1. Urinary pyrrole (Mauve Factor): marker for oxidative stress in behavioral disorders Woody McGinnis MD mcginnis@mind.net Seattle, 6 November 2004

2. CH 3 C2H5C2H5 The Mauve Factor OHHPL (hydroxyhemopyrrolin-2-one)

3. OHHPL (Mauve Factor) In human urine, blood and CSF Mistakenly identified as kryptopyrrole, a persistent erroneous term Chemically similar to kryptopyrrole, which can be used for OHHPL assay

4. CH 3 C2H5C2H5 C2H5C2H5 HPKP CH 3 C2H5C2H5 C2H5C2H5 OHHPLOHKPL

5. Mauve history Discovered in urine in 1957 Named for lilac-colored appearance on paper chromatograms developed with Erhlich's reagent Labile and elusive Abram Hoffer is the father of Mauve

6. Mauve Hall of Fame Hoffer Irvine Osmond Pfeiffer Sohler Cutler O'Reilly Graham Riordan Jackson Walsh Audhya Europe

7. Hoffer J Neuropscyh 1961 Qualitative Mauve assay All normals mauve-negative 27/39 early schizophrenics positive All 7 who recovered on niacinamide converted to negative

8. Hoffer 1961 Relapses associated with reappearance of Mauve Apparent role in other behaviors: ETOH, depression. A "mentally retarded" mauve-positive child responded dramatically to niacinamide

9. Hoffer and Mauve Heat and light sensitive Relatives should be tested Preventive potential 10/14 criminal / deviant positives Report on 740 patients in 1966 All recovered schizophrenics negative, unrecovered 50% positive

10. O'Reilly 1965 Report on 850 behavioral patients 25% of "disturbed children" mauve- positive, vs 12% of well children First documented observation of Mauve association with stress

11. Mauve in schizophrenia Hoffer 1961, 1963, 1966 Yutwiller 1962 O'Reilly 1965 Sohler 1967 x 2

12. High-Mauve and behavior Down syndrome 70% Schizophrenia 40-70% Autism 50% ADHD 30% ETOH 20-80%

13. Carl Pfeiffer 1972 "Sara" 15 y.o. with four years of unreality spells, insomnia, seizures, attempted suicides, knee problems; quite well on B6 1000 mg, Zn 160 mg, Mn 8 mg. Signs, symptoms, and clinical response imply high B6 / zinc need in high-Mauves. B6 and zinc quickly recognized by clinicians as main-stay treatment.

14. Mauve levels Clinicians: behavioral symptoms in individuals correlate with level Irvine 1972: likelihood of depressive reactions correlate with level Cutler 1974: B6 dose needed to normalize Mauve proportional to level McCabe 1983: Mauve can be normalized with high-dose B6 only

15. Pfeiffer 1983 Symptoms may improve in 24 hours, usually within 1 week May need months for full recovery Relapse within days or weeks if no nutrients Changing needs

16. OHHPL Levels and B6 (10mg/kg/day) + Zn (25mg) + Mg (400mg) in Autism

17. Pfeiffer correlates Nail spots Stretch marks Pale skin Poor tanning Knees and joints Constipation Dream recall Morning nausea Light and sound Odor intolerance Migraines Stitch-in-side

18. Walsh Low stress tolerance Anxious, overly pessimistic Explosive anger Hyperactivity Kruesi Social withdrawal Emotionally labile Loss of appetite Fatiguability

19. Mauve and stress Mauve is associated with stress, including, emotional stress. Audhya 1992. Cold-immersion increased Mauve <1 hour The correlation is well- documented over decades

20. Mauve and stress O'Reilly 1965 Sohler 1971 Pfeiffer "stress-dosing" 1973 Ward says across all diagnoses 1975 Hippchem 1978 McCabe 1983 Jaffe and Kruesi 1992

21. Non-behavioral Mauve Acute Intermittent Porphyria Cutler 1974: High-mauve obesity and abnormal glucose tolerance Hoffer 1966: 33/99 Cancer patients, 7/8 lung cancer patients Riordan and Jackson: 43% of general medical patients: arthritis, chronic fatigue, heart disease, hypertension, irritable bowel, migraine. Range 20-40 mcg%.

22. Mauve bumps in the road U Michigan 1962: no pathological importance in schizophrenia because found 34% Mauve-negative. U Cal 1969: their "simplified" assay produced phenothiazine false-positive India 1971: No Mauve in 120 psych patients--used HCl, 24° collection.

23. More bumps in the road.. Am J Psychiatr 1978: "Pyroluria a poor marker in chronic schizophrenia" (based on 2/9 Mauve-positives) J Nutr 1979: "..urinary kryptopyrrole..proved invalid as screening test for vitamin dependent disorders.." (based on 6/20 Mauve- positives, all borderline. And no zinc.)

24. Bumps U Cal 1975: "Non-occurrence of kryptopyrrole and hemopyrrole in urine of schizophrenics by GC-MS" UC Berkeley 1978: GC/MS shows no kryptopyrrole or hemopyrrole in schizophrenics or controls Irvine 1977-78 confirms Mauve is OHHPL by synthesis.

25. Bumps.. Irvine, Nature 1969: Mauve identified unequivocally as kryptopyrrole Irvine, in landmark Orthomolecular Psychiatry, "Mauve is kryptopyrrole" Irvine 1974: lactam of kryptopyrrole is the "identity of the natural kryptopyrrole"

26. Was Pfeiffer right about Mauve and low zinc? Walsh. 1148 ADHD patients: Plasma Zn vs colorimetric Mauve Strong negative correlation 0.974 significance (F test)

27. OHHPL vs. RBC Zinc Correlation Coefficient -.985

28. Correlation Coefficient -.743 Mauve vs WBC Zinc

29. Mauve as clinical tool Careful specimen collection Mild 20-30, moderate 30-40, severe over 40 mcg% Elevations imply zinc and B6 need Titrate nutrients to suppress Mauve Individualize adjunctive nutrients

33. Mauve is OHHPL Graham, Univ Glasgow 1978 quantified normal range by GLC Audhya 1994-present: commercial OHHPL by HPLC/MS and synthetic standard. Strong logical imperative to cease "kryptopyrrole" terminology

34. OHHPL facts Irvine 1977: levels correlate with emotional withdrawal, motor retardation, blocked affect and severe depression; IP to rats: ptosis, locomotor aberration, hypothermia Cutler 1990: IP to mice increased backward locomotion and head- twitching (as with psychotomimetics)

35. OHHPL facts Photo, heat, and acid-labile Urinary half-life 10-12 hours Nearly 100% urinary clearance intact after IP administration Daily excretion up to 1 mg Urine, Blood, CSF; animal brain Graham: similarity to kainic acid and pyroglutamate suggests excitoxicity

36. Why do they both work? Niacinamide (B3) OR Vitamin B6 (P5P) and Zinc

37. Thinking points B3, Zn and B6 are anti-oxidant Strong stress / Mauve association Emotional stress clearly causes oxidative stress The behavioral and somatic high- Mauve disorders feature high oxidative stress

38. High Oxidative Biomarkers Down Syndrome Schizophrenia Autism ADHD Emotional Stress Cancer and Inflammatory Disease Hyperglycemia

39. Zinc is anti-oxidant Shields -SH groups Blocks lipid peroxidation and PLA 2 Induces metallothionein Constituent of SOD Maintains vitamin A Deficiency increases intestinal NO˙

40. Zinc deficiency increases oxidative stress Lower glutathione, vitamin E, GST, GSHPx and SOD Increased reactive species and lipid peroxides in tissue, membranes and mitochondria

41. Oxidants mobilize zinc Oxidants release complexed zinc from zinc-binding proteins, including metallothionein and albumin It is likely--but unproven--that zinc retention is reduced in direct relationship to oxidative stress

42. Oxidative stress Low zinc

43. B6 is anti-oxidant P5P for Glutathione, Metallothionein, CoQ10 and Heme synthesis With Zn, cofactor for GAD P5P protects vulnerable lysinyl groups, as in GSHPx

44. Marginal B6 deficiency: Lowers GSHPx Lowers glutathione reductase Promotes mitochondrial decay Raises lipid peroxide levels

45. B6 and oxidative stress Binding of P5P-dependent enzymes is subject to carbonyl inhibition Binding of key P5P-dependent enzymes such as GAD impaired by oxidants generally OH˙ and 1 O 2 attack B6 vitamers

46. B6 and Mauve B6 levels are normal Pfeiffer alluded to lower P5P and EGOT activity in high-Mauves Lower zinc may impair B6 activation Oxidative stress affects activation of B6 and binding of B6-dependent enzymes

47. B3 is anti-oxidant NADPH for reduction of glutathione Potent free-radical quencher: protects both lipids and proteins from oxidation Blocks NO˙ neurotoxicity High tissue levels: better lipoxidation prevention than ascorbate

48. B3 is anti-oxidant Niacin antagonists increase lipoxidation Low B3 decreases MT and increases apoptosis in brain cells Neuroprotective in experimental mitochondrial toxicity

49. Reciprocal relationships Oxidative stress Poor energetics Excitotoxicty

50. Require heme Cystathionine synthase Catalase Heme-hemopexin for MT translation Pyrrolase Guanylate cyclase Cytochromes Sulfite reductase NOS

51. OHHPL and heme Durko 1970. Oxidized kryptopyrrole binds heme in vitro Graham 1979. Microsomal levels 48 hours after IP OHHP to rats: Heme down 42% Cytochrome p450 down 50%

52. Regulatory heme Sustains zinc, vitamin A and melatonin levels Differentiation, response to growth factors and resistance to viruses Levels lowered by toxins: gasoline, benzene, arsenic and cadmium

53. Low Heme is pro-oxidant Ames 2002. Experimental heme depression in cultured brain cells: Decreased Complex IV 50% reduction in intracellular Zn Increased intracellular Fe Increased NOS (x 7), NO 2 and NO 3

54. Heme depression Neuroblastoma cells fail to differentiate, axons degenerate Astrocytoma cells fail to proliferate Heme synthesis itself depends on sufficient B6 and Zn

55. Hypothesis Mauve may be a significant contributer to oxidative stress. So, it may be a good biomarker for oxidative stress. Heme depression, with impaired energetics, zinc, and detox Mauve excitotoxicity

56. Mauve vs. RBC Glutathione Correlation Coefficient -.408

57. Mauve vs. GST Correlation Coefficient -.65087 p<.02

58. OHHPL vs. Biotin Correlation Coefficient -.973

59. A second hypothesis Mauve may form as a result of oxidative stress. Sequential lipid oxidation, protein adduction, oxidative side-chain scission and dissociation. Models: Isolevuglandin pyrroles and urinary pyrroles from hexane

60. Proposed mechanism Sequential lipid oxidation, protein adduction, oxidative side-chain scission and dissociation. Models: Isolevuglandin pyrroles and urinary pyrroles from hexane

62. Oxidative by-product? Some consistencies: In the high-Mauve schizophrenics, membrane arachidonate, presumed substrate, is lower. Formation of the pyrrolic adduct occurs at lysinyl groups, so may impair pyridoxal kinase and the B6- dependent enzymes.

63. More Mauve research! Controlled clinical trials Other therapeutic and toxic modulators Pro-oxidant and excitotoxic properties Prove origin Genetic predisposers

64. The Oxidative Stress in Autism Study Principle Investigator: William Walsh NIH Author: Robert Salomon Collaborators: Bruce Ames, Allen Lewis, George Perry, Domenico Pratico, Aristo Vojdani Sponsors: Alexander and Bo MacInnis