Norman G. Hord

Food sources of nitrates and nitrites: the physiologic context for potential health benefits

The presence of nitrates and nitrites in food is associated with an increased risk of gastrointestinal cancer and, in infants, methemoglobinemia. Despite the physiologic roles for nitrate and nitrite in vascular and immune function, consideration of food sources of nitrates and nitrites as healthful dietary components has received little attention. Approximately 80% of dietary nitrates are derived from vegetable consumption; sources of nitrites include vegetables, fruit, and processed meats. Nitrites are produced endogenously through the oxidation of nitric oxide and through a reduction of nitrate by commensal bacteria in the mouth and gastrointestinal tract. As such, the dietary provision of nitrates and nitrites from vegetables and fruit may contribute to the blood pressure-lowering effects of the Dietary Approaches to Stop Hypertension (DASH) diet. We quantified nitrate and nitrite concentrations by HPLC in a convenience sample of foods. Incorporating these values into 2 hypothetical dietary patterns that emphasize high-nitrate or low-nitrate vegetable and fruit choices based on the DASH diet, we found that nitrate concentrations in these 2 patterns vary from 174 to 1222 mg. The hypothetical high-nitrate DASH diet pattern exceeds the World Health Organizations Acceptable Daily Intake for nitrate by 550% for a 60-kg adult. These data call into question the rationale for recommendations to limit nitrate and nitrite consumption from plant foods; a comprehensive reevaluation of the health effects of food sources of nitrates and nitrites is appropriate. The strength of the evidence linking the consumption of nitrate- and nitrite-containing plant foods to beneficial health effects supports the consideration of these compounds as nutrients.

Leptin, Insulin-Like Growth Factor-1, and Insulin-Like Growth Factor-2 Are Mitogens in ApcMin/+ but not Apc+/+ Colonic Epithelial Cell Lines

The obese state is associated with elevated circulating levels of insulin, insulin-like growth factors (IGF), and leptin. Research is contradictory regarding the role of these elevated growth factors in colon cancer risk. We hypothesized that colonic epithelial cells that were Apc deficient (ApcMin/+) but not those expressing wild-type Apc (Apc+/+) would experience a hyperproliferative and antiapoptotic phenotype when exposed to these growth factors. This hypothesis was addressed using two nontumorigenic murine colonic epithelial cell lines with distinct Apc genotypes: Apc+/+ YAMC cells and ApcMin/+ IMCE cells. Cells were treated for 48 hours with various concentrations of leptin (0.001-50 ng/mL), IGF-1 (0.1-200 ng/mL), or IGF-2 (0.1-600 ng/mL). In YAMC cells, leptin caused a significant decrease in cell proliferation (P < 0.01) compared with controls due to induction of caspase activity and apoptosis. In contrast, in the IMCE cells, leptin induced a 75% increase in cell proliferation compared with controls (P < 0.0001). IGF-1 and IGF-2 also induced 50% greater proliferation in the IMCE cells (P < 0.001) compared with controls. Cotreatment of IMCE cells with leptin and either IGF-1 or IGF-2 induced greater proliferation than either growth factor alone (P < 0.0001). IMCE cell proliferation caused by leptin only treatment was associated with activation of p42/44 mitogen-activated protein kinase (MAPK), p38 MAPK, and nuclear factor-κB nuclear translocation but not with MAPK kinase or Janus-activated kinase/signal transducers and activators of transcription activation. These data provide the first evidence that leptin may interact with IGFs to promote survival and expansion of colonic epithelial cells that were Apc deficient (ApcMin/+) but not those expressing wild-type Apc (Apc+/+).

Adiponectin blocks multiple signaling cascades associated with leptin-induced cell proliferation in ApcMin/+ colon epithelial cells

We previously demonstrated that leptin, an adipose‐derived hormone, induces cell proliferation in a model of preneoplastic (IMCE (ApcMin/+), but not normal (YAMC (Apc+/+), colon epithelial cells by inducing autocrine IL‐6 production and trans‐IL‐6 signaling. Low serum adiponectin is associated with colon, prostate and breast cancer. Adiponectin is secreted by white adipose tissue; the levels of adiponectin in the blood decrease as body mass index (and leptin) increases. In our study, we tested whether murine recombinant globular adiponectin (gArcp30) could modulate leptin‐induced cell proliferation, autocrine IL‐6 production, trans‐IL‐6 signaling and other leptin‐induced cell signaling events previously observed in IMCE cells but not YAMC cells. Under serum‐free conditions, adiponectin (1 μg/ml) inhibited leptin‐induced autocrine IL‐6 production, soluble IL‐6 receptor shedding, trans‐IL‐6 signaling and subsequent STAT3 phosphorylation in IMCE cells. Adiponectin inhibited leptin‐induced cell proliferation in the IMCE cells and this inhibition was associated with IκB‐α phosphorylation, IκB‐α degradation and decreased NF‐κB p65 DNA activation and binding. These data indicate that adiponectin acts on preneoplastic colon epithelial cells to regulate cell growth via 2 distinct pathways inhibiting leptin‐induced NF‐κB‐dependent autocrine IL‐6 production and trans‐IL‐6 signaling. We hypothesize that adiponectin may be an important regulator of colon epithelial cell homeostasis by linking the observed reduced risk for cancer in populations with high serum adiponectin concentrations to specific mechanisms of cell number homeostasis in a model of preneoplastic colon epithelial cells. These data may have broad implications for diet and lifestyle strategies for the prevention and treatment of obesity‐associated cancers.

Eukaryotic-Microbiota Crosstalk: Potential Mechanisms for Health Benefits of Prebiotics and Probiotics

The ability to link dietary consumption of prebiotic food ingredients and probiotic microorganisms to health benefits rests, in part, on our ability to identify both the extent to which these factors alter human microbiome activity and/or structure and the ability to engage eukaryotic cells necessary to transduce signals originating from the microbiome. The human microbiome consists of bacterial, archaeal, and fungal components that reside in mucosal surfaces of the gut, the airways, and the urogenital tract. Characterization of the symbiotic nature of the relationship between eukaryotic cells and the bacterial and archaeal components of the microbiota has revealed significant contributions in energy balance, bowel function, immunologic function, sensory perception, glycemic control, and blood pressure regulation. Elucidating the complex interactions between the microbiota and their associated epithelial, immune, and neural cells may provide mechanistic insights and a rational basis for our belief that dietary consumption of probiotic microorganisms and prebiotics produces health benefits.

Diet‐induced adiposity alters the serum profile of inflammation in C57BL/6N mice as measured by antibody array

Morbid obesity is considered a systemic inflammatory state. The objective of this project was to characterize the adipokine, cytokine and chemokine protein profile in serum from control, lean and obese mice. We hypothesized that chemokines and cytokines are altered by caloric restriction and diet‐induced obesity as a function of changes in body composition. Six‐week‐old female C57BL/6N mice (n = 12 per group) were randomized to one of three diets: control (fed ad libitum); lean (30% calorie‐restricted regimen relative to control) and diet‐induced obese (DIO; high calorie diet, fed ad libitum). Body weight, body composition and food intake were monitored throughout the study. After 10 weeks on the diets, blood samples were collected, and adipokine/cytokine/chemokine serum profiles were measured by antibody array. Lean mice, relative to the control group, displayed increased concentrations of insulin‐like growth factor (IGF) binding protein‐3, ‐5 and ‐6 and adiponectin and decreased IGF‐1. These mice also showed increased concentrations of interleukin (IL)‐10, IL‐12 p40/p70, eotaxin, monocyte chemoattractant protein‐5 and SDF‐1. In contrast, DIO mice displayed increased leptin, IL‐6 and LPS‐induced chemokine and decreased concentrations of all chemokines/cytokines measured relative to control mice. As such, these data indicate that DIO may lead to an inflammatory state characterized as a shift towards a T helper lymphocyte type 1–skewed responsiveness. The demonstration of differential adipokine, cytokine and chemokine protein profile in control, lean and DIO mice may have implications for immune responsiveness and risk of disease.

Immunomodulation by dietary long chain omega-3 fatty acids and the potential for adverse health outcomes

Recommendations to consume fish for prevention of cardiovascular disease (CVD), along with the U.S. Food and Drug Administration-approved generally recognized as safe (GRAS) status for long chain omega-3 fatty acids, may have had the unanticipated consequence of encouraging long-chain omega-3 (ω-3) fatty acid [(eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] supplementation and fortification practices. While there is evidence supporting a protective role for EPA/DHA supplementation in reducing sudden cardiac events, the safety and efficacy of supplementation with LCω-3PUFA in the context of other disease outcomes is unclear. Recent studies of bacterial, viral, and fungal infections in animal models of infectious disease demonstrate that LCω-3PUFA intake dampens immunity and alters pathogen clearance and can result in reduced survival. The same physiological properties of EPA/DHA that are responsible for the amelioration of inflammation associated with chronic cardiovascular pathology or autoimmune states, may impair pathogen clearance during acute infections by decreasing host resistance or interfere with tumor surveillance resulting in adverse health outcomes. Recent observations that high serum LCω-3PUFA levels are associated with higher risk of prostate cancer and atrial fibrillation raise concern for adverse outcomes. Given the widespread use of supplements and fortification of common food items with LCω-3PUFA, this review focuses on the immunomodulatory effects of the dietary LCω-3PUFAs, EPA and DHA, the mechanistic basis for potential negative health outcomes, and calls for biomarker development and validation as rational first steps towards setting recommended dietary intake levels.

Treatment of glioma patients with ketogenic diets: report of two cases treated with an IRB-approved energy-restricted ketogenic diet protocol and review of the literature.

BackgroundBased on the hypothesis that cancer cells may not be able to metabolize ketones as efficiently as normal brain cells, the ketogenic diet (KD) has been proposed as a complementary or alternative therapy for treatment of malignant gliomas.Case presentationWe report here our experience in treating two glioma patients with an IRB-approved energy-restricted ketogenic diet (ERKD) protocol as monotherapy and review the literature on KD therapy for human glioma patients. An ERKD protocol was used in this pilot clinical study. In addition to the two patients who enrolled in this study, we also reviewed findings from 30 other patients, including 5 patients from case reports, 19 patients from a clinical trial reported by Rieger and 6 patients described by Champ. A total of 32 glioma patients have been treated using several different KD protocols as adjunctive/complementary therapy. The two patients who enrolled in our ERKD pilot study were monitored with twice daily measurements of blood glucose and ketones and daily weights. However, both patients showed tumor progression while on the ERKD therapy. Immunohistochemistry reactions showed that their tumors had tissue expression of at least one of the two critical mitochondrial ketolytic enzymes (succinyl CoA: 3-oxoacid CoA transferase, beta-3-hydroxybutyrate dehydrogenase 1). The other 30 glioma patients in the literature were treated with several different KD protocols with varying responses. Prolonged remissions ranging from more than 5 years to 4 months were reported in the case reports. Only one of these patients was treated using KD as monotherapy. The best responses reported in the more recent patient series were stable disease for approximately 6 weeks. No major side effects due to KD have been reported in any of these patients.ConclusionsWe conclude that 1. KD is safe and without major side effects; 2. ketosis can be induced using customary foods; 3. treatment with KD may be effective in controlling the progression of some gliomas; and 4. further studies are needed to determine factors that influence the effectiveness of KD, whether as a monotherapy, or as adjunctive or supplemental therapy in treating glioma patients.Trial registrationClinicalTrials.gov# NCT01535911

Expression of prostaglandin endoperoxide H synthase-2 induced by nitric oxide in conditionally immortalized murine colonic epithelial cells

Increased expression of prostaglandin endoperoxide H synthase‐2 (PGHS‐2) has been implicated in pathological conditions such as inflammatory bowel diseases and colon cancer. Recently, it has been demonstrated that inducible nitric oxide syn‐thase (NOS II) expression and nitric oxide (NO) production are up‐regulated in these diseases as well. However, the apparent link between PGHS‐2 and NOS II has not been thoroughly investigated in nontransformed and nontumorigenic colonic epithelial cells. In the present study, we examined the concomitant expression of PGHS‐2 and NOS II as well as the production of prostaglandin E2 (PGE2) and NO in conditionallyimmortalized mouse colonic epithelial cells, namely YAMC (Apc+/+). We found that the induction of PGHS‐2 and generation of PGE2 in these cells by IFN‐γ and lipopolysaccharide (LPS) were greatly reduced by two selective NOS II inhibitors, L‐NIL and SMT. To ascertain the effect of NO on PGHS‐2 overexpression, we tested NO‐releasing compounds, NOR‐1 and SNAP, and found that they caused PGHS‐2 expression and PGE2 production. This effect was abolished by hemoglobin, a NO scavenger. Using electrophoretic mobility shift assays, we found that both NOR‐1 and SNAP caused β‐catenin/LEF‐1 DNA complex formation. Super‐shift by anti‐β‐catenin antibody confirmed the presence of β‐catenin in the complex. Cell fractionation studies indicated that NO donors caused an increase in free soluble cytoplasmic β‐catenin. This is further corroborated by the immunocytochemistry data showing the redistribution of β‐catenin from the predominantly membrane localization into the cytoplasm and nucleus after treatment with NO donors. To further explore the possible connection between PGHS‐2 expression and β‐catenin/LEF‐1 DNA complex formation, we studied IMCE (ApcMin/+) cells, a sister cell line of YAMC with similar genetic background but differing in Apc genotype and, consequently, their β‐catenin levels. We found that IMCE cells, in comparison with YAMC cells, had markedly higher β‐catenin/LEF‐1 DNA complex formation under both resting conditions as well as after induction with NO. In parallel fashion, IMCE cells expressed significantly higher levels of PGHS‐2 mRNA and protein, and generated more PGE2. Overall, this study suggests that NO may be involved in PGHS‐2 overexpression in conditionally immortalized mouse colonic epithelial cells. Although the molecular mechanism of the link is still under investigation, this effect of NO appears directly or indirectly to be a result of the increase in free soluble β‐catenin and the formation of nuclear β‐catenin/LEF‐1 DNA complex.—Mei, J. M., Hord, N. G., Winterstein, D. F., Donald, S. P., and Phang, J. M. Expression of prostaglandin endoperoxide H synthase‐2 induced by nitric oxide in conditionally immortalized murine colonic epithelial cells. FASEB J. 14, 1188–1201 (2000)

Dietary Nitrates, Nitrites, and Cardiovascular Disease

Dietary nitrate (NO3), nitrite (NO2), and arginine can serve as sources for production of NOx (a diverse group of metabolites including nitric oxide, nitrosothiols, and nitroalkenes) via ultraviolet light exposure to skin, mammalian nitrate/nitrite reductases in tissues, and nitric oxide synthase enzymes, respectively. NOx are responsible for the hypotensive, antiplatelet, and cytoprotective effects of dietary nitrates and nitrites. Current regulatory limits on nitrate intakes, based on concerns regarding potential risk of carcinogenicity and methemoglobinemia, are exceeded by normal daily intakes of single foods, such as soya milk and spinach, as well as by some recommended dietary patterns such as the Dietary Approaches to Stop Hypertension diet. This review includes a call for regulatory bodies to consider all available data on the beneficial physiologic roles of nitrate and nitrite in order to derive rational bases for dietary recommendations.

Nitrate and Nitrite Content of Human, Formula, Bovine, and Soy Milks: Implications for Dietary Nitrite and Nitrate Recommendations

BACKGROUND Estimation of nitrate and nitrite concentrations of milk sources may provide insight into potential health risks and benefits of these food sources for infants, children, and adults. The World Health Organization and American Academy of Pediatrics recommends exclusive consumption of human milk for the first 6 months of life. Human milk is known to confer significant nutritional and immunological benefits for the infant. Consumption of formula, cows, and soy milk may be used as alternatives to human milk for infants. METHODS We sought to estimate potential exposure to nitrate and nitrite in human, formula, bovine, and soy milk to inform total dietary exposure estimates and recommendations. Using sensitive quantitative methodologies, nitrite and nitrate were analyzed in different samples of milk. RESULTS Human milk concentrations of colostrum (expressed days 1-3 postpartum; n=12), transition milk (expressed days 3-7 postpartum; n=17), and mature milk (expressed >7 days postpartum; n=50) were 0.08 mg/100 mL nitrite and 0.19 mg/100 mL nitrate, 0.001 mg/100 mL nitrite and 0.52 mg/100 mL nitrate, and 0.001 mg/100 mL nitrite and 0.3 mg/100 mL nitrate, respectively, revealing that the absolute amounts of these anions change as the composition of milk changes. When expressed as a percentage of the World Health Organizations Acceptable Daily Intake limits, Silk® Soy Vanilla (WhiteWave Foods, Broomfield, CO) intake could result in high nitrate intakes (104% of this standard), while intake of Bright Beginnings Soy Pediatric® formula (PBM Nutritionals, Georgia, VT) could result in the highest nitrite intakes (383% of this standard). CONCLUSIONS The temporal relationship between the provision of nitrite in human milk and the development of commensal microbiota capable of reducing dietary nitrate to nitrite supports a hypothesis that humans are adapted to provide nitrite to the gastrointestinal tract from birth. These data support the hypothesis that the high concentrations of breastmilk nitrite and nitrate are evidence for a physiologic requirement to support gastrointestinal and immune homeostasis in the neonate.