A High-Throughput NMR Method for Lipoprotein-X Quantification.

Lipoprotein X (LP-X) is an abnormal cholesterol-rich lipoprotein particle that accumulates in patients with cholestatic liver disease and familial lecithin-cholesterol acyltransferase deficiency (FLD). Because there are no high-throughput diagnostic tests for its detection, a proton nuclear magnetic resonance (NMR) spectroscopy-based method was developed for use on a clinical NMR analyzer commonly used for the quantification of lipoproteins and other cardiovascular biomarkers. The LP-X assay was linear from 89 to 1615 mg/dL (cholesterol units) and had a functional sensitivity of 44 mg/dL. The intra-assay coefficient of variation (CV) varied between 1.8 and 11.8%, depending on the value of LP-X, whereas the inter-assay CV varied between 1.5 and 15.4%. The assay showed no interference with bilirubin levels up to 317 mg/dL and was also unaffected by hemolysis for hemoglobin values up to 216 mg/dL. Samples were stable when stored for up to 6 days at 4 °C but were not stable when frozen. In a large general population cohort (n = 277,000), LP-X was detected in only 50 subjects. The majority of LP-X positive cases had liver disease (64%), and in seven cases, had genetic FLD (14%). In summary, we describe a new NMR-based assay for LP-X, which can be readily implemented for routine clinical laboratory testing.

Plasma lipoprotein-X quantification on filipin-stained gels: monitoring recombinant LCAT treatment ex vivo.

Familial LCAT deficiency (FLD) patients accumulate lipoprotein-X (LP-X), an abnormal nephrotoxic lipoprotein enriched in free cholesterol (FC). The low neutral lipid content of LP-X limits the ability to detect it after separation by lipoprotein electrophoresis and staining with Sudan Black or other neutral lipid stains. A sensitive and accurate method for quantitating LP-X would be useful to examine the relationship between plasma LP-X and renal disease progression in FLD patients and could also serve as a biomarker for monitoring recombinant human LCAT (rhLCAT) therapy. Plasma lipoproteins were separated by agarose gel electrophoresis and cathodal migrating bands corresponding to LP-X were quantified after staining with filipin, which fluoresces with FC, but not with neutral lipids. rhLCAT was incubated with FLD plasma and lipoproteins and LP-X changes were analyzed by agarose gel electrophoresis. Filipin detects synthetic LP-X quantitatively (linearity 20-200 mg/dl FC; coefficient of variation <20%) and sensitively (lower limit of quantitation <1 mg/ml FC), enabling LP-X detection in FLD, cholestatic, and even fish-eye disease patients. rhLCAT incubation with FLD plasma ex vivo reduced LP-X dose dependently, generated HDL, and decreased lipoprotein FC content. Filipin staining after agarose gel electrophoresis sensitively detects LP-X in human plasma and accurately quantifies LP-X reduction after rhLCAT incubation ex vivo.

LCAT Enzyme Replacement Therapy Reduces LpX and Improves Kidney Function in a Mouse Model of Familial LCAT Deficiency.

Familial LCAT deficiency (FLD) is due to mutations in lecithin:cholesterol acyltransferase (LCAT), a plasma enzyme that esterifies cholesterol on lipoproteins. FLD is associated with markedly reduced levels of plasma high-density lipoprotein and cholesteryl ester and the formation of a nephrotoxic lipoprotein called LpX. We used a mouse model in which the LCAT gene is deleted and a truncated version of the SREBP1a gene is expressed in the liver under the control of a protein-rich/carbohydrate-low (PRCL) diet-regulated PEPCK promoter. This mouse was found to form abundant amounts of LpX in the plasma and was used to determine whether treatment with recombinant human LCAT (rhLCAT) could prevent LpX formation and renal injury. After 9 days on the PRCL diet, plasma total and free cholesterol, as well as phospholipids, increased 6.1 ± 0.6-, 9.6 ± 0.9-, and 6.7 ± 0.7-fold, respectively, and liver cholesterol and triglyceride concentrations increased 1.7 ± 0.4- and 2.8 ±0.9-fold, respectively, compared with chow-fed animals. Transmission electron microscopy revealed robust accumulation of lipid droplets in hepatocytes and the appearance of multilamellar LpX particles in liver sinusoids and bile canaliculi. In the kidney, LpX was found in glomerular endothelial cells, podocytes, the glomerular basement membrane, and the mesangium. The urine albumin/creatinine ratio increased 30-fold on the PRCL diet compared with chow-fed controls. Treatment of these mice with intravenous rhLCAT restored the normal lipoprotein profile, eliminated LpX in plasma and kidneys, and markedly decreased proteinuria. The combined results suggest that rhLCAT infusion could be an effective therapy for the prevention of renal disease in patients with FLD.

High density lipoproteins and type 2 inflammatory biomarkers are negatively correlated in atopic asthmatics.

Blood eosinophil counts and serum periostin levels are biomarkers of type 2 inflammation. Although serum levels of HDL and apoA-I have been associated with less severe airflow obstruction in asthma, it is not known whether serum lipids or lipoprotein particles are correlated with type 2 inflammation in asthmatics. Here, we assessed whether serum lipids and lipoproteins correlated with blood eosinophil counts or serum periostin levels in 165 atopic asthmatics and 163 nonasthmatic subjects with and without atopy. Serum lipids and lipoproteins were quantified using standard laboratory assays and NMR spectroscopy. Absolute blood eosinophils were quantified by complete blood counts. Periostin levels were measured using the Elecsys® periostin assay. In atopic asthmatics, blood eosinophils negatively correlated with serum HDL cholesterol and total HDL particles measured by NMR spectroscopy (HDLNMR). Serum periostin levels negatively correlated with total HDLNMR In contrast, blood eosinophil counts positively correlated with serum triglyceride levels. This study demonstrates for the first time that HDL particles were negatively correlated, whereas serum triglycerides were positively correlated, with blood eosinophils in atopic asthmatics. This supports the concept that serum levels of HDL and triglycerides may be linked to systemic type 2 inflammation in atopic asthma.

Creation of Apolipoprotein C-II (ApoC-II) Mutant Mice and Correction of Their Hypertriglyceridemia with an ApoC-II Mimetic Peptide.

Apolipoprotein C-II (apoC-II) is a cofactor for lipoprotein lipase, a plasma enzyme that hydrolyzes triglycerides (TGs). ApoC-II deficiency in humans results in hypertriglyceridemia. We used zinc finger nucleases to create Apoc2 mutant mice to investigate the use of C-II-a, a short apoC-II mimetic peptide, as a therapy for apoC-II deficiency. Mutant mice produced a form of apoC-II with an uncleaved signal peptide that preferentially binds high-density lipoproteins (HDLs) due to a 3-amino acid deletion at the signal peptide cleavage site. Homozygous Apoc2 mutant mice had increased plasma TG (757.5 ± 281.2 mg/dl) and low HDL cholesterol (31.4 ± 14.7 mg/dl) compared with wild-type mice (TG, 55.9 ± 13.3 mg/dl; HDL cholesterol, 55.9 ± 14.3 mg/dl). TGs were found in light (density < 1.063 g/ml) lipoproteins in the size range of very-low-density lipoprotein and chylomicron remnants (40-200 nm). Intravenous injection of C-II-a (0.2, 1, and 5 µmol/kg) reduced plasma TG in a dose-dependent manner, with a maximum decrease of 90% occurring 30 minutes after the high dose. Plasma TG did not return to baseline until 48 hours later. Similar results were found with subcutaneous or intramuscular injections. Plasma half-life of C-II-a is 1.33 ± 0.72 hours, indicating that C-II-a only acutely activates lipolysis, and the sustained TG reduction is due to the relatively slow rate of new TG-rich lipoprotein synthesis. In summary, we describe a novel mouse model of apoC-II deficiency and show that an apoC-II mimetic peptide can reverse the hypertriglyceridemia in these mice, and thus could be a potential new therapy for apoC-II deficiency.

Metabolite profiling of a NIST Standard Reference Material for human plasma (SRM 1950): GC-MS, LC-MS, NMR, and clinical laboratory analyses, libraries, and web-based resources.

Recent progress in metabolomics and the development of increasingly sensitive analytical techniques have renewed interest in global profiling, i.e., semiquantitative monitoring of all chemical constituents of biological fluids. In this work, we have performed global profiling of NIST SRM 1950, "Metabolites in Human Plasma", using GC-MS, LC-MS, and NMR. Metabolome coverage, difficulties, and reproducibility of the experiments on each platform are discussed. A total of 353 metabolites have been identified in this material. GC-MS provides 65 unique identifications, and most of the identifications from NMR overlap with the LC-MS identifications, except for some small sugars that are not directly found by LC-MS. Also, repeatability and intermediate precision analyses show that the SRM 1950 profiling is reproducible enough to consider this material as a good choice to distinguish between analytical and biological variability. Clinical laboratory data shows that most results are within the reference ranges for each assay. In-house computational tools have been developed or modified for MS data processing and interactive web display. All data and programs are freely available online at http://peptide.nist.gov/ and http://srmd.nist.gov/ .

Cell-free, high-density lipoprotein-specific phospholipid efflux assay predicts incident cardiovascular disease.

BACKGROUNDCellular cholesterol efflux capacity (CEC) is a better predictor of cardiovascular disease (CVD) events than HDL-cholesterol (HDL-C) but is not suitable as a routine clinical assay.METHODSWe developed an HDL-specific phospholipid efflux (HDL-SPE) assay to assess HDL functionality based on whole plasma HDL apolipoprotein-mediated solubilization of fluorescent phosphatidylethanolamine from artificial lipid donor particles. We first assessed the association of HDL-SPE with prevalent coronary artery disease (CAD): study I included NIH severe-CAD (n = 50) and non-CAD (n = 50) participants, who were frequency matched for sex, BMI, type 2 diabetes mellitus, and smoking; study II included Japanese CAD (n = 70) and non-CAD (n = 154) participants. We also examined the association of HDL-SPE with incident CVD events in the Prevention of Renal and Vascular End-stage Disease (PREVEND) study comparing 340 patients with 340 controls individually matched for age, sex, smoking, and HDL-C levels.RESULTSReceiver operating characteristic curves revealed stronger associations of HDL-SPE with prevalent CAD. The AUCs in study I were as follows: HDL-SPE, 0.68; apolipoprotein A-I (apoA-I), 0.62; HDL-C, 0.63; and CEC, 0.52. The AUCs in study II were as follows: HDL-SPE, 0.83; apoA-I, 0.64; and HDL-C, 0.53. Also longitudinally, HDL-SPE was significantly associated with incident CVD events independent of traditional risk factors with ORs below 0.2 per SD increment in the PREVEND study (P < 0.001).CONCLUSIONHDL-SPE could serve as a routine clinical assay for improving CVD risk assessment and drug discovery.TRIAL REGISTRATIONClinicalTrials.gov NCT01621594.FUNDINGNHLBI Intramural Research Program, NIH (HL006095-06).

Endothelial expression of human ABCA1 in mice increases plasma HDL cholesterol and reduces diet-induced atherosclerosis.

The role of endothelial ABCA1 expression in reverse cholesterol transport (RCT) was examined in transgenic mice, using the endothelial-specific Tie2 promoter. Human ABCA1 (hABCA1) was significantly expressed in endothelial cells (EC) of most tissues except the liver. Increased expression of ABCA1 was not observed in resident peritoneal macrophages. ApoA-I-mediated cholesterol efflux from aortic EC was 2.6-fold higher (P < 0.0001) for cells from transgenic versus control mice. On normal chow diet, Tie2 hABCA1 transgenic mice had a 25% (P < 0.0001) increase in HDL-cholesterol (HDL-C) and more than a 2-fold increase of eNOS mRNA in the aorta (P < 0.04). After 6 months on a high-fat, high-cholesterol (HFHC) diet, transgenic mice compared with controls had a 40% increase in plasma HDL-C (P < 0.003) and close to 40% decrease in aortic lesions (P < 0.02). Aortas from HFHC-fed transgenic mice also showed gene expression changes consistent with decreased inflammation and apoptosis. Beneficial effects of the ABCA1 transgene on HDL-C levels or on atherosclerosis were absent when the transgene was transferred onto ApoE or Abca1 knockout mice. In summary, expression of hABCA1 in EC appears to play a role in decreasing diet-induced atherosclerosis in mice and is associated with increased plasma HDL-C levels and beneficial gene expression changes in EC.

A Simple Fluorescent Cholesterol Labeling Method to Cryoprotect and Detect Plasma Lipoprotein-X.

Lipoprotein-X (LpX) are abnormal nephrotoxic lipoprotein particles enriched in free cholesterol and phospholipids. LpX with distinctive lipid compositions are formed in patients afflicted with either familial LCAT deficiency (FLD) or biliary cholestasis. LpX is difficult to detect by standard lipid stains due to the absence of a neutral lipid core and because it is unstable upon storage, particularly when frozen. We have recently reported that free cholesterol-specific filipin staining after agarose gel electrophoresis sensitively detects LpX in fresh human plasma. Herein, we describe an even more simplified qualitative method to detect LpX in both fresh and frozen-thawed human FLD or cholestatic plasma. Fluorescent cholesterol complexed to fatty-acid-free BSA was used to label LpX and was added together with trehalose in order to cryopreserve plasma LpX. The fluorescent cholesterol bound to LpX was observed with high sensitivity after separation from other lipoproteins by agarose gel electrophoresis. This methodology can be readily developed into a simple assay for the clinical diagnosis of FLD and biliary liver disease and to monitor the efficacy of treatments intended to reduce plasma LpX in these disease states.

Nuclear Magnetic Resonance Derived Biomarkers for Evaluating Cardiometabolic Risk in Youth and Young Adults Across the Spectrum of Glucose Tolerance.

Youth with obesity have an increased risk for cardiometabolic disease, but identifying those at highest risk remains a challenge. Four biomarkers that might serve this purpose are "by products" of clinical NMR LipoProfile® lipid testing: LPIR (Lipoprotein Insulin Resistance Index), GlycA (inflammation marker), BCAA (total branched-chain amino acids), and glycine. All are strongly related to insulin resistance and type 2 diabetes (T2DM) in adults (glycine inversely) and are independent of biological and methodological variations in insulin assays. However, their clinical utility in youth is unclear. We compared fasting levels of these biomarkers in 186 youth (42 lean normal glucose tolerant (NGT), 88 obese NGT, 23 with prediabetes (PreDM), and 33 with T2DM. All four biomarkers were associated with obesity and glycemia in youth. LPIR and GlycA were highest in youth with PreDM and T2DM, whereas glycine was lowest in youth with T2DM. While all four were correlated with HOMA-IR (Homeostatic Model Assessment for Insulin Resistance), LPIR had the strongest correlation (LPIR: r = 0.6; GlycA: r = 0.4, glycine: r = -0.4, BCAA: r = 0.2, all P < 0.01). All four markers correlated with HbA1c (LPIR, GlycA, BCAA: r ≥ 0.3 and glycine: r = -0.3, all P < 0.001). In multi-variable regression models, LPIR, GlycA, and glycine were independently associated with HOMA-IR (Adjusted R2 = 0.473, P < 0.001) and LPIR, glycine, and BCAA were independently associated with HbA1c (Adjusted R2 = 0.33, P < 0.001). An LPIR index of >44 was associated with elevated blood pressure, BMI, and dyslipidemia. Plasma NMR-derived markers were related to adverse markers of cardiometabolic risk in youth. LPIR, either alone or in combination with GlycA, should be explored as a non-insulin dependent predictive tool for development of insulin resistance and diabetes in youth. Clinical Trial Registration: Clinicaltrials.gov, identifier NCT:02960659.

LCAT protects against Lipoprotein-X formation in a murine model of drug-induced intrahepatic cholestasis.

Familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is a rare genetic disease characterized by low HDL-C levels, low plasma cholesterol esterification, and the formation of Lipoprotein-X (Lp-X), an abnormal cholesterol-rich lipoprotein particle. LCAT deficiency causes corneal opacities, normochromic normocytic anemia, and progressive renal disease due to Lp-X deposition in the glomeruli. Recombinant LCAT is being investigated as a potential therapy for this disorder. Several hepatic disorders, namely primary biliary cirrhosis, primary sclerosing cholangitis, cholestatic liver disease, and chronic alcoholism also develop Lp-X, which may contribute to the complications of these disorders. We aimed to test the hypothesis that an increase in plasma LCAT could prevent the formation of Lp-X in other diseases besides FLD. We generated a murine model of intrahepatic cholestasis in LCAT-deficient (KO), wild type (WT), and LCAT-transgenic (Tg) mice by gavaging mice with alpha-naphthylisothiocyanate (ANIT), a drug well known to induce intrahepatic cholestasis. Three days after the treatment, all mice developed hyperbilirubinemia and elevated liver function markers (ALT, AST, Alkaline Phosphatase). The presence of high levels of LCAT in the LCAT-Tg mice, however, prevented the formation of Lp-X and other plasma lipid abnormalities in WT and LCAT-KO mice. In addition, we demonstrated that multiple injections of recombinant human LCAT can prevent significant accumulation of Lp-X after ANIT treatment in WT mice. In summary, LCAT can protect against the formation of Lp-X in a murine model of cholestasis and thus recombinant LCAT could be a potential therapy to prevent the formation of Lp-X in other diseases besides FLD.

Development of a novel fluorescent activity assay for lecithin:cholesterol acyltransferase.

Background Lecithin:cholesterol acyltransferase (LCAT) is a plasma enzyme that esterifies cholesterol. Recombinant human LCAT (rhLCAT) is now being developed as an enzyme replacement therapy for familial LCAT deficiency and as a possible treatment for acute coronary syndrome. The current 'gold standard' assay for LCAT activity involves the use of radioisotopes, thus making it difficult for routine clinical use. Methods We have developed a novel and more convenient LCAT activity assay using fluorescence-labelled cholesterol (BODIPY-cholesterol), which is incorporated into proteoliposomes as a substrate instead of radiolabelled cholesterol. Results The apparent Km and Vmax were 31.5 µmol/L and 55.8 nmol/h/nmoL, rhLCAT, respectively, for the 3H-cholesterol method and 103.1 µmol/L and 13.4 nmol/h/nmol rhLCAT, respectively, for the BODIPY-cholesterol method. Although the two assays differed in their absolute units of LCAT activity, there was a good correlation between the two test assays ( r = 0.849, P < 1.6 × 10-7, y = 0.1378x + 1.106). The BODIPY-cholesterol assay had an intra-assay CV of 13.7%, which was superior to the intra-assay CV of 20.8% for the radioisotopic assay. The proteoliposome substrate made with BODIPY-cholesterol was stable to storage for at least 10 months. The reference range ( n = 20) for the fluorescent LCAT activity assay was 4.6-24.1 U/mL/h in healthy subjects. Conclusions In summary, a novel fluorescent LCAT activity assay that utilizes BODIPY-cholesterol as a substrate is described that yields comparable results to the radioisotopic method.

Detection and quantitation of serum mesothelin, a tumor marker for patients with mesothelioma and ovarian cancer.

PURPOSE: To determine whether mesothelin, a cell surface protein highly expressed in mesothelioma and ovarian cancer, is shed into serum and if so to accurately measure it. EXPERIMENTAL DESIGN: We developed a sandwich ELISA using antibodies reacting with two different epitopes on human mesothelin. To quantitate serum mesothelin levels, a standard curve was generated using a mesothelin-Fc fusion protein. Sera from 24 healthy volunteers, 95 random hospital patients, 56 patients with mesothelioma, and 21 patients with ovarian cancer were analyzed. Serum mesothelin levels were also measured before and after surgical cytoreduction in six patients with peritoneal mesothelioma. RESULTS: Elevated serum mesothelin levels were noted in 40 of 56 (71%) patients with mesothelioma and in 14 of 21 (67%) patients with ovarian cancer. Serum mesothelin levels were increased in 80% and 75% of the cases of mesothelioma and ovarian cancer, respectively, in which the tumors expressed mesothelin by immunohistochemistry. Out of the six patients with peritoneal mesothelioma who underwent surgery, four had elevated serum mesothelin levels before surgery. Out of these four patients, three had cytoreductive surgery and the serum mesothelin level decreased by 71% on postoperative day 1 and was undetectable by postoperative day 7. CONCLUSIONS: We developed a serum mesothelin assay that shows that mesothelin is elevated in patients with mesothelioma and ovarian cancer. The rapid decrease in mesothelin levels after surgery in patients with peritoneal mesothelioma suggests that serum mesothelin may be a useful test to monitor treatment response in mesothelin-expressing cancers.

CUSUM-Logistic Regression analysis for the rapid detection of errors in clinical laboratory test results.

OBJECTIVE: The main drawback of the periodic analysis of quality control (QC) material is that test performance is not monitored in time periods between QC analyses, potentially leading to the reporting of faulty test results. The objective of this study was to develop a patient based QC procedure for the more timely detection of test errors. METHOD: Results from a Chem-14 panel measured on the Beckman LX20 analyzer were used to develop the model. Each test result was predicted from the other 13 members of the panel by multiple regression, which resulted in correlation coefficients between the predicted and measured result of >0.7 for 8 of the 14 tests. A logistic regression model, which utilized the measured test result, the predicted test result, the day of the week and time of day, was then developed for predicting test errors. The output of the logistic regression was tallied by a daily CUSUM approach and used to predict test errors, with a fixed specificity of 90%. RESULTS: The mean average run length (ARL) before error detection by CUSUM-Logistic Regression (CSLR) was 20 with a mean sensitivity of 97%, which was considerably shorter than the mean ARL of 53 (sensitivity 87.5%) for a simple prediction model that only used the measured result for error detection. CONCLUSION: A CUSUM-Logistic Regression analysis of patient laboratory data can be an effective approach for the rapid and sensitive detection of clinical laboratory errors.

Polyphenol rich olive oils improve lipoprotein particle atherogenic ratios and subclasses profile: A randomized, crossover, controlled trial.

SCOPE: Lipoprotein particle measures performed by nuclear magnetic resonance (NMR), and associated ratios, may be better markers for atherosclerosis risk than conventional lipid measures. The effect of two functional olive oils, one enriched with its polyphenols (FVOO, 500 ppm), and the other (FVOOT) with them (250 ppm) and those of thyme (250 ppm), versus a standard virgin olive oil (VOO), on lipoprotein particle atherogenic ratios and subclasses profiles was assessed. METHODS AND RESULTS: In a randomized, double-blind, crossover, controlled trial, 33 hypercholesterolemic individuals received 25 mL/day of VOO, FVOO, and FVOOT. Intervention periods were of 3 weeks separated by 2-week washout periods. Lipoprotein particle counts and subclasses were measured by NMR. Polyphenols from olive oil and thyme modified the lipoprotein subclasses profile and decreased the total LDL particle/total HDL particle (HDL-P), small HDL/large HDL, and HDL-cholesterol/HDL-P ratios, and decreased the lipoprotein insulin resistance index (LP-IR) (p < 0.05). CONCLUSION: Olive oil polyphenols, and those from thyme provided benefits on lipoprotein particle atherogenic ratios and subclasses profile distribution. Polyphenol-enriched olive oil is a way of increasing the olive oil healthy properties while consuming the same amount of fat, as well as a useful and complementary tool for the management of cardiovascular risk individuals.

Endothelial Expression of Scavenger Receptor Class B, Type I Protects against Development of Atherosclerosis in Mice.

The role of scavenger receptor class B, type I (SR-BI) in endothelial cells (EC) was examined in several novel transgenic mouse models expressing SR-BI in endothelium of mice with normal C57Bl6/N, apoE-KO, or Scarb1-KO backgrounds. Mice were also created expressing SR-BI exclusively in endothelium and liver. Endothelial expression of the Tie2-Scarb1 transgene had no significant effect on plasma lipoprotein levels in mice on a normal chow diet but on an atherogenic diet, significantly decreased plasma cholesterol levels, increased plasma HDL cholesterol (HDL-C) levels, and protected mice against atherosclerosis. In 8-month-old apoE-KO mice fed a normal chow diet, the Tie2-Scarb1 transgene decreased aortic lesions by 24%. Mice expressing SR-BI only in EC and liver had a 1.5 ± 0.1-fold increase in plasma cholesterol compared to mice synthesizing SR-BI only in liver. This elevation was due mostly to increased HDL-C. In EC culture studies, SR-BI was found to be present in both basolateral and apical membranes but greater cellular uptake of cholesterol from HDL was found in the basolateral compartment. In summary, enhanced expression of SR-BI in EC resulted in a less atherogenic lipoprotein profile and decreased atherosclerosis, suggesting a possible role for endothelial SR-BI in the flux of cholesterol across EC.

Rapid measurement of insulin using the Abbott IMx: application to the management of insulinoma.

BACKGROUND: Patients with multiple endocrine neoplasia (type 1) often present with multiple pancreatic endocrine tumours, such as insulinomas. A major difficulty in the surgical treatment of such patients is identifying which tumours are functionally active and therefore need to be resected for a cure. The objective of this study was to develop a rapid insulin assay that could be used intraoperatively for identifying insulinomas from pancreatic aspirates. METHODS: By reducing the incubation time and by increasing the sample volume, a rapid insulin assay was developed on the IMx analyser. This was used to measure insulin from tissue aspirates collected from suspected insulinomas under ultrasound guidance. RESULTS: The rapid insulin assay (y) could be performed in 14 min and showed a good correlation with the standard IMx (x) insulin assay (y = 0.808x + 0.04; r(2) = 0.986). Using the rapid insulin assay on pancreatic tissue aspirates, insulinomas could be readily distinguished from normal pancreatic tissue or from non-functional adenomas based on a marked increase in insulin content. CONCLUSION: In summary, a new rapid assay for insulin is described that compares favourably to the standard IMx insulin assay and can potentially be used intraoperatively on pancreatic aspirates for identifying functionally active insulinomas.