A digital microfluidic method for in situ formation of porous polymer monoliths with application to solid-phase extraction.

We introduce the marriage of two technologies: digital microfluidics (DMF), a technique in which droplets are manipulated by application of electrostatic forces on an array of electrodes coated by an insulator, and porous polymer monoliths (PPMs), a class of materials that is popular for use for solid-phase extraction and chromatography. In this work, circular PPM discs were formed in situ by dispensing and manipulating droplets of monomer solutions to designated spots on a DMF device followed by UV-initiated polymerization. We used PPM discs formed in this manner to develop a digital microfluidic solid-phase extraction (DMF-SPE) method, in which PPM discs are activated and equilibrated, samples are loaded, PPM discs are washed, and the samples are eluted, all using microliter droplets of samples and reagents. The new method has extraction efficiency (93%) comparable to that of pipet-based ZipTips and is compatible with preparative sample extraction and recovery for on-chip desalting, removal of surfactants, and preconcentration. We anticipate that DMF-SPE may be useful for a wide range of applications requiring preparative sample cleanup and concentration.

Molecularly imprinted spongy columns for Angiotensin(II) recognition from human serum.

Angiotensin II (AngII), the effector peptide of the renin angiotensin system and has an important role in regulating cardiovascular hemodynamics and structure. AngII is an important biomarker for certain diseases that are associated with cardiovascular disorders, i.e., influenza, SARS-CoV-2, tumors, hypertension, etc. However, AngII presents in blood in very low concentrations and they are not stable due to their reactivity, therefore spontaneous detection of AngII is a big challenge. In this study, AngII-imprinted spongy columns (AngII-misc) synthesized for AngII detection from human serum, and characterized by surface area measurements (BET), swelling tests, scanning electron microscopy (SEM), FTIR studies. AngII binding studies were achieved from aqueous environment and maximum binding capacity was found as 0.667 mg/g. It was calculated that the AngII-miscs recognized AngII 8.27 and 14.25 times more selectively than competitor Angiotensin I and Vasopressin molecules. Newly produced AngII-misc binds 60.5 pg/g AngII from crude human serum selectively. It has a great potential for spontaneous detection of AngII from human serum for direct and critical measurements in serious diseases, that is, heart attacks, SARS-CoV-2, etc.

A human neutrophil-dependent pathway for generation of angiotensin II. Purification of the product and identification as angiotensin II.

A human neutrophil lysosomal protease interacts at physiologic pH with a 62,000--67,000-mol wt plasma protein substrate to generate a vasoactive, smooth muscle-contracting "neutral" peptide. The peptide product of this system, previously designated the "neutral" peptide-generating pathway, was generated from purified components and purified by Bio-Gel P2 gel filtration and reverse-phase high performance liquid chromatography with a 50--60% yield of starting activity. The purified peptide had an amino acid composition of Asx, Pro, Val, Ile, Tyr, Phe, His, Arg, a composition identical to that of angiotensin II. The peptide and synthetic angiotensin II each filtered at 48--52% bed volume on Bio-Gel P2, had an isoelectric point of Ph 7.8--8.1 at 4 degrees C, migrated 3 cm toward the cathode during pH 6.4 low-voltage paper electrophoresis, and had a retention time of 44.8 min during reverse-phase high-performance liquid chromatography. In addition, the functional activity of the peptide at each purification step correlated with angiotensin II content determined by specific radioimmunoassay. The amino acid sequence of 25 nmol of the peptide was Asp-Arg-Val-Try-Ile-His-Pro-Phe, the same covalent structure as that of angiotensin II. Therefore, under physiologic conditions, in the absence of renin or angiotensin converting enzyme, a human neutrophil neutral protease cleaves a plasma protein to yield angiotensin II. This pathway provides a mechanism through which the neutrophil may alter local blood flow during inflammation by generation of a potent vasoactive peptide.

Angiotensin II formation in the intact human heart. Predominance of the angiotensin-converting enzyme pathway.

It has been proposed that the contribution of myocardial tissue angiotensin converting enzyme (ACE) to angiotensin II (Ang II) formation in the human heart is low compared with non-ACE pathways. However, little is known about the actual in vivo contribution of these pathways to Ang II formation in the human heart. To examine angiotensin II formation in the intact human heart, we administered intracoronary 123I-labeled angiotensin I (Ang I) with and without intracoronary enalaprilat to orthotopic heart transplant recipients. The fractional conversion of Ang I to Ang II, calculated after separation of angiotensin peptides by HPLC, was 0.415 +/- 0.104 (n = 5, mean +/- SD). Enalaprilat reduced fractional conversion by 89%, to a value of 0.044 +/- 0.053 (n = 4, P = 0.002). In a separate study of explanted hearts, a newly developed in vitro Ang II-forming assay was used to examine cardiac tissue ACE activity independent of circulating components. ACE activity in solubilized left ventricular membrane preparations from failing hearts was 49.6 +/- 5.3 fmol 125I-Ang II formed per minute per milligram of protein (n = 8, +/- SE), and 35.9 +/- 4.8 fmol/min/mg from nonfailing human hearts (n = 7, P = 0.08). In the presence of 1 microM enalaprilat, ACE activity was reduced by 85%, to 7.3 +/- 1.4 fmol/min/mg in the failing group and to 4.6 +/- 1.3 fmol/min/mg in the nonfailing group (P < 0.001). We conclude that the predominant pathway for angiotensin II formation in the human heart is through ACE.

Lowering the concentration limits of detection by on-line solid-phase extraction-capillary electrophoresis-electrospray mass spectrometry.

The use of solid-phase extraction coupled on-line to capillary electrophoresis using electrospray mass spectrometry detection (SPE-CE-ESI-MS) is described for the analysis of peptides in dilute solutions. A SPE microcartridge or analyte concentrator containing C(18) derivatized silica particles as the extraction sorbent was easily constructed near the inlet of the separation capillary using commercially available materials. The reversed-phase sorbent selectively retained the target peptides, enabling large volumes of the sample to be introduced (>100muL). The captured analytes were eluted in a small volume of an appropriate solution (20-50nL). This resulted in sample clean-up and concentration enhancement, with minimum sample handling. As the SPE-CE conditions were compatible with on-line ESI-MS detection, the potential for identifying and characterizing the preconcentrated analytes by SPE-CE-ESI-MS using a sheath-flow CE-ESI-MS interface is also shown. Using separation electrolytes containing N-[carbamoylmethyl]-2-aminoethanesulfonic acid (ACES) at pH 7.4, an elution plug of 80:20 (v/v) (25mM of formic acid in MeCN):H(2)O and a sheath liquid of 20mM of acetic acid in 50:50 (v/v) methanol:H(2)O the concentration limits of detection for the analyzed peptides in the positive ion mode were lowered to nanogram per milliliter levels. The systematic optimization of the operational parameters involved in the development of the SPE-CE method is described in detail, in order to promote robust and quantitative SPE-CE-ESI-MS analysis and facilitate the widespread use of the technique.

Renal cathepsin G and angiotensin II generation.

BACKGROUND: Alternative pathways of angiotensin II biosynthesis play a significant role in the renin-angiotensin system. In this study porcine renal tissue was investigated for angiotensin II-generating enzymes. METHODS AND RESULTS: Protein extracts from porcine renal tissue were fractionated by liquid chromatography and tested for their angiotensin II-generating activity by the mass-spectrometry-assisted enzyme screening system (MES) and the active fractions were purified to near homogeneity. In one of these active fractions, inhibitable by an angiotensin-converting enzyme specific inhibitor, purified by anion-exchange chromatography, followed by hydroxyapatite chromatography, lectin affinity chromatography, size-exclusion chromatography and two-dimensional electrophoresis, angiotensin-converting enzyme was identified by a tryptic peptide matrix-assisted-laser-desorption/ionization (MALDI) mass fingerprint analysis. In a second active fraction, which was inhibited by chymostatin and antipain, yielded by anion-exchange chromatography, followed by hydroxyapatite chromatography, lectin affinity chromatography, chymostatin-antipain chromatography and one-dimensional electrophoresis, cathepsin G was identified by electro-spray ionization (ESI)-ion-trap mass spectrometry. The angiotensin-generating activities of the fraction containing angiotensin-converting enzyme and the fraction containing cathepsin G were in the same order of magnitude, thus showing that the contribution of cathepsin G towards the production of angiotensin II is significant. CONCLUSION: This is the first time that cathepsin G has been identified in mammalian renal tissue.

Ultra-rapid separation of an angiotensin mixture in nanochannels using shear-driven chromatography.

The present paper reports on the separation of a mixture of fluorescein isothiocyanate-labeled angiotensin I and II peptides in a shear-driven nanochannel with a C18-coating and using an eluent consisting of 5% acetonitrile in 0.02 M aqueous phosphate buffer at pH 6.5. The flat-rectangular nanochannel in fused silica consisted of an etched structure in combination with a flat moving wall. The very fast separation kinetics that can be achieved in a nanochannel allowed to separate the angiotensin peptides in less then 0.2 s in a distance of only 1.8 mm. Plate heights as small as 0.4 microm were calculated after substraction of the injection effect.

Ionic liquids as novel solvent additives to separate peptides.

A novel analytical approach involving the addition of an ionic liquid into the mobile phase of the thin-layer chromatography (TLC) system during the optimization of chromatographic separation of peptides was demonstrated. Different behavior of peptides in the TLC sytem was observed after the addition of 1,3-dimethylimidazolium methyl sulfate to the eluent in comparison to the system without the ionic liquid. The objective of the work was to study the effect of the addition of different contents of ionic liquid to the mobile phase comprising mostly water and to observe the behavior of peptides' retention. The potential usefulness of environmentally friendly ionic liquids for the optimization of separation of peptides was demonstrated. An increase of R(f) values was observed with increasing the ionic liquid content in the mobile phase. The benefits of the used approach were related to the separation achieved. Finally, quantitative structure-retention relationships (QSRR) were used for the studies on the predictions of peptides' retention in the TLC systems with the addition of ionic liquid in terms of the predictions performed recently in HPLC systems.

Preparation and immunoreactive properties of monoiodinated angiotensin labelled at high specific activity.

Monoiodinated angiotensin II at a specific activity in the order of 1 C/mg can be prepared with high yields by controlling the pH of iodination and can be purified on G-25 Sephadex. The fractions from the head to the centre of the single peak of radioactivity obtained by gel filtration of the iodination mixture contain pure monoiodinated angiotensin. The monoiodinated derivative shows an immunoreactivity very close to that of the native hormone; in contrast, the diiodinated derivative has a low immunoreactivity and its use as a tracer results in a loss of sensitivity in the radioimmunoassay of angiotensin II. It is suggested that the use of the monoiodinated derivative in pure form should be recommended whenever a small hormonal polypeptide labelled at high specific activity is to be prepared for radioimmunoassay purposes.

Influence of polyfluorination of the phenylalanine ring of angiotensin II on conformation and biological activity.

[Phe(F5)8]angiotensin II was synthesized by the solid phase method and purified by reverse-phase HPLC. In rat uterus and rabbit aorta bioassays the analogue had 10 and 50%, respectively, of the contractile activity of angiotensin II and demonstrated antagonist properties. These findings illustrate that inversion of the Phe8 ring quadrupole moment in angiotensin II decreases agonist activity and invokes antagonist properties. 1H-NMR studies at 400 MHz in DMSO-d6 demonstrated the presence of cis and trans isomers in the ratio 1:3 due to restricted rotation of the His-Pro bond. Downfield shifts of the His C2 and C4 protons in [Phe(F5)]ANG II compared to ANG II suggest that the Phe(F5) residue may be involved in a parallel-plate ring pairing interaction with the imidazole group. However heteronuclear NOE studies, carried out by measuring the proton difference spectrum before and after saturation of the fluorine resonances, showed the absence of any NOE enhancement illustrating that electrostatic influences of the Phe(F5) ring occur at relatively long range.

Dynamic pH junction technique for on-line preconcentration of peptides in capillary electrophoresis.

A method based on the presence of a dynamic pH junction within the capillary to induce band narrowing for enhanced detection sensitivity for some peptides is presented. This technique is predicated on a sharp reduction in an analyte's migration velocity following a reversal of its electrophoretic direction from the acidic sample zone to the basic BGS zone. Larger-than-usual injection volumes of samples in relatively high-conductivity matrices were enabled, without degrading peak shape, resolution and efficiency. The size of the original sample plug was reduced by as much as 38-fold, and improvement in detector response in terms of peak height by as much as 124-fold was obtained. The effects of pH and concentration of the sample matrix, and the length of sample injection on the efficiency of the technique are discussed.

Zone Sharpening of Peptides in Pressurized Capillary Electrochromatography Using Dynamic pH Junction.

In HPLC, analytes injected into a separation column broaden naturally during the separation procedure. In this paper, analyte zone sharpening of peptides was achieved in pressurized capillary electrochromatography, which is a separation method that combines capillary HPLC and capillary electrophoresis (CE), by employing dynamic pH junction for CE. When the pH of the mobile phase was altered from basic to acidic in a step gradient, the analyte peptides were focused at the basic/acidic interface with the application of voltage. The effect of both pH and pressurized flow velocity on the zone sharpening was investigated. With the proposed method, the peak height of angiotensin II, [Asn(1), Val(5)]-angiotensin II, and angiotensin III were enhanced 12, 10, and 12 times, respectively. Selective peak zone sharpening for angiotensin II was also demonstrated.

Identification of "angiotensin immunoreactive material" in rat kidney.

The reported presence of large quantities of a high molecular weight form of angiotensin I and II in renin granules from rat kidney cortex was investigated. Subcellular fractionation by differential centrifugation and isopycnic gradient centrifugation confirmed the presence of 'angiotensin I immunoreactive material,' but the distribution resembled that of lysosomes rather than renin granules, mitochondria or protein. The material did not possess pressor properties, was stable to incubation at 37 C and was precipitated by ethanol. Incubation of subcellular fractions with peptidase inhibitors known to inhibit the activity of renal angiotensinases (EDTA, diisopropyl phosphorofluoridate and 2,3-dimercaptopropanol) decreased the level of 'angiotensin I immunoreactive material' in kidney fractions treated by radioimmunoassay. By paper chromatography it was apparent that subcellular fractions were capable of degrading 125I-labelled angiotensin I used as tracer in the radioimmunoassay. The degree of degradation followed the distribution of lysosomes among the fractions and was decreased by the angiotensinase inhibitors. The apparent molecular weight of the major portion of angiotensinase activity persisting despite the presence of angiotensinase inhibitors was 75,000, a value similar to that of 'angiotensin immunoreactive material'inkidney fractions treated with non-ionic detergent. On the basis of the present findings it is suggested that 'angiotensin immunoreactive material' may be largely artifactual, beingcreated by the hydrolysis of tracer by angiotensinase enzymes during radioimmunoassay to form fragments that are no longer capable of binding to the specific antibody. This would produced results that would appear the same as those produced had genuine angiotensin immunoreactivity indeed been present in the samples. Such an effect could, in principle, occur in any competitive protein binding assay and should be tested.

Subcellular localization of angiotensin II immunoreactivity in the rat cerebellar cortex.

We localized angiotensin II (Ang II) immunoreactivity in the rat cerebellar cortex with immunogold staining methods. Perfusion fixation with high amounts of glutaraldehyde and the use of cryoultramicrotomy caused remarkable changes in immunostaining versus formaldehyde/picric acid fixation. With the use of monoclonal and polyclonal anti-Ang II, Ang II immunoreactivity was prominent in cerebellar neurons such as Purkinje, granule, basket, and stellate cells. At the subcellular level, the peptide was clearly localized in nuclei, and in some cell types, such as endothelial and granule cells, it was nearly exclusively present in the transcriptionally active euchromatin. Intracellular Ang II immunoreactivity was also detected in vesicle-like structures in cytoplasm and mitochondria and at cell-cell contacts. Additional experiments with liver and adrenal tissue confirmed the nuclear localization of Ang II immunoreactivity, suggesting a role of Ang II in the regulation of gene transcription.

Angiotensin formation in the isolated rat hindlimb.

Local vascular generation of angiotensin was investigated in isolated perfused rat hindquarters. Extraction and combined high-performance liquid chromatography (HPLC)/radioimmunoassay analysis of hindlimb perfusate showed a spontaneous release of angiotensin I (Ang I; 5.0 +/- 3.4 fmol/h) and angiotensin II (Ang II; 31.8 +/- 7.9 fmol/h). Angiotensin converting enzyme (ACE) inhibition with captopril abolished Ang II release while Ang I levels increased more than 10-fold. Perfusion with purified hog renin caused a dose-dependent angiotensin release and vasoconstriction. The renin inhibitor H-142 abolished all effects of renin whereas ACE inhibition prevented Ang II formation and vasoconstriction but increased Ang I levels. Metabolism and pressor effects of synthetic tetradecapeptide renin substrate (TDP), Ang I and Ang II were studied using a recirculating rat hindlimb perfusion system. TDP-dependent formation of Ang I and II, and an increase in perfusion pressure was shown; ACE inhibition reduced but did not abolish Ang II formation and vasoconstriction. Ang I was converted to Ang II by about 50% during one pass through a hindlimb. This conversion was abolished by ACE inhibition. These data add support to the presence of a functional vascular renin-angiotensin system.

Structural features of angiotensin II which are important for biologic activity.

In summary, the aromatic ring of phenylalanine (COOH-terminus) is the key to the intrinsic activity with Tyr4, also involved in activation of a response. The NH2-terminal residue is a determinant of duration of action. The side chains of Tyr4 and His6 are key determinants for affinity, and the amino acids in the No. 1 and No. 2 position also contribute to receptor binding. Val3, Ile5, and Pro7 contain neutral side chains which probably establish and/or help maintain the appropriate distances among the key sidechains of Tyr4, His6, and Phe8. In other words, Val3, Ile5 (or Val), and Pro7 are key determinants of structural conformation.

Angiotensin II immunoreactivity in push-pull perfusates from the paraventricular nucleus.

Push-pull perfusion of the hypothalamic paraventricular nucleus in sodium pentobarbital anesthetized Sprague-Dawley rats indicates the release of angiotensin II-immunoreactive material in this area. Attempts to demonstrate a neuronal origin of this material by chemical depolarization with perfusate containing either 40 or 120 mM K+ were unsuccessful. However, this material does appear to be of central origin since intravenous infusion of arginine-vasopressin, a similar sized peptide, did not result in the appearance of increased levels of this substrate in the perfusate, indicating that the integrity of the blood-brain barrier was not compromised by the perfusion.

Measurement of angiotensin II: use of two injectors to minimize HPLC shadowing.

We investigated the use of two HPLC injectors, one reserved for standards and the other for blanks or biological samples, to minimize shadowing in the measurement of angiotensin II (ANGII). HPLC carryover of standard ANGII to blank with a one-injector and a two-injector system were 47.0 +/- 5.0 and 2.4 +/- 0.5 fmol/ml, respectively, a 19.6-fold reduction. Measured normal canine left ventricular myocardium ANGII level by the two-injector HPLC-RIA system was 22.3 +/- 2.4 fmol/g, with a signal-to-noise ratio of 11.7, an improved signal-to-noise ratio of 29.3 fold vs. the one injector. This innovation reduced the incidence of false-positive ANGII results, and thus can be applied to other compounds that exhibit HPLC-derived shadowing.

Adrenal, kidney, and heart angiotensins in female murine Ren-2 transfected hypertensive rats.

We analyzed by high-performance liquid chromatography and radioimmunoassay angiotensin I (Ang I), Ang II, Ang-(1-7), and metabolites in the adrenal, kidney and heart of normotensive female Sprague-Dawley (SD) and transgenic hypertensive [TGR(mRen-2)27] rats carrying the murine Ren-2d renin gene. The monogenetic model of hypertensive rats had significant increases in adrenal Ang II; whereas in the kidney Ang II was unchanged, but Ang I and Ang-(1-7) were significantly lower. Cardiac Ang I, Ang II, and Ang-(2-10) were significantly reduced in transgenic rats, while Ang-(2-7) was increased. In SD and transgenic rats kidney and adrenal angiotensins increased primarily during estrus or proestrus. In female transgenic rats the increased adrenal Ang II and the sustained renal Ang II may contribute to the established phase of hypertension.

An immunocytochemical comparison of the angiotensin and vasopressin hypothalamo-neurohypophysial systems in normotensive rats.

In the present study we investigated the possibility that angiotensin II/III and vasopressin coexist in the hypothalamo-neurohypophysial pathway. For our experiments 8-week-old male rats not treated with colchicine were used. The anatomical orientation of the entire pathway for angiotensin and vasopressin was facilitated by examining a series of subsequent coronal, horizontal and sagittal sections. Arching fibre tracts are formed mainly by projections emanating from cell bodies in the paraventricular nucleus, the accessory magnocellular nuclei, the supraoptic nucleus and the retrochiasmatic part of the supraoptic nucleus. The majority extend as far as the median eminence and the neurohypophysis, where major terminal fields exist. However, there is a difference between the staining pattern within the suprachiasmatic nucleus and the hypophysis. The results clearly show the colocalization of angiotensin and vasopressin in neurones as well as in fibres of the hypothalamo-neurohypophysial system.