Buffalo yogurt fermented with commercial starter and Lactobacillus plantarum originating from breast milk lowered blood pressure in pregnant hypertensive rats.

Nutritional therapy, which may have advantages over medication, is being investigated as a novel treatment for pregnancy-induced hypertension. Several studies have shown that probiotic yogurt supplementation during pregnancy has beneficial effects on maternal and fetal health. In this study, fermented buffalo milk was produced with yogurt culture and Lactobacillus plantarum B, a probiotic isolated from healthy breast milk with high angiotensin-converting enzyme inhibitory activity. The fermentation conditions under which the angiotensin-converting enzyme (ACE) inhibitory activity reached 84.51% were optimized by the response surface method as follows: 2 × 106 cfu/mL of L. plantarum B, yogurt culture 2.5 × 105 cfu/mL, and 8 h at 37°C. The distribution of ACE inhibitory peptides from fermented buffalo milk and fermented cow milk were further analyzed by liquid chromatography-mass spectrometry. By searching according to the structural features of ACE inhibitory peptides, 29 and 11 peptides containing ACE inhibitory peptide features were found in fermented buffalo milk and fermented cow milk, respectively. To investigate the in vivo antihypertensive activity of fermented buffalo milk, 18 pregnant rats were divided into 3 groups (n = 6 in each group) and administered 10 mL of normal saline, yogurt (20 mg/kg), or labetalol hydrochloride (4 mg/kg) daily from the beginning of pregnancy to parturition. To induce hypertension, methyl nitrosoarginine (125 mg/kg) was injected subcutaneously every day from d 15 of pregnancy to the day of delivery. Blood pressure was not significantly changed in the yogurt and labetalol groups after induction of hypertension and was lower compared with the normal saline group, but there was no difference between the yogurt and labetalol groups. This implied that the buffalo yogurt had a preventive and antihypertensive effect in the pregnancy-induced hypertensive rat model. Further studies to determine the mechanism of action, as well as a randomized control trial, are warranted.

Differential behavior of Lactobacillus helveticus B1929 and ATCC 15009 on the hydrolysis and angiotensin-I-converting enzyme inhibition activity of fermented ultra-high-temperature milk and nonfat dried milk powder.

Consumers' growing interest in fermented dairy foods necessitates research on a wide array of lactic acid bacterial strains to be explored and used. This study aimed to investigate the differences in the proteolytic capacity of Lactobacillus helveticus strains B1929 and ATCC 15009 on the fermentation of commercial ultra-pasteurized (UHT) skim milk and reconstituted nonfat dried milk powder (at a comparable protein concentration, 4%). The antihypertensive properties of the fermented milk, measured by angiotensin-I-converting enzyme inhibitory (ACE-I) activity, were compared. The B1929 strain lowered the pH of the milk to 4.13 ± 0.09 at 37°C after 24 h, whereas ATCC 15009 needed 48 h to drop the pH to 4.70 ± 0.18 at 37°C. Two soluble protein fractions, one (CFS1) obtained after fermentation (acidic conditions) and the other (CFS2) after the neutralization (pH 6.70) of the pellet from CFS1 separation, were analyzed for d-/l-lactic acid production, protein concentration, the degree of protein hydrolysis, and ACE-I activity. The CFS1 fractions, dominated by whey proteins, demonstrated a greater degree of protein hydrolysis (7.9%) than CFS2. On the other hand, CFS2, mainly casein proteins, showed a higher level of ACE-I activity (33.8%) than CFS1. Significant differences were also found in the d- and l-lactic acid produced by the UHT milk between the 2 strains. These results attest that milk casein proteins possessed more detectable ACE-I activity than whey fractions, even without a measurable degree of hydrolysis. Findings from this study suggest that careful consideration must be given when selecting the bacterial strain and milk substrate for fermentation.

Effects of ultra-high pressure treatment on angiotensin-converting enzyme (ACE) inhibitory activity, antioxidant activity, and physicochemical properties of milk fermented with Lactobacillus delbrueckii QS306.

The present study investigated the influence of ultra-high pressure (UHP) treatment on angiotensin-converting enzyme inhibitory (ACEI) activity and quality of milk fermented with Lactobacillus delbrueckii QS306 after storage. By varying treatment pressure, duration of pressure treatment, and duration of fermentation, optimal process parameters for the UHP treatment of milk fermented with QS306 to enhance ACEI activity were found to be 400 MPa, 10 min, and 48 h, respectively. The degree of ACE inhibition of the fermented milk was 91.63 ± 0.02%. After UHP treatment, ACEI activity, apparent viscosity, concentrations of polypeptides and volatile aromatic substances, umami, and richness had increased significantly, bitterness and astringency were significantly reduced, and antioxidant properties were maintained. In addition, UHP fermented milk maintained a high level of ACEI activity and good quality during storage. Thus, the data represent a valuable reference for improving the storage quality of fermented milk and research for the future development of dairy products with high ACEI activity.

Development of a Biocompatible Solid Phase Microextraction Thin Film Coating for the Sampling and Enrichment of Peptides.

While currently available methods for peptide sample preparation are mostly suitable for ex situ analysis via exhaustive extraction techniques, these techniques do not allow for in situ extraction of peptides from biological samples, such as blood or plasma collected from patients for routine clinical applications. Biocompatible solid phase microextraction (Bio-SPME) has shown great potential in metabolomics for in situ extraction of metabolites including labile compounds from biological matrices in a biocompatible and non-exhaustive fashion, thus facilitating even in vivo sampling. However, the amounts of peptides extracted by such Bio-SPME chemical biopsy tools are deemed too low for quantification when porous polyacrylonitrile (PAN)-based biocompatible thin film sorbent coatings are used, since such materials have been commonly applied as means to restrict access of high molecular weight compounds such as proteins. Aiming to improve peptide extraction by the SPME sorbent while still preventing protein adsorption, thin films with nanoscale irregularities and mesopores were prepared by inclusion of the porogen lithium perchlorate in the slurries of the coatings. The novel thin film coating method significantly improved extraction of a range of angiotensins known to possess important roles in blood pressure regulation and electrolyte balance. Model low abundance peptides covering a wide range of hydrophobicities were successfully extracted from physiological buffers and human plasma using the increased porosity coating, while the SPME protocol on the tryptic digestion of a protein supported that enzymes were excluded during peptide extraction. Surface rheological analysis, which displayed mesopores on the C18/PAN coatings, confirmed that the porosity of the coating facilitated the mass transport of peptides through the PAN layer, thus enabling extraction of high amounts of peptides by the new C18/PAN coating.

Enhanced Ion Yields Using High Energy Water Cluster Beams for Secondary Ion Mass Spectrometry Analysis and Imaging.

Previous studies have shown that the use of a 20 keV water cluster beam as a primary beam for the analysis of organic and bio-organic systems resulted in a 10-100 times increase in positive molecular ion yield for a range of typical analytes compared to C60 and argon cluster beams. This resulted in increased sensitivity to important lipid molecules in the bioimaging of rat brain. Building on these studies, the present work compares 40 and 70 keV water cluster beams with cluster beams composed of pure argon, argon and 10%CO2, and pure CO2. First, as previously, we show that for E/nucleon about 0.3 eV/nucleon water and nonwater containing cluster beams generate very similar ion yields, but below this value, the water beams yields of BOTH negative and positive "molecular" ions increase, in many cases reaching a maximum in the <0.2 region, with yield increases of ∼10-100. Ion fragment yields in general decrease quite dramatically in this region. Second, for water cluster beams at a constant E/nucleon, "molecular" ion yield increases with beam energy and hence cluster size due to increased sputter yield (ionization probability is constant). Third, as a consequence of the increased ion yield and the improved focusability using high-energy cluster beams, imaging in the 1 µm spatial resolution region is demonstrated on HeLa cells and rat brain tissue, monitoring molecules that were previously difficult to detect with other primary beams. Finally, the suggestion that the secondary ion emission zone has quasi-aqueous character seems to be sustained.

Detecting Low-Abundance Molecules at Single-Cell Level by Repeated Ion Accumulation in Ion Trap Mass Spectrometer.

Low-abundance metabolites or proteins in single-cell samples are usually undetectable by mass spectrometry (MS) due to the limited amount of substances in single cells. This limitation inspired us to further enhance the sensitivity of commercial mass spectrometers. Herein, we developed a technique named repeated ion accumulation by ion trap MS, which is capable of enhancing the sensitivity by selectively and repeatedly accumulating ions in a linear ion trap for up to 25 cycles. The increase in MS sensitivity was positively correlated with the number of repeated cycles. When ions were repeatedly accumulated for 25 cycles, the sensitivity of adenosine triphosphate detection was increased by 22-fold within 1.8 s. Our technique could stably detect low-abundance ions, especially MSn ions, at the single-cell level, such as 5-methylcytosine hydrolyzed from sample equivalent to ∼0.2 MCF7 cell. The strategy presented in this study offers the possibility to aid single-cell analysis by enhancing MS detection sensitivity.

Kidney damage induced by repeated fine particulate matter exposure: Effects of different components.

BACKGROUND: Exposure to fine particulate matter with an aerodynamic diameter of ≤2.5 µm (PM2.5) is associated with adverse health effects. This study aimed to evaluate the toxic effects of the constituents of PM2.5 on mouse kidneys. METHODS: We collected PM2.5 near an industrial complex located in southern Kaohsiung, Taiwan, that was divided into water extract and insoluble particles. Male C57BL/6 mice were divided into five groups: control, low- and high-dose insoluble particle exposure, and low- and high-dose water extract exposure. Biochemical analysis, Western blot analysis, histological examination, and immunohistochemistry were performed to evaluate the impact of PM2.5 constituents on mice kidneys. RESULTS: PM2.5 was collected from January 1, 2021, to February 8, 2021, from an industrial complex in Kaohsiung, Taiwan. Metallic element analysis showed that Pb, Ni, V, and Ti were non-essential metals with enrichment factors >10. Polycyclic aromatic hydrocarbon and nitrate polycyclic aromatic hydrocarbon analyses revealed that the toxic equivalents are, in the order, benzo(a)pyrene (BaP), indeno(1,2,3-cd) pyrene (IP), dibenzo(a,h)anthracene (DBA), and benzo(b)fluoranthene (BbF), which are potential carcinogens. Both water extract and insoluble particle exposure induced inflammatory cytokine upregulation, inflammatory cell infiltration, antioxidant activity downregulation, and elevation of kidney injury molecule 1 (KIM-1) level in mouse kidneys. A dose-dependent effect of PM2.5 water extract and insoluble particle exposure on angiotensin converter enzyme 2 downregulation in mouse kidneys was observed. CONCLUSION: We found that water-soluble extract and insoluble particles of PM2.5 could induce oxidative stress and inflammatory reactions, influence the regulation of renin-angiotensin system (RAS), and lead to kidney injury marker level elevation in mouse kidneys. The lowest-observed-adverse-effect level for renal toxicity in mice was 40 µg water-soluble extract/insoluble particle inhalation per week, which was approximately equal to the ambient PM2.5 concentration of 44 µg/m3 for mice.

Definition of chemical markers for Hancornia speciosa Gomes by chemometric analysis based on the chemical composition of extracts, their vasorelaxant effect and α-glucosidase inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: Hancornia speciosa Gomes (Apocynaceae) is a tree found in the Brazilian savannah, traditionally used to treat several diseases, including diabetes and hypertension. The anti-hypertensive activity of H. speciosa leaves (HSL) has been demonstrated in different models and is credited to the vasodilator effect and ACE (angiotensin-converting enzyme) inhibition. The hypoglycemic effect of HSL has been also reported. AIM OF THE STUDY: To establish correlations between the biological activities elicited by H. speciosa extracts and the contents of their major compounds, aiming to define chemical markers related to the potential antihypertensive and antidiabetic effects of the species. Additionally, it aimed to isolate and characterize the chemical structure of a marker related to the α-glucosidase inhibitory effect. MATERIALS AND METHODS: Extracts of a single batch of H. speciosa leaves were prepared by extraction with distinct solvents (ethanol/water in different proportions; methanol/ethyl acetate), employing percolation or static maceration as extraction techniques, at different time intervals. The contents of chlorogenic acid, rutin and FlavHS (a tri-O-glycoside of quercetin) were quantified by a developed and validated HPLC-PDA method. Bornesitol was determined by HPLC-PDA after derivatization with tosyl chloride, whereas total flavonoids were measured spectrophotometrically. Identification of other constituents in the extracts was performed by UPLC-DAD-ESI-MS/MS analysis. The vasorelaxant activity was assayed in rat aortic rings precontracted with phenylephrine, and α-glucosidase inhibition was tested in vitro. Principal component analysis (PCA) was employed to evaluate the contribution of each marker to the biological responses. Isolation of compound 1 was carried out by column chromatography and structure characterization was accomplished by NMR and UPLC-DAD-ESI-MS/MS analyses. RESULTS: The contents of the chemical markers (mean ± s.d. % w/w) varied significantly among the extracts, including total flavonoids (2.68 ± 0.14 to 5.28 ± 0.29), bornesitol (5.11 ± 0.26 to 7.75 ± 0.78), rutin (1.46 ± 0.06 to 1.97 ± 0.02), FlavHS (0.72 ± 0.05 to 0.94 ± 0.14) and chlorogenic acid (0.67 ± 0.09 to 0.91 ± 0.02). All extracts elicited vasorelaxant effect (pIC50 between 4.97 ± 0.22 to 6.48 ± 0.10) and α-glucosidase inhibition (pIC50 between 3.49 ± 0.21 to 4.03 ± 0.10). PCA disclosed positive correlations between the vasorelaxant effect and the contents of chlorogenic acid, rutin, total flavonoids, and FlavHS, whereas a negative correlation was found with bornesitol concentration. No significant correlation between α-glucosidase inhibition and the contents of the above-mentioned compounds was found. On the other hand, PCA carried out with the areas of the ten major peaks from the chromatograms disclosed positive correlations between a peak ascribed to co-eluted triterpenes and α-glucosidase inhibition. A triterpene was isolated and identified as 3-O-ß-(3'-R-hydroxy)-hexadecanoil-lupeol. CONCLUSION: According to PCA results, the vasorelaxant activity of H. speciosa extracts is related to flavonoids and chlorogenic acid, whereas the α-glucosidase inhibition is associated with lipophilic compounds, including esters of lupeol like 3-O-ß-(3'-R-hydroxy)-hexadecanoil-lupeol, described for the first time for the species. These compounds can be selected as chemical markers for the quality control of H. speciosa plant drug and derived extracts.

Synthesis of d-labeled and unlabeled benzoyloxysuccinimides and application to quantitative analysis of peptides and a protein by isotope differential mass spectrometry.

Benzoyloxysuccinimide and its d(5)-labeled version, which react with amino groups in the N-termini and lysine side chains in proteins, were synthesized and applied to quantitative analysis of peptides and a commercially available protein in combination with a MALDI mass spectrometer.

[Direct incorporation of 18O isotopes into peptides and proteins for quantative analysis by mass spectrometry method].

The method of direct introduction 18O isotopes in peptides and proteins carboxylic groups through the exchange with H218O in the presence of TFA is shown. The isotope label is steady enough in awide range of pH. Because the labeled compounds retain their physical and chemical characteristics, they can be used as an internal standard in quantitative determination of authentic compounds in the analyzed sites by mass spectrometry methods. The technique may be applicable for quantitative analysis of peptides and proteins in biological environments, for quantitative study of the kinetics of metabolism and enzymatic activity. For polypeptides and proteins the quantitative analysis is combined with trypsinolysis. If necessary, the isotope label may be introduced simultaneously in all peptides and proteins control bioassays, making it suitable for use as a standard for the comparative analysis of experimental bioassays.

Quantum dots laser desorption/ionization MS: multifunctional CdSe quantum dots as the matrix, concentrating probes and acceleration for microwave enzymatic digestion for peptide analysis and high resolution detection of proteins in a linear MALDI-TOF MS.

We report the first use of functionalized cadmium selinide quantum dots (CdSe QDs) with 11-mercaptoundecanoic acid (MUA) as the matrix for the selective ionization of proteins with high resolution and rapid analysis of amino acids and peptides by using quantum dots laser desorption/ionization mass spectrometry (QDLDI-MS). The mercaptocarboxylic groups of CdSe QDs have been known to be an effective affinity probe to interact with the biomolecules at low abundance level. Using these QDs as the matrix, sensitivity of the method was greatly enhanced and the LOQ of peptides was found to be 100 pM with RSD <10%. The QDLDI-MS is capable for the selective ionization of insulin, lysozyme and myoglobin with high resolution, which is not observed with sinapic acid (SA) as the matrix. The QDLDI-MS technique offers many advantages for the analysis of amino acids, peptides and proteins with regard to simplicity, rapidity, sensitivity and the mass spectra were generated in the presence of signal suppressors such as urea and Trition X-100. In addition, the CdSe QDs have been successfully applied as preconcentrating probes for the analysis of digested peptides in lysozyme from chicken egg white by microwave-assisted enzymatic digestion. This indicates that the QDs are able to absorb radiation from microwave and their ability to trap peptides from microwave-digested lysozyme. These results demonstrate that the CdSe QDs are promising candidates for the selective ionization of the analytes with an accurate platform to the rapid screening of biomolecules.

Selective covalent bond formation in polypeptide ions via gas-phase ion/ion reaction chemistry.

Primary amines present in protonated polypeptides can be covalently modified via gas-phase ion/ion reactions using bifunctional reagent ions. The use of reagent anions with a charge-bearing site that leads to strong interactions with the polypeptide, such as sulfonic acid, gives rise to the formation of a long-lived adduct. A distinct reactive functional group, an aldehyde in the present case, can then undergo reaction with the peptide. Collisional activation of the adduct ion formed from a reagent with an aldehyde group and a peptide ion with a primary amine gives rise to water loss in conjunction with imine (Schiff base) formation. The covalently bound modification is retained upon subsequent collisional activation. This work demonstrates the ability to selectively modify polypeptide ions in the gas phase within the context of a multistage mass spectrometry experiment.

TiO(2) printed aluminum foil: single-use film for a laser desorption/ionization target plate.

Single-use aluminum foil-based laser desorption/ionization (LDI) target plates have been developed for mass spectrometry (MS) analysis and provide detection results comparable to those of commercial stainless steel plates while offering a convenient way to avoid the time-consuming surface cleaning process. Additionally, arrays of TiO(2) nanoparticle spots are coated on the foil either by screen-printing or rotogravure-printing followed by sintering to form a mesoporous layer spot to act as an anchor for sample deposition. These TiO(2) spots offer further functions to the Al foil, such as matrix-free laser desorption/ionization or specific affinity for in situ enrichment of phosphopeptides. The single-use TiO(2)-Al foils are cheap to produce, easy to use, and well suited for high-throughput proteomics research. They can also be of interest for protein post-translational modifications study.

Gradient elution in microchannel electrochromatography.

There is great interest in using microfluidic channels packed with a stationary phase for chemical separations of complex mixtures. A key advantage of such techniques is the use of electroosmotic flow (EOF), controlled simply by applying electrical potentials between reservoirs. A disadvantage for this technique, however, is a lack of compatibility with gradient elution separations. This limitation arises from the dependence of EOF velocity on run buffer content (including the concentration of organic modifier). Here, we introduce a method for implementing gradient elution in electrochromatography in which multiple run buffers are velocity-matched, such that the elution profile resembles that found in conventional HPLC. This method is driven entirely with EOF, meaning that pumps, valves, and pressure fittings are not required. The method was validated by application to separations of peptide standards and protein digests. These results suggest that microfluidic electrochromatography may be compatible with a wide range of applications that have previously been unexplored.

Renin and angiotensin: the complete system within the neuroblastoma x glioma cell.

Cells of the homogeneous hybrid line neuroblastoma x glioma (NG108-15) have many neuronal properties. Immunocytochemical tests show that they contain both immunoreactive renin and angiotensin; direct radioimmunoassays show that they are positive for renin, angiotensin I, and angiotensin II; enzymatic assays show that they contain angiotensinogen and converting enzyme as well. The renin appears to be present in an enzymatically inactive form that can be activated by trypsin and then blocked by antiserum to purified mouse submaxillary renin. Renin concentration and activity are increased by enhancing cellular differentiation with dibutyryl cyclic adenosine monophosphate or by serum withdrawal. These findings demonstrate a complete renin-angiotensin system within these neuron-like cells, and suggest that activation of intracellular renin could generate angiotensin II.

Characterization of a local renin-angiotensin system in rat gingival tissue.

BACKGROUND: The systemic renin-angiotensin system (RAS) promotes the plasmatic production of angiotensin (Ang) II, which acts through interaction with specific receptors. There is growing evidence that local systems in various tissues and organs are capable of generating angiotensins independently of circulating RAS. The aims of this study were to investigate the expression and localization of RAS components in rat gingival tissue and evaluate the in vitro production of Ang II and other peptides catalyzed by rat gingival tissue homogenates incubated with different Ang II precursors. METHODS: Reverse transcription-polymerase chain reaction assessed mRNA expression. Immunohistochemical analysis aimed to detect and localize renin. A standardized fluorimetric method with tripeptide hippuryl-histidyl-leucine was used to measure tissue angiotensin-converting enzyme (ACE) activity, whereas high performance liquid chromatography showed products formed after the incubation of tissue homogenates with Ang I or tetradecapeptide renin substrate (TDP). RESULTS: mRNA for renin, angiotensinogen, ACE, and Ang II receptors (AT(1a), AT(1b), and AT(2)) was detected in gingival tissue; cultured gingival fibroblasts expressed renin, angiotensinogen, and AT(1a) receptor. Renin was present in the vascular endothelium and was intensely expressed in the epithelial basal layer of periodontally affected gingival tissue. ACE activity was detected (4.95 +/- 0.89 nmol histidyl-leucine/g/minute). When Ang I was used as substrate, Ang 1-9 (0.576 +/- 0.128 nmol/mg/minute), Ang II (0.066 +/- 0.008 nmol/mg/minute), and Ang 1-7 (0.111 +/- 0.017 nmol/mg/minute) were formed, whereas these same peptides (0.139 +/- 0.031, 0.206 +/- 0.046, and 0.039 +/- 0.007 nmol/mg/minute, respectively) and Ang I (0.973 +/- 0.139 nmol/mg/minute) were formed when TDP was the substrate. CONCLUSION: Local RAS exists in rat gingival tissue and is capable of generating Ang II and other vasoactive peptides in vitro.

Detection of 1 zmol injection of angiotensin using capillary zone electrophoresis coupled to a Q-Exactive HF mass spectrometer with an electrokinetically pumped sheath-flow electrospray interface.

An electrokinetically pumped sheath-flow nanoelectrospray interface provides an efficient means of transferring ions from a capillary electrophoresis system into a mass spectrometer. To characterize its performance, we analyzed angiotensin solutions prepared in a background of 0.25 mg/mL of a BSA tryptic digest. Calibration curves were prepared from 10 zmol (10-21 mol) to 10 fmol (10-14 mol) of angiotensin injected into the capillary. A parallel reaction monitoring approach was used; MS1 was set to m/z = 523.8 ±â€¯2, and fragment ion signals at 263.1389 (y2+) and 784.4095 (b6+) were used to generate selected ion electropherograms. Calibration curves based on peak areas were linear (log-log slope of 0.94 for the y2+ fragment and 0.98 for the b6+ fragment). We then injected 1-zmol (600 copies) of angiotensin in the BSA background using a 10-µm ID separation capillary. Triplicate analyses consistently produced co-migrating peaks for the two fragment ions. The b6+ electropherogram showed no background signal, whereas the y2+ electropherogram showed a few noise spikes that were smaller than the peak maximum. Bienayme-Tchebycheff inequality was used to estimate detection limits of 230  ymol (140 ions) injected into the separation capillary. To the best of our knowledge, these electropherograms present the smallest number of molecules detected using mass spectrometry coupled with a separation.

Angiotensinergic Innervation of the Human Right Atrium: Implications for Cardiac Reflexes.

BACKGROUND: The right atrium is densely innervated and provides sensory input to important cardiocirculatory reflexes controlling cardiac output and blood pressure. Its angiotensin (Ang) II-expressing innervation may release Ang II as a neuropeptide cotransmitter to modulate reflexes but has not yet been characterized. METHODS: Intraoperative surgical biopsies from human right atria (n = 7) were immunocytologically stained for Ang II, tyrosine hydroxylase (TH), and synaptophysin (SYN). Tissue angiotensins were extracted and quantified by radioimmunoassay. RESULTS: Angiotensinergic fibers were frequent in epicardial nerves and around vessels with variable TH co-localization (none to >50%/bundle). Fibers were also widely distributed between cardiomyocytes and in the endocardium where they were typically nonvaricose, TH/SYN-negative and usually accompanied by varicose catecholaminergic fibers. In the endocardium, some showed large varicosities and were partially TH or SYN-positive. A few endocardial regions showed scattered nonvaricose Ang fibers ending directly between endothelial cells. Occasional clusters of thin varicose terminals co-localizing SYN or TH were located underneath, or protruded into, the endothelium. Endocardial density of Ang and TH-positive fibers was 30-300 vs. 200-450/mm2. Atrial Ang II, III, and I concentrations were 67, 16, and 5 fmol/g (median) while Ang IV and V were mostly undetectable. CONCLUSIONS: The human right atrium harbors an abundant angiotensinergic innervation and a novel potential source of atrial Ang II. Most peripheral fibers were noncatecholaminergic afferents or preterminal vagal efferents and a minority was presumably sympathetic. Neuronal Ang II release from these fibers may modulate cardiac and circulatory reflexes independently from plasma and tissue Ang II sources.

Improving fragmentation of poorly fragmenting peptides and phosphopeptides during collision-induced dissociation by malondialdehyde modification of arginine residues.

Despite significant technological and methodological advancements in peptide sequencing by mass spectrometry, analyzing peptides that exhibit only poor fragmentation upon collision-induced dissociation (CID) remains a challenge. A major cause for unfavorable fragmentation is insufficient proton 'mobility' due to charge localization at strongly basic sites, in particular, the guanidine group of arginine. We have recently demonstrated that the conversion of the guanidine group of the arginine side chain by malondialdehyde (MDA) is a convenient tool to reduce the basicity of arginine residues and can have beneficial effects for peptide fragmentation. In the present work, we have focused on peptides that typically yield incomplete sequence information in CID-MS/MS experiments. Energy-resolved tandem MS experiments were carried out on angiotensins and arginine-containing phosphopeptides to study in detail the influence of the modification step on the fragmentation process. MDA modification dramatically improved the fragmentation behavior of peptides that exhibited only one or two dominant cleavages in their unmodified form. Neutral loss of phosphoric acid from phosphopeptides carrying phosphoserine and threonine residues was significantly reduced in favor of a higher abundance of fragment ions. Complementary experiments were carried out on three different instrumental platforms (triple-quadrupole, 3D ion trap, quadrupole-linear ion trap hybrid) to ascertain that the observation is a general effect.

Measurement of angiotensin metabolites in organ bath and cell culture experiments by liquid chromatography - electrospray ionization - mass spectrometry (LC-ESI-MS).

The metabolism of renin-angiotensin system (RAS) is more complicated than previously expected and understanding the biological phenomena regulated by variety of angiotensin metabolites requires their precise and possibly comprehensive quantitation. Physiological concentrations of angiotensins (Ang) in biological fluids are low, therefore their accurate measurements require very sensitive and specific analytical methods. In this study we developed an accurate and reproducible method of quantitation of angiotensin metabolites through coupling of liquid chromatography and electrospray ionization - mass spectrometry (LC-ESI-MS). With this method main angiotensin metabolites (Ang I, II, III, IV, 1-9, 1-7, 1-5) can be reliably measured in organ bath of rat tissues (aorta, renal artery, periaortal adipose tissue) and in medium of cultured endothelial cells (EA.hy926), exposed to Ang I for 15 minutes, in the absence or in the presence of angiotensin converting enzyme inhibitor, perindoprilat. Presented LC-ESI-MS method proved to be a quick and reliable solution to comprehensive analysis of angiotensin metabolism in biological samples.