The results of the present study provide the first in vivo evidence that chronic exposure to the KD increases concentrations of KYNA in discrete brain regions (i.e., the hippocampus and striatum, but not cortex) in rats. This finding proposes a potential mechanism involved in the therapeutic effects of the KD, especially in neuropsychiatric disorders associated with KYNA hypofunction.

The methodology of chronic exposure to the KD used in the present study resulted in the development of chronic ketosis with corresponding hypoglycemia. These metabolic changes were consistent with those reported when the KD was studied for its anticonvulsant properties in animals (Appleton and De Vivo 1973; Greene et al. 2001; Hartman et al. 2008; Samala et al. 2011). Also, KYNA concentrations in brain structures of animals when fed a regular diet in the present study corresponded with previously published data (Gramsbergen et al. 1992). In contrast to previous reports, however, there was no age-dependent increase in brain KYNA concentrations in young versus adult rats. This lack of difference was likely due to a small difference in age in the groups used in the present study (approximately, 5–7-week difference) in comparison to earlier studies (approximately, 18–21 months difference) that reported age-dependent increases in KYNA concentrations (Moroni et al. 1988; Gramsbergen et al. 1992).

In the present study, concentrations of KYNA in the KD-exposed rats increased 1.9- and 3.8-fold in the striatum and hippocampus in comparison to control rats, respectively; concentrations of KYNA in the cortex did not changed significantly. This differential effect suggests that the KD affects specific brain structures in a unique way rather than producing a global state of KYNA elevation. Alternatively, different brain regions may have different capacities and/or sensitivities to respond to the KD with resultant increases in KYNA concentrations. Of note, brain-specific changes in KYNA concentrations due to differential sensitivities of KATs to various modulators (e.g., metabolic substrates, pH, pharmacological inhibitors, or convulsive stimulations) have been previously reported (Moroni et al. 2005; Maciejak et al. 2009; Han et al. 2010; Szyndler et al. 2011).

The effects of external factors on KYNA concentrations in vivo have rarely been reported. Thus, it is difficult to compare the magnitude of the KD-induced increases in KYNA in the present study to responses produced by other means. Administration of kainic acid at doses that produced convulsions resulted in 200–500% increases in KYNA concentrations in the piriform cortex, amygdala, and cerebellum in rats (Baran et al. 1995). An approximately 50% increase in KYNA production was reported after incubation of rat cortical slices and primary glial cultures with β-hydroxybutyrate (Chmiel-Perzynska et al. 2011). Under similar experimental conditions, increases in KYNA production did not exceed 100% after incubations with several approved antiepileptic drugs (Kocki et al. 2006). Likewise, a reduction of Na+ concentration resulted in a 20–30% increase in KYNA production (Turski et al. 1989). Probably, the greatest increases in KYNA production in vitro (approximately, 155–170%) were reported during the incubation of neuronal and glial preparations with ammonia (Saran et al. 2004; Wejksza et al. 2006).

Since excessive increases in KYNA concentration may have detrimental behavioral effects on, for example, cognition or ambulatory activity (Vecsei and Beal 1990; Potter et al. 2010), it is important to note that the KD is generally well tolerated by the animals and no gross behavioral abnormalities have been reported. In contrast, exposure to the KD has been reported to produce generally positive effects on spontaneous or disease-altered behavioral outcomes (e.g., Murphy et al. 2004; Ziegler et al. 2005; Mantis et al. 2009; Ruskin et al. 2011). Thus, increases in KYNA concentrations produced by the KD do not appear to reach levels that would result in behavioral side effects.

The exact mechanism whereby an exposure to the KD would result in increases in KYNA concentrations is not known. In vitro studies demonstrated an increased activity of KYNA-producing KATs in cultured glial cells incubated with β-hydroxybutyrate; this effect, however, was not confirmed in cortical homogenates (Chmiel-Perzynska et al. 2011). Note that KYNA concentrations were not changed by the KD in the present study. Furthermore, KYNA production was enhanced in vitro by the addition of substrates that increase cellular metabolism (Hodgkins and Schwarcz 1998). Of note, exposure to the KD, among others, increases cellular metabolism (Bough et al. 2006), thus providing a potential mechanistic explanation of the KD-induced increases in KYNA concentrations reported here.

In summary, chronic exposure to the KD in rats resulted in several-fold increases in KYNA concentrations in discrete brain structures. The magnitude of these increases was generally greater than increases produced by any other modulators (e.g., antiepileptic drugs) studied so far under physiological conditions. Given the proposed role of KYNA in the pathophysiology of several neuropsychiatric disorders, further studies in this area are justified. Also, the development of small molecule-based approaches targeting the kynurenine pathway to increase KYNA (e.g., via the inhibition of kynurenine hydroxylase or kynureninase) remains at the stage of pre-clinical testing. Thus, the KD may offer an alternative and expedited way of obtaining the clinical proof-of-concept evidence to test whether exogenously induced increases in KYNA concentrations in the brain can translate into clinically significant improvements in neuropsychiatric diseases associated with KYNA hypofunction. Testing of this hypothesis is supported mechanistically, since an increased cellular energy metabolism has been implicated in the effects produced by the KD and as a factor enhancing KYNA production. Further support comes from a long history of clinical benefit of the KD in drug-resistant epilepsy syndromes and the proposed clinical utility of the KD in other neuropsychiatric disorders (Gasior et al. 2006; Freeman and Kossoff 2010).