Beyond sports: Efficacy and safety of creatine supplementation in pathological or paraphysiological conditions of brain and muscle.

Creatine is pivotal in energy metabolism of muscle and brain cells, both in physiological and in pathological conditions. Additionally, creatine facilitates the differentiation of muscle and neuronal cells. Evidence of effectiveness of creatine supplementation in improving several clinical conditions is now substantial, and we review it in this paper. In hereditary diseases where its synthesis is impaired, creatine has a disease-modifying capacity, especially when started soon after birth. Strong evidence, including a Cochrane meta-analysis, shows that it improves muscular strength and general well-being in muscular dystrophies. Significant evidence exists also of its effectiveness in secondary prevention of statin myopathy and of treatment-resistant depression in women. Vegetarians and vegans do not consume any dietary creatine and must synthesize all they need, spending most of their methylation capacity. Nevertheless, they have a lower muscular concentration of creatine. Creatine supplementation has proved effective in increasing muscular and neuropsychological performance in vegetarians or vegans and should, therefore, be recommended especially in those of them who are athletes, heavy-duty laborers or who undergo intense mental effort. Convincing evidence also exists of creatine effectiveness in muscular atrophy and sarcopenia in the elderly, and in brain energy shortage (mental fatigue, sleep deprivation, environmental hypoxia as in mountain climbing, and advanced age). Furthermore, we review more randomized, placebo-controlled trials showing that creatine supplementation is safe up to 20 g/d, with a possible caveat only in people with kidney disease. We trust that the evidence we review will be translated into clinical practice and will spur more research on these subjects.

Circulating Biomarkers in Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy Patients.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited cerebral microangiopathy presenting with variable features, including migraine, psychiatric disorders, stroke, and cognitive decline and variable disability. On neuroimaging, CADASIL is characterized by leukoencephalopathy, multiple lacunar infarcts, and microbleeds. Previous studies suggest a possible role of endothelial impairment in the pathogenesis of the disease. METHODS: We assessed plasma levels of von Willebrand factor (vWF) and thrombomodulin (TM) and the blood levels of endothelial progenitor cells (EPCs) and circulating progenitor cells (CPCs) in 49 CADASIL patients and 49 age-matched controls and their association with clinical/functional and neuroimaging features. RESULTS: In multivariate analysis, CADASIL patients had significantly higher vWF and lower EPC levels. TM levels were similar in the 2 groups. CADASIL patients with a more severe clinical phenotype (history of stroke or dementia) presented lower CPC levels in comparison with patients with a milder phenotype. On correlation analysis, lower CPC levels were associated with worse performances on neuropsychological, motor and functional tests, and with higher lesion load on brain magnetic resonance imaging (degree of leukoencephalopathy and number of lacunar infarcts). CONCLUSIONS: This is the first CADASIL series in which multiple circulating biomarkers have been studied. Our findings support previous studies on the presence and the possible modulating effect of endothelial impairment in the disease. Furthermore, our research data suggest that blood CPCs may be markers of disease severity.

Potential of creatine or phosphocreatine supplementation in cerebrovascular disease and in ischemic heart disease.

Creatine is of paramount importance for maintaining and managing cellular ATP stores in both physiological and pathological states. Besides these "ergogenic" actions, it has a number of additional "pleiotropic" effects, e.g., antioxidant activity, neurotransmitter-like behavior, prevention of opening of mitochondrial permeability pore and others. Creatine supplementation has been proposed for a number of conditions, including neurodegenerative diseases. However, it is likely that creatine's largest therapeutic potential is in those diseases caused by energy shortage or by increased energy demand; for example, ischemic stroke and other cerebrovascular diseases. Surprisingly, despite a large preclinical body of evidence, little or no clinical research has been carried out in these fields. However, recent work showed that high-dose creatine supplementation causes an 8-9 % increase in cerebral creatine content, and that this is capable of improving, in humans, neuropsychological performances that are hampered by hypoxia. In addition, animal work suggests that creatine supplementation may be protective in stroke by increasing not only the neuronal but also the endothelial creatine content. Creatine should be administered before brain ischemia occurs, and thus should be given for prevention purposes to patients at high risk of stroke. In myocardial ischemia, phosphocreatine has been used clinically with positive results, e.g., showing prevention of arrhythmia and improvement in cardiac parameters. Nevertheless, large clinical trials are needed to confirm these results in the context of modern reperfusion interventions. So far, the most compelling evidence for creatine and/or phosphocreatine use in cardiology is as an addition to cardioplegic solutions, where positive effects have been repeatedly reported.

Effects of amide creatine derivatives in brain hippocampal slices, and their possible usefulness for curing creatine transporter deficiency.

The creatine/phosphocreatine system carries ATP from production to consumption sites and buffers the intracellular content of ATP at times of energy deprivation. The creatine transporter deficiency syndrome is an X-linked disease caused by a defective creatine transporter into the central nervous system. This disease is presently untreatable because creatine lacking its carrier cannot cross neither the blood-brain barrier nor the cell plasma membranes. Possible strategies to cure this condition are to couple creatine to molecules which have their own carrier, to exploit the latter to cross biological membranes or to modify the creatine molecule to make it more lipophilic, in such a way that it may more easily cross lipid-rich biological membranes. Such molecules could moreover be useful for treatment of stroke or other ischemic brain syndromes of normal (transporter working) tissue. In this paper we tested four molecules in in vitro hippocampal slices experiments to investigate whether or not they had a neuroprotective effect similar to that of creatine. On two of them we also performed biochemical measurements to investigate whether or not they were able to increase the creatine and phosphocreatine content of the hippocampal slices with and without block of the transporter. We found that these molecules increase levels of creatine after block of the transporter, and significantly increased the levels of phosphocreatine. Both significantly increased the total creatine content in both conditions of active and blocked transporter. This shows that these molecules are capable of entering cells through biological membranes without using the creatine transporter. By contrast, neither of them was able to delay synaptic block during anoxia of normal (transporter functioning) tissue. We conclude that these compounds might possibly be useful for therapy of creatine transporter deficiency, but further research is needed to understand their possible role in anoxia/ischemia of normal tissue.

Selective Alteration of the Left Arcuate Fasciculus in Two Patients Affected by Creatine Transporter Deficiency.

(1) Background: In hereditary creatine transporter deficiency (CTD), there is an absence of creatine in the brain and neurological symptoms are present, including severe language impairment. However, the pathological changes caused by creatine deficiency that generate neuropsychological symptoms have been poorly studied. (2) Aims: To investigate if the language impairment in CTD is underpinned by possible pathological changes. (3) Methods: We used MRI tractography to investigate the trophism of the left arcuate fasciculus, a white matter bundle connecting Wernicke's and Broca's language areas that is specifically relevant for language establishment and maintenance, in two patients (28 and 18 y.o.). (4) Results: The T1 and T2 MRI imaging results were unremarkable, but the left arcuate fasciculus showed a marked decrease in mean fractional anisotropy (FA) compared to healthy controls. In contrast, the FA values in the corticospinal tract were similar to those of healthy controls. Although white matter atrophy has been reported in CTD, this is the first report to show a selective abnormality of the language-relevant arcuate fasciculus, suggesting a possible region-specific impact of creatine deficiency.

A new method to synthesize creatine derivatives.

Creatine is an amino acid that has a pivotal role in energy metabolism of cells. Creatine acts as an "ATP shuttle", carrying ATP to the sites where it is utilized, through its reversible phosphorylation by creatine kinase. Moreover, the creatine-phosphocreatine system delays ATP depletion during anoxia or ischemia, thus exerting a neuroprotective role during those pathological conditions. Thus, its administration has been advocated as a treatment or prevention of several conditions involving the central nervous system. However, creatine crosses poorly the blood-brain barrier and the cell plasma membrane, thus its administration has but a limited effect. The use of more lipophilic creatine derivatives has thus been suggested. However, such a synthesis is complicated by the intrinsic characteristics of the creatine molecule that hardly reacts with other molecules and easily cyclizes to creatinine. We obtained amide derivatives from creatine starting from a new protected creatine molecule synthesized by us, the so-called (Boc)2-creatine. We used a temporary protection only on the creatine guanidine group while allowing a good reactivity on the carboxylic group. This temporary protection ensured efficient creatine dissolution in organic solvents and offered simultaneous protection of creatine toward intramolecular cyclization to creatinine. In this manner, it was possible to selectively conjugate molecules on the carboxylic group. The creatine guanidine group was easily released from the protection at the end of the reaction, thus obtaining the desired creatine derivative.

In vitro study of uptake and synthesis of creatine and its precursors by cerebellar granule cells and astrocytes suggests some hypotheses on the physiopathology of the inherited disorders of creatine metabolism.

BACKGROUND: The discovery of the inherited disorders of creatine (Cr) synthesis and transport in the last few years disclosed the importance of blood Cr supply for the normal functioning of the brain. These putatively rare diseases share a common pathogenetic mechanism (the depletion of brain Cr) and similar phenotypes characterized by mental retardation, language disturbances, seizures and movement disorders. In the effort to improve our knowledge on the mechanisms regulating Cr pool inside the nervous tissue, Cr transport and synthesis and related gene transcripts were explored in primary cultures of rat cerebellar granule cells and astrocytes. METHODS: Cr uptake and synthesis were explored in vitro by incubating monotypic primary cultures of rat type I astrocytes and cerebellar granule cells with: a) D3-Creatine (D3Cr) and D3Cr plus ß-guanidinopropionate (GPA, an inhibitor of Cr transporter), and b) labelled precursors of Guanidinoacetate (GAA) and Cr (Arginine, Arg; Glycine, Gly). Intracellular D3Cr and labelled GAA and Cr were assessed by ESI-MS/MS. Creatine transporter (CT1), L-arginine:glycine amidinotransferase (AGAT), and S-adenosylmethionine:guanidinoacetate N-methyltransferase (GAMT) gene expression was assessed in the same cells by real time PCR. RESULTS: D3Cr signal was extremely high in cells incubated with this isotope (labelled/unlabelled Cr ratio reached about 10 and 122, respectively in cerebellar granule cells and astrocytes) and was reduced by GPA. Labelled Arg and Gly were taken up by the cells and incorporated in GAA, whose concentration paralleled that of these precursors both in the extracellular medium and inside the cells (astrocytes). In contrast, the increase of labelled Cr was relatively much more limited since labelled Cr after precursors' supplementation did not exceed 2,7% (cerebellar granule cells) and 21% (astrocytes) of unlabelled Cr. Finally, AGAT, GAMT and SLC6A8 were expressed in both kind of cells. CONCLUSIONS: Our results confirm that both neurons and astrocytes have the capability to synthesize and uptake Cr, and suggest that at least in vitro intracellular Cr can increase to a much greater extent through uptake than through de novo synthesis. Our results are compatible with the clinical observations that when the Cr transporter is defective, intracellular Cr is absent despite the brain should be able to synthesize it. Further research is needed to fully understand to what extent our results reflect the in vivo situation.

Effects of Delivering Guanidinoacetic Acid or Its Prodrug to the Neural Tissue: Possible Relevance for Creatine Transporter Deficiency.

The creatine precursor guanidinoacetate (GAA) was used as a dietary supplement in humans with no adverse events. Nevertheless, it has been suggested that GAA is epileptogenic or toxic to the nervous system. However, increased GAA content in rodents affected by guanidinoacetate methyltransferase (GAMT) deficiency might be responsible for their spared muscle function. Given these conflicting data, and lacking experimental evidence, we investigated whether GAA affected synaptic transmission in brain hippocampal slices. Incubation with 11.5 µM GAA (the highest concentration in the cerebrospinal fluid of GAMT-deficient patients) did not change the postsynaptic compound action potential. Even 1 or 2 mM had no effect, while 4 mM caused a reversible decrease in the potential. Guanidinoacetate increased creatine and phosphocreatine, but not after blocking the creatine transporter (also used by GAA). In an attempt to allow the brain delivery of GAA when there was a creatine transporter deficiency, we synthesized diacetyl guanidinoacetic acid ethyl ester (diacetyl-GAAE), a lipophilic derivative. In brain slices, 0.1 mM did not cause electrophysiological changes and improved tissue viability after blockage of the creatine transporter. However, diacetyl-GAAE did not increase creatine nor phosphocreatine in brain slices after blockage of the creatine transporter. We conclude that: (1) upon acute administration, GAA is neither epileptogenic nor neurotoxic; (2) Diacetyl-GAAE improves tissue viability after blockage of the creatine transporter but not through an increase in creatine or phosphocreatine. Diacetyl-GAAE might give rise to a GAA-phosphoGAA system that vicariates the missing creatine-phosphocreatine system. Our in vitro data show that GAA supplementation may be safe in the short term, and that a lipophilic GAA prodrug may be useful in creatine transporter deficiency.

Lack of Epileptogenic Effects of the Creatine Precursor Guanidinoacetic Acid on Neuronal Cultures In Vitro.

The creatine precursor Guanidinoacetic Acid (GAA) accumulates in the genetic deficiency of the GuanidinoAcetate Methyl Transferase (GAMT) enzyme and it is believed to cause the seizures that often occur in this condition. However, evidence that it is indeed epileptogenic is scarce and we previously found that it does not cause neuronal hyperexcitation in in vitro brain slices. Here, we used Micro-Electrode Arrays (MEAs) to further investigate the electrophysiological effects of its acute and chronic administration in the networks of cultured neurons, either neocortical or hippocampal. We found that: (1) GAA at the 1 µM concentration, comparable to its concentration in normal cerebrospinal fluid, does not modify any of the parameters we investigated in either neuronal type; (2) at the 10 µM concentration, very similar to that found in the GAMT deficiency, it did not affect any of the parameters we tested except the bursting rate of neocortical networks and the burst duration of hippocampal networks, both of which were decreased, a change pointing in a direction opposite to epileptogenesis; (3) at the very high and unphysiological 100 µM concentration, it caused a decrease in all parameters, a change that again goes in the direction opposite to epileptogenesis. Our results confirm that GAA is not epileptogenic.

A novel hypothesis about mechanisms affecting conduction velocity of central myelinated fibers.

The hypothesis that gap junctions are implicated in facilitating axonal conduction has not yet been experimentally demonstrated at the electrophysiological level. We found that block of gap junctions with oleammide slows down axonal conduction velocity in the hippocampal Schaffer collaterals, a central myelinated pathway. Moreover, we explored the possibility that support by the oligodendrocyte to the axon involves energy metabolism, a hypothesis that has been recently proposed by some of us. In agreement with this hypothesis, we found that the effect of oleammide was reversed by pretreatment with creatine, a compound that is known to increase the energy charge of the tissue. Moreover, conduction velocity was also slowed down by anoxia, a treatment that obviously decreases the energy charge of the tissue, and by ouabain, a compound that blocks plasma membrane Na/K-ATPase, the main user of ATP in the brain. We hypothesize that block of gap junctions slows down conduction velocity in central myelinated pathways because oligodendrocytes synthesize ATP and transfer it to the axon through gap junctions.

Creatine as a Candidate to Prevent Statin Myopathy.

Statins prevent cardiovascular diseases, yet their use is limited by the muscle disturbances they cause. Rarely, statin-induced myopathy is autoimmune, but more commonly it is due to direct muscle toxicity. Available evidence suggests that statin-induced creatine deficiency might be a major cause of this toxicity, and that creatine supplementation prevents it. Statins inhibit guanidinoacetate methyl transferase (GAMT), the last enzyme in the synthesis of creatine; thus, they decrease its intracellular content. Such decreased content could cause mitochondrial impairment, since creatine is the final acceptor of the phosphate group of adenosine triphosphate (ATP) at the end of mitochondrial oxidative phosphorylation. Decreased cellular synthesis of ATP would follow. Accordingly, ATP synthesis is decreased in statin-treated cells. In vitro, creatine supplementation prevents the opening of the mitochondrial permeability transition pore that is caused by statins. Clinically, creatine administration prevents statin myopathy in statin-intolerant patients. Additional research is warranted to hopefully confirm these findings. However, creatine is widely used by athletes with no adverse events, and has demonstrated to be safe even in double-blind, placebo-controlled trials of elderly individuals. Thus, it should be trialed, under medical supervision, in patients who cannot assume statin due to the occurrence of muscular symptoms.

Statin-induced myopathy prevented by creatine administration.

A 66-year-old woman with chronic myeloid leukaemia in nilotinib-induced remission was diagnosed with amaurosis fugax, caused by carotid stenosis. Serum cholesterol was 316 mg/dL (Low-Density Lipoprotein (LDL) cholesterol 213 mg/dL). Nilotinib was discontinued and replaced by interferon. Antiplatelet therapy and atorvastatin 40 mg/day were prescribed. Muscle pain and elevation of serum creatine kinase (CK) occurred; thus, atorvastatin was replaced by ezetimibe. Afterwards, muscle pain subsided and CK reverted to normal, but 2 years later serum cholesterol was still elevated at 218 mg/dL with LDL cholesterol 126 mg/dL. Simvastatin 5 mg/day was then started, but again muscle pain occurred and CK rose to 267 U/L. Simvastatin was stopped and serum cholesterol climbed to 252 mg/dL. Creatine was prescribed and simvastatin was reintroduced. Two months later, cholesterol was 171 mg/dL, CK was 72 U/L and there was no muscle pain. This case supports the view that creatine may prevent statin-induced myopathy.

A Novel Method to Synthesize Phosphocreatine and Phosphocreatine Prodrugs.

BACKGROUND: Adenosine triphosphate (ATP) is the energy currency of the body; it takes part in various and indispensable metabolic processes for the maintenance of cell homeostasis, degrading to its hydrolysis product, adenosine diphosphate (ADP). Efficient ways to restore ATP are therefore necessary in the cells. When the cell lacks energy due to ischemic conditions or high ATP demand, phosphocreatine gives its phosphate group to ADP that converts to ATP, in a reaction catalyzed by the enzyme creatine kinase. For this reason, phosphocreatine is utilized as a pharmacological treatment in human diseases that involve a failure of the cellular energy, most notably in coronary artery disease. OBJECTIVE: Commercially available phosphocreatine is currently synthesized using different methods, each of one characterized by a rather low yield of the final product, probably due to the low reactivity of the guanylating reagent. The aim of this work is to overcome the drawbacks of the synthetic methods currently employed, devising a novel synthetic route to obtain phosphocreatine and phosphocreatine prodrugs in higher yields and purity. METHOD: To obtain a higher yield of the final product and a lower number of sub-products, this method utilizes a new guanylating agent characterized by high reactivity, endowed with a protecting group t-Boc on one of the two nitrogen atoms of the guanidinic function and a protected phosphate on the other one; that compound is then conjugated with an opportune secondary amine. The obtained product is cleaved first with acidic conditions to obtain the phosphocreatine prodrug (phosphocreatine ethyl ester) and then with an enzymatic method to obtain the phosphocreatine. RESULT: Have been obtained in good yield and purity as demonstrated by HPLC and mass spectrometry analysis. CONCLUSION: This novel synthetic route permits to obtain the phosphocreatine molecule in higher yield and purity compared to the methods currently employed with a combination of chemical and enzymatic methods.

Di-acetyl creatine ethyl ester, a new creatine derivative for the possible treatment of creatine transporter deficiency.

Creatine is pivotal in energy metabolism of the brain. In primary creatine deficiency syndromes, creatine is missing from the brain. Two of them (AGAT and GAMT deficiency) are due to impaired creatine synthesis, and can be treated by creatine supplementation. By contrast, creatine transporter deficiency cannot be treated by such supplementation, since creatine crossing of biological membranes (plasma membrane and blood-brain barrier) is dependent on its transporter. This problem might be overcome by modifying the creatine molecule to allow it to cross biological membranes independently of its transporter. Thus, we designed and synthesized di-acetyl creatine ethyl ester (DAC), a compound that should cross biological membranes independently of the transporter due to its very high lipophilicity. We investigated its ability to increase intracellular creatine levels even after block of creatine transporter, and to counter cell damage induced by transporter block. In our experiments after block of the creatine transporter, DAC was able both to prevent electrophysiological failure and to increase intracellular creatine. Interestingly, it did so in micromolar concentrations, at variance with all the other creatine derivatives that we know of.

Support of Nerve Conduction by Respiring Myelin Sheath: Role of Connexons.

Recently, we have demonstrated that myelin conducts an extramitochondrial oxidative phosphorylation, hypothesizing a novel supportive role for myelin in favor of the axon. We have also hypothesized that the ATP produced in myelin could be transferred thought gap junctions. In this work, by biochemical, immunohistochemical, and electrophysiological techniques, the existence of a connection among myelin to the axon was evaluated, to understand how ATP could be transferred from sheath to the axoplasm. Data confirm a functional expression of oxidative phosphorylation in isolated myelin. Moreover, WB and immunohistochemistry on optic nerve slices show that connexins 32 and 43 are present in myelin and colocalize with myelin basic protein. Interestingly, addition of carbenoxolone or oleamide, two gap junction blockers, causes a decrease in oxidative metabolism in purified myelin, but not in mitochondria. Similar effects were observed on conduction speed in hippocampal Schaffer collateral, in the presence of oleamide. Confocal analysis of optic nerve slices showed that lucifer yellow (that only passes through aqueous pores) signal was found in both the sheath layers and the axoplasma. In the presence of oleamide, but not with oleic acid, signal significantly decreased in the sheath and was lost inside the axon. This suggests the existence of a link among myelin and axons. These results, while supporting the idea that ATP aerobically synthesized in myelin sheath could be transferred to the axoplasm through gap junctions, shed new light on the function of the sheath.