Radioimmunoassay of haloperidol in human serum

Immunization of rabbits with a haloperidol hydrazone-bovine serum albumin conjugate elicited the formation of antibody to haloperidol. With this antiserum, concentrations of haloperidol as low as 1 ng/ml can be easily measured by radioimmunoassay of unextracted human serum. None of the known major metabolites of haloperidol displayed any significant cross-reactivity. Psychiatric patients receiving daily oral doses of 20 to 200 mg haloperidol had serum levels ranging between 5.8 and 245 ng/ml.     

Selective determination of haloperidol in human serum: surface ionization mass spectrometry and gas chromatography with surface ionization detection

Surface ionization organic mass spectrometry (SIOMS) has been performed on the clinically important drug haloperidol using quadrupole mass spectrometry in which the thermal ion source has a rhenium oxide emitter. The surface ionization (SI) mass spectrum is presented, interpreted in a purely empirical way by means of evidence from previous investigations, and then compared to results from conventional electron impact (EI) ionization. An approach to detection of this drug in serum by gas chromatography (GC) with a surface ionization detector (SID) and GC-SIOMS is described. This approach demonstrates that (a) haloperidol is efficiently surface-ionized, giving a unique SI mass spectrum, (b) experimental results rationalize the combined sensitivity and selectivity of the GC-SID for the examined drug, (c) the detection limit for haloperidol in serum is 1.1 ng/ml (S/N = 3) by GC-SID (the coefficients of variation of the assay are generally low, i.e. below 8.5%) and (d) the GC-SIOMS coupling can be used for sensitive and selective detection of haloperidol in serum.     

Megabore capillary gas-liquid chromatographic method with nitrogen-phosphorus selective detection for the assay of haloperidol and reduced haloperidol in serum: results of therapeutic drug-monitoring during acute therapy of eight schizophrenics

A gas chromatographic method using a HP-5 megabore capillary and nitrogen-phosphorus selective detection for the quantitative analysis of haloperidol (H) and reduced haloperidol (RH) in human serum or plasma is described. A 3-step liquid-liquid extraction is applied. The extraction yield of this procedure is 63% for haloperidol at 20 ng/ml. The limits of detection are 0.4 ng/ml for haloperidol and 1.0 ng/ml for the metabolite if 2 ml of body fluid are applied. At 10 ng/ml the within-day precision is 4.5% for H and 8.3% for RH. Serum levels of eight schizophrenic patients have been monitored weekly over a therapeutic period of six weeks. Seven patients mainly had metabolite ratios RH/H < 1 over the entire period of investigation. They exhibited a linear correlation between dose and serum concentration of haloperidol. In contrast, one patient had metabolite ratios RH/H > 1 over the entire period of the study. Due to considerable increased serum concentrations this patient did not show a linear correlation between the dose and the serum level of haloperidol.     

Determination of aldosterone in serum by liquid chromatography-tandem mass spectrometry

Measurement of serum aldosterone is clinically important in the diagnosis of hypertension. While isotope dilution gas chromatography-mass spectrometry (ID-GC-MS) provides reliable results, it requires derivatization and is lengthy and time-consuming. Detection by liquid chromatography-mass spectrometry (LC-MS) is a potentially superior method. The analysis utilizes 0.5mL of serum. The samples were extracted with dichloromethane-ether. The extract was evaporated to dryness and aldosterone was analyzed by LC-MS/MS operating in the negative mode ESI after separation on a reversed-phase column. Aldosterone was also measured by RIA. The calibration curves for analysis of serum aldosterone exhibited consistent linearity and reproducibility in the range of 60-3000pmol/L. Interassay CVs were 4.3-7.5% at aldosterone concentrations of 97-993pmol/L. The lower limit of quantitation (LOQ) was 30pmol/L (signal to noise ratio=10). The mean recovery of the analyte added to serum ranged from 95 to 102%. The regression equation by LC-MS/MS (x) and RIA (y) method was: y=1.33x+185 (r=0.95; n=124). Sensitivity and specificity of the LC-MS/MS method for serum aldosterone offer advantages over GC-MS by eliminating derivatization. The novel method is rapid, reliable and simple to perform with a routine LC-MS/MS spectrometer. The sensitivity is adequate for patient samples. Aldosterone concentrations reported by nonextraction RIA were consistently higher than those produced by LC-MS/MS.     

Comparison of serum estradiol to urinary estrone conjugates in the rhesus macaque (Macaca mulatta)

Paired urine and serum samples were collected daily during fourteen nonconceptive (7 females) and ten conceptive (9 females) ovarian cycles from a total of 12 female rhesus monkeys (Macaca mulatta). Daily urine samples were analyzed for concentrations of estrone conjugates (Ei Conj). Serum samples were evaluated for concentrations of estradiol (E2) and progesterone (P) by radioimmunoassay (RIA), and bioactive luteinizing hormone (bLH) and monkey chorionic gonadotropin (mCG) were analyzed by a mouse Leydig cell bioassay. Linear correlation (r) between urinary E1 Conj and serum E2 (r range = -0.176-0.948) during nonconceptive cycles aligned by the preovulatory E1 Conj peak (Day 0) improved when daily hormone values were realigned to account for an approximately 24-h delay in the excretion of hormonal metabolites in urine (r range = 0.465-0.967). Similarly, correlation between urinary E1 Conj and serum E2 during conceptive cycles aligned by Day 0 (r range = 0.300-0.824) improved when values were offset by 24 h (r range = 0.408-0.876). When conceptive cycles were compared to nonconceptive cycles, serum P levels were significantly elevated over nonconceptive levels by Day +12 (p less than 0.001), and urinary E1 Conj levels by Day +13 (p less than 0.02), whereas serum E2 and bLH were both significantly elevated by Day +14 (p less than 0.0006 and p less than 0.01, respectively). In both nonconceptive and conceptive cycles, urinary E1 Conj paralleled serum E2 and demonstrated incremental increases above baseline levels, which were greater than for serum E2.     

A non-chromatographic radioimmunoassay of prugesterone in serum

Convenient methodology based on separation of progesterone from alcoholic neutral steroids by means of a sulfation-procedure has been developed for the radioimmunoassay (RIA) of progesterone in male and female serum. When coordinated with our previously published nonchromatographic procedure for the RIA of estrone and estradiol in serum, all 3 seteroids can be determined in the same specimen. Validation of the procedure was based on: 1. Agreement between results obtained using TLC and sultation to fractionate progesterone (r=0.98; b=0.86), 2. accurate recovery of different quantities of progesterone added to serum, 3. independence of the concentration of progesterone and volume of serum used for assay, 4. low procedural blanks (3.6 ± 1.3 pg), 5. low intraassay (9.7 – 10.3%) and interassay (11.0 – 11.6%) variability and 6. correspondence of observed values for progesterone in male serum (108 ± 20 pg/ml) and in female serum (follicular, 285 ± 149 pg/ml; luteal, 3.46±1.45 ng/ml) with those reported previously by others.     

Seasonal changes in serum leptin of the feral raccoon (Procyon lotor) determined by canine-leptin-specific ELISA

Several reports have been published on blood leptin concentrations in feral animals, including members of the Carnivora, using a commercially available multi-species radioimmunoassay (RIA) kit with anti-human leptin antibody. However, we observed weak immunoreactivity between recombinant canine leptin and anti-human leptin antibody, suggesting a limitation in the applicability of the RIA kit for leptin assays in Carnivora species. We tested the applicability of RIA and sandwich enzyme-linked immunosorbent assay (ELISA) with anti-canine leptin antibody to assay blood leptin in the dog (Canis familiaris) and the raccoon (Procyon lotor). When RIA was used for recombinant canine leptin and dog sera, values were much lower than those determined by ELISA at higher concentrations (>10 ng/ml), while rather higher at lower concentrations (<2 ng/ml). A similar discrepancy between the two methods was found for serum leptin concentrations in raccoons. Clear seasonal variations were observed by ELISA, but not by RIA, with high values in autumn (3.46+/-0.45 ng/ml) and low values in spring and summer (0.71+/-0.07 ng/ml). Serum leptin concentrations in raccoons correlated positively with their body weight (r=0.753) and body mass index (r=0.755), corroborating our previous findings of a strong positive correlation between serum leptin concentrations and body fat content in dogs. Thus, the canine leptin ELISA is useful for assays of dog and raccoon leptin, and blood leptin is a good marker of nutritional condition in the species of Carnivora assayed in this study.     

Quantification of the antipsychotics flupentixol and haloperidol in human serum by high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatography (HPLC) method was developed for quantification of both isomers of the thioxanthene neuroleptic flupentixol and of the butyrophenone derivative haloperidol in human serum. After extraction with diethyl ether–n-heptane (50:50, v/v), an isocratic normal-phase HPLC system with a Hypersil cyanopropyl silica column (250×4.6 mm, 5 μm particle size) was used with ultraviolet detection at 254 nm and elution with a mixture of 920 ml acetonitrile, 110 ml methanol, 30 ml 0.1 M ammonium acetate, and 50 μl triethylamine. The limit of quantitation of 0.5 ng/ml and 0.3 ng/ml for flupentixol and haloperidol, respectively, was sufficient to quantify both compounds in serum after administration of clinically adjusted doses. The suitability of the described method for therapeutic drug monitoring and clinical pharmacokinetic studies was assessed by analysis of more than 100 trough level serum samples.     

Relationships between serum IGF-I concentrations and piglet development or neonatal viability following porcine somatotropin (pST) and insulin administration to gestating gilts

This study was designed to evaluate the effects of exogenous treatment with pST, insulin and their combination on periparturient serum IGF-I levels, prenatal piglet development and viability. Pregnant Yorkshire gilts were injected daily with either 5 ml of saline (C), n = 23; 5 mg pST (P), n = 23; 0.50 IU/kg of insulin (I), n = 23; or pST plus insulin at the above dosage, according to 2 x 2 factorial design from Day 30 to 70 of gestation. All gilts were sacrificed on Day 113 of gestation. Peripartum IGF-I serum levels were determined by RIA following a 36 h incubation in 0.2 M gly-gly HCL. Recovery of human rIGF-I standard was > 95%, while the intra-assay CV was 5.74%. Measures of piglet viability were compared using Randall's adaptation of Apgar score for human neonates. Piglet weight and crown-to-rump length were determined prior to dissection. Treatments (P, I and P + I) elicited highly significant increases in maternal serum IGF-I concentrations (321.9, 337.0, 375.0 vs 247.6 ng/ml; P = 0.0001) on Day 113 of gestation. Nonsignificant differences were detected in piglet serum IGF-I (94.8, 102.6, 110.2 vs 92.2 ng/ml; P = 0.06). These results revealed no relationship between piglet weight and maternal (r = 0.03) or piglet serum IGF-I levels (r = 0.08). Only a weak association between gilt and piglet serum IGF-I concentrations (r = 0.26) was detected. Injections with pST and or insulin did not influence piglet viability scores (P = 0.74). Viability scores were highly correlated with piglet weight (r = 0.66), crown -rump length (r = 0.70), but not with IGF-I concentrations in gilt (r = 0.09) and piglet serum (r = 0.16). Body weights, crown -rump length and visceral organ weights of piglets did not differ between control and hormone -treated gilts (P >/= 0.05). These results indicate 1) that there was no direct treatment effects on in utero piglet development or neonatal viability; 2) that IGF-I production in gilt and piglet compartment is independent, suggesting fetal endocrine autonomy regarding IGF-I production; and 3) that the usual low IGF-I concentrations in mid-late gestation in swine could be replenished by maternal pST and or insulin therapy.     

Superiority of gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol for monitoring of aromatase inhibitor therapy

Currently available radioimmunoassay methods for estradiol in serum lack sufficient sensitivity and precision to monitor estradiol levels in patients placed on third generation aromatase inhibitors. We recently validated a gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol and determined estrogen levels in normal post-menopausal women and in women with breast cancer before and during administration of aromatase inhibitors. Validation of the GC/MS/MS assay in human plasma and human serum included determination of assay sensitivity (<0.63 pg/ml), precision (all CVs less than 17.8%), recovery (98-103%), and linearity of recovery (R=0.998). Levels of estradiol were lower when assayed by GC/MS/MS compared to RIA under all conditions (7.26+/-4.82 pg/ml versus 11.9+12.0 pg/ml in normal post-menopausal women; 5.88+/-3.43 pg/ml versus 13.8+/-7.5 pg/ml in breast cancer patients prior to treatment; and<0.63 pg/ml versus 5.8+/-4.1 pg/ml during aromatase inhibitor therapy). Fifty-five women treated either with atamestane/toremiphene or letrozole/placebo were monitored for estradiol levels at 4, 8 and 12 weeks of therapy. The mean levels of estradiol during aromatase inhibitor therapy was 5.8+/-4.1 pg/ml as measured by RIA and <0.63 pg/ml by GC/MS/MS. The degree of suppression with the aromatase inhibitors as detected by RIA was 58% versus >89% by GC/MS. These results suggest that most RIA methods detect cross-reacting estrogen metabolites and yield higher measured levels than GC/MS/MS. Several pharmacological and clinical considerations suggest that GC/MS/MS should become the preferred method for monitoring aromatase inhibitor therapy.     

Time-resolved fluorescence immunoassay of thyroxine in serum: immobilized antigen approach

With T(4)-bovine IgG as a solid-phase antigen, we have developed a direct competitive-type immunoassay for serum total thyroxine (TT(4)), which depends on the competitive distribution of europium-labeled anti-T(4) monoclonal antibody between solid-phase-bound T(4) and the T(4) in the sample or standard. The captured fraction of the tracer was measured after a dissociation-enhancement step. Four different T(4) protein conjugates were synthesized, of which T(4)-bovine IgG was selected as the most favorable for the preparation of solid-phase antigen. The sensitivity was 3.5 ng/ml with a sample volume of 20 microl. T(4) values obtained by this procedure agreed well with those obtained by RIA (r = 0.967, n = 38) and EG&G Wallac TRFIA (r = 0.926, n = 64). All other quality criteria was also fulfilled with respect to precision, accuracy, and dynamic range.     

An enzymeimmunoassay method for thyroxine determination in serum samples

Here we describe an enzymeimmunoassay (EIA) for thyroxine (T4) in serum, whose performance is comparable to that of a sensitive T4 radioimmunoassay (RIA). In this assay, specific T4 antibody adsorbed on polystyrene beads is used along with T4-horseradishperoxidase as the tracer and omicron-phenylenediamine as chromogen. Several samples were analysed both by this T4 EIA and by using a commercial RIA kit for T4. The results correlate well with a correlation coefficient r = 0.9 and slope = 0.93 (n = 50).     

Radioimmunoassay for non-enzymatically glycated serum proteins

We have developed a radioimmunoassay (RIA) for nonenzymatically glycated serum proteins. The polyclonal antibodies prepared against reduced glycated human albumin were specific for the glucitollysine residues of serum proteins. Serum proteins from diabetic patients (n = 25) contained 5.3 +/- 2.8 nmoles of glucitollysine/mg protein, compared to 2.0 +/- 0.2 in controls (n = 20). The intra- and inter-assay variables were 3.2-6.2% and 4.4-8.6%, respectively. Results from this assay procedure correlated well with those from the boronate affinity chromatography procedure (r = 0.94; P less than 0.001). The data suggested that diabetic serum proteins contained at least 2.5 times as much immunochemically detectable glucitollysine residures as normal serum proteins after reduction of the proteins with sodium borohydride.     

Direct radioimmunoassay (RIA) of salivary testosterone: correlation with free and total serum testosterone

Simple and sensitive direct RIA for determination of salivary testosterone was developed by using RSL NOSOLVEX TM (125 1) kit produced by Radioassay System Laboratories (Carson, California). In addition, a relationship between salivary and serum free and total testosterone concentrations was studied in randomly selected 45 healthy subjects, 5 females on oral contraceptive pills and 28 hypertensive patients on various treatment regimens. The lowest weight of testosterone detectable by our modified method was equivalent to 1 pg/ml of saliva, taking into account analytical variability. Intra- and interassay coefficients of variation were 5.09 +/- 2.7% and 8.2 +/- 5.9% respectively. Statistically significant correlations were found between salivary and serum free testosterone (r = 0.97) and salivary and serum total testosterone concentrations (r = 0.70-0.87). The exception to this was a group of hypertensive females in which no correlation (r = 0.14) between salivary and total serum testosterone was found. It is also of interest that, while salivary testosterone was significantly increased in subjects taking oral contraceptives and most of the hypertensive patients the total serum testosterone concentration was in normal range. Our findings suggest that determination of salivary testosterone is a reliable method to detect changes in the concentration of available biologically active hormone in the circulation.     

Serum prolactin and growth hormone determined by radioimmunoassay and a two-site immunoradiometric assay: comparison with the Nb2 cell bioassay

Serum levels of the two lactogenic hormones prolactin (PRL) and growth hormone (GH) were compared when determined by radioimmunoassay (RIA) and two-site immunoradiometric (IRMA) assays in 83 normal premenopausal women. The mean values for the PRL and GH results determined by RIA were higher than those obtained by IRMA, despite strong correlations between the two (PRL, r = 0.92; GH, r = 0.79). The lactogenic hormones were also determined together by the Nb2 cell bioassay (BA) in 38 of these same women, and the results compared with the sum of the PRL and GH immunoassays. There was a strong correlation between the BA and RIA (r = 0.75), and the BA/PRL+GH RIA ratio averaged 1.6 +/- 0.5. Corresponding values for IRMA were r = 0.66, and BA/PRL + GH IRMA 3.3 +/- 1.1. Thus, the polyclonal RIA antisera appeared to recognize bioactive hormone components not determined by the double monoclonal antibody IRMA. Another 23 women at risk for familial breast cancer, and 14 cystic breast disease patients were also studied. High BA, but normal RIA results, giving mean ratios of 2.4 +/- 1.1 and 3.6 +/- 3.0 respectively, suggest the presence of a further variant with high bioactivity not detected by RIA in these two clinical situations.     

Nitric oxide, superoxide, and hydrogen peroxide production in brain mitochondria after haloperidol treatment.

Inhibition of mitochondrial respiration and free radical induction have been suggested to be involved in haloperidol neurotoxicity. In this study, mice were injected i.p. with haloperidol, according to two different treatments: (a) a single injection (1 mg/kg), sacrificed 1 h after the injection (single-dose model); and (b) two injections (1 mg/kg each), sacrificed 24 h after the first dose (double-dose model). Determinations of oxygen consumption and hydrogen peroxide (H2O2) production rate were carried out in isolated brain mitochondria. Nitric oxide (NO) and superoxide (O2-) production rates were measured in submitochondrial particles (SMP). Single-dose haloperidol treatment produced a 33% inhibition in malate-glutamate-dependent respiration, while no significant changes were found after double-dose treatment. NO production was inhibited by 39 and 54% in SMP from haloperidol-treated mice (single- and double-dose treatments, respectively) (control value: 1.6 +/- 0.2 nmol/min mg protein). NO steady-state concentration was estimated at about 16.5 nM and was decreased by 40% by haloperidol treatment. Increases of 105 and 54% were found in succinate-supported O2- and H2O2 production rates, respectively, after haloperidol single-dose treatment. Haloperidol treatment generated a 248% increase in SMP O2- production rate when measured in the presence of NADH plus rotenone. Our results suggest that haloperidol neurotoxicity would be mediated by a decreased mitochondrial NO production, a decreased intramitochondrial NO steady-state concentration, and by an inhibition of mitochondrial electron transfer with enhancement of O2- and H2O2 production. This inhibition does not seem to be caused by increased NO or ONOO- formation.     

Influence of inactivation of trypsin on immunoreactivity and serum immunoreactive trypsin concentration measured by radioimmunoassay

The influences of various active site-specific reagents of trypsin and protease inhibitors on the immunoreactivity of trypsin and serum trypsin concentration have been studied by radioimmunoassay (RIA). The RIA using inactivated 125I-trypsin as tracer showed lower Bo/T than the RIA using active 125I-trypsin, but the coefficient of variance of the former was smaller than that of the latter. Normal serum trypsin concentrations were 26.12-36.38 ng/ml with the RIA using inactivated 125I-trypsin as antigen tracer, and 201.15 ng/ml with the RIA using active 125I-trypsin as tracer. The recovery experiment showed that the difference was due to the interaction of serum protease inhibitors and labeled active trypsin.     

Tissue levels of haloperidol by radioreceptor assay and behavioral effects of haloperidol in the rat

A radioreceptor assay verified by independent biochemical methods was used to evaluate tissue levels of neuroleptic activity in serum and brain extracts after injections of haloperidol in the rat. The assay detected activity between doses of 0.1 and 10 mg/kg at times between 0.25 and 12 hrs. Tissue levels in blood and brain were highly correlated and corresponded well with a behavioral test of catalepsy at one hour after drug administration. This relationship between brain levels and behavior persisted but changed quantitatively over time.     

The correlation of serum immunoglobulin free light chain levels and selected biological markers in multiple myeloma

Aims: the presented study is aimed at the evaluation of correlation of free light chains serum levels - kappa, lambda and their relation (K/L ratio) and serum levels of selected biological markers in a group of patients with multiple myeloma examined at the time of the diagnosis. Methods: 102 patients with multiple myeloma were included in this prospective study. Free light chains serum levels were determined by Freelite Binding Site system, for determination of serum levels of selected parameters the following methods were used: REA thymidinekinase (TK), RIA (beta(2)microglobulin (beta(2)m), ICTP, PINP), enzymoimmunoassay (sIL-6R, sVCAM-1, sICAM-1, sOPG) and quantitative enzymatic immunoassay (sHGF, sVEGF, sSyndecan-1 (sSyn-1) a sFas). Results: There was found a correlation in the kappa-group of the dominant kappa chain and serum readings of beta(2)m (r = 0.344, p = 0.005), TK (r = 0.263, p = 0.035), ICTP (r = 0.402, p = 0.001), PINP (r = 0.264, p = 0.039), sOPG (r = 0.328, p = 0.028), sSyn-1 (r = 0.255, p = 0.046) and sFas (r = 0.418, p = 0.001). In case of K/L ratio there was found a statistically significant association of levels of beta(2)m (r = 0.316, p = 0.01), TK (r = 0.274, p = 0.027), ICTP (r = 0.346, p = 0.006), PINP (r = 0.261, p = 0.042), sSyn-1 (r = 0.283, p = 0.026) and sFas (r = 0.377, p = 0.002). In the lambda-group the analysis confirmed mutual dependence of the dominant lambda chain levels on beta(2)m (r = 0.476, p = 0.003), ICTP (r = 0.375, p = 0.022), sVCAM-1 (r = 0.383, p = 0.019), sHGF (r = 0.441, p = 0.006) and sFas (r = 0.334, p = 0.040). In addition we ascertained a correlation of L/K ratio and levels of beta(2)m (r = -0.473, p = 0.003), TK (r = -0.412, p = 0.011), ICTP (r = -0.331, p = 0.045), PINP (r = -0.409, p = 0.012), sHGF (r = -0.357, p = 0.028), sSyn-1 (r = -0.449, p = 0.005) a sFas (r = - 0.371, p = 0.022). Conclusions: The study confirmed mutual correlation of FLC serum levels and the levels of several selected biological markers, in particular beta(2)m, TK, ICTP, PINP, sSyn-1 a sFas at time of the diagnosis. It referred to the mutual relation of bone marrow microenvironment, biological qualities of clonal plasmocytes and the intensity of the free light chains production by the tumour cell population.     

Effects of caerulein + haloperidol on amphetamine-induced locomotor stereotypy in rats

The combination of haloperidol + caerulein has been reported to produce a long-lasting reduction of amphetamine-induced hyperlocomotions in rats. This study was designed to replicate those findings and to determine whether haloperidol + caerulein produce any unique effect on amphetamine-induced locomotor stereotypy. In two experiments, haloperidol + caerulein failed to produce a long-lasting reduction in amphetamine-induced hyperlocomotions. Although haloperidol reduced the locomotor stereotypy produced by higher doses of amphetamine, caerulein had no effect, either alone or combined with haloperidol.