Continuous-flow Dialysis Research Articles

Ultrafast continuous flow-dialysis for nanoparticle-based drug delivery systems via microfluidic-multiple buffer injector

Nanoparticle-based drug delivery systems (NP-DDS) have emerged as promising carriers for bioactive organic molecules due to their potential in enhancing therapeutic effects. However, traditional purification methods like batch dialysis and diafiltration can alter NP-DDS bio-physicochemical properties due to the lengthy process, prolonged coexistence with organic solvents, and external forces. To address this challenge, we developed an ultrafast flow-dialysis microfluidic module with dual channels separated by a membrane layer. The module leverages multiple buffer injectors (MBIs) to accelerate dialysis by injecting fresh buffer into multiple dialysis zones of bottom channel, refreshing the concentration gradients over the mixture sample in the upper channel to maximize mass transfer efficiency. Using this method for a dual drug-loaded liposome sample containing doxorubicin and curcumin, impurities (such as ethanol and un-trapped drugs) removal and buffer exchange were rapidly terminated within 15 min, continuously operating for ∼ 6 hrs with sequential membrane regeneration process. Importantly, unlike traditional methods, the MBI process maintains the initial uniformity in the size and distribution of liposomes with no leakage of the encapsulated drugs. In vitro HeLa cell tests reveal that the liposomes purified by MBI exhibited significantly higher cellular uptake (1.13 ∼ 1.67 fold), presumably due to the small and uniform size of liposomes, leading to improved anti-tumor effects. Notably, the MBI platform’s versatility was further verified by purifying a mixture of laccase enzyme and by removing the used organic solvents from various carriers (e.g., lipid-based, polymeric, and protein nanoparticles). It is envisioned that the outstanding performance of ultrafast dialysis will enables a continuous-flow downstream process for diverse nano-carriers by intensifying with extra steps in the manufacturing of nanomedicnes, including targeted drug delivery and cancer therapy.

Biomass-based mixed matrix membrane adsorbers for removal of creatinine in dialysate fluid

Dialysate fluid is a vital component in hemodialysis therapy. Processing and reusing spent dialysate in hemodialysis significantly reduces costs and produces environmentally friendly hemodialysis. In this research, mixed matrix membrane adsorbers (MMMAs), which have the potential to recycle spent dialysate, have been developed. MMMA is composed of cellulose acetate (CA)-based polymers synthesized from cellulose isolated from oil palm empty fruit bunches (OPEFB) and integrated with silica (Si) bio-adsorbent from rice husk ash waste. The membrane was cast using the phase inversion method to form MMMAs CA-Si. The MMMAs CA-Si has a porous structure with evenly distributed silica particles. The pristine CA membranes had swelling and porosity degrees of 38.89 % and 21.23 %, while the MMMAs CA-Si had swelling and porosity degrees of 41.43 % and 27.81 %, respectively. The pristine CA membrane had a clean water flux of 86.86 L/m2h, while the MMMAs CA-Si membrane had a 96.25 L/m2h clean water flux. The MMMAs CA-Si has a tensile strength of 15.89 (Kgf/mm2) and an elongation of 14.01 %. The pristine CA membrane had a creatinine adsorption capacity of 2.8 mg/g, while the CA-Si MMMAs membrane had a maximum adsorption capacity of 9.98 mg/g. The maximum adsorption capacity (qm) is 9.98 mg/g MMMAs, equivalent to 24.95 mg creatinine/g silica in MMMAs and the dissociation constant (Kd) value is 1.91 mg/L. Creatinine can be removed from the dialysate through a continuous flow dialysis process using MMMAs CA-Si with a total removal of 10.48 mg/g or above 66.45 %. Creatinine can also be filtered by cross-flow circulation, and the total creatinine removal is above 81.0 %. MMMAs CA-Si can be an alternative separation medium to remove creatinine in the dialysate.

From monomer to micelle: a facile approach to the multi-step synthesis of block copolymers via inline purification.

A one-pass continuous flow strategy to form block copolymer nanoaggregates directly from monomers is presented. A key development towards such a sophisticated continuous flow setup is a significant improvement in continuous flow dialysis. Often impurities or solvent residues from polymerizations must be removed before block extensions or nanoaggregate formation can be carried out, typically disrupting the workflow. Hence, inline purification systems are required for fully continuous operation and eventual high throughput operation. An inline dialysis purification system is developed and exemplified for amphiphilic block copolymer synthesis from thermal and photoiniferter reversible addition fragmentation chain transfer (RAFT) polymerization. The inline dialysis system is found to be significantly faster than conventional batch dialysis and the kinetics are found to be very predictable with a diffusion velocity coefficient of 4.1 × 10-4 s-1. This is at least 4-5 times faster than conventional dialysis. Moreover, the newly developed setup uses only 57 mL of solvent for purification per gram of polymer, again reducing the required amount by almost an order of magnitude compared to conventional methods. Methyl methacrylate (MMA) or butyl acrylate (BA) was polymerized in a traditional flow reactor as the first block via RAFT polymerization, followed by a 'dialysis loop', which contains a custom-built inline dialysis device. Clearance of residual monomers is monitored via in-line NMR. The purified reaction mixture can then be chain extended in a second reactor stage to obtain block copolymers using poly(ethylene glycol) methyl ether acrylate (PEGMEA) as the second monomer. In the last step, nano-objects are created, again from flow processes. The process is highly tuneable, showing for the chosen model system a variation in nanoaggregate size from 34 nm to 188 nm.

Development and Validation of an in vitro Experimental GastroIntestinal Dialysis Model with Colon Phase to Study the Availability and Colonic Metabolisation of Polyphenolic Compounds.

The biological effects of polyphenols depend on their mechanism of action in the body. This is affected by bioconversion by colon microbiota and absorption of colonic metabolites. We developed and validated an in vitro continuous flow dialysis model with colon phase (GastroIntestinal dialysis model with colon phase) to study the gastrointestinal metabolism and absorption of phenolic food constituents. Chlorogenic acid was used as model compound. The physiological conditions during gastrointestinal digestion were mimicked. A continuous flow dialysis system simulated the one-way absorption by passive diffusion from lumen to mucosa. The colon phase was developed using pooled faecal suspensions. Several methodological aspects including implementation of an anaerobic environment, adapted Wilkins Chalgren broth medium, 1.10(8) CFU/mL bacteria suspension as inoculum, pH adaptation to 5.8 and implementation of the dialysis system were conducted. Validation of the GastroIntestinal dialysis model with colon phase system showed a good recovery and precision (CV < 16 %). Availability of chlorogenic acid in the small intestinal phase (37 ± 3 %) of the GastroIntestinal dialysis model with colon phase is comparable with in vivo studies on ileostomy patients. In the colon phase, the human faecal microbiota deconjugated chlorogenic acid to caffeic acid, 3,4-dihydroxyphenyl propionic acid, 4-hydroxybenzoic acid, 3- or 4-hydroxyphenyl acetic acid, 2-methoxy-4-methylphenol and 3-phenylpropionic acid. The GastroIntestinal dialysis model with colon phase is a new, reliable gastrointestinal simulation system. It permits a fast and easy way to predict the availability of complex secondary metabolites, and to detect metabolites in an early stage after digestion. Isolation and identification of these metabolites may be used as references for in vivo bioavailability experiments and for investigating their bioactivity in in vitro experiments.

Bioaccessibility of ochratoxin A from red wine in anin vitro dynamic gastrointestinal model

Ochratoxin A (OTA) is a mycotoxin produced byAspergillus andPenicillium species with immunosuppressive, teratogenic, and carcinogenic properties. It has been determined that wine is the second largest source of OTA (10% of total OTA intake) in the European diet and that its presence, even in small doses, can be a problem in terms of long-term toxicity. In this paper, we evaluated the bioaccessibility of OTA in a spiked red wine sample under human fasting conditions using anin vitro dynamic digestion model that includes a continuous-flow dialysis system to simulate intestinal passage. To the best of our knowledge, this report is the first examining the bioaccessibility of OTA in wine. A liquid-liquid method was used to extract the OTA and ochratoxin alpha (OTα) from gastrointestinal juices, and the extracts were analysed by HPLC with a fluorescence detector. The bioaccessibility of OTA from the spiked red wine (1.0, 2.0 and 4 μg/l) was high in the gastric compartment (102.8, 128.3 and 122.3%, respectively), whereas in the simulated intestine, it did not exceed 26%, and the amount of OTA that crossed the dialysis membrane was very low (&lt;3.3%). The amount of OTα in gastric chyme ranged from 5.1 to 19.1% of the spiked OTA, whereas in the intestinal compartment it did not exceed 5%. In conclusion, in thein vitro system assayed, OTA exhibited a high bioaccessibility in the simulated stomach, but it decreased after the intestinal digestion and passage through the dialysis membrane.

Evaluation of protein disulfide conversion in vitro using a continuous flow dialysis system

Recombinant therapeutic proteins are heterogeneous due to chemical and physical modifications. Understanding the impact of these modifications on drug safety and efficacy is critical for optimal process development and for setting reasonable specification limits. In this study, we describe the development of an in vitro continuous flow dialysis system to evaluate potential in vivo behavior of thiol adducted species and incorrectly disulfide bonded species of therapeutic proteins. The system is capable of maintaining the low-level cysteine concentrations found in human blood. Liabilities of cysteamine adducted species, incorrectly disulfide bonded species, and the correctly disulfide bonded form of an Fc-fusion protein were studied using this system. Results showed that 90% of the cysteamine adduct converted into the correctly disulfide bonded form and incorrectly disulfide bonded species in approximately 4h under physiological conditions. Approximately 50% of incorrectly disulfide bonded species converted into the correctly bonded form in 2days. These results provide valuable information on potential in vivo stability of the cysteamine adduct, incorrectly disulfide bonded species, and the correctly bonded form of the Fc-fusion protein. These are important considerations when evaluating the criticality of product quality attributes.

Inductively coupled plasma mass spectrometry with a continuous-flow dialysis simulated gastrointestinal digestion for study of arsenic bioaccessibility in shrimp

A hyphenated method of continuous-flow dialysis and on-line inductively coupled plasma mass spectrometry (ICP-MS), with and without the use of hydride generation sample introduction, was developed for the study of arsenic bioaccessibility in shrimp. The method was based on a simulated gastric digestion in a batch system followed by a continuous-flow intestinal digestion. The simulated intestinal digestion was performed in a dialysis bag placed inside a channel in a flowing stream of dialyzing solution (NaHCO 3). The pH of the dialysate was monitored on-line to ensure pH changes similar to the situation in the gastrointestinal tract. The concentrations of total arsenic and inorganic arsenic in the dialysate were determined by ICP-MS without and with hydride generation, respectively. With the parameters used in the hydride generation as follows: 8% (v/v) of HCl; 1% (m/v) of NaBH 4; and a pre-reduction solution (10% (w/v) KI in 5% (w/v) ascorbic acid), only inorganic arsenic can form arsine, implying that hydride generation-ICP-MS can be used to detect inorganic arsenic only. The method was applied for the estimation of arsenic bioaccessibility in shrimp samples, by which the bioaccessibility from shrimp was found to be approximately 55%, contributed from inorganic arsenic only 1% or 2%, and mostly from organic arsenic. Further, the effect of ascorbic acid and fruit juices on arsenic bioaccessibility was investigated which was found not to cause any significant changes in bioaccessibility.

In vitro bioaccessibility of intrinsically zinc-enriched egg and effect of cooking

Adequate dietary zinc is essential for appropriate growth, neurological development, and immune function in all age groups, especially in children. Eggs are a potentially attractive means of delivering dietary zinc. This study used an in vitro continuous-flow dialysis (CFD) method to determine the effect of cooking (boiling or frying) on bioaccessibility of zinc from eggs which had been biofortified in vivo. The intrinsically zinc-enriched eggs with the highest level of zinc (18 μg/g) provided the highest available zinc at 75%. There was no statistically significant difference in the bioaccessibility of zinc from eggs enriched with inorganic zinc versus enrichment with organic zinc. The intrinsically zinc-enriched eggs from hens fed both organic and inorganic forms with the maximum available zinc (about 13.5 μg zinc/g) were selected to study the effect of cooking. Boiling and frying did not have any adverse effect on zinc bioaccessibility in the eggs intrinsically enriched with inorganic zinc. However, boiling reduced by about 6% and frying reduced by about 10% the bioaccessible zinc in the control and eggs intrinsically enriched with organic zinc. One intrinsically enriched egg could provide 142% of the daily zinc requirement for a child aged one year.

5-Fluorouracil-Loaded BSA Nanoparticles: Formulation Optimization and In Vitro Release Study

Over the past few decades, there has been considerable interest in developing protein nanoparticles as drug delivery devices. The underlying rationale is their exceptional characteristics, namely biodegradability and nonantigenicity. Herein, phase separation method was used to prepare 5-fluorouracil-loaded bovine serum albumin (BSA) nanoparticles. Drug release was tracked by continuous flow dialysis technique. Effect of process variables on loading efficiency of 5-fluorouracil was investigated and optimized through Taguchi's M16 design with the amount of entrapped drug as response. Optimum condition was found to be 2 mg/mL of 5-fluorouracil, 3.7 mL of added ethanol, 176 microL of glutaraldehyde, drug-protein incubation time of 30 min, and pH of 8.4 for 200 mg of BSA in 2 mL drug solution. pH had the most noticeable effect on the amount of entrapped drug, but glutaraldehyde had the least. Mean diameter and zeta potential of fabricated nanoparticles under these conditions were 210 nm and -31.7 mV, respectively. Drug-loaded BSA nanoparticles suspension maintained constant release of drug for 20 h under experimental conditions, so this colloidal drug carrier is capable of releasing drug in a sustained manner.

Towards better understanding of in vitro bioavailability of iron through the use of dialysis profiles from a continuous-flow dialysis with inductively coupled plasma spectrometric detection

A continuous-flow dialysis (CFD) method with an on-line inductively coupled plasma optical emission spectrometric (ICP-OES) simultaneous multielement measurement for the study of in vitro mineral bioavailability was previously reported. The method was based on a simulated gastric digestion in a batch system followed by a continuous-flow intestinal digestion—dialysis with on-line measurement of dialysed mineral concentration by ICP-OES. This study demonstrates how the dialysis profiles obtained could be exploited to understand differences of mineral dialysability and the effect of enhancers and inhibitors. The graphical plot of time-dependent cumulative dialysed mineral concentrations and percent dialysis was efficiently used for these purposes. Iron fortificants in various chemical forms were used to demonstrate the effect of their anionic parts on dialysability together with enhancement and inhibition effects from food acids.

Enhancement Effect Study of Some Organic Acids on the Calcium Availability of Vegetables: Application of the Dynamic in Vitro Simulated Gastrointestinal Digestion Method with Continuous-Flow Dialysis

The effect of added organic acids on the calcium availability of vegetables was investigated using the dialysis profiles obtained from an in vitro simulated gastrointestinal digestion with continuous-flow dialysis method. Citric acid was the most effective enhancer followed by tartaric, malic, and ascorbic acids. For amaranth, which has a low calcium availability (5.4%), a significant increase of availability was observed with increasing concentrations of all acids studied. With the continuous-flow dialysis approach, organic acids could be observed to promote the dialyzability even at an elevated intestinal pH. An enhancement effect from added organic acids was not clearly observed for Chinese kale, which itself contains a high amount of available calcium (52.9%).

Dynamic continuous-flow dialysis method to simulate intestinal digestion for in vitro estimation of mineral bioavailability of food

A system for dynamic continuous-flow dialysis during intestinal digestion for an in vitro simulation of gastrointestinal digestion is presented as an alternative to human and animal in vivo methods for estimation of the bioavailability of minerals. The method is based on the in vitro batch dialysis method described by Miller, which was developed into a continuous-flow system of a simple design to perform dynamic dialysis in the intestinal digestion stage. A flow dialysis system has the advantages of simulation being close to in vivo physiological conditions because pH change during dialysis is gradual and dialyzed components are continuously removed. The proposed new design performed dialysis during a continuous flow of dialyzing solution (NaHCO 3) around a dialysis bag containing peptic digest, which is placed inside a glass dialysis chamber. Gradual change of dialysis pH, similar to that occurring in the gastrointestinal tract, was obtained by optimization of flow rate and concentration of NaHCO 3. The dialysate collected in fractions was analyzed to determine dialyzed minerals and pH change in the course of dialysis. The method was tested by determination of calcium bioavailability of powder milk and calcium carbonate tablets.

A dynamic continuous-flow dialysis system with on-line electrothermal atomic-absorption spectrometric and pH measurements for in-vitro determination of iron bioavailability by simulated gastrointestinal digestion

A dynamic continuous-flow dialysis (CFD) method with on-line electrothermal atomic absorption spectrometric (ETAAS) and pH measurements for study of simulated gastrointestinal digestion has been developed for prediction of iron bioavailability. The method used to estimate mineral bioavailability was based on gastric digestion in a batch system then dynamic continuous-flow intestinal digestion. The intestinal digestion was performed in a dialysis bag placed inside a chamber containing a flowing stream of dialyzing solution. Mineral concentration and dialysate pH were monitored by ETAAS and use of a pH meter, respectively. The amount of dialyzed minerals in the intestinal digestion stage was used to evaluate the dialyzability. The dialysis profile and pH change can be used to understand or examine differences between the dialyzability of different food samples. To test the proposed system it was used to estimate the iron dialyzability of different kinds of milk. Iron dialyzability for powdered cow milk, cereal milk, and two brands of soymilk was found to be 1.7, 20.4, 24.9, and 37.7%, respectively. The developed CFD-ETAAS-pH system is a simple, rapid, and inexpensive tool for bioavailability studies, especially for minerals at ultratrace levels.

A continuous-flow dialysis system with inductively coupled plasma optical emission spectrometry for in vitro estimation of bioavailability

A continuous-flow dialysis (CFD) method with on-line inductively coupled plasma optical emission spectrometric (ICP-OES) simultaneous multielement measurement for the study of in vitro mineral bioavailability was developed. The method was based on a simulated gastric digestion in a batch system followed by a continuous-flow intestinal digestion. The simulated intestinal digestion was performed in a dialysis bag placed inside a channel in a flowing stream of dialyzing solution (NaHCO3). The mineral concentrations in the dialysate were determined by ICP-OES using Y and Sc as internal standards. The pH of the dialysate was also monitored on-line to ensure pH changes similar to the situation in the gastrointestinal tract.The developed system was applied to determining the dialysability of five essential elements (Ca, Mg, P, Fe, Zn) for various kinds of foods, i.e., milk-based infant formula reference material (NIST SRM 1846), milk powder, kale, mungbean, chicken meat, jasmine rice, and Acacia pennata. The dialysis profiles of elements and pH change profiles can be useful in understanding the dialysis change and factors affecting dialysability. All studied elements were rapidly dialysed in the first 30 min of simulated intestinal digestion. It is expected that this system will be useful for estimation of dialysability and for studying the mutual effects of components in food.

Dissolved Organic Nitrogen Measurement Using Dialysis Pretreatment

Dissolved organic nitrogen (DON) is important for ecological and engineering researches. Quantification of low DON concentrations in waters with elevated dissolved inorganic nitrogen (DIN) using existing methods is inaccurate. In this study, a dialysis-based pretreatment technique was optimized and adopted to reduce the interference from DIN to the quantification of DON in natural water. A cellulose ester dialysis tube (nominal molecular weight cutoff = 100 Da) was used in batch and continuous-flow dialysis steps with model compounds, natural organic matter isolates, and bulk waters to develop a dialysis pretreatment approach that selectively reduces DIN from solutions containing DON. By reducing DIN concentrations, propagation of analytical variance in total dissolved nitrogen (TDN) and DIN species concentrations allows more accurate determination of DON (DON = TDN - NO3 - NO2- - NH3/NH4+). Dialysis for 24 h against continuously flowing distilled water reduced DIN species by 70%. With dialysis pretreatment, DON recoveries of more than 95% were obtained for surface water and finished drinking water, but wastewater experienced a slight loss (approximately 10%) of DON possibly due to the adsorption of organics onto the dialysis membrane, permeation of low molecular weight fractions, or biodegradation. Dialysis experiments using surface water spiked with different DIN/TDN ratios concluded that dialysis pretreatment leads to more accurate DON determination than no dialysis when DIN/TDN ratios exceed 0.6 mg of N/mg of N.

Do thickening properties of locust bean gum affect the amount of calcium, iron and zinc available for absorption from infant formula? In vitro studies

Locust bean gum acts as a milk thickener in infant formula because of its high apparent viscosity. The effects of such thickening agents on metabolic and physiologic responses during infancy have not been clarified sufficiently. Due to the increased volume of the digest and the bulking and trapping effects, digestion and absorption of nutrients may be influenced in presence of locust bean gum. The central question addressed in this paper is whether the thickening properties of locust bean gum affect the availability of calcium, iron, and zinc. Increasing amounts of powdered locust bean gum were homogenised with infant formula and samples were diluted to 0.14, 0.27, 0.42 and 0.71 g/100 ml. Viscosity of the samples was measured by a Carrie-Med CSL100 rheometer. Available amounts of calcium, iron, and zinc were evaluated using a continuous-flow dialysis model with preliminary digestion. Elemental contents of samples and dialysates were analysed with atomic absorption spectrometry. The first set of experiments showed that addition of locust bean gum to infant formulas increased the viscosity of the luminal contents. Correlations between the locust bean gum concentration and the viscosity of the samples before and after gastric or intestinal digestion were highly significant (0.97). In the second set of experiments, the correlations between the locust bean gum concentration and the amounts of calcium trapped by the locust bean gum fraction also showed high significance (0.93). In the third experimental design, again strong correlations were found between the viscosity of the intestinal digest and the amounts of calcium trapped by the fibre fraction (0.90). For iron and zinc, no such relationships were found. From this experimental set-up it appears that locust bean gum influences calcium availability in infant formulas by means of its physical properties to act as thickening agent, rather than its chemical ability to form complexes as demonstrated earlier with respect to iron and zinc.

Calcium, iron and zinc availability from weaning meals.

During the weaning period, solid foods are introduced in the diet to complement human milk or infant formula. Consequently, marginal deficiencies may easily develop because of inappropriate feeding and low bioavailability of nutrients. The objective of this study was to determine calcium, iron and zinc availability from weaning foods that contained a variety of ingredients but almost the same dietary fibre content, and to describe dietary factors that influence availability in a linear matter. Availability measurements from eight meals were made in vitro by a continuous-flow dialysis system with preliminary intraluminal digestion. Calcium, iron and zinc were analysed by flame atomic absorption spectrometry; ascorbic acid content was taken from food-composition tables; and macronutrient and mineral contents were obtained from the manufacturer. Calcium availability was higher in fruits (mean +/- SD, 39.2 +/- 4.1%) than vegetables (31.7 +/- 5.8%) (p < or = 0.001). Iron availability was marginally significant higher in vegetables (13.0 +/- 4.7%) than fruits (10.2 +/- 1.6%) (p = 0.027). Zinc availability showed higher availability in fruits (52.3 +/- 22.3%) than vegetables (22.1 +/- 5.6%) (p < or = 0.001). A major portion (47.8%) of the variance in calcium availability, as determined by multiple regression, could be explained by protein (p = 0.058, beta = -0.691). Major predictors of iron availability among the nutrients were iron content of the meal (p = 0.014, beta = -0.838), carbohydrates (p = 0.044, beta = -0.610) and dietary fibre (p = 0.235, beta = -0.285). Zinc availability was merely influenced by the protein (p = 0.010, beta = -0.876) and fat content of the meals (p = 0.295, beta = 0.255) in relation to other nutrients.

Calcium, iron and zinc availability from weaning meals

During the weaning period, solid foods are introduced in the diet to complement human milk or infant formula. Consequently, marginal deficiencies may easily develop because of inappropriate feeding and low bioavailability of nutrients. The objective of this study was to determine calcium, iron and zinc availability from weaning foods that contained a variety of ingredients but almost the same dietary fibre content, and to describe dietary factors that influence availability in a linear matter. Availability measurements from eight meals were made in vitro by a continuous‐flow dialysis system with preliminary intraluminal digestion. Calcium, iron and zinc were analysed by flame atomic absorption spectrometry; ascorbic acid content was taken from food‐composition tables; and macronutrient and mineral contents were obtained from the manufacturer. Calcium availability was higher in fruits (mean ± SD, 39.2 ± 4.1%) than vegetables (31.7 ±5.8%) (p 0.001). Iron availability was marginally significant higher in vegetables (13.0 ±4.7%) than fruits (10.2 ± 1.6%) (p= 0.027). Zinc availability showed higher availability in fruits (52.3 ± 22.3%) than vegetables (22.1 ± 5.6%) (p± 0.001). Conclusion: A major portion (47.8%) of the variance in calcium availability, as determined by multiple regression, could be explained by protein (p= 0.058, β=–0.691). Major predictors of iron availability among the nutrients were iron content of the meal (p= 0.014, β=–0.838), carbohydrates (p= 0.044, β=–0.610) and dietary fibre (p= 0.235, β=–0.285). Zinc availability was merely influenced by the protein (p= 0.010, β=–0.876) and fat content of the meals (p= 0.295, β= 0.255) in relation to other nutrients.

A method for in vitro determination of calcium, iron and zinc availability from first-age infant formula and human milk.

A method for in vitro determination of available calcium, iron and zinc content from infant food after digestion was evaluated. This method introduced an intraluminal digestive phase, adapted to the gastrointestinal conditions of infants younger than 6 months of age, prior to continuous flow dialysis of the resultant gastric digest. Precautions handling the method were discussed and enzymatic parameters were defined. Ruggedness of the method was determined from the availability of calcium, iron and zinc at different gastrointestinal conditions. Availability of all three elements was higher at gastric pH of 2 (20.0 +/- 1.1% for calcium, 4.06 +/- 0.66% for iron and 17.5 +/- 1.3% for zinc), than from the normal procedure (pH 4) (15.6 +/- 1.2% for calcium, 1.18 +/- 0.26% for iron and 8.2 +/- 0.9% for zinc). At pH 5, however, calcium availability appeared to be lower (11.7 +/- 1.0%) (P < 0.05). The intestinal pH also had a major influence on the availability. At low intestinal pH (5.5), availability was 40.5 +/- 2.3% for calcium, 3.01 +/- 0.58% for iron and 26.8 +/- 1.8% for zinc, which was higher compared with the normal procedure (P < 0.05). Moreover, other factors, such as digestion time, mixing and filtration pressure, also affected the availability. Recovery tests yielded mean values of 94 +/- 3% for calcium, 109 +/- 9% for iron and 106 +/- 4% for zinc. Mean intra- and inter-batch precision of the availability procedure was 4.1 CV% and 6.6 CV% for calcium, 14.5 CV% and 19.2 CV% for iron, and 4.0 CV% and 13.6 CV% for zinc. The method provides adequate accuracy, acceptable precision and good recovery. It offers the advantage of being simple, rapid and inexpensive, since it takes only 1 day to run the whole availability procedure (including four replicates per sample), and the low costs of the dialysis equipment. It can therefore be considered as suitable for predicting the availability of essential elements from foods used during the first months of infancy.

A method for in vitro determination of calcium, iron and zinc availability from first-age infant formula and human milk

A method for in vitro determination of available calcium, iron and zinc content from infant food after digestion was evaluated. This method introduced an intraluminal digestive phase, adapted to the gastrointestinal conditions of infants younger than 6 months of age, prior to continuous flow dialysis of the resultant gastric digest. Precautions handling the method were discussed and enzymatic parameters were defined. Ruggedness of the method was determined from the availability of calcium, iron and zinc at different gastrointestinal conditions. Availability of all three elements was higher at gastric pH of 2 (20.0 ± 1.1% for calcium, 4.06 ± 0.66% for iron and 17.5 ± 1.3% for zinc), than from the normal procedure (pH 4) (15.6 ± 1.2% for calcium, 1.18 ± 0.26% for iron and 8.2 ± 0.9% for zinc). At pH 5, however, calcium availability appeared to be lower (11.7 ± 1.0%) (P < 0.05). The intestinal pH also had a major influence on the availability. At low intestinal pH (5.5), availability was 40.5 ± 2.3% for calcium, 3.01 ± 0.58% for iron and 26.8 ± 1.8% for zinc, which was higher compared with the normal procedure (P < 0.05). Moreover, other factors, such as digestion time, mixing and filtration pressure, also affected the availability. Recovery tests yielded mean values of 94 ± 3% for calcium, 109 ± 9% for iron and 106 ± 4% for zinc. Mean intra- and inter-batch precision of the availability procedure was 4.1 CV% and 6.6 CV% for calcium, 14.5 CV% and 19.2 CV% for iron, and 4.0 CV% and 13.6 CV% for zinc. The method provides adequate accuracy, acceptable precision and good recovery. It offers the advantage of being simple, rapid and inexpensive, since it takes only 1 day to run the whole availability procedure (including four replicates per sample), and the low costs of the dialysis equipment. It can therefore be considered as suitable for predicting the availability of essential elements from foods used during the first months of infancy.