BIOCHEMICAL SOCIETY TRANSACTIONS
1060
Hypoxanthine output is increased by ATP use in man
G . M. McCREANOR and R. A. HARKNESS
Division of Inherited Metabolic Diseases,
M . R.C. Clinical Research Centre, Watford Road,
Harrow, Middlesex H A 1 3UJ, U . K .
Hypoxanthine is a central metabolite in ATP metabolism
and its salvage to IMP is catalysed by hypoxanthine
phosphoribosyltransferase (HPRT: EC 2.4.2.8.).
Since deficiency of this enzyme in man causes profound
neurological dysfunction and testicular atrophy, a regular
supply of substrate should be available. However, only ATP
depletion has been proven to produce a marked increase in
hypoxanthine output (Harkness et al., 1983), but ATP
depletion rarely occurs in human tissues under normal conditions. This study was set up to investigate hypoxanthine
output under conditions of ATP turnover rather than
depletion. This was achieved by exercising at low-tomoderate levels of ATP use and examining hypoxanthine,
xanthine and urate output.
Exercise was performed for 2 min on a bicycle ergometer
by ten normal volunteers (five men and five women) at five
different work rates on five consecutive days of one week.
To avoid any effects of training, a latin-square design was
used. Urine was collected overnight before each exercise and
at three two hourly intervals after the morning exercise. The
overnight outputs were used as a baseline. Hypoxanthine
and xanthine were extracted from urine by ion-exchange
column chromatography on Zerolit 225 and determined by
reverse phase h.p.1.c. (Simmonds & Harkness, 1981).
Urinary urate was measured spectrophotometrically.
The results in Table 1 show that there is a positive correlation between the amount of exercise performed and the
excretion of hypoxanthine at low-to-moderate exercise
levels. There was no increase in xanthine or urate excretion
except at the highest level of work which was near to fatigue.
Hyperuricaemia occurs after strenuous exercise, e.g. 5000 m
and 42 km races (Sutton et al., 1980).
Hypoxanthine output in control subjects increased with
low levels of exercise. Thus, HPRT has a role in purine
salvage at low levels of ATP turnover. In actively growing
HPRT-deficient cells - lymphoblasts - there is a significant reduction in the intracellular concentration of ATP
(McCreanor et al., 1987); in contrast, ATP concentrations
Table 1. The increased excretion ofhypoxanthine, xanthine and
urate in,five men a n d j v e women after exercise
Results are mean + s . E . M . (n = 5).
Exercise level
(kJ)
Men
9
12
15
Hypoxanthine
(nmol/h . kg)
Xanthine
(nmol/h. kg)
f 1I
f 15
f 32
f 99
f 351
12.3
20.3
5.9
14.7
32.3
k4
k5
21
29.2
45.8
87.2
258.4
823.4
Women
9
12
15
18
21
39.3
38.3
110.2
359.5
953.0
f 16
f 23
f 16
f 49
f 230
10.9
8.1
12.7
13.6
28.6
k7
18
*f 42
f9
*k 45
f3
k
10
Urate
(pmol/h . kg)
1.43 f 0.33
1.39 f 0.54
1.22 & 0.17
1.01 0.46
1.66 0.70
+
0.90
0.77
1.00
0.87
1.77
& 0.38
rf; 0.58
rf; 0.23
rf; 0.17
& 0.56
in fibroblasts, which are less active cells, are not consistently
reduced (Rosenbloom et al., 1968). Thus, HPRT has a role
in ATP conservation in active cells. Our findings suggest
that the effects of HPRT deficiency will become greater with
increasing ATP use, especially in tissues which normally
have high levels of HPRT; testis and the highly active
human brain. Since the brain protects itself from marked
ATP depletion by rapidly reducing its function (Duffy et al.,
1972), the neurological dysfunction without structural
damage found in HPRT deficiency could be explained by
our findings if hypoxanthine output by the brain is also
related to ATP turnover.
Duffy, T. E., Nelson, S. R. & Lowry, 0. H. (1972) J . Neurochem. 19,
959-977
Harkness, R. A., Simmonds, R. J. & Coade, S. B. (1983) Clin. Sci. 64,
333-340
McCreanor, G. M., Harkness, R. A. & Watts, R. W. E. (1987)
Biochem. Soc. Trans. in the press
Rosenbloom, F. H., Henderson, J. F., Caldwell, I. C., Kelley, W. N.
& Seegmiller, J. E. (1968) J . Biol. Chem. 243, 11661 173
Simmonds, R. J. & Harkness, R. A. (1981) J . Chromatogr. 226, 36938 1
Sutton, J. R., Toews, C. J., Ward, G. R. & Fox, I. H. (1980) Me/aho/i.sm
29 (3), 254 259
~
Abbreviations used: HPRT, hypoxanthine phosphoribosyltransferase; h.p.l.c., high pressure liquid chromatography.
Received 1 April 1987
Immunoassay of the cardiac-specific isoform of troponin-I in the diagnosis of
heart muscle damage
BERNADETTE CUMMINS and PETER CUMMINS
Molecular Cardiology Unit, Department of Cardiovascular
Medicine, University of Birm ingham , Birm ingham ,
B15 2 T H , U . K .
The clinical diagnosis of heart muscle damage relies in
general on non-cardiac-specific methods involving electrocardiographic, patient history and serum enzyme criteria.
Serum levels of cardiac isoforms of creatine kinase and
lactate dehydrogenase have been determined but significant
levels may be present in skeletal muscles precluding unambiguous source identification. A highly specific marker of
cardiac cell damage is required.
Abbreviations used: RIA, radioimmunoassay; ELISA, enzymelinked immunosorbent assay.
Troponin-I, a myofibrillar regulatory protein, exists in
fast and slow skeletal and cardiac isoforms (Wilkinson &
Grand, 1978). Cardiac troponin-I is uniquely located, and
is the only isoform present in the heart (Humphreys &
Cummins, 1984). Its diagnostic potential as a cardiac specific
marker of cell damage has previously been examined in
patients after myocardial infarction by measuring serum
troponin-I levels using a 24 h radioimmunoassay (RIA)
(Cummins et al., 1987). The RIA sensitivity of 10 ng/ml did
not allow precise measurement of normal serum levels. To
improve both speed and sensitivity, an enzyme-linked
immunosorbent assay (ELISA) has been established for use
in a canine model (Cummins & Cummins, 1985) and clinically post-myocardial infarction.
Plastic microtitre plates were coated with 50 pl of primate
or canine cardiac troponin-I at 0.5 pg/ml. Standard cardiac
1987