Introduction:

The important role played by calcium in mammalian organisms is now well recognized. Calcium is the major cation within bone mineral. Calcium also plays an important role as an intracellular messenger in many systems and cells^[1,2]. Although calcium is a microelement closely related to the health of the human body, the average actual calcium intake in China is, on average, 405 mg/d, which is only approximately 50% of the recommended dietary allowance^[3,4]. The calcium deficiency situation is especially serious in children, adolescents, and pregnant women, as reported by the Third National Nutritional Investigation in China (1992). However, recent studies have shown that some diseases may be caused by excess calcium supplementation (especially in patients with osteoporosis)^[5-8]. Therefore, the accuracy of calcium absorptive measurements is very meaningful for reasonable calcium supplementation and prevention of diseases caused by calcium metabolism. Due to the poor distinction between calcium sources, lower measurement sensitivity, and radioactive damage that results from traditional calcium absorptivity measurement techniques, it is difficult to carry out accurate, systematic, and long-term research on this topic^[9-11].

A common method of determining calcium absorptivity is using the relation ζ = (I − F)/I, where ζ is the apparent calcium absorptivity, I is the calcium intake, and F is the calcium content in feces. However, F can be divided into two parts according to the relation F = F_i+ F_e, where F_i is calcium of dietary origin and F_e is calcium of endogenous origin^[12-14]. Thus, ζ cannot be used to replace calcium absorptivity accurately. We used a more reliable relation, η = (I − F_i)/I = (I − F + F_e)/I, to measure the calcium absorptivity of rats, where η is the true calcium absorptivity. The primary difficulty in the determination is the measurement of F_e. A tracer should be used to label the calcium of endogenous origin and calculate the proportion of F_e in F. Because the radiation dose is small and it is not present in biological systems, ⁴¹Ca can be used for biological tracer studies^[15-17]. Currently, AMS (accelerator mass spectrometry) is the most effective method of measuring ⁴¹Ca. Based on the high sensitivity of ⁴¹Ca measurements made using CIAE-AMS and the innovative method of labeling endogenous calcium with ⁴¹Ca, the calcium absorptivity of rats was studied in this work.

Experimental Section:

Measurement Method

Detailed AMS conditions for ⁴¹Ca measurements are described elsewhere^[22]. Briefly, the ⁴¹CaF₃⁻ ions were selected by the injection system and injected into the accelerator. The negative molecular ions were accelerated by tandem terminal voltage (8.27 MV). Stripping foil (5 μg/cm²) was employed to break up the molecular ions and to produce atomic ions with high charge states. The resulting positively charged ions were further accelerated at the same terminal voltage. A 90^o double focusing high energy analyzing magnet was used to select ⁴¹Ca⁷⁺ with an energy of 61.39 MeV. After a switching magnet, the ⁴¹Ca⁷⁺ was transported to the AMS beam line, and then the ⁴¹Ca⁷⁺ was selected using a 15^o electrostatic detector and finally detected with an ionization chamber. The ⁴¹Ca optical guidance in the accelerator was simulated with a ⁴⁰Ca beam using the magnetic rigidity of ⁴¹Ca.

The samples were disposed of by ashing in a muffle (the sample was prepared using an identical chemical procedure to that of the ⁴¹Ca sample), and then processed in a Type AA700CRT atom absorption spectrophotometer (Perkin ELMER, Singapore) to determine the Ca²⁺ contents in the feces. The results of the measurement and a linear fitting curve of standard samples are shown in Fig. 1.

Fig. 1.Full-screen View

Linear fitting curve of standard samples

A linear fit was obtained in Fig. 1:

$y=34.726x,$ (1)

where y is the calcium content, x is the absorbance, and the linear regression coefficient was R² = 0.9973; this equation was used to calculate the calcium content in the actual sample.

Results and Discussion:

Fig. 2 shows the ratio of ⁴¹Ca to ⁴⁰Ca in the rat vs feces after injecting ⁴¹Ca. The ratio of ⁴¹Ca to ⁴⁰Ca in feces tended to reach equilibrium on the 28th day after injection. As a result, the average value of ⁴¹Ca/⁴⁰Ca in feces can be used to represent the actual value from the 35th to 49th day (including the 42nd day) after injection. On the 42nd day after injection, the rats were exclusively fed a Ca²⁺-poor basic diet and deionized water to obtain the ratio of ⁴¹Ca to ⁴⁰Ca in feces without calcium intake. This value can be used as the ⁴¹Ca/⁴⁰Ca ratio of endogenous origin.

Fig. 2.Full-screen View

The ratio of ⁴¹Ca/⁴⁰Ca in the rat vs feces after injecting ⁴¹Ca

The calcium absorption rate of each rat was replaced by the absorption rate of calcium per 100 g of rat to account for the differences in weight between rats. The results of these measurements are shown in Table 2.

Using these data, we obtained the following results in Table 3.

Conclusion:

Generally, the actual calcium absorptivity is significantly higher than the apparent calcium absorptivity in rats (p < 0.05). Based on the actual calcium absorptivity data, the absorption rates of calcium carbonate and calcium citrate are approximately equal at the same dose, indicating that the calcium absorption rate in SD rats is not influenced by the chemical formula of calcium supplements. On the other hand, the amount of absorbed calcium in the intestine depended on the calcium intake. When intake is low, active transcellular calcium transport in the duodenum is upregulated, and a larger proportion of calcium is absorbed by the active process than by the passive paracellular process that prevails in the jejunum and ileum. Bioavailability of the calcium source—digestibility and solubilization—plays a role under low calcium intake conditions but is relatively unimportant when calcium intake is high. Our experimental results are consistent with this theory within a certain range: the greater the calcium intake, the lower the calcium absorption rate, and small doses of calcium can be nearly completely absorbed. This match between theory and experiment validates the method of measuring calcium absorptivity by ⁴¹Ca labeling endogenous calcium.

In conclusion, a monitoring method for endogenous calcium absorptivity using rats and ⁴¹Ca tracing was developed based on CIAE-AMS, and encouraging results were obtained. The method is currently restricted by factors such as limitations in biological diversity and sample size. A more detailed experimental program is being developed, and further exploration will be implemented in the near future.