Permanent intravenous access in experimental surgery ­ our experience

Introduction: Permanent intravenous access is usually required in pigs used for surgical experiments, not only to enable repeated blood sample collections. The aim of this study was to evaluate the patency and complications of vascular access ports (VAP) implanted in pigs included in different surgical experiments. Methods: VAPs were implanted via the external jugular vein in a total of 211 pigs from 7 different experiments. All observed complications were retrospectively evaluated. Results: No complications were observed in 157 animals (74.4%). Complications of the least severity were edema or seroma around the port which were observed in 12 (5.7%) and 3 (1.4%) animals, respectively. Temporary problems with aspiration of blood via the port occurred in 13 animals (6.2%). The most severe complications which prevented the use of the VAP for aspiration and application were recorded in 26 animals (12.3%). These complications included: abscess formation around the port (12 animals), skin necrosis over the port (2 animals), partial wound dehiscence (2 animals) and loss of the VAP function due to an unspecified cause (10 animals). Removal of the VAP was not needed in any of the animals and none of the animals had to be excluded from the experiment due to the complications. The VAP can also be used for safe administration of iodine contrast agent during CT examination. Conclusion: Despite the observed complications the VAP is suitable as permanent intravenous access in pigs used for surgical experiments. This method helps to minimize the stress of the animals in the postoperative period and to reduce the number of experimental animals.

Influence of dihydropyrimidine dehydrogenase gene (DPYD) coding sequence variants on the development of fluoropyrimidine-related toxicity in patients with high-grade toxicity and patients with excellent tolerance of fluoropyrimidine-based chemotherapy.

Alterations in dihydropyrimidine dehydrogenase gene (DPYD) coding for the key enzyme (DPD) of fluoropyrimidines (FPs) catabolism contribute to the development of serious FPs-related toxicity. We performed mutation analysis of DPYD based on cDNA sequencing in 76 predominantly colorectal cancer patients treated by FPs with early development of high (grade 3-4) hematological and/or gastrointestinal toxicity. Six previously described [85T>C (C29R), 496A>G (M166V), 775A>G (K259E), 1601G>A (S534N), 1627A>G (I543V), IVS14+1G>A, 2194G>A (V732I)] and two novel [187A>G (K63E) and 1050 G>A (R357H)] non-synonymous DPYD variants were found in 56/76 (73.7%) high-toxicity patients. Subsequently, these alterations were analyzed in 48 patients with excellent long-term tolerance of FPs and in 243 controls and were detected in 37/48 (77.1%) and 166/243 (68.3%) cases, respectively. Analysis of these alterations as risk factors for development of toxicity in pooled FPs-treated population demonstrated that C29R negatively correlated with overall gastrointestinal toxicity (OR = 0.48; 95%CI 0.23-1.0) and M166V in women protected against overall hematological toxicity and neutropenia (both OR = 0.26; 95%CI 0.07-0.89), whereas IVS14+1G>A (found in five high-toxicity patients only) increased risk of mucositis in overall population (OR = 7.0; 95%CI 1.1-44.53), and thrombocytopenia in women (OR = 10.8; 95%CI 1.24-93.98). Moreover, we identified a strong association of V732I with leucopenia (OR = 8.17; 95%CI 2.44 - 27.31) and neutropenia (OR=2.78; 95% CI 1.03-7.51). Our data enabled characterization of "high risk" haplotypes (carriers of IVS14+1G>A or V732 lacking M166V) representing small (22% female and 11% male patients), population in high risk of serious hematological toxicity development, and in patients with "lower risk" that unlikely develop serious hematological toxicity [carriers of M166V without IVS14+1G>A and V732I in females (32% women), and non-carriers of C29R, M166V, IVS14+1G>A, and V732I in males (46% men)]. Our results indicate that genotyping of several DPYD variants may lead to stratification of patients with respect to the risk of serious hematological toxicity development during FPs treatment.

Comparison of active sites of some microbial ribonucleases: structural basis for guanylic specificity.

Crystallographic studies of enzymes complexed with suitable ligands are an important tool to aid our understanding of biological catalysis. To this goal, a contribution is made by analysing structures of complexes formed by three guanyl-specific ribonucleases with guanosine 3'-monophosphate.

Long-term BRCA1 down-regulation by small hairpin RNAs targeting the 3' untranslated region.

Mutations in the BRCA1 gene are responsible for the majority of hereditary breast/ovarian cancers. The functional significance of many mutations/splicing variants identified during the screening of high-risk individuals is difficult to predict due to the lack of in vitro functional tests correlating sequence variants with a risk of cancer development. RNA interference is a promising tool in analyzing functional properties of BRCA1 mutations. Here we designed and functionally analyzed shRNAs directed to 3'-UTR of BRCA1 mRNA that may be used to knock-down expression of endogenous BRCA1. Using retroviral infection, we achieved long-term down-regulation of BRCA1 in a cell-type specific manner. We propose that 3'-UTR-directed shRNAs, coupled with up-regulation of exogenous mutated BRCA1 variants, may constitute a versatile system for the functional analysis of BRCA1 gene alterations.

Intrinsically disordered tau protein in Alzheimer's tangles: a coincidence or a rule?

Tau protein, the major constituent of neurofibrillary tangles in Alzheimer's disease (AD) and related tauopathies, is classified as intrinsically disordered protein (IDP). IDPs in contrast to globular proteins contain high proportion of polar and charged amino acids in their sequence, which results in the absence of a well-defined three-dimensional structure of the free protein. Structural flexibility of IDPs is required to perform their important role in many cellular processes. In the course of tauopathies, highly soluble disordered tau protein acquires rigid fold and forms highly insoluble filaments. Beneficial intrinsic disorder transforms into a fatal order: is it a coincidence, or is there an underlying reason for preferential IDPs assembly? In this review we present the structural characteristics of tau protein filamentous lesions in AD and discuss the tendency of IDPs to assembly and to form amyloid deposits (Ref: 65).

Mutation analysis of the PALB2 gene in unselected pancreatic cancer patients in the Czech Republic.

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis among common solid cancer diagnoses. It has been shown that up to 10% of PDAC cases have a familial component. Characterization of PDAC-susceptibility genes could reveal high-risk individuals and patients that may benefit from tailored therapy. Hereditary mutations in PALB2 (Partner and Localizer of BRCA2) gene has been shown to predispose, namely to PDAC and breast cancers; however, frequencies of mutations vary among distinct geographical populations. Using the combination of sequencing, high-resolution melting and multiplex ligation-dependent probe amplification analyses, we screened the entire PALB2 gene in 152 unselected Czech PDAC patients. Truncating mutations were identified in three (2.0%) patients. Genotyping of found PALB2 variants in 1226 control samples revealed one carrier of PALB2 truncating variant (0.08%; P = 0.005). The mean age at PDAC diagnosis was significantly lower among PALB2 mutation carriers (51 years) than in non-carriers (63 years; P = 0.016). Only one patient carrying germline PALB2 mutation had a positive family breast cancer history. Our results indicate that hereditary PALB2 mutation represents clinically considerable genetic factor increasing PDAC susceptibility in our population and that analysis of PALB2 should be considered not only in PDAC patients with familial history of breast or pancreatic cancers but also in younger PDAC patients without family cancer history.

Tyrosine hydrogen bonds make a large contribution to protein stability.

The aim of this study was to gain a better understanding of the contribution of hydrogen bonds by tyrosine -OH groups to protein stability. The amino acid sequences of RNases Sa and Sa3 are 69 % identical and each contains eight Tyr residues with seven at equivalent structural positions. We have measured the stability of the 16 tyrosine to phenylalanine mutants. For two equivalent mutants, the stability increases by 0.3 kcal/mol (RNase Sa Y30F) and 0.5 kcal/mol (RNase Sa3 Y33F) (1 kcal=4.184 kJ). For all of the other mutants, the stability decreases with the greatest decrease being 3.6 kcal/mol for RNase Sa Y52F. Seven of the 16 tyrosine residues form intramolecular hydrogen bonds and the average decrease in stability for these is 2.0(+/-1.0) kcal/mol. For the nine tyrosine residues that do not form intramolecular hydrogen bonds, the average decrease in stability is 0.4(+/-0.6) kcal/mol. Thus, most tyrosine -OH groups contribute favorably to protein stability even if they do not form intramolecular hydrogen bonds. Generally, the stability changes for equivalent positions in the two proteins are remarkably similar. Crystal structures were determined for two of the tyrosine to phenylalanine mutants of RNase Sa: Y80F (1.2 A), and Y86F (1.7 A). The structures are very similar to that of wild-type RNase Sa, and the hydrogen bonding partners of the tyrosine residues always form intermolecular hydrogen bonds to water in the mutants. These results provide further evidence that the hydrogen bonding and van der Waals interactions of polar groups in the tightly packed interior of folded proteins are more favorable than similar interactions with water in the unfolded protein, and that polar group burial makes a substantial contribution to protein stability.

Conformational stability and thermodynamics of folding of ribonucleases Sa, Sa2 and Sa3.

Ribonucleases Sa, Sa2, and Sa3 are three small, extracellular enzymes produced by different strains of Streptomyces aureofaciens with amino acid sequences that are 50% identical. We have studied the unfolding of these enzymes by heat and urea to determine the conformational stability and its dependence on temperature, pH, NaCl, and the disulfide bond. All three of the Sa ribonucleases unfold reversibly by a two-state mechanism with melting temperatures, Tm, at pH 7 of 48.4 degrees C (Sa), 41.1 degrees C (Sa2), and 47.2 degrees C (Sa3). The Tm values are increased in the presence of 0.5 M NaCl by 4.0 deg. C (Sa), 0.1 deg. C (Sa2), and 7.2 deg. C (Sa3). The Tm values are decreased by 20.0 deg. C (Sa), 31.5 deg. C (Sa2), and 27.0 deg. C (Sa3) when the single disulfide bond in the molecules is reduced. We compare these results with similar studies on two other members of the microbial ribonuclease family, RNase T1 and RNase Ba (barnase), and with a member of the mammalian ribonuclease family, RNase A. At pH 7 and 25 degrees C, the conformational stabilities of the ribonucleases are (kcal/mol): 2.9 (Sa2), 5.6 (Sa3), 6.1 (Sa), 6.6 (T1), 8.7 (Ba), and 9.2 (A). Our analysis of the stabilizing forces suggests that the hydrophobic effect contributes from 90 to 110 kcal/mol and that hydrogen bonding contributes from 70 to 105 kcal/mol to the stability of these ribonucleases. Thus, we think that the hydrophobic effect and hydrogen bonding make large but comparable contributions to the conformational stability of these proteins.

Cloning and sequencing of the gene encoding a ribonuclease from Streptomyces aureofaciens CCM3239.

A ribonuclease-encoding gene (rnaSa3) from Streptomyces aureofaciens CCM3239 has been isolated and sequenced. The deduced amino acid sequence shows 77% homology with RNase Sa from S. aureofaciens.

The evolution of starch-binding domain.

Amylolytic enzymes belonging to three distinct families of glycosidases (13, 14, 15) contain the starch-binding domain (SBD) positioned almost exclusively at the C-terminus. Detailed analysis of all available SBD sequences from 43 different amylases revealed its independent evolutionary behaviour with regard to the catalytic domains. In the evolutionary tree based on sequence alignment of the SBDs, taxonomy is respected so that fungi and actinomycetes form their own separate parts surrounded by bacteria that are also clustered according to taxonomy. The only known N-terminal SBD from Rhizopus oryzae glucoamylase is on the longest branch separated from all C-terminal SBDs. The 3-dimensional (3-D) structures of fungal glucoamylase and bacterial CGTase SBDs are compared and used to discuss the interesting SBD evolution.

Amino acid sequence determination of guanyl-specific ribonuclease Sa from Streptomyces aureofaciens.

Using automated Edman degradation of two nonfractionated peptide mixtures of tryptic and staphylococcal protease digests of the protein, the complete amino acid sequence of the guanyl-specific ribonuclease Sa from Streptomyces aureofaciens was established. Ribonuclease Sa contains 96 amino acid residues (Mr 10,566). A 50% sequence homology of ribonuclease Sa to the guanyl-specific ribonuclease St from S. erythreus was found.

An attempt to localize the site of action of different agents within cholinergic motor neurones of the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum by the triple bath method.

The site of action of cholinergic, adrenergic, peptidergic and opioid agents was studied in myenteric plexus-longitudinal muscle strips from the guinea pig ileum. A preparation in a special triple bath was drawn through two rubber membranes, dividing the strip into three segments. Neurogenic stimulation of the oral segment, set up nerve action potentials also in the neurones projecting axons up to the aboral segment. These axons, turning into varicose nerve terminals, conducted action potentials aborally across the middle segment, that was up to 10 mm wide. Finally, the nerve terminals, extending into the aboral segment, might be also invaded triggering twitches. Agents were added, either to the oral segment, to affect the genesis and spread of action potentials in the proximal parts of cholinergic neurones (cell bodies, axon hillocks, initial segments and axon preterminals) or they were added to the middle segment to affect propagation of action potentials in varicose nerve terminals. As a result, the amplitude of aboral twitches reflected their effects at each site, quantitatively. Noradrenaline and ethylketocyclazocine were more effective at the site of varicose nerve terminals, whereas substance P, acetylcholine and oxotremorine were more effective at the proximal parts; pilocarpine and nicotine were effective at both sites. Changes in membrane polarization might be the final common effect in the mechanism of action of all the stimulatory agents used.

The topographical basis of cholinergic transmission in guinea-pig ileum myenteric plexus.

Myenteric plexus-longitudinal muscle strips were used to study nerve action potential propagation and transmission and their differences between the proximal and the distal regions of cholinergic terminals. Neurogenic twitches of a portion of the strip were evoked by focal electrical stimulation. Twitches mediated by the distal regions of cholinergic nerve terminals were more influenced by drugs affecting Ca2+ "utilization" (Bay K 8644, kappa opiate ligand ethylketocyclazocine, changes in extracellular Ca2+ or Co2+ concentration) in contrast to twitches mediated by proximal regions of these terminals which were more influenced by drugs affecting sodium-potassium spike (tetrodotoxin, dendrotoxin, 4-aminopyridine, tetraethylammonium). Post-tetanic potentiation of twitches was prominent with that portion of the strip where the distal regions of nerve terminals were involved. Drugs interfering with Na+/K+ spikes indiscriminately influenced both the twitch height and post-tetanic potentiation whereas changes in extracellular Ca2+ concentration affected selectively only post-tetanic potentiation. Release of [3H]acetylcholine from pre-labelled strips evoked by 1 Hz continuous stimulation or by train stimulation at 30 Hz was measured selectively from portions containing either proximal and distal or only distal regions of nerve terminals. The release from portions containing the distal regions was relatively higher when evoked by 30 Hz than by 1 Hz. The distal regions of nerve terminals might be thus recruited to participate in transmission by a frequency-dependent process. Nerve impulses were recorded from strands of nerve fibres in the myenteric plexus. At 1 and 5 mm distance from the stimulation focus nerve impulses were completely suppressed by tetrodotoxin. At 5 mm, in some strands the amplitude of nerve impulses was also subject to the effect of drugs affecting Ca2+ "utilization"; facilitation of nerve impulse amplitude during 30 Hz train stimulation was always influenced by drugs affecting Ca2+ "utilization". Propagation of nerve impulses in the distal region of cholinergic nerve terminals was found to be Ca-sensitive and frequency-dependent; this might form the basis for facilitation and post-tetanic potentiation of muscarinic transmission.

Effects of the central analgesic tramadol and its main metabolite, O-desmethyltramadol, on rat locus coeruleus neurones.

1. Tramadol is a centrally acting analgesic with low opioid receptor affinity and, therefore, presumably additional mechanisms of analgesic action. Tramadol and its main metabolite O-desmethyltramadol were tested on rat central noradrenergic neurones of the nucleus locus coeruleus (LC), which are involved in the modulation of nociceptive afferent stimuli. 2. In pontine slices of the rat brain the spontaneous discharge of action potentials of LC cells was recorded extracellularly. (-)-Tramadol (0.1-100 microM), (+)-tramadol (0.1-100 microM), (-)-O-desmethyl-tramadol (0.1-100 microM) and (+)-O-desmethyltramadol (0.01-1 microM) inhibited the firing rate in a concentration-dependent manner. (+)-O-desmethyltramadol had the highest potency, while all other agonists were active at a similar range of concentrations. 3. (-)-Tramadol (10, 100 microM) was less inhibitory in brain slices of rats pretreated with reserpine (5 mg kg-1, 5 h before decapitation) than in controls. 4. The effect of (-)-tramadol (10 microM) was abolished in the presence of the alpha 2-adrenoceptor antagonist, rauwolscine (1 microM), whilst that of (+)-O-desmethyltramadol (0.3 microM) virtually disappeared in the presence of the opioid antagonist, naloxone (0.1 microM). (+)-Tramadol (30 microM) and (-)-O-desmethyl-tramadol (10 microM) became inactive only in the combined presence of naloxone (0.1 microM) and rauwolscine (1 microM). 5. In another series of experiments, the membrane potential of LC neurones was determined with intracellular microelectrodes. (-)-Tramadol (100 microM) inhibited the spontaneous firing and hyper-polarized the cells; this effect was abolished by rauwolscine (1 microM). (+)-O-desmethyltramadol (10 microM)had a similar but somewhat larger effect on the membrane potential than (-)-tramadol. The (+)-O-desmethyltramadol-(10 microM) induced hyperpolarization was abolished by naloxone (0.1 microM).6. The hyperpolarizing effect of noradrenaline (30 microM) was potentiated in the presence of (-)-tramadol(100 microM), but not in the presence of (+)-O-desmethyltramadol (10 microM). There was no potentiation of the noradrenaline (30 microM) effect, when the cells were hyperpolarized by current injection to an extent similar to that produced by (-)-tramadol (100 microM).7. Both noradrenaline (100 microM) and (- )-tramadol (100 microM) decreased the input resistance.8. The results confirm that the analgesic action of tramadol involves both opioid and non-opioid components. It appears that (-)-tramadol inhibits the uptake of noradrenaline and via a subsequent increase in the concentration of endogenous noradrenaline indirectly stimulates alpha2-adrenoceptors. (+)-0-desmethyltramadol seems to stimulate directly opioid micro-receptors. The effects of (+)-tramadol and(-)-O-desmethyltramadol consist of combined micro-opioid and alpha2-adrenergic components.

Effects of potassium channel openers and their antagonists on rat locus coeruleus neurones.

1. Intracellular recordings were obtained from a pontine slice preparation of the rat brain containing the locus coeruleus (LC). Two openers of ATP-sensitive potassium (K(ATP)) channels, RO 31-6930 (10 microM) and cromakalim (100 microM) decreased the spontaneous discharge of action potentials without altering their amplitude or duration. Neither compound changed the resting membrane potential. 2. Of two K(ATP) channel blockers, tolbutamide (300 microM) increased the firing rate, while glibenclamide (3 microM) only tended to do so. In addition, both compounds antagonized the effect of RO 31-6930 (10 microM). Neither glibenclamide (3 microM) nor tolbutamide (300 microM) altered the resting membrane potential. 3. Tetrodotoxin (0.5 microM) depressed the firing, but did not influence the inhibitory action of RO 31-6930 (10 microM). The excitatory amino acid antagonist, kynurenic acid (500 microM), did not change the spontaneous discharge of action potentials. 4. Small shifts (2-4 mV) of the membrane potential by hyper- or depolarizing current injections markedly decreased and increased the firing rate, respectively. 5. Noradrenaline (100 microM) hyperpolarized the cells and decreased their input resistance. This effect was not antagonized by glibenclamide (3 microM) or tolbutamide (300 microM). Ba2+ (2 mM), a blocker of both ATP-sensitive and inwardly rectifying potassium channels, abolished the effects of RO 31-6930 (10 microM) and noradrenaline (100 microM). 6. These data suggest that K(ATP) channels are present on the noradrenergic LC neurones, but are not coupled to alpha 2-adrenoceptors.

Galanin receptors inhibit the spontaneous firing of locus coeruleus neurones and interact with mu-opioid receptors.

Electrophysiological experiments were performed in a pontine slice preparation of rat brain containing the locus coeruleus (LC). The extracellular part of this study showed that galanin (0.003-0.1 mumol/l), [Met5]enkephalin (0.01-10 mumol/l) and noradrenaline (0.1-100 mumol/l) concentration dependently inhibited the firing rate. Noradrenaline (1 and 3 mumol/l) had the same effect both before and during the application of galanin (0.001 or 0.01 mumol/l). Similarly, [Met5]enkephalin (0.03 and 0.1 mumol/l) produced identical inhibition, regardless of the presence or absence of 0.01 mumol/l galanin. Whereas rauwolscine (1 mumol/l) potentiated the effect of galanin (0.03 mumol/l), idazoxan (1 mumol/l) was inactive. In contrast, both naloxone (0.1 mumol/l) and beta-funaltrexamine (0.1 mumol/l) facilitated the galanin-induced inhibition. In the intracellular experiments, galanin (0.3 mumol/l) abolished the spontaneous discharge of action potentials, hyperpolarized the cells and decreased their input resistance. In conclusion, galanin may depress the firing rate by increasing a potassium permeability. Moreover, galanin receptors appear to interact with mu-opioid receptors but not with alpha 2-adrenoceptors.

The effect of substance P on nerve action potential propagation and cholinergic transmission in the myenteric plexus of the guinea-pig ileum.

The effect of substance P on nerve terminals in myenteric plexus of the guinea-pig ileum was investigated. Neurogenic twitches of the myenteric plexus longitudinal muscle strip were recorded. Twitches of the strip portion where excitation involved the most distal parts of cholinergic nerve terminals were more increased by local application of substance P (0.1 and 0.4 nmol/l) than twitches of the portion where excitation involved both distal and proximal parts of nerve terminals. Substance P addition to a portion of the strip conducting nerve action potentials to invade the neighbouring strip portion also augmented twitches of the latter portion so that the interference with the propagation process was considered. The effect of substance P was poorly antagonized by the addition of a substance P antagonist, (D-Arg1, D-Pro2, D-Trp7,9, Leu11)-substance P. Compound nerve action potentials were evoked in strands of fibres of the myenteric plexus by low-frequency train stimulation (1 Hz). The addition of substance P prevented a decrease of the amplitude of responses observed under control conditions. Using high-frequency train stimulation (30 Hz) the amplitude of responses to impulses 2-7 was augmented over that to the first impulse; substance P further increased such facilitation regularly. It seems that substance P might promote nerve action potential invasion of the distal parts of nerve terminals.

Modulation of locus coeruleus neurons by extra- and intracellular adenosine 5'-triphosphate.

The cell membrane of rat locus coeruleus (LC) neurons is sensitive to both extra- and intracellular ATP. Extracellular ATP or its enzymatically stable analogues activate membrane receptors of the P2 type. These receptors inhibit a persistent potassium current and simultaneously activate a nonselective cationic conductance. The resulting depolarization increases the spontaneous firing rate. A decrease in the concentration of intracellular ATP during hypoxia or hypoglycemia opens ATP-sensitive K+ (KATP) channels of LC neurons. The resulting hyperpolarization depresses the discharge of action potentials and conserved energy. The hypoxia-induced hyperpolarization is additionally due to the release of adenosine from neighboring neurons or glial cells. A certain class of compounds, termed potassium channel openers, also decrease the firing, while sulphonylurea antidiabetics known to block KATP channels increase it. Sulphonylurea antidiabetics antagonize the excitability decrease induced both by potassium channel openers and metabolic damage.

A rapid and simple screening method for detection of orotic aciduria by capillary zone electrophoresis.

A rapid and selective capillary zone electrophoretic method for screening of patients with orotic aciduria is described. The method is based on direct measurement of orotic acid in untreated urine. Total analysis time is 5 min with the limit of quantification of 10 mumol/l. Selectivity of the method is given by the use of a low pH of background electrolyte at which practically no interferences from native urine appeared, as well as by using a fast scanning detector which enables the identification of orotic acid via its characteristic UV spectra. This method can be used for quantitative assessment of orotic acid present in urine at physiological conditions after an ion exchange chromatography-clean up and preconcentration procedure. Urine samples containing orotic acid at pathological concentrations (typically more than 1 mol/mol creatinine) could be successfully analysed. The method described was applied to urine specimens collected from both healthy volunteers and patients with orotic acidurias of various origin.

A fast and simple screening method for detection of 2,8-dihydroxyadenine urolithiasis by capillary zone electrophoresis.

2,8-Dihydroxyadenine urolithiasis is an inherited disorder caused by adenine phosphoribosyltransferase deficiency. A fast, simple, sensitive and selective capillary zone electrophoretic method for diagnosis of 2,8-dihydroxyadenine urolithiasis in adenine phosphoribosyltransferase deficiency is described. The method is based on direct measurement of 2,8-dihydroxyadenine in untreated urine in phosphate buffer at pH 3.0 within 8 min. Under the given separation conditions 2,8-dihydroxyadenine is very well separated from other purine and pyrimidine substances and presents characteristic UV spectra which enable identification in case of doubt. The urine samples containing pathological 2,8-dihydroxyadenine could be successfully analysed in levels approaching those relevant for bioanalytical applications. The reliability of the method presented for screening of patients with adenine phosphoribosyltransferase deficiency is demonstrated on a urine sample of a patient with the defect who was already treated with allopurinol at the time of obtaining the sample. No interfering substances were found in 50 urine samples from healthy infants under the analytical condition described.