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Urinary pyrrole (Mauve Factor): marker for oxidative stress in behavioral disorders Woody McGinnis MD [email protected] Seattle, 6 November 2004 The Mauve Factor CH3 C2H5 OHHPL (hydroxyhemopyrrolin-2-one) 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 HP CH3 C2H5 OHHPL CH3 C2H5 KP C2H5 CH3 OHKPL C2H5 CH3 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 Mauve Hall of Fame Hoffer Irvine Osmond Pfeiffer Sohler Cutler O'Reilly Graham Riordan Jackson Walsh Audhya Europe 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 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 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 O'Reilly 1965 • Report on 850 behavioral patients • 25% of "disturbed children" mauvepositive, vs 12% of well children • First documented observation of Mauve association with stress Mauve in schizophrenia • Hoffer 1961, 1963, 1966 • Yutwiller 1962 • O'Reilly 1965 • Sohler 1967 x 2 High-Mauve and behavior • Down syndrome 70% • Schizophrenia 40-70% • Autism 50% • ADHD 30% • ETOH 20-80% 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. 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 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 OHHPL Levels and B6 (10mg/kg/day) + Zn (25mg) + Mg (400mg) in Autism micromoles/100ml 120 100 80 60 40 20 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Months of Treatment 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 Walsh • Low stress tolerance • Anxious, overly pessimistic • Explosive anger • Hyperactivity Kruesi • Social withdrawal • Emotionally labile • Loss of appetite • Fatiguability Mauve and stress • Mauve is associated with stress, including, emotional stress. Audhya 1992. Cold-immersion increased Mauve <1 hour • The correlation is welldocumented over decades 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 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%. 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. 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 Mauvepositives, all borderline. And no zinc.) 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. 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" 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) 18 16 OHHPL vs. RBC Zinc RBC Zinc 14 12 10 8 Correlation Coefficient -.985 6 4 2 0 0 50 100 OHHPL 150 7 Mauve vs WBC Zinc 6.5 WBC Zinc 6 5.5 5 Correlation Coefficient -.743 4.5 4 3.5 0 10 20 30 40 Mauve 50 60 70 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 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 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 headtwitching (as with psychotomimetics) 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 Why do they both work? Niacinamide (B3) OR Vitamin B6 (P5P) and Zinc Thinking points • B3, Zn and B6 are anti-oxidant • Strong stress / Mauve association • Emotional stress clearly causes oxidative stress • The behavioral and somatic highMauve disorders feature high oxidative stress High Oxidative Biomarkers • Down Syndrome • Schizophrenia • Autism • ADHD • Emotional Stress • Cancer and Inflammatory Disease • Hyperglycemia Zinc is anti-oxidant • Shields -SH groups • Blocks lipid peroxidation and PLA2 • Induces metallothionein • Constituent of SOD • Maintains vitamin A • Deficiency increases intestinal NO˙ Zinc deficiency increases oxidative stress • Lower glutathione, vitamin E, GST, GSHPx and SOD • Increased reactive species and lipid peroxides in tissue, membranes and mitochondria 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 Oxidative stress Low zinc 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 Marginal B6 deficiency: Lowers GSHPx Lowers glutathione reductase Promotes mitochondrial decay Raises lipid peroxide levels 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 1O2 attack B6 vitamers 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 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 B3 is anti-oxidant • Niacin antagonists increase lipoxidation • Low B3 decreases MT and increases apoptosis in brain cells • Neuroprotective in experimental mitochondrial toxicity Reciprocal relationships Oxidative stress Poor energetics Excitotoxicty Require heme • Cystathionine synthase • Catalase • Heme-hemopexin for MT translation • Pyrrolase • Guanylate cyclase • Cytochromes • Sulfite reductase • NOS 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% 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 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), NO2 and NO3 Heme depression • Neuroblastoma cells fail to differentiate, axons degenerate • Astrocytoma cells fail to proliferate • Heme synthesis itself depends on sufficient B6 and Zn 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 5,000.00 Mauve 4,500.00 Glutathione 4,000.00 vs. RBC Glutathione 3,500.00 3,000.00 2,500.00 2,000.00 1,500.00 Correlation Coefficient -.408 1,000.00 500.00 0.00 0.00 5.00 10.00 Mauve 15.00 20.00 2.00 1.80 Mauve vs. GST 1.60 1.40 GST 1.20 1.00 0.80 Correlation Coefficient -.65087 0.60 0.40 0.20 p<.02 0.00 0.00 5.00 10.00 Mauve 15.00 20.00 500 450 OHHPL vs. Biotin 400 350 Biotin 300 250 200 Correlation Coefficient -.973 150 100 50 0 0 20 40 60 80 100 OHHPL 120 140 160 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 Proposed mechanism • Sequential lipid oxidation, protein adduction, oxidative side-chain scission and dissociation. • Models: Isolevuglandin pyrroles and urinary pyrroles from hexane IsoLevuglandin E2 protein-NH Schiff base protein-NH O OH O OH COOH COOH OH protein-- N COOH O OH COOH protein-- N O IsoLevuglandin E2 protein-- N Schiff base Pyrrole protein-NH Schiff base OH COOH O HO protein-- N protein-- N O OH OH protein-- N COOH COOH COOH protein-- N Schiff base Hydroxylactam OH Pyrrole Pyrrole rea is HPL (Hydroxyhemopyrrolin-2-one), putative Mauve Factor OH OH 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 B6dependent enzymes. More Mauve research! • Controlled clinical trials • Other therapeutic and toxic modulators • Pro-oxidant and excitotoxic properties • Prove origin • Genetic predisposers 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