Feasibility of a Nutritional Supplement As Treatment for Childhood Mood Dysregulation

Feasibility of a Nutritional Supplement as Treatment for Childhood Mood Dysregulation

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Elisabeth Anne Frazier, B.S.

Graduate Program in Psychology

The Ohio State University

2009

Thesis Committee:

Mary Fristad, Ph.D., ABPP, Advisor

Steven Beck, Ph.D., ABPP

Michael Vasey, Ph.D.

Elisabeth Anne Frazier

2009

Abstract

Current treatments for childhood mood dysregulation rely on psychotropic medications that are associated with significant adverse events (Kowatch et al.,

2005). Human nutrition research suggests nutrients play an important role in physical and mental health and may be useful in treating mood dysregulation without the side effects of contemporary pharmaceuticals (Kaplan, Crawford, Field,

& Simpson 2007). The current open-label study explored the feasibility of testing possible therapeutic effects of a multinutrient supplement, EMPowerplus (EMP+), as treatment for childhood mood dysregulation. Ten children, age 6-12 with mood dysregulation were recruited. All received EMP+ treatment. Mood symptoms were assessed seven times over an eight week trial. Blood draws were taken at baseline and final visits for nutrient analyses. Hypotheses included: 1) The sample can be recruited in 5 months; 2) Children aged 6-12 can swallow the supplement with >80% compliance; 3) Micronutrient levels of iron, copper, magnesium and zinc and vitamins B1, B6, B12, E and folate will increase after eight weeks of supplementation; 4) Depression scores, measured by the KDRS, will show a decreasing trend over the course of supplement treatment; and 5) Mania scores, measured by the KMRS, will show a decreasing trend over the course of supplement treatment. ii Results showed recruitment was completed in 6.5 months. Three participants terminated due to palatability and compliance issues. The mean sample compliance rate was approximately 91%. Of the seven study completers, all maintained at least 93% compliance and two maintained 100% compliance.

Twelve, one-tailed Fisher Randomization Tests were computed, showing significant increases in blood levels of vitamins A, B6, D, E (alpha tocopherol) and folate from pre- to post-supplementation for the seven study completers (p<0.05).

Two, one-tailed Fisher Randomization Tests showed significant decreasing trends in depression and mania scores for the seven study completers from baseline to the final visit, suggesting improvement and possible treatment response (p<0.05).

Overall, results of this feasibility trial suggest recruitment for future studies is possible and may improve with summer recruitment. Also, children who meet swallowing inclusion criteria will likely have high medication compliance, but those who struggle swallowing capsules may not benefit from this intervention.

This trial also shows children tolerate fasting and blood draw procedures well.

Lastly, although open-label, significant decreasing trends in depression and mania scores throughout supplementation suggest future randomized, placebo-controlled trials of EMP+ are warranted. Suggestions for future research and limitations of the current study are discussed.

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Acknowledgments

Thank you to the many mentors, faculty members, family and friends who have helped me complete this thesis. I am forever grateful for your continued support and encouragement.

I would like to thank each of my committee members for helping me throughout this process. First, my sincere gratitude to my advisor, Dr. Mary

Fristad, for introducing me to this exciting project and guiding me through each step along the way to its completion. She created the perfect balance of independence and advice over the course of multiple editing and feedback sessions as my thesis evolved. I cannot imagine having a better mentor. To Dr. Steven

Beck, thank you for your excitement and support of this project. You have provided encouragement in the face of skepticism. Also, thank you to Dr. Michael

Vasey for challenging me to think critically and helping to make me a better scientist.

Thank you to all of the staff and faculty at The Ohio State University that helped make this study possible. Thank you to Dr. L. Eugene Arnold for sharing

iv your knowledge of childhood nutrition and mental health as well as for taking the time to edit and guide my research. I am forever indebted to Dr. Mark Failla, Julie, and the rest of the human nutrition research team for teaching me the intricacies of human nutrition and guiding me in the interpretation of blood assays. I have learned more from you than I ever expected. A special thanks to the nurses, dieticians, and staff of the research clinic at The Ohio State University Center for

Clinical and Translational Research. This project would not have been possible without your excellent care during blood draw and dietary monitoring visits. Also, thank you to Dr. Thomas Nygren and Dr. Joseph Verducci for your consultation regarding the statistical analyses for this thesis.

Last but certainly not least, many thanks to my family and friends for their support throughout this crazy ride. Thank you all for pretending to understand and be interested in what I was talking about when I tried to explain my research. Also, thank you for your understanding during times when it seemed like I disappeared off the face of the Earth. A special thanks to Rob for calming me down in times of crisis and tracking down my work when I thought my computer had eaten it all.

Finally, to Ryan and my parents, thank you for your encouragement and interest in this project. I love you all and truly appreciate your love and support.

Vita

June 2002 ...... Chagrin Falls High School Chagrin Falls, Ohio

May 2006...... Bachelor of Science, Psychology Denison University

2006 to present ...... Graduate Research Associate The Ohio State University

Publications

Fristad, M.A. & Frazier, E.A. (2008). Special treatment issues. In R. Findling, M. Fristad, & R. Kowatch (Eds.), Clinical Manual for the Management of Bipolar Disorder in Children and Adolescents (pp. 273-290). Arlington, VA: American Psychiatric Publishing, Inc.

Fields of Study

Major Field: Psychology

Specialty: Clinical Child

Table of Contents

Abstract ...... ii

Acknowledgments ...... iv

Vita ...... vi

List of Tables ...... ix

List of Figures ...... x

Chapter 1: Introduction ...... 01

Chapter 2: Method ...... 23

Chapter 3: Results ...... 45

Chapter 4: Discussion ...... 57

References ...... 72

Appendix A: EMPowerplus (EMP+) 36-ingredient List ...... 80

Appendix B: Pill Swallowing Desensitization Protocol ...... 82

Appendix C: Demographic Form ...... 83

Appendix D: Medical History ...... 85

Appendix E: Physical Exam ...... 86

Appendix F: Side Effects Form ...... 87

vii Appendix G: Investigator’s Brochure ...... 88

Appendix H: Medication Accountability Form ...... 136

Appendix I: 24 Hour Recall/Typical Diet Form ...... 137

Appendix J: Individual Nutrient Blood Levels Pre- and Post- Supplementation. 138

Appendix K: Individual Mood Ratings Over Time ...... 145

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List of Tables

Table 1. Time course for data collection for EMP+ open-label pilot ...... 21

Table 2. Participant medication compliance over time ...... 46

Table 3. Mean nutrient blood levels pre- and post-EMP+ supplementation with normal range comparisons...... 47

List of Figures

Figure 1. Mean nutrient levels pre- and post- supplementation for vitamin B6, copper, vitamin D, ferritin, and magnesium...... 48

Figure 2. Mean nutrient levels pre- and post- supplementation for vitamin A, carotenoids, vitamin E (alpha-tocopherol [a-TC] and gamma-tocopherol [g-TC]), folate, transferrin receptor (TfR), and zinc ...... 49

Figure 3. Depression ratings (KDRS) for all participants over time...... 51

Figure 4. Mean depression ratings (KDRS) for study completers over time . . . . . 52

Figure 5. Mean intent to treat depression ratings (KDRS) over time ...... 53

Figure 6. Mania ratings (KMRS) for all participants over time ...... 54

Figure 7. Mean mania ratings (KMRS) for study completers over time...... 55

Figure 8. Mean intent to treat mania ratings (KMRS) over time ...... 56

Figure 9. Participant 1 nutrient levels pre- and post- supplementation for vitamin B6, copper, vitamin D, ferritin, and magnesium ...... 138

Figure 10. Participant 1 nutrient levels pre- and post- supplementation for vitamin A, carotenoids, vitamin E (alpha-tocopherol [a-TC] and gamma- tocopherol [g-TC]), folate, transferrin receptor (TfR), and zinc ...... 138

Figure 11. Participant 3 nutrient levels pre- and post- supplementation for vitamin B6, copper, vitamin D, ferritin, and magnesium ...... 139

Figure 12. Participant 3 nutrient levels pre- and post- supplementation for vitamin A, carotenoids, vitamin E (alpha-tocopherol [a-TC] and gamma- tocopherol [g-TC]), folate, transferrin receptor (TfR), and zinc ...... 139 x

Figure 13. Participant 5 nutrient levels pre- and post- supplementation for vitamin B6, copper, vitamin D, ferritin, and magnesium ...... 140

Figure 14. Participant 5 nutrient levels pre- and post- supplementation for vitamin A, carotenoids, vitamin E (alpha-tocopherol [a-TC] and gamma- tocopherol [g-TC]), folate, transferrin receptor (TfR), and zinc ...... 140

Figure 15. Participant 6 nutrient levels pre- and post- supplementation for vitamin B6, copper, vitamin D, ferritin, and magnesium ...... 141

Figure 16. Participant 6 nutrient levels pre- and post- supplementation for vitamin A, carotenoids, vitamin E (alpha-tocopherol [a-TC] and gamma- tocopherol [g-TC]), folate, transferrin receptor (TfR), and zinc ...... 141

Figure 17. Participant 7 nutrient levels pre- and post- supplementation for vitamin B6, copper, vitamin D, ferritin, and magnesium ...... 142

Figure 18. Participant 7 nutrient levels pre- and post- supplementation for vitamin A, carotenoids, vitamin E (alpha-tocopherol [a-TC] and gamma- tocopherol [g-TC]), folate, transferrin receptor (TfR), and zinc ...... 142

Figure 19. Participant 9 nutrient levels pre- and post- supplementation for vitamin B6, copper, vitamin D, ferritin, and magnesium ...... 143

Figure 20. Participant 9 nutrient levels pre- and post- supplementation for vitamin A, carotenoids, vitamin E (alpha-tocopherol [a-TC] and gamma- tocopherol [g-TC]), folate, transferrin receptor (TfR), and zinc ...... 143

Figure 21. Participant 10 nutrient levels pre- and post- supplementation for vitamin B6, copper, vitamin D, ferritin, and magnesium ...... 144

Figure 22. Participant 10 nutrient levels pre- and post- supplementation for vitamin A, carotenoids, vitamin E (alpha-tocopherol [a-TC] and gamma- tocopherol [g-TC]), folate, transferrin receptor (TfR), and zinc ...... 144

Figure 23. Participant 1 depression and mania ratings over time...... 145

Figure 24. Participant 2 depression and mania ratings over time...... 145

Figure 25. Participant 3 depression and mania ratings over time...... 146

Figure 26. Participant 4 depression and mania ratings over time...... 146 xi

Figure 27. Participant 5 depression and mania ratings over time...... 147

Figure 28. Participant 6 depression and mania ratings over time...... 147

Figure 29. Participant 7 depression and mania ratings over time...... 148

Figure 30. Participant 8 depression and mania ratings over time...... 148

Figure 31. Participant 9 depression and mania ratings over time...... 149

Figure 32. Participant 10 depression and mania ratings over time...... 149

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Chapter 1: Introduction

Feasibility of a Nutritional Supplement as Treatment for Childhood Mood

Dysregulation

Childhood-onset mood dysregulation (bipolar spectrum disorders [BPSD] +

Longitudinal Assessment of Manic Symptoms [LAMS] study defined subthreshold bipolar disorder-not otherwise specified [BP-NOS]) is a major health concern for children and a significant challenge for clinicians to work with effectively (APA

Working Group on Psychoactive Medications for Children and Adolescents, 2006;

McClellan, Kowatch, Findling, the Work Group on Quality Issues, & AACAP

Staff, 2007). The disorders captured under the title of mood dysregulation follow the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-

IV) criteria except for subthreshold BP-NOS. Subthreshold BP-NOS is operationalized as follows: (a) participant must have one to 3 manic-like episodes of moderate to severe intensity lasting 4 or more hours that does not recur or two or more brief episodes (i.e., lasting a total of 2 to 4 hours in a month); (b) any of the following symptoms - elated mood, grandiosity, decreased need for sleep, pressured speech, racing thoughts, distractibility, increased goal-directed activity,

excessive involvement in pleasurable activities with high potential for painful consequences (c) there is no evidence the behavior is drug- or health-induced;

(d) interference is present and associated with these manic-like episodes; (e) parent and/or child can provide clear behavioral descriptors of manic-like activity that does not appear to have any environmental trigger; and (f) manic-like activity can be differentiated from attention deficit hyperactivity disorder (ADHD) symptoms with reasonable certainty.

Morbidity and mortality are of significant concern, yet pharmacologic interventions are not well tested and those agents found efficacious are associated with risk for adverse events (Kowatch et al., 2005). While recent clinical trials have found efficacy in using atypical antipsychotics for BP-I, treatment literature is lacking for children with BP-II, BP-NOS and cyclothymia, despite the functional impairment inherent to these diagnoses (Kowatch, Fristad, Findling & Post 2009).

Medications recommended in current treatment guidelines (Kowatch et al. 2005;

McClellan et al. 2007) appear beneficial but carry significant risk for adverse events. The Food and Drug Administration (FDA) recently approved the use of

Risperidone in children with bipolar disorder, although many children experience significant metabolic side effects on therapeutic doses (Slatko, 2007). This trend may continue after a June 2009 article from the Associated Press indicated a psychiatric expert panel from the Food and Drug Administration (FDA) voted to endorse the use of Zyprexa, Seroquel, and Geodon as treatments for bipolar disorder and schizophrenia in youth age 10-17 (Perrone, 2009).

A recent retrospective study compared medical and pharmacy claims from a cohort of 4,140 youth prescribed one of five types of atypical antipsychotics or two conventional antipsychotics compared to a random sample of 4,500 youth not treated with psychotropics (McIntyre & Jerrell, 2008). The treated cohort had higher rates of a variety of metabolic and cardiovascular side effects, including greater risk of obesity, type 2 diabetes mellitus, cardiovascular conditions, dyslipidemia and orthostatic hypotension (McIntyre & Jerrell 2008). Researchers found metabolic and cardiovascular risks increased for youths taking multiple antipsychotics (McIntyre & Jerrell 2008).

In another study monitoring side effects of atypical neuroleptics including clozapine, olanzapine, and risperidone, all three drugs caused drowsiness and hypoactivity. Thirty to sixty percent of children and adolescents taking clozapine experienced constipation, increased salivation, orthostatic hypotension, and nasal congestion. These side effects were seen in patients taking olanzapine and risperidone less often, but 5% to 15% of participants taking olanzapine or risperidone suffered from rigidity, tremor, and dystonia. Participants in all three atypical neuroleptic conditions gained weight during the study, but those in the olanzapine group gained significantly more weight than those in the other two treatment groups (4.6 ± 1.9 kg; Fleischhaker et al. 2006).

Recent clinical trials of depression and bipolar disorders in youth show approximately 20%-25% of participants dropped out of psychotropic medication treatment (Biederman et al. 2007; DelBello, Adler, Whitsel, Stanford, and

Strakowski 2007). DelBello and colleagues (2007) conducted a single-blind, 12- week study of quetiapine in adolescents age 12-18 in which researchers observed a

25% drop out rate of adolescents whose diagnoses included dysthymia, BP II, BP-

NOS, major depressive disorder, or cyclothymia. Biederman and colleagues (2007) conducted an 8 week, open-label trial of aripiprazole in children age 6-17 with bipolar disorder in which they observed a 21% drop out rate. Approximately 14% of adolescent participants age 13-20 years (3 out of 21) dropped out of a recent effectiveness and tolerability study of quetiapine. Of the 18 adolescents who completed this study, about 28% (5 out of 18) were unable to maintain stable mood without relapse and required additional pharmacotherapy (Duffy, Milin and Grof,

2009). Additionally, a recent study of an anticonvulsant mood stabilizer in children failed to show any superiority to placebo (Wagner et al. 2006).

Within the diagnosis of bipolar disorder, more effective treatments have been identified that quell manic symptoms rather than ameliorate depressive symptoms; unfortunately, it is the depressive phase that is associated with longer duration of illness and greater functional impairment (Kowatch et al., 2005).

Research suggests that earlier age of BPSD onset results in heavier genetic loading as well as more severe symptom representation, making advances in childhood onset bipolar disorder research an immediate necessity (APA Working Group on

Psychoactive Medications for Children and Adolescents, 2006).

Dietary Supplementation and Mental Health

Increasing evidence suggests that nutrition affects the structure and functioning of the brain due to the high percentage of human metabolic activity accounted for by this organ. In adulthood, the brain accounts for 20% of the human basal metabolic rate; as a neonate, this number is as high as 44% (Benton 2008).

Previous research on diet and nutrition suggests the possibility that multinutrient supplements may have a beneficial effect on mood, which might provide either a primary or adjunctive treatment with a more favorable risk-benefit ratio for children suffering from BPSD than currently available pharmacologic interventions

(Kaplan, Crawford, Field, and Simpson 2007; Kaplan, Crawford, Gardner, and

Farrelly 2002; Kaplan, Fisher, Crawford, Field, and Kolb 2004; Kaplan, Simpson,

Ferre, Gorman, McMullen, and Crawford 2001; Popper 2001).

Individual Vitamins and Minerals

The neuropsychiatric effects of certain individual nutrients have gained recent attention. Specific nutrients linked to mental health include: iron, copper, magnesium, zinc, vitamins B1, B6, B12, D, E and folate (Kaplan et al. 2007). Iron deficiencies may subtly affect general mental functioning. Iron helps produce adenosine triphosphate (ATP) brain energy, works with hemoglobin to maintain appropriate oxygen levels in the brain, increases binding of serotonin and dopamine in the frontal cortex, and plays a role in producing several neurotransmitters including dopamine, serotonin, epinephrine and norepinephrine (Kaplan et al.

2007; Velez-Pardo, Jimenez del Rio, Ebinger, and Vauquelin, 1995). Copper is

involved in neurotransmitter production; if the ratio of copper to zinc is unbalanced in the body, metabolic functions may be disrupted. Zinc is involved in over 200 enzymatic reactions, many of which occur in the brain, and plays a central role in metabolism. Zinc is also found in glial cells and neurons, and it is involved in protein synthesis and the regulation of gene expression. Certain brain areas, such as the hippocampus, appear particularly sensitive to dietary changes in zinc and may malfunction without proper zinc levels (Kaplan et al. 2007; Takeda, 2001).

B vitamin deficiencies have been shown to lead to brain dysfunction.

Vitamin B1, thiamine, is necessary for neuronal health, and deficiencies of this nutrient may negatively impact the cardiovascular and/or peripheral nervous system, leading to Wernicke’s encephalopathy. B1 can also act like acetylcholine in the brain (Kaplan et al. 2007; Meador et al., 1993). Vitamin B6 is a precursor to the active form, pyridoxal 5’-phosphate (PLP), which is essential to numerous bodily enzymes, neurotransmitter synthesis, and neuronal health. Deficiencies in B6 may result in lower levels of serotonin and gamma-Aminobutyric acid (GABA) in the brain (Kaplan et al. 2007; McCarty, 2000). Vitamin B12 is essential to brain and nervous system functioning, maintains myelin sheaths, helps synthesize monoamine neurotransmitters, and can alter folate levels (Hutto, 1997; Kaplan et al. 2007). Deficiencies in vitamin D are associated with mental illness, specifically depression, and abnormal brain development. Vitamin E is an anti-oxidant, protecting cell membranes from oxidation and potentially effecting brain functioning. Lastly, folate is important for neurotransmitter synthesis, brain

metabolism, and influencing the relationship between tryptophan and serotonin

(Hutto, 1997). Folate deficiencies may be related to dementia and mood disorders

(Kaplan et al. 2007).

Mild effects of subtle nutritional deficiencies are being increasingly recognized, possibly related either to genetic variations in which some patients may be more vulnerable or to historical changes in diet composition. Nutritional supplements have been related to a wide range of human health factors from neuronal development to depression (Hibbeln, 1998; Noaghiul and Hibbeln, 2003).

In addition to more recently studied nutrients and their role in brain function and mental health, it is important to note that lithium, a natural chemical nutrient, is one of the common contemporary treatments for BPSD.

Multinutrient Combinations

While past nutritional research focuses on individual vitamins and minerals, more recent research turns to multi-ingredient supplements, as there is plausible reasoning to support the concept that if one nutrient is deficient, a grouping of nutrients are deficient, and the level of one nutrient can affect the adequacy of others (Benton, 2008; Kaplan et al. 2007). Nutritional interventions, particularly multi-ingredient multinutrient supplements, have several possible mechanisms of action to explain their association with clinical improvement in mood (Kaplan et al., 2007).

Theoretical models. Kaplan and colleagues (2007) provide four theories on how multinutrient supplementation may alter human mood. First, they suggest

mood dysregulation may be a result of innate metabolism malfunctions that ultimately affect brain functioning by influencing enzymatic reactions. Genetic defects resulting in dysfunctional enzymatic binding play a role in many genetic disorders (e.g., cardiovascular disease, migraines, rages, anemia, alcohol intolerance), the majority of which can be corrected with high concentration vitamin supplementation. Appropriate vitamin supplementation adds cofactors and coenzymes that improve enzymatic activity and correct the original genetic mutation (Ames, Elson-Schwab, and Silver, 2002). Kaplan and colleagues (2007) theorize mood dysregulation may result from a similar genetic mutation that may be corrected by nutrient supplementation, which returns metabolic functioning to normal.

Second, they state mood instability may result from deficiencies in methylation of molecules. Methylation reactions link nutrients and gene expression. These reactions are made possible by methyl donors and are responsible for completing DNA transcription, switching on genes, regulating protein generation, activating enzymes, and synthesizing neurotransmitters.

Recently, researchers have used this methylation theory as a basis for examining the relationship between depression and S-adenosyl-L-methionine (SAMe), a methyl donor used in synthesizing neurotransmitters and other reactions in the central nervous system. The relationship between methylation and mood may also be bidirectional due to the physiological influence of stress from mental disorders

(Kaplan et al., 2007).

Third, Kaplan and colleagues (2007) propose nutrition deficiencies may alter gene expression and lead to mood instability. This is again theorized to be linked to metabolism and methylation of nucleotides in the human genome. Kaplan and colleagues (2007) cite a study of 31 colorectal cancer patients who were able to alter gene expression and reverse deficiencies in DNA methylation after folic acid supplementation (Pufulete et al., 2005). Research in this area suggests genotyping may be necessary to determine these types of deficiencies and altered genes since the study of plasma nutrient levels may not be sensitive enough (Kaplan et al.,

2007).

Fourth, Kaplan and colleagues (2007) suggest unstable mood may result from long-latency effects of nutrient deficiencies which alter brain development directly or by way of dysfunctional nutrient absorption. This theory suggests that similar to the process of osteoporosis developing after years of inadequate calcium intake and absorption, perhaps mood disorders develop over time due to deficiencies or altered gene expression from in vitro or early life experiences.

Although these four pathways require considerable empirical evaluation, they provide a series of possible mechanisms through which nutrient supplementation may affect mood symptoms (Kaplan et al., 2007).

Multinutrients and behavior. Gesch, Hammond, Hampson, Eves, and

Crowder (2002) examined the effects of vitamins, minerals, and essential fatty acids on antisocial behavior in a randomized, double-blind, placebo-controlled study of 231 prisoners. Participants spent, on average, 142 days on the

recommended daily dose of two supplements (one capsule of Forceval, a multinutrient supplement that contains 25 vitamins and minerals, and four capsules of Efamol Marine, an essential fatty acid supplement containing omega-6 and omega-3 essential fatty acids) or placebo (identically appearing oil-based gelatin capsules). The Efamol Marine dose contained 1260mg linoleic acid, 160mg gamma linolenic acid, 80mg eicosapentaenoic acid and 44mg docosahexaenoic acid

(Gesch et al., 2002).

Participants’ antisocial behavior was measured throughout the study period using disciplinary reports. Results revealed a decrease in overall antisocial behavior for participants taking nutritional supplements compared to placebo.

Neither group reported notable side effects. Overall infringements resulting in disciplinary reports decreased by 35.1% in the active condition for participants who took the supplement for at least two weeks compared to placebo participants, whose disciplinary reports decreased by 6.7% (p<.001). Intent-to-treat analyses showed active condition participants experienced a 26% decrease in overall infringements resulting in disciplinary reports compared to placebo participants

(p<.03). This research suggests that multinutrient supplements and essential fatty acids may decrease antisocial behavior; the researchers speculate that physiological changes caused by dietary intervention affect mental health, and warrant further clinical investigation (Gesch et al., 2002).

Schoenthaler and Bier (2000) examined the impact of low-dose multinutrient tablets (not EMP+) on rates of violent and antisocial behavior in

school children. Participants were 468 children aged 6 to 12 from two working class primarily Hispanic elementary schools in the Southwestern US. Using a stratified randomized double-blind, placebo-controlled design, half the sample received daily multinutrient supplementation at 50% of the US recommended daily allowance (RDA) for 4 months and the other half received placebo. Of the 468 children, 80 were disciplined at least once during the September 1 to May 1 study interval; their results were analyzed. The 40 who had received supplementation had a 47% lower mean rating of antisocial behavior than the 40 who had been on placebo (1 vs. 1.875 disciplinary actions). Children on supplementation had lower ratings of antisocial behavior for every type of recorded infraction: threats/fighting, vandalism, being disrespectful, disorderly conduct, assault/battery, defiance, obscenities, refusal to work or serve, endangering others, and nonspecified offenses.

EMPowerplus (EMP+: multinutrient supplement). The multinutrient supplement of focus in this pilot study is EMPowerplus (EMP+; Truehope

Nutritional Support Ltd., Raymond, Alberta, Canada), a 36-ingredient supplement consisting of sixteen minerals, fourteen vitamins, three amino acids and three antioxidants. The full list of ingredients can be found in Appendix A. One reason to study this supplement is that it is being used currently in over 2,000 children to treat mood dysregulation (personal communication, TrueHope, 5/7/2008) with anecdotal suggestion of benefit (Frazier, Fristad & Arnold, in press) but randomized controlled trials have not yet been conducted in children.

Prior to participant recruitment, an Investigational New Drug (IND) approval was obtained from the FDA for the 36-ingredient supplement formulation, indicating quality assurance acceptable to the FDA has been implemented (IND#

102,467). Preliminary case studies and open-label trials have been conducted in youth and adults as well as one animal study with this multinutrient supplement.

Results suggest some benefit for depression, irritability and mood stability, which might provide either a primary or adjunctive treatment with a more favorable risk- benefit ratio for children suffering from mood dysregulation than currently available pharmacologic interventions (Kaplan et al., 2002; Kaplan et al., 2001;

Simmons, 2003).

Animal study. Halliwell & Kolb (2003) studied newborn rats who received frontal or posterior parietal lesions on Day 3, then subsequently were fed either normal rat chow or rat chow enhanced with a rodent-appropriate dose of EMP+. At

Day 60, the supplemented animals exhibited reversal of behavioral deficits (e.g., performance on spatial learning tasks) and had significant re-growth of cortical tissue compared to un-supplemented rats. Behaviorally, the animals were significantly calmer than un-supplemented rats.

Adult studies. Initial studies were conducted with adult patients resistant to conventional treatments. Most recently, Rucklidge (2009) examined the effect of

EMP+ in a case study of an 18-year-old male with obsessive-compulsive disorder

(OCD) and Asperger’s Disorder who previously showed partial response to cognitive-behavioral therapy (CBT). Prior to CBT, he scored a 29 on the Yale-

Brown Obsessive Compulsive Scale (Y-BOCS; Goodman et al., 1989), and after 27 session of CBT over the course of a year, his Y-BOCS score decreased to 16. After this modest response to CBT, this individual’s anxiety symptoms returned to a severe level one year post-treatment termination and he developed co-morbid major depression. His Y-BOCS score returned to a 24. He entered an ABAB design trial of EMP+. After eight weeks on the multinutrient supplement, his anxiety decreased, his mood stabilized, and his obsessions remitted. He then discontinued treatment for eight weeks, during which all symptoms of anxiety, mood, and obsessions worsened. Anxiety and mood symptoms improved again with reintroduction of EMP+ treatment (Rucklidge, 2009).

Another recent article by Gately and Kaplan (2009) analyzed a database of adults who voluntarily completed self-report mood rating forms provided by

TrueHope when they purchased EMP+. TrueHope created a database to track the progress of individuals taking EMP+ who completed self-report forms consisting of

16 DSM-specified symptoms of mood disorders rated on a scale of 0 to 3 (not at all to very much). Forms were returned by 682 adults between January, 2001 and

August, 2007 who indicated they had been diagnosed with bipolar disorder.

Researchers excluded respondents with additional diagnoses other than depression, as well as respondents who returned fewer than 60 self-report forms in the first 180 days of EMP+ supplementation. This left 358 respondents for analysis.

Approximately 81% of this reduced sample was taking psychotropic medications at the start of EMP+ supplementation. Symptom severity scores decreased 41% after

three months of supplementation compared to baseline [t (349) = 14.5, p < 0.001; effect size = 0.78] and decreased 45% after six months [t (242) = 12.7, p < 0.001; effect size = 0.76]. Approximately 18% of the sample deteriorated but 53% reported at least 50% improvement in mood symptoms after six months (Gately and Kaplan, 2009).

Regression analyses showed 72% of the variance in mood symptom severity was accounted for by EMP+ dose, and decreasing symptom severity was significantly associated with increasing EMP+ supplementation. A second regression analysis showed decreasing medication over six months was significantly associated with decreasing mood symptom severity. There were no significant differences in self-reported treatment response based on gender.

Respondents in all medication categories (no medication, low level of medication, or high level of medication assigned at baseline) experienced significant decreases in mood symptom severity from pre- to six months post-supplementation, but those in the high level of medication category appeared to have moderate responses

(approximately 35% improvement) compared to respondents with no or low levels of medication (44%-56% improvement; Gately and Kaplan, 2009).

Open-label trials were first reported on adults diagnosed with bipolar disorder (Kaplan et al., 2001). Kaplan and colleagues studied 11 patients aged 19-

46 years for 6-21 months (2001). The effects of EMP+ on symptoms of bipolar disorder were assessed using the Hamilton Depression Rating Scale (HAM-D:

Hamilton, 1960), the Young Mania Rating Scale (YMRS: Young et al., 1978), and

the Brief Psychiatric Rating Scale (BPRS; Overall & Gorham, 1962). Participants could continue the use of concurrent psychiatric medications under the supervision of their psychiatrist. Results indicated a 55-66% reduction in symptoms reported on the HAM-D, YMRS and BPRS as well as a 50% decrease in the need for psychotropic medications. These results were achieved with only one mild side effect, infrequent/transitory nausea, which occurred most commonly when participants took their supplement without food (Kaplan et al., 2001). The researchers recommended further empirical investigation of EMP+ by other researchers.

Simmons (2003) described his use of EMP+ in private clinical practice. He reported that twelve out of nineteen treatment-resistant adult patients diagnosed with bipolar I (n=14) and bipolar II (n=5) who began what is now an outdated version of EMP+ displayed marked improvement, three appeared moderately improved, and one person showed mild improvement after a mean of 5.3 weeks on the supplement. Thirteen participants completely stopped taking their original psychiatric medications after an average of 5.2 weeks on EMP+ (range = 3-10 weeks) and remained stable. Side effects included mild gastrointestinal problems, but a majority of patients (11 of 19, 58%) continued using this multinutrient supplement instead of their previously prescribed psychopharmacological medications (Simmons, 2003). While placebo-controlled trials are currently being conducted by Kaplan and colleagues in adults, placebo-controlled trials have not

yet commenced with children (Kaplan et al. 2001). However, several case series and open trial studies have been conducted, reviewed below.

Child studies. Several studies have examined the efficacy of EMP+. In a case study using an ABACB design, Popper (2001) reported on a 10-year old boy diagnosed with bipolar disorder who experienced sever temper tantrums for multiple hours each day for the previous four months. After two days of taking the full dose of EMP+ the boy’s behavior was significantly better. Within five days, all tantrums and irritability ceased. After 14 days, EMP+ was discontinued.

Within two days of discontinuation, the tantrums began again. The boy was then put on a different supplement, which, according to parent and teacher reports, provided 60% of the benefit noted on EMP+. EMP+ was resumed a second time, which resulted in resolution of the boy’s tantrums and irritability. In a follow-up study, Popper conducted an open trial on 22 patients with bipolar disorder (2001).

Participants included ten adults, nine adolescents and three preadolescents.

Although mild side effects were common (e.g., headache), a majority of participants (19/22, 86%) responded positively. Further, 11 of the 15 patients

(73%) who previously were on psychiatric medications remained stable without resumption of these medications at both 6-month and 9-month follow-up assessments (Popper, 2001).

Kaplan and associates also have conducted an open-label ABAB trial with two boys aged 8 and 12 (Kaplan et al., 2002). These participants displayed irritability, mood lability, and explosive rage at baseline. The 8-year old boy had

diagnoses of atypical obsessive-compulsive disorder (OCD) and ADHD while the

12-year old boy was diagnosed with pervasive developmental disorder (PDD). The

8-year old displayed consistent explosive rage, irritability and obsessions with guns

(but no compulsions) during baseline and each withdrawal phase. When on EMP+, these behaviors were almost completely eliminated. His obsessive thoughts ceased, the frequency and duration of his temper outbursts decreased significantly, and his mood fluctuations minimized. After more than two years of treatment, the boy remained well and free of side-effects while taking 25% of his initial dose of

EMP+. The second boy, 12 years old, displayed consistent irritability, negative attitude, temper outbursts, and extremely disruptive behavior in school at baseline.

These behaviors subsided markedly while on EMP+. His mood impairment and temper levels returned to baseline status during treatment withdrawal. He demonstrated marked improvement in mood stabilization and behavior when treatment was reintroduced. Stimulant medication was still required in addition to

EMP+ to control ADHD symptoms. After almost three years of treatment, this boy also maintained wellness without adverse side-effects on 25% of his original dose

(Kaplan et al., 2002).

Kaplan and colleagues then completed a case series to further test the impact of EMP+ in eleven children ranging in age from 8 to 15 (Kaplan et al.,

2004). All had mood and/or behavioral problems. Diagnoses at intake included: bipolar disorder (n=3; 1 with comorbid anxiety, 1 with behavior disorders);

Asperger’s + an anxiety disorder (n=2); ADHD + comorbid anxiety and/or

behavior disorders (n=5); and Praeder-Willi syndrome, oppositional defiant disorder (ODD) + anxiety (n=1). Nine of the 11 (82%) completed the open-label trial. Two children withdrew from the trial before completion. One girl, who was diagnosed with a severe pervasive developmental disorder and generalized anxiety, withdrew due to increases in anxiety as the supplement dose was increased. The other non-completer, who was diagnosed with attention deficit hyperactivity disorder and mood swings, reported increases in anger and mood lability, and he chose to withdraw instead of decrease his 40mg dose of concurrent methylphenidate as was suggested by the study physician. Intent-to-treat analyses indicated significantly decreased scores on the Youth Outcome Questionnaire

(YOQ: p<.001) and Young Mania Rating Scale (YMRS: p<.01) from baseline to final visit. For the 9 completers, improvement was significant on 7/8 (88%) Child

Behavior Checklist scales, YOQ and YMRS (p values ranged from <.05 to <.001).

The authors concluded that results from this study support the need for formal clinical trials of nutritional interventions in children with mood and behavioral dysregulation (Kaplan et al., 2004).

While such open data are intriguing, further research using systematic data collection from randomized, controlled trials is necessary to empirically evaluate the safety and efficacy of EMP+ as a treatment for mood dysregulation. In addition, it is important to note that all of the above publications except for the

2009 Rucklidge study used a now-outdated version of EMP+, one which often caused gastrointestinal side effects. In the currently-marketed version, the

ingredients have been processed differently. In particular, the minerals have been pulverized so that the individual particle size has been dramatically reduced to as low as 14-15 microns. Consequently, the ingredients now look like a powder and pack better into fewer capsules. There have also been some small reductions in quantities of several vitamins (e.g., vitamin A).

Summary

Mood dysregulation in children represents a significant public health concern. Relatively few clinical trials have been conducted and treatments with demonstrated safety and efficacy are lacking. Studies to date have focused on BP-

I; there is essentially no treatment literature available for children with BP-II, BP-

NOS and cyclothymia, despite the functional impairment inherent to these diagnoses. Medications recommended in current treatment guidelines (Kowatch et al., 2005; McClellan, et al., 2007) appear beneficial but carry with them significant risk for adverse events. Additionally, very little is known about treating the depressive phase of the illness. Limited studies have been conducted with a multinutrient complex, EMPowerplus (EMP+). Clinical trials suggest some benefit for depression, irritability and mood stability; however, double-blind placebo controlled trials have not yet begun in children. This open-label trial lays the groundwork for undertaking a large randomized clinical trial by testing feasibility and estimating potential treatment response in a pilot study of EMP+ for children with mood dysregulation.

Current Study

This open-label study explored the feasibility of testing possible therapeutic effects of a multinutrient nutritional intervention, EMPowerplus (EMP+), in ten children with mood dysregulation (MD: Bipolar I, Bipolar II, Bipolar Not

Otherwise Specified [BP-NOS], subthreshold BP-NOS as operationalized in the current Longitudinal Assessment of Manic Symptoms [LAMS] protocol, and cyclothymia). This was a feasibility study in which doses recommended and utilized by current consumers and closely mimicked methods from previous literature were used to examine the following primary goals: 1) determine feasibility of sample recruitment for a larger nutritional study and compliance with ingestion of the nutritional intervention; and 2) collect preliminary data regarding ability of the supplement to raise blood levels of relevant nutrients. Secondary goals were to observe clinical response in an exploratory manner in preparation for a subsequent double-blinded, randomized clinical trial comparing EMP+ multinutrient supplement and placebo.

Ten children aged 6-12 years with mood dysregulation were recruited to participate in this 2-month, open-label trial. This study explored the feasibility of conducting a nutritional study in this population as well as preliminary descriptive efficacy of the multinutrient complex, EMP+. All ten children were given EMP+ treatment. The study design included seven assessments with each parent-child pair over eight weeks. Main outcomes (KDRS and KMRS scores) were collected

weekly at the beginning of the trial and then biweekly at the last three study visits.

Table 1 summarizes the time course for data collection.

Start 8 week trial Task Screen EMP+ Ongoing Endpoint Assessment Week -1 to -3 0 1,2, 4, 6 8 Visit 1 2 3 ,4, 5,6 7 Treat- No treatment ment (Washout of previous med; 3 EMP+ EMP+ weeks), Complete pill N = 10 swallowing evaluation (training, as necessary) Assess- DSM-IV by ChIPS/P-ChIPS, KDRS & KMRS, KDRS & KMRS, CDRS-R & ments WASI, clinical eval., med. Vital signs, DM, Vital signs, C- YMRS, KDRS & history, demographics, CDRS- C-GAS, CGI- GAS, CGI-S/I KMRS, R & YMRS, KDRS & KMRS, S/I, blood draw Vital signs, PE, Vital signs, PE, DM, C-GAS, C-GAS, CGI-S, NE CGI-S/I, NE, blood draw

Table 1. Time course for data collection for EMP+ open-label pilot. Note: P-ChIPS = Children’s Interview for Psychiatric Syndromes, parent version; ChIPS = Children’s Interview for Psychiatric Syndromes; PE = Physical Exam ; DM = Dietary Monitoring; WASI = Wechsler Abbreviated Scales of Intelligence; CDRS-R = Children’s Depression Rating Scale-Revised; YMRS = Young Mania Rating Scale; KDRS = Depression section of the Kiddie Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present Episode-Depression Rating Scale; KMRS = Kiddie Schedule for Affective Disorders and Schizophrenia for School-Age Children-Mania Rating Scale; C-GAS = Children's Global Assessment Scale; CGI-S/I = Clinical Global Impressions Severity & Improvement Scales; NE = Neurological Evaluation.

I explored the following three hypotheses for this open trial examining feasibility measures of sample recruitment, supplementation compliance, and biological effects on micronutrient levels of various vitamins and minerals. 21

Although definitive inferences cannot be made about treatment response due to the open-label design and small sample size, descriptive data were graphed and exploratory non-parametric analyses were completed to examine possible trends in treatment response. Two secondary hypotheses regarding trends in treatment response were examined.

Feasibility of Sample Recruitment

1. The sample can be recruited in 5 months.

Feasibility of Supplementation Compliance

2. Children aged 6-12 can swallow the supplement with >80% compliance

Feasibility of Increasing Micronutrient Levels

3. Micronutrient levels of iron, copper, magnesium and zinc and vitamins

B1, B6, B12, E and folate will increase after eight weeks of supplementation.

Trends in Treatment Response

4. Depression scores, measured by the KDRS, will show a decreasing trend over the course of supplement treatment.

5. Mania scores, measured by the KMRS, will show a decreasing trend over the course of supplement treatment.

Chapter 2: Method

Participants

Eleven children were screened and ten children aged 6-12 years with mood dysregulation participated in this open-label trial. One child screened did not meet symptom criteria to participate. Each child was accompanied by at least one parent or guardian (hereafter referred to as parent). Participants were recruited through continuous enrollment over a 6.5 month period until ten eligible participants were enrolled. Participants had to meet seven inclusion criteria to participate: 1) aged 6-

12 years (boys and girls); 2) DSM-IV-TR (APA, 2000) diagnosis of bipolar spectrum disorder (BPSD: BP-I, BP-II, BP-NOS, subthreshold BP-NOS, cyclothymia) as determined by consensus conference, as described below; 3) full scale IQ>70; 4) child and at least one parent must be able to complete all assessments, which consist of structured interviews, clinical rating scales and self- report inventories; 5) child must be able to swallow multiple capsules (training in swallowing was offered, see Appendix B); 6) child must appear able to tolerate being off psychotropic medication(s) for a minimum of 11 weeks (three week washout + eight week trial) and the child and parent must agree to this; 7) child must be off psychotropic medication(s) for at least three weeks prior to starting treatment.

Participants had to not have the following criteria: 1) major medical disorders (e.g., diabetes, epilepsy, metabolic disorder) or other contraindication to the supplement; 2) current psychotropic medication within 3 weeks prior to beginning study treatment; 3) inability to communicate in English; 4) lack of access via phone contact; 5) intellectual disability or autism; 6) psychotic symptoms; 7) active suicidal concern (i.e., the child might endorse passive suicidal ideation, such as “the world would be a better place without me” or “I wish I were dead” but may not have serious active suicidal ideation, e.g., “I want to die”, a plan for suicide, or an attempt in the past month).

Participants were allowed to continue any psychosocial interventions that were on-going at the time of study enrollment. They were asked not to begin any other psychosocial intervention during the trial. Their usage of psychosocial interventions was monitored throughout the study using the Service Provider Grid described in detail below. They were not allowed to receive psychotropic pharmacologic intervention during the washout period (other than the medication from which they were still weaning), or the rest of the trial. Three participants

(30%) were taking psychotropic medications at the time of enrollment and completed the minimum three-week washout period of these medications. Seven participants (70%) were not taking psychotropic medications at study enrollment.

All participants were receiving psychological services throughout the study, though one participant switched providers at the beginning of study participation and did not begin working with his new provider until the end of the study. One participant

continued taking omega-3 fatty acids throughout the study and one continued taking omega-3 fatty acids plus melatonin. These non-psychotropic medications were allowed, but participants were asked not to make changes to these medications from the time they were enrolled in the study until completion. The participant who continued taking omega-3 fatty acids did not follow this request and began taking 10mg of Focalin between study Visits 5 and 6 through the remainder of the study because of increased inattention and impulsivity that was impairing his school functioning.

Participants were between the ages of 6 and 12 at study entry (M = 8.9, SD

= 2.02). Nine of the children were White (90%) and one was Hispanic (10%). A majority (60%) were male. Participants’ IQ, assessed by the two-subscale estimation from the Wechsler Abbreviated Scales of Intelligence (WASI), ranged from 86 to 117 (M = 98.7, SD = 9.53). Family income of the sample ranged from

$20,000 to over $100,000. Participants’ waist to hip ratio ranged from 0.71-0.86

(M = 0.82, SD = 0.05) and body mass index (BMI) ranged from 15.0-26.5 (M =

18.69, SD = 3.44). All participants in the study were diagnosed with mood dysregulation. One child was diagnosed with BP-I (10%), three children were diagnosed with BP-NOS (30%), and six were diagnosed with subthreshold BP-

NOS (60%). Of the six participants diagnosed with subthreshold BP-NOS, two met DSM-IV diagnostic criteria for major depressive disorder (20%) and one met diagnostic criteria for dysthymia (10%). All participants were diagnosed with a comorbid behavior disorder (i.e., ADHD, ODD, or conduct disorder [CD]) and six

participants (60%) were diagnosed with a comorbid anxiety disorder (i.e. specific phobia, social phobia, SAD, GAD, or OCD). One participant met diagnostic criteria for enuresis (10%). This study sample appears to be representative of the population of youth with bipolar spectrum disorders in that the majority of participants were male, those who were previously on psychopharmacologic regimens were taking multiple medications, and all children met criteria for comorbid diagnoses (Kowatch et al., 2009).

All participants were given EMP+ supplementation and remained on supplementation for an average of 46.4 days (SD = 23.29, median = 55). Seven participants completed the entire open-label trial (70%); three dropped out prior to completion (30%). The three drop-outs were the three participants who had difficulty swallowing capsules and tried mixing the supplement contents with food.

One participant dropped out after Visit 3 due to difficulties swallowing the supplement. Another participant dropped out after Visit 2 due to refusal to swallow the supplement. These children were given the option to mix the supplement into foods when they continued having difficulty swallowing capsules after completing the pill swallowing desensitization protocol (see Appendix B). However, these participants reported the taste was unpleasant and they no longer wished to participate. The third participant who dropped out did not have trouble eating the supplement mixed with food in the beginning of the study. However, he ended participation after Visit 6 when his parents reported increased refusal to take the supplement. He was estimated by his parents to have consumed only one dose of

EMP+ that week; they decided he needed more intensive (and compliant) treatment and put him on an antidepressant medication.

Measures

All measures used in this study are described below.

Demographic Form. A demographic form was used to collect the child’s sex, birth date, socio-economic status, contact information, and parent information at baseline (see Appendix C).

Medical History. As part of the initial assessment, a medical history was obtained that included current and past psychiatric and medical history, height, weight, body mass index (BMI), waist circumference, developmental history, past and current medications, family history, and a mental status exam (see Appendix

D).

Physical Exam. A physical exam was conducted by a certified pediatric nurse practitioner with authorization to do physical exams; the exam included focus on signs of thyroid or other endocrine disorder (see Appendix E).

Height, Weight, Waist/Hip Circumference and Body-Mass Index (BMI).

Height, weight, waist and hip circumference were measured at each visit in addition to vital signs, as recommended by the American Diabetes Association in conjunction with the American Psychiatric Association (2004). From this, the child’s BMI and waist-hip ratio were calculated per standard procedures.

The Children’s Interview for Psychiatric Syndromes-Child and Parent

Forms (ChIPS/P-ChIPS: Weller et al, 1999a; Weller et al, 1999b) are structured

psychiatric interviews designed to assess psychopathology according to DSM-IV criteria in clinical and epidemiological research with children and adolescents ages

6-18 years (Fristad et al, 1998b; Fristad et al, 1998d; Fristad et al, 1998e; Teare et al, 1998a; Teare et al, 1998b). The ChIPS and P-ChIPS assess twenty behavioral, anxiety, mood, and other syndromes as well as psychosocial stressors, including abuse, which the child might have experienced. Symptoms are assessed using a

“yes/no” question format. Within each diagnostic section cardinal questions are asked and a multiple “skip” procedure is used (Rooney, Fristad, Weller, and

Weller, 1999). If a child answers “no” to a certain number of questions, the rest of the questions can be skipped. Onset, offset and duration information is gathered for each disorder.

Reliability and validity in both inpatient and outpatient populations for both the ChIPS and P-ChIPS have been demonstrated with high test-retest reliability and moderate to high correlations with discharge diagnoses (Fristad et al, 1998b;

Fristad et al, 1998d; Fristad et al, 1998e; Teare et al, 1998a; Teare et al, 1998b).

Results from these five psychometric studies were combined to calculate sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) (Weller et al, 2000). ChIPS’ overall sensitivity, compared to clinician diagnoses, was .66 (range: .44-.79) and P-ChIPS was .83. Overall specificity for

ChIPS was .88 (range: .77 to .995) and .78 for P-ChIPS. PPV for ChIPS was .72 while NPV was .96. ChIPS was chosen as the structured interview of choice for this study because its psychometric properties mimic those of other structured

interviews while it offers several pragmatic advantages, including: shorter administration time; a more detailed training manual; greater ease of administration; and a concise response booklet that decreases storage requirements

(Fristad, 1999). Additionally, ChIPS has been suggested as a useful tool in studies of childhood bipolar disorder because of its potential for discriminating between

ADHD and mania (Nottelmann & Jensen, 1998). The ChIPS and P-ChIPS were administered at baseline to document the lifetime (LT) and current (CUR) presence/absence of psychiatric symptoms and syndromes.

Kiddie Schedule for Affective Disorders and Schizophrenia for School-Age

Children-Present Episode-Depression Rating Scale (KDRS). Depressive symptom severity was assessed using the KDRS. The KDRS is a 12-item semi-structured interview with depression symptoms rated on a 6-point scale from none to severe.

The KDRS has been shown to be a reliable measure of symptom severity

(Chambers et al., 1985; Ambrosini et al., 1989). The KDRS was administered at baseline, the first two weeks of treatment, and at each successive 2-week assessment throughout the 8-week study.

Kiddie Schedule for Affective Disorders and Schizophrenia for School-Age

Children-Present Episode-Mania Rating Scale (KMRS: Axelson et al., 2003) contains 21 items that assess the severity of manic symptoms in children and adolescents. The KMRS shows high internal consistency (α = 0.94), sensitivity

(0.87), and specificity (0.81) (Axelson et al., 2003). This scale was used in the

COBY grant, (MH 59929-01), “Improving the Assessment of Juvenile Psychiatric

Disorders” (R01 MH066647-01A1, P.I. Eric Youngstrom, Ph.D.), the “Pediatric

Bipolar Collaborative Mood Stabilizer Trial: (R01 MH60814-01A1, P.I. Robert

Kowatch, M.D.), and the LAMS grant, (R01 MH073801-01), The KMRS was administered at baseline, the first two weeks of treatment, and at each successive 2- week assessment throughout the 8-week study.

Wechsler Abbreviated Scales of Intelligence (WASI: The Psychological

Corp., 1999) is a brief standardized intelligence test that provides estimates of verbal and nonverbal cognitive ability. The WASI has several important features for this project: (a) its norms are recent; (b) it provides accurate estimates of ability by virtue of its development using item response theory; and (C) the Wechsler scales are the most widely used instruments in clinical practice and research, thereby maximizing the generalizability of findings from the present study.

Service Provider Grid (Goldberg-Arnold & Fristad, 1999) is a semistructured interview designed to record and track past and current experiences with treatment/service providers and educational services. The primary informant parent is asked to recall those who have been involved with their child’s care. The age at which a given treatment was initiated, length of the intervention, perceived effectiveness, how it was helpful, and the reason for termination or addition of the intervention is recorded onto a grid. As with the medication grid, perceived effectiveness is rated on a 1 to 5 scale with 1 being “not at all helpful” and 5 being

“very helpful”. Services are coded into the following categories: psychiatrist; psychologist; pediatrician/family physician; other (e.g., social worker, family

therapist); school services; school psychologist/counselor; cognitive/diagnostic testing; inpatient hospitalization; residential treatment; respite care; bibliotherapy; online support groups; crisis management (e.g., emergency department visit); and none. After generating the service provider grids during the initial interview, they will be reviewed at each time period to assess any changes in service provision.

Initial psychometric evaluation of this instrument suggests the Service Provider

Grid is a reliable way to gather information about mental health and educational services (the correlation between information gathered with the Service Provider

Grid and information found in the patients’ medical charts was .92, Davidson,

Mendenhall & Fristad, in preparation).

Children's Global Assessment Scale (CGAS: Shaffer et al, 1983) is a clinical rating scale used to document children's overall functional capacity at home, school, and with peers. Scores range from 1 (indicating a severely impaired child) to 100 (indicating a child with superior functioning). Reliability and validity are adequate. The CGAS was completed at each visit to provide a severity of impairment index.

Clinical Global Impressions Severity & Improvement Scales (CGI-S, CGI-I:

NIMH, 1985). The CGI (NIMH, 1985) severity and improvement scales were used to assess severity and improvements of symptomatology at each visit throughout the duration of the study. CGI Severity items are rated from 1 (normal, not ill) to 7

(very, severely ill). Symptom improvement items on the CGI are rated from 1

(very much improved) to 7 (very much worse).

Neurological Evaluation. Researchers observed degree of alertness and ability to converse to look for magnesium sedation.

Procedures

Assessments were conducted at the Ohio State University Childhood Mood

Disorders Research Program, physical exams were conducted at the Ohio State

University Nisonger Center, and blood draws were completed at the Ohio State

University Center for Clinical and Translation Science (CCTS) Clinical Research

Center (CRC). Participants were recruited first using letters and flyers sent to a previously developed referral network, which included local psychiatrists, psychologists, pediatricians, family physicians, school psychologists, and other mental health professionals. Recruitment flyers were also distributed to families who potentially fit study criteria; these families were located from the LAMS (R01

MH073801, Longitudinal Assessment of Manic Symptoms) study, for which OSU is a data collection site. Third, Dr. Fristad maintains a lengthy list of families who initiate contact with her for clinical services and agree to be called if future studies become available; these families were contacted regarding the current investigation.

(These are families who call for clinical services with Dr. Fristad and/or for studies closed to enrollment. They are provided with clinical referrals and the option to leave contact information in case future clinical trials materialize.) Lastly, an announcement was posted regarding study information on the Child and

Adolescent Bipolar Foundation (CABF) website (www.bpkids.org). Five study participants were recruited through the referrals of community mental health

professionals and physicians. One child was recruited from Dr. Fristad’s research study waitlist. Two participants were recruited after contacting Dr. Fristad for general information related to treatment of childhood mood dysregulation, and two participants were recruited by contacting the study coordinator after seeing the study announcement on the CABF website.

Upon being contacted by the parent/caregiver, I conducted a pre-screening interview by phone to determine potential study eligibility/interest. The phone prescreening procedure was designed to address the following questions: Does the child have a high likelihood of meeting current DSM-IV diagnostic criteria for a bipolar spectrum disorder? Does the child have a diagnosed metabolic disorder?

Does the child currently experience psychotic or active suicidal symptoms? (If either psychosis or suicidality was endorsed, the family was asked if they wish to receive a clinical referral as they would not be study eligible.) Does the child live with one or more parents/caregivers? Is it likely the child and one or more parent(s)/caregiver(s) will be interested in participating in the research protocol

(after hearing a brief description—this included awareness of the three week washout of all psychotropic medication and the eight subsequent weeks of no psychotropic medication and no starting of psychological intervention)? If this prescreening interview indicated possible interest in and eligibility to participate in the study, the child and parent(s)/caregiver(s) were invited to the screening appointment, at which time informed assent, parental permission, and consent were obtained and the screening assessment was completed. Parents of children not

eligible for study participation were offered referral information to seek other mental health services as appropriate.

The study design included seven assessments with each parent-child pair over eight weeks. Visit 1 (screen visit) consisted of screening and assessment procedures, including a brief physical exam, and lasted approximately 3-4 hours.

Visit 2 included assessment procedures, vital sign collection, dietary monitoring and a blood draw. This visit lasted about 2-3 hours. Visits 3-6 consisted of assessment procedures and vital sign collection. These visits were approximately 1 hour. The seventh and final visit involved assessment procedures, including final blood draws, physical exam, and dietary monitoring and lasted approximately 2-3 hours. Children and their parent were paid upon completion of each assessment.

Parents received $15 per visit and a parking voucher to partially defray transportation costs. Children chose from an assortment of “prizes” with a value of

$5 or less at the end of each visit. Total participant reimbursement was $105 for parents and prizes equivalent to <$35 for children summing to $140 per family.

I completed standard Ohio State University IRB ethics training and interviewer training in preparation for this research. The training program included extensive instructions on child psychiatric syndromes; how to administer questionnaires; information regarding clinical and ethical issues involved in interviewing; practicing, observing and rating interviews; performing mock interviews and receiving feedback from experienced study personnel; and undergoing reliability checks. I obtained, consent, assent, and parental permission

and conducted initial evaluations with primary parental informants and children.

Then, I conduct all subsequent assessments. Two of the investigators on the research team, a physician and a research pharmacist, monitored dosages and distribution of all supplement capsules. One co-investigator, a nutritionist, reviewed nutritional health information for general safety purposes.

At Visit 1, children and their parent(s)/caregiver(s) provided written informed assent/consent/parental permission and authorization to use personal health information in research (HIPAA). Then they completed the screening assessment. Information obtained was reviewed to verify study eligibility. The children’s pill swallowing ability was determined at this time. Children who could swallow pills the size used in the study at screening required no further intervention. Children unable to swallow pills proceeded with the desensitization protocol (see Appendix B). Up to three weekly visits could be scheduled, if needed, for clinical supervision of the desensitization, and/or parents could work with their children at home to complete the desensitization. Three children (30%) had difficulty swallowing capsules after completing the desensitization protocol and were given the option to mix the supplement into their food. Children currently taking psychotropic medications needed to cease their medications after this visit using the standard tapering strategy recommended for the medication they were prescribed. Three children (30%) completed this tapering process in order to participate in the study. This tapering process was supervised by each participant’s prescribing physician. Children tapering off psychotropic medication were

scheduled for their baseline treatment visit-Visit 2, three weeks after the taper was complete. Children not currently taking psychotropic medication who were able to swallow study-sized pills were scheduled for their baseline visit within a week.

Participants took the daily dosage of EMP+ recommended by the manufacturer. They began treatment by taking 1 capsule three times a day for 2 days. They increased their dosage to 2 capsules three times a day for two more days, then 3 capsules three times a day for two more days, then 4 capsules three times a day (the target dose). Participants returned to the clinic for re-evaluation after an average of 9.78 days. If side-effects were minimal to non-existent and more treatment response was desired, they increased their dosage to 5 capsules three times per day. If side-effects were moderate or greater, the dose was not increased. Dosage could be reduced at any time (or titration slowed) by phone for side effects (see Side Effects Form in Appendix F). No participants experienced moderate or severe side-effects. The maximum dose for children is 5 capsules three times a day for a total of 15 capsules each day. Not including the two participants who dropped out of the study prior to reaching a full dose, four took 12 capsules a day and four took the maximum 15 capsules a day. For specific amounts of each ingredient included in the EMP+ capsule, see Appendix A.

Safety data appear in the Investigator Brochure, in Appendix G. The EMP+ capsules were donated by Truehope Nutritional Support Ltd. (Raymond, Alberta,

Canada).

Four participants reported mild nausea, one of whom reported vomiting once, when taking the supplement without food. One child reported mild difficulty falling asleep at one visit. Another child reported taking naps during the day and mild difficulty falling asleep twice. This child also reported slight increased appetite at one visit. A third child reported mild difficulty falling asleep and waking in the morning at one visit, but his mother reportedly felt this was due to the time change that occurred during this week for Daylight Savings Time. One child wet the bed one night during study participation, but her mother reportedly felt this was unrelated to supplementation. This mother did not report a history of enuresis for this child.

Participant compliance was checked by standard pill counts of returned unused dosage forms. Data was recorded on a Medication Accountability Form

(Appendix H). Compliance below 86% resulted in re-instruction, troubleshooting possible barriers to compliance, and emphasis on the importance of taking the capsules regularly. Compliance below 70% without good reason (such as intercurrent gastroenteritis) resulted in further assistance with planning where to keep the doses, when to take them, and how to remember. Compliance/palatability is one of the outcomes of this pilot study.

The assessment team (Dr. Fristad and I) met within 48 hours of screening assessments. During these meetings, study eligibility decisions were finalized and consensus diagnoses were determined. Consensus conference diagnoses utilized all information obtained during the screening evaluation. Data obtained from ChIPS

and/or P-ChIPS used an "either" strategy (i.e., counting a symptom as positive if either the child or parent endorses it) unless there were clear and compelling reasons to disregard information obtained from an informant (e.g., if the child refused to seriously answer questions, responding "yes" to everything asked of him/her; if the parent/caregiver provided clearly exaggerated responses to all questions asked).

I completed all interviews except one, which the post-doctoral study coordinator in the Childhood Mood Disorders Research Program completed. To maintain reliability, 10% of all interviews were videotaped. Graduate research associates rated a series of these videotaped interviews and correlations were calculated between my ratings and the ratings of the other five graduate researchers to examine reliability of mood ratings during assessments. Two of the graduate researchers rated two of the seven different videos and the other three graduate researchers rated one of the seven different videos. Intraclass correlations were calculated using ratings on each question in the KDRS and KMRS that correspond to DSM-IV-TR criteria for depression and mania. This included twelve depression items from the KDRS and 13 mania items from the KMRS. Study reliability for K-

SADS depression and mania ratings was 0.799 (p<0.001).

Blood draws were completed by Center for Clinical and Translational

Research (CCTR) nurses trained in pediatric blood draws who used a topical analgesic to numb the blood draw site. This was done to complete the micronutrient assays, directed by Mark Failla, PhD. Blood was drawn at the

baseline visit (Visit 2) and at eight weeks (Visit 7). Members of Dr. Failla’s laboratory examined the effect of supplementation on the levels of several micronutrients for which some literature supports beneficial influences on neurological activities, as well as serving as components of the antioxidant network. These included the minerals iron, copper, magnesium and zinc and vitamins A, B6, D, E and folate. C-reactive protein (CRP) levels were examined by enzyme-linked immunosorbent assay (ELISA) kit as a measure of inflammation and carotenoids (lutein [LUT], zeaxanthin [ZEA], beta cryptoxanthin [B-Cryp], alpha-carotene [AC], beta-carotene [BC], lycopene [LYC]) were examined as part of the antioxidant network. Iron status was determined in two ways: 1) grossly from the hematological parameters (hemoglobin concentration, hematocrit, packed cell volume, etc.) in the standard screening performed by the hospital laboratory; and 2) biochemically from assay of plasma transferrin receptor (sTfR) and ferritin by enzyme immunoassay (Ramco Lab., Inc.). sTfR is a sensitive indicator of whole body iron status that is not subject to conditions that can affect classic biochemical indices such as serum iron, transferring saturation and ferritin (Cook,

Skikne, & Baynes, 1993; World Health Organization). The sTfR/ferritin ratio is particularly useful for determining iron deficiency in absence of anemia in children

(Vazquez-Lopez, Carracedo, Lendinez, Munoz, & Lopez, 2006). Serum ceruloplasmin activity was measured as a marker for plasma copper using PPD as substrate since the assay requires less sample than atomic absorption spectroscopy

(AAS; Rice, 1962). Magnesium level was determined using an inductive coupled

plasma-mass spectrometry (ICP-MS) technique (Shaole, Feng, & Wittmeier, 1997).

Plasma zinc was measured by atomic absorption spectrophotometry. Plasma pyridoxal -5-phosphate, the active isomer of vitamin B6, was determined using a commercially available radioassay kit (Buhlmann Laboratories AG, Switzerland).

Vitamin A, vitamin D, carotenoids, and plasma α- and γ-tocopherols (vitamin E) were determined by high-performance liquid chromatography (HPLC) using a method requiring only 0.10 mL plasma (Podda, Weber, Traber, & Packer, 1996).

Diet was monitored pre- and post-supplementation (Visits 2 and 7) with the standardized USDA Automated Multiple Pass Method (Conway et al, 2003) in addition to Food Frequency Questionnaires (FFQ). A research dietician well practiced in using a 5-step multiple-pass method interviewed children and their parents about the child’s food/beverage consumption for the previous 24-hour period (Wilson & Lewis, 2004; Weber et al., 2004). The 24 Hour Recall or Typical

Diet Form (Appendix I) supplemented by samples of plastic and paper food models was used to increase precision in the data gathered. This method, which uses multiple passes to elicit information about an individual’s food and beverage consumption, has been found to reduce underreporting of intake due to repeated opportunities to recall foods and beverages (Conway et al., 2003).

Dietary records were entered into the Nutrition Data System for Research

(NDS-R) software (Nutrition Data System for Research, 2005). The Nutrition Data

System for Research is a nutrient analysis program maintained by the Nutrition

Coordinating Center (NCC) at the University of Minnesota’s School of Public

Health. The NCC maintains a database containing 139 nutrients, nutrient ratios, and food components. The NDS-R software provides data for 18,000 foods and

8,000 brand names of foods. The NCC database is derived from the USDA database, scientific literature, food manufacturers and foreign food composition tables. Individual and group data and comparisons to national recommendations can be exported into spreadsheet or database analysis programs for further statistical comparisons. Food groupings can be reviewed and scored according to the method developed by Kant, Schatzkin, & Ziegler (1995) using the data generated by NDS-R to determine if diet pattern has changed over the study.

Likewise, the diversity score can be determined by the number of food groups consumed daily (Kant, Schatzkin, Harris, Ziegler & Block, 1993) and compared to food groups recommended by the Dietary Guidelines for Americans (Nutrition

Data System for Research 2005; Cullen, et al., 2004; Weber et al., 2001). Dietary records were kept to account for vitamin and mineral intake from daily foods in addition to the supplement.

Data Analysis

Feasibility of Sample Recruitment

Length of the sample recruitment period was used to determine recruitment feasibility. Length was determined by calculating the number of months from the initial distribution of recruitment materials until the final eligible participant who enrolled in the study attended his first appointment. Sample recruitment was hypothesized to be completed in five months.

Feasibility of Supplementation Compliance

Compliance was calculated by dividing the number of missed doses by the total number of doses since the last visit, subtracting this number from one, and then multiplying by 100 to create a compliance percentage. It was hypothesized that children could swallow the supplement with >80% compliance.

Feasibility of Increasing Micronutrient Levels

A series of twelve nonparametric, one-tailed Fisher’s Randomization Tests were completed to analyze levels of various nutrients pre- and post- supplementation (Visit 2 and Visit 7). These analyses included the seven participants who completed the entire study. Post-supplementation blood analyses were not conducted on the three participants who dropped out prior to study completion. Originally, blood analyses were to be conducted for the following nutrients: iron, copper, magnesium, zinc, and vitamins B1, B6, B12, E and folate.

However, due to assay kit availability, analyses could not be completed for vitamins B1 and B12. Analyses were actually conducted for iron (ferritin and transferrin receptor [TfR]), copper (ceruloplasmin), magnesium, zinc, C-reactive protein (CRP), and vitamins A (retinol), B6 (pyridoxal phosphate [PLP]), D, E

(gamma tocopherol [g-TC] and alpha tocopherol [a-TC]), carotenoids (lutein

[LUT], zeaxanthin [ZEA], beta cryptoxanthin [B-Cryp], alpha-carotene [AC], beta- carotene [BC], lycopene [LYC]), and folate. Blood analysis data was graphed for each participant to illustrate nutrient response to treatment on an individual basis.

This is important in nutritional research, because what is statistically significant

may not be biologically relevant, and individuals tend to have unique biological responses to changes in dietary intake, which may not be captured by analyzing the entire sample as a whole. It was hypothesized that all nutrient levels would be higher post-supplementation compared to pre-supplementation levels.

Trends in Treatment Response

Descriptive data were obtained and trends in treatment response were summarized. KDRS and KMRS scores were graphed over time to inspect the time course of effect for individual participants and for group means. It was expected that differences in KDRS and KMRS scores would exist between visits. Two

Friedman Analysis of Variance tests were completed to identify any differences in

KDRS and KMRS scores across visits for the seven participants who completed the study. It was hypothesized that depression (KDRS) and mania (KMRS) scores would decrease over time from pre- to post-supplementation. Two one-tailed

Fisher’s Randomization Tests were completed to further examine trends in treatment response for depression and mania scores pre- and post-supplementation in the seven study completers (Visit 2 and Visit 7).

Missing Data

Three participants could not be included in blood analyses and two of these participants could not be included in nonparametric analyses of mood symptoms due to missing data after they dropped out of the study. Additionally, there was one case pre-supplementation and four cases post-supplementation where

carotenoid nutrient levels of study completers did not register on blood assay equipment and therefore could not be measured during blood analyses.

Chapter 3: Results

Feasibility of Sample Recruitment

Sample recruitment was hypothesized to be completed in five months.

Unfortunately, IRB approval was not obtained to allow recruitment during the summer months, which anecdotally appeared to affect time to recruit, as numerous families who inquired about the study were willing for their child to wean off current medications during the summer months, but not during the school year.

Actual recruitment took 6.5 months. Recruitment efforts began at the end of

August, 2008, and the first participant was enrolled at the beginning of September.

Six more participants were enrolled by the end of the year. Recruitment slowed during the holiday season with only one participant enrolled in January, 2009 and the final two participants enrolled in March after a recruitment notice was posted on the CABF website.

Five study participants were recruited through the referrals of community mental health professionals and physicians. One child was recruited from Dr.

Fristad’s research study waitlist. Two participants were recruited after contacting

Dr. Fristad for general information related to treatment of childhood mood dysregulation, and two participants were recruited by contacting the study coordinator after seeing the study announcement on the CABF website.

Feasibility of Supplementation Compliance

It was hypothesized that children could swallow the supplement with >80% compliance. Of those who completed the study, average compliance was 96.7%

(SD = 5.1%). Average compliance for the entire sample was 91.2% (SD = 22.1%).

See Table 2 for medication compliance of each participant across study visits.

Participant Visit 3 Visit 4 Visit 5 Visit 6 Visit 7 Ss Total M (SD) 1 0.95 1 0.77 0.95 1 0.934 (0.095) 2 1 0.5 ------0.750 (0.354) 3 1 1 0.95 0.95 0.97 0.974 (0.025) 4 0 ------0.000 (---) 5 1 0.95 0.98 0.96 1 0.978 (0.023) 6 0.94 1 1 0.92 0.9 0.950 (0.046) 7 1 1 1 1 1 1.000 (0.000) 8 0.95 1 0.98 0.95 --- 0.776 (0.434) 9 0.87 0.98 0.86 1 0.95 0.930 (0.064) 10 1 1 1 1 1 1.000 (0.000) Visit Total 0.871 0.937 0.943 0.966 0.853 0.912 M (SD) (0.309) (0.165) (0.084) (0.030) (0.346) (0.221)

Table 2. Participant medication compliance over time.

Feasibility of Increasing Micronutrient Levels

It was hypothesized that all nutrient levels would be higher at post supplementation compared to pre-supplementation levels. See Table 3 for pre- and post-supplementation levels of examined nutrients and CRP levels in the seven study completers with normal range comparisons. Blood levels of all nutrients were within normal ranges except ceruloplasmin levels. It is unclear why pre- and

post-supplementation levels of ceruloplasmin are below the given normal range.

Perhaps this is a result of lacking a normal range for ceruloplasmin specific to children or there may have been problems with the analysis of this nutrient.

Nutrient Normal Range Average Pre- Average Post- EMP+ Level EMP+ Level M (SD) M (SD) Vit A: Retinol >0.70 μmol/L 3.25 (1.16) 4.96 (1.58) Vit B6:PLP 20.0-120.0 nmol/L 54.28 (16.61) 104.03 (41.24) Carotenoids ---- 0.70 (0.23) 0.64 (0.22) Copper-Ceruloplasmin 100.0-180.0 μg/mL 65.67 (7.94) 66.10 (13.69) Vit D > 20.0 ng/L 125.49 (24.07) 154.94 (18.74) Vit E: a-TC 6-12 μg/mL 6.57 (2.07) 10.56 (3.00) Vit E: g-TC ---- 0.22 (0.20) 0.20 (0.27) Ferritin 20.0-400.0 ng/mL 138.29 (104.31) 90.5 (69.97) Folate 3.8-23.2 μg/mL 3.90 (1.72) 5.88 (0.75) Mg 15.0-30.0 μg/mL 17.31 (1.02) 17.72 (0.49) TfR 2.9-8.3 μg/mL 3.86 (0.82) 3.56 (0.46) Zinc >0.8 μg/mL 1.73 (0.42) 1.81 (0.47) CRP low: <1.0 mg/L 9.88 (21.54) 8.43 (17.42) medium: 1.0-3.0 mg/L high: >3.0 mg/L

Table 3. Mean nutrient blood levels pre- and post-EMP+ supplementation with normal range comparisons. Note: Vit = vitamin; EMP+ = EMPowerPlus; PLP = pyridoxal phosphate; a-TC = alpha tocopherol; g-TC = gamma tocopherol; CRP = C-reactive protein; Mg = magnesium; TfR = transferrin receptor; μmol/L = micromoles per liter; nmol/L = nanomoles per liter; μg/mL = microgram per milliliter; mg/L = milligram per liter; ng/mL = nanogram per liter.

Results of twelve nonparametric, one-tailed Fisher’s Randomization Tests show five of the nutrients tested increased significantly in the sample from pre- to post-supplementation: vitamin A-retinol Di(N=128)=-515.45; vitamin B6

Di(N=128)=-348.24; vitamin D Di(N=128)=-206.18; vitamin E-α-TC Di(N=128)=-

27.93; and folate Di(N=128)=-13.85, p<0.05. Blood levels of carotenoids, copper, vitamin E-γ-TC, iron (ferritin and TfR), magnesium, and zinc did not increase significantly from pre- to post-supplementation. Mean nutrient assay results for the seven study completers were split into two figures based on range of measurement for each nutrient (see Figures 1 and 2).

180

160

140

120

B6 100 copper D ferritin 80 Blood Level Mg

0 Pre Post Timepoint

Figure 1. Mean nutrient levels pre- and post- supplementation for vitamin B6, copper, vitamin D, ferritin, and magnesium.

A carotenoids E-a-TC 6 E-g-TC folate

Blood Level Blood TfR zinc

0 Pre Post Timepoint

While statistical analyses needed to be conducted with these sample nutrients to determine feasibility, the results must be interpreted with caution.

These data reflect changes in nutrient blood levels over a short period of time from only two time points. These results are likely more a reflection of dietary intake than actual chemical changes affecting brain and central nervous system functioning. In addition, biological relevance should be examined on an individual basis instead of by group means so that individual trends are not lost. Therefore, blood analysis data was also graphed for each participant to illustrate individualized

nutrient response to treatment since individuals tend to have unique biological responses to dietary changes, which may not be captured by analyzing the sample as a whole. See Figures 9-22 in Appendix J for blood analysis results separated by individual participants.

Trends in Treatment Response

Descriptive data were obtained and trends in treatment response were summarized. Each outcome measure was graphed over time to inspect the time course of effect for individual participants (see Figures 3 and 6 below and Figures

23-32 in Appendix K) and for group means of study completers (see Figures 4 and

7 below). Results of intent to treat analyses were also graphed with the last observation carried forward for study drop outs (see Figures 5 and 8). It was hypothesized that differences in KDRS and KMRS scores would exist between visits. Results of two Friedman Analysis of Variance tests indicated significant differences in KDRS and KMRS scores across visits for the seven participants who completed the study, KDRS Χ2(6, N = 7) = 19.02, p = 0.004; KMRS Χ2(6, N = 7) =

20.26, p = 0.002.

It was hypothesized that depression (KDRS) and mania (KMRS) scores would decrease over time from pre to post-supplementation. Two, one-tailed

Fisher’s Randomization Tests were completed to examine trends in KDRS and

KMRS scores over the course of treatment. Results show a significant decreasing trend in depression and mania scores over the course of the study for the seven study completers, KDRS Di(N = 128) = 44, p<0.05; KMRS Di(N = 128) = 79,

p<0.05. Results of intent to treat Fisher Randomization Tests including all study participants show a decreasing trend in depression scores approaching significance and a statistically significant decreasing trend in mania scores over the course of treatment, KDRS Di(N = 256) = 37, p<0.06; KMRS Di(N = 512) = 81, p<0.01.

P 1 20 P 2 P 3 P 4

15 P 5 P 6

KDRS Score KDRS P 7 P 8 10 P 9 P 10

0 1234567 Timepoint

Figure 3. Depression ratings (KDRS) for all participants over time. Note: KDRS = Depression section of the Kiddie Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present Episode-Depression Rating Scale; P=participant.

6 Mean KDRS Score Mean

0 visit 1 visit 2 visit 3 visit 4 visit 5 visit 6 visit 7 Timepoint

Figure 4. Mean depression ratings (KDRS) for study completers over time. Note: KDRS = Depression section of the Kiddie Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present Episode-Depression Rating Scale.

6 Mean ITTKDRS Score

0 visit 1 visit 2 visit 3 visit 4 visit 5 visit 6 visit 7 Timepoint

Figure 5. Mean intent to treat depression ratings (KDRS) over time. Note: KDRS = Depression section of the Kiddie Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present Episode-Depression Rating Scale; ITT = Intent to Treat.

30 P 1 P 2 25 P 3 P 4

20 P 5 P 6

KMRS Scores KMRS P 7 15 P 8 P 9 P 10 10

0 1234567 Timepoint

Figure 6. Mania ratings (KMRS) for all participants over time. Note: KMRS = Mania section of the Kiddie Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present Episode-Mania Rating Scale; P = participant

10 Mean KMRS Score KMRS Mean

0 visit 1 visit 2 visit 3 visit 4 visit 5 visit 6 visit 7 Timepoint

Figure 7. Mean mania ratings (KMRS) for study completers over time. Note: KMRS = Mania section of the Kiddie Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present Episode-Mania Rating Scale.

8 Mean ITT KMRS Score

0 visit 1 visit 2 visit 3 visit 4 visit 5 visit 6 visit 7 Timepoint

Figure 8. Mean intent to treat mania ratings (KMRS) over time. Note: KMRS = Mania section of the Kiddie Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present Episode-Mania Rating Scale; ITT = intent to treat.

Chapter 4: Discussion

Summary

Childhood mood dysregulation represents a group of severe, chronic, and highly comorbid mental health problems with a poor prognosis and deleterious affects on family, school, and peer functioning. Pharmacological intervention is considered the first-line treatment for children with bipolar spectrum disorders, and recent reviews of the research suggest mood stabilizers or atypical antipsychotics should be the first psychotropics considered for children with BP-I (Kowatch, et al.,

2009). Unfortunately, research on other psychotropic medications and varying presentations of pediatric bipolar disorder (BP-II, BP-NOS, cyclothymia) and childhood mood dysregulation is limited, and those agents found efficacious are associated with risk for adverse events (Kowatch et al. 2005). Additionally, it may take an average of nine months to two years until an efficacious drug combination is found to stabilize mood in youth, and relapse rates are high (Kowatch et al,

2009).

Novel treatments that are safe and effective are sorely needed. Previous nutritional studies suggest more rigorous evaluation of the multinutrient supplement, EMPowerplus (EMP+) is warranted in children with mood dysregulation. This open-label feasibility trial provides important methodological

information and preliminary data for a future double-blind, placebo-controlled study of EMP+ in children aged 6 to 12 with mood dysregulation.

Feasibility of Sample Recruitment

While study recruitment took 1.5 months longer than hypothesized, it appears as though all methods of recruitment were useful (i.e. flyers and letters to community mental health professionals and physicians, maintaining a waitlist for people interested in ongoing research, referring possible participants who contact the office for general information, posting on the CABF website), and the sample was still recruited in a reasonable amount of time. Several factors must be considered when adjusting study methodology for a randomized, placebo- controlled trial of this supplement. First, the Ohio State University Childhood

Mood Disorders Research Program is a well-established research center for children with mood dysregulation and is located in the Ohio State University

Medical Center, which is also well known for research studies. Additionally, Dr.

Fristad has developed a well-respected history of work in the field of childhood mood disorders. These variables may have made recruitment easier at this site compared to other locations that may wish to conduct such a study but do not have a similar established reputation or referral network.

Another factor to consider regarding recruitment feasibility is the requirement that participants taper off any and all psychotropic medications. It became apparent that finding children with notable mood dysregulation symptoms but without any psychotropic medications was a difficult task at times. There were

multiple potential participants who expressed interest in participating in this study but did not want to taper their children off of certain medications. Many of these medications were to treat symptoms of ADHD. There were also many families who were willing to taper children off of medication during the summer but did not want to remove medications during the school year for fear of negatively impacting academic and classroom performance. Participants likely could be recruited at a faster rate over the summer when school is not in session. In fact, some families may potentially take medication breaks from stimulants during summer months anyway, making the tapering process much easier. Participant recruitment may also be easier if stimulant medications, which should not directly affect mood symptoms, were allowed during the study. This potential change to study methodology might add an element of increased potential risk and would require more monitoring and coordination between the psychiatrist and pharmacist working on the study as well as with the participants’ physicians.

There were anecdotal reports from one study participant post-study that the child continued taking EMP+ with a concomitant stimulant to treat residual ADHD symptoms and was functioning very well. We were also able to briefly monitor the safety and response of one study participant who began taking a stimulant medication in addition to EMP+ after Visit 5. This participant tolerated the combination of EMP+ and stimulant very well and showed improvement in ADHD symptoms while maintaining improvements in mood. This suggests removing stimulant medication from the exclusion criteria for study participation may be a

reasonable way to increase feasibility of recruitment for a large-scale placebo- controlled trial of EMP+ in the future.

Feasibility of Supplementation Compliance

Of the three participants who dropped out of the study prior to completion, two terminated participation specifically due to compliance and tolerability issues.

Tolerability and compliance also appears to play a role in the third drop out as well.

However, of the seven participants who completed the study, all maintained high compliance rates. All study completers had compliance rates of 93% or higher, and two participants maintained 100% compliance throughout the study. These results suggest that children who have difficulty swallowing the EMP+ supplement even after completing the swallowing desensitization protocol are likely not good candidates for EMP+ treatment. When these children attempted mixing the supplement powder from the EMP+ capsules with food instead of swallowing the capsule whole, they continued to have palatability issues due to the taste of the supplement powder. This feasibility study suggests future researchers should adhere to the original inclusion criterion that children must be able to swallow supplement capsules in order to participate. Of the children who fit this criterion, all displayed excellent adherence to dosage instructions, suggesting treatment adherence in future studies of EMP+ should be high.

Feasibility of Increasing Micronutrient Levels

Many micronutrients are essential for neurological development and integrity (Shils, Shike, Ross, Caballero, & Cousins, 2006). These vitamins and

minerals are active participants in biochemical processes associated with synthesis of neurotransmitters, cognition, and protection against oxidative stress. Although frank micronutrient deficiencies are relatively infrequent in developed countries, the possibility that the anecdotal benefits of supplementation with EMP+ offsets one or more defects in the absorption, cellular metabolism or whole body retention of selective vitamins and minerals merits consideration.

It is important to acknowledge that nutritional status is very difficult to assess, and what is considered an optimal level of a nutrient for one individual may be a completely different amount from what is considered optimal in another individual. Determination of nutritional status is very individualized and requires clinical, chemical, dietary, and anthropometric information (Kaplan et al., 2007).

Even when looking at all of these aspects of human nutrition, it may be difficult to determine the actual amount of various nutrients affecting brain functioning. The analyses used in this study examined serum and plasma levels of various nutrients from participant blood samples. These nutrient levels are likely better reflections of dietary intake as opposed to the status of each nutrient actively working in the brain. However, the information collected shows that the sample appeared to respond to supplementation for vitamins A-retinol, B6, D, E (α-TC), and folate.

Unfortunately, it is difficult to draw any conclusions from this preliminary data other than participants appeared to respond to supplementation for certain nutrients, and responses varied in unique biochemical ways among individual participants. Regarding nutrients that did not appear to respond to

supplementation, perhaps these nutrients were at homeostatic levels, and therefore they did not change. The human body is very skilled at maintaining homeostasis by storing excess nutrients in tissue until they are needed and maintaining a steady level of each nutrient in balance with other nutrients in the blood stream.

In future studies of EMP+, it will be important to continue looking at biomarkers to further the understanding of how various nutrients may be related to central nervous system functioning and mood. However, it is important to remember that statistical significance does not necessarily equal biological relevance. Determining biological relevance requires multiple analyses of nutrient levels in an individual over time to determine biochemical changes unique to the individual. This feasibility pilot shows children are able to tolerate fasting and blood draws with the aid of nurses trained in pediatric blood draws and the use of a topical analgesic to numb the blood draw site. It is recommended that future studies increase the number of blood draws throughout the study to allow for individualized analyses which could not be completed with the limited pre- and post-supplementation data in this trial. While increasing the number of blood draws may increase potential participant discomfort, children in the current study tolerated this discomfort well and the potential gains in individualized nutritional analysis appear to outweigh this slight discomfort.

Trends in Treatment Response

Exploratory analyses of mood symptoms throughout this pilot trial suggest significant decreases in depression and mania symptoms over the course of EMP+

supplementation. However, the results must be interpreted with caution for many reasons. The study design was open-label, so there is no way to control for placebo effects. There are also difficulties in studying mood symptoms over the relatively short time period of this pilot trial due to the natural waxing and waning of symptoms inherent to mood disorders.

In fact, a recent review article on randomized placebo-controlled trials for acute bipolar mania in adults showed that although participants responded better to psychotropic interventions compared to placebo, there were also many placebo- responders in these studies (Smith, Cornelius, Warnock, Tacchi, and Taylor, 2007).

Smith and colleagues (2007) reported ratios of the number of events when participants showed at least 50% improvement in YMRS scores relative to the number of participants analyzed who took active psychotropic medication and those who took placebo. They found placebo response ranges from 18.7% to

33.6% in studies of haloperidol, olanzapine, quetiapine, risperidone, aripiprazole, lithium, valproate semisodium, and carbamazepine (Smith et al., 2007).

Considering such placebo-response rates, it is possible that some participants in this study showed decreases in mood symptoms due to placebo effects, not actually in response to EMP+ supplementation.

There is clearly no way to determine if the participants in the current study displayed a placebo response or if their mood symptoms did in fact improve as a result of EMP+ supplementation. However, the significant decrease in depression

and mania scores in this trial suggests that future randomized placebo-controlled trials are warranted to answer such questions.

Limitations and Future Directions

The current investigation provides a solid foundation from which to build regarding continued examination of EMP+ supplementation as a treatment for childhood mood dysregulation. However, there were several limitations in this feasibility pilot trial; they are discussed below. These limitations should be addressed in future randomized, placebo-controlled trials of EMP+ in children with mood dysregulation.

Open-Label Design

As mentioned above, the open-label design of this feasibility trial greatly limits what can be inferred from results. Exploratory mood symptom results must be interpreted with extreme caution since this study was un-blinded and did not include a placebo control. Based on a review of pharmacologic randomized placebo-controlled treatment studies in adult bipolar disorder, it is possible that as many as a third of participants would appear to respond due to placebo-effects if a placebo-controlled trial of EMP+ is conducted. The cyclical nature of mood disorders may contribute to this concern, as it is unclear if mood symptoms responded to EMP+ supplementation or if participants were experiencing a natural decrease in symptomotology. However, the purpose of this study was not to analyze mood symptoms in response to EMP+ treatment. Rather, the purpose of this study was to determine feasibility of conducting a randomized placebo-

controlled trial using EMP+. Overall, results of this trial suggest such a study is warranted, and the exploratory look at mood symptoms provide further support to examine this supplement in a more scientifically rigorous methodological design.

Length of Study

Although similar in length to many medication trials, this pilot feasibility trial is relatively brief. One participant’s mother anecdotally commented at her last visit that she felt like she “just got the hang of taking EMP+” and the study was already ending. Future studies would greatly benefit by extending the length of the study to at least 16 weeks and add monthly follow-up visits for a year. A cross- over design could be used for placebo participants to reduce the potential harm of being on no medications for too long. This extension would allow for a more detailed look at changes in mood over time and hopefully reduce the likelihood of mistaking a natural decrease in symptomotology for true treatment response. An increase in study length would also allow for more blood draws to increase the number of nutrient data collection points, thereby allowing for meaningful analyses of individual biochemical responses to EMP+ treatment. Lastly, the addition of monthly follow-up visits for a year would provide some knowledge of the effects of

EMP+ over an extended period of time.

Sample Size

Using data from only ten participants limited the types of analyses and interpretation of data in this study. While the sample in this trial provided helpful information on feasibility of recruitment, medication compliance, and pre- and

post-supplementation nutrient levels, future studies will need to conduct power analyses to determine the sample size necessary to achieve adequate power in a larger, placebo-controlled clinical trial.

Mood Rating Methodology

As previously noted, I completed all interviews and mood ratings in this study except for one interview completed by the Ohio State University Childhood

Mood Disorders Research Program study coordinator. Using mostly one rater in an open-label study design may limit interpretation of mood ratings. However, this limitation was minimized by video-recording 10 percent of all interviews and having graduate research associates who were blind to all study conditions independently rate mood symptoms in these interviews. Computing reliability scores between the interviewer’s depression and mania ratings and the ratings of the other graduate students who rated the videos suggest the interviewer’s mood ratings were generally reliable.

Blood Analyses

Interpretation of the blood analyses in this study was limited due to the collection of nutrient data at only two time points. Definitive conclusions regarding nutrient levels cannot be drawn due to the uniqueness of human biochemical responses to dietary changes. Therefore, to truly learn about possible biological responses to EMP+ treatment, future studies should focus on individual patterns of change in nutrients levels over time. It is essentially impossible to determine detailed patterns in changing nutrient levels with only two data points.

As serum and plasma levels of nutrients are easily influenced by dietary intake, changes in these levels could be interpreted as simply related to what a person ate that day instead of a biochemical trend changing in response to supplementation.

Without more blood samples over time, it is impossible to identify true trends in nutrient levels in response to EMP+ treatment. This limitation may be resolved by increasing the length of study participation and administering more blood draws over time as described above.

In addition to collecting more blood samples to assess response trends, future studies will need to look at the relationship between various nutrients and changes in biochemistry in much greater detail. Kaplan and colleagues (2007) began to introduce foundations of possible mechanisms of action relating nutrients to biochemical changes that may affect mood. However, current scientific understanding of the brain and central nervous system appears much more complex than simply a matter of globally increasing or decreasing neurotransmitter levels.

Perhaps of more importance is the ratio balance of various nutrients and how that affects the balance of enzymes and neurotransmitter efficiency. It is possible that certain nutrients should be increased while others are low in order to reach optimal neuro-oscillations and have beneficial effects on regulating mood. Scientists continue to learn about the complexities of various neurotransmitter pathways with neurotransmitters that have multiple different receptors. These complexities suggest treatments may need to be tailored very specifically to create an optimal ratio of nutrients that creates the appropriate balance of neurotransmitters in the

correct brain area at the right time. Determining optimal combinations of nutrients and how they alter biochemical functioning in such specific terms will require extensive future research.

Multi-ingredient Formulation

The current 36-ingredient formulation of EMP+ may create skepticism regarding which of these ingredients play an active role in altering biochemical functioning, if any. Due to the large number of ingredients, dismantling studies would be very difficult and tedious. However, it seems necessary to trim the number of ingredients in some way to better examine which nutrients actively alter central nervous system functioning that may relate to mood regulation. One way to break down the EMP+ ingredients for more scientifically rigorous investigation may be to group participants a-priori based on different metabolic profiles. Future researchers could examine sets of nutrients in the supplement believed to be related to each metabolic profile and examine possible interaction effects.

As investigations of EMP+ continue, it is important to note there is no information on the long-term effects, positive or negative, of the high doses of the nutrients found in EMP+. Therefore, future studies must continue with caution and maintain careful safety monitoring. It will also be important to determine how one supplement can appear to improve symptoms of multiple disorders. Studies have been published suggesting EMP+ may improve symptoms of mood disorders, anxiety, and disruptive behavior. These disorders have overlapping but unique biological pathways. Therefore, it seems curious that one intervention could

improve all of these symptoms. However, antipsychotics and selective serotonin reuptake inhibitors (SSRIs) are currently used in a similar way for treating schizophrenia, mood, anxiety, aggression and other mental health problems

(American Psychiatric Association, 2009; 2007; 2004; 2002; 2000). Randomized placebo-controlled trials will be necessary to determine if these results are simply a large placebo effect, a result of people having an overall feeling of improved health after improving their nutrition, or if EMP+ is truly making biochemical changes that improve the symptoms of these disorders.

Exclusion Criteria

Lastly, the strict exclusion criterion requiring participants to stay off of all psychotropic medications created a type of limitation in this trial. While this criterion was included for safety purposes and methodologically to explore how

EMP+ may affect mood as a monotherapy, it may have impeded recruitment and excluded some potential participants who may have benefitted from EMP+ treatment. Future studies should consider the risks and benefits of allowing certain psychotropic medications during study participation. For example, perhaps children could be allowed to continue stimulant medication under careful observation while participating in future EMP+ trials. The use of a stimulant should not directly affect mood symptoms, so it should not interfere with the investigation of EMP+ as a treatment for mood dysregulation, but would allow for children with comorbid ADHD to receive benefit from a proven treatment for that condition. One participant in this trial began stimulant medication after Visit 5 and

a boy in Kaplan and colleagues’ case study (2002) also had concomitant stimulant medication added to his regimen. Neither showed any negative side effects and experienced improvement in ADHD symptoms and overall general functioning following addition of the stimulant. However, allowing additional medications during a trial of EMP+ also adds additional potential risks to participants since the nutrients in EMP+ tend to change the rate of absorption of other medications. This must be taken into consideration when determining exclusion criteria for future studies of the EMP+ supplement.

Conclusions

In summary, although this pilot feasibility trial lacked the scientific rigor of a randomized controlled trial, it provided important information regarding the feasibility of studying EMP+ in the future. While study recruitment took slightly longer than hypothesized, steps have been identified to enhance study recruitment in future trials, particularly if summer recruitment occurs and if exclusion criteria are adjusted to allow for concomitant stimulant medication for children in the trial.

Results of medication compliance and tolerability of the EMP+ supplement suggest if a child cannot swallow supplement capsule without difficulty after completing the pill swallowing desensitization protocol, EMP+ treatment is probably not a good choice. However, if a child does not have any problems swallowing the supplement capsules, the child will likely be able to follow dosing instructions and maintain a high level of medication compliance. In regard to collecting blood samples to explore biomarkers in future EMP+ trials, this study shows that children

tolerate blood draws well with the aid of nurses trained in pediatric blood draws as well as topical analgesics. Lastly, although definitive conclusions cannot be made about mood symptom improvement due to EMP+ treatment in this open-label trial, decreasing trends in depression and mania scores suggest further, more scientifically rigorous examinations of the effect of EMP+ on mood dysregulation in children is warranted. While further investigation of the nutrients in EMP+ as treatment for mood dysregulation appears warranted, researchers should note there are numerous unanswered questions regarding treatment response, mechanisms of action, and long-term effects of supplementation. Therefore, research on the effects of nutrients on mood dysregulation should continue and advance, but future studies should proceed with caution and experimental rigor.

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Appendix A: EMPowerplus (EMP+) 36-ingredient List

1 cap 4 caps 8 caps 15 caps 384.0 IU 1536.0 IU 3072.0 IU 5760 IU Vitamin A 40.0 mg 160.0 mg 320.0 mg 600 mg Vitamin C 96.0 IU 384.0 IU 768.0 IU 1440 IU Vitamin D 24.0 IU 96.0 IU 192.0 IU 360 IU Vitamin E 1.2 mg 4.8 mg 9.6 mg 18 mg Vitamin B1 0.9 mg 3.6 mg 7.2 mg 13.5 mg Vitamin B2 6.0 mg 24.0 mg 48.0 mg 90 mg Vitamin B3 1.4 mg 5.8 mg 11.5 mg 21.6 mg Vitamin B5 2.4 mg 9.6 mg 19.2 mg 36 mg Vitamin B6 96.0 ug 384.0 ug 768.0 ug 1440 ug Vitamin B9 60.0 ug 240.0 ug 480.0 ug 900 ug Vitamin B12 72.0 ug 288.0 ug 576.0 ug 1080 ug Vitamin H 88.0 mg 352.0 mg 704.0 mg 1320 mg Calcium 0.9 mg 3.7 mg 7.3 mg 13.74 mg Iron 56.0 mg 224.0 mg 448.0 mg 840 mg Phosphorus 13.6 ug 54.4 ug 108.8 ug 204 ug Iodine 40.0 mg 160.0 mg 320.0 mg 600 mg Magnesium 3.2 mg 12.8 mg 25.6 mg 48 mg Zinc 13.6 ug 54.4 ug 108.8 ug 204 ug Selenium 0.5 mg 1.9 mg 3.8 mg 7.2 mg Copper 0.6 mg 2.6 mg 5.1 mg 9.6 mg Managnese 41.6 ug 166.4 ug 332.8 ug 624 ug Chromium 9.6 ug 38.4 ug 76.8 ug 144 ug Molybdenum 16.0 mg 64.0 mg 128.0 mg 240 mg Potassium 24.0 mg 96.0 mg 192.0 mg 360 mg dl-phenylalanine 12.0 mg 48.0 mg 96.0 mg 180 mg glutamine 16.0 mg 64.0 mg 128.0 mg 240 mg citrus bioflavanoids 3.0 mg 12.0 mg 24.0 mg 45 mg grape seed 36.0 mg 144.0 mg 288.0 mg 540 mg choline bitartrate 12.0 mg 48.0 mg 96.0 mg 180 mg inositol 2.4 mg 9.6 mg 19.2 mg 36 mg ginkgo biloba 4.0 mg 16.0 mg 32.0 mg 60 mg methionine 1.4 mg 5.5 mg 11.0 mg 20.7 mg germanium sesquioxide 160.0 ug 640.0 ug 1280.0 ug 2400 ug boron 80

2.0 ug 7.8 ug 15.7 ug 29.4 ug nickel 79.6 ug 318.4 ug 636.8 ug 1194 ug vanadium

111.0 444.1 888.2 1665.3 Proprietary Total

Appendix B: Pill Swallowing Desensitization Protocol (Kaplan, 2006)

1. Preparation. In a child genuinely fearful of swallowing pills, it is important to have a rapport-building chat. • Find out his/her favorite food. Discuss what it means that every time they eat this (e.g., pizza), they swallow chunks of food larger than a capsule. They need to understand that this really is a “mental” or conceptual problem, and not a physical one. • Point out that gelatin capsules are slippery when wet (unlike some pressed tablets), and that they will need to be able to swallow gelatin capsules for this study. • Teach them how ducks swallow (cute for younger children especially). They get the food to the back of their throat, stretch out their necks, and sort of shake their bodies as they swallow. Demonstrate! • Demonstrate the 5 head positions: center (conventional), left, right, up, and down. For each position, it is important that the pill/candy plus water be shaken to the back of the tongue first, before attempting to swallow. Point out that when you swallow with your head turned about 45 degrees to the side, most people can actually feel a difference – their throat opens in a spasm that goes “gunk” (vocalize this).

2. Shaping. Go from small to large candies before trying even a small pill. • E.g.,Tic Tacs, Wonka Nerds, Mini M&M’s, Candy Hearts, and Micro-Mints. It is possible that a child will immediately be able to swallow the smallest, and can begin with the larger ones. • Next use placebos—both EMP+ and omega-3.

3. Head position. Combine experimenting with head position with every size candy. For example, when you introduce the smallest candy, have the child try all 5 head positions. Then, because novel positions are usually rejected immediately, require some home practice in each position. • Remember: get the water and pill/candy to the back of the tongue before rotating the head. • Do not use extreme positions. Each one is about a 45 degree move, not 90 degrees. • Demonstrate each one, give lots of praise for success, etc. • Require 5 minutes of home practice every day, with a parent present (safety feature: in case of choking --- also available to provide effusive praise for success). Reassure the child that no one likes the alternative positions at first --- they need to practice and make up their own minds. • Ask the child (or parent) to write down their opinion or ranking of each of the 5 positions each night. This fosters awareness/perceptiveness. • If/when they settle on a strongly preferred position, they can maintain that while they continue working up to the large sized capsules.

Appendix C: Demographic Form

Referral Source:______

Birth Dates: Primary: __/__/____ Secondary: __/__/____ Child: __/__/____

Child’s Sex: M F Child’s grade in school: ______Child’s School: ______Name of teacher: ______

Family Structure: Married biological parents Single adoptive parents Single-parent (mother) Foster family Single-parent (father) Group home Step-family Residential treatment center Married adoptive parents Other

Contact with Child: Primary: Secondary: Full Full Partial Partial Episodic Family Episodic Family Episodic Non-family Episodic Non-Family

Relationship to Child: Primary: Secondary: Bio parent Bio parent Step-parent Step-parent Grandparent Grandparent Aunt/Uncle Aunt/Uncle Foster parent Foster parent Adoptive parent Adoptive parent

# of children in the home: ______# of full siblings: ______# of half-siblings: ______# of step-siblings: ______

Ethnicity: Primary: Secondary: White, non-Hispanic White, non-Hispanic Black, non-Hispanic Black, non-Hispanic Hispanic Hispanic Asian/Pacific Islander Asian/Pacific Islander Native American Native American Mixed Mixed Other Other Child: White, non-Hispanic Black, non-Hispanic Hispanic Asian/Pacific Islander Native American Mixed Other Education of Caregivers: Primary: Secondary: Graduate/professional Graduate/professional Standard college/university Standard college/university Partial college Partial college High school diploma/GED High school diploma/GED Partial high school Partial high school Junior high school Junior high school

Occupation of caregivers: Primary: ______Secondary: ______

Who lives with child? : Primary Secondary Neither Other (______)

Income: Less than 20,000 20,000-39,000 40,000-59,000 60,000-79,000 80,000-99,000 Over 100,000

Appendix D: Medical History

Height: ______in.

Weight:______lbs.

BMI:______

Waist Cicumference:______in. Hip Circumference:______in

Developmental History:

Medication Dosage Start Date End Date

Family History:

Mental Status:

Appendix E: Physical Exam

ID #: ______Date: __/__/____ Completer’s Initials:____

System Not Examined Normal Abnormal General Appearance Head, neck, face Nose & Sinuses Mouth & Throat Ears Eyes Lungs & Chest Heart Vascular System Abdomen Genitourinary System Endocrine & Metabolic System Musculoskeletal System Skin & Lymphatics Neurological System Other

If abnormal please list system and comment below:

Appendix F: Side Effects Form

Side Effect Absent Mild Moderate Severe Insomnia 0 1 2 3 Tired during day 0 1 2 3 Difficulty waking 0 1 2 3 Tremors 0 1 2 3 Twisting tongue movements 0 1 2 3 Stiff muscles 0 1 2 3 Eyes appearing “stuck” 0 1 2 3 Headaches 0 1 2 3 Dizziness 0 1 2 3 Constipation 0 1 2 3 Diarrhea 0 1 2 3 Dyspepsia (acid stomach) 0 1 2 3 Nausea or vomiting 0 1 2 3 Anxiety 0 1 2 3 Excessive saliva/drooling 0 1 2 3 Excessive appetite 0 1 2 3 Dry mouth 0 1 2 3 Blurred vision 0 1 2 3 Enuresis 0 1 2 3 Rhinitis 0 1 2 3 Gynecomastia 0 1 2 3 Galactorrhea 0 1 2 3 Menstrual problems 0 1 2 3 Coughing 0 1 2 3 Tachycardia 0 1 2 3 Seizures 0 1 2 3 Skin rash 0 1 2 3 Tinnitus (ringing ears) 0 1 2 3 Weight gain 0 1 2 3 Other:______0 1 2 3

Bedtime: ___:___ Wake time: ___:___

Has the subject had any change in eating habits since the baseline visit?

Appendix G: Investigator’s Brochure

TABLE OF CONTENTS 1.0 SUMMARY 1 2.0 INTRODUCTION 1 3.0 PHYSICAL, CHEMICAL, AND PHARMACEUTICAL PROPERTIES; FORMULATION 2 5.0 EFFECTS IN HUMANS 2 5.1 Open-label trial in adults with bipolar disorder 2 5.2 Open-label trial in children with anxiety/mood problems 2 5.3 Children studied with reversal design 3 5.4 Patients monitored clinically 3 6.0 SAFETY 3 6.1 Pilot study in children 3 6.2 Safety survey conducted by Truehope Nutritional Support Ltd 5 7.0 SAFETY AND TOXICITY OF INDIVIDUAL MCN36 INGREDIENTS 8 7.1 VITAMIN A 9 7.2 VITAMIN C 13 7.3 VITAMIN D 14 7.4 VITAMIN E 15 7.5 VITAMIN B1 17 7.6 VITAMIN B2 18 7.7 VITAMIN B3 19 7.8 VITAMIN B5 20 7.9 VITAMIN B6 21 7.10 VITAMIN B9 23 7.11 VITAMIN B12 24 7.12 VITAMIN H 26 7.13 CALCIUM 26 7.14 PHOSPHORUS 28 7.15 MAGNESIUM 29 7.16 POTASSIUM 30 7.17 IODINE 31 7.18 ZINC 32 7.19 SELENIUM 34 7.20 COPPER 36 7.21 MANGANESE 37 7.22 CHROMIUM 38 7.23 MOLYBDENUM 40 7.24 IRON 41 7.25 dl-PHENYLALANINE 43 7.26 GLUTAMINE 43 7.27 CITRUS BIOFLAVONOIDS 44 7.28 GRAPE SEED 44 7.29 CHOLINE 45 7.30 INOSITOL 46 88

7.31 GINKGO BILOBA 47 7.32 METHIONINE 48 7.33 GERMANIUM 49 7.34 BORON 50 7.35 VANADIUM 51 7.36 NICKEL 52 8.0 REFERENCES 53

1.0 SUMMARY

This Investigator’s Brochure has been prepared according to the guidance provided in the Natural Health Products Regulations, Part 4, section 66 (e). The ingredients of MCN36 are listed below. The information collected thus far on the safety and toxicity of each of the ingredients in MCN36 is also contained in this document. Most of these ingredients have been used by the public for centuries with few safety concerns as long as the dosages were below tolerable upper limits. This combination, however, is novel. The existing data on the combination of all 36 ingredients together is presented in section 6. The data in section 6 have not been collected in a systematic way, but do suggest that people taking this supplement for up to about three years are generally remaining well. In the proposed clinical trial, participants will be monitored systematically from pre-exposure through to the end of the trial.

2.0 INTRODUCTION

Although many studies of the pharmacological treatment for mood lability demonstrate some therapeutic benefit, there currently is no single ideal medication and adverse medication effects are often burdensome. A “natural” nutrient-based treatment of mood problems would be welcomed by many, but to date none appears as effective as the many psychiatric medications that are available. The current investigations of micronutrient supplements for the treatment of mood lability appear to be promising.

The supplement approach was developed over several years by David Hardy and Anthony Stephan in Alberta, and eventually was manufactured and distributed from the U.S. Their formulation was based in part on agricultural knowledge of the treatment of stress reactions and behavioral problems in cattle and hogs: transport and handling of livestock have been shown to have a particularly large adverse impact on chloride, potassium, calcium, and magnesium, thus altering normal electrolyte balance and overall health (Schaefer, Jones, & Stanley, 1997). The animal supplement has been developed empirically over generations of farming by nutritional consultants to agribusiness, where changes in health translate into large financial gains.

After about 5 years of development by these two men (who are now part of the Synergy Group of Canada/Truehope Nutritional Support, Ltd), in April 2000 the formulation of the supplement was finalized and given the name E.M.Power+ (“Essential Mineral (E.M.) Power+” where the plus refers to the presence of ingredients other than dietary minerals). The evidence supporting the importance of minerals and vitamins in central nervous system functioning (reviewed below) provided some scientific support for this supplement, which is gaining considerable clinical attention in Canada and in many areas of the United States. In December 2002, the company modified the formula (generally reducing the minerals by a third, and crushing the powder to a finer grain) and renamed it Empowerplus to indicate the change. MCN36, the topic of the current proposal, is similar to Empowerplus.

Several principles of nutrition and nutrition guidelines warrant brief mention. First, all national and international nutrient guidelines are established for normal, healthy individuals; in contrast, the 89

people who participate in clinical trials of MCN36 are not normal, healthy subjects. Because the information on the individual nutrients in the following pages is based on data collected for ‘healthy populations,’ its relevance to these study groups is uncertain.

A second point to mention is that guidelines are established for the general population at large, whereas the participants in this clinical trial will be under relatively close medical supervision.

3.0 PHYSICAL, CHEMICAL, AND PHARMACEUTICAL PROPERTIES; FORMULATION

Please refer to section 7.0 below for details on each ingredient in MCN36. The full adult dose is six capsules taken three times a day, on a full stomach. Further details of the method of titration and use of the formulation can be found in the Protocol.

5.0 EFFECTS IN HUMANS

The published literature on MCN36 is provided in this section. Two open-label case series have been completed (Kaplan et al., 2001) (Kaplan, Fisher, Crawford, Field, & Kolb, in press). In addition, two children were studied in ABAB reversal designs, showing on-off control of their symptoms (Kaplan, Crawford, Gardner, & Farrelly, 2002). Also, two psychiatrists have independently reported case series monitored in their private practices (Popper, 2001; Simmons, 2002). These five publications are summarized.’

5.1 Open-label trial in adults with bipolar disorder The results from a 6-month study were published in 2001 (Kaplan et al., 2001). In brief, the findings were: • all 11 patients benefited from the supplement • all outcome measures showed a statistically significant reduction in symptoms (measured with the Hamilton-Depression scale, the Brief Psychiatric Rating Scale, the Young Mania Rating Scale, and the Outcome Questionnaire) • the effect size was large ( >.8) for each measure • a 50% reduction in need for psychiatric medication was also statistically significant • side effects were minor and transitory • patients generally preferred to take the supplement rather than their previous doses of psychotropic medication

5.2 Open-label trial in children with anxiety/mood problems Nine children ages 8-15 years participated in this 10-week protocol (Kaplan, Fisher et al., in press). Their other diagnoses varied widely (mood and anxiety disorders, ADHD, PDD, Prader-Willi syndrome, etc.), but the symptoms they all had in common were significant irritability, mood swings, and explosive rage. In brief, the findings were: • all 9 patients benefited from the supplement, 8 of them significantly • all outcome measures showed a statistically significant reduction in symptoms (measured with the Child Behavior Checklist and the Youth Outcome Questionnaire) • the effect size was large ( >.8) for each measure • side effects were minor and transitory 5.3 Children studied with reversal design During the pilot work leading up to the open-label case series, two children with significant mood problems and explosive rage were studied with ABAB designs (Kaplan et al., 2002). Baseline (A) involved just monitoring, intervention (B) was the use of the supplement, withdrawal (A) involved stopping the supplement, and B was subsequent reintroduction of the supplement. In both cases, the reason for supplement withdrawal was that the child had changed schools, so that there was an 90

interest in assessing whether the behavioural improvements were due to the supplement or due to the environmental changes. In both cases, withdrawal was associated with significant exacerbation of the mood symptoms, and reintroduction led to amelioration of the problem once again. 5.4 Patients monitored clinically Dr. Popper recently achieved complete symptom remission in less than two weeks in a medication- naive child with bipolar disorder, using this supplement. Subsequently, he monitored 22 private practice patients and described his experience in a commentary published in 2001 (Popper, 2001). Nineteen of the 22 patients benefited from the supplement, representing a response rate of about 86%. Similarly, Dr. Miles Simmons, a private practice psychiatrist in Brunswick, Maine, monitored 19 patients clinically (Simmons, 2002). Sixteen of the patients (84%) were said to have improved on the supplement; in 12 of them (63%) the improvement was “marked.” 6.0 SAFETY 6.1 Pilot study in children

In a pilot study with children with ADHD conducted in 1997-98 at the University of Calgary, each child had a blood and urine sample collected at each clinic visit while participating in the research. Heart rate and blood pressure were also measured.

6.1.1 Heart/blood pressure results. During participation in the study, the mean heart rate for the patients was 84.5 (sd=11.4), the mean systolic blood pressure was 103.3 (sd=13.3), and the mean diastolic blood pressure was 65.0 (sd = 9.0). The physician who carried out the repeated physical exams (Dr. Fisher) had no concerns about the health of any of the children.

6.1.2 Blood and urine sample results. The table below shows the results from 27 blood and urine samples. Again, the physician reviewing the lab results (Dr. Fisher) had no concerns about the health of any of the participants.

Mean SD Reference Ranges Hematology WBC 6.6 1.4 4.0-12.0 x 109/L RBC 4.79 0.38 3.90-5.50 x 1012/L HGB 133.7 9.5 113-150 g/L HCT/PCV 0.389 0.027 0.340-0.450 MCV 81.3 3.9 77.0-90.0 fl MCH 28.0 1.6 25.0-30.0 pg MCHC 332.4 61.6 315-350 g/l RDW 13.0 0.8 11.5-17.0 % Platelet 274.4 50.4 150-400x109/L MPV 8.7 0.8 7.2-11.1

WBC Diff. Neutrophils 3.44 1.02 1.10-7.20 x 109 /L Lymphocytes 2.49 0.54 1.30-7.20 x 109 /L Monocytes 0.33 0.13 0.10-1.10 x 109 /L Eosinophils 0.16 0.13 0.00-0.70 x 109 /L 91

Basophils 0.07 0.02 0.00-0.10 x 109 /L LUC 0.16 0.06 0.00-0.80 x 109 /L Clinical Chemistry Sodium 139.0 1.3 135-143 mmol/L Potassium 4.1 0.2 3.5-5.5 mmol/L Chloride 102.9 2.0 100-110 mmol/L Urea 4.2 0.9 1.8-6.4 mmol/L Creatinine 53.1 9.2 32-75 umol/L Calcium 2.40 0.08 2.20-2.70 mmol/L Magnesium 0.82 0.04 0.70-0.95 mmol/L Phosphate 1.59 0.12 1.07-1.71 mmol/L ALT 29.6 5.6 5-40 IU/L AST 32.9 5.4 13-46 IU/L Alkaline Phos. 219.6 46.4 75-300 IU/L Protein Total** 71.0 0 55-75 g/L CO2 27.1 1.7 18-27 mmol/L Urate 244.5 61.5 100-410 umol/L Urinalysis Chemical: Clarity Clear Colour Yellow Protein Negative, but one* patient had 1+ on one test (repeat was normal) PH 6.06 0.88 Bilirubin Negative Blood Negative, but one* patient had 3+ on one test (repeat was normal) Urobilinogen 3.69 2.51 Specific Gravity 1.02 0.01 Ketones Negative, but one* patient had a trace on one test (repeat was normal) Glucose Negative Nitrates Negative Leucocytes Negative

*Note: The elevated protein and blood occurred in the same patient; the trace ketones were in a different patient. All were normal at repeat testing. ** Note: Only one patient was assessed for “protein total,” so there is no SD.

6.2 Safety survey conducted by Truehope Nutritional Support Ltd In the fall of 2003, Truehope Nutritional Support Ltd wrote to about 80 Canadian participants in the Truehope program, asking them to report any health problems that might conceivably be related to their use of Empowerplus. They were also asked to seek a specified series of blood tests, to be obtained from the participant’s physician, to assess for possible indications of vitamin or mineral toxicity. An accompanying letter was included, written to the participant’s physician, requesting a CBC with differential; TSH; fasting blood glucose; AST, ALT, bilirubin-total; albumin; ferritin; calcium; magnesium; electrolyte panel (or potassium and sodium); creatinine; PTT and PT (or INR); 92

and a routine urinalysis. The mailing to the participants also included a questionnaire regarding the history of use of the Truehope supplement, as well as a medication history and some general health questions.

Lab results were sent by the participants’ physicians to Truehope, and an independent physician reviewed all the laboratory reports. Truehope has permitted the inclusion of these data in this Investiagor’s Brochure. All participants signed consent forms to release their lab test results.

Eventually, 27 participants had provided the requested information. The sample consisted of 10 males and 16 females (gender missing in 1), aged 20-74 years, who had been taking the supplement for approximately one to three years, primarily for bipolar disorder.

In this sample of 27 patients, there were a few abnormalities evident in the laboratory results (cf. table below). Most of the aberrant values pertained to one 61-year-old male who had the highest ALT (110), ferritin (499), INR (1.7), and fasting blood glucose (6.9). This patient was being treated concurrently with warfarin for blood clots (likely explaining the high INR) as well as with thyroid hormone. In addition, this patient had been taking nefazodone (Serzone), which is known to be associated occasionally with elevated liver function tests and rarely liver failure, at times resulting in liver transplantation or death. (Note: On November 10, 2003 Health Canada announced the market withdrawal by the manufacturer of this drug because of liver-related adverse effects.) Without pre- exposure baseline laboratory tests, it is not known whether this patient’s high ALT is attributable to the nutritional supplement, to exposure to nefazodone, or to his other medical problems; however, none of the other 26 patients showed any liver abnormalities on the supplement. It is also worth noting that this patient had the very lowest Exposure Index of the entire group.

The following is a detailed listing of all laboratory abnormalities in this sample: N for this Group Range N (%) % laboratory variable Mean outside lab abnormalities (SD) reference outside action range limits Age at Blood 26 46.0 20.9 - 74.1 NA NA test (in years) (14.6) Exposure 23 350.8 125.1 - 868.9 Index* (203.0) NA NA Months on 23 28.0 10 - 40.7 NA NA supplement (8.7) Capsules/day 24 13.0 4 - 24 NA NA (5.8) CBC: HGB 26 144.00 120 - 167 1 (3.8%) 0 (12.74) RBC 25 4.62 4 - 5.28 2 (8%) 0 (0.38) WBC 26 6.43 4.1 - 10.3 1 (3.8%) 0 (1.59) INR 22 1.03 1 – 2 1 (4.5%) 0 (0.16) PTT 21 29.20 24 – 35 1 (4.8%) 0 (2.59) TSH 23 1.76 .03 - 3.57 1 (4.3%) 0 (0.80) Fasting blood 25 5.02 3.9 - 6.9 2 (8%) 0

glucose (0.68) AST 17 24.29 15 – 43 1 (5.9%) 0 (7.10) ALT 26 36.12 14 – 110 4 (15.4%) 1 (22.14) Bilirubin 23 9.57 3 – 20 0 0 (3.51) Albumin 24 42.86 31 – 48 1 (4.2%) 0 (4.43) Ferritin 24 114.12 7 – 499 3 (12.5%) 0 (116.34) Calcium 25 2.38 2.17 - 2.57 0 0 (.10) Magnesium 25 0.86 .74 - 1.02 0 0 (.07) Potassium 25 4.23 3.5 - 4.9 0 0 (.34) Sodium 25 140.28 136 – 146 1 (4%) 0 (2.15) Creatinine 23 79.83 57 – 118 0 0 (16.18) Urinalyses: 16 0 0 All reported as normal

*Exposure Index is defined as the number of capsules taken daily (as reported by the patient) times the number of months they had taken the supplement at the time of the blood test.

Complete list of incidental findings: HGB: 136, reference range of 140-175 RBC: 4.0, reference range of 4.1-5.2 4.6, reference range 4.7-5.8 WBC: 4.7, reference range of 4.8-10.8 INR: 1.7, reference range 0.8-1.2, in a 61-year-old male taking warfarin because of a prior medical history TSH: 0.03, reference range 0.2-4.0, in a 54-year-old woman being treated for pre- existing thyroid condition Fasting glucose: 6.6 and 6.9, reference range 3.3-6.0, in two males (ages 44 and 61 years) AST: 43, reference range <40, in a 26-year-old woman ALT: 54, reference <50 80, reference range <60 60, reference range 9-52 110, reference range <50, in the 61-year-old patient described above Albumin: 31, reference range 37-51 Ferritin: 339, reference range 10-244 7, reference range 25-250 499, reference range 12-300 Sodium: 146, reference range 133-146

The patients’ subjective reports of their health were all very positive: they attributed good physical and mental health to the supplement. The people who responded to a request for blood samples would presumably be biased toward those who feel grateful to the Truehope people, so these positive comments must be interpreted cautiously.

Since some questions have been raised about the presence of germanium (albeit on the basis of erroneous information), we estimated the amount of germanium consumed by the 24 people who provided data on the number of pills they consumed. At 1.15 mg of germanium sesquioxide per capsule, the mean daily dosage was 15 mg (SD=6.7), range from 4.6 to 27.6.

Finally, two women reported healthy pregnancies and births of full-term infants while taking the supplement.

7.0 SAFETY AND TOXICITY OF INDIVIDUAL MCN36 INGREDIENTS

Concerns about the potential for adverse effects are not equal across the ingredients. For example, many of the ingredients are believed to be entirely safe at up to 100 times the recommended nutrient intakes, because they are water soluble or are ubiquitous in our food environment(Marks, 1989). Some of the ingredients that are entirely safe even at very high levels are vitamins B1 {thiamine}, B2 {riboflavin}, B9 {folic acid}, B12 {cobalamins}, C {ascorbic acid}, biotin, and pantothenic acid. Even vitamin E {tocopherols} can be ingested safely at very high levels, according to Marks. In some cases such as riboflavin, it appears “that it is not possible to achieve a toxic dose by the oral route”.

In contrast, some ingredients such as vitamin A {retinol}, vitamin D {calciferols}, and vitamin B6 {pyridoxine} bear closer scrutiny. For these, more extensive information about potential toxicity is provided. Again, the data used by governments for the development of dietary guidelines are from case studies and research on ‘healthy individuals,’ not people with mental disorders.

Not all nutrition experts agree on all issues of safety and toxicity. The information below comes from several established sources: especially, National Institutes of Health (NIH), the American Society for Nutritional Sciences, Modern Nutrition in Health and Disease, by Shils et al., and Textbook of Natural Medicine, by Murray and Pizzorno. These sources are not always in complete agreement about safe levels for healthy individuals. Sometimes a range of views is presented, with summary comment at the end of each ingredient that attempts to synthesize the available information in relationship to the formula of MCN36.

RDAs and DRIs. The primary websites that provided useful information tended to use the term RDA. At the present time, the American and Canadian governments are collaborating on a new system called Dietary Reference Intakes (DRIs). Although not yet finalized, the DRIs are being used extensively in both countries. A complete history of the development of the DRIs can be found on the Health Canada webpage at http://www.hc-sc.gc.ca/hppb/nutrition/factsheets/index.html

The following excerpt from the new text on DRIs by the National Academy of Sciences explains the various terms(Medicine, 2001):

“Dietary Reference Intakes (DRIs) comprise a set of four nutrient-based reference values, each of which has special uses. The development of DRIs expands on the periodic reports called Recommended Dietary Allowances, which have been published since 1941 by the National Academy of Sciences and the Recommended Nutrient Intakes of Canada. This 95

comprehensive effort is being undertaken by the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes of the Food and Nutrition Board, Institute of Medicine, National Academy of Sciences, with the active involvement of Health Canada.

WHAT ARE DIETARY REFERENCE INTAKES? The reference values, collectively called the Dietary Reference Intakes (DRIs), include the Estimated Average Requirement (EAR), Recommended Dietary Allowance (RDA), Adequate Intake (Al), and Tolerable Upper Intake Level (UL). A requirement is defined as the lowest continuing intake level of a nutrient that will maintain a defined level of nutriture in an individual.”

The strongest evidence that these ingredients are safe when ingested over the long term is the evidence from thousands of years of human food habits. Although one cannot truly know the cumulative effects of human ingestion of psychiatric medication over decades of use, nor are pharmaceutical companies expected to provide such information when applying for government approval, what is known is that eating most of the natural ingredients in MCN36 constitutes how humans have fed and supplemented themselves for millennia. Most humans consume 33 of these 36 ingredients virtually every day of their lives, often at higher doses than found in MCN36. If they eat citrus fruit, then that number increases to 34. The only two that are not part of the normal human diet are ginkgo and grape seed.

7.1 VITAMIN A (as retinyl palmitate) (full dose of MCN36 = 5760 IU = 1730 mcg)

7.1.1 What is it ? From the NIH website: Vitamin A is a family of fat-soluble vitamins. Retinol is one of the most active, or usable, forms of vitamin A, and is found in animal foods such as liver and eggs. It can be converted to retinal and retinoic acid, other active forms of the vitamin A family. Some plant foods contain orange pigments called provitamin A carotenoids that the liver can convert to retinol. Beta- carotene is a provitamin A carotenoid found in many foods. Lycopene, lutein, and zeaxanthin are also carotenoids commonly found in food, but your body cannot convert them to vitamin A.

Vitamin A plays an important role in vision, bone growth, reproduction, cell division and cell differentiation, which is the process by which a cell decides what it is going to become. It also maintains the surface linings of your eye and your respiratory, urinary, and intestinal tracts. When those linings break down, bacteria can enter your body and cause infection. Vitamin A also helps your body regulate its immune system. The immune system helps prevent or fight off infections by making white blood cells that destroy harmful bacteria and viruses. Vitamin A may help lymphocytes, a type of white blood cell that fights infections, function more effectively. Vitamin A also may help prevent bacteria and viruses from entering your body by maintaining the integrity of skin and mucous membranes.

Some carotenoids, in addition to serving as a source of vitamin A, have been shown to function as antioxidants in laboratory tests. However, this role has not been consistently demonstrated in humans. Antioxidants protect cells from free radicals, which are potentially damaging by-products of the body’s metabolism that may contribute to the development of some chronic diseases.

7.1.2 Recommended intake The RDA is the average daily dietary intake level that is sufficient to meet the nutrient requirements of nearly all (97-98%) healthy individuals in each life-stage and gender group. The 1989 RDAs for vitamin A for adults and children are:

Life-Stage Children Men Women Pregnancy Lactation Ages 1-3 400 RE or 2000 IU* Ages 4-6 500 RE or 2500 IU Ages 7-10 700 RE or 3,500 IU Ages 11-18 1000 RE or 800 RE or 800 RE or 800 RE or 4000 IU 4000 UI 4000 IU 4000 IU Ages 19 + 1000 RE or 800 RE or 800 RE or 800 RE or 5000 IU 4000 IU 4000 IU 4000 IU

Results of the third National Health and Nutrition Examination survey suggested that the dietary intake of some Americans does not meet recommended levels. The Continuing Survey of Food Intakes of Individuals suggested that diets of many Americans provide less than 75% of recommended intake levels. These surveys highlight the importance of encouraging Americans to include dietary sources of vitamin A in their daily diets.

There is no separate RDA for beta-carotene or other carotenoids. The Institute of Medicine (IOM) report suggests that consuming 3 to 6 mg of beta-carotene daily will maintain plasma B-carotene blood levels in the range associated with a lower risk of chronic diseases. This concentration can be achieved by a diet that provides five or more servings of fruits and vegetables per day.

7.1.3 Toxicity information Hypervitaminosis A refers to high storage levels of vitamin A in the body that can lead to toxic symptoms. Toxicity can result in dry, itchy skin, headache, fatigue, hair loss, loss of appetite, vomiting, and liver damage. When toxic symptoms arise suddenly, which can happen after consuming very large amounts of vitamin A over a short period of time, signs of toxicity include dizziness, blurred vision, and muscular uncoordination.

Although hypervitaminosis A can occur when very large amounts of liver are regularly consumed, most cases of vitamin A toxicity result from an excess intake of vitamin A in supplements. A generally recognized safe upper limit of intake for vitamin A from diet and supplements is 1,600 to 2,000 RE (8,000 to 10,000 IU) per day. The Institute of Medicine is currently reviewing the scientific literature on vitamin A. They are considering revising the RDAs and establishing an Upper Limit (UL) of safe intake for vitamin A.

Vitamin A toxicity also can cause severe birth defects. Women of child-bearing age are advised to limit their total daily intake of vitamin A (retinol) from foods and supplements combined to no more than 1,600 RE (8,000 IU) per day. {For this reason women who are pregnant or planning to become pregnant are excluded from the clinical trial.}

Retinoids are compounds that are chemically similar to vitamin A. Over the past 15 years, synthetic retinoids have been prescribed for acne, psoriasis, and other skin disorders. Isotretinoin (Roaccutane® or Accutane®) is considered an effective anti-acne therapy. At very high doses,

however, it can be toxic, which is why this medication is usually saved for the most severe forms of acne. The most serious consequence of this medication is birth defects.

It is extremely important for sexually active females who may become pregnant and who take these medications to use an effective method of birth control. Women of childbearing age who take these medications are advised to undergo monthly pregnancy tests to make sure they are not pregnant.

Nutrient toxicity traditionally refers to adverse health effects from a high intake of a particular vitamin or mineral. For example, large amounts of the active form of vitamin A (naturally found in animal foods such as liver but also available in dietary supplements) can cause birth defects.

Provitamin A carotenoids such as beta-carotene are generally considered safe because they are not traditionally associated with specific adverse health effects. The conversion of provitamin A carotenoids to vitamin A decreases when body stores are full, which naturally limits further increases in storage levels. A high intake of provitamin A carotenoids can turn the skin yellow, but this is not considered dangerous to health.

Recent clinical trials that suggested a greater incidence of lung cancer and total mortality (death) in current smokers who supplemented their diet with 20 mg of beta-carotene per day have raised concern about the safety of beta-carotene supplements. However, conflicting studies make it difficult to interpret the health risk. For example, the Physicians’ Health Study compared the effects of taking 50 mg beta-carotene every other day to a placebo (sugar pill) in over 22,000 male physicians and found no adverse health effects. Also, a trial that tested the ability of four different nutrient combinations to inhibit the development of esophageal and gastric cancers in 30,000 men and women in China suggested that after 5 years those participants who took a combination of beta- carotene, selenium and vitamin E had a 13% reduction in cancer deaths. One point to consider is that there may be a relationship between alcohol and beta-carotene because “only those men who consumed more than 11 g per day of alcohol (approximately one drink per day) showed an adverse response to B-carotene supplementation” in the lung cancer trial.

The Institute of Medicine did not set a Tolerable Upper Intake Level (UL), the highest level of daily nutrient intake that is likely to pose no risk of adverse health effects, for B-carotene or carotenoids. Instead, they concluded that B-carotene supplements are not advisable for the general population. As stated earlier, however, they may be appropriate as a provitamin A source or for the prevention of vitamin A deficiency in specific populations.

There are different manifestations of acute and chronic vitamin A toxicity. The following is from a book chapter by Marks: “The most common manifestations of vitamin A overdose are skin redness and desquamation, disturbed hair growth, and nausea and anorexia; liver damage has been noted occasionally, mainly in those with reduced renal function….Retinol is one of the few vitamins in which the incidence of adverse reactions is very much influenced by the health of the individual and particularly by the functional integrity of the liver. In the presence of liver damage, there is an increased level of unbound circulating retinol and a higher incidence of adverse reactions…..When considered in terms of dosage, retinol adverse effects are extremely rare at doses below 9000 mcg RE (30,000 IU) daily and the majority arise at a much higher dose when there is no hepatic damage…..Hence, the safety margin may probably be placed at 10 times the RDA.”

Olson explained that there are three toxic syndromes that can be caused by excessive vitamin A: acute, chronic, and teratogenic. An acute dose of about 12 g of vitamin A is presumed to be the median lethal dose. Chronic toxicity is believed to be possible by recurrent intake of at least 33,300 IU for an adult (10 times the RDA). Spontaneous abortion and fetal abnormalities are believed to be possible at long-term intakes of 25,000 IU/day during early pregnancy. 98

Years ago, there was great concern about toxicity of even small amounts of vitamin A. This information has been retained by the public in general, but the fact is that toxic effects of chronic ingestion are uncommon at dosages less than 100,000 IU/day. The most common symptoms of excessive vitamin A intake are nausea and vomiting, fatigue, and headache. They concluded that doses of 5-10,000 IU are safe; these are the doses found in most commercial preparations of multivitamins.

The recent publication that is part of the transition to DRIs remarks that the Tolerable Upper Limit of vitamin A for adults is 3000 mcg per day (120,000 IU). The amount in MCN36 is a small portion (<5%) of that.

7.1.4 Summary comment The Vitamin A content of a previous version of this supplement exceeded 10,000 IU/day; in MCN36 it has now been decreased to 5760 IU/day. Acute hypervitaminosis A has been reported to occur in children after the administration of a single does of very high amounts of the Vitamin (>50,000 IU). Thus, the acute level provided in the supplement would be unlikely to be associated with hypervitaminosis, even in children. Chronic toxic reactions are more frequent than acute toxic reactions to Vitamin A, but they are uncommon at dosages less than 100,000 IU/d in adults with normal liver function. A recent review of the literature found <20 reported cases of excessive dietary intakes of Vitamin A being associated with teratogenicity in humans. They concluded to avoid any possibly negative effect on fetal development that daily doses of >10,000 IU of Vitamin A should not be provided during pregnancy.

The RDA for an adult male is 5,000 IU. Hence, the amount in MCN36 represents less than 1/6th the level that has been associated with toxicity. Regardless of whether one looks at acute or chronic effects, the amount of Vitamin A in MCN36 appears to be in the range of 1/6th to 1/10th of the level that might cause concern, thus providing a wide margin of safety. According to the 2001 Tolerable Upper Limit published by the National Academy of Sciences, the amount in MCN36 is <5% of the UL. Because of the potential concern about high intakes of this vitamin during pregnancy, no pregnant women are entered into this trial.

7.2 VITAMIN C (full dose of MCN36 = 600 mg)

7.2.1 What is it? Vitamin C is also known as ascorbic acid, L-ascorbic acid, dehydroascorbic acid and the antiscorbutic vitamin. Chemically, it is called L-xyloascorbic acid and L-threo-hex-2-uronic acidy- lactone. The very highest concentrations of vitamin C are found in the adrenal and pituitary glands. High levels are also found in liver, leukocytes, brain, kidney and pancreas. Most of the vitamin C is found in liver and skeletal muscle because of their size relative to the rest of the body.

The best characterized function of vitamin C is in the synthesis of collagen connective tissue protein at the level of hydroxylation of prolyl and lysyl residues of procollagen. Vitamin C also plays important roles in the synthesis of neurotransmitters, steroid hormones, carnitine, conversion of cholesterol to bile acids, tyrosine degradation and metal ion metabolism. This vitamin also may enhance iron bioavailability. The role of ascorbic acid as a biological reducing agent may be linked to its prevention of degenerative diseases, such as cataracts, certain cancers and cardiovascular diseases.

7.2.2 Recommended intake From the American Society for Nutritional Sciences web site: www.nutrition.org:

The RDA for adults is 60 mg/day in the US, but may range from 30-75 mg/day in other Western countries. Intakes of 75-95 mg/day are recommended for pregnant and lactating women. The RDA is 35 mg/day in infants and 40 mg/day in children, ages 1-3 yr. About 10 mg/day is required to prevent scurvy. Increased intake of vitamin C is recommended for stress situations such as trauma, infection, strenuous exercise, or elevated environmental temperatures. The requirement in smokers may be 100 mg/day. Recent kinetic analyses suggest that intakes of 150-200 mg/day, but below 400 mg/day, obtained from the diet, may offer the most benefit in normal, healthy individuals.

7.2.3 Toxicity information From American Society for Nutritional Sciences website: “Megadoses of vitamin C of 1000-2000 mg have commonly been associated with gastrointestinal disturbances (nausea, abdominal cramps and diarrhea). In general, megadoses of vitamin C should be avoided in individuals with a history of renal stones due to oxalate formation or hemochromatosis or other diseases related to excessive iron accumulation. Excess vitamin C may predispose premature infants to hemolytic anemia due to the fragility of their red blood cells. In healthy individuals, it appears that megadoses of up to 1000 mg/day of vitamin C are well tolerated and not associated with any consistent adverse effects. Concern of its pro-oxidant properties is stimulating renewed interest in its potential long-term toxicity.”

Marks deals with a number of the allegations regarding high levels of vitamin C. For instance, he argues that even at 10 g/day, ascorbic acid conversion to oxalate stones does not reach significant levels unless the person has evidence of renal insufficiency. He dismisses several other allegations about high dose vitamin C as being equally unfounded: e.g., that it is mutagenic, that it elevates iron absorption to dangerous levels. He concludes that a safe daily level is at least 100 times the RDA. For an adult male, this would be 6 grams/day; the amount in MCN36 is 1/10th that level.

The text by Shils provides further information on toxicity: “Jacob addressed the issue of whether vitamin C intake at greater than 1 g/day would contribute to oaxalate accumulation and kidney stone formation. He concluded that ‘Most studies show that increased ….(ascorbic acid)…intakes do not significantly increase body oaxalate levels, and reports of stone formation linked directly to excess …(ascorbic acid)… intake are rare.’ Of the many other controversies regarding high vitamin C intake, Jacob concludes that the evidence is scanty. The one exception is systemic conditioning of high intake, followed by a rebound deficiency after withdrawal of high intake. He concludes that people wishing to stop taking high doses of vitamin C would be well-advised to withdraw gradually over 2-4 weeks, to avoid this systemic conditioning and rebound deficiency.”

An excellent review article in the Archives of Internal Medicine article stated that “…toxic reactions are rare at dosages less than 4 g/day.” He also points out that the intestines probably cannot absorb more than about 3 g/day. “Given the scarcity of case reports {of adverse effects} involving a vitamin consumed by 35% of the US population, the incidence of adverse effects must be extremely low.”

7.2.4 Summary comment The dose of 600 mg/day appears to be well within the safe limit for vitamin C ingestion.

7.3 VITAMIN D (as cholecalciferol) (full dose of MCN36 = 1000 IU = 25 mcg)

100

From the NIH website, a fact sheet published by the Clinical Nutrition Service, Warren Grant Magnuson Clinical Center, National Institutes of Health (NIH), Bethesda, MD, in conjunction with the Office of Dietary Supplements (ODS) in the Office of the Director of NIH

7.3.1 What is it? Vitamin D, calciferol, is a fat-soluble vitamin. It is found in food, but also can be made in your body after exposure to ultraviolet rays from the sun. Vitamin D exists in several forms, each with a different activity. Some forms are relatively inactive in the body, and have limited ability to function as a vitamin. The liver and kidney help convert vitamin D to its active hormone form.

The major biologic function of vitamin D is to maintain normal blood levels of calcium and phosphorus.Vitamin D aids in the absorption of calcium, helping to form and maintain strong bones. It promotes bone mineralization in concert with a number of other vitamins, minerals, and hormones. Without vitamin D, bones can become thin, brittle, soft, or misshapen. Vitamin D prevents rickets in children and osteomalacia in adults, which are skeletal diseases that result in defects that weaken bones.

7.3.2 Recommended intake The RDA is the average daily dietary intake level that is sufficient to meet the nutrient requirements of nearly all (97-98%) healthy individuals in each life-stage and gender group. There is insufficient evidence to establish a RDA for vitamin D. Instead, an Adequate Intake (AI), a level of intake sufficient to maintain healthy blood levels of an active form of vitamin D, has been established. The 1998 Ais for vitamin D for adults, in micrograms (mcg) and International Units (IUs) are:

Life-Stage Men Women Ages 19-50 5 mcg* or 200 IU 5 mcg* or 200 IU Ages 51-69 10 mcg* or 400 IU 10 mcg* or 400 IU Ages 70 + 15 mcg* or 600 IU 15 mcg* or 600 IU 1 mcg vitamin D = 40 International Units (IU)

7.3.3 Toxicity information There is a high health risk associated with consuming too much vitamin D. Vitamin D toxicity can cause nausea, vomiting, poor appetite, constipation, weakness, and weight loss. It can also raise blood levels of calcium, causing mental status changes such as confusion. High blood levels of calcium also can cause heart rhythm abnormalities. Calcinosis, the deposition of calcium and phosphate in soft tissues like the kidney can be caused by vitamin D toxicity.

Consuming too much vitamin D through diet alone is not likely unless you routinely consume large amounts of cod liver oil. It is much more likely to occur from high intakes of vitamin D in supplements. The Food and Nutrition Board of the Institute of Medicine considers an intake of 25 mcg (1,000 IU) for infants up to 12 months of age and 50 mcg (2,000 IU) for children, adults, pregnant, and lactating women to be the tolerable upper intake level (UL). A daily intake above the UL increases the risk of adverse health effects and is not advised.

The newly-developed Dietary Reference Intakes (DRIs) specify a Tolerable Upper Intake Level (UL) of 50 ug/day.

7.3.4 Summary comment In adults Vitamin D toxicity usually does not occur unless intakes exceed 50,000 IU per day. There have been a few cases of adverse reactions reported at daily intakes around 12,000 IU/d. 101

The 1000 IU of Vitamin D consumed in the full dose of MCN36 is below the tolerable upper limit set by the U.S. Government, and is well below the 12,000 IU level at which adverse reactions have been reported in the literature.

7.4 VITAMIN E (as d-alpha tocopheryl succinate) (full dose of MCN36 = 360 IU = 240 mg) From the NIH website, a fact sheet published by the Clinical Nutrition Service, Warren Grant Magnuson Clinical Center, National Institutes of Health (NIH), Bethesda, MD, in conjunction with the Office of Dietary Supplements (ODS) in the Office of the Director of NIH

7.4.1 What is it? Vitamin E is a fat-soluble vitamin that exists in eight different forms. Each form has its own biological activity, the measure of potency or functional use in the body. Alpha-tocopherol is the most active form of vitamin E in humans, and is a powerful biological antioxidant. Antioxidants such as vitamin E act to protect your cells against the effects of free radicals, which are potentially damaging by-products of the body’s metabolism. Free radicals can cause cell damage that may contribute to the development of cardiovascular disease and cancer. Studies are underway to determine whether vitamin E might help prevent or delay the development of those chronic diseases.

7.4.2 Recommended intake The RDA is the average daily dietary intake level that is sufficient to meet the nutrient requirements of nearly all (97-98%) healthy individuals in each life-stage and gender group. The 2000 RDAs for vitamin E for adults, in milligrams (mg) and International Units (IUs) are: Life-Stage Men and Women Pregnancy Lactation Ages 19 + 15 mg* or 22 IU All Ages 15 mg* or 22 IU 19mg* or 28 IU *1 mg alpha-tocopherol equivalents = 1.5 IU

The RDA for vitamin E is based on the alpha-tocopherol form because it is the most active, or usable, form. Unlike other vitamins, the form of alpha-tocopherol made in the laboratory and found in supplements is not identical to the natural form, and is not quite as active as the natural form.

7.4.3 Toxicity information The health risk of too much vitamin E is low. A recent review of the safety of vitamin E in the elderly indicated that taking vitamin E supplements for up to four months at doses of 530 mg or 800 IU (35 times the current RDA) had no significant effect on general health, body weight, levels of body proteins, lipid levels, liver or kidney function, thyroid hormones, amount or kinds of blood cells, and bleeding time. Even though this study provides evidence that taking a vitamin E supplement containing 530 mg or 800 IU for four months is safe, the long term safety of vitamin E supplementation has not been tested. The Institute of Medicine has set an upper tolerable intake level for vitamin E at 1,000 mg (1,500 IU) for any form of supplementary alpha-tocopherol per day because the nutrient can act as an anticoagulant and increase the risk of bleeding problems. Upper tolerable intake levels “represent the maximum intake of a nutrient that is likely to pose no risk of adverse health effects in almost all individuals in the general population”.

Extracted from the Merck Manual website: http://www.merck.com/pubs/mmanual/ “Adults have taken relatively large amounts of vitamin E (400 to 800 mg/day of d—tocopherol) for months to years without any apparent harm. Occasionally, muscle weakness, fatigue, nausea, and diarrhea have occurred in persons taking 800 to 3200 mg/day. The most significant toxic effect of vitamin E

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at > 1000 mg/day is antagonism to vitamin K action and enhancement of the effect of oral coumarin anticoagulants, which may result in overt hemorrhage.”

Marks points out that doses of 2-3.5 g (3000 IU – 5,250 IU) have been administered for up to 11 years with no adverse reactions. He suggests that the safety factor is “substantially” in excess of 100 times the RDA. The RDA for an adult male is 22 IU. The amount provided by MCN36 is less than 20% that level (2200 IU).

Meyers et al. state that “case reports {of toxic reactions} are few at dosages less than 3200 mg/day.”

7.4.4 Summary comment The 360 IU (240 mg) of vitamin E contained in the full adult dose of MCN36 is well below the level that any of sources report to be potentially harmful. It is less than 10% of what Meyers reports as having been associated with toxic reactions.

7.5 VITAMIN B1 (as thiamine mononitrate) (full dose of MCN36 = 18 mg) From the website of the American Society for Nutritional Sciences Extracted from the American Society for Nutritional Sciences web site: www.nutrition.org

7.5.1 What is it? Thiamin (vitamin B-1) is a water-soluble substance, consisting of thiazole and pyrimidine rings joined by a methylene bridge. Thiamin is found in high concentrations in skeletal muscle, the heart, liver, kidneys and brain. The total amount in an adult is about 30 mg and the biologic half-life in the body is about 15 days. It is not surprising that a state of severe depletion can be seen in patients on a strict thiamin-deficient diet in 18 days. Similarly, the rate of flux of thiamin across the blood-brain barrier in rats is equivalent to the turnover in brain, indicating no spare thiamin capacity for the brain.

Thiamin diphosphate (ThDP) is the active form of thiamin. ThDP serves as a cofactor for several enzymes involved in carbohydrate catabolism, including pyruvate dehydrogenase, transketolase, and a-ketoglutarate, and for the branched-chain a-keto acid dehydrogenase complex that is involved in amino acid catabolism. The former enzymes play crucial roles in the tricarboxylic acid cycle and the pentose phosphate pathway. They are important in the biosynthesis of a number of cell constituents, including the neurotransmitters acetylcholine and gamma-aminobutyric acid (GABA), and in the production of reducing equivalents (NADPH) for biosyntheses and pentoses for nucleic acid synthesis. Because of its involvement in the synthesis of precursors of DNA, thiamin utilization is increased in tumors. Limiting thiamin can inhibit tumor cell proliferation. A neurochemical role for thiamin triphosphate (ThTP) has been postulated; ThTP is localized to neuronal cells and is implicated in neuronal conduction.

Thiamin uptake by the small intestines and by cells within various organs is mediated at physiological concentrations by a saturable, high affinity transport system. The requirement for cellular energy (ATP) for thiamin uptake is a secondary event and results from the rapid diphosphorylation of imported thiamin by thiamin diphosphokinase. Alcohol affects various aspects of thiamin transport/uptake, and these effects may contribute to the prevalence of thiamin deficiency in alcoholics. Alcohol also reduces cellular thiamin diphosphokinase activity.

7.5.2 Dietary intake The Recommended Dietary Allowance is as follows: 0-5 month infants, 0.2 mg/day; 6 to 11 months, 0.3 mg/day; 1-3 years of age, 0.5 mg/day; 4-8 years, 0.6 mg/day; 9-13 years, 0.9 mg/day; males 14 103

years old and older, 1.2 mg/day; females 14-18 years of age, 1.0 mg/day; and females 19 years and older, 1.1 mg/day. During pregnancy, the RDA is increased to 1.4 mg/day, and during lactation, 1.5 mg/day. The RDAs are based on the Dietary Reference Intakes of the National Academy of Sciences.

7.5.3 Toxicity information From American Society for Nutritional Sciences website: There is no reported toxicity with oral thiamin. There are only a few reports of toxic reactions to intravenous thiamin, resulting mainly in an anaphylactic reaction.

In the recent National Academy of Sciences document supporting the transition to DRIs, the Academy did not set a Tolerable Upper Level for thiamin. They point out that there are no reports of adverse effects from thiamin in either food or supplement.

Marks is very clear that aside from idiosyncratic hypersensitivity reactions, doses for chronic oral administration can safely exceed 100 mg/day.

7.5.4 Summary comment There appears to be no potential toxicity concern from oral ingestion of thiamine.

7.6 VITAMIN B2 (as riboflavin) (full dose of MCN36 = 13.5 mg) Extracted from the American Society for Nutritional Sciences web site: www.nutrition.org

7.6.1 What is it? Riboflavin (vitamin B-2) is chemically known as 7,8-dimethyl-10 (1’-D-ribityl) isoalloxazine. In tissues there is a broad distribution of flavin but little is present as free riboflavin. The majority is found in flavocoenzymes (mainly flavin adenine dinucleotide (FAD)) and lesser amounts are in a mononucleotide (riboflavin-5’-phosphate (FMN)). Levels of flavin range from a few µg/g wet weight in skeletal muscle and intestine, and up to 30 to 35 µg/g in liver and kidney. Flavocoenzymes are largely non-covalently associated within diverse flavoproteins, but some exist as 8-a-linked FAD.

As the catalytically operating moiety within bound flavocoenzymes, riboflavin participates in oxidation-reduction reactions in numerous metabolic pathways and in energy production via the respiratory chain. Flavoproteins participate in both one- and two-electron transfers. They operate in pyridine nucleotide-dependent and independent and independent dehydrogenations, reactions with sulfur-containing compounds, hydroxylation, oxidative decarboxylations, deoxygenations, and reduction of oxygen to hydrogen peroxide following abstraction of hydrogen from substrates.

7.6.2 Dietary intake Suggested amounts range from an Adequate Intake of 0.3 mg/day for early infants to RDAs of 1.1 mg/day for women and 1/3 mg/day for men. An additional 0.3 mg/day is recommended during pregnancy and 0.4 mg/day for lactation.

7.6.3 Toxicity information The limited capacity to absorb orally administered riboflavin precludes its potential for harm. Riboflavin intake of many times the RDA is without demonstrable toxicity.

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In the recent document supporting the transition to DRIs, the Academy concluded that the data on adverse effects of riboflavin were not sufficient to set a UL. They review many studies employing doses from 60-400 mg/day, reporting no adverse effects.

Marks concluded that “it is not possible to achieve a toxic dose by the oral route”.

7.6.4 Summary comment The dose of riboflavin in MCN36 precludes any potential for toxicity with chronic use(Shils, Olson, & Shike, 1994).

7.7 VITAMIN B3 (as niacinamide) (full dose of MCN36 = 90 mg) Extracted from the American Society for Nutritional Sciences web site: www.nutrition.org

7.7.1 What is it? Niacin (nicotinic acid or nicotinamide) is essential in the form of the coenzymes NAD and NADP in which the nicotinamide moiety acts as electron acceptor or hydrogen donor in many biological redox reactions. NAD functions as an electron carrier for intracellular respiration as well as a codehydrogenase with enzymes involved in the oxidation of fuel molecules. NADP functions as a hydrogen donor in reductive biosyntheses such as in fatty acid and steroid syntheses and, like NAD, as a co-dehydrogenase. NAD, in its only non-redox role, is the substrate for three classes of enzymes that transfer ADP-ribose units to proteins involved in DNA processing, cell differentiation, and cellular calcium mobilization.

Nicotinic acid and nicotinamide are rapidly absorbed from the stomach or the intestine. Nicotinamide, the major form in the bloodstream, arises from enzymatic hydrolysis of NAD(P) in the intestinal mucosa and liver, and is transported to tissues that synthesize their own NAD as needed. Niacin and NAD are biosynthesized from dietary tryptophan via the kynurenine pathway and quinolinic acid. Excess niacin is excreted in the urine primarily as Nl-methylnicotinamide and Nl-methyl-2-pyridone-5-carboxamide.

7.7.2 Dietary intake The RDA is expressed in mg niacin equivalents (NE) in which 1 mg NE = 1 mg niacin or 60 mg tryptophan. For individuals above 13 years of age, the 1998 RDA is 16 mg/d for males and 14 mg/d for females, with an additional allowance of 4 mg/d during pregnancy and 3 mg/d during lactation. The RDAs range from 6-12 mg/d for children 1-13 years and 2-3 mg/d for infants to one year.

7.7.3 Toxicity information From the American Society for Nutritional Sciences website: “Large doses of nicotinic acid given to lower cholesterol may produce flushing of the skin, hyperuricemia, and hepatic abnormalities. These effects are reversed if the drug is reduced in amount or discontinued. The 1998 Tolerable Upper Intake Level (UL) of niacin, based on flushing produced by nicotinic acid, is 35 mg/d for you to use window.”

In the recent document supporting the transition to DRIs (National Academy of Sciences), a tolerable UL of 35 mg/day is established solely on the basis of skin flushing. As usual, they point out that the UL is not meant to apply to individuals taking higher amounts under medical supervision for treatment of a condition. They also acknowledge that other than skin flushing, other symptoms of excess niacin ingestion are not reported in normal, healthy individuals. On the other hand, those with certain other conditions such as hepatic dysfunction may be susceptible to adverse effects of large doses. 105

Marks states that the vasodilation caused by niacin is not considered to be significant as an adverse reaction. He also points out that doses of 200 mg-10 g daily have been given for periods of at least 10 years with few difficulties that could be attributed to the vitamin. He concludes that 100 times the RDA represents a safe chronic dose. This would be about 1600 mg/day in males. MCN36 provides only 5% of this amount.

7.7.4 Summary comment The amount of niacin provided by MCN36 (90 mg/day) is above the upper tolerable level, established on the basis of the possibility of the ‘niacin flush’ symptom. However, very high doses of this vitamin (1.5 – 3 g) have been safely used to treat hypercholesterolemia. In relation to this clinical use, the amount provided in the supplement is unlikely to produce serious adverse effects in the subjects, even with chronic use. Perhaps it is of interest to note that in the preliminary research conducted by the university, no patient has reported experiencing a niacin flush.

7.8 VITAMIN B5 (as d-calcium pantothenate) (full dose of MCN36 = 21.6 mg) Extracted from the American Society for Nutritional Sciences web site: www.nutrition.org

7.8.1 What is it? Pantothenic acid (PA), a B-complex vitamin, is essential for humans and animals for growth, reproduction, and normal physiological functions. It is a precursor of the coenzymes, CoA and acl carrier protein of fatty acid synthase, which are involved in more than 100 different metabolic pathways including energy metabolism of carbohydrates, proteins and lipids, and the synthesis of lipids, neurotransmitters, steroid hormones, porphyrins and hemoglobin.

7.8.2 Dietary intake The Dietary Reference Intakes for pantothenic acid are 1.7 mg for infants 0-0.5 yr; 1.8 mg for children 0.5-1 yrs; 2 mg for children 1 – 3 yrs; 3 mg for children 4 – 8 yrs; 4 mg for children 9 –13 yrs, 5 mg for adults, 6 mg for pregnant women, and 7 mg for lactating women. The average North American diet provides 2-3 mg PA/1000 kcal or 4-6 mg PA/2000 kcal, which is within the range of the suggested intake.

7.8.3 Toxicity information According to the American Society for Nutritional Sciences website, in humans, the only reported symptom after intakes of 10 to 20 g calcium pantothenic acid was diarrhea.

In the recent document supporting the transition to DRIs, the National Academy of Sciences concluded that there was insufficient information to set a tolerable UL. As they say: “No reports of adverse effects of oral pantothenic acid in humans or animals were found.”

Marks draws a parallel to biotin: there is no adverse reaction reported even at high doses (10-12 g/daily). He again recommends an upper limit of 100 times the probable RDA. For a man, the RDA is 5 mg/day. The amount of pantothenic acid in MCN36 constitutes less than 5% that amount.

7.8.4 Summary comment There are no toxicity concerns with the chronic use of pantothenic acid, particularly at the low dose (21.6 mg/day) provided in MCN36.

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7.9 VITAMIN B6 (as pyridoxine hydrochloride) (full dose of MCN36 = 36 mg) Extracted from a fact sheet published by the Clinical Nutrition Service, Warren Grant Magnuson Clinical Center, National Institutes of Health (NIH), Bethesda, MD, in conjunction with the Office of Dietary Supplements (ODS) in the Office of the Director of NIH. It is copied here from their website.

7.9.1 What is it? Vitamin B6 is a water-soluble vitamin that exists in three major chemical forms: pyridoxine, pyridoxal, and pyridoxamine. It performs a wide variety of functions in your body and is essential for your good health. For example, vitamin B6 is needed for more than 100 enzymes involved in protein metabolism. It is also essential for red blood cell metabolism. The nervous and immune systems need vitamin B6 to function efficiently, and it is also needed for the conversion of tryptophan (an amino acid) to niacin (a vitamin).

7.9.2 Dietary intake The RDA is the average daily dietary intake level that is sufficient to meet the nutrient requirements of nearly all (97 to 98 percent) healthy individuals in each life-stage and gender group.

The 1998 RDAs for vitamin B6 for adults, in milligrams, are: Life-Stage Men Women Pregnancy Lactation Ages 19-50 1.3 mg 1.3 mg Ages 51 + 1.7 mg 1.5 mg All Ages 1.9 mg 2.0 mg

Results of two national surveys, the National Health and Nutrition Examination Survey and the Continuing Survey of Food Intakes by Individuals, indicated that diets of most Americans meet current intake recommendations for vitamin B6.

7.9.3 Toxicity information Too much vitamin B6 can result in nerve damage to the arms and legs. This neuropathy is usually related to high intake of vitamin B6 from supplements, and is reversible when supplementation is stopped. According to the Institute of Medicine, “Several reports show sensory neuropathy at doses lower than 500 mg per day”. As previously mentioned, the Food and Nutrition Board of the Institute of Medicine has established an upper tolerable intake level (UL) for vitamin B6 of 100 mg per day for all adults. “As intake increases above the UL, the risk of adverse effects increases.”

The UL for pyridoxine has been set at 100 mg/day for adults. Hence, the amount in MCN36 is only about a third of the amount believed to be safe for daily ingestion.

Extracted from the Merck Manual website: http://www.merck.com/pubs/mmanual/ “ The ingestion of megadoses (2 to 6 g/day for 2 to 40 mo) of pyridoxine, mistakenly taken for premenstrual tension, may cause progressive sensory ataxia and profound lower limb impairment of position and vibration sense. Senses of touch, temperature, and pain are less affected. The motor and central nervous systems are unimpaired. Recovery is slow and, in some patients, is only partial after pyridoxine ingestion is stopped.”

The following is taken from the article by Marks, who summarizes the toxicity information on chronic use quite clearly: 107

“This is one of the vitamins about which there is dispute on the question of the safety margin. It is abundantly clear that doses in the range of 500 mg--6 g daily on chronic administration can provoke a reversible neuropathy….On the other hand, doses up to 500 mg have been given for months or years without adverse effects….The main point at issue is whether adverse reactions occur at 200- 500 mg in women being treated long term for premenstrual tension. In my opinion, the balance of evidence favours safety of doses of at least 500 mg used chronically, though caution should be exercises at present above 200 mg daily. I therefore suggest on present evidence that we can define the safe level as being at least 100 times the RDA (200 mg daily).”

7.9.4 Summary comment This vitamin is not safe when taken chronically at high doses, particularly above 500 mg/day. The Merck Manual reports toxicity effects at 2g (2000 mg) per day. Marks cautiously suggests a safety level of about 200 mg/day for chronic use. Thus, the current thought is that the amount of pyridoxine in MCN36 is only 18% the amount that is safe to take chronically on a longterm basis. It is about one-third of the amount set as the Tolerable Upper Level for daily ingestion.

7.10 VITAMIN B9 (as folic acid) (full dose of MCN36 = 1000 mcg) From a fact sheet was published by the Clinical Nutrition Service, Warren Grant Magnuson Clinical Center, National Institutes of Health (NIH), Bethesda, MD, in conjunction with the Office of Dietary Supplements (ODS) in the Office of the Director of NIH. The content is extracted from their website.

7.10.1 What is it? Folate and folic acid are forms of a water-soluble B vitamin. Folate occurs naturally in food. Folic acid is the synthetic form of this vitamin that is found in supplements and fortified foods. Folate gets its name from the Latin word “folium” for leaf. A key observation of researcher Lucy Wills nearly 70 years ago led to the identification of folate as the nutrient needed to prevent the anemia of pregnancy. Dr. Wills demonstrated that the anemia could be corrected by a yeast extract. Folate was identified as the corrective substance in yeast extract in the late 1930s and was extracted from spinach leaves in 1941. Folate is necessary for the production and maintenance of new cells. This is especially important during periods of rapid cell division and growth such as infancy and pregnancy. Folate is needed to make DNA and RNA, the building blocks of cells. It also helps prevent changes to DNA that may lead to cancer. Both adults and children need folate to make normal red blood cells and prevent anemia.

7.10.2 Dietary intake The RDA is the average daily dietary intake level that is sufficient to meet the nutrient requirements of nearly all (97 to 98 percent) healthy individuals in each life-stage and gender group. The 1998 RDAs for folate are expressed in a term called the Dietary Folate Equivalent. The Dietary Folate Equivalent (DFE) was developed to help account for the differences in absorption of naturally occurring dietary folate and the more bioavailable synthetic folic acid. The 1998 RDAs for folate expressed in micrograms (mcg) of DFE for adults are:

Life Stage Men Women Pregnancy Lactation

Ages 19 + 400 mcg 400 mcg

All Ages 600 mcg 500 mcg

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1 mcg of food folate = 0.6 mcg folic acid from supplements and fortified foods

The National Health and Nutrition Examination Survey and the Continuing Survey of Food Intakes by Individuals indicated that most adults did not consume adequate folate. However, the folic acid fortification program has increased folic acid content of commonly eaten foods such as cereals and grains, and as a result diets of most adults now provide recommended amounts of folate equivalents.

7.10.3 Toxicity information The risk of toxicity from folic acid is low. The Institute of Medicine has established a tolerable upper intake level (UL) for folate of 1,000 mcg for adult men and women, and a UL of 800 mcg for pregnant and lactating (breast-feeding) women less than 18 years of age. Supplemental folic acid should not exceed the UL to prevent folic acid from masking symptoms of vitamin B12 deficiency.

In the document supporting the transition to DRIs, the National Academy of Sciences sets the UL as 1000 mcg/day. This was derived from the lowest-observed-adverse-event level (LOAEL) of 5000 mcg/day. In fact, at doses above 5000 mcg/day there have been over 100 cases of adverse neurological effects. In contrast, at doses below 5000 mcg/day, chronic daily doses of 330-2500 mcg/day have been associated with only eight cases in the literature of adverse neurological effects.

Marks points out that high doses of folic acid have not been studied very much, however 400 mg/day (275 times the amount in MCN36) has been associated with no adverse reactions when ingested over 5 months. Again, Marks recommends a safety level at 50-100 times the RDA. For an adult male, this would be up to 40,000 mcg/day; the amount in MCN36 would represent <4% of the safe level for chronic use.

Herbert and Das review the toxicity data of folic acid and conclude that it is nontoxic in humans at doses that exceed the RDA by several hundred times. It is a water soluble vitamin and the excess is simply excreted. Oral doses as high as 15 mg daily have resulted in no harm. Theoretically, a very high concentration could have a convulsant effect, although none has been reported. Reduced absorption of zinc can occur if folic acid is administered orally in isolation.

7.10.4 Summary comment Deficiency of this nutrient appears to be a much greater concern than toxicity. The major concern about exceeding the upper limit recommended by the National Academy of Sciences appears to apply only to individuals taking folic acid in isolation, because high doses can mask vitamin B12 deficiency and reduce zinc absorption. The amount provided by MCN36 appears to be less than 4% of the upper level that Marks believes would be safe for chronic use. Additionally, the supplement contains generous amounts of vitamin B12 and zinc.

7.11 VITAMIN B12 (as cyanocobalamin) (full dose of MCN36 = 900 mcg) From a fact sheet was published by the Clinical Nutrition Service, Warren Grant Magnuson Clinical Center, National Institutes of Health (NIH), Bethesda, MD, in conjunction with the Office of Dietary Supplements (ODS) in the Office of the Director of NIH. This information was extracted from the website.

7.11.1 What is it? Vitamin B12, also called cobalamin, is important to good health. It helps maintain healthy nerve cells and red blood cells, and is also needed to make DNA, the genetic material in all cells. Vitamin B12 is bound to the protein in food. Hydrochloric acid in the stomach releases B12 from protein

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during digestion. Once released, B12 combines with a substance called intrinsic factor (IF) before it is absorbed into the bloodstream.

7.11.2 Dietary intake The RDA is the average daily dietary intake level that is sufficient to meet the nutrient requirements of nearly all (97 to 98 percent) healthy individuals in each life-stage and gender group. The 1998 RDAs for vitamin B12 (in micrograms) for adults are:

Life-Stage Men Women Pregnancy Lactation

Ages 19 + 2.4 mcg 2.4 mcg

All Ages 2.6 mcg 2.8 mcg

Results of two national surveys, the National Health and Nutrition Examination Survey and the Continuing Survey of Food Intakes by Individuals found that most adult men and women consume recommended amounts of vitamin B12.

7.11.3 Toxicity information Vitamin B12 has a very low potential for toxicity. The Institute of Medicine states that “no adverse effects have been associated with excess vitamin B12 intake from food and supplements in healthy individuals.” The Institute recommends that adults over 50 years of age get most of their vitamin B12 from supplements or fortified food because of the high incidence of impaired absorption of B12 from unfortified foods in this population.

In the recent document supporting the transition to DRIs, the Academy states that “No adverse effects have been associated with excess B12 intake from food or supplements in healthy individuals.”

Marks points out that the safety margin for B12 is “large”: chronic doses as high as 30 mg/day have been given safely. The dose provided by MCN36 is less than 1/30 that high. Additionally, the level of absorption that would occur in this trial’s subjects is not known.

Herbert and Das conclude that vitamin B12 is safe in humans when ingested at a level that exceeds the RDA by 10,000 times. They state “Daily doses of up to 15 mg.…in healthy humans without convulsive disorders are without known toxic effects.”

As the body stores of this vitamin increases, absorption of additional amounts decreases. There has been no reported toxicity associated with intakes of Vitamin B12 at levels 1000 times the recommended daily intake of 6 μg/d. Animal studies have demonstrated no adverse effects of Vitamin B12, even when administered by injection in very large doses.

7.11.4 Summary comment Toxicity is obviously not a concern for Vitamin B12 at 900 mcg/day, as provided by MCN36, even when used chronically.

7.12 VITAMIN H (as biotin) (full dose of MCN36 = 1080 mcg) Extracted from the American Society for Nutritional Sciences web site: www.nutrition.org

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7.12.1 What is it? Biotin is also known as vitamin H and coenzyme R (Hexahydro-2-oxo-1H-thienal(3,4-d)- imidazole-4-pentatonic acid). It is found primarily in liver, kidney and muscle. Biotin functions as an essential cofactor for four carboxylases that catalyze the incorporation of cellular bicarbonate into the carbon backbone of organic compounds. Acetyl-CoA carboxylase (ACC) is located in the cytosol where it catalyzes the formation of malonyl-CoA which then serves as a substrate for fatty acid elongation. The other three enzymes are located in the mitochondria. Pyruvate carboxylase (PC) catalyzes the incorporation of bicarbonate into pyruvate to form oxaloacetate, an intermediate in the tricarboxylic acid cycle. In gluconeogenic tissues such as the liver and kidney, oxaloacetate can be converted to glucose. Methcrotonyl-CoA carboxylase (MCC) catalyzes the incorporation of bicarbonate into propionyl-CoA to form methylmalonyl-CoA which, in turn, is metabolized to other compounds that eventually enter the tricarboxylic acid cycle.

7.12.2 Dietary intake The estimated safe and adequate dietary intake values for biotin for different age groups are as follows: 5, 6, 8, 12, 20, 25 and 30 ug/day for ages of 0-0.5, 0.5-1, 1-3, 4-8, 9-13, 14-18 and >19 years, respectively. The values for pregnacy and lactation were estimated to be 30 and 35 ug/day, respectively.

7.12.3 Toxicity information Oral and intravenous doses up to 200 mg have not produced frank toxicity in human subjects. In animal studies, even higher doses per kilogram body weight have not produced toxicity.

In the recent document associated with the transition to DRIs, the Academy concluded that a tolerable UL could not be established due to lack of information. As they said, “No reported adverse effects of biotin in humans or animals were found.”

Marks emphasizes the low toxicity of this vitamin even at very high doses . He suggests that “For practical purpose …..a figure of at least 100 times the probable RDA may be regarded as safe.” Since the dietary recommendation for an adult is 30 mcg, 3,000 mcg/day may be assumed to be safe for chronic use. The amount in MCN36 is about one-third of this dose.

7.12.4 Summary comment Toxicty is not a concern for chronic use of biotin, particularly at the low dose of 1080 mcg/day provided by MCN36. Some sources do not even discuss it as a possibility.

7.13 CALCIUM (full dose of MCN36 = 1320 mg) Extracted from the American Society for Nutritional Sciences web site: www.nutrition.org

7.13.1 What is it? Calcium is the most common mineral in the human body. Calcium is a nutrient in the news because adequate intakes are an important determinant of bone health and risk of fracture or osteoporosis. Our nation suffers from approximately 1.5 million fractures annually with an associated health care cost of $13.8 billion.

Approximately 99% of total body calcium is in the skeleton and teeth and 1% in blood and soft tissues. Calcium has four major biological functions: 1) structural as stores in the skeleton, 2) electrophysiological – carries charge during an action potential across membranes, 3) intracellular regulator, and 4) as a cofactor for extracellular enzymes and regulatory proteins. Calcium is present

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in variable amounts in all the foods and water we consume, although the main sources are dairy products and vegetables.

7.13.2 Dietary intake The dietary recommendations set by the 1997 National Academy of Science Panel on Calcium and Related Nutrients are: 210 mg/d for 0-6 month olds, 270 mg/d for 6-12 month olds, 500 mg/d for 1- 3 year olds, 800 mg/d for 4-8 year olds, 1300 mg/d for individuals aged 9-18 years, 1000 mg/d for individuals aged 19-50 years, and 1200 mg/d for individuals over the age of 51 years. No alterations for pregnancy or lactation were recommended. The recommended upper level of calcium is 2.5 g/day.

7.13.3 Toxicity information From American Society for Nutritional Sciences website Symptoms of calcium toxicity are largely anecdotal. Excess calcium supplementation has been associated with some mineral imbalances such as zinc.

Other sources confirm that ingestion up to 2400 mg daily has not resulted in any adverse effects, except constipation in individuals sensitive to that effect. High intakes can inhibit iron absorption. Allen and Wood go on to make two additional points regarding toxicity: 1) That daily intakes above 2400 mg may put a person at risk for impaired renal function, and that 2) “Calcium supplementation carries no increased risk for stone formation in normal adults although individuals with renal problems or hyperparathyroidism may be susceptible.

The tolerable Upper Level (UL) for calcium has been set at 2500 mg/day for adults, both male and female.

7.13.4 Summary comment MCN36 provides 1320 mg/day, which is <55% the tolerable Upper Level of the new DRIs. The level in participants’ diets and their absorption of this mineral will not be known in this trial. Calcium absorption is related to calcium status and this would be lower if the patients had sufficient calcium status. The level in the supplement has been safely used under medical prescription for women with osteoporosis. To our knowledge there are no adverse side effects associated with a supplement of this level.

7.14 PHOSPHORUS (full dose of MCN36 = 840 mg) Extracted from the American Society for Nutritional Sciences web site: www.nutrition.org

7.14.1 What is it? Phosphorus (P) is an essential mineral that is found in all cells within the body. The body of the human adult contains about 400-500 g. The greatest amount of body phosphorus can be found primarily in bone (85%) and muscle (14%). Phosphorus is primarily found as phosphate (PO4 2-). The major building blocks of biology are covalent molecules comprising proteins, polysaccharides, and nucleic acids. The nucleic acids deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are polymers based on phosphate ester monomers. The high-energy phosphate bond of ATP is the major energy currency of living organisms. Cell membranes are composed largely of phospholipids. The inorganic constituents of bone are primarily a calcium phosphate salt called hydroxyapatite. A variety of enzymatic activities are controlled by alternate phosphorylation and dephosphorylation of proteins. The metabolism of all major metabolic substrates depends on the functioning of phosphorus as a cofactor in a variety of enzymes and as the principal reservoir for metabolic energy. 112

7.14.2 Dietary intake New recommendations for dietary phosphorus include a value, the RDA, which an individual should aim to meet, and a value, the Tolerable Upper Level (UL), which should not be exceeded. Intakes between the RDA and the UL ensure that serum phosphorus levels will be maintained in the normal range. Values for infants are called Adequate Intake (AI) levels, and are based on a significant portion of intake being from breast milk. It should also be noted that there are no additional requirements for P during pregnancy or lactation. The Recommended intake levels for P (mg per day) are set based on life stage groups. For infants 0 to 6, and 6 to 12 months, the RDA is 100 and 275 mg, respectively. No UL has been set for these ages as supplementation would be unlikely. For children 1 to 3 and 4 to 8 years, the RDA is 460 and 500, respectively, and the UL is 3000 mg. For youth 9 to 18 years, the RDA is 1250 mg, which indicates the higher need for phosphorus during the adolescent growth spurt; the UL for youth is 4000 mg. Adults 19 years and older have an RDA of 700 mg. The UL is 4000 mg up to age 70, then declines to 3000 mg after age 70 years.

7.14.3 Toxicity information From American Society for Nutritional Sciences website A diet containing a 2:1 dietary ratio of phosphorus to calcium can cause low blood calcium (hypocalcemia) and secondary hyperparathyroidism with excess bone resorption and bone loss in animals. Human breast milk, with a lower phosphorus content than cow milk, is considered better suited to the needs of the infant. For older ages, typical diets in the United States frequently exceed the recommended ratio; however, these diets are not believed to be harmful unless calcium intake is also very low. As intake of phosphorus rises, so does serum phosphorus. Elevated serum phosphorus levels (hyperphosphatemia) can occur in patients with renal failure due to a poor ability to excrete phosphorus in the urine. As indicated by UL values, intake of phosphorus exceeding 3 to 4 grams may be harmful in healthy individuals.

Other sources confirm that diets containing greater than a 2:1 phosphorus:calcium ratio have been shown to be harmful in lab animals. However, they also state that ordinary daily diets frequently deviate from this ratio and the diets are not believed to be causing harm.

7.14.4 Summary comment As indicated above, the new Dietary Referent Intakes specify a Tolerable Upper Level (UL) of 4000 mg/day. The amount in MCN36 is <25% that amount. The amount ingested in the daily food intake of the trial participants will not be known. Perhaps most importantly, however, is the fact that the phosphorus-to-calcium ratio in MCN36 does not exceed the ratio considered safe.

7.15 MAGNESIUM (full dose of MCN36 = 600 mg) From a fact sheet was published by the Clinical Nutrition Service, Warren Grant Magnuson Clinical Center, National Institutes of Health (NIH), Bethesda, MD, in conjunction with the Office of Dietary Supplements (ODS) in the Office of the Director of NIH.

7.15.1 What is it? Magnesium is a mineral needed by every cell of your body. About half of your body’s magnesium stores are found inside cells of body tissues and organs, and half are combined with calcium and phosphorus in bone. Only 1 percent of the magnesium in your body is found in blood. Your body works very hard to keep blood levels of magnesium constant. Magnesium is needed for more than 300 biochemical reactions in the body. It helps maintain normal muscle and nerve function, keeps heart rhythm steady, and bones strong. It is also involved in energy metabolism and protein synthesis.

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7.15.2 Dietary intake The RDA is the average daily dietary intake level that is sufficient to meet the nutrient requirements of nearly all (97-98 percent) individuals in each life-stage and gender group. The 1999 RDAs for magnesium for adults, in milligrams (mg), are:

Life-Stage Men Women Pregnancy Lactation Ages 14 – 18 410mg 360mg 400mg 360mg Ages 19 – 30 400mg 310mg 350mg 310mg Ages 31 + 420mg 320mg 360mg 320mg

Results of two national surveys, the National Health and Nutrition Examination Survey and the Continuing Survey of Food Intakes of Individuals, indicated that the diets of most adult men and women do not provide the recommended amounts of magnesium. The surveys also suggested that adults age 70 and over eat less magnesium than younger adults, and that non-Hispanic black subjects consumed less magnesium than either non-Hispanic white or Hispanic subjects.

7.15.3 Toxicity information from the NIH website: Dietary magnesium does not pose a health risk, however very high doses of magnesium supplements, which may be added to laxatives, can promote adverse effects such as diarrhea. Magnesium toxicity is more often associated with kidney failure, when the kidney loses the ability to remove excess magnesium. Very large doses of laxatives also have been associated with magnesium toxicity, even with normal kidney.The elderly are at risk of magnesium toxicity because kidney function declines with age and they are more likely to take magnesium-containing laxatives and antacids.

Signs of excess magnesium can be similar to magnesium deficiency and include mental status changes, nausea, diarrhea, appetite loss, muscle weakness, difficulty breathing, extremely low blood pressure, and irregular heartbeat.

The Institute of Medicine of the National Academy of Sciences has established a tolerable upper intake level (UL) for supplementary magnesium for adolescents and adults at 350 mg daily. As intake increases above the UL, the risk of adverse effects increases.

The newly-specified DRIs include a Tolerable UL for magnesium of 350 mg/day. As indicated above, higher levels are not a serious health risk, but may be associated with nausea and diarrhea.

According to Shils, magnesium-containing drugs can elevate serum magnesium and cause serious symptoms of toxicity ranging from hypotension, nausea and vomiting all the way to cardiac arrest. However, when a person’s kidneys are normal, even injected magnesium can be rapidly cleared. Dietary ingestion does not appear to pose a potential risk, especially if balanced with calcium, as indicated by the NIH statement (above).

7.15.4 Summary comment The amount of magnesium in MCN36 (600 mg/day) exceeds the newly-specified UL. Several variables need to be considered when evaluating the implications: a) as stated by the NIH review (above), most diets have been shown to be insufficient in magnesium; b) excess magnesium is not believed to be associated with any serious health risk; and c) magnesium is needed for the metabolism of the calcium in the supplement. The calcium:magnesium ratio in MCN36 (1320:600, or approximately 1:0.45) was established to mirror the ratio in the RDA (800:350, or approximately 1: 0.44).

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7.16 POTASSIUM (full dose of MCN36 = 240 mg) Extracted from the American Society for Nutritional Sciences web site: www.nutrition.org

7.16.1 What is it? Potassium (K) in the form of K+ is the most essential cation of the cells. Its high intracellular concentration is regulated by the cell membrane through the sodium-potassium pump. Most of the total body potassium is found in muscle tissue. Total body potassium has been used as a measure of lean body mass, of muscle mass, or (more accurately) of cell mass. Because of its association with the metabolizing, oxygen-consuming portion of the body, a decline in total body potassium is usually interpreted as a loss of muscle mass due to a catabolic condition. Potassium exists in nature in three isotopes: 39K (93.26%), 40K (0.0117%) and 41K (6.73%). 40K is radioactive and responsible for most of the naturally occurring internal radioactivity in the body. This property enables investigators to monitor total body potassium values as a function of age and disease.

7.16.2 Dietary intake The Estimated Minimum Requirement for potassium for adolescents and adults is 2000 mg or 50 mEq/day. The usual dietary intake for adults is about 100 mEq/day. For hypertension patients using diuretic medications, it is recommended often to supplement their diet with orange juice, bananas and vegetables which contain high amounts of potassium. Increased potassium intake helps maintain normal plasma levels. However, the blood level of potassium (which is sensitive to diet) is not indicative of total body potassium which is an index of cell mass and muscle.

7.16.3. Toxicity information from American Society for Nutritional Sciences website The fraction of potassium which is present outside the cells plays an active role in the propagation of electrical signals between neurons, skeletal muscle function and regulation of blood pressure. Urinary excretion protects against the accumulation of high levels of potassium. However, acute hyperkalemia can be lethal by causing cardiac arrest. {Note, however, that urinary excretion protects against the accumulation of high levels of potassium from dietary intake.}

7.16.4 Summary comment Accumulation of high levels of potassium from diet or supplementation is unlikely because of urinary excretion of excess amounts. The dose of potassium provided by MCN36 (240 mg/day) is insignificant in terms of potential toxicity.

7.17 IODINE (full dose of MCN36 = 204 mcg) Extracted from the American Society for Nutritional Sciences web site: www.nutrition.org

7.17.1 What is it? Iodine (I2) is a nonmetallic element belonging to the halogen family. Iodine is converted to iodide in the gut, efficiently absorbed in the digestive tract and carried into circulation by serum proteins. Most iodide is actively trapped by the thyroid gland where, as I2, it forms an essential component of the thyroid hormones, thyroxin (T4) and triiodothyronine. Thyroid hormones regulate cell activity and growth in virtually all tissues and are, therefore, essential for both normal embryonic and postnatal development.

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7.17.2 Dietary intake The Recommended Dietary Allowance for iodine in the United States is as follows: infants, 40-50 µg; 1-3 yrs, 70 µg; 4-6 yrs, 90 µg; 7-10 yrs, 120 µg; over 11 yrs, 150 µg; pregnancy, 175 µg; and lactation, 200 µg. Globally, the per capita iodine requirement is 150-200 µg per day.

7.17.3 Toxicity information From American Society for Nutritional Sciences website: A wide range of iodine intake is tolerated by individuals. For this reason, long term consumption of iodine through iodized salt is considered safe. Chronic, excessive iodine intakes may occasionally lead to goiter and hypothyroidism. A small increase in thyrotoxicosis (<0.1%) may occur from increasing the iodine intake of a population which has had a low intake for many generations.

The recent document supportive of the transition to DRIs places the Tolerable Upper Limit at 1.1 mg/day. At 204 mcg, the amount in MCN36 is roughly 18% the UL.

The World Health Organization expert panel on trace elements and human health pointed out that 20 mg/day of iodine (referred to as “a grossly excessive intake”) results in endemic goitre and hypothyroidism. Even at 5 mg/day (still considered to be excessive), no ill effects have been found. On the other hand, they cite some research suggesting that 2000 mcg/day should be considered to be potentially harmful.

7.17.4 Summary comment Like certain other ingredients of MCN36 (notably, iron), the primary concern about iodine in human health the world over has been deficiency syndromes rather than excessive ingestion. The 204 mcg/day contained in the full adult dose of MCN36 is less than 1/6th the dose that might become problematic with long term use, according to the World Health Organization.

7.18 ZINC (full dose of MCN36 = 48 mg) This fact sheet was published by the Clinical Nutrition Service, Warren Grant Magnuson Clinical Center, National Institutes of Health (NIH), Bethesda, MD, in conjunction with the Office of Dietary Supplements (ODS) in the Office of the Director of NIH. This information was extracted from their website.

7.18.1 What is it? Zinc has been known for more than 50 years to be an essential mineral. It is found in almost every cell in the body and is contained within more than 200 enzymes, substances needed for biochemical reactions. Zinc is important for a healthy immune system, for healing cuts and wounds, and for maintaining your sense of taste and smell. Zinc also supports normal growth and development during pregnancy, childhood, and adolescence.

7.18.2 Dietary intake “The RDA is the average daily dietary intake level that is sufficient to meet the nutrient requirements of nearly all (97 – 98%) individuals in each life-stage and gender group.” The 1989 RDAs for zinc for adults, in milligrams (mg), are:

Life-Stage Men Women Pregnancy Lactation Ages 19 + 15mg 12mg All Ages 15mg 19mg (first six months)

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16mg (second six months)

Results of two national surveys, the National Health and Nutrition Examination Survey and the Continuing Survey of Food Intakes of Individuals indicated that the diets of many adults, especially older Americans and women, do not provide the recommended amounts of zinc.

7.18.3 Toxicity information From the NIH website: The health risk of taking too much zinc is moderate to high. Zinc toxicity has been seen in both acute and chronic forms. Intakes of 150 to 450 mg of zinc per day have been associated with low copper status, altered iron function, reduced immune function, and reduced levels of high-density lipoproteins (the good cholesterol). One case report cited severe nausea and vomiting within 30 minutes after the person ingested four grams of zinc gluconate (570 mg elemental zinc). The 1989 RDA committee stated that “chronic ingestion of zinc supplements exceeding 15 mg/day is not recommended without adequate medical supervision.” The National Academy of Sciences is currently reviewing recent research and considering new recommendations on zinc intake and risk.

In the recent document supporting the transition to DRIs, the Academy sets the UL at 40 mg/day. It is important to note that the endpoint upon which this UL was establish was solely the effect on copper status in individuals who did not simultaneously receive copper supplementation. In other words, the UL established by the Academy does not pertain to MCN36 which provides both copper and zinc in balance. In fact, this UL illustrates well the inability to use the Academy guidelines to evaluate a complex nutritional supplement: of necessity, all toxicity data from the Academy are based on single-ingredient supplementation.

In addition, it should be noted that the Academy inserted its usual statement regarding the possibility of studying this ingredient at dosages that exceed the UL: “…intake above the UL may be appropriate for investigation within well controlled clinical trials.”

Stressed once again is the fact that all these guidelines are established for normal, healthy individuals. As the Academy states with respect to zinc, “The UL is not meant to apply to individuals who are receiving zinc for treatment purposes.”

The World Health Organization report on trace elements in human health expresses the opinion that the primary issue of chronic exposrre to high zinc levels is the resulting interference with copper utilization. Because MCN36 at the full adult dose provides 48 mg of zinc, it is worth quoting the WHO precisely on this topic:

“The limited human data available indicate that clinically detectable changes or functional impairments can occur at an average zinc intake of 150 mg/day or more. Interactions with nutrients influencing their absorption and utilization have been detected biochemically at total zinc intakes as low as 60 mg/day when zinc was given in the form of a supplement to a diet that, it is reasonable to assume, already provided 10 mg zinc/day.”

“To ensure that very few individuals in a population have an intake of zinc of 60 mg or higher, the Expert Consultation recommended that the adult population mean intake should not exceed 45 mg if a 20% variation in intake is assumed.”

There is much confirmation in the literature that zinc toxicity is an unusual occurrence. There is additional confirmation that the major adverse effect of administering zinc alone is copper deficiency, because of their competititve interactions. King and Keen report that “Levels of Zn supplements as low as 25 mg per day have been reported to induce Cu deficiency. The long-term 117

consumption of Zn supplements in excess of 150 mg per day has also been reported to result in low serum HDL levels, gastric erosion, and depressed immune function.”

Because we are stressing the fact that there is a difference between intakes appropriate for the general population, and intakes appropriate for use in treatment in carefully monitored patients, we note that many studies of zinc supplementation exceed the UL. For instance, researchers involved in a 1994 Canadian randomized, double-blind, placebo-controlled trial gave a daily dose of 100 mg of zinc or a placebo to 35 female anorexia patients until they achieved a 10 per cent increase in body mass index (BMI). The rate of increase in BMI of the zinc-supplemented group was twice that of the placebo group.

7.18.4 Summary comment It appears to be safe to conclude that the zinc in MCN36 is not a concern even with chronic use. Zinc supplementation at that level without copper supplementation might pose some risk. However, not only is the zinc in MCN36 below the WHO’s concern regarding toxicity at 150 mg/day, but also it is balanced appropriately with copper in this supplement.

7.19 SELENIUM (full dose of MCN36 = 204 mcg) From a fact sheet was published by the Clinical Nutrition Service, Warren Grant Magnuson Clinical Center, National Institutes of Health (NIH), Bethesda, MD, in conjunction with the Office of Dietary Supplements (ODS) in the Office of the Director of NIH.

7.19.1 What is it? Selenium is an essential trace mineral in the human body. This nutrient is an important part of antioxidant enzymes that protect cells against the effects of free radicals that are produced during normal oxygen metabolism. The body has developed defenses such as antioxidants to control levels of free radicals because they can damage cells and contribute to the development of some chronic diseases. Selenium is also essential for normal functioning of the immune system and thyroid gland.

7.19.2 Dietary intake The RDA is the average daily dietary intake level that is sufficient to meet the nutrient requirements of nearly all (97-98%) individuals in each life-stage and gender group. The 2000 RDAs for selenium for adults, in micrograms (mcg), are:

Life-Stage Men Women Pregnancy Lactation Ages 19 + 55mcg 55mcg All Ages 60mcg 70mvg

Results of the Total Diet Study, a national survey conducted by the U.S. Food and Drug Administration (1982-86), indicated that the diets of most adult men and women provide recommended amounts of selenium.

7.19.3 Toxicity information From the NIH website: There is a moderate to high health risk of too much selenium. High blood levels of selenium can result in a condition called selenosis. Symptoms include gastrointestinal upsets, hair loss, white blotchy nails, and mild nerve damage. Selenium toxicity is rare in the United States and the few reported cases have been associated with industrial accidents and a manufacturing error that led to an excessively high dose of selenium in a supplement. The Institute of Medicine has set a tolerable upper intake level for selenium at 400 micrograms per day for adults to prevent the risk of developing selenosis. “Tolerable upper intake levels represent the maximum 118

intake of a nutrient that is likely to pose no risk of adverse health effects in almost all individuals in the general population”.

Extracted from the Merck Manual website: http://www.merck.com/pubs/mmanual/: “Selenium (Se) is a part of the enzyme glutathione peroxidase, which metabolizes hydroperoxides formed from polyunsaturated fatty acids. Selenium is also a part of enzymes that deiodinate thyroid hormones. Generally, selenium functions as an antioxidant that works in conjunction with vitamin E. Plasma levels vary from 8 to 25 µg/dL (1.0 to 3.2 µmol/L), depending on selenium intake. In a recent study of patients with a history of basal or squamous cell skin cancer, selenium 200 µg/day appeared to reduce mortality from all cancers and the incidence of lung, colorectal, and prostate cancers. However, it did not prevent the appearance of skin cancer nor significantly affect all-cause mortality. These findings require further study. At high doses (> 900 µg/day), selenium produces a toxic syndrome consisting of dermatitis, loose hair, diseased nails, and peripheral neuropathy associated with plasma levels >100 µg/dL (> 12.7 µmol/L).”

The World Health Organization report on trace elements and human health reviewed studies around the world, as well as reports of community outbreaks of selenosis. From these results, they concluded that daily intakes of 900 mcg/day could definitely result in selenosis. The Expert Panel then set 400 mcg/day as the level believed to be safe for daily intake. As they state, this figure “was derived arbitrarily by dividing the mean marginal level of daily safe dietary selenium intake….by two.”

7.19.4 Summary comment Dietary selenium intakes that cause toxicity are less well defined than those required to prevent deficiency as endpoints of selenium overexposure are less clear. Studies have indicated that there are no clinical signs of selenosis at intakes of 853 μg/. There are scientists who feel the current recommendation is too conservative and should be reassessed by basing it not on prevention of deficiency symptoms but rather on the daily dose needed to prevent chronic diseases. Supplementation with 200 μg/d in patients with histories of basal cell or squamous cell carcinomas reduced the total cancer mortality and total cancer incidence.

The 204 mcg/day of selenium provided by MCN36 is within the guidelines of the fairly conservative Institute of Medicine (above), which recently set 400 mcg/day as its Tolerable Upper Limit, as well as the identical figure derived by the WHO (above). It is well below the level indicated in research showing that more than twice that amount does not result in clinical signs of selenosis. Hence, the amount contained in MCN36 is unlikely to be a problem even with chronic use.

7.20 COPPER (full dose of MCN36 = 7.2 mg) Extracted from the American Society for Nutritional Sciences web site: www.nutrition.org

7.20.1 What is it? Copper (Cu) is a trace element that is essential for most animals, including humans. The influence of copper upon human health is due to the fact it is part of enzymes, which are proteins that help biochemical reactions occur in every cell. Copper is involved in the absorption, storage and metabolism of iron. The symptoms of a copper deficiency are similar to iron deficiency anemia. Copper may be absorbed by both the stomach and small intestinal mucosa, with most absorbed by the small intestine. Copper is found in the blood bound to proteins.

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7.20.2 Dietary intake The estimated safe and adequate intake for copper is 1.5 – 3.0 mg/day. Many survey studies show that Americans consume about 1.0 mg or less of copper per day. Copper is found in foods such as nuts {0.2 to 0.5 mg/28 g (1 Tbsp.)}, shellfish (1.0 to 3.7 mg/serving), organ meats (3.8 mg/serving of beef liver) and legumes (0.2 mg/serving). Grains, grain products and chocolate have appreciable levels of copper. While these food items are good to excellent sources of copper, the absolute amount of copper absorbed may be influenced by other dietary components.

Copper absorption may be decreased by excess dietary iron or zinc. Conversely, too much copper may cause an iron deficiency. Vitamin C supplementation results in decreased copper status. In rats, large doses of vitamin C can lead to copper deficiency. Other dietary components have an influence upon copper status, but not necessarily absorption. Feeding rats either sucrose or fructose, as opposed to glucose or cornstarch, decreases copper status and exacerbates the signs of copper deficiency.

7.20.3 Toxicity information From American Society for Nutritional Sciences website: Cases of copper toxicity are rare but may occur. Excess copper consumption may lead to liver damage. Intake of supplements exceeding 3 mg copper/day for a protracted period of time may be cause for concern. Doses of 10 mg/day over several weeks may lead to toxic symptoms, such as weakness and nausea.

The recent text that provides the supporting information for the transition to DRIs places the Tolerable Upper Limit of copper at 10 mg/day. They also point out, however, that at doses above 5 mg/day, less than 20% is absorbed. At 7.5 mg/day, only 12% is absorbed. And finally, when zinc is contained in the same supplement, even less copper is absorbed. They conclude that “intake above the UL may be appropriate for investigation within well controlled clinical trials.”

The World Health Organization report on trace minerals in human health concluded that the upper limit for adults should be set at 12 mg/day for men and 10 mg/day for women. These levels were set with the assumption that people ingest up to 10 mg additionally in their diet every day. Note that these limits differ slightly from the recommendations (above) from the American Society of Nutritional Sciences.

Turnlund reports that “The amount of oral copper required to produce toxic effects is not well established….”.

7.20.4 Summary comment. The copper in MCN36 may sound rather high, but copper binds so readily to zinc that it is not absorbed well. The high level of zinc in this supplement is needed to balance the copper.

The 7.2 mg/day of copper provided by a full adult dose of MCN36 is less that the upper limit established by the WHO. It essential to recognize, in addition, that those guidelines pertain to supplementation with isolated ingredients. In contrast, MCN36 has been formulated to balance zinc and copper in a safe ratio, which is usually considered to be in the range of 5-10:1. The proper ratio can mitigate toxicity concerns. In this context, it is also important to recognize that copper is one of the ingredients for which a significant portion of the population is deficient: according to the American Society for Nutritional Sciences website, Americans consume less than 1.0 mg/day in general, indicating a significant dietary deficiency.

7.21 MANGANESE (full dose of MCN36 = 9.6 mg) 120

Extracted from the American Society for Nutritional Sciences web site: www.nutrition.org

7.21.1 What is it? Manganese (Mn) is an essential trace mineral that is concentrated primarily in the bone, liver, pancreas, and brain. This mineral is a component of several enzymes: (1) Mn-superoxide dismutase which prevents tissue damage due to lipid (fat) oxidation; (2) pyruvate carboxylase which helps break down carbohydrates; and (3) arginase which is important for nitric oxide synthesis and the formation of urea in the urine. Manganese also activates numerous enzymes, particularly glycosyltransferases which are involved with the formation of cartilage in bone and skin.

7.21.2 Dietary intake The Estimated Safe and Adequate Dietary Intakes (ESADDIs) for Mn each day are 2.0-5.0 mg for adults. For children, ESADDIs are 1.0-1.5 mg for ages 1-3 yrs; 1.5- 2.0 mg for ages 4-6 yrs, 2.0-3.0 mg for ages 7-10 yrs, and 2.0-5.0 for ages 11-14 yrs. Recent research suggests that recommendations for formula-fed infants are 0.005 mg/day and 0.030 mg/day for breast-fed infants.

Usual dietary intakes in the U.S. are about 2.2 and 2.8 mg/day for adult women and men, respectively. However, much higher intakes (10-18 mg) are found with vegetarian diets and those based on whole-grain products. Thus, the current ESADDI may be too conservative for adults.

7.21.3 Toxicity information from American Society for Nutritional Sciences website: Toxicity has occurred from industrial exposure, such as miners breathing manganese dust and drinking contaminated well water. Symptoms of toxicity are the development of a schizophrenia with nervous disorders resembling Parkinson’s disease. The reference dose (RfD) set by the EPA in 1993 is 10 mg/day for a 70 kg body weight; this dietary level is considered to be without significant risk of a deleterious effect for a lifetime of exposure. There is no evidence of toxicity occurring from ingestion of typical diets. For drinking water, the RfD is 0.2 mg Mn/L.

The inadequacy of research to justify Tolerable Upper Limits is nowhere more apparent than in the area of manganese. The UL was recently set at 11 mg/day, but the Academy was quite candid in giving the rationale: the NOAEL, no-observed-adverse-effect level, and the LOAEL, lowest- observed-adverse-effect level. On the basis of a single study, 15 mg/day was accepted as the LOAEL; on the basis of one other study, 11 mg/day was set as the NOAEL.

Recognizing the inadequacy of their data, the Acadamy also provided a risk assessment. They concluded that, “The risk of an adverse effect resulting from excess intake of manganese from food and supplements appears to be low at the highest intakes noted above.” (It is noted that manganese in water is absorbed more readily and carries with it a higher risk.)

Finally, the Academy inserted its usual statement regarding the possibility of studying this ingredient at dosages that exceed the UL: “…intake above the UL may be appropriate for investigation within well controlled clinical trials.”

The World Health Organization panel on trace elements and human health expressed little concern about toxicity. In fact, they stated that “Manganese is often considered to be among the least toxic of the trace elements when administered orally.” Industrial exposure seems to account for the only reported cases of human toxicity.

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The expert panel refused to set a safe range of population intake, because of insufficient data. However they did cite work that suggested that 3.5 mg/day was a minimal level for daily intake for good health.

Nielsen’s chapter on ultratrace minerals states that “Managanese is often considered among the least toxic of the trace elements through oral intake … Reported cases of human toxicity caused by oral ingestion of high amounts of manganese are essentially nonexistent.”He reviews the data and arguments for the 3.5 mg/day minimum level, and recommends that it be reconsidered, suggesting that 10 mg would be more realistic for the upper level, but that “Toxicity does not really enter into the picture in setting this value.”

7.21.4 Summary comment All the evidence seems to indicate that manganese deficiency is a far greater problem than manganese toxicity. The level in MCN36 (9.6 mg/day) is below the estimate of an upper tolerable limit set by the National Academy of Science.

7.22 CHROMIUM (full dose of MCN36 = 624 mcg) Extracted from the American Society for Nutritional Sciences web site: www.nutrition.org

7.22.1 What is it? Chromium (Cr) is an essential nutrient required for normal sugar and fat metabolism. Chromium functions primarily by potentiating the action of insulin. Chromium occurs primarily in the trivalent and hexavalent forms; the form in higher organisms is trivalent. This mineral occurs throughout the body with highest concentrations in the liver, kidney, spleen and bone.

7.22.2 Dietary intake The Estimated Safe and Adequate Daily Dietary Intake (ESADDI) for adults is 50 to 200 µg. Usual dietary intakes in the U.S. are about 25 µg/day for women and 33 µg/day for men. Breast-fed infants consume less than 1 µg Cr/day and the ESADDI for infants is 10 to 40 µg/day. The current ESADDI for chromium needs to be reevaluated.

7.22.3 Toxicity information From the American Society for Nutritional Sciences website: Both solubility and oxidation state affect the potential for toxicity; furthermore, the type of complex may impact toxicity. Toxic effects are limited primarily to industrial exposure to hexavalent chromium, which is much more toxic than the trivalent form. The hexavalent chromium compounds may be carcinogenic. The acidity of the stomach promotes reduction of hexavalent chromium to the trivalent form. Most of the chromium absorbed from the gastrointestinal tract is trivalent. The Reference Dose (RfD) for trivalent chromium is 1 mg/kg/day. This level is more than 300-fold the upper limit of the ESADDI, making trivalent chromium one of the least toxic nutrients.

The World Health Organization on trace elements and human health once again expresses far greater concern with deficiency syndromes than with toxicity. In fact, they state that, “Chromium toxicity as a result of oral ingestion is very unlikely.”

When making an effort to set a safe upper limit, they did so (at 250 μg/day) based essentially on no evidence: “The relatively non-toxic nature of chromium as found in food indicates that the tolerable limit for chromium is quite high. Findings that supplements of 125-200 mcg of chromium/day, in addition to the usual dietary intake, can in some cases reverse hypoglycaemia and impaired glucose tolerance, and improve both circulating insulin levels and the lipid profile, suggest that the upper 122

limit of the safe range of population mean intakes could be above 250 μg/day (italics added).” In other words, they know it is safe to take at least 250 mcg/day, but do not know how high a level is safe.

7.22.4 Summary comment Chromium is a transition element that can occur in a number of valence states including 0, +2, +3 and +6. Chromium +6 is a strong oxidizing agent that comes primarily from industrial sources (usually air bound). Toxicity is associated with this form. Chromium +3 is the most stable form in biological systems and the form found in MCN36. Even in a chelated form, it is not well absorbed and very high oral intakes would be necessary to attain anything that might be considered toxic. As there are not case studies available on toxicity, the maximum upper limit for a safe intake and dietary supplementation with chromium is generally not considered a risk.

The National Academy of Sciences publication that supports the transition to DRIs points out that “Few serious adverse effects have been associated with excess intake of chromium from food. Therefore, a Tolerable Upper Limit was not established.” As Nielsen said in the Shils et al text, “Chromium toxicity through oral ingestion, however, is not a practical concern for humans.”

7.23 MOLYBDENUM (full dose of MCN36 = 144 mcg)

7.23.1 What is it? Extracted from the American Society for Nutritional Sciences web site: www.nutrition.org: Molybdenum (Mo) is an essential nutrient for animals and humans. Tissue content of molybdenum is low, with the highest concentrations in the liver, kidney, adrenal gland and bone. It is a component of a number of enzymes, including sulfite oxidase (involved in the metabolism of sulfur amino acids), xanthine oxidase (involved in the oxidation of purines and pyrimidines and the production of uric acid), and aldehyde oxidase (involved in the oxidation of aldehydes). These enzymes share a common “molybdenum cofactor.”

7.23.2 Dietary intake The Estimated Safe and Adequate Dietary Intakes of molybdenum (µg/day) are: 15-30 at age 0-6 months, 20-40 for 6-12 months, 25-50 for 1-3 years, 30-75 for 4-6 years, 50-150 for 7-10 years, and 75-250 for adolescents and adults. This range is based on the usual dietary intake, about 75 to 240 µg/day by adults. The range was extrapolated for other age groups on the basis of body weight.

7.23.3 Toxicity information From from the American Society for Nutritional Sciences: Molybdenum toxicity is much more likely than deficiency. Toxicity is common in cattle grazing in pastures with high molybdenum soil. A high incidence of gout has been reported in humans with intakes of 10-15 mg/day.

The recent document that supports the transition to DRIs states that the Tolerable Upper Limit for molybdenum is 2 mg/day. In other words, the amount in MCN36 is about 7% the amount considered safe for ongoing ingestion.

The World Health Organization’s report on trace elements and human health stated that molybdenum intoxication’s symptoms are in part due to a secondary deficiency of copper. Molybdenum intake of 0.14-0.20 mg/kg of body weight was found to be acceptable; this translates to 140-200 mcg/kg. In the adult range of perhaps 45-90 kg body weight, the WHO is thus comfortable with daily ingestion of 6300-18000 mcg of molybdenum. MCN36 provide only 144 mcg/day, which is less than 1% of the safe limit. 123

7.23.4 Summary comment Secondary deficiency of copper with MCN36 is unlikely because it contains high levels of copper along with the molybdenum. In addition, 144 mcg/day is a very small amount (less than 1%) of the levels considered to be unsafe by the WHO, and is only 7% of the UL very recently set by the National Academy of Sciences. Nielsen argues that molybdenum is a “relatively nontoxic element” in which 100-5000 mg/kg of food or water would be necessary to elicit clinical symptoms.

7.24 IRON (full dose of MCN36 = 13.74 mg) From a fact sheet was published by the Clinical Nutrition Service, Warren Grant Magnuson Clinical Center, National Institutes of Health (NIH), Bethesda, MD, in conjunction with the Office of Dietary Supplements (ODS) in the Office of the Director of NIH.

7.24.1 What is it? Iron is an essential mineral and an important component of proteins involved in oxygen transport and metabolism. Almost two-thirds of the iron in your body is found in hemoglobin, the protein in red blood cells that carries oxygen to your body’s tissues. Smaller amounts of iron are found in myoglobin, a protein that helps supply oxygen to muscle, and in enzymes that assist biochemical reactions in cells. About 15 percent of your body’s iron is stored for future needs and mobilized when dietary intake is inadequate. The remainder is in your body’s tissues as part of proteins that help your body function. Adult men and post-menopausal women lose very little iron except through bleeding. Women with heavy monthly periods can lose a significant amount of iron. Your body usually maintains normal iron status by controlling the amount of iron absorbed from food.

7.24.2 Dietary intake The RDA is the daily dietary intake level that is sufficient to meet the nutrient requirements of nearly all (97-98%) healthy individuals in each life-stage and gender group. The 2001 RDAs for iron (in milligrams) for infants ages 7 to 12 months, children and adults are:

Age Infants, Males Females Pregnancy Lactation Children 7 – 12 Months 11mg 1 – 3 years 7mg 4 – 8 years 10mg 9 – 13 years 8mg 8mg 14 – 18 years 11mg 15mg 27 mg 10mg 19 – 50 years 8mg 18mg 27mg 9mg 51 + 8mg 8mg

Normal full term infants are born with a supply of iron that lasts for 4 to 6 months. Evidence is not available to establish a RDA for iron for infants from birth through 6 months of age. Recommended iron intake for infants from 0 to 6 months is based on an Adequate Intake (AI) of 0.27 milligrams (mg) per day that reflects the average iron intake of breastfed infants. Iron in human milk (breast milk) is well absorbed by infants. It is estimated that infants can use greater than 50% of the iron in breast milk as compared to typically less than 12% of the iron in infant formula. Cow milk is not only low in iron and poorly absorbed by infants, its use in infancy can cause gastrointestinal bleeding and iron loss from the body. For these reasons, cow milk should not be fed to infants until after age 1. The American Academy of Pediatrics recommends that infants who are not breastfed or who are partially breastfed should receive an iron-fortified formula from birth to 12 months. Formulas that contain between 4.0 to 12 milligrams of iron per liter of formula are considered iron- fortified. 124

Results of two national surveys, the National Health and Nutrition Examination Survey and the Continuing Survey of Food Intakes by Individuals indicate that diets of most adult men and post- menopausal women provide recommended amounts of iron.Diets of females of childbearing age, pregnant women, and women who breast-feed generally do not provide recommended amounts of iron.

7.24.3 Toxicity information from the NIH website: Iron has a moderate to high potential for toxicity because very little iron is excreted from the body. Thus, iron can accumulate in body tissues and organs when normal storage sites are full. In children, acute toxicity can occur from overdoses of medicinal iron. Ingestion of as few as five or six high-potency tablets can provide amounts of iron that can be fatal to a child of 22 pounds. Consuming 1 to 3 grams of iron can be fatal to children under six and lower doses can cause severe symptoms such as vomiting and diarrhea. It is important to keep iron supplements tightly capped and away from children’s reach. Any time excessive iron intake is suspected, immediately call your physician or Poison Control Center, or visit your local emergency room. In adults high intakes of iron supplements are associated with constipation, nausea, vomiting, and diarrhea, especially when the supplements are taken on an empty stomach.

In 2001, the Institute of Medicine set a tolerable upper intake level (UL) of 40 mg per day for infants and children through age 13 and 45 mg per day for adolescents ages 14 to 18 years and adults 19 years of age and older. The upper limit does not apply to individuals who receive iron under medical supervision. There may be times when a medical doctor prescribes an intake higher than the upper limit, such as when individuals with iron deficiency anemia need higher doses of iron until their iron stores return to normal.

The recent document in support of the transition to DRIs specifies a Tolerable Upper Limit of 45 mg/day. The amount in MCN36 is less than one-third of that. Long-term ingestion of 13.74 mg/day is well within a safe limit.

Other sources confirm the concern about iron overload effects, but the majority of such occurrences are due to heritary hemochromatosis or chronic liver disease. Only one form of dietary excess seems to be recognized, and has been labeled the African Bantu siderosis, after the people in whom it was first described. In that tribe, as a result of cooking in iron pots and using steel barrels for fermenting alcoholic beverages, the intake of iron was believed to exceed 100 mg/day. As Fairbanks points out, however, even in this situation it is not clear whether the ensuing clinical symptoms are due to excess iron intake, to chronic alcoholism, or to associated nutritional “disturbances”.

7.24.4 Summary comment The worldwide concern about iron is generally related to deficiency syndromes, rather than excess. In North American there would be very little risk associated with pre-menopausal women taking an additional 14 mg of iron per day. In the case of men and post-menopausal women, the 13.74 mg/day provided by MCN36, even with the level in the habitual diet added to it, is below the amount that the U.S. Institute of Medicine has set as a tolerable limit for chronic ingestion.

7.25 dl-PHENYLALANINE (full dose of MCN36 = 360 mg) From the report of the Center for Food Safety and Applied Nutrition in the Food and Drug Administration of the Department of Health and Human Services, Washington, D.C.

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7.25.1 What is it? Phenylalanine is another indispensible amino acid that must be ingested: the dietary requirement of an adult male is 980 mg/day, and the amount consumed in 100 g/day of protein is 4600 mg.

L-phenylalanine is converted to tyrosine in the liver. Much of the research on the possible toxic effects of phenylalanine in humans are found in the research on aspartame, because phenylalanine constitites 50% by weight of aspartame. In fact, a 12-oz serving of a diet soft drink with aspartame contains 100 mg of phenylalanine. The information on D-phenylalanine is less complete. Side effects such as nausea, increased bowel activity and drowsiness have been reported with doses of 750-1000 mg/day in several studies, though not in others.

7.25.2 Toxicity information The FDA document cited above concludes that doses of d-phenylalanine up to 1000 mg/day for periods as long as 6 months have been associated with side effects such as nausea, and drowsiness. It concludes that “it is not possible to estimate a maximal safe level of oral intake of D- phenylalanine”.

This government report concludes that the safety of chronic ingestion of L-phenylalanine as a dietary supplement (usually occurring in the range of 1-10 g) could not be determined. Adverse effects have not been reported for single acute oral doses as high as 10 g daily.

7.25.3 Summary comment There is of course a risk of giving phenylalanine to patients with phenylketonuria (PKU), but anyone with this disorder would have become aware of it by adulthood. Hence, the medical history and exclusion criterion for metabolic disorders will eliminate having a participant with PKU in this trial.

It is difficult to know whether the 360 mg/day om MCN36 poses a problem without knowing the dietary intakes of the patients in the trial. The FDA appears to be more concerned with extremely large doses (up to 10 g/day). The participants are monitored for the adverse effects that might occur in response to excessive phenylalanine.

7.26 GLUTAMINE (full dose of MCN36 = 180 mg) Based on information in the report of the Center for Food Safety and Applied Nutrition in the Food and Drug Administration of the Department of Health and Human Services, Washington, D.C.

7.26.1 What is it? “Glutamine is the most abundant amino acid in the body, accounting for more than 60% of the entire free amino acid pool in skeletal muscle and 20% of the total circulating free amino acid pool.”

A daily intake of only 100 g of protein provides about 9.6 g of glutamine in a daily diet. Glutamine has many functions in the human body: it is a vehicle for the transfer of nitrogen between tissues; it regulates acid-base homeostasis in the kidney; it regulates skeletal muscle catabolism; it regulates pancreatic function, and so on. Typical studies of glutamine supplementation in humans have employed doses of 3-47 g/day. No adverse effects have been noted.

Ammonia and glutamate are two by-products of glutamine metabolism. The potential neurotoxicity of glutamine and these by-products have been studied in healthy adults, using doses of 20-40 g/day. No changes have been found on mental status, mood, or performance. This government report concludes that there has been no report of adverse effects at the doses studied (3-47 g/day). 126

Although it makes no recommendations for safe upper limit, it would seem that 600 mg/day (roughly 6% of what would be obtained by eating 100 g of protein) is quite small.

7.26.2 Summary comment The amount of glutamine contained in MCN36 is quite small. In fact, it is only about 2% of the amount that would be obtained in a serving of 100g of protein.

7.27 CITRUS BIOFLAVONOIDS (full dose of MCN36 = 240 mg) From The Textbook of Natural Medicine, by Murray and Pizzorno.

7.27.1 What is it? The term “flavonoids” refers to a group of plant pigments that contribute significantly to the colors of many plants and fruits. There are over 4,000 that have been characterized and studied. One group that has been shown to have significant clinical benefit is the one known as proanthocyanidins, or PCOs (discussed below). According to Murray and Pizzorno, it was the Nobel-prize winning Alberta Szent-Gyorgyi who discovered flavonoids and demonstrated that scurvy is due to a deficiency in not only vitamin C but also flavonoids.

Citrus bioflavonoids often include hesperidin and naringin, the two major ones in MCN36. These are natural compounds with no adulteration or preservative of any kind.

7.27.2 Summary comment The small amount of citrus bioflavonoids in MCN36 is no more than would be ingested by an individual eating citrus fruit as part of their daily diet. It is unlikely to represent any threat to health.

7.28 GRAPE SEED (full dose of MCN36 = 45 mg) From the Textbook of Natural Medicine, by Murray and Pizzorno.

7.28.1 What is it? Grape seed is rich in a category of plant flavonoids known collectively as as procyanidolic oligomers (PCOs). PCOs include proanthocyanidins (sometimes called procyanidins) in various size molecules. Grape seed is not the only source of PCOs, but it is a common one. The less scientific term for PCOs or proanthocyanidin complexes is “pycnogenol.” Pycnogenol is actually a registered trademark in the U.S. and refers specifically to the PCO from the bark of a type of pine tree.

Over the last 30 years, PCOs have been shown to contribute to several biochemical functions in the human body, including the support of collagen, free radical scavenging (hence, its primary reputation as an antioxidant), assistance in visual vunction, threatment of atherosclerosis, etc. Most of these functions actually depend upon the antioxidant effects of PCOs, which Murray and Pizzorno report are 50 times greater than that of vitamins C and E.

Because of its antioxidant effectiveness, PCOs are being used and studied for their potential clinical applications in the following areas: venous and capillary disorders including venous insufficiency; varicose veins; capillary fragility; disorders of the retina; including diabetic retinopathy and macular degeneration.

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7.28.2 Toxicity information Murray and Pizzorno report that “PCO extracts are without known side-effects”. They suggest that a daily dose of 50 mg is appropriate for general antioxidant support, and that therapeutic purposes call for 150-300 mg/day.

In the American Pharmaceutical Association’s text of 1999, there is confirmatory information regarding the clinical benefits of PCOs for the treatment of a variety of vascular problems. Regarding toxicity, they state that, “The recent medical literature contains no reports of significant adverse reactions to grape seed extracts. Its toxicity does not appear to have been carefully examined, however.” PCOs have, however, been used therapeutically for decades in Europe, with apparently no toxicity concerns.

7.28.3 Summary comment PCOs such as grape seed apparently are without danger in terms of toxicity. Studies evaluating the effect of PCOs on visual function and on atherosclerosis routinely employ chronic doses of 200 mg/day. The amount provided by MCN36 is 45 mg/day at the full adult dose, and is unlikely to pose a danger even with chronic use.

7.29 CHOLINE (full dose of MCN36 = 540 mg) From the text Modern Nutrition in Health and Disease, by Shils et al.

7.29.1 What is it? Choline is an essential nutrient and is ubiquitous in our food supply. It is in most common foods, including fruit, dairy products, red meat, grains, etc. It exists as both free choline and phosphatidylcholine (also called lecithin).

Choline is a precursor to acetylcholine, the neurotransmitter. It plays many other roles in the human body: e.g., it is a component of phospholipids, it is required for the synthesis of many components of cell membranes, and so on.

7.29.2 Toxicity information In the recent document associated with the transition to DRIs, the Academy specified 3.5 g/day as the tolerable UL. The LOAEL (lowest-observed-adverse-event level) was 7.5 g/day. This level was associated with nausea, and a small amount of hypotension with chronic administration.

7.29.3 Summary comment The toxicity of choline ingestion appears not even to be a topic worthy of consideration in many texts. It is such a common nutrient that even a deficiency syndrome has been difficult to demonstrate, and toxicity appears to be nonexistent. The National Academy of Sciences recently set 3.5 g/day as its UL; the amount in MCN36 represents about 15% of the UL considered to be safe for chronic use.

7.30 INOSITOL (full dose of MCN36 = 180 mg) From the text Modern Nutrition in Health and Disease, by Shils et al.

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7.30.1 What is it? Inositol is essential for cellular growth. It is widely available in most food sources, including plants, animals, and fungi. Inositol-containing phospholipids function in cellular mediators of signal transduction, as well as metabolic regulation.

In plant food sources, inositol exists preiminantly as phytate. It is broken down in the gut by phytase, and then actively transported across the intestinal wall

In human tissue, inositol exists as both free inositol and as inositol phospholipids, each of which has different biological functions. Inositol phospholipids have been studied the most, especially in relationship to the biosynthesis of the major brain neurotransmitters: catecholamines, dopamine, and norepinephrine.

7.30.2 Dietary intake Inositol is not listed as an essential nutrient and does not have an RDA. Aukema reviews the data on possible therapeutic uses, and suggests it has potential for benefit in diabetes mellitus, chronic renal failure, and glactosemia.

7.30.3 Toxicity information Inositol appears to be so safe that once again, very little about toxicity potential is even acknowledged in the texts. As Aukema says, “Excess dietary inositol appears not to be toxic, except in certain clinical situations where inositol metabolism is impaired”.

7.30.4 Treatment of mental disorders Because levels of inositol in cerebrospinal fluid have been reported to be low in patients with depression, Benjamin et al. (1995) administered inositol to patients with major depression both in open trials and in double-blind placebo-controlled trials. Results showed that inositol was beneficial, and was equally as effective as antidepressants. Some investigators have conducted randomized clinical trials of inositol in treating depression as well as bipolar disorder. Inositol was found to have a significant antidepressant effect in double-blind controlled trial in patients with major depression. In another randomized controlled trial, eight of twelve patients treated with inositol had a 50% or greater decrease in the baseline depression scores compared to four of twelve subjects assigned to placebo (p = 0.10). Patients in this study were taking medications like lithium, valproate, and/or carbamazepine in stable doses and at therapeutic levels at study entry, and continued taking their medications throughout the study. Chengappa et al. suggested that a controlled study with a more adequate sample size may demonstrate efficacy for inositol in bipolar depression.

7.30.5 Summary comment In recent years, there has been quite a lot of research on the use of inositol in the treatment of mental disorders. From the toxicity perspective, it is worth noting that those studies are typically employing doses of 12-18 g/day. The amount of inositol provided by the full adult dose of MCN36 is only 180 mg, and all the resources found suggest that there would be no concerns about toxicity.

7.31 GINKGO BILOBA (full dose of MCN36 = 36 mg) From the Textbook of Natural Medicine.

7.31.1 What is it? It is believed that ginkgo biloba is the oldest living tree species on earth. Its medicinal use stretches back at least 5000 years to the oldest surving Chinese documents on medicine. The leaves were used 129

to “benefit the brain….and relieve the symptoms of asthma and coughs….” In modern days, there have been more than 400 clinical and experimental studies reported on the Ginkgo biloba extract (GBE).

The following is a list of some of the effects of GBE that have been well-documented: antioxidant effect, cellular membrane stabilization, scavenging of free radicals (especially in the brain), enhanced oxygen utilization in neurons. It also “promotes increase nerve transmission rate, improves synthesis and turnover of brain neurotransmitters, and normalizes acetylcholine receptors in the hippocampus”. It promotes vasodilation by directly stimulating the release of chemicals that accomplish this.

It is understandable, then, that an important clinical use of GBE has been to treat vascular insufficiency. Murray and Pizzorno state that over 50 double-blind clinical trials have shown benefits both centrally and peripherally. In the studies showing central effects, many mental symptoms have displayed significant regression, including depression, memory loss, headaches, male impotence, and tinnitus. Some of these effects have been shown in the elderly, and there has been a great deal of research on the use of GBE in age-related mental symptoms, as well as dementia such as Alzheimer’s.

7.31.2 Toxicity information GBE is very safe. Most clinical studies use 40 mg three times a day (i.e., total daily dose of 120 mg). Some studies, especially those focusing on the mental symptom of depression, use 80 mg three times a day (i.e., total daily dose of 240 mg). Side effects are quite uncommon.

“GBE is extremely safe and side-effects are uncommon. In 44 double-blind studies involving 9,772 patients taking GBE, the number of side effects reported was extremely small. The most common side-effect, gastrointestinal discomfort, occurred in only 21 cases, followed by headache (seven cases) and dizziness (six cases).”

The American Pharmacetical Association considers a daily dose of GBE to be 120-160 mg. They indicate that side effects are rare and not serious.

7.31.3 Summary comment At 36 mg/day, the Ginkgo in MCN36 is presumably exerting some moderate effect on depression and on central blood flow in general. It is below the usual dose, however, and is unlikely to be a problem in terms of toxicity.

7.32 METHIONINE (full dose of MCN36 = 60 mg) From the report of the Center for Food Safety and Applied Nutrition in the Food and Drug Administration of the Department of Health and Human Services, Washington, D.C.

7.32.1 What is it? Methionine is one of the indispensible, sulfur amino acids: its ingestion is essential to enable humans to fix inorganic sulfur into organic molecules in order to synthesize protein and other important compounds such as taurine. Research has shown that of the two isomers (D- and L- isomers), it is the latter that is more effective in humans and is utilized more efficiently. A daily intake of only 100 g of protein provides about 1.4 g of methionine in a daily diet.

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7.32.2 Toxicity information Most studies of toxicity in humans have involved more than 10,000 mg/day. For instance, some studies in patients suffering from chronic schizophrenia reported the exacerbation of symptoms when the patients were given from 10-20 g/day. Endocrine studies also have used around 30 g/day to examine the effects of methionine on insulin. Examination of biochemical changes in normal humans has employed daily doses of 12-15 g/day.

This government summary concludes that daily supplements of 3 g/day might result in adverse effects such as appetite suppression, and elevation of plasma homocystein concentrations. The full dose of MCN36 provides only 2% of the dose they consider to be potentially unsafe.

7.32.3 Summary comment The full dose of MCN36 provides only about 2% of the level that the FDA considers to be unsafe. In fact, it provides less than 1% of the amount obtained in a single serving of 100 g of protein.

7.33 GERMANIUM (as germanium sesquioxide) (full dose of MCN36 = 20.7 mg)

7.33.1 What is it? Germanium Sesquioxide is an organic form of germanium. It is the active ingredient in garlic and is found in other botanicals including aloe vera and comfrey.

The inclusion of germanium sesquioxide in this supplement is frequently questioned but the reasoning behind this questioning is based on erroneous information in the scientific literature. This is primarily due to a single error reported in 1987, subsequently corrected in a 1988 study. But the 1987 error has been repeatedly cited in the subsequent literature and has inaccurately coloured the general understanding about germanium and led to premature termination of valid scientific inquiry into its potential value in clinical treatment. A revised view of the safety of germanium, taking these historical errors into account, has been offered in recent reviews (Kaplan, Andrus, & Parish, in press; Kaplan, Parish, Andrus, Simpson, & Field, in press). The abstracts from those two papers are reproduced below. These papers indicate that the misunderstanding in the literature is based, in part, on the inappropriate generalizations about the toxicity of some forms of germanium to all other forms, including the nontoxic form present in MCN36.

Kaplan, Andrus, & Parish: Germane Facts about Germanium Sesquioxide. I Chemistry and anticancer properties. In press Abstract: This article reviews the history, chemistry, safety, toxicity, and anticancer effects of the organogermanium compound bis (2-carboxyethylgermanium) sesquioxide (referred to as CEGS). A companion review follows, discussing the inaccuracies in the scientific record which have prematurely terminated research on clinical uses of CEGS. CEGS is a unique organogermanium compound first made by Mironov and coworkers in Russia, and shortly thereafter popularized by Asai and his colleagues in Japan. Low concentrations of germanium occur in nearly all soils, plants and animal life; natural occurrence of the CEGS form is postulated but not yet demonstrated. The literature demonstrating its anticancer effect is particularly strong: CEGS induces interferon gamma (IFN-γ), enhances Natural Killer cell activity, and inhibits tumour and metastatic growth, effects often detectable after a single oral dose. In addition, oral consumption of CEGS is readily assimilated and rapidly cleared from the body without evidence of toxicity. Given these findings, the absence of human clinical trials of CEGS is unexpected. Possible explanations of why the convincing findings from animal research

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have not been used to support clinical trials are discussed. Clinical trials of CEGS are recommended (Kaplan, Parish et al., in press).

Kaplan, Parish, Andrus, Simpson, & Field: Germane Facts about Germanium Sesquioxide. II. Scientific error and misrepresentation. In press Abstract. The previous article reviewed the anticancer properties and safety of bis (2- carboxyethylgermanium) sesquioxide (CEGS). An examination of those data leads one to question why this information has not stimulated clinical trials in cancer patients. The answer is discussed in this paper, which traces the history to an error published in the scientific literature in 1987. The reliance by subsequent authors on secondary sources, citing only the error and not the correction published in 1988, constitutes part of the explanation for why CEGS has been neglected. A second factor is also considered: careless reporting about any germanium-based compound as if the many thousands of germanium compounds were all the same. This combination of publication error, careless writing, and the reliance on secondary sources appears to be responsible for the neglect of the potential clinical use of this unique germanium compound (Kaplan, Andrus et al., in press).

7.33.2 Summary comment The toxicity of germanium sesquioxide in milligram quantities is low. It is estimated that North Americans consume about 0.4-1.5 mg/day of this nontoxic form of the mineral. The metalloid germanium has found widespread application in electronics, nuclear sciences and in medicine, without indication of toxicity or danger. Germanium is not carcinogenic and there is substantive evidence that it may even in inhibit cancer development. Germanium compounds have no mutagenic activity and may, under certain conditions, inhibit the mutagenic activity of other substances. Germanium, in most of its forms, may thus be considered an element of rather low risk to man.

It is most important to note that germanium sesquioxide, the form of germanium contained in MCN36, is not a toxic form, and there has never been any direct evidence in the scientific literature to question its safety in milligram quantities. Those articles that have reviewed the toxicity of germanium have tended to neglect the distinction between germanium sesquioxide (known to be safe) and other forms (some of which are hazardous). The form and quantity of germanium in MCN36 appear to be safe, even for chronic ingestion.

7.34 BORON (full dose of MCN36 = 2400 mcg) Summarized from a World Health Organization expert panel report:

7.34.1 What is it? Although little is known about boron’s function in humans, it is certain that it affects steroid hormone metabolism. There is some evidence that a low boron diet results in excessive excretion of calcium and magnesium. The major dietary sources are plants, such as fruits, leafy vegetables, nuts and legumes. Various studies of dietary intake have suggested that the normal adult diet in the USA provides only 2-6 mg of boron per day.

7.34.2 Toxicity information The recent document in support of the transition to DRIs places a Tolerable Upper Limit of 20 mg/day for adults. It also points out that the reports of adverse effects suggest low toxicity, even at doses of 2.5 mg/kg/day (perhaps over 150 mg/day for an adult).

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There is very little information on the toxicity of chronic supplementation with boron. This WHO expert panel recommends that a safe range might be anywhere from 1-13 mg/day. Elsewhere in the same article, they suggest that 20 mg/day is the “threshold for toxicity.”

Nielson points out that boron’s toxicity is of such a low order that from 1870-1920 it was used routinely as a preserver for fish, shellfish, meat, and dairy products. They also cite a study from 1904 in which volunteers fed daily doses of 500 mg of boron, some symptoms of toxicity occurred such as appetite and digestion changes; 400 mg per day appeared to be without toxic effects.

7.34.3 Summary comment The 2.4 mg/day provided by the full adult dose of MCN36 is well below the level where toxicity would become a concern with chronic use (20 mg/day). It is also < 1% of the amount shown to result in adverse symptom reports in humans.

7.35 VANADIUM (full dose of MCN36 = 1194 mcg) From a World Health Organization expert panel report:

7.35.1 What is it? Vanadium seems to serve an important function as an enzyme cofactor, and in contributing to metabolic pathways involving hormones, glucose, lipid, bones, and teeth. Low intake of vanadium has been associated with cardiovascular disease, in broad population-based epidemiological studies.

Estimates of absorption rates range from 5-10%; in either case, it is clear that the majority of the vanadium consumed by humans is excreted in the feces.

7.35.2 Toxicity information Signs of vanadium toxicity seem to include depressed growth, diarrhea, and anorexia. In humans specifically, gastrointestinal disturbances and “green tongue” have been reported. There are a couple of reports of toxicity at chronic daily doses of 10,000 mcg. The Expert Panel did not set a threshold toxicity level, because of insufficient evidence.

Nielsen points out that vanadium can be relatively toxic at extremely high doses, and that a few human studies have demonstrated this. These human studies employed daily doses ranging from 13,500 mcg/day to 22,500 mcg/day. Studies have followed patients for up to five months; signs of toxicity at these high levels of ingestion included green tongue and gastrointestinal disturbance.

Although athletes take large doses of vanadium with apparently no adverse effects, and in fact there has not been a report of acute toxicity from vanadium supplementation, the Academy did set a UL based on available data from kidney research. The UL of 1.8 mg/day was established for adults. When characterizing the risk from overdose, they stated that “The risk of adverse effects resulting from food is very unlikely.” With respect to supplementation, they noted that there is ongoing research in individuals with diabetes to determine whether larger doses of vanadium can be useful. And they concluded that “The UL is not meant to apply to individuals who are being treated with vanadium under close medical supervision.”

7.35.3 Summary comment Vanadium is relatively toxic when provided to animals in large doses. Human studies indicate that long-term daily intakes of vanadium greater than 10 mg/day (10,000μg/d) might lead to toxicological consequences. Large doses of vanadium have been studied in patients with diabetes and there are no reports of negative effects at doses less than 4.5 mg/d. The vanadium in a full daily 133

dose of MCN36 (1194 mcg) is < 1/6th the amount that has been reported to be associated with clinical signs of toxicity. It is also below the level (1.8 mg/day) established recently as the UL.

7.36 NICKEL (full dose of MCN36 = 29.4 mcg) From a World Health Organization expert panel report:

7.36.1 What is it? The biochemical function of nickel in humans is not well-understood, although at least four important enzymes found in plants and lower organisms are dependent upon nickel: urease, hydrogenase, methylcoenzyme M reductase and carbon monoxide dehydrogenase. In farm animals, nickel deficiency results in growth impairment, as well as poor iron utilization.

Most nickel in food remains unabsorbed in the human gastrointestinal track. Typically, diets provide less than 150 mcg/day. Depending on the food ingested at the same time, absorption is thought to be less than 10% of that. Requirements for nickel have been difficult to establish.

7.36.2 Toxicity information In the recent document supporting the transition to DRIs, the Academy calculates a NOAEL (no- observed-adverse-effect level) at 5 mg/kg body weight/day, which they then used to derive a UL of 1.0 mg/day for healthy adults.

The WHO expert panel suggests a “threshold level for toxicity” to be set at less than 600 mcg/day. The amount ingested in our daily diet is often less than 150 mcg/day, according to studies from the U.K. (range for adults of 140-150 mcg), the U.S. (range for adults of 69-162 mcg), and Denmark (range for adults of 60-260 mcg).

Nielsen states that “Life-threatening toxicity of nickel through oral intake is unlikely. Because of excellent homeostatic regulation, nickel salts exert their toxic action mainly by gastrointestinal irritation and not by inherent toxicity.” He also states that it would take a daily dose of 250 mg to produce toxic symptoms, although nickel sulfate at much lower doses (600 mcg) has resulted in a positive skin reaction in individuals allergic to nickel.

7.36.3 Summary comment Toxic effects from exposure to nickel fall into two categories: industrial accidents and individuals with unusual nickel sensitivity. If one were to add the possibly-typical dietary intake of 150 mcg/day to the 29.4 mcg/day provided by a full adult dose of MCN36, the total (179.4 mcg/day) is still well below the WHO’s threshold level of toxicity of 600 mcg/day. It is also far below the recently established UL set for safe chronic ingestion.

8.0 REFERENCES

Benjamin, A., Agam, G., Levine, J., Bersudsky, Y., Korman, O., & Belmaker, R. H. (1995). Inositol treatment in psychiatry. Psychopharmacology Bulletin, 31(1), 167-175. Halliwell, C., & Kolb, B. (2003). Diet can stimulate functional recovery and cerebral plasticity after perinatal cortical injury in rats. Society for Neuroscience Abstracts, 29, 459.11., 29, 459.411. Kaplan, B. J., Andrus, G. M., & Parish, W. W. (in press). Germane facts about germanium sesquioxide. II. Scientific error and misrepresentation. Journal of Alternative and Complementary Medicine.

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Kaplan, B. J., Crawford, S. G., Gardner, B., & Farrelly, G. (2002). Treatment of mood lability and explosive rage with minerals and vitamins: Two case studies in children. Journal of Child and Adolescent Psychopharmacology, 12(3), 203-218. Kaplan, B. J., Fisher, G. C., Crawford, S. G., Field, C. J., & Kolb, B. (in press). Improved mood and behavior during treatment with a mineral-vitamin supplement: An open-label case series. Journal of Child and Adolescent Psychopharmacology. Kaplan, B. J., Parish, W. W., Andrus, G. M., Simpson, J. S. A., & Field, C. J. (in press). Germane facts about germanium sesquioxide. I. Chemistry and anticancer properties. Journal of Alternative and Complementary Medicine. Kaplan, B. J., Simpson, J. S. A., Ferre, R. C., Gorman, C., McMullen, D., & Crawford, S. G. (2001). Effective mood stabilization in bipolar disorder with a chelated mineral supplement. Journal of Clinical Psychiatry, 62, 936-944. Marks, J. (1989). The safety of the vitamins: An overview. In P. Walter, G. Brubacher & H. Stahelin (Eds.), Elevated dosages of vitamins: Benefits and hazards (pp. 12-20). Toronto: Hans Huber. Medicine, I. o. (2001). Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. Washington, D.C.: National Academy Press. Popper, C. W. (2001). Do vitamins or minerals (apart from lithium) have mood-stabilizing effects? [Commentary]. Journal of Clinical Psychiatry, 62, 933-935. Schaefer, A. L., Jones, S. D. M., & Stanley, R. W. (1997). The use of electrolyte solutions for reducing transport stress. Journal of Animal Science, 75, 258-265. Shils, M. E., Olson, J. A., & Shike, M. (Eds.). (1994). Modern nutrition in health and disease (8 ed. Vol. 1). Philadelphia: Lea & Febiger. Simmons, M. (2002). Letter to the Editor. Journal of Clinical Psychiatry, 64, 338.

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Appendix H: Medication Accountability Form

Subject ID#: ______Date: __/__/____ Visit #: ______

EMPowerplus Dispensation

Date Dispensed: __/__/____ Date Returned: __/__/____

Number Dispensed: ____ Number Returned: ____ Number Lost: ____

Compliance: ____

Start Date: __/__/____ Stop Date: __/__/____

Comments:

136

Appendix I: 24 Hour Recall/Typical Diet Form

Name: ______Study ID______Date: __/__/____

Step 1: Quick List: reports an uninterrupted listing of all foods and beverages consumed Time/Place Food Eaten Food Prep and Details (see right) Amount

Step 2: Forgotten Foods List: answers a series of 9 food category questions for additional foods Step 3: Time and Occasion: answers the time they consumed foods and what they called eating occasions Step 4: Detail Cycle: provides descriptions and amounts of each food reported, reviews each occasion and times between occasions Step 5: Final Review Probe: a final probe for anything else consumed Some other things to think about: Do you have any food or beverages at your desk, in your car, or in your book bag that you may have snacked on? What did you do when you got home from work/school?

Foods Bread: White/Wheat/type of wheat? Reg/low cal/high fiber? Cake: Home-made/store? Frosting? Frosting flavor? Cheese: Natural/processed? Low fat/Na+ Milk & other dairy: % fat? Fats: Type? Regular/light? Salted? Fish: Pre-breaded/fresh/frozen/smoked/canned/dried? Type/breed? Fruit: Fresh/frozen/canned/cooked/dried? Skin eaten? Grains: Salted water? Fat or salt added? Meat cut: % fat? Visible fat eaten? Skin eaten? How prepared? Fat/salt added? Breaded/marinated? Nuts/seeds: Raw/roasted? Oil/fry roast? Salted? Vegetables: Raw/blanched/cooked from fresh or frozen/canned? Fat or salt added?

Beverages Coffee: Regular/decaf? Ground/instant/vend? Sugar? Creamer? Juice: One juice/blend? 100% cocktail? Fortified? Ice? Soda: Regular/diet? Caffeine free? Ice? Tea: Brewed/herbal/green/instant? Ice? Caffeine? Water: Tap/bottled? Iced?

137

Appendix J: Individual Nutrient Blood Levels Pre- and Post- Supplementation

180.00

160.00

140.00

120.00

B6 100.00 copper D

80.00 ferritin Blood Level Mg

60.00

40.00

20.00

0.00 Pre Post Timepoint Figure 9. Participant 1 nutrient levels pre- and post- supplementation for vitamin B6, copper, vitamin D, ferritin, and magnesium.

14.00

12.00

10.00

A 8.00 carotenoids E-a-TC E-g-TC folate

Blood Level 6.00 TfR zinc

4.00

2.00

0.00 Pre Post

Timepoint

Figure 10. Participant 1 nutrient levels pre- and post- supplementation for vitamin A, carotenoids, vitamin E (alpha-tocopherol [a-TC] and gamma-tocopherol [g-TC]), folate, transferrin receptor (TfR), and zinc. 138

180.00

160.00

140.00

120.00

B6 100.00 copper D

80.00 ferritin Blood Level Mg

60.00

40.00

20.00

0.00 Pre Post

Timepoint

Figure 11. Participant 3 nutrient levels pre- and post- supplementation for vitamin B6, copper, vitamin D, ferritin, and magnesium.

14.00

12.00

10.00

A 8.00 carotenoids E-a-TC E-g-TC folate

Blood Level 6.00 TfR zinc

4.00

2.00

0.00 Pre Post

Timepoint

Figure 12. Participant 3 nutrient levels pre- and post- supplementation for vitamin A, carotenoids, vitamin E (alpha-tocopherol [a-TC] and gamma-tocopherol [g-TC]), folate, transferrin receptor (TfR), and zinc. 139

160.00

140.00

120.00

100.00 B6 copper 80.00 D ferritin

Blood Level Mg 60.00

40.00

20.00

0.00 Pre Post

Timepoint

Figure 13. Participant 5 nutrient levels pre- and post- supplementation for vitamin B6, copper, vitamin D, ferritin, and magnesium.

16.00

14.00

12.00

10.00 A carotenoids E-a-TC 8.00 E-g-TC folate

Blood Level TfR 6.00 zinc

4.00

2.00

0.00 Pre Post

Timepoint

Figure 14. Participant 5 nutrient levels pre- and post- supplementation for vitamin A, carotenoids, vitamin E (alpha-tocopherol [a-TC] and gamma-tocopherol [g-TC]), folate, transferrin receptor (TfR), and zinc. 140

350.00

300.00

250.00

200.00 B6 copper D ferritin

Blood Level 150.00 Mg

100.00

50.00

0.00 Pre Post

Timepoint

Figure 15. Participant 6 nutrient levels pre- and post- supplementation for vitamin B6, copper, vitamin D, ferritin, and magnesium.

7.00

6.00

5.00

A 4.00 carotenoids E-a-TC E-g-TC folate

Blood Level 3.00 TfR zinc

2.00

1.00

0.00 Pre Post

Timepoint

Figure 16. Participant 6 nutrient levels pre- and post- supplementation for vitamin A, carotenoids, vitamin E (alpha-tocopherol [a-TC] and gamma-tocopherol [g-TC]), folate, transferrin receptor (TfR), and zinc. 141

200.00

180.00

160.00

140.00

120.00 B6 copper 100.00 D ferritin

Blood Level Mg 80.00

60.00

40.00

20.00

0.00 Pre Post

Timepoint

Figure 17. Participant 7 nutrient levels pre- and post- supplementation for vitamin B6, copper, vitamin D, ferritin, and magnesium.

12.00

10.00

8.00 A carotenoids E-a-TC 6.00 E-g-TC folate

Blood Level TfR zinc 4.00

2.00

0.00 Pre Post

Timepoint

Figure 18. Participant 7 nutrient levels pre- and post- supplementation for vitamin A, carotenoids, vitamin E (alpha-tocopherol [a-TC] and gamma-tocopherol [g-TC]), folate, transferrin receptor (TfR), and zinc. 142

160.00

140.00

120.00

100.00 B6 copper 80.00 D ferritin

Blood Level Mg 60.00

40.00

20.00

0.00 Pre Post

Timepoint

Figure 19. Participant 9 nutrient levels pre- and post- supplementation for vitamin B6, copper, vitamin D, ferritin, and magnesium.

8.00

7.00

6.00

5.00 A carotenoids E-a-TC 4.00 E-g-TC folate

Blood Level TfR 3.00 zinc

2.00

1.00

0.00 Pre Post

Timepoint

Figure 20. Participant 9 nutrient levels pre- and post- supplementation for vitamin A, carotenoids, vitamin E (alpha-tocopherol [a-TC] and gamma-tocopherol [g-TC]), folate, transferrin receptor (TfR), and zinc.

143

250.00

200.00

150.00 B6 copper D ferritin

Blood Level Mg 100.00

50.00

0.00 Pre Post

Timepoint

Figure 21. Participant 10 nutrient levels pre- and post- supplementation for vitamin B6, copper, vitamin D, ferritin, and magnesium.

12.00

10.00

8.00 A carotenoids E-a-TC 6.00 E-g-TC folate

Blood Level TfR zinc 4.00

2.00

0.00 Pre Post

Timepoint

Figure 22. Participant 10 nutrient levels pre- and post- supplementation for vitamin A, carotenoids, vitamin E (alpha-tocopherol [a-TC] and gamma-tocopherol [g-TC]), folate, transferrin receptor (TfR), and zinc.

144

Appendix K: Individual Mood Ratings Over Time

Depression Mania

Mood Rating 10

0 1234567 Timepoint

Figure 23. Participant 1 depression and mania ratings over time.

Depression 15 Mania Mood Rating

0 1234567 Timepoint

Figure 24. Participant 2 depression and mania ratings over time. 145

10 Depression Mania 8 Mood Rating Mood

0 1234567 Timepoint

Figure 25. Participant 3 depression and mania ratings over time.

10 Depression Mania 8 Mood Rating

0 1234567 Timepoint

Figure 26. Participant 4 depression and mania ratings over time. 146

10 Depression Mania 8 Mood Rating Mood

0 1234567 Timepoint

Figure 27. Participant 5 depression and mania ratings over time.

Depression Mania

Mood Rating 10

0 1234567 Timepoint

Figure 28. Participant 6 depression and mania ratings over time. 147

Depression 20 Mania Mood Rating Mood 15

0 1234567 Timepoint

Figure 29. Participant 7 depression and mania ratings over time.

Depression 8 Mania Mood Rating 6

0 1234567 Timepoint

Figure 30. Participant 8 depression and mania ratings over time. 148

10 Depression Mania 8 Mood Rating Mood

0 1234567 Timepoint

Figure 31. Participant 9 depression and mania ratings over time.

10 Depression Mania 8 Mood Rating

0 1234567 Timepoint

Figure 32. Participant 10 depression and mania ratings over time. 149