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Background There is a trend towards decentralisation of laboratory tests by means of Point-of-Care testing (POCT). Within hospitals, Belgian law requires a POCT policy, coordinated by the clinical laboratory. There is however no legal framework for POCT performed outside the hospital: no reimbursement, no compulsory quality monitoring and no limits nor control on the prices charged to the patient. Uncontrolled use of POCT can have negative consequences for individual and public health. Proposal We propose that POCT outside hospitals would only be reimbursed for tests carried out within a legal framework, requiring evidence-based testing and collaboration with a clinical laboratory, because clinical laboratories have procedures for test validation and quality monitoring, are equipped for electronic data transfer, are familiar with logistical processes, can provide support when technical issues arise and can organise and certify training. Under these conditions the government investment will be offset by health benefits, e.g. fall in antibiotic consumption with POCT for CRP in primary care, quick response to SARS-CoV2-positive cases in COVID-19 triage centres. Priorities 1 degrees extension of the Belgian decree on certification of clinical laboratories to decentralised tests in primary care; 2 degrees introduction of a separate reimbursement category for POCT; 3 degrees introduction of reimbursement for a limited number of specified POCT; 4 degrees setup of a Multidisciplinary POCT Advisory Council, the purpose of which is to draw up a model for reimbursement of POCT, to select tests eligible for reimbursement and to make proposals to the National Institute for Health and Disability Insurance (RIZIV/INAMI). No funding was received for this work. Meetings of the members of the Working Group POCT with external experts on POCT were facilitated by Roche Diagnostics Belgium (Machelen, Belgium).

Clinicians trust medical laboratories to provide reliable results on which they rely for clinical decisions. Laboratories fulfil their responsibility for accurate and consistent results by utilizing an arsenal of approaches, ranging from validation and verification experiments to daily quality control procedures. All these procedures verify, on different moments, that the results of a certain examination procedure have analytical performance characteristics (APC) that meet analytical performance specifications (APS) set for a particular intended use. The APC can in part be determined by estimating the measurement uncertainty component under conditions of within-laboratory precision (u Rw), which comprises all components influencing the measurement uncertainty of random sources. To maintain the adequacy of their measurement procedures, laboratories need to distinguish aspects that are manageable vs. those that are not. One of the aspects that may influence u Rw is the momentary significant bias caused by shifts in reagent and/or calibrator lots, which, when accepted or unnoticed, become a factor of the APC. In this paper, we postulate a model for allocating a part of allowable u Rw to between-reagent lot variation, based on the need for long-term consistency of the measurement variability for that specific measurand. The allocation manages the ratio between short-term and long-term variation and indicates laboratories when to reject or correct certain variations due to reagent lots.

The ISO 15189:2012 standard section 5.9.1 requires laboratories to review results before release, considering quality control, previous results, and clinical information, if any, and to issue documented procedures about it. While laboratory result reporting is generally regarded as part of the post-analytical phase, the result release process requires a general view of the total examination process. Reviewing test results may follow with troubleshooting and test repetition, including reanalyzing an individual sample or resampling. A systematic understanding of the result release may help laboratory professionals carry out appropriate test repetition and ensure the plausibility of laboratory results. In this paper, we addressed the crucial steps in the result release process, including evaluation of sample quality, critical result notification, result reporting, and recommendations for the management of the result release, considering quality control alerts, instrument flags, warning messages, and interference indexes. Error detection tools and plausibility checks mentioned in the present paper can support the daily practice of results release.

ISO15189:2012 requires medical laboratories to document metrological traceability of their results. While the ISO17511:2003 standard on metrological traceability in laboratory medicine requires the use of the highest available level in the traceability chain, it recognizes that for many measurands there is no reference above the manufacturer’s selected measurement procedure and the manufacturer’s working calibrator. Some immunoassays, although they intend to measure the same quantity and may even refer to the same reference material, unfortunately produce different results because of differences in analytical selectivity as manufacturers select different epitopes and antibodies for the same analyte. In other cases, the cause is the use of reference materials, which are not commutable. The uncertainty associated with the result is another important aspect in metrological traceability implementation. As the measurement uncertainty on the clinical samples is influenced by the uncertainty of all steps higher in the traceability chain, laboratories should be provided with adequate and appropriate information on the uncertainty of the value assignment to the commercial calibrators that they use. Although the between-lot variation in value assignment will manifest itself as part of the long-term imprecision as estimated by the end-user, information on worst-case to be expected lot-lot variation has to be communicated to the end-user by the IVD provider. When laboratories use ancillary equipment that potentially could have a critical contribution to the reported results, such equipment needs verification of its proper calibration and criticality to the result uncertainty could be assessed by an approach based on risk analysis, which is a key element of ISO15189:2012 anyway. This paper discusses how the requirement for metrological traceability as stated in ISO15189 should be met by the medical laboratory and how this should be assessed by accreditation bodies.

This document is based on the original recommendation of the Expert Panel on the Theory of Reference Values of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), updated guidelines were recently published under the auspices of the IFCC and the Clinical and Laboratory Standards Institute (CLSI). This document summarizes proposals for recommendations on: (i) The terminology, which is often confusing, noticeably concerning the terms of reference limits and decision limits. (ii) The method for the determination of reference limits according to the original procedure and the conditions, which should be used. (iii) A simple procedure allowing the medical laboratories to fulfill the requirements of the regulation and standards. The updated document proposes to verify that published reference limits are applicable to the laboratory involved. Finally, the strengths and limits of the revised recommendations (especially the selection of the reference population, the maintenance of the analytical quality, the choice of the statistical method used…) will be briefly discussed.

Accreditation is a valuable resource for medical laboratories. The development of quality systems based on ISO 15189 has taken place in many laboratories in the European countries but data about accreditation remain scarce. The EFLM Working Group “Accreditation and ISO/CEN standards” conducted a survey that reviews the current state of the accreditation process in European countries.An on-line questionnaire was addressed to delegates of 39 EFLM scientific societies in March 2014. One answer by country was taken into account. The survey was dealing with mandatory status, number of accredited medical laboratories in each country, possibility of flexible scope and concerned medical fields. The status of point-of-care testing (POCT) in each country was also studied.Twenty-nine responses (74%) were registered. All the assessed countries (100%) have begun an accreditation process in various ways. All the national accreditation bodies (NAB) offer or are working to offer an ISO 15189 accreditation. The accreditation process most often concerns all phases of the examination and various medical fields. Medical laboratories are responsible for POCT in 20 (69%) countries. The accreditation process for POCT, according to ISO 15189 and ISO 22870, is also developing.While there are several variations in the approaches to accreditation of medical laboratories in the European countries, the ISO 15189 accreditation project has been widely accepted. The use of a unique standard and the cooperation among countries due to scientific societies, EFLM, accreditation bodies and EA enable laboratory professionals to move toward uniform implementation of the accreditation concept.

ispartof: Clinical Chemistry and Laboratory Medicine vol:54 issue:3 pages:E97-E100 ispartof: location:Germany status: published

BACKGROUNDSystematic evidence of the contribution made by laboratory medicine to patient outcomes and the overall process of healthcare is difficult to find. An understanding of the value of laboratory medicine, how it can be determined, and the various factors that influence it is vital to ensuring that the service is provided and used optimally.CONTENTThis review summarizes existing evidence supporting the impact of laboratory medicine in healthcare and indicates the gaps in our understanding. It also identifies deficiencies in current utilization, suggests potential solutions, and offers a vision of a future in which laboratory medicine is used optimally to support patient care.SUMMARYTo maximize the value of laboratory medicine, work is required in 5 areas: (a) improved utilization of existing and new tests; (b) definition of new roles for laboratory professionals that are focused on optimizing patient outcomes by adding value at all points of the diagnostic brain-to-brain cycle; (c) development of standardized protocols for prospective patient-centered studies of biomarker clinical effectiveness or extraanalytical process effectiveness; (d) benchmarking of existing and new tests in specified situations with commonly accepted measures of effectiveness; (e) agreed definition and validation of effectiveness measures and use of checklists for articles submitted for publication. Progress in these areas is essential if we are to demonstrate and enhance the value of laboratory medicine and prevent valuable information being lost in meaningless data. This requires effective collaboration with clinicians, and a determination to accept patient outcome and patient experience as the primary measure of laboratory effectiveness.

The recent revision of ISO15189 has further strengthened its position as the standard for accreditation for medical laboratories. Both for laboratories and their customers it is important that the scope of such accreditation is clear. Therefore the European co-operation for accreditation (EA) demands that the national bodies responsible for accreditation describe the scope of every laboratory accreditation in a way that leaves no room for doubt about the range of competence of the particular laboratories. According to EA recommendations scopes may be fixed, mentioning every single test that is part of the accreditation, or flexible, mentioning all combinations of medical field, examination type and materials for which the laboratory is competent. Up to now national accreditation bodies perpetuate use of fixed scopes, partly by inertia, partly out of fear that a too flexible scope may lead to over-valuation of the competence of laboratories, most countries only use fixed scopes. The EA however promotes use of flexible scopes, since this allows for more readily innovation, which contributes to quality in laboratory medicine. In this position paper, the Working Group Accreditation and ISO/CEN Standards belonging to the Quality and Regulation Committee of the EFLM recommends using an approach that has led to successful introduction of the flexible scope for ISO15189 accreditation as intended in EA-4/17 in The Netherlands. The approach is risk-based, discipline and competence-based, and focuses on defining a uniform terminology transferable across the borders of scientific disciplines, laboratories and countries.

Off-resonance lactate magnetization transfer (MT experiments were performed on the in situ rat liver under perfused and ischemic conditions. A significant MT effect for lactate methyl protons was observed. The effect was larger for the ischemic condition than for the perfused condition, and was largest in the blood-filled ischemic livers. The size of the motionally restricted lactate pool, determined using a two-pool model fit, was estimated to be about 1% in perfused livers and about 1.8-2.5% after more than 1 hr of onset of ischemia, suggesting that lactate in liver is almost fully NMR-visible. The MT data for both the perfused and the ischemic condition appeared to be better approximated when assuming a superLorentzian lineshape for the immobile pool rather than a Gaussian lineshape. Finally, the experiments demonstrated a coupling between the lactate methyl and water protons, which may be mediated by macromolecules. (C) 2002 Wiley-Liss, Inc. ispartof: Magnetic resonance in medicine vol:47 issue:5 pages:880-887 ispartof: location:United States status: published

Abstract —Glucose-insulin-potassium solutions exert beneficial effects on the ischemic heart by reducing infarct size and mortality and improving postischemic left ventricular function. Insulin could be the critical protective component of this mixture, although the insulin response of the ischemic and postischemic myocardium has not been systematically investigated. The aim of this work was to study the insulin response during ischemia by analyzing insulin signaling. This was evaluated by measuring changes in activity and/or phosphorylation state of insulin signaling elements in isolated perfused rat hearts submitted to no-flow ischemia. Intracellular pH (pH i ) was measured by NMR. No-flow ischemia antagonized insulin signaling including insulin receptor, insulin receptor substrate-1, phosphatidylinositol 3-kinase, protein kinase B, p70 ribosomal S6 kinase, and glycogen synthase kinase-3. These changes were concomitant with intracellular acidosis. Perfusing hearts with ouabain and amiloride in normoxic conditions decreased pH i and insulin signaling, whereas perfusing at pH 8.2 counteracted the drop in pH i and the inhibition of insulin signaling by ischemia. Incubation of cardiomyocytes in normoxic conditions, but at pH values below 6.75, mimicked the effect of ischemia and also inhibited insulin-stimulated glucose uptake. Finally, the in vitro insulin receptor tyrosine kinase activity was progressively inhibited at pH values below physiological pH i , being abolished at pH 6.0. Therefore, ischemic acidosis decreases kinase activity and tyrosine phosphorylation of the insulin receptor thereby preventing activation of the downstream components of the signaling pathway. We conclude that severe ischemia inhibits insulin signaling by decreasing pH i .

Intact human erythrocytes catalyse the conversion of fructose into fructose 3-phosphate with an apparent K(m) of 30 mM [Petersen, Kappler, Szwergold and Brown (1992) Biochem. J. 284, 363-366]. The physiological significance of this process is still unknown. In the present study we report that the formation of fructose 3-phosphate from 50 mM fructose in intact erythrocytes is inhibited by 1-deoxy-1-morpholinofructose (DMF), a synthetic fructosamine, with an apparent K(i) of 100 microM. (31)P NMR analysis of cell extracts incubated with DMF indicated the presence of an additional phosphorylated compound, which was partially purified and shown to be DMF 3-phosphate by tandem MS. Radiolabelled DMF was phosphorylated by intact erythrocytes with an apparent K(m) ( approximately 100 microM) approx. 300-fold lower than the value reported for fructose phosphorylation on its third carbon. These results indicate that the physiological function of the enzyme that is able to convert fructose into fructose 3-phosphate in intact erythrocytes is probably to phosphorylate fructosamines. This suggests that fructosamines, which are produced non-enzymically from glucose and amino compounds, may be metabolized in human erythrocytes.

We examined the route of uptake of 2-aminoethylphosphonate (NEthPo) and of phenylphosphonate (PhePo; 10 mM each) in perfused liver by (31)P-NMR. Uptake of NEthPo was concentrative. The rate of uptake was reduced to 21 +/- 2% (n = 3; all percentages refer to control rates) by substituting choline for Na(+), and to 21 +/- 4% (n = 3), 32 +/- 6% (n = 5) and 70 +/- 5% (n = 3) by replacing Cl(-) by gluconate, SO(4)(2-) or NO(3)(-), respectively. Taurine (20 mM) reduced NEthPo uptake to 38 +/- 6% (n = 3). The data are consistent with uptake of NEthPo by the Na(+)-coupled Cl(-)-dependent beta-amino acid transporter. A small fraction of NEthPo was incorporated into phospholipid. PhePo uptake evolved over 1 h towards levels of the membrane-permeant volume marker dimethyl methylphosphonate. Uptake depended on H(+), and was inhibited by 4, 4'-diisothiocyanato-stilbene-2,2'-disulphonic acid (100 microM), bumetanide and furosemide (1 mM each) and alpha-cyano-4-OH-cinnamic acid (5 mM) to 31 +/- 4% (n = 4), 28 +/- 4% (n = 4), 27 +/- 5% (n = 6) and 40 +/- 7% (n = 4), respectively. These characteristics of PhePo uptake are reminiscent of H(+)-coupled monocarboxylate transport. The monocarboxylates, lactate and acetate (20 mM), and the substrate analogue, phenylalanine (20 mM), were not inhibitory, while benzoic acid (20 mM) slightly inhibited (to 82 +/- 5%; n = 4) PhePo uptake. The tested phosphonates (10 mM) did not significantly affect hepatic extraction of [(3)H]-cholate or [(3)H]-taurocholate (25 microM each; 1:3 bile salt:albumin). The monocarboxylate analogue, PhePo (10 mM), did not significantly interfere with disposal of lactate (0.3-5 mM).

Inhibition of hormone-stimulated hepatic glycogenolysis by fructose (Fru) has been attributed to accumulation of the competitive inhibitor Fru1P and/or to the associated depletion of the substrate phosphate (Pi). To evaluate the relative importance of either factor, we used the Fru analogue 2,5-anhydro-D-mannitol (aHMol). This analogue is avidly phosphorylated, traps Pi, and inhibits hormone-stimulated glycogenolysis, but it is not a gluconeogenic substrate, and hence does not confound glycogenolytic glucose production. Livers were continuously perfused with dibutyryl-cAMP (100 microM) to clamp phosphorylase in its fully activated a form. We administered aHMol (3.8 mM), and studied changes in glycogenolysis (glucose, lactate and pyruvate output) and in cytosolic Pi and phosphomonoester (PME), using in situ 31P-NMR spectroscopy (n = 4). Lobes of seven livers perfused outside the magnet were extracted for evaluation, by high-resolution 31P-NMR, of the evolution of aHMol1P and of aHMol(1,6)P2. After addition of aHMol, both glycogenolysis and the NMR Pi signal dropped precipitously, while the PME signal rose continuously and was almost entirely composed of aHMol1P. Inhibition of glycogenolysis in excess of the drop in Pi could be explained by continuing accumulation of aHMol1P. A subsequent block of mitochondrial ATP synthesis by KCN (1 mM) caused a rapid increase of Pi. Despite recovery of Pi to values exceeding control levels, glycogenolysis only recovered partially, attesting to the Pi-dependence of glycogenolysis, but also to inhibition by aHMol phosphorylation products. However, KCN resulted in conversion of the major part of aHMol1P into aHMol(1,6)P2. Residual inhibition of glycogenolysis was due to aHMol1P. Indeed, the subsequent withdrawal of aHMol caused a further gradual decrease in the proportion of aHMol1P (being converted into aHMol(1,6)P2, in the absence of de novo aHMol1P synthesis), and this resulted in a gradual de-inhibition of glycogenolysis, in the absence of marked changes in Pi. Glycogenolytic rates were consistently predicted by a model assuming non-saturated Pi kinetics and competition by aHMol1P exclusively: In conclusion, limited Pi availability and the presence of competitive inhibitors are decisive factors in the control of the in situ catalytic potential of phosphorylase a.

Oral creatine supplementation has been shown to improve power output during high intensity intermittent muscle contractions. Facilitated muscle phosphocreatine (PCr) resynthesis, by virtue of elevated intracellular PCr concentration, might contribute to this ergogenic action. Therefore, the effect of creatine loading (C: 25 g X d(-1) for 5 d) on muscle PCr breakdown and resynthesis and muscle performance during high intensity intermittent muscle contractions was investigated.A double-blind randomized cross-over study was performed in young healthy male volunteers (N = 9). 31P-NMR spectroscopy of the m. gastrocnemius and isokinetic dynamometry of knee-extension torque were performed before and after 2 and 5 d of either placebo (P) or C administration.Compared with P, 2 and 5 d of C increased (P0.05) resting muscle PCr concentration by 11% and 16%, respectively. Furthermore, torque production during maximal intermittent knee extensions, including the first bout of contractions, was increased (P0.05) by 5-13% by either 2 or 5 d of C. However, compared with P, the rate of PCr breakdown and resynthesis during intermittent isometric contractions of the calf was not significantly affected by C.Creatine loading raises muscle PCr concentration and improves performance during rapid and dynamic intermittent muscle contractions. Creatine loading does not facilitate muscle PCr resynthesis during intermittent isometric muscle contractions.

The dynamics of size-selected fractions of glycogen in solution have been investigated by proton NMR spectroscopy, using a recently described relaxation study method which relies on strong offresonance irradiation. The dependence of the steady-state magnetisation on angle and intensity of the effective radio-frequency field was measured and compared to theoretical curves derived from different models of motion. Absence or presence of contributions to relaxation from molecular motions on the microsecond time scale can be tested with this method, without having to resort to models. We found that glycogen dipolar relaxation did not result from isotropic Brownian rotation, and despite some contribution from slow motion (1 microsecond) to relaxation in glycogen alpha-particles extracted from rat liver, bulk movement of the molecules did not appear to participate in averaging the dipolar term to zero. Whereas hepatic glycogen rat beta-particles and commercial oyster glycogen displayed very similar relaxation properties, alpha-particles showed significantly different behaviour. However, all results were compatible with a diversity of movements within the molecule, ranging from freely rotating pyranoside rings through collective chain motion and possibly to bulk movement of the beta sub-units within the alpha-particle.

We evaluated phosphonates (Po) as markers of the extra-and intracellular space in perfused rat liver, (i) In-and outwash behaviour of phenylphosphonate (PhePo), 3-amino-propylphosphonate (NProPo) and methyl phosphonate (MePo) was compared with that of creatine phosphate (CrP), a marker of the extracellular space, and of dimethyl methylphosphonate (MePoMe2), a marker of the total water-accessible space, In-and outwash of CrP was accurately predicted by the time constant (similar to 12 s) for the in-and outwash of inulin, a standard marker of the extracellular space, MePoMe2 rapidly distributed over the total liver volume (about three times the CrP accessible space), PhePo, NProPo and MePo washed rapidly into the extracellular space with CrP, and then steadily spilled over into the MePoMe2-accessible space, Upon outwash, Po signals rapidly declined in phase with that of CrP. Residual Po (PhePo much greater than NProPo similar or equal to MePo) reflected the amount internalized during prolonged (60 min) inwash, Proportional amounts of residual Po were found in extracts of livers harvested after outwash of perfusate and extracellular markers, Consistent with exclusion from the cells, CrP went undetected in these extracts, (ii) The resonance frequency of residual PhePo after outwash of the extracellular fraction corresponded with the pH reported by cytosolic Pi and responded to transient changes of the intracellular pH, induced by perfusion with and withdrawal of 20 mM NH4Cl, (iii) MePoMe2 homogeneously distributed over perfusate, parenchyma and bile, consistent with unrestricted permeability, Other Po were transported transcellularly and excreted in bile. CrP was virtually excluded from the bile, attesting to a minimal role for 'bulk-phase pinocytotic' transcellular transport, or for 'paracellular' leakage, In summary, charged Po can be used as extracellular markers in liver, provided experimental conditions are adjusted to minimize their internalization. Some Po (e.g. PhePo) can reach intracellular concentrations which suffice for the compound to act as a reporter molecule of the cytosolic pH. (C) 1997 John Wiley & Sons, Ltd.

Background: With the use of a traditional blood gas analyzer (BGA) (ABL800 Radiometer) for blood glucose monitoring, tight glucose control (TGC) reduced in-hospital mortality and morbidity of surgical intensive care unit (ICU) adult and pediatric patients. Such BGAs are now superseded by cassette-based BGAs. We assessed the analytical reliability of cassette-based BGAs to monitor patient's metabolic status in an ICU setting. Methods: We evaluated recovery/linearity, imprecision/repeatability and relative accuracy vs. comparison methods for glucose [coupled hexokinase glucose-6-phosphate dehydrogenase (HK/G6PD) assay] and lactate (lactate dehydrogenase assay) in ICU patient samples with two cassette-based BGAs [RP500 (Siemens) and ABL90 (Radiometer)] and with the ABL800 BGA. Results: Recovery of spiked glucose up to 348 mg/dL (19.3 mmol/L) was complete for all BGAs. Repeatability of ABL800 and ABL90 was comparable with the comparison method (about 1%), but higher for RP500 (about 2.4%). All BGAs were in agreement with the comparison method, with all glucose measurements falling within preset 10% criteria suggested by Karon. Recovery of spiked lactate (up to 25 mmol/L) was incomplete at all levels. Repeatability of ABL800 and ABL90 was comparable with the comparison method (about 4%), and 5.5% for RP500. All BGAs were in agreement with the comparison method, with >98% of the lactate measurements falling within 30% biological-variation-based criteria. Conclusions: The cassette-based BGAs quantified blood glucose and lactate levels in ICU patients within the acceptable error ranges. Cassette-based BGAs can be used for monitoring patient's metabolic status in an ICU setting. ispartof: Clinical Chemistry and Laboratory Medicine vol:51 issue:7 pages:1417-1427 ispartof: location:Germany status: published

1. Iodogen (tetrachloro-diphenyl glycoluril) dissolved in DMSO (dimethyl sulphoxide) appears indispensable in radioiodination of hypericin for a new anticancer strategy. We studied the safety of intravenously administered iodogen/DMSO in mice (n = 132). 2. Median lethal dose (LD50) of iodogen/DMSO was determined with doses of 40.0, 50.0, 55.0, 60.0, 65.0 and 70.0 mg/kg. Next, toxicity of iodogen/DMSO at 30.0 mg/kg was evaluated using saline and DMSO as controls. Changes in behaviour, body weight and serum biochemistry were evaluated. Histopathology of lungs, heart, liver and kidney was performed. 3. LD50 values of iodogen/DMSO were 59.5 mg/kg (95% confidence limits (CI): 54.1-65.4 mg/kg) and 61.0 mg/kg (95%CI: 56.2-66.2 mg/kg) for female and male mice, respectively. Similar to that of control groups, no animal deaths were encountered after iodogen/DMSO administration at 30.0 mg/kg. Body weights over 24 h were not altered in all groups, but significantly higher in iodogen/DMSO and DMSO groups (p 0.05) 14 d post-injection. Blood urea nitrogen and alkaline phosphatase increased (p 0.05) in iodogen/DMSO group without clinical symptoms. No pathologies were found by gross and microscopic inspection. 4. A single dose of iodogen/DMSO up to 30.0 mg/kg, over 3000 times the dose in potential human applications, appears safe, with an LD50 doubling that dose in mice.

The 13 C-1 NMR peak in proton-decoupled spectra of liver glycogen solution was quantitatively analyzed by three types of model-function fitting algorithms: iterative line-fitting in the frequency domain (MDCON); iterative least-squares fitting (VARPRO) in the time domain; and noniterative singular value decomposition-based analysis (HTLS), also in the time domain. Quantification results were compared with manual integration values. Performance of the algorithms was tested at different signal-to-noise ratios (S/N) of the glycogen C-1 peak. This was achieved by varying the number of scans summed prior to analysis. Since T 2 relaxation in glycogen has been shown to be multiexponential [Overloop, K. et al.Magn. Reson. Med. 36, 45-51 (1996], the exact quantification of the C-1 glycogen signal requires a model function comprising a sum of Lorentzian components, each with a different broadening at the glycogen frequency. This paper focuses on the performances of the above methods to fit such a multicomponent resonance line. In the frequency domain, line fitting with two Lorentz lines gives good results at sufficiently high S/N. In the time domain, VARPRO performs better than HTLS because fixed values can be imposed to the linewidth of the components at the common C-1 frequency, thereby reducing convergence problems at low S/N.

In search for a nonmetabolized, superior glucose analogue to study the mechanism of glucose-induced glycogen synthesis, we have tested 2-deoxy-2-fluoro-alpha-D-glucopyranosyl fluoride, which inhibits muscle phosphorylase beta 10-fold better than dose glucose (Street, I.P., Armstrong, C.R., and Withers, S.G. (1986) Biochemistry 25, 6021-6027). In a gel-filtered liver extract, 0.6 mM analogue and 10 mM glucose equally accelerated the inactivation of phosphorylase and shortened the latency before the activation of glycogen synthase. The analogue was not measurably defluorinated or phosphorylated by intact hepatocytes, as monitored by 19F NMR. When added to isolated hepatocytes, 10 mM analogue inactivated phosphorylase more extensively than did 50 mM glucose, but unlike glucose, it did not result in the activation of glycogen synthase. Therefore, the binding of glucose to phosphorylase alpha can account for the inactivation of phosphorylase, but the metabolism of glucose (probably to Glc-6-P) appears to be required to achieve activation of glycogen synthase. The livers of overnight-fasted, anesthetized mice contained appreciable amounts of both phosphorylase alpha and glycogen synthase alpha, without net glycogen accumulation. Likewise, hepatocytes isolated from fasted rats and incubated with 10 mM glucose contained 41% of phosphorylase and 32% of glycogen synthase in the alpha form, and these values remained stable for 1 h, while glycogen accumulated at only 22% of the rate expected from the glycogen synthase activity. The addition of 10 mM analogue decreased phosphorylase alpha to 10% without significant change in glycogen synthase alpha (38%), but with a 4-fold increased rate of glycogen accumulation. These findings imply that synthase alpha is fully active in the liver of the fasted animal and that the absence of net glycogen synthesis is due to continuous glycogenolysis by phosphorylase alpha.

OBJECTIVE: Serum creatinine (Scr) reflects to a certain extent glomerular filtration rate in neonates, but postnatal observations also depends on the technique used to quantify Scr (Jaffe colorimetry or enzymatic quantification). METHODS: In an attempt to quantify differences between these techniques, we compared postnatal Scr trends in two consecutive cohorts of extremely low birth-weight (ELBW) neonates before and following a switch from uncompensated Jaffe to enzymatic Scr quantification. Postnatal Scr (Days 1, 2, 3, 4, 5, 6, 7, 8, 9, 14, 21, 28, and 42) in 151 ELBW neonates (uncompensated Jaffe) was compared to 116 more recently admitted ELBW neonates (enzymatic). RESULTS: Although clinical characteristics were similar between both cohorts, median postnatal Jaffe Scr values were significantly higher compared to enzymatic quantification (all days, at least p < 0.001) throughout postnatal life. While both cohorts displayed a similar trend with an initial increase with a Scr peak on Days 3 and 4 and a subsequent decrease, the difference in within-day median values fluctuated between 11 and 24 mmol.L(-1). There is neither fixed nor relative difference in Scr between both techniques. CONCLUSIONS: When using Scr to estimate renal function in ELBW neonates, clinicians should in addition to the postnatal changes and other covariates of renal function, also consider the technique applied. We provide reference values and comparison between both techniques. ispartof: Journal of Maternal-Fetal & Neonatal Medicine vol:25 issue:9 pages:1678-1681 ispartof: location:England status: published

BACKGROUND Experiments were carried out using the new Na(+)-H+ exchange inhibitor (3-methylsulfonyl-4-piperidinobenzoyl)guanidine methanesulfonate (HOE 694) to assess the role of Na(+)-H+ exchange in myocardial ischemic and reperfusion injury. METHODS AND RESULTS Three groups of rabbit hearts (n = 5 in each) were perfused with blood and were subjected to 45 minutes of global normothermic (37 degrees C) ischemia, followed by 1 hour of reperfusion. Group 1 was the control group (vehicle only); in group 2, HOE 694 (1 mumol/L) was administered before ischemia (pretreatment group); and in group 3, HOE 694 was given only during reperfusion to separate actions exerted during ischemia from those specifically obtained during reperfusion. End-diastolic pressure rise at 1 hour of reperfusion was reduced by administration of HOE 694 starting before ischemia (from 52.2 +/- 8.5 mm Hg in group 1 to 17.6 +/- 4.5 mm Hg in group 2, P < .01) or starting on reperfusion (28.8 +/- 5.4 mm Hg in group 3, P < .05 versus group 1). Left ventricular developed pressure (LVDP) and its derivative (dP/dt) recovered better in HOE 694-pretreated hearts (LVDP, 79 +/- 9.9 mm Hg in group 2 versus 24.8 +/- 10 mm Hg in group 1; dP/dt, 1580 +/- 198 mm Hg/s versus 340 +/- 221 mm Hg/s, P < .01). In hearts treated only on reperfusion, some improvement was observed, which, however, did not reach statistical significance. Coronary flow on reperfusion was higher in groups 2 and 3 compared with controls, and no "no-reflow" was observed. Two additional groups of hearts were perfused with phosphate-free Krebs-Henseleit solution to enable studies with 31P nuclear magnetic resonance (NMR). ATP was better preserved in HOE 694-pretreated (62 +/- 4.9% of preischemic value) than in control hearts (44 +/- 3.3%) at the end of 30 minutes of reperfusion, and phosphocreatine resynthesis was higher (109 +/- 3.7% versus 86 +/- 5.4%). HOE 694 did not affect the time course of intracellular acidosis during ischemia but suppressed a small alkaline overshoot occurring early in reperfusion (pH 6.96 +/- 0.02 in HOE 694-pretreated hearts versus 7.14 +/- 0.05 in control hearts). Electron microscopy with Ca2+ staining of the blood-perfused hearts showed that clumping of Ca2+ aggregates in mitochondria was prevented by HOE 694. CONCLUSIONS Postischemic dysfunction was associated with a rise in end-diastolic pressure. This rise was effectively blocked by HOE 694. The drug was most effective when hearts were treated before ischemia, although partial protection was observed when administration was started on reperfusion. The action of HOE 694 strengthens the idea that Na(+)-H+ exchange during both ischemia and reperfusion contributes to contractile dysfunction.

Using in vivo 31P-nuclear magnetic resonance spectroscopy, we studied the skeletal muscle metabolism of 17 anesthetized malignant hyperthermia-susceptible piglets and 25 control piglets during and after a halothane stress test. At rest, the phosphocreatine- (PCr) to-ATP ratio was 12% higher in the anesthetized piglets than in the control piglets, which may reflect a higher proportion of fast glycolytic fibers in the former. About 15 min of halothane administration sufficed to provoke onset of a reaction, which was characterized by a reciprocal drop in PCr and an increase in Pi with commencing intracellular acidosis. Halothane was withdrawn after a 20% drop in PCr. Within the next few minutes, intracellular pH dropped sharply and phosphomonoesters (PME) accumulated excessively. ATP was observed to decrease in 8 of the 17 animals. Halothane inhalation provoked a switch of metabolism toward glycolysis. Accumulation of PME suggests a mismatch between glycogenolysis and glycolysis. Despite severe acidification, glycolysis was not completely halted. Recovery of PCr and Pi started approximately 5 min after halothane withdrawal, with a longer time constant for recovery of the former. PME and intracellular pH aberrations lingered and started to recover later. Lost ATP was never restored within the observed recovery period of approximately 20 min.

To the Editor: The introduction of biological criteria has transformed quality control from descriptive process statistics to control of fulfillment of quality requirements on the basis of normally observed biological variation of analytes (1). Because drugs are xenobiotic, it is difficult to determine “biological” variation. Quality requirements remain contingent on achievable instrument performance, in the most optimistic case based on best practice criteria, or on arbitrary rules. An article by Fraser, published in 1987 (2), tried to address this gap (3, 4). Fraser estimated the biological variation for the drug concentration on the basis of fundamental pharmacokinetic theory, assuming a first order kinetics of elimination, rapid absorption as compared to elimination, a single compartmental system, and constant half-life and dosing regimens. For drugs with >2 …

Rat livers perfused at constant flow via the portal vein with dibutyryl cyclic AMP produced glucose equivalents at a steady maximal rate (6 mumol/min per g of liver). Addition of adenosine (150 microM) caused a biphasic effect. (i) First, the glycogenolytic rate rose transiently, to a mean peak of 150% of control levels after 2 min. This glycogenolytic burst was reproduced by two P1-receptor agonists, but not by ATP, and was blocked by a P1-antagonist (8-phenyltheophylline), as well as by inhibitors of eicosanoid synthesis (indomethacin, ibuprofen or aspirin). It did not occur in phosphorylase-kinase-deficient livers. The adenosine-induced glycogenolytic burst coincided with moderate and transient changes in portal pressure (+6 cmH2O) and O2 consumption (-20%), but it could not be explained by an increase in cytosolic Pi, since the n.m.r. signal fell precipitously. (ii) Subsequently, the rate of glycogenolysis decreased to one-third of the preadenosine value, in spite of persistent maximal activation of phosphorylase. The decrease could be linked to the decline in cytosolic Pi: both changes were prevented by the adenosine kinase inhibitor 5-iodotubercidin, whereas they were not affected by ibuprofen or 8-phenyltheophylline, and were not reproduced by non-metabolized adenosine analogues. In comparison with adenosine, ATP caused a slower decrease of Pi and of glycogenolysis. The fate of the cytosolic Pi was unclear, especially with administered ATP, which did not increase the n.m.r.-detectable intracellular ATP.

Glycogenolysis was studied in glycogen-rich perfused livers in which glycogen phosphorylase was fully converted into the a form by exposure of the livers to dibutyryl cyclic AMP. We monitored intracellular Pi by 31P n.m.r. Perfusion with Pi-free medium during 30 min caused a progressive decrease of the Pi signal to 50% of its initial value. In contrast, exposure of the livers to KCN and/or 2,4-dinitrophenol resulted in a rapid doubling of the Pi signal. Alterations in the intracellular Pi coincided with proportional changes in the rate of hepatic glycogenolysis (measured as the output of glucose plus lactate). The results indicate that the rate of glycogenolysis catalysed by phosphorylase a depends linearly on the hepatic Pi concentration. Hence the Km of phosphorylase a for its substrate Pi must be considerably higher than the concentrations that occur in the cytosol, even during hypoxia.

Glucocorticoid (GC) administration before preterm birth reduces neonatal morbidity but may restrain growth. Here we explored the effect of antenatal GC on nutrient substrates [glucose, FFA, amino acids (AA)], and on IGF-I and IGF-binding protein-1 (IGFBP-1). We analyzed umbilical vein (UV) plasma obtained at birth from 91 preterm newborns that received one course of GC (last exposure 1-1358 h before birth) and 49 newborns that did not. We found that recent GC exposure (-48 h) raised glucose, FFA, and AA concentrations, and the homeostasis model assessment of insulin resistance (HOMA-IR) index, but lowered IGF-I concentrations. The AA surge was greater in newborns with a birth weight z score 0. Although all AA were transiently increased, the increment was most robust for glutamine and alanine. Shorter duration since GC administration and lower IGF-I concentrations independently predicted AA levels. In conclusion, an antenatal course of GC elicited a transient catabolic state encompassing all nutrient substrates, and a temporary drop in IGF-I concentrations. These changes may explain the growth-inhibitory effects of repeated antenatal GC administration. Future research should clarify the role of IGF-I in the protein-catabolic response to GC. ispartof: Pediatric research vol:62 issue:3 pages:295-300 ispartof: location:United States status: published

Magnetic resonance spectroscopic signals analyzed by time-domain models in order to retrieve estimates of the model parameters usually require prior knowledge about the model order. For multi-exponential signals where a superpo- sition of peaks occurs at the same resonance frequency, but with different damping values, model order selection criteria from information theory can be used. In this study, several generalized versions of information criteria are compared using Monte-Carlo simulation signals. The best criterion is further applied for selecting the model order of experimental 13 C glycogen signals. I. INTRODUCTION Quantification of Magnetic Resonance Spectroscopic (MRS) signals can be done parametrically in the time domain by modeling the signal as a superposition of exponentially damped sinusoids. This supposes the model order to be known, which in practice is not the case. The determination of the model order is particularly difficult in the case that some of the exponentially damped sinusoids have the same frequency. This problem is encountered in the analysis of 13 C MRS signals where glycogen (GLY) is a superposition of an unknown number of exponentially damped sinusoids with the same frequency but with different damping factors: a multi-exponential signal (9). Model order selection criteria would allow an objective evaluation of the model order rather than the operator-biased evaluation of the residue. The glycogen signals were obtained during a 13 C-1 pulse- chase experiment, which followed the glycogen synthesis in a perfused rat liver and mainly consists of two pulse phases and a chase phase (1). Processing these signals demonstrated that a sum of exponentials was necessary to accurately quantify the changing glycogen signals during the experiment. Section II shortly describes some background about the considered NMR experiment, while section III mentions several concepts regarding the provided prior knowledge and model order selection. This analysis is then applied to simulation signals as well as experimental signals from the glycogen experiment. The simulation and experimental results are described and discussed in section IV. This includes the evaluation of the model order selection criteria, the optimal model order and the evolution of the signal parameters during the experiment.

Fructosamine 3-kinase (FN3K), an enzyme initially identified in erythrocytes, catalyses the phosphorylation of fructosamines on their third carbon, leading to their destabilization and their removal from protein. We show that human erythrocytes also contain FN3K-related protein (FN3K-RP), an enzyme that phosphorylates psicosamines and ribulosamines, but not fructosamines, on the third carbon of their sugar moiety. Protein-bound psicosamine 3-phosphates and ribulosamine 3-phosphates are unstable, decomposing at pH 7.1 and 37 °C with half-lives of 8.8 h and 25 min respectively, as compared with 7 h for fructosamine 3-phosphates. NMR analysis indicated that 1-deoxy-1-morpholinopsicose (DMP, a substrate for FN3K and FN3K-RP), like 1-deoxy-1-morpholinofructose (DMF, a substrate of FN3K), penetrated erythrocytes and was converted into the corresponding 3-phospho-derivative. Incubation of erythrocytes with 50 mM allose, 200 mM glucose or 10 mM ribose for 24 h resulted in the accumulation of glycated haemoglobin, and this accumulation was approx. 1.9–2.6-fold higher if DMP, a competitive inhibitor of both FN3K and FN3K-RP, was present in the incubation medium. Incubation with 50 mM allose or 200 mM glucose also caused the accumulation of ketoamine 3-phosphates, which was inhibited by DMP. By contrast, DMF, a specific inhibitor of FN3K, only affected the glucose-dependent accumulation of glycated haemoglobin and ketoamine 3-phosphates. These data indicate that FN3K-RP can phosphorylate intracellular, protein-bound psicosamines and ribulosamines, thus leading to deglycation.

We studied glycogen synthesis from glucose in perfused livers of fed (n = 4) and 24 h starved (n = 7) rats. Glycogenolysis was inhibited by BAY R3401 (150 microM) and proglycosyn (100 microM). After 60 min, we replaced 99% (13)C-1 glucose by natural abundance glucose. This pulse-chase design allowed us to recognize residual ongoing futile glycogen turnover from the release of initially deposited (13)C-label, into the (13)C-free chase medium. Net residual turnover was less than 2 +/- 0.7% and 0.6 +/- 0.2% of 1-(13)C glycogen deposition rates of 0.31 +/- 0.04 and 0.99 +/- 0.04 micromol glucose g(-1) min(-1), in starved and fed livers, respectively. The 1-(13)C glycogen signal was monitored throughout the experiment with proton-decoupled (13)C NMR spectroscopy and analyzed in the time domain using AMARES. We noticed progressive line-broadening in any single experiment in the chase phase. One or a sum of two to three overlapping Lorentzians, with different exponential damping factors, were fitted to the signal. When the S/N was better than 40, the fit always delivered a small and a broad component. In the chase phase, the fit with a single Lorentzian resulted in a decline of glycogen signal by about 15 +/- 4 and 12 +/- 2% in starved and fed rats, respectively. This apparent decline in 1-(13)C glycogen signal could not be accounted for by the appearance of equivalent amounts of (13)C-labeled metabolites in the perfusate. The fit with a sum of two Lorentzians resulted in a decline of glycogen signal intensity of 7 +/- 5 and 5 +/- 3% in starved and fed rats, respectively, which reduced the apparent turnover to 8 +/- 9% and 6 +/- 4%, respectively. Quantification of the growing (13)C-1 glycogen signal requires a model function that accommodates changes in line shape throughout the period under study.

Background Serum creatinine (Scr) reflects to a certain extent GFR in neonates, but postnatal observations also depends on the technique used (Jaffe colorimetry or enzymatic quantification) as recently quantified in ELBW neonates (1.2). We aimed to assess the impact of enzymatic versus Jaffe quantification and to describe postnatal Scr trends for both techniques in neonates with higher birth weight (3). Methods Scr values quantified by Jaffe in 1140 neonates were compared to values obtained by enzymatic quantification in 1023 neonates in one NICU. All Scr values collected in the first 42 days of postnatal life were included and postnatal trends for cohorts 3 kg were compared. Results Postnatal patterns were similar between both techniques, with an initial increase of Scr (highest and last in the smallest neonates) in early postnatal life, and a subsequent decrease, most delayed in the smallest neonates. For all consecutive postnatal observations, Jaffe always resulted in higher Scr compared to the enzymatic technique, but the differences in median values between both techniques (0.1–0.26 mg/dl, equal to 8.8–23 µmol/l), were not a fixed value. Conclusions When using Scr to estimate renal function in neonates, clinicians should in addition to postnatal changes and other covariates of renal function, also consider the technique applied. There is no fixed conversion factor to correct for differences between both techniques. Allegaert, J Matern Fetal Neonatal Med DOI 10.3109/14767058.2012.657277. Kuppens, J Matern Fetal Neonatal Med DOI 10.3109/14767058.2011.602144. Bueva, Pediatr Res 1994.

The racemic prodrug BAY R3401 suppresses hepatic glycogenolysis. BAY W1807, the active metabolite of BAY R3401, inhibits muscle glycogen phosphorylase a and b. We investigated whether BAY R3401 reduces hepatic glycogenolysis by allosteric inhibition or by phosphatase-catalyzed inactivation of phosphorylase. In gel-filtered liver extracts, racemic BAY U6751 (containing active BAY W1807) was tested for inhibition of phosphorylase in the glycogenolytic (in which only phosphorylase a is active) and glycogen-synthetic (for the evaluation of a:b ratios) directions. Phosphorylase inactivation by endogenous phosphatase was also studied. In liver extracts, BAY U6751 (0.9-36 micromol/l) inhibited glycogen synthesis by phosphorylase b (notwithstanding the inclusion of AMP), but not by phosphorylase a. Inhibition of phosphorylase-a-catalyzed glycogenolysis was partially relieved by AMP (500 micromol/l). BAY U6751 facilitated phosphorylase-a dephosphorylation. Isolated hepatocytes and perfused livers were tested for BAY R3401-induced changes in phosphorylase-a:b ratios and glycogenolytic output. Though ineffective in extracts, BAY R3401 (0.25 micromol/l-0.5 mmol/l) promoted phosphorylase-a dephosphorylation in hepatocytes. In perfused livers exposed to dibutyryl cAMP (100 micromol/l) for maximal activation of phosphorylase, BAY R3401 (125 micromol/l) inactivated phosphorylase by 63% but glucose output dropped by 83%. Inhibition of glycogenolysis suppressed glucose-6-phosphate (G6P) levels. Activation of glycogen synthase after phosphorylase inactivation depended on the maintenance of G6P levels by supplementing glucose (50 mmol/l). We conclude that the metabolites of BAY R3401 suppress hepatic glycogenolysis by allosteric inhibition and by the dephosphorylation of phosphorylase a.

Fructosamines are thought to play an important role in the development of diabetic complications. Little is known about reactions that could metabolize these compounds in mammalian tissues, except for recent indications that they can be converted to fructosamine 3-phosphates. The purpose of the present work was to identify and characterize the enzyme responsible for this conversion. Erythrocyte extracts were found to catalyze the ATP-dependent phosphorylation of 1-deoxy-1-morpholinofructose (DMF), a synthetic fructosamine. The enzyme responsible for this conversion was purified approximately 2,500-fold by chromatography on Blue Sepharose, Q Sepharose, and Sephacryl S-200 and shown to copurify with a 35,000-M(r) protein. Partial sequences of tryptic peptides were derived from the protein by nanoelectrospray-ionization mass spectrometry, which allowed for the identification of the corresponding human and mouse cDNAs. Both cDNAs encode proteins of 309 amino acids, showing 89% identity with each other and homologous to proteins of unknown function predicted from the sequences of several bacterial genomes. Both proteins were expressed in Escherichia coli and purified. They were shown to catalyze the phosphorylation of DMF, fructoselysine, fructoseglycine, and fructose in order of decreasing affinity. They also phosphorylated glycated lysozyme, though not unmodified lysozyme. Nuclear magnetic resonance analysis of phosphorylated DMF and phosphorylated fructoseglycine showed that the phosphate was bound to the third carbon of the 1-deoxyfructose moiety. The physiological function of fructosamine-3-kinase may be to initiate a process leading to the deglycation of fructoselysine and of glycated proteins.

This study is the first report on the multiexponential T2 relaxation of the 13C-1 carbon of glycogen. In contrast to T1 relaxation, which does not display observable multiexponential decay behavior, T2 relaxation is described by a continuous distribution of T2 times. Changes in molecular weight and sample viscosity, which affect the overall mobility of the glycogen particle have little influence on T1 and T2 relaxation times. This is in contradiction with earlier results that T2 is dominated by the overall motion of the glycogen particles [L.-H. Zang Biochemistry 29, 6815-6820 (1990)]. T1 depends strongly on the external field Bo and is almost temperature independent in the range 23-37 degrees C whereas T2 is field independent and varies appreciably with temperature. The experimental T1 and T2 relaxation data are shown to be consistent with existing theoretical models for relaxation, suitably modified to include a distribution of correlation times for the internal motions. The presence of fast decaying components (short T2) in the FID implies broad line components in the frequency spectrum and the corresponding need to appropriately set the integration limits for the quantification of the glycogen peak.

We perfused livers from fed rats with a balanced salt solution containing 1 mmol/L glucose. Under these conditions a low steady rate of glycogenolysis was observed (approximately 1.7 micromol glucose equivalents/g/min; 20% of the maximal glycogenolytic activity). Nitric oxide (NO) transiently stimulated hepatic glucose production. A maximal response (on average doubling basal glucose output) was observed with 34 micromol/L NO. The same concentration of nitrite (NO 2 - ) was ineffective. Half-maximal effects were seen at 8 to 10 micromol/L NO, irrespective of the flow direction (portocaval or retrograde). This glycogenolytic response to NO corresponded to a partial activation of phosphorylase. The NO effect was not additive to maximal stimulation of glycogenolysis (7.7 +/- 0.2 micromol hexose equivalents/g/min; n = 4) by 100 micromol/L dibutyryl cyclic adenosine monophosphate (Bt 2 cAMP). The requirement for activation of phosphorylase was also evidenced by the ineffectiveness of NO in phosphorylase-kinase-deficient livers of gsd/gsd rats. The NO effect was blocked by co-administration of cyclooxygenase inhibitors (50 micromol/L ibuprofen, 50 micromol/L indomethacin, or 2 mmol/L aspirin), suggesting a mediatory role of prostanoids from nonparenchymal cells. This conclusion was confirmed by the fact that NO did not activate phosphorylase in isolated hepatocytes. Moreover, NO was no longer glycogenolytic in livers perfused with Ca 2 + -free medium, in agreement with the known mediatory role of Ca 2 + in prostanoid-mediated responses. Surprisingly, in Ca 2 + - free medium NO inhibited the basal glucose production. This coincided with an increased elution of cyclic guanosine monophosphate (cGMP). Inhibition of glycogenolysis by NO under these conditions was blocked by 1 mmol/L theophylline, suggestive for involvement of cGMP-stimulated cAMP phosphodiesterase. However, we could not confirm that an increase in cGMP resulted in a drop in cAMP. In conclusion, NO recruits opposing mechanisms with respect to modulation of basal hepatic glycogenolysis. In the presence of Ca 2 + , activation of phosphorylase with stimulation of glycogenolysis dominates. Cyclooxygenase inhibitors abolish this effect. Activation by NO of the cyclooxygenase in nonparenchymal cells is a distinct possibility. In the absence of Ca 2 + , inhibition of basal glycogenolysis becomes observable. It remains to be established whether this results from cGMP-mediated stimulation of hydrolysis of cAMP. (Hepatology 1996 Jun;23(6):1564-71)

This study aimed to compare the effects of oral creatine (Cr) supplementation with creatine supplementation in combination with caffeine (Cr+C) on muscle phosphocreatine (PCr) level and performance in healthy male volunteers (n = 9). Before and after 6 days of placebo, Cr (0.5 g x kg-1 x day-1), or Cr (0.5 g x kg-1 x day-1) + C (5 mg x kg-1 x day-1) supplementation, 31P-nuclear magnetic resonance spectroscopy of the gastrocnemius muscle and a maximal intermittent exercise fatigue test of the knee extensors on an isokinetic dynamometer were performed. The exercise consisted of three consecutive maximal isometric contractions and three interval series of 90, 80, and 50 maximal voluntary contractions performed with a rest interval of 2 min between the series. Muscle ATP concentration remained constant over the three experimental conditions. Cr and Cr+C increased (P < 0.05) muscle PCr concentration by 4-6%. Dynamic torque production, however, was increased by 10-23% (P < 0.05) by Cr but was not changed by Cr+C. Torque improvement during Cr was most prominent immediately after the 2-min rest between the exercise bouts. The data show that Cr supplementation elevates muscle PCr concentration and markedly improves performance during intense intermittent exercise. This ergogenic effect, however, is completely eliminated by caffeine intake.

31P-NMR spectroscopy has become the major tool for studying myocardial high energy phosphates. Conflicting results concerning NMR visibility of ATP in ischaemic myocardium were reported. A detailed study was undertaken to resolve this controversy. After cardioplegic arrest, canine hearts were excised and preserved for 24 h at 1 degree C (group 1) or for 6h at 23 degrees C (group 2). ATP breakdown was followed by 31P-NMR spectroscopy in a transmural piece of the anterior wall introduced in the NMR magnet, and by HPLC analysis using serial transmural biopsies from the rest of the anterior wall. At both temperatures, identical relative ATP decay curves were obtained, whether measured by NMR or by HPLC. Absolute quantification of ATP was carried out after varying periods of ischaemia at 1 degree C. The NMR-measured ATP concentration was 106 +/- 8% of the ATP concentration determined by HPLC. From our experiments, we conclude that ATP visibility for 31P-NMR spectroscopy is complete and constant during prolonged periods of hypothermic ischaemia in canine hearts.

We report on the 13C NMR visibility of the C-1 glycosidic carbon of alpha-particulate glycogen in perfused rat liver. We used rats fed ad libitum, animals refed after a 48 h fast with a sucrose supplement with or without glucocorticoid treatment, and gsd/gsd rats with a hepatic glycogen storage disease due to phosphorylase kinase deficiency. Thus we studied a wide range of glycogen levels (25-140 mg/g liver). All livers were perfused with 15 mM glucose, to maintain constant glycogen levels. Failure to activate glycogen phosphorylase ensures stable glycogen levels in gsd/gsd livers. Natural abundance 13C NMR signals were calibrated against a phantom containing a fixed amount of glycogen. Accumulated free induction decays were analysed after Fourier transformation by numerical integration, or by direct analysis of the signal in the time domain using a non-iterative method based on singular value decomposition. NMR quantification of the glycogen correlated well with the chemical determination over the whole concentration range. However, the precision (reproducibility) of glycogen determinations (be it by chemical methods or by NMR spectroscopy) may pose problems. Authors should be encouraged to report systematically on the precision of their methods.

Ex vivo cardiac preservation was evaluated by measuring the catabolism of high-energy phosphate (ATP and creatine phosphate, CrP) using 31P-NMR spectroscopy. After cardioplegic arrest St. Thomas' Hospital cardioplegic solution (group A), and University of Wisconsin (UW) preservation solution (group B) were tested. The hearts were mounted in the 4.7 T horizontal bore magnet of the NMR spectrometer and were continuously perfused with the test solution under 25 cm H2O pressure for 6 h at 10 degrees C. Peak heights of the beta-phosphate of ATP and CrP were measured and expressed as percentages of the initial value. For both group A and group B. ATP declined less rapidly during preservation than CrP. In group A, ATP remained constant for 60 min while CrP decreased from the onset of preservation. After 6 h of preservation 28.3% of ATP and 24.5% of CrP remained (group A). On the other hand, in group B, levels of both ATP and CrP remained much more stable: CrP did not decrease during the first 3 h of preservation, while ATP started to decrease after 5 h. At the end of preservation 76.1% of ATP and 71.5% of CrP were still present. We conclude that UW solution is superior to St. Thomas' Hospital solution for the preservation of high-energy phosphates during 6 h cardiac preservation with continuous hypothermic low-flow perfusion.

A radioassay for specific determination of the rates of UDP-glucuronic acid-dependent conversion of bilirubin into the two isomeric (C-8, C-12) bilirubin monoglucuronides and bilirubin diglucuronide is described and illustrated by its application to rat liver microsomes. The method is based on measurement of the relative amounts of radiolabel in unesterified bilirubin and its mono- and di-esterified reaction products after incubation with [14C]bilirubin as substrate. This analysis is performed by the alkaline-methanolysis procedure, combined with one of two t.l.c. systems developed in order to enhance the sensitivity, accuracy and precision of the radioassay. Results for rates of total bilirubin glucuronide formation obtained with the new assay and the standard enzyme assay based on the ethyl anthranilate diazo-method were identical. However, the sensitivity of the latter technique is approx. 10-fold lower than that of the radioassay.

1. The mechanism that underlies the induction of glycogen synthesis in the foetal rat liver by glucocorticoids was reinvestigated in conditions where the accumulation of glycogen is either precociously induced with dexamethasone or inhibited by steroid deprivation. It appears that glucocorticoids act as the physiological trigger for glycogen synthesis by inducing both glycogen synthase (a known effect) and its activating enzyme, glycogen synthase phosphatase. 2. The activity of glycogen synthase phosphatase in adult liver stems from the interaction of two protein components [Doperé, Vanstapel & Stalmans (1980) Eur. J. Biochem. 104, 137–146]. Two independent experimental approaches indicate that the cytosolic ‘S-component’ is already well developed in the foetal liver before the onset of glycogen synthesis. The manifold glucocorticoid-dependent increase in synthase phosphatase activity during late gestation must be attributed to the specific development of the glycogen-bound ‘G-component’.

We examined regulatory properties of bilirubin UDP-glucuronyltransferase in sealed RER (rough endoplasmic reticulum)- and SER (smooth endoplasmic reticulum)-enriched microsomes (microsomal fractions), as well as in nuclear envelope from rat liver. Purity of membrane fractions was verified by electron microscopy and marker studies. Intactness of RER and SER vesicles was ascertained by a high degree of latency of the lumenal marker mannose-6-phosphatase. No major differences in the stimulation of UDP-glucuronyltransferase by detergent or by the presumed physiological activator, UDPGlcNAc, were observed between total microsomes and RER- or SER-enriched microsomes. Isolated nuclear envelopes were present as a partially disrupted membrane system, with approx. 50% loss of mannose-6-phosphatase latency. The nuclear transferase had lost its latency to a similar extent, and the enzyme failed to respond to UDPGlcNAc. Our results underscore the necessity to include data on the integrity of the membrane permeability barrier when reporting regulatory properties of UDP-glucuronyltransferase in different membrane preparations.

Administration of 0.1 or 1 mg of prednisolone to fed mice caused a 5-fold activation of glycogen synthase in the liver after 3 h, without significant changes in the circulating levels of glucose or insulin, or in the hepatic concentration of cyclic AMP. Adrenalectomized fasted rats responded to Cortisol (10 mg) with an increased glycaemia and a progressive activation of hepatic glycogen synthase after 2–4 h, but without an increase in the very low insulinaemia. These results are incompatible with the prevailing hypothesis that glucocorticoids provoke hepatic glycogen synthesis through an extra secretion of insulin. It is discussed that the acute effect of glucocorticoids is to inhibit rather than stimulate the release of insulin.