Enzyme reference composition

A stable enzyme reference composition having an excellent shelf-life. The composition comprises at least one enzyme of known value; about 20 to about 40 weight percent of at least one alkylene polyol having from 2 to 5 carbon atoms; about 3 to about 8 grams (gm) per deciliter (dl) total protein present in a human serum albumin matrix; and about 60 to about 80 weight percent water.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a laboratory material and, more particularly, to 
a stable enzyme reference composition. 
2. Description of the Prior Art 
Various compositions capable of being employed in conjunction with the 
analysis of enzymes, to either calibrate a instrument or to periodically 
verify that the instrument is still operating within the tolerances 
desired, are known to those skilled in the art. 
For example, U.S. Pat. No. 3,466,249 (hereinafter referred to as Anderson) 
discloses a blood serum reference standard comprising a first container of 
freeze-dried blood serum and a second container of aqueous ammonium 
bicarbonate. The final pH of Anderson's reconstituted serum is 7.5.+-.0.5. 
This pH range is felt by Anderson to be within an acceptable range for 
test systems as well as for stability of the reconstituted serum's 
components, particularly the enzymes. 
U.S. Pat. No. 3,629,142 (hereinafter referred to as Marbach) discloses a 
freeze-dried serum reference standard comprising blood serum and a 
carbonate or bicarbonate of tris(hydroxymethyl)aminomethane which is 
reconstituted by the addition of distilled water. The tris carbonate 
imparts a normal pH to the serum so as to provide for the stability of its 
constituents, and particularly the enzymes. (For normal serum and plasma 
that are collected under routine conditions, the normal pH range is 
generally considered to be about 7.3 to about 7.45. See Tietz, Fundamental 
of Clinical Chemistry, W. B. Saunders Co., Philadelphia, Pa. (1970), pg. 
634; and Henry et al., Clinical Chemistry, Principles and Technics, 2nd 
Edition, Harper & Row, New York, N.Y. (1974), pgs. 774-778.) 
U.S. Pat. No. 3,876,375 and 4,121,905 (hereinafter referred to Maurukas I 
and Maurukas II) disclose a biological reference control composition 
comprising in its non-biological compound from about 60 to about 80 weight 
percent water and from about 20 to about 40 weight percent of at least one 
alkylene polyol having from 2 to 5 carbon atoms, the remainder being 
chiefly at least one natural biological material selected from the group 
consisting of blood serum, enzyme, metabolites, electrolytes, and 
hormones. Although the pH of this composition is not disclosed in either 
Maurukas I or Maurukas II, experiments have shown the pH to be from about 
8.3 to about 8.5. 
One short coming of the above prior art composition is that their enzyme 
constituents are highly unstable. For example, the manufacturer of a 
commercial composition believed to be within the scope of Marbach, supra, 
recommends that determinations of enzyme constituents of its distilled or 
deionized water reconstituted serum be made only on the day that the serum 
is reconstituted. 
Similarly, the manufacturer of a commercial composition within the scope of 
Anderson also recommends that the determinations of its reconstituted 
composition be made only on the day that the serum is reconstituted. 
Both of the above reconstituted compositions are recommended by their 
manufacturer to be stored between 2.degree. and 8.degree. C. 
SUMMARY OF THE INVENTION 
The instant invention encompasses an enzyme reference composition having an 
improved shelf life. The composition comprises at least one enzyme of 
known value; about 20 to about 40 weight percent of at least one alkylene 
polyol having from 2 to 5 carbon atoms; about 3 to about 8 gm/dl total 
protein present is a human serum albumin matrix; and about 60 to about 80 
weight percent water. 
The enzyme constituents of a composition within the scope of this invention 
retains 90% of their initial value, based upon an Arrhenius Plot, for the 
periods set forth in Table I. 
TABLE I 
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ARRHENIUS PLOT FOR 90% LIFE, 
AT 4.degree. C. STORAGE 
Enzyme Constitution Shelf-Life 
______________________________________ 
Amylase 6.8 Years 
.gamma.-Glutamyl Transpeptidase 
206.9 Years 
Lactic Dehydrogenase 1.0 Years 
Creatine Kinase 145.9 Years 
Alkaline Phosphatase 9.9 Months 
Aspartate Amino Transferase 
2.4 Years 
Alanine Amino Transferase 
6.6 Years 
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The above Arrhenius Plot shows that the enzyme constituents of the enzyme 
reference compositions within the scope of this invention are stable for 
periods of time far exceeding the shelf life of enzyme constituents 
present in prior art reference compositions. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The enzyme reference composition of the instant invention comprises at 
least one enzyme constituent of known value. Any enzyme of clinical 
significance can be present therein. Typical enzymes which are currently 
assayed in clinical laboratories include acid phosphatase (ACP); aldolase; 
alkaline phosphatase (ALP); amylase; cholinesterase; creatine kinase (CK; 
also known as creatine phosphokinase (CPK)); .alpha.-glutamyl 
transpeptidase (.gamma.-GR; GGT); .alpha.-hydroxybutyric dehydrogenase 
(.alpha.-HBD;HBD); isocitric dehydrogenase (ICD); lactic dehydrogenase 
(LDH); leucine aminopeptidase (LAP); lipase; alanine amino transferase 
(SGPT; ALT; GPT; glutamic-pyruvic transaminase); and aspartate amino 
transferase (SGOT; AST: GOT: glutamic-oxalacetic transaminase). 
The activity of each enzyme present in the enzyme reference composition is 
not critical. Preferably, the activity of each enzyme should be within a 
range covering both normal and abnormal values. 
The source of each enzyme is also not critical. However, the preference 
source of several enzymes are set forth in Table II. 
TABLE II 
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Enzyme Constituent Preferred Source 
______________________________________ 
ACP Human Seminal Fluid 
ALP Calf Intestine 
Amylase Pig Pancreas 
CK Monkey Muscle 
SGOT Pig Heart 
SGPT Pig Heart 
.gamma.-GT Pig Intestine 
LDH Chicken Heart 
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The enzyme reference composition of the instant invention also comprises 
from about 20 to about 40 weight percent of at least one alkylene polyol 
having from 2 to 5 carbon atoms. Preferably, the alkylene polyol comprises 
from about 30 to about 34 weight percent of the enzyme reference 
composition. 
Suitable alkylene polyols which can be used include, but are not limited 
to, ethylene glycol, propylene glycol, butylene glycol, pentanediol, and 
glycerol. The alkylene polyol material is preferably selected from a group 
consisting of ethylene glycol, propylene glycol, glycerol, and mixtures 
thereof. 
The enzyme reference composition of the instant invention also comprises 
from about 3 to about 8 gm/dl total protein. Preferably, from about 4 to 
about 5 gm/dl total protein is present in the enzyme reference 
composition. The total protein is present in a human serum albumin matrix. 
The enzyme reference composition of the instant invention also comprises 
from about 60 to about 80 weight percent water. Preferably, water 
comprises from about 66 to about 70 weight percent of the enzyme reference 
composition. 
The water can be part of the human serum albumin material and/or can be 
added as a separate constituent. 
The pH of enzyme reference composition of the instant is not critical. The 
optimum pH of the enzyme reference composition will vary depending upon 
the particular enzymes present therein. In general, the pH can be from 
about 5.0 to about 8.7. Preferably, the pH can be from about 6 to about 
8.5. More preferably, the pH can be from about 6 to about 7. Usually, the 
preferred pH will be from about 6.4 to about 6.6. However, ACP has an 
optional pH of about 6.0. 
The pH can be adjusted by any conventional means employed by those skilled 
in the art, e.g., by the addition of hydrochloric acid (HCl) or sodium 
hydroxide (NaOH) to the composition. 
The enzyme reference composition of the instant invention can also further 
comprise metabolites, electrolytes, and hormones of known value. 
Preferably, the enzyme reference composition of the instant invention will 
further comprise sodium, potassium, calcium, phosphorous, magnesium and 
chloride in amounts of interest to the clinical chemist. 
The enzyme reference composition of the instant invention can be stored in 
any suitable container, such as a glass ampule. 
In general, the enzyme composition of the instant invention can be prepared 
via the following procedure. A solution having a desired percentage of 
albumin therein is made by dissolving an appropriate amount of albumin in 
distilled or deionized water. To this albumin solution is then added a 
sufficient amount of at least one alkylene polyol having 2 to 5 carbons so 
that the alkylene polyol-albumin solution comprises from about 20 to about 
40 weight percent alkylene polyol, from about 60 to about 80 weight 
percent water, and from about 3 to about 8 gms/dl total protein in a human 
serum albumin matrix. The pH of the alkylene polyol-albumin solution is 
then adjusted to a desirable level. After adjusting the pH, the alkylene 
polyol-albumin solution is assayed for enzyme activity and salt content to 
obtain baseline values for each constituent of each of the solution's 
desired constituents. An amount of each of the constituents is then added 
to the solution so that the enzymes and salts are present in their desired 
amount. 
If necessary, the pH of the solution is then readjusted to its optimum 
level with respect to the enzymes present therein. 
The stable enzyme reference composition of the instant invention can be 
employed as an enzyme reference standard or as an enzyme reference 
control, i.e., the composition can be employed to either calibrate an 
instrument or can be employed to periodically verify that the instrument 
is still operating within the tolerances desired. For the above uses, the 
enzyme reference composition of the instant invention can contain at least 
one enzyme in amounts typical of those of interest to those in the 
clinical laboratory.