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PubMed1990

Effect of lactose on mineral absorption in preterm infants.

To determine whether a reduction of dietary lactose affects mineral absorption in preterm infants, a controlled, randomized, 72-hour balance study was conducted with very low birth weight infants (birth weights less than 1400 gm) fed Similac Special Care formula with a 50:50 carbohydrate blend of glucose polymers and lactose (n = 8) or a nearly identical formula with 100% carbohydrate as lactose (n = 10). The studies were conducted after at least 3 consecutive days of full oral feeds at a mean age of approximately 23 days. There was no difference between groups in percentage of apparent absorption of calcium, phosphorus, magnesium, zinc, copper, or manganese. Retention of these nutrients was similar to or greater than that which occurs in utero. Calcium absorption was 75% of intake. These results indicate that reducing the lactose content of premature-infant formulas to 50% of the carbohydrate does not impair mineral absorption.

Calcium, Dietary↗
PubMed2004

Preparation of lactose-silk fibroin conjugates and their application as a scaffold for hepatocyte attachment.

We prepared glycoconjugates (Lac-CY-SF) by the homogeneous chemical modification of solubilized silk fibroin (SF) with lactose bearing the galactose residue using cyanuric chloride (CY) as a coupling spacer and examined the usefulness of their application as a scaffold for hepatocyte attachment. The covalent immobilization of lactose into SF was assessed by several criteria including 1H-NMR measurements and reactions with lectins such as Ricinus communis agglutinin (RCA(120)) and fluorescein isothiocyanate-labeled RCA(120) (FITC-RCA(120)). The 1H-NMR spectrum of Lac-CY-SF showed new broad peaks attributed to methine and methylene protons of lactose, and by the integrated intensities of the peaks the weight ratio of lactose to SF in Lac-CY-SF was determined to be 0.20. Addition of RCA(120) to Lac-CY-SF solution caused an increase in the turbidity of the Lac-CY-SF solution. Incubation of Lac-CY-SF films with FITC-RCA(120) showed that the fluorescence emitted from the whole surface of Lac-CY-SF films. Furthermore, we examined the effect of the Lac-CY-SF conjugate-coating onto polystyrene culture dishes on the attachment of rat hepatocytes and their morphology. Attachment of hepatocytes onto the 0.1% (w/v) conjugate-coated dishes showed about an 8-fold increase as compared with that on uncoated dishes, being comparable to that on collagen-coated dishes, whereas the attachment on the SF-coated dishes was lower than that on uncoated dishes. Hepatocytes cultured onto the conjugate-coated dishes for 2.5 h showed smaller round-shaped morphology compared to those on collagen-coated dishes. After 2 days of culture in medium containing 100 nM insulin and 100 nM dexamethasone, hepatocytes on the conjugate-coated dishes formed monolayer islands with a slightly dispersed morphology, while hepatocytes cultured on collagen-coated dishes were uniformly spread flat. These results indicate that the Lac-CY-SF conjugates are useful as a scaffold for hepatocyte attachment, but the morphology of hepatocytes cultured on the conjugate-coated dishes is different from that on collagen-coated dishes.

Animals↗
PubMed1998

Recombinant gene expression in Escherichia coli cultivation using lactose as inducer.

The use of lactose as inducer of foreign gene expression under control of the lac UV5 promoter was investigated in recombinant Escherichia coli. Chicken muscle troponin C (TnC) gene was transcripted by T7 RNA polymerase and expressed in bioreactor cultivations after a feed-forward controlled fed-batch growth phase. Cell concentrations of 22 g l-1 dry cell weight (DCW)--before induction started--were used to achieve a TnC content of 19.5% of total cell protein through an induction strategy that combined the addition of a specific lactose amount of 4.7 g g-1 DCW divided into three pulses and the addition of yeast extract (1 g l-1) together with the second and the third lactose pulses. The results presented suggest that the residual lactose concentration plays an important role on the production of the heterologous protein.

Animals↗
PubMed2001

The use of inverse phase gas chromatography to study the change of surface energy of amorphous lactose as a function of relative humidity and the processes of collapse and crystallisation.

The purpose of this study was to assess the effect of relative humidity (RH) on the surface energy of amorphous lactose. Two samples of amorphous lactose were investigated; a spray dried 100% amorphous material and a ball milled sample of crystalline lactose. The milled sample had less than 1% amorphous content by mass, but on investigation at 0% RH, yielded surface energies comparable to those obtained from the 100% amorphous material, indicating that the surface was amorphous. The effect of increasing humidity was to reduce the dispersive surface energy of the two samples from 36.0 +/- 0.14 and 41.6 +/- 1.4 mJ m(-2) at 0% RH for the spray dried and milled samples respectively, to a value comparable to that obtained for the crystalline alpha-lactose monohydrate of 31.3 +/- 0.41 mJ m(-2). The change in surface energy due to water sorption was only reversible up to 20% RH; after exposure to higher RH values subsequent drying did not result in a return to the original surface energy of the amorphous form. This shows that the surface is reorganising as the glass transition temperature (Tg) is reduced, even though the sample has not collapsed or crystallised. It was possible to follow the collapse behaviour in the column with ease, using a number of different methods.

Absorption↗
PubMed2003

Quantitative determination of crystallinity of alpha-lactose monohydrate by Near Infrared Spectroscopy (NIRS).

The purpose of this study was to determine quantitatively the crystallinity in crystalline/amorphous powder mixtures of lactose, to asses the capability of Near Infrared Spectroscopy (NIRS) for quantitative determination of crystallinity and to compare the accuracy of the NIRS method with that of conventional X-ray powder diffraction (XRPD). Amorphous lactose was prepared by spray drying. Samples with different crystallinity were prepared by physical mixing of 100% amorphous and 100% crystalline materials. The samples were characterized by XRPD and NIRS. Analysis was performed on the data sets by multiple linear regression (MLR). There is a close correlation between the predicted and the actual crystallinity of physical mixtures of crystalline and amorphous lactose, determined by NIRS (R(2)=0.9994). NIRS results were compared to the XRPD using the same sample sets. The correlation coefficients was 0.9981. The results showed that NIRS is an useful method for accurately determining low quantities of the crystalline lactose in a physical mixture. Therefore, NIRS can be used for the quantitative determination of crystallinity of materials during pharmaceutical procedures.

Crystallization↗
PubMed1999

Relationships between the effective interparticulate contact area and the tensile strength of tablets of amorphous and crystalline lactose of varying particle size.

The aim of this study was to experimentally evaluate an equation for describing tablet tensile strength by studying relationships between the measured tensile strength and the calculated effective interparticulate contact area in tablets prepared from varying size fractions of amorphous and crystalline lactose at varying pressures. Amorphous lactose produced tablets of higher tensile strength than crystalline lactose and there was a tendency for reduced particle size to increase tablet strength. The tablet tensile strength correlated reasonably well with the effective area of contact for each material after compensation for a particle size-related intercept. It was thus concluded that the tensile strength equation under investigation reflects a structural property of the tablet which correlates with the tensile strength of the tablet. The slope of the tensile strength-contact area relationship differed between the materials and original particle size also had a small effect on this parameter. The results indicate thus also that the differences in compactability between amorphous and crystalline lactose are mainly the result of differences in bonding capacity, although differences in particle deformability also play a limited part.

Chemistry, Pharmaceutical↗
PubMed1999

Lactose-derived oligosaccharides in the milk of elephants: comparison with human milk.

Human milk is commonly considered to be unique when compared with the milk of other species with regard to its high content of complex fucosylated and sialylated lactose-derived oligosaccharides. We describe the application of high-pH anion-exchange chromatography with pulsed amperometric detection and TLC to characterize and quantitate neutral and sialylated lactose-derived oligosaccharides in milk from three Asian elephants and human milk. The lactose contents of elephant and human milks were 25-30 g/l and about 66 g/l respectively, whereas total oligosaccharide concentration was about three times higher in elephant milk and comprised up to 40% (10% in human milk) of the carbohydrate content. The ratio neutral: acidic components was different in the milk of the two species; in elephant milk, the N-acetylneuraminic acid-containing oligosaccharides made up almost half of the total amount v. 30% in human milk. Most oligosaccharides in elephant milk were more fucosylated and/or sialylated compared with human milk components. By mild acid hydrolysis, fucose and N-acetylneuraminic acid were cleaved off from complex components, and this resulted in increased amounts of fucose, galactose, N-acetylneuraminic acid, lactose and lacto-N-neo-tetraose. Unique to elephant milk are the high levels of 3'-galactosyllactose (up to 4 g/l) and lacto-N-neo-tetraose which are present in human milk only in trace amounts. Elephant and human milks have high levels and unique patterns of oligosaccharides which may reflect the relative importance of these components in neonatal host defence, in endothelial leucocyte interactions or in brain development.

Animals↗
PubMed1991

Construction of a functional lactose permease devoid of cysteine residues.

By use of oligonucleotide-directed, site-specific mutagenesis, a lactose (lac) permease molecule was constructed in which all eight cysteinyl residues were simultaneously mutagenized (C-less permease). Cys154 was replaced with valine, and Cys117, -148, -176, -234, -333, -353, and -355 were replaced with serine. Remarkably, C-less permease catalyzes lactose accumulation in the presence of a transmembrane proton electrochemical gradient (interior negative and alkaline). Thus, in intact cells and right-side-out membrane vesicles containing comparable amounts of wild-type and Cys-less permease, the mutant protein catalyzes lactose transport at a maximum velocity and to a steady-state level of accumulation of about 35% and 55%, respectively, of wild-type with a similar apparent Km (ca. 0.3 mM). As anticipated, moreover, active lactose transport via C-less permease is completely resistant to inactivation by N-ethylmaleimide. Finally, C-less permease also catalyzes efflux and equilibrium exchange at about 35% of wild-type activity. The results provide definitive evidence that sulfhydryl groups do not play an essential role in the mechanism of lactose/H+ symport. Potential applications of the C-less mutant to studies of static and dynamic aspects of permease structure/function are discussed.

Amino Acid Sequence↗
PubMed1992

Site-directed mutagenesis of lysine 319 in the lactose permease of Escherichia coli.

Lys319, which is on the same face of putative helix X as His322 and Glu325 in the lactose permease of Escherichia coli, has been replaced with Leu by oligonucleotide-directed, site-specific mutagenesis. Although previous experiments suggested that the mutation does not alter permease activity, we report here that K319L permease is unable to catalyze active lactose accumulation or lactose efflux down a concentration gradient. The mutant does catalyze facilitated influx down a concentration gradient at a significant rate; however, the reaction occurs without concomitant H+ translocation. The mutant also catalyzes equilibrium exchange at about 50% of the wild-type rate, but it exhibits poor counterflow activity. Finally, flow dialysis and photoaffinity labeling experiments with p-nitrophenyl alpha-D-galactopyranoside indicate that K319L permease probably has a markedly decreased affinity for substrate. The alterations described are not due to diminished levels of the mutated protein in the membrane, since immunological studies reveal comparable amounts of permease in wild-type and K319L membranes. It is proposed that Lys319, like Arg302, His322, and Glu325, plays an important role in active lactose transport, as well as substrate recognition.

Affinity Labels↗
PubMed1993

Cysteine scanning mutagenesis of putative helix XI in the lactose permease of Escherichia coli.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid in putative transmembrane helix XI was individually replaced with Cys (from Ala347 to Ser366). Fifteen of the 20 mutants are highly functional and accumulate lactose to > 60% of the level achieved by C-less permease, and an additional three mutants, all located at the cytoplasmic end of the helix, exhibit lower but significant lactose accumulation. Cys replacements for Thr348 or Lys358 result in virtually inactive permease. Lys358, however, is not essential for active lactose transport but plays a role in permease folding or membrane insertion by interacting with Asp237. Immunoblots reveal that all mutant proteins are present in the membrane in amounts comparable to C-less with the exception of Lys358-->Cys which is hardly detectable, as expected. The results highlight Thr348 as a potentially important residue for further analysis. Finally, all active mutants were assayed after treatment with the sulfhydryl reagent N-ethyl-maleimide, and results range from nearly complete inhibition to almost 2-fold stimulation. Remarkably, all of the strongly inhibited positions lie on one face of helix XI. The implications of the findings for packing of transmembrane helices in the C-terminal half of the permease are discussed.

Amino Acid Sequence↗
PubMed1975

The interaction of N-acetylglucosamine and an affinity-label analogue with alpha-lactalbumin and lactose synthetase.

We have attempted to detect binding of N-acetylglucosamine (NAG) to alpha-lactalbumin, the B protein of lactose synthetase, under conditions in which binding of NAG to lysozyme, a protein to which alpha-lactalbumin has a significant sequence homology, is observed. Using 1H nuclear magnetic resonance spectroscopy, uv difference spectroscopy, competition of NAG with N-methylnicotinamide chloride, and fluorescence spectroscopy, no binding was detected. The synthesis of a NAG analogue, N-diazoacetyl-glucosamine (diazoNAG), was carried out, and the molecule was demonstrated to be an active galactose acceptor in the lactose synthetase reaction. Use of this molecule in photochemical labeling experiments resulted in a large amount of nonspecific labeling of alpha-lactalbumin, lactose synthetase A protein, ribonuclease, and lysozyme, but competition experiments in the presence of an excess of NAG revealed some specific labeling in the case of A protein and lysozyme, but not with alpha-lactalbumin or a ribonuclease control. Thus, it is highly questionable that a NAG binding site is retained in alpha-lactalbumin; furthermore, it appears that the galacyosyl acceptor makes significant contacts with the A protein rather than alpha-lactalbumin in the lactose synthetase complex.

Acetylglucosamine↗
PubMed1996

Purification and functional characterization of the C-terminal half of the lactose permease of Escherichia coli.

The lactose permease has been expressed in contiguous, non-overlapping polypeptide fragments containing the N-terminal (N6) and C-terminal (C6) transmembrane domains of the protein [Bibi, E., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4325; Zen, K., et al. (1994) Biochemistry 33, 8198]. When expressed individually, N6 and C6 are unstable and do not catalyze active transport. However, when expressed simultaneously, the polypeptides stabilize each other and form a complex that catalyzes active lactose transport. Moreover, a deletion construct containing the first transmembrane domain and the six C-terminal transmembrane domains mediates downhill lactose translocation [Bibi et al. (1991) proc. Natl. Acad. Sci. U.S.A. 88, 7271]. Here we report that C6 can be expressed independently in a relatively stable form that binds monoclonal antibodies 4B1 and 4B11, which interact with conformationally dependent epitopes on the periplasmic and cytoplasmic surfaces of the membrane, respectively. In addition, C6 retains the ability to catalyze lactose translocation down a concentration gradient in a specific manner. Finally, as observed with full-length Val331Cys permease, beta-D-galactopyranosyl 10thio-beta-D-galactopyranoside quenches the fluorescence of 2-(4'-maleimidylanilino)naphthalene- 6-sulfonic acid (MIANS)- labeled C6 with a single-Cys residue in place of Val331, exhibiting as apparent Kd of 0.2 mM. Unlike full-length Val331Cys permease, however, ligand does not induce a chance in the position of the emission maximum of MIANS-labeled C6(Val331Cys) permease not in the reactivity of C6 (Val331Cys) permease with MINAS. the results indicate that C6 retains a conformation similar to that on the native permease and that most of the structure required of high-affinity binding and substrate translocation is located in the C-terminal half of the molecule.

Amino Acid Sequence↗
PubMed2002

Quantification of exopolysaccharide, lactic acid, and lactose concentrations in culture broth by near-infrared spectroscopy.

Near-infrared spectroscopy (NIRS) was used for the simultaneous prediction of exopolysaccharide (EPS; 0-3 g/L) and lactic acid (0-59 g/L) productions as well as lactose (0-68 g/L) concentration in supernatant samples from pH-controlled batch cultures of Lactobacillus rhamnosus RW-9595M in supplemented whey permeate medium. To develop calibration equations, the correlation between the second derivative of 164 NIRS transmittance spectra and concentration data obtained with reference methods was calculated at the wavelength between 1653-1770 and 2041-2353 nm, using a partial least-squares method (PLS). The lactic acid and lactose concentrations were measured by HPLC, and the EPS concentration was estimated by a new ultrafiltration method. The PLS correlation coefficient (R(2)) and the standard error of cross-validation for the calibrations were 91% and 0.26 g/L for EPS, 99% and 2.54 g/L for lactic acid, and 98% and 3.32 g/L for lactose, respectively. The calibration equations were validated with 45 randomly selected culture samples from 6 cultures that were not used for calibration. A high agreement between data of the reference methods and those of NIRS was observed, with correlation coefficients and standard errors of prediction of 99% and 1.64 g/L for lactic acid, 99% and 4.5 g/L for lactose, and 91% and 0.32 g/L for EPS. The results suggest that NIRS could be a useful method for rapid monitoring and control of EPS lactic fermentations.

Chromatography, High Pressure Liquid↗
PubMed2006

Production of prebiotic galacto-oligosaccharides from lactose using beta-galactosidases from Lactobacillus reuteri.

The beta-galactosidases (beta-Gals) of Lactobacillus reuteri L103 and L461 proved to be suitable biocatalysts for the production of prebiotic galacto-oligosaccharides (GOS) from lactose. Maximum GOS yields were 38% when using an initial lactose concentration of 205 g/L and at approximately 80% lactose conversion. The product mixtures were analyzed by capillary electrophoresis (CE) and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Disaccharides other than lactose and trisaccharides made up the vast majority of GOS formed. The main products were identified as beta-d-Galp-(1-->6)-d-Glc (allolactose), beta-d-Galp-(1-->6)-d-Gal, beta-d-Galp-(1-->3)-d-Gal, beta-d-Galp-(1-->6)-Lac, and beta-d-Galp-(1-->3)-Lac. There were no major products with beta1-->4 linkages formed. Both intermolecular and intramolecular transgalactosylation were observed. d-Galactose proved to be a very efficient galactosyl acceptor; thus, a relatively large amount of galactobioses was formed. Monosaccharides could be conveniently separated from the mixture by chromatography using a strong cation-exchange resin.

Carbohydrate Conformation↗
PubMed1998

Maillard reaction of lactose and fluoxetine hydrochloride, a secondary amine.

Analysis of commercially available generic formulations of fluoxetine HCl revealed the presence of lactose as the most common excipient. We show that such formulations are inherently less stable than formulations with starch as the diluent due to the Maillard reaction between the drug, a secondary amine hydrochloride, and lactose. The Amadori rearrangement product was isolated and characterized; the characterization was aided by reduction with sodium borohydride and subsequent characterization of this reduced adduct. The lactose-fluoxetine HCl reaction was examined in aqueous ethanol and in the solid state, in which factors such as water content, lubricant concentration, and temperature were found to influence the degradation. N-Formylfluoxetine was identified as a major product of this Maillard reaction and it is proposed that N-formyl compounds be used as markers for this drug-excipient interaction since they are easy to prepare synthetically. Many characteristic volatile products of the Maillard reaction have been identified by GC/MS, including furaldehyde, maltol, and 2,3-dihydro-3,5-dihydroxy-6-methyl-4 H-pyran-4-one. Close similarity between the degradation products of simple mixtures and formulated generic products was found; however, at least one product decomposed at a rate nearly 10 times that predicted from the simple models. Maillard products have also been identified in unstressed capsules. The main conclusion is that drugs which are secondary amines (not just primary amines as sometimes reported) undergo the Maillard reaction with lactose under pharmaceutically relevant conditions. This finding should be considered during the selection of excipients and stability protocols for drugs which are secondary amines or their salts, just as it currently is for primary amines.

Antidepressive Agents, Second-Generation↗
PubMed2000

Spray-dried lactose composite particles containing an ion complex of alginate-chitosan for designing a dry-coated tablet having a time-controlled releasing function.

PURPOSE: The properties of novel spray-dried lactose composite particles suitable for the coating filler of a dry-coated tablet having a long induction period in drug release were investigated. METHODS: To prepare spray-dried composite particles containing alginate-chitosan complex (SD(L/AL-CS)), an aqueous solution of lactose and sodium alginate and the acetic acid solution of chitosan were concomitantly fed into the rotary atomizer of a spray-dryer. The formation of the alginate-chitosan complex was confirmed by measuring the weight of insoluble portion in the mixture of sodium alginate and chitosan solutions. The dissolution properties of the dry-coated tablet were measured with the JP specified paddle method. RESULTS: The micromeritic properties of SD(L/AL-CS) were compared to those of the SD composite particles of lactose-sodium alginate, having a good compacting property. The drug release profiles of dry-coated tablet with SD(L/AL-CS) contained a long induction period followed by a rapid drug release phase in the artificial intestinal fluid. The induction period for drug release to occur was increased with an increase in the degree of deacetylation of chitosan and in the amount of chitosan in the formulation. The prolongation of induction period was attributed to the formation of an insoluble ion complex between sodium alginate and chitosan in the composite particles, which could form a rigid gel structure on the tablet surface. CONCLUSIONS: A time-controlled release tablet was designed with the composite particles of lactose containing the alginate-chitosan ion complex. The induction period of the dry-coated tablet could be prolonged in order to deliver the drug to the colon by controlling the type and amount of chitosan formulated in the composite particles.

Alginates↗
PubMed1999

Compaction properties of composite particles consisting of lactose with sodium alginate prepared by spray-drying.

PURPOSE: Composite particles of lactose with a small amount of sodium alginate were prepared by spray-drying (SD) in an effort to improve the compactibility of the polymer for direct compression. The compaction behavior of the SD composite particles with a range of polymer contents was investigated. METHODS: Composite particles were prepared by spray-drying an aqueous solution of lactose and sodium alginate at various formulating ratios. Improvement in the compactibility of the composite particles was evaluated by measuring the tablet tensile strength, porosity-applied pressure profiles, stress relaxation, elastic recovery of the compressed powder, and surface properties of the tablets by scanning electron microscopy. RESULTS: The tensile strength of compacts formed from the SD composite particles containing sodium alginate (< or = 10 wt%) was as high as that of spray-dried amorphous lactose. The improved compaction was attributed to the higher relaxation pressure and lower elastic recovery of the composite particles compared with alpha-lactose monohydrate. However, increasing the sodium alginate content of the SD composite particles above 10 wt% led to a marked reduction in the tensile strength of the resultant tablets. Scanning electron micrographs revealed that composite particles with a good compactibility fused totally in the tablets while composite particles containing 15% or more sodium alginate retained their shape, even after compression. The presence of sodium alginate layered uniformly on the surface of the particles and the increase in the glass transition temperature of the particles, possibly due to interpolation of sodium alginate are responsible for the reduction in the fusion property of the composite particles on compression. CONCLUSIONS: Although increasing the sodium alginate content of SD composite particles led to an increase in their plastic deformation, fusion on compression was prevented by the presence of sodium alginate. The reduced compactibility of SD composite particles with an excess amount of sodium alginate was attributed to reduced cohesion and fusion of the particles during compression.

Alginates↗
PubMed2003

Effect of polymer content and molecular weight on the morphology and heat- and moisture-induced transformations of spray-dried composite particles of amorphous lactose and poly(vinylpyrrolidone).

PURPOSE: The aim was to investigate the influence of polymer content and molecular weight on the morphology and heat- and moisture-induced transformations, as indicators of stability, of spray-dried composite particles of amorphous lactose and poly(vinylpyrrolidone) (PVP). METHODS: Amorphous lactose and composite particles of amorphous lactose with different contents and molecular weights of PVP were prepared by spray drying. The nanostructure of the particles was analyzed by x-ray powder diffractometry, the morphology by light microscopy and SEM, the glass transition temperatures (Tg), crystallization temperatures (Tc), heats of crystallization and melting temperatures by differential scanning calorimetry, and moisture-induced crystallizations gravimetrically and by microcalorimetry. RESULTS: All the types of particles prepared were amorphous. The Tg was unchanged or only marginally increased as a result of the inclusion of PVP. However, crystallization temperature, time to moisture-induced crystallization, and particle morphology were affected by both content and molecular weight of PVP. CONCLUSIONS: Increased content and molecular weight of PVP may have the potential to increase the physical stability of amorphous lactose. However, Tg seems not to be a relevant indicator for the stability of this type of amorphous composite materials.

Drug Stability↗