Chronic Dialysis Patients Are Depleted of Creatine: Review and Rationale for Intradialytic Creatine Supplementation.

There is great need for the identification of new, potentially modifiable risk factors for the poor health-related quality of life (HRQoL) and of the excess risk of mortality in dialysis-dependent chronic kidney disease patients. Creatine is an essential contributor to cellular energy homeostasis, yet, on a daily basis, 1.6-1.7% of the total creatine pool is non-enzymatically degraded to creatinine and subsequently lost via urinary excretion, thereby necessitating a continuous supply of new creatine in order to remain in steady-state. Because of an insufficient ability to synthesize creatine, unopposed losses to the dialysis fluid, and insufficient intake due to dietary recommendations that are increasingly steered towards more plant-based diets, hemodialysis patients are prone to creatine deficiency, and may benefit from creatine supplementation. To avoid problems with compliance and fluid balance, and, furthermore, to prevent intradialytic losses of creatine to the dialysate, we aim to investigate the potential of intradialytic creatine supplementation in improving outcomes. Given the known physiological effects of creatine, intradialytic creatine supplementation may help to maintain creatine homeostasis among dialysis-dependent chronic kidney disease patients, and consequently improve muscle status, nutritional status, neurocognitive status, HRQoL. Additionally, we describe the rationale and design for a block-randomized, double-blind, placebo-controlled pilot study. The aim of the pilot study is to explore the creatine uptake in the circulation and tissues following different creatine supplementation dosages.

Creatine Supplementation for Patients with Inflammatory Bowel Diseases: A Scientific Rationale for a Clinical Trial

Based on theoretical considerations, experimental data with cells in vitro, animal studies in vivo, as well as a single case pilot study with one colitis patient, a consolidated hypothesis can be put forward, stating that "oral supplementation with creatine monohydrate (Cr), a pleiotropic cellular energy precursor, is likely to be effective in inducing a favorable response and/or remission in patients with inflammatory bowel diseases (IBD), like ulcerative colitis and/or Crohn's disease". A current pilot clinical trial that incorporates the use of oral Cr at a dose of 2 × 7 g per day, over an initial period of 2 months in conjunction with ongoing therapies (NCT02463305) will be informative for the proposed larger, more long-term Cr supplementation study of 2 × 3-5 g of Cr per day for a time of 3-6 months. This strategy should be insightful to the potential for Cr in reducing or alleviating the symptoms of IBD. Supplementation with chemically pure Cr, a natural nutritional supplement, is well tolerated not only by healthy subjects, but also by patients with diverse neuromuscular diseases. If the outcome of such a clinical pilot study with Cr as monotherapy or in conjunction with metformin were positive, oral Cr supplementation could then be used in the future as potentially useful adjuvant therapeutic intervention for patients with IBD, preferably together with standard medication used for treating patients with chronic ulcerative colitis and/or Crohn's disease.

Intradialytic creatine supplementation: A scientific rationale for improving the health and quality of life of dialysis patients.

The CK/PCr-system, with creatine (Cr) as an energy precursor, plays a crucial role in cellular physiology. In the kidney, as in other organs and cells with high and fluctuating energy requirements, energy-charged phospho-creatine (PCr) acts as an immediate high-energy source and energy buffer, and as an intracellular energy transport vehicle. A maximally filled total Cr (Cr plus PCr) pool is a prerequisite for optimal functioning of the body and its organs, and health. Skeletal- and cardiac muscles of dialysis patients with chronic kidney disease (CKD) are depleted of Cr in parallel with the duration of dialysis. The accompanying accumulation of cellular damage seen in CKD patients lead to a deterioration of musculo-skeletal and neurological functioning and poor quality of life (QOL). Therefore, to counteract Cr depletion, it is proposed to supplement CKD patients with Cr. The anticipated benefits include previously documented improvements in the musculo-skeletal system, brain and peripheral nervous system, as well as improvements in the common comorbidities of CKD patients (see below). Thus, with a relatively simple, safe and inexpensive Cr supplementation marked improvements in quality of life (QOL) and life span are likely reached. To avoid Cr and fluid overload by oral Cr administration, we propose intradialytic Cr supplementation, whereby a relatively small amount of Cr is added to the large volume of dialysis solution to a final concentration of 1-10mM. From there, Cr enters the patient's circulation by back diffusion during dialysis. Because of the high affinity of the Cr transporter (CRT) for Cr affinity for Cr (Vmax of CRT for Cr=20-40µM Cr), Cr is actively transported from the blood stream into the target cells and organs, including skeletal and cardiac muscle, brain, proximal tubules of kidney epithelial cells, neurons, and leukocytes and erythrocytes, which all express CRT and depend on the CK/PCr system. By this intradialytic strategy, only as much Cr is taken up by the body as is needed to fill the tissue Cr pools and no excess Cr has to be excreted, as is the case with oral Cr. Because aqueous solutions of Cr are not very stable, Cr must be added immediately before dialysis either as solid Cr powder or from a frozen Cr stock solution to the dialysate, or alternatively, Cr could become an additional component of a novel dry dialysate mixture in a cartridge device.

A short review on creatine-creatine kinase system in relation to cancer and some experimental results on creatine as adjuvant in cancer therapy.

The creatine/creatine kinase (CK) system plays a key role in cellular energy buffering and transport. In vertebrates, CK has four isoforms expressed in a tissue-specific manner. In the process of creatine biosynthesis several other important metabolites are formed. The anticancer effect of creatine had been reported in the past, and recent literature has reported low creatine content in several types of malignant cells. Furthermore, creatine can protect cardiac mitochondria from the deleterious effects of some anticancer compounds. Previous work from our laboratory showed progressive decrease of phosphocreatine, creatine and CK upon transformation of skeletal muscle into sarcoma. It was convincingly demonstrated that prominent expression of creatine-synthesizing enzymes L-arginine: glycine amidinotransferase and N-guanidinoacetate methyltransferase occurs in sarcoma, Ehrlich ascites carcinoma and sarcoma 180 cells; whereas, both these enzymes are virtually undetectable in skeletal muscle. Creatine transporter also remained unaltered in malignant cells. The anticancer effect of methylglyoxal had been known for a long time. The present work shows that this anticancer effect of methylglyoxal is significantly augmented in presence of creatine. On creatine supplementation the effect of methylglyoxal plus ascorbic acid was further augmented and there was no visible sign of tumor. Moreover, creatine and CK, which were very low in sarcoma tissue, were significantly elevated with the concomitant regression of tumor.

Dietary phytoestrogens activate AMP-activated protein kinase with improvement in lipid and glucose metabolism.

OBJECTIVE: Emerging evidence suggests that dietary phytoestrogens can have beneficial effects on obesity and diabetes, although their mode of action is not known. Here, we investigate the mechanisms mediating the action of dietary phytoestrogens on lipid and glucose metabolism in rodents. RESEARCH DESIGN AND METHODS: Male CD-1 mice were fed from conception to adulthood with either a high soy-containing diet or a soy-free diet. Serum levels of circulating isoflavones, ghrelin, leptin, free fatty acids, triglycerides, and cholesterol were quantified. Tissue samples were analyzed by quantitative RT-PCR and Western blotting to investigate changes of gene expression and phosphorylation state of key metabolic proteins. Glucose and insulin tolerance tests and euglycemic-hyperinsulinemic clamp were used to assess changes in insulin sensitivity and glucose uptake. In addition, insulin secretion was determined by in situ pancreas perfusion. RESULTS: In peripheral tissues of soy-fed mice, especially in white adipose tissue, phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase was increased, and expression of genes implicated in peroxisomal fatty acid oxidation and mitochondrial biogenesis was upregulated. Soy-fed mice also showed reduced serum insulin levels and pancreatic insulin content and improved insulin sensitivity due to increased glucose uptake into skeletal muscle. Thus, mice fed with a soy-rich diet have improved adipose and glucose metabolism. CONCLUSIONS: Dietary soy could prove useful to prevent obesity and associated disorders. Activation of the AMPK pathway by dietary soy is likely involved and may mediate the beneficial effects of dietary soy in peripheral tissues.

Creatine treatment promotes differentiation of GABA-ergic neuronal precursors in cultured fetal rat spinal cord.

Creatine is a substrate of cytosolic and mitochondrial creatine kinases. Its supplementation augments cellular levels of creatine and phosphocreatine, the rate of ATP resynthesis, and improves the function of the creatine kinase energy shuttle. High cytoplasmatic total creatine levels have been reported to be neuroprotective by inhibiting apoptosis. In addition, creatine has direct antioxidant effects, which may be of importance in amyotrophic lateral sclerosis. In the present study, we investigated the effects of creatine [5 mM] on survival and differentiation of cultured GABA-immunoreactive (-ir) and choline acetyltransferase (ChAT)-ir rat spinal cord neurons. Furthermore, we addressed the neuroprotective potential of creatine supplementation against 3-nitropropionic acid (3-NP) induced toxicity. General cell survival and total neuronal cell density were not altered by chronic creatine treatment. We found, however, after chronic creatine and short-term creatine exposure a significantly higher density of GABA-ir neurons hinting to a differentiation-inducing mechanism of creatine. This notion is further supported by a significant higher content of GAD after creatine exposure. Creatine supplementation also exerted a partial, but significant neuroprotection for GABA-ir neurons against 3-NP induced toxicity. Interestingly, chronic creatine treatment did not alter cell density of ChAT-ir neurons but promoted their morphologic differentiation. Cell soma size and number of primary neurites per neuron were increased significantly after creatine supplementation. Taken together, creatine supplementation promoted the differentiation or the survival of GABAergic neurons and resulted in partial neuroprotection against 3-NP induced toxicity. The data suggest that creatine may play a critical role during development of spinal cord neurons.

Creatine promotes the GABAergic phenotype in human fetal spinal cord cultures.

In the present study, we investigated the expression pattern of cytosolic brain specific-BB-CK and ubiquitous mitochondrial-creatine kinases (uMt-CK) in developing human spinal cord. Consequently, we studied the effects of creatine treatment on cultured fetal human spinal cord tissue. We found that both CK isoforms were expressed in fetal spinal cord at all time points investigated (5 to 11.5 weeks post conception) and correspondingly specific CK activity was detected. Chronic creatine exposure resulted in significantly higher densities of GABA-immunoreactive neurons in the cultures, while total neuronal cell density was not altered, suggesting a differentiation inducing mechanism of creatine supplementation. Taken together, our observations favour the view that the creatine phosphocreatine system plays an important role in the developing CNS.

Improved reperfusion and neuroprotection by creatine in a mouse model of stroke.

Stroke leads to energy failure and subsequent neuronal cell loss. Creatine and phosphocreatine constitute a cellular energy buffering and transport system, and dietary creatine supplementation was shown to protect neurons in several models of neurodegeneration. Although creatine has recently been found to reduce infarct size after cerebral ischemia in mice, the mechanisms of neuroprotection remained unclear. We provide evidence for augmented cerebral blood flow (CBF) after stroke in creatine-treated mice using a magnetic resonance imaging (MRI)-based technique of CBF measurement (flow-sensitive alternating inversion recovery-MRI). Moreover, improved vasodilatory responses were detected in isolated middle cerebral arteries obtained from creatine-treated animals. After 3 weeks of dietary creatine supplementation, minor changes in brain creatine, phosphocreatine, adenosine triphosphate, adenosine diphosphate and adenosine monophosphate levels were detected, which did not reach statistical significance. However, we found a 40% reduction in infarct volume after transient focal cerebral ischemia. Our data suggest that creatine-mediated neuroprotection can occur independent of changes in the bioenergetic status of brain tissue, but may involve improved cerebrovascular function.

Acute and moderate-term creatine monohydrate supplementation does not affect creatine transporter mRNA or protein content in either young or elderly humans.

Animal studies have shown that supra-physiological creatine monohydrate (Cr-mH) supplementation for 3 months reduced skeletal muscle creatine transporter (CRT) content. The doses of Cr-mH (1-2 g/kg/day) used in these studies were between 5 and 10 times those usually used in human studies, and it is unclear whether a down-regulation of CRT would occur in humans at the recommended doses of 0.1-0.2 g/kg/day. We measured CRT, and citrate synthase (CS) protein content using Western blotting before and after 2 months of Cr-mH supplementation and weight training in young men (N = 11 Cr-mH (0.125 g/kg/ day); N = 8 placebo). CRT and CS were also measured before and after 4 months of Cr-mH supplementation and weight training in elderly (> 65 years) men and women (N = 14 Cr-mH (0.075 g/kg/day); N = 14 placebo). Finally, CRT mRNA was measured using competitive RT-PCR before and after 8-9 days of Cr-mH loading in young men and women (N = 14, CR-mH (mean = 0.18 g/kg/day); N = 13, PL). Total creatine content was significantly elevated after the Cr-mH supplementation period as compared to placebo in each of the studies. Neither Cr-mH supplementation, nor exercise training resulted in measurable alterations in CRT protein content and acute Cr-mH loading did not alter CRT mRNA. There were no gender differences in CRT mRNA or total creatine content in the young subjects and no gender differences in total creatine content or CRT protein content in the elderly subjects. Weight training in young men did not increase CS protein content, however, in the elderly there was a significant increase in CS protein content after exercise training (p < 0.05). These results demonstrated that Cr-mH supplementation during weight training resulted in increases in skeletal muscle total creatine without reductions in CRT protein and acute Cr-mH loading did not decrease CRT mRNA content.

Inhibition of the mitochondrial permeability transition by creatine kinase substrates. Requirement for microcompartmentation.

Mitochondria from transgenic mice, expressing enzymatically active mitochondrial creatine kinase in liver, were analyzed for opening of the permeability transition pore in the absence and presence of creatine kinase substrates but with no external adenine nucleotides added. In mitochondria from these transgenic mice, cyclosporin A-inhibited pore opening was delayed by creatine or cyclocreatine but not by beta-guanidinopropionic acid. This observation correlated with the ability of these substrates to stimulate state 3 respiration in the presence of extramitochondrial ATP. The dependence of transition pore opening on calcium and magnesium concentration was studied in the presence and absence of creatine. If mitochondrial creatine kinase activity decreased (i.e. by omitting magnesium from the medium), protection of permeability transition pore opening by creatine or cyclocreatine was no longer seen. Likewise, when creatine kinase was added externally to liver mitochondria from wild-type mice that do not express mitochondrial creatine kinase in liver, no protective effect on pore opening by creatine and its analog was observed. All these findings indicate that mitochondrial creatine kinase activity located within the intermembrane and intercristae space, in conjunction with its tight functional coupling to oxidative phosphorylation, via the adenine nucleotide translocase, can modulate mitochondrial permeability transition in the presence of creatine. These results are of relevance for the design of creatine analogs for cell protection as potential adjuvant therapeutic tools against neurodegenerative diseases.

Creatine transporter activity and content in the rat heart supplemented by and depleted of creatine.

The intracellular creatine concentration is an important bioenergetic parameter in cardiac muscle. Although creatine uptake is known to be via a NaCl-dependent creatine transporter (CrT), its localization and regulation are poorly understood. We investigated CrT kinetics in isolated perfused hearts and, by using cardiomyocytes, measured CrT content at the plasma membrane or in total lysates. Rats were fed control diet or diet supplemented with creatine or the creatine analog beta-guanidinopropionic acid (beta-GPA). Creatine transport in control hearts followed saturation kinetics with a K(m) of 70 +/- 13 mM and a V(max) of 3.7 +/- 0.07 nmol x min(-1) x g wet wt(-1). Creatine supplementation significantly decreased the V(max) of the CrT (2.7 +/- 0.17 nmol x min(-1) x g wet wt(-1)). This was matched by an approximately 35% decrease in the plasma membrane CrT; the total CrT pool was unchanged. Rats fed beta-GPA exhibited a >80% decrease in tissue creatine and increase in beta-GPA(total). The V(max) of the CrT was increased (6.0 +/- 0.25 nmol x min(-1) x g wet wt(-1)) and the K(m) decreased (39.8 +/- 3.0 mM). The plasma membrane CrT increased about fivefold, whereas the total CrT pool remained unchanged. We conclude that, in heart, creatine transport is determined by the content of a plasma membrane isoform of the CrT but not by the total cellular CrT pool.

Effects of creatine supplementation in cystic fibrosis: results of a pilot study.

BACKGROUND: The CF transmembrane conductance regulator (CFTR), whose mutations cause cystic fibrosis (CF), depends on ATP for activation and transport function. Availability of ATP in the cell and even more in specific cellular microcompartments often depends on a functional creatine kinase system, which provides the 'energy buffer' phosphocreatine. Creatine supplementation has been shown to increase phosphocreatine levels, thus promoting muscle growth and strength in athletes and having protective effects in neuromuscular disorders. AIM: To test clinically, if creatine supplementation improves maximal isometric muscle strength (MIMS), lung function and CFTR channel activity in patients with CF, and to determine enzymatic activity of creatine kinase in respiratory epithelial cells. METHODS: In an open-label pilot study 18 CF patients (8-18-year-old) with pancreatic insufficiency and mild to moderate lung disease received daily creatine supplementation during 12 weeks. Patients were monitored during 24-36 weeks. Enzymatic activity of creatine kinase was measured in primary epithelial cell cultures. RESULTS: After creatine supplementation, there was no change in lung function and sweat electrolyte concentrations, possibly due to the very low creatine kinase activities detected in respiratory epithelia. However, the patients consistently showed significantly increased MIMS (18.4%; P < 0.0001), as well as improved general well-being, as assessed by a standardized questionnaire. Except for one patient with transient muscle pain, no side effects were reported. CONCLUSIONS: Our pilot study suggests, that creatine supplementation should be further evaluated as a possible clinically beneficial adjuvant therapy for patients with CF to increase muscle strength, body-weight and well-being.

Reduced creatine kinase activity in transgenic amyotrophic lateral sclerosis mice.

Creatine (Cr), the substrate of the creatine kinase (CK) isoenzymes, was shown to be neuroprotective in several models of neurodegeneration, including amyotrophic lateral sclerosis (ALS). In order to investigate the mechanism of this beneficial effect, we determined CK activities and mitochondrial respiration rates in tissues from G93A transgenic mice, which overexpress a mutant form of human superoxide dismutase associated with familial ALS (FALS). While respiration rates of mitochondria from G93A transgenic or wild-type control mice isolated from spinal cord showed no difference, a significant and dramatic loss of CK activity could be detected in these tissues. In homogenates from spinal cord of G93A transgenic mice, CK activity decreased to 49% and in mitochondrial fractions to 67% compared to CK activities in wild-type control mice. Feeding the G93A transgenic mice with 2% Cr, the same tissues showed no statistically significant increase of CK activity compared to regular fed G93A transgenic mice. Experiments with isolated mitochondria, however, showed that Cr and adenosine triphosphate (ATP) protected mitochondrial CK activity against peroxynitrite-induced inactivation, which may play a role in tissue damage in neurodegeneration. Our data provide evidence for oxidative damage to the CK system in ALS, which may contribute to impaired energy metabolism and neurodegeneration.

Creatine supplementation reduces skeletal muscle degeneration and enhances mitochondrial function in mdx mice.

The mdx mouse serves as animal model for Duchenne muscular dystrophy. Energy status in muscles of mdx mice is impaired and we have demonstrated recently that the energy precursor creatine exerts beneficial effects on mdx skeletal muscle cells in culture. Here we show that feeding a creatine-enriched diet to new-born mdx mice strongly reduced the first wave of muscle necrosis four weeks after birth. Necrosis of the fast-twitch muscle extensor digitorum longus was inhibited by 63+/-14% (P<0.0001) while necrosis of the slow-twitch soleus muscle was not significantly decreased. In addition, using chemically skinned muscle fibres, we found that mitochondrial respiration capacity was decreased by about 25% in mdx-derived fibres and that long-term creatine-feeding restored respiration to wild-type levels. These results provide evidence that creatine supplementation in mdx mice improves muscle health and may provide a scientific basis for its use as adjuvant therapy in Duchenne muscular dystrophy.

Neuroprotection of creatine supplementation in neonatal rats with transient cerebral hypoxia-ischemia.

We hypothesized that creatine (Cr) supplementation would preserve energy metabolism and thus ameliorate the energy failure and the extent of brain edema seen after severe but transient cerebral hypoxia-ischemia (HI) in the neonatal rat model. Six-day-old (P6) rats received subcutaneous Cr monohydrate injections for 3 consecutive days (3 g/kg body weight/day), followed by 31P-magnetic resonance spectroscopy (MRS) at P9. In a second group, P4 rats received the same Cr dose as above for 3 days prior to unilateral common carotid artery ligation followed 1 h later by 100 min of hypoxia (8% O2) at P7. Rats were maintained at 37 degrees C rectal temperature until magnetic resonance imaging was performed 24 h after HI. Cr supplementation for 3 days significantly increased the energy potential, i.e. the ratio of phosphocreatine to beta-nucleotide triphosphate (PCr/betaNTP) and PCr/inorganic phosphate (PCr/Pi) as measured by 31P-MRS. Rats with hemispheric cerebral hypoxic-ischemic insult that had received Cr showed a significant reduction (25%) of the volume of edemic brain tissue compared with controls as calculated from diffusion-weighted images (DWI). Thus, prophylactic Cr supplementation demonstrated a significant neuroprotective effect 24 h after transient cerebral HI. We hypothesize that neuroprotection is probably due to the availability of a larger metabolic substrate pool leading to a reduction of the secondary energy failure because DWI has been reported to correlate with the PCr/Pi ratio in the acute phase of injury. Additional protection by Cr may be related to prevention of calcium overload, prevention of mitochondrial permeability transition pore opening and direct antioxidant effects.