Comparative assessment of favipiravir and remdesivir against human coronavirus NL63 in molecular docking and cell culture models.

Human coronavirus NL63 (HCoV-NL63) mainly affects young children and immunocompromised patients, causing morbidity and mortality in a subset of patients. Since no specific treatment is available, this study aims to explore the anti-SARS-CoV-2 agents including favipiravir and remdesivir for treating HCoV-NL63 infection. We first successfully modelled the 3D structure of HCoV-NL63 RNA-dependent RNA polymerase (RdRp) based on the experimentally solved SARS-CoV-2 RdRp structure. Molecular docking indicated that favipiravir has similar binding affinities to SARS-CoV-2 and HCoV-NL63 RdRp with LibDock scores of 75 and 74, respectively. The LibDock scores of remdesivir to SARS-CoV-2 and HCoV-NL63 were 135 and 151, suggesting that remdesivir may have a higher affinity to HCoV-NL63 compared to SARS-CoV-2 RdRp. In cell culture models infected with HCoV-NL63, both favipiravir and remdesivir significantly inhibited viral replication and production of infectious viruses. Overall, remdesivir compared to favipiravir is more potent in inhibiting HCoV-NL63 in cell culture. Importantly, there is no evidence of resistance development upon long-term exposure to remdesivir. Furthermore, combining favipiravir or remdesivir with the clinically used antiviral cytokine interferon-alpha resulted in synergistic effects. These findings provided a proof-of-concept that anti-SARS-CoV-2 drugs, in particular remdesivir, have the potential to be repurposed for treating HCoV-NL63 infection.

Adaptive landscape flattening allows the design of both enzyme: Substrate binding and catalytic power.

Designed enzymes are of fundamental and technological interest. Experimental directed evolution still has significant limitations, and computational approaches are a complementary route. A designed enzyme should satisfy multiple criteria: stability, substrate binding, transition state binding. Such multi-objective design is computationally challenging. Two recent studies used adaptive importance sampling Monte Carlo to redesign proteins for ligand binding. By first flattening the energy landscape of the apo protein, they obtained positive design for the bound state and negative design for the unbound. We have now extended the method to design an enzyme for specific transition state binding, i.e., for its catalytic power. We considered methionyl-tRNA synthetase (MetRS), which attaches methionine (Met) to its cognate tRNA, establishing codon identity. Previously, MetRS and other synthetases have been redesigned by experimental directed evolution to accept noncanonical amino acids as substrates, leading to genetic code expansion. Here, we have redesigned MetRS computationally to bind several ligands: the Met analog azidonorleucine, methionyl-adenylate (MetAMP), and the activated ligands that form the transition state for MetAMP production. Enzyme mutants known to have azidonorleucine activity were recovered by the design calculations, and 17 mutants predicted to bind MetAMP were characterized experimentally and all found to be active. Mutants predicted to have low activation free energies for MetAMP production were found to be active and the predicted reaction rates agreed well with the experimental values. We suggest the present method should become the paradigm for computational enzyme design.

Burkholderia pseudomallei d-alanine-d-alanine ligase; detailed characterisation and assessment of a potential antibiotic drug target.

Burkholderia pseudomallei is a serious, difficult to treat Gram-negative pathogen and an increase in the occurrence of drug-resistant strains has been detected. We have directed efforts to identify and to evaluate potential drug targets relevant to treatment of infection by B. pseudomallei. We have selected and characterised the essential enzyme d-alanine-d-alanine ligase (BpDdl), required for the ATP-assisted biosynthesis of a peptidoglycan precursor. A recombinant supply of protein supported high-resolution crystallographic and biophysical studies with ligands (AMP and AMP+d-Ala-d-Ala), and comparisons with orthologues enzymes suggest a ligand-induced conformational change occurring that might be relevant to the catalytic cycle. The detailed biochemical characterisation of the enzyme, development and optimisation of ligand binding assays supported the search for novel inhibitors by screening of selected compound libraries. In a similar manner to that observed previously in other studies, we note a paucity of hits that are worth follow-up and then in combination with a computational analysis of the active site, we conclude that this ligase represents a difficult target for drug discovery. Nevertheless, our reagents, protocols and data can underpin future efforts exploiting more diverse chemical libraries and structure-based approaches.

Bovine lactoferrin promotes energy expenditure via the cAMP-PKA signaling pathway in human reprogrammed brown adipocytes.

Lactoferrin (LF) is a multifunctional protein in mammalian milk. We previously reported that enteric-coated bovine LF reduced the visceral fat in a double-blind clinical study. We further demonstrated that bovine LF (bLF) inhibited adipogenesis and promoted lipolysis in white adipocytes, but the effect of bLF on brown adipocytes has not been clarified. In this study, we investigated the effects of bLF on energy expenditure and cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway using human reprogrammed brown adipocytes generated by gene transduction. bLF at concentrations of ≥ 100 µg/mL significantly increased uncoupling protein 1 (UCP1) mRNA levels, with the maximum value observed 4 h after bLF addition. At the same time point, bLF stimulation also significantly increased oxygen consumption. Signaling pathway analysis revealed rapid increases of intracellular cAMP and cAMP response element-binding protein (CREB) phosphorylation levels beginning 5 min after bLF addition. The mRNA levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) were also significantly increased after 1 h of bLF stimulation. H-89, a specific PKA inhibitor, abrogated bLF-induced UCP1 gene expression. Moreover, receptor-associated protein (Rap), an antagonist of low-density lipoprotein receptor-related protein 1 (LRP1), significantly reduced bLF-induced UCP1 gene expression in a dose-dependent manner. These results suggest that bLF promotes UCP1 gene expression in brown adipocytes through the cAMP-PKA signaling pathway via the LRP1 receptor, leading to increased energy expenditure.

Using two-site binding models to analyze microscale thermophoresis data.

The emergence of microscale thermophoresis (MST) as a technique for determining the dissociation constants for bimolecular interactions has enabled these quantities to be measured in systems that were previously difficult or impracticable. However, most models for analyses of these data featured the assumption of a simple 1:1 binding interaction. The only model widely used for multiple binding sites was the Hill equation. Here, we describe two new MST analytic models that assume a 1:2 binding scheme: the first features two microscopic binding constants (KD(1) and KD(2)), while the other assumes symmetry in the bivalent molecule, culminating in a model with a single macroscopic dissociation constant (KD,M) and a single factor (α) that accounts for apparent cooperativity in the binding. We also discuss the general applicability of the Hill equation for MST data. The performances of the algorithms on both real and simulated data are assessed, and implementation of the algorithms in the MST analysis program PALMIST is discussed.

In silico identification of AMPylating enzymes and study of their divergent evolution.

AMPylation is a novel post-translational modification (PTM) involving covalent attachment of an AMP moiety to threonine/tyrosine side chains of a protein. AMPylating enzymes belonging to three different families, namely Fic/Doc, GS-ATase and DrrA have been experimentally characterized. Involvement of these novel enzymes in a myriad of biological processes makes them interesting candidates for genome-wide search. We have used SVM and HMM to develop a computational protocol for identification of AMPylation domains and their classification into various functional subfamilies catalyzing AMPylation, deAMPylation, phosphorylation and phosphocholine transfer. Our analysis has not only identified novel PTM catalyzing enzymes among unannotated proteins, but has also revealed how this novel enzyme family has evolved to generate functional diversity by subtle changes in sequence/structures of the proteins. Phylogenetic analysis of Fic/Doc has revealed three new isofunctional subfamilies, thus adding to their functional divergence. Also, frequent occurrence of Fic/Doc proteins on highly mobile and unstable genomic islands indicated their evolution via extensive horizontal gene transfers. On the other hand phylogenetic analyses indicate lateral evolution of GS-ATase family and an early duplication event responsible for AMPylation and deAMPylation activity of GS-ATase. Our analysis also reveals molecular basis of substrate specificity of DrrA proteins.

Classification of the adenylation and acyl-transferase activity of NRPS and PKS systems using ensembles of substrate specific hidden Markov models.

There is a growing interest in the Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) of microbes, fungi and plants because they can produce bioactive peptides such as antibiotics. The ability to identify the substrate specificity of the enzyme's adenylation (A) and acyl-transferase (AT) domains is essential to rationally deduce or engineer new products. We here report on a Hidden Markov Model (HMM)-based ensemble method to predict the substrate specificity at high quality. We collected a new reference set of experimentally validated sequences. An initial classification based on alignment and Neighbor Joining was performed in line with most of the previously published prediction methods. We then created and tested single substrate specific HMMs and found that their use improved the correct identification significantly for A as well as for AT domains. A major advantage of the use of HMMs is that it abolishes the dependency on multiple sequence alignment and residue selection that is hampering the alignment-based clustering methods. Using our models we obtained a high prediction quality for the substrate specificity of the A domains similar to two recently published tools that make use of HMMs or Support Vector Machines (NRPSsp and NRPS predictor2, respectively). Moreover, replacement of the single substrate specific HMMs by ensembles of models caused a clear increase in prediction quality. We argue that the superiority of the ensemble over the single model is caused by the way substrate specificity evolves for the studied systems. It is likely that this also holds true for other protein domains. The ensemble predictor has been implemented in a simple web-based tool that is available at http://www.cmbi.ru.nl/NRPS-PKS-substrate-predictor/.

The platelet P2Y(12) receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [(3)H]PSB-0413.

Various radioligands have been used to characterize and quantify the platelet P2Y(12) receptor, which share several weaknesses: (a) they are metabolically unstable and substrates for ectoenzymes, (b) they are agonists, and (c) they do not discriminate between P2Y(1) and P2Y(12). We used the [(3)H]PSB-0413 selective P2Y(12) receptor antagonist radioligand to reevaluate the number of P2Y(12) receptors in intact platelets and in membrane preparations. Studies in humans showed that: (1) [(3)H]PSB-0413 bound to 425 ± 50 sites/platelet (K (D) = 3.3 ± 0.6 nM), (2) 0.5 ± 0.2 pmol [(3)H]PSB-0413 bound to 1 mg protein of platelet membranes (K (D) = 6.5 ± 3.6 nM), and (3) competition studies confirmed the known features of P2Y(12), with the expected rank order of potency: AR-C69931MX > 2MeSADP ≫ ADPßS > ADP, while the P2Y(1) ligand MRS2179 and the P2X(1) ligand α,ß-Met-ATP did not displace [(3)H]PSB-0413 binding. Patients with severe P2Y(12) deficiency displayed virtually no binding of [(3)H]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y(12) receptor had normal binding. Studies in mice showed that: (1) [(3)H]PSB-0413 bound to 634 ± 87 sites/platelet (K (D) = 14 ± 4.5 nM) and (2) 0.7 pmol ± 0.3 [(3)H]PSB-0413 bound to 1 mg protein of platelet membranes (K (D) = 9.1 ± 5.3 nM). Clopidogrel and other thiol reagents like pCMBS or DTT abolished the binding both to intact platelets and membrane preparations. Therefore, [(3)H]PSB-0413 is an accurate and selective tool for radioligand binding studies aimed at quantifying P2Y(12) receptors, to identify patients with P2Y(12) deficiencies or quantify the effect of P2Y(12) targeting drugs.

A pharmacy practice laboratory exercise to apply biochemistry concepts.

OBJECTIVES: To develop exercises that allow pharmacy students to apply foundational knowledge discussed in a first-professional year (P1) biochemistry course to specific disease states and patient scenarios. DESIGN: A pharmacy practice laboratory exercise was developed to accompany a lecture sequence pertaining to purine biosynthesis and degradation. The assignment required students to fill a prescription, provide patient counseling tips, and answer questions pertaining to the disease state, the underlying biochemical problem, and the prescribed medication. ASSESSMENT: Students were graded on the accuracy with which they filled the prescription, provided patient counseling, and answered the questions provided. Overall, students displayed mastery in all of these areas. Additionally, students completed a course survey on which they rated this exercise favorably, noting that it helped them to integrate basic science concepts and pharmacy practice. CONCLUSION: A laboratory exercise provided an opportunity for P1 students to apply foundational pharmacy knowledge to a patient case and can serve as a template for the design of additional exercises.

Anaerobic energy expenditure and mechanical efficiency during exhaustive leg press exercise.

Information about anaerobic energy production and mechanical efficiency that occurs over time during short-lasting maximal exercise is scarce and controversial. Bilateral leg press is an interesting muscle contraction model to estimate anaerobic energy production and mechanical efficiency during maximal exercise because it largely differs from the models used until now. This study examined the changes in muscle metabolite concentration and power output production during the first and the second half of a set of 10 repetitions to failure (10RM) of bilateral leg press exercise. On two separate days, muscle biopsies were obtained from vastus lateralis prior and immediately after a set of 5 or a set of 10 repetitions. During the second set of 5 repetitions, mean power production decreased by 19% and the average ATP utilisation accounted for by phosphagen decreased from 54% to 19%, whereas ATP utilisation from anaerobic glycolysis increased from 46 to 81%. Changes in contraction time and power output were correlated to the changes in muscle Phosphocreatine (PCr; r = -0.76; P<0.01) and lactate (r = -0.91; P<0.01), respectively, and were accompanied by parallel decreases (P<0.01-0.05) in muscle energy charge (0.6%), muscle ATP/ADP (8%) and ATP/AMP (19%) ratios, as well as by increases in ADP content (7%). The estimated average rate of ATP utilisation from anaerobic sources during the final 5 repetitions fell to 83% whereas total anaerobic ATP production increased by 9% due to a 30% longer average duration of exercise (18.4 ± 4.0 vs 14.2 ± 2.1 s). These data indicate that during a set of 10RM of bilateral leg press exercise there is a decrease in power output which is associated with a decrease in the contribution of PCr and/or an increase in muscle lactate. The higher energy cost per repetition during the second 5 repetitions is suggestive of decreased mechanical efficiency.

Rapid and ultra-sensitive detection of AMP using a fluorescent and magnetic nano-silica sandwich complex.

We report here a novel AMP biosensor based on the aptamer-induced disassembly of fluorescent and magnetic nano-silica sandwich complexes with a direct detection limit of 0.1 microM.

Assessment of enzyme induction and aerenchyma formation as mechanisms for flooding tolerance in Trifolium subterraneum 'Park'.

The objective of this study was to evaluate the role of enzyme induction and aerenchyma formation in prolonged tolerance to soil flooding in a variety of underground clover (Trifolium subterraneum 'Park') previously selected for resistance. Seedlings were grown in hydroponic tanks, initially with aeration for 3 weeks and subsequently in the absence of aeration for up to 3 weeks. After 1 h in the absence of aeration, the oxygen concentration in the hydroponic medium had decreased to 1.5 %. During the 3 weeks of extreme oxygen deficiency, primary roots died and were replaced by considerable numbers of adventitious roots. Activities of many glycolytic and fermentative enzymes increased in adventitious roots. Excised adventitious roots were capable of immediate induction of ethanol in the absence of lactate production, in association with energy charge higher than that in excised roots of aerobically maintained controls. Energy charge was even higher when measured in adventitious roots in planta. Interestingly, haemoglobin protein could be correlated with energy charge. Aerenchyma was readily visualized in adventitious roots by optical microscopy of longitudinal and transverse sections. We conclude that avoidance of root anoxia via aerenchyma is the major mechanism for prolonged root tolerance in Trifolium subterraneum 'Park'.

Differences in nucleotide compartmentation and energy state in isolated and in situ rat heart: assessment by 31P-NMR spectroscopy.

Free cytosolic concentrations of ATP, PCr, ADP and 5'-AMP, and the cytosolic [ATP]/[ADP].[Pi] ratio, were determined in isolated and in situ rat hearts using 31P-NMR spectroscopy. Total tissue metabolite concentrations were determined by HPLC analysis of freeze-clamped, perchloric acid-extracted tissue. In in situ myocardium the PCr/ATP ratio was 2.7 +/- 0.2 determined from 31P-NMR data (using either PCr/beta-NTP or PCr/gamma-NTP), and 1.9 +/- 0.1 (P < 0.01) determined from total tissue concentrations. 31P-NMR-determined and total tissue [PCr] were in excellent agreement (49.6 +/- 8.4 and 49.5 +/- 1.0 mumol.g-1 dry wt, respectively), whereas 31P-NMR-determined [ATP] (18.6 +/- 3.2 mumol.g-1 dry wt) was only 71% of the total tissue concentration (26.1 +/- 1.7 mumol.g-1 dry wt, P < 0.01). Isolation and Langendorff perfusion of rat hearts with glucose as substrate reduced total tissue [ATP] and [PCr] and the 31P-NMR-determined PCr/ATP ratio fell to 1.5 +/- 0.1. This value agreed well with the total tissue ratio of 1.4 +/- 0.1, and there was excellent agreement between 31P-NMR-determined and total tissue [PCr] and [ATP] values in the perfused heart. Addition of pyruvate to perfusate increased the 31P-NMR-determined PCr/ATP ratio to 1.7 +/- 0.1 due to elevated [PCr], and there remained excellent agreement between NMR-determined and total tissue [PCr] and [ATP] values. Free cytosolic [ADP] (from the creatine kinase equilibrium) was 5% of total tissue ADP, and free cytosolic [5'-AMP] (from the adenylate kinase equilibrium) ranged from 0.2-0.3% of total tissue 5'-AMP. Bioenergetic state, indexed by [ATP]/[ADP].[Pi], was much lower in isolated perfused hearts (30 mM-1) vs. in situ myocardium (approximately 150 mM-1). In summary, we observe a substantial disproportionality between total tissue PCr/ATP and 31P-NMR-determined PCr/ATP in highly energised in situ myocardium but not in isolated perfused hearts. This appears due to an NMR invisible ATP compartment approximating 29% of total tissue ATP in situ. Additionally, more than 95% of ADP and more than 99% of 5'-AMP exist in bound forms in perfused and in situ myocardium. The physiological significance of these observations is unclear. However, substantial differences between 31P-NMR visible and total tissue [ATP] introduces significant errors in conventional estimation of free cytosolic [ADP], [5'-AMP] and [ATP]/[ADP].[Pi] from in vivo 31P-NMR data.

Pre-transplantation assessment of renal viability with 31P magnetic resonance spectroscopy.

As acute tubular necrosis (ATN) is still an important cause for postoperative malfunction of renal grafts, it would be useful to have a method predicting such a complication. We investigated the possibility to predict ATN by measuring the ratio of phosphomonoesters (PME, largely consisting of adenosine monophosphate) and inorganic phosphate (Pi) in the renal tissue, using 31P magnetic resonance spectroscopy (MRS) during the cold ischemia period. Assuming that this ratio reflects the tissue high-energy phosphate status, we studied five kidneys from living related donors (LRD), 28 kidneys from heart beating donors (HBD) and nine kidneys from non-heart beating donors (non-HBD). All kidneys were preserved with a phosphate free solution. We found an inverse relation between the time of 31P MRS and the PME/Pi ratio, suggesting a graded decay of tissue high energy phosphates during cold ischemia. The PME/Pi ratio was highest in grafts from LRD (2.65 +/- 0.50, no ATN), intermediate in grafts from HBD (1.65 +/- 0.41, 21% ATN) and lowest in those derived from non-HBD (1.05 +/- 0.47, 56% ATN). The differences in PME/Pi ratio between the groups was statistically significant (P < 0.01). Moreover, the ratio was significantly lower in grafts developing ATN (1.73 +/- 0.41 vs. 1.35 +/- 0.29 in the HBD group, 1.41 +/- 0.24 vs. 0.76 +/- 0.36 in the non-HBD group, P < 0.05). These observations point to a general relation between the pre-transplant kidney PME/Pi ratio and the development of ATN. However, the predictive value of a low PME/Pi ratio was too low (36%) to reliably predict development of ATN in individual cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of two experimental models for assessment of cardiac preservation.

Previous studies from this institution using human cell cultures have suggested that University of Wisconsin solution is preferred for prolonged hypothermic storage for cardiac transplantation. The primary objective of this study was to evaluate the effectiveness of extended cardiac preservation with University of Wisconsin solution by assessing the time-related changes of purine metabolites using two different models of cold storage. Isolated rat hearts (n = 6/group) or human ventricular myocyte cultures (n = 7 dishes/group) were assessed after 0, 6, 12, and 24 hours in University of Wisconsin solution at 0 degrees C using high-performance liquid chromatography. Adenosine triphosphate content decreased from 18.1 +/- 5.4 to 9.6 +/- 2.7 mumol/g dried weight by 12 hours and to 1.0 +/- 0.6 mumol/g by 24 hours (p < 0.0001 by analysis of variance) in the rat model. Adenosine triphosphate content decreased from 0.64 +/- 0.42 to 0.14 +/- 0.11 nmol/micrograms DNA at 6 hours and to 0.04 +/- 0.03 nmol/micrograms DNA by 24 hours (p < 0.00001) in the cardiomyocytes. Inosine monophosphate content increased from 0.1 +/- 0.2 to 10.8 +/- 1.0 by 24 hours (p < 0.0001) in the rat studies. Inosine monophosphate values tended to increase up to 12 hours (p = 0.06) in the cell cultures and then declined. Adenosine concentration increased from 0.3 +/- 0.3 to 2.3 +/- 0.9 mumol/g at 6 hours and declined thereafter (p < 0.0005) in the rodent hearts. Adenosine concentration increased from 0.03 +/- 0.02 to 1.53 +/- 0.72 nmol/micrograms DNA at 6 hours (p < 0.0001) in the cardiomyocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional responses within the kidney to ischemia: assessment of adenine nucleotide and catabolite profiles.

UNLABELLED: Renal cortex (C) has predominantly aerobic metabolism, whereas inner medulla (IM) has both aerobic and anaerobic capacities. This study was undertaken (1) to assess how well rat IM anaerobic metabolism maintains this region's ATP content during ischemia; and (2) to determine whether regional variations in adenylate pool/catabolite responses to ischemia exist, obscuring interpretation of cellular energetics in rat studies of acute renal failure (ARF). Adenine nucleotides/catabolites were measured in rat C, IM and outer medulla (OM) after 15 and 45 min of ischemia. After 15 min, all regions showed profound ATP depletion, although the IM maintained slightly higher (by 0.23 mumol/g) absolute ATP levels than C/OM tissues (normal ATP value = 8.7 mumol/g). By 45 min, significant differences in regional ATP levels did not exist. Striking regional catabolite differences were apparent at both 15 and 45 min. Most prominent were: (1) intrarenal purine base/inosine gradients, levels falling approx. 22-50% from C to IM; and (2) preferential OM AMP/IMP/adenosine accumulation. To assess whether more homogeneous results might be found in rabbit kidney, possibly making this animal preferable to rats for studies of renal ischemia, rabbit C, OM and IM adenylate pools were analyzed after 15 min of ischemia. C vs. IM ATP differences were greater (approx. 1.3 mumol/g) and large catabolite concentration differences were still apparent. CONCLUSIONS: (1) anaerobic mechanisms support IM ATP levels during ischemia but, in terms of normal concentrations, the impact is small, particularly in the rat; and (2) marked regional differences in adenylate catabolite levels exist within ischemic kidneys. These need to be recognized when analyzing adenylate pool responses in ischemic ARF.

The energy budget of Tetrahymena and the material fluxes into and out of the adenylate pool.

The material budget of the adenylate pool deals with all processes which physically establish and maintain this pool, while the energy budget is concerned with the intracompartmental ATP recycling. Both budgets were analysed in Tetrahymena thermophila exposed to various energy and material demands. Some of the general conclusions are: at a maximum growth rate the overall ATP consumption during one cell cycle is 10(-10) mol ATP; the contribution of osmoregulation and ciliary motion to the budget is about 1% each; at zero net growth, energy is consumed because of a continuous recycling of matter between the monomer and the polymer compartment. The rate of ATP production is about 1000-fold greater than the rate of adenylate monomer influx. The residence time of adenylate monomers within the pool is about 30 min, but for ATP molecules it is only 2 sec.

Porcine malignant hyperthermia. IX: Changes in the concentrations of intramuscular high-energy phosphates, glycogen and glycolytic intermediates.

A technique of obtaining muscle biopsies with a liquid nitrogen cryoprobe was used to study intramuscular metabolites in MH-susceptible and unsusceptible pigs. There was no significant difference in muscle metabolite values obtained from susceptible and unsusceptible pigs in the resting state. During MH the changes in metabolites were a result of rapid glycogenolysis and no abnormality of glycolytic control was observed.

Urid acid accumulated in isolated kidney grafts: an assessment of agonal ischaemia.

Hypoxanthine-xanthine washed out from kidney tissue during preservation has previously been found to be a reliable measure of the in vitro assessment of the deleterious effect of ischaemia on the functional regenerative ability of the graft. We have now studied the question: can uric acid accumulation in the isolated kidney graft be employed as a retrospective measure of the agonal ischaemia? We have found that uric acid alone accumulates in renal tissue during the agonal phase, in an amount that remains unchanged during the subsequent in vitro ischaemia. The determination of hypoxanthine-xanthine and uric acid in kidney perfusate samples during preservation must thus be presumed to be an optimal clinical-chemical method of assessing the graft ischaemia.