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Biomedical subjects

N A Guzman

Publications and source records attributed to N A Guzman.

At least 19 recordsLinked to original sources
PubMed2001

The use of selective adsorbents in capillary electrophoresis-mass spectrometry for analyte preconcentration and microreactions: a powerful three-dimensional tool for multiple chemical and biological applications.

Much attention has recently been directed to the development and application of online sample preconcentration and microreactions in capillary electrophoresis using selective adsorbents based on chemical or biological specificity. The basic principle involves two interacting chemical or biological systems with high selectivity and affinity for each other. These molecular interactions in nature usually involve noncovalent and reversible chemical processes. Properly bound to a solid support, an "affinity ligand" can selectively adsorb a "target analyte" found in a simple or complex mixture at a wide range of concentrations. As a result, the isolated analyte is enriched and highly purified. When this affinity technique, allowing noncovalent chemical interactions and biochemical reactions to occur, is coupled on-line to high-resolution capillary electrophoresis and mass spectrometry, a powerful tool of chemical and biological information is created. This paper describes the concept of biological recognition and affinity interaction on-line with high-resolution separation, the fabrication of an "analyte concentrator-microreactor", optimization conditions of adsorption and desorption, the coupling to mass spectrometry, and various applications of clinical and pharmaceutical interest.

Absorption↗
PubMed2000

Determination of immunoreactive gonadotropin-releasing hormone in serum and urine by on-line immunoaffinity capillary electrophoresis coupled to mass spectrometry.

The need for urgent diagnoses has propelled the development of automated analyses that can be performed in a short time at reasonable cost. One such method is immunoaffinity capillary electrophoresis. This emerging hybrid technology employs two powerful techniques coupled on-line for the direct and rapid determination of analytes present in biological fluids. The first technique, immunoaffinity, is used for the selective extraction of a molecule present in a complex matrix, utilizing a microscale-format chamber affinity device. An analyte (affinity target) present in serum or urine is captured by an immobilized molecular recognition antibody molecule (affinity ligand) bound to a solid support constituent (glass beads or an appropriate porous structure) of a microchamber affinity device. The second technique, capillary electrophoresis, is used for the high-resolution analytical separation of the purified and concentrated affinity target material after elution from the microchamber affinity device. In this work, immunoaffinity capillary electrophoresis was developed for the identification and characterization of a single constituent of a complex matrix. Immunoreactive gonadotropin-releasing hormone was determined in serum and urine specimens derived from a normal individual and from a patient suffering from benign prostatic hyperplasia. Furthermore, the on-line immuno-separation system was coupled in tandem to mass spectrometry to obtain molecular mass information of the affinity isolated and CE separated neuropeptide. This hybrid immuno-analytical technology is simple, rapid, selective and sensitive. In addition, an attempt was also made to characterize other urinary constituents by CE-MS that may lead to marker activity in the urine of the diseased subject. The hyphenation of analytical techniques has proved valuable in enhancing their individual features. The future of bioanalysis using miniaturized affinity systems is discussed in this paper.

Electrophoresis, Capillary↗
PubMed1998

Affinity capillary electrophoresis: important application areas and some recent developments.

Affinity capillary electrophoresis (ACE) is a broad term referring to the separation by capillary electrophoresis of substances that participate in specific or non-specific affinity interactions during electrophoresis. The interacting molecules can be found free in solution or can be immobilized to a solid support. Every ACE mode has advantages and disadvantages. Each can be used for a wide variety of applications. This paper focuses on applications that include purification and concentration of analytes present in diluted solutions or complex matrices, quantitation of analytes based on calibration curves, and estimation of binding constants from direct and derived binding curves based on quantitation of analytes or on analyte migration shifts. A more recent chemicoaffinity strategy in capillary electrophoresis/capillary electrochromatography (CE/CEC) termed molecular imprinting ('plastic antibodies') is discussed as well. Although most ACE studies are aimed at characterizing small-molecular mass analytes such as drugs, hormones, and peptides, some efforts have been pursued to characterize larger biopolymers including proteins, such as immunoglobulins. Examples of affinity interactions that have been studied are antigen-antibody, hapten-antibody, lectin-sugar, drug-protein, and enzyme-substrate complexes using ultraviolet, laser-induced fluorescence, and mass spectrometer detectors. This paper also addresses the critical issue of background electrolyte selection and quantitation of analytes. Specific examples of bioaffinity applications are presented, and the future of ACE in the biomedical field is discussed.

Electrophoresis, Capillary↗
PubMed1997

New approaches in clinical chemistry: on-line analyte concentration and microreaction capillary electrophoresis for the determination of drugs, metabolic intermediates, and biopolymers in biological fluids.

The use of capillary electrophoresis (CE) for clinically relevant assays is attractive since it often presents many advantages over contemporary methods. The small-diameter tubing that holds the separation medium has led to the development of multicapillary instruments, and simultaneous sample analysis. Furthermore, CE is compatible with a wide range of detectors, including UV-Vis, fluorescence, laser-induced fluorescence, electrochemistry, mass spectrometry, radiometric, and more recently nuclear magnetic resonance, and laser-induced circular dichroism systems. Selection of an appropriate detector can yield highly specific analyte detection with good mass sensitivity. Another attractive feature of CE is the low consumption of sample and reagents. However, it is paradoxical that this advantage also leads to severe limitation, namely poor concentration sensitivity. Often high analyte concentrations are required in order to have injection of sufficient material for detection. In this regard, a series of devices that are broadly termed 'analyte concentrators' have been developed for analyte preconcentration on-line with the CE capillary. These devices have been used primarily for non-specific analyte preconcentration using packing material of the C18 type. Alternatively, the use of very specific antibody-containing cartridges and enzyme-immobilized microreactors have been demonstrated. In the current report, we review the likely impact of the technology of capillary electrophoresis and the role of the CE analyte concentrator-microreactor on the analysis of biomolecules, present on complex matrices, in a clinical laboratory. Specific examples of the direct analysis of physiologically-derived fluids and microdialysates are presented, and a personal view of the future of CE in the clinical environment is given.

Biopolymers↗
PubMed1995

Degradation of lyophilized and reconstituted MACROSCINT (DTPA-IgG): precipitation vs. glucosylation.

Diethylenetriaminepentaacetic anhydride (DTPA) conjugated to IgG (DTPA-IgG) and labeled with 111In is useful for detecting focal sites of infection and inflammation (R.H. Rubin, A.J. Fischman, R.J. Callahan, B. Khaw, F. Keech, M. Ahmad, R. Wilkinson and H.W. Strauss, 111In-labeled nonspecific immunoglobulin scanning in the detection of focal infection, N. Engl. J. Med., 321 (1989) 935-940). MACROSCINT contains DTPA-IgG formulated as a lyophile from a citrate buffer containing maltose. Exposure of both reconstituted and lyophilized MACROSCINT to intense light resulted in degradation primarily via formation of precipitating aggregates. However, lyophilized and reconstituted MACROSCINT responded differently to thermal stress. Reconstituted MACROSCINT subjected to thermal stress (65 degrees C) also degraded through formation of precipitating aggregates. In contrast, exposure of lyophilized MACROSCINT to thermal stress (65 degrees C) resulted primarily in an increase in the molecular size of the MACROSCINT DTPA-IgG monomer. This increase in molecular size was a function of both the moisture content in the vial and the amount of time for which the sample was stressed, but was not a function of the conjugation with DTPA. Monosaccharide analysis of the samples demonstrated that this increase in molecular size corresponded to an increase in the amount of glucose covalently attached to the IgG. These data suggest that the increase in molecular size as a function of thermal stress is due to the covalent attachment of maltose, which is a glucose disaccharide present in the lyophile as an excipient, to the IgG. This degradation pathway was only observed in the lyophile.

Chemical Precipitation↗
PubMed1992

Effect of buffer constituents on the determination of therapeutic proteins by capillary electrophoresis.

Capillary electrophoresis has proved to be a versatile method for the determination of proteins, peptides and amino acids in pharmaceutical formulations. For quantification of the capillary electrophoresis data, however, significant errors may result if the analysis is performed using improper separation conditions. The peak area response for protein analytes, which is generally low in conventional UV detection, may also vary dramatically depending on the nature of the buffer used in the separation. This paper describes the effects of various buffer constituents and analytical conditions on the capillary electrophoretic separation and quantification of a humanized monoclonal antibody in bulk form and in a typical therapeutic formulation. For optimum peak area response and reproducibility, protein derivatization with an appropriate chromophore (e.g., fluorescamine) and separation in the presence of a moderate ionic strength buffer containing lithium chloride, tetramethylammonium chloride or trimethylammonium propylsulfonate is recommended. General guidelines for the determination of proteins by capillary electrophoresis and a rationale for the use of internal standards to improve the quantification of data are also discussed.

Antibodies, Monoclonal↗
PubMed1992

Assay of protein drug substances present in solution mixtures by fluorescamine derivatization and capillary electrophoresis.

A method is described to enhance the resolution and detection sensitivity of proteins, peptides, and amino acids in capillary electrophoretic analysis of solution mixtures. The method consists of derivatizing the analytes with fluorescamine, which is normally used as a fluorogenic reagent for compounds containing a reactive primary amine functional group, and then using the derivative as an ultraviolet chromophore to enhance detection sensitivity (measured at 280 nm) in capillary electrophoresis. The results demonstrated a significant improvement in the separation and detection sensitivity of the derivatized analytes as compared to their underivatized counterparts. The use of chromophores, such as fluorescamine, in capillary electrophoresis facilitates the analysis of components of solution mixtures, such as pharmaceutical formulations, that could not be resolved and/or detected by conventional capillary electrophoresis procedures.

Antibodies, Monoclonal↗
PubMed1992

Comparison between avian and human prolyl 4-hydroxylases: studies on the holomeric enzymes and their constituent subunits.

Prolyl 4-hydroxylase, a key enzyme in collagen biosynthesis, catalyzes the conversion of selected prolyl residues to trans-hydroxyproline in nascent or completed pro-alpha chains of procollagen. The enzyme is a tetramer composed of two nonidentical subunits, designated alpha and beta. To compare the enzyme and its subunits from different sources, the chick embryo and human placental prolyl 4-hydroxylases were purified to homogeneity and their physicochemical and immunological properties were determined. Both enzymes were glycoproteins with estimated apparent molecular weights ranging between 400 and 600 kDa. Amino acid and carbohydrate analyses showed slight differences between the two holomeric enzymes, consistent with their deduced amino acid sequences from their respective cDNAs. Human placental prolyl 4-hydroxylase contained more tightly bound iron than the chick embryo enzyme. Immunodiffusion of the human placental enzyme with antibodies raised against the purified chick embryo prolyl 4-hydroxylase demonstrated partial identity, indicating different antigenic determinants in their tertiary structures. The enzymes could be separated by high-resolution capillary electrophoresis, indicating differential charge densities for the native chick embryo and human placental proteins. Electrophoretic studies revealed that the human prolyl 4-hydroxylase is a tetrameric enzyme containing two nonidentical subunits of about 64 and 62 kDa, in a ratio of approximately 1 to 2, designated alpha and beta, respectively. In contrast, the chick embryo alpha and beta subunit ratio was 1 to 1. Notably, the human alpha subunit was partially degraded when subjected to electrophoresis under denaturing conditions. Analogously, when the chick embryo enzyme was subjected to limited proteolysis, selective degradation of the alpha subunit was observed. Finally, only the alpha subunit was bound to Concanavalin A demonstrating that the alpha subunits of prolyl 4-hydroxylase in both species were glycosylated. Using biochemical techniques, these results demonstrated that the 4-trans-hydroxy-L-proline residues in human placental collagens are synthesized by an enzyme whose primary structure and immunological properties differ from those of the previously well-characterized chick embryo enzyme, consistent with their recently deduced primary structures from cDNA sequences.

Amino Acids↗
PubMed1991

Bidirectional microdialysis in vivo shows differential dopaminergic potency of cocaine, procaine and lidocaine in the nucleus accumbens using capillary electrophoresis for calibration of drug outward diffusion.

Cocaine and two other local anesthetics were applied directly into the nucleus accumbens for 20 min by diffusion from a 4 mm microdialysis probe in freely moving rats. Cocaine (7.3 mM) increased the extracellular concentration of dopamine (DA). Equimolar procaine did also, but was not as potent as cocaine. Equimolar lidocaine had no effect. The concentration of these drugs outside the probe as measured by capillary electrophoresis in vitro was about 28% of that inside the probe, i.e. 72% remained inside. However, an in vivo test showed that about 53% cocaine and procaine, and 37% lidocaine remained in the perfusion fluid after passing through a probe inserted in the brain. This suggests that in vivo about 68 nmol cocaine diffused into the nucleus accumbens (NAC) during the 20 min. Five conclusions are drawn: (1) this confirms our earlier finding that local injection of cocaine increases extracellular DA, but in this case the cocaine was infused via the probe without disturbing the animal; (2) the action of cocaine on dopamine terminals in the accumbens is independent of local anesthesia; (3) procaine may enhance mood by a cocaine-like effect; (4) capillary electrophoresis has potential for measuring cocaine levels in small samples and (5) in vitro calibrations are of limited value to evaluate in vivo performance of microdialysis probes.

Animals↗
PubMed1984

Human alpha-L-iduronidase. I. Purification and properties of the high uptake (higher molecular weight) and the low uptake (processed) forms.

Two major forms of human alpha-L-iduronidase have been individually purified over 175,000-fold to apparent homogeneity by sequential anion exchange, lectin affinity, and gel filtration chromatography. The two forms, initially designated as soluble and membrane-associated, were extracted from human lung in approximately equal amounts. Optimal solubilization of the membrane-associated form was facilitated by use of a non-ionic detergent or mannose 6-phosphate and saponin. Following detergent homogenization, the two forms were separated by anion exchange chromatography and then individually purified. The more electronegative form was membrane-associated, had a pI of approximately 5.9, and was selectively taken up (high uptake) by cultured Hurler syndrome fibroblasts; the more electropositive soluble form had a pI of about 6.6 and was incorporated into Hurler fibroblasts at a markedly lower rate (low uptake). After treatment with alkaline phosphatase, the pI values of both enzymes were about 7.8. Using 4-methylumbelliferyl-alpha-L-iduronide as substrate, the low and high uptake forms were each purified in milligram quantities to specific activities of 284,000 and 202,000 units/mg, respectively, with a combined yield greater than 35%. Each purified enzyme form migrated as a single protein band which also stained for enzymatic activity when electrophoresed in 7% native polyacrylamide disc gels at pH 4.3. By gel filtration, the high uptake form had an Mr = 85,000 whereas the Mr for the low uptake form was 68,000. Molecular weight estimates by analytical polyacrylamide gel electrophoresis were 82,000 and 70,000 for the high and low uptake forms, respectively. Rabbit anti-human low uptake alpha-L-iduronidase antibodies cross-reacted with the high uptake form as demonstrated by both immunotitration and Ouchterlony double immunodiffusion. Amino acid analysis revealed that the high uptake (higher molecular weight) form contained more arginine, glycine, alanine, glutamate or glutamine, leucine, isoleucine, histidine, and proline residues per molecule than the low uptake (lower molecular weight) form. Automated Edman degradation determined that the NH2-terminal residues of both forms were blocked. Both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high performance liquid chromatography demonstrated that each purified form was composed of several components; each post-high performance liquid chromatographic component retained catalytic activity and was immunologically cross-reactive with antibodies against the low uptake form.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1984

Human alpha-L-iduronidase. II. Comparative biochemical and immunologic properties of the purified low and high uptake forms.

The physicokinetic and immunologic properties of the purified low and high uptake forms of the human lysosomal hydrolase, alpha-L-iduronidase, have been determined and compared. The apparent Km and Vmax values for the low and high uptake forms were similar toward two artificial substrates, 4-methylumbelliferyl-alpha-L-iduronide (0.07 and 0.06 mmol/l; 16.15 and 14.85 mumol/min/mg, respectively), and phenyl-alpha-L-iduronide (1.42 and 1.66 mmol/l; 0.83 and 1.05 mumol/min/mg, respectively), and one natural substrate, anhydro-[3H]-mannitol-iduronide (0.86 and 1.04 mmol/l; 2.50 and 2.79 mumol/min/mg, respectively). The pH optima for both purified forms also were similar for each of the three substrates ( approximately 3.50, approximately 3.50, and approximately 4.50, respectively). Heparin markedly inhibited the 4-methylumbelliferyl-alpha-L-iduronide activities of both the low and high uptake forms, while dermatan sulfate and heparan sulfate were more inhibitory toward the low uptake activity. EDTA was a potent inhibitor of both enzyme forms; the divalent cations, Mg2+ and Ca2+, could recover up to 30% of the enzymatic activities after EDTA treatment. p-Chloromercuribenzoate and maleate also were inhibitory, whereas dithiothreitol and 2-mercaptoethanol were stimulatory. Both enzyme forms had similar thermostabilities ; the half-lives at 45, 52, and 60 degrees C were about 38, 24 and 12 min, respectively. The low and high uptake forms were immunologically cross-reactive as demonstrated by Ouchterlony double immunodiffusion and immunotitration studies using anti-human low uptake antibodies.

Endocytosis↗