Andrew L. Milkowski

Ingested nitrate and nitrite and stomach cancer risk: An updated review

Nitrite and nitrate are naturally occurring molecules in vegetables and also added to cured and processed meats to delay spoilage and pathogenic bacteria growth. Research over the past 15 years has led to a paradigm change in our ideas about health effects of both nitrite and nitrate. Whereas, historically nitrite and nitrate were considered harmful food additives and listed as probable human carcinogens under conditions where endogenous nitrosation could take place, they are now considered by some as indispensible nutrients essential for cardiovascular health by promoting nitric oxide (NO) production. We provide an update to the literature and knowledge base concerning their safety. Most nitrite and nitrate exposure comes from naturally occurring and endogenous sources and part of the cell signaling effects of NO involve nitrosation. Nitrosation must now be considered broadly in terms of both S- and N-nitrosated species, since S-nitrosation is kinetically favored. Protein S-nitrosation is a significant part of the role of NO in cellular signal transduction and is involved in critical aspects of cardiovascular health. A critical review of the animal toxicology literature of nitrite indicates that in the absence of co-administration of a carcinogenic nitrosamine precursor, there is no evidence for carcinogenesis. Newly published prospective epidemiological cohort studies indicate that there is no association between estimated intake of nitrite and nitrate in the diet and stomach cancer. This new and growing body of evidence calls for a reconsideration of nitrite and nitrate safety.

Nutritional epidemiology in the context of nitric oxide biology: A risk–benefit evaluation for dietary nitrite and nitrate

The discovery of the nitric oxide (NO) pathway in the 1980s represented a critical advance in understanding cardiovascular disease, and today a number of human diseases are characterized by NO insufficiency. In the interim, recent biomedical research has demonstrated that NO can be modulated by the diet independent of its enzymatic synthesis from l-arginine, e.g., the consumption of nitrite- and nitrate-rich foods such as fruits, leafy vegetables, and cured meats along with antioxidants. Regular intake of nitrate-containing food such as green leafy vegetables may ensure that blood and tissue levels of nitrite and NO pools are maintained at a level sufficient to compensate for any disturbances in endogenous NO synthesis. However, some in the public perceive that dietary sources of nitrite and nitrate are harmful, and some epidemiological studies reveal a weak association between foods that contain nitrite and nitrate, namely cured and processed meats, and cancer. This paradigm needs revisiting in the face of undisputed health benefits of nitrite- and nitrate-enriched diets. This review will address and interpret the epidemiological data and discuss the risk-benefit evaluation of dietary nitrite and nitrate in the context of nitric oxide biology. The weak and inconclusive data on the cancer risk of nitrite, nitrate and processed meats are far outweighed by the health benefits of restoring NO homeostasis via dietary nitrite and nitrate. This risk/benefit balance should be a strong consideration before there are any suggestions for new regulatory or public health guidelines for dietary nitrite and nitrate exposures.

The role of red and processed meat in colorectal cancer development: a perspective

This paper is based on a workshop held in Oslo, Norway in November 2013, in which experts discussed how to reach consensus on the healthiness of red and processed meat. Recent nutritional recommendations include reducing intake of red and processed meat to reduce cancer risk, in particular colorectal cancer (CRC). Epidemiological and mechanistic data on associations between red and processed meat intake and CRC are inconsistent and underlying mechanisms are unclear. There is a need for further studies on differences between white and red meat, between processed and whole red meat and between different types of processed meats, as potential health risks may not be the same for all products. Better biomarkers of meat intake and of cancer occurrence and updated food composition databases are required for future studies. Modifying meat composition via animal feeding and breeding, improving meat processing by alternative methods such as adding phytochemicals and improving our diets in general are strategies that need to be followed up.

Activation of bovine epididymal sperm respiration by caffeine. Its transient nature and relationship to the utilization of acetyl carnitine.

Abstract Caffeine, which stimulates the motility of freshly extruded bovine epididymal spermatozoa, caused a large but transient increase in the respiratory activity of these cells incubated in a modified Ringer buffer without exogenously added substrate. In spermatozoa that were incubated without added substrate for 2 h at 30 °C or for 15 min at 37 °C, caffeine addition failed to increase respiratory activity even transiently. However, subsequent addition of pyruvate to these aged and caffeine-treated cells resulted in a rapid increase in the respiratory rate, nearly equal to that observed after caffeine addition to fresh cells or to cells stored at 4 °C. These observations indicate that the loss in metabolic response to caffeine is a result of the active metabolism of the spermatozoa. In freshly prepared sperm that were incubated without added substrate, the acetyl carnitine content declined and the free carnitine content of the sperm increased in amounts sufficient to account for the entire respiratory increment produced by caffeine addition. Respiratory stimulation by caffeine was sustained in the presence of those exogenously added substrates that are capable of entering the acetyl carnitine pool, such as acetate, pyruvate, l (+)-lactate, glucose, fructose or β-hydroxybutyrate. Tricarboxylic acid cycle intermediates were not effective. These observations clarify the relationship between the stimulatory effects of caffeine and the metabolic state of the spermatozoan and suggest the importance of the acetyl carnitine pool to the activation of sperm motility and oxidative metabolism.

Determining the impact of varying levels of cherry powder and starter culture on quality and sensory attributes of indirectly cured, emulsified cooked sausages

Indirect curing is a process that utilizes ingredients high in naturally occurring nitrate and a nitrate reducing bacterial starter culture (SC) to provide quality and sensory attributes similar to nitrite-added cured meats. The objective of this study was to determine the effects varying concentrations of starter culture and the addition of cherry powder (CP) had on improving quality and sensory attributes of indirectly cured sausages. Four treatments (TRTs) (TRT 1: low SC+no CP; TRT 2: low SC+CP; TRT 3: high SC+no CP; and TRT 4: high SC+CP) and a sodium nitrite-added (156 ppm) control were investigated. Residual nitrite levels throughout storage declined most rapidly in TRTs 2 and 4 (P<0.05). Few differences existed between TRTs and C for pH, objective color, or cured pigment concentrations. Consumer sensory panel scores revealed all treatment combinations were comparable (P>0.05) to the C for all sensory attributes.

Dietary nitrate and nitrite: Benefits, risks, and evolving perceptions

Consumers have an illogical relationship with nitrite (and its precursor, nitrate) in food. Despite a long history of use, nitrite was nearly banned from use in foods in the 1970s due to health concerns related to the potential for carcinogenic nitrosamine formation. Changes in meat processing methods reduced those potential risks, and nitrite continued to be used in foods. Since then, two opposing movements continue to shape how consumers view dietary nitrate and nitrite. The discovery of the profound physiological importance of nitric oxide led to the realization that dietary nitrate contributes significantly to the nitrogen reservoir for nitric oxide formation. Numerous clinical studies have also demonstrated beneficial effects from dietary nitrate consumption, especially in vascular and metabolic health. However, the latest wave of consumer sentiment against food additives, the clean-label movement, has renewed consumer fear and avoidance of preservatives, including nitrite. Education is necessary but may not be sufficient to resolve this disconnect in consumer perception.

Factors affecting the redox state of bovine epididymal spermatozoa

Abstract The oxidation-reduction state of bovine epididymal spermatozoa was determined in vitro by fluorescence spectroscopy and by direct chemical analysis. Enhanced NADH fluorescence in sperm was observed with the onset of anaerobiosis in the sample cuvette. However, part of this increased fluorescence was temporary and a stable pyridine nucleotide fluorescence was not reached until 25 min after the onset of anaerobiosis. The transient was not paralleled by an equivalent increase in cellular NADH as measured by absorption spectroscopy. Hypotonic treatment of sperm, which removed the plasma membrane, liberated greater than 50% of the cellular NAD and that remaining was reduced by rotenone addition, indicating its mitochondrial location. Hypotonically treated sperm did not demonstrate a transient fluorescence above that due to the increases in NADH from anaerobiosis. Addition of pyruvate to anaerobic sperm resulted in a rapid decrease in fluorescence that corresponded to NADH oxidation coupled with the reduction of pyruvate to lactate. The duration of this oxidized state was dependent on the amount of pyruvate added. Analysis of cellular NAD under similar conditions confirmed this result. The pyridine nucleotides of hypotonically treated cells were also oxidized by pyruvate but were not reduced by added glucose as in untreated sperm. These results indicate that pyruvate reduction served to balance reducing equivalents and temporarily reoxidized the intracellular milieu of the anaerobic spermatozoon. The data also support the hypothesis that pyruvate and lactate can serve as reducing equivalent carriers between cytosol and mitochondria.

Inhibition of Clostridium perfringens growth by potassium lactate during an extended cooling of cooked uncured ground turkey breasts.

The U.S. Department of Agricultures Food Safety and Inspection Service compliance guideline known as Appendix B specifies chilling time and temperature limits for cured and uncured meat products to inhibit growth of spore-forming bacteria, particularly Clostridium perfringens. Sodium lactate and potassium lactate inhibit toxigenic growth of Clostridium botulinum, and inhibition of C. perfringens has been reported. In this study, a cocktail of spores of three C. perfringens strains (ATCC 13124, ATCC 12915, and ATCC 12916) were inoculated into 100-g samples of ground skinless, boneless turkey breast formulated to represent deli-style turkey breast. Three treatment groups were supplemented with 0 (control), 1, or 2% potassium lactate (pure basis), cooked to 71 °C, and assayed for C. perfringens growth during 10 or 12 h of linear cooling to 4 °C. In control samples, populations of C. perfringens increased 3.8 to 4.7 log CFU/g during the two chilling protocols. The 1% potassium lactate treatment supported only a 2.5- to 2.7-log increase, and the 2% potassium lactate treatment limited growth to a 0.56- to 0.70-log increase. When compared with the control, 2% potassium lactate retarded growth by 2.65 and 4.21 log CFU/g for the 10- and 12-h cooling protocols, respectively. These results confirm that the addition of 2% potassium lactate inhibits growth of C. perfringens and that potassium lactate can be used as an alternative to sodium nitrite for safe extended cooling of uncured meats.

Comparison of the Effect of Curing Ingredients Derived from Purified and Natural Sources on Inhibition of Clostridium perfringens Outgrowth during Cooling of Deli-Style Turkey Breast.

The antimicrobial impact of purified and natural sources of both nitrite and ascorbate were evaluated against Clostridium perfringens during the postthermal processing cooling period of deli-style turkey breast. The objective of phase I was to assess comparable concentrations of nitrite (0 or 100 ppm) and ascorbate (0 or 547 ppm) from both purified and natural sources. Phase II was conducted to investigate concentrations of nitrite (50, 75, or 100 ppm) from cultured celery juice powder and ascorbate (0, 250, or 500 ppm) from cherry powder to simulate alternative curing formulations. Ground turkey breast (75% moisture, 1.2% salt, pH 6.2) treatments were inoculated with C. perfringens spores (three-strain mixture) to yield 2.5 log CFU/g. Individual 50-g portions were vacuum packaged, cooked to 71.1°C, and chilled from 54.4 to 26.7°C in 5 h and from 26.7 to 7.2°C in 10 additional hours. Triplicate samples were assayed for growth of C. perfringens at predetermined intervals by plating on tryptose-sulfite-cycloserine agar; experiments were replicated three times. In phase I, uncured, purified nitrite, and natural nitrite treatments without ascorbate had 5.3-, 4.2-, and 4.4-log increases in C. perfringens, respectively, at 15 h, but <1-log increase was observed at the end of chilling in treatments containing 100 ppm of nitrite and 547 ppm of ascorbate from either source. In phase II, 0, 50, 75, and 100 ppm of nitrite and 50 ppm of nitrite plus 250 ppm of ascorbate supported 4.5-, 3.9-, 3.5-, 2.2-, and 1.5-log increases in C. perfringens, respectively. In contrast, <1-log increase was observed after 15 h in the remaining phase II treatments supplemented with 50 ppm of nitrite and 500 ppm of ascorbate or ≥75 ppm of nitrite and ≥250 ppm of ascorbate. These results confirm that equivalent concentrations of nitrite, regardless of the source, provide similar inhibition of C. perfringens during chilling and that ascorbate enhances the antimicrobial effect of nitrite on C. perfringens at concentrations commonly used in alternative cured meats.

Validation of pepperoni process for control of Shiga toxin-producing Escherichia coli.

The objective of this study was to compare the survival of non-O157 Shiga toxin-producing Escherichia coli (STEC) with E. coli O157:H7 during pepperoni production. Pepperoni batter was inoculated with 7 log CFU/g of a seven-strain STEC mixture, including strains of serotypes O26, O45, O103, O111, O121, O145, and O157. Sausages were fermented to pH ≤4.8, heated at 53.3°C for 1 h, and dried for up to 20 days. STEC strains were enumerated at designated intervals on sorbitol MacConkey (SMAC) and Rainbow (RA) agars; enrichments were completed in modified EC (mEC) broth and nonselective tryptic soy broth (TSB). When plated on SMAC, total E. coli populations decreased 2.6 to 3.5 log after the 1-h heating step at 53.3°C, and a 4.9- to 5-log reduction was observed after 7 days of drying. RA was more sensitive in recovering survivors; log reductions on it were 1.9 to 2.6, 3.8 to 4.2, and 4.6 to 5.3 at the end of cook, and at day 7 and day 14 of drying, respectively. When numbers were less than the limit of detection by direct plating on days 14 and 20 of drying (representing a 5-log kill), no more than one of three samples in each experiment was positive by enrichment with mEC broth; however, STEC strains were recovered in TSB enrichment. Freezing the 7-day dried sausage for 2 to 3 weeks generated an additional 1- to 1.5-log kill. Confirmation by PCR revealed that O103 and O157 had the greatest survival during pepperoni productions, but all serotypes except O111 and O121 were occasionally recovered during drying. This study suggests that non-O157 STEC s trains have comparable or less ability than E. coli O157 to survive the processing steps involved in the manufacture of pepperoni. Processes suitable for control of E. coli O157 will similarly inactivate the other STEC strains tested in this study.