J Clin Pathol: first published as 10.1136/jcp.36.1.1 on 1 January 1983. Downloaded from J Clin Pathol 1983;36: 1-8 Cerebrospinal fluid,concentrations of hypoxanthine, xanthine, uridine and inosine: high concentrations of the ATP metabolite, hypoxanthine, after hypoxia RA HARKNESS, RJ LUND* From the Division ofPerinatal Medicine, MRC Clinical Research Centre, and * Department of Paediatrics Northwick Park Hospital, Watford Road, Harrow SUMMARY CSF obtained for clinical purposes from newborn, children and adults has been analysed by high pressure liquid chromatography for hypoxanthine, xanthine, inosine, uridine and urate. Large rises in hypoxanthine and to a lesser extent xanthine occur for about 24 h after hypoxia. High concentrations were associated with later evidence of brain damage or subsequent death. Changes in CSF could be independent of those in plasma. Small or negligible rises were associated with localised and generalised infections including bacterial meningitis, fits, or both. Marked and rapid rises were found after death. These estimations may "predict" the extent of brain damage or brain death. copyright. Damage in many diseases is due to hypoxia. rises could be separated from the slight or negligible Intrapartum hypoxia is recognised as a major cause of changes associated with infection or fits. Brain morbidity and mortality,' 2 yet objective methods for damage or brain death proven by clinical, confirming the diagnosis lack specificity and electrophysiological and ultrasonic methods was comparative measurements are not available.3" The associated with high concentrations. CNS is the most vulnerable system to damage by http://jcp.bmj.com/ hypoxia.5 The central processes in metabolic damage Methods due to hypoxia involve ATP,6 the universal energy currency of cells. A reduction in intracellular ATP CLINICAL can be a measure of the metabolic damage due to Northwick Park is a large general hospital with a hypoxia. Means of estimating ATP breakdown by perinatal department delivering about 3400 infants non-invasive methods are needed in clinical practice per year. The CSF samples analysed were all aliquots are not available. Since clinical indications because serial tissue samples of samples taken for conventional on September 23, 2021 by guest. Protected some of the uncharged products of ATP breakdown, from 1978-1982. In the last two years of the study hypoxanthine, xanthine, and inosine, can escape these aliquots were immediately centrifuged to from cells, concentrations of these oxypurines in remove blood cells and then stored at -200C. This extracelluar fluids like CSF can reflect ATP was advisable because blood staining of samples was breakdown. The compounds hypoxanthine, frequent in newborn CSF in contrast to that from xanthine, and inosine can now be estimated older children and adults. Contamination of over specifically by methods of sufficient sensitivity.' Since about 109 erythrocytes per litre or fluid which was metabolic events in brain will be reflected in its obviously blood stained, resulted in high oxypurine extracellular fluid,8 we have used CSF samples to concentrations and such specimens were not analysed study ATP breakdown in the CNS after hypoxia. or the results were discarded. Clinical records were Results obtained from 1978 to 1982 show raised surveyed to determine the final diagnoses, the timing concentrations of hypoxanthine and xanthine in CSF of clinical abnormalities such as an asphyxial episode, at least 24 h after serious hypoxia in newborn. These and the time of the CSF sample. The clinical criteria *Present address: Dr RJ Lund, 21 Norfolk Street, Harfield Village, for intrapartum asphyxia and some aspects of Charemont 7700, South Africa. the neurological assessment have already been Accepted for publication 16 July 1982 described.2 Neurological status at the time of the CSF 1 J Clin Pathol: first published as 10.1136/jcp.36.1.1 on 1 January 1983. Downloaded from 2 Harkness, Lund sample was specifically noted by JL in 1981. We are 9- indebted to Dr M O'Connor for post-mortem samples of vitreous humour and CSF. For a set of results to be accepted for a normal 8- range no abnormality apart from mild fever or poor feeding had to exist at the time of the sample and subsequent investigation and progress had to be 7-~ I essentially negative. In newborn, the majority ofsuch 0 specimens were taken during a search for evidence of infection. The adult normal range was determined 6- S from a series of CSF samples from patients without marked evidence of metabolic or other disease, 0 stored at -20°C by the Clinical Chemistry 5- Department at Northwick Park Hospital. However, S R-E both sets of "normal" ranges are probably falsely high 0 because some abnormality existed to prompt' the 4- . : *- - taking of the CSF sample. : * BIOCHEMICAL 3- Trichloracetic acid extracts of CSF were analysed by * 0 high pressure liquid chromatography by the methods _ * 0 | of Simmonds and Harkness.7 The results of 2- . bacteriological examinations, cell counts, protein and glucose concentrations by conventional methods . 1- -. were obtained from the case notes. C- Nucleosides, inosine and adenosine, were specifically measured using boronate affinity column copyright. chromatography followed by high performance liquid 0 - i . .~~~~~ chromatography (HPLC) using a gradient of c,2*~~~Hi's scAeeo methanol in phosphate buffer on a C18 Hypersil column. A similar method has been used to measure adenosine and inosine in plasma.9 The nucleoside, Fig. I Normal concentrations in CSFfrom newborn of uridine, the central compound in pyrimidine http://jcp.bmj.com/ metabolism, was measured by the simple and by the hypoxanthine, xanthine, uridine and inosine. The longer a measure of the bars indicate arithmetic means with shorter bars to indicate selective method and this provided +2 SD limits exceptforinosine in which only an upper limit is overall recovery of nucleosides which was used to shown. correct the final analytical results. Geometric mean and range of +2 SD of the Results logarithmically transformed data are therefore shown in Fig. 2. The mean and ranges (,umol/l) were NORMAL INFANTS AND ADULTS hypoxanthine 1-8 (0 S6-5 1), xanthine 1 7 (0 6-4 7), on September 23, 2021 by guest. Protected In 18 "normal" CSF samples from 16 newborn infants uridine 1 6 (0 3 8- 2), and inosine 0 2 (0(1-0). the concentrations of hypoxanthine, xanthine, uridine, and inosine are shown in Fig. 1; the dis- CONCENTRATIONS IN PATHOLOGICAL tribution of values are symmetrical about a mean. An CONDITIONS: HYPOXIA arithmetic mean and range of ±2 SD were therefore In cases of hypoxia CSF concentrations of used. Values (p.mol/l) were for hypoxanthine 3-6 hypoxanthine and xanthine were raised most with (1 8-5-5), xanthine 5 0 (0-9-9 1), uridine 3-3 (0 6-- lesser rises in uridine and inosine. Results for uridine 6 3) inosine 0 7 (0-2 0). Urate, the catabolic product and inosine are not therefore shown with the pattern of hypoxanthine and xanthine, was also estimated in of results in Fig. 3 but are given in Table 1. The upper nine samples which had a mean (+SD) concentration limits of the normal ranges for hypoxanthine and of 30 (+25) ,umol/l. xanthine are shown as a line in Fig. 3. In the first two In a "normal control" series of 29 adults the days after an episode of hypoxia there was a marked concentrations in CSF of hypoxanthine, xanthine, rise in the concentration of hypoxanthine. We have uridine, inosine are shown in Fig. 2. The results little evidence after this but the trend in the data showed an approximately log-normal distribution. suggests no abnormalities are to be expected on day 3 J Clin Pathol: first published as 10.1136/jcp.36.1.1 on 1 January 1983. Downloaded from Raised CSF oxypurines after hypoxia 3 9.0 1000_ Hypoxonthine in CSF Xanthine in CSF 0 500 - 50- 400- 0 300- 40- 200 - 30- a * 100 - 20 *. X X 50 - a - 40- 30- 10 ? -a a 0 E I 20- a * a 0~~~~~ 0 09 0* . 10 0-5- o 04- . ui8~~ o o o 5 . 4- 0 3- 0 02 2- i- copyright. 012 34 5 6 0 1 2 3 4 5 6 0.1 Days after hypoxia _I I * Fig. 3 Concentrations ofhypoxanthine and xanthine in ,,,o.-Ir &-Fs ;5iv s#? CSFafter hypoxia. Symbols show subsequent major clinical findings: * = died, * = brain damaged, 0 = normal, O = congenital hypothyroidism, A = chloramphenical toxicity. The are http://jcp.bmj.com/ Fig. 2 Normal concentrations in CSFfrom adults of horizontal lines the +2 SD limit ofconcentration. hypoxanthine, xanthine, uridine and inosine. The longer bars indicate geometric means with shorter bars to indicate the predetermined criteria. Two of three of our ±2 SD oflogarithmetically transformed data exceptfor survivors with cerebral damage and high hypo- inosine in which only an upper limit is shown. xanthine concentrations were observed to be markedly cyanosed and apnoeic in postnatal after an episode. The three children who survived episodes, one had subsequent renal tubular necrosis. after showing high concentrations of hypoxanthine The third had sustained low Apgar scores, one at 1 on September 23, 2021 by guest. Protected and xanthine have cerebral damage as shown in one min, four at 5 min and subsequent fits. One infant was by electrophysiological evidence of cerebral cortical shown to have brain death by conventional criteria. blindness and in another by ultrasonographic evid- This infant had a cardiac arrest at the age of 12 min; ence of severe cerebral atrophy. The third is an the heart then continued after a later respiratory athetoid spastic. Five infants died with clinical arrest sustaining normal plasma concentrations of evidence of asphyxia and had high concentrations of ATP metabolites, hypoxanthine 1-5, xanthine 1-2, hypoxanthine in their CSF.