Current immunoassays for the chemokine RANTES (regulated on activation, normal T-cell expressed and secreted) are not tailored for specific isoforms that exist endogenously, despite the fact that variants with modified activity are known to exist. This is surprising in view of this protein’s ubiquitous increased presence in many diseases and that the 2 established isoforms are truncated by enzymes also correlated to disease. An in-depth population survey of RANTES heterogeneity in the context of multiple diseases via a mass spectrometric immunoassay (MSIA) may resolve this issue.
We developed an MSIA for RANTES and endogenous variants apparent in human plasma. Samples from multiple cohorts of individuals (type 2 diabetes, congestive heart failure, history of myocardial infarction, and cancer patients) were run in parallel with samples from healthy individuals (239 people total). We used 230 μL of plasma per individual and tabulated relative percent abundance (RPA) values for identified isoforms.
We detected at least 19 variants, including the dipeptidyl peptidase IV (DPP-IV)-truncated variant. The majority of variants were unreported in the literature. Identifiable modifications included N- and/or C-terminal truncations, oxidation, glycation, and glycosylation. We observed statistically significant differences in RPA values for multiple variants between disease cohorts and recognized prospective disease-specific protein profiles for RANTES.
Because of widespread interest in the clinical value of RANTES, the protein diversity established here may aid in the design of future, fully quantitative assays. Equally important, an inclusive qualitative understanding of RANTES heterogeneity may present new insights into the relationship between RANTES and disease.
Accumulating evidence links higher circulating asymmetric dimethylarginine (ADMA) to greater risk of cardiovascular disease (CVD). Relatively small differences in ADMA concentrations between healthy individuals and those with disease underscore the need to formulate reference intervals that may aid risk stratification of individuals.
We formulated reference intervals for plasma ADMA concentrations using a community-based reference sample from the Framingham Offspring Study consisting of 1126 nonsmoking individuals [mean (SD) age 56 (9) years; 60% women] who were free of clinical CVD, hypertension, diabetes, and obesity and who attended a routine examination at which ADMA was assayed. ADMA concentrations were determined using a validated tandem mass spectrometry–liquid chromatography assay.
In the study sample, the mean ADMA concentration was 0.52 (0.11) μmol/L, and the reference limits were 0.311 and 0.732 (2.5th and 97.5th percentile). The sex-specific reference limits were 0.310 and 0.745 in men and 0.313 and 0.721 μmol/L in women. In multivariable regression analysis, ADMA plasma concentrations were positively correlated with age and total plasma homocysteine (both P < 0.001).
Reference limits calculated for circulating ADMA in our large community-based healthy reference sample confirm the previous observation of a relatively narrow distribution of concentrations. This suggests a tight physiological control of ADMA plasma concentrations, presumably by dimethylarginine dimethylaminohydrolase (DDAH) metabolism of ADMA.
A direct assay for small dense low density lipoprotein cholesterol (sdLDL-C) has been developed. Our goal was to establish normal ranges for this assay as well as to measure values in patients with established coronary heart disease (CHD) versus control subjects.
Direct LDL-C and sdLDL-C analyses were carried out on samples from 3,188 male and female participants of the Framingham Offspring Study, which included 173 male and 74 female CHD cases.
Male gender and female postmenopausal status were both associated with significantly (p<0.0001) higher sdLDL-C values. Use of cholesterol-lowering medications was significantly (p<0.0001) higher in CHD cases than in controls (46.8% versus 11.4% in men, and 35.1% versus 8.8% in women). Direct LDL-C levels were significantly lower in male CHD patients than in male controls (3.22 versus 3.51 mmol/L, p<0.0001), but their mean sdLDL-C levels were similar to those in controls (0.83 versus 0.84 mmol/L, p=0.609). Female CHD patients had similar LDL-C values to female controls (3.53 versus 3.46 mmol/L, p=0.543), but had significantly higher sdLDL-C values (0.83 versus 0.68 mmol/L, p=0.0015). Both male and female cases also had significantly (p<0.01) higher percentages of LDL-C as sdLDLC than controls.
Despite four fold greater cholesterol lowering therapy use, CHD patients had mean LDL-C values well above the LDL-C goal of < 2.6 mmol/L or 100 mg/dl, and male CHD cases had similar sdLDL C values and female CHD cases had significantly higher values than controls. These findings may explain some of the high residual risk of future CHD events in CHD patients.
small dense LDL-cholesterol; coronary heart disease; risk factor; Framingham Offspring Study; obesity
Adiponectin may have a protective role in the development of obesity-related metabolic and vascular disorders including hypertension. We conducted a prospective, nested case-control study to investigate the relationship between baseline plasma adiponectin, measures of adiposity, and subsequent risk of hypertension.
We selected 400 White and 400 Black postmenopausal women, aged <70 years, who have developed incident hypertension during 5.9-year follow-up and an equal number of age and race matched controls in the Women's Health Initiative Observational Study. We measured plasma concentrations of total adiponectin in their baseline bloods.
In crude matched models, plasma adiponectin was inversely associated with risk of hypertension among both White and Black women. The association appeared to be non-linear in White women but dose-related in Black women. Adjustment for lifestyle factors, measures of obesity, and obesity-related clinical factors attenuated these associations. The multivariable relative risks (95% confidence interval) of hypertension across increasing quartiles of plasma adiponectin were 1.00, 0.98 (0.66-1.46), 0.63 (0.41-0.97), and 0.92 (0.60-1.42) in White women (p, trend: 0.38) and 1.00, 0.96 (0.64-1.46), 0.83 (0.53-1.29), and 0.58 (0.36-0.94) in Black women (p, trend: 0.02). Further adjustment for inflammatory markers and endothelial markers eliminated the association in White, but not Black, women.
In this prospective, nested case-control study, we found an inverse association between plasma adiponectin and risk of hypertension in White and Black postmenopausal women. The reduced risk of hypertension was limited to intermediate levels of adiponectin in White women while was graded across quartiles of adiponectin in Black women.
adiponectin; hypertension; epidemiology; prospective study; postmenopausal women
The repertoire of serologic tests for identifying a monoclonal gammopathy includes serum and urine protein electrophoresis (PEL), serum and urine immunofixation electrophoresis (IFE), and quantitative serum free light chain (FLC). Although there are several reports on the relative diagnostic contribution of these assays, none has looked at the tests singly and in combination for the various plasma cell proliferative disorders (PCPDs).
Patients with a PCPD and all 5 assays performed within 30 days of diagnosis were included (n = 1877). The diagnoses were multiple myeloma (MM) (n = 467), smoldering multiple myeloma (SMM) (n =191), monoclonal gammopathy of undetermined significance (MGUS) (n = 524), plasmacytoma (n = 29), extramedullary plasmacytoma (n = 10), Waldenström macroglobulinemia (WM) (n = 26), primary amyloidosis (AL) (n = 581), light chain deposition disease (LCDD) (n =18), and POEMS syndrome (n = 31).
Of the 1877 patients, 26 were negative in all assays. Omitting urine from the panel lost an additional 23 patients (15 MGUS, 6 AL, 1 plasmacytoma, 1 LCDD), whereas the omission of FLC lost 30 patients (6 MM, 23 AL, and 1 LCDD). The omission of serum IFE as well as urine lost an additional 58 patients (44 MGUS, 7 POEMS, 5 AL, 1 SMM, and 1 plasmacytoma).
The major impact of using a simplified screening panel of serum PEL plus FLC rather than PEL, IFE, and FLC is an 8% reduction in sensitivity for MGUS, 23% for POEMS (7 patients), 4% for plasmacytoma (1 patient), 1% for AL, and 0.5% for SMM. There is no diminution in sensitivity for detecting MM, macroglobulinemia, and LCDD.
In 2008, the US Food and Drug Administration (FDA) issued a Guidance for Industry statement formally recognizing (during drug development) the conjoined nature of type 2 diabetes (T2D) and cardiovascular disease (CVD), which has precipitated an urgent need for panels of markers (and means of analysis) that are able to differentiate subtypes of CVD in the context of T2D. Here, we explore the possibility of creating such panels using the working hypothesis that proteins, in addition to carrying time-cumulative marks of hyperglycemia (e.g., protein glycation in the form of Hb A1c), may carry analogous information with regard to systemic oxidative stress and aberrant enzymatic signaling related to underlying pathobiologies involved in T2D and/or CVD.
We used mass spectrometric immunoassay to quantify, in targeted fashion, relative differences in the glycation, oxidation, and truncation of 11 specific proteins.
Protein oxidation and truncation (owing to modified enzymatic activity) are able to distinguish between subsets of diabetic patients with or without a history of myocardial infarction and/or congestive heart failure where markers of glycation alone cannot.
Markers based on protein modifications aligned with the known pathobiologies of T2D represent a reservoir of potential cardiovascular markers that are needed to develop the next generation of antidiabetes medications.
Findings regarding the association of lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and mass with incident cardiovascular disease (CVD) have been inconsistent, and their role in risk prediction is uncertain.
A case-cohort sample from the Women’s Health Initiative Observational Study (WHI-OS) comprised 1,821 CVD cases and a subcohort of 1,992. Cox regression models with inverse sampling weights assessed the association of Lp-PLA2 mass and activity with CVD (myocardial infarction [MI], stroke, and CVD mortality).
Subcohort means were 184.3 mmol/min/mL for Lp-PLA2 activity and 499.2 ng/mL for Lp-PLA2 mass, with 99% having mass above 200 ng/mL, the clinically recommended cut-point. Both activity and mass were positively associated with incident CVD in age- and race/ethnicity-adjusted analyses. Following adjustment by CVD risk factors, the association with activity became null (hazard ratio [HR] = 1.02 for top vs. bottom quartile, 95% confidence interval [CI] = 0.79-1.33, p-trend=0.65), but the association with mass remained (HR = 1.84, 95% CI = 1.45-2.34, p-trend <0.0001). In contrast to blood pressure, HDL, and hsCRP, reclassification statistics for Lp-PLA2 mass did not suggest improvement for overall CVD after full adjustment.
In the WHI-OS Lp-PLA2 mass, but not activity, was independently associated with CVD. However, model fit did not significantly improve with Lp-PLA2, and assay calibration remains a clinical concern.
We analyzed serial data in patients with clinically stable monoclonal gammopathy to determine the total variation of serum M-spikes [measured with serum protein electrophoresis (SPEP)], urine M-spikes [measured with urine protein electrophoresis (UPEP)], and monoclonal serum free light chain (FLC) concentrations measured with immunoassay.
Patients to be studied were identified by (a) no treatment during the study interval, (b) no change in diagnosis and <5 g/L change in serum M-spike over the course of observation; (c) performance of all 3 tests (SPEP, UPEP, FLC immunoassay) in at least 3 serial samples that were obtained 9 months to 5 years apart; (d) serum M-spike ≥10 g/L, urine M-spike ≥200 mg/24 h, or clonal FLC ≥100 mg/L. The total CV was calculated for each method.
Among the cohort of 158 patients, 90 had measurable serum M-spikes, 25 had urine M-spikes, and 52 had measurable serum FLC abnormalities. The CVs were calculated for serial SPEP M-spikes (8.1%), UPEP M-spikes (35.8%), and serum FLC concentrations (28.4%). Combining these CVs and the interassay analytical CVs, we calculated the biological CV for the serum M-spike (7.8%), urine M-spike (35.5%), and serum FLC concentration (27.8%).
The variations in urine M-spike and serum FLC measurements during patient monitoring are similar and are larger than those for serum M-spikes. In addition, in this group of stable patients, a measurable serum FLC concentration was available twice as often as a measurable urine M-spike.
Identification of von Willebrand factor (vWF) abnormalities in a variety of conditions is hampered by the limitations of currently available diagnostic tests. Although direct multimer visualization by immunoelectrophoresis is a commonly used method, it is impractical as a routine clinical test. In this study, we used a biophysical analysis tool, fluorescence correlation spectroscopy (FCS), to measure vWF distributions. The goals were to develop a method that is quicker and simpler than vWF gel electrophoresis and to evaluate the potential of FCS as a clinical diagnostic technique.
We analyzed plasma from 12 patients with type 1 von Willebrand disease (vWD), 14 patients with type 2 vWD, and 10 healthy controls using a fluctuation-based immunoassay approach.
FCS enabled identification and proper classification of type 1 and type 2 vWD, producing quantitative results that correspond to qualitative gel multimer patterns. FCS required minimal sample preparation and only a 5-min analysis time.
This study represents the first implementation of FCS for clinical diagnostics directly on human plasma. The technique shows potential for further vWF studies and as a generally applicable laboratory test method.
Selecting controls that match cases on risk factors for the outcome is a pervasive practice in biomarker research studies. Yet, such matching biases estimates of biomarker prediction performance. The magnitudes of bias are unknown.
We examined the prediction performance of biomarkers and improvements in prediction gained by adding biomarkers to risk factor information. Data simulated from bivariate normal statistical models and data from a study to identify critically ill patients were used. We compared true performance with that estimated from case-control studies that do or do not use matching. Receiver operating characteristic curves quantified performance. We propose a new statistical method to estimate prediction performance from matched studies when data on the matching factors are available for subjects in the population.
Performance estimated with standard analyses can be grossly biased by matching especially when biomarkers are highly correlated with matching risk factors. In our studies, the performance of the biomarker alone was underestimated while the improvement in performance gained by adding the marker to risk factors was overestimated by 2 to 10 fold. We found examples where the relative ranking of two biomarkers for prediction was inappropriately reversed by use of a matched design. The new approach to estimation corrected for bias in matched studies.
To properly gauge prediction performance in the population or the improvement gained by adding a biomarker to known risk factors, matched case-control studies must be supplemented with risk factor information from the population and must be analyzed with nonstandard statistical methods.
design; diagnosis; prediction; prognosis; receiver operating characteristic curve
Δ9-Tetrahydrocannabinol (THC) is the most frequently observed illicit drug in investigations of accidents and driving under the influence of drugs. THC-glucuronide has been suggested as a marker of recent cannabis use, but there are no blood data following controlled THC administration to test this hypothesis. Furthermore, there are no studies directly examining whole-blood cannabinoid pharmacokinetics, although this matrix is often the only available specimen.
Participants (9 men, 1 woman) resided on a closed research unit and smoked one 6.8% THC cannabis cigarette ad libitum. We quantified THC, 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-THC (THCCOOH), cannabidiol (CBD), cannabinol (CBN), THC-glucuronide and THCCOOH-glucuronide directly in whole blood and plasma by liquid chromatography/ tandem mass spectrometry within 24 h of collection to obviate stability issues.
Median whole blood (plasma) observed maximum concentrations (Cmax) were 50 (76), 6.4 (10), 41 (67), 1.3 (2.0), 2.4 (3.6), 89 (190), and 0.7 (1.4) μg/L 0.25 h after starting smoking for THC, 11-OH-THC, THCCOOH, CBD, CBN, and THCCOOH-glucuronide, respectively, and 0.5 h for THC-glucuronide. At observed Cmax, whole-blood (plasma) detection rates were 60% (80%), 80% (90%), and 50% (80%) for CBD, CBN, and THC-glucuronide, respectively. CBD and CBN were not detectable after 1 h in either matrix (LOQ 1.0 μg/L).
Human whole-blood cannabinoid data following cannabis smoking will assist whole blood and plasma cannabinoid interpretation, while furthering identification of recent cannabis intake.
Sativex®, a cannabis extract oromucosal spray containing Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), is currently in phase III trials as an adjunct to opioids for cancer pain treatment, and recently received United Kingdom approval for treatment of spasticity. There are indications that CBD modulates THC’s effects, but it is unclear if this is due to a pharmacokinetic and/or pharmacodynamic interaction.
Cannabis smokers provided written informed consent to participate in this randomized, controlled, double-blind, double-dummy institutional review board–approved study. Participants received 5 and 15 mg synthetic oral THC, low-dose (5.4 mg THC and 5.0 mg CBD) and high-dose (16.2 mg THC and 15.0 mg CBD) Sativex, and placebo over 5 sessions. CBD, THC, 11-hydroxy-THC, and 11-nor-9-carboxy-THC were quantified in plasma by 2-dimensional GC-MS. Lower limits of quantification were ≤0.25 μg/L.
Nine cannabis smokers completed all 5 dosing sessions. Significant differences (P < 0.05) in maximum plasma concentrations (Cmax) and areas under the curve from 0–10.5 h postdose (AUC0→10.5) for all analytes were found between low and high doses of synthetic THC and Sativex. There were no statistically significant differences in Cmax, time to maximum concentration or in the AUC0→10.5 between similar oral THC and Sativex doses. Relative bioavailability was calculated to determine the relative rate and extent of THC absorption; 5 and 15 mg oral THC bioavailability was 92.6% (13.1%) and 98.8% (11.0%) of low- and high-dose Sativex, respectively.
These data suggest that CBD modulation of THC’s effects is not due to a pharmacokinetic interaction at these therapeutic doses.
We measured Δ9-tetrahydrocannabinol (THC), 11-nor-9-carboxy-THC (THCCOOH), cannabidiol (CBD), and cannabinol (CBN) disposition in oral fluid (OF) following controlled cannabis smoking to evaluate whether monitoring multiple cannabinoids in OF improved OF test interpretation.
Cannabis smokers provided written informed consent for this institutional review board–approved study. OF was collected with the Quantisal™ device following ad libitum smoking of one 6.8% THC cigarette. Cannabinoids were quantified by 2-dimensional GC-MS. We evaluated 8 alternative cutoffs based on different drug testing program needs.
10 participants provided 86 OF samples −0.5 h before and 0.25, 0.5, 1, 2, 3, 4, 6, and 22 h after initiation of smoking. Before smoking, OF samples of 4 and 9 participants were positive for THC and THCCOOH, respectively, but none were positive for CBD and CBN. Maximum THC, CBD, and CBN concentrations occurred within 0.5 h, with medians of 644, 30.4, and 49.0 μg/L, respectively. All samples were THC positive at 6 h (2.1–44.4 μg/L), and 4 of 6 were positive at 22 h. CBD and CBN were positive only up to 6 h in 3 (0.6–2.1 μg/L) and 4 (1.0–4.4 μg/L) participants, respectively. The median maximum THCCOOH OF concentration was 115 ng/L, with all samples positive to 6 h (14.8–263 ng/L) and 5 of 6 positive at 22 h.
By quantifying multiple cannabinoids and evaluating different analytical cutoffs after controlled cannabis smoking, we determined windows of drug detection, found suggested markers of recent smoking, and minimized the potential for passive contamination.
3,4-Methylendioxymethamphetamine (MDMA) is excreted in human urine as unchanged drug and phase I and II metabolites. Previous urinary excretion studies after controlled oral MDMA administration have been performed only after conjugate cleavage. Therefore, we investigated intact MDMA glucuronide and sulfate metabolite excretion.
We used LC–high-resolution MS and GC-MS to reanalyze blind urine samples from 10 participants receiving 1.0 or 1.6 mg/kg MDMA orally. We determined median Cmax, tmax, first and last detection times, and total urinary recovery; calculated ratios of sulfates and glucuronides; and performed in vitro–in vivo correlations.
Phase II metabolites of 3,4-dihydroxymeth-amphetamine (DHMA), 4-hydroxy-3-methoxymeth-amphetamine (HMMA), 3,4-dihydroxyamphetamine (DHA), and 4-hydroxy-3-methoxyamphetamine were identified, although only DHMA sulfates, HMMA sulfate, and HMMA glucuronide had substantial abundance. Good correlation was observed for HMMA measured after acid hydrolysis and the sum of unconjugated HMMA, HMMA glucuronide, and HMMA sulfate (R2 = 0.87). More than 90% of total DHMA and HMMA were excreted as conjugates. The analyte with the longest detection time was HMMA sulfate. Median HMMA sulfate/glucuronide and DHMA 3-sulfate/4-sulfate ratios for the first 24 h were 2.0 and 5.3, respectively, in accordance with previous in vitro calculations from human liver microsomes and cytosol experiments.
Human MDMA urinary metabolites are primarily sulfates and glucuronides, with sulfates present in higher concentrations than glucuronides. This new knowledge may lead to improvements in urine MDMA and metabolite analysis in clinical and forensic toxicology, particularly for the performance of direct urine analysis.
Defining cannabinoid stability in authentic oral fluid (OF) is critically important for result interpretation. There are few published OF stability data, and of those available, all employed fortified synthetic OF solutions or elution buffers; none included authentic OF following controlled cannabis smoking.
An expectorated OF pool and a pool of OF collected with Quantisal™ devices were prepared for each of 10 participants. Δ9-Tetrahydrocannabinol (THC), 11-nor-9-carboxy-THC (THCCOOH), cannabidiol (CBD), and cannabinol (CBN) stability in each of 10 authentic expectorated and Quantisal-collected OF pools were determined after storage at 4 °C for 1 and 4 weeks and at −20 °C for 4 and 24 weeks. Results within ±20% of baseline concentrations analyzed within 24 h of collection were considered stable.
All Quantisal OF cannabinoid concentrations were stable for 1 week at 4 °C. After 4 weeks at 4 °C, as well as 4 and 24 weeks at −20 °C, THC was stable in 90%, 80%, and 80% and THCCOOH in 89%, 40%, and 50% of Quantisal samples, respectively. Cannabinoids in expectorated OF were less stable than in Quantisal samples when refrigerated or frozen. After 4 weeks at 4 and −20 °C, CBD and CBN were stable in 33%–100% of Quantisal and expectorated samples; by 24 weeks at −20 °C, CBD and CBN were stable in ≤44%.
Cannabinoid OF stability varied by analyte, collection method, and storage duration and temperature, and across participants. OF collection with a device containing an elution/stabilization buffer, sample storage at 4 °C, and analysis within 4 weeks is preferred to maximize result accuracy.
Low-level mutations in clinical tumor samples often reside below mutation detection limits, thus leading to false negatives that may impact clinical diagnosis and patient management. COLD-PCR is a technology that magnifies unknown mutations during PCR, thus enabling downstream mutation detection. However, a practical difficulty in applying COLD-PCR has been the requirement for strict control of the denaturation temperature for a given sequence, to within ±0.3°C. This precludes simultaneous mutation enrichment in sequences of substantially different melting-temperature (Tm) and limits the technique to a single sequence at a time. We present a temperature-tolerant (TT-COLD-PCR) approach that reduces this obstacle.
Thermo-cycling programs featuring a gradual increase of the denaturation temperature during COLD-PCR are described. This approach enables enrichment of mutations when the cycling achieves the appropriate critical denaturation temperature of each DNA amplicon that is being amplified. Validation is provided for KRAS and TP53 exons 6–9 using dilutions of mutated DNA, clinical cancer samples and plasma-circulating DNA.
A single thermocycling program with a denaturation-temperature window of 2.5–3.0°C enriches mutations in all DNA amplicons simultaneously, despite their different Tms. Mutation enrichments of 6–9-fold were obtained using TT-full-COLD-PCR. Higher mutation enrichments were obtained for the other two forms of COLD-PCR, fast-COLD-PCR and ice-COLD-PCR.
Low-level mutations in diverse amplicons with different Tm can be mutation-enriched via TT-COLD-PCR provided that their Tms fall within the denaturation-temperature window applied during amplification. This approach enables simultaneous enrichment of mutations in several amplicons, and increases significantly the versatility of COLD-PCR.
COLD-PCR; mutation detection; mutation enrichment; low-abundance mutations; cancer
The use of serum human chorionic gonadotropin (hCG) and progesterone to identify patients with ectopic pregnancy (EP) has been shown to have poor clinical utility. Pregnancy-associated circulating microRNAs (miRNAs) have been proposed as potential biomarkers for the diagnosis of pregnancy-associated complications. This proof-of concept study examined the diagnostic accuracy of various miRNAs to detect EP in an emergency department (ED) setting.
This was a retrospective case-control analysis of 89 women who presented to the ED with vaginal bleeding and/or abdominal pain/cramping, and were diagnosed with viable intrauterine pregnancy (VIP), spontaneous abortion (SA), or EP. Serum hCG and progesterone concentrations were determined by immunoassays. Serum miR-323-3p, miR-517a, miR-519d, and miR-525-3p concentrations were measured using TaqMan real-time PCR. Statistical analysis was performed to determine the clinical utility of these biomarkers as single markers and as multimarker panels for EP.
Concentrations of serum hCG, progesterone, miR-517a, miR-519d, and miR-525-3p were significantly lower in EP and SA than in VIP. In contrast, the concentration of miR-323-3p was significantly elevated in EP as compared to SA and VIP. As a single marker, miR-323-3p had the highest sensitivity of 37.0% (at a fixed-specificity of 90%). Comparatively, combined hCG, progesterone, and miR-323-3p panel yielded the highest sensitivity of 77.8% (at a fixed-specificity of 90%). A stepwise analysis using hCG, then progesterone, and then miR-323-3p resulted in 96.3% sensitivity and 72.6% specificity.
Pregnancy-associated miRNAs, especially miR-323-3p, added significant diagnostic accuracy to a panel including hCG and progesterone for the diagnosis of EP.
Ectopic pregnancy; MicroRNAs; Biomarkers; Diagnostic accuracy
The diagnosis of galactosemia usually involves the measurement of galactose-1-phosphate uridyltransferase (GALT) activity. Traditional radioactive and fluorescent GALT assays are nonspecific, laborious, and/or lack sufficient analytical sensitivity. We developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS)–based assay for GALT enzyme activity measurement.
Our assay used stable isotope-labeled α-galactose-1-phosphate ([13C6]-Gal-1-P) as an enzyme substrate. Sample cleanup and separation were achieved by reversed-phase ion-pair chromatography, and the enzymatic product, isotope-labeled uridine diphosphate galactose ([13C6]-UDPGal), was detected by MS/MS at mass transition (571 > 323) and quantified by use of [13C6]-Glu-1-P (265 > 79) as an internal standard.
The method yielded a mean (SD) GALT enzyme activity of 23.8 (3.8) µmol · (gHgb)−1 · h−1 in erythrocyte extracts from 71 controls. The limit of quantification was 0.04 µmol · (g Hgb)−1 · h−1 (0.2% of normal control value). Intraassay imprecision was determined at 4 different levels (100%, 25%, 5%, and 0.2% of the normal control values), and the CVs were calculated to be 2.1%, 2.5%, 4.6%, and 9.7%, respectively (n = 3). Interassay imprecision CVs were 4.5%, 6.7%, 8.2%, and 13.2% (n = 5), respectively. The assay recoveries at the 4 levels were higher than 90%. The apparent Km of the 2 substrates, Gal-1-P and UDPGlc, were determined to be 0.38 mmol/L and 0.071 mmol/L, respectively. The assay in erythrocytes of 33 patients with classical galactosemia revealed no detectable activity.
This LC-MS/MS–based assay for GALT enzyme activity will be useful for the diagnosis and study of biochemically heterogeneous patients with galactosemia, especially those with uncommon genotypes and detectable but low residual activities.
Mass spectrometric assays have the potential to replace protein immunoassays in basic science, clinical research, and clinical care. Previous studies have demonstrated the utility of assays using multiple-reaction monitoring mass spectrometry (MRM-MS) for the quantification of proteins in biological samples and many examples of the accuracy of these approaches to quantify spiked analytes have been reported. However, a direct comparison of multiplexed assays using liquid chromatography-tandem mass spectrometry with established immunoassays to measure endogenous proteins has not been reported.
We purified the HDL from the plasma of 30 human subjects enrolled in a clinical nutrition research study and used label-free shotgun proteomics approaches to analyze each sample. We then developed two different 6-plex assays that used isotope dilution MRM-MS: one assay used stable isotope labeled peptides and the other used stable isotope labeled apolipoprotein A-I (apoA-I), the most abundant protein in HDL, as internal standards to control for matrix effects and mass spectrometer performance. The shotgun and MRM-MS assays were then compared with commercially available immunoassays for each of the six analytes.
Quantification by shotgun proteomics approaches correlated poorly with the six protein immunoassays. However, the MRM-MS approaches that used internal standard peptide or a single internal standard protein correlated well. In addition, MRM-MS approaches had good repeatability (<10% CV) and linearity.
Multiplexed MRM-MS assays correlate well with immunochemical measurements and have acceptable operating characteristics in complex samples. Our results support the proposal that MRM-MS could be used to replace immunoassays in a variety of settings.
Mass spectrometry; multiple reaction monitoring; endogenous; proteins; high density lipoprotein; targeted proteomics
Genomic research in cardiovascular disease (CVD) has progressed rapidly over the last five years. However, in most cases these ground-breaking observations have not yet been accompanied by clinically applicable tools for risk prediction, diagnosis, or therapeutic interventions.
We reviewed the English literature for novel methods and promising genomic targets that will permit large-scale screening and follow-up of recent genomic findings for CVD. We anticipate that advances in three key areas will be critical for the success of these projects. First, exome-centered and whole genome next generation sequencing will identify rare and novel genetic variants associated with CVD and its risk factors. Improvements in methods will also greatly advance the field of epigenetics and gene expression in humans. Second, research increasingly acknowledges that static DNA sequence variation explains only a fraction of the inherited phenotype. Therefore we expect that multifold epigenetic and gene expression signatures will be related to CVD in experimental and clinical settings. Leveraging existing large-scale consortia and clinical biobanks combined with electronic health records holds promise to integrate epidemiological and clinical genomics data. Finally, a systems biology approach will be needed to integrate the accumulated multidimensional data.
Novel methods in sequencing, epigenetics and transcriptomics, and unprecedented large-scale cooperative efforts promise to generate insights into complex CVD. The rapid accumulation and integration of knowledge will shed light onto the considerable proportion of the missing heritability of CVD.
genomics; sequencing; epigenetics; transcriptomics
New high-performance liquid chromatography/ tandem mass spectrometry (LC-MS/MS) methods are among the most successful approaches to improve specificity problems inherent in many immunoassays.
We emphasize problems with immunoassays for the measurement of steroids and review the emerging role of LC-MS/MS in the measurement of clinically relevant steroids. The latest generation of tandem mass spectrometers has superior limits of quantification, permitting omission of previously employed derivatization steps. The measurement of steroid profiles in the diagnosis and treatment of congenital adrenal hyperplasia, adrenal insufficiency, chronic pelvic pain and prostatitis, oncology (breast cancer), and athletes has important new applications.
LC-MS/MS now affords the specificity, imprecision, and limits of quantification necessary for the reliable measurement of steroids in human fluids, enhancing diagnostic capabilities, particularly when steroid profiles are available.