357
Clinica Chimica Acta, 69 (1976) 357-359 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
SHORT COMMUNICATION CGA 7807
A SEARCH FOR METHYL UREA IN THE BLOOD AND URINE OF PATIENTS WITH CHRONIC RENAL DISEASE
R.T. EVANS
*
Department of Clinical Chemistry, Southmead Bristol BSIO 5NB (U.K.) (Received
January
Hospital,
6, 1976)
Summary No methyl urea was detected in either blood or urine of uraemic patients by a specific gas-liquid chromatographic method in contrast to previous reports based on thin-layer chromatography and high voltage electrophoretis. It is concluded that methyl urea is unlikely to be implicated in the clinical manifestations of chronic renal disease.
Introduction The possibility that methyl urea might be a normal constituent of human urine was first suggested by Folin in 1907 [ 11. However, no further investigation of his hypothesis was made until 1968 when Giordano and his colleagues reported the presence of a substance in normal urine which stained with Ehrlich’s reagent and which had the same chromatographic mobility as methyl urea [2]. These observations were interpreted as indicating the presence of methyl urea, but no more rigorous tests involving isolation and chemical analysis were performed. On applying their methods to urine from uraemic subjects they found an increase in the amount of this material, a finding which they attributed to an unspecified disturbance of methylation caused by the renal disease
131. Further studies by the same group into the toxic effects of methyl urea in growing mice [4] led them to conclude that methyl urea might be responsible for some of the symptoms of uraemia and in particular the anaemia. * Present 7TF,
address:
U.K.
Department
of
Chemical
Pathology.
St.
James’s
University
Hospital.
Leeds
IS9
358
If these findings could be confirmed, then clearly an important new biochemical fact would have been discovered with signficant implications for the study of renal disease. Materials and methods Assay of methyl urea The analytical system for methyl urea described by Evans [ 51 has been applied, based upon the gas-liquid chromatography of its trifluoroacetyl derivative [6] . Collection of samples Blood and urine samples were collected from 22 uraemic patients during the course of routine visits to the Nephrology Department of Southmead Hospital, Bristol. All were selected on the basis of their being in severe renal failure with repeated creatinine clearances of less than 15 ml per minute. None had experienced either peritoneal or haemodialysis although several were ready for inclusion in the haemodialysis programme. Heparinised blood samples were taken during the course of routine collections for biochemical and haematological investigations. Urine samples were taken from 24-h collections where such urines were available. When this was not the case, random urines were provided. Of the 22 patients 11 were male and 11 female. The mean age was 50 years, range 16-79. The average plasma urea was 25 mmol/l range 14.5-46.5 while plasma creatinine varied from 290 to 1300 pmol/l with a mean of 765. Blood and urine specimens were stored without preservative at 4°C prior to analysis. Most analyses were performed within three days of receipt of the specimens, and all analyses within ten days, Results and discussion In the 22 blood and 21 urine samples collected from these patients no evidence could be produced from any of them for the presence of methyl urea at concentrations in excess of the minimum detectable, 4 mg (54 pmol)/l. These findings could not be attributed to loss of methyl urea during storage before analysis since it showed a high level of stability in both blood and urine. The recovery of methyl urea added to blood at a concentration of 100 mg (1.3 mmol)/l was 93% after 4 weeks storage, and after addition to urine at the same concentration, 96% after 8 weeks. Both recoveries are within the error of the method [5]. It has thus not been possible to confirm the findings of Giordano and his colleagues. One possible explanation of this discrepancy could be the higher sensitivity of their method for methyl urea compared with the method of Evans [ 51, but no quantitative data were reported by these workers and the use of 200 mg quantities of methyl urea in their recovery experiments would suggest that this is not the case. It must be assumed, therefore, that the material which they supposed to be methyl urea was in fact a different substance but with similar chemical and chromatographic properties.
359
These findings therefore indicate that the possibility that methyl urea is implicated in the clinical manifestations of uraemia is remote unless it could be shown to be extraordinarily highly toxic in man, for which there is, at present, no evidence. Acknowledgements Grateful patients,
thanks are due to Dr. J.C. Mackenzie for permission to investigate his and to Dr. J.B. Holton for the provision of laboratory facilities.
References 1 Folin, 0. (1907) J. Biol. Chem. 3. 83-86 2 Balestrieri, C., Raw. R., De Pascale, C.. Esposito, R.. De Santa. N.G. and Giordano. C. (1968) 3 4 5 6
Boll.
Sot. Ital. Biol. Sper. 44, 2224-2226 Giordsno, C.. De Pascale, C.. Esposito. R., Balestrier& C.. Demma, G., Cittadini. D. and De Santa. N.G. (1968) BOIL Sot. Ital. Biol. Sper. 44. 2229-2232 Giordano, C.. Minale, C., Esposito, R., Balestrieri. C., De Santa, N.G. and De Pascale, C. (1968) Boll. Sot. Ital. Biol. Sper. 44. 2227-2229 Evans. R.T. (1976) J. Chromatogr. 118.357-362 Evans, R.T. (1974) J. Chromatogr. 88. 398402
Clinica Chimica Acta, 69 (1976) 357-359 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
SHORT COMMUNICATION CGA 7807
A SEARCH FOR METHYL UREA IN THE BLOOD AND URINE OF PATIENTS WITH CHRONIC RENAL DISEASE
R.T. EVANS
*
Department of Clinical Chemistry, Southmead Bristol BSIO 5NB (U.K.) (Received
January
Hospital,
6, 1976)
Summary No methyl urea was detected in either blood or urine of uraemic patients by a specific gas-liquid chromatographic method in contrast to previous reports based on thin-layer chromatography and high voltage electrophoretis. It is concluded that methyl urea is unlikely to be implicated in the clinical manifestations of chronic renal disease.
Introduction The possibility that methyl urea might be a normal constituent of human urine was first suggested by Folin in 1907 [ 11. However, no further investigation of his hypothesis was made until 1968 when Giordano and his colleagues reported the presence of a substance in normal urine which stained with Ehrlich’s reagent and which had the same chromatographic mobility as methyl urea [2]. These observations were interpreted as indicating the presence of methyl urea, but no more rigorous tests involving isolation and chemical analysis were performed. On applying their methods to urine from uraemic subjects they found an increase in the amount of this material, a finding which they attributed to an unspecified disturbance of methylation caused by the renal disease
131. Further studies by the same group into the toxic effects of methyl urea in growing mice [4] led them to conclude that methyl urea might be responsible for some of the symptoms of uraemia and in particular the anaemia. * Present 7TF,
address:
U.K.
Department
of
Chemical
Pathology.
St.
James’s
University
Hospital.
Leeds
IS9
358
If these findings could be confirmed, then clearly an important new biochemical fact would have been discovered with signficant implications for the study of renal disease. Materials and methods Assay of methyl urea The analytical system for methyl urea described by Evans [ 51 has been applied, based upon the gas-liquid chromatography of its trifluoroacetyl derivative [6] . Collection of samples Blood and urine samples were collected from 22 uraemic patients during the course of routine visits to the Nephrology Department of Southmead Hospital, Bristol. All were selected on the basis of their being in severe renal failure with repeated creatinine clearances of less than 15 ml per minute. None had experienced either peritoneal or haemodialysis although several were ready for inclusion in the haemodialysis programme. Heparinised blood samples were taken during the course of routine collections for biochemical and haematological investigations. Urine samples were taken from 24-h collections where such urines were available. When this was not the case, random urines were provided. Of the 22 patients 11 were male and 11 female. The mean age was 50 years, range 16-79. The average plasma urea was 25 mmol/l range 14.5-46.5 while plasma creatinine varied from 290 to 1300 pmol/l with a mean of 765. Blood and urine specimens were stored without preservative at 4°C prior to analysis. Most analyses were performed within three days of receipt of the specimens, and all analyses within ten days, Results and discussion In the 22 blood and 21 urine samples collected from these patients no evidence could be produced from any of them for the presence of methyl urea at concentrations in excess of the minimum detectable, 4 mg (54 pmol)/l. These findings could not be attributed to loss of methyl urea during storage before analysis since it showed a high level of stability in both blood and urine. The recovery of methyl urea added to blood at a concentration of 100 mg (1.3 mmol)/l was 93% after 4 weeks storage, and after addition to urine at the same concentration, 96% after 8 weeks. Both recoveries are within the error of the method [5]. It has thus not been possible to confirm the findings of Giordano and his colleagues. One possible explanation of this discrepancy could be the higher sensitivity of their method for methyl urea compared with the method of Evans [ 51, but no quantitative data were reported by these workers and the use of 200 mg quantities of methyl urea in their recovery experiments would suggest that this is not the case. It must be assumed, therefore, that the material which they supposed to be methyl urea was in fact a different substance but with similar chemical and chromatographic properties.
359
These findings therefore indicate that the possibility that methyl urea is implicated in the clinical manifestations of uraemia is remote unless it could be shown to be extraordinarily highly toxic in man, for which there is, at present, no evidence. Acknowledgements Grateful patients,
thanks are due to Dr. J.C. Mackenzie for permission to investigate his and to Dr. J.B. Holton for the provision of laboratory facilities.
References 1 Folin, 0. (1907) J. Biol. Chem. 3. 83-86 2 Balestrieri, C., Raw. R., De Pascale, C.. Esposito, R.. De Santa. N.G. and Giordano. C. (1968) 3 4 5 6
Boll.
Sot. Ital. Biol. Sper. 44, 2224-2226 Giordsno, C.. De Pascale, C.. Esposito. R., Balestrier& C.. Demma, G., Cittadini. D. and De Santa. N.G. (1968) BOIL Sot. Ital. Biol. Sper. 44. 2229-2232 Giordano, C.. Minale, C., Esposito, R., Balestrieri. C., De Santa, N.G. and De Pascale, C. (1968) Boll. Sot. Ital. Biol. Sper. 44. 2227-2229 Evans. R.T. (1976) J. Chromatogr. 118.357-362 Evans, R.T. (1974) J. Chromatogr. 88. 398402
