Maaike J. Bruins

Consumption of gluten with gluten-degrading enzyme by celiac patients: a pilot-study.

AIM To assesses the safety and efficacy of Aspergillus niger prolyl endoprotease (AN-PEP) to mitigate the immunogenic effects of gluten in celiac patients. METHODS Patients with initial diagnosis of celiac disease as confirmed by positive serology with subtotal or total villous atrophy on duodenal biopsies who adhere to a strict gluten-free diet (GFD) resulting in normalised antibodies and mucosal healing classified as Marsh 0 or I were included. In a randomised double-blind placebo-controlled pilot study, patients consumed toast (approximately 7 g/d gluten) with AN-PEP for 2 wk (safety phase). After a 2-wk washout period with adherence of the usual GFD, 14 patients were randomised to gluten intake with either AN-PEP or placebo for 2 wk (efficacy phase). Measurements at baseline included complaints, quality-of-life, serum antibodies, immunophenotyping of T-cells and duodenal mucosa immunohistology. Furthermore, serum and quality of life questionnaires were collected during and after the safety, washout and efficacy phase. Duodenal biopsies were collected after the safety phase and after the efficacy phase. A change in histological evaluation according to the modified Marsh classification was the primary endpoint. RESULTS In total, 16 adults were enrolled in the study. No serious adverse events occurred during the trial and no patients withdrew during the trial. The mean score for the gastrointestinal subcategory of the celiac disease quality (CDQ) was relatively high throughout the study, indicating that AN-PEP was well tolerated. In the efficacy phase, the CDQ scores of patients consuming gluten with placebo or gluten with AN-PEP did not significantly deteriorate and moreover no differences between the groups were observed. During the efficacy phase, neither the placebo nor the AN-PEP group developed significant antibody titers. The IgA-EM concentrations remained negative in both groups. Two patients were excluded from entering the efficacy phase as their mucosa showed an increase of two Marsh steps after the safety phase, yet with undetectable serum antibodies, while 14 patients were considered histologically stable on gluten with AN-PEP. Also after the efficacy phase, no significant deterioration was observed regarding immunohistological and flow cytometric evaluation in the group consuming placebo compared to the group receiving AN-PEP. Furthermore, IgA-tTG deposit staining increased after 2 wk of gluten compared to baseline in four out of seven patients on placebo. In the seven patients receiving AN-PEP, one patient showed increased and one showed decreased IgA-tTG deposits. CONCLUSION AN-PEP appears to be well tolerated. However, the primary endpoint was not met due to lack of clinical deterioration upon placebo, impeding an effect of AN-PEP.

Dietary Arachidonic Acid Dose-Dependently Increases the Arachidonic Acid Concentration in Human Milk

Lactation hampers normalization of the maternal arachidonic acid (AA) status, which is reduced after pregnancy and can further decline by the presently recommended increased consumption of (n-3) long-chain PUFA [(n-3) LCPUFA]. This may be unfavorable for breast-fed infants, because they also require an optimum supply of (n-6) LCPUFA. We therefore investigated the LCPUFA responses in nursing mothers upon increased consumption of AA and (n-3) LCPUFA. In a parallel, double-blind, controlled trial, lactating women received for 8 wk no extra LCPUFA (control group, n = 8), 200 (low AA group, n = 9), or 400 (high AA group, n = 8) mg/d AA in combination with (n-3) LCPUFA [320 mg/d docosahexaenoic acid (DHA), 80 mg/d eicosapentaenoic acid, and 80 mg/d other (n-3) fatty acids], or this dose of (n-3) LCPUFA alone [DHA + eicosapentaenoic acid group, n = 8]. Relative concentrations of AA, DHA, and sums of (n-6) and (n-3) LCPUFA were measured in milk total lipids (TL) and erythrocyte phospholipids (PL) after 2 and 8 wk and changes were compared by ANCOVA. The combined consumption of AA and (n-3) LCPUFA caused dose-dependent elevations of AA and total (n-6) LCPUFA concentrations in milk TL and did not significantly affect the DHA and total (n-3) LCPUFA increases caused by (n-3) LCPUFA supplementation only. This latter treatment did not significantly affect breast milk AA and total (n-6) LCPUFA concentrations. AA and DHA concentrations in milk TL and their changes were strongly and positively correlated with their corresponding values in erythrocyte PL (r(2) = 0.27-0.50; P </= 0.002). We thus concluded that the consumption by lactating women of AA in addition to extra (n-3) LCPUFA dose dependently increased the AA concentration of their milk TL.

Randomised clinical study: Aspergillus niger-derived enzyme digests gluten in the stomach of healthy volunteers.

Aspergillus niger prolyl endoprotease (AN‐PEP) efficiently degrades gluten molecules into non‐immunogenic peptides in vitro.

Bioavailability of iron, zinc, folic acid, and vitamin A from fortified maize.

Several strategies appear suitable to improve iron and zinc bioavailability from fortified maize, and fortification per se will increase the intake of bioavailable iron and zinc. Corn masa flour or whole maize should be fortified with sodium iron ethylenediaminetetraacetate (NaFeEDTA), ferrous fumarate, or ferrous sulfate, and degermed corn flour should be fortified with ferrous sulfate or ferrous fumarate. The choice of zinc fortificant appears to have a limited impact on zinc bioavailability. Phytic acid is a major inhibitor of both iron and zinc absorption. Degermination at the mill will reduce phytic acid content, and degermed maize appears to be a suitable vehicle for iron and zinc fortification. Enzymatic phytate degradation may be a suitable home‐based technique to enhance the bioavailability of iron and zinc from fortified maize. Bioavailability experiments with low phytic acid–containing maize varieties have suggested an improved zinc bioavailability compared to wild‐type counterparts. The bioavailability of folic acid from maize porridge was reported to be slightly higher than from baked wheat bread. The bioavailability of vitamin A provided as encapsulated retinyl esters is generally high and is typically not strongly influenced by the food matrix, but has not been fully investigated in maize.

Plasma oxylipin profiling identifies polyunsaturated vicinal diols as responsive to arachidonic acid and docosahexaenoic acid intake in growing piglets

The dose-responsiveness of plasma oxylipins to incremental dietary intake of arachidonic acid (20:4n-6; ARA) and docosahexaenoic acid (22:6n-3; DHA) was determined in piglets. Piglets randomly received one of six formulas (n = 8 per group) from days 3 to 27 postnatally. Diets contained incremental ARA or incremental DHA levels as follows (% fatty acid, ARA/DHA): (A1) 0.1/1.0; (A2) 0.53/1.0; (A3–D3) 0.69/1.0; (A4) 1.1/1.0; (D1) 0.66/0.33; and (D2) 0.67/0.62, resulting in incremental intake (g/kg BW/day) of ARA: 0.07 ± 0.01, 0.43 ± 0.03, 0.55 ± 0.03, and 0.82 ± 0.05 at constant DHA intake (0.82 ± 0.05), or incremental intake of DHA: 0.27 ± 0.02, 0.49 ± 0.03, and 0.81 ± 0.05 at constant ARA intake (0.54 ± 0.04). Plasma oxylipin concentrations and free plasma PUFA levels were determined at day 28 using LC-MS/MS. Incremental dietary ARA intake dose-dependently increased plasma ARA levels. In parallel, ARA intake dose-dependently increased ARA-derived diols 5,6- and 14,15-dihydroxyeicosatrienoic acid (DiHETrE) and linoleic acid-derived 12,13-dihydroxyoctadecenoic acid (DiHOME), downstream metabolites of cytochrome P450 expoxygenase (CYP). The ARA epoxide products from CYP are important in vascular homeostatic maintenance. Incremental DHA intake increased plasma DHA and most markedly raised the eicosapentaenoic acid (EPA) metabolite 17,18-dihydroxyeicosatetraenoic acid (DiHETE) and the DHA metabolite 19,20-dihydroxydocosapentaenoic acid (DiHDPE). In conclusion, increasing ARA and DHA intake dose-dependently influenced endogenous n-6 and n-3 oxylipin plasma concentrations in growing piglets, although the biological relevance of these findings remains to be determined.

Influence of dietary components on Aspergillus niger prolyl endoprotease mediated gluten degradation.

Celiac disease (CD) is caused by intolerance to gluten. Oral supplementation with enzymes like Aspergillus niger propyl-endoprotease (AN-PEP), which can hydrolyse gluten, has been proposed to prevent the harmful effects of ingestion of gluten. The influence of meal composition on AN-PEP activity was investigated using an in vitro model that simulates stomach-like conditions. AN-PEP optimal dosage was 20 proline protease units (PPU)/g gluten. The addition of a carbonated drink strongly enhanced AN-PEP activity because of its acidifying effect. While fat did not affect gluten degradation by AN-PEP, the presence of food proteins slowed down gluten detoxification. Moreover, raw gluten was degraded more efficiently by AN-PEP than baked gluten. We conclude that the meal composition influences the amount of AN-PEP needed for gluten elimination. Therefore, AN-PEP should not be used to replace a gluten free diet, but rather to support digestion of occasional and/or inadvertent gluten consumption.

Addressing the risk of inadequate and excessive micronutrient intakes: traditional versus new approaches to setting adequate and safe micronutrient levels in foods

Fortification of foods consumed by the general population or specific food products or supplements designed to be consumed by vulnerable target groups is amongst the strategies in developing countries to address micronutrient deficiencies. Any strategy aimed at dietary change needs careful consideration, ensuring the needs of at-risk subgroups are met whilst ensuring safety within the general population. This paper reviews the key principles of two main assessment approaches that may assist developing countries in deciding on effective and safe micronutrient levels in foods or special products designed to address micronutrient deficiencies, that is, the cut-point method and the stepwise approach to risk–benefit assessment. In the first approach, the goal is to shift population intake distributions such that intake prevalences below the Estimated Average Requirement (EAR) and above the Tolerable Upper Intake Level (UL) are both minimized. However, for some micronutrients like vitamin A and zinc, a narrow margin between the EAR and UL exists. Increasing their intakes through mass fortification may pose a dilemma; not permitting the UL to be exceeded provides assurance about the safety within the population but can potentially leave a proportion of the target population with unmet needs, or vice versa. Risk–benefit approaches assist in decision making at different micronutrient intake scenarios by balancing the magnitude of potential health benefits of reducing inadequate intakes against health risks of excessive intakes. Risk–benefit approaches consider different aspects of health risk including severity and number of people affected. This approach reduces the uncertainty for policy makers as compared to classic cut-point methods.

Prenatal arachidonic acid exposure and selected immune-related variables in childhood.

Arachidonic acid (AA) is considered essential in fetal development and some of its metabolites are thought to be important mediators of the immune responses. Therefore, we studied whether prenatal exposure to AA is associated with some immune-related clinical conditions and plasma markers in childhood. In 280 children aged 7 years, atopy, lung function and plasma inflammation markers were measured and their relationships with early AA exposure were studied by linear and logistic regression analyses. AA exposure was deduced from AA concentrations in plasma phospholipids of the mothers collected at several time points during pregnancy and at delivery, and in umbilical cord plasma and arterial and venous wall phospholipids. In unadjusted regression analyses, significant positive associations were observed between maternal AA concentrations at 16 and 32 weeks of pregnancy (proxies for fetal AA exposure) and peak expiratory flow decline after maximal physical exercise and plasma fibrinogen concentrations of their children, respectively. However, after correction for relevant covariables, only trends remained. A significant negative relationship was observed between AA concentrations in cord plasma (reflecting prenatal AA exposure) and the average daily amplitude of peak expiratory flow at rest, which lost significance after appropriate adjustment. Because of these few, weak and inconsistent relationships, a major impact of early-life exposure to AA on atopy, lung function and selected plasma inflammation markers of children at 7 years of age seems unlikely.

Maximizing the benefits and minimizing the risks of intervention programs to address micronutrient malnutrition: symposium report

Abstract Interventions to address micronutrient deficiencies have large potential to reduce the related disease and economic burden. However, the potential risks of excessive micronutrient intakes are often not well determined. During the Global Summit on Food Fortification, 9–11 September 2015, in Arusha, a symposium was organized on micronutrient risk–benefit assessments. Using case studies on folic acid, iodine and vitamin A, the presenters discussed how to maximize the benefits and minimize the risks of intervention programs to address micronutrient malnutrition. Pre‐implementation assessment of dietary intake, and/or biomarkers of micronutrient exposure, status and morbidity/mortality is critical in identifying the population segments at risk of inadequate and excessive intake. Dietary intake models allow to predict the effect of micronutrient interventions and their combinations, e.g. fortified food and supplements, on the proportion of the population with intakes below adequate and above safe thresholds. Continuous monitoring of micronutrient intake and biomarkers is critical to identify whether the target population is actually reached, whether subgroups receive excessive amounts, and inform program adjustments. However, the relation between regular high intake and adverse health consequences is neither well understood for many micronutrients, nor do biomarkers exist that can detect them. More accurate and reliable biomarkers predictive of micronutrient exposure, status and function are needed to ensure effective and safe intake ranges for vulnerable population groups such as young children and pregnant women. Modelling tools that integrate information on program coverage, dietary intake distribution and biomarkers will further enable program makers to design effective, efficient and safe programs.

Symposium report: effective and safe micronutrient interventions, weighing the risks against the benefits.

Micronutrient fortification of staple foods can be an effective strategy to combat micronutrient malnutrition. When planning on fortification, challenges faced include the collection of essential information on population food and nutrient intake patterns, as well as the use of this information in a method to select appropriate fortification levels. A symposium was organized aimed at discussing the existing approaches to set effective and safe micronutrient fortification levels and to outline the challenges and needs in this area. Two different approaches to establish effective and safe fortification levels for food fortification were presented. In the first approach, the Estimated Average Requirement (EAR) and Tolerable Upper Intake Level (UL) are used as cut-points in the micronutrient intake distribution to evaluate and simulate effective and safe micronutrient intakes. Review Article Bielderman et al.; EJNFS, 5(4): 202-228, 2015; Article no.EJNFS.2015.019 203 This was exemplified by challenges encountered in Guatemala and Cameroon towards unequal vitamin A intake distribution and the impact of the food vehicle choice. Secondly, the risk-benefit approach was presented as an approach in which risks and benefits of micronutrient intakes can be quantified and balanced in order to optimize fortification benefits with the least risks and to allow decision making. This was illustrated by a case on folic acid fortification in The Netherlands. Irrespective of the approach, food and nutrient intake data are required to identify potential vehicles for fortification, quantify the nutrient gap to be addressed, and set the appropriate level of fortification based on consumption pattern. Such information is rarely available to the quality and extent ideal to set fortification levels and requires regular updating, as exemplified in the case of sugar fortification in Guatemala. While the EAR cut-point method can be used to determine the proportion of the population meeting their required and safe nutrient intakes and set goals, riskbenefit assessment may offer an answer to commonly-asked questions as to whether, and at which levels, the benefits of increasing micronutrient intakes outweigh the risks.