BIOMEDICAL CHROMATOGRAPIIY, VOL. 5 , 235-239 (1991)
RE VIEW PAPER
The Measurement of Nucleoside Deaminases by High Performance Liquid Chromatography and their Use in Clinical Chemistry R. A. Sherwood Department of Clinical Biochemistry, Kings College School of Medicine and Dentistry, Denmark Hill, London SE5 9RS, UK
The measurement of the nucleoside deaminases-cytidine deaminase, guanosine deaminase and adenosine deaminase-by reversed phase high performance liquid chromatography is reviewed. The clinical value of assaying the enzyme activity is discussed for each of these enzymes. Both cytidine deaminase and adenosine deaminase measurements have proven clinical value, although the use of the assay of cytidine deaminase in the diagnosis of pre-eclampsia is probably not helpful. ~
INTRODUCTION The nucleoside deaminases are a family of enzymes involved in the purine pathways, which catalyse important steps in the formation and degradation of DNA. The value to clinical chemists of measuring these enzymes has only been investigated relatively recently as methods have become available for the specific determination of their activities. The assay of three of the deaminases in particular-cytidine deaminase, guanosine deaminase and adenosine deaminase-has been reported to be of clinical value. Initially the methods developed to determine the activity of these enzymes were based on the measurement of the ammonia produced during the deamination of the enzyme substrate, using the Berthelot method. This procedure has a number of drawbacks and therefore high Performance liquid chromatographic (HPLC) methods have been developed. These are based on the measurement of the change in the nucleoside concentrations, thus reducing the likelihood of contamination from exogenous ammonia. The development of column packings for reversed phase HPLC has resulted in methods becoming available for the rapid separation of the nucleosides and nucleotides commonly encountered in body fluids. These methods have been extensively used to study the purine and pyrimidine pathways in man and animals. Application of the technique to the assay of the activity of the enzymes involved in these pathways was a logical advance. In this paper the methods developed for the assay of the above-mentioned enzymes are reviewed and evidence is given for a clinical role for the measurements.
METHODS AND RESULTS Cytidine deaminase Cytidine deaminase (CD) (E.C. 3.5.4.5) catalyses the conversion of cytidine to uridine and deoxycytidine to deoxyuridine. It also catalyses the hydrolytic deamina0269-3879/91/060235-05 $OS.OO
01991 by John Wiley & Sons. Ltd.
tion of some of the pharmacological analogues of cytidine. Although its exact physiological role is not known, CD is a cytoplasmic enzyme primarily found in liver and polymorphonuclear leucocytes. CD leaks out of damaged cells and can be detected in the blood o r synovial fluid in a number of disease states (Jones and Roberts, 1984). In particular, inflammatory diseases such as ulcerative colitis and rheumatoid arthritis (RA) show increased CD activity. Thompson et al. (1986a) reported that serum CD activity was raised in R A compared with osteoarthritis (OA). The enzyme activity in synovial fluid was also measured and found to be less than that in serum in patients with OA but up to 22 times higher than in serum in RA. This group have also demonstrated that there is some relationship between serum CD activity and other well-established indices of joint inflammation such as erthyrocyte sedimentation rate (ESR), Ritchie articular index score, etc. (Thompson et al. 1986b). These results suggest that neutrophils in the synovial fluid release CD into the synovial fluid from which it diffuses into the blood. This group have subsequently used serum CD measurements in their assessment of RA patients and have shown a sharp increase in serum CD activity within two days of withdrawal of non-steroidal anti-inflammatory drugs (NSAIDs) from patients with RA, which pre-
Tablel. Conditions for the measurement of CD by HPLC (Richards et al., 1987) Column Eluent
ODs-Hypersil 5p (125 m m x4.6 mm id.) 3% Methanol in 0.1 M potassium phosphate buffer, pH 5.5 1 mL/min, isocratic Flow rate Injection volume 20 p.L Internal standard 5-Fluorouridine SarnDle volume 100 WLserum incubated with cytidine for 2 h at 37 “C Detection UV 260 nm Retention times Cytidine 2.8 rnin Uridine 4.1 rnin 5-Fluorouridine 5.9 min
Received 14 September 1990 Accepted 30 October 1990
R. A . SHERWOOD
236
0
1
2
3
4
5
6
7
0
1
2
11
5
6
Figure 1. Chromatogram of CD assay by reversed phase HPLC. The chromatographic conditions are given in Table 1. Peaks are (1) cytidine, (2) uridine, (3)5-fluorouracil. (a) Aqueous standard solution of cytidine, uridine and 5-fluorouridine; (b) serum (100 pL) incubated with cytidine for 2 h at 37 "C.
cedes the change in other parameters by 2-3 days (Thompson et al. 1988). They postulated that this might be due to an influx of polymorphs into joint spaces because of the removal of the inhibitory effects of NSAlDs resulting in increased CD release. The early work on CD was carried out using the method first described by Jones et al. (1982). This involved incubation of serum (100 pL) with deoxycytidine in carbonate/bicarbonate buffer (pH 9.2) for 18 h at 22 "C. The ammonia produced was estimated using the Berthelot reaction. These workers had previously measured deoxycytidylate deaminase (E.C. 3.5.4.12) and had demonstrated elevated dCMP deaminase activity in patients with pre-eclampsia and other causes of high-risk pregnancies (Williams and Jones, 1975,1982). CD is more stable than deoxycytidylate deaminase and Jones et al. (1982) claimed that the discrimination between normal and high-risk pregnancies was as good when CD was measured as when dCMP deaminase was used. Baines and Clark (1985) also investigated the use of serum CD as a marker for pre-eclampsia using a similar method but with the ammonia determined after incubation of serum with substrate for only 2 h at 37 "C, using the glutamate dehydrogenase end-point method Table2. Conditions for the measurement of CD activity by HPLC with ion pairing (James et al., 1989) ODs-Hypersil 5 p (100 mm ~ 4 . mm 6 i.d.) 1% Methanol in 0.1 M ammonium acetate with 1 mM 1-octanesulphonic acid 1.2 mllmin, isocratic Flow rate Injection volume 20 FL Internal standard Allopurinol Sample volume 100 pL serum incubated with cytidine for 20 min at 56 "C Detection UV 262 nm Retention times Uridine 1.9 min Allopurinol 2.8 min Cytidine 4.3 min Column Eluent
described by Targett-Adams et al. (1975). There was a significant overlap in their study between serum CD activity in normal pregnancies and pre-eclampsia. There were a large number of false positive elevations of CD activity in normal pregnancies (8.6%) while only 42% of samples from patients with pre-eclampsia had increased serum CD activity. Measurement of the ammonia, by whatever method, is subject to potential interference from exogenous ammonia from the environment. In an attempt to eliminate this possible error an HPLC assay was developed for CD (Richards et al., 1987). As in the original spectrophotometric method serum (100 pL) was incubated with cytidine as substrate but the incubation used was reduced to 2 h at 37 "C. The uridine produced was separated from the substrate using reversed phase HPLC with an ODs-Hypersil column, isocratic elution and UV detection at 260 nm. The parameters used are given in Table 1 and representative chromatograms are shown in Fig. 1. The HPLC method was used to assay serum CD activity in 45 women with normal pregnancies and 10 with pre-eclampsia (Sherwood et al., 1987). The results obtained were similar to those obtained by Baines and Clark in that, although the mean CD activity of the preeclampsia group was higher (18.0) than that of the normal group (10.7), there was a significant overlap between the two populations. Measurement of CD would not appear to offer any additional benefits in the detection and monitoring of pre-eclampsia. A reversed phase HPLC method for the measurement of CD activity in synovial fluid, which was very similar to that of Richards et al. (1987), was reported by Herbert et al. (1989). The incubation time had been shortened to 10 min at 56 "C and detection was by UV absorption at 280 nm. This group have since improved on existing methods by the use of an ion pairing agent (1-octanesulphonic acid, OSA) with reversed phase HPLC (James et al., 1989). The addition of OSA caused the retention order of cytidine and uridine to
237
HPLC OF NUCLEOSIDE DEAMTNASE
l3
2 0.0051
-
N Q N
JI
P
0
1
2
3
5
4
6
0
1
2
4
3
5
5
Figure 2. Chromatograms for the assay of CD by HPLC by ion pair reversed phase HPLC. The chromatographic conditions are given in Table 2. The peaks are (1) uridine, (2) allopurinol, (3) cytidine. (a) Standard mixture of uridine, allopurinol and cytidine; (b) serum (100 pL) incubated with cytidine for 20 min a t 56 "C.
reverse so that uridine, which is the product, eluted before the much larger cytidine peak, the substrate. The conditions used are given in Table 2 and representative chromatograms are shown in Fig. 2. The method
has considerably improved the limit of detection for uridine and hence for CD. A clear role has been established for the measurement of CD in serum and synovial fluid for the assess-
1
3
3
f
0
.n
N
v a
m
D
L 0
W 4 5 0)
0
m D 0
D
=c
2
10
Figure 3. Chromatograms for the assay of guanosine deaminase by HPLC. The chromatographic conditions are as given in Table 1 except the flow rate was 1.2 mL/min. Peaks are (1) 5-fluorouridine. (2) xanthosine, (3) guanosine. (a) Standard mixture of 5fluorouridine, xanthosine and guanosine; (b) serum (100 $1 incubated with guanosine for 2 h at 37 "C.
R. A . SHERWOOD
238 ~
~
~
Table 3. Conditions for the measurement of adenosine deaminase activity by HPLC (Hartwick et d.,1978) ODS-Hypersil 10 p (300 m m x 4 mm i.d.1 14% Methanol in 0.01 M phosphate buffer, pH unadjusted Flow rate 2 mLlmin, isocratic Injection volume 5 pL Internal standard Not used Sample volume 200 pL packed erythrocytes added to 200 pL water incubated with 1 mL adenosine (3.5 mM) for 15 min at 25 "C Detection UV 254 nm Retention times Hypoxanthine 2.0 min lnosine 2.5 min Adenosine 6.5 min
Column Eluent
ment of RA. The HPLC methods described above are superior to the original methodology employed, with a considerably reduced risk of Contamination.
Guanosine deaminase Guanosine deaminase (E.C. 3.5.4.15) is an interconverting enzyme in DNA metabolism which catalyses the deamination of guanosine to xanthosine which is then converted to xanthine and on to uric acid (Jones and Roberts, 1984). Few studies have been carried out on this enzyme, but Jones et al. (1983) stated that guanosine deaminase activity was normal in those cases of pre-eclampsia and intrauterine death where CD activity was elevated. This group reported that guanosine deaminase activity was elevated in certain liver diseases, particularly infective hepatitis and metastatic
carcinoma but normal in alcoholic liver disease and obstructive jaundice. They proposed that it might prove to be a useful "liver function test". The method used was based on the method for CD with the ammonia released after incubation of serum with guanosine for 18 h at 22 "C, measured by the Berthelot reaction. The reversed phase HPLC method for CD described by Richards et al. (1987) was adapted to permit the measurement of guanosine deaminase activity. Serum (100 pL) was incubated with guanosine (2.6 mM, 100 pL) for 2 h at 37 "C. The guanosine consumed in the reaction was measured using the HPLC conditions shown in Table 1, with the exception of the flow rate which was increased to 1.2mL/min. The retention times for the three compounds of interest were as follows: 5-fluorouridine, 4.9 min; xanthosine, 7.8 min; guanosine, 8.1 min. A typical chromatogram for the standards and a sample after incubation with substrate is shown in Fig. 3. For this enzyme, in contrast to CD, the elution order of product (hypoxanthine, xanthine and xanthosine) followed by substrate (guanosine) is optimal and there is no need to use ion pairing agents. The performance characteristics of the method are currently under evaluation.
Adenosine deaminase Adenosine deaminase has a wide distribution in tissues and is an important enzyme in the metabolic pathway of the adenosine nucleotides, catalysing the conversion of adenosine to inosine, and in the production of hypoxanthine which is utilized in the purine salvage pathways. Deficiency of adenosine deaminase
c
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
Figure 4. Chromatograms for the assay of adenosine deaminase by HPLC. The chromatographic conditions are given in Table 3. Peaks are (1) hypoxanthine, (2) inosine, (3) adenosine. (a) Standard solution of hypoxanthine, inosine and adenosine; (b) erythrocytes incubated with adenosine for 15 min at 25 "C.
HPIC OF NUCLEOSTDE DEAMINASE
(E.C. 3.5.4.4) has been found in up to one third of subjects with inherited severe combined immunodeficiency (SCID) since it was first reported in 1972 (Giblett et af.,1972). The measurement of adenosine deaminase activity is obviously essential for the diagnosis of the condition. There is some evidence for the use of adenosine deaminase measurements in the differential diagnosis of tuberculous pericarditis (Piras et al., 1978). Adenosine deaminase activity has also been reported to be increased three-fold in subjects with HIV infection (Delia ef al., 1987). Various methods have been described for the measurement of adenosine deaminase based on either the spectrophotometric (Hirschhorn et al., 1975) or radioisotopic (Coleman and Hutton, 1975) measurement of the inosine produced. Hartwick et af. (1978) described an HPLC method for adenosine deaminase based on the measurement of the decrease in adenosine concentration after incubation with the sample. These workers were measuring adenosine deaminase in erythrocytes (200 pL) which were lysed by the addition of an equal volume of water and then incubated with 1 m L of adenosine ( 3 . 5 m ~ for ) 15 min at 25°C at which time the reaction was stopped by raising the temperature to 100 "C for 45 s. The chromatographic conditions used are shown in Table 3 and representative chromatograms are shown in Fig. 4. As in the assay of guanosine deaminase the order of separation is optimal with the reaction productshypoxanthine and inosine-both eluting before the substrate peak. In this case the decrease in substrate concentration was measured rather than the sum of the reaction products.
239
DISCUSSION HPLC is a very valuable technique for the study of the metabolic pathways involving purines and pyrimidines. In particular, HPLC has been applied to the measurement of the activity of many of the enzymes involved in these pathways, including those described in this paper. The various modes available such as normal phase, reversed phase and ion exchange plus the use of ion pairing agents permit the separation of most of the nucleosides in the desired order. The value of this was demonstrated by the use of ion pair reversed phase HPLC to improve the CD method as described. Either the appearance of the product can be measured, as in the CD method, or alternatively the disappearance of the substrate can be monitored, as in the case of reactions with complex product formation, e.g., the adenosine deaminase and guanosine deaminase reactions where the primary products, inosine and xanthosine, are further metabolized by endogenous purine nucleoside phosphorylase, These techniques allow HPLC to be used to determine the activity of almost any enzyme on these pathways. The clinical value of measuring adenosine deaminase in patients with inherited immunodeficiency is well proven and its possible use in other areas of clinical medicine has been advocated. The use of CD mcasurements in the assessment of RA has been shown to be beneficial but the value of CD in the diagnosis of pre-eclampsia must now be considered questionable. Whether guanosine deaminase is useful in the differentiation of liver disease is currently under investigation.
REFERENCES Baines, T. J. and Clark, A. (1985). Ann. Clin. Biochem. 22, 420. Coleman, M. S. and Hutton, J. J. (1975). Biochem. Med. 13, 46. Delia, S., Mastroianni, C. M., Massetti, A. P., Turbessi, G., Cirelli, A,, Catania, S. and Vullo, V. (1987). Clin. Chem. 33, 1675. Giblett, E. R., Anderson, J. E., Cohen, F., Pollara, B. and Meuwissen, H. J. (1972). Lancet2, 1067. Hartwick, R., Jeffries, A.. Krstulovic, A. and Brown, P. R. (1978). J. Chromatogr. Sci. 16, 427. Herbert, K. E., Scott, D. L. and Perrett, 0. (1989). J. Pharm. Biomed. Anal. 7 , 737. Hirschhorn, R., Beratis, N., Rosen, F. S., Parkman, R., Stern, R. and Polmar, S. (1975). Lancet 1,73. James, 1. T., Herbert, K., Perrett. D. and Thompson, P. W. (1989). J. Chromatogr. Biomed. Appl. 495, 105. Jones, D. D. and Roberts, E. L. (1984). Enzymes of DNA Metabolism in Clinical Diagnosis. Chancery Publications, Cambridge. Jones, D. D., Bahijri, S., Roberts, E. L. and Williams, G. F. (1982). Brit. J. Obstet. Gynaecol. 89, 314.
Jones, D. D., Roberts, E. L. and Davies, A. G. (1983). J. Clin. Chem. Clin. Biochem. 21, 835. Piras, M. A., Gakis, C., Budroni, M. and Andreoni, G. (1978). Brit. Med. J. 11, 1751. Richards, 0. A., Sherwood, R. A., Ndebele, D. and Rocks, B. F. (1987). Biomed. Chromatogr. 2, 148. Sherwood, R. A., Richards, D. A,, Ndebele, D. and Rocks, B. F. (1987). Ann. Clin. Biochem. 24, (Suppl. 1) 217. Targett-Adams, L., Jones, D. D. and Williams, G. F. (1975). Clin. Chim. Acta 63, 377. Thompson, P. W., Jones, D. D. and Currey, H. L. F. (1986a). Ann. Rheum. Dis. 45. 9. Thompson, P. W., Jones, D. D. and Currey, H. L. F. (1986b). Brit J. Rheumatol. 25, 97. Thompson, P. W., Kirwan, J. R., Jones, D. D. and Currey H. L. F. (1988). Ann. Rheum. Dis. 47, 308. Williams, G. F. and Jones, D. D. (1975). Brit. Med. J. ii, 10. Williams, G. F. and Jones, D. D. (1982). Brit. J. Obstet. Gynaecol. 39. 309.
RE VIEW PAPER
The Measurement of Nucleoside Deaminases by High Performance Liquid Chromatography and their Use in Clinical Chemistry R. A. Sherwood Department of Clinical Biochemistry, Kings College School of Medicine and Dentistry, Denmark Hill, London SE5 9RS, UK
The measurement of the nucleoside deaminases-cytidine deaminase, guanosine deaminase and adenosine deaminase-by reversed phase high performance liquid chromatography is reviewed. The clinical value of assaying the enzyme activity is discussed for each of these enzymes. Both cytidine deaminase and adenosine deaminase measurements have proven clinical value, although the use of the assay of cytidine deaminase in the diagnosis of pre-eclampsia is probably not helpful. ~
INTRODUCTION The nucleoside deaminases are a family of enzymes involved in the purine pathways, which catalyse important steps in the formation and degradation of DNA. The value to clinical chemists of measuring these enzymes has only been investigated relatively recently as methods have become available for the specific determination of their activities. The assay of three of the deaminases in particular-cytidine deaminase, guanosine deaminase and adenosine deaminase-has been reported to be of clinical value. Initially the methods developed to determine the activity of these enzymes were based on the measurement of the ammonia produced during the deamination of the enzyme substrate, using the Berthelot method. This procedure has a number of drawbacks and therefore high Performance liquid chromatographic (HPLC) methods have been developed. These are based on the measurement of the change in the nucleoside concentrations, thus reducing the likelihood of contamination from exogenous ammonia. The development of column packings for reversed phase HPLC has resulted in methods becoming available for the rapid separation of the nucleosides and nucleotides commonly encountered in body fluids. These methods have been extensively used to study the purine and pyrimidine pathways in man and animals. Application of the technique to the assay of the activity of the enzymes involved in these pathways was a logical advance. In this paper the methods developed for the assay of the above-mentioned enzymes are reviewed and evidence is given for a clinical role for the measurements.
METHODS AND RESULTS Cytidine deaminase Cytidine deaminase (CD) (E.C. 3.5.4.5) catalyses the conversion of cytidine to uridine and deoxycytidine to deoxyuridine. It also catalyses the hydrolytic deamina0269-3879/91/060235-05 $OS.OO
01991 by John Wiley & Sons. Ltd.
tion of some of the pharmacological analogues of cytidine. Although its exact physiological role is not known, CD is a cytoplasmic enzyme primarily found in liver and polymorphonuclear leucocytes. CD leaks out of damaged cells and can be detected in the blood o r synovial fluid in a number of disease states (Jones and Roberts, 1984). In particular, inflammatory diseases such as ulcerative colitis and rheumatoid arthritis (RA) show increased CD activity. Thompson et al. (1986a) reported that serum CD activity was raised in R A compared with osteoarthritis (OA). The enzyme activity in synovial fluid was also measured and found to be less than that in serum in patients with OA but up to 22 times higher than in serum in RA. This group have also demonstrated that there is some relationship between serum CD activity and other well-established indices of joint inflammation such as erthyrocyte sedimentation rate (ESR), Ritchie articular index score, etc. (Thompson et al. 1986b). These results suggest that neutrophils in the synovial fluid release CD into the synovial fluid from which it diffuses into the blood. This group have subsequently used serum CD measurements in their assessment of RA patients and have shown a sharp increase in serum CD activity within two days of withdrawal of non-steroidal anti-inflammatory drugs (NSAIDs) from patients with RA, which pre-
Tablel. Conditions for the measurement of CD by HPLC (Richards et al., 1987) Column Eluent
ODs-Hypersil 5p (125 m m x4.6 mm id.) 3% Methanol in 0.1 M potassium phosphate buffer, pH 5.5 1 mL/min, isocratic Flow rate Injection volume 20 p.L Internal standard 5-Fluorouridine SarnDle volume 100 WLserum incubated with cytidine for 2 h at 37 “C Detection UV 260 nm Retention times Cytidine 2.8 rnin Uridine 4.1 rnin 5-Fluorouridine 5.9 min
Received 14 September 1990 Accepted 30 October 1990
R. A . SHERWOOD
236
0
1
2
3
4
5
6
7
0
1
2
11
5
6
Figure 1. Chromatogram of CD assay by reversed phase HPLC. The chromatographic conditions are given in Table 1. Peaks are (1) cytidine, (2) uridine, (3)5-fluorouracil. (a) Aqueous standard solution of cytidine, uridine and 5-fluorouridine; (b) serum (100 pL) incubated with cytidine for 2 h at 37 "C.
cedes the change in other parameters by 2-3 days (Thompson et al. 1988). They postulated that this might be due to an influx of polymorphs into joint spaces because of the removal of the inhibitory effects of NSAlDs resulting in increased CD release. The early work on CD was carried out using the method first described by Jones et al. (1982). This involved incubation of serum (100 pL) with deoxycytidine in carbonate/bicarbonate buffer (pH 9.2) for 18 h at 22 "C. The ammonia produced was estimated using the Berthelot reaction. These workers had previously measured deoxycytidylate deaminase (E.C. 3.5.4.12) and had demonstrated elevated dCMP deaminase activity in patients with pre-eclampsia and other causes of high-risk pregnancies (Williams and Jones, 1975,1982). CD is more stable than deoxycytidylate deaminase and Jones et al. (1982) claimed that the discrimination between normal and high-risk pregnancies was as good when CD was measured as when dCMP deaminase was used. Baines and Clark (1985) also investigated the use of serum CD as a marker for pre-eclampsia using a similar method but with the ammonia determined after incubation of serum with substrate for only 2 h at 37 "C, using the glutamate dehydrogenase end-point method Table2. Conditions for the measurement of CD activity by HPLC with ion pairing (James et al., 1989) ODs-Hypersil 5 p (100 mm ~ 4 . mm 6 i.d.) 1% Methanol in 0.1 M ammonium acetate with 1 mM 1-octanesulphonic acid 1.2 mllmin, isocratic Flow rate Injection volume 20 FL Internal standard Allopurinol Sample volume 100 pL serum incubated with cytidine for 20 min at 56 "C Detection UV 262 nm Retention times Uridine 1.9 min Allopurinol 2.8 min Cytidine 4.3 min Column Eluent
described by Targett-Adams et al. (1975). There was a significant overlap in their study between serum CD activity in normal pregnancies and pre-eclampsia. There were a large number of false positive elevations of CD activity in normal pregnancies (8.6%) while only 42% of samples from patients with pre-eclampsia had increased serum CD activity. Measurement of the ammonia, by whatever method, is subject to potential interference from exogenous ammonia from the environment. In an attempt to eliminate this possible error an HPLC assay was developed for CD (Richards et al., 1987). As in the original spectrophotometric method serum (100 pL) was incubated with cytidine as substrate but the incubation used was reduced to 2 h at 37 "C. The uridine produced was separated from the substrate using reversed phase HPLC with an ODs-Hypersil column, isocratic elution and UV detection at 260 nm. The parameters used are given in Table 1 and representative chromatograms are shown in Fig. 1. The HPLC method was used to assay serum CD activity in 45 women with normal pregnancies and 10 with pre-eclampsia (Sherwood et al., 1987). The results obtained were similar to those obtained by Baines and Clark in that, although the mean CD activity of the preeclampsia group was higher (18.0) than that of the normal group (10.7), there was a significant overlap between the two populations. Measurement of CD would not appear to offer any additional benefits in the detection and monitoring of pre-eclampsia. A reversed phase HPLC method for the measurement of CD activity in synovial fluid, which was very similar to that of Richards et al. (1987), was reported by Herbert et al. (1989). The incubation time had been shortened to 10 min at 56 "C and detection was by UV absorption at 280 nm. This group have since improved on existing methods by the use of an ion pairing agent (1-octanesulphonic acid, OSA) with reversed phase HPLC (James et al., 1989). The addition of OSA caused the retention order of cytidine and uridine to
237
HPLC OF NUCLEOSIDE DEAMTNASE
l3
2 0.0051
-
N Q N
JI
P
0
1
2
3
5
4
6
0
1
2
4
3
5
5
Figure 2. Chromatograms for the assay of CD by HPLC by ion pair reversed phase HPLC. The chromatographic conditions are given in Table 2. The peaks are (1) uridine, (2) allopurinol, (3) cytidine. (a) Standard mixture of uridine, allopurinol and cytidine; (b) serum (100 pL) incubated with cytidine for 20 min a t 56 "C.
reverse so that uridine, which is the product, eluted before the much larger cytidine peak, the substrate. The conditions used are given in Table 2 and representative chromatograms are shown in Fig. 2. The method
has considerably improved the limit of detection for uridine and hence for CD. A clear role has been established for the measurement of CD in serum and synovial fluid for the assess-
1
3
3
f
0
.n
N
v a
m
D
L 0
W 4 5 0)
0
m D 0
D
=c
2
10
Figure 3. Chromatograms for the assay of guanosine deaminase by HPLC. The chromatographic conditions are as given in Table 1 except the flow rate was 1.2 mL/min. Peaks are (1) 5-fluorouridine. (2) xanthosine, (3) guanosine. (a) Standard mixture of 5fluorouridine, xanthosine and guanosine; (b) serum (100 $1 incubated with guanosine for 2 h at 37 "C.
R. A . SHERWOOD
238 ~
~
~
Table 3. Conditions for the measurement of adenosine deaminase activity by HPLC (Hartwick et d.,1978) ODS-Hypersil 10 p (300 m m x 4 mm i.d.1 14% Methanol in 0.01 M phosphate buffer, pH unadjusted Flow rate 2 mLlmin, isocratic Injection volume 5 pL Internal standard Not used Sample volume 200 pL packed erythrocytes added to 200 pL water incubated with 1 mL adenosine (3.5 mM) for 15 min at 25 "C Detection UV 254 nm Retention times Hypoxanthine 2.0 min lnosine 2.5 min Adenosine 6.5 min
Column Eluent
ment of RA. The HPLC methods described above are superior to the original methodology employed, with a considerably reduced risk of Contamination.
Guanosine deaminase Guanosine deaminase (E.C. 3.5.4.15) is an interconverting enzyme in DNA metabolism which catalyses the deamination of guanosine to xanthosine which is then converted to xanthine and on to uric acid (Jones and Roberts, 1984). Few studies have been carried out on this enzyme, but Jones et al. (1983) stated that guanosine deaminase activity was normal in those cases of pre-eclampsia and intrauterine death where CD activity was elevated. This group reported that guanosine deaminase activity was elevated in certain liver diseases, particularly infective hepatitis and metastatic
carcinoma but normal in alcoholic liver disease and obstructive jaundice. They proposed that it might prove to be a useful "liver function test". The method used was based on the method for CD with the ammonia released after incubation of serum with guanosine for 18 h at 22 "C, measured by the Berthelot reaction. The reversed phase HPLC method for CD described by Richards et al. (1987) was adapted to permit the measurement of guanosine deaminase activity. Serum (100 pL) was incubated with guanosine (2.6 mM, 100 pL) for 2 h at 37 "C. The guanosine consumed in the reaction was measured using the HPLC conditions shown in Table 1, with the exception of the flow rate which was increased to 1.2mL/min. The retention times for the three compounds of interest were as follows: 5-fluorouridine, 4.9 min; xanthosine, 7.8 min; guanosine, 8.1 min. A typical chromatogram for the standards and a sample after incubation with substrate is shown in Fig. 3. For this enzyme, in contrast to CD, the elution order of product (hypoxanthine, xanthine and xanthosine) followed by substrate (guanosine) is optimal and there is no need to use ion pairing agents. The performance characteristics of the method are currently under evaluation.
Adenosine deaminase Adenosine deaminase has a wide distribution in tissues and is an important enzyme in the metabolic pathway of the adenosine nucleotides, catalysing the conversion of adenosine to inosine, and in the production of hypoxanthine which is utilized in the purine salvage pathways. Deficiency of adenosine deaminase
c
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
Figure 4. Chromatograms for the assay of adenosine deaminase by HPLC. The chromatographic conditions are given in Table 3. Peaks are (1) hypoxanthine, (2) inosine, (3) adenosine. (a) Standard solution of hypoxanthine, inosine and adenosine; (b) erythrocytes incubated with adenosine for 15 min at 25 "C.
HPIC OF NUCLEOSTDE DEAMINASE
(E.C. 3.5.4.4) has been found in up to one third of subjects with inherited severe combined immunodeficiency (SCID) since it was first reported in 1972 (Giblett et af.,1972). The measurement of adenosine deaminase activity is obviously essential for the diagnosis of the condition. There is some evidence for the use of adenosine deaminase measurements in the differential diagnosis of tuberculous pericarditis (Piras et al., 1978). Adenosine deaminase activity has also been reported to be increased three-fold in subjects with HIV infection (Delia ef al., 1987). Various methods have been described for the measurement of adenosine deaminase based on either the spectrophotometric (Hirschhorn et al., 1975) or radioisotopic (Coleman and Hutton, 1975) measurement of the inosine produced. Hartwick et af. (1978) described an HPLC method for adenosine deaminase based on the measurement of the decrease in adenosine concentration after incubation with the sample. These workers were measuring adenosine deaminase in erythrocytes (200 pL) which were lysed by the addition of an equal volume of water and then incubated with 1 m L of adenosine ( 3 . 5 m ~ for ) 15 min at 25°C at which time the reaction was stopped by raising the temperature to 100 "C for 45 s. The chromatographic conditions used are shown in Table 3 and representative chromatograms are shown in Fig. 4. As in the assay of guanosine deaminase the order of separation is optimal with the reaction productshypoxanthine and inosine-both eluting before the substrate peak. In this case the decrease in substrate concentration was measured rather than the sum of the reaction products.
239
DISCUSSION HPLC is a very valuable technique for the study of the metabolic pathways involving purines and pyrimidines. In particular, HPLC has been applied to the measurement of the activity of many of the enzymes involved in these pathways, including those described in this paper. The various modes available such as normal phase, reversed phase and ion exchange plus the use of ion pairing agents permit the separation of most of the nucleosides in the desired order. The value of this was demonstrated by the use of ion pair reversed phase HPLC to improve the CD method as described. Either the appearance of the product can be measured, as in the CD method, or alternatively the disappearance of the substrate can be monitored, as in the case of reactions with complex product formation, e.g., the adenosine deaminase and guanosine deaminase reactions where the primary products, inosine and xanthosine, are further metabolized by endogenous purine nucleoside phosphorylase, These techniques allow HPLC to be used to determine the activity of almost any enzyme on these pathways. The clinical value of measuring adenosine deaminase in patients with inherited immunodeficiency is well proven and its possible use in other areas of clinical medicine has been advocated. The use of CD mcasurements in the assessment of RA has been shown to be beneficial but the value of CD in the diagnosis of pre-eclampsia must now be considered questionable. Whether guanosine deaminase is useful in the differentiation of liver disease is currently under investigation.
REFERENCES Baines, T. J. and Clark, A. (1985). Ann. Clin. Biochem. 22, 420. Coleman, M. S. and Hutton, J. J. (1975). Biochem. Med. 13, 46. Delia, S., Mastroianni, C. M., Massetti, A. P., Turbessi, G., Cirelli, A,, Catania, S. and Vullo, V. (1987). Clin. Chem. 33, 1675. Giblett, E. R., Anderson, J. E., Cohen, F., Pollara, B. and Meuwissen, H. J. (1972). Lancet2, 1067. Hartwick, R., Jeffries, A.. Krstulovic, A. and Brown, P. R. (1978). J. Chromatogr. Sci. 16, 427. Herbert, K. E., Scott, D. L. and Perrett, 0. (1989). J. Pharm. Biomed. Anal. 7 , 737. Hirschhorn, R., Beratis, N., Rosen, F. S., Parkman, R., Stern, R. and Polmar, S. (1975). Lancet 1,73. James, 1. T., Herbert, K., Perrett. D. and Thompson, P. W. (1989). J. Chromatogr. Biomed. Appl. 495, 105. Jones, D. D. and Roberts, E. L. (1984). Enzymes of DNA Metabolism in Clinical Diagnosis. Chancery Publications, Cambridge. Jones, D. D., Bahijri, S., Roberts, E. L. and Williams, G. F. (1982). Brit. J. Obstet. Gynaecol. 89, 314.
Jones, D. D., Roberts, E. L. and Davies, A. G. (1983). J. Clin. Chem. Clin. Biochem. 21, 835. Piras, M. A., Gakis, C., Budroni, M. and Andreoni, G. (1978). Brit. Med. J. 11, 1751. Richards, 0. A., Sherwood, R. A., Ndebele, D. and Rocks, B. F. (1987). Biomed. Chromatogr. 2, 148. Sherwood, R. A., Richards, D. A,, Ndebele, D. and Rocks, B. F. (1987). Ann. Clin. Biochem. 24, (Suppl. 1) 217. Targett-Adams, L., Jones, D. D. and Williams, G. F. (1975). Clin. Chim. Acta 63, 377. Thompson, P. W., Jones, D. D. and Currey, H. L. F. (1986a). Ann. Rheum. Dis. 45. 9. Thompson, P. W., Jones, D. D. and Currey, H. L. F. (1986b). Brit J. Rheumatol. 25, 97. Thompson, P. W., Kirwan, J. R., Jones, D. D. and Currey H. L. F. (1988). Ann. Rheum. Dis. 47, 308. Williams, G. F. and Jones, D. D. (1975). Brit. Med. J. ii, 10. Williams, G. F. and Jones, D. D. (1982). Brit. J. Obstet. Gynaecol. 39. 309.
