Sex-dependent associations of maternal androgen levels with offspring BMI and weight trajectory from birth to early childhood.

CONTEXT: In preclinical studies, high androgen levels during pregnancy are associated with low birth weight and rapid postnatal weight gain in the offspring. However, human data linking prenatal androgens with birth weight and early life weight gain in the offspring are scarce. DESIGN: We evaluated 516 mother-child pairs enrolled in the New England birth cohorts of the Collaborative Perinatal Project (1959-1966). We assayed androgen bioactivity in maternal sera during third-trimester using a receptor-mediated luciferase expression bioassay. Age and sex-specific BMI Z-scores (BMIz), defined using established standards, were assessed at birth, 4 months, 1 year, 4 years, and 7 years. We used linear mixed models to evaluate the relation of maternal androgens with childhood BMIz overall and by sex. We examined the association of maternal androgens with fetal growth restriction. The association of weight trajectories with maternal androgens was examined using multinomial logistic regression. RESULTS: Higher maternal androgen levels associated with lower BMIz at birth (ß = - 0.39, 95% CI: - 0.73, - 0.06); this relation was sex-dependent, such that maternal androgens significantly associated with BMIz at birth in girls alone (ß = - 0.72, 95% CI: - 1.40, - 0.04). The relation of maternal androgens with fetal growth restriction revealed dose threshold effects that differed by sex. There was no significant association between maternal androgens and weight trajectory overall. However, we found a significant sex interaction (p = 0.01); higher maternal androgen levels associated with accelerated catch-up growth in boys (aOR = 2.14, 95% CI: 1.14, 4.03). CONCLUSION: Our findings provide evidence that maternal androgens may have differential effects on the programming of intrauterine growth and postnatal weight gain depending on fetal sex.

Capillary electrophoretic assay of human acetyl-coenzyme A carboxylase 2.

Human acetyl-coenzyme A carboxylase 2 catalyzes the carboxylation of acetyl coenzyme A to form malonyl coenzyme A, along with the conversion of magnesium-adenosine triphosphate complex to magnesium-adenosine diphosphate complex. A simple off-column capillary electrophoresis assay for human acetyl-coenzyme A carboxylase 2 was developed based on the separation of magnesium-adenosine triphosphate complex, magnesium-adenosine diphosphate complex, acetyl coenzyme A and malonyl coenzyme A with detection by ultraviolet absorption at 256 nm. When Mg2+ was absent from the separation buffer, the zones due to magnesium-adenosine triphosphate complex and magnesium-adenosine diphosphate complex both split and migrated as two separate peaks. With Mg2+ added to the separation buffer, magnesium-adenosine triphosphate complex and magnesium-adenosine diphosphate complex produced single peaks, and the reproducibility of peak shape and area improved for human acetyl-coenzyme A carboxylase 2 assay components. The final separation buffer used was 30.0 mM HEPES, 3.0 mM MgCl2 , 2.5 mM KHCO3 , and 2.5 mM potassium citrate at pH 7.50. The same buffer was used for the enzyme-catalyzed reaction (off-column). Inhibition of human acetyl-coenzyme A carboxylase 2 by CP-640186, a known inhibitor, was detected using the capillary electrophoresis assay.

Predicting suicides after outpatient mental health visits in the Army Study to Assess Risk and Resilience in Servicemembers (Army STARRS).

The 2013 US Veterans Administration/Department of Defense Clinical Practice Guidelines (VA/DoD CPG) require comprehensive suicide risk assessments for VA/DoD patients with mental disorders but provide minimal guidance on how to carry out these assessments. Given that clinician-based assessments are not known to be strong predictors of suicide, we investigated whether a precision medicine model using administrative data after outpatient mental health specialty visits could be developed to predict suicides among outpatients. We focused on male nondeployed Regular US Army soldiers because they account for the vast majority of such suicides. Four machine learning classifiers (naive Bayes, random forests, support vector regression and elastic net penalized regression) were explored. Of the Army suicides in 2004-2009, 41.5% occurred among 12.0% of soldiers seen as outpatient by mental health specialists, with risk especially high within 26 weeks of visits. An elastic net classifier with 10-14 predictors optimized sensitivity (45.6% of suicide deaths occurring after the 15% of visits with highest predicted risk). Good model stability was found for a model using 2004-2007 data to predict 2008-2009 suicides, although stability decreased in a model using 2008-2009 data to predict 2010-2012 suicides. The 5% of visits with highest risk included only 0.1% of soldiers (1047.1 suicides/100 000 person-years in the 5 weeks after the visit). This is a high enough concentration of risk to have implications for targeting preventive interventions. An even better model might be developed in the future by including the enriched information on clinician-evaluated suicide risk mandated by the VA/DoD CPG to be recorded.

Contributions of the social environment to first-onset and recurrent mania.

In treated cohorts, individuals with bipolar disorder are more likely to report childhood adversities and recent stressors than individuals without bipolar disorder; similarly, in registry-based studies, childhood adversities are more common among individuals who later become hospitalized for bipolar disorder. Because these types of studies rely on treatment-seeking samples or hospital diagnoses, they leave unresolved the question of whether or not social experiences are involved in the etiology of bipolar disorder. We investigated the role of childhood adversities and adulthood stressors in liability for bipolar disorder using data from the National Epidemiologic Survey on Alcohol and Related Conditions (n=33 375). We analyzed risk for initial-onset and recurrent DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition) manic episodes during the study's 3-year follow-up period. Childhood physical abuse and sexual maltreatment were associated with significantly higher risks of both first-onset mania (odds ratio (OR) for abuse: 2.23; 95% confidence interval (CI)=1.71, 2.91; OR for maltreatment: 2.10; CI=1.55, 2.83) and recurrent mania (OR for abuse: 1.55; CI=1.00, 2.40; OR for maltreatment: 1.60; CI=1.00, 2.55). In addition, past-year stressors in the domains of interpersonal instability and financial hardship were associated with a significantly higher risk of incident and recurrent mania. Exposure to childhood adversity potentiated the effects of recent stressors on adult mania. Our findings demonstrate a role of social experiences in the initial onset of bipolar disorder, as well as in its prospective course, and are consistent with etiologic models of bipolar disorder that implicate deficits in developmentally established stress-response pathways.

Food insecurity and mental illness: disproportionate impacts in the context of perceived stress and social isolation.

OBJECTIVES: Food insecurity is associated with elevated risk of mental illness. This risk may be further compounded by stressful life events and by social isolation. This study investigated whether the risk of mental illness is higher among individuals experiencing food insecurity along with greater stress and social isolation. STUDY DESIGN: Cross-sectional self-report survey data from the 2009-10 Canadian Community Health Survey (N = 100,401). METHODS: We estimated prevalence differences of the risk of self-reported mental illness associated with food insecurity alone and in combination with stressful life events and social isolation. Sensitivity analyses were conducted on a sub-sample who completed a structured diagnostic interview. RESULTS: Overall, the prevalence of mental illness was 18.4% [95% CI 16.7-20.1] higher for women and 13.5% higher [95% CI 11.9, 15.2] for men in severely food insecure households compared to those reporting food security. The increased risk of mental illness associated with food insecurity was more pronounced among females and those reporting higher stress and social isolation. CONCLUSIONS: Individuals reporting food insecurity are at increased risk of mental illness. This increased risk is further exacerbated in high stress and socially isolated environments. Policies, clinical and public health interventions must address broader constellations of risks that exist when food insecurity is present.

Occupational differences in US Army suicide rates.

BACKGROUND: Civilian suicide rates vary by occupation in ways related to occupational stress exposure. Comparable military research finds suicide rates elevated in combat arms occupations. However, no research has evaluated variation in this pattern by deployment history, the indicator of occupation stress widely considered responsible for the recent rise in the military suicide rate. METHOD: The joint associations of Army occupation and deployment history in predicting suicides were analysed in an administrative dataset for the 729 337 male enlisted Regular Army soldiers in the US Army between 2004 and 2009. RESULTS: There were 496 suicides over the study period (22.4/100 000 person-years). Only two occupational categories, both in combat arms, had significantly elevated suicide rates: infantrymen (37.2/100 000 person-years) and combat engineers (38.2/100 000 person-years). However, the suicide rates in these two categories were significantly lower when currently deployed (30.6/100 000 person-years) than never deployed or previously deployed (41.2-39.1/100 000 person-years), whereas the suicide rate of other soldiers was significantly higher when currently deployed and previously deployed (20.2-22.4/100 000 person-years) than never deployed (14.5/100 000 person-years), resulting in the adjusted suicide rate of infantrymen and combat engineers being most elevated when never deployed [odds ratio (OR) 2.9, 95% confidence interval (CI) 2.1-4.1], less so when previously deployed (OR 1.6, 95% CI 1.1-2.1), and not at all when currently deployed (OR 1.2, 95% CI 0.8-1.8). Adjustment for a differential 'healthy warrior effect' cannot explain this variation in the relative suicide rates of never-deployed infantrymen and combat engineers by deployment status. CONCLUSIONS: Efforts are needed to elucidate the causal mechanisms underlying this interaction to guide preventive interventions for soldiers at high suicide risk.

Understanding the elevated suicide risk of female soldiers during deployments.

BACKGROUND: The Army Study to Assess Risk and Resilience in Servicemembers (Army STARRS) has found that the proportional elevation in the US Army enlisted soldier suicide rate during deployment (compared with the never-deployed or previously deployed) is significantly higher among women than men, raising the possibility of gender differences in the adverse psychological effects of deployment. METHOD: Person-month survival models based on a consolidated administrative database for active duty enlisted Regular Army soldiers in 2004-2009 (n = 975,057) were used to characterize the gender × deployment interaction predicting suicide. Four explanatory hypotheses were explored involving the proportion of females in each soldier's occupation, the proportion of same-gender soldiers in each soldier's unit, whether the soldier reported sexual assault victimization in the previous 12 months, and the soldier's pre-deployment history of treated mental/behavioral disorders. RESULTS: The suicide rate of currently deployed women (14.0/100,000 person-years) was 3.1-3.5 times the rates of other (i.e. never-deployed/previously deployed) women. The suicide rate of currently deployed men (22.6/100,000 person-years) was 0.9-1.2 times the rates of other men. The adjusted (for time trends, sociodemographics, and Army career variables) female:male odds ratio comparing the suicide rates of currently deployed v. other women v. men was 2.8 (95% confidence interval 1.1-6.8), became 2.4 after excluding soldiers with Direct Combat Arms occupations, and remained elevated (in the range 1.9-2.8) after adjusting for the hypothesized explanatory variables. CONCLUSIONS: These results are valuable in excluding otherwise plausible hypotheses for the elevated suicide rate of deployed women and point to the importance of expanding future research on the psychological challenges of deployment for women.

Theoretical limitations of quantification for noncompetitive sandwich immunoassays.

Immunoassays exploit the highly selective interaction between antibodies and antigens to provide a vital method for biomolecule detection at low concentrations. Developers and practitioners of immunoassays have long known that non-specific binding often restricts immunoassay limits of quantification (LOQs). Aside from non-specific binding, most efforts by analytical chemists to reduce the LOQ for these techniques have focused on improving the signal amplification methods and minimizing the limitations of the detection system. However, with detection technology now capable of sensing single-fluorescence molecules, this approach is unlikely to lead to dramatic improvements in the future. Here, fundamental interactions based on the law of mass action are analytically connected to signal generation, replacing the four- and five-parameter fittings commercially used to approximate sigmoidal immunoassay curves and allowing quantitative consideration of non-specific binding and statistical limitations in order to understand the ultimate detection capabilities of immunoassays. The restrictions imposed on limits of quantification by instrumental noise, non-specific binding, and counting statistics are discussed based on equilibrium relations for a sandwich immunoassay. Understanding the maximal capabilities of immunoassays for each of these regimes can greatly assist in the development and evaluation of immunoassay platforms. While many studies suggest that single molecule detection is possible through immunoassay techniques, here, it is demonstrated that the fundamental limit of quantification (precision of 10 % or better) for an immunoassay is approximately 131 molecules and this limit is based on fundamental and unavoidable statistical limitations.

Capillary electrophoresis-based assay of phosphofructokinase-1.

An assay was developed for phosphofructokinase-1 (PFK-1) using capillary electrophoresis (CE). In the glycolytic pathway, this enzyme catalyzes the rate-limiting step from fructose-6-phosphate and magnesium-bound adenosine triphosphate (Mg-ATP) to fructose-1,6-bisphosphate and magnesium-bound adenosine diphosphate (Mg-ADP). This enzyme has recently become a research target because of the importance of glycolysis in cancer and obesity. The CE assay for PFK-1 is based on the separation and detection by ultraviolet (UV) absorbance at 260 nm of Mg-ATP and Mg-ADP. The separation was enhanced by the addition of Mg²âº to the separation buffer. Inhibition studies of PFK-1 by aurintricarboxylic acid and palmitoyl coenzyme A were also performed. An IC50 value was determined for aurintricarboxylic acid, and this value matched values in the literature obtained using coupled spectrophotometric assays. This assay for PFK-1 directly monitors the enzyme-catalyzed reaction, and the CE separation reduces the potential of spectral interference by inhibitors.

Sociodemographic and career history predictors of suicide mortality in the United States Army 2004-2009.

BACKGROUND: The US Army suicide rate has increased sharply in recent years. Identifying significant predictors of Army suicides in Army and Department of Defense (DoD) administrative records might help focus prevention efforts and guide intervention content. Previous studies of administrative data, although documenting significant predictors, were based on limited samples and models. A career history perspective is used here to develop more textured models. METHOD: The analysis was carried out as part of the Historical Administrative Data Study (HADS) of the Army Study to Assess Risk and Resilience in Servicemembers (Army STARRS). De-identified data were combined across numerous Army and DoD administrative data systems for all Regular Army soldiers on active duty in 2004-2009. Multivariate associations of sociodemographics and Army career variables with suicide were examined in subgroups defined by time in service, rank and deployment history. RESULTS: Several novel results were found that could have intervention implications. The most notable of these were significantly elevated suicide rates (69.6-80.0 suicides per 100 000 person-years compared with 18.5 suicides per 100 000 person-years in the total Army) among enlisted soldiers deployed either during their first year of service or with less than expected (based on time in service) junior enlisted rank; a substantially greater rise in suicide among women than men during deployment; and a protective effect of marriage against suicide only during deployment. CONCLUSIONS: A career history approach produces several actionable insights missed in less textured analyses of administrative data predictors. Expansion of analyses to a richer set of predictors might help refine understanding of intervention implications.

A capillary electrophoretic assay for acetyl coenzyme A carboxylase.

A simple off-column capillary electrophoretic (CE) assay for measuring acetyl coenzyme A carboxylase holoenzyme (holo-ACC) activity and inhibition was developed. The two reactions catalyzed by the holo-ACC components, biotin carboxylase (BC) and carboxyltransferase (CT), were simultaneously monitored in this assay. Acetyl coenzyme A (CoA), malonyl-CoA, adenosine triphosphate (ATP), and adenosine diphosphate (ADP) were separated by capillary electrophoresis, and the depletion of ATP and acetyl-CoA as well as the production of ADP and malonyl-CoA were monitored. Inhibition of holo-ACC by the BC inhibitor, 2-amino-N,N-dibenzyloxazole-5-carboxamide, and the carboxyltransferase inhibitor, andrimid, was confirmed using this assay. A previously reported off-column CE assay for only the CT component of ACC was optimized, and an off-column CE assay for the BC component of ACC also was developed.

Psychosocial stressors and the prognosis of major depression: a test of Axis IV.

BACKGROUND: Axis IV is for reporting 'psychosocial and environmental problems that may affect the diagnosis, treatment and prognosis of mental disorders'. No studies have examined the prognostic value of Axis IV in DSM-IV. METHOD: We analyzed data from 2497 participants in the National Epidemiologic Survey on Alcohol and Related Conditions (NESARC) with major depressive episode (MDE). We hypothesized that psychosocial stressors predict a poor prognosis of MDE. Secondarily, we hypothesized that psychosocial stressors predict a poor prognosis of anxiety and substance use disorders. Stressors were defined according to DSM-IV's taxonomy, and empirically using latent class analysis (LCA). RESULTS: Primary support group problems, occupational problems and childhood adversity increased the risks of depressive episodes and suicidal ideation by 20-30%. Associations of the empirically derived classes of stressors with depression were larger in magnitude. Economic stressors conferred a 1.5-fold increase in risk for a depressive episode [95% confidence interval (CI) 1.2-1.9]; financial and interpersonal instability conferred a 1.3-fold increased risk of recurrent depression (95% CI 1.1-1.6). These two classes of stressors also predicted the recurrence of anxiety and substance use disorders. Stressors were not related to suicidal ideation independent from depression severity. CONCLUSIONS: Psychosocial and environmental problems are associated with the prognosis of MDE and other Axis I disorders. Although DSM-IV's taxonomy of stressors stands to be improved, these results provide empirical support for the prognostic value of Axis IV. Future work is needed to determine the reliability of Axis IV assessments in clinical practice, and the usefulness of this information to improving the clinical course of mental disorders.

Influence of immobilized biomolecules on magnetic bead plug formation and retention in capillary electrophoresis.

Significant changes in the formation and retention of magnetic bead plugs in a capillary during electrophoresis were studied, and it was demonstrated that these effects were due to the type of biological molecule immobilized on the surface of these beads. Three biological molecules, an antibody, an oligonucleotide, and alkaline phosphatase (AP), were attached to otherwise identical streptavidin-coated magnetic beads through biotin-avidin binding in order to isolate differences in bead immobilization in a magnetic field resulting from the type of biological molecule immobilized on the bead surface. AP was also attached to the magnetic beads using epoxy groups on the bead surfaces (instead of avidin-biotin binding) to study the impact of immobilization chemistry. The formation and retention of magnetic bead plugs were studied quantitatively using light scattering detection of magnetic particles eluting from the bead plugs and qualitatively using microscopy. Both the types of biomolecule immobilized on the magnetic bead surface and the chemistry used to link the biomolecule to the magnetic bead impacted the formation and retention of the bead plugs.

Separation and detection of individual Aß aggregates by capillary electrophoresis with laser-induced fluorescence detection.

The separation and detection of individual amyloid beta (Aß) aggregates by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was demonstrated. Samples were prepared with either Aß (1-40) or Aß (1-42) peptides and were characterized by CE with ultraviolet (UV) absorbance detection and transmission electron microscopy (TEM). Using thioflavin T (ThT) in the electrophoresis buffer, electrophoresis of aggregate-containing samples (5.0-s injection) produced up to several hundred narrow (< 20 ms FWHM [full width at half maximum]) fluorescence peaks. Injection of Aß (1-40) monomer samples resulted in no additional peaks compared with controls. The CE-LIF results were validated by bulk ThT fluorescence measurements for the same samples. The potential of laser-induced fluorescence anisotropy (LIFA) with CE to characterize individual Aß aggregates also was investigated.

Classes of conduct disorder symptoms and their life course correlates in a US national sample.

BACKGROUND: Population data on conduct disorder (CD) symptoms can help determine whether hypothesized subtypes of CD are sufficiently disparate in their familial, psychiatric and life course correlates to distinguish separate diagnostic entities. METHOD: Latent class analysis (LCA) of CD symptoms occurring before age 15 was conducted in a national sample of adults aged 18-44 years from the National Epidemiological Study of Alcohol and Related Conditions. Associations of latent class membership with parental behavior problems, onset of psychiatric disorders and anti-social behaviors after age 15, adolescent life events (e.g. high school drop-out), and past-year life events (e.g. divorce/separation, bankruptcy) were estimated. RESULTS: LCA identified a no-CD class with low prevalence of all symptoms, three intermediate classes - deceit/theft, rule violations, aggression - and a severe class. The prevalence of CD, according to DSM-IV criteria, was 0% in the no-CD class, between 13.33% and 33.69% in the intermediate classes and 62.20% in the severe class. Latent class membership is associated with all the familial, psychiatric and life course outcomes examined. Among the intermediate classes, risk for subsequent mood/anxiety disorders and anti-social behavior was higher in the deceit/theft and aggressive classes than in the rule violations class. However, risk for adolescent life events is highest in the rule violations class. CONCLUSIONS: CD symptoms tend to occur in a partially ordered set of classes in the general population. Prognostically meaningful distinctions can be drawn between classes, but only at low levels of symptoms.

Manipulation and capture of Aß amyloid fibrils and monomers by DC insulator gradient dielectrophoresis (DC-iGDEP).

Here we report a novel method for the manipulation and concentration of Aß amyloid fibrils, implicated in Alzheimer's disease, using DC insulating gradient dielectrophoresis (DC-iGDEP). Fibril enrichment was found to be ∼400%. Simulations suggest that capture of the full range of amyloid protein aggregates is possible with optimized device design.

Manifestation of the Jahn-Teller effect subject to quadraticT⊗e+t2problem in ultrasonic attenuation. Case study of CaF2:Cr crystal with isovalent and aliovalent substitution.

A new approach is introduced to obtain information about the adiabatic potential energy surface of the Jahn-Teller (JT) complexes subject to theT⊗e+t2problem in doped crystals. It employs simulation of the contribution of the JT sub-system to ultrasonic attenuation with account of three mechanisms of relaxation and subsequent comparison with the experimental data on temperature dependence of the attenuation obtained with the use of doped crystal and nominally pure one. The approach has been applied to the CaF2crystal doped with chromium. The experiment has been carried out at the frequencies of (13-158) × 106Hz at low temperatures (4-170 K). Peak for all the ultrasonic normal modes propagating along the [110] axis has been observed at about 8 K. It has been considered as the JT effect manifestation by the sub-system of Cr2+F8-complexes. Symmetry properties of the global minima and saddle points have been identified, the JT stabilization energies and coordinates of the extrema points in the five-dimensional space of symmetrized displacements have been evaluated. Besides, a broad shoulder has been found at 15-40 K and attributed to contribution of the Cr3+F8-sub-system.

TDP-43 pathology occurs infrequently in multiple system atrophy.

AIMS AND METHODS: The α-synucleinopathy multiple system atrophy (MSA) and diseases defined by pathological 43-kDa transactive response DNA-binding protein (TDP-43) or fused in sarcoma (FUS) aggregates such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration show overlapping clinico-pathological features. Consequently, we examined MSA for evidence of TDP-43 or FUS pathology utilizing immunohistochemical studies in autopsy material from 29 MSA patients. RESULTS: TDP-43 pathology was generally rare, and there were no FUS lesions. The TDP-43 lesions were located predominantly in medio-temporal lobe and subcortical brain areas and were comprised mainly of dystrophic processes and perivascular (and subpial) lesions. CONCLUSIONS: The multisystem clinical symptoms and signs of MSA, and in particular the neurobehavioural/cognitive and pyramidal features, appear not to result from concomitant TDP-43 or FUS pathology, but rather from widespread white matter α-synuclein positive glial cytoplasmic inclusions and neurodegeneration in keeping with a primary α-synuclein-mediated oligodendrogliopathy. The gliodegenerative disease MSA evidently results from different pathogenetic mechanisms than neurodegenerative diseases linked to pathological TDP-43.

Race/ethnic differences in exposure to traumatic events, development of post-traumatic stress disorder, and treatment-seeking for post-traumatic stress disorder in the United States.

BACKGROUND: To identify sources of race/ethnic differences related to post-traumatic stress disorder (PTSD), we compared trauma exposure, risk for PTSD among those exposed to trauma, and treatment-seeking among Whites, Blacks, Hispanics and Asians in the US general population. METHOD: Data from structured diagnostic interviews with 34 653 adult respondents to the 2004-2005 wave of the National Epidemiologic Survey on Alcohol and Related Conditions (NESARC) were analysed. RESULTS: The lifetime prevalence of PTSD was highest among Blacks (8.7%), intermediate among Hispanics and Whites (7.0% and 7.4%) and lowest among Asians (4.0%). Differences in risk for trauma varied by type of event. Whites were more likely than the other groups to have any trauma, to learn of a trauma to someone close, and to learn of an unexpected death, but Blacks and Hispanics had higher risk of child maltreatment, chiefly witnessing domestic violence, and Asians, Black men, and Hispanic women had higher risk of war-related events than Whites. Among those exposed to trauma, PTSD risk was slightly higher among Blacks [adjusted odds ratio (aOR) 1.22] and lower among Asians (aOR 0.67) compared with Whites, after adjustment for characteristics of trauma exposure. All minority groups were less likely to seek treatment for PTSD than Whites (aOR range: 0.39-0.61), and fewer than half of minorities with PTSD sought treatment (range: 32.7-42.0%). CONCLUSIONS: When PTSD affects US race/ethnic minorities, it is usually untreated. Large disparities in treatment indicate a need for investment in accessible and culturally sensitive treatment options.

Improved peak capacity for CE separations of enzyme inhibitors with activity-based detection using magnetic bead microreactors.

A technique for separating and detecting enzyme inhibitors was developed using CE with an enzyme microreactor. The on-column enzyme microreactor was constructed using NdFeB magnet(s) to immobilize alkaline phosphatase-coated superparamagnetic beads (2.8 microm diameter) inside a capillary before the detection window. Enzyme inhibition assays were performed by injecting a plug of inhibitor into a capillary filled with the substrate, AttoPhos. Product generated in the enzyme microreactor was detected by LIF. Inhibitor zones electrophoresed through the capillary, passed through the enzyme microreactor, and were observed as negative peaks due to decreased product formation. The goal of this study was to improve peak capacities for inhibitor separations relative to previous studies, which combined continuous engagement electrophoretically mediated microanalysis and transient engagement electrophoretically mediated microanalysis to study enzyme inhibition. The effects of electric field strength, bead injection time and inhibitor concentrations on peak capacity and peak width were investigated. Peak capacities were increased to >or=20 under optimal conditions of electric field strength and bead injection time for inhibition assays with arsenate and theophylline. Five reversible inhibitors of alkaline phosphatase (theophylline, vanadate, arsenate, L-tryptophan and tungstate) were separated and detected to demonstrate the ability of this technique to analyze complex inhibitor mixtures.