Sex-dependent effects of dietary phosphorus source on bone histomorphometry and calcium balance in rats

Excess dietary phosphorus, particularly from highly bioaccessible inorganic sources, has been shown to adversely affect calcium homeostasis and bone health. However, the extent to which sex differences may interact with the effects of dietary phosphorus on calcium balance and bone has not been well characterized. Our objective was to determine sex differences and interaction effects with dietary phosphorus source on calcium balance and bone parameters in a pilot study in healthy rats. We hypothesized a sex*diet interaction where a high bioaccessible phosphorus (HBP) diet compared with a low bioaccessible phosphorus (LBP) diet would negatively affect females to a greater extent than males. This investigation was ancillary within a pilot study that had its primary aim to determine sex differences and dietary phosphorus source effects and their interaction on intestinal phosphorus absorption and balance. Sprague-Dawley rats (N=24 total) were studied in a 2x2 factorial design experiment where male (n=12) and female (n=12) rats were randomized by sex to one of two diets (HBP or LBP) at 8-9 weeks-of-age, and continued on the experimental diets for 2 weeks. Thus, end-of-study outcomes were assessed around 10-11 weeks-of-age. The HBP and LBP diets both contained 0.7% P and 1.0% Ca, but differed in their main sources of phosphorus (casein for HBP and grain/soy for LBP). The diets were analyzed for P bioaccessibility, which confirmed high bioaccessibility for the HBP diet and low bioaccessibility for the LBP diet (80% and 35%, respectively). At the end of the study, a four-day metabolic cage study was performed to assess calcium balance (dietary calcium intake minus urine and fecal calcium, mg/d). Calcium was measured in diet, urine, and feces by microwave plasma-atomic emission spectrometry. Bone was double-labeled with 1% calcein solution (1.5 mg/100 g body weight) by subcutaneous injection for dynamic histomorphometry of the tibia. Results showed expected sex differences in the dynamic bone parameters of mineralizing surface normalized to bone surface (MS/BS), mineral apposition rate (MAR, μm/d), and bone formation rate normalized to bone surface (BFR/BS, μm/d) in males compared with females (all, p< 0.001), prior to adjusting for body weight. However, adjusting for body weight eliminated sex differences on all three parameters, and revealed a marginally significant sex*diet interaction effect (p=0.047) where males on the HBP diet had higher values compared with males on the LBP diet (p=0.03), and females did not differ by diet. Calcium balance results showed patterns consistent with the histomorphometry findings, with a significant main effect for sex prior to adjusting for body weight (M >F), that was eliminated after adjusting for body weight. There was a significant sex*diet interaction for percent net Ca absorption, where males on the HBP diet had greater values compared with females on the HBP diet (p=0.038). There was also a significant sex*diet interaction for urine Ca excretion, where females on the LBP diet had greater urine Ca compared with females on the HBP diet (P=0.025) and males on either diet (P< 0.02), and there was no effect of diet on urine Ca in males. These effects largely persisted after adjusting for net phosphorus absorption. These pilot study findings suggest sex-specific effects of dietary phosphorus source on calcium balance components and bone parameters, but the pilot nature of the study and small sample size necessitate further studies to confirm these effects and implication for bone health. This study was funded by a seed grant from the University of Minnesota College of Food, Agricultural and Natural Resource Sciences. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.