8. Bjorkhem I, Blomstrand R, Lantto 0, et al. Towards absolute 15. Wide L. Radioimmunoassays employing immunosorbents.

Acta
methods in clinical chemistry: Application of mass fragmentogra-
phy to high-accuracy analyses. Clin Chem 22, 1789-1801 (1976).
9. Siekniann L. Isotope dilution-mass spectrometry of steroid hor-
mones-a definitive method in clinical chemistry. In Quantitative
Mass Spectromet,y in Life Sciences 2, AP De Leenheer, RR Ron-
cucci, C Van Peteghem, Eds., Elsevier, Amsterdam, The Nether-
lands, 1978, pp 3-16.
10. Finlay EM}i, Gaskell Si. Determination of testosterone
plasma from men by gas chromatography/mass spectrometry, with
high-resolution selected-ion monitoring and metastable peak moni-
toring. 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
Endocrinol, Suppl. 142, 207-221 (1969).
16. Seth J, Brown LM. A simple radioimmunoassay for plasma
cortisol. Clin Chim Acta 86, 109-120 (1978).
17. Aringer L, Eneroth P, Gustaisson J-A. Trimethylbromosilane
catalyzed trimethylsilylation of slow-reacting hydroxy- and oxoster-
oids in gas chromatographic-mass spectrometric analysis. Steroids
17, 377-398 (1971).
in 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 com-
parison of seven assays. Gun Chim Acta 100, 39-46 (1980).
20. Lantto 0, Bjorkhem I, Blomstrand R, Kallner A. Interlabora-
tory 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).

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(1979). 21. Lantto 0, Aakvaag A, Damkjaer-Nielsen M, et al. Assay of
13. Mattingly D. A simple fluot-imetric method for the estimation of cortisol with a radioimmunoassay method calibrated by isotope
free il-hydroxycorticoids in human plasma. J Gun Pathol 15, 374- dilution-mass spectrometry. A pilot study. Scand J Clin Lab Invest,
379 (1962). in press.
14. Fahmy D, Read GF, Hillier SG. Some observations on the 22. Bj#{246}rkhem I, Blomstrand R, Lantto 0, et a!. Plasma cortisol
determination of cortisol in human plasma by radioimmunoassay determination by mass fragmentography. Clin Chim Acta 56, 241-
using antisera against cortisol-3-BSA. Steroids 26, 267-280 (1975). 248 (1974).

CLIN. CHEM. 29/5, 867-869 (1983)

Micromethod for Determining Plasma Ammonia Nitrogen with Use of an Ion-

Selective Electrode
Richard J. Cooke and Robert L. Jensen

A micromethod for measuring the ammonia nitrogen content through adapter, Model 95-10 (Orion Research, Inc., Cam-
of plasma by use of an ion-selective electrode is evaluated bridge, MA 02139).
and described. The effectof storing plasma for 24,48, and 72 Syringe pump, Model 341 (Sage Instruments, Division of
h at -70 #{176}C
was evaluated. Values for newborns and fasting Orion Research, Inc.).
adults were 880 g/L (SD, 210 g/L) and 620 ,ug/L (SD, 170 Electrometer, Model 801A (Orion Research, Inc.).
gfL), respectively. Tuberculin syringes, 1-mL.
Plastic cups, 3-mL capacity.
Additional Keyphrases: pediatric chemistry . newborns - hy-
perammonemia reference interval
Reagents
“Titrisol” buffer, pH 10.00 (boric acid 1.546 g/L, potassium
Availability of the aminonium ion-selective electrode has chloride 1.864 g/L, sodium hydroxide 476 mg/L; E. Merck &
simplified determination of ammonia in whole blood and Co., Darmstadt, F.R.G.).
plasma (1-3). However, the large samples required (6.0 mL) “Titrisol” buffer, pH 12.0 (disodium phosphate 2.225 g/L,
make the method impractical for use in newborns and small sodium hydroxide 446 mg/L) (E. Merck & Co.).
children. We adapted the method of Attili et al. (2) and Buffer, pH 10.4: Combine 81.0 mL of pH 10 buffer with 19
obtained accurate and reproducible results for 200-L sam- mL of pH 12 buffer.
ples of plasma, the measurement being made in a virtually
anaerobic fashion. Thus, newborns at risk for developing Procedures
hyperammonemia (4, 5) may be evaluated serially without
excessive withdrawal of blood.
Preparation of ammonium (NH4) standards. Ammonium
chloride 1.00 g of ammonia
standard, nitrogen per liter:
Materials and Methods Dilute 71.4 mL of ammonium chloride reference standard
(100 mmol/L; Orion no. 95-10-06) to 100 mL with distilled
Equipment water (standard I).
Ammonium-selective ion electrode probe with flow- Dilute 1.0 mL of standard I to 100 mL with distilled water
to give a final concentration of 10 mg of ammonia nitrogen
per liter (standard II).
Dilute 2, 1, and 0.5 mL of standard H to 8, 9, and 9.5 mL
University of Iowa, Department of Pediatrics, Iowa City, IA. with distilled water, to give final concentrations of 2.00,
Received Oct. 15, 1982; accepted Feb. 18, 1983. 1.00, and 0.50 mg/L, respectively.

CLINICAL CHEMISTRY, Vol. 29, No. 5, 1983 867

 Dilute 1.0 mL of the 500 ,ugtL standard with 1.0 mL of we found that it became stable for all concentrations tested
distilled water to give a final concentration of 250 g/L. by 8 mm. Because the standards and samples are matched
Preparation of sample. Collect 0.6-0.8 mL of whole blood for ionic strength, the response time is the same for both.
by venipuncture into a heparinized tuberculin syringe (sy- Add 0.2 mL of sample to 0.4 mL of pH 12.0 buffer in a
ringe 1). Pierce the barrel and plunger of the syringe with a similar fashion. Recalibrate the electrode with the ammoni-
21-gauge needle, and seal the tip of the syringe with a 25- urn standards after 1 h or four sample analyses, whichever
gauge needle with bent shaft (Figure la). Snip off the end of comes first. The membrane of the electrode is changed every
the plunger and the hub of the 21-gauge needle (Figure lb), second day to minimize the possibility of error from mem-
and immediately place the sample in ice. Then centrifuge brane deterioration.
the specimen, with the bent 25-gauge needle pointing Cakulation. The ammonia concentration in the sample is
upward, in a refrigerated centrifuge (0#{176}C,
5 mm, 12 000 x calculated from comparison with a semilog plot of the
g). Keep the tuberculin syringe upright when it is removed readings of the standards, noted before the sample readings
from the centrifuge, and remove the 25- and 21-gauge were obtained.
needles. Insert the 25-gauge needle of tuberculin syringe 2
into the barrel of syringe 1 as illustrated in Figure lc, and Results
separate the plasma up into syringe 2 by advancing the Analytical recovery was assessed on three different occa-
plunger of syringe 1. Refrigerate the sample and analyze it sions by adding 0.1 mL of various standard solutions (10, 5,

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within 4 h or store it at - 70#{176}C for analysis within 48 h. and 1 mgIL) to samples of plasma. The mean recoveries were
Preparation of standard curve and sample analysis. Add 98, 100, and 103% respectively, for the three (standard
0.2 mL of each of the five working-standard solutions to 0.4 deviations 4.6, 3.7, and 5.0%).
mL of pH 10.4 buffer in a 3-mL plastic cup immediately A single sample of plasma was divided into 10 portions
before analysis. Aspirate the solution into a tuberculin and ammonia nitrogen was measured. The mean value was
syringe (after mixing by drawing the solution up and down 0.92 mg/L, range 0.84-0.96, standard deviation 0.022, and
twice in the tuberculin syringe). Then place the syringe on CV 2.4%. The procedure was repeated on seven portions of a
the pump, which is set to deliver the solution at 0.06 mu different sample. The mean value was 0.70 mg/L, range
miii. Set the electrometer at 0 mV, and make the final 0.68-0.74, standard deviation 0.014, and CV 2%. The varia-
reading at 8 mm. Although the response time on all samples tion in reproducibility attributable to the electrode itself is
may vary somewhat, depending on ammonia concentration, ±2% (6), so the error incorporated by using the microtech-
nique seems negligible.
To investigate the effect of storage, we determined ammo-
nia nitrogen concentrations of 10, 16, and 12 samples within
Whole Blood 4 h and again after 24, 48, and 72 h, of storage at -70#{176} C.
The mean difference in result between the initial determin-
Plasma
tion and determination after 24 h was -8 s.g/L (not signifi-
Cells cant) 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 <
.05), range 190 to -80 g/L. We conclude that analyses
should be done within 48 h of sampling.
Synnge Syringe Syringe Having obtained informed consent,we analyzed plasma of
2 28 normal fasting adults and 26 three-day-old infants. The
25 gauge mean concentration of ammonia nitrogen in the adults was
needle 620 .tg/L (SD 170 g/L) with a range of 310 to 820 tg/L. We
sampled blood from the infants 2.5 h postprandially. The
mean concentration of ammonia nitrogen was 880 pg/L (SD
210 sg/L), the range 600-1300 g/L.

Discussion
Plasma ammonia nitrogen values reported for fasting
adults include: Dienst and Morris (7), mean 630 g/L (SD
100 g!L) and range 450-800 p.g/L (ion-exchange tech-
nique); Forman (8), mean 530 g/L, range 240-690 ug/L
(ion-exchange technique); and Da Fonesca-Wollheim (9),
mean 510 g/L (SD 170 g/L), range 190-1020 L.g/L. These
values are in the same general range as ours. However, with
Syringe use of the ammonia ion-selective electrode and the relative-
ly aerobic macromethod, lower values, mean 280 g/L (1)
(b) and 240 g/L (2) have been reported, possibly reflecting the
greater loss of ammonia in that aerobic macromethod.
In the case of our three-day-old infants, the values for
plasma ammonia nitrogen compare favorably to those re-
ported by Meites (10), with a range of 790-1290 g/L (ion-
exchange), and Rubaltelli et al. (11), mean 900 pgfL and
(a) standard deviation of 150 j.i.g/L (ion-exchange). Thus, this
micromethod not only provides a rapid, reliable assessment
(c) of plasma ammonia nitrogen, but plasma samples may be
Figure 1 stored at -70 #{176}C
for as long as 48 h before analysis.

Fig. 1. Sample collection procedure Supported in part by USPHS Grant No. 1 P01 HD 07578.

868 CLINICAL CHEMISTRY, Vol. 29, No. 5, 1983

References 6. Instruction Manual, Ammonia Electrode Model 95-10, p 22,
(1978). Orion Research, Inc., Cambridge, MA 02139.
7. Dienst SG, Morris B. Plasma ammonia by ion exchange. J Lab
1. Proelss FIF, Wright BS. Rapid determination of ammonia in a Clin Med 64, 495-500 (1964).
perchioric acid supernate from blood, by use of an ammonia-specific 8. Forman DT. Rapid determination of plasma ammonia by an ion
electrode. Clin Chem 19, 1162-1169 (1973). exchange technique. Clin Chem 10, 497-508 (1964).
2. Attili AF, Autizi D, Capocallin L, et al. Rapid determination of 9. Da-Fonesca-Wollheim Von F. Direkte Plasmaammonia-Bestim-
plasma ammonia using an ion specific electrode. Biomedicine 14, mung ohne Enteiweissung. Z Kim Chem Kim Biochem 11,426-431
109-1 16 (1975). (1973).
3. Coleman RL. Clin Chem 18, 867 (1972). Letter. 10. Meites S. Pediatric Clinical Chemistry. Am Assoc Clin Chem,
4. Batahaw ML, Brusilow SW. Asymptomatic hyperammonemia in Washington, DC, 1977, p 36.
low birth weight babies. Pediatr Res 12, 221-224 (1978). 11. Rubaltelli FF, Formentin PA, Tate L. Ammonia nitrogen, urea
5. Ballard RA, Vinocur B, Reynolds JW. Transient hyperammone- and uric acid blood levels in normal and hypodystrophic newborns.
mis of the preterm infant. N Engi J Med 299, 920-925 (1978). Biol Neonate 15, 129-134 (1970).

Downloaded from https://academic.oup.com/clinchem/article/29/5/867/5667644 by guest on 26 October 2020

CLIN. CHEM. 29/5, 869-870 (1983)

Characteristics of High-Affinity Folate Binding in Human Leukocytes

Jan HoIm,’ Steen lngemann Hansen,2and JOrgen Lyngbye2

High-affinity binding of [3H]folate in leukocytes from normal separation medium composed of five volumes of sodium
subjects was studied in equilibrium dialysis experiments (pH metrizoate (in premixed kit form; Nycomed, Oslo, Norway)
7.4, 37 #{176}C).
Binding displayed positive cooperativity, and the and 11 volumes of a 60 g/L solution of dextran (Pharmacia,
binding affinity increased with decreasing concentration of Hilleroed, Denmark) in isotonic NaCl (3). The upper leuko-
the binding protein. Both phenomena could be interpreted in cyte layer, which could be pipetted off after 1 h, was
terms of ligand binding to a polymerizing system where the centrifuged and washed several times in isotonic NaCI to
affinity of ligand for the oligomer is greater than its affinity for remove contaminating plasma. The albumin concentration
the polymer prevailing at higher concentrations of the binding in the last wash solution was <0.5 g/L as determined by
protein. rocket immunoelectrophoresis (7) with monospecific anti-
body against albumin (Dakopatts, Copenhagen, Denmark).
The leukocyte suspensions, which contained from 10 x iO
High folate binding activity in granulocytes was first
to 20 x i09 cells per liter, were subjected to freeze-thaw
demonstrated in some patients with chronic myelogenous
leukemia (1) and subsequently in some women who were procedures, then the serine protease inhibitor phenylmethyl
sulfonyl fluoride (B.D.H., Poole, England) was added to give
pregnant or taking oral contraceptives (2). However, we
a concentration of 1 mmol/L. The cells were homogenized
have recently reported that even granulocytes from normal
persons of both sexes were in several cases very active in
and solubilized in 1 g/L Triton X-100 surfactant (Merck,
binding folate (3,4). Lymphocytes and monocytes contained
Darmstadt, F.R.G.), then dialyzed against Tris buffer (0.17
molfL, pH 7.4) for 24 h at 5 #{176}C
to remove endogenous folate,
less than 1% of the total folate binding activity (3).
Here, we characterize the mechanism of folate binding in and finally centrifuged. The supernatant fluids were pooled
human leukocytes containing large amounts of binding and 600-.iL aliquots of the pool were dialyzed to equilibrium
protein. Binding studies were performed by means of a (20 h) against [3H]folate in 200 mL of the Tris buffer at
standardized equilibrium dialysis technique we previously 37#{176}C.
One gram of Triton X-100 per liter was added to the
used to characterize high-affinity folate binding in milk and fluid on both sides of the dialysis membrane (8). Radioactiv-
serum (5, 6). ity was measured as previously described (9).

Materials and Methods Results and Discussion

[3H]Folic acid with specific activity of 4.8 kCilmol and 26-
Folatebinding in the supernatant fluid from leukocyte
lysate was studied in equilibrium dialysis experiments
45 kCi/mol was purchased from Amersham International
pH 7.4) at three different
(37 #{176}C, concentrations of the
Ltd., Amersham, U.K. Venous blood (EDTA-stabilized) was
binding protein (Table 1). The binding data were analyzed
sampled from three healthy women (B.V., I.P. and A.M.H.
in ref 3), whose hematological values were normal. Blood in Scatchard and Hill plots. Three conclusions could be
drawn from the data shown in Table 1.
samples (5 mL) were layered on top of equal volumes of
#{149}
Maximum folate bound is proportional to the concentra-
separation medium composed of five volumes of sodium
tion of binding protein.
#{149}
A decrease in the concentration of folate binding protein
resulted in a decrease of S05, the external folate concentra-
tion required for half saturation of binding; that is, there
‘Department of Clinical Chemistry, Skive Hospital, DK-7800
was a parallel increase in the binding affinity (itS05).
Skive, Denmark.
2Department of Clinical Chemistry, Central Hospital Hilleroed, However, this dependence of ligand affinity on concentra-
DK-3400 Hilleroed, Denmark. tion of binding protein seemed to level off in more diluted
Received Dec. 28, 1982; accepted Feb. 18, 1983. lysate solutions.

CLINICALCHEMISTRY, Vol. 29, No. 5, 1983 869