Lactulose as a novel template for anticancer drug development targeting galectins.

Galectins are carbohydrate binding proteins (lectins), which characteristically bind ß-galactosides. Galectins play a role in tumour progression through involvement in proliferation, metastasis, angiogenesis, immune evasion and drug resistance. There is need for inhibitors (antagonists) that are specific for distinct galectins and that can interfere with galectin-carbohydrate interactions during cancer progression. Here, we propose that lactulose, a non-digestible galactose-fructose disaccharide, presents a novel inhibitor scaffold for design of inhibitors against galectins. Thermodynamic evaluation displays binding affinity of lactulose against the galectin-1 and galectin-3 carbohydrate recognition domain (CRD). Crystal structures of galectin-1 and galectin-3 in complex with lactulose reveal for the first time the molecular basis of the galectin-lactulose interactions. Molecular modelling was implemented to propose novel lactulose derivatives as potent anti-cancer agents.

Heterofunctional hydrophilic-hydrophobic porous silica as support for multipoint covalent immobilization of lipases: application to lactulose palmitate synthesis.

Lipase-catalyzed synthesis of sugar esters, as lactulose palmitate, requires harsh conditions, making it necessary to immobilize the enzyme. Therefore, a study was conducted to evaluate the effect of different chemical surfaces of hierarchical meso-macroporous silica in the immobilization of two lipases from Pseudomonas stutzeri (PsL) and Alcaligenes sp. (AsL), which exhibit esterase activity. Porosity and chemical surface of silica supports, before and after functionalization and after immobilization, were characterized by gas adsorption and Fourier transform infrared (FTIR) spectroscopy. PsL and AsL were immobilized in octyl (OS), glyoxyl (GS), and octyl-glyoxyl silica (OGS). Hydrolytic activity, thermal and solvent stability, and sugar ester synthesis were evaluated with those catalysts. The best support in terms of expressed activity was OS in the case of PsL (100 IU g(-1)), while OS and OGS were the best for AsL with quite similar expressed activities (60 and 58 IU g(-1), respectively). At 60 °C in aqueous media the more stable biocatalysts were GS-PsL and OGS-AsL (half-lives of 566 and 248 h, respectively), showing the advantage of a heterofunctional support in the latter case. Lactulose palmitate synthesis was carried out in acetone medium (with 4% of equilibrium moisture) at 40 °C obtaining palmitic acid conversions higher than 20% for all biocatalysts, being the highest of those obtained with OGS-AsL and OS-PsL. Therefore, screening of different chemical surfaces on porous silica used as supports for lipase immobilization allowed obtaining active and stable biocatalyst to be employed in the novel synthesis of lactulose palmitate.

[Prebiotic properties of lactite and lactulose under creation of synbiotics of bacillus genus bacteria].

The influence of lactite and lactulose on growth of probiotic strains B. subtilis UKM 5139 and B. subtilis UKM 5140 and conditionally pathogenic microorganisms has been studied. It is shown that the extent of influence of these substances depends on their concentration. Addition of lactite or lactulose in the nutrient medium stimulated growth of probiotic bacilli strains in concentration of 15-20 % and inhibited growth of conditionally pathogenic microorganisms. It specifies the prebiotic properties of the studied disaccharides. It is established that the extent of inhibition of P. vulgaris, S. flexnery, S. enterica abony, E. coli 028 by lactulose was higher in comparison with the extent of their inhibition by lactite, but in turn lactite was more effective in relation to the strains E coli ATCC 25922, C. albicans, S. derby and S. aureus.

Inhibition of prostate cancer bone metastasis by synthetic TF antigen mimic/galectin-3 inhibitor lactulose-L-leucine.

Currently incurable, prostate cancer metastasis has a remarkable ability to spread to the skeleton. Previous studies demonstrated that interactions mediated by the cancer-associated Thomsen-Friedenreich glycoantigen (TF-Ag) and the carbohydrate-binding protein galectin-3 play an important role in several rate-limiting steps of cancer metastasis such as metastatic cell adhesion to bone marrow endothelium, homotypic tumor cell aggregation, and clonogenic survival and growth. This study investigated the ability of a synthetic small-molecular-weight nontoxic carbohydrate-based TF-Ag mimic lactulose-L-leucine (Lac-L-Leu) to inhibit these processes in vitro and, ultimately, prostate cancer bone metastasis in vivo. Using an in vivo mouse model, based on intracardiac injection of human PC-3 prostate carcinoma cells stably expressing luciferase, we investigated the ability of Lac-L-Leu to impede the establishment and growth of bone metastasis. Parallel-flow chamber assay, homotypic aggregation assay, modified Boyden chamber assay, and clonogenic growth assay were used to assess the effects of Lac-L-Leu on tumor cell adhesion to the endothelium, homotypic tumor cell aggregation, transendothelial migration, and clonogenic survival and growth, respectively. We report that daily intraperitoneal administration of Lac-L-Leu resulted in a three-fold (P < .05) decrease in metastatic tumor burden compared with the untreated control. Mechanistically, the effect of Lac-L-Leu, which binds and inhibits galectins by mimicking essential structural features of the TF-Ag, was associated with a dose-dependent inhibition of prostate cancer cell adhesion to bone marrow endothelium, homotypic aggregation, transendothelial migration, and clonogenic growth. We conclude that small-molecular-weight carbohydrate-based compounds targeting ß-galactoside-mediated interactions could provide valuable means for controlling and preventing metastatic prostate cancer spread to the skeleton.

Sugar recognition by CscB and LacY.

The sucrose permease (CscB) and lactose permease (LacY) of Escherichia coli belong to the oligosaccharide/H(+) symporter subfamily of the major facilitator superfamily, and both catalyze sugar/H(+) symport across the cytoplasmic membrane. Thus far, there is no common substrate for the two permeases; CscB transports sucrose, and LacY is highly specific for galactopyranosides. Determinants for CscB sugar specificity are unclear, but the structural organization of key residues involved in sugar binding appears to be similar in CscB and LacY. In this study, several sugars containing galactopyranosyl, glucopyranosyl, or fructofuranosyl moieties were tested for transport with cells overexpressing either CscB or LacY. CscB recognizes not only sucrose but also fructose and lactulose, but glucopyranosides are not transported and do not inhibit sucrose transport. The findings indicate that CscB exhibits practically no specificity with respect to the glucopyranosyl moiety of sucrose. Inhibition of sucrose transport by CscB tested with various fructofuranosides suggests that the C(3)-OH group of the fructofuranosyl ring may be important for recognition by CscB. Lactulose is readily transported by LacY, where specificity is directed toward the galactopyranosyl ring, and the affinity of LacY for lactulose is similar to that observed for lactose. The studies demonstrate that the substrate specificity of CscB is directed toward the fructofuranosyl moiety of the substrate, while the specificity of LacY is directed toward the galactopyranosyl moiety.

Identification and site-specific relative quantification of beta-lactoglobulin modifications in heated milk and dairy products.

During milk processing, proteins can be severely modified by oxidation, condensation, and Maillard reaction, leading to changes in their nutritional and technological properties. In this study, major modifications of beta-lactoglobulin, formed during the heating and processing of milk, were screened by mass spectrometry. For this purpose, beta-lactoglobulin was isolated from the milk samples by gel electrophoresis and analyzed by matrix-assisted laser desorption/ionization mass spectrometry after in-gel digestion with endoproteinase AspN. In heated milk, lactulosyllysine was detected at lysine 47 and 138 or 141 as well as methionine sulfoxide at methionine 7, 24, and 145. All these modifications increased gradually when raw milk was heated for 20, 40, and 60 min at 120 degrees C. The major modifications were also relatively quantified in dairy products, such as raw, high-temperature, ultra-high-temperature, sterilized, and condensed milk as well as infant formulas. The highest contents of lactulosyllysine at Lys47 were detected in powdered infant formulas, whereas lactulosyllysine at Lys138/141 was predominant in condensed milk samples. Methionine sulfoxide at Met7 and Met24 showed a trend toward higher modification rates in more severely processed products.

An oral load of the early glycation compound lactuloselysine fails to accumulate in the serum of uraemic patients.

BACKGROUND: It has been hypothesized that in renal failure, exogenous glycation compounds from food accumulate and play a major pathogenetic role when renal excretion is impaired. METHODS: To address this, a diet containing a defined amount of the lysine Amadori product (AP) lactuloselysine was used. Plasma concentrations and cumulative urinary excretion of AP were assessed in 16 healthy subjects, 12 renal failure patients and 6 continuous ambulatory peitoneal dialysis (CAPD) patients. Amadori product was measured as furosine using reverse phase high performance liquid chromatography (RP-HPLC) after acid hydrolysis. RESULTS: A diet low in glycation compounds significantly decreased excretion of APs in healthy subjects. In healthy individuals, ingestion of lactuloselysine bound to food proteins caused only a minor acute increase (8.24+/-1.11 mg/day, 2% of the administered dose) of AP excretion in the urine; in patients with renal failure not yet on dialysis, the increase in AP excretion in the urine was significantly less (4.0+/-0.51 mg/day) and the same was true in CAPD patients (0.21+/-0.09 mg/day). The plasma concentration of total APs, i.e. the sum of APs as free amino acids and residues bound to plasma proteins, did not change in any of the three groups, however. CONCLUSION: Dietary APs do not accumulate in the blood even in advanced renal failure. The amount of APs measured as furosine excreted in the urine is significantly less, however, in renal failure and CAPD patients compared with healthy subjects. Although the findings exclude accumulation of lactuloselysine in renal failure, they do not generally exclude accumulation of other food-derived advanced glycation end products (AGEs).

Nutritional and metabolic consequences of the early Maillard reaction of heat treated milk in the pig. Significance for man.

BACKGROUND: During the processing of foods, the Maillard reaction may occur contributing to altering the nutritional value of proteins. In dairy products the formation of lactuloselysine reduces the availability of lysine but the effects on the other nutrients are not very well known. AIM OF THE STUDY: Determination of the consequences of a high level of lactuloselysine in milk on the bioavailability of skim milk nutrients and the kinetics of their appearance in the portal blood and of their urinary and faecal excretions and extrapolation to lower heat treatments and to man, using the pig model. METHODS: Sub-adult pigs were fitted, under anaesthesia, with permanent catheters in the portal vein, carotid artery and urethra, and with an electromagnetic flow probe around the portal vein. Each animal was successively fed with two experimental meals containing an equal amount of dried skim milk (SM), either lyophilised or heat treated to obtain an intense Maillard reaction, (M-SM) resulting in a 50% lysine blockage. Portal and arterial concentrations and flux of individual amino acids (AA), glucose, galactose and fructoselysine were measured for a period of 12h after the meals. Lysine, fructoselysine and AA excreted in the urine and faeces within 72h were also determined. RESULTS: In M-SM containing 50% blocked lysine, no other AA was chemically modified. Fructoselysine appeared in the portal blood very late compared to amino acids resulting from a very slow release and corresponded to 8.2 and 18.6% of the ingested amount after 12 and 72h, respectively. Significant changes of the appearance in the portal blood were observed only for lysine (-60%), alanine (-17%) and cystine (+37%). A small decrease in the digestibility of most AA during the same period was observed, which was significant after 48h for lysine, phenylalanine, cystine, aspartic acid, glycine and total AA (-6%). CONCLUSION: It was confirmed that lactuloselysine was not bioavailable. The loss in protein nutritive value was mainly due and proportional to the deterioration of lysine and, to a lesser extent, to the decrease in the digestibility of some essential AA. Taking into account the very high level of lactuloselysine in the M-SM sample studied, it may be concluded that in common foods such as milk, infant formulas, biscuits, bread, pasta, containing lower levels of blocked lysine, the nutritional loss is primarily due to the loss of lysine and to a less extent to the decrease in the digestibility of other essential AA.

Convenient synthesis of lactuloselysine and its use for LC-MS analysis in milk-like model systems.

The synthesis of the Amadori product lactuloselysine [N(epsilon)-(1-deoxy-D-lactulosyl-1)-L-lysine] was obtained starting from FMOC-lysine-OH (N(alpha)-9-fluorenylmethoxy-carbonyl-N(epsilon)H(2)-L-lysine-OH) and lactose. Compound identity was confirmed by MALDI-ToF, electrospray, and NMR analysis. A selective LC-MS procedure which allowed the detection of lactuloselysine up to 10 ng mL(-)(1) was set up and used to follow the formation of the compound in a lactose-lysine model system; quantification of this molecule after complete enzymatic hydrolysis of whey-proteins from milk samples was also performed.

Evaluation of the extent of the early Maillard-reaction in milk products by direct measurement of the Amadori-product lactuloselysine.

A new method for the evaluation of the extent of lysine modification caused by the Maillard reaction is presented, based on the direct determination of the Amadori product lactuloselysine plus unmodified lysine after complete enzymatic hydrolysis by ion-exchange chromatography. Using a simple mathematical relation, the amount of modified lysine in milk products can be estimated without knowledge of the initial lysine value before heating or storage.

Determination of beta-galactosidase activity in the intestinal tract of mice by ion-exchange high-performance liquid chromatography using epsilon-N-1-(1-deoxylactulosyl)-L-lysine as substrate.

epsilon-N-1-(1-Deoxylactulosyl)-L-lysine was synthesized and used as a substrate to assay beta-galactosidase activity. epsilon-N-1-(1-Deoxylactulosyl)-L-lysine and its degradation product epsilon-N-1-(1-deoxyfructosyl)-L-lysine were detected by high-voltage paper electrophoresis and ion-exchange high-performance liquid chromatography. The beta-galactosidase activity in different parts of the intestinal tract of germ-free and control mice was determined and compared with a beta-galactosidase activity which degrades lactose at pH 8.5 and 5.0 and which corresponded with bacterial and host enzymatic activities, respectively.