Search

Background: Proprotein convertase subtilisin kexin type 9 (PCSK9) is gaining attention as a key regulator of serum LDL-cholesterol (LDLC). This novel serine protease causes the degradation of hepatic LDL receptors by an unknown mechanism. In humans, gain-of-function mutations in the PCSK9 gene cause a form of familial hypercholesterolemia, whereas loss-of-function mutations result in significantly decreased LDLC and decreased cardiovascular risk. Relatively little is known about PCSK9 in human serum.Methods: We used recombinant human PCSK9 protein and 2 different anti-PCSK9 monoclonal antibodies to build a sandwich ELISA. We measured PCSK9 and lipids in 55 human serum samples and correlated the results. We used the anti-PCSK9 antibodies to assay lipoprotein particle fractions separated by sequential flotation ultracentrifugation.Results: Serum concentrations of PCSK9 ranged from 11 to 115 μg/L and were directly correlated with serum concentrations of LDLC (r = 0.45, P = 0.001) and total cholesterol (r = 0.50, P = 0.0003), but not with triglycerides (r = 0.15, P = 0.28) or HDL cholesterol concentrations (r = 0.13, P = 0.36). PCSK9 was not detectable in any lipoprotein particle fraction, including LDL.Conclusions: PCSK9 is present in human serum, likely not associated with specific lipoprotein particles. The circulating concentrations of human PCSK9 are directly correlated with LDL and total cholesterol concentrations.

Monoclonal antibody therapeutics typically have relatively long half-lives and can be dosed at high levels. Although formation of anti-drug antibodies (ADA) is relatively rare, detection of these antibodies can be very difficult in the presence of high circulating levels of drug. Typically these ADA are detected by bridging ELISAs which can be very sensitive to even low levels of drug. We describe an ELISA method based on affinity capture of ADA on solid-phase drug followed by removal of excess free drug, release and transfer of bound ADA and subsequent detection using biotinylated drug. The assay is both sensitive and highly tolerant to free drug with detection of 500 ng/ml of ADA readily achieved in the presence of 500 mug/ml of drug.

Background The role of apolipoprotein A5 (ApoA5) in modulating triglyceride levels in humans is incompletely understood. Some researchers have reported modest positive correlations of ApoA5 with triglycerides while others have reported negative correlations. A recent report suggested that ApoA5 gene expression may be influenced by insulin. In type 2 diabetes, some groups have reported higher levels of ApoA5 compared to normals while others have reported lower levels. Methods To better understand the relationships between ApoA5, apolipoprotein C3 (ApoC3), and triglycerides in type 2 diabetes, ApoA5 levels were measured and correlated with triglyceride, insulin, and HbA1c levels. ApoC3 levels were measured and correlated with triglycerides. Results In patients with type 2 diabetes, ApoA5 levels were elevated compared to normals, with several patients having markedly increased levels confirmed by Western blotting. ApoA5 levels were positively correlated with triglycerides ( r = 0.60) but were not correlated with either HbA1c or serum insulin levels. ApoC3 levels were highly positively correlated with triglycerides ( r = 0.88). Conclusions These data indicate that in patients with type 2 diabetes ApoA5 levels are positively correlated with triglycerides but are not correlated with HbA1c or insulin levels. ApoC3 levels are strongly positively correlated with triglycerides in these patients.

Objective Hepcidin reduces iron absorption by binding to the intestinal iron transporter ferroportin, thereby causing its degradation. Although short-term administration of testosterone or growth hormone (GH) has been reported to decrease circulating hepcidin levels, little is known about how hepcidin is influenced in human endocrine conditions associated with anemia. Research design and methods We used a sensitive and specific dual–monoclonal antibody sandwich immunoassay to measure hepcidin-25 in patients (a) during initiation of in vitro fertilization when endogenous estrogens were elevated vs. suppressed, (b) with GH deficiency before and after 12 months substitution treatment, (c) with hyperthyroidism before and after normalization, and (d) with hyperprolactinemia before and after six months of treatment with a dopamine agonist. Results In response to a marked stimulation of endogenous estrogen production, median hepcidin levels decreased from 4.85 to 1.43 ng/mL (p < 0.01). Hyperthyroidism, hyperprolactinemia, or GH substitution to GH-deficient patients did not influence serum hepcidin-25 levels. Conclusions In humans, gonadotropin-stimulated endogenous estrogen markedly decreases circulating hepcidin-25 levels. No clear and stable correlation between iron biomarkers and hepcidin-25 was seen before or after treatment of hyperthyroidism, hyperprolactinemia or growth hormone deficiency.

One of the most prominent symptoms in substance use disorders is a strong desire to consume a particular substance or to show a certain behaviour (craving). The aim of the present project was to modify craving-related neuronal responses in patients with alcohol use disorder (AUD) using neurofeedback (real-time fMRI). Patients with AUD and healthy subjects participated in the study; neurofeedback was an add-on to other therapeutic interventions. Neutral and alcohol-associated pictures were presented during functional MRI measurements. Alcohol-associated BOLD-responses in the frontal cortex were used as a region of interest (ROI). During the neurofeedback sessions, participants were instructed to reduce their neuronal activity in the specific ROI during the presentation of alcohol-associated pictures. The results indicated decreased brain responses after real-time fMRI sessions in alcohol-associated brain areas, especially the anterior cingulate cortex, the insula, inferior temporal gyrus, medial frontal gyrus and cuneus. In addition, the neuronal activity was reduced in the individual ROIs after the neurofeedback training in patients. In future, real-time fMRI may offer a therapeutic strategy to be used in addition to other psychotherapeutic strategies for patients with psychiatric and psychosomatic disorders. This study was supported by LMUexcellent

Background Increased emphasis on the development of biologics has placed a significant focus on anti-drug antibody (ADA) detection. To address this need, several immunoassay formats have been described for use in characterizing potential immune responses. Two commonly utilized methods include the affinity capture elution (ACE) and bridging formats. While these approaches have been effective in supporting many clinical initiatives, both possess potential disadvantages. Here, we compare these standard methods to a novel format that addresses these noted drawbacks. Results A novel assay format has been designed to incorporate the benefits of the ACE and bridging methods while overcoming the disadvantages incurred with each approach. The described ACE–Bridge format exhibits excellent sensitivity and precision while providing superior drug tolerance when compared to bridging formats. Further, this assay format is not susceptible to the endogenous target interference that can be an issue in the ACE format. Conclusions The ACE–Bridge format provides an often superior option as a screening method to monitor patient ADA responses. This method is unique in its ability to measure ADA in the presence of high circulating endogenous target concentrations (> 100 ng/mL) while demonstrating very high drug tolerance.

Pseudotumor cerebri is an idiopathic increase in intracranial pressure, which commonly affects obese women of fertile age. The diagnostic criteria according to the guidelines of the German Society for Neurology are increased cerebrospinal fluid (CSF) pressure, symptoms of increased CSF pressure, normal results of CSF examination, no relevant medication and a lack of structural and vascular lesions in magnetic resonance imaging (MRI).This article presents the case of a 39-year-old male patient who presented at hospital with visual obscuration and recently occurred double vision. Except for a recently diagnosed thrombosis of the left jugular vein of unknown origin, there was nothing else of note in the medical history. Biomicroscopic examination showed papilledema with hemorrhages and cotton wool spots. The CSF opening pressure was initially 50 cmH2O. During therapy by lumbar puncture (three times), oral carbonic anhydrase inhibitors and loop diuretics, the abducens nerve palsy and papilledema receded. Anticoagulation therapy (initially with coumarin derivatives, then with low molecular weight heparins) was unsuccessful in eliminating the thrombosis of the jugular vein. Surgical intervention was not recommended by the vascular surgeons.This case report demonstrates the unusual combination of (idiopathic) intracranial hypertension and thrombosis of the jugular vein, which occurred spontaneously and without any detectable coagulation disorders.

Streptozotocin (STZ) inhibits O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase (O-GlcNAcase), the enzyme that removes O-GlcNAc from proteins. The active site of the enzyme was recently proposed to include aspartates 174, 175, and 177, with STZ inhibition via a transition state analog. We explored the effect of STZ on the tryptic peptide digest pattern of O-GlcNAcase. LC/MS/MS analysis demonstrated that STZ modified two areas of the enzyme. One peptide, LGCFEIAK (894-901), in a C-terminal region previously proposed to possess O-GlcNAcase activity, was methylated by STZ. Another peptide, EYEIEFIYIASPGLDITFSNPK (128-149), was detected only after treatment with STZ and was in an N-terminal region, overlapping a glutamate-rich area containing an adjacent phenylalanine residue. No covalent modification of this peptide could be demonstrated. Detection of this peptide after treatment with STZ was accompanied by the simultaneous inability to detect the nearby peptide KLDQVSQFGCR (157-167), which contains a cysteine residue recently shown to be essential for enzymatic activity. To determine which of the first two peptides might also be important for O-GlcNAcase activity, site-specific mutagenesis was performed. Mutation of the N-terminal phenylalanine and serine residues resulted in almost complete inhibition of activity. In contrast, mutation of conserved C-terminal glycine and cysteine residues caused little inhibition of enzymatic activity. Together, these data extend the region of the active site N-terminally and give independent evidence to support the idea that STZ inhibits O-GlcNAcase through formation of a transition state analog that resides in the active site of the enzyme and in doing so alters its conformation and ensuing tryptic digest pattern.

Purpose Measurements of cytokine release in whole blood after ex vivo stimulation are useful in drug development. The components contributing to variation within such assays have not been clearly defined. Therefore, we characterized the sources of variability within an ex vivo stimulation assay for TNF-α release. Method Fresh whole blood or mononuclear cells from a cell preparation tube were added to silanized, screw-top tubes with a final concentration of 1 μg/mL lipopolysaccharide (LPS). Each tube was purged with 95% air/5%CO 2 and incubated 4 or 6 h at 37 °C in a metabolic water bath. Plasma TNF-α was next measured in supernatants by immunoassay. Total method variability was assessed in 10 normal donors each drawn in the morning and afternoon over 3 days. Four additional samples were pre-treated with dexamethasone to investigate inhibition of TNF-α release. Results Our analysis indicated precise temperature control, the timing and duration of stimulation, and the surface properties of the stimulation vessel most significantly influenced assay performance. A comparison of multiple anticoagulants indicated that careful consideration should be taken in selecting the optimal anticoagulant. The estimated total assay CV for all anticoagulants tested was less than 33.81%. The analytical variability (stimulation and measurement) was less than 25.88% CV. The one exception was mononuclear cells collected in sodium heparin. The total variability estimate incorporated day-to-day, diurnal, inter-donor, tube-to-tube and immunoassay variability. Using our optimized conditions, TNF-α release was inhibited by dexamethasone with a mean IC 50 of 33.3 ± 4.6 nM. Conclusions We have described an optimal set of conditions for collection, storage and processing of an ex vivo cytokine stimulation assay. These conditions were selected for operational feasibility, minimal imprecision and elimination of potential confounding factors. The end result is a more robust method that can be applied to clinical drug development.

Background: Apolipoprotein A5 (ApoA5) originally gained attention as a regulator of serum triglyceride concentrations through transgenic mouse studies. Our group recently developed the first assay to quantify serum ApoA5 protein concentrations and demonstrated that they are increased by administration of a potent peroxisome proliferator-activated receptor-α agonist. Methods: To better characterize the circulating ApoA5, the protein was purified from human serum, and a definitive N-terminal protein sequence was obtained. In light of previous observations that ApoA5 was present in VLDL and not LDL, plasma infranatant and intermediate-density lipoprotein (IDL) were analyzed for ApoA5. Because the mature protein contains a single unpaired cysteine, ApoA5 in human serum was immunoprecipitated, and its migration pattern was examined via Western blotting under reducing and nonreducing conditions to determine whether the protein circulates as a disulfide-linked homodimer or heterodimer. Results: Definitive N-terminal protein sequences obtained from ApoA5 purified from human serum indicated that cleavage of the signal peptide occurs in vivo at the predicted site. We found ApoA5 in VLDL, HDL, and chylomicrons but not in LDL, IDL, or plasma infranatant. Under both reducing and nonreducing conditions, ApoA5 migrated mainly as a single band with a relative molecular mass (Mr) of ∼39 000, indicating that the protein exists in serum as a monomer and not as a disulfide-linked homodimer or heterodimer. Conclusions: Our data help characterize ApoA5 by defining its lipoprotein particle distribution, by determining its N-terminal protein sequence, and by demonstrating that the mature protein circulates mainly as a monomer and not as a disulfide-linked homodimer or heterodimer.

The double antigen bridging immunoassay has been used extensively for detection of immunogenicity responses to therapeutic monoclonal antibodies. We have analyzed parameters affecting performance of this type of immunoassay including microtiter plate antigen coating concentration, enzyme-labeled antigen conjugate dilution and assay format (one-step versus two-step). We present results demonstrating that the format of the assay has a significant impact on the optimal parameters to maximize assay performance. A one-step assay format achieves maximal sensitivity across a broad range of coating concentrations and at a lower concentration of conjugate than that in a two-step format. In contrast, a two-step format requires very low coating concentrations and higher conjugate concentrations to achieve maximal sensitivity and suffers from significantly reduced sensitivity at higher coating concentrations. Together, these findings indicate that a one-step assay format can greatly reduce the effect of coating concentration variation on assay performance.

Development of immunogenicity assays for assessment of human antibodies to therapeutic proteins requires a quantitative determination of assay sensitivity. In the absence of true human positive controls, this is usually accomplished by utilizing affinity-purified antibodies from non-human primates or monoclonal antibodies. In the former case, it is generally considered that non-human primate antibodies will be recognized equally to human antibodies by secondary anti-human immunoglobulin reagents used in immunogenicity assays. We present results here demonstrating that this is not the case. In reality, anti-human immunoglobulin secondary antibodies do not recognize primate immunoglobulins as well as human immunoglobulins. As a result, the use of affinity purified primate antibodies to determine the sensitivity of an immunogenicity assay will likely result in the true sensitivity of the assay being underestimated.

Background: The recently discovered apolipoprotein A5 (ApoA5) is fast gaining attention as a key regulator of serum triglyceride concentrations. An ApoA5 mouse knock-out model produced an approximately fourfold increase in serum triglycerides, whereas a knock-in model with human ApoA5 produced 50–70% lower concentrations of mouse serum triglycerides. In addition, peroxisome proliferator-activated receptor-α agonists, which are used clinically to lower serum triglyceride concentrations, cause increased ApoA5 mRNA expression. Despite these compelling molecular biology data, relatively little is known about ApoA5 protein in human serum. Methods: To better understand circulating concentrations and lipoprotein particle distribution of ApoA5, we expressed the recombinant human ApoA5 protein and raised antibodies against both the NH2 and COOH termini. Results: Using the above reagents, we demonstrate for the first time that ApoA5 is present in human serum, although at much lower concentrations than other apolipoproteins such as ApoA1. Using a dual-antibody sandwich ELISA that we developed, we observed ApoA5 concentrations in human serum ranging from 24 to 406 μg/L compared with ∼1 g/L for ApoA1. We also examined the lipoprotein particle distribution of ApoA5 and found that ApoA5 was detectable in VLDL, HDL, and chylomicrons, but not LDL. Conclusions: These data demonstrate for the first time that ApoA5 is a secreted protein present in human serum and is associated with specific lipoprotein particles. In addition, our data indicate that the circulating concentration of human ApoA5 is very low compared with other apolipoproteins.

Objectives Previously, we demonstrated glucose-induced beta-cell tyrosine phosphorylation of p130Cas, a protein containing 15 YXXP repeats that can become tyrosine phosphorylated and bind Src-homology 2 (SH2)-containing proteins. In light of the importance of p130Cas in other cell types, we determined which beta-cell proteins exhibited glucose-induced association with p130Cas. Methods beta cells were stimulated with glucose and/or the muscarinic agonist carbachol to determine which SH2-containing adapter proteins underwent glucose-induced association with p130Cas. Results The SH2-containing adapter protein Crk underwent glucose-induced association with p130Cas, while other SH2-containing proteins such as grb2, PI3 kinase, Shp-2, paxillin, and pyk2 did not. Glucose-induced Crk-p130Cas association was rapid and sustained and was maximal with the combination of glucose and carbachol, paralleling insulin secretion. There was no increased tyrosine phosphorylation of Crk itself. The expression of Crk in isolated rat islets was also demonstrated. Conclusion beta cells contain the SH2-containing adapter protein Crk, which undergoes glucose-induced association with p130Cas.

We have previously shown that diabetogenic antibiotic streptozotocin (STZ), an analog of N-acetylglucosamine (GlcNAc), inhibits the enzyme O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase (O-GlcNAcase) which is responsible for the removal of O-GlcNAc from proteins. Alloxan, another beta-cell toxin is a uracil analog. Since the O-GlcNAc transferase (OGT) uses UDP-GlcNAc as a substrate, we investigated whether alloxan might interfere with the process of protein O-glycosylation by blocking OGT, a very abundant enzyme in beta-cells. In isolated pancreatic islets, alloxan almost completely blocked both glucosamine-induced and STZ-induced protein O-GlcNAcylation, suggesting that alloxan indeed was inhibiting (OGT). In order to show definitively that alloxan was inhibiting OGT activity, recombinant OGT was incubated with 0-10 mM alloxan, and OGT activity was measured directly by quantitating UDP-[(3)H]-GlcNAc incorporation into the recombinant protein substrate, nucleoporin p62. Under these conditions, OGT activity was completely inhibited by 1 mM alloxan with half-maximal inhibition achieved at a concentration of 0.1 mM alloxan. Together, these data demonstrate that alloxan is an inhibitor of OGT, and as such, is the first OGT inhibitor described.

Economic feasibility is a major factor in the viability of established acute inpatient palliative medicine. Several clinical, administrative, and financial parameters determine the financial health of inpatient care. Financial management metrics include case mix index (CMI) (as determined by the Federal Register as an assigned relative weight to the diagnosis-related group (DRG) reflecting resource consumption), direct costs, indirect costs, contribution margin, and in the future of all patient revised-DRG (APR-DRG). Both census and length of stay will have a major impact on these financial metrics. The type of patient referral and clinical decisions will influence direct costs and revenues. In the future, an international CMI or APR-DRG will allow palliative inpatient units to compare disease severity, resource utilization, and outcome measures.

Pancreatic lipase is produced and stored in pancreatic acinar cells, and is normally secreted into the duct system. In disorders that cause pancreatic damage, pancreatic lipase enters the circulation, and serum lipase activity becomes useful in the diagnosis and evaluation of pancreatitis. During the last decade, many lipase assays have used the Imamura method, in which a series of enzymatic reactions utilizing a diglyceride substrate leads to the formation of a colorimetrically detected product. Historically, this method has been prone to significant interference from various substances, including glycerol itself. In the light of these limitations, we evaluated an automated enzymatic rate assay that uses a non‐diglyceride‐based, pancreatic lipase‐specific substrate. Precision, linearity, and potential interference were assessed, and when compared to the Imamura method, the non‐diglyceride‐based assay exhibited a slope of 0.475, y‐intercept of 15.89, r‐value of 0.9516, and S(y,x) of 12.96. Similar results were also observed when the two assays were compared using samples with markedly elevated creatinine levels. Between‐day coefficients of variance (CVs) ranged from 5.0% to 5.5%, which compared well with the diglyceride‐based method, and linearity spanned a range of 3–156 U/L. Evaluation of over 2,000 patient results collected during a 6‐month period suggested that the manufacturer’s upper reference limit of 51 U/L may be too conservative. No significant interference was identified with bilirubin, triglyceride, or glycerol itself, and significant interference from hemoglobin was observed only at concentrations of 116 mg/dL or greater. As a result, the non‐diglyceride‐based method provides an acceptable alternative for the routine laboratory measurement of lipase activity. J. Clin. Lab. Anal. 16:52–55, 2002. © 2002 Wiley‐Liss, Inc.

Gut-derived hormones, such as GLP-1, have been proposed to relay information to the brain to regulate appetite. GLP-1 receptor agonists, currently used for the treatment of type 2 diabetes (T2DM), improve glycemic control and stimulate satiety, leading to decreases in food intake and body weight. We hypothesized that food intake reduction after GLP-1 receptor activation is mediated through appetite- and reward-related brain areas. Obese T2DM patients and normoglycemic obese and lean individuals (n = 48) were studied in a randomized, crossover, placebo-controlled trial. Using functional MRI, we determined the acute effects of intravenous administration of the GLP-1 receptor agonist exenatide, with or without prior GLP-1 receptor blockade using exendin 9-39, on brain responses to food pictures during a somatostatin pancreatic-pituitary clamp. Obese T2DM patients and normoglycemic obese versus lean subjects showed increased brain responses to food pictures in appetite- and reward-related brain regions (insula and amygdala). Exenatide versus placebo decreased food intake and food-related brain responses in T2DM patients and obese subjects (in insula, amygdala, putamen, and orbitofrontal cortex). These effects were largely blocked by prior GLP-1 receptor blockade using exendin 9-39. Our findings provide novel insights into the mechanisms by which GLP-1 regulates food intake and how GLP-1 receptor agonists cause weight loss.

Homozygous carriers of the apolipoprotein 62 allele are at risk of type III hyperlipidaemia, but do not necessarily develop this lipid disorder. In the present study, we have investigated the role of circulating PCSK9 (pro-protein convertase subtilisin kexin type 9), an important regulator of LDL (low-density lipoprotein) receptor expression, in the development of this hyperlipidaemic phenotype. In an observational study, plasma PCSK9 was measured in homozygous carriers of apolipoprotein epsilon 2 (epsilon 2/epsilon 2; n=12), normal controls (n = 72) and hypertriglyceridaemic patients with FCHL (familial combined hyperlipidaemia; n = 38), who served as a hyperlipidaemic reference group. Cholesterol, triacylglycerols (triglycerides) and apolipoprotein B content in VLDL (very-low-density lipoprotein) and LDL particles was determined by ultracentrifugation in epsilon 2/epsilon 2 and FCHL patients. Median circulating PCSK9 levels did not differ between epsilon 2/epsilon 2 carriers compared with controls and hypertriglyceridaemic FCHL patients (84.5 compared with 82.0 and 84.9 ng/ml; P = 0.2 and 0.6 respectively). Circulating PCSK9 was associated with total cholesterol and triacylglycerols levels in epsilon 2/epsilon 2 carriers (P

Proprotein convertase subtilisin-kexin type 9 (PCSK9) is a secreted protein which regulates serum LDL cholesterol. It circulates in human and rodent serum in an intact form and a major truncated form. Previous in vitro studies involving the expression of human PCSK9 genetic variants and in vivo studies of furin knockout mice suggest that the truncated form is a furin cleavage product. However, the circulating truncated form of PCSK9 has not been isolated and characterized. Utilizing antibodies which bind to either the catalytic domain or the C-terminal domain of PCSK9, the truncated PCSK9 was isolated from serum. MS was used to determine that this form of PCSK9 is a product of in vivo cleavage at Arg218 resulting in pyroglutamic acid formation of the nascent N terminus corresponding to Gln219 of intact PCSK9. We also determined that the truncated PCSK9 in serum lacked the N-terminal segment which contains amino acids critical for LDL receptor binding. A truncated PCSK9, expressed and purified from HEK293 cells with identical composition as the circulating truncated protein, was not active in inhibition of LDL uptake by HepG2 cells. These studies provide a definitive characterization of the composition and activity of the truncated form of PCSK9 found in human serum.

We have previously shown that rat pancreatic islets contain a predominant 135 kDa O-glycosylated protein (p135) that is recognized by immunoprecipitation and Western blotting with anti-O-GlcNAc antibody. In this paper, we show that p135 is also detectable in other rat tissues including brain, heart, liver, spleen, and lung, but not kidney. To identify p135, the protein was purified from rat brain using a multistep procedure including selective absorption with anti-O-GlcNAc antibody. After electrophoresis, and Coomassie staining, the protein was excised from the gel for tryptic digestion. Next, O-methylisourea was used to convert lysine residues to homoarginine to increase the sequence coverage, and MALDI-TOF mass spectrometry detection was performed. MALDI-TOF identified p135 as rat O-GlcNAc transferase (OGT), an identity confirmed by LC/MS of individual peptides. The identification of p135 as OGT is consistent with previous reports of the tissue distribution of OGT, as well as reports that OGT is itself O-glycosylated.

Streptozotocin (STZ), an analogue of GlcNAc, inhibits purified rat spleen O-GlcNAc-selective N-acetyl-β-d-glucosaminidase (O-GlcNAcase), the enzyme that removes O-GlcNAc from protein. We have shown previously that STZ increases pancreatic islet O-linked protein glycosylation. In light of these data, we investigated the possibility further that STZ causes β-cell death by inhibiting O-GlcNAcase. In isolated islets, the time course and dose curve of STZ-induced O-glycosylation correlated with β-cell toxicity. STZ inhibition of rat islet O-GlcNAcase activity also paralleled that of its β-cell toxicity, with significant inhibition occurring at a concentration of 1mM. In contrast, STZ inhibition of rat brain O-GlcNAcase and β-TC3 insulinoma cell O-GlcNAcase was significantly right-shifted compared with islets, with STZ only significantly inhibiting activity at a concentration of 5mM, the same concentration required for β-TC3 cell toxicity. In comparison, N-methyl-N-nitrosourea, the nitric oxide-donating portion of STZ, did not cause increased islet O-glycosylation, β-cell toxicity or inhibition of β-cell O-GlcNAcase. Enhanced STZ sensitivity of islet O-GlcNAcase compared with O-GlcNAcase from other tissues or an insulinoma cell line suggests why actual islet β-cells are particularly sensitive to STZ. Confirming this idea, STZ-induced islet β-cell toxicity was completely blocked by GlcNAc, which also prevented STZ-induced O-GlcNAcase inhibition, but was not even partially blocked by glucose, glucosamine or GalNAc. Together, these data demonstrate that STZ's inhibition of β-cell O-GlcNAcase is the mechanism that accounts for its diabetogenic toxicity.

Background: Anemia and vitamin D deficiency are highly prevalent in critical illness, and vitamin D status has been associated with hemoglobin concentrations in epidemiologic studies. We examined the effect of high-dose vitamin D therapy on hemoglobin and hepcidin concentrations in critically ill adults. Materials and Methods: Mechanically ventilated critically ill adults (N = 30) enrolled in a pilot double-blind, randomized, placebo-controlled trial of high-dose vitamin D3 (D3) were included in this analysis. Participants were randomized to receive placebo, 50,000 IU D3, or 100,000 IU D3 daily for 5 days (totaling 250,000 IU D3 and 500,000 IU D3, respectively). Blood was drawn weekly throughout hospitalization for up to 4 weeks. Linear mixed-effects models were used to assess change in hemoglobin and hepcidin concentrations by treatment group over time. Results: At enrollment, >75% of participants in all groups had plasma 25-hydroxyvitamin D (25(OH)D) concentrations 85% of participants ac...

Item does not contain fulltext BACKGROUND AND AIMS: Two recent independent studies showed that patients with familial combined hyperlipidemia (FCHL) have elevated plasma levels of proprotein convertase subtilisin kexin type 9 (PCSK9) and markers of cholesterol synthesis. Both PCSK9 expression and cholesterol synthesis are downstream effects of hepatic activation of sterol regulatory element binding protein 2 (SREBP2). The present study was conducted to study the relationship between plasma PCSK9 and markers of cholesterol synthesis in FCHL. METHODS AND RESULTS: Markers of cholesterol synthesis (squalene, desmosterol, lathosterol), cholesterol absorption (campesterol, sitosterol, cholestanol) and PCSK9 were measured in plasma of FCHL patients (n = 103) and their normolipidemic relatives (NLR; n = 240). Plasma PCSK9, lathosterol and desmosterol levels were higher in FCHL patients than their NLR (p < 0.001, age and sex adjusted). Heritability calculations demonstrated that 35% of the variance in PCSK9 levels could be explained by additive genetic effects (p < 0.001). Significant age- and sex-adjusted correlations were observed for the relationship between PCSK9 and lathosterol, both unadjusted and adjusted for cholesterol, in the overall FCHL population (both p < 0.001). Multivariate regression analyses, with PCSK9 as the dependent variable, showed that the regression coefficient for FCHL status decreased by 25% (from 0.8 to 0.6) when lathosterol was included. Nevertheless, FCHL status remained an independent contributor to plasma PCSK9 (p < 0.001). CONCLUSIONS: The present study confirms the previously reported high and heritable PCSK9 levels in FCHL patients. Furthermore, we now show that high PCSK9 levels are, in part, explained by plasma lathosterol, suggesting that SREBP2 activation partly accounts for elevated PCSK9 levels in FCHL.

Our goal was to consolidate different methodologies in the clinical chemistry laboratory and replace aging analyzers; therefore, we evaluated the newly available Beckman Coulter LX20 (Brea, CA). Results were obtained for linearity, within- and between-day precision, correlation, interference, and serum-vs-plasma studies. Satisfactory precision results were obtained, with most assays demonstrating within-day coefficients of variation less than 2% and between-day coefficients of variation less than 5%. The linearity for all assays was acceptable over the range tested. Correlation results were adequate. The major difference in serum-vs-plasma studies was potassium. The only significant interferences noted were that lipemia decreased uric acid and bilirubin results, while hemolysis increased potassium results. We conclude that the LX20 demonstrates good performance capabilities, making this instrument suitable for a medium- to high-volume laboratory.

Objective. The objective of this study was to compare the cost (measured as resource utilization by the institution) and clinical effectiveness of transcatheter coil occlusion and surgical patent ductus arteriosus (PDA) closure. Similar comparisons have been made previously with other devices no longer in use in the United States. No such comparison has been made for coil occlusion, which has been performed increasingly since 1992. Methods. All patients who underwent either coil or surgical closure of uncomplicated PDA at our institution between August 1993 and June 1996 were retrospectively identified. Patients were included in the study if they were eligible for either closure technique. Thus, they had a restrictive PDA (not associated with pulmonary hypertension) and no overt evidence of congestive heart failure. Patients were excluded if they had other significant cardiac or noncardiac problems. Total procedural and recovery costs (including labor, material, equipment, and overhead) incurred by the provider were determined using a cost accounting system called Transition Systems, Inc. To define further how costs differed for the two techniques, total costs were subdivided into the categories of professional, technical, inpatient hospital stay, postprocedure testing, and supplies and other miscellaneous costs. PDA closure rates and associated complications also were compared. Follow-up information was sought from outpatient visits to our institution or by contacting the referring physicians. Results. A total of 39 patients were identified, 3 of whom were excluded because of coexisting medical problems. The study group consisted of 36 patients; 24 underwent PDA coil occlusion and 12 surgical closure. Mean age and weight were 8.8 years and 28.5 kg for the coil patients, and 7.3 years and 32.8 kg for the surgical patients. Median procedural duration was 150 minutes for the coil group and 165 minutes for the surgical group. The total cost to the institution of coil occlusion was significantly lower than that of surgical closure ($5273 vs $8509). The largest difference lay in the cost of hospital stay ($398 vs $2566) and in the professional costs ($1506 vs $2782). Technical costs were similar ($2156 for coil, $2151 for surgery), although use of the catheterization laboratory per unit of time was more expensive than use of the operating room ($800 vs $400 per hour). Additional technical costs of the surgical procedure related to general anesthesia and postoperative care made up the difference. No patient in either group had a residual PDA murmur at hospital discharge or thereafter. Follow-up echocardiograhy was performed in all coil occlusion patients, and tiny residual leaks were detected in 17%. Only 42% of the surgical patients had postoperative echocardiography; none had residual leaks. There were no deaths or major complications in either group. Conclusions. Transcatheter coil occlusion is as effective and less costly than surgical closure if silent residual leaks are not considered clinically significant. This information may be used increasingly in patient care decisions in the current era of managed medical care.

BACKGROUND: Hepcidin‐25 regulates iron homeostasis by binding the iron transporter ferroportin, causing its degradation. Increased hepcidin‐25 causes decreased intestinal iron absorption and release from intracellular stores. Our objective in this study was to measure hepcidin‐25 levels in patients with chronic kidney disease (CKD) to determine if they might contribute to the anemia of CKD. METHODS: We used a hepcidin‐25‐specific enzyme‐linked immunosorbent assay to measure hepcidin‐25 in 103 CKD patients and 100 healthy individuals. We assessed in CKD subjects the correlation of hepcidin‐25 with creatinine, estimated glomerular filtration rate (eGFR), hemoglobin, blood urea nitrogen, serum iron, transferrin, and ferritin. RESULTS: Hepcidin‐25 concentrations in CKD patients were significantly increased compared to healthy subjects (60.4 ± 6.1 μg/l vs. 3.0 ± 0.5 μg/l, P < 0.001). Hepcidin‐25 concentrations were directly correlated with creatinine (R = 0.28, P = 0.004) and inversely correlated with eGFR (R = −0.32, P = 0.001). Hepcidin‐25 levels were also correlated with transferrin (R = −0.28, P = 0.004) and ferritin (R = 0.80, P < 0.001). CONCLUSION: The direct correlation of hepcidin‐25 with creatinine and its inverse correlation with eGFR suggest that hepcidin‐25 levels increase as renal function deteriorates, possibly due to decreased hepcidin‐25 renal clearance.

The aim of this study was to validate new assays for measurement of amyloid-β (Aβ) peptides in cerebrospinal fluid (CSF) and plasma specimens in clinical studies of solanezumab according to current regulatory recommendations. Four assays based on the INNOTEST® β-AMYLOID(1-42) and prototype INNOTEST β-AMYLOID(1-40) kits were developed and validated. To render these assays 'solanezumab-tolerant', excess drug was added to calibrators, quality control, and test samples via a 2-fold dilution with kit diluent. Validation parameters were evaluated by repeated testing of human CSF and EDTA-plasma pools containing solanezumab. Calibration curve correlation coefficients for the four assays were ≥0.9985. Intra- and inter-assay coefficients of variation for Aβ1-40 and Aβ1-42 were ≤13 and ≤15%, respectively for both matrices. Dilutional linearity, within and between assays, was demonstrated for both analytes in CSF and plasma at clinically relevant dilution factors. This dilution regimen was successfully applied during Phase 3 clinical sample analysis. Aβ1-40 and Aβ1-42 were stable in CSF and plasma containing solanezumab at 2-8°C and room temperature for up to 8 h and during 5 additional freeze-thaw cycles from ≤-20 and ≤-70°C. Results of parallel tests on stored clinical samples using INNOTEST methods and proprietary ELISA methods were closely correlated (r2 > 0.9), although bias in reported concentrations was observed between assays. In conclusion, the modified INNOTEST assays provided (relatively) accurate and precise quantification of Aβ1-40 and Aβ1-42 in CSF and plasma containing solanezumab according to established consensus validation criteria. The clinical experience with these assays post validation has shown them to be robust and reliable.

Information on the influence of the mass of the helmet on the pattern of head injuries suffered by motorcyclists involved in collisions is scarce. This study was undertaken to verify a possible connection between the weight of the helmet worn and the occurrence of a ring fracture of the base of the skull surrounding the foramen magnum.One hundred twenty-two fatally injured motorcyclists were studied retrospectively. In all cases, an autopsy had been performed. Data, including the autopsy report, were obtained from official police files. All helmets were studied in a technical laboratory. Statistical tests were performed using ANOVA, Fisher's exact test, Student's t test, and the chi 2 test. A p0.05 was considered significant.The overall incidence of this type of injury was 9.2%. There was a positive correlation between the incidence of complete or partial circular fractures of the base of the skull and the weight of the involved helmet. There was a significant increase (p = 0.012) in the incidence of this type of fracture when the helmet weighed more than 1,500 grams. An increase in the dynamic active mass caused by the combination of head and helmet leads to a supramaximal stress load during a collision, resulting in such injuries.In accidents with axial load shift, helmets weighing more than 1,500 grams increase the risk of a basal skull fracture. Therefore high-weight helmets should be avoided.

There has been a large amount of recent literature suggesting that omega-3 unsaturated fatty acids found in fish oils should be incorporated into the diet for the purpose of decreasing serum cholesterol levels. Inclusion of these fatty acids in the diet has been shown to decrease total serum cholesterol as well as low-density lipoprotein cholesterol. Some of these trials have been complicated by the fact that many of the subjects are afflicted with non-insulin-dependent diabetes mellitus. Unfortunately, the effects of omega-3 unsaturated fatty acids on insulin secretion have not been well characterized. In this study, we have examined the effect of a common omega-3 unsaturated fatty acid, eicosapentaenoic acid (C20:5), on insulin secretion. Using the beta-TC3 insulinoma cell line as a model system for studying insulin exocytosis, C20:5 selectively potentiated glucose-induced insulin secretion. At the same concentration at which it significantly increased glucose-induced insulin secretion, C20:5 did not affect glucose metabolism or intracellular free calcium concentrations. C20:5 also augmented potassium-induced insulin secretion. These data suggest that C20:5, an abundant omega-3 unsaturated fatty acid, acts to augment insulin secretion in a glucose-dependent manner.

In this study, we demonstrate that stimulation of beta cells with carbachol and glucose causes increased tyrosine phosphorylation of a 125-kDa protein concurrently with increased insulin secretion. The effect was observed in two different insulin-secreting cell lines and in rat pancreatic islets. Tyrosine phosphorylation was largely calcium independent and occurred within 2 min after stimulation of beta cells with glucose and the muscarinic agonist carbachol. In islets, the effect of glucose was greatly diminished by the addition of mannoheptulose, a seven-carbon sugar that inhibits glucokinase, suggesting that glucose metabolism is required for tyrosine phosphorylation of the protein to occur. Neither insulin nor insulin-like growth factor I significantly increased tyrosine phosphorylation of the 125-kDa protein, suggesting that it was not an autocrine effect. Depolarization of beta cells with glyburide or 50 mM potassium dramatically increased insulin secretion but had no significant effect on tyrosine phosphorylation. Addition of phorbol ester caused a less than 2-fold increase in tyrosine phosphorylation, whereas the calcium ionophore A23187 had no effect. Among the various fuel secretagogues tested, only D-glucose stimulated tyrosine phosphorylation, both alone and in combination with carbachol. Finally, the tyrosine kinase inhibitor AG879 inhibited both tyrosine phosphorylation and insulin secretion in a dose-dependent manner. Taken together, these data demonstrate the presence of a novel signaling pathway in glucose-induced insulin secretion: tyrosine phosphorylation of beta cell p125, which is a proximal step in insulin secretion. Our current working hypothesis is that glucose stimulation of beta cell p125 tyrosine phosphorylation is an essential step for insulin secretion.

Glucose is the primary stimulus for insulin secretion by pancreatic β-cells, and it triggers membrane depolarization and influx of extracellular Ca2+. Cholinergic agonists amplify insulin release by several pathways, including activation of phospholipase C, which hydrolyzes membrane polyphosphoinositides. A novel phospholipid, phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3[, a product of phosphatidylinositol 3-kinase (PI 3-kinase), has recently been found in various cell types. We demonstrate by immunoblotting that PI 3-kinase is present in both cytosolic and membrane fractions of insulin-secreting β-TC3 cells and in rat islets. The catalytic activity of PI 3-kinase in immunoprecipitates of islets and β-TC3 cells was measured by the production of radioactive phosphatidylinositol 3-monophosphate from phosphatidylinositol (Ptdlns) in the presence of [γ-32P[ATP. Wortmannin, a fungal metabolite, dose dependency inhibited PI 3-kinase activity of both islets and P-TC3 cells, with an IC50 of 1 nmol/l and a maximally effective concentration of 100 nmol/l, when it was added directly to the kinase assay. However, if intact islets were incubated with wortmannin and PI 3-kinase subsequently was determined in islet immunoprecipitates, ∼50% inhibition of PI 3-kinase activity (but no inhibition of glucose- and carbachol-stimulated insulin secretion) from intact islets was obtained at wortmannin concentrations of 100 µmol/l. Wortmannin, at higher concentrations (1 and 10 µmol/l), inhibited glucose- and carbachol-induced insulin secretion of intact rat islets by 58 and 92%, respectively. Wortmannin had no effect on the basal insulin release from rat islets. A similar dose curve of inhibition of glucose- and carbachol-induced insulin secretion by wortmannin was obtained when β-TC3 cells were used. Cellu-lar metabolism was not changed by any wortmannin concentrations tested (0.01–10 µmol/l). Both basal cytosolic [Ca2+]1 and carbamyl choline-induced increases of [Ca2+]1 were unaffected by wortmannin in the presence of 2.5 mmol/l Ca2+, while Ca2+ mobilization from intracellular stores was partially decreased by wortmannin. Together, these data suggest that wortmannin at concentrations that inhibit PI 3-kinase does not affect insulin secretion. PI 3-kinase is unlikely to have a major role in insulin secretion induced by glucose and carbachol.Diabetes 45:854–862, 1996

The aim of this study was to validate the INNO-BIA plasma amyloid-β (Aβ) forms assay for quantification of Aβ1-40 and Aβ1-42 according to regulatory guidance for bioanalysis and demonstrate its fitness for clinical trial applications. Validation parameters were evaluated by repeated testing of human EDTA-plasma pools. In 6 separate estimates, intra-assay coefficients of variation (CV) for repeated testing of 5 plasma pools were ≤9% and relative error (RE) varied between -35% and +22%. Inter-assay CV (n = 36) ranged from 5% to 17% and RE varied from -17% to +8%. Dilutional linearity was not demonstrated for either analyte using diluent buffer, but dilution with immuno-depleted plasma by 1.67-fold gave results within 20% of target. Analyte stability was demonstrated in plasma at 2-8 °C for up to 6 h. Stability during frozen storage up to 12 months and through 3 freeze-thaw cycles at ≤ -70 °C was also demonstrated in 5 of 6 individuals but deteriorated thereafter. Neither semagacestat nor LY2811376 interfered with the assay but solanezumab at 500 mg/L reduced recovery of Aβ1-42 by 53%. Specimens from a Phase I human volunteer study of the β-secretase inhibitor LY2811376 were tested at baseline and at intervals up to 12 h after single oral doses, demonstrating a clear treatment effect. During 1,041 clinical assay runs from semagacestat studies over 10 months, the CV for plasma quality control pools at three levels were ≤15% and RE were

Objectives To develop a novel, dual-monoclonal sandwich immunoassay with superior sensitivity that provides a rapid and convenient method for measuring glucagon. Glucagon is a 29-amino acid polypeptide hormone produced in the pancreas by the α-cells of the islets of Langerhans. Working in concert with insulin, glucagon is involved in regulating circulating glucose concentrations. Design and methods The immunoassay utilizes Meso Scale Discovery (MSD) electrochemiluminescence (ECL) technology and two affinity-optimized monoclonal antibodies. A series of experiments was performed to determine the linear range of the assay and to evaluate sensitivity, accuracy, recovery, precision, and linearity. Results The sandwich assay was specific for glucagon and did not recognize the closely related peptide oxyntomodulin or other incretin peptides. The assay demonstrated excellent recovery, precision, and linearity, and a broad dynamic range of 0.14 pmol/L to 1950 pmol/L. In addition, assay results were highly correlated with those obtained using a previously described competitive RIA employing polyclonal antiserum. Conclusion The use of affinity-optimized monoclonal antibodies in a sandwich immunoassay format provides a robust, sensitive, and convenient method for measuring concentrations of glucagon that is highly sensitive and specific. This immunoassay should help to improve our understanding of the role of glucagon in the regulation of glucose metabolism.

BACKGROUND Hepcidin-25 reduces iron absorption by binding to the intestinal iron transporter ferroportin and causing its degradation. Currently, little is known about the basal regulation of circulating hepcidin-25. In addition, although erythropoietin administration has been reported to decrease the circulating hepcidin concentration, information is limited regarding how other stimulators of erythropoiesis, such as growth hormone (GH), might alter hepcidin-25 concentrations. METHODS We used a sensitive and specific hepcidin-25 dual–monoclonal antibody sandwich immunoassay to measure hepcidin-25 in healthy human volunteers at various time points throughout the day and during 3 days of fasting and subsequent refeeding. We also measured hepcidin-25 concentrations in healthy volunteers after GH administration. RESULTS In healthy individuals, hepcidin-25 concentrations displayed a diurnal variation, with concentrations being lowest in the early morning and steadily increasing throughout the day before declining during the evening hours, a pattern that was not influenced by food intake. Prolonged fasting produced statistically significant increases in hepcidin-25 concentrations. Refeeding reversed this process, and GH administration markedly decreased hepcidin-25 concentrations. CONCLUSIONS Our results indicate that in humans, hepcidin-25 exhibits diurnal changes that can be altered by prolonged fasting, which increases hepcidin-25 concentrations approximately 3-fold after 3 days of fasting, possibly owing to a suppression of erythropoiesis that may occur during the fasting state to preserve tissue iron concentrations. In contrast, GH administration decreased hepcidin-25 concentrations by approximately 65%, presumably by stimulating erythropoiesis. These results indicate that circulating hepcidin-25 concentrations display much more dynamic and rapid variation than might have been anticipated previously.

There is strong experimental evidence associating cathepsin S with the pathogenesis of atherosclerosis, with emerging data to support its role in diseases such as abdominal aortic aneurysm, obesity, and type 2 diabetes. To further our understanding of cathepsin S, we have developed a novel sandwich immunoassay to measure the mature form of cathepsin S in plasma (mean values from 12 healthy donors of 53 ± 17 ng/ml, range = 39–102). We also developed a targeted liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay to measure in vitro cathepsin S activity to compare activity levels with the protein mass levels determined by enzyme-linked immunosorbent assay (ELISA). Interestingly, we observed that only 0.4 to 1.1% of circulating cathepsin S was enzymatically active. We subsequently demonstrated that the attenuated activity we observed resulted from binding between cathepsin S and its endogenous inhibitor cystatin C in plasma. These data were obtained through immunoprecipitation coupled with either Western blotting analysis or in-gel tryptic digestion and LC–MS/MS characterization of Coomassie-stained gel bands. Although many laboratories have explored the relationship between cathepsin S and cystatin C, this is the first study to demonstrate their association in human circulation, a finding that could prove to be important in furthering our understanding of cathepsin S biology.

Low-density lipoprotein cholesterol (LDL-C) is a major risk factor for atherosclerosis and coronary artery disease (CAD). The role of LDL-C in CAD has been established through experimental studies, epidemiological and genetic studies and the elucidation of the lowdensity lipoprotein receptor (LDLR) pathway (Brown and Goldstein, 1986). LDLR deficient mice develop frank hypercholesterolemia and atherosclerosis on western diet (Ishibashi et al., 1994). In humans, people with low LDL-C have very low risk of developing CAD and plasma LDL-C is positively associated with CAD (Stamler et al., 2000). Mutations in LDLR in humans form the molecular basis for familial hypercholesterolemia and patients with this disease develop premature CAD (Hobbs et al., 1990). Statins are a class of small molecule compounds that inhibit HMG-CoA reductase, a rate-limiting enzyme in cholesterol biosynthesis, thereby reducing LDL-C in humans. Statins effectively reduce LDL-C and the associated cardiovascular disease risk by about 30%, but residual risks for developing cardiovascular disease remain. In recent years, clinical studies have suggested that further lower circulating LDL-C is closely associated with additional reduction of cardiovascular risk. The majority of the high risk patients often fail to reach their LDL-C goal, and thus, novel targets and medications are highly desirable for the prevention and treatment of cardiovascular disorders. In the last few years, novel regulators of LDLR and/or circulating LDL-C have emerged that suggest potentially more therapeutic opportunities. These include proprotein convertase subtilisin/kexin type 9 (PCSK9) and the inducible degrader of LDLR (Idol) that regulate LDLR and LDL-C levels. Novel therapeutic modalities thus may provide additional clinical options to further reduce LDL-C and cardiovascular disease risk.

Glycerolipid acyltransfereases play important roles in physiological and pathophysiological processes of triglyceride (TAG) metabolism and energy balance. Glycerol-3-phosphate acyltransferases (GPATs) are key enzymes in the triglyceride biosynthetic pathway. In addition to the mitochondrial GPAT1 that was first cloned and studied, novel microsomal enzyme isoforms have been discovered in recent years. The potential function of one of the GPATs, GPAT4, was studied in GPAT4 deficient mice that suggested its role in TAG synthesis in multiple tissues. Monoacylglycerol and diacylglycerol acyltransferases (MGAT2 and DGAT1) are important enzymes involved in intestinal triglyceride absorption, and studies in recent years from knockout mice have revealed their important role in whole body energy metabolism through changes in intestinal TAG absorption kinetics. Both MGAT2 and DGAT1 mice are resistant to dietinduced obesity and have improved insulin sensitivity and hepatic TAG accumulation. These data suggest that these enzymes are intimately involved in TAG metabolism and whole body energy homeostasis and that inhibition of these enzymes may provide therapeutic benefits for metabolic disorders such as obesity, metabolic syndrome, and type 2 diabetes.

Tau measurements in cerebrospinal fluid (CSF) are gaining acceptance as aids to diagnosis of Alzheimer's disease (AD) and differentiation from other dementias. Two ELISA assays, the INNOTEST® hTAU Ag and the INNOTEST® PHOSPHO-TAU(181P) for quantification of t-tau and p-tau181 respectively, have been validated to regulatory standards. Validation parameters were determined by repeated testing of human CSF pools. Specimens from Phase 2 studies of the γ-secretase inhibitor semagacestat and the therapeutic antibody solanezumab at baseline and following 12-14 weeks of treatment were also tested. Estimated intra-assay CV for repeated testing of 3 CSF pools were ≤11.5% and RE varied between -14.1% and +6.4%. Inter-assay CV for t-tau was

BACKGROUND Glucose-dependent insulinotropic peptide (GIP) is an incretin peptide secreted by intestinal K cells that stimulates insulin secretion in a glucose-dependent manner. It is secreted as an active, intact 42–amino acid peptide GIP1-42, which is rapidly degraded by dipeptidyl peptidase 4 to GIP3-42, which is inactive. There is currently no described monoclonal antibody–based sandwich immunoassay to quantify concentrations of GIP1-42, the active form of the peptide. METHODS To create a sandwich ELISA for GIP1-42, we generated a monoclonal antibody specific for the intact N-terminus of the peptide, which was further optimized to increase its affinity. We used this antibody as a conjugate antibody in a sandwich ELISA and paired it with an anti–total GIP capture monoclonal antibody to create a dual monoclonal sandwich ELISA for GIP1-42. RESULTS The sandwich ELISA was highly specific for GIP1-42 and did not recognize GIP3-42. The ELISA demonstrated a broad dynamic range and a lower limit of quantification of 5 ng/L. Using the ELISA, we were able to show that GIP1-42 concentrations in healthy volunteers increased dramatically in the postprandial state compared to the fasting state. GIP1-42 values were correlated with total GIP values overall; however, there was substantial interindividual variation. CONCLUSIONS The use of an N-terminal–specific monoclonal antibody in a sandwich ELISA format provides a robust and convenient method for measuring concentrations of GIP1-42, the active form of the incretin hormone. This ELISA should help to improve our understanding of the role of GIP1-42 in regulating glucose-dependent insulin secretion.

Objective Type 2 diabetes mellitus (T2DM) is associated with elevated plasma apolipoprotein B and triglycerides levels, reduced HDL cholesterol and the presence of small-dense LDL particles. The present study was conducted to investigate the role of plasma proprotein convertase subtilisin kexin type 9 (PCSK9) levels, a regulator of LDL-receptor expression, in the occurrence of diabetic dyslipidemia. Methods Plasma PCSK9 was measured in a cohort of subjects with normal glucose metabolism (NGM; n = 288), impaired glucose metabolism (IGM; n = 121) and type 2 diabetes mellitus (T2DM; n = 139) to study whether its relation with plasma apolipoprotein B, triglycerides, total cholesterol, non-HDL cholesterol, LDL cholesterol and HDL cholesterol differed by levels of glucose metabolism status. Results Plasma PCSK9 levels were not different between the three groups (82, 82 and 80 ng/mL in NGM, IGM and T2DM, respectively). PCSK9 was positively associated with total cholesterol, non-HDL cholesterol, LDL cholesterol, apolipoprotein B and triglycerides levels in all subgroups. The regression slopes for the associations with non-HDL cholesterol were steeper among individuals with T2DM than with NGM ( β = 0.016 versus β = 0.009, p -interaction = 0.05). Similar results were obtained for the relation with apolipoprotein B ( β = 0.004 versus β = 0.002, p -interaction = 0.09). Conclusions Although glucose metabolism status per se is not associated with plasma PCSK9 levels, the presence of T2DM may modify the relation between plasma PCSK9 and non-HDL cholesterol and apolipoprotein B. These observations should be regarded as hypothesis generating for further studies aimed at elucidating the role of PCSK9 in the pathogenesis and treatment of diabetic dyslipidemia.

The TAP1 and TAP2 genes, located in the HLA class II region, encode subunits of a peptide transporter. Both genes display limited genetic variability; four different nucleotide substitutions have been found in the TAP2 gene. Here studies on linkage disequilibrium between TAP2 variants and HLA class II alleles are reported, in an attempt to evaluate whether TAP2 variants are associated with insulin-dependent diabetes mellitus (IDDM). As reported previously, a significant decrease of homozygosity for TAP2 alleles encoding alanine at residue 665 (665 Ala) and glutamine at 687 (687 Gln) paralleled by an increase in homozygosity for TAP2 alleles encoding threonine at residue 665 (665 Thr) and a stop codon at 687 (687 Stop), was found in both Finnish and Norwegian IDDM patients compared to random controls. However, a strong linkage disequilibrium between these TAP2 polymorphisms and given HLA-DR and -DQ genes was observed among healthy controls. The frequent 665 Thr and 687 Stop variants were in linkage disequilibrium both with the DR4-DQ8 and the DR3-DQ2 haplotypes, haplotypes which are strongly associated with IDDM. In contrast, the DR1-DQ5 and DR13-DQ6 (e.g. DQB1*0603) haplotypes, which are decreased among IDDM patients, were associated with the 665 Ala and 687 Gln variants. Thus, when DR- and DQ-matched patients and controls were compared, associations of the investigated TAP2 variants and IDDM were no longer detectable. These data, therefore, indicate that the associations previously found between certain TAP2 variants and IDDM are secondary to a primary association between this disease and particular DQ alpha beta heterodimers.

It has been previously demonstrated that glucose stimulation of islets of Langerhans causes an accumulation of unesterified arachidonic acid that correlates with insulin secretion. In addition, it is well established that glucose metabolism is essential for insulin secretion. We show that non-metabolizable analogs of glucose which do not stimulate insulin secretion fail to cause significant accumulation of unesterified arachidonic acid. In addition, mannoheptulose, an inhibitor of glucose metabolism, completely blocks the glucose-induced increase in arachidonic acid accumulation. Among the nutrient secretagogues tested, only alpha-ketoisocaproic acid causes a significant increase in unesterified arachidonic acid accumulation. Mannose, fructose, and glyceraldehyde, in particular, failed to elicit a significant increase in unesterified arachidonic acid accumulation. Our data, taken together with previous studies, suggests that glucose must be metabolized to induce accumulation of unesterified arachidonic acid in pancreatic islets.

Triglyceride (TAG) absorption involves its initial hydrolysis to fatty acids and monoacylglycerol (MAG), which are resynthesized back to diacylglycerol (DAG) and TAG within enterocytes. The resynthesis of DAG is facilitated by fatty acyl-CoA dependent monoacylglycerol acyltransferases (MGATs). Three MGAT enzymes have been isolated in humans and the expression of MGAT2 and MGAT3 in the intestines suggests their functional role in the TAG absorption. In this paper, we report that the Mogat3 gene appears to be a pseudogene in mice while it is a functional gene in rats. Examination of the mouse genomic Mogat3 sequence revealed multiple changes that would result in a translational stop codon or frameshifts. The rat Mogat3 gene, however, is predicted to encode a functional enzyme of 362 amino acids. Expression of rat MGAT3 in human embryonic kidney 293 (HEK293) cells led to the formation of a 36-kDa protein that displayed significant MGAT but not DGAT activity. Tissue expression analysis of rat MGAT3 by real-time PCR analysis indicated that rat MGAT3 has a high level of expression in intestines and pancreas. Our results thus provide the molecular basis to understand the relative functional role of MGAT2 and MGAT3 and also for future exploration of MGAT3 function in animal models.

Objective— To gain insight into the function of proprotein convertase subtilisin kexin type 9 (PCSK9) in humans by establishing whether circulating levels are influenced by diurnal, dietary, and hormonal changes. Methods and Results— We monitored circulating PCSK9 in a set of dynamic human experiments and could show that serum PCSK9 levels display a diurnal rhythm that closely parallels that of cholesterol synthesis, measured as serum lathosterol. In contrast to these marked diurnal changes in cholesterol metabolism, serum low-density lipoprotein (LDL) cholesterol levels remained stable during the diurnal cycle. Depletion of liver cholesterol by treatment with the bile acid–binding resin, cholestyramine, abolished the diurnal rhythms of both PCSK9 and lathosterol. Fasting (>18 hours) strongly reduced circulating PCSK9 and lathosterol levels, whereas serum LDL levels remained unchanged. Growth hormone, known to be increased during fasting in humans, reduced circulating PCSK9 in parallel to LDL cholesterol levels. Conclusion— Throughout the day, and in response to fasting and cholesterol depletion, circulating PCSK9 displays marked variation, presumably related to oscillations in hepatic cholesterol that modify its activity in parallel with cholesterol synthesis. In addition to this sterol-mediated regulation, additional effects on LDL receptors may be mediated by hormones directly influencing PCSK9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that regulates cholesterol metabolism through low-density lipoprotein receptor (LDLR) degradation. Gain-of-function and loss-of-function mutations within PCSK9 gene lead to hypercholesterolemia or hypocholesterolemia respectively. Studies in the U.S. and Canada reported a correlation between multiple metabolic factors and circulating PCSK9 concentrations. However, there is no data available on circulating PCSK9 levels in Chinese. A sandwich ELISA assay was applied to measure serum PCSK9 levels in a Chinese population of 2719 adults from Nanjing district, China, which represents a large and uniform ethnic population of Han Chinese. Serum PCSK9 levels ranged from 12.85 to 222.50 ng/ml with a mean concentration of 69.35 ng/ml in this population. Serum PCSK9 levels were slightly higher in women than in men. Compared to premenopausal women, postmenopausal women had significantly higher PCSK9 levels. Serum PCSK9 levels were correlated with multiple metabolic variables including age, BMI, total cholesterol, LDL cholesterol, triglycerides, fasting blood glucose, systolic blood pressure (SP) and diastolic blood pressure (DP) in this population. After stepwise regression analysis, there was a significant positive association between serum PCSK9 levels and total cholesterol, triglycerides and SP in men. In women, there was a positive correlation between PCSK9 levels and total cholesterol, age and DP. Our study indicates that the serum PCSK9 level may be a biomarker of metabolic status and cardiovascular disease.