BHB rises with acute hyperketonemia
These data demonstrate that plasma BHB levels can be quickly increased with the acute infusion protocol used, rising from overnight conditions (0.20 ± 0.10 mm) to 2.12 ± 0.30 mm. These elevated plasma levels of BHB are higher than that reached after 48 h of fasting (1.67 ± 0.34 mm). During the hyperketonemic infusion, the tissue BHB increased by approximately 47%, from 0.16 ± 0.07–0.24 ± 0.04 mm. This may be compared with the 48 h fasted data (Pan et al. 2000a), which found the tissue BHB concentration to be 0.6 ± 0.26 mm, significantly correlated to the plasma BHB concentration with a slope of 0.26.
Methodologically, the present data acquired with the double adiabatic editing sequence represent an improvement over the previous method used to acquire the fasting data. This does not preclude comparison between the previous and present data; however, the present method has decreased variability, with the standard deviation in the (present) baseline lactate data at 0.09 mm, previously at 0.16 mm. However, a part of the variation in the 48 h fasting BHB data (0.60 ± 0.26 mm) is most likely due to inter-individual differences in the speed with which each subject adjusted to ketosis (by 72 h of fasting, the group measure of brain BHB reached 0.98 ± 0.16 mm). Thus a strict comparison of 48 h fasted and non-fasted data is complex. However, using an unpaired t-test, a direct comparison between the 48 h fasted and acute hyperketonemic data showed the latter to be significantly lower (p < 0.02) despite its higher plasma BHB level.
As measured by in vivo MR, the BHB signal represents both intra- and extracellular components. If the relaxation parameters and visibility of blood and CSF BHB are equivalent to that of brain, we can determine a lower bound to the brain BHB concentration. Using a CSF–blood partition factor of 0.15 (Lamers et al. 1987; measured under fasting conditions), at a concentration of 2.12 ± 0.3 mm and a non-brain volume contribution of less than 10%, at a minimum, the brain BHB would be 0.12 mm. This value is similar to the baseline BHB measurement at 0.16 ± 0.07 mm. However, if the relaxation values of blood BHB are shorter than those of tissue (due to the susceptibility effects of deoxyhemoglobin), and given the long echo time of this acquisition (Te = 148 ms), brain BHB will be higher than 0.12 mm. Thus the concentration of 0.24 mm represents an upper limit to the brain BHB.
Whether steady state was actually reached may be of concern in the physiological analysis of the data. Although there was some variability in the rapidity with which individual subjects reached steady state, all subjects reached their steady state BHB measurements within 45–60 min of the infusion start. This is consistent with earlier rodent data, which used ketone infusions of approximately 30 min to 1 h to reach steady state (Hawkins et al. 1971; Ruderman et al. 1974; Corddry et al. 1982). As demonstrated in Fig. 4(b), there is a suggestion of a slight rise in the brain BHB with time; however, given the error in the measurement, ad hoc comparisons of the intermediate time points to the final time point did not show any significant differences.
To evaluate the acquired data in terms of fasting induced changes to the monocarboxylic acid transporter, it is important to consider that the steady state BHB concentration, whether non-fasting or fasting, necessarily represents a balance of transport and oxidative clearance (Eqns 1 and 2).
Jin is the plasma to brain BHB flux, Jout is brain to plasma BHB flux, CMRbhb is the cerebral consumption rate of BHB, Bbhb is the brain concentration of BHB, Pbhb is the plasma concentration of BHB.
If induction is hypothesized not to occur in this reversible symmetric Michaelis–Menten model (i.e. neither Vmax nor Km change with fasting), the inward flux Jin in acute hyperketonemia should be close to that in the 2 day fasted case since similar Pbhb values were obtained. Thus, in order to explain the higher brain BHB in the fasted case, the clearance of BHB (through oxidation CMRkb and/or efflux Jout) would have to be lower. However, CMRbhb has been reported to be elevated in fasting by Owen et al. (1967) and by Hasselbalch et al. (1994, 1996), with the latter studies reporting that ketone oxidation more than doubled with fasting (8.1 ± 4.7 µm/kg/min non-fasted versus 20.0 ± 15.7 µm/kg/min fasted). Thus, if induction does not occur, this implies that Jout should decrease in the fasted case. However this is also unlikely, since the higher brain BHB concentrations imply that Jout must be increased in the fasted case relative to the acute hyperketonemic state. Notably, inclusion of a diffusion (net) efflux Jdiff=D(Bbhb − Pbhb) would similarly increase with elevated Bbhb. Thus, the presence of a higher brain BHB level in the fasted case compared with the non-fasted case suggests that transport of ketones is up-regulated in humans with fasting. These considerations can be analytically described by solving Eqn 2 (assuming a reversible symmetric Michaelis–Menten facilitated carrier model) to give the concentration of BHB at steady state (Eqn 3):
From Eqn 3 it is evident that without any changes in CMRbhb, the transport parameters Km or Vmax, for a given value of Pbhb, Bbhb is fixed.
A decrease in Jout can occur without alteration in transport parameters if an asymmetric model of BHB transport is used. Physiologically, this describes the case where the affinity for BHB is characterized by two values, on the luminal side, Km,l and on the abluminal side Km,a. In this model, the concentration of lactate may affect the value of Km,a since lactate may serve as a competitive inhibitor of BHB. As we had reported (Pan et al. 2000a), by the second day of fasting, lactate nearly doubled from a baseline of 0.69 ± 0.17 mm to 1.31 ± 0.26 mm. Thus, with the competing presence of lactate, Km,a would increase from its original value Km,a,o as in Eqn 4.
where Ki is the affinity of the transporter for lactate.
Equation 5 describes the brain BHB with a reversible asymmetric Michaelis–Menten model:
Equation 5 indicates that in order to explain a 2.5-fold higher Bbhb in the fasted case relative to the non-fasted case (0.6 mm, 0.24 mm, respectively) without altering other components, Km,a must increase by 2.5-fold. However, with literature values for the Ki binding of lactate to the MCT of approximately 1.5–2 mm (1.8 mm, Oldendorf 1973), a two-fold lactate increase (from 0.7 to 1.3 mm) can at most result in a 24% increase in brain BHB. Thus, competition from lactate appears to be insufficient to explain the 2.5-fold BHB rise under the fasting conditions.
The rodent studies by Gjedde and Crone 1975 have shown that the Km and apparent Vmax for ketone transport increase, rather than decrease, with fasting. Taken together with studies showing increased ketone metabolism with fasting, our finding of elevated Bbhb for similar Pbhb for the fasted relative to the non-fasted state provides strong evidence that the transport activity for BHB is increased with fasting.
Notably, the ratio of the tissue BHB concentrations between the fasted and non-fasted cases is 0.60/0.24, or approximately 2.5. This may be compared directly with the ratio of cerebral ketone consumption rates as cited above (Hasselbalch et al. 1994, 1996), at 20.0/8.1, or approximately 2.5. Such a correlation suggests that under these conditions and at this level of ketonemia, cerebral ketone consumption is a function of tissue ketone levels. Greater information as to the contribution of oxidative flux from ketones can be obtained through the infusion of labeled glucose and ketones.
No significant differences in tissue lactate were detected with the acute hyperketonemia. This differs from that seen from the fasting hyperketonemia, where tissue lactate rose from 0.69 ± 0.17 mm at baseline to 1.31 ± 0.26 mm after 48 h of fasting. If, in the fasted case, the elevation of lactate were due to metabolic displacement of glucose by ketones, the absence of an increase in cerebral lactate in the non-fasted case suggests that the oxidative flux by ketones is relatively small, consistent with limited ketone transport.
Under normal conditions, elevations in plasma BHB are accompanied by increases in plasma acetoacetate. However, in this initial study of brain BHB metabolism, we did not simultaneously infuse acetoacetate. As measured in rodents, the relative rate of transport between BHB and acetoacetate is not significantly different between fasted and non-fasted states (Hawkins et al. 1986). Thus, although the interconversion of BHB and acetoacetate has been reported to be fast (Barton 1973), to account for both ketones, these BHB values may be scaled to provide an upper bound measure of their combined transport.
These data imply that the way the brain handles BHB in fasting induced ketosis is not identical to that in acute hyperketonemia. Given that it is unlikely that ketone body oxidation is greater in the non-fasted compared to the fasted state, it is likely that transport is up-regulated in the fasted state, consistent with rodent data and Hasselbalch et al. 1995. Additional studies over a range of plasma ketone levels in the fasted and non-fasted state will provide data as to the values for the parameters of transport.