Use of tumor markers for differential diagnosis of mesothelioma and secondary pleural malignancies.

STUDY OBJECTIVE: The aim of the study was to assess diagnosis value of tumor markers for differential diagnosis between mesothelioma and other pleural tumors. DESIGN AND METHODS: Prospective study of 85 patients attending our hospital with malignant pleural effusion. The diagnostic approach involved routine pleurocentesis followed by pleural needle. When precise diagnosis was not achieved, thoracoscopy with pleural biopsies was performed. Carcinoembryonic antigen (CEA), hyaluronic acid, tissue polypeptide antigen and cyfra 21 to 1 were measured in serum and pleural fluid. RESULTS: By using receiver operating characteristics curves and area under curves, the best diagnostic characteristics were obtained with pleural and serum CEA concentrations. The area under the curve was larger for pleural ACE than for serum ACE. The sensitivity and specificity of a pleural CEA level exceeding 3 ng/mL for ruling out the diagnosis of mesothelioma were 100% and 77%, respectively. CONCLUSION: A CEA level above 3 ng/mL in pleural fluid eliminated the diagnosis of mesothelioma, whereas the other markers were not sufficiently discriminant. However, despite a negative predictive value of 100% at a cutoff of 3 ng/mL, CEA assay in pleural fluid only avoids a small number of diagnostic thoracoscopies.

Expression of the genetic variants of human alpha-1-acid glycoprotein in cancer.

OBJECTIVES: We have investigated the AAG and its genetic variants concentrations in plasma samples of 61 patients suffering from different types of cancers. DESIGN AND METHODS: The patients were shared out in three groups, breast, lung, and ovary cancers groups. AAG concentration was measured by an immunonephelometric method and the phenotype was determined, after desialylation of plasma by analytical isoelectric focusing. Detection of AAG variants was made by immunoblotting and their proportions were determined by laser densitometry analysis. A population of 74 healthy individuals served as controls. RESULTS: The plasma concentrations of AAG in the breast and lung cancer groups were 2.5 times increased, while in the ovary cancer group, the concentrations were 1.6 times increased. AAG concentrations in the cancer population ranged between 0.45 and 2.85 g/L (mean value 1.12 +/- 0.51 g/L). The proportions of the ORM1 and ORM2 variants were similar to those in the healthy population. In breast and lung cancer groups, the relative concentrations of genetic variants were increased more than 2.5 fold, whereas a 1. 6-fold increase was observed in the ovary cancer group. CONCLUSIONS: These results show that AAG plasma concentrations are increased in these types of cancers and that changes in the expression of the genetic variants of AAG could also occur according to the type of cancer.

In vitro binding and partitioning of irinotecan (CPT-11) and its metabolite, SN-38, in human blood.

The binding of CPT-11 and SN-38 to human plasma proteins was studied by ultrafiltration at 37 degrees C and pH 7.4. In plasma, CPT-11 was 66-60% bound in the range 100-4000 ng/ml and SN-38 was 94-96% bound in the range 50-200 ng/ml. At these concentrations the plasma binding of CPT-11 was slightly saturable, but the plasma binding of SN-38 was concentration-independent. Albumin was the main carrier of CPT-11 and SN-38 in plasma. In blood, the binding of CPT-11 was moderate (80%), mainly to plasma proteins (47%) and erythrocytes (33%). The binding of SN-38 was high (99%) and most of SN-38 in blood was located in blood cells (approximately 66%) The simulation of a grade 3 hematotoxicity (according to National Cancer Institute's Common Toxicity Criteria grading) on the SN-38 blood distribution yielded an increase in fu (free fraction of drug in plasma) from 1.05 to 2.08 and a decrease in C(Bl)/C(P) from 1.66 to 1.14 (both resulting from a decreased cell binding).

Lack of relevance of the acetylator status on dapsone response in chronic autoimmune thrombocytopenic purpura.

Dapsone provides an alternative treatment for patients with chronic autoimmune thrombocytopenic purpura (AITP) who had inadequate response to conventional therapy. However, the efficacy of this treatment is achieved in only 50% of patients. Dapsone is partly metabolized by the polymorphic N-acetyltransferase 2% and 50% of Caucasian patients show a genetically determined slow acetylator phenotype. The aim of our study was to investigate the influence of the acetylator status on dapsone efficacy in patients with chronic AITP. Nineteen caucasian adults with chronic AITP, previously treated by dapsone, were included in the study. Acetylator phenotype was determined by using a caffeine urinary test. Among the fourteen fast acetylator patients, eight patients exhibited positive response to dapsone and six patients did not. Among the five slow acetylator patients, one patient displayed a positive response to dapsone. Comparison of data by using the Fisher's exact test did not reach statistical significance. Our results do not support a relationship between dapsone efficacy and acetylator status in adults with chronic AITP.

Study of the expression of the genetic variants of human alpha1-acid glycoprotein in healthy subjects using isoelectric focusing and immunoblotting.

Human alpha1-acid glycoprotein (AAG) exists as an heterogeneous population of two or three genetic variants (ORM1 F1 and/or S and ORM2 A) in the plasma of most individuals. The ORM1 and ORM2 variants have a separate genetic origin. AAG belongs to the acute-phase proteins, which, under conditions of inflammation, increase several-fold in concentration. Additionally, there is evidence to suggest that it is not only the concentration but also the distribution of the two gene products of AAG (ORM1 and ORM2) that alter in such conditions. Variations of the relative concentrations of the AAG variants in certain diseases, such as cancer, can only be shown by reference to data collected in healthy people. In this study, we have investigated a group of 74 healthy subjects (42 men and 32 women) for AAG concentrations, AAG phenotypes and relative proportions of genetic variants in plasma. The specific assay of AAG was carried out by an immunonephelometric method and the phenotyping was performed, after desialylation of AAG, by analytical isoelectric focusing. Detection of the AAG variants was made by immunoblotting and their relative proportions were determined by laser densitometry analysis. The AAG plasma concentrations in the healthy group ranged between 0.28 and 0.92 g/l (mean value 0.50+/-0.14 g/l). The relative proportions of the variants derived from the two genes of AAG were variable, depending on the individual, but the amount of ORM1 variants almost always exceeded that of the ORM2 variant. No sex-related differences were observed in respect either in the total AAG level nor the relative proportions of the ORM1 and ORM2 variants. The data collected in this study may serve as a reference towards the investigation of possible changes in the expression of the genetic variants of AAG in chronic inflammatory diseases.

Changes in expression and microheterogeneity of the genetic variants of human alpha1-acid glycoprotein in malignant mesothelioma.

Human alpha1-acid glycoprotein (AAG), an acute-phase plasma protein, is heterogeneous in the native state and polymorphic in the desialylated state. The AAG heterogeneity is mainly explained by a variable glycan chain composition in its five glycosylation sites. The AAG polymorphism is due to the presence of genetic variants. Three main variants are observed for AAG, ORM1 F1, ORM1 S and ORM2 A, which have a separate genetic origin. In this paper, we have used different isoelectric focusing (IEF) methods and chromatography on immobilized metal affinity adsorbent to study the relative occurrence of the genetic variants of AAG in relation to changes in microheterogeneity, in plasma and pleural effusions of patients with malignant mesothelioma (MM). The results were compared to those obtained with the variants in plasma of healthy individuals. Significant changes in variant distribution were observed in the MM samples, that corresponded to a rise in the proportion of the ORM1 variants and a fall in that of the ORM2 variant. However, the concentration in MM plasma increased for both variants. The AAG in MM plasma and effusion fluids was found to be more heterogeneous on IEF than AAG of healthy plasma. The evidence of stronger concentrations of both the high and low pI forms of AAG in the MM samples suggested two kinds of changes in charge heterogeneity. These two changes were shown to be attributed to different variants--i.e. the high pI forms to ORM1 F1 and S and the low pI forms to ORM2 A, after fractionation of AAG by chromatography on immobilized copper(II) ions. These results indicate specific changes in both the expression and glycosylation for each AAG variant, according to its separate genetic origin, in MM.

Ligand specificity of the genetic variants of human alpha1-acid glycoprotein: generation of a three-dimensional quantitative structure-activity relationship model for drug binding to the A variant.

Human alpha1-acid glycoprotein (AAG) is a mixture of at least two genetic variants: the A variant and the F1 and/or S variant or variants, which are encoded by two different genes. In a continuation of previous studies indicating specific drug transport roles for each AAG variant according to its separate genetic origin, this work was designed to (1) determine the affinities of the two main gene products of AAG (i.e., the A variant and a mixture of the F1 and S variants) for 35 chemically diverse drugs and (2) to obtain meaningful 3D-QSARs for each binding site. Affinities were obtained by displacement experiments, leading to qualitative indications about binding site characteristics. In particular, drugs binding selectively to the A variant displayed some common structural features, but this was not seen for the F1*S variants. Three-dimensional QSAR analyses using the CoMFA method yielded a steric model for binding to the A variant, from which a simplified haptophoric model was derived. In contrast, no statistically sound model was found for the F1*S variants, possibly due (among other reasons) to an insufficient number of high affinity ligands in the set.

The genetic variant A of human alpha 1-acid glycoprotein limits the blood to brain transfer of drugs it binds.

The objective of this work was to check the effects of alpha-1 acid glycoprotein (AAG) and of its components, A and F1/S genetic variants, on the brain transfer of drugs they bind in plasma. The relevant extractions of six basic drugs, highly bound to AAG, were measured. We chose three drugs selectively bound to the A variant, disopyramide, imipramine and methadone, one drug mainly bound to the mixture F1/S, mifepristone, and two drugs which were simultaneously bound to the variant A and the mixture F1/S, propranolol and chlorpromazine. Their brain extraction were investigated in rats using the carotid injection technique and the capillary depletion method. Injected drugs were dissolved either in buffer, either in native AAG containing the three variants (A, F1 and S), either in variant A or in variant F1/S solutions. Brain extractions of disopyramide, imipramine and methadone were significantly reduced by native AAG and by variant A. Drug's plasma retention was related to their preferential and almost exclusive binding to A variant, both of them exhibiting the same decrease in brain transfer as compared to a buffered solution. At the opposite, there were no significative differences between the extraction either in buffer, either in AAG or in F1/S solutions, of drugs both bound to A variant and F1/S mixture (chlorpromazine and propranolol) or to the F1/S mixture (mifepristone). In serum, the retentional effect of the A variant on the extraction of disopyramide and imipramine was counteracted by the presence of albumin and lipoproteins, which simultaneously bind these two drugs at a high extent and act as permissive binders. We conclude that AAG binding decreases brain drug transfer when the A variant is mainly and almost exclusively involved in the binding. On the contrary, the entire fraction of the tested drugs when bound exclusively or partly to the mixture F1/S is available for transfer into the brain.

Trimetazidine does not modify blood levels and immunosuppressant effects of cyclosporine A in renal allograft recipients.

AIMS: In renal allograft recipients, trimetazidine (Vastarel) was proposed to be associated with the classic immunosuppressant treatments because it displays anti-ischaemic effects which may protect against cyclosporine A nephrotoxicity. The objective of this work was to assess the possibility of coadministering cyclosporin A, Sandimmun, and trimetazidine. METHODS: Twelve renal transplant patients were selected on the basis of the stability of their cyclosporine A blood concentrations for the previous 3 months. They received trimetazidine, 40 mg twice daily orally for 5 days. Other coadministered drugs were kept unchanged during the study. Before and after trimetazidine administration, cyclosporine A blood concentrations, plasma interleukin-2 and soluble interleukin-2 receptor levels were measured. RESULTS: The data showed that neither cyclosporin A blood pharmacokinetic parameters, Cmax, tmax, AUC, nor the concentrations of interleukin-2 and soluble interleukin-2 receptors were significantly modified. CONCLUSIONS: Therefore, it was suggested that trimetazidine may be coadministered with cyclosporine A without cyclosporine A dosage adjustment.

Binding of disopyramide, methadone, dipyridamole, chlorpromazine, lignocaine and progesterone to the two main genetic variants of human alpha 1-acid glycoprotein: evidence for drug-binding differences between the variants and for the presence of two separate drug-binding sites on alpha 1-acid glycoprotein.

Human alpha 1-acid glycoprotein (AAG), a plasma drug transport protein, has three main genetic variants, the A variant and the F1 and S variants, which are encoded by two different genes. The binding of disopyramide, methadone, dipyridamole, chlorpromazine, lignocaine and progesterone to the two main gene products of AAG-the A variant and a mixture of the F1 and S variants (60% F1 and 40% S)-separated by chromatography from native commercial AAG, a mixture of almost equal proportions of the F1, S and A variants, was studied by equilibrium dialysis. A selective binding of disopyramide and methadone to the A variant and a preferential binding of dipyridamole to the F1S variant mixture were found. Lignocaine and chlorpromazine had a slight preference for binding to the A variant and to the F1S mixture, respectively, but progesterone showed no selectivity with regard to any of the variants of AAG. The differences in drug-binding demonstrated between the A variant and the F1S mixture confirmed those of a previous study, in which a selective binding of imipramine to the A variant and of warfarin and mifepristone to the F1S mixture have been found. These results indicate specific drug transport roles for each AAG variant, according to its separate genetic origin. The results of control binding experiments performed with (unfractionated) commercial AAG and the series of tested ligands concurred with that for the separate AAG variants, with respect to the proportion of the A variant (27%) and that of the F1 and S variants (73%) in the commercial protein. In addition, disopyramide, methadone, dipyridamole, chlorpromazine, lignocaine and progesterone were used in equilibrium dialysis displacement experiments to study interactions on binding sites labelled with imipramine for the A variant and with warfarin for the F1S variant mixture. The four latter ligands were found to competitively inhibit the binding of warfarin to the F1S variant mixture and all of them that of imipramine to the A variant. The ligands association constants to each AAG variant obtained from such inhibitory experiments were comparable to those determined in the direct binding studies. As the stochlometry of the interactions of the A variant and the F1S variants, respectively, with their specific ligands was approximately one (1), it was concluded that these ligands bind to each of these variants via a single common binding site. These results indicate that the AAG molecule would have for its ligands at least two separate binding sites, showing different specificity and localization, and not one site, as it is generally assumed. The possible pharmacological and clinical consequences of the binding results with the separate AAG variants are discussed.

Two-step chromatographic purification of human plasma alpha(1)-acid glycoprotein: its application to the purification of rare phenotype samples of the protein and their study by chromatography on immobilized metal chelate affinity adsorbent.

Alpha1-Acid glycoprotein (AAG) or orosomucoid was purified to homogeneity from human plasma by a separate two-step method using chromatography on immobilized Cibacron Blue F3G-A to cross-linked agarose and chromatography on hydroxyapatite. The conditions for the pre-purification of AAG by chromatography on immobilized Cibacron Blue F3G-A were first optimized using different buffer systems with different pH values. The overall yield of the combined techniques was 80% and ca. 12 mg of AAG were purified from an initial total amount of ca. 15 mg in a ca. 40 ml sample of human plasma. This method was applied to the purification of AAG samples corresponding to the three main phenotypes of the protein (FI*S/A, F1/A and S/A), from individual human plasma previously phenotyped for AAG. A study by isoelectric focusing with carrier ampholytes showed that the microheterogeneity of the purified F1*S/A, F1/A and S/A AAG samples was similar to that of AAG in the corresponding plasma, thus suggesting that no apparent desialylation of the glycoprotein occurred during the purification steps. This method was also applied to the purification of AAG samples corresponding to rare phenotypes of the protein (F1/A*AD, S/A*X0 and F1/A*C1) and the interactions of these variants with immobilized copper(II) ions were then studied at pH 7, by chromatography on an iminodiacetate Sepharose-Cu(II) gel. It was found that the different variants encoded by the first of the two genes coding for AAG in humans (i.e. the F1 and S variants) interacted non-specifically with the immobilized ligand, whereas those encoded by the second gene of AAG (i.e. the A, AD, X0 and C1 variants) strongly bound to immobilized Cu(II) ions. These results suggested that chromatography on an immobilized affinity Cu(II) adsorbent could be helpful to distinguish between the respective products of the two highly polymorphic genes which code for human AAG.

[Reference intervals and biological profile in a group of healthy elderly population in the Paris region]. / Intervalles de référence et profil biologique d'une population de sujets âgés "en bonne santé" habitant la région parisienne. (Group d'évaluation et de recherche des biologistes de l'APHP).

The superimposition of pathological processes on ageing-related metabolic changes makes the interpretation of laboratory data difficult in the elderly. Therefore, in order to establish reference values of anthropometric, hematological and biochemical variables, we have carefully selected fit, health-conscious elderly subjects on the basis of clinical and biological criteria. We observed a trend for a wider range of values than in young adults. For some variables, the values were shifted down (eg: albumin, vitamin D) or up (eg: glucose, urea, cholesterol, ferritin), as a result of slow metabolic changes or progressive functional decline of different organs. The high prevalence of hypovitaminosis D was worthy of note, particularly in women, suggesting a high risk of deficiency.

Extensive laboratory assessment of nutritional status in fit, health-conscious, elderly people living in the Paris area. Research Group on Aging.

OBJECTIVE: Nutritional status-related biological indexes were measured in fit, health-conscious elderly subjects in order to establish reference values for people over 70 years. SUBJECTS: 103 men and 90 women aged 70-89 years living freely in the Paris area volunteered to participate. METHODS: Nutritional status was assessed by anthropometric and biochemical methods. RESULTS: Serum protein and amino acid status was similar to that of young adults, with only 5.2% of the elderly subjects showing transthyretin concentrations < 0.20 g/L, as well as decreased essential amino acid levels. Iron status, assessed in terms of serum and erythrocyte ferritin levels, total iron binding capacity and erythrocyte protoporphyrin tended to be satisfactory, but iron depletion was detected in 8.8% of the subjects. Serum ferritin levels were elevated in 19.7% of the subjects. Folate and vitamin B12 status was satisfactory, while hypovitaminosis D was observed in 48.2% of cases. CONCLUSION: Our findings suggest that, in aging uncomplicated by disease, nutritional status is similar to that in younger adults, although the range of values tended to be wider, with a higher risk of certain nutrient deficiencies.

Drug binding in plasma. A summary of recent trends in the study of drug and hormone binding.

The ligands are generally bound in plasma to a significant extent by several transport proteins (both high and low affinity), irrespective of their endogenous or exogenous origin. The protein binding of endogenous compounds (such as hormones) exhibits higher affinity and specificity than those of exogenous compounds (such as drugs). For plasma proteins that bind the same ligand(s), structural similarities or a common genetic origin may be found, although this is not a general rule. Alterations in ligand binding may be due to modifications of either the structure or the level of the binding protein. These modifications may result from genetic make up, physiology or pathology. In some situations, plasma binding may impair the distribution of drugs to tissues, with drug distribution then mainly restricted to the distribution compartment of the drug-binding protein. In other instances, the plasma drug-binding is permissive, and does not limit drug distribution to tissues. A given drug-transport protein system may have either a permissive or a restrictive effect on the drug distribution, depending on the tissue. The physiological significance of the high-affinity transport proteins is not completely understood. These proteins may increase the plasma concentration of poorly hydrosoluble ligands, ensure a more uniform tissue distribution and increase the life of the ligands. The life of the protein may also be increased by ligand binding. High-affinity transport proteins are also involved in some specific carrier mediated transfer mechanisms. It is possible to demonstrate structure-binding relationships or binding selectivity for the plasma transport proteins, but these are quite independent of relationships observed at the receptor level.

Evidence for differences in the binding of drugs to the two main genetic variants of human alpha 1-acid glycoprotein.

1. Human alpha 1-acid glycoprotein (AAG), a plasma transport protein, has three main genetic variants. F1. S and A. Native commercial AAG (a mixture of almost equal proportions of these three variants) has been separated by chromatography into variants which correspond to the proteins of the two genes which code for AAG in humans: the A variant and a mixture of the F1 and S variants (60% F1 and 40% S). Their binding properties towards imipramine, warfarin and mifepristone were studied by equilibrium dialysis. 2. The F1S variant mixture strongly bound warfarin and mifepristone with an affinity of 1.89 and 2.06 x 10(6) l mol-1, respectively, but had a low affinity for imipramine. Conversely, the A variant strongly bound imipramine with an affinity of 0.98 x 10(6) l mol-1. The low degree of binding of warfarin and mifepristone to the A variant sample was explained by the presence of protein contaminants in this sample. These results indicate specific drug transport roles for each variant, with respect to its separate genetic origin. 3. Control binding experiments performed with (unfractionated) commercial AAG and with AAG isolated from individuals with either the F1/A or S/A phenotypes, agreed with these findings. The results for the binding of warfarin and mifepristone by the AAG samples were similar to those obtained with the F1S mixture: the mean high-affinity association constant of the AAG samples for each drug was of the same order as that of the F1S mixture: the decrease in the number of binding sites of the AAG samples, as compared with the F1S mixture, was explained by the smaller proportion of variants F1 and/or S in these samples. Conversely, results of the imipramine binding study with the AAG samples concurred with those for the binding of this basic drug by the A variant, with respect to the proportion of the A variant in these samples.

Dextromethorphan O-demethylation and dextrorphan glucuronidation in a French population.

The dextromethorphan (DMP) O-demethylation and the dextrorphan (DRP) glucuronidation distributions were studied in 120 French Caucasian subjects. After a single 25 mg DMP oral administration, DMP, free and total DRP concentrations were measured in 8h-urine collection, using an HPLC technique with fluorescent detection. The DMP and free DRP concentrations ratio, in log form, was used to estimate the oxidative demethylation phenotype of the subjects. Two different populations were found. The first one consisted of the extensive metabolizers (90.8%, 95% confidence interval from 85.6 to 95.9%) and the second one consisted of poor metabolizers (9.2%, 95% confidence interval from 4.0 to 14.4%). The antimode value of the distribution was estimated at approximately 0.7 corresponding to a ratio of 5. Moreover, this ratio was compared to the DMP and total DRP concentrations ratio, usually defined to DMP O-demethylation phenotyping. On the other hand, the glucuronide DRP concentration was calculated by subtracting the free DRP concentration from the total DRP concentration. Consequently, the free DRP and glucuronide DRP concentrations ratio was also used to estimate the DRP glucuronidation in the present population. This ratio in log form reflected the UDP-glucuronyltransferase(s) capacity(ies). This log ratio appeared to be normally distributed in the population studied. These results show that log DMP/free DRP ratio can be used, as well as log DMP/total DRP ratio, to determine the oxidative phenotype of subjects and that the DRP conjugation does not exhibit any apparent genetic polymorphism.

Fractionation of the genetic variants of human alpha 1-acid glycoprotein in the native form by chromatography on an immobilized copper(II) affinity adsorbent. Heterogeneity of the separate variants by isoelectrofocusing and by concanavalin A affinity chromatography.

Fractionation of the three main genetic variants (F1, S and A) of human alpha 1-acid glycoprotein (AAG), in their native (sialylated) form, by chromatography on immobilized copper(II) affinity adsorbent was investigated. This chromatographic method had been previously developed to fractionate the desialylated protein variants. For that purpose, the three main AAG phenotypes samples (F1S/A, F1/A and S/A), which had been previously isolated from individual human plasma samples, and an AAG sample from commercial source (a mixture of the phenotypes) were used in the native form. Affinity chromatography of these different samples on an iminodiacetate Sepharose-copper(II) gel at pH 7 resolved two protein peaks, irrespective of the origin of the native AAG sample used. The unbound peak 1 was found to consist of the F1, the S or both variants, depending on the phenotype of the AAG sample used in the chromatography. The bound peak 2 was found to consist of the A variant in a pure form. The fractionation results obtained with native AAG were found to be the same as those originally yielded by the desialylated protein. However, comparison of the interactions of native and desialylated AAG with immobilized copper(II) ions, using an affinity chromatographic method and a non-chromatographic equilibrium binding technique, respectively, showed that desialylation increased the non-specific interactions of the protein with immobilized copper(II) ions. The AAG variants were not fractionated when affinity chromatography was performed using immobilized zinc, nickel or cobalt(II) ions, instead of copper. After purification of each variant in the sialylated form (F1, S and A), their respective heterogeneity was studied by analytical isoelectrofocusing with carrier ampholytes in the pH range 2.5-4.5. In addition, the lectin-binding behaviour of the separate sialylated AAG variants was investigated by affinity chromatography on immobilized concanavalin A.

Orosomucoid system: 17 additional orosomucoid variants and proposal for a new nomenclature.

There are two forms of orosomucoid (ORM) in the sera of most individuals. They are encoded by two separate but closely linked loci, ORM1 and ORM2. A number of variants have been identified in various populations. Duplication and nonexpression are also observed in some populations. Thus, the ORM system is very complicated and its nomenclature is very confusing. In order to propose a new nomenclature, ORM variants detected by several laboratories have been compared and characterized by isoelectric focusing (IEF) followed by immunoprinting. A total of 57 different alleles including 17 new ones were identified. The 27 alleles were assigned to the ORM1 locus, and the others to the ORM2 locus. The designations ORM*F1, ORM1*F2, ORM1*S and ORM2*M were adopted for the four common alleles instead of ORM1*1, ORM1*3, ORM1*2 and ORM2*1 (ORM2*A), respectively. The variants were designated alpha numerically according to their relative mobilities after IEF in a pH gradient of 4.5-5.4 with Triton X-100 and glycerol. For the duplicated genes a prefix is added to a combined name of two alleles, e.g. ORM1*dB9S. Silent alleles were named ORM1*Q0 and ORM2*Q0 conventionally. In addition, the effects of diseases to ORM band patterns after IEF are also discussed.

pH titration curves of the desialylated human alpha 1-acid glycoprotein variants by combined isoelectrofocusing-electrophoresis: utilization in the development of a fractionation method for the protein variants by chromatography on immobilized metal affinity adsorbent.

Using a two-dimensional isoelectrofocusing (IEF)-electrophoresis technique, the pH titration curves of the three main desialylated variants (F1, S and A) of human alpha 1-acid glycoprotein (AAG) were studied to assist in the development of a fractionation method for the AAG variants. For this purpose, different AAG samples, each corresponding to one of the three main phenotypes of the protein (F1S/A, F1/A and S/A), were first purified by chromatographic separation of individual human plasma samples on immobilized Cibacron Blue F3G-A. The purified AAG samples were then disialylated and their heterogeneity was checked by analytical IEF. The pH-mobility curves of the desialylated AAG samples were displayed in polyacrylamide gel slabs, under a constant set of experimental conditions, by carrying out electrophoresis of the protein samples perpendicularly to two stationary pH gradients: a large gradient (pH 3.5-9.5) and a narrow gradient (pH 5-8). The curves showed that all the desialylated variants of AAG exhibited small charge differences and moved closely together between about pH 3.5-5.5 and pH 7.5-9.5. However, the variants were found to show microheterogeneity in their total charge between about pH 5.5 and 7.5 due to the titrated ionizable groups involved along this pH zone. This microheterogeneity was assumed to be accounted for by the existence of differences between the titratable histidyl residues of the AAG variants. Consequently, the interactions of the variants with immobilized transition metal ions were studied at pH 7, using affinity chromatography on an iminodiacetate Sepharose-Cu(II) gel. It was found that the A variant was strongly bound by immobilized Cu(II) ions, whereas the F1 and S variants interacted non-specifically with the immobilized ligand. This finding allowed the development of a rapid and effective fractionation method for desialylated AAG into its A and F1 or S variants, depending on the AAG phenotype, by chromatography on an immobilized affinity Cu(II) adsorbent. The quantitative relationships between immobilized Cu(II) ions and desialylated AAG (the apparent association constant and gel protein-binding capacity) were also determined using a non-chromatographic equilibrium binding technique.