Julia E. Raftos

Glutathione Synthesis and Turnover in the Human Erythrocyte ALIGNMENT OF A MODEL BASED ON DETAILED ENZYME KINETICS WITH EXPERIMENTAL DATA

The erythrocyte is exposed to reactive oxygen species in the circulation and also to those produced by autoxidation of hemoglobin. Consequently, erythrocytes depend on protection by the antioxidant glutathione. Mathematical models based on realistic kinetic data have provided valuable insights into the regulation of biochemical pathways within the erythrocyte but none have satisfactorily accounted for glutathione metabolism. In the current model, rate equations were derived for the enzyme-catalyzed reactions, and for each equation the nonlinear algebraic relationship between the steady-state kinetic parameters and the unitary rate constants was derived. The model also includes the transport processes that supply the amino acid constituents of glutathione and the export of oxidized glutathione. Values of the kinetic parameters for the individual reactions were measured predominately using isolated enzymes under conditions that differed from the intracellular environment. By comparing the experimental and simulated results, the values of the enzyme-kinetic parameters of the model were refined to yield conformity between model simulations and experimental data. Model output accurately represented the steady-state concentrations of metabolites in erythrocytes suspended in plasma and the changing glutathione concentrations in whole and hemolyzed erythrocytes under specific experimental conditions. Analysis indicated that feedback inhibition of γ-glutamate-cysteine ligase by glutathione had a limited effect on steady-state glutathione concentrations and was not sufficiently potent to return glutathione concentrations to normal levels in erythrocytes exposed to sustained increases in oxidative load.

Role of N-acetylcysteine and cystine in glutathione synthesis in human erythrocytes

Abstract Glutathione is an intracellular antioxidant that often becomes depleted in pathologies with high oxidative loads. We investigated the provision of cysteine for glutathione synthesis to the human erythrocyte (red blood cell; RBC). Almost all plasma cysteine exists as cystine, its oxidized form. In vitro, extracellular cystine at 1.0 mM sustained glutathione synthesis in glutathione-depleted RBCs, at a rate of 0.206 ± 0.036 μmol (L RBC)−1min−1 only 20% of the maximum rate obtained with cysteine or N-acetylcysteine. In plasma-free solutions, N-acetylcysteine provides cysteine by intracellular deacetylation but to achieve maximum rates of glutathione synthesis by this process in vivo, plasma N-acetylcysteine concentrations would have to exceed 1.0 mM, which is therapeutically unattainable. 1H-NMR experiments demonstrated that redox exchange reactions between NAC and cystine produce NAC-cysteine, NAC-NAC and cysteine. Calculations using a mathematical model based on these results showed that plasma concentrations of N-acetylcysteine as low as 100 μM, that are attainable therapeutically, could potentially react with plasma cystine to produce ∼50 μM cysteine, that is sufficient to produce maximal rates of glutathione synthesis. We conclude that the mechanism of action of therapeutically administered N-acetylcysteine is to reduce plasma cystine to cysteine that then enters the RBC and sustains glutathione synthesis.

The effects of long-term storage of human red blood cells on the glutathione synthesis rate and steady-state concentration

BACKGROUND: Banked red blood cells (RBCs) undergo changes that reduce their viability after transfusion. Dysfunction of the glutathione (GSH) antioxidant system may be implicated. We measured the rate of GSH synthesis in stored RBCs and applied a model of GSH metabolism to identify storage‐dependent changes that may affect GSH production.

Triethyl phosphate as an internal 31P NMR reference in biological samples

Since the early 1970s 31P NMR has been widely applied to the study of biological systems. Many of the applications have entailed monitoring the variation in chemical shifts that arise as a result of various physical and chemical phenomena. The binding of a number of biological phosphates to macromolecules (e.g., 2,3-bisphosphoglycerate to deoxyhemoglobin) has been shown to cause a variation in the “P NMR chemical shifts of the phosphate groups (I-3). The variation in the “P NMR chemical shifts of titratable phosphorus compounds with pK, values in the physiological pH range has been used to estimate the pH in intact cells (4-8). More recently, the cell volume dependence of the chemical-shift separation between the intraand extracellular 31P NMR resonances of dimethyl methylphosphonate added to a red blood cell suspension has been used to monitor time-dependent changes in cell volume (9). The traditional chemical-shift standard for “P NMR is 85% phosphoric acid; however, it has a number of shortcomings (10, II): (i) it is restricted to use as an external reference; (ii) it gives rise to an inconveniently broad resonance (10); and (in) it has a bulk susceptibility very different from that of aqueous solutions (II). The tetrahydroxyphosphonium ion has been suggested as a variable alternative to phosphoric acid (10) but it too is restricted to use as an external reference. Although the use of an external shift reference eliminates all interactions between components of the sample and the reference compound it often necessitates the introduction of chemical-shift corrections to account for bulk susceptibility differences between the reference and sample (12). Such corrections are particularly important for samples containing hemoglobin, the susceptibility of which has been shown to vary widely with the degree of oxygenation (13, 7). The need for susceptibility difference corrections is obviated by using an internal reference compound, i.e., one added directly to the sample. Any such compound should fulfill a number of criteria (II): (i) it should be water soluble; (ii) it should preferably give rise to a single, narrow resonance, occurring well away from those of the sample; (iii) it should be chemically inert; and (iv), its chemical shift should be independent of the nature of the sample. Triethyl phosphate (TEP, (CH3CH20)3P0, BDH Chemicals, U.K.) has been shown to be suitable for use as an internal reference compound, particularly in the application of “P NMR to the study of red blood cell suspensions and lysates. TEP is an inexpensive, neutral, nontitratable compound which, with proton decoupling, gives rise

Glutamine and α‐ketoglutarate as glutamate sources for glutathione synthesis in human erythrocytes

Glutathione (GSH) is an intracellular antioxidant synthesized from glutamate, cysteine and glycine. The human erythrocyte (red blood cell, RBC) requires a continuous supply of glutamate to prevent the limitation of GSH synthesis in the presence of sufficient cysteine, but the RBC membrane is almost impermeable to glutamate. As optimal GSH synthesis is important in diseases associated with oxidative stress, we compared the rate of synthesis using two potential glutamate substrates, α‐ketoglutarate and glutamine. Both substrates traverse the RBC membrane rapidly relative to many other metabolites. In whole RBCs partially depleted of intracellular GSH and glutamate, 10 mm extracellular α‐ketoglutarate, but not 10 mm glutamine, significantly increased the rate of GSH synthesis (0.85 ± 0.09 and 0.61 ± 0.18 μmol·(L RBC)−1·min−1, respectively) compared with 0.52 ± 0.09 μmol·(L RBC)−1·min−1 for RBCs without an external glutamate source. Mathematical modelling of the situation with 0.8 mm extracellular glutamine returned a rate of glutamate production of 0.36 μmol·(L RBC)−1·min−1, while the initial rate for 0.8 mmα‐ketoglutarate was 0.97 μmol·(L RBC)−1·min−1. However, with normal plasma concentrations, the calculated rate of GSH synthesis was higher with glutamine than with α‐ketoglutarate (0.31 and 0.25 μmol·(L RBC)−1·min−1, respectively), due to the substantially higher plasma concentration of glutamine. Thus, a potential protocol to maximize the rate of GSH synthesis would be to administer a cysteine precursor plus a source of α‐ketoglutarate and/or glutamine.

Further investigation of the use of dimethyl methylphosphonate as a 31P-NMR probe of red cell volume

We have refined a method for measuring erythrocyte volume using the 31P-NMR spectrum of a probe molecule, dimethyl methylphosphonate. This compound, when added to an erythrocyte suspension, gives rise to two 31P-NMR resonances, and the frequency separation between them is linearly dependent on the intracellular haemoglobin concentration. If, for a given cell sample (under standard conditions), the separation of the two dimethyl methylphosphonate peaks has been measured and an independent estimation of the mean cell haemoglobin content and concentration has been obtained, then changes in the mean cell volume due to altered experimental conditions may be estimated from the peak separation measured under the new conditions. Although the peak separation was independent of extracellular pH, it did vary with (i) a range of extracellular suspension media, (ii) temperature, (iii) dimethyl methylphosphonate concentration, (iv) haemoglobin ligand state and (v) different blood donors.

Glutathione synthesis by red blood cells in type 2 diabetes mellitus.

Abstract Oxidative stress is implicated in the pathogenesis and complications of type 2 diabetes mellitus (NIDDM). Glycoxidation may damage the enzymes that synthesise glutathione (GSH), an endogenous intracellular antioxidant. Erythrocytes (RBCs) taken from NIDDM subjects, and non-diabetic controls, were GSH-depleted using 1-chloro-2,4-dinitrobenzene, incubated in a solution containing GSH-rebuilding substrates, and sampled for GSH using a 5,5′-γ-dithiobis-(2-nitrobenzoic acid)/enzymatic recycling procedure. NIDDM subjects, on average, had the same GSH concentration and synthesising ability as non-diabetic controls, indicating normal function of the synthesis enzymes. A positive correlation between synthesis and concentration of GSH seen in non-diabetic controls did not exist in NIDDM, due to their putatively larger oxidative load. The results, to the best of our knowledge, provide the first evidence that, despite a higher oxidative load, intact RBCs from NIDDM subjects are able to synthesise GSH normally. It is hypothesised that increased rates of GSH synthesis would maintain a normal steady-state GSH concentration.

The relationship between glucose concentration and rate of lactate production by human erythrocytes in an open perfusion system.

A thermodynamically open system, based on an assembly of capillaries with semi-permeable walls was constructed in order to study glycolysis in human erythrocytes in high haematocrit suspensions. A phenomenological expression for the rate of lactate production as a function of glucose concentration was obtained. The rate was measured under steady-state conditions with low substrate concentrations (approx. 50 mumol/l). In a corresponding closed system, this concentration of glucose would be exhausted within a few minutes. A mathematical model of the whole system consisted of five differential equations, and involved parameters relating to flow rates, volumes of reaction chambers, the rates of lactate efflux from erythrocytes and the expression for the rate of lactate production by red cells. The binding of [14C]pyruvate to haemoglobin and the rate of efflux of [14C]lactate from red cells were measured to yield additional information for the model. The concentrations of ATP and 2,3-bisphosphoglycerate were measured during the perfusion experiments, and a detailed analysis of a model of red cell hexokinase was carried out; the former two compounds inhibit hexokinase and alter the apparent Km and Vmax for glucose in vivo. These steady-state parameters were similar to the glucose concentration at the half-maximal rate of lactate production and the maximal rate, respectively. These findings are consistent with the known high control-strength for hexokinase in glycolysis in human red cells. The practical and theoretical validation of this perfusion system indicates that it will be valuable for NMR-based studies of red cell metabolism using a flow-cell in the spectrometer.

Oxidative Stress in Type II Diabetes Mellitus and the Role of the Endogenous Antioxidant Glutathione

Oxidative stress appears to be involved in aging and a great many diseases, including diabetes mellitus (Lang, Naryshin et al. 1992; Fletcher and Fletcher 1994; Julius, Lang et al. 1994; Richie, Skowronski et al. 1996; Nuttall, Martin et al. 1998; Lang, Mills et al. 2000; Erden-Inal, Sunal et al. 2002; Junqueira, Barros et al. 2004; Gil, Siems et al. 2006). ‘Oxidative stress’ is a term that was introduced by Sies in 1985 and refers to any situation where there is a serious imbalance between the production of free radicals (FR) or reactive oxygen species (ROS), called the oxidative load, and the antioxidant defense system. The oxidative load is described as “a measure of the steady-state level of reactive oxygen or oxygen radicals in a biological system” (Baynes 1991). Oxidative stress has been defined as “a disturbance in the pro-oxidant-antioxidant balance in favour of the former, leading to potential damage” (Sies 1985). Because it is hard to measure oxidative stress directly, it is inferred from the accumulation of oxidation products, such as plasma O2●radicals or high levels of peroxidation products such as thiobarbituric acid-reactive substances (TBARS) in plasma (Dominguez, Ruiz et al. 1998). Cells can tolerate moderate oxidative loads by increasing gene expression to up-regulate their reductive defense systems and restore the oxidant/antioxidant balance. But when this increased synthesis cannot be achieved due to damage to enzymes, or substrate limitations, or when the oxidative load is overwhelming, an imbalance persists and the result is oxidative stress. Persisting imbalance leads to damage to DNA, proteins and lipids, and cell death. Oxidative stress has been implicated in over 100 diseases, more as a consequence of the pathology than as the causative factor (Halliwell 2005). Glutathione, the tripeptide ┛-L-glutamyl-L-cysteinyl-glycine (GSH), is an antioxidant molecule synthesised in almost all living cells from prokaryote organisms to the eukaryote kingdoms (Griffith and Mulcahy 1999). Glutathione is able to protect cells from oxidation by virtue of the reducing power of the thiol group on the cysteine portion of the molecule. Normally ~98% of the total GSH in healthy human cells exists in the reduced form (Griffith 1981; Kennett, Bubb et al. 2005). When GSH is oxidised to glutathione disulphide (GSSG), the enzyme GSH reductase (GR; EC 1.6.4.2) rapidly reduces it back to GSH using NADPH as an electron donor, thus ensuring that the cycling of ROS does not alter the GSH to GSSG