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.
Vitamin B1 consists of free thiamine, a pyrimidyl substituted thiazole, and its phosphate esters thiamine monophosphate (TMP), thymidine diphosphate (TDP) and thiamine triphosphate [
In humans, thiamine deficiency (TD) causes brain dysfunctions known as Wernicke’s Encephalopathy and Wernicke-Korsakoff Syndrome (WKS), respectively considered by some authors as the acute and chronic stages of the same disorder. Symptoms observed in WKS include oculomotor disturbance, ataxia and confusion (acute symptoms), and amnesia and confabulation (chronic symptoms) [
Our group [
TD in rodents is a relevant model to study cellular and molecular mechanisms that lead to a gradual progression of selective neuronal loss that resembles clinical neurodegenerative diseases [
Therefore, the quantification of thiamine and its phosphate esters in rodent brain areas has been of great interest and there are continual attempts to accurately measure their concentrations under physiological and pathological conditions. A multitude of methods for the determination of thiamine levels through microbiological and biochemical techniques such as chromatography (e.g. high- performance liquid chromatography―HPLC), spectrophotometry and fluorescence by ultraviolet exposure were described [
HPLC grade methanol was obtained from J. T. Baker (Phillipsburg, NJ, USA). Trichloroacetic acid (TCA), potassium ferricyanide (K3Fe(CN)6), thiamine hydrochloride (vitamin B1), thiamine pyrophosphate (TDP), thiamine monophosphate chloride (TMP) and hydrochloric acid were purchased by Sigma-Al- drich (St. Louis, MO, USA). Diethyl ether was obtained from Cromoline (Diadema, SP, Brazil), disodium hydrogen phosphate (Na2HPO4・7H2O), sodium dihydrogen phosphate (NaH2PO4・H2O), sodium hydroxide and triethylamine (TEA) were obtained from Reagen (Colombo, PN, Brazil). Ethylenediaminetetraacetic acid (EDTA) was purchased from Vetec (Duque de Caxias, RJ, Brazil). All reagents were of the highest purity available. For all solutions in which water had to be added, Milli-Q deionized water was used.
The HPLC system was a Shimadzu chromatograph (LC-10AD, Tokyo, Japan) with a fluorescence detector (FLD-Shimadzu spectrofluorometric detector RF- 551, Tokyo, Japan), a 200 µL loop (Rheodyne 7725-I, CA, USA) and a LC-10 AD PUMP. This system was equipped with a 5 µm particle size analytical column (Purospher Star RP-18 end capped −250 mm × 4.6 mm, ID-Merck, Darmstadt E.R., Germany) and a pre-packed column (Purospher Star RP-18 −4 × 4 mm- Merck, Darmstadt E.R., Germany). An integrator (Shimadzu C-R7Ae plus) was used to analyze the chromatographic data.
The mobile phase consisted of an equimolar buffer solution of 0.14 M NaH2PO4・H2O and Na2HPO4・7H2O and methanol (70:30, v/v), pH 7.0, plus 0.1% TEA. Before the chromatographic analyses, the phase was filtered through 0.45 µm membrane filters (Millipore Durapore) and vacuum degassed prior to use. Chromatographic analyses were performed at 25˚C ± 2˚C and the compounds were eluted isocratically over 15 min runtime at a flowrate of 1 mL/min. The fluorescence detector was set at an excitation wavelength of 367 nm and an emission wavelength of 435 nm, high sensitivity and range of 1.5. Both peak height and area are proportional to concentration.
One milligram per milliliter stock solutions of TDP, TMP and B1 were prepared in 0.1 M hydrochloric acid and aliquoted out for keeping at −20˚C. Standard solutions (100, 325, 550, 775, 1000 ng/mL for TDP; 50, 100, 175, 250 ng/mL for TMP and 2, 10, 50, 80, 100, 150 ng/mL for B1) were prepared daily by dilutions of the stock solutions, aliquoted out and stored at 4˚C until derivatization and analysis. 150 µL of standards were derivatized by addition of 150 µL of freshly prepared potassium ferricyanide in 15% (w/v) sodium hydroxide. A blank sample was used containing 150 µL of water and 150 µL of potassium ferricyanide solution.
Sixteen male Wistar rats weighing 250 - 300 g were collected from the Centro de Bioterismo da Universidade Federal de Minas Gerais (CEBIO-ICB-UFMG vivarium). The rats were housed in plastic cages in groups of four, maintained on a 12:12 h light-dark cycle and fed ad libitum. Animals were killed by decapitation in random order, blood samples were collected, their brains were removed and the tissues were separated and processed as described in Section 2.5. The present study was approved by the Ethics Committee for Care and Use of Laboratory Animals of the Universidade Federal de Minas Gerais (CEUA-UFMG protocol number: 107/2010) and the care and use of animals were done according to the National Institutes of Health Guide for Care and Use of Laboratory Animals [
As mentioned above, the brains were rapidly removed from the cranium and prefrontal cortex, thalamus and hippocampus were dissected out on an ice-cold plate. The tissues were weighed and placed in 1.5 mL microcentrifuge tubes, and stored at −80˚C until analysis. Samples of 500 µL of blood were collected in tubes containing EDTA 6% (w/v) and stored at −20˚C until the day of the assay. All assays were done within one week. On the day of the assay the samples were thawed, homogenized and the thiamine and its phosphate esters extracted. The assays were then carried out in the supernatant extract within one minute after derivatization. All these methods are described below.
The brain tissue and blood samples were processed as described in [
The assay validations were carried out with hippocampus samples of male Wistar rats and standards of thiamine and its phosphate esters.
Linearity and detection limit: The linearity of the detector response to standard solutions of TDP, TMP and B1 were determined. Calibration curves obtained from standard solutions of 100, 325, 550, 775 and 1000 ng/mL of TDP; 10, 50, 100, 175 and 250 ng/mL of TMP, and 10, 50, 80, 100 and 150 ng/mL of B1 were done.
Recovery: For evaluating the recovery of TDP, TMP and B1, a mixture of these compounds was added to samples of hippocampus at the moment of homogenization, at final concentrations of 100, 250, 400, 550 and 700 ng/mL of TDP; 25, 75, 125, 175 and 250 ng/mL of TMP and 20, 40, 60, 80 and 100 ng/mL of B1. Recovery was calculated as: [(final concentration-initial concentration)/ added concentration].
Stability: Experiments were done to determine the stability of thiamine and its phosphate esters in two kinds of samples/conditions: 1) hippocampus homogenate and 2) supernatant extract after derivatization process. In the first case, homogenized hippocampus samples were stored at −80˚C and assayed 7, 15 and 30 days after homogenization. In the latter, the stability of the thiochrome derivatives in the supernatant extract was assessed 1, 30, 60, 120 and 240 minutes after potassium ferricyanide oxidation. For the two conditions, the data obtained were expressed as nmol/g of tissue for each compound separately or by adding the three obtained values in a single representative total value (TDP + TMP + P = Total, nmol/g of tissue).
Experimental variation: Intraday reproducibility was calculated from 10 consecutive injections of hippocampus samples and interday reproducibility was obtained by comparing the means of 10 replicates over two consecutive days.
Hippocampus samples from male Wistar rat brains were used in the assays to validate the method. Mean elution times for TDP, TMP and B1 were 3.82, 4.28 and 11.67 min, respectively. Thus, in a total run time of less than 12 min the thiamine and its phosphorylated forms were eluted from the HPLC system.
The linearity of the detector response to standard solutions of TDP, TMP and B1 were determined by calibration curves and the obtained data are shown in Figures 2(a)-(c), respectively. The result of each concentration is the average of triplicate assays. Linear correlation between the peak areas and concentrations was demonstrated for the three parameters, with r-values ranging from 0.993 to 0.996. Both peak height and area are proportional to concentration.
The accuracy of the method was estimated by recovery assays.
The data show that after the derivatization process the brain extract samples have to be analyzed in less than 30 min. In the present study, all samples were
Concentration added (ng/mL) | Concentration found (ng/mL) | % Recovery | ||||||
---|---|---|---|---|---|---|---|---|
TDP | TMP | B1 | TDP | TMP | B1 | TDP | TMP | B1 |
0 | 0 | 0 | 167.25 | 17.63 | 1.03 | - | - | - |
100 | 25 | 20 | 271.8 | 40.38 | 21.17 | 104.55 | 90.98 | 100.67 |
250 | 75 | 40 | 417.76 | 87.52 | 38.18 | 100.2 | 93.18 | 92.86 |
400 | 125 | 60 | 579.19 | 135.03 | 59.92 | 102.99 | 93.92 | 98.14 |
550 | 175 | 80 | 721.9 | 188.39 | 82.34 | 100.85 | 97.57 | 101.63 |
700 | 250 | 100 | 867.14 | 279 | 97.34 | 99.99 | 104.55 | 96.3 |
injected into the chromatographic system within 1 min. Comparing to data obtained by other study, 30 min can be considered a lower stability [
The experimental variation of the analyses is shown in
Following the validation of the method using hippocampus sample, as described above, we also determined the levels of TDP, TMP and B1 in the thalamus, PFC and blood samples from male Wistar rats.
Relative Standard Deviations (%) | ||
---|---|---|
Intraday | Interday | |
TDP | 2.66 | 37.54 |
TMP | 4.50 | 25.39 |
B1 | 7.43 | 25.87 |
Sample | Retention time (min) | Concentration (nmol/g of tissue or nmol/L blood) | |
---|---|---|---|
Hippocampus | TDP | 3.89 ± 0.023 | 8.78 ± 0.66 |
TMP | 4.68 ± 0.81 | 7.38 ± 0.41 | |
B1 | 11.79 ± 0.070 | 0.55 ± 0.03 | |
Prefrontal Cortex | TDP | 3.83 ± 0.004 | 11.19 ± 0.53 |
TMP | 4.28 ± 0.005 | 6.02 ± 0.66 | |
B1 | 11.67 ± 0.021 | 0.62 ± 0.05 | |
Thalamus | TDP | 3.71 ± 0.010 | 9.63 ± 0.37 |
TMP | 4.17 ± 0.014 | 3.91 ± 0.50 | |
B1 | 11.25 ± 0.042 | 1.33 ± 0.91 | |
Blood samples | TDP | 3.78 ± 0.016 | 1127.11 ± 54.87 |
TMP | 4.28 ± 0.027 | 718.02 ± 89.97 | |
B1 | 11.51 ± 0.095 | 187.80 ± 72.89 |
blood. In addition, it also shows the retention time, in minutes, for each of the three assessed variables. The results are expressed as the average of two independent experiments. In each experiment the measurements were done in triplicate for hippocampus, PFC and blood. The measurements for thalamus were done in duplicate.
Extraction procedures of B1 and its phosphate esters and HPLC quantifications after derivatization under alkaline conditions to form fluorescent thiochrome derivatives were previously reported by other authors who used biological samples obtained from different kinds of tissues [
The TDP concentrations shown here for each of the three rat brain areas are within the same range, from 6 to 15 nmol/g of brain tissue, described by other authors [
The methods described here are also useful for measuring levels of B1 and its phosphate esters in rat blood. The average concentration found for the total thiamine (TDP + TMP + B1) in rat blood was 203.3 ± 8.7 μg/L (n = 8). This value is similar to that described by others authors: 283.1 ± 0.75 μg/L (n = 7) [
This study has limitations, including the stability of thiamine and its phosphate esters, and the amount of the sample.
Thiamine and its phosphate esters are temperature and light-sensitive and their stability is low. Therefore, samples should be storage in −80˚C for 1 week and the analysis should be carry out in the same day and until one minute after the derivatization.
Since the thalamic sample obtained is very small, it only allowed for a duplicate run rather than the standard triplicate.
Due to our difficulty in obtaining a standard for thiamine triphosphate (ThTP), we were unable to perform analysis for the compound.
In short, when compared to the diversity of methods in the literature, the present data are the first to represent the validation of an efficient HPLC method for determining the vitamin B1concentrations and those of its phosphorylated forms in hippocampus from rats. It is known that rats are an important and widely used experimental model to study the molecular mechanism of neurodegeneration and behavioral deficits induced by thiamine deficiency. In addition, the present work is the first to show validated results of the concentrations of these parameters in the hippocampus as well as their levels in the thalamus, PFC and blood samples. Moreover, it 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.
This work was supported by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and FAPEMG (Fundação de Amparo à Pesquisa de Minas Gerais). Patricia da Silva Oliveira received scholarship from CAPES. The authors wish to thank Aparecida Guerra de Jesus for technical assistance. She and Polliana Toledo Nunes received scholarship from FAPEMG.
Nunes, P.T., da Silva Oliveira, P., Ferraz, V. and Ribeiro, A.M. (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. https://doi.org/10.4236/jbbs.2017.72009