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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.

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Presentation on theme: "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."— Presentation transcript:

1 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?

2 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?

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

4 Production of thyroid hormones

5 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

6 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

7 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.

8 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

9 Frequent Fouling of MS

10 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

11 Mass Spec parameters AnalyteQ1 (Da)Q3 (Da)DwellCECXP T3/rT3651.8507.81003314 T3/rT3651.8605.81003118 IS657.8513.81003314 IS657.8611.81003118 CAD: 6 CUR: 25 GS1: 50 GS2: 50 IS: 4200 TEM: 700 ihe: ON DP 55 EP 10

12 0.51.01.52.02.53.03.5 Time, min 5000.0 1.5e4 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, rT3 6.66 ug/dL

13 IS signal record MS Cleaned

14 Suspected causes for MS fouling and tackling them

15 496.3/184.3: 1-palmitoyl-2-hydroxy-sn-glycero-3 phosphocholine 524.37/184.3: 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

16 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

17 CX SPE method 1. 400ul sample + 100 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.

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

19 C) ICP-MS of Quad washes

20 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)

21 Source temp 700 o C. Q1 scan 100-400 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. 0.51.01.52.02.53.03.54.0 Time, min Intensity, cps 0.50 4.33 2.66 0.75 3.01 1.03 1.39 1.42 +Q1: 1.407 to 2.194 min from Sample 4 (Prime 700) of Source temp test.wiff (Turbo Spray) Max. 1.1e6 cps. 100120140160180200220240260280300320340360380400 m/z, Da Intensity, cps 122.9 141.1 142.1 124.2 122.1 327.3 372.2 391.4 333.1 399.4 2.0e5

22 Source temp 700 o C. Q1 scan 100-400 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. 0.51.01.52.02.53.03.54.0 Time, min 8.0e8 Intensity, cps 0.50 3.62 4.33 3.49 2.66 2.81 3.08 3.01 +Q1: 2.479 to 3.266 min from Sample 4 (Prime 700) of Source temp test.wiff (Turbo Spray)Max. 1.7e6 cps. 100120140160180200220240260280300320340360380400 m/z, Da Intensity, cps 124.2 327.3 126.0 399.4 372.2 391.3 385.3 2.0e5

23 Source temp 300 o C. Q1 scan 100-400 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. 0.51.01.52.02.53.03.54.0 Time, min Intensity, cps 0.51 3.62 4.30 3.84 2.82 2.54 +Q1: 1.508 to 1.692 min from Sample 9 (Prime 300) of Source temp test.wiff (Turbo Spray)Max. 1.7e6 cps. 100120140160180200220240260280300320340360380400 m/z, Da Intensity, cps 124.0 122.9 132.0 122.3 142.2 125.0 349.1 111.1 163.3 371.2 391.0 334.8 363.2

24 Source temp 300 o C. Q1 scan 100-400 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. 0.51.01.52.02.53.03.54.0 Time, min Intensity, cps 0.51 3.62 4.30 3.84 2.82 3.05 2.67 2.54 +Q1: 2.496 to 3.501 min from Sample 9 (Prime 300) of Source temp test.wiff (Turbo Spray)Max. 3.8e6 cps. 100120140160180200220240260280300320340360380400 m/z, Da Intensity, cps 124.2 131.9 123.1 126.0 383.4 349.1 399.4 114.1 371.4 14.0

25 Accuracy between 700 o C and 300 o C source temperatures

26 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

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

28 Samples extracted using current production method

29 Samples extracted using new wash method

30 Possibility of Deconvolution of Data T3/rT3651.8605.8Quant T3/rT3651.8507.8Qual 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

31 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 125.15105.12330015500041002680023775.224422.02219%0.15325.1525 25.6 Cal 250.8998.43560012100064502150035948.526686.1719%0.29750.8950 50.8 Cal 3248.1199.62450001730004710030300241884.444988.7919%1.398248.11250 247 Cal 4500.85100.24060001420007900024800398907.674193.9919%2.809500.85500 501 rT3 C3 point0.00 15200 10300 rT3 5 QC Low67.55101.955400141000105002470055001.3510229.8619%0.39067.55 167.5456968 6 QC Norm130.9693.81160001550002190028700115339.321452.2919%0.744130.96 1130.9603130 7 QC High339.2895.52640001380004950024400263190.748951.6119%1.907339.28 1339.2809336 8 QC Neg-0.57503.02120200000474364001957.899364.155419%0.010-0.57 1-0.570160.994 14310123325103.57101.01040001740001990030400102867.619132.6419%0.591103.57 1103.568104 1431413076787.20101.052700103000104001860051483.319575.5319%0.50087.20 187.204989.3 14314121297105.0598.160900101000115001790060548.0511261.5119%0.599105.05 1105.0533105 1431412571896.32104.182200147000159002550080956.4415057.3219%0.55196.32 196.3193797 14314125721101.41106.782900139000166002410080497.4514971.9519%0.579101.41 1101.4052104 1431413081080.52106.563600135000124002300062438.8811613.1919%0.46380.52 180.5189581.9 14314127025149.49100.785400100000162001730084756.8315764.1719%0.848149.49 1149.4887149 14314133102118.84100.81160001690002240029700114322.321263.1419%0.676118.84 1118.8412120 1431511046974.88106.470400160000138002770068963.7812826.7719%0.43174.88 174.8792276.3 rT3 ratio0.68 T3 ratio0.19 Slope0.006 y-intersept0.013 r0.999982 Expected Conc Ratio, m/z T3 qual/quantC1/A Cal 125 0.176 0.175965665 Cal 250 0.181 0.181179775 Cal 3250 0.192 0.192244898 Cal 4200 0.195 0.194581281

32 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 12.82127.912901240084082701265.7835.366%0.1022.822.5 2.62 Cal 25.4083.921109530133082301975.81303.966%0.2075.405 5.65 Cal 323.67101.31520016600103001050015778.510412.666%0.95123.6725 23.6 Cal 450.61100.0288001470019600925030077.119848.566%2.04650.6150 50.6 T3 C4 point0.00406000 79000 T3 5 QC Low8.14138.33340.0014300277089804556.03006.666%0.3198.14 18.1389745.93 6 QC Norm23.75105.212900.00144008900894013731.79061.866%0.95423.75 123.7495423 7 QC High56.3693.729000.001240018800791028274.318658.866%2.28056.36 156.3626560.2 8 QC Neg0.57726.7182.001980013411400211.9139.866%0.0110.57 10.5694570.172 1431012332528.3798.019400.0016800127001060019179.812657.266%1.14228.37 128.3729529.7 1431413076724.71114.38560.009860622059209787.26458.866%0.99324.71 124.7090522.3 1431412129720.88104.47410.009240504057507732.35102.766%0.83720.88 120.8791320.6 1431412571835.40114.820900.001420013500740020271.813377.866%1.42835.40 135.4023737.8 1431412572118.06104.08880.0013100613084109460.06242.966%0.72218.06 118.0595217.3 143141308107.44108.83580.0012400237078503596.12373.166%0.2907.44 17.4359557.37 1431412702515.73114.66230.0010100416056806334.64180.466%0.62715.73 115.7253515.8 1431413310216.72114.410300.00166007160969011079.77311.766%0.66716.72 116.7150716 rT3 ratio0.66 T3 ratio0.19 Slope0.041 y-intersept-0.012 r0.999180 Expected conc Ratio, m/z rT3 qual/quantC1/A Cal 12.5 0.651 0.6511628 Cal 25 0.630 0.6303318 Cal 325 0.678 0.6776316 Cal 450 0.681 0.6805556

33 rT3 levels to interpret Blood Brain Barrier Integrity?

34 Blood Brain Barrier (BBB)

35 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 9.1-14Slight impairment 14.1-30Moderate impairment 30.1-100Severe impairment >100Complete breakdown

36 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

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

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

39 Astrocyte Blood vessel Neuron

40 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.

41 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

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