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The erythropoietic activity of coprogen
LETTERS
TO
THE
EDITORS
475
3O’C. After cent,rifugation, the amount of B12 in the supernatant was determined microbiologically with Lactobacill~s leichnLannii as assay organism. The distribution of t,hr B&inding factor as seen from Table I is very similar to the distribut,ion of thcl “intrinsic factfor” of Castle (4-g).
1. 2. 3. 1. 5. 6. 7.
I)AVIH? IS. II., AND MINGIOLI,E.S.,J.BUC~.~O, 17 (1950). HOFF-JORGENSEN, E., Arch. Biochem. and Biophys. 36, 235 (1952). HURKHOLDER, P. It., Arch. Biochem. and Biophys. 39, 32’2 (19521. MEULES(:RACHT, E., Acfa Med. &and. 86, 70 (1935). UNGLEY, C. C., AND MOFFETT, R., Lancet 230, 1232 (19%). Fox, II. J., ANI) CASTLE, W. B., ~VI. J. Mel]. Sci. 203, 18 (1942). I,ANI,BOE-(~HRI~TENVSEN. IS., .4Nl) Borrs, C. L. S., ;lcta Marl. Scar~tl.
137, 116
(1017).
Departments I ‘niversity (loper¥, Received
of Biochemistry of Copenhagen, Derunark October 21, 1952
and of :t rmtc:rrr,u.
The Erythropoietic
Activity of Coprogen
The isolation of coprogen, a new iron-containing pigment which is high15 active as a growt.h factor for several members of the genus Pilobollts, has been reported b,v Hesseltine and co-workers (1). Hematin has been shown to have :t very slight activity for this species (1, 2). In a variety of experiments, conducted to determine whether coprogen has any function for higher animals, it was found that, a stimulation of the erythropoietic system could be induced by this factor when the bone marrow was irrhibited by one of several methods described below. ,411 result,s were obtained t)> removal and histological examination of the bone marrow and other tissues \>J methods previously described (3). Weanling rats fed a synthetic diet and given 10 pg. of aminopterin orally pel day for 5 days, will show a marked aplasia of the bone marrow, as well as fibrosis and degeneration of the thymus and spleen (3, 4). In thirteen experiments in which such aminopterin-treated rats were given daily intramuscular injections of 0.5 or 1 .O mg. of coprogen, a very marked degree of protection against t,he depression of red-cell production in the bone marrow and some protection against the degenerative changes in the thymus and spleen were obtained. Splenic erythroIjoiesis was stimulated in the normal as well as in the aminopterin-treatred rats. Weanling rats fed a synthetic diet, containing 1% sulfaguanidine will show varying degrees of hemorrhage and degeneration of t,he erythroid elements of the bone marrow in 2-3 weeks. In two experiments the administration of 1 mg. of coprogen per day prevented the development of these changes. In two additional rasperiments in \vhich the bone-marrow changes werr first :rllo\vrd to develop, as
476
LETTERS
TO
THE
TABLE The
Effect
Treatment
A.
APFa-dejicient chicks
1. Folic acid-deficient, diet
of Coprogen
EDITORS
I
on Chick
Criteria
SCOR
Macrocytosie Hemorrhage Fibrosis
4+ 4+ 4+
Degeneratior RBC b islandr
4f 0
Bone
Marrow
Histology:
Macrocytosis If Hemorrhage 1+ 1+ Fibrosis Degeneratior 1 I Slight RBC islands 2-3+
Slight Few areas Few areas Few RBC Small but
3. As 1 + 1 mg coprogen/day
Hemorrhage
4. As 2 + 1 mg.
days
Seen throughout RBC islands Estensive throughout marrow Replacing hemorrage and degeneration areas Any remaining cells Finally leading to aplasia
2. As 1 + 10 pg. folic acid and 1 PR.Bn/day
Slight
femur @ lo-21
evident evident pycnotic definite RBC areas
RBC islands
3+
No evidence of fibrosis, macrocytosis, and rarely any degenerating RBC Large and numerous
RBC islands
4+
Apparently
normal marrow
coprogenlday B.
As A-2
New Hampshire Red chicks
1. Folic acid-deficient diet 2. As I + 1 mg. folic acid/kg. 3. As 2 + 20 mg. coprogen/kg 4. As 2 + 500 pg. aminopterin/ kg. 5. As 4 + 20 mg. coprogen/kg.
As ‘4-2 -4s A-3, large RBC islands As A-l
-
and numerous
As A-3, increased erythropoiesis
stimulat,ion
a APF = animal protein factor. b RBC = red blood cells. determined by a biopsy technique (5), treatment with 1 mg. of coprogen per day for 1 week resulted in a very pronounced erythrocytic stimulation. Day-old chicks fed a folic acid-deficient diet, with or without the addition of an antagonist, will develop nearly complete inhibition of red-cell formation in the bone marrow (6). In a series of ten experiments in which deficient. birds were
LETTERS
TO
THE
4si
EDITORS
1rratted with coprogen, alone or in combination \vit,h folic acid, vit,arnin Blz, and .~-:tminopt,eroplaspartic acid, coprogen markedly stimulated erythropoiesis in all cases and appeared 1.0 act synergistically with the folic acid and vitamin Bls in overcoming the inhibitory effects of the antagonists OII the marrow. The eft’ect of coprogen on chick hone marrow is shown in Table I. These trsperiments demon& rate that coprogen stimulates erythropoirsis in the bone marrow of ratts and chicks, and will prevent rrrtain of the t osir rfrects of folic acid antagonists.
C. W., PIL)ACI(S,C.,WHITEHII,I,, A.R., BOHONO~, N..IIUTCIIIN~;S. B. I,.. ASII WILLIAMS, J. H.. .I. .+l?>~.Clhenl. Sot. 74, 362 (1952). 2. I’.%GE, 1~. M., paper presented at Am. Inst. Biol. Sci. Meetings. I’. of Minn.. Sept., 1951. 1. LIESSEI,TI~~E,
:<. RITTER,
fI.
k%., Am
OL%ON,J.J.,
A?&.t%cfh.47,%j
(1949).
J. J., Hrit. J. Erptl. Path. 32, 458 (1951). 5. RITTER, H. H., ANI) OLESOS~ J. J., A&. Path. 46, 498 (19483. 6. WOLL, l’., l’,ans. N. I’. Ad. Sci. 10, 83 (1048). 4. WOLL,
b;., .~R'I, ()LESON,
Lederle Laboratories Division, .Imericnn Qanamid Compaq, Pearl IZiuer. New York
Received
LVfrzwtfber
3,
1959
II. B. RITTER ,I. .J. OLESOS B. I,. HUTCHINUS J. II. WILLIAMS
Synthesis of C14-17-Hydroxy-ll-Desoxycorticosterone 17-Hydroxycorticosterone by Fractionated Extracts Adrenal Homogenates’
and of
We have prcvious1.v reported the presence of enzyme systems in beef adrenal homogenates which will oxidize the 21-methyl group of progesterone to a primary alcohol group, and introduce a l7-hydroxyl group into the progesterone molecule (1). This conversion of progesterone to 17.hydroxy-ll-desosycorticosterone (Reichstein’s compound S} was originally found to require adenosine t!riphosphat,r (ATP) activation; more recent work by us indicat’es that diphosphopyridine nucleot,idc (Dl’K;) or triphosphopyridine nucleotide (Tl’?J) will also ‘activate t.his conversion. Both enzymes have been found in the supernatant fraction of beef adrenal homogenates centrifuged at 20,000 X [I for 0.5 hr. This met)hod has been applied to the biosynthesis of compound S from C’“labeled progesterone, the compound S being further converted to compound F by taking advantage of the excellent methods which have heen developed in other laboratories using beef adrenal mitochondrial preparations for the ll-hydroxylation (2, 31. Fresh heifer adrenals, frozen in Dry Ice at t,he slaught8erhouse, were homogenized in Krebs-Ringer bicarbonate buffer, pH 7.4, and centrifuged at 20,090 X f, L This work was supported Cancer Soriety.
in part under a research grant from the American
TO
THE
EDITORS
475
3O’C. After cent,rifugation, the amount of B12 in the supernatant was determined microbiologically with Lactobacill~s leichnLannii as assay organism. The distribution of t,hr B&inding factor as seen from Table I is very similar to the distribut,ion of thcl “intrinsic factfor” of Castle (4-g).
1. 2. 3. 1. 5. 6. 7.
I)AVIH? IS. II., AND MINGIOLI,E.S.,J.BUC~.~O, 17 (1950). HOFF-JORGENSEN, E., Arch. Biochem. and Biophys. 36, 235 (1952). HURKHOLDER, P. It., Arch. Biochem. and Biophys. 39, 32’2 (19521. MEULES(:RACHT, E., Acfa Med. &and. 86, 70 (1935). UNGLEY, C. C., AND MOFFETT, R., Lancet 230, 1232 (19%). Fox, II. J., ANI) CASTLE, W. B., ~VI. J. Mel]. Sci. 203, 18 (1942). I,ANI,BOE-(~HRI~TENVSEN. IS., .4Nl) Borrs, C. L. S., ;lcta Marl. Scar~tl.
137, 116
(1017).
Departments I ‘niversity (loper¥, Received
of Biochemistry of Copenhagen, Derunark October 21, 1952
and of :t rmtc:rrr,u.
The Erythropoietic
Activity of Coprogen
The isolation of coprogen, a new iron-containing pigment which is high15 active as a growt.h factor for several members of the genus Pilobollts, has been reported b,v Hesseltine and co-workers (1). Hematin has been shown to have :t very slight activity for this species (1, 2). In a variety of experiments, conducted to determine whether coprogen has any function for higher animals, it was found that, a stimulation of the erythropoietic system could be induced by this factor when the bone marrow was irrhibited by one of several methods described below. ,411 result,s were obtained t)> removal and histological examination of the bone marrow and other tissues \>J methods previously described (3). Weanling rats fed a synthetic diet and given 10 pg. of aminopterin orally pel day for 5 days, will show a marked aplasia of the bone marrow, as well as fibrosis and degeneration of the thymus and spleen (3, 4). In thirteen experiments in which such aminopterin-treated rats were given daily intramuscular injections of 0.5 or 1 .O mg. of coprogen, a very marked degree of protection against t,he depression of red-cell production in the bone marrow and some protection against the degenerative changes in the thymus and spleen were obtained. Splenic erythroIjoiesis was stimulated in the normal as well as in the aminopterin-treatred rats. Weanling rats fed a synthetic diet, containing 1% sulfaguanidine will show varying degrees of hemorrhage and degeneration of t,he erythroid elements of the bone marrow in 2-3 weeks. In two experiments the administration of 1 mg. of coprogen per day prevented the development of these changes. In two additional rasperiments in \vhich the bone-marrow changes werr first :rllo\vrd to develop, as
476
LETTERS
TO
THE
TABLE The
Effect
Treatment
A.
APFa-dejicient chicks
1. Folic acid-deficient, diet
of Coprogen
EDITORS
I
on Chick
Criteria
SCOR
Macrocytosie Hemorrhage Fibrosis
4+ 4+ 4+
Degeneratior RBC b islandr
4f 0
Bone
Marrow
Histology:
Macrocytosis If Hemorrhage 1+ 1+ Fibrosis Degeneratior 1 I Slight RBC islands 2-3+
Slight Few areas Few areas Few RBC Small but
3. As 1 + 1 mg coprogen/day
Hemorrhage
4. As 2 + 1 mg.
days
Seen throughout RBC islands Estensive throughout marrow Replacing hemorrage and degeneration areas Any remaining cells Finally leading to aplasia
2. As 1 + 10 pg. folic acid and 1 PR.Bn/day
Slight
femur @ lo-21
evident evident pycnotic definite RBC areas
RBC islands
3+
No evidence of fibrosis, macrocytosis, and rarely any degenerating RBC Large and numerous
RBC islands
4+
Apparently
normal marrow
coprogenlday B.
As A-2
New Hampshire Red chicks
1. Folic acid-deficient diet 2. As I + 1 mg. folic acid/kg. 3. As 2 + 20 mg. coprogen/kg 4. As 2 + 500 pg. aminopterin/ kg. 5. As 4 + 20 mg. coprogen/kg.
As ‘4-2 -4s A-3, large RBC islands As A-l
-
and numerous
As A-3, increased erythropoiesis
stimulat,ion
a APF = animal protein factor. b RBC = red blood cells. determined by a biopsy technique (5), treatment with 1 mg. of coprogen per day for 1 week resulted in a very pronounced erythrocytic stimulation. Day-old chicks fed a folic acid-deficient diet, with or without the addition of an antagonist, will develop nearly complete inhibition of red-cell formation in the bone marrow (6). In a series of ten experiments in which deficient. birds were
LETTERS
TO
THE
4si
EDITORS
1rratted with coprogen, alone or in combination \vit,h folic acid, vit,arnin Blz, and .~-:tminopt,eroplaspartic acid, coprogen markedly stimulated erythropoiesis in all cases and appeared 1.0 act synergistically with the folic acid and vitamin Bls in overcoming the inhibitory effects of the antagonists OII the marrow. The eft’ect of coprogen on chick hone marrow is shown in Table I. These trsperiments demon& rate that coprogen stimulates erythropoirsis in the bone marrow of ratts and chicks, and will prevent rrrtain of the t osir rfrects of folic acid antagonists.
C. W., PIL)ACI(S,C.,WHITEHII,I,, A.R., BOHONO~, N..IIUTCIIIN~;S. B. I,.. ASII WILLIAMS, J. H.. .I. .+l?>~.Clhenl. Sot. 74, 362 (1952). 2. I’.%GE, 1~. M., paper presented at Am. Inst. Biol. Sci. Meetings. I’. of Minn.. Sept., 1951. 1. LIESSEI,TI~~E,
:<. RITTER,
fI.
k%., Am
OL%ON,J.J.,
A?&.t%cfh.47,%j
(1949).
J. J., Hrit. J. Erptl. Path. 32, 458 (1951). 5. RITTER, H. H., ANI) OLESOS~ J. J., A&. Path. 46, 498 (19483. 6. WOLL, l’., l’,ans. N. I’. Ad. Sci. 10, 83 (1048). 4. WOLL,
b;., .~R'I, ()LESON,
Lederle Laboratories Division, .Imericnn Qanamid Compaq, Pearl IZiuer. New York
Received
LVfrzwtfber
3,
1959
II. B. RITTER ,I. .J. OLESOS B. I,. HUTCHINUS J. II. WILLIAMS
Synthesis of C14-17-Hydroxy-ll-Desoxycorticosterone 17-Hydroxycorticosterone by Fractionated Extracts Adrenal Homogenates’
and of
We have prcvious1.v reported the presence of enzyme systems in beef adrenal homogenates which will oxidize the 21-methyl group of progesterone to a primary alcohol group, and introduce a l7-hydroxyl group into the progesterone molecule (1). This conversion of progesterone to 17.hydroxy-ll-desosycorticosterone (Reichstein’s compound S} was originally found to require adenosine t!riphosphat,r (ATP) activation; more recent work by us indicat’es that diphosphopyridine nucleot,idc (Dl’K;) or triphosphopyridine nucleotide (Tl’?J) will also ‘activate t.his conversion. Both enzymes have been found in the supernatant fraction of beef adrenal homogenates centrifuged at 20,000 X [I for 0.5 hr. This met)hod has been applied to the biosynthesis of compound S from C’“labeled progesterone, the compound S being further converted to compound F by taking advantage of the excellent methods which have heen developed in other laboratories using beef adrenal mitochondrial preparations for the ll-hydroxylation (2, 31. Fresh heifer adrenals, frozen in Dry Ice at t,he slaught8erhouse, were homogenized in Krebs-Ringer bicarbonate buffer, pH 7.4, and centrifuged at 20,090 X f, L This work was supported Cancer Soriety.
in part under a research grant from the American