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DETECTION OF FORMIMINOGLUTAMIC ACID IN URINE WITHOUT HISTIDINE LOADING
359 and to enhance glycogen synthesis by heart muscle.It is circulating N.E.F.A. levels folanticipated that the depressed lowing a fructose infusion 10 would be association with increased glucose oxidation and a decreased rate of glycogen storage in muscle. Another consequence of the lowered N.E.F.A. levels would be the deprivation of heart and skeletal muscle of a major substrate for oxidative metabolism." The role of fructose in muscle metabolism is still open to speculation, but there is little experimental evidence to suggest that fructose itself would have any beneficial Such or harmful influence in McArdle’s syndrome. of of the the result are more effects likely products hepatic metabolism of fructose, such as pyruvate or lactate. LIONEL H. OPIE Department of Medicine, JOHN R. EVANS. University of Toronto. Baker Clinic Research Laboratory, ALBERT E. RENOLD. Harvard Medical School.
DETECTION OF FORMIMINOGLUTAMIC ACID IN URINE WITHOUT HISTIDINE LOADING SIR,-We have received a number of requests for fuller information on our method 12 for detecting formiminoglutamic acid without histidine loading. These are the details of the method which we are now using with success. A paper on our findings is being prepared for
publication. Reagents 1. Chloramine T 7-5% chloramine T prepared immediately before use, the solid to be stored in a cool place protected from light and kept dry. Batches vary: our best results have been with the chemical supplied by Hopkin and Williams Ltd. 2. Citric buffer pH 4,7 at 18’C Citric acid up to 500 ml. with NazHPO 13-639g.j distilled water. 3. Ninhydrin 200 mg. ninhydrin 6 ml. of ethanol 94 ml. of diethylether. 4. Electrophoretic buffer pH 5,4 20 ml. glacial acetic acid made up to 4 litres with 50 ml. pyridine distilled water. 5. Zeo-Carb 225 Preparation and regeneration of resin see Chromatographic Techniques by Ivor Smith.13 Test A random specimen of urine is collected, preserved at a pH of 2-3 and stored at 4°C with a crystal of thymol. For testing, dilute the urine to a creatinine 14 concentration of 0-5 g. per litre. Minimum dilution 1 in 2, regardless of creatinine
10’718g.made
level. 1. Removal of Glutamic Acid 15 16 To 10 ml. of diluted urine add 2N KOH until pH is between 4 and 5 by indicator paper. 2 ml. of 7.5% chloramine T (freshly prepared) are added and the mixture thoroughly shaken. Readjust the pH with 2N HCL until the pH again is between 4 and 5. 4 ml. of citric buffer is added and the mixture shaken well. Incubate at 40°C for 30 min. with vigorous shaking at 4-minute intervals. Keep at 4°C for at least 30 min. or overnight. This allows the precipitation of the unused chloramine T and the reaction product, toluene sulphonamide. 2. Ion-exchange A column of resin 12 x1 cm. is prepared in a 25 ml. burette and washed with distilled water. The urine mixture is filtered directly onto the column through a Whatman no. 1 11 cm. paper and run through the resin at approximately 0-5 ml. per min. until the resin is only just covered. Then wash the column thoroughly with 50-75 ml. of distilled water at 2 ml. per min. When column is only just covered add about 15 ml. of concentrated ammonia and elute the 9.
Shipp, J. C., Opie, 1018.
10. 11. 12. 13. 14. 15. 16.
L.
H., Challoner, D. C. Nature, Lond. 1961, 189,
Stuhlfauth, K., Zollner, N. Klin. Wschr. 1959, 37, 1162. Fritz, I. B. Phys. Rev, 1961, 41, 52. Lewis, F. J. W., Moore, G. R. Lancet, 1962, i, 305. Smith, I. Chromatographic Techniques 52-53, London, 1958. Bonsnes, R. W., Taussky, R. H., J. Biol. Chem. 1945, 158, 581. Dakin, H. D. Biochem. J. 1917, 11, 79. Cohen, P. P. ibid. 1939, 33, 551.
aminoacids at a rate of 0’5 ml. per min. The moment the ammonia appears through the column (tested with pH paper) collect the next 10 ml. into an evaporating basin. 3. Concemration The evaporating basin is placed in a current of hot air (a hairdrycr has been found suitable) until the volume is reduced in consecutive sequence to represent the following concentrations: 5 g. of creatinine per litre. 10 g. " " " 25 g. 50 g.
"
33 33
"
"
"
I
These, for example, with the urine diluted to contain 0-5 g. of creatinine per litre, will be volumes of 1 ml., 0-5 ml., 0-2 ml., and 0-1 ml.
respectively, and will be referred to below as the g. creatinine concentration. Approximately 0-005 ml. from each is taken into capillary pipettes for clectrophoresis. 4. Electrophoresis This is as published by Kohn and Mollin " except that we now use paper (Whatman no. 1) instead of cellulose-acetate strips. After dipping the strip in the ninhydrin incubate at 100°C±5"C for 11/x minutes. Longer periods of heating will produce background colours. 5. Control of Method A urine from a normal subject is made alkaline with 2N KOH (pH 10) arid incubated at 40’C for 24 hours to destroy any existing F.I.G.L.U. Acidify and add L-glutamic acid to a concentration of 2000 jjLg per ml. This may be stored at 4°C and used when required. A sample is put up with each batch of tests and treated in the same way.
No spot at all should be visible in the glutamic acid region at a concentration equivalent to 50 g. creatinine per litre-if a spot does appear the tests are repeated using fresh chloramine T from a new batch. At intervals positive controls of urines of known titre are also put up.
Results Visible spots in the glutamic-acid region with the 25 g. creatinine concentration are regarded as positive. Of our 140 pregnancy cases which were selected arbitrarily to include every suspected folic-acid-deficiency anaemia, those proven to be megaloblastic have all been positive at the 10 g. creatinine concentration, many of them at the 5 g. creatinine concentration; approximately two-thirds of the twin pregnancies and half the remainder have been positive in the third trimester. Almost all our 60 normal controls have been negative at the 50 g. creatinine concentration, one, however, a prenursing candidate whom we are unable to follow up, was positive at the 25 g. creatinine concentration. One of the theoretical advantages of the method is that there is no artificially produced disturbance of metabolic balance such as might conceivably happen after histidine loading and which might result in a false positive. In this connection, it is of interest that 6 out of 7 cases of pernicious anaemia were negative, the positive (25 g. creatinine concentration) being a man of 77 who had also developed a severe infection. If, after a significant number of investigations, a satisfactory normal baseline can be established, it may be possible to devise a simple histidine-tolerance test which could demonstrate abnormalities not demonstrable by the simple urinary excretion of F.I.G.L.U. alone, even after concentration; bearing in mind that the quantitation is only approximate and the specificity of the test not established. Empirically the clinical agreement has so far been remarkable. F. J. W. LEWIS Department of Pathology, Southmead Hospital, Bristol.
G. R. MOORE.
FLUOCINOLONE ACETONIDE IN THE TREATMENT OF CHRONIC DISCOID LUPUS ERYTHEMATOSUS SIR,-A patient with discoid lupus erythematosus of ten years’ duration responded surprisingly well to the local application of fluocinolone as ’Synalar ’ cream. We have therefore investigated the results of such treatment in a small series of cases. Fluocinolone acetonide is a relatively new corticosteroid. Its
pharmacology and the clinical effect of its topical application
17.
Kohn, J., Mollin, D. L., Rosenbach, L. M. J. clin. Path. 1961, 14,
345.
DETECTION OF FORMIMINOGLUTAMIC ACID IN URINE WITHOUT HISTIDINE LOADING SIR,-We have received a number of requests for fuller information on our method 12 for detecting formiminoglutamic acid without histidine loading. These are the details of the method which we are now using with success. A paper on our findings is being prepared for
publication. Reagents 1. Chloramine T 7-5% chloramine T prepared immediately before use, the solid to be stored in a cool place protected from light and kept dry. Batches vary: our best results have been with the chemical supplied by Hopkin and Williams Ltd. 2. Citric buffer pH 4,7 at 18’C Citric acid up to 500 ml. with NazHPO 13-639g.j distilled water. 3. Ninhydrin 200 mg. ninhydrin 6 ml. of ethanol 94 ml. of diethylether. 4. Electrophoretic buffer pH 5,4 20 ml. glacial acetic acid made up to 4 litres with 50 ml. pyridine distilled water. 5. Zeo-Carb 225 Preparation and regeneration of resin see Chromatographic Techniques by Ivor Smith.13 Test A random specimen of urine is collected, preserved at a pH of 2-3 and stored at 4°C with a crystal of thymol. For testing, dilute the urine to a creatinine 14 concentration of 0-5 g. per litre. Minimum dilution 1 in 2, regardless of creatinine
10’718g.made
level. 1. Removal of Glutamic Acid 15 16 To 10 ml. of diluted urine add 2N KOH until pH is between 4 and 5 by indicator paper. 2 ml. of 7.5% chloramine T (freshly prepared) are added and the mixture thoroughly shaken. Readjust the pH with 2N HCL until the pH again is between 4 and 5. 4 ml. of citric buffer is added and the mixture shaken well. Incubate at 40°C for 30 min. with vigorous shaking at 4-minute intervals. Keep at 4°C for at least 30 min. or overnight. This allows the precipitation of the unused chloramine T and the reaction product, toluene sulphonamide. 2. Ion-exchange A column of resin 12 x1 cm. is prepared in a 25 ml. burette and washed with distilled water. The urine mixture is filtered directly onto the column through a Whatman no. 1 11 cm. paper and run through the resin at approximately 0-5 ml. per min. until the resin is only just covered. Then wash the column thoroughly with 50-75 ml. of distilled water at 2 ml. per min. When column is only just covered add about 15 ml. of concentrated ammonia and elute the 9.
Shipp, J. C., Opie, 1018.
10. 11. 12. 13. 14. 15. 16.
L.
H., Challoner, D. C. Nature, Lond. 1961, 189,
Stuhlfauth, K., Zollner, N. Klin. Wschr. 1959, 37, 1162. Fritz, I. B. Phys. Rev, 1961, 41, 52. Lewis, F. J. W., Moore, G. R. Lancet, 1962, i, 305. Smith, I. Chromatographic Techniques 52-53, London, 1958. Bonsnes, R. W., Taussky, R. H., J. Biol. Chem. 1945, 158, 581. Dakin, H. D. Biochem. J. 1917, 11, 79. Cohen, P. P. ibid. 1939, 33, 551.
aminoacids at a rate of 0’5 ml. per min. The moment the ammonia appears through the column (tested with pH paper) collect the next 10 ml. into an evaporating basin. 3. Concemration The evaporating basin is placed in a current of hot air (a hairdrycr has been found suitable) until the volume is reduced in consecutive sequence to represent the following concentrations: 5 g. of creatinine per litre. 10 g. " " " 25 g. 50 g.
"
33 33
"
"
"
I
These, for example, with the urine diluted to contain 0-5 g. of creatinine per litre, will be volumes of 1 ml., 0-5 ml., 0-2 ml., and 0-1 ml.
respectively, and will be referred to below as the g. creatinine concentration. Approximately 0-005 ml. from each is taken into capillary pipettes for clectrophoresis. 4. Electrophoresis This is as published by Kohn and Mollin " except that we now use paper (Whatman no. 1) instead of cellulose-acetate strips. After dipping the strip in the ninhydrin incubate at 100°C±5"C for 11/x minutes. Longer periods of heating will produce background colours. 5. Control of Method A urine from a normal subject is made alkaline with 2N KOH (pH 10) arid incubated at 40’C for 24 hours to destroy any existing F.I.G.L.U. Acidify and add L-glutamic acid to a concentration of 2000 jjLg per ml. This may be stored at 4°C and used when required. A sample is put up with each batch of tests and treated in the same way.
No spot at all should be visible in the glutamic acid region at a concentration equivalent to 50 g. creatinine per litre-if a spot does appear the tests are repeated using fresh chloramine T from a new batch. At intervals positive controls of urines of known titre are also put up.
Results Visible spots in the glutamic-acid region with the 25 g. creatinine concentration are regarded as positive. Of our 140 pregnancy cases which were selected arbitrarily to include every suspected folic-acid-deficiency anaemia, those proven to be megaloblastic have all been positive at the 10 g. creatinine concentration, many of them at the 5 g. creatinine concentration; approximately two-thirds of the twin pregnancies and half the remainder have been positive in the third trimester. Almost all our 60 normal controls have been negative at the 50 g. creatinine concentration, one, however, a prenursing candidate whom we are unable to follow up, was positive at the 25 g. creatinine concentration. One of the theoretical advantages of the method is that there is no artificially produced disturbance of metabolic balance such as might conceivably happen after histidine loading and which might result in a false positive. In this connection, it is of interest that 6 out of 7 cases of pernicious anaemia were negative, the positive (25 g. creatinine concentration) being a man of 77 who had also developed a severe infection. If, after a significant number of investigations, a satisfactory normal baseline can be established, it may be possible to devise a simple histidine-tolerance test which could demonstrate abnormalities not demonstrable by the simple urinary excretion of F.I.G.L.U. alone, even after concentration; bearing in mind that the quantitation is only approximate and the specificity of the test not established. Empirically the clinical agreement has so far been remarkable. F. J. W. LEWIS Department of Pathology, Southmead Hospital, Bristol.
G. R. MOORE.
FLUOCINOLONE ACETONIDE IN THE TREATMENT OF CHRONIC DISCOID LUPUS ERYTHEMATOSUS SIR,-A patient with discoid lupus erythematosus of ten years’ duration responded surprisingly well to the local application of fluocinolone as ’Synalar ’ cream. We have therefore investigated the results of such treatment in a small series of cases. Fluocinolone acetonide is a relatively new corticosteroid. Its
pharmacology and the clinical effect of its topical application
17.
Kohn, J., Mollin, D. L., Rosenbach, L. M. J. clin. Path. 1961, 14,
345.