The High Performance Liquid Chromatography of Enzyme Systems Relating to Purine and Pyrimidine Metabolism: an Overview David Perrett* Department of Medicine, St Bartholomew's Hospital Medical College, West Smithfield, London EClA 7BE, U K
H. Anne Simmonds Purine Research Laboratories, UMDS of Guy's and St Thomas' Hospitals, London SE1 9RT, UK
Purines and pyrimidines are now routinely separated by HPLC. By careful selection of chromatographic conditions which match the expected changes in hydrophobicity and/or ionic nature of the substrate and products most enzymes of the purine and pyrimidine salvage pathways can be routinely and accurately determined. Ion-paired reversed-phase systems are often the most advantageous. The relevance of such assays to biomedical analysis including their role in the diagnosis of inborn errors of metabolism is stressed.
INTRODUCTION The metabolism of purine and pyrimidine bases is fundamental to life processes since these compounds are the components of DNA and RNA as well as being intimately involved in most energy-requiring metabolic reactions. Nucleotides, nucleosides and bases are the principal purine and pyrimidine derivatives integral to these processes although many other related compounds, such as ADP-ribose, NAD, NADP as well as DNA and RNA can be involved either directly or indirectly. The metabolic pathways involving the inter* Author to whom correspondence should be addressed.
conversion of these compounds are complex and diverse ranging from the de novo synthetic pathway through the interconversion of nucleotides to the catabolic pathways (Henderson and Paterson, 1973). At least 100 enzymes are involved in these different metabolic pathways (Fig. 1). Nucleotides can be formed de no00 from simpler components including ribose-5-phosphate and glycine but the pathways are complex and energetically expensive. The energetically more favourable enzyme systems are those that salvage pre-formed heterocyclic rings; these predominate in many tissues, particularly the erythrocyte. High performance liquid chromatography (HPLC) can be employed in the context of many different enzyme systems in a variety of ways:
DNA
(b)
't
dGTP
i" 8JT
dATP
GDP
t 1
UTP
d C D P c CDP
OA
UDP-
"
t
t
":!
1
&q
de n o v o m
CiP.
dCTP ATP
RNd
1
dGDP
dUDP
""
dTY +d T M P
dGMP Cytidine
Uridine
Cytosine
Uracil
71
deoxy Adenosine
9i
p -alanine
I
Thymidine
4
Thymine
9: V
e-aib
yGuanine
Xanthine
1.
Uric Acid
l5
Allantoin
Figure 1. Major pathways of nucleotide salvage and interconversion in man. (a) purines 1. 5'-nucleotidase 2. adenosine dearninase 3. purine nucleoside phosphorylase 4. xanthine oxidase 5. allantoinase 6. guanase 7. AMP deaminase 8. ribonucleoside-diphosphate reductase 9. (deoxy)nucleoside kinase 6). hypoxanthine-guanine phosphoribosyltransferase. (b) pyrimidines. 1 . cytidine deaminase 2. OMP decarboxylase 3. U M P hydrolase 4. CTP synthetase 5. orotic acid phosphoribosyltransferase6. uridine-cytidine kinase 7. uridine phosphorylase 8. thymidine phosphofylase 9. dihydropyrimidine dehydrogenase. N.B.Not all pathways are shown and some of those shown d o not operate in all tissue types. 0269-3879/90/0267-0272$5.00 @ 1990 by John Wiley & Sons, Ltd. 1990
BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 6, 1990 267
D. PERRETT AND H. A. SIMMONDS
(a) Estimation of enzyme activities for quantitative purposes may be required in a number of situations such as enzyme purification or biochemical characterization of tissues and cells. (b) Quantification may also be useful in disease diagnosis where enzyme leakage may be a marker of a clinical condition, e.g., plasma cytidine deaminase levels have been proposed as a marker for pre-eclampsia in pregnancy (Jones et al., 1982), adenosine deaminase for tuberculosis and guanase for liver failure. (c) The direct estimation of the affected enzyme is essential for confirmation of inborn errors of metabolism plus antenatal and carrier detection to enable the necessary genetic counselling. (d) The estimation of certain enzyme activities may be useful in understanding drug metabolism in certain pharmacogenetic studies, e.g., the variations in the metabolism of azothioprine (Lennard et al., 1990) and allopurinol (Reiter et al., 1990) in man. (e) Preparative protein HPLC can be used to isolate and purify enzymes. (f) Coupling of enzymes to the stationary phase can produce useful chiral columns. General features of the application of HPLC to enzyme systems have been reviewed previously, mainly by Rossomodo (1987,1989). From the examples mentioned above it is clear that most of these areas of application have already been employed in the field of purine and pyrimidine metabolism. The following discussion will deal only with the first four areas of application mentioned above and attempt to stress aspects not covered in the above reviews. DISCUSSION HPLC of nucleotides, nucleosides and bases The separation of this group of compounds is one of the oldest application areas of liquid chromatography and a substantial literature has developed. It would be inappropriate to even attempt to review the subject here. The relevant literature has been summarized in at least two books (Brown, 1984; Krustolovic, 1987) and in various chapters and reviews (e.g., Perrett, 1986a). The following therefore only briefly summarizes the approaches to the HPLC of this group of compounds. Nucleotides are strongly ionic with one, two or three phosphate groups and their separation has traditionally been performed on anion exchange HPLC columns. Recently reversed phase liquid chromatography (RPLC) and RPLC with both ion pairing and zwitterion pairing have also been employed. We have found that for complex biological mixtures the anion exchange systems give the better separations (Perrett et al., 1989). Attention to the pH gradient and the quality of the reagents has meant that the anion exchange system is capable of resolving at least 22 nucleotides in 13 minutes on a 10 cm column of APS Hypersil (Perrett, 1982). Nucleosides and bases, which possess varying degrees of hydrophobic character, are well separated on reversed phase packings. In general a simple methanol/buffer gradient will give excellent separations although for more complex separations ternary gradients may be 268 BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 6, 1990
necessary (Morris et al., 1985). With the introduction of high efficiency 3 pm materials it is possible to achieve very similar resolution isocratically. By the correct choice of buffer pH and percent organic modifier, rapid separations of small numbers of compounds can be easily developed empirically. Halfpenny and Brown (1986) used simplex optimization to develop methods for PNP and HGPRTase.* The purine ring absorbs strongly in the region 240290nm and 254nm is commonly employed to detect purine and pyrimidines. With modern UV detectors this enables quantitation down to about 50 pmol injected. This level of sensitivity is adequate for the quantitation of nucleotides in most tissue extracts but may not be sufficient for many nucleosides and bases in similar samples. It is certainly adequate for appropriately designed enzyme assays. Variable wavelength HPLC detectors allow monitoring at the absorbance maxima of compounds but more importantly for enzyme assays they may allow enhanced selectivity by reducing the sensitivity towards the substrate whilst enhancing the response to the product by appropriate wavelength choice. It is unusual for enzyme assays to require high sensitivity detection but occasionally other detectors may offer not only higher sensitivity but also increased selectivity towards substrate and/or products. Over the pH range normally associated with HPLC eluates, the majority of purine bases, etc., do not fluoresce. Pre- and post-column derivatization can be employed to introduce fluorophors into adenine- or cytosine-based compound molecules either for analysis (Perrett, 1986b) or as potential enzyme substrates. Purines containing hydroxyl ring substituents, e.g., guanine, xanthine and urate, are electroactive at moderate to low oxidative potentials (Perrett, 1986). Electrochemical detectors equipped with glassy carbon electrodes possibly in series with UV detection can be used to monitor the eluate from isocratic separations. The EC detector is significantly more sensitive than the UV detector as well as being more selective. Nucleotides, nucleosides and bases are usually released from biological samples by an appropriate degree of physical homogenization in the presence of acids which serve to terminate any enzyme activity. The acid most commonly used is trichloroacetic acid, the excess being removed with either water-saturated ether or an insoluble amine/immiscible solvent mixture. Perchloric acid is also frequently used. The same procedures can be used with most enzyme assays. With suitably dilute protein concentrations it is possible to inject a small quantity of reaction mixture directly into the HPLC without causing significant loss of column performance. Extraction techniques have been comprehensively reviewed (Perrett, 1987). Design of HPLC assays for purine metabolism For clinically meaningful assays consideration must be given to many non-chromatographic factors prior to the analysis step. These include incubation conditions, particularly phosphate concentrations in media, substrate/enzyme concentrations and the degree of cellular
* See Table 2 for abbreviations. @ 1990 by John Wiley & Sons, Ltd. 1990
HPLC OF ENZYMES OF PURINE A N D PYRIMIDINE MET BOLISM
ischaemia that may be permissible (Dean and Perrett, 1976). However, under appropriate substrate and incubation conditions many pathways can be and have been determined in intact cells, tissue homogenates and even whole organs using HPLC with UV detection. Enzyme assays, which involve the separation and quantitation of substrates from products and possibly side-reactants are relatively easy for modern HPLC techniques. The analytical step should therefore be designed to be simple, rapid and specific. This can only be achieved by devising isocratic separations and the use of sensible substrate and enzyme concentrations. Because of the serial nature of HPLC analyses, HPLC does not readily allow realtime kinetic studies, although if the assay is rapid enough many time-points may be automatically injected without extraction if protein loads are low enough. When enzyme activity is low and little product is formed from even large amounts of substrate, conditions should be chosen so that the small peak due to the product appears before the large peak due to the substrate. This may be achieved by standard variations of elution conditions or by more dramatic means such as changing the column type or using ion pairs. For example, cytidine deaminase activity can be best monitored by including octanesulfonic acid in the eluent to elute uridine prior to cytidine (Fig. 2) (James et al., 1989). Although the pathways of purine and pyrimidine metabolism are complex, they fall into a limited number of types which can be summarized as base-nucleosidenucleotide interconversions, base-base degradation, nucleoside interconversions and nucleotide interconversions as well as de no00 synthetic pathways. However, it is the chemical nature of the various types of compounds that dictates the approach to their individual and group chromatography. Therefore enzymes that simdv interconvert can usually be assayed using suitable liqiid chromatographic conditions: for example
I
4 4 4
0
8
0
8
0
8
0
8
0
2
0
4
min
I-
min
Figure 2. Assay of cytidine deaminase in serum by ion-paired RPLC (Assay as per James et a/ 1989). (a) RPLC separation of cytidine and uridine standards without ion-pair (b) separation of cytidine and formed uridine with allopurinol (internal standard). Column: 100 ~ 4 . mm 6 5 prn ODS-Hypersil; eluent: 100 rnM ammonium acetate, pH 5 , l m M octanesulphonic acid; flow rate: 1.2 mL/min; detection 262 nm.
@ 1990 by John Wiley & Sons, Ltd. 1990
I T
c
0
8
adenosine deaminase is assayed by monitoring the conversion of adenosine to inosine or hypoxanthine by isocratic RPLC. However, enzymes that involve phosphorylation, or phosphorylation plus degradation, may require multiple analyses using different liquid chromatographic separations. We have exploited the ability of ion pairs to retain nucleotides on ODS columns whilst leaving nucleoside and base chromatography relatively
.-
I . . . -
0
Figure 3. Assay of red cell purine enzymes by ion-paired RPLC. Separation conditions; Column: 250 x4.6 mm 5 p m ODSII-Hypersil; eluent: 40 m M sodium acetate, pH 2.75, 5 rnM tetrabutylammonium ion; flow rate: 1.2 mL/min; detection: 254 nrn (lower trace), 280 nm (upper trace). (A) adenine phosphoribosyltransferase assay with adenine (A) as substrate, ( B ) hypoxanthine-guanine phosphoribosyltransferase with hypoxanthine (H) as substrate, (C) adenosine deaminase with adenosine (AR) as substrate, (D) purine nucleoside phosphorylase with inosine (HR) as substrate. Chrornatograms show 0 and 15 rnin incubations.
B
A
8
--
7
8
Ti
0 '
8
(mins)
Figure 4. Assay of uridine monophosphate hydrolyase by ion-paired RPLC with UMP as substrate and, uridine as product. Column: 250 X4.6 m m 5 p m ODSII-hypersil; eluent: 40 mM sodium acetate, pH 2.75, 5 m M tetrabutylammonium ion; flow rate: 1.2 mL/min; detection: 254 nm (lower trace), 280 nrn (upper trace). Chromatogram shows 0 and 15 min incubations of red cells.
BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 6, 1990 269
D. PERRETT A N D H. A. SIMMONDS
Table 1. Enzyme interconversions and their polarity changes Polarity change
Example
base+ base base c* nucleoside NMP+ NMP
xanthine oxidase PNP AMP deaminase
small medium small
NMP+ nucleoside
5 nucleotidase
large
nucleoside+ nucleotide base + nucleotide NTP+ NDP- NMP
AK APRTase Apyrase
large v. large large
HPLC type
RPLC RPLC IEC or IP-RPLC RPLC or IP-RPLC IP-RPLC IP-RPLC IEC or IP-RPLC
NMP, NDP, NTP: nucleotide mono-, di- and tri-phosphates. RPLC: reversed phase HPLC. IP-RPLC: reversed phase HPLC with ion pair. IEC: anion exchange HPLC.
unchanged. Both tetrabutylammonium (Hoffman and Liao, 1977) and triethylamine ion (Willis et af., 1986) can be employed to retain nucleotides on RPLC columns. By appropriate adjustments to the percent organic modifier overall analysis time can be controlled. Figure 3 illustrates this very useful approach for a series of purine salvage enzymes of clinical interest. Figure 4 shows the same approach applied to an enzyme of pyrimidine metabolism. The relationship between enzymatic change and the HPLC approach is summarized in Table 1. Pathways where little product is formed or gross interferences are present on the UV chromatogram have required the use of more specific detectors. Xanthine oxidase activity is very low in most human tissues (Watts et af., 1965), but can be assayed by electrochemical detection which can be both highly sensitive to the product urate and relatively insensitive toward the substrate xanthine (Perrett, 1986) (Fig. 5 ) . Poly-ADP ribosylation can be measured by chloroacetaldehyde derivatization of the product, followed by anion exchange HPLC with fluorescent detection. If none of these methods are possible, then the use of radiochemicals should be considered. Chromatographic considerations remain the same, but the determination of radioactivity 3Omin
15min
in the separated product can be performed either offor on-line. Trace levels of adenosine deaminase and adenosine kinase were monitored by collecting fractions from an anion exchange column, addition of scintillant and counting (Perrett and Dean, 1977). More satisfactory for some situations when activities are lower is the use of in-line radioactivity detectors (Fairbanks et af., 1987).
Disorders of purine and pyrimidine metabolism in man Abnormalities of purine and pyrimidine metabolism are related to either inborn errors of metabolism (Table 2) or disorders of normal metabolism due to disease or drug therapy (Table 3). Clinically the most interesting group with regard to enzyme and metabolite assays are the inborn errors.
Table 2. Inborn errors of purine and pyrimidine metabolism in man Purine metabolism
1. Hypoxanthine-Guanine phosphoribosyl transferase (HGPRTase; EC 2 . 4 2 8 ) deficiency (Lesch-Nyhan Syndrome) 2. Adenosine deaminase SClD (ADA; EC 3.5.4.4) deficiency 3. Purine nucleoside phosphorylase (PNP; EC 2.4.2.1) deficiency 4. Adenine phosphoribosyl transferase (APRTase; EC 2.4.2.7) deficiency 5. Phosphoribosyl pyrophosphate synthetase (PPRP-S; EC 2.7.6.1) superactivity 6. Xanthine oxidase (XO; EC 1.2.3.2) deficiency 7. Combined xanthine oxidase and sulphite oxidase deficiency 8. Partial HGPRTase 9. Gout 10. ITP-pyrophosphohydrolase (ITP-ase; EC 3.6.1.19) deficiency 1 1 . Myoadenylate deaminase (AMPDA; EC 3.5.4.6) deficiency 12. Adenylosuccinase (ASAse; EC 4.3.2.2) deficiency Pyrimidine metabolism
13. Oroticaciduria (UMP synthase; EC 2.4.2.10/ODC EC 4.1.1.23) deficiency 14. Pyrimidine-5-nucleotidase (UMP hydrolyase 1 ; EC 3.1.3.5) deficiency 15. Dihydropyrimidine dehydrogenase (DiHpyDH; EC 1.3.1.2) deficiency
0
Table 3. Some causes of decreased ATP levels in tissues 1 . Increased ATP turnover Fructose, xylitol infusion. etc. Exercise Ethanol Glycogen storage disease Type 1 Hereditary fructose intolerance Fructose 1.I-diphosphatase deficiency Figure 5. Assay of xanthine oxidase in liver homogenate with electrochemical detection. Incubate a t pH 6 with xanthine (X) substrate. The uric acid (UA) formed was measured a t the shown intervals. Column: 100 X4.6 mm 3 p m ODSII-hypersil eluent: 50 mM ammonium acetate, pH 5.5/MeOH (98:2); flow rate: 1.2 mL/min; detection: oxidative electrochemical a t +0.7 vV. Ag/AgCI a t glassy carbon electrode.
270 BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 6, 1990
2. Decrease ATP synthesis Tissue hypoxia a) lschaemia b) Hypoxia Metabolic myopathies Hypophosphatemia
@ 1990 by John Wiley & Sons, Ltd. 1990
HPLC OF ENZYMES OF PURINE A N D PYRIMIDINE METABOLISM
Fifteen genetic disorders involving essential enzymes of purine metabolism and three of pyrimidine metabolism have now been identified. Some are associated with clinicopathological features, such as the severe neurological abnormalities of complete Lesch-Nyhan syndrome, a fatal immunodeficiency syndrome in ADA and PNP deficiency. Other features are inherited nerve deafness as well as severe muscular hypotonicity in severe PP-ribose-P synthetase superactivity, urolithiasis in APRTase deficiency or XO deficiency and megaloblastic or heamolytic anaemia in hereditary oroticaciduria and pyrimidine-5‘-nucleotidase deficiency, respectively. For a complete description of these defects see Simmonds (1987) and the appropriate chapters in ‘The Metabolic Basis of Inherited Disease’ (Scriver et aL, 1989). There is considerable genetic heterogeneity, which means that in some instances the defect may be relatively benign or of late onset. The broad spectrum of the clinical symptoms makes recognition difficult and could be equally indicative of the enzyme affecting other metabolic pathways. Treatment is available for some disorders which, if unrecognised, can otherwise progress to dialysis, transplantation or death. Early identification is thus imperative. Bone marrow transplant therapy for ADA and PNP deficiency has been successful in only a minority of cases. Consequently carrier detection and prenatal diagnosis are equally essential and are now possible in the first and second trimester, but it is important to be aware of the possible pitfalls in chorionic villus sampling for diagnosis. Such early and efficient recognition has only been made possible by considerable advances which have taken place in HPLC in the past 15 years. HPLC has enabled not only the rapid diagnosis of the underlying enzyme defect using simple liquid chromatographic systems but also their detection from the presence/absence of abnormal/normal metabolites in cells and body fluids. In every instance where there has been clinical evidence of cellular toxicity, abnormal erthrocyte nucleotide patterns have been found (Simmonds et aL, 1988). These patterns have proved useful in diagnosis as ‘fingerprints’ characteristic of the particular disorder (Simmonds et al., 1988). They may also provide some clue to the metabolic basis for the disorder and thus can be important as a research tool, as well as a diagnostic one. However, identification of these patterns depends on the efficiency of the separation obtained. In the anion exchange system used for nucleotides, constant attention to the pH and concentration of the eluents employed is essential to obtain reproducible results as the column
ages (Perrett et a/., 1989). It is equally important to recognise that no two columns will give exactly the same separation and that specific changes may also be masked in the presence of severe renal impairment. Nucleotide patterns in children also differ slightly from those in adult erythrocytes and appropriate control ranges must be established. Where the appropriate enzyme is expressed in the erythrocyte, deletion of the defect can be established from investigation of activity in lysed cells using ion pair HPLC coupled with intact cell studies using radiolabelled substrate (Fairbanks et al., 1987). Additional to this group is gout, where the genetic error leads to the precipitation of uric acid crystals in gouty tophi. The disease at present appears to include a number of different but related conditions. The largest group is composed of men aged over 40 with hyperuricemia. However, even for this classical group, the exact nature of this defect is still unknown and may relate to urate transport either in the kidney or intestine, some abnormality of minor pathways of purine production or defective urate binding to plasma proteins. Many diseases affect purine metabolism indirectly by causing (a) increased turnover of nucleotide synthetic pathways, (b) gross losses of intracellular nucleotides or (c) leakage of purine-metabolizing enzymes. Renal failure not only leads to the accumulation of trace purine and pyrimidine end-products in plasma, but also to elevated levels of nucleotides within red and white blood cells. The elevated levels of nucleotides within RBCs correlates with the degree of renal failure and the concentration of plasma phosphate in man (Rejman et aL, 1985). It is likely that such increases are also found in the intracellular phosphate pool but such data has not yet been documented.
CONCLUSIONS Over the last 15 years HPLC has become a complementary technique to the many dozens of traditional spectrometric enzyme assays already available to the purine biochemist. In many areas, such as prenatal diagnosis of inborn errors of purine metabolism, it is a superior technique and should be the technique of choice when sample amounts are limited. However, it must always be remembered that it is only an analytical technique and therefore cannot give good results from poorly designed enzyme assays.
REFERENCES Brown, P. R. (1984) (ed.). HPLC in Nucleic Acid Research, Methods and Applications, Vol. 28, Chromatographic Sciences Series. Marcel Dekker, New York. Dean, B. M. and Perrett, D. (1976). Biochem. Biophys. Acta 437, 1. Fairbanks, L. D., Simmonds, H. A. and Webster, D. R. (1987). Inherit. Metab. Dis. 10, 174. Halfpenny, A. P. and Brown, P. R. (1986). J. Liq. Chromatogr. 9,2585. Henderson, J. F. and Paterson, A. R. P. (1973). Nucleotide Metabolism, an Introduction. Academic Press, New York. Hoffman, N. E. and Liao, J. C. (1977). Anal. Chem. 49, 2231.
@ 1990 by John Wiley & Sons, Ltd. 1990
James, I. T., Herbert, K. E., Perrett, D. and Thompson, P. W. (1989). J. Chromatogr. 495, 105. Jones, D. D., Bahjri, S.. Roberts, E. L. and Williams, G. F. (1982). Brit. J. Obstet. Gynaecol. 89, 314. Krustolovic, A. M. (1987) (ed.). CRC Handbook of Chromatography of Nucleic Acids and Related Compounds, Vols I and II. CRC Press, Boca Raton. Lennard, L., Lilleyman, J. S., Van Loon, J. and Weinshilboum, R. M. (1990). Lancet 336,225. Morris, G. and Simmonds. H. A. (1985). J. Chrornatogr. 344,101.
BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 6, 1990 271
D. PERRETT A N D H. A. SIMMONDS Perrett, D. (1982). Chromatographia 16, 21 1. Perrett, D. (1986a). In HPLC of Small Molecules, C. K. Lim, ed., 221-259, IRL Press, Oxford. Perrett, D. (1986b). J. Chromatogr. 386,289. Perrett, D. (1987). In Handbook of Chromatography of Nucleic Acids and Related Compounds, ed. by A. M. Krustolovic, Vol. I, Part 8, pp. 3-29. CRC Press, Boca Raton. Perrett, D. and Dean, B. M. (1977). Biochem. Biophys. Res. Commun. 77,374. Perrett, D., Herbert, K. E.. Morris, G. and Simmonds, H. A. (1989). In Human Purine and Pyrimidine Metabolism. ed. by K. Mikanagi, K. Nishioka and W. N. Kelly, Vol. VI, Part B, pp. 463468. Plenum Press, New York. Reiter, S., Simmonds, H. A,, Zollner, N.. Braun, S. L. and Kneldel, M. (1990). Clin. Chim. Acta 187, 221. Rejman, A. S. M., Mansell, M.A., Grimes, A. J. and Joekes, A. M. (1985). Brit. J. Haematol. 61, 433.
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Rossomondo, E. F. (1987). High Performance Liquid Chromatography in Enzymatic Analysis. Wiley, New York. Rossomondo, E. F. (1989). J. Chromatogr. 492, 361. Scriver, C. R.. Beaudet, A. L., Sly, W. S. and Valle, D., (1989). The Metabolic Basis of Inherited Disease, 6th Ed. McGraw-Hill, New York. Simmonds. H. A. (1987). In The Inherited Metabolic Diseases, ed. by J. B. Holton, pp. 215-255. Churchill Livingstone, Edinburgh. Simmonds, H. A., Fairbanks, L. D., Morris, G. S., Webster, D. R. and Harley, E. H. (1988). Clin. Chim. Acta 171, 197. Watts, R. W. E., Watts, J. E. M. and Seegmiller, J. E. (1965). J. Lab. Clin. Med. 66, 688. Willis, C. L., Lim, C. K. and Peters, T. J. (1986). J. Pharm. Biomed. Anal. 4. 247.
Received 22 August, 1990; accepted 5 September, 1990.
0 1990 by John Wiley & Sons, Ltd.
1990
H. Anne Simmonds Purine Research Laboratories, UMDS of Guy's and St Thomas' Hospitals, London SE1 9RT, UK
Purines and pyrimidines are now routinely separated by HPLC. By careful selection of chromatographic conditions which match the expected changes in hydrophobicity and/or ionic nature of the substrate and products most enzymes of the purine and pyrimidine salvage pathways can be routinely and accurately determined. Ion-paired reversed-phase systems are often the most advantageous. The relevance of such assays to biomedical analysis including their role in the diagnosis of inborn errors of metabolism is stressed.
INTRODUCTION The metabolism of purine and pyrimidine bases is fundamental to life processes since these compounds are the components of DNA and RNA as well as being intimately involved in most energy-requiring metabolic reactions. Nucleotides, nucleosides and bases are the principal purine and pyrimidine derivatives integral to these processes although many other related compounds, such as ADP-ribose, NAD, NADP as well as DNA and RNA can be involved either directly or indirectly. The metabolic pathways involving the inter* Author to whom correspondence should be addressed.
conversion of these compounds are complex and diverse ranging from the de novo synthetic pathway through the interconversion of nucleotides to the catabolic pathways (Henderson and Paterson, 1973). At least 100 enzymes are involved in these different metabolic pathways (Fig. 1). Nucleotides can be formed de no00 from simpler components including ribose-5-phosphate and glycine but the pathways are complex and energetically expensive. The energetically more favourable enzyme systems are those that salvage pre-formed heterocyclic rings; these predominate in many tissues, particularly the erythrocyte. High performance liquid chromatography (HPLC) can be employed in the context of many different enzyme systems in a variety of ways:
DNA
(b)
't
dGTP
i" 8JT
dATP
GDP
t 1
UTP
d C D P c CDP
OA
UDP-
"
t
t
":!
1
&q
de n o v o m
CiP.
dCTP ATP
RNd
1
dGDP
dUDP
""
dTY +d T M P
dGMP Cytidine
Uridine
Cytosine
Uracil
71
deoxy Adenosine
9i
p -alanine
I
Thymidine
4
Thymine
9: V
e-aib
yGuanine
Xanthine
1.
Uric Acid
l5
Allantoin
Figure 1. Major pathways of nucleotide salvage and interconversion in man. (a) purines 1. 5'-nucleotidase 2. adenosine dearninase 3. purine nucleoside phosphorylase 4. xanthine oxidase 5. allantoinase 6. guanase 7. AMP deaminase 8. ribonucleoside-diphosphate reductase 9. (deoxy)nucleoside kinase 6). hypoxanthine-guanine phosphoribosyltransferase. (b) pyrimidines. 1 . cytidine deaminase 2. OMP decarboxylase 3. U M P hydrolase 4. CTP synthetase 5. orotic acid phosphoribosyltransferase6. uridine-cytidine kinase 7. uridine phosphorylase 8. thymidine phosphofylase 9. dihydropyrimidine dehydrogenase. N.B.Not all pathways are shown and some of those shown d o not operate in all tissue types. 0269-3879/90/0267-0272$5.00 @ 1990 by John Wiley & Sons, Ltd. 1990
BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 6, 1990 267
D. PERRETT AND H. A. SIMMONDS
(a) Estimation of enzyme activities for quantitative purposes may be required in a number of situations such as enzyme purification or biochemical characterization of tissues and cells. (b) Quantification may also be useful in disease diagnosis where enzyme leakage may be a marker of a clinical condition, e.g., plasma cytidine deaminase levels have been proposed as a marker for pre-eclampsia in pregnancy (Jones et al., 1982), adenosine deaminase for tuberculosis and guanase for liver failure. (c) The direct estimation of the affected enzyme is essential for confirmation of inborn errors of metabolism plus antenatal and carrier detection to enable the necessary genetic counselling. (d) The estimation of certain enzyme activities may be useful in understanding drug metabolism in certain pharmacogenetic studies, e.g., the variations in the metabolism of azothioprine (Lennard et al., 1990) and allopurinol (Reiter et al., 1990) in man. (e) Preparative protein HPLC can be used to isolate and purify enzymes. (f) Coupling of enzymes to the stationary phase can produce useful chiral columns. General features of the application of HPLC to enzyme systems have been reviewed previously, mainly by Rossomodo (1987,1989). From the examples mentioned above it is clear that most of these areas of application have already been employed in the field of purine and pyrimidine metabolism. The following discussion will deal only with the first four areas of application mentioned above and attempt to stress aspects not covered in the above reviews. DISCUSSION HPLC of nucleotides, nucleosides and bases The separation of this group of compounds is one of the oldest application areas of liquid chromatography and a substantial literature has developed. It would be inappropriate to even attempt to review the subject here. The relevant literature has been summarized in at least two books (Brown, 1984; Krustolovic, 1987) and in various chapters and reviews (e.g., Perrett, 1986a). The following therefore only briefly summarizes the approaches to the HPLC of this group of compounds. Nucleotides are strongly ionic with one, two or three phosphate groups and their separation has traditionally been performed on anion exchange HPLC columns. Recently reversed phase liquid chromatography (RPLC) and RPLC with both ion pairing and zwitterion pairing have also been employed. We have found that for complex biological mixtures the anion exchange systems give the better separations (Perrett et al., 1989). Attention to the pH gradient and the quality of the reagents has meant that the anion exchange system is capable of resolving at least 22 nucleotides in 13 minutes on a 10 cm column of APS Hypersil (Perrett, 1982). Nucleosides and bases, which possess varying degrees of hydrophobic character, are well separated on reversed phase packings. In general a simple methanol/buffer gradient will give excellent separations although for more complex separations ternary gradients may be 268 BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 6, 1990
necessary (Morris et al., 1985). With the introduction of high efficiency 3 pm materials it is possible to achieve very similar resolution isocratically. By the correct choice of buffer pH and percent organic modifier, rapid separations of small numbers of compounds can be easily developed empirically. Halfpenny and Brown (1986) used simplex optimization to develop methods for PNP and HGPRTase.* The purine ring absorbs strongly in the region 240290nm and 254nm is commonly employed to detect purine and pyrimidines. With modern UV detectors this enables quantitation down to about 50 pmol injected. This level of sensitivity is adequate for the quantitation of nucleotides in most tissue extracts but may not be sufficient for many nucleosides and bases in similar samples. It is certainly adequate for appropriately designed enzyme assays. Variable wavelength HPLC detectors allow monitoring at the absorbance maxima of compounds but more importantly for enzyme assays they may allow enhanced selectivity by reducing the sensitivity towards the substrate whilst enhancing the response to the product by appropriate wavelength choice. It is unusual for enzyme assays to require high sensitivity detection but occasionally other detectors may offer not only higher sensitivity but also increased selectivity towards substrate and/or products. Over the pH range normally associated with HPLC eluates, the majority of purine bases, etc., do not fluoresce. Pre- and post-column derivatization can be employed to introduce fluorophors into adenine- or cytosine-based compound molecules either for analysis (Perrett, 1986b) or as potential enzyme substrates. Purines containing hydroxyl ring substituents, e.g., guanine, xanthine and urate, are electroactive at moderate to low oxidative potentials (Perrett, 1986). Electrochemical detectors equipped with glassy carbon electrodes possibly in series with UV detection can be used to monitor the eluate from isocratic separations. The EC detector is significantly more sensitive than the UV detector as well as being more selective. Nucleotides, nucleosides and bases are usually released from biological samples by an appropriate degree of physical homogenization in the presence of acids which serve to terminate any enzyme activity. The acid most commonly used is trichloroacetic acid, the excess being removed with either water-saturated ether or an insoluble amine/immiscible solvent mixture. Perchloric acid is also frequently used. The same procedures can be used with most enzyme assays. With suitably dilute protein concentrations it is possible to inject a small quantity of reaction mixture directly into the HPLC without causing significant loss of column performance. Extraction techniques have been comprehensively reviewed (Perrett, 1987). Design of HPLC assays for purine metabolism For clinically meaningful assays consideration must be given to many non-chromatographic factors prior to the analysis step. These include incubation conditions, particularly phosphate concentrations in media, substrate/enzyme concentrations and the degree of cellular
* See Table 2 for abbreviations. @ 1990 by John Wiley & Sons, Ltd. 1990
HPLC OF ENZYMES OF PURINE A N D PYRIMIDINE MET BOLISM
ischaemia that may be permissible (Dean and Perrett, 1976). However, under appropriate substrate and incubation conditions many pathways can be and have been determined in intact cells, tissue homogenates and even whole organs using HPLC with UV detection. Enzyme assays, which involve the separation and quantitation of substrates from products and possibly side-reactants are relatively easy for modern HPLC techniques. The analytical step should therefore be designed to be simple, rapid and specific. This can only be achieved by devising isocratic separations and the use of sensible substrate and enzyme concentrations. Because of the serial nature of HPLC analyses, HPLC does not readily allow realtime kinetic studies, although if the assay is rapid enough many time-points may be automatically injected without extraction if protein loads are low enough. When enzyme activity is low and little product is formed from even large amounts of substrate, conditions should be chosen so that the small peak due to the product appears before the large peak due to the substrate. This may be achieved by standard variations of elution conditions or by more dramatic means such as changing the column type or using ion pairs. For example, cytidine deaminase activity can be best monitored by including octanesulfonic acid in the eluent to elute uridine prior to cytidine (Fig. 2) (James et al., 1989). Although the pathways of purine and pyrimidine metabolism are complex, they fall into a limited number of types which can be summarized as base-nucleosidenucleotide interconversions, base-base degradation, nucleoside interconversions and nucleotide interconversions as well as de no00 synthetic pathways. However, it is the chemical nature of the various types of compounds that dictates the approach to their individual and group chromatography. Therefore enzymes that simdv interconvert can usually be assayed using suitable liqiid chromatographic conditions: for example
I
4 4 4
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8
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8
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4
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I-
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Figure 2. Assay of cytidine deaminase in serum by ion-paired RPLC (Assay as per James et a/ 1989). (a) RPLC separation of cytidine and uridine standards without ion-pair (b) separation of cytidine and formed uridine with allopurinol (internal standard). Column: 100 ~ 4 . mm 6 5 prn ODS-Hypersil; eluent: 100 rnM ammonium acetate, pH 5 , l m M octanesulphonic acid; flow rate: 1.2 mL/min; detection 262 nm.
@ 1990 by John Wiley & Sons, Ltd. 1990
I T
c
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adenosine deaminase is assayed by monitoring the conversion of adenosine to inosine or hypoxanthine by isocratic RPLC. However, enzymes that involve phosphorylation, or phosphorylation plus degradation, may require multiple analyses using different liquid chromatographic separations. We have exploited the ability of ion pairs to retain nucleotides on ODS columns whilst leaving nucleoside and base chromatography relatively
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Figure 3. Assay of red cell purine enzymes by ion-paired RPLC. Separation conditions; Column: 250 x4.6 mm 5 p m ODSII-Hypersil; eluent: 40 m M sodium acetate, pH 2.75, 5 rnM tetrabutylammonium ion; flow rate: 1.2 mL/min; detection: 254 nrn (lower trace), 280 nm (upper trace). (A) adenine phosphoribosyltransferase assay with adenine (A) as substrate, ( B ) hypoxanthine-guanine phosphoribosyltransferase with hypoxanthine (H) as substrate, (C) adenosine deaminase with adenosine (AR) as substrate, (D) purine nucleoside phosphorylase with inosine (HR) as substrate. Chrornatograms show 0 and 15 rnin incubations.
B
A
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--
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Figure 4. Assay of uridine monophosphate hydrolyase by ion-paired RPLC with UMP as substrate and, uridine as product. Column: 250 X4.6 m m 5 p m ODSII-hypersil; eluent: 40 mM sodium acetate, pH 2.75, 5 m M tetrabutylammonium ion; flow rate: 1.2 mL/min; detection: 254 nm (lower trace), 280 nrn (upper trace). Chromatogram shows 0 and 15 min incubations of red cells.
BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 6, 1990 269
D. PERRETT A N D H. A. SIMMONDS
Table 1. Enzyme interconversions and their polarity changes Polarity change
Example
base+ base base c* nucleoside NMP+ NMP
xanthine oxidase PNP AMP deaminase
small medium small
NMP+ nucleoside
5 nucleotidase
large
nucleoside+ nucleotide base + nucleotide NTP+ NDP- NMP
AK APRTase Apyrase
large v. large large
HPLC type
RPLC RPLC IEC or IP-RPLC RPLC or IP-RPLC IP-RPLC IP-RPLC IEC or IP-RPLC
NMP, NDP, NTP: nucleotide mono-, di- and tri-phosphates. RPLC: reversed phase HPLC. IP-RPLC: reversed phase HPLC with ion pair. IEC: anion exchange HPLC.
unchanged. Both tetrabutylammonium (Hoffman and Liao, 1977) and triethylamine ion (Willis et af., 1986) can be employed to retain nucleotides on RPLC columns. By appropriate adjustments to the percent organic modifier overall analysis time can be controlled. Figure 3 illustrates this very useful approach for a series of purine salvage enzymes of clinical interest. Figure 4 shows the same approach applied to an enzyme of pyrimidine metabolism. The relationship between enzymatic change and the HPLC approach is summarized in Table 1. Pathways where little product is formed or gross interferences are present on the UV chromatogram have required the use of more specific detectors. Xanthine oxidase activity is very low in most human tissues (Watts et af., 1965), but can be assayed by electrochemical detection which can be both highly sensitive to the product urate and relatively insensitive toward the substrate xanthine (Perrett, 1986) (Fig. 5 ) . Poly-ADP ribosylation can be measured by chloroacetaldehyde derivatization of the product, followed by anion exchange HPLC with fluorescent detection. If none of these methods are possible, then the use of radiochemicals should be considered. Chromatographic considerations remain the same, but the determination of radioactivity 3Omin
15min
in the separated product can be performed either offor on-line. Trace levels of adenosine deaminase and adenosine kinase were monitored by collecting fractions from an anion exchange column, addition of scintillant and counting (Perrett and Dean, 1977). More satisfactory for some situations when activities are lower is the use of in-line radioactivity detectors (Fairbanks et af., 1987).
Disorders of purine and pyrimidine metabolism in man Abnormalities of purine and pyrimidine metabolism are related to either inborn errors of metabolism (Table 2) or disorders of normal metabolism due to disease or drug therapy (Table 3). Clinically the most interesting group with regard to enzyme and metabolite assays are the inborn errors.
Table 2. Inborn errors of purine and pyrimidine metabolism in man Purine metabolism
1. Hypoxanthine-Guanine phosphoribosyl transferase (HGPRTase; EC 2 . 4 2 8 ) deficiency (Lesch-Nyhan Syndrome) 2. Adenosine deaminase SClD (ADA; EC 3.5.4.4) deficiency 3. Purine nucleoside phosphorylase (PNP; EC 2.4.2.1) deficiency 4. Adenine phosphoribosyl transferase (APRTase; EC 2.4.2.7) deficiency 5. Phosphoribosyl pyrophosphate synthetase (PPRP-S; EC 2.7.6.1) superactivity 6. Xanthine oxidase (XO; EC 1.2.3.2) deficiency 7. Combined xanthine oxidase and sulphite oxidase deficiency 8. Partial HGPRTase 9. Gout 10. ITP-pyrophosphohydrolase (ITP-ase; EC 3.6.1.19) deficiency 1 1 . Myoadenylate deaminase (AMPDA; EC 3.5.4.6) deficiency 12. Adenylosuccinase (ASAse; EC 4.3.2.2) deficiency Pyrimidine metabolism
13. Oroticaciduria (UMP synthase; EC 2.4.2.10/ODC EC 4.1.1.23) deficiency 14. Pyrimidine-5-nucleotidase (UMP hydrolyase 1 ; EC 3.1.3.5) deficiency 15. Dihydropyrimidine dehydrogenase (DiHpyDH; EC 1.3.1.2) deficiency
0
Table 3. Some causes of decreased ATP levels in tissues 1 . Increased ATP turnover Fructose, xylitol infusion. etc. Exercise Ethanol Glycogen storage disease Type 1 Hereditary fructose intolerance Fructose 1.I-diphosphatase deficiency Figure 5. Assay of xanthine oxidase in liver homogenate with electrochemical detection. Incubate a t pH 6 with xanthine (X) substrate. The uric acid (UA) formed was measured a t the shown intervals. Column: 100 X4.6 mm 3 p m ODSII-hypersil eluent: 50 mM ammonium acetate, pH 5.5/MeOH (98:2); flow rate: 1.2 mL/min; detection: oxidative electrochemical a t +0.7 vV. Ag/AgCI a t glassy carbon electrode.
270 BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 6, 1990
2. Decrease ATP synthesis Tissue hypoxia a) lschaemia b) Hypoxia Metabolic myopathies Hypophosphatemia
@ 1990 by John Wiley & Sons, Ltd. 1990
HPLC OF ENZYMES OF PURINE A N D PYRIMIDINE METABOLISM
Fifteen genetic disorders involving essential enzymes of purine metabolism and three of pyrimidine metabolism have now been identified. Some are associated with clinicopathological features, such as the severe neurological abnormalities of complete Lesch-Nyhan syndrome, a fatal immunodeficiency syndrome in ADA and PNP deficiency. Other features are inherited nerve deafness as well as severe muscular hypotonicity in severe PP-ribose-P synthetase superactivity, urolithiasis in APRTase deficiency or XO deficiency and megaloblastic or heamolytic anaemia in hereditary oroticaciduria and pyrimidine-5‘-nucleotidase deficiency, respectively. For a complete description of these defects see Simmonds (1987) and the appropriate chapters in ‘The Metabolic Basis of Inherited Disease’ (Scriver et aL, 1989). There is considerable genetic heterogeneity, which means that in some instances the defect may be relatively benign or of late onset. The broad spectrum of the clinical symptoms makes recognition difficult and could be equally indicative of the enzyme affecting other metabolic pathways. Treatment is available for some disorders which, if unrecognised, can otherwise progress to dialysis, transplantation or death. Early identification is thus imperative. Bone marrow transplant therapy for ADA and PNP deficiency has been successful in only a minority of cases. Consequently carrier detection and prenatal diagnosis are equally essential and are now possible in the first and second trimester, but it is important to be aware of the possible pitfalls in chorionic villus sampling for diagnosis. Such early and efficient recognition has only been made possible by considerable advances which have taken place in HPLC in the past 15 years. HPLC has enabled not only the rapid diagnosis of the underlying enzyme defect using simple liquid chromatographic systems but also their detection from the presence/absence of abnormal/normal metabolites in cells and body fluids. In every instance where there has been clinical evidence of cellular toxicity, abnormal erthrocyte nucleotide patterns have been found (Simmonds et aL, 1988). These patterns have proved useful in diagnosis as ‘fingerprints’ characteristic of the particular disorder (Simmonds et al., 1988). They may also provide some clue to the metabolic basis for the disorder and thus can be important as a research tool, as well as a diagnostic one. However, identification of these patterns depends on the efficiency of the separation obtained. In the anion exchange system used for nucleotides, constant attention to the pH and concentration of the eluents employed is essential to obtain reproducible results as the column
ages (Perrett et a/., 1989). It is equally important to recognise that no two columns will give exactly the same separation and that specific changes may also be masked in the presence of severe renal impairment. Nucleotide patterns in children also differ slightly from those in adult erythrocytes and appropriate control ranges must be established. Where the appropriate enzyme is expressed in the erythrocyte, deletion of the defect can be established from investigation of activity in lysed cells using ion pair HPLC coupled with intact cell studies using radiolabelled substrate (Fairbanks et al., 1987). Additional to this group is gout, where the genetic error leads to the precipitation of uric acid crystals in gouty tophi. The disease at present appears to include a number of different but related conditions. The largest group is composed of men aged over 40 with hyperuricemia. However, even for this classical group, the exact nature of this defect is still unknown and may relate to urate transport either in the kidney or intestine, some abnormality of minor pathways of purine production or defective urate binding to plasma proteins. Many diseases affect purine metabolism indirectly by causing (a) increased turnover of nucleotide synthetic pathways, (b) gross losses of intracellular nucleotides or (c) leakage of purine-metabolizing enzymes. Renal failure not only leads to the accumulation of trace purine and pyrimidine end-products in plasma, but also to elevated levels of nucleotides within red and white blood cells. The elevated levels of nucleotides within RBCs correlates with the degree of renal failure and the concentration of plasma phosphate in man (Rejman et aL, 1985). It is likely that such increases are also found in the intracellular phosphate pool but such data has not yet been documented.
CONCLUSIONS Over the last 15 years HPLC has become a complementary technique to the many dozens of traditional spectrometric enzyme assays already available to the purine biochemist. In many areas, such as prenatal diagnosis of inborn errors of purine metabolism, it is a superior technique and should be the technique of choice when sample amounts are limited. However, it must always be remembered that it is only an analytical technique and therefore cannot give good results from poorly designed enzyme assays.
REFERENCES Brown, P. R. (1984) (ed.). HPLC in Nucleic Acid Research, Methods and Applications, Vol. 28, Chromatographic Sciences Series. Marcel Dekker, New York. Dean, B. M. and Perrett, D. (1976). Biochem. Biophys. Acta 437, 1. Fairbanks, L. D., Simmonds, H. A. and Webster, D. R. (1987). Inherit. Metab. Dis. 10, 174. Halfpenny, A. P. and Brown, P. R. (1986). J. Liq. Chromatogr. 9,2585. Henderson, J. F. and Paterson, A. R. P. (1973). Nucleotide Metabolism, an Introduction. Academic Press, New York. Hoffman, N. E. and Liao, J. C. (1977). Anal. Chem. 49, 2231.
@ 1990 by John Wiley & Sons, Ltd. 1990
James, I. T., Herbert, K. E., Perrett, D. and Thompson, P. W. (1989). J. Chromatogr. 495, 105. Jones, D. D., Bahjri, S.. Roberts, E. L. and Williams, G. F. (1982). Brit. J. Obstet. Gynaecol. 89, 314. Krustolovic, A. M. (1987) (ed.). CRC Handbook of Chromatography of Nucleic Acids and Related Compounds, Vols I and II. CRC Press, Boca Raton. Lennard, L., Lilleyman, J. S., Van Loon, J. and Weinshilboum, R. M. (1990). Lancet 336,225. Morris, G. and Simmonds. H. A. (1985). J. Chrornatogr. 344,101.
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D. PERRETT A N D H. A. SIMMONDS Perrett, D. (1982). Chromatographia 16, 21 1. Perrett, D. (1986a). In HPLC of Small Molecules, C. K. Lim, ed., 221-259, IRL Press, Oxford. Perrett, D. (1986b). J. Chromatogr. 386,289. Perrett, D. (1987). In Handbook of Chromatography of Nucleic Acids and Related Compounds, ed. by A. M. Krustolovic, Vol. I, Part 8, pp. 3-29. CRC Press, Boca Raton. Perrett, D. and Dean, B. M. (1977). Biochem. Biophys. Res. Commun. 77,374. Perrett, D., Herbert, K. E.. Morris, G. and Simmonds, H. A. (1989). In Human Purine and Pyrimidine Metabolism. ed. by K. Mikanagi, K. Nishioka and W. N. Kelly, Vol. VI, Part B, pp. 463468. Plenum Press, New York. Reiter, S., Simmonds, H. A,, Zollner, N.. Braun, S. L. and Kneldel, M. (1990). Clin. Chim. Acta 187, 221. Rejman, A. S. M., Mansell, M.A., Grimes, A. J. and Joekes, A. M. (1985). Brit. J. Haematol. 61, 433.
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Rossomondo, E. F. (1987). High Performance Liquid Chromatography in Enzymatic Analysis. Wiley, New York. Rossomondo, E. F. (1989). J. Chromatogr. 492, 361. Scriver, C. R.. Beaudet, A. L., Sly, W. S. and Valle, D., (1989). The Metabolic Basis of Inherited Disease, 6th Ed. McGraw-Hill, New York. Simmonds. H. A. (1987). In The Inherited Metabolic Diseases, ed. by J. B. Holton, pp. 215-255. Churchill Livingstone, Edinburgh. Simmonds, H. A., Fairbanks, L. D., Morris, G. S., Webster, D. R. and Harley, E. H. (1988). Clin. Chim. Acta 171, 197. Watts, R. W. E., Watts, J. E. M. and Seegmiller, J. E. (1965). J. Lab. Clin. Med. 66, 688. Willis, C. L., Lim, C. K. and Peters, T. J. (1986). J. Pharm. Biomed. Anal. 4. 247.
Received 22 August, 1990; accepted 5 September, 1990.
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1990
