The research, published in Environmental Science and Pollution Research, was led by Vahid Ghasemzadeh-Mohammadi of Hamadan University of Medical Sciences together with colleagues from several Iranian institutions, including Shahid Beheshti University of Medical Sciences and the National Nutrition and Food Technology Research Institute. The team collected breast milk samples from 93 lactating mothers whose infants were between zero and six months old. Rather than treating all newborns as a single group, the researchers divided the infants into three age bands — zero to two months, two to four months, and four to six months — a decision that matters, because both milk consumption patterns and body weight change rapidly across the first half-year, and both variables directly shape how much of any given chemical an infant actually ingests per kilogram of body weight.

To understand why nitrate and nitrite attract regulatory attention in the first place, it helps to trace their journey through the environment and the body. Nitrogen compounds are ubiquitous: they cycle through soil, water, and crops, and human activity — above all intensive fertilizer use — has amplified their presence in the food supply. Leafy vegetables, cured meats, and contaminated drinking water are the dominant dietary sources for adults. In the body, nitrate can be reduced to nitrite, and nitrite in turn can react with amines to form N-nitroso compounds, some of which are carcinogenic in animal studies. For infants, the classic toxicological concern is methemoglobinemia, a condition in which nitrite oxidizes the iron in hemoglobin so that the molecule can no longer carry oxygen efficiently. Infant hemoglobin is chemically more susceptible to this oxidation, and the enzymes that normally reverse it are less active in the first months of life, which is why nitrate limits for drinking water are set with formula-fed infants specifically in mind.

But the story is not purely one of hazard. Nitrate and nitrite are also physiological molecules with genuine roles in human biology. They serve as precursors to nitric oxide, a signaling gas that regulates vascular tone, supports blood pressure control, and participates in gastrointestinal and immune defenses. The enterosalivary circulation — in which nitrate is concentrated in saliva, reduced to nitrite by oral bacteria, and converted to nitric oxide in the acidic stomach — is now recognized as an important homeostatic pathway. Some researchers have even argued that the nitrite naturally present in human milk may contribute to neonatal gastric and cardiovascular physiology, particularly in the early weeks when an infant’s own nitric oxide production pathways are still maturing. Risk assessment for these compounds therefore involves a delicate balance between their potential harms at high doses and their physiological functions at the low doses typical of most diets.

Against this backdrop, the Hamadan team set out to quantify what exclusively breastfed infants actually receive. The analytical backbone of the study was ion chromatography, a technique that separates charged species in a liquid sample and quantifies them with high specificity, allowing nitrate and nitrite to be measured independently even in a biologically complex matrix like milk. The researchers paired these measurements with interview-based data on each infant’s age, body weight, and daily milk consumption, obtained from the mothers themselves. From these inputs they calculated the estimated daily intake of each compound for every age group, expressing exposure relative to body weight so that the results could be compared against established health-based guidance values.

A distinctive strength of the study lies in its statistical approach. Rather than relying on a single point estimate — which would assume, unrealistically, that every infant drinks the same amount of milk with the same contaminant concentration — the team used Monte Carlo simulation, a probabilistic method that samples repeatedly from the measured distributions of concentrations, consumption rates, and body weights. Each iteration of the simulation represents one plausible infant, and running the model thousands of times produces a full distribution of possible exposures rather than a single average. This matters because risk decisions based on averages can miss the sensitive tail of the population: the small infant who drinks relatively more milk per kilogram of body weight, or the mother whose milk carries higher concentrations. Probabilistic assessment reveals how likely it is that any individual infant would exceed a safety threshold, which is a far more informative question for public health.

The verdict from the simulations was consistent across the board. Nitrate and nitrite were detectable in every one of the 93 milk samples, confirming that exposure is essentially universal among the infants studied. However, the estimated daily intakes remained below the acceptable thresholds for both non-carcinogenic and carcinogenic risk in all three age groups. In other words, even accounting for the variability in milk composition and infant consumption captured by the Monte Carlo model, the exposure of exclusively breastfed infants in this population to nitrate and nitrite through milk alone does not appear to reach levels of toxicological concern. The authors also note that, given the role of these compounds as nitric oxide precursors, the concentrations observed may even contribute to normal physiological processes rather than undermining them.

The study also probed whether maternal characteristics influenced the contaminant burden in milk. Maternal body mass index, age, and educational level showed no significant association with nitrate or nitrite concentrations. This null result is itself informative: it suggests that, within this population, the variation in milk nitrate and nitrite is not easily explained by the demographic factors most often collected in biomonitoring studies, and that other determinants — perhaps dietary patterns, water sources, or metabolic factors — would need to be examined to understand what drives concentration differences between mothers. The work builds on earlier research by overlapping teams, including a 2025 probabilistic assessment of potentially toxic elements in breastfed infants in the same city, indicating a sustained effort to map the early-life exposome in an Iranian urban population.

Context from the broader literature helps calibrate these findings. Previous analyses have compared the nitrite and nitrate content of human milk with infant formula and bovine milk, finding that human milk contains measurable amounts of both compounds and that these may be biologically meaningful rather than incidental. Regulatory bodies such as the European Food Safety Authority have re-evaluated nitrate and nitrite as food additives, weighing genotoxicity concerns against physiological benefits, while epidemiological work has linked high nitrate in drinking water to adverse birth outcomes and cancer risk in adults. The Hamadan study adds a specific and previously underexplored data point: the exposure of exclusively breastfed infants via milk, which is distinct from the formula-and-water pathway that dominates most infant nitrate risk assessments.

The authors are careful to frame their conclusions within the limits of a pilot design. Ninety-three mother-infant pairs from a single city cannot represent an entire country, let alone the diverse exposure conditions that different geographies and water systems create. The team explicitly recommends further biomonitoring studies with larger sample sizes and broader geographic coverage to better characterize exposure pathways and long-term health implications. Still, for the parents lying awake at three in the morning wondering whether every trace chemical in breast milk is a threat, the study offers a measured dose of perspective: one of the most scrutinized nitrogen contaminants of the modern food system is present in human milk, but at levels that fall comfortably within safe limits — and may, in small physiological amounts, be part of what makes early human development work as it should.

Subject of Research: Nitrate and nitrite exposure through breast milk and probabilistic health risk assessment in exclusively breastfed infants

Article Title: A pilot study on nitrate and nitrite concentration in breast milk: probabilistic risk assessment in breastfed infants

Article References: Ghasemzadeh-Mohammadi, V., Moslemi, M., Behbahan, S. E. B., Vahidinia, A., Sharifi, A., & Abedi, A.-S. (2026). A pilot study on nitrate and nitrite concentration in breast milk: probabilistic risk assessment in breastfed infants. Environmental Science and Pollution Research. https://doi.org/10.1007/s11356-026-38283-w

Image Credits: AI Generated

DOI: 10.1007/s11356-026-38283-w

Keywords: breast milk, nitrate, nitrite, infant exposure, probabilistic risk assessment, Monte Carlo simulation, ion chromatography, methemoglobinemia, nitric oxide, breastfeeding, environmental contaminants, public health

The new analysis, published in BMC Pediatrics by Yılmaz Seçilmiş, Gülşah Kartal and Muhammet Sami Kayan of Erciyes University in Kayseri, Türkiye, takes the form of a scoping review. Following the Joanna Briggs Institute methodology and reporting to the PRISMA-ScR standard, the team searched PubMed, Scopus and Web of Science on 14 March 2026 without date or language restrictions, supplementing the search with hand-searching that included regional Turkish journals. Their PROSPERO-registered protocol (CRD420261340727) focused on a very specific triad: individual cases of methemoglobinemia following prilocaine administered for circumcision in infants aged zero to twelve months. Two reviewers screened studies independently, achieving an inter-rater agreement of kappa equal to 0.849, a level conventionally regarded as very strong.

The search identified twenty-four studies published between 1989 and 2026 that together documented thirty-eight cases. Strikingly, thirty-three of those cases—86.8 percent—came from Türkiye, a concentration the authors caution reflects the geography of the published literature rather than any measured difference in incidence. No reliable incidence rate can be extracted from case reports, which are subject to publication bias: dramatic recoveries get written up, routine events do not. Still, the consistency of the clinical picture across reports is what makes the review valuable. In every single case that reported presentation details, the defining feature was the same: cyanosis that did not respond to oxygen therapy, the classic fingerprint of methemoglobinemia.

The biochemistry explains why oxygen fails. Under normal conditions, hemoglobin carries iron in its ferrous (Fe2+) state, binding oxygen in the lungs and releasing it in tissues. Oxidizing agents can convert some of that iron to the ferric (Fe3+) state, producing methemoglobin, which binds oxygen tightly but cannot unload it. Worse, methemoglobin shifts the oxygen-dissociation curve of the remaining normal hemoglobin to the left, compounding the tissue hypoxia. Infants are especially vulnerable because their red cells contain fetal hemoglobin, which oxidizes more readily, and because the NADH-dependent reductase pathway that normally reduces methemoglobin back to hemoglobin is not fully mature in the first months of life. Glucose-6-phosphate dehydrogenase (G6PD) deficiency, which is more common in some populations, adds a further layer of susceptibility.

Prilocaine enters this picture because it is an amide local anesthetic whose metabolites, notably o-toluidine, are known oxidizing agents capable of overwhelming red-cell defenses. In the reviewed cases, prilocaine was given by injection—typically as a dorsal penile nerve block or subcutaneously—in 89.5 percent of reports, while 10.5 percent involved topical application of EMLA, the eutectic mixture of lidocaine and prilocaine that is widely marketed for procedural pain relief in infants. Both routes have been implicated, though dosing detail was surprisingly scarce: the prilocaine dose was reported in only 44.7 percent of cases, a gap the authors highlight as a failure of case-report completeness with direct consequences for understanding dose-response relationships.

The severity of the episodes was considerable. Among thirty-seven cases with laboratory data, the mean methemoglobin fraction was 33.6 percent, with individual values ranging from 11.9 to 62.0 percent. Normal levels sit below about one to two percent, and clinical signs such as cyanosis generally appear above ten to fifteen percent, with levels above roughly thirty percent considered clinically significant and above fifty percent potentially life-threatening. That the reported episodes reached such heights underscores how quickly a routine analgesic injection in a healthy infant can escalate into a pediatric emergency requiring immediate recognition and intervention.

Treatment in the published cases followed two main paths. Methylene blue, the established first-line antidote for clinically significant acquired methemoglobinemia, was administered in twenty cases. The drug works as an exogenous electron carrier, accepting electrons from NADPH via the hexose monophosphate shunt and reducing methemoglobin back to functional hemoglobin—essentially providing the infant red cell with an enzymatic shortcut it cannot yet perform efficiently on its own. Ascorbic acid, a slower-acting reducing agent described mainly in isolated reports and sometimes used as adjunctive therapy, was given in twenty-one cases, either alone or in combination with methylene blue. Every published case ended in full recovery, but the authors are careful to note that uncontrolled case reports cannot disentangle the effects of treatment from spontaneous recovery, supportive care, or the natural course of the condition, and no claim of comparative efficacy between antidotes can be made from such data.

To widen the lens beyond the formal case literature, the team also queried three major pharmacovigilance databases: the World Health Organization’s VigiBase, the US Food and Drug Administration’s FAERS, and the European EudraVigilance system. These spontaneous-report databases contained reports extending beyond the circumcision-specific literature, including some with fatal outcomes. The authors treat these as contextual reference only, stressing that spontaneous reports cannot confirm causality, cannot verify the procedural context, and must not be used as a quantitative comparator. The distinction matters: pharmacovigilance signals can justify scrutiny, but they are not evidence of risk magnitude, and conflating them with verified clinical case series distorts the risk picture for both clinicians and parents.

The completeness assessment is one of the review’s most practically useful contributions. Methemoglobin level and treatment were each documented in 97.4 percent of cases, reflecting awareness that both are central to the diagnosis and management narrative. But the prilocaine dose was reported in fewer than half the cases, and other details—timing of onset, use of screening for G6PD deficiency, cumulative exposure, and follow-up—varied widely. The authors argue that better-structured case reporting would allow future syntheses to move beyond description toward genuine risk characterization. Their quality appraisal used the Murad et al. tool for case reports, and the full charted dataset, search strategy and PRISMA-ScR checklist are available as supplementary files with the open-access article.

For clinical practice, the authors’ conclusions are measured rather than alarmist. The published reports describe a recurring, recognizable presentation—cyanosis poorly responsive to oxygen in a recently anesthetized infant—but cannot establish how often it occurs or which treatment is superior. Methylene blue remains the established first-line antidote for clinically significant acquired methemoglobinemia. Depending on the clinical context, indication and dose, minimizing prilocaine exposure and observing infants for post-procedure cyanosis may be considered, with clinical decisions individualized. The uniformly positive outcomes recorded in the literature, the team warns, may well reflect publication and survivorship bias rather than an inherently benign course. In other words, the absence of documented deaths in the case series should not be read as reassurance; it may simply mean that the worst outcomes go unreported. The review received no specific funding, and the authors declare no competing interests. Its message for clinicians is simple and urgent: when a circumcised infant turns blue and oxygen does not help, think methemoglobinemia, check a methemoglobin level, and act fast.

Subject of Research: Prilocaine-associated methemoglobinemia in neonates and infants undergoing circumcision

Article Title: Prilocaine-associated methemoglobinemia after neonatal and infant circumcision: a scoping review of published cases with pharmacovigilance context

Article References: Seçilmiş, Y., Kartal, G., & Kayan, M. S. (2026). Prilocaine-associated methemoglobinemia after neonatal and infant circumcision: a scoping review of published cases with pharmacovigilance context. BMC Pediatrics. https://doi.org/10.1186/s12887-026-07741-2

Image Credits: AI Generated

DOI: 10.1186/s12887-026-07741-2

Keywords: prilocaine, methemoglobinemia, circumcision, neonates, infants, methylene blue, ascorbic acid, EMLA cream, pharmacovigilance, scoping review, cyanosis, pediatric emergency