Determination of amino acids in foods by reversed-phase HPLC with new precolumn derivatives,butylthiocarbamyl, and benzylthiocarbamyl derivatives compared to the phenylthiocarbamyl derivative and ion exchange chromatography

New precolumn derivatizing reagents for analysis of amino acids by HPLC—butylisothiocyanate (BITC) and benzylisothiocyanate (BZITC)—reacted quantitatively with 22 standard amino acids and the amino acids in the acid hydrolysate of food and protein standard, bovine serum albumin (BSA), at 40°C for 30 min to yield butylthiocarbamyl (BTC) amino acids and at 50°C for 30 min to yield benzylthiocarbammyl (BZTC) amino acids. BTC and BZTC amino acids were successfully separated in 35 min on the reversed-phase Nova-Pak C18 column (30 cm × 3.9 mm, 4 μm). The optimum wavelengths for determination of BTC and BZTC derivatives were 240 nm and 246 nm, respectively. Analysis of the results obtained with BSA and food samples as BTC and BZTC derivatives showed good agreement with those determined as ion-exchange chromatography and data presented in the literature. The advantage of BITC reagent over the phenylisothiocyanate (PITC) and BZITC was that it had high volatility, so the excess reagent and by-products were easily removed in about 10 min, compared to about 1 h in the PITC and BZITC reagents. In the BTC and BZTC derivatives, cystine and cysteine were determined separately, but in the PTC amino acids derivatized with PITC reagent they were resolved into single peak.     

On the synthesis of a 32P-labelled Edman reagent for the sensitive identification of amino-acid derivatives

A phosphorus-32 containing derivative of phenylisothiocyanate was prepared to increase the sensitivity of amino-acid sequence determination. The respective compound 2-(4-isothiocyanatophenoxy)-1,3,2-dioxaphosphinane 2-oxide showed about the same reactivity, stability, and polarity as the Edman reagent itself. A repetitive yield of 94% was obtained in the stepwise degradation of insulin B chain using a solid phase sequencer. The synthesis of this radioactive reagent was achieved within 5 h but with a specific activity of 1 Ci/mol. Eight amino acids were reacted with the 32P-labelled reagent and identified by autoradiography after two dimensional thin-layer chromatography.     

A new condensing reagent, 1-(2,4,6-triisopropylbenzenesulfonyl)-5-(pyridin-2-yl)tetrazolide and its use in the synthesis of lambda cro binding heptadecanucleotide on a polymer support.

A new condensing reagent 1-(2,4,6,-triisopropylbenzenesulfonyl)-5-(pyridin-2-yl)tetrazolide (TPSPy) was found to give one diastereoisomer of dinucleoside monophosphate aryl esters. Several oligodeoxynucleotide blocks were prepared using this reagent. A heptadecanucleotide, dTATCCCTTGCGGTGATA, which had the same sequence as the lambda cro binding DNA sequence was synthesized by condensing mono-, tri- and dodecanucleotide blocks using this reagent on a polystyrene support.     

Effect of naturally occurring isothiocyanates in the inhibition of cyclophosphamide-induced urotoxicity.

Cyclophosphamide-induced urotoxiciy was reduced in Swiss albino mice by the treatment of naturally occurring isothiocyanates such as AITC or PITC (25 microg/dose/animal, i.p.) for 5 days along with CTX (1.5 mmol/kg body wt.; i.p.). Severely inflamed and dark coloured urinary bladders of the CTX alone treated animals were found to be normalized on morphological analysis by the treatment of AITC or PITC. Urine protein levels were reduced by the treatment with AITC (6.2 +/- 0.37 g/l) and PITC (6.56 +/- 1.56 g/l), which was elevated by CTX administration (8.66 +/- 0.47 g/l). Urine urea N2 that was enhanced significantly by CTX administration (26.87 +/- 1.86 g/l) was reduced by treatment with both AITC (17.38 +/- 0.06 g/l) and PITC (15.85 +/- 1.56 g/l). GSH content, which was drastically reduced by CTX administration in both bladder (0.87 +/- 0.1 nmol/mg protein) and liver (2.47 +/- 0.6 nmol/mg protein) was enhanced by treatment with AITC and PITC both in bladder (AITC- 3.65 +/- 0.18 nmol/mg protein; PITC- 2.8 +/- 0.15 nmol/mg protein) and in liver (AITC- 4.10 +/- 0.81 nmol/mg protein; PITC- 4.70 +/- 0.44 nmol/mg protein). Histopathology of the bladders of CTX alone treated group showed severe necrosis of the tissue whereas AITC and PITC treated group showed normal bladder pathology.     

Modification of bovine pancreatic ribonuclease A with pyridoxal 5'-phosphate. Isolation and identification of derivatives.

Bovine pancreatic ribonuclease A was allowed to react with pyridoxal 5'-phosphate at pH 8 and 4 degrees. After reduction with sodium borohydride, the principal products formed in the initial stages of modification were separated by successive chromatography on CM-cellulose and SP-Sephadex. The isolated derivatives were identified as Nalpha-(P-pyridoxyl)-Lys-1-,Nepsilon-(P-pyridoxyl)-Lys-7-, and Nepsilon-(P-pyridoxyl)-Lys-41-ribonuclease A. These results are interpreted in terms of the specificity of pyridoxal-P as a protein reagent.     

Technetium labeling of monoclonal antibodies with functionalized BATOs. 1. TcCl(DMG)3PITC

BATO (boronic acid adduct of technetium dioximes) complexes, TcCl(dioxime)3BR, were prepared in which the boron substituent (R) was the protein-reactive m-phenyl isothiocyanate (PITC). The 99TcCl(dioxime)3PITC complexes [dioxime = dimethylglyoxime (DMG) or cyclohexanedione dioxime (CDO)] were prepared from 99Tc(dioxime)3(mu-OH)SnCl3 and characterized. The X-ray crystal structure of 99TcCl(DMG)3PITC was determined. The 99mTc complexes were prepared from 99mTcO4- in a process using a freeze-dried kit, either in a one-step procedure or via 99mTcCl(dioxime)3. Initial labeling studies with 99mTcCl(dioxime)3PITC were performed on glycine and polylysine and, subsequently, on mouse IgG and the B72.3 monoclonal antibody. Covalent attachment of 99mTcCl(DMG)3PITC to B72.3 was demonstrated by SDS-PAGE electrophoresis. B72.3 labeled with 99mTcCl(DMG)3PITC displayed high binding to a TAG 72 affinity column and had a distribution in normal mice similar to that reported for iodine-labeled B72.3.     

Fluorometric determination of guanidino compounds by new postcolumn derivatization system using reversed-phase ion-pair high-performance liquid chromatography

A new postcolumn derivatization system using 1,2-naphthoquinone-4-sulfonate as fluorogenic reagent for the fluorometric determination of guanidino compounds is described. The guanidino compounds were separated by reversed-phase ion-pair high-performance liquid chromatography with an isocratic mobile phase containing the fluorogenic reagent and octane-sulfonate as the counterion. Fluorophors were derived from a condensation of guanidino compounds with the fluorogenic reagent in an alkaline solution. The chromatographic system using the mobile phase containing the fluorogenic reagent was simplified because only two pumps were required to deliver the mobile phase and the alkaline solution. Separation of guanidino compounds was completed within 25 min using a Nucleosil C8 column (5 microns, 15 cm X 4.6 mm i.d.). This method was applied to serum obtained from patients on hemodialysis therapy.     

Microquantitative determination of Pi-ATP and ADP-ATP exchange kinetics using thin-layer chromatography on silica gel

A new method for determination of 32Pi-ATP and [14C]ADP-ATP exchange rates is described. It is based upon separation of nucleotides and Pi by thin-layer chromatography on commercial aluminum or plastic sheets precoated with silica gel. The method permits avoiding special procedures for stopping of the reaction and for preparation of aliquots for thin-layer chromatography separation. It also allows to separate all adenine nucleotides and Pi in one chromatography procedure, and to work with a double label. The volume of the reaction mixture was 50-100 microliters. Aliquots (2-6 microliters) of the reaction mixture were taken at various moments without stopping the reaction and were layered immediately on a heated silica gel sheets. The nucleotides and Pi were separated in a solvent system which consisted of dioxane, isopropanol, 25% ammonia, and water (4:2:3:4, v/v). The nucleotide spots were detected in ultraviolet light, cut out, and their radioactivity was measured with a liquid scintillation counter. The method for measurement of kinetics of exchange reactions catalyzed by reconstituted into liposomes H+-ATPase complexes from beef heart mitochondria and high plant chloroplasts, is described.     

Anti-HBs antibodies from rabbits and their use as reagents for serological tests in the diagnosis of hepatitis B

By the method of affinity chromatography a partially purified antigen was obtained after passing the plasma of an asymptomatic carrier of HBsAg through a column of Sepharose 4B linked to angi-HBs. This antigen was inoculated in rabbits using a schedule of 1,0 mg in the first dose and 4 other doses of 0,5 mg with intervals of approximately 15 days. Observing that blood samples collected after the 5th inoculation showed no change in antibody levels, the animals were bled on the 62th day and these immune sera were standardized with the following tests for the detection of HBsAg: Reverse passive hemagglutination (R-PHA) - using specific gamma globulin that was obtained from rabbit sera by affinity chromatography and reaching an optimal concentration of 10 micrograms/ml to sensitise SRBC at 5% fixed in glutaraldehyde. Counter immuno electrophoresis (CIEP) - using the rabbit immune sera diluted to 1/20 as a reagent for the detection of HBsAg. The immune sera was also used to conjugate new Sepharose 4B for affinity chromatography and was found having a linking capacity of approximately 0,5 to 1,0 mg of HBsAg per ml of Sepharose after complete saturation.     

3-([3-125I]iodo-4-hydroxyphenyl)propionyl carbohydrazide,a new radioiodination reagent for ultrasensitive detection and determination of periodate-oxidized nucleoside derivatives and other carbonyl compounds

A new radioiodination reagent for the identification and quantitation of periodate-oxidized ribonucleosides was developed. The reagent, 3-([3-¹²⁵I]iodo-4-hydroxyphenyl)propionyl carbohydrazide, was prepared by radioiodination of 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester in the presence of chloramine T, followed by reduction of the latter with sodium arsenite and treatment of the radioiodinated ester with an excess of carbohydrazide. The reagent reacted quantitatively with periodate-oxidized nucleosides to form ¹²⁵I-labeled morpholine derivatives which were separated by thin-layer chromatography and quantitated by liquid scintillation counting. The reagent was found to react also with other carbonyl compounds and thus may find more general application in the qualitative and quantitative ultramicroanalysis of aldehydes and ketones.     

Provider-Initiated HIV Testing and Counselling in Rwanda: Acceptability among Clinic Attendees and Workers,Reasons for Testing and Predictors of Testing

Introduction

Routine provider-initiated HIV testing and counselling (PITC) may increase HIV testing rates, but whether PITC is acceptable to health facility (HF) attendees is unclear. In the course of a PITC intervention study in Rwanda, we assessed the acceptability of PITC, reasons for being or not being tested and factors associated with HIV testing.

Methods

Attendees were systematically interviewed in March 2009 as they left the HF, regarding knowledge and acceptability of PITC, history of testing and reasons for being tested or not. Subsequently, PITC was introduced in 6 of the 8 HFs and a second round of interviews was conducted. Independent factors associated with testing were analysed using logistic regression. Randomly selected health care workers (HCWs) were also interviewed.

Results

1772 attendees were interviewed. Over 95% agreed with the PITC policy, both prior to and after implementation of PITC policy. The most common reasons for testing were the desire to know one’s HIV status and having been offered an HIV test by an HCW. The most frequent reasons for not being tested were known HIV status and test not being offered. In multivariable analysis, PITC, age ≥15 years, and not having been previously tested were factors significantly associated with testing. Although workload was increased by PITC, HIV testing rates increased and HCWs overwhelmingly supported the policy.

Conclusion

Among attendees and HCWs in Rwandan clinics, the acceptability of PITC was very high. PITC appeared to increase testing rates and may be helpful in prevention and early access to treatment.     

Location of Chemically Modified Lysine 41 in the Structure of Bacteriorhodopsin by Neutron Diffraction

Purple membranes were prepared in which bacteriorhodopsin was labeled at lysine 41 with phenylisothiocyanate (PITC) and with perdeuterated PITC. The in-plane position of this small label containing only five deuterons was determined from the differences between the neutron diffraction intensities of the two samples. At 8.7-Å resolution the Fourier difference map revealed a well-defined site between helices 3 and 4. This position was confirmed by a refinement procedure in reciprocal space. Model calculations showed that the observed difference density had the right amplitude for the label. Thus it is possible to locate a small group in a large protein structure by replacing as few as five hydrogens by deuterium. The observed location of PITC restricts the number of possibilities for the assignment of helix B in the sequence (to which lysine 41 is attached) to one of the seven helices of the structure. Taking into account the size of the label and the length of the lysine side chain our result excludes helices 1, 2, and 7 as candidates for B.     

Phenylisothiocyanate and dansyl chloride precolumn derivatizations for the high-performance liquid chromatography analysis of the antitumoral agent ES-285 in dog plasma

Chromophor and fluorophor addition reactions involving phenylisothiocyanate (PITC) and dansyl chloride (DC) were optimized to adapt two high-performance liquid chromatography (HPLC) procedures designed for the accurate determination of the novel antitumoral agent ES-285 in Beagle dog plasma. ES-285 was reacted with PITC at 60 degrees C for 15 min in the presence of triethylamine. The dansyl derivative was obtained by reaction of ES-285 with dansyl chloride in a basic medium at 80 degrees C for 20 min. Both reactions also worked for ES-299, a compound structurally related to ES-285 which was used as internal standard. The treatment of ES-285 and ES-299 spiked plasma samples with a basic phosphate buffer and MeOH permitted the extraction of the drug from the matrix. The purification of the analytes was carried out by solid-phase extraction followed by precolumn derivatization with PITC and DC. The phenylisothiocyanate adducts were analyzed by isocratic HPLC with UV detection at 254 nm. The ES-285 and ES-299 derivatives were eluted from a C(18) column at approximately 6.9 and approximately 8.1 min, respectively. The eluent ACN-water (95:5, v/v) was delivered to the column at a flow-rate of 1 ml/min and the analysis was completed in 15 min. The dansyl derivatives were analysed by a two-HPLC column system with fluorescence detection and gradient elution. The column temperature was maintained at 40 degrees C. The analysis lasted 55 min with the elution of ES-285 and ES-299 derivatives at approximately 35.2 and approximately 37.9 min, respectively. The PITC- and DC-based procedures were characterized by limits of quantification of 20 and 15 ng/ml, respectively. Both procedures were validated according to the ICH and FDA guidelines. They were selective for ES-285 and provided accurate, precise and reproducible results. ES-299 was shown to be a suitable internal standard. The HPLC procedure involving derivatization with DC was more sensitive and permitted to process plasma sample volumes as low as 100 microl. On the other hand, the PITC-based procedure characterised by a quite similar LOQ permitted a higher throughput but implied the processing of a 500-microl plasma volume.     

Manual N-terminal microsequencing of proteins electroeluted from polyacrylamide gel slices

A simple and rapid procedure for preparation of proteins for manual microsequencing using sodium dodecyl sulfate gel electrophoresis is described. The procedure involves pre-electrophoretic labeling of the protein amino groups with a coloured Edman reagent, disk electrophoresis for purification or fractionation of the proteins, and reversed electrophoretic transfer of the separated protein from gel slices into a small volume of buffer (100 to 150 microliter) using a discontinuous conductivity gradient to recover the proteins. The pre-electrophoretic labeling facilitates location of the separated proteins in the gel and the monitoring of their complete electroelution. The isolated proteins are separated from excess of salts by acetone precipitation and solvent partitioning in pyridine/water (1:1) and subjected to manual DABITC/PITC degradation.     

Isolation and characterization of a C12-lipopeptide produced by Bacillus subtilis HSO 121.

A new lipopeptide with C12 fatty acid has been isolated from the cell broth of Bacillus subtilis HSO121 by chromatographic methods, which is believed to be the homologue of lipopeptides. The fatty acid portion was methylated and analyzed by GC/MS, ESI Q-TOF MS and 1H-NMR. The peptide portion, of which the amino acid composition was obtained by HPLC combined with a phenyl isothiocyanate (PITC) derivatization methods, was analyzed by ESI Q-TOF MS. Comparing the obtained results with surfactin C13 showed that the new lipopeptide has a peptide moiety similar to that of surfactin and the difference exists in the fatty acid portion, which is an iso-C12 beta-hydroxy fatty acid. The critical micelle concentration (CMC) of this new homologue is estimated to be 6.27 x 10(-5) mol/l in 10 mmol/l phosphate buffer solution (PBS, pH 8.0) at 30 degrees C, and the surface tension at CMC (gamma CMC) achieved is as little as 27.71 mN/m. The hemolytic activities of the C12-lipopeptide on 2% human erythrocytes showed a HC50 of 26.5 micromol/l.     

N-(3-(p-azido-m-[125I]iodophenyl)propionyl)-succinimide--a heterobifunctional reagent for the synthesis of radioactive photoaffinity ligands: synthesis of a carrier-free 125I-labeled cardiac glycoside photoaffinity label

A new heterobifunctional reagent, N-(3-(p-azido-m-iodophenyl)propionyl)-succinimide (AIPPS), was synthesized and chemically characterized. The radiochemical form of the reagent, [125I]AIPPS, should be of general use as a photoactive reagent for the derivatization of free amino groups on a large variety of biologically active compounds, including many hormones. Amino-containing ligands can be derivatized with [125I]AIPPS in a method which is similar to that used for the 125I-labeled Bolton-Hunter reagent (N-(3-(p-hydroxyphenyl)propionyl)-succinimide). The added advantage with [125I]AIPPS, however, is that the ligand derivative is made both photoactive and radioactive in a single step. As an example of how this reagent can be used, we have prepared carrier-free [125I]AIPPS and reacted it with the amino-containing cardiac glycoside, 4-amino-4,6-dideoxyglucosyl digitoxigenin (GluD). The radioiodinated cardiac glycoside, [125I]AIPP-GluD, was purified by thin-layer chromatography and was carrier-free with a specific radioactivity of 2175 Ci/mmol. [125I]AIPP-GluD was an effective photoaffinity label for Na,K-ATPase as shown by specific photoaffinity labeling of purified canine kidney enzyme and human erythrocyte enzyme.     

Isolation of the glutamyl peptide labeled by the nucleotide analogue 2-(4-bromo-2,3-dioxobutylthio)-1,N(6)-ethenoadenosine 2',5'-biphosphate in the active site of NADP+-specific isocitrate dehydrogenase

2-(4-Bromo-2,3-dioxobutylthio)-1,N(6)-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) is an affinity label for the coenzyme-binding site of pig heart NADP+-dependent isocitrate dehydrogenase. Specific reaction occurs at the coenzyme site with an incorporation of 0.5 mol of reagent/mol of enzyme subunit (i.e. modification of only one subunit of the dimeric enzyme) (Bailey, J.M., and Colman, R.F. (1985) Biochemistry 24, 5367-5377). Modified enzyme, prepared by incubating 1 mg/ml isocitrate dehydrogenase with 75 microM 2-BDB-T epsilon A-2',5'-DP in the absence and presence of substrate or coenzyme, was reduced with NaBH4, carboxymethylated, and digested with trypsin. Nucleotidyl peptides were isolated by chromatography on DEAE-cellulose, followed by treatment with acid phosphatase (to decrease the negative charge by removing the phosphate groups from covalently bound reagent) and rechromatography on the same DEAE-cellulose column. The isolated peptides were characterized by amino acid analysis, dansylation, and gas-phase sequencing. A single triskaidekapeptide corresponding to modification of the coenzyme site by 2-BDB-T epsilon A-2',5'-DP was identified as: Asp-Leu-Ala-Gly-X-Ile-His-Gly-Leu-Ser-Asn-Val-Lys. Additional evidence indicated that X is a glutamate residue derivatized by 2-BDB-T epsilon A-2',5'-DP.     

Pharmacokinetic study of three cardiovascular drugs by high-performance liquid chromatography using pre-column derivatization with 9,10-anthraquinone-2-sulfonyl chloride

A new method was developed to analyze three cardiovascular drugs in rat plasma, Mexiletine hydrochloride (MXL), Methoxamine hydrochloride (MTX), and Metaraminol bitartrate (MTR), by high-performance liquid chromatography (HPLC) using 9,10-anthraquinone-2-sulfonyl chloride (ASC) as the derivatization reagent. The derivatization modes and conditions for this method were optimized. The quantitative analysis was achieved using a C18 column at room temperature (25 degrees C), with various volume ratios of methanol-water as the mobile phase and a detection wavelength at 256 nm. Analytical linearity was obtained for the method over the concentration range of 0.04-8.0 microg mL(-1) for all the three drugs. The lower limit of quantification (LLOQ) was 0.04 microg mL(-1). This method was successfully applied to the analysis of the three drugs in rat plasma and their pharmacokinetic studies. The t1/2 values of the three drugs in rats were found to be 5.38+/-0.61, 4.49+/-0.53, and 3.70+/-0.19 h for MXL, MTX, and MTR, respectively.     

[125I]diiodofluorescein isothiocyanate: Its synthesis and use as a reagent for labeling proteins and cells to high specific radioactivity

We have synthesized a radioactive derivative of fluorescein isothiocyanate (PITC) by lactoperoxidase-catalyzed iodination of fluorescein amine using ¹²⁵I. The iodinated amine was purified by thin-layer chromatography and converted to the isothiocyanate by reaction with thiophosgene. The product was inferred to be the diiodo derivative of FITC by comparing its absorbance and fluorescence emission spectra with those of known standards. This reagent, [¹²⁵I]diI-FITC, shares many of the useful features of its congener, FITC. Specifically, it may be used to label under mild conditions of temperature and pH, and it is chemically stable. When erythrocytes were labeled with [¹²⁵I]diI-FITC, radioactivity was found principally in a major exposed protein of the cell surface, and very little hemoglobin was labeled. [¹²⁵I]diI-FITC may prove generally useful as a means of labeling proteins and cell surfaces to high specific radioactivity.     

Synthesis of a reagent for fluorescence-labeling of vitamin D and its use in assaying vitamin D metabolites

The fluorogenic dienophile 1,2,4-triazoline-3,5-dione with a highly fluorescent quinoxalinone group at the 4-position (DMEQ-TAD) was synthesized and exploited as a reagent to assay vitamin D metabolites. 25-Hydroxyvitamin D3, 1 alpha,25-dihydroxyvitamin D3, and 24(R),25-dihydroxyvitamin D3 reacted quantitatively with DMEQ-TAD when the two substrates were mixed in dichloromethane at room temperature to yield the corresponding 6,19-cycloadduct. The reaction was very fast so that 1 alpha,25-dihydroxyvitamin D3 at a concentration as low as 10(-8) M could be quantitatively labeled with the fluorescent reagent within 30 min at room temperature. With this reagent, down to 10 fmol of vitamin D metabolites could be quantified linearly. The detection limit of the labeled vitamin D using high-performance liquid chromatography was usually about 1 fmol. Thus, it was shown in a model system that the fluorometric method using the new reagent (DMEQ-TAD) can be applied to the assay of the three major vitamin D metabolites in 1 ml of plasma. This is the first practical fluorometric method for assaying the active vitamin D metabolite.