Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
A brief review of rat thyroid-stimulating hormone (TSH) and introduction to Shibayagi’s rat TSH ELISA KIT Katsumi WAKABAYASHI, Ph.D. Prof. emer. Gunma University Technical consultant, Shibayagi Co. Ltd. What is TSH? TSH is produced and secreted by basophilic cells called “thyrotrophs” in the anterior pituitary gland of vertebrates from fishes to mammals. TSH is also called thyrotropic hormone or thyrotropin. TSH acts on thyroid gland, adipose tissue, eye, and so on. TSH receptor penetrates the cell membrane 7 times and is coupled with G-protein-PKA system. TSH increases the release of thyroid hormone, and causes morphological changes in thyroid tissue, and promotes biosynthesis of the thyroid hormones through enhancement of inorganic iodide uptake, iodination of tyrosine in thyroglobulin, and changes of iodotyrosine to thyroid hormones. TSH promotes metabolism of glucose, phospholipids, and nucleic acids in the thyroid. In the adipose tissue, TSH enhances glucose uptake, degradation of lipids, and oxygen consumption. TSH and TSH metabolites bind to cell membrane of retroorbital tissue and increases adenylate cyclase activity, causing exophthalmos. The most characteristic syndrome of TSH deficiency is the reduction of the thyroid function, in other words, decreases in thyroid hormone production and secretion. Diseases showing low blood TSH levels: Hyperthyroidism ( Basedow or Graves disease, Plummer disease), panhypopituitarism ( Simmonds Syndrome ) , partial hypopituitarism, isolated TSH deficiency, Sheehan’s syndrome, etc. Diseases showing high blood TSH levels: Pituitary tumor, ectopic TSH-producing tumor, hypothyroidism (cretinism, iodine deficiency), thyroid hormone refractoriness, etc. Regulation of TSH secretion Factors for enhancement of secretion TRH (Thyrotropin releasing hormone): TRH, pyroglutamylhistidylprolinamide, originated in the hypothalamus, directly stimulates TSH secretion. TRH, at a concentration of 10pg/ml, increased TSH release from rat anterior pituitary in vitro. An intravenous injection of 0.1-1ng of TRH caused an elevation of blood TSH in mice within 2 minutes. The cold exposure stress and thyroidectomy increase blood TSH levels. Such 1 elevation of TSH is blocked by anti-TRH serum pretreatment, indicating the involvement of TRH (1). Estrogens: Estrogens are reported to enhance the TSH-releasing response of pituitary cells for TRH-stimulation. In human subjects, women show larger TSH-releasing response to TRH than men. The estrogen-pretreated men responded more to TRH stimulation than control. Estrogen is considered to increase the number of TRH receptors in thyrotrophs (2). Factors for suppression of secretion Various factors have been reported to inhibit TSH secretion. Somatostatin (Somatotropin or growth hormone release-inhibiting factor, SRIF): Injection of anti-somatostatin causes an increase of TSH blood levels. Injection of synthetic somatostatin to hypothyroidism patients lowers blood levels of TSH. In healthy human subjects, somatostatin blocks the increase of TSH during sleeping. Somatostatin suppressed the TSH-releasing action of TRH. These facts indicate the direct action of somatostatin on TSH secretion in the pituitary. It was also reported that somatostatin suppressed TRH action both in vivo and in vitro. Also in rats, somatostatin has reported to inhibit TSH surge in early morning (3). Thyroid hormones (Triiodothyronin, T3, Thyroxin, T4): As an important factor, thyroid hormones play the negative feedback role in hypothalamic-pituitary-thyroid system, and also reported to directly suppress the action of TRH in the pituitary gland. Growth hormone: Administration of GH lowers the reactivity of thyrotrophs to TRH. Glucocorticoids: Cortisol injection suppresses TSH secretion in human. In human, the blood levels of cortisol are low during 18:00~24:00 clock time, and TSH shows high levels during this period, suggesting a relationship of circadian rhythm of TSH to that of cortisol (4). This is supported by the fact that TSH releasing response to TRH at night is higher than daytime (5). On the other hand, in rats, blood corticosterone levels are high during dark period with lower TSH levels, and the lighting is started, TSH levels go up, indicating suppressive effect of glucocorticoids on TSH secretion (6). Melatonin: In rats, melatonin injection lowers blood TSH, T4 and T3 levels. Opioid-peptides (Endorphins, Enkephalins), sympathetic stress, and starvation were reported to decrease blood levels of TSH and thyroid hormones. Structure and nature of TSH TSH is a heterodimer glycoprotein hormone with molecular weight about 28,000, and is consisted of α-subunit (96 amino acids in case of rat and mouse, with common structure to LH and FSH) and β-subunit (118 amino acids, specific to TSH). Both 2 subunits are glycosylated (α-Subunit: at 56 and 82 asparagine, and β-subunit: at 23 asparagine). Amino acid sequences of subunits of rat, mouse and dog TSH are shown below. Boldface letters in the sequences indicate amino acid different from rat subunits. Rat TSH α-subunit lpdgdliiqg cpecklkenk yfsklgapiy qcmgccfsra yptparskkt mlvpknitse atccvaksft katvmgnarv enhtdchcst cyyhks ↑Glycosylated ↑Glycosylated Disulfide bonds: 11-35, 14-64, 32-86, 36-88, 63-91 Mouse TSH α-subunit lpdgdfiiqg cpecklkenk yfsklgapiy qcmgccfsra yptparskkt mlvpknitse atccvakaft katvmgnarv enhtechcst cyyhks Homology: 94/96 (97.9%) Dog TSH α-subunit fpdgeftmqg cpecklkenk yfsklgapiy qcmgccfsra yptparskkt mlvpknitse atccvakaft katvmgnakv enhtechcst cyyhks Homology: 90/96 (93.8%) Rat TSH β-subunit fcipteymmy vdrrecaycl tintticagy cmtrdingkl flpkyalsqd vctyrdftyr ↑Glycosylated tveipgcphh vapyfsypva lsckcgkcnt dysdctheav ktnyctkpqt fylggfsg Disulfide bonds: 2-52, 16-67, 19-105, 27-83, 31-85, 88-95 Mouse TSH β-subunit fcipteytmy vdrrecaycl tintticagy cmtrdingkl flpkyalsqd vctyrdfiyr tveipgcphh vtpyfsfpva vsckcgkcnt dnsdciheav rtnyctkpqs fylggfsv Homology 108/118 (91.5%) Dog TSH β-subunit fcfpteytmh verkecaycl tintticagy cmtrdingkl flpkyalsqd vctyrdfmyk tveipgcprh vtpyfsypva vsckcgkcnt dysdciheai ktnyctkpqk syvvgfsi Homology: 103/118 (87.3%) Homology of amino acid sequences are very high between rat and mouse, and also dog TSH shows a high homology to rat TSH. Amino acid sequences of TSH of other species are shown in the last part of this text. 3 Nature of TSH TSH shows microheterogeneity due to the differences in oligosaccharide chains, and at least five components with different isoelectric points (9). The author’s laboratory (Hormone Assay Center, Inst. of Endocrinology, Gunma Univ., in collaboration with the 1st Dept. of Int. Med., Sch. of Med., Gunma Univ., found an interesting nature of TSH (9). After thyroidectomy of rats, the blood levels of immunoreactive (IR) TSH rose up to 3 times after 2 days, 10 times after 1 week, and 20 times after 2 weeks compared with control levels (such a big increase is not seen with LH after castration), and pituitary TSH contents decreased until 1 week, and then recovered to nearly control levels after 2 weeks, and increased to 150% after 4 weeks. Usually, β-subunit is present only in very small amount in the pituitary of normal rat. But the amount of IR-β-subunit gradually increased after thyroidectomy, and exceeded the amount of TSH after 4 weeks. A gel-filtration analysis of the pituitary homogenates of thyroidectomized rats followed by β-subunit RIA showed a large peak in the fractions with larger molecular weight than TSH, and this peak was low when assayed by TSH RIA, indicating this big molecular weight component is not TSH but β-subunit-related substance. If animals were given daily T4 injections after thyroidectomy at a dose (1.5μg/100g body weight) that kept normal T4 levels, this β-subunit-related substance did not appear. Such phenomenon was never seen with LH after castration, though LH has similar structure to TSH. Bioassay for TSH A classical bioassay method by McKenzie (Endocrinology, 62; 372-382, 1958) was based on the increase of radioiodine in the blood of immature mouse pretreated with radioiodine and T4, while a recent in vitro method using cultured FRTL-5 cell, which is derived from normal rat thyroid epithelial cell, with cAMP as a marker has been widely used because of small variation (Sho, et al., Endocrinology; 124: 589-604, 1989, Pckles, et al., J Mol Endocrinol, 9: 251-256, 1992) Immunoassay for TSH RIA and IRMA (immunoradiometric assay) kits have been commercially available for human TSH. As for animal TSH, NIDDK’s assay kits for some animal species have been supplied though they are not ready for use because users themselves must carry out radioiodination. An ELISA system for rat TSH is supplied by Shibayagi Co, Ltd., which will be introduced in detail later. 4 Standard preparation and international unit of TSH The amount of human TSH is expressed using international unit (IU). The first international standard was 1st IRP human TSH (68/38), and defined as one ampoule contained 150mIU. Then, 2nd IRP human TSH (80/558) was provided, one ampoule of which contained 37mIU, and the weight of TSH was 7.5μg, and when compared with 1st IRP. The assay value for TSH in clinical diagnosis is expressed as mIU/dl or μIU/ml even immunoassay is used. The USP unit is almost equal to IU. For animal TSH, a biological standard preparation, NIH-TSH-S1, of ovine origin, was supplied by NIH. But as RIA has become more popular, immunoassay standards were distributed and used widely. A rat TSH RIA kit has been provided by NIDDK belonging to NIH. The kit consists of NIDDK rat TSH I-X, a highly purified preparation for radioiodine labeling, NIDDK anti-rat TSH S-X, rabbit antiserum against rat TSH, and NIDDK rat TSH RP-X, a standard preparation (X: lot number starting from 1). RP-1 was a very crude material, and the potency was only 1/160 of the preparation for labeling. In such case, it was nonsense to express the assay values in terms of the weight of RP-1 as some old reports did. After RP-1, RP-2 and RP-3 were provided, and these preparations were prepared from I-series, i.e. highly purified for radioiodination, by adding inert protective protein and buffer component, and if we reconstitute the solution by adding purified water, we can get a standard solution of a fixed concentration of the highly purified preparation. In this case we can use the weight of RP-3 for assay results. The biological potency of NIDDK rat TSH I-9 (=RP-2 or 3) is 35IU/mg. Some suggestive information for sampling Circadian rhythm of blood TSH levels Circadian rhythm is very well known with glucocorticoids and melatonin, but TSH blood level shows also circadian rhythm, and considerable changes are seen during a day. Changes in blood TSH levels will influence blood thyroid hormone levels. As a rat is a nocturnal animal, blood TSH pattern is different from that of human TSH. The representative glucocorticoid in human is cortisol, while in rat corticosterone is the only glucocorticoid due to the absence of 17α-hydroxylase. Presence of circadian rhythm means that the assay values will change depending on the time of blood sampling. In female rats, under the condition of 12 hours light-12 hours dark ( light on: 7:30, off: 19:30 ), circadian changes were seen with TSH, T4 and T3. The peak of TSH was 5 seen immediately after the start of lighting, while peaks of T4 and T3 appeared 3-4 hours later, and corticosterone and prolactin increased soon after the light off. If the light and dark periods are reversed, i.e. light off: 7:30 and light on: 19:30, for 3 weeks, all the hormone peaks shifted 12 hours (6), suggesting that the circadian changes of hypothalamic-pituitary-thyroid system are depending on the light and dark cycle. According to this report, under the normal lighting condition, low blood level of TSH during dark period started to increase after lighting to reach the maximum level about 2 hours later and kept the high level until 14:00, and then decreased until 16:00, and the level remained low until next morning. T4 and T3 levels gradually increased after onset of lighting, and reach the maximum at 16:00. So, the descending of TSH level after 14:00 may be due to the negative feedback effect of thyroid hormones. But such changes of TSH were not seen under reversed lighting condition. The relationship to melatonin is also suggested. Another report (8) described that, in 60 days old male rats, pituitary TSH content and blood TSH level change in the opposite way, and the peak of the content and troughs of blood TSH, T4, and T3 were seen at 24:00, while the trough of TSH content and peaks of blood TSH, T4, and T3 were seen at 12:00. From these data, it would be better to avoid sampling around the time of switching on and off, and the sampling should be carried out at fixed period of time like 10:00 to 14:00 to stabilize control value. Circadian rhythm may change slightly depending on the strain and sex of rats, I recommend checking the circadian change with your rats. In human, blood TSH shows circadian rhythm, however, as human is not nocturnal like rats, the situation is reverse. Cortisol shows the lowest level from 22:00, and start increasing from 3:00, reaching the maximum level at 7:00-8:00. Blood TSH remains low in the daytime, and starts ascending about 21:00 and remains high through the night. Melatonin levels were low in the daytime, and rises from 23:00. In women, blood levels of TSH were reported to increase from 21:00, reaching the peak at 24:00, then decrease until 9:00, and stayed low until 21:00 (10). Influence of stress and anesthesia on blood TSH level I would like to introduce a report telling the effect of anesthesia on blood TSH which appeared in Endocrinology, 1975 (13) Anesthetics examined were Chloral hydrate 300mg/kg,i.p., Pentobarbitone i.p., Thiopentone Ether inhalation 50mg/kg i.p., Urethane (Deep Methoxyflurane 1.5% and light 1.5g/kg (1/2 ip,1/2 sc) anesthesia), inhalation. 6 50mg/kg Halothane 2% inhalation, Observations were made on simple administration, effects on TSH upraise by cold exposure, and effects on TSH secretion by TRH administration. After anesthetization, blood TSH levels were similar to those of controls, however significantly decreased after 30 min. Increase in TSH level by cold exposure was minimized by all anesthetics examined. Especially ether, urethane and chloral hydrate completely blocked TSH increase, while halothane and methoxyflurane retarded the increase. TSH secretion by TRH was inhibited only by deep ether anesthesia, while thiopentone, pentobarbitone hydrate and chloral enhanced TRH action. Cold exposure causes TRH release from hypothalamus, and thus secreted TRH stimulate TSH release from thyrotrophs in anterior pituitary. The results mean that all the anesthetics acted on hypothalamus and block TRH release. So, it is very risky to use anesthetics in experiments which include hypothalamic function. On the other hand, inhibition of TRH action was seen only by deep ether anesthesia, and some anesthetics seemed to enhance TRH action. In TRH experiments, all the anesthetics including light ether anesthesia, except deep ether anesthesia, showed no significant changes of TSH levels for the first 10 min after drug administration. The deep ether anesthesia lowered TSH levels at 10 min after anesthesia. Another report (14) told the results as follows. Non-anesthetized rats showed 5-fold increase in blood TSH level after 25 minutes’ cold exposure, and ether anesthesia completely blocked this increase. 7 Pentobarbital anesthesia inhibited the effect of cold exposure by more than 90%. Three minutes of ether anesthesia before cold exposure inhibited or retarded the TSH-upraising reaction. During 2 hours’ ether anesthesia at room temperature, blood TSH levels were decreased. Pentobarbital anesthesia at room temperature did not decrease blood TSH levels. TRH administration under pentobarbital anesthesia increased TSH level more than non-anesthetized control. The response of blood TSH to TRH administration under ether anesthesia was less than non-anesthetized control. The slope of dose-response curve of blood TSH increase by TRH was larger under pentobarbital anesthesia and smaller under ether anesthesia than that in non-anesthetized control. A report (15) told that immobilization stress for 10 minutes caused a decrease in blood TSH, and further immobilization for 60 minutes the level went down to a half and the level became 1/4 by 300 minutes’ immobilization. Another report (16) showed that administration of apomorphine caused a decrease of blood TSH level. Infusion with apomorphine (50μg/20μl/min.) lowered the TSH releasing response by TRH. These results may suggest that apomorphine works directly on pituitary. Extraction of anterior pituitary hormones with high efficiency. In estimation of the pituitary contents of hormones, their complete extraction from the gland is indispensable. All the hormones are stored in secretory granules, and some are hydrophobic. We examined various methods for the simple and complete extraction of hormones (11). We found that prolactin was the most difficult to extract, and that immunoreactivty of GH was lost if high concentration of ethanol is present under neutral pH, and that simple homogenization with neutral phosphate buffer is not enough even for glycoprotein hormones. As our conclusion, we found two methods: 1) to homogenize the gland with neutral phosphate buffered saline (PBS) containing 1~2% urea followed by freezing and thawing, and then centrifugation, and 2) to sonicate the gland with PBS containing about 0.1% Triton X-100, followed by freezing and thawing, then centrifugation. The centrifugation at 4,000rpm for 15 minutes will give a clear supernatant fluid. Dilution of the pituitary extract differed depending on hormones and also sex. So, preliminary 8 test for the best dilution for assay is absolutely necessary. Introduction to Rat TSH ELISA KIT (R-type) (AKRTS-010R) Shibayagi is providing an ELISA (Enzyme Linked ImmunoSorbent Assay) kit for measurement of rat TSH (thyroid-stimulating hormone) with high sensitivity using Sandwich assay principle. Features (1) Highly sensitive assay with the standard range of 0.288~36ng/ml. (2) This kit is for TSH in rat serum, culture medium and tissue extract. (3) Assay sample volume is 10μl in the standard procedure. (4) Assay format is 96 wells. (5) Standard preparation is rat origin. (6) Components of the kit are provided ready to use or in concentrated form. Antibodies used in this kit A monoclonal antibody that recognizes TSH β-subunit is solidified on the wells and used as capture antibody, and a monoclonal antibody that recognizes α-subunit is used as detection antibody. Assay principle In Shibayagi’s Rat TSH ELISA Kit (R-type), biotin-conjugated anti-TSH and standard or sample are incubated in monoclonal anti-TSH antibody-coated wells. After 15~18 hours’ incubation and washing, HRP (horse radish peroxidase)-conjugated avidin is added, and incubated for 30 minutes. After washing, HRP-complex remaining in wells are reacted with a chromogenic substrate (TMB) for 30 minutes, and reaction is stopped by addition of acidic solution, and absorbance of yellow product is measured spectrophotometrically at 450 nm(sub-wavelength is 620nm). The absorbance is nearly proportional to TSH concentration. The standard curve is prepared by plotting absorbance against standard TSH concentrations. TSH concentrations in unknown samples are determined using this standard curve. Assay range The range of standard curve:0.288~36ng/ml 9 Reagents supplied Components State Amount Use after washing 96 wells/1 plate Concentrated. Use after dilution 200 μl/1 vial (A)Anti-TSH-coated plate (B)Standard rat TSH (360ng/ml) (C)Buffer solution Ready for use. 60 ml/1 bottle (D)Biotin-conjugated anti-TSH Concentrated. Use after dilution. 50 μl/1 vial (E)HRP-conjugated avidin Concentrated. Use after dilution. 100 μl/1 vial (F)Chromogenic substrate reagent (TMB) Ready for use. 12 ml/1 bottle (H)Reaction stopper(1M H2SO4) Ready for use. 12 ml/1 bottle Concentrated. Use after dilution. 100 ml/1 bottle Plate cover - 1 plate Instruction Manual - 1 copy (I)Concentrated washing buffer (10x) Preparation of assay samples This kit is principally intended to measure TSH in rat serum and plasma. Tissue extracts and incubation or culture media can be also assayed for TSH if confirmed by assay validation tests. For preparation of plasma, we recommend to use EDTA-2Na at final concentration of 1mg/ml. Deep ether anesthesia at blood sampling may lower blood TSH level, we recommend barbiturates as anesthesia, though they also influence hypothalamus to minimize TRH release (Endocrinology 99: 875-880, 1975). High hemolysis (more than 120mg/dl hemoglobin) may influence assay results. If sample is turbid or contains insoluble materials, centrifuge and use clear supernatant fluid. Organic solvents may influence assay results. Assay samples soon after preparation. Sample dilution should be carried out with the buffer solution of the kit using small test tubes before assay. Mix well, and pipette 50 μl of diluted sample into a well. In the standard assay procedure, the dilution rate is 5x. The minimum dilution rate is 2.5x. Undiluted serum and plasma are not suitable because their pH and high protein content may influence assay results. Frozen stored samples should be thawed just before assay and mixed well to make them homogenous. If the presence of any interfering substances is suspected, confirm dilution linearity 10 using more 2 different dilutions or more. Stability and storage of samples In immediate assay, samples can be kept in a refrigerator, and brought to room temperature just before assay. If they have to be kept for a long period, tightly close the container and store lower than –35oC. Avoid repeated freezing and thawing. Summary of Assay Procedure Day 1 Anti-TSH-coated plate ↓Washing 4 times* Biotin-conjugated anti-TSH 50μl ↓Shaking** Diluted sample or standard solution 50μl ↓Shaking**, Reaction at 2~8℃,15~18hours(Standing***) Day 2 Dilution of HRP-conjugated avidin with buffer of room temperature to 200x ↓Washing 4 times* HRP-conjugated avidin 100μl ↓Shaking**, reaction at room temp. 30min (standing***) ↓Washing 4 times* Chromogenic substrate reagent (TMB) 100μl ↓Shaking**, reaction at room temp. 20min (standing***) Reaction stopper(1M H2SO4)※Careful 100μl ↓Shaking** Measurement of absorbance(450nm, sub 620nm) Room temp:20~25℃ *Washing buffer volume:300 μl/well Plate washer pressure:5~25ml/min(depending on nozzle diameter) Be careful not to try wells after removal of liquid. **Guideline of shaking:800rpm-10sec.×3 times Sub-wavelength(reference wavelength):600~650nm ***Put a plate cover on the plate while the reaction after shaking. 11 Standard curve and assay validation An example of standard curve . Precision of assay Intra-assay variation (3 samples, 5 replicates assay,) Mean CV is less than 5 %. Reproducibility Inter-assay variation (3 samples, 4 days, assayed in 4 replicates ) Mean CV is less than 5 % Spike Recovery test Standard TSH was added in 3 concentrations to 2 serum samples and were assayed in duplicates. The recoveries were 98.8 ~103% Dilution test Two serum samples were serially diluted and assayed in triplicates. The dilution curves showed excellent linearity with R2 of 0.9977 and 0.9998. Reference assay data Rat TSH assay data: Mean 3.43 ng/ml, SD 2.34ng/ml Serum samples obtained between 14:00 to 16:00 clock hour from 8 adult CD(SD) male rats. Species specificity of the kit This kit showed 100% cross-reactivity with highly purified canine TSH preparation, BRC canine TSH (12). This indicated theoretically that canine TSH could be measured with this kit. Mouse TSH amino acid sequence showed the homology of 97% with α-subunit and 91% with β-subunit to those of rat TSH, and mouse pituitary homogenate 12 showed a parallel dilution curve to the rat TSH standard curve suggesting the possibility that mouse TSH may be also measurable by this kit. Of course, careful and enough examinations are necessary to apply this kit to canine or mouse TSH assay. Hormone specificity of the kit We examined hormone and species specificity of the monoclonal antibodies used in this kit. The results are shown in the table below. The capture antibody did not react with the family hormones, LH, FSH, or CG. So, these hormones are washed off after the first reaction. The capture antibody cross-reacted with mouse TSH and, to some extent, with human TSH. Reactivity of the capture antibody examined by dot blot test. Dot sample Rat TSH Capture antibody Detection antibody +++ +++ - ++ +++ +++ - ++ ++ ++ Rat LH/FSH/CG Mouse TSH Mouse LH/FSH/CG Human TSH +++ :Well reacted, ++ :Reacted,+ :Weakly reacted,-:Not reacted Reactivity in ELISA system Reactivity was examined using the kit by adding hormones to the system and checked if they give any assay value. Hormones were added in an amount of 100ng/ml. Hormones examined Reactivity Rat TSH 100% Rat LH Less than detection limit Rat FSH Less than detection limit Rat GH Less than detection limit 85%(*) Human TSH *: Preparaton used: Acris Antibodies GmbH/PA1199 (Recombinant human TSH) Potency>4 IU/mg (This potency seems to be very low compared with highly purified rat TSH that has the potency of 35IU/mg. But this is a recombinant TSH without glycosylation. Deglycosylated glycoprotein hormone can bind the receptor, and immunologically active, however, its action is weak.). . 13 Amino acid sequence of TSH subunits with their homology to rat TSH Bold letters shows different amino acids from those of rat TSH α-Subunit Bull TSH α-subunit fpdgeftmqg cpecklkenk yfskpdapiy qcmgccfsra yptparskkt mlvpknitse atccvakaft katvmgnvrv enhtechcst cyyh Homology: 84/96 (87.5%) Pig TSH α-subunit fpdgeftmqg cpecklkenk yfsklgapiy qcmgccfsra yptparskkt mlvpknitse atccvakaft katvmgnarv enhtechcst cyyhks Homology: 90/96 (93.8%) Cat TSH α-subunit fpdgeftmqg cpecklkenk yfsklgapiy qcmgccfsra yptparskkt mlvpknitse atccvakaft katvmgnakv enhtechcst cyhhki Homology: 86/96 (89.6%) Horse TSH α-subunit fpdgefttqd cpecklrenk yffklgvpiy qckgccfsra yptparsrkt mlvpknitse stccvakafi rvtvmgnikl enhtqcycst cyhhki Homology: 77/96 (80.2%) Human TSH α-subunit apdvqdcpec tlqenpffsq pgapilqcmg ccfsrayptp lrskktmlvq knXXXXvtss etccvaksyn rvtvmggfkv enhtachcst cyyhks Homology: 33/96 (34.4%) β-Subunit Bull TSH β-subunit fcipteymmh verkecaycl tinttvcagy cmtrdvngkl flpkyalsqd vctyrdfmyk taeipgcprh vtpyfsypva isckcgkcnt dysdciheai ktnyctkpqk symvgfsi Homology: 102/118 (86.4%) Pig TSH β-subunit fcipteymmh verkecaycl tintticagy cmtrdfngkl flpkyalsqd vctyrdfmyk tveipgcphh vtpyfsypva isckcgkcnt dysdciheai ktnyctkpqk syvlefsi Homology: 103/118 (87.3%) Cat TSH β-subunit fcfpteymmh verkecaycl tintticagy cmtrdingkl flpkyalsqd vctyrdflyk tveipgcphh vtpyfsypva vsckcgkcnt dysdciheai ktndctkpqk sdvvgvXX 14 Homology: 18/118 (84.7%) Horse TSH β-subunit fcipteymmh verkecaycl tintticagy cmtrdingkl flpkyalsqd vctyrdfmyk tveipgcpdh vtpyfsypva vsckcgkcnt dysdciheai kanyctkpqk syvvefsi Homology: 102/118 (86.4%) Human TSH β-subunit fcipteytmh ierrecaycl tintticagy cmtrdingkl flpkyalsqd vctyrdfiyr tveipgcplh vapyfsypva lsckcgkcnt dysdciheai ktnyctkpqk sylvgfsv Homology: 106/118 (89.8%) References 1. Mori, M., Kobayashi, I., and Wakabayashi, K. Suppression of serum thyrotropin (TSH) concetrations following thyroidectomy and cold exposure by passive immunization with antiserum to thyrotropin-releasing hormone (TRH) in rats. Metabolism 27:1485-1490, 1978 2. Woeber, K. A., and Braverman, L. E. ”Contemporary Endocrinology”, Ingbar, S. H.(Ed.) Vol. 1, Plenum Press, N.W., 1979, pp.77-117. 3. Guillemin, R. Recent Prog. Horm. Res., 28, 229-286, 1977 4. Wilber, J. F., Utiger, R. D. The effect of glucocorticoids on thyrotropin secretion. J. Clin. Invest., 48, 2096, 1969 5. Weeke, J. Scand. J. Clin. Lab. Invest., 33, 17, 1974 6. Ottenweller, J. E., and Hedge, G. A. Diurnal variations of plasma thyrotropin, thyroxine, and triiodothyronine in female rats are phase shifted after inversion of the photoperiod Endocrinology, 111: 509-514, 1982 7. Ozturk, G., Coskun, S., Erbas, D., and Hasanoglu, E. The effect of melatonin on liver superoxide dismutase activity, serum nitrate and thyroid hormone levels. Jpn J. Physiol.;50:149-53, 2000 8. Ooka-Souda, S., Draves, D. J., Timiras, P. S. 15 Diurnal rhythm of pituitary-thyroid axis in male rats and the effect of adrenalectomy. Endocri. Res. Commun. 6:43-56, 1979 9. Mori, M., Oshima, K., Fukuda, H., Kobayashi, I. , and Wakabayashi K. Changes in the multiple components of rat pituitary TSH and TSHβ subunit following thyroidectomy. Acta Endocrinol., 105: 49-56, 1984 10. Spontaneous diurnal TSH secretion is enhanced in proportion to circulating leptin in obese premenopausal women. Petra Kok, P., Ferdinand Roelfsema , F., Frölich, M., Meinders, A., E. , Pijl, H. J. Clin. Endocr. Metab. August 9, 2005 as doi:10.1210/jc.2005-0003 11. Ishikawa J., Fuse, Y., and Wakabayashi, K. Choice of extraction procedure for estimation of anterior pituitary hormone content. Endocrinol. Japon. 34, 755-767, 1987 12. Chiba, K., Kobayashi, H., and Wakabayashi, K. Isolation and partial characterization of LH, FSH and TSH from canine pituitary gand. Endocrine J., 44: 205-218,1997 13. Mannisto, P. T., Saarinen, A., and Ranta, T. Anesthetics and thyrotropin secretion in the rat Endocrinology 99: 875-880, 1976 14. Ohtake M, Bray GA. Effects of pentobarbital anesthesia on thyroid function in the rat. Horm Metab Res. 9: 146-149, 1977 15. Immobilization stress and prolactin secretion in male rats. Kawakami, M., Higuchi, T., Matsuura, M. Neuroendocrinology 29: 262-269, 1079 16. Studies on the inhibitory effect of apomorphine and bromocryptine basal and TRH induced level of TSH and PRL in hypothyroid rats under pentobarbiturate anesthesia. Langer, P., Mess, B., Foldes, O., Ruzsas, C., Brozmanova, H., Straussove, K., and Gschwendtova, K. Exp. Clin. Endocrinol., 83: 269-274, 1984 (10/03/04) 16