Calcium-rich dairy matrix protects better than mineral calcium against colonic luminal haem-induced alterations in male rats

Epidemiological studies strongly link red meat consumption to increased colorectal cancer (CRC) risk. Hemoglobin in red meat, specifically its heme iron content, is implicated in this risk. Heme iron, unlike non-heme iron, catalyzes lipid oxidation in the colon, producing reactive aldehydes like 4-hydroxy-2-nonenal (4-HNE). These aldehydes cause free radical release, cytotoxicity, genotoxicity, gut barrier defects, and preneoplastic lesions. Previous research using rodent models demonstrated that calcium salts can prevent heme-induced colorectal carcinogenesis by reducing heme iron bioavailability. This study aimed to investigate whether calcium delivered through a dairy matrix, a naturally occurring calcium-rich food source, offers similar or enhanced protection compared to calcium salts against the negative effects of heme iron.

Existing literature establishes a strong link between red meat consumption and colorectal cancer risk. Heme iron, a component of red meat, is believed to be a key driver of this increased risk due to its ability to initiate lipid peroxidation in the colon. This process leads to the formation of reactive aldehydes, causing cellular damage and potentially promoting carcinogenesis. Studies have shown that calcium salts can mitigate these effects by reducing heme iron bioavailability. However, the impact of calcium delivered via a food matrix, such as dairy products, remained unclear. This study addresses this knowledge gap by comparing the protective effects of calcium from both sources.

A three-week intervention study was conducted using male Fischer 344 rats. Rats were assigned to six groups (n=8 per group) and fed diets varying in iron source (ferric citrate or haemoglobin) and calcium source (control, mineral calcium, or dairy calcium). Dietary composition was carefully controlled, with differences primarily in the iron and calcium sources. Body weight, food and water intake were monitored weekly. Faecal and urinary samples were collected to measure biomarkers of lipid peroxidation (TBARS, DHN-MA). Intestinal permeability was assessed using ⁵¹Cr-EDTA. Colonic mucosal gene expression of relevant genes (Hmox1, IL-18, Slc7a11, Akr1b8) was analyzed via qPCR. Faecal microbiota composition was analyzed via 16S rRNA gene sequencing and bioinformatic analysis, including alpha and beta diversity analysis and correlation analysis with physiological parameters. Statistical analysis using two-way ANOVA with post-hoc tests was performed to compare groups.

The haemoglobin-enriched diet (compared to ferric citrate) increased faecal, mucosal, and urinary lipoperoxidation biomarkers, indicating increased heme iron bioavailability and oxidative stress. This was accompanied by gut microbiota dysbiosis. The addition of calcium to the haemoglobin-enriched diet reduced haem iron bioavailability and attenuated lipoperoxidation. Importantly, calcium provided through the dairy matrix was more effective than mineral calcium in reducing haem iron bioavailability and preventing lipoperoxidation. Microbiota analysis revealed significant alterations driven by both iron form and calcium source. Specific bacterial families (Peptococcaceae, *Eubacterium coprostanoligenes* group, Bifidobacteriaceae) showed correlations with lipoperoxidation biomarkers. The dairy matrix diet also promoted the growth of specific bacterial communities (*Frisingicoccus caecimuris*, *Macrococcus* spp., *Exigobacterium* spp.) not observed in other groups.

The study findings confirm the pro-oxidant effects of heme iron in the gut and the protective role of dietary calcium. The superior efficacy of dairy matrix-derived calcium suggests that the dairy matrix itself may contain additional bioactive components contributing to its protective effect. The observed changes in the gut microbiota composition in response to different diets also highlight the complex interplay between diet, gut microbiota, and host health. The correlations between specific bacterial communities and lipoperoxidation biomarkers further support the link between dietary components, gut microbiota, and oxidative stress. These results provide valuable insights into the protective effects of dairy products against the adverse effects of red meat consumption and could inform dietary recommendations for colorectal cancer prevention.

This study demonstrates that dietary calcium from a dairy matrix is more effective than mineral calcium in mitigating the negative effects of heme iron on the gut. This superior protection is likely due to a combination of reduced heme iron bioavailability and potential synergistic effects of other bioactive components within the dairy matrix. Further research should investigate the specific mechanisms behind this enhanced protection, focusing on the interaction of dairy components with heme iron and the modulation of gut microbiota.

This study utilized a rodent model, which may not fully replicate human physiology and dietary patterns. The relatively short duration of the intervention (3 weeks) might limit the detection of long-term effects. The specific components of the dairy matrix responsible for the enhanced protection require further investigation. The study focused on male rats, limiting generalizability to females.