My major project #2

The Comparative Determination of Gamma-Aminobutyric Acid (GABA) between Ultraviolet and Fluorescence Detection by High-Performance Liquid Chromatography (HPLC)

         For the ultraviolet (UV) detection, Khuhawar and Rajper (2003) conducted a study to determine an amount of GABA derivatized with 2-hydroxynapthaldehyde (HN) in a sample from central nervous system (CNS). The samples were collected from patients at Liaquat Medical University Hospital, Jamshoro. This study had three meningitis subjects, one patient suffered from tuberculous meningitis and the others suffered from septic meningitis. The sample was centrifuged and mixed with HN and borax buffer. After that, the sample was heated and cool down. The solution was injected on column C₁₈ and eluted with methanol: water with a flow-rate 1 ml/min. The detection was 330 nm. The results showed that a linear calibration curve of GABA was in the range of 1.12–28.0 mg/ml with a coefficient of correlation = 0.998. Moreover, the derivative of GABA and HN is stable more than 12 hours. The researchers suggested that this might indicate that the high amount of GABA in CSF by the meningitis patients.

For the fluorescent detection, de Freitas Silva et al. (2009) conducted a study to determine an amount of GABA derivatized with o-phthaladehyde (OPA) in a sample from prefrontal cortex, thalamus and hippocampus samples. The samples were collected from male Wistar rats. The total number of samples in this study was twenty. Before the analysis of GABA, the sample was centrifuged. Then, the sample was mixed with OPA, borate buffer and 3-mercaptopropionic acid. The solution was vortexed and incubated 1-min. The solution was injected on column C₁₈, and eluted with sodium acetate, tetrahydrofuran and methanol, adjusted to pH 4.0, with a flow-rate 1 ml/min. The excitation and emission wavelengths were 337 nm and 454 nm respectively. The results showed that a linear calibration curve of GABA was in the range from 0.1 to 0.75 μg/ml with a correlation coefficient < 0.990. In addition, the derivative of GABA and OPA is stable less than 30 minutes. The researchers noted that this method might perform successfully developed an isocratic HPLC method coupled to fluorescent detection for the determination of GABA and OPA derivatives.

These studies provide scientific evidence of the quantitative analysis of GABA in brain sample. However, there are some limitations.

For the UV detection

1)         The small group of sample could be distorted the real result in amount of GABA in human CSF so that the researchers should be collecting the data from the more people according to the FDA guideline etc. Moreover, the participants should be a normal people having not a disease because of preventing the result from bias or higher than normal condition of GABA in CSF (Rizzo et al. 1996)

2)         The sensitivity of analysis of a GABA derivative by UV detection was lower than fluorescent detection (de Freitas Silva et al. 2009) so when the study was wanted to detect a GABA in a low level, it should select the method of the fluorescent detection.

           

            For the fluorescent detection

1)         The stability of a GABA and OPA derivative was too low. It could be degraded in thirty minutes. Thus, the good planning lab would help to overcome this problem.

2)         This mobile phase used a buffer system. After the detection was finish, the operator had to always clean the column with water such as ultra-pure water or HPLC grade water preventing from salting in column and HPLC.

The strength of these two studies is that the analysis reagents of the two techniques can be easily to purchase and not expensively. Furthermore, HPLC technique used is widely accepted for research in the same area. Another advantage of these two studies is that the results were clearly described with illustrated figures and conclusions were made according to the results in their studies.   

References

Khuhawar, M. Y., & Rajper, A. D. (2003). Liquid chromatographic determination of γ-aminobutyric acid in cerebrospinal fluid using 2-hydroxynaphthaldehyde as derivatizing reagent. Journal of Chromatography B, 788(2), 413-418.

de Freitas Silva, D. M., Ferraz, V. P., & Ribeiro, A. M. (2009). Improved high-performance liquid

            chromatographic method for GABA and glutamate determination in regions of the rodent

brain. J Neurosci Methods, 177(2), 289-293.

Rizzo, V., Anesi, A., Montalbetti, L., Bellantoni, G., Trotti, R., & Melzi d'Eril, G. V. (1996). Reference values of neuroactive amino acids in the cerebrospinal fluid by high-performance liquid chromatography with electrochemical and fluorescence detection. Journal of Chromatography A, 729(1–2), 181-188.

My major project

The Comparative Determination of Gamma-Aminobutyric Acid (GABA) between Ultraviolet and Fluorescence Detection by High-Performance Liquid Chromatography (HPLC)

         For the ultraviolet (UV) detection, Khuhawar and Rajper (2003) conducted a study to determine an amount of GABA derivatized with 2-hydroxynapthaldehyde (HN) in a sample from central nervous system (CNS). The samples were collected from patients at Liaquat Medical University Hospital, Jamshoro. The total number of samples in this study was three, one patient suffering from tuberculous meningitis and two patients suffering from septic meningitis. Before the analysis of GABA, the sample was centrifuged. Then, the sample was mixed with HN and borax buffer pH 8.00. After that, the sample solution was heated on a water bath at 80 C° for 10 min and was allowed to cool at room temperature. The final volume was adjusted to 5 ml with methanol. The solution (5 μl) was injected on short column C₁₈, 5 mm and eluted with methanol: water (62:38 v/v) with a flow-rate 1 ml/min. The detection UV was 330 nm. The results showed that a linear calibration curve was obtained for GABA in the range of 1.12–28.0 mg/ml with a coefficient of correlation (r) = 0.998 and detection limit of 2.8 ng/injection (5 μl). In cerebral spinal fluid (CSF) samples, the result was shown 19.0 to 22.4 mg/ml with coefficient of variation 2.4% . Moreover, the derivative of GABA and HN is stable more than 12 hours. The researchers suggested that this might indicate that the high amount of GABA in CSF by the meningitis patients.

For the fluorescent detection, de Freitas Silva et al. (2009) conducted a study to determine an amount of GABA derivatized with o-phthaladehyde (OPA) in a sample from prefrontal cortex, thalamus hippocampus samples. The samples were collected from male Wistar rats. The total number of samples in this study was twenty. Before the analysis of GABA, the sample was centrifuged. Then, the sample was mixed with OPA, borate buffer pH 9.9 and 3-mercaptopropionic acid (MPA). The resulting solution was vortexed and analyzed after 1-min at room temperature. The solution was injected 10 μl on short column C₁₈, 3 mm and eluted with 0.05 M sodium acetate, tetrahydrofuran and methanol (50:1:49, v/v) adjusted to pH 4.0 with a flow-rate 1 ml/min and the temperature = 25 ± 2◦C. The fluorescent detector was set at an excitation wavelength of 337 nm and an emission wavelength of 454 nm. The results showed that a linear calibration curve was obtained for GABA in the range from 0.1 to 0.75 μg/ml by hippocampus samples of male Wistar rats with a correlation coefficient of not less than 0.990. In addition, the derivative of GABA and OPA is stable less than 30 minutes. The researchers noted that this method might perform successfully developed an isocratic HPLC method coupled to fluorescent detection for the determination of GABA and OPA derivatives.

These studies provide scientific evidence of the quantitative analysis of GABA in brain sample. However, there are some limitations.

For the UV detection

1)         The small group of sample could be distorted the real result in amount of GABA in human CSF so that the researchers should be collecting the data from the more people according to the FDA guideline etc. Moreover, the participants should be a normal people having not a disease because of preventing the result from bias or higher than normal condition of GABA in CSF (Rizzo et al. 1996)

2)         The sensitivity of analysis of a GABA derivative by UV detection was lower than fluorescent detection (de Freitas Silva et al. 2009) so when the study was wanted to detect a GABA in a low level, it should select the method of the fluorescent detection.

           

            For the fluorescent detection

1)         The stability of a GABA and OPA derivative was too low. It could be degraded in thirty minutes. Thus, the good planning lab would help to overcome this problem.

2)         This mobile phase used a buffer system. After the detection was finish, the operator had to always clean the column with water such as ultrapure water or HPLC grade water preventing from salting in column and HPLC.

The strength of these two studies is that the analysis reagents of the two techniques can be easily to purchase and not expensively. Furthermore, HPLC technique used is widely accepted for research in the same area. Another advantage of these two studies is that the results were clearly described with illustrated figures and conclusions were made according to the results in their studies.   

References

Khuhawar, M. Y., & Rajper, A. D. (2003). Liquid chromatographic determination of γ-aminobutyric acid in cerebrospinal fluid using 2-hydroxynaphthaldehyde as derivatizing reagent. Journal of Chromatography B, 788(2), 413-418.

de Freitas Silva, D. M., Ferraz, V. P., & Ribeiro, A. M. (2009). Improved high-performance liquid

            chromatographic method for GABA and glutamate determination in regions of the rodent

brain. J Neurosci Methods, 177(2), 289-293.

Rizzo, V., Anesi, A., Montalbetti, L., Bellantoni, G., Trotti, R., & Melzi d'Eril, G. V. (1996). Reference values of neuroactive amino acids in the cerebrospinal fluid by high-performance liquid chromatography with electrochemical and fluorescence detection. Journal of Chromatography A, 729(1–2), 181-188.

Minor project

Minor project

           My research question is whether performing analysis techniques of gamma-aminobutyric acid (GABA) between photodiode array and fluorescence detector by high-performance liquid chromatography (HPLC). GABA is a small molecule and has polarity so that a detection of GABA is problematic. Moreover, some research studies, such as food and drugs, want to detect amount of GABA in low and/or sensitive levels, for example in levels of milligram, microgram and nanogram. HPLC coupled with detectors, photodiode array detector and/or fluorescence detector, can be detected a chemical compound in a solution. In addition, amino acids, like GABA, have a small molecule and do not have fluorescent or ultraviolet absorbance itself. Therefore, one of the solutions to resolve an obstruction is the pre-column derivatization. The pre-column derivatization will use an agent providing strong ultraviolet (UV) or fluorescent absorbance combined with the sample having amino acids before used in HPLC. This issue is claimed that some researchers use an UV derivatizing agent and others use a fluorescent derivatizing agent.

            Researchers using an UV derivatizing agent, 2-hydroxynapthaldehyde (HN), are Khuhawar and Rajper and others use a fluorescent derivatizing agent, o-phthaladehyde (OPA), are de Freitas Silva et al. The former reveal that they are the first group to study about GABA derivatized with HN and the latter notice that they have developed the method to quantify the amount of GABA with OPA and 3-mercaptopropionic acid (MPA)   

            Khuhawar and Rajper (2003) argue that they find an amount of GABA derivatized with HN in a sample from central nervous system (CNS) and have a standard curve ranged from 1.12 to 28 μg/ml by a short C₁₈ column. The derivative is stable more than 12 hours.  

            De Freitas Silva et al. (2009) argue that they have modified a method to detect GABA derivatized with OPA and used MPA as a stabilizer in samples from the rodent brain. They use a short C₁₈ column and run with an isocratic system. A standard curve is ranged from 0.1 to 0.75 μg/ml and the derivative is stable approximately 30 minutes.

           

            Debate centers on the basic issue if the comparison between photodiode array detector and fluorescence detector shows an effective analysis of GABA because it has not been studied in one study yet on this point.

            My work will be closer to Khuhawar and Rajper and de Freitas Silva et al. for the season that I will be conducting the two experiments to show whether each of techniques can be interpreted results in terms of sensitivity, reliability and reproducibility.

Hopefully my contribution will be the first study to give an idea of the result between two techniques in the one research. Furthermore, this study could be represented as information to guide other researchers selecting the suitable and sensitive experiment to doing their research in the future.

             

Reference List

Khuhawar, M. Y., & Rajper, A. D. (2003). Liquid chromatographic determination of γ-aminobutyric acid in cerebrospinal fluid using 2-hydroxynaphthaldehyde as derivatizing reagent. Journal of Chromatography B, 788(2), 413-418.

de Freitas Silva, D. M., Ferraz, V. P., & Ribeiro, A. M. (2009). Improved high-performance liquid chromatographic method for GABA and glutamate determination in regions of the rodent brain.  Journal of Neuroscience Methods, 177(2), 289-293.

Assignment 2 : Writing an introduction

The Comparative Determination of Gamma-Aminobutyric Acid (GABA) between Ultraviolet and Fluorescence Detection by High-Performance Liquid Chromatography (HPLC)

                                              Kritchapol  Panrod

Stage 1 : Gamma-aminobutyric acid (GABA) is a non-protein amino acid. GABA is a one of the neurotransmission agents which can inhibit over stimulating of the brain and reduce hypertension from the stress (Zhang et al., 2006). Moreover, GABA generally finds in human such as the cortex, hippocampus, hypothalamus, central nervous system (CNS), plants and animals. 

Thus, an analysis of GABA in the quantitation usually use the high-performance liquid chromatography (HPLC) to measure the amount of GABA in unknown sample. HPLC is the technique that can detect an agent in solution by it’s detector (such as ultraviolet light or fluorescence light) which analysts the agent when it passes through the HPLC column and interprets the results in terms of graphs and area under the peak. However, a detection of GABA has a problem when using HPLC because it has a small molecule and does not has fluorescent or ultraviolet absorbance (Shah et al., 2002). Therefore, one of the technique to resolve that problem is the pre-column derivatisation. The pre-column derivatisation will use an agent providing strong ultraviolet (UV) or fluorescent absorbance mixing with the sample before used in HPLC  (Shah et al., 1999) 

             Stage 2 : In UV detection, GABA is analysed by derivatizing agents including phenylisothiocyanate (PITC) (Jarry et al., 1992), 2-hydroxynapthaldehyde (HN) (Khuhawar and Rajper, 2003; Bor et al., 2009; Jannoey et al., 2010 and Hayat et al., 2014), 4-dimethyl-aminoazobenzene-4-sulfonyl chloride (DABSYL-Cl) (Varanyanond et al., 2005), naproxen acyl chloride (NAC) (Hsieh et al., 2006) and 1-fluoro-2,4-dinitrobenzene (FDNB) (Ishikawa et al., 2009). 

            In fluorescence detection, GABA is commonly analysed by a derivatizing agent which is o-phthaladehyde (OPA). OPA reacts with primary amines in the presence of thiol and generates derivatives which are fluorescent (Devall et al., 2007, Zhang et al., 2005, Fekkes et al., 1995 and Sheng et al., 2005). Unfortunately, the deravatives of OPA are not stable especially in the acid solution so they have developed to improve the stability and more sensitivity of derivatives by added naphthalene-2,3-dicarboxaldehyde (NDA) (Clarke et al., 2007), 2-mercaptoethanol (Iwaki and Kitada, 2007) and 3-mercaptopropionic acid (MPA) (Freitas Silva et al., 2009) 

   

            Stage 3 : Many of papers have a single analysis GABA but none of them has not comparative two techniques in one paper.

            Stage 4 & 5 : Thus, this paper will compare between UV detection and fluorescence detection. The sample of UV derivatizing agent is HN, an inexpensive agent and the fluorescence derivatizing agent is OPA because it normally uses in a detection. Therefore, this paper will point out that each of the technique can present the result in quantitation of GABA, retention time, limit of detection and limit of quantitation. 

CitatioN

Wound Healing Activity of Gamma-Aminobutyric Acid (GABA) in Rats

 Abstract :

Gamma-aminobutyric acid (GABA) is a non-protein amino acid. It is well known for its role as an inhibitory neurotransmitter of developing and operating nervous systems in brains. In this study, a novel function of GABA in the healing process of cutaneous wounds was presented regarding anti-inflammation and fibroblast cell proliferation. The cell proliferation activity of GABA was verified through an MTT assay using murine fibroblast NIH3T3 cells. It was observed that GABA significantly inhibited the mRNA expression of iNOS, IL-1beta, and TNF-alpha, in LPS-stimulated RAW 264.7 cells. To evaluate in vivo activity of GABA in wound healing, excisional open wounds were made on the dorsal sides of Sprague-Dawley rats under anesthesia, and the healing of the wounds was apparently assessed. The molecular aspects of the healing process were also investigated by hematoxylineosin staining of the healed skin, displaying the degrees of reepithelialization and linear alignment of the granulation tissue, and immunostaining and RT-PCR analyses of fibroblast growth factor and platelet-derived growth factor, implying extracellular matrix synthesis and remodeling of the skin. The GABA treatment was effective to accelerate the healing process by suppressing inflammation and stimulating reepithelialization, compared with the epidermal growth factor treatment. The healing effect of GABA was remarkable at the early stage of wound healing, which resulted in significant reduction of the whole healing period.

Reference :

Dongoh, Han., Kim, Hee-Young., Lee, Hye-Jung., Shim, Insop. and Hahm, Dae-Hyun. (2007). Wound Healing Activity of Gamma-Aminobutyric Acid (GABA) in Rats. J. Microbiol. Biotechnol. 2007, 17(10), 1661-1669.

Results/findings :

1. GABA stimulated the most of the cell proliferation activity in NIH3T3 cells from Swiss albino mouse embryo tissue by MTT assay.

            2. In NIH3T3 cells,  GABA inhibited the expression of primary inflammatory mediators for example TNF-α, IL-1β and the most was an iNOS using an LPS(lipopolysaccharide)-induced in vitro cell line.

             3. GABA reduced time healing on wound after 5^th day in adult male Sprague-Dawley(SD) and the result was similar to treating with EGF (epidermal growth factor).

             4. Not only treating with GABA found that wound skin had more fibroblast and collagenous fibers in dermis by histological analysis but also protein expressions of %FGF(fibroblast growth factor) and %PDGF(platelet-derived growth factor) were high in the immature fibroblasts of dermis and epithelial cells.

Citations: 

Dongoh et al. (2007) noted that GABA was trended to be a drug for promoting the wound healing effect especially in open wounds (1661-1669).

Dongoh et al. (2007) reported that GABA was many activities such as increasing cell proliferation activity in NIH3T3 cells,  decreasing time healing on wound in adult male rats and enriching fibroblast and collagen in dermis on wounds (1661-1669).