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Contribution of diet to the dosing time-dependent change of vitamin D3-induced hypercalcemia in rats
Life Sciences 68 (2000) 579–589
Contribution of diet to the dosing time-dependent change of vitamin D3-induced hypercalcemia in rats Shuichi Tsuruoka*, Koh-ichi Sugimoto, Akio Fujimura Department of Clinical Pharmacology, Jichi Medical School, Tochigi 329-0498, Japan Received 25 October 1999; accepted 10 August 2000
Abstract We have recently reported that the degree of hypercalcemia as an adverse effect induced by a single large-dose of active vitamin D3 varied with its dosing time without alteration in therapeutic effect for secondary hyperparathyroidism in patients with chronic renal failure. The present study was conducted to elucidate an effect of intestinal calcium (Ca) absorption on the chronopharmacological profiles of vitamin D3. 1, 25-dihydroxy-cholecalciferol (D3, 2 mg/kg) or vehicle alone was orally administered at two different times (2 and 14 hours after lights on; HALO) to male Wistar rats (n510) kept in rooms with a 12 h light-dark cycle. Blood samples for serum Ca concentration were taken before and 3, 6, 9, and 12 hours after the administration. Urine was collected for 6 hours after dosing. An identical protocol was repeated using the same animals after 16 hours fasting by a cross-over fashion. Under free-fed condition, basal concentration of serum Ca was higher at a resting period (lights on) than during an active period (lights off). Serum Ca reached its peak at 6 hours after dosing in both timings, while the value was significantly higher in the 2 HALO trial than in the 14 HALO trial. Area under the serum Ca concentration-time curve from 0 to 12 hours (AUC0-12h) and urinary excretion of Ca for 6 hours were also significantly higher in the 2 HALO trial than in the 14 HALO trial. When fasted, basal Ca concentration was reduced compared with the free-fed condition, while the daily variation was maintained. Serum Ca concentration profiles from 3 to 12 hours after dosing were not significantly different between the 2 HALO and 14 HALO trials. The AUC0-12h of serum Ca or its urinary excretion was not different between both trials. Serum concentrations of parathyroid hormone and total protein, measured before and 6 hours after the dosing were not affected by the dosing schedule. We have concluded that intestinal Ca absorption is a major factor for the chronopharmacological phenomenon of D3-induced hypercalcemia in intact rats, while intestinal and renal involvement may be relatively small in the mechanism of the intrinsic diurnal variation of serum Ca. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Vitamin D; Chronopharmacology; Hypercalcemia; Ca absorption
* Corresponding author: Department of Clinical Pharmacology, Jichi Medical School, 3311 Yakushiji, Minamikawachi, Kawachi, Tochigi 329-0498, Japan. Tel.: 182-285-58-7388; fax: 181-285-44-7562. E-mail address: [email protected] (S. Tsuruoka) 0024-3205/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 9 6 4 -4
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Introduction Vitamin D is widely prescribed for the treatment of osteoporosis and secondary hyperparathyroidism in patients with chronic renal failure. Especially for secondary hyperparathyroidism, an intermittent high-dose treatment (namely “pulse therapy”) with active vitamin D3 is performed to reduce hyperplasia of parathyroid gland as well as parathyroid hormone (PTH) concentration in blood [1, 2], while hypercalcemia induced by vitamin D sometimes interrupts the treatment [3, 4]. On the other hand, serum calcium (Ca) concentration possesses a circadian rhythm which shows a peak in the morning and a trough at evening in both human and rats [5–8]. We have recently reported the chronopharmacological profiles of vitamin D3 in chronic renal failure patients with secondary hyperparathyroidism. This study showed that the elevation in serum Ca concentration following a single oral administration of vitamin D3 for pulse therapy is significantly smaller by dosing in the evening than in the morning without any loss of treatment efficacy [9]. Regarding the mechanism of the chronopharmacological effect of vitamin D3, we also found that the profile of serum 1, 25-dihydroxy vitamin D3 concentration was not different between the dosing times, suggesting that pharmacokinetic-unrelated factor(s) might be responsible for the phenomenon. The mechanism, however, is not fully understood. The purpose of this study was to evaluate 1) the chronopharmacological effect of vitamin D3 on serum Ca concentration in normal rats, 2) the contribution of intestinal Ca absorption to this phenomenon by fasting the animals, and 3) the influence of a dosing time of vitamin D3 on renal Ca excretion, serum concentrations of PTH and total protein. Methods Animals Male Wistar rats (350–400 g; Japan Clea Co. Ltd., Tokyo, Japan) were maintained for more than two weeks in two separate rooms under a 12 h light-dark cycle (n55 in each). In room 1, lights were on at 07:00 and off at 19:00. In room 2, lights were on at 19:00 and off at 07:00 h, respectively [10]. Rats were given free access to standard rat chow (CE-2, containing 1.18 % Ca and 2.5 IU/g vitamin D3, Japan Clea Co. Ltd., Tokyo, Japan) and deionized water. Experimental protocol On the experimental day, chow was removed and the animals were placed into other cages to measure body weight about one hour prior to drug dosing. 1,25-dihydroxy-cholecalciferol (D3; Sigma, St. Louis, MO, USA, 2 mg/Kg) or vehicle was given by gavage at 2 and 14 hours after lights on (HALO). The dose of the drug was selected on the basis of significant raise in serum Ca concentration in our preliminary study (data not shown). Blood samples for serum Ca concentration were taken from the tail vein before and 3,6,9 and 12 h after dosing. To minimize stress by blood loss, sample volume was restricted to 200ml in each point. Serum concentrations of PTH and total protein were also measured before and 6 hours after the dosing. Then the room was exchanged and identical protocols were performed in a cross-over fashion after more than two-weeks acclimatization period. It is reported that most physiolog-
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ical parameters, such as neuronal, humoral, motor, and behavioral functions are completely re-synchronized within two weeks after changing lighting schedule in the animals [10–14]. For collection of urine samples, 3 % body weight (vol./wt.) of deionized water was given to the animals by gavage at 30 min. after dosing of D3 or vehicle, and the animals were placed individually in metabolic cages for 6 h. To determine a contribution of intestinal Ca absorption, the identical protocols were repeated using same rats which were fasted for more than 16 h before the administration. Each protocol was performed after a two-weeks acclimatization period in a cross-over fashion. Serum and urine samples were stored at 2208C until assay. Experiments were conducted in accordance with the Jichi Medical School Guide for Laboratory Animals. Assay methods Ca concentrations in serum and urine were measured by orthocresolphthalein complexone method [15]. Urine creatinine concentration was measured by Jaffe’s reaction [16]. Area under the serum Ca concentration-time curve from 0 to 12 h (AUC0-12h) was calculated by trapezoidal rule. Serum PTH concentration was determined by immunoradiometric assay (Rat PTH IRMA kit, Immutopics, Inc. San Clemente, CA, USA). Its normal range of the concentration was 10–40 pg/ml. Total protein concentration was determined by Biuret’s method. Statistics Data were expressed as the mean 6 SE. Comparisons were performed with ANOVA and Student’s t-test as appropriate. P,0.05 was regarded as significant. Results Serum Ca concentration profile under a free-fed condition The protocols were completed in all animals without major difficulties. Figure 1 shows the serum Ca concentration profiles in the free-fed state. The mean serum Ca concentration before dosing was significantly higher during the resting period (10.70 6 0.05 mg/dL at 2 HALO) than during the active period (10.43 6 0.04 mg/dL at 14 HALO, P,0.01; one-way ANOVA). Following single oral dosing of D3, the serum Ca concentration significantly increased and reached its peak at 6 h postdose in both dosing trials. The maximum concentration of serum Ca was significantly (P,0.01) higher in the morning trial (11.67 6 0.13 mg/dL, dosed at 2 HALO) than in evening (11.36 6 0.12 mg/dL, dosed at 14 HALO). The concentration gradually declined after 9 h but still remained higher than the basal level up to 12 h in both trials. The serum Ca concentration showed a diurnal variation after dosing of vehicle alone. In the morning trial, the serum Ca concentration peaked at 5 HALO and thereafter declined gradually toward 14 HALO. On the other hand, this parameter gradually increased from 14 HALO to 2 HALO in the evening trial. Serum Ca concentration profile under a fasting condition The serum Ca concentration profile after 16 h fasting was significantly different from that of under the free-fed condition (Figure 2). Before D3 dosing, the serum Ca concentrations
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Fig. 1. Serum calcium (Ca) concentration (A) and the change of Ca concentration (B) profiles after a single oral dosing of 1, 25-dihydroxy vitamin D3 (D3, 2mg/Kg) or vehicle at 2 HALO (morning) or 14 HALO (evening) to Wistar rats freely fed. Mean 6 SE, n510. HALO, hours after lights on.
were lower than those at 2 and 14 HALO under the free-fed condition. However, the difference between 2 and 14 HALO was still observed (10.47 6 0.05 mg/dL at 2 HALO, 10.21 6 0.03 mg/dL at 14 HALO, P,0.01). Although serum Ca concentration increased following D3 in both trials , the time to peak concentration was significantly delayed while fasting. Maximum Ca concentrations in the fasted trial were significantly lower than those in the free-fed trial. This parameter at the 2 and 14 HALO didn’t differ significantly. Serum Ca concentrations while fasting were lower than during the free-fed condition after vehicle alone, and the daily variation was still observed.
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Fig. 2. Serum calcium (Ca) concentration (A) and the change of Ca concentration (B) profile following a single oral dosing of 1, 25-dihydroxy vitamin D3 (D3, 2mg/Kg) or vehicle at 2 HALO (morning) or 14 HALO (evening) to Wistar rats fasted for 16 h before treatment. Mean 6 SE, n510. HALO, hours after lights on.
The AUC0-12h data are summarized in Figure 3 and Table. Under free-fed condition, the AUC0-12h following D3 was significantly higher in the 2 HALO than in the 14 HALO trials, while the difference was diminished under the fasting condition. This result indicates that intestinal Ca absorption is the major factor for the change of AUC. Urinary Ca excretion Urinary excretion of Ca was also different between the free-fed and fasted conditions (Figure 4 and Table). D3 enhanced the Ca excretion under both dietary conditions. Under the free-fed condition, Ca excretion for 6 h after dosing of D3 at 2 HALO (1.43 6 0.16 mg/6 h)
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Fig. 3. Area under the serum calcium concentration-time curve from 0 to 12 h (AUC0-12h) following a single oral dosing of 1, 25-dihydroxy vitamin D3 (D3, 2mg/Kg) or vehicle. These agents were given at 2 HALO (morning) or 14 HALO (evening) to Wistar rats freely fed or fasted for 16 h before dosing. Mean 6 SE, n510. HALO, hours after lights on.
was higher than that of 14 HALO (1.11 6 0.14 mg/6 h, P,0.01; one-way ANOVA), while it was not different during the fasted condition (0.61 6 0.07 mg/6 h at 2 HALO, 0.55 6 0.07 mg/6 h at 14 HALO, P.0.1; one-way ANOVA). Urinary Ca excretion after vehicle tended to be higher in the morning trial (dosed at 2 HALO) than during the evening (dosed at 14 HALO). In addition, it was higher during the free-fed period than under the fasted condition. Thus, the change of Ca excretion by D3 wasn’t significantly different between morning and evening under both feeding conditions (Table). Similar results were obtained for urinary Ca concentration corrected by urine creatinine concentration. Serum concentrations of PTH and total protein In order to further examine the mechanisms of the chronopharmacological findings, serum concentrations of PTH and total protein were measured at before and 6 hours after the dosing (Figure 5, 6) . Basal PTH concentration tended to be higher (P,0.1) in the evening. By administration of D3, it was similarly reduced in both trials. Total protein concentration was not changed by D3 although it tended (P,0.1) to be higher in the evening.
Table Change of AUC0-12h and urinary Ca excretion Free-fed Delta AUC0-12h (mg/dl. h) Delta urinary Ca excretion (mg/6h) * P,0.05 vs evening.
Fasted
Morning
Evening
Morning
Evening
8.0 6 1.0* 0.77 6 0.14
4.5 6 0.9 0.60 6 0.08
5.5 6 1.1 0.30 6 0.11
5.4 6 1.2 0.35 6 0.11
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Fig. 4. Amount of Ca excretion into urine following a single oral dosing of 1,25-dihydroxy vitamin D3 (D3, 2mg/ Kg) or vehicle. These agents were given at 2 HALO (morning) or 14 HALO (evening) to Wistar rats freely fed or fasted for 16 h before dosing. Mean 6 SE, n510. HALO, hours after lights on.
Discussion We found that intestinal Ca absorption is a major factor involved in the dosing time-dependent change in D3-induced hypercalcemia in intact rats. When fasted, the dosing time-dependent differences in the maximum serum Ca concentration, AUC0-12h, and urinary Ca excretion were diminished. In addition, as the diurnal variation of serum Ca concentration was maintained even under a fasting condition, the intestinal and renal contribution may be relatively small for the intrinsic diurnal variation of serum Ca. We have recently reported the possible advantage of chronotherapy of D3 in chronic renal failure patients with secondary hyperparathyroidism under maintenance hemodialysis [9]. As there was no significant difference in the profile of serum 1, 25-dihydroxy vitamin D3 concentration in blood between the morning and the evening dosings, we think that pharmacokinetic-unrelated factors might be responsible for the phenomenon. In general, Ca homeostasis in the body is regulated by bone resorption, renal secretion and intestinal absorption. Of these, Ca absorption from the gut is completely different between active and resting period [17]. We thus have tried to evaluate the intestinal contribution to this interesting phenomenon. Similar to our previous findings in patients [9], we observed that serum Ca concentration after a single dosing of D3 varied with its dosing time in normal rats. Basal Ca concentration was higher in the resting period (lights on), which is compatible with previous reports in rats [5, 17, 18]. Our result indicates that absorption of Ca from the gut which is mainly taken during an active period, is facilitated by the dosing of D3 at an early resting period, resulting in the higher elevation of serum Ca concentration at this period. Although basal Ca concentration was decreased after fasting for 16 h, the daily variation was maintained, which is also similar to the previous report [18]. Under a fasting condition, serum Ca concentration after D3 increased in both trials, but its value at 3 to 12 hours after dosing didn’t differ between morning
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Fig. 5. Serum parathyroid hormone (PTH) concentration before and 6 hours following a single oral dosing of 1, 25-dihydroxy vitamin D3 (D3, 2mg/Kg) or vehicle. These agents were given at 2 HALO (morning) or 14 HALO (evening) to Wistar rats freely fed (A) or fasted for 16 h (B) before dosing. Mean 6 SE, n510. HALO, hours after lights on.
and evening trials. These results suggest that intestinal Ca absorption is involved in the mechanism of chronopharmacological phenomenon of D3. The Tmax of D3 was significantly increased when fasting. They were 7.2 6 1.1 and 7.5 6 0.9 hours (morning and evening trial, respectively) under a free-fed condition, while 9.8 6 1.7 and 9.4 6 1.5 hours in fasting (morning and evening trial, respectively). It is not certain about the reason of this change. It might be that the time to affect intestinal Ca absorption and bone mineralization by vit D are varied. Because basal circadian rhythm of serum Ca concentration was maintained even under a fasting condition, it is likely that other factors than diet contribute to the formation of the
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Fig. 6. Serum total protein concentration before and 6 hours following a single oral dosing of 1, 25-dihydroxy vitamin D3 (D3, 2mg/Kg) or vehicle. These agents were given at 2 HALO (morning) or 14 HALO (evening) to Wistar rats freely fed (A) or fasted for 16 h before (B) dosing. Mean 6 SE, n510. HALO, hours after lights on.
rhythmicity. It has been reported that the light induces some factors in brain to increase bone resorption and serum Ca concentration [19] and this contribute to the diurnal rhythm in serum Ca concentration. The diurnal variation in urinary excretion of Ca after vehicle is in agreement with a previous report in normal rats [20]. When hypercalcemia by D3 was greater (resting period), urinary Ca excretion was also larger. It is likely that the renal Ca filtration may be influenced by the concurrent blood Ca concentration regulated by non-renal factors. While fasting, D3 increased urinary Ca excretion and serum concentration without difference between dosing time. These results suggest that the time-dependent renal Ca excretion is apparently not a major factor for D3-induced hypercalcemia in normal rats. As shown in the results, PTH and to-
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tal protein concentration were not varied with the dosing time although both parameters showed diurnal changes which are good agreement with previous reports [21,22]. Other humoral factors such as calcitonin could affect the pharmacological effects of D3. Furthermore, we found that the change of Ca excretion was varied with the dosing-time under a free-fed, but not fasted condition. This is somehow different from that of the change of AUC because in free fed condition, the change of AUC is higher in the morning while the change of Ca excretion was similar. One of the possibility to explain this discrepancy is that GFR is higher in the evening and total filtered load of Ca is similar. We didn’t measure phosphate concentration mainly due to limited volume of the samples. Because Ca concentration was directly affected by its concentration, it might be possible that intestinal phosphate in the food affects the Ca absorption. Further studies involving measurement of these parameters are required to evaluate the mechanism(s) for this dosing-time dependent phenomenon of D3. In addition, as there are no appropriate animal models of secondary hyperparathyroidism in severe chronic renal failure, we thus used normal rats to determine our hypothesis. This may limit apprehension of this study to evaluate the merit of chronotherapy for renal failure patients. In summary, we showed that 1) the dosing time-dependent difference in D3-induced hypercalcemia may be related to a time-dependent increase in intestinal Ca absorption, but not the time-dependent excretion from kidney in normal rats, and 2) the basal diurnal variation of serum Ca concentration appears to be regulated by other factors than renal excretion and intestinal absorption. These results may be helpful for better understanding of the mechanisms of vitamin D chronotherapy in patients. Acknowledgments We thank Ms. Mariko Hojo for her technical assistance. References 1. Llach F, Bover J. Renal osteodystrophy. In: Brenner B, editor. Brenner and Rector’s The Kidney. Philadelphia WB Saunders, 1996. pp 2187–2273. 2. Slatopolsky E, Weerts C, Thielan J, Horst R, Harter H, Martin KJ. Marked suppression of secondary hyperparathyroidism by intravenous administration of 1,25-dihydroxy-cholecalciferol in uremic patients. Journal of Clinical Investigation 1984; 74: 2136–43. 3. Tsukamoto Y, Nomura M, Takahashi Y, Takagi Y, Yoshida A, Nagaoka T, et al. The oral 1,25-dihydroxyvitamin D3 pulse therapy in hemodialysis patients with severe secondary hyperparathyroidism. Nephron 1991; 57: 23–8. 4. Fukagawa M, Okazaki R, Takano K, Kaname S, Ogata E, Kitaoka M, et al. Regression of parathyroid hyperplasia by calcitriol-pulse therapy in patients on long-term dialysis. New England Journal of Medicine 1990; 323: 421–2. 5. Calvo NS, Eastell R, Offord KP, Bergstralh EJ, Burritt, MP. Circadian variation in ionized calcium and intact parathyroid hormone: Evidence for sex differences in calcium homeostasis. Journal of Clinical Endocrinology and Metabolism 1991; 72: 69–76. 6. el Hajj Fuleihan G, Klerman EB, Brown EN, Choe Y, Brown EM, Czeisler CA. The parathyroid hormone circadian rhythm is truly endogenous -a general clinical research center study. Journal of Clinical Endocrinology and Metabolism 1997; 82: 281–6.
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7. Kishikawa T, Takahashi H, Shimazawa E, Ogata E. Diurnal changes in calcium and phosphate metabolism in rats. Hormone and Metabolism Research. 1980; 12: 545–51. 8. Staub J, Peraut-Staub AM, Milhaud G. Endogenous nature of circadian rhythms in calcium metabolism. American Journal of Physiology. 1979; 237: R311–R317. 9. Tsuruoka S, Sugimoto K, Ohmori M, Kawaguchi A, Saito T, Fujimura, A. Chronotherapy of high-dose 1,25dihydroxyvitamin D3 in hemodialysis patients with secondary hyperparathyroidism: a single dose study. Clinical Pharmacology and Therapeutics 1999; 66: 609–616. 10. Yamauchi H, Kobayashi E, Sugimoto K, Tsuruoka S, Yabana M, Ishii M,et al. Time-dependent cyclosporine A-induced nephrotoxicity in rats. Clinical and Experimental Pharmacology and Physiology. 1998; 25: 435–40. 11. Turek FW. Circadian neural rhythms in mammals. Annual Review of Physiology 1985; 47: 49–64. 12. Takahashi JS, Zatz M. Regulation of circadian rhythmicity. Science(wash) 1982; 217:1104–1111. 13. Mrosovsky N, Salmon PA. A behavioural method for accelerating re-entrainment of rhythms to new lightdark cycles. Nature(Lond) 1987; 330:372–373. 14. Takamure M, Murakami N, Takahashi K, Kuroda H, Etoh T. Rapid reentrainment of the circadian clock itself, but not the measurable activity rhythms, to a new light-dark cycle in the rat. Physiology and Behavior 1991; 50:443–449. 15. Connerty HV, Briggs AR. Determination of serum calcium by means of orthocresolphtalein complexone. American Journal of Clinical Pathology 1966; 45 290–6. 16. Taussky HH. A microcalorimetric determination of creatinine in urine by the Jaffe reaction. Journal of Biological Chemistry. 1954; 208: 853–861. 17. Shinoda H, Seto H. Diurnal rhythms in calcium and phosphate metabolism in rhodents and their relations to lighting and feeding schedules. Mineral and Electrolyte Metabolism. 1985; 11: 158–66. 18. Shinoda, H. Diurnal rhythms in calcium metabolism in rats and their relations to the effect of blockers of bone resorption. In Ogura H, editor. Pharmacological approach to the study of the formation and resorption mechanism of hard tissue. St Louis: Ishiyaku EuroAmerica, 1994. pp 43–46. 19. Silver R, Romero MT, Besmer HR, Leak R, Nunez JM, LeSauter J. Calbindin-D28K cells in the hamster SCN express light-induced Fos. Neuroreport 1996; 7; 1224–8. 20. Roelfsema F, van der Heide D, Smeenk D. Circadian rhythms of urinary electrolyte excretion in freely moving rats. Life Sciences 1980; 27: 2303–2309. 21. Wong SE, Dohler KD, Atkinson MJ, Geerlings H, Hersh RD, von dur Muhlen A. Influence of age, strain and season on diurnal periodicity of thyroid stimulating hormone, thyroxine, triiodothyronine and parathyroid hormone in the serum of male laboratory rats. Acta Endocrinology (copenh) 1983; 102: 377–385. 22. Calvano SE, Reynolds RW. Circadian fluctuations in plasma corticosterone, corticosterone-binding activity and total protein in male rats: possible disruption by serial blood sampling. Endocrine Research 1984; 10: 11–25.
Contribution of diet to the dosing time-dependent change of vitamin D3-induced hypercalcemia in rats Shuichi Tsuruoka*, Koh-ichi Sugimoto, Akio Fujimura Department of Clinical Pharmacology, Jichi Medical School, Tochigi 329-0498, Japan Received 25 October 1999; accepted 10 August 2000
Abstract We have recently reported that the degree of hypercalcemia as an adverse effect induced by a single large-dose of active vitamin D3 varied with its dosing time without alteration in therapeutic effect for secondary hyperparathyroidism in patients with chronic renal failure. The present study was conducted to elucidate an effect of intestinal calcium (Ca) absorption on the chronopharmacological profiles of vitamin D3. 1, 25-dihydroxy-cholecalciferol (D3, 2 mg/kg) or vehicle alone was orally administered at two different times (2 and 14 hours after lights on; HALO) to male Wistar rats (n510) kept in rooms with a 12 h light-dark cycle. Blood samples for serum Ca concentration were taken before and 3, 6, 9, and 12 hours after the administration. Urine was collected for 6 hours after dosing. An identical protocol was repeated using the same animals after 16 hours fasting by a cross-over fashion. Under free-fed condition, basal concentration of serum Ca was higher at a resting period (lights on) than during an active period (lights off). Serum Ca reached its peak at 6 hours after dosing in both timings, while the value was significantly higher in the 2 HALO trial than in the 14 HALO trial. Area under the serum Ca concentration-time curve from 0 to 12 hours (AUC0-12h) and urinary excretion of Ca for 6 hours were also significantly higher in the 2 HALO trial than in the 14 HALO trial. When fasted, basal Ca concentration was reduced compared with the free-fed condition, while the daily variation was maintained. Serum Ca concentration profiles from 3 to 12 hours after dosing were not significantly different between the 2 HALO and 14 HALO trials. The AUC0-12h of serum Ca or its urinary excretion was not different between both trials. Serum concentrations of parathyroid hormone and total protein, measured before and 6 hours after the dosing were not affected by the dosing schedule. We have concluded that intestinal Ca absorption is a major factor for the chronopharmacological phenomenon of D3-induced hypercalcemia in intact rats, while intestinal and renal involvement may be relatively small in the mechanism of the intrinsic diurnal variation of serum Ca. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Vitamin D; Chronopharmacology; Hypercalcemia; Ca absorption
* Corresponding author: Department of Clinical Pharmacology, Jichi Medical School, 3311 Yakushiji, Minamikawachi, Kawachi, Tochigi 329-0498, Japan. Tel.: 182-285-58-7388; fax: 181-285-44-7562. E-mail address: [email protected] (S. Tsuruoka) 0024-3205/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 9 6 4 -4
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Introduction Vitamin D is widely prescribed for the treatment of osteoporosis and secondary hyperparathyroidism in patients with chronic renal failure. Especially for secondary hyperparathyroidism, an intermittent high-dose treatment (namely “pulse therapy”) with active vitamin D3 is performed to reduce hyperplasia of parathyroid gland as well as parathyroid hormone (PTH) concentration in blood [1, 2], while hypercalcemia induced by vitamin D sometimes interrupts the treatment [3, 4]. On the other hand, serum calcium (Ca) concentration possesses a circadian rhythm which shows a peak in the morning and a trough at evening in both human and rats [5–8]. We have recently reported the chronopharmacological profiles of vitamin D3 in chronic renal failure patients with secondary hyperparathyroidism. This study showed that the elevation in serum Ca concentration following a single oral administration of vitamin D3 for pulse therapy is significantly smaller by dosing in the evening than in the morning without any loss of treatment efficacy [9]. Regarding the mechanism of the chronopharmacological effect of vitamin D3, we also found that the profile of serum 1, 25-dihydroxy vitamin D3 concentration was not different between the dosing times, suggesting that pharmacokinetic-unrelated factor(s) might be responsible for the phenomenon. The mechanism, however, is not fully understood. The purpose of this study was to evaluate 1) the chronopharmacological effect of vitamin D3 on serum Ca concentration in normal rats, 2) the contribution of intestinal Ca absorption to this phenomenon by fasting the animals, and 3) the influence of a dosing time of vitamin D3 on renal Ca excretion, serum concentrations of PTH and total protein. Methods Animals Male Wistar rats (350–400 g; Japan Clea Co. Ltd., Tokyo, Japan) were maintained for more than two weeks in two separate rooms under a 12 h light-dark cycle (n55 in each). In room 1, lights were on at 07:00 and off at 19:00. In room 2, lights were on at 19:00 and off at 07:00 h, respectively [10]. Rats were given free access to standard rat chow (CE-2, containing 1.18 % Ca and 2.5 IU/g vitamin D3, Japan Clea Co. Ltd., Tokyo, Japan) and deionized water. Experimental protocol On the experimental day, chow was removed and the animals were placed into other cages to measure body weight about one hour prior to drug dosing. 1,25-dihydroxy-cholecalciferol (D3; Sigma, St. Louis, MO, USA, 2 mg/Kg) or vehicle was given by gavage at 2 and 14 hours after lights on (HALO). The dose of the drug was selected on the basis of significant raise in serum Ca concentration in our preliminary study (data not shown). Blood samples for serum Ca concentration were taken from the tail vein before and 3,6,9 and 12 h after dosing. To minimize stress by blood loss, sample volume was restricted to 200ml in each point. Serum concentrations of PTH and total protein were also measured before and 6 hours after the dosing. Then the room was exchanged and identical protocols were performed in a cross-over fashion after more than two-weeks acclimatization period. It is reported that most physiolog-
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ical parameters, such as neuronal, humoral, motor, and behavioral functions are completely re-synchronized within two weeks after changing lighting schedule in the animals [10–14]. For collection of urine samples, 3 % body weight (vol./wt.) of deionized water was given to the animals by gavage at 30 min. after dosing of D3 or vehicle, and the animals were placed individually in metabolic cages for 6 h. To determine a contribution of intestinal Ca absorption, the identical protocols were repeated using same rats which were fasted for more than 16 h before the administration. Each protocol was performed after a two-weeks acclimatization period in a cross-over fashion. Serum and urine samples were stored at 2208C until assay. Experiments were conducted in accordance with the Jichi Medical School Guide for Laboratory Animals. Assay methods Ca concentrations in serum and urine were measured by orthocresolphthalein complexone method [15]. Urine creatinine concentration was measured by Jaffe’s reaction [16]. Area under the serum Ca concentration-time curve from 0 to 12 h (AUC0-12h) was calculated by trapezoidal rule. Serum PTH concentration was determined by immunoradiometric assay (Rat PTH IRMA kit, Immutopics, Inc. San Clemente, CA, USA). Its normal range of the concentration was 10–40 pg/ml. Total protein concentration was determined by Biuret’s method. Statistics Data were expressed as the mean 6 SE. Comparisons were performed with ANOVA and Student’s t-test as appropriate. P,0.05 was regarded as significant. Results Serum Ca concentration profile under a free-fed condition The protocols were completed in all animals without major difficulties. Figure 1 shows the serum Ca concentration profiles in the free-fed state. The mean serum Ca concentration before dosing was significantly higher during the resting period (10.70 6 0.05 mg/dL at 2 HALO) than during the active period (10.43 6 0.04 mg/dL at 14 HALO, P,0.01; one-way ANOVA). Following single oral dosing of D3, the serum Ca concentration significantly increased and reached its peak at 6 h postdose in both dosing trials. The maximum concentration of serum Ca was significantly (P,0.01) higher in the morning trial (11.67 6 0.13 mg/dL, dosed at 2 HALO) than in evening (11.36 6 0.12 mg/dL, dosed at 14 HALO). The concentration gradually declined after 9 h but still remained higher than the basal level up to 12 h in both trials. The serum Ca concentration showed a diurnal variation after dosing of vehicle alone. In the morning trial, the serum Ca concentration peaked at 5 HALO and thereafter declined gradually toward 14 HALO. On the other hand, this parameter gradually increased from 14 HALO to 2 HALO in the evening trial. Serum Ca concentration profile under a fasting condition The serum Ca concentration profile after 16 h fasting was significantly different from that of under the free-fed condition (Figure 2). Before D3 dosing, the serum Ca concentrations
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Fig. 1. Serum calcium (Ca) concentration (A) and the change of Ca concentration (B) profiles after a single oral dosing of 1, 25-dihydroxy vitamin D3 (D3, 2mg/Kg) or vehicle at 2 HALO (morning) or 14 HALO (evening) to Wistar rats freely fed. Mean 6 SE, n510. HALO, hours after lights on.
were lower than those at 2 and 14 HALO under the free-fed condition. However, the difference between 2 and 14 HALO was still observed (10.47 6 0.05 mg/dL at 2 HALO, 10.21 6 0.03 mg/dL at 14 HALO, P,0.01). Although serum Ca concentration increased following D3 in both trials , the time to peak concentration was significantly delayed while fasting. Maximum Ca concentrations in the fasted trial were significantly lower than those in the free-fed trial. This parameter at the 2 and 14 HALO didn’t differ significantly. Serum Ca concentrations while fasting were lower than during the free-fed condition after vehicle alone, and the daily variation was still observed.
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Fig. 2. Serum calcium (Ca) concentration (A) and the change of Ca concentration (B) profile following a single oral dosing of 1, 25-dihydroxy vitamin D3 (D3, 2mg/Kg) or vehicle at 2 HALO (morning) or 14 HALO (evening) to Wistar rats fasted for 16 h before treatment. Mean 6 SE, n510. HALO, hours after lights on.
The AUC0-12h data are summarized in Figure 3 and Table. Under free-fed condition, the AUC0-12h following D3 was significantly higher in the 2 HALO than in the 14 HALO trials, while the difference was diminished under the fasting condition. This result indicates that intestinal Ca absorption is the major factor for the change of AUC. Urinary Ca excretion Urinary excretion of Ca was also different between the free-fed and fasted conditions (Figure 4 and Table). D3 enhanced the Ca excretion under both dietary conditions. Under the free-fed condition, Ca excretion for 6 h after dosing of D3 at 2 HALO (1.43 6 0.16 mg/6 h)
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Fig. 3. Area under the serum calcium concentration-time curve from 0 to 12 h (AUC0-12h) following a single oral dosing of 1, 25-dihydroxy vitamin D3 (D3, 2mg/Kg) or vehicle. These agents were given at 2 HALO (morning) or 14 HALO (evening) to Wistar rats freely fed or fasted for 16 h before dosing. Mean 6 SE, n510. HALO, hours after lights on.
was higher than that of 14 HALO (1.11 6 0.14 mg/6 h, P,0.01; one-way ANOVA), while it was not different during the fasted condition (0.61 6 0.07 mg/6 h at 2 HALO, 0.55 6 0.07 mg/6 h at 14 HALO, P.0.1; one-way ANOVA). Urinary Ca excretion after vehicle tended to be higher in the morning trial (dosed at 2 HALO) than during the evening (dosed at 14 HALO). In addition, it was higher during the free-fed period than under the fasted condition. Thus, the change of Ca excretion by D3 wasn’t significantly different between morning and evening under both feeding conditions (Table). Similar results were obtained for urinary Ca concentration corrected by urine creatinine concentration. Serum concentrations of PTH and total protein In order to further examine the mechanisms of the chronopharmacological findings, serum concentrations of PTH and total protein were measured at before and 6 hours after the dosing (Figure 5, 6) . Basal PTH concentration tended to be higher (P,0.1) in the evening. By administration of D3, it was similarly reduced in both trials. Total protein concentration was not changed by D3 although it tended (P,0.1) to be higher in the evening.
Table Change of AUC0-12h and urinary Ca excretion Free-fed Delta AUC0-12h (mg/dl. h) Delta urinary Ca excretion (mg/6h) * P,0.05 vs evening.
Fasted
Morning
Evening
Morning
Evening
8.0 6 1.0* 0.77 6 0.14
4.5 6 0.9 0.60 6 0.08
5.5 6 1.1 0.30 6 0.11
5.4 6 1.2 0.35 6 0.11
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Fig. 4. Amount of Ca excretion into urine following a single oral dosing of 1,25-dihydroxy vitamin D3 (D3, 2mg/ Kg) or vehicle. These agents were given at 2 HALO (morning) or 14 HALO (evening) to Wistar rats freely fed or fasted for 16 h before dosing. Mean 6 SE, n510. HALO, hours after lights on.
Discussion We found that intestinal Ca absorption is a major factor involved in the dosing time-dependent change in D3-induced hypercalcemia in intact rats. When fasted, the dosing time-dependent differences in the maximum serum Ca concentration, AUC0-12h, and urinary Ca excretion were diminished. In addition, as the diurnal variation of serum Ca concentration was maintained even under a fasting condition, the intestinal and renal contribution may be relatively small for the intrinsic diurnal variation of serum Ca. We have recently reported the possible advantage of chronotherapy of D3 in chronic renal failure patients with secondary hyperparathyroidism under maintenance hemodialysis [9]. As there was no significant difference in the profile of serum 1, 25-dihydroxy vitamin D3 concentration in blood between the morning and the evening dosings, we think that pharmacokinetic-unrelated factors might be responsible for the phenomenon. In general, Ca homeostasis in the body is regulated by bone resorption, renal secretion and intestinal absorption. Of these, Ca absorption from the gut is completely different between active and resting period [17]. We thus have tried to evaluate the intestinal contribution to this interesting phenomenon. Similar to our previous findings in patients [9], we observed that serum Ca concentration after a single dosing of D3 varied with its dosing time in normal rats. Basal Ca concentration was higher in the resting period (lights on), which is compatible with previous reports in rats [5, 17, 18]. Our result indicates that absorption of Ca from the gut which is mainly taken during an active period, is facilitated by the dosing of D3 at an early resting period, resulting in the higher elevation of serum Ca concentration at this period. Although basal Ca concentration was decreased after fasting for 16 h, the daily variation was maintained, which is also similar to the previous report [18]. Under a fasting condition, serum Ca concentration after D3 increased in both trials, but its value at 3 to 12 hours after dosing didn’t differ between morning
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Fig. 5. Serum parathyroid hormone (PTH) concentration before and 6 hours following a single oral dosing of 1, 25-dihydroxy vitamin D3 (D3, 2mg/Kg) or vehicle. These agents were given at 2 HALO (morning) or 14 HALO (evening) to Wistar rats freely fed (A) or fasted for 16 h (B) before dosing. Mean 6 SE, n510. HALO, hours after lights on.
and evening trials. These results suggest that intestinal Ca absorption is involved in the mechanism of chronopharmacological phenomenon of D3. The Tmax of D3 was significantly increased when fasting. They were 7.2 6 1.1 and 7.5 6 0.9 hours (morning and evening trial, respectively) under a free-fed condition, while 9.8 6 1.7 and 9.4 6 1.5 hours in fasting (morning and evening trial, respectively). It is not certain about the reason of this change. It might be that the time to affect intestinal Ca absorption and bone mineralization by vit D are varied. Because basal circadian rhythm of serum Ca concentration was maintained even under a fasting condition, it is likely that other factors than diet contribute to the formation of the
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Fig. 6. Serum total protein concentration before and 6 hours following a single oral dosing of 1, 25-dihydroxy vitamin D3 (D3, 2mg/Kg) or vehicle. These agents were given at 2 HALO (morning) or 14 HALO (evening) to Wistar rats freely fed (A) or fasted for 16 h before (B) dosing. Mean 6 SE, n510. HALO, hours after lights on.
rhythmicity. It has been reported that the light induces some factors in brain to increase bone resorption and serum Ca concentration [19] and this contribute to the diurnal rhythm in serum Ca concentration. The diurnal variation in urinary excretion of Ca after vehicle is in agreement with a previous report in normal rats [20]. When hypercalcemia by D3 was greater (resting period), urinary Ca excretion was also larger. It is likely that the renal Ca filtration may be influenced by the concurrent blood Ca concentration regulated by non-renal factors. While fasting, D3 increased urinary Ca excretion and serum concentration without difference between dosing time. These results suggest that the time-dependent renal Ca excretion is apparently not a major factor for D3-induced hypercalcemia in normal rats. As shown in the results, PTH and to-
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tal protein concentration were not varied with the dosing time although both parameters showed diurnal changes which are good agreement with previous reports [21,22]. Other humoral factors such as calcitonin could affect the pharmacological effects of D3. Furthermore, we found that the change of Ca excretion was varied with the dosing-time under a free-fed, but not fasted condition. This is somehow different from that of the change of AUC because in free fed condition, the change of AUC is higher in the morning while the change of Ca excretion was similar. One of the possibility to explain this discrepancy is that GFR is higher in the evening and total filtered load of Ca is similar. We didn’t measure phosphate concentration mainly due to limited volume of the samples. Because Ca concentration was directly affected by its concentration, it might be possible that intestinal phosphate in the food affects the Ca absorption. Further studies involving measurement of these parameters are required to evaluate the mechanism(s) for this dosing-time dependent phenomenon of D3. In addition, as there are no appropriate animal models of secondary hyperparathyroidism in severe chronic renal failure, we thus used normal rats to determine our hypothesis. This may limit apprehension of this study to evaluate the merit of chronotherapy for renal failure patients. In summary, we showed that 1) the dosing time-dependent difference in D3-induced hypercalcemia may be related to a time-dependent increase in intestinal Ca absorption, but not the time-dependent excretion from kidney in normal rats, and 2) the basal diurnal variation of serum Ca concentration appears to be regulated by other factors than renal excretion and intestinal absorption. These results may be helpful for better understanding of the mechanisms of vitamin D chronotherapy in patients. Acknowledgments We thank Ms. Mariko Hojo for her technical assistance. References 1. Llach F, Bover J. Renal osteodystrophy. In: Brenner B, editor. Brenner and Rector’s The Kidney. Philadelphia WB Saunders, 1996. pp 2187–2273. 2. Slatopolsky E, Weerts C, Thielan J, Horst R, Harter H, Martin KJ. Marked suppression of secondary hyperparathyroidism by intravenous administration of 1,25-dihydroxy-cholecalciferol in uremic patients. Journal of Clinical Investigation 1984; 74: 2136–43. 3. Tsukamoto Y, Nomura M, Takahashi Y, Takagi Y, Yoshida A, Nagaoka T, et al. The oral 1,25-dihydroxyvitamin D3 pulse therapy in hemodialysis patients with severe secondary hyperparathyroidism. Nephron 1991; 57: 23–8. 4. Fukagawa M, Okazaki R, Takano K, Kaname S, Ogata E, Kitaoka M, et al. Regression of parathyroid hyperplasia by calcitriol-pulse therapy in patients on long-term dialysis. New England Journal of Medicine 1990; 323: 421–2. 5. Calvo NS, Eastell R, Offord KP, Bergstralh EJ, Burritt, MP. Circadian variation in ionized calcium and intact parathyroid hormone: Evidence for sex differences in calcium homeostasis. Journal of Clinical Endocrinology and Metabolism 1991; 72: 69–76. 6. el Hajj Fuleihan G, Klerman EB, Brown EN, Choe Y, Brown EM, Czeisler CA. The parathyroid hormone circadian rhythm is truly endogenous -a general clinical research center study. Journal of Clinical Endocrinology and Metabolism 1997; 82: 281–6.
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