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To the Editor:

We read with interest the article by Nylen et al. (1), “A Comparison of the Ability of a 4:1 Ketogenic Diet and a 6.3:1 Ketogenic Diet to Elevate Seizure Thresholds in Adult and Young Rats.” The authors show that the 6.3:1 ketogenic diet (KD) significantly elevated the intravenous pentylenetetrazol (PTZ) seizure threshold in the immature, but not in the adult rats. The authors proposed several explanations for the observed age-dependent results including differences in β-hydroxybutyrate levels. They also proposed an interesting hypothesis regarding differences in the metabolic pathway (blood acetone levels) between human and rat species as a possible mechanism of the action of the KD.

The authors challenge the usefulness of intravenous PTZ test to evaluate the effectiveness of KD, which we feel merits further discussion. The authors propose that differences in the weight of young animals on the KD artificially elevate the threshold dose of PTZ because they do not weigh as much as their control diet counterparts. With pregnant rats, Ramzen and Levy (2) reported that weight was not a modifying factor.

Nylen et al. (3) further reported, “The time period between the start of PTZ infusion and the onset of the seizure—in the adults were <60 s, and in young rats were <30 s.” The authors suggest therefore that calculating the dose (because it is a function of weight) becomes unnecessary. The rapid onset can be understood when considering the cardiac outputs in adult and juvenile rats as 112 ± 9 ml/min and 55 ± 4 ml/min, respectively. Because the blood volume of a rat is 65 ml/kg of body weight (4), the total blood volume in the rats used in the present study is reached in <11 s in the adult (300 g) and 5 s in the young (60 g) by using the reported infusion rate of 1 ml/min. With this rate, other parameters can become more important for PTZ to pass through the blood–brain barrier such as molecular weight, lipid solubility, serum concentration, and the plasma protein binding (5). Because the molecular weight of PTZ is 138.17, its structural features assure good lipid solubility, and its extent of protein-binding is <10% (6), we believe that the application of a slower infusion can make these characteristics of PTZ less relevant in the appearance of the first myoclonus/generalized seizure, such that the threshold dose may become a useful measurement.

Although we recognize that the infusion rate used in the present study is used by other investigators, we reemphasize that the isolated myoclonic jerks, followed by the clonic phase (the end point used for screening antiepileptic drugs for antiabsence effect) may not be discernible if the infusion rate is too high. Because of the facile physicochemical properties of the molecule, a slower rate can still afford a comparable achievement of blood, and therefore brain concentration for rapid equilibration into CNS tissue.

Finally, we suggest caution in attempting to compare the results from PTZ testing in rodents treated with the KD to the impact of KD in human epilepsies. Many discrepancies are found when one attempts to correlate pharmacologic screening data for AEDs by using the PTZ model in rodents to the utility of those AEDs in human epilepsy (7).

REFERENCES

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  2. REFERENCES
  • 1
    Nylen K, Likhodii S, Abdelmalik PA, et al. A comparison of the ability of a 4:1 ketogenic diet and a 6.3:1 ketogenic diet to elevate seizure thresholds in adult and young rats. Epilepsia 2005;46: 11981204.
  • 2
    Ramzan IM, Levy G. Kinetics of drug action in disease states. XV: Effect of pregnancy on the convulsive activity of pentylenetetrazol in rats. J Pharm Sci 1985;74: 12331235.
  • 3
    Delp MD, Evans MV, Duan C. Effects of aging on cardiac output, regional blood flow, and body composition in Fischer-344 rats. J Appl Physiol 1998;85: 18131822.
  • 4
    BVA/FRAME/RSPCA/UFAW Joint Working Group on Refinement. Removal of blood from laboratory mammals and birds: first report of the BVA/FRAME/RSPCA/UFAW Joint Working Group on Refinement. Lab Anim 1993;27: 122.
  • 5
    Pardridge WM. Blood-brain barrier genomics and the use of endogenous transporters to cause drug penetration into the brain. Curr Opin Drug Discov Dev 2003;6: 683691.
  • 6
    Ramzan IM, Levy G. Kinetics of drug action in disease states, XIV: Effect of infusion rate on pentylenetetrazol concentrations in serum, brain and cerebrospinal fluid of rats at onset of convulsions. J Pharmacol Exp Ther 1985;234: 624628.
  • 7
    Sankar R. Animal model of cortical dysplasia for screening candidate AEDs. Epilepsy Curr 2003;3: 67.