Discussion of Nutritional/Metabolic Changes
In general, although we focus on averages in the following sections, it is important to realize the breadth of the distributions. So, although children with autism may (for example) have average levels of vitamin B1, there is a subset with lower levels, so increases in the average level of vitamin B1 may be more beneficial to those with lower levels. Similarly, although children with autism tend to have lower levels of (for example) glutathione, some children have normal levels, but many have low levels; improvements in the average level are probably most beneficial to those with lower levels.
Also, it is important to point out that "average" levels of the neurotypical group may not necessarily be optimal, as they were on typical western diets that are probably not nutritionally optimal.
Overall, the supplement increased the level of most vitamins, including vitamins B1, B3, B5, B6, folic acid, B12, C, E, and biotin. It appears that higher levels of vitamin B2 are needed in the supplement to affect blood levels. Carotene levels improved, but were still somewhat low, so higher amounts are needed. It is interesting that supplementation with carotenes and modest amounts of vitamin A did not significantly alter vitamin A levels, which remained normal; this is consistent with the body only converting carotenes to vitamin A if vitamin A levels are low.
The supplement also improved two functional biomarkers in urine, FIGLU and methylmalonic acid, indicating the supplement decreased the need for folic acid and vitamin B12, respectively. Levels of FIGLU and methylmalonic actually decreased somewhat below the levels of the neurotypical controls; this may be a good result, as some typical children do not have optimal nutritional intake. SAM levels normalized, and uridine levels improved but did not normalize, suggesting that more vitamin B12 and/or folinic might be needed.
Vitamin C levels in the autism group were initially somewhat above that of the neurotypical group, and the supplement raised those levels significantly. This is probably beneficial, as the children with autism initially had high oxidative stress, and the supplement significantly decreased the level of oxidative stress, probably in part due to the vitamin C in the supplement.
Vitamin D decreased in both the treatment and placebo group - this was apparently a seasonal effect, as the study began in the summer/fall, and ended in the fall/winter, and most vitamin D in the body is produced by sunlight. It appears that much higher levels of vitamin D are needed to affect blood levels of vitamin D.
Caution needs to be used in interpreting the results for minerals, as absolute levels are not necessarily the best way to measure body stores and the need for minerals. Also, there is some debate over which compartment (WB, RBC, serum, urine, etc) is the best to use in measuring a given mineral. For a full discussion of these complex issues, see Gibson 2005 [32
Overall, the supplement tended to increase the levels of many essential minerals, including calcium, iodine, lithium, manganese, molybdenum, and selenium. The increase in lithium levels was large (this form of lithium was very well absorbed), so less lithium may be needed in future studies. Magnesium levels in whole blood significantly increased and normalized, but there was a possible decrease in RBC levels, and no change in serum levels, which is somewhat inconsistent; however, overall, it seems that the increase in whole blood levels was the most significant/important.
The supplement also normalized RBC iron, from slightly (but significantly) higher initial levels compared to the neurotypical average, to levels close to that of the neurotypical group. RBC iron is a measure of the total iron in the RBC, and about 65% of the body's iron is in the RBC [33
], so RBC iron may be a reasonable indicator of total body stores of iron. The importance of elevations in RBC iron (statistically significant), serum ferritin and serum iron (non-significant) are unclear; supplementation resulted in all three declining to the neurotypical average level. Increases in serum ferritin and altered iron metabolism are known to occur with inflammation. This may also hold for increased oxidative stress. In the baseline evaluations the regression analysis found that RBC iron was significantly associated with all three assessments of autism severity (p < 0.01) [20
]. Based on these findings, further evaluation of iron metabolism in autism is warranted.
Levels of copper, and zinc were not significantly affected (note that copper is not included in the supplement since children with autism seem to generally have adequate or slightly high levels of it). It should be pointed out that zinc levels began and ended in the normal range. It is possible that increased zinc supplementation would normalize the slightly elevated copper levels. Chromium decreased to a normal level, but the change was not significant.
Regarding the placebo group, there was a significant increase in manganese, but other changes appeared to be small fluctuations around the average level in neurotypical children.
The supplement substantially improved sulfate status, but sulfate levels were still low, suggesting that higher levels of MSM or other sources of sulfate such as Epsom salt (magnesium sulfate) baths are needed. Sulfur is the third most common mineral in the body [34
]. Most sulfate is produced in vivo
by metabolism of cysteine [35
]. Sulfation is important for many reactions including detoxification, inactivation of catecholamines, synthesis of brain tissue, sulfation of mucin proteins which line the gastrointestinal tract, and more. The measurement of total plasma sulfate involves many substances in the plasma, including neurotransmitters, steroids, glycosaminoglycans, phenols, amino acids, peptides, and other molecules. Low free and total plasma sulfate in children with autism has been previously reported in two studies [36
], and is consistent with four studies [36
] which found that children with ASD had a significantly decreased sulfation capacity compared to controls, based on decreased ability to detoxify paracetamol (acetaminophen). The finding of low plasma sulfate is also consistent with a large study that found high sulfate in the urine of children with autism [41
], as sulfate wasting in the urine partly explains low levels in the plasma. ATP is required for the kidneys to resorb sulfate, and the accompanying study [20
] found that ATP was moderately correlated with levels of free and total plasma sulfate (r = 0.32 and 0.44, respectively), so this suggests that low levels of ATP are a contributor to decreased sulfate in children with autism. One study [41
] also reported high levels of urinary sulfite in children with autism, suggesting that there was a problem of converting sulfite to sulfate in the mitochondria. In 38% of cases (14/38) urinary sulfite and sulfate levels improved by giving 50 mcg of molybdenum, presumably since the enzyme for converting sulfite to sulfate (sulfite oxidase) contains molybdenum. The vitamin/mineral supplement in this study contained molybdenum (150 mcg for a 30 kg child), so this may also have contributed to increases in sulfate levels.
Methylation, Glutathione and Oxidative Stress
Methylation improved to near-normal levels, as indicated by improvements in SAM and uridine. SAM is the primary methyl donor for methylation of DNA, RNA, proteins, phospholipids, and neurotransmitters. The improvement in SAM may in part be due to improvements in ATP, since that is the co-factor needed to convert methionine to SAM. The methylation pathway is diagrammed in Figure .
Conversion of Methionine to SAM to SAH to Homocysteine. Homocysteine is then either recycled to methionine or converted into cystathionine.
The supplement also substantially improved glutathione (an important anti-oxidant and defense against toxic metals). The supplement substantially reduced oxidative stress to near-normal levels, as evidenced by improved ratio of GSSG:GSH and improved levels of nitrotyrosine. NADPH is the co-factor needed to recycle GSSG to GSH (see Figure ), so normalizing the level of NADPH probably was the major factor in improving the GSSG:GSH ratio.
Reduction of GSSG to GSH (net result of a more complex process which involves FADH).
Previous studies [9
] have demonstrated that oral folinic acid, oral trimethylglycine, and subcutaneous injections of methyl Vitamin B12 were able to greatly improve methylation, glutathione, and oxidative stress, similar to the results here. This suggests that the oral supplement used in this study may be a reasonable alternative to subcutaneous injections of methyl-B12. Oral intake of vitamin B12 has a complex absorption mechanism involving "intrinsic factor", and typically only 1% of oral vitamin B12 is absorbed, so it is interesting that the levels used in this study were sufficient to substantially improve methylation, glutathione, and oxidative stress. The vitamin C in the present supplement probably also helped reduce oxidative stress.
The current observed improvements in methylation and GSH are similar to effects of treatment with NADH [42
] and ribose [42
], but neither ribose nor NADH had significant effect on improving levels of GSSG after two weeks.
ATP, NADH, NADPH, CoQ10
ATP, NADH, NADPH, and CoQ10 are important co-factors for many metabolic processes in the body. ATP is a primary energy source for the body and the brain. The CoQ10 in the supplement was very well absorbed, so that the relatively modest dosage resulted in a large, significant increase in CoQ10 levels. The supplement significantly increased the plasma levels of ATP, NADH, and NADPH, from about 25% below normal to normal levels. Plasma ATP may be a biomarker of general ATP status in the body, and may be related to overall level of ATP, and/or the ability to recycle ATP, and/or the ability to transport ATP where needed - more research is needed to interpret the importance of plasma ATP. Many children with autism have low muscle tone and impaired endurance, and it is interesting to hypothesize if those symptoms relate to decreased ATP levels, and if improvements in plasma ATP will result in improvements in muscle tone and endurance - those symptoms were not assessed in this study, but would be interesting to assess in future.
The results of vitamin/mineral supplementation on ATP, NADH, NADPH is similar to the results of supplementation with NADH [42
] and ribose [42
], since NADH is easily converted to NADPH, which is a co-factor for making ribose, which is a building block of ATP, NADH, NADPH, and many other important substances.
Primary and Secondary Amino Acids
There were no significant or marginally significant changes. Most small changes in primary and secondary essential amino acids appeared to involve modest fluctuations around the average level of the neurotypicals. One possible exception is the slight decrease in serine coupled due to increase in glycine. Serine is converted to glycine in an enzymatic reaction requiring tetrahydrofolate as a co-factor - this may suggest a small increase in production of tetrahydrofolate.
A previous paper [20
] reports on a comparison of the nutritional and metabolic status of the participants taking medications vs. those not taking medications. The only differences with a p-value less then 0.01 were lower RBC copper (-9% lower, p = 0.001) and higher plasma methionine sulfoxide (+35% higher, p = 0.002) for the autism medication group compared to the autism no-medication group. The sample size in this paper is too small to determine if medications had an effect on changes in the nutritional and metabolic status of the treatment group, but since no changes in medication were made during the study, this was probably a minor effect at most.
Discussion of Placebo
The placebo group had a few significant changes, including significant increases in biotin, CoQ10, WB manganese, and SAM. In all four cases the supplement group had similar changes (biotin, WB manganese) or larger changes (CoQ10, SAM). Some of these findings might be due to laboratory error (drift in standards), but that seems unlikely. Some of the changes may be due to random fluctuations in diet, or possibly due to seasonal effects (i.e., baseline values were measured in summer/fall (June-October) and final values in fall/winter (September to January). Finally, it may be that the natural plant-based flavorings used in the placebo (not in the supplement) contained modest amounts of biotin and other nutrients.
Discussion of Effect on Symptoms
The supplement group had significantly greater improvement that the placebo group on the Average Change of the PGI-R. The supplement group had greater improvement than the placebo group on all of the subscales, with several of the results being significant (p < 0.005), marginally significant (p < 0.01), or possibly significant (p < 0.05). Although the magnitude of the effects were modest, the supplement group reported roughly twice the improvement as did the placebo group on the Average Change score (the average of all the PGI-R scores). Since the supplement resulted in many significant improvements in nutritional and metabolic status after three months, we hypothesize be that the child's overall health and learning ability is improved at that point, but that more time may be needed for the increase in learning ability to fully translate into greater skills in language, social understanding, and behavior.
For the other three assessment tools, the supplement group also had a slightly greater improvement than did the placebo group, but the effect was not significant. This suggests that the PGI-R is more sensitive at detecting changes, which is what it was designed for, whereas the other scales measure overall autism severity. It should be noted that the PGI-R uses a 7-point scale, whereas the ATEC and PDD-BI use a 3 to 4 point scale, and that may be part of the reason why the PGI-R appears to be more sensitive. More importantly, the PGI-R directly assesses the degree of improvement, whereas the other assessment tools only indirectly assess the degree of improvement by calculating a small difference between two large numbers (initial and final), which leads to a greater uncertainty in the degree of improvement.
Correlation with Biomarkers
The correlation of three biomarkers with the Average Change of the PGI-R is interesting. The correlations need to be interpreted cautiously, because the sample size (only the Arizona treatment group) is small. The autism group had lower levels of biotin than did the neurotypical group (-20%, p = 0.001) at the start of the study, and the supplement significantly increased levels of biotin (+51%, p = 0.008) in the treatment group. So, it makes sense that children with low levels of biotin would be more likely to benefit from supplementation. Biotin is an important co-factor for four carboxylases that regulate gluconeogenesis (generation of glucose from non-carbohydrate sources), fatty acid synthesis, and the Krebs cycle.
The autism group initially had levels of vitamin K that were similar to the control group. Vitamin K was the only vitamin not included in the supplement, and the level did not significantly change (+15%, n.s.) during the study. The primary role of vitamin K is in blood coagulation, which is not reported as a common problem in autism, which is why it was not included in the supplement in this study. However, a previous study [20
] found by regression analysis that levels of vitamin K were somewhat associated with variation in the severity of autism. So, the correlation of vitamin K with degree of improvement is puzzling.
What biotin and vitamin K have in common is that both are made in substantial amounts by beneficial intestinal bacteria. It is estimated that approximately half of the biotin and half of the vitamin K in humans comes from their intestinal bacteria [43
]. One of the common causes of biotin or vitamin K deficiency is antibiotic usage, because some antibiotics destroy the beneficial bacteria that produce them [43
]. Several studies have reported that one major difference in the medical history of children with autism compared to neurotypical children is a much higher usage of oral antibiotics antibiotic [44
]. So, it could be that excessive oral antibiotic usage contributed to lower levels of biotin and vitamin K in some children. Vitamin K occurs in two natural forms, vitamin K1 (phylloquinone) produced by plants, and vitamin K2 (menaquinone) produced by intestinal bacteria. In this study we measured total vitamin K (K1 plus K2); in future studies it would be interesting to measure both forms individually.
We analyzed the possible correlation of levels of vitamin K and biotin in the autism group at the start of the study, and found that they were significantly correlated (r = 0.44, p < 0.001). This is consistent with both being partially produced by beneficial intestinal bacteria. One study found a very high correlation of GI problems with autism severity (r = 0.59, p < 0.001) [48
]. So, it appears that the correlation of improvement in autism symptoms with biotin and vitamin K may relate to a lack of beneficial bacteria which produce biotin and vitamin K, so that supplementation with biotin was beneficial. This suggests that supplementation with vitamin K would be beneficial, especially for those with low levels of vitamin K (the standard deviation of vitamin K levels in the autism group was large).
The negative correlation of lipoic acid with the Average Change of the PGI-R is interesting. The supplement did not contain lipoic acid, and it did not affect levels of lipoic acid, so it appears that children were more likely to improve if they already had sufficient lipoic acid, whereas a lower level of lipoic acid seemed to be associated with less improvement. However, this correlation is not as strong as that for biotin and vitamin K. More research into supplementation with lipoic acid may be warranted.
The regression analysis found that the Average Change of the PGI-R was very strongly associated with several biomarkers, with vitamin K and biotin being the most significant. This suggests that children with low biotin or low vitamin K were most likely to benefit from the multi-vitamin/mineral supplement, for reasons discussed in the preceeding section. This suggests that vitamin K should be added to future formulations.
It is important to realize that vitamin levels are not independent variables, but are usually significantly correlated with one another, because they often occur in the same foods. So, in interpreting these results, it may be that biotin and vitamin K are also markers of overall nutritional status, and their individual importance may be less important.
At the start of the study the children with autism had many statistically significant differences (p < 0.001) in their nutritional and metabolic status compared to the neurotypical group [20
], including: Low levels of biotin, glutathione, SAM, plasma ATP, NADH, NADPH, plasma sulfate (free and total), and plasma tryptophan; also high levels of oxidative stress biomarkers and evidence of impaired methylation (high uridine). By the end of the treatment study, these biomarkers had all improved or even normalized. Also, the baseline study [20
] found that levels of several vitamins, minerals, and amino acids were strongly associated with variation in autism severity. Vitamins and minerals act as enzymatic co-factors for hundreds of important enzymatic reactions in the body, so low levels of them can result in impaired metabolic functioning. Also, many genetic variations result in impaired enzymatic activity, resulting in an increased need for vitamin/mineral co-factors for normal metabolic functioning. This study was only able to assess a limited portion of human metabolism, and it is likely that other metabolic problems exist in children with autism and possible that the vitamin/mineral supplement could improve other problems as well as those reported here. For example, vitamins and minerals are required co-factors for the production of many neurotransmitters and their pre-cursors, so vitamin/mineral supplementation may have also affected neurotransmitter status, and that may have contributed to improvements in autism severity and overall symptoms. So, it is not surprising that nutritional supplementation would improve metabolic functioning in some children with autism, and it is very interesting that nutritional supplementation also resulted in significant improvements in the Average Score of the PGI-R, as well as improvements in several of its subscores. Some children improved much more than others, presumably because some had a greater need for nutritional supplementation.
This study is consistent with several other studies that reported that vitamin/mineral supplementation is beneficial in treating children with autism. A 30-week, double-blind, placebo-controlled study [12
] of high-dose vitamin C (110 mg/ kg) found a reduction in autism severity. One open-label study [49
] found that micronutrient supplementation was comparable or more effective than treatment with pharmaceuticals in terms of improvements in the Childhood Autism Rating Scale, Childhood Psychiatric Rating Scale, Clinical Global Impressions, and Self-Injurious Behavior. A small randomized, double-blind, placebo-controlled study of a moderate-dose vitamin/mineral supplement was found to be beneficial to children with autism, primarily in the areas of sleep and gastrointestinal symptoms [14
]. There have been many studies of high dose vitamin B6 therapy in children with autism [13
], with most showing beneficial effects; those studies investigated very high dosages, generally 500-1000 mg, compared to the current study which investigated a lower dosage (40 mg for a 30 kg child) which is still substantially higher than the RDA (0.6 mg), and was sufficient to substantially increase P5P levels inside RBC (+187%, p < 0.001). Some children and adults may benefit from adding high-dosage vitamin B6 to a broad-spectrum vitamin/mineral supplement such as investigated in this study.
Compared to other treatments, the administration of a vitamin/mineral supplement requires only a few minutes a day, is relatively inexpensive, and is very safe. Although it will not help all children and adults with autism, it appears that a significant percentage are likely to improve to some degree after only three months, and longer-term use is likely very safe and may result in even greater benefits. Also, the vitamin/mineral supplement improved many nutritional and metabolic problems. So, vitamin/mineral therapy seems to be a reasonable adjunct therapy for helping some children and adults with autism, and can be easily used in conjunction with other therapies (behavior therapy, speech therapy, etc.).
Limitations of this study
1) The diagnosis of an autism spectrum disorder by a qualified medical professional was verified in writing, but there no additional verification. Similarly, for the neurotypical children, no additional verification was made beyond the parental report. The supplement group included a somewhat higher fraction of individuals with classic autism than did the placebo group, since random assignment was done and severity of diagnosis was not controlled for. However, the effect on the results is probably small, since the analysis investigated the change in symptoms, not the final symptoms only.
2) The sample size was large enough to observe many major significant differences between the two groups; but a larger sample size is needed for appropriate statistical power for more subtle, possibly significant differences.
3) The formulation of the supplement was very good; the present data suggests ways to further improve composition and dosage optimization and titration.
4) Seasonal changes slightly affected some results (vitamin D) and possibly others.
5) The placebo contained small amounts of natural plant-based extracts that may have slightly affected some results.
6) Some of the children (45%) were taking various types of medications, which did not change during the study. A comparison of the baseline levels of the autism groups taking and not-taking medications revealed little difference between the two groups in their nutritional and metabolic status [20
]. There was a trend that the medicated group had less improvement than the unmedicated group in the Average Score of the PGI-R.
7) The length of the study (three months) may not have been long enough to observe the full-effect of the supplement, and longer treatment may result in larger effect.