Methods in Nutrition & Gut Microbiome Research: An American Society for Nutrition Satellite Session [13 October 2022].

The microbial cells colonizing the human body form an ecosystem that is integral to the regulation and maintenance of human health. Elucidation of specific associations between the human microbiome and health outcomes is facilitating the development of microbiome-targeted recommendations and treatments (e.g., fecal microbiota transplant; pre-, pro-, and post-biotics) to help prevent and treat disease. However, the potential of such recommendations and treatments to improve human health has yet to be fully realized. Technological advances have led to the development and proliferation of a wide range of tools and methods to collect, store, sequence, and analyze microbiome samples. However, differences in methodology at each step in these analytic processes can lead to variability in results due to the unique biases and limitations of each component. This technical variability hampers the detection and validation of associations with small to medium effect sizes. Therefore, the American Society for Nutrition (ASN) Nutritional Microbiology Group Engaging Members (GEM), sponsored by the Institute for the Advancement of Food and Nutrition Sciences (IAFNS), hosted a satellite session on methods in nutrition and gut microbiome research to review currently available methods for microbiome research, best practices, as well as tools and standards to aid in comparability of methods and results. This manuscript summarizes the topics and research discussed at the session. Consideration of the guidelines and principles reviewed in this session will increase the accuracy, precision, and comparability of microbiome research and ultimately the understanding of the associations between the human microbiome and health.

Impact of Maternal Vitamin D Supplementation during Breastfeeding on Infant Serum Vitamin D Levels: A Narrative Review of the Recent Evidence.

Vitamin D supplementation for breastfed infants is recommended due to low levels of vitamin D in human milk and the high prevalence of vitamin D deficiency. The relationship between maternal vitamin D supplementation while breastfeeding and infant serum vitamin D levels is beginning to be described. A literature review was conducted that investigated the impact of maternal supplementation, with at least 4000 IU of vitamin D, on infant serum vitamin D levels. Inclusion criteria were publication between 2016-2022, primary research, exclusively breastfed infants, and mothers taking vitamin D supplements while breastfeeding. Exclusion criteria were publication prior to 2016, review articles, results that did not include infant serum vitamin D levels, and research using participants already included in this review. Over 90% of infants whose mothers took vitamin D supplements while breastfeeding had adequate serum vitamin D levels. The final mean serum vitamin D of all infant participants whose mothers consumed vitamin D supplementation was 66.7 nmol/L, while mean serum vitamin D in those whose mothers did not consume supplements was 33.5 nmol/L. Consumption of vitamin D supplements by lactating women exclusively breastfeeding their infants can lead to adequate serum vitamin D levels in their infants.

Vitamin D Supplementation in Exclusively Breastfed Infants Is Associated with Alterations in the Fecal Microbiome.

Breastfeeding and introduction of solid food are the two major components of infant feeding practices that influence gut microbiota composition in early infancy. However, it is unclear whether additional factors influence the microbiota of infants either exclusively breastfed or not breastfed. We obtained 194 fecal samples from infants at 3-9 months of age, extracted DNA, and sequenced the V4 region of the 16S rRNA gene. Feeding practices and clinical information were collected by questionnaire and abstraction of birth certificates. The gut microbiota of infants who were exclusively breastfed displayed significantly lower Shannon diversity (p-adjust < 0.001) and different gut microbiota composition compared to infants who were not breastfed (p-value = 0.001). Among the exclusively breastfed infants, recipients of vitamin D supplements displayed significantly lower Shannon diversity (p-adjust = 0.007), and different gut microbiota composition structure than non-supplemented, breastfed infants (p-value = 0.02). MaAslin analysis identified microbial taxa that associated with breastfeeding and vitamin D supplementation. Breastfeeding and infant vitamin D supplement intake play an important role in shaping infant gut microbiota.

Disparities in Risks of Inadequate and Excessive Intake of Micronutrients during Pregnancy.

BACKGROUND: Inadequate or excessive intake of micronutrients in pregnancy has potential to negatively impact maternal/offspring health outcomes. OBJECTIVE: The aim was to compare risks of inadequate or excessive micronutrient intake in diverse females with singleton pregnancies by strata of maternal age, race/ethnicity, education, and prepregnancy BMI. METHODS: Fifteen observational cohorts in the US Environmental influences on Child Health Outcomes (ECHO) Consortium assessed participant dietary intake with 24-h dietary recalls (n = 1910) or food-frequency questionnaires (n = 7891) from 1999-2019. We compared the distributions of usual intake of 19 micronutrients from food alone (15 cohorts; n = 9801) and food plus dietary supplements (10 cohorts with supplement data; n = 7082) to estimate the proportion with usual daily intakes below their age-specific daily Estimated Average Requirement (EAR), above their Adequate Intake (AI), and above their Tolerable Upper Intake Level (UL), overall and within sociodemographic and anthropometric subgroups. RESULTS: Risk of inadequate intake from food alone ranged from 0% to 87%, depending on the micronutrient and assessment methodology. When dietary supplements were included, some women were below the EAR for vitamin D (20-38%), vitamin E (17-22%), and magnesium (39-41%); some women were above the AI for vitamin K (63-75%), choline (7%), and potassium (37-53%); and some were above the UL for folic acid (32-51%), iron (39-40%), and zinc (19-20%). Highest risks for inadequate intakes were observed among participants with age 14-18 y (6 nutrients), non-White race or Hispanic ethnicity (10 nutrients), less than a high school education (9 nutrients), or obesity (9 nutrients). CONCLUSIONS: Improved diet quality is needed for most pregnant females. Even with dietary supplement use, >20% of participants were at risk of inadequate intake of ≥1 micronutrients, especially in some population subgroups. Pregnancy may be a window of opportunity to address disparities in micronutrient intake that could contribute to intergenerational health inequalities.

Dietary Intake Influences Metabolites in Healthy Infants: A Scoping Review.

Metabolites are generated from exogenous sources such as diet. This scoping review will summarize nascent metabolite literature and discriminating metabolites for formula vs. human- milk-fed infants. Using the PICOS framework (P-Patient, Problem or Population; I-Intervention; C-Comparison; O-Outcome; S-Study Design) and PRISMA item-reporting protocols, infants less than 12 months old, full-term, and previously healthy were included. Protocol was registered with Open Science Framework (OSF). Publications from 1 January 2009-2019 were selected, for various biofluids, study designs, and techniques (such as high-performance liquid chromatography (HPLC)). From 711 articles, blinded screening of 214 articles using Abstrackr® software, resulted in 24 for final review. Strengthening the Reporting of Observational studies in Epidemiology (STROBE) guidelines were adopted, which included a 24-point checklist. Articles were stratified according to biofluid. Of articles reporting discriminating metabolites between formula- and human milk-fed infants, 62.5% (5/8) of plasma/serum/dried blood spot, 88% (7/8) of urine and 100% (6/6) of feces related articles reported such discriminating metabolites. Overall, no differences were found between analytical approach used (targeted (n = 9) vs. un-targeted (n = 10)). Current articles are limited by small sample sizes and differing methodological approaches. Of the metabolites reviewed herein, fecal metabolites provided the greatest distinction between diets, which may be indicative of usefulness for future diet metabolite-focused work.

Compliance to Prenatal Iron and Folic Acid Supplement Use in Relation to Low Birth Weight in Lilongwe, Malawi.

Prenatal iron and folic acid (IFA) supplements are offered free to all pregnant women in Malawi to reduce maternal anemia and improve birth outcomes. We investigated the association between self-reported compliance to IFA intake and risk of low birth weight (LBW). Pregnant women who attended Bwaila Maternity Wing of Lilongwe District Hospital for delivery were recruited (n = 220). We used a questionnaire to collect self-reported information on IFA use and maternal sociodemographic data. Before delivery, blood samples for maternal hemoglobin (Hb) and folate status, and upon delivery, birth weight, and other newborn anthropometrics were measured. We used multivariable logistic regression to determine risk of LBW by prenatal IFA intake. The self-reported number of IFA pills taken during pregnancy was positively associated with Hb, but not serum and RBC folate concentration: <45, 45⁻89 and ≥90 pills taken corresponded with mean (SD) Hb 10.7 (1.6), 11.3 (1.8), and 11.7 (1.6) g/dL, respectively (p = 0.006). The prevalence of LBW was 20.1%, 13.5% and 5.6% for those who reported taking IFA pills <45, 45⁻89, and ≥90 pills, respectively (p = 0.027). Taking >60 IFA pills reduced risk of LBW delivery (OR (95% CI) = 0.15 (0.03⁻0.70), p = 0.033) than taking ≤30 pills. Self-reported compliance to IFA use is valid for assessing prenatal supplement program in Malawi, especially Hb status, and can reduce the rate of LBW.

Short-term consumption of n-3 PUFAs increases murine IL-5 levels, but IL-5 is not the mechanistic link between n-3 fatty acids and changes in B-cell populations.

N-3 polyunsaturated fatty acids (PUFAs) exert immunomodulatory effects on B cells. We previously demonstrated that n-3 PUFAs enhanced the relative percentage and/or frequency of select B2 cell subsets. The objectives here were to determine if n-3 PUFAs (a) could boost cytokines that target B-cell frequency, (b) enhance the frequency of the B1 population and (c) to identify the mechanism by which n-3 PUFAs modify the proportion of B cells. Administration of n-3 PUFAs as fish oil to C57BL/6 mice enhanced secretion of the Th2 cytokine IL-5 but not IL-9 or IL-13. N-3 PUFAs had no influence on the percentage or frequency of peritoneal B1 or B2 cells. Subsequent experiments with IL-5(-/-) knockout mice showed n-3 PUFAs decreased the percentage of bone marrow B220(lo)IgM(hi) cells and increased the proportion and number of splenic IgM(+)IgD(lo)CD21(lo) cells compared to the control. These results, when compared with our previous findings with wild-type mice, suggested IL-5 had no role in mediating the effect of n-3 PUFAs on B-cell populations. To confirm this conclusion, we assayed IL-5 secretion in a diet-induced obesity model in which n-3 PUFAs enhanced the frequency of select B-cell subsets. N-3 PUFA supplementation as ethyl esters to obesogenic diets did not alter circulating IL-5 levels. Altogether, the data establish that n-3 PUFAs as fish oil can increase circulating IL-5 in lean mice, which has implications for several disease end points, but this increase in IL-5 is not the mechanistic link between n-3 PUFAs and changes in B-cell populations.

Dietary bovine lactoferrin increases intestinal cell proliferation in neonatal piglets.

Lactoferrin is a bioactive milk protein that stimulates cell proliferation in vitro; however, limited in vivo evidence exists to allow lactoferrin to be incorporated into infant formula. Herein, the effect of dietary bovine lactoferrin (bLF) on neonatal intestinal growth and maturation was investigated guided by the hypothesis that bLF would increase cellular proliferation leading to functional differences in neonatal piglets. Colostrum-deprived piglets were fed formula containing 0.4 [control (Ctrl)], 1.0 (LF1), or 3.6 (LF3) g bLF/L for the first 7 or 14 d of life. To provide passive immunity, sow serum was provided orally during the first 36 h of life. Intestinal cell proliferation, histomorphology, mucosal DNA concentration, enzyme activity, gene expression, and fecal bLF content were measured. Intestinal enzyme activity, DNA concentration, and villus length were unaffected by bLF. However, crypt proliferation was 60% greater in LF1- and LF3-fed piglets than in Ctrl piglets, and crypt depth and area were 20% greater in LF3-fed piglets than in Ctrl piglets. Crypt cells from LF3-fed piglets had 3-fold higher ß-catenin mRNA expression than did crypt cells from Ctrl piglets. Last, feces of piglets fed bLF contained intact bLF, suggesting that some bLF was resistant to digestion and could potentially affect intestinal proliferation through direct interaction with intestinal epithelial cells. This study is the first to our knowledge to show that dietary bLF stimulates crypt cell proliferation in vivo. The increased ß-catenin expression indicates that Wnt signaling may in part mediate the stimulatory effect of bLF on intestinal cell proliferation.

Natural killer cell populations and cytotoxic activity in pigs fed mother's milk, formula, or formula supplemented with bovine lactoferrin.

BACKGROUND: Natural killer (NK) cells are components of the innate immune defense system, and their levels differ between breast and formula-fed (FF) infants. Lactoferrin (Lf) modulates NK cell cytotoxicity ex vivo. We hypothesized that dietary bovine Lf (bLf) would increase NK cell populations and cytotoxicity. METHODS: Piglets were sow-reared (SR), FF, or 1 g/l bLf-fed (LF) for 21 d. NK cells (CD3(-)CD4(-)CD8(+)) in blood (peripheral blood mononuclear cells (PBMCs)), spleen, and mesenteric lymph node (MLN) were determined by flow cytometry. PBMC NK cells were tested for cytotoxic activity against target K562 cells ex vivo in the presence of media (unstimulated), interleukin-2, or bLf. NK cell mRNA expression was determined by reverse transcription-quantitative PCR. RESULTS: SR and LF piglets had more NK cells in MLN (P = 0.0097) and spleen (P = 0.0980) than FF piglets. In PBMCs, SR piglets had more NK cells than FF piglets (P = 0.0072); LF piglets were intermediate and not different from FF or SR piglets. NK cell intelectin-2 mRNA expression was 2.5-fold higher (P = 0.0095) in LF than SR or FF piglets. NK cells in SR piglets exhibited greater (P < 0.0001) cytotoxic activity than those in LF or FF piglets, which was supported by greater perforin mRNA expression. CONCLUSION: Dietary bLf increased blood NK cell populations and NK Lf receptor expression but not NK cell cytotoxicity.

Dietary (1,3/1,6)-ß-D-glucan decreases transforming growth factor ß expression in the lung of the neonatal piglet.

Identification and characterization of compounds that enhance the growth, development, and health of infants who are not breastfed continues to be a goal for nutritional science. This study explored the effects of one dietary component, (1,3/1,6)-ß-D-glucan (Wellmune WGP), on lung immune development in the neonatal piglet. The hypothesis was that supplementation with WGP, a pathogen-associated molecular pattern, would enhance pathogen-responsive elements of the immune system, for instance, by increasing the size of the cytotoxic T-cell population or the expression of inflammatory cytokines. Piglets were fed a control formula or formula plus WGP at 1.8, 18, or 90 mg/kg body weight per day. Serum, thoracic lymph nodes (TLNs), mediastinal lymph nodes, and lung were collected at days 7 or 21. Immune parameters including tissue messenger RNA (mRNA) expression and T-cell phenotypes were analyzed. Normal developmental changes were observed, with a decrease in T-helper cells and an increase in cytotoxic T cells in both TLN and mediastinal lymph node, but there was no effect of WGP. Dietary WGP reduced the mRNA expression of transforming growth factor (TGF) ß2 and tended to reduce the mRNA expression of TGF-ß1 in lung tissue. With the exception of reducing TGF-ß mRNA in the lung and tending to decrease the ratio of T helper to cytotoxic T cell in the TLN, dietary WGP did not affect lung-associated adaptive immunity in piglets.

Effect of walnut (Juglans regia) polyphenolic compounds on ovalbumin-specific IgE induction in female BALB/c mice.

English walnuts are implicated in severe, IgE-mediated food allergy in humans. We sought to determine if polyphenolic compounds extracted from the edible nut could promote IgE production to a coadministered allergen. BALB/c mice were sensitized to ovalbumin (OVA) with or without alum (AL) or polyphenolic-enriched extract via intraperitoneal injection. Serum was analyzed for total IgE and OVA-specific IgE, IgG(1,) and IgG(2a/2b). Coadministration of walnut polyphenolic-enriched extract with antigen and AL increased serum concentrations of antigen-specific IgE and IgG(1). When AL was excluded from the injections, polyphenolic extract tended to enhance OVA-specific IgE and IgG(1) over levels induced by OVA alone, but the increase did not reach significance. Serum IgG(2a/2b) levels were similar between mice receiving OVA/AL and OVA/AL with polyphenolics. Thus, walnut polyphenolic extract enhanced the Th2-skewing effect of an aluminum hydroxide adjuvant. This indicates that walnut polyphenolic compounds may play a role in allergic sensitization of genetically predisposed individuals.

Immunoglobulin E-reactive proteins in cashew (Anacardium occidentale) apple juice concentrate.

Cashew apple juice has the potential to be a natural source of vitamin C and sugar in processed foods. The juice of the cashew apple is obtained by pressing the fleshy peduncle or receptacle, which forms a rounded apple that sits above the true fruit, the cashew nut. Cashew nut allergy is the second most commonly reported tree nut allergy in the United States. To determine if cashew apple juice contains cashew nut allergens, immunoblotting was performed using a cashew apple juice 6X concentrate that was extracted and further concentrated through dialysis, lyophilization, and resuspension. Serum IgE of individuals allergic to cashew nut bound proteins in the cashew apple juice concentrate extract. For some serum samples, IgE reactivity could be inhibited by preincubation of the serum with cashew nut extract, suggesting the presence of cashew nut-related allergens. Using monoclonal antibodies specific for cashew nut allergens, the concentrate was found to contain Ana o 1 (vicilin) and Ana o 2 (legumin). Neither IgE from cashew nut allergic sera nor the monoclonal antibodies bound any peptides in 5 kDa filtered cashew apple juice concentrate. The cashew apple juice concentrate used in these studies contains proteins with IgE-reactive epitopes, including cashew nut legumin and vicilin. No IgE-binding peptides remained after 5 kDa filtration of the concentrate.