LETTERS TO THE EDITORS
Br. J. clin. Pharmac. (1975), 2
475
DETERMINATION OF ACETYLATOR STATUS IN URAEMIA There is a bimodal distribution of the ability to inactivate sulphadimidine in a normal population (Evans & White, 1964). This has been established by measuring various parameters assumed to represent rates of metabolism. The most widely accepted criteria (Rao, Mitchison, Nair, Prema & Tripathy, 1970) define fast acetylators as those who have a proportion of acetylated sulphadimidine greater than 70% in urine and 25% in blood after oral ingestion. Most of the previous work in this area has been based on measurements in patients with normal renal function. The renal clearance of acetyl sulphadimidine exceeds that of the parent drug. Consequently in severe uraemia there might be expected to be a relatively greater retention of the metabolite in the body which will tend to invalidate these criteria upon which acetylator status is defined in normals. We have measured metabolic clearance rates (MCR) of sulphadimidine in normals in order to compare the results obtained with the simpler tests. Similar measurements were performed in functionally anephric patients. Twenty-five healthy, white test subjects and ten patients on chronic intermittent haemodialysis were studied. Sulphadimidine (2 g) was ingested in the fasting state. Blood samples were taken hourly for 10 h and at 24 hours. Urine was collected for 24 h and an aliquot at 7-8 h taken. Blood and urine blanks were estimated for each subject. Free and acetylated sulphadimidine were measured by the Bratton and Marshall technique (Varley,
1962). The plasma concentration data were fitted to a function of the form A(e-Kt - e-K2t ) using a Hewlett Packard 91 OOB programmable calculator. K1 and K2 are the rate constants of absorption and elimination although it is not possible to identify them uniquely. A is a constant which depends on the dose administered, the distribution volume, K1 and K2Then total clearance = Dose administered Area under plasma concentration curvee from 0-0 Renal clearance = Amount excreted in 24 h Area under plasma concentration curve from 0-24 h and metabolic clearance = total clearance - renal
clearance. The area under the curve from the time of
.
* 0.
80F 0
60
0
*0
CD
0 0
Q40 0
20O *
*
0.
: so@ 0*
*
a 20
40
60
80
100
% AcS in urine Figure 1 Plot of % acetylated sulphadimidine (Ac S) in urine and in blood of twenty-five normal subjects.
administration to infinity was calculated from the formula A ( I /K1 - /K2 ). Ten normal subjects had greater than 70% of the total urine sulphadimidine present as acetyl sulphadimidine and greater than 43% in blood. The MCR in this group ranged from 70-342 ml/ minute. Fifteen subjects had less than 70% acetylated drug in urine and fourteen of these had less than 30% acetylated in blood. The range of MCR was 2-37 ml/minute. The remaining subject had 45% acetylated drug in blood and had a MCR of 48 ml/minute. Acetylator phenotype in normals was therefore defined by measurement of MCR. A plot of % acetylated drug in wine and in blood in twenty-five normal subjects is shown in Figure 1. The renal clearance of free drug was 29 ± 14 ml/min in fast acetylators and 19 ± 11 ml/min in slow acetylators (NS, Student's t test). The corresponding figures for. acetyl sulphadimidine were 67 ± 30 ml/min and 47 ± 24 ml/min respectively (NS, Student's t test). The above results are expressed as mean ± s.d. Four of the uraemic patients had MCRs of between 107-276 ml/min and consequently were fast acetylators. In three of these, the urine contained less than 70% acetylated drug. Six patients had MCRs of between 10-30 ml/min and were slow acetylators of which two had greater than 30o present as the acetylated metabolite in blood (Table 1). The total renal excretion of sulphadimidine was
476
Br. J.
LETTERS TO THE EDITORS
clin. Pharmac. (1975), 2
Table 1 Individual results of sulphadimidine acetylation tests in ten dialysis patients. Only five of these patients produced urine
Patient C.R. T.McG. W.J. J.B. J.McM. J.M. C.M. J.T. T.S. J.J.
% acetylated in blood % acetylated in urine (8 h) (6 h) 28 42 26 23 11 44 62 66 76 84
negligible in these uraemic patients, amounting to less than 52 mg excreted throughout the duration of the test in the five patients who produced urine. On the basis of the single urine and blood tests. our results accurately define acetylator phenotype with values obtained closely approximating those of other workers (Evans, 1969; Rao et al., 1970). Calculation of MCR defines acetylator status with a degree of accuracy at least equal to these simpler tests. The proportion of fast acetylators found in this series (40%) closely approximates the incidence of 32.1% found in a normal large Glasgow population (Smith, Tyrell, Gow, Allan & Lees, 1972). Consequently there is no need to perform this more elaborate test in patients with normal renal function. Conventional criteria would have incorrectly defined the acetylator status of 50% of our uraemic patients. The relatively greater retention of the metabolite in functionally anephric patients leads to an increased ratio of acetylated drug in the blood and a diminished ratio in urine. It is apparent, therefore, that acetylator status, when required, can only be consistently defined in severe uraemic patients by estimation of the metabolic clearance rates. It has been found that acetylation is induced in those patients with prolonged and severe uraemia and that this trend is apparently reversed by long term haemodialysis (Fine & Sumner, 1974). Consequently the determination of acetylator status by estimating the metabolic clearance rate assumes greater significance in these patients.
30 42 -
20 55 -
62
Metabolic clearance rate (ml/min) 10 12 18 27 29 30 107 155 179 276
We wish to thank Mr J. McCulloch for his invaluable technical assistance. A. FINE & D.J. SUMNER Renal Unit, Glasgow Royal Infirmary and West of Scotland Health Boards, Department of Clinical Physics and Bioengineering, Glasgow, Scotland
Received March 14,1975
References EVANS, D.A.P. (1969). An improved and simplifiled method of determination of acetylator phenotype. J.
med. Genet., 6, 405407. EVANS, D.A.P. & WHITE, T.A. (1964). Human acetylation polymorphism. J. lab. clin. Med., 63 394403. FINE, A. & SUMNER, D.J. (1974). Alteration of hepatic acetylation in uraemia. Proceedings of European Dialysis and Transplant Association, Tel Aviv (in press). RAO, K.V.N., MITCHISON, D.A., NAIR, N.G.K., PREMA, K. & TRIPATHY, S.P. (1970). Sulphadimidine acetylation test for classification of patients as slow or rapid acetylators of isoniazid. Br. med. J., 3,
495497. SMITH, J., TYRELL, W.F., GOW, A., ALLAN, G.W. & LEES, A.W. (1972). Hepatotoxicity in RifampicinIsoniazid treated patients related to their rates of
inactivation. Chest, 61, 587-588. VARLEY, H. (1962). Practical Clinical Biochemistry, 3rd ed., p. 634. London: Heinemann.
Br. J. clin. Pharmac. (1975), 2
475
DETERMINATION OF ACETYLATOR STATUS IN URAEMIA There is a bimodal distribution of the ability to inactivate sulphadimidine in a normal population (Evans & White, 1964). This has been established by measuring various parameters assumed to represent rates of metabolism. The most widely accepted criteria (Rao, Mitchison, Nair, Prema & Tripathy, 1970) define fast acetylators as those who have a proportion of acetylated sulphadimidine greater than 70% in urine and 25% in blood after oral ingestion. Most of the previous work in this area has been based on measurements in patients with normal renal function. The renal clearance of acetyl sulphadimidine exceeds that of the parent drug. Consequently in severe uraemia there might be expected to be a relatively greater retention of the metabolite in the body which will tend to invalidate these criteria upon which acetylator status is defined in normals. We have measured metabolic clearance rates (MCR) of sulphadimidine in normals in order to compare the results obtained with the simpler tests. Similar measurements were performed in functionally anephric patients. Twenty-five healthy, white test subjects and ten patients on chronic intermittent haemodialysis were studied. Sulphadimidine (2 g) was ingested in the fasting state. Blood samples were taken hourly for 10 h and at 24 hours. Urine was collected for 24 h and an aliquot at 7-8 h taken. Blood and urine blanks were estimated for each subject. Free and acetylated sulphadimidine were measured by the Bratton and Marshall technique (Varley,
1962). The plasma concentration data were fitted to a function of the form A(e-Kt - e-K2t ) using a Hewlett Packard 91 OOB programmable calculator. K1 and K2 are the rate constants of absorption and elimination although it is not possible to identify them uniquely. A is a constant which depends on the dose administered, the distribution volume, K1 and K2Then total clearance = Dose administered Area under plasma concentration curvee from 0-0 Renal clearance = Amount excreted in 24 h Area under plasma concentration curve from 0-24 h and metabolic clearance = total clearance - renal
clearance. The area under the curve from the time of
.
* 0.
80F 0
60
0
*0
CD
0 0
Q40 0
20O *
*
0.
: so@ 0*
*
a 20
40
60
80
100
% AcS in urine Figure 1 Plot of % acetylated sulphadimidine (Ac S) in urine and in blood of twenty-five normal subjects.
administration to infinity was calculated from the formula A ( I /K1 - /K2 ). Ten normal subjects had greater than 70% of the total urine sulphadimidine present as acetyl sulphadimidine and greater than 43% in blood. The MCR in this group ranged from 70-342 ml/ minute. Fifteen subjects had less than 70% acetylated drug in urine and fourteen of these had less than 30% acetylated in blood. The range of MCR was 2-37 ml/minute. The remaining subject had 45% acetylated drug in blood and had a MCR of 48 ml/minute. Acetylator phenotype in normals was therefore defined by measurement of MCR. A plot of % acetylated drug in wine and in blood in twenty-five normal subjects is shown in Figure 1. The renal clearance of free drug was 29 ± 14 ml/min in fast acetylators and 19 ± 11 ml/min in slow acetylators (NS, Student's t test). The corresponding figures for. acetyl sulphadimidine were 67 ± 30 ml/min and 47 ± 24 ml/min respectively (NS, Student's t test). The above results are expressed as mean ± s.d. Four of the uraemic patients had MCRs of between 107-276 ml/min and consequently were fast acetylators. In three of these, the urine contained less than 70% acetylated drug. Six patients had MCRs of between 10-30 ml/min and were slow acetylators of which two had greater than 30o present as the acetylated metabolite in blood (Table 1). The total renal excretion of sulphadimidine was
476
Br. J.
LETTERS TO THE EDITORS
clin. Pharmac. (1975), 2
Table 1 Individual results of sulphadimidine acetylation tests in ten dialysis patients. Only five of these patients produced urine
Patient C.R. T.McG. W.J. J.B. J.McM. J.M. C.M. J.T. T.S. J.J.
% acetylated in blood % acetylated in urine (8 h) (6 h) 28 42 26 23 11 44 62 66 76 84
negligible in these uraemic patients, amounting to less than 52 mg excreted throughout the duration of the test in the five patients who produced urine. On the basis of the single urine and blood tests. our results accurately define acetylator phenotype with values obtained closely approximating those of other workers (Evans, 1969; Rao et al., 1970). Calculation of MCR defines acetylator status with a degree of accuracy at least equal to these simpler tests. The proportion of fast acetylators found in this series (40%) closely approximates the incidence of 32.1% found in a normal large Glasgow population (Smith, Tyrell, Gow, Allan & Lees, 1972). Consequently there is no need to perform this more elaborate test in patients with normal renal function. Conventional criteria would have incorrectly defined the acetylator status of 50% of our uraemic patients. The relatively greater retention of the metabolite in functionally anephric patients leads to an increased ratio of acetylated drug in the blood and a diminished ratio in urine. It is apparent, therefore, that acetylator status, when required, can only be consistently defined in severe uraemic patients by estimation of the metabolic clearance rates. It has been found that acetylation is induced in those patients with prolonged and severe uraemia and that this trend is apparently reversed by long term haemodialysis (Fine & Sumner, 1974). Consequently the determination of acetylator status by estimating the metabolic clearance rate assumes greater significance in these patients.
30 42 -
20 55 -
62
Metabolic clearance rate (ml/min) 10 12 18 27 29 30 107 155 179 276
We wish to thank Mr J. McCulloch for his invaluable technical assistance. A. FINE & D.J. SUMNER Renal Unit, Glasgow Royal Infirmary and West of Scotland Health Boards, Department of Clinical Physics and Bioengineering, Glasgow, Scotland
Received March 14,1975
References EVANS, D.A.P. (1969). An improved and simplifiled method of determination of acetylator phenotype. J.
med. Genet., 6, 405407. EVANS, D.A.P. & WHITE, T.A. (1964). Human acetylation polymorphism. J. lab. clin. Med., 63 394403. FINE, A. & SUMNER, D.J. (1974). Alteration of hepatic acetylation in uraemia. Proceedings of European Dialysis and Transplant Association, Tel Aviv (in press). RAO, K.V.N., MITCHISON, D.A., NAIR, N.G.K., PREMA, K. & TRIPATHY, S.P. (1970). Sulphadimidine acetylation test for classification of patients as slow or rapid acetylators of isoniazid. Br. med. J., 3,
495497. SMITH, J., TYRELL, W.F., GOW, A., ALLAN, G.W. & LEES, A.W. (1972). Hepatotoxicity in RifampicinIsoniazid treated patients related to their rates of
inactivation. Chest, 61, 587-588. VARLEY, H. (1962). Practical Clinical Biochemistry, 3rd ed., p. 634. London: Heinemann.
