Urinary Excretion - Toxic Metals
One participant's results were excluded from the general analyses in this section and elsewhere because she had extremely high levels of many toxic metals both at baseline and after taking DMSA, including extremely high baseline aluminum (63× average of other participants), antimony (45× average), bismuth (40× average), cadmium (7× average), lead (12× average), tin (12× average) and uranium (65× average). After DMSA, her excretion of antimony increased to 24× her baseline (1000× the average baseline), lead increased to 2.4× her baseline, and excretion of other metals was less affected. This participant did not continue into phase 2 so that she could pursue private treatment.
For all other participants, Table and Figure shows the level of urinary excretion of toxic metals (normalized per gram creatinine) at baseline (beginning of Phase 1, before taking any DMSA), and after the first and 9th doses of DMSA in the Phase 1 challenge round. The metals are listed in order of effect of DMSA on excretion. There was a very large and extremely statistically significant increase in excretion of lead. There were also large and significant increases in excretion of tin and bismuth. There was a large increase in excretion of uranium, but it was not statistically significant (only 14-15 participants had detectable excretion of uranium before or after taking DMSA). There was a large and highly significant initial increase in excretion of mercury, but at the 9th dose the excretion was only slightly higher than baseline and not statistically significant. There were significant increases in excretion of thallium, antimony, and tungsten. There was little change in levels of aluminum or cadmium. There was a small initial decrease in excretion of nickel, and a small decrease in excretion of arsenic at the 9th dose only. Due to multiple comparisons (12 metals), only p-values below 0.004 should be considered significant, and p values between 0.05 and 0.004 should be considered marginally significant.
Urinary excretion of toxic metals in Phase 1, at baseline, after 1st dose, and after 9th dose, in mcg/g creatinine.
Figure 2 Percentage change in urinary excretion of toxic metals. a: Percentage Change in Urinary Excretion of Toxic Metals after 1st and 9th Dose of DMSA in Phase 1. (n = 63). b: Percentage Change in Urinary Excretion of Toxic Metals after 1st and 9th dose of (more ...)
The effect of the glutathione lotion on excretion of toxic metals was analyzed by comparing the groups receiving the DMSA + glutathione lotion vs. DMSA + placebo lotion. In general there were no statistically significant differences in the two groups when comparing the average excretion after dose 1 plus after dose 9 vs. baseline. However, the excretion of mercury was possibly higher (p = 0.06) for the group receiving the DMSA + glutathione (average excretion of 1.38 vs 0.91, vs. baseline of 0.73 and 0.80, units of mcg/g creatinine, respectively).
Table and Figure shows the data for urinary excretion of toxic metals for the participants who were in the treatment group in Phase 2 (n = 20). The table lists both their Phase 1 results (1st and 9th dose) and their Phase 2 results (after round 2, 4, and 6 of DMSA in Phase 2). Table lists the metals in the same order as in Table for convenience. Note that the number of participants in this group is smaller than in Table (n = 20 vs. n = 63, respectively), which affects which results are statistically significant. In general, the results for phase 2 were similar to those for phase 1. Overall, there was a very large increase in excretion of lead, some increases in excretion of tin, bismuth, mercury, and thallium, possible ncreases in excretion of uranium, antimony, tungsten, nickel, and decreases in excretion ofi cadmium and arsenic. The increased excretion of lead persisted throughout the entire study, and mercury excretion also generally continued with some fluctuations.
Comparison of change in average urine excretion compared to baseline for participants who completed six rounds of phase 2 (n = 20).
Table lists the correlations of urinary metal excretion with one another at baseline, Table lists the correlations at 9th dose of Phase 1, and Table lists the correlations at 9th dose vs. Baseline. Most of the metals have one or more significant correlations with other metals, so that regression analysis had some difficulty in distinguishing the effect of one metal from another. Since multiple comparisons were made, only p values less than 0.01 are listed.
Significant Correlations of Baseline Urinary Metal Excretion with Baseline Urinary Metal Excretion (only correlations >0.31 in magnitude are listed, corresponding to p = 0.01 on an individual comparison basis)
Significant Correlations of Phase 1, 9th dose Urinary Metal Excretion with Phase 1, 9th dose Urinary Metal Excretion (only correlations >0.31 in magnitude are listed, corresponding to p = 0.01 on an individual comparison basis)
Significant Correlations of Phase 1, 9th dose Urinary Metal Excretion with Baseline Urinary Metal Excretion (only correlations >0.31 in magnitude are listed, corresponding to p = 0.01 on an individual comparison basis)
Urinary Excretion of Essential and Other Minerals
Table and Figure show the average urinary excretion of essential and other metals for Phase 1, including the values at baseline, and the percentage change after the 1st dose of DMSA and after the 9th dose of DMSA in Phase 1. There was a large and statistically significant excretion of copper, potassium, and manganese. Excretion of iron increased and was marginally statistically significant. There was a moderate initial increase of excretion of vanadium, chromium, sodium, and boron, but then they were similar to baseline by the 9th dose. The excretions of zinc and magnesium were initially similar to baseline and then had a moderate increase by the 9th dose. There were large average increases in zirconium and lithium (due to a few outliers), but the increases were not statistically significant. The excretion of barium, sulfur, selenium, cobalt, calcium, strontium, and phosphorus were not significantly affected. There was a moderate decrease in the excretion of molybdenum at the 9th dose which was marginally significant. Since multiple comparisons were made, only p values below 0.003 should be considered significant.
Comparison of average urine excretion of essential and other minerals compared to baseline.
Loss of Essential Minerals, averaged over the 3 days of DMSA therapy in Phase 1, in units of the % RDA. The only major losses are chromium and potassium.
Table also lists the extra loss of essential minerals, in terms of the fraction of the Recommended Daily Allowance (RDA) lost each day, based on two reasonable assumptions: 1) averaging the loss between the 1st and 9th dose, and 2) assuming a daily urinary excretion of 500 mcg of creatinine, which is typical of a 60-pound child. On that basis, there was a large daily loss of chromium (88% of the RDA) and potassium (57% of the RDA). The amount of loss of other essential minerals was negligible (less than 6% of the RDA).
It should be noted that all the participants were taking a vitamin/mineral supplement for at least two months prior to and throughout the study, to minimize effects due to possible loss of essential minerals.
The level of glutathione in the Red Blood Cells (RBC) was measured at baseline and at the beginning of Phase 2 (for those who continued on to phase 2), which was typically 1.5 months (42 days +/- 33) after taking the DMSA. The values are given in Table and Figure . Initially there was a very broad distribution of levels, with some being lower or higher than the lab's reference range for adults, defined as +/- 2 standard deviations around the mean. (It should be noted that children tend to have lower levels than adults, but the laboratory did not have a pediatric reference range.) After administration of DMSA, there was little change in the average level (a small decrease, not statistically significant), but the standard deviation became dramatically smaller. Figure plots the average change in glutathione vs. the initial level. The change in glutathione level had an extremely high inverse correlation (-0.96, p < 0.0001) to the initial glutathione. Specifically, the children with unusually low initial levels had a large increase in glutathione towards the average value, those children with unusually high initial levels had a large decrease towards the average value, and those with initially average values had little change. Note that the change was very similar regardless of whether the participants were receiving the glutathione lotion or the placebo lotion, suggesting that the effect was due to the DMSA.
Glutathione levels before and 1-2 months after 1st round of DMSA in Phase 1 (just prior to Phase 2).
Figure 4 RBC glucathione distributions. a: Initial RBC Glutathione Distribution (pre-treatment). Many autistic children have levels below and above the laboratory's reference range of 427-714 micromolar. Each histogram corresponds to the frequency between that (more ...)
RBC Glutathione and Heavy Metals
Correlation analysis found that baseline RBC glutathione (before DMSA) had significant positive correlations with urinary excretion of several metals (at baseline and after 9th dose of DMSA), including baseline Sb (r = 0.26), and post-9th dose urinary excretion of Pb (r = 0.25), Al-9 (r = 0.29), and Cd (r = 0.30), suggesting that increased levels of these metals correlated with increased RBC glutathione. This suggests that the body responds to increased levels of most toxic metals by increasing the production of RBC glutathione. However, there was a negative correlation of RBC glutathione with excretion of mercury at the 9th dose (r = -0.27), which suggests that mercury has an adverse effect on RBC glutathione, presumably by both causing it to be excreted from the body after the glutathione binds to mercury, and perhaps more importantly by inhibiting production of glutathione. It is very interesting that mercury, and only mercury, has a significant negative correlation with RBC glutathione, and again suggests the special importance and toxicity of mercury.
A stepwise linear regression analysis for predicting initial glutathione was conducted based on the urinary excretion of toxic metals at baseline and after the 9th dose of DMSA. The results are shown in Table . It was found that the initial glutathione could be partially predicted (adjusted R2 = 0.25, p = 0.003) based primarily on excretion of cadmium, lead, tin, thallium, and arsenic.
Regression analysis of initial glutathione and change in glutathione (from before and after phase 1).
A similar analysis was conducted for the change in glutathione between baseline and after the first round of DMSA, and it was found that the change in glutathione could be partially predicted (adjusted R2 = 0.29, p = 0.002) based primarily on excretion of cadmium after the 9th dose in phase 1 (see Table ). This is consistent with the correlation analysis above.
Complete Blood Count
Table shows the results of the initial complete blood count for 77 participants, prior to any treatment. The only somewhat unusual feature at the initial blood draw was that 18% had elevated platelets (reference range of 130-450 k/mm3), a marker of inflammation. The % Lymphocytes tended to be slightly above or below the reference range, but the absolute lymphocytes were almost all within the reference range.
Complete Blood Count and Chemistry Panel at baseline (N = 77).
Table shows the changes in Complete Blood Count (CBC), measured before and 1.5 months (42 +/- 33 days) after the first round of DMSA. The only significant change was a 6% decrease in platelet count (p = 0.02). The decrease in platelet count was primarily in the group with an initial level above the reference range (decrease of 70 k/mm3), whereas the group initially in the reference range had much less change (decrease of 24 k/mm3), and the one child with an initially low level had an increase of 26 k/mm3 The correlation of the. initial platelet level and the change was -0.32, p = 0.05. Figure shows the initial distribution of platelet count, Figure shows the value 1-2 months after the first round of DMSA, and Figure shows the values at the end of phase 2 for both groups. Overall, the many children with initially high levels had a decrease towards the normal range, and the one child with an initially low value had an increase towards the normal range. It is interesting to note that one child had an extremely high initial value of 1996 k/mm3, and it decreased by the end of the study to 1195 k/mm3.
Complete Blood Count and Chemistry Panel at baseline and after 1 round of DMSA (N = 41).
Figure 5 Platelet count. a: Platelet Count before DMSA. Note many children have values above the reference range (130-450 k/mm3). One child had a very high value (1996 k/mm3) which is not shown on this chart. b: Platelet Count 1-2 months after first round of DMSA (more ...)
White blood cell count was investigated carefully, due to concerns about the possible effect of DMSA on it. For the 7-round group there was a 7% decrease in white blood cell count (not significant), with 1 child initially having a high level, and at the end 1 child having a high level and one child ending with a level slightly below the reference range (3.9 k/mm3, compared to a reference range of 4.0-12.0 k/mm3). The child who ended with a slightly low level had started with a level of 4.9 k/mm3; at the beginning of phase 2 it was 3.6, then in the middle of the study it was 5.2 k/mm3, so the total white blood cell count fluctuated near the bottom of the reference range during the study, but at no point was the participant either neutropenic or lymphocytopenic. For the 1-round group, there was an 8% decrease (not significant) in white blood cell count with one child initially having an elevated level, and all being normal at the end of the study.
A stepwise linear regression analysis of initial platelet count did not reveal any significant relationship with urinary excretion of toxic metals (at baseline and after 9th dose) and initial glutathione. However, a similar analysis for the change in platelet count from beginning of phase 1 to the beginning of phase 2 was performed, with excretion of toxic metals after the 9th dose of DMSA and change in glutathione as the dependent variables. It was found that the change in platelet count could be partially explained (adjusted R2 = 0.41, p = 0.02), with the major factors being Tl (p = 0.002), As (p = 0.01), Cd (p = 0.03), and change in glutathione (p = 0.04). Table
Stepwise linear regression analysis of change in platelet count (from baseline to beginning of phase 2, 1-2 months after DMSA therapy), based on excretion of metals after the 9th dose in phase one, and the change in glutathione.
Table shows the results of the initial blood chemistry panel for 77 participants in Phase 1. The major unusual feature was the abnormally high Blood Urea Nitrogen (BUN)/Creatinine ratio, an indicator of kidney function. Initially, 43% of the children had a value above the laboratory's reference range, and none had a level below the reference range. This was largely due to low serum creatinine (22% of the children had levels below the reference range, and none had levels above it). The BUN values were almost all within the reference range, but tended to be in the higher end of it.
Table also shows the changes in blood chemistry, measured before and 1.5 months (42 +/- 33 days) after the first round of DMSA. There were no statistically significant changes in blood chemistry. However, it is interesting to note that initially 17/42 participants had elevated Blood Urea Nitrogen (BUN)/Creatinine ratios, and after the DMSA it decreased 6% (not significant), so that only 14/42 had elevated levels, and 1/42 had low levels. The reason for the initial elevated ratio is largely due to low creatinine (in 8/42 children), whereas none had high initial BUN values. Low creatinine was defined as less than 0.4 (younger) or 0.5 (older) mg/dl. Overall, this suggests there was no worsening of kidney function.
Stepwise linear regression analysis found that the variations in initial BUN/Creatinine ratio could be partially predicted by urinary metal excretion at baseline and after the 9th dose of Phase 1, with an adjusted R2 = 0.35, p = 0.0001, with the most significant factors being baseline Sn (p = 0.003), baseline W (p = 0.02), and 9th dose Sb (p = 0.05).
A similar analysis found that the changes in BUN/Creatinine from beginning of phase 1 to beginning of phase 2 could be partially predicted (adjusted R2 = 0.51, p = 0.05, with the most significant factors being 9th dose Hg (p = 0.01), change in glutathione (p = 0.02), 9th dose Tl (p = 0.04), and 9th dose Pb (p = 0.05).
At the end of phase 2, the 7-round treatment group did not have any significant changes in blood chemistry. There was a possible trend of a small decrease in eosinophils (-18%, p = 0.08), with initially 3/21 high, and only 2/21 high at the end of the study. There was also a possible trend of an increase in triglycerides (+47%, p = 0.06) - initially all were normal, and at the end of the study 2/21 were elevated. In contrast, in the 1-round treatment group, there was a 15% decrease (not significant) in triglycerides levels. It should be noted that these were not fasting measurements, so the fluctuations could be diet-related.
It is interesting to note that there was a 12% increase (not significant) in alkaline phosphatase, with initially 0/42 having abnormal levels, and 5/42 having elevated levels at the end of the study. Elevated alkaline phosphatase is associated with growth spurts, and it is expected that children in the study age range will normally have growth spurts.
Heavy Metal Exposure Questionnaire
Table lists the medical history data that relates to toxic metal exposure, as reported by the parents. Some of these factors were analyzed to determine if they correlated with severity of autism. Maternal fish consumption (a primary source of mercury) and total oral antibiotic use (which inhibits excretion of mercury) over the first 3 years of life did not significantly correlate with any of the autism severity scales at baseline. The number of maternal mercury amalgam fillings did not correlate with the SAS, PDD-BI autism scale or the ADOS total, but there was a negative correlation (-0.33) of the ATEC total with maternal mercury dental fillings - this is unexpected, as it suggests that a higher number of mercury fillings was associated with less severe autism.
Medical History, as reported by parents (n = 73)
Table shows the strong correlation of "eating/licking paint" and pica with the autism severity scores and with metal excretion. It should be noted that the correlation scores are generally quite similar for both the autism severity scales and for metal excretion.
Correlations of symptoms of "Eating/licking paint" and "pica" with symptoms of autism and urinary excretion of metals (at baseline or immediately after the 9th dose of DMSA in Phase 1).