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Comparative metabolism of strontium and calcium in man
International Journal of AppliedRadiation and Isotopes, 1959, Vol. 4, pp. 144-153. Pergamon Press Ltd. Printed in Northern Ireland
Comparative Metabolism of Strontium and Calcium in Man** A R T H U R R. SCHULERT and EDWIN A. PEETS L a m o n t Geological Observatory of Columbia University, Palisades, New York
and DANIEL LASZLO,~ HERTA SPENCER, MARTIN CHARLES, and JOSEPH SAMACHSON Division of Neoplastic Diseases, Montefiore Hospital, Bronx, N e w York
(Received 11 May 1958) T h e metabolic fate of intravenously administered Sr s5 a n d Ca 45 was studied in m a n . It was found that the isotopes were s o m e w h a t equally divided between bone a n d soft tissue shortly after administration, but that after four months, more t h a n 99 per cent of the fraction of each isotope remaining in the b o d y resided in bone. T h e initial specific activity (isotope concentration per g r a m m e calcium) was far greater in soft tissue t h a n bone with the values a p p r o a c h i n g uniformity in a b o u t four months. T h e retention a m o n g the bones in the interval studied was greatest in vertebrae and least in long bone shaft and skull. T h e bone exhibits no m a r k e d preference in the uptake of the isotopes initially. However, as Sr s5 is preferentially excreted by the kidneys, the relative ratio of Ca 45 to Sr s5 remaining in bone gradually increases. T h e net retention of Ca 45 is a b o u t 60 per cent whereas that for Sr s5 is a b o u t 25 per cent.
LE MI~TABOLISME C O M P A R A T I F E N T R E LE S T R O N T I U M C A L C I U M DANS L ' H O M M E
ET
Le r6sultat m6tabolique de Sr s5 et Ca a~ administrd intraveineusement rut 6tudid dans l ' h o m m e . I1 fut ddcouvert que les isotopes dtaient divisds prSsque 6galement entre l'os et le tissulaire peu apr&s l'administration, mais aprSs quatre mois, plus de 99 p o u r cent de la fraction de chaque isotope restant dans le corps se trouvait dans l'os. A u d~but, la radioactivitd spdcifique (la concentration d'isotope p a r g r a m m e de calcium) 6tait plus grande dans le tissulaire que dans l'os, les valeurs a p p r o c h a n t de l'uniformit6 apres environ quatre mois. La retention entre les os dans l'interval 6tudi6, 6tait la plus grande dans les vert~bres et dtait la m o i n d r e dans les longs os et le cr~ne. L'os ne m o n t r e aucune pr6f6rence initialement entre les isotopes. Ndanmoins c o m m e le strontium est excretd par les reins de prdf6rence, le r a p p o r t r61atif entre le Ca 45 et le Sr ss restant dans l'os a u g m e n t e graduellement. La retention nette d u Ca 45 est pros de 60 p o u r cent et prSs de 25 pour cent pour le strontium.
C P A B H 1 4 T E . J I b H M ~ O B M E H C T P O H K H H H HA.JIBKHH B HE.;'IOBEHE BHyTpIIBOHHO CTpOHILI4It-85 14 l~aabi414n-45. 1]oga3aHO, qT0 BeKope rioe.~Ie BBe/~eItHH o~a H3OTOHa paenpe;IeJImOTCg 6os~ee mli4 MeHee panHoMepHo Me~,~y HOCTHMHH MHFHHMHTHaHItMH. O,~HaHO, qepe3 4 Mee~lta noe~e BBe~ettIIIt
gsyqaJiacb cyabSa BBO~IeHHI~IX qe.~oBeKy
* A report of the early phases of this work was presented before the 3rd annual meeting of the Society of Nuclear Medicine at Salt Lake City in June 1956. 1" This work was conducted under U.S. Atomic Energy Commission contract number AT (30-1) 1656 with Columbia University (Lamont contribution No. 312) and contract number AT (30-1) 1763 with Montefiore Hospital. **Deceased 7 June, 1958. 144
Comparative metabolism of strontium and calcium in man
145
B •OCTHX oTHapymHBaeTca 6o~ee 99% OT oSmero RoaHqeCTBa~aovona, co~epmameroca BO BceM were. HaqaaI,Haa y~eJibHau aHTI4BHOCTI, (HOHI~eHTpalu4flnaovona Ha rpaMM Ha~b~Ha) B MFIFHHXTHaHAX 3HaqIlTeJISrI0BBIme, tleM B HOCTHX, 0~HaH0 nO HcTeqertl414 4-x MecfIILeB y~eabHBie aRTHBHOCTH IlpaHTI4qecHH BBIpaBHI4BaIOTCH. l l p r t cpaBHeHI,II4 coAepH~aHI4H I43OTOl]a B pa3JIHtIHt,IX HOCTYIX OTMeqeH0 caMoe Bt~ICOH0e IlOrOlOli~eHHe B II03BOH0qHI4He H caMoe HHBHOe--B ~JIHHHI:,IX TpySqaTblX HOCTflX I4 B qepelIe. B HatIa.lIbHBIl~ IlepI4oA ~.11~ HOCTe~i He HaT~io~aeTeH 3aMeTH01~ H86HpaTeJIBHOCTH IIOF~IOII~eHHH BBO~FIMBIX H3OTOHOB; O~HaH0, C TeqeHI4eM BpeMeHH 0THOIIIeHHe coAepmaHn~ B HOCTHXHaJIBIIHH-45 H cTp0HI~14IO-85 IIOCTeHeHHO yBeaIlqHBaeTeH, TaR Hall qepe3 noqHrI HpeHMyIKeeTBeHHO BBiReafleTefl CTpOHI~H~]-85. IIpH aTOM n o r ~ o ~ e H ~ e n ~OCT~X H a a b ~ - 4 5 ~ocwr~raew np~tT~i~3~we.~no 6 0 % i~ c w p o m l H ~ - 8 5 - -
o~oao 25%. METABOLISCHER VERGLEICI-I VON STRONTIUM UND CALCIUM IM MENSCHLICHEN KORPER Der metabolische Ablauf von intraven6s verabreichten Sr s5 und Ca~5 wurde erprobt im menschlichen K6rper. Es wurde festgestellt, dass die Isotopen kurz nach Verabreichung gleichm/issig verteilt waren zwischen Knochen uns Geweben, jedoch nach vier Monaten, mehr als 99 prozent jedes einzelnen Isotopen die im K6rper verblieben, waren in den Knochen enthalten. Die ursprfingliche T~itigkeit (Isotopen Konzentration per Gramm Calcium) war bedeutend gr6sser in weichen Geweben als in den Knochen; nach vier Monaten waren die Auswertungen beinahe einheitlich. Die Behaftung zwischen den Knochen w~ihrend der Versuchszeit war am gr6ssten in der Wirbels~iule und am geringsten in R6hren- und Sch/idelknochen. Ursprfinglich zeigt der Knochen keine lange markante Bevorzugung in der Aufnahme von Isotopen. Jedoch, da Sr 85 durch die Nieren bevorzugt ausgeschieden wird, vergr6ssert sich das relative Verh/iltnis von Ca 45 zu Sr ss allm~ihlich. Der Netto-Rfickstand von Ca45 ist etwa 60 prozent wogegen der des Sr sa etwa 25 prozent betr~igt. INTRODUCTION STRONTIUM represents a t r a c e e l e m e n t in the h u m a n b o d y , h a v i n g a n a v e r a g e conc e n t r a t i o n o f 4-5 parts p e r 10,000 p a r t s of c a l c i u m in bone. a) W i d e s p r e a d interest in s t r o n t i u m t o d a y is d u e to the presence o f the long-lived radioisotope Sr 9° a m o n g the p r o d u c t s o f n u c l e a r fission. ~ S t r o n t i u m - 9 0 is g e n e r a l l y r e g a r d e d as the p r i n c i p a l h e a l t h h a z a r d a m o n g the fission p r o d u c t s since it is selectively t a k e n u p b y the h u m a n skeleton w h e r e it r e m a i n s deposited for m a n y years. ~3)
EXPERIMENTAL
1. Clinical design and sampling T r a c e r quantities o f Sr 85 [T½ 65 days] a n d C a 45 [T½ 163 days] in chloride f o r m were a d m i n i s t e r e d in a single dose simultaneously to ten t e r m i n a l c a n c e r patients b y the i n t r a v e n o u s r o u t e [ T a b l e 1]. T h e a v e r a g e dose was 1 . 5 / t c / k g for Sr 85 a n d 0.4/~c/kg for C # 5. T h e i n t r a v e n o u s route
I n o r d e r to be able to e v a l u a t e the radiotoxicity o f Sr 9° in m a n , the k n o w l e d g e o f the m e t a b o l i s m o f the e l e m e n t in h u m a n s is o f paramount importance. Extensive w o r k has b e e n d o n e to d e t e r m i n e the a b s o r p t i o n a n d excretion o f a d m i n i s t e r e d s t r o n t i u m a n d c a l c i u m isotopes. ~4~ T h e present s t u d y was designed to show the relative r e t e n t i o n o f s t r o n t i u m c o m p a r e d to c a l c i u m in m a n a n d to d e t e r m i n e the distribution o f administered isotopes within the b o d y including b o t h b o n e a n d soft tissue. PROCEDURES o f a d m i n i s t r a t i o n was selected since it excludes the p a r a m e t e r o f gastro-intestinal absorption. Since a u t o p s y analyses w e r e e m p l o y e d , the patients were, o f necessity, o f limited life e x p e c t a n c y w i t h c a n c e r i n v o l v e m e n t , a n d c a n n o t be considered as n o r m a l h e a l t h y adults. Survival times o f patients studied v a r i e d f r o m 3 h r to 124
146
Arthur R. Schulert et al. TABLE 1. List of patients studied
q Patient
H.H.
S.K.
S.J. C.C.
J.Z. G.B. S.G. T.M. A.W. M.S. J.T. I.J.
Age
Sex
Wt. (kg)
62 50 60 59 72 70 49 70 63 70 67
F
45
M
54
F F M F M M M F M
i
54 73 73 54 50 43 61 56 76
1
53
i 55
v
] ~
Time interval isotope admin.-death (days)
1/8 2 3 :3 6 18 30 39 65 124. 251 960
Clinical diagnosis
Carcinoma of pancreas Hodgkins disease Carcinoma of colon Carcinoma of breast Carcinoma of lung ] Carcinoma of breast Multiple myeloma ] Carcinoma of lung Carcinoma of kidney Carcinoma of lung Multiple myeloma Multiple myeloma
Skeletal involvement
None All vertebrae Moderate Diffuse Diffuse None Moderate Single area of skull Thoracic vertebrae None Moderate Diffuse
i
days. In addition, samples were obtained from one patient who received Sr s5 only, 251 days prior to death and from one patient who received Ca 4~ only, 960 days before death• At autopsy, samples of many soft tissues and of bone were taken for analysis. As many as twenty-five soft tissue samples were obtained in the early studies while later sampling and analysis of soft tissues were restricted to four major organs, namely, muscle, liver, kidney, and lung. Except for muscle, these organs were obtained in toto and only small sections were removed for histologic analysis. In regard to bone, samples of vertebrae and ribs were obtained from all patients and in seven of the ten cases either femur shaft, iliac crest, and skull were obtained. Particular attention was given to the variation among bones of the skeleton as well as the distribution within single bones• 2. Radiochemical assay
Strontium-85 is readily assayed, since it emitS a 7-ray of moderate energy. The sample is merely dried, ashed, and counted in a well-crystal y-ray spectrometer. The isotope counted with an efficiency of approximately 10 per cent with an associated background of about 10 counts per min. (Slight variations in both of these values were constantly checked by appropriate blanks and standards.)
Two chemical procedures were used in the Ca 45 assay, one adapted to bone and the other for soft tissue. Ashed bone is dissolved in HC1 after which the p H is adjusted to 3.0-3.5 with ammonium hydroxide. A mixture of oxalic acid and ammonium oxalate is then added to precipitate the calcium oxalate. (Care is taken to avoid a p H higher than 3.5 since this precipitates some calcium as the phosphate.) The calcium oxalate is filtered and converted to the oxide by heating in a muffle furnace. The calcium oxide is weighed, dissolved in HC1, and diluted to 100 ml in a volumetric flask. A convenient aliquot is taken, the p H adjusted, and the calcium again precipitated as the oxalate. After digestion the precipitate is filtered on 2 9 m m diameter No. 42 W h a t m a n filter paper which is then placed on a brass planchet, covered with a thin piece of pliofilm and placed in a low-level end window fLcounter. For the determination of Ca 45 in soft tissue, the sample is ashed in platinum and then fused with sodium carbonate at 1200°C. The fusion mixture is leached with water, filtered to remove sodium phosphate and soluble carbonates, and the residue dissolved in hot HC1. After cooling, ammonium hydroxide is added to p H 7, precipitating ferric hydroxide which also occludes remaining phosphate ion. The precipitate
Comparative metabolism of strontium and calcium in man
is filtered with the calcium remaining in the filtrate. To remove traces of calcium which may have been carried with the ferric hydroxide, the precipitate is redissolved in HCI and again precipitated, the filtrate being combined with the previous one. Excess ammonia is expelled by boiling. Oxalic acid and ammonium hydroxide are added to precipitate the calcium oxalate. After digestion and cooling, the oxalate is filtered and counted in the same manner as the bone samples. The fl-counter in which the Ca 45 samples
are counted is equipped with an anticoincidence ring so that background is about 2 counts per min. The counting efficiency for Ca 45 varies with particular counters from 8 to 12 per cent so that 1 0 - e # c can be adequately detected in a sample. The Sr 85 which follows the calcium through the chemistry, registers on the t-counter with an efficiency of only 0.3 per cent. As the Sr 85 concentration is known from the 7-assay, this small correction for the Sr 85 is made in the t-count.
PRECISION ANALYSIS AND TREATMENT
In order to properly evaluate the experimental findings, it is necessary to know the relative precision of each particular result. The problem may be considered in three categories :
147
OF D A T A
fions within bone are noted.) The average o f the mean deviations for the Ca 45 determinations i n these groups is 22.0 per cent for soft tissue and 16.4 per cent for bone. The corresponding values for Sr 85 are (1) the precision of the radiochemical 14.5 per cent for soft tissue and 17.1 per determinations, cent for bone. (2) the biological precision of different There is insufficient direct information to portions of the same tissue, and assess the biological variation from one individual to another. However, the relative (3) the precision between the tissues of distribution of isotopes in the skeletons of two individuals treated in an identical different individuals gives some indication. manner. Thus the standard deviation in the vertebra/ There is also the matter of statistical rib concentration ratio for Sr 85 is 30 per error of counting but the counting time was cent showing, as expected, that variation almost always sufficient to reduce this to among individuals is somewhat greater below 1 per cent, making it negligible than variation in representative samples in compared to the other variations. a single tissue. The concentration ratio The chemical precision is of concern of one bone to another is changing in time, primarily in the Ca 4s determination, since as subsequently discussed, but the change essentially no chemistry is involved in the is slight over a four month interval. Sr 85 assay. Duplicate and in some cases These facts account for a large a m o u n t triplicate samples were analysed. The of the "scatter" in the data and underline average of the mean deviations for the the need for caution in its interpretation. Ca 45 determinations in these small groups Nevertheless, certain generalizations and is 4.8 per cent and 7.5 per cent in bone and a' number of significant facts can be drawn from the experimental results. soft tissue respectively. In comparing the biological variation in In order to give a gross overall picture two or more parts of the same tissue, an of the behavior of the isotopes in the human effort was made to obtain representative body in time, the total body load is calcusamples, e.g. a cross section of bone or the lated for each patient. A standard procedure, half of a kidney cut along its major axis. which admittedly involves considerable (In subsequent discussion, systematic varia- approximation, is employed as follows:
148
Arthur R. Schulert et al.
Soft tissues
(1) The weights of lung, liver, and kidney were obtained at autopsy. Muscle weight is estimated from the total body weight using the body composition data of MITCHELL et (2) The total content of each tissue is then calculated by multiplying the estimated tissue weight by the tissue concentration. (3) Accordingly, these four tissues constitute 40.3 per cent of the total body weight, whereas the total soft tissue comprises 85.2 per cent. On the assumption that the tissues analysed are representative of all the soft tissue, the total isotopic content of these 4 tissues is multiplied by 85.2]40.3 to give the total estimated isotopic content for soft tissue. Bones
The bones are divided into three groups, which according to the data of TROTTER, ~6~ comprise the following percentages of the total skeleton: RESULTS
AND
The distribution of Sr 85 and C # a in the body, the percentage of the isotopes accounted for, and the ratios of each isotope contained in bone and soft tissues are listed in Table 2. Also shown are the ratios of Ca45/Sr 85 in bone and soft tissues. In spite of the problems of representative sampling the approximations entering in the calculations, and the fact that the extent and degree of the cancer involvement varied markedly, a fairly consistent pattern is obtained. The limitations of this analysis are demonstrated most sharply by the calculated C # 5 retention in patient C. C. at three days (127 per cent). There is a partial explanation for this since the patient (a female weighing 72.6 kg) had a higher percentage of adipose tissue than normal. Thus the standard assumption that the skeleton represents 14-8 per cent of the body undoubtedly gives an estimated bone
I " L o n g " bone: limb, hip, skull (and mandible) 70.6% II Rib 5.7% I I I Vertebra and sternum 8.9% Total 85.2% (1) The weight of each bone analysed is estimated from the body weight and the data of TROTTERC8~ together with the data of MITCHELL et al. C5~ for the percentage of total skeletal mass. (2) Bones within a group which are not represented are assumed to have the same value as the average of the others in order to estimate the total isotopic content for each group. (3) When an entire group is not represented, its value is estimated by using the femur/rib ratio (for "long" bone) and the vertebra/rib ratio (for the vertebra/sternum group) as obtained from the average on all patients. (4) The total estimated isotopic content of the three groups is multiplied by 100185.2 to get the estimated total skeletal content. DISCUSSION
weight in this case which is in excess of the true value. Since the concentration of the isotopes per gramme of bone is much greater than of soft tissue, the standard procedure results in a high value for total body retention. It is noted that both isotopes are somewhat equally divided between bone and soft tissue shortly after their administration and that the proportion gradually shifts so that less than one-half per cent of the body load of each isotope is retained in the soft tissues by 124 days. This is primarily due to the more rapid removal of the isotopes from the soft tissues. The data also suggest some translocation from soft tissue to bone, particularly of Ca 45 as previously noted by others in animals over short intervals. ¢7) This translocation is shown graphically in Fig. 1. Whereas the skeletal Sr 8~ retention begins to decline within the first few days and
149
Comparative metabolism of strontium and calcium in man TABLE 2. Distribution of Sr s5 a n d Ca 45 in soft tissues a n d bone; tissue concentrations expressed in per cent of administered dose Patient days*
H.H. 1/8
S. K .
s.j.
2
3
c 3
J. Z. 6
G. B. 18
S. G. 3O
Y. M. 39
A.W. 65
M. S, 124
c
Soft tissue Bone
55 33
31 47
44 36
35 45
26 42
22 41
1"2 20
1-4 26
1.5 30
0"15 26
Total Sr ss ratio bone/soft tissue
88
78
80
80
68
63
21
27
32
26
0.60
1-5
0.82
1.3
1.6
1-8
17
19
20
173
So~ tissue Bone
33 30
28 42
28 55
53 74
32 59
14 36
3-0 35
5.5 71
1-9 79
0"27 6O
Total Ca 45 ratio bone/so~ tissue
33
70
83
127
91
50
38
76
81
6O
0.91
1-5
2.0
1.4
1.8
2.6
12
14
42
222
0.60 0.91
0-90 0.89
0.64 1-5
1.5 1.6
1.2 1-4
0.64 0.88
2.5 1.8
3-9 2-7
1-3 2.6
1.8 2"3
SrS5
Ca45
Ca45/ Soft tissue Sr ss Bone
* Days elapsed between the injection of the isotopes a n d exitus of patients.
b3 O0
0 8O h
o
6c bJ
Ld n~
r0
~h- 4C
o
O
I
I
20
I
I 4O
J
I 6O
I 80
I
I
I00
DAYS FIG. 1. ISotopic content of the total skeleton.
•
C a 4~
0 Sr ss
I 120
Arthur R. Schulert et al.
150
approaches a plateau at about 25 per cent, the C # 5 appears to rise for the first month and levels off at about 65 per cent. Three experimental points are badly off the C # 5 curve, but these are the three least valid values of the ten: the high 3 day point [C. C.] is subject to question as previously mentioned, and the low points at 18 and 30 days represent the only skeletons for which just two bones were obtained for analysis. The retention values for the two isotopes are consistent with those obtained in balance studies over shorter intervals by other investigators. (4, 8, 9, 10~ The ratios of Ca45/Sr s5 of the individual tissues are listed in Table 3. Although these ratios are somewhat more variable for soft tissues than for bone, both show an increase with time; the values for bone being generally 2 or above after 30 days. This is a reflection of the fact that Sr s5 is preferentially eliminated from the body and thus has a shorter average biological residence time than calcium. The enrichment in favor of calcium is not apparent in the bones until several days after the administration of the isotopes. Thus the bone tissue as a whole shows no preference in accepting both isotopes immediately after administration in agreement with the conclusion of BAUER et al. (n) and COMAR et al. a2) The subsequent increase in the Ca4a/Sr 85 ratio in time reflects the preferential excretion of strontium by the
kidneys. It has been stated that the overall calcium enrichment from diet to bone is governed primarily by the calcium/strontium discrimination factor across the intestine and by that of urinary excretion. (13,14) In the course of time, the renal discrimination factor is reflected by the retention of the isotopes in the body, i.e. primarily in the skeleton. Whereas the intestinal discrimination is essentially a one step process (disregarding the endogenous fecal strontium) in that the isotopes which reach the intestinal tract are either taken across the intestinal wall or eliminated from the body, the kidney discrimination is a cyclic process in which ions that have been retained in the body during one circulatory cycle and via the renal tubules are presented to the kidney again during the next circulatory cycle. This repetitive renal process leads to continued preferential excretion of radio-strontium and therefore to increased relative retention of radio-calcium. The factor of net effective enrichment from blood to bone for C # ~ in the two to four month interval is approximately 2-3. An appreciable increase in the enrichment over longer periods of time is not expected since the loci of isotope deposition become more and more inaccessible with time due to the fact that: (t) The isotope is cleared early from the more accessible sites and (2) Some accessible sites will be made
TABLE 3. Ca45[Sr s5 ratios in bone and soft tissues as a function of time Soft tissue
Kidney Liver Lung Muscle Bone Femur Iliac crest Skull Sternum Rib Vertebra
Days 1/8
2
3
3
6
18
30
39
65
124
0'46 0"34 0.28 0"72
0.86 0"84 0'68
0.81 0"63 0"67 0.88
0.74 1.3 0-74
0.91 1.6 1.4 1.7
1.2 1.7 0.50 0.70
1.3
1.5 1.9 1-2 8.0
0.58 1,2 1,1 1,4
0.87 3'0 0.44 1"7
1.8
2.1
0"86 0'94 0.74 0'86
1.9
0"81 1"0 0"90 0"82
0.94
4.6 0.28
1.1 0.76 2'1
1.7 1.4 1.4 1.5
1.3 1.3
1.0 1.3 0.94
2-4 1-0
2'2 3.6 2-7
3-5 2-2 3.8
2.7 2.3 2.0 1.9
Comparative metabolism of strontium and calcium in man
unavailable by the continued deposition of new bone mineral. Although extensive data on the excretion of Sr s5 and Ca 45 in man have been obtained, t4,15) these excretions have not been followed for prolonged periods of time. One would expect the elimination of administered Sr 85 and Ca 45 to follow a power function as in the case of radium, a6) While the present data on bone retention of the isotopes are inconclusive, they sug.gest that only slight changes are occurnng after the second month, compatible with such an elimination pattern. Since the Ca45/Sr 85 retention ratio reaches two in two months, and since these isotopes present in bone remain there for years, it is concluded that this enrichment will also occur under continuous intake of Sr 9° due to fallout. A comparison of average Sr a° content of diet and human bone in northeastern United States by ECKELMANN et al. a7) indicates that the overall calcium/ strontium discrimination from diet to bone is four. Since the discrimination against strontium across the intestinal barrier has been reported to be two t4) and the discrimination from blood to bone is also two, the present data are in general agreement with those reported in the literature. The specific activity of Sr s5 (concentration o f Sr s5 per gramme stable calcium) of bones and of the major soft tissues are listed in Table 4. The average specific activity
151
of vertebrae and rib are respectively 5.4 and 2.3 times that of femur. This distribution must clearly change in time as evidenced by the fact that common strontium is uniformly distributed throughout the skeleton.m However, this approach to uniformity proceeds very slowly since Sr 9°, which has been in our environment for several years, is reported to be more greatly concentrated in vertebrae and rib than in long-bone shaft by factors of about four and two respectively, tlv) It is apparent that while the isotopes are largely concentrated in the skeletal tissue, the specific activity of the soft tissue greatly exceeds that of bone throughout the initial two month period following injection. It is noted in the 124 day and 251 day samples, however, that the specific activities are of the same order of magnitude. This indicates that about four months are required for the soft tissue to be cleared of the strontium initially deposited therein. Subsequently the isotopic content of the soft tissue probably reflects that which is resorbed from the bone into the blood stream, (the blood probably being in near equilibrium with t h e soft tissue). Further evidence that this must be the case comes from the data from one patient who was given Ca 45 alone, 960 days before death. Samples of the four major soft tissues were obtained and showed an average specific activity of one-twentieth that shown by the 124 day
TABLE 4. Specific activity of Sr s5 in bone and soft tissue (% administered dose/g Ca)
1/8
Kidney Liver Lung Muscle Femur Iliac crest Skull Sternum Rib Vertebra
21 8-0 8"0 11 0"048 0"18 0"071 0"17
2
8"1 12 9'5 3"3 0"036
0"070 0'031 0"067 0"035 0"095
3
3
6
8"8 7"4 1"8 12
7"6 1'.7 5"1 4'0
0'29 5'8 4'8 2'9
0"017 0"10 0"035 0"13
0'012
0"21 0"077 0"16
Days 18
30
39
65
124
251
2"3
0-69
3'2 4"1
1"6 0"24
0"47 0"54 1"4 0"25 0"024
0'57 0"25 0"36 0'42 0'016
0'17 0'070 0"32 0'053
0.092 0.043 0.019 0-056
0"11 0-027 0'099
0'073
0'25 0"052 0"16
0"14
0'052 0"070
0-067 0"078 0"16
0.062 0.056 0.116
0-14
0.14 0.044 0"11
152
Arthur R. Schulert et al.
case. Therefore it may be concluded that at intervals greater than four months, urinary excretion reflects bone resorption. The excretion may also give an index of the isotopic bone concentration, although the relationship is complex. For example, animal experiments have shown that the specific activity of soft tissue may ultimately become much less than that of bone. (ls,ag) Thus the relation is largely an empirical one. It is evident that in the practical experimental evaluation of agents for their effect on removing strontium from the skeleton, the most meaningful results would be obtained at long periods after strontium administration when the specific activity of the soft tissue approximates that of bone, or at least does not greatly exceed it. The induced enhancement of urinary radiostrontium excretion achieved in the early phase (2°) will largely represent clearing of soft tissues, whereas a sustained effect produced at later periods will represent removal of the isotope from bone. Studies carried out in our laboratories on the relationship of urinary radio-strontium excretion and bone concentration determined at time intervals of varying length following the administration of the tracer are in progress at the present. STERNAL END
~3.~,so
4
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I 75 I 54 ~ $ 5 ~ 12cm.
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2. V a r i a t i o n s o f Sr s5 concentration w i t h i n section.
a rib
Fig. 2 shows that the isotope concentration varies greatly within a single bone. For example, Sr 8~ concentration decreases b y a factor of greater than two in a section of rib as one proceeds from the sternal end for 12 cm toward the axillary portion of the SUMMARY
rib. This finding indicates the biological variation one may expect to encounter in rib samples taken at random. Table 5 lists the data of radio-strontium concentration in successive one mm layers of the femur which were taken from the "FABLE 5. Sr
85
concentratmn m various layers of the femur "
"
Time
D e p t h of bone mm
Concentration in % of dose/kg bone
3 hr
1 2 3 4 5 Marrow 1 2 3 4 5 Marrow 1 2 3 4 5
4'I1 1"64 1"72 1 "84 2"25 ll'16 6'00 3'96 3"95 4"48 4"57 7"41 2'91 2'01 1'57 1"35 1"21
2 days
65 days
external surface and were followed towards the marrow cavity. It is noted that the samples taken after three hours and two days show high activity in the outermost layer followed by a marked decline in the second and third mm. The value then gradually rises with the layer adjacent to the marrow having the highest concentration of all. However, the samples obtained 65 days after the administration of the isotopes show a progressive unidirectional decrease of activity from the outermost layer which has the highest concentration to the inner layer which has the lowest. This may suggest a greater resorption of the isotope through the marrow cavity than from the outer surface of the bone.
AND CONCLUSIONS
(1) Tracer quantities of Sr s5 and Ca 45 were administered simultaneously to ten cancer patients by the intravenous route and samples of bone and four major soft
tissue organs were obtained at autopsy for radio-assay. (2) The isotopes are approximately equally distributed between bone and soft
Comparative metabolism of strontium and calcium in man
tissue for the first few d a y s after a d m i n i s t r a t i o n ; after f o u r m o n t h s a b o u t 99.5 p e r c e n t o f the isotopes w h i c h are r e t a i n e d in the b o d y are f o u n d in b o n e . (3) T h e n e t r e t e n t i o n o f t h e isotopes a p p e a r s to level o f f at a b o u t 60 p e r c e n t for C a 45, a n d 25 p e r c e n t for Sr sS. (4) T h e r e is a g r a d u a l increase in the Ca45/Sr s5 ratio in b o n e , w h i c h r e a c h e s a v a l u e o f t w o o r m o r e in the t w o to f o u r m o n t h interval. (5) T h e specific a c t i v i t y o f the isotopes
153
is m u c h g r e a t e r in soft tissue t h a n in b o n e t h r o u g h o u t t h e first t w o m o n t h s after a d m i n i s t r a t i o n , b u t the values a p p r o a c h t h e s a m e o r d e r o f m a g n i t u d e at f o u r m o n t h s . Acknowledgements--The authors gratefully acknowledge
the role of J. LAURENCEKULP, who along with DANIEL LASZLO,initiated this cooperative effort and gave valuable counsel throughout the investigation. WALTERECKELMANN was integrally associated with the early phases of the work. Important assistance in the radiochemical analysis was provided by MARIAN WAMPLER and DOROTHY WALTON.
REFERENCES
1. THURBER D. L., KULP J. L., HODGES E., GAST P. W. and WAMPLER J. M. Science 28, 256 (1958). 2. Hearings before the Special Subcommittee on Radiation of the Joint Committee on Atomic Energy, The nature
3. 4. 5.
6. 7. 8. 9. 10.
of radioactive fallout and its effects on man, May 27-29-June 3-7, (1957), U.S. Government Printing Office, Washington, D.C. (1957). HAMILTONJ . G . Rev. Mud. Phys. 20, 718 (1948). SPENCER H., LASZLO D., and BROTHERS M. or. Clin. Invest. 36, 680 (1957). MITCHELL H. H., HAMILTON T. S., STEGGERDA F. R. and BEAN H . W . J. Biol. Chem. 158, 625 (1945). TROTTER M. Personal communication. NORRISW. P. and KISlELESKIW. Cold Spr. Harb. S~mp. Quant. Biol. 13, 164 (1948). BELLIN J. and LASZLO D. Science 117, 331 (1953). BRONNER F., HARRIS R. S., MALETSKOS C. J. and BENDA C. E. J. Clin. Invest. 35, 78 (1956). HARRISON G. E., RAYMOND W. H. A. and TRETHEWAY H. C. Clin. Sci. 14, 681 (1955).
11. BAUERG. C. H., CARLSSONA. and LINDQUISTB. Acta Physiol. Scan& 35, 56 (1955). 12. COMAR C. L., WHITNEY I. B. and LENGEMANN F. W . Proc. Soc. Expl. Biol. Med. 88, 232 (1955). 13. COMMAR C. L., RUSSEL R. S. a n d WASSERMAN R. H. Science 126, 485(1957). 14. Deposition and retention of ingested strontium-90 in the skeleton. Committee Report Washington, D.C., April 23, (1957). Official use only. 15. SPENCER H., SAMACHSON J . and LASZLO D. Fed. Proc. 17, 154 (1958). 16. NORRISW. P., SPECKMANT. W. and GUSTAFSON P. W . Amer. J. Roentgenol. 73, 661 (1955). 17. EC•ELMANN W. R., KULP J. L. and SCHULERT A.R. Science 127, 266 (1958). 18. NEUMAN W. F. and NEUMAN M. W. The Chemical Dynamics of Bone Minerals, U. of Chicago Press (1958). 19. COMAR C. L. Radioisotopes in Biology and Agriculture, McGraw-Hill (1955). 20. SPENCER H., SAMACHSONJ., KABAKOWB. and LASZLO D. Clin. Sci. 17, 291 (1958).
Comparative Metabolism of Strontium and Calcium in Man** A R T H U R R. SCHULERT and EDWIN A. PEETS L a m o n t Geological Observatory of Columbia University, Palisades, New York
and DANIEL LASZLO,~ HERTA SPENCER, MARTIN CHARLES, and JOSEPH SAMACHSON Division of Neoplastic Diseases, Montefiore Hospital, Bronx, N e w York
(Received 11 May 1958) T h e metabolic fate of intravenously administered Sr s5 a n d Ca 45 was studied in m a n . It was found that the isotopes were s o m e w h a t equally divided between bone a n d soft tissue shortly after administration, but that after four months, more t h a n 99 per cent of the fraction of each isotope remaining in the b o d y resided in bone. T h e initial specific activity (isotope concentration per g r a m m e calcium) was far greater in soft tissue t h a n bone with the values a p p r o a c h i n g uniformity in a b o u t four months. T h e retention a m o n g the bones in the interval studied was greatest in vertebrae and least in long bone shaft and skull. T h e bone exhibits no m a r k e d preference in the uptake of the isotopes initially. However, as Sr s5 is preferentially excreted by the kidneys, the relative ratio of Ca 45 to Sr s5 remaining in bone gradually increases. T h e net retention of Ca 45 is a b o u t 60 per cent whereas that for Sr s5 is a b o u t 25 per cent.
LE MI~TABOLISME C O M P A R A T I F E N T R E LE S T R O N T I U M C A L C I U M DANS L ' H O M M E
ET
Le r6sultat m6tabolique de Sr s5 et Ca a~ administrd intraveineusement rut 6tudid dans l ' h o m m e . I1 fut ddcouvert que les isotopes dtaient divisds prSsque 6galement entre l'os et le tissulaire peu apr&s l'administration, mais aprSs quatre mois, plus de 99 p o u r cent de la fraction de chaque isotope restant dans le corps se trouvait dans l'os. A u d~but, la radioactivitd spdcifique (la concentration d'isotope p a r g r a m m e de calcium) 6tait plus grande dans le tissulaire que dans l'os, les valeurs a p p r o c h a n t de l'uniformit6 apres environ quatre mois. La retention entre les os dans l'interval 6tudi6, 6tait la plus grande dans les vert~bres et dtait la m o i n d r e dans les longs os et le cr~ne. L'os ne m o n t r e aucune pr6f6rence initialement entre les isotopes. Ndanmoins c o m m e le strontium est excretd par les reins de prdf6rence, le r a p p o r t r61atif entre le Ca 45 et le Sr ss restant dans l'os a u g m e n t e graduellement. La retention nette d u Ca 45 est pros de 60 p o u r cent et prSs de 25 pour cent pour le strontium.
C P A B H 1 4 T E . J I b H M ~ O B M E H C T P O H K H H H HA.JIBKHH B HE.;'IOBEHE BHyTpIIBOHHO CTpOHILI4It-85 14 l~aabi414n-45. 1]oga3aHO, qT0 BeKope rioe.~Ie BBe/~eItHH o~a H3OTOHa paenpe;IeJImOTCg 6os~ee mli4 MeHee panHoMepHo Me~,~y HOCTHMHH MHFHHMHTHaHItMH. O,~HaHO, qepe3 4 Mee~lta noe~e BBe~ettIIIt
gsyqaJiacb cyabSa BBO~IeHHI~IX qe.~oBeKy
* A report of the early phases of this work was presented before the 3rd annual meeting of the Society of Nuclear Medicine at Salt Lake City in June 1956. 1" This work was conducted under U.S. Atomic Energy Commission contract number AT (30-1) 1656 with Columbia University (Lamont contribution No. 312) and contract number AT (30-1) 1763 with Montefiore Hospital. **Deceased 7 June, 1958. 144
Comparative metabolism of strontium and calcium in man
145
B •OCTHX oTHapymHBaeTca 6o~ee 99% OT oSmero RoaHqeCTBa~aovona, co~epmameroca BO BceM were. HaqaaI,Haa y~eJibHau aHTI4BHOCTI, (HOHI~eHTpalu4flnaovona Ha rpaMM Ha~b~Ha) B MFIFHHXTHaHAX 3HaqIlTeJISrI0BBIme, tleM B HOCTHX, 0~HaH0 nO HcTeqertl414 4-x MecfIILeB y~eabHBie aRTHBHOCTH IlpaHTI4qecHH BBIpaBHI4BaIOTCH. l l p r t cpaBHeHI,II4 coAepH~aHI4H I43OTOl]a B pa3JIHtIHt,IX HOCTYIX OTMeqeH0 caMoe Bt~ICOH0e IlOrOlOli~eHHe B II03BOH0qHI4He H caMoe HHBHOe--B ~JIHHHI:,IX TpySqaTblX HOCTflX I4 B qepelIe. B HatIa.lIbHBIl~ IlepI4oA ~.11~ HOCTe~i He HaT~io~aeTeH 3aMeTH01~ H86HpaTeJIBHOCTH IIOF~IOII~eHHH BBO~FIMBIX H3OTOHOB; O~HaH0, C TeqeHI4eM BpeMeHH 0THOIIIeHHe coAepmaHn~ B HOCTHXHaJIBIIHH-45 H cTp0HI~14IO-85 IIOCTeHeHHO yBeaIlqHBaeTeH, TaR Hall qepe3 noqHrI HpeHMyIKeeTBeHHO BBiReafleTefl CTpOHI~H~]-85. IIpH aTOM n o r ~ o ~ e H ~ e n ~OCT~X H a a b ~ - 4 5 ~ocwr~raew np~tT~i~3~we.~no 6 0 % i~ c w p o m l H ~ - 8 5 - -
o~oao 25%. METABOLISCHER VERGLEICI-I VON STRONTIUM UND CALCIUM IM MENSCHLICHEN KORPER Der metabolische Ablauf von intraven6s verabreichten Sr s5 und Ca~5 wurde erprobt im menschlichen K6rper. Es wurde festgestellt, dass die Isotopen kurz nach Verabreichung gleichm/issig verteilt waren zwischen Knochen uns Geweben, jedoch nach vier Monaten, mehr als 99 prozent jedes einzelnen Isotopen die im K6rper verblieben, waren in den Knochen enthalten. Die ursprfingliche T~itigkeit (Isotopen Konzentration per Gramm Calcium) war bedeutend gr6sser in weichen Geweben als in den Knochen; nach vier Monaten waren die Auswertungen beinahe einheitlich. Die Behaftung zwischen den Knochen w~ihrend der Versuchszeit war am gr6ssten in der Wirbels~iule und am geringsten in R6hren- und Sch/idelknochen. Ursprfinglich zeigt der Knochen keine lange markante Bevorzugung in der Aufnahme von Isotopen. Jedoch, da Sr 85 durch die Nieren bevorzugt ausgeschieden wird, vergr6ssert sich das relative Verh/iltnis von Ca 45 zu Sr ss allm~ihlich. Der Netto-Rfickstand von Ca45 ist etwa 60 prozent wogegen der des Sr sa etwa 25 prozent betr~igt. INTRODUCTION STRONTIUM represents a t r a c e e l e m e n t in the h u m a n b o d y , h a v i n g a n a v e r a g e conc e n t r a t i o n o f 4-5 parts p e r 10,000 p a r t s of c a l c i u m in bone. a) W i d e s p r e a d interest in s t r o n t i u m t o d a y is d u e to the presence o f the long-lived radioisotope Sr 9° a m o n g the p r o d u c t s o f n u c l e a r fission. ~ S t r o n t i u m - 9 0 is g e n e r a l l y r e g a r d e d as the p r i n c i p a l h e a l t h h a z a r d a m o n g the fission p r o d u c t s since it is selectively t a k e n u p b y the h u m a n skeleton w h e r e it r e m a i n s deposited for m a n y years. ~3)
EXPERIMENTAL
1. Clinical design and sampling T r a c e r quantities o f Sr 85 [T½ 65 days] a n d C a 45 [T½ 163 days] in chloride f o r m were a d m i n i s t e r e d in a single dose simultaneously to ten t e r m i n a l c a n c e r patients b y the i n t r a v e n o u s r o u t e [ T a b l e 1]. T h e a v e r a g e dose was 1 . 5 / t c / k g for Sr 85 a n d 0.4/~c/kg for C # 5. T h e i n t r a v e n o u s route
I n o r d e r to be able to e v a l u a t e the radiotoxicity o f Sr 9° in m a n , the k n o w l e d g e o f the m e t a b o l i s m o f the e l e m e n t in h u m a n s is o f paramount importance. Extensive w o r k has b e e n d o n e to d e t e r m i n e the a b s o r p t i o n a n d excretion o f a d m i n i s t e r e d s t r o n t i u m a n d c a l c i u m isotopes. ~4~ T h e present s t u d y was designed to show the relative r e t e n t i o n o f s t r o n t i u m c o m p a r e d to c a l c i u m in m a n a n d to d e t e r m i n e the distribution o f administered isotopes within the b o d y including b o t h b o n e a n d soft tissue. PROCEDURES o f a d m i n i s t r a t i o n was selected since it excludes the p a r a m e t e r o f gastro-intestinal absorption. Since a u t o p s y analyses w e r e e m p l o y e d , the patients were, o f necessity, o f limited life e x p e c t a n c y w i t h c a n c e r i n v o l v e m e n t , a n d c a n n o t be considered as n o r m a l h e a l t h y adults. Survival times o f patients studied v a r i e d f r o m 3 h r to 124
146
Arthur R. Schulert et al. TABLE 1. List of patients studied
q Patient
H.H.
S.K.
S.J. C.C.
J.Z. G.B. S.G. T.M. A.W. M.S. J.T. I.J.
Age
Sex
Wt. (kg)
62 50 60 59 72 70 49 70 63 70 67
F
45
M
54
F F M F M M M F M
i
54 73 73 54 50 43 61 56 76
1
53
i 55
v
] ~
Time interval isotope admin.-death (days)
1/8 2 3 :3 6 18 30 39 65 124. 251 960
Clinical diagnosis
Carcinoma of pancreas Hodgkins disease Carcinoma of colon Carcinoma of breast Carcinoma of lung ] Carcinoma of breast Multiple myeloma ] Carcinoma of lung Carcinoma of kidney Carcinoma of lung Multiple myeloma Multiple myeloma
Skeletal involvement
None All vertebrae Moderate Diffuse Diffuse None Moderate Single area of skull Thoracic vertebrae None Moderate Diffuse
i
days. In addition, samples were obtained from one patient who received Sr s5 only, 251 days prior to death and from one patient who received Ca 4~ only, 960 days before death• At autopsy, samples of many soft tissues and of bone were taken for analysis. As many as twenty-five soft tissue samples were obtained in the early studies while later sampling and analysis of soft tissues were restricted to four major organs, namely, muscle, liver, kidney, and lung. Except for muscle, these organs were obtained in toto and only small sections were removed for histologic analysis. In regard to bone, samples of vertebrae and ribs were obtained from all patients and in seven of the ten cases either femur shaft, iliac crest, and skull were obtained. Particular attention was given to the variation among bones of the skeleton as well as the distribution within single bones• 2. Radiochemical assay
Strontium-85 is readily assayed, since it emitS a 7-ray of moderate energy. The sample is merely dried, ashed, and counted in a well-crystal y-ray spectrometer. The isotope counted with an efficiency of approximately 10 per cent with an associated background of about 10 counts per min. (Slight variations in both of these values were constantly checked by appropriate blanks and standards.)
Two chemical procedures were used in the Ca 45 assay, one adapted to bone and the other for soft tissue. Ashed bone is dissolved in HC1 after which the p H is adjusted to 3.0-3.5 with ammonium hydroxide. A mixture of oxalic acid and ammonium oxalate is then added to precipitate the calcium oxalate. (Care is taken to avoid a p H higher than 3.5 since this precipitates some calcium as the phosphate.) The calcium oxalate is filtered and converted to the oxide by heating in a muffle furnace. The calcium oxide is weighed, dissolved in HC1, and diluted to 100 ml in a volumetric flask. A convenient aliquot is taken, the p H adjusted, and the calcium again precipitated as the oxalate. After digestion the precipitate is filtered on 2 9 m m diameter No. 42 W h a t m a n filter paper which is then placed on a brass planchet, covered with a thin piece of pliofilm and placed in a low-level end window fLcounter. For the determination of Ca 45 in soft tissue, the sample is ashed in platinum and then fused with sodium carbonate at 1200°C. The fusion mixture is leached with water, filtered to remove sodium phosphate and soluble carbonates, and the residue dissolved in hot HC1. After cooling, ammonium hydroxide is added to p H 7, precipitating ferric hydroxide which also occludes remaining phosphate ion. The precipitate
Comparative metabolism of strontium and calcium in man
is filtered with the calcium remaining in the filtrate. To remove traces of calcium which may have been carried with the ferric hydroxide, the precipitate is redissolved in HCI and again precipitated, the filtrate being combined with the previous one. Excess ammonia is expelled by boiling. Oxalic acid and ammonium hydroxide are added to precipitate the calcium oxalate. After digestion and cooling, the oxalate is filtered and counted in the same manner as the bone samples. The fl-counter in which the Ca 45 samples
are counted is equipped with an anticoincidence ring so that background is about 2 counts per min. The counting efficiency for Ca 45 varies with particular counters from 8 to 12 per cent so that 1 0 - e # c can be adequately detected in a sample. The Sr 85 which follows the calcium through the chemistry, registers on the t-counter with an efficiency of only 0.3 per cent. As the Sr 85 concentration is known from the 7-assay, this small correction for the Sr 85 is made in the t-count.
PRECISION ANALYSIS AND TREATMENT
In order to properly evaluate the experimental findings, it is necessary to know the relative precision of each particular result. The problem may be considered in three categories :
147
OF D A T A
fions within bone are noted.) The average o f the mean deviations for the Ca 45 determinations i n these groups is 22.0 per cent for soft tissue and 16.4 per cent for bone. The corresponding values for Sr 85 are (1) the precision of the radiochemical 14.5 per cent for soft tissue and 17.1 per determinations, cent for bone. (2) the biological precision of different There is insufficient direct information to portions of the same tissue, and assess the biological variation from one individual to another. However, the relative (3) the precision between the tissues of distribution of isotopes in the skeletons of two individuals treated in an identical different individuals gives some indication. manner. Thus the standard deviation in the vertebra/ There is also the matter of statistical rib concentration ratio for Sr 85 is 30 per error of counting but the counting time was cent showing, as expected, that variation almost always sufficient to reduce this to among individuals is somewhat greater below 1 per cent, making it negligible than variation in representative samples in compared to the other variations. a single tissue. The concentration ratio The chemical precision is of concern of one bone to another is changing in time, primarily in the Ca 4s determination, since as subsequently discussed, but the change essentially no chemistry is involved in the is slight over a four month interval. Sr 85 assay. Duplicate and in some cases These facts account for a large a m o u n t triplicate samples were analysed. The of the "scatter" in the data and underline average of the mean deviations for the the need for caution in its interpretation. Ca 45 determinations in these small groups Nevertheless, certain generalizations and is 4.8 per cent and 7.5 per cent in bone and a' number of significant facts can be drawn from the experimental results. soft tissue respectively. In comparing the biological variation in In order to give a gross overall picture two or more parts of the same tissue, an of the behavior of the isotopes in the human effort was made to obtain representative body in time, the total body load is calcusamples, e.g. a cross section of bone or the lated for each patient. A standard procedure, half of a kidney cut along its major axis. which admittedly involves considerable (In subsequent discussion, systematic varia- approximation, is employed as follows:
148
Arthur R. Schulert et al.
Soft tissues
(1) The weights of lung, liver, and kidney were obtained at autopsy. Muscle weight is estimated from the total body weight using the body composition data of MITCHELL et (2) The total content of each tissue is then calculated by multiplying the estimated tissue weight by the tissue concentration. (3) Accordingly, these four tissues constitute 40.3 per cent of the total body weight, whereas the total soft tissue comprises 85.2 per cent. On the assumption that the tissues analysed are representative of all the soft tissue, the total isotopic content of these 4 tissues is multiplied by 85.2]40.3 to give the total estimated isotopic content for soft tissue. Bones
The bones are divided into three groups, which according to the data of TROTTER, ~6~ comprise the following percentages of the total skeleton: RESULTS
AND
The distribution of Sr 85 and C # a in the body, the percentage of the isotopes accounted for, and the ratios of each isotope contained in bone and soft tissues are listed in Table 2. Also shown are the ratios of Ca45/Sr 85 in bone and soft tissues. In spite of the problems of representative sampling the approximations entering in the calculations, and the fact that the extent and degree of the cancer involvement varied markedly, a fairly consistent pattern is obtained. The limitations of this analysis are demonstrated most sharply by the calculated C # 5 retention in patient C. C. at three days (127 per cent). There is a partial explanation for this since the patient (a female weighing 72.6 kg) had a higher percentage of adipose tissue than normal. Thus the standard assumption that the skeleton represents 14-8 per cent of the body undoubtedly gives an estimated bone
I " L o n g " bone: limb, hip, skull (and mandible) 70.6% II Rib 5.7% I I I Vertebra and sternum 8.9% Total 85.2% (1) The weight of each bone analysed is estimated from the body weight and the data of TROTTERC8~ together with the data of MITCHELL et al. C5~ for the percentage of total skeletal mass. (2) Bones within a group which are not represented are assumed to have the same value as the average of the others in order to estimate the total isotopic content for each group. (3) When an entire group is not represented, its value is estimated by using the femur/rib ratio (for "long" bone) and the vertebra/rib ratio (for the vertebra/sternum group) as obtained from the average on all patients. (4) The total estimated isotopic content of the three groups is multiplied by 100185.2 to get the estimated total skeletal content. DISCUSSION
weight in this case which is in excess of the true value. Since the concentration of the isotopes per gramme of bone is much greater than of soft tissue, the standard procedure results in a high value for total body retention. It is noted that both isotopes are somewhat equally divided between bone and soft tissue shortly after their administration and that the proportion gradually shifts so that less than one-half per cent of the body load of each isotope is retained in the soft tissues by 124 days. This is primarily due to the more rapid removal of the isotopes from the soft tissues. The data also suggest some translocation from soft tissue to bone, particularly of Ca 45 as previously noted by others in animals over short intervals. ¢7) This translocation is shown graphically in Fig. 1. Whereas the skeletal Sr 8~ retention begins to decline within the first few days and
149
Comparative metabolism of strontium and calcium in man TABLE 2. Distribution of Sr s5 a n d Ca 45 in soft tissues a n d bone; tissue concentrations expressed in per cent of administered dose Patient days*
H.H. 1/8
S. K .
s.j.
2
3
c 3
J. Z. 6
G. B. 18
S. G. 3O
Y. M. 39
A.W. 65
M. S, 124
c
Soft tissue Bone
55 33
31 47
44 36
35 45
26 42
22 41
1"2 20
1-4 26
1.5 30
0"15 26
Total Sr ss ratio bone/soft tissue
88
78
80
80
68
63
21
27
32
26
0.60
1-5
0.82
1.3
1.6
1-8
17
19
20
173
So~ tissue Bone
33 30
28 42
28 55
53 74
32 59
14 36
3-0 35
5.5 71
1-9 79
0"27 6O
Total Ca 45 ratio bone/so~ tissue
33
70
83
127
91
50
38
76
81
6O
0.91
1-5
2.0
1.4
1.8
2.6
12
14
42
222
0.60 0.91
0-90 0.89
0.64 1-5
1.5 1.6
1.2 1-4
0.64 0.88
2.5 1.8
3-9 2-7
1-3 2.6
1.8 2"3
SrS5
Ca45
Ca45/ Soft tissue Sr ss Bone
* Days elapsed between the injection of the isotopes a n d exitus of patients.
b3 O0
0 8O h
o
6c bJ
Ld n~
r0
~h- 4C
o
O
I
I
20
I
I 4O
J
I 6O
I 80
I
I
I00
DAYS FIG. 1. ISotopic content of the total skeleton.
•
C a 4~
0 Sr ss
I 120
Arthur R. Schulert et al.
150
approaches a plateau at about 25 per cent, the C # 5 appears to rise for the first month and levels off at about 65 per cent. Three experimental points are badly off the C # 5 curve, but these are the three least valid values of the ten: the high 3 day point [C. C.] is subject to question as previously mentioned, and the low points at 18 and 30 days represent the only skeletons for which just two bones were obtained for analysis. The retention values for the two isotopes are consistent with those obtained in balance studies over shorter intervals by other investigators. (4, 8, 9, 10~ The ratios of Ca45/Sr s5 of the individual tissues are listed in Table 3. Although these ratios are somewhat more variable for soft tissues than for bone, both show an increase with time; the values for bone being generally 2 or above after 30 days. This is a reflection of the fact that Sr s5 is preferentially eliminated from the body and thus has a shorter average biological residence time than calcium. The enrichment in favor of calcium is not apparent in the bones until several days after the administration of the isotopes. Thus the bone tissue as a whole shows no preference in accepting both isotopes immediately after administration in agreement with the conclusion of BAUER et al. (n) and COMAR et al. a2) The subsequent increase in the Ca4a/Sr 85 ratio in time reflects the preferential excretion of strontium by the
kidneys. It has been stated that the overall calcium enrichment from diet to bone is governed primarily by the calcium/strontium discrimination factor across the intestine and by that of urinary excretion. (13,14) In the course of time, the renal discrimination factor is reflected by the retention of the isotopes in the body, i.e. primarily in the skeleton. Whereas the intestinal discrimination is essentially a one step process (disregarding the endogenous fecal strontium) in that the isotopes which reach the intestinal tract are either taken across the intestinal wall or eliminated from the body, the kidney discrimination is a cyclic process in which ions that have been retained in the body during one circulatory cycle and via the renal tubules are presented to the kidney again during the next circulatory cycle. This repetitive renal process leads to continued preferential excretion of radio-strontium and therefore to increased relative retention of radio-calcium. The factor of net effective enrichment from blood to bone for C # ~ in the two to four month interval is approximately 2-3. An appreciable increase in the enrichment over longer periods of time is not expected since the loci of isotope deposition become more and more inaccessible with time due to the fact that: (t) The isotope is cleared early from the more accessible sites and (2) Some accessible sites will be made
TABLE 3. Ca45[Sr s5 ratios in bone and soft tissues as a function of time Soft tissue
Kidney Liver Lung Muscle Bone Femur Iliac crest Skull Sternum Rib Vertebra
Days 1/8
2
3
3
6
18
30
39
65
124
0'46 0"34 0.28 0"72
0.86 0"84 0'68
0.81 0"63 0"67 0.88
0.74 1.3 0-74
0.91 1.6 1.4 1.7
1.2 1.7 0.50 0.70
1.3
1.5 1.9 1-2 8.0
0.58 1,2 1,1 1,4
0.87 3'0 0.44 1"7
1.8
2.1
0"86 0'94 0.74 0'86
1.9
0"81 1"0 0"90 0"82
0.94
4.6 0.28
1.1 0.76 2'1
1.7 1.4 1.4 1.5
1.3 1.3
1.0 1.3 0.94
2-4 1-0
2'2 3.6 2-7
3-5 2-2 3.8
2.7 2.3 2.0 1.9
Comparative metabolism of strontium and calcium in man
unavailable by the continued deposition of new bone mineral. Although extensive data on the excretion of Sr s5 and Ca 45 in man have been obtained, t4,15) these excretions have not been followed for prolonged periods of time. One would expect the elimination of administered Sr 85 and Ca 45 to follow a power function as in the case of radium, a6) While the present data on bone retention of the isotopes are inconclusive, they sug.gest that only slight changes are occurnng after the second month, compatible with such an elimination pattern. Since the Ca45/Sr 85 retention ratio reaches two in two months, and since these isotopes present in bone remain there for years, it is concluded that this enrichment will also occur under continuous intake of Sr 9° due to fallout. A comparison of average Sr a° content of diet and human bone in northeastern United States by ECKELMANN et al. a7) indicates that the overall calcium/ strontium discrimination from diet to bone is four. Since the discrimination against strontium across the intestinal barrier has been reported to be two t4) and the discrimination from blood to bone is also two, the present data are in general agreement with those reported in the literature. The specific activity of Sr s5 (concentration o f Sr s5 per gramme stable calcium) of bones and of the major soft tissues are listed in Table 4. The average specific activity
151
of vertebrae and rib are respectively 5.4 and 2.3 times that of femur. This distribution must clearly change in time as evidenced by the fact that common strontium is uniformly distributed throughout the skeleton.m However, this approach to uniformity proceeds very slowly since Sr 9°, which has been in our environment for several years, is reported to be more greatly concentrated in vertebrae and rib than in long-bone shaft by factors of about four and two respectively, tlv) It is apparent that while the isotopes are largely concentrated in the skeletal tissue, the specific activity of the soft tissue greatly exceeds that of bone throughout the initial two month period following injection. It is noted in the 124 day and 251 day samples, however, that the specific activities are of the same order of magnitude. This indicates that about four months are required for the soft tissue to be cleared of the strontium initially deposited therein. Subsequently the isotopic content of the soft tissue probably reflects that which is resorbed from the bone into the blood stream, (the blood probably being in near equilibrium with t h e soft tissue). Further evidence that this must be the case comes from the data from one patient who was given Ca 45 alone, 960 days before death. Samples of the four major soft tissues were obtained and showed an average specific activity of one-twentieth that shown by the 124 day
TABLE 4. Specific activity of Sr s5 in bone and soft tissue (% administered dose/g Ca)
1/8
Kidney Liver Lung Muscle Femur Iliac crest Skull Sternum Rib Vertebra
21 8-0 8"0 11 0"048 0"18 0"071 0"17
2
8"1 12 9'5 3"3 0"036
0"070 0'031 0"067 0"035 0"095
3
3
6
8"8 7"4 1"8 12
7"6 1'.7 5"1 4'0
0'29 5'8 4'8 2'9
0"017 0"10 0"035 0"13
0'012
0"21 0"077 0"16
Days 18
30
39
65
124
251
2"3
0-69
3'2 4"1
1"6 0"24
0"47 0"54 1"4 0"25 0"024
0'57 0"25 0"36 0'42 0'016
0'17 0'070 0"32 0'053
0.092 0.043 0.019 0-056
0"11 0-027 0'099
0'073
0'25 0"052 0"16
0"14
0'052 0"070
0-067 0"078 0"16
0.062 0.056 0.116
0-14
0.14 0.044 0"11
152
Arthur R. Schulert et al.
case. Therefore it may be concluded that at intervals greater than four months, urinary excretion reflects bone resorption. The excretion may also give an index of the isotopic bone concentration, although the relationship is complex. For example, animal experiments have shown that the specific activity of soft tissue may ultimately become much less than that of bone. (ls,ag) Thus the relation is largely an empirical one. It is evident that in the practical experimental evaluation of agents for their effect on removing strontium from the skeleton, the most meaningful results would be obtained at long periods after strontium administration when the specific activity of the soft tissue approximates that of bone, or at least does not greatly exceed it. The induced enhancement of urinary radiostrontium excretion achieved in the early phase (2°) will largely represent clearing of soft tissues, whereas a sustained effect produced at later periods will represent removal of the isotope from bone. Studies carried out in our laboratories on the relationship of urinary radio-strontium excretion and bone concentration determined at time intervals of varying length following the administration of the tracer are in progress at the present. STERNAL END
~3.~,so
4
,3.2
I 75 I 54 ~ $ 5 ~ 12cm.
Fio,
)4
2. V a r i a t i o n s o f Sr s5 concentration w i t h i n section.
a rib
Fig. 2 shows that the isotope concentration varies greatly within a single bone. For example, Sr 8~ concentration decreases b y a factor of greater than two in a section of rib as one proceeds from the sternal end for 12 cm toward the axillary portion of the SUMMARY
rib. This finding indicates the biological variation one may expect to encounter in rib samples taken at random. Table 5 lists the data of radio-strontium concentration in successive one mm layers of the femur which were taken from the "FABLE 5. Sr
85
concentratmn m various layers of the femur "
"
Time
D e p t h of bone mm
Concentration in % of dose/kg bone
3 hr
1 2 3 4 5 Marrow 1 2 3 4 5 Marrow 1 2 3 4 5
4'I1 1"64 1"72 1 "84 2"25 ll'16 6'00 3'96 3"95 4"48 4"57 7"41 2'91 2'01 1'57 1"35 1"21
2 days
65 days
external surface and were followed towards the marrow cavity. It is noted that the samples taken after three hours and two days show high activity in the outermost layer followed by a marked decline in the second and third mm. The value then gradually rises with the layer adjacent to the marrow having the highest concentration of all. However, the samples obtained 65 days after the administration of the isotopes show a progressive unidirectional decrease of activity from the outermost layer which has the highest concentration to the inner layer which has the lowest. This may suggest a greater resorption of the isotope through the marrow cavity than from the outer surface of the bone.
AND CONCLUSIONS
(1) Tracer quantities of Sr s5 and Ca 45 were administered simultaneously to ten cancer patients by the intravenous route and samples of bone and four major soft
tissue organs were obtained at autopsy for radio-assay. (2) The isotopes are approximately equally distributed between bone and soft
Comparative metabolism of strontium and calcium in man
tissue for the first few d a y s after a d m i n i s t r a t i o n ; after f o u r m o n t h s a b o u t 99.5 p e r c e n t o f the isotopes w h i c h are r e t a i n e d in the b o d y are f o u n d in b o n e . (3) T h e n e t r e t e n t i o n o f t h e isotopes a p p e a r s to level o f f at a b o u t 60 p e r c e n t for C a 45, a n d 25 p e r c e n t for Sr sS. (4) T h e r e is a g r a d u a l increase in the Ca45/Sr s5 ratio in b o n e , w h i c h r e a c h e s a v a l u e o f t w o o r m o r e in the t w o to f o u r m o n t h interval. (5) T h e specific a c t i v i t y o f the isotopes
153
is m u c h g r e a t e r in soft tissue t h a n in b o n e t h r o u g h o u t t h e first t w o m o n t h s after a d m i n i s t r a t i o n , b u t the values a p p r o a c h t h e s a m e o r d e r o f m a g n i t u d e at f o u r m o n t h s . Acknowledgements--The authors gratefully acknowledge
the role of J. LAURENCEKULP, who along with DANIEL LASZLO,initiated this cooperative effort and gave valuable counsel throughout the investigation. WALTERECKELMANN was integrally associated with the early phases of the work. Important assistance in the radiochemical analysis was provided by MARIAN WAMPLER and DOROTHY WALTON.
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1. THURBER D. L., KULP J. L., HODGES E., GAST P. W. and WAMPLER J. M. Science 28, 256 (1958). 2. Hearings before the Special Subcommittee on Radiation of the Joint Committee on Atomic Energy, The nature
3. 4. 5.
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of radioactive fallout and its effects on man, May 27-29-June 3-7, (1957), U.S. Government Printing Office, Washington, D.C. (1957). HAMILTONJ . G . Rev. Mud. Phys. 20, 718 (1948). SPENCER H., LASZLO D., and BROTHERS M. or. Clin. Invest. 36, 680 (1957). MITCHELL H. H., HAMILTON T. S., STEGGERDA F. R. and BEAN H . W . J. Biol. Chem. 158, 625 (1945). TROTTER M. Personal communication. NORRISW. P. and KISlELESKIW. Cold Spr. Harb. S~mp. Quant. Biol. 13, 164 (1948). BELLIN J. and LASZLO D. Science 117, 331 (1953). BRONNER F., HARRIS R. S., MALETSKOS C. J. and BENDA C. E. J. Clin. Invest. 35, 78 (1956). HARRISON G. E., RAYMOND W. H. A. and TRETHEWAY H. C. Clin. Sci. 14, 681 (1955).
11. BAUERG. C. H., CARLSSONA. and LINDQUISTB. Acta Physiol. Scan& 35, 56 (1955). 12. COMAR C. L., WHITNEY I. B. and LENGEMANN F. W . Proc. Soc. Expl. Biol. Med. 88, 232 (1955). 13. COMMAR C. L., RUSSEL R. S. a n d WASSERMAN R. H. Science 126, 485(1957). 14. Deposition and retention of ingested strontium-90 in the skeleton. Committee Report Washington, D.C., April 23, (1957). Official use only. 15. SPENCER H., SAMACHSON J . and LASZLO D. Fed. Proc. 17, 154 (1958). 16. NORRISW. P., SPECKMANT. W. and GUSTAFSON P. W . Amer. J. Roentgenol. 73, 661 (1955). 17. EC•ELMANN W. R., KULP J. L. and SCHULERT A.R. Science 127, 266 (1958). 18. NEUMAN W. F. and NEUMAN M. W. The Chemical Dynamics of Bone Minerals, U. of Chicago Press (1958). 19. COMAR C. L. Radioisotopes in Biology and Agriculture, McGraw-Hill (1955). 20. SPENCER H., SAMACHSONJ., KABAKOWB. and LASZLO D. Clin. Sci. 17, 291 (1958).