Supplementary Material Fundamental role of Methylenetetrahydrofolate Reductase 677 C->T genotype and Flavin compounds in biochemical phenotypes for schizophrenia and schizoaffective psychosis. Stephanie Fryar-Williams1,2,3 Youth in Mind Research Institute, Norwood, SA, Australia. 2 The Queen Elizabeth Hospital, Woodville, SA, Australia. 3 Basil Hetzel Institute for Translational Health Research, Woodville, SA.
1
* Correspondence: Stephanie Fryar-Williams MBBS BSc. FRANZCP. Youth in Mind Research Institute 106 The Parade, Norwood, South Australia, 5067. Phone +61 411310449. E mail
[email protected] Supplementary Sections S1-S13. S1 Pilot Study Summary S2 Exclusion criteria S3. Severity and disability outcome measures, with citations. S4. Methods for candidate biochemical markers, including equipment and citations. S5. Further methods for candidate biochemical markers, including equipment and citations S6. Summary of biomarker results. S7. Sample characteristics for recruitment outcomes, medications, data imputation. S8. Compound biomarkers of further theoretical interest S1 Pilot study summary A case series pilot study was conducted in 2007-2009. In this study, 10 out of 15 cases with schizophrenia (67%, P 0.002) and 7 out of 8 cases with auditory processing disorder (87%, P0.0002) had accumulated three or more abnormal biochemistry markers, when compared with 15 in-clinic selected control cases. These abnormal markers were for homocysteine, hydroxyhaempyrroline-2-one (HPL), folate, zinc, percent free copper and histamine. From these need for a minimum number of 60 cases and 60 matched controls, was estimated for 95% confidence interval power for a phase II casecontrol study. S2 Exclusion criteria Exclusion criteria included medication with Clozapine, Olanzapine which are frequently-prescribed medications for ward and outpatient clinic patients with repeated admissions for psychosis. Together with antihistamines, the following medications have prominent histamine- binding effects, so were excluded as histamine was a candidate biomarker. Patients taking antipsychotic-agents such as Zuclopenthixol, Modecate, Amisulpride, Quetiapine and Risperidone were included. Persons on mood stabilizing medications were allowed. Persons with active or unremitted use of alcohol or other substance abuse were excluded, since this can confound neurotransmitter results. Persons with organic cerebral damage as evidenced by a clinically documented, investigated or descriptive history of hospitalized head injury, unconsciousness or central nervous system disease were also excluded, as were persons with upper respiratory tract infections, middle ear congestion or known sensory or
learning disability. Persons with extra-pyramidal signs in ocular, arm or hand muscles were excluded prior to consent. Persons receiving vitamin therapy were also excluded due to the inclusion of vitamins as candidate markers. It was not possible to exclude smoking and have any chance of patient recruitment. Persons taking anti-histamine medication or vitamin supplementation similar to candidate markers, were also xcluded. S3. Severity and disability outcome measures, with citations. Functional Rating Scale
Citation
The Brief Psychiatric Rating Scale (BPRS) Positive and Negative Syndrome Scale for schizophrenia (PANSS)
Clinical Global Impression of Severity (CGI)
Overall JE and Gorham DR (1962) The Brief Psychiatric Rating Scale. Kay SR, Fiszbein A and10(3): Opler799-812. LA (1987) The positive and Psychological Report negative syndrome scale (PANSS) for schizophrenia. Schizophrenia Bulletin 13(2): 261-276. Guy W (1976) Clinical global impressions [CGI]. ECDEU assessment manual for psychopharmacology. National Institute of Mental Health, Early Clinical Drug Evaluation, Psychopharmacology Research Branch, Rev. Rockville, MD, U.S.
Global assessment of Function (GAF)
Frances A, Incus HA and First MB (1994) Diagnostic and Statistical Manual of Mental Disorders, 4th edition, American Psychiatric Association, Washington, DC.
Social and Occupational Functioning Assessment Scale (SOFAS)
Goldman HH, Skodol AE and Lave TR (1992) Revising axis V for DSM-IV: a review of measures of social functioning. American Journal Psychiatry 149(9): 1148-1156.
S4. Methods for candidate biochemical markers, including equipment and citations. Nutrition-Biochemistry
Vitamin D ( 25-OH)
Serum total Vitamin B12
Plasma Red Cell Folate
Serum Vitamin B6 (Pyridoxal- 5’-phosphate coenzyme form)
All fasting blood samples, collected between Laboratory/Reference 9 and 11 am daily. By arrangement, transported directly to the laboratory with no storage. Diasorin Liason assay kit, for use on the Clinpath Laboratories, 19 Fullarton Rd, Kent Liaison platform. (nmol/L) Town. South Australia 5067 +61 8 8366 2000. Competitive Electrochemiluminescent Clinpath Laboratories. As above. Immunoassay. Roche Modular E 170 Automated Immunoassay Analyser, using Roche Vitamin B12 Reagent. (nmol/L) Competitive Electrochemiluminescent Protein Clinpath Laboratories. As above. Binding Assay, using Roche Modular E 170, using Roche Folate Red Blood Cell (RBC) Reagent and Roche Folate RBC Haemolysing Reagent on Automated Immunoassay Analyser. (nmol/L) Whole blood High Pressure Liquid Sullivan Nicolaides Pathology Chromatography with fluorescent detection. 143 Whitmore St, Taringa. Queensland 4068. Chromsystems Vitamin B6 in Whole Blood Australia. +61 7 337 8666 High pressure Liqid Chromatography Reagent Kit. Waters Alliance 2695 Separations Module. Waters 474 Fluorescence Detector.(nmol/L)
Serum Copper
Flame Atomic Absorption Spectrophotometry. Douglass Hanly Moir Pathology Varian AA-240FS. (umol/L) 14 Griffnock Avenue, Macquarie Park. New South Wales 2113. +61 2 9855 5222. Plasma Red Cell Zinc Inductively coupled plasma mass spectroscopy Sullivan Nicolaides Pathology. (ICP-MS), using 6% n-Butanol reagent and Serum Ceruloplasmin Immunoturbidimetric method, using 6K91-30 Douglass Hanly Moir Pathology. Agileny ICP-MS 7500ce analyser. (umol/L) Multignet Caeruloplamin Kit and Abbott Percentage Free Copper/Red Percentage of free copper in the serum Calculated by authors Architect analyser. (g/L)on the Cell Zinc calculatedci16000 by an equation based molecular and atomic weights of ceruloplasmin and copper (one ceruloplasmin molecule binds to six copper atoms). The ratio of the percentage free copper to red cell zinc was calculated as “percentage free copper” / “Red cell zinc umol/L”.
Intermediate substrates and enzymes MTHFR Ala222Val (C677T) Real time PCR analysis methyl tetrahydrofolate Roche Diagnostics Light-Cycler 480 kit. Using reductase polymorphism TecnoBiol reagents, Sigma probes and primers on Roche LC480 analyser. Plasma homocysteine Ice transported EDTA sample. Competitive Chemiluminescent Immunoassay, using Seimens Homocysteine reagent on Seimens Advia centaur Automated Immunoassay.(umol/L) Serum histamine Beckman Coulter Radio Immunoassay, using Beckman Coulter R.I.A. Kit on Perkin Elmer Wizard 1470 Automated Gamma Counter. (umol/L)
Douglass Hanly Moir Pathology.
SA Pathology. +61 8 8222 3000
Sullivan and Nicolaides.
S 5. Further methods for biochemistry markers, including equipment and citations. Test Neurotransmitters
Method, Analyzer, Reagents.
Laboratory/Reference
Biogenic amines: Dopamine, Noradrenaline and Adrenaline,
Spot-baseline (fasting) urinary neurotransmitter testing (second void morning), snap-frozen to minus 30 degrees and analysed by mass spectrometry, using nanomols per millimol of urinary creatinine as a standard.
SA Pathology, Adelaide, South Australia.
Creatinine
Whiting MJ. 2009. Simultaneous measurement of urine metanephrines and catecholamines by liquid chromatography with tandem mass spectrometric detection. Annals of Clinical Biochemistry, 46:129-136
Spot urine specimen from the same void as SA Pathology, Adelaide SA. biogenic amines, expressed in (millimols per Litre) Oxidative stress: Fasting urine sample collected whilst patient at rest, separated from blood drawing by Urinary hydroxyhemopyrro minimum of 2 hours.
line-2- one
S6. Summary of single biomarker results (XLSTAT, alpha 0.05).
AUC
p-value (Twotailed)
Sensitivity
Specificity
PPV
NPV
Accuracy
DA (n 133)
0.687
< 0.0001
0.364
0.94
0.058
0.993
0.661
NA (n 133)
0.835
< 0.0001
0.792
0.791
0.037
0.997
0.792
Adrenaline (n 133)
0.836
< 0.0001
0.74
0.821
0.04
0.997
0.778
Free Cu:Zn (n 133)
0.599
0.032
0.468
0.761
0.019
0.993
0.603
Vitamin B6 (n 129)
0.647
0.001
0.808
0.484
0.016
0.996
0.662
RC Folate (n 133)
0.659
0.000
0.577
0.731
0.021
0.994
0.648
Vit D (n132)
0.664
0.000
0.61
0.687
0.019
0.994
0.646
HPL/Creatinine (n 133)
0.696
