Attention-deficit hyperactivity disorder (ADHD) as a pyridoxine-dependent condition: urinary diagnostic biomarkers.

The data obtained in children with different forms of epilepsy allowed us to consider epilepsy as an inborn error of pyridoxine (vitamin B6) metabolism (Dolina et al., 2012). Mutual interconnections between ADHD and epilepsy indicate that such an approach is reasonable for ADHD. To check such an assumption we analyzed in ADHD patients the same parameters of pyridoxal phosphate (PLP)-dependent tryptophan (TRP) degradation, which were analyzed in epileptic children. The level of TRP and concentrations of compounds formed or metabolized by TRP degradation, the ratios between some of them, and the level of 4-pyridoxic acid were HPLC detected in ADHD children and healthy controls. The data obtained, including low values of 4PA/TRP, IND/TRP and IND/KYN ratios, have evidenced dramatically impaired activity of pyridoxine-dependent enzymes in ADHD patients. Ritalin treatment did not change the general pattern of TRP degradation, but still created a kind of balance between some of detected metabolites. However, the 4PA/TRP, IND/TRP and IND/KYN ratios remained as low as in untreated patients, keeping the importance of diagnostic markers. Almost identical parameters of TRP degradation in untreated ADHD and epileptic patients allow to assume that inborn disorders of vitamin B6 metabolism are the common biochemical background of both diseases. The disturbed activity of PLP dependent enzymes apparently forms those profound disturbances of neurotransmitter systems, which are inherent in ADHD: low concentrations of monoamines and disordered amino acid metabolism. If vitamin B6 disorders are the core biochemical disturbances inherent in ADHD, then the long-term pyridoxine treatment is pathogenetically based replacement therapy of the disease. According to our data, multi-year pyridoxine treatment normalizes completely the pattern of ADHD behavior, without causing any serious side effects.

A recombinant decoy comprising EGFR and ErbB-4 inhibits tumor growth and metastasis.

Epidermal growth factor (EGF)-like growth factors control tumor progression as well as evasion from the toxic effects of chemotherapy. Accordingly, antibodies targeting the cognate receptors, such as EGFR/ErbB-1 and the co-receptor HER2/ErbB-2, are widely used to treat cancer patients, but agents that target the EGF-like growth factors are not available. To circumvent the existence of 11 distinct ErbB ligands, we constructed a soluble fusion protein (hereinafter: TRAP-Fc) comprising truncated extracellular domains of EGFR/ErbB-1 and ErbB-4. The recombinant TRAP-Fc retained high-affinity ligand binding to EGF-like growth factors and partially inhibited growth of a variety of cultured tumor cells. Consistently, TRAP-Fc displayed an inhibitory effect in xenograft models of human cancer, as well as synergy with chemotherapy. Additionally, TRAP-Fc inhibited invasive growth of mammary tumor cells and reduced their metastatic seeding in the lungs of animals. Taken together, the activities displayed by TRAP-Fc reinforce critical roles of EGF-like growth factors in tumor progression, and they warrant further tests of TRAP-Fc in preclinical models.

Novel derivatives of 6-mercaptopurine: synthesis, characterization and antiproliferative activities of S-allylthio-mercaptopurines.

Biologically active S-allylthio derivatives of 6-mercaptopurine (6-MP) and 6-mercaptopurine riboside (6-MPR) were synthesized. The products, S-allylthio-6-mercaptopurine (SA-6MP) and S-allylthio-6-mercaptopurine riboside (SA-6MPR) were characterized. The antiproliferative activity of the new prodrugs was tested on human leukemia and monolayer cell lines, and compared to that of their parent reactants. The new prodrugs acted by a concentration-dependent mechanism. They inhibited cell proliferation and induced-apoptosis more efficiently than the parent molecules. Leukemia cell lines were more sensitive to the new prodrugs than monolayer cell lines. Higher hydrophobicity of the derivatives improves their penetration into cells, where upon reaction with glutathione, S-allylthioglutathione (GSSA) is formed, and 6-MP or 6-MPR is released for further processing.

[3H]Allicin: preparation and applications.

Allicin (diallylthiosulfinate), the active substance of garlic, has been shown to possess a variety of biological activities. Mechanistic and pharmacokinetic studies of allicin and its derivatives raise the need for a labeled compound. However, labeling of this volatile and unstable liquid requires delicate handling. Here, we describe a simple method for the preparation of (3)H-labeled allicin. This was achieved by applying synthetic [(3)H]alliin ([2,3-(3)H]allylcysteine sulfoxide) to a column containing immobilized alliinase [EC 4.1.1.4.] from garlic. Purification of [(3)H]allicin was done by differential adsorbtion of the reaction components on a neutral polystyrene resin, Porapak Q. Thiol-containing compounds are known to be the main target of allicin. In this work we demonstrated that [(3)H]allicin can be used for the synthesis of labeled [(3)H]allylmercapto derivatives of SH peptides and proteins. Thus, we prepared [(3)H]S-allylmercaptoglutathione which can be used in metabolic studies. Moreover, we showed that incubation of alliinase with [(3)H]allicin led to modification of 1.4 cysteine residues per subunit of the enzyme.

The effects of allicin and enalapril in fructose-induced hyperinsulinemic hyperlipidemic hypertensive rats.

The effects of a synthetic preparation of an active constituent of garlic, allicin, were studied on blood pressure (BP), triglycerides, and insulin levels in Sprague-Dawley rats in which high fructose feeding elicited hyperinsulinemia, hypertension, and hypertriglyceridemia. Results were compared with those of the antihypertensive drug enalapril. Three groups of male Sprague-Dawley rats were fed a fructose-enriched diet for 5 weeks. During the last 2 weeks 10 animals received only fructose, 10 received allicin, and 10 received enalapril. Blood pressure, insulin level, and triglyceride levels were measured at the beginning of the experiment and after 3 and 5 weeks on the fructose diet, fructose/allicin diet, or fructose/enalapril diet. Allicin lowered BP from the maximal level (after 3 weeks of fructose) of 153.4 +/- 8 mm Hg to 139.7 +/- 12 mm Hg after 2 weeks on allicin; insulin from 11.7 +/- 3.7 ng/mL on fructose diet to 6.92 +/- 3.3 ng/mL on allicin; and triglycerides from 132.8 +/- 18 mg/dL on fructose to 59.6 +/- 27 mg/dL on allicin. The similar effect of allicin and enalapril on BP, insulin, and triglycerides reinforces the trend toward combining the nonpharmacologic approach with drug therapy.

S-Allylmercaptoglutathione: the reaction product of allicin with glutathione possesses SH-modifying and antioxidant properties.

The reaction between allicin (diallylthiosulfinate), the active component of garlic and reduced glutathione was investigated. The product of this reaction, mixed disulfide S-allylmercaptoglutathione (GSSA) was separated by high performance liquid chromatography and identified by 1H and (13)C nuclear magnetic resonance and mass spectroscopy. The reaction is fast (with an apparent bimolecular reaction rate constant of 3.0 M(-1) s(-1)). It is pH-dependent, which reveals a direct correlation to the actual concentration of mercaptide ion (GS(-)). Both GSSA and S-allylmercaptocysteine (prepared from allicin and cysteine) reacted with SH-containing enzymes, papain and alcohol dehydrogenase from Thermoanaerobium brockii yielding the corresponding S-allylmercapto proteins, and caused inactivation of the enzymes. The activity was restored with dithiothreitol or 2-mercaptoethanol. In addition, GSSA also exhibited high antioxidant properties. It showed significant inhibition of the reaction between OH radicals and the spin trap 5,5'-dimethyl-1-pyroline N-oxide in the Fenton system as well as in the UV photolysis of H2O2. In ex vivo experiments done with fetal brain slices under iron-induced oxidative stress, GSSA significantly lowered the production levels of lipid peroxides. The similar activity of GSSA and allicin as SH-modifiers and antioxidants suggests that the thioallyl moiety has a key role in the biological activity of allicin and its derivatives.

Natural antibodies against alliinase in human serum and polyclonal antibodies elicited in rabbit share the same immunogenic determinants.

Human serum contains natural antibodies against alliinase, a protein abundantly found in garlic (Allium sativum) cloves. In order to study the epitope(s) of this protein recognized by anti-alliinase antibodies, we used a random hexapeptide library displayed on filamentous M13 phage. Analysis of the phagotopes selected on rabbit anti-alliinase antibodies revealed that the motif-GKXVXX- was common for all peptides. The most frequent phage displaying -GKHVAV- sequence has a 50% identity with the original alliinase sequence (amino acid residues 156-161). The position of this epitope is only nine amino acids apart from the oligosaccharide chain attached to the N146. The rabbit anti-alliinase immunoglobulin G (IgG), which bound the phages displaying this phagotope, also bound the corresponding peptide derived from the alliinase sequence. Affinity-purified natural antibodies against alliinase, present in normal human serum (which can specifically recognize the native and denaturated protein) also bound the selected phagotope. Thus, our results indicate that specific natural anti-dietary protein antibodies presented in human serum can have the same. or overlapping. epitopes with the IgG evoked during the active (experimental) immunization in animals.

The mode of action of allicin: its ready permeability through phospholipid membranes may contribute to its biological activity.

Allicin (diallyl thiosulfinate) is the main biologically active component of the freshly crushed garlic extracts. In the present work the ability of allicin to cross through membranes (artificial and biological) was studied. Partition coefficients of allicin in water/octanol, water/hexadecane and water/phospholipids mixtures were determined. Using phospholipid vesicles loaded with hydrophilic thiols (reduced glutathione or 2-nitro-5-thiobenzoate), we observed that allicin freely permeates through phospholipid bilayers and interacts with the SH groups. The reaction rate of allicin with SH containing molecules after crossing the membrane was the same as in solution. Fast diffusion and permeation of allicin across human red blood cell membranes was also demonstrated. Allicin does not induce leakage, fusion or aggregation of membrane. The high permeability of allicin through membranes may greatly enhance the intracellular interaction with thiols.

Effect of purified allicin, the major ingredient of freshly crushed garlic, on cancer cell proliferation.

The diverse health benefit effects of garlic include its anticancer activity. However, very little is known about such activity of isolated garlic compounds, among which allicin (the major ingredient of crushed garlic) has been the least studied. The aim of this work was to determine whether pure allicin exhibits the antiproliferative effect reported for garlic in in vitro models. Allicin, but not its precursor alliin, inhibited proliferation of human mammary (MCF-7), endometrial (Ishikawa), and colon (HT-29) cancer cells (50% inhibitory concentration = 10-25 microM). Two of three tested primary lines of human fibroblasts displayed a similar response to allicin (50% inhibitory concentration = 16-40 microM), whereas the third line was almost unaffected by this compound. The pure allicin and water extract of garlic powder with equivalent allicin concentrations displayed a similar potency, suggesting that allicin is responsible for the antiproliferative effect of the extract. The growth inhibition was accompanied by accumulation of cells in the G0/G1 and G2/M phases of the cell cycle (MCF-7 cells) and not by a significant increase in cell death. Allicin caused a transient drop in the intracellular glutathione (GSH) level, the magnitude and kinetics of which significantly varied depending on cell type. The extent of the decrease in GSH levels correlated well (r = 0.75) with the growth inhibitory activity of allicin. On the basis of these findings, we suggest that allicin plays a major role in the antiproliferative effect of water-soluble garlic preparations and that this effect may be attributed to the ability of allicin to transiently deplete the intracellular GSH level.

Allicin-induced decrease in formation of fatty streaks (atherosclerosis) in mice fed a cholesterol-rich diet.

BACKGROUND: Garlic (Allium sativum) has been considered to exhibit therapeutic features for many years. The effects of garlic on levels of serum lipids and on atherosclerosis have been investigated extensively. We have previously demonstrated that allicin, an active component of garlic, exerts a beneficial effect on lipid profile in hyperlipidemic rabbits. OBJECTIVE: To investigate the effects of allicin on formation of fatty streaks (atherosclerosis) and lipid profile in mice. METHODS: Allicin was extracted from garlic and kept in a buffer citrate solution at 4 degrees C. Sixty C57BL/6 mice were fed Paigen diet (17% fat, 1.25% cholesterol) for 15 weeks. Thirty randomly selected animals were administered allicin solution (9 mg/kg) and 30 were administered placebo. Blood lipid profile was evaluated five times during the study. At the end of the 15-week period, the animals were killed and the aortic sinus was evaluated for formation of fatty streaks (atherosclerosis). RESULTS: We observed no statistically significant differences between blood lipid profiles of groups. Microscopic evaluation of aortic sinus formation of fatty streaks (atherosclerosis), however, showed that values for mice in the allicin-treated group were significantly lower: areas of formation of fatty streaks (atherosclerosis) were 13,440 +/- 3310 and 23,410 +/- 3723 micron 2, respectively, for allicin-treated and control mice (means +/- SEM; P = 0.023). CONCLUSIONS: These results indicate that allicin reduces formation of fatty streaks (atherosclerosis) in hyperlipidemic mice. These changes do not seem to occur through an alteration in blood lipid profile.

The mode of action of allicin: trapping of radicals and interaction with thiol containing proteins.

Allicin (thio-2-propene-1-sulfinic acid S-allyl ester) is the main biologically active component of garlic clove extracts. Its biological activity was attributed to either antioxidant activity or thiol disulfide exchange. Antioxidant properties of both allicin and its precursor, alliin (+S-allyl-L-cysteine sulfoxide), were investigated in the Fenton oxygen-radical generating system [H2O2-Fe(II)]. Using the spin trapping technique and ESR, it was found that both compounds possessed significant antioxidant activity. The reaction between allicin and L-cysteine was studied by 1H and 13C-NMR, and a S-thiolation product, S-allylmercaptocysteine, was identified. Allicin irreversibly inhibited SH-protease papain, NADP(+)-dependent alcohol dehydrogenase from Thermoanaerobium brockii (TBAD), and the NAD(+)-dependent alcohol dehydrogenase from horse liver (HLAD). All the three enzymes could be reactivated with thiol containing compounds. Papain could be reactivated with glutathione, TBAD with dithiothreitol or 2-mercaptoethanol (2-ME) but not by glutathione, while HLAD could be reactivated only with 2-ME. This study demonstrates that in addition to its antioxidant activity, the major biological effect of allicin should be attributed to its rapid reaction with thiol containing proteins.

A spectrophotometric assay for allicin and alliinase (Alliin lyase) activity: reaction of 2-nitro-5-thiobenzoate with thiosulfinates.

Allicin (diallylthiosulfinate) is the main biologically active component of freshly crushed garlic cloves. It is produced upon the interaction of the nonprotein amino acid alliin with the enzyme alliinase (alliin lyase, EC 4.4.1.4). A simple and rapid spectrophotometric procedure for determination of allicin and alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin, is described. NTB reacts with the activated disulfide bond --S(O)-S--; of allicin, forming the mixed-disulfide allylmercapto-NTB, as characterized by NMR. The method can be used for determination of allicin and total thiosulfinates in garlic preparations and garlic-derived products. The method was applied for determination of pure alliinase activity and for the activity of the enzyme in crude garlic extracts.

Allicin from garlic strongly inhibits cysteine proteinases and cytopathic effects of Entamoeba histolytica.

The ability of Entamoeba histolytica trophozoites to destroy monolayers of baby hamster kidney cells is inhibited by allicin, one of the active principles of garlic. Cysteine proteinases, an important contributor to amebic virulence, as well as alcohol dehydrogenase, are strongly inhibited by allicin.

Salicylaldehyde-metal-amino acid ternary complex: a new tool for immobilized metal affinity chromatography.

An immobilized salicylaldehyde (sal) was used to build various salicylaldehyde-copper-amino acid (Sal-Cu-AA) complexes which are stable at a range of pH values (2.0-11.0). The complexes were found to bind protein molecules as IMAC resins. Thirteen proteins were examined for their binding to a Sal-Cu-Gly column. The efficacy of the Sal-Cu-AA resin for protein separation were demonstrated by two examples. The first was a new purification process for garlic lectins from garlic crude extract. It seems that in this case the Sal-Cu-AA resins were more selective than IDA resin. The second was immobilization of concanavalin A (Con A) on the resin and using the immobilized Con A for affinity chromatography of mannose-rich glycoprotein ovalbumin. The Con A could be later eluted with EDTA or imidazole and the Sal-containing polymer could be recharged again for further use.

Alteration of lipid profile in hyperlipidemic rabbits by allicin, an active constituent of garlic.

BACKGROUND: The effect of garlic on the serum lipid profile has been the subject of controversy. This study was therefore designed to examine the effects of allicin, an active constituent of garlic, on the lipid profile in a rabbit model. METHODS: Allicin was produced by reacting alliin, synthesized in our laboratory, with purified alliinase. Nineteen New Zealand White rabbits were fed a cholesterol-rich diet (0.25% cholesterol) for 18 weeks. Ten rabbits received freshly produced allicin (3 mg/kg orally) starting at 8 weeks, and nine received placebo. There was no significant difference between the lipid profiles of the two groups at baseline up to 8 weeks. RESULTS: From day 28 of allicin supplementation a significant difference was found between the allicin and placebo groups in the graph regression lines describing the influence of allicin on serum cholesterol: Y = 41.39 + 8.69 multiplied by day (control) versus Y = -877.24 + 17.67 multiplied by day (allicin). The same trend was found for low-density lipoprotein concentrations: Y = 10.3 + 8.4 multiplied by (control) versus Y = -750.4 + 15.7 multiplied by day (allicin). The serum high-density lipoprotein levels also differed significantly between the groups: Y = 20.29 + 0.24 multiplied by day (control) versus Y = -109.9 + 1.65 multiplied by day (allicin). CONCLUSIONS: Our results indicate that allicin has a beneficial effect on the serum lipid profile in hyperlipidemic rabbits, and should be further tested clinically.

Alliinase (alliin lyase) from garlic (Alliium sativum) is glycosylated at ASN146 and forms a complex with a garlic mannose-specific lectin.

Alliinase (EC 4.4.1.4) catalyses the production of allicin (thio-2-propene-1-sulfinic acid S-allyl ester), a biologically active compound which is also responsible for the characteristic smell of garlic. It was demonstrated that alliinase which contains 5.5-6% of neutral sugars, gives clear PAS-staining, binds to Con A and can form a complex with garlic mannose-specific lectin (ASA). Evidence that the formation of such a complex is mediated by the interaction of the carbohydrate of the glycoprotein enzyme with the lectin was obtained from a radioligand assay which demonstrated the binding of alliinase to ASA and competitive inhibition of this binding by methyl alpha-D-mannoside. ASA I was shown as the lectin mainly present in the complex with alliinase. The results of this study also demonstrate that alliinase is glycosylated at Asn146 in the sequence Asn146-Met147-Thr148.

Natural antibodies to dietary proteins: the existence of natural antibodies to alliinase (Alliin lyase) and mannose-specific lectin from garlic (Allium sativum) in human serum.

It is known that human serum contains natural antibodies to self and non-self proteins. We wished to determine whether normal human serum contains antibodies to dietary proteins that were never injected. We found that human serum contains antibodies to the two major proteins from cloves of garlic (Allium sativum) which is used as a flavorigard dietary food additive. The antibodies found were directed against alliinase and mannose-specific Allium sativum agglutinin (ASA). The antibodies were purified by affinity chromatography on their corresponding antigens. The purified immunoglobulins were mainly of the IgG and IgM classes and could be divided into two categories--specific and crossreactive. The anti-alliinase antibodies were highly specific, while anti-ASA antibodies were polyreactive. Some of the possible reasons for this difference in specificity are suggested.

Alliin lyase (Alliinase) from garlic (Allium sativum). Biochemical characterization and cDNA cloning.

The garlic plant (Allium sativum) alliinase (EC 4.4.1.4), which catalyzes the synthesis of allicin, was purified to homogeneity from bulbs using various steps, including hydrophobic chromatography. Molecular and biochemical studies showed that the enzyme is a dimer of two subunits of MW 51.5 kDa each. Its Km using synthetic S-allylcysteine sulfoxide (+ isomer) as substrate was 1.1 mM, its pH optimum 6.5, and its isoelectric point 6.35. The enzyme is a glycoprotein containing 6% carbohydrate. N-terminal sequences of the intact polypeptide chain as well as of a number of peptides obtained after cyanogen bromide cleavage were obtained. Cloning of the cDNAs encoding alliinase was performed by a two-step strategy. In the first, a cDNA fragment (pAli-1-450 bp) was obtained by PCR using a mixed oligonucleotide primer synthesized according to a 6-amino acid segment near the N-terminal of the intact polypeptide. The second step involved screening of garlic lambda gt11 and lambda ZAPII cDNA libraries with pAli-1, which yielded two clones; one was nearly full length and the second was full length. These clones exhibited some degree of DNA sequence divergence, especially in their 3' noncoding regions, suggesting that they were encoded by separate genes. The nearly full length cDNA was fused in frame to a DNA encoding a signal peptide from alpha wheat gliadin, and expressed in Xenopus oocytes. This yielded a 50 kDa protein that interacted with the antibodies against natural bulb alliinase. Northern and Western blot analyses showed that the bulb alliinase was highly expressed in bulbs, whereas a lower expression level was found in leaves, and no expression was detected in roots. Strikingly, the roots exhibited an abundant alliinase activity, suggesting that this tissue expressed a distinct alliinase isozyme with very low homology to the bulb enzyme.