Table 1.

¹³C Isotopic turnover kinetics and quasi-steady state enrichments of amino acids in forebrain nerve terminals and homogenate and cortical tissue from anesthetized rats infused intravenously with [1,6-¹³C₂]glucose

		k (min−1)	τ1/2 (min)	ESS′ (%)a	r2	Ctot/NAA	Ctotb (μmol·g−1)	Fluxf (μmol·g−1·min−1)
GluC4	NT	0.0318*,## (0.0037)	21.8	48.5***,### (2.4)	0.97	0.976 (0.138)	10.3c (2.6)	0.33 (0.09)
	Hom	0.0487 (0.0063)	14.2	59.4$ (2.8)	0.94		12.4d (1.4)	0.60 (0.10)
	Ctx	0.0559 (0.0071)	12.4	64.0 (3.6)	0.93		13.3e (1.2)	0.74 (0.08)
GluC3	NT	0.0168 (0.0030)	41.4		0.97
	Hom	0.0116 (0.0041)	59.5		0.93
	Ctx	0.0209 (0.0038)	33.1		0.96
GABAC2	NT	0.0330 (0.0059)	21.0	36.4 (4.3)	0.92	0.458 (0.061)	4.8c (1.2)	0.16 (0.05)
	Ctx	0.0477 (0.0084)	14.5	41.7 (4.8)	0.89		2.5e (0.29)	0.12 (0.02)
GlnC4	Ctx	0.0268 (0.0054)	25.8	39.1 (3.7)	0.92		6.5 (0.74)
AspC3	NT	0.0261 (0.0132)	26.5	28.3*,## (11)	0.67	0.225 (0.092)	2.3c (0.94)
	Hom	0.0224 (0.0035)	31.0	41.6 (3.6)	0.96		NA
	Ctx	0.0257 (0.0050)	27.0	50.1 (7.4)	0.93		1.3e (0.51)

Turnover parameters (mean (SE)) were estimated by non-linear least squares analysis of amino acid ¹³C percentage enrichments vs. time data to a single exponential function using 12 to 15 rats per data set. Turnover half-times (τ_½) were calculated as τ_½ = ln(2)/k.

P values for rate constant (k) group comparisons were estimated from the probability distributions calculated by Monte-Carlo analysis(see Fig. 2) and multiplied by 3 to adjust for comparisons between the three groups (NT, Hom, Ctx). P values for ESS′ group comparisons were determined by one-way ANOVA with repeated measures and Tukey’s test to adjust for multiple comparisons. * P < 0.05, ** P < 0.001 and *** P < 0.0001 for comparisons of NT vs. Hom; # P < 0.05, ^## P < 0.001 and ^### P < 0.0001 for comparisons of NT vs. Ctx; $ P < 0.05 for comparisons of Hom vs. Ctx.

GluC4 glutamate-C4, GluC3 glutamate-C3, GABAC2 γ-aminobutyrate-C2, GlnC4 glutamine-C4, AspC3 aspartate-C3, NT nerve terminals, Hom homogenate, Ctx cortex, NA not available

^aE_ss′ is the apparent steady-state ¹³C percentage enrichment averaged over 60 and 120 min (n = 5 to 7). Amino acid ¹³C enrichments reflect excess above natural (1.1%) abundance, and were normalized by the respective animal’s plasma glucose-C1 enrichment before computing group means. Equivalency of plasma glucose-C1 and -C6 ¹³C enrichment was assumed.

^bC_tot is the respective total (¹²C+¹³C) amino acid (AA) concentration (mean (SD)) as measured for cerebral cortex or estimated relative to N-acetylaspartate (NAA) in the nerve terminals.

^cIn nerve terminals, [NAA] was assumed to be equal to the average cortical concentration (11.32 ± 2.17 μmol·g⁻¹) multiplied by the forebrain-to-cortex ratio for NAA (f^fb/ctx_NAA = 0.93) determined from Wang et al. [63].

^dIn homogenate, C_tot was calculated from the average cortical concentrations of glutamate (13.32 ± 1.22 μmol·g⁻¹) and GABA (2.50 ± 0.29 μmol·g⁻¹) multiplied by the respective forebrain-to-cortex ratio for glutamate (f^fb/ctx_Glu = 0.93) or GABA (f^fb/ctx_Gaba = 1.29) determined from Wang et al. [63]. NAA, which is present only in neurons, was assumed to be uniformly distributed in neuronal cytoplasm (see [48] for a discussion).

^eIn cortex, the respective amino acid concentrations (C_tot) were measured in the extract.

^fFlux was calculated as the product, k x C_tot. The flux value for homogenate was determined using amino acid concentrations in cerebral cortex, and is considered an upper limit.

Definitions: Glu_C4, glutamate-C4; Glu_C3, glutamate-C3; GABA_C2, γ-aminobutyrate-C2; Gln_C4, glutamine-C4; Asp_C3, aspartate-C3; NT, Nerve Terminals; Hom, Homogenate; Ctx, Cortex; NA, not available; P-values for rate constant (k) group comparisons were estimated by t-test of the probability distributions calculated by Monte-Carlo analysis (see Fig. 2) and multiplied by 3 to adjust for comparisons between the three groups (NT, Hom, Ctx). P-values for E_SS′ group comparisons were determined by one-way ANOVA with repeated measures and Tukey’s test to adjust for multiple comparisons. * P<0.05, ** P<0.001 and *** P<0.0001 for comparisons of NT vs. Hom; # P<0.05, ^## P<0.001 and ^### P<0.0001 for comparisons of NT vs. Ctx; $ P<0.05 for comparisons of Hom vs. Ctx.