Timothy J. Maher

Changes in Brain Levels of Acidic, Basic, and Neutral Amino Acids After Consumption of Single Meals Containing Various Proportions of Protein

Abstract: Rats fasted overnight were allowed to consume single meals containing 0, 18, or 40% protein or continued to fast; after 2 h, brains and sera were taken and assayed for various amino acids. In general, serum levels of most amino acids were reduced by the 0% protein meal and elevated by the high‐protein meal when compared with those associated with fasting conditions. Exceptions were those not diminished by the 0% protein meal (tryptophan, methionine, proline) and those increased (alanine) or decreased (glycine) by all of the test meals. Amino acids exhibiting the broadest normal ranges (estimated by comparing their serum levels after 40% protein with those after 0% protein) were tyrosine, leucine, valine, isoleucine, and proline; serum lysine and histidine, two basic amino acids, also varied more than threefold. Brain levels of lysine, histidine, and some of the large neutral amino acids (LNAAs) also exhibited clear relationships to the protein content of the test meal: those of valine, leucine, and isoleucine were depressed by the 0% protein but increased (compared with 0% protein) when protein was added to the meal: brain tyrosine was increased by all of the test meals in proportion to their protein contents; tryptophan, phenylalanine, and glutamate were increased after the 0% protein meal but not by protein‐containing meals; brain lysine, histidine, and methionine were increased after the high‐protein meal, and brain alanine was increased slightly by all of the meals. For each of the LNAAs, significant correlations were observed between its brain level in any animal and the ratio of its serum concentration to the sum of the concentrations of its LNAA competitors (for blood‐brain barrier transport). For valine, tyrosine, lysine, and histidine, significant correlations were obtained between their brain and serum levels.

Restorative effects of uridine plus docosahexaenoic acid in a rat model of Parkinson's disease.

Administering uridine-5-monophosphate (UMP) and docosahexaenoic acid (DHA) increases synaptic membranes (as characterized by pre- and post-synaptic proteins) and dendritic spines in rodents. We examined their effects on rotational behavior and dopaminergic markers in rats with partial unilateral 6-hydroxydopamine (6-OHDA)-induced striatal lesions. Rats receiving UMP, DHA, both, or neither, daily, and intrastriatal 6-OHDA 3 days after treatment onset, were tested for d-amphetamine-induced rotational behavior and dopaminergic markers after 24 and 28 days, respectively. UMP/DHA treatment reduced ipsilateral rotations by 57% and significantly elevated striatal dopamine, tyrosine hydroxylase (TH) activity, TH protein and synapsin-1 on the lesioned side. Hence, giving uridine and DHA may partially restore dopaminergic neurotransmission in this model of Parkinsons disease.

L-threonine administration increases glycine concentrations in the rat central nervous system

Abstract Intraperitoneal administration of L-threonine increased the glycine and threonine concentrations in rat spinal cord. Glycine contents also increased in synaptosomes prepared from spinal cords from threonine-pretreated animals. These findings suggest that plasma threonine concentrations normally might affect production of glycine by central nervous system neurons, and also that exogenous threonine might be useful in modifying glycinergic transmission.

Oral l-glutamine increases GABA levels in striatal tissue and extracellular fluid

We explored the possibility that circulating glutamine affects γ‐aminobutyric acid (GABA) levels in rat striatal tissue and GABA concentrations in striatal extracellular fluid (ECF). Striatal microdialy‐sates, each collected over a 20 min interval, were obtained after no treatment, oral L‐glutamine (0.5 g/kg), or glutamine followed by NMDA (administered via the microdialysis probe). GABA concentrations were measured by HPLC using a stable OPA/sulfite precolumn derivatization and an electrochemical detection method. L‐Glutamine administration significantly increased ECF GABA concentrations by 30%, and en‐hanced the response evoked by NMDA alone (70%) to 120% over baseline (all P<0.05). Striatal GABA levels increased significantly 2.5 h after oral L‐glutamine (e.g., from 1.76 ± 0.04 μmol/g in vehicle‐treated rats to 2.00 ± 0.15 μmol/g in those receiving 2.0 g/kg of glutamine). Striatal glutamine levels also increased significantly, but not those of glutamate. These data suggest that GABA synthesis in, and release from, rat striatum may be regulated in part by circulating glu‐tamine. Hence, glutamine administration may provide a useful adjunct for treating disorders (e.g., anxiety, seizures) when enhanced GABAergic transmission is desired. Moreover, the elevation in plasma and brain glutamine associated with hepatic failure may, by increasing brain GABA release, produce some of the manifestations of hepatic encephalopathy.—Wang, L., Maher, T. J., Wurtman, R. J. Oral L‐glutamine increases GABA levels in striatal tissue and extracellular fluid. FASEB J. 21, 1227–1232 (2007)

Plasma choline concentration varies with different dietary levels of vitamins B 6 , B 12 and folic acid in rats maintained on choline-adequate diets

Choline is an important component of the human diet and is required for the endogenous synthesis of choline-containing phospholipids, acetylcholine and betaine. Choline can also be synthesised de novo by the sequential methylation of phosphatidylethanolamine to phosphatidylcholine. Vitamins B6, B12 and folate can enhance methylation capacity and therefore could influence choline availability not only by increasing endogenous choline synthesis but also by reducing choline utilisation. In the present experiment, we determined whether combined supplementation of these B vitamins affects plasma choline concentration in a rat model of mild B vitamin deficiency which shows moderate increases in plasma homocysteine. To this end, we measured plasma choline and homocysteine concentrations in rats that had consumed a B vitamin-poor diet for 4 weeks after which they were either continued on the B vitamin-poor diet or switched to a B vitamin-enriched diet for another 4 weeks. Both diets contained recommended amounts of choline. Rats receiving the B vitamin-enriched diet showed higher plasma choline and lower plasma homocysteine concentrations as compared to rats that were continued on the B vitamin-poor diet. These data underline the interdependence between dietary B vitamins and plasma choline concentration, possibly via the combined effects of the three B vitamins on methylation capacity.

5-Hydroxy-L-tryptophan suppresses food intake in food-deprived and stressed rats

Giving L-tryptophan, serotonins circulating precursor, or a serotonin-releasing drug can decrease food intake and body weight. Giving 5-hydroxy-L-tryptophan (5-HTP), serotonins immediate intracellular precursor, has been thought to be ineffective in enhancing brain serotonin synthesis unless it is coadministered with a dopa decarboxylase inhibitor to protect 5-HTP from destruction outside the brain. We have examined the effect of 5-HTP on food consumption and tissue 5-HTP levels among rats subjected to two different hyperphagic stimuli, food deprivation and a standardized stress (tail pinch), and on plasma 5-HTP levels in humans. In rats, 5-HTP (3-200 mg/kg ip) suppressed food intake in a dose-dependent manner in both models, but was at least eight times more effective in our stress-hyperphagia model. (Differences in the two procedures might have contributed to the observed differences in potencies.) This suppression was blocked by coadministration of another large neutral amino acid (LNAA), L-valine. Brain 5-HTP levels correlated significantly with peak plasma 5-HTP (r(2)=.69) or 5-HTP/LNAA (r(2)=.81) levels. Additionally, among humans, oral 5-HTP (1.2-2.0 mg/kg) produced, after 1 and 2 h, a significant increase in plasma 5-HTP (1.5- to 2.3-fold). These observations suggest that 5-HTP may be useful in controlling the excessive food intake sometimes generated by stress, even if given without decarboxylase inhibitors or other drugs.

Tyrosine's pressor effect in hypotensive rats is not mediated by tyramine

Tyrosine, the amino acid precursor of catecholamines, increases blood pressure (BP) in hemorrhaged hypotensive rats. Since tyrosine may also be decarboxylated to form tyramine, which releases norepinephrine from sympathetic terminals, we tested the hypothesis that tyramine formation might mediate tyrosines ability to increase BP. Three lines of evidence indicate that tyrosine does not act via this mechanism: pretreatment with reserpine blocked tyramines but not tyrosines pressor activity; pretreatment with hexamethonium left tyramines effect intact but blocked the pressor response to tyrosine; and plasma tyramine did not increase after an hemodynamically-active dose of tyrosine (100 mg/kg).

Tyrosine's vasoactive effect in the dog shock model depends on the animal's starting blood pressure

We examined the effect of tyrosine (10–200 mg/kg given intravenously) or placebo on blood pressure (BP) in dogs made hypotensive (systolic BP=50 mm Hg) by bleeding one hour previously. Animals which, prior to induction of hypotension, had been normotensive (mean arterial pressures, [MAP]≦145 mm Hg) subsequently exhibited a dose-related increase in BP after tyrosine administration. In contrast, dogs which had beenhypertensive prior to bleeding exhibited afall in BP after tyrosine. These observations indicated that prior cardiovascular status may be an important factor influencing responses to exogenous tyrosine, and to endogenous catecholamines produced from the tyrosine.

Effects of running the Boston marathon on plasma concentrations of large neutral amino acids

Plasma large neutral amino acid concentrations were measured in thirty-seven subjects before and after completing the Boston Marathon. Concentrations of tyrosine, phenylalanine, and methionine increased, as did their “plasma ratios” (i.e., the ratio of each amino acids concentration to the summed plasma concentrations of the other large neutral amino acids which compete with it for brain uptake). No changes were noted in the plasma concentrations of tryptophan, leucine, isoleucine, nor valine; however, the “plasma ratios” of valine, leucine, and isoleucine all decreased. These changes in plasma amino acid patterns may influence neurotransmitter synthesis.

Use of a Hemoglobin-Trapping Approach in the Determination of Nitric Oxide in In Vitro and In Vivo Systems

We describe methods for measuring the release of nitric oxide (NO) derived from organic nitrates in vitro, using triple wavelength and difference spectrophotometry in the presence and absence of concentric microdialysis probes. These methods are based on the ability of NO to oxidize oxyhemoglobin (OxyHb) to methemoglobin (MetHb) quantitatively in aqueous solution. Isosorbide dinitrate (ISDN), a thiol-dependent organic nitrate, increased MetHb concentration in 45 min from 2.47 ± 0.47 to 4.15 ± 0.12 μM (p < 0.05) and decreased OxyHb concentration from 2.13 ± 0.35 to 0.33 ± 0.26 μM (p < 0.05) at 37°C. At 27°C, the OxyHb concentration was not significantly altered (2.04 ± 0.23 to 1.60 ± 0.04 μM) by ISDN, nor was the MetHb concentration (from 2.68 ± 0.50 to 2.59 ± 0.25 μM). Sodium nitroprusside (SNP), a thiol-independent organic nitrate, increased MetHb concentrations in 30 min from 4.21 ± 0.26 to 6.00 ± 0.56 μM (p < 0.05) at 37°C, and from 4.23 ± 0.39 to 5.90 ± 0.43 μM (p < 0.01) at 27°C. SNP also decreased OxyHb concentrations in 30 min from 1.99 ± 0.32 to 0.13 ± 0.12 μM (p < 0.01) at 37°C, and from 2.25 ± 0.31 to 0.13 ± 0.09 μM (p < 0.01) at 27°C. Difference spectrophometry indicated that 0.25-5 mM SNP significantly increased NO production in a dose-dependent fashion. This hemoglobin-trapping technique was also useful in quantifying the concentrations of NO released from SNP in aqueous solution in vitro, using concentric microdialysis probes. The NO concentration following exposure to SNP was 530 ± 50 nM, as determined using the difference spectrophotometric technique. To demonstrate the applicability of this technique to in vivo microdialysis, we implanted concentric microdialysis probes into hippocampus and cerebellum of conscious and anesthetized rats. Baseline NO concentrations in hippocampus of conscious and anesthetized rats were 11 ± 2 nM and 23 ± 9 nM, respectively, while in the cerebellum NO concentrations were 28 ± 9 nM and 41 ± 20 nM, respectively. These results demonstrate that microdialysis using a novel hemoglobin-trapping technique possesses adequate sensitivity to measure the NO levels produced from organic nitrates in aqueous solutions, and further document the applicability of this approach to in vivo systems.