There is a substantial list of pre‐analytical variables that can alter the analysis of blood‐derived samples. We have undertaken studies on some of these issues including choice of sample type, ...stability during storage, use of protease inhibitors, and clinical standardization. As there is a wide range of sample variables and a broad spectrum of analytical techniques in the HUPO PPP effort, it is not possible to define a single list of pre‐analytical standards for samples or their processing. We present here a compendium of observations, drawing on actual results and sound clinical theories and practices. Based on our data, we find that (1) platelet‐depleted plasma is preferable to serum for certain peptidomic studies; (2) samples should be aliquoted and stored preferably in liquid nitrogen; (3) the addition of protease inhibitors is recommended, but should be incorporated early and used judiciously, as some form non specific protein adducts and others interfere with peptide studies. Further, (4) the diligent tracking of pre‐analytical variables and (5) the use of reference materials for quality control and quality assurance, are recommended. These findings help provide guidance on sample handling issues, with the overall suggestion being to be conscious of all possible pre‐analytical variables as a prerequisite of any proteomic study.
Human plasma and serum proteins are subject to intrinsic proteolytic degradation both during and after blood collection. By monitoring peptides, we investigated the stability of plasma and serum ...samples and the effects of anticoagulants and protease inhibitors on the plasma samples. Serum and plasma were subjected to time-course incubation, and the peptides (750−3200 Da) were extracted and analyzed with MALDI-TOF MS. Peptides of interest were further identified by MALDI-TOF/TOF MS and ESI−MS/MS analyses. Our observations indicate that plasma peptides are significantly different from serum peptides. Intrinsic proteases cause these differences between plasma and serum samples, as well as the differences among three plasma samples using either EDTA, sodium citrate, or heparin as the anticoagulant, which accounts for partial inhibitory effects on plasma proteolytic activities. Proteases and peptidases, including both aminopeptidases and carboxypeptidases, also cause time-dependent, sequential generation and digestion of the peptides in serum and all three plasmas, specifically during early sample collection and processing. Protease inhibitors within an EDTA-plasma-collection device inhibit both intrinsic plasma peptidases and proteases and moderate the time-dependent changes of the plasma peptides, including bradykinin, and complement C4- and C3- derived peptides. Our results suggest that mixing protease inhibitors immediately with blood during blood collection provides enhanced stabilization of the plasma proteome. Keywords: bradykinin • C3 peptides • inhibition • mass spectrometry • proteolysis • stabilization • serum and plasma
Prevention of bronchopulmonary dysplasia (BPD) in premature-birth babies continues to be an unmet medical need. Intramuscular vitamin A is currently employed in preterm neonates to prevent BPD but ...requires intramuscular injections in fragile neonates. We hypothesized that noninvasive inhaled delivery of vitamin A, targeted to lung, would be a more effective and tolerable strategy. We employed our well-established hyperoxia-injury neonatal rat model, exposing newborn rats to 7 days of constant extreme (95% O
) hyperoxia, comparing vitamin A dosed every 48 h via either aerosol inhalation or intramuscular injection with normoxic untreated healthy animals and vehicle-inhalation hyperoxia groups as positive and negative controls, respectively. Separately, similar vitamin A dosing of normoxia-dwelling animals was performed. Analyses after
included characterization of alveolar histomorphology and protein biomarkers of alveolar maturation surfactant protein C (SP-C), peroxisome proliferator-activated receptor (PPAR) γ, cholinephosphate cytidylyl transferase, vascular endothelial growth factor and its receptor, FLK-1, and retinoid X receptors (RXR-α, -β, and -γ, apoptosis (Bcl2 and Bax) key injury repair pathway data including protein markers (ALK-5 and β-catenin) and neutrophil infiltration, and serum vitamin A levels. Compared with intramuscular dosing, inhaled vitamin A significantly enhanced biomarkers of alveolar maturation, mitigated hyperoxia-induced lung damage, and enhanced surfactant protein levels, suggesting that it may be more efficacious in preventing BPD in extremely premature infants than the traditionally used IM dosing regimen. We speculate lung-targeted inhaled vitamin A may also be an effective therapy against other lung damaging conditions leading to BPD or, more generally, to acute lung injury.
Background: During clotting, α thrombin cleaves fibrinogen releasing fibrinopeptide A (FPA). FPA is easily identified in serum using matrix-assisted laser desorption/ionization time-of-flight ...(MALDI-TOF) mass spectrometry (MS). Using MALDI-TOF MS, we observed multiple, progressively shorter fragments of serum FPA. Following ambient incubation of serum, variations in the content of FPA fragments occur over time. Denaturation of α thrombin by heating the serum sample appears to minimize this variation. These observations suggest that intrinsic proteolytic and peptidolytic activity is elevated in serum and perhaps originates from the coagulation cascade enzymes themselves, especially α thrombin. Methods: Extrinsic addition of α thrombin to a subset (3–30 kDa) of plasma proteins was carried out to induce proteolysis and to examine the resultant peptides to reveal α thrombin susceptible parent proteins. One of these identified proteins, hemopexin, was directly digested by α thrombin and the peptides examined to confirm the observations from the initial plasma protein digestion. Results: Extrinsic addition of α thrombin to a subset (3–30 kDa) of plasma proteins results in wide-spread digestion of proteins unrelated to coagulation, revealing a substrate range encompassing more than fibrinogen. Direct digestion of one of these proteins, hemopexin, by α thrombin confirms these observations. Conclusions: The resulting peptides indicate broad tolerance beyond the consensus R-G cleavage site of fibrinogen; in fact, there appears to be no bias for the amino acid following the R/K residue. These data support our hypothesis that the enzymatic activities inherent to coagulation, or at least to thrombin, contribute to destabilization of the protein and peptide content of serum. Clin Chem Lab Med 2009;47:685–93.
The abasic site in DNA may arise spontaneously, as a result of nucleotide base damage, or as an intermediate in glycosylase-mediated DNA-repair pathways. It is the most common damage found in DNA. We ...have examined the consequences of this lesion and its sequence context on DNA duplex structure, as well as the thermal and thermodynamic stability of the duplex, including the energetic origins of that stability. To this end, we incorporated a tetrahydrofuran abasic site analogue into a family of 13-mer DNA duplexes, wherein the base opposite the lesion (A, C, G, or T) and the base pairs neighboring the lesion (C·G or G·C) were systematically varied and characterized by a combination of spectroscopic and calorimetric techniques. The resulting data allowed us to reach the following conclusions: (i) the presence of the lesion in all sequence contexts studied does not alter the global B-form conformation characteristic of the parent undamaged duplex; (ii) the presence of the lesion induces a significant enthalpic destabilization of the duplex, with the magnitude of this effect being dependent on the sequence context; (iii) the thermodynamic impact of the lesion is dominated by the identity of the neighboring base pairs, with the cross strand partner base exerting only a secondary thermodynamic effect on duplex properties. In the aggregate, our data reveal that even in the absence of lesion-induced alterations in global structure, the abasic lesion can significantly alter the thermodynamic properties of the host duplex, with the magntiude of this impact being strongly dependent on sequence context.
Human plasma and serum samples, including protein and peptide biomarkers, are subjected to preanalytical variations and instability caused by intrinsic proteases. In this study, we directly ...investigated the stability of peptide biomarkers by spiking an isotopically labeled peptide into human plasma and serum samples and then monitoring its time-dependent change. Fibrinogen peptide A (FPA) was used as a model substrate, and its degradation in a conventional serum and plasma either with citrate, heparin, or EDTA as the anticoagulant, or EDTA plus protease inhibitors (inhibited plasma), was measured using time-course MALDI-TOF MS analysis. The FPA and other peptides tested in this study vary in these samples. However, the peptides are most stable in the inhibited plasma followed by, in general order, EDTA plasma, citrate plasma, heparin plasma and serum, demonstrating the benefit of plasma versus serum, and protease inhibitors for biomarker stabilization. Kinetic analysis indicates that intrinsic peptidases cause an observed first-order Sequential Multiple-Step Reaction (SMSR) in digestion of the peptide. Modeling analysis of the SMSR demonstrates that step reactions differ in their kinetic rate constants, suggesting a significant contribution of the truncated end residue on the substrate specificity of the intrinsic peptidase(s). Our observations further show that synthetic peptides introduced into plasma as internal controls can also be degraded, and thus, their (in)stability as a preanalytical variable should not be overlooked.
We used stopped-flow calorimetry to measure the overall enthalpy change associated with template-directed nucleotide insertion and DNA extension. Specifically, we used families of hairpin ...self-priming templates in conjunction with an exonuclease-free DNA polymerase to study primer extension by one or more dA or dT residues. Our results reveal exothermic heats between -9.8 and -16.0 kcal/bp for template-directed enzymatic polymerization. These extension enthalpies depend on the identity of the inserting base, the primer terminus, and/or the preceding base. Despite the complexity of the overall process, the sign, magnitude, and sequence dependence of these insertion and extension enthalpies are consistent with nearest-neighbor data derived from DNA melting studies. We recognize that the overall process studied here involves contributions from a multitude of events, including dNTP to dNMP hydrolysis, phosphodiester bond formation, and enzyme conformational changes. It is therefore noteworthy that the overall enthalpic driving force per base pair is of a magnitude similar to that expected for addition of one base pair or base stack per insertion event, rather than that associated with the rupture and/or formation of covalent bonds, as occurs during this catalytic process. Our data suggest a constant sequence-independent background of compensating enthalpic contributions to the overall process of DNA synthesis, with discrimination expressed by differences in noncovalent interactions at the template-primer level. Such enthalpic discrimination underscores a model in which complex biological events are regulated by relatively modest energy balances involving weak interactions, thereby allowing subtle mechanisms of regulation.
Objective
β‐Blocker use can be associated with adverse effects that may have an impact on adherence or harm patients. The commonly prescribed β‐blocker metoprolol is metabolized by the polymorphic ...cytochrome P450 (CYP) 2D6 enzyme, resulting in widely variable drug exposure. We investigated whether metoprolol plasma concentrations, CYP2D6 polymorphisms, or genotype‐derived phenotype was associated with adverse effects or efficacy in patients with hypertension.
Methods
Fifty hypertensive patients received metoprolol by use of a dose‐titration algorithm until target blood pressure was reached, intolerable side effects occurred, or maximal daily dose was achieved. CYP2D6 genotype was determined by methods based on polymerase chain reaction–restriction fragment length polymorphism and included 19 allelic variants. Patients were assigned to standard phenotype groups on the basis of genotype. Patients were also assigned activity scores based on functional activity of the alleles. General and dose‐limiting adverse events and blood pressure responses were analyzed in relation to metoprolol steady‐state pharmacokinetic profile and CYP2D6 genotype–derived phenotype.
Results
Poor metabolizers had a significantly longer elimination half‐life, higher S‐metoprolol area under the plasma concentration–time curve (AUC), and lower oral clearance (P ≤ .007 for all parameters). There was a 29.6‐fold variability in AUC among extensive metabolizers, which was largely explained by CYP2D6 activity scores (P = .032 for ordered differences in AUC by activity score among extensive metabolizers). Overall general and dose‐limiting adverse event rates were 46% and 14%, respectively. General adverse event rates did not differ by AUC quartile (66.7% 95% confidence interval (CI), 35.4%‐88.7% and 41.7% 95% CI, 16.5%‐71.4% in the lowest and highest quartiles, respectively; P = .09 among all quartiles). Dose‐limiting adverse event rates were also not different by AUC quartile (16.7% 95% CI, 2.9%‐49.1% and 8.3% 95% CI, 0.4%‐40.2% in the lowest and highest quartiles; P = .35 among all quartiles). Furthermore, adverse event rates did not differ by activity %scores or between extensive, intermediate, or poor metabolizers. Antihypertensive response rate and blood pressure changes also were not influenced by differences in plasma concentrations or CYP2D6 genotypes.
Conclusions
As expected, CYP2D6 genotype‐phenotype correlates with differences in metoprolol pharmacokinetics. However, there was no association between variable pharmacokinetics or CYP2D6 genotype and β‐blocker–induced adverse effects or efficacy.
Clinical Pharmacology & Therapeutics (2004) 76, 536–544; doi: 10.1016/j.clpt.2004.08.020
Many cancers are characterized by chromosomal aberrations that may be predictive of disease outcome. Human neuroblastomas are characterized by somatically acquired copy number changes, including loss ...of heterozygosity (LOH) at multiple chromosomal loci, and these aberrations are strongly associated with clinical phenotype including patient outcome. We developed a method to assess region-specific LOH by genotyping multiple SNPs simultaneously in DNA from tumor tissues. We identified informative SNPs at an average 293-kb density across nine regions of recurrent LOH in human neuroblastomas. We also identified SNPs in two copy number neutral regions, as well as two regions of copy number gain. SNPs were PCR-amplified in 12-plex reactions and used in solution-phase single-nucleotide extension incorporating tagged dideoxynucleotides. Each extension primer had 5' complementarity to one of 2000 oligonucleotides on a commercially available tag-array platform allowing for solid-phase sorting and identification of individual SNPs. This approach allowed for simultaneous detection of multiple regions of LOH in six human neuroblastoma-derived cell lines, and, more importantly, 14 human neuroblastoma primary tumors. Concordance with conventional genotyping was nearly absolute. Detection of LOH in this assay may not require comparison to matched normal DNAs because of the redundancy of informative SNPs in each region. The customized tag-array system for LOH detection described here is rapid, results in parallel assessment of multiple genomic alterations, and may speed identification of and/or assaying prognostically relevant DNA copy number alterations in many human cancers.
Background Dual anti-platelet therapy consisting of aspirin (ASA) and a P2Y12 inhibitor is a standard treatment to prevent thrombotic events in patients with acute coronary syndrome or myocardial ...infarction.
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