Hello Fellow Researchers!

•February 6, 2010 • 1 Comment

This blog is to help all of you keep up with our research. Please feel free to comment or ask questions. In fact, I invite you to respond.

Update 7.17.11

1. I investigated the potential role of copper in Dimentia. The brain is particularly vulnerable to oxidative damage induced by unregulated redox-active metals such as copper and iron, and the brains of AD patients display evidence of metal dyshomeostasis and increased oxidative stress. The colocalisation of copper and amyloid β (Aβ) in the glutamatergic synapse during NMDA-receptor-mediated neurotransmission provides a microenvironment favouring the abnormal interaction of redox-potent Aβ with copper under conditions of copper dysregulation thought to prevail in the AD brain, resulting in the formation of neurotoxic soluble Aβ oligomers. Interactions between Aβ oligomers and copper can further promote the aggregation of Aβ, which is the core component of extracellular amyloid plaques, a central pathological hallmark of AD. Copper dysregulation is also implicated in the hyperphosphorylation and aggregation of tau, the main component of neurofibrillary tangles, which is also a defining pathological hallmark of AD. Therefore, tight regulation of neuronal copper homeostasis is essential to the integrity of normal brain functions.

Ya Hui Hung, Ashley I. Bush and Robert Alan Cherny Copper in the brain and Alzheimer’s disease JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY 2010;15:61-76.

Tetrathiomolybdate (TTM) is a copper chelator that has also demonstrated antiangiogenic, antifibrogenic and anti-inflammatory actions in preclinical studies. phase II and phase III clinical trials were ongoing in patients with Alzheimer’s disease and primary biliary cirrhosis, respectively. The most common clinical side effects observed for TTM over the range of indications have been anemia, neutropenia, leukopenia and transaminase elevations. These side effects were generally resolved with either a dose adjustment or temporary suspension of the dosing regimen. TTM is predicted to most likely find a niche in the therapy of Wilson disease and therefore possibly in patients with copper toxicity who don’t respond to zinc therapy.

Medici V, Sturniolo GC Tetrathiomolybdate, a copper chelator for the treatment of Wilson disease, pulmonary fibrosis and other indications. IDrugs. 2008 Aug;11(8):592-606.

Zinc-thiolate clusters in Zn7MT-3 can efficiently silence the redox-active free Cu2 ions and may play a protective role of Zn7MT-3 from the Cu2 toxicity in AD and other neurodegenerative disorders.

Gabriele Meloni‡, Peter Faller§, and Milan Vasˇ a´ k‡1 Redox Silencing of Copper in Metal-linked Neurodegenerative Disorders THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 22, pp. 16068 –16078, June 1, 2007

2. I began to organize data for presentation in the paper, Elevated HMGB1 in Autistic Children Correlates With Perceived Symptom Severity.

Plasma HMGB1 (Box Protein) is significantly elevated in autistic children compared to controls (p=0.020).

Autistic children with GI disease have significantly lower HMGB1 than non GI autistic children and controls (p=0.022).

Cu levels correlate with HMGB1 plasma concentration (p=0.05).

HMGB1 levels correlate with symptom severity.

Table 1. Correlation between Plasma HMGB1 Concentration and Perceived Symptom Severity

Hyperactivity   Corr         P-value
-0.579     0.026

Focus/Attention Corr         P-value
-0.386     0.014

Rocking/Pacing  Corr     P-value
-0.635     0.034

Hand Flapping/ Finger Stimming  Corr        P-value
-0.324     0.033

Overall, HMGB1 is an important inflammatory marker and it’s strong correlation with symptom severity (particularly hyperactivity symptoms) suggests the association between inflammation and behavior in autism.

3. I continued to write the paper (above) based on this data.

4.. I analyzed correlation between individual Cu, Zn and Cu/Zn and Pragmatic Language Severity and Focus/Attention Severity in autistic children (N=100).

We did not find a significant correlation between Cu, Zn or Cu/Zn and Pragmatic Language Severity. We found a significant correlation between decreased zinc and focus attention severity (p=0.015) and increased copper/zinc and focus attention severity (p= 0.033).

This continues to support the relationship between zinc and copper levels and behavior in our patients.

5. I began to organize data which supports normalization of zinc and copper as it is associated with improved perceived symptoms in our patients.

Update 7.6.11

1. I continued to analyze the correlation between Cu, Zn and Cu/Zn and outcomes in autistic patients associated with the GI/probiotic study, and found the following correlation with stimming, obsession/fixation and sound sensitivity.

Cu      Improved Stimming
Correlation     0.376
p-value (1 sided)       0.050
Zn      Stimming
p-value (1 sided)       0.080

Zn      Improved Obsession/Fixation
Correlation     -0.498
p-value (1 sided)       0.020
Cu/Zn   Improved Obsession/Fixation
Correlation     0.3228
p-value (1 sided)       0.111

Zn      Improved Sound Sensitivity
Correlation     -0.286
p-value (1 sided)       0.132

2. I analyzed outcomes in autistic patients with respect to those taking probiotics, with and without GI disease., pre and post zinc therapy.

I found a improvement in awareness, receptive language and hyperactivity after zinc therapy.

Awareness Pre Therapy   Awareness Post Therapy
Mean = 2.884    Mean = 1.757
Standard Deviation = 1.416      Standard Deviation = 1.195
Standard Error = 0.392  Standard Error = 0.205

p = 0.019

ANOVA
p=0.14

Receptive Language Pre Therapy  Receptive Language Post Therapy
Mean = 2.916    Mean = 2.059
Standard Deviation = 1.311      Standard Deviation = 1.288
Standard Error = 0.378  Standard Error = 0.227

p = 0.06684

ANOVA p=0.030

Hyperactivity Pre Therapy       Hyperactivity Post Therapy
Mean = 2.964    Mean = 1.821
Standard Deviation = 1.802      Standard Deviation = 1.471
Standard Error = 0.481  Standard Error = 0.252

p = 0.04821

ANOVA p=0.29

And a difference in improvement of symptoms in the GI group taking probiotics with respect to expressive language, receptive language and rocking/pacing (ANOVA).

Expressive Language Pre Therapy Expressive Language Post Therapy
Mean = 3.423    Mean = 2.81
Standard Deviation = 1.483      Standard Deviation = 1.761
Standard Error = 0.411  Standard Error = 0.306

p = 0.24308

ANOVA p=0.0084

Receptive Language Pre Therapy  Receptive Language Post Therapy
Mean = 2.916    Mean = 2.059
Standard Deviation = 1.311      Standard Deviation = 1.288
Standard Error = 0.378  Standard Error = 0.227

p = 0.066

ANOVA p=0.030

Rocking/Pacing Pre Therapy      Rocking/Pacing Post Therapy
Mean = 1.46429  Mean = 1.46094
Standard Deviation = 1.94604    Standard Deviation = 1.58934
Standard Error = 0.5201 Standard Error = 0.28096

p = 0.99553

ANOVA p=0.050

3. I focused analysis on GABA levels and how they correlate with other markers and outcomes. GABA correlates significantly with Hyperactivity, Hand Flapping/ Finger Stimming and Light Sensitivity.

Hyperactivity
Overall
Corr    P-value
0.470   0.008
Hand Flapping/ Finger Stimming
Overall
Corr    P-value
0.424   0.014
Light Sensitivity
Overall
Corr    P-value
0.743   0.000

High Cu, low Zn and high Cu/Zn also correlate with Hyperactivity

Cu      Hyperactivity
Correlation     0.213
p-value (1 sided)       0.090
Zn      Hyperactivity
Correlation     -0.259
p-value (1 sided)       0.046
Cu/Zn   Hyperactivity
Correlation     0.335
p-value (1 sided)       0.014

And Stimming

Cu      Improved Stimming
Correlation     0.376
p-value (1 sided)       0.050
Zn      Stimming
p-value (1 sided)       0.080

We have also found that high GABA correlates with lower Cu (p=0.05).

GABA levels correlate with Hyperactivity, and lower copper. Hyperactivity improves after zinc therapy (copper normalization). We believe that this supports the theory that GABA levels are associated with hyperactivity as well as copper levels in autistic children and suggests that both are connected to a common etiology. Theoretically, in patients with severe hyperactivity symptom, high copper blocks GABA receptor function, which causes high GABA in this group and low functional GABA (bound GABA), resulting in hyperactivity.

4. I continued to analyze Cu, Zn and CuZn indvividual values and their correlate with outcomes of autistic patients (N=100) done on the same day (as blood draw). This is our most accurate way of performing this correlation. I completed analysis of receptive language there is a significant correlation between high receptive language severity and low Zn (p=0.034) and high Cu/Zn (p=0.003). Whereas high expressive language severity correlates with high Cu levels (p=0.037), and high Cu/Zn (p=0.024), but not with zinc concentration.

The more data we collect, the more confident we are that Cu and Zinc levels correlate with symptom severity in our autistic group (and most likely on the adult groups as well) and that GABA levels are playing a particularly important role in the etiology of hyperactivity symptoms in these same patients.

5. I continued to write the paper (high GABA levels in autistic children), incorporating the data above.

These results (along with previous correlations)suggest different roles for zinc and copper as thy are associated

Update 6.28.11

1.Based on recent data presented by Jim Adams at Arizona State  showing that SAM levels are significantly lower in autistic children and we are searching for a better biomarker for methylation, I investigated potential labs who assay SAM.

Both Health Diagnostics and Research Institute (formerly Vitamin Diagnostics) and Doctor’s Data offer the testing.

http://www.doctorsdata.com

http://www.europeanlaboratory.com/

2. Based on an inquiry by Maizie and Lakshmi, I investigated the long term effects of high doses of zinc.

Zinc toxicity can occur in both acute and chronic forms. Acute adverse effects of high zinc intake include nausea, vomiting, loss of appetite, abdominal cramps, diarrhea, and headaches [1]. One case report cited severe nausea and vomiting within 30 minutes of ingesting 4 g of zinc gluconate (570 mg elemental zinc) [2]. Intakes of 150–450 mg of zinc per day have been associated with such chronic effects as low copper status, altered iron function, reduced immune function, and reduced levels of high-density lipoproteins [3]. Reductions in a copper-containing enzyme, a marker of copper status, have been reported with even moderately high zinc intakes of approximately 60 mg/day for up to 10 weeks [1]. The doses of zinc used in the AREDS study (80 mg per day of zinc in the form of zinc oxide for 6.3 years, on average) have been associated with a significant increase in hospitalizations for genitourinary causes, raising the possibility that chronically high intakes of zinc adversely affect some aspects of urinary physiology [4].

The FNB has established ULs for zinc (Table). Long-term intakes above the UL increase the risk of adverse health effects [2]. The ULs do not apply to individuals receiving zinc for medical treatment, but such individuals should be under the care of a physician who monitors them for adverse health effects.

Table: Tolerable Upper Intake Levels (ULs) for Zinc [2]
Age      Male    Female  Pregnant        Lactating
0–6 months      4 mg    4 mg
7–12 months     5 mg    5 mg
1–3 years       7 mg    7 mg
4–8 years       12 mg   12 mg
9–13 years      23 mg   23 mg
14–18 years     34 mg   34 mg   34 mg   34 mg
19+ years       40 mg   40 mg   40 mg   40 mg

1.Institute of Medicine, Food and Nutrition Board. Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. Washington, DC: National Academy Press, 2001.

2. Lewis MR, Kokan L. Zinc gluconate: acute ingestion. J Toxicol Clin Toxicol 1998;36:99-101.

3. Hooper PL, Visconti L, Garry PJ, Johnson GE. Zinc lowers high-density lipoprotein-cholesterol levels. J Am Med Assoc 1980;244:1960-1.

4. Johnson AR, Munoz A, Gottlieb JL, Jarrard DF. High dose zinc increases hospital admissions due to genitourinary complications. J Urol 2007;177:639-43.

http://ods.od.nih.gov/factsheets/Zinc-HealthProfessional/

To my knowledge there is not any data specifically indicating harm from high doses of zinc in patients who are zinc deficient. My educated guess is that since we monitor zinc and copper levels, this will give us the opportunity to monitor side effects. With that said, the data below, indicates that there is a possibility of side effects. Have you seen side effects in our patients? Here is more data from the Linus Pauling Center at Oregon State:http://lpi.oregonstate.edu/infocenter/minerals/zinc/

Zinc Toxicity

Acute toxicity

Isolated outbreaks of acute zinc toxicity have occurred as a result of the consumption of food or beverages contaminated with zinc released from galvanized containers. Signs of acute zinc toxicity are abdominal pain, diarrhea, nausea, and vomiting. Single doses of 225 to 450 mg of zinc usually induce vomiting. Milder gastrointestinal distress has been reported at doses of 50 to 150 mg/day of supplemental zinc. Metal fume fever has been reported after the inhalation of zinc oxide fumes. Specifically, profuse sweating, weakness, and rapid breathing may develop within eight hours of zinc oxide inhalation and persist 12-24 hours after exposure is terminated (4, 5).

Adverse effects

The major consequence of long-term consumption of excessive zinc is copper deficiency. Total zinc intakes of 60 mg/day (50 mg supplemental and 10 mg dietary zinc) have been found to result in signs of copper deficiency. In order to prevent copper deficiency, the U.S. Food and Nutrition Board set the tolerable upper level of intake (UL) for adults at 40 mg/day, including dietary and supplemental zinc (4).

Tolerable Upper Intake Level (UL) for Zinc
Age Group        UL (mg/day)
Infants 0-6 months      4
Infants 7-12 months     5
Children 1-3 years      7
Children 4-8 years       12
Children 9-13 years      23
Adolescents 14-18 years 34
Adults 19 years and older       40

3. I continued investigating the possible correlation between HMGB1, GABA, MPO, HGF and symptom severity of autistic children in our MPO/Probiotics study. We found that in addition to correlation with Hyperactivity and Focus/Attention, GABA levels correlate with improved (difference between first and last severity rating) impulsivity (p= 0.036; correlation 0.35), improved Fine Motor Skills (p= 0.006; correlation -0.693), improved Rocking/Pacing (p=0.1; correlation -0.391), Hand Flapping/Finger Stimming (p=0.013; correlation 0.423), Improved Eye Contact (p=0.037; correlation -0.510) and overall Eye Contact (p=0.07; correlation 0.303), Sound Sensitivity (p=0.05; correlation 0.317), Light Sensitivity (p=0.0005; correlation 0.742).

We also found that HMGB1 correlates with Hyperactivity (p=0.025; correlation -0.579) and Focus/Attention (p=0.070; correlation -0.414), improved Fine Motor Skills (p=0.078; correlation 0.383), improved Motor Skills (correlation 0.346), improved Tip Toeing (correlation -0.345), improved and overall Rocking/Pacing (p=0.074; correlation 0.444 and p= 0.034; correlation -0.634, respectively), improved and overall Hand Flapping/ Finger Stimming (p=0.10; correlation 0.337 and p= 0.032; correlation -0.323, respectively), Light Sensitivity (p=0.0.; correlation -0.291)

HGF correlates with improved Hyperactivity (correlation -0.362), improved and overall Tactile Sensitivity (correlation -0.299 and  -0.459, respectively), improved Obsession/Fixation (correlation -0.346).

MPO levels correlate with Hypotonia (p= 0.035; correlation -0.278), Improved Rocking/Pacing (p=0.010; correlation 0.551), improved Rocking/Pacing (p=0.091; correlation 0.294).

Observations: GABA and HMGB1 continue to be the most significant markers associated with symptom severity in autistic children. They appear to be associated with symptoms which have hyperactivity and sense dysfunction as components.

4. I analyzed the correlation between Cu, Zn and Cu/Zn in these same patients.

Cu correlates with Receptive Language (p=.038; correlation -0.287), Gross Motor Skills (p=0.043; correlation 0.267), Hypotonia (p=0.019; correlation 0.329),

Zn correlates with Focus/Attention (p=0.027; correlation -0.294), Hyperactivity (p=0.046; correlation -0.259), Impulsivity (p=0.068; correlation -0.235, Perseveration (p=0.042; correlation -0.264), Tactile Sensitivity (p=0.080; correlates 0.226), Stimming (p=0.080; correlation 0.212)

Cu/Zn correlates with Focus Attention (p=0.043; correlation 0.266), Hyperactivity (p=0.014; correlation 0.335), Gross Motor Skills (p=0.035; correlation 0.281), Hypotonia (p=0.021; correlation 0.317).

Observations: Zinc levels and Cu/Zn are also associated with hyperactivity related symptoms.

5. I analyzed Cu, Zn, Cu/Zn with respect to correlation to the biomarkers above.  Copper levels correlate with HMGB1 levels (p=0.027; correlation -0.336). Copper levels also correlate with HGF levels (p=0.066; correlation 0.291). Cu/Zn correlates with HGF levels (p=0.072; correlation 0.282).

We will continue to analyze this data,  as we strengthen our overall theory that ultimately zinc and copper levels dictate neurotransmitter and growth factor levels, which, in turn, affect behavior in our patients.

Update 6.10.11

1. I have been analyzing the group of autistic children in our MPO/Probiotics study (N=50)  for possible correlation between symptom severity and biomarkers (MPO, HMGB1, GABA and HGF). We found a significant correlation between High GABA and hyperactivity severity (p=0.015) and improved hyperactivity and lower GABA (p=0.05), increased HMGB1 and lower hyperactivity (p=0.05). Increased HMGB1 and better focus/attention (p=0.05). Higher GABA and higher impulsivity (p=0.03). Improved Motor skills and high GABA (p=0.012).

Although we haven’t finished analyzing all symptoms, it is interesting that this data supports the relationship between GABA and symptoms of our autistic patients. Autistic children have higher levels of GABA than controls and, depending on the patient’s symptoms, it may be important to lower GABA levels in some individuals in order to improve symptoms.

2. With the help of Kyle and Laurie, we are collecting outcomes data from the Mood Questionnaire for Bipolar Disorder (filled out by our patients over the past 8 months). This data will be used to correlate biomarkers and therapy as they relate to perceived symptom severity in our bipolar patients.

3. I wrote and submitted the following update  to the Autism Research Institute of our funded projects:

ARI Grants Update
Preliminary Data and Observations
A.J. Russo, Ph.D.

Taurine, Taurine Transport Protein and GABA Levels in Autistic Children

We have now measured GABA levels in 60 autistic patients and 32 neurotypical, age and gender similar controls. We find that autistic patients have significantly higher plasma GABA concentration than controls.

GABA Autism     GABA Controls
Mean = 2121.54  Mean = 1386.22
Standard Deviation = 1347.58    Standard Deviation = 667.21
Standard Error = 200.88 Standard Error = 119.83

p = 0.002

We also find that before zinc therapy, GABA levels of autistic children without GI disease are significantly lower than post therapy.

GABA Pre Therapy        GABA Post Therapy
Mean = 1188.32  Mean = 2169.16
Standard Deviation = 638.38     Standard Deviation = 1207.63
Standard Error = 260.61 Standard Error = 241.52

p = 0.015

We still don’t have enough patients to evaluate differences between GABA levels in autistic children with GI disease vs without GI disease. We are seeking more of this patient group.

We are currently correlating MPO levels with GABA levels in these same patients and are establishing a taurine ELISA to compare taurine levels in these patients and controls.

To study why increased copper in individuals with autism normalizes post zinc therapy in individuals with concurrent GI disease

We have measured copper and zinc in 100 autistic children and 35 age and gender similar neurotypical controls.

Autistic individuals had significantly elevated plasma levels of copper and Cu/Zn and lower, but not significantly lower, plasma Zn compared to neurotypical controls.

Zn levels increased significantly in autistic individuals with and without GI disease after zinc therapy. Cu decreased significantly after zinc therapy in the GI disease group but not in the autistic group without GI disease.

Autistic children significantly improved with respect to hyperactivity and stimming after zinc therapy in autistic children with GI disease. Autistic children without GI disease did not improve in these symptoms after the same therapy.

We wrote and have had accepted the paper, Increased Copper in Individuals With Autism Normalizes Post Zinc Therapy More Efficiently in Individuals With Concurrent GI Disease in the journal Nutrition and Metabolism.

We have measured GABA concentration in 60 of these patients and 35 controls and found a significant correlation between Increased GABA and decreased copper in these patients.

Preliminary Data:

Overall, GABA levels don’t increase significantly post zinc therapy

GABA Autism Pre Zinc Therapy    GABA Autism Post Therapy
Mean = 2039.81    Mean = 2141.97
Standard Deviation = 1535.46   Standard Deviation = 1319.81
Standard Error = 511.82      Standard Error = 219.96

p = 0.857

However, GABA levels in autistic children without GI disease increase significantly post zinc therapy.

GABA Pre Therapy        GABA Post Therapy
Mean = 1188.32       Mean = 2169.16
Standard Deviation = 638.38    Standard Deviation = 1207.63
Standard Error = 260.61      Standard Error = 241.52

p = 0.014

We are currently gathering and testing blood from autistic children with GI disease before zinc therapy to compare GABA concentration with those who have already had zinc therapy.

We did not found a correlation between Cu and GABA concentration in these patients (p=0.797) or between Zn and GABA (p=0.805).

To study the Relationship Between Decreased Hepatocyte Growth Factor (HGF) and Glutamate Excitotoxicity in Autistic Children

A. HGF is significantly lower in autistic children (total and those with GI disease). Those without GI disease do not have significantly less HGF. This suggests that HGF levels are most affected in autistic children with GI disease, which could be associated with selective MET gene/receptor dysfunction in the GI tract. Copper inhibits HGF-MET receptor interaction. Autism-GI patients have significantly higher copper. These patients respond to zinc therapy by significantly lowering copper. Higher copper correlates with higher HGF in these same patients, hypothetically because of copper blocking HGF –MET interaction resulting in higher free (plasma) HGF. As copper decreases, HGF decreases in these same patients (HGF significantly decreases after zinc therapy/lowering copper) and lower HGF correlates with improved symptom severity, suggesting that lower Cu levels, which results in lower HGF may be part of autism etiology.

The receptor tyrosine kinase Met and its ligand, hepatocyte growth factor (HGF), are clustered at excitatory synapses, especially in hippocampal neurons, and may regulate aspects of excitatory synaptic function. HGF increases expression of excitatory synaptic proteins, enhances their clustering at sites along dendrites, and increases current through the NMDA receptor. It is likely that the lowering of HGF, in turn causes decreased excitatory synaptic function, which is related to improved symptoms such as hyperactivity.

Tyndall, S. J. and Walikonis, R. S. (2007), Signaling by hepatocyte growth factor in neurons is induced by pharmacological stimulation of synaptic activity. Synapse, 61: 199–204.

Stephanie Joyce Tyndall, “A role for hepatocyte growth factor and Met at excitatory synapses” (January 1, 2005). Dissertations Collection for University of Connecticut. Paper AAI3205766.

More specifically, HGF stimulates Ca2+ influx into dendrites through the NMDA receptor and that this effect is necessary for the changes in dendritic morphology induced by HGF.

Stephanie J. Tyndall, Sandip J. Patel, Randall S. Walikonis Hepatocyte growth factor-induced enhancement of dendritic branching is blocked by inhibitors of N-methyl-D-aspartate receptors and calcium/calmodulin-dependent kinases Journal of Neuroscience Research 2007;85: 2343–2351.

HGF and c-Met, can promote formation of neurites and enhance elaboration of dendrites in mature neurons, particularly during early stages of maturation.

Lim CS, Walikonis RS. Hepatocyte growth factor and c-Met promote dendritic maturation during hippocampal neuron differentiation via the Akt pathway. Cell Signal. 2008 May;20(5):825-35. Epub 2007 Dec 27.

Lowering HGF, therefore, may decrease expression of excitatory synaptic proteins and decrease current through the NMDA receptors, thereby improving symptom severity in these patients.

Glutamate treatment decreases the amount of pro HGF and increases the amount of the proteolytically-activated HGF.

Preliminary Data:

HGF Total Data
Autistic        Controls
Mean = 469.49        Mean = 545.64
Standard Deviation = 183.85  Standard Deviation = 173.19
Standard Error = 23.34       Standard Error = 27.04
N=63    N=56

p = 0.035

HGF GI
Autism GI       Controls
Mean = 450.88        Mean = 545.64
Standard Deviation = 158.11   Standard Deviation = 173.19
Standard Error = 37.26       Standard Error = 27.04
N=18    N=56

p = 0.047

Autism GI Pre Therapy   Autism GI Post Therapy
Mean = 441.26   Mean = 454.58
Standard Deviation = 142.79   Standard Deviation = 169.01
Standard Error = 63.85       Standard Error = 46.87
N=5     N=13

p = 0.870

Autism Non-GI Pre Therapy       Autism Non-GI Post Therapy
Mean = 561.50        Mean = 455.41
Standard Deviation = 255.49  Standard Deviation = 173.65
Standard Error = 85.16       Standard Error = 29.35

p = 0.133

B. HMBG1 is significantly higher in autistic children. HMBG1 levels increase significantly, post zinc therapy in autistic children with GI disease, but not in the non-GI disease group. Symptoms of this group (GI) significantly improve after therapy, suggesting that Box Protein may play a role in this improvement.

Preliminary Data:

HMBG1 (pg/ml)
Autistic        Controls
Mean = 102.36        Mean = 52.85
Standard Deviation = 101.60  Standard Deviation = 52.93
Standard Error = 13.22       Standard Error = 15.28

p = 0.020

HMBG1
Autistic GI     Controls
Mean = 86.32   Mean = 52.85
Standard Deviation = 71.95   Standard Deviation = 52.93
Standard Error = 17.45       Standard Error = 15.28

p = 0.160

HMBG1
Autistic GI     Autistic Non-GI
Mean = 86.32   Mean = 106.70
Standard Deviation = 71.95   Standard Deviation = 108.82
Standard Error = 17.45       Standard Error = 16.22

p = 0.396

HMBG1
Autistic GI Pre Therapy   Autistic GI Post Therapy
Mean = 42.34    Mean = 93.84
Standard Deviation = 29.59   Standard Deviation = 68.96
Standard Error = 13.23       Standard Error = 15.42

p = 0.022

HMBG1
Autistic Non-GI Pre Therapy     Autistic Non-GI Post Therapy
Mean = 117.34   Mean = 106.21
Standard Deviation = 106.83    Standard Deviation = 114.45
Standard Error = 33.78       Standard Error = 20.23

p = 0.780

High mobility group box 1 (HMGB1) can be released from neurons after glutamate excitotoxicity, and induces glutamatergic release from glial (gliosomes) cells. So, the cytokine could act as a modulator of glutamate homeostasis in adult brain.

We find HMGB1 correlates directly with HGF. Post zinc therapy HGF decreases and HMGB1 increases and both are associated with improved symptom severity. We suggest that increased HMGB1 results in increased glutamate. This in turn may result in

C. GABA plasma levels are significantly higher in autistic children.

GABA Autism     GABA Controls
Mean = 2121.54       Mean = 1386.22
Standard Deviation = 1347.58    Standard Deviation = 667.21
Standard Error = 200.88      Standard Error = 119.83

p = 0.002

Overall GABA levels don’t increase significantly post zinc therapy

GABA Autism Pre Zinc Therapy    GABA Autism Post Therapy
Mean = 2039.81   Mean = 2141.97
Standard Deviation = 1535.46    Standard Deviation = 1319.81
Standard Error = 511.82      Standard Error = 219.96

p = 0.857

However, GABA levels in autistic children without GI disease increase significantly post zinc therapy.

GABA Pre Therapy        GABA Post Therapy
Mean = 1188.32       Mean = 2169.16
Standard Deviation = 638.38     Standard Deviation = 1207.63
Standard Error = 260.61      Standard Error = 241.52

p = 0.014

Copper has high affinity for GABAa receptor, blocking GABA attachement, elevating plasma GABA. This could also be why GABA is higher in autism with higher copper.

Sharonova IN, Vorobjev VS, Haas HL High-affinity copper block of GABA(A) receptor-mediated currents in acutely isolated cerebellar Purkinje cells of the rat. Eur J Neurosci. 1998 Feb;10(2):522-8.

Increased GABA correlates with decreased HMGB1 (p=0.009) as ell as decreased HGF (p=0.03) in our autistic patients.

Summary:

In summary, post zinc therapy, copper levels decrease, GABA levels increase significantly in the non-GI group, but not the GI group; HMGB1 increases significantly in the GI group but not the non-GI group; and HGF decreases in the non-GI group significantly but not the GI group.

4. I wrote and submitted the following grant proposal to ARI

Grant submission to Autism Research Institute

A.J. Russo, Ph.D.
Research Director

Health Research Institute/Pfeiffer Treatment Center
4575 Weaver Parkway
Warrenville, IL  60555
Phone: 630-505-0300 X207
Fax: 630-836-0667
ajrusso@hriptc.org

To study the Relationship Between Myeloperoxidase (MPO) Deficiency and Probiotic Therapy in Autistic Children

Introduction and Rationale

Oxidants are thought to be key components of the neutrophil host defense system. Upon contact with a pathogen, particularly bacteria and fungi, neutrophils produce a respiratory burst, characterized by intense uptake of oxygen. The resulting superoxide dismutates into hydrogen peroxide (H2O2). The toxicity of H2O2 to microbe pathogens is greatly enhanced by the heme enzyme myeloperoxidase (MPO), found in the azurophilic granules of neutrophils, which uses H2O2 to convert chloride (Cl−) into hypochlorous acid (HOCl). Although the exact mechanism is not completely understood, MPO also kills by directly chlorinating phagocytosed bacteria. [1].

In addition to killing bacteria, the products of the MPO-hydrogen peroxide-Cl system are believed to play a role in killing fungi, parasites, protozoa, viruses, tumor cells, natural killer (NK) cells, red cells, and platelets, and they may be involved in terminating the respiratory burst, because individuals with MPO deficiency have a prolonged reaction [2].
Other functions of MPO include tyrosyl radical production, generation of tyrosine peroxide, mediation of the adhesion of myeloid cells via b2-integrins, and oxidation of serum lipoproteins [2].

Myeloperoxidase (MPO) is an inflammatory marker. We have found it to be decreased in autistic children, particularly those with GI disease (3).

The cytokine, High mobility group box 1 (HMGB1) functions as an activator of the immune response, an inflammatory marker, and can be released from neurons after glutamate excitotoxicity. Serum levels of HMGB1 have been found to be significantly higher in patients with autistic disorder (4).

We have found that zinc is lower and copper is significantly higher than normal in autistic children and normalization of zinc and copper levels, after zinc therapy, correlates significantly with perceived symptom severity improvement (5.6)

Copper has high affinity for GABAa receptor. GABA is higher in autism with higher copper (7). Copper also inhibits HGF-MET receptor interaction. Our lab has found that HGF is significantly decreased in autistic children (8).

Our preliminary data demonstrates that probiotic therapy may be associated with reduced inflammation, as evidenced by reduced HMGB1 and MPO, and subsequent increased zinc absorption in autistic children with GI disease. The funds from this proposal would help us confirm this observation and evaluate the efficacy of specific probiotics at reducing inflammation.

Preliminary Data

Plasma from 79 autistic individuals, and 48 age and gender similar neurotypical controls, were tested for plasma zinc and copper concentration using inductively-coupled plasma-mass spectrometry, and for plasma HMGB1, GABA, HGF and MPO using ELISAs.

Copper levels were significantly higher in autistic children, particularly those with GI disease (p=0.013).

After zinc therapy, copper levels normalized (decreased to the level of controls) significantly in autistic children with GI disease (p=0.024), but not in autistic children without GI disease.

Parents perceived that severity of hyperactivity and stimming in autistic children with GI disease improved significantly (p=0.005; p=0.05, respectively), whereas those without GI disease did not improve significantly.

When Zn, Cu, HMGB1, GABA, HGF and MPO plasma levels in autistic children with GI disease and without, who are taking probiotic therapy, were compared to those not taking probiotics, zinc levels were significantly higher in the GI group taking probiotics (p=0.008) compared to the GI group not taking probiotics (p=0.725) and copper levels were significantly lower in the GI group taking probiotics, compared to the group not taking probiotics (p=0.04). Whereas, there was not a difference in copper levels in the non GI group taking probiotics, compared to the non GI group not taking probiotics (p=0.890).

HMGB1 levels were lower and MPO levels were significantly lower in the GI group taking probiotics (p=0.002) (Table below).

These results suggest that zinc is absorbed better in the GI group taking probiotics (influencing the lowering of copper levels) and decreased inflammation in this group (as evidenced by lower HMGB1 and MPO levels, as well as the correlation between these inflammatory markers) may be associated with better zinc absorption.

HMGB1 correlates with MPO levels (p=0.04; Pearson Correlation), further supporting involvement of inflammation.

MPO concentration correlates with decreasing GABA (p=0.05 Pearson Correlation). This supports a relationship between decreased MPO and increased GABA.

In summary, plasma zinc levels are lower and copper levels are significantly higher in autistic children. Copper levels normalize (decrease) significantly after zinc therapy in autistic children with GI disease. Symptom severity (hyperactivity and stimming) decreases (behavior improves) in these autistic children with GI disease. Myeloperoxidase (marker for inflammation) levels are significantly lower in autistic children with GI disease, particularly those on probiotic therapy. Zinc is significantly higher and copper significantly lower in the GI group taking probiotics. HMGB1 levels correlate with MPO levels in this group of autistic children supporting the hypothesis that increased zinc absorption may be associated with improved inflammation.

Methods

Assays:

We will use ELISA’s to measure MPO HMGB1, IL-1, Il-6 and TNF-alpha concentration. Briefly, the procedure for all the assays is as follows:
1. Add Capture Antibody: Dilute one vial of Capture Antibody with 10 ml of Capture Antibody Dilution Buffer (Solution A).  Add 100 ml of capture antibody solution to each well and incubate at 4°C overnight.
2. Prepare Standard Dilutions: The recommended standard range is 1.6-100 ng/ml.  Dilute one vial of HMGB1 Standard with 950 ml of Sample/Standard Dilution Buffer (Solution B) – 100 ng/ml.  Prepare serial dilutions of the standard by mixing 250 ml of the 100 ng/ml standard with 250 ml of Solution B – 50 ng/ml.  Then repeat this procedure to make fi ve more serial dilutions of standard – 25, 12.5, 6.25, 3.1 and 1.6 ng/ml solutions. 3. Prepare Sample Dilutions: Centrifuge samples at 10,000 rpm at 4°C for 3 minutes to remove insoluble materials and lipids, and use the supernatant as samples.  If the HMGB1 level is more than 100 ng/mL, re-assay the sample at a higher dilution.
4. Prepare Detection Antibody:  Dissolve one vial of Detection Antibody in 5 ml of Detection Antibody Dilution Buffer (Solution C).
5. Dilute Wash Buffer: Dilute 50 ml of 20X wash buffer in 950 ml of distilled water (1X wash buffer).   Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer.  Empty the plate by inverting it and blot on a paper towel to remove excess liquid.  Do not allow the plate to dry out.
6. Add Standards, Samples and Detection Antibody: Mix standards, samples and detection antibody tubes well.  Add 50 ml of Solution B (blank), standards and samples to appropriate wells (Figure 1).  Add 50 ml of diluted detection antibody solution to all wells.  Mix detection antibody solution and sample/standard solutions in all wells by pipettiwith a plate sealer and incubate at 37°C for 1 hour, then settle at 4°C overnight.
7. Prepare Streptavidin Peroxidase: Dilute one vial of Streptavidin Peroxidase in 10 ml of Streptavidin Peroxidase Dilution Buffer (Solution D).
8. Wash: Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer.  Empty the plate by inverting it and blot on a paper towel to remove excess liquid.
9. Add Streptavidin Peroxidase: Add 100 ml of streptavidin peroxidase solution to each well and incubate at room temperature for 30 minutes.  Do not incubate the plate more than 60 minutes.
10. Wash: Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer.  Empty the plate by inverting it and blot on a paper towel to remove excess liquid.
11. TMB: Add 100 ml of TMB Solution to all wells immediately after washing the plate.  Incubate for 30 minutes at room temperature.
12. Stop: Add 50 ml of 2N sulfuric acid (Stop Solution) to each well.
13. Read Plate: Read the OD values within 30 minutes by dual wavelength at 450 nm (sample) and 630 nm (reference)

Hypothesis and Design:

Based on this data, as well as the research of others, we hypothesize that, lower inflammation (evidenced by lower HMGB1 and MPO), promoted by probiotics, enhances intestinal absorption. Enhanced intestinal absorption then facilitates zinc absorption, which, in turn, promotes decreased copper. Decreased copper may be associated with changes in growth factor concentration (such as HGF) (HGF increases post zinc therapy in GI/probiotic group), neurotransmitter levels (such as GABA) (GABA levels increase post zinc therapy/increased GABA levels correlate with decreased MPO) and inflammatory cytokine production (HMGB1) (HMGB1 levels correlate with MPO levels). Collectively, normalization or modulation of these molecules may be associated with decreased symptom severity.

To confirm the association between probiotics, decreased inflammation, improved response to zinc therapy and improved symptoms, we propose to measure MPO, HMGB1, as well as the inflammatory cytokines, IL-1, Il-6 and TNF-alpha in 100 additional autistic children and 100 neurotypical controls. These numbers (172 autistic children) will allow us to assess the effect of the type of probiotic therapy on inflammation change.

We will test serum from 100 (50 taking probiotics and 50 not taking probiotics) autistic children and 100 neurotypical, age and gender similar controls for HMGB1, IL-1, Il-6 and TNF-alpha. We will then compare this data to probiotic therapy taken by these patients. We will also assess this data by comparing the autistic groups pre and post zinc therapy (we don’t have enough samples from these groups in our preliminary data to make this assessment), and we will assess the effect of each type of probiotic therapy on inflammatory marker levels.

We hypothesize that autistic children with GI disease, taking probiotics, will have reduced inflammatory cytokine levels. Plasma zinc levels will be significantly increased and copper levels will be significantly decreased post zinc therapy in this group, indicating improved absorption of zinc. We also hypothesize that symptom severity will decrease (improve) in this group and this improvement will correlate with zinc and copper normalization. It is also possible that we will be able to find probiotic therapies, which are more effective at affecting normalization.

Budget

MPO ELISA Kits (5) eBiosciences, Inc. $495.00/kit Total = $2475.00

HMGB1 ELISA Kits (5) Chondrex, Inc. $398.00/kit Total = $1990.00

IL-1 ELISA Kits (5) R&D $495.00/kit Total = $2475.00

IL-6 ELISA Kits (5) R&D $495.00/kit Total = $2475.00

TNF-alpha ELISA Kits (5) R&D $495.00/kit Total = $2475.00

Total = $11890.

References

1. Dale DC, Boxer L, Liles WC. The phagocytes: neutrophils and monocytes. Blood. Aug 2008;112:935-945.

2. Nauseef WM. Insights into myeloperoxidase biosynthesis from its inherited deficiency. J Mol Med. Sep 1998;76(10):661-8.

3. Russo,A.J.,  et al Low serum myeloperoxidase in autistic children with gastrointestinal disease Clinical and Experimental Gastroenterology 2009;2 85–94.

4. Emanuele E et al Increased serum levels of high mobility group box 1 protein in patients with autistic disorder. Prog Neuropsychopharmacol Biol Psychiatry. 2010;34(4):681-3.

5. Russo A.J., deVito R., and Filer S. Analysis of Copper and Zinc Plasma Concentration and the Efficacy of Zinc Therapy in Individuals with Asperger’s Syndrome, Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS) and Autism. Accepted for publication Biomarkers April, 2011.

6. Russo A.J. Increased Copper in Individuals With Autism Normalizes Post Zinc Therapy More Efficiently in Individuals With Concurrent GI Disease Accepted for publication Nutrition and Metabolism February, 2011.

7. Sharonova IN, Vorobjev VS, Haas HL High-affinity copper block of GABA(A) receptor-mediated currents in acutely isolated cerebellar Purkinje cells of the rat. Eur J Neurosci. 1998 Feb;10(2):522-8.

8. Russo,A.J.,  et al Decreased Serum Hepatocyte Growth Factor (HGF) in Autistic Children with Severe Gastrointestinal Disease Biomarker Insights 2009:4:181-190.

5. I continued to analyze copper, zinc and Cu/Zn levels as they correlate with each of the autistic symptoms. The unique aspect of this analysis is that each patient visit and outcome is part of the correlation. So far we have analyzed Awareness and Expressive Language, and found that copper levels correlate with both Awareness (p=0.08) and Expressive Language (p=0.037). Cu/Zn correlates with Expressive Language (p=0.02). Zinc levels do not correlate with either symptom. This supports the importance of Cu levels as they relate to symptoms of autistic children.

Update 5.27.11

1. Began a more extensive study on correlation between individual symptom severity outcomes and Cu and Zinc. In this study we are comparing each Cu and Zn concentration with severity rating done on that same day.

2. Wrote and submitted an update of grant related projects to Autism Research Institute.

3. Completed revision of paper, Analysis of Copper and Zinc Plasma Concentration and the Efficacy of Zinc Therapy in Individuals with Asperger’s Syndrome, Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS) and Autism

4. Analyzed a correlation between MPO, GABA, HMGB1 levels and probiotics in autistic children.

We found a significant correlation between HMGB1 and MPO levels in autistic children (p=0.05).

We also found a significant correlation between increased GABA and decreased MPO (p=0.05).

5. When we analyzed MPO, GABA and HMGB1 levels in autistic children we found no significant difference between levels in autistic children with GI disease and those without. However,  when we compare these groups with those who are taking probiotics and those that are not, we found that MPO plasma concentration in autistic children with GI disease and taking probiotics is significantly lower than those without GI disease and taking probiotics, as well as those not taking probiotics with and without GI disease (ANOVA p=0.002). The significance of this is that probiotics may play an important role in affecting biomarkers such as MPO as well as symptom severity (although this still needs to be evaluated).

6. I developed my talk to be presented at Autism One this Sunday.

7. I continued to write and edit the manuscripts Increased GABA in Bipolar Disorder and Increased GABA in Schizophrenia.

8. I began to summarize levels of HGF, HMGB1, HGF and GABA levels and symptom severity in our youth groups and adult groups.

Update 5.20.11

1. Because we have found correlation between HGF and HMGB1 and GABA, I investigated the possible relationship between these molecules (below).

2. Based in a question by Maizie and Kim, I investigated B-6 toxicity.

This (below) is from the Linus Pauling Institute at Oregon State U.  I appreciate that we exceed upper limits for B-6 intake so that B-6 and zinc can work effectively as cofactors to help module neurotransmitter levels.

Pyridoxal phosphate-dependent enzymes play a role in the biosynthesis of four important neurotransmitters: serotonin, epinephrine, norepinephrine and gamma-aminobutyric acid (3).

With that said, we may want to reconsider our upper limit of B-6, particularly for those who experience neuropathies. I couldn’t find any documentation separating pyridoxine and P5P intake requirements. Hope it helps.

Toxicity

Because adverse effects have only been documented from vitamin B6 supplements and never from food sources, safety concerning only the supplemental form of vitamin B6 (pyridoxine) is discussed. Although vitamin B6 is a water-soluble vitamin and is excreted in the urine, long-term supplementation with very high doses of pyridoxine may result in painful neurological symptoms known as sensory neuropathy. Symptoms include pain and numbness of the extremities and in severe cases, difficulty walking. Sensory neuropathy typically develops at doses of pyridoxine in excess of 1,000 mg per day. However, there have been a few case reports of individuals who developed sensory neuropathies at doses of less than 500 mg daily over a period of months. Yet, none of the studies in which an objective neurological examination was performed reported evidence of sensory nerve damage at intakes below 200 mg pyridoxine daily (1). To prevent sensory neuropathy in virtually all individuals, the Food and Nutrition Board of the Institute of Medicine set the tolerable upper intake level (UL) for pyridoxine at 100 mg/day for adults (see table below) (2). Because placebo-controlled studies have generally failed to show therapeutic benefits of high doses of pyridoxine, there is little reason to exceed the UL of 100 mg/day.

Tolerable Upper Intake Level (UL) for Vitamin B6

Age Group        UL (mg/day)
Infants 0-12 months     Not possible to establish*
Children 1-3 years      30
Children 4-8 years       40
Children 9-13 years      60
Adolescents 14-18 years 80
Adults 19 years and older       100
*Source of intake should be from food and formula only.

1. Bender DA. Non-nutritional uses of vitamin B6. Br J Nutr. 1999;81(1):7-20.

Abstract: Vitamin B6 is a water-soluble vitamin, and is readily metabolized and excreted, so it has generally been assumed to have negligible toxicity, although at very high levels of intake it can cause peripheral nerve damage. Nutritional deficiency disease is extremely rare, although a significant proportion of the population shows biochemical evidence of inadequate status, despite apparently adequate levels of intake. The vitamin has been used to treat a wide variety of conditions, which may or may not be related to inadequate intake. In some conditions use of vitamin B6 supplements has been purely empirical; in other conditions there is a reasonable physiological or metabolic mechanism to explain why supplements of the vitamin many times greater than average requirements may have therapeutic uses. However, even in such conditions there is little evidence of efficacy from properly conducted controlled trials.

2. Food and Nutrition Board, Institute of Medicine. Vitamin B6. Dietary Reference Intakes: Thiamin, Riboflavin, Niacin, Vitamin B6, Vitamin B12, Pantothenic Acid, Biotin, and Choline. Washington D.C.: National Academies Press; 1998:150-195.

http://www.nap.edu/openbook.php?isbn=0309065542

3. Combs, G.F. The Vitamins: Fundamental Aspects in Nutrition and Health. 2008. San Diego: Elsevier

Here is another good site for B-6 information:

http://ods.od.nih.gov/factsheets/vitaminb6/

3. Over the past few months we have found in autistic children :

A. HGF is significantly lower in autistic children (total and those with GI disease). Those without GI disease do not have significantly less HGF. This suggests that HGF levels are most affected in autistic children with GI disease, which could be associated with selective MET gene/receptor dysfunction in the GI tract. Copper inhibits HGF-MET receptor interaction. Autism-GI patients have significantly higher copper. These patients respond to zinc therapy by significantly lowering copper. Higher copper correlates with higher HGF in these same patients, hypothetically because of copper blocking HGF –MET interaction resulting in higher free (plasma) HGF. As copper decreases, HGF decreases in these same patients (HGF significantly decreases after zinc therapy/lowering copper). Lower HGF correlates with improved symptom severity.

The receptor tyrosine kinase Met and its ligand, hepatocyte growth factor (HGF), are clustered at excitatory synapses, especially in hippocampal neurons, and may regulate aspects of excitatory synaptic function. HGF increases expression of excitatory synaptic proteins, enhances their clustering at sites along dendrites, and increases current through the NMDA receptor.

Tyndall, S. J. and Walikonis, R. S. (2007), Signaling by hepatocyte growth factor in neurons is induced by pharmacological stimulation of synaptic activity. Synapse, 61: 199–204.

Stephanie Joyce Tyndall, “A role for hepatocyte growth factor and Met at excitatory synapses” (January 1, 2005). Dissertations Collection for University of Connecticut. Paper AAI3205766.

More specifically, HGF stimulates Ca2+ influx into dendrites through the NMDA receptor and that this effect is necessary for the changes in dendritic morphology induced by HGF.

Stephanie J. Tyndall, Sandip J. Patel, Randall S. Walikonis Hepatocyte growth factor-induced enhancement of dendritic branching is blocked by inhibitors of N-methyl-D-aspartate receptors and calcium/calmodulin-dependent kinases Journal of Neuroscience Research 2007;85: 2343–2351.

HGF and c-Met, can promote formation of neurites and enhance elaboration of dendrites in mature neurons, particularly during early stages of maturation.

Lim CS, Walikonis RS. Hepatocyte growth factor and c-Met promote dendritic maturation during hippocampal neuron differentiation via the Akt pathway. Cell Signal. 2008 May;20(5):825-35. Epub 2007 Dec 27.

Lowering HGF, therefore, may decrease expression of excitatory synaptic proteins and decrease current through the NMDA receptors, thereby improving symptom severity in these patients.

Glutamate treatment decreases the amount of pro HGF and increases the amount of the proteolytically-activated HGF.

HGF Total Data
Autistic        Controls
Mean = 469.49756        Mean = 545.64693
Standard Deviation = 183.85044  Standard Deviation = 173.1958
Standard Error = 23.34903       Standard Error = 27.04864
N=63    N=56

p = 0.03583

HGF GI
Autism GI       Controls
Mean = 450.88167        Mean = 545.64693
Standard Deviation = 158.11342  Standard Deviation = 173.1958
Standard Error = 37.26769       Standard Error = 27.04864
N=18    N=56

p = 0.04701

Autism GI Pre Therapy   Autism GI Post Therapy
Mean = 441.2612 Mean = 454.58185
Standard Deviation = 142.79181  Standard Deviation = 169.01646
Standard Error = 63.85844       Standard Error = 46.87673
N=5     N=13

p = 0.87033

Autism Non-GI Pre Therapy       Autism Non-GI Post Therapy
Mean = 561.50378        Mean = 455.41271
Standard Deviation = 255.49463  Standard Deviation = 173.65236
Standard Error = 85.16488       Standard Error = 29.35261

p = 0.13311

B. HMBG1 is significantly higher in autistic children. HMBG1 levels increase significantly, post zinc therapy in autistic children with GI disease, but not in the non-GI disease group. Symptoms of this group (GI) significantly improve after therapy, suggesting that Box Protein may play a role in this improvement.

HMBG1
Autistic        Controls
Mean = 102.36846        Mean = 52.85925
Standard Deviation = 101.60183  Standard Deviation = 52.93824
Standard Error = 13.22743       Standard Error = 15.28195

p = 0.02027

HMBG1
Autistic GI     Controls
Mean = 86.32029 Mean = 52.85925
Standard Deviation = 71.95985   Standard Deviation = 52.93824
Standard Error = 17.45283       Standard Error = 15.28195

p = 0.16072

HMBG1
Autistic GI     Autistic Non-GI
Mean = 86.32029 Mean = 106.70964
Standard Deviation = 71.95985   Standard Deviation = 108.8237
Standard Error = 17.45283       Standard Error = 16.22248

p = 0.39683

HMBG1
Autistic GI Pre Therapy Autistic GI Post Therapy
Mean = 42.3462  Mean = 93.8469
Standard Deviation = 29.59381   Standard Deviation = 68.9618
Standard Error = 13.23475       Standard Error = 15.42033

p = 0.02208

HMBG1
Autistic Non-GI Pre Therapy     Autistic Non-GI Post Therapy
Mean = 117.3499 Mean = 106.21234
Standard Deviation = 106.83099  Standard Deviation = 114.45062
Standard Error = 33.78293       Standard Error = 20.2322

p = 0.78092

High mobility group box 1 (HMGB1) can be released from neurons after glutamate excitotoxicity, and induces glutamatergic release from glial (gliosomes) cells. So, the cytokine could act as a modulator of glutamate homeostasis in adult brain.

We find HMGB1 correlates directly with HGF. Post zinc therapy HGF decreases and HMGB1 increases and both are associated with improved symptom severity. We suggest that increased HMGB1 results in increased glutamate. This in turn may result in

C. GABA plasma levels are significantly higher in autistic children.

GABA Autism     GABA Controls
Mean = 2121.54313       Mean = 1386.225
Standard Deviation = 1347.58082 Standard Deviation = 667.21071
Standard Error = 200.88549      Standard Error = 119.83458

p = 0.00247

Overall GABA levels don’t increase significantly post zinc therapy

GABA Autism Pre Zinc Therapy    GABA Autism Post Therapy
Mean = 2039.817 Mean = 2141.97467
Standard Deviation = 1535.46701 Standard Deviation = 1319.81002
Standard Error = 511.82234      Standard Error = 219.96834

p = 0.8578
However, GABA levels in autistic children without GI disease increase significantly post zinc therapy.

GABA Pre Therapy        GABA Post Therapy
Mean = 1188.32633       Mean = 2169.16824
Standard Deviation = 638.38549  Standard Deviation = 1207.63572
Standard Error = 260.61978      Standard Error = 241.52714

p = 0.0146

Copper has high affinity for GABAa receptor, blocking GABA attachement, elevating plasma GABA. This could also be why GABA is higher in autism with higher copper.

Sharonova IN, Vorobjev VS, Haas HL High-affinity copper block of GABA(A) receptor-mediated currents in acutely isolated cerebellar Purkinje cells of the rat. Eur J Neurosci. 1998 Feb;10(2):522-8.

Increased GABA correlates with decreased HMGB1 (p=0.009) as ell as decreased HGF (p=0.03) in our autistic patients.

Summary:

In summary, post zinc therapy, lowering copper, GABA levels increase significantly in the non-GI group, but not the GI group; HMGB1 increases significantly in the GI group but not the non-GI group; and HGF decreases in the non-GI group significantly but not the GI group.

Role of Copper:

1. Inhibits HGF-MET receptor interaction. Raising free plasma HGF (Pro-HGF)

2. Copper has high affinity for GABAa receptor. Raising Plasma GABA

Role of Glutamate:

1. Glutamate treatment decreases the amount of pro HGF and increases the amount of the proteolytically-activated HGF.

2. High mobility group box 1 (HMGB1) can be released from neurons after glutamate excitotoxicity, and induces glutamatergic release from glial (gliosomes) cells.

3. GABA is the major inhibitory neurotransmitter in the mature brain, but, in the developing brain GABA can be excitatory. Glutamate may play an inhibitory role in modulating the calcium-elevating actions of GABA that may affect neuronal migration, synapse formation, neurite outgrowth, and growth cone guidance during early brain development.

It is possible that in these autistic groups:

1.Lowering copper levels, lowers HGF plasma levels (associated with improved symptoms) because of enhanced HGF-MET receptor interaction.

2.Higher HMGB1 levels (especially in the GI group), associated with improved symptom severity, possibly associated with inflammation, results in high glutamine (seen in autistic groups), which in turn, may result in higher levels of proteolytically-activated HGF (and then enhanced HGF-MET interaction).

3. Higher plasma GABA may be the result of faulty receptor (genetic) in autistic children as well as high copper, which competes for the receptor site with GABA. GABA levels may increase in the non-GI disease group, and be associated with improved symptoms, as a feedback mechanism resulting from glutamate increase.

Héja L, Barabás P, Nyitrai G, Kékesi KA, Lasztóczi B, et al. (2009) Glutamate Uptake Triggers Transporter-Mediated GABA Release from Astrocytes. PLoS ONE 4(9): e7153.

These results suggest that we measure glutamate in these autistic patients to confirm an association between glutamate, GABA, HGF and HMGB1.

Update 5.13.11

1. I continued analysis of GABA levels in bipolar patients.

GABA levels in all Bipolar patients  (N=34) are significantly higher than controls (N=22).

GABA Bipolar    GABA Controls
Mean = 1772.19078       Mean = 1259.35136
Standard Deviation = 766.77262  Standard Deviation = 713.13474
Standard Error = 135.54753      Standard Error = 152.04084

p = 0.01525

GABA levels increase  post zinc therapy

GABA Pre Zinc Therapy   GABA Post Zinc Therapy
Mean = 1751.12905       Mean = 2161.2524
Standard Deviation = 633.56356  Standard Deviation = 582.69651
Standard Error = 141.66912      Standard Error = 184.26482

p = 0.04663

HGF increases post zinc therapy in Bipolar Patients

HGF Pre Therapy HGF Post Therapy
Mean = 287.29333        Mean = 548.35818
Standard Deviation = 81.32084   Standard Deviation = 269.24318
Standard Error = 27.10695       Standard Error = 81.17987

p = 0.00994

Pre Therapy decreasing HGF correlates with increasing GABA in Bipolar patients (p=0.036).

Pre Therapy, increasing zinc correlates with decreasing GABA (p=0.025).

2. I analyzed new data of GABA levels in schizophrenic patients.

We found that schizophrenic patients (N=22)  have significantly higher plasma GABA than controls (N=22).

GABA Schizophrenic    GABA Controls
Mean = 1511.85214       Mean = 1177.5481
Standard Deviation = 471.0455   Standard Deviation = 615.96203
Standard Error = 100.42724      Standard Error = 134.41393
p = 0.05

GABA levels do not change significantly post zinc therapy in these patients.

GABA Pre Therapy        GABA Post Therapy
Mean = 1626.7616        Mean = 1416.09425
Standard Deviation = 447.97025  Standard Deviation = 487.23602
Standard Error = 141.66063      Standard Error = 140.65292
p = 0.30401

Higher GABA correlates with lower HGF in these schizophrenic patients (p=0.03).

Higher GABA correlates with higher HMGB1 (p=0.08).

Increased GABA correlates with decreased symptom severity in schizophrenia (p=0.02).

3. I analyzed GABA levels of Bipolar patients with respect to drug therapy used by patients in this study. Patients taking drug therapy have lower GABA than Bipolar patients who not on drug therapy (p=0.08). Those on anticonvulsants (p=0.035) and antipsychotics (p=0.09) had particularly lower GABA compared to those not taking drug therapy. Those taking antidepressants and anti anxiety medication had lower, but not significantly lower GABA. Post zinc therapy, however, those taking drugs had the same increase in GABA and HGF as those not taking any drugs. Both groups (taking and not taking drugs) had a significant increase in zinc and decrease in copper and both groups had significant improvement in symptom severity, post zinc therapy. This suggests that it is the zinc therapy that is associated with patient improvement and not drug therapy.

4. I began to revise the manuscipt, Analysis of Copper and Zinc Plasma Concentration and the Efficacy of Zinc Therapy in Individuals with Asperger’s Syndrome, Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS) and Autism for resubmission to the Journal Biomarkers.

5. I continued to write the manuscript, Increased HMGB1 in Autistic Children Correlates with Symptom Severity.

6. I began to write a manuscript, Increased GABA in Autistic Children Correlates with Decreased HMGB1 and Decreased Copper.

Update 5.3.11

1. I analyzed updated GABA data in autistic children compared to controls.

2. Based on now studying  65 autistic children (N=65),  our autistic patients, before receiving therapy, have significantly higher plasma GABA (mmole/L) than controls (this is in contrast to my previous report of preliminary data):

GABA Autism     GABA Controls
Mean = 2121.54313       Mean = 1386.225
Standard Deviation = 1347.58082 Standard Deviation = 667.21071
Standard Error = 200.88549      Standard Error = 119.83458

p = 0.00247

This supports previous reports.
Dhossche D, et al Elevated plasma gamma-aminobutyric acid (GABA) levels in autistic youngsters: stimulus for a GABA hypothesis of autism. Med Sci Monit. 2002 Aug;8(8):PR1-6.

Abnormalities in the GABAergic system have been found the platelets of all of the autistic children in one study, which makes some researchers hypothesize that the elevated plasma GABA levels result because of hyposensitivity of a subset of GABA receptors. It is plausable that presynaptic cells increase GABA release to compensate for the subsets’ decreased sensitivity.

The GABAergic receptor system is significantly reduced in autism.

Schmitz, C. et. al. Autism: neuropathology, alterations of the GABAergic system, and animal models. Int Rev Neurobiol. 2005;71:1-26.

There are decreased GABA receptors and a decrease in benzodiazepine binding sites in the anterior cingulate cortex in autistic patients, as compared to typically developing controls. These findings may result from increased GABA innervation and/or release which disturbs the balance of excitation/inhibition of principle neurons and their output to other targets in the limbic system.

Oblak A et. al. Decreased GABAA receptors and benzodiazepine binding sites in the anterior cingulate cortex in autism. Autism Res. 2009 Aug;2(4):205-19.

A reduction of the plasma GABA (by administrating a GABA-T agonist, Imipramine) probably results in more axon(s)-to-oligodendrocyte signaling in the corpus callosum and it is postulated that this could result in a reduction of the autistic features.

Cohen BI Use of a GABA-transaminase agonist for treatment of infantile autism. Med Hypotheses. 2002 Jul;59(1):115-6.

3. Post Zinc therapy, in our patients, GABA levels increase, but not significantly:

GABA Autism Pre Zinc Therapy    GABA Autism Post Therapy
Mean = 2039.817 Mean = 2141.97467
Standard Deviation = 1535.46701 Standard Deviation = 1319.81002
Standard Error = 511.82234      Standard Error = 219.96834

p = 0.8578

4. We analyzed correlation between GABA and HGF, HMGB1, Cu/Zn SOD, Cu and Zn in these same autistic children. We found that increased GABA correlates with decreased HMGB1 (p=0.035) and decreased copper (p=0.08).

We previously found that autistic children have higher levels of HMGB1.

HMBG1
Autistic        Controls
Mean = 102.36846        Mean = 52.85925
Standard Deviation = 101.60183  Standard Deviation = 52.93824
Standard Error = 13.22743       Standard Error = 15.28195

p = 0.02027

High mobility group box 1 (HMGB1) functions as an activator of the immune response and can be released from neurons after glutamate excitotoxicity. Serum levels of HMGB1 have been found to be significantly higher in patients with autistic disorder.

Emanuele E et al Increased serum levels of high mobility group box 1 protein in patients with autistic disorder. Prog Neuropsychopharmacol Biol Psychiatry. 2010;34(4):681-3.

Copper is a potent inhibitor of GABA-evoked responses in rat Purkinje cells and may be an endogenous synaptic modulating factor. Copper toxicity, notably in Wilson’s disease, could result to some extent from chronic GABAA receptor blockade.

Sharonova IN, Vorobjev VS, Haas HL. High-affinity copper block of GABA(A) receptor-mediated currents in acutely isolated cerebellar Purkinje cells of the rat. Eur J Neurosci. 1998 Feb;10(2):522-8.

Copper toxicity, seen in many of our patients, may be causing receptor blockade, which is measured as increased plasma GABA. Lowering copper may reduce this blockade and decrease GABA levels.

5. Most interestingly, I studied the correlation between plasma GABA and symptom severity and found that increased GABA correlates significantly with increased severity of receptive language (p=0.05), hyperactivity (p=0.001), impulsivity (p=0.005), fine motor skills (p=0.04), sound sensitivity (p=0.004) and light sensitivity (p=0.025). This supports the relationship between plasma GABA and symptoms, and strongly suggests that the regulation of GABA is important in decreasing symptom severity in our autistic patients.

6. I began to investigate the potential relationship between Box Protein (HMGB1) and GABA.

7. I began to analyze update of data of plasma GABA in Bipolar Disorder and Schizophrenia. More to come later. Recent postmortem studies have provided consistent evidence that a defect of GABAergic neurotransmission probably plays a role in both schizophrenia and bipolar disorder.

Benes FM, Berretta S. GABAergic interneurons: implications for understanding schizophrenia and bipolar disorder. Neuropsychopharmacology. 2001 Jul;25(1):1-27.

8. I began to revise the manuscript, Analysis of Copper and Zinc Plasma Concentration and the Efficacy of Zinc Therapy in Individuals with Asperger’s Syndrome, Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS) and Autism.

9. I continued to edit the manuscript, Increased HMGB1 in autistic children.

Update 4.18.11

1. Analyzed results of HGF and HMGB1 assays and found a direct correlation between HGF  and HMGB1 (p=0.05) in autistic childen (N=68). In this same autistic group, increased HGF correlates with Lower Zn (p=0.01). Increased HMGB1 also correlates with lower zinc (p=0.03).

2. I investigated the relationship between age and HGF, HMGB1 and GABA levels. HGF and GABA levels are not associated with age. However, HMGB1 is significantly related to age (p=0.003), higher in older age groups.

3. I assessed the correlation between HGF, HMGB1 and severity of perceived symptoms in 68 autistic patients and found that increased HMGB1 correlates with decreased awareness (p=0.03), receptive language (p=0.02), focus/attention (p=0.01),  hyperactivity (p=0.02), rocking/pacing (p=0.02), and sound sensitivity (p=0.03). increased HGF levels correlate with decreased hypotonia (p=0.08) and sound sensitivity (p=0.06). This suggests that HMGB1 (Box protein) may be a good marker for symptom severity in autistic children.

4. We found that autistic children (n=30) had significantly higher HMGB1 levels compared to neurotypical controls (n=10) (p = 0.00237) and HGF levels are significantly decreased in autistic children (p=0.0001) (Biomarker Insights 2009:2 181–190).

5. When assessing HGF and HMGB1 levels, pre and post therapy, in these same autistic children (n=68). HGF levels decrease (although not significantly) post zinc/B-6 therapy (mean 518 pg/ml pre; 455 pg/ml post), HMGB1 levels increase after therapy (mean 92 ng/ml pre; 106 ng/m post) and GABA levels decrease post therapy (860 pg/ml pre; 1221 pg/ml post).

6. We assessed differences in autistic children with and without GI disease, pre and post therapy, and found that, before zinc/B-6 therapy, HMGB1 levels are significantly lower in autistic children with GI disease than without (p=0.05). HGF levels were not significantly lower in the GI group, pre therapy.

7. I continued to edit manuscript on HMGB1 levels in autistic children.

Update 4.8.11

1.  My grant proposal, To study the Relationship Between Decreased Hepatocyte Growth Factor (HGF) and Glutamate Excitotoxicity in Autistic Children was approved for $7228.00.

Introduction and Rationale

Recent studies have shown that HGF supplementation restores GABAergic interneurons and corrects reversal-learning deficits (1). It also modulates GABAergic inhibition and seizure susceptibility (2), and has been found to be an enhancer of nmda currents and synaptic plasticity in the hippocampus (3). Recently HGF has also been found to decrease glutamatergic neurotoxicity on motoneurons, as it has been found to increase levels of EAAT2/GLT1 (glutamate transporter), which suggests the possible improvement of glutamate clearance by HGF overexpression (4). We suggest that decreased levels of HGF would, in turn, lower glutamate transporter levels, resulting in increased glutamate availability.

High mobility group box 1 (HMGB1) functions as an activator of the immune response and can be released from neurons after glutamate excitotoxicity. Serum levels of HMGB1 have been found to be significantly higher in patients with autistic disorder (5).

We found decreased levels of HGF in autistic children (10) and adults with depression, anxiety, bipolar disorder and schizophrenia (6-9) and suggest that this is associated with lower levels of glutamate transporter and higher levels of glutamate in these individuals.

We suggest that one of the ways that HGF may be involved in the etiology of autism is through glutamate neurotoxicity. More specifically, since high glutamate has been associated with autism (11), and decreased HGF may lead to decreased glutamate transporter, it is possible that decreased HGF is associated with higher glutmate.

We intend to measure HMGB1, glutamate and EAAT2/GLT1 (glutamate transporter), to see if levels correlate with HGF levels. We hypothesize that decreased levels of HGF will be associated with higher HMGB1 and glutamate, and lower EAAT2/GLT1 (glutamate transporter), suggesting a role for HGF in glutamate modulation.

Methods

Assays:

We will use ELISA’s to measure HMGB1, glutamate and glutamate transporter serum concentration. Briefly, the procedure for both assays is as follows:
1. Add Capture Antibody: Dilute one vial of Capture Antibody with 10 ml of Capture Antibody Dilution Buffer (Solution A).  Add 100 ml of capture antibody solution to each well and incubate at 4°C overnight.
2. Prepare Standard Dilutions: The recommended standard range is 1.6-100 ng/ml.  Dilute one vial of HMGB1 Standard with 950 ml of Sample/Standard Dilution Buffer (Solution B) – 100 ng/ml.  Prepare serial dilutions of the standard by mixing 250 ml of the 100 ng/ml standard with 250 ml of Solution B – 50 ng/ml.  Then repeat this procedure to make fi ve more serial dilutions of standard – 25, 12.5, 6.25, 3.1 and 1.6 ng/ml solutions. 3. Prepare Sample Dilutions: Centrifuge samples at 10,000 rpm at 4°C for 3 minutes to remove insoluble materials and lipids, and use the supernatant as samples.  If the HMGB1 level is more than 100 ng/mL, re-assay the sample at a higher dilution.
4. Prepare Detection Antibody:  Dissolve one vial of Detection Antibody in 5 ml of Detection Antibody Dilution Buffer (Solution C).
5. Dilute Wash Buffer: Dilute 50 ml of 20X wash buffer in 950 ml of distilled water (1X wash buffer).   Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer.  Empty the plate by inverting it and blot on a paper towel to remove excess liquid.  Do not allow the plate to dry out.
6. Add Standards, Samples and Detection Antibody: Mix standards, samples and detection antibody tubes well.  Add 50 ml of Solution B (blank), standards and samples to appropriate wells (Figure 1).  Add 50 ml of diluted detection antibody solution to all wells.  Mix detection antibody solution and sample/standard solutions in all wells by pipettiwith a plate sealer and incubate at 37°C for 1 hour, then settle at 4°C overnight.
7. Prepare Streptavidin Peroxidase: Dilute one vial of Streptavidin Peroxidase in 10 ml of Streptavidin Peroxidase Dilution Buffer (Solution D).
8. Wash: Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer.  Empty the plate by inverting it and blot on a paper towel to remove excess liquid.
9. Add Streptavidin Peroxidase: Add 100 ml of streptavidin peroxidase solution to each well and incubate at room temperature for 30 minutes.  Do not incubate the plate more than 60 minutes.
10. Wash: Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer.  Empty the plate by inverting it and blot on a paper towel to remove excess liquid.
11. TMB: Add 100 ml of TMB Solution to all wells immediately after washing the plate.  Incubate for 30 minutes at room temperature.
12. Stop: Add 50 ml of 2N sulfuric acid (Stop Solution) to each well.
13. Read Plate: Read the OD values within 30 minutes by dual wavelength at 450 nm (sample) and 630 nm (reference).

Design:

We will test serum from 50 autistic children and 50 neurotypical, age and gender similar controls for HMGB1, glutamate and EAAT2/GLT1 (glutamate transporter). We will then compare this data to HGF serum concentration previously established in these patients by our laboratory.

Budget

HMGB1 ELISA Kits (6) Chondrex, Inc. $398.00/kit Total = $2388.00.00

Glutamate ELISA Kits(6) Rocky Mountain Diagnostics $490.00/kit Total = $2940.00

EAAT2/GLT1 (glutamate transporter) ELISA materials (polyclonal and monoclonal antibodies, secondary enzyme conjugated antibodies), buffers, blocking solution, substrate kits = $1900.00

Total = $7228.00

References

1.Bissonette G et al Astrocyte-mediated HGF/SF supplementation restores GABAergic interneurons and corrects reversal learning deficits in mice J Neurosci. 2010;30(8): 2918–2923.

2. Bae MH et al Hepatocyte growth factor (HGF) modulates GABAergic inhibition and seizure susceptibility. Exp Neurol. 2010;221(1):129-35.

3. Akimoto M et al Hepatocyte growth factor as an enhancer of nmda currents and synaptic plasticity in the hippocampus Neuroscience 2004;128:155-162.

4. Woong S Overexpression of HGF Retards Disease Progression and Prolongs Life Span in a Transgenic Mouse Model of ALS The Journal of Neuroscience 2002;22(15):6537-6548.

5. Emanuele E et al Increased serum levels of high mobility group box 1 protein in patients with autistic disorder. Prog Neuropsychopharmacol Biol Psychiatry. 2010;34(4):681-3.

6. Russo,A.J. Decreased serum Hepatocyte Growth Factor (HGF) in Individuals with Bipolar Disorder normalizes after Zinc and Anti-oxidant Therapy Nutrition and Metabolic Insights 2010:3 49–55.

7. Russo,A.J. and deVito,R Decreased Serum Hepatocyte Growth Factor (HGF) in Individuals with Schiphrenia Normalizes after Zinc and B-6 Therapy Proteomics Insights 2010:3 71–77.

8. Russo,A.J. Decreased Serum Hepatocyte Growth Factor (HGF) in Individuals with Anxiety Increases After Zinc Therapy Nutrition and Metabolic Insights 2010:3 1–6.

9. Russo,A.J. Decreased Serum Hepatocyte Growth Factor (HGF) in Individuals with Depression Correlates with Severity of Disease Biomarker Insights 2010:5 1–5.

10. Russo,A.J.,  et al Decreased Serum Hepatocyte Growth Factor (HGF) in Autistic Children with Severe Gastrointestinal Disease Biomarker Insights 2009:4:181-190.

11. Jamain S Linkage and association of the glutamate receptor 6 gene with autism Nature 2002; 7:302-310.

2.Submitted the following preliminary grant proposal to Organization for Autism Research:

To study the effect of iPad applications (Apps) on conversational language skills in autistic children

Background

In 1983, Rapin and Allen referred to a group of children who presented with mild autistic features and specific semantic pragmatic language problems when they described the communicative behavior of children as pathological talkativeness, deficient access to vocabulary and discourse comprehension, atypical choice of terms and inappropriate conversational skills (1). More recently, the term “pragmatic language impairment” (PLI) has been proposed to describe this trait (2,3).

Children with PLI have difficulty in understanding the meaning of words, grammar, syntax, prosody, eye gaze, body language, gestures, or social context.

Language disorders have been extensively reported in autistic children (6-8). Most autistic children have difficulty in communication and effectively using language, although the degree of these probelems vary greatly (9,10).

In preliminary research, we studied the symptom severity of 79 autistic and 29 Asperger’s individuals and 18 controls, before and after nutrient therapy, and found that symptoms decreased after therapy in autistic individuals with respect to awareness (p = 0.039), receptive language (p = 0.014), focus and attention (p = 0.011), Hyperactivity (p = 0.002), Tip Toeing (p = 0.002), Eye Contact (p = 0.085), Sound Sensitivity (p = 0.098), Tactile Sensitivity (p = 0.012) and seizures (p = 0.057), but not with respect to other measured symptoms including pragmatic language (p = 0.19608) . None of the measured symptoms worsened after therapy. None of the symptoms in the Asperger’s patients improved after therapy, including pragmatic language (p = 0.85655). It is also worth noting that in the autistic group, the mean severity of dysfunctional pragmatic language was higher than any other symptom measured, and it was the second most severe (behind focus/attention) in the Asperger’s group.

Our data suggests that PLI is an important symptom in autism and Asperser’s and nutrient therapy is not effective enough in alleviating the problem.

Methodology

Applications (Apps) designed for iPAD technology (described below) will be used to engage children in the process of improving their speech and communication skills.

20 iPADs will be distributed to autistic children (meeting DSM-IV criteria for diagnosis) and 20 iPADs will be distributed to neurotypical age and gender similar controls. Each iPAD will have Apps installed.

Technicians will meet with each participant. Baseline scores on the CCC will be established for each and iPAD instruction will be given to participants and parents (guardians).

Participants will be asked to use the 3 apps, 15 minutes each, each day (total 45 minutes/day).

**The Pfeiffer Center will maintain the iPADS for future use in experiments (possibly measuring the effect of new software as it is developed).

Software (Apps)

Articulate it!

This application allows speech therapists and parents to work toward improving the speech of children. The software contains over one thousand images in all sounds of the English language. Main Features include: 1. Over one thousand pictures 2. Audio recordings for every word on the application. The child can practice saying sounds on their own, repeating the voice on the app. 3. Contains all phonemes of the English language 4. Has the ability to track multiple students with separate goals 5. Ability to group sounds by manner of articulation or phonological processes 6. Built in voice recording allowing children to compare their productions with the audio recording 7. Extremely specific results screens- providing percentage based on phonemes/position associated with missing words. 8. Note taking capability, which allows professionals and parents to take notes during practice. 9. Allows user to skip images they don’t want to practice.

Match2Say

Match2Say is the matching game for children that have difficulties producing some sounds. While they play, they say the words and improve their articulation skills at the same time. The software was developed to be used by parents to help children learn to pronounce their sounds quicker and more accurately.

Expressive

Expressive was developed to help improve the communication abilities of individuals with a communication disorder. Expressive will give that person the ability to express themselves through the use of pictured images and corresponding audio.
 The best thing about Expressive is its ease of use. It does not require any knowledge of programming or the use of complicated manuals. The software is flexible and its intuitive customization allows users to select from a pool of images with audio files, create their own folders and arrange the images as they please.

* All three Apps were developed by Barbara Fernandes, M.S, CCC-SLP. She is a practicing speech and language pathologist in Dallas, Texas.

Evaluation/Data Analysis

PLI can be measured best using the Children’s Communication Checklist  (CCC) (4), and this tool is useful in a research setting for the assessment of PLI (5).

CCC will be given to each participant once per month, preferably online, for a total of six months. Correlation analysis will be performed, comparing improvement in language skills of controls with experimental (autism) group.

Outcome Recommendations

We propose to study and ultimately propose the use of other methodology for helping to improve pragmatic language skills in autistic patients. We hypothesize that the combined use of the iPAD touch/manipulation screen technology and appropriate learning software with help to improve pragmatic language skills (as measured by the CCC) in autistic children.

Budget

40 iPADs @ $499.00 ea = $19960.00
40 Articulate it! By Smarty Ears @ $49.99 ea = $1999.60
40 Match2Say By Smarty Ears @ $34.99 ea = $1399.60
40 Expressive By Smarty Ears @ $34.99 ea = $1399.60
12 Children’s Communication Checklist by Pearson @ $176.00/kit (25)= $2112.00
Part Time Technician (for administration and data analysis) – $3,000.00

Total = $29,870.80

References

1.Rapin I, Allen D (1983). Developmental language disorders: Nosologic considerations. In U. Kirk (Ed.), Neuropsychology of language, reading, and spelling (pp. 155–184). : Academic Press.

2.Conti-Ramsden G, Botting N (1999). “Classification of children with specific language impairment: longitudinal considerations”. J. Speech Lang. Hear. Res. 42 (5): 1195–204.

3. Bishop DVM (2000). Pragmatic language impairment: A correlate of SLI, a distinct subgroup, or part of the autistic continuum? In DVM Bishop & LB Leonard (Eds.), Speech and language impairments in children: Causes, characteristics, intervention and outcome (pp. 99–113). Hove, UK: Psychology Press.

4. Volden J and Phillips L Measuring Pragmatic Language in Speakers With Autism Spectrum Disorders: Comparing the Children’s Communication Checklist—2 and the Test of Pragmatic Language American Journal of Speech-Language Pathology 2010;19:204-212.

5. Geurts H et al Can the Children’s Communication Checklist differentiate between children with autism, children with ADHD, and normal controls? Journal of Child Psychology and Psychiatry 2004;45:1437-1453.

6. Sahyoun CP, Belliveau JW, Soulieres I, Schwartz S, Mody M. Neuroimaging of the functional and structural networks underlying visuospatial vs. linguistic reasoning in high-functioning autism. Neuropsychologia. 2009.

7. Pickett E, Pullara O, O’Grady J, Gordon B. Speech acquisition in older nonverbal individuals with autism: a review of features, methods, and prognosis. Cogn Behav Neurol. 2009;22(1):1–21.

8. Herbert MR, Kenet T. Brain abnormalities in language disorders and in autism. Pediatr Clin North Am. 2007;54(3):563–83, vii.

9. Landa R, Garrett-Mayer E. Development in infants with autism spectrum disorders: a prospective study. J Child Psychol Psychiatry. 2006;47(6): 629–38.

10. Tager-Flusberg H, Caronna E. Language disorders: autism and other pervasive developmental disorders. Pediatr Clin North Am. 2007;54(3):469–81.

3. I investigated the RTN4 gene. It was recently reported that the RTN4 gene is associated with intermediate phenotypes for anxiety disorder, according to findings presented at the Anxiety Disorders Association of America conference. The product of this gene is a potent neurite outgrowth inhibitor, which may also help block the regeneration of the central nervous system in higher vertebrates.

4. I analyzed experiment measuring GABA levels in autistic children. GABA levels in autistic children (N=30) were significantly lower than neurotypical controls (N=10) (p = 0.016). We found a correlation between increased GABA and decreased HGF (p = 0.0256) and between increased GABA and decreased Cu, but not between HMGB1 and GABA  (p = 0.445), Cu/Zn SOD and GABA (p = 0.376), or Zn and GABA (p = 0.386).

Post zinc/B-6 therapy, in these same patients, GABA and HMGB1 levels increase (although not significantly), and HGF levels decrease significantly (p = 0.0455).

Increased HGF correlates with increased Cu in the autistic group (p=0.03), but not zinc (p=0.14).

Those autistic children with GI disease (N=8) have lower levels of GABA and HMGB1 than those without GI disease (although not significantly lower). HGF levels were similar in both GI and non GI groups.

5. I did an analysis of GABA levels and individual symptom severity in these autistic children. Higher GABA levels correlated with higher severity of receptive language impairment (p=0.05),  hyperactivity (p=0.03),  focus/attention (p=0.035), hypotonia (p=0.04). Lower GABA levels correlated with higher severity of conversational language (p=0.03), hand flapping/stimming (p=0.05), and rocking/pacing (p=0.01).

6. I analyzed experiments of GABA levels in bipolar patients.  Bipolar patients (n=18) had significantly higher levels of GABA than neurotypical controls (n=22) (p=0.008). In Bipolar patients, higher GABA levels correlated with decreased HGF (p=0.09), decreased Cu/Zn SOD (p=0.05), decreased Zn (p=0.08). There was also a direct correlation between increased zinc and increased HGF (p=0.07).

Post Zinc/B-6 therapy, in bipolar patients, GABA levels remained constant, but HGF levels increased significantly (p=0.009). Overall symptom severity significantly improved in this same group, post therapy (p=0.0015).

7. I continued to write and edit the manuscript, increased Plasma HMGB1 in Autistic Children.

8. Continued to write and edit the manuscript, increased Plasma HMGB1 in Individuals with Schizohrenia.

Update 3.19.11

1. I began to write a paper increased Plasma HMGB1 in Autistic Children.

2. Analyzed HMGB1 levels and outcomes in individuals with schizophrenia. Did not find a correlation between HMGB1 levels and symptom severity (p=0.81), nor was there a correlation between Cu/Zn SOD and overall symptom severity (p=0.48).

3. Analyzed HMGB1 and HGF levels in schizophrenic individuals. HMGB1 and HGF levels do not correlate with each other in schizophrenic individuals (p=0.52). However, HMGB1 levels in schizophrenics do correlate with Cu/Zn SOD (p=0.0002) and borderline in controls (p=0.06)

4. HGF levels in schizophrenics did correlate with overall symptom severity (p=0.01) and particularly with anxiety (p=0.04).

5. We also found that HMGB1 levels significantly decreased (normalized) post therapy in our schizophrenic patients (p=0.03) and Cu/Zn SOD levels also decrease (p=0.02).

6. I began to write a paper , Increased HMGB1 in Individuals with Schizophrenia.

7. I resubmitted (after revision) paper, Increased Copper in Individuals With Autism Normalizes Post Zinc Therapy More Efficiently in Individuals With Concurrent GI Disease.

8. I responded to reviewer’s comments about my recent ARI grant proposal. Here are reviewer’s comments and my responses.

Reviewer #2

I think this is a very strong proposal.  Anthony has already conducted several studies showing that HGF is lower in autism, bipolar, schizophrenia, anxiety, and depression, and he has demonstrated that it is treatable with vitamin/mineral therapy for bipolar, schizophrenia, and anxiety.  He has also shown that it is strongly linked to GI problems.

The proposed study will further investigate possible mechanisms of action of HGF, including measuring HMGB1 (activator of immune response), glutatmate, and glutamate transport.

My one concern is that serum levels of glutamate and EAAT2/GLT1 (glutamate transporter) are probably only moderately correlated with CSF levels; I suspect platelet levels would be better, but I don’t know if that is available.

This is a good suggestion. We will attempt to measure both plasma and platelet levels of glutamate and EAAT2/GLT1.

In measuring glutamate levels, I suggest using fasting levels, and processing samples immediately/freezing immediately, since the glutamine/glutamate ratio in plasma is very unstable and very sensitive to shipping conditions.

Another good suggestion. We will use fasting levels and process samples immediately after blood is drawn.

The budget is very modest, and the participant numbers are high.  He is taking advantage of previous measurements of HGF, which is good.

Since his previous work demonstrated that HGF correlates with GI problems, I recommend he add to this study an assessment of GI severity if he does not already have that data.  Similarly, since one study found a correlation of autism severity with levels of HMGB, I suggest he also collect data on autism severity using a standardized tool (ATEC, etc) and investigate correlations.

We will add to the study an assessment of GI severity and correlate HGF levels with this severity.

In summary, I strongly recommend fully funding this study, due to its very modest cost and high impact – I think he is very likely to have positive results, and it may then indicate new avenues for treatment.

It would also be interesting for him to consider doing a treatment study for autism, as he did for other disorders.

Reviewer #1

ARI Grant Review

Investigator:  A.J. Russo PhD
Title:  Relationship between decreased hepatocyte growth factor and glutamate excitotoxicity in autistic children
Critique:  This is a straight-forward pilot study using commercially available kits to determine whether or not low HGF previously observed in children with autism is associated with elevated glutamate, and HMGB1 (an immune activator released from neurons after glutamate toxicity), and lower levels of the glutamate transporter, EAAT2/GLT1.  This is an interesting hypothesis loosely on a rodent study and a small study in children with autism.  Specific concerns and questions are outlined below:
The hypothesis is based on a transgenic mouse study of HGF over-expression which was associated with increased levels of glutamate transporter.  Did this study find lower glutamate levels associated with HGF over-expression or is that an assumption?

We intend to confirm other studies which have already found glutamatergic excitotoxicity as etiologically significant in autism, reviewed and found in:

Rossignol DA  Novel and emerging treatments for autism spectrum disorders: a systematic review Ann Clin Psychiatry. 2009 Oct-Dec;21(4):213-36.

Yap IK, Angley M, Veselkov KA, Holmes E, Lindon JC, Nicholson JK Urinary metabolic phenotyping differentiates children with autism, from their unaffected siblings and age-matched controls J Proteome Res. 2010 Jun 4;9(6):2996-3004.

Moreno-Fuenmayor, H., Borjas, L., Arrieta, A., Valera, V., & Socorro-Candanoza, L. Plasma excitatory amino acids in autism. The Journal of Clinical Investigation, 1996;37(2):113-128.

Aldred, S., Moore, K.M., Fitzgerald, M., & Waring, R.H. Plasma amino acid levels in children with autism and their families. Journal of Autism and Developmental Disorders, 2003;33:93-97.

Both the postulated increase in HMGB1 and decrease in EAAT2/GLT1 are dependent on the finding of elevated glutamate in this cohort of children.  If elevated glutamate is not found, is there any reason to pursue the other endpoints? Glutamate levels should be done first.

I agree with this. We will confirm elevated glutamate levels before moving ahead with the rest of the study. If glutamate levels are not increased, this will be an important, although unexpected, finding. As such, this might necessitate a re-evaluation of our patient population. Regardless, measuring HMGB1 and EAAT2/GLT1 levels and correlating these levels with HGF, as well as symptom severity, will provide a better understanding of the mechanism of HGF action, as these proteins may be associated with HGF but not result in changes in glutamate levels.

The suggestion that HGF may be involved in the “etiology” of autism is not a valid assumption since these samples were taken after the diagnosis of autism.  It is not possible to know if HGF is involved in the cause of autism or is simply a epiphenomenon.  This needs to be clarified in studies of children already diagnosed with autism.

I agree. Further studies (prospective) will need to be done to confirm whether or not HGF is involved in the etiology of autism. This would be a next logical step in our research plans.

Update 3.13.11

1.Analyzed experiment measuring HMGB1 in autistic children. We found that autistic children (n=30) had significantly higher HMGB1 levels compared to neurotypical controls (n=10) (p = 0.00237).

2. I analyzed results (above) with previously measured HGF and Cu/Zn SOD levels and a correlation between increased HMGB1, decreased HGF (p = 0.046) and increased Cu/Zn SOD (p = 0.010).

3. I also analyzed HMGB1, HGF and Cu/Zn SOD levels with respect to individual symptom severity in these same individuals. We found that increasing HMGB1 levels correlate with improved awareness (p= 0.045), receptive language (p= 0.010), conversational language (p=0.05), focus attention (p=0.012), hyperactivity (p=0.03), impulsivity (p=0.03), gross motor skills (p=0.05), rocking/pacing (p=0.05), obsession/fixations (p=0.06), sound sensitivity (p=0.007)).

I found that decreasing HGF levels correlate with improved receptive language (p=0.03), conversational language (p=0.05), hyperactivity (p=0.03), impulsivity (p=0.014), fine motor skills (p=0.05), gross motor skills (p=0.09), rocking/pacing (p=0.02), hand flapping (p=0.03), obsession/fixation (p=0.05), sound sensitivity (p=0.015)

I found that increased Cu/Zn SOD correlated with obsession/fixati0on (p=0.04) and sound sensitivity (p=0.03).

4. Updated outcomes of ASD, Asperger’s, autism and PDD-NOS patients.

5. Used updated outcomes (4) to correlate HMGB1, HGF and Cu/Zn SOD levels with outcomes of each symptom in autistic children.

6. Jim Adams (Arizona State U) and I will collaborate on a major 1-year multi-treatment study, including vitamins, minerals, EFA’s, carnitine, digestive enzymes, and GFCF diet.

We are doing many pre/post behavioral and biochemical measurements.

We are intrigued by measurements of HGF, and the association with GI symptoms and hypothesize that the treatment will improve many symptoms including GI.  We will be doing an intestinal permeability study (with Doctor’s Data).

We will measure HGF pre/post in 50 children/adults with autism, and 1x measurement in 50 child/adult neurotypicals.

Dr. Adams will be supplying us with $2000.00 for the cost of the HGF assays.

7. My abstract, entitled “Increased Copper In Individuals with Autism Normalizes Post Zinc Therapy More Efficiently In Individuals with Concurrent GI Disease,” has been accepted for presentation as a poster or oral presentation at the International Meeting for Autism Research in San Diego this Spring, 2011.

8. Analyzed experiment HMGB1 and schizophrenia experiment. We found that individuals with schizophrenia (n=20) had significantly higher HMGB1 levels compared to neurotypical controls (n=24) (p = 0.0221).

9. My paper, Analysis of Plasma Zinc and Copper Concentration, and Perceived Symptoms, in Individuals With Depression, Post Zinc and B-6 Therapy (resubmitted) was accepted for publication in Nutrition and Metabolic Insights. What we found was that depressed individuals, with and without secondary anxiety, had decreased plasma Zn and elevated plasma Cu compared to controls. Zn normalized (increased to the level of normal controls) but Cu increased in individuals with depression (with and without secondary anxiety), after Zn therapy, whereas both plasma Zn increased and Cu levels decreased in anxiety, with and without secondary depression, after Zn therapy. Individuals with depression, with and without secondary anxiety, had significantly higher symptom severity when compared to neurotypical controls. Symptom severity in individuals with anxiety (both with and without secondary depression) significantly decreased after Zn therapy, whereas symptoms remained the same in individuals with primary depression. These data show an association between Zn and Cu plasma levels and clinically depressed individuals, and suggest that high Cu levels are associated with high symptom severity and that lowering copper, particularly in patients who don’t respond well to zinc therapy, might be important.

Update 2.28.11

1. Compared Cu, Zn, and Ferritin levels in all young patients (with no secondary disease) ADD, ADHD, PDD, ASD, autism and neurotypical controls. Overall, the autistic group had the lowest mean Ferritin concentration. All the groups have lower Ferritin, lower zinc and higher copper than controls, but none of the groups are significantly lower than the other groups.

2. Investigated HGF and its relationship to neurotransmission. Recent studies have shown that HGF supplementation restores GABAergic interneurons and corrects reversal learning deficits and that it modulates GABAergic inhibition and seizure susceptibility It has also been found to be an enhancer of nmda currents and synaptic plasticity in the hippocampus. Recently it has also been found to decrease glutamatergic neurotoxicity on motoneurons, in that HGF has been found to retain or even increase the levels of EAAT2/GLT1 (glutamate transporter), which suggests the improvement of glutamate clearance by HGF overexpression.

3. Compared Cu, Zn, and Ferritin levels in the older patient groups (with no secondary disease) anxiety, depression, bipolar, schizophrenia, and neurotypical controls. All the groups have similar Ferritin levels, lower zinc and higher copper than controls. Groups collectively have significantly higher copper than controls (p=0.06), with the depression group having considerably higher copper than the rest.

4. My abstract, Increased Copper in Individuals With Autism Normalizes Post Zinc Therapy More Efficiently in Individuals With Concurrent GI Disease, was accepted by Autism One, and I will be presenting on Sunday (May 29th) at the Autism One Conference.

5. Investigated the biology of HMGB1. HMGB1 was first described as a ubiquitous and highly expressed nuclear non-histone protein. In the nucleus, HMGB1 stabilizes nucleosome formation and facilitates transcription factor binding by bending DNA. Outside the cell it may function as a potent cytokine with the ability to trigger inflammatory mediators and can be released from neurons after glutamate excitotoxicity. Serum levels of HMGB1 are significantly higher in patients with autistm.

6. Based on investigations above, I wrote and submitted a grant (below) to Autism Research Institute (ARI), To study the Relationship Between Decreased Hepatocyte Growth Factor and Glutamate  Excitotoxicity in Autistic Children

Introduction and Rationale

Recent studies have shown that HGF supplementation restores GABAergic interneurons and corrects reversal-learning deficits (1). It also modulates GABAergic inhibition and seizure susceptibility (2), and has been found to be an enhancer of nmda currents and synaptic plasticity in the hippocampus (3). Recently HGF has also been found to decrease glutamatergic neurotoxicity on motoneurons, as it has been found to increase levels of EAAT2/GLT1 (glutamate transporter), which suggests the possible improvement of glutamate clearance by HGF overexpression (4). We suggest that decreased levels of HGF would, in turn, lower glutamate transporter levels, resulting in increased glutamate availability.

High mobility group box 1 (HMGB1) functions as an activator of the immune response and can be released from neurons after glutamate excitotoxicity. Serum levels of HMGB1 have been found to be significantly higher in patients with autistic disorder (5).

We found decreased levels of HGF in autistic children (10) and adults with depression, anxiety, bipolar disorder and schizophrenia (6-9) and suggest that this is associated with lower levels of glutamate transporter and higher levels of glutamate in these individuals.

We suggest that one of the ways that HGF may be involved in the etiology of autism is through glutamate neurotoxicity. More specifically, since high glutamate has been associated with autism (11), and decreased HGF may lead to decreased glutamate transporter, it is possible that decreased HGF is associated with higher glutmate.

We intend to measure HMGB1, glutamate and EAAT2/GLT1 (glutamate transporter), to see if levels correlate with HGF levels. We hypothesize that decreased levels of HGF will be associated with higher HMGB1 and glutamate, and lower EAAT2/GLT1 (glutamate transporter), suggesting a role for HGF in glutamate modulation.

Methods

Assays:

We will use ELISA’s to measure HMGB1, glutamate and glutamate transporter serum concentration. Briefly, the procedure for both assays is as follows:
1. Add Capture Antibody: Dilute one vial of Capture Antibody with 10 ml of Capture Antibody Dilution Buffer (Solution A).  Add 100 ml of capture antibody solution to each well and incubate at 4°C overnight.
2. Prepare Standard Dilutions: The recommended standard range is 1.6-100 ng/ml.  Dilute one vial of HMGB1 Standard with 950 ml of Sample/Standard Dilution Buffer (Solution B) – 100 ng/ml.  Prepare serial dilutions of the standard by mixing 250 ml of the 100 ng/ml standard with 250 ml of Solution B – 50 ng/ml.  Then repeat this procedure to make fi ve more serial dilutions of standard – 25, 12.5, 6.25, 3.1 and 1.6 ng/ml solutions. 3. Prepare Sample Dilutions: Centrifuge samples at 10,000 rpm at 4°C for 3 minutes to remove insoluble materials and lipids, and use the supernatant as samples.  If the HMGB1 level is more than 100 ng/mL, re-assay the sample at a higher dilution.
4. Prepare Detection Antibody:  Dissolve one vial of Detection Antibody in 5 ml of Detection Antibody Dilution Buffer (Solution C).
5. Dilute Wash Buffer: Dilute 50 ml of 20X wash buffer in 950 ml of distilled water (1X wash buffer).   Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer.  Empty the plate by inverting it and blot on a paper towel to remove excess liquid.  Do not allow the plate to dry out.
6. Add Standards, Samples and Detection Antibody: Mix standards, samples and detection antibody tubes well.  Add 50 ml of Solution B (blank), standards and samples to appropriate wells (Figure 1).  Add 50 ml of diluted detection antibody solution to all wells.  Mix detection antibody solution and sample/standard solutions in all wells by pipettiwith a plate sealer and incubate at 37°C for 1 hour, then settle at 4°C overnight.
7. Prepare Streptavidin Peroxidase: Dilute one vial of Streptavidin Peroxidase in 10 ml of Streptavidin Peroxidase Dilution Buffer (Solution D).
8. Wash: Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer.  Empty the plate by inverting it and blot on a paper towel to remove excess liquid.
9. Add Streptavidin Peroxidase: Add 100 ml of streptavidin peroxidase solution to each well and incubate at room temperature for 30 minutes.  Do not incubate the plate more than 60 minutes.
10. Wash: Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer.  Empty the plate by inverting it and blot on a paper towel to remove excess liquid.
11. TMB: Add 100 ml of TMB Solution to all wells immediately after washing the plate.  Incubate for 30 minutes at room temperature.
12. Stop: Add 50 ml of 2N sulfuric acid (Stop Solution) to each well.
13. Read Plate: Read the OD values within 30 minutes by dual wavelength at 450 nm (sample) and 630 nm (reference)

Design:

We will test serum from 50 autistic children and 50 neurotypical, age and gender similar controls for HMGB1, glutamate and EAAT2/GLT1 (glutamate transporter). We will then compare this data to HGF serum concentration previously established in these patients by our laboratory.

Budget

HMGB1 ELISA Kits (6) Chondrex, Inc. $398.00/kit Total = $2388.00.00

Glutamate ELISA Kits(6) Rocky Mountain Diagnostics $490.00/kit Total = $2940.00

EAAT2/GLT1 (glutamate transporter) ELISA materials (polyclonal and monoclonal antibodies, secondary enzyme conjugated antibodies), buffers, blocking solution, substrate kits = $1900.00

Total = $7228.00

References

1.Bissonette G et al Astrocyte-mediated HGF/SF supplementation restores GABAergic interneurons and corrects reversal learning deficits in mice J Neurosci. 2010;30(8): 2918–2923.

2. Bae MH et al Hepatocyte growth factor (HGF) modulates GABAergic inhibition and seizure susceptibility. Exp Neurol. 2010;221(1):129-35.

3. Akimoto M et al Hepatocyte growth factor as an enhancer of nmda currents and synaptic plasticity in the hippocampus Neuroscience 2004;128:155-162.

4. Woong S Overexpression of HGF Retards Disease Progression and Prolongs Life Span in a Transgenic Mouse Model of ALS The Journal of Neuroscience 2002;22(15):6537-6548.

5. Emanuele E et al Increased serum levels of high mobility group box 1 protein in patients with autistic disorder. Prog Neuropsychopharmacol Biol Psychiatry. 2010;34(4):681-3.

6. Russo,A.J. Decreased serum Hepatocyte Growth Factor (HGF) in Individuals with Bipolar Disorder normalizes after Zinc and Anti-oxidant Therapy Nutrition and Metabolic Insights 2010:3 49–55.

7. Russo,A.J. and deVito,R Decreased Serum Hepatocyte Growth Factor (HGF) in Individuals with Schiphrenia Normalizes after Zinc and B-6 Therapy Proteomics Insights 2010:3 71–77.

8. Russo,A.J. Decreased Serum Hepatocyte Growth Factor (HGF) in Individuals with Anxiety Increases After Zinc Therapy Nutrition and Metabolic Insights 2010:3 1–6.

9. Russo,A.J. Decreased Serum Hepatocyte Growth Factor (HGF) in Individuals with Depression Correlates with Severity of Disease Biomarker Insights 2010:5 1–5.

10. Russo,A.J.,  et al Decreased Serum Hepatocyte Growth Factor (HGF) in Autistic Children with Severe Gastrointestinal Disease Biomarker Insights 2009:4:181-190.

11. Jamain S Linkage and association of the glutamate receptor 6 gene with autism Nature 2002; 7:302-310.

Update 2.25.11

1. I assessed the relationship between iron levels and copper levels in autistic children and their response to zinc therapy. Correlation between the difference between pre therapy and post therapy ferritin and copper levels – p=0.007.

2. I finished revision of the paper, Analysis of Plasma Zinc and Copper Concentration, and Perceived Symptoms, in Individuals With Depression, Post Zinc and B-6 Therapy, for resubmission.

3. Assessed correlation between changes in ferritin, Cu and Zn levels and changes in hyperactivity severity in autistic children, pre and post therapy; and first and last visit. In summary, improvement in hyperactivity correlates significantly with decreased change in ferritin pre-post therapy (p=0.05) and first-last visit (p=0.08), but not with decreased Cu or increased Zn

4. Assessed correlation between changes in ferritin, Cu and Zn levels and changes in focus/attention severity  in autistic children, pre and post therapy; and first and last visit. Improvement in focus/attention correlates significantly with decreased change in ferritin pre-post therapy (p=0.006) and decreased change in Cu (p=0.09), but not increased Zn.

5. Assessed correlation between changes in ferritin, Cu and Zn levels and changes in tactile sensitivity severity in autistic children, pre and post therapy; and first and last visit. Improvement in focus/attention correlates significantly with decreased change in ferritin pre-post therapy (p=0.08), Cu (p=0.05) and increased Zn first-last visit (p=0.05).

Overall, a theme developing with these observations is that ferritin (stored iron) is associated with symptoms in autism more than we thought. It’s possible that the correlation between copper and ferritin levels is associated with this. It is also interesting that copper and zinc levels (changes after therapy) are related to improvement of different symptoms. This could be associated with changes in the physiology of different areas of the CNS.

6. Began to assess correlation between changes in ferritin, Cu and Zn levels and eye contact severity in autistic children, pre and post therapy; and first and last visit.

7. Began revision of paper, Increased Copper in Individuals With Autism Normalizes Post Zinc Therapy More Efficiently in Individuals With Concurrent GI Disease.

8. As part of the revision if the paper above (7), I analyzed the types of GI disease of our autistic patient’s with respect to zinc and copper levels. In summary, our patients have (approximately the same percentages), generalized GI disease, GERD, Gluten Intolerance and Constipation (p=0.74 Cu levels; p=0.84 Zn levels). Smaller percentages have IBS, Colitis and Celiac disease. The individual groups are too small to analyze separately with respect to changes in Cu and Zn post therapy, but collectively, the post therapy Cu and Zn levels were similar (p=0.28 Cu levels; p=0.38 Zn levels) and there was no significant difference between groups with respect to improved symptoms after therapy.

Update 2.9.11

1. We assessed our autistic groups, pre and post zinc therapy, with respect to Zinc (increased and not increased) and copper (decreased and not decreased) levels compared to outcomes for those groups. Interestingly, as we previously reported, overall, our autistic patients perceive that they improve significantly with respect to hyperactivity. The only group that improves significantly with respect to hyperactivity among the groups above is the group where zinc increases and copper decreases. This suggests that with respect to this symptom it may be necessary to look carefully at tweaking our therapy to make sure zinc increases and copper decreases in our autstic patients.


When we assess the individual patients symptoms (with data from first visit-pre therapy) and compare pre and post therapy symptom severity of individual patients in the groups above, we find that in 12 of 14 symptoms, the mean symptom severity is lower (perceive less severe symptom) in the individuals whose zinc levels normalized. Positive mean values below indicate an overall improvement in the symptom among patients in that group.

Mean Symptom Severity Difference (Pre vs Post Therapy)
Zinc Normalized     Zinc Did Not Normalize
Focus, Attention        0.11                  0.8
Hyperactivity           0.65               -0.38
Perseveration           0.42       -1.63
Fine Motor Skills       0                  -0.25
Gross Motor Skills      0.3                      0
Hypotonia               0.81              -1.33
Tip Toeing              1.76                    0
Rocking/Pacing  -0.05     -0.75
Stimming                -1.17     -1.25
Fixations                       -0.67     -0.75
Eye Contact              0.72     -0.17
Sound Sensitivity        0.13           0
Light Sensitivity       -0.11         0.5
Tactile Sensitivity      1.28       1.75

Zinc Normalized                             Zinc Did Not Normalize

Mean = 0.29857                            Mean = -0.24714
Standard Deviation = 0.74487    Standard Deviation = 0.89642
Standard Error = 0.19907           Standard Error = 0.23958

p = 0.09158

2. I assessed our Asperger’s groups, pre and post zinc therapy, with respect to Zinc (increased and not increased) and copper (decreased and not decreased) levels compared to outcomes for those groups. Although the number of Asperger’s patients in these groups is small, the severity of symptoms is higher in the group whose Zn levels did not normalize after therapy with respect to conversational language skills, perseveration, fine motor skills, obsessions and fixation, eye contact, sound sensitivity, light sensitivity. These differences are not significant (the number of patients in each group are too small) but they may signify a trend to keep an eye on. Like the autistic group, we might find that the group of Asperger’s patients who don’t respond to zinc therapy (raising zinc levels) are the most symptomatic. Keep in mind that in our previous assessment of ALL Asperger’s patients, there was no improvement in any of the symptoms after therapy.

3. I analyzed the relationship between Cu/Zn SOD levels and HGF levels in Bipolar patients (N=17). There is a correlation between Cu/Zn SOD levels and HGF (p=0.03), particularly post zinc therapy, suggesting that zinc levels are associated with rising Cu/Zn SOD levels and that this anti-oxidant is reducing oxidative stress and possibly influencing the rise in HGF levels.

There is also a negative correlation between HGF levels and symptom severity in these same patients (higher the HGF levels, lower the perceived symptom severity; p=0.01). This suggests that HGF may be a marker for disease severity and may be associated with disease etiology.

4. I began to write a grant to the International Bipolar Association, based on the findings above.

5. I began to revise the manuscript, Analysis of Plasma Zinc and Copper Concentration, and Perceived Symptoms, in Individuals With Depression, Post Zinc and B-6 Therapy.

Update 2.4.11

1. I continued to edit a grant proposal to study the effectiveness of iPAD language Apps at improving conversational skills in autistic and Asperger’s individuals. This proposal will be sent to the Organization for Autism Research (OAR) as a pre-proposal.

2. Began to do a thorough search for potential granting agencies to support our research. Starting with groups supporting autism research, the most promising groups, but most competitive, are NIH and the Simon Foundation (LOI due early January). Both will support research with budgets over $100,000.00, but tend to support projects involving multiple sites, including high profile institutions. Autism Speaks (LOI due mid-December) is in this same category, except that their primary awards, pilot and full-level, are for treatment based research and their new TrailBlazer Award, designed for quick review, requires that the primary investigator be a faculty member of a major educational institution. The Autism Research Institute continues to be our best source of funding. We already have a strong relationship with them and so far, they seem to be impressed with our work. On the negative side, their grants are limited to $30,000.00, and even so, they prefer smaller proposals seemingly to have the opportunity to spread their resources. The Organization for Autism Research (OAR) (LOI due in March) funds behavioral and educational proposals (see proposal above). The Dan Marino Foundation funds research, but through four pre-selected universities. The Doug Flutie Foundation funds educational and family services, but their focus is on geographic areas in the Northeast. Grants for Schizophrenia Research are outlined here from the Schizophrenia research Forum:http://www.schizophreniaforum.org/res/gra/default.asp Grants for Bipolar Disorder: Internationalbipolarfoundation (submission deadline Dec. 15, 2011) $50,000.00 limit. Based in California but is not limited to applicants from California.http://www.internationalbipolarfoundation.org/grant-opportunities NARSAD (Brain and Behavioral Research Fund) $30,000-$100,000. http://www.narsad.org/?q=node/164/apply_for_grants

3. Submitted abstract for presentation at the Ninth International Conference on Bipolar Disorder 9-11 June 2011 David L. Lawrence Convention Center, Pittsburgh, PA, U.S.A.

Decreased Serum Hepatocyte Growth Factor (HGF) in Individuals With Bipolar Disorder Normalizes after Zinc and Anti-oxidant Therapy, Russo AJ and deVito R

Abstract
Aim: To assess serum HGF concentration in individuals with bipolar disorder and investigate the efficacy of zinc therapy on these levels. Subjects and Methods: Serum from 35 individuals diagnosed with bipolar disorder and 19 age and gender similar controls were tested for HGF concentration using ELISAs, and copper and zinc plasma levels using inductively-coupled plasma-mass spectrometry. Results: HGF serum levels of individuals with bipolar disorder were significantly lower than age and gender similar controls (p=0.0021). HGF serum concentration was significantly lower in Bipolar patients pre-therapy (p=0.0009) and HGF levels normalized post-therapy. Zinc levels in these same individuals also normalized (p=0.0046) and patient’s perceived severity of Bipolar symptoms significantly decreased after therapy (p=0.0003). We also found a significant direct correlation between Zinc and HGF serum concentration in the bipolar patients (p=0.04). Discussion: These results suggest an association between low HGF levels and bipolar disorder and also demonstrate that zinc therapy may be associated with the normalization of HGF levels and decrease in severity of disease.

3. I continued to edit paper, Plasma Zinc and Copper Concentration, and Perceived Symptoms, in Individuals With Obsessive Compulsive Disorder (OCD), Post Zinc and B-6 Therapy

4. I assessed Cu and zinc levels in schizophrenia patients (n=30) vs neurotypical controls (n=19). Overall, our schizophrenic patients have less, but not significantly less Zn than controls, and more but not significantly more Cu than controls. Interestingly, when we separate out Schizophrenics without any other observable secondary neurological disease (N=11), there is no difference in zinc or copper between patients and controls. This suggests that secondary disease influences Zn and Cu metabolism. Related to this, all our schizophrenic patients (including those with secondary disease) tend to get significantly better (overall symptoms) after zinc therapy, whereas those with no secondary disease do not perceive they’re getting better.

5. I continued analysis of value of plasma histamine levels as a marker for methylation. Quest is now only reporting values >1.5ng/µl plasma histamine. None (or very few) of our patients have high histamine (above this value). Therefore, there appears to be no value in using this as a marker for methylation. We will assess the value of measuring SAMe as a marker for methylation (Great Plains Labs) instead.

6. I assessed Cu and Zn levels in all our patient groups, comparing patients who don’t have any other secondary or tertiary conditions (ie depression without anxiety or other conditions) with controls and those who have secondary neurological disease. More to come from this analysis soon.

7. I analyzed autism, PDD and Asperger’s patients with respect to the biochemistry of those patients who don’t respond to zinc therapy (lowering zinc) and analyzed those patients who respond to zinc therapy (zinc significantly increases), but copper doesn’t decrease appropriately. This analysis is ongoing, but there is preliminary indiction that there may be an association between ferritin (stored iron) levels and those that don’t respond to zinc therapy correctly. We are investigating whether those that have a dysfunctional response are receiving iron supplements. More to come soon.

8. My manuscript, Analysis of Plasma Zinc and Copper Concentration, and Perceived Symptoms, in Individuals With Depression, Post Zinc and B-6 Therapy, has been accepted for publication (with needed revision). The significance of this paper is that it suggests that, in our patients with depression, the inability of zinc to help normalize copper levels may be associated with the fact that our depression patients don’t perceive that they are getting better (as a group). This is compared to our anxiety patients, whose copper normalizes and they perceive they are getting better after zinc therapy. It may suggest that we need to look at additional ways of lowering copper in the depression group.

Update 1.21.11

**We received grant approval for the proposal, To Study why increased copper in individuals with autism normalizes post zinc therapy in individuals with concurrent GI disease for $12,435.00 from the Autism Research Institute.

1.We performed outcomes analysis of Asperger’s and autism individuals. Organized a chart of symptom severity of autism and Asperger’s patients, pre and post zinc therapy, for use as a figure in the paper, Analysis of Copper and Zinc Plasma Concentration and the Efficacy of Zinc Therapy in Individuals With Asperger’s Syndrome, Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS) and Autistic Disorder. I also continued to write and edit this paper. The abstract is as follows:

Aim: To assess plasma zinc and copper concentration in individuals with Asperger’s Syndrome, Pervasive Developmental Disorder-Not Otherwise Specified (PDD-NOS) and Autistic Disorder, and to analyze the efficacy of zinc therapy on the normalization of zinc and copper levels and symptom severity.

Subjects and Methods: Plasma from 79 autistic individuals, 52 individuals with PDD-NOS, 21 individuals with Asperger’s Syndrome (all meeting DSM-IV diagnostic criteria), and 18 age and gender similar neurotypical controls, were tested for plasma zinc and copper using inductively-coupled plasma-mass spectrometry.

Results: Autistic and PDD-NOS individuals had significantly elevated plasma levels of copper (p = 0.0133; p = 0.04556, respectively) None of the groups (autism, Asperger’s or PDD-NOS) had significantly lower plasma zinc concentration (Table 1).

Post zinc and B-6 therapy, individuals with autism and PDD-NOS had significantly lower levels of copper (p = 0.00972; p = 0.04139, respectively), but individuals with Asperger’s did not have significantly lower copper (p = 0.66915). Individuals with autism, PDD-NOS and Asperger’s all had significantly higher zinc levels (Table 2).

Severity of symptoms decreased in autistic individuals following zinc and B-6 therapy with respect to awareness (p = 0.039), receptive language (p = 0.014), focus and attention (p = 0.011), Hyperactivity (p = 0.002), Tip Toeing (p = 0.002), Eye Contact (p = 0.085), Sound Sensitivity (p = 0.098), Tactile Sensitivity (p = 0.012) and seizures (p = 0.057). None of the measured symptoms worsened after therapy. None of the symptoms in the Asperger’s patients improved after therapy (Figure 1).

Discussion: These results suggest an association between copper and zinc plasma levels and individuals with autism, PDD-NOS and Asperger’s Syndrome. The data also indicates that copper levels normalize (decrease to levels of controls) in individuals with autism and PDD-NOS, but not in individuals with Asperger’s. These same Asperger’s patients do not improve with respect to symptoms after therapy. This may indicate an association between copper levels and symptom severity.

2. We analyzed plasma histamine vs whole blood histamine as markers for methylation. After speaking with scientists at Quest and searching the literature, we determined that plasma histamine should be more accurate than whole blood histamine. However, we are still unsure whether there is a correlation between plasma histamine levels and methylation. This month we will correlate plasma histamine levels and perceived histadelia in our patients. We are currently analyzing whether methionine (which can be assayed by Quest) would be a good marker for methylation. We inquired about the accuracy of B-6 measurement by Quest. Scientists assured us that B-6 is measured at Quest by liquid chromatography and mass spec. and therefore the data should be very accurate.

3. I responded to reviewer’s questions concerning our grant proposal to ARI. After this response, we received grant approval for the proposal, To Study why increased copper in individuals with autism normalizes post zinc therapy in individuals with concurrent GI disease for $12,435.00. This grant will give us an opportunity to begin to study the association between copper toxicity/regulation and neurotransmitter concentration in autism (and other diseases).

4. I wrote and submitted another grant to The Johnson Foundation, to  study the effect of iPad applications (Apps) on conversational language skills in autistic children ($36,000.00).

The Rationale and Preliminary Research

In 1983, Rapin and Allen referred to a group of children who presented with mild autistic features and specific semantic pragmatic language problems when they described the communicative behavior of children as pathological talkativeness, deficient access to vocabulary and discourse comprehension, atypical choice of terms and inappropriate conversational skills (1). More recently, the term “pragmatic language impairment” (PLI) has been proposed to describe this trait (2,3).

Children with PLI have difficulty in understanding the meaning of words, grammar, syntax, prosody, eye gaze, body language, gestures, or social context.

PLI can be measured best using the Children’s Communication Checklist  (CCC) (4), and this tool is useful in a research setting for the assessment of PLI (5).

In preliminary research, we studied the symptom severity of 79 autistic and 29 Asperger’s individuals and 18 controls before and after nutrient therapy and found that symptoms decreased after therapy in autistic individuals with respect to awareness (p = 0.039), receptive language (p = 0.014), focus and attention (p = 0.011), Hyperactivity (p = 0.002), Tip Toeing (p = 0.002), Eye Contact (p = 0.085), Sound Sensitivity (p = 0.098), Tactile Sensitivity (p = 0.012) and seizures (p = 0.057), but not with respect to other measured symptoms including pragmatic language (p = 0.19608) . None of the measured symptoms worsened after therapy. None of the symptoms in the Asperger’s patients improved after therapy, including pragmatic language (p = 0.85655). It is also worth noting that in the autistic group, the mean severity of dysfunctional pragmatic language was higher than any other symptom measured, and it was the second most severe (behind focus/attention) in the Asperger’s group.

Our data suggests that PLI is an important symptom in autism and Asperser’s and nutrient therapy is not effective enough in alleviating the problem.

It is because of this that we propose to study other means of helping to improve pragmatic language skills in these patients.

5. We gathered current data on medication (drug) use by our anxiety (n=50) and depression (n=100) patients. We will continue to analyze this data with respect to outcomes (perceived symptom severity). The purpose of this study is to determine the effect of drugs taken outside of Pfeiffer on patient biochemistry and symptom severity.

6. I continued to write and edit the manuscript, Plasma Zinc and Copper Concentration, and Perceived Symptoms, in Individuals With Obsessive Compulsive Disorder (OCD), Post Zinc and B-6 Therapy.

Update 1.2.11

1.We submitted a grant, to study why increased copper in individuals with autism normalizes post zinc therapy in individuals with concurrent GI disease, to the Autism Research Institute – Total -  $12,435.00.

2. We gathered, formatted and analyzed outcomes of Asperger’s and Autism Patients for manuscript (Analysis of Zinc and Copper Levels in Individuals With Autism, PDD-NOS and Aspergers).

3. I wrote first draft of the manuscript, Analysis of Zinc and Copper Levels in Individuals With Autism, PDD-NOS and Asperger’s. In summary our findings indicate that although zinc levels are lower in all three groups, they are not significantly lower than controls. However, copper levels are significantly higher in the autistic and PDD groups, but not in the Asperger’s group. Post zinc therapy, copper levels go down significantly in the Autism and PDD groups, but not the Asperger’s group. Zinc increases significantly in all groups. Perceived symptoms of Asperger’s patients are generally lower than the autistic group, but the autistic individuals improve significantly in hyperactivity and stimming, whereas there is no equivalent improvement in the Asperger’s group. This suggests that copper levels may play an important role in symptomotology in these groups.

4. After peer review, I re-wrote the manuscript, Increased Copper and Decreased Zinc in Individuals With Depression, and changed it to, Analysis of Plasma Zinc and Copper Concentration, and Perceived Symptoms, in Individuals With Depression, Post Zinc and B-6 Therapy. This was a major change designed to include effect of therapy on outcomes in depression. In summary, what we found is that individuals with depression (with and without secondary anxiety) have high copper, which does not decrease (as expected) after zinc therapy. Symptoms of these same individuals don’t improve. Individuals with primary anxiety also have low zinc and high copper, but after zinc therapy, copper levels decrease and these patients have symptom improvement. This suggests that the reduction of copper levels in depression patients may help them improve. Here is the new abstract.

Aim: To assess plasma zinc and copper levels in individuals with depression.

Subjects and Methods: Plasma from 73 clinically depressed individuals and 16 controls were tested for plasma zinc and copper concentration using inductively-coupled plasma-mass spectrometry.

Results: Depressed individuals with secondary anxiety and without secondary anxiety had significantly lower plasma levels of zinc and elevated Cu compared to controls. Plasma zinc normalized (increased to the level of normal controls) but copper plasma concentration increased in individuals with depression (with and without secondary anxiety, after zinc therapy. Whereas both plasma zinc increased and copper levels decreased in anxiety, with and without secondary depression, after zinc therapy.

As expected, individuals with depression and secondary anxiety and without secondary anxiety had significantly higher symptom severity when compared to neurotypical controls. Symptom Severity in individuals with anxiety (both with and without secondary depression) significantly decreased after zinc therapy, whereas symptoms remained the same in individuals with depression.

Discussion: These data show an association between zinc and copper plasma levels and clinically depressed individuals, and suggest that high copper levels are associated with symptom severity.

5. We began to analyze biochemistry of OCD patients (N=18), compared to neurotypical controls (N=16). In summary, zinc levels are significantly lower and copper significantly higher in OCD patients. Zinc increases significantly in these patients after zinc therapy, but copper doesn’t significantly decrease. We are currently gathering outcomes data on these patients to analyze whether they tend to perceive improvement.

Update 12.21.10

Wrote and submitted abstract for presentation at the International Meeting For Autism Research (IMFAR) this coming spring (May 2011).

Aim: To assess plasma zinc and copper concentration in individuals with autism.

Subjects and Methods: Plasma from 79 autistic individuals (diagnosed by the Autism Diagnostic Interview-Revised – ADI-R), and 18 age and gender similar neurotypical controls, were tested for plasma zinc and copper using inductively-coupled plasma-mass spectrometry.

Results: Autistic individuals had significantly elevated plasma levels of copper and Cu/Zn and lower, but not significantly lower, concentration of plasma Zn compared to neurotypical controls.

Zn levels increased significantly in autistic individuals with GI Disease and without GI disease after zinc therapy. Cu decreased significantly after zinc therapy in the GI disease group but not in the autistic group without GI disease.

Autistic children significantly improved with respect to hyperactivity and stimming after zinc therapy in autistic children with GI disease. Autistic children without GI disease did not improve in hyperactivity or stimming after the same therapy.

Discussion: These results suggest an association between zinc and copper plasma levels and autistic children, and that zinc therapy may be most effective at lowering copper levels in autistic children with GI disease.

We analyzed 22 Asperger’s patients with respect to biochemistry and outcomes compared to controls, and compared these results to autism group.

I began to write manuscript, Analysis of Copper and Zinc Plasma Concentration and the Efficacy of Zinc Therapy in Individuals With Asperger Syndrome, Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS) and Autistic Disorder.

We began an updated analysis of drugs and depression (N=99)/anxiety (N=83) patients, correlating type drugs with markers (Particularly Cu and Zn and outcomes). We assessed individuals with primary anxiety and primary depression. Overall, we found no significant difference in Cu, Zn plasma concentration and outcomes, when comparing those who presented to Pfeiffer while taking drug therapy (anti-depressants, Benzodiazepines, etc) to those not on drug therapy. As reported previously, since our patients tend to perceive they are getting better after our therapy, this probably can’t be contributed to the addition of drug therapy.

I wrote the final edit and submitted the manuscript, Increased Copper and Decreased Zinc in Individuals With Depression, for publication.

Aim: To assess plasma zinc and copper levels in individuals with depression.

Subjects and Methods: Plasma from 73 clinically depressed individuals and 16 controls were tested for plasma zinc and copper using inductively-coupled plasma-mass spectrometry.

Results: Depressed individuals had significantly lower plasma levels of zinc (p= 0.04218) and elevated Cu (p= 0.00288) and Cu/Zn (p= 0.00996), compared to controls. Zn levels increased significantly (p= 0.0002) and Cu/Zn decreased significantly (p=0.02877) post zinc therapy, but copper levels did not decrease significantly (p=0.09851).

Discussion: These data show an association between zinc and copper plasma levels and clinically depressed individuals.

I began to prepare manuscript, Analysis of Zinc and Copper levels, and Perceived Outcomes, in Individuals With Depression and Anxiety Post Zinc and B-6 Therapy

Update 12.1.10

We worked on gathering data on anxiety patients with respect to drugs they were/are taking, prescribed outside of Pfeiffer. Did analysis of effect of these drugs on biomarker levels and outcomes. When comparing anxiety patients who were not taking drug therapy with those taking therapy (SSRIs, anti-anxiety), I found no significant difference in HGF (p = 0.16729), Cu/Zn SOD (p = 0.0971), Cu (p = 0.63632), Zn (p = 0.14904), Cu/Zn (p = 0.55586), overall symptoms (p = 0.46747) or specific anxiety symptoms (p = 0.84182). The significance being that the improvement of our anxiety patients post therapy is due to our therapy and not concurrent drug therapy.

We also began analysis of the relationship between drugs and depression patients, correlating type of drugs with biomarkers and outcomes of these patients. More to come next update.

We analyzed Zn, Cu and Cu/Zn in Autistic Individuals with and without GI disease Pre and Post Zinc Therapy.

Based, in part on this analysis we wrote a paper, Increased Copper in Individuals With Autism Normalizes Post Zinc Therapy More Efficiently in Individuals With Concurrent GI Disease, to be submitted to the Journal Molecular Autism.

Aim: To assess plasma zinc and copper concentration in individuals with autism.

Subjects and Methods: Plasma from 79 autistic individuals (diagnosed by the Autism Diagnostic Interview-Revised – ADI-R), and 18 age and gender similar neurotypical controls, were tested for plasma zinc and copper using inductively-coupled plasma-mass spectrometry.

Results: Autistic individuals had significantly elevated plasma levels of copper and Cu/Zn and lower, but not significantly lower, concentration of plasma Zn compared to neurotypical controls.

Zn levels increased significantly in autistic individuals with Gi Disease and without GI disease after zinc therapy. Cu decreased significantly after zinc therapy in the GI disease group but not in the autistic group without GI disease after zinc therapy.

Autistic children significantly improved with respect to hyperactivity and stimming after zinc therapy in autistic children with GI disease. Autistic children without GI disease did not improve in hyperactivity or stimming after the same therapy.

Discussion: These results suggest an association between zinc and copper plasma levels and autistic children, and that zinc therapy may be most effective at lowering copper levels in autistic children with GI disease.

Our paper, Decreased Zinc and Increased Copper in Individuals With Anxiety was accepted (with revision) for publication in the journal, Nutrition and Metabolic Insights. Here is the abstract:

Aim: To assess plasma zinc and copper levels in individuals with anxiety and to test the hypothesis that there is a relationship between these levels and improved symptoms.

Subjects and Methods: Serum from 38 individuals with anxiety and 16 neurotypical age, gender and size similar controls were tested for plasma zinc and copper concentration using inductively-coupled plasma-mass spectrometry. Zinc and copper levels, pre and post therapy, were compared and assessed for perceived anxiety symptoms.

Results: Individuals with anxiety had significantly higher serum levels of Cu (p=0.0348), Cu/Zn (p=0.0493) and lower Zn (p= 0.0294) compared to controls. Zn levels normalized (increased to the normal range) and Cu/Zn significantly decreased after zinc therapy (p= 0.0004, p= 0.0033, respectively), but Cu did not significantly decrease (0.3577). These same patients improved significantly with respect to perceived overall symptoms after zinc and therapy (p=0.013).

Discussion: These results suggest an association between Zn plasma levels and individuals with anxiety, demonstrate that zinc therapy is effective in increasing zinc plasma levels, and show that zinc supplementation may play a role in improved symptoms.

The significance of this paper is that it is the first time we have shown (compared to neurotypical controls) a relationship between zinc and copper levels and our patients. This also supports the efficacy of our zinc therapy.

We revised the paper, Decreased Zinc and Increased Copper in Individuals With Anxiety (described above), for resubmission.

I visited Dr. Goldrosen at NIH to discuss ideas for a basic research grant. I began to outline a grant to be submitted in early 2011. This grant will likely investigate the hypothesis that Low Zinc and High Copper levels in autistic patients is associated with GABA and Norepinephrine/Dopamine levels.

We Investigated the comparison between plasma and whole blood histamine values in light of the fact that Quest only does plasma histamine. All of our previous data comes from whole blood histamine. As expected, most reports concur with preliminary experiments we performed which show that plasma and whole blood concentrations may not correlate with one another. It is likely that whole blood histamine has been preferred for histamine because the concentration of histamine there is considerably higher than in plasma. With that said, newer technology allows for the ability to accurately measure smaller concentrations of histamine. If we have to measure plasma histamine as a marker for methylation, we should probably initially find out the normal expected range from Quest and use that as a reference. Without the opportunity to run whole blood histamines along side plasma histamines in the same patients it will be hard to correlate the two tests.

We continued to edit the manuscript, Increased Copper and Decreased Zinc in Individuals With Depression.

Update 11.8.10

We finished analysis of zinc and copper’s relationship to outcomes in depression and anxiety patients. I found that copper levels of patients with anxiety (no secondary depression) correlated significantly with symptom severity (higher copper-higher severity) (p= 0.034). Interestingly, in depression patients with no secondary anxiety, zinc levels correlated (but not quite significantly) with symptom severity (low zinc-high severity) (p= 0.075).

The paper by Dr. deVito and I  Decreased Serum Hepatocyte Growth Factor (HGF) in Individuals With Schizophrenia Normalizes after Zinc and B-6 Therapy was accepted for publication in  the Journal Proteomics Insights. The abstract is as follows:

Aim: To assess serum HGF concentration in individuals with schizophrenia and investigate the efficacy of zinc and B-6 therapy on these levels.

Subjects and Methods: Serum from 18 individuals diagnosed with schizophrenia and 19 age and gender similar controls (p=0.18) were tested for HGF concentration using ELISAs, and tested for copper and zinc plasma levels using inductively-coupled plasma-mass spectrometry.

Results: HGF serum levels of individuals with schizophrenia, before zinc and B-6 therapy, were significantly lower than age and gender similar controls (p=0.016), and significantly lower in schizophrenia patients pre-therapy compared to post therapy (p=0.028). HGF levels normalized (reached levels similar to controls) post-therapy. Zinc levels in these same individuals also normalized, and perceived symptoms, particularly anxiety (p=0.03), improved significantly after therapy.

Discussion: These results suggest an association between low HGF levels and schizophrenia and demonstrate that zinc and B-6 therapy may be associated with the normalization of HGF levels and perceived improvement in symptoms.

We (with much help from Tony) revised the research page of HRIPTC web site.

In the analysis of depression and anxiety patients, we discovered that copper levels of anxiety patients without secondary depression correlated with severity of symptoms (p=0.03).  When we compared the levels of Cu and Zn to outcomes (symptom severity) pre and post zinc and B-6 therapy, depression patients don’t get significantly better, whereas anxiety patients do. Zinc levels normalized post therapy in anxiety group(s), but copper went up (not down as expected) in the depression group(s).

The anxiety group with no secondary depression improved significantly with respect to overall symptoms (p=0.001) and anxiety symptoms (p=0.004), post therapy. In the anxiety group, there was also a significant improvement (lowering) in Cu levels (p=0.03).

The fact that depression patient’s symptoms didn’t improve post therapy and copper levels didn’t normalize (actually went up), whereas in the anxiety group copper levels significantly improved (decreased) and so did symptoms, we suggest a relationship between copper levels and symptoms in both anxiety and depression patients.

We are currently investigating possible reasons why copper levels increased in depression patients, despite zinc therapy. Ceruloplasm levels in these same patients increased slightly after therapy as well. This is most likely a response to increased copper and probably doesn’t explain the copper levels.

We began to study the possible relationship between HGF, Cu/Zn SOD, Cu, Zn levels and outcomes associated with our autism patients.  We analyzed the following symptoms before and after MT promotion: Awareness, Expressive Language, Receptive Language, (Conversational) Pragmatic Language, Focus, Attention, Hyperactivity, Impulsivity, Perseveration, Fine Motor Skills, Gross Motor Skills, Hypotonia (low muscle tone), Tip Toeing, Rocking/Pacing, Hand Flapping/Finger Stimming, Obsessions/Fixations, Eye Contact, Sound Sensitivity, Light Sensitivity, Tactile Sensitivity, Pica/ eats dirt, metal, Tics and Seizures.

We found a significant decrease in HGF (p = 0.034) in autistic patients post MTP and a significant lowering in Ferritin (p = 0.025) post MTP. No other significant differences pre and post therapy were found (although the groups are small). Most symptoms improved after MTP, but hyperactivity (p = 0.001) and (Conversational) Pragmatic Language (p = 0.036) were the only two that significantly improved (again, the numbers in these groups were relatively small).

When we assessed symptom outcomes in our first 46 autism patients (since collecting blood/urine), 15 pre therapy and 31 post therapy, including those we haven’t yet tested for HGF concentration, we found that Receptive Language (p=0.0461) and Hyperactivity (p=0.016) symptoms got significantly better, and Sound Sensitivity (p = 0.07078) as well as Focus, Attention (p=0.081) symptoms improved to a level which was almost significant.

Update 10.25.10

We analyzed biochemistry of our Schiz patients with biochem of neurotypical controls. Found no significant difference between Cu levels (p = 0.24262), Zn levels (p = 0.57231) or Cu/Zn (p = 0.86774) in Schizophrenia patients compare to neurotypical controls. This is different than the other groups suggesting a difference in etiology.

We analyzed Zn, Cu, Cu/Zn in anxiety patients with depression and depression with anxiety, anxiety alone and depression alone. In both groups, Zn is significantly decreased, Cu and Cu/Zn are significantly increased and Zn, Cu and Cu/Zn normalize after our zinc therapy. However, when we analyzed outcomes of anxiety and depression groups. we found that our anxiety patients perceive they are getting significantly better after therapy, whereas our depression patients are not (they get better, but not significantly better).

When we analyze anxiety patients with no secondary depression, depression patients with no secondary anxiety, anxiety patients with secondary depression and depression patients with secondary anxiety, interestingly, depression patients with no secondary anxiety had significantly higher copper than all the other groups. Zinc levels were not significantly different in these groups. This suggests that copper levels (and not necessarily zinc levels) may be playing a role in symptomotology of depression patients. When we compare perceived severity of symptoms of these groups, those with primary depression have significantly worse symptoms than those with primary anxiety. We are now analyzing the correlation between copper and zinc levels and perceived level of symptom severity in individual patients of these groups.

We analyzed the role of copper in dopamine and epinephrine pathway. High copper is associated with high dopamine and epinephrine, leading to high anxiety. Copper doesn’t significantly decrease after therapy in depression patients and these patients don’t significantly get better. Does this suggest that we need to find better ways of decreasing copper in our depression patients?

We analyzed ceruloplasmin levels in the above groups and found that generally ceruloplasm levels correlate with copper levels. Since ceruloplasm is the primary copper transporting protein, this makes sense. There is not a significant difference between ceruloplasm levels in any of these groups suggesting that level of this protein is not associated with the etiology of depression.

We began to analyze Zn, Cu, Cu/Zn, ceruloplasm levels in 73 autistic patients vs controls. More to come about this analysis.

I did a final edit and submitted the paper, Decreased Zinc and Increased Copper in Individuals With Anxiety to the journal Nutrition and Metabolism. The importance of this paper is that it is the first time we have been able to confirm that our patient groups are zinc deficient (and have increased copper) by being able to compare data to neurotypical controls.

Update 10.10.10

1. We analyzed the relationship between bipolar patients and drug therapy. In summary, the data indicates that there is not a significant improvement in symptoms when those on drug therapy (n=18) are compared to those not on drug therapy (n=11) (p = 0.19806). Also, there is no significant difference in Zn, Cu, Cu/Zn or the markers HGF and Cu/Zn SOD between these groups. When drug group is separated into the following: Those taking-Antidepressants (n=7), Anticonvulsant (n=8), Antipsychotic (n=12), Antianxiety (10) and Antimania (3), had no significant differences in Zn, Cu, Cu/Zn or symptom severity.

This suggests that these drugs are not affecting zinc and copper levels in our bipolar patients and improved symptoms in our patients is not associated with the action of these drugs.

2. We revised the editorial to Am J Psych, Hepatocyte Growth Factor (HGF)/GABA Modulation: Hypothesis for a Common Neurological Disease Etiology, and submitted it again.

4. We analyzed biochemistry of our PDD (n=52) patients and compared data to biochem of neurotypical controls.

5. We analyzed biochemistry of our ASD (n=30) patients and compared data to biochem of neurotypical controls.

6. We analyzed biochemistry of our Asp (n=20) patients and compared data to biochem of neurotypical controls.

Interestingly, zinc levels are significantly lower in individuals diagnosed with autism (p = 0.032), but not in patients with PDD (p=0.108) and Asperger’s (o.350). This suggests that zinc levels might be associated with the severity and type of autistic symptoms.

7. Revised and finished paper Decreased Zinc in Individuals With Anxiety and began paper Decreased Zinc in Individuals with Depression. In summary, in the depression group, zinc is significantly lower and copper and Cu/Zn are higher in this group as well. Zn and Cu/Zn normalize after therapy whereas Cu doesn’t.

Update 9.25.10

I wanted to share a letter to the editor Dr. DeVito and I are sending to The American Journal of Psychiatry.

Letter to the Editor

Hepatocyte Growth Factor (HGF)/GABA Modulation: Hypothesis for a Common Neurological Disease Etiology

A.J. Russo, PhD and Robert deVito, MD

Health Research Institute/Pfeiffer Treatment Center

4575 Weaver Parkway

Warrenville, Illinois 60555

Corresponding Author: A.J. Russo, Ph.D., Research Director, Health Research Institute/Pfeiffer Treatment Center, 4575 Weaver Parkway, Warrenville, Illinois 60555

ajrusso@hriptc.org

Based on neurobiological findings and location within a chromosome 7q31 autism candidate gene region (1), Campbell et al. analyzed the MET receptor tyrosine kinase gene in a family- based study of autism and found a functional variant of MET in autistic children, with a calculated relative risk of 2.27 (2).

Recently, reported in the American Journal of Psychiatry, Burdick and colleagues examined the relationship between 21 single-nucleotide polymorphisms (SNPs) in MET and schizophrenia in 173 patients and 137 normal volunteers. They found that several varieties of MET influenced the risk for schizophrenia, as well as general cognitive ability. The authors were then able to replicate their findings in a second sample of 107 patients and 112 healthy volunteers (3).

The MET gene codes for the production of the membrane protein, cMET, found on many cell types, particularly in the CNS and GI tract. The protein, hepatocyte growth factor (HGF), secreted by mesenchymal cells and ligand for cMET, regulates cell growth, cell motility, morphogenesis and inflammatory responses by activating a tyrosine kinase signaling cascade in a wide variety of cells after binding to the c-MET receptor. Its ability to stimulate branching morphogenesis, cell migration, survival, and proliferation gives it a central role in angiogenesis, tissue regeneration, as well as tumorogenesis (4-10).

Signaling by HGF seems to be particularly active in the nervous system, where it has been found to have neurotrophic and angiogenetic activity on CNS neurons, promote both the survival of neurons and the regeneration of injured nerves, and function as a target-derived axonal chemoattractant, guiding axons to their target. As a result, it plays significant roles in the development of the CNS (11), and abnormal synthesis of HGF has been found to be associated with many diseases of the CNS (12-14)

GABA (γ-Aminobutyric acid) is the chief inhibitory neurotransmitter in the CNS (15). During development it regulates the proliferation of neural progenitor cells (16,17) the migration (18) and differentiation (19,20) the elongation of neurites (21) and the formation of synapses (22). Genes involved in GABAergic neurotransmission (23,24) and decreased levels of serum GABA have been associated with neurological diseases, such as Bipolar Disorder (24).

HGF has been shown to modulate GABAergic activity (25) and enhance NMDA currents in the hippocampus (26).

Our research has resulted in the discovery that HGF serum concentration is significantly decreased in individuals with autism (30), clinical depression (27), anxiety (28), bipolar disorder (29) and schizophrenia (unpublished data), suggesting that this common deficiency, possibly related to a MET gene variant, may be associated with the etiology of all these diseases. We have also found, in anxiety and bipolar individuals, HGF levels normalize after zinc and vitamin B-6 therapy (28,29), suggesting that, nutrient therapy may help improve symptoms of these disorders and this improvement is, at least in part, associated with increased HGF.

Although it is possible that zinc and B-6 supplementation may cause an increase in GABA directly, since increased HGF often results in increased GABA levels, and zinc, as well as vitamin B-6, are associated with raising both HGF and GABA, we hypothesize that zinc and B-6 supplementation causes increased GABA by increasing HGF levels, which, in turn, plays an integral part in the perceived improvement in symptoms seen in these patients. We are currently studying this premise.

1. Sousa I, Clark TG, Toma C, Kobayashi K, Choma M, Holt R, et al. MET and autism susceptibility: family and case-control studies. Eur J Hum Genet 2009;17(6):749–58.

2. Campbell D, et alA genetic variant that disrupts MET transcription is associated with autism PNAS 2006;103:16834-16839.

3. Burdick et al. Association of Genetic Variation in the MET Proto-Oncogene With Schizophrenia and General Cognitive Ability. Am J Psychiatry 2010; 167:436-443.

4. Nakamura Tet al. Purification and characterization of a growth factor from rat platelets for mature parenchymal hepatocytes in primary cultures. Proc. Natl. Acad. Sci. USA. 1986;83:6489.

5. NakamuraTet al Partial purification and characterization in hepatocyte growth factor from serum of hepatectomized rats.  Biochem. Biophys. Res. Commun. 1994;122:1450.

6. Sasaki, M, .et al. Identification of mouse mammary fibroblast-derived mammary growth factor as hepatocyte growth factor. Biochem. Biophys. Res. Commun. 1994;199:772.

7. Michalopoulos, G.et al. Control of hepatocyte replication by two serum factors. Cancer Res. 1994;44:4414.

8. Thaler, FJMichalopoulos G,Hepatopoietin A: Partial characterization and trypsin activation of a hepatocyte growth factor (1985). Cancer Res. 45:2545.

9. Zarnegar RMichalopoulos GH, The many faces of hepatocyte growth factor: from hepatopoiesis to hematopoiesis. J. Cell Biol. 1995;129:1177.

10. Weidner KMet al. Evidence for the identity of human scatter factor and human hepatocyte growth factor. Proc. Natl. Acad. Sci. USA, 1991;88(16):7001-5.

11. Hamanoue M, et al. Neurotrophic effect of hepatocyte growth factor on central nervous system neurons in vitro. J Neurosci Res. 1996;43(5):554-64.

12. Tsuboi Y,et al. Increased hepatocyte growth factor level in cerebrospinal fluid in Alzheimer’s disease. Acta neurologica scandinavica. 2003;107:81-86.

13. Sun W, et al. Overexpression of HGF retards disease progression and prolongs lifespan in a transgenic mouse model of ALS. The Journal of Neuroscience 2002;22(15):6537-6548.

14. H Koike, et al. Prevention of onset of Parkinson’s disease by in vivo gene transfer of human hepatocyte growth factor in rodent model: a model of gene therapy for Parkinson’s disease. Gene Therapy. 2006;13:1639-1644.

15. Watanabe M, Maemura K, Kanbara K, Tamayama T, Hayasaki H (2002). “GABA and GABA receptors in the central nervous system and other organs”. Int. Rev. Cytol. 213: 1–47.

16. LoTurco JJ, Owens DF, Heath MJ, Davis MB, Kriegstein AR (December 1995). “GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis”. Neuron 15 (6): 1287–98.

17. Haydar TF, Wang F, Schwartz ML, Rakic P (August 2000). “Differential modulation of proliferation in the neocortical ventricular and subventricular zones”. J. Neurosci. 20 (15): 5764–74.

18. Behar TN, Schaffner AE, Scott CA, O’Connell C, Barker JL (August 1998). “Differential response of cortical plate and ventricular zone cells to GABA as a migration stimulus”. J. Neurosci. 18 (16): 6378–87.

19. Barbin G, Pollard H, Gaïarsa JL, Ben-Ari Y (April 1993). “Involvement of GABA A receptors in the outgrowth of cultured hippocampal neurons”. Neurosci. Lett. 152 (1-2): 150–4.

20. Ganguly K, Schinder AF, Wong ST, Poo M (May 2001). “GABA itself promotes the developmental switch of neuronal GABAergic responses from excitation to inhibition”. Cell 105 (4): 521–32.

21. Maric D, Liu QY, Maric I, Chaudry S, Chang YH, Smith SV, Sieghart W, Fritschy JM, Barker JL (April 2001). “GABA expression dominates neuronal lineage progression in the embryonic rat neocortex and facilitates neurite outgrowth via GABA(A) autoreceptor/Cl- channels”. J. Neurosci. 21 (7): 2343–60.

22. Ben-Ari Y (September 2002). “Excitatory actions of gaba during development: the nature of the nurture”. Nat. Rev. Neurosci. 3 (9): 728–39.

23. Kelsoe JR et al Possible locus for bipolar disorder near the dopamine transporter on chromosome 5 American Journal of Medical Genetics Part B: Neuropsychiatric Genetics 1998;67:533-540.

24. Petty F et al Low plasma GABA is a trait-like marker for bipolar illness Neuropsychopharmacology 1993;9(2):125-32.

25. Bae MH et al Hepatocyte growth factor (HGF) modulates GABAergic inhibition and seizure susceptibility Experimental Neurology 2010;221:129-135.

26. Akimoto M et al Hepatocyte growth factor as an enhancer of nmda currents and synaptic plasticity in the hippocampus Neuroscience 2004;128:155-162.

27. Russo,A.J. Decreased Serum Hepatocyte Growth Factor (HGF) in Individuals with Depression Correlates with Severity of Disease Biomarker Insights 2010:5 1–5.

28. Russo,A.J. Decreased Serum Hepatocyte Growth Factor (HGF) in Individuals with Anxiety Increases After Zinc Therapy Nutrition and Metabolic Insights 2010:3 1–6.

29. Russo,A.J. Decreased Serum Hepatocyte Growth Factor (HGF) in Individuals With Bipolar Disorder Normalizes after Zinc and Anti-oxidant Therapy, accepted for publication Journal of Central Nervous System Disease June, 2010.

30. Russo A.J. et al Decreased Serum Hepatocyte Growth Factor (HGF) in Autistic Children with Severe Gastrointestinal Disease Biomarker Insights 2009:2 181–190.

Recently:

We analyzed HGF data, pre and post MT therapy, in autistic children with and without GI disease. In summary, the mean HGF of GI disease in autistic children before and after therapy correlate with mean zinc and Cu/Zn but not Cu alone. Interestingly, HGF levels in this group significantly decreases after MT promotor therapy. Zinc levels are lower in GI disease group compared to non GI autistic group. Controls have significantly higher zinc than the GI group.

We analyzed depression, bipolar and schizophrenia patient biochemistry, and compared these data to controls. Overall, there are similarities in these groups. As in the anxiety group, Zinc is significantly lower than controls, Cu is significantly higher as is Cu/Zn ratios compared to levels of controls. Zinc and Cu/Zn levels tend to normalize in these groups after Zn/B-6 therapy, but copper levels are not affected significantly by primer.

We wrote and submitted progress report to Johnson Foundation for grant, Study of Hepatocyte Growth Factor in Autistic children with GI disease.

We wrote first draft of paper, Decreased Zinc in Individuals With Anxiety. In summary: Aim: I assessed serum zinc and copper levels in individuals with anxiety to test the hypothesis that there is a relationship between zinc therapy and improved symptoms. Serum from 38 individuals with anxiety and 16 neurotypical age and gender similar controls were tested for serum Zinc. Zinc and copper levels in anxiety patients, pre and post therapy, were also measured and compared and these same patients were assessed for perceived anxiety symptoms. Individuals with anxiety had significantly higher serum levels of Cu (p= 0.0348), Cu/Zn (p= 0.0493) and lower Zn (p= 0.0294) compared to controls. Zn levels normalized (increased to the normal range) and Cu/Zn significantly decreased after zinc and B-6 Therapy (p= 0.0004, p= 0.0033, respectively), but Cu did not significantly decrease (0.3577). These same patients improved significantly with respect to perceived overall symptoms after zinc and B-6 therapy (p=0.013). These results suggest an association between Zn serum levels and individuals with anxiety, demonstrate that zinc and B-6 therapy is effective in increasing zinc, and zinc and B-6 supplementation may play a role in improved symptoms.

We analyzed biochemistry of Aspergers patients (N=25) and compared to controls. Interestingly, Zn, Cu and Cu/Zn are not significantly different than controls in this group, which may speak to the importance of Zn levels in the other neurological disorders.

We assessed the relationship between zinc and N-acetylcysteine.

There is evidence to suggest that zinc absorption is facilitated by N-acetylcysteine:

Journal of Trace Elements in Medicine and Biology

Volume 20, Issue 3, 26 September 2006, Pages 197-204

Co-administration of zinc and n-acetylcysteine prevents arsenic-induced tissue oxidative stress in male rats

J Nutr. 1986 Nov;116(11):2171-9.

Zinc intestinal absorption in rats: specificity of amino acids as ligands.

Anticancer Res. 2009 Nov;29(11):4571-4.

Reduction of oxidative DNA fragmentation by ascorbic acid, zinc and N-acetylcysteine in nasal mucosa tissue cultures.

There seems to be confusion about N-acetylcysteine causing increased excretion of zinc. Blogs (most unscientific I would add) seem to preach that there is increased zinc excretion with N-acetylcysteine. However there seems to evidence to the contrary:

Does NAC cause increased excretion of zinc? Maybe not:

http://www.springerlink.com/content/40q1617580005144/

http://www.ncbi.nlm.nih.gov/pubmed/2276385

Historically, there are probably some secondary reasons for N-acetylcysteine supplementation. First N-Acetyl Cysteine is rapidly metabolized to intracellular glutathione. This would provide an anti-oxidant effect.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC436956/pdf/jcinvest00153-0204.pdf

Second, it is involved in chelation. Heavy metals like lead, mercury and arsenic are detoxified and removed from the body by N-Acetyl Cysteine.

Environ Health Perspect. 1998 May; 106(5): 267–271.

N-acetylcysteine as an antidote in methylmercury poisoning.

If our patients are getting both zinc and N-Acetyl-Cysteine, I can tell you from our data that a very high percentage of those who come in with zinc deficiency wind up with normal zinc levels, which stabilize with continued therapy. Overall, in these cases, those receiving N-Acetyl-Cysteine don’t seem to be affected by abnormally high zinc excretion.

Update 9.15.10

I just received a grant for 12K from the Autism Research Institute to establish a control blood/urine bank for blood and urine for studies in autism. So far (since May 2009) we have been able to secure grants totaling approximately $61,000.00. See the grants page for more detail.

Updated 8.31.10

Recent data analysis:

We have been interested in studying the efficacy of Kryptopyroles as a biomarker for Zinc and B-6 deficiency.

We analyzed the relationship between Kp and Zn levels in all patients of various groups (anxiety, depression, bipolar, schizophrenia, autism, ADHD) and discovered that Kp values decrease significantly -post therapy- in only the ADHD group (p=0.02) and the schizophrenia group (p=0.05), although the numbers in this group are small (n=19). Kp values do not correlate with zinc levels in any of the groups.

We also analyzed the relationship between Kp and Zn levels in ADHD individuals with respect to high, med and low Kp levels-pre and post therapy. Still no association between Kp levels and Zn in any of these groups.

We have also been interested in assessing whether Histamine levels are the best marker for methylation.

We analyzed histamine levels with respect to methionine and folic acid therapy and outcomes. Out of 77 patients studied, 20 were treated for low histamine with folic acid and 16 were treated for high histamine with methionine. Of the 20 low histamine group, histamine levels were assessed post therapy 57 times. Of the 16 high histamine group, histamine levels were assessed after treatment 61 times. Within the low histamine group (57 assessments) the sum of increase or decrease of histamine levels was +58, for an average effect on histamine levels of approximately 1 µg/ml per treatment. Within the high histamine group (60 assessments) the sum of increase or decrease of histamine levels was -5 for an average effect of -0.08 µg/ml histamine per treatment.

It appears that histamine levels are not significantly affected by treatment with methionine or folic acid and may not be a good marker for methylation.

We analyzed 22 neurotypical patients (Zn, Cu, C/Zn Kp etc, before and after therapy) who have presented to Pfeiffer for general health or otherwise non-neurological conditions. We may be able to use this group as a control group. Overall, the treatment of these individuals parallels our normal patients (Zn, Biotin, B-6, P5P Methionine, Folic acid, vitamin D). Only two received primer. Only 3 received MT promotor. Overall, post therapy, zinc levels significantly increased, copper levels decreased slightly, copper/zinc decreased significantly and Kp levels started high and stayed high after therapy.

This finding raises some interesting points. First, it confirms the insignificance of high Kp levels (since controls may also have high Kp). Second, although this is a neurotypical group, these individuals still present with underlying symptoms. The post therapy group has more severe perceived symtoms than the group presenting for the first time (pre-therapy), suggesting that this group may not be helped by our therapy. This is in contrast with outcomes assessment of our patient groups, where all have less severe symptoms post therapy.

The publication page has been updated to include recent articles as well as articles accepted for publication, but in the revision stage.

Also, as our research has progressed, it has become clear that oxidative stress is associated with patients who are autistic, hyperactive, have depression, anxiety, bipolar disorder as well as shizophrenia, as evidenced by abnormal concentration of Cu/Zn SOD–a mitochondrial anti-oxidant, in these patient groups (see publications). Also, primer (zinc and B-6)  therapy is associated with the normalization of oxidative stress, particularly in patients who are clinically depressed (see publications).

In 2006, scientists discovered that a variant of the MET gene, which produces the membrane protein, c-MET, was present in about 15% of autistic children.

Campbell D, et al. A genetic variant that disrupts MET transcription is associated with autism. PNAS. 2006;103:16834–9.

Then in 2010, scientists discovered that a variant of the same gene was present in a significant number of patients with schizophrenia.

Burdick K et al Association of Genetic Variation in the MET Proto-Oncogene With Schizophrenia and General Cognitive Ability Am J Psychiatry 2010;167:436-443.

Hepatocyte Growth factor is the signaling molecule which attaches to c-MET and causes a host of things to happen in cells, including cell division, cell differentiation, cell morphology changes and migration changes.

We have been studying the concentration of HGF in our patient groups and have found, in general, that it is decreased.

Developing HGF/GABA Hypothesis and Questions:

Decreased HGF has been found to be associated with decreased GABA.

Decreased Zinc and  B-6 are associated with decreased GABA.

We have recently found in Bipolar patients a dose relationship between zinc and HGF

Could HGF deficiency cause modulation of GABA in our patient group?

Is our zinc/B-6 primer therapy associated with raising GABA and HGF?

We have shown that our primer therapy is directly associated with an increase in HGF. HGF, in turn, increases GABA. Our primer therapy could also be having a direct effect on GABA levels (as well as norepinephrine production).

Update 6.13.10

Check out updates to research on the Projects Page (click on Projects)!

Thanks to “The Team” for the tremendous response to our Research Blood Drive. As many of you know, we will use this control blood to compare results of our studies with predominantly adult populations, such as patients with depression, anxiety, bipolar disorder, schizophrenia, and early stage dementia. In fact, we have already used control plasma for experiments studying oxidative stress in depression.

Presentations and invites:

I was invited and presented our research on decreased hepatocyte growth factor in autistic children with GI disease at the upcoming International Meeting For Autism Research (IMFAR) in Philadelphia in May, 2010.

I also presented this data at the upcoming Autism One meeting in Chicago in May, 2010.

I was invited to submit a paper to the journal Autism Files. The paper, Hepatocyte Growth Factor (HGF): Marker for Neurological Disease including Autism?, is scheduled to appear in the July issue of the journal, Autism Files.

I took part in the GastroImmune Think Tank on Saturday at Autism One in May.

On March 31st at 11:00 am CT I was interviewed on radio by Teri Arranga of Autism One on VoiceAmerica Health and Wellness Channel. The topics to be discussed were : 1) autoimmunity and gastrointestinal disease; 2) biomarkers for GI disease (e.g., Cu/Zn SOD, MPO, and HGF); 3) Autism Insights; 4) work at HRI/PTC; 5) future research directions; and 6) resources for further information.

You can listen to the broadcast at:

http://www.voiceamerica.com/voiceamerica/vepisode.aspx?aid=45359

This past month or so we have been investigating two important markers, Cu/Zn SOD and Hepatocyte Growth Factor in Pfeiffer patients with depression, anxiety, bipolar disorder and schizophrenia. We published one papers over this period (see publications page for downloads) and two more were accepted for publication and are awaiting final typesetting by the publishers.

Increased Serum Cu/Zn SOD in Individuals With Clinical Depression Normalizes After Zinc and Anti-oxidant Therapy, was published in the journal Nutrition and Metabolism.

Decreased Serum Hepatocyte Growth Factor (HGF) in Individuals With Depression Correlates With Severity of Disease, has been accepted for publication and has gone to the typesetter at the journal Biomarker Insights.

Increased Serum Cu/Zn SOD in Individuals With Anxiety has been accepted for publication and has gone to the typesetter at the journal Proteomics.

Three additional papers have been submitted for publication.

Decreased Serum Hepatocyte Growth Factor (HGF) in Individuals With Anxiety Increases After Zinc Therapy

Increased Serum Cu/Zn SOD in Individuals With Bipolar Disorder

Decreased Serum Hepatocyte Growth Factor (HGF) in Individuals With Bipolar Disorder Normalizes after Zinc and Anti-oxidant Therapy

Overall, we have found, after primer therapy …

In Anxiety – Cu/Zn SOD goes up; HGF goes up  : Zn goes up, Cu stays same, Cu/Zn goes down. Patients perceive they are getting better.

In Depression – Cu/Zn SOD goes down; HGF no change : Zn goes up, Cu stays same Cu/Zn goes down. Patients perceive they are getting better.

In Bipolar – Cu/Zn SOD stays the same; HGF goes up  : Zn goes up, Cu stays same, Cu/Zn goes down. Patients perceive they are getting better.

We have not tested enough Schizophrenia patients to assess results.

 
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