BIOCHEMICAL
MEDICINE
14, 147-151
A Modified
GERALD Blood
(1975)
Fluorometric Assay for Adenine in Plasma and Urine
L. MOORE AND MARY EDITH
Research Division, Letterman Presidio of San Francisco, Received
September
LEDFORD
Army Institute of Research, Calfornia 94129 30, 1975
There is current interest in the United States of adding adenine to the anticoagulant into which human blood is drawn for transfusion purposes. Adenine has been used in this capacity for several years in certain European countries. This additive increases the levels of red cell ATP during cold storage, which correlates with the red cell viability and extends the theoretical storage time from 21 to 42 days (1). With these developments occurring in blood banking, it is important to have a sensitive, specific assay for adenine in plasma and urine samples from recipients of the adenine fortified blood. The older spectrophotometric assays for adenine are lacking in sensitivity and specificity, particularly when applied to biological fluids containing many interfering substances; for example, the calorimetric complex method of Davis (2), while excellent for aqueous solutions, gives very high background values when used in plasma and urine samples. Yuki et al. (3) have developed a fluorometric assay for aqueous adenine samples which is lOO-fold more sensitive than the spectrophotometric methods. The assay is specific for adenine and its nucleotides, and will not react with other purines, their nucleotides, or substituted adenine. This assay involves complexation of the adenine with glyoxal hydrate trimer in acid media to give a fluorescent aldehyde addition compound; which, when excited at 328 nM, will emit fluorescence at 382 nM. Initial attempts to measure adenine in plasma and urine were made by adding aliquots of these fluids containing known adenine concentrations to glyoxal reagent, according to Yuki. Although the standard curve (aqueous standards) appeared reasonable, the plasma samples showed scattering and the urine samples were discolored and quenched. In order to use the fluorometric assay in plasma and urine, it was necessary to modify the sample preparation, standard curve composition and preparation of the glyoxal reagent. This report presents the required modifications. 147 Copyright All right-
0 1975 by Academic Press. Inc. of reproduction in any form reserved.
148
MOORE
MATERIALS
AND
LEDFORD
AND METHODS
Glyoxal hydrate trimer was obtained from either Matheson, Coleman & Bell or Sigma Chemical Company. Glacial acetic acid and reagent grade trichloroacetic acid were from Allied and Baker & Adamson Co., respectively. Adenine was from the Sigma Chemical Company. All water was deionized, glass distilled. Glassware was washed in 40% nitric acid. Initial studies were made on an Aminco-Bowman Spectrophotofluorometer. but for speed and convenience the procedure was adapted to a Farrand Ratio Fluorometer using a 333 nm primary filter and a 5-59 secondary filter. The glyoxal reagent was prepared by adding 105 g of glyoxal hydrate trimer to 400 ml of distilled water and dissolving with the aid of gentle heating and stirring. Three grams of Norit A were added to the hot solution, stirred 10 min, then filtered through Whatman No. 1 paper. The cooled. filtered solution was brought up to 500 ml volume. The volume of this reagent needed for a given day was mixed with 3 vol of glacial acetic acid. The preparation of plasma samples was carried out by diluting the plasma 1: 10 with water, and adding an equal volume of 10% (w/v) trichloroacetic acid solution to precipitate the plasma proteins. These solutions were maintained at 0°C for 15 min then centrifuged at 0°C for 15 min at 27,OOOg. Aliquots of 1.O ml of supernatant were mixed with 4.0 ml of glyoxal-acetic acid solution in 15 x 150mm test tubes, the tubes were capped with marbles then placed in a boiling water bath for 3 hr. Boiling action should be gentle so as not to drive solvent from the tubes, but the bath temperature must be above 98°C for complete reaction. The tubes were removed from the bath, cooled, remixed, and read in the fluorometer within 5 hr against a plasma standard curve. Adenine in urine can be assayed by a modified procedure. Urine was diluted 1:20 with water: 1.O ml of the diluted sample was mixed with 4.0 ml glyoxal-acetic acid solution and treated as above, using a urine standard curve. Development of a standard curve for this assay started with carefully weighed additions of adenine to water. When assaying adenine in plasma. it was found best to use pooled plasma as the solvent, and likewise when assaying in urine to use pooled urine as solvent. Several concentrations between 0 and 60pg/ml were used in plasma and 0 to lOO~g/ml were added to pooled urine. Large volumes of the standard solutions were made up and frozen as aliquots which were thawed as needed for the standard curve used with each day’s analysis. Plasma or urine samples or standards may be stored frozen for at least 4 months at -20°C prior to assay with no loss of recovery. RESULTS AND DISCUSSION Since the fluorometric adenine assay of Yuki could not be applied directly to samples of plasma and urine, it wah necessary to modify the
ADENINE
IN PLASMA
AND
149
URINE
samples prior to complexation with glyoxal. When plasma was the sample solution, the interference of fluorescence was eliminated by preparing a protein-free filtrate using an equal volume of 10% trichloroacetic acid (4). When the adenine was contained in urine, total quenching was observed following the glyoxal reaction. This was apparently an interaction of two substances, one in urine and one in the glyoxal reagent itself. If the urine sample was diluted at least 1:20 and the glyoxal processed with charcoal, this interaction was reduced to negligible levels, producing good recoveries. Standard curves, madeup in water, pooled plasma, or pooled urine are shown in Fig. 1. Linearity is good in all of the curves. The concentration range chosen was most useful for adenine toxicity studies being done in our laboratory (5). If desired, the concentration range can be scaled down at least IO-fold. When low concentrations of adenine were measured in toxicity studies (0.1 to 5pg/ml), the standard curve was set up to reflect full scale between 0 and OSO~g/ml. Total initial dilutions of the plasma were 1: 10 for on-scale heading. When running the plasma adenine assay daily, there were some days when recovery of standard adenine plasma samples was low compared to a standard curve using aqueous adenine. This was felt to be due to variations in the protein precipitation step. This problem was eliminated by making the standard curve from adenine dissolved in a pool of several plasma samples. The standards (0, 15, 30, 50, 60 pg/ml) were treated in the same manner as the samples, resulting in consistent high recovery of adenine. In a similar fashion, when adenine was assayed in urine, the standard curve was made from adenine dissolved in a pool of several urine samples. Recovery studies were done by adding known concentrations of adenine solutions to random individual plasma or urine samples and assaying as above. The results are shown in Table 1 where it can be seen that recovery from plasma was near 100% down to at least 20 pg/ml and above 90% as low as 5 pg/ml. Recoveries in urine average
IAd.nin.1
,,qo/ml
(Ad.nin.)
,,g,ml
FIG. I. Sample standard curves obtained from the Farrand fluorometer where standard concentrations of adenine were dissolved in (A)---water, (Btpooled human plasma, and (C)--pooled human urine. Each point represents mean offour measurements with coefficients of variation t 2.0 or less.
150
MOORE
.4denine
added
AND
L.EDFORD
4denine
pglml
recovered
pLgi.ml”
Plasma (Mean
Z.O(R; 70.0 LV 25.0 (‘V 50.0 60.0 100.0
(!v = CR: = (V =
” Values recoveries
~. IO) = X) = IO) IO) IO) I I)
given also
Q)
‘4.53
f
12.367
19.7
2
I.OIci
60.7
as mean
t
with
It
2.34r;
coefficient
i. i~ne I’.; )
( hlean
f
(21
(‘J 1 .-
90.6 9x i -.. IOO. 1 .-.
of variation
23. 46.’
q
94 7 ^
a\ percentage
lO.l?r 5,731,
94.x ‘)3.-t -..
-I.h?,
‘!I’
-
I(.)).
Percentage
presented.
93-95s. Precision is shown by the coefficients of variations indicated fog each set of values. The preparation of the glyoxal reagent is critical to the performance of this assay. If prepared according to Yuki (3). it cannot be used with urine samples because of quenching. Also some lots of glyoxal hydrate trimer have a yellow coloration when dissolved in water, instead of being colorless. The presence of this color will cause loss of precision in the plasma assay as well as partial quenching if the final samples are diluted prior to reading in the fluorometer. By treating the hot aqueous glyoxal solution with charcoal and filtering, the color is removed from this solution yielding a reagent that gives high precision with plasma and is also effective with urine samples. This modified fluorometric adenine assay is accurate, precise. and specific when used on plasma and urine samples. In our laboratory. the assay was set up for use in the 10 to 60pgiml range. By changing the standard curve and the initial dilutions, the assay can be dropped to a IO-fold lower range. The presence of 7,8-dioxyadenine. uric acid. hypoxanthine, or xanthine does not interfere with the assay. While the assay was set up to study the metabolism and toxicity of adenine when used as part of a blood anticoagulant solution, it may also be useful in monitoring patients being treated by therapeutic administration of adenine for nucleotide metabolism deficiencies such as Lesch-Nyhan syndrome (6). SUMMARY A fluorometric adenine assay, using glyoxal trimer to form an adenine fluorophore. was modified to allow measurement of adenine in sample\ of
ADENINE
IN PLASMA
AND
URINE
151
plasma and urine. The assay was set up to be accurate and precise in the 10-60 &ml range, but can be used at IO-fold lower concentrations. Recoveries of adenine added to plasma were above 98%, and similar additions of adenine to urine gave 93-95% recovery in the 25-100 p&ml range. The technique may be of value in monitoring the plasma and urine of patients receiving adenine as part of a blood transfusion, or as a therapeutic treatment for metabolic diseases involving purine nucleotides. REFERENCES 1. 2. 3. 4. 5. 6.
Simon, E. R., Transfusion 7, 395 (1967). Davis. J., and Morris, R.. Anal. Biochem. 5, 64 (1963). Yuki. H., Sempuku, C., Park, M., and Takiura, K., Anal. Biochem. 46, 123 (1972). Beutler. E., “Red Cell Metabolism,” Chap. 3. Grune and Stratton, New York, 1971 Roth, G., Moore, G., Kline, W., and Poskitt, T., Transfusion 15, 116 (1975). Beyer, P., Bieth, R., Lutz, D., and Geisert, J., Arch. Fr. Ped. 32, 293 (1975).
MEDICINE
14, 147-151
A Modified
GERALD Blood
(1975)
Fluorometric Assay for Adenine in Plasma and Urine
L. MOORE AND MARY EDITH
Research Division, Letterman Presidio of San Francisco, Received
September
LEDFORD
Army Institute of Research, Calfornia 94129 30, 1975
There is current interest in the United States of adding adenine to the anticoagulant into which human blood is drawn for transfusion purposes. Adenine has been used in this capacity for several years in certain European countries. This additive increases the levels of red cell ATP during cold storage, which correlates with the red cell viability and extends the theoretical storage time from 21 to 42 days (1). With these developments occurring in blood banking, it is important to have a sensitive, specific assay for adenine in plasma and urine samples from recipients of the adenine fortified blood. The older spectrophotometric assays for adenine are lacking in sensitivity and specificity, particularly when applied to biological fluids containing many interfering substances; for example, the calorimetric complex method of Davis (2), while excellent for aqueous solutions, gives very high background values when used in plasma and urine samples. Yuki et al. (3) have developed a fluorometric assay for aqueous adenine samples which is lOO-fold more sensitive than the spectrophotometric methods. The assay is specific for adenine and its nucleotides, and will not react with other purines, their nucleotides, or substituted adenine. This assay involves complexation of the adenine with glyoxal hydrate trimer in acid media to give a fluorescent aldehyde addition compound; which, when excited at 328 nM, will emit fluorescence at 382 nM. Initial attempts to measure adenine in plasma and urine were made by adding aliquots of these fluids containing known adenine concentrations to glyoxal reagent, according to Yuki. Although the standard curve (aqueous standards) appeared reasonable, the plasma samples showed scattering and the urine samples were discolored and quenched. In order to use the fluorometric assay in plasma and urine, it was necessary to modify the sample preparation, standard curve composition and preparation of the glyoxal reagent. This report presents the required modifications. 147 Copyright All right-
0 1975 by Academic Press. Inc. of reproduction in any form reserved.
148
MOORE
MATERIALS
AND
LEDFORD
AND METHODS
Glyoxal hydrate trimer was obtained from either Matheson, Coleman & Bell or Sigma Chemical Company. Glacial acetic acid and reagent grade trichloroacetic acid were from Allied and Baker & Adamson Co., respectively. Adenine was from the Sigma Chemical Company. All water was deionized, glass distilled. Glassware was washed in 40% nitric acid. Initial studies were made on an Aminco-Bowman Spectrophotofluorometer. but for speed and convenience the procedure was adapted to a Farrand Ratio Fluorometer using a 333 nm primary filter and a 5-59 secondary filter. The glyoxal reagent was prepared by adding 105 g of glyoxal hydrate trimer to 400 ml of distilled water and dissolving with the aid of gentle heating and stirring. Three grams of Norit A were added to the hot solution, stirred 10 min, then filtered through Whatman No. 1 paper. The cooled. filtered solution was brought up to 500 ml volume. The volume of this reagent needed for a given day was mixed with 3 vol of glacial acetic acid. The preparation of plasma samples was carried out by diluting the plasma 1: 10 with water, and adding an equal volume of 10% (w/v) trichloroacetic acid solution to precipitate the plasma proteins. These solutions were maintained at 0°C for 15 min then centrifuged at 0°C for 15 min at 27,OOOg. Aliquots of 1.O ml of supernatant were mixed with 4.0 ml of glyoxal-acetic acid solution in 15 x 150mm test tubes, the tubes were capped with marbles then placed in a boiling water bath for 3 hr. Boiling action should be gentle so as not to drive solvent from the tubes, but the bath temperature must be above 98°C for complete reaction. The tubes were removed from the bath, cooled, remixed, and read in the fluorometer within 5 hr against a plasma standard curve. Adenine in urine can be assayed by a modified procedure. Urine was diluted 1:20 with water: 1.O ml of the diluted sample was mixed with 4.0 ml glyoxal-acetic acid solution and treated as above, using a urine standard curve. Development of a standard curve for this assay started with carefully weighed additions of adenine to water. When assaying adenine in plasma. it was found best to use pooled plasma as the solvent, and likewise when assaying in urine to use pooled urine as solvent. Several concentrations between 0 and 60pg/ml were used in plasma and 0 to lOO~g/ml were added to pooled urine. Large volumes of the standard solutions were made up and frozen as aliquots which were thawed as needed for the standard curve used with each day’s analysis. Plasma or urine samples or standards may be stored frozen for at least 4 months at -20°C prior to assay with no loss of recovery. RESULTS AND DISCUSSION Since the fluorometric adenine assay of Yuki could not be applied directly to samples of plasma and urine, it wah necessary to modify the
ADENINE
IN PLASMA
AND
149
URINE
samples prior to complexation with glyoxal. When plasma was the sample solution, the interference of fluorescence was eliminated by preparing a protein-free filtrate using an equal volume of 10% trichloroacetic acid (4). When the adenine was contained in urine, total quenching was observed following the glyoxal reaction. This was apparently an interaction of two substances, one in urine and one in the glyoxal reagent itself. If the urine sample was diluted at least 1:20 and the glyoxal processed with charcoal, this interaction was reduced to negligible levels, producing good recoveries. Standard curves, madeup in water, pooled plasma, or pooled urine are shown in Fig. 1. Linearity is good in all of the curves. The concentration range chosen was most useful for adenine toxicity studies being done in our laboratory (5). If desired, the concentration range can be scaled down at least IO-fold. When low concentrations of adenine were measured in toxicity studies (0.1 to 5pg/ml), the standard curve was set up to reflect full scale between 0 and OSO~g/ml. Total initial dilutions of the plasma were 1: 10 for on-scale heading. When running the plasma adenine assay daily, there were some days when recovery of standard adenine plasma samples was low compared to a standard curve using aqueous adenine. This was felt to be due to variations in the protein precipitation step. This problem was eliminated by making the standard curve from adenine dissolved in a pool of several plasma samples. The standards (0, 15, 30, 50, 60 pg/ml) were treated in the same manner as the samples, resulting in consistent high recovery of adenine. In a similar fashion, when adenine was assayed in urine, the standard curve was made from adenine dissolved in a pool of several urine samples. Recovery studies were done by adding known concentrations of adenine solutions to random individual plasma or urine samples and assaying as above. The results are shown in Table 1 where it can be seen that recovery from plasma was near 100% down to at least 20 pg/ml and above 90% as low as 5 pg/ml. Recoveries in urine average
IAd.nin.1
,,qo/ml
(Ad.nin.)
,,g,ml
FIG. I. Sample standard curves obtained from the Farrand fluorometer where standard concentrations of adenine were dissolved in (A)---water, (Btpooled human plasma, and (C)--pooled human urine. Each point represents mean offour measurements with coefficients of variation t 2.0 or less.
150
MOORE
.4denine
added
AND
L.EDFORD
4denine
pglml
recovered
pLgi.ml”
Plasma (Mean
Z.O(R; 70.0 LV 25.0 (‘V 50.0 60.0 100.0
(!v = CR: = (V =
” Values recoveries
~. IO) = X) = IO) IO) IO) I I)
given also
Q)
‘4.53
f
12.367
19.7
2
I.OIci
60.7
as mean
t
with
It
2.34r;
coefficient
i. i~ne I’.; )
( hlean
f
(21
(‘J 1 .-
90.6 9x i -.. IOO. 1 .-.
of variation
23. 46.’
q
94 7 ^
a\ percentage
lO.l?r 5,731,
94.x ‘)3.-t -..
-I.h?,
‘!I’
-
I(.)).
Percentage
presented.
93-95s. Precision is shown by the coefficients of variations indicated fog each set of values. The preparation of the glyoxal reagent is critical to the performance of this assay. If prepared according to Yuki (3). it cannot be used with urine samples because of quenching. Also some lots of glyoxal hydrate trimer have a yellow coloration when dissolved in water, instead of being colorless. The presence of this color will cause loss of precision in the plasma assay as well as partial quenching if the final samples are diluted prior to reading in the fluorometer. By treating the hot aqueous glyoxal solution with charcoal and filtering, the color is removed from this solution yielding a reagent that gives high precision with plasma and is also effective with urine samples. This modified fluorometric adenine assay is accurate, precise. and specific when used on plasma and urine samples. In our laboratory. the assay was set up for use in the 10 to 60pgiml range. By changing the standard curve and the initial dilutions, the assay can be dropped to a IO-fold lower range. The presence of 7,8-dioxyadenine. uric acid. hypoxanthine, or xanthine does not interfere with the assay. While the assay was set up to study the metabolism and toxicity of adenine when used as part of a blood anticoagulant solution, it may also be useful in monitoring patients being treated by therapeutic administration of adenine for nucleotide metabolism deficiencies such as Lesch-Nyhan syndrome (6). SUMMARY A fluorometric adenine assay, using glyoxal trimer to form an adenine fluorophore. was modified to allow measurement of adenine in sample\ of
ADENINE
IN PLASMA
AND
URINE
151
plasma and urine. The assay was set up to be accurate and precise in the 10-60 &ml range, but can be used at IO-fold lower concentrations. Recoveries of adenine added to plasma were above 98%, and similar additions of adenine to urine gave 93-95% recovery in the 25-100 p&ml range. The technique may be of value in monitoring the plasma and urine of patients receiving adenine as part of a blood transfusion, or as a therapeutic treatment for metabolic diseases involving purine nucleotides. REFERENCES 1. 2. 3. 4. 5. 6.
Simon, E. R., Transfusion 7, 395 (1967). Davis. J., and Morris, R.. Anal. Biochem. 5, 64 (1963). Yuki. H., Sempuku, C., Park, M., and Takiura, K., Anal. Biochem. 46, 123 (1972). Beutler. E., “Red Cell Metabolism,” Chap. 3. Grune and Stratton, New York, 1971 Roth, G., Moore, G., Kline, W., and Poskitt, T., Transfusion 15, 116 (1975). Beyer, P., Bieth, R., Lutz, D., and Geisert, J., Arch. Fr. Ped. 32, 293 (1975).
