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SERUM-VITAMIN B12 DURING MAINTENANCE THERAPY IN PERNICIOUS ANÆMIA WITH A DEPOT PREPARATION OF VITAMIN B12
739
Gordon 1951, McFadzean and Choa 1953). It is most SERUM-VITAMIN B12 other infecinfection salmonella that and, perhaps, likely DURING MAINTENANCE THERAPY IN tions in people with SS or SC genotype predispose them PERNICIOUS ANÆMIA WITH A to much greater haemolysis and, therefore, to a much more DEPOT PREPARATION OF VITAMIN B12 severe crisis. Pyrexia from any cause can increase the rate of red blood-cell destruction and the rate of sickling P. BASTRUP-MADSEN S. NØRREGAARD M.D. Copenhagen M.D. Copenhagen (Basu et al. 1963). Although jaundice is thought to be hxmolytic it would M. SCHWARTZ T. HANSEN seem from the first case that an obstructive element may M.D. Copenhagen Cand. Pharm. Copenhagen have been present. This might be due to swelling of the E. MEULENGRACHT liver cells as a result of the infection. M.D. Copenhagen, L.L.D. Fullerton and Watson-Williams (1962) and Edington From the University Department of Medicine of Aarhus Amts(1963) thought that folic-acid deficiency was the com- sygehus, Aarhus, Medical Department B, Frederiksberg monest factor precipitating crises in sickle-cell disease. Hospital, Copenhagen, Medical Department F, Glostrup The role of folic-acid deficiency in the precipitation of Hospital, Copenhagen, and the Research Laboratory, Dumex Ltd., the crises was not investigated in our three patients but it Copenhagen is unlikely that there was any significant deficiency. PosTHERE are two reasons for using long-acting vitamin-B12 sibly, however, in addition to an antibiotic these patients preparations in pernicious anasmia. In a life-long therapy should be given folic acid in the usual dose of 15-30 mg. it is an advantage for the patient to have as few injections daily in divided doses. as possible, and long-acting preparations may produce a The hip-joint finds in the first case are interesting. The more rapid and complete replenishment of the tissue dislocation of the hip while the patient was still confined stores. Since a very large proportion of an injected to bed was thought to be due to an infective arthritis with amount of aqueous cyanocobalamin is excreted in the softening of the ligaments and consequent displacement urine (Chesterman et al. 1951, Lang et al. 1952, Sokoloff of the head of the femur. Cockshott (1961), Barton and et al. 1952, Heinrich and Gabbe 1961, Adams 1962) it Cockshott (1962), and Middlemiss (1964) reviewed the may be doubted whether treatment with conventional X-ray appearances in SS and SC disease but did not doses can build up a tissue store of 4 mg. vitamin B12 mention dislocation of the hip or of other joints. It seems which is believed to be the normal content. that this is the first reported case. Infarction of the head Hydroxocobalamin (Glass et al. 1962) is more suitable and neck of the femur is seen not infrequently in SC than cyanocobalamin because it is more readily bound to disease and to a lesser extent in SS disease. We feel that the serum-proteins (Hertz et al. 1964); the loss in the the destruction of the head and neck of the femur some urine is reduced to about 25%. weeks after the dislocation of the hip-joint was, in part at We present our results with a preparation of vitamin least, due to infarction. It is difficult, however, to exclude B12 which is more long-acting than hydroxocobalamin. the role of the infective arthritis in the pathogenesis. This preparation is a suspension of a cyanocobalaminPrognosis is said to be much brighter in SC disease tannin complex in a sesame oil/aluminium monostearate than in SS disease. This is certainly true for patients gel 0 7Biz-T.A.M/, ’Betolvex ’). With this preparation under 10, since the manifestations of SC disease do not it is possible to ensure a continuous release of a suitable usually appear before the second decade. One of the amount of the vitamin into the body, resulting eventually lessons to be learned from these 3 cases is that the gener- in complete replenishment, at the same time meeting the ally complacent attitude to SC disease in non-pregnant daily requirements. The depot effect has made it possible adults is not wholly justifiable. It seems that the same to increase the intervals between the injections to two or precautionary measures taken in SS disease should be three months. Even though preliminary results have been taken in SC disease-viz., all patients should receive folic favourable (Schwartz et al. 1962, Meulengracht 1963, acid, 5-10 mg. daily and antimalarial drugs in the usual Gough et al. 1964), conclusive evidence of the value of this compound cannot be established until its ability to prophylactic dosage. maintain clinical remission and a high level of vitamin Bl2 Summary in the blood has been demonstrated in a sufficiently large 3 cases of severe crises with jaundice in sickle-cell of patients treated for a long time. Our investigahasmoglobin-C disease in young non-pregnant Nigerian number tion is an attempt to provide such information. adults are described. The role of salmonella infection in the precipitation of crises is emphasised. Cases of severe crisis should probably be treated with chloramphenicol even before the results of the blood, stool, and urine cultures are known. The normal precautionary measures taken in SS disease should be extended to SC diseases: all patients should be given folic acid and antimalarial drugs. We thank the department of pathology for the necropsy reports and Prof. W. P. Cockshott for radiological reports and his interest in this paper. We are also grateful to the department of haematology for determining the genotype of the patients and to Mr. D. Simmonds and Mr. F. Speed for preparing the illustrations. Requests for reprints should be addressed to Prof. G. Onuaguluchi, Department of Pharmacology and Therapeutics, University of
Ibadan, Nigeria. References
at
foot of next column
Material and Methods Our series comprises 169 patients with addisonian pernicious anaemia, in all of whom haematological and neurological PROF.
ONUAGULUCHI,
DR. AKANDE: REFERENCES
Barton, C. J., Cockshott, W. P. (1962) Am. J. Reontogenol, 88, 523. Basu, A. K., Woodruff, A. W., Pettitt, L. E. (1963) Lancet, ii, 1088. Cockshott, W. P. (1961) in Tropical Radiology (edited by J. H. Middlemiss); p. 75. London. Edington, G. M. (1957) J. clin. Path. 10, 182. (1963) Ghana med. J. 2, 83. Fullerton, W. T., Watson-Williams, E. J. (1962) J. Obst. Gynœ. Br. Commonw. 69, 729. Hendrickse, R. G., Collard, P. (1960) Lancet, i, 80. Hook, E. W., Campbell, C. G., Wreens, H. S., Cooper, G. R. (1957) New Engl. J. Med. 257, 403. McFadzean, A. J. S., Choa, G. H. (1953) Br. med. J. ii, 360. Middlemiss, H. (1964) Trans. R. Soc. trop. Med. Hyg. 58, 197. Raper, A. B. (1956) Br. med. J. i, 965. Shaw, A. B. (1951) Lancet, ii, 813. Smith-Gordon, C. E. (1951) ibid. p. 1020. Tuttle, A. H., Kock, B. (1960) J. Pediat. 56, 331. —
740 values. Betolvex was administered by deep intramuscular injections of 1 ml. containing 1 mg.
cyanocobalamin.
Results Haematological and neurological remission was maintained in all patients: no fresh neurological manifestations developed during the treatment. No local or general
Fig. 1—Serum-vitamin-Bj levels in 127 patients with pernicious anaemia during maintenance therapy with 1 mg. betolvex every second month.
The ordinates are shown on a logarithmic scale. The the course of the arithmetic mean.
curve
shows
examinations and determinations of serum-vitamin-Bl2 levels continued for two to four years. The vitamin-B,2 analyses were carried out by the Lactobacillus leichmannii method. Blood-samples were taken immediately before each injection; vitamin-B,,levels in these samples represented the lowest
were
side-effects were observed. The serum-vitamin-B12 levels in 127 patients treated with betolvex 1 ml. (containing 1 mg. vitamin B12) at intervals of two months are shown in fig. 1. The period of observation for these patients was two to three and a half years. A few patients were observed for four years and in these the serum-vitamin-B12 levels remained at the same level as after three and a half years. Since previouslv untreated and treated patients are included in this series, the initial values on the graph include both subnormal and normal values. The curve represents average values. Levels increased initially but then levelled off, and the curve became almost horizontal. Most patients had serum-vitamin-B12 concentrations at the upper limit of the normal range; a few reached levels above normal (average 800 pg. per ml.).
Fig. 2 shows the serum-vitamin-B12 levels in 42 patients who received 1 ml. betolvex at intervals of three months. This treatment caused an increase in serum-vitamin-B12 during the first year to almost the same level as did injections every second month. Some of the patients had previously been treated with oral preparations or injections of aqueous cyanocobalamin. In these, the serum-vitamin-Bl2 levels did not differ from those of patients treated with betolvex only.
Fig. 2-Serum-vitamin-B" levels in 42 patients with pernicious anaemia during maintenance therapy with 1 mg. betolvex every
To investigate how a higher initial dosage might effect the serum-vitamin-B12 level, 10 patients were given, during the first month, 4 ml. betolvex divided into two or four injections (fig. 3). Their serum-vitamin-B12 levels were compared with those of 10 patients who from the beginning of treatment had received only 1 ml. betolvex every second month (fig. 4). The high serum-vitamin-B12 levels were reached in about four months after the larger initial doses, compared with the approximately twelve months with the lower dosage.
third month.
Fig. 3—Serum-vitamin-B, levels in 10 patients with pernicious anaemia receiving as initial therapy 4 mg. betolvex during the first month and then maintenance second month.
therapy with
1 mg. every
Fig. 4-Serum-vitamin-B" levels in 10 patients with pernicious anaemia who from the beginning of treatment received only 1 mg. betolvex every second month.
741
Discussion
A suspension cyanocobalamin-tannin complex in a sesame-oil/aluminium-monostearate gel differs from other preparations of vitamin-Bl2 in that liberation of the vitamin from the intramuscular injection site is extremely protracted and the loss of the vitamin in the urine is only of
a
slight. In our series the serum-vitamin-B12 values after injection of betolvex were much higher than those reported for any other form of treatment. The high levels of the vitamin were maintained throughout the observation period of two to four years by administering 1 ml. betolvex as much as three months apart. Vitamin B12may be liberated from the intramuscular depot in a non-cyanide form that is, perhaps, more readily bound to the plasma-proteins (as with the form of vitamin B12 that appears in the plasma after injection of hydroxocobalamin). The protein-bound state of the vitamin formed from both cyanocobalamin or hydroxocobalamin is equally physiologically active because either form can be assumed to be converted ultimately into the active coenzyme. Betolvex is thus acting as a depot preparation in two ways: first, by prolonged continuous liberation of the vitamin from the injection site and secondly by bringing about a continued high concentration in the plasma, the protein-bound vitamin itself acting as a depot. A few of our patients reached unusually high-serumvitamin B12 levels of several thousand pg. per ml. We did not see any side-effects of these high levels, nor were any expected, for vitamin B12 is thought to be non-toxic. No local reactions after intramuscular injection were noted. The action of betolvex is so protracted that an injection of 1 ml. every third month will supply the body with adequate amounts of the vitamin throughout this period. Thus, the depot effect far exceeds that of hydroxocobalamin and that of another depot preparation, ’Depinar ’ (Kristensen and Hansen 1966).
Summary The value of the
depot preparation ’Betolvex ’ (cyanoa sesame-oil/ in the maintenance aluminium-monostearate gel) therapy of pernicious ansemia was investigated in 169 patients for
cobalamin-tannin complex suspended in
four years. When 1 mg. vitamin B12 was given in this form intramuscularly every second or third month, the serumvitamin-B12 values gradually increased to a constant level of about 800 pg. per ml. When 4 mg. vitamin B12 were given during the first month of therapy, the high constant level was reached earlier. This level, which seems to indicate that the body stores had been replenished, was maintained throughout the observation period. Clinical remission was also maintained and there were no side-effects. two to
CUSHING’S SYNDROME WITH ADRENAL MEDULLARY INSUFFICIENCY AND ADRENAL AUTOANTIBODIES LAURENCE C. WEGIENKA M.D. Michigan LOUIS E. KOMARMY M.D. California
KIRK D. WUEPPER M.D.
Michigan PETER H. FORSHAM M.A. Cantab, M.D. Harvard
From the Metabolic Research Unit and Departments of Medicine (Division of Dermatology) and Pathology, University of California School of Medicine, San Francisco
ANTIBODIES to adrenocortical-cell cytoplasm have been found in human sera (Anderson et al. 1957, Blizzard et al. 1962, Irvine 1963). The most sensitive method for detecting adrenal autoantibodies is by immunofluorescence. By this method Blizzard and Kyle (1963) found adrenal antibodies in the sera of 51 % of 71 patients with Addison’s disease. Adrenal antibodies were not demonstrated in the sera of 7 patients with Cushing’s syndrome due to bilateral adrenocortical hyperplasia (5 cases) or carcinoma (2 cases) or in patients with virilising adrenal hyperplasia
(20 cases). We describe here a patient with Cushing’s syndrome due to adrenal hyperplasia with impaired ability to secrete epinephrine from the adrenal medulla, evidence of prominent mononuclear-cell infiltration into the medullary tissue, and associated circulating adrenal and thyroid antibodies. Materials and Methods Fresh and stored sera from 10 patients with Cushing’s syndrome due to bilateral adrenocortical hyperplasia or tumour were kept at - 20° C until use. Tissue autoantibodies were detected by the methods of Holborow et al. (1959) for thyroid antibodies, Taylor et al. (1962) for gastric antibodies, and Blizzard et al. (1962) for adrenal antibodies: all methods were modified so that all three tissue slices could be examined simultaneously on a single miscrocope slide. The tissue was allowed to react with patient’s serum for 30 minutes at room temperature, and the slices were washed three times in phosphate-buffered saline solution (pH 7-2). Rabbit antisera, conjugated with fluorescein isothiocyanate by the method of McKinney et al. (1964), was allowed to react with the slices for 30 minutes. After three more washes in phosphate-buffered saline solution, the slides were mounted in 50% glycerol in buffer at pH 7’8 and examined under a Zeiss ultraviolet photomicroscope, using an HBO-200 Hg-vapour bulb, BG 38 and BG 3 exciter filters, and 440 and 470 m[L barrier filters. Fig. 1
REFERENCES F. (1962) in Vitamin B12 und Intrinsic Factor; p. 628. Stuttgart. Chesterman, D. C., Cuthbertson, W. F. J., Pegler, H. F. (1951) Biochem. J.
Adams, J.
48,11.
Glass,
G. B. J., Skeggs, H. R., Lee, D. H., Jones, E. L., Hardy, W. W. (1962) Vitamin B12 und Intrinsic Factor; p. 673. Stuttgart. Gough, J., Israëls, M. C. G., Bottomley, A. G. (1964) Lancet, ii, 1311. Heinrich, H. C., Gabbe, E. E. (1961) Arzneimittel-Forsch. 11, 1087. Hertz, H., Kristensen, H. P. Østergaard, Hoff-Jørgensen, E. (1964) Scand. J.Hœmat. 1, 5. Kristensen, K., Hansen, T. (1966) J. pharm. Sci. (in the press). Lang, C., Harte, R. A., Conley, C. L., Chow, B. F. (1952) J. Nutr. 46, 215. Meulengracht, E. (1963) Lancet, i, 354. Schwartz, M., Bastrup-Madsen, P., Nørregaard, S., Kristensen, K. (1962) ibid. ii, 1181. Sokoloff, M. F., Sannemann, E. H., Jr., Beard, M. F. (1952) Blood, 7, 243.
Fig. 1-Specific cytoplasmic fluorescence is visible cells but not in adrenal capsule.
in adrenocortical
Gordon 1951, McFadzean and Choa 1953). It is most SERUM-VITAMIN B12 other infecinfection salmonella that and, perhaps, likely DURING MAINTENANCE THERAPY IN tions in people with SS or SC genotype predispose them PERNICIOUS ANÆMIA WITH A to much greater haemolysis and, therefore, to a much more DEPOT PREPARATION OF VITAMIN B12 severe crisis. Pyrexia from any cause can increase the rate of red blood-cell destruction and the rate of sickling P. BASTRUP-MADSEN S. NØRREGAARD M.D. Copenhagen M.D. Copenhagen (Basu et al. 1963). Although jaundice is thought to be hxmolytic it would M. SCHWARTZ T. HANSEN seem from the first case that an obstructive element may M.D. Copenhagen Cand. Pharm. Copenhagen have been present. This might be due to swelling of the E. MEULENGRACHT liver cells as a result of the infection. M.D. Copenhagen, L.L.D. Fullerton and Watson-Williams (1962) and Edington From the University Department of Medicine of Aarhus Amts(1963) thought that folic-acid deficiency was the com- sygehus, Aarhus, Medical Department B, Frederiksberg monest factor precipitating crises in sickle-cell disease. Hospital, Copenhagen, Medical Department F, Glostrup The role of folic-acid deficiency in the precipitation of Hospital, Copenhagen, and the Research Laboratory, Dumex Ltd., the crises was not investigated in our three patients but it Copenhagen is unlikely that there was any significant deficiency. PosTHERE are two reasons for using long-acting vitamin-B12 sibly, however, in addition to an antibiotic these patients preparations in pernicious anasmia. In a life-long therapy should be given folic acid in the usual dose of 15-30 mg. it is an advantage for the patient to have as few injections daily in divided doses. as possible, and long-acting preparations may produce a The hip-joint finds in the first case are interesting. The more rapid and complete replenishment of the tissue dislocation of the hip while the patient was still confined stores. Since a very large proportion of an injected to bed was thought to be due to an infective arthritis with amount of aqueous cyanocobalamin is excreted in the softening of the ligaments and consequent displacement urine (Chesterman et al. 1951, Lang et al. 1952, Sokoloff of the head of the femur. Cockshott (1961), Barton and et al. 1952, Heinrich and Gabbe 1961, Adams 1962) it Cockshott (1962), and Middlemiss (1964) reviewed the may be doubted whether treatment with conventional X-ray appearances in SS and SC disease but did not doses can build up a tissue store of 4 mg. vitamin B12 mention dislocation of the hip or of other joints. It seems which is believed to be the normal content. that this is the first reported case. Infarction of the head Hydroxocobalamin (Glass et al. 1962) is more suitable and neck of the femur is seen not infrequently in SC than cyanocobalamin because it is more readily bound to disease and to a lesser extent in SS disease. We feel that the serum-proteins (Hertz et al. 1964); the loss in the the destruction of the head and neck of the femur some urine is reduced to about 25%. weeks after the dislocation of the hip-joint was, in part at We present our results with a preparation of vitamin least, due to infarction. It is difficult, however, to exclude B12 which is more long-acting than hydroxocobalamin. the role of the infective arthritis in the pathogenesis. This preparation is a suspension of a cyanocobalaminPrognosis is said to be much brighter in SC disease tannin complex in a sesame oil/aluminium monostearate than in SS disease. This is certainly true for patients gel 0 7Biz-T.A.M/, ’Betolvex ’). With this preparation under 10, since the manifestations of SC disease do not it is possible to ensure a continuous release of a suitable usually appear before the second decade. One of the amount of the vitamin into the body, resulting eventually lessons to be learned from these 3 cases is that the gener- in complete replenishment, at the same time meeting the ally complacent attitude to SC disease in non-pregnant daily requirements. The depot effect has made it possible adults is not wholly justifiable. It seems that the same to increase the intervals between the injections to two or precautionary measures taken in SS disease should be three months. Even though preliminary results have been taken in SC disease-viz., all patients should receive folic favourable (Schwartz et al. 1962, Meulengracht 1963, acid, 5-10 mg. daily and antimalarial drugs in the usual Gough et al. 1964), conclusive evidence of the value of this compound cannot be established until its ability to prophylactic dosage. maintain clinical remission and a high level of vitamin Bl2 Summary in the blood has been demonstrated in a sufficiently large 3 cases of severe crises with jaundice in sickle-cell of patients treated for a long time. Our investigahasmoglobin-C disease in young non-pregnant Nigerian number tion is an attempt to provide such information. adults are described. The role of salmonella infection in the precipitation of crises is emphasised. Cases of severe crisis should probably be treated with chloramphenicol even before the results of the blood, stool, and urine cultures are known. The normal precautionary measures taken in SS disease should be extended to SC diseases: all patients should be given folic acid and antimalarial drugs. We thank the department of pathology for the necropsy reports and Prof. W. P. Cockshott for radiological reports and his interest in this paper. We are also grateful to the department of haematology for determining the genotype of the patients and to Mr. D. Simmonds and Mr. F. Speed for preparing the illustrations. Requests for reprints should be addressed to Prof. G. Onuaguluchi, Department of Pharmacology and Therapeutics, University of
Ibadan, Nigeria. References
at
foot of next column
Material and Methods Our series comprises 169 patients with addisonian pernicious anaemia, in all of whom haematological and neurological PROF.
ONUAGULUCHI,
DR. AKANDE: REFERENCES
Barton, C. J., Cockshott, W. P. (1962) Am. J. Reontogenol, 88, 523. Basu, A. K., Woodruff, A. W., Pettitt, L. E. (1963) Lancet, ii, 1088. Cockshott, W. P. (1961) in Tropical Radiology (edited by J. H. Middlemiss); p. 75. London. Edington, G. M. (1957) J. clin. Path. 10, 182. (1963) Ghana med. J. 2, 83. Fullerton, W. T., Watson-Williams, E. J. (1962) J. Obst. Gynœ. Br. Commonw. 69, 729. Hendrickse, R. G., Collard, P. (1960) Lancet, i, 80. Hook, E. W., Campbell, C. G., Wreens, H. S., Cooper, G. R. (1957) New Engl. J. Med. 257, 403. McFadzean, A. J. S., Choa, G. H. (1953) Br. med. J. ii, 360. Middlemiss, H. (1964) Trans. R. Soc. trop. Med. Hyg. 58, 197. Raper, A. B. (1956) Br. med. J. i, 965. Shaw, A. B. (1951) Lancet, ii, 813. Smith-Gordon, C. E. (1951) ibid. p. 1020. Tuttle, A. H., Kock, B. (1960) J. Pediat. 56, 331. —
740 values. Betolvex was administered by deep intramuscular injections of 1 ml. containing 1 mg.
cyanocobalamin.
Results Haematological and neurological remission was maintained in all patients: no fresh neurological manifestations developed during the treatment. No local or general
Fig. 1—Serum-vitamin-Bj levels in 127 patients with pernicious anaemia during maintenance therapy with 1 mg. betolvex every second month.
The ordinates are shown on a logarithmic scale. The the course of the arithmetic mean.
curve
shows
examinations and determinations of serum-vitamin-Bl2 levels continued for two to four years. The vitamin-B,2 analyses were carried out by the Lactobacillus leichmannii method. Blood-samples were taken immediately before each injection; vitamin-B,,levels in these samples represented the lowest
were
side-effects were observed. The serum-vitamin-B12 levels in 127 patients treated with betolvex 1 ml. (containing 1 mg. vitamin B12) at intervals of two months are shown in fig. 1. The period of observation for these patients was two to three and a half years. A few patients were observed for four years and in these the serum-vitamin-B12 levels remained at the same level as after three and a half years. Since previouslv untreated and treated patients are included in this series, the initial values on the graph include both subnormal and normal values. The curve represents average values. Levels increased initially but then levelled off, and the curve became almost horizontal. Most patients had serum-vitamin-B12 concentrations at the upper limit of the normal range; a few reached levels above normal (average 800 pg. per ml.).
Fig. 2 shows the serum-vitamin-B12 levels in 42 patients who received 1 ml. betolvex at intervals of three months. This treatment caused an increase in serum-vitamin-B12 during the first year to almost the same level as did injections every second month. Some of the patients had previously been treated with oral preparations or injections of aqueous cyanocobalamin. In these, the serum-vitamin-Bl2 levels did not differ from those of patients treated with betolvex only.
Fig. 2-Serum-vitamin-B" levels in 42 patients with pernicious anaemia during maintenance therapy with 1 mg. betolvex every
To investigate how a higher initial dosage might effect the serum-vitamin-B12 level, 10 patients were given, during the first month, 4 ml. betolvex divided into two or four injections (fig. 3). Their serum-vitamin-B12 levels were compared with those of 10 patients who from the beginning of treatment had received only 1 ml. betolvex every second month (fig. 4). The high serum-vitamin-B12 levels were reached in about four months after the larger initial doses, compared with the approximately twelve months with the lower dosage.
third month.
Fig. 3—Serum-vitamin-B, levels in 10 patients with pernicious anaemia receiving as initial therapy 4 mg. betolvex during the first month and then maintenance second month.
therapy with
1 mg. every
Fig. 4-Serum-vitamin-B" levels in 10 patients with pernicious anaemia who from the beginning of treatment received only 1 mg. betolvex every second month.
741
Discussion
A suspension cyanocobalamin-tannin complex in a sesame-oil/aluminium-monostearate gel differs from other preparations of vitamin-Bl2 in that liberation of the vitamin from the intramuscular injection site is extremely protracted and the loss of the vitamin in the urine is only of
a
slight. In our series the serum-vitamin-B12 values after injection of betolvex were much higher than those reported for any other form of treatment. The high levels of the vitamin were maintained throughout the observation period of two to four years by administering 1 ml. betolvex as much as three months apart. Vitamin B12may be liberated from the intramuscular depot in a non-cyanide form that is, perhaps, more readily bound to the plasma-proteins (as with the form of vitamin B12 that appears in the plasma after injection of hydroxocobalamin). The protein-bound state of the vitamin formed from both cyanocobalamin or hydroxocobalamin is equally physiologically active because either form can be assumed to be converted ultimately into the active coenzyme. Betolvex is thus acting as a depot preparation in two ways: first, by prolonged continuous liberation of the vitamin from the injection site and secondly by bringing about a continued high concentration in the plasma, the protein-bound vitamin itself acting as a depot. A few of our patients reached unusually high-serumvitamin B12 levels of several thousand pg. per ml. We did not see any side-effects of these high levels, nor were any expected, for vitamin B12 is thought to be non-toxic. No local reactions after intramuscular injection were noted. The action of betolvex is so protracted that an injection of 1 ml. every third month will supply the body with adequate amounts of the vitamin throughout this period. Thus, the depot effect far exceeds that of hydroxocobalamin and that of another depot preparation, ’Depinar ’ (Kristensen and Hansen 1966).
Summary The value of the
depot preparation ’Betolvex ’ (cyanoa sesame-oil/ in the maintenance aluminium-monostearate gel) therapy of pernicious ansemia was investigated in 169 patients for
cobalamin-tannin complex suspended in
four years. When 1 mg. vitamin B12 was given in this form intramuscularly every second or third month, the serumvitamin-B12 values gradually increased to a constant level of about 800 pg. per ml. When 4 mg. vitamin B12 were given during the first month of therapy, the high constant level was reached earlier. This level, which seems to indicate that the body stores had been replenished, was maintained throughout the observation period. Clinical remission was also maintained and there were no side-effects. two to
CUSHING’S SYNDROME WITH ADRENAL MEDULLARY INSUFFICIENCY AND ADRENAL AUTOANTIBODIES LAURENCE C. WEGIENKA M.D. Michigan LOUIS E. KOMARMY M.D. California
KIRK D. WUEPPER M.D.
Michigan PETER H. FORSHAM M.A. Cantab, M.D. Harvard
From the Metabolic Research Unit and Departments of Medicine (Division of Dermatology) and Pathology, University of California School of Medicine, San Francisco
ANTIBODIES to adrenocortical-cell cytoplasm have been found in human sera (Anderson et al. 1957, Blizzard et al. 1962, Irvine 1963). The most sensitive method for detecting adrenal autoantibodies is by immunofluorescence. By this method Blizzard and Kyle (1963) found adrenal antibodies in the sera of 51 % of 71 patients with Addison’s disease. Adrenal antibodies were not demonstrated in the sera of 7 patients with Cushing’s syndrome due to bilateral adrenocortical hyperplasia (5 cases) or carcinoma (2 cases) or in patients with virilising adrenal hyperplasia
(20 cases). We describe here a patient with Cushing’s syndrome due to adrenal hyperplasia with impaired ability to secrete epinephrine from the adrenal medulla, evidence of prominent mononuclear-cell infiltration into the medullary tissue, and associated circulating adrenal and thyroid antibodies. Materials and Methods Fresh and stored sera from 10 patients with Cushing’s syndrome due to bilateral adrenocortical hyperplasia or tumour were kept at - 20° C until use. Tissue autoantibodies were detected by the methods of Holborow et al. (1959) for thyroid antibodies, Taylor et al. (1962) for gastric antibodies, and Blizzard et al. (1962) for adrenal antibodies: all methods were modified so that all three tissue slices could be examined simultaneously on a single miscrocope slide. The tissue was allowed to react with patient’s serum for 30 minutes at room temperature, and the slices were washed three times in phosphate-buffered saline solution (pH 7-2). Rabbit antisera, conjugated with fluorescein isothiocyanate by the method of McKinney et al. (1964), was allowed to react with the slices for 30 minutes. After three more washes in phosphate-buffered saline solution, the slides were mounted in 50% glycerol in buffer at pH 7’8 and examined under a Zeiss ultraviolet photomicroscope, using an HBO-200 Hg-vapour bulb, BG 38 and BG 3 exciter filters, and 440 and 470 m[L barrier filters. Fig. 1
REFERENCES F. (1962) in Vitamin B12 und Intrinsic Factor; p. 628. Stuttgart. Chesterman, D. C., Cuthbertson, W. F. J., Pegler, H. F. (1951) Biochem. J.
Adams, J.
48,11.
Glass,
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Fig. 1-Specific cytoplasmic fluorescence is visible cells but not in adrenal capsule.
in adrenocortical