Enzyme determination method

An improved colorimetric aldolase method in which the quantity of aldolase in a sample is determined by the rate at which it enzymatically cleaves fructose-1, 6-diphosphate (FDP) into glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP); the phosphate group is hydrolyzed from the GAP and DHAP; and one of the hydrolysis products is measured. The initial enzymatic reaction is carried out in a reaction mixture containing a concentrated tris-(hydroxymethyl)-aminomethane buffer, pH 7.0, and a small quantity of sodium fluoride, but without added hydrazine or other compound to prevent the enzymatic conversion of a great majority of GAP to DHAP in the reaction mixture. Hydrolysis of the reaction products and conversion to a colored reaction product is carried out by a procedure which yields intense, long-lasting, reproducible color. An improved standard includes dihydroxyacetone carried through the same procedure.

BACKGROUND OF THE INVENTION 
This invention relates to an improved method for determining aldolase, EC 
4.1.2.13, in serum and other biological fluids, and to related improved 
methods for determining other species. 
Aldolase belongs to a group of enzymes, termed lyases, which reversibly 
cleave substrates into two compounds without hydrolysis. Aldolase splits 
the hexose fructose-1, 6-disphosphate (FDP) into the triose phosphates 
glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). 
The term "triose" as used throughout this specification and claims is 
limited to the two species glyceraldehyde (GA) and dihydroxyacetone (DHA). 
The enzyme is present in virtually all cells of the body, especially in 
the muscles. Circulating blood normally contains comparatively little 
aldolase. Serum levels of aldolase increase in conditions which involve 
cell destruction, and it is believed that the normal level arises from 
physiologic breakdown of tissue. Clinically, the measurement of aldolase 
is useful, for example, in cases of muscular diseases such as progressive 
muscular dystrophy in which serum aldolase levels rise to five or ten 
times the upper limit of normal, and in prostate cancer in which the 
elevation of serum aldolase levels is less pronounced. Aldolase is also a 
commercial product, and tests are required for determining aldolase levels 
during the isolation of the enzyme. 
Four well-known approaches to determining aldolase in serum (or 
equivalently, in plasma), by measuring the cleavage products of FDP, have 
been proposed. As set out in the article on aldolase by Bruns & Bergmeyer, 
in Methods of Enzymatic Analysis, ed. H. S. Bergmeyer (New York 1965), p. 
724, the methods are as follows: 
(1) DETERMINATION OF THE ALKALI LABILE TRIOSE PHOSPHATE FORMED. Meyerhoff & 
Lohmann, Biochem. Z. 271, 89 (1934) and 273, 73, 413 (1934). 
(2) COLORIMETRIC ESTIMATION OF THE TRIOSE PHOSPHATE FORMED BY THE METHOD 
ORIGINALLY DESCRIBED FOR LACTATE. Dounce and Beyer, J. Biol. Chem, 173, 
159 (1948). 
(3) MEASUREMENT OF ACTIVITY BY THE SPECTROPHOTOMETRIC METHOD OF Warburg. 
Warburg and Christian, Biochem. Z. 314, 399 (1943). 
(4) another colorimetric method in which the dinitrophenylhydrazones of the 
free trioses are determined. Sibley and Lehninger, J. Biol. Chemistry 177, 
859 (1949). 
A fifth approach, by Jagannathan et al, Biochem. J. 63, 94 (1956), adds 
hydrazine to the incubation mixture and measures absorbance at 240 nm. 
Meyerhoff and Lohmann's early studies were made largely on aldolase 
obtained from muscle extract dialysates. The high blanks, variability and 
technical difficulties of alkali labile phosphorus determination have led 
not only to its being ignored as a routine method for determining aldolase 
in serum or other biological fluids, but also to numerous proposals for 
replacing it as a primary standard. No completely satisfactory primary or 
secondary standard has yet been devised. See Beck, J. Biol. Chem, 212, 847 
(1955). Among other things, Meyerhoff and Lohmann found that the enzyme is 
active between pH's of about 6 and 10 (in a carbonate-bicarbonate buffer), 
and that it is not influenced by certain materials, such as iodoacetic 
acid, sodium fluoride and sodium oxalate, which are known to inhibit some 
other enzymes. In later work (Bull. Soc, Chimie Biol. 20, 1033) (1938)), 
Meyerhoff used hydrazine to fix the relative proportions of the triose 
phosphates GAP and DHAP in a 1:1 ratio to permit their polarimetric 
determination. Fixing the proportions of GAP and DHAP is necessary because 
certain enzymes which may be present in biological fluids selectively 
catalyze destruction of one of the trioses. In particular, triosephosphate 
isomerase (TPI), EC 5.3.1.1, which has a wide distribution in animal 
tissues, catalyzes the conversion of GAP to DHAP. 
The colorimetric method of Dounce and Beyer degraded DHA and GA to 
acetaldehyde and added p-hydroxydiphenyl to produce a color, but their 
procedure suffered from frequent and variable high blanks and from the 
inconvenience of using concentrated sulfuric acid and heat. After the 
publication of the Sibley and Lehninger method, these workers modified 
their method by reducing the concentration of FDP substrate, adding 
hydrazine and adding collidine buffer pH 7.2. Dounce et al, J. Biol. Chem. 
185, 769 (1950). 
The method of Warburg and Christian, and other similar methods using 
coupled enzyme systems, suffer from several disadvantages, not the least 
of which is their need for an expensive ultraviolet spectrophotometer. 
The colorimetric method of Sibley and Lehninger, with or without minor 
variations, is widely used for the determination of aldolase in serum. 
Sibley and Lehninger carried out the incubation of sample and FDP in Tris 
buffer at pH 8.6 and added hydrazine to the enzymatic incubation mixture 
to prevent the enzymatic conversion of GAP and DHAP through the action of 
TPI. After an alkaline hydrolysis step, they added an acidified 
2,4-dinitrophenylhydrazine reagent to the alkaline hydrolysis mixture, 
then added more alkali to develop a characteristic color. The colored 
product is presumed to be hydrazone, and will be so characterized herein. 
Some later investigators, such as Dounce et al, J. Biol. Chem., 185, 769 
(1950), modified the sibley and Lehninger procedure by using a collidine 
buffer at a pH of 7.2 for the incubation mixture, and adding iodoacetate 
to the enzymatic incubation mixture to reduce side reactions and to lower 
the absorbance readings of the blanks used in the procedure. Friedman and 
Lapan, J. Clin. Lab. Med. 51, 745 (1958), recommended the use of DHA as a 
(secondary) standard, and the reporting of aldolase values in "DHA units". 
Although the methods based on Sibley and Lehninger's method are regarded as 
the most reliable and suitable for routine laboratory use, all of these 
methods suffer from a lack of sensitivity, reliability and accuracy, from 
high blanks, from the instability of the colored compounds measured, and 
from the difficulty of preparing reliable standard solutions. 
SUMMARY OF THE INVENTION 
One of the objects of this invention is to provide an improved aldolase 
procedure which is considerably more sensitive than previously known 
procedures, and which, therefore, gives more meaningful normal and 
borderline values for aldolase in serum. 
Another object is to provide such a procedure which is more accurate and 
reproducible than previously known procedures. 
Another object is to provide such a procedure which is relatively simple 
and requires relatively simple equipment. 
Another object is to provide such a procedure in which blanks are less 
variable and generally lower than with the previously known procedures. 
Another object is to provide such a procedure which may utilize a reliable 
standard. 
Another object is to provide a modification of the procedure of Sibley and 
Lehninger which produces a stable color proportional to aldolase 
concentration and is therefore readily adaptable to automatic or multiple 
simultaneous determinations of aldolase. 
Another object is to provide such a modified procedure which is applicable 
to determinations of other enzymes which produce triose phosphates or 
trioses, and to determinations of the phosphate esters of the trioses 
dihydroxyacetone and glyceraldehyde and of the trioses themselves in 
biological fluids such as human serum. 
Other objects will become apparent in light of the following description. 
In accordance with one aspect of this invention, generally stated, an 
improved aldolase procedure is provided which includes an enzymatic 
incubation step in which fructose-1,6-diphosphate is incubated in a 
reaction mixture containing a buffer, sufficient to an inhibitor to 
inhibit side reactions, and a sample of human serum containing an unknown 
quantity of aldolase, and in which the incubation mixture is free of 
hydrazine or other triose "fixing" compound and the major part of the 
glyceraldehyde-3-phosphate (GAP) formed is enzymatically converted to 
dihydroxyacetone phosphate (DHAP) by triosephosphate isomerase (TPI). The 
DHAP is then converted to a colored hydrazone. It has been found that the 
quantity of TPI in human serum is sufficient to carry out the conversion 
of GAP to DHAP essentially as fast as GAP is formed. 
In accordance with another aspect of the invention, a procedure is provided 
which is an improvement of the method of Sibley and Lehninger, supra, and 
involves alkali hydrolysis of triose phosphate at a pH of from about 11 to 
about 12.25 in a solution containing at least a 25 millimolar 
concentration of an activating amino alcohol, addition of 
2,4-dinitrophenylhydrazine and acid, and alkaline color development of the 
reaction solution to develop a color which is proportional to aldolase 
concentration in the sample. In the presence of the amino alcohol and the 
absence of hydrazine, a color is produced which is far more intense and 
resistant to fading than the color produced in previous methods. The 
method is also useful for the determination of triose phosphates and of 
trioses in other systems, including systems in which the triose phosphate 
or triose is formed by the action of an enzyme other than aldolase. 
In the preferred embodiment, the enzymatic incubation mixture includes FDP, 
a human serum sample, an inhibiting compound to inhibit side reactions, 
and a color-reaction activiting amino alcohol buffer in a concentration of 
at least about 0.1 molar, to maintain a pH in about the 6-9 range which 
favors the enzymatic cleavage. The incubation mixture does not contain 
hydrazine or other reactant to fix the ratio of DHAP to GAP at 1:1. The 
inhibiting compound is preferably sodium fluoride, and the buffer is 
preferably tris-(hydroxymethyl)-aminomethane (Tris) at a pH of about 7. 
Careful control of the alkaline hydrolysis step has been found to be 
critical to the formation of the colored product. The conditions are not 
those described by Fleisher in Standard Methods of Clinical Chemistry, 
Vol. 3 (New York 1961) p. 14. In particular, a pH of from about 11 to 
about 12.25 has been found to favor rapid and intense color development. 
The preferred Tris buffer has been unexpectedly found to have considerable 
buffering capacity, both at the pH of the enzymatic reaction and at the pH 
of the hydrolysis mixture. It is also an activating amino alcohol in the 
color forming reaction. The procedure reduces side reactions. It also 
produces a color which is about five times as intense as the color 
produced with previous procedures, and is capable of far better resolution 
of normal and marginal levels of aldolase in serum. 
The suppression of side reactions greatly reduces the coloration of the 
blanks. 
The new procedure also simplifies the assay of multiple samples because it 
produces a colored end product which is much more stable than those 
previously formed, and also because it permits the procedure to be 
interrupted after the incubation step, if necessary. 
The procedure also is considerably more reproducible than previously known 
procedures, and utilizes a more reliable standard. 
Since the work of Sibley and Lehninger, hydrazine has been universally 
included in the reaction mixtures of (colorimetric) procedures which 
hydrolyze GAP and DHAP. By eliminating hydrazine, the present procedure 
permits conversion of at least 90% of the GAP produced in the incubation 
step to DHAP. This conversion is advantageous because it has been found, 
in accord with the work of others (e.g. Sibley and Lehninger; see also 
Riddle and Lorenz, J. Biol. Chem. 243, 2718 (1968)), that DHAP forms a 
colored reaction product in the subsequent steps of the procedure much 
faster than GAP; it now appears that upon hydrolysis of the trioses, GA 
must isomerize to DHA before it can react further. It also now has been 
found that DHA is converted to hydroxypyruvic aldehyde (HPA) which in turn 
reacts with DNPH to form the colored reaction product. The elimination of 
hydrazine from the reaction system thus appears to accomplish more than 
merely permitting the nearly complete conversion of GAP to DHAP. Hydrazine 
also seems to encourage the enzymatic phosphorylation of Tris, especially 
in the presence of alkaline phosphatase and FDP, thereby changing the 
concentrations of the reactants and also giving rise to interfering 
colored products. It likewise competes with DNPH to form triose hydrazones 
and destabilizes the final colored product. 
In the absence of hydrazine, a color enhancing compound is needed to 
produce a colored product from the reaction products of the incubation 
reaction. Besides Tris, other amino alcohol buffers have been found to be 
effective, such as Tricine, TAPS, AMPD, aminoethylpropanediol, 
Bis-tris-propane and AMP, and to a lesser extent serine, ethanolamine, 
diethanolamine, and TES. Other buffers, such as Bicine, EPPS, 
triethanolamine, HEPES, MOPS and PIPES were found not to be effective 
under the color development conditions of the preferred embodiments. The 
term "color enhancing" is used herein to indicate a material which, in the 
absence of hydrazine, produces a color at least one-quarter as intense as 
that produced in the presence of an equimolar concentration of Tris, under 
the same color formation reaction conditions. 
In the absence of hydrazine, side reactions must be inhibited by an 
inhibiting reagent to provide accurate results and reduce the color of 
blanks. For example, 3-phosphoglyceraldehyde dehydrogenase may reduce the 
amount of color-forming reaction products in the incubation mixture. 
Beside sodium fluoride, other known enzyme blocking agents, such as 
iodoacetate, iodoacetic acid, potassium fluoride and sodium arsenite are 
usable. The term "inhibiting compound" is used herein to designate a 
material which inhibits enzymatic side reactions but does not materially 
interfere with either aldolase activity or TPI activity. 
In the preferred embodiment, the Tris buffer is used at a pH (7.0) which is 
normally below the range at which it possesses sufficient buffering 
capacity in the enzymatic incubation step, and at a pH (about 12) which 
has heretofore been thought to be far above the range at which it 
possesses any substantial buffering capacity. Therefore, a substantially 
higher-than-normal concentration of buffer at both steps of the method is 
beneficial. 
The alkaline hydrolysis step has also been found to be of great importance. 
In the preferred embodiment, the hydrolysis of the deproteinized TCA 
supernatant is carried out at a temperature of 25.degree. C. for a timed 
period of twenty minutes. At this temperature, the reaction is about 90% 
complete, and the reaction rate after twenty minutes is low enough to make 
precise timing of the step less critical. The amount of color formed with 
DNPH is great enough to provide a sensitive procedure for even normal 
human serum samples. 
If pure DHA is treated with alkali (pH 11-12.25) in the presence of Tris, 
at a temperature of 25.degree. the reaction is more than half complete and 
is proceeding quite slowly after about fifteen minutes; at a temperature 
of 37.degree. C. the reaction is essentially complete after ninety 
minutes, as evidenced by the color formed with DNPH. Pure GA, treated in 
the same manner, reacts more slowly. The reaction at pH 11-12.25 in the 
presence of Tris or other activating amino alcohol is many times greater 
than that in the absence of an activating amino alcohol.

DESCRIPTION OF THE PREFERRED EMBODIMENT 
The following examples are illustrative of the preferred methods of the 
present invention. 
In these examples, aldolase in a serum sample catalyzes the splitting of 
FDP to DHAP and GAP. TPI present in serum catalyzes the almost complete 
conversion of GAP to DHAP. The reaction product is hydrolyzed at room 
temperature to the free triose. The free triose under alkaline conditions 
and in the presence of Tris, is converted to hydroxypyruvic aldehyde, 
which reacts with 2,4-dinitrophenylhydrazine in acid to form a hydrazone. 
On addition of alkali, a stable, intense purple color forms with an 
absorbance maximum at about 560 nm. The intensity of the color is 
proportional to triose concentration, hence to triose phosphate 
concentration, hence to aldolase activity. 
EXAMPLE 1 
Preparation of Reagents 
The following reagents are prepared: 
Reagent A. FDP Substrate Solution. 1.0 gm D-Fructose-1,6-Diphosphate, 
Trisodium salt, is dissolved in 40 ml deionized water. The solution is 
stable for two weeks at 0.degree.-5.degree. C. or several months frozen. 
Reagent B. Tris/Fluoride Buffer Solution. Tris-(hydroxymethyl)-aminomethane 
(0.3 mol/liter, pH 7.0 at 37.degree. C.) and sodium fluoride (0.0015 
mol/liter) are dissolved in water. Chloroform may be added as a 
preservative. The solution is stable at room temperature. 
Reagent C. Color Reagent. 2,4-Dinitrophenylhydrazine (1 gm/liter) is 
dissolved in dilute (about 1.3 molar) hydrochloric acid. Stored in dark at 
0.degree.-5.degree. C. 
Reagent D. Standard Solution. Dihydroxyacetone (0.20 gm/liter) is dissolved 
in water. The solution is stored below 0.degree. C. and mixed thoroughly 
after thawing. 
Reagent E. Sodium Hydroxide Solution. Anhydrous Sodium Hydroxide (48 
gms/liter) is dissolved in water. The solution is accurately standardized 
at 1.20 N and stored in tightly closed small containers. 
Reagent F. Trichloroacetic Acid Solution. TCA (approximately 100 gm/liter) 
is dissolved in water to form a 0.6 N solution. 
EXAMPLE 2 
Preparation of Calibration Curve (Determination of DHA) 
Two 100 ml Erlenmeyer flasks are labeled STANDARD and BLANK. To the 
STANDARD flask is added 0.4 ml calibration solution (Reagent D) and to the 
BLANK flask is added 0.4 ml water. To both flasks are added 4.8 ml 
Tris/Fluoride Buffer Solution (Reagent B), 0.6 ml substrate solution 
(Reagent A), and 7.7 ml water. Both flasks are placed in a 25.degree. C. 
water bath for about 5 minutes to reach temperature. To each flask is 
added 4.5 ml sodium hydroxide solution (Reagent E), and after gentle 
mixing, the solutions are allowed to stand at 25.degree. C. for exactly 
twenty minutes. To each is then added 6.0 ml color reagent (Reagent C.), 
the solutions are again mixed gently, and the flasks are placed in a 
37.degree. C. water bath. After thirty minutes, the flasks are removed 
from the water bath and to each is added 24 ml sodium hydroxide solution 
(Reagent E). The reaction mixtures are mixed by swirling. The resulting 
STANDARD solution color is stable for about thirty minutes, after which it 
fades about 2% per hour. The color of the STANDARD solution is measured 
against the BLANK solution at 560 .+-. 10 nm. If desired, a calibration 
curve may be prepared plotting known dilutions of the final STANDARD 
solution (using the BLANK solution as diluent) against absorbance. 
EXAMPLE 3 
Determination of Aldolase in Serum 
Blood is drawn into a plain tube and allowed to clot. Serum is separated 
within one hour and promptly assayed or stored frozen. Hemolyzed samples 
are avoided. 
Into two test tubes, labeled TEST and BLANK are pipetted 1.6 ml 
Tris-Fluoride Buffer Solution (Reagent B) and 0.2 ml serum. Both tubes are 
placed in a 37.degree. C. water bath for about five minutes to reach 
temperature. To the tube labeled TEST is added 0.2 ml substrate solution 
(Reagent A). The tube is gently swirled and replaced in the water bath. 
After exactly thirty minutes, 2.0 ml Trichloroacetic Acid (Reagent F) is 
added to each tube. To the tube labeled BLANK is then added 0.2 ml 
substrate solution (Reagent A), and the tubes are shaken well. Both tubes 
are centrifuged to obtain clear supernatant, 2.0 ml of which is removed 
from each tube to a corresponding tube labeled TEST and BLANK. Both tubes 
are placed in a water bath at 25.degree. C. for about five minutes to 
reach bath temperature. To each tube is then added 1.0 ml of sodium 
hydroxide solution (Reagent E), and after gentle mixing the tubes are 
allowed to stand in the 25.degree. C. water bath for exactly twenty 
minutes. To each tube is added 1.0 ml color reagent (Reagent C). After 
gentle mixing the tubes are placed in the 37.degree. C. water bath for 
thirty minutes. The tubes are removed from the bath and to each is added 
4.0 ml sodium hydroxide solution (Reagent D). After mixing by inversion, 
the tubes are allowed to stand at room temperature for about five minutes. 
The contents are transferred to cuvets, and the absorbance of the TEST 
solution is read against the BLANK as reference, using the same instrument 
at the same wavelength as in the preceding example. The reading should be 
completed within thirty minutes after the final addition of sodium 
hydroxide solution. 
Aldolase activity in the serum sample is determined from the calibration 
curve of the previous experiment. An absorbance reading equal to the 
absorbance of the undiluted reference indicates the splitting of 20 
nanomoles (millimicromoles) of FDP per minute under the cnditions of this 
Example. If the splitting of one nanomole of FDP under the conditions of 
this Example is taken as one "unit", normal serum contains about 2 to 8 
units per ml. Because the absorbance in a typical colorimeter changes more 
than 0.030 per unit of activity, the normal range and the borderline 
between normal and elevated values are easily determined. 
Reproducibility studies on three serum pools assayed on ten separate 
occasions over a period of three weeks showed mean aldolase activities of 
3.2, 6.0 and 43.8 units per ml respectively. Standard deviations were 
0.06, 0.21 and 0.60, respectively. A series of fourteen sera assayed by 
the foregoing procedure and in terms of alkali-labile phosphate yielded 
aldolase values ranging from 2 to 50 units per ml and a correlation 
coefficient of 0.998. 
If desired, the centrifuged TCA supernatants of this Example 3 may be 
stored frozen until the remainder of the procedure is carried out. The 
TEST supernatant contains both DHAP and a small amount (less than 5% of 
total triose) of GAP. When assayed, the thawed supernatants yield aldolase 
activities identical with freshly assayed samples. 
Numerous variations in the methods of the present invention, within the 
scope of the following claims, will occur to those skilled in the art in 
light of this disclosure.