Endogenous Metabolite

Thiamine (vitamin B1) is required in the diet of animals, and thiamine deficiency leads to diseases such as beri-beri and the Wernicke-Korsakoff syndrome. Dietary thiamine (vitamin B1) consists mainly of thiamine pyrophosphate (TPP), which is transformed into thiamine by gastrointestinal phosphatases before absorption. It is believed that TPP itself cannot be transported across plasma membranes in significant amounts. We have identified a partial loss-of-function mutation in the Caenorhabditis elegans gene (tpk-1) that encodes thiamine pyrophosphokinase, which forms TPP from thiamine at the expense of ATP inside cells. The mutation slows physiological rhythms and the phenotype it produces can be rescued by TPP but not thiamine supplementation. tpk-1 functions cell nonautonomously, as the expression of wild-type tpk-1 in one tissue can rescue the function of other tissues that express only mutant tpk-1. These observations indicate that, in contrast to expectation from previous evidence, TPP can be transported across cell membranes. We also find that thiamine supplementation partially rescues the phenotype of partial loss-of-function mutants of the Na/K ATPase, providing genetic evidence that thiamine absorption, and/or redistribution from the absorbing cells, requires the full activity of this enzyme[3].

In this study, the biochemical and histopathological effects of thiamine and thiamine pyrophosphate on ischemia-reperfusion induced oxidative damage in rat ovarian tissue were investigated. Animals were divided into four groups of six rat each, ovarian ischemia-reperfusion (IR), 25 mg/kg thiamine + ovarian ischemia-reperfusion (TIR), 25 mg/kg thiamine pyrophosphate + ovarian ischemia-reperfusion (TPIR) and Sham group (SG). The results of the biochemical experiments have shown that the rat ovarian tissue with thiamine treatment, the level of MDA, GSH and the 8-hydroxyguanine are almost the same as the IR group; while in the group with thiamine pyrophosphate treatment, the level of MDA, GSH and the 8-hydroxyguanine are almost the same as the SG. Ovarian tissue of rats in the IR group were congested and dilated vessels, edema, hemorrhage, necrotic and apoptotic cells. In this group, the migration and the adhesion of the polymorphonuclear leucocytes to the endothelium were observed. Both ovaries in TPIR group, there was no difference according to the SG. Histopathology of ovarian tissues in the TIR group was almost the same with the IR group. Our results indicate that thiamine pyrophosphate significantly prevents the ischemia-reperfusion induced oxidative damage in ovarian tissue, whereas thiamine has no effect. In conclusion, we have found that thiamine pyrophosphate prevents oxidative damage due to ischemia-reperfusion injury, whereas thiamine does not have this effect. Furthermore, we have confirmed that the results of our biochemical analyses are in concordance with the histopathological findings[1].

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This study investigated the effect of thiamine pyrophosphate on oxidative damage associated with cardiotoxicity caused by cisplatin (CIS), an antineoplastic agent, in rats, and compared this with thiamine. Animals used in the study were divided into four groups of 6 rats each. These represented a control group receiving 5 mg/kg of CIS, study groups receiving 20 mg/kg of thiamine pyrophosphate plus 5 mg/kg of cisplatin (CTPG) or 20 mg/kg of thiamine plus 5 mg/kg of cisplatin and a healthy (H) group. All doses were administered intraperitoneally once a day for 14 days. Malondialdehyde, total glutathione and products of DNA injury results were similar in the CTPG and H groups (p > 0.05). Creatinine kinase, creatine kinase MB and troponin 1 levels were similar in the CTPG and H groups (p > 0.05). Thiamine pyrophosphate prevented CIS-associated oxidative stress and heart injury, whereas thiamine did not prevent these.[2]

Biochemical analysis of ovarian tissue[1]
In this part, 0.2 g of whole ovarian tissue was weighed for each ovary. The samples were homogenized in ice with 2 ml buffers consisting of 1.15 % potassium chloride solution for MDA analysis and pH 7.5 phosphate buffer for the other analyses. Then, they were centrifuged at 4 °C, 10,000 rpm for 15 min. The supernatant part was used as the analysis sample. For all the measurements the tissue-protein estimation was performed according to Bradford’s method (Bradford 1976).[1]

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Total glutathione (tGSH) determination[1]
The amount of GSH in the total homogenate was measured according to the method of Sedlak and Lindsay with some modifications (Sedlak and Lindsay 1968). The sample was weighed and homogenized in 2 ml of 50 mM Tris–HCl buffer containing 20 mM EDTA and 0.2 mM sucrose at pH 7.5. The homogenate was immediately precipitated with 0.1 ml of 25 % trichloroacetic acid, and the precipitate was removed after centrifugation at 4,200 rpm for 40 min at 4 °C and the supernatant was used to determine GSH level. 1,500 μl of measurement buffer (200 mM Tris–HCl buffer containing 0.2 mM EDTA at pH 7.5), 500 μl supernatant, 100 μl DTNB (10 mM) and 7,900 μl methanol were added to a tube and vortexed and incubated for 30 min in 37 °C. 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) was used as an chromogen and it formed a yellow-colored complex with SH groups. The absorbance was measured at 412 nm using a spectrophotometer. The standard curve was obtained by using reduced glutathione.[1]

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MDA determination[1]
The concentrations of ovarian lipid peroxidation were determined by estimating MDA using the thio barbituric acid test (Ohkawa et al. 1979). The rat ovaries were rinsed with cold saline. The corpus mucosa was scraped, weighed and homogenized in 10 ml of 100 g/l KCl. The homogenate (0.5 ml) was added to a solution containing 0.2 ml of 80 g/l sodium lauryl sulfate, 1.5 ml of 200 g/l acetic acid, 1.5 ml of 8 g/l 2-thiobarbiturate, and 0.3 ml distilled water. The mixture was incubated at 98 °C for 1 h. Upon cooling, 5 ml of n-butanol:pyridine (15:l) was added. The mixture was vortexed for 1 min and centrifuged for 30 min at 4,000 rpm. The absorbance of the supernatant was measured at 532 nm. The standard curve was obtained by using 1,1,3,3-tetramethoxypropane.

General procedure[1]
Experimental animals were randomly divided into four groups of six rat each, ovarian ischemia–reperfusion (IR), 25 mg/kg thiamine + ovarian ischemia–reperfusion (TIR), 25 mg/kg thiamine pyrophosphate + ovarian ischemia–reperfusion (TPIR) and SG before surgical and pharmacological processes. Sham-operated group named SG group was performed only laparotomy.

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Surgical and pharmacological processes[1]
One hour before anesthesia with thiopental sodium, TIR and TPIR groups were injected i.p. thiamine and thiamine pyrophosphate respectively. IR and SG rat groups were administered distilled water as a solvent in the same way. Surgical procedures on rats was performed under sterile conditions, following anesthesia with intraperitoneal (i.p.) administration of thiopental sodium (25 mg/kg). One hour after administration drugs, the ovaries of all rat groups were obtained under anesthesia by performing a 2–2.5 cm lower abdominal vertical incision. Then TIR, TPIR and IR group except the SG groups ischemia for 3 h was created in rats by applying vascular clips to the lower part of the right ovary and reperfusion for 2 h was achieved. At the end of this period, all animals were killed with an overdose of anesthesia and their right and left ovaries were removed and histopathological and biochemical examinations were conducted. Pathological and biochemical results were compared among the TIR, TPIR, and IR groups, as well as with the SG group (Kurt et al. 2011).

[1]. A comparative investigation of biochemical and histopathological effects of thiamine and thiamine pyrophosphate on ischemia-reperfusion induced oxidative damage in rat ovarian tissue. Arch Pharm Res. 2013 Sep;36(9):1133-9.

[2]. The protective effect of thiamine pyrophosphate, but not thiamine, against cardiotoxicity induced with cisplatin in rats. Drug Chem Toxicol. 2014 Jul;37(3):290-4.

[3]. Thiamine pyrophosphate biosynthesis and transport in the nematode Caenorhabditis elegans. Genetics. 2004 Oct;168(2):845-54.

Thiamine(1+) diphosphate chloride is an organic chloride salt of thiamine(1+) diphosphate. It is an organic chloride salt and a vitamin B1. It contains a thiamine(1+) diphosphate.
The coenzyme form of Vitamin B1 present in many animal tissues. It is a required intermediate in the pyruvate dehydrogenase complex and the ketoglutarate dehydrogenase complex.
Thiamine pyrophosphate is a metabolite found in or produced by Escherichia coli (strain K12, MG1655).
The coenzyme form of Vitamin B1 present in many animal tissues. It is a required intermediate in the PYRUVATE DEHYDROGENASE COMPLEX and the KETOGLUTARATE DEHYDROGENASE COMPLEX.

C12H19CLN4O7P2S

460.7674

460.013

C, 31.28; H, 4.16; Cl, 7.69; N, 12.16; O, 24.31; P, 13.44; S, 6.96

154-87-0

9068

White to off-white solid powder

4

12

8

27

568

0

YXVCLPJQTZXJLH-UHFFFAOYSA-N

InChI=1S/C12H18N4O7P2S.ClH/c1-8-11(3-4-22-25(20,21)23-24(17,18)19)26-7-16(8)6-10-5-14-9(2)15-12(10)13;/h5,7H,3-4,6H2,1-2H3,(H4-,13,14,15,17,18,19,20,21);1H

2-[3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-4-methyl-1,3-thiazol-3-ium-5-yl]ethyl phosphono hydrogen phosphate;chloride

Thiamine pyrophosphate chloride, Thiamine pyrophosphate chloride, Aneurinepyrophosphoric acid, Cocarboxylase, Cocarboxylase Aneurinepyrophosphoric acid; Cocarboxilasa; Cocarboxylasum; Berolase;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)