Oct 15, 1982 - Richard J. Cooke and Robert L. Jensen. A micromethod for measuring the ammonia nitrogen content of plasma by use of an ion-selective ...
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Mass Spectromet,y in Life Sciences 2, AP De Leenheer, RR Roncucci, C Van Peteghem, Eds., Elsevier, Amsterdam, The Netherlands, 1978, pp 3-16. 10. Finlay EM}i, Gaskell Si. Determination of testosterone in plasma from men by gas chromatography/mass spectrometry, with high-resolution selected-ion monitoring and metastable peak monitoring. Clin Chem 27, 1165-1170 (1981). 11. Gaskell Si, Pike AW, Finlay EMH. New techniques in gas chromatography-mass spectrometry and their use in the validation of routine steroid assays. In ref. 2, pp 67-72. 12. Riad-Fahmy D, Read GF, Gaskell Si, et al. A simple direct radioimmunoassay for plasma cortisol, featuring a 1I radioligand and a solid-phase separation technique. Gun Chem 25, 665-668 (1979). 13. Mattingly D. A simple fluot-imetric method for the estimation of free il-hydroxycorticoids in human plasma. J Gun Pathol 15, 374379 (1962). 14. Fahmy D, Read GF, Hillier SG. Some observations on the determination of cortisol in human plasma by radioimmunoassay using antisera against cortisol-3-BSA. Steroids 26, 267-280 (1975).
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17, 377-398 (1971). 18. Bjarkhem I, Lantto 0, Svensson L. Use of isotope dilution-mass spectrometry as a reference technique in clinical endocrinology. In ref. 2, pp 61-66. 19. Thijssen JHH, van den Berg JHM, Adlercreutz H, et al. The determination of cortisol in human plasma: Evaluation and comparison of seven assays. Gun Chim Acta 100, 39-46 (1980). 20. Lantto 0, Bjorkhem I, Blomstrand R, Kallner A. Interlaboratory evaluation of four RIA kits for determination of plasma cortisol with special reference to accuracy: Influence of matrix in calibration standards. Clin C/tern 26, 1899-1901 (1980). 21. Lantto 0, Aakvaag A, Damkjaer-Nielsen M, et al. Assay of cortisol with a radioimmunoassay method calibrated by isotope dilution-mass spectrometry. A pilot study. Scand J Clin Lab Invest, in press. 22. Bj#{246}rkhem I, Blomstrand R, Lantto 0, et a!. Plasma cortisol determination by mass fragmentography. Clin Chim Acta 56, 241248 (1974).
CLIN. CHEM. 29/5, 867-869 (1983)
Micromethod for Determining Plasma Ammonia Nitrogen with Use of an IonSelective Electrode Richard J. Cooke and Robert L. Jensen A micromethod for measuring the ammonia nitrogen content of plasma by use of an ion-selective electrode is evaluated
and described. The effectof storing plasma for 24,48, and 72 h at -70 #{176}C was evaluated. Values for newborns and fasting adults were 880 g/L (SD, 210 g/L) and 620 ,ug/L (SD, 170 gfL), respectively. Additional Keyphrases: pediatric perammonemia
chemistry
.
newborns
hy-
-
reference interval
Availability of the aminonium ion-selective electrode has simplified determination of ammonia in whole blood and plasma (1-3). However, the large samples required (6.0 mL) make the method impractical for use in newborns and small children. We adapted the method of Attili et al. (2) and
obtained accurate and reproducible results for 200-L samples of plasma, the measurement being made in a virtually anaerobic fashion. Thus, newborns at risk for developing hyperammonemia (4, 5) may be evaluated serially without excessive withdrawal of blood.
Materials and Methods Equipment Ammonium-selective
through adapter, Model
95-10 (Orion Research, Inc., Cambridge, MA 02139). Syringe pump, Model 341 (Sage Instruments, Division of Orion Research, Inc.). Electrometer, Model 801A (Orion Research, Inc.). Tuberculin syringes, 1-mL. Plastic cups, 3-mL capacity.
Reagents “Titrisol” buffer, pH 10.00 (boric acid 1.546 g/L, potassium chloride 1.864 g/L, sodium hydroxide 476 mg/L; E. Merck & Co., Darmstadt, F.R.G.). “Titrisol” buffer, pH 12.0 (disodium phosphate 2.225 g/L, sodium hydroxide 446 mg/L) (E. Merck & Co.). Buffer, pH 10.4: Combine 81.0 mL of pH 10 buffer with 19 mL of pH 12 buffer.
Procedures of ammonium (NH4) standards. Ammonium 1.00 g of ammonia nitrogen per liter: Dilute 71.4 mL of ammonium chloride reference standard (100 mmol/L; Orion no. 95-10-06) to 100 mL with distilled Preparation
chloride
standard,
water (standard ion
electrode
probe
with
flow-
University of Iowa, Department of Pediatrics, Iowa City, IA. Received Oct. 15, 1982; accepted Feb. 18, 1983.
I).
Dilute to give a per liter Dilute
with
1.0 mL of standard I to 100 mL with distilled water final concentration of 10 mg of ammonia nitrogen (standard II). 2, 1, and 0.5 mL of standard H to 8, 9, and 9.5 mL distilled water, to give final concentrations of 2.00,
1.00, and 0.50 mg/L, respectively. CLINICAL CHEMISTRY, Vol. 29, No. 5, 1983
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Dilute 1.0 mL of the 500 ,ugtL standard with 1.0 mL of distilled water to give a final concentration of 250 g/L. Preparation of sample. Collect 0.6-0.8 mL of whole blood by venipuncture into a heparinized tuberculin syringe (sy-
ringe 1). Pierce the barrel and plunger of the syringe with a 21-gauge needle, and seal the tip of the syringe with a 25gauge needle with bent shaft (Figure la). Snip off the end of the plunger and the hub of the 21-gauge needle (Figure lb), and immediately place the sample in ice. Then centrifuge the specimen, with the bent 25-gauge needle pointing upward, in a refrigerated centrifuge (0#{176}C, 5 mm, 12 000 x g). Keep the tuberculin syringe upright when it is removed from the centrifuge, and remove the 25- and 21-gauge needles. Insert the 25-gauge needle of tuberculin syringe 2 into the barrel of syringe 1 as illustrated in Figure lc, and separate the plasma up into syringe 2 by advancing the plunger of syringe 1. Refrigerate the sample and analyze it within 4 h or store it at - 70#{176} C for analysis within 48 h. Preparation of standard curve and sample analysis. Add 0.2 mL of each of the five working-standard solutions to 0.4 mL of pH 10.4 buffer in a 3-mL plastic cup immediately before analysis. Aspirate the solution into a tuberculin syringe (after mixing by drawing the solution up and down twice in the tuberculin syringe). Then place the syringe on the pump, which is set to deliver the solution at 0.06 mu miii. Set the electrometer at 0 mV, and make the final reading at 8 mm. Although the response time on all samples may vary somewhat, depending on ammonia concentration,
Whole Blood Plasma
Cells
Synnge
Syringe
Syringe 2
25 gauge needle
we found that it became stable for all concentrations tested by 8 mm. Because the standards and samples are matched for ionic strength, the response time is the same for both. Add 0.2 mL of sample to 0.4 mL of pH 12.0 buffer in a similar fashion. Recalibrate the electrode with the ammoniurn standards after 1 h or four sample analyses, whichever comes first. The membrane of the electrode is changed every second day to minimize the possibility of error from membrane deterioration. Cakulation. The ammonia concentration in the sample is calculated from comparison with a semilog plot of the readings of the standards, noted before the sample readings were obtained.
Results Analytical recovery was assessed on three different occasions by adding 0.1 mL of various standard solutions (10, 5, and 1 mgIL) to samples of plasma. The mean recoveries were 98, 100, and 103% respectively, for the three (standard deviations 4.6, 3.7, and 5.0%). A single sample of plasma was divided into 10 portions and ammonia nitrogen was measured. The mean value was 0.92 mg/L, range 0.84-0.96, standard deviation 0.022, and CV 2.4%. The procedure was repeated on seven portions of a different sample. The mean value was 0.70 mg/L, range 0.68-0.74, standard deviation 0.014, and CV 2%. The variation in reproducibility attributable to the electrode itself is ±2% (6), so the error incorporated by using the microtechnique seems negligible. To investigate the effect of storage, we determined ammonia nitrogen concentrations of 10, 16, and 12 samples within 4 h and again after 24, 48, and 72 h, of storage at -70#{176} C. The mean difference in result between the initial determintion and determination after 24 h was -8 s.g/L (not significant) with a range of 40 to -30 g’L; after 48 h, 17 pgfL (not significant), range 40 to -90 g/L; after 72 h, 57 g/L (p
