Quantitative determination of the peptide retention of polymeric substrates using matrix-assisted laser desorption/ionization mass spectrometry.
(41/3036)
Polymer surface-peptide binding interactions have been shown previously to lead to reductions in peptide matrix assisted laser desorption/ionization (MALDI) ion signals. In previous studies, increases in surface-peptide binding were characterized by the increases in both the initially adsorbed and retained quantities of 125I-radiolabeled peptides. The present studies establish a specific correlation between the peptide retention properties of the polymer surface and the reduction in the peptide MALDI ion signal. This correlation is demonstrated by obtaining MALDI mass spectra of angiotensin I applied to various polymer surfaces having a range of peptide adsorption and retention properties. In addition, the use of a MALDI based method of standard additions is shown to allow the quantitation of the polymer surface-peptide retention affinity for angiotensin I and porcine insulin. The MALDI standard additions method for measurement of surface-peptide retention affinities offers a number of significant advantages over conventional radiolabeled peptide binding methods and promises to be a valuable tool for the determination of this important biomaterial characteristic. (+info)
Proteome analysis using selective incorporation of isotopically labeled amino acids.
(42/3036)
A method is described for identifying intact proteins from genomic databases using a combination of accurate molecular mass measurements and partial amino acid content. An initial demonstration was conducted for proteins isolated from Escherichia coli (E. coli) using a multiple auxotrophic strain of K12. Proteins extracted from the organism grown in natural isotopic abundance minimal medium and also minimal medium containing isotopically labeled leucine (Leu-D10), were mixed and analyzed by capillary isoelectric focusing (CIEF) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR). The incorporation of the isotopically labeled Leu residue has no effect on the CIEF separation of the protein, therefore both versions of the protein are observed within the same FTICR spectrum. The difference in the molecular mass of the natural isotopic abundance and Leu-D10 isotopically labeled proteins is used to determine the number of Leu residues present in that particular protein. Knowledge of the molecular mass and number of Leu residues present can be used to unambiguously identify the intact protein. Preliminary results show the efficacy of this method for unambiguously identifying proteins isolated from E. coli. (+info)
Approaches to sequence analysis of 125I-labeled RNA.
(43/3036)
A method is described for the initial steps of sequence analysis of RNase T1-and pancreatic RN-ase-resistant oligonucleotides of RNA containing cytidylate residues labeled in vitro with 125I. In many cases an oligonucleotide sequence can be deduced from a consideration of (i) its relative position in the two-dimensional fingerprint (with DEAE thin layer homochromatographic second dimension), (ii) its electrophoretic mobility on DEAE paper at pH 1.9, and (iii) identification of its products of further enzymatic digestion by comparison with a set of marker oligonucleotides. Additional methods including analysis of oligonucleotides following chemical blocking of uridylate residues with CMCT and analysis of products of incomplete enzymatic digestion are also discussed. (+info)
Metabolism and pharmacokinetics, in the rat, of (R)-N-(2-Heptyl)Methyl-propargylamine (R-2HMP), a new potent monoamine oxidase inhibitor and antiapoptotic agent.
(44/3036)
(R)-N-(2-Heptyl)-N-methylpropargylamine (R-2HMP) is a monoamine oxidase inhibitor and putative antiapoptotic agent analogous to (R)-deprenyl. In the rat, the major amine metabolites of R-2HMP have been identified as (R)-N-2-heptylmethylamine (R-2HMA), (R)-N-2-heptylpropargylamine (R-2HPA), and (R)-2-heptylamine (R-2HA). After R-2HMP was administered s.c. to male Wistar rats, it was observed that the greatest concentration was of the original drug followed in decreasing order by R-2HMA, R-2HPA, and R-2HA in brain, liver, and plasma at all times after administration. The greatest concentrations of the three metabolites were found in brain followed by liver and plasma, and the peak concentrations occurred between 15 and 30 min after administration. After oral administration, the liver contained the greatest concentrations of drug and metabolites, and, again, the peak concentrations occurred at about 15 min. In all cases, depropargylation appears to occur at a faster rate than demethylation. After s.c. administration, R-2HMP and its metabolites exhibited biexponential redistribution and elimination losses. Half-lives of the compounds in brain for the redistribution phase were: R-2HMP, 10 min; R-2HMA, 11 min; R-2HPA, 16 min; and R-2HA, 15 min. (+info)
99mTc-labeled vasoactive intestinal peptide analog for rapid localization of tumors in humans.
(45/3036)
In recent years, imaging tumors with receptor-specific biomolecules has been the focus of increasing interest. Vasoactive intestinal peptide (VIP) has a high affinity for specific receptors that are expressed in high density on a large number of malignant tumors. VIP was modified (TP 3654) without compromising its biologic activity and labeled with 99mTc. Pharmacokinetics and feasibility studies were performed in 3 healthy volunteers and 11 patients with a history of cancer. Imaging was performed for up to 2 h after injection. Within 24 h after injection of 99mTc-TP 3654 (370-555 MBq/5 microg), approximately 70% of the tracer cleared through the kidneys and 20% through the liver. Blood clearance was rapid. No adverse reaction was noted in any subject. All known tumors were clearly delineated within 20 min. Findings were compared with the results of 99mTc-methoxyisobutyl isonitrile, CT, MRI, or histology. There was concordance in 9 patients. In the other 2 patients, only the VIP scan was positive for tumors known to express VIP receptors. The early results of imaging tumors with 99mTc-VIP are promising and warrant further study. (+info)
Imaging vascular thrombosis with 99mTc-labeled fibrin alpha-chain peptide.
(46/3036)
An agent that permits scintigraphic detection of chronic deep venous thrombosis (DVT) or pulmonary embolism (PE) would be a welcome addition to the armamentarium of nuclear medicine. Because fibrin is the integral part of each clot, old or fresh, we hypothesized that a 99mTc-labeled fibrin alpha-chain N-terminal peptide, Gly-Pro-Arg-Pro-Pro, that binds to the C-terminal portion of the gamma-chain of fibrin can detect DVT and PE. METHODS: The peptide was modified to Gly-Pro-Arg-Pro-Pro-Aba-Gly-Gly-(D)-Ala-Gly to permit efficient binding of 99mTc (99mTc-TP 850). The stability of the peptide was examined in vitro as well as in vivo. The ability of the agent to bind to rabbit, dog, and human fibrin and to inhibit adenosine diphosphate-induced platelet aggregation was examined. Blood clearance and 3-h tissue distribution were studied. DVT was induced in 8 rabbits using a stimulating electrode and in 2 rabbits by inserting a thrombin-soaked suture. PE was induced in 6 additional rabbits by introducing tantalum-impregnated blood clots into the right atrium, and the rabbits were radiographed to locate the emboli. 99mTc-TP 850 was then injected through a lateral ear vein, and each rabbit was imaged for up to 3 h. The rabbits were then killed, the heart and lungs were dissected and radiographed and the clots were harvested so that clot-to-blood radioactivity ratios could be determined. RESULTS: The peptide analog permitted efficient incorporation of 99mTc, which was stable in vitro and in vivo. The blood clearance was biphasic, with an alpha phase half-life of approximately 4 min (20%) and a beta phase half-life of approximately 13 min (88%). The mean binding of 99mTc-TP 850 to human, dog, and rabbit fibrin was 46% +/- 2%, 60% +/- 3%, and 56% +/- 2.5%, respectively, and the inhibitory concentration of 50% for dog and rabbit platelet aggregation was 236 pm and 167 pm, respectively. All clots, including 24-h-old pulmonary emboli, were delineated. The radioactivity associated with clots varied from 0.01 to 0.09 %ID/g, with clot-to-blood radioactivity ratios ranging from 1.2 to 12.0. However, 48-h-old pulmonary emboli had lysed and were seen neither by radiography nor by scintigraphy. CONCLUSION: A fibrin alpha-chain, N-terminal peptide that binds to the C-terminal portion of the gamma-chain of fibrin has been modified and labeled with 99mTc. The resultant peptide is stable in vitro and in vivo; binds to human, dog, and rabbit fibrin in large quantities; and inhibits platelet aggregation. The peptide clears rapidly from the blood and delineates experimental DVT and PE in rabbits. This agent is worthy of further investigation. (+info)
Pharmacokinetics and renal handling of 99mTc-labeled peptides.
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99mTc-labeled peptides, particularly those of a lipophilic nature, are often excreted through the hepatobiliary system, and the subsequent accumulation in the intestine may obscure receptor-mediated uptake in tumor sites in the pelvis. We have therefore explored the route and rate of excretion of a small series of Tc-labeled peptides to shed some light on the mechanisms that influence the clearance of these agents. METHODS: Pharmacokinetic parameters, biodistribution, routes of elimination of 99mTc-complexes of 3 model tetrapeptides--namely, acetyl-N-Gly-Gly-Cys-Gly (AGGCG), acetyl-N-Ser-Ser-Cys-Gly (ASSCG), and acetyl-N-Gly-Gly-Cys-Lys (AGGCL)--were determined in rats in vivo. Renal handling of the complexes was studied in the perfused rat kidney. RESULTS: After intravenous injection, a relatively fast disappearance of the complexes from blood was found. Although the parameters of distribution in all 3 chelates were very similar, the elimination rate of 99mTc-AGGCG was higher than those of 99mTc-ASSCG and 99mTc-AGGCL. The Tc complexes under study were distributed mainly to the excretory organs (kidneys and liver), and no specific accumulation in other organs or tissues was found. Most of the radioactivity after intravenous administration of the chelates was rapidly eliminated through the urine, but a significant amount was also excreted through the feces, in the following order among the 3 chelates: 99mTc-AGGCL < 99mTc-ASSCG < 99mTc-AGGCG. Different proportions of glomerular filtration and secretion in renal tubules of the complexes were found in the perfused rat kidney. Elimination by glomerular filtration was dominant only in the case of 99mTc-AGGCL, whereas the rate of filtration of 99mTc-AGGCG was very low because of its high protein binding. Various rates of secretion into renal tubules were shown for all 3 agents. This renal excretion pathway was decisive in 99mTc-AGGCG and lowest in 99mTc-AGGCL. 99mTc-ASSCG was eliminated by both mechanisms at similar rates. CONCLUSION: These studies show that increasing the hydrophilic nature or reducing the negative charge of the peptides will reduce their hepatobiliary excretion, whereas the incorporation of suitable peptide sequences permits them to exploit efficient routes of renal excretion, such as tubular secretion, thereby optimizing the pattern of biodistribution of these radiopharmaceuticals. (+info)
Comparison of estimates of zinc absorption in humans by using 4 stable isotopic tracer methods and compartmental analysis.
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BACKGROUND: Adjustment of gastrointestinal absorption is the primary means of maintaining zinc homeostasis; however, a precise, accurate method for measuring zinc absorption in humans has not been identified. OBJECTIVE: The purpose of this study was to compare the estimates of the fraction of dietary zinc absorbed (FZA) by using 4 stable isotopic tracer methods: mass balance (MB) corrected for endogenous secretion, fecal monitoring (FM), deconvolution analysis (DA), and the double isotopic tracer ratio (DITR) method. DESIGN: All 4 methods were applied to a single data set for each of 6 women. FZA was also determined for each subject by using a detailed compartmental model of zinc metabolism, and that value was used as the reference with which the simpler methods were compared. RESULTS: The estimates of FZA (&xmacr; +/- SD) determined by DA (0.27 +/- 0. 08) and the DITR technique in plasma (0.30 +/- 0.10), 24-h urine samples (0.29 +/- 0.09), and spot urine samples (0.291 +/- 0.089) all compared well with the FZA reference value from the compartmental model (0.30 +/- 0.10). The MB and FM methods tended to overestimate FZA compared with the reference value. CONCLUSIONS: The determination of FZA by MB or FM is laborious, is sensitive to subject compliance, and may result in an overestimate. DA, although relatively accurate, has the disadvantage of requiring multiple blood drawings over several days. In contrast, the DITR technique applied to a spot urine specimen obtained >/=3 d after tracer administration provides an accurate measure of FZA and is easy to implement; therefore, it is the recommended method for determination of FZA. (+info)