High-performance liquid chromatography determination of alpha1-acid glycoprotein in small volumes of plasma from neonates

In order to investigate how the alpha1-acid glycoprotein (AGP) concentrations of neonates change in response to surgical stress, a simple high-performance liquid chromatography (HPLC)-assay for the measurement of alpha1-acid glycoprotein levels was developed. A fraction containing alpha1-acid glycoprotein was isolated from the bulk of plasma protein by addition of 0.6M perchloric acid and was then analysed directly on a short PLRP-S 4000A reversed phase column column. The method was validated by analysis of pooled plasma from healthy adults both in comparison with a calibration curve and by standard additions. The procedure was able to isolate alpha1-acid glycoprotein rapidly (<30 min) and required only 50 microl of plasma. The mean extraction recovery was 79.1% (CV 6.4%). The within-run precision for the analysis of three replicates of quality control sample ranged from +/-1.2 to +/-3.8% and the between-run precision was +/-6.1%. The method was linear (r(2)=0.988) over a concentration range from 6 to 100.0 mg/100 ml. The AGP levels in neonatal samples ranged from 25 to 93 mg/100 ml.     

Structure and characterisation of a duplicated human alpha 1 acid glycoprotein gene

Human alpha 1-acid glycoprotein (AGP), also known as orosomucoid, is a major acute-phase plasma protein. The amino acid sequence of AGP, which was determined by sequencing from protein isolated from pooled plasma, contained amino acid substitutions in 21 different positions. Genomic and cDNA clones which correspond to one of the possible amino acid sequences have been previously reported. In this paper we present the complete nucleotide sequence of a second gene, AGP2 which is located approx. 3.3 kb downstream from AGP1. The derived amino acid sequence of AGP2 contains 19 of the possible alternative amino acid substitutions as well as two additional differences. It is clear from the results presented here that the AGP in human plasma is the product of two separate gene loci.     

Plasma protein binding study of oxybutynin by high-performance frontal analysis

Plasma protein binding of oxybutynin (OXY) was investigated quantitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system which consists of HPFA column, extraction column and analytical column was developed to determine the unbound concentrations of OXY enantiomers in human plasma, in human serum albumin (HSA) solutions, and in human alpha1-acid glycoprotein (AGP) solutions. OXY is bound in human plasma strongly and enantioselectively. The bound drug fraction in human plasma containing 2-10 microM (R)- or (S)-OXY was higher than 99%, and the unbound fraction of (R)-OXY was 1.56 times higher than that of (S)-isomer. AGP plays the dominant role in this strong and enantioselective plasma protein binding. The total binding affinities (nK) of (R)- and (S)-OXY to AGP were 6.86 x 10(6) and 1.53 x 10(7) M(-1), respectively, while the nK values of (R)- and (S)-OXY to HSA were 2.64 x 10(4) and 2.19 x 10(-4) M(-1), respectively. The binding affinity of OXY to AGP is much higher than that to HSA, and shows high enantioselectivity (SIR ratio of nK values is 2.2). It was found that both enantiomers are bound competitively at the same binding site on an AGP molecule. The binding property between OXY and low density lipoprotein (LDL) was investigated by using the frontal analysis method incorporated in high-performance capillary electrophoresis (HPCE/FA). It was found the binding is non-saturable and non-enantioselective.     

Respective role of mifepristone (RU486) binding to blood and tissue proteins in its quantitative distribution in the body]

In plasma, mifepristone (RU486)-alpha 1-glycoprotein acid (AGP) interaction follows a quickly saturable process within the range of therapeutic concentrations. The high affinity to AGP, Kap = 8 x 10(6) M-1, suggests that the tissue distribution of the drug is possibly impaired. Checking this hypothesis was done by simulating its body distribution between plasma proteins and tissues. Two methods were used. The first was to calculate the number of available binding sites in both plasma (Np) and tissues (NT), to measure the relevant association constants Kap and KaT, and to consider that mifepristone partitioned according to their ratios, expressed as [NpKap]/[NpKap + NTKaT] for the plasma and [NTKaT]/ NpKap + NTKaT] for the tissues. The second method used equations relating the apparent volume of distribution and the plasma unbound fraction of mifepristone with volumes of physiologic spaces to calculate the percentage of drug located in plasma, extracellular and intracellular fluids. The results yielded by the two methods are close. They show that with AGP levels within a normal range (18.4 microM), only 18% of mifepristone is bound to AGP and the remaining part 82% is located in tissues. By contrast, when AGP level dramatically increases (300 microM) most of the dose (77%) is retained in plasma. These results suggested that when AGP concentration increases, the efficacy of therapeutic concentration of mifepristone may be partially decreased if only the unbound fraction of this synthetic hormone were biologically active.     

Measurement of drug-protein binding by immobilized human serum albumin-HPLC and comparison with ultrafiltration

An HPLC method employing CHIRAL-I (150 mm x 3 mm), 5 microm column from Chrom. Tech., immobilized with human serum albumin (HSA), was used to determine in vitro protein binding of several compounds. Experimentally obtained plasma protein data exhibited good correlation with the reported values. The method was compared with the conventional ultra filtration technique and both yielded similar results. Proprietary compounds that could not be analyzed by ultra filtration due to high non-specific binding to filter membrane were successfully analyzed by HSA-HPLC method. On the other hand, two proprietary compounds did not elute from HSA column due to strong binding, but were successfully analyzed by ultra filtration. This proves that both the techniques have their own merits and demerits and should be exploited judiciously as per the requirement. The plasma protein binding studies conducted on four gyrase inhibitors in rat and human plasma exhibited no interspecies difference via ultra filtration method. Further, it was also observed that the protein binding obtained for the four gyrase inhibitors by HSA-HPLC method was not only similar to that obtained by ultra filtration in human plasma but was also in accordance with ex vivo and in vitro protein binding obtained for rat plasma after ultra filtration because these compounds predominantly bind to HSA The binding of several compounds to alpha1-acid glycoprotein (AGP), another important plasma protein, was also examined using AGP immobilized column. However, the data could not be relied upon since some anti-bacterials and non-steroidal anti-inflammatory drugs (NSAIDS), which are known to predominantly bind to HSA, were also found to bind to AGP.     

Enantioselective plasma protein binding of propafenone: mechanism, drug interaction, and species difference

The interaction of propafenone (PPF) enantiomers with human plasma, human serum albumin (HSA), alpha(1)-acid glycoprotein (AGP), as well as with plasma from rat, rabbit, and cow was investigated using indirect chiral high performance liquid chromatography (HPLC) and ultrafiltration techniques. The stronger binding of the S-PPF found in human plasma was due to AGP. Two classes of binding sites in AGP were identified: one with high-affinity and small binding capacity (K(1(S)) = 7.65 x 10(6) M(-1), n(1(S)) = 0.50; K(1(R)) = 2.81 x 10(6) M(-1), n(1(R)) = 0.46), which revealed stereoselectivity; the other with low-affinity and high-binding capacity (n(2(S)) K(2(S)) = 9.95 x 10(3) M(-1); n(2(R)) K(2(R)) = 9.74 x 10(3) M(-1)). The binding to HSA was found to be weak and not enantioselective (nK(S) = 2.08 x 10(3) M(-1), nK(R) = 2.05 x 10(3) M(-1)). The interaction between enantiomers observed in human plasma was confirmed as a competitive type interacting at the high-affinity site in AGP. The binding mode of both enantiomers with AGP was mainly hydrophobic bond. PPF enantiomers had higher-binding affinity for the F-S variant of human AGP. Drug-drug binding interaction studies showed that verapamil, diazepam, nifedipine, furosemide, nitrendipine, and nimodipine did not affect the binding of PPF enantiomers except quinidine and aprindine at the therapeutic concentration. Comparative studies indicated considerable species-dependent binding stereoselectivity between plasma of the four species investigated.     

Stereoselective protein binding of tetrahydropalmatine enantiomers in human plasma,HSA, and AGP,but not in rat plasma

Tetrahydropalmatine (THP) is one of the active alkaloid ingredients of Rhizoma Corydalis. THP has a chiral center, and the stereoselective pharmacokinetics and tissue distribution have been reported. The aim of the present article is to study the stereoselective protein binding of THP using equilibrium dialysis followed by HPLC‐UV analysis. The results showed that THP stereoselectively binds to human serum albumin (HSA), α₁‐acid glycoprotein (AGP), and proteins in human plasma. The fraction binding of (+)‐THP was significantly higher than that of (?)‐THP, whereas such stereoselectivity was not found in rat plasma. The affinity of HSA and AGP to (+)‐THP, expressed as nK_A, were 9.0 × 10³ M^?1 and 2.34 × 10⁵ M^?1, respectively, which were notablely higher than to (?)‐THP, with the nK_A of 3.4 × 10³ M^?1 and 1.44 × 10⁵ M^?1, respectively. The binding site of HSA for (?)‐THP was Site I, whereas for (+)‐THP was both Site I and Site II. The F1/S variants of AGP were proved to be the key variants (?)‐ and (+)‐THP binding to both. Finally, the AGP binding drugs, such as mifepristone, were demonstrated to reduce the fraction binding of (?)‐ and (+)‐THP with pure AGP (1 mg/ml) but did not affect the fraction binding of both (?)‐ and (+)‐THP with proteins in human plasma. It can be concluded that protein binding of THP is species dependent and stereoselective, both HSA and AGP contribute to the stereoselective binding to THP enatiomers, and AGP binding drugs may not cause the drug–drug interaction on THP in healthy human plasma. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Identification of alpha-1 acid glycoprotein as a lysophospholipid binding protein: a complementary role to albumin in the scavenging of lysophosphatidylcholine

Alpha-1 acid glycoprotein (AGP, orosomucoid), a major acute phase protein in plasma, displays potent cytoprotective and anti-inflammatory activities whose molecular mechanisms are largely unknown. Because AGP binds various exogenous drugs, we have searched for endogenous ligands for AGP. We found that AGP binds lysophospholipids in a manner discernible from albumin in several ways. First, mass spectrometric analyses showed that AGP isolated from plasma and serum contained lysophosphatidylcholine (LPC) enriched in mono and polysaturated acyl chains, whereas albumin contained mostly saturated LPC. Second, AGP bound LPC in a 1:1 molar ratio and with a higher affinity than free fatty acids, whereas albumin bound LPC in a 3:1 ratio but with a lower affinity than that of free fatty acids. Consequently, free fatty acids displaced LPC more avidly from albumin than from AGP. Competitive ligand displacement indicated the highest affinity for AGP to LPC20:4, 18:3, 18:1, and 16:0 (150-180 nM), lysophosphatidylserine (Kd 190 nM), and platelet activating factor (PAF) (Kd 235 nM). The high affinity of AGP to LPC in equilibrium was verified by stopped-flow kinetics, which implicated slow dissociation after fast initial binding, being consistent with an induced-fit mechanism. AGP also bound pyrene-labeled phospholipids directly from vesicles and more efficiently than albumin. AGP prevented LPC-induced priming and PAF-induced activation of human granulocytes, thus indicating scavenging of the cellular effects of the lipid ligands. The results suggest that AGP complements albumin as a lysophospholipid scavenging protein, particularly in inflammatory conditions when the capacity of albumin to sequester LPC becomes impaired.     

Separation of alpha-acid glycoprotein glycoforms using affinity-based reversed micellar extraction and separation

A new method for the preparation of the glycoforms of bovine alpha(1)-acid glycoprotein (AGP) is described relying on affinity-reversed micellar extraction and separation (ARMES). This method has proven effective in separating structurally similar glycoproteins and separating glycoproteins from nonglycosylated proteins from natural sources. In this method, individual glycoforms complex with the lectin, concanavalin A (ConA) are extracted into an organic-phase reversed micellar solution formed by Aerosol OT (AOT). The purity of three AGP glycoforms isolated was assessed by hydroxyapatite high-performance liquid chromatography (HPLC), gel-permeation chromatography and SDS-PAGE. The glycan structure of the pure glycoforms was analyzed. Oligosaccharide mapping using capillary electrophoresis (CE) and PAGE showed the glycans obtained from each glycoform to be distinctly different. ARMES can be used for the semi-preparative scale resolution of the glycoforms of bovine AGP or other therapeutic glycoproteins.     

Stereoselective distribution of acenocoumarol enantiomers in human plasma: Chiral chromatographic analysis of the ultrafiltrates

Stereoselectivities for the binding of rac-acenocoumarol to human serum albumin (HSA), α₁-acid glycoprotein (AGP), and human plasma were determined by chiral HPLC analysis of the ultrafiltrates on a Chiral-AGP column. The results confirmed the previously detected inverse stereoselectivities; for HSA the ratio of the enantiomeric constants was K^R/K^S ～ 2, while for AGP it was K^R/K^S ～ 0.3. In plasma the contribution of HSA dominates, although in pathological states, elevated AGP levels may compensate for stereoselective distribution. © 1993 Wiley-Liss, Inc.     

One-step purification of rat plasma α1-acid glycoprotein by antibody affinity chromatography: Application to normal and inflamed rat sera

Most purification procedures used previously to isolate α₁-acid glycoprotein (AGP) from plasma can lead to some alterations in its carbohydrate moiety. An immunoaffinity chromatographic method is proposed for purifying in one step rat plasma AGP without any detectable modification of its glycan moiety. Crossed immunoaffinoelectrophoresis with concanavalin A before and after purification showed identical patterns, suggesting no glycan selection during the purification. In the same way no desialylation occurred during the purification step. This immunoaffinity chromatographic procedure provided evidence of a decreased level of fucosyl residues in turpentine oil rat plasma AGP compared with normal rat plasma AGP.     

Immobilization of alpha1-acid glycoprotein for chromatographic studies of drug-protein binding II. correction for errors in association constant measurements

A new method for the immobilization of α₁-acid glycoprotein (AGP) in high-performance liquid chromatography (HPLC) columns was recently described for applications such as drug binding studies. Part of this earlier work used self-competition zonal elution studies to measure association equilibrium constants between immobilized AGP and R- or S-propranolol. It was later found that analysis of these data by a common equation derived for linear elution conditions gave erroneous values for experiments actually conducted under nonlinear conditions. This report discusses the nature of this error and uses frontal analysis to estimate the true binding strength between R- and S-propranolol and HPLC columns containing immobilized AGP.     

Tomato LeAGP-1 is a plasma membrane-bound, glycosylphosphatidylinositol-anchored arabinogalactan-protein

Arabinogalactan-proteins (AGPs) are a class of highly glycosylated, hydroxyproline-rich glycoproteins that function in plant growth and development. Tomato LeAGP-1 represents a major AGP expressed in cultured cells and plants. Based on cDNA and amino acid sequence analyses along with carbohydrate and other biochemical analyses, tomato LeAGP-1 is hypothesized to be a classical AGP localized to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. Here, this hypothesis was tested and supported with the following experiments. First, tomato ( Lycopersicon esculentum , cv. UC82B) cotyledon protoplasts were isolated following cell wall digestion with cellulase and pectinase, and LeAGP-1 was immunolocalized to the plasma membrane with a LeAGP-1 antibody. Second, LeAGP-1 was shown to be a major AGP component in plasma membrane vesicles from tomato cv. Bonnie Best suspension-cultured cells by Western blot analysis with the LeAGP-1 antibody. Third, fluorescence microscopy of plasmolysed, transgenic tobacco ( Nicotiana tabacum BY-2) suspension-cultured cells expressing a green fluorescent protein (GFP)-LeAGP-1 fusion product demonstrated localization to the plasma membrane and Hechtian threads. Fourth, the GFP-LeAGP-1 fusion protein was present in plasma membrane preparations from these transgenic tobacco cells by Western blot analysis with a GFP antibody. Fifth, GFP-LeAGP-1 secreted into the culture media contained ethanolamine, presumably attached to the C-terminal amino acid residue, consistent with its processing and release from the plasma membrane. Thus, these data support the hypothesis that LeAGP-1 is localized to the plasma membrane via a GPI anchor and suggest possible roles for LeAGP-1 in cellular signalling and matrix remodelling.     

Structure and expression of the genes coding for human alpha 1-acid glycoprotein.

alpha 1-acid glycoprotein (alpha AGP) is a well-characterized human plasma protein. Its structural properties have been studied for many years but little is known about its function. Amino acid sequence analysis of purified human alpha AGP from plasma pooled from several individuals showed considerable heterogeneity. We have cloned the genomic DNA segment encoding alpha AGP and we show that it contains three adjacent alpha AGP coding regions, AGP-A, B and B', identical in exon--intron organization but with slightly different coding potential. These results account for the heterogeneity observed by protein sequencing. Southern blot analysis indicates that the cloned cluster contains all the alpha AGP coding sequences present in the human genome. The larger majority of alpha AGP mRNA in human liver is transcribed from AGP-A, whose promoter and cap site have been determined while the level of AGP-B and B' mRNA in human liver is very low. Using Hep3B hepatoma cells as a model system for the in vitro study of the acute phase reaction, we show that only AGP-A is strongly induced by treatment with culture medium of LPS stimulated monocytes.     

A carbohydrate component of alpha1-acid glycoprotein from normal and abortive blood

alpha 1-Acid glycoprotein (AGP) samples, viz., normal AGP (nAGP) and embryonic AGP (eAGP), were isolated from plasma of normal donor blood and abortive human blood, respectively. The materials possess similar amino acid composition and immunochemical properties. However, structural patterns of their sugar moieties differ substantially. nAGP contains only N-glycosidically bound sugar chains, whereas on eAGP some sugar chains are linked through O-glycosidic bonds. Disaccharide GlcNAc beta 1----3Gal was identified in considerable amounts in HF-solvolysate of eAGP but only as traces in nAGP.     

Immobilization of alpha(1)-acid glycoprotein for chromatographic studies of drug-protein binding

A new method for preparing immobilized alpha1-acid glycoprotein (AGP) for use in drug-protein binding studies was developed and optimized. In this approach, periodate was used under mild conditions to oxidize the carbohydrate chains in AGP for attachment to a hydrazide-activated support. The final conditions chosen for this oxidation involved the reaction of 5.0 mg/mL AGP at 4 degrees C and pH 7.0 with 5-20 mM periodic acid for 10 min. These conditions helped maximize the immobilization of AGP without significantly affecting its activity. This method was evaluated by using it to attach AGP to silica for use in high-performance affinity chromatography and self-competition zonal elution studies. In work with R- and S-propranolol, only one type of binding site was observed for both enantiomers on the immobilized AGP, in agreement with previous studies using soluble AGP. The association equilibrium constants measured for the immobilized AGP with R- and S-propranolol at pH 7.4 and 37 degrees C were 2.7 x 10(6) and 4.2 x 10(6) M(-1), respectively, with linear van't Hoff plots being obtained between 5 and 37 degrees C. Work performed with other drugs also gave good agreement between the behavior seen for immobilized AGP and that for soluble AGP. The same immobilization method described in this work could be used to attach AGP to other materials, such as those used for surface plasmon resonance or alternative biosensors.     

Altered glycosylation and expression of plasma alpha-1-acid glycoprotein and haptoglobin in rheumatoid arthritis

Altered glycosylation patterns in plasma proteins are found to be associated with the pathogenesis of various malignancies and autoimmune disorders. Our previous studies demonstrated the occurrence of some differentially glycosylated plasma proteins in rheumatoid arthritis (RA) patients. The current study was conducted to evaluate the alterations in expression and glycosylation of major acute phase proteins from wheat germ agglutinin enriched RA patients' plasma. Immunoblotting studies revealed a significant enhancement in the plasma levels of alpha-1 acid glycoprotein (AGP) and haptoglobin (Hp) in RA patients with respect to healthy controls. Monosaccharide analysis by high performance anion exchange-chromatography with pulse amperometric detection showed significant variations in the relative percentage of galactose, glucosamine and mannose in AGP and of mannose in Hp in RA patients. Altered patterns of mannosylation in AGP and Hp were also established by enzyme linked immunosorbent assay and Western blotting using Concanavalin-A lectin. These results could give information for understanding the disease pathogenesis and may provide an insight into the development and progression of the disease.     

Alteration in amino-glycerophospholipids levels in the plasma of children with autism: a potential biochemical diagnostic marker

Currently, there is no biochemical test to assist in the behavioral diagnosis of autism. We observed that levels of phosphatidylethanolamine (PE) were decreased while phosphatidylserine (PS) were increased in the erythrocyte membranes of children with autism as compared to their non-autistic developmentally normal siblings. A new method using Trinitrobenezene sulfonic acid (TNBS) for the quantification of PE and PS (amino-glycerophospholipids, i.e., AGP) in the plasma of children was developed and standardized. Wavelength scans of TNBS-PE and TNBS-PS complexes gave two peaks at 320 nm and 410 nm. When varying concentrations of PS and PE were used, a linear regression line was observed at 410 nm with TNBS. Using this assay, the levels of AGP were found to be significantly increased in the plasma of children with autism as compared to their non-autistic normal siblings. It is proposed that plasma AGP levels may function as a potential diagnostic marker for autism.     

Acute phase mediated change in glycosylation of rat alpha 1-acid glycoprotein in transgenic mice.

Transgenic mouse lines carrying the gene for rat alpha 1-acid glycoprotein (AGP) express the protein in the plasma at concentrations equal to or exceeding that of acute phase rats. Owing to the high basal level, these transgenic mice represent a unique experimental system for defining the largely unknown function of AGP. Since the carbohydrate moiety of AGP has been found to be changed during acute phase and the oligosaccharide structure to be important for immunomodulating activity of the protein, the rat AGP in transgenic mice was characterized by lectin-affinity immuno-electrophoresis. Unlike in the rat, the AGP in the transgenic mouse plasma consisted primarily of strongly concanavalin A-reactive forms. Acute phase mediated a several-fold increase in the total plasma concentration of AGP concomitant with a shift toward moderately concanavalin A-reactive forms. A similar change in concanavalin A-reactive forms was observed for the endogenous acute phase plasma protein haptoglobin. To define the role of inflammatory factors in AGP production, primary cultures of hepatocytes were prepared. In contrast to in vivo, the AGP recovered from tissue culture medium represented primarily the concanavalin A-non-reactive form. Treatment of the cells with recombinant human interleukin-1, interleukin-6 and dexamethasone stimulated the production of concanavalin A-reactive AGP forms. The data indicate that the glycosylation pattern of plasma-resident AGP is modulated by acute phase, but that the profile of AGP forms does not coincide with that secreted by hepatocytes in tissue culture. This finding demands an assessment of which of the possible glycosylated forms of AGP is functionally significant in vivo.     

Determination of warfarin enantiomers and hydroxylated metabolites in human blood plasma by liquid chromatography with achiral and chiral separation

An assay comprising two simple, selective and isocratic HPLC methods with UV detection was developed and validated for measuring warfarin enantiomers and all five warfarin monohydroxylated metabolites in patient blood plasma. Following liquid/liquid extraction from 1 ml of blood plasma a baseline separation of analytes was achieved on chiral (alpha(1) acid glycoprotein - AGP) and achiral (C(18)) column. Both methods were consistent (R.S.D.<6.9% for warfarin enantiomers and<8.9% for monohydroxylated metabolites) and linear (r>0.998). The limits of detection were 25 ng/ml for warfarin enantiomers, 25 ng/ml for 4'-, 10-, 6- and 7-hydroxywarfarin, 35 ng/ml for 8-hydroxywarfarin and 50 ng/ml for racemic warfarin. In a clinical study in 204 patients, it was confirmed that the assay is appropriate for evaluation of influences of genetic polymorphisms, demographic factors and concomitant drug treatment on warfarin metabolism.