J. Biochem. Biophys.

Methods 69 (2006) 79 – 87
www.elsevier.com/locate/jbbm

Determination of glucose metabolites in stored

erythrocytes and in erythrocytes from patients with
thalassemia by analytical isotachophoresis
Zyrafete Kuçi a , Jürgen Hins b , Selim Kuçi a , Susanne Renner a ,
Dirk Flottmann b , Gernot Bruchelt c,⁎
a
Children's University Hospital, Hoppe-Seyler-Str.1, D-72076 Tuebingen, Germany
b
Aalen University of Applied Sciences, D-73430 Aalen, Germany
c
Children's University Hospital Tuebingen / Medical University Graz, A-8010 Graz, Austria
Received 31 October 2005; received in revised form 23 February 2006; accepted 1 March 2006

Abstract

Glycolysis is for some cells, such as erythrocytes, neutrophil granulocytes and many cancer cells, the
only or most important source of energy (ATP) production. Based on previous studies we developed an
isotachophoretic (ITP) method which allows, in principle, the simultaneous determination of all metabolites
of glycolysis. Since glucose metabolites are small anions, mobility of some of them may overlap in
isotachophoresis and, therefore, partial mixed zones are generated. By variation of the leading/terminating
system, however, it is possible to separate the compounds of interest. In this communication, we describe a
method for analysis of glucose metabolites in erythrocytes from healthy donors during storage in blood
bags, and from patients with thalassemia, with special respect to intracellular 2,3 bisphosphoglycerate,
lactate and ATP/ADP. The well known characteristic changes of glycolysis in erythrocytes during blood
storage and in erythrocytes from thalassemia patients, which are often analysed by separate enzymatic
assays, could be confirmed with this isotachophoretic procedure. The method is currently adapted for
analysis of glycolysis in neutrophil granulocytes and cancer cells which requires some modifications of
sample preparation and performance of the isotachophoretic analysis.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Isotachophoresis; Glycolysis; Erythrocytes; 2,3 bisphosphoglycerate; Thalassemia; Erythrocyte storage

⁎ Corresponding author. Tel.: +49 70712984710; fax: +49 7071295482.

E-mail address: gernot.bruchelt@med.uni-tuebingen.de (G. Bruchelt).

0165-022X/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbbm.2006.03.002
80 Z. Kuçi et al. / J. Biochem. Biophys. Methods 69 (2006) 79–87

1. Introduction

In most human cells generation of cellular energy (production of ATP) is carried out by
oxidative phosphorylation: Glucose is metabolised to pyruvate which is further oxidized to
CO2 (citric acid cycle) and H2O. In this way more than 30 molecules of ATP are generated
from one molecule glucose (see textbooks of biochemistry). However, glycolysis (oxygen-
independent metabolism of glucose to lactate; in this way only 2 molecules of ATP are
generated from 1 molecule glucose) is the main pathway to generate ATP in some cells.
Among them are erythrocytes, neutrophil granulocytes [1], and many cancer cells which prefer
glycolysis even in the presence of oxygen (“aerobe glycolysis”, Warburg effect) [2]. In this
study we investigated glycolysis in erythrocytes during blood storage and from patients with
thalassemia by analytical isotachophoresis (ITP) [3–5]. Starting with glucose-6-phosphate, all
compounds of glucose metabolism are small anions which can, in principle, be analysed
simultaneously with this method [6]. Due to the high differences of their cellular
concentrations (μM to mM range) and similar electrophoretic mobilities of some of the
compounds (e.g. ATP and free phosphate), formation of mixed zones of unseparated
compounds are possible. Depending on a selected leading (L) and terminating (T) electrolyte,
it is possible to separate the molecules of main interest. Based on many data available in the
literature concerning the biochemistry [7] and analysis of glycolysis in erythrocytes [8–10],
we established recently a method for determination of glycolysis in erythrocytes from healthy
donors [11]. In the present study, we investigated glycolysis (with special respect to 2,3
bisphosphoglycerate, lactate and ATP/ADP) in erythrocytes from patients with thalassemia, a
hemoglobinopathy that in its severe form (e.g. β-thalassemia) needs lifelong blood
transfusions [12]. Additionally, we analysed the pattern of glycolysis in erythrocyte
concentrates stored in blood bags for up to 35 days. In the case of glycolysis in erythrocytes,
the most interesting molecule is 2,3 bisphosphoglycerate (2,3 BPG). Formation of this
substance is a specialty of erythrocytes; it is formed in a side pathway from 1,3
bisphosphoglycerate to 3-phosphoglycerate (the Lubbering–Rappaport pathway, see textbooks
of biochemistry). 2,3 BPG binds to hemoglobin, thereby facilitating the release of oxygen.
This is important for sufficient supply of tissue cells with oxygen.
The aim of our study was therefore to investigate whether these well known facts concerning
changes in glucose metabolites during blood storage or in cases of anemia [13,14] can be analysed
and confirmed by isotachophoretic analysis.

2. Materials and methods

2.1. Chemicals

Methylhydroxyethylcellulose (MHEC): Roth, (Karlsruhe, Germany); Terminating electrolyte

(caproic acid/histidine, pH ∼ 6.0): J and M (Aalen, Germany). ATP, ADP, lactate and 2,3 BPG
were from Sigma, Deisenhofen, Germany. All other chemicals were purchased from Merck,
Darmstadt, Germany.

2.2. Blood samples

Whole blood samples were either from healthy male donors (20–30 years old), or from
patients suffering from β-thalassemia, who have been routinely treated with transfusion of
 Z. Kuçi et al. / J. Biochem. Biophys. Methods 69 (2006) 79–87 81

erythrocyte concentrates at the Children's University Hospital in Tuebingen. Stored erythrocyte

concentrates (stabilisator: SAG-M; saline, adenine, glucose–mannitol) were obtained from the
Department of Transfusion Medicine, University of Tuebingen.

2.3. Isolation of erythrocytes and preparation of lysates

Erythrocyte concentrates: 20 ml erythrocyte concentrates (0.7 vol. erythrocytes + 0.3 vol.

SAG-M) were taken from blood bags and mixed with 30 ml 0.9% (m/v) NaCl solution. After
estimation of erythrocytes on the blood cell analyser ADVIA 120 (Bayer, Fernwald,
Germany), samples were centrifuged for 4 min at 1200 g at room temperature. Cell pellets
were resuspended in PBS (phosphate buffered saline), centrifuged, washed and finally adjusted
to 4.1 × 106 cells/μl 0.9% NaCl solution. After centrifugation, cell pellets were lysed (see
below).
Twenty millilitres of erythrocyte concentrates were taken from blood bags on days 1, 2, 5, 7,
12, 19, 23, 30 and 35. After drawing, blood bags were further stored at 4 °C. This procedure was
carried out with two separate blood bags showing similar results. Time-dependent changes during
storage are shown here only from one experiment.
Isolation of erythrocytes from blood samples: Heparinised blood samples were counted on
ADVIA 120, and subsequently centrifuged at room temperature (4 min, 1200 g). Cell pellets were
washed with 0.9% NaCl solution; finally the red cell pellets, contaminated with a neglectable amount
of leucocytes, were resuspended in phosphate buffered saline (1 vol. erythrocyte pellet + 2 vol.
buffer) and counted again. After centrifugation, the cell pellets were lysed.
Lysis of erythrocyte pellets: One volume of erythrocyte's pellet was mixed with 2 volumes of
double distilled water in Eppendorf cups, boiled for 2 min and centrifuged for 5 min at 14,000 g
(4 °C). After centrifugation the supernatant was ultrafiltrated two times (Filter 1: cut off 30,000 D,
followed by filter 2: cut off 5000 D; Ultrafree Centrifugal Filter units, Millipore, Beford, MA,
USA). Ultrafiltrates were further diluted with double distilled water 1:1 and immediately analysed
by ITP or stored at − 70 °C till analysis.

2.4. Analytical isotachophoresis (ITP)

ITP was carried out on an ITA-Chrom EA 101 (J and M, Aalen, Germany). Preseparation
column: capillary 90 × 0.8 mm I.D. × 1.2 mm O.D.; analytical column: 160 × 0.3 mm I.D. × 0.7 mm
O.D. Preseparation was carried out at 200 μA, analysis at 50 μA. Leading electrolyte (L): 10 mM
HCl-β-alanine + 0.1% (m/v) MHEC, pH 3.21; terminating electrolyte (T): 5 mM caproic acid–
histidine, pH 6.0.
For separation of ATP from phosphate, 10 mM HCl-6-aminohexanoic acid + 0.1% (m/v)
MHEC, pH 3.6 was used as leading electrolyte, and 10 mM caproic acid as terminating
electrolyte. Preseparation and analytical column: 160 mm; preseparation was carried out at
250 μA, analytical separation at 50 μA.
30 μl samples were injected for analysis. Signals were registered by UV detection at 254 nm
and by conductivity detection.

2.5. Other methods

Extracellular glucose and lactate were measured on an ABL-705 (Radiometer, Copenhagen,

Denmark).
82 Z. Kuçi et al. / J. Biochem. Biophys. Methods 69 (2006) 79–87

3. Results

3.1. Separation conditions

Metabolites of glucose (glucose-6-phosphate → lactate) are small anions with partly very
similar electrophoretic mobilities. Therefore, it is not possible to separate all of them
because some mixed zones occur in dependence on the L/T system used. However, in most
cases it is only important to quantify a restricted number of selected compounds. Beside
measurements of lactate and ATP, analysis of 2,3 BPG is of special interest for
characterisation of the metabolism in erythrocytes. Using the L/T system HCl/β-alanine,
pH 3.2 (L) and caproic acid/histidine (T), these three compounds can easily be separated
from each other (see tachograms in Fig. 1a–d; upper part of the tachogram: UV-signal at
254 nm; lower part: R: resistance). However, mixed zones between ATP (strong absorbance
at 254 nm) and phosphate (no absorbance at 254 nm) may occur. Nevertheless,
quantification of ATP in phosphate-containing samples is possible in different ways. First,
zone length of pure ATP (strong UV-signal) proved to be proportional to the length of the
total mixed zone (measured by addition of different amounts of ATP to lysates, Fig. 2a).
Furthermore, it is possible to separate ATP and phosphate by using another L/T system: Fig.
2b shows that a clear separation of ATP/phosphate is possible using HCl/6-aminohexanoic
acid, pH 3.6. However, under these conditions mixed zones between ADP and lactate are
formed.

Fig. 1. Isotachograms of lysates obtained from erythrocyte concentrates stored in blood bags at 4 °C for up to 35 days.
Signal registration by UV absorption (upper signals) and resistance (lower signals). A254: absorption at 254 nm; R:
resistance; 1: 2,3 BPG; 2: ATP; 3: ADP; 4: lactate. L: leading electrolyte; T: terminating electrolyte.
 Z. Kuçi et al. / J. Biochem. Biophys. Methods 69 (2006) 79–87 83

Fig. 2. Determination of ATP in the presence of phosphate: (a) isotachograms using the standard L/T system (pH 3.21).
Zone length of pure ATP (strong signal at 254 nm) was measured after addition of different amounts of ATP to lysates and
proved to be proportional to the total zone length (ATP and phosphate). (b) Part of isotachogram (cell lysate spiked with
ATP, ADP, lactate and phosphate): leading electrolyte: HCl/6-aminohexanoic acid, pH 3.6; terminating electrolyte: caproic
acid. One volume of cell lysate was mixed with one volume ATP + ADP, lactate and phosphate in water. Final
concentration of each added substance before injection was 350 μM. 1: 2,3 BPG; 2: phosphate; 3: ATP; 4: mixed zone of
ADP and lactate. Signal registration by UV absorption (upper signals) and resistance (lower signals). A254: absorption at
254 nm; R: resistance; L: leading electrolyte; T: terminating electrolyte.

3.2. Changes of erythrocyte metabolites during storage in blood bags

Metabolic changes during blood storage at 4 °C were estimated in the extracellular fluid as
well as within the erythrocytes. Glucose and lactate were measured with specific electrodes after
enzymatic oxidation to H2O2 on a Radiometer 705. The design of these electrodes allows a
separation of cellular compounds from extracellular fluid prior to enzymatic oxidation of glucose
and lactate, respectively. As shown in Fig. 3, a continuous consumption of glucose and
production of lactate was detected in the extracellular fluid during the storage period of five
weeks. Fig. 1 gives an overview of the metabolic changes within the erythrocyte during this

Fig. 3. Consumption of glucose (squares) and production of lactate (dots) in the extracellular fluid of erythrocyte
concentrates during storage of erythrocyte concentrates at 4 °C. Before measurements on the ABL 705, the erythrocyte
concentrates were diluted 1:1 (v:v) with 0.9% NaCl solution.
84 Z. Kuçi et al. / J. Biochem. Biophys. Methods 69 (2006) 79–87

Fig. 4. Quantification of intracellular increase of lactate (a) and decrease of 2,3 BPG (b) during storage of erythrocyte
concentrates at 4 °C.

storage period, showing the well known rapid decreases of 2,3 BPG (Fig. 4b) and ATP and a
significant increase of lactate concentration within the cells (up to 1.5 mM after 3 weeks; Fig. 4a).
ATP and lactate can be best identified by registration of UV-signals. Although lactate does not
absorb at 254 nm, its zone is located between two compounds which absorb at 254 nm. Increase
of the lactate during storage is clearly shown.

3.3. Tachogram of erythrocyte lysates from a patient with thalassemia

Thalassemia is a highly heterogeneous hemoglobinopathy with defects in the α- or β-

globin chain (α/β-thalassemia), characterised by a reduced production rate of erythrocytes in
bone marrow as well as by hemolysis of released erythrocytes. Some severe forms need
lifelong blood transfusions every few weeks. Characteristically, erythrocytes from the
thalassemia patient shown in Figs. 5b and 6 were characterised by microcytosis [mean
cellular volume (MCV) of erythrocytes: 77 × 10− 15 L; normal range: 83–96 × 10− 15 L], and
hypochromia [Hemoglobin (Hb) concentration: 8.2 g/dl; normal range: 11–15 g/dl)]. Fig. 5
shows the isotachophoretic pattern of erythrocyte lysates of the patient immediately before
blood transfusion (b), compared to a healthy person (a). Although the overall isotachograms
are similar in both cases, an increased zone of 2,3 BPG is present in the patient with
thalassemia (4.8 mM) compared to the healthy person (3.6 mM) (Fig. 6). These elevated levels

Fig. 5. Isotachogram of erythrocyte lysates from a person with β-thalassemia (b) compared to a lysate from a healthy donor
(a). [1: 2,3 BPG; 2: ATP (ATP + mixed zone ATP/phosphate); 3: ADP; 4: lactate].
 Z. Kuçi et al. / J. Biochem. Biophys. Methods 69 (2006) 79–87 85

Fig. 6. 2,3 BPG levels in erythrocyte lysates from a healthy donor (1), from a patient with β-thalassemia before transfusion
(2), and from the same patient some hours after transfusion (3) with erythrocyte concentrates stored at 4 °C for 14 days (4).
Concentration in erythrocytes was calculated on the basis of MCV (mean cellular volume) values measured on the Bayer
ADVIA 120. MCVof the healthy person: 88 × 10− 15 L; MCVof the patient with thalassemia: 77 × 10− 15 L; MCVof stored
erythrocytes: 96 × 10− 15 L.

are typical for patients with thalassemia and allow a better oxygen release as compensation for
the reduced number and minor quality of erythrocytes. Since blood transfusion for patients
with thalassemia is not an emergency case, older erythrocyte concentrates (stored for about up
to 14 days before transfusion associated with a loss of 2,3 BPG, see Fig. 4b) are routinely
used. Therefore, a decrease in the mean 2,3 BPG levels in the blood (erythrocytes) of the
patient was observed immediately after blood transfusion (Fig. 6/3). However, it is well known
that 2,3 BPG synthesis starts again rapidly in the transfused erythrocytes during circulation at
37 °C [12].

4. Discussion

Analytical isotachophoresis proved to be a suitable method for analysis of glucose

metabolites. Using a selected leading/terminating electrolyte system, relative step heights of
conductivity (resistance) signals can be established [11] which are characteristic for mobility
of a metabolite. Since molecular weight and negative charge of some metabolites are very
similar (e.g. glucose-6-P and fructose-6-P) they may have almost identical mobilities by using
a certain L/T system, leading to the formation of mixed zones. Many of the glucose
metabolites are only of minor analytical and biological interest, and mixed zones in these
cases can be neglected. However, if one compound present in a mixed zone is especially
important, separation is possible in most cases by changing the L/T system, as it is shown in a
representative isotachogram for ATP/phosphate (Fig. 2b). On the other hand, quantification of
ATP using the standard leading electrolyte system used in this study (HCl/β-alanine at pH 3.2)
was also possible using calibration curves of lysates supplemented with ATP. Part of ATP is
clearly separated from phosphate, but part of it is present in a phosphate/ATP mixed zone
(Figs. 1 and 5) which is proportional to the separated ATP zone.
Erythrocyte concentrates stored in blood bags and blood from a patient with thalassemia
were analysed with ITP and showed the characteristic pattern as it could be predicted. 2,3
BPG, e.g., which has the highest mobility of all glucose metabolites, decreased rapidly
86 Z. Kuçi et al. / J. Biochem. Biophys. Methods 69 (2006) 79–87

during storage of erythrocyte concentrates in blood bags and was elevated in the patient
with thalassemia. ATP also decreased in a time-dependent manner during blood storage (Fig.
1), whereas lactate levels increased in the extracellular fluids as well as within the
erythrocytes.
Our further goal is to use this isotachophoretic method for the determination of the energy
status in cancer cells and neutrophilic granulocytes. These latter cells generate energy almost
exclusively by glycolysis [1] in order to use all oxygen available for production of reactive
oxygen compounds necessary for killing of microbes [15]. Concerning cancer cells, it was only
recently found that, in accordance with the Warburg's theory, inhibition of glycolysis may be an
effective new therapeutical approach in cancer treatment [16].

5. Simplified description of the method and future applications

An isotachophoretic method was used for analysis of glycolysis in erythrocytes which, in

principle, allows a simultaneous determination of all anions of this pathway (from glucose-
6-phosphate to lactate). By variation of the leading/terminating electrolyte systems it is
possible to separate substances in mixed zones which should be estimated quantitatively.
The method, originally used for characterisation of glycolysis in erythrocytes from healthy
persons, was applied for characterisation of erythrocytes from patients with thalassemia and
for quality control of erythrocyte concentrates prepared for blood transfusions. Future
applications will comprise characterisation of glycolysis in neutrophil granulocytes and
cancer cells.

Acknowledgements

This work was supported by the Fortüne Programme, University of Tuebingen, Project 660-
01. The authors thank Dr. Neumeister and Dr. Weinstock, University Tuebingen, Department of
Transfusion Medicine, for the kind supply of blood bags.

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