AU2019101853A4

AU2019101853A4 - Application of MIT and/or DIT as thyroid cancer marker and kit

Info

Publication number: AU2019101853A4
Application number: AU2019101853A
Authority: AU
Inventors: Hongyang CUI; Yi Wan
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2019-04-09
Filing date: 2019-11-29
Publication date: 2024-05-02
Anticipated expiration: 2027-11-29
Also published as: AU2019440527A1; WO2020207037A1

Abstract

Application of MIT and/or DIT as thyroid cancer marker and kit are disclosed. MIT and DIT are the precursors of thyroid hormone 3,3',5-Triiodothyronine (13) and thyroxine (14). The concentrations of MIT and DIT are stable and constant in thyroid. In accordance with this invention, and we demonstrated for the first time that concentrations of MIT, DIT were significantly lower (100-200 times) in the cancerous tissues than in the corresponding paracancerous tissues (p < 0.05). The thyroid levels of the iodized-tyrosine (MIT and DIT), therefore, appear to be sensitive marker of thyroid cancer tissue. The analysis method can be used to prepare kits for the detection of thyroid cancer, which provides a new technology for detection, diagnosis, treatment and prognosis evaluation of thyroid cancer. In this invention, the needle puncture thyroid samples can be accurately analyzed. Moreover, this invention also possesses numerous other advantages, such as high efficiency, and accurate determination. Standards Normal thyroid tissue 100 100 8.67 MIT 652.04>363.12 8.67 MIT 65204>363.12 2.44e5 1 2.82e6 to100778 >331 100. 10.84 DIT 777.8>32 5 10.84 DIT 777.88 > 363.12 2.69e6 0 0 6 8 10 12 14 16 18 20 6 8 10 12 14 16 18 20 Retention time (min) Retention time (min) Fig. 1 2.0x105 *MIT 1.0x101 0.0 20 40 60 80 100 120 Time (h) 2.0x105 * DIT 1.0x1001 0 20 40 60 80 100 120 Time (h) Fig. 2 1/3

Description

Standards Normal thyroid tissue 100 100 8.67 MIT 652.04>363.12 2.44e5 1 8.67 MIT 65204>363.12 2.82e6

to100778 >331 100. 10.84 DIT 777.8>32 5 10.84 DIT 777.88 > 363.12 2.69e6

0 6 8 10 12 14 16 18 20 6 8 10 12 14 16 18 20

Retention time (min) Retention time (min)

Fig. 1

2.0x105 *MIT 1.0x101

0.0 20 40 60 80 100 120 Time (h) 2.0x10 5 * DIT 1.0x1001

0 20 40 60 80 100 120 Time (h)

Fig. 2

1/3

APPLICATION OF MIT AND/OR DIT AS THYROID CANCER MARKER AND KIT

Technical Field

This invention belongs to the field of biochemical analysis and biomedical sciences, and in

particular, to application and kits for the specific analysis of MIT and DIT as the marker for

thyroid cancer.

Background Arts

Thyroid carcinoma is the most common endocrine malignancy in clinical practice and has

increased rapidly over the past few decades. Current medical studies have confirmed that thyroid

carcinoma led to the metabolic changes. The incidence of thyroid carcinoma has increased rapidly

over the past few decades. At present, the palpation, imaging tests, and cytological examinations

were commonly used in clinical diagnosis of thyroid cancer. However, the diagnostic accuracy is

still low and need multiple diagnosis criteria. Palpation cannot identify the actual incidence of

cancer. Imaging examination can only obtain images of early or advanced cancer and cannot

confirm the diagnosis of cancer. Cytological examination has obvious diagnosis effect on most

advanced cancer but is difficult for the tumor diagnosis. Furthermore, little pathological evidence

is available regarding the T3 and T4 dysregulation on thyroid cancer progression, and they cannot

reflect the incidence of cancer. Generally, at present, there are many diagnostic methods for

thyroid cancer, but each method has some limitations, which makes the diagnosis of thyroid

cancer more difficult.

Therefore, exploration of a biomarker for rapid clinical diagnosis and prognosis of thyroid cancer

has become the key to achieve the accurate diagnosis of thyroid cancer.

Summary of the invention

MIT and DIT are the precursors of thyroid hormone 3,3',5-Triiodothyronine (T3) and thyroxine

(T4). The concentrations of MIT and DIT are stable and constant in thyroid. In accordance with

this invention, and we demonstrated for the first time that concentrations of MIT, DIT were

significantly lower (100-200 times) in the cancerous tissues than in the corresponding

paracancerous tissues (p < 0.05). Similarly, the concentrations of MIT, DIT, were significantly

lower in the thyroid cancer cells than in the normal cells. Therefore, thyroid MIT and DIT were identified as promising indicators of PTC. The disclosed methods and kits can be applied to the detection of thyroid cancer, which provides a new technology for detection, diagnosis, treatment and prognosis evaluation of thyroid cancer.

The technical solution of the present invention can include the following:

In one aspect, the present invention provides an application and a kit for MIT and/or DIT as the

marker for thyroid cancer. This method includes the extraction of MIT and DIT, the derivation of

MIT and DIT with SPTPP, and the analysis of MIT and DIT derivatives by UPLC-MS/MS. Based

on the above methods, so that MIT and DIT can be simultaneously and accurately determined.

It should be noted that the extraction method of MIT and DIT mentioned above are listed below.

In some embodiments, the MIT and DIT were extracted from 0.1-1 mg of homogenized thyroid

tissues. The tissue samples were spiked with 50 pL of IS working solution and homogenized then

kept on ice for 15 min for deproteination before centrifugation at 8, 000 xg and 4 °C for 5 min.

The supernatants were collected in a 2-mL glass vial. Methanol (45 pL) was added to each

precipitate, and the centrifugation and extraction steps were repeated twice. All supernatants were

combined for subsequent SPTPP derivatization. 3 Further, the IS working solution is C6 -MIT dissolved in methanol at the concentration of 100

ng/L.

In some embodiments, the MIT and DIT derivatization procedure includes the following steps: 30

pL of derivatization buffer and 20 pL of derivatization reagent are added to the extracts of MIT

and DIT; after vortex mixing, the mixed solution is heated at 40 °C for 5 min; and 600 pL of a

termination solution is added to the reaction mixture.

In some embodiments, the derivatization reagent is SPTPP dissolved in DMSO to a concentration

of 30 mM. The derivatization buffer is 100 mM pH 8.0 phosphate-buffered saline (PBS). The

termination solution is 1 M NaOH solution.

In all embodiments, in this invention, after the SPTPP derivation, the derivates should be

conducted on the steps of purification, extraction and concentration prior to UPLC-MS/MS

analysis. Specifically, ethyl acetate is employed to purify the derivates after the addition of the

termination solution. After purification, an acidifier is added to the purified derivates reaction

solution. Then, ethyl acetate is used to extract the derivatized MIT and DIT from the purified

derivates reaction solution. Next, the extracts are dried with nitrogen and redissolved in methanol for UPLC-MS/MS analysis.

In some embodiments, the specific steps of UPLC-MS/MS analysis can include the follows:

calibration standards of MIT and DIT are prepared in methanol; calibration standards are

derivatized with SPTPP; all of the samples are analyzed using UPLC-MS/MS; calibration curves

are constructed by plotting the area ratio of MIT and DIT to the IS versus the respective analyte

concentrations, and these data are fitted using linear regression; and the amount of MIT and DIT

in the thyroid tissues are then interpolated using this linear function.

In some embodiments, this invention provides a thyroid cancer detection kit, which includes MIT

and/or DIT detection reagents.

In some embodiments, the present invention also provides a kit for prognostic assessment of

thyroid cancer that includes MIT and/or DIT detection reagents.

In some embodiments, the reagents for MIT and/or DIT detection described in the kit include:

PBS (pH 8.0), the standard stock solution, extraction reagents, and IS working solution 1(3 C6 -MIT

at 100 ng/L dissolved in methanol), and SPTPP (30 mM dissolved in DMSO).

The disclosed methods and kits can include one or more of the following advantages:

(1) It is for the first time that MIT and DIT in thyroid cancer tissue show a significant

downregulation compared with thyroid normal tissues. Furthermore, this invention shows that

MIT and DIT is a new indicator for reliable and specific discrimination of thyroid cancer and its

effectors.

(2) Compared with traditional methods, this invention requires only a small sample amount and

simple pretreatment and provides accurate determination and strong analytical specificity.

Moreover, SPTPP derivatives are very stable and the quantitative analysis results remain

unchanged for at least five days (Figure 2). The SPTPP derivatives can be stored stably at -20 °C

for at least two months. Meanwhile, this invention provides high efficiency, simple sample

preparation, high accuracy and efficiency, and is highly recommended. Furthermore, the

corresponding reagents and methods for detecting MIT and DIT, such as SPTPP reagent, can be

employed to make thyroid cancer detection kit, which provides a new idea and means for tumor

detection, diagnosis, treatment and prognosis evaluation. In this invention, MIT and DIT are

derivatized by developed MIT and DIT ultra-sensitive derivatization reagent SPTPP, and the

ultra-sensitive detection is performed by UPLC-MS/MS. This newly established analytical method required only 0.1-1 mg thyroid tissue and simple liquid-liquid purifications, realizing the rapid detection of MIT and DIT with extremely high sensitivity. This invention provides a basis for medical detection and disease diagnosis.

(3) This invention is also suitable for the analysis of thyroid puncture tissues, and achieve the

purpose of ultra-high efficiency and high sensitivity detection of this kit.

Description of the drawings

FIG.1 shows UPLC-MS/MS chromatographic profiles of the SPTPP-derivatized MIT and DIT in

standards and thyroid tissue sample.

FIG. 2 shows the stability of MIT and DIT derivative. LC-MS/MS analysis were taken at 0.5 to

120 hours, and the analyses were conducted once every two hours.

FIG. 3 shows the concentrations of detected DIT in thyroid cancer cells and normal cells.

FIG. 4 shows the concentrations of detected MIT in thyroid cancer tissue and normal tissue.

FIG. 5 shows the concentrations of detected DIT in thyroid cancer tissue and normal tissue.

Detailed descripton of the invention

To make the purpose, technical solutions and advantages of the embodiments of the invention

more clear, the technical solution in the embodiments of the invention is clearly and completely

described below. Where specific conditions are not specified in the embodiments, they are clearly

and completely described in accordance with the general conditions or the conditions suggested by

the manufacturer. For reagents not specified, conventional commercially available products can be

used. The instruments, equipment and consumables used can be used after simple debugging of

the common instruments and consumables purchased on the market.

In the following, the technical scheme of the present invention will be described in more detail by

taking the detection of the concentration of thyroid tumor markers in normal thyroid tissues and

thyroid cancer tissues as examples.

Example 1. Determination of tumor marker concentrations in thyroid normal cells and thyroid

cancer cells

1. Instruments and reagents

ACQUITY Ultra Performance LC system (Waters, Milford, MA, USA). Waters Xevo TQ-XS

Triple Quadrupole Mass Spectrometer equipped with an ESI source (Waters, USA). Refrigerated

centrifuge (Hermle Labortechnik, Wehingen, Germany). Analytical balance (Mettler, USA). N

EVAP analytical evaporator (Berlin, MA, USA). Vortex mixer (Vortex-Genie 2).

Methanol and ethyl acetate (LC/MS grade) and ultrapure water was acquired from a Milli-Q water

purification system (Millipore Corporation, Hayward, CA, USA) operating at 18.2 MQ-cm.

2. Pretreatment of normal thyroid cells and thyroid cancer cells

(1) 0.1-1 mg normal and cancer thyroid cells are accurately weighed to be detected. The tissue

samples were spiked with 50 pL of IS working solution and homogenized then kept on ice for 15

min for deproteination before centrifugation at 8, 000 xg and 4 °C for 5 to 15 min. The

supernatants were collected in a 2-mL glass vial. Methanol was added to each precipitate, and the

centrifugation and extraction steps were repeated twice. All supernatants were combined for

subsequent SPTPP derivatization. The supernatants are the extracts of MIT and DIT.

(2) Derivatization reaction: Place 30 pL of the PBS buffer into 2 mL brown bottle. Then add the

extracts of MIT and DIT from the step (1) and 20 pL of derivatization reagent. After vortex mixing,

the mixed solution is heated at 40 °C for 5 min; and 600 pL of a termination solution is added to

the reaction mixture. The reaction mixture is extracted twice with 800 pL of ethyl acetate. After

purification, 200 pL of acidifying agents (HCl) is added to the purified reaction mixture. The

acidified purified reaction mixture is extracted twice with 800 800 pL of ethyl acetate, and the

extracts are transferred to a new 2 mL glass vial. Next, the extracts were dried with nitrogen and

redissolved in 50 pL of methanol. Finally, the sample containing the MIT and DIT derivates are

transferred into a glass vial for UPLC-MS/MS analysis.

(3) UPLC-MS/MS analysis

The parameters used for UPLC-MS/MS analysis are as follows: 1) UPLC is performed on a

Poroshell HPH-C18 column (2.1 x 100 mm, 1.9 im, Agilent). The mobile phase, operating at a

flow rate of 0.3 mL/min, consisted of methanol as solvent A and Milli-Q water as solvent B. A

total of 2-20 pL of each sample is injected onto the column. The column temperature is set as

35 °C. The gradient elution started at 30% A and was held for 1 min and then increased to 60% A

at 15 min, 75% A at 17 min, and 100% A at 18 min. After washing with 100% A for 8 min, the

column was re-equilibrated with 30% A for 4 min prior to the next injection. 2) the mass

spectrometer is operated in the positive ESI mode with MRM. The conditions for ESI-MS/MS

detection are optimized to obtain the highest signal intensity using an optimization program and

are as follows: desolvation temperature: 500 °C; capillary voltage: 3 KV; desolvation gas flow rate:

1000 L/h; source temperature: 150 °C. The MRM operating conditions are presented in Table 1.

Table 1. MRM operating conditions

No. Analyte RT Cone Transition for Collision Transition(m/z) Collision

Voltage(V) quantification energy energy

(m/z) (eV) (eV)

1 MIT 8.67 88 652.04->363.12 40 652.04->262.16 54

2 DIT 10.84 44 778.88->363.12 48 778.88->262.16 58

(4) Calibration curves and detection limits

1) The working standard solutions are diluted to different concentrations. To obtain the calibration

curves, 50 pL of IS working solution, 30 pL of derivatization buffer, and 20 pL of derivatization

reagent are added to 50 pL of each working solutions. After vortex mixing, the mixed solutions are

heated at 40 °C for 20 min, and then 600 pL of1 M NaOH solution is added to each sample. Then,

each reaction mixture is extracted twice with 800 pL of ethyl acetate. After purification, 200 pL of

5 M HCl is added to each purified reaction mixture. Each acidified purified reaction mixture is

extracted twice with 800 tL of ethyl acetate, and the extracts are transferred to new 2 mL glass

vials. Next, the extracts are dried with nitrogen and redissolved in 50 pL of methanol. Finally, the

samples containing the SPTPP-derivatized MIT and DIT are transferred into glass vials for

UPLC-MS/MS analysis. The concentrations of the calibration standard samples are 5.00 ng/L,

50.00 ng/L, 200.00 ng/L, 500.00 ng/L, 1000.00 ng/L, and 3000.00 ng/L.

2) 2 pL of each calibration standard sample was injected into the UPLC-MS/MS system.

Calibration curves are constructed by plotting the area ratio of each analyte relative to its IS versus

the respective analyte concentrations, and these data are fitted using linear regression. All of the

analytes displayed good linearity in the range of 0.5-300 ng/L and the coefficients of

determination typically exceeded 0.99. The signal-to-noise (S/N) ratios were used to obtain the

limits of detection (LODs) (Table 2).

Table 2. Calibration curves and LODs for MIT and DIT

No. Analyte Linear equation Coefficient (Y2) LOD (pg/mL)

1 MIT Y=98.71X-1.84 0.9999 0.07

2 DIT Y=155.24X-0.09 0.9999 0.14

Example 2. Determination of tumor marker concentrations in thyroid normal tissue and thyroid

cancer tissue

1. Instruments and reagents

Same as described in example 1.

2. Pretreatment of normal thyroid tissue and thyroid cancer tissue

(1) 0.1-1 mg normal and cancer thyroid tissues are accurately weighed to be detected. The tissue

acidified purified reaction mixture is extracted twice with 800 pL of ethyl acetate, and the extracts

are transferred to a new 2 mL glass vial. Next, the extracts were dried with nitrogen and

transferred into a glass vial for UPLC-MS/MS analysis.

(3) UPLC-MS/MS analysis

Same as described in example 1.

The results from the example 1 and 2 show that the concentration of DIT is 0.035 ng/g in thyroid

normal cells, and 0.010 ng/g in thyroid cancer cell, respectively, indicating that the concentration of DIT is significantly lower in the thyroid cancer cells than in the normal cells. In thyroid normal tissues, the mean concentration of MIT is 4.2 tg/g, and the mean concentration of DIT is 3.4 tg/g

(Figure 4 and 5). Whereas, in thyroid cancer tissues, the mean concentration of MIT is 220 pg/g,

and the mean concentration of DIT is 367 pg/g (Figure 4 and 5). These results indicated that MIT

and DIT in thyroid cancer tissues are 100 to 200 times lower than these in thyroid normal tissues

(Figure 3 and 4). The thyroid levels of the iodized-tyrosine (MIT and DIT), therefore, appear to be

sensitive marker of thyroid cancer tissue. Based on the developed method, 0.1-1 mg thyroid

tissues could be accurately analyzed, and provide basis for the diagnosis of thyroid cancer.

These examples describe the exemplified embodiment of this invention. However, the scope of the

invention is not limited to these two examples. The invention is amenable to any modifications,

alterations, or substitutes of the embodiment without departing from the spirit and scope of the

invention. The appended claim is intended to cover such modifications, alterations, or substitutes.

Claims

Claims

1. An application for iodotyrosine (MIT) and/or 3,5-diiodotyrosine (DIT) as the marker for thyroid

cancer.

2. The application according to claim 1, comprising:

1) extracting MIT and DIT from thyroid normal/cancer tissues;

2)derivatizing extracted MIT and DIT with

(5-N-succinimidoxy-5-oxopentyl)triphenylphosphonium bromide (SPTPP); and

3) analyzing MIT and DIT SPTPP derivatives by ultra-high-performance liquid

chromatography-tandem mass spectrometry (UPLC-MS/MS) to accomplish analysis of MIT and

DIT in the tissues.

3. The application according to claim 2, where the samples include thyroid tissues and serum

samples.

4. The application according to claim 2, the MIT and DIT are extracted by the following steps:

0.1-1 mg tissues or 5-20 pL serum samples are spiked with 50 pL of IS working solution and

homogenized then kept on ice for 15 min for deproteination before centrifugation at 8, 000 xg and

4 °C for 5 to 15 min. The supernatants were collected in a 2-mL glass vial. Methanol was added to

each precipitate, and the centrifugation and extraction steps were repeated twice. All supernatants

were combined for subsequent SPTPP derivatization. The supernatants are the extracts of MIT and 3 DIT. The IS working solution is C6-MIT with the concentration of 100 ng/L

5. The application according to claim 2, wherein the step of derivatizing MIT and DIT with SPTPP

includes the following steps: adding 30 pL of a derivatization buffer and 20 pL of derivatization

reagent to the extracts; after vortex mixing, heating the mixed solution at 40 °C for 5 min; and

adding the termination solution to the reaction mixture.

6. The application according to claim 5, wherein the derivatization reagent is SPTPP dissolved in

DMSO, wherein the derivatization buffer is 0.1 M pH 8.0 phosphate-buffered saline, wherein the

termination solution is 1 M NaOH solution.

7. The application according to claim 2, further comprising:

prior to UPLC-MS/MS analysis, purifying the reaction mixture using ethyl acetate after the

addition of the termination solution; wherein 800 tL of ethyl acetate is used to purify the reaction mixture two to three times after the addition of the termination solution; adding an acidifier to the purified reaction mixture; wherein the acidifier is 5M HCl solution; extracting the SPTPP derivatives using ethyl acetate from the purified reaction mixture; and drying the extracts with nitrogen and redissolved in 50 pL methanol for UPLC-MS/MS analysis.

8. The application according to claim 2, wherein the step of analyzing SPTPP derivatives using

UPLC-MS/MS comprises the steps of:

preparing calibration standards containing MIT and DIT in water as dilution series;

derivatizing calibration standards samples with SPTPP;

analyzing the samples using UPLC-MS/MS;

constructing calibration curves by plotting the are ratio of each analyte relative to its IS versus the

respective analyte concentrations, which is fitted using linear regression; and interpolating amount

of MIT and DIT in samples using the linear function.

9. A kit for the determination of thyroid cancer, comprising: the reagents and materials for the

analysis of MIT and/or DIT.

10. A kit for the prognostic assessment of thyroid cancer, comprising: the reagents and materials

for the analysis of MIT and/or DIT.

11. The kit according to claim 9 and 10, further comprising:

1) a derivatization buffer: PBS (pH 8.0)

2) a derivatization reagent: SPTPP dissolved in DMSO

3) IS working solution containing 'C 6-MIT (100 ng/L) dissolved in methanol

4) a termination solution and

) an acidifier

INSURANCE

Standards Normal thyroid tissue 100 100 8.67 652.04 > 363.12 8.67 8.67 MIT MIT 652.04 > 363.12 2.44e5 2.82e6 % % 0 0

100 DIT 777.88 > 363.12 100 10.84 10.84 DIT 777.88 > 363.12 1.27e5 2.69e6 % 2019101853

0 0 6 8 10 12 14 16 18 20 6 8 10 12 14 16 18 20

Retention time (min) Retention time (min)

Fig. 1 Fig. 1

5 2.0x10 2.0x10 MIT 5 1.0x103 1.0x105

0.0 0 20 40 60 80 100 120

Time (h) 5 5 2.0x105 2.0x10 DIT 5 1.0x10

0.0 0 20 40 60 80 80 100 120

Time (h)

Fig. 22 Fig.

1 /3 1/3

Thyroid normal cell

0.04 Thyroid cancer cell

0.03

0.02 0.02 * 2019101853

0.01

0.00

DIT Fig. 3 Fig. 3

Thyroid normal cell 6000 Thyroid cancer cell

5000 5000

4000

3000 200 times

2000

1000

0

MIT Fig. 44 Fig.

2/3 2/3

5000 Thyroid normal cell

Thyroid cancer cell

4000 4000

3000 100 times

2000 2019101853

1000

0

DIT Fig. 555 Fig. Fig.

3/3 3/3