Quantitative determination of the main glucose metabolic fluxes in human erythrocytes by 13C- and 1H-MR spectroscopy

J. Biochem. Biophys. Methods 39 (1999) 63–84

Quantitative determination of the main glucose metabolic fluxes in human erythrocytes by 13 C- and 1 H-MR spectroscopy a ,b ,

c

d

Irene Messana *, Francesco Misiti , Said el-Sherbini , Bruno Giardina a ,b , Massimo Castagnola b ,c a

Istituto di Chimica e Chimica Clinica, Facolta` di Medicina e Chirurgia, Universita` Cattolica, Roma, Italy b Centro di Studio per la Chimica dei Recettori e delle Molecole Biologicamente Attive, C.N.R., Roma, Italy c Istituto di Chimica Biologica, Universita` di Cagliari, Cagliari, Italy d Faculty of Veterinary Medicine, Mansoura University, Mansoura, Egypt Received 9 October 1998; received in revised form 15 December 1998; accepted 16 December 1998

Abstract Information displayed by homonuclear and heteronuclear spin-coupling patterns in 1 3 C- and H-MR spectra allowed us to identify the major lactate isotopomers produced either from [1- 1 3 C]-glucose or from [2- 1 3 C]-glucose by human erythrocytes. Relative concentrations of detectable isotopomers were determined by integrating the corresponding MR signals. The interpretation of these data in terms of the fractional glucose metabolised through glycolysis and pentose phosphate pathway was performed by a computer simulation of the metabolism that took into account metabolic schemes pertaining to glycolysis and to the F-type of pentose phosphate pathway. The simulation was organised in a way to anticipate the populations of the isotopomers produced from any precursor at a priori established metabolic steady state. By the simulation, 1

Abbreviations: B3, band 3 protein; CDB3, cytoplasmic domain of band 3; MR, magnetic resonance; MRS, magnetic resonance spectroscopy; PC, pentose cycle; PPP, pentose phosphate pathway; EMP, Embden– Meyerhof pathway (glycolysis); ISS, isotopomeric steady state; GIc6P, glucose 6-phosphate; Fru6P, fructose 6-phosphate; Fru(1,6)P2 , fructose 1,6-biphosphate; GraP, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; Rul5P, ribulose 5-phosphate; Rib5P, ribose 5-phosphate; Xul5P, xylulose 5-phosphate; Sed7P, sedoheptulose 7-phosphate; Ery4P, erythrose 4-phosphate; other abbreviations used are reported in Table 1. The two conventions suggested by London [1] were adopted for isotopomer nomenclature: i.e. the notations [1,2- 13 C]-lactate and / or L 12 indicate the lactate molecule containing two carbon-13 nuclei at positions 1 and 2; the notation L 0 indicates the unlabelled isotopomer *Corresponding author. Tel.: 139-06-30154215; fax: 139-06-3053598; e-mail: [email protected] 0165-022X / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S0165-022X( 99 )00005-6

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isotopomer populations were determined according to different values of pentose cycle, defined as the flux of glyceraldehyde 3-phosphate originating from pentose phosphate pathway at unitary glucose uptake. The populations of the isotopomers originating from [2- 1 3 C]-glucose were described by polynomials, and ratios between the polynomials were used in conjunction with 13 Cand 1 H-MR data to determine pentose cycle values. The knowledge of glucose uptake and of pentose cycle value allowed us to perform accurate measurement of the pentose phosphate pathway flux, of the hexokinase and phosphofructokinase fluxes as well as, indirectly, of the carbon dioxide production.  1999 Elsevier Science B.V. All rights reserved. Keywords: Erythrocytes; Glycolysis; Pentose phosphate pathway; Nuclear magnetic resonance; Metabolic simulation

1. Introduction Recently, we have accumulated experimental evidence suggesting that the oxygendependent modulation of erythrocyte metabolism is mediated by the competition of several glycolytic enzymes and deoxy-hemoglobin for the cytoplasmic domain of band 3 (CDB3) [2]. At high oxygen saturation, glycolytic enzymes are bound to CDB3 and, with respect to deoxygenated erythrocytes, more glucose is addressed towards pentose phosphate pathway (PPP) in order to produce high quantities of NADPH and to protect red cells against oxidative molecular modifications. On going towards erythrocyte deoxygenation, deoxy-hemoglobin binds to CDB3 allowing the release and the activation of glycolytic enzymes with a contemporary higher ATP production in order to provide proper quantities for the necessity of ion-pumps and enzymes [3]. In order to verify this oxygen-dependent modulation we needed a reliable and accurate method to establish even small differences in the erythrocyte glucose metabolic fluxes. Early methods quantified main erythrocyte glucose fluxes by using 14 C-labelled glucose precursors and by detecting either radio-labelled carbon dioxide or radiolabelled lactate [4]. These methods are characterised by a great error (more than 10%) and oblige the researcher to the precaution necessary for the manipulation of radioactive compounds. Although characterised by a low sensitivity with respect to methods that utilise radio-labelled compounds, 13 C-MR spectroscopy has been successfully applied to the study of cell metabolism [5,6]. The comprehensive view provided by MR spectroscopy permits to follow the course of specifically labelled atoms through metabolic pathways without isolating and degrading metabolites, such as required by radio-tracing experiments. Isotopomers generated in branched systems may provide information on metabolic fluxes if a quantitative relationship between labelling fate and pathway fluxes is established. This approach was utilised by Schrader et al. [7] that utilised incubations with [2- 1 3 C]-glucose for the determination of glucose fluxes in erythrocytes from normal subjects. The method is based on the experimental determination of the ratio between the MR signals of C-3 and C-2 of lactate and its connection to the fluxes of the glucose pathways by the following relationship:

I. Messana et al. / J. Biochem. Biophys. Methods 39 (1999) 63 – 84 13 C23 2PC ]]] 5 ]]] 13 1 1 2PC C22

65

(1)

where PC represents the pentose cycle, i.e. the flux of glyceraldehyde 3-phosphate originating from pentose phosphate pathway at unitary glucose uptake. This relationship was derived from that of Wood et al. [4] early established for [2- 1 4 C]-glucose and it is not strictly applicable to 13 C-MR data. In fact, the various lactate isotopomers having either C-3 or C-2 labelled positions may not be magnetically equivalent and thus may give different signals. For instance, C-3 of L 23 and L 123 isotopomers resonate distinctly from C-3 of L 3 and L 13 isotopomers, due to homonuclear C-coupling between C-2 and C-3. Moreover, lacking of opportune isotopomer simulation, the Schrader and colleagues [7] approach did not take profit of MR heteronuclear spin–spin couplings. Thus, in this study we describe a method that allows the measurement of erythrocyte principal glucose metabolic fluxes by 13 C-labelled glucose incubation. The method is based on the determination of different isotopomer MR signal ratios that are connected to PC values by a simulation that establishes their relationships. The simulation achieves this aim contemporarily using input–output equations and convergent series [5]. Since several ratios may be contemporarily determined from the same spectrum, this method allowed us to obtain measurements characterised by reliability and low experimental error.

2. Materials and methods

2.1. Materials [1- 1 3 C]- and [2- 1 3 C]-glucose were purchased either from Sigma (St. Louis, MO) or from CIL (Cambridge Isotope Laboratories, Wobum, MA). Methylene blue, Hepes and D 2 O containing 0.75% sodium-3-(trimethylsilyl)-[2,2,3,3-H 4 ]-1-propionate were purchased from Sigma (St. Louis, MO). All other analytical grade compounds were purchased either from Farmitalia-Carlo Erba (Milan, Italy) or from Sigma. Erythrocyte samples were obtained from informed healthy adult volunteers. Freshly drawn heparinized venous blood (10–25 ml) was treated within 20 min after collection.

2.2. Erythrocyte incubation Plasma separation was followed by four washings (1500 g) of erythrocytes with Hepes 25 mmol / l, NaH 2 PO 4 1 mmol / l, NaCl 110 mmol / l, KCl 5 mmol / l, MgCl 2 2 mmol / l and buffer adjusted with NaOH to pH 7.40 and 29065 mOsm / kg, measured by an Osmostat OM-6020 apparatus (Daiichikagakuco Ltd., Kyoto, Japan). The buffy coat together with part of the upper erythrocyte layer were removed and discarded after each washing. Washed erythrocytes were suspended in the buffer solution supplemented with 30 mmol / l labelled glucose to give 50% hematocrit. During vigorous stirring, the erythrocyte suspension was divided into various 1.0 ml samples (5–6 samples). The samples were incubated in a thermostatic shaker at 378C. Erythrocyte incubations in the presence of methylene blue were performed on samples supplemented 10 min after

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labelled glucose addition with 3.5 ml of 150 mmol / l methylene blue (final concentration 0.5 mmol / l). At fixed times, controls and methylene blue-treated samples, were checked for pH and then immediately mixed with an equal volume of cold 12% (w / w) HClO 4 solution. Denatured material was centrifuged for 10 min (2000 g) and the upper solution was submitted to 13 C-MR analysis. Subsequently, the solution neutralised with potassium carbonate was lyophilised and dissolved in 0.7 ml of D 2 O containing 0.75% sodium-3-(trimethylsilyl)-[2,2,3,3- 2 H 4 ]-1-propionate (used as reference peak, chemical shift50 ppm) and adjusted to pH 9.0 for 1 H- and 13 C-MR analysis. Before experiments plasticware, glassware and buffers were autoclaved. Each step of sample preparation was performed in a sterile chamber in order to avoid sample contamination.

2.3. MR spectroscopy MR measurements were performed by a Gemini 300 spectrometer (Varian, Palo Alto, CA) at 258C using a 5-mm diameter tube. 1 3 C-MR measurements were performed at 74.62 MHz and spectra were broadband decoupled from protons. A 458 pulse, an acquisition time of 0.8 and 1 s recovery between pulses were used. A line broadening of 1 Hz was applied before Fourier transformation. Under these conditions, i.e. fast pulsing and continuous decoupling, a correction must be made for differences in NOE and relaxation times. By measuring the peak areas from a sample of 5 mM [1,2,3- 1 3 C]lactate in D 2 O brought to pH 9.0 by potassium carbonate and using the pulsing conditions described above, we have found that peak areas of 1 3 C-3 and 1 3 C-2 signals were in the ratio of 0.9, and this factor was used to correct the peak area ratio ( 13 C-3 / 13 C-2) in all spectra. 1 H-MR measurements were performed at 300 MHz, using 2 s acquisition time, 578 pulse, 12 s repetition time, spectral width 15 ppm. Irradiation was applied between acquisitions in order to pre-saturate the residual water peak. An accumulation of about 120 scans was required to achieve a satisfactory signal to noise ratio. No line broadening was applied before Fourier transformation. To calibrate 1 H-MR spectra, a sample containing 1:1 mixture of [1,2,3- 13 C]-lactate (2 mg / ml) and unlabelled lactate (2 mg / ml) dissolved in D 2 O (pH 9.0; potassium carbonate) was 13 12 13 12 prepared. Peak areas of the proton signals of CH 3 , CH 3 , CHOH and CHOH of lactate were measured on spectra recorded using different interpulse delays. 1 H-MR peak areas measured using 1 s of interpulse delay gave an overestimated (2%) 13 C / 12 C ratio for the methyl group and an underestimated one (20%) for the CHOH group. The difference between expected versus measured values decreased according to delay increase, becoming negligible when an interpulse delay of 12 s was used. To determine glucose uptake, a sealed capillary containing a 50% D 2 O / methanol (v / v) solution was used (methanol resonance was also used as a chemical shift reference, 49.0 ppm). Glucose uptake (G) was measured on perchloric acid extracts by following the timecourse of 13 C-MR signals of a and b anomers of labelled glucose. 1 3 C-MR glucose signals were integrated and normalised with respect to the integral of the methanol used as external standard. Since the nanomoles of glucose initially present in the sample were known, it was possible to measure the nanomoles of glucose still present at different incubation times. Glucose uptake was noted to be approximately linear until the end of

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incubation. Pentose cycle was determined after 30 h of incubation on control erythrocytes and after 20 h on methylene blue-treated erythrocytes, in consideration of the different glucose uptake shown in the two different conditions.

2.4. Simulation of the isotopomer generation According to a simplified metabolic scheme, erythrocytes convert glucose into lactate using two different pathways, the glycolysis (or Embden–Meyerhof pathway) and the F-type pentose phosphate pathway [8]. Fig. 1 shows the metabolic network utilised to perform the simulation. The scheme considers only the reactions that, providing a transformation of the carbon skeleton, can be held responsible for labelled atom randomisation. In this respect, the relevant enzymatic reactions are those of aldolase (Ald), 6-phospho-gluconate dehydrogenase (6 PGLDH ), transketolase I and II (TKI and TKII) and transaldolase (TA). In Fig. 1 the double-headed arrow, connecting all the triose sources to the triose utilised by transaldolase, indicates that this enzyme may also utilise triose isotopomers produced by the action of aldolase as substrates. Thus, this is the main reaction responsible for labelling randomisation. According to the network of Fig. 1, five pools containing metabolic intermediates, and three tanks containing glucose (G), carbon dioxide (C) and lactate (L) were considered. Each pool included all the metabolites having the same carbon atom chain (S: sedoheptulose; H: hexoses; P: pentoses; E: erythrose; T: trioses), corresponding to the assumption that the molecules of a pool are equilibrated faster than they are transferred from one pool to another. The symbols used to define the pools and tanks are

Fig. 1. Schematic presentation of the metabolic network and metabolite isotopic transformations considered in the simulation of the erythrocyte metabolism. In the scheme molecules are represented as numerical series with a number of terms corresponding to the chain length. Aldolase: Ald; 6-phosphogluconate dehydrogenase: 6 -PGLDH; transketolase I and II: TK I and TK II; transaldolase: TA. The other symbols are explained in Table 1.

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Table 1 Pools and tanks of metabolites considered in the simulation Abbreviation

Class

The class comprises

G H

Initial tank Hexose pool

T

Triose pool

P S E L C

Pentose pool Sedoheptulose pool Erythrose pool Final tank Final tank

Glucose Glc6P, Fru6P, Fru(1,6)P2 , all the hexose Phosphates of the oxidative part of PPP GraP, DHAP, all the trioses of the ATP-generating part of glycolysis, except lactate Rul5P, Rib5P and XuI5P Sed7P Ery4P Lactate Carbon dioxide

Metabolites with the same number of carbon atoms were included in the same pool.

summarised in Table 1. Fluxes at steady state of the different metabolic intermediates as a function of pentose cycle are reported in Table 2. They were derived by considering the model formulated in 1960 by Wood et al. [4], which defined the pentose cycle as the flux of glyceraldehyde 3-phosphate generated by pentose phosphate pathway at unitary glucose uptake (Fig. 1). On the basis of this definition, pentose phosphate pathway flux corresponds to three times the pentose cycle. In fact, two thirds of the glucose molecules metabolised in the oxidative portion of pentose phosphate pathway are re-transformed into hexose phosphates, and only one third produces glyceraldehyde 3-phosphate and carbon dioxide in the ratio 1:3. Assuming that G is the observed glucose uptake and that glucose is exclusively metabolised through the Embden–Meyerhof pathway and pentose phosphate pathway, glucose fluxes through the two pathways correspond to (1-PC)?G and 3?PC?G, respectively. The total flux of hexose phosphates corresponds to (112? PC)?G. Simulation was performed in order to establish the isotopomeric distribution of each

Table 2 Total fluxes throughout the pools Pool (p) or tank (t)

At steady state

G (t) H (p) T (p) P (p) S (p) E (p) C (t) L (t)

G (nmoles / min / ml) (112PC)?G (2-PC)?G 3?PC?G PC?G PC?G 3?PC?G (2-PC)?G

The table reports the flux of the molecules between the pools at steady state and does not consider the distribution among the different isotopomers.

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metabolite produced by any labelled precursor at imposed pentose cycle values. A glucose uptake of 1 000 000 molecules / min, corresponding approximately to the mean number of molecules per minute per mean erythrocyte, established on the basis of the haematological data reported in Table 3, was assumed (but in the simulation may be freely changed). The simulation was organised on a spreadsheet with a number of pages equal to the total number of pools and tanks. Each page contained a number of columns corresponding to the number of isotopomers of the pool (2 n , where n is the number of carbon atoms) and a number of rows, which was variable and imposed by the number of metabolic transformations (steps) necessary to reach the ISS. The passage from the n-row to the (n11)-row corresponded to the simultaneous occurrence of all the metabolic transformations considered in the network. The simulation is equivalent to a set of differential equations solved at integer differences. The simulation started with the introduction of a set number of glucose molecules having the desired labelling into the proper box in the first row. The number of steps necessary to reach ISS depended principally upon the pentose cycle value. Each new row was created by filling the appropriate boxes with new glucose molecules derived from the external tank and with the isotopomers generated from the metabolites present in the previous row. The quantity of each isotopomeric reaction product was established on the basis of the following considerations: 1. the fractional glucose flux through the two pathways considered in the network (established on the basis of the PC value) 2. the stoichiometry of the reactions; 3. the populations of the various isotopomers of each pool in the previous rows; 4. the topological characteristic of the reactions responsible for the formation of the particular isotopomer. We have utilised the simulation to determine the population of each isotopomeric metabolite at different pentose cycle values, which was varied from 0 up to 1 by 0.01 unit steps. The simulation utilised in this study can be requested by e-mail from the corresponding author.

Table 3 Mean hematological values utilised in the simulation Erythrocyte volume Packed cell volume Total volume Total erythrocytes Glucose concentration Total glucose molecules Glucose molecules / erythrocyte Mean glucose consumption (pH 7.4) Glucose uptake (G)

87 fl (87310 215 l) 50% 1.0 ml 6.0310 9 15 mM 9.0310 1 8 1.5310 9 20 nmoles /(min?ml (packed cells)) 1310 6 molecules /(min?erythrocyte)

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3. Results

3.1. MR spectra: [2 - 1 3 C] -glucose In Figs. 2 and 3, the 13 C- and 1 H-MR spectra of the lyophilised extract of erythrocytes incubated in the presence of [2- 13 C]-glucose are shown. The major peaks detectable in the 13 C-spectrum were assigned to C-2 of [2- 13 C]-D-glucose a and b anomers (d 574.31 and 76.97 ppm, respectively) and to C-1, C-2, C-3 (d 5185.32, 71.34 and 22.95 ppm, respectively) of the most abundant labelled lactates, namely L 2 , L 3 and L 13 . Moreover, signals due to Hepes natural abundance carbon-13 (d 561.60, 60.98, 54.95, 54.76, 54.23,

Fig. 2. Typical 1 3 C-MR spectrum of perchloric acid extracts of erythrocytes incubated with [2- 1 3 C]-glucose. Acid extract was neutralised, lyophilised and dissolved in D 2 O. In the figure the signals of the methyl group of L 3 and L 13 , of the carboxylic group of L 13 are evident, as well as the signal of the methyne group of L 2 , that are the major labelled isotopomers present in the sample.

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Fig. 3. Typical 1 H-MR spectrum of perchloric acid extracts of erythrocytes incubated with [2- 1 3 C]-glucose. Acid extract was neutralised, lyophilised and dissolved in D 2 O. In the figure the signals of the methyl group of L 0 , L 2 , L 3 and L 13 are evident as well as the signals of the methyne group of L 0 and L 2 , that are the major isotopomers present in the sample.

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50.36 ppm) were present. 1 H-MR spectra (registered at pH 9.0) appeared more complex. In the spectrum the predominant peaks were those of Hepes (d 3.74 ppm, triplet, J56.2 Hz; d 3.13 ppm, multiplet; d 2.82 ppm, multiplet; d 2.59 ppm, triplet, J56.2 Hz) and of residual [2- 1 3 C 1 ]-glucose. H-1 of a and b glucose anomers resonated at 5.24 (doublet, J53.6 Hz) and 4.65 ppm (doublet, J57.9 Hz), respectively, whereas H-3, H-4, H-5 and H 2 -6 protons appeared as multiplets in region 3.7–3.9 ppm (partially overlapped by Hepes triplet) and 3.4–3.5 ppm. H-2 of glucose anomers should be split by additional heteronuclear coupling with 13 C-2 (see Fig. 3), but only half of the signal was observed, due to overlapping with other signals. They appeared as a double-doublet at 3.30 ppm (JH-2,H-1 53.6 and JH-2,H-3 59.5 Hz, a anomer) and as a broad triplet at 3.00 ppm (JH-2,H-1 and JH-2,H-3 59.0 Hz, b anomer). The remaining signals of the spectrum belonged to erythrocyte metabolites and were mainly represented by the protons of lactate isotopomers. In the region around 1–2 ppm, where the methyl group resonates, a double-triplet centred at 1.33 ppm (Fig. 3C) and two double-triplets centred at 1.12 and 1.54 ppm (Fig. 3D and E) were detected. The double-triplet at 1.33 ppm originated from the overlapping of the signals of all the C-3 unlabelled lactates. In fact, the lactate methyl group appears as a doublet (JH-3,H-2 56.9 Hz) when it does not undergo additional coupling with either 13 C-1 or 1 3 C-2 (L 0 ), as a double-doublet if either C-2 or C-1 atoms are labelled (L 2 or L 1 , JH-3,C-2 5 JH-3,C-1 54.4 Hz, respectively) and as a double-triplet if C-2 and C-1 are both labelled (L 12 ). The two double-triplets at 1.12 and 1.54 ppm (Fig. 3E and D), were assigned to the methyl group of all the C-3 labelled lactates, since the signal was additionally split by coupling with 13 C-3 (JH-3,C-3 5128.0 Hz). The signals of the lactate methyne proton were observed in the region 4.1–4.4 ppm. In this case only two quartets appeared, namely a quartet at 4.11 ppm (JH-2,H-3 56.9 Hz) belonging to L 0 , the main isotopomer unlabelled at C-2 position, and a quartet at 4.35 ppm, corresponding to half of the signal due to L 2 , the main isotopomer labelled at C-2 position (JH-2,C-2 5158.0 Hz). The higher-field quartet of L 2 in fact overlapped with the glucose signals. In order to better explain these signal attributions, Figs. 4 and 5 report the complete coupling patterns of methyl and methyne protons that should be observed analysing a mixture of equal amounts of all the possible lactate isotopomers. Finally, the doublet at 1.42 ppm and the singlet at 1.92 ppm were assigned to alanine and acetate methyl groups, respectively. Quantification of the various isotopomers of lactate generated by erythrocyte metabolism were determined by integrating the corresponding 1 3 C- and 1 H-MR peaks. Data were not corrected for natural abundance of carbon-13 (1.1%). The following 1 H-MR signals were integrated: a. the signal centred at 4.35 ppm (wide 14.1 Hz), originating from all the C-2 labelled lactates, namely L 2 , L 12 , L 23 , L 123 ; b. the signal centred at 4.11 ppm (wide 14.1 Hz), originating from all the C-2 unlabelled lactates (L 0 , L 1 , L 3 , L 13 ); c. the 1.12 and 1.54 ppm signals (each one wide 15.7 Hz), originating from all the C-3 labelled lactates, namely L 3 , L 13 , L 23 , L 123 ; d. the signal centred at 1.33 ppm (wide 15.7 Hz), originating from all the C-3 unlabelled lactates (L 0 , L 1 , L 2 , L 12 ); e. the two doublets centred at 1.32 and 1.34 ppm, originating from the mono-labelled lactates either at C-1 or at C-2 position, namely L 1 , L 2 ;

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Fig. 4. The scheme reports the complete coupling patterns of methyl protons that are observed analysing a mixture of equal amounts of L 0 , L 1 , L 2 and L 12 , the signal is centred at 1.33 ppm. L 3 , L 1 3 , L 2 3 and L 1 2 3 show pattern of coupling identical to L 0 , L 1 , L 2 and L 1 2 , respectively, but they are further split into two signals centred at 1.12 and 1.54 ppm by coupling with 1 3 C-3 (JH3-C3 5128.0 Hz, see Fig. 3) and they are not shown. The coupling constant values are: JH2-H3 56.9 Hz; JH3-C1 54.4 Hz; JH3-C2 54.4 Hz.

f. the doublet centred at 1.33 ppm, originating from the unlabelled lactate (L 0 ) plus one half of bi-labelled lactates at C-1 and C-2 positions (L 12 ) (since only half of the signal of bi-labelled lactates is overlapped by that of unlabelled lactates). The integration of these MR signals allowed us multiple isotopomer determinations that permitted redundant connections to the metabolic fluxes. Since we observed that systematic errors might originate either from the manipulation of the sample or the presence of high reticulocyte amounts, the integrals were not directly connected to absolute isotopomer concentrations, but used as ratios to calculate their relative concentrations. A generic integral ratio was defined as u. It is usually obtained from the ratio between the integrals of the isotopomer of a particular carbon atom with respect to

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Fig. 5. The scheme reports the complete coupling patterns of methyne protons that are observed analysing a mixture of equal amounts of L 0 , L 1 , L 3 and L 1 3 , the signal is centred at 4.11 ppm. L 2 , L 12 , L 2 3 and L 1 2 3 show coupling pattern identical to L 0 , L 1 , L 3 and L 1 3 , respectively, but they are further split into two signals centred at 4.35 and 3.87 ppm by coupling with 1 3 C-2 (JH2-C2 5158.0 Hz, see Fig. 3) and they are not shown. The coupling constant values are: JH2-H3 56.9 Hz; JH2-C1 53.6 Hz; JH2-C3 53.6 Hz.

the proper unlabelled isotopomers. From 1 H-MR data of [2- 13 C]-glucose incubations, the following ratios were calculated: 2 u2total obtained by dividing the doubled integral of signal (a), since only one satellite quartet was detectable, by that of signal (b): (L 2 1 L 12 1 L 23 1 L 123 ) 2 ? (a) u2total 5 ]] 5 2 ? ]]]]]]] L 0 1 L 1 1 L 3 1 L 13 (b)

2

2

u3total obtained by dividing the integral of signal (c) by that of signal (d): (c) L 3 1 L 13 1 L 23 1 L 123 u3total 5 ] 5 ]]]]]]] L 0 1 L 1 1 L 2 1 L 12 (d)

2

2

u1,2 obtained by dividing the integral of signal (e) by that of signal (f): L1 1 L2 (e) u1,2 5 ] 5 ]]]]. (f) L 0 1 1 / 2 ? L 12

2

The subscript of the symbol recalls the lactate isotopomers present in the numerator of

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the ratio, whereas the superscript recalls glucose labelling. Moreover, from 13 C-MR spectra the ratio between the integrals of the signals at 22.95 ppm and 71.34 ppm was calculated and named 1 3 C-3 / 1 3 C-2 ratio.

3.2. MR spectra: [1 - 13 C] -glucose The major peaks detectable in the 13 C-MR spectrum were those pertaining to C-1 of [1- 13 C]-D-glucose a and b anomers (d 593.11 and 96.90 ppm, respectively) and to C-3 of [3- 13 C]-lactate. In the 1 H-MR spectrum the signals due to residual glucose, to Hepes and to lactates labelled at C-3 (L 3 ) and unlabelled ones (L 0 ) were observed. Thus, the unique ratio measured was 1u3 .

3.3. Determination of metabolic fluxes from [2 - 1 3 C] -glucose incubation data MR data obtained from erythrocytes incubated with [2- 13 C]-glucose and simulation results offered different ways of determining the relative fluxes of glucose through the Embden–Meyerhof pathway and pentose phosphate pathway. The first relied on the measurement of the ratio of lactate labelled at C-3 and C-2 ( 13 C-3 / 13 C-2) from 13 C-MR data (Fig. 2). Wood et al. [4] indicated that the 14 C-1 / 14 C-2 ratio of hexoses 6-P produced in experiments carried out using radio-labelled [2- 14 C]-glucose can be related to pentose cycle (and thus to pentose phosphate pathway flux) by simple formulations. Schrader et al. [7] used a similar approach to determine pentose cycle by measuring the integrals of the 13 C-3 and 13 C-2-MR signals of the lactates produced by the metabolism. Since C-1 of hexose 6-P corresponded to C-3 of lactate, the relationship used was the Eq. (1) reported in Section 1. Simulation results indicated that this relationship is precisely respected if all the lactates labelled at C-3 and at C-2 are considered, as happens in degradative radio-tracing 1 4 C experiments. Simulation indicated that relationship (1) may be used to produce satisfactory results only when pentose cycle assumes values lower than 0.05. A more correct relationship that connects pentose cycle values to the 1 3 C-3 / 1 3 C-2 ratio is: 13 C23 1.856 ? PC ]]] 5 ]]]]. 13 C 2 2 1 1 1.817 ? PC

(2)

This relationship was obtained by applying multiparametric least-square fitting procedures to the theoretical curve (Fig. 6) that describes the dependence of the 13 C-3 / 1 3 C-2 ratio from the pentose cycle value. This curve was obtained by the simulation, incrementing the pentose cycle value by 0.01 units and considering only the lactate isotopomers that contribute to the considered 13 C-MR signals, namely L 3 and L 13 for the 1 3 C-3 signal and L 2 and half of L 123 for the 1 3 C-2 signal. Table 4 reports the pentose cycle values obtained by utilising this equation and 13 C-MR data from a typical incubation experiment. PC values are affected by an approximate 10% error estimated from the integral errors. As already reported, due to the higher sensitivity of 1 H-MR spectroscopy with respect to 13 C-MR spectroscopy, 1 H-MR spectra allowed us to determine several additional

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Fig. 6. Theoretical 1 3 C-3 / 1 3 C-2 lactate ratios from [2- 1 3 C]-glucose calculated according to different discrete pentose cycle values (0.01 increments). Continuous line was obtained by considering only magnetically equivalent isotopomers (see text) and is described by Eq. (2). Dashed line was obtained considering all C-3 and C-2 labelled isotopomers and is described by Eq. (1).

ratios ( yuz ) between isotopomeric populations, namely 2u2total , 2u3total and 2u1,2 . Nonetheless, these ratios were complex functions of the pentose cycle value and could not be described by simple relationships like Eqs. (1) and (2). By best-fitting procedures applied to the populations determined by the simulation as a function of pentose cycle at unitary glucose uptake, we found that either each isotopomeric population or the sum of Table 4 Pentose cycle and D values obtained from

13

C- and 1 H-MR data and simulation results

Ratios obtained from MR signals

Methylene blue-treated

Control

13

0.3160.03 0.3660.03 0.09060.005 0.4160.03 0.3360.02

0.1660.02 0.5660.04 0.05460.002 0.6160.04 0.5460.04

0.2460.03 0.24460.002 0.3160.01

0.1060.02 0.10260.002 0.2560.01

8.160.2

7.960.2

13

C-3 / C-2 u2total 2 u3total 2 u1,2 1 u3 Calculated PC and D values Pentose cycle from 1 3 C-3 / 1 3 C-2 (Eq. 2) Pentose cycle (systems (7–8)) D (systems (7–8)) Intra-erythrocyte metabolites at the start of incubation (mmol / l) (Eq. 9) 2

I. Messana et al. / J. Biochem. Biophys. Methods 39 (1999) 63 – 84

77

various isotopomeric populations might be satisfactorily expressed by a third degree polynomial:

O l ? PC . i

l5

i

(3)

i

The coefficients of the polynomial (Eq. 3) were determined for the following populations (L 2 1L 12 1L 23 1L 123 ), (L 0 1L 1 1L 3 1L 13 ), (L 3 1L 13 1L 23 1L 123 ), (L 0 1 L 1 1L 2 1L 12 ), (L 1 1L 2 ), (L 0 11 / 2?L 12 ), by applying least-square fitting procedures on the curves obtained by the simulation and reported in Figs. 7 and 8. The coefficients obtained (l i ) are reported in Table 5. These coefficients were used in connection with 1 H-MR ratios to solve equation systems that allowed us to perform multiple measurements of the pentose cycle, as described below. Since molecules deriving from precursors present within the erythrocyte at the start of incubation (mainly 2,3-biphosphoglycerate) also contributed to 1 H-MR signals pertaining to unlabelled lactate, the intensity of these signals was incremented by a factor that we call D. Unlike the simulation data, experimental ratios (u ) included the contribution of D. Thus, the polynomial (Eq. 3) was modified in the expression (Eq. 4), and this polynomial was used to express the populations of unlabelled metabolites:

O u ? PC 1 D. i

u5

i

(4)

i

Fig. 7. Moles of lactate isotopomers produced per mole of [2- 1 3 C]-glucose consumed as a function of pentose cycle values (0.01 increments), calculated by the simulation. The curves comprise the isotopomers indicated.

I. Messana et al. / J. Biochem. Biophys. Methods 39 (1999) 63 – 84

78

Fig. 8. Moles of lactate isotopomers produced per mole of [2- 1 3 C]-glucose consumed as a function of pentose cycle values (0.01 increments), calculated by the simulation. The curves comprise the isotopomers indicated.

On these bases, the expression used to indicate a generic isotopomeric ratio was:

O

l i ? PC i ]]]]] ux 5 . u i ? PC i 1 D

y

O

(5)

which, upon algebraic transformation became:

OS]lu 2 u D ? PC 5 D. i

i

y

(6)

i

x

Table 5 Coefficients of polynomials (Eqs. 3 and 4) obtained by fitting the curves reported in Figs. 7 and 8 Lactate isotopomers from [2- 1 3 C]-glucose

l 3 or u 3

l 2 or u 2

l 1 or u 1

l 0 or u 0

L 2 1L 12 1L 23 1L 123 L 0 1L 1 1L 3 1L 13 L 3 1L 1 3 1L 2 3 1L 123 L 0 1L 1 1L 2 1L 12 L 1 1L 2 L 0 11 / 2?L 12

21.000 11.034 10.972 20.961 21.007 10.018

12.257 22.276 21.974 11.956 12.248 20.212

22.249 11.234 10.990 21.985 22.234 10.196

10.976 11.025 10.031 11.969 10.975 10.995

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79

This equation contains the two unknowns PC and D, which could be determined by simultaneously using two different experimental ratios:

OS]lu 2 u D ? PC 5 D l9 OS] 2 u 9D ? PC 5 D u

5

i

i

y

i

x

i

(7)

i

y

i

x9

This system could be solved by the following polynomial i grade equation: l9 O FS]lu 2 ] 2 (u 2 u 9 ) G ? PC 5 0. u D i

i

y

y

x

i

i

(8)

i

x9

The values of pentose cycle and D determined by Eqs. (7) and (8) and 1 H-MR data are reported in Table 4. Three ratios could be calculated from 1 H-MR data of [2- 1 3 C]-glucose incubation experiments, and thus three different equation systems and three different pentose cycle measurements could be obtained. The D value is inversely proportional to incubation time and to glucose uptake and increases according to the pentose cycle value, since it represents the contribution of unlabelled metabolites present within the erythrocyte at the beginning of the experiment to lactate production. This concentration can be calculated from D (Table 4) by the following equation: nmol initial (intra 2 erythrocyte) 5 D ? G ? t ? (2 2 PC)

(9)

The use of the solution of different systems displayed values characterised by a low percentage error and a good agreement between the concentration of the intra-erythrocyte metabolites in control and blue methylene-treated erythrocytes (7.9 and 8.1 mmol / l, respectively). D factor can be reduced by pre-incubating erythrocytes with labelled glucose and by discarding the products of pre-incubation. Even though this procedure was expensive in terms of labelled precursor waste and analysis time, we tested it, and the results obtained are reported in Table 6. As expected, under these experimental conditions the D factor was practically reduced to zero.

3.4. Determination of metabolic fluxes from [1 - 1 3 C] -glucose incubation data Using the simulation, we determined hexose fluxes at isotopomeric steady state in erythrocytes incubated with [1- 1 3 C]-glucose. It was found that the H 1 flux corresponds Table 6 Pentose cycle and D values obtained from erythrocyte samples pre-incubated with [2- 1 3 C]-labelled glucose Preincubated erythrocytes 2

u3total u1,2 Pentose cycle D 2

0.07360.004 0.7960.04 0.12060.009 0.00160.0003

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80

to G, and H 6 and H 0 total flux is equal to 2?PC?G (H 6 50.461?PC?G and H 0 51.539? PC?G, respectively). All the reactions of the pentose phosphate pathway responsible for the randomisation produced hexose labelled at C-6 position. Since the H 6 isotopomer generates only L 3 lactate, the latter was the sole labelled isotopomer produced by [1- 1 3 C]-glucose. At unitary glucose uptake, the flux of labelled carbon dioxide corresponded to: 13

3PC CO 2 5 ]]]. 1 1 2PC

(10)

The other labelled isotopomeric intermediates produced by the metabolism were P5 , S 7 , E 4 and T 3 . Thus, in the lactate tank only L 0 and L 3 were present, in a quantity corresponding to: 1 1 4PC 2 2PC 2 ]]]]] L0 5 1 1 2PC

(11)

1 2 PC L 3 5 ]]]. 1 1 2PC

(12)

and

Eqs. (11) and (12) were analytically derived, and the values assumed by L 0 and L 3 for different PC values are reported in Fig. 9. In incubation experiments performed using

Fig. 9. Moles of lactate isotopomers produced per mole of [1- 1 3 C]-glucose consumed as a function of pentose cycle values (0.01 increments), calculated by the simulation. The curves comprise the isotopomers indicated.

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81

Table 7 Metabolic fluxes of normal erythrocyte determined by MR measurements in a typical incubation experiment Metabolic fluxes (nmoles / min?ml a)

Control

Methylene blue-treated

Flux through hexokinase Flux through pentose phosphate pathway Flux through phosphofructokinase Triose flux CO 2 production Glyceraldehyde from pentose phosphate Pathway (pentose cycle flux)

9.360.2 2.760.1 8.460.3 17.760.5 2.760.1 0.960.1

12.460.4 8.860.3 9.560.3 21.960.7 8.860.3 2.960.2

a

Packed erythrocytes.

[1- 1 3 C]-glucose, L 3 was the sole lactate isotopomer detectable by 1 3 C-MR in acceptable experimental times. On the contrary, 1 H-MR spectra allowed us to determine the ratio between labelled and unlabelled atoms at C-3 position ( 1u3 ). Since the signal belonging to unlabelled lactates at C-3 comprised the unknown D factor, this ratio alone could not be used to determine the pentose cycle value. Nonetheless, in paired incubation experiments performed with [1- 13 C] and [2- 13 C]-glucose, it represented a fourth experimental MR-signal ratio that was used to generate six equation systems (7–8). The pentose cycle and D values obtained by applying these six systems to the data collected either from erythrocytes incubated under normal conditions or in the presence of methylene blue are reported in Table 4. The use of redundant measurements allowed a sensible reduction of percentage error. In fact the use of the six possible combinations of the four isotopomeric ratios provided a measurement characterised by a percentage error about five times lower than that obtained utilizing the 13 C-3 / 13 C-2 ratio alone (about 2 against 10%). Thereby, this experimental approach should be utilised when small variations are expected and, in particular, can be used to assess the metabolic modification produced by different incubation conditions (different ion activities, oxygenation–deoxygenation cycle). Table 7 reports phosphofructokinase, 6-phosphogluconolactone dehydrogenase and triose flux values together with CO 2 production, determined indirectly, and measured in a typical experiment performed on normal and methylene blue-treated erythrocytes. The values obtained indicate that the use of different precursors and different ratios produce comparable results. In particular, the method allowed us a very reliable measurement of the flux throughout the pentose phosphate pathway (6-phosphogluconolactone dehydrogenase flux) in erythrocytes submitted to a high oxidative stress. Thus, this method could be also of particular interest in the study of widespread erythrocyte pathologies deriving from glucose-6phosphate dehydrogenase defect.

4. Discussion The analysis of 13 C- and 1 H-MR signals assisted by a computer simulation of the metabolism allowed us to measure principal erythrocyte glucose fluxes with an experimental percentage error of about 2% (Table 4). Erythrocyte metabolism has been

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I. Messana et al. / J. Biochem. Biophys. Methods 39 (1999) 63 – 84

already studied using MR spectroscopy by various researchers [9–11]. Oxley et al. [9] have used multinuclear MR spectroscopy and [1- 1 3 C]-glucose in order to evaluate the flux through 2,3-biphosphoglycerate. These authors did not profit from isotopomeric analysis and indirectly determined an approximate value of the pentose phosphate pathway flux. Berthon et al. [10] and McIntyre et al. [11] utilised 31 P- and 13 C-MR spectroscopy in order to investigate thoroughly isotopic exchange reactions of the nonoxidative portion of the pentose phosphate pathway. Their study was performed on human erythrocyte hemolysates incubated with a mixture of universally 13 C-labelled and unlabelled glucose 6-phosphate and fructose 1,6-biphosphate. Unlike these researches [10,11], our study was devoted to MR measurements of the relative fluxes of Embden– Meyerhof and pentose phosphate pathway on intact human erythrocytes. Our experimental conditions were thus optimised to achieve this aim. Schrader et al. [7] have already used [2- 13 C]-glucose and MR spectroscopy to measure erythrocyte metabolic fluxes of glucose, but the proposed equation did not allow us to obtain sufficiently accurate measurement of the pentose cycle for values higher than 0.05 (see Fig. 6). This is the case of incubation experiments performed both on unstimulated erythrocytes and on cells exposed to substances like methylene blue, which enhance the pentose phosphate pathway [12]. Moreover, they did not profit from 1 H-MR information displayed by heteronuclear spin–spin coupling, whereas the present approach also permitted us to measure metabolic fluxes from 1 H-MR data. The simulated isotopomeric populations allowed us to determine the coefficients of third degree polynomials reported in Table 5, which can be utilised together with 1 H-MR data to determine pentose cycle and dilution (D) values. The consistency of the results obtained utilising different isotopomer ratios suggested that the network in Fig. 1 is satisfactory to describe principal glucose metabolic pathways in human erythrocyte. In other words, our results confirmed that the erythrocyte pentose phosphate pathway is adequately described by the F-type and that a contribution of either the L-type of pentose phosphate pathway or other metabolic schemes [8,13,14] can be considered negligible [10]. For example, we experimentally | 2% for a PC of 0.07) determined the quantity of L 12 produced by [2- 13 C]-glucose ( 5 and the value substantially agreed with the quantity anticipated by the simulation. The different approaches utilised in this study were not equivalent with respect to the difficulties connected to data collection and measurement sensitivity. Fig. 4 shows how the determination performed by 13 C-3 / 1 3 C-2 ratio becomes less sensitive as pentose cycle increases. The sensitivity of the measurements performed using 1 H-MR data depends on pentose cycle and D values. 2u1,2 , 2u2total and 1u3 ratios shows similar trends, progressively decreasing as a function of pentose cycle values, as suggested by the curves reported in Figs. 7–9. This behaviour may be explained by considering that 2u1,2 and 2u2total although obtained from distinct 1 H-MR signals, differs only in the contribution of minor isotopomers. 2u2total and 1u3 shows similar trends since they are obtained by taking into account the isotopomers originating throughout glycolysis from [2- 1 3 C]- and [1- 1 3 C]-glucose, respectively. On the contrary, 2u3total increases to about 0.3 pentose cycle values and subsequently slowly decreases. As a consequence, the most sensitive equation systems were those that coupled 2u3total with the other ratios. Among them, 2u1,2 was the easiest to determine since it was obtained from more intense signals. Thus, the best option was represented by the coupling of 2u3total and 2u1,2 ratios, especially when high pentose cycle values were expected.

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Dilution (D) produces lower ratios, since unlabelled isotopomers are always present in the denominator. The value of D obtained under different incubation conditions ranged between 45 and 20% of unlabelled lactate, depending upon incubation time, glucose consumption and PC value (Eq. 9). Global metabolite concentration within the erythrocyte calculated by Eq. (9) was approximately 8 mM. Since 2,3-biphosphoglycerate intra-erythrocyte concentration is about 4 mM, the results obtained indicated that the pool of other essential metabolites present within the erythrocyte and not eliminable by washing procedures accounted for 4–5 mM. Simulation showed that all the isotopomers of any metabolite, although in a very different percentage with respect to those originating from [2- 13 C]-glucose, can be generated from [3- 1 3 C]-glucose. The main lactate isotopomers generated from pentose phosphate pathway were L 2 and L 12 , whereas L 0 and L 1 were derived principally from the Embden–Meyerhof pathway. The high labelling accumulation at C-1 in lactate discouraged the use of [3- 1 3 C]-glucose as substrate, since the 1 3 C-MR signal intensity of the carboxylate atom is strongly affected by relaxation time. Moreover, C-1 is not proton-bound, and thus information deriving from heteronuclear couplings in 1 H-MR spectra is not obtainable. For these reasons we limited experimental study to incubation with [1- 13 C]- and [2- 1 3 C]-glucose. Moreover simulation indicated that the major lactate isotopomers produced by [4- 1 3 C]-, [5- 1 3 C]- and [6- 1 3 C]-glucose were L 1 , L 2 and L 3 , respectively. A small percentage of H 0 derived from sedoheptulose and unlabelled triose by the action of transaldolase. Isotopomeric lactate flux corresponded exactly to G, whereas L 0 flux was equal to G?(1-PC). Since labelled carbon dioxide is derived from the upper three carbon atoms of glucose, no labelled carbon dioxide was produced. Due to these properties, the use of [4- 1 3 C]-, [5- 1 3 C]- and [6- 1 3 C]-glucose does not seem to be advantageous for the study of human erythrocyte metabolism.

References [1] London RE. 13 C labelling in studies of metabolic regulation. Prog NMR Spectr 1988;20:337–83. [2] Messana I, Orlando M, Cassiano L, et al. Human erythrocyte metabolism is modulated by the O 2 -linked transition of hemoglobin. FEBS Lett 1996;390:25–8. [3] Giardina B, Scatena R, Messana I, et al. The multiple functions of hemoglobin. Crit Rev Biochem Mol Biol 1995;30:165–96. [4] Wood HG, Katz J, Landau BR. Estimation of pathways of carbohydrate metabolism. Biochem Z 1963;338:809–47. [5] Kunnecke B. Application of 1 3 C NMR spectroscopy to metabolic studies on animals. In: Beckmann N, editor. Carbon-13 NMR spectroscopy of biological systems. New York: Academic Press, 1995:159–267. [6] Katz J, Grunnet N. In: Kornberg HL editor. Techniques in metabolic research. Amsterdam: Elsevier, 1979:1–18. [7] Schrader MC, Eskey CJ, Simplaceanu V, et al. A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated with glucose metabolism in human erythrocytes. Biochim Biophys Acta 1993;1182:162–78. [8] Horecker BL. Unravelling the pentose phosphate pathway. In: Kornberg A, Cornudella L, Horecker BL, et al., editors. Reflections on Biochemistry. Great Britain: Pergamon, 1976:65–72. [9] Oxley ST, Porteous R, Brindle KM, et al. A multinuclear NMR study of 2,3-diphosphoglycerate metabolism in the human erythrocyte. Biochim Biophys Acta 1984;805:19–24. [10] Berthon HA, Bubb WA, Kuchel PW. 1 3 C NMR isotopomer and computer simulation of the non-oxidative pentose phosphate pathway of human erythrocytes. Biochem J 1993;296:379–87.

84

I. Messana et al. / J. Biochem. Biophys. Methods 39 (1999) 63 – 84

[11] McIntyre LM, Thorburn DR, Bubb WA, et al. Comparison of computer simulations of the F-type and L-type non oxidative hexose monophosphate shunts with 31 P-NMR experimental data from human erythrocytes. Eur J Biochem 1989;180:399–420. [12] Keitt AS. In: Stanbury JB, Wyngaargen JB, Frederickson DS, editors. The metabolic basis of inherited disease. New York: McGraw-Hill, 1972:1389–1397. [13] Williams JF, Clark MG, Blackmore PF. The fate of 14 C in glucose 6-phosphate synthesized from [1- 14 C]Ribose 5-phosphate by enzymes of rat liver. Biochem J 1978;176:241–56. [14] Williams JF, Blackmore PF, Clark MG. New reaction sequences for the nonoxidative pentose phosphate pathway. Biochem J 1978;176:257–82.