Journal of Neurochemistry
`Raven Press, New York
`© 1987 International Society for Neurochemistry
`
`Evidence for the Participation of a Cytosolic NADP+(cid:173)
`Dependent Oxidoreductase in the Catabolism of
`')'-Hydroxybutyrate In Vivo
`
`Elaine E. Kaufman and Thomas Nelson
`
`Laboratory of Cerebral Metabolism, National Institute of Mental Health, U.S. Public Health Service,
`Department of Health and Human Services, Bethesda, Maryland, U.S.A.
`
`Abstract: The concentration of -y-hydroxybutyrate (GHB)
`in brain, kidney, and muscle as well as the clearance of [1-
`14C)GHB in plasma have been found to be altered by the
`administration of a number of metabolic intermediates and
`drugs that inhibit the NADP+ -dependent oxidoreductase,
`"GHB dehydrogenase," an enzyme that catalyzes the oxida(cid:173)
`tion of GHB to succinic semialdehyde. Administration of
`valproate, salicylate, and phenylacetate, all inhibitors of
`GHB dehydrogenase, significantly increased the concentra(cid:173)
`tion ofGHB in brain; salicylate increased GHB concentra(cid:173)
`tion in kidney, and <:r-ketoisocaproate increased GHB levels
`in kidney and muscle. The half-life of [l-14C]GHB in
`
`plasma was decreased by D-glucuronate, a compound that
`stimulates the oxidation of GHB by this enzyme and was
`increased by a competitive substrate of the enzyme, L-gulo(cid:173)
`nate. The results of these experiments suggest a role for
`GHB dehydrogenase in the regulation of tissue levels of en(cid:173)
`dogenous GHB. Key Words: -y-Hydroxybutyrate dehydro(cid:173)
`genase-Succinic semialdehyde dehydrogenase-o-Glu(cid:173)
`curonate-Sodium valproate. Kaufman E. E. and Nelson T.
`Evidence for the participation of a cytosolic NADP+ -depen(cid:173)
`dent oxidoreductase in the catabolism of -y-hydroxybutyr(cid:173)
`ate in vivo. J. Neurochem. 48, 1935-1941 ( 1987).
`
`Studies on the metabolic fate of /'-hydroxybutyric
`acid (GHB), a naturally occurring compound present
`in both brain (Roth and Giarman, 1969; Roth, 1970)
`and peripheral tissues (Nelson et al., 1981 ), have es(cid:173)
`tablished that this compound is largely disposed of by
`oxidation to C02 and water (Walkenstein et al., 1964 ).
`Furthermore, it has been found that most ofthe car(cid:173)
`bon skeleton enters the citric acid cycle as succinate
`(Doherty et al., 1975; Mohler et al., 1976) rather than
`as acetyl-CoA derived from ,6-oxidation as previously
`proposed by Walkenstein et al. (1964). The recent dis(cid:173)
`covery of a metabolic disease in which GHB and suc(cid:173)
`cinic semialdehyde (SSA) are markedly elevated in
`both blood and urine due to a block in SSA dehydro(cid:173)
`genase (Jakobs et al., 1981; Gibson et al., 1983) adds
`evidence to support a degradative pathway in which
`GHB is oxidized to SSA, which in tum is oxidized to
`succinate.
`These findings strongly suggest that the main degra(cid:173)
`dative pathway for GHB proceeds through the follow(cid:173)
`ing series of steps.
`
`SSA
`(1) GHB
`succinate
`(2) SSA
`(3) succinate___,___,___,___, C02 + H20
`
`The reactions in step (3) are catalyzed by the enzymes
`of the citric acid cycle and in step (2) by SSA dehydro(cid:173)
`genase, the enzyme that is either low or missing in pa(cid:173)
`tients with GHB aciduria (Gibson et al., 1983). Al(cid:173)
`though step ( 1) is always depicted as an essential part
`of this scheme, until recently an enzyme or enzymes
`that could catalyze this reaction had not been identi(cid:173)
`fied.
`We have reported the purification and characteriza(cid:173)
`tion of a cytosolic NADP+ -dependent oxidoreduc(cid:173)
`tase, "GHB dehydrogenase," which can catalyze the
`oxidation ofGHB to SSA (step 1) in vitro under con(cid:173)
`ditions that approximate those in the cytosol of both
`brain and some peripheral tissues (Kaufman et al.,
`1979; Kaufman and Nelson, 1981). The physical
`characteristics as well as substrate and inhibitor speci(cid:173)
`ficity of this enzyme indicate that the ability to cata-
`
`Received August 5, 1986; revised manuscript accepted December
`15, 1986.
`Address correspondence and reprint requests to Dr. E. E. Kauf(cid:173)
`man at Laboratory of Cerebral Metabolism, National Institute of
`
`Mental Health, 36/1A-05, 9000 Rockville Pike, Bethesda, MD
`20205 U.S.A.
`Abbreviations used: GHB, ')'-hydroxybutyric acid; SSA, succinic
`semialdehyde.
`
`1935
`
`Ranbaxy Ex. 1016
`IPR Petition - USP 8, 772,306
`
`

`
`1936
`
`E. E. KAUFMAN AND T. NELSON
`
`lyze the oxidation ofGHB may represent a previously
`unreported activity for the NADP+ -dependent oxi(cid:173)
`doreductase (EC 1.1.1.19), commonly known as D-
`glucuronate reductase (York et al,., 1 ?61 ).
`.
`Whether this enzyme plays a stgmficant role m the
`oxidation of GHB to SSA in vivo has been studied
`by the administration to animals of compounds th~t
`either inhibit or increase the activity of this enzyme m
`vitro. Determination of tissue levels ofGHB or of the
`half-life ofGHB in plasma was used to assess the effect
`of the inhibition of this enzyme on the catabolism of
`GHB in vivo. The results suggest that factors that
`affect the activity of this enzyme in vitro alter both
`tissue levels ofGHB and the half-life ofGHB in vivo
`in directions consistent with their effects on the en(cid:173)
`zyme in vitro.
`
`MATERIALS AND METHODS
`Materials and animals
`Sodium a-ketoisocaproate, sodium phenylacetate, 'Y(cid:173)
`aminobutyric acid (GABA), and sodium GHB were pur(cid:173)
`chased from Sigma Chemical (St. Louis, MO, U.S.A.). Val(cid:173)
`proic aci4 was obtained from Saber Laboratories (~orf:on
`Grove, IL, U.S.A.) and salicylic acid from Fisher Scientific
`(Fairlawn, NJ, U.S.A.). Sprague-Dawley male rats weighing
`350-450 g were obtained from Taconic Farms (Ger(cid:173)
`mantown, NY, U.S.A.). [l-14C]GHB was obtained from
`Research Products
`International
`(Mount Prospect,
`IL, U.S.A.).
`
`Effects of inhibitors of GHB dehydrogenase
`In studies in which the animals were infused with solu(cid:173)
`tions of either a-ketoisocaproate, phenyl acetate, or 0.9%
`sodium chloride, a catheter was implanted in the femoral
`vein under light halothane anesthesia. The animals were al(cid:173)
`lowed to recover from the anesthesia for at least 2 h before
`the experiment was started. The solutions were infused for
`2 h intravenously at constant rates with a Harvard infusion
`pump, model number 600 (Harvard Apparatus, ~over,
`MA, U.S.A.). Immediately after the infusions the ammals
`were killed by decapitation and the brains, kidneys, and
`quadriceps muscle were removed and quickly frozen in liq(cid:173)
`uid nitrogen. The organs were stored at -80°C until assayed
`for GHB content as described by Nelson eta!. ( 1981 ).
`In the studies in which either salicylate or valproate was
`administered, the drug or physiological saline solution was
`administered intra peritoneally to the experimental and con(cid:173)
`trol animals, respectively. The animals were killed by decap(cid:173)
`itation I h after receiving salicylate and 2 h after valproate.
`Brain, kidney, and quadriceps muscle were rapidly frozen
`in liquid N2 and assayed for GHB as described above.
`
`Effects of D-glucuronate or L-gulonate on the t112 of
`(t-14C]GHB in plasma
`.
`Catheters were implanted in both the femoral vem and
`artery of Sprague-Dawley male rats under halothane anes(cid:173)
`thesia and the animals were allowed to recover from the sur(cid:173)
`gery and anesthesia for at least 2 h. The experiment w~s
`started by the injection into the venous catheter of approxi(cid:173)
`mately 4 ~Ci of[l-14C]GHB (sp act 4.5 ~Ci/~mol) in 0.4 ml
`of 0.9% NaCI. In one series of experiments o-glucuronate
`(333 mg) was administered (intravenously) as a bolus in sa(cid:173)
`line just prior to the start of the experiment followed by a
`
`J. Neurochem., Vol. 48, No.6, 1987
`
`constant infusion (556 mg/h) during the experiment. In a
`second series of experiments, L-gulonate (240 mg) was given
`as a bolus just prior to the start of the experiment followed
`by a constant infusion of 320 mg/h fo~ 3 h: Control r~ts
`received a matching bolus and constant mfus10n of physiO(cid:173)
`logical saline. Blood samples were wi~hdrawn at various
`times into heparinized tubes and centnfuged. The plasma
`was deproteinized by the addition of I 00 ~I of ice-cold abso(cid:173)
`lute ethanol to 50 ~1 of plasma. Sixty microliters of the de(cid:173)
`proteinized plasma were counted in Aquasol (New England
`Nuclear, Boston, MA, U.S.A.), and the remainder was used
`for paper chromatography. Chromatographic separation of
`GHB was carried out by spotting approximately 20 ~1 of the
`deproteinized plasma on Whatman 3MM Chr paper. The
`solvent system was ethanolfH20/NH40H, 98:2:1, by vol.
`The chromatogram was developed in the ascending direc(cid:173)
`tion for approximately 18 h, cut into !-em strips, and
`counted in a Packard Tri-carb scintillation counter, Model
`3375 (Packard Instrument, Downers Grove, IL, U.S.A.) in
`10 ml of Aquasol and I ml of H 20. The fraction oftotal of
`14C in each plasma sample that migrated with authentic
`GHB was measured and used to determine the concentra(cid:173)
`tion of [14C]GHB found in the plasma sample in the pres(cid:173)
`ence of radioactive metabolites of GHB. The plasma
`[14C]GHB concentrations were plotted on semilo~arithmic
`paper against time. The straight line that was obtamed after
`the initial equilibration with the tissues was extrapolated to
`zero time and used to calculate the t1;z for the disappearance
`ofGHB from plasma.
`
`Preparation and assay of GHB dehydrogenase and of
`SSA dehydrogenase
`GHB dehydrogenase was prepared from the livers of
`adult male golden Syrian hamsters as previously described
`by Kaufman et al. (1979). Inasmuch as the previou~ work
`on the kinetics of GHB dehydrogenase was done with the
`enzyme purified from hamster liver, it is important to note
`that the GHB dehydrogenase from rat brain and from rat
`kidney has been shown to cross-react with a.n antibody to
`the purified hamster liver enzyme (~np.ubhshed. resul~s).
`GHB dehydrogenase was assayed at 37 C m a reactiOn mix(cid:173)
`ture containing 0.08 M potassium phosphate, pH 7.6,
`0.0025 M NADP+, purified enzyme, and 0.01 M sodium
`GHB. Rat brain SSA dehydrogenase was prepared accord(cid:173)
`ing to Whittle and Turner ( 1978); the purific~tion .was car(cid:173)
`ried out through the ammonium sulfate fractiOnatiOn step,
`and was assayed as described by Whittle and Turner ( 1978).
`Rat brain and kidney cytosol (I 00,000 g supernatant frac(cid:173)
`tion) were prepared according to the method of Sokoloff and
`Kaufman ( 1961 ).
`
`RESULTS
`Effects of a-ketoisocaproate or phenyl acetate on
`tissue levels of GHB
`Phenylacetate and a-ketoisocaproate, metabolic
`products of phenylalanine and leucine, are potent in(cid:173)
`hibitors of GHB dehydrogenase (Table 1 ). If this en(cid:173)
`zyme plays a role in the disposition of GHB in viv?,
`then these inhibitors, when administered to an am(cid:173)
`mal, should decrease the rate of degradation of GHB
`and thereby increase the tissue level of GHB. T~e
`effects of infusions ofthese compounds are shown m
`Table 2. a-Ketoisocaproate produced a small but not
`
`

`
`REGULATION OF ENDOGENOUS 'Y-HYDROXYBUTYRATE
`
`1937
`
`TABLE 1. Comparison ofK1 values for compounds
`that are inhibitors of both GHB dehydrogenase
`and SSA dehydrogenase
`
`K; values
`
`Compound
`
`SSA dehydrogenase
`
`GHB dehydrogenase
`
`Valproate
`
`4.0 X 10-3 M•
`4.8 X 10-3 M•
`5.7 X 10-5 M
`1.15 X 10-4 M
`1.2 X 10-3 M
`Salicylate
`1.7 X 10-4 Mb
`1.7 X 10-3 M
`a-Ketoisocaproate
`5.0 X 10-4 Mb
`3.5 X 10-3 M
`Phenyl acetate
`a The K, of 4.8 X I o- 3 M for sodium valproate for SSA dehydroge(cid:173)
`nase is taken from Whittle and Turner ( 1978), and the value of 4.0
`X 10-3 Mfrom Maitre eta!. (1976).
`b The K; values of a-ketoisocaproate and phenyl acetate for GHB
`dehydrogenase are taken from Kaufman eta!. (1983).
`
`statistically significant increase in the level ofGHB in
`the brain. It did, however, produce a twofold increase
`in the level of GHB in both kidney and muscle. Phe(cid:173)
`nyl acetate, by contrast, increased the level ofGHB in
`brain by 2.4-fold but did not increase the level in kid(cid:173)
`ney or muscle.
`
`Effects of sodium valproate and sodium salicylate on
`tissue levels of GHB
`The drugs sodium salicylate and sodium valproate
`are also excellent inhibitors of GHB dehydrogenase
`(Table 1) and therefore might also be expected to alter
`tissue levels ofGHB. Administration (intraperitoneal)
`of sodium salicylate to rats produced a twofold in(cid:173)
`crease in the level of GHB in brain and a 1.5-fold in(cid:173)
`crease in kidney (Table 3). Sodium valproate pro(cid:173)
`duced a 1.4-fold increase in the GHB level in brain
`and a small but insignificant decrease in the kidney.
`Three of the four inhibitors that were used produced
`statistically significant increases in the concentration
`of GHB in brain. The fourth inhibitor, 'Y-ketoisoca(cid:173)
`proate, produced a small increase that did not reach
`statistical significance (Tables 2 and 3). These data
`support a role for GHB dehydrogenase in brain.
`In contrast to the effects of these inhibitors ofGHB
`
`dehydrogenase on GHB levels in brain, two of these
`did not increase GHB levels in kidney and quadriceps
`muscle and therefore raise questions about the contri(cid:173)
`bution of GHB dehydrogenase to the metabolism of
`GHB in these particular peripheral tissues. One expla(cid:173)
`nation would be that there are different concentra(cid:173)
`tions of valproate or phenyl acetate in the cytosol of
`these tissues. Secondly, the question of whether equal
`concentrations of these inhibitors would exhibit
`different magnitudes of inhibitory effects in these tis(cid:173)
`sues was addressed by testing the ability of these
`compounds to inhibit GHB dehydrogenase activity in
`rat brain cytosol and rat kidney cytosol. The inhibi(cid:173)
`tion found in cytosol was compared to that found with
`the purified enzyme (Table 4). All four inhibitors,
`when tested at two concentrations, produced a degree
`of inhibition of GHB dehydrogenase activity in the
`cytosol from both brain and kidney that was compara(cid:173)
`ble to that found with the purified enzyme (Table 4).
`These data provide further evidence of the identity of
`the enzyme in rat brain cytosol and in rat kidney cyto(cid:173)
`sol with purified hamster liver enzyme, an identity
`previously established by titration with an antibody to
`the purified enzyme. Finally, it should be noted that
`since tissue levels represent a balance between synthe(cid:173)
`sis and degradation, an effect of these compounds on
`the rate of synthesis could either magnify or obscure
`an effect on the rate of degradation.
`
`Effects ofD-glucuronate and L-gulonate on the t 112 of
`(l-14qGHB in plasma
`Since the results obtained by examining the effects
`of inhibitors ofGHB dehydrogenase on the tissue con(cid:173)
`centrations ofGHB in specific peripheral tissues such
`as kidney were not conclusive, a method that would
`measure the sum ofGHB metabolism occurring in the
`whole animal, namely, a measure ofthe t 112 ofGHB
`in plasma, was also included.
`In vitro experiments in which the oxidation ofGHB
`was coupled to the reduction of D-glucuronate dem(cid:173)
`onstrated that the addition of D-glucuronate mark(cid:173)
`edly accelerates the oxidation of GHB catalyzed by
`GHB dehydrogenase under conditions approximat-
`
`TABLE 2. Effects of a-ketoisocaproate and of phenyl acetate on tissue levels ofGHB
`
`Saline
`infusion
`
`nmolGHB/g
`tissue
`
`2.6 ± 0.3 (5)
`27.8 ± 3.2 (5)
`22.2 ± 3.0 (6)
`
`Tissue
`
`Brain
`Kidney
`Muscle
`
`a-Ketoisocaproate infusion
`
`Phenyl acetate infusion
`
`nmolGHB/g
`tissue
`
`3.2 ± 0.1 (3)
`55.0 ± 8.2 (4)"
`46.4 ± 9.9 (4)"
`
`Percent of control
`
`123
`198
`209
`
`nmolGHB/g
`tissue
`
`6.1 ± 1.0 (4)h
`18.4 ± 1.3 (4)"
`16.7 ± 7.1 (4)
`
`Percent of control
`
`235
`66
`75
`
`Phenyl acetate ( 1.0 M) was given intravenously as an initial bolus of 1.5 ml followed by a constant infusion of ;:;:2.0 ml/h for 2 h.
`£~<-Ketoisocaproate (0.5 M) was given intravenously as an initial bolus of0.6 ml followed by a constant infusion of;:;:2.0 ml/h for 2 h. At the
`end of the infusion the animals were killed and the tissues were removed and assayed for GHB as described under Materials and Methods. All
`values are means± SEM, numbers of animals in parentheses.
`a p < 0.05.
`bp<O.OI.
`
`J. Neurochern., Vol. 48, No.6, 1987
`
`

`
`1938
`
`E. E. KAUFMAN AND T. NELSON
`
`TABLE 3. Effects of salicylate and of va/proate on tissue levels ofGHB
`
`Saline
`(control)
`
`nmolGHB/g
`tissue
`
`2.9 ± 0.5 (6)
`2.6 ±0.2 (4)
`34.5 ± 5.7 (6)
`30.0 ± 4.1 (4)
`
`Tissue
`
`Brain
`
`Kidney
`
`Salicylate
`
`Valproate
`
`nmolGHB/g
`tissue
`
`5.8 ± 0.5 (4)b
`
`52.8 ± 2.5 (5)a
`
`Percent of control
`
`200
`
`153
`
`nmolGHB/g
`tissue
`
`3.7 ± 0.2 (4)b
`
`23.7 ± 3.8 (4)
`
`Percent of control
`
`142
`
`79
`
`Sodium valproate ( 100 mgjkg, i.p.) was given 2 h prior to decapitation; sodium salicylate (500 mgjkg, i.p.) was given 1 h prior to decapitation.
`Tissues were removed and assayed as described under Materials and Methods. All values are means± SEM, numbers of animals in parentheses.
`Qp<0.05.
`bp<O.Ol.
`
`ing those existing in the cytosol of brain, kidney, and
`muscle (Kaufman and Nelson, 1981 ). L-Gulonate, the
`product of o-glucuronate reduction, competes with
`GHB as a substrate for GHB dehydrogenase and
`therefore inhibits GHB oxidation. We have examined
`the effects of both of these compounds on the t1 12 of
`[1-14C]GHB in plasma and found that the administra(cid:173)
`tion ofo-glucuronate in vivo decreases the t112 by 33%
`whereas administration of L-gulonate increases it by
`33% (Fig. 1). The decrease in the t112 is consistent with
`an increased rate of oxidation ofGHB by GHB dehy(cid:173)
`drogenase in the presence ofo-glucuronate; similarly,
`the increase in t1 12 caused by L-gulonate suggests a de(cid:173)
`crease in the rate of oxidation of GHB. These results
`are consistent with our in vitro findings.
`
`Inhibition of SSA dehydrogenase and of GHB
`dehydrogenase by valproate, salicylate,
`a-ketoisocaproate, and phenyl acetate
`Since an increase in tissue levels ofGHB produced
`by phenyl acetate, a-ketoisocaproate, valproate, and
`salicylate could be attributed to either an increase in
`the rate of synthesis of GHB or to a decrease in its
`
`rate of degradation or to a combination of both, we
`examined the inhibition of both SSA dehydrogenase
`and GHB dehydrogenase by valproate, salicylate, a(cid:173)
`ketoisocaproate, and phenyl acetate. Inhibition of
`SSA dehydrogenase could theoretically lead to higher
`tissue levels of SSA and, therefore, an increased rate
`of synthesis of GHB, whereas inhibition of the GHB
`dehydrogenase activity could increase GHB levels di(cid:173)
`rectly. Valproate, salicylate, a-ketoisocaproate, and
`phenyl acetate are all competitive inhibitors for GHB
`dehydrogenase with GHB as the variable substrate
`(Kaufman et al., 1983). ~values for SSA dehydroge(cid:173)
`nase were similarly determined for salicylate, phenyl
`acetate, and a-ketoisocaproate with SSA as the vari(cid:173)
`able substrate. All of these compounds were competi(cid:173)
`tive inhibitors of SSA dehydrogenase (data not
`shown). Whittle and Turner ( 1978) have reported that
`sodium valproate is a noncompetitive inhibitor of
`SSA dehydrogenase.
`The ~ values for these compounds with both en(cid:173)
`zymes are given in Table 1. In every case the Ki of
`these inhibitors for GHB dehydrogenase was one to
`two orders of magnitude lower than that for SSA de(cid:173)
`hydrogenase.
`
`TABLE 4. Effects of inhibitors of purified NADP+ -dependent
`GHB dehydrogenase on the NADP+-dependent oxidation
`ofGHB in rat brain cytosol and in rat kidney cytosol
`
`Percent inhibition
`
`Inhibitor
`
`Concentration
`(rnM)
`
`Purified GHB
`dehydrogenase
`
`Rat brain Rat kidney
`cytosol
`cytosol
`
`a-Ketoisocaproate
`a-Ketoisocaproate
`
`Valproate
`
`Salicylate
`
`Phenyl acetate
`
`2
`5
`
`2
`5
`
`2
`5
`
`2
`5
`
`82
`96
`
`93
`100
`
`92
`94
`
`67
`93
`
`100
`100
`
`87
`100
`
`90
`100
`
`51
`72
`
`76
`93
`
`91
`100
`
`84
`91
`
`47
`84
`
`GHB dehydrogenase activity was assayed as described in Materials and Methods. In(cid:173)
`hibitors were added to the reaction mixture in the concentrations indicated in the table.
`
`J. Neurochem., Vol. 48, No.6, 1987
`
`

`
`REGULATION OF ENDOGENOUS 'Y-HYDROXYBUTYRATE
`
`1939
`
`10,000
`
`<(
`
`;:)5
`
`N
`
`<(
`...J
`"(cid:173)
`"-
`0
`~
`' m
`J:
`'-"
`"
`;;:
`
`(.)
`
`1,000
`
`100
`
`COMPOUND
`
`INFUSED
`
`SALINE
`D-GLUCURONATE
`L-GULONATE
`
`(nJ
`
`t 112 for[14c)GHB (mini
`mean :t S.E.M.
`60 ± 3.6
`40 ± 9
`•
`81 ± 0.5 * •
`
`(6)
`(2)
`(2)
`
`A·--···-.!> l-GULONATE
`
`~ NaCL
`s---s 0-GLUCURONATE
`
`0
`
`20
`
`40
`
`60
`
`80
`
`100
`
`120
`
`140
`
`160
`
`180
`
`200
`
`TIME (min)
`
`FIG. 1. Animals that received o-glucuronate received a bolus containing 240 mg followed by an infusion of 320 mgjh for 3 h. Animals
`receiving L-gulonate received a bolus of 333 mg and an infusion of 556 mg/h for 3 h. The t112 for the disappearance of [14C]GHB from
`plasma was determined as described under Materials and Methods. *p < 0.05; **p < 0.025.
`
`DISCUSSION
`
`This investigation was designed to determine
`whether GHB dehydrogenase, an enzyme that cata(cid:173)
`lyzes the oxidation of GHB to SSA in vitro, plays a
`significant role in the disposition ofGHB in vivo. We
`have also examined the possibility that the informa(cid:173)
`tion obtained in this study might provide a reason(cid:173)
`able explanation for the significant increases in GHB
`levels in brain brought about by the acute administra(cid:173)
`tion of sodium valproate and other drugs used in the
`treatment of petit mal epilepsy (Snead et al., 1980).
`This would be of interest since investigations of the
`inhibition of aminobutyrate aminotransferase (EC
`2.6.1.19), SSA dehydrogenase (EC 1.2.1.16), as well
`as an NADPH-dependent aldehyde reductase (EC
`1.1.1.2) by sodium valproate (Godin et al., 1969; Har(cid:173)
`vey et al., 1975; Sawaya et al., 1975; Whittle and
`Turner, 1978), have failed to produce a satisfactory
`explanation for the elevated GHB levels following an
`acute dose of sodium valproate.
`Marked inhibition of SSA dehydrogenase might be
`expected to lead to increased tissue levels of SSA and
`thereby to increased synthesis ofGHB. Indeed, in pa(cid:173)
`tients with GHB aciduria, a genetic disease in which
`this dehydrogenase is low or missing (Gibson et al.,
`1983), exactly such increases do occur. Little or no
`inhibition of SSA dehydrogenase would be expected
`
`to result from relatively high doses (100 mg/kg) of so(cid:173)
`dium valproate since the ~ of sodium valproate for
`SSA dehydrogenase has been reported to be 4.0 X 10-3
`Mby Maitre et al., (1976) or 4.8 X 10-3 Mby Whittle
`and Turner (1978). At tissue concentrations of 0.1
`rnM-1.0 mM, the highest concentrations likely to be
`found in clinical use (Sawaya et al., 1975), inhibition
`would vary from a negligible amount at 0.1 mM to
`12-15% inhibition at 1.0 mM.
`In vitro studies of GHB dehydrogenase (Kaufman
`et al., 1979) have provided the first clue to the meta(cid:173)
`bolic basis for the increased level ofGHB in brain fol(cid:173)
`lowing acute administration of sodium valproate
`(Snead et al., 1980). The activity of SSA dehydroge(cid:173)
`nase in the brain of the adult rat (Pitts and Quick,
`1967) is approximately 1,000 times greater than that
`of GHB dehydrogenase (Kaufman et al., 1979).
`Therefore, in the sequence of steps leading to the for(cid:173)
`mation of succinate from GHB, GHB dehydrogenase
`would catalyze the rate-limiting step. If GHB dehy(cid:173)
`drogenase is the first enzyme in one of the quantita(cid:173)
`tively significant pathways for the degradation of
`GHB, then administration of inhibitors of this en(cid:173)
`zyme might lead to increased tissue levels of GHB.
`Phenyl acetate, a-ketoisocaproate, valproate, and sali(cid:173)
`cylate, all potent inhibitors of GHB dehydrogenase
`with~ values in the range of w-4-10-s M, were se(cid:173)
`lected to test this hypothesis. Indeed, administration
`
`J. Neurochem., Vol. 48, No.6, 1987
`
`

`
`1940
`
`E. E. KAUFMAN AND T. NELSON
`
`of these compounds increased the endogenous con(cid:173)
`centration of GHB in one or more of the tissues that
`were assayed (Tables 2 and 3). The failure of a-keto(cid:173)
`isocaproate to increase brain levels significantly may
`indicate that it does not readily cross the blood-brain
`barrier.
`The effect of sodium valproate on SSA dehydroge(cid:173)
`nase and GHB dehydrogenase can now be compared
`under conditions seen in the clinical use of sodium
`valproate as an anticonvulsant drug. Sawaya et al.
`( 1975) have reported plasma concentrations of 1.1
`rnM-0.07 mM in clinical use. In addition, LOscher
`and Frey ( 1977) have reported that brain levels, in
`mice, are approximately 30% of the plasma levels. We
`can therefore calculate that, at a tissue concentration
`of 0.3 mM sodium valproate, GHB dehydrogenase
`would be 85% inhibited, a very marked inhibition
`compared to the 5% inhibition ofSSA dehydrogenase
`which would occur at a 0.3 rnMtissue concentration
`ofthe drug. 1
`Even at a tissue concentration of0.05 mM sodium
`valproate, a tissue concentration at which there would
`be essentially no inhibition of SSA dehydrogenase,
`GHB dehydrogenase still would be 48% inhibited. 1
`Inasmuch as SSA levels could be elevated if phenyl
`acetate, a-ketoisocaproate, or salicylate were potent
`inhibitors of SSA dehydrogenase, K;. values for these
`compounds were determined for SSA dehydrogenase.
`It is apparent from a comparison of the K; values (Ta(cid:173)
`ble l) that, like valproate, these compounds are also
`much better inhibitors of GHB dehydrogenase than
`of SSA dehydrogenase. In each case the K; for GHB
`dehydrogenase is such that if the tissue level of any of
`these compounds reached a concentration of 5-l 0
`X 10-4 M, there would be significant inhibition of that
`portion of the degradative pathway that goes through
`GHB dehydrogenase step with very little inhibition of
`SSA dehydrogenase.
`It is likely, therefore, that the increased tissue levels
`of GHB that follow the acute administration of val(cid:173)
`proate and of other compounds of which salicylate,
`a-ketoisocaproate, and phenyl acetate are examples,
`result from inhibition of the oxidation of GHB to
`SSA, the first step in the degradative pathway to succi(cid:173)
`nate and ultimately to C02 , rather than from the inhi(cid:173)
`bition of SSA dehydrogenase. The data presented in
`Tables 2 and 3 are consistent with a significant role
`for GHB dehydrogenase in the overall degradation of
`GHB in brain.
`
`1 The relative velocities with and without inhibitor were calcu(cid:173)
`lated from the Michaelis-Menten equation in which the Km term
`has been multiplied by the factor (I + (1]/ Ki) in the presence of a
`competitive inhibitor. In the case of SSA dehydrogenase an arbi(cid:173)
`trary value of I X 10-6 M was assumed for substrate concentration
`and 5 X 10-6 M for the Km for SSA at pH 7.6 (unpublished results
`from this laboratory). This value is similar to the Km of2.7 X 10-6
`for SSA at pH 8.4 found by Albers and Koval ( 1961 ). In the case of
`GHB dehydrogenase, values of 4.5 x 10-4 M for the Km for GHB
`were used. The K, values are those given in Table I.
`
`J. Neurochem., Vol. 48, No.6, 1987
`
`Another in vivo reflection of the action of com(cid:173)
`pounds that stimulate or inhibit the rate of a degrada(cid:173)
`tive enzyme such as GHB dehydrogenase would be a
`change in the t 112 for GHB in plasma after the admin(cid:173)
`istration of such compounds. This method has the ad(cid:173)
`vantage of examining the effect of such compounds
`on GHB metabolism in the whole body rather than in
`any one specific tissue; furthermore it can reveal
`effects on degradation uncomplicated by effects on
`synthesis since a trace dose of[ 14C]GHB is used to fol(cid:173)
`low the rate of degradation. In vitro, D-glucuronate
`markedly stimulated the GHB dehydrogenase-cata(cid:173)
`lyzed oxidation of GHB by participating in a coupled
`oxidation-reduction reaction (Kaufman and Nelson,
`1981 ). In vivo, the infusion of D-glucuronate led to a
`33% decrease in the half-life of GHB in plasma, a re(cid:173)
`sult consistent with an increased rate of degradation
`of GHB in body tissues. On the other hand, infusion
`ofL-gulonate, which we have shown to be a competi(cid:173)
`tive substrate of GHB for GHB dehydrogenase (un(cid:173)
`published data), produced a 33% increase in the half(cid:173)
`life for the plasma disappearance of GHB, indicating
`a decreased rate of degradation.
`Preliminary evidence from this laboratory indicates
`that in both brain and kidney there is at least one other
`enzyme, in addition to GHB dehydrogenase, that can
`convert GHB to SSA. The relative contribution of
`these two enzymes to the metabolism ofGHB has not
`yet been determined. However, both the increase in
`tissue levels of GHB following administration of in(cid:173)
`hibitors ofGHB dehydrogenase and the change in the
`t 112 for the plasma disappearance curve after infusion
`of either an inhibitor or a compound that stimulates
`the reaction are findings that are consistent with a role
`for GHB dehydrogenase in the overall degradation of
`GHBinvivo.
`
`Acknowledgment: We would like to thank Dr. Louis So(cid:173)
`koloff for his most generous help and support in carrying
`out this project. The expert technical assistance of John
`Kline is gratefully acknowledged.
`
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