Introduction

Epilepsy, or recurrent seizures, is reported to be the most common neurologic condition in dogs.^1,2 By definition, seizures are uncontrolled synchronous discharges of neurons that originate within the cerebral cortex.¹ Although seizures are common in dogs (prevalence is ∼0.5–5.7% of the canine population), there are only a limited number of successful treatment options in existence for canine patients.³ Of the dogs affected with seizures, 20–30% are considered to be pharmacoresistant, requiring more than anticonvulsant to maintain adequate seizure control.² Thus, alternative canine epilepsy treatments are needed.

Recent human literature indicates that the hormone melatonin may have anticonvulsant effects.^4–7 Melatonin is synthesized from tryptophan within the parenchyma cells of the pineal gland.⁶ Following its production in the brain, it is released into the blood and cerebrospinal fluid.⁸ Although the majority of melatonin in mammals is synthesized in brain pinealocytes, this hormone is also produced in astrocytes, glial cells, lymphocytes, retinal cells, the gut, testes, ovaries, placenta, and skin cells.⁹

The main function of melatonin is circadian rhythm regulation. However, recently, melatonin has been proved to have anticonvulsant effects on lab animals.^5,7,10–12 This hormone increases the brain gamma-aminobutyric acid concentration and the affinity for this neurotransmitter in the brain, reduces the excitatory effect of N-methyl-D-aspartate, prevents lipid peroxidation, and protects the brain from oxidative damage via hydroxyl and peroxyl radical scavenging.^{5–7,10,12,13} A study by Mevissen et al. showed that the use of melatonin in rats reduced the current threshold for inducing epileptic discharges; seizure duration and severity were reduced in these rats.¹¹ Yamamoto et al. showed that melatonin prevented cyanide-induced seizures in a population of mice.⁷ Meanwhile, Kabuto et al. showed that certain doses of melatonin completely suppressed iron-induced epileptic discharges in rats as recorded with electroencephalography.¹⁴ Another study by Lapin et al. showed that in mice, melatonin had antagonistic properties against several exogenous and endogenous convulsants.⁵

In addition to these lab animal studies, there are several case reports in human medicine discussing the successful use of melatonin as an antiseizure therapy.^6,15 To the authors’ knowledge, there are no published studies of the use of melatonin to control seizures in the veterinary literature.

The central hypothesis of this study is that serum levels of melatonin will be lower in dogs with seizures than in normal dogs. The study’s aim will be to compare mean serum melatonin levels in normal dogs versus dogs with seizures. We hope to use the data obtained in this study to design future studies that examine the use of melatonin supplementation in animals with seizures.

Materials and Methods

Results

The majority of dogs in both groups had values that were not readable by melatonin assay (i.e., <0.5 pg/mL). In Group 1 (Table 1), there were no significant differences between the mean values in any of the three time points. Of the dogs in Group 1, nine dogs (31%) had at least one readable serum melatonin value; four of those dogs (44%) had a readable level at one time point, none of them (0%) at two time points, and five of them (56%) at all three time points.

TABLE 1 Readable Serum Melatonin Values in Normal Dogs (Group 1)

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In Group 2 (Table 2), there were no significant differences between mean values in any of the three times points. Of the dogs in Group 2, eight dogs (24%) had at least one readable serum melatonin value; three of those dogs (9%) had a readable level at one time point, one of them (3%) at two time points, and four of them (12%) at all three time points.

TABLE 2 Readable Serum Melatonin Values in Dogs with Seizures (Group 2)

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In Group 2, 25 dogs (76%) were medicated with at least one anticonvulsant at the time of the study. Of those 25, 20 (80%) did not have readable values of serum melatonin at any time point. Of those with readable values (Table 2), 1 (4%) had readable values at one time point, 1 (4%) at two time points, and 3 (12%) at all three time points.

When the groups were compared with respect to time of blood draw (hour of day and time of year) and length of time sample was frozen prior to performing assay, there were no significant differences between the groups. When comparing anticonvulsant therapy (presence of medication and type of medication), no significant difference in the distribution of readable melatonin values was found. Of 33 dogs in the seizure group, 19 were on phenobarbital, 9 on levetiracetam, 5 on potassium bromide, and 4 on zonisamide. When comparing prednisone therapy (presence of medication), no significant difference in the distribution of readable melatonin values was found.

Discussion

Canine serum melatonin levels have historically been reported with success in the literature.^16,17 A study by Dunlap et al. successfully measured serum melatonin in a group of sled dogs using a chloroform extraction method followed by RIA.¹⁷ Sampling in the Dunlap study was performed during early-morning hours: 2:00, 8:00, 10:30 a.m., and 5:00 p.m., during the summer and winter solstices in two different states (New York and Alaska).¹⁷ Mean serum values in the sled dogs living in New York were 1.76 ± 0.40 pg/mL at 10:30 a.m. versus 0.24 ± 0.36 pg/mL for dogs living in Alaska.¹⁷ In a separate study, serum melatonin values were measured over a 24 hr period in a single dog. All sampling was done in the month of August. In this study, the conditions in which the sample was obtained and the method of sample processing were not specified. All values obtained from this one dog were <0.3 pg/mL. Serum melatonin peaked at 2:00 a.m.; the values were lowest between 9:00 a.m. and 6:00 p.m.¹⁶

In our study, 76% of the dogs in Group 1 and 69% of dogs in Group 2 did not have readable values (serum level <0.5 pg/mL) on the melatonin RIA. We found that there was no significant difference between serum melatonin values in dogs with or without seizures. The extraction method used in our study and the actual RIA assay was identical to that used in the Dunlap study.¹⁷ We tested our extraction method and RIA on four pilot dogs who were housed in a dark room and supplemented with oral melatonin ∼20 min prior to an evening blood draw (performed at 9:00 p.m.). Each of these four dogs had recordable melatonin values, all reading >5000 pg/mL. Therefore, it would seem unlikely that the type of assay or extraction method was the source of the high numbers of unreadable values we encountered in study.

Time of blood draw, however, should be considered. In humans, it is well known that serum melatonin levels follow a circadian rhythm. A study by Sääf et al. showed that canine serum melatonin also follows a diurnal rhythm, remaining low during the day and peaking at 2:00 a.m..¹⁶ The study by Dunlap et al. also showed seasonal and diurnal changes in serum melatonin levels.¹⁷ Melatonin levels were higher at 2:00 a.m. and during winter months than during daytime hours. To be clinically relevant, we intentionally obtained serum samples at times when veterinary clinics would normally be fully operational (between 8:00 a.m. and 4:00 p.m.). As it was a clinical study, in order to get the desired sample size, we obtained blood throughout the entire calendar year. That being said, there was no statistically significant difference between groups when time of year or time of day of sampling was compared.

The assay used in our study was identical to that used in the study by Dunlap et al.; however, the sensitivity of our assay was 0.5 pg/mL, whereas it was 0.2 pg/mL in the Dunlap et al. study.¹⁷ In that study, dogs living in Alaska had mean serum melatonin values of 0.24 ± 0.36 pg/mL; this value would have been recorded as <0.5 pg/mL based on the sensitivity with which the RIA was performed in our study. The majority of our values were below the level of sensitivity for the RIA. Finally, the Dunlap et al. study indicated that only “selected means” were reported.¹⁷ Therefore, it may be that they only reported and calculated means from values that were successfully detected by the assay.

Another possible explanation for the low number of readable values obtained in our study could be the length of time samples were stored prior to RIA analysis. The cost of the assay in addition to its short expiration date dictated that samples had to be batched. As obtaining clinical case samples can take months, samples were stored in the freezer from anywhere between 6 and 12 mo prior to RIA analysis. In the four pilot dogs supplemented with oral melatonin, high serum melatonin levels were easily obtained. These pilot samples were frozen prior to analysis but for a shorter average time than most other samples analyzed in the study. Comparing freeze time in our study with the two previous canine studies is difficult, as the freeze time was not specified in either of the previous studies.^16,17

The effect of oral anticonvulsants and prednisone on serum melatonin levels was also considered to have a possible effect on serum melatonin values; 76% of seizure dogs in this study were on at least one anticonvulsant at the time of the study, whereas 38% were on prednisone. Melatonin is highly protein-bound in both rat and human plasma; between 60 and 78% of melatonin in these species is bound in plasma.¹⁸ The majority of popularly prescribed anticonvulsants alter protein binding and thus may alter melatonin levels. That being said, we found no significant differences in melatonin values in seizure dogs who were and were not currently taking an anticonvulsant at the time of the study. The use of concurrent anticonvulsant medications and prednisone therapy was assessed in Group 2 at the time of the study. Owners were specifically questioned about the use of anticonvulsants and steroids, as these specific medications are likely to be the most commonly used medications in a population of dogs with seizures.

It may be that a difference in serum melatonin values between seizure dogs and normal dogs does exist but at a level below the detectable range in the assay use. Several human studies indicate that serum melatonin values are transiently elevated directly following seizure activity but generally lower in patients with epilepsy.^4,19,20 If this data in human patients can be extrapolated to veterinary patients, there still may be use in supplementing seizure dogs with melatonin. It may be that increased levels of melatonin could affect seizure frequency and/or severity and clinically benefit canine seizure patients.

Conclusion

There were no notable differences in daytime serum melatonin values in normal dogs versus dogs with seizures. It was difficult to obtain readable values of serum melatonin during the daytime hours in dogs with and without seizures.