261
Clinica Chimica Acta, @ Elsevier/North-Holland
CCA
81 (1977) 261-265 Biomedical Press
8882
SERUM ASPIRIN ESTERASE ASPIRIN INTAKE
J.D.
GUPTA
a** and
VATSALA
ACTIVITY
GUPTA
IN WOMEN WITH HABITUAL
b***
a Children’s Medical Research Foundation, Royal Alexandra Hospital for Children, Memorial Research Institute, Camperdown, N.S. W. 2050 (Australia) and b Kanematsu Sydney Hospital, Sydney, N.S. W. 2000 (Australia) (Received
May
6th,
1977)
Summary
A method for the determination of aspirin esterase activity in serum is described. Sera from 59 pregnant women who were habitual aspirin users were found to have a mean enzyme activity value statistically lower than those of 68 non-pregnant women controls or of 12 pregnant women controls who were either occasional users of the drug or were non-users. The distribution of enzyme activity in the experimental group was also significantly different from that of the control group. It is postulated that the low enzyme activity may further aggravate the injurious effects of high intake of aspirin.
Introduction
Aspirin or acetyl salicylic acid (ASA) is probably the most widely used drug in the world either singly or in combination with other constituents. The rate of ingestion of aspirin varies in individuals although a small but significant fraction of the population, especially in the western world, are heavy users of aspirin or preparations containing ASA. It was observed by Collins and Turner [l] that there was an increased incidence of maternal anaemia, ante-pa&urn haemorrhage, prolonged gestation, complicated deliveries and peri-natal mortality in a group of pregnant women who were habitual aspirin users as compared to non-users. It is also known that the injurious effects of ASA are mostly due to the intact molecule rather than to its breakdown products [2,3], and thus possible that any defect in aspirin metabolism may contribute to such injuries. We therefore determined the aspirin esterase activity in the sera of the users as compared with those of normal controls. * To whom correspondence should be addressed. ** Present address: Department of Medicine, Prince Henry Hospital. Little Bay. N.S.W. 2036. Australia.
Aspirin e&erase degrades ASA to acetate and salicylate and is present in plasma, liver and kidney 141. The presence of this enzyme has also been reported recently in the gastric mucosa [5]. For the assay of the enzyme in plasma or serum we developed a simple technique which gave higher values for normal controls than reported earlier [6], was highly reproducible and which corrected for non-enzymatic hydrolysis of ASA on incubation and for the presence of pre-existing salicylate in the samples tested. Materials and methods Sera from a group of 144 pregnant women at term who were habitual users of aspirin or ASA containing prep~ations were collected by Collins and Turner in their study of the maternal and fetal effects of regular salicylate ingestion in pregnancy. Details have been previously described [l]. 59 of these 144 samples were available at random to us for the present study. The control group consisted of 68 women of which 64 were less than forty years old and who took aspirin or ASA-containing preparations only occasionally or not at all. Serum samples from 12 women at term who were not habitual users of aspirin were also available for comparison. Sera were also collected from 19 adult male volunteers none of whom was a regular user. The frozen sera from the experimental group had probably been thawed and refrozen at least once for the earlier study, but such treatment was found to have no significant effect on the enzyme activity as tested with the control sera. Assay of aspirin e&erase The method of Trinder [7] for the determination of salicylate in biological fluids was adapted after suitable modifications for the assay of aspirin esterase activity in serum or plasma and may also be used for tissue homogenate or its sub-cellular fractions. With plasma, however, clotting may occur on incubation after incubation of the and require defibrination before use. Essentially, enzyme source with soluble aspirin, the mixture is treated with acidic mercuric chloride-ferric nitrate solution which precipitates the proteins and also forms coloured ferric salicylate in the supernate in direct proportion to the salicylate released by enzymic activity. To 0.5 ml serum were added 0.5 ml aspirin solution and 2 ml of 0.06 M Tris/ HCI buffer, pH 7.4 and the mixture incubated for 1 h at 37°C in a water bath with occasional shaking. 3 ml of acidic HgC12-Fe(NO,), solution was then added to each tube, shaken vigorously, centrifuged at 3000 rev./min for 10 min and the colour of the supernate read immediately at 540 nm against a reagent blank (using 1 ml water instead of serum plus aspirin), The absorbance is denoted by A,. For each sample, a blank prepared as follows was used to correct for the non-enzymatic hydrolysis of aspirin during incubation and for pre-existing salicylate ion in the serum. To 0.5 ml of aspirin solution was added 2 ml buffer and the mixture incubated for 1 h at 37°C. 0.5 ml of serum was then added followed by the immediate addition of 3 ml of HgC~~-~e(NO~)~ solution. The absorbance of the supernate is designed as AZ. The corrected absorbance A, (A, -A,) for each sample was then used for
263
the determination of the equivalent salicylic acid from a standard graph. Aspirin esterase activity was expressed as pug salicylic acid released/h/ml of serum tested. The standard curve was prepared by adding 3 ml of HgC12-Fe(N03)3 solution to each 3 ml solution containing 100-800 lug salicylic acid (116-928 pg sodium salicylate). The colour produced was read at 540 nm after 10 to 15 min (to correct for the centrifugation time of experimental sample) against a reagent blank. The absorbances were linear (0.16-1.28) in the range of salicylic acid used. Preparation of aspirin solution Aspirin solution was freshly prepared immediately before use. One 300 mg tablet of commercially available soluble aspirin (Disprin, Reckitts Pty. Ltd., Australia) was added to 100 ml distilled water, shaken vigorously and the homogeneous mixture was used as a substrate without centrifugation. Centrifuged supernatant solution may also be used as substrate, but gave comparable results. Different batches of the commercial aspirin used were tested for free salicylic acid and was found to contain either traces or none at all. Preparation of mercuric chloride-ferric nitrate solution 10 g mercuric chloride (A.R.) were dissolved in 160-170 ml hot distilled water. The solution was cooled and 60 ml of 1 M HCl and 10 g Fe(NO,), . 9H20 were added. When all the ferric nitrate had dissolved the volume was made up to 250 ml with distilled water. The solution is stable indefinitely. Results In the assay procedure described, the colour produced by standard solutions of sodium salicylate was linear over a wide range. The same standard curve could be used with one set of reagents but it is advisable to include one standard in each assay. The test gave reproducible results in replicate determinations and with assays on the same sample on different days. The coefficient of
TABLE
I
SERUM
ASPIRIN
ESTERASE
ACTIVITY
Group
IN HABITUAL
ASPIRIN
USER
NO.
Aspirin
tested
(pg salicylic Mean
1. Pregnant
women
2. Non-pregnant 3. Pregnant 4. Adult
female
(heavy
female
control
male
Statistical
significance:
1 vs. 2, P < 0.01.. 1 vs. 3, P
Clinica Chimica Acta, @ Elsevier/North-Holland
CCA
81 (1977) 261-265 Biomedical Press
8882
SERUM ASPIRIN ESTERASE ASPIRIN INTAKE
J.D.
GUPTA
a** and
VATSALA
ACTIVITY
GUPTA
IN WOMEN WITH HABITUAL
b***
a Children’s Medical Research Foundation, Royal Alexandra Hospital for Children, Memorial Research Institute, Camperdown, N.S. W. 2050 (Australia) and b Kanematsu Sydney Hospital, Sydney, N.S. W. 2000 (Australia) (Received
May
6th,
1977)
Summary
A method for the determination of aspirin esterase activity in serum is described. Sera from 59 pregnant women who were habitual aspirin users were found to have a mean enzyme activity value statistically lower than those of 68 non-pregnant women controls or of 12 pregnant women controls who were either occasional users of the drug or were non-users. The distribution of enzyme activity in the experimental group was also significantly different from that of the control group. It is postulated that the low enzyme activity may further aggravate the injurious effects of high intake of aspirin.
Introduction
Aspirin or acetyl salicylic acid (ASA) is probably the most widely used drug in the world either singly or in combination with other constituents. The rate of ingestion of aspirin varies in individuals although a small but significant fraction of the population, especially in the western world, are heavy users of aspirin or preparations containing ASA. It was observed by Collins and Turner [l] that there was an increased incidence of maternal anaemia, ante-pa&urn haemorrhage, prolonged gestation, complicated deliveries and peri-natal mortality in a group of pregnant women who were habitual aspirin users as compared to non-users. It is also known that the injurious effects of ASA are mostly due to the intact molecule rather than to its breakdown products [2,3], and thus possible that any defect in aspirin metabolism may contribute to such injuries. We therefore determined the aspirin esterase activity in the sera of the users as compared with those of normal controls. * To whom correspondence should be addressed. ** Present address: Department of Medicine, Prince Henry Hospital. Little Bay. N.S.W. 2036. Australia.
Aspirin e&erase degrades ASA to acetate and salicylate and is present in plasma, liver and kidney 141. The presence of this enzyme has also been reported recently in the gastric mucosa [5]. For the assay of the enzyme in plasma or serum we developed a simple technique which gave higher values for normal controls than reported earlier [6], was highly reproducible and which corrected for non-enzymatic hydrolysis of ASA on incubation and for the presence of pre-existing salicylate in the samples tested. Materials and methods Sera from a group of 144 pregnant women at term who were habitual users of aspirin or ASA containing prep~ations were collected by Collins and Turner in their study of the maternal and fetal effects of regular salicylate ingestion in pregnancy. Details have been previously described [l]. 59 of these 144 samples were available at random to us for the present study. The control group consisted of 68 women of which 64 were less than forty years old and who took aspirin or ASA-containing preparations only occasionally or not at all. Serum samples from 12 women at term who were not habitual users of aspirin were also available for comparison. Sera were also collected from 19 adult male volunteers none of whom was a regular user. The frozen sera from the experimental group had probably been thawed and refrozen at least once for the earlier study, but such treatment was found to have no significant effect on the enzyme activity as tested with the control sera. Assay of aspirin e&erase The method of Trinder [7] for the determination of salicylate in biological fluids was adapted after suitable modifications for the assay of aspirin esterase activity in serum or plasma and may also be used for tissue homogenate or its sub-cellular fractions. With plasma, however, clotting may occur on incubation after incubation of the and require defibrination before use. Essentially, enzyme source with soluble aspirin, the mixture is treated with acidic mercuric chloride-ferric nitrate solution which precipitates the proteins and also forms coloured ferric salicylate in the supernate in direct proportion to the salicylate released by enzymic activity. To 0.5 ml serum were added 0.5 ml aspirin solution and 2 ml of 0.06 M Tris/ HCI buffer, pH 7.4 and the mixture incubated for 1 h at 37°C in a water bath with occasional shaking. 3 ml of acidic HgC12-Fe(NO,), solution was then added to each tube, shaken vigorously, centrifuged at 3000 rev./min for 10 min and the colour of the supernate read immediately at 540 nm against a reagent blank (using 1 ml water instead of serum plus aspirin), The absorbance is denoted by A,. For each sample, a blank prepared as follows was used to correct for the non-enzymatic hydrolysis of aspirin during incubation and for pre-existing salicylate ion in the serum. To 0.5 ml of aspirin solution was added 2 ml buffer and the mixture incubated for 1 h at 37°C. 0.5 ml of serum was then added followed by the immediate addition of 3 ml of HgC~~-~e(NO~)~ solution. The absorbance of the supernate is designed as AZ. The corrected absorbance A, (A, -A,) for each sample was then used for
263
the determination of the equivalent salicylic acid from a standard graph. Aspirin esterase activity was expressed as pug salicylic acid released/h/ml of serum tested. The standard curve was prepared by adding 3 ml of HgC12-Fe(N03)3 solution to each 3 ml solution containing 100-800 lug salicylic acid (116-928 pg sodium salicylate). The colour produced was read at 540 nm after 10 to 15 min (to correct for the centrifugation time of experimental sample) against a reagent blank. The absorbances were linear (0.16-1.28) in the range of salicylic acid used. Preparation of aspirin solution Aspirin solution was freshly prepared immediately before use. One 300 mg tablet of commercially available soluble aspirin (Disprin, Reckitts Pty. Ltd., Australia) was added to 100 ml distilled water, shaken vigorously and the homogeneous mixture was used as a substrate without centrifugation. Centrifuged supernatant solution may also be used as substrate, but gave comparable results. Different batches of the commercial aspirin used were tested for free salicylic acid and was found to contain either traces or none at all. Preparation of mercuric chloride-ferric nitrate solution 10 g mercuric chloride (A.R.) were dissolved in 160-170 ml hot distilled water. The solution was cooled and 60 ml of 1 M HCl and 10 g Fe(NO,), . 9H20 were added. When all the ferric nitrate had dissolved the volume was made up to 250 ml with distilled water. The solution is stable indefinitely. Results In the assay procedure described, the colour produced by standard solutions of sodium salicylate was linear over a wide range. The same standard curve could be used with one set of reagents but it is advisable to include one standard in each assay. The test gave reproducible results in replicate determinations and with assays on the same sample on different days. The coefficient of
TABLE
I
SERUM
ASPIRIN
ESTERASE
ACTIVITY
Group
IN HABITUAL
ASPIRIN
USER
NO.
Aspirin
tested
(pg salicylic Mean
1. Pregnant
women
2. Non-pregnant 3. Pregnant 4. Adult
female
(heavy
female
control
male
Statistical
significance:
1 vs. 2, P < 0.01.. 1 vs. 3, P
