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A homogeneous substrate-labeled fluorescent immunoassay has been developed to measure amikacin levels in human serum. Amikacin is covalently labeled with the fluorogenic enzyme substrate beta-galactosyl-umbelliferone. This beta-galactosyl-umbelliferone-amikacin conjugate is nonfluorescent under assay conditions until it is hydrolyzed by beta-galactosidase to yield a fluorescent product. When antiserum to amikacin binds the substrate-labeled drug, the antibody complex formation inhibits hydrolysis of the fluorogenic substrate. Reaction mixtures containing a constant level of substrate-labeled amikacin and a limiting amount of antiserum enable labeled and unlabeled amikacin to compete for the antibody-binding sites. Unbound substrate-labeled drug is hydrolyzed by the enzyme to release a fluorescent product that is proportional to the unlabeled amikacin concentration. The amikacin levels found in clinical serum samples with this method were comparable (r = 0.987) to those obtained by radioimmunoassay. The fluorescent immunoassay is rapid and simple to perform and requires only 2 microliters of serum.

1. The relationship between thirst perception and plasma osmolality was studied during hypertonic and physiological saline infusion in ten healthy volunteers. 2. Thirst perception was quantified using a linear visual analogue scale which volunteers marked at intervals during the infusion periods. 3. Infusion of hypertonic saline caused a steady rise in plasma osmolality together with a progressive linear increase in thirst perception and also plasma arginine vasopressin. No significant changes in thirst, plasma osmolality or plasma arginine vasopressin occurred during infusion of physiological saline. 4. Linear regression analysis of the results defined the functions. Thirst (cm) = 0.3 (plasma osmolality −281) (r = + 0.92, P < 0.001) and plasma arginine vasopressin (pmol/l) = 0.4 (plasma osmolality −285) (r = + 0.96, P < 0.001). The osmolar threshold for thirst onset thus defined (281 mosmol/kg) was much lower than in previous studies and similar to the theoretical osmolar threshold for vasopressin release (285 mosmol/kg). 5. We conclude that thirst perception rises in a progressive fashion throughout a wide range of plasma osmolality and that the osmolar threshold for thirst onset is similar to the theoretical osmolar threshold for vasopressin release. 6. The results are compatible with the concept of either a single osmoreceptor subserving both thirst and vasopressin release, or two osmoreceptors sharing similar functional characteristics.

Infusion of hypertonic saline into six normal volunteers caused an increase in plasma osmolality from 286·8 ± 1·7 (mean ± s.e.m.) to 307·6 ± 2·6 mosmol/kg (P<0·001), a 7·1% increase in estimated blood volume, a rise in plasma immunoreactive arginine vasopressin (AVP) concentrations from 1·3 ± 0·2 to 12·7 ±3·6 pmol/l (P<0·001) but no change in plasma AVP concentrations (2·1 ±0·9 and 1·9 ± 1·3 pmol/l) as measured by a cytochemical technique based on the ability of AVP to stimulate rat renal medullary Na+/K+-ATPase activity. Addition of synthetic AVP to plasma obtained before, during and after hypertonic saline infusion also failed to stimulate Na+/K+-ATPase activity. The results suggest that infusion of hypertonic saline interfered with the cytochemical assay for AVP by inhibiting AVP-stimulated medullary Na+/K+-ATPase activity. We conclude that the use of this cytochemical method to detect plasma AVP has severe limitations under these experimental conditions.J. Endocr.(1986) 111,495–499

Abstract. The presence of the human placental enzyme, oxytocinase, in blood samples taken during pregnancy causes major methological problems in the radioimmunoassay for plasma oxytocin. Inadequate inhibition of the enzyme activity may lead to spuriously high or low values of plasma oxytocin. This study systematically investigates a variety of enzyme inhibitors. The optimum inhibitory system was obtained by the addition of 10 μl of cold 125 mmol/l 1,10 phenanthrolene and 1 mol/l EDTA per ml of whole blood into the syringe. Complete enzyme inhibition was maintained for up to 60 min, during which time the lithium heparinized plasma samples were extracted by the Florisil method. Following extraction there was no enzyme activity in the extract residue. Concentrations of phenanthrolene and EDTA necessary to eliminate enzyme activity were 50- and 10-fold greater, respectively, than in any previously reported method. Recovery of synthetic oxytocin added to pregnancy plasma with inhibitors was 80% or higher, over the concentration range 1–100 pmol/l. Extract residue could be stored at −20°C for up to 7 weeks. Dilutions of pregnancy plasma extracts ran parallel to the oxytocin standard curve. Studies on plasma concentrations of oxytocin (OT) during the first stage of labour in 6 patients showed that 3 had pulsatile plasma OT, peak values ranging from 4–10 pmol/l in phase with uterine concentrations, but 2 who had regular uterine activity had no episodic changes in plasma OT. One patient with hypocontractile labour had low non-fluctuating plasma OT.