1. Introduction

The thyroid is a hormone regulation organ. It plays an important role in protein synthesis, the rate of energy source utilization, calcium homeostasis and stimulation of other organ hormones. These functions are particularly essential for animal growth and development. The primary cellular level structure of the thyroid consists of a number of follicular cells spherically surrounding a colloid (Fig. 1). This is the follicular lumen. Physiologically, iodide ions move from extracellular (e.g. blood vessels) to intracellular (follicular cell). This movement relies on the concentration gradient generated by sodium ions in the follicular cell. Inside a follicular cell and the colloid, two iodide ions are synthesized into iodine by thyroid peroxidase and the iodine iodinates the tyrosine to form monoiodotyrosine (MIT) and diiodotyrosine (DIT). The thyroglobulin (Tg) protein is synthesized in the thyroid epithelial cell and released to the colloid. The “MIT” and “DIT” molecules are attached to the Tg protein. Via a conjugation process in the Tg protein, two moieties of DIT are conjugated together to produce thyroxine (T4). Also, one molecule of MIT and one molecule of DIT are conjugated together to produce triiodothyronine (T3). T3 and T4 are important hormones for regulating metabolism.

 

Fig. 1 Iodide ion movement from blood vessels to thyroid follicular cells relies on the concentration gradient of sodium ions in the cells. In the colloid, two iodide ions are synthesized into iodine by thyroid peroxidase and the iodine iodinates the tyrosine to form monoiodotyrosine (MIT) and diiodotyrosine (DIT). The thyroglobulin (Tg) protein is synthesized in the thyroid epithelial cell and released to the colloid. The iodinated tyrosine molecules “MIT” and “DIT” are attached to the Tg protein. Via a conjugation process in the Tg protein, two moieties of DIT are conjugated together to produce thyroxine (T4). Also, one molecule of MIT and one molecule of DIT are conjugated together to produce triiodothyronine (T3).

Download Full Size | PDF

Recent cellular level studies of the thyroid have typically grown thyroid cell cultures or isolated the thyroid cell from the tissue histologically [1, 2]. These studies have typically been performed with the use of biochemical assays. Using traditional methods, the sample preparation time can be long. Raman spectroscopy is a vibrational spectroscopy technique that is particularly suited for biological samples [3–5], including thyroid tissue, because it can be applied with minimal sample preparation. Raman spectroscopy is based on the Raman effect [6], where a small fraction of photons scattered by a molecule are inelastically scattered at (usually) longer wavelength than that of the incident photons. Cell studies with Raman spectroscopy have recently attracted great interest [7, 8] because Raman provides information about the structure of bio-molecules in situ. This information can be used to identify the hormone or signal molecule variation in the cell. Another advantage of using Raman to study cells is minimal interference from water molecules [9]. Therefore, Raman spectroscopy is an excellent complement to existing laboratory methods for studying thyroid cells.

Raman spectroscopy has been employed to study the thyroid at the bulk tissue level [10] and at the cellular level in vitro [11]. In this study, we develop a novel Raman spectroscopy method for in situ cellular level analysis of the thyroid. Thyroids are harvested from mice and specially prepared for Raman. Raman is performed with a confocal setup and the laser spot is placed on a follicular lumen. We also treat a second group of mice with lithium and employ Raman to examine changes in the lumen.

2. Materials and methods

2.1 Animal subjects

All aspects of this study were approved by the animal research ethics committees of the City University of Hong Kong, the University of Hong Kong, and the Department of Health of the Hong Kong Special Administrative Region. A total of ten C57 strain male mice (6 – 8 weeks old, 25 – 30 g) were provided by the Laboratory Animal Unit of the University of Hong Kong. Five subjects were housed in one cage under a constant temperature of 25 °C and humidity of 60 to 70%. Subjects were housed in 12/12 hour light/dark cycles and had access to regular chow food and drinking water ad libitum. Subjects were acclimated to the housing environment for one day prior to the experiment.

2.2 Lithium treatment

Lithium carbonate (Li₂CO₃) was purchased from Sigma Aldrich (USA). All chemicals and solvents used in this study were of analytical grade and obtained through commercial sources. The subjects were divided into control and lithium treated groups, each with five mice. The water bottles in cages housing control subjects were filled with 250 mL of water. For lithium treated subjects, a stock solution (7.5 mM Li₂CO₃) was first prepared, which was equivalent to 554 mg of Li₂CO₃ added to each liter of water. Fifty mL of stock solution was mixed with 200 mL of water, resulting in a 1.5 mM concentration solution of Li₂CO₃. At this concentration, Li₂CO₃ dissolved thoroughly in water. All subjects were housed for 14 days in the above conditions. The water bottles were refilled on a weekly basis such that the subjects had a continuous supply of water. The water consumption rate of each cage was recorded.

2.3 Sample preparation

On day 14, subjects were euthanized by 1 mL/kg body weight of 20% Dorminal Tropfen via intraperitoneal injection. Since the size of the mouse thyroid is around 1 mm x 1 mm (length x width), the thyroid extraction process was performed under light microscopy with 60X magnification. The thyroid, which is located superior to the trachea (Fig. 2), was extracted from the animal. The organ was immersed in saline (0.9% Sodium Chloride) to remove the existing blood. The organ was then placed in a 5 mL test tube and immersed in liquid nitrogen for several seconds followed by storage in a −20 °C freezer. Before performing Raman spectroscopy, the thyroid epithelial cells and smooth muscle were removed and the remaining tissue was flattened into a thin layer tissue. This prepared thyroid specimen was now ready for Raman spectroscopy. A thyroid tissue component containing follicular cells surrounding a colloid (follicular lumen) was determined under a high power microscope (Fig. 3) integrated with the Raman system.

 

Fig. 2 A) The location of the thyroid is posterior to the trachea. B) The morphology of the mouse thyroid. C) The same location about the trachea after the thyroid has been extracted.

Download Full Size | PDF

 

Fig. 3 The colloid and the follicular cell as seen under 500X magnification. This photograph was acquired with a commercial light microscope (Olympus, Japan).

Download Full Size | PDF

2.4 Raman spectroscopy

A schematic of the Raman spectroscopy setup employed for this study is shown in Fig. 4. The setup was adapted from a Renishaw inViaTM QontorTM system (United Kingdom). In principle, the methodology presented in this paper can be implemented with any confocal Raman spectrometer coupled to a light microscope. Prior to Raman, the samples were soaked in physiologic saline (0.9% Sodium Chloride) at room temperature. The Raman spectrometer employed a continuous excitation laser at 514 nm. The Raman scattered light was recorded by a thermoelectrically cooled (−70 °C) CCD detector coupled to a spectrograph (1800 l/mm). Light collection was performed from 400 to 2000 cm⁻¹. Laser line specific notch filters were employed to reduce elastically scattered light reaching the detector. The laser and spectrograph were focused onto the sample using a standard confocal setup. The laser spot size on the sample was 20.2 µm. Raman spectra were acquired with a 10 s exposure time. The Raman spectroscopy setup was coupled to a standard microscope setup that had a 50X objective (Nikon, Japan). The microscope was used to locate the cells of interest.

 

Fig. 4 Raman spectroscopy setup. The thyroid sample was mounted on the specimen table. The microscope with a 50X objective lens was used to target a single thyroid follicular lumen. Raman spectroscopy was performed on the target using a standard confocal setup and 514 nm laser excitation.

Download Full Size | PDF

To distinguish the thyroid follicular lumen from other cells and structures in the thyroid, we first identified a colloid like structure, which is transparent under light microscopy without staining (Fig. 3). The colloid is the center of the follicular lumen. Raman spectroscopy was then performed with the laser spot set on the colloid like structure. If minimal Raman signal was collected, the laser spot was moved to another colloid like structure. Lack of signal indicated the target was an air bubble rather than a follicular lumen.

2.5 Data analysis

Analysis was performed with custom software written in Matlab (Mathworks, USA) along with the optical spectroscopy software Essential FTIR version 3.5 (Operant LLC, USA). To subtract the background fluorescence along with other background signals from the data, quadratic (2nd order polynomial) baseline correction was performed. A quadratic function was fit to the spectrum using regression and then subtracted away. Vector normalization was than performed by taking the difference between the average amplitude of the spectrum and the spectrum. The spectrum was then divided by the square root of the sum of squares of the amplitudes. This normalization was performed to adjust the amplitude differences between the control spectra and the lithium treated spectra to facilitate comparison. Only unambiguous peaks in the spectra were chosen for further analysis. Refer to Table 1 for the wavenumbers of peaks observed from the samples.

Table 1. Raman peaks

View Table

Statistical analysis of the spectra was performed with the Microsoft Excel Program. T-tests were performed across the two subject groups (controls and lithium treated) using the two-tailed distribution and unpaired testing. A p-value threshold of 0.05 was considered statistically significant.

3. Results

The subjects on average consumed 100 mL of water during the two weeks experiment. Each of the lithium treated subjects consumed on average 11.1 mg of lithium carbonate before they were sacrificed.

The Raman spectra of the thyroid contained seven main peaks at 563, 1087, 1265, 1301, 1440, 1656 and 1746 cm⁻¹ (Fig. 5). According to the peak description from Lambert et. al. [12], the peak at 563 cm⁻¹ corresponds to the organic compound side chain of the C-I stretch in Iodine compounds and the C_nH_{2n + 1} in alkyl groups. The peak at 1087 cm⁻¹ corresponds to the C-O-C in ether and the carbon ring in cyclic compounds. The peak at 1265 cm⁻¹ corresponds to the deformation of C-C-H and C-O-H and vibration of Amide III (peptide bond). The peak at 1301 cm⁻¹ corresponds to the COO- group in carboxylic acid salts. The peak at 1440 cm⁻¹ corresponds to the OH in carboxylic acids with the formation of in-plane OH Bending. The 1656 and 1746 cm⁻¹ peaks correspond to the functional group of the phenol and the carboxylic acid, respectively, which make up the tyrosine molecule.

 

Fig. 5 Group averaged (N = 5) Raman spectrum acquired from control subjects. The spectra have undergone baseline subtraction and normalization. Seven clear Raman peaks are observed.

Download Full Size | PDF

The Raman spectra from lithium treated thyroids also contain seven main peaks at the same wavenumbers (Fig. 6). The peak at 1087 cm⁻¹ is lower following lithium treatment when compared with controls (p < 0.05, see Table 1). The peaks at 1440, 1656 and 1746 cm⁻¹ were significantly higher following treatment with p < 0.05 (Table 1). The three significantly different peaks at 1440, 1656 and 1746 cm⁻¹ are related to carboxylic acid and phenol, both of which are significant components of tyrosine. Therefore, the Raman spectroscopy data indicates increased tyrosine in the follicular lumen following lithium treatment.

 

Fig. 6 Group averaged (N = 5) Raman spectra acquired from control (green) and lithium treated (blue) subjects. The control spectrum is the same as that in Fig. 5. Peaks corresponding to the phenol and carboxylic acid functional groups of tyrosine are significantly higher in lithium treated subjects.

Download Full Size | PDF

4. Discussion

In this study, we developed a novel Raman Spectroscopy method for cellular level analysis of the thyroid. The spectra recorded from follicular lumens in the thyroid contained seven main peaks at 563, 1087, 1265, 1301, 1440, 1656 and 1746 cm⁻¹. These peaks correspond to bonds in iodine compounds, alkyl group, cyclic compounds, amide, carboxylic acid and phenol. When subjects were treated for two weeks with lithium, the Raman spectra showed significant increases at 1440, 1656 and 1746 cm⁻¹. These changes suggest elevated tyrosine levels in the lumen following Lithium treatment.

4.1 Raman spectroscopy of the thyroid

Raman spectroscopy has been applied to examine normal and cancerous thyroid tissues ex vivo and thyroid cell lines in vitro [10, 11, 13, 14]. Teixeira’s study on benign human thyroid tissue and malignant human thyroid tissue employed 1064 nm excitation and observed significant peaks at 540 – 800 cm⁻¹, 856 – 930 cm⁻¹, 960 cm⁻¹, 1087 – 1090 cm⁻¹, 1246 – 1270 cm⁻¹, 1337/1339 cm⁻¹ and 1552 – 1585 cm⁻¹ [10]. Harris’ study on the discrimination of human thyroid normal and cancerous cell lines in vitro with Raman spectroscopy employed 783 nm excitation and observed significant peaks at 717 cm⁻¹, 777 cm⁻¹, 824 cm⁻¹, 851 cm⁻¹, 931 cm⁻¹, 970 cm⁻¹, 1002 cm⁻¹, 1072 cm⁻¹, 1178 cm⁻¹, 1268 cm⁻¹, 1299 cm⁻¹, 1446 cm⁻¹, 1579 cm⁻¹ and 1656 cm⁻¹ [11]. The Raman peaks employed in this in situ cellular level thyroid study (Table 1) are overall in agreement with the peaks observed in earlier ex vivo and in vitro studies. It is important to note that in general, thyroid tissue is very complex as there are a large number of functional groups vibrating at the same time [10, 11]. Therefore, there is significant value in performing Raman spectroscopic analysis on specific cellular level structures within the thyroid.

4.2 Lithium drugs

Lithium is the atomic number = 3 element and lithium ions are the key component in many drugs that treat bipolar disorder [15–18]. Lithium drugs treatment is a chronic treatment that reduces suicide risk and prevents manic and depressive episodes [19, 20]. The exact mechanisms for how lithium acts on the brain are presently not fully understood. One side effect of lithium treatment is thyroid dysfunction, particularly hyperthyroidism or hypothyroidism have been reported on many bipolar patients receiving the drug [21–23]. Deficiency of the thyroid can result in lung and kidney dysfunction, growth retardation, water retention and autoimmune disorder. Similar to in the brain, the exact mechanisms for how lithium leads to thyroid dysfunction are not fully understood. Therefore, Raman spectroscopy can help to advance this understanding.

4.3 Lithium and thyroid dysfunction

Raman spectroscopy of the follicular lumen in control and lithium treated subjects show significantly elevated peaks at 1440, 1656 and 1746 cm⁻¹. These peaks correspond to carboxylic acid and phenol, both present in the amino acid tyrosine. This indicates elevated tyrosine levels in the follicular lumen following lithium treatment. Tyrosine is one of the fundamental molecules for producing the important T3 and T4 hormones. The structure of the tyrosine consists of three major functional groups: carboxylic acid, phenol and amine. While the tyrosine in the colloid combines with the iodine, the thyroglobulin will diffuse the synthetic tyrosine into the follicular cell to synthesize into T3 and T4 hormones (Fig. 1). We hypothesize that lithium can disrupt this process in the following manner (Fig. 7). Lithium has the ability to enter from the extracellular environment to the intracellular environment through the cation ion channel, such as the sodium/potassium ion pump and the sodium ion channel. Excess lithium ions in the intracellular environment interrupts the function of the pump, which indirectly limits the iodide influx from extracellular to intracellular [19]. Lack of the iodine in the colloid would disable the iodination of the tyrosine to form MIT and DIT. Consequently, the conjugation processes that convert MIT and DIT into T3 and T4 would also be shutdown, resulting in failure of hormone production and overall thyroid dysfunction. Since tyrosine is not iodinated into MIT and DIT, there will be an accumulation of tyrosine in the follicular lumen, which was observed by Raman. The end result is thyroid dysfunction in bipolar patients treated with lithium ion drugs.

 

Fig. 7 Excess lithium ion in the follicular cell may interrupt the function of the sodium ion pump, which indirectly limits the iodide influx from the blood vessel to the cell. Lack of the iodine in the colloid would disable the iodination and conjugation process, resulting in failure of the T3 and T4 hormone production, and a buildup of tyrosine.

Download Full Size | PDF

Further in support of this hypothesis, the Raman peaks at 563 and 1087 cm⁻¹ along with the small peak at 1365 cm⁻¹ were slightly higher in the control group. These peaks correspond to C-I, C-O-C and CH₃. Though this difference was not statistically significant, it should be noted that these compounds are found in T3 and T4. In particular, C-I and C-O-C are the major functional groups in the T3 and T4 hormones. Therefore, the reduction in peaks at 563, 1087 and 1365 cm⁻¹ suggest reduced T3 and T4 in the follicular lumen. This was likely due to lithium blocking the T3 and T4 production process.

4.4 Technical considerations

The peaks at wavenumbers 1265 cm⁻¹, 1301 cm⁻¹ and 1440 cm⁻¹ in Figs. 5 and 6 correspond to the C-C-H, C-O-H, COO- and OH- groups. As the tyrosine, T3 hormone and T4 hormone all contain the C-C-H, C-O-H, COO- and OH- groups, those peaks cannot be specifically related to the tyrosine molecule, T3 hormone or T4 hormone. Therefore, the peaks at 1656 and 1746 cm⁻¹ are more specific to tyrosine.

5. Conclusion

We have developed a novel Raman Spectroscopy method for cellular level analysis of the thyroid. The spectra recorded from follicular lumens in the thyroid contained seven main peaks. These peaks correspond to bonds in iodine compounds, alkyl group, cyclic compounds, amide, carboxylic acid and phenol. When subjects were treated with lithium, the key component in many drugs that treat bipolar disorder, the spectra showed significant increases at peaks corresponding to tyrosine. This novel Raman method can be used to study the impact of many exogenous treatments on thyroid biochemistry.