Abstract

For many years, magnesium has been described as a crucial factor for cellular activity. In this chapter, a brief overview of pharmacology and genetics of magnesium transport will be followed by a review of clinical and biological studies of Mg-vitamin B6 supplementation in attention deficit/hyperactivity disorder (ADHD) and autism (autistic spectrum disorders family, ASD) in children. Although no study carried out on a rational basis has been published to date, some experimental and/or clinical works support a positive effect of such therapy in these pathologies. All the individual observations report a decrease in hyperactivity and a stabilization of scholarly behaviour with treatment. These data strongly support the need for a controlled study to confirm or invalidate these assumptions.

Introduction

Magnesium is the second most abundant intra- cellular cation in the body. Its main action is to regulate enzyme activity, to control the activity of various calcium and potassium channels, and to promote membrane stabilization. It is also responsible for the maintenance of the trans- membrane gradients of sodium and potassium. Magnesium depletion is known to be associated with many clinical diseases including hypo- calcemia, hypokalemia, cardiac arrhythmias, neuromuscular excitability, hypertension, athero- sclerosis, and osteoporosis. Some evidence indicates that magnesium could also be involved in neurological diseases such as attention deficit/hyperactivity disorder and autism. However, no direct study has been published to confirm this assumption.

Attention deficit/hyperactivity disorder (ADHD) and autism (autistic spectrum disorders, ASD; pervasive developmental disorders, PDD) are different neurological disorders which have been described for many years, and in which the involvement of a deficient magnesium pathway could be suspected given the presence of active transport for such cations through transient receptor potential melastatin (TRPM) channels in the brain.

ADHD is the most common neurobehavioral disorder presenting for treatment in youth.

Children with ADHD are “a group at risk” as far as their further emotional and social development and educational possibilities are concerned (Spencer et al., 2002). An effective intervention for many hyperactive children, besides methyl- phenidate and other psycho-stimulant drugs, is the use of vitamin B6 (pyridoxine) and magnesium (Mg). For over 30 years, parents have given high doses of pyridoxine and Mg to their children and have observed decreased physical aggression and improved social responsiveness. However, up until today, very few studies have reported a possible association between magnesium supplementation, ADHD symptoms, and the Mg status of the children. The first such study (Liebscher and Liebscher, 2003) suggested that patients with ADHD should be considered as potentially Mg-deficient as opposed to an incorrect interpretation of the serum Mg test (tetanic patients have lower Mg values than normals). Other studies from Kozielec et al., for the first time reported an intra-erythrocyte magnesium deficiency in ADHD children (Kozielec and Starobrat-Hermelin, 1997). We also published similar data (Mousain-Bosc et al., 2004, 2006). However, it is not a true “Mg deficiency” with clinically associated respiratory repletion, as is observed in familial hypomagnesaemia with secondary hypocalcemia (Shalev et al., 1998; Mousain-Bosc et al., 2004). More precisely, it may be called “intracellular Mg deficiency” affecting mainly neural transmission, which is very sensitive to such ionic variations.

Another group of neuronal diseases in which magnesium has been implicated is ASD/PDD/ autism. Studies from 18 different research groups have shown that vitamin B6 and Mg are beneficial to about half of autistic individuals, with no significant adverse effects. Eleven of these studies involved a double-blind, placebo controlled design and have documented decreases in behavioural problems, improvements in appropriate behaviour, and normalisation of brain wave activity and urine biochemistry. There is also evidence that B6 and Mg may reduce seizure activity. Parent reports confirm improvement in attention, learning, speech/language, and visual contact. More recently, in a pilot study of a moderate dose, multivitamin/mineral supplement for children with autistic spectrum disorder, Adams and Holloway (2004) found significant improvements in sleep and gastrointestinal problems compared to the placebo group. Despite all these data, the intervention using Mg-B6 remains controversial and contradictory studies have been published (Vink, 2001; Helpern, 1993; Mousain-Bosc et al., 2006; Shalev et al., 1998).

New data reporting a possible association between Mg-B6 supplementation, neurobehav- ioural symptoms, and Mg status of children have subsequently been published, opening a new way for research in this domain. This chapter will present a review of essential works published in this domain.

Magnesium transport and pharmacology of magnesium

Clinical symptoms of ADHD syndrome

Clinical cases

ADHD has been described since the beginning of the twentieth century and was called hyperkinesia or “psycho-motor instability”. It affects 3 to 9% of the population of children and adults (Feillet-Coudray, 2005; Biederman, 2006). The clinical picture is that of a hyperirritable, impulsive, aggressive child with an attention deficit. The diagnosis is basically done at six years of age when the child presents at primary school. These symptoms impair emotional development and can lead to serious social and family disorders. In adults, statistics show a powerful link between ADHD and serious problems in life. These findings support the idea that when diagnosed in the community, ADHD is a clinically significant and highly disabling disorder in adults and must be treated very early in children. However, parents’ and children’s histories should be studied. In parents, behavioural disorders such as hyperactivity, aggressiveness, emotional lability, stress, or maternal spasmophilia may be present. During pregnancy, several symptoms can be observed including weariness, stress, anxiety and breakdown, arterial hypertension, muscle cramps, contractions, sleeping disorders, diet deficiency, and twin pregnancy.

The first signs of ADHD generally appear during the first year of life and include sleeping disorders (short sleeping and waking to a small intensity noise), spasm of the sob, tremor of the hands and the arms, intense emotionality, frequent tears, hyper-excitability, frequent fidgeting or squirming, inability to keep one's seat, inclination to put their life in danger, climbing, and an inability to see potential danger. The adaptation to nursery school at three or four years of age becomes rapidly difficult, with hyper-excitability, impulsivity and difficulty in being a good listener.

At six years of age, all the clinical symptoms of ADHD as described in the DMS-IV manual (Findling et al., 1997) are present, including:

• hyperactivity: fidgets with hands or feet, or squirms in seat; gets up from seat when expected to remain seated; runs about or climbs when it is not appropriate; is "on the go" or often acts as if "driven by a motor"; talks excessively; blurts out answers before questions have been finished; has difficulty in awaiting their turn; interrupts or intrudes on others; has an inclination to put their life in danger.
• impulsivity: becomes aggressive, doesn’t control their movements; gets angry and has difficulty stopping this aggression.
• attention deficit: symptoms of inattention should be present for at least six months and when children are attending primary school; does not give close attention to detail or makes careless mistakes in schoolwork or other activities; has trouble maintaining attention on tasks or play activities; does not follow instructions and fails to finish schoolwork, chores or duty in the work place (not due to opposite behaviour or lack of understanding instructions but due to slowness to execute the tasks); has trouble organizing activities; avoids dislikes or does not want to do things that take a lot of mental effort for a long period of time; loses things needed for tasks and activities (toys, pencils, books or tools); is often easily inattentive; is often forgetful in daily activities and has memory difficulties.

In addition, other clinical symptoms frequently occur and must been considered as main symptoms. These include:

• sleeping disorders: mild sleeping problems; cannot put to sleep; has nightmares, broken sleep; is frightened by the night and awakes weary; very frequent abdominal pains occurring with stress.
• unexplained weariness: weariness begins in the morning with slowness to stand up, to get dressed, and abnormal weariness in sports activities.
• feeling of faintness with or without losing consciousness; twitches in the face or in the breathing reflect hyper-excitability; gnaws one's nails; anxiety, stress, mood disorders, easily upset; loss of trust and loss of respect of oneself; often breaks down; is afraid of people and frightened to die. The medical examination reveals a Chvostek's signs in 66% sign of hyper-excitable children.

In the clinical description of ADHD, biological data are usually not evoked. However, for instance, hyper-excitability can be revealed by an intracellular Mg deficiency since Mg has been shown to be involved in the control of some nervous system processes.

Clinical Case Study 1

In the first months of her life, patient OCE presented signs of hyper-excitability. She twiddled her fingers, jumped to a lower level noise and woke often. A prescribed calcium therapy was not effective and the tremors persisted. Alternate calcium and Mg therapy improved all the signs.

At five years, before beginning primary school, patient OCE again presented signs of hyper- excitability. She was fidgety and didn’t stay in her seat. Sleeping disorders reappeared where she awoke very often and slept very little. She was impulsive and slapped children, although it was not intentional. She didn’t control herself and was subject to terrible fits of anger. The possibility of Mg deficiency was raised since her mother has suffered from "spasmophilia" for some years. Both OCE and her mother showed a decrease in erythrocyte Mg levels.

After two months of Mg-B6 treatment, OCE felt better. She became cool, good, less capricious, didn’t play the fool, and her sleeping was calm. Erythrocyte Mg increased and the therapy was accordingly stopped. Some months later, all the ADHD signs rapidly reappeared including attention deficit, hyperactivity, sleeping disorders and impulsiveness. Biological control confirms the diagnosis of Mg metabolism disorder (Mg imbalance), a “genetic disease". The Mg-B6 treatment was recommenced with symptoms resolving. However, each time OCE stopped taking the treatment, ADHD symptoms returned! OCE received Mg therapy over the eight-year period made up of 6mg/kg/day Mg and 0.6 mg/kg/day vitamin B6 (Figure 1).

Figure 1.

Evolution of biological parameters for magnesium in the case of patient OCE (born February, 1997) during the follow-up (ADHD syndrome). Ser-Mg: total serum magnesium concentrations in mmol/l; Erc-Mg: magnesium concentrations in erythrocytes (obtained after red cell lysis) in mmol/l.

Clinical Case Study 2

Patient NIC was seventeen years old when he first came to the consulting office. In the first years of his life, he demonstrated sleeping disorders, restless sleep, frequent tears, had a delay with communication, was an emotional child, uneasy, over-excitable, and a little aggressive. Upon admission to secondary school, behavioural disorders persisted. He was impulsive, didn’t control himself, was aggressive and was dyslexic.

A diagnosis of ADHD was made and psycho- stimulant treatment was prescribed over three years. He never took the prescribed dosage because his physician (his grandfather) and his pharmacist had warned the parents of the risks of this medication (methylphenidate). Rapidly, he presented side effects to this treatment, including sleeping disorders, muscular pains, weariness, "feeling lethargic", behavioural disorders, "became as a zombie", headaches, palpitations, anxiety attacks, breakdowns, and "was frightened to die". His parents were worried by these side effects, and read our publications at the beginning of 2007. The levels of erythrocyte Mg, calcium, and ionized calcium had dramatically decreased. The parents decided to stop the psychostimulant treatment. A Mg supplementation (300 mg) spectacularly improved patient NIC's behaviour. In some weeks, NIC became cool and more relaxed. An excellent participation in scholarly activities and a better concentration in tasks resulted in excellent scholastic results in four months. NIC received the "congratulations of the schoolboard" at the end of 2007! NIC could control himself, was not aggressive, had no behavioural disorders, and had self-confidence. In six months, all the biological disturbances were normalized. At the school of “masonry for historic monuments", NIC has excellent assessments – "a good student and hardworker"! NIC received a Mg supplementation for more than three years and each time when he tried to decrease the dose, clinical symptoms reappeared.

The disease, called hypomagnesemia with secondary hypocalcemic, is heritable. NIC’s sister was also emotional and hypersensitive while his brother was hyper-excitable, always "on the go " and had both sleeping and behavioural disorders. She had Mg deficiency and now feels better with Mg therapy. His mother presented with cramps, muscular pains, pins and needles in the hands, stress and migraines. A biological profile showed an erythrocyte Mg deficiency. After six months of Mg therapy, the signs disappeared. The maternal grandfather suffers from Alzheimer’s disease. NIC’s father was emotionally stressed, uneasy and for several years experienced sleeping disorders, heart disease, attention deficit, and above all memory disorders. He is only forty years of age! He had an erythrocyte Mg deficiency and after six months of the same treatment, their memory improved (Figure 2).

Figure 2.

Evolution of biological parameters for magnesium and calcium in the case of patient NIC during the follow-up (ADHD syndrome). Ser-Mg: total serum magnesium concentrations in mmol/l; Erc-Mg: magnesium concentrations in eryth- rocytes (obtained after red cell lysis) in mmol/l; Total Ca: total serum calcium concentrations in mmol/l; Ionized calcium concentrations in mmol/l in serum.

In March 2007, the methylphenidate treatment was stopped and the magnesium therapy was set up. A clear behavioral improvement was observed. However, after the three years of magnesium therapy, all biological data remain stable. This result could suggest an alteration of the Mg transport. It encouraged us to perform additional experiments on Mg channels in order to check their expression in various tissues.

Experimental Study Number 1

In order to study relationships between hyperactivity symptoms and Erc-Mg levels, we designed an open study on 40 children with ADHD syndrome. For ethical reasons, we chose not to perform a double-blind study against either psychostimulants (methylphenidate) or placebo. We felt that parents would not support such a design. In addition, psychostimulants were found to alter Mg homeostasis (Tolbert et al., 1993). Our results (Mousain-Bosc et al., 2004) showed a statistically significant improvement of the symptoms after Mg-B6 supplementation, together with a rise in Erc-Mg values.

In this study, a slight but significant intra- erythrocyte Mg depletion was evident in ADHD patients together with a concomitant decrease in intracellular calcium concentrations. As we know, Mg is essential for normal central activity and Erc- Mg could be representative of intracellular Mg²⁺ concentrations. A decrease in Erc-Mg without changes in serum Mg concentrations could be interpreted as an alteration in a Mg transporter (Na⁺/Mg²⁺ exchanger) in erythrocytes with concomitant effects on neuronal Mg concentrations. The difficulty in achieving normal Erc-Mg values under Mg-B6 treatment supports this hypothesis. In addition, supplementation with Mg pidolate was found to decrease Na⁺/Mg²⁺ exchanger activity with a concomitant rise in Mg and K⁺ contents of erythrocytes in sickle cell disease (DMS-IV). Erc-Mg was described as a controversial biological parameter for the monitoring of Mg deficiency, in contrast to others (Borella et al., 1993; Vink et al., 2009) who consider Erc-Mg as a suitable index. Moreover, Basso et al., (2000) and Helpern et al., (1998) present Erc-Mg as not useful for the monitoring of individual changes. We think that in this last study, a 3-week treatment of Mg without B6 was too short to induce a durable increase in Erc-Mg (vitamin B6 was described as enhancing Mg entry into the cell). In any case, in our hands, Erc-Mg measurements were standardized and it appears as a potent indicator of cellular Mg deficiency.

Calcium and Mg cellular contents classically follow the same pathway – when Mg increased, calcium also increased. This may explain the significant correlation between Erc-Mg and intracellular calcium values as well as the fact that in children who have low intracellular calcium values, Mg therapy increased intracellular calcium levels. It can be hypothesized that a genetic factor, which modulates Na⁺/Mg²⁺ exchanger activity, may be important in the regulation of Mg metabolism (Heath and Vink, 1998).

We also found that increased hyperactivity and decreased scholastic attention were associated with decreased Erc-Mg values. This observation was supported by the fact that Mg-B6 supple- mentation induced a rise in Erc-Mg values and a concomitant improvement of the clinical symptoms. What are the respective roles of pyridoxine and Mg in these observations? It was classically accepted that Mg is administered with pyridoxine to decrease irritable side-effects of the B6 therapy. We show here evidence of the role of Mg itself in this therapy. Previous data support this observation. In ADHD disorders, in which disruptive behaviour with hyperactivity was found, psychostimulants are used to improve mental health, probably by increasing synaptic noradrenaline activity. In children who received methylphenidate, a significant increase (6%) in plasma Mg concentrations was found depending on the dosage of the drug, showing a relationship between improvement of hyperactivity and Mg metabolism (Schmidt et al., 1994 (Tolbert et al., 1993). More recently, in autistic children with behavioural disorders and hyperactivity (Zilbovicius et al., 2000; Schmidt and Taylor, 1988; Chakraborti et al., 2002), positive emission tomography (PET) has shown a significant decrease in cerebral blood flow localized at the temporal lobe level in 76% of the children examined. Taken together with the fact that intra- erythrocyte free Mg²⁺ is associated with increased blood pressure (Zilbovicius et al., 2000) and that brain from rats fed with low Mg diets are more susceptible to permanent brain focal ischemia (Gervais et al., 2004), we can hypothesize that intracellular Mg²⁺ deficiency could be responsible, at least in part, of some central activity disorders observed in these children.

The duration of the treatment necessary to get significant improvement seems to be about 8 weeks. Since the cause of this deficiency is yet unknown, and since the symptoms reappeared when the Mg-B6 diet was stopped, the treatment must be maintained for a long time. In addition, while it was difficult to find an evident biological link between central disorders and Erc-Mg values, this biological parameter could be used to select, among the large population of children with hyperactive symptoms, a small population with behavioural abnormalities that is relevant for a Mg-B6 diet. It is evident that another accessible Mg store, more significant for central disorders, has to be found.

In conclusion, this study provides additional information about the therapeutic role of a Mg-B6 regimen in children with ADHD. This effect seems to be associated, at least in part, to a cellular Mg²⁺ deficiency, as evidenced by intra-erythrocyte Mg measurements. Installing a Mg-B6 supplem- entation for some weeks restored higher intra- erythrocyte Mg²⁺ values and significantly reduced the clinical symptoms of these diseases. As chronic Mg deficiency was shown to be associated to hyperactivity, irritability, sleep disturbances, and low scholar attention, besides other traditional therapeutic treatment, a Mg supplementation could be required in children with ADHD.

Treatment of ADHD

Conventional therapy is often multimodal including behavioural therapies and medications. Approved drugs for ADHD are psychostimulants(amphetamine derivatives), including methyl- phenidate. Psychostimulant medications approved by the U.S. Food and Drug Administration (FDA) include methylphenidate (Ritalin^®, Concerta^®), and, more recently, atomoxetine (Strattera^®).

Figure 3.

Biological data obtained at the first visit for 65 ADHD and 22 non-ADHD children. Ser-Mg: total serum magnesium concentrations in mmol/l; Erc-Mg: magnesium concentrations in erythrocyte (obtained after red cell lysis) in mmol/l. Only Erc- Mg appeared to be significantly lower than before treatment (statistically significant at p<0.05).

Figure 4.

Biological data obtained 3 months after the first visit for the same children. Ser-Mg: total serum magnesium concentrations in mmol/l; Erc- Mg: magnesium concentrations in erythrocyte (obtained after red cell lysis) (in mmol/l). Erc-Mg recovered normal values (statistically significant at p<0.05).

In 2008, a study by the National Centre of Scientific Research (CNRS) in France (Gonon, 2009) concluded that "although psychostimulants alleviate the core symptoms of attention deficit hyperactivity disorder (ADHD), recent studies confirm that their impact on the long-term outcomes of ADHD children is null. Psychostimulants enhance extracellular dop- amine”. Numerous review articles assert that they correct an underlying dopaminergic deficit of genetic origin. This dopamine-deficit theory of ADHD is often based upon an overly simplistic dopaminergic theory of reward. Psychostimulant medication does not improve long-term academic outcomes of the ADHD children. Therefore, this hypothesis should not be put forward to bias ADHD management towards psychostimulants. The American Journal of Psychiatry in June 2009 published a study entitled "Sudden death and use of stimulant medications in youths” funded, in part, by the FDA and the National Institute for Mental Health (Gould et al., 2009). Side effects are described for psychostimulant medications over some years.

As we have seen previously, dietary factors and Mg deficiency can play a significant role in the etiology of ADHD syndrome. In our study, Mg deficiency was found in 89% of children with ADHD, whereas only 23% of non-ADHD children present with low erythrocyte Mg deficiency (Table1).

Table 1.

Clinical symptoms of autistic spectrum disorders (ASD)

The prevalence of autism increases continuously and remains an extreme challenge to clinical researchers. It is a neurobiological condition having its origins in a disturbance of the cellular structure of the brain during pregnancy. Generally speaking, there is no "cure" for such children and the holy grail of finding a neuropharmacological reversal of symptoms is currently being researched worldwide.

For some years, one must insist on the screening of the early signs of autism during the two first years of the life as described in the DMS-IV manual:

• Relationship disorders; child is “too good” with very few smiles.
• Visual contact disorders; missing or poor visual attention; doesn't look at the parents; look is shifty.
• Difficulty in listening; seems not to hear; delayed communication; doesn't repeat words.
• Motor affectation; doesn't take the toys; stereotyped movements of the hands; sits and walks belatedly; hypo-tonicity or hyper-tonicity.
• Sleeping disorders; broken sleep; crying.
• Feeding difficulties; slobbers; cannot swallow; refuses food.

The major feature of autism spectrum disorder is that the symptoms only manifest themselves after the age of eighteen months and before the age of three. It is considered like a regression of development (Burn, 2009). We can explain that ASD are disorders of brain function and not of brain structure.

After three years, the clinical picture of ASD becomes typical:

• Impairment of social interactions.
  • visual contact: doesn't look at the parents and the siblings; gives a blank look; look is shifty.
  • connection with equals: has no interest with the parents, with the friends at the primary school; seems to be "in their bubble".
  • delight partitions: has no emotional relationship with the family, cannot express their pleasure for the event; does not show social reciprocity in the form of an answer to a human presence.
• Loss of communication.
  • delayed communication: speaks belatedly; some sounds or some syllables are emitted late; few spontaneous verbal exchanges; lack of creativity in thought.
  • no communication: doesn't repeat words.
  • stereotypical language: repeats the same word sometimes without significance.
  • social mimicking: cannot reproduce some- thing after a long time earnestly trying.
• Stereotyped restricted behavior.
  • stereotyped interest: has very poor interest or repeats the same answer for something.
  • customs.
  • handling things: difficulty to take the things, to take toys, and can have abnormal movements.
  • motor affectation: to walk, moves with difficulty; to bring something to somebody, cannot take initiative.
• Abnormal or delayed functioning.
  • language.
  • social interactions.
  • symbolic games: doesn't understand the rules of the game; cannot dream up the game.
  • behavioural disorders like sleeping disorders, aggressiveness, impulsivity; attention deficit; cannot listen.

Magnesium metabolism could be involved in autism, autism spectrum disorder or pervasive developmental disorder (Rimland et al., 1978). Magnesium prevents against encephalopathy and developmental delay (Doyle et al., 2008).

Clinical Case Study 3

Patient ARN, three years old, presents all the clinical signs of ASD, an autistic disorder that began after two years of age. The adaptation at the nursery school became difficult. His mother describes behavioural disorders, with incessant restlessness, doesn't listen, puts own life in danger, let's go the mother's hand in the street, crosses the street without looking around, has important sleeping and communication disorders, doesn't speak, is always squealing, and his glance is shifty. At school, the headmaster's evidence was spectacular – "ARN become isolated, doesn't draw, has no interest with the activities, doesn't listen about the forbidden subjects. He doesn't speak, expresses himself by shouting, and his glance is shifty”. During the examination, patient ARN was nervous, restless, tetanized, he clenched his fists and shook his hands. In the face of all these clinical symptoms, the possibility of a Mg deficiency is raised. His mother has presented a Mg deficiency for several years.

With Mg-B6 therapy, (6 mg/kg/day of Mg and 0.6 mg/kg/day of vitamin B6), ARN improved quickly month after month, probably because Mg supplementation was set up very early in life before the age of four years. ARN becomes cool, obedient, wants to get dressed alone, put his shoes on, takes interest in the family activities, is looking to manipulate the computer, begins drawing, doesn't cry, says some words and some sentences. At school, he begins to participate in the games, takes a book, shows the pictures and repeats the words. The improvements are spectacular. After six months of treatment, he can pronounce complete sentences and draws. After eighteen months of therapy, the parents stopped therapy, however the behavioural disorders reappeared with incessant restlessness at home and school in two weeks. Another attempt to stop the treatment was also unsuccessful where he became tired and depressed. The parents now do not want to stop the treatment anymore.

Today, patient ARN is twelve, doesn't present a behavioural disorder and communication and language is almost normal. He begins high school and has taken Mg-B6 supplementation for eight years (additional biological data: karyotype is 46 XY; fragile X negative).

Clinical Case Study 4

Patient FLO was four years old when the diagnosis of typical autism was made. When he was seven years old, the parents were informed about our work on Mg and autism. He presented communication disorders, could not emit sounds, didn't talk, had abnormal visual reactions, didn't look someone in the eyes, had a blank look and his look was shifty. The behavioural disorders were important with many anxiety attacks, a difficulty to calm, impulsiveness and constant movement with fits of anger. He had no creativity for games and didn't know how to play. Erythrocyte Mg was low. The same observation was made in his parents (Figures 5-7), despite them having a balanced diet including fishes, meats, fruits and vegetables.

Figures 5.

Evolution of biological parameters for magnesium and calcium in the case of patient FLO (born November 1999) during the follow-up (ASD/PED syndrome). Ser-Mg: total serum magnesium concentrations in mmol/l; Erc-Mg: magnesium concentrations in erythrocytes (obtained after red cell lysis) in mmol/l; Total Ca: total serum calcium concentrations in mmol/l; Ionized calcium concentrations in mmol/l in serum.

Figure 6.

Evolution of biological parameters for magnesium and calcium in the case of patient FLO’s father during the follow-up. Ser-Mg: total serum magnesium concentrations in mmol/l; Erc- Mg: magnesium concentrations in erythrocytes (obtained after red cell lysis) in mmol/l; Total Ca: total serum calcium concentrations in mmol/l; Ionized calcium concentrations in mmol/l in serum.

Figure 7.

Evolution of biological parameters for magnesium and calcium in the case of patient FLO’s mother during the follow-up. Ser-Mg: total serum magnesium concentrations in mmol/l; Erc- Mg: magnesium concentrations in erythrocytes (obtained after red cell lysis) in mmol/l; Total Ca: total serum calcium concentrations in mmol/l; Ionized calcium concentrations in mmol/l in serum.

After two months of treatment with 6 mg/kg/day magnesium and 0.6 mg/kg/day vitamin B6, patient FLO was more cool, stayed at school with pleasure, began to talk with little sentences, was less in “his bubble”, and was looking for his father when he came back from work. He was beginning to play with friends. After three years of treatment, FLO talks appropriately, understands the language, goes to a normal school where his reading skills have developed. During the first discussion, the parents advise that they want a third child. They both have evidence of important Mg depletion. A preventive Mg supplementation both before the desired pregnancy and during the pregnancy seemed to be essential, and indeed allowed good development of the baby without baby blues in the mother. The little sister is now two years old, bright, very much smiling and is always in a good mood! (Additional biological data: search for mutation in the gene neuroligine NLGN3 was negative; search for mutation in the gene neuroligine NLGN 4 X was also negative).

Experimental Study Number 2

In order to study the effect of Mg-B6 for treating social, communication and behavioural responses of children with pervasive developmental disorders (PDD) or autism in connection with the Mg/calcium status of the child, we designed an open study on 33 children with PDD syndrome. Our results showed a statistically significant improvement of the symptoms after Mg-B6 supplementation together with a rise in Erc-Mg values (Mousain-Bosc et al., 2006).

Intraerythrocyte Mg (Erc-Mg), serum Mg (s-Mg) and blood ionized calcium (i-Ca) were measured at different times. Clinical symptoms of PDD were scored (0 to 4). In contrast to s-Mg or i-Ca, PDD children exhibited significantly lower Erc-Mg values than controls (1.26 times; 16/33). The Mg- B6 regimen led to an increase in Erc-Mg values (1.18 times, 11/17) and this supplementation improved PDD symptoms in the large majority of children with no adverse effects: social interactions (23/33, p<0.0001), communication (24/33, p<0.0001), stereotypical restricted behaviour (18/33, p<0.0001), and abnormal/ delayed functioning (17/33, p<0.0001); 15/33 children improved in the first three groups of symptoms. When the Mg-B6 treatment was stopped, PDD symptoms reappeared in a few weeks. A statistically significant relationship was found in Erc-Mg values from children before treatment and their mothers.

The neurobiological basis of a Mg/vit B6 supplementation supposes the existence of an impaired neuronal Mg pathway which could be reversed with Mg-B6 therapy. As we have previously discussed, Mg acts as an ionic membrane regulator and modulator of ion transfer through membrane channels. In brain, it has been shown that traumatic injury causes a decline in Mg²⁺ concentrations, focally as well as in the blood circulation, and contributes to the development of neurologic deficit (Vink et al., 2009). Similarly, brain ischaemia caused a decline in intracellular free Mg²⁺ concentrations (Basso et al., 2000) and Mg salt administration improves motor outcome in this situation (Ebel and Gunther, 2005). One of its most important modes of action is to inhibit the glutamate N-methyl-D- aspartate (NMDA) channel (Zilbovicius et al., 2000). The activity of this channel generates an influx of calcium and, in turn, leads to excitotoxic cell death and apoptosis (Borella et al., 1993). In the same way, abnormal dietary deficiency of Mg as well as abnormalities in Mg metabolism play important roles in different types of heartdiseases, and Mg influences catecholamine signalling in such diseases (Gervais et al., 2004).

Recently, in primary autistic children, positive emission tomography (PET) has been used to demonstrate a significant decrease in cerebral blood flow localized to the temporal lobes in 16/21 of children (Zilbovicius et al., 2000; Macdonald et al., 2004; Demougeot et al., 2004). Taken together with the fact that Mg has been shown to increase blood pressure (Macdonald et al., 2004) and that brain from rats fed with low Mg diets are more susceptible to permanent brain focal ischaemia (Demougeot et al., 2004), we can hypothesize that intracellular Mg²⁺ depletion could be responsible, at least in part, of some central activity disorders observed in PDD/autistic children.

In our study, an intra-erythrocyte Mg depletion was evidenced in almost half of the PDD children. To explain such a phenomenon, two hypotheses can be proposed: (i) a metabolic inhibition of membrane Na⁺/K⁺ ATPase (observed in autism (Kurup and Kurup, 2003) with concomitant rise in intracellular calcium and decrease in intracellular Mg²⁺; (ii) a genetic defect in magnesium transport through plasma membrane (Na⁺-Mg²⁺ exchanger (Ebel et al., 2004; Ebel and Gunther, 2005) or TRPM chanzymes (Montell, 2005). As Erc-Mg can be considered as representative of some intracellular Mg²⁺ concentrations, a decrease in Erc-Mg without changes in serum Mg concentration could be interpreted as an alteration of Mg²⁺ transport through the plasma membrane. The demonstration that TRPM7 is critical for Mg² homeostasis evoked the possibility that mutation of TRPM channels may cause disease in humans as a result of reduced intracellular Mg²⁺ levels. Indeed, mutations were found in the case of hypomagnesemia with secondary hypocalcemia (Schlingmann et al., 2008) and, in this case, symptoms associated with TRPM6 mutations were improved by supplementation with high Mg doses, in agreement with increased Mg entry through a passive mode of Mg influx. This genetic hypothesis was also supported by our own data showing a positive correlation between low Erc- Mg values in PDD children and their mothers. Similarly, Feillet-Coudray et al., (2006) have found that, in mice genetically selected for low magnesium levels, Mg efflux from erythrocytes was significantly increased. The genetic regulation of erythrocyte Mg content depends on the modification of Mg influx (Schlingmann et al., 2002). To confirm such a hypothesis, a genetic study of a PDD child’s family has clearly to be developed.

When PDD children were supplemented with Mg- B6 treatment, Erc-Mg values more or less increased in only 65% of children. The impair- ment to get normal Erc-Mg values under Mg- vitB6 treatment supports the hypothesis of a defect in Mg transport in erythrocytes. In sickle cell disease, Mg pidolate supplementation was found to decrease Na⁺/Mg²⁺ exchanger activity with a partial rise in Mg and K⁺ content of eryth- rocytes (De Franceschi et al., 2000). Doses of Mg- B6 and the duration of treatment, which have not been taken into account in our study, could also explain such an observation. Concerning the respective roles of pyridoxine and Mg in these observations, it was classically accepted that Mg is associated with pyridoxine to decrease irritable side-effects of the B6 therapy and that B6 is the main factor involved in the improvement of clinical symptoms in autistic patients. Following Erc-Mg values during Mg-B6 treatment, we bring here evidence of the role of Mg itself in this therapy.

Mg-B6 treatment of PDD children was shown to improve symptoms of the disease. Three of the four main groups of clinical signs described in DSM-IV were significantly reduced and, for the first time, we found that 8/12 of children who improved under treatment showed higher Erc-Mg values. Persons only slightly deficient in Mg become irritable, highly-strung, sensitive to noise, hyperexcitable, apprehensive and beligerent. If the deficiency was more severe or prolonged, they may develop twitching, tremors, irregular pulse, insomnia, muscle weakness, jerkiness, and leg and foot cramps. These symptoms can also be found in some cases of PDD/autism. Although this study was an open, non-controlled study, we found a relationship between clinical signs of PDD/autism and a biological parameter, namely Erc-Mg. However, we were unable to establish any correlation between improvement of symptoms and increase in Erc-Mg. Various possibilities might explain this lack of correlation. Firstly, Erc-Mg is probably not the best biological parameter to follow the relationship between Mg homeostasis and the neurological dysfunction of PDD/autism. Contradictory reports have been published on the use of Erc-Mg as index of Mg²⁺ status (Borella et al., 1993, Basso et al., 2000) and new biological tests that could help to study genetic alterations of Mg transport (lymphocytes, etc.) have to be tested. Secondly, other neurofunctional disorders may be involved in autism, such as a decrease in temporal blood flow. Even if low Erc-Mg levels have been correlated with a decrease in blood pressure, there is no evidence to associate blood pressure and cerebral blood flow in all cases.

In conclusion, this study presents new information about the therapeutic role of a Mg- B6 regimen in children with PDD syndrome. This effect seems to be associated, at least in part, to a cellular Mg depletion as evidenced by intraeythrocyte Mg measurements. Children with pervasive developmental disorders (including autism) exhibit low Erc-Mg levels. Parents frequently showed similar low Erc-Mg values suggesting a genetic defect in Mg transport. Installing a Mg-B6 supplementation for some weeks restored intraerythrocyte Mg values and significantly reduced the clinical symptoms of these diseases.

Etiology: role of magnesium during pregnancy

Since developmental disorders appear early during fetal development, Mg therapy could be justified even during pregnancy. The efficacy of Mg supplementation is more important if treatment is in the early years of life. Magnesium activates protein and amino acid synthesis and Mg deficiency leads to fetal growth retardation by reducing the nutritive utilization of protein as a result of decreased protein absorption and synthesis. Another study found a low cerebral blood flow in the temporal lobe probably due to low Mg levels in the cells (Zilbovicius et al., 2000).

Effect of magnesium in fragile-X syndrome

Fragile-X syndrome is an X-linked disorder characterized primarily by speech delay and moderate mental retardation and neuro- behavioural disorders. The incidence of fragile X syndrome is estimated at 1/4000-1/6000 in males and half that for females (Wiesner et al., 2004). This disease is linked to the mutation of the FMR1 gene located on the X chromosome, characterized by expansion (CGG amplification) at the FRAXA site (Xq27.3) in the non-coding region of the first exon (Weisman-Shomer et al., 2002). These authors also showed that two factors were indispensable for the stabilization of the CGG tetraplex: magnesium and ATP.

We report a single case of a child referred to our pediatrics unit for a behavioural disorder and for whom Fragile X syndrome had previously been genetically confirmed. Considering the genetic origin of this disease, performing the clinical and biological evaluation of the patient’s family completed our work. Both the patient and his affected family members were given a Mg and vitamin B6 supplementation and evaluated over three years. The family were followed clinically and biologically for the three years, including the children with the mental retardation, pervasive development disorder or attention deficit hyperactivity disorder. All children carried a mutation produced by expansion at the FRAXA site at Xq 27.3 and showed Mg and calcium disorders (normal serum Mg concentration, but decreased erythrocyte Mg and plasma ionized Ca²⁺). The mother presented a premutation by expansion at the FRAXA site at Xq 27.3 with symptoms of Mg depletion (emotivity, asthenia, stress). Mg-B6 supplementation (6mg/kg/day Mg and 0.6 mg/kg/day vitamin B6, orally) for three years reduced clinical symptoms in the mother and improved the behaviour of the children (aggressiveness, lack of attention at school) concomitant with an increase of intraerythrocyte Mg. When the Mg^-B6 treatment was stopped for two months in one of the children, clinical symptoms reappeared.

In 2007, the hypothesis was put forward that Fragile X syndrome (Fra-X) is caused by the transcriptional silencing of the FMR1 gene that encodes the Fra-X mental retardation protein (FMRP) (Dolen et al., 2007; Hayashi et al., 2007). However, the pathogenesis of this disease is unknown. According to one proposal, many psychiatric and neurological symptoms of Fra-X result from the unchecked activation of mGluR5, a metabotropic glutamate receptor. To test this hypothesis, FMR1 mutant mice presenting a 50% reduction in mGluR5 expression were generated and studied in terms of the range of phenotypes with relevance to the human disorder. Results demonstrated that mGluR5 contributes significantly to the pathogenesis of the disease, a finding that has significant therapeutic implications for Fragile X and related developmental disorders. In line with this observation, Hou et al., (2006) demonstrated that mGluR-LTD induces a transient, translation- dependent increase in FMRP that is rapidly degraded by the ubiquitin-proteasome pathway.

The successful management of this disease by Mg- B6 supplementation observed in the present study, with an early and late evaluation at 3 years, supports the argument that a deficit in Mg influences the appearance of Fra-X and that by replacing this Mg, the disease symptoms may be managed. Berthelot’s group (Martin et al., 2007) has previously demonstrated in rats that a deficit in Mg acts by reducing the level of proteasomes, protein complexes that act at the level of the mGluR5 receptor already implicated in this disease. Conversely, Picado et al., (1994) noted an increase in the levels of Mg/NA in patients with behavioural problems and for whom hypertension was diagnosed. It has been demonstrated that for behavioural disorders in which hyperactivity and autism in particular are involved, for which current treatment regimens are difficult to standardize, management of disease symptoms with Mg-B6 supplementation is possible (Mousain-Bosc et al., 2006). Magnesium depletion represents one possible explanation for the appearance of Fra-X syndrome. Studies show that Mg-B6 supplementation given to subjects improved their behavioural disorders. The presence of a genetic mutation together with Mg deficiency in mothers may constitute an indication for regular evaluation of Mg status, and supplementation with Mg-B6 during pregnancy. We present herein some unpublished data obtained with 3 cases of Fra-X abnormality (poster presentation in Gordon Research Conference, Ventura, CA, March 2008).

Conclusions

This review brings additional information about the therapeutic role of a Mg-B6 regimen in children with ADHD or ASD/autism syndrome. This effect seems to be associated, at least in part, to a cellular Mg depletion as evidenced by intraeythrocyte Mg measurements. Children with ADHD or PDD/ASD (pervasive developmental disorders/autistic spectrum disorders), including autism, exhibit low Erc-Mg levels. Parents frequently showed similar low Erc-Mg values suggesting a genetic defect in Mg transport. Installing a Mg-B6 supplement-ation for some weeks restored higher intraerythrocyte Mg values and significantly reduced the clinical symptoms of these diseases.

However, in a recent meta-analysis of all studies, Nye et al., (2010) arrived at the same conclusion as what they published in 2005: “Due to the small number of studies, and the methodological quality of studies, no recommendation can be advanced regarding the use of B6-Mg as a treatment for autism. There is simply not sufficient evidence to demonstrate treatment efficacy”. Together with the fact that both the American Psychiatric Association and the American Academy of Pediatrics have stated that megavitamin treatment for learning disabilities and autism is not justified, it helps explain the dearth of clinical studies investigating the use of magnesium and vitamin B6 (Mg-B6) in the treatment of autism and autistic spectrum disorders.

Magnesium is known to be crucial for brain activity and its involvement in the prevention of neurobehavioural diseases seems to be established. As a clinical double-blind study with Mg-B6 treatment over placebo cannot be accepted for regulatory and ethical reasons, it was suggested to put children under an “alternative treatment” before the conventional drug therapy. We hope that the combined use of new tools to measure intracerebral Mg²⁺ levels as proposed by the group of Iotti et al., (2008), and a more specific clinical evaluation, will help to improve the outcome of children with these pathologies.

Acknowledgements

The authors would like to express their thanks to Professor J.P. Rapin (Dijon) who help them in the conception of the work.

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