High-performance Liquid Chromatography Radioimmunoassay Research Articles

LC–MS/MS coupled with immunoaffinity extraction for determination of estrone, 17β-estradiol and estrone 3-sulfate in human plasma

Determination of estrogens in plasma is important in evaluation of effects of some anticancer drugs, such as aromatase inhibitors. However, as reported previously, high performance liquid chromatography–radio immunoassay (HPLC–RIA) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) with chemical derivatization require complicated sample preparation. In this study, a highly sensitive and simple method for determination of estrone (E1), 17β-estradiol (E2) and estrone 3-sulfate (E1S) in human plasma has been developed. Following diethylether extraction from plasma, analytes were purified by immunosorbents and then determined by LC–MS/MS using electrospray ionization (ESI). Immunosorbents were prepared by immobilization of specific antibodies raised against each analyte onto solid support. Use of selective immunosorbents in sample preparation removed interference in plasma samples that would cause ionization suppression, and markedly improved the sensitivity of LC–MS/MS for these analytes, without derivatization. Calibration curves of each analyte showed good linearity and reproducibility over the range of 0.05–50 pg/injection for E1, 0.2–50 pg/injection for E2 and 0.05–300 pg/injection for E1S, respectively. The mean values of lower limits of quantification (LLOQ) in human plasma corrected by recovery of deuterated estrogens (internal standard, I.S.) were 0.1892 pg/mL for E1, 0.7064 pg/mL for E2 and 0.3333 pg/mL for E1S, respectively. These LLOQ values were comparable to those previous reported using HPLC–RIA and LC–MS/MS. Using this method, the normal levels of three estrogens in healthy female plasma ( n = 5) were determined. The mean values of E1, E2 and E1S were 38.0 pg/mL (range 24.8–53.0), 34.3 pg/mL (22.6–46.6) and 786 pg/mL (163–2080), respectively. The immunoaffinity LC–MS/MS described here allows sensitive and accurate quantification of E1, E2 and E1S without laborious sample preparation.

Dermal fibroblasts pretreated with a sterol Δ 7-reductase inhibitor produce 25-hydroxyvitamin D 3 upon UVB irradiation

As dermis is a physiological site of vitamin D 3 photoproduction, the photo-endocrine vitamin D 3 system was studied in dermal fibroblasts. Dermal fibroblasts contain the vitamin D receptor and induce 1α,25-dihydroxyvitamin D 3-24-hydroxylase [CYP24] mRNA upon stimulation with 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3]. In addition, dermal fibroblasts contain mRNA of the vitamin D 3-25-hydroxylases (CYP2R1 and CYP27A1). However, we could not detect any 25-hydroxyvitamin D 3 [25OHD 3]-1α-hydroxylase mRNA in dermal fibroblasts and no CYP24 mRNA was induced upon ultraviolet [UVB] irradiation, even when endogenous 7-dehydrocholesterol content was elevated by pretreatment with the sterol Δ 7-reductase inhibitor BM15766. Nevertheless, dermal fibroblasts produce inactive vitamin D 3 metabolites that can be activated by epidermal keratinocytes as CYP24 mRNA is induced in epidermal keratinocytes but not in dermal fibroblasts after transfer of medium or cellular suspensions from BM15766-pretreated, UVB-irradiated fibroblasts. This CYP24 induction was UVB-dose dependent and was inhibited by ketoconazole. As revealed in a competitive binding assay, BM15766-pretreated dermal fibroblasts are able to produce 25OHD 3 upon UVB irradiation, but no 1,25(OH) 2D 3 was detected via combined high-performance liquid chromatography radioimmunoassay. The physiological relevance of dermal vitamin D 3 photoproduction and its subsequent conversion into 25OHD 3 remains elusive.

UVB‐induced 1,25(OH)2D3 production and vitamin D activity in intestinal CaCo‐2 cells and in THP‐1 macrophages pretreated with a sterol Δ7‐reductase inhibitor

Epidermal keratinocytes are able to produce 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and induce vitamin D activity upon UVB irradiation. To find out whether this property is keratinocyte specific, we investigated this characteristic in two other cell types, namely intestinal CaCo-2 cells and the macrophage-like differentiated THP-1 cells. THP-1 macrophages and preconfluent CaCo-2 cells contain the vitamin D receptor (VDR), possess 25-hydroxylase (CYP2R1 and CYP27A1) and 1alpha-hydroxylase (CYP27B1) activity, and survive the low UVB doses essential for vitamin D3 photoproduction. Upon irradiation, 24-hydroxylase (CYP24) mRNA is induced in both cell types pretreated with the sterol Delta7-reductase inhibitor BM15766 whereby the 7-dehydrocholesterol (7-DHC) content was increased. Transfection studies in CaCo-2 cells with a vitamin D response element-containing construct revealed the involvement of the VDR in this UVB-dependent CYP24 induction. The CYP24 inducing activity in BM15766-pretreated UVB-irradiated CaCo-2 cells and THP-1 macrophages was identified as 1,25(OH)2D3 by combined high-performance liquid chromatography radioimmunoassay. Addition of vitamin D binding protein to the CaCo-2 cells attenuated UVB-induced CYP24 induction suggesting the possibility of a paracrine or autocrine role for the photoproduced 1,25(OH)2D3. In conclusion, preconfluent CaCo-2 cells and THP-1 macrophages are able to induce vitamin D activity upon UVB irradiation and hence combine all parts of the vitamin D photoendocrine system, a characteristic which is therefore not keratinocyte specific.

HPLC–RIA analysis of steroid hormone profile in a virilizing stromal tumor of the ovary

The pathological steroid biosynthesis of a virilizing ovarian tumor was examined via high performance liquid chromatography–radioimmunoassay (HPLC–RIA) determination of the intratissular steroid concentrations. Sex cord-stromal tumor of the ovary was obtained surgically from an 18-year-old female patient with extremely high androst-4-ene-3,17-dione (4-en-dione) and testosterone (Test) blood serum levels. The tissue specimen was extracted with ethyl acetate and the extract was then purified on a C 18 mini-column with methanol–water eluents. Steroids were isolated by reversed-phase HPLC on a C 18-silica gel column with 51%, 55% and 64% v/v methanol–water eluents. Steroids in the collected eluent fractions were detected by the radioactivity of tritiated internal standards and then quantified by specific RIAs. In the tumor specimen, very high 17α-hydroxyprogesterone (17-OH-Prog; 6300 fmol/g), dehydro-epiandrosterone (2870 fmol/g), androst-4-ene-3,17-dione (3000 fmol/g), testosterone (5700 fmol/g) concentrations, and less progesterone (PROG; 320 fmol/g) and androst-5-ene-3β,17β-diol (5-en-diol; 320 fmol/g), were determined. Tissue levels of 5α-dihydrotestosterone (DHT), 5α-androstane-3α,17β-diol (3α-diol), 5α-androstane-3β,17β-diol (3β-diol), and 17β-estradiol were found to be 71, 20, 28, and 12 fmol/g, respectively. Steroid profile analysis verified a pathological steroid biosynthesis in the ovarian tumor and suggested that the 17α-hydroxylase (17α-H), 17,20-lyase (17,20-L), and 3β-hydroxysteroid dehydrogenase/Δ 5–4-isomerase (Δ 5-3β-HSD) activities were particularly elevated in this tumorous tissue. Present data demonstrate that the analysis of intratissular steroid profile by a HPLC–RIA method may valuably contribute to the steroidal pathophysiology of endocrine tumors.

Radioimmunoassay for a novel lignan-related hypocholesterolemic agent, S-8921, in human plasma after high-performance liquid chromatography purification and in human urine after immunoaffinity extraction

The novel compound methyl-1-(3,4-dimethoxyphenyl)-3-(3-ethylvaleryl)-4-hydroxy-6,7,8-trimethoxy-2-naphthoate (S-8921) has hypocholesterolemic activity in animals and is expected to exhibit a similar activity in human. Reversed-phase high-performance liquid chromatography (HPLC) separation followed by radioimmunoassay (RIA) for human plasma samples (HPLC–RIA) and immunoaffinity extraction (IAE) followed by RIA for human urine samples (IAE–RIA) were developed for investigation of S-8921 behavior in clinical studies. For the RIA, antisera from rabbit and a radioiodine-labelled S-8921 were prepared by immunizing a conjugate of S-8921 with bovine serum albumin and by the Bolton and Hunter method, respectively. HPLC–RIA using a semi-micro column was very sensitive, that is a 0.05 ng/ml limit of quantitation in human plasma, and specific for unchanged form of S-8921. IAE–RIA using a centrifugal filtration tube completely eliminated the matrix effect of human urine, and was very feasible. The limit of quantitation was 0.10 ng/ml. RIA detection following HPLC or IAE proved to be very useful for the pharmaceutical analysis of extremely low drug concentrations in body fluids.

Exemestane experience in breast cancer treatment

Exemestane is a very potent, orally active, selective and long-lasting steroidal irreversible inhibitor of aromatase. It is 150 times more potent than aminoglutethimide (AG) in inhibiting human placental aromatase (Ki of 4.3 and 671 nM, respectively). The compound is presently under phase III evaluation in Europe and the U.S.A. for the treatment of postmenopausal advanced breast cancer (ABC). Clinical pharmacology studies have been carried out with single doses ranging from 0.5 to 800 mg and repeated doses of up to 600 mg a day, in 132 postmenopausal healthy volunteers and in 185 post-menopausal women with ABC. Results obtained using a very specific and sensitive analytical method (high performance liquid chromatography—radioimmunoassay; HPLC—RIA) indicated that exemestane is extremely effective in inhibiting plasma estrogens levels. Estrogen inhibition is clearly evident at 5 mg a day and maximal suppression for E2, E1 and E1S (>85%, >90% and >90%, respectively) is obtained at 10–25 mg a day. Data from non-controlled phase II studies involving more than 400 patients indicated a clear anti-tumour activity in postmenopausal ABC patients failing multiple hormonal treatments. In 62 patients progressing on AG (≥500 mg a day) exemestane treatment resulted in an objective response rate of approximately 24%; disease stabilization ≥24 weeks was observed in an additional 24% of cases. With regard to safety, although daily doses up to 600 mg were administered, the maximal tolerated dose was not achieved; reported symptoms were mainly related to the pharmacological action of the compound and were usually mild to moderate in severity, resulting in the discontinuation of therapy in less than 3% of cases. In conclusion, the available results suggest that exemestane treatment is associated with minimal toxicity, and may be of significant benefit for ABC women who have exhausted conventional therapy.

Exemestane Experience in Breast Cancer Treatment

Exemestane is a very potent, orally active, selective and long-lasting steroidal irreversible inhibitor of aromatase. It is 150 times more potent than aminoglutethimide (AG) in inhibiting human placental aromatase (K i of 4.3 and 671 nM, respectively). The compound is presently under phase III evaluation in Europe and the U.S.A. for the treatment of postmenopausal advanced breast cancer (ABC). Clinical pharmacology studies have been carried out with single doses ranging from 0.5 to 800 mg and repeated doses of up to 600 mg a day, in 132 postmenopausal healthy volunteers and in 185 post-menopausal women with ABC. Results obtained using a very specific and sensitive analytical method (high performance liquid chromatography—radioimmunoassay; HPLC—RIA) indicated that exemestane is extremely effective in inhibiting plasma estrogens levels. Estrogen inhibition is clearly evident at 5 mg a day and maximal suppression for E 2 , E 1 and E 1 S (>85%, >90% and >90%, respectively) is obtained at 10–25 mg a day. Data from non-controlled phase II studies involving more than 400 patients indicated a clear anti-tumour activity in postmenopausal ABC patients failing multiple hormonal treatments. In 62 patients progressing on AG (≥500 mg a day) exemestane treatment resulted in an objective response rate of approximately 24%; disease stabilization ≥24 weeks was observed in an additional 24% of cases. With regard to safety, although daily doses up to 600 mg were administered, the maximal tolerated dose was not achieved; reported symptoms were mainly related to the pharmacological action of the compound and were usually mild to moderate in severity, resulting in the discontinuation of therapy in less than 3% of cases. In conclusion, the available results suggest that exemestane treatment is associated with minimal toxicity, and may be of significant benefit for ABC women who have exhausted conventional therapy.

Quantification of zidovudine and individual zidovudine phosphates in peripheral blood mononuclear cells by a combined isocratic high performance liquid chromatography radioimmunoassay method

Zidovudine (ZDV, AZT) is the first clinically effective drug licensed for use in the treatment of human immunodeficiency virus (HIV) infection. Activation of ZDV requires phosphorylation to ZDV triphosphate by cellular kinases. It is important, therefore, to determine the intracellular levels of the active form because measurement of ZDV concentrations in plasma have not reflected any direct relationship with activity or toxicity. In this paper a validated assay for the measurement of both ZDV and its three phosphorylated anabolites, ZDV mono-, diand triphosphate, in peripheral blood mononuclear cells (PBMCs) is described. The method consisted of a combination of isocratic high performance liquid chromatography (HPLC) separation and radioimmunoassay (RIA). The PBMCs were separated from whole blood and ZDV and ZDV nucleotides were extracted and separated by isocratic elution with an ion-pairing mobile phase on a reversed-phase HPLC column. The collected ZDV and individual ZDV nucleotide fractions were dephosphorylated to ZDV, cleaned by solid phase extraction and assayed by a commercially available RIA kit. The assay developed was successfully used to determine intracellular ZDV and anabolite concentrations of 10 PBMC samples taken from HIV positive patients on ZDV treatment.

Alterations in peptide levels in Parkinson's disease and incidental Lewy body disease.

The levels of the neuropeptides Met- and Leu-enkephalin (MET-ENK, LEU-ENK), substance P and neurotensin were measured by a combined high performance liquid chromatography/radioimmunoassay (HPLC/RIA) method in postmortem samples of basal ganglia from Parkinson's disease patients, incidental Lewy body disease patients (pre-symptomatic Parkinson's disease) and matched controls. Dopamine (DA) levels were reduced in the caudate nucleus and putamen in Parkinson's disease, but unaltered in incidental Lewy body disease. The levels of MET-ENK were reduced in the caudate nucleus, putamen and substantia nigra in Parkinson's disease. Met-enkephalin levels were reduced in the caudate nucleus and in the putamen in incidental Lewy body disease. Leu-enkephalin levels were decreased in the putamen and were undetectable in the substantia nigra in Parkinson's disease. Leu-enkephalin levels were unchanged in incidental Lewy body disease, although there was a tendency to a reduction in putamen. Substance P levels were reduced in the putamen in Parkinson's disease. No significant changes in substance P content were observed in incidental Lewy body disease. Neurotensin levels were increased in the substantia nigra in Parkinson's disease. Neurotensin levels in incidental Lewy body disease were not altered significantly, but tended to parallel the changes in Parkinson's disease. The changes in basal ganglia peptide levels in incidental Lewy body disease generally followed a trend similar to those seen in Parkinson's disease, but were less marked. This suggests that they are an integral part of the pathology of the illness and not secondary to DA neuronal loss or a consequence of prolonged drug therapy.

Ecdysteroid titers during postembryonic development of Dermanyssus gallinae (Acari: Dermanyssidae).

Ecdysteroid titers in methanolic extracts of protonymphal and deutonymphal stages of the chicken mite, Dermanyssus gallinae (De Geer), were measured using radioimmunoassay. Highest titers of ecdysteroids were present 24 h after feeding. Further studies using high-performance liquid chromatography radioimmunoassay showed that ecdysteroid immunopositive material had the same retention time as 20-hydroxyecdysone and ecdysone. Ecdysteroid conjugates appear to be present in polar and apolar fractions.

Influence of Estrogen on Prostacyclin and Thromboxane Balance in Postmenopausal Women

Abstract Although the cardioprotective effect of estrogen is well recognized, the mechanism by which this effect occurs is not well understood. One possible mechanism may involve an alteration in the local vascular equilibrium between prostacyclin (PGI2) and thromboxane A2 (TxA2), favoring PGI2. To test this hypothesis, we studied the estrogenic effect on urinary PGI2/TxB2 balance, both acutely and long term, in postmenopausal women. Seven subjects received estradiol (E2) during one visit and only the vehicle during another visit (control). Both were administered intravenously for 8 h. Serum E2 levels of 200 pg/ml were attained after E2 treatment. An additional eight subjects were treated subcutaneously with two 25-mg E2 pellets for 24 weeks; the serum E2 levels averaged between 80 and 120 pg/ml. Also, 12 premenopausal women were used for comparison of baseline prostanoid values with those of postmenopausal women. A highly specific and sensitive high-performance liquid chromatography/radioimmunoassay (HPLC-RIA) was used to quantitate PGI2 and TxA2 by measuring their stable metabolites, 6-keto-prostaglandin F1 (6-keto-PGF1 and thromboxane B2 (TxB2), respectively, in urine. The 6-keto-PGF1 and TxB2 levels in postmenopausal women were 35.6 ± 6.5 and 20.5 ± 3.8 ng/g of creatinine compared with 32.2 ± 5.3 and 16.7 ± 3.9 ng/g in premenopausal women, respectively. After E2 infusion, there was a significant increase (41%) in mean 6-keto-PGF1 levels, a 35% drop in mean TxB2 levels, and a significantly greater (65%) 6-keto-PGF1/TxB2 ratio, compared to the controls. After long-term E2 treatment, there was a progressive decrease in mean TxB2 levels and a transient increase in 6-keto-PGF1 levels. The results of our study suggest that the anticipated effects of E2 treatment on PGI2 and TxA2 metabolism in postmenopausal women do occur.

Measurement of canine urinary thromboxanes by GC-MC and HPLC-RIA

Immunoaffinity extraction/gas chromatography-mass spectrometry (IA/GC-MS) and high-performance liquid chromatography-radioimmunoassay (HPLC-RIA) methods were developed to analyse a major human urinary thromboxane metabolite 2,3-dinor thromboxane B 2, in the urine of dogs, a species commonly used for functional studies of thromboxane pharmacology. The β-metabolite 2,3-dinor TXB 2 was unequivocally identified in pooled normal canine urine by IA/GC-MS, and its excretion measured in 6 anesthetized dogs over a 7h period as 2001 ± 132 pg 2,3-dinor TXB 2/mg creatinine (range 624–4493 pg/mg). Thromboxane immunoreactivity co-eluting with synthetic 2,3-dinor TXB 2 was also identified by HPLC-RIA and similarly determined (2585 ± 276 pg/mg creatinine). Exogenous 2,3-dinor TXB 2 could be quantitatively recovered by both methodologies over a wide range of concentrations (50–5000 pg/mL), although with better precision by IA/GC-MS (added vs recovered; m = 1.05, r = 0.99) compared with HPLC-RIA (added vs recovered; m = 0.89, r = 0.89). The cyclooxygenase inhibitor indomethacin given by infusion in anaesthetized dogs (2.5, 8 and 25 μg/kg/min) dose-dependently inhibited 2,3-dinor TXB 2 excretion measured by IA/GC-MS, with maximal inhibition (83.0 ± 4.2%) being achieved after 6 h (25 μg/kg/min). Similar results were obtained by HPLC-RIA, with a correlation of 0.88 (slope = 0.9) between the methodologies in samples after drug treatment. These data suggest that the profile of metabolism and excretion of thromboxanes in dogs resembles that of man, and provide a useful animal model for the non-invasive in vivo assessment of inhibitors of thromboxane biosynthesis.

Urinary leukotriene E4 excretion in exercise-induced asthma.

Recent evidence suggests that the cysteinyl-leukotrienes (LTC4, LTD4, and LTE4) may be important in the pathogenesis of exercise-induced asthma. To evaluate the role of these mediators further, nine asthmatic subjects with exercise-induced bronchoconstriction were studied on two occasions. On visit 1, subjects performed 6 min of treadmill exercise; the mean maximal percent fall in FEV1 was 38.0 +/- 5.3%. On visit 2, maximal bronchoconstriction observed after exercise was matched with aerosolized methacholine. Urine was collected in two 90-min fractions (0-90 and 90-180 min) after challenges and analyzed by high-performance liquid chromatography-radioimmunoassay for LTE4. There were no significant differences in urinary LTE4 excretion between exercise and methacholine challenges for the periods 0-90 min (16.9 +/- 5.4 vs. 20.4 +/- 4.2 ng/mmol urinary creatinine), 90-180 min (24.9 +/- 8.2 vs. 20.1 +/- 5.5), or 0-180 min (21.5 +/- 6.5 vs. 18.8 +/- 4.1). Thus in contrast to allergen-induced bronchoconstriction, there is little evidence for enhanced cysteinyl-leukotriene generation in exercise-induced bronchoconstriction as assessed by urinary LTE4. If local release and subsequent participation of functionally active cysteinyl-leukotrienes in the pathways that ultimately lead to bronchoconstriction after exercise challenge do occur, these are of insufficient magnitude to perturb urinary LTE4 excretion.

Profile of embryonic ecdysteroids in the decapod crustacean,Macrobrachium rosenbergii

Summary The ecdysteroid composition of embryos of M. rosenbergii at various stages of embryogenesis has been studied by means of radioimmunoassay (RIA), high-performance liquid chromatography—RIA (HPLC-RIA) and gas chromatography/mass spectrometry (selected ion monitoring; GC/MS(SIM). In newly-laid eggs, only free ecdysteroids (ecdysone and 20-hydroxyecdysone) were detected, whereas eggs at an early stage of embryogenesis contained ecdysone and also low levels of apolar ecdysteroid conjugates. Mid stage embryonating eggs contained comparatively high levels of both free ecdysteroid (ecdysone, 18.3 ng/g) and apolar conjugates (29.9ng/g). Late embryos contained by far the highest overall level of ecdysteroid, indicating the ability of the developing embryo to synthesize ecdysteroid. In contrast to the other stages, late embryos contained 26-hydroxyecdysone, ecdysonoic acid and 20-hydroxyecdysonoic acid at relatively low levels and 2, 3-diacetylecdysone 22-phosphate at a comparatively high level (324.0 ng/g)....

Level of Abscisic Acid in Integuments, Nucellus, Endosperm, and Embryo of Peach Seeds (Prunus persica L. cv Springcrest) during Development

Free abscisic acid (ABA) in integuments, nucellus, endosperm, and embryo was determined throughout seed development of peach (Prunus persica L. cv Springcrest). Quantification of ABA was performed using combined high performance liquid chromatography-radioimmunoassay based on a monoclonal antibody raised against free (S)-ABA. In the integuments and endosperm, ABA concentration remained constant during the first 100 days after anthesis and rose in the following days when fresh weight was rapidly decreasing. In the nucellus, the ABA concentration variation pattern paralleled that of tissue growth. ABA concentration in the embryo increased constantly with the growth of the tissues to reach a maximum at the last growth stage. The role of ABA in peach seeds is discussed in relation to the development of the different seed tissues.

Analysis of Hemolymph Ecdysteroids in the Female Earwig:Labidura riparia

Summary The nature of hemolymph ecdysteroids was analyzed by high performance liquid chromatography-radioimmunoassay, during the two main phases of the reproductive cycles of Labidura riparia. 20-hydroxyecdysone and ecdysone were present together with polar and apoiar products. Changes in the ecdysone/20-hydroxyecdysone ratio were evidenced according to the ovarian development. During natural (feeding and sexual phase of the cycle) or induced ovarian development, an increase of the ratio occurred whereas, during ovarian rest (fasting and/or parental phase), a significant decrease was observed. Up to 40 days after ovariectomy, immunoreactive compounds were observed in the hemolymph which corresponded partly to 20-hydroxyecdysone, ecdysone, and apoiar products. However, a large increase was observed in the proportions of the polar products which were mainly resolved, on the action of Helix enzymes, into 20-hydroxyecdysone and ecdysone.

Comparative Analyses of Cyclosporine in Whole Blood and Plasma by Radioirmmunoassay, Fluorescence Polarization Immunoassay, and High-Performance Liquid Chromatography

Cyclosporine (CsA) concentrations were prospectively monitored for 6 months after transplantation in 66 consecutive renal transplant recipients. Analysis for CsA was performed with polyclonal radioimmunoassay (RIA), high-performance liquid chromatography (HPLC), and fluorescence polarization immunoassay (FPIA) in whole blood and plasma, and with specific and nonspecific monoclonal RIA in whole blood. Precision within and between runs was best with FPIA, followed by HPLC and RIA. A strong correlation was observed between the results obtained with HPLC and specific monoclonal RIA (r = 0.98). Correlation coefficients between the other assays ranged from 0.56 (plasma HPLC versus blood polyclonal RIA) to 0.91 (blood nonspecific monoclonal RIA versus blood FPIA). The highest values for CsA concentrations in blood were found with nonspecific monoclonal RIA, followed by FPIA and polyclonal RIA (mean ratio versus specific monoclonal RIA was 3.3, 2.9, and 2.4, respectively). Specific monoclonal RIA had 7% higher values than HPLC. There was a more than threefold interindividual variation in the mean ratio between specifically and nonspecifically measured CsA concentrations in whole blood and plasma. The ratio between CsA concentrations, determined with specific versus nonspecific methods in whole blood, decreased significantly during the first month after transplantation (from 0.42 to 0.35, as assayed with specific monoclonal RIA versus polyclonal RIA in whole blood; p less than 0.05). The concentrations in blood were, on the average, three times higher than those in plasma, although there was a more than fourfold interindividual variation in the mean values.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropeptide K is present in human cerebrospinal fluid

Neurokinin A-like immunoreactivity (NKALI) in human cerebrospinal fluid (CSF) was determined by radioimmuno assay (RIA) combined with high performance liquid chromatography (HPLC). the major immunoreactive component did not coelute with NKA, but coeluted with neuropeptide K (NPK), which contains the NKA sequence in its C-terminus. Trypsin treatment of this component from human CSF and of synthetic NPK, produced a substance which coeluted with NKA in the HPLC system. When the NKALI was oxidized with hydrogen peroxide and rechromatographed, the immunoreactivity coeluted with NPK sulfoxide. The results indicate that the main part of the NKALI in CSF is identical with NPK. The mean concentration of NPK measured in CSF from 6 healthy subjects by HPLCRIA was 23 ± 11 (SD) pmol/L.

Fractional conversion of thromboxane A 2 and B 2 to urinary 2,3-dinor-thromboxane B 2 and 11-dehydrothromboxane B 2 in the cynomolgus monkey

Following the intravenous administration of thromboxane (TX) B 2, the stable hydration product of TXA 2, to human and nonhuman primates the most abundant urinary metabolites are 2,3-dinor-TXB 2 and 11-dehydro-TXB 2. However, it is not known ▪ of TXB 2 to its enzymatic metabolites is an accurate representation of TXA 2 metabolism. Thus, we have compared the metabolic disposition of synthetic TXA 2 and TXB 2 via the β-oxidation and 11-OH-dehydrogenase pathways in vivo in the monkey. TXA 2 or TXB 2 (20 ng/kg) was intravenously administered to four cynomolgus monkeys pretreated with aspirin in order to suppress endogenous TXA 2 production. Urinary TXB 2, 2,3-dinor-TXB 2 and 11-dehydro-TXB 2 were measured before, during and up to 24 h after thromboxane administration by means of reversed-phase high-performance liquid chromatography radioimmunoassay. Aspirin treatment suppressed urinary 2,3-dinor-TXB 2 and 11-dehydro-TXB 2 by approx. 75%. A similar fractional conversion of TXA 2 and TXB 2 into 2,3-dinor-TXB 2 and 11-dehydro-TXB 2 was found. These results suggest that TXA 2 is hydrolyzed to TXB 2 prior to enzymatic degradation and that metabolites of the latter represent reliable indices of TXA 2 biosynthesis. Due to the variability in the conversion of thromboxanes into 2,3-dinor-TXB 2 and 11-dehydro-TXB 2, the measurement of both metabolites seems to represent a more reliable index of acute changes in TXA 2 production.

Improvement of radioimmunoassays for prostaglandins in bovine blood plasma and their application to monitor reproductive functions

Efficient RIA procedures are required for determination of prostaglandins (PGF 2α, PGE 2, PGI 2 and their metabolites) in bovine blood plasma to elucidate their significance in reproductive endocrinology. A new rapid efficient prepurification was developed using commercial octadecyl silicagel cartridges. Prepurification is especially necessary for the determination of 13,14-dihydro-15-keto-PGE 2 (PGEM). After prepurification, PGEM was first converted into the more stable 13,14-dihydro-15-keto-PGA 2 (PGAM) and measured in a RIA-system for PGAM. For PGF 2α, 13,14-dihydro-15-keto-PGF 2α (PGFM), PGE 2 and 6-keto-PGF 1α direct tests using 50 μl plasma per tube were elaborated. The validity of the tests was monitored by high performance liquid chromatography radioimmunoassay ( HPLC RIA ). Infusion studies using PGF 2α and PGE 2 showed that about 10% of these hormones remained unmetabolized after the first passage through the lungs. The biological half life of the metabolites PGFM and PGEM in bovines was estimated to be 4 min. Thus, PGFM and PGEM measurements in the peripheral circulation reflect even short-term secretory changes of PGF 2α and PGE 2. During the infusion of PGF 2α the levels of progesterone decreased but were not affected by PGE 2. Both prostaglandins caused increased oxytocin secretion. In the cow peripartum first PGEM elevations were measured 5 to 8 d ante partum, whereas PGFM increased 1 to 2 d ante partum. Then both prostaglandins increased simultaneously until parturition. In the postpartal phase PGFM was higher than PGEM, and both prostaglandins remained elevated for several days. Prostacyclin levels remained unchanged during the peripartal period.