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An unknown metabolite of D -penicillamine found in the urine of patients receiving the drug has been isolated by ion-exchange chromatography. It was identified as S-methyl- D -penicillamine by mass spectrometry and its structure was confirmed by synthesis. In subjects receiving D -penicillamine for treatment of cystinuria and rheumatoid arthritis 4 and 8 per cent respectively of the dose was methylated. The total percentage of the D -penicillamine dose excreted (as cysteine-penicillamine plus penicillamine disulphide plus S-methyl- D -penicillamine) was 40 and 34 per cent in cystinuria and rheumatoid arthritis respectively. The findings in two cases of Wilson's Disease were similar to those in rheumatoid arthritis. In studies on four cystinuria patients an average of 12 per cent of the administered D -penicillamine was recovered in the faeces mainly as penicillamine disulphide. However, only between 42 and 53 per cent of the dose was identified in the urine and faeces, so that approximately 50 per cent of the administered drug was unaccounted for. D -Penicillamine caused a 32 per cent reduction in the urinary excretion of cysteine residues in cystinuria but a 400 per cent increase in their excretion in rheumatoid arthritis.

Ileal transport of dibasic amino acids has not previously been studied in the intestine of healthy volunteers or cystinuric patients. Experiments have therefore been designed to compare ileal and jejunal absorption of lysine and arginine both in normal subjects and cystinuric patients. In addition, jejunal perfusion experiments have been carried out to investigate absorption of the dipeptide L-arginyl-L-leucine. The results indicate that, at the concentrations studied (4.2 mm lysine, 1 mm arginine), severe transport defects exist throughout the whole small intestine for both amino acids in cystinuria. Despite the transport defect for free arginine, cystinuric patients absorbed the dipeptide l-arginyl-l-leucine normally. Because of the transport defect for free arginine, it has been possible to show that during absorption of l-arginyl-l-leucine in cystinuria approximately 30% of dipeptide-bound arginine can be recovered from the gut lumen in the free form. These findings indicate that the prime function of specific amino acid transport systems during the absorption of protein digestion products may be as a "recapture mechanism" for amino acids liberated as a result of mucosal cell peptide hydrolysis.

An in-situ isolated rat adrenal perfusion technique has been devised to study the opioid control of neurally mediated adrenomedullary catecholamine release. Adrenomedullary catecholamine secretion was induced by electrical stimulation of the cut end of the left descending thoracic sympathetic chain on platinum electrodes. The half-maximal stimulatory potential (ED50) of the system was 8 V, 20 Hz, with 300μs pulse width. Basal release of catecholamine from the adrenal was constant using a perfusion flow rate of 100–300μl/min, but increased significantly with increasing perfusion temperature over the range 36–38 °C. Following repetitive 30-s stimulation of the left thoracic sympathetic chain, and 3-min fraction collections, the total amount of catecholamine released per fraction remained within 80–100% of the maximum release for up to eight consecutive stimuli. The release of catecholamines was completely blocked by hexamethonium (0·1 mmol/l), but recovered to preblockade values within two further stimuli. Using the ED50 and the first three stimuli as control, the effects of morphine (10 nmol/l–1 mmol/l), d-Ala2-MePhe4-Met-enkephalin-(O5)-ol (DAMME; 10 nmol/l–0·1 mmol/l) and naloxone (10 nmol/l– 10 μmol/l) on the response to the next three stimuli were compared. Morphine, DAMME or naloxone did not significantly alter the amount of catecholamine released by this form of stimulation. Therefore in the rat, under the conditions used, there is no evidence for mu (μ) or delta (†) opiate modulation of neurally mediated catecholamine release from the rat adrenal medulla. J. Endocr. (1986) 111, 7–15

We report on a systematic investigation into some frequently encountered problems in the estimation of catecholamines in plasma and biological fluids using high-performance liquid chromatography with electrochemical detection. The kinetics of adsorption and desorption of catecholamines from plasma on to alumina has been studied using laboratory-prepared and some commercial aluminas with various acids. Chromatographic conditions yielding optimal resolution and sensitivity have been characterised. Some possible interfering peaks have been identified. The stability of catecholamines in plasma has been studied.

1. An intestinal perfusion technique has been used to study absorption in vivo of l-cystine and l-cysteine in six normal human subjects and three patients with homozygous cystinuria. 2. No significant absorption of l-cystine was detected during perfusion of the cystinuric patients, whereas l-cysteine absorption was normal. These results imply that l-cystine and l-cysteine are normally absorbed by different transport processes. 3. No significant reduction of l-cystine to l-cysteine occurred in the gut lumen during the perfusion experiments. No more oxidation of l-cysteine to l-cystine occurred in the gut lumen during the perfusion experiments than in the test solutions which were simultaneously incubated in vitro.

Adenine and adenosine metabolism has been studied in intact human erythrocytes in vitro using high performance liquid chromatography, isotopic labelling and electrophoresis. Their metabolism to nucleotides was controlled by phosphoribose diphosphate synthesis which was phosphate dependent. Adenosine formed hypoxanthine or IMP depending upon P i concentration, but adenosine kinase and deaminase activities were not affected by P i levels. Free [ 14 C]adenine and [ 14 C]hypoxanthine were found in cellular extracts. Rapid interconversions occurred to give a distribution for ATP : ADP : AMP of 10 : 1 : 0.1. Marked decomposition of ATP to ADP and AMP occurred during incubations in plasma and Earle's media in air on nitrogen, but ATP levels remained stable in phosphate buffers and in the presence of oxygen. At physiological P i (1 mM) adenosine kinase activity grossly exceeded adenine phosphoribosyltransferase activity. The latter was approximately 7 fold that of hypoxanthine phosphoribosyltransferase activity. These differences decreased with increasing P i levels. No significant increase in corresponding nucleotides was obtained by incubation with high levels (0.5 mM) of adenine, guanine or guanosine at physiological P i , ATP increased by 10% independently of the substrate employed and significant amounts of IMP and GTP were formed from adenosine and guanosine, respectively. The existence of a bound intracellular pool of ATP is suggested.

dATP, dADP, and dAMP equalled or exceeded the depleted levels of ATP, ADP, and AMP in erythrocytes from two children with adenosine deaminase (ADA; EC 3.5.4.4) deficiency. dATP and dADP were identified in the mononuclear cells of only one child. The levels of deoxyadenosine compounds fell dramatically after enzyme replacement therapy and were no longer detectable in the urine or in mononuclear cells. Erythrocyte adenosine nucleotide levels showed a corresponding increase. Intact erythrocytes prior to treatment contained adenine, presumed to be from deoxyadenosine degraded during extraction. Adenosine at high concentrations in vitro increased both dATP and ATP levels and decreased intracellular deoxyadenosine levels. There was no significant deamination of either [8-14C]adenosine or deoxyadenosine by intact ADA-deficient erythrocytes. About 90% of adenosine was metabolized to ATP at substrate concentrations from 10-100 microM, compared to 40-60% of deoxyadenosine metabolized to dATP. These studies suggest that (i) high intracellular deoxyadenosine levels may be necessary in vivo to sustain the raised dATP levels in ADA deficiency. (ii) When ADA is inhibited or absent, deoxyadenosine is removed rapidly from the circulation by the human erythrocyte utilizing an adenosine transport system linked to both ADA and adenosine kinase (EC 2.7.1.20).

1 The metabolites of thiopurinol have been investigated in intact human and pig erythrocytes using [6-(14)C]-thiopurinol, high voltage electrophoresis and automated cation exchange chromatography. 2 Pre-incubation of human erythrocytes in vitro with thiopurinol increased the formation of hypoxanthine from [8-(14)C]-inosine and simultaneously reduced IMP synthesis. 3 Reduced inosine formation, following one week of thiopurinol therapy, has also been observed in vitro using the intact red cells from three patients suffering from gout. 4 Binding of thiopurinol, and to lesser extents of 6-mercaptopurine, uric acid, oxipurinol and allopurinol, has been demonstrated electrophoretically to human and pig serum proteins. 5 Thiopurinol is shown to have a greater binding capacity for purified human serum albumin than uric acid or 6-mercaptopurine at varying substrate and albumin concentrations covering the physiological range. Under these conditions the binding of uric acid is reduced in the presence of thiopurinol. 6 A comparison of the distribution of thiopurinol, 6-mercaptopurine, allopurinol and oxipurinol in whole blood has revealed that allopurinol and oxipurinol are not irreversibly bound to cellular proteins; whereas 30% and 13% of the total cellular uptake was irreversibly bound in the case of thiopurinol and 6-mercaptopurine respectively at substrate levels of 1 nmol per μl blood.

Summary The metabolism of adenosine and adenine by the intact human erythrocyte has been studied over the range 0.1 to 180 μmol/l. At physiological levels ( 90% of the adenosine was deaminated. Over the physiological range adenine and adenosine were incorporated into nucleotides at identical rates. The relevance of these findings to human purine metabolism and their implications to adenosine deaminase deficient patients is discussed.

1. 1. Langendorff perfused rat hearts show synchronous, statistically significant, systematic variations in ATP and ADP. 2. 2. Here we show that AMP and IMP also vary in register with ATP and ADP and we suggest that the synchronizing trigger for these oscillations may be ischaemia. 3. 3. Oscillations in the ATP/ADP ratio were found to be significantly correlated with creatine phosphate content but by contrast these quantities vary quite differently from the GTP/GDP ratio. 4. 4. Cyclic GMP oscillations showed a significant negative correlation with variations in ADP. 5. 5. Epinephrine raised mean cyclic AMP content and stabilized cyclic GMP oscillations, but had little other effect on the purine nucleotide variations.

1. Deoxyadenosine metabolism was compared in intact erythrocytes from two children with severe combined immunodeficiency: one had normal adenosine deaminase (ADA; EC 3.5.Lt.4) levels and the other, a homozygote for ADA deficiency, had (following a bone-marrow graft) 15% of normal activity in lysed erythrocytes. 2. In contrast to previous studies with adenosine, deamination was the principal route of deoxyadenosine metabolism, under all incubation conditions, in intact erythrocytes from both patients and control subjects. 3. When the same studies were performed together with the ADA inhibitor EHNA (erythro-9[2-hyd roxy3-nonyl]adenine) , further differences between the metabolism of deoxyadenosine and adenosine became apparent: (i) deamination of deoxyadenosine was not completely inhibited by 5 pM EHNA even at the lowest substrate concentrations; and (ii) significant deoxynucleotide formation occurred only at unphysiological phosphate levels (18 mM Pi ) and most of the substrate remained unmetabolized. 4. In these in vitro studies at high phosphate concentrations deoxyadenosine was incorporated into the deoxynucleotides dAMP:dADP:dATP in the same ratio (10:l.0:0.1) as had been found in the ADA-deficient erythrocytes prior to bone-marrow graft. These studies demonstrate that under physiological conditions deoxyadenosine- unlike adenosine- is deaminated~ not phosphorylated, and is thus dependent on intact ADA activity for its normal metabolism. The fact that even low levels of ADA (as assessed by lysate activity) are adequate in intact ceils to perform the important detoxifying function of this enzyme may explain the existence of immunocompetent children deficient in erythrocyte ADA. Intact cells may thus give a better indication of prognosis in suspected cases of ADA deficiency.

A modified double lumen perfusion method has been used to study dibasic amino acid absorption in human jejunum. Results using this method have been compared with those from oral load studies. Using the perfusion method, kinetic constants (Kt and Vmax) for lysine were: Kt 4.9 mM; V max 51.3 μmoles min-1 30 cm and for arginine were: Kt 3.8 mM, V max 46.3 μmoles min-1 30 cm Lysine absorption was compared in normal and cystinuric subjects. A significant difference was seen between these groups when studied by perfusion but not using the oral load method. Furthermore, oral load studies using mixtures of lysine, ornithine, and arginine showed a difference between the groups for arginine but no difference for lysine and ornithine. All three dibasic amino acids appeared to be well absorbed in the cystinuric patients. Possible explanations for these differences and the failure of oral tolerance tests to demonstrate the known transport defect in cystinuria are discussed.

The role of pH in the gradient elution of nucleotides and related compounds from short high efficiency microparticulate anion-exchange columns has been evaluated. Only at pH 3 could good resolution be obtained and a system capable of the separation of 22 nucleotides from physiological extracts in 13 minutes has been described. Other factors affecting the satisfactory operation of the system are discussed.

1. Nucleosides and bases in physiological fluids result from metabolism of nucleic acids and nucleotides and from dietary sources. As nucleotide catabolism increases during tissue injury, nucleosides and bases could serve as useful biochemical markers in arthritis. 2. We have quantified nucleosides and bases in synovial fluid and plasma by high-performance liquid chromatography in order to examine whether nucleotide metabolism is increased in patients with rheumatoid arthritis and osteoarthritis. 3. At least ten u.v.-absorbing compounds were detected in plasma and synovial fluid; only urate, creatinine, hypoxanthine and uridine were present in identifiable and quantifiable amounts. 4. In synovial fluid from patients with rheumatoid arthritis the concentration of hypoxanthine was increased and that of urate decreased compared with osteoarthritis. 5. These data suggest that there is an increase in purine metabolism in the rheumatoid arthritis joint and that hypoxanthine is a potential marker of synovitis.

Ten patients with active rheumatoid arthritis (RA) were entered into a pilot study to evaluate the effectiveness of thiomalic acid as a disease modifying agent and to assess its toxicity. Oral thiomalic acid (100 mg) was given daily for up to six months. Changes in disease activity were recorded monthly and all side effects noted. No patient recorded any improvement in subjective well being, pain score, or duration of early morning stiffness. No significant change occurred in articular index or haemoglobin (Hb); the erythrocyte sedimentation rate (ESR) showed a tendency to increase. Only three patients completed six months' treatment; six withdrew because of toxic reactions (three with rashes and three with severe gastrointestinal upset) and one because of lack of effect. Thiomalic acid alone appears to have no significant antirheumatic activity and is associated with an unacceptably high incidence of adverse reactions.

The chromatographic resolution of fluorescent 1-N 6 -etheno derivatives of adenine and adenosine ribo- and deoxyribonucleotides on reversed-phase columns has been optimised by a systematic study of the effect of ion-pair concentration, pH, eluent molarity and methanol concentration. Using tetrabutylammonium hydrogne sulphate as the ion-pair separation of up to eight derivatives could be achieved in ca. 10 min. Conditions of derivatisation with chloroacetaldehyde have been investigated in order to reduce hydrolysis of the nucleotides.

1. Glycogen, nucleotides and glycolytic intermediates and products were measured before and during tetanus in the hamstrings-muscle groups of normal (C3H) and phosphorylase kinase-deficient (ICR/IAn) mice. 2. Phosphorylase kinase-deficient muscles contained 3-4-fold more glycogen and sustained a larger (approx. 2-fold), more rapid (11 +/- 2 ng/s faster) and more prolonged glycogenolysis during 120s tetanus despite their lack of phosphorylase a. 3. No significant change in total adenine nucleotide contents occurred during tetanus in either strain, but there was a 60-100-fold rise in IMP concentration to approx. 2mM in both strains. The initial rate of IMP formation was 6-fold more rapid (112 nmol/s per g) in phosphorylase kinase-deficient muscle. 4. Adenylosuccinate content rose to 36 nmol/g in phosphorylase kinase-deficient muscle and to 9 nmol/g in normal muscle at 45s tetanus, but then fell. 5. In phosphorylase kinase-deficient muscle, glucose 6-phosphate, a powerful phosphorylase inhibitor, was 56% of that in normal muscle. 6. The mass-action ratio of the phosphoglucomutase-catalysed reaction [glucose 6-phosphate]/[glucose 1-phosphate] was markedly lower than Keq. (approx. 17) in relaxed muscle of both strains (approx. 5-7), but rose significantly during tetanus to the value for Keq. 7. The data for IMP satisfy the criteria put forward by Rahim, Perrett & Griffiths [(1976) FEBS Lett. 69, 203-206] for a nucleotide activator of phosphorylase b: it should be present at a higher concentration in phosphorylase kinase-deficient muscle, its concentration should rise during muscle work, and it should attain a concentration comparable with its activation constant for phosphorylase b.

A scoring system using computerised analysis of digital stored images of knee radiographs has been developed. Measurement is based on the assessment of joint space size. It is sensitive, rapid, and reproducible. Plain radiographs are positioned on an acetate grid; a computer generated grid is superimposed on a digital image of the radiograph viewed on a closed circuit television monitor and the joint space measured automatically. Area and distance have been assessed; area measurements are more reproducible. Application of microcomputer based digital image analysis to radiological scoring systems is an important step in understanding the nature and progression of arthritis.

Fibronectin, an opsonic glycoprotein has been shown to exist in fragmented forms in serum and synovial fluid. Some fragments in synovial fluid appear to be polyethylene glycol (PEG) precipitable, suggesting incorporation into immune complexes (IC). PEG precipitation, SDS-PAGE and immunoblotting were used to determine whether PEG precipitable fragments are real or artefactual. Disease specificity of fragmentation and IC incorporation of fibronectin and other proteins were also studied using these techniques. PEG precipitable fragments do not appear to be artefactual, although some fibronectin fragments are cryoprecipitable. Protein fragments showed similar distributions in whole serum and synovial fluid, disease specific differences being confined to PEG precipitates. Rheumatoid arthritis (RA) synovial fluid PEG precipitates displayed the greatest array of fragmented immunoglobulins and fibronectin. No PEG precipitates contained albumin fragments. Protein fragments in IC may impair their effective removal from RA joints. Accumulated IC could lead to tissue damage via complement activation.

We have investigated the distribution of cells responsive to faces within the macaque temporal cortex and their sensitivity to different face attributes. We found a functional organization of cells responsive to the sight of different views of the head. Cells of a similar type were grouped together both vertically down through the cortex, and horizontally in patches extending 0.5-2.0 mm across the surface of the cortex. A substantial proportion of cells responsive to faces were found to be sensitive to biologically important characteristics such as identity or expression. Cells were found to be highly selective for particular individuals that were familiar to the monkey with selectivity persisting across a great variety of viewing conditions such as changing face expression, orientation, colour, distance and size. Data suggested that sensitivity to identity arises at the level of specific views of the individual (e.g. full face). Information about different views may then be pooled to allow recognition independent of view. Visual transformations that make it difficult for humans to perceive faces (e.g., contrast reversal, isoluminant colour, coarsely quantized images, rotation or inversion) reduced the magnitude or increased the latency of cells' responses to faces. In this way, cell responses were related to perception and not simply to visual qualities of the image.

GTP levels were low and NAD + levels high in purine nucleoside phosphorylase (PNP) deficient erythrocytes, in addition to the raised deoxy-GTP (dGTP) levels previously noted by others. dGTP was also identified in the PNP deficient child's lymphocytes. A further novel finding was the conversion of hypoxanthine to inosine by the PNP deficient red cells, as compared to inosine monophosphate (IMP) in controls. This has been attributed to IMP formation with subsequent breakdown, and raises interesting questions regarding the controls which normally maintain erythrocyte nucleotide pools. These findings may also explain the gross purine overproduction seen in this defect; they may likewise be related to the associated immunodeficiency, anaemia, and other clinical manifestations. The results may also have important implications for the development and clinical use of PNP inhibitors.

Cysteamine, an amino thiol, was separated by rapid isocratic cation exchange chromatography and detected by electrochemical oxidation at a platinum electrode maintained at +0.45 V relative to an Ag/AgCl reference electrode. Eluent pH and electrode working potentials were optimized and the effects of alternative buffers and organic modifiers have been examined. On column sensitivity for cysteamine was 1.5 pmol at a signal-to-noise ratio of 5. Although the specificity was good, plasma samples required maximal sensitivity whereas urine samples required greater selectivity, which was achieved by use of lower working potentials. Cysteamine concentrations were determined in serial samples of plasma and urine from volunteers who had received a single oral dose of 200 mg of the drug. Cysteamine was rapidly oxidized in vivo, and detection required prior reduction with dithiothreitol before analysis.

1. We have studied the metabolism of adenine and adenosine in intact erythrocytes from three children and four adults of two separate families. The children were homozygotes for a deficiency of AMP-pyrophosphate phosphoribosyltransferase (adenine phosphoribosyltransferase, EC 2.4.2.7), and the parents heterozygotes. 2. In this intact cell system at physiological concentrations of inorganic phosphate (Pi) and adenine heterozygote adenine phosphoribosyltransferase activity was normal, whereas at higher adenine concentrations (1.0–10 μmol/l) the four heterozygotes showed approximately 50% of control activity. At high concentrations of Pi (18 mmol/l) and adenine (10 μmol/l) and with extended incubation heterozygote activity was again indistinguishable from normal. Activity of the enzyme in homozygotes was negligible under all these conditions. 3. Formation of inosine 5′-monophosphate from [8-14C]hypoxanthine at high Pi concentrations was normal in both homozygotes and heterozygotes. Thus no abnormality of either of the enzymes IMP—pyrophosphate phosphoribosyltransferase (hypoxanthine phosphoribosyltransferase, EC 2.4.2.8) or phosphoribosyl pyrophosphate synthetase (ribose phosphate pyrophosphokinase, EC 2.7.6.1) was found. 4. To ascertain whether deficiency of adenine phosphoribosyltransferase was linked with enhanced conversion of adenosine into adenine nucleotides at the expense of its deamination pathways, the complete metabolism of [8-14C]adenosine was studied over the range 0.1–9.0 μmol/l. No such increase was observed for either heterozygotes or homozygotes. 5. Insignificant formation of either [8-14C]-adenine from [8-14C]adenosine or vice versa in the erythrocytes of homozygotes lacking adenine phosphoribosyltransferase but capable of forming hypoxathine from adenosine, supports the concept that adenine and adenosine are not directly interconvertible and do not utilize purine nucleoside phosphorylase (EC 2.4.2.1) in erythrocytes. 6. Measurement of erythrocyte nucleotide concentrations by high-performance liquid chromatography revealed no obvious abnormalities. ATP:ADP:AMP proportions were normal (approximately 10:1:0.1) and the total adenine nucleotide pool was at the lower limit of the normal range for both homozygotes and heterozygotes. These observations imply that deficiency of adenine phosphoribosyltransferase does not grossly affect adenine nucleotides synthesis in the erythrocyte.

1. A family is reported with an unusual type of cystinuria. 2. The propositus presented with a cystine renal stone; the renal tubular reabsorption of cystine was grossly abnormal but the tubular reabsorption of ornithine, lysine and arginine was only slightly less than normal. 3. One of the children of the propositus excreted cystine and lysine in increased amounts typical of type II heterozygotes for cystinuria. 4. The renal transport defect in this family may represent one end of the spectrum of cystinuria or it may be a form akin to isolated hypercystinuria.

We previously reported severe ATP depletion in the erythrocytes of an adenosine deaminase (ADA: EC 3.5.4.4) deficient child and also found raised dAMP levels1 in addition to the raised dATP and dADP levels noted by others.2 Post-marrow graft ATP levels returned to normal and the three deoxyadenosine nucleotides rapidly 2 disappeared. No ATP depletion had been noted in earlier reports. Similar findings have now been obtained in the erythrocytes of two further untreated ADA deficient children. Enzyme replacement therapy also resulted in a rapid return of nucleotide levels toward normal.3

For suitable compounds, HPLC with electrochemical detection offers excellent sensitivity with relatively good selectivity; but accurate and reliable operation, particularly at levels of a few pmol, relies upon both correct selection and good matching of equipment and its operation. Often the detection of trace compounds requires the instrumentation to give satisfactory results at signal-to-noise ratios of below 10 at fmol levels. The ability of some commercially available detectors, a number of different pumps, and various electrodes to meet these requirements has been investigated in connection with measuring the levels in plasma of catecholamines and a number of thiols including thiol drugs.

Radioactivity from [14C] adenosine was linearly incorporated into tissue nucleotides in perfused rat hearts. All the TCA-extractable 14C was confined to the purine nucleoside phosphates for up to 1 h of perfusion. Radioactivity was also incorporated linearly into the TCA-insoluble fraction, which by 40 min accounted for 24% of the tissue 14 C. Estimates based on precursor specific radioactivity suggest that at least 0.6 micro mol/g of the mononucleotide is in this stable insoluble form. Following 2 min total ischaemia, the tissue nucleotide content and soluble radioactivity decreased while the insoluble radioactivity showed a corresponding increase to account now for 35% of the tissue radiolabel. This redistribution was rapidly reversed by post-ischaemic reperfusion. A possible function for the rapid reversible sequestration of adenine nucleotides in ischaemia is proposed.

1. The contents of the major purine nucleotides in the isolated non-working perfused rat heart varied systematically during 80min of perfusion. In particular the amounts of ATP, ADP, GTP, cyclic AMP and cyclic GMP in the well-oxygenated myocardium showed changes ranging from 25 to 60% of the mean concentrations. The apparent periodicity was about 30min for some and about 60min for other nucleotides. 2. These data are in contrast with measurements of parameters reflecting heart performance, which remained constant over this period of perfusion. 3. The ATP/ADP ratio, the cyclic AMP content, the GTP content and the GTP/GDP ratio in the tissue bore a constant relationship to one another, and all showed the same temporal variation. 4. Increasing the energy demand on the heart by administration of bovine somatotropin (1μg/ml) tended to damp the variations, and generally lower the content of all the nucleotides. 5. The total extractable adenine nucleotide pool also showed systematic temporal variations of as much as 1.3μmol/g wet wt. of tissue within 10min. 6. These variations could not be accounted for as inter-conversion with adenosine, other purine nucleotides, nucleosides or purine-degradation products either in the tissue or in the perfusion medium. No evidence was found in this preparation of the purine nucleotide oscillations described by Lowenstein and his co-workers [see Tornheim & Lowenstein (1975) J. Biol. Chem.250, 6304–6314]. 7. Further, the pool size increases cannot be satisfactorily explained by either synthesis de novo or the breakdown of any purine macromolecular species in the cell. Thus it is suggested that an unsuspected substantial storage form of purine nucleotide may exist in heart.

The alpha and beta adrenoceptor blocking drug labetalol is a potent antihypertensive agent in widespread clinical use. Its interference in the classical chemical estimations of urinary catecholamines and their metabolites has been the subject of several reports. Factitiously raised values have been noted in both the fluorimetric catecholamine assay, and the standard spectrophotometric procedure for total (free and conjugated) metadrenalines. To avoid such drug interference, modification of these methods is required in the estimation of catecholamines and their o-methylated metabolites. Alternatively, VMA estimations or plasma/urinary catecholamine measurements by radioenzymatic assay may be used in patients on labetalol. Although high performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) methods for estimation of plasma catecholamines are now in widespread use, the interference of labetalol in this method has not been reported. We now report that significant direct interference of labetalol in the HPLC-ECD assay does indeed occur, and can yield spuriously raised adrenaline levels.

During collaborative studies of purine metabolism in PNP and ADA deficiency2, we investigated ‘lymphocyte’ (peripheral blood mononuclear cells-PBMs) nucleotide levels and found consistently low ATP:ADP ratios in both patient and controls. Values quoted in the literature for total nucleotides and ATP/ADP ratios also varied widely3,4. For this reason experiments to investigate these differences and establish a range for PBM nucleotide levels in the local population were undertaken.

A reciprocal relationship between erythrocyte ATP and deoxy-ATP levels has been noted in an immunodeficient child with adenosine deaminase (ADA) deficiency during therapy with red cell transfusions. The sum of red cell ATP plus deoxy-ATP equalled the normal complement of ATP prior to any form of therapy. dATP, dADP and dAMP levels were found in the same ratio (10:1:0.1) as the adenine nucleotides ATP, ADP and AMP. Red cell ATP levels were low, not high or normal as found by others in ADA deficiency, but no deoxyadenosine nucleotides could be found in peripheral blood mononuclear cells. Erythrocyte ATP depletion has recently been identified as a serious consequence of anti-leukaemic therapy with ADA inhibitors; it may thus be an important but hitherto unrecognised contributing factor in the clinical expression of inherited ADA deficiency.

A quantitative perfusion method has been used to study intestinal absorption of two dipeptides-glycyl-glycine and glycyl-l-alanine-in normal subjects. In each case, the constituent amino acids were absorbed faster when presented as dipeptides than as free amino acids, suggesting intact dipeptide transport. During absorption constituent amino acids were measured within the lumen and it is suggested that these represent amino acids which have diffused back to the lumen after absorption as dipeptide. Portal blood analyses during absorption of a third dipeptide, glycyl-l-lysine, have shown that this dipeptide, known to be transported intact from the intestinal lumen, is hydrolysed to its constitutent amino acids before it reaches portal venous blood.

1. A double lumen perfusion technique has been used in man to study the absorption of the two neutral amino acids glycine and l-alanine from the two dipeptides, l-alanylglycine and glycyl-l-alanine and from an equivalent amino acid mixture. 2. Glycine was absorbed faster from the dipeptides than from the equivalent amino acid mixture, and the difference in absorption rates of glycine and alanine seen when the equimolar mixture of the amino acids was perfused, was abolished when either dipeptide was perfused. This suggests that dipeptides are taken up by the mucosal cell by a mechanism independent of the amino acid-transport system. 3. The presence of free amino acids in the lumen during perfusion of both dipeptides suggests that hydrolysis occurs at some stage in the uptake process. Intraluminal hydrolysis was insufficient to account for the concentration of the amino acids seen, and their presence is thought to be due to hydrolysis of the dipeptides at the brush border. 4. It is suggested that these results confirm that at least two modes of peptide absorption occur simultaneously, namely, direct peptide uptake, and peptide hydrolysis with subsequent absorption of the released amino acids by the amino acid transport system.

1. The absorption of lysine and two lysine-containing dipeptides was studied in normal and cystinuric subjects by jejunal perfusion. Absorption of each dipeptide was compared with that of its constituent amino acids. 2. in normal subjects lysine was better absorbed from the dipeptide glycyl-l-lysine than from the equivalent amino acid mixture. in cystinuric patients lysine was absorbed very poorly in its free form but was absorbed normally from the dipeptide. in cystinuric patients lysine was also absorbed faster as l-lysyl-l-lysine than as free lysine. 3. During perfusion of both dipeptides significantly greater quantities of free lysine were found within the intestinal lumen in cystinuric subjects than in normal subjects. This is thought to be due to back-diffusion after hydrolysis of dipeptide by the mucosa. 4. These observations confirm the existence of dipeptide transport systems independent of those for free amino acids.

1. Eight normal subjects were given, in double-blind, random order L-tyrosine 50, 250 and 500 mg kg-1 and placebo orally. Plasma tyrosine concentrations rose in a dose-dependent manner, without affecting the concentrations of the other large neutral amino acids. Tyrosine stimulated the secretion of prolactin and thyrotrophin (TSH) but had no effect on the plasma concentrations of adrenocorticotrophic hormone (ACTH), cortisol, growth hormone or the gonadotrophins. 2. The lack of a stimulant effect of tyrosine on ACTH secretion was presumed to be due to activation of one of the negative feedback mechanisms that control the rate of synthesis and release of the catecholamines, and this hypothesis was tested by examining the effects of the alpha 2-adrenoceptor antagonist idazoxan on the actions of tyrosine. 3. Seven normal males were given on 6 separate occasions tyrosine 250 and 500 mg kg-1 and placebo orally following pretreatment with saline and idazoxan (0.1 mg kg-1 i.v.). Following pretreatment with idazoxan, tyrosine stimulated the secretion of ACTH and noradrenaline in a dose-dependent manner, although neither tyrosine nor idazoxan on their own had any effect on the secretion of either substance. 4. The lack of effect of tyrosine when given on its own appears to be due, to partly, to activation of alpha 2-adrenoceptors, which inhibit the release of noradrenaline. Idazoxan caused a small increase in systolic blood pressure, both when given on its own and in combination with tyrosine. Neither tyrosine nor idazoxan had any significant effect on the state of behavioural arousal, as measured by visual analogue scales, or on the secretion of growth hormone or the gonadotrophins.