Clin. Biochem. 9, (4) 192-194 (1976)
A Comparative Study of Micro.Ammonia Determinations in Plasma Using Two Different Methods ERIC J. SAMPSON and LAURENCE M. DEMERS Division of Clinical Pathology, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania 17033, USA (Accepted March 30, 1976)
MATERIALS AND METHODS CLBIA 9, (4) 192-194 (1976)
Clim Biochem. Sampson, Eric J. and Demers, Laurence M.
Division of Cli~zical Pathology, The Milto?z S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pa 17033 USA. A C O M P E T I T I V E S T U D Y OF MICRO-AMMONIA D E T E R M I N A T I O N S IN PLASMA U S I N G TWO D I F F E R E N T METHODS Two commercially available kits for determining blood ammonia were compared, an enzymatic method requiring a sample volume of 100~l and a nonenzymatic method requiring 1 ml of sample. Calibration curves for both kits were linear to 270 ~mol/I, however correlation studies on plasma samples revealed a lack of agreement between methods (r ~ 0.825, Y ---- 0.76X -t- 51). hnproved correlation occurs when portein interference is eliminated from the enzy-matic procedure. Finally, the nonenzymatic method is modified to accommodate 100 ~l of sample and compared to the original method.
I'VIOST METHODS FOR DETERMINING BLOOD AMMONIA have r e q u i r e d sample volumes in excess of one ml to ach i ev e a d e q u a t e precision. In n e w b o r n i n f a n t s , this p r e s e n t s a problem i f cap i l l a r y s a m p l i n g is used. Recently, a c o m m e r c i a l l y available k it f o r d e t e r m i n ing blood a m m o n i a levels on 100 /zl of s e r u m was introduced wh i ch employs the enzyme g l u t a m i c dehydrogenase to catalyze the f o l lo w i n g r e a c t i o n : H ÷ + o~-ketoglutarate -F N A D H + NH.~ ~ L - g l u t a m a t e -~- N A D ÷ -+- H..,O. T h e e q u i l i b r i u m of this reaction lies f a r to the r i g h t , so t h a t c h a n g e s in a b s o r b a n c e at 340 nm are directly p r o p o r t i o n a l to the a m m o n i a concentration. A n o t h e r k i t involves the use of an ion e x c h a n g e r e s i n w h i c h selectively adsorbs a m m o n i a f r o m plasma. T h e a m m o n i a is t h e n eluted and r e a c te d w i t h hypoc h l o r i t e and phenol to yield an indophenol ( B e r t h e l o t ' s r e a c t i o n ) . T h i s m e t h o d is c o m p a r a b l e to the recommended p r o c e d u r e described by K i n g s l e y and T a g e r ('). We e x a m i n e d both kits, w i t h a d a p t a t i o n s , f o r possible use in t h e d e t e r m i n a t i o n of a m m o n i a on capillary samples. Correspondence: Dr. L. Demers, Department of Pathology, The M. S. Hershey Medical Center, Hershey, Pennsylvania 17033, USA.
"Blood Ammonia Test" kit (Hyland, Costa, Mesa, Calif. 92626) included the following reagents: 4 mol/l sodium chloride, phenol color reagent composed of 5 g phenol and 0.55/~ (w/v) sodium nitroferricyanide, a solution of (15 g/l) sodium hypochlorite and 178 g / l sodium hydroxide, ion exchange resin, and nitrogen standard 707 mg/l. "Eskalab Reagent for Determination of Ammonia in Blood" (Smith Kline Instruments, Inc., Palo Alto, California 94306) included two tablets labeled "Ammonia" and "Ammonia No. 2". The tablet labeled "Ammonia" contained : 7 ~mol a-ketoglutarate, 0.28 ~mol reduced nicotinamide adenine dinucleotide, 4.2 .~mol adenosine diphosphate, 126 .~mol tris(hydroxymethyl) aminomethane, 28~mol succinic acid, and 48 ~mol potassium bicarbonate. The tablet labeled "Ammonia No. 2" contained 0.42 IU of glutamic dehydrogenase. Additional reagents included: reagent grade tris base (Schwartz/Mann), perchloric acid (Baker), and distilled deionized water. Disposable plastic containers and MLA pipettes with disposable tips were used to avoid ammonia contamination. Absorbance measurements were performed on a Gilford Model 240 spectrophotometer equipped with a temperature controlled cell block and on a Gilford Model 300 N spectrophotometer, with a flow through cuvette. Standard ammonia solutions were prepared from the Hyland nitrogen standard. Patient samples were collected in heparinized tubes and the separated plasma allowed to stand at room temperature for several hours prior to running comparative analyses to avoid large changes in ammonia concentration reported to occur shortly after collection':' :" Hyland Macro-Method. The Hyland method was performed according to the manufacturer's instructions. One ml of standard or sample, 2 ml of water, and five drops of resin were added to a 15 ml plastic tube. The tubes were capped and mixed by inversion for five minutes, after which the supernatant was removed from the resin and the resin-washed twice with 10 ml portions of water. After thoroughly decanting the final wash, 1 ml of 4 mol/l NaCl and 1 ml of phenol color reagent were added to each tube and allowed to stand for 3 minutes. One ml of alkali hypochlorite reagent was then added to initiate the Berthelot reaction and the mixture incubated at 37" for 15 minutes. Finally, 3 ml of water were added and the resulting absorbance measured at 635 nm. Hyland Micro-Method. The sample and all reagent volumes were reduced to 1/10th of the original volume employed with the Hyland Macro Procedure described above. Only 1 drop of resin was used and for the Berthelot reaction the mixture was incubated at 56" for 6 minutes. Similarly, the size of the container was reduced to a Technicon T M sample cup (4 ml) to accommodate the smaller volume. Eskalab Method. This method was performed according to the manufacturer's instructions: 0.1 ml of standard or sample, 1.4 ml of water, and one "Ammonia" tablet were added to a cuvette. A f t er 10 minutes at room temperature, the absorbance was measured at 340 nm and "Ammonia
BLOOD AMMONIA D E T E R M I N A T I O N S
193
200
.6 t
.5'
E =
,1-
.4 ¸ ',s oo
A Comparative Study of Micro.Ammonia Determinations in Plasma Using Two Different Methods ERIC J. SAMPSON and LAURENCE M. DEMERS Division of Clinical Pathology, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania 17033, USA (Accepted March 30, 1976)
MATERIALS AND METHODS CLBIA 9, (4) 192-194 (1976)
Clim Biochem. Sampson, Eric J. and Demers, Laurence M.
Division of Cli~zical Pathology, The Milto?z S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pa 17033 USA. A C O M P E T I T I V E S T U D Y OF MICRO-AMMONIA D E T E R M I N A T I O N S IN PLASMA U S I N G TWO D I F F E R E N T METHODS Two commercially available kits for determining blood ammonia were compared, an enzymatic method requiring a sample volume of 100~l and a nonenzymatic method requiring 1 ml of sample. Calibration curves for both kits were linear to 270 ~mol/I, however correlation studies on plasma samples revealed a lack of agreement between methods (r ~ 0.825, Y ---- 0.76X -t- 51). hnproved correlation occurs when portein interference is eliminated from the enzy-matic procedure. Finally, the nonenzymatic method is modified to accommodate 100 ~l of sample and compared to the original method.
I'VIOST METHODS FOR DETERMINING BLOOD AMMONIA have r e q u i r e d sample volumes in excess of one ml to ach i ev e a d e q u a t e precision. In n e w b o r n i n f a n t s , this p r e s e n t s a problem i f cap i l l a r y s a m p l i n g is used. Recently, a c o m m e r c i a l l y available k it f o r d e t e r m i n ing blood a m m o n i a levels on 100 /zl of s e r u m was introduced wh i ch employs the enzyme g l u t a m i c dehydrogenase to catalyze the f o l lo w i n g r e a c t i o n : H ÷ + o~-ketoglutarate -F N A D H + NH.~ ~ L - g l u t a m a t e -~- N A D ÷ -+- H..,O. T h e e q u i l i b r i u m of this reaction lies f a r to the r i g h t , so t h a t c h a n g e s in a b s o r b a n c e at 340 nm are directly p r o p o r t i o n a l to the a m m o n i a concentration. A n o t h e r k i t involves the use of an ion e x c h a n g e r e s i n w h i c h selectively adsorbs a m m o n i a f r o m plasma. T h e a m m o n i a is t h e n eluted and r e a c te d w i t h hypoc h l o r i t e and phenol to yield an indophenol ( B e r t h e l o t ' s r e a c t i o n ) . T h i s m e t h o d is c o m p a r a b l e to the recommended p r o c e d u r e described by K i n g s l e y and T a g e r ('). We e x a m i n e d both kits, w i t h a d a p t a t i o n s , f o r possible use in t h e d e t e r m i n a t i o n of a m m o n i a on capillary samples. Correspondence: Dr. L. Demers, Department of Pathology, The M. S. Hershey Medical Center, Hershey, Pennsylvania 17033, USA.
"Blood Ammonia Test" kit (Hyland, Costa, Mesa, Calif. 92626) included the following reagents: 4 mol/l sodium chloride, phenol color reagent composed of 5 g phenol and 0.55/~ (w/v) sodium nitroferricyanide, a solution of (15 g/l) sodium hypochlorite and 178 g / l sodium hydroxide, ion exchange resin, and nitrogen standard 707 mg/l. "Eskalab Reagent for Determination of Ammonia in Blood" (Smith Kline Instruments, Inc., Palo Alto, California 94306) included two tablets labeled "Ammonia" and "Ammonia No. 2". The tablet labeled "Ammonia" contained : 7 ~mol a-ketoglutarate, 0.28 ~mol reduced nicotinamide adenine dinucleotide, 4.2 .~mol adenosine diphosphate, 126 .~mol tris(hydroxymethyl) aminomethane, 28~mol succinic acid, and 48 ~mol potassium bicarbonate. The tablet labeled "Ammonia No. 2" contained 0.42 IU of glutamic dehydrogenase. Additional reagents included: reagent grade tris base (Schwartz/Mann), perchloric acid (Baker), and distilled deionized water. Disposable plastic containers and MLA pipettes with disposable tips were used to avoid ammonia contamination. Absorbance measurements were performed on a Gilford Model 240 spectrophotometer equipped with a temperature controlled cell block and on a Gilford Model 300 N spectrophotometer, with a flow through cuvette. Standard ammonia solutions were prepared from the Hyland nitrogen standard. Patient samples were collected in heparinized tubes and the separated plasma allowed to stand at room temperature for several hours prior to running comparative analyses to avoid large changes in ammonia concentration reported to occur shortly after collection':' :" Hyland Macro-Method. The Hyland method was performed according to the manufacturer's instructions. One ml of standard or sample, 2 ml of water, and five drops of resin were added to a 15 ml plastic tube. The tubes were capped and mixed by inversion for five minutes, after which the supernatant was removed from the resin and the resin-washed twice with 10 ml portions of water. After thoroughly decanting the final wash, 1 ml of 4 mol/l NaCl and 1 ml of phenol color reagent were added to each tube and allowed to stand for 3 minutes. One ml of alkali hypochlorite reagent was then added to initiate the Berthelot reaction and the mixture incubated at 37" for 15 minutes. Finally, 3 ml of water were added and the resulting absorbance measured at 635 nm. Hyland Micro-Method. The sample and all reagent volumes were reduced to 1/10th of the original volume employed with the Hyland Macro Procedure described above. Only 1 drop of resin was used and for the Berthelot reaction the mixture was incubated at 56" for 6 minutes. Similarly, the size of the container was reduced to a Technicon T M sample cup (4 ml) to accommodate the smaller volume. Eskalab Method. This method was performed according to the manufacturer's instructions: 0.1 ml of standard or sample, 1.4 ml of water, and one "Ammonia" tablet were added to a cuvette. A f t er 10 minutes at room temperature, the absorbance was measured at 340 nm and "Ammonia
BLOOD AMMONIA D E T E R M I N A T I O N S
193
200
.6 t
.5'
E =
,1-
.4 ¸ ',s oo
