optimized, sensitive LC–MS/MS method, with a fast and simple sample preparation and a short run time, can be applied to future studies especially in different mushroom species with variable growing conditions. This will improve our knowledge about the vitamin D2 content in mushrooms. Graphical abstract

Objectives Living kidney donors provide a unique setting to study functional and metabolic consequences after organ donation. Since the lack of data of the homoeostasis of numerous vitamin D metabolites in these healthy subjects, the aim of this study was to assess the vitamin D metabolism before and after kidney donation. Methods We investigated the 25-dihydroxyvitamin D2 (25[OH]D2), 25-dihydroxyvitamin D3 (25[OH]D3), 1,25-dihydroxyvitamin D3 (1,25[OH]2D3), 24,25-dihydroxyvitamin D3 (24,25[OH]2D3), 25,26-dihydroxyvitamin D3 (25,26[OH]2D3), and the native vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol) in a well characterized study cohort of 32 healthy living kidney donors before and after organ donation. Results Thirty-two healthy subjects after kidney donation had significantly lower median (interquartile range) 1,25(OH)2D3 serum concentrations (88.6 [62.6–118.8] vs. 138.0 [102.6–152.4] pmol/L, p2 serum levels (1.80 [1.19–2.19] vs. 1.11 [0.74–1.59] nmol/L, p=0.019) than before donation. Similar serum concentrations of 25(OH)D3 and 25,26(OH)2D3 were observed before and after donation. The 24,25(OH)2D3 blood levels distinctly decreased after organ donation (4.1 [2.3–5.3] vs. 5.3 [2.2–6.9] nmol/L, p=0.153). Native vitamin D2 (0.10 [0.08–0.14] vs. 0.08 [0.06–0.12] nmol/L, p=0.275) was slightly increased and vitamin D3 (1.6 [0.6–7.2] vs. 2.5 [0.9–8.6] nmol/L, p=0.957) decreased after kidney donation. Conclusions Living kidney donors were found with decreased 1,25(OH)2D3 and 24,25(OH)2D3, increased 25(OH)D2 and consistent 25(OH)D3 and 25,26(OH)2D3 serum concentrations after organ donation. The current study advances the understanding on vitamin D metabolism suggesting that altered hydroxylase-activities after donation is accompanied by compensatory elevated dietary-related 25(OH)D2 blood concentrations.

Objectives Vitamin K and metabolites have a beneficial role in blood coagulation, bone metabolism and growth. However, the determination of vitamin K concentrations in the blood in patients consuming a diet with naturally occurring vitamin K is currently challenging. We aim to develop a cost-effective and rapid method to measure vitamin K metabolites with potential application for clinics and research. Methods We developed a simple liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of vitamin K1, menaquinone-4 (MK-4), menaquinone-7 (MK-7) and vitamin K1-2,3 epoxide in human serum and validated the method in a study cohort of 162 patients tested for carbohydrate malabsorption and in 20 patients with oral phenprocoumon intake. Results The overall precision (CVs) ranged between 4.8 and 17.7% in the specified working range (0.06–9.0 nmol/L for all analytes except for MK-7 with 0.04–6.16 nmol/L). In the malabsorption cohort samples, measured values were obtained for all different vitamin K metabolites except for vitamin K1-2,3 epoxide. This metabolite could be detected only in patients with phenprocoumon intake. The good performance of the method is especially achieved by the interaction of three factors: the use of lipase in the sample preparation, the use of an atypical fluorinated reversed phase column, and a logarithmic methanol gradient. Conclusions The described method is able to determine the concentration of four vitamin K metabolites in a time-efficient, simple and cost-effective manner. It can be suitable for both routine clinics and research.

Background Immune dysregulation and inflammation in patients with SARS-CoV-2 is associated with a poor clinical outcome. We investigated the value of the inflammatory markers tryptophan and kynurenine in predicting the survival outcome of patients with SARS-CoV-2. Methods The study included 252 inpatients with a SARS-CoV-2 infection hospitalized between August 2020 and April 2021. Two groups were generated based on disease survival (survival group: n = 199; deceased group: n = 53). Plasma concentrations of tryptophan, kynurenine and interleukin-6 (IL-6) were measured on admission. In a subset of patients (n = 105; 81 survivors and 24 deceased) concentrations of tryptophan and kynurenine were checked 7 days after admission. The kynurenine/tryptophan ratio (TRP/KYN ratio) was calculated. Results On admission, the deceased group showed significantly higher concentrations of kynurenine and a significantly higher KYN/TRP ratio compared to the survival group (p-values

While myeloperoxidase (MPO) serves as an indicator of both neutrophil and innate-immune-system function, the potential suppression of the innate immune system in patients with acute myocardial infarction (AMI)-induced depression might be evidenced by a decrease in MPO serum levels. The aim of this prospective study was to (1) determine whether serum concentrations of MPO vary immediately and 6 months after AMI and (2) to investigate whether MPO concentrations at the time of the AMI are significant predictors of AMI-induced depression and the depression-associated suppression of the innate immune system. A total of 109 AMI patients were assessed with the Hamilton Depression Scale (HAMD-17) immediately after admission to the hospital and 6 months later. The MPO status was assessed with serum samples, which were also collected immediately and 6 months after AMI. The depressive patients showed significantly lower MPO blood levels immediately and 6 months after the AMI compared to the patients without depression (ANCOVA: MPO (depression) F = 4.764, df = 1, p = 0.031). The baseline MPO was observed as a significant predictor (p = 0.027) of AMI-induced depression 6 months after AMI. MPO is a potential biomarker for AMI-induced depression, indicating a depression-associated suppression of the innate immune system.

The scale and the course of antibody production in patients with SARS-CoV-2 is highly variable. Factors involved in the immune regulation during the infection may play a major role in the antibody response. We investigated the relationship between the inflammatory markers of the kynurenine pathway and the concentration of antibodies against SARS-CoV-2 in infected patients 8 - 11 days after admission.The study included 72 SARS-CoV-2 - positive inpatients hospitalized between August 2020 and April 2021. The plasma concentrations of tryptophan, kynurenine, anti-SARS-CoV-2 antibodies and the leucocyte count were measured 8 - 11 days after admission. The kynurenine/tryptophan ratio (KYN/TRP ratio) was calculated. Tertiles based on the values for tryptophan, kynurenine, KYN/TRP ratio and the leucocytes were generated.Statistically significant correlations were observed between anti-SARS-CoV-2 antibodies and tryptophan, kynurenine, KYN/TRP ratio and the leucocytes (p-values 0.001-0.007). The high kynurenine and KYN/TRP ratio tertiles showed significantly lower antibody titers compared to the low tertiles (p-values 0.017 and 0.001). The low tryptophan and leucocytes tertiles showed significantly lower antibody titers compared to the high tertiles (p-values 0.001 and 0.008).Patients with higher activation levels of the kynurenine pathway tended to develop lower anti-SARS-CoV-2 antibody titers.

Monogenic mutations of the hepatocyte nuclear factor 1 homeobox A maturity onset diabetes of the young (HNF1A-MODY) is characterized by early onset, typically before the age of 25 years. Patients are often not clinically recognized; however, the identification of HNF1A-MODY patients is crucial because they require different antihyperglycemic medical treatment than patients with type 1 or type 2 diabetes mellitus. We describe two adult patients with monogenic diabetes, both identified as HNF1A-MODY, genetically c.815G>A, p.Arg272His and c675delC, p.Ser225Argfs*8, respectively. They were misdiagnosed as having type 1 diabetes mellitus, and consequently, initiating insulin therapy led to hypoglycemia and unstable blood glucose control. Usually, sulfonylureas represent the basis of antidiabetic treatment in patients with HNF1A-MODY; however, all medical personnel involved in diabetes care should be aware of monogenic diabetes mellitus and the possibilities for genetic testing. The patients observed have shown the necessity of the identification and appropriate genetic diagnosis of HNF1A-MODY in order to discontinue insulin therapy and to initiate adjusted diabetes management.

In westernized countries, adverse reactions to ingested foods are reported to affect up to 20% of the population. Functional, nonspecific, non-allergic gastrointestinal complaints are mainly due to the intolerance/malabsorption of carbohydrates (lactose and fructose), proteins (gluten), and biogenic amines (histamine). Food intolerance/malabsorption is defined by one or several of the above mentioned food components not being degraded and/or absorbed properly within the gastrointestinal tract. Food intolerance/malabsorption causes variable, functional, nonspecific, non-allergic gastrointestinal and extra-intestinal complaints, and a detailed diagnostic workup for all possible etiologic factors in individual patients is essential. Usually, evaluation for histamine intolerance is not included in differential diagnoses of patients with functional, nonspecific, non-allergic gastrointestinal complaints. A targeted dietary intervention for single or possibly combined intolerance/malabsorption is required. In this article, we review currently discussed differential diagnoses and available tests for intolerance/malabsorption. Accordingly, we aim to outline why including histamine and, histamine intolerance, should be considered in differential diagnoses of patients with functional, nonspecific, non-allergic gastrointestinal complaints.

Inflammatory bowel disease (IBD) involves two clinically defined entities, namely Crohn’s disease and ulcerative colitis. Fecal calprotectin (FCAL) is used as a marker to distinguish between organic IBD and functional bowel disease in disorders of the irritable bowel syndrome (IBS) spectrum. Food components may affect digestion and cause functional abdominal disorders of the IBS spectrum. In this retrospective study, we report on FCAL testing to search for IBD in 228 patients with disorders of the IBS spectrum caused by food intolerances/malabsorption. Included were patients with fructose malabsorption (FM), histamine intolerance (HIT), lactose intolerance (LIT), and H. pylori infection. We found elevated FCAL values in 39 (17.1%) of 228 IBS patients with food intolerance/malabsorption and H. pylori infection. Within these, fourteen patients were lactose intolerant, three showed fructose malabsorption, and six had histamine intolerance. The others had combinations of the above conditions: five patients had LIT and HIT, two patients had LIT and FM, and four had LIT and H. pylori. In addition, there were individual patients with other double or triple combinations. In addition to LIT, IBD was suspected in two patients due to continuously elevated FCAL, and then found via histologic evaluation of biopsies taken during colonoscopy. One patient with elevated FCAL had sprue-like enteropathy caused by the angiotensin receptor-1 antagonist candesartan. When screening for study subjects concluded, 16 (41%) of 39 patients with initially elevated FCAL agreed to voluntarily control FCAL measurements, although symptom-free and -reduced, following the diagnosis of intolerance/malabsorption and/or H. pylori infection. After the initiation of a diet individualized to the symptomatology and eradication therapy (when H. pylori was detected), FCAL values were significantly lowered or reduced to be within the normal range.

Background Acute myocardial infarction is not only a somatic disease but potentially triggers psychological effects, too. Post-traumatic stress disorder (PTSD) is a common stress-related disorder. It is characterized by numerous symptoms, such as flashbacks, intrusions, nightmares and severe anxiety, as well as uncontrollable, intense and disturbing thoughts and feelings related to the traumatic experience. However, with regard to the development of PTSD, individual stress perception might be crucial since not every serious traumatic experience leads to PTSD. To date, almost no biological correlates of an individual's perception of stress have been identified as being associated with the long-term development of PTSD. Objective The aim of the study was to determine whether blood levels of TMAO vary immediately after AMI (1) in patients with or without depression, and (2) in patients with AMI induced PTSD symptomatology (subsyndromal PTSD and full PTSD). Furthermore, we investigated whether TMAO is a potential biomarker that might be useful in the prediction of PTSD symptomatology in the long term. Method A total of 114 AMI patients were assessed with standardized clinical psychiatric interviews based on the Hamilton Depression Scale (HAMD-17) after admission to the hospital and 6 months later. In addition, the CAPS-5 was used to explore PTSD symptoms (subsyndromal PTSD and full PTSD) 6 months after AMI. To assess patients' TMAO status, serum samples were collected at hospitalization and 6 months after AMI. Results Study participants with post-myocardial infarction PTSD symptomatology (subsyndromal PTSD and full PTSD) had significantly higher TMAO levels immediately after AMI than patients without PTSD symptoms (ANCOVA: TMAO (PTSD x time), F = 4.544, df = 1, p=0.035). In contrast, depressive symptomatology 6 months after AMI had no influence on TMAO levels (TMAO (depression x time), F = 0.083, df = 1, p=0.774). With the inclusion of additional clinical predictors in a hierarchical logistic regression model, TMAO becomes a significant predictor of PTSD symptomatology. Conclusions An elevated TMAO level immediately after AMI might reflect severe stress in PTSD-vulnerable patients, which might also lead to a short-term increased gut permeability to trimethylamine (TMA), the precursor of TMAO. Thus, elevated TMAO might be a biological correlate for stress that is associated with vulnerability to PTSD and might help to identify patients at increased risk. Funding Acknowledgement Type of funding sources: None.

Objectives In-house developed liquid-chromatography mass spectrometry (LC-MS/MS) methods are used more and more frequently for the simultaneous quantification of vitamin D metabolites. Among these, 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) is of clinical interest. This study assessed the agreement of this metabolite in two validated in-house LC-MS/MS methods. Methods 24,25(OH)2D3 was measured in 20 samples from the vitamin D external quality assurance (DEQAS) program and in a mixed cohort of hospital patients samples (n=195) with the LC-MS/MS method at the Medical University of Graz (LC-MS/MS 1) and at the University of Liège (LC-MS/MS 2). Results In DEQAS samples, 24,25(OH)2D3 results with LC-MS/MS 1 had a proportional bias of 1.0% and a negative systemic difference of −0.05%. LC-MS/MS 2 also showed a proportional bias of 1.0% and the negative systemic bias was −0.22%. Comparing the EQA samples with both methods, no systemic bias was found (0.0%) and the slope was 1%. The mean difference of 195 serum sample measurements between the two LC-MS/MS methods was minimal (−0.2%). Both LC-MS/MS methods showed a constant bias of 0.31 nmol/L and a positive proportional bias of 0.90%, respectively. Conclusions This study is the first to assess the comparability of 24,25(OH)2D3 concentrations in a mixed cohort of hospitalized patients with two fully validated in-house LC-MS/MS methods. Despite different sample preparation, chromatographic separation and ionization, both methods showed high precision measurements of 24,25(OH)2D3. Furthermore, we demonstrate the improvement of accuracy and precision measurements of 24,25(OH)2D3 in serum samples and in the DEQAS program.

Histamine intolerance (HIT) is thought to be caused by a disproportionate amount of histamine in the body. The enzyme diamine oxidase (DAO) is considered for the gastrointestinal degradation of histamine. For this open-label interventional pilot study, we identified 28 patients with HIT. For 4 weeks, they were instructed to take DAO capsules before meals. Then, throughout a follow-up period, they were instructed not to take the DAO. We used a questionnaire that included 22 symptoms, which were divided into 4 categories, as well as a symptom severity score. All symptoms improved significantly during the oral supplementation of DAO. During the follow-up period, without DAO supplementation, the symptoms sum scores increased again. The symptom intensity score was reduced for all symptoms. We have demonstrated, a significant reduction of every HIT-related symptom and its intensity due to DAO oral supplements. The ClinicalTrials.gov identifier (NCT number) is NCT03298568.

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 (pH. 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.

Oxidative stress describes an imbalance of reactive oxygen species (ROS) and antioxidative defence systems. Recently, the consequences of oxidative stress have become a central field of research and have been linked to the genesis of multiple psychiatric diseases. Some oxidative stress parameters have not been investigated before in anorexia nervosa (AN) patients, including the gut microbiota-derived metabolite trimethylamine N-oxide (TMAO) and polyphenols (PPm). In this cross-sectional pilot study, we evaluated these markers together with total peroxides (TOC), antioxidative capacity (TAC), endogenous peroxidase activity (EPA) and antibodies against oxidized LDL (oLAb) in serum samples of 20 patients with AN compared to 20 healthy controls. The antioxidative capacity was significantly decreased in AN patients, with a mean TAC of 1.57 mmol/L (SD: ±0.62); t (34) = −2.181, p = 0.036) compared to HC (mean = 1.91 mmol/L (SD: ±0.56), while the other investigated parameters were not significantly different between the two groups. In AN patients, TAC correlated with EPA (rsp = −0.630, p = 0.009). This study suggests that there is an antioxidative deficiency in AN patients. In this respect, there is a demand for interventional studies to determine whether antioxidants can be used as add-on therapy in the treatment of AN.

Objectives Recently, the fully automated flow cytometry-based UF-5000 (Sysmex Corboration, Kobe, Japan) urine sediment analyzer was developed providing bacteria (BACT) info flags for more accurate bacterial discrimination of urinary tract infections (UTIs). This study aimed to compare the reliability of the UF-5000 BACT-info flags with manual Gram stain and urine culture as the gold standard method. Methods A total of 344 urine samples were analyzed on the UF-5000 and compared with manual microscopic Gram stain and urine cultures. Agreement was assessed by Cohen’s kappa (κ) analysis. The Youden index was used to determine the optimal BACT and white blood cell (WBC) cut-off points for discriminating positive and negative urine cultures. Results Overall 98/344 (28.5%) samples were urine culture positive at a cut-off of ≥105 CFU/mL. “Gram-negative?” UF-5000 BACT-Info flags showed a better concordance of 25/40 (62.5%) with urine culture compared to Gram stain with 30/50 (60%). The results for UF-5000 discrimination of Gram-positive and Gram-negative microorganisms demonstrated a substantial (κ = 0.78) and fair (κ = 0.40) agreement with urine culture. Optimal cut-off points detecting positive urine cultures were 135 BACT/µL (sensitivity [SE]: 92.1%, specificity [SP]: 85.4%, positive predictive value [PPV]: 71%, negative predictive value [NPV]: 96%) and 23 WBC/µL (SE: 73.5%, SP: 84.1%, PPV: 65%, NPV: 89%). Conclusions The UF-5000 analyzer (Sysmex) is a reliable diagnostic tool for UTI screening. The displayed BACT-Info flags allow a quick diagnostic orientation for the clinician. However, the authors suggest verifying the automated Gram categories with urine culture.

Objectives The mucoprotein uromodulin is considered to correlate with glomerular filtration rates (GFR) in patients with chronic kidney disease (CKD). Here we investigated how serum uromodulin is associated with measured GFR using inulin-clearance and GFR estimated by CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) equation in healthy subjects. Methods We assessed possible correlations between uromodulin serum concentrations, inulin-GFR and CKD-EPI-GFR in a well characterized study cohort of 112 healthy living kidney donors with two kidneys before and 64 with one kidney after kidney donation. A subgroup of 32 individuals, which presented data before and after nephrectomy, was assessed separately. Results All 112 healthy living kidney donors with two kidneys revealed individual serum uromodulin concentrations between 60.1 and 450.5 µg/L. Sixty-four healthy kidney donors after nephrectomy had significantly lower median (interquartile range) serum uromodulin concentrations (124 [101–166] vs. 185 [152–238] µg/L), inulin-GFR (67.3 [60.6–74.6] vs. 93.5 [82.1–104.4] mL/min/1.73 m2), and CKD-EPI-GFR (61.2 [53.1–69.7] vs. 88.6 [80.0–97.1] mL/min/1.73 m2) as compared to the 112 donors before donation (p Conclusions These novel findings indicate that – in contrast to patients with CKD – serum uromodulin concentrations are not correlated with measured and estimated GFR in healthy individuals.

Vitamin D status is typically assessed by the measurement of 25-hydroxyvitamin D (25(OH)D). However, in selected patient groups the sole determination of 25(OH)D has been proven insufficient for this purpose. The simultaneous measurement of additional vitamin D metabolites may provide useful information for a better evaluation of the vitamin D status. Therefore, we developed and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of 25(OH)D3, 25(OH)D2, 24,25(OH)2D3 and additionally 25,26(OH)2D3, which was identified with a synthesized pure substance. Pure and deuterated substances were used to prepare calibrators and internal standards for all target metabolites. Pre-analytical sample preparation comprised protein precipitation followed by liquid-liquid-extraction and derivatization with 4-Phenyl-1,2,4-triazole-3,5-dione (PTAD) using 50 µL sample volume. Samples were analyzed on an Agilent HPLC 1260 system equipped with a silica-based Kinetex® 5 µm F5 100 A core-shell column (150 × 4.6 mm) coupled to a Sciex 4500 mass spectrometer. For all four metabolites, limit of detection (LoD) and limit of quantification (LoQ) ranged from 0.3 to 1.5 nmol/L and 1.0 to 3.1 nmol/L, respectively. Recovery varied between 76.1 % and 84.3 %. Intra- and inter-assay imprecision were

Sugars including lactose and fructose, or proteins (gluten), or biogenic amines (histamine), and combinations thereof may cause food intolerance/malabsorption. However, in usually asymptomatic patients with rare diseases, who present with functional, non-specific, non-allergic gastrointestinal (GI) complaints the etiologic factors of food intolerance/malabsorption need to be evaluated. We summarize patients with rare diseases, such as primary epiploic appendagitis, beta-thalassemias minor, Gullo syndrome and anomaly of the inferior vena cava, who presented functional, non-specific, non-allergic GI complaints. As conclusion, these GI symptoms in patients with otherwise asymptomatic, rare diseases were due to fructose malabsorption, histamine-, lactose intolerance and Helicobacter pylori (H.p.) infection. A registered and experienced dietician was employed to design an individually-tailored diet which ensured effective treatments and H.p. infection was accordingly eradicated.

Background As trimethylamine-N-oxide (TMAO) is considered to be associated with various diseases, rapid determination of serum TMAO concentration is of clinical interest. This study is aimed at evaluating the analytical performance of a simple isocratic liquid chromatography-tandem mass-spectrometry (LC-MS/MS) method for TMAO quantification. Methods TMAO measurements were performed on a tandem mass spectrometer, SCIEX QTRAP 4500 (Applied Biosystems, Framingham, MA, USA), coupled with an Agilent 1260 Infinity HPLC system (Agilent Technologies, Santa Clara, CA, USA). The separation was performed on a Hypercarb Porus Grahitic Carbon (PGC) column (ThermoFisher Scientific, Waltham, MA, USA) by isocratic elution mode. Linearity, precision, recovery, and pre-analytical requirements of the TMAO LC-MS/MS method were evaluated. The imprecision acceptance criteria were defined 15%. We investigated sample stability at room temperature (RT) and assessed the serum TMAO concentrations of 188 healthy adults. Results The TMAO LC-MS/MS method was linear over the concentration range of 0.5 - 80 μmol/L. Intra- and inter-day precision ranged between 2.24 - 3.37% and 6.95 - 9.97%, recovery between 106 - 114%, respectively. At RT, serum samples were stable for 8 days. The median serum TMAO concentration of 188 healthy adults was 2.27 μmol/L (2.5th and 97.5th percentile: 0.75 - 10.46 μmol/L), respectively. Conclusions The isocratic TMAO LC-MS/MS shows a broad analytical range and meets the imprecision acceptance criteria of 15%. This method is a robust and reliable diagnostic tool for the assessment of the human TMAO status. Serum samples are stable at RT for at least 8 days.

This work aimed at investigating demographic, clinical, and genetic characteristics of individuals experiencing their first familial Mediterranean fever (FMF) attack at age ≥40 years in a very large cohort of Armenian FMF patients. In total, 10,370 Armenian patients diagnosed with FMF based on the Tel Hashomer criteria and carrying at least one MEFV mutant allele were included in this study. A total of 354 (3.40%) patients had late-onset FMF. Of these, 194 (54.80%) were female and 160 (45.20%) were male. The following genotypes were significantly associated with the late-onset variant: M680I/E148Q (P = 0.004), M694V/E148Q (P

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.

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.

The brain’s supply with vitamin D is poorly understood. Therefore, the present study aimed to determine 25-hydroxy vitamin D3 (25(OH)D) and 24,25-dihydroxy vitamin D (24,25(OH)2D3) in serum and cerebrospinal fluid (CSF) from individuals with intact and disturbed brain-CSF-barrier (BCB) function. In 292 pairs of serum and CSF samples the vitamin D metabolites were measured with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). CSF/serum ratios (QALB, Q25(OH)D, Q24,25(OH)2D3) were calculated. Median (IQR) serum concentrations of 25(OH)D and 24,25(OH)2D3 were 63.8 (43.4–83.9) nmol/L and 4.2 (2.2–6.2) nmol/L. The CSF concentrations of both metabolites accounted for 3.7 and 3.3% of the respective serum concentrations. Serum 25(OH)D correlated inversely with Q25(OH)D and Q24,25(OH)2D3 implying a more efficient transport of both metabolites across the BCB when the serum concentration of 25(OH)D is low. In patients with BCB dysfunction, the CSF concentrations and the CSF/serum ratios of both vitamin D metabolites were higher than in individuals with intact BCB. The CSF concentrations of 25(OH)D and 24,25(OH)2D3 depend on BCB function and the respective serum concentrations of both metabolites. Higher vitamin D metabolite concentrations in CSF of patients with impaired BCB function may be due to passive diffusion across the BCB.

Food intolerance/malabsorption is caused by food ingredients, carbohydrates (mainly lactose and fructose), proteins (gluten), and biogenic amines (histamine) which cause nonspecific gastrointestinal and extra-intestinal symptoms. Here we focus on possible etiologic factors of intolerance/malabsorption especially in people with non-celiac gluten sensitivity (NCGS) or the so-called people without celiac disease avoiding gluten (PWCDAG) and histamine intolerance. Recognizing the recently described symptoms of NCGS (PWCDAG) we review correlations and parallels to histamine intolerance (HIT). We show that intestinal and extra-intestinal NCGS (PWCDAG) symptoms are very similar to those which can be found in histamine intolerance. After a detailed diagnostic workup for all possible etiologic factors in every patient, a targeted dietary intervention for single or possibly combined intolerance/malabsorption might be more effective than a short-term diet low in fermentable oligo-, di- and monosaccharides and polyols (FODMAP) or the untargeted uncritical use of gluten-free diets.

Background This study aimed to evaluate the diagnostic performance of a novel nonquantitative methylation-specific reverse hybridization (MSRH) assay to detect secreted frizzled-related protein 2 ( SFRP2) promotor methylation in fecal DNA. Methods SFRP2 promoter methylation was investigated in stool DNA isolated from 18 colorectal cancer (CRC) patients and 22 healthy controls using the MSRH assay based on methylation-specific DNA amplification followed by reverse hybridization of biotinylated amplicons to sequence-specific methylation detection probes, with MethyLight serving as a reference method. Results SFRP2 promotor methylation as determined by MSRH vs. MethyLight showed a sensitivity and specificity of 61.1% and 86.3% vs. 77.7% and 77.3%, respectively. Moderate agreement (κ = 0.54, 95% confidence interval [95% CI], 0.29-0.80, pConclusions Our findings, although limited by the small sample size, do not support the use of the MSRH assay for CRC screening in stool.

Objectives Helicobacter pylori infection is considered to alter the bacterial flora in the upper gastrointestinal tract. This study aimed at investigating the presence of small intestinal bacterial overgrowth (SIBO) in patients with active H. pylori infection assessed by functional breath testing. Design and methods A total of 109 outpatients, who were referred for the H. pylori 13C-urea breath test (13C-UBT) by general practitioners and specialists, were also tested for the presence of SIBO by the glucose hydrogen (H2)/methane (CH4) breath test (HMBT). A detailed anamnesis was carried out about the history of H. pylori infection, eradication therapies, proton pump inhibitor intake, and comorbidities. Results In total, 36/109 (33.0%) patients had a positive H. pylori 13C-UBT, and 35/109 (32.1%) patients had a positive glucose HMBT, the latter being indicative of SIBO. Interestingly, individuals with a positive H. pylori 13C-UBT were significantly more often associated with a positive glucose HMBT (p = 0.002). Cohen's κ measuring agreement between the 13C-UBT and the glucose HMBT was 0.31 (confidence intervals: 0.12–0.50) (p = 0.001). Altogether, 19 of 54 (35.2%) patients, who had completed up to four eradication therapies, were diagnosed with SIBO by HMBT. Conclusions H. pylori infection was found to be significantly associated with the presence of SIBO as determined by functional breath testing. In addition, SIBO rates appeared to have increased after completed eradication therapies. However, further longitudinal studies are warranted to fully elucidate the relationship and treatment modalities of coincident H. pylori infection and SIBO.

Introduction: The aim of this study was to assess pneumatic tube system (PTS) alteration on platelet function by the light transmission aggregometry (LTA) and whole blood aggregometry (WBA) method, and on the results of platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen. Materials and methods: Venous blood was collected into six 4.5 mL VACUETTE® 9NC coagulation sodium citrate 3.8% tubes (Greiner Bio-One International GmbH, Kremsmünster, Austria) from 49 intensive care unit (ICU) patients on dual anti-platelet therapy and immediately hand carried to the central laboratory. Blood samples were divided into 2 Groups: Group 1 samples (N = 49) underwent PTS (4 m/s) transport from the central laboratory to the distant laboratory and back to the central laboratory, whereas Group 2 samples (N = 49) were excluded from PTS forces. In both groups, LTA and WBA stimulated with collagen, adenosine-5’-diphosphate (ADP), arachidonic acid (AA) and thrombin-receptor-activated-peptide 6 (TRAP-6) as well as platelet count, PT, APTT, and fibrinogen were performed. Results: No statistically significant differences were observed between blood samples with (Group 1) and without (Group 2) PTS transport (P values from 0.064 – 0.968). The AA-induced LTA (bias: 68.57%) exceeded the bias acceptance limit of ? 25%. Conclusions: Blood sample transportation with computer controlled PTS in our hospital had no statistically significant effects on platelet aggregation determined in patients with anti-platelet therapy. Although AA induced LTA showed a significant bias, the diagnostic accuracy was not influenced.

Diagnosis of histamine intolerance (HIT) has been based on low serum diamine oxidase (DAO) values, functional gastrointestinal disorders and improvement of symptoms with a histamine-reduced diet (HRD). In a retrospective analysis of outpatients' charts we identified 101 patients with HIT. After a median of 13 months, a questionnaire was distributed to the patients so that they could be classified into four diet-compliance groups. Calculated with all 101 patients we found an increase of serum DAO values due to a HRD. In the 63 patients that completed the questionnaire, we found that 50 patients had improvement of symptoms or no continuing symptoms. A significant increase of serum DAO levels was found in the patients with strict and occasional diet compliance. Therefore, we demonstrate that a HRD is not only improving symptoms in HIT, but is causing an increase in serum DAO values that correlates with the degree of diet compliance.

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.

Background Recently, several manufacturers have launched automated urinalysis platforms. This study aimed to compare the diagnostic performance of the UF-5000 (Sysmex Corporation, Kobe, Japan) and the cobas® u 701 (Roche Diagnostics, Rotkreuz, Switzerland) urine sediment analyzers with manual phase-contrast microscopy as the reference method. Methods A total of 195 urine samples were analyzed on both automated platforms and subjected to manual microscopic examination. Agreement was assessed by Cohen’s kappa (κ) analysis. Sensitivities and specificities were calculated. Results The agreement of the UF-5000 with manual microscopy was almost perfect (κ > 0.8) for red (RBC) and white blood cells (WBC), renal tubular epithel cells, hyaline casts, bacteria (BACT) and yeast (YLC), substantial (κ = 0.61–0.80) for squamous epithel cells (SEC) and pathologic casts, and moderate (κ = 0.41–0.60) for transitional epithel cells. The cobas® u 701 showed substantial agreement (κ = 0.61–0.80) for WBC, moderate agreement (κ = 0.41–0.60) for hyaline casts, and fair agreement (κ = 0.21–0.40) for RBC, SEC, non-squamous epithel (NEC), pathologic casts, BACT and YLC. The UF-5000 sensitivities ranged between 98.5% for RBC and 83.3% for pathological casts. The cobas® u 701 showed sensitivities between 83.0% for WBC and 31.6% for YLC. Conclusions The UF-5000 (Sysmex) analyzer showed a better diagnostic agreement with manual phase-contrast microscopy compared to the cobas® u 701 (Roche) module. The Sysmex platform showed reliable results for urine sediment analysis. However, pathological samples should be verified with manual microscopy.

Healthy human blood contains only a trace amount of methemoglobin (Hb M), less than 1%. In Hb M iron is present in the oxidized ferric state (Fe3+) not in the reduced ferrous form (Fe2+) and this reduces the ability of hemoglobin to bind oxygen. The described rare hemoglobin variant Hb M‑Hyde Park (also known as Hb M-Akita) results from the substitution of amino acid tyrosine by histidine at position 93 of the beta-globin chain of hemoglobin. The rare Hb variant Hb M‑Hyde Park (Hb M‑Akita) is mainly inherited autosomal dominant and causes methemoglobinemia. Due to the low frequency of inherited Hb M variants, the diagnosis is challenging. Here, we here report on a family with Hb M‑Hyde Park (Hb M‑Akita) whose members demonstrated Hb M > 10%, but were, asymptomatic except for chronic cyanosis. Due to human mobility and migration other hemogobin variants, such as beta-thalassemia minor have spread to Austria . A genetic combination of two different hemoglobin variants may result in severe anemia. Genetic counseling for patients with hemoglobin variants, including Hb M‑Hyde Park (Hb M‑Akita) and beta-thalassemia minor, is essential.

Background and pilot study Celiac disease (CD) or gluten malabsorption is a well-defined autoimmune disorder characterized by mucosal gastrointestinal reaction to ingested gluten proteins. The necessary treatment for CD is a gluten-free diet. However, up to 30% of celiac patients experience persistent or recurring abdominal complaints despite following an exact gluten-free diet. This condition was named refractory, non-responsive celiac disease. Other food ingredients, such as carbohydrates and biogenic amines, also influence and impair digestion, and may cause these abdominal symptoms. In this retrospective pilot study, we have reported on 20 non-responsive, celiac disease patients, with persistent abdominal complaints, for longer than 6 months. These patients were evaluated for extra food intolerance/malabsorption, including fructose malabsorption, histamine-, lactose intolerance, and Helicobacter pylori (H.p.) infection. Results and conclusions The results demonstrate that 18 of the 20 refractory, non-responsive celiac disease patients presented various, additional food intolerance/malabsorption and/or H.p. infection. Seven NRCD patients demonstrated lactose intolerance, 7 showed fructose malabsorption, 11 had additional histamine intolerance and 6 had signs of H.p. infection or combinations thereof. If present, then eradication of H.p. was performed. Histamine intolerance, was found in more than 50% of patients, and it seems to play an important role in non-responsive celiac disease. A registered dietician continued to help with, and to improve, the patients’ gluten-free diet. Furthermore, additional food intolerance/malabsorption considerations were included in the individual, dietary recommendations.

Cardiovascular risk is increased in obese subjects. Nevertheless, some overweight and obese remain cardiometabolically healthy (CMH), and normal-weight persons develop cardiovascular disease (CVD). Herein, we investigate the potential of branched-chain amino acids (BCAAs) to identify an increased CVD risk in a cross-sectional study of 666 adults and juveniles (age 25.3±12.8years), classified as lean, overweight or obese. Cardiometabolic groups were defined by cutoffs of systolic blood pressure130mmHg, diastolic blood pressure85mmHg, glucose125mg/dl, triglycerides150mg/dl, HDL-cholesterol40mg/dl (males), HDL-cholesterol50mg/dl (females) and HOMA-IR5. CMH had ≤1 cutoff, and cardiometabolically abnormal (CMA) had ≥2 cutoffs. Amino acids were measured by high-pressure lipid chromatography after precipitation of serum with perchloric acid and derivatization with o-phthalaldehyde. Valine correlated with 5, leucine correlated with 3 and isoleucine correlated with 5 of the cardiac risk classification factors. Valine and leucine were significantly higher in the obese (P.001, P=.015, respectively), overweight (P.001, P=.015, respectively) and lean (P=.024, P=.012, respectively) CMA compared to CMH subjects. Isoleucine showed except of the lean group the same results. Taken together, BCAAs, especially valine and leucine, are proposed as a cardiometabolic risk marker independent of body mass index (BMI) category.

BACKGROUND: The branched-chain amino acids (BCAAs) valine, leucine and isoleucine are reported to influence erythropoiesis and the human iron status. Large study cohorts encompassing biomarkers of iron metabolism and BCAAs are still lacking. METHODS: We investigated potential interactions between blood concentrations of all three BCAAs valine, leucine and isoleucine and biomarkers of iron metabolism [i.e., hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), iron, transferrin, ferritin, transferrin saturation, soluble transferrin receptor (sTfR)] in 430 outpatients referred for a medical health check-up. Linear regression models were performed to assess possible associations between variables. RESULTS: All three BCAAs were positively correlated with Hb, ferritin and the sTfR (r-values: 0.145–0.382; P values

Background Branched-chain amino acids (BCAAs) are reported to influence erythropoiesis and the human iron status. This study is aimed at investigating potential interactions between blood concentrations of all three BCAAs valine, leucine and isoleucine and indicators of human iron status. Methods Overall, 430 outpatients referred for a medical health check-up were included in this study. They underwent investigation of valine, leucine and isoleucine and biomarkers of iron metabolism (i.e., hemoglobin [Hb], mean corpuscular volume [MCV], mean corpuscular hemoglobin [MCH], iron, transferrin, ferritin, transferrin saturation, soluble transferrin receptor [sTfR]). Linear regression models were performed to assess possible associations between variables. Results All three BCAAs were positively correlated with Hb, ferritin and the sTfR (r-values: 0.145 – 0.382; P-values: Conclusions These data indicate a pathophysiological link between the three BCAAs valine, leucine and isoleucine and the human iron indicators hemoglobin and ferritin. Anemic individuals had significantly lowered serum BCAA concentrations compared to non-anemic subjects and lowered BCAA concentrations were observed with lowered ferritin levels.

Statin-induced myopathy is reported to be significantly associated with the SCLO1B1 c.521TC polymorphism. To date, SLCO1B1 c.521Tgt;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.521Tgt;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.521Tgt;C polymorphism in a comparative manner using the SLCO1B1 c.521Tgt;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.521Tgt;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.521Tgt;C genotyping tools in clinical routine.