Comparison of pre-analytical conditions for quantification of serotonin in platelet-poor plasma

Background: Reported concentrations of serotonin in platelet-poor plasma (PPP) in healthy subjects vary widely due to different pre-analytical procedures. Aim: To examine how different pre-analytical conditions affect the measured concentration of serotonin in PPP. Method: Six pre-analytical protocols were compared for preparation of PPP from EDTA whole blood for quantification of serotonin from nine healthy individuals. Three combinations of centrifugation with a mild centrifugation of gel-free EDTA tubes followed by a stronger centrifugation were compared to single-stage centrifugation of EDTA tubes with separator gel and heat shock treatment of blood prior to centrifugation. All samples were analysed using the same enzyme linked immunosorbent assay (ELISA) method. Results: Findings show that two consecutive centrifugations; first a mild centrifugation at 100 or 200×g followed by centrifugation at 4500 or 14500×g resulted in the lowest serotonin concentration in PPP. Conclusion: Two successive centrifugations to produce PPP for serotonin analysis; first a mild centrifugation to avoid mechanical stress on the platelets, and next a stronger centrifugation to remove platelets, is superior to the use of gel tubes and heat shock treatment.publishedVersio

Plasma levels of guanylins are reduced in patients with Crohn’s disease

Background: Guanylin (GN) and uroguanylin (UGN) are endogenous ligands for the intestinal receptor guanylate cyclase C (GC-C), an important regulator of intestinal fluid homeostasis. Gene expression and protein levels of GN are suppressed in inflamed intestinal tissue from patients with inflammatory bowel disease (IBD), but knowledge about plasma levels of guanylins in these conditions is sparse. We aimed to investigate the fasting plasma levels of the prohormones proGN and proUGN in patients with Crohn’s Disease (CD) and relate these to levels found in persons with other diarrheal conditions, as well as persons with normal bowel habits. Methods: Plasma from patients with CD, patients with Familial GUCY2C Diarrheal Disease (FGDS), diarrhea-predominant irritable bowel syndrome (IBS-D) and healthy controls (HC) was analyzed using ELISA assays. Results: Significantly lower fasting plasma levels of proguanylins were found in CD and FGDS patients, compared to HC. In CD patients, plasma proGN levels correlated negatively with Harvey Bradshaw Index and with number of stools/24 h. Conclusion: Our data indicate that diarrhea may be a determinant for levels of proGN in plasma, and should be further explored in studies of different diarrheal disorders.acceptedVersio

The intronic BRCA1 c.5407-25T>A variant causing partly skipping of exon 23—a likely pathogenic variant with reduced penetrance?

Rare sequence variants in the non-coding part of the BRCA genes are often reported as variants of uncertain significance (VUS), which leave patients and doctors in a challenging position. The aim of this study was to determine the pathogenicity of the BRCA1 c.5407-25T>A variant found in 20 families from Norway, France and United States with suspected hereditary breast and ovarian cancer. This was done by combining clinical and family information with allele frequency data, and assessment of the variant’s effect on mRNA splicing. Mean age at breast (n = 12) and ovarian (n = 11) cancer diagnosis in female carriers was 49.9 and 60.4 years, respectively. The mean Manchester score in the 20 families was 16.4. The allele frequency of BRCA1 c.5407-25T>A was 1/64,566 in non-Finnish Europeans (gnomAD database v2.1.1). We found the variant in 1/400 anonymous Norwegian blood donors and 0/784 in-house exomes. Sequencing of patient-derived cDNA from blood, normal breast and ovarian tissue showed that BRCA1 c.5407-25T>A leads to skipping of exon 23, resulting in frameshift and protein truncation: p.(Gly1803GlnfsTer11). Western blot analysis of transiently expressed BRCA1 proteins in HeLa cells showed a reduced amount of the truncated protein compared with wild type. Noteworthily, we found that a small amount of full-length transcript was also generated from the c.5407-25T>A allele, potentially explaining the intermediate cancer burden in families carrying this variant. In summary, our results show that BRCA1 c.5407-25T>A leads to partial skipping of exon 23, and could represent a likely pathogenic variant with reduced penetrance.publishedVersio

Elevated cerebrospinal fluid protein in POLG-related epilepsy : Diagnostic and prognostic implications

Objective: Epilepsy is common in individuals with mutations in POLG, the gene encoding the catalytic subunit of the mitochondrial DNA polymerase gamma. Early recognition and aggressive seizure management are crucial for patient survival. Disruption of the blood-brain barrier (BBB) is implicated in various neurological disorders including epilepsy. The aim of this study was to assess whether POLG-related disease is associated with BBB dysfunction and what clinical implications this has for patients. Methods: Our retrospective study used data from 83 patients with pathogenic POLG mutations from 4 countries-Norway, Sweden, Finland, and the United Kingdom. Data were collected using a structured questionnaire. We used the presence of raised cerebrospinal fluid (CSF) protein and a raised CSF/serum ratio of albumin (Q-alb) to evaluate the integrity of the blood-CSF bather. Results: Raised CSF protein was found in 70% of patients (n = 58/83) and appeared to be associated with the most severe phenotypes. In those in whom it was measured, the Q-alb ratio was markedly elevated (n = 18). The majority of those with epilepsy (n = 50/66, 76%) had raised CSF protein, and this preceded seizure debut in 75% (n = 15/20). The median survival time from symptom onset for those with raised CSF protein was decreased (13 months) compared to those with normal CSF protein (32 months). Significance: Our results indicate that there is disruption of the BBB in POLG-related disease, as evidenced by a raised CSF protein and Q-alb ratio. We also find that raised CSF protein is a common finding in patients with POLG disease. Our data suggest that the presence of BBB dysfunction predicts a poorer outcome, and elevated CSF protein may therefore be an additional biomarker both for early diagnosis and to identify those at high risk of developing epilepsy.Peer reviewe

Neue Untersuchungsergebnisse zum alter von dunkelbäuchigen Ringelgänsen Branta b. bernicla

info:eu-repo/semantics/publishe

Comparison of pre-analytical conditions for quantification of serotonin in platelet-poor plasma

Background: Reported concentrations of serotonin in platelet-poor plasma (PPP) in healthy subjects vary widely due to different pre-analytical procedures. Aim: To examine how different pre-analytical conditions affect the measured concentration of serotonin in PPP. Method: Six pre-analytical protocols were compared for preparation of PPP from EDTA whole blood for quantification of serotonin from nine healthy individuals. Three combinations of centrifugation with a mild centrifugation of gel-free EDTA tubes followed by a stronger centrifugation were compared to single-stage centrifugation of EDTA tubes with separator gel and heat shock treatment of blood prior to centrifugation. All samples were analysed using the same enzyme linked immunosorbent assay (ELISA) method. Results: Findings show that two consecutive centrifugations; first a mild centrifugation at 100 or 200×g followed by centrifugation at 4500 or 14500×g resulted in the lowest serotonin concentration in PPP. Conclusion: Two successive centrifugations to produce PPP for serotonin analysis; first a mild centrifugation to avoid mechanical stress on the platelets, and next a stronger centrifugation to remove platelets, is superior to the use of gel tubes and heat shock treatment