Is post-hypoxic-ischemic cell damage associated with excessive ATP consumption rather than a failure of ATP production?

Secondary cell damage after ATP depletion due to hypoxia or ischemia is clinically important because it correlates with residual effects; post-hypoxic-ischemic fits can be associated with later cerebral palsy. The mechanisms involved in delayed secondary cell damage are not clear, possibly because extensive relevant evidence is often fragmented. However, a sequence of changes can be suggested; this cross-linked sequence is tentatively outlined in this review. The outline suggests explanations for otherwise ill-understood clinical disturbances such as the loss of inhibitory control in damaged cells and the well documented reduction of cellular ATP. Loss of control may be due to reduced synthesis of control proteins and the reduced ATP concentration may be due to increased energy consumption.

The importance of the measurement of ATP depletion and subsequent cell damage with an estimate of size and nature of the market for a practicable method: a review designed for technology transfer.

ATP is the energy currency of cells. ATP depletion is a central process in pathogenesis, in particular ischaemia, hypoxia and hypoglycaemia. ATP depletion in cells can be indirectly measured from the increased concentrations of extracellular hypoxanthine, a central intermediate in the metabolism of ATP. Cell damage secondary to ATP depletion can also be measured from extracellular hypoxanthine. The relevant biochemistry and physiology is briefly reviewed. Since market size is needed for investment decisions that would allow technology transfer, the numbers of hypoxanthine analyses that are clinically justified from the extensive published evidence are calculated per million population from UK, Norwegian and other evidence. The concentration of oxygen in blood is measured to estimate whether mitochondrial oxidative phosphorylation is adequate. Measurements of bicarbonate are used to estimate anaerobic glycolysis. Since the indirect estimation of ATP depletion is a major objective of blood gas and acid-base analyses, the number of such analyses per million population provides a good estimate of potential market size for a more direct method of estimating ATP depletion. A method is required for the rapid, dispersed emergency analyses needed clinically. Routes for method development are indicated. Competition, risks, acceptability, consumer motivation and timetables are indicated for the development phase. There are medicolegal pressures, especially in the USA, for the proposed advances to be widely used.

Lesch-Nyhan syndrome and its pathogenesis: normal nicotinamide-adenine dinucleotide but reduced ATP concentrations that correlate with reduced poly(ADP-ribose) synthetase activity in HPRT-deficient lymphoblasts.

In hypoxanthine (guanine) phosphoribosyltransferase- (HPRT; EC 2.4.2.8) deficient lymphoblasts, ATP but not nicotinamide-adenine dinucleotide coenzyme concentrations are reduced by limited nutrition. Such reduced ATP concentrations are correlated with reduced poly(ADP-ribose) synthetase (polyADPRT; EC 2.4.2.30) activity; this reduces the breakdown of nicotinamide-adenine dinucleotide coenzymes and thus explains their normal intracellular concentrations. Since reductions in poly(ADP-ribose) synthetase activity reduce DNA repair, alterations in DNA could accumulate even in non-multiplying cells such as neurons, especially in the continuously active 'respiratory centre'. Our Lesch-Nyhan patients suffered respiratory deaths between 15 and 20 years of age.

Introduction to the age-related diagnosis (ARD) index: an age at presentation related index for diagnostic use.

Development and, therefore, age is important in paediatrics. Diagnostically useful data from this journal has been related to age and organized to form an age-related diagnostic (ARD) index. The ARD index is designed for non-expert clinical and laboratory workers to use in the early phases of diagnosis and as an addition to existing diagnostic schemes. Entry to the index is from the age at clinical presentation. Each entry is a sequence starting with clinical and laboratory presentations, clinical course, laboratory key investigations and finally diagnosis with volume and page numbers of the original article, the primary source. Within age groups, entries are grouped by diagnoses with the commonest diagnosis first; this has the effect of roughly but not precisely grouping similar clinical and laboratory findings.

Muscle ATP loss and lactate accumulation at different work intensities in the exercising Thoroughbred horse.

The effect of 2 min treadmill exercise, at speeds of 6-12 m.s-1 on an incline of 5 degrees, upon muscle adenine nucleotide loss and lactate accumulation was studied in six Thoroughbred horses. Minimal change occurred in the adenosine triphosphate (ATP) content of the middle gluteal muscle at speeds of 10 m.s-1 or less, but significant loss (up to 47%) had occurred in all horses by 12 m.s-1. The decline in ATP significantly correlated with the accumulation of muscle lactate, beginning shortly after the accumulation of 40 mmol.kg-1 dry muscle lactate. Decline in muscle ATP was mirrored closely by the appearance of ammonia, and to a lesser extent, hypoxanthine and uric acid in plasma. The results suggest that peak accumulation of any of these, or simply the concentration at a specified recovery time, may be used as a measure of ATP loss in the musculature as a whole. This was not so in the case of xanthine, which may also be formed from the degradation of guanidine nucleotides. An In-In plot of plasma ammonia against treadmill speed indicated a break point in accumulation between 8 and 9 m.s-1. The kinetics of ammonia accumulation with speed differed from those of lactate.

The pathogenesis of the Lesch-Nyhan syndrome: ATP use is positively related to hypoxanthine supply to hypoxanthine guanine phosphoribosyltransferase.

In order to explain features of severe hypoxanthine guanine phosphoribosyltransferase (HPRT) deficiency, the Lesch-Nyhan syndrome, a continuous supply of substrate, hypoxanthine, for the enzyme must be generated. This supply must be increased in association with increased ATP turnover. We have shown that ATP turnover continuously supplies hypoxanthine for recycling by the enzyme HPRT and that this supply increases curvilinearly with increasing ATP turnover. The effects of increasing exercise on ATP turnover were examined using a Latin square experimental design. The outputs of hypoxanthine, xanthine, urate and creatinine were measured. The data were then examined statistically.

Hypoxanthine phosphoribosyltransferase activity in tissues and hypoxanthine concentrations in plasma and CSF of the horse in comparison with other species.

1. Plasma hypoxanthine and xanthine concentrations are very low in the horse and low in rat, mouse and greyhound compared to concentrations in beagles, man, sheep and rabbit. 2. Activities in erythrocytes of the main enzyme metabolizing hypoxanthine, hypoxanthine phosphori-bosyltransferase, show a similar pattern (Tax et al., 1976, Comp. Biochem. Physiol. 54B, 209-212); thus low activities have been found where plasma concentrations were low. 3. Hypoxanthine phosphoribosyltransferase activities in horse tissue other than erythrocytes are similar to those in man and rabbit with high activities in brain; this enzyme may therefore be functionally important in equine brain.

Distribution of xanthine dehydrogenase and oxidase activities in human and rabbit tissues.

The activity of xanthine dehydrogenase in human postmortem tissues is surprisingly high in brain and heart; activity was found in most tissue samples, whereas many samples contained little or no oxidase activity. We have confirmed the high level of oxidase activity in liver in which tissue conversion of dehydrogenase to oxidase appears complete. We have also confirmed the virtual absence of either activity in fresh human placenta. Fresh rabbit tissues similarly show considerable dehydrogenase activity in brain and heart. In view of the stability and generalised distribution of dehydrogenase activity, our results suggest that some modification of existing ideas on the physiological and pathological roles of the enzyme may be needed.

Cerebrospinal fluid nucleotide metabolites following short febrile convulsions.

Cerebrospinal fluid (CSF) markers of cerebral energy depletion were measured in 32 infants and children following short (less than 10 minutes) febrile convulsions, and in 19 controls. Specific and sensitive indices of high-energy phosphate compound depletion (hypoxanthine, xanthine and uridine) showed no marked changes. Values for patients and febrile controls were significantly higher than for afebrile controls, which is consistent with increased cerebral metabolism in febrile patients. There were no differences in pH, lactate or creatine kinase levels in the CSF of patients and controls. The results suggest that short febrile convulsions are benign and that in the absence of risk factors for the subsequent development of epilepsy, prophylactic anticonvulsant treatment is not indicated.

Cerebrospinal fluid nucleotide metabolites following non-convulsive status epilepticus.

The authors studied specific and sensitive indicators of neuronal adenosine triphosphate (ATP) depletion--hypoxanthine, xanthine and uridine levels--in the cerebrospinal fluid (CSF) of nine children during non-convulsive status epilepticus. No evidence of ATP depletion was found and CSF pH and creatine kinase levels were similar to those of controls. Hypoxanthine, xanthine and uridine had a tendency to be low, but this was significant only for xanthine. The authors speculatively link this reduction to a reduction in neuronal protein synthesis. This might be a mechanism whereby non-convulsive status epilepticus could lead to intellectual deterioration and dementia.

Antenatal fetal heart rate variation in relation to the respiratory and metabolic status of the compromised human fetus.

Three groups of women were delivered by caesarean section before labour: for an abnormal fetal heart rate (FHR) trace (21 cases, group 1), or for maternal deterioration in severe pre-eclampsia without gross fetal heart rate abnormalities (20 cases, group 2), or to avoid mechanical difficulties in labour at term (30 cases, group 3). The mean gestational ages of the first two groups were 32 weeks with a high proportion of infants small-for-gestational-age. In group 1, FHR variation (mean range of pulse intervals) was less than half (20.6 SE 1.2 ms) of the normal value at the same age (44.4 SE 1.5 ms). This was associated with hypoxaemia (mean umbilical artery PO2 of 6 mmHg at delivery), with evidence of compensation shown by an elevated amniotic fluid erythropoietin. The fetuses were hypoglycaemic and had greater umbilical artery blood alanine concentrations, but no large changes in adenine nucleotide or endorphin plasma concentrations. Although there was a minor degree of respiratory acidaemia at birth, there was not significant metabolic acidaemia. The results demonstrate that the reduced variation of 'suboptimal' and 'decelerative' fetal heart rate records is associated with fetal hypoxaemia and evidence of nutritional deprivation, but not with asphyxia.

Hypoxanthine, xanthine and uridine in body fluids, indicators of ATP depletion.

Measurements of hyp, xan and urd in body fluids can provide evidence of energy, ATP, depletion in the body, in organs or in cells. Such information is clinically useful in the many diseases in which cellular energy supplies cannot be maintained like perinatal asphyxia, hydrocephalus and vascular insufficiency in brain, heart, limbs, kidneys or other organs. Similar HPLC methods using reversed-phase C18 columns and quantitation by UV absorption have been employed in a variety of centres to yield almost identical results. These have been assembled in this review to form a series of reference values. The current analytical problems are reviewed. Since concentrations of hyp and xan may alter independently situations are discussed in which separate measurements rather than their summed, total oxypurine concentrations are needed. The biochemistry and physiology underlying the use of such analyses is examined to guide sampling of the appropriate body fluid at a relevant time and to avoid oversimplified interpretation of results as well as unnecessary arguments. Specifically: (1) Intracellular concentrations of hyp and xan are inversely related to adenylate energy change and therefore to the energy currency of the cell ATP. Uridine in tissues is similarly 'controlled'. (2) There is extensive evidence that large increases in hyp, xan and urd in body fluids indicate ATP depletion. (3) Small changes in hyp probably reflect alterations of ATP turnover. (4) Xanthine arises mainly from guanine and can change independently of hyp. (5) Clinically useful information is obtainable from hyp and xan concentrations in CSF, amniotic fluid, urine and plasma. Extensive clinical correlations are reviewed. At present we are in a development phase for which HPLC is ideal but the most efficient way to perform and use such analyses in routine clinical practice remains to be established.

Clinical and biochemical assessments of damage due to perinatal asphyxia: a double blind trial of a quantitative method.

Using conventional criteria, a series of 26 infants was selected for intrapartum asphyxia from about 4000 deliveries over one year at a single hospital to assess the efficacy of a new biochemical method. Tissue damage was estimated from urinary excretion of hypoxanthine, an important and central intermediate in purine metabolism. The overall pattern showed agreement between the grading (by previously accepted methods) of asphyxia in the perinatal period and our new biochemical approach. The association with handicap at one year of age following asphyxia was complex. This biochemical technique could be used to exclude postasphyxial damage as a cause of clinical disturbances and to select a small group (0.1% of all births) who require further investigation for rarer disorders which may also cause long term handicap.

Lesch-Nyhan syndrome and its pathogenesis: purine concentrations in plasma and urine with metabolite profiles in CSF.

Purine metabolism in the Lesch-Nyhan syndrome has been re-examined in 10 patients. Hypoxanthine and xanthine concentrations in plasma and CSF and urinary excretion have been studied, on and off allopurinol treatment, using high performance liquid chromatographic methods. Accumulation of the substrate, hypoxanthine, of the missing hypoxanthine guanine phosphoribosyltransferase (HPRT) enzyme, is more marked in urine and in CSF than in plasma. The greater increase in CSF is consistent with the most metabolically active tissue, brain, showing the most marked functional changes. The function of HPRT seems to be the recycling of hypoxanthine which is released from tissues in increasing quantities as energy use, ATP 'turnover', in the tissue increases. The existing screening method for HPRT deficiency, the ratio of the urinary concentration of urate to that of creatinine, shows overlap between the values in severe HPRT deficiency and in controls; this overlap is not found with a urinary hypoxanthine/creatinine molar concentration ratio.

The effects of fetal energy depletion on amniotic fluid concentrations of amino acids, organic acids and related metabolites.

Concentrations of amino and organic acids, phosphate, sulphate, gluconic acid and gluconolactone were measured in amniotic fluid samples which contained either normal or raised hypoxanthine concentrations. In this way, the effect of mild fetal ATP depletion could be determined. The effects of this mild asphyxia were to raise concentrations of phenylalanine, tyrosine, lysine, glycine, phosphate, sulphate, gluconic acid and glucono-1,5-lactone. However, concentrations of a variety of other metabolites were unchanged; thus no diagnostic confusion should arise with organic acidurias in mild asphyxia in contrast to the biochemical mimickry produced by severe asphyxia. Since clinically normal parturition can produce changes in amniotic fluid, urine from newborn or cord blood may not reflect the metabolic balance in utero.