Quantification of 5-methylcytosine, 5-hydroxymethylcytosine and 5-carboxylcytosine from the blood of cancer patients by an enzyme-based immunoassay

Background

Genome-wide aberrations of the classic epigenetic modification 5-methylcytosine (5mC), considered the hallmark of gene silencing, has been implicated to play a pivotal role in mediating carcinogenic transformation of healthy cells. Recently, three epigenetic marks derived from enzymatic oxidization of 5mC namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), have been discovered in the mammalian genome. Growing evidence suggests that these novel bases possess unique regulatory functions and may play critical roles in carcinogenesis.

Methods

To provide a quantitative basis for these rare epigenetic marks, we have designed a biotin–avidin mediated enzyme-based immunoassay (EIA) and evaluated its performance in genomic DNA isolated from blood of patients diagnosed with metastatic forms of lung, pancreatic and bladder cancer, as well as healthy controls. The proposed EIA incorporates spatially optimized biotinylated antibody and a high degree of horseradish-peroxidase (HRP) labeled streptavidin, facilitating signal amplification and sensitive detection.

Results

We report that the percentages of 5mC, 5hmC and 5caC present in the genomic DNA of blood in healthy controls as 1.025 ± 0.081, 0.023 ± 0.006 and 0.001 ± 0.0002, respectively. We observed a significant (p < 0.05) decrease in the mean global percentage of 5hmC in blood of patients with malignant lung cancer (0.013 ± 0.003%) in comparison to healthy controls.

Conclusion

The precise biological roles of these epigenetic modifications in cancers are still unknown but in the past two years it has become evident that the global 5hmC content is drastically reduced in a variety of cancers. To the best of our knowledge, this is the first report of decreased 5hmC content in the blood of metastatic lung cancer patients and the clinical utility of this observation needs to be further validated in larger sample datasets.     

Incorporation of methamphetamine and amphetamine in human hair following controlled oral methamphetamine administration

Background

Although hair testing is well established for the assessment of past drug exposure, uncertainties persist about mechanisms of drug incorporation into hair and interpretation of results. The aim of this study was to administer methamphetamine (MAMP) under controlled conditions as a model drug to investigate drug incorporation into human hair.

Material and methods

Seven volunteers with a history of stimulant use received 4 × 10 mg (low) doses of sustained release S-(+)-MAMP HCl within 1 week, with weekly head hair samples collected by shaving. 3 weeks later, 4 of them received 4 × 20 mg (high) doses. After extensive isopropanol/phosphate buffer washing of the hair, MAMP and its metabolite amphetamine (AMP) concentrations were determined in all weekly hair samples by LC–MS–MS in selected reaction monitoring mode with the undeca- and deca-deuterated drugs, respectively, as internal standards (LLOQ, 0.005 ng mg⁻¹).

Results

MAMP T_max occurred from 1 to 2 weeks after both doses, with C_max ranging from 0.6 to 3.5 ng mg⁻¹ after the low and 1.2 to 5.3 ng mg⁻¹ after the high MAMP doses. AMP C_max in hair was 0.1–0.3 ng mg⁻¹ and 0.2–0.5 ng mg⁻¹, respectively, for low and high doses. Highly dose-related concentrations within subjects, but large variability between subjects were observed. MAMP concentrations were above the 0.2 ng mg⁻¹ cut-off for at least 2 weeks following administration of both low and high doses. The overall AMP/MAMP ratio ranged from 0.07 to 0.37 with a mean value of 0.15 ± 0.07, and a median of 0.13. The percentage of MAMP and AMP removed with the washing procedure decreased with time after administration. A strong correlation was found between area under the curve of MAMP (r² = 0.90, p = 0.00) and AMP (r² = 0.94, p = 0.00) concentrations calculated for the 3-week period following administration and the total melanin concentration in hair. Significant correlations were observed also between C_max and melanin.

Conclusions

This study demonstrated that despite large inter-individual differences, the incorporation of MAMP and AMP into hair is dose-related with much of the observed scatter of MAMP and AMP concentrations explained by melanin concentration in hair.     

Online trapping and enrichment ultra performance liquid chromatography–tandem mass spectrometry method for sensitive measurement of “arginine-asymmetric dimethylarginine cycle” biomarkers in human exhaled breath condensate

Background

Exhaled breath condensate (EBC) is a biofluid collected non invasively that, enabling the measurement of several biomarkers, has proven useful in the study of airway inflammatory diseases, including asthma, COPD and cystic fibrosis. To the best of our knowledge, there is no previous report of any analytical method to detect ADMA in EBC.

Objectives

Aim of this work was to develop an online sample trapping and enrichment system, coupled with an UPLC–MS/MS method, for simultaneous quantification of seven metabolites related to “Arginine-ADMA cycle”, using the isotopic dilution.

Methods

Butylated EBC samples were trapped in an online cartridge, washed before and after each injection with cleanup solution to remove matrix components and switched inline into the high pressure analytical column. Multiple reaction monitoring in positive mode was used for analyte quantification by tandem mass spectrometry.

Results

Validation studies were performed in EBC to examine accuracy, precision and robustness of the method. For each compound, the calibration curves showed a coefficient of correlation (r²) greater than 0.992. Accuracy (%Bias) was <3% except for NMMA and H-Arg (<20%), intra- and inter-assay precision (expressed as CV%) were within ±20% and recovery ranged from 97.1 to 102.8% for all analytes.Inter-day variability analysis on 20 EBC of adult subjects did not demonstrate any significant variation of quantitative data for each metabolite. ADMA and SDMA mean concentrations (μmol L⁻¹), measured in EBC samples of asthmatic adolescents are significantly increased (p < 0.0001) than in normal controls (0.0040 ± 0.0021 vs. 0.0012 ± 0.0005 and 0.0020 ± 0.0015 vs. 0.0002 ± 0.0001, respectively), as well the ADMA/Tyr (0.34 ± 0.09 vs. 0.12 ± 0.02, p < 0.0001) and the SDMA/Tyr ratio (0.10 ± 0.04 vs. 0.015 ± 0.004, p < 0.0001).

Conclusions

The proposed method features simple specimen preparation, maintenance of an excellent peak shape of all metabolites and reduced matrix effects as well mass spectrometer noise. Moreover, the possibility to perform different cycles of enrichment, using large injection volumes, compensated for the low concentration of analytes contained in EBC, leading to a good analytical sensitivity. Preliminary data obtained from asthmatic and healthy adolescents, demonstrated that the analytical method applied to EBC seems suitable not only for research purposes, but also for clinical routinely analysis.     

Synthesis and properties of a new ketoiminate derivative of the 2,2,6,6-tetramethylheptane-3,5-dionate ligand to prepare volatile CVD precursors

The β-ketoimine (CH₃)₃CC(NH₂)CHC(O)C(CH₃)₃ (1) was synthesized by amination with dry ammonia in the presence of TiCl₄. M.p. = 131 °C. IR and ¹H and ¹³C NMR spectroscopic characterization indicates that the structure of a solution of 1 is the ketone form, and a single-crystal X-ray diffraction study shows that the structure of 1 is the enaminoketone form. The reaction of 1 with copper(II) and nickel(II) salts in solution gave chelate metal complexes: Cu[(CH₃)₃CC(NH)CHC(O)C(CH₃)₃]₂ (2), M.p. = 209 °C, and Ni[(CH₃)₃CC(NH)CHC(O)C(CH₃)₃]₂ (3), M.p. = 267 °C. These complexes are volatile and sublime at 180–190 °C at 5 × 10⁻³ Torr. An X-ray diffraction study reveals that these metal complexes are monomeric and isostructural in the solid state. In compound 2, the Cu atom has a square coordination environment: Cu–O ≈ Cu–N = 1.91 Å, ∠O–Cu–N = 91.81 Å.     

A synthetic approach to palmerolides via Negishi cross coupling. The challenge of the C15–C16 bond formation

The esterification of fragment C1–C8 (2) with fragment C16–C23 (3) to give iodo derivative 4, followed by a Pd-catalysed coupling with a C9–C15 fragment (7 or 8), may provide a common precursor of most palmerolides. Ligands and reaction conditions were exhaustively examined to perform the C15–C16 bond formation via Negishi reaction. With simple models, pre-activated Pd–Xantphos and Pd–DPEphos complexes were the most efficient catalysts at RT. Zincation of the C9–C15 fragment (8) and cross coupling with 4 required 3 equiv of t-BuLi, 10 mol % of Pd–Xantphos and 60 °C.     

Syntheses, structures, and dynamic properties of M(CO)2(η-C3H5)(L-L)(NCBH3) (M = Mo, W; L-L = dppe, bipy, en)

Compounds M(CO)₂(η³-C₃H₅)(L-L)(NCBH₃) (L-L = dppe, M = Mo(1), W(2); L-L = bipy, M = Mo(3), W(4); L-L = en, M = Mo(5), W(6)) were prepared and characterized. The single crystal X-ray analyses of 2-6 revealed that the cyanotrihydroborate anion bonds to the metal through a nitrogen atom, the open face of the allyl group being pointed toward the two carbonyls (endo-isomer). In compounds 2, 5, and 6, the two donor atoms of the bidentate ligand occupy equatorial and axial positions, respectively. In the solid state structures of compounds 3 and 4 both nitrogen atoms of the bipy ligand occupy equatorial positions. The NMR spectroscopy reveals a fluxional behavior of compounds 1, 2, 5, and 6 in solution. Although the fluxional behavior of compounds 5 and 6 ceased at about −40 °C, that of compound 1 could not be stopped even at −90 °C. Their low temperature conformations are consistent with their solid state structures. Both the endo- and exo-isomers coexist in solution for compounds 3 and 4.     

Structural studies of two-coordinate complexes of tris(2-methoxylphenyl)phosphine and tris(4-methoxyphenyl)phosphine with gold(I) halides

A series of five gold(I) halide complexes with the two isomeric methoxy-substituted triarylphosphines, tris(2-methoxyphenyl)phosphine [P(oanis)₃], [AuP(oanis)₃X] [for X = Cl, (1); X = Br, (2) and X = I, (3)] and tris(4-methoxyphenyl)phosphine [P(panis)₃], [AuP(panis)₃X] [for X = Br (4) and X = I (5)] have been synthesized and characterized by single crystal X-ray diffraction and solution ³¹P{¹H} NMR spectroscopy. The structure determinations confirm the expected presence of linear two-coordination about the gold centres in all five complexes with bond distance and angle data typical of this type of compound [Au–P, 2.239(2)–2.259(3) Å; Au–Cl, 2.294(2) Å; Au–Br, 2.385(2)–2.402(2) Å; Au–I, 2.546(1)–2.554(1) Å; P–Au–X; 175.3(1)–180°]. All analogues except the iodo complex 5 crystallize with one complex molecule in the crystallographic asymmetric unit. The bromo and iodo complexes 2 and 3 constitute a trigonal isomorphous set while the bromo complex 4 is also isomorphous with the previously determined chloro complex [AuP(panis)₃Cl]. The 2-methoxy analogues are stabilized by significant methoxy-O?Au interactions.     

Preparation of 2,2-difluoro-1-trialkylsilylethenylstannanes and their cross-coupling reactions

Reaction of 2 with bis(tributyltin) in the presence of 3 mol % Pd₂(dba)₃, 6 mol % XPhos, and 30 equiv of LiBr in wet and air bubbled THF at reflux for 8 h afforded the desired products 3 in 73–74% yields. The cross-coupling reaction of 3a with aryl iodides in the presence of 10 mol % Pd(PPh₃)₄ and 10 mol % CuI afforded the coupled products 4a–p in 47–90% yields. The coupling reaction of 3b with various alkynyl bromides having aryl-, alkyl, or trialkylsilyl group also afforded the corresponding 1,3-enynes 5a–g in 61–77% yields.     

Si-C coupling reaction of polychloromethanes with HSiCl3 in the presence of Bu4PCl: Convenient synthetic method for bis(chlorosilyl)methanes

Coupling reaction of polychloromethanes CH_4−nCl_n (n = 2-4) with HSiCl₃ in the presence of tetrabutylphosphonium chloride (Bu₄PCl) as a catalyst occurred at temperatures ranging from 30 °C to 150 °C. The reactivity of polychloromethanes increases as the number of chlorine-substituents on the carbon increases. In the reactions of CCl₄ with HSiCl₃, a variety of coupling products such as bis(chlorosilyl)methanes CH₂(SiCl₃)(SiXCl₂) [X = Cl (1a), H (1b)], (chlorosilyl)trichloromthanes Cl₃CSiXCl₂ [X = Cl (2a), H (2b)], and (chlorosilyl)dichloromthanes Cl₂HCSiXCl₂ [X = Cl (3a), H (3b)] were obtained along with reductive dechlorination products such as CHCl₃ and CH₂Cl₂ depending on the reaction temperature. In the reaction of CCl₄, 2a is formed at the initial stage of the coupling reaction and converted to give CHCl₃ at low temperature of 30 °C, to give 1a, 3a, and CHCl₃ at 60 °C, and to afford 1a as major product and CH₂Cl₂ in competition above 100 °C. Si-H bond containing silylmethanes can be formed by the H-Cl exchange reaction with HSiCl₃. Reaction of CHCl₃ with HSiCl₃ took placed at 80 °C to give three compounds 1a, 3a, and CH₂Cl₂, and finally 3a was converted to give 1a and CH₂Cl₂ at longer reaction time. While the condition for the reaction of CH₂Cl₂ with HSiCl₃ required a much higher temperature of 150 °C. Under the optimized conditions for synthesizing bis(chlorosilyl)methanes 1a,b, a mixture of 1a and 1b were obtained as major products in 65% (1a:1b = 64:1) and 47% (42:5) yields from the reaction of CCl₄ and CHCl₃ at 100 °C for 8 h, respectively, and in 41% (34:7) yield from that of CH₂Cl₂ at 170 °C for 12 h. In the Si-C coupling reaction of polychloromethanes with HSiCl₃, it seems likely that a trichlorosilyl anion generated from the reaction of HSiCl₃ with Bu₄PCl is an important key intermediate.     

Differential scanning calorimetric examination of the tracheal cartilage after primary reconstruction with differential suturing techniques

Objective

Long segmental tracheal defects often lead to life threatening clinical conditions. Treatment of these lesions still represents an unsolved surgical challenge. The aim of the study was to test the effect of continuous and simple interrupted suturing in the replacement of long segment tracheal defects using polytetrafluoroethylene (PTFE) vascular prosthesis in a rabbit model.

Methods

2 cm long segment of the cervical trachea was resected in 20 New Zealand rabbits. The trachea was replaced with reinforced polytetrafluoroethylene vascular graft. The anastomoses were performed telescopically using continuous (group I, n = 10) or simple interrupted sutures (group II, n = 10). Laser Doppler measurements were taken before the resection and following the anastomoses. Length of survivals were noted, the patency and microscopical pattern of the anastomoses were evaluated. Calorimetric examinations were performed to detect possible structural changes in the tracheal cartilage.

Results

Following the resection local microcirculation decreased by 9 ± 4%. The anastomoses caused a significant decrease of 29% in group I (p = 0.02) and 13% in group II. The mean survival was 58 ± 17 and 135 ± 25 days, respectively. Calorimetric results showed no change after the resection, but significant shift in melting temperature and calorimetric enthalpy proved the presence of structural changes of the cartilage in group II.

Conclusions

We saw significant lowering of microcirculation following continuous sutures, while simple interrupted stitches produced only moderate decrease. We found that interrupted suture technique is superior to the continuous technique causing only moderate damage to the tracheal anastomosis.     

Efficient green procedures for the preparation of novel tetraalkynyl-substituted phthalocyanines

This work provides a successful, easy and efficient process for the preparation of metal-free 2(3),9(10),16(17),23(24)-octamethoxyphthalocyanine, [(OMe)₈PcH₂] (2), and its metal complexes [(OMe)₈PcM] (3–11) (M = Zn, Cu, Ni, Mg, Co, Fe, Ru, TiCl and RhCl) by using green energy techniques such as exposure to UV-irradiation as well as microwave irradiation. Two different routes have been used, which involve modifications to that reported in the literature. The results suggest that these techniques drastically reduce the reaction time of metallophthalocyanine [(OMe)₈PcM] (3–11) formation from 5–96 h to 5–11 min. The prepared octamethoxyphthalocyanines [(OMe)₈PcM] (2–4) (M = H₂, Zn, Cu) are used as key materials to synthesize the corresponding novel tetraalkynyl-substituted phthalocyanines 15–17.     

Thermodynamic and kinetic parameters associated with the fluxional behavior of 2-methyl- and 2,6-dimethyltroponeiron tricarbonyl complexes

The kinetic and thermodynamic parameters for regioisomerisation of 2-methyl- and 2,6-dimethyl-derivatives of tricarbonyl[η⁴-tropone]iron complexes have been studied by ¹H NMR spectrometry over a range of 40 °C. Regioisomerisation of these complexes proceeds by an intramolecular first-order process and results in the almost complete conversion of the less stable complexes (4, 8) to more stable regioisomers (1, 5). The activation energies and half lifes for the conversion (4 → 1) and (8 → 5) were found to be ΔG^#=92 kJ mol⁻¹; τ_1/2=12.8 h, and ΔG^#=107 kJ mol⁻¹; τ_1/2=26.8 h, respectively, at 23 °C. Complex 1 reacts with (1R,2S,5R)-menthol in sulphuric acid solution, followed by neutralisation with sodium carbonate to give a separable mixture of diastereomeric tricarbonyl[(2,3,4,5-η)-(1R^′,2S^′,5R^′)-6-menthyloxy-2-methyltropone]iron complexes, 9 and 10. The corresponding dimethylated complex 5 fails to react under these conditions.     

A review of recent trends in electrospray ionisation-mass spectrometry for the analysis of metal-organic ligand complexes

Background

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide (NO) formation inhibitor, has emerged as a promising biomarker of NO-associated endothelial dysfunction in cardiovascular diseases as well in chronic renal failure. The interest in potentially fundamental role of this metabolite, in basic and clinical research, led to the development of numerous analytical methods for the quantitative determination of ADMA and dimethylarginines in biological systems, notably plasma, serum and urine.

Objectives

The aim of this work was to present a simple, fast and accurate UPLC-tandem-MS-based method for the simultaneous determination and quantification of arginine, ADMA, SDMA, NMMA, homo-arginine and citrulline. This method is designed for high sample throughput of only 10 μL of human plasma, serum or urine.

Methods

The analysis time is reduced to 1.9 min by an ultrahigh-performance liquid chromatography run coupled with electrospray ionization (ESI) in the positive mode tandem mass spectrometry detection.

Results

The method was validated in plasma, serum and urine. Correlation coefficients (r²) of the calibration curves in all matrices considered ranged from 0.9810 to 0.9993. Inter- and intra-assay precision, accuracy, recovery and carry-over were evaluated for validation. The LOD was 0.01 μM for all compounds in water, plasma and serum and 0.1 μM in urine. The LOQ was 0.05 μM for ADMA, SDMA, NMMA and H-Arg and 0.5 μM for Arg and Cit in water, plasma and serum; while in urine was 0.1 μM for ADMA, SDMA, NMMA and H-Arg and 0.5 μM for Arg and Cit.The precision was ranged from 1% to 15% expressed as CV% and the accuracy (bias %) was <±7% for all added concentrations with the exception of NMMA (−10%).ADMA mean plasma levels, measured in healthy adults and newborns, were in accord with literature data published: (M ± SD) 0.56 ± 0.10 μM and 0.84 ± 0.21 μM, respectively, showing that ADMA levels in plasma decreased with age. In serum we have similar data (0.54 ± 0.18 μM and 1.14 ± 0.36 μM), while in neonatal urine ADMA was 11.98 ± 7.13 μmol mmol⁻¹ creatinine.

Conclusions

Data from calibration curves and method validation reveal that the method is accurate and precise. The fast run time, the feasibility of high sample throughput and the small amount of sample required make this method very suitable for routine analysis in the clinical setting.     

Single stranded helical supramolecular architecture with a left handed helical water chain in ternary copper(II) tryptophan/diamine complexes

The ternary copper(II) complexes [Cu(l-trp)(bpy)](ClO₄) (1) and [Cu(l-trp)(phen)] (ClO₄) · 3H₂O (2) (where l-trp = l-tryptophan, bpy = bipridyl, phen = phenanthroline) have been synthesized. The single crystal X-ray structures for these complexes revealed that the monocationic Cu^II-units are interlinked through Cu–OCO–Cu connectivity and exist as helical coordination polymers. The two different helical strands composed with Cu1 and Cu2 independently, possess a similar pitch distance of 7.713 Å in complex 1. For complex 2, existing in the hydrated form, the Cu(II) polymeric strand and the hydrated water molecules have gained a supramolecular helical architecture with a similar pitch distance of 8.133 Å. The two helical strands in complex 1 are associated with right handed (PP) supramolecular chirality, while the helical water chain and the Cu^II-strand in 2 are self assembled into left handed (MM) helicity in the solid state. The solid state CD recorded for 1 and the dehydrated form of 2 exhibit a positive optical sign at their respective d–d band [λ_max = 667 nm, 1; λ_max = 630 nm, 2], the solution state CD for both these complexes are found to be inverted into a negative optical sign, which could be attributed to inversion of their associated supramolecular helicity. The TGA curve illustrates two distinct weight losses at 60 °C and 87 °C, equivalent to one and two water molecules, respectively. The PXRD pattern for the hydrated and dehydrated forms of 2 indicated a change, on comparison with the simulated diffractograph. The fluorescence properties of both these complexes, possessing tryptophan and bipy/phen, were investigated.     

A short-cut from 1-acetyl adamantane to 2-(1-adamantyl)pyrroles

The oxime of 1-acetyl adamantane 2 is added to acetylene (KOH/DMSO, 70 °C, initial acetylene pressure 13 atm, 30 min) to afford the corresponding O-vinyl oxime 5 in 80% yield. The latter upon heating (DMSO, 120 °C, 1 h) gives 2-(1-adamantyl)pyrrole 3, 1-acetyl adamantane 1, and adamantane (6:3:1 mass ratio), the yield of the pyrrole 3 being 83% (based on 1-acetyl adamantane 1 consumed). Under harsher conditions (NaOH/DMSO, 130 °C, atmospheric pressure of acetylene, 4 h) oxime 2 reacts with acetylene to furnish pyrrole 3, 1-acetyl adamantane 1, 1-vinyl adamantane 9, and adamantane (6:7:3:1 mass ratio), with the isolated yield of pyrrole 3 reaching 34%. Under pressure (NaOH/DMSO, 120 °C, initial acetylene pressure 14 atm, 1 h) the same reaction leads to 2-(1-adamantyl)-1-vinylpyrrole 4 and ketone 1 in 48% (based on consumed ketone 1) and 24% yields, respectively. The pyrrole 4 is easily deprotected to the corresponding 1H-pyrrole 3 in 77% yield by treatment (aqueous MeCN) with Hg(OAc)₂ and NaBH₄.