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Uptake of radiosulphate and radiophosphate in various tissues of normal and scorbutic guinea pigs
E.rperimeninl
UPTAKE
Cell Research,
OF
6, 527-528
527
(1954)
RADIOSULPHATE
VARIOUS
TISSUES
AND OF NORMAL
GUINEA
of
Histology,
AND
IN
SCORBUTIC
PIGS
and N. R. RIKGERTZ
U. FRIBERG Department
RADIOPHOSPHATE
Karolinska
Received
January
Institutrt,
Stockholm,
Sweden
19, 1954
THErate
of collagen formation is diminished in scurvy (2, 4). The effect of C-avitaminosis on the mucopolysaccharides in the ground substance of the connective tissue has not yet been completely clarified but histochemical investigations tend to indicate a reduced formation of polysaccharides (5, 6), eventually with an accumulation of products of a lower degree of polymerization (3, 7). Observations on the fato of radiosulphate in animal organisms seem to support the view that the uptake in the connective tissue and cartilage is mainly due to an incorporation in the sulphomucopolysaccharides (1). The metabolism of these substances can thus preferably be studied with labelled sulphate. Reddi and Norstriim (8) recently found that the incorporation of radio-sulphate is considerably reduced in chondroitin sulphuric acid, in scorbutic guinea pigs. The aim of the present investigation is to study the uptake of sulphate in scurvy and to relate this uptake to the specific scorbutic bone changes. A simultaneous determination of the uptake of radiophosphate to some extent gives a comparison as to the effect of C-avitaminosis on the mineral part of the bone tissue. L%Zethod. Young female guinea pigs, weighing about 200 g each, were given a modified HGjer scorbutogenous diet (4). Within three weeks they had developed a typical clinical scurvy and did not survive five weeks. Control animals which were given an adequate addition of C-vitamin showed normal weight increase and vitality. Each individual in one group of animals was given an injection of carrier-free Ss5labelled sodium sulphate. The dose was 3.0 me/kg in 0.9 per cent saline with 0.02 per cent sodium sulphate as a carrier. Another group received P3a-labelled carrierfree orthophosphate in a dose of 0.5 me/kg in 0.9 per cent saline at pH 6.7 containing 0.01 per cent phosphate. All injections were given intraperitoneally. The animals were killed 48 hours after the injections. Small portions (approx. 2 x 10 x 10 mm) of the various organs were excised. Blood samples were also taken. The sections were dried for at least 4X hrs. at about 5O”C, and repeated weighing revealed that they had attained constant weight. The radioactivity was measured in a standard way all through the experiment. The activity was corrected for background and decay and was expressed as counts/min./mg wet tissue. Results. The table discloses that, with the exception of cartilage, the radiosulphate activity in various organs of the scorbutic animals was only about one third of the activity in normal animals’ tissues. This difference is statistically significant. Cartilage showed the highest sulphate uptake but also the greatest inherent variations. In cartilage from scorbutic animals the activity was slightly lower than in the controls but this decrease can not be regarded as significant. No significant difference Esperimenfal
Cell Research
6
U. Friberg
528
and N. R. Ringertz
I Activity of radioactive samples. Counts/min./m:: wet tissue. TABLE
Radiosulphate Organs
Normal i animals i n=4
Blood 0.52+0.06 Liver ’ l.OG+O.17 Spleen. . 1.3110.17 Brain . 0.20+0.03 Kidney . IO 94+0.13 I 0.58 kO.05 Skin. Muscle. ~0.2420.03 Cartilage 6.50 +2.18 none . i 1.51 i-O.27
Radiophosphate
Stu- 1, Normal 1
animals n=G
n==6
O.lGf0.03 0.26+_0.06 0.45t0.06 I 0.05+0.01 0.33LO.06 O.lS? 0.02 10.09+0.02 I 5.17 t 1.39 (0.55+0.08
Scorbutici llifference / Stuanimals n=9
0.36+0.(16 9.0-t 0.5 8.3+_10.8 O.SOiO.15 62.9+ 3.7 74.7& 7.3 O.SGi0.15 5.54*** 7X.6? 6.5 G7.8? 8.4 6.8G*** 6.9$- 0.5 7.ti+ 0.8 ’ 0.15+0.02 10.61 i0.13 , 4.57** ~ 47.2i 5.5 5G.9+ 7.2 0.40 i 0.05 ~ s.35*** 15.9& 2.2 13.x-t 1.1 0.15+0.04 4.34** 1X9+- 2.6 20.1 z 1.0 11.33 i2.45 0.34 52.2 -t 11.7 G-1.2+ 6.4 i 0.96i-0.24 1 4.06** l 142.4-t 17.1 ’ 92.6+17.0,
of means
I
0.7& 1.1 ll.S+ 9.6 lO.S&1l.G 0.7i 1.0 9.7 & 9.9 2.1 i 2.2 0.2-t 2.4 12.0 +12.3 49.8i17.0 (
dent’s I 0.68 1.23 0.93 0.68 0.97 0.95 0.09 0.98 2.93*
between the radiophosphate uptake in normal and scorbutic animals could be established. In bone, however, the radiophosphate uptake in the scorbutic animals showed a probably significant decrease of about one third. Discussion. A considerable decrease in the uptake of radioactive sulphate in the various organs of scorbutic animals was found. Cartilage, however, showed no significant diminution in the uptake of sulphate, but~the manifest tendency is in agreement with earlier observations (8). ,4t present it is not possible to give an exact explanation of these findings. If present views on the fate of radiosulphate in the animal organism are valid, it is justifiable to assume that our results at least partially reflect a diminished sulphomucopolysaccharide formation. If that is the case, vitamin C is of importance for the synthesis of these polysaccharides, as well as for the formation of collagen. It would then be tempting to assume that the sulphomucopolysaccharides are in some way involved in the synthesis of the fibrillar proteins. On the other hand there was no apparent, essential change in the metabolism of phosphate. Bone alone showed appreciable lowering of the radiophosphate uptake in the scorbutic animals. It is not yet possible to state whether this decrease-or the decrease in radiosulphate uptake-is due to a reduced incorporation in the organic and/or mineral parts of bone. The experiments proceed and a full account of the work will be published elsewhere. REFERENCES 1. UOSTRBM,
2. 3. 4.
BOURNE, GERSH,
H., Arkiv G. H.,
I., and
Kemi,
6, 43
(1953).
Lancet, 243, 661 (1942). CATCHPOLE, H. R., Am.
J. Aaat.,
85, 457 (1949).
HBJER, J. A., Acla Paediat., 3, suppl. (1924). 5. MEYER, A., Z. vifaminforsch., 14, 332 (1943-44). 6. PENNEY, J. R., and BALFOUR, B. M., J. Pathol. Bacterial., 7. PERSSOS, B. H., Upsala Ltikaref. Fiirhandl., 58, suppl. 2 8. REDDI, K. K., and NORSTRBM, A. (to be published).
Experimental
Cell Research
6
61, 171 (1949). (1953).
UPTAKE
Cell Research,
OF
6, 527-528
527
(1954)
RADIOSULPHATE
VARIOUS
TISSUES
AND OF NORMAL
GUINEA
of
Histology,
AND
IN
SCORBUTIC
PIGS
and N. R. RIKGERTZ
U. FRIBERG Department
RADIOPHOSPHATE
Karolinska
Received
January
Institutrt,
Stockholm,
Sweden
19, 1954
THErate
of collagen formation is diminished in scurvy (2, 4). The effect of C-avitaminosis on the mucopolysaccharides in the ground substance of the connective tissue has not yet been completely clarified but histochemical investigations tend to indicate a reduced formation of polysaccharides (5, 6), eventually with an accumulation of products of a lower degree of polymerization (3, 7). Observations on the fato of radiosulphate in animal organisms seem to support the view that the uptake in the connective tissue and cartilage is mainly due to an incorporation in the sulphomucopolysaccharides (1). The metabolism of these substances can thus preferably be studied with labelled sulphate. Reddi and Norstriim (8) recently found that the incorporation of radio-sulphate is considerably reduced in chondroitin sulphuric acid, in scorbutic guinea pigs. The aim of the present investigation is to study the uptake of sulphate in scurvy and to relate this uptake to the specific scorbutic bone changes. A simultaneous determination of the uptake of radiophosphate to some extent gives a comparison as to the effect of C-avitaminosis on the mineral part of the bone tissue. L%Zethod. Young female guinea pigs, weighing about 200 g each, were given a modified HGjer scorbutogenous diet (4). Within three weeks they had developed a typical clinical scurvy and did not survive five weeks. Control animals which were given an adequate addition of C-vitamin showed normal weight increase and vitality. Each individual in one group of animals was given an injection of carrier-free Ss5labelled sodium sulphate. The dose was 3.0 me/kg in 0.9 per cent saline with 0.02 per cent sodium sulphate as a carrier. Another group received P3a-labelled carrierfree orthophosphate in a dose of 0.5 me/kg in 0.9 per cent saline at pH 6.7 containing 0.01 per cent phosphate. All injections were given intraperitoneally. The animals were killed 48 hours after the injections. Small portions (approx. 2 x 10 x 10 mm) of the various organs were excised. Blood samples were also taken. The sections were dried for at least 4X hrs. at about 5O”C, and repeated weighing revealed that they had attained constant weight. The radioactivity was measured in a standard way all through the experiment. The activity was corrected for background and decay and was expressed as counts/min./mg wet tissue. Results. The table discloses that, with the exception of cartilage, the radiosulphate activity in various organs of the scorbutic animals was only about one third of the activity in normal animals’ tissues. This difference is statistically significant. Cartilage showed the highest sulphate uptake but also the greatest inherent variations. In cartilage from scorbutic animals the activity was slightly lower than in the controls but this decrease can not be regarded as significant. No significant difference Esperimenfal
Cell Research
6
U. Friberg
528
and N. R. Ringertz
I Activity of radioactive samples. Counts/min./m:: wet tissue. TABLE
Radiosulphate Organs
Normal i animals i n=4
Blood 0.52+0.06 Liver ’ l.OG+O.17 Spleen. . 1.3110.17 Brain . 0.20+0.03 Kidney . IO 94+0.13 I 0.58 kO.05 Skin. Muscle. ~0.2420.03 Cartilage 6.50 +2.18 none . i 1.51 i-O.27
Radiophosphate
Stu- 1, Normal 1
animals n=G
n==6
O.lGf0.03 0.26+_0.06 0.45t0.06 I 0.05+0.01 0.33LO.06 O.lS? 0.02 10.09+0.02 I 5.17 t 1.39 (0.55+0.08
Scorbutici llifference / Stuanimals n=9
0.36+0.(16 9.0-t 0.5 8.3+_10.8 O.SOiO.15 62.9+ 3.7 74.7& 7.3 O.SGi0.15 5.54*** 7X.6? 6.5 G7.8? 8.4 6.8G*** 6.9$- 0.5 7.ti+ 0.8 ’ 0.15+0.02 10.61 i0.13 , 4.57** ~ 47.2i 5.5 5G.9+ 7.2 0.40 i 0.05 ~ s.35*** 15.9& 2.2 13.x-t 1.1 0.15+0.04 4.34** 1X9+- 2.6 20.1 z 1.0 11.33 i2.45 0.34 52.2 -t 11.7 G-1.2+ 6.4 i 0.96i-0.24 1 4.06** l 142.4-t 17.1 ’ 92.6+17.0,
of means
I
0.7& 1.1 ll.S+ 9.6 lO.S&1l.G 0.7i 1.0 9.7 & 9.9 2.1 i 2.2 0.2-t 2.4 12.0 +12.3 49.8i17.0 (
dent’s I 0.68 1.23 0.93 0.68 0.97 0.95 0.09 0.98 2.93*
between the radiophosphate uptake in normal and scorbutic animals could be established. In bone, however, the radiophosphate uptake in the scorbutic animals showed a probably significant decrease of about one third. Discussion. A considerable decrease in the uptake of radioactive sulphate in the various organs of scorbutic animals was found. Cartilage, however, showed no significant diminution in the uptake of sulphate, but~the manifest tendency is in agreement with earlier observations (8). ,4t present it is not possible to give an exact explanation of these findings. If present views on the fate of radiosulphate in the animal organism are valid, it is justifiable to assume that our results at least partially reflect a diminished sulphomucopolysaccharide formation. If that is the case, vitamin C is of importance for the synthesis of these polysaccharides, as well as for the formation of collagen. It would then be tempting to assume that the sulphomucopolysaccharides are in some way involved in the synthesis of the fibrillar proteins. On the other hand there was no apparent, essential change in the metabolism of phosphate. Bone alone showed appreciable lowering of the radiophosphate uptake in the scorbutic animals. It is not yet possible to state whether this decrease-or the decrease in radiosulphate uptake-is due to a reduced incorporation in the organic and/or mineral parts of bone. The experiments proceed and a full account of the work will be published elsewhere. REFERENCES 1. UOSTRBM,
2. 3. 4.
BOURNE, GERSH,
H., Arkiv G. H.,
I., and
Kemi,
6, 43
(1953).
Lancet, 243, 661 (1942). CATCHPOLE, H. R., Am.
J. Aaat.,
85, 457 (1949).
HBJER, J. A., Acla Paediat., 3, suppl. (1924). 5. MEYER, A., Z. vifaminforsch., 14, 332 (1943-44). 6. PENNEY, J. R., and BALFOUR, B. M., J. Pathol. Bacterial., 7. PERSSOS, B. H., Upsala Ltikaref. Fiirhandl., 58, suppl. 2 8. REDDI, K. K., and NORSTRBM, A. (to be published).
Experimental
Cell Research
6
61, 171 (1949). (1953).