Fermented date residue extract mix containing gamma-aminobutyric acid augments the immune function of mouse splenocytes.

An extract of date (fruit of a palm tree) residue plus food-grade glutamate, acetic acid, and yeast extract (date residue extract mix, DREM) has been successfully fermented with using Lactobacillus brevis JCM 1059T to produce gamma-aminobutyric acid (GABA). Here, mouse splenocytes were found to be viable when supplemented with DREM and fermented DREM containing GABA (fDREM). The addition of DREM and fDREM resulted in the secretion of tumor necrosis factor (TNF)-α from the splenocytes, fDREM being more effective than DREM. The TNF-α secretion with DREM was elevated by exogenous addition of GABA and that with fDREM was in part mediated via A-type GABA receptors. Contrary to general understanding of the suppressive effects of GABA on various biological functions, our findings suggest that GABA-containing fDREM arguments the immune function as a food and pharmaceutical material.

NMR-Based Metabolomic Analyses to Identify the Effect of Harvesting Frequencies on the Leaf Metabolite Profile of a Moringa oleifera Cultivar Grown in an Open Hydroponic System.

Moringa oleifera Lam. is one of the world's most useful medicinal plants. Different parts of the M. oleifera tree contain a rich profile of important minerals, proteins, vitamins, and various important bioactive compounds. However, there are differences in the phytochemical composition of the medicinal plant's raw materials due to seasonal variation, cultivation practices, and post-harvest processing. The main objective of this study was therefore to determine the effect of harvesting frequencies on selected bioactive compounds of a M. oleifera cultivar (PKM1) grown in a hydroponic system under a shade net structure. Three harvesting frequency treatments were applied in the study, with the plants harvested at every 30 days (high frequency), 60 days (intermediate frequency), and 90 days (low frequency) respectively. 1H-NMR was used for data acquisition, and multivariate data analysis by means of principal component analysis (PCA), partial least square discriminatory analysis (PLS-DA), and orthogonal partial least square discriminatory analysis (OPLS-DA) were applied to determine the changes in the leaf metabolite profile, and also to identify the spectral features contributing to the separation of samples. Targeted metabolite analysis was used to match the NMR peaks of the compounds with the NMR chemical shifts of the contribution plot. The contribution plot showed that the increase in concentration of some compounds in aliphatic, sugar and aromatic regions contributed to the separation of the samples. The results revealed that intermediate and low harvesting frequencies resulted in a change in the leaf metabolite profile. Compounds such as chlorogenic acid, ferulic acid, vanillic acid, wogonin, esculetin, niazirin, and gamma-aminobutyric acid (GABA) showed an increase under intermediate and low harvesting frequencies. These results provide insight into the effect of harvesting frequencies on the metabolite profile and associated medicinal activity of M. oleifera.

Beneficial effect of GABA-rich fermented milk on insomnia involving regulation of gut microbiota.

Insomnia is a common health problem in modern societies. GABA, an inhibitory neurotransmitter, can promote relaxation and reduce anxiety. In this study, milk was fermented with Lactobacillus brevis DL1-11, a strain with high GABA-producing capacity. The potential beneficial effects of this fermented milk on anxiety and sleep quality were evaluated in animal experiments. Sixty mice were divided into control, non-GABA fermented milk (NGFM), low-dose GABA fermented milk (LGFM, 8.83 mg/kg.bw), medium-dose GABA fermented milk (MGFM, 16.67 mg/kg.bw), high-dose GABA fermented milk (HGFM, 33.33 mg/kg.bw) and diazepam groups. The results of open field test and elevated plus-maze test indicated decreases in anxiety behavior after oral HGFM administration. Moreover, mice in the HGFM group exhibited a significantly prolonged sleep time after an intraperitoneal injection of sodium pentobarbital and a shortened sleep latency after an intraperitoneal injection of sodium barbital. These results indicate a beneficial effect of HGFM on sleep. Additionally, significant increases in the relative abundances of Ruminococcus, Adlercreutzia and Allobaculum and the levels of some short-chain fatty acids (SCFAs), such as butyric acid, were observed in the HGFM group. The results suggest that GABA-fermented milk may improve sleep and the protective pathways may involve in regulation of gut microbiota and increase of SCFAs level.

Use of Streptococcus thermophilus for the in situ production of γ-aminobutyric acid-enriched fermented milk.

γ-Aminobutyric acid (GABA) is a potentially bioactive ingredient with health-promoting properties that is added to functional foods. Streptococcus thermophilus was selected to produce naturally GABA-enriched fermented milk. This strain can yield a GABA concentration of 2.8 g/L after a 48-h fermentation. In the presence of 1 g/L food-grade casein hydrolysate as a nitrogen source, S. thermophilus yielded GABA concentrations as high as 5.4 g/L or even 8.3 g/L when cocultured with Lactobacillus rhamnosus. In other words, both of these added conditions promoted GABA enrichment. The GABA dose achieved with fermented milk was comparable to the doses of commercially available GABA supplements. Additionally, the in situ use of S. thermophilus to produce GABA-enriched fermented milk was cost effective. The complete genomic sequence of S. thermophilus GABA has been published and will be highly useful to other researchers studying the regulation of genes related to GABA accumulation. In conclusion, the S. thermophilus GABA-producing strain reported herein represents a natural method for the production of fermented milk containing high GABA concentrations.

Characterization of bioactive film from pectin incorporated with gamma-aminobutyric acid.

Gamma-aminobutyric acid (GABA) is a nutritious bioactive compound that is also an antioxidant. The effect of GABA contents (5-15%) on pectin film properties were investigated and compared to those obtained by adding 10% glycerol, with pure pectin film as the control. The addition of 5% GABA led to similar film tensile strength and elastic modulus with lower film solubility and water vapor permeability compared with film plasticized with 10% glycerol. Additionally, 5% GABA had higher film flexibility compared with the control film. Moreover, GABA content slightly affected the color and moisture content of pectin films with a high antioxidant activity. Furthermore, thermal properties of film plasticized with GABA supported the changes in mechanical properties of pectin film. Therefore, this small amount of GABA provided a desirable film with functional properties similar to those obtained by using the higher amount of glycerol. Thus, GABA not only acted as an alternative plasticizer but also showed antioxidant activity as a bioactive film.

Glutamate Decarboxylase (GAD) Extracted from Germinated Rice: Enzymatic Properties and Its Application in Soymilk.

Glutamate decarboxylase (GAD) is an important enzyme in biological metabolisms acting on catalyzing the irreversible α-decarboxylation of L-glutamic acid to γ-aminobutyric acid (GABA) and CO2, which was focused in this study. Three rice varieties different in color were germinated at different times and used for crude GAD extraction. Crude GADs with an optimal germination time from germinated black (GBR), red (GRR), and white (GWR) rice were evaluated for enzymatic properties, including the effect of pHs, temperatures, and concentrations of both L-glutamic acid and pyridoxal 5'-phosphate (PLP). Crude GAD with optimum enzymatic properties was selected to be partially purified using ammonium sulfate (AMS) precipitation. The obtained GAD was supplemented to soymilk and determined for GABA content. All crude GADs from germinated rice at 10 germination days presented the highest enzyme activity. For enzymatic properties, crude GADs showed the highest activity at pH in a range of 5.6-6.0 at 60ºC. The Km values of crude GADs were in the range of 7.68-8.06 mM for L-glutamic acid and 0.15-0.20 µM for PLP and were the lowest in crude GAD from GBR. GAD from GBR presented the highest enzyme activity in the fraction with 50% saturation (v/v) after AMS precipitation and it was purified for 14.61 folds. The addition of this GAD (1.0%, v/v) resulted in the increasing of GABA content in soymilk to 53.79 mg/100 mL, accounted for 1.23 times compared with control.

Antioxidant Activity and Main Chemical Components of a Novel Fermented Tea.

In the present study, we aimed to develop a novel fermented tea (NFT) product and to evaluate their in vitro antioxidant potential and chemical composition. We found that NFT contained a high level of total phenolic compounds (102.98 mg gallic acid equivalents/g extract) and exhibited diverse antioxidant activities, such as scavenging of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and hydroxyl radicals, as well as reducing power. The total catechins in NFT were comparable to those of Lipton black tea (LBT), but lower than those of Boseong green tea (BGT) or Tieguanyin oolong tea (TOT). Among all catechins tested, epigallocatechin (EGC) and epigallocatechin-3-O-gallate (EGCG) were the predominant compounds in NFT. In particular, the contents of total theaflavins (TFs), theaflavin (TF), theaflavin-3-gallate (TF3G), and theaflavin-3'-gallate (TF3'G) in NFT were significantly higher than that of BGT, TOT, or LBT. NFT had the highest level of total essential amino acid and γ-aminobutyric acid (GABA) compared with BGT, TOT and LBT. Furthermore, the sensory evaluation results showed that NFT had satisfactory color, aroma, taste, and overall acceptability scores. Our results highlight the potential usefulness of this novel fermented tea as a nutraceutical food/ingredient with special functional activities.

A Randomized Clinical Efficacy Trial of Red Yeast Rice (Monascus pilosus) Against Hyperlipidemia.

Red yeast rice (RYR) has been used as an alternative treatment for hyperlipidemia. According to the previous studies, other compounds, besides monacolin K in RYR, may also reduce the serum lipid level. This study aims at examining the efficacy of monacolin K-rich and Gamma-Aminobutyric Acid (GABA)-rich RYR (Monascus pilosus) with regards to treating hyperlipidemia in a randomized control, double-blind clinical trial. In the research, we assigned 50 eligible subjects to monacolin K-rich RYR, GABA-rich RYR and placebo groups ( n=16 , 17, 17, respectively). The concentrations of TC, LDL-C, HDL, TG and blood biochemical data were evaluated at different phases: before applying (visit 1), after 1-month (visit 2), 2-month (visit 3), 3-month (visit 4) of providing the intervention and 1-month after ending the test food (visit 5) among three groups. During the 3-month intervention, the serum TC and LDL-C levels decreased significantly in the monacolin K group compared to the baseline and the other two groups. The Serum TG level declined steadily but was not statistically significant. Meanwhile, no marked differences in the serum HDL level were revealed among the three groups. Most safety assessment data had minor variation except two subjects (in monacolin K and GABA group separately) reported elevated liver enzymes. Monacolin K-rich RYR can reduce cholesterol as expected, while the GABA-rich RYR performed non-significant reduction on serum triglyceride. The research results demonstrate that using different concentrations and ratios between monacolin K and GABA could be beneficial for antihyperlipidemia.

Effect of esters based on terpenoids and GABA on fluidity of phospholipid membranes.

The influence of esters based on gamma-aminobutyric acid (GABA) and mono-/bicyclic terpenoids on membrane structure was investigated. The mechanism of action for terpenoid esters on phospholipids of artificial membranes and lipids isolated from the rat stratum corneum was studied by fluorescence and FT-IR spectroscopy. We report here, that inclusion of monocyclic terpenoid esters in phospholipid liposomes leads to growth of excimer to monomer ratio (IE/IM) indicating a decrease of membrane microviscosity. Another mechanism of influence on biomembranes was proposed for ester of bicyclic borneol - in this case a high ratio of vibronic peak intensities (I1/I3) was revealed. The addition of terpenoid esters appears in the FT-IR spectra as intensity reduction of absorption bands associated with C = O, P = O and P-O-С groups of lecithin phospholipids. Similar results were obtained after esters addition to lipids isolated from stratum corneum indicating a decrease of hydrogen bonds number between polar groups of lipids. Thus, the influence of terpenoid esters on molecular organization of the lipid matrix substantiates the feasibility of their use after transdermal delivery in vivo.

Amino acid substitutions in the human homomeric ß3 GABAA receptor that enable activation by GABA.

GABAA receptors (GABAARs) are pentameric ligand-gated ion channels that mediate synaptic inhibition throughout the central nervous system. The α1ß2γ2 receptor is the major subtype in the brain; GABA binds at the ß2(+)α1(-) interface. The structure of the homomeric ß3 GABAAR, which is not activated by GABA, has been solved. Recently, four additional heteromeric structures were reported, highlighting key residues required for agonist binding. Here, we used a protein engineering method, taking advantage of knowledge of the key binding residues, to create a ß3(+)α1(-) heteromeric interface in the homomeric human ß3 GABAAR that enables GABA-mediated activation. Substitutions were made in the complementary side of the orthosteric binding site in loop D (Y87F and Q89R), loop E (G152T), and loop G (N66D and A70T). The Q89R and G152T combination enabled low-potency activation by GABA and potentiation by propofol but impaired direct activation by higher propofol concentrations. At higher concentrations, GABA inhibited gating of ß3 GABAAR variants containing Y87F, Q89R, and G152T. Reversion of Phe87 to tyrosine abolished GABA's inhibitory effect and partially recovered direct activation by propofol. This tyrosine is conserved in homomeric GABAARs and in the Erwinia chrysanthemi ligand-gated ion channel and may be essential for the absence of an inhibitory effect of GABA on homomeric channels. This work demonstrated that only two substitutions, Q89R and G152T, in ß3 GABAAR are sufficient to reconstitute GABA-mediated activation and suggests that Tyr87 prevents inhibitory effects of GABA.

Gastroretentive raft liquid delivery system as a new approach to release extension for carrier-mediated drug.

Gabapentin (GBP), an antiepileptic and anti-neuropathic agent, suffers from short half-life (5-7 h), has narrow absorption window, and is absorbed via carrier-mediated mechanism resulting in frequent dosing, poor compliance, and poor bioavailability (<60%). Moreover, GBP is a freely water-soluble drug, thus it is considered a challenging candidate to be formulated as extended release dosage form. In this study, raft forming systems were investigated as a potential drug delivery system for prolonging gastric residence time of GBP. A 23 full factorial design was adopted to study the effect of formulation variables (% gellan gum, % GMO, and % LM-pectin 101), on the percent of GBP released at different time intervals (1, 5, and 8 h) as well as the gel strength, and thus was achieved an optimized formula with zero-order release profile suitable for once-daily administration. In vivo assessment was performed in rats to evaluate gastric residence of the gel formed. In addition, the oral bioavailability of GBP relative to commercially available Neurontin® immediate release oral solution was also investigated. Significant increase was observed for Cmax, AUC(0-t), and AUC(0-∞). The increase in relative bioavailability of GBP from the optimized formula was 1.7 folds.

Rapid analysis of glutamate, glutamine and GABA in mice frontal cortex microdialysis samples using HPLC coupled to electrospray tandem mass spectrometry.

In vivo measurement of multiple neurotransmitters is highly interesting but remains challenging in the field of neuroscience. GABA and l-glutamic acid are the major inhibitory and excitatory neurotransmitters, respectively, in the central nervous system, and their changes are related to a variety of diseases such as anxiety and major depressive disorder. This study described a simple method allowing the simultaneous LC-MS/MS quantification of l-glutamic acid, glutamine and GABA. Analytes were acquired from samples of the prefrontal cortex by microdialysis technique in freely moving mice. The chromatographic separation was performed by hydrophilic interaction liquid chromatography (HILIC) with a core-shell ammonium-sulfonic acid modified silica column using a gradient elution with mobile phases consisting of a 25 mM pH 3.5 ammonium formate buffer and acetonitrile. The detection of l-glutamic acid, glutamine and GABA, as well as the internal standards [d6]-GABA and [d5]-glutamate was performed on a triple quadrupole mass spectrometer in positive electrospray ionization and multiple reaction monitoring mode. The limit of quantification was 0.63 ng/ml for GABA, 1.25 ng/ml for l-glutamic acid and 3.15 ng/ml for glutamine, and the intra-day and inter-day accuracy and precision have been assessed for the three analytes. Therefore, the physiological relevance of the method was successfully applied for the determination of basal extracellular levels and potassium-evoked release of these neuroactive substances in the prefrontal cortex in adult awake C57BL/6 mice.

Accumulating pathways of γ-aminobutyric acid during anaerobic and aerobic sequential incubations in fresh tea leaves.

The roles of the γ-aminobutyric acid (GABA) shunt and polyamine (PA) degradation pathway on GABA accumulation were investigated in fresh tea leaves under anaerobic and aerobic sequential incubations. The GABA accumulation was mainly completed in the first anaerobic incubation through the interaction of the GABA shunt with the PA degradation pathway. When treated with aminoguanidine, the diamine oxidase and polyamine oxidase activities were almost completely inhibited, and the GABA contents decreased by 40.7%, 46.4%, 41.0% and 37.5% in the tea leaves of the four cultivars 'Fuyun No. 6', 'Fudingdabai', 'Longjing No. 43' and 'Pingyangtezao', respectively. The glutamate decarboxylase activity decreased significantly, and the accumulation of GABA may increase or decrease because of varietal differences, which occurred mainly relying on the PA degradation pathway during the second anaerobic incubation. Thus, approximately 37%-47% of the GABA formed in fresh tea leaves under hypoxia was supplied by the PA pathway.

Improvement of Chia Seeds with Antioxidant Activity, GABA, Essential Amino Acids, and Dietary Fiber by Controlled Germination Bioprocess.

Chia (Salvia hispanica L.) plant is native from southern Mexico and northern Guatemala. Their seeds are a rich source of bioactive compounds which protect consumers against chronic diseases. Germination improves functionality of the seeds due to the increase in the bioactive compounds and associated antioxidant activity. The purpose of this study was to obtain functional flour from germinated chia seeds under optimized conditions with increased antioxidant activity, phenolic compounds, GABA, essential amino acids, and dietary fiber with respect to un-germinated chia seeds. The effect of germination temperature and time (GT = 20-35 °C, Gt = 10-300 h) on protein, lipid, and total phenolic contents (PC, LC, TPC, respectively), and antioxidant activity (AoxA) was analyzed by response surface methodology as optimization tool. Chia seeds were germinated inside plastic trays with absorbent paper moisturized with 50 mL of 100 ppm sodium hypochlorite dissolution. The sprouts were dried (50 °C/8 h) and ground to obtain germinated chia flours (GCF). The prediction models developed for PC, LC, TPC, and AoxA showed high coefficients of determination, demonstrating their adequacy to explain the variations in experimental data. The highest values of PC, LC, TPC, and AoxA were obtained at two different optimal conditions (GT = 21 °C/Gt = 157 h; GT = 33 °C/Gt = 126 h). Optimized germinated chia flours (OGCF) had higher PC, TPC, AoxA, GABA, essential amino acids, calculated protein efficiency ratio (C-PER), and total dietary fiber (TDF) than un-germinated chia seed flour. The OGCF could be utilized as a natural source of proteins, dietary fiber, GABA, and antioxidants in the development of new functional beverages and foods.

Quality components and antidepressant-like effects of GABA green tea.

Gamma (γ)-aminobutyric acid (GABA) green tea, with high GABA content, is a kind of special green tea. The goals of this study are to analyze the changes in quality components of green tea during anaerobic treatment, and to investigate whether or not the extract of GABA present in green tea can prevent depression or improve the depressive state of animals. Results showed that GABA content in green tea had increased significantly after anaerobic treatment. The contents of tea polysaccharides, total free amino acids, and water extracts were also increased whereas tea polyphenols were reduced. More importantly, the extract of GABA green tea could alleviate mouse depression and stress from desperate environments through the forced swim test (FST), tail suspension test (TST), mRNA and protein expression levels of GABAA receptors. Therefore, these results indicate that GABA green tea may have a health effect on prevention and alleviation of depression, and it works on the GABAergic neurotransmission of mouse cerebral cortex via up-regulating expression of the GABAA receptor α1 subunit, thus ameliorating depression.

Activation volume vs BOLD signal change as measures of fMRI activation - Its impact on GABA - fMRI activation correlation.

PURPOSE: To explore the relative robustness of functional MRI (fMRI) activation volume and blood oxygen level-dependent (BOLD) signal change as fMRI metric, and to study the effect of relative robustness on the correlation between fMRI activation and cortical gamma amino butyric acid (GABA) in healthy controls and patients with multiple sclerosis (MS). METHODS: fMRI data were acquired from healthy controls and patients with MS, with the subjects peforming self paced bilateral finger tapping in block design. GABA spectroscopy was performed with voxel placed on the area of maximum activation during fMRI. Activation volume and BOLD signal changes at primary motor cortex (M1), as well as GABA concentration were calculated for each patient. RESULTS: Activation volume correlated with BOLD signal change in healthy controls, but no such correlation was observed in patients with MS. This difference was likely the result of higher intersubject noise variance in the patient population. GABA concentration correlated with M1 activation volume in patients but not in controls, and did not correlate with any fMRI metric in patients or controls. CONCLUSION: Our data suggest that activation volume is a more robust measure than BOLD signal change in a group with high intersubject noise variance as in patients with MS. Additionally, this study demonstrated difference in correlation behavior between GABA concentration and the 2 fMRI metrics in patients with MS, suggesting that GABA - activation volume correlation is more appropriate measure in the patient group.

Recent advances in γ-aminobutyric acid (GABA) properties in pulses: an overview.

Beans, peas, and lentils are all types of pulses that are extensively used as foods around the world due to their beneficial effects on human health including their low glycaemic index, cholesterol lowering effects, ability to decrease the risk of heart diseases and their protective effects against some cancers. These health benefits are a result of their components such as bioactive proteins, dietary fibre, slowly digested starches, minerals and vitamins, and bioactive compounds. Among these bioactive compounds, γ-aminobutyric acid (GABA), a non-proteinogenic amino acid with numerous reported health benefits (e.g. anti-diabetic and hypotensive effects, depression and anxiety reduction) is of particular interest. GABA is primarily synthesised in plant tissues by the decarboxylation of l-glutamic acid in the presence of glutamate decarboxylase (GAD). It is widely reported that during various processes including enzymatic treatment, gaseous treatment (e.g. with carbon dioxide), and fermentation (with lactic acid bacteria), GABA content increases in the plant matrix. The objective of this review paper is to highlight the current state of knowledge on the occurrence of GABA in pulses with special focus on mechanisms by which GABA levels are increased and the analytical extraction and estimation methods for this bioactive phytochemical. © 2017 Society of Chemical Industry.

2-Pyrrolidone synthesis from γ-aminobutyric acid produced by Lactobacillus brevis under solid-state fermentation utilizing toxic deoiled cottonseed cake.

There is an increasing demand of γ-aminobutyric acid (GABA) as drug and food additive, as well as feedstock to produce 2-pyrrolidone, a precursor for the synthesis of nylon 4. 2-Pyrrolidone is a petrochemical and depleting reserve which raises concern for its bio-based production. The study herein describes bio-based economical GABA production from Lactobacillus brevis by solid-state fermentation (SSF) using toxic deoiled cottonseed cake (CSC) as substrate. In general, the use of cottonseed cake remains restricted due to the presence of toxic gossypols. Thus, simultaneous detoxification observed during fermentation also widens the scope of utilization of this residual seedcake for feed use vis-a-vis production of other value added chemicals. The SSF conditions were optimized for maximum GABA production, viz., 19.7 mg/g, CSC of GABA was obtained at 6th day of fermentation with 70 % degradation of gossypols simultaneously. The potential of this bio-based GABA as a platform chemical is demonstrated in the synthesis of 2-pyrrolidone. Thus, a simple and cost-effective strategy for utilizing toxic biomass has been developed as an alternate to chemical synthetic route.

Cholesterol-Lowering Effect of Octaarginine-Appended ß-Cyclodextrin in Npc1-Trap-CHO Cells.

Niemann-Pick disease type C (NPC) is an autosomal recessive lysosomal storage disorder, which is an inherited disease characterized by the accumulation of unesterified cholesterol in endolysosomes. Recently, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CyD) has been used for the treatment of NPC, and ameliorated a hepatosplenomegaly in the patients. However, to obtain the treatment efficacy, a high dose of HP-ß-CyD was necessary. Therefore, the decrease in dose by using active intracellular delivery system of ß-CyD to NPC cells is expected. In this study, to efficiently deliver ß-CyD to NPC-like cells, we newly synthesized octaarginine (R8)-appended ß-CyD with a spacer of γ-aminobutyric acid (R8-ß-CyD) and evaluated its cytotoxicity, intracellular distribution, endocytosis pathway and cholesterol-lowering effect in Npc1-trap-Chinese hamster ovary (CHO) cells, cholesterol-accumulated cells through the impairment of NPC1 function. R8-ß-CyD did not show cytotoxicity in the cells. In addition, Alexa568-labeled R8-ß-CyD was actively internalized into Npc1-trap-CHO cells, possibly through micropinocytosis. Notably, R8-ß-CyD significantly decreased intracellular cholesterol content compared with HP-ß-CyD. These results suggest that R8-ß-CyD may be a promising therapeutic agent for ameliorating cholesterol accumulation in NPC.

Synthesis and inhibition potency of novel ureido benzenesulfonamides incorporating GABA as tumor-associated carbonic anhydrase IX and XII inhibitors.

New ureido benzenesulfonamides incorporating a GABA moiety as a linker between the ureido and the sulfonamide functionalities were synthesized and their inhibition potency determined against both the predominant cytosolic (hCA I and II) and the transmembrane tumor-associated (hCA IX and XII) isoforms of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The majority of these compounds were medium potency inhibitors of the cytosolic isoform hCA I and effective hCA II inhibitors, whereas they showed strong inhibition of the two transmembrane tumor-associated isoforms hCA IX and XII, with KIs in nanomolar range. Only one derivative had a good selectivity for inhibition of the tumor-associated hCA IX target isoform over the cytosolic and physiologically dominant off-target hCA I and II, being thus a potential tool to develop new anticancer agents.