Non-invasive Scores and Serum Biomarkers for Fatty Liver in the Era of Metabolic Dysfunction-associated Steatotic Liver Disease (MASLD): A Comprehensive Review From NAFLD to MAFLD and MASLD.

PURPOSE OF REVIEW: The prevalence of non-alcoholic fatty liver disease (NAFLD) is rapidly increasing worldwide, making it the leading cause of liver related morbidity and mortality. Currently, liver biopsy is the gold standard for assessing individuals with steatohepatitis and fibrosis. However, its invasiveness, sampling variability, and impracticality for large-scale screening has driven the search for non-invasive methods for early diagnosis and staging. In this review, we comprehensively summarise the evidence on the diagnostic performance and limitations of existing non-invasive serum biomarkers and scores in the diagnosis and evaluation of steatosis, steatohepatitis, and fibrosis. RECENT FINDINGS: Several non-invasive serum biomarkers and scores have been developed over the last decade, although none has successfully been able to replace liver biopsy. The introduction of new NAFLD terminology, namely metabolic dysfunction-associated fatty liver disease (MAFLD) and more recently metabolic dysfunction-associated steatotic liver disease (MASLD), has initiated a debate on the interchangeability of these terminologies. Indeed, there is a need for more research on the variability of the performance of non-invasive serum biomarkers and scores across the diagnostic entities of NAFLD, MAFLD and MASLD. There remains a significant need for finding valid and reliable non-invasive methods for early diagnosis and assessment of steatohepatitis and fibrosis to facilitate prompt risk stratification and management to prevent disease progression and complications. Further exploration of the landscape of MASLD under the newly defined disease subtypes is warranted, with the need for more robust evidence to support the use of commonly used serum scores against the new MASLD criteria and validation of previously developed scores.

Anti-osteoporotic treatments in the era of non-alcoholic fatty liver disease: friend or foe.

Over the last years non-alcoholic fatty liver disease (NAFLD) has grown into the most common chronic liver disease globally, affecting 17-38% of the general population and 50-75% of patients with obesity and/or type 2 diabetes mellitus (T2DM). NAFLD encompasses a spectrum of chronic liver diseases, ranging from simple steatosis (non-alcoholic fatty liver, NAFL) and non-alcoholic steatohepatitis (NASH; or metabolic dysfunction-associated steatohepatitis, MASH) to fibrosis and cirrhosis with liver failure or/and hepatocellular carcinoma. Due to its increasing prevalence and associated morbidity and mortality, the disease-related and broader socioeconomic burden of NAFLD is substantial. Of note, currently there is no globally approved pharmacotherapy for NAFLD. Similar to NAFLD, osteoporosis constitutes also a silent disease, until an osteoporotic fracture occurs, which poses a markedly significant disease and socioeconomic burden. Increasing emerging data have recently highlighted links between NAFLD and osteoporosis, linking the pathogenesis of NAFLD with the process of bone remodeling. However, clinical studies are still limited demonstrating this associative relationship, while more evidence is needed towards discovering potential causative links. Since these two chronic diseases frequently co-exist, there are data suggesting that anti-osteoporosis treatments may affect NAFLD progression by impacting on its pathogenetic mechanisms. In the present review, we present on overview of the current understanding of the liver-bone cross talk and summarize the experimental and clinical evidence correlating NAFLD and osteoporosis, focusing on the possible effects of anti-osteoporotic drugs on NAFLD.

Retinol-binding protein 4 (RBP4) circulating levels and gestational diabetes mellitus: a systematic review and meta-analysis.

Background: Gestational diabetes mellitus (GDM) is a prevalent condition where diabetes is diagnosed during pregnancy, affecting both maternal and fetal outcomes. Retinol-binding protein 4 (RBP4) is a circulating adipokine which belongs to the lipocalin family and acts as a specific carrier protein that delivers retinol (vitamin A) from the liver to the peripheral tissues. Growing data indicate that circulating RBP4 levels may positively correlate with GDM. Thus, this systematic review and meta-analysis aimed to investigate the potential relationship between circulating RBP4 levels and GDM when measured at various stages of pregnancy. Methods: MEDLINE, CINAHL, EMCARE, EMBASE, Scopus, and Web of Science databases were searched to identify studies comparing pregnant women with and without GDM, whose circulating RBP4 levels were measured in at least one pregnancy trimester. Findings were reported using standardized mean difference (SMD) and random-effects models were used to account for variability among studies. Furthermore, the risk of bias was assessed using the RoBANS tool. Results: Out of the 34 studies identified, 32 were included in the meta-analysis (seven with circulating RBP4 levels measured in the first trimester, 19 at 24-28 weeks, and 14 at >28 weeks of pregnancy). RBP4 levels were statistically higher in the GDM group than in controls when measured during all these pregnancy stages, with the noted RBP4 SMD being 0.322 in the first trimester (95% CI: 0.126-0.517; p < 0.001; 946 GDM cases vs. 1701 non-GDM controls); 0.628 at 24-28 weeks of gestation (95% CI: 0.290-0.966; p < 0.001; 1776 GDM cases vs. 1942 controls); and 0.875 at >28 weeks of gestation (95% CI: 0.252-1.498; p = 0.006; 870 GDM cases vs. 1942 non-GDM controls). Significant study heterogeneity was noted for all three pregnancy timepoints. Conclusion: The present findings indicate consistently higher circulating RBP4 levels in GDM cases compared to non-GDM controls, suggesting the potential relevance of RBP4 as a biomarker for GDM. However, the documented substantial study heterogeneity, alongside imprecision in effect estimates, underscores the need for further research and standardization of measurement methods to elucidate whether RBP4 can be utilized in clinical practice as a potential GDM biomarker. Systematic review registration: PROSPERO (CRD42022340097: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022340097).

The DAPA-DIET study: Metabolic response to Dapagliflozin combined with dietary carbohydrate restriction in patients with Type 2 Diabetes Mellitus and Obesity-A longitudinal cohort study.

OBJECTIVE: The cardio-renal benefits of sodium glucose-like transporter 2 inhibitor (SGLT2i) therapies have been demonstrated in patients with and without type 2 diabetes. However, no studies have explored the long-term metabolic effects of SGLT2i, combined with dietary carbohydrate restriction. Our primary objective was to describe long-term changes in weight, energy expenditure, appetite and body composition after 12 months of Dapagliflozin therapy, with carbohydrate restriction, in people with type 2 diabetes and obesity. Our secondary objective was to assess changes in adiponectin and leptin. METHOD: This was a 12-month cohort study in a secondary care setting. Participants (n = 18) with type 2 diabetes (T2D) and class 3 obesity underwent baseline indirect calorimetry for determination of 24-h energy expenditure, body composition, fasting serum leptin and adiponectin levels, and appetitive assessments. Following initiation of Dapagliflozin (and dietary carbohydrate restriction), measurements were repeated at monthly intervals up to 12 months. RESULTS: Mean starting weight of participants was 129.4 kg (SD 25.9), mean BMI 46.1 kg/m2 (SD 8.3) and mean HbA1c 53.9 mmol/mol (14.1). Seventeen participants completed the study; after 12 months of Dapagliflozin and dietary carbohydrate restriction, mean weight loss was 8.1 kg (SD 11.3 kg; p = .009). This was mediated by reduced fat mass (mean loss, 9.9 kg; SD 10.4 kg; p = .002) associated with reduced serum leptin at 12 months (mean reduction 11,254 pg/ml; SD 16,075; p = .011). There were no significant changes in self-reported appetite, 24-h energy expenditure or serum adiponectin during follow-up. CONCLUSION: In this study, combined Dapagliflozin therapy and carbohydrate restriction in patients with T2D and obesity resulted in a significant reduction of body weight and fat mass at 12 months without any discernible changes in energy expenditure or appetite. These results offer a scientific and clinical rationale to conduct an exploratory trial investigating the effects of a low carbohydrate diet combined with SGLT2 inhibitors in patients with T2D.

Cardiovascular Biomarkers: Lessons of the Past and Prospects for the Future.

Cardiovascular diseases (CVDs) are a major healthcare burden on the population worldwide. Early detection of this disease is important in prevention and treatment to minimise morbidity and mortality. Biomarkers are a critical tool to either diagnose, screen, or provide prognostic information for pathological conditions. This review discusses the historical cardiac biomarkers used to detect these conditions, discussing their application and their limitations. Identification of new biomarkers have since replaced these and are now in use in routine clinical practice, but still do not detect all disease. Future cardiac biomarkers are showing promise in early studies, but further studies are required to show their value in improving detection of CVD above the current biomarkers. Additionally, the analytical platforms that would allow them to be adopted in healthcare are yet to be established. There is also the need to identify whether these biomarkers can be used for diagnostic, prognostic, or screening purposes, which will impact their implementation in routine clinical practice.

COVID­19 and SARS­CoV­2 host cell entry mediators: Expression profiling of TMRSS4 in health and disease.

Severe acute respiratory syndrome (SARS) coronavirus­2 (SARS­CoV­2), the causative viral agent for the ongoing COVID­19 pandemic, enters its host cells primarily via the binding of the SARS­CoV­2 spike (S) proteins to the angiotensin­converting enzyme 2 (ACE2). A number of other cell entry mediators have also been identified, including neuropilin­1 (NRP1) and transmembrane protease serine 2 (TMPRSS2). More recently, it has been demonstrated that transmembrane protease serine 4 (TMPRSS4) along with TMPRSS2 activate the SARS­CoV­2 S proteins, and enhance the viral infection of human small intestinal enterocytes. To date, a systematic analysis of TMPRSS4 in health and disease is lacking. In the present study, using in silico tools, the gene expression and genetic alteration of TMPRSS4 were analysed across numerous tumours and compared to controls. The observations were also expanded to the level of the central nervous system (CNS). The findings revealed that TMPRSS4 was overexpressed in 11 types of cancer, including lung adenocarcinoma, lung squamous cell carcinoma, cervical squamous cell carcinoma, thyroid carcinoma, ovarian cancer, cancer of the rectum, pancreatic cancer, colon and stomach adenocarcinoma, uterine carcinosarcoma and uterine corpus endometrial carcinoma, whilst it was significantly downregulated in kidney carcinomas, acute myeloid leukaemia, skin cutaneous melanoma and testicular germ cell tumours. Finally, a high TMPRSS4 expression was documented in the olfactory tubercle, paraolfactory gyrus and frontal operculum, all brain regions which are associated with the sense of smell and taste. Collectively, these data suggest that TMPRSS4 may play a role in COVID­19 symptomatology as another SARS­CoV­2 host cell entry mediator responsible for the tropism of this coronavirus both in the periphery and the CNS.

Empagliflozin Attenuates Non-Alcoholic Fatty Liver Disease (NAFLD) in High Fat Diet Fed ApoE(-/-) Mice by Activating Autophagy and Reducing ER Stress and Apoptosis.

AIMS/HYPOTHESIS: SGLT-2 inhibitors (SGLT-2i) have been studied as potential treatments against NAFLD, showing varying beneficial effects. The molecular mechanisms mediating these effects have not been fully clarified. Herein, we investigated the impact of empagliflozin on NAFLD, focusing particularly on ER stress, autophagy and apoptosis. METHODS: Five-week old ApoE(-/-) mice were switched from normal to a high-fat diet (HFD). After five weeks, mice were randomly allocated into a control group (HFD + vehicle) and Empa group (HFD + empagliflozin 10 mg/kg/day) for five weeks. At the end of treatment, histomorphometric analysis was performed in liver, mRNA levels of Fasn, Screbp-1, Scd-1, Ppar-γ, Pck-1, Mcp-1, Tnf-α, Il-6, F4/80, Atf4, Elf2α, Chop, Grp78, Grp94, Χbp1, Ire1α, Atf6, mTor, Lc3b, Beclin-1, P62, Bcl-2 and Bax were measured by qRT-PCR, and protein levels of p-EIF2α, EIF2a, CHOP, LC3II, P62, BECLIN-1 and cleaved CASPASE-8 were assessed by immunoblotting. RESULTS: Empagliflozin-treated mice exhibited reduced fasting glucose, total cholesterol and triglyceride serum levels, as well as decreased NAFLD activity score, decreased expression of lipogenic enzymes (Fasn, Screbp-1c and Pck-1) and inflammatory molecules (Mcp-1 and F4/80), compared to the Control group. Empagliflozin significantly decreased the expression of ER stress molecules Grp78, Ire1α, Xbp1, Elf2α, Atf4, Atf6, Chop, P62(Sqstm1) and Grp94; whilst activating autophagy via increased AMPK phosphorylation, decreased mTOR and increased LC3B expression. Finally, empagliflozin increased the Bcl2/Bax ratio and inhibited CASPASE-8 cleavage, reducing liver cell apoptosis. Immunoblotting analysis confirmed the qPCR results. CONCLUSION: These novel findings indicate that empagliflozin treatment for five weeks attenuates NAFLD progression in ApoE(-/-) mice by promoting autophagy, reducing ER stress and inhibiting hepatic apoptosis.

Neuropilin­1 as a new potential SARS­CoV­2 infection mediator implicated in the neurologic features and central nervous system involvement of COVID­19.

Infection by the severe acute respiratory syndrome (SARS) coronavirus­2 (SARS­CoV­2) is the cause of the new viral infectious disease (coronavirus disease 2019; COVID­19). Emerging evidence indicates that COVID­19 may be associated with a wide spectrum of neurological symptoms and complications with central nervous system (CNS) involvement. It is now well­established that entry of SARS­CoV­2 into host cells is facilitated by its spike proteins mainly through binding to the angiotensin­converting enzyme 2 (ACE­2). Preclinical studies have suggested that neuropilin­1 (NRP1), which is a transmembrane receptor that lacks a cytosolic protein kinase domain and exhibits high expression in the respiratory and olfactory epithelium, may also be implicated in COVID­19 by enhancing the entry of SARS­CoV­2 into the brain through the olfactory epithelium. In the present study, we expand on these findings and demonstrate that the NRP1 is also expressed in the CNS, including olfactory­related regions such as the olfactory tubercles and paraolfactory gyri. This furthers supports the potential role of NRP1 as an additional SARS­CoV­2 infection mediator implicated in the neurologic manifestations of COVID­19. Accordingly, the neurotropism of SARS­CoV­2 via NRP1­expressing cells in the CNS merits further investigation.

Is There a Link between Bisphenol A (BPA), a Key Endocrine Disruptor, and the Risk for SARS-CoV-2 Infection and Severe COVID-19?

Infection by the severe acute respiratory syndrome (SARS) coronavirus-2 (SARS-CoV-2) is the causative agent of a new disease (COVID-19). The risk of severe COVID-19 is increased by certain underlying comorbidities, including asthma, cancer, cardiovascular disease, hypertension, diabetes, and obesity. Notably, exposure to hormonally active chemicals called endocrine-disrupting chemicals (EDCs) can promote such cardio-metabolic diseases, endocrine-related cancers, and immune system dysregulation and thus, may also be linked to higher risk of severe COVID-19. Bisphenol A (BPA) is among the most common EDCs and exerts its effects via receptors which are widely distributed in human tissues, including nuclear oestrogen receptors (ERα and ERß), membrane-bound oestrogen receptor (G protein-coupled receptor 30; GPR30), and human nuclear receptor oestrogen-related receptor gamma. As such, this paper focuses on the potential role of BPA in promoting comorbidities associated with severe COVID-19, as well as on potential BPA-induced effects on key SARS-CoV-2 infection mediators, such as angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). Interestingly, GPR30 appears to exhibit greater co-localisation with TMPRSS2 in key tissues like lung and prostate, suggesting that BPA exposure may impact on the local expression of these SARS-CoV-2 infection mediators. Overall, the potential role of BPA on the risk and severity of COVID-19 merits further investigation.

Co­expression of peripheral olfactory receptors with SARS­CoV­2 infection mediators: Potential implications beyond loss of smell as a COVID­19 symptom.

Severe acute respiratory syndrome (SARS) coronavirus­2 (SARS­CoV­2) enters into human host cells via mechanisms facilitated mostly by angiotensin­converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2). New loss of smell (anosmia/hyposmia) is now recognized as a COVID­19 related symptom, which may be caused by SARS­CoV­2 infection and damage of the olfactory receptor (OR) cells in the nasal neuro­epithelium and/or central involvement of the olfactory bulb. ORs are also expressed peripherally (e.g., in tissues of the gastrointestinal and respiratory systems) and it is possible that their local functions could also be impaired by SARS­CoV­2 infection of these tissues. Using Gene Expression Profiling Interactive Analysis, The Cancer Genome Atlas, Genotype­Tissue Expression, cBioPortal and Shiny Methylation Analysis Resource Tool, we highlight the expression of peripheral ORs in both healthy and malignant tissues, and describe their co­expression with key mediators of SARS­CoV­2 infection, such as ACE2 and TMPRSS2, as well as cathepsin L (CTSL; another cellular protease mediating SARS­CoV­2 infection of host cells). A wide expression profile of peripheral ORs was noted, particularly in tissues such as the prostate, testis, thyroid, brain, liver, kidney and bladder, as well as tissues with known involvement in cardio­metabolic disease (e.g., the adipose tissue, pancreas and heart). Among these, OR51E2, in particular, was significantly upregulated in prostate adenocarcinoma (PRAD) and co­expressed primarily with TMPRSS2. Functional networks of this OR were further analysed using the GeneMANIA interactive tool, showing that OR51E2 interacts with a plethora of genes related to the prostate. Further in vitro and clinical studies are clearly required to elucidate the role of ORs, both at the olfactory level and the periphery, in the context of COVID­19.

Polycystic ovary syndrome (PCOS) and COVID-19: an overlooked female patient population at potentially higher risk during the COVID-19 pandemic.

BACKGROUND: In women of reproductive age, polycystic ovary syndrome (PCOS) constitutes the most frequent endocrine disorder. Women with PCOS are considered to typically belong to an age and sex group which is at lower risk for severe COVID-19. MAIN BODY: Emerging data link the risk of severe COVID-19 with certain factors such as hyper-inflammation, ethnicity predisposition, low vitamin D levels, and hyperandrogenism, all of which have known direct associations with PCOS. Moreover, in this common female patient population, there is markedly high prevalence of multiple cardio-metabolic conditions, such as type 2 diabetes, obesity, and hypertension, which may significantly increase the risk for adverse COVID-19-related outcomes. This strong overlap of risk factors for both worse PCOS cardio-metabolic manifestations and severe COVID-19 should be highlighted for the clinical practice, particularly since women with PCOS often receive fragmented care from multiple healthcare services. Comprehensively informing women with PCOS regarding the potential risks from COVID-19 and how this may affect their management is also essential. CONCLUSION: Despite the immense challenges posed by the COVID-19 outbreak to the healthcare systems in affected countries, attention should be directed to maintain a high standard of care for complex patients such as many women with PCOS and provide relevant practical recommendations for optimal management in the setting of this fast moving pandemic.

Pan­cancer analysis of transmembrane protease serine 2 and cathepsin L that mediate cellular SARS­CoV­2 infection leading to COVID-19.

Severe acute respiratory syndrome (SARS) coronavirus­2 (SARS­CoV2) is the cause of a new disease (COVID­19) which has evolved into a pandemic during the first half of 2020. Older age, male sex and certain underlying diseases, including cancer, appear to significantly increase the risk for severe COVID­19. SARS­CoV­2 infection of host cells is facilitated by the angiotensin­converting enzyme 2 (ACE­2), and by transmembrane protease serine 2 (TMPRSS2) and other host cell proteases such as cathepsin L (CTSL). With the exception of ACE­2, a systematic analysis of these two other SARS­CoV2 infection mediators in malignancies is lacking. Here, we analysed genetic alteration, RNA expression, and DNA methylation of TMPRSS2 and CTSL across a wide spectrum of tumors and controls. TMPRSS2 was overexpressed in cervical squamous cell carcinoma and endocervical adenocarcinoma, colon adenocarcinoma, prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), uterine corpus endometrial carcinoma and uterine carcinosarcoma, with PRAD and READ exhibiting the highest expression of all cancers. CTSL was upregulated in lymphoid neoplasm diffuse large B­cell lymphoma, oesophageal carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, lower grade glioma, pancreatic adenocarcinoma, skin cutaneous melanoma, stomach adenocarcinoma, and thymoma. Hypo­methylation of both genes was evident in most cases where they have been highly upregulated. We have expanded on our observations by including data relating to mutations and copy number alterations at pan­cancer level. The novel hypotheses that are stemming out of these data need to be further investigated and validated in large clinical studies.

Chemerin in human cardiovascular disease.

Adipose tissue, initially known only for storing excess fat, produces a number of active cytokine-like hormones, collectively known as adipokines or adipocytokines. These molecules are further known to elicit auto-, para- and endocrine functions in the body. In healthy bodies, the cardiovascular endothelium maintains vascular health by critically controlling the interplay between various factors. However, in diseased states such as obesity, owing to numerous metabolic malfunctions, this vascular homeostasis is disrupted. The dysregulated metabolic stimuli perturb vascular homeostasis via initiating or exacerbating the pre-existing inflammatory processes. These inflammatory processes further recruit immune cells to the site of injury, alter cell adhesion molecule expression, and reduce Nitric Oxide levels. These altered mechanisms result in increased blood pressure, endothelial cell migration, proliferation, and apoptosis. In this review article, we aim to evaluate the current literature in understanding the role of Chemerin in vascular health and furthermore, its role in maintaining vascular homeostasis with respect to inflammation, obesity and associated Metabolic Syndrome (MetS) risk factors. For over fifteen years, a growing body of research has been published studying Chemerin in the disease states associated with obesity, MetS and cardiovascular disease. Chemerin appears to form an integral link between obesity and related dysfunctional cardiometabolic states as well as in inflammation and immune-system related complications. These combine to suggest a significant Chemerin role in human vascular health and disease.

Distinct patterns of hepcidin and iron regulation during HIV-1, HBV, and HCV infections.

During HIV type-1 (HIV-1), hepatitis C virus (HCV), and hepatitis B virus (HBV) infections, altered iron balance correlates with morbidity. The liver-produced hormone hepcidin dictates systemic iron homeostasis. We measured hepcidin, iron parameters, cytokines, and inflammatory markers in three cohorts: plasma donors who developed acute HIV-1, HBV, or HCV viremia during the course of donations; HIV-1-positive individuals progressing from early to chronic infection; and chronically HIV-1-infected individuals (receiving antiretroviral therapy or untreated). Hepcidin increased and plasma iron decreased during acute HIV-1 infection, as viremia was initially detected. In patients transitioning from early to chronic HIV-1 infection, hepcidin in the first 60 d of infection positively correlated with the later plasma viral load set-point. Hepcidin remained elevated in individuals with untreated chronic HIV-1 infection and in subjects on ART. In contrast to HIV-1, there was no evidence of hepcidin up-regulation or hypoferremia during the primary viremic phases of HCV or HBV infection; serum iron marginally increased during acute HBV infection. In conclusion, hepcidin induction is part of the pathogenically important systemic inflammatory cascade triggered during HIV-1 infection and may contribute to the establishment and maintenance of viral set-point, which is a strong predictor of progression to AIDS and death. However, distinct patterns of hepcidin and iron regulation occur during different viral infections that have particular tissue tropisms and elicit different systemic inflammatory responses. The hypoferremia of acute infection is therefore a pathogen-specific, not universal, phenomenon.

Laboratory database population surveillance to improve detection of progressive chronic kidney disease.

BACKGROUND: Some patients with chronic kidney disease are still referred late for specialist care despite the evidence that earlier detection and intervention can halt or delay progression to end-stage kidney disease (ESKD). OBJECTIVES: To develop a population surveillance system using existing laboratory data to enable early detection of patients at high risk of ESKD by reviewing cumulative graphs of estimated glomerular filtration rate (eGFR). METHODS: A database was developed, updated daily with data from the laboratory computer. Cumulative eGFR graphs containing up to five years of data are reviewed by clinical scientists for all primary care patients or out-patients with a low eGFR for their age. For those with a declining trend, a report containing the eGFR graph is sent to the requesting doctor. A retrospective audit was performed using historical data to assess the predictive value of the graphs. RESULTS: In nine months, we reported 370,000 eGFR results, reviewing 12,000 eGFR graphs. On average 60 graphs per week were flagged as 'high' or 'intermediate' risk. Patients with graphs flagged as high risk had a significantly higher mortality after 3.5 years and a significantly greater chance of requiring renal replacement therapy after 4.5 years of follow-up. Five patients (7%) with graphs flagged as high risk had a sustained >25% fall in eGFR without evidence of secondary care referral. Feedback about the service from requesting clinicians was 73% positive. CONCLUSIONS: We have developed a system for laboratory staff to review cumulative eGFR graphs for a large population and identify patients at highest risk of developing ESKD. Further research is needed to measure the impact of this service on patient outcomes.

National UK audit of the short synacthen test.

UNLABELLED: We present the first national audit of the Short Synacthen Test (SST), identifying the clinical, analytical and interpretative procedures adopted by 89 laboratories. BACKGROUND: The SST has replaced the insulin stress test as the first-line test to assess adrenal insufficiency and has received considerable attention regarding its sensitivity and specificity. Concerns regarding this test include the bias of cortisol methods, cut-off values used, contraindications and the limitations of the test in diagnosing recent, mild secondary adrenal insufficiency. The audit took into consideration the protocols used by laboratories, the advice provided prior and after the SST and the analytical bias of the methods used. METHODS: A web-based questionnaire using Microsoft FrontPage(TM) was prepared to collect data from laboratories and provided drop-down lists and other form-field elements to capture additional comments. The resultant data were exported to Microsoft Excel(TM) for data clean-up and analysis. RESULTS: The workloads were highly variable; however, most laboratories were in general agreement to the indications, contraindications, timing and reference ranges. In contrast, there was variability in the bias of the cortisol methods, which had not been translated to the cut-off values used by the majority of laboratories. CONCLUSIONS: The audit has shown that though the preanalytical procedures were similar in most laboratories, there is a requirement to recognize the effect that method bias may have on the reference ranges and consequently on the diagnosis of adrenal insufficiency. There is a need to develop consensus guidelines, which can aid both clinicians and laboratories.

Methylation of the imprinted GNAS1 gene in cell-free plasma DNA: equal steady-state quantities of methylated and unmethylated DNA in plasma.

BACKGROUND: Genomic DNA sequences in cell-free plasma are biomarkers of cancer prognosis, where characteristic changes in methylation of tumour suppressor or oncogene DNA regions are indicative of changes in gene activity. Also, cell-free fetal DNA can be distinguished, by its methylation status, from the maternal DNA in the plasma of pregnant women, hence providing DNA biomarkers for the proposed minimally-invasive diagnosis of fetal aneuploidies, including Down's syndrome. However, the production and clearance of cell-free DNA from plasma in relation to its methylation status, are poorly understood processes. METHODS: We studied the methylation status of DNA derived from the imprinted GNAS1 locus, in cell-free plasma DNA of healthy adults. Heterozygotes were identified that carried the SNP rs1800905 in the imprinted region. The parent-of-origin-dependent DNA methylation was analysed by bisulfite conversion, followed by cloning and sequencing. RESULTS: Genomic DNA molecules derived from both the methylated, maternal, allele and the unmethylated, paternal, allele were found in plasma. Methylated and unmethylated DNA molecules were present in equal numbers. CONCLUSIONS: Our data indicate that the methylation status of a DNA sequence has no effect on its steady state concentration in the cell-free DNA component of plasma, in healthy adults.