Determination of Ammonia in Biological Solutions by SecondDerivative Spectrometry R. D. Hunt," R. Cade, and D. T. Williams Departments of Physiology, Medicine, and Engineering Sciences, University of Florida, Gainesville, Fla. 326 10
Modifications are reported which improve accuracy and versatility for a method of blood ammonia assay by derivative spectrometry first descrlbed by Hager in Anal. Chem., 42, 14 (1970). The modified assay is proven for plasma and whole blood and may be used with cerebrospinal fluid and deproteinated filtrates. Fifleen minutes is requlred for a single assay in plasma. After 20 minutes an alkali generated artifact on the order of 0.01 pg/ml/min is observed. Recovery of ammonia was 104.4 f 2 % and 100 f 2.7%, respectively,for plasma and whole blood with cells lysed. In non-lysed whole blood 84.5 f 4.2% recovery was observed. Ammonia found in plasma of nine normal humans was 0.81 f 0.29 pg/ml, and is comparable to that assayed by the method of Seligson and Hihara, J. Lab. Clin. Med. (1957).
Hager et al. ( I ) first applied the second-derivative (d2) spectrometer to measure total blood ammonia. The method consisted of measuring partial pressure of ammonia over an alkalized deproteinated filtrate of blood with and without an ammonium standard added. We were, however, unable satisfactorily to apply the method as published. Inadequate recoveries and poor repeatability occurred frequently. Two inadequacies are seen: 1)when anhydrous potassium carbonate is used as the alkali, an exothermic reaction occurs on addition of sample and 2) optical cells loaded with alkali and sample were not rotated on a continuous basis for equilibration. Because of its unique sensitivity and simplicity, refinement of the method was thought warranted, and is the purpose of this report.
EXPERIMENTAL Reagents. A saturated potassium carbonate solution is prepared by dissolving reagent grade anhydrous KzC03 in ammonia free water; saturation and absence of ammonia is ensured by boiling. Reagent grade crystalline KzC03-1%H20and ("&SO4 or NHdCl is used for alkalization and preparation of standards. Deionized distilled water maintained ammonia free with Hyland Company resin is used for preparation of all reagents. Apparatus. The d2 spectrometer and quartz absorption cells for sample containment are as described by Hager ( I ) . Additionally required is a device for rotation of sample cells after loading. We attached a 6.35-cm radius wheel with spring clamps on the periphery to a motor driven shaft for this purpose. Rotation of samples at 45 rpm was thus maintained during equilibration. A tool for transferring 0.8-g aliquots of K2C0r11hH20 to the sample cells, made by cutting the tip off a plastic disposable 1-ml tuberculin syringe, is also useful. Procedure. Plasma, whole blood, cerebrospinal fluid (CSF), saline, and deproteinated filtrates of whole blood may be assayed. Whole blood is drawn anaerobically in heparinized vacutainers and iced. Samples are centrifuged for 10 min, and plasma separated for storage or for immediate assay. Deproteinated filtrates are prepared as described by Hager ( 1 ) . Two ml of saturated K ~ C O solution J and approximately 1.6 g of KLCOyl%HpO is added to ensure saturation of cell contents when up to 1ml of sample is added. One ml of sample is loaded into one cell, and 1 ml of sample plus a known quantity of ammonia standard into
Mailing address, University of Florida, Department of Physiology, P.O. Box 5-274, J.H.M.H.C., Gainesville, Fla. 32610.
a second cell. Both cells are placed on the rotor and a timer set. Twenty min later the d2 signal a t 204.6 mp resulting from partial pressure of ammonia in each of the two cells is recorded and ammonia concentration calculated. Instrument sensitivity is first determined,
+
(Amplitude for sample standard added) - (Amplitude for sample) Sensitivity = pg of standard added
(1)
from which sample ammonia content is Amplitude for sample pg NH3-N/ml sample = (2) Sensitivity Average d2 instument sensitivities calculated for saline, plasma, and cerebrospinal fluid on several days are shown in Table I. An average sensitivity of 18.28 f 19.3%(95% confidence interval) occurred. Approximately 5.3% is instrument error and the balance method error. It is noteworthy that in clinical screening for high ammonia, the accuracy for the four days shown is such that the sensitivity could be applied directly to signal amplitude for calculation of ammonia concentration and simultaneous standards would not have to be run.
ANALYSIS OF METHOD AND DISCUSSION A calibration with diluted ammonia gas confirmed the linear response for the d 2 instrument of 31.5 X lo2 divisions/ mm Hg up to 0.1 mm Hg. A similar linear response of 21.0 divisions/yg ammonia was observed for 1-ml samples of saline with ammonia added. It is desirable to use smaller sample volumes if concentrations greater than 15 yglml are to be measured. Time for Equilibration. Signal amplitudes after rotating 10-yl aliquots of aqueous ammonium sulfate, saline with ammonia added, plasma, and whole blood for various periods of time are shown in Figure 1. An equilibrium for ammonia between the gas phase and solution is indicated by approach ~ ,an asymptote in each case. An of signal amplitude, or P N H to initial rapid rise with subsequent decrease in P N H ?to an asymptote is seen for 10-yl samples. The very small sample apparently becomes completely alkalized and saturated on contact with the KzC03 solution. Ammonia gas thus created diffuses into the gas phase, redistributing after 15min to the solution. One-ml saline samples with ammonia added behave differently. Because of the greater volume, saline retains a variable capacity for ammonium and ammonia gas until equilibrium; P N Hthus ~ rises from zero to an asymptote during equilibration. The amplitude of an equilibrated saline or aqueous standard remains constant within an instrument error of f5.3% over a 12-h period. Plasma and whole blood equilibrate much like saline. All solutions appear to equilibrate in about 15 min, though a time of 22 min is more appropriate for whole blood. The longer time for blood is attributed to inhibition of diffusion of ammonia from solution by protein ( 2 ) . The similarity of appearance for curve forms of saline, plasma, and whole blood is significant. Many authors (3-5) have cited the rise from an apparent zero ammonia content, Le., zero for the plasma and whole blood as evidence that there is no free ammonia in the sample a t zero time, but that ammonia is created as a result of alkalization. This is clearly not the case, since it occurs for saline as well. ANALYTICAL CHEMISTRY, VOL. 49, N O . 1, J A N U A R Y 1977
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c Table I. Sensitivity for Ammonia Assays in Plasma, CSF, and Saline Solution Sensitivity and no. of for 1-ml volume (/%) samples Plasma (6) 18.05 Plasma (5) 16.61 CSF ( 5 ) 17.08 18.57 Saline (6) CSF (6) 19.02 Plasma (5) 20.07 CSF (5) 18.57 Av = 18.28
Date (1974) 5 Aug. 21 Aug. 21 Aug. 10 Sept. 10 Sept. 17 Sept. 17 Sept.
Standard error
E
-1.0
2
LL LL W
1.94
m
W
.8
>-
2.12
c
2.62 .97 .87 2.05 1.65 1.76
2 .6
t-
v)
2 W
u)
a4
W
I
3 -I
0
.2
> 50] 40
0
0
20
U
3
c .E 30
t
E
a -
22 2.4 2.6 2.8 TOTAL VOLUME in CELL h l )
-+Q2 Sa t. K2C03
20
.-CI,
v)
' 0t , O
I
IO
20 30 30 40 50 60 Miptes After Exposure to Alkali
1 .o
0.4 0.6 0.8 SAMPLE VOLUME (mi)
Figure 2. Effect of sample volume on signal response as determined by Equation 4 for A V = 0 (- - -), for Equation 4 (-), and as determined empirically (0).Standard errors are shown as vertical bars, and number
IO
t-x";rxI'x
3.0
70
of samples are shown in parentheses where applicable
Figure 1. Equilibration as between gaseous ammonia and dissolved ammonia with time for 10-pl aliquots of aqueous ammonia standard ((0)each each point with standard error shown as vertical bar is 4 samples),1 ml of saline with ammonia added (0). 1 ml of human plasma (X), and 1 ml of human whole blood with lysed red blood cells (A)
a
a a
B
0
a
Effect of Sample Volume. We have found in our own work that it is often necessary to compare sensitivities for quartz cells containing different volumes, the greater volume being placed in the cell without standard. This is often the case, for example, when serial CSF samples are drawn from dogs and it is desired to keep samples small. Instrument sensitivity is more than adequate for smaller volumes but, when calculated by Equation 1,varies inversely with volume of sample in the quartz cell. A relation correlating sensitivities for differing volumes is therefore useful. Conveniently, in this respect, concentration of ammonium ion is negligible in saturated K2C03, virtually all being converted to ammonia gas by the high pH. Distribution of ammonia in the cell may thus be simply described by the relation
where P N H is ~ proportional to signal amplitude and cy' is solubility for ammonia in saturated KzC03 solution. V = 2.0 ml KzCO:~solution, and V, is the sample volume, AV is change in volume resulting from solution of the crystalline KzCOy 11/2H20, and is approximately 0.3 ml when V, = 1.0 ml. for 1.0 pg Comparing P";j for 1.0 pg ammonia in V, to when V, = 0, we have "Volume Sensitivity Coefficient" =
P N H=~ 2.0 84
2.0
+ 1.3V,
(4)
ANALYTICAL CHEMISTRY, VOL. 49, NO. 1, JANUARY 1977
O
l 0
. IO
,
. 20
,
.
, , , . , 50 60 70 Minutes After Exposure to Alkali 30
,
. 40
,
,
Figure 3. Ammonia concentration in a single human whole blood sample with time after exposure to alkali Samples prepared as follows: whole blood: 1) plain (O), 2) under 100% COz after 4 h (a), 3) plus 3.37 pg/ml (a),and 4) under 100% COz plus 3.37 fig/mi after 4 h (0); whole blood with dry ice lysed red blood cells: 5) plain (O),6) under 100% COn after 4 h (B), 7) plus 3.37 pg/ml (a),and 8)under 100% COz plus 3.37 fig/rnl after 4 h (h).Lines are visual fits of the data
This is plotted as the solid curve in Figure 2 for sample volumes up to 1ml. T o test assumptions inherent to Equation 4,different volumes of saline with 1.0 pg ammonia each were analyzed. was assumed as that observed for 1O-wl samples and divided into P",j observed for larger volumes. The data, plotted as open circles in Figure 2, agree very well with prediction. Of interest from information given above is that a' is only about 0.055 1NH3/l. saturated KzCO3/mm Hg at 25 "C. Also approximately 87% of the total ammonia in the 27-ml volume of the quartz cell remains in solution. K & 0 3 as a n Alkalizing Agent. The use of KzCO3 as an alkalizing agent for blood ammonia determinations has been
Table 11. Recoveries of Ammonia from Plasma and Whole Blood (Cells Lysed) Added, Pg
Total, Yg
Recovered Pg
%
Table 111. Recoveries of Ammonia from Whole Blood
0.65 pg/ml 0.62
2.61 pg/ml
3.07 5.98 24.76 12.39 49.44 2.00 2.00 1.33
5.24 4.97 94.8 8.15 8.72 107.0 26.93 27.71 102.9 14.56 14.64 100.5 51.61 58.40 113.2 4.61 5.06 110.0 4.61 4.70 102.0 3.84 4.12 105.0 Av = 104.4 f s.e. 2.03%
Total
Recovered
%
2.63 2.75 3.37 3.37
3.28 3.37 4.47 4.44
2.91 3.44 3.87 4.25
89.0 102.0 87.0 96.0
2.40 2.40 2.86 2.75
4.11 2.79 68.0 4.69 3.23 69.0 4.86 3.96 81.0 5.07 4.22 84.0 Av = 84.5 f s.e. 4.2%
Human blood
Plasmas 2.17 pg/ml
Added
1.10
1.07 Dog blood
1.71 2.29 2.11 2.32
Whole blood (lysed red blood cells) Human blood 1.56 pg/ml
1.60 pg/ml
2.10 3.16 2.00 3.00 0.40 0.80 1.60
3.66 4.72 3.56 4.56 2.00 2.40 3.20
3.68 4.50 3.58 3.87 1.78 2.30 3.29
2.00 2.00 2.00 2.00
4.10 4.76 116.0 4.00 4.15 104.0 4.10 4.64 113.0 4.09 97.5 4.20 Av = 100.0 f s.e. 2.7%
100.0 97.0 100.0 85.0 89.0 96.0 103.0
Dog blood
2.10 pg/ml 2.00 2.10 2.20
criticized by many authors because of its high pH and the liberation of loosely bonded or labile ammonia from protein (6) thought to occur as a result. We have consistently found with human and dog plasmas an alkali-generated artifact on the order of Conway's ( 3 ) of about 0.01 fig/min after 20 min of equilibration. Incomplete recoveries of added ammonia from whole blood have also been attributed to use of KzC03 as an alkalizing agent (2). Recovery of ammonia from a single blood sample prepared in four ways, as shown in Figure 3, does not bear this out. It is clear that about 26% more ammonia is made available by lysing the cells. The cell wall apparently acts as a barrier to the exchange of ammonia after alkalization, or more logically, perhaps as a barrier to alkalization of the red cell interior. It is noteworthy in this respect that the red blood cell is extremely crenated in the hypertonic medium and that membrane properties in this circumstance would be unpredictable. A barrier to ammonia exchange would also be likely when preparing deproteinated filtrates of whole blood, probably explaining why ammonia added to whole blood before deproteination cannot be recovered (7). Clearly, red blood cell lysis is desirable when assaying whole blood. Cell lysis may be accomplished with saponin or by freezing; prompt assay is essential. Notice in Figure 3, that maintenance of whole blood lysed and non-lysed
under 100%carbon dioxide preserves ammonia content up to 4 h, confirming a conclusion to this effect of Preuss et al. (8).
Ammonia Assay i n Plasma a n d Whole Blood. New assay techniques may be proven by recovering amounts of ammpnia added to a sample. Hager ( I ) , though testing his method against the method of Seligson and Hihara; reported only two recoveries. Table I1 shows recoveries by the present method. Recoveries of 104.4 f 2% are shown for 8 plasmas and 100.0 f 2.7% for 11lysed whole blood samples. Incomplete recoveries occur when cells are not lysed as shown in Table 111. Though complete recoveries are claimed for all methods, ammonia concentrations reported in normal human plasma differ by several-fold; 0.4-0.5 fig/ml as found in most ion exchange techniques (9, 10) to 0.78-3.0 pg/ml for alkaline diffusion techniques (2,3,11).I found levels in normal humans of 0.81 f 0.29 pg/ml in 9 plasma samples and 1.12 f 0.43 pg/ml in 8 non-lysed whole blood samples; 2.21 f 0.87 figlml was found in 14 whole blood samples when the cells were lysed. Non-lysed whole blood values compare with those reported by Hager et al. ( 1 )for deproteinated filtrates. Average plasma and whole blood ammonia levels for normal humans are similar to those reported by Seligson and Hihara (2). LITERATURE CITED (1)R. N. Hager, Jr., D. R. Clarkson, and J. Savory, Anal. Chem., 42, 1813 (1970). (2) D. Seligson and K. Hihara, J. Lab. Clin. Med.,49, 962 (1957). (3)E. J. Conway, "Microdiffusion Analysis and Volumetric Error," 3d ed., Crosby, Lockwood and Son, Ltd., London, 1950. (4) L. P. White, E. A. Phear, W. H. J. Summerskill, and S. Sherlock, J. Clin. lnvest., 34, 158 (1955). (5) H. Kaprowski and H. Uninski, Biochern. J., 33, 747 (1939). (6)J. G.Rheinhold and C. C. Chung, Clin. Chem. ( Winston-Salem, N.C.), 7,
54 (1961). (7) G. N. Nathan and F. L. Rodkey, J. Lab. Clin. Med., 45, 779 (1957). (8)H. G.Preuss, 6.B. Bise, and G. E. Schreiner, Clin. Chem. ( Winston-Salem, N.C.), 12, 329 (1966). (9)R. Schwartz, G.B. Phillips, G. J. Gabuzda, and C. S. Davidson, J. Lab. Clin., Med., 42, 499 (1953). (IO)J. H. Hutchinson and D. H. Labby, J. Lab. Clin. Med., 60, 170 (1962). (11) G.E. Miller and J. D. Rice, Jr., Am. J. Clin. Pathol., 39, 97 (1963)
RECEIVEDfor review July 16, 1976. Accepted October 1, 1976. Research supported by NIH Grant No. HL 05979 and NIH GM 20237-01.
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