Quantification of myo-inositol, 1,5-anhydro- D-sorbitol, and D-chiro-inositol using high-performance liquid chromatography with electrochemical detection in very small volume clinical samples.

Inositol is a six-carbon sugar alcohol and is one of nine biologically significant isomers of hexahydroxycyclohexane. Myo-inositol is the primary biologically active form and is present in higher concentrations in the fetus and newborn than in adults. It is currently being examined for the prevention of retinopathy of prematurity in newborn preterm infants. A robust method for quantifying myo-inositol (MI), D-chiro-inositol (DCI) and 1,5-anhydro- D-sorbitol (ADS) in very small-volume (25 µL) urine, blood serum and/or plasma samples was developed. Using a multiple-column, multiple mobile phase liquid chromatographic system with electrochemical detection, the method was validated with respect to (a) selectivity, (b) accuracy/recovery, (c) precision/reproducibility, (d) sensitivity, (e) stability and (f) ruggedness. The standard curve was linear and ranged from 0.5 to 30 mg/L for each of the three analytes. Above-mentioned performance measures were within acceptable limits described in the Food and Drug Administration's Guidance for Industry: Bioanalytical Method Validation. The method was validated using blood serum and plasma collected using four common anticoagulants, and also by quantifying the accuracy and sensitivity of MI measured in simulated urine samples recovered from preterm infant diaper systems. The method performs satisfactorily measuring the three most common inositol isomers on 25 µL clinical samples of serum, plasma, milk, and/or urine. Similar performance is seen testing larger volume samples of infant formulas and infant formula ingredients. MI, ADS and DCI may be accurately tested in urine samples collected from five different preterm infant diapers if the urine volume is greater than 2-5 mL.

Novel nutritional immune formula maintains host defense mechanisms.

Military combat and training stress induce immune changes that increase the risk of infection and ultimately influence soldiers' performance and readiness. Strenuous military training/assessment provides a uniform stress and the opportunity to evaluate nutritional strategies to minimize stress-induced immune changes that predispose soldiers to infection. Immunological changes and effects of a novel nutritional immune formula (NNIF) were examined prospectively in a double-blind, controlled study of 200 soldiers attending Special Forces Assessment and Selection School. Immune function was measured by skin delayed-type hypersensitivity, lymphocyte phenotyping, mitogenic proliferative responses, and granulocyte function. Approximately 50% of soldiers completed the study (control, n = 57; NNIF, n = 50). Several stress-induced lymphocyte changes were observed (decreased mitogen-induced proliferation, T and total lymphocytes, and interferon-gamma-producing lymphocytes and increased percentage of neutrophils). NNIF modified several changes, including delayed-type hypersensitivity responses (NNIF, 78%; control, 59%; p < 0.05), increased proportions of helper T cells, activation of B cells, enhanced neutrophil phagocytosis, and attenuation of declines in certain functional subpopulations (i.e., cytotoxic/ suppressor lymphocytes). Soldiers who consumed NNIF experienced less stress-induced immune impairment, thereby lowering the risk of infection.

Effect of dietary ribonucleotides on infant immune status. Part 2: Immune cell development.

The objective of this study was to determine whether dietary ribonucleotides alter immune cell phenotypes or function in the first year of life. Newborn term infants in a double-blind, 12-mo, multicenter trial were randomized to cow milk formula groups with (FN, n = 138) or without (F, n = 147) 72 mg/L supplemental ribonucleotides. A nonrandomized HMF cohort (n = 192) was concurrently enrolled. Eighty-eight immune blood cell types were characterized by flow cytometry. Data were analyzed by multivariate ANOVA (MANOVA), ANOVA, and repeated measures analysis (RMA), with adjustments made for multiple comparisons. Ribonucleotide feeding changed subpopulations of T and natural killer (NK) cells. FN had higher numbers and percentages of memory/effector (M/E) cytotoxic/suppressor (CD45R0(+)CD8(+), RMA) T, Fas(+) M/E (CD45R0(+)CD95(+)CD3(+), 6 mo) T, and CD56(+)CD16(-) NK cells (CD56(+)CD16(-)CD3(-)CD8(-), 12 mo), and higher percentages of M/E helper (CD45R0(+)CD4(+), RMA) T, Tc1 (IFN gamma(+)CD4(-)CD3(+), RMA), total interferon (IFN)gamma T (IFN gamma(+)CD4(+/-)CD3(+), RMA), Th2 (IL-4(+)CD4(+)CD3(+), 7 mo), and CD57(+) NK-T cells (CD57(+)CD56(-)CD3(+), 6 mo, 7 mo) compared with F. Percentages of naive helper T (CD45RA(+)CD4(+), 12 mo) and numbers and percentages of CD56(+) NK-T cells (CD56(+)CD16(-)CD3(+)CD8(-), 2 mo, 6 mo) were lower in FN than F. Percentages of M/E cytotoxic/suppressor, Th2, and CD56(+)CD16(-) NK cells in FN were significantly higher than F but were not different from HMF, whereas F was significantly lower than HMF. Ribonucleotide supplementation of infant formula supported increased T-cell maturation and affected immunoregulatory NK cell subsets. These FN-associated immune cell profiles either did not differ from those infants fed HMF or tended to be more like those fed HMF than those fed F.

Effect of dietary ribonucleotides on infant immune status. Part 1: Humoral responses.

The objective of this study was to further explore previously identified effects of supplemental ribonucleotides on infant immune status as measured by antibody responses to routine infant immunizations. Infants were randomized to a milk-based formula with (FN, n = 138) or without (F, n = 147) 72 mg ribonucleotides/L. A cohort of human milk-fed (HMF, n = 192) infants was also followed. Subjects were given Haemophilus influenzae type b (Hib), diphtheria tetanus acellular pertussis, and oral poliovirus vaccinations at 2, 4, and 6 mo of age, and specific antibody responses were assessed at 2, 6, 7, and 12 mo. Growth and safety data were also monitored. Using a two-group repeated measures analysis (RMA), FN-fed infants had significantly higher poliovirus type 1 neutralizing antibody (PV-VN1) responses than F-fed infants (p = 0.045). Using three-group RMA, PV-VN1 responses in HMF infants were not different from FN-fed infants, while HMF-fed infant PV-VN1 responses were significantly higher than F-fed infants at 6 (p = 0.0004) and 12 mo (p = 0.0001). FN-fed infants had responses to Hib Farr, diphtheria, tetanus toxoid, oral poliovirus-specific IgA, and PV-VN3 not significantly different from those of F and HMF infants. Growth, gastrointestinal tolerance, and adverse events were equivalent among the three groups. The FN-associated increase in PV-VN1 response and nonstatistically significant trends toward increased Hib and diphtheria antibody responses were consistent with observations from earlier studies, indicating immune benefits of nucleotide supplementation of infant formula.

Soy protein allergy: incidence and relative severity.

Food allergy is a relatively rare and sometimes violent reaction of the immune system to food proteins. The first report characterizing soy allergy appeared in 1934. The Food and Agriculture Organization of the United Nations includes soy in its list of the 8 most significant food allergens. At least 16 potential soy protein allergens have been identified but their relative clinical significance is unknown. Conversely, soy has a long history of successful use in managing cow's milk allergies in infants. To better predict the utility of soy proteins for controlling food allergy, it is important to understand the relative allergenic reactivity of soy compared with other major food proteins. This can be studied using clinical data, animal models, and biochemical approaches; all show diminished reactivity for soy. Clinical studies using in vitro methods and blinded food challenges have generated substantial information. Study populations include high-risk asymptomatic infants and patients with atopic symptoms, positive food challenges, and specific milk allergies. Generally, these studies show lower allergic reactivity for soy proteins vs. other food allergens. Comparisons of food allergen dose-response relationships for triggering allergic symptoms also demonstrate a higher protein concentration threshold for soy (approximately 100 times), indicating lower allergenic reactivity. Extensive investigations of soy immunological reactivity have also been carried out using animal models. Consistent with clinical results, all of these data show substantially diminished immunological reactivity for soy proteins. Biochemical and immunochemical analyses indicate no striking differences between soy and other food proteins that would explain these unexpected differences in allergenic reactivity.

Immune status of infants fed soy-based formulas with or without added nucleotides for 1 year: part 1: vaccine responses, and morbidity.

BACKGROUND: Immunologic development of soy-fed infants has not been extensively studied. Early studies of soy flour-based formulas showed decreased immunoglobulin production when soy protein intake was limited. However, there were no significant differences in rotavirus vaccine responses between breast-fed and soy protein isolate-based formula-fed infants. Nucleotides added to milk-based formula benefit infant immune status, but reports of the immunologic effects of adding nucleotides to soy-based formula are not available. This study evaluated immune status and morbidity of infants fed soy protein isolate formulas with and without added nucleotides for 1 year. METHODS: Newborn, term infants enrolled in a masked 12-month feeding trial were assigned randomly to groups fed soy formula with or without added nucleotides (n = 94, n = 92). A nonrandomized human milk/formula cohort (n = 81) was concurrently enrolled. Recommended immunizations were administered at 2, 4, and 6 months. Immune status was determined from antibody responses to Haemophilus influenzae type b, tetanus, diphtheria, and poliovirus vaccines at 6, 7, and 12 months. Parents and physicians reported morbidity data. RESULTS: All vaccine responses were within normal ranges. No response differences were observed between infants fed soy formula and those fed nucleotide-supplemented soy. However, antibody to H. influenzae type b at 7 and 12 months was higher in infants fed nucleotide-supplemented soy than in infants fed human milk/formula ( P = 0.007, P = 0.008, respectively). Human milk/formula-fed infants had higher poliovirus neutralizing antibody at 12 months than did soy-fed infants ( P = 0.016). Morbidity analyses showed that only physician-reported diarrhea was different among groups (groups fed human milk/formula had less diarrhea than did soy groups, P = 0.011). CONCLUSIONS: Term infants fed soy protein isolate-based formulas have normal immune development as measured by antibody responses to childhood immunizations.

Immune status of infants fed soy-based formulas with or without added nucleotides for 1 year: part 2: immune cell populations.

BACKGROUND: Infants fed a soy protein isolate-based formula have immunization responses similar to breast-fed infants. However, cellular aspects of the immunologic development of soy-fed infants have not been studied extensively. Nucleotides added to milk-based formula benefit infant immune status, but reports of the immunologic effects of adding nucleotides to soy-based formula are not available. This study examines immune cell populations of infants fed soy protein isolate formulas with and without added nucleotides for 1 year. METHODS: Newborn, term infants studied in a masked 12-month feeding trial were assigned randomly to soy formula groups with and without added nucleotides (n = 94, n = 92). A nonrandomized human milk/formula-fed cohort (n = 81), was concurrently enrolled. Blood samples were collected at 6, 7, and 12 months. Thirty-two immune cell populations were characterized using three-color flow cytometry. Cellular markers were chosen to assess general pediatric immune status, emphasizing maturation and activation of B, T, and NK lymphocytes. RESULTS: All cell populations, number and percentages, were within age-related normal ranges. The only significant difference found between soy formula and human milk/formula-fed infants was the percentage of CD57 + NK T cells at 12 months (human milk/formula > soy formula, P = 0.034). There were significant differences at some time points between human milk/formula-fed and nucleotide-supplemented soy formula-fed infants in populations of lymphocytes, eosinophils, total T, helper T, naive helper, memory/effector helper, CD57 - T, and CD11b + CD8 + NK cells. None of the cell populations differed between infants fed soy formula versus soy plus nucleotides. CONCLUSIONS: Infants fed this commercial soy formula demonstrated immune cell status similar to human milk/formula-fed infants, consistent with normal immune system development. The addition of nucleotides to soy formula did not significantly change specific individual immune cell populations but tended to increase numbers and percentages of T cells and decreased numbers and percentages of NK cells.