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An improved method for determination of uric acid in serum
ANALYTICAL
4, 24-27 (1962)
BIOCHEMISTRY
An
Improved
Method Uric
MARIE From
the
Acid
H. CARR’
Research
and Administration
for in
Determination Serum1
AND BERTON
Radioisotope Hospital, Received
C. PRESSMAN3
Laboratories, Kansas City,
January
of
11,
Kansas Missouri
City
Veterans
1962
The large numbers of papers dealing with the determination of uric acid which have appeared in the literature in recent years imply a dissatisfaction of analysts with current methods (l-7). Prevalent colorimetric procedures lack specificity and hence reliability, while the enzymic methods employing uricase are complicated by technical problems arising from the ultraviolet absorption of serum protein. This is particularly troublesome in the case of experimental animals having low serum uric acid levels. Since one of our studies involved the following of slight changes in serum uric acid levels in dogs, we were motivated to develop a reliable method giving good precision at low uric acid concentrations. METHOD
Principle
Separation of uric acid from prot.ein is accomplished by passage of serum or other biological fluid through a strong anion-exchange resin in the acetate form. It has been found that such a column removes the uric acid from the fluid with greater ease and consequently is more reliable than the chloride form previously recommended (4, 6). Following the removal of extraneous UV absorbing materials by washing, the uric acid is eluted from the column and determined by enzymic assay. Materials
and Reagents
Ion-Exchange Resin. Dowex 2-X8 (100-206 mesh) in the acetate form. This resin is purchased in the chloride form and converted to the acetate ‘Supported in part by Research Grant C-4715 for U. S. Public Health Service. A preliminary account of this work was presented at the 139th National Meeting of the American Chemical Society at St. Louis, Missouri, April, 1961. ’ Deceased, April 28, 1962. 3 Present address: Johnson Foundation, University of Pennsylvania, Philadelphia 4, Pennsylvania.
24
URIC
ACID
IN
25
SERUM
form by washing on a column with 10% sodium acetate until tests for chloride in the effluent are not more than faintly positive. Standards. Uric acid stock, 1 mg/ml, is prepared by dissolving uric acid4 in M/15 glycine buffer. This solution is stable almost indefinitely if stored frozen in small vials. For a working standard, the stock solution is diluted 0.5 ml to 10 ml wit,h water. For assay purposes, 0.5 ml of the working standard is diluted to 6 ml with &1/15 glycine buffer to correspond to the dilution of the biological fluids analyzed. Eluant. To 500 ml of 0.04 M HCl add 500 ml M NaCl. Glycine Buffer, 95 M, pH 9.35. Glycine Buffer, M/15. This solution is prepared by diluting 10 ml of the sJSM buffer to 100 ml with water. Uricase,” purified, 125 units/ml. Dilute the amount required for a day’s work 1:20 with M/15 glycine buffer. Procedure 1. Prepare resin columns (2 cm in height X 0.4 cm in diameter). 2. Pass 0.5 ml biological fluid containing 2 to 100 pg uric acid through the column. If whole blood is to be analyzed, a volume of diluted hemolyzed blood equivalent to 0.5 ml whole blood is used. 3. Wash twice with 10 ml H,O. Discard the washings. 4. Elute the uric acid with 5 ml of the HCl-NaCl eluant. 5. Add 1 ml of 2/3M glycine buffer, pH 9.35, to which has been added 6.25 ml 10N NaOH per liter to compensate for the HCl in the eluant. 6. Measure the absorption of the buffered eluate at 293 nip. 7. To a 3.5-ml aliquot of buffered eluate, add 0.1 ml of diluted uricase described above, and incubate at 45°C for 2 hr. 8. Redetermine the 293 mp absorption. The decrease following incubation is a measure of the uric acid present,. A standard containing 5.00 mg % uric acid in 1M/15 glycine buffer gave an absorption change of 0.306 OD in t,he enzymic assay. RESULTS
AND
DISCUSSION
The results obtained by this method in the analysis of human and canine subjects are given in Table 1. Reproducibility was 0.013 mg % in dogs at a level of 0.30 mg ‘$ and 0.063 mg % in humans at a level of 4.31 mg %I. Concentrations of HCl in 0.5 M NaCl of 0.050 to 0.005 ii/T were tested ‘The reagent-grade found to have the Associates (5). ‘Sigma Chemical
product purchased from Matheson, Coleman same absorption as that purified by the method Company,
St. Louis,
Missouri.
and Bell of Liddle
was and
26
CARR
AND
PRESSMAN
as eluting agents. The level of 0.02 M HCl as described above was found optimal. An incubation period of 1 hr at 45°C was found to be adequate for the oxidation of uric acid by the amount of uricase used. A 2-hr period is used as a safety factor. TABLE NORMAL
1 VALUES
Dog serum
Number Mean S.D.
Human
64 0.30 0.155
serum
38 4.19 1.22
Assays for uric acid were linear with concentration over a wide range. Following passage of aqueous uric acid solutions through the columns the values obtained were somewhat low. This error was decreased when assaying uric acid solutions prepared in deionized serum and no longer appeared significant when uric acid was dissolved in untreated dog serum (see Table 2). TABLE RECOVERY
OF URIC
ACID
2 FROM
VARIOUS Recovery
Added h3 %I 10.0
7.5 5.0 2.5
Water 93
90 89 83
SOLVENTS (% )
Deionized SCX”Xl
Dog SW”ll
98 96 96 94
102 98 98 98
It is evident that the presence of other anions in plasma, as well as the protein, plays a significant role in facilitating subsequent elution of uric acid from the resin. Comparison of elution graphs of both aqueous uric acid and a serum solution in the same concentration range of uric acid shown in Fig. 1 further illustrates this point. Accordingly, the use of this method for additional types of biological fluids should be checked out by running recovery determinations in the presence of the fluid. Dowex 2-X8 in the chloride form has been used by several investigators (4, 6) to separate uric acid from interfering components present in biological fluids. Since the resin is known to have a much stronger affinity for chloride ion t.han for organic ions, it follows that uric acid should displace acetate with greater ease than it can chloride. Accordingly, urate ions are bound with greater ease by the acet,ate column with
URIC
actn
IN
Aqueous
SERUM
Solution
,?/c
I
FIG.
1. Effect
of extraneous
2 3 ml of Elude
ions
4
on the elution
5
of uric
acid
from
resin
no sacrifice of their susceptibility to subsequent elution by acidic chloride solutions. This reasoning has been corroborated by us experimentally; under otherwise identical conditions, a given specimen giving 95% recovery from acetate columns resulted in an 85% recovery of uric acid from equivalent chloride columns. SUMMARY
A method is described for the determination of uric acid in serum which is sufficiently precise and reliable to follow small changes at low levels. The advantage of the use in the acet,ate form of the anion exchange resin Dowex 2-X8 is pointed out. The influence of protein and ot,her anion components of serum on the absorbance of uric acid and on its elution from the resin is noted. Normal values of serum uric acid in dogs as well as man, with sampling deviations, and reproducibility of the method at both levels is demonstrated. REFERENCES Chem. 167, 429 (1947). E., AND POULSON, H., Stand. J. Clin. & Lab. Invest. 5, 273 (1953). T. V., AND WRENN. T. W., Am. J. Clin. Path. 25, 833 (1955). SHAPIRO, B., SELIGSON, D., AND JESSAR, R., Ch. Chem. 3, 169 (1957). LIDDLE, L., SEEGMILLER, J. E., AND LASTER, L., J. Lab. C&n. Med. 54, 903 (1959). SAMBHI, M. P., AND GROLLMAN, A., Clin. Chem. 5, 623 (1959). IVHARTON, M. L., AND WHARTON, D. R. A., Ad. Biochcm. 1, 213 (1960).
1. KALCK.IR, 2. PRAETORIUS, 3. FEICHTMEIR, .I. 5. 6.
7.
H.
M.,
J. Biol.
4, 24-27 (1962)
BIOCHEMISTRY
An
Improved
Method Uric
MARIE From
the
Acid
H. CARR’
Research
and Administration
for in
Determination Serum1
AND BERTON
Radioisotope Hospital, Received
C. PRESSMAN3
Laboratories, Kansas City,
January
of
11,
Kansas Missouri
City
Veterans
1962
The large numbers of papers dealing with the determination of uric acid which have appeared in the literature in recent years imply a dissatisfaction of analysts with current methods (l-7). Prevalent colorimetric procedures lack specificity and hence reliability, while the enzymic methods employing uricase are complicated by technical problems arising from the ultraviolet absorption of serum protein. This is particularly troublesome in the case of experimental animals having low serum uric acid levels. Since one of our studies involved the following of slight changes in serum uric acid levels in dogs, we were motivated to develop a reliable method giving good precision at low uric acid concentrations. METHOD
Principle
Separation of uric acid from prot.ein is accomplished by passage of serum or other biological fluid through a strong anion-exchange resin in the acetate form. It has been found that such a column removes the uric acid from the fluid with greater ease and consequently is more reliable than the chloride form previously recommended (4, 6). Following the removal of extraneous UV absorbing materials by washing, the uric acid is eluted from the column and determined by enzymic assay. Materials
and Reagents
Ion-Exchange Resin. Dowex 2-X8 (100-206 mesh) in the acetate form. This resin is purchased in the chloride form and converted to the acetate ‘Supported in part by Research Grant C-4715 for U. S. Public Health Service. A preliminary account of this work was presented at the 139th National Meeting of the American Chemical Society at St. Louis, Missouri, April, 1961. ’ Deceased, April 28, 1962. 3 Present address: Johnson Foundation, University of Pennsylvania, Philadelphia 4, Pennsylvania.
24
URIC
ACID
IN
25
SERUM
form by washing on a column with 10% sodium acetate until tests for chloride in the effluent are not more than faintly positive. Standards. Uric acid stock, 1 mg/ml, is prepared by dissolving uric acid4 in M/15 glycine buffer. This solution is stable almost indefinitely if stored frozen in small vials. For a working standard, the stock solution is diluted 0.5 ml to 10 ml wit,h water. For assay purposes, 0.5 ml of the working standard is diluted to 6 ml with &1/15 glycine buffer to correspond to the dilution of the biological fluids analyzed. Eluant. To 500 ml of 0.04 M HCl add 500 ml M NaCl. Glycine Buffer, 95 M, pH 9.35. Glycine Buffer, M/15. This solution is prepared by diluting 10 ml of the sJSM buffer to 100 ml with water. Uricase,” purified, 125 units/ml. Dilute the amount required for a day’s work 1:20 with M/15 glycine buffer. Procedure 1. Prepare resin columns (2 cm in height X 0.4 cm in diameter). 2. Pass 0.5 ml biological fluid containing 2 to 100 pg uric acid through the column. If whole blood is to be analyzed, a volume of diluted hemolyzed blood equivalent to 0.5 ml whole blood is used. 3. Wash twice with 10 ml H,O. Discard the washings. 4. Elute the uric acid with 5 ml of the HCl-NaCl eluant. 5. Add 1 ml of 2/3M glycine buffer, pH 9.35, to which has been added 6.25 ml 10N NaOH per liter to compensate for the HCl in the eluant. 6. Measure the absorption of the buffered eluate at 293 nip. 7. To a 3.5-ml aliquot of buffered eluate, add 0.1 ml of diluted uricase described above, and incubate at 45°C for 2 hr. 8. Redetermine the 293 mp absorption. The decrease following incubation is a measure of the uric acid present,. A standard containing 5.00 mg % uric acid in 1M/15 glycine buffer gave an absorption change of 0.306 OD in t,he enzymic assay. RESULTS
AND
DISCUSSION
The results obtained by this method in the analysis of human and canine subjects are given in Table 1. Reproducibility was 0.013 mg % in dogs at a level of 0.30 mg ‘$ and 0.063 mg % in humans at a level of 4.31 mg %I. Concentrations of HCl in 0.5 M NaCl of 0.050 to 0.005 ii/T were tested ‘The reagent-grade found to have the Associates (5). ‘Sigma Chemical
product purchased from Matheson, Coleman same absorption as that purified by the method Company,
St. Louis,
Missouri.
and Bell of Liddle
was and
26
CARR
AND
PRESSMAN
as eluting agents. The level of 0.02 M HCl as described above was found optimal. An incubation period of 1 hr at 45°C was found to be adequate for the oxidation of uric acid by the amount of uricase used. A 2-hr period is used as a safety factor. TABLE NORMAL
1 VALUES
Dog serum
Number Mean S.D.
Human
64 0.30 0.155
serum
38 4.19 1.22
Assays for uric acid were linear with concentration over a wide range. Following passage of aqueous uric acid solutions through the columns the values obtained were somewhat low. This error was decreased when assaying uric acid solutions prepared in deionized serum and no longer appeared significant when uric acid was dissolved in untreated dog serum (see Table 2). TABLE RECOVERY
OF URIC
ACID
2 FROM
VARIOUS Recovery
Added h3 %I 10.0
7.5 5.0 2.5
Water 93
90 89 83
SOLVENTS (% )
Deionized SCX”Xl
Dog SW”ll
98 96 96 94
102 98 98 98
It is evident that the presence of other anions in plasma, as well as the protein, plays a significant role in facilitating subsequent elution of uric acid from the resin. Comparison of elution graphs of both aqueous uric acid and a serum solution in the same concentration range of uric acid shown in Fig. 1 further illustrates this point. Accordingly, the use of this method for additional types of biological fluids should be checked out by running recovery determinations in the presence of the fluid. Dowex 2-X8 in the chloride form has been used by several investigators (4, 6) to separate uric acid from interfering components present in biological fluids. Since the resin is known to have a much stronger affinity for chloride ion t.han for organic ions, it follows that uric acid should displace acetate with greater ease than it can chloride. Accordingly, urate ions are bound with greater ease by the acet,ate column with
URIC
actn
IN
Aqueous
SERUM
Solution
,?/c
I
FIG.
1. Effect
of extraneous
2 3 ml of Elude
ions
4
on the elution
5
of uric
acid
from
resin
no sacrifice of their susceptibility to subsequent elution by acidic chloride solutions. This reasoning has been corroborated by us experimentally; under otherwise identical conditions, a given specimen giving 95% recovery from acetate columns resulted in an 85% recovery of uric acid from equivalent chloride columns. SUMMARY
A method is described for the determination of uric acid in serum which is sufficiently precise and reliable to follow small changes at low levels. The advantage of the use in the acet,ate form of the anion exchange resin Dowex 2-X8 is pointed out. The influence of protein and ot,her anion components of serum on the absorbance of uric acid and on its elution from the resin is noted. Normal values of serum uric acid in dogs as well as man, with sampling deviations, and reproducibility of the method at both levels is demonstrated. REFERENCES Chem. 167, 429 (1947). E., AND POULSON, H., Stand. J. Clin. & Lab. Invest. 5, 273 (1953). T. V., AND WRENN. T. W., Am. J. Clin. Path. 25, 833 (1955). SHAPIRO, B., SELIGSON, D., AND JESSAR, R., Ch. Chem. 3, 169 (1957). LIDDLE, L., SEEGMILLER, J. E., AND LASTER, L., J. Lab. C&n. Med. 54, 903 (1959). SAMBHI, M. P., AND GROLLMAN, A., Clin. Chem. 5, 623 (1959). IVHARTON, M. L., AND WHARTON, D. R. A., Ad. Biochcm. 1, 213 (1960).
1. KALCK.IR, 2. PRAETORIUS, 3. FEICHTMEIR, .I. 5. 6.
7.
H.
M.,
J. Biol.