Stabilized Liquid Coenzyme Compositions

1. Field of the Invention

This invention relates to the stabilization of labile coenzymes in liquid media.

2. Description of the Prior Art

It has recently been estimated that 25% of all in vitro diagnostic tests conducted annually in this country are not reliable. Unreliable tests can result in unnecessary medical treatment, the withholding of necessary treatment and lost income. Because of their high specificity, the use of enzyme determinations has significantly increased during the last few years and indications are that this trend will continue. However, rigorous quality control measures are required to assure the accuracy and consistency of results. This requirement stems from the fact that the exact nature of enzymes, as well as the mechanisms of their action, remain unknown for the most part. At present, the greatest limitation on the enzyme reagent manufacturer, by far, lies in the unstable characteristics of his products. Current methodologies require the use of numerous labile ingredients, and these ingredients are more likely to increase, rather than decrease, in number.

The present commercial state of the art used for stabilizing the reactive ability of enzymes or coenzymes is by locking them into a solid matrix either by freeze drying, dry blending such as used for tableting dried powders, primarily in the pharmaceutical diagnostic and related industries and immobilization by locking the chemical structure of the enzyme into a solid matrix. Contrary to the sophistication these terms imply, these approaches are neither practical nor desirable and are also expensive. The manufacturer is forced to remove the water and supply a partial product, thus relinquishing part of the quality control cycle in the dilution and use of the final product. Laboratories are forced to pay the high cost of packaging, reagent waste, freeze drying and dry blending, and usefulness of the product is further limited by packaging modes and sizes.

Furthermore, good product uniformity is difficult to achieve. This condition is exemplified by the fact that most commercial freeze dried control sera (reference serum) list the acceptable bottle to bottle variation of enzyme constituents at .+-.10% of the mean.

In the clinical diagnostic field the commercial application is represented by, but not limited to, the diagnostic reagents used to determine and quantitate the following constituents in biological fluids:

1. Glutamic-oxalacetic transaminase (SGOT);

2. glutamic-pyruvic transaminase (SGPT);

3. lactic dehydrogenase (LDH);

4. creatine Phosphokinase (CPK);

5. .alpha.-hydroxybuteric dehydrogenase (.alpha.-HBD)

6. glucose (via Hexokinase-G-6-PDH). These reagents react similarily, contain some common labile ingredients, and some of the chemical reactions involved are common. The following chemical reaction scheme is presented as a model to illustrate the general nature of the reactions involved: ##STR1##

All enzymatic reactions listed above will follow this general scheme, where reaction (2.) is usually referred to as the coupling reaction, reactions (2.) or (3.) are the measuring reactions, and reaction (1.) may be characterized as the primary reaction. It is understood however, that not all three reactions are required for measurement; in fact, they may be limited to two, or one. In the case of the ultraviolet measurement of lactic dehydrogenase (LD) activity, only reaction (2.) is involved, as follows:

Conversely, more than the three reactions listed may be involved as in the case of Creatine phosphokinase (CPK):

cp=creatine phosphate

Adp=adenosine-5'-diphosphate

Atp=adenosine triphosphate

Hk=hexokinase

Nad=nicotinamide-adenine dinucleotide

Nadh.sub.2 =nicotinamide-adenine dinucleotide, reduced

G-6-pdh=glucose-6-phosphate dehydrogenase

Int=tetrazolium salt

Pms=phenazine methosulfate

In this case, reactions (2.) and (3.) may be considered the coupling reactions, reactions (3.) or (4.) the measuring reactions, and reaction (1.) the primary reaction.

Referring to REACTION SCHEME 1,--GENERAL MODEL, it becomes obvious and is general knowledge that the use of the reaction sequence permits the analytical quantitation of either the reaction substrates/products or the catalyzing enzymes.

The quantitation of these constituents in biological fluids is a well accepted and widely used diagnostic tool in diagnosis and treatment of human and animal disease states.

Enzymes are large molecular weight, complex protein molecules, usually of unknown chemical structure. They are presently classified by their catalytic activity and extreme substrate specificity. Enzymes may be redefined as biological catalysts, capable of catalyzing a reaction of a single substrate, or a reaction of a similar group of substrates.

Coenzymes are lower molecular weight organic chemicals of well-defined structure, whose reactions or interactions are necessary for specific enzyme assay or reaction. They are catalyzed resulting in an irreversible change in the coenzyme's structure and/or atomic composition. Coenzymes are very useful in clinical assay procedure. Some have strong absorbance, their reactions are stoichiometric with the substrate and therefore the creation or disappearance of the absorbing form can be followed photometrically. Nicotinamide adenide dinucleotide (NAD) and its reduced form (NADH.sub.2) are used in many important clinical assays such as the S.G.O.T., S.P.G.T. and LDH assays previously described. NAD and NADH.sub.2 have a molecular weight of about 700 and are very complex organic molecules. NADH.sub.2 absorbs strongly at 340 nm whereas NAD does not absorb at this wavelength.

NADH.sub.2 is extremely unstable in water solution or in dry form when exposed to humid environments. Even when frozen NADH.sub.2 must be kept free of moisture. Stability is better at alkaline pH, whereas at acid pH NADH.sub.2 decomposes very rapidly in a matter of minutes. Neither the exact mechanism, nor the end products are of significance except that decomposed NADH.sub.2 can no longer effectively function as a coenzyme nor does it possess the extinction coefficient at 340 nm. The typical commercial form is a dry dessicated package or freeze dried stored under nitrogen. NADH.sub.2 is classically insoluble in organic solvents.

Labile coenzymes are treated according to the invention resulting in long term stability without affecting coenzymatic reactivity or photometric absorptivity. The invention provides reagents where quality control is assured throughout manufacturing, packaging, storage and use. The inconvenience of rigid package size is eliminated as is the high cost of packaging, freeze drying and reagent waste. Liquid enzyme and coenzyme systems provide application flexibility and separation of the ingredients is easily accomplished with negligible manufacturing cost providing the flexibility of triggering the desired reaction after all side reactions have been dissipated.

The stabilized coenzymes of the invention have been assessed in studies which compared liquid coenzyme reagents with fresh reagents. The studies show a 1:1 correlation between aged liquid and fresh reagents with comparable sensitivity and precision. Providing coenzyme reagents in a stable liquid form enhances the colorimetric applicability of present day NAD/NADH coupled methodologies primarily because the separation of ingredients is easily accomplished. Stable liquid reagents are especially advantageous where NADH consumption is the basis of measurement and the color reagent must be separated from NADH and the reaction main. In the ultraviolet mode, the liquid system offers better reagent homogeneity and packaging, as well as flexibility in usage, in contrast to the freeze-dried or dry media preparations.

In diagnostic enzymology, the stabilization of enzyme reagents in a ready-to-use liquid media is a new and exciting approach to satisfy the needs of the clinical laboratory and the reliability demands of the regulatory authorities. The flexibility of liquid enzyme systems insures their applicability to automated instrumentation, as well as their convenience in manual testings.

Stabilization of labile coenzymes is accomplished in accordance with the invention by dissolving the coenzymes in organic solvent. After solution is achieved at least 1% V/V of inert, hygroscopic solid is added and the container closed. The suspension is maintained at room temperature at least 1 hour, usually 1 to 2 days with occasional mixing to remove water from the mixture down to a level of no more than 0.05%. The solution may then be dispensed into amber-glass bottles containing at least 1% V/V hygroscopic agent which are sealed airtight and stored under refrigeration. Projected shelf life is up to 4 years under these conditions without appreciable degradation.

Surprisingly the coenzyme NADH.sub.2 exhibits good solubility and stability in the aqueous miscible organic solvent even though solvents such as 1,2-propanediol are hygroscopic. Evidently the solvent molecules efficiently solvate the coenzyme protecting it from water and the solvent media acts as an efficient transfer media, delivering the absorbed water to the solid hygroscopic agent where it is irreversibly bound.

These and many other features and attendant advantages of the invention will become apparent as the invention becomes better understood by reference to the following detailed description.

The organic solvent should have the following characteristics:

1. Low water content (trace <0.1%);

2. Neutral or alkaline pH;

3. liquid at room and refrigerator temperatures;

4. Does not react with NADH.sub.2 other than forming electrostatic (i.e. hydrogen) bonds;

5. Miscible with water;

6. Standard free energy of solvolysis is low (normal resonance is established).

Non-reactive organic solvents of neutral or alkaline pH, such as alcohols especially liquid polyols containing from 2-4 hydroxyl groups and 2-10 carbon atoms are preferred, such as glycerol, ethylene glycol, propylene glycol or butane diol. Propylene glycol, 1,2-propanediol, was found to possess all these qualities and is the solvent of choice.

The inert hygroscopic solid maintains the desired low water content i.e. below 0.5% preferably below 0.1%. The hygroscopic solid must be an efficient water adsorber non-reactive with the coenzyme and of neutral or alkaline pH. The solid is preferably a high area hygroscopic agent such as a natural or synthetic molecular sieve having a particle size from 2-16 mesh present in an amount of at least 1% V/V typically from 5-20% V/V. The amount of surface area is important since the material acts to adsorb water into the pores.

Molecular sieves are zeolites or similar materials whose atoms are arranged in a crystal lattice in such a way that there are a large number of small cavities interconnected by smaller openings or pores of precisely uniform size. Normally these cavities contain water molecules, but upon heating, this water is driven off without any change in the remaining crystal lattice. The network of cavities and pores may occupy 50% of the total volume of the crystals. Molecular sieves have a strong tendency to readsorb water.

A few natural zeolites exhibit molecular sieve characteristics to a limited degree. Synthetic zeolites are available in several sizes (pore openings 3, 4, 5 and 10 angstrom units in diameter) with high capacity for adsorption and regeneration even when used at elevated temperatures.

The coenzyme can be present up its solubility limit and is preferably as concentrated as possible since propylene glycol is a strong enzyme inhibitor and can interfere with the primary or coupling enzymatic activity if too much is carried into the test from the coenzyme reagent. Typical NADH.sub.2 compositions according to the invention contain about 2-15 g/l typically about 7 g/l. Hydrated NADH.sub.2 can be utilized for speedier solution in the polyol solvent. Example: 6.65 g/l of NADH.sub.2 was dissolved in spectroquality 1,2-propanediol in a closed amber glass container. After complete solution is attained, 10% V/V of molecular sieves (4 mesh) were added and the container was closed and left at room temperature for 24 hours with occasional mixing to reduce water in the mixture below 0.01%. The solution was dispensed into final marketing amber-glass bottles containing fresh 4 mesh molecular sieves (10% v/v). The containers were sealed airtight and stored under refrigeration. An Arrhenius plot depicting the temperature stability profile of NADH.sub.2 degradation in this media indicates storage stability of up to 4 years without significant degradation. The data was obtained at three storage temperatures of 60.degree. C., room temperature (225.degree. C.), and at refrigerated temperatures of 2.degree.-8.degree. C. (The mean of 4.degree. C. was used.) The maximum allowable loss is less than 10% after 360 days storage at 27.degree. C.

When propylene glycol is utilized without hygroscopic agent, NADH.sub.2 degrades quite quickly in use when subject to opening and closing the container.

It is to be realized that only preferred embodiments of the invention have been described and that numerous substitutions, modifications and alterations are permissible without departing from the spirit and scope of the invention as defined in the following claims.