A new and specific assay for ammonia and glutamine sensitive to 100 pmol

ANALYTICAL

BIOCHEMISTRY

90,

47-57 (1978)

A New and Specific Assay for Ammonia Sensitive to 100 pm01

and Glutamine

V.F. KALB JR.,’ T.J. DONOHUE, M.G. CORRIGAN,AND R.W. BERNLOHR The Department

of Biochemistry University

& Biophysics, The Pennsylvania Park, Pennsylvania 16802

Sfafe

Universiry,

Received September 6, 1977 A new enzymatic-radiochemical technique of NH,+ determination has been developed that is sensitive and specific. The reaction of cy-[1-W]ketoglutarate with NH4+ yields [ l-‘4C]glutamate as a direct measure of the NH,+ over a range of 0.1 to 10.0 nmol. By the measurement of the NH,+ present in a sample before and after glutamine hydrolysis the assay also allows the determination of glutamine.

The existing methods for NH4+ determination fall into two general classes, chemical and enzymatic. Chemical methods (I-7) measure most amines in addition to NH4+ and, if used for NH4+ determination in physiological fluids require microdiffusion to separate NH,+ from other compounds. During microdiffusion glutamine is hydrolyzed to glutamate and NH,+ so specificity for NH,+ is lost. Enzymatic methods (8- 11) are specific for NH,+ but either lack sensitivity or use enzymes that are not commercially available. We have developed a new enzymatic-radiochemical assay for NH,+ that is sensitive, specific, and uses commercially available enzymes. Our procedure is based on the following reactions. a-[ I-‘“ClKetoglutarate

+ NAD(P)H

+ NH4+

GDH _

[I-‘“Clglutamate

+ NAD(P)+.

[I]

The cr-[l-14C]ketoglutarate and the NADPH are in excess so that NH,+ is the limiting reactant. Remaining [1-14C]-cu-ketoglutarate is then specifically decarboxylated by treatment with H,O,. a-[ l-‘4C]Ketoglutarate

+ H,O, + 14C0, + succinate.

121 The volatile 14C0, formed is removed from the solution by heating and the [l-‘4C]glutamate formed in Eq. [l] is a direct measure of the amount of ’ Present address: Department of Biological Chemistry, College of Medicine, University of Cincinnati, Cincinnati. Ohio 45267. 47

0003-2697/78/0901-0047$02.00/O Copyright All nghfs

‘cl 1978 by Academic Press. Inc. of reproduction m any form reserved.

48

KALB

ET AL.

NH,+ present in the original sample. Since L-glutamate dehydrogenase (GDH)” catalyzes a reversible reaction, the presence of NADP+ and an excess of nonradioactive glutamate can lead to erroneously high values for NH,+. To prevent this, any NADP formed is removed by coupling the glucose-6-phosphate dehydrogenase reaction to the assay. Glucose-6-phosphate

+ NADP+ G1c-6-PDH,

6-phosphogluconate

This assay can be modified for use with product or a reactant. For example, we glutamine with the determination of NH,+ both NH,+ and glutamine pools in plasma,

+ NADPH.

[3]

any system in which NH,+ is a have coupled the hydrolysis of and have been able to determine urine, and bacterial cell extracts.

MATERIALS

L-Glutamic dehydrogenase (EC 1.4.1.3, type II) from bovine liver, MES buffer, ADP, a-ketoglutarate, glucose 6-phosphate, NADP, NADH, yeast glucosed-phosphate dehydrogenase (EC 1.1.1.49, type XV), beef liver catalase (EC 1.11.1.6, > 10,000 units/ml), and Escherichia coli glutaminase (EC 3.5.1.2, Grade V) were purchased from the Sigma Chemical Company. The catalase was dialyzed overnight against 1000 vol of 10 mM phosphate buffer (pH 7.4) containing 10 mM NaCl and the glutaminase was dialyzed overnight against 2000 vol of 10 mM phosphate buffer (pH 7.4) to reduce NH4+ contamination. The a-[1-14C]ketoglutarate (approximately 53.7 mCi/mmol) was a product of the New England Nuclear Company. Pentachloroacetone was purchased from the Aldrich Chemical Company (Metuchen, N. J.). Double-distilled water was used throughout and all solutions were neutralized to approximately pH 7 before use. Other chemicals were of reagent grade. METHODS

This NH,+ assay gives reproducible results in both simple and complex systems. A simple system is one in which all the components in the sample to be analyzed are known and inhibition or exchange reactions are not expected. A complex system is one containing unknown amounts of different compounds which could interfere with the assay. An example of this type of system is the determination of NH4+ in a cell extract. Ammonia

Assay -Simple

When assaying for NH4+ in a simple system the reaction mixture contains 300 mM MES buffer (pH 7.0), 2.5 mM ADP [a stabilizer of GDH 2 Abbreviations used: GDH, L-glutamate dehydrogenase; phate dehydrogenase; MES, 2[N-Morpholinolethane sulfonic

Glc-6-PDH, acid.

glucose-6-phos-

AMMONIA

AND

GLUTAMINE

ASSAY

49

(12)], 0.3 mM a-ketoglutarate, 0.2 mg/ml bovine glutamate dehydrogenase (8 to 9 units) and 2 mM NADH in double-distilled water. The NADH is added to this solution from a stock concentrate dissolved in 100 mM carbonate buffer (pH 10.6). This concentrated NADH solution is heated at 100°C for 5 min. before use to destroy any NAD present. The reaction mixture is incubated at room temperature for 1 h to allow for the utilization of endogenous NH,+ present in the stock solutions. This incubation decreases the blank values obtained in the absence of added NH4+ by a factor of 2. Concurrent with this l-h incubation of the reaction mixture, the samples to be analyzed are dispensed into scintillation minivials (15 x 45 mm, Yorktown Research, S. Hackensack. N. J.). An NH,+ standard stock solution is prepared and known volumes are delivered to each vial to provide standards ranging from 0.1 to 10.0 nmol of NH,+. Final sample volumes are adjusted to 0.1 ml with double-distilled water. After the l-h incubation of the reaction mixture, approximately 20 nmol (1.125 &i) of a-[ l-14C]ketoglutarate is added per ml of reaction mixture, the solution is mixed, and 50 ~1 is then delivered to each scintillation vial. Total radioactivity in 50 ~1 of reaction mixture is determined separately. The vials are incubated at room temperature (21°C) for 2 h to allow the enzymatic reaction to go to completion, the reaction is stopped by adding 100 ~1 of acetone to each, and the samples are heated for 10 min at 100°C to inactivate the catalase present in commercial GDH preparations. To release radioactivity from the excess cY-[1-‘“Clketoglutarate, 50 ~1 of fresh 4 M H,O, is added and the vials are heated for 10 min at 100°C. To decrease the solubility of any remaining 14C0, and decrease any chemiluminescence from residual H,Oz, 100 ~1 of 1 N HCl is added to each vial and the vials are heated for an additional 20 min at 100°C. The vials are allowed to cool and 3 ml of a modified Tritisol (13) scintillation fluid (ethylene glycol monoethyl ether is used instead of ethylene glycol) is added to each. The radioactivity in the blank (no added NH,+), standard and sample vials is determined in a liquid scintillation spectrometer. After subtracting the blank values from the others, nanomoles of NH4+ can be calculated on the basis of the original specific activity of the cY-ketoglutarate or from the standard curve. Ammonia

Assay -Complex

When assaying for NH, + in cell extracts or other complex mixtures, sample solutions will contain varying amounts of compounds such as NAD(P), glutamate, or a-ketoglutarate which have been found to interfere with the accuracy of the observed results. Any cw-ketoglutarate present in a sample dilutes the radioactive a-ketoglutarate and leads to erroneously low values for NH,+. In addition, since the reaction catalyzed by GDH

50

KALB

ET AL.

(Eq. [l]) is reversible, NAD(P) in the presence of an excess of nonradioactive glutamate is reduced with the simultaneous formation of an equivalent amount of NH,+ and nonradioactive a-ketoglutarate. If not controlled this exchange reaction catalyzed by GDH results in erroneously high values for NH,+ in samples. However, the assay has been modified to circumvent these problems. In order to prevent interference, a-ketoglutarate is removed from samples by treatment with H202. NAD(P) is removed by treating the samples with pentachloroacetone, a halogenated ketone which has been shown to irreversibly react at the 4 position on the nicotinamide ring of NAD(P) (14). To prevent any H,Oz from decarboxylating cu-[lJ4C]ketoglutarate during the course of the enzymatic reaction, a large excess of catalase is included in the reaction mixture. NAD(P) formed during the course of analysis is removed by the addition of an excess of glucose 6-phosphate and glucose-6-phosphate dehydrogenase (Eq. [3]). In the complex system the reaction mixture contains 300 mM MES buffer (pH 7.0), 2.5 mM ADP, 0.3 mM cY-ketoglutarate, 1 mM NADP, 0.5 mM dithiothreitol, 10 mM Mg (acetate),, 30 mM glucose 6-phosphate, 0.2 mg/ml GDH, 1.25 mg/ml catalase, and 0.1 mg/ml glucose-6-phosphate dehydrogenase (20 units) in double-distilled water. This mixture is incubated at room temperature for 1 h to allow for the utilization of endogenous NH,+ present in the stock solutions and to reduce the NADP to NADPH. Then approximately 20 nmol(l.125 &i) of a-[ lJ4C]ketoglutarate is added per ml of reaction mixture. Samples and standards are prepared in a manner identical to that outlined for the simple system, except that 10 ~1 of 4.4 mM pentachloroacetone is added to each vial approximately 30 min before the addition of the above reaction mixture. In addition, 10 min before the addition of the above reaction mixture 5 ~1 of fresh 220 mM H,O, is added to each vial. After the addition of the 50 ~1 of reaction mixture to each vial the treatment of all samples is identical to that described for the simple system. Glutamine

Assay

Glutamine was determined by measuring the additional NH,+ found in samples after releasing NH,+ from glutamine using either the complex or simple NH,+ assay. Samples containing glutamine were divided and one-half assayed for NH,+. To the other half, 1.O M phosphate buffer (pH 6.5) was added to a final concentration of 0.1 M and heated at 100°C for 90 min. Hamilton (15) has shown that this treatment quantitatively removes the e-amino group from glutamine. As a check on this procedure, glutamine was hydrolyzed to glutamate and NH4+ using glutaminase (16). Glutamine solutions were standardized by the procedure of Lund (16).

AMMONIA

Gro++xth of Bncteriu

AND

and Preparation

GLUTAMINE

ASSAY

51

of Cell Extracts

Bacillus licheniformis was grown in the A salts medium (17) supplemented with 15 mM glucose and the indicated nitrogen sources at 10 mM. Salmonella typhimurium and E. coli were grown in the minimal medium of Makman and Sutherland (18) using glucose as the carbon source and (NH&SO, as the nitrogen source. Cell extracts were prepared from exponentially growing cultures using the filtration and perchloric acid extraction procedure of Siegel et al. (17), except that the cells were washed with 50 ml of prewarmed medium before acid extraction. Samples of cells (approximately 100 ml) were removed for filtration when the culture had reached a density of approximately 150 pg of cell protein/ml of culture medium. Calculations of pool sizes were performed as described previously (19). Preparation

of Plasmu

and Urine Samples

For the determination of the ammonia and glutamine content of plasma, approximately 10 ml of freshly drawn blood was mixed with 0.1 ml of a 10% EDTA solution (9). This suspension was centrifuged for 10 min at 12,000g (2-4°C) and the plasma carefully removed with a Pasteur pipet. Both plasma and urine samples were deproteinized before analysis for ammonia and glutamine. Perchloric acid was added to the samples to a final concentration of 0.3 N (2-4°C). After cooling for 10 min the suspensions were centrifuged at 12,000g for 10 min to remove precipitated proteins (2-4°C). The pH of the supernatant fraction was adjusted to approximately 6 with a known volume of 2 M KHC03 and this solution was then centrifuged for 10 min at 12,OOOgto remove insoluble KClO,. The supernatant fraction of this centrifugation was stored at -20°C until analysis for ammonia and glutamine. Deproteinization is necessary when using the Hamilton method (15) of glutamine hydrolysis described above because release of ammonia from proteins present in the solution during the hydrolysis step leads to erroneously elevated glutamine values. RESULTS Characteristics

of the NH,+

Assuy

Typical standard curves (Fig. 1) using the simple (a) and the complex (0) methods exhibit linearity between [ l-14C]glutamate formation and NH,+ concentrations in the range of 100 pmol to 10 nmol of NH,+. Ammonia contamination from the reagents and glassware used and radioactivity not released from the (Y-[lJ4C]ketoglutarate with H,O, result in blank values of approximately 2300 cpm. This represents 3.8% of the total radioactivity added (approximately 60,000 cpm) to each vial. a-Keto acids are known

52

KALB

ET AL.

nmoles NH. FIG. 1. Standard curves for the ammonia assays. All conditions were as described under Methods for both the simple (A) and complex (0) assay systems. Lines shown were constructed after subtraction of blank values for the amount of radioactivity present in a vial containing no added NH,+ (approximately 2300 cpm). The range of variability of results of replicate standard curves is depicted by the size of the points on the lines.

to undergo dimerization in solution so that this background increases slightly when using older lots of radioactive cY-ketoglutarate. Because NH,+ contamination from these different sources can vary slightly on a day to day basis it is imperative that standard NH,+ samples be included with each set of assays. The range of variability of the results of replicate standard curves is shown by the size of the points in Fig. 1 and does not exceed ~5.1% in the range of 0.5 to 10 nmol of NH,+. One-hundred picomoles of NH,+ can be measured but contamination of the reagents and glassware used (approximately 50 pmol) increases the variability to ?7.7%. Time-course studies using either of the assay systems and standard ammonia solutions have shown that the reaction is complete after approximately 1 h (data not shown). Bovine liver GDH is subject to inhibition by a variety of compounds (12). Therefore when assaying NH,+ in samples which might contain varying amounts of inhibitory compounds (i.e., a cell extract) it is necessary to allow the reaction sufficient time to go to completion. It is for this reason that the incubation time has been set at 2 h under standard assay conditions. The pH of the reaction, 7.0, was chosen for kinetic considerations (20).

AMMONIA

Determination

of NH,+

AND GLUTAMINE

and Glutamine

53

ASSAY

in Various Samples

Results of NH,+ and glutamine determinations on cell extracts of B. licheniformis are shown in Table 1. The values presented for NH,+ are the averages of 12 determinations from six separate sets of extracts and have associated with them a variability of ? 15% in the worst cases. An internal NH,+ standard added to cell extracts (2.0 nmol) is recovered with an efficiency of 94%. The values presented for glutamine are the averages of 8 determinations from four separate sets of extracts and have been found to be accurate to ?20%. An internal glutamine standard (2.0 nmol) is recovered with an efficiency of 98%. The method used for glutamine hydrolysis was that of Hamilton (1.5). yielding at least 92% hydrolysis of a standard in all cases. We obtained similar data when glutaminase was used for NH,+ release (16); thus the hydrolysis procedure described in the methods section is preferred because of its simplicity and economy. Table 2 presents a compilation of ammonia and glutamine levels in plasma, urine, and extracts of S. typhimurium and E. co/i. It can be seen that the values obtained using the new radiochemical technique are similar to those reported previously. Thus it appears that the new method is applicable for use with a wide variety of specimens of biological interest. In addition the sensitivity of the assay allows for the specific analysis of ammonia and glutamine using a significantly smaller sample size than other techniques while maintaining a comparable level of precision. Since glutamine is determined after release of ammonia from the e-amino group the precision of the assay for the determination of glutamine decreases when the ratio of ammonia to glutamine approaches or exceeds 10 to 1. In these instances this problem can be circumvented by the removal of amTABLE AMMONIA

AND GROWN

GLUTAMINE ON VARIOUS

1

POOLS IN EXTRACTS OF CELLS NITROGEN SOURCES

Pool (rnM) Nitrogen source

(NH&SO, L-Glutamine L-Glutamate KNO, L-Alanine L-Omithine L-Arginine

Concentration (mhf)

5 10 10 10 10 10 10

Ammonia 3.49 2.58 0.54 0.64 0.55 2.46 2.53

2 + r k 2 k 2

0.11 0.27 0.02 0.05 0.05 0.26 0.27

Glutamine 1.55 3.70 5.81 5.75 5.10 7.41 6.90

2 0.13 + 0.58 r 0.43 _f 1.10 t 0.51 2 0.75 k 1.10

KALB ET AL.

54

TABLE

2

COMPARISONOFAMMONIAANDGLUTAMINELEVELSDETERMINEDBYTHE RADIOCHEMICAL ASSAY WITH PREVIOUSLY PUBLISHED

Previously published values Source

Ammonia

Human plasma

25.9 /AM 6.5-35 jbM 10-47 /LM 19-31 /.LM 40-72 /LM

Human urine

30-32

mM

Reference (28) (9) (10)

Glutamine 510-580 /.LM 429 ” 39/.~M 430 PM

VALUES

Values obtained using the new assay Reference (21) (24)

Ammonia

Glutamine

45 “_ l@LM

425 k 45 jLM

22 ” 2 rnM

0.2 2 0.1 rnM

(22)

(2% (21) (30)

0.21 rnM

Salmonella typhimurium

Not reported

2.5

Escherichia coli

Not reported

3.0 rnM

mM

(22)

mM 3.0 2 0.3 mM

(27)

4.7 rt 0.3

(31)

7.0 2 0.5 mM 3.2 2 0.3 mM

monia from the sample to be analyzed for glutamine prior to glutamine hydrolysis (21). A comparison of the quantities of NH,+ and glutamine in extracts leads to a very important caveat. Many methods used for NH,+ determination either measure NH,+ plus glutamine or release NH,+ from glutamine during analysis. Methods that include microdiffusion of NH,+ from strong basic solutions hydrolyze glutamine and the resulting data are inflated. Since the levels of glutamine in plasma and bacterial extracts are equal to or higher than ammonia levels, care must be exercised to prevent glutamine hydrolysis in ammonia assays. DISCUSSION

As can be seen from Fig. 1, the standard curves obtained using the conditions described for both the simple and complex systems are very similar. By incorporating the few minor modifications described under Methods for use of the assay with a complex system, the error introduced into the NH,+ determination by the exchange reaction of GDH in the presence of an excess of nonradioactive glutamate has been minimized. Without these modifications the presence of as little as 100 nmol of nonradioactive glutamate in the assay vial will result in the incorporation of all the radioactive a-ketoglutarate (approximately 60,000 cpm) into radioactive glutamate in the absence of added NH,+ (data not shown). By using the conditions described under Methods for the complex system the ad-

AMMONIA

AND

GLUTAMINE

ASSAY

55

dition of glutamate has no measurable effect on the slope of the standard curve (0 in Fig. 1) until the ratio of glutamate to NH,+ in the assay exceeds 1250 to 1. Based on previously published intracellular glutamate pools in B. ficheniformis (19) and the NH,+ pools presented in this paper (Table I), the ratio of glutamate to NH,+ in the cell extracts is between 50 and 300 to 1. Considering the fact that B. licheniformis has been found to have significantly higher glutamate pools than other microorganisms (19) this exchange phenomenon should not present a problem in the analysis of NH,+ in other bacterial cell extracts. However, in those instances where the excess of glutamate in a sample is significantly higher than that found in extracts of B. licheniformis the contribution of the exchange reaction of GDH to the amount of radioactive glutamate formed might have to be determined separately. Glutamine samples were hydrolyzed by Hamilton’s method (15) and the results are shown in Table 1. We find that Hamihon’s method has the advantages of simplicity and economy over the use of glutaminase (16). This method yields 98% recovery of internal glutamine standards added to cell extracts. Less than 0.1% of an asparagine standard or an asparagine standard added to a cell extract was hydrolyzed using this technique. Results similar to this for asparagine and other free a-amino acids had been originally reported by Hamilton (15). Overall, the enzymatic-radiochemical method described is a simple, highly sensitive, specific, and reproducible method of NH,+ and glutamine determination. A principle advantage of this NH,+ assay is the precision in the range of 100 pmol to 10 nmol of NH,+. Combined with this level of sensitivity is a specificity for NH,+ in simple or complex systems. Many other methods of NH,+ determination have been reported in the literature. Chemical methods exhibit good sensitivity but lack specificity for NH,+ ( l-7). The modified phenol-hypochlorite technique (1) has been the most sensitive chemical technique developed (measuring 2.5 nmol/ml). The NH,+ electrode (2,30) reports a sensitivity of 1 nmol/ml, but requires at least 3 ml of solution and is subject to interference by K+ and volatile amines. Nessler’s method (3) is subject to interference by amines, inorganic ions, and organic solvents. The ninhydrin method (4,5) is subject to interference by amines, as is the phthalaldehyde-ammonia fluorometric method (6). The procedure described here for NH,+ determination in complex systems is not subject to interference by these types of compounds. Other existing methods of NH,+ determination are based on the use of NH,+ specific enzymes (8- 11). Glutamine synthetase (EC 6.3.1.2) has been used by Hersch et ul. (8), but, there is no commercial source of glutamine synthetase. GDH has been used to quantitate NH,+ by either spectrophotometric (9.10) or fluorescent (11,28) techniques based on the oxidation of NADH. While these methods are specific for NH,+, sen-

56

KALB

ET AL

sitivities are not good, ranging from 20 to 47 nmol of NH,+/ml of sample. Our new NH,+ assay combines the sensitivity of an isotopic method with the specificity of an enzymatic method to measure NH,+ in the lOO-pm01 range. An additional preparative step enables one to measure glutamine at this same level of precision. Others (16,21) have used this principle to measure glutamine but have been limited by the shortcomings of the existing NH,+ assays discussed above. Hamilton’s technique of hydrolyzing glutamine to NH,+ and pyrrolidonecarboxylic acid is excellent, but the method of measuring the pyrrolidonecarboyxlic acid is sensitive to only 3.4 pmol(15). Ion-exchange chromatography and automated amino acid analysis have been employed to obtain sensitivity to 10 to 30 nmol (22,31), but this method is expensive and lengthy if only glutamine is of interest. Glutamine-specific enzymes such as glutamine synthetase (23) and glutamate synthase (EC 2.6.1.53) have been used to measure glutamine (24). A disadvantage of these methods is that there is no commercial source of these enzymes. In addition, strongly colored ions, phosphate concentrations greater than 100 mM, and substances that complex with iron interfere with the glutamine synthetase system. Glutamine aminotransferase (EC 2.6.1.15) has been used in glutamine determinations but it is not specific for glutamine as phenylalanine, tyrosine, and methionine can replace glutamine as the amino donor (25). Furthermore, due to the high K, of the aminotransferase the reaction does not go to completion. The new enzymatic-radiochemical NH,+ assay uses only commercially available enzymes and is more sensitive than these enzyme-based glutamine assays. More recent methods of glutamine determination have achieved sensitivity in the 4-400 pmol range using a glutamine binding protein (26) or by monitoring radioactive citrulline (27) formation. Again, the glutamine binding protein and the enzymes necessary for citrulline formation were purified from bacterial sources. In conclusion, we feel that the new enzymatic-radiochemical NH,+ assay offers several advantages. The procedure is simple, specific, and sensitive, and all of the materials needed are commercially available. The method can be extended for the convenient determination of NH,+-releasing compounds such as glutamine. ACKNOWLEDGMENT This study Foundation.

was supported

1. Kaplan. 2. Huang,

A. (1969) Methods Y.-Z. (1974) Anal.

in part

by Grant

PCM74-22671

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AND GLUTAMINE

ASSAY

57

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