Competitive Protein Binding Assay Research Articles

125I-RIA kit cannot distinguish vitamin D deficiency as well as a more specific assay for 25-hydroxyvitamin D

Objectives: To compare a new radioimmunoassay (RIA), measuring vitamin D metabolites, against an established, competitive-protein binding assay (CPBA) that uses rat vitamin D binding protein. Methods: For the CPBA, we first isolated 25-hydroxyvitamin D (25(OH)D) by eluting sample through silica cartridges. For the RIA, serum was added to acetonitrile and centrifuged, followed by RIA using 125I-25(OH)D 3. Results: The within-run CV was 1.5% to 4.1% by RIA, compared to 8.1% to 15.0% by CPBA. For both methods, analytical recovery was not significantly different from 100%, and both methods were appropriately linear with sample dilution. We compared RIA values (y axis) from 90 subjects with 25(OH)D ranging from 11 to 232 nmol/L by CPBA (x axis). The slope was 0.9992 (not significantly different from 1). However, the RIA exhibited a bias of 11.15 nmol/L ( p < 0.05 vs 0), and a correlation coefficient of r = 0.923. When the comparison was confined to the 37 samples in the lower half of our reference range, the RIA did not compare well: slope, 0.764 ( p < 0.05 vs 1.0); intercept 18.7 nmol/L ( p < 0.05 vs 0); r = 0.517. For the samples below 36 nmol/L by CPBA, there was no discrimination by RIA between the 11 values below, and the 11 above the decision level (25 nmol/L by CPBA) for vitamin D deficiency. Conclusions: We conclude that the RIA is not optimized to detect vitamin D deficiency, and for this purpose it is not a valid substitute for conventional 25(OH)D methods.

A Simple Competitive Protein-Binding Experiment

This experiment was developed for teaching the concept of competitive protein-binding assays, but with the use of inexpensive reagents and commonly available laboratory equipment in place of specialized immunoassay reagents and radiation counting equipment.

Receptor-ligand interactions between serum amyloid P component and model soluble immune complexes.

We report here that isolated human serum amyloid P (SAP) binds aggregates of human IgG (AAg), which are model soluble immune complexes. The binding interaction was specific with regard to AAg compared to monomeric IgG, saturable, of high affinity, and reversible. With SAP adsorbed to microtiter plate wells, protein binding assays of AAg preparations with m.w. values of 1.05 to 2.25 x 10(6) were performed. The assay results yielded binding isotherms and estimates of the dissociation constant values of SAP:AAg binding ranging between 0.60 and 1.9 nM. Using the same method, the dissociation constant of SAP binding of biotinylated AAg was estimated to be approximately 3.0 nM. Competitive protein binding assays using biotinylated AAg and unlabeled AAg or monomeric IgG were performed to determine the relative affinity (IC50) of SAP binding. The IC50 values ranged from 22 to 51 nM for different sizes of AAg. The IC50 determined for monomeric IgG was 10 microM, demonstrating a relative specificity of SAP binding for AAg. The binding interaction was reversible, the SAP:AAg complexes dissociating with a t1/2 of 125 min. AAg binding to solid phase-adsorbed SAP was not dependent upon the presence of several cations, including calcium, but was not supported by cuprous ion. The differential binding of AAg vs monomeric IgG reported here for SAP is similar to the other human immune complex binding proteins C1q, rheumatoid factor, and cellular Ig C-region receptors. These results indicate that SAP:AAg binding has the hallmarks of a receptor:ligand interaction and may have implications for the disposal of immune complexes in vivo.

Human plasma free activin and inhibin levels during the menstrual cycle.

The role of inhibin in gonadal function and reproduction has been confirmed by the measurement of plasma inhibin levels, but there has been no clinical data available on activin because of the lack of a good assay method. We measured plasma free activin levels during the normal menstrual cycle using a newly developed competitive protein binding assay with follistatin as the binding protein. Plasma inhibin levels were measured simultaneously using an alpha-subunit N-terminal fragment RIA with recombinant inhibin as the reference standard. Four normal women, aged 23-29 years, were investigated by obtaining plasma at 3-day intervals. Plasma inhibin levels showed some variation during the follicular phase, but a parallel rise in inhibin and progesterone was observed during the luteal phase. These findings confirmed those of previous studies. In contrast, plasma free activin levels did not show any substantial changes during the menstrual cycle. This study suggests that activin has no endocrine role in modulating the pituitary-gonadal axis during the normal menstrual cycle, while changes of inhibin reflect cyclic gonadal function and indicate an endocrine role for this protein in modulating gonadal activity.

Modification of a Competitive Protein Binding Assay for Determination of Inositol 1,4,5-Trisphosphate

A modification of a competitive protein-binding method for the determination of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), facilitated by the use of a cell harvester, is described. The method is based on the competition of [3H]Ins(1,4,5)P3 with Ins(1,4,5)P3 in the sample for binding to a binding protein prepared from bovine adrenal cortex. The assay is carried out in 96-well microtiter plates in a final volume of 100 μl and free [3H]Ins(1,4,5)P3 is separated from bound by filtration using a cell harvester. This allows the rapid measurement of large numbers of samples, with high reproducibility. Ins(1,4,5)P3 bound to a single class of high-affinity receptors with a KD of 2.3 ± 0.2 nM and a Bmax of 289 ± 7 fmol/mg of protein. The binding was rapid, reaching equilibrium after 20 min, and reversible. The sensitivity of the assay allows the determination of amounts of Ins(1,4,5)P3 as small as 0.1 pmol, which corresponds to a concentration of 5 nM in the sample. The application of the method to measure the formation of Ins(1,4,5)P3 in DDT1 MF-2 smooth muscle cells activated with ATP or bradykinin is described. The modification is a rapid, sensitive, convenient, and relatively inexpensive method for the determination of Ins(1,4,5)P3 in large numbers of samples.

オスニホンザルにおける30分間隔による24時間内繰り返し強制保定採血が血中ホルモン値に及ぼす影響

We investigated effects of frequent blood collections under the compulsory restraints on serum testosterone, LH and glucocorticoids in adult male Japanese monkeys. Blood samples were withdrawn from three animals at 30-min intervals and two animals at 4-hr intervals during 24 hr. Serum testosterone and LH were measured by a radioimmunoassay, and glucocorticoids was determined by a competitive protein binding assay. Sampling at 4-hr intervals during 24 hr revealed diurnal changes in serum testosterone and glucocorticoids. Levels of testosterone were high throughout the night and low at the day time, conversely serum glucocorticoids were high levels in the morning and low in the evening. On the other hand, 30-min intervals sampling, serum testosterone levels decreased and glucocorticoids levels increased, respectively, immediately after the start of blood sampling. And then, low in testosterone and high in glucocorticoids levels were continually maintained. But serum LH levels had the pulsatile pattern and did not change markedly during 24 hr. After ACTH administration in five animals, serum glucocorticoids levels increased markedly and also testosterone levels increased slightly, but LH levels did not change. These data indicated that the every 30-min restraint stress caused the increment of glucocorticoids levels and the suppression of testosterone levels, but did not affect the serum LH levels. The increased glucocorticoids might inhibit the testicular steroidogenesis, without suppressing the LH secretion from the pituitary.

Activin and inhibin secretion by cultured porcine granulosa cells is stimulated by FSH and LH.

Follistatin-free activin and inhibin levels were measured in the culture medium of porcine granulosa cells by a competitive protein binding assay and N-fragment RIA, respectively. Cultures were maintained for 6 days in MEM containing 10% fetal calf serum. FSH and HCG (1-1,000 IU/L) were added on day 2 to cultures containing 1 x 10(5) cells/ml and the medium was changed every 2 days. Granulosa cells secreted a large amount of progesterone, which indicated their luteinization. Activin and inhibin were both secreted throughout the 6 day culture period. Both of them increased equally in response to FSH and HCG in a dose-dependent manner on day 4, but their responses became discrepant on day 6, when the activin response decreased while the response of inhibin increased. This study demonstrated that cultured porcine granulosa cells secrete both activin and inhibin under the control of FSH and LH. The activin response was lost before that of inhibin, suggesting the role of activin at an earlier stage of luteinization.

Enzyme immunoassay for plasma 25-hydroxyvitamin D 3 employing immunoaffinity chromatography as a pretreatment method

An enzyme immunoassay (EIA) of 25-hydroxyvitamin D 3 [25(OH)D 3] has been developed as a new methodology for measuring its plasma levels. Three anti-25(OH)D 3 antibodies elicited against 25(OH)D 3 3-hemisuccinate or -hemiglutarate conjugated with bovine serum albumin were used for this purpose. Two enzyme-labeled antigens were prepared by linking these haptens to β-galactosidase employing the N-succinimidyl ester method. An EIA system, selected from several combinations of the antibodies and labeled antigens, exhibited higher sensitivity and specificity than those of the conventional competitive protein binding assay. However, direct measurement of plasma specimens gave lower values than those obtained from the ones which were pretreated with a Sephadex® LH-20 column followed by a normal-phase high-performance liquid chromatography. This problem has been overcome by employing immunoaffinity chromatography (IAC). In IAC the homologous anti-25(OH)D 3 antibody with that used in the EIA was immobilized as a pretreatment method. The IAC/EIA system developed in this study afforded reliable plasma 25(OH)D 3 levelswhich confirmed by serial dilution study and the recovery test. The 25(OH)D 3 levels of healthy volunteers in the summer measured by this method were 25.2 ± 6.2 ng/ml ( n = 10).

Progesterone increases basal 3',5'-cyclic adenosine monophosphate formation and down-regulates the agonist-induced inositol phosphates generation in human term placenta.

Whether the placenta is a target tissue for estrogens and progesterone, and their putative mechanism of action, is still a controversial question in the literature. The effect of progesterone and estradiol on 3',5'-cyclic adenosine monophosphate (cAMP) and inositol phosphates generation in human term placenta was investigated. Placental explants were incubated in vitro for up to 48 h in the absence and in the presence of estradiol, progesterone or both steroids (0.1 mumol/l final concentration in all cases), and were stimulated with terbutaline, a beta-adrenergic agonist, (0.1 mmol/l) or angiotensin II (1 mumol/l). The cAMP content was measured by a competitive protein binding assay, and the generation of labelled inositol phosphates formation in explants prelabelled with 3H-myo-inositol was measured by anion exchange chromatography. Progesterone increased significantly basal cAMP concentrations in comparison with control or estradiol-treated tissues (169 +/- 13, 72 +/- 8, and 69 +/- 2 pmol/g wet wt tissue, mean +/- SEM, respectively). However, following terbutaline stimulation cAMP levels (mean +/- SEM) increased to similar values under all conditions (182 +/- 33, 197 +/- 36, and 237 +/- 17 pmol/g wet wt tissue for control, estradiol-, and progesterone-treated tissues, respectively). Angiotensin II stimulated inositol phosphates generation in placental explants by an average of fivefold, but this increase was significantly reduced in the presence of progesterone (5.2 +/- 0.7, 3.7 +/- 0.4, and 2.2 +/- 0.3 fold increase vs non-angiotensin-stimulated tissues, for control, estradiol-, and progesterone-treated placenta, mean- +/- SEM, respectively). These data suggest that progesterone modulates the formation of second messengers in human placenta at term.

The use of octadecyl-bonded microparticulate silica in the separation of free and bound fractions during saturation analysis of vitamin D metabolites.

The use of octadecyl-bonded microparticulate silica to separate free and bound fractions during the saturation analysis of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D has been investigated. A slurry of octadecyl-bonded silica in an appropriate incubation buffer was prepared and used in parallel with a conventional dextran-coated charcoal suspension in several assay procedures. Standard curves, non-specific binding and plasma values were compared. A competitive protein binding assay for 25-hydroxyvitamin D and two radioreceptor assays and one radioimmunoassay for 1,25-dihydroxyvitamin D were investigated. In most cases the octadecyl-bonded silica preparation gave the more favourable results; its action was rapid, time- and temperature-independent, and it produced low non-specific binding and higher B0 values in all the assays examined. It was in our hands easier to use than dextran-coated charcoal. The use of octadecyl-bonded silica is recommended as an efficient agent for the separation of free and bound fractions in the saturation analysis of vitamin D metabolites.

Evaluation of solubilizing agents for 25-hydroxyvitamin D 3 immunoassays

The serum or plasma levels of 2Shydroxyvitamin Ds [25(OH)Ds], the major circulating metabolite of vitamin D, are useful as an index of the overall vitamin D status of the body [l-3]. These levels are now usually measured by competitive protein binding assays (CPBA) employing serum vitamin D binding protein (DBP) [4121. However, tedious and time-consuming pretreatment of biological samples is necessary to remove some cross-reactive metabolites [4,5]. On the other hand, immunoassays using highly specific antibodies are expected as an alternative methodology which is more simple and feasible for routine use. The antibodies having much higher specificity than DBP would be producible by the immunization using well-designed haptenic derivative [2]. However, the limited solubility of 25(OH)Ds and its tendency to be adsorbed by glass walls pose a serious problem in common with CPBA methodology, namely, the binding reaction does not proceed to completion. Thus the addition of a proper solubilizing agent to the assay buffer is essential to the development of sensitive and reproducible assay systems. Although several solubilizing compounds have been recommended for CPBA [6-121, this has not been studied in immunoassays. From these points of view, we examined various compounds for their usefulness as a solubilizing agent in the radioimmunoassay (RIA) of 25(OH)D3.

Competitive protein binding assay for activin [formula omitted] using follistatin determination of activin levels in human plasma

A sensitive and specific protein binding assay for activin A EDF (activin) was developed using follistatin as a binding protein and [ 125I] labelled activin as a tracer. As 50% acetonitrile (CH 3CN) separated free and follistatin-bound activin, plasma pretreated with an equal volume of CH 3CN was used as the assay sample and B F separation was also done with 50% CH 3CN. The recovery of the assay was 85.0% and its sensitivity was 0.5 ng/ml. Crossreactivity with inhibin A was 1.8%. The mean plasma level of follistatin-free activin in normal subjects was 1.3±0.7 (M±SD) ng/ml. Plasma free activin levels were generally elevated in patients with chronic renal failure or hematological diseases associated with anemia.

A method for the simultaneous determination of vitamins D 2,D 3 and their metabolites in plasma and its application to plasma samples obtained from normal subjects and patients

A method has been established for the simultaneous determination of vitamins D 2,D 3 and their metabolites in plasma. Vitamin D 2 D 3 , 25-hydroxyvitamin D 2 D 3 24 R,25-dihydroxyvitamin D 2 D 3 [ 24,25( OH) 2 D 2 D 3 ] and 1α,25-dihydroxyvitamin D 2 D 3 [ 1,25( OH) 2 D 2 D 3 ] in 1.5 ml of a plasma sample were simultaneously assayed. The method includes extraction of lipid, followed by three steps of high-performance liquid chromatography (HPLC) for clean-up and separation. Vitamin D 2 D 3 and 25- OH- D 2 D 3 were quantified by a UV detector, while 24,25( OH) 2 D 2 D 3 and 1,25( OH) 2 D 2 D 3 were assayed by competitive protein binding assay (CPBA) and radioreceptor assay (RRA) methods, respectively. Existing interfering substances in a sample could be effectively eliminated by HPLC and reliable results with small coefficients of variation were obtained. The method was applied to plasma samples obtained from normal subjects and patients with epilepsy, liver cirrhosis, leukaemia, diabetes, sensorineural deafness and hypophosphataemic osteomalacia; satisfactory results were obtained.

Determination of cyclosporine by a competitive binding assay with cyclophilin

A competitive protein-binding assay for cyclosporine based on use of the intracellular cyclosporine-binding protein cyclophilin (CYP) was used to measure cyclosporin A (CsA) and its bioactive metabolites in whole blood. CYP from cytoplasmic extracts or erythrocyte lysates was applied in the binding assay with use of [3H]CsA as tracer and charcoal adsorption for separating bound from free tracer. Binding affinities of various CsA analogs and metabolites correlated well with their reported in vitro immunosuppressive activities. The assay detected as little CsA as 50 micrograms/L (1 g = 0.832 mmol of CsA), analytical recovery was greater than 80%, and CVs were less than 8% for intra-assay and less than 11% for interassay precision in the range of 150-1000 micrograms/L. We used this assay to measure CsA concentrations in blood and compared the results with those measured by HPLC or by CsA-specific (monoclonal) and CsA-nonspecific (polyclonal) radioimmunoassays. Binding assay results were, in nearly all cases, less than those measured by the nonspecific RIA and frequently greater than 20% above the values determined by the CsA-specific assays. Individual patients had pronounced differences in the relative proportions of CsA, CYP-binding (bioactive) metabolites, and cross-reacting CsA metabolites. Because the presence of bioactive metabolites may considerably contribute to the immunosuppressive activity of CsA, we consider the binding assay clinically useful for measuring CsA in biological fluids.

腎癌患者におけるビタミンD代謝の検討

Recently it has been known that the active form of vitamin D, 1 alpha, 25-dihydroxyvitamin D (1 alpha, 25-(OH)2D), plays some immunological roles in controlling cell differentiation and carcinogenesis. Moreover, 1 alpha, 25-(OH)2D is said to have some effects in the changes of several numbers of oncogenes. In this study, the serum concentrations of 1 alpha, 25(OH)2D and 25-hydroxyvitamin D (25-(OH)2D) were measured in 30 patients with renal cell carcinoma. The two vitamin D metabolites were separated and purified using high performance liquid chromatography, and each fraction was subjected to competitive protein binding assay using vitamin D deficient rat serum for 25-(OH)2D and chick embryo intestinal receptors for 1 alpha, 25-(OH)2D as the binder. The average concentration of serum 1 alpha, 25-(OH)2D was 28.9 +/- 5.2 pg/ml for controls whereas the average in patients with renal cell carcinoma was 19.7 +/- 5.9 pg/ml. The concentration of 1 alpha, 25-(OH)2D was significantly lower in the patients with renal cell carcinoma compared to in the controls (p less than 0.01). The average concentrations of serum 25-(OH)2D had no significant differences between the two groups. Comparison between the stage I + II and stage III + IV, T1 + T2 and T3 + T4, rapid type and slow type groups showed that the average serum 1 alpha, 25-(OH)2D concentrations was significantly lower in the stage III + IV, T3 + T4, and rapid type groups compared to the other groups (p less than 0.01, p less than 0.05, p less than 0.01). And besides there were no significant correlations between the concentration of serum 1 alpha, 25-(OH)2D and creatinine clearance in patients with renal cell carcinoma. Taken together, it is suggested that the serum concentration of 1 alpha, 25-(OH)2D is in some way correlated with the progression of renal cell carcinoma.

Hypothalamic paraventricular nucleus lesions do not prevent anorectic effect of exercise in male rats

The effects of a hypothalamic paraventricular nucleus (PVN) lesion on energy balance were investigated in exercise-trained rats. Male Wistar rats weighing initially 250 g were divided into four groups. Two groups of rats underwent a bilateral PVN lesion, whereas the two remaining groups were sham operated. The PVN lesions were done electrolytically. One group from each surgical treatment was exercised, while the other group was kept in sedentary conditions. Rats were exercised on a rodent motor-driven treadmill at moderate intensity, 1 h/day for 21 consecutive days. Food intake and body weight were measured each day during the study. At the end of the treatment period, rats were killed, and carcasses were analyzed for their energy content. Serum corticosterone was measured by a competitive protein-binding assay. Energy gain and energy intake were lower in exercised rats than in sedentary controls, regardless of whether they were sham or PVN lesioned. Concurrently, there was no difference in the energy gain between PVN-lesioned and sham-operated rats, despite the fact that PVN-lesioned rats ended the experiment with a larger body weight than the sham-lesioned animals. Serum corticosterone levels were lower in PVN-lesioned rats than in sham-lesioned rats. In conclusion, the present results indicate that the PVN, the hypothalamic nucleus predominantly controlling the pituitary-adrenal axis activity, is not a prominent structure in the regulation of energy balance in exercised male Wistar rats.

Production of insulin‐like growth factor II (IGF‐II) and different forms of IGF‐binding proteins by HT‐29 human colon carcinoma cell line

The serum-free medium conditioned by the human colon cancer cell line HT-29 contains insulin-like growth factors (IGF) that are entirely complexed to binding proteins (IGF-BP). Gel filtration in acid conditions of the cell-conditioned medium permits separation of IGF-BP from two molecular forms of IGF of 15,000 and 7,500 Mr. As determined by ligand blotting, IGF-BP are heterogeneous and constituted of three molecular forms of 31,000, 28,000, and 26,000 Mr. Using IGF-I and IGF-II radioreceptor assays, IGF-I radioimmunoassay (RIA), and competitive protein-binding assay specific for IGF-II, it is shown that the IGF-type eluting in 15 K and 7.5 K position from gel filtration is restricted to IGF-II. Its concentration is approximately 6 ng/10(6) HT-29 cells with 60% present as a high-molecular-weight form of IGF-II. This large 15 K IGF molecule is devoided of any IGF-binding activity and might represent incomplete processing of pro-IGF-II peptide. By contrast, the level of IGF-I detected by RIA is barely measurable and considered negligible (0.57 pg/10(6) HT-29 cells). Although these IGF-II-like peptides exhibit a growth-promoting activity on FR3T3 fibroblasts, they cannot stimulate, as recombinant IGF-I or IGF-II, 3H-thymidine incorporation into DNA of HT-29 cells, whatever the experimental conditions used. Finally, we have shown that IGF binding is restricted predominantly to the basolateral domain of the cell membrane by using HT-29-D4 clonal cells, derived from the parental HT-29 cell line, maintained in a differentiated state by culture in a medium in which glucose is replaced by galactose.

Correction of Vitamin D Deficiency in Elderly Long-stay Patients by Sunlight Exposure

The relationship between plasma 25-OHD and exposure to ultraviolet B (UVB) radiation in natural sunlight was measured in elderly long-stay patients from April to July 1984. Polysulphone film badges, calibrated in energy units, were worn by all subjects to measure accumulated UVB dose, and plasma 25-OHD was measured fortnightly by a competitive protein binding assay. A linear relationship between changes in plasma 25-OHD and UVB exposure was found. The result shows that exposure to sunlight for a few hours each week is sufficient to improve the vitamin D status of the elderly.

Plasma dexamethasone concentrations and the dexamethasone suppression test

Altered bioavailability or altered pharmacokinetics of dexamethasone (dex) may contribute to a positive Dexamethasone Suppression Test (DST) in psychiatric patients. We measured plasma dex and plasma cortisol concentrations in 32 patients with primary major depressive disorder (MDD), 14 patients with other psychiatric disorder, and 16 normal controls. Cortisol was measured by the competitive protein binding (CPB) assay and dex by RIA (IgG Corp.). Additionally, cortisol was measured by a fluorescent polarization immunoassay (FPIA) available on the Abbott TDx analyzer in an attempt to validate this method for the use in the DST. The agreement between FPIA and CPB cortisol results was excellent. Depressed nonsuppressors, by definition, had significantly higher mean plasma cortisol concentrations than depressed suppressors, psychiatric controls and normal volunteers at 8:00 AM, 3:00 PM, and 10:00 PM postdex. When DST nonsuppressors and suppressors were compared regardless of diagnostic group, plasma dex concentrations were significantly lower (p < 0.01) in the DST nonsuppressors. There was a significant negative correlation between plasma cortisol levels and plasma dex levels across all subjects at 8:00 AM (r = −0.365, n = 44, p < 0.05). When the subjects were sorted by diagnostic category, there was a strong, but not statistically significant, trend toward lower plasma dex concentrations in the melancholic nonsuppressors versus the melancholic suppressors and between the psychiatric control non-suppressors and the corresponding suppressors group. These relationships disappeared when we restricted our analyses to an empirically derived middle range of plasma dex concentrations within which the DST results were considered to be valid. We conclude that bioavailability or pharmacokinetics of dex may significantly contribute to DST results. Further investigation is needed to determine whether or not the quantification of dex and its metabolites and their determination at which specific timepoints during the DST will enhance the predictive or interpretive value of the DST in psychiatric patients.

A phase I clinical and pharmacological study of weekly intravenous infusions of piritrexim (BW301U)

Thirty-eight patients with advanced resistant cancers were enrolled on this study of piritrexim (PTX; BW 301U) administered intravenously weekly for 4 weeks. Of 50 courses of treatment begun, 39 evaluable 4-week courses of the drug were completed by this group of patients. Dosages ranged from 44 to 530 mg/m2/week. One patient at each dosage level received an initial weekly dose of PTX in oral form accompanied by pharmacokinetic blood sampling after the oral dose and also after a subsequent intravenous dose.Toxicities included mild nausea and vomiting, and moderate to severe peripheral vein phlebitis. Anemia and thrombocytopenia were the dominant hematological toxicities. One patient with pulmonary metastases from malignant fibrous histiocytoma experienced a 12-week partial response to PTX treatment at a dosage of 400 mg/m2/week.Pharmacokinetic analysis of plasma for PTX concentrations was accomplished utilizing a competitive protein binding assay. The estimated total body clearance ranged from 136 to 173 ml/min/1.73 m2. Mean terminal half-life after intravenous administration was 5.61 ± 2.38 h (S.D.), and after oral administration was 5.72 ± 2.04 h. Mean systemic bioavailability after oral administration was 75 ± 56%.