Hormonale, Hopital Saint-Louis,. 1 ave. Claude Vellefaux,. 75010 Paris, France) ... Nature. (London) 1989;341:167-8. 2. Hart HE, Greenwald EB. Scintillation.
References
1. Eichhorn JH. Accuracy and comparisons in blood gas measurements. Chest 1988;94:1-2. 2. Burnett RW, Covington AK, Mans AHJ, et al. IFCC method
(1988) for tonometryofblood: referencematerials forPco2andp. J Cliii Chem Clin Biochem 1989;27:403-8. 3. Sprokholt R, Mans AHJ. Some experiences with commercially available tonometers. In: Oeseburg B, Zijlstra WG, eds. Methodology and physiology of blood gases and pH. Proc., 6th meeting of IFCC Expert Panel on pH and blood gases, GrOmngen, August 23-25, 1981: 28-50. 4. ABL 500 blood gas system user’s handbook. Copenhagen: Radiometer, 1989: book 4: 10.27-10.30.
were evaluated as the quantities of cold steroid displacing 50% of the radioactive ligands. With technique 1, 50% ligand displacement was obtained with 75 pg of A, 130 pg of DHEA, and 100 pg of 11f3OHA. With technique 2, this required 93 pg ofA, 180 pg ofDHEA, and 85 pg of 11f3OHA. Reproducibility of results for standard and patients’sample duplicates was better with SPA reagent (CV = 1.6-6%) than with technique 1 (CV = 2.6-8%). Comparison of the results of 130 patients’ plasma concentrations obtained with the two techniques yielded the following regression equations (x = technique 1, y = technique 2): y = 0.92x + 0.19 (A); y = 0.97x + 0.17 (DHEA); y = 0.97x 0.05 (11f3OHA). The CVs obtained for the same plasma control sample measured by the two techniques were very similar: 8.9% (A), 5.9% (DHEA), and 7.8% (11/3OHA) for technique 1 and 9.0%, 6.7%, and 4.7%, respectively, for technique 2. Overall, the analytical performances of the assays for the three steroids carried out with the SPA reagent appear very similar to those obtained by using the classical technique involving a charcoal-dextran reagent. However, substantial time was saved by using the-SPA reagent technique, because filling vials with scintillation liquid, adding charcoal-dextran reagent to each assay tube, waiting before centrifugation, and decantation into the vials were all eliminated. -
Application of Scintillation Proximity Assay to Homogeneous Radloimmunoassay of Androstenedione, Dehydroeplandrosterone, and 11 p-Hydroxyandrostenedlone, J. Fiet, Ph. Boudou, J. M. Burthier, and J. M. Villette (Lab. de Biol. Hormonale,
Hopital
Saint-Louis,
1 ave. Claude Vellefaux,
75010 Paris, France) The final step in steroid radioimmunoassay consists in separating the free (F) from the immunologically bound (B)
radioactive steroid. This separation is usually carried out with a charcoal-dextran reagent. Although widely used, this charcoal-dextran separation step is very time-consuming. This is why the introduction of scintillation proximity assay (SPA) reagent (1-3), which eliminates the need for both charcoal-dextran reagent and scintillation liquid, may represent considerable progress in the daily practice of steroid radioimmunoassay. We compared the results of the radioimmunoassay of three steroids-androstenedione (A), dehydroepiandrosterone (DHEA), and 1113-hydroxyandrostenedione (11/3OHA)by a technique used for several years in our laboratory (4), which includes a dextran-charcoal separation step, with the results obtained by using the newly introduced SPA reagent. The SPA reagent consists of fluorescer-impregnated microspheres coated with anti-rabbit antiserum; it was obtained from Amersham France. The working principle is as follows: During radioimmunoassay with 3H-labeled steroids, bound labeled ligand (B) is in close proximity to the microspheres, allowing the emitted energy to activate the
fluorescer and producelight. Becausethe assay is carried out in 0.4 mL of buffer, most of the free labeled steroid molecules (F) are too far from the microspheres to promote the transfer of energy.
Briefly, simultaneous radioimmunoassay of A-DHEAwas carried out after extraction, followed by Celite partition chromatography (4). We used androstene11pOHA
dione-613-hemisuccunate/bovine
serum
albumin (BSA), and 11f3OHA-3-Ocarboxymethyloxime/BSA antibodies (4) along with the corresponding tritiated steroids. The classical technique (technique 1) involves overnight incubation at 4#{176}C, followed by charcoal-dextran separation (4). In the new procedure with SPA reagent (technique 2), a chromatographic eluate (0.1 mL), specific antibodies (0.1 mL), tritiated steroids (0.1 mL), and SPA reagent (0.1
DHEA-7-O-carboxymethyloxime/BSA,
mL) are mixed together in minivials overnight at ambient temperature
Sensitivities
before counting
the bound radioactivity.
of the standard curves of the two techniques
References 1. Bosworth N, Towero P. Scintillation proximity assay. Nature (London) 1989;341:167-8. 2. Hart HE, Greenwald EB. Scintillation proximity assay (SPA)new method of immunoassay. Mol Imniunol 1979;16:265-7. 3. Udenfriend 5, Dieckmann GL, Brink L, Spector S. Scintillation proximity radioiinmunoassay utilizing ‘251-labeled ligand. Proc Natl Acad Sci USA 1985;82:8672-6. 4. Fiet J, Gourmel B, Villette JM, et al. Simultaneous radioimmunoassay of androstenedione, dehydroepiandrosterone and 11beta-hydroxy-androstenedione in plasma. Horm Res (Basel) 1980; 13:133-49.
Effect of Hyperglycemia on Plasma Sodium and Potassium Concentration Revisited, Robert A. Shalwitz/ Ronald L. Gingerich,’ Janet B. McGill,2 and Jay M. McDonalct3 (Depts. of’ Pediatrics, 2Med, and 3Pathol., Washington Univ. School of Med., St. Louis, MO 63110) During intravascular
hyperglycemia,
intracellular
water shifts to the
space, which results in a decrease in the
concentration of plasma sodium. This phenomenon was classically described by Seldun and Tarail (1). Katz (2) analyzed the specifics of the relationship, showing that for each 1000 mgfL (5.6 mmol/L) increment in serum glucose, there is an expected decrease of 1.6 mniollL in serum sodium. However, a recent study by Strand et al. (3) found a poor correlation (r = -0.556) between simultaneous glucose and sodium concentrations in a cross section of patients with spontaneous hyperglycemia. These results were in contrast to those of McNair et al. (4), who found a consistent decrease in sodium (r = -0.61) and an increase in potassium (r = 0.37) with hyperglycemia in patients with diabetes mellitus. Therefore, we undertook this study to analyze the consistency of the effects of hyperglycemia on plasma sodium and potassium under controlled condi-
CLINICAL CHEMISTRY, Vol. 37, No. 2, 1991 293
0
0
ci
ci
‘en
Surgery, Univ. of Texas Med. School at Houston, 6431 Fannin, SUite 6.240, Houston, TX 77030)
0
ci
Rapamycin (RAPA), an atypical triene macrolide antibiotic containing
Sodium
(mmoilL)
Giuco5e (mgldL)
Fig. 1. Normalized
High-Performance Liquid Chromatography of Rapamycin, Kimberly L. Napoli and Barry D. Kahan (Div. of Immunol. and Organ Transplantation, Dept. of
glucose and sodium plasma concentrations
during controlled
hyperglycemia in six patients with 10DM Normalization was performed by subtracting the value of the glucose or sodium concentration for an individual patient at time 0 mm from each subsequent value. Note that 100 mg/dL = 5.6 mmol/L for glucose
tions in patients with insulin-dependent
diabetes mellitus
(IDDM). Six otherwise healthy patients with IDDM fasted overnight and had their morning insulin withheld. Each patient was given an oral dose of Sustacal (6 mL/kg), and plasma venous samples for sodium and potassium (deter-
mined potentiometrically with a Nova Analyzer) and glucose(determined with a Beckman Glucose Analyzer) were obtained at 10-mm intervals for 130 mm. The samples from all of the subjects were analyzed in one batch. Linear-regression analysis of normalized sodium and glucose concentrations (seeFigure 1) showed a very significant correlation (r = 0.67, P
