Acetaldehyde trapping system

The invention provides a reagent system for the enzymatic determination of an oxidizable substrate in a fluid sample; the system includes an active amine trap for inactivating high concentrations of aldehyde and ketone oxidation products comprising a combination of at least one primary amine and at least one alpha-effect amine. An example of the oxidizable substrate is alcohol.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
Diagnostic procedures based on the enzymatic determination of substances in 
biological fluids are well-known. In the enzymatic determination of 
alcohol or other dehydrogenase/oxidase substrates in body fluids, enzyme 
assay systems comprising a dehydrogenase and a cofactor for the enzyme 
(usually NAD+) are typically employed to oxidize the substrate and 
simultaneously reduce the cofactor to a readily assayable product. 
In the case of an alcohol, selective oxidation by such enzyme systems 
results in the production of the corresponding free aldehyde or ketone. 
The presence of these oxidation products are very detrimental to the 
assay, as firstly, they tend to inactivate the enzymes employed in the 
system, and, secondly, they unfavorably shift the dehydrogenation reaction 
equilibrium so that oxidation of the substrate does not tend to go to 
completion. Since assay results under these conditions are unreliable, 
enzyme assay systems for the determination of alcohol now typically 
require dilution of the substrate and/or include complexing or trapping 
agents for the oxidation products which in essence remove free aldehyde or 
ketone from the reaction. 
2. Description of Related Art 
A variety of agents for trapping alcohol oxidation products, especially 
acetaldehyde produced by enzymatic dehydrogenation of ethanol, have been 
proposed. In commercial applications, the class of agents generally 
employed comprises primary amines, which react with free aldehyde to form 
the corresponding imine. Since the product imine is less reactive than the 
precursor aldehyde or ketone, enzyme deactivation is reduced, and the 
dehydrogenation reaction equilibrium is favorably shifted. Even with the 
primary amine trapping agents, however, the alcohol dehydrogenation 
typically still does not go substantially to completion, with often less 
than about 20% of the alcohol converted. Further, assay systems employing 
a primary amine as trapping agent for alcohol oxidation products are 
generally only useful for assaying fluids having a low alcohol content and 
a correspondingly low production of aldehyde or ketone oxidation product. 
In general, the usefulness of these enzyme assay systems is limited to 
samples producing less than about 5 mM acetaldehyde, more typically less 
than about 1 mM acetaldehyde, as the known primary amine trapping agents 
cannot effectively remove larger amounts from the assay system. For 
instance, acetaldehyde in concentration greater than 1 mM inhibits the 
enzyme alcohol dehydrogenase (ADH). Since analysis of body fluid samples 
potentially containing in excess of about 150 mM alcohol is routine, such 
samples must be diluted prior to assay to reduce target concentrations to 
an assayable level. For example, in situations where 21 mM blood ethanol 
is the legal definition of intoxication, and the sample is to be tested 
for this concentration, the sample must be diluted sufficiently to reduce 
the alcohol concentration thereof to within the assay range; generally to 
less than about 5 mM, depending upon the particular assay system. This 
must be done very carefully to ensure an accurate determination. 
An example of known systems is the Abbott TDX Analyzer. This device 
requires extensive dilution of the sample to be tested so that only 1 mM 
of acetaldehyde is produced since otherwise the undesirable results 
discussed above will take place. The Abbott Analyzer uses an acetaldehyde 
trap, 2-amino-2-methyl-1,3-propanediol ("Tris amino" or "Tris") as a 
buffer. 
Another illustration of this type of assay is illustrated by U.S. Pat. No. 
3,926,736 to Buccolo (Calbiochem), 1975. The patent discloses the use of 
Tris buffer as a trapping agent. The samples to be analyzed are diluted so 
that only about 0.5 mM acetaldehyde is produced. 
U.S. Pat. No. 4,481,292 to Raymond (The Coca-Cola Company), 1984, also 
shows the use of Tris buffer as trapping agent for high concentrations of 
acetaldehyde. The reactor is adapted for and the process is a continuous 
flow type. 
The patent describes the difficulties encountered in a practical 
enzyme-catalyzed process for converting ethanol to acetaldehyde, including 
equilibrium considerations which favor acetaldehyde conversion to alcohol. 
Although concentrations of acetaldehyde on the order of 85 mM are 
disclosed, it is essential in accordance with the process (and apparatus) 
that the alcohol dehydrogenase (ADH) be separated and confined during the 
reaction sequence. The ADH is separated by a semi-permeable membrane from 
the acetaldehyde and the system permits the passage of the starting 
materials as well as the products formed so that a liquid flow away from 
the enzyme takes place on a continuous basis. Thus, the enzyme is never 
exposed to high concentrations of acetaldehyde. Even under such 
conditions, the yield of acetaldehyde is quite limited, the conversion of 
the alcohol being about 18%. The efficiency of the amine buffer system is 
not a feature of the process, and "Tris" is not capable by itself of 
reacting with and removing such high concentrations of acetaldehyde. 
The process and the system of the present invention does not call for a 
flow system, twice the amount of acetaldehyde can be trapped and 100% of 
the alcohol can be converted to acetaldehyde. 
The system of the invention can be embodied and used as a dry film rather 
than a large continuous flow reactor. Other differences between the device 
of the invention and the prior art will become apparent in the description 
of the invention as presented herein. 
Owing to the inability of these known systems to assay for routinely 
encountered concentrations of alcohols without sample dilution, the assay 
is of necessity a liquid assay with the reagents supplied in liquid form, 
usually as a kit. Sigma Chemical, for example, markets the following 
individual liquid reagents for the enzymatic determination of ethyl 
alcohol in blood samples: NAD-ADH; ethanol standard solution; hydrazine; 
glycine buffer reagent; trichloroacetic acid solution. These liquid 
reagents are bulky, and must be carefully combined in the proper amounts 
for each assay, refrigerated during storage, and protected against 
contamination and spillage. 
Bostick and Overton (Biotechnol. Bioengin. 22:2383-92, 1980) also describe 
the addition of hydrazine to an enzymatic alcohol test, which increases 
the effectiveness of the measuring range to 3 mM, and the test format is 
liquid. 
In Biochem. J., 104 p. 165 (1967), Dickinson and Dalziel, The Specificities 
and Configurations of Ternary Complexes of Yeast and Liver Alcohol 
Dehydrogenases, discuss the use of Tris as an acetaldehyde trapping agent. 
The authors note that Tris is an unsuitable buffer for equilibrium or 
initial-rate measurements with some carbonyl compounds. The authors state, 
however, that Tris does not afford a convenient buffer for the enzymatic 
estimation of small amounts of ethanol. Additional differences over that 
art will become apparent from the detailed discussions which follow. 
It is accordingly desirable and there is a need to provide an assay in a 
fluid for the enzymatic determination of alcohol concentration, which is 
capable of reliably converting substantially 100% of the substrate, 
trapping high concentrations of ketone or aldehyde (in excess of 5 mM, 
e.g. in excess of 150 mM), does not deactivate enzyme reagents, is useful 
for determining high alcohol concentrations without sample dilution (e.g. 
in the range of 20 to 150 mM, or higher), which can be provided in easily 
useable and storable form such as a dry film format, and which is highly 
reliable in use, even for inexperienced assayers. 
SUMMARY OF THE INVENTION 
The invention provides an assay for the enzymatic determination of the 
alcohol content of a fluid, particularly a body fluid of a mammal, based 
on enzyme-catalyzed oxidation of the alcohol present to a corresponding 
aldehyde or ketone (generically "carbonyl compound"), with simultaneous 
reduction of NAD+ to NADH. The assay includes a reagent system preferably 
comprising NAD+, alcohol dehydrogenase (ADH), buffering agent, and a 
non-volatile trapping agent for the alcohol dehydrogenation product. In a 
particular embodiment, the invention provides a diagnostic kit comprising 
a film-forming reagent system including a marker for NADH, applied to 
support means for physically supporting the film. An ideal embodiment is a 
dry film. 
It is also envisioned that this trapping system can be used with other 
enzyme systems which oxidize alcohol to form inactivating carbonyl 
compounds, for example oxidase enzymes, and for a specific example, 
alcohol oxidase, which will convert methanol and ethanol to the 
inactivating respective carbonyl compounds, formaldehyde and acetaldehyde. 
The trapping agent is selected for non-volatility, good solubility, and 
effective, preferably substantially complete, inactivation of the 
dehydrogenation product, at sample alcohol concentrations up to about 200 
mM. With appropriate selection of trapping agent, the assay provides 
reliable end-point determinations (alcohol conversion of about 100%), even 
at high alcohol concentrations, and full enzyme activity over the course 
of the reaction. 
In the below-described invention, the term cofactor describes any second 
substrate to the enzyme oxidase on dehydrogenase which is capable of 
accepting electrons derived upon oxidation of the first substrate, such 
oxidation of the first substrate resulting in production of a ketone or 
aldehyde. 
DETAILED DESCRIPTION OF THE INVENTION 
The assay method of the invention is based on known assays wherein the 
alcohol content of a fluid sample is enzymatically determined by 
enzyme-catalyzed oxidation of the alcohol employing a reagent system 
including a dehydrogenase or oxidase and a cofactor for the enzyme which 
is simultaneously reduced as the alcohol is oxidized; the amount of 
reduced cofactor present in the sample after completion of the reaction is 
then determined, typically by colorimetric analysis; the amount of alcohol 
originally present in the sample may be determined against a standard for 
the analysis, or a simple end-point determination employed. 
The basic principles of this reaction are well-known, as discussed above, 
and the general process is widely used for the determination of ethanol 
content of blood or saliva. Typically, the ethanol content of the sample 
is dehydrogenated in the presence of ADH, which is a highly specific 
enzyme for this reaction. The reducible cofactor employed is usually NAD+, 
which is simultaneously reduced as the ethanol is oxidized on a mole 
equivalent basis. The amount of ADH employed is an amount sufficient to 
obtain the desired sample enzyme activity, and sufficient NAD+ is employed 
to ensure that the reaction goes to completion. The known systems are 
conventionally buffered to a pH which promotes maximum enzyme activity. 
In accordance with the present invention, the reagent system for the 
enzymatic determination of alcohol further includes a trapping system for 
trapping and removing aldehyde or ketone oxidation products from the 
reaction mixture comprising a non-volatile active amine trap including at 
least one primary amine and at least one alpha-effect amine, wherein each 
of the amines is a) non-volatile and b) characterized by a pKa of at least 
about 7.5. Though the independent use of alpha-effect amines as aldehyde 
traps in kinetic reactions is known in the art, the end product (e.g. the 
aldehyde) is present in low concentrations generally below 5 mM, commonly 
below 1 mM. The results obtained by the us of the amines in combination in 
accordance with the invention as described herein is quite unexpected. 
For reasons which are not well understood at the present time, the use of a 
combination of alpha-effect and primary amines according to the present 
invention dramatically increases the aldehyde/ketone trapping capacity of 
the amine trap, as compared to prior art amine traps consisting 
essentially of primary amine or of alpha-effect amine. Increases in 
trapping capacity, as measured by effective inactivation of 
aldehyde/ketone oxidation product in the reaction mixture, of up to about 
100-fold over known prior art traps are typical. Since alpha-effect amines 
do not function well alone as traps in standard ethanol ADH/NAD+ reaction 
systems, and primary amines alone have a limited trapping capacity in such 
systems, it is postulated that the primary amine according to the present 
invention catalyzes or otherwise activates the alpha-effect amine trapping 
function, and that the alpha-effect amine is the ultimate trap. 
Primary and alpha-effect amines suitable for use in the practice of the 
present invention are selected for their non-volatility, pKa, solubility, 
and ability to function in combination as an active amine trap in 
enzymatic determinations of alcohol according to the invention at the 
biological pH range of the enzyme, without denaturation or substantial 
deactivation of the enzyme. An "active amine trap" according to the 
present invention is a trap capable of substantially inactivating at least 
about 5 mM acetaldehyde from a standard enzyme-catalyzed oxidation of 
ethanol employing an ADH/NAD+ reagent system; preferably the trap is 
capable of substantially inactivating at least about 20 mM acetaldehyde 
from this system, and more preferably, at least about 50 mM acetaldehyde. 
Removal of acetaldehyde up to about 150 mM concentrations and greater, are 
contemplated. It is noted that, while the term "active amine trap" is 
defined herein with reference to an ADH/NAD+ reagent system with ethanol 
as the substrate, the broad use of the active amine trap of the invention 
in a range of enzymatic determinations of alcohols or other enzymatically 
oxidizable substrates producing aldehyde or ketone oxidation products, 
such as acids, is contemplated. 
With proper selection of amines, typical substrate conversions of about 
100% are obtainable without dilution of the sample alcohol concentrations 
up to about 150 mM, thus permitting accurate and reliable end-point 
determinations. Such conversions are obtained by selection of amines for 
trap efficiency so that accumulation of active oxidation product is 
avoided. This ensures that a favorable reaction equilibrium is 
established, and that the catalyzing enzyme is not deactivated by 
oxidation product; the use of amines in the practice of the invention 
which tend to deactivate the catalyzing enzyme should, of course, be 
avoided. 
The amines employed in the active amine trap of the invention are 
preferably substantially non-volatile amines having a boiling point above 
about 200.degree. C. (1 atm), a molecular weight greater than about 150, 
or both. However, in some instances, slightly more volatile amines may be 
employed, as long as the trapping combination of amines is substantially 
non-volatile (bp&gt;200.degree. C. at 1 atm). Preferably, the amines employed 
have a high solubility, preferably at least a solubility in water of at 
least about 100 mM at room temperature (25.degree. C.). 
Non-volatile amines having the requisite pKa useful in the practice of the 
present invention are conveniently and easily selected from such known 
prior art amines. Exemplary suitable primary amines for use in the present 
invention comprise amines of the following categories having the pKa, 
volatility, and solubility characteristics described above and carrying at 
least one primary amino substituent: 
1) unsubstituted or substituted naturally-occurring amino acids such as 
lysine, arginine, alanine, serine, proline, glycine, glycylglycine, and 
glycine methyl or ethyl ester; 
2) non-naturally occurring amino acids, such as amino C.sub.2 -C.sub.10 
-carboxylic acids, particularly aminobutyric acid or aminocaprylic acid; 
3) compounds containing both a primary amino substituent and a non-carboxy 
acid-functional moiety, such as amino-C.sub.1 -C.sub.10 -alkyl-substituted 
inorganic acids, particularly aminoethyl phosphoric acid or aminoethyl 
sulfonic acid; 
4) compounds containing a primary amino substituent and an additional 
functional group bearing a fixed charge, such as amino-tetraalkylammonium 
salts or amino-tetraalkylphosphonium salts; 
5) primary amines having a molecular weight of more than about 150 or a 
boiling point greater than about 200.degree. C. (at 1 atm), or both, and 
which are accordingly substantially non-volatile, such as C.sub.8 
-C.sub.20 -alkyl or -cycloalkyl-amines, or C.sub.6 -C.sub.20 
-alkylenediamines such as 1,6-diaminohexane; and 
6) primary amines containing hydrogen bonding sites which act to increase 
their boiling point and render them non-volatile, such as Tris hydroxyl 
methylamine methane. 
Alpha-effect amines and their use as aldehyde traps in kinetic studies is 
known in the art, as described, for example, in a report by W. P. Jencks, 
Catalysis in Chemistry and Enzymology, McGraw Hill, N.Y. (1969), 
incorporated herein by reference. Suitable exemplary alpha-effect amines 
for use in the practice of the present invention comprise alpha-effect 
amines of the following categories having the pKa, volatility, and 
solubility characteristics set forth above: 
1) substituted C.sub.2 -C.sub.8 -hydrazines such as carboxymethylhydrazine, 
nitrophenyl hydrazine, ethylhydrazine acetate, 2-hydrazine-2-imidazole, 
and 1,3-dihydroxyl-2-hydrazinopropane; and 
2) substituted C.sub.2 -C.sub.8 -alkylhydroxylamines, including salts 
thereof, such as carboxymethoxyamine salts, carboxy-C.sub.2 -C.sub.8 
-alkylhydroxyl-amines, benzylhydroxylamine, and substituted 
benzylhydroxyl-amines, especially nitrobenzylhydroxylamines. 
Generally, alpha-effect hydroxylamines suitable for use in the practice of 
the invention comprise hydroxylamines containing at least 2 carbon atoms 
(i.e., are substantially non-volatile), and have at least two hydroxyl 
substituents (i.e., have good solubility). 
Again, alpha-effect and primary amines useful in the practice of the 
present invention are readily identified by one of ordinary skill in the 
art, for example by reference to any standard text such as the Chemistry 
and Physics, CRC Press for pKa, solubility, and volatility 
characteristics. The ratio of primary amine to alpha-effect amine in the 
amine trap is not critical, as long as the activity of the trap meets the 
above-specified parameters. Generally, however, at least about 10% w/w of 
total primary amine to total alpha effect amine is employed, and 
preferably no more than about 60% w/w primary amine. A range of from about 
20% by weight to about 50% by weight total primary amine, based on the 
weight of total alpha-effect amine, is most preferred. 
In a further embodiment of the invention, it has been discovered 
unexpectedly that a suitable trap for the practice of the invention can be 
the diamine, 1,6-hexanediamine. This diamine can be used alone without the 
alpha-effect amine. For reasons which are not well understood at the 
present time, very satisfactory results are obtained by the use of this 
diamine alone as trapping agent according to the invention. However, this 
diamine may also be used with at least one other primary amine as 
described above. Generally, 1,6-hexanediamine is used as the active amine 
trap of the invention in an amount ranging from about 20% by weight up to 
100% by weight, based on the total weight of amines present. 
In the practice of the process of the invention, the active amine trap is 
added to a conventional reaction system comprising a dehydrogenase or 
oxidase, a reducible cofactor, and an alcohol substrate for the enzyme, in 
an amount which will substantially inactivate alcohol oxidation product 
and remove it as a reactant from the system. Generally a concentration of 
primary amine plus alpha-effect amine, or 1,6-hexanediamine plus 
additional primary amine if used, of at least about 4 times that of the 
alcohol substrate is effective to achieve maximum trap activity. Lower 
amine concentrations of at least about 1.5 times that of the substrate 
concentration may, however, be effective in particular applications. 
Excess amine does not appear to generally adversely affect the reaction. 
Preferably, the reaction mixture is buffered to between about pH 9 and 10, 
which maximizes the extent and rate of the reaction, without substantially 
inhibiting enzyme activity. 
The concentration of alcohol in the sample is conveniently determined by 
colorimetric quantitation of reduced cofactor, according to conventional 
processes. Typically, NADH is determined by ultraviolet colorimetry at 340 
nm, at which wavelength NADH absorbs ultraviolet light, but NAD does not. 
Alternatively, markers for NADH which allow the quantitative determination 
thereof are employed, typically chromogens reactive with NADH. 
In a preferred embodiment of the invention, the reagent system of the 
invention for the enzymatic determination of alcohol is disposed as a film 
on a solid support, and dried. Owing to the low volatility of the amine 
trap, the trapping system of the invention is retained as a film-forming 
component, without evaporation during the drying process. To use, a fluid 
sample is placed in contact with the dried reagent film, and the alcohol 
content of the sample determined by quantitating reduced cofactor present, 
preferably NADH, typically by colorimetric analysis. Conveniently, the 
reagent system includes a chromogen quantitatively reactive with the 
reducible cofactor, and the chromogen/cofactor product is either 
quantitated against a standard to determine alcohol content of the sample, 
or is selected to provide an end-point determination, as known in the art 
and as described, for example, in U.S. patent applications Ser. Nos. 
943,414 and 075,817, of common assignment herewith, and incorporated 
herein by reference. 
Suitable solid supports are also described at length in these applications, 
and include sheets, rods, webs, sticks, or strips of paper, glass, 
cellulose, wood, metal, or polymers such as polyalkylenes or 
polycarbonates. In a particularly preferred embodiment, a bibulous 
material such as filter or blotting paper capable of adsorbing a fixed 
amount of liquid per unit is incorporated into the reagent system in a 
predetermined amount to thereby provide a standardized sample for 
enzymatic determination according to the invention. 
The components of the assay system of the invention are preferably prepared 
in a liquid form for deposit upon the support member. Once placed on the 
support member, the components in solution are dried to adhere the 
compositions to the support member. Generally, adhesion of the reactant 
compositions to the support member is conveniently effected when the 
support member is a bibulous material. Certain binders such as resin or 
gums are advantageously incorporated into the reactant compositions to 
assist in adhering them to non-porous support members such as metal, glass 
or non-porous polymeric materials. Conventionally employed inert filters, 
binders, surfactants and the like may also be incorporated into the 
reagent compositions when desired. The assay device is preferably kept 
under conditions that do not cause the deactivation of the enzyme, e.g. 
avoiding high temperature.

The following Examples illustrate the invention and are not intended to be 
a limitation in any way whatever. 
EXAMPLE 1 
To a reaction mixture containing 5 IU ADH, 100 mM NAD, pH 9.5 and 65 mM 
ethanol was added a trapping mixture containing: 
400 mM Tris, pH 9.5 
200 mM Glycine, pH 9.5 
50 mM Carboxymethoxyamine, pH 10 
All of the alcohol was oxidized to produce 65 mM acetaldehyde and NADH. 
When 650 mM borate buffer was substituted for the amine containing buffers 
listed above, less than 4 mM of alcohol was oxidized, and the ADH enzyme 
was inactivated by the free aldehyde present in the reaction. Use of 650 
mM of Tris buffer in the absence of alpha-effect amine did not result in 
complete alcohol oxidation. Use of 650 mM carboxymethoxyamine and borate 
buffer, without added primary amine, again did not result in the complete 
oxidation of the alcohol. 
EXAMPLE 2 
A trapping mixture containing: 
120 mM Tris, pH 9.5 
120 mM Glycine, pH 9.8 
150 mM Carboxymethoxyamine, pH 10 
was substituted for the trap in Example 1. Identical results were obtained. 
EXAMPLE 3 
A trapping mixture containing: 
100 mM Hexanediamine, pH 10 
100 mM Glycine, pH 9.8 
150 mM Carboxymethoxyamine, pH 10 
was substituted for the trap in Example 1. Identical results were obtained. 
EXAMPLE 4 
A trapping mixture containing: 
120 mM Tris, pH 9.5 
120 mM Glycine, pH 9.8 
150 mM Allylhydroxylamine, pH 9.8 
was substituted for the trap in Example 1. Identical results were obtained. 
EXAMPLE 5 
A trapping mixture containing: 
120 mM Tris, pH 9.8 
120 mM Glycine, pH 9.8 
100 mM Nitrobenzylhydroxylamine, pH 10 
was substituted for the trap in Example 1. Identical results were obtained. 
EXAMPLE 6 
To a reaction mixture containing 5 IU/ml ADH, 200 mM NAD pH 9.5, and 150 mM 
ethanol was added a trapping formulation containing: 
240 mM Tris, pH 9.8 
240 mM Glycine, pH 10 
200 mM Carboxymethoxyamine, pH 10 
Complete oxidation of all of the alcohol was observed, indicating that this 
formulation was capable of trapping 150 mM of acetaldehyde. Once again, 
elimination of either the primary amine or the alpha-effect amine resulted 
in failure to observe complete alcohol oxidation. This failure was not due 
to lack of sufficient buffer, as the addition of a non-amine containing 
borate buffer did not result in complete oxidation. 
EXAMPLE 7 
To the reaction mixture described in Example 6 was added: 
120 mM Hexanediamine, pH 10 
200 mM Glycine, pH 10 
200 mM Carboxymethoxyamine, pH 10 
EXAMPLE 8 
To the reaction mixture described in Example 6 was added: 
100 mM Hexanediamine, pH 9.8 
200 mM Glycine, pH 10 
120 mM Nitrobenzylhydroxylamine, pH 10 
Identical results as described in Example 6 were observed. 
EXAMPLE 9 
The combined reaction and trapping mixtures of Example 1, further including 
1 mM MTT chromogen [2-(2.sup.1 -triazolyl)-3,5-diphenyl tetrazolium 
bromide], are applied to an absorbent paper in an amount sufficient to 
saturate the paper. The paper is then dried and cut into 0.5 inch diameter 
circles. To all but one of the circles, 20 ul of fluid sample containing 
unknown concentrations of ethanol are applied; the remaining circle serves 
as a control. A color change from the pale yellow shade of the control 
paper to a bright blue in the presence of the sample fluid indicates a 
sample alcohol concentration of at least 20 mM alcohol. 
The relative equilibrium constants for trapping agents are listed below in 
Table 1. 
Their relative equilibrium constants were determined by measuring the NADH 
production by alcohol dehydrogenase after 10 minutes in the presence of 
each of the trapping agents. 
The reaction formulation was as follows: 
200 mM Glycine, pH 9.5, primary amine 
10 mM NAD 
5 mM ethanol 
5 mM trapping agent 
125 IU ADH 
TABLE 1 
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RELATIVE 
TRAP EQUILIBRIUM CONSTANT 
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Control (no trapping agent) 
.05 
Methoxyamine 11.7 
Ethylhydroxylamine 
11.8 
Tris .2 
Nitrobenzylhydroxylamine 
202.0 
O-Benzylhydroxylamine 
40.9 
Carboxymethoxyamine 
38.6 
Allylhydroxylamine 
25.3 
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