Automated microanalysis of urinary ammonia

Clin. Biochem. 2, 381-388 (1969)

AUTOMATED MICROANALYSIS OF URINARY AMMONIA

C. R. DOBBS, H. B. C A S T L E B E R R Y AND E. G. S H A W &L~;AF School of Aerospace Medicine, Aerospace Medical Division ( A FSC),

Brooks Air Force Base, Texas, U.S.A. (Received March 10, t06"9)

SUMMARY

1. An application of the Berthelot reaction to the analysis of microgram quantities of ammoniuna in urine has been described in terms of a newly developed reagent system incorporating ferrocyanide as a catalyst, and the procedure has I~een adapted for the AutoAnalyzer. 2. Criteria of sensitivity of the reaction conditions, studies on dialysis conditions, analytical recoveries and the contributions of urea and creatinine as ammonogenic substances under the reaction conditions are presented.

T H E REACTION I:IETWF.I,:N AMMONIA, PHENOL, AND HYPOCHLORITE in an alkaline

mediuna yields a blue color, as described by Berthelot (1) in 1859. The speed of conversion of ammonia to the blue chromogen was later found to be enhanced by sodium nitroprusside ('2) and acetone (3). A detailed study of the Berthelot reaction with regard to incubation times and optimum reagent concentrations was recently presented by \Veatherburn (.~). T he resulting increase in sensitivity of the reaction allows the determination of ammonia in microgram quantities. Ammonia, at the pH of most biological materials, exists ahnost exclusively as anlnloniunl ion (NH4+). An adaptation of this reaction to biological samples utilizing the Technicon AutoAnalyzer was presented by Logsdon (:5), who measured anamonium in microbiological media. The procedure has been adapted in our laboratory for the determination of ammonia in urine (6). Assous, l)reux, and (;irard (7) utilized the same reaction to determine plasma ammonia levels, and their reaction conditions were later modified by l)ropsv and Bob" (8). I n our efforts to improve the sensitivity and reagent stability of the autonmted procedure, we found that the reaction was enhanced by ferrocyanide. \Ve have incorporated this catalyst into a reagent system similar to that reported earlier (~/) to be optimum. The results of our experience in adapting this reagent system to the AutoAnalyzer are presented in this report, along with comparative data on the analysis of ammonium in urine, and studies on the contributions of urea and creatinine as ammonogenic substances under the reaction conditions.

382

DOBBS, CASTLEBERRY & SHAW

~IATERIALS

Phenol- Ferrocyanide Reagent Phenol, CsH~OH 15 gm Potassium Ferrocyanide l{aFe(CN)e 3Ho.O 2 gm Distilled water q.s. to 1000 ml Stored in a brown plastic container at refrigerator temperature

A lkaline Hypochlorite Reagent Sodium Hydroxide, NaOH 7.5 gm Sodium Hypochlorite solution, NaOCI, 5.25% Available Chlorine 75 ml Distilled water q.s. to 1000 ml Stored in a brown plastic container at refrigerator temperature

Saline, 0.9% Sodium Chloride, NaCI 9 gm Distilled water q.s. to 1000 ml Stable in glass or plastic containers at room temperature

Ammonium Standard, Stock (1 ml = 1 nag NH~+) Ammonium Chloride (anhydrous) NH4CI 2.967 gm Distilled water q.s. to 1000 ml Stored in a glass container at room temperature.

Ammonium Standards, Working Dilutions of the stock anamonium standard were prepared with distilled water to yield final standard concentrations from 0.50 to 15.0 micrograms per milliliter (#g/ml NH4+), All water was glass distilled, and all reagents were ACS grade unless indicated otherwise. The final reactant concentrations were within the range found to be optimum in the final reaction mixture according to Weatherburn (4). The phenol-ferrocyanide and alkaline hypochlorite reagents, while routinely stored at refrigerator temperatures, retained usable stability for up to five days at room temperature. I ETHOD l)ilutions of urine were prepared in ammonia-free water such t h a t the final concentration of ammoniunl was from 1-10 #g/nil. Thus, 24-hour urine samples were conveniently diluted 1:100 or 1:200 prior to analysis. Biological fluids which normally contain only lnicrogram quantities of anm3onium, such as blood plasma, cerebrospinal fluid, parotid fluid, etc., m a y not require dilution prior to analysis. T h e A u t o A n a l y z e r manifold designed for use in this procedure was part of the overall analytical schenla depicted in Fig. 1. T h e solution to be analyzed was diluted with saline to form the donor stream, which was dialyzed against saline at ,37° . T h e recipient saline stream was mixed in turn with the phenol-ferrocyanide and alkaline hypochlorite reagents. Following reaction development in a single-coil heating bath at 9,5°, the color was measured in a 15-ram tubular flow-cell at 6'30 nm. T h e air lines leading to the manifold and serving the reagent containers were " s c r u b b e d " by a 1 N H_~SO4 wash. Sampling rate was 60 s a m p l e s / h r ; the samples were interrupted by a water-air-water wash, giving a net analysis rate of ,'30 samples/hr.

383

AUTOMATED MICROANALYSIS OF URINARY AMMONIA

~,,#, ae,-*Raa#

-o.-~d]

..........

I

i

~

', ~,,.,a.

i 0 ~,.oso I ,*m

I

g

0 o.,~o

(~

(a,~

o.or. .I

"

"

~A~P.xJezr

~u,v="

--.-

tO',, g

O o.oa°

: At./r,t.IN4" #y.a~c~v, oa,/r~" ~[A<,4"#/"

[

.,~o.o~¢,,

!

•

a'/.ot..'.'ct.~

L_~__

f

~,~,

:I

I I

* AI,~~SH

OO

(OLOlP/PtC,?'C.~ / f . . ~ r¢I~dI.AR pL OtV,
I:IG. 1.

~f¢c~CP~

Flow scheme for NH4 + procedure. RESULTS

T h e r e c o r d e r t r a c i n g of a series of a q u e o u s a m n l o n i u m s t a n d a r d s

( f r o m 0.5

to 1;3.0 mcg/ml NH4 +) in duplicate followed by analyses of a series of 1:200 dilutions of five individual urine samples (peaks A through E) is presented in Fig. 2. The tracing demonstrated a logarithmic recorder response to concentration, thus giving maxinaum sensitivity in the lower ranges of amnlonium concentration.

oF '° I ,o~

,o!

1Z.5

AQUEOUS AMMONIUM STANDARDS

,,q/m,

tO.O

! SO-

URINE SAMPLES DILUTED 1:200

40 A

IO0 L _ I

Fie;,. "2. AutoAnah'zer tracing of a series of ammoniunl analyses, duplicate .~tandards followed by a series of urine dilutions (A through E).

DOBBS, CASTLEBERRY & SHAW

384

S a m p l e reproducibility, recorder response to c o n t i n u o u s aspiration, and r e t u r n to the baseline were good. A s t a n d a r d c u r v e resulting from a q u e o u s a m m o n i u n l s t a n d a r d s p r e p a r e d from the r e c o r d e r t r a c i n g (see Fig. 3) I)y c o n v e r t i n g t r a n s m i t t a n c e to c o r r e s p o n d i n g optical d e n s i t y was p r e p a r e d to d e t e r m i n e the linearity 1.000 0.900

0.800 0.700 )-

P- 0.600 Z "

0.500

-I

ca

0.400 0.300 0.200 0.100

O,OOC 0

t

I

I

[

I

I

Z

3

4

5

I

I

I

I

I

6 7 8 9 10 AMMONIUM CONC.(pg/ml NH4 ÷)

I

,[

[

11

12

15

i 14

I 15

FIC;. 3. Standard curve prepared from .-~utoAnalyzer tracing. of tile r e a c t i o n with respect to c o n c e n t r a t i o n . T h e c u r v e itself was derived as a line of a v e r a g e relationship b e t w e e n the c o n c e n t r a t i o n s of the s t a n d a r d s and the c o r r e s p o n d i n g c o m p u t e d optical d e n s i t y . T h e a n a l y s i s of 24 hr urine s a m p l e s b y this p r o c e d u r e was c o , n p a r e d with the m e t h o d of L o g s d o n (5). T h e results are s h o w n in "l'al)le 1, where each value TABLE 1 ~fiRINARY AMMONIA VALUES. :\ COMPARISON OF METHODS

Spec. No.

I.ogsdon (5)

Present Method

1 2

103.') (it)5

I()l-) ti 16

3 4 :) 6 7 S

!104 68!) 7,~7 659 772 7()4

.~2! 7:36 776 5S!) 772 7()4

All values in mg NH4+/24 hrs and are averages of duplicate~.

AUTOMATED MICROANALYSIS OF URINARY AMMONIA

385

represents the mean of duplicate determinations, expressed as milligrams of NH4 + per day. Analysis of this d a t a as a series of paired observations revealed no significant difference between the two analytical procedures (0.4 > p > 0.3). A s t u d y of recoveries of aqueous a m n m n i u m s t a n d a r d s from urine dilutions was performed. T o each of two separate urine dilutions was added an equal a m o u n t of each of two aqueous a m m o n i u m standards. T h e urine dilutions and recovery mixtures were analyzed five successive times and the recovery data are presented in Table 2. TABLE 2 RECOVI~R'¢ OF .~M.MONIUM FROM [,~RINI-2 DILUTIONS

Nil4 + added, mcg

NH4 + recovered, mcg

Percent P, e c o v e r y

1:100

(}. 2;5 1.0(} 2.00 5.00

o . 26 0 . 9 {} 2.01 5. (}0

104.0 99. (} 10{}.3 100.0

1 : 200

0.25 1.00 2,00 5.0{}

{}. 25 0.99 2.00 4.99

1 {}{}. {} 99.0 10(}. 0 !}9.5

A v e r a g e ~£~, S.D,

1{}{}.;] 1 .{3

1 "rine I)ilu tion

The effects of urea and creatinine as a m m o n o g e n i c substances trader tile present reaction conditions were evaluated. Aqueous solutions of urea containing from 1,000 to 5,000 rag/100 urea (A('S grade) were prepared and treated with a cation-exchange resin (Dowex ,'50X-10) for removal of preformed ammouia, and then analyzed for amnmniunl I}y the present method. T h e contribution of urea as an ammonogenic substance was thus estimated for a range of urea concentrations spanning those expected in nornlal urine, as presented in Table 3. Tile contribution of color by urea was minimal in urine samples but not linear with respect to concentration when analyzed I}.x the present procedure. T h e contribution of urea as an a m m o n o g e n i c substance is thus negligible in urine samples diluted as suggested in the present procedure. ()ur studies showed that aqueous creatinine standards spanning the urinary physiologic range were ammonogenically insignificant, as was expected. TABLE 3 EFFECT OF ['RI-A ON AMMONIA ANAI.YSIS

I_'rea Concentration, ..\queous Solution hi nlg/l {){}

Value, as NI-IC, in Present method mcg/nll

10{}0

{1. O19

2000 300{} 40{}1} 5000

{}.02S O. 033 0.1136 0. {}39

DOBBS, CASTLEBERRY & SHAW

386

T o determine the dialysis characteristics of aqueous anamonium under the experimental conditions, both the recipient and donor streams of a series of aqueous a m m o n i u m standards were analyzed as they emerged from the dialysis unit over a range of a m m o n i u m concentration from 0.5 m c g / m l to 1,5 m c g / m l . Similarly, the recipient and donor streams of a 1 : 200dilution of urine (in replicate) were analyzed and compared with the respective s t a n d a r d curves. T h e analyzed values and percentage dialysis, presented in Table 4, d e m o n s t r a t e d that dialysis TABLE 4 DIALYSIS OF AMMONIUM FROM AQUEOUS STANDARDS AND URINE DILUTIONS

mcg NH4+/ml Sample Standards:

Donor Stream

Recipient Stream

Percent Dialysis

0,5

0.3 0.7 0.9 1.3 1.6 2.9 4.7 6.3 7.9 8.5

37.5 41.2 37.5 39,4 39,O 36,7 38,5 38.7 38.7 36.1

1.0

1,5 2,0 2.5 5.0 7.5 10.0 12.5 15.0

Average

Urine Dilutions (1:200)

S.D. 2.7 2.8 2.8 2.9

1.6 1.7 1.7 1.7 S.D.

6.2 6.2 6.3

1.5

37.2 37.8 37.8 37.0 Average

Urine Dilutions (1:100)

38.3

2.9 3.3 3.3

37.5

0.4 31.9 34.7 34.4

Average

S.D.

33.7 1.5

of a m m o n i u m from the aqueous a m m o n i u m s t a n d a r d s was 38.3 ± 1.5%, from 1 : 200 urine dilutions was 37.5 4- 0.9%, and from 1 : 100 urine dilutions was 33.7 41.5%. T h u s tile percentage dialysis of a m m o n i u m from the 1:200 urine dilution a p p r o x i m a t e d t h a t of the aqueous anamonium s t a n d a r d s more closely than did the 1:100 urine dilution. I t is possible t h a t tile a p p a r e n t l y lower dialysis of a m m o n i u m from urine dilutions was not due to a lower dialysis constant, t)ut to other chromogenic c o n t a m i n a n t s in the donor stream. I)ISCUSSION In order to measure a m m o n i u m in biological materials, it is necessary to use at reaction of high molecular specificity and to remove or compensate for potentially

AUTOMATED MICROANALYSIS OF URINARY AMMONIA

387

interfering substances. Searcy, et al. (9) and W e a t h e r b u r n (4) have shown that the chemical specificity of the Berthelot reaction depends on the composition and sequence of addition of the reagents, with the addition of phenol followed by alkaline hypochlorite offering o p t i m u m specificity for ammonium. Che,nical reaction specificity alone cannot compensate for those constituents of biological materials which produce additional a m m o n i u m (ammonogenic substances) under the reaction conditions. Dialysis, under conditions similar to those reported here, had been reported to eliminate the majority of biologically derived ammonogenic substances from aqueous solution, particularly glutamine (2). Our observations have been limited to urine, specifically to the contribution of urea and creatinine as ammonogenic substances under these reaction conditions. T h e dialysis characteristics of such an a u t o m a t e d system were of particular interest in terms of developing maximum sensitivity. Other workers (7) have reported 50070 dialysis of a m m o n i u m and of urea from aqueous standards; our results with such standards have been presented. Since optimum reaction criteria in terms of reactant concentrations can be obtained by either direct (i.e., vs. phenate) (8, 6, 8) or indirect (i.e., vs. saline) (7) dialysis, it was felt that saline dialysis was more desirable. Searcy, et al. (10) have pointed out the deleterious effect of phenolic solutions on the Lucite dialyzer plates, a fact which we experienced in our modification of the Logsdon (6) procedure. Saline dialysis, on the other hand precluded damage to the plates with no demonstrable effect on reaction sensitivity. In addition, direct dialysis was thought less desirable in view of the ammonogenic effect of v e r y basic solutions on biological materials (11). T h e estimation of ammonium in plasma has been accomplished by m a n y different workers, and there is today no real agreement as to what constitutes a normal vahle for this constituent, nor whether different biological sources of ammonium are measured to different degrees with the various methods of analysis. T h e r e are m a n y advantages to an a u t o m a t e d approach to plasma anamonium analysis, chief of which is minimization of contamination. Our approach to the estimation of a m m o n i u m in plasma has included analysis of refrigerated whole plasma, and of protein-free filtrates, based on the method of sample preparation described by .~IcCullough (12). A t this time, our observations on the specificity" of plasma a m m o n i u m analyzed under these conditions are as yet incomplete; however, we employ plasma obtained in heparinized Vacutainers in a direct analysis to obtain an estimation of anamoniuna concentration. Insufficient d a t a prevent comparisons with other methods at this time, and we are engaged in further studies on this problem. REFERENCES Violet d'aniline. R~pertoire de Chimie Appliqu~e. 1,284 (18.39). LUBOCHINSKY,B. & ZALTA,J.P. Microdosage colorhn~trique de I'azote ammoniacal. Bull. Soc. Chim. Biol. (Paris) 36, 1363 (19,54). CROWTHER,A. B. & LARGE, R.S. Improved conditions for the sodium phenoxide-sodium hypochlorite determination of ammonia. Analyst 81, 64 (1956). ~VEATHERBURN,~lI. W. Phenol-hypochlorite reaction for determination of ammonia. Anal. Chem. 3?, 971 (1967). LOGSDON,E.E. A method for the determination of ammonia in biological materiMs on the AutoAnalyzer. Ann. N.Y. Acad. Sci. 87, 801 (1960).

1. BERTHELOT, ~I.

2. 3. .~.

5.

388

DOBBS, C A S T L E B E R R Y & SHAW

6. GLATTE, H. G. & CASTLEBERRY, H . B . Unpublished observations. 7. Assous, E., DREUX, C. ~¢.GIRARD, M. Application nouvelle de la dialyse a la d6termiuation de Fammoni(~mie. Ann. Biol. Clin. (Paris) 18, 319 (1960). 8. DROPSY, G. & BoY, J. Determination de I'ammoni6mie. Ann. Biol. Clin. (Paris) 19, 313 (1961). 9. SEARCY, R. L., et al. A study of the specificity of the Berthelot colour reaction. Clin. Chhn. Acta 12, 170 (1965). 10. SEARCY,R. L., et al. A new automated method for urea nitrogen analysis. Amer. J. Clin. Path. 47, 677 (1967). I I . REINHOLD, J. G. & CHUNG, C . C . Formation of artifactual ammonia in blood by action of alkali: Its significance for the measurement of blood ammonia. Clin. Chem. 7, 54 (1961). 12. McCULLOUGH, H. The determination of ammonia in whole blood by a direct colorimetric method. Clin. Chim. Acta 17, 297 (1967).