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INHIBITION BY SERA OF URÆMIC PATIENTS
906
hssmatological findings in 34 patients with other chronic infections, such as pulmonary tuberculosis and pyogenic lung abscess, with those in 41 subjects in apparent good health. Our results will be reported more fully elsewhere. Mean values in the patients with chronic infection were: Hb 9-6 g. per 100 ml. (range 4-8-13-5 g. per 100 ml.), M.C.H.C. 29.4% (range 25,0-32,5%). In the controls the means were: Hb 14-9 g. per 100 ml. (range 12-0-16-8 g. per 100 ml.), M.C.H.C. 33-3% (range 31-0-35-0%). Our belief that the hypochromic anaemia which is commonly associated with chronic infection is not due to irondeficiency is borne out by our finding 24 patients out of 34 in this series (and 15 among the 31 with amoebic liver abscess) with Hb under 12-0 g. per 100 ml., and M.C.H.C. values of 30% or less. Similar findings have been reported in Seattle,4although the proportion of patients with hypochromic anaemia was lower. the
Department of Medicine, University of Natal, and
E. B. ADAMS F. G. H. MAYET.
King Edward VIII Hospital, Durban, South Africa.
UROCANIC-ACID AND FOLIC-ACID ESTIMATION SIR,-The normal metabolic degradation of histidine to glutamic acid takes place via urocanic acid (imidazoleacrylic acid), an unstable intermediary, and formininoglutamic acid (FIGLU). Bennett and Chanarinfound that in states of folicacid deficiency, after a histidine load, FIGLU is often excreted in urine, together with its precursor, urocanic acid. Later, in describing their spectrophotometric method 6 for the combined estimation of FIGLU and urocanic acid, these workers stressed that urocanic acid may comprise more than 80% of the histidine derivatives appearing in the urine of patients with folic-acid deficiency. Thus the customary estimation of FIGLU, after a histidine load, which does not include urocanic acid, may be completely misleading in trying to prove folicacid deficiency, since FIGLU may comprise as little as 3% of the total histidine derivatives excreted; particularly in longstanding folic acid deficiency, mainly urocanic acid is excreted.’We present here a simple method for the estimation of urocanic acid, based on " two-solutions electrophoresis ". A comprehensive result may be obtained within a few hours. No additional equipment is required when FIGLU is examined
by electrophoresis. Procedure Untreated urine is separated by low-voltage " two-solutions paper-electrophoresis ". Reagents and apparatus are the same as described by us for estimation of vanilmandelic acid8 3,4-dihydroxyphenyl alanine (DOPA), and dopamine.99 An aliquot of urine corresponding to 0-075 mg. creatinine is on an electrophoretic paper-strip (30x4 cm.) 7 cm. from the anode-end of the strip, and run for two hours. Urocanic acid migrates about 8 cm. towards the cathode in this time. After air-drying and heating of the strip, it is dyed with diazotised p-nitroaniline. Urocanic acid gives a purple colour. A standard of urocanic acid can be run simultaneously on a second strip, to facilitate its identification, in addition to the urine. We also use another dye procedure, which is our modification of Pauly’s sulphanilic acid reagent. One part of 0-1% sulphanilic acid in 1% HCI v/v and, one part of 0-2% sodium nitrite are mixed under cooling; after 3 minutes one part of 0-025% ammonium sulphamate, and then two parts of 10% sodium carbonate, are added. The paper strips are immediately dipped through this mixture. Urocanic acid gives a roseyellow colour, next to histidine which migrates a little further and is dyed an intense orange. When viewed, after being airdried, under ultraviolet light (Wood’s light), urocanic acid gives a bright red fluorescence.
applied
4. 5. 6. 7. 8. 9.
Bainton, D. F., Finch, C. A. Bennett, M. C., Chanarin, I. Chanarin, I., Bennett, M. C. Bennett, M. C., Chanarin, I. Eichhorn, F., Rutenberg, A. Eichhorn, F., Rutenberg, A.
Am. J. Med. 1964, 37, 62. Lancet, 1961, ii, 1095. J. 1962, i, 27. Nature, Lond. 1962, 196, 171. Clin. Chem. 1963, 9, 615. ibid. 1965, 11, 563. Br. med.
Interpretation As urocanic acid may not be increased in some cases of folicacid deficiency, a negative result should be followed by an estimation of FIGLU. In the same way, a FIGLU estimation alone would not indicate a folic-acid deficiency in those cases where only urocanic acid is increased." Part of this project was supported by a grant from Lady Edith Wolfson’s Research Fund, through the Israel Cancer Association. Rappaport Laboratories, Biochemical Section, F. EICHHORN Beilinson Hospital, A. RUTENBERG. Petah Tiqva, Israel.
INHIBITION BY SERA OF URÆMIC PATIENTS SiR,ňThe preliminary communication by Dr. Emerson and her colleagues (Sept. 18) deals with the interesting problem of the interaction between ursemic serum and enzyme activities. We 11 have investigated the glutathione-reductase (G.R.) activity of erythrocytes in 53 patients with renal insufficiency, most of them with hyperazotsemia, and we found, in contrast with reports by other workers," considerably raised enzyme activity. Mean values of G.R. activity, in units per g. of Hb, for two groups of patients with chronic and acute renal insufficiency, were respectively 86-4 units (range 43-9-140-0) and 75-1 units (range 49-3-97-0), while in a group of control subjects the mean value was 37-9 units (range 21-0-49-3). G.R. activity increases with urea concentration, but it never reaches values higher than 100-150 units. In acute renal insufficiency, the increase in G.R. activity generally follows the rise of azotaemia, but high G.R. values may be found long after the resolution of the acute episode. Moreover, plasma from ursmic patients with high erythrocytic G.R. values has the remarkable property of inducing in normal erythrocytes a sharp elevation of G.R. activity, far above the normal range, in an incubation system at 37’C constituted by washed erythrocytes from normal blood-compatible donors and urxmic plasma, with the addition of glucose as follows:
Thus, plasma from urxmic patients seems to contain a factor of stimulating erythrocyte G.R.-activity, in agreement with the report 13 of high reduced-glutathione concentrations in red blood-cells of hyperazotxmic patients. Activation seems to be due to a stable and perhaps irreversible modification of the structure of the enzyme, since it is shown after haemolysis and successive dialysis of the haemolysate. Uraemic plasma completely loses its activating power after dialysis against saline, and, almost completely, after heat denaturation at 70-100°C. Further studies are needed to clarify the kinetics of the activation, and to establish the nature of the activating factor. PAOLO BRUNETTI GIUSEPPE G. NENCI Medical Clinic of the ALBERTO PARMA. of University Perugia, Italy.
capable
LACTATE DEHYDROGENASE IN URÆMIA SIR,-We are able to confirm the preliminary communication of Dr. Emerson and her colleagues (Sept. 18) concerning the increase of serum lactate dehydrogenase (L.D.H.) which is often found immediately after haemodialysis in urxmic patients. The possible removal of an easily diffusible inhibitor, as they stress, needs due consideration. We wish to draw attention, however, to other factors which might also play a part. Slight haemolysis, undetected to the naked eye, easily induces an increase of the order of 10% in a normal total serum L.D.H.; one then expects a shift towards the two fastest L.D.H.S (L.D.H. 1 10. 11.
Knowles, J. P. Lancet, 1961, ii, 1149. Brunetti, P., Parma, A., Nenci, G. G. Hœmatologica, 1965, 50, 415. Loehr, G. W., Waller, H. D. Schweiz. med. Wschr. 1962, 92, 1. Wernze, H., Koch, W. Klin. Wschr. 1965, 43, 451.
12. Bock, H. E., 13.
hssmatological findings in 34 patients with other chronic infections, such as pulmonary tuberculosis and pyogenic lung abscess, with those in 41 subjects in apparent good health. Our results will be reported more fully elsewhere. Mean values in the patients with chronic infection were: Hb 9-6 g. per 100 ml. (range 4-8-13-5 g. per 100 ml.), M.C.H.C. 29.4% (range 25,0-32,5%). In the controls the means were: Hb 14-9 g. per 100 ml. (range 12-0-16-8 g. per 100 ml.), M.C.H.C. 33-3% (range 31-0-35-0%). Our belief that the hypochromic anaemia which is commonly associated with chronic infection is not due to irondeficiency is borne out by our finding 24 patients out of 34 in this series (and 15 among the 31 with amoebic liver abscess) with Hb under 12-0 g. per 100 ml., and M.C.H.C. values of 30% or less. Similar findings have been reported in Seattle,4although the proportion of patients with hypochromic anaemia was lower. the
Department of Medicine, University of Natal, and
E. B. ADAMS F. G. H. MAYET.
King Edward VIII Hospital, Durban, South Africa.
UROCANIC-ACID AND FOLIC-ACID ESTIMATION SIR,-The normal metabolic degradation of histidine to glutamic acid takes place via urocanic acid (imidazoleacrylic acid), an unstable intermediary, and formininoglutamic acid (FIGLU). Bennett and Chanarinfound that in states of folicacid deficiency, after a histidine load, FIGLU is often excreted in urine, together with its precursor, urocanic acid. Later, in describing their spectrophotometric method 6 for the combined estimation of FIGLU and urocanic acid, these workers stressed that urocanic acid may comprise more than 80% of the histidine derivatives appearing in the urine of patients with folic-acid deficiency. Thus the customary estimation of FIGLU, after a histidine load, which does not include urocanic acid, may be completely misleading in trying to prove folicacid deficiency, since FIGLU may comprise as little as 3% of the total histidine derivatives excreted; particularly in longstanding folic acid deficiency, mainly urocanic acid is excreted.’We present here a simple method for the estimation of urocanic acid, based on " two-solutions electrophoresis ". A comprehensive result may be obtained within a few hours. No additional equipment is required when FIGLU is examined
by electrophoresis. Procedure Untreated urine is separated by low-voltage " two-solutions paper-electrophoresis ". Reagents and apparatus are the same as described by us for estimation of vanilmandelic acid8 3,4-dihydroxyphenyl alanine (DOPA), and dopamine.99 An aliquot of urine corresponding to 0-075 mg. creatinine is on an electrophoretic paper-strip (30x4 cm.) 7 cm. from the anode-end of the strip, and run for two hours. Urocanic acid migrates about 8 cm. towards the cathode in this time. After air-drying and heating of the strip, it is dyed with diazotised p-nitroaniline. Urocanic acid gives a purple colour. A standard of urocanic acid can be run simultaneously on a second strip, to facilitate its identification, in addition to the urine. We also use another dye procedure, which is our modification of Pauly’s sulphanilic acid reagent. One part of 0-1% sulphanilic acid in 1% HCI v/v and, one part of 0-2% sodium nitrite are mixed under cooling; after 3 minutes one part of 0-025% ammonium sulphamate, and then two parts of 10% sodium carbonate, are added. The paper strips are immediately dipped through this mixture. Urocanic acid gives a roseyellow colour, next to histidine which migrates a little further and is dyed an intense orange. When viewed, after being airdried, under ultraviolet light (Wood’s light), urocanic acid gives a bright red fluorescence.
applied
4. 5. 6. 7. 8. 9.
Bainton, D. F., Finch, C. A. Bennett, M. C., Chanarin, I. Chanarin, I., Bennett, M. C. Bennett, M. C., Chanarin, I. Eichhorn, F., Rutenberg, A. Eichhorn, F., Rutenberg, A.
Am. J. Med. 1964, 37, 62. Lancet, 1961, ii, 1095. J. 1962, i, 27. Nature, Lond. 1962, 196, 171. Clin. Chem. 1963, 9, 615. ibid. 1965, 11, 563. Br. med.
Interpretation As urocanic acid may not be increased in some cases of folicacid deficiency, a negative result should be followed by an estimation of FIGLU. In the same way, a FIGLU estimation alone would not indicate a folic-acid deficiency in those cases where only urocanic acid is increased." Part of this project was supported by a grant from Lady Edith Wolfson’s Research Fund, through the Israel Cancer Association. Rappaport Laboratories, Biochemical Section, F. EICHHORN Beilinson Hospital, A. RUTENBERG. Petah Tiqva, Israel.
INHIBITION BY SERA OF URÆMIC PATIENTS SiR,ňThe preliminary communication by Dr. Emerson and her colleagues (Sept. 18) deals with the interesting problem of the interaction between ursemic serum and enzyme activities. We 11 have investigated the glutathione-reductase (G.R.) activity of erythrocytes in 53 patients with renal insufficiency, most of them with hyperazotsemia, and we found, in contrast with reports by other workers," considerably raised enzyme activity. Mean values of G.R. activity, in units per g. of Hb, for two groups of patients with chronic and acute renal insufficiency, were respectively 86-4 units (range 43-9-140-0) and 75-1 units (range 49-3-97-0), while in a group of control subjects the mean value was 37-9 units (range 21-0-49-3). G.R. activity increases with urea concentration, but it never reaches values higher than 100-150 units. In acute renal insufficiency, the increase in G.R. activity generally follows the rise of azotaemia, but high G.R. values may be found long after the resolution of the acute episode. Moreover, plasma from ursmic patients with high erythrocytic G.R. values has the remarkable property of inducing in normal erythrocytes a sharp elevation of G.R. activity, far above the normal range, in an incubation system at 37’C constituted by washed erythrocytes from normal blood-compatible donors and urxmic plasma, with the addition of glucose as follows:
Thus, plasma from urxmic patients seems to contain a factor of stimulating erythrocyte G.R.-activity, in agreement with the report 13 of high reduced-glutathione concentrations in red blood-cells of hyperazotxmic patients. Activation seems to be due to a stable and perhaps irreversible modification of the structure of the enzyme, since it is shown after haemolysis and successive dialysis of the haemolysate. Uraemic plasma completely loses its activating power after dialysis against saline, and, almost completely, after heat denaturation at 70-100°C. Further studies are needed to clarify the kinetics of the activation, and to establish the nature of the activating factor. PAOLO BRUNETTI GIUSEPPE G. NENCI Medical Clinic of the ALBERTO PARMA. of University Perugia, Italy.
capable
LACTATE DEHYDROGENASE IN URÆMIA SIR,-We are able to confirm the preliminary communication of Dr. Emerson and her colleagues (Sept. 18) concerning the increase of serum lactate dehydrogenase (L.D.H.) which is often found immediately after haemodialysis in urxmic patients. The possible removal of an easily diffusible inhibitor, as they stress, needs due consideration. We wish to draw attention, however, to other factors which might also play a part. Slight haemolysis, undetected to the naked eye, easily induces an increase of the order of 10% in a normal total serum L.D.H.; one then expects a shift towards the two fastest L.D.H.S (L.D.H. 1 10. 11.
Knowles, J. P. Lancet, 1961, ii, 1149. Brunetti, P., Parma, A., Nenci, G. G. Hœmatologica, 1965, 50, 415. Loehr, G. W., Waller, H. D. Schweiz. med. Wschr. 1962, 92, 1. Wernze, H., Koch, W. Klin. Wschr. 1965, 43, 451.
12. Bock, H. E., 13.