For more than 30 years, the Equine Pharmacology & Forensic Research Program, a collaboration between Penn Vet’s New Bolton Center and the Pennsylvania Equine Toxicology & Research Center in West Chester, has been working to develop drug detection tests for banned substances in race horses at both Thoroughbred and Standardbred race tracks. This work is vital to the health and well being of equine athletes in both racing disciplines. According to the Racing Medication and Testing Consortium (RMTC), testing in horse racing is superior to that in human athletics as equine testing laboratories analyze samples for a far wider variety of drugs and many more samples are tested. Every winner of each race in the U.S. has a post-race sample collected (either blood or urine – often both), and race stewards are empowered to send additional horses to the test barn following a race. Annually, the industry spends approximately $30 million overall for drug testing.
Dr. Lawrence Soma, with Dr. Cornelius Uboh, has led the work of the PETR Laboratory and has been instrumental in developing drug tests that are infinitely more accurate and reliable than anything in the past. The Pennsylvania Equine Toxicology and Research Laboratory (PETRL) is one of 18 laboratories nationwide handling the entire volume of drug testing for horse racing. Only six of these laboratories are accredited, and PETRL is one of those six. Dr. Soma presented an overview of this work in a “First Tuesday” lecture at New Bolton Center on April 2, 2013. A summary of Dr. Soma’s presentation follows:
What Do Tests Consist Of? The primary mediums used for drug testing are blood (plasma) and urine. Saliva is rarely used. Following the death of a horse at a PA racing facility, a post-mortem examination is conducted and tissues are submitted to the laboratory for drug testing. The order of drug testing procedures is (1) screening for drugs in plasma and urine to determine if a drug is present, (2) identifying the presence of a suspect drug in the sample, (3) identification and confirmation of the illegal substance, and (4) determining the concentration (quantification) of the drug in the sample.
What Methods Are Used to Screen Samples?
Two methods are used for the initial screening – enzyme-linked immunosorbent assay (ELISA) and instrumental screening using mass-spectrometry.
ELISA is an antibody-based screening method that has been in use for over 40 years. It is primarily used for the screening of drugs in urine. This is a biological-based system in that antibodies are produced in an animal against the drug, and the antibodies produced are then used to detect the drug. The production of antibodies is similar to vaccination. An animal is vaccinated using a protein-drug complex, the body produces antibodies against this complex, and following a number of booster injections, blood is collected and antibodies are harvested. The animal now has a rich source of antibodies against a drug or specific group of drugs. The advantage is that the method is simple to use and will detect drugs in the same group. For example, antibodies produced against morphine will detect other drugs related to morphine. The disadvantages are that it is not specific and is subject to false-positives. In addition, the drug has to be confirmed by other methods, the sensitivity of the test can vary, and if a new drug comes along, it takes an average of a year to develop a test using antibodies.
Over the last ten years, the development of liquid chromatography mass-spectrometry has revolutionized drug detection and the use of instruments for screening a large number of drugs at the same time. Sensitivity, speed, and versatility have led to the development of screening methods for hundreds of drugs in a plasma or urine sample in less than five minutes. Currently, Pennsylvania screens every post-race sample submitted to the laboratory for many drugs. For example, methods have been developed for simultaneous screening and identification of 60 anabolic and androgenic steroids in equine plasma. In the past, only specific groups of drugs were targeted on a rotational basis due to the limitations of instrumentation and sensitivity.
How Are Illegal Substances Identified or Confirmed?
Drug confirmation establishes the “fingerprint” of the specific drug in question. Each drug, when subjected to high energy within the mass-spectrometer, will disintegrate or break into specific ions or pieces based on its chemical structure. Further energy will produce secondary ions or pieces. This “fingerprint,” when matched to a known certified authentic standard of that drug, will confirm the unknown drug. Confirmation of a drug is a rigorous, well-documented process in which a number of criteria must be met before a positive is reported to the Horse or Harness Racing Commissions. The rigorous nature of the procedure is such that it resembles the human fingerprint and will withstand challenges in court.
How Are the Amounts of Drugs in a Specimen Quantified?
Quantification of the drug is the last step in the process and determines the quantity of the drug in plasma, urine, or tissue. Using a calibration curve, the identified drug in the test sample is compared to a verified, authentic standard of that drug. The concentrations are measured in duplicate or triplicate, and the slight variability around the average concentration is multiplied by two. This number is known as the “degree of uncertainty in the measurement.”
Accuracy of drug quantifications is very important when studies are done to determine how drugs are removed from the body of horses. Pharmacokinetic is the science of the effect of the horse on the drug and pharmacodynamic determines the effect of the drug on the horse. These studies are conducted to determine how rapidly the drug is eliminated from the horse and its effect on the horse. These are important in determining when therapeutic medications have cleared from the horse’s body, allowing the horse to compete drug-free in an official race sanctioned by the PA Racing Commission.
What Are Peptides, Polypeptides and Proteins?
Proteins are the body’s functional machinery and are made according to the DNA blueprints that carry out many cell functions in the human and animal body. All naturally occurring proteins are made from 20 amino acids found in all higher mammals. Peptides comprise a fewer number of amino acids assembled from the body’s amino acids. Insulin is a larger polypeptide (multiple amino acids linked together) and recombinant erythropoietin (EPO) is a much larger protein-based drug. Peptides or protein-based drugs are substitutes for a specific group of proteins produced by the body. Peptides and recombinant drugs are replicates of naturally occurring proteins and can be made to behave like “the real thing” and stimulate body functions.
Recombinant EPO can be used to stimulate the production of red blood cells, thereby increasing transport of oxygen to the tissues and hence improving performance. Polypeptides and protein-based drugs are considered large molecules and are therefore more difficult to detect and recover from blood and urine. Polypeptides have to be broken or cut into smaller pieces before they can be confirmed by mass-spectrometry. At Penn Vet, we have had great success developing methods to detect and confirm these drugs.
Future Testing Methods – What’s Next?
If a drug is identified in a sample collected from a horse after the conclusion of a race, the positive drug finding is reported to the PA Horse Racing Commission. But what happens if the drug rapidly disappears from plasma or urine yet still has the ability to affect performance? Studies are underway to establish alternative ways of identifying the effects of drug administration or manipulation of the horse.
One form of “manipulation” is Extracorporeal Shock-Wave Therapy (ESWT). Shockwaves are high-energy sound waves that have therapeutic value when transmitted to injured sections of the body. They can be used to enhance the repair of tendons, ligaments, stress fractures, and non-union fractures. ESWT offers pain relief to the treated area for up to four days. Pennsylvania Racing Commission Regulations state that no ESWT should be used within seven days of racing, but there is no way of enforcing this regulation. Unlike drug administration, where there is a method for detection, there is no method for detecting ESWT. Many protein-based drugs cannot be detected in blood or urine hours or days following their administrations, but they still have an effect on the horse’s performance.
Biomarker Discovery Using Equine DNA
The description of the equine genome has led to the development and the production of an equine chip containing 20,000 equine genes. This has opened the door for the determination of changes in DNA, up regulation or down regulation, as a possible marker for describing alterations in the genome produced by diseases, chronic disorders, or extracorporeal shock-wave therapy. Currently, there is no method to determine if ESWT has been administered prior to racing. Using noted DNA changes in the horse as a means to identify the use of illegal substances or treatments is one of our current approaches. RNA is isolated from white blood cells before and after the administration of shock wave therapy. The main job of RNA is to transfer the genetic code from the DNA needed for the creation of proteins. Proteins are the workhorses of the cell, carrying out a variety of functions. Following ESWT, changes in candidate biomarkers (RNA) have been seen across a 72-hour time frame. These changes are now being validated so that we can use them as “tellers” or “pointers” for the use of ESWT in horses.
Biomarker Discovery Using Serum Proteome Analysis
Plasma contains thousands of proteins, of which only a small percentage carries out body functions. The field of protein study (proteomics) is rapidly growing, attempting to identify and quantify many of the proteins produced by living organisms. The primary focus of proteome analysis in humans is to define changes produced by diseases, chronic disorders, and for cancer diagnosis. It is essential for these methods to be developed and applied to biomarker discovery in the horse. Our current focus is on how biomarker discovery can benefit the drug testing community.
Biomarker Discovery Using Serum Cytokine Analysis
Cytokines are proteins that are released by tissue cells in response to trauma, disease, pain, and various stimuli. Shock wave therapy produces inflammation and an increase in regional blood flow. This speeds up the healing process. Many cytokines remain at the local level within the tissues and some are released into the blood stream. We are seeking those that are released into plasma and can be identified. We have developed enzyme-linked immunosorbent assay (ELISA) for nine equine cytokine proteins, which are both inflammatory and anti-inflammatory proteins. We have found significant changes in three of the nine cytokines we have examined following ESWT.
The studies underway are the work of a number of talented researchers who each have special expertise in analytic chemistry, protein chemistry, molecular biology, pharmacology, equine pharmacology, and veterinary medicine. The work is funded by the Pennsylvania Harness and Horse Racing Commissions, through the Pennsylvania Department of Agriculture, with additional support from Standardbred and Thoroughbred horsemen’s organizations in Pennsylvania.