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A new 75Se-analogue with superior properties over 131I-hippuran
Intemarional Journal of Applied Radiarion & Isoropr% Vol. 0 Pcrgemon Press Ltd 1980. Printed in Great Britain 002(r708X/80/%01-0379SO2.00/0
31. pp. 379 to 381
A New 7SSe-Analoguewith Superior Properties over ’ 3’ I-Hippuran ANDRE J. VAN WYK*, PIETER J. FOURIE and WILLEM H. VAN ZYL Chemistry Division, Atomic Energy Board, Private Bag X256, Pretoria 0001, South Africa,* and National Hospital, Bloemfontein 9300, South Africa
in
(Received 13 August 1979; revised form 19 November 1979)
IlltroductiOll ALTHOUGH
'5Se has desirable physical characteristics for in
viw
imaaina. very few ‘%e-radiopharmaceuticals have been dedo&d, despite the availability of new methods for labellina with 75Se.“’ At nresent 75Se-methionine and 6-methyl (75Se) selenomethyl-l9-norcholest-5(1O)-en-3~-ol are probably the 75Se-radiopharmaceuticals most often used routinely. The purpose of this particular study was to develop a ‘5Se-analogue of “‘I-hippuran. Such a ‘5Secompound has certain advantages over the “‘I equivalent: (a) “Se has improved physical properties over r3iI, viz. the /l-particle absorbed dose is about 7% of that of 13’I; between 100 and 400 KeV there are 1.74 y rays per disintegration as compared to 0.91 for ‘“‘I!” (b) Due to these improved physical properties less activitv would be needed to.obtain the same scan quality.@’ (cj Since the ‘%e-compound contains no 1311masin the case of ‘“‘I-hiDDUraD. which could give rise to free 1311with subseque;ithyroid uptake, no blocking of the thyroid is needed. (d) The concentration of free 13rI- present also has an adverse effect on renogram quality’“’ and necessitates special purification of 13iI-hippuran. (e) The overall stability of the 75Se-analogue appears to be‘better than i3’I-hippuran, e.g. after one month no significant change from the original quality was observed by means of chromatography. In the case of l3 ’ I-hippuran up to 6% free 13’1- was seen over the same period.(4i (f) Due to the long physical half-life of “Se (118.5 d), the 75Se-analogue has a definite advantage over “‘I-hippuran (13’1 half-life 8.1 d) for the purpose of routine production. The long physical half-life of “Se could also be a disadvantage if the ‘“Se-analogue had a very long biological half-life which would give rise to an undesirably high absorbed radiation dose. However, in this particular case the ‘5Se-analogue has a fast renal excretion and short biological half-life which thus eliminates this problem in cases where some renal function is still operative. In cases where total renal obstruction is present in both kidneys and where there is no renal function at all, an undesirably high absorbed radiation dose can occur with the 75Se-analogue, but it would be a relatively small problem compared with the serious situation as a whole.
l
Address for correspondence.
(g) From a dosimetric viewpoint “Se has an advantage over la11 giving a lower in vioo absorbed radiation dose. This can be seen by comparing their respective absorbed dose per unit cumulated activity (S-values) when the kidney is both source and target organ; 75Se: 0.00033 rad/&i-h 13iI: 0.0015 rad/&i-h”) These kidney S-values could possibly be used for a reasonably valid in vioo comparison provided (i) the biological half-lives and excretion rates are similar for the ‘5Se- and 13’1-renal compounds. (ii) both have low and acceptable levels of impurities, viz. 75Se-methionine and “‘I- respectively. (iii) total renal obstruction is not present in both kidneys and therefore some renal function still remains. The same rationalec2) which was used to develop 6-methyl (75Se) selenomethyl-l9-norcholest-5(1O)-en-3~-ol (available from the Radiochemical Centre Amersham) as an improvement on 13’Itholesterol for adrenal studies was followed for the development of the “Se-renal agent. Basically the approach entailed analysing the structure of “‘I-hippuran and making an adaptation. The structure of ‘“‘I-hippuran consists of an amide bond formed between (0) 13’1- benzoyl chloride and an amino acid, viz. glycine. It was thought that the “Se-amino acid, “Se-methionine which is freely available, should react with benzoyl chloride in the same manner as glycine, giving rise to a new “Se-compound with similar in uiw properties as 13’I-hippuran. It is known that when 9g”Tc is incorporated into a molecule by means of a chelating group, a relatively large molecular change can occur due to the formation of biscomplexes, compared to the original unlabelled compound. This results in changed in uiw properties with different biodistribution and organ uptake for the 99”%compounds compared to the unlabelled compounds.‘6) Since ‘5Se like “‘1 does not require a chelating group to label a compound; no big molecular disturbance should occur when 75Se is incorporated into a molecule. In uiuo properties analogous to those of r3*I-hippuran could thus be expected for the ‘5Se-renal agent.
Methad 75Se-methionine (Radiochemical Centre Amersham) (0.5 mCi/S ml, specific activity 320 mCi/mg) was mixed with L-methionine (104 mg) and sodium hydroxide (55 mg) in a 10 ml vial. This solution reacted with benzoyl chloride (70 mg) on shaking for 10-15 min. The mixture was transferred to a centrifugation tube and acidified with cold 6 M hydrochloric acid (1 ml). At this point the actual radioactive mixture was seeded with some crystals of the N-benzoylmethionine product amide inactive C6H,CONHCH(CH,CH,SCH3)COOH obtained from a similar experiment where only inactive L-methionine had been used. The structure of this carrier product was verified by NMR studies. After seeding the radioactive mixture and stirring with a glass rod-coprecipitation was induced N-benzoyl- ‘%e-methionine: ‘5Se-product : Of the C6HsCONHCH (CH,CH275SeCH3) COOH. The mixed oroduct was isolated by centrifugation and the mother liquor decanted from theprecipitate. The yields which were obtained in the precipitate itself directly after the acidification step, varied depending on the starting activities used, e.g. starting with 50@/0.5 ml of 75Se-methionine, a yield 379
380
Technical Note
FIG. 1. (a) Renogram of left kidney, without probenecid (tl = 82%, Tmi, = 2 mm). (b) Renogram of left kidney, with probenecid (9 = 68%, Tmi. = 3.5 min).
of 6@-70% was obtained. Starting with 500 &i/5 ml a yield of 4060% was obtained. However, at this point significant free 75Se-methionine was still present and further purification was thus essential. The precipitate was well washed with cold water (2 ml) while stirring with a glass rod. Final centrifugation and decantation were carried out. The precipitate was dissolved in 1 M sodium hydroxide (l-2 ml) and the pH adjusted to 6-7. After millipore filtration a final relative yield of 2530% could be obtained of the pure oroduct. The absolute reaction yield of “Se-nroduct was determined on the reaction mixture itself by means of chromatography before any purification had commenced. The final radiochemical purity was also established through chromatography. The following different chromatography systems were evaluated for these purposes:
(b) was preferred. Compared with the original starting activity, the absolute reaction yield was 30-40% as determined by means of chromatography system (b). The final radiochemical purity of the purified product was also established with chromatography system (b), and 2-3% of free/unbound 75Se-methionine was found to be present. Using chromatography system (b) no significant radiochemical degradation was seen 1 month after production. After injecting the 75Se-compound, good renal uptake was already obtained at SO-200 s and at 8-10 min the biggest percentage of activity had cleared into the bladder. After 12-15 min all the activity appeared to be in the bladder. Except for initial transient activity in the heart directly after injection, the only activity detected thereafter was sequentially in the kidneys and bladder with no significant uptake in other organs. In order to test whether the new compound shows definite tubular renal excretion, probenecid was administered beforehand followed by injection of the ‘?Se-compound and the renograms compared with those of the “Secompound alone. Probenecid is known to interfere with compounds which show tubular excretion, e.g. “‘I-hip puran, by suppressing the rate of renal clearance while having no effect on compounds which excrete by glomerular filtration.@) The renal transit efficiency and minimum renal transit time as determined by the BRITWN and BROWNmethod”) changed from 82% and 2min. respectively (without probenecid), to 68% and 3.5 min. (with probenecid) while the renogram curve flattened significantly (Fig. I), thus positively indicating tubular excretion. Other convincing evidence for this excretion mode was the closely analogous renal transit efficiency (n) and minimum renal transii time (Tmin) found for both the 7sSe-compound (mean n 78%. mean T,;. 2.7 min) and ‘“‘I-hionuran (mean 1182%. mean’T,i, 2.7 r&), including the very similar‘renogram curves @) obtained for these compounds in the same rabbit (Fig. 2). These observations indicated that the new ‘?Ie-renal agent has in uiuo properties very similar to that of ‘311-hippuran. Unbound “Se-methionine would be retained for a long time in oiuo and would exhibit mainly liver and pancreas uptake.“” Such uptake was not seen with the new agent
(a) Gelman ITLC (SG):9 chloroform: 1 acetic acid R,: “Se-product: 0.84.9 Rf. . 7sSe-methionine: 0.2. (b) Whatman 1 paper: 4 butanol: 1 acetic acid: 1 water RI:75Se-product:0.8-0.9 R,:75Se-methionine:0.4-0.5 All in vim measurements were carried out on healthy rabbits bv means of a Nuclear Chicago Phogamma III scintillationOcamera in line with a PDPI8 computer system. Renograms were initially done separately for the “Secompound and ‘“‘I-hippuran using different rabbits for each agent. At a later stage both labelled compounds were directly compared in the same rabbit during several experiments. BRITTONand BROWN’Smethod”) was used to determine mean renal parameter values, viz. renal transity efficiency_ (n) .,. and minimum renal transit time (7X over a study period of 20min. using time intervals of 40s. A 2% solution of probenecid and 30-60 &i of ?Se-analogue or ‘3’I-hippuran per rabbit were used for injections.
It was found that although chromatography system (a) (ITLC) was faster it was also somewhat less dependable and repeatable than system (b) (paper), possibly because of undesirable tailing effects and thus chromatography system
FIG. 2. (a) Renogram of left kidney, rabbit A injected with “Se-analogue, 6O&i/ml (mean rl = 78%. Tmin= 2.7 min). (b) Renogram of left kidney, rabbit A injected with ‘311-Hippuran, 50 pCi/O.S ml (mean rl = 82%, Tmi, = 2.7 min).
Technical Note which indicated the absence of significant free/unbound “Se-methionine in oiuo. Since 73Se-methionine has already been developed” I) and especially since 13Se has some useful physical properties(12) viz. suitable enemies for both Y_(Y= 360KeV. 95.j%) and positron imaging (6’ = l.is’ MeV, 51 i KeV = 130%) including a half-life of 7.2 h, the corresponding 73!Se-labelled renal agent should also be develoued. It co%ld be expected that ‘3Se-methionine would react in a similar way with benzoyl chloride as 75Se-methionine, to give the 73Se-labelled renal agent with similar in oivo properties as the ‘%e-analogue.
It is clear that the new ‘%e-labelled analogue of ‘“‘I-hippuran shows promise as a renal agent and warrants further investigation. Limited clinical evaluation is envisaged after the usual requirements towards toxicity, sterility and pyrogenicity have been fulfilled. Development of the corresponding 73Se-renal agent is also intended in future.
References 1. BASMADJIAN G. P., HETZELK. R. and ICE R. D. Inc. J. awl. . . Radiat. Isotooes . 26., 695 1197%. . ,
381
CHARBONNELB. and GIJIHARDD. Abstr. 2nd Int. Cona. World Federation of Nuclear Medicine and Biology, i. 152. Washington, !&ptember 1978.
2. CHATALJ. E.,
3. BIANCHIG., HEGE~IPPEE., MEGZZI A., ROSA U. and Sos~ S. Minerua Nucl. 9, I52 (1965). 4. Analytical Control of Radiopharmaceuticals, pp. 143, 182. IAEA, Vienna (1970). 5. SNYDERW. S., FORD M. R., WARNERG. G. and WATSONS. B. MIRD pamphlet No. 11, pp. l-257. Society of Nuclear Medicine, New York (1975). 6. HEIDELN. D., BURNS H. D., HONDA T. and BRADY L. W. (Editors) The Chemistry of Radiopharmaceuticals, p. 278. Masson, New York (1978). 7. BRIAN K. E. and BROWNN. J. G. Clinical Renography, p. 87. Lloyd-Luke Medical Books, London (197 I). 8. KLOPPERJ. F., HAUSER W., ATKINS H. L., ECKELMAN W. C. and RICHARDSP. J. Nucl. Med. 13, 107 (19X!). 9. FREEMAN L. M. and JOHNSON P. M. (Editors) Clinical Scintillation Imaging, pp. 40 and 42. Grune & Stratton, New York (1975). IO. FREEMAN L. M. and JOHNSON P. M. (Editors) Clinical Scintillation Imaging, p. 54. Grune & Stratton, New York (1975). I I. OGAWA K., TAKI K., NOZAK~T., OKANA S. and AMBE S. J. lab. Comp. Radiopharmac 13, 214 (1977). 12. GUILWUMEM., LAMBRECHT R. M. and WOLFA. P. Inc. J. appl. Radiat. Isotopes 29, 41 I (1978).
31. pp. 379 to 381
A New 7SSe-Analoguewith Superior Properties over ’ 3’ I-Hippuran ANDRE J. VAN WYK*, PIETER J. FOURIE and WILLEM H. VAN ZYL Chemistry Division, Atomic Energy Board, Private Bag X256, Pretoria 0001, South Africa,* and National Hospital, Bloemfontein 9300, South Africa
in
(Received 13 August 1979; revised form 19 November 1979)
IlltroductiOll ALTHOUGH
'5Se has desirable physical characteristics for in
viw
imaaina. very few ‘%e-radiopharmaceuticals have been dedo&d, despite the availability of new methods for labellina with 75Se.“’ At nresent 75Se-methionine and 6-methyl (75Se) selenomethyl-l9-norcholest-5(1O)-en-3~-ol are probably the 75Se-radiopharmaceuticals most often used routinely. The purpose of this particular study was to develop a ‘5Se-analogue of “‘I-hippuran. Such a ‘5Secompound has certain advantages over the “‘I equivalent: (a) “Se has improved physical properties over r3iI, viz. the /l-particle absorbed dose is about 7% of that of 13’I; between 100 and 400 KeV there are 1.74 y rays per disintegration as compared to 0.91 for ‘“‘I!” (b) Due to these improved physical properties less activitv would be needed to.obtain the same scan quality.@’ (cj Since the ‘%e-compound contains no 1311masin the case of ‘“‘I-hiDDUraD. which could give rise to free 1311with subseque;ithyroid uptake, no blocking of the thyroid is needed. (d) The concentration of free 13rI- present also has an adverse effect on renogram quality’“’ and necessitates special purification of 13iI-hippuran. (e) The overall stability of the 75Se-analogue appears to be‘better than i3’I-hippuran, e.g. after one month no significant change from the original quality was observed by means of chromatography. In the case of l3 ’ I-hippuran up to 6% free 13’1- was seen over the same period.(4i (f) Due to the long physical half-life of “Se (118.5 d), the 75Se-analogue has a definite advantage over “‘I-hippuran (13’1 half-life 8.1 d) for the purpose of routine production. The long physical half-life of “Se could also be a disadvantage if the ‘“Se-analogue had a very long biological half-life which would give rise to an undesirably high absorbed radiation dose. However, in this particular case the ‘5Se-analogue has a fast renal excretion and short biological half-life which thus eliminates this problem in cases where some renal function is still operative. In cases where total renal obstruction is present in both kidneys and where there is no renal function at all, an undesirably high absorbed radiation dose can occur with the 75Se-analogue, but it would be a relatively small problem compared with the serious situation as a whole.
l
Address for correspondence.
(g) From a dosimetric viewpoint “Se has an advantage over la11 giving a lower in vioo absorbed radiation dose. This can be seen by comparing their respective absorbed dose per unit cumulated activity (S-values) when the kidney is both source and target organ; 75Se: 0.00033 rad/&i-h 13iI: 0.0015 rad/&i-h”) These kidney S-values could possibly be used for a reasonably valid in vioo comparison provided (i) the biological half-lives and excretion rates are similar for the ‘5Se- and 13’1-renal compounds. (ii) both have low and acceptable levels of impurities, viz. 75Se-methionine and “‘I- respectively. (iii) total renal obstruction is not present in both kidneys and therefore some renal function still remains. The same rationalec2) which was used to develop 6-methyl (75Se) selenomethyl-l9-norcholest-5(1O)-en-3~-ol (available from the Radiochemical Centre Amersham) as an improvement on 13’Itholesterol for adrenal studies was followed for the development of the “Se-renal agent. Basically the approach entailed analysing the structure of “‘I-hippuran and making an adaptation. The structure of ‘“‘I-hippuran consists of an amide bond formed between (0) 13’1- benzoyl chloride and an amino acid, viz. glycine. It was thought that the “Se-amino acid, “Se-methionine which is freely available, should react with benzoyl chloride in the same manner as glycine, giving rise to a new “Se-compound with similar in uiw properties as 13’I-hippuran. It is known that when 9g”Tc is incorporated into a molecule by means of a chelating group, a relatively large molecular change can occur due to the formation of biscomplexes, compared to the original unlabelled compound. This results in changed in uiw properties with different biodistribution and organ uptake for the 99”%compounds compared to the unlabelled compounds.‘6) Since ‘5Se like “‘1 does not require a chelating group to label a compound; no big molecular disturbance should occur when 75Se is incorporated into a molecule. In uiuo properties analogous to those of r3*I-hippuran could thus be expected for the ‘5Se-renal agent.
Methad 75Se-methionine (Radiochemical Centre Amersham) (0.5 mCi/S ml, specific activity 320 mCi/mg) was mixed with L-methionine (104 mg) and sodium hydroxide (55 mg) in a 10 ml vial. This solution reacted with benzoyl chloride (70 mg) on shaking for 10-15 min. The mixture was transferred to a centrifugation tube and acidified with cold 6 M hydrochloric acid (1 ml). At this point the actual radioactive mixture was seeded with some crystals of the N-benzoylmethionine product amide inactive C6H,CONHCH(CH,CH,SCH3)COOH obtained from a similar experiment where only inactive L-methionine had been used. The structure of this carrier product was verified by NMR studies. After seeding the radioactive mixture and stirring with a glass rod-coprecipitation was induced N-benzoyl- ‘%e-methionine: ‘5Se-product : Of the C6HsCONHCH (CH,CH275SeCH3) COOH. The mixed oroduct was isolated by centrifugation and the mother liquor decanted from theprecipitate. The yields which were obtained in the precipitate itself directly after the acidification step, varied depending on the starting activities used, e.g. starting with 50@/0.5 ml of 75Se-methionine, a yield 379
380
Technical Note
FIG. 1. (a) Renogram of left kidney, without probenecid (tl = 82%, Tmi, = 2 mm). (b) Renogram of left kidney, with probenecid (9 = 68%, Tmi. = 3.5 min).
of 6@-70% was obtained. Starting with 500 &i/5 ml a yield of 4060% was obtained. However, at this point significant free 75Se-methionine was still present and further purification was thus essential. The precipitate was well washed with cold water (2 ml) while stirring with a glass rod. Final centrifugation and decantation were carried out. The precipitate was dissolved in 1 M sodium hydroxide (l-2 ml) and the pH adjusted to 6-7. After millipore filtration a final relative yield of 2530% could be obtained of the pure oroduct. The absolute reaction yield of “Se-nroduct was determined on the reaction mixture itself by means of chromatography before any purification had commenced. The final radiochemical purity was also established through chromatography. The following different chromatography systems were evaluated for these purposes:
(b) was preferred. Compared with the original starting activity, the absolute reaction yield was 30-40% as determined by means of chromatography system (b). The final radiochemical purity of the purified product was also established with chromatography system (b), and 2-3% of free/unbound 75Se-methionine was found to be present. Using chromatography system (b) no significant radiochemical degradation was seen 1 month after production. After injecting the 75Se-compound, good renal uptake was already obtained at SO-200 s and at 8-10 min the biggest percentage of activity had cleared into the bladder. After 12-15 min all the activity appeared to be in the bladder. Except for initial transient activity in the heart directly after injection, the only activity detected thereafter was sequentially in the kidneys and bladder with no significant uptake in other organs. In order to test whether the new compound shows definite tubular renal excretion, probenecid was administered beforehand followed by injection of the ‘?Se-compound and the renograms compared with those of the “Secompound alone. Probenecid is known to interfere with compounds which show tubular excretion, e.g. “‘I-hip puran, by suppressing the rate of renal clearance while having no effect on compounds which excrete by glomerular filtration.@) The renal transit efficiency and minimum renal transit time as determined by the BRITWN and BROWNmethod”) changed from 82% and 2min. respectively (without probenecid), to 68% and 3.5 min. (with probenecid) while the renogram curve flattened significantly (Fig. I), thus positively indicating tubular excretion. Other convincing evidence for this excretion mode was the closely analogous renal transit efficiency (n) and minimum renal transii time (Tmin) found for both the 7sSe-compound (mean n 78%. mean T,;. 2.7 min) and ‘“‘I-hionuran (mean 1182%. mean’T,i, 2.7 r&), including the very similar‘renogram curves @) obtained for these compounds in the same rabbit (Fig. 2). These observations indicated that the new ‘?Ie-renal agent has in uiuo properties very similar to that of ‘311-hippuran. Unbound “Se-methionine would be retained for a long time in oiuo and would exhibit mainly liver and pancreas uptake.“” Such uptake was not seen with the new agent
(a) Gelman ITLC (SG):9 chloroform: 1 acetic acid R,: “Se-product: 0.84.9 Rf. . 7sSe-methionine: 0.2. (b) Whatman 1 paper: 4 butanol: 1 acetic acid: 1 water RI:75Se-product:0.8-0.9 R,:75Se-methionine:0.4-0.5 All in vim measurements were carried out on healthy rabbits bv means of a Nuclear Chicago Phogamma III scintillationOcamera in line with a PDPI8 computer system. Renograms were initially done separately for the “Secompound and ‘“‘I-hippuran using different rabbits for each agent. At a later stage both labelled compounds were directly compared in the same rabbit during several experiments. BRITTONand BROWN’Smethod”) was used to determine mean renal parameter values, viz. renal transity efficiency_ (n) .,. and minimum renal transit time (7X over a study period of 20min. using time intervals of 40s. A 2% solution of probenecid and 30-60 &i of ?Se-analogue or ‘3’I-hippuran per rabbit were used for injections.
It was found that although chromatography system (a) (ITLC) was faster it was also somewhat less dependable and repeatable than system (b) (paper), possibly because of undesirable tailing effects and thus chromatography system
FIG. 2. (a) Renogram of left kidney, rabbit A injected with “Se-analogue, 6O&i/ml (mean rl = 78%. Tmin= 2.7 min). (b) Renogram of left kidney, rabbit A injected with ‘311-Hippuran, 50 pCi/O.S ml (mean rl = 82%, Tmi, = 2.7 min).
Technical Note which indicated the absence of significant free/unbound “Se-methionine in oiuo. Since 73Se-methionine has already been developed” I) and especially since 13Se has some useful physical properties(12) viz. suitable enemies for both Y_(Y= 360KeV. 95.j%) and positron imaging (6’ = l.is’ MeV, 51 i KeV = 130%) including a half-life of 7.2 h, the corresponding 73!Se-labelled renal agent should also be develoued. It co%ld be expected that ‘3Se-methionine would react in a similar way with benzoyl chloride as 75Se-methionine, to give the 73Se-labelled renal agent with similar in oivo properties as the ‘%e-analogue.
It is clear that the new ‘%e-labelled analogue of ‘“‘I-hippuran shows promise as a renal agent and warrants further investigation. Limited clinical evaluation is envisaged after the usual requirements towards toxicity, sterility and pyrogenicity have been fulfilled. Development of the corresponding 73Se-renal agent is also intended in future.
References 1. BASMADJIAN G. P., HETZELK. R. and ICE R. D. Inc. J. awl. . . Radiat. Isotooes . 26., 695 1197%. . ,
381
CHARBONNELB. and GIJIHARDD. Abstr. 2nd Int. Cona. World Federation of Nuclear Medicine and Biology, i. 152. Washington, !&ptember 1978.
2. CHATALJ. E.,
3. BIANCHIG., HEGE~IPPEE., MEGZZI A., ROSA U. and Sos~ S. Minerua Nucl. 9, I52 (1965). 4. Analytical Control of Radiopharmaceuticals, pp. 143, 182. IAEA, Vienna (1970). 5. SNYDERW. S., FORD M. R., WARNERG. G. and WATSONS. B. MIRD pamphlet No. 11, pp. l-257. Society of Nuclear Medicine, New York (1975). 6. HEIDELN. D., BURNS H. D., HONDA T. and BRADY L. W. (Editors) The Chemistry of Radiopharmaceuticals, p. 278. Masson, New York (1978). 7. BRIAN K. E. and BROWNN. J. G. Clinical Renography, p. 87. Lloyd-Luke Medical Books, London (197 I). 8. KLOPPERJ. F., HAUSER W., ATKINS H. L., ECKELMAN W. C. and RICHARDSP. J. Nucl. Med. 13, 107 (19X!). 9. FREEMAN L. M. and JOHNSON P. M. (Editors) Clinical Scintillation Imaging, pp. 40 and 42. Grune & Stratton, New York (1975). IO. FREEMAN L. M. and JOHNSON P. M. (Editors) Clinical Scintillation Imaging, p. 54. Grune & Stratton, New York (1975). I I. OGAWA K., TAKI K., NOZAK~T., OKANA S. and AMBE S. J. lab. Comp. Radiopharmac 13, 214 (1977). 12. GUILWUMEM., LAMBRECHT R. M. and WOLFA. P. Inc. J. appl. Radiat. Isotopes 29, 41 I (1978).