Absent expansion of AXIN2+ hepatocytes and altered physiology in Axin2CreERT2 mice challenges the role of pericentral hepatocytes in homeostatic liver regeneration.

BACKGROUND & AIMS: Mouse models of lineage tracing have helped to describe the important subpopulations of hepatocytes responsible for liver regeneration. However, conflicting results have been obtained from different models. Herein, we aimed to reconcile these conflicting reports by repeating a key lineage-tracing study from pericentral hepatocytes and characterising this Axin2CreERT2 model in detail. METHODS: We performed detailed characterisation of the labelled population in the Axin2CreERT2 model. We lineage traced this cell population, quantifying the labelled population over 1 year and performed in-depth phenotypic comparisons, including transcriptomics, metabolomics and analysis of proteins through immunohistochemistry, of Axin2CreERT2 mice to WT counterparts. RESULTS: We found that after careful definition of a baseline population, there are marked differences in labelling between male and female mice. Upon induced lineage tracing there was no expansion of the labelled hepatocyte population in Axin2CreERT2 mice. We found substantial evidence of disrupted homeostasis in Axin2CreERT2 mice. Offspring are born with sub-Mendelian ratios and adult mice have perturbations of hepatic Wnt/ß-catenin signalling and related metabolomic disturbance. CONCLUSIONS: We find no evidence of predominant expansion of the pericentral hepatocyte population during liver homeostatic regeneration. Our data highlight the importance of detailed preclinical model characterisation and the pitfalls which may occur when comparing across sexes and backgrounds of mice and the effects of genetic insertion into native loci. IMPACT AND IMPLICATIONS: Understanding the source of cells which regenerate the liver is crucial to harness their potential to regrow injured livers. Herein, we show that cells which were previously thought to repopulate the liver play only a limited role in physiological regeneration. Our data helps to reconcile differing conclusions drawn from results from a number of prior studies and highlights methodological challenges which are relevant to preclinical models more generally.

Changes in hematological indices and lymphocyte subsets in response to whole blood donation in healthy male donors.

Whole blood donation has immunomodulatory effects, and most of these have been observed at short intervals following blood donation. This study aimed to investigate the impact of whole blood donation on lymphocyte subsets over a typical inter-donation interval. Healthy male subjects were recruited to study changes in complete blood count (CBC) (n = 42) and lymphocyte subsets (n = 16) before and at four intervals up to 106 days following blood donation. Repeated measures ANOVA were used to compare quantitative variables between different visits. Following blood donation, changes in CBC and erythropoietin were as expected. The neutrophil count increased by 11.3% at 8 days (p < .001). Novel changes were observed in lymphocyte subsets as the CD4/CD8 ratio increased by 9.2% (p < .05) at 8 days and 13.7% (p < .05) at 22 days. CD16-56 cells decreased by 16.2% (p < .05) at 8 days. All the subsets had returned to baseline by 106 days. Regression analysis showed that the changes in CD16-56 cells and CD4/CD8 ratio were not significant (Wilk's lambda = 0.15 and 0.94, respectively) when adjusted for BMI. In conclusion, following whole blood donation, there are transient changes in lymphocyte subsets. The effect of BMI on lymphocyte subsets and the effect of this immunomodulation on the immune response merit further investigation.

Assessment of Becton Dickinson Plain and Serum Separator Tubes in Measurement of 25-Hydroxyvitamin D3 (25OHD3) by HPLC and Immunoassay Methods.

BACKGROUND: The accuracy of 25-hydroxyvitamin D3 (25OHD3) measurement on specimens collected into serum separator tubes (SSTs) has been questioned because of possible interference by the gel. Possible interference was investigated in SSTs from Becton Dickinson (BD). DESIGN AND METHODS: Blood specimens were collected simultaneously from 50 normal subjects into plain tubes and SSTs. 25OHD3 was assayed on serum using high performance liquid chromatography (Chromsystems), and Architect (Abbott) and Liaison (Diasorin) immunoassays. RESULTS: There were no significant differences between 25OHD3 results (means ± SE, nmol/l) obtained from specimens collected into plain tubes and SSTs assayed by HPLC (39.0 ± 2.7 vs. 39.3 ± 2.7), Liaison (32.9 ± 2.2 vs. 32.8 ± 2.3), or Architect (43.1 ± 2.8 vs. 43.2 ± 2.8). In specimens collected into plain tubes and SSTs, 25OHD3 measurements by HPLC correlated significantly (P < 0.0001) with those from the Architect (r = 0.895, r = 0.908) and Liaison (r = 0.907, r = 0.913), respectively. CONCLUSIONS: The gel in SSTs (BD) does not interfere with the measurement of 25OHD3 by HPLC or common immunoassays. This important finding may enable clinical laboratories to make cost savings by using SSTs without concerns about inaccuracy.

Insulin-like growth factor-II: its role in metabolic and endocrine disease.

Insulin-like growth factor-II (IGF-II) is a widely expressed 7·5 kDa mitogenic peptide hormone. Although it is abundant in serum, understanding of its physiological role is limited compared with that of IGF-I. IGF-II regulates foetal development and differentiation, but its role in adults is less well understood. Evidence suggests roles in a number of tissues including skeletal muscle, adipose tissue, bone and ovary. Altered IGF-II expression has been observed in metabolic conditions, notably obesity, diabetes and the polycystic ovary syndrome. This article summarizes what is known about the actions of IGF-II and its dysregulation in metabolic and endocrine diseases. The possible causes and consequences of dysregulation are discussed along with the implications for diagnostic tests and future research.

Insulin-like growth factor-I (IGF-I) and clinical nutrition.

IGF-I (insulin-like growth factor-I) is a peptide hormone, produced predominantly by the liver in response to pituitary GH (growth hormone), which is involved in a wide variety of physiological processes. It acts in an endocrine, paracrine and autocrine manner to promote growth. The production of IGF-I signals the availability of nutrients needed for its anabolic actions. Recently, there has been growing interest in its role in health and disease. IGF-I has long been known to be regulated by nutrition and dysregulated in states of under- and over-nutrition, its serum concentrations falling in malnutrition and responding promptly to refeeding. This has led to interest in its utility as a nutritional biomarker. A considerable evidence base supports utility for measurement of IGF-I in nutritional contexts. Its concentration may be valuable in providing information on nutritional status, prognosis and in monitoring nutritional support. However, it is insufficiently specific for use as a screening test for under nutrition as its serum concentration is influenced by many factors other than nutritional status, notably the APR (acute-phase response) and endocrine conditions. Concentrations should be interpreted along with clinical findings and the results of other investigations such as CRP (C-reactive protein). More recently, there has been interest in free IGF-I which holds promise as a nutritional marker. The present review covers nutritional regulation of IGF-I and its dysregulation in disease, then goes on to review recent studies supporting its utility as a nutritional marker in clinical contexts. Although not currently recommended by clinical guidelines, it is likely that, in time, measurement of IGF-I will become a routine part of nutritional assessment in a number of these contexts.

Reference change values for insulin and insulin-like growth factor binding protein-1 (IGFBP-1) in individuals with varying degrees of glucose tolerance.

BACKGROUND: The reference change value (RCV) is an important parameter of biological variation used to assess the significance of differences between consecutive results obtained in a single individual. This study evaluated the RCV for insulin-like growth factor binding protein-1 (IGFBP-1) and fasting serum insulin (FSI) in individuals with different degrees of glucose tolerance. METHODS: IGFBP-1 and FSI concentrations were measured in 33 fasting subjects who had two blood samples taken 10 days apart. Subjects were distributed in the following categories: Normal glucose tolerance (NGT), n = 15, impaired fasting glucose (IFG), n = 9 and impaired glucose tolerance (IGT), n = 9. RESULTS: The RCV values for IGFBP-1 were 59.9%, 83.2% and 93.0% and for FSI were 68.5%, 79.0% and 93.4% for subjects with NGT, IFG and IGT respectively. CONCLUSIONS: The RCVs for IGFBP-1 and FSI increase with deteriorating glucose tolerance. When monitoring IGFBP-1 and FSI, changes in concentrations should be interpreted using glycaemic category-specific RCV values.

Selection of the appropriate method for the assessment of insulin resistance.

Insulin resistance is one of the major aggravating factors for metabolic syndrome. There are many methods available for estimation of insulin resistance which range from complex techniques down to simple indices. For all methods of assessing insulin resistance it is essential that their validity and reliability is established before using them as investigations. The reference techniques of hyperinsulinaemic euglycaemic clamp and its alternative the frequently sampled intravenous glucose tolerance test are the most reliable methods available for estimating insulin resistance. However, many simple methods, from which indices can be derived, have been assessed and validated e.g. homeostasis model assessment (HOMA), quantitative insulin sensitivity check index (QUICKI). Given the increasing number of simple indices of IR it may be difficult for clinicians and researchers to select the most appropriate index for their studies. This review therefore provides guidelines and advices which must be considered before proceeding with a study.

The relationship between glycosylated haemoglobin (HbA1c) and measures of insulin resistance across a range of glucose tolerance.

AIM: This study aimed to assess the correlation between HbA1c and insulin resistance as measured by a variety of different indices in subjects from across the glycaemic spectrum. METHODS: Subjects with normal glucose tolerance (NGT; n = 24), impaired fasting glucose (IFG; n = 12), impaired glucose tolerance (IGT; n = 12), and type 2 diabetes (DM; n = 13) were studied. All had specimens taken in the context of a standard oral glucose tolerance test at their first visit and had the insulin sensitivity parameter (Si) determined by frequently-sampled intravenous glucose tolerance test at a second visit. RESULTS: HbA1c was more strongly associated with Si in NGT (r = - 0.65) than in IFG (r = - 0.48). Compared to other indices of insulin resistance HbA1c has minimal overlap in values (0.0%) between NGT and subjects with type 2 diabetes. CONCLUSIONS: HbA1c can be used as a simple and reliable marker of insulin resistance in NGT adults with relatively high insulin sensitivity.

Insulin sensitivity (Si) assessment in lean and overweight subjects using two different protocols and updated software.

BACKGROUND: The standard frequently-sampled intravenous glucose tolerance test (FSIVGTT) is an alternative procedure to the clamp technique for estimating the insulin sensitivity (Si) parameter. The goal of this study was to compare Si in lean and overweight individuals in addition to assessing intra-individual reproducibility using two different protocols and updated software. METHODS: FSIVGTT was carried out in 14 lean (BMI 25 kg/m(2)) subjects using two different protocols; full (29 samples) and short (12 samples). For reproducibility assessment four normal subjects (triplicate on three and twice on one) were recruited to undergo the same procedure at 1-week intervals. Data analysis was performed using COMAL and Minmod Millennium software. RESULTS: Mean Si (10(-4)min(-1)[pmol/l](-1)) values were significantly different between lean and overweight subjects (p < 0.001) but not between the two protocols using both software packages. For the full and short protocols, Si values were more closely related in lean versus overweight subjects using either COMAL (r = 0.98, p < 0.001), (r = 0.89, p < 0.001) or Minmod Millennium (r = 0.99, p < 0.001), (r = 0.85, p < 0.001) software respectively. The intra-individual reproducibility (%CV) of Si (COMAL) in full versus short protocol was 18.3 +/- 11.1% and 13.7 +/- 1.9% respectively. Reproducibility values for Si (Minmod Millenium) in full versus short protocols were 14.3 +/- 3.8 and 14.9 +/- 1.9% respectively. CONCLUSIONS: Si can be assessed accurately by a short protocol FSIVGTT in normal individuals. The short protocol may give less acceptable results for insulin sensitivity in individuals who have normal glucose tolerance but high BMI.

Serum insulin-like growth factor binding protein-1: an improvement over other simple indices of insulin sensitivity in the assessment of subjects with normal glucose tolerance.

BACKGROUND: Insulin resistance is associated with an increased risk of cardiovascular disease and diabetes. It can be assessed using complex reference techniques, such as clamp or frequently sampled intravenous glucose tolerance test (FSIVGTT). Therefore, simple indices derived from fasting insulin and glucose concentrations have been proposed. The aim of this study is to assess fasting serum insulin-like growth factor binding protein-1 (IGFBP-1) as a simple index of insulin sensitivity compared with other simple indices and FSIVGTT. METHODS: Fasting serum IGFBP-1, fasting plasma insulin (FPI), homeostasis model assessment (HOMA-IR), quantitative insulin check index (QUICKI), fasting glucose to insulin ratio (FGIR), Raynaud and insulin glycaemic index (ISI-gly) were correlated with FSIVGTT (Si) in 22 subjects with normal glucose tolerance (NGT) and nine with impaired fasting glucose (IFG). RESULTS: In NGT individuals, IGFBP-1 correlated more strongly with Si than did any other index both before (r = 0.76) and after (r = 0.79) natural logarithm (ln) transformation. In subjects with IFG, IGFBP-1 was weakly correlated with Si before and after ln-transformation (r = 0.55, r = 0.56, respectively), but ISI-gly was the index most strongly correlated with Si (r = 0.77, r = 0.85, respectively). CONCLUSIONS: In subjects with NGT, fasting serum IGFBP-1 could be used as a simple reliable marker of insulin sensitivity. For more accurate estimation of insulin sensitivity in normal subjects and those with IFG, ln-transformation is preferred over raw data.

Bromide-sulfur interchange: ion chromatographic determination of total reduced thiol levels in plasma.

Plasma thiol concentration has long been recognised as a potential indicator for assessing the severity of oxidative stress processes within physiological systems. While such measurements are normally restricted to research studies, this communication has sought to develop and characterise a novel approach through which this parameter could be exploited within routine clinical settings. The protocol is based on the rapid derivatisation of reduced thiol functionalities (protein and monomolecular moieties) through the homogenous reaction of a naphthoquinone bromide derivative. Bromide released in the reaction can be easily quantified through ion chromatography (Isocractic Dionex DX-120 incorporating an IonPac AS14 anion exchange column and a 25 microL sample loop with conductivity detector. Mobile phase consisted sodium carbonate/bicarbonate (3.5 mM/1 mM) at a flow rate of 1.5 mL/min). Method selectivity and sensitivity has been critically evaluated. The technique covers the range 15 microM-3.5 mM PSH with a detection limit of 9 microM PSH and analysis time of 5 min. The efficacy of the approach for the analysis of human plasma from five volunteers was assessed (ranging from 49 to 72 microM with an intra assay variation of less than 5% in all cases). The responses were validated through comparison with the standard Ellman colorimetric technique.

Serum zinc and copper status in dyslipidaemic patients with and without established coronary artery disease.

INTRODUCTION: Aspects of trace element status have previously been investigated as possible contributory factors to atherosclerosis. In this present study a more comprehensive approach has been taken, looking at the relationship between dietary macro- and micronutrient intake, serum concentrations of zinc and copper, and markers of inflammation in dyslipidaemic patients with or without established coronary artery disease (CAD) and healthy controls, so that a clearer understanding of the potential relationship between copper and zinc status and coronary disease may be ascertained. METHODS AND MATERIALS: Dyslipidaemic patients (n = 238) were recruited from the local General Hospital in Guildford, UK. Fifty-five of these patients had established CAD. Control subjects (n = 135) were recruited from among employees at the local University and Hospital. A validated food frequency questionnaire was used for estimating the dietary intake of zinc and copper. RESULTS: Serum copper, copper/caeruloplasmin ratio, zinc/copper ratio, and C-reactive protein (CRP) were significantly different in the patient groups compared to controls [serum copper: 17.20 +/- 0.2 v 15.91 +/- 0.29 micromol/L, p < 0.001; copper/caeruloplasmin ratio: 111.37 +/- 2.18 v 100.63 +/- 2.93 micromol/g, p < 0.01; zinc/copper ratio: 0.85 +/- 0.01 v 0.90 +/- 0.01, p < 0.05; and CRP: 1.25 (0.42-3.26) v 0.58 (0.17-1.42) mg/L, p < 0.001]. Dietary protein, total fat, starch, fibre, monounsaturated fat, zinc, and zinc/copper ratio were also significantly higher in the patients compared to controls. Patients with established CAD had significantly higher serum CRP (p < 0.05) and lower serum zinc (p < 0.01) and zinc/copper ratio (p < 0.01) compared to both patients without CAD and healthy controls. CONCLUSION: Significant differences in copper and zinc status, dietary intake and markers of inflammation were observed in patients with dyslipidaemia, with or without established CAD, compared with control subjects. Differences in serum CRP, copper and caeruloplasmin may be related to a heightened state of inflammation. The imbalance in zinc/copper metabolism may either contribute to the CAD risk or be a consequence of an acute phase response.

Manganese Provision in Parenteral Nutrition: An Update.

Manganese (Mn) is an essential micronutrient required for the activity of metalloenzymes. It is an essential component of parenteral nutrition (PN), but requirements are low. Mn status is difficult to assess, with the commonest method being measurement of its concentration in whole blood. This method has limitations, including artifactually high concentrations resulting from contamination of specimen tubes. Mn toxicity is a well-recognized complication of PN, the risk of which increases if there is cholestasis or if the patient has received high doses. It usually presents with parkinsonian-like symptoms but may be detected presymptomatically as hypermanganesemia or as increased signal intensity of the basal ganglia upon T1-weighted magnetic resonance imaging. Caution is necessary when providing Mn for patients on long-term PN (>1 month). It is advisable to withhold supplementation if hypermanganesemia or cholestasis develops. Deficiency of Mn is rare in patients treated with PN. PN regimens are contaminated with Mn in amounts likely to meet requirements. Consequently, it is debated whether PN should be routinely supplemented with Mn. The currently recommended dose of Mn in adults treated with PN is 55 µg/d, but the doses provided by most currently available multi-trace element products exceed this. In response to calls for new products to be developed, 2 new multi-trace element products are currently available in Europe that provide Mn doses of 55 µg/d. Once these products are in general use, it is likely that the incidence of Mn toxicity will decrease.

Solid state differentiation of plasma thiols using a centrifugally activated mercaptobenzothiazole disulfide exchange indicator.

The solid state interaction of mono and macromolecular thiols at a disulfide heterocycle is shown to provide a versatile pathway for their speciation.

The biochemical assessment of insulin resistance.

Insulin resistance is a common condition, recognized to be a central feature of the metabolic syndrome, and strongly associated with an increased risk of cardiovascular disease and diabetes. The quantitative assessment of insulin sensitivity is not used for routine clinical purposes, but the emerging importance of insulin resistance has led to its wider application to research studies that have examined its pathogenesis, aetiology and consequences. The gold standard method for the determination of insulin sensitivity is the euglycaemic hyperinsulinaemic clamp from which indices of insulin sensitivity can be derived. The clamp technique is both expensive and complex to undertake and has prompted the use of surrogate methods, notably the insulin tolerance test and frequently sampled intravenous glucose tolerance test. Indices may be derived from these methods and correlate well with those derived from clamp studies. Indices can also be derived from measurements made during a standard oral glucose tolerance test and from one-off fasting specimens (e.g. homeostasis model assessment and quantitative insulin sensitivity check index). These indices lend themselves for use in large population studies where a relatively simple, inexpensive assessment is necessary. However, these tests all suffer from important limitations, including poor precision. Insulin resistance is increasingly being assessed in clinical situations, where relatively simple markers are required. Insulin-like growth factor binding protein-1 is an emerging marker which may be useful in this context.

A chromatographic tool for preparing combinatorial quinone-thiol conjugate libraries.

Quinones are well established as key players in the production of reactive oxygen species within cellular environments. Many factors govern their cytotoxicity but most studies have been restricted to a few, core, derivatives. A new strategy for the in situ production of quinone derivatives has been developed such that libraries of diverse functionality can be rapidly created without recourse to extensive synthetic procedures. The approach relies upon nucleophilic addition by reduced thiol derivatives to the quinone core within a pre-culture assay mixture and provides a generic strategy that exploits the large reservoir of commercial thiols currently available. A readily accessible chromatographic method has been developed that allows the derivatisation process to be easily monitored and the purity of the resulting one pot preparation to be assessed. The viability of the combinatorial approach has been fully validated through comparison with a range of quinone-S-conjugates prepared using conventional bench synthesis. The latter have been fully characterised.

Review of Copper Provision in the Parenteral Nutrition of Adults [Formula: see text].

The essential trace element copper (Cu) is required for a range of physiologic processes, including wound healing and functioning of the immune system. The correct amount of Cu must be provided in parenteral nutrition (PN) if deficiency and toxicity are to be avoided. While provision in line with the standard recommendations should suffice for most patients, Cu requirements may be higher in patients with increased gastrointestinal losses and severe burns and lower in those with cholestasis. The tests of Cu status that are currently available for clinical use are unreliable. Serum Cu concentration is the most commonly ordered test but is insensitive to Cu deficiency and toxicity and is misleadingly increased during the acute phase response. These limitations make it difficult for prescribers to assess Cu status and to decide how much Cu to provide. There is a need for better tests of Cu status to be developed to decrease uncertainty and improve individualization of Cu dosing. More information is needed on Cu requirements in disease and Cu contamination of PN components and other intravenous fluids. New multi-trace element products should be developed that provide Cu doses in line with the 2012 American Society for Parenteral and Enteral Nutrition recommendations. This article discusses the evaluation and treatment of Cu deficiency and toxicity in patients treated with PN.

Review of Copper Provision in the Parenteral Nutrition of Adults.

The essential trace element copper (Cu) is required for a range of physiologic processes, including wound healing and functioning of the immune system. The correct amount of Cu must be provided in parenteral nutrition (PN) if deficiency and toxicity are to be avoided. While provision in line with the standard recommendations should suffice for most patients, Cu requirements may be higher in patients with increased gastrointestinal losses and severe burns and lower in those with cholestasis. The tests of Cu status that are currently available for clinical use are unreliable. Serum Cu concentration is the most commonly ordered test but is insensitive to Cu deficiency and toxicity and is misleadingly increased during the acute phase response. These limitations make it difficult for prescribers to assess Cu status and to decide how much Cu to provide. There is a need for better tests of Cu status to be developed to decrease uncertainty and improve individualization of Cu dosing. More information is needed on Cu requirements in disease and Cu contamination of PN components and other intravenous fluids. New multi-trace element products should be developed that provide Cu doses in line with the 2012 American Society for Parenteral and Enteral Nutrition recommendations. This article discusses the evaluation and treatment of Cu deficiency and toxicity in patients treated with PN.

Wolman's disease and cholesteryl ester storage disorder: the phenotypic spectrum of lysosomal acid lipase deficiency.

Lysosomal acid lipase deficiency is a rare, autosomal recessive condition caused by mutations in the gene encoding lysosomal acid lipase (LIPA) that result in reduced or absent activity of this essential enzyme. The severity of the resulting disease depends on the nature of the underlying mutation and magnitude of its effect on enzymatic function. Wolman's disease is a severe disorder that presents during infancy, resulting in failure to thrive, hepatomegaly, and hepatic failure, and an average life expectancy of less than 4 months. Cholesteryl ester storage disorder arises later in life and is less severe, although the two diseases share many common features, including dyslipidaemia and transaminitis. The prevalence of these diseases has been estimated at one in 40 000 to 300 000, but many cases are undiagnosed and unreported, and awareness among clinicians is low. Lysosomal acid lipase deficiency-which can be diagnosed using dry blood spot testing-is often misdiagnosed as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hereditary dyslipidaemia, or cryptogenic cirrhosis. There are no formal guidelines for treatment of these patients, and treatment options are limited. In this Review we appraise the existing literature on Wolman's disease and cholesteryl ester storage disease, and discuss available treatments, including enzyme replacement therapy, oral lipid-lowering therapy, stem-cell transplantation, and liver transplantation.

Lithium: a review of its metabolic adverse effects.

Treatment with lithium has long been recognized to be associated with metabolic adverse effects notably hypothyroidism, hyperparathyroidism, weight gain and nephrogenic diabetes insipidus. It is important that clinicians prescribing lithium are aware of these adverse effects and have a strategy for their detection and management. We review aspects of these actions of lithium including their prevalence, risk factors, biochemical changes involved and management, and discuss some advances that have been made in their understanding in recent years.