ALT Bioscience Oral Toxicity Testing

ALT Bioscience uses state-of-the-art photoaffinity labeling to determine toxicity levels of extracted root canal teeth, avital teeth and osteonecrotic and/or osteomyelitic bone lesions.  The approach used by our laboratory is similar to the approach of the coal miners of old who carried a canary in a cage into the mines with them.  In that day, there were no detectors to alert the miners when oxygen levels were becoming dangerously low or when carbon dioxide or methane gas levels were approaching toxic levels.  Instead, they relied on the canary, which would show signs of toxicity before the miners too were overcome.  Likewise, the toxicity test performed by ALT relies on the use of several sensitive nucleotide binding enzymes which indicate the presence of a toxic compound or compounds in a sample by a decrease in their ability to interact with their respective nucleotides.  Decreases in enzyme activity can be accurately measured by nucleotide photoaffinity labeling which uses radioactive and photoactive analogs of the nucleotide adenosine triphosphate or ATP.  Using this technology, inhibition of these ATP binding enzymes can be detected and quantified using recognized scientific laboratory techniques.
                The human body contains many sensitive nucleotide binding enzymes crucial for normal cellular functioning.  Impairment of any of these enzymes would have dire consequences for the affected cells in the tissues or organs of the individual.  ALT has chosen a combination of 6 ATP binding enzymes that have one very important thing in common; each of these five enzymes is directly involved in the production of ATP.  The body's ability to produce and maintain ATP levels is absolutely essential for life because every cellular process is driven either directly or indirectly by ATP.  These enzymes include the following:

1. Phosphorylase Kinase: Phosphorylase kinase (PhosK) is responsible for converting Phosphorylase b, the inactive form of the enzyme, to Phosphorylase a, which is the active form of the enzyme by the transfer of a high energy phosphate group from ATP.
2. Phosphorylase a:  Phosphorylase a is the controlling enzyme in the breakdown of glycogen to glucose.  Glucose is the primary fuel the body uses for the production of ATP, which is the body's source of energy for virtually all cellular processes.  This includes everything from muscle contraction to nerve impulse conduction.
3. Pyruvate kinase:  Pyruvate kinase (PK) is one of the glycolytic enzymes which functions in the breakdown of glucose to ultimately yield energy in the form of ATP.  The three enzymatic pathways involved in this complex process are glycolysis, followed by the tricarboxylic acid (TCA) or citric acid cycle and finally the electron transport chain or oxidative phosphorylation.  The pyruvate produced by PK can then enter the TCA cycle to begin the second phase of the energy production cycle.  In addition to its role as one of the enzymes involved in the breakdown of glucose to pyruvate, PK also functions directly in the production of ATP in a process referred to as substrate level phosphorylation (as opposed to oxidative phosphorylation).
4. Phosphoglycerate kinase:  Phosphoglycerate kinase (PGK) is another of the enzymes which functions in the glycolytic pathway involving the conversion of 1 molecule of glucose to 2 molecules of pyruvate which can then enter the TCA cycle.  Like pyruvate kinase, PGK also functions directly in the substrate level production of ATP.
5. Creatine kinase:  Tissues which have a high demand for energy in the form of ATP such as the brain and muscle utilize creatine kinase (CK) to regenerate ATP.  CK makes phosphocreatine during times of low energy demand.  When needed, creatine kinase converts this phosphocreatine to ATP which can then be used as energy.
6. Adenylate kinase:  Adenylate kinase (AK) converts 2 molecules of ADP, a low energy molecule, into one molecule of ATP, a high energy molecule.  Thus, AK kinase serves to maintain ATP levels constant when the energy demands of the body exceed the rate at which ATP can be produced from the breakdown of carbohydrates (e.g. glucose) or fats.

               
These 6 enzymes are commercially available and can be treated with a toxic sample (i.e. the water rinse of a tooth or cavitation sample) then photolabeled together in the same test tube.  While each of these enzymes is sensitive to a variety of toxic compounds, the degree of sensitivity to a given toxin often differs among the enzymes.  For example, a level of a given bacterial toxin, such as hydrogen sulfide, butyric acid or cadaverine, which completely inhibits one enzyme may only partially inhibit the other enzymes. Thus by using 6 different enzymes, this procedure detects varying levels of many different toxins in a sample at one time.  It is important to keep in mind that just because a particular sample does not adversely affect any of these 6 particular enzymes, it does not rule out the possibility that other body enzymes could be inhibited by these same toxins.
The laboratory results of the testing are given as the percentage of inhibition of each of the enzymes caused by toxins present in the oral samples.  An overall average level of toxicity of the sample on a scale of 1-5 (1=slight toxicity to 5=extreme toxicity) is also provided. 
Since most of these enzymes used in the ALT toxicity test are found in virtually every cell in the body, inhibiting the activity of one of these would certainly prove detrimental to the particular tissue or organ affected.  However, the effect of inhibiting the activity of any of these enzymes on the overall health and well being of the patient depends, to a large extent, where in the body each of these toxins accumulates.  For example, a toxin or combination of toxins, which accumulates primarily in the neurons of the brain and impairs the activity of these enzymes would produce nervous system pathology.  On the other hand, a different toxin which accumulates mainly in the cardiac muscle, causing enzyme inhibition, would impair heart functioning.  It is important to realize that how an individual responds to a given level of toxin or combination of toxins may vary depending on the persons genetic predisposition, clinical history, age, nutritional status, dental history, etc.  Therefore, while the assay performed by ALT can detect if toxins are present in the extract from a particular root canal tooth or cavitation, this assay cannot be used to diagnosis or predict the clinical outcome of a particular disease.  The results of this in vitro assay do suggest that the presence of these toxins in the body could certainly exacerbate or hasten the progression of any ongoing disease process.

Instructions for Preparation and Shipping of and Cavitational Biopsy Materials