Phosphorus Balance in Rats with Hypophosphatemia Induced by Lanthanum Carbonate ()
1. Introduction
Phosphorus is a major essential mineral in human and animal nutrition. Inadequate phosphorus intake is expressed as hypophosphatemia and induces anorexia, anemia, muscle weakness, bone pain, rickets and osteomalacia, general debility, increased susceptibility to infection, paresthesias, ataxia, confusion, and even death [1]. To prepare an animal model with a low phosphorus status, several kinds of commercial low phosphorus diet are used [2,3]; however, the detailed composition of these commercial low phosphorus diets is unclear as information on the ingredients of the diet except for phosphorus is not available. Accordingly, it has not been evaluated whether nutrients other than phosphorus are associated with phenomena observed in the experimental low phosphorus status. In an experimental nutrition study using rats, to estimate the intake of each nutrient precisely, a semi-purified diet such as the AIN93G or AIN93M diet is used as the standard diet [4]. When a low phosphorus diet is prepared based on the AIN93 diets, potassium should be supplemented because potassium phosphate is removed from the mineral mixture; it is not possible to avoid changing mineral composition other than phosphorus. In addition, a major source of phosphorus in the AIN93 diets is casein-bound phosphate; about 30% to 50% of phosphorus is derived from casein; therefore, a severely low phosphorus diet cannot be prepared based on the AIN93 diets.
Lanthanum, a rare-earth metal, has been used industrially as a raw material of ceramic capacitors, optical lenses and semiconductors. On the other hand, trivalent lanthanum ion has high affinity to phosphate ion and has been used as a chemical reagent to suppress the interference of phosphate in the determination of calcium in atomic absorption spectrometry [5]. Recently, attention has focused on the high affinity between lanthanum and phosphate, and the use of lanthanum carbonate as a phosphate-binder to decrease the absorption of dietary phosphate in patients with chronic renal failure receiving hemodialysis [6]. This indicates that oral administration of lanthanum carbonate can induce a low-phosphorus status in normal animals who have ingested the AIN93 diets; however, because lanthanum carbonate was developed as a phosphate-binder for human patients with renal failure, the purpose of animal studies using lanthanum carbonate is not to induce hypophosphatemia in normal animals but to prevent hyperphosphatemia and its complications in animals with experimental renal failure [7,8].
Although serious adverse side effects have not been observed in any human cases of long-term lanthanum administration [9], some researchers have pointed out the adverse effects of lanthanum and the harmfulness of long-term lanthanum administration mainly based on animal experiments; lanthanum decreased liver function [10,11]. Generally, adverse effects induced by chemical substances may occur more readily in animals fed semipurified diets than those fed commercial conventional diets composed of natural products containing several phytochemicals. Accordingly, it is necessary to examine the effect of lanthanum carbonate administration on liver function in rats fed semi-purified diets when lanthanum carbonate is used as an inducer of a low phosphorus status.
In the present study, to examine whether lanthanum carbonate can be used to induce a low phosphorus status in experimental animals fed a casein-based semi-purified diet, we measured urinary and fecal phosphorus excretion and phosphorus balance in normal rats fed an AIN93M diet supplemented with lanthanum carbonate and discussed the interaction between lanthanum and phosphorus in the gut. In addition, we examined the effect of lanthanum carbonate administration on liver function in these rats.
2. Materials and Methods
2.1. Reagents
Lanthanum carbonate (La2(CO3)3·xH2O) was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Lanthanum content in this reagent was 50%. The ingredients of animal diets were purchased from Oriental Yeast Co. (Tokyo, Japan). Lanthanum and rhodium standard solution, as well as metal-free grade HNO3 and HClO4 were purchased from Wako Pure Chemical Industries (Osaka, Japan).
2.2. Animal Experiments
The experimental protocol was reviewed and approved by the Animal Ethics Committee of Kansai Medical University and followed the “Guide for the Care and Use of Experimental Animals” of the Prime Minister’s Office of Japan. Experimental animals were housed in plastic cages in a room under a controlled 12 h light (8:00 to 20:00) and dark cycle at a temperature of 23˚C to 24˚C and humidity of 60%. The animals were given experimental diets and deionized water ad libitum during the entire experimental period.
Eighteen male 8-week-old Wistar rats (Shimizu Lab. Supplies Co. Ltd., Kyoto, Japan) were divided into three groups. The mean and SD of the initial body weights of the eighteen rats were 316 and 12 g, respectively. One group was fed a casein-based semi-purified basal diet (AIN93M diet [4]) and the other two groups were fed the basal diet supplemented with 0.45% or 0.90% (0.032 or 0.065 mmol/g, respectively) of lanthanum as lanthanum carbonate for 4 weeks. Analysis showed that the basal AIN93M diet contained phosphorus at a level of 3.24 mg/g (0.105 mmol/g). Lanthanum could not be detected in the basal diet. In the third week of feeding, the rats were transferred to metabolic cages and urine and feces samples were collected for 3 days. After the end of the feeding periods, the rats were anesthetized with sodium pentobarbital, blood was collected from the abdominal aorta, and the liver, kidney, brain, spleen, small intestine and femur were excised, washed with saline, blotted and weighed A part of each blood sample was transferred to a heparinized tube and subjected to hemoglobin determination. Serum was separated from the remainder of the blood and subjected to serum biochemical tests (total protein, triglyceride, total cholesterol, alanine aminotransferase, aspartate aminotransferase, urea nitrogen, creatinine, iron, total iron-binding capacity, calcium, magnesium and phosphorus). The hemoglobin measurement and serum biochemical tests were performed by a commercial service system (Japan Medical Laboratory, Osaka, Japan).
2.3. Analysis of Lanthanum and Phosphorus
Up to 2 g of the organ samples was heated with 10 mL metal-free HNO3 until the disappearance of insoluble components, and then 2 mL metal-free HClO4 was added to the digestion mixture. The digestion mixture was further heated until the appearance of the white vapor of HClO4. The volume of the digest was made up to 10 mL with pure water. Approximately 1 g basal diet or feces was digested with metal-free HNO3 and HClO4 similarly to the digestion of organs. Urine was diluted with pure water.
Lanthanum in the digests and the diluted urine was determined by inductively coupled plasma mass spectrometry (ICPMS) with direct nebulization. The ICPMS operating conditions were as follows: instrument, ICPM- 8500 (Shimadzu, Kyoto, Japan); forward power, 1200 W; coolant gas flow rate, 7.0 L/min; auxiliary gas flow rate, 1.5 L/min; nebulizer gas flow rate, 0.58 L/min; sampling depth, 5.0 mm; integration time, 2.0 s; number of run, 20; mode of analysis, pulse; isotopes monitored, 139La. A rhodium isotope (103Rh) was used as an internal standard. In the organ samples, the detection limit was 4 ng/g wet tissue.
Phosphorus in the digested samples and the diluted urine was determined by colorimetric method using vanadomolybdate [12].
2.4. Statistics
Experimental data were assessed by one-way analysis of variance. When the F value was significant (p < 0.05), the Tukey-Kramer multiple range test was performed to determine which pairs of the means were significantly different (p < 0.05). When the analytical values were below the detection limit, a half-value of the detection limit was used. These statistical tests were performed using a personal computer (eMac; Apple Computer, Cupertino, CA) with the operating system Mac OS 9.2 and statistical program package StatView-J version 5.0 (Abacus Concept, Berkeley, CA).
3. Results
Table 1 shows whole body and several organ weights of rats fed experimental diets with or without lanthanum carbonate for 4 weeks. No influence of lanthanum administration was observed.
Lanthanum concentrations in several organs are summarized in Table 2. Lanthanum was not detected in the organs of rats fed the basal diet without lanthanum. In rats administered lanthanum, the highest lanthanum concentration was observed in the liver followed by the femur and kidney. For the brain, lanthanum was detected in
Table 1. Effect of dietary lanthanum on body and organ weights of rats.
Table 2. Lanthanum contents in several organs of rats fed experimental diets with or without lanthanum carbonate for 4 weeks.
two of six rats fed the 0.90% lanthanum diet but the difference in the lanthanum concentration between the rats fed 0.90% lanthanum diet and other diets was not significant. An increase in the dietary lanthanum level showed a tendency for the concentration of organ lanthanum to increase but the difference between the 0.45% and the 0.90% lanthanum administration groups was not significant.
Table 3 shows the effects of lanthanum administration on hemoglobin and serum biochemical tests of the rats. Serum phosphorus was significantly decreased by lanthanum administration and the decrease was dependent on the dose of lanthanum administered; serum phosphorus was decreased 20% or 36% by dietary lanthanum at 0.45% or 0.90%, respectively. On the other hand, serum calcium was significantly increased in rats fed the 0.90% lanthanum diet. Other serum biochemical tests and hemoglobin were not influenced by lanthanum administration.