Neutrophil extracellular trap biomarkers in aneurysmal subarachnoid hemorrhage: early decline of DNase 1 activity associated with delayed cerebral ischemia.

Introduction: High-mobility group box 1 (HMGB1) protein is a critical mediator of neutrophil extracellular trap (NET) formation (NETosis). Myeloperoxidase (MPO)-DNA complexes, a biomarker of NETs, and HMGB1 have been associated with delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (aSAH). Additional mechanistic NET-related biomarkers and their role in the neuroinflammatory cascade surrounding DCI remain to be explored. Methods: A post-hoc analysis of a prospective, blinded, single-center biomarker observational study was performed. De novo measurements of serum citrullinated histone H3-DNA complexes (H3Cit-DNA), peptidylarginine deiminase 4 (PAD4), cell-free DNA (cf-DNA), and DNase 1 activity were conducted on admission (D0) and day 4 (D4). Delayed cerebral infarction (DCI) was defined as new cerebral infarction on CT head not present on the post-treatment scan. Results: H3Cit-DNA, PAD4, cf-DNA, and DNase 1 activity were within quantifiable ranges in all serum samples analyzed at D0 and D4. Admission biomarker levels were not associated with DCI development. From D0 to D4, in both the DCI and the non-DCI groups, H3Cit-DNA levels significantly decreased, cf-DNA levels significantly increased, and PAD4 levels remained stable. In contrast, DNase 1 activity significantly decreased from D0 to D4 in the DCI group (p < 0.001) but not in the non-DCI group. Conclusion: This exploratory analysis demonstrated NET-related biomarkers such as H3Cit-DNA, PAD4, cf-DNA, and DNase 1 activity in all aSAH patients. A decline of systemic DNase 1 activity in the early phase might increase the risk of delayed cerebral ischemia.

Plasma Concentrations of Trimethylamine-N-Oxide, Choline, and Betaine in Patients With Moderate to Advanced Chronic Kidney Disease and Their Relation to Cardiovascular and Renal Outcomes.

OBJECTIVES: Trimethylamine N-oxide (TMAO) is a gut bacteria-mediated liver metabolite of dietary betaine, choline, and carnitine, which is excreted by glomerular filtration. We studied whether TMAO is excreted by cardiovascular disease (CVD) in patients with chronic kidney disease (CKD). METHODS: Among 478 patients with CKD stage G2 (n = 104), G3a (n = 163), G3b (n = 123), and G4 (n = 88), we studied the association between fasting plasma concentrations of TMAO, choline, or betaine at baseline and kidney function, prevalent CVD, and future renal outcomes during a mean follow-up of 5.1 years. RESULTS: Decreased glomerular filtration rate was associated with higher plasma concentrations of TMAO, choline, and betaine. Baseline concentrations of TMAO were higher in participants with preexisting CVD compared to those without CVD (8.4 [10.1] vs. 7.8 [8.0] µmol/L; P = .047), but the difference was not significant after adjusting for confounders. During the follow-up, 147 participants experienced CVD or died, and 144 reached the predefined renal endpoint. In the adjusted regression analyses, TMAO or choline concentrations in the upper three quartiles (vs. the lowest quartile) were not associated with any of the study's clinical endpoints. In contrast, the adjusted hazard ratio of plasma betaine in the highest quartile versus the lowest quartile was 2.14 (1.32, 3.47) for the CVD endpoint and 1.64 (1.00, 2.67) for the renal endpoint. CONCLUSIONS: Elevated plasma TMAO concentrations were explained by impaired kidney function. Elevated plasma concentrations of betaine, but not those of TMAO or choline, constituted a risk factor for adverse outcomes. TMAO might not be an appropriate target to reduce CVD or renal outcomes in patients with preexisting CKD.

ELOVL2-methylation and renal and cardiovascular event in patients with chronic kidney disease.

BACKGROUND: Methylation of the Elongation Of Very Long Chain Fatty Acids-Like 2 (ELOVL2) gene promoter may predict premature ageing and cardiovascular risk. METHODS: We studied the cross-sectional associations between blood ELOVL2-methylation and cardiovascular risk factors in 350 patients with chronic kidney disease (CKD) stage G2-G4 aged between 22 and 90 years. In a follow-up study for a mean of 3.9 years, we investigated the association between baseline ELOVL2-methylation and renal or cardiovascular events including death. RESULTS: ELOVL2-methylation at seven CpG cites increased with age (the correlation coefficients between 0.67 and 0.87, p < 0.001). The ELOVL2-CpGs methylation was lower in patients with CKD stage G2 versus those in stage G3a, G3b and G4, but the differences were explained by age. ELOVL2-CpGs methylation showed no correlations with cardiovascular risk factors after adjusting for age. During the follow-up, 64 patients showed deterioration in renal function or died and 77 showed cardiovascular events or died. The hazard ratio and 95% confidence intervals for renal or cardiovascular events according to baseline ELOVL2-CpGs methylation were not significant after adjustment for covariates. CONCLUSIONS: ELOVL2-hypermethylation showed a strong association with age, but was not independently associated with cardiovascular risk factors or with future renal or cardiovascular events in patients with CKD. ELOVL2 gene methylation is not likely to be itself a cause for ageing or illnesses, but it could be rather influenced by other upstream processes that deserve investigation.

Expression of TRAIL, IP-10, and CRP in children with suspected COVID-19 and real-life impact of a computational signature on clinical decision-making: a prospective cohort study.

PURPOSE: We evaluated the host-response marker score "BV" and its components TRAIL, IP-10, and CRP in SARS-CoV-2 positive children, and estimated the potential impact on clinical decision-making. METHODS: We prospectively analyzed levels of TRAIL, IP-10, CRP, and the BV score, in children with suspected COVID-19. Classification of infectious etiology was performed by an expert panel. We used a 5-point-questionnaire to evaluate the intention to treat with antibiotics before and after receiving test results. RESULTS: We screened 111 children, of whom 6 (5.4%) were positive for SARS-CoV-2. A total of 53 children were included for the exploratory analysis. Median age was 3.1 years (interquartile range [IQR] 1.3-4.3), and 54.7% (n = 29) were girls. A viral and a bacterial biomarker pattern was found in 27/53 (50.9%) and 15/53 (28.3%), respectively. BV scores differed between COVID-19, children with other viral infections, and children with bacterial infections (medians 29.5 vs. 9 vs. 66; p = 0.0006). Similarly, median TRAIL levels were different (65.5 vs. 110 vs. 78; p = 0.037). We found no differences in IP-10 levels (555 vs. 504 vs. 285; p = 0.22). We found a concordance between physicians' "unlikely intention to treat" children with a viral test result in most cases (n = 19/24, 79.2%). When physicians expressed a "likely intention to treat" (n = 15), BV test revealed 5 bacterial, viral, and equivocal scores each. Antibiotics were withheld in three cases (20%). Overall, 27/42 (64%) of pediatricians appraised the BV test positively, and considered it helpful in clinical practice. CONCLUSION: Host-response based categorization of infectious diseases might help to overcome diagnostic uncertainty, support clinical decision-making and reduce unnecessary antibiotic treatment.

Concentrations of Soluble Angiotensin Converting Enzyme 2 (sACE2) in Children and Adults with and without COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the coronavirus disease 2019 (COVID-19) pandemic, leads to illness and death. Various risk factors for a severe course, such as higher age, male gender and pre-existing illnesses are known. However, pathophysiological risk factors are largely unclear. Notably, the mild course of disease in children is conspicuous. Angiotensin converting enzyme 2 (ACE2) serves as a receptor for SARS-CoV-2 and is a key enzyme in infection. Differences in the distribution of ACE2 can provide insights into different courses of COVID-19. Our aim was to elucidate the role of ACE2 as a pathophysiological risk factor by measuring soluble ACE2 (sACE2) via ELISA in blood samples (lithium-heparin-plasma or serum) of 367 individuals including children and adults with and without COVID-19. sACE2-levels were compared between the groups according to age and sex. In adults and children with COVID-19, sACE2-concentrations are significantly higher compared to healthy individuals. sACE2-levels increase with age and are lower in children compared to adults with COVID-19. Sex doesn't significantly influence sACE2-concentration. It remains unclear whether sACE2 concentrations increase because of the infection and what factors could influence this response. In conclusion, the increase of sACE2-concentration with age could indicate that ACE2 concentrations mirror increased COVID-19 severity.

TNF-related apoptosis-inducing ligand, interferon gamma-induced protein 10, and C-reactive protein in predicting the progression of SARS-CoV-2 infection: a prospective cohort study.

BACKGROUND: Early prognostication of COVID-19 severity will potentially improve patient care. Biomarkers, such as TNF-related apoptosis-inducing ligand (TRAIL), interferon gamma-induced protein 10 (IP-10), and C-reactive protein (CRP), might represent possible tools for point-of-care testing and severity prediction. METHODS: In this prospective cohort study, we analyzed serum levels of TRAIL, IP-10, and CRP in patients with COVID-19, compared them with control subjects, and investigated the association with disease severity. RESULTS: A total of 899 measurements were performed in 132 patients (mean age 64 years, 40.2% females). Among patients with COVID-19, TRAIL levels were lower (49.5 vs 87 pg/ml, P = 0.0142), whereas IP-10 and CRP showed higher levels (667.5 vs 127 pg/ml, P <0.001; 75.3 vs 1.6 mg/l, P <0.001) than healthy controls. TRAIL yielded an inverse correlation with length of hospital and intensive care unit (ICU) stay, Simplified Acute Physiology Score II, and National Early Warning Score, and IP-10 showed a positive correlation with disease severity. Multivariable regression revealed that obesity (adjusted odds ratio [aOR] 5.434, 95% confidence interval [CI] 1.005-29.38), CRP (aOR 1.014, 95% CI 1.002-1.027), and peak IP-10 (aOR 1.001, 95% CI 1.00-1.002) were independent predictors of in-ICU mortality. CONCLUSIONS: We demonstrated a correlation between COVID-19 severity and TRAIL, IP-10, and CRP. Multivariable regression showed a role for IP-10 in predicting unfavourable outcomes, such as in-ICU mortality. TRIAL REGISTRATION: Clinicaltrials.gov, NCT04655521.

German federal-state-wide seroprevalence study of 1st SARS-CoV-2 pandemic wave shows importance of long-term antibody test performance.

Background: Reliable data on the adult SARS-CoV-2 infection fatality rate in Germany are still scarce. We performed a federal state-wide cross-sectional seroprevalence study named SaarCoPS, that is representative for the adult population including elderly individuals and nursing home residents in the Saarland. Methods: Serum was collected from 2940 adults via stationary or mobile teams during the 1st pandemic wave steady state period. We selected an antibody test system with maximal specificity, also excluding seroreversion effects due to a high longitudinal test performance. For the calculations of infection and fatality rates, we accounted for the delays of seroconversion and death after infection. Results: Using a highly specific total antibody test detecting anti-SARS-CoV-2 responses over more than 180 days, we estimate an adult infection rate of 1.02% (95% CI: [0.64; 1.44]), an underreporting rate of 2.68-fold (95% CI: [1.68; 3.79]) and infection fatality rates of 2.09% (95% CI: (1.48; 3.32]) or 0.36% (95% CI: [0.25; 0.59]) in all adults including elderly individuals, or adults younger than 70 years, respectively. Conclusion: The study highlights the importance of study design and test performance for seroprevalence studies, particularly when seroprevalences are low. Our results provide a valuable baseline for evaluation of future pandemic dynamics and impact of public health measures on virus spread and human health in comparison to neighbouring countries such as Luxembourg or France.

Neutrophil Extracellular Traps and Delayed Cerebral Ischemia in Aneurysmal Subarachnoid Hemorrhage.

IMPORTANCE: Myeloperoxidase (MPO)-DNA complexes, biomarkers of neutrophil extracellular traps (NETs), have been associated with arterial and venous thrombosis. Their role in aneurysmal subarachnoid hemorrhage (aSAH) is unknown. OBJECTIVES: To assess whether serum MPO-DNA complexes are present in patients with aSAH and whether they are associated with delayed cerebral ischemia (DCI). DESIGN SETTING AND PARTICIPANTS: Post-hoc analysis of a prospective, observational single-center study, with de novo serum biomarker measurements in consecutive patients with aSAH between July 2018 and September 2020, admitted to a tertiary care neuroscience ICU. MAIN OUTCOMES AND MEASURES: We analyzed serum obtained at admission and hospital day 4 for concentrations of MPO-DNA complexes. The primary outcome was DCI, defined as new infarction on brain CT. The secondary outcome was clinical vasospasm, a composite of clinical and transcranial Doppler parameters. We used Wilcoxon signed-rank-test to assess for differences between paired measures. RESULTS: Among 100 patients with spontaneous subarachnoid hemorrhage, mean age 59 years (sd ± 13 yr), 55% women, 78 had confirmed aSAH. Among these, 29 (37%) developed DCI. MPO-DNA complexes were detected in all samples. The median MPO-DNA level was 33 ng/mL (interquartile range [IQR], 18-43 ng/mL) at admission, and 22 ng/mL (IQR, 11-31 ng/mL) on day 4 (unpaired test; p = 0.015). We found a significant reduction in MPO-DNA levels from admission to day 4 in patients with DCI (paired test; p = 0.036) but not in those without DCI (p = 0.17). There was a similar reduction in MPO-DNA levels between admission and day 4 in patients with (p = 0.006) but not in those without clinical vasospasm (p = 0.47). CONCLUSIONS AND RELEVANCE: This is the first study to detect the NET biomarkers MPO-DNA complexes in peripheral serum of patients with aSAH and to associate them with DCI. A pronounced reduction in MPO-DNA levels might serve as an early marker of DCI. This diagnostic potential of MPO-DNA complexes and their role as potential therapeutic targets in aSAH should be explored further.

Admission serum high mobility group box 1 (HMGB1) protein predicts delayed cerebral ischemia following aneurysmal subarachnoid hemorrhage.

High mobility group box 1 protein (HMGB1) is a prototypical damage associated particle and acts as a key player in aseptic inflammation. HMGB1 appears critical for the crosstalk of a prothrombotic and proinflammatory state that is implicated in mediating and exacerbating ischemic brain injury. The role of HMGB1 in aneurysmal subarachnoid hemorrhage (aSAH) remains to be elucidated. A prospective, single blinded observational study was designed to investigate the role of HMGB1 in aSAH. Serial serum HMGB1 level quantification on admission day 0, 4, 8, and 12 was performed. Primary outcome measures were delayed cerebral ischemia (DCI - new infarction on CT) and poor functional outcome (90-day modified Rankin Scale 4-6). The role of HMGB1 levels for DCI, functional outcome and radiological vasospasm prediction was analyzed. Collectively, 83 aSAH patients were enrolled. Five patients died within 48 h. In 29/78 patients (37.2%), DCI was identified. In multivariable analysis, radiological vasospasm and admission HMGB1 were independent predictors for DCI. Younger age and higher white blood cell count, but not insult burden (World Federation of Neurosurgical Societies scale, modified Fisher scale, intraparenchymal or intraventricular hematoma existence) correlated with admission HMGB1 levels. Serial HMGB1 levels did not differ between patients with or without DCI, poor functional outcome or radiological vasospasm development. Admission serum HMGB1 does not reflect initial insult burden but serves as an independent biomarker predictive of DCI. Further studies are warranted to disentangle the role of HMGB1 surrounding the sequelae of aSAH.

Combined antibiotic stewardship and infection control measures to contain the spread of linezolid-resistant Staphylococcus epidermidis in an intensive care unit.

BACKGROUND: The unrestricted use of linezolid has been linked to the emergence of linezolid-resistant Staphylococcus epidermidis (LRSE). We report the effects of combined antibiotic stewardship and infection control measures on the spread of LRSE in an intensive care unit (ICU). METHODS: Microbiological data were reviewed to identify all LRSE detected in clinical samples at an ICU in southwest Germany. Quantitative data on the use of antibiotics with Gram-positive coverage were obtained in defined daily doses (DDD) per 100 patient-days (PD). In addition to infection control measures, an antibiotic stewardship intervention was started in May 2019, focusing on linezolid restriction and promoting vancomycin, wherever needed. We compared data from the pre-intervention period (May 2018-April 2019) to the post-intervention period (May 2019-April 2020). Whole-genome sequencing (WGS) was performed to determine the genetic relatedness of LRSE isolates. RESULTS: In the pre-intervention period, LRSE were isolated from 31 patients (17 in blood cultures). The average consumption of linezolid and daptomycin decreased from 7.5 DDD/100 PD and 12.3 DDD/100 PD per month in the pre-intervention period to 2.5 DDD/100 PD and 5.7 DDD/100 PD per month in the post-intervention period (p = 0.0022 and 0.0205), respectively. Conversely, vancomycin consumption increased from 0.2 DDD/100 PD per month to 4.7 DDD/100 PD per month (p < 0.0001). In the post-intervention period, LRSE were detected in 6 patients (4 in blood cultures) (p = 0.0065). WGS revealed the predominance of one single clone. CONCLUSIONS: Complementing infection control measures by targeted antibiotic stewardship interventions was beneficial in containing the spread of LRSE in an ICU.

The Erythrocyte Sedimentation Rate and Its Relation to Cell Shape and Rigidity of Red Blood Cells from Chorea-Acanthocytosis Patients in an Off-Label Treatment with Dasatinib.

BACKGROUND: Chorea-acanthocytosis (ChAc) is a rare hereditary neurodegenerative disease with deformed red blood cells (RBCs), so-called acanthocytes, as a typical marker of the disease. Erythrocyte sedimentation rate (ESR) was recently proposed as a diagnostic biomarker. To date, there is no treatment option for affected patients, but promising therapy candidates, such as dasatinib, a Lyn-kinase inhibitor, have been identified. METHODS: RBCs of two ChAc patients during and after dasatinib treatment were characterized by the ESR, clinical hematology parameters and the 3D shape classification in stasis based on an artificial neural network. Furthermore, mathematical modeling was performed to understand the contribution of cell morphology and cell rigidity to the ESR. Microfluidic measurements were used to compare the RBC rigidity between ChAc patients and healthy controls. RESULTS: The mechano-morphological characterization of RBCs from two ChAc patients in an off-label treatment with dasatinib revealed differences in the ESR and the acanthocyte count during and after the treatment period, which could not directly be related to each other. Clinical hematology parameters were in the normal range. Mathematical modeling indicated that RBC rigidity is more important for delayed ESR than cell shape. Microfluidic experiments confirmed a higher rigidity in the normocytes of ChAc patients compared to healthy controls. CONCLUSIONS: The results increase our understanding of the role of acanthocytes and their associated properties in the ESR, but the data are too sparse to answer the question of whether the ESR is a suitable biomarker for treatment success, whereas a correlation between hematological and neuronal phenotype is still subject to verification.

Acanthocyte Sedimentation Rate as a Diagnostic Biomarker for Neuroacanthocytosis Syndromes: Experimental Evidence and Physical Justification.

(1) Background: Chorea-acanthocytosis and McLeod syndrome are the core diseases among the group of rare neurodegenerative disorders called neuroacanthocytosis syndromes (NASs). NAS patients have a variable number of irregularly spiky erythrocytes, so-called acanthocytes. Their detection is a crucial but error-prone parameter in the diagnosis of NASs, often leading to misdiagnoses. (2) Methods: We measured the standard Westergren erythrocyte sedimentation rate (ESR) of various blood samples from NAS patients and healthy controls. Furthermore, we manipulated the ESR by swapping the erythrocytes and plasma of different individuals, as well as replacing plasma with dextran. These measurements were complemented by clinical laboratory data and single-cell adhesion force measurements. Additionally, we followed theoretical modeling approaches. (3) Results: We show that the acanthocyte sedimentation rate (ASR) with a two-hour read-out is significantly prolonged in chorea-acanthocytosis and McLeod syndrome without overlap compared to the ESR of the controls. Mechanistically, through modern colloidal physics, we show that acanthocyte aggregation and plasma fibrinogen levels slow down the sedimentation. Moreover, the inverse of ASR correlates with the number of acanthocytes (R2=0.61, p=0.004). (4) Conclusions: The ASR/ESR is a clear, robust and easily obtainable diagnostic marker. Independently of NASs, we also regard this study as a hallmark of the physical view of erythrocyte sedimentation by describing anticoagulated blood in stasis as a percolating gel, allowing the application of colloidal physics theory.

Antibiotic therapeutic drug monitoring in intensive care patients treated with different modalities of extracorporeal membrane oxygenation (ECMO) and renal replacement therapy: a prospective, observational single-center study.

BACKGROUND: Effective antimicrobial treatment is key to reduce mortality associated with bacterial sepsis in patients on intensive care units (ICUs). Dose adjustments are often necessary to account for pathophysiological changes or renal replacement therapy. Extracorporeal membrane oxygenation (ECMO) is increasingly being used for the treatment of respiratory and/or cardiac failure. However, it remains unclear whether dose adjustments are necessary to avoid subtherapeutic drug levels in septic patients on ECMO support. Here, we aimed to evaluate and comparatively assess serum concentrations of continuously applied antibiotics in intensive care patients being treated with and without ECMO. METHODS: Between October 2018 and December 2019, we prospectively enrolled patients on a pneumological ICU in southwest Germany who received antibiotic treatment with piperacillin/tazobactam, ceftazidime, meropenem, or linezolid. All antibiotics were applied using continuous infusion, and therapeutic drug monitoring of serum concentrations (expressed as mg/L) was carried out using high-performance liquid chromatography. Target concentrations were defined as fourfold above the minimal inhibitory concentration (MIC) of susceptible bacterial isolates, according to EUCAST breakpoints. RESULTS: The final cohort comprised 105 ICU patients, of whom 30 were treated with ECMO. ECMO patients were significantly younger (mean age: 47.7 vs. 61.2 years; p < 0.001), required renal replacement therapy more frequently (53.3% vs. 32.0%; p = 0.048) and had an elevated ICU mortality (60.0% vs. 22.7%; p < 0.001). Data on antibiotic serum concentrations derived from 112 measurements among ECMO and 186 measurements from non-ECMO patients showed significantly lower median serum concentrations for piperacillin (32.3 vs. 52.9; p = 0.029) and standard-dose meropenem (15.0 vs. 17.8; p = 0.020) in the ECMO group. We found high rates of insufficient antibiotic serum concentrations below the pre-specified MIC target among ECMO patients (piperacillin: 48% vs. 13% in non-ECMO; linezolid: 35% vs. 15% in non-ECMO), whereas no such difference was observed for ceftazidime and meropenem. CONCLUSIONS: ECMO treatment was associated with significantly reduced serum concentrations of specific antibiotics. Future studies are needed to assess the pharmacokinetic characteristics of antibiotics in ICU patients on ECMO support.

Vitamin B12-fortified toothpaste improves vitamin status in elderly people: a randomized, double-blind, placebo-controlled study.

BACKGROUND: Elderly people are at risk for vitamin B12 deficiency. AIMS: We studied the ability of vitamin B12-enriched toothpaste vs. placebo to increase vitamin B12 status in elderly subjects. METHODS: We conducted a randomized double-blind placebo-controlled intervention in 103 elderly subjects. Serum concentrations of vitamin B12, holotranscobalamin (holoTC), methylmalonic acid (MMA), and plasma total homocysteine (tHcy) were measured at baseline and after 3 months. RESULTS: 92 subjects met the inclusion criteria, completed the 3 months study, and were included in the data analysis. After the intervention, concentrations of vitamin B12 were higher [mean (SD) = 368 (123) vs. 295 (123) pmol/L; p = 0.005] and holoTC tended to be higher [112 (48) vs. 91 (68) pmol/L; p = 0.088] in the vitamin B12 group compared with the placebo group. The changes of serum vitamin B12 [54 (74) vs. 3 (60) pmol/L, p < 0.001], holoTC [21 (34) vs. 2 (32) pmol/L, p = 0.007], and tHcy [- 0.9 (2.3) vs. 0.3 (1.9) µmol/L, p = 0.010] were significantly different between the intervention groups. Mean percentage increase of serum vitamin B12 (+ 23% corresponds to + 54 pmol/L) in the vitamin B12 toothpaste group suggests that the intervention had provided an additional daily intake of approximately + 7 µg oral B12. Common diseases and drugs did not predict the change of blood markers in the vitamin group. No side effects were observed. CONCLUSIONS: The toothpaste enriched with 100 µg cyanocobalamin/g has increased vitamin B12 status and can thus be used for preventing vitamin B12 depletion in elderly people. The trial was registered at ClinicalTrials.gov: NCT02679833.

Nucleated red blood cells and serum lactate values on days 2 and 5 are associated with mortality and morbidity in VLBW infants.

AIM: To correlate nucleated red blood cell counts and serum lactate concentrations on day 2 and 5 of life with morbidity and mortality in very low birth weight infants and to determine corresponding cutoff values. METHODS: Retrospective analysis in a cohort of very low birth weight infants. RESULTS: 250 very low birth weight infants were included in this study. Gestational age ranged from 23 to 35 weeks (mean 29.04) and birth weight was 320-1500 g (mean 1047.9). 55 (22%) patients developed intraventricular hemorrhage, 55 (22%) bronchopulmonary dysplasia, 12 (4.8%) periventricular leukomalacia, 93 (37.2%) retinopathy of prematurity, and 1 (0.4%) necrotizing enterocolitis. Mortality rate was 25/250 (10%). Nucleated red blood cells and serum lactate on day 2 of life were associated with mortality (p < 0.001). Serum lactate on day 5 of life demonstrated an association with retinopathy of prematurity (p = 0.017), bronchopulmonary dysplasia (p = 0.044), and intraventricular hemorrhage (p < 0.001). Cutoff values predicting mortality were >89.5 nucleated red blood cells/100 leucocytes (sensitivity 68.2%, specificity 89.0%) and serum lactate concentrations >8.5 mmol/l (sensitivity 69.6%, specificity 93.5%) on day 2 of life. CONCLUSION: We conclude that both nucleated red blood cell count and serum lactate concentration are valuable biomarkers in predicting important outcome parameters in very low birth weight infants.

Glucose and Fat Tolerance Tests Induce Differential Responses in Plasma Choline Metabolites in Healthy Subjects.

Plasma choline shows associations with plasma glucose and lipids. We studied changes of choline metabolites after oral glucose tolerance test (OGTT) and fat tolerance test (OFTT). Eighteen healthy subjects (mean age 54.3 years; BMI 26.8 kg/m²) underwent 2 tests. First, OFTT (80 g fat) was applied and blood was collected at baseline and 4 h after OFTT. Seven days later, 75 g glucose was applied and blood was collected at baseline and 2 h after OGTT. Plasma concentrations of choline, betaine, trimethylamine N-oxide (TMAO), dimethylglycine, S-adenosylmethionine (SAM), lipids and glucose were measured. After OFTT, plasma choline declined (10.6 to 9.2 µmol/L; p = 0.004), betaine declined (33.4 to 31.7 µmol/L; p = 0.003), TMAO slightly increased (4.1 to 5.6 µmol/L; p = 0.105), glucose declined (5.39 to 4.98 mmol/L; p < 0.001), and triglycerides increased (1.27 to 2.53 mmol/L; p < 0.001). After OGTT, plasma choline increased (10.1 to 11.1 µmol/L; p < 0.001), TMAO declined (4.0 to 3.5 µmol/L; p = 0.029), dimethylglycine declined (2.0 to 1.7 µmol/L; p = 0.005), SAM declined (103 to 96 nmol/L; p = 0.041), but betaine, glucose, and SAM were unchanged. In conclusion, OFTT lowered plasma betaine and choline and caused heterogeneous changes in plasma TMAO. OGTT reduced the flow of methyl groups and plasma TMAO.

Selenium increases hepatic DNA methylation and modulates one-carbon metabolism in the liver of mice.

The average intake of the essential trace element selenium (Se) is below the recommendation in most European countries, possibly causing sub-optimal expression of selenoproteins. It is still unclear how a suboptimal Se status may affect health. To mimic this situation, mice were fed one of three physiologically relevant amounts of Se. We focused on the liver, the organ most sensitive to changes in the Se supply indicated by hepatic glutathione peroxidase activity. In addition, liver is the main organ for synthesis of methyl groups and glutathione via one-carbon metabolism. Accordingly, the impact of Se on global DNA methylation, methylation capacity, and gene expression was assessed. We observed higher global DNA methylation indicated by LINE1 methylation, and an increase of the methylation potential as indicated by higher S-adenosylmethionine (SAM)/S-adenosylhomocysteine (SAH) ratio and by elevated mRNA expression of serine hydroxymethyltransferase in both or either of the Se groups. Furthermore, increasing the Se supply resulted in higher plasma concentrations of triglycerides. Hepatic expression of glycolytic and lipogenic genes revealed consistent Se-dependent up-regulation of glucokinase. The sterol regulatory element-binding transcription factor 1 (Srebf1) was also up-regulated by Se. Both effects were confirmed in primary hepatocytes. In contrast to the overall Se-dependent increase of methylation capacity, the up-regulation of Srebf1 expression was paralleled by reduced local methylation of a specific CpG site within the Srebf1 gene. Thus, we provided evidence that Se-dependent effects on lipogenesis involve epigenetic mechanisms.

Reference values for nucleated red blood cells and serum lactate in very and extremely low birth weight infants in the first week of life.

STUDY PURPOSE: To provide reference values for nucleated red blood cells and serum lactate concentrations in very and extremely low birth weight (VLBW/ELBW) infants in the first week of life. PATIENTS AND METHODS: Retrospective data analysis of serial, daily measurements of NRBC counts and serum lactate during the first 6days of life in VLBW and ELBW infants. RESULTS: In total, 250 infants<1500g were included in this study. Intrauterine growth retardation (IUGR) was seen in 87 (34.8%) patients. Gestational age (GA) ranged from 23 to 35weeks (mean 29.0weeks) and birth weight (BW) was 320-1499g (mean 1047.9g). During hospital stay, 55 (22%) patients developed IVH and 55 children (22%) BPD. PVL was seen in 12 (4.8%) cases, ROP in 93 (37.2%) and NEC in only 1 (0.4%) patient. NRBC counts as well as serum lactate concentrations depended significantly on birth weight (p<0.01) and presence respectively absence of IUGR (p<0.01). Both NRBC counts and serum lactate concentrations declined constantly during the 6-day period (p<0.01), and both were highly inter-correlated (p<0.01). CONCLUSIONS: This is one of only a very few studies that systematically and serially evaluated both NRBC counts and serum lactate concentration in VLBW and ELBW infants in the first 6days of life. Both parameters were significantly dependent on birth weight and the presence of IUGR. Moreover, a significant correlation between NRBC counts and serum lactate concentrations in this early period of life could be demonstrated. In future studies, the role of these readily available biomarkers in predicting important neonatal outcome parameters needs to be evaluated in a prospective manner.

One-carbon metabolites and telomere length in a prospective and randomized study of B- and/or D-vitamin supplementation.

BACKGROUND: Vitamin B deficiency is common in elderly people and has been associated with an increased risk of developing age-related diseases. B-vitamins are essential for the synthesis and stability of DNA. Telomers are the end caps of chromosomes that shorten progressively with age, and short telomers are associated with DNA instability. OBJECTIVE: In the present randomized intervention study, we investigated whether the one-carbon metabolism is related to telomere length, a surrogate marker for cellular aging. DESIGN: Sixty-five subjects (>54 years) were randomly assigned to receive either a daily combination of vitamin D3 (1200 IU), folic acid (0.5 mg), vitamin B12 (0.5 mg), vitamin B6 (50 mg) and calcium carbonate (456 mg) (group A) or vitamin D3 and calcium carbonate alone (group B). Blood testing was performed at baseline and after 1 year of supplementation. The concentrations of several metabolites of the one-carbon pathway, as well as relative telomere length (RTL) and 5,10-methylenetetrahydrofolate reductase C677T genotype, were analyzed. RESULTS: At baseline, age- and gender-adjusted RTL correlated with total folate and 5-methyltetrahydrofolate (5-methylTHF). Subjects with RTL above the median had higher concentrations of total folate and 5-methylTHF compared to subjects below the median. At study end, gender- and age-adjusted RTL correlated in group A with methylmalonic acid (MMA; r = -0.460, p = 0.0012) and choline (r = 0.434, p = 0.0021) and in group B with 5,10-methenyltetrahydrofolate (r = 0.455, p = 0.026) and dimethylglycine (DMG; r = -0.386, p = 0.047). Subjects in the group A with RTL above the median had lower MMA and higher choline compared to subjects below the median. CONCLUSIONS: The present pilot study suggests a functional relationship between one-carbon metabolism and telomere length. This conclusion is supported by several correlations that were modified by B-vitamin supplementation. In agreement with our hypothesis, the availability of nucleotides and methylation groups seems to impact telomere length. Due to the small sample size and the limitations of the study, further studies should confirm the present results in a larger cohort.