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VITAMIN-B12 ASSAY
862 disease conditions, but far more work must be done before any positive assertions can be made. On one further point we should like to take issue with Dr. Fleming. His letter refers to " heat-labile " and " stable " forms of serum-vitamin-Bis, his " labile " form apparently corresponding to the difference between vitamin Bla extractable without added cyanide and total vitamin B12 (i.e., Smith’s
Obituary
some
"
hydroxocobalamin ").6It was shown some years ago that no destroyed if serum is extracted by heating with acetate buffer, at pH about 4-5, in the absence of cyanide. Though the assay result is low if added cyanide is absent, this is due to some of the vitamin B12 remaining in association with the precipitate of serum proteins-not to heat lability.78 Even if extraction is by autoclaving with buffer or assay medium (which was not used in the method8 used by Smith and ourselves) it is most unlikely that appreciable vitamin B12 is destroyed. We mention this in case an already confusing subject should become further confused through misunderstandings about what happens in the relevant extraction procedure.
vitamin B12 is
Department of Chemical Pathology, Westminster Medical School, London. M.R.C. Clinical Genetics Research Unit at the Institute of Neurology, the National Hospital,
D. M. MATTHEWS.
Queen Square, London, and the Institute of Child Health, Guilford Street, London.
J. WILSON.
VITAMIN-B12 ASSAY SIR,-In recent months there has been in your columns expression of a lively interest in the assay of plasma vitamin B12 by radioisotope dilution. My colleagues and I have tried two such methods extensively and found them to give either erratic or falsely low results. We are evaluating several modifications and the results of others.
at
least
one
of them may be useful
to
The basis for all published methods is the competition bevitamin B12 in the solution being assayed and a known amount of 5’Co-labelled vitamin B12 for the vitamin-B12-binding sites on a protein such as intrinsic factor. The procedure cannot work unless, as is assumed, the affinities of the proteins in the system are the same for the plasma vitamin B12 and the 57Co-labelled vitamin. Not only is this assumption unsupported but there is evidence to the contrary. The vitamin B12 in the processed plasma is in an unknown form, while the 57COlabelled vitamin B12 is cyanocobalamin. If the plasma vitamin B12 is hydroxocobalamin it is more reactive with several proteins than is cyanocobalamin. My colleagues and I have therefore added a trace of cyanide to the reaction in order to convert all vitamin B12 to cyanocobalamin-the form of the 57COlabelled vitamin.
tween
The basic procedure is that of Rothenberg 9 modified by: (1) removal of plasma-proteins after the vitamin B12 has been released from them; (2) preparation of the standard curve in vitamin-B12-free and protein-free plasma; (3) the use of cyanide. The mean serum vitamin B12 of 27 geriatric patients and normal patients was 216 pg. per ml., as compared with 221 pg. per ml. by Euglena gracilis assay. 26 of these sera were also assayed by the same isotope-dilution method, but without cyanide, and the mean vitamin-B12 concentration was 85 pg. per ml. The use of cyanide gave higher values in 24 out of 26 patients, with an average increase of 137 pg. per ml. The results of these preliminary studies suggest a further trial of cyanide in this and other methods of vitamin-B12 assay based on similar principles. Albany Medical College of Union University, CHARLES A. HALL. Albany, New York. 6. 7. 8. 9.
Smith, A. D. M. Lancet, 1961, i, 1001. Matthews, D. M. ibid. p. 1289. Matthews, D. M. Clin. Sci. 1962, 22, 101. Rothenberg, S. P. Proc. Soc. exp. Biol. Med. 1961, 108, 45.
GORDON ROY CAMERON
Kt., M.B., D.Sc., LL.D. Melb., LL.D. Edin., P.C.Path., F.R.C.P., F.R.S. Sir Roy Cameron, emeritus professor of morbid anatomy in the University of London at University College Hospital, died on Oct. 7 at the age of 67. He was the first president of the College of Pathologists. to
He was the son of a methodist minister who had emigrated Australia from Aberdeen, and he was educated in Victoria
village school, Kyneton High School, and the University of Melbourne, where he graduated in medicine in 1922. Among his conat a
temporaries
at
Melbourne
were
Sir Macfarlane Burnet and Prof. Rupert Willis, who have remained valued friends throughout his life. At that time S. W. Patterson was the director of the Walter and Eliza Hall Institute in Melbourne, which had been founded a few years earlier. He helped Cameron to carry out his first piece of research as a house-physician and encouraged him to perform necropsies in his spare time. At the end of the year in this appointment Cameron was made Stewart lecturer in pathology to Sir Harry Allen, who held the chair at Melbourne University. From then Cameron’s allegiance to pathology never faltered. Charles Kellaway arrived in Melbourne at the end of 1923 to become director of the Walter and Eliza Hall Institute. He was a pathologist who was an outstanding experimentalist, and under his influence Cameron became convinced that this was the way in which pathology would develop in the future. He continued to do necropsies, however, and in 1925 he was appointed morbid anatomist and deputy director of the Institute.
In 1928 he left for what was intended to be a few years in Europe-with Aschoff at Freiburg and with Boycott in London at University College Hospital Medical School. But the world financial depression had among other curious results the effect of restricting development of the Institute in Melbourne, and Cameron stayed on at U.C.H. as Graham scholar, Beit fellow, reader, and, from 1937, professor of morbid anatomy. In these posts he played his part in teaching and routine morbid anatomy, but his heart was in experimental investigations. If the response of the liver to injury was the centre of these, his interest was nevertheless wide, and during the war years, while attached as pathologist to Barcroft’s research team at Porton, the problem of pulmonary oedema was perhaps paramount, and his Sidney Ringer lecture in 1947 dealt with this problem. After his return to U.C.H. in 1945 Cameron continued work on his book, Pathology of the Cell, which was published in 1952, while organising experimental investigations, as well as being responsible for teaching and the routine hospital service. In the autumn of 1956 he suffered his first severe illness, and after this he curtailed his work to some extent, but his courage in pursuing research even after a second severe illness in the first part of 1963 was remarkable.
During these years he was also full of committee responsibilities. He was a member of the Agricultural Research Council (1948-56) and the Medical Research Council (1952-56); he was secretary of the Beit fellowships for Medical Research (1959-64) and chairman of the zoological committee fOI the Nuffield Institute of Comparative Medicine (1960-64),to name but a few of his charges. He was made F.R.C.P. in 1941 and F.R.S. in 1946, and he felt that the award of a Royal Medal in 1960 was a recognition not only of his own work, but
Obituary
some
"
hydroxocobalamin ").6It was shown some years ago that no destroyed if serum is extracted by heating with acetate buffer, at pH about 4-5, in the absence of cyanide. Though the assay result is low if added cyanide is absent, this is due to some of the vitamin B12 remaining in association with the precipitate of serum proteins-not to heat lability.78 Even if extraction is by autoclaving with buffer or assay medium (which was not used in the method8 used by Smith and ourselves) it is most unlikely that appreciable vitamin B12 is destroyed. We mention this in case an already confusing subject should become further confused through misunderstandings about what happens in the relevant extraction procedure.
vitamin B12 is
Department of Chemical Pathology, Westminster Medical School, London. M.R.C. Clinical Genetics Research Unit at the Institute of Neurology, the National Hospital,
D. M. MATTHEWS.
Queen Square, London, and the Institute of Child Health, Guilford Street, London.
J. WILSON.
VITAMIN-B12 ASSAY SIR,-In recent months there has been in your columns expression of a lively interest in the assay of plasma vitamin B12 by radioisotope dilution. My colleagues and I have tried two such methods extensively and found them to give either erratic or falsely low results. We are evaluating several modifications and the results of others.
at
least
one
of them may be useful
to
The basis for all published methods is the competition bevitamin B12 in the solution being assayed and a known amount of 5’Co-labelled vitamin B12 for the vitamin-B12-binding sites on a protein such as intrinsic factor. The procedure cannot work unless, as is assumed, the affinities of the proteins in the system are the same for the plasma vitamin B12 and the 57Co-labelled vitamin. Not only is this assumption unsupported but there is evidence to the contrary. The vitamin B12 in the processed plasma is in an unknown form, while the 57COlabelled vitamin B12 is cyanocobalamin. If the plasma vitamin B12 is hydroxocobalamin it is more reactive with several proteins than is cyanocobalamin. My colleagues and I have therefore added a trace of cyanide to the reaction in order to convert all vitamin B12 to cyanocobalamin-the form of the 57COlabelled vitamin.
tween
The basic procedure is that of Rothenberg 9 modified by: (1) removal of plasma-proteins after the vitamin B12 has been released from them; (2) preparation of the standard curve in vitamin-B12-free and protein-free plasma; (3) the use of cyanide. The mean serum vitamin B12 of 27 geriatric patients and normal patients was 216 pg. per ml., as compared with 221 pg. per ml. by Euglena gracilis assay. 26 of these sera were also assayed by the same isotope-dilution method, but without cyanide, and the mean vitamin-B12 concentration was 85 pg. per ml. The use of cyanide gave higher values in 24 out of 26 patients, with an average increase of 137 pg. per ml. The results of these preliminary studies suggest a further trial of cyanide in this and other methods of vitamin-B12 assay based on similar principles. Albany Medical College of Union University, CHARLES A. HALL. Albany, New York. 6. 7. 8. 9.
Smith, A. D. M. Lancet, 1961, i, 1001. Matthews, D. M. ibid. p. 1289. Matthews, D. M. Clin. Sci. 1962, 22, 101. Rothenberg, S. P. Proc. Soc. exp. Biol. Med. 1961, 108, 45.
GORDON ROY CAMERON
Kt., M.B., D.Sc., LL.D. Melb., LL.D. Edin., P.C.Path., F.R.C.P., F.R.S. Sir Roy Cameron, emeritus professor of morbid anatomy in the University of London at University College Hospital, died on Oct. 7 at the age of 67. He was the first president of the College of Pathologists. to
He was the son of a methodist minister who had emigrated Australia from Aberdeen, and he was educated in Victoria
village school, Kyneton High School, and the University of Melbourne, where he graduated in medicine in 1922. Among his conat a
temporaries
at
Melbourne
were
Sir Macfarlane Burnet and Prof. Rupert Willis, who have remained valued friends throughout his life. At that time S. W. Patterson was the director of the Walter and Eliza Hall Institute in Melbourne, which had been founded a few years earlier. He helped Cameron to carry out his first piece of research as a house-physician and encouraged him to perform necropsies in his spare time. At the end of the year in this appointment Cameron was made Stewart lecturer in pathology to Sir Harry Allen, who held the chair at Melbourne University. From then Cameron’s allegiance to pathology never faltered. Charles Kellaway arrived in Melbourne at the end of 1923 to become director of the Walter and Eliza Hall Institute. He was a pathologist who was an outstanding experimentalist, and under his influence Cameron became convinced that this was the way in which pathology would develop in the future. He continued to do necropsies, however, and in 1925 he was appointed morbid anatomist and deputy director of the Institute.
In 1928 he left for what was intended to be a few years in Europe-with Aschoff at Freiburg and with Boycott in London at University College Hospital Medical School. But the world financial depression had among other curious results the effect of restricting development of the Institute in Melbourne, and Cameron stayed on at U.C.H. as Graham scholar, Beit fellow, reader, and, from 1937, professor of morbid anatomy. In these posts he played his part in teaching and routine morbid anatomy, but his heart was in experimental investigations. If the response of the liver to injury was the centre of these, his interest was nevertheless wide, and during the war years, while attached as pathologist to Barcroft’s research team at Porton, the problem of pulmonary oedema was perhaps paramount, and his Sidney Ringer lecture in 1947 dealt with this problem. After his return to U.C.H. in 1945 Cameron continued work on his book, Pathology of the Cell, which was published in 1952, while organising experimental investigations, as well as being responsible for teaching and the routine hospital service. In the autumn of 1956 he suffered his first severe illness, and after this he curtailed his work to some extent, but his courage in pursuing research even after a second severe illness in the first part of 1963 was remarkable.
During these years he was also full of committee responsibilities. He was a member of the Agricultural Research Council (1948-56) and the Medical Research Council (1952-56); he was secretary of the Beit fellowships for Medical Research (1959-64) and chairman of the zoological committee fOI the Nuffield Institute of Comparative Medicine (1960-64),to name but a few of his charges. He was made F.R.C.P. in 1941 and F.R.S. in 1946, and he felt that the award of a Royal Medal in 1960 was a recognition not only of his own work, but