The effect of oral creatine supplementation on brain metabolite concentrations was investigated in gray matter, white matter, cerebellum, and thalamus of healthy young volunteers by means of quantitative localized proton magnetic resonance spectroscopy in vivo (2.0 T, stimulated echo acquisition mode sequence; repetition time = 6,000 ms, echo time = 20 ms, middle interval = 10 ms, automated spectral evaluation). Oral consumption of 4 × 5 g creatine-monohydrate/day for 4 wk yielded a statistically significant increase (8.7% corresponding to 0.6 mM,P < 0.001) of the mean concentration of total creatine (tCr) when averaged across brain regions and subjects (n = 6). The data revealed considerable intersubject variability (3.5–13.3%), with the smallest increases observed for the two male volunteers with the largest body weights. A regional analysis resulted in significant increases of tCr in gray matter (4.7%), white matter (11.5%), and cerebellum (5.4%) and was most pronounced in thalamus (14.6% corresponding to 1.0 mM). Other findings were significant decreases ofN-acetyl-containing compounds in cerebellum and thalamus as well as of choline-containing compounds in thalamus. All cerebral metabolic alterations caused by oral Cr were reversible, as evidenced by control measurements at least 3 mo after the diet. This work demonstrates that excess consumption of Cr yields regionally dependent increases of the tCr concentration in human brain over periods of several weeks.
- brain metabolism
- magnetic resonance spectroscopy
creatine (Cr) and phosphocreatine (PCr) play essential roles in the storage and transmission of phosphate-bound energy. In tissues with a high energy demand, the enzyme creatine kinase catalyzes the one-step reversible exchange of a phosphate group between Cr and ATP The creatine-kinase-PCr system serves both as a temporal buffer for ATP homeostasis when rates of synthesis are transiently exceeded by rates of consumption and as a spatial buffer by which PCr is efficiently transported from sites of ATP synthesis to sites of ATPases (11). In humans, the Cr pool is maintained by nutritional intake and de novo synthesis. The latter takes place in liver, kidney, pancreas, and to a smaller extent also in brain by means of arginine, glycine, andS-adenosyl-l-methionine as substrates, and arginine:glycine amidinotransferase and guanidinoacetate methyltransferase (GAMT) as enzymes (20). Tissue uptake of Cr transported via the blood into muscle and brain is accomplished with the use of a specific Cr transporter (6).
Oral supplementation of Cr has been shown to increase the Cr blood plasma level in a dose-dependent manner (10). Continuous consumption of Cr-monohydrate, e.g., 4 × 5 g/day for 5 days, elevated the total creatine (tCr) content in skeletal muscle by ∼20% and improved muscle performance during high-intensity exercise (1, 10) but not endurance exercise (2).
Studies of cerebral metabolism after oral Cr have been carried out in brain slices and animal models mainly to assess the putative neuroprotective potential ascribed to excess availability of Cr. For example, feeding pregnant mice with Cr and examining brainstem preparations of their offspring showed an almost twofold increase of PCr and, more importantly, higher PCr and ATP levels were maintained after anoxia in animals pretreated with Cr (21). Similar beneficial effects were observed in an animal model of Huntington’s disease (14). On the other hand, few studies addressed the effect of Cr consumption on the human brain. In a child with an inborn error of Cr biosynthesis and total absence of brain tCr, continuous supplementation of Cr (400 mg ⋅ kg−1 ⋅ day−1) markedly improved the clinical symptoms and rose cerebral tCr pools to ∼50% of normal values within 3 mo (18, 19).
The purpose of this work was to examine the extent of cerebral metabolite alterations induced by oral Cr in healthy human subjects under physiological conditions. Investigations involved follow-up proton magnetic resonance spectroscopy (MRS) examinations for up to 8 h after a single dose of Cr-monohydrate as well as in response to a chronic protocol of continuous Cr administration over a period of 4 wk.
All studies were conducted at 2.0 T with the standard imaging head coil (Siemens Magnetom Vision, Erlangen, Germany). Fully relaxed short-echo time proton MR spectra were acquired by means of a single-voxel stimulated echo acquisition mode localization sequence as described previously (7). Volumes of interest (VOI) were selected on the basis of three sets of orthogonal T1-weighted 3D gradient-echo images. As indicated in Fig. 1, the study included fourstandardized locations in paramedian parietal gray matter (12.5 ml VOI), parietooccipital white matter (4.1 ml VOI), central cerebellum (8.0 ml VOI), and deep gray matter mainly containing thalamus (5.1 ml VOI). Total duration of the examination was ∼70 min.
The experimental conditions were chosen to facilitate conversion of spectral resonance areas into metabolite concentrations by eliminating or minimizing the effects of T1 saturation and T2 attenuation. This particularly applied to the choice of repetition time (TR, 6,000 ms) and echo time [TE, 20 ms; middle interval (TM), 10 ms]. Typically, 64 accumulations resulted in an acquisition time of 6.5 min. Spectral evaluation was accomplished with use of LCModel, a user-independent fitting routine based on a library of calibrated model spectra of all individual compounds taking differences in coil loading into account (15-17). Major cerebral metabolites include the neuronal markers (3) N-acetylaspartate and N-acetylaspartylglutamate (tNAA), glutamate, glutamine, Cr and PCr, choline-containing compounds (Cho), the glial marker (4) myo-inositol (Ins), and glucose. Regional differences and age dependencies of pertinent metabolites in human brain have recently been addressed (16, 17). Concentrations are expressed as mmol/l VOI without corrections for CSF contributions and residual T2 relaxation effects. Ratios of metabolite concentrations were employed to test for changes in water content and/or cell density. Putative changes of the water content of the thalamus VOI were monitored by spectral integration (software supplied by the manufacturer) of the localized unsuppressed water resonance routinely acquired before metabolite MRS.
A total of 9 different young healthy volunteers (no medication, no previous Cr intake) participated in two separate studies designed to assess a short-term effect of a single dose of oral Cr (feasibility study) and a long-term effect after continuous consumption (main study). In all cases informed written consent was obtained before all examinations. Initially, each volunteer was studied twice in independent sessions to determine baseline values of metabolite concentrations. In the acute study, six subjects (4 female/2 male, age range 26–28 yr, mean 26.3 ± 1.4 years, mean wt 68 ± 6 kg) ingested a single dose of 20 g Cr-monohydrate (SKW, Trostberg, Germany) with proton MRS examinations 0.5, 2.5, 5.5, and 8.5 h after intake. Blood was drawn from one participant before and 2, 5, and 8 h after intake. The observed plasma Cr level increased from a basal value of 37 to 3,200 μM 2 h after consumption and subsequently decreased to 1,100 and 230 μM after 5 and 8 h, respectively. These findings are in agreement with a reported 20-fold increase 1 h after 5 g of Cr-monohydrate and a rapid clearance with a half-life of 1.5 h (10).
In the main study six subjects (4 female/2 male, age range 24–30 yr, mean age 26.8 ± 2.2 years, mean wt 70 ± 12 kg) received 4 × 5 g of Cr-monohydrate/day for 4 wk. Proton MRS was performed after 1, 2, 3, and 4 wk during continuous Cr intake and at least 3 mo after the end of the study. Blood was drawn from one volunteer before the start of the study, during several days of the first week of Cr ingestion, and before each MRS session to confirm the elevation of blood plasma levels in response to this protocol. The plasma Cr level increased from 50 μM to a mean value of 800 μM averaged over 4 wk.
To assess a putative increase of the brain tCr concentration after oral consumption, statistical comparisons were based on a one-sided pairedt-test. A two-sided pairedt-test was employed for all other metabolites.
Application of a single high dose of Cr-monohydrate revealed a trend for elevated tCr in human brain. Mean concentrations (averaged across subjects and time points) increased in gray matter (3.1%), white matter (3.1%), and thalamus (7.7%). Figures2 and 3 show the effect of 4 wk of continuous Cr consumption on localized proton MR spectra of a single subject in gray matter and thalamus, respectively. In both regions the elevation of the tCr methyl resonance is readily observable despite marked differences in spectral line widths caused by unavoidable magnetic field inhomogeneities in basal ganglia. Figure4 depicts time courses of mean tCr concentrations averaged across subjects for gray matter, white matter, cerebellum, and thalamus. Although most regions revealed a tendency for an early increase of brain tCr, the effects became more pronounced after 3 and 4 wk of Cr diet. As evidenced by control measurements performed at least 3 mo after the end of oral Cr, all cerebral metabolic alterations were reversible in all regions investigated.
Absolute metabolite concentrations before and after 4 wk of oral Cr are summarized in Tables 1 and2 emphasizing tCr increases in individual subjects and mean metabolite levels in the investigated brain regions, respectively. Despite pronounced intersubject differences, the mean tCr increase in human brain of 8.7% was highly statistically significant (P = 0.0008). Within experimental accuracy, the mean brain concentrations of tNAA (8.7 vs. 8.4 mM), Cho (1.7 vs. 1.6 mM), and Ins (4.3 vs. 4.4 mM) remained unchanged.
Although of lower statistical significance, Table 2 reveals regional differences of tCr increases yielding 4.7% (P = 0.05) in gray matter, 11.5% (P = 0.02) in white matter, 5.4% (P = 0.03) in cerebellum, and 14.6% (P = 0.01) in thalamus. Other findings were a significant decrease of tNAA in cerebellum and thalamus as well as of Cho in thalamus after 4 wk of Cr. In contrast, however, the concentration ratios of tNAA/Cho (3.6 ± 0.6 vs. 3.7 ± 0.4 in cerebellum and 4.8 ± 0.4 vs. 5.0 ± 0.6 in thalamus), tNAA/Ins (1.6 ± 0.2 vs. 1.4 ± 0.1 in cerebellum and 2.6 ± 0.2 vs. 2.6 ± 0.6 in thalamus), and Cho/Ins (0.55 ± 0.07 vs. 0.53 ± 0.15 in thalamus) showed no significant changes. Moreover, the brain water concentration in thalamus did not change in response to oral Cr. Taking the mean value before Cr as a reference (1.00 ± 0.06), the relative water concentration after 4 wk of Cr was 0.99 ± 0.06.
The main finding of this work is that excess oral Cr consumption increases the physiological concentration of tCr in the intact human brain. The observation of completely reversible effects on cerebral metabolite levels over periods of several weeks confirms and extends previous studies of Cr uptake in brain slices (5) and animal models of brain disorders (14, 21) as well as sparse reports of therapeutic Cr supplementation in children with mitochondrial encephelopathy, lactic acidosis, and strokelike episodes (MELAS) (8) and GAMT deficiency (18,19). In the latter case, the observation of a 50% replenishment of normal tCr levels in gray and white matter within 3 mo of oral Cr is in accordance with present indications that cerebral uptake of Cr (and washout of excess Cr) seems to span time periods of several weeks.
The present study reveals considerable intersubject variability of total brain tCr increases. Although the number of subjects is too low for unambiguous correlations with gender, age, and body weight, the smallest effects were observed for the two male subjects, who were the oldest and heaviest participants.
The observed alteration of brain tCr is not homogeneous but shows a regional dependency with mild percentage increases in gray matter and cerebellum and strong increases in white matter and thalamus. In absolute concentrations tCr increased by 0.3–0.5 mmol/l VOI in gray matter, white matter, and cerebellum but by 1.0 mmol/l VOI in thalamus. In general, one might expect that cerebral Cr uptake is most pronounced in regions with high energy demands, such as cerebellum and thalamus, which have been shown to be rich in creatine kinase (11, 13) and the specific Cr transporter (9). However, any assessment of tCr increases is complicated by the existence of two different Cr pools in brain (12). Because only one of these compartments has been shown to be accessible by the Cr analog β-guanidinopropionic acid (GPA) and connected to the Cr transporter, the GPA-inaccessible pool seems to be supplied by de novo synthesis. Thus without detailed knowledge about the relative contributions of these pools in different brain systems, it is impossible to accurately estimate the overall uptake capacity in a particular region.
Apart from changes in tCr, the data indicate reduced tNAA in cerebellum and thalamus. Because this finding is not supported by changes in the respective tNAA/Cho and tNAA/Ins concentration ratios, it is unlikely to represent a specific metabolic alteration. Instead, the data seem to suggest an increase of the brain water content or cerebrospinal fluid contribution in thalamus, as these effects would result in a lower cell density decreasing the observable metabolite concentrations in the selected VOI. Although Cr ingestion has previously been shown to cause a gain in body weight ascribed to increased tissue water content in muscle (2), the tentative assignment of the reduced tNAA level to an osmotic effect in the brain region with the strongest tCr elevation is contradicted by the fact that no change of the thalamic water concentration could be detected by a direct proton MRS analysis of the water signal. In contrast to the tNAA findings, the altered tCr/Cho and tCr/Ins concentration ratios in thalamus are even more enhanced (29.2% and 24.5%, respectively) than the tCr concentration and therefore indicate a true modulation of cellular composition or metabolism.
The present findings in humans together with the neuroprotective function of Cr and PCr suggested in animals broaden the basis for new therapeutic applications of Cr supplementation. So far, a benefit of excess Cr has been reported in a patient with GAMT deficiency (18, 19) and in animal studies of hypoxic and hypoxic-ischemic events (14, 21). The effect is ascribed to increased PCr, which buffers the cellular ATP concentration and delays its depletion under situations of energy compromise. Thus oral administration of Cr may contribute to maintaining cellular integrity and normal brain function.
Ongoing clinical trials in our institution involve the use of Cr after cardiac surgery with extracorporal oxygenation, in preterm infants, and in children with mitochondrial disorders. Notwithstanding these promising possibilities, additional studies are needed to further detail the physiological effects of elevated brain tCr. This particularly applies to the tNAA findings and the observation of variations among different brain regions. Current knowledge does not provide enough evidence to demonstrate either a beneficial or a harmful effect of increased tCr in the normal human brain.
The authors thank X. Mao for competent assistance in data analysis and F. Langer for statistical advice.
Address for reprint requests and other correspondence: J. Frahm, Biomedizinische NMR Forschungs GmbH, D-37070 Göttingen, Germany (E-mail:).
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- Copyright © 1999 the American Physiological Society