Calcif. Tiss. Res. 24, 41-46 (1977)
Calcified Tissue Research 9 by Springer-Verlag 1977
Mechanism of Induction of Hypercalcemia and Hyperphosphatemia by Lead Acetate in the Rat Yuzo Kato, Shoichiro Takimoto, and Hideaki Ogura Department of Pharmacology,Faculty of Dentistry, Tokyo Medical and Dental University, Yushima 1 chome, Bunkyo ku, Tokyo 113, Japan
Summary. The present study is an investigation of the mechanism of hypercalcemia and hyperphosphatemia induced by the intravenous injection of lead acetate (Pb-Ac). A total of 118 male rats were injected with 30 mg/kg of Pb-Ac, or with 16.5 mg/kg of sodium acetate as the control. The levels of serum calcium, phosphorus and lead were then determined at various time periods after the injections. Serum calcium and phosphorus levels increased with time after Pb-Ac injection and the maximum values of calcium (17 rag%) were found after 1 h and of phosphorus (13.5 rag%) after 30 rain. Both calcium and phosphorus levels reverted to the normal range after 12 h. The maximum net rates of increase of calcium and phosphorus were found immediately after Pb Ac injection. At that time, deposition of lead at the calcifying sites of bone and incisor dentin was demonstrated by a histochemical examination. In other experiments the changes in the calcium and phosphorus contents in the medium after shaking bone powder in serum with Pb-Ac in an in vitro system were studied. It was confirmed that the calcium and phosphorus were displaced from the bone mineral, the extent of the displacement being correlated with the concentration of the Pb-Ac added to the medium, and that these displacements were very rapid reactions. These results suggest that hypercalcemia and hyperphosphatemia following Pb-Ac injection results from a direct action of lead on the bone mineral.
hard tissues (Neuman and Neuman, 1958a). Many biological effects are known to result from the absorption of lead in measurable quantities by animals and man (Karpatkin, 1961; de Bruin, 1971). One of these is an alteration in the mineral content of blood following the intravenous (i.v.) injection of lead salt (Kumagai and Sakai, 1966; Johannsson et al., 1968; Kato, 1970). Even though these data suggest indirectly that the abnormalities in the behavior of calcium and phosphorus may be induced by a direct action of the lead salt on bone, the mechanism is still a matter of conjecture. The present study is an attempt to investigate the mechanism of interaction between lead acetate (Pb-Ac) and the bone minerals. Experiments with the following purposes were carried out: (1) to follow the changes in the levels of serum calcium, phosphorus, and lead at various time periods following i.v. injection of Pb-Ac, or sodium acetate (Na-Ac) as the control, (2) to compare the changes in the calcium and phosphorus contents in the medium in an in vitro system after shaking bone powder in serum with Pb-Ac under various conditions, and (3) to ascertain the site of deposition of lead in the hard tissues immediately after i.v. injection of Pb-Ac by a histochemical method.
Key words: Lead salt 1 Bone - - Mobilization - Hypercalcemia - - Hyperphosphatemia.
In Vivo Experiments: A total of 118 male rats of the Wistar strain
Introduction Lead ion is one of the nonphysiological cations termed "bone seekers" which concentrate selectively in the Send offprint requests to Y. Kato at the above address
Materials and Methods
weighing from 155 to 175 g were used. Groups of 6 to 10 rats were injected intravenously with a dose of 30 mg of Pb-Ac (0.75% aqueous solution)/kg body weight. As the control for acetate, groups of five rats were injected with a dose of Na-Ac (16.5 mg/kg, 0.28% aqueous solution) equivalent to the acetate content of the PbAc solution. Groups of rats were killed at 0.5, 5, 15, and 30 min and 1, 2, 6, and 12 h after the injections by taking blood samples from the carotid artery under ether anesthesia. The blood samples from the 0.5-rain group after Pb-Ac injection were carefully timed so that the interval between the end of the Pb-Ac injection and the cutting of the carotid artery was precisely 0.5 rain. In addition, 10 s
42 were required for injecting the reagent and for taking the blood sample. Nine untreated rats were used to obtain the normal values of total calcium and phosphorus in serum. Each serum sample was deproteinated with 10% trichloroacetic acid and centrifuged. The supernatant solutions were used for the chemical analyses. Both calcium (Willis, 1961) and lead (Sprague and Slavin, 1966) were determined by the atomic absorption spectrophotometer (Model 303, Perkin-Elmer Co., Norwalk, Conn.) and the content of phosphorus (Eastoe, 1965) was determined using a photoelectric spectrophotometer (Model 624, Hitachi, Ltd., Tokyo, Japan).
Histoehemieal Experiments: Five rats were injected two times a week with Pb-Ac at the same dose as mentioned above. The rats were decapitated 30 s after the last injection. The mandibulae with teeth were then excised and fixed for several days in 10% formalin buffered with phosphate at pH 7.3. These specimens were prepared for staining of lead in hard tissues (Okada and Mimura, 1938; Schneider, 1968), as follows: After washing out the formalin in tap water for 1 day, the samples were demineralized in 0.2 N hydrochloric acid saturated with hydrogen sulfide gas from a Kipp generator for about 1 week. These specimens were immersed in tap water for 1 day to wash out residual hydrogen sulfide and embedded in gelatin. The gelatin blocks were hardened and fixed in 10% formalin at 4 ~ for several days. The blocks were washed in tap water for a few h and frozen sections were cut at 15/an thickness. The sections were immersed in 0.1% gold chloride until the purple or black lead line became recognizable and then dipped in 5% sodium thiosulfate to remove excess gold chloride. These sections were finally mounted with glycerin jelly (Pearse, 1954) in the wet condition and observed microscopically.
Y. Kato et al.: Hypercalcemia and Hyperphosphatemia Induced by Lead effect of different concentrations of Pb-Ac on the changes of the contents of calcium and phosphorus in the media. Bone powder, serum, and Pb-Ac solution, in different concentrations, were shaken for 30 min and then centrifuged. The Pb-Ac solutions were prepared so that 0.2 ml of the reagent contained 100, 200, 400, 600, 800, or 1000 r of lead. A third experiment was conducted to determine the time required for the beginning of displacement of calcium and phosphorus from the bone to the medium. The bone powder and serum were preequilibrated in each test tube by shaking for 30 min. To four groups of test tubes, 0.2 ml of the Pb-Ac solution (Pb: 600 pg, samples 2 and 4) or 0.2 ml of distilled water for control (samples 1 and 3) was added. The tubes were centrifuged after each shaking period (samples 1 and 2, I0 s; samples 3 and 4, 30 rain). The 10 s trials were briefly centrifuged (5 to 10 s) to stop the reaction between the bone and medium as quickly as possible. Each supernatant fluid was then pipetted to another test tube and centrifuged again. In all experiments in vitro, the samples were finally centrifuged at 2500 rpm for 10 min and the total calcium and phosphorus contents of the supernatant fluids were determined.
Results
In Vivo Experiments: As shown in Figure 1, the levels of both serum calcium and phosphorus increased and decreased with time after the injection of Pb-Ac in an
30
In Vitro Experiments: Samples of serum and bone were obtained from groups of normal rats weighing 450 to 550 g. The sera were stored in a refrigerator at - 3 0 ~ until used. The bone sample was prepared by freezing in a wet condition to prevent a change of the physicochemicai properties from those in vivo (Termine and Posner, 1967; Neuman and Mulryan, 1967; Kato and Ogura, 1975), as follows: Both femora and tibiae were excised immediately after sacrificing the animals, cleaned of superficial soft tissues, and quick-frozen in a polyethylene bag in a dry ice-acetone bath, avoiding contact of the bones directly with the freezing mixture. The ends of the bones and the marrow were removed under freezing and only the cortices of the shafts were used in the experiments. The cleaned shafts were crushed in a stainless steel mortar to 50--100 mesh particle size and 100 mg wet weight of the samples due to the weighed as quickly as possible to prevent loss of weight due to the evaporation of water. As changes in pH and temperature in the media have been known to affect the equilibrium of the water-bone system (Neuman and Neuman, 1958b), the pH of the serum was stabilized to about 7.4 by bubbling with 5% CO 2 in O 5 (Toribara et al., 1957) for 20-30 min before being used. To 100 mg of the bone powder, 2 ml of pooled serum and/or 0.2 ml of the Pb Ac solution in various concentrations were put in turn into a test tube. The test tubes were again gassed with 5% CO 2 in 02 and sealed with glass stoppers coated with vaseline. To monitor the actual pH in the media, other groups of test tubes were prepared in much the same way as in the experimental groups. The actual pH in the media was thus ascertained, being in the range of 7.3 to 7.5 throughout the in vitro experiments. All experiments, however, were performed at room temperature, since it was difficult to maintain the temperature of the media in the sealed test tubes at body temperature. The first experiment was carried out to measure the time required for attainment of equilibrium of calcium and phosphorus between the bone powder and serum. The test tubes and contents were shaken with a mechanical shaker for 15, 30, and 60 min and centrifuged. A second experiment was performed to observe the
25
--o--
Ca
~o~
P
~e~
Pb
20 (9)
E 2
,,
9~- 15
:I0) I'I'
"
I
{
(10)
6
t2h
10
0.5 5 15
30
60rain 2 Time after injection
Fig. 1. Calcium, phosphorus and lead contents in serum after the intravenous injection of lead acetate (30 mg/kg). Normal values are shown at the extreme left not connected by lines. Numbers in parentheses are the number of animals. Means and standard deviations are shown
Y. Kato et al.: Hypercalcemiaand HyperposphatemiaInduced by Lead almost similar fashion, except for the 30-s period. In this latter case, only the phosphorus level showed a significant increase (P < 0.01) in comparison with the normal concentration. The maximum values were found after 1 h with calcium and 30 min with phosphorus. Both calcium and phosphorus levels began to decrease after 1 h and reverted to the normal range after 12 h. The level of serum lead decreased with time, rapidly during the first 15 min, moderately and constantly up to 6 h, and more slowly up to 12 h, there being three phases in the overall experimental period. As shown in Figure 2, the levels of serum calcium and phosphorus at 30 s after the i.v. injections of NaAc showed a significant decrease in comparison with the normal levels (Ca: P < 0.001, P: P < 0.05) and reverted to the normal levels at 30 min after the injection9 After that, these levels did not show significant alterations in any group of rats. As the levels of both serum calcium and phosphorus in the Pb-Ac group were always greater than those in the Na-Ac group (P < 0.05-43.001) at the same time after the injections, the differences between the calcium and phosphorus concentrations at the same time in the experimental and control groups were calculated. In Figure 3, each gradient between two points shows the mean rate of increase over each portion of the experimental period. For both calcium and phosphorus, the greatest rate of augmentation of serum concentrations was in the first 0 - 3 0 s and it then decreased with time. The rate of increment of phosphorus was greater than that of calcium only in the first experimental period and this tendency was reversed in the other experimental periods.
Histochemical Experiments: Two lead lines induced by each injection of Pb-Ac were recognized in both the incisor dentin and bone (Fig. 4a-b). The lead line due to the first injection of Pb-Ac was found to be dense within each hard tissue9 The lead line due to the second injection was clearly situated at the calcifying site of the periosteal surface of the bone and a lighter deposition of lead was found at the calcifying site in the incisor dentin and at the endosteal surface of the bone. It was thus ascertained that lead began to deposit in the hard tissues immediately after the Pb-Ac injections.
43
(5) ....I_
"(9 I0.0
.}(
/t t
2 8.o
{
E ~ 0
E 6.0
~
CO
m
9
p
1.0 ! !
0.5 5
I'5Time 3=0after injection 610 (min)
120
Fig. 2. Calcium and phosphorus contents in serum after the intravenous injection of sodium acetate (16.5 mg/kg). Normal values taken from Figure 1 are shown at the extreme left not connected by lines. Numbers in parentheses are the number of animals. Means and standard deviations are shown
phosphorus and lead ( r = 0 . 9 9 ) , and calcium and phosphorus (r = 0.99) were all significantly different from zero (P < 0.05). The results in Table 1 show that the displacement of calcium and phosphorus from the bone due to the addition of Pb-Ac was induced very rapidly. The increments of both the calcium and phosphorus in the media after shaking for 10 s with PbAc were about 40% for calcium and 50% for phosphorus, the maximum increase in both being observed after 30 min of shaking.
6.0
~ 4.0 ~o ttl
~2.0
~o--
Co
~e--
P
|
In Vitro Experiments: The contents of calcium and phosphorus in the media reached a dissolution equilibrium after shaking for 30 min under the experimental conditions (Fig. 5). As shown in Figure 6, the contents of calcium and phosphorus displaced from the bone mineral to the media correlated with the amount of Pb-Ac added to the in vitro system. The correlation coefficients between calcium and lead (r -- 0.99),
0 5 15
I0
30 Time after injection (min)
!
120
Fig. 3. Net increase of calcium and phosphorus in serum after the intravenous injection of lead acetate (30 mg/kg). Each value was calculated as follows: Calculated value = (mean in Fig. 1)(mean in Fig. 2)
44
Y. Kato et al.: Hypercalcemia and Hyperphosphatemia Induced by Lead
Fig. 4a and b. Two lead lines induced by the intravenous injection of lead acetate (30 mg/kg) are shown in both the incisor dentin (a) and the mandible (b) in the rat. P: pulp, D: dentin, B: bone, (1) lead line due to the first injection, (2) lead line due to the second injection. The width between two lead lines indicates the growth in each hard tissue in 1 week
Discussion
In the in vivo experiments, it was arranged to use PbAc in a smaller dose than employed previously (Kumagai and Sakai, 1966; Kato, 1970) to moderate the harmful effect of lead on the animal organism.
1o.or\ /
- - 0 - - Ca ~O~
P
\(sl
8.0
(5)
~__
-}
6.0
4.0
o[, 0
I
I
I
15
30
60
Duration of shaking(min) Fig. 5. Changes in the contents of calcium and phosphorus in the media after shaking bone powder (100 rag) in serum (2 ml, pH 7.4) for various periods of time. Numbers in parentheses are the number of specimens. Means and standard deviations are shown. If the standard deviation is not indicated, it was too small to be illustrated. No difference is shown between 30 min and 60 min
After the i.v. injection of Pb-Ac, it has been reported that lead in the blood is distributed between cells and plasma (Castellino and Aloj, 1964) and that only lead in the plasma deposits in hard tissues (Okabe, 1970). Accordingly, pooled rat serum was used for the media in our in vitro study. Since no precipitate was recognized macroscopically in the media containing Pb-Ac, a decrease of the mineral contents in the media caused by the spontaneous precipitation of lead phosphate reported previously (Gabbiani, 1964; Bachra and van Harskamp, 1970), was not a factor in our experiments. The blood volume of the rat has been reported to be about 6.7 ml/100 g body weight (Cartland and Koch, 1928). Since the lead-containing solution was injected at a volume of 0.59 ml/100 g body weight, the blood was expected to be diluted immediately after the injection, at least temporarily. The decrease in the serum calcium and phosphorus contents immediately after the Na-Ac injections (Fig. 2) may, therefore, be due to dilution of the circulating blood volume caused by the injected volume of sodium acetate solution. For that reason, the Na-Ac group seemed to be more suitable than normal animals as the controls for the PbAc group. By comparison with the Na-Ac group, both the hypercalcemia and hyperphosphatemia were shown to be induced immediately after the Pb-Ac injection. These observations are new findings, in that the previous studies (Kumagai and Sakai, 1966; Kato, 1970) could not show similar findings by comparison with a normal group. There is a latent period before increments in serum calcium and phosphorus contents related to the
Y. Kato et al.: Hypercalcemia and Hyperphosphatemia Induced by Lead
thyroparathyroid appratus (Copp, 1960) and/or kidney damage (Jastak, 1968). In the present experiments, no latent period was recognized before the concentration changes in the serum calcium and phosphorus after Pb-Ac treatment, in vivo as well as in vitro. These facts and the histochemical observations (Fig. 4) suggest that these phenomena, at least in the early stage, may result not from participation of thyroparathyroid apparatus or from kidney damage but from some direct action of Pb-Ac on the bone surface. A large concentration of lead produces hemolysis (Waldron, 1966) which results in the increment of the serum phosphorus content. Following the Pb-Ac injection in the present study, the degree of hemolysis observed macroscopically decreased with time and could not be observed after 1 h, while conversely the phosphorus content in the serum increased with time up to 30 min. The hemolysis may be, at least partially, one of the factors that resulted in an increase in the content of phosphorus in the serum in the early stages of our experiments. It is known that bone is a highly vascular tissue (Shim, 1968; Kelly, 1968) and the rate of the bone blood flow has been estimated with lSF to be about 17% of the plasma volume per min in rat (Van Dyke et al., 1965). Lead in blood may permeate easily into the extracellular fluid (ECF) through the walls of the capillaries supplying bone. Lead in ECF may pass between the lining cells of the bone surface to the bone extracellular fluid (BEF) (Neuman and Ramp, 1971) by a concentration gradient and become deposited on the crystal surface of bone. These phenomena appeared to occur quickly since the deposition of lead was demonstrated on the surface of the bone within 30 s after the Pb-Ac injections (Fig. 4). It seems likely that the mechanism of lead deposition in bone is not simple. By X-ray diffraction studies, synthetic crystals of hydroxyapatite have been reported to change to lead-apatite when immersed in a buffer with Pb-Ac for 5 to 45 days (Moriwaki et al., 1975). The results suggested that calcium ions were displaced by lead ions equivalently on the synthetic crystals. In the present short-term experiments, however, the concentrations of both calcium and phosphorus increased simultaneously in vivo as well as in vitro. In the hydration shells of crystals, 1 mol of calcium should be displaced by 1 mol of lead. However, in the lattice surface of crystals, it would be expected that 1 mol of lead may displace more than 1 mol of calcium because of the difference in ionic radii (Ca++: 0.99 A, Pb++: 1.32 A) (Neuman and Neuman, 1953). In this case, the altered charge equivalence may reduce the positive charge on the crystal surface, resulting in a corresponding loss of boundary phosphate. By this
45
15.0
/, 2
"~ lO.O
.c 5.0 --o--
I
I
0
I
I
l
200 4.00 600 800 Pb Qdded (/..t,g)
P
I
1000
Fig. 6. Increments in the contents of calcium and phosphorus in the media after shaking bone powder (100 rag) in the serum (2 ml, p H 7.4) for 30 min with the various amounts of lead acetate (0.2 ml). The increments of contents of calcium and phosphorus correlate with the amount of lead acetate added
mode of action, a rapid increase in concentrations of calcium and phosphorus in the BEF may result from a continuous influx of lead. If the concentrations of calcium and phosphorus in the BEF become greater than those in ECF, calcium and phosphorus may be expected to efflux from BEF to ECF according to the concentration gradient and enter the plasma in the numerous small vessels. As to the mechanism of calcium efflux from BEF to ECF, two theories have been presented. One is that calcium is transported out of bone actively by an intraceUular transport mechanism (Talmage, 1975). The other is that calcium is transported between the lining cells (Rasmussen and Bordier, 1974). Even if the active transport theory of Table 1. Contents of calcium and phosphorus in media shaken with bone for different periods of time Reagent
DW" Pb-Ac u DW Pb-Ac
Time of shaking
I0 10 30 30
s s min min
No. of experiments
5 5 5 5
Mineral content/100 ml of medium Ca (rag)
P (rag)
6.43 7.75 6.51 9.80
4.48 5.54 4.63 6.74
(0.16) c (0.22) (0.23) (0.41) a
(0.24) (0.40) (0.38) (0.71) e
a Distilled water as control b Lead acetate (Pb: 600 pg) c Mean and standard deviation (in parentheses) are shown a 10 s vs. 30 min, P < 0.001 e 10 s vs. 30 min, P < 0.02 The differences are all significant between D W and P b - A c (P < 0.001)
46 c a l c i u m is c o r r e c t physiologically, c a l c i u m efltux by intracellular t r a n s p o r t w o u l d p r o b a b l y be restrained rather t h a n activated after the P b - A c injection. T h i s is b e c a u s e the astringent effect o f a metal salt results in interference with the p h y s i o l o g i c a l action o f cells ( C a w s o n and Spector, 1975). Since the functions and the m o r p h o l o g i c c h a n g e s o f the o s t e o g e n i c cells were n o t e x a m i n e d , it c a n n o t be stated w h e t h e r o r not these cells t o o k p a r t in the mineral c h a n g e s in serum. All d a t a in the present experiments, h o w e v e r , indicate that the m o v e m e n t o f the lead f r o m the b l o o d to b o n e and o f c a l c i u m and p h o s p h o r u s f r o m the b o n e to the b l o o d w e r e rapid. T h e s e facts suggest t h a t the m o v e m e n t o f the minerals following the P b - A c injection m a y p r o c e e d b y passing between the lining cells o f the b o n e surface rather t h a n f r o m a c t i v a t i o n o f the native t r a n s p o r t m e c h a n i s m s o f the cells.
References Bachra, B.N., van Harskamp, G.A.: The effect of polyvalent metal ions on the stability of a buffer system for calcification in vitro. Calcif. Tiss. Res. 41, 359-365 (1970) Cart.land, G.F., Koch, C.: A micro-modification of the KeithRowntree plasma-dye method for the estimation of blood volume in the rat. Amer. J. Physiol. 85, 540-545 (1928) Castellino, N., Aloj, S.: Kinetics of the distribution and excretion of lead in the rat. Brit. J. Industr. Med. 21, 308-314 (1964) Cawson, R.A., Spector, R.G.: Clinical pharmacology in dentistry, p. 55. Edinburgh-London-New York: Churchill Livingstone 1975 Copp, D.H.: Parathyroids and homeostasis of blood calcium. In: Bone as a tissue (K. Rodahl, J.T. Nicholson, E.M. Brown, eds.), pp. 289-299. New York-Toronto-London: McGraw-Hill 1960 de Bruin, A.: Certain biological effects of lead upon the animal organism. Arch. Environ. Health 23, 249-264 (1971) Eastoe, J.E.: Methods for determination of phosphate, calcium and protein in small portions of mineralized tissues. In: Proc. Second European Symposium on Calcified Tissues (L.J. Richelle, M.J. Dallemagne, eds.), pp. 265~274. Liege: Universitb de Liege 1965 Gabbiani, G.: Relationship between the calcifying power of various substances in vivo and their solubility in vitro. Experientia (Basel) 20, 514 (1964) Jastak, J.T., Morrison, A.B., Raisz, L.G.: Effects of renal insufficiency on the parathyroid gland and calcium homeostasis. Amer. J. Physiol. 215, 84-89 (1968) Johannsson, O., Perrault, G., Savoie, L., Tuchweber, B.: Action of various metallic chlorides on calcaemia and phosphataemia. Br. J. Pharm. Chemother. 33, 91-97 (1968) Karpatkin, S.: Lead poisoning after taking Pb-Ac with suicidal intent. Arch. Environ. Health 2, 679-684 (196 I) Kato, Y.: Analytical studies on experimental skin calcification in the rat after administration of lead acetate and subsequent
Y. Kato et al.: Hypercalcemia and Hyperphosphatemia Induced by Lead administration of polymyxin B sulfate. Bull. Tokyo Med. Dent. Univ. 17, 53-63 (1970) Kato, Y., Ogura, H.: Low-temperature ashing of bovine dentine. CalciL Tiss. Res. 18, 141-148 (1975) Kelly, P.J.: Anatomy, physiology, and pathology of the blood supply of bones. J. Bone Joint Surg. 50-A, 766-783 (1968) Kumagai, A., Sakai, T.: Effect of lead acetate on serum calcium and phosphorus in rats. Arch. int. Pharmacodyn. Thtr. 160, 248-252 (1966) Moriwaki, Y., Ida, K., Yamaga, R.: Effect of diverse ions on the crystallinity of carbonate-containing hydroxyapatite. (In Japanese, with English abstract.) J. Chem. Soc. Jap. No. 5, 801-807 (1975) Neuman, W. F., Mulryan, B.J.: Synthetic hydroxyapatite crystals III. The carbonate system. Calcif. Tiss. Res, 1, 94-104 (1967) Neuman, W.F., Neuman, M.W.: The nature of the mineral phase of bone. Chem. Rev. 53, 1-45 (1953) Neuman, W.F., Neuman, M.W.: The chemical dynamics of bone mineral, p. 94 (a), pp. 31-33 (b). Chicago: University of Chicago Press 1958 Neuman, W.F., Ramp. W.K.: The concept of a bone membrane; some implications. In: Cellular mechanisms for calcium transfer and homeostasis (G. Nichols, Jr., R.H. Wasserman, eds.), pp. 197-209. New York-London: Academic 1971 Okabe, T.: Studies on deposition mechanism of lead salt in hard tissues. (In Japanese.) Ochanomizu Medical J. 18, 175-185 (1970) Okada, M., Mimura, T.: Zur Physiologie and Pharmakologie der Hartgewebe. I. Mitteilung: Eine Vitalfiirbungsmethode mit Bleisalzen und ihre Anwendung bei den Untersuchungen fiber die rhythmische Streifenbildung der barten Zahngewebe. Jap. J. Med. Sci., IV. Pharmacol. 11, 166-170 (1938) Pearse, A.G.E.: Histochemistry, p. 405. London: Churchill 1954 Rasmussen, H., Bordier, P.: The physiological and cellular basis of metabolic bone disease, pp. 29-31. Baltimore: Williams & Wilkins 1974 Schneider, B.J.: Lead acetate as a vital marker for the analysis of bone growth. Amer. J. Phys. Anthrop. 29, 197-200 (1968) Shim, S.S.: Physiology of blood circulation of bone. J. Bone Joint Surg. 50-A, 812-824 (1968) Sprague, S., SIavin, W.: A simple method for the determination of lead in blood. Atomic Absorption Newsletter 5, 9-10 (1966) Talmage, R.V.: Effect of fasting and parathyroid hormone injection on plasma 45Ca concentrations in rats. Calcif. Tiss. Res. 17, 103-112 (1975) Termine, J.D., Posner, A.S.: Amorphous/crystalline interrelationships in bone mineral. Calcif. Tiss. Res. 1, 8-23 (1967) Toribara, T.Y., Terepka, A.R., Dewey, P.A.: The ultrafiltrable calcium of human serum. I. Ultrallltration methods and normal values. J. Clin. Invest. 36, 738-748 (1957) Van Dyke, D., Anger, H.O., Yano, Y., Bozzini, C.: Bone blood flow shown with ~SF and the positron camera. Amer. J. Physiol. 209, 65-70 (1965) Waldron, H.A." The anaemia of lead poisoning. Brit. J. Industr. Meal. 23, 83-100 (1966) Willis, J.B.: Determination of calcium and magnesium in urine by atomic absorption spectroscopy. Anal. Chem. 33, 556-559 (1961) Received March 14/A ceepted April 2 7, 1977
Calcified Tissue Research 9 by Springer-Verlag 1977
Mechanism of Induction of Hypercalcemia and Hyperphosphatemia by Lead Acetate in the Rat Yuzo Kato, Shoichiro Takimoto, and Hideaki Ogura Department of Pharmacology,Faculty of Dentistry, Tokyo Medical and Dental University, Yushima 1 chome, Bunkyo ku, Tokyo 113, Japan
Summary. The present study is an investigation of the mechanism of hypercalcemia and hyperphosphatemia induced by the intravenous injection of lead acetate (Pb-Ac). A total of 118 male rats were injected with 30 mg/kg of Pb-Ac, or with 16.5 mg/kg of sodium acetate as the control. The levels of serum calcium, phosphorus and lead were then determined at various time periods after the injections. Serum calcium and phosphorus levels increased with time after Pb-Ac injection and the maximum values of calcium (17 rag%) were found after 1 h and of phosphorus (13.5 rag%) after 30 rain. Both calcium and phosphorus levels reverted to the normal range after 12 h. The maximum net rates of increase of calcium and phosphorus were found immediately after Pb Ac injection. At that time, deposition of lead at the calcifying sites of bone and incisor dentin was demonstrated by a histochemical examination. In other experiments the changes in the calcium and phosphorus contents in the medium after shaking bone powder in serum with Pb-Ac in an in vitro system were studied. It was confirmed that the calcium and phosphorus were displaced from the bone mineral, the extent of the displacement being correlated with the concentration of the Pb-Ac added to the medium, and that these displacements were very rapid reactions. These results suggest that hypercalcemia and hyperphosphatemia following Pb-Ac injection results from a direct action of lead on the bone mineral.
hard tissues (Neuman and Neuman, 1958a). Many biological effects are known to result from the absorption of lead in measurable quantities by animals and man (Karpatkin, 1961; de Bruin, 1971). One of these is an alteration in the mineral content of blood following the intravenous (i.v.) injection of lead salt (Kumagai and Sakai, 1966; Johannsson et al., 1968; Kato, 1970). Even though these data suggest indirectly that the abnormalities in the behavior of calcium and phosphorus may be induced by a direct action of the lead salt on bone, the mechanism is still a matter of conjecture. The present study is an attempt to investigate the mechanism of interaction between lead acetate (Pb-Ac) and the bone minerals. Experiments with the following purposes were carried out: (1) to follow the changes in the levels of serum calcium, phosphorus, and lead at various time periods following i.v. injection of Pb-Ac, or sodium acetate (Na-Ac) as the control, (2) to compare the changes in the calcium and phosphorus contents in the medium in an in vitro system after shaking bone powder in serum with Pb-Ac under various conditions, and (3) to ascertain the site of deposition of lead in the hard tissues immediately after i.v. injection of Pb-Ac by a histochemical method.
Key words: Lead salt 1 Bone - - Mobilization - Hypercalcemia - - Hyperphosphatemia.
In Vivo Experiments: A total of 118 male rats of the Wistar strain
Introduction Lead ion is one of the nonphysiological cations termed "bone seekers" which concentrate selectively in the Send offprint requests to Y. Kato at the above address
Materials and Methods
weighing from 155 to 175 g were used. Groups of 6 to 10 rats were injected intravenously with a dose of 30 mg of Pb-Ac (0.75% aqueous solution)/kg body weight. As the control for acetate, groups of five rats were injected with a dose of Na-Ac (16.5 mg/kg, 0.28% aqueous solution) equivalent to the acetate content of the PbAc solution. Groups of rats were killed at 0.5, 5, 15, and 30 min and 1, 2, 6, and 12 h after the injections by taking blood samples from the carotid artery under ether anesthesia. The blood samples from the 0.5-rain group after Pb-Ac injection were carefully timed so that the interval between the end of the Pb-Ac injection and the cutting of the carotid artery was precisely 0.5 rain. In addition, 10 s
42 were required for injecting the reagent and for taking the blood sample. Nine untreated rats were used to obtain the normal values of total calcium and phosphorus in serum. Each serum sample was deproteinated with 10% trichloroacetic acid and centrifuged. The supernatant solutions were used for the chemical analyses. Both calcium (Willis, 1961) and lead (Sprague and Slavin, 1966) were determined by the atomic absorption spectrophotometer (Model 303, Perkin-Elmer Co., Norwalk, Conn.) and the content of phosphorus (Eastoe, 1965) was determined using a photoelectric spectrophotometer (Model 624, Hitachi, Ltd., Tokyo, Japan).
Histoehemieal Experiments: Five rats were injected two times a week with Pb-Ac at the same dose as mentioned above. The rats were decapitated 30 s after the last injection. The mandibulae with teeth were then excised and fixed for several days in 10% formalin buffered with phosphate at pH 7.3. These specimens were prepared for staining of lead in hard tissues (Okada and Mimura, 1938; Schneider, 1968), as follows: After washing out the formalin in tap water for 1 day, the samples were demineralized in 0.2 N hydrochloric acid saturated with hydrogen sulfide gas from a Kipp generator for about 1 week. These specimens were immersed in tap water for 1 day to wash out residual hydrogen sulfide and embedded in gelatin. The gelatin blocks were hardened and fixed in 10% formalin at 4 ~ for several days. The blocks were washed in tap water for a few h and frozen sections were cut at 15/an thickness. The sections were immersed in 0.1% gold chloride until the purple or black lead line became recognizable and then dipped in 5% sodium thiosulfate to remove excess gold chloride. These sections were finally mounted with glycerin jelly (Pearse, 1954) in the wet condition and observed microscopically.
Y. Kato et al.: Hypercalcemia and Hyperphosphatemia Induced by Lead effect of different concentrations of Pb-Ac on the changes of the contents of calcium and phosphorus in the media. Bone powder, serum, and Pb-Ac solution, in different concentrations, were shaken for 30 min and then centrifuged. The Pb-Ac solutions were prepared so that 0.2 ml of the reagent contained 100, 200, 400, 600, 800, or 1000 r of lead. A third experiment was conducted to determine the time required for the beginning of displacement of calcium and phosphorus from the bone to the medium. The bone powder and serum were preequilibrated in each test tube by shaking for 30 min. To four groups of test tubes, 0.2 ml of the Pb-Ac solution (Pb: 600 pg, samples 2 and 4) or 0.2 ml of distilled water for control (samples 1 and 3) was added. The tubes were centrifuged after each shaking period (samples 1 and 2, I0 s; samples 3 and 4, 30 rain). The 10 s trials were briefly centrifuged (5 to 10 s) to stop the reaction between the bone and medium as quickly as possible. Each supernatant fluid was then pipetted to another test tube and centrifuged again. In all experiments in vitro, the samples were finally centrifuged at 2500 rpm for 10 min and the total calcium and phosphorus contents of the supernatant fluids were determined.
Results
In Vivo Experiments: As shown in Figure 1, the levels of both serum calcium and phosphorus increased and decreased with time after the injection of Pb-Ac in an
30
In Vitro Experiments: Samples of serum and bone were obtained from groups of normal rats weighing 450 to 550 g. The sera were stored in a refrigerator at - 3 0 ~ until used. The bone sample was prepared by freezing in a wet condition to prevent a change of the physicochemicai properties from those in vivo (Termine and Posner, 1967; Neuman and Mulryan, 1967; Kato and Ogura, 1975), as follows: Both femora and tibiae were excised immediately after sacrificing the animals, cleaned of superficial soft tissues, and quick-frozen in a polyethylene bag in a dry ice-acetone bath, avoiding contact of the bones directly with the freezing mixture. The ends of the bones and the marrow were removed under freezing and only the cortices of the shafts were used in the experiments. The cleaned shafts were crushed in a stainless steel mortar to 50--100 mesh particle size and 100 mg wet weight of the samples due to the weighed as quickly as possible to prevent loss of weight due to the evaporation of water. As changes in pH and temperature in the media have been known to affect the equilibrium of the water-bone system (Neuman and Neuman, 1958b), the pH of the serum was stabilized to about 7.4 by bubbling with 5% CO 2 in O 5 (Toribara et al., 1957) for 20-30 min before being used. To 100 mg of the bone powder, 2 ml of pooled serum and/or 0.2 ml of the Pb Ac solution in various concentrations were put in turn into a test tube. The test tubes were again gassed with 5% CO 2 in 02 and sealed with glass stoppers coated with vaseline. To monitor the actual pH in the media, other groups of test tubes were prepared in much the same way as in the experimental groups. The actual pH in the media was thus ascertained, being in the range of 7.3 to 7.5 throughout the in vitro experiments. All experiments, however, were performed at room temperature, since it was difficult to maintain the temperature of the media in the sealed test tubes at body temperature. The first experiment was carried out to measure the time required for attainment of equilibrium of calcium and phosphorus between the bone powder and serum. The test tubes and contents were shaken with a mechanical shaker for 15, 30, and 60 min and centrifuged. A second experiment was performed to observe the
25
--o--
Ca
~o~
P
~e~
Pb
20 (9)
E 2
,,
9~- 15
:I0) I'I'
"
I
{
(10)
6
t2h
10
0.5 5 15
30
60rain 2 Time after injection
Fig. 1. Calcium, phosphorus and lead contents in serum after the intravenous injection of lead acetate (30 mg/kg). Normal values are shown at the extreme left not connected by lines. Numbers in parentheses are the number of animals. Means and standard deviations are shown
Y. Kato et al.: Hypercalcemiaand HyperposphatemiaInduced by Lead almost similar fashion, except for the 30-s period. In this latter case, only the phosphorus level showed a significant increase (P < 0.01) in comparison with the normal concentration. The maximum values were found after 1 h with calcium and 30 min with phosphorus. Both calcium and phosphorus levels began to decrease after 1 h and reverted to the normal range after 12 h. The level of serum lead decreased with time, rapidly during the first 15 min, moderately and constantly up to 6 h, and more slowly up to 12 h, there being three phases in the overall experimental period. As shown in Figure 2, the levels of serum calcium and phosphorus at 30 s after the i.v. injections of NaAc showed a significant decrease in comparison with the normal levels (Ca: P < 0.001, P: P < 0.05) and reverted to the normal levels at 30 min after the injection9 After that, these levels did not show significant alterations in any group of rats. As the levels of both serum calcium and phosphorus in the Pb-Ac group were always greater than those in the Na-Ac group (P < 0.05-43.001) at the same time after the injections, the differences between the calcium and phosphorus concentrations at the same time in the experimental and control groups were calculated. In Figure 3, each gradient between two points shows the mean rate of increase over each portion of the experimental period. For both calcium and phosphorus, the greatest rate of augmentation of serum concentrations was in the first 0 - 3 0 s and it then decreased with time. The rate of increment of phosphorus was greater than that of calcium only in the first experimental period and this tendency was reversed in the other experimental periods.
Histochemical Experiments: Two lead lines induced by each injection of Pb-Ac were recognized in both the incisor dentin and bone (Fig. 4a-b). The lead line due to the first injection of Pb-Ac was found to be dense within each hard tissue9 The lead line due to the second injection was clearly situated at the calcifying site of the periosteal surface of the bone and a lighter deposition of lead was found at the calcifying site in the incisor dentin and at the endosteal surface of the bone. It was thus ascertained that lead began to deposit in the hard tissues immediately after the Pb-Ac injections.
43
(5) ....I_
"(9 I0.0
.}(
/t t
2 8.o
{
E ~ 0
E 6.0
~
CO
m
9
p
1.0 ! !
0.5 5
I'5Time 3=0after injection 610 (min)
120
Fig. 2. Calcium and phosphorus contents in serum after the intravenous injection of sodium acetate (16.5 mg/kg). Normal values taken from Figure 1 are shown at the extreme left not connected by lines. Numbers in parentheses are the number of animals. Means and standard deviations are shown
phosphorus and lead ( r = 0 . 9 9 ) , and calcium and phosphorus (r = 0.99) were all significantly different from zero (P < 0.05). The results in Table 1 show that the displacement of calcium and phosphorus from the bone due to the addition of Pb-Ac was induced very rapidly. The increments of both the calcium and phosphorus in the media after shaking for 10 s with PbAc were about 40% for calcium and 50% for phosphorus, the maximum increase in both being observed after 30 min of shaking.
6.0
~ 4.0 ~o ttl
~2.0
~o--
Co
~e--
P
|
In Vitro Experiments: The contents of calcium and phosphorus in the media reached a dissolution equilibrium after shaking for 30 min under the experimental conditions (Fig. 5). As shown in Figure 6, the contents of calcium and phosphorus displaced from the bone mineral to the media correlated with the amount of Pb-Ac added to the in vitro system. The correlation coefficients between calcium and lead (r -- 0.99),
0 5 15
I0
30 Time after injection (min)
!
120
Fig. 3. Net increase of calcium and phosphorus in serum after the intravenous injection of lead acetate (30 mg/kg). Each value was calculated as follows: Calculated value = (mean in Fig. 1)(mean in Fig. 2)
44
Y. Kato et al.: Hypercalcemia and Hyperphosphatemia Induced by Lead
Fig. 4a and b. Two lead lines induced by the intravenous injection of lead acetate (30 mg/kg) are shown in both the incisor dentin (a) and the mandible (b) in the rat. P: pulp, D: dentin, B: bone, (1) lead line due to the first injection, (2) lead line due to the second injection. The width between two lead lines indicates the growth in each hard tissue in 1 week
Discussion
In the in vivo experiments, it was arranged to use PbAc in a smaller dose than employed previously (Kumagai and Sakai, 1966; Kato, 1970) to moderate the harmful effect of lead on the animal organism.
1o.or\ /
- - 0 - - Ca ~O~
P
\(sl
8.0
(5)
~__
-}
6.0
4.0
o[, 0
I
I
I
15
30
60
Duration of shaking(min) Fig. 5. Changes in the contents of calcium and phosphorus in the media after shaking bone powder (100 rag) in serum (2 ml, pH 7.4) for various periods of time. Numbers in parentheses are the number of specimens. Means and standard deviations are shown. If the standard deviation is not indicated, it was too small to be illustrated. No difference is shown between 30 min and 60 min
After the i.v. injection of Pb-Ac, it has been reported that lead in the blood is distributed between cells and plasma (Castellino and Aloj, 1964) and that only lead in the plasma deposits in hard tissues (Okabe, 1970). Accordingly, pooled rat serum was used for the media in our in vitro study. Since no precipitate was recognized macroscopically in the media containing Pb-Ac, a decrease of the mineral contents in the media caused by the spontaneous precipitation of lead phosphate reported previously (Gabbiani, 1964; Bachra and van Harskamp, 1970), was not a factor in our experiments. The blood volume of the rat has been reported to be about 6.7 ml/100 g body weight (Cartland and Koch, 1928). Since the lead-containing solution was injected at a volume of 0.59 ml/100 g body weight, the blood was expected to be diluted immediately after the injection, at least temporarily. The decrease in the serum calcium and phosphorus contents immediately after the Na-Ac injections (Fig. 2) may, therefore, be due to dilution of the circulating blood volume caused by the injected volume of sodium acetate solution. For that reason, the Na-Ac group seemed to be more suitable than normal animals as the controls for the PbAc group. By comparison with the Na-Ac group, both the hypercalcemia and hyperphosphatemia were shown to be induced immediately after the Pb-Ac injection. These observations are new findings, in that the previous studies (Kumagai and Sakai, 1966; Kato, 1970) could not show similar findings by comparison with a normal group. There is a latent period before increments in serum calcium and phosphorus contents related to the
Y. Kato et al.: Hypercalcemia and Hyperphosphatemia Induced by Lead
thyroparathyroid appratus (Copp, 1960) and/or kidney damage (Jastak, 1968). In the present experiments, no latent period was recognized before the concentration changes in the serum calcium and phosphorus after Pb-Ac treatment, in vivo as well as in vitro. These facts and the histochemical observations (Fig. 4) suggest that these phenomena, at least in the early stage, may result not from participation of thyroparathyroid apparatus or from kidney damage but from some direct action of Pb-Ac on the bone surface. A large concentration of lead produces hemolysis (Waldron, 1966) which results in the increment of the serum phosphorus content. Following the Pb-Ac injection in the present study, the degree of hemolysis observed macroscopically decreased with time and could not be observed after 1 h, while conversely the phosphorus content in the serum increased with time up to 30 min. The hemolysis may be, at least partially, one of the factors that resulted in an increase in the content of phosphorus in the serum in the early stages of our experiments. It is known that bone is a highly vascular tissue (Shim, 1968; Kelly, 1968) and the rate of the bone blood flow has been estimated with lSF to be about 17% of the plasma volume per min in rat (Van Dyke et al., 1965). Lead in blood may permeate easily into the extracellular fluid (ECF) through the walls of the capillaries supplying bone. Lead in ECF may pass between the lining cells of the bone surface to the bone extracellular fluid (BEF) (Neuman and Ramp, 1971) by a concentration gradient and become deposited on the crystal surface of bone. These phenomena appeared to occur quickly since the deposition of lead was demonstrated on the surface of the bone within 30 s after the Pb-Ac injections (Fig. 4). It seems likely that the mechanism of lead deposition in bone is not simple. By X-ray diffraction studies, synthetic crystals of hydroxyapatite have been reported to change to lead-apatite when immersed in a buffer with Pb-Ac for 5 to 45 days (Moriwaki et al., 1975). The results suggested that calcium ions were displaced by lead ions equivalently on the synthetic crystals. In the present short-term experiments, however, the concentrations of both calcium and phosphorus increased simultaneously in vivo as well as in vitro. In the hydration shells of crystals, 1 mol of calcium should be displaced by 1 mol of lead. However, in the lattice surface of crystals, it would be expected that 1 mol of lead may displace more than 1 mol of calcium because of the difference in ionic radii (Ca++: 0.99 A, Pb++: 1.32 A) (Neuman and Neuman, 1953). In this case, the altered charge equivalence may reduce the positive charge on the crystal surface, resulting in a corresponding loss of boundary phosphate. By this
45
15.0
/, 2
"~ lO.O
.c 5.0 --o--
I
I
0
I
I
l
200 4.00 600 800 Pb Qdded (/..t,g)
P
I
1000
Fig. 6. Increments in the contents of calcium and phosphorus in the media after shaking bone powder (100 rag) in the serum (2 ml, p H 7.4) for 30 min with the various amounts of lead acetate (0.2 ml). The increments of contents of calcium and phosphorus correlate with the amount of lead acetate added
mode of action, a rapid increase in concentrations of calcium and phosphorus in the BEF may result from a continuous influx of lead. If the concentrations of calcium and phosphorus in the BEF become greater than those in ECF, calcium and phosphorus may be expected to efflux from BEF to ECF according to the concentration gradient and enter the plasma in the numerous small vessels. As to the mechanism of calcium efflux from BEF to ECF, two theories have been presented. One is that calcium is transported out of bone actively by an intraceUular transport mechanism (Talmage, 1975). The other is that calcium is transported between the lining cells (Rasmussen and Bordier, 1974). Even if the active transport theory of Table 1. Contents of calcium and phosphorus in media shaken with bone for different periods of time Reagent
DW" Pb-Ac u DW Pb-Ac
Time of shaking
I0 10 30 30
s s min min
No. of experiments
5 5 5 5
Mineral content/100 ml of medium Ca (rag)
P (rag)
6.43 7.75 6.51 9.80
4.48 5.54 4.63 6.74
(0.16) c (0.22) (0.23) (0.41) a
(0.24) (0.40) (0.38) (0.71) e
a Distilled water as control b Lead acetate (Pb: 600 pg) c Mean and standard deviation (in parentheses) are shown a 10 s vs. 30 min, P < 0.001 e 10 s vs. 30 min, P < 0.02 The differences are all significant between D W and P b - A c (P < 0.001)
46 c a l c i u m is c o r r e c t physiologically, c a l c i u m efltux by intracellular t r a n s p o r t w o u l d p r o b a b l y be restrained rather t h a n activated after the P b - A c injection. T h i s is b e c a u s e the astringent effect o f a metal salt results in interference with the p h y s i o l o g i c a l action o f cells ( C a w s o n and Spector, 1975). Since the functions and the m o r p h o l o g i c c h a n g e s o f the o s t e o g e n i c cells were n o t e x a m i n e d , it c a n n o t be stated w h e t h e r o r not these cells t o o k p a r t in the mineral c h a n g e s in serum. All d a t a in the present experiments, h o w e v e r , indicate that the m o v e m e n t o f the lead f r o m the b l o o d to b o n e and o f c a l c i u m and p h o s p h o r u s f r o m the b o n e to the b l o o d w e r e rapid. T h e s e facts suggest t h a t the m o v e m e n t o f the minerals following the P b - A c injection m a y p r o c e e d b y passing between the lining cells o f the b o n e surface rather t h a n f r o m a c t i v a t i o n o f the native t r a n s p o r t m e c h a n i s m s o f the cells.
References Bachra, B.N., van Harskamp, G.A.: The effect of polyvalent metal ions on the stability of a buffer system for calcification in vitro. Calcif. Tiss. Res. 41, 359-365 (1970) Cart.land, G.F., Koch, C.: A micro-modification of the KeithRowntree plasma-dye method for the estimation of blood volume in the rat. Amer. J. Physiol. 85, 540-545 (1928) Castellino, N., Aloj, S.: Kinetics of the distribution and excretion of lead in the rat. Brit. J. Industr. Med. 21, 308-314 (1964) Cawson, R.A., Spector, R.G.: Clinical pharmacology in dentistry, p. 55. Edinburgh-London-New York: Churchill Livingstone 1975 Copp, D.H.: Parathyroids and homeostasis of blood calcium. In: Bone as a tissue (K. Rodahl, J.T. Nicholson, E.M. Brown, eds.), pp. 289-299. New York-Toronto-London: McGraw-Hill 1960 de Bruin, A.: Certain biological effects of lead upon the animal organism. Arch. Environ. Health 23, 249-264 (1971) Eastoe, J.E.: Methods for determination of phosphate, calcium and protein in small portions of mineralized tissues. In: Proc. Second European Symposium on Calcified Tissues (L.J. Richelle, M.J. Dallemagne, eds.), pp. 265~274. Liege: Universitb de Liege 1965 Gabbiani, G.: Relationship between the calcifying power of various substances in vivo and their solubility in vitro. Experientia (Basel) 20, 514 (1964) Jastak, J.T., Morrison, A.B., Raisz, L.G.: Effects of renal insufficiency on the parathyroid gland and calcium homeostasis. Amer. J. Physiol. 215, 84-89 (1968) Johannsson, O., Perrault, G., Savoie, L., Tuchweber, B.: Action of various metallic chlorides on calcaemia and phosphataemia. Br. J. Pharm. Chemother. 33, 91-97 (1968) Karpatkin, S.: Lead poisoning after taking Pb-Ac with suicidal intent. Arch. Environ. Health 2, 679-684 (196 I) Kato, Y.: Analytical studies on experimental skin calcification in the rat after administration of lead acetate and subsequent
Y. Kato et al.: Hypercalcemia and Hyperphosphatemia Induced by Lead administration of polymyxin B sulfate. Bull. Tokyo Med. Dent. Univ. 17, 53-63 (1970) Kato, Y., Ogura, H.: Low-temperature ashing of bovine dentine. CalciL Tiss. Res. 18, 141-148 (1975) Kelly, P.J.: Anatomy, physiology, and pathology of the blood supply of bones. J. Bone Joint Surg. 50-A, 766-783 (1968) Kumagai, A., Sakai, T.: Effect of lead acetate on serum calcium and phosphorus in rats. Arch. int. Pharmacodyn. Thtr. 160, 248-252 (1966) Moriwaki, Y., Ida, K., Yamaga, R.: Effect of diverse ions on the crystallinity of carbonate-containing hydroxyapatite. (In Japanese, with English abstract.) J. Chem. Soc. Jap. No. 5, 801-807 (1975) Neuman, W. F., Mulryan, B.J.: Synthetic hydroxyapatite crystals III. The carbonate system. Calcif. Tiss. Res, 1, 94-104 (1967) Neuman, W.F., Neuman, M.W.: The nature of the mineral phase of bone. Chem. Rev. 53, 1-45 (1953) Neuman, W.F., Neuman, M.W.: The chemical dynamics of bone mineral, p. 94 (a), pp. 31-33 (b). Chicago: University of Chicago Press 1958 Neuman, W.F., Ramp. W.K.: The concept of a bone membrane; some implications. In: Cellular mechanisms for calcium transfer and homeostasis (G. Nichols, Jr., R.H. Wasserman, eds.), pp. 197-209. New York-London: Academic 1971 Okabe, T.: Studies on deposition mechanism of lead salt in hard tissues. (In Japanese.) Ochanomizu Medical J. 18, 175-185 (1970) Okada, M., Mimura, T.: Zur Physiologie and Pharmakologie der Hartgewebe. I. Mitteilung: Eine Vitalfiirbungsmethode mit Bleisalzen und ihre Anwendung bei den Untersuchungen fiber die rhythmische Streifenbildung der barten Zahngewebe. Jap. J. Med. Sci., IV. Pharmacol. 11, 166-170 (1938) Pearse, A.G.E.: Histochemistry, p. 405. London: Churchill 1954 Rasmussen, H., Bordier, P.: The physiological and cellular basis of metabolic bone disease, pp. 29-31. Baltimore: Williams & Wilkins 1974 Schneider, B.J.: Lead acetate as a vital marker for the analysis of bone growth. Amer. J. Phys. Anthrop. 29, 197-200 (1968) Shim, S.S.: Physiology of blood circulation of bone. J. Bone Joint Surg. 50-A, 812-824 (1968) Sprague, S., SIavin, W.: A simple method for the determination of lead in blood. Atomic Absorption Newsletter 5, 9-10 (1966) Talmage, R.V.: Effect of fasting and parathyroid hormone injection on plasma 45Ca concentrations in rats. Calcif. Tiss. Res. 17, 103-112 (1975) Termine, J.D., Posner, A.S.: Amorphous/crystalline interrelationships in bone mineral. Calcif. Tiss. Res. 1, 8-23 (1967) Toribara, T.Y., Terepka, A.R., Dewey, P.A.: The ultrafiltrable calcium of human serum. I. Ultrallltration methods and normal values. J. Clin. Invest. 36, 738-748 (1957) Van Dyke, D., Anger, H.O., Yano, Y., Bozzini, C.: Bone blood flow shown with ~SF and the positron camera. Amer. J. Physiol. 209, 65-70 (1965) Waldron, H.A." The anaemia of lead poisoning. Brit. J. Industr. Meal. 23, 83-100 (1966) Willis, J.B.: Determination of calcium and magnesium in urine by atomic absorption spectroscopy. Anal. Chem. 33, 556-559 (1961) Received March 14/A ceepted April 2 7, 1977
