Julie A Ray, PhD ARUP Institute for Clinical and Experimental Pathology® Recurrent Need for a Robust Method for Measuring T3/rT3 by LC-MS/MS: An Exercise in Madness or a New Beginning for a Misused Marker?

Outline 1. Introduction to Thyroid hormones 2. Tackling frequent fouling of the MS 3. Possibility of Eliminating LC using Deconvolution of Data? 4. rT3 as a new Bio Marker?

Effect of Thyroid Hormones 1. Metabolism (lipid, carbohydrate) 2. Growth 3. Development 4. Cardiovascular system 5. Reproductive system 6. Central Nervous System

Production of thyroid hormones

TSI Antithyroid Antibody Test TSH abnormal high low HypothyroidismHyperthyroidism Additional tests T4 Bound T4Free T4 High => Hyperthyroidism T3 Suspected hyperthyroidism with normal fT4 Normal fT4, high fT3

Why/when do we measure rT3? Controversy of measuring rT3 in the thyroid community Conventional endocrinology dismisses the value of RT3 measurement in diagnosing, treating and managing hypothyroidism. Integrative and holistic medical community, and in particular those physicians who focus on optimal hormone balance, consider RT3 to be an essential test in the diagnosis and management of an underactive thyroid

Integrative physicians believe that elevated levels of RT3 -- even though TSH, Free T4 and Free T3 values may be normal– reflect a thyroid problem at the cellular level -- "cellular hypothyroidism." T3 = active thyroid; stimulates energy and metabolism. rT3 = mirror image of T3, goes to the receptors, sticks there, and blocks T3’s ‘thyroid effect’. In times of stress and chronic illness, it lowers metabolism.

Normal T3/rT3 Ratio ~ 10 High levels of rT3 caused by Stress Dieting Chronic inflammation Fibromyalgia Obesity Insulin resistance High levels of cortisol prevents T4  T3; >>> rT3

Frequent Fouling of MS

Current Sample Preparation 400µL sample 50µL H 3 PO 4 100µL IS 700µL water SPE (Cation exchange)Washes: 1.0.1% HCOOH in water 2.700µL MeOH Elute (600µL 2.5% NH 4 OH in MeOH) Reconstitute (50µL; 80:20 Water: MeOH ) LC-MS/MS

Mass Spec parameters AnalyteQ1 (Da)Q3 (Da)DwellCECXP T3/rT T3/rT IS IS CAD: 6 CUR: 25 GS1: 50 GS2: 50 IS: 4200 TEM: 700 ihe: ON DP 55 EP 10

Time, min e4 2.5e4 3.5e4 4.5e4 5.5e4 6.5e4 7.5e4 8.5e4 9.5e4 Intensity, cps Patient serum sample: T3 65.5ug/dL, rT ug/dL

IS signal record MS Cleaned

Suspected causes for MS fouling and tackling them

496.3/184.3: 1-palmitoyl-2-hydroxy-sn-glycero-3 phosphocholine /184.3: 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine 758.6/184.3: 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-2-(9Z,12Z- octadecadienoyl)-glycero phosphocholine 806.6/184.3: 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine 786.6/184.4: C 44 H 84 NO 8 P A) Phospholipids

AX SPE method 1. Load 400µl sample in 1:1 ACN: Water + 100µL IS 2. Wash plate with 1:1 ACN: water 3. Wash plate with 50mM Ammonium acetate 4. Wash plate with MeOH 5. Wash with 1mL 2% FA in DCM 6. Elute 5% FA in MeOH 7. Dry at 50 o C 8. Reconstitute Results: Unusual elevation of T3 and rT3 B) SPE stationary phase

CX SPE method ul sample µl IS 2. Pretreat w/ 500 µl of 80:20 4% Formic Acid (aqueous): Acetonitrile. 3. Load sample 4. Wash 2% Formic Acid (aqueous). 5. Wash Dry columns 6. Elute w/ 500 µl of either: 1. Mix of Methanol: NH 4 OH 2. Mix of Acetonitrile: Methanol:NH 4 OH 7. Evaporate, Reconstitute Results: Unusual elevation of T3 and rT3 Conclusion: Under acidic conditions there is conversion of T4 to T3 and rT3.

Thankfully no conversion of T4 in our current CX method! If not Phospholipids and SPE phase then ???

C) ICP-MS of Quad washes

Ion Source temperature dependency of oxidized iodine 1. [IO] - Hypoiodite (144) 2. [IO2] - Iodite (160) 3. [IO3] - Iodate (176) 4. [Na(IO3) 2 ] - Sodium iodate adduct ion (372)

Source temp 700 o C. Q1 scan amu. Iodine salts at retention time of analyte TIC of +Q1: from Sample 4 (Prime 700) of Source temp test.wiff (Turbo Spray)Max. 8.4e8 cps Time, min Intensity, cps Q1: to min from Sample 4 (Prime 700) of Source temp test.wiff (Turbo Spray) Max. 1.1e6 cps m/z, Da Intensity, cps e5

Source temp 700 o C. Q1 scan amu. Iodine salts during high organic wash TIC of +Q1: from Sample 4 (Prime 700) of Source temp test.wiff (Turbo Spray)Max. 8.4e8 cps Time, min 8.0e8 Intensity, cps Q1: to min from Sample 4 (Prime 700) of Source temp test.wiff (Turbo Spray)Max. 1.7e6 cps m/z, Da Intensity, cps e5

Source temp 300 o C. Q1 scan amu. Iodine salts at retention time of analyte TIC of +Q1: from Sample 9 (Prime 300) of Source temp test.wiff (Turbo Spray)Max. 5.2e8 cps Time, min Intensity, cps Q1: to min from Sample 9 (Prime 300) of Source temp test.wiff (Turbo Spray)Max. 1.7e6 cps m/z, Da Intensity, cps

Source temp 300 o C. Q1 scan amu. Iodine salts during high organic wash TIC of +Q1: from Sample 9 (Prime 300) of Source temp test.wiff (Turbo Spray)Max. 5.2e8 cps Time, min Intensity, cps Q1: to min from Sample 9 (Prime 300) of Source temp test.wiff (Turbo Spray)Max. 3.8e6 cps m/z, Da Intensity, cps

Accuracy between 700 o C and 300 o C source temperatures

D) Pigmentation in 30% of samples 0.1% formic acid in water 2.5% NH 4 OH in water 5% NH 4 OH in water 2.5% NH 4 OH in water, 1 mL DCM 1% formic acid in DCM 100% MeOH Diluted in 1mL water + 100uL H 3 PO 4 SPE (CX) Washes Evaluate Cleanliness of extract Bilirubin

Visual evaluation of washes 1% formic acid in DCM Courtesy: Phenomenex

Samples extracted using current production method

Samples extracted using new wash method

Possibility of Deconvolution of Data T3/rT Quant T3/rT Qual 1. Peak area qual: peak area quant in T3 and rT3 are different 2. Use the uniqueness of T3 and rT3 fragmentation pattern to mathematically remove one as an interference from the other 3. This could allow for very fast run time without separating them by HPLC

File name Calc.Conc. By deconv All Ratios T3 quant A- Pk.Area MMA 119 T3 quant IS- Pk.Area T3 qual C1- Pk.Area MMA 175 IS qual C2- Pk.Area T3 quant PAMMA 119 T3 qual PA MMA 175 Ratio T3 qual/Quant Ratio MMA 119/MMA 175 T3 quant/IS T3 quant MMA 119/IS Quant pk area MMA/ISTD Conc calculated by the deconvoluti on algorithm File NameSample dilution Corrected for dilution LC MSMS results for T3 Cal % Cal % Cal % Cal % rT3 C3 point rT3 5 QC Low % QC Norm % QC High % QC Neg % % % % % % % % % % rT3 ratio0.68 T3 ratio0.19 Slope0.006 y-intersept0.013 r Expected Conc Ratio, m/z T3 qual/quantC1/A Cal Cal Cal Cal

File name Calc.Conc. By deconv All Ratios rT3 quant A- Pk.Area rT3 quant IS-Pk.Area rT3 qual C1- Pk.Area rT3 IS qual C2- Pk.Area rT3 quant PA rT3 qual PA Ratio rT3 qual/Quant rT3 quant/IS T3 quant Conc calculated by the deconvolutio n algorithm File Name Sample dilution Corrected for dilution LC MSMS results for rT3 Cal % Cal % Cal % Cal % T3 C4 point T3 5 QC Low % QC Norm % QC High % QC Neg % % % % % % % % % rT3 ratio0.66 T3 ratio0.19 Slope0.041 y-intersept r Expected conc Ratio, m/z rT3 qual/quantC1/A Cal Cal Cal Cal

rT3 levels to interpret Blood Brain Barrier Integrity?

Blood Brain Barrier (BBB)

CSF Albumin Index Interpretation CSF/serum albumin ratio is a test performed to compare the levels of albumin in the cerebrospinal fluid and the serum. It is useful as a measure of the integrity of the blood–brain barrier (BBB). Mostly used for MS diagnosis. CSF ALBUMIN INDEXBBB Status < 9BBB intact Slight impairment Moderate impairment Severe impairment >100Complete breakdown

34 serum and CSF matched samples from brain injury patients were analyzed for T3, rT3 and T4 Complete and moderate breakdown of BBB showed normal serum T4, T3, rT3 levels

CSF T4 low CSF T3 low CSF T3 and T4 in complete, moderate or severe breakdown of BBB

Samples related to severe/complete breakdown of BBB showed abnormally high concentrations of CSF rT3

Astrocyte Blood vessel Neuron

Conclusions 1. Improvements were made to the current LC-MS/MS method 2. Preliminary investigation for deconvolution works and might reduce the need for chromatography 3. Reverse T3 measurement in CSF samples was evaluated as a possible brain injury biomarker and correlated with blood-brain barrier integrity.

Acknowledgements Marcie Traballoni Steve McGuire David McPhie Greg Gillespie Seyed Sadjadi (Phenomenex) Sonia La’ulu Natalie Rasmussen Mark Kushnir Dr Fred Strathmann Dr Alan Rockwood Dr Joely Straseski Dr A.W. Meikle