Thursday, April 16, 2015

What is Cobalt?

What is Cobalt?

By:  Clara Fenger, DVM, PhD, DACVIM and Pete Sacopulos, JD

Cobalt is a mineral which is present in animal systems predominantly as part of Vitamin B12, which is a key cofactor in DNA synthesis. It is a nutritionally required element with the lowest dietary requirement of all of the required trace minerals.   Recommended daily allowances for humans is about 0.1 µg/day in the form of Vitamin B12 (Unice et al., 2012).  The National Research Council, which determines daily requirements of nutrients for animals, has determined a dietary cobalt requirement of 0.5 to 1.1 mg/day in horses and 1.2 to 2.4 mg/day for a cow, depending upon weight and metabolic state (Lawrence et al., 2007).  Maximum tolerable amount is 25 mg/kg of dry matter of feed or forage (Lawrence et al., 2007).

Cobalt concentrations in drinking water typically range from 0.1–5 ng/mL, and daily cobalt intake ranges from 11–580 µg/day in humans (Unice et al, 2012; Schroeder, 1967). Legumes like alfalfa and clover are the principle sources of cobalt in the natural diet of horses, and there can be widely varied concentrations of cobalt in these forages across different geographical locations in the United States and internationally (Kubota et al, 1987, Herbert, 1996). Deficient soils can result in profound deficiencies in cattle and sheep, resulting in anemia, weight loss and general unthriftiness.  Deficiencies in these species are likely under identified, but readily corrected by supplementation (Merck Veterinary Manual, accessed 1/22/15).  In contrast, deficiency has not been identified in horses.

Oral absorption of cobalt salts in humans varies depending on the dose, compound, nutritional status of the individual and proximity in time to a meal (Tvermoes et al., 2013).  For example, less than 5% of an oral dose of cobalt oxide is absorbed, compared with 30% of an oral dose of cobalt chloride, as determined from rodent studies. Increasing the dose of cobalt does not tend to cause significant accumulation as increasing doses result in a smaller proportion of the dose being absorbed. In humans, approximately 80% of cobalt is excreted in the urine and about 15% in the feces (Lison, 2007).  No studies evaluating the oral absorption of cobalt have been done in horses.

Why is cobalt used in athletes?

Human recombinant erythropoietin (EPO) became available in the 1990’s, and rapidly became adopted into covert doping programs for human athletes.  EPO stimulates the bone marrow to produce red blood cells, increasing the oxygen carrying capacity of the blood.

The ability to test for EPO was developed in the early 2000’s, curbing its abuse, but it was pointed out in the medical literature (Lippi et al., 2005, 2006) that cobalt, the original treatment used to increase human hematocrits in the treatment of anemia, also had potential to be used/misused in athletics in much the same way as administration of EPO. The potential illicit use of cobalt to improve athletic performance is based on the cellular actions of cobalt, leading to its recent and reportedly widespread use in horse racing (Paulick, 2014; Merkeberg, 2013).

A high dose of cobalt triggers a series of events which increases endogenous blood concentrations of EPO. Cobalt increases a protein in the cell called Hypoxia Inducible Factor (HIF-1α).  High intracellular HIF-1α causes direct activation of the erythropoietin gene resulting in increased plasma concentrations of erythropoietin, which would then drive the increase in red cell formation and an equivalent increase in the animal’s hematocrit (Semenza, 2014).  In addition to its EPO effects, other effects include increased blood supply to the muscle, and increased efficiency of energy utilization.

The theory is interesting but does this really work in horses?  The hematological effects at high levels of cobalt can be impressive:  in laboratory animals (Gluhcheva et al., 2011), dogs (Fisher, 1959) and humans (Lippi et al., 2006) chronic daily administration of large quantities of cobalt chloride have resulted in significant increases in red blood cell counts and hemoglobin.  Lower doses appear to have no hematological effect.  In horses, single doses of 22 mg or 49 mg of Co2+  (Kynch et al., 2014), or up to 449 mg of Co2+  (pers. Comm. Mary Scollay) had no effect on red blood cell parameters.   The consensus from the literature including multiple studies in multiple species is that chronic blood concentrations of 300 ppb and less are not associated with hematological or toxicological effects, whereas chronic concentrations in excess of 300 ppb are associated with both hematological and toxicological effects (Finley et al, 2012).

In racehorses, cobalt is typically administered a day or two before racing.  It seems unlikely that cobalt directed changes in EPO gene transcription or capillarity of muscles could be in effect at the time of the race if cobalt is administered only a few days before competition.  However, a small subset of horses, specifically among Standardbreds, seem to have impressive racing performances pursuant to Co2+ administration two days pre-race.  One possible mechanism of action of this cation is its effect as a calcium channel blocker (Simonsen et al., 2012).  Standardbreds are unique among the racing breeds in that they are susceptible to “tying-up” during racing (Isgren et al., 2010).  Other racing disciplines do not suffer this condition during racing, but may “tie-up” before or after racing.  The underlying mechanism of tying-up is unclear, except that it is probably a heritable condition (Collinder et al., 1997), and likely mediated by calcium channels in the muscles (Lopez et al., 1995).  Certainly some of the preventative therapies for tying-up involve calcium channel blockers, such as Dantrolene (Lopez et al., 1995) or another divalent cation, magnesium (Fenger, unpublished observation).  Since tying-up is prevalent among Standardbreds, and other preventative treatments for this condition are banned on raceday, the purported performance enhancing effect of Co2+ administration two days before racing may simply be prevention of tying-up.   

Why should cobalt be regulated?

Other than a possible mechanism to prevent tying-up, or muscle cramping in some horses, there appears to be no effect of cobalt on horses in the doses most commonly used.  On the other hand, there are many reports of cobalt toxicity in people.  In a mining town in Peru, excessive exposure caused chronic excessive red blood cell production in miners to the point of sludging of blood (Jefferson, 2002) and the US Environmental Protection Agency has set safety levels for cobalt for the protection of workers at high risk, such as mining and some industrial jobs. 

Typical adverse reactions to chronic cobalt administration in humans include nausea, vomiting, heart failure, low thyroid hormone levels and goiter, with neurological symptoms being reported less frequently (Jelkmann, 2012).  Large circulating concentrations of cobalt interfere with the uptake of iodine into the thyroid gland, resulting in low thyroid hormone levels.  In the late 1960’s, cobalt was added to beer as an anti-foaming agent, and resulted in a number of cases of heart failure. Cobalt is taken up in high concentrations into the heart muscle, likely in its role as a calcium channel blocker, and has been linked to an epidemic of heart failure in a group of heavy beer drinkers from the cobalt-containing beer (Alexander, 1972).  This specific syndrome was clearly multi-factorial, as the specific disease syndrome did not match any others associated with excessive cobalt.

It is likely that these chronic high doses which are required to cause thyroid dysfunction or cardiac failure are not achievable in horses.  However, at the extremely high doses that are suspected of being in use, horses can exhibit impressive adverse effects, such as tremors, sweating and colic (pers. comm Mary Scollay).  All of these effects are transient, and the horses appeared to be completely normal within a few hours.  Nonetheless, there has been suspicion that some incidents of sudden death on the racetrack have been associated with high serum cobalt levels, suggesting that there may be a relationship.

What are current regs?

Hong Kong has had a cobalt threshold longer than any other jurisdiction, and the ‘in-house’ urine cobalt threshold is 100 ng/mL.  A recent paper out of Hong Kong suggests 2 ppb as an appropriate threshold in plasma (Ho et al., 2014).  Among the criticisms of this threshold by the Hong Kong authors is that one of the study populations was a group of 109 horses in the Emirates.  This population races under strict security and yet 6 horses were considered to be outliers and had to be eliminated from the population before the balance of the group would fit the 2 ppb threshold.  Thresholds for naturally occurring substances must be carefully considered, and elimination of data must be done only where considerable investigation is performed. 

In Australia, Harness racing officials have introduced a urinary threshold of 200 ng/mL after samples in certain harness horses there reportedly exceeded 3,500 ng/mL (Bartley, 2014).  Similar to the Hong Kong analysis, Hibbert (2014) examined a group of post-race samples and identified a “natural break” in the data, ultimately requiring the researchers to eliminate 17 horses from a population of 80 in order to make the proposed threshold fit.  Again, the elimination of 20% of the population without further investigation into alternative explanations, such as feed or hay sources is inappropriate for establishing a threshold for which the penalty is on par with erythropoietin.

Indiana is the first US jurisdiction to regulate and implement a cobalt rule.   Indiana’s rule regulating cobalt establishes a threshold of 25 ppb.   The regulation that was implemented, by way of emergency rule, became effective October 1, 2014, for horses in competition and January 1, 2015, for out-of-competition testing.   Indiana’s cobalt rule states:  “Cobalt – not to exceed twenty-five (25) parts per billion of cobalt in serum or plasma.  A sample from a horse tested and found by the Commission’s primary lab to have cobalt in excess of this threshold shall be placed and remain on the veterinarian’s list until the concentration of cobalt in serum or plasma has fallen below the designated threshold.”   71 IAC 8-1-9(a)(3)   Indiana’s rule regulating cobalt makes a positive test a category “A” penalty, as established by the Recommended Penalties and Model Rule, regardless of its presence in a post-race or out-of-competition sample.    71 IAC-8-1-7(b)

Interestingly, the Indiana Horse Racing Commission has issued a supplemental notice relative to the new emergency rule on cobalt that suggests leniency for those receiving a positive test for cobalt wherein the positive detected level is more than 25 ppb but less than 50 ppb. 

Indiana’s cobalt rule for in-competition testing became effective during Indiana’s 2014 season and, significantly, with only weeks left in that season.   Despite that being the case, there have been positive test results for cobalt all of which, as of this date, have been/are for in-competition testing.

Several other states have been closely observing Indiana’s lead to regulate cobalt.   The State of California is one of those states.   In fact, the State of California’s Horse Racing Board recently discussed a proposal by its Medications and Track Safety Committee to add Board rules to regulate the use of cobalt.   In October of last year, California’s Equine Medical Director, Dr. Rick Arthur, addressed the issue before the State of California Horse Racing Board.   In doing so, Dr. Arthur noted a study that the Board had performed at Maddy Laboratory that examined the results of Cobalt Chloride at low doses.   Dr. Arthur reported that: “. . .  what was found is that after the first elimination where probably 80% of it is eliminated in the first 48 hours . . . it takes weeks to get rid of the rest of it . . . that’s actually good news because it allows us to set a threshold that would eliminate its use . . . .”   (State of California Horse Racing Board Meeting Minutes for October, 2014)   Based on Dr. Arthur’s report and other considerations, the Executive Director of the State of California’s Horse Racing Board,  Rick Baedecher, stated that the Board would pursue a rule regulating cobalt at: “ . . . the 25 nanogram level.   And if a horse is above that level (the horse) will be placed on the vet’s list until . . . it tests lower than the threshold . . . .”  (State of California Horse Racing Board Meeting Minutes for October, 2014)    Dr. Arthur, at the October, 2014 meeting of the Board, also recommended the proposed rule regulating cobalt include out-of-competition testing.

Another state contemplating a rule regulating cobalt is Minnesota.   In the Fall of last year the Minnesota Racing Commission discussed the status of cobalt regulation in racing.    The chief veterinarian of the Minnesota Racing Commission, Dr. Lynn Hovda, provided a briefing to the Commission.  Dr. Hovda discussed both Indiana and California’s efforts in this regard and the reason for needed regulation.   The reasons given are those that are normally cited with regard to regulating cobalt, being the potential for toxicity to equine athletes if Cobalt Chloride is administered at high levels and a second reason being that it is believed that excessive amounts  of Cobalt Chloride may be performance enhancing.  The Minnesota Racing Commission has been and continues to move forward with adopting a rule for regulating cobalt.   It will likely closely follow the Indiana rule.   One exception may be the threshold level.    Discussions have included setting the threshold level for cobalt between 50 and 70 ppb, slightly higher than Indiana’s current threshold of 25 ppb.   (Minnesota Horse Racing Commission Meeting Minutes for September and October, 2014)

On March 4, 2014, the California Horse Racing Board (CHRB) issued a notification that they were about to commence monitoring cobalt concentrations, and they implemented a regulatory threshold of 25 ppb.  California, similar to the Hong Kong and Australian studies performed preliminary survey studies, which also excluded outliers in order to achieve their threshold.  The trend is clear:  in order to set the regulatory thresholds, horses with higher cobalt levels must be eliminated from the study data because they fall outside what looks “right.”  This is hardly appropriate science, nor the appropriate method of regulating a multi-million dollar industry.

On May 20, 2014, the Maryland Racing Commission decided that Maryland will begin testing horses for cobalt. The chairman of the board indicated that there was ‘no definitive threshold’ concentration for cobalt at that time. There are also no specific rules in place in Maryland relating to cobalt, and no comments were made on how a horse presenting with high concentrations of cobalt might be addressed (Vespe, 2014). 

In the absence of a state regulation, the Meadowlands Racetrack owner, Gural implemented a “house rule” regarding cobalt, using the current Hong Kong threshold of 10 ppb, as an out of competition threshold.

There is marked confusion over the regulation of cobalt, because experts disagree on an appropriate thresholds (Popot, 2014).  Surveys of horses have resulted in the wholesale elimination of “outlier” horses based on the assumption that these horses must have been treated with exogenous cobalt.  In a study where different laboratories across the globe tested the same samples, the variation between testing laboratories was as high as 82% for the testing in serum and 23% in urine.  Because of the greater agreement between lab testing methods using urine, the international community has settled on a urine threshold of 100 mcg/mL raceday threshold for uniformity.

No dose-response studies have been performed to determine at what level cobalt has any effect on horses.  However, a review of the literature in the animals which have been studied indicates that a sustained cobalt level above 300 ppb is required for cobalt to exert its hematopoietic and other effects (Finley, 2012).  Current regulatory focus on thresholds have sought levels which reflect likely exogenous administration of a naturally occurring substance which may or may not have been with intent, and not levels which actually reflect any performance enhancing effect.  This lack of valid scientific basis for the cobalt thresholds which have been adopted has led to headlines and career-threatening accusations across the turf media.

Conclusions

The regulation of cobalt in North America has come about with great fanfare and headlines, but the science has yet to catch up.  The thresholds adopted fail to hold up to scientific scrutiny, and, like so many other regulations in this day and age, are more likely to trap innocent horsemen than those actually cheating.

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