JBBS  Vol.7 No.2 , February 2017
Validation of a HPLC Method for Quantification of Thiamine and Its Phosphate Esters in Rat Brain Tissue
Abstract: The present data show a fast and efficient biological sample processing method for the extraction of thiamine (vitamin B1) and its mono-(TMP) and di-(TDP) phosphate esters from hippocampus, thalamus and prefrontal cortex (PFC) and blood sample of the rodents. In addition, using the hippocampus and standards of these three compounds we validated an isocratic fluorescence HPLC procedure for a simultaneous detection of them in a single chromatogram within a total run time of about 12 min. Reproducibility for TDP, TMP and B1 was 2.66%, 4.50% and 7.43% (intraday) and 37.54%, 25.39% and 25.87% (interday), respectively. Recovery assays were between 96.0% and 101.7%. The calibration curves were linear and the concentrations of the three compounds, all in nanomolar range, were determined in the brain areas and in the blood samples. When compared to the current methods in the literature, this new method provides information on essential variables, such as linearity range and limit of detection, reproducibility and stability of thiamine, TMP and TDP in rat brain samples. The present data on sample processing and B1 and its phosphate ester level determinations are the first to be validated using hippocampus samples of rats.
Cite this paper: Nunes, P. , da Silva Oliveira, P. , Ferraz, V. and Ribeiro, A. (2017) Validation of a HPLC Method for Quantification of Thiamine and Its Phosphate Esters in Rat Brain Tissue. Journal of Behavioral and Brain Science, 7, 79-93. doi: 10.4236/jbbs.2017.72009.

[1]   Lu, J. and Frank, E.L. (2008) Rapid HPLC Measurement of Thiamine and Its Phosphate Esters in Whole Blood. Clinical Chemistry, 54, 901-906.

[2]   Gibson, G.E. and Zhang, H. (2002) Interactions of Oxidative Stress with Thiamine Homeostasis Promote Neurodegeneration. Neurochemistry International, 40, 493- 504.

[3]   Lonsdale, D. (2006) A Review of the Biochemistry, Metabolism and Clinical Benefits of Thiamin (e) and Its Derivatives. Evidence-Based Complementary and Alternative Medicine, 3, 49-59.

[4]   Manzetti, S., Zhang, J. and van der Spoel, D. (2014) Thiamin Function, Metabolism, Uptake, and Transport. Biochemistry, 53, 821-835.

[5]   Mayr, J.A., Freisinger, P., Schlachter, K., Rolinski, B., Zimmermann, F.A., Scheffner, T., Sperl, W., et al. (2011) Thiamine Pyrophosphokinase Deficiency in Encephalopathic Children with Defects in the Pyruvate Oxidation Pathway. The American Journal of Human Genetics, 89, 806-812.

[6]   Harper, C. (2009) The Neuropathology of Alcohol-Related Brain Damage. Alcohol and Alcoholism, 44, 136-140.

[7]   Kopelman, M.D., Thomson, A.D., Guerrini, I. and Marshall, E.J. (2009) The Korsakoff Syndrome: Clinical Aspects, Psychology and Treatment. Alcohol and Alcoholism, 44, 148-154.

[8]   Thomson, A.D., Baker, H. and Leevy, C.M. (1970) Patterns of 35S-Thiamine Hydrochloride Absorption in the Malnourished Alcoholic Patient. Journal of Laboratory and Clinical Medicine, 76, 34-45.

[9]   Gibson, G.E., Hirsch, J.A., Fonzetti, P., Jordan, B.D., Cirio, R.T. and Elder, J. (2016) Vitamin B1 (Thiamine) and Dementia. Annals of the New York Academy of Sciences, 1367, 21-30.

[10]   Zubaran, C., Fernandes, J.G. and Rodnight, R. (1997) Wernicke-Korsakoff Syndrome. Postgraduate Medical Journal, 73, 27-31.

[11]   Troncoso, J.C., Johnston, M.V., Hess, K.M., Griffin, J.W. and Price, D.L. (1981) Model of Wernicke’s Encephalopathy. Archives of Neurology, 38, 350-354.

[12]   Pires, R.G., Pereira, S.R., Pittella, J.E.H., Franco, G.C., Ferreira, C.L., Fernandes, P.A. and Ribeiro, A.M. (2001) The Contribution of Mild Thiamine Deficiency and Ethanol Consumption to Central Cholinergic Parameter Dysfunction and Rats’ Open-Field Performance Impairment. Pharmacology Biochemistry and Behavior, 70, 227-235.

[13]   Pires, R.G., Pereira, S.R., Oliveira-Silva, I.F., Franco, G.C. and Ribeiro, A.M. (2005) Cholinergic Parameters and the Retrieval of Learned and Re-Learned Spatial Information: A Study Using a Model of Wernicke-Korsakoff Syndrome. Behavioural Brain Research, 162, 11-21.

[14]   Carvalho, F.M., Pereira, S.R., Pires, R.G., Ferraz, V.P., Romano-Silva, M.A., Oliveira-Silva, I.F. and Ribeiro, A.M. (2006) Thiamine Deficiency Decreases Glutamate Uptake in the Prefrontal Cortex and Impairs Spatial Memory Performance in a Water Maze Test. Pharmacology Biochemistry and Behavior, 83, 481-489.

[15]   Vigil, F.A.B., de Fátima Oliveira-Silva, I., Ferreira, L.F., Pereira, S.R.C. and Ribeiro, A.M. (2010) Spatial Memory Deficits and Thalamic Serotonergic Metabolite Change in Thiamine Deficient Rats. Behavioural Brain Research, 210, 140-142.

[16]   de Freitas-Silva, D.M., de Souza Resende, L., Pereira, S.R.C., Franco, G.C. and Ribeiro, A.M. (2010) Maternal Thiamine Restriction during Lactation Induces Cognitive Impairments and Changes in Glutamate and GABA Concentrations in Brain of Rat Offspring. Behavioural Brain Research, 211, 33-40.

[17]   de Fátima Oliveira-Silva, I., Pereira, S.R.C., Fernandes, P.A., Ribeiro, A.F., Pires, R.G. and Ribeiro, A.M. (2015) Mild Thiamine Deficiency and Chronic Ethanol Consumption Modulate Acetylcholinesterase Activity Change and Spatial Memory Performance in a Water Maze Task. Journal of Molecular Neuroscience, 55, 217- 226.

[18]   Ferreira-Vieira, T.H., de Freitas-Silva, D.M., Ribeiro, A.F., Pereira, S.R.C. and Ribeiro, Â.M. (2016) Perinatal Thiamine Restriction Affects Central GABA and Glutamate Concentrations and Motor Behavior of Adult Rat Offspring. Neuroscience Letters, 617, 182-187.

[19]   Pannunzio, P., Hazell, A.S., Pannunzio, M., Rao, K.V. and Butterworth, R.F. (2000) Thiamine Deficiency Results in Metabolic Acidosis and Energy Failure in Cerebellar Granule Cells: An in Vitro Model for the Study of Cell Death Mechanisms in Wernicke’s Encephalopathy. Journal of Neuroscience Research, 62, 286-292.<286::AID-JNR13>3.0.CO;2-0

[20]   Savage, L.M., Hall, J.M. and Vetreno, R.P. (2011) Anterior Thalamic Lesions Alter Both Hippocampal-Dependent Behavior and Hippocampal Acetylcholine Release in the Rat. Learning & Memory, 18, 751-758.

[21]   Nakagawasai, O. (2005) Behavioral and Neurochemical Alterations Following Thiamine Deficiency in Rodents: Relationship to Functions of Cholinergic Neurons. Yakugaku Zasshi, 125, 549-554.

[22]   Nakagawasai, O., Murata, A., Arai, Y., Ohba, A., Wakui, K., Mitazaki, S., Tadano, T., et al. (2007) Enhanced Head-Twitch Response to 5-HT-Related Agonists in Thiamine-Deficient Mice. Journal of Neural Transmission, 114, 1003-1010.

[23]   Savage, L.M., Chang, Q. and Gold, P.E. (2003) Diencephalic Damage Decreases Hippocampal Acetylcholine Release during Spontaneous Alternation Testing. Learning & Memory, 10, 242-246.

[24]   Vetreno, R.P., Anzalone, S.J. and Savage, L.M. (2008) Impaired, Spared, and Enhanced ACh Efflux across the Hippocampus and Striatum in Diencephalic Amnesia is Dependent on Task Demands. Neurobiology of Learning and Memory, 90, 237- 244.

[25]   Roland, J.J. and Savage, L.M. (2009) The Role of Cholinergic and GABAergic Medial Septal/Diagonal Band Cell Populations in the Emergence of Diencephalic Amnesia. Neuroscience, 160, 32-41.

[26]   Savage, L.M., Hall, J.M. and Resende, L.S. (2012) Translational Rodent Models of Korsakoff Syndrome Reveal the Critical Neuroanatomical Substrates of Memory Dysfunction and Recovery. Neuropsychology Review, 22, 195-209.

[27]   Langlais, P.J., Mandel, R.J. and Mair, R.G. (1992) Diencephalic Lesions, Learning Impairments, and Intact Retrograde Memory Following Acute Thiamine Deficiency in the Rat. Behavioural Brain Research, 48, 177-185.

[28]   Hazell, A.S. and Butterworth, R.F. (2009) Update of Cell Damage Mechanisms in Thiamine Deficiency: Focus on Oxidative Stress, Excitotoxicity and Inflammation. Alcohol and Alcoholism, 44, 141-147.

[29]   Jhala, S.S. and Hazell, A.S. (2011) Modeling Neurodegenerative Disease Pathophysiology in Thiamine Deficiency: Consequences of Impaired Oxidative Metabolism. Neurochemistry International, 58, 248-260.

[30]   Langlais, P.J. and Savage, L.M. (1995) Thiamine Deficiency in Rats Produces Cognitive and Memory Deficits on Spatial Tasks That Correlate with Tissue Loss in Diencephalon, Cortex and White Matter. Behavioural Brain Research, 68, 75-89.

[31]   Langlais, P.J., Zhang, S.X. and Savage, L.M. (1996) Neuropathology of Thiamine Deficiency: An Update on the Comparative Analysis of Human Disorders and Experimental Models. Metabolic Brain Disease, 11, 19-37.

[32]   Langlais, P.J. and Zhang, S.X. (1997) Cortical and Subcortical White Matter Damage without Wernicke’s Encephalopathy after Recovery from Thiamine Deficiency in the Rat. Alcoholism: Clinical and Experimental Research, 21, 434-443.

[33]   Dror, V., Eliash, S., Rehavi, M., Assaf, Y., Biton, I.E. and Fattal-Valevski, A. (2010) Neurodegeneration in Thiamine Deficient Rats—A Longitudinal MRI Study. Brain Research, 1308, 176-184.

[34]   Noble, J.M., Mandel, A. and Patterson, M.C. (2007) Scurvy and Rickets Masked by Chronic Neurologic Illness: Revisiting “Psychologic Malnutrition”. Pediatrics, 119, e783-e790.

[35]   Pincus, J.H. and Grove, I. (1970) Distribution of Thiamine Phosphate Esters in Normal and Thiamine-Deficient Brain. Experimental Neurology, 28, 477-483.

[36]   Rindi, G. and De Giuseppe, L. (1961) A New Chromatographic Method for the Determination of Thiamine and Its Mono-, Di- and Tri- Phosphates in Animal Tissues. Biochemical Journal, 78, 602.

[37]   Rindi, G., Patrini, C., Comincioli, V. and Reggiani, C. (1980) Thiamine Content and Turnover Rates of Some Rat Nervous Regions, Using Labeled Thiamine as a Tracer. Brain Research, 181, 369-380.

[38]   Adamolekun, B. (2010) Etiology of Konzo, Epidemic Spastic Paraparesis Associated with Cyanogenic Glycosides in Cassava: Role of Thiamine Deficiency? Journal of the Neurological Sciences, 296, 30-33.

[39]   Basoglu, M., Yetimalar, Y., Gürgör, N., Büyükçatalbas, S., Kurt, T., Seçil, Y. and Yeniocak, A. (2006) Neurological Complications of Prolonged Hunger Strike. European Journal of Neurology, 13, 1089-1097.

[40]   Warnock, L.G., Prudhomme, C.R. and Wagner, C. (1978) The Determination of Thiamin Pyrophosphate in Blood and Other Tissues, and Its Correlation with Erythrocyte Transketolase Activity. Journal of Nutrition, 108, 421-427.

[41]   Basiri, B., Sutton, J.M., Hanberry, B.S., Zastre, J.A. and Bartlett, M.G. (2016) Ion pair Liquid Chromatography Method for the Determination of Thiamine (Vitamin B1) Homeostasis. Biomedical Chromatography, 30, 35-41.

[42]   Bettendorff, L., Peeters, M., Jouan, C., Wins, P. and Schoffeniels, E. (1991) Determination of Thiamin and Its Phosphate Esters in Cultured Neurons and Astrocytes Using an Ion-Pair Reversed-Phase High-Performance Liquid Chromatographic Method. Analytical Biochemistry, 198, 52-59.

[43]   Héroux, M. and Butterworth, R.F. (1995) Regional Alterations of Thiamine Phosphate Esters and of Thiamine Diphosphate-Dependent Enzymes in Relation to Function in Experimental Wernicke’s Encephalopathy. Neurochemical Research, 20, 87-93.

[44]   Losa, R., Sierra, M.I., Fernández, A., Blanco, D. and Buesa, J.M. (2005) Determination of Thiamine and Its Phosphorylated Forms in Human Plasma, Erythrocytes and Urine by HPLC and Fluorescence Detection: A Preliminary Study on Cancer Patients. Journal of Pharmaceutical and Biomedical Analysis, 37, 1025-1029.

[45]   National Research Council Guide for the Care and Use of Laboratory Animals (1985) A Report of the Institute of Laboratory Animal Resources Committee on Care and Use of Laboratory Animals. U.S. Department of Health and Human Services, Washington DC.

[46]   Batifoulier, F., Verny, M.A., Besson, C., Demigne, C. and Remesy, C. (2005) Determination of Thiamine and Its Phosphate Esters in Rat Tissues Analyzed as Thiochromes on a RP-Amide C16 Column. Journal of Chromatography B, 816, 67-72.

[47]   Stuetz, W., Carrara, V.I., McGready, R., Lee, S.J., Biesalski, H.K. and Nosten, F.H. (2012) Thiamine Diphosphate in Whole Blood, Thiamine and Thiamine Monophosphate in Breast-Milk in a Refugee Population. PloS ONE, 7, e36280.

[48]   Gangolf, M., Czerniecki, J., Radermecker, M., Detry, O., Nisolle, M., Jouan, C., Grisar, T., et al. (2010) Thiamine Status in Humans and Content of Phosphorylated Thiamine Derivatives in Biopsies and Cultured Cells. PLoS ONE, 5, e13616.

[49]   Roser, R.L., Andrist, A.H., Harrington, W.H., Naito, H.K. and Lonsdale, D. (1978) Determination of Urinary Thiamine by High-Pressure Liquid Chromatography Utilizing the Thiochrome Fluorescent Method. Journal of Chromatography B: Biomedical Sciences and Applications, 146, 43-53.

[50]   Baker, H., Frank, O., Fennelly, J.J. and Leevy, C.M. (1964) A Method for Assaying Thiamine Status in Man and Animals. The American Journal of Clinical Nutrition, 14, 197-201.

[51]   Vedder, L.C., Hall, J.M., Jabrouin, K.R. and Savage, L.M. (2015) Interactions between Chronic Ethanol Consumption and Thiamine Deficiency on Neural Plasticity, Spatial Memory, and Cognitive Flexibility. Alcoholism: Clinical and Experimental Research, 39, 2143-2153.

[52]   Rindi, G., De Giuseppe, L. and Sciorelli, G. (1968) Thiamine Monophosphate, a Normal Constituent of Rat Plasma. Journal of Nutrition, 94, 447-454.

[53]   Kimura, M., Fujita, T. and Itokawa, Y. (1982) Liquid-Chromatographic Determination of the Total Thiamin Content of Blood. Clinical Chemistry, 28, 29-31.