Development and validation of a high-performance liquid chromatographic method for the determination of gamma-hydroxybutyric acid in rat plasma.

A method for the determination of gamma-hydroxybutyric acid (GHB) in rat plasma was developed using solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with UV detection. GHB was isolated from plasma using strong anion-exchange SPE columns. The chromatographic separation was performed on a C18 Aqua column. The lower limit of quantification was 10 microg/ml using 60 microl of plasma. The linearity of the calibration curves was satisfactory as indicated by correlation coefficients of >0.990. The within-day and between-day precision were <10% (n=24), the accuracy was nearly 101%. Plasma concentrations in rats after GHB infusion determined by HPLC-UV were compared with the corresponding concentrations determined with a validated gas chromatographic-mass spectrometric method by orthogonal distance regression. A good correlation was observed and a t-test indicated no significant differences from 0 and 1 for the intercept and slope, respectively.

2-oxo-3-hydroxy-LSD: an important LSD metabolite?

Recently, certain studies have indicated that 2-oxo-3-hydroxy-LSD is present in urine at much higher concentrations than LSD. We determined LSD and 2-oxo-3-hydroxy-LSD in urine by GC-MS (Hewlett Packard 5970) after solid phase extraction on SPEC.PLUS MP1 disks (3 mL, 30 mg, Ansys, Irvine, CA, USA), using the method recommended for amphetamines and derivatisation with BSTFA. For 2-oxo-3-hydroxy-LSD-bis-TMS, the ions with m/z of 309 and 499 were used as quantification and confirmation ion respectively. In four samples containing between 561 and 7007 pg/mL of LSD, 2-oxo-3-hydroxy-LSD concentrations between 8021 and 28466 pg/mL were found, i.e. 4 to 41 times higher than the LSD concentrations. 2-oxo-3-hydroxy-LSD does not seem to be conjugated. These results indicate that 2-oxo-3-hydroxy-LSD, is present in urine in much higher concentrations than LSD, which could facilitate confirmation of positive screenings and increase detection times.

Glycan microheterogeneity of alpha 1-acid glycoprotein in sera and dialysates of patients on continuous ambulatory peritoneal dialysis.

Total concentration and concanavalin (Con A) dependent microheterogeneity of alpha 1-acid glycoprotein (AGP) were studied in sera of eight chronic renal failure patients before the start of continuous ambulatory peritoneal dialysis (CAPD), and in sera and dialysate fluids for up to 6 months of CAPD. The glycan heterogeneity of AGP in the samples was expressed as a reactivity coefficient, i.e. the ratio of the Con A-reactive AGP components to the Con A non-reactive AGP component. Concentrations of AGP in serum and reactivity coefficients were markedly elevated in non-dialysed uraemic patients. AGP concentrations in serum increased further during the first week on CAPD, and then gradually decreased to pre-dialysis values, which were reached after 1 to 6 mth. The reactivity coefficients did not change significantly during the CAPD treatment. Dialysate AGP concentrations were low in comparison to those in serum, and there was a good correlation between the reactivity coefficients in the dialysate fluids and those in the corresponding sera. The effect of peritonitis was evaluated in a separate group of eight CAPD patients. Serum and dialysate AGP concentrations were significantly higher during than after peritonitis while the corresponding reactivity coefficients were only slightly elevated.

Molecular forms of dopamine beta-hydroxylase in rat superior cervical ganglion and adrenal gland.

Dopamine beta-hydroxylase (DBH) enzyme activity was associated in rat superior cervical ganglion with tetrameric DBH-A (294,000 D) and dimeric DBH-B (147,000 D) and in rat adrenal gland with DBH-A and a novel molecular form of DBH, defined as DBH-C, with a molecular weight of 125,000 D. Pretreatment of the rats with cycloheximide markedly reduced DBH activity without altering the molecular heterogeneity.

Influence of continuous ambulatory peritoneal dialysis on serum alpha 1-acid glycoprotein concentration and drug binding.

The influence of continuous ambulatory peritoneal dialysis (CAPD) on the concentrations of alpha 1-acid glycoprotein in serum and dialysate and on the serum binding of oxprenolol, propranolol and phenytoin has been studied. Before starting CAPD treatment, the serum binding of oxprenolol and propranolol was higher and that of phenytoin lower than in healthy volunteers, and the serum alpha 1-AGP concentration was higher. During the first days to weeks after starting CAPD, the serum alpha 1-AGP concentration rose with a concomitant increase in the binding of oxprenolol and propranolol. Subsequently, the alpha 1-AGP level and the binding of oxprenolol and propranolol decreased to the values found before starting CAPD. The binding of phenytoin showed little change. The concentration of alpha 1-AGP in dialysate was 2 to 5% of that in serum.

A monoclonal antibody-based enzyme-linked immunosorbent assay of alpha 1-acid glycoprotein.

A "sandwich"-type enzyme-linked immunosorbent assay for determining concentrations of human alpha 1-acid glycoprotein (AGP) is described. Microtiter plates coated with a polyclonal rabbit antibody to human AGP were subsequently incubated with the antigen, with a specific murine monoclonal antibody, and with goat anti-mouse immunoglobulins conjugated to alkaline phosphatase. To evaluate the method for assay of AGP in human sera, we compared it with single radial immunodiffusion and "rocket" electroimmunoassay. The respective correlations were r = 0.988 (n = 45) and r = 0.973 (n = 47). Repeated assays of a human serum sample with an average AGP concentration of 859 mg/L yielded within-day and between-day CVs of 1.4% (n = 5) and 6.3% (n = 10), respectively. Because of its low detection limit (4.4 micrograms/L), this assay is also suitable for determination of AGP concentrations in other biological fluids, such as dialysates of patients being treated by continuous ambulatory peritoneal dialysis.

A study of the heterogeneity of human alpha 1-acid glycoprotein with monoclonal antibodies.

Monoclonal antibodies (MABS) were raised against human alpha 1-acid glycoprotein (AGP) and their reaction with the polymorphic forms of this plasma protein were evaluated. Spleen cells of BALB/c mice, immunized with native or desialylated human AGP, were fused with NSO mouse myeloma cells. The hybridoma products were screened with a direct ELISA test, in which the immunoplates were coated with a mixture of native and desialylated AGP. In this test, 14 anti-AGP antibody producing clones were retained. Coating the wells with either native or desialylated AGP showed that eleven clones reacted with both types of AGP ('Type I' MABS), while three MABS reacted specifically with the desialylated form ('Type II' MABS). Precoating the immunoplates with polyclonal anti-human AGP followed by incubation with native or desialylated AGP before the addition of hybridoma supernatant (indirect ELISA), confirmed the specificities observed in the direct ELISA. The molecular heterogeneity of both native AGP and desialylated AGP, based on Concanavalin A reactivity and isoelectric point, was not reflected by any specificity in the antibody reactions. Thus 'Type I' MABS reacted with all molecular forms present in native and desialylated AGP while 'Type II' MABS reacted with all molecular forms present in desialylated AGP.

Release of dopamine, noradrenaline and dopamine B-hydroxylase from mouse neuroblastoma.

The murine C1300 neuroblastoma tumor was found to secrete dopamine, noradrenaline and dopamine B-hydroxylase into the circulation of tumor-bearing A/J mice. The plasma levels of dopamine, noradrenaline and dopamine B-hydroxylase increased with the size of the tumor, and the increase in noradrenaline paralleled the increase in dopamine B-hydroxylase (r = 0.86). The vesicular storage of dopamine and noradrenaline in the tumor was evidenced by a decrease of the tissue content of dopamine and noradrenaline 24 hours after the administration of reserpine (5 micrograms/g) respectively to 17.6% and 7.8% of control values. A similar observation could be made for the levels of dopamine and noradrenaline in the plasma of reserpinized C1300 mice. The total activity of dopamine B-hydroxylase in the tumor and in plasma was unaffected by the reserpine treatment. Chronic administration of 6-hydroxydopamine (100 micrograms/g for 8 days) had no effect on the tissue contents of dopamine, noradrenaline or dopamine B-hydroxylase. The release of catecholamines and dopamine B-hydroxylase from the C1300 neuroblastoma was studied in vitro on superfused tumor slices. Stimulation of these slices with 56 mM KC1 or with 5.10(-5) M tyramine failed to induce the release of endogenous dopamine, noradrenaline or dopamine B-hydroxylase above the basal outflow levels. These results are suggestive for a non-exocytotic release of catecholamines and dopamine B-hydroxylase from the neuroblastoma tumor.

Multiple molecular forms of dopamine beta-hydroxylase in the C1300 mouse neuroblastoma tumor and in the serum of tumor-bearing mice.

The distribution of the enzymatic activity of dopamine beta-hydroxylase (DBH) in linear sucrose gradients was studied for a soluble fraction of the C1300 mouse neuroblastoma tumor, for the serum of tumor-bearing A/J mice, and for adrenal tissue and serum of control mice. In controls (adrenal gland and serum of A/J mice), about 75% of the DBH activity was associated with a high-molecular-weight form, denoted as DBHA, with an apparent sedimentation coefficient of 11.3 S. About 25% of the DBH activity was attributable to a slower-sedimenting species (7.1 S), denoted as DBHB. In tumor supernatants and in the serum of tumor-bearing mice, about 55% of the DBH activity was present as the 7.1 S species (DBHB), while only 35% was recovered as the high-molecular-weight form (DBHA). Approximately 5% of the activity could be attributed to a separate form, with a sedimentation coefficient of about 4.5 S. This form is designated DBHC. The ratio DBHB/DBHA is significantly higher in tumor tissue and in serum of tumor-bearing mice than in controls. The three enzymically active forms of DBH in the C1300 tumor are considered to represent the tetrameric (DBHA), dimeric (DBHB), and monomeric (DBHC) forms of the enzyme.