Biantennary glycans as well as genetic variants of alpha1-acid glycoprotein control the enantioselectivity and binding affinity of oxybutynin.

PURPOSE: The purpose of this study was to investigate the role of biantennary branching glycans of alpha1-acid glycoprotein (AGP) and its genetic variants in the enantioselective binding of oxybutynin (OXY). METHOD: Human native AGP was separated using imminodiacetate-copper (II) affinity chromatography into two fractions, the A variant and a mixture of the F1 and S variants (F1-S). These fractionated AGPs were further separated by concanavalin A affinity chromatography into two fractions, with and without biantenarry glycans. An on-line high-performance liquid chromatography (HPLC) system consisting of a high-performance frontal analysis column, an extraction column, and an analytical HPLC column was developed to determine the binding affinities of OXY enantiomers for respective AGP species. RESULTS: The total binding affinity as well as the enantiomeric selectivity of OXY in the F1-S mixed variant was significantly higher than that for the A variant, indicating that the chiral recognition ability of native AGP for the OXY enantiomers highly depends on the F1-S mixed variant. Furthermore, not only the genetic variants but also bianntenary glycans of AGP affect the binding affinity of OXY and are also responsible for the enantioselectivity. CONCLUSIONS: Both genetic variants and glycan structures significantly contribute to the enantioselectivity and the binding affinity of OXY.

Simultaneous determination of unbound thyroid hormones in human plasma using high performance frontal analysis with electrochemical detection.

A direct injection HPLC method in combination with high-performance frontal analysis (HPFA) and electrochemical detection (ECD) was developed for the simultaneous and sensitive determination of unbound thyroid hormones (thyroxine, triiodothyronine, and reverse triiodothyronine) in human plasma. The present on-line HPLC/HPFA system consists of an HPFA column, an extraction column and an analytical HPLC column connected through a column-switching device, and the eluent from the analytical column was monitored by ECD. The calibration lines showed good linearity (rsq.>0.999) within 7.4-148.2 pM for T4 and 1.5-74.1 pM for T3 and rT3. Unbound T4 and T3 concentrations determined by the present system were 16.4+/-2.4 pM (n=15) and 7.14+/-1.04 pM (n=15), which were in agreement with those determined by the EIA method. The unbound rT3 concentration was 2.30+/-0.27 pM (n=15). The CV% values of intra-day and inter-day assays (n=15) were less than 14.9% for T4, 14.5% for T3 and 13.2% for rT3. The present system was also applied to a competitive binding study of these thyroid hormones in human plasma.

High-performance frontal analysis of the binding of thyroxine enantiomers to human serum albumin.

PURPOSE: The aim of this study was to characterize the binding property between thyroxine and human serum albumin (HSA) qualitatively and enantioselectively using high-performance frontal analysis (HPFA). METHODS: An on-line HPLC system consisting of an HPFA column, an extraction column, and an analytical HPLC column was developed to be used to determine the unbound concentrations of thyroxine enantiomers. RESULTS: Both enantiomers were bound to human serum albumin at two high-affinity sites with similar affinities. The binding constant (K) and the number of binding sites on an HSA molecule (n) evaluated from Scatchard plot analysis were K = 1.01 x 10(6)m(-1) and n = 1.90 for L: -thyroxine, and K = 9.71 x 10(5) m(-1) and n = 1.97 for D: -thyroxine. The binding sites were identified using phenylbutazone and diazepam as site-specific probes for sites I and II, respectively, and each enantiomer was found to bind to both sites. Incorporation of a chiral HPLC column into the on-line system permitted the investigation of enantiomer-enantiomer interactions, which revealed that both enantiomers competitively bind to the same binding sites without significant allosteric effects.

On-line capillary isoelectric focusing-mass spectrometry for quantitative analysis of peptides and proteins.

On-line capillary isoelectric focusing-mass spectrometry (cIEF-MS) was applied to determine concentrations of peptides and proteins using angiotensin II and human tetrasialo-transferrin as the model samples. The concentration of the carrier ampholyte was optimized for both resolution and ion intensity. cIEF-MS employing 1% Pharmalyte 3-10 and a sheath liquid containing water/methanol/acetic acid (50/49/1) resolved angiotensin I and II (5 microM each, DeltapI=0.2) at an Rs value of 2.29. The determined concentration of angiotensin II (0.1-5 microM) well correlated (R=0.999) with that obtained by the conventional RP-HPLC method. The limit of detection was 0.22 microM, which was about 10 times lower than that by UV detection (2 microM). The repeatability and accuracy were <15 and <11%, respectively. cIEF-MS was also applied to determine human tetrasialo-transferrin concentration. The good linearity (R2=0.998) was also observed between the transferrin concentration (0.5-1.2 g/L) and peak area ratio (IS; beta-lactoglobulin B) with acceptable accuracy (<1.9%) and repeatability ( approximately 10% at 1g/L).

Capillary electrophoretic study on pH dependence of enantioselective disopyramide binding to genetic variants of human alpha1-acid glycoprotein.

A high-performance frontal analysis-capillary electrophoresis (HPFA-CE) method was applied to investigate the effect of pH on the drug binding properties of genetic variants of human alpha1-acid glycoprotein (AGP), A variant and a mixture of F1S variants. The unbound concentrations of a model basic drug, disopyramide (DP), in A variant solutions and in F1S variant solutions were measured by HPFA-CE to evaluate binding constants at pH 4.0, 5.0, 6.0 and 7.4. The binding between DP and A variant was gradually weakened by acidification of background buffer (from pH 7.4 to 4.0), while the binding between DP and FIS variants decreased at first (from pH 7.4 to 6.0), and then gained (from pH 6.0 to 4.0). Consequently, DP was more strongly bound to A variant than to FIS variants at pH 7.4, while at pH 4.0 DP was more strongly bound to F1S variants. At any pH (S)-DP was bound more strongly than (R)-DP, and the enantioselectivity of A variant was significantly higher than that of F1S variants. Electrophoretic mobilities of the AGP genetic variants decreased along with a decrease in pH. Fluorescent emission of these genetic variants indicated a distinct conformational change between pH 5.0 and 4.0. However, there was no significant difference in the electrophoretic mobility and the fluorescent emission spectrum between these variants at any pH. On the other hand, circular dichroism analyses revealed that beta-sheet content in FIS variants diminished as pH decreased, while that in A variant increased. These results suggest that the conformational change induced by acidification of background buffer differs between these genetic variants, and this causes the difference in DP bindability.

[Drug binding analysis of human alpha 1-acid glycoprotein using capillary electrophoresis].

Drug-plasma protein binding analysis is indispensable for drug development and clinical use. However, conventional methods for binding analyses were not suitable for small amounts of proteins because of large sample requirements. On the other hand, high-performance frontal analysis/capillary electrophoresis (HPFA/CE) consumes very small sample volumes, and is useful for ligand-binding study of small amounts of proteins. In this study, HPFA/CE was used in a drug-binding study of alpha 1-acid glycoprotein (AGP) subtypes in which plasma concentrations change dynamically to elucidate the effects of structural variation on drug binding. Binding study on desialyrated AGP revealed that (S)-enantiomer selectivity in propranolol-AGP binding was caused by sialic acid residues, while neither sialic acid nor galactose caused the enantioselectivity of verapamil binding to AGP. Biantennary glycans slightly suppressed disopyramide binding to AGP, whereas the glycans did not have any influence on propranolol and verapamil binding. Disopyramide and verapamil were selectively bound to the A variant rather than the F1S variant. The A variant showed larger enantioselective binding to disopyramide, but not to verapamil.

Role of phospholipids in drug-LDL bindings as studied by high-performance frontal analysis/capillary electrophoresis.

The binding study between basic drugs ((S)-verapamil (VER) and (S)-propranolol (PRO)) and phospholipid liposomes was performed by using high-performance frontal analysis/capillary electrophoresis (HPFA/CE) in order to investigate the effect of oxidative modification of low-density lipoprotein (LDL) upon drug-binding affinity from molecule-based viewpoint. 1-Palmitoyl-2-oleoyl-phosphatidylcholine (POPC, 16:0, 18:1), 1-palmitoyl-2-linoleoyl-phosphatidylcholine (PLPC, 16:0, 18:2), dilauloyl-phosphatidylcholine (DLaPC, 12:0, 12:0), 1-palmitoyl-2-oleoyl-phosphatidyl-glycerol (POPG, 16:0, 18:1), and 1-palmitoyl-sn-glycero-3-phosphocholine (monoPPC, 16:0) were used to prepare the model liposomes. At physiological pH (pH 7.4), the model liposome prepared from POPG+POPC had negative net charges, while the total net charge of the other model liposomes (POPC liposome, PLPC liposome, DLaPC liposome, and monoPPC+POPC liposome) was zero. The drug and the model liposome mixed solutions were subjected to HPFA/CE, and the total binding affinities (nK) were calculated. The nK values of VER and PRO to POPG+POPC liposome were more than six and 10 times higher than those of other liposomes, respectively. On the other hand, the nK values of the model drugs to POPC liposome, PLPC liposome, DLaPC liposome and monoPPC+POPC liposome showed small differences less than twice. These results indicate that the electrostatic interaction plays an important effect on drug-liposome binding, and suggest that the increase in the negative charge of LDL phospholipids gives more significant effect on the drug-binding affinity of the basic drugs than the acyl-chain structure.

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.

Frontal analysis of drug-plasma lipoprotein binding using capillary electrophoresis.

High performance frontal analysis coupled with capillary electrophoresis (HPFA/CE) was applied to the ultramicroanalysis of enantioselective binding of drug to plasma lipoproteins. A small volume (ca. 80 nl) of (R)- or (S)-propranolol (PRO, 25-150 microM) and human high-density lipoprotein (HDL, 2.63 g/l) or human low-density lipoprotein (LDL, 4.37 g/l) mixed solution, which was in the state of binding equilibrium, was introduced hydrodynamically into a non-coated fused silica capillary. Positively charged unbound PRO enantiomers migrated toward cathodic end much faster than negatively charged lipoproteins and the bound form. Once unbound PRO migrated apart from lipoprotein, the bound PRO was quickly released from the lipoprotein to maintain the binding equilibrium. Thus, PRO migrated as a zone in the capillary, giving a peak with a plateau region, where the concentration is the same as the unbound PRO concentration in the original sample solution. The unbound PRO concentration calculated form the plateau height agreed with that determined by a conventional ultrafiltration method used as a reference method. It was found that the bindings of PRO to HDL and PRO to LDL were not enantioselective, while the total binding affinity of PRO to LDL (4.01 x 10(5) per M) was 17 times higher than that of PRO-HDL binding (2.38 x 10(4) per M).