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Fe59 utilization during heme biosynthesis
Report of meeting with combined hepatic and renal disease, one cerebellar ataxia treated with vitamin BI~, one acquired erythroid hypoplasia of bone marrow, and one case of macrocytic anemia due to intestinal anastomosis. These cases will be discussed in detail.
Studies
127 on
the
Biosynthesis
of
Heme:
H. C. SCHWARTZ, A. GOLDBERG, G. E. CARTWRIGHT, and M. M. WINTROBE, Salt Lake City, Utah.
A method, based on the uptake of radioiron into meme, has been utilized for the measurement of heme synthesis in a hemoT h e U s e of the S c h i l l i n g T e s t for the lysate of chicken erythrocytes. By this method it has been possible to study various E v a l u a t i o n of I n t r i n s i c - F a c t o r P r e p a r a factors which stimulate and other factors tion: STEVEN SCHWARTZ, WM. MASLOW, ROBERT STOREY, and D. L. TABERN, which inhibit heme synthesis. The addition of glycine, A-aminolevulinic Chicago, Illinois. acid, porphobilinogen, or protoporphyrin 9, The current official assay method for stimulated heme synthesis. The addition of intrinsic factor based upon observing the coproporphyrin I or III, or uroporphyrin I clinical response in P.A. patients in relapse or III, did not augment the uptake of is so unsatisfactory, and the number of cases radioiron into heme. These studies lend support to the concept which may be carried out with it is so small, that the Schilling test is being actively that glycine, A-aminole vulinic acid, porphobilinogen, and protoporphyrin 9, are studied as a replacement. A co-operative project of Cook County intermediates in the synthesis of heme, but Hospital and Abbott has carried out, during they do not lend support to the concept that the last two years, more than 600 determi- uroporphyrin and coproporphyrin are internations on various research and production mediates in heme synthesis. Evidence obtained suggests that the inlots of I.F. Before this method could be employed with adequate confidence, it was corporation of iron into protop0rphyrin necessary to study (1) the relation of the in the synthesis of heme is an enzymatic size of the radiocyanocobalamin dose to reaction. This reaction is inhibited by the urinary output response, (2) the efficiency K C N and heating to 56°C. Preliminary of various counting systems, (3) the effect studies on the purification of this enzyme are of various levels and purities of intrinsic described. The synthesis of heme in the chicken factor on output, (4) the safety of the proposed method, (5) the possible need for erythrocyte hemolysate was potentiated by repeated flushing doses and. (6) the corre- citrate in the presence of glycine, but citrate lation between assays carried out by the had no effect in the presence of porphobilinogen. Succinate, in the presence of two procedures. Data is presented indicating that 0.5 to glycine, did not enhance heine synthesis. 0.7/zg of B12 containing 0"25 or 0"5/~c Inhibition of heme synthesis occurred on the provides a safe dosage level and a urinary addition of malonate or lead. Co 6° B12 output that may be counted with equipment m most hospital isotope units. Fe 59 U t i l i z a t i o n D u r i n g H e m e B i o s y n t h e s i s : RICHARD A. NEVE, ROBERT F. LABBE, The method is applicable to the types of and ROBERT A. ALDRICH, Portland, preparation now being produced and used Oregon. in medical practice. Providing "standardized" patients are used and one has deterThe pathway of porphyrin biosynthesis mined that the responses are on the rising has been elucidated in considerable detail portion of the dose output curve, the results in recent years, but the incorporation of iron in every case where intercomparison has into porphyrin to produce heme has received been possible, have checked those of the little attention. Knowledge of the mechanclinical evaluation test. ism by which iron is inserted into the
128
Report of meeting
porphyrin ring may have important clinical implications in the group of anemias characterized as being "refractory." In studies to determine the site of iron incorporation, results have been obtained which indicate that reduced uroporphyrin I I I ( R U P - - a tetrapyrrole related to protoporphyrin), in the presence of Fe 59, increased the radioactivity of the hemin up to tenfold over the control, indicating increased biosynthesis. O f several tetrapyrroles tested R U P was the most active heine precursor. Further data will be presented to show that R U P is a direct intermediate in heme biosynthesis. F e r r o k i n e t i c s in N o r m a l and A n e m i c S w i n e :
G. E. CARTWRIGHT,J. A. BUSH, W. N. JENSEN and M. M. WINTROBE,Salt Lake City, Utah. Ferrokinetic studies have been performed in normal, pyridoxine-deficient, pteroylglutamic-acid-deficient, and copper-deficient swine, and in swine given phenylhydrazine. In the normal pigs, the average "apparent" red-cell life span was 62 days. The mean plasma iron turnover rate was 1-11 mg/kg/day and the red cell iron incorporation rate, 1.10 mg/kg/day. In the animals given phenylhydrazine, the mean erythrocyte survival time was 5 days. The plasma iron turnover rate was increased about fourfold, and the rate of erythropoiesis was four to five times greater than in the control pigs. In the pyridoxine-deficient swine, the mean erythrocyte survival time was normal. The plasma iron turnover rate was increased fourfold, but the rate of erythropolesis was approximately one fourth the normal mean value. These data are interpreted" as indicating that the anemia associated with this deficiency is the result of inability of the bone marrow to produce a normal number of erythrocytes. In the pteroylglutamic acid.deficient swine, the mean ery throcyte survival time was 17 days. The plasma iron turnover rate was 5 times the normal mean value. The rate of erythropoiesis was 1.6 times greater
than the mean value in the control pigs. These data are interpreted as indicating that anemia develops in this deficiency as the result of a combination of shortening of the erythr0cyte survival time and limitation of the capacity of the bone marrow to increase red cell production to the same degree as normal marrow can accomplish. In the copper-deficient pigs, the mean erythrocyte survival time was 17 days. The plasma iron turnover rate was 1.6 times normal. The rate of erythropoiesis was 1-2 times normal. It is concluded that copper-deficiency anemia results from both shortened erythrocyte survival time and limited capacity of the bone marrow to produce cells. B o n e Salt K i n e t i c s in M a n : GORAN C. H.
BAUER and ROBERT D. RAY, Seattle, Washington. Results of tracer studies using Ca 45 suggest that from a kinetic standpoint the skeletal calcium exists in two principally different fractions. One comparatively small fraction is in equilibrium with body fluid calcium while the major fraction is not. When radioactive calcium is introduced into the blood stream, it is picked up by the skeleton as a result of (1) rapid equilibration of the exchangeable calcium fraction with calcium of the body fluids, and (2) incorporation into the nonexchangeable skeletal calcium coincident with formation of new bone. After the isotope concentration has reached equilibrium between the body fluids and the skeletal exchangeable calcium, the fall in blood-calcium activity will b e determined by (1) the rate of excretion of the isotope, and (2) the rate of incorporation of the isotope into the nonexchangeable bone salt. Provided the isotope incorporated into the nonexchangeable skeletal calcium does not re-enter the blood stream during the experimental period, it is possible to determine the magnitude of the exchangeable body calcium pool and the rate of renewal of the skeleton through anabolism-katabolism. Using Ca 45 and other "bone-seeking" isotopes, these quantities have been calculated
Studies
127 on
the
Biosynthesis
of
Heme:
H. C. SCHWARTZ, A. GOLDBERG, G. E. CARTWRIGHT, and M. M. WINTROBE, Salt Lake City, Utah.
A method, based on the uptake of radioiron into meme, has been utilized for the measurement of heme synthesis in a hemoT h e U s e of the S c h i l l i n g T e s t for the lysate of chicken erythrocytes. By this method it has been possible to study various E v a l u a t i o n of I n t r i n s i c - F a c t o r P r e p a r a factors which stimulate and other factors tion: STEVEN SCHWARTZ, WM. MASLOW, ROBERT STOREY, and D. L. TABERN, which inhibit heme synthesis. The addition of glycine, A-aminolevulinic Chicago, Illinois. acid, porphobilinogen, or protoporphyrin 9, The current official assay method for stimulated heme synthesis. The addition of intrinsic factor based upon observing the coproporphyrin I or III, or uroporphyrin I clinical response in P.A. patients in relapse or III, did not augment the uptake of is so unsatisfactory, and the number of cases radioiron into heme. These studies lend support to the concept which may be carried out with it is so small, that the Schilling test is being actively that glycine, A-aminole vulinic acid, porphobilinogen, and protoporphyrin 9, are studied as a replacement. A co-operative project of Cook County intermediates in the synthesis of heme, but Hospital and Abbott has carried out, during they do not lend support to the concept that the last two years, more than 600 determi- uroporphyrin and coproporphyrin are internations on various research and production mediates in heme synthesis. Evidence obtained suggests that the inlots of I.F. Before this method could be employed with adequate confidence, it was corporation of iron into protop0rphyrin necessary to study (1) the relation of the in the synthesis of heme is an enzymatic size of the radiocyanocobalamin dose to reaction. This reaction is inhibited by the urinary output response, (2) the efficiency K C N and heating to 56°C. Preliminary of various counting systems, (3) the effect studies on the purification of this enzyme are of various levels and purities of intrinsic described. The synthesis of heme in the chicken factor on output, (4) the safety of the proposed method, (5) the possible need for erythrocyte hemolysate was potentiated by repeated flushing doses and. (6) the corre- citrate in the presence of glycine, but citrate lation between assays carried out by the had no effect in the presence of porphobilinogen. Succinate, in the presence of two procedures. Data is presented indicating that 0.5 to glycine, did not enhance heine synthesis. 0.7/zg of B12 containing 0"25 or 0"5/~c Inhibition of heme synthesis occurred on the provides a safe dosage level and a urinary addition of malonate or lead. Co 6° B12 output that may be counted with equipment m most hospital isotope units. Fe 59 U t i l i z a t i o n D u r i n g H e m e B i o s y n t h e s i s : RICHARD A. NEVE, ROBERT F. LABBE, The method is applicable to the types of and ROBERT A. ALDRICH, Portland, preparation now being produced and used Oregon. in medical practice. Providing "standardized" patients are used and one has deterThe pathway of porphyrin biosynthesis mined that the responses are on the rising has been elucidated in considerable detail portion of the dose output curve, the results in recent years, but the incorporation of iron in every case where intercomparison has into porphyrin to produce heme has received been possible, have checked those of the little attention. Knowledge of the mechanclinical evaluation test. ism by which iron is inserted into the
128
Report of meeting
porphyrin ring may have important clinical implications in the group of anemias characterized as being "refractory." In studies to determine the site of iron incorporation, results have been obtained which indicate that reduced uroporphyrin I I I ( R U P - - a tetrapyrrole related to protoporphyrin), in the presence of Fe 59, increased the radioactivity of the hemin up to tenfold over the control, indicating increased biosynthesis. O f several tetrapyrroles tested R U P was the most active heine precursor. Further data will be presented to show that R U P is a direct intermediate in heme biosynthesis. F e r r o k i n e t i c s in N o r m a l and A n e m i c S w i n e :
G. E. CARTWRIGHT,J. A. BUSH, W. N. JENSEN and M. M. WINTROBE,Salt Lake City, Utah. Ferrokinetic studies have been performed in normal, pyridoxine-deficient, pteroylglutamic-acid-deficient, and copper-deficient swine, and in swine given phenylhydrazine. In the normal pigs, the average "apparent" red-cell life span was 62 days. The mean plasma iron turnover rate was 1-11 mg/kg/day and the red cell iron incorporation rate, 1.10 mg/kg/day. In the animals given phenylhydrazine, the mean erythrocyte survival time was 5 days. The plasma iron turnover rate was increased about fourfold, and the rate of erythropoiesis was four to five times greater than in the control pigs. In the pyridoxine-deficient swine, the mean erythrocyte survival time was normal. The plasma iron turnover rate was increased fourfold, but the rate of erythropolesis was approximately one fourth the normal mean value. These data are interpreted" as indicating that the anemia associated with this deficiency is the result of inability of the bone marrow to produce a normal number of erythrocytes. In the pteroylglutamic acid.deficient swine, the mean ery throcyte survival time was 17 days. The plasma iron turnover rate was 5 times the normal mean value. The rate of erythropoiesis was 1.6 times greater
than the mean value in the control pigs. These data are interpreted as indicating that anemia develops in this deficiency as the result of a combination of shortening of the erythr0cyte survival time and limitation of the capacity of the bone marrow to increase red cell production to the same degree as normal marrow can accomplish. In the copper-deficient pigs, the mean erythrocyte survival time was 17 days. The plasma iron turnover rate was 1.6 times normal. The rate of erythropoiesis was 1-2 times normal. It is concluded that copper-deficiency anemia results from both shortened erythrocyte survival time and limited capacity of the bone marrow to produce cells. B o n e Salt K i n e t i c s in M a n : GORAN C. H.
BAUER and ROBERT D. RAY, Seattle, Washington. Results of tracer studies using Ca 45 suggest that from a kinetic standpoint the skeletal calcium exists in two principally different fractions. One comparatively small fraction is in equilibrium with body fluid calcium while the major fraction is not. When radioactive calcium is introduced into the blood stream, it is picked up by the skeleton as a result of (1) rapid equilibration of the exchangeable calcium fraction with calcium of the body fluids, and (2) incorporation into the nonexchangeable skeletal calcium coincident with formation of new bone. After the isotope concentration has reached equilibrium between the body fluids and the skeletal exchangeable calcium, the fall in blood-calcium activity will b e determined by (1) the rate of excretion of the isotope, and (2) the rate of incorporation of the isotope into the nonexchangeable bone salt. Provided the isotope incorporated into the nonexchangeable skeletal calcium does not re-enter the blood stream during the experimental period, it is possible to determine the magnitude of the exchangeable body calcium pool and the rate of renewal of the skeleton through anabolism-katabolism. Using Ca 45 and other "bone-seeking" isotopes, these quantities have been calculated