Application of lithium cationization tandem mass spectrometry for structural analysis of lignin model oligomers with ß-ß' and ß-O-4' linkages.

Structural elucidation of lignin degradation products is a requirement for successfully developing lignin valorization technology. Most of mass spectrometry-based techniques have utilized negative ion mode mass spectrometry for structural elucidation of lignin-derived compounds. Unfortunately, simple deprotonation can lead to in-source fragmentation and may not be suitable for condensed lignin structures without acidic moieties. Herein, we present a lithium cationization methodology for mass spectrometry sequencing of advanced lignin oligomers having ß-ß' and ß-O-4' bonding motifs. To do so, two advanced lignin oligomers were first synthesized through a step-by-step synthetic route, and then subjected to two different ESI mass spectrometry techniques in positive ion mode using lithium cations for ionization. An orbitrap mass spectrometer was used to obtain exact mass information, and higher-energy collisional dissociation (HCD) was used to sequence the lignin model oligomers. Based on the sequence-specific fragment ions, sequence rules were proposed. Multi-stage (MSn) collision-induced dissociation (CID) using an ion trap mass spectrometer provided data to investigate the origin of each fragment ion and to further confirm proposed fragmentation pathways. In addition to ß-O-4' bond cleavage, the presented lithium cationization approach led to cleavage of ß-ß' bonds on the model oligomers in both ion trap and orbitrap mass spectrometry experiments. Additionally, MSn experiments were used to investigate possible lithium cationization sites on the model oligomers. Lithium cationization in positive ion mode mass spectrometry proved to be a robust tool for characterization and sequencing of advanced lignin oligomers with different bonding motifs.

Strategy for Conjugating Oligopeptides to Mesoporous Silica Nanoparticles Using Diazirine-Based Heterobifunctional Linkers.

Successful strategies for the attachment of oligopeptides to mesoporous silica with pores large enough to load biomolecules should utilize the high surface area of pores to provide an accessible, protective environment. A two-step oligopeptide functionalization strategy is examined here using diazirine-based heterobifunctional linkers. Mesoporous silica nanoparticles (MSNPs) with average pore diameter of ~8 nm and surface area of ~730 m2/g were synthesized and amine-functionalized. Tetrapeptides Gly-Gly-Gly-Gly (GGGG) and Arg-Ser-Ser-Val (RSSV), and a peptide comprised of four copies of RSSV (4RSSV), were covalently attached via their N-terminus to the amine groups on the particle surface by a heterobifunctional linker, sulfo-succinimidyl 6-(4,4'-azipentanamido)hexanoate (sulfo-NHS-LC-diazirine, or SNLD). SNLD consists of an amine-reactive NHS ester group and UV-activable diazirine group, providing precise control over the sequence of attachment steps. Attachment efficiency of RSSV was measured using fluorescein isothiocyanate (FITC)-tagged RSSV (RSSV-FITC). TGA analysis shows similar efficiency (0.29, 0.31 and 0.26 mol peptide/mol amine, respectively) for 4G, RSSV and 4RSSV, suggesting a generalizable method of peptide conjugation. The technique developed here for the conjugation of peptides to MSNPs provides for their attachment in pores and can be translated to selective peptide-based separation and concentration of therapeutics from aqueous process and waste streams.

Complexation of Lignin Dimers with ß-Cyclodextrin and Binding Stability Analysis by ESI-MS, Isothermal Titration Calorimetry, and Molecular Dynamics Simulations.

Lignin derived from lignocellulosic biomass is the largest source of renewable bioaromatics present on earth and requires environmentally sustainable separation strategies to selectively obtain high-value degradation products. Applications of supramolecular interactions have the potential to isolate lignin compounds from biomass degradation fractions by the formation of variable inclusion complexes with cyclodextrins (CDs). CDs are commonly used as selective adsorbents for many applications and can capture guest molecules in their internal hydrophobic cavity. The strength of supramolecular interactions between CDs and lignin model compounds that represent potential lignocellulosic biomass degradation products can be characterized by assessing the thermodynamics of binding stability. Consequently, the inclusion interactions of ß-CD and lignin model compounds G-(ß-O-4')-G, G-(ß-O-4')-truncG (guaiacylglycerol-ß-guaiacyl ether), and G-(ß-ß')-G (pinoresinol) were investigated empirically by electrospray ionization mass spectrometry and isothermal titration calorimetry, complemented by molecular dynamics (MD) simulations. Empirical results indicate that there are substantial differences in binding stability dependent on the linkage type. The lignin model ß-ß' dimer showed more potential bound states including 1:1, 2:1, and 1:2 (guest:host) complexation and, based on binding stability determinations, was consistently the most energetically favorable guest. Empirical results are supported by MD simulations that reveal that the capture of G-(ß-ß')-G by ß-CD is promising with a 66% probability of being bound for G-(ß-O-4')-truncG compared to 88% for G-(ß-ß')-G (unbiased distance trajectory and explicit counting of bound states). These outcomes indicate CDs as a promising material to assist in separations of lignin oligomers from heterogeneous mixtures for the development of environmentally sustainable isolations of lignin compounds from biomass fractions.

Pharmacokinetic and metabolic analysis of an Alzheimer's disease therapeutic in rat serum via microfluidic CZE-MS.

Sensitive, high-throughput methods for pharmacokinetic (PK) profiling are essential for potential therapeutics during critical stages of clinical trials. The application of a microfluidic capillary zone electrophoresis mass spectrometry (CZE-MS) method for PK profiling allows for rapid, sensitive and in-depth analysis of multiple samples within a short timeframe. Here, a CZE-MS approach for PK analysis was compared with a traditional UHPLC-MS approach when analyzing serum extracts from rats treated with a potential Alzheimer's disease therapeutic, BNC-1. Resulting PK data generated from both methods displayed statistical similarities. Additionally, the separation efficiency attributed to the use of the CZE-MS method provided substantial metabolic regulation data that was not apparent in the UHPLC-MS method. Additionally, the coupling of the CZE-MS method to the data processing software, MZmine2, was used to monitor changes in metabolism and observe putative BNC-1-derived metabolites. The ability to perform fast analyses without sacrificing sensitivity or metabolic information suggests that this CZE-MS method is ideal for metabolomics-inclusive, high-throughput PK profiling.

Interaction of lignin dimers with model cell membranes: A quartz crystal microbalance and molecular dynamics simulation study.

A study of the interaction between cell membranes and small molecules derived from lignin, a protective phenolic biopolymer found in vascular plants, is crucial for identifying their potential as pharmacological and toxicological agents. In this work, the interactions of model cell membranes [supported 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid bilayers] are compared for three ßO4 dimers of coniferyl alcohol (G lignin monomer): guaiacylglycerol guaiacol ester with a hydroxypropenyl (HOC3H4-) tail (G-ßO4'-G), a truncated GG dimer without HOC3H4- (G-ßO4'-truncG), and a benzylated GG dimer (benzG-ßO4'-G). The uptake of the lignin dimers (per mass of lipid) and the energy dissipation (a measure of bilayer disorder) are higher for benzG-ßO4'-G and G-ßO4'-truncG than those for G-ßO4'-G in the gel-phase DPPC bilayer, as measured using quartz crystal microbalance with dissipation (QCM-D). A similar uptake of G-ßO4'-truncG is observed for a fluid-phase bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine, suggesting that the effect of the bilayer phase on dimer uptake is minimal. The effects of increasing lignin dimer concentration are examined through an analysis of density profiles, potential of mean force curves, lipid order parameters, and bilayer area compressibilities (disorder) in the lipid bilayers obtained from molecular dynamics simulations. Dimer distributions and potentials of mean force indicate that the penetration into bilayers is higher for benzG-ßO4'-G and G-ßO4'-truncG than that for G-ßO4'-G, consistent with the QCM-D results. Increased lipid tail disorder due to dimer penetration leads to a thinning and softening of the bilayers. Minor differences in the structure of lignin derivatives (such as truncating the hydroxypropenyl tail) have significant impacts on their ability to penetrate lipid bilayers.

The study of the chromatographic behavior and a mass spectrometric approach to differentiating the diastereomer pair of the ß-O-4 lignin dimer series.

Lignin and lignans are natural products found in plant cell walls. Lignin research has historically focused on lignin degradation techniques in the hope of converting lignin into useful aromatic carbon feedstocks. In contrast, investigations of lignans existing as natural product dimers, have been focused on thier interesting biological activities. Many lignan compounds are chemically identical to dimers derived from lignin, and both lignin and lignan dimers can possess multiple chiral centers leading to observations of diastereomer pairs where one diastereomer exhibits the bulk of the activity. For example, the G-(ß-O-4')-G dimer was reported to have a pro-angiogenic activity with one diastereomer of the pair showing enhanced pro-angiogenic activity. Traditional analytical techniques such as nuclear magnetic resonance (NMR) can differentiate the diastereomer pairs of ß-O-4 compounds; however, isolation of a pure sample is often required for analysis. This work was aimed at exploring the potential use of tandem mass spectrometry to differentiate diastereomer pairs in the ß-O-4 dimer series. Each diastereomer pair in the nine-dimer series was separated by HPLC and interrogated by tandem mass spectrometry. To understand the chromatographic behavior of the diastereomer pair in the ß-O-4 dimer series, three commercially available reverse phase HPLC columns were evaluated. A temperature programming experiment using water/acetonitrile isocratic elution showed that the chromatographic retention mechanism of these diastereomers was hydrophobically driven with analytes having more methoxy groups exhibiting larger ΔH0 and higher octanol-water partition coefficient values. Tandem mass spectrometry performed on each of the diastereomers produced fragment ions having different ion abundances. A mechanistic study based on the ion abundance of "sequence-specific ions" and "-48 ions" was used to assign a configuration to each of the pairs of diastereomers in the nine-dimer series.

Interaction of lignin-derived dimer and eugenol-functionalized silica nanoparticles with supported lipid bilayers.

The potential to impart surfaces with specific lignin-like properties (i.e. resistance to microbes) remains relatively unexplored due to the lack of well-defined lignin-derived small molecules and corresponding surface functionalization strategies. Here, allyl-modified guaiacyl ß-O-4 eugenol (G-eug) lignin-derived dimer is synthesized and attached to mesoporous silica nanoparticles (MSNPs) via click chemistry. The ability of G-eug lignin-dimer functionalized particles to interact with and disrupt synthetic lipid bilayers is compared to that of eugenol, a known natural antimicrobial. Spherical MSNPs (∼150 nm diameter with 4.5 nm pores) were synthesized using surfactant templating. Post-synthesis thiol (SH) attachment was performed using (3-mercaptopropyl) trimethoxysilane and quantified by Ellman's test. The resultant SH-MSNPs were conjugated with the G-eug dimers or eugenol by a thiol-ene reaction under ultraviolet light in the presence of a photo initiator. From thermogravimetric analysis (TGA), attachment densities of approximately 0.22 mmol eugenol/g particle and 0.13 mmol G-eug dimer/g particle were achieved. The interaction of the functionalized MSNPs with a phospholipid bilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (representing model cell membranes) supported on gold surface was measured using Quartz Crystal Microbalance with Dissipation monitoring (QCM-D). Eugenol-grafted MSNPs in PBS (up to 1 mg/mL) associated with the bilayer and increased the mass adsorbed on the QCM-D sensor. In contrast, MSNPs functionalized with G-eug dimer show qualitatively different behavior, with more uptake and evidence of bilayer disruption at and above a particle concentration of 0.5 mg/mL. These results suggest that bio-inspired materials with conjugated lignin-derived small molecules can serve as a platform for novel antimicrobial coatings and therapeutic carriers.

Experimental and Molecular Dynamics Simulation Study of the Effects of Lignin Dimers on the Gel-to-Fluid Phase Transition in DPPC Bilayers.

High resolution differential scanning calorimetry (DSC) and molecular dynamics (MD) simulations were used to investigate the effect of three lignin dimers on the gel to fluid phase transition in DPPC lipid bilayers. The goal of this research is to begin to understand the partitioning of model lignin dimers into lipid bilayers and its effects on the gel to fluid transition temperature (Tm). The long-term objective is to establish structure-function relationships for well-defined lignin derivatives at biologically relevant surfaces. This work uses a newly synthesized guiacylglycerol guaiacol ester with a hydroxypropenyl (HOC3H4-) group resembling natural lignin (GG dimer), compared with a truncated GG dimer without the HOC3H4- and benzyl-modified GG dimers. The DSC results show that the dimer most like natural lignin (with a hydroxypropenyl tail) has log K = 2.72 ± 0.05, and MD simulations show that it associates with the headgroups of the lipid but does not penetrate strongly into the interior of the bilayer. Therefore, this dimer has little effect on the Tm value. In contrast, the truncated dimer, which has been used as a representative GG dimer in prior studies, partitions into the bilayer, as seen in MD simulations, and shifts Tm because of its increased lipophilicity (DSC log K = 3.45 ± 0.20). Similarly, modification of the natural GG dimer by benzylation of the phenol makes it lipophilic (DSC log K = 3.38 ± 0.28), causing it to partition into the bilayer, as seen in MD simulations and shift Tm. In MD, we capture the transition from gel to fluid phase by defining and analyzing a normalized deuterium order parameter averaged over all carbon atoms located in the middle of the lipid tails. In this way, the phase transition can be clearly observed and, importantly, MD results show the same trend of transition temperature shifts as the DSC results. Furthermore, we compare partition coefficients estimated from free energy profiles calculated in MD to those obtained from experiment and they are in qualitative agreement. The success at predicting the structural effects of lignin dimers on lipid bilayers suggests that MD simulations can be used in the future to screen the interactions of lignin oligomers and their derivatives with lipid bilayers.

Microfluidic capillary zone electrophoresis mass spectrometry analysis of alkaloids in Lobelia cardinalis transgenic and mutant plant cell cultures.

Application of a microfluidic CE* device for CZE-MS allows for fast, rapid, and in-depth analysis of large sample sets. This microfluidic CZE-MS device, the 908 Devices ZipChip, involves minimal sample preparation and is ideal for small cation analytes, such as alkaloids. Here, we evaluated the microfluidic device for the analysis of alkaloids from Lobelia cardinalis hairy root cultures. Extracts from wild-type, transgenic, and selected mutant plant cultures were analyzed and data batch processed using the mass spectral processing software MZmine2 and the statistical software Prism 8. In total 139 features were detected as baseline resolved peaks via the MZmine2 software optimized for the electrophoretic separations. Statistically significant differences in the relative abundance of the primary alkaloid lobinaline (C27 H34 N2 ), along with several putative "lobinaline-like" molecules were observed utilizing this approach. Additionally, a method for performing both targeted and untargeted MS/MS experiments using the microfluidic device was developed and evaluated. Coupling data-processing software with CZE-MS data acquisition has enabled comprehensive metabolomic profiles from plant cell cultures to be constructed within a single working day.

A comparative study of the electrospray ionization response of ß-O-4' lignin model compounds.

Electrospray ionization mass spectrometry has recently become the technique of choice for rapid characterization of lignin degradation products. However, the fundamental question of the relationship between lignin structure and ionization efficiency has not been explored. In this work, we studied the electrospray ionization response of five structurally similar ß-O-4' model lignin compounds using lithium cationization in the positive electrospray ionization mode. The studied compounds have the same ß-O-4' backbone structure but differ at the α-position by increasing nonpolar side chains. Our results show a correlation between the ionization response and the length of the nonpolar side chain, with analytes having the longest side chain recording the highest ESI response in the full scan mode. Factors affecting the formation of analyte ions and analyte cluster ions were also studied. We have shown for the first time in this work that the introduction of a nonpolar group onto a ß-O-4' lignin compound can increase the lithium cationization ESI response in the positive ion mode.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis for characterization of lignin oligomers using cationization techniques and 2,5-dihydroxyacetophenone (DHAP) matrix.

RATIONALE: Effective analytical techniques are needed to characterize lignin products for the generation of renewable carbon sources. Application of matrix-assisted laser desorption/ionization (MALDI) in lignin analysis is limited because of poor ionization efficiency. In this study, we explored the potential of cationization along with a 2,5-dihydroxyacetophenone (DHAP) matrix to characterize model lignin oligomers. METHODS: Synthesized lignin oligomers were analyzed using the developed MALDI method. Two matrix systems, DHAP and α-cyano-4-hydroxycinnamic acid (CHCA), and three cations (lithium, sodium, silver) were evaluated using a Bruker UltraFlextreme time-of-flight mass spectrometer. Instrumental parameters, cation concentration, matrix, sample concentrations, and sample spotting protocols were optimized for improved results. RESULTS: The DHAP/Li+ combination was effective for dimer analysis as lithium adducts. Spectra from DHP and ferric chloride oligomers showed improved signal intensities up to decamers (m/z 1823 for the FeCl3 system) and provided insights into differences in the oligomerization mechanism. Spectra from a mixed DHP oligomer system containing H, G, and S units showed contributions from all monolignols within an oligomer level (e.g. tetramer level). CONCLUSIONS: The DHAP/Li+ method presented in this work shows promise to be an effective analytical tool for lignin analysis by MALDI and may provide a tool to assess lignin break-down efforts facilitating renewable products from lignin.

Modulating Mechanical Properties of Collagen-Lignin Composites.

Three-dimensional matrices of collagen type I (Col I) are widely used in tissue engineering applications for its abundance in many tissues, bioactivity with many cell types, and excellent biocompatibility. Inspired by the structural role of lignin in a plant tissue, we found that sodium lignosulfonate (SLS) and an alkali-extracted lignin from switchgrass (SG) increased the stiffness of Col I gels. SLS and SG enhanced the stiffness of Col I gels from 52 to 670 Pa and 52 to 320 Pa, respectively, and attenuated shear-thinning properties, with the formulation of 1.8 mg/mL Col I and 5.0 mg/mL SLS or SG. In 2D cultures, the cytotoxicity of collagen-SLS to adipose-derived stromal cells was not observed and the cell viability was maintained over 7 days in 3D cultures. Collagen-SLS composites did not elicit immunogenicity when compared to SLS-only groups. Our collagen-SLS composites present a case that exploits lignins as an enhancer of mechanical properties of Col I without adverse cytotoxicity and immunogenicity for in vitro scaffolds or in vivo tissue repairs.

Characterization and sequencing of lithium cationized ß-O-4 lignin oligomers using higher-energy collisional dissociation mass spectrometry.

The prospect of developing new bio-based products from lignin partially depends on the development of effective analytical techniques to characterize the end products from lignin degradation experiments. To date, the most utilized mass spectrometric technique for characterizing lignin oligomers has been negative ion mass spectrometry. Positive ion mass spectrometry remains a relatively unexplored approach for lignin sequencing. Here we report on the sequencing of lignin oligomers using lithium cationization positive ion electrospray followed by higher-energy collisional dissociation (HCD) tandem mass spectrometry on a high-resolution accurate-mass Orbitrap mass spectrometer. Thirteen synthetic lignin model oligomers containing ß-O-4 and 4-O-α linkages were analyzed and sequenced based on their HCD tandem mass spectrometry fragmentation patterns. Proposed sequence-specific fragmentation pathways are provided. The method presented in this study provides a potential application of lithium cationization tandem mass spectrometry for the analysis of lignin degradation products.

A novel method for the rapid detection of post-translationally modified visinin-like protein 1 in rat models of brain injury.

BACKGROUND: Although elevated serum levels of visinin-like protein 1 (VILIP-1), a neuron-specific calcium sensor protein, are associated with ischaemic stroke, only a single study has evaluated VILIP-1 as a biomarker of traumatic brain injury (TBI). The current proof-of-concept study was designed to determine whether serum VILIP-1 levels increase post-injury in a well-characterized rat unilateral cortical contusion model. METHODS: Lateral flow devices (LFDs) rapidly (< 20 min) detected trace serum levels (pg/mL) of VILIP-1 in a small input sample volume (10 µL). Temporal profiles of serum levels at baseline and post-injury were measured in male Sprague Dawley rats subjected to very mild-, mild unilateral-cortical contusion, or naïve surgery and in male Sprague Dawley rats following a diffuse TBI or sham surgery. RESULTS: Mean serum levels were significantly elevated by 0.5 h post-injury and remained so throughout the temporal profile compared with baseline in very mild and mild unilateral contusions but not in naïve surgeries. Serum levels were also elevated in a small cohort of animals subjected to a diffuse TBI injury. CONCLUSIONS: Overall, the current study demonstrates that the novel LFD is a reliable and rapid point-of-care diagnostic for the detection and quantification of serum levels of UB-VILIP-1 in a clinically relevant time frame.

Correlating the potentiometric selectivity of cyclosporin-based electrodes with binding patterns obtained from electrospray ionization-mass spectrometry.

Electrospray ionization mass spectrometry ESI-MS is a powerful technique for the characterization of macromolecules and their noncovalent binding with guest ions. We herein evaluate the feasibility of using ESI-MS as a screening tool for predicting potentiometric selectivities of ionophores. Ion-selective electrodes based on the cyclic peptide, cyclosporin A, were developed, and their potentiometric selectivity pattern was evaluated. Optimized electrodes demonstrated near-Nernstian slopes with micromolar detection limits toward calcium. ESI-MS and ESI-MS/MS were employed to determine the relative association strengths of cyclosporin A with various cations. The observed MS intensities of ion-ionophore complexes correlate favorably with the potentiometric selectivity pattern that was demonstrated by cyclosporin-based electrodes. This correlation was found to hold true for other established ionophores, such as valinomycin and benzo-18-crown-6. Taken together, these experiments demonstrate that mass spectrometry could be used to predict the selectivity patterns of new ionophores for potentiometric and optical ion sensors. Further, this approach could be useful in screening mixtures or libraries of newly-synthesized compounds to identify selective ionophores.

A Novel Small Molecule Modulator of Amyloid Pathology.

Because traditional approaches to drug development for Alzheimer's disease are becoming increasingly expensive and in many cases disappointingly unsuccessful, alternative approaches are required to shift the paradigm. Following leads from investigations of dihydropyridine calcium channel blockers, we observed unique properties from a class of functionalized naphthyridines and sought to develop these as novel therapeutics that minimize amyloid pathology without the adverse effects associated with current therapeutics. Our data show methyl 2,4-dimethyl-5-oxo-5,6-dihydrobenzo[c][2,7]naphthyridine-1-carboxylate (BNC-1) significantly decreases amyloid burden in a well-established mouse model of amyloid pathology through a unique mechanism mediated by Elk-1, a transcriptional repressor of presenilin-1. Additionally, BNC-1 treatment leads to increased levels of synaptophysin and synapsin, markers of synaptic integrity, but does not adversely impact presenilin-2 or processing of Notch-1, thus avoiding negative off target effects associated with pan-gamma secretase inhibition. Overall, our data show BNC-1 significantly decreases amyloid burden and improves markers of synaptic integrity in a well-established mouse model of amyloid deposition by promoting phosphorylation and activation of Elk-1, a transcriptional repressor of presenilin-1 but not presenilin-2. These data suggest BNC-1 might be a novel, disease-modifying therapeutic that will alter the pathogenesis of Alzheimer's disease.

A Novel Plasma Based Biomarker of Alzheimer's Disease.

Specific biomarkers in a readily accessible biological fluid, such as blood, could aid in the identification, characterization, validation, and routine monitoring of Alzheimer's disease (AD) progression. In the current study, levels of the previously described novel cerebrospinal fluid aberrant protein complex composed of prostaglandin-D-synthase (PDS) and transthyretin (TTR) were quantified in plasma by a custom two-probe sandwich ELISA and compared to amyloid-ß (Aß)(1-42) as a standard plasma biomarker of AD. Plasma was analyzed from 140 probable AD subjects, 135 subjects with mild cognitive impairment (MCI), 74 normal control subjects (NC) prior to MCI transition, 23 diseased control (DC) subjects with either frontotemporal dementia or dementia with Lewy bodies, and 182 normal control (NC) subjects who did not progress to MCI or dementia. Levels of Aß(1-42) were significantly elevated in NC subjects prior to MCI conversion but significantly reduced in probable AD subjects compared to NC subjects. Similarly, levels of the PDS-TTR complex were significantly reduced in both MCI and probable AD subjects compared to NC subjects. Furthermore, levels of Aß(1-42) and the PDS-TTR complex were not significantly different in DC subjects compared to NC subjects. MMSE scores were weakly but significantly correlated with plasma levels of the PDS-TTR complex and Aß(1-42). Trail B scores were weakly but significantly correlated with plasma levels of Aß(1-42). Comparison of receiver operating curves shows the PDS-TTR complex is comparable to Aß(1-42) in both MCI and probable AD subjects.

Calcium Channel Blockers, Progression to Dementia, and Effects on Amyloid Beta Peptide Production.

Previous epidemiologic studies suggest that antihypertensive drugs may be protective against cognitive decline. To determine if subjects enrolled in the University of Kentucky longitudinal aging study who used antihypertensive drugs showed diminished progression to dementia, we used a 3-parameter logistic regression model to compare the rate of progression to dementia for subjects who used any of the five common categories of antihypertensive drugs to those with similar demographic characteristics but who did not use antihypertensives. Regression modeling showed that subjects who used calcium channel blockers (CCBs) but not the other classes of antihypertensives showed a significant decrease in the rate of progression to dementia. Significantly, use of CCBs ameliorated the negative effects of the presence of APOE-4 alleles on cognitive decline. To determine if CCBs could minimize amyloid beta peptide (Aß(1-42)) production, H4 neuroglioma cultures transfected to overexpress APP were treated with various CCBs and Aß(1-42) levels and levels of proteins involved in Aß production were quantified. Results show that treatment with nifedipine led to a significant decrease in levels of Aß(1-42), with no significant decrease in cell viability. Collectively, these data suggest that use of CCBs significantly diminishes the rate of progression to dementia and may minimize formation of Aß(1-42).