During the 4–6 years of follow-up of nine boys with the CRTR defect, we did not find enough evidence to conclude that treatment with creatine monohydrate, L-arginine, and glycine effectively improves cerebral creatine and/or development, as assessed by neuropsychological tests and 1H-MRS.
Nonetheless, subjective improvements were reported by parents and caretakers during the first months of treatment. Also formal neuropsychological testing did indeed show significant improvements in motor and social skills in the whole group during the first assessments, while most patients were still on creatine monohydrate plus L-arginine treatment without glycine. However this effect did not last. It is unlikely that this is caused by the addition of glycine because scores also decreased in patients who were not (yet) started on glycine. Scores on the hearing and speech and the hand-eye coordination subscales even declined during treatment. Probably this is mainly age-related and not due to treatment because older patients already had lower scores at the start of treatment. Statistical analysis confirmed a significant negative correlation with age. An age-related decline in IQ scores is also known to occur in Down’s syndrome and fragile X syndrome (Fisch et al. 2007
) and might be common to more mental retardation disorders. This does not necessarily mean that these conditions are progressive. When the rate of development slows down and reaches an early ceiling, the IQ score (ratio of developmental age and chronological age) declines while the patient remains unchanged. As the developmental ages did not decrease, we saw in our cohort no indication for regression. An age-related decline in IQ scores is also consistent with the fact that IQ scores described in adult patients with the creatine transporter defect are lower than described in affected children (Hahn et al. 2002
). Speech and coordination appear to be specific weaknesses in CRTR-deficient patients and a ceiling appears to be reached earlier than for the other subscales. The age-related decline in IQ scores complicates the evaluation of treatment in children, and further studies of the natural course of the CRTR defect are therefore needed.
No significant increase in brain creatine content upon treatment was seen in 1
H-MRS. It should be noted that creatine measurements varied inter- and intraindividually, which can be temporarily (mis)taken for improvements. Creatine measurements also differed over the various brain regions, being significantly higher in the basal ganglia and cerebellum than in cortex and white matter. These differences must be taken into account when comparing H1
-MRS creatine levels in a single individual or in small numbers of observations. The regional differences are comparable to those in healthy subjects (Pouwels et al. 1999
There are several possible explanations why treatment failed to increase cerebral creatine content. It is possible that the uptake of arginine and glycine through the blood-brain barrier was insufficient. In future trials, this could be monitored by measurements of cerebrospinal fluid concentrations of amino acids. Other explanations are related to the central question why the CRTR defect leads to cerebral creatine deficiency to begin with if the brain is capable of endogenous creatine synthesis. It is possible that the cerebral creatine synthesis is limited and that the expected upregulation of the AGAT reaction, the rate-limiting step in creatine synthesis, by decreasing creatine (Wyss and Kaddurah-Daouk 2000
) or by supplementation of precursors L-arginine and glycine, does not occur in the brain. This is, however, in contrast with the hypothesis that the CNS mainly derives its creatine from endogenous synthesis (Braissant et al. 2001
) because the permeability of the blood-brain barrier for creatine appears limited (Wyss and Schulze 2002
), and astrocytes, contacting the capillary endothelial cells forming the blood-brain barrier, do not express CRTR (Braissant et al. 2001
). In addition, CRTR might also be important in the cerebral creatine synthesis. Braissant et al. found that AGAT and GAMT, although expressed in all CNS cell types, are rarely co-expressed within the same cell and hypothesized that GAA must be transported by CRTR between brain cells for creatine synthesis to occur (Braissant and Henry 2008
; Braissant et al. 2007
). In this model GAA accumulation would be expected in cases of CRTR deficiency (Braissant and Henry 2008
), which was indeed suggested in one patient (Sijens et al. 2005
). Arginine and glycine supplementation would then not lead to the aimed for creatine increase but to further GAA accumulation, an adverse effect because GAA is considered to be epileptogenic (Tachikawa et al. 2008
). However, we saw no GAA accumulation in our cohort of CRTR-deficient patients nor an increase during treatment.
It is possible that our treatment has been effective outside the brain, for instance on muscle function. The temporary improvements in motor skills may have been caused by improved muscle function. Unfortunately, formal muscle function tests were not performed. An increase in body weight has been noted upon creatine monohydrate treatment in patients with the CRTR defect (Anselm et al. 2006
; Poo-Arguelles et al. 2006
). Five patients in our study showed an increase of more than one to two SD scores in height and weight, although not statistically significant in the whole group.
Our follow-up has been too short to decide whether the treatment might prevent complications later in life, such as myopathy and intestinal dysfunction, which have been described in adult patients (Kleefstra et al. 2005
; Hahn et al. 2002
). Personal observations of creatine monohydrate plus L-arginine and glycine treatment in four male adult patients aged between 17 and 54 years registered positive effects with improved behavior in all, amelioration of severe constipation in one, and achievement of urinary continence in another (Mancini, unpublished).
Though this study represents the largest and longest treated cohort so far and includes repeated neuropsychological assessments and repeated brain 1H-MRS, there are still several limitations that should be addressed in future studies. The cohort was heterogeneous in age at the start of treatment. This complicates the evaluation of treatment because neuropsychological assessments may be less reliable in the first years and IQ scores may decline with age as we noticed for certain subscales. Furthermore it is possible that treatment starting at a younger age is more successful. Due to the sample size, this could not be evaluated in this study. The treatment conditions in the cohort differed in the addition of glycine, and neuropsychological assessments and brain 1H-MRS were performed at different treatment durations. This limited the power of the statistical analyses. The analyses were performed irrespective of compliance because the compliance could not be optimally monitored.
In future therapeutic trials, additional or other treatment options need to be considered. S-adenosylmethionine (SAMe) supplementation might be another way to strengthen the cerebral creatine synthesis. SAMe acts as methyl donor in the GAMT reaction, which in fact accounts for the main percentage of total utilization of methyl groups in the body (Wyss and Kaddurah-Daouk 2000
). SAMe crosses the blood-brain barrier and increases cerebral phosphocreatine (Moxon-Lester et al. 2009
). Folic acid and vitamins B6 and B12 could be added to increase methionine synthesis and maintain low concentrations of S-adenosylhomocysteine, which inhibits GAMT (Moxon-Lester et al. 2009
). Other alternative treatment could be with lipophilic creatine analogs, which cross the blood-brain barrier independent of the CRTR. Uptake of lipophilic creatine analogs was found in CRTR-blocked mouse hippocampal slices (Lunardi et al. 2006
) and in human CRTR-deficient and control fibroblasts, however no effect of treatment with creatine ethyl ester (CEE) was found in CRTR patients (Fons et al. 2010
It is possible that creatine is released from central neurons and acts as a neuromodulator (Almeida et al. 2006
). Therefore CRTR might also be essential for creatine reuptake and termination of synapsis (Peral et al. 2010
) and/or for release of creatine from neurons (Mak et al. 2009
). Treatment directed at increasing the levels of creatine might not solve disturbed neuromodulation in CRTR deficiency.