Multiplexed LC-MS/MS analysis of methylsuccinic acid, ethylmalonic acid, and glutaric acid in plasma and urine.

Low molecular-mass aliphatic carboxylic acids are critically important for intermediate metabolism and may serve as important biomarkers for metabolic homeostasis. Here in, we focused on multiplexed method development of aliphatic carboxylic analytes, including methylsuccinic acid (MSA), ethylmalonic acid (EMA), and glutaric acid (GA). Also assessed was their utility in a population's health as well as metabolic disease screening in both plasma and urine matrices. MSA, EMA, and GA are constitutional isomers of dicarboxylic acid with high polarity and poor ionization efficiency, resulting in such challenges as poor signal intensity and retention, particularly in reversed-phase liquid chromatography with electrospray mass spectrometry (RP-LC-ESI-MS/MS). Derivatization using n-butanol was performed in the sample preparation to enhance the signal intensity accompanied with a positive charge from ionization in complicated biomatrices as well as to improve the separation of these isomers with optimal retention. Fit-for-purpose method validation results demonstrated quantitative ranges for MSA/EMA/GA from 5/10/20 ng/mL to 400 ng/mL in plasma analysis, and 100/200/100 ng/mL to 5000/10000/5000 ng/mL in urine analysis. This validated method demonstrates future utility when exploring population health analysis and biomarker development in metabolic diseases.

A Clinical Prediction Rule for Bacterial Musculoskeletal Infections in Children with Monoarthritis in Lyme Endemic Regions.

STUDY OBJECTIVE: Children with a bacterial musculoskeletal infection (MSKI) require prompt identification and treatment. In Lyme disease endemic areas, children with an MSKI can present similarly to those with Lyme arthritis. Our goal was to derive a clinical prediction rule to accurately identify children at a low risk for an MSKI. METHODS: We enrolled children with monoarthritis presenting to 1 of 6 Pedi Lyme Net centers and performed a procalcitonin (PCT) and a first-tier Lyme C6 enzyme immunoassay (EIA) test. Our primary outcome was an MSKI (septic arthritis, osteomyelitis, or pyomyositis). Using recursive partitioning with k-fold cross validation, we derived a clinical prediction rule to identify children at a low risk of an MSKI. We calculated the accuracy of our novel rule in a derivation cohort. RESULTS: Of the 735 children in the derivation cohort with an available research biosample, 39 (5%) had an MSKI (18 had septic arthritis, 20 had osteomyelitis, and 1 had pyomyositis), 260 (37%) had Lyme arthritis, and 436 (53%) had other inflammatory arthritis. Children with a PCT level of more than or equal to 0.50 ng/mL and those with a C-reactive protein (CRP) level of more than or equal to 0.6 mg/dL with a negative Lyme C6 EIA were classified as not low risk for an MSKI. Of the 451 (61%) children categorized as low risk, none had an MSKI (sensitivity 100%, 95% confidence interval 91.0% to 100%; specificity 74.2%, 95% confidence interval 70.5% to 77.6%). CONCLUSION: A novel clinical decision rule that includes PCT, CRP, and a first-tier Lyme EIA was highly sensitive for MSKIs. Although broader external validation is required, the application of this rule may safely reduce invasive testing, procedures, and treatment for low risk children.

Moderate doses of commercial preparations of Ginkgo biloba do not alter markers of liver function but moderate alcohol intake does: A new approach to identify and quantify biomarkers of 'adverse effects' of dietary supplements.

It is difficult to determine if certain dietary supplements are safe for human consumption. Extracts of leaves of Ginkgo biloba trees are dietary supplements used for various purported therapeutic benefits. However, recent studies reported they increased risk of liver cancer in rodents. Therefore, this study assessed the association between ginkgo consumption and liver function using NHANES 2001-2012 data (N = 29,684). Since alcohol is known to adversely affect liver function, association of its consumption with liver function was also assessed. Alcohol and ginkgo extract intake of adult consumers and clinical markers of liver function (alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transferase, lactate dehydrogenase, bilirubin) were examined. Moderate consumers of alcohol (0.80 ± 0.02 drinks/day) had higher levels of aspartate aminotransferase and gamma glutamyl transferase than non-consumers (P < 0.001). There was no difference (P > 0.01) in levels of markers of liver function in 616 ginkgo consumers (65.1 ± 4.4 mg/day intake) compared to non-consumers. While moderate alcohol consumption was associated with changes in markers of liver function, ginkgo intake as typically consumed by U.S. adults was not associated with these markers. Biomarkers measured by NHANES may be useful to examine potential adverse effects of dietary supplements for which insufficient human adverse event and toxicity data are available. TRIAL REGISTRATION NUMBER: Not applicable, as this is secondary analysis of publicly released observational data (NHANES 2001-2012).

Multicenter evaluation of the thermo scientific prelude for measurement of immunosuppressant drugs using sample preparation liquid chromatography-tandem mass spectrometry.

Immunosuppressant drugs (ISDs) are commonly prescribed to solid organ transplant patients. Their narrow therapeutic index and potential for toxicity necessitates careful monitoring of blood concentrations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods are increasingly used for ISD measurement. However, there remain many challenges with this methodology, particularly regarding interassay variability. The Thermo Scientific Prelude is an online extraction/liquid chromatography platform that uses turbulent flow technology coupled with MS/MS. A multicenter evaluation of the Prelude for the measurement of cyclosporine A, tacrolimus, and sirolimus is described. ISDs were measured at each site using standardized protocols. Sample preparation liquid chromatography-MS/MS was performed using the Prelude coupled to a TSQ Vantage. Chromatography was achieved with a Cyclone-P TurboFlow/Accucore C8 column combination using a multisolvent loading and eluting pump system. Mass spectrometry acquisitions were performed in selective reaction monitoring mode and data processed using TraceFinder (version 3.1). Multisite mean imprecision for cyclosporine A ranged from 8.8% (54 mcg/L) to 9.8% (450 mcg/L); for tacrolimus, 4.7% (15.5 mcg/L) to 12.6% (2.5 mcg/L); for sirolimus, 7.4% (19.9 mcg/L) to 16.5% (2.6 mcg/L). Approximately 110 specimens were used for method comparison. For cyclosporine A, mean bias against the multisite mean ranged from -18% to 1%; for tacrolimus, values ranged from -7% to 4%; for sirolimus, values ranged from -4% to 2%. Comparisons of multisite mean Prelude results with routine ISD method results was also performed for cyclosporine A (slope = 0.7878, intercept = 24.16, r = 0.98), tacrolimus (slope = 0.9391, intercept = 0.1017, r = 98), and sirolimus (slope = 0.9618, intercept = 0.1483, r = 0.97). The Prelude ISD method offers acceptable and comparable multisite performance. This study has also highlighted the importance of adopting standardized protocols and LC-MS/MS methods for better comparability between ISD assays.

Liquid chromatography-tandem mass spectrometry enzyme assay for UDP-galactose 4'-epimerase: use of fragment intensity ratio in differentiation of structural isomers.

BACKGROUND: Distinction between asymptomatic and potentially clinically significant forms of galactosemia due to UDP-galactose 4'-epimerase (GALE) deficiency requires enzyme measurement in erythrocytes and other cells. We sought to develop a GALE assay using a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method. METHODS: The reversible GALE assay was conducted with UDPGal as a substrate. The coeluting reaction product, uridine diphosphate glucose (UDPGlc), and its isomeric substrate, uridine diphosphate galactose (UDPGal), were detected by MS/MS at mass transitions 565 > 280, 565 > 241 and 565 > 403. The UDPGal was enriched in mass transition 565 > 403 compared with UDPGlc, whereas the UDPGlc was enriched in the mass transition 565 > 241 compared with UDPGal. The percentage of UDPGal in the reaction mixture was calculated by use of the ratio of ion intensities of the 2 daughter ions and a fourth-order polynomial calibrator curve. RESULTS: The method yielded a mean (SD) GALE activity of 9.8 (2.2) µmol · g(-1) hemoglobin · h(-1) in erythrocyte extracts from 27 controls. The apparent Km of the substrate, UDPGal, was 0.05 mmol/L. The GALE activity ranged from 433 to 993 µmol · g(-1) protein · h(-1) in control lymphoblast extracts. In a blinded test of 22 subjects suspected of GALE deficiency, we identified 6 individuals whose residual activities were below the range of controls, compatible with intermediate GALE deficiency. CONCLUSIONS: This assay can be used to distinguish the different forms of GALE deficiency. From an analytical standpoint, differentiating isomers on the basis of fragment intensity ratios should also prove useful for analogous enzymatic studies involving substrates and products that are structural isomers.

Serum citrulline as a biomarker of gastrointestinal function during hematopoietic cell transplantation in children.

OBJECTIVES: We sought to determine whether serum citrulline (CIT), an amino acid produced by small bowel enterocytes, was associated with clinical and biochemical markers of gastrointestinal function in children undergoing hematopoietic cell transplantation (HCT). METHODS: We conducted a multicenter, prospective cohort study of 26 children to define time-related changes in serum CIT during the course of HCT. Markers of gastrointestinal function including oral energy intake, emesis, stool volume, presence of graft-versus-host disease (GVHD), oral mucositis severity, and cytokine and neurohormone levels were measured. Weekly serum CIT concentrations were obtained from 10 days prior until 30 days after HCT. RESULTS: Mean baseline CIT concentration was 22.7 µmol/L (95% confidence interval [CI] 17.7-27.6) on day -10, which decreased to a nadir of 7.5 µmol/L (95% CI 3.1-18.0, P = 0.017) on day 8 following HCT before returning to baseline by day 30. After adjustment for IL-6 level (1.0% lower CIT per 10% increase in interleukin-6, P = 0.004), presence of acute GVHD (27% lower CIT, P = 0.025), and oral energy intake (2.1% lower CIT per 10% decrease in energy intake, P = 0.018), the nadir shifted to day 10, when mean CIT concentration was lower in patients with severe oral mucositis (6.7 µmol/L, 95% CI 3.4-13.1) than in those without severe mucositis (11.9 µmol/L, 95% CI 5.8-24.4, P = 0.003). Change in CIT was not correlated with stool volume, C-reactive protein, tumor necrosis factor-α, leptin, or ghrelin. CONCLUSIONS: In children undergoing HCT, serum CIT correlates with measures of gastrointestinal function (oral mucositis severity, dietary intake, acute GVHD) and may reflect mucosal injury to the gastrointestinal tract.

Filamin A Is a Prognostic Serum Biomarker for Differentiating Benign Prostatic Hyperplasia from Prostate Cancer in Caucasian and African American Men.

Prostate cancer represents a significant health risk to aging men, in which diagnostic challenges to the identification of aggressive cancers remain unmet. Prostate cancer screening is driven by the prostate-specific antigen (PSA); however, in men with benign prostatic hyperplasia (BPH) due to an enlarged prostate and elevated PSA, PSA's screening utility is diminished, resulting in many unnecessary biopsies. To address this issue, we previously identified a cleaved fragment of Filamin A (FLNA) protein (as measured with IP-MRM mass spectrometry assessment as a prognostic biomarker for stratifying BPH from prostate cancer and subsequently evaluated its expanded utility in Caucasian (CA) and African American (AA) men. All men had a negative digital rectal examination (DRE) and PSA between 4 and 10 ng/mL and underwent prostate biopsy. In AA men, FLNA serum levels exhibited diagnostic utility for stratifying BPH from patients with aggressive prostate cancer (0.71 AUC and 12.2 OR in 48 men with BPH and 60 men with PCa) and outperformed PSA (0.50 AUC, 2.2 OR). In CA men, FLNA serum levels also exhibited diagnostic utility for stratifying BPH from patients with aggressive prostate cancer (0.74 AUC and 19.4 OR in 191 men with BPH and 109 men with PCa) and outperformed PSA (0.46 AUC, 0.32 OR). Herein, we established FLNA alone as a serum biomarker for stratifying men with BPH vs. those with high Gleason (7-10) prostate cancers compared to the current diagnostic paradigm of using PSA. This approach demonstrates clinical actionability of FLNA alone without the requirement of prostate volume measurement as a test with utility in AA and CA men and represents a significant opportunity to decrease the number of unnecessary biopsies in aggressive prostate cancer diagnoses.

Identification and validation of N-acetylputrescine in combination with non-canonical clinical features as a Parkinson's disease biomarker panel.

Parkinson's disease is a progressive neurodegenerative disorder in which loss of dopaminergic neurons in the substantia nigra results in a clinically heterogeneous group with variable motor and non-motor symptoms with a degree of misdiagnosis. Only 3-25% of sporadic Parkinson's patients present with genetic abnormalities that could represent a risk factor, thus environmental, metabolic, and other unknown causes contribute to the pathogenesis of Parkinson's disease, which highlights the critical need for biomarkers. In the present study, we prospectively collected and analyzed plasma samples from 194 Parkinson's disease patients and 197 age-matched non-diseased controls. N-acetyl putrescine (NAP) in combination with sense of smell (B-SIT), depression/anxiety (HADS), and acting out dreams (RBD1Q) clinical measurements demonstrated combined diagnostic utility. NAP was increased by 28% in Parkinsons disease patients and exhibited an AUC of 0.72 as well as an OR of 4.79. The clinical and NAP panel demonstrated an area under the curve, AUC = 0.9 and an OR of 20.4. The assessed diagnostic panel demonstrates combinatorial utility in diagnosing Parkinson's disease, allowing for an integrated interpretation of disease pathophysiology and highlighting the use of multi-tiered panels in neurological disease diagnosis.

A rapid host-protein test for differentiating bacterial from viral infection: Apollo diagnostic accuracy study.

Objectives: To determine the diagnostic accuracy of a rapid host-protein test for differentiating bacterial from viral infections in patients who presented to the emergency department (ED) or urgent care center (UCC). Methods: This was a prospective multicenter, blinded study. MeMed BV (MMBV), a test based on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), interferon gamma-inducible protein-10 (IP-10), and C-reactive protein (CRP), was measured using a rapid measurement platform. Patients were enrolled from 9 EDs and 3 UCCs in the United States and Israel. Patients >3 months of age presenting with fever and clinical suspicion of acute infection were considered eligible. MMBV results were not provided to the treating clinician. MMBV results (bacterial/viral/equivocal) were compared against a reference standard method for classification of infection etiology determined by expert panel adjudication. Experts were blinded to MMBV results. They were provided with comprehensive patient data, including laboratory, microbiological, radiological and follow-up. Results: Of 563 adults and children enrolled, 476 comprised the study population (314 adults, 162 children). The predominant clinical syndrome was respiratory tract infection (60.5% upper, 11.3% lower). MMBV demonstrated sensitivity of 90.0% (95% confidence interval [CI]: 80.3-99.7), specificity of 92.8% (90.0%-95.5%), and negative predictive value of 98.8% (96.8%-99.6%) for bacterial infections. Only 7.2% of cases yielded equivocal MMBV scores. Area under the curve for MMBV was 0.95 (0.90-0.99). Conclusions: MMBV had a high sensitivity and specificity relative to reference standard for differentiating bacterial from viral infections. Future implementation of MMBV for patients with suspected acute infections could potentially aid with appropriate antibiotic decision-making.

Sweetened beverage consumption, incident coronary heart disease, and biomarkers of risk in men.

BACKGROUND: Sugar-sweetened beverage consumption is associated with weight gain and risk of type 2 diabetes mellitus. Few studies have tested for a relationship with coronary heart disease (CHD) or intermediate biomarkers. The role of artificially sweetened beverages is also unclear. METHODS AND RESULTS: We performed an analysis of the Health Professionals Follow-Up Study, a prospective cohort study including 42 883 men. Associations of cumulatively averaged sugar-sweetened (eg, sodas) and artificially sweetened (eg, diet sodas) beverage intake with incident fatal and nonfatal CHD (myocardial infarction) were examined with proportional hazard models. There were 3683 CHD cases over 22 years of follow-up. Participants in the top quartile of sugar-sweetened beverage intake had a 20% higher relative risk of CHD than those in the bottom quartile (relative risk=1.20; 95% confidence interval, 1.09-1.33; P for trend <0.01) after adjustment for age, smoking, physical activity, alcohol, multivitamins, family history, diet quality, energy intake, body mass index, pre-enrollment weight change, and dieting. Artificially sweetened beverage consumption was not significantly associated with CHD (multivariate relative risk=1.02; 95% confidence interval, 0.93-1.12; P for trend=0.28). Adjustment for self-reported high cholesterol, high triglycerides, high blood pressure, and diagnosed type 2 diabetes mellitus slightly attenuated these associations. Intake of sugar-sweetened but not artificially sweetened beverages was significantly associated with increased plasma triglycerides, C-reactive protein, interleukin-6, and tumor necrosis factor receptors 1 and 2 and decreased high-density lipoprotein, lipoprotein(a), and leptin (P<0.02). CONCLUSIONS: Consumption of sugar-sweetened beverages was associated with increased risk of CHD and some adverse changes in lipids, inflammatory factors, and leptin. Artificially sweetened beverage intake was not associated with CHD risk or biomarkers.

Effects of theobromine and caffeine on mood and vigilance.

Like caffeine, theobromine crosses the blood-brain barrier and binds to adenosine receptors, suggesting it might share caffeine's beneficial effects on mood and vigilance. Therefore, the purpose of this study was to assess the effect of theobromine doses commonly found in foods on mood and vigilance parameters sensitive to caffeine. Caffeine was tested as a positive control. Twenty-four men (age, 23 [3] years) completed 6 double-blind trials during which they consumed experimental beverages, assessed their mood using standardized self-report questionnaires, and completed a 2-hour visual vigilance task. Three experimental doses (100, 200, and 400 mg theobromine) were delivered in a cocoa-based beverage; 3 matched control treatments (0 mg theobromine, 400 mg theobromine, and 100 mg caffeine) were delivered in a non-cocoa beverage. Mean salivary concentrations of theobromine exhibited significant dose-dependent differences (400 mg trials > 200 mg trial > 100 mg trial > 0 mg trials; P < 0.005). At every dose tested, theobromine failed to consistently affect mood state or vigilance (P > 0.05), but 100-mg caffeine significantly decreased lethargy/fatigue and increased vigor (P = 0.006 and 0.011, respectively). These findings indicate theobromine does not influence mood and vigilance when administered in nutritionally relevant doses, despite sharing many of caffeine's structural characteristics.

Whey protein supplementation during resistance training augments lean body mass.

UNLABELLED: Compared to soy, whey protein is higher in leucine, absorbed quicker and results in a more pronounced increase in muscle protein synthesis. OBJECTIVE: To determine whether supplementation with whey promotes greater increases in muscle mass compared to soy or carbohydrate, we randomized non-resistance-trained men and women into groups who consumed daily isocaloric supplements containing carbohydrate (carb; n = 22), whey protein (whey; n = 19), or soy protein (soy; n = 22). METHODS: All subjects completed a supervised, whole-body periodized resistance training program consisting of 96 workouts (~9 months). Body composition was determined at baseline and after 3, 6, and 9 months. Plasma amino acid responses to resistance exercise followed by supplement ingestion were determined at baseline and 9 months. RESULTS: Daily protein intake (including the supplement) for carb, whey, and soy was 1.1, 1.4, and 1.4 g·kg body mass⁻¹, respectively. Lean body mass gains were significantly (p < 0.05) greater in whey (3.3 ± 1.5 kg) than carb (2.3 ± 1.7 kg) and soy (1.8 ± 1.6 kg). Fat mass decreased slightly but there were no differences between groups. Fasting concentrations of leucine were significantly elevated (20%) and postexercise plasma leucine increased more than 2-fold in whey. Fasting leucine concentrations were positively correlated with lean body mass responses. CONCLUSIONS: Despite consuming similar calories and protein during resistance training, daily supplementation with whey was more effective than soy protein or isocaloric carbohydrate control treatment conditions in promoting gains in lean body mass. These results highlight the importance of protein quality as an important determinant of lean body mass responses to resistance training.

Biomarkers for Pediatric Bacterial Musculoskeletal Infections in Lyme Disease-Endemic Regions.

OBJECTIVES: Bacterial musculoskeletal infections (MSKIs) are challenging to diagnose because of the clinical overlap with other conditions, including Lyme arthritis. We evaluated the performance of blood biomarkers for the diagnosis of MSKIs in Lyme disease-endemic regions. METHODS: We conducted a secondary analysis of a prospective cohort study of children 1 to 21 years old with monoarthritis presenting to 1 of 8 Pedi Lyme Net emergency departments for evaluation of potential Lyme disease. Our primary outcome was an MSKI, which was defined as septic arthritis, osteomyelitis or pyomyositis. We compared the diagnostic accuracy of routinely available biomarkers (absolute neutrophil count, C-reactive protein, erythrocyte sedimentation rate, and procalcitonin) to white blood cells for the identification of an MSKI using the area under the receiver operating characteristic curve (AUC). RESULTS: We identified 1423 children with monoarthritis, of which 82 (5.8%) had an MSKI, 405 (28.5%) Lyme arthritis, and 936 (65.8%) other inflammatory arthritis. When compared with white blood cell count (AUC, 0.63; 95% confidence interval [CI], 0.55-0.71), C-reactive protein (0.84; 95% CI, 0.80-0.89; P < .05), procalcitonin (0.82; 95% CI, 0.77-0.88; P < .05), and erythrocyte sedimentation rate (0.77; 95% CI, 0.71-0.82; P < .05) had higher AUCs, whereas absolute neutrophil count (0.67; 95% CI, 0.61-0.74; P < .11) had a similar AUC. CONCLUSIONS: Commonly available biomarkers can assist in the initial approach to a potential MSKI in a child. However, no single biomarker has high enough accuracy to be used in isolation, especially in Lyme disease-endemic areas.

Thiocyanate toxicity: a teaching case.

Introduction: Thiocyanate can cause gastrointestinal, neurologic, and cardiovascular toxicity. Additionally, it interferes with multiple laboratory assays. We present a case of acute thiocyanate toxicity. Case: A 17-year-old female presented with an intentional thiocyanate ingestion. Her course was notable for delirium, wide complex tachycardia, and presumed seizure activity with concurrent lactatemia, acidemia, and narrowing of her arterio-venous oxygen gradient. She received lipid emulsion therapy (LET). While hemodialysis was considered, she recovered without additional treatment. After resolution of her critical illness, a serum cyanide concentration was 0.21 mcg/mL. She had laboratory testing notable for hyperchloremia, hypocalcemia, hypokalemia, and an elevated salicylate concentration attributed to interference by thiocyanate. The thiocyanate was eliminated via first-order kinetics with a half-life of 61.6 hours. Discussion: Thiocyanate poisoning may cause cardiac and neurologic toxicity. Laboratory evidence of impaired cellular respiration in this case suggests possible in vivo conversion to cyanide, however this is not proven. Cyanide antidotal treatment was not administered for this patient, however LET was given based on thiocyanate's lipophilicity. Hemodialysis is known to effectively remove thiocyanate from the blood, however the patient improved without it. The patient's laboratory derangements were due to thiocyanate interference with ion selective electrode and colorimetric analyzer technology. Conclusions: Thiocyanate can cause cardiac and neurologic toxicity, and interferes with several laboratory assays. Theoretically, LET and cyanide antidotal treatment may be useful, but this requires further investigation. Thiocyanate toxicity should be managed supportively, and hemodialysis may be used in severe cases.

Clinical utility of Abbott Precision Xceed Pro® ketone meter in diabetic patients.

BACKGROUND: Diagnosis and management of diabetic ketoacidosis (DKA) often rely on the measurement of urine ketones along with blood glucose, anion gap, and pH. These values, however, do not reliably reflect the severity of ketoacidosis. The Abbott Precision Xceed Pro® meter is an FDA-approved device that quantitatively measures ß-hydroxybutyrate (BOH) in whole blood. This study was undertaken to determine whether the ketone meter meets the analytical criteria to aid DKA diagnosis and management in the hospital. METHODS: 54 heparinized venous whole blood BOH concentrations from 27 diabetic patients were measured by the Abbott meter, and compared with the plasma BOH concentrations measured with Stanbio reagent (reference method). Measurements were done in the hospital central laboratory. RESULTS: Of the 54 pairs of specimens analyzed, 17 pairs displayed a difference of >15% between the two methods. Nearly all discrepant points occurred when BOH >5 mmol/L (reference method). Linearity evaluation revealed that the meter is not linear from 0.0 to 8.0 mmol/L, contrary to the claim by the manufacturer. Further, we identified acetoacetate, a metabolite commonly present in DKA patients, as a potential interfering substance for the meter BOH measurement. CONCLUSIONS: BOH measurements by the Abbott meter up to 3 mmol/L correlate well with the reference method, but become discrepant above that point. While this characteristic may be useful in the diagnosis of DKA, it may not allow clinicians to serially follow the response to therapy in hospitalized DKA patients with BOH values greater than 5 mmol/L (reference method).

Development and validation of an immunoassay for quantification of NCAM-1 in human plasma.

BACKGROUND: Neural Cell Adhesion Molecule 1 (NCAM-1), a multifunctional member of the immunoglobulin superfamily, is expressed on the surface of neurons, glia, skeletal muscle, and natural killer cells. NCAM-1 has been implicated as having a role in cell-cell adhesion, involved in development of the nervous system, and for cells involved in the expansion of T cells and dendritic cells which play an important role in immune surveillance. Sensitive and specific methods to quantify non-surface bound NCAM-1 are not available. METHOD: A sandwich ligand binding assay was developed for quantification of NCAM-1 in plasma and validated using an electro-chemiluminescent (ECL) technology. RESULTS: The data presented here demonstrated that the validated method met all prespecified criteria for precision, linearity, and accuracy in the range of 62.5 ng/mL to 4000.0 ng/mL, the range believed to be most relevant for plasma. The bioanalytical validation of the assay established the inter-assay coefficient of variation <8 % for calibration points, <2 % for high quality control (HQC), <8 % for medium quality control (MQC) and <19 % for low quality control (LQC) samples. Purified NCAM-1 spike-recovery experiment in plasma was used to determine assay accuracy; nominal concentrations (%) of NCAM-1 ranged from 91 % to 112 % for high and low spike level, respectively. Assay performance was subsequently evaluated for parallelism, selectivity, interference, and stability. CONCLUSION: NCAM-1 assay has been developed and validated in human plasma and met all assay validation parameters pre-determined during development. Clinical testing of human plasma samples indicated that NCAM-1 does not seem to be influenced by age and was slightly influenced by gender. NCAM-1 assay has potential to be used as a biomarker assay once the assay is subjected to appropriate clinical assessment and diagnostic thresholds are established.

Clinical utility of a serum biomarker panel in distinguishing prostate cancer from benign prostate hyperplasia.

Prostate-specific antigen (PSA) screening for prostate cancer (PCa) is limited by the lack of specificity but is further complicated in the benign prostatic hyperplasia (BPH) population which also exhibit elevated PSA, representing a clear unmet need to distinguish BPH from PCa. Herein, we evaluated the utility of FLNA IP-MRM, age, and prostate volume to stratify men with BPH from those with PCa. Diagnostic performance of the biomarker panel was better than PSA alone in discriminating patients with negative biopsy from those with PCa, as well as those who have had multiple prior biopsies (AUC 0.75 and 0.87 compared to AUC of PSA alone 0.55 and 0.57 for patients who have had single compared to multiple negative biopsies, respectively). Of interest, in patients with PCa, the panel demonstrated improved performance than PSA alone in those with Gleason scores of 5-7 (AUC 0.76 vs. 0.56) and Gleason scores of 8-10 (AUC 0.74 vs. 0.47). With Gleason scores (8-10), the negative predictive value of the panel is 0.97, indicating potential to limit false negatives in aggressive cancers. Together, these data demonstrate the ability of the biomarker panel to perform better than PSA alone in men with BPH, thus preventing unnecessary biopsies.