Dr Gunnar Ronquist, Department of Clinical Chemistry, University Hospital, S-75185 Uppsala, Sweden (Fax: +46 018 5525 62).
Abstract. Ronquist G, Rudolphi O, Engström I, Waldenström A (University Hospital, Uppsala; and Norrlands University Hospital, Umeå; Sweden). Familial phosphofructokinase deficiency is associated with a disturbed calcium homeostasis in erythrocytes. J Intern Med 2001; 249: 85–95.
Objectives. To critically evaluate whether an altered calcium homeostasis in erythrocytes could be contributing to the symptomatology of the Tarui's disease, which is an inherited phosphofructokinase (PFK) deficiency of the muscle isoenzyme. PFK is a tetrameric enzyme with three different isoenzymes, muscle (M), liver (L), and platelet (P). Erythrocytes contain a 50 : 50 hybrid of M and L type. The deficiency of the muscle isoenzyme displays a symptomatology which is mainly characterized by myopathy, and a compensated haemolytic anaemia.
Design. Erythrocyte deformability was assessed before and after autoincubation. Energy related metabolites and energy charge was determined in erythrocytes under various experimental conditions.
Setting. The clinical part of the study was performed at the Departments of Cardiology and Clinical Chemistry, Umeå University Hospital, and the experimental investigation was carried out at the Department of Clinical Chemistry of the University Hospital of Uppsala, Sweden.
Subjects. Four family members with Tarui's disease participated in the study: the proband (patient 1), a 39-year-old male and two siblings, patient 2 (male, aged 46 years) and patient 3 (female, 30 years). Patient 4 (male, 16 years) was the son of the patient 2. Five healthy persons served as controls (controls 1–5).
Main outcome measures. Cell-physiological variables were determined after autoincubation of erythrocytes (i.e. incubation in their own plasma at 37 °C) and after incubation in a composite buffered medium.
Results. Erythrocyte deformability as assessed by the erythrocyte fluidity was substantially decreased in patients compared to the moderate decrease in the control after 24 h of autoincubation, in presence of endogenous Ca2+ (heparin plasma). Moreover, autoincubation of erythrocytes shows that the patient's erythrocytes, although being moderately deficient in PFK activity, exhibit a normal (or slightly increased) lactate production compared to controls. Despite this, we show an increased ATP turnover with an Ca2+-induced AMP deaminase (and 5′-nucleotidase) activation leading to an increase in hypoxanthine content in patients' erythrocytes of about 100% after 24 h of autoincubation in heparin plasma, when compared to controls. A loss of volume in patient's erythrocytes after 24 h of autoincubation (in presence of Ca2+), as revealed by a diminished MCV, was consistent with an increased metabolic pool of intracellular calcium ions with a selective loss of K+ due to the activation of the K+ channel by intracellular Ca2+ (Gardos-effect).
Conclusion. We conclude that the different calcium ion-induced effects on energy metabolism, structure and function of patients' erythrocytes are due to an augmented membrane leakage of Ca2+ and therefore an accumulated intracellular Ca2+ pool. This will result in an increased energy demand by the Ca2+-stimulated ATPase (calcium pump) to compensate for the dissipated Ca2+ gradient across the plasma membrane. The concomitant haemolysis may be explained by a diminished erythrocyte deformability due to Ca2+ overload.
Phosphofructokinase (PFK, EC 18.104.22.168), subjected to allosteric regulation in glycolysis, is a tetrameric enzyme under the control of three structural loci coding for three unique subunits, muscle (M), liver (L), and platelet (P) types . Hence, human muscle and liver express homotetramers (M4 and L4, respectively) whilst erythrocytes contain the five isoenzymes M4, M3L, M2L2, ML3, and L4. Inherited deficiency of PFK-M in man is associated with myopathy and/or haemolysis or an asymptomatic state . The most common type, glycogenosis VII (Tarui's disease), is characterized by the coexistence of muscle disease and moderate haemolysis . The genetic defect was recently identified in a Swedish family with affected individuals in two generations [4, 5].
Glycolysis is the most important source of energy in erythrocytes and fast muscle fibres. Therefore, any defect of glycolytic flow can cause haemolytic anaemia and myopathy with exercise intolerance. The mechanism of haemolysis, being a defect in glycolytic flow, can deprive ATP production to such an extent that ion homeostasis and cell volume regulation can not be kept in balance and the erythrocyte goes into lysis. The severity of each of these principal manifestations may vary depending on which particular step in the glycolytic pathway is involved and on residual enzyme activity, the expression of tissue-specific isoenzymes, and physicochemical properties of the afflicted enzyme.
Erythrocytes contain two different types of ATPases (ion pumps), the Na+ and K+-dependent ATPase and the Ca2+-stimulated ATPase (25% and 75% activity, respectively) . A metabolic coupling may exist in human erythrocytes between cation-transport ATPases and glycolytic rate [7–13]. An increase in intracellular Ca2+ concentration will activate the Ca2+-stimulated ATPase, pumping Ca2+ out of the cell and thus restoring the Ca2+ homeostasis . Hence, an augmented Ca2+-stimulated ATPase activity can deplete intracellular ATP stores , due to an imbalance between the limited metabolic capacity of the erythrocytes and the powerful hydrolytic activity of the Ca2+saturated pump [16, 17].
ATP hydrolysis and metabolic sustenance of ATP levels are difficult to study simultaneously. For example, investigating isolated membranes may only permit the study of hydrolysis of ATP under given experimental conditions, and it should be pointed out that membrane isolation may even cause loss of Ca2+-stimulated ATPase activity .
The aim of the present investigation was to demonstrate, with minimum perturbation of the cell systems, various calcium ion-induced effects by incubation, on structure, energy metabolism and function, due to a proposed increased leak of calcium ions in erythrocytes from patients with a PFK-M deficiency.
Materials and methods
This study was approved by the ethical committe of the medical faculty at Umeå University.
Patients and controls
The proband (patient 1) was a 39-year-old male with an inherited PFK deficiency of the muscle isoenzyme type. He was originally referred to the hospital because of unexplained jaundice, diagnosed as morbus Gilbert, and an earlier episode of acute erythropoetic failure. It became evident that the patient's icterus was caused by a nonspherocytic haemolytic disorder, as his erythrocytes showed normal osmotic fragility and extracorpuscular causes of jaundice were ruled out. The patient also complained of premature fatigue and muscle pain after jogging and heavy exercise. Since childhood he had suffered from excessive fatigue but during recent years he had been troubled with increasing weakness, and even short walks resulted in aching calf muscles. Two of his siblings were also investigated, patient 2 (male, 46 years) and patient 3 (female, 30 years), and also patient 4 (male, 16 years) who was the son of patient 2 [4, 5]. Five healthy persons were used as controls: control 1 (female 49 years), control 2 (male 27 years), control 3 (male 41 years), control 4 (female 31 years) and control 5 (male 25 years). Patients 1–4 had a median age of 34.5 (range 16–46 years) and controls 1–5 had a median age of 31 (range 25–45 years).
Fresh sodium-heparin (plasma with endogenous calcium ions) and EDTA (plasma mainly lacking calcium ions) blood samples from patients and healthy controls were autoincubated (i.e. incubated in their own plasma) in duplicate at 37 °C for different times and terminated by the addition of perchloric acid (final concentration 0.5 mol L−1), followed by centrifugation.
Preparation of samples and incubation media
Freshly drawn heparinized venous blood was used for the experiments. Separation of plasma was followed by two washings of erythrocytes in an isotonic NaC1 solution. The buffy coat, together with part of the upper layer of erythrocytes, was removed by aspiration in each washing procedure. The third washing was performed with phosphate buffer (10 mmol L−1, pH 7.4) made isotonic with NaCl, phosphate-buffered saline (PBS), 290 mosmol L−1. Solutions were kept at 4 °C. The washed erythrocytes were resuspended in PBS containing 5 mmol L−1 KCl and 1 mmol L−1 MgCl2 (basic incubation medium) which was enriched with 1 mmol L−1 CaCl2 or 1 mmol L−1 EGTA. In some experiments the incubation medium additionally contained 0.5 mmol L−1 ouabain. The erythrocyte suspension was adjusted to an erythrocyte volume fraction (EVF) of 0.05 and total incubation volume was 0.5 mL (samples were run in triplicate). All incubations were carried out at 37 °C and terminated after different times by addition of perchloric acid (final concentration: 0.5 mol L−1) followed by centrifugation.
The supernatant (acid extracts, both from autoincubated, and washed and incubated erythrocytes) was treated with the same volume of DOMA (N,N-dioctylmethylamine, 1 mol L−1; Fluca Chemie AG, Buchs, Switzerland) dissolved in chloroform and the neutral aqueous phase was recovered and stored at − 20 °C. Nucleotides were determined by high performance liquid chromatography (HPLC) according to a previously described method . Due to interference by EDTA, the adenylates (except IMP, which it was not possible to detect) were determined with Sellevold's modified method  in all samples containing EDTA plasma. The eluent was, in this latter case, ammonium dihydrogen phosphate buffer, 0.2 mol L−1, pH 6.0, containing TBAHS (tetrabutyl ammonium hydrogen sulphate, 3 mmol L−1; Fluca Chemie) and acetonitrile, 4.5%. The nucleotide content was given in µmol g Hb−1. In order to achieve a good resolution in the separation of nucleosides and purine bases (adenosine, inosine, hypoxanthine and xanthine) from charged nucleotides, a preseparation procedure was performed on minicolumns (Bond Elute SAX, 1 mL size, containing 100 mg anion exchange material; Varian, Harbor City, CA, USA) . The separation of nucleosides and purine bases was performed in a 2-stage procedure according to Brown . Lactate concentration was determined by an enzymatic method (Sigma Chemical Company, St. Louis, MO, USA) and expressed in µmol g Hb−1. 2,3-Bisphosphoglycerate (2,3-BPG) was analysed enzymatically (Boehringer Mannheim GMBH, Mannheim, Germany; Cat. no. 148 334) and expressed in mmol L Ery−1.
For calculation of energy charge (EC), Atkinson's equation  was adopted:
All chemicals were of analytical grade. Chloroform, acetonitrile and methanol were of LiChrosol grade from Merck (Darmstadt, Germany).
The haemorheological variables were assessed at 37 °C using a Low Shear 30 rotational viscometer (Contraves AG, Zurich, Switzerland) . Plasma viscosity was analysed at a shear rate of 38 s−1 and apparent whole-blood viscosity at 100 s−1. The whole-blood viscosity at a low shear rate is essentially dependent on erythrocyte aggregation . In order to measure the erythrocyte aggregation tendency, the whole-blood viscosity was analysed at a shear rate of 1 s−1. Corrections for patient EVF were made by dividing the whole-blood viscosity by a reference value  at a corresponding shear rate and EVF. Corrections for plasma viscosity were carried out in a similar way. Thus, the unit for erythrocyte aggregation becomes dimensionless. Inverse viscosity is termed fluidity. The erythrocyte fluidity has been shown to reflect the erythrocyte deformability . It was assessed after separation of erythrocytes from plasma by centrifugation and after resuspension of erythrocytes in PBS containing glucose, 5 mmol L−1, at pH 7.4. to an erythrocyte volume fraction (EVF) of 0.55. The erythrocyte fluidity was calculated as the inverse viscosity of the suspension, at a shear rate of 1 s−1.
The significance of differences was established by analysis of variance (anova) combined with Bonferroni t-test. This t-test was chosen when more than two groups were subjected to comparison. Welch's unpaired, two-tailed t-test was used when only two groups were compared. A P-value of less than 0.05 was considered statistically significant.
Table 1 displays the different erythrocyte enzyme activities and the reticulocyte count in the four patients and in one control. The enzyme activities were generally within their normal ranges except for a decreased PFK activity in the patients and a slightly elevated hexokinase activity in patient 1. This latter finding conforms with the elevated reticulocyte count in patient 1 (who is considered to be the most affected patient), indicative of ongoing haemolysis, whilst the reticulocyte counts in patients 2–4 were only somewhat increased.
Table 1. Erythrocyte enzyme activities and reticulocyte count (109 L–1; mean ± SD, from two different occasions) in the four affected family members (patients 1–4) and one control. Enzyme activity was expressed in kat 10−18 erythrocyte–l at 25 °C, pH 7.5
Glucose 6 phosphate dehydrogenase
3.4 (0 57)
Purine nucleoside phosphorylase
Different rheological variables of erythrocytes from the four patients and one control are shown in Table 2. Autoincubation for 24 h in the presence of endogenous Ca2+ (heparin plasma) rendered a 8.3% lower EVF mean value for patients, contrary to a corresponding increase of 11.0% in the controls. Erythrocyte deformability, as assessed by the erythrocyte fluidity, was substantially decreased in patients, compared to a moderate decrease in the controls after 24 h of autoincubation in the presence of endogenous Ca2+ (heparin plasma).
Table 2. EVF and rheological variables (mean values and ranges) of erythrocytes (E), plasma (P) and blood (B) from patients (male) and one control (female) at 37 °C, before and after autoincubation for 24 h (heparin plasma)
Various energy-related metabolites were demonstrated in the erythrocytes of the patients and the controls before and after autoincubation in the presence of endogenous Ca2+ (heparin plasma) (Table 3). Zero time values revealed a moderately decreased ATP content and distinctly elevated contents of IMP and NAD+ in erythrocytes of patients compared to controls. Further, the 2,3-BPG concentration was less than half that of control erythrocytes. Autoincubation for 24 h resulted in very low concentrations of ADP and AMP in erythrocytes of patients. IMP values were also lower in patients. Most strikingly, the EC value did not decrease as much as in control erythrocytes and the hypoxanthine content was about twice as high as in controls after 24 h of autoincubation. The ratio between the sum of AMP and IMP over hypoxanthine was only 0.130 in patients' erythrocytes compared to 0.991 in those of controls, indicating elevated activities of erythrocyte AMP deaminase and 5′-nucleotidase in patients. Most conspicuously, the lactate production was significantly higher at 24 h of autoincubation of erythrocytes from patients in spite of the slight PFK deficiency (Table 3).
Table 3. Energy-related metabolites in erythrocytes from patients and controls at +37 °C, before and after autoincubation (heparin plasma)
α Statistical difference from controls (P = < 0.00l). Results are expressed in b µmol g Hb –l and cmmol L Ery–l. dSamples were run with only control 1 and not in duplicate (all other samples were run in duplicate). eSamples were run with only control 1. Each value represents mean ± SD.
The erythrocytes from patient 1 and controls were further investigated during autoincubation in both EDTA (endogenous Ca2+ absent) and heparin plasma (endogenous Ca2+ present) for 0, 5, 10 and 24 h. Figure 1 demonstrates that the hydrolysis of ATP was faster in erythrocytes of the patient, regardless of plasma type, but more pronounced in heparin plasma. It is also seen that the production of IMP was elevated for the patient, compared to controls (in case of heparin plasma), reflecting an increased AMP deaminase activity in the presence of Ca2+. The EC value increased in the patient's erythrocytes between 10 and 24 h of autoincubation, most probably due to a shift in the adenylate kinase equilibrium as a consequence of enhanced activities of AMP deaminase and 5′-nucleotidase, resulting in a lower AMP concentration.
A rapid production of lactate was observed in the erythrocytes of the patient and a maximum was reached at 10 h of autoincubation, concomitant with a minimum level of glucose (Fig. 2). This pattern was more obvious during autoincubation in heparin plasma. This lactate production was slower in controls and the lactate level of patient 1 was significantly higher already after 5 h of autoincubation in heparin plasma (Fig. 2).
Figure 3 illustrates that autoincubation in EDTA plasma resulted in a successive increase in mean corpuscular volume (MCV) and a corresponding decrease in mean corpuscular haemoglobin concentration (MCHC) over time, in erythrocytes of both patient 1 and controls. Such a uniform pattern was not noted during autoincubation in heparin plasma. Hence, a decrease in MCV and an increase in MCHC were observed between 10 and 24 h in the erythrocytes of patient 1 only (compare with B-EVF values in Table 2), whilst controls demonstrated a reversed pattern concordant with the one observed in EDTA plasma.
Incubation of washed erythrocytes (from all patients and controls) in basic incubation medium supplemented with 1 mmol L−1 of CaCl2 for up to 90 min resulted in a decrease in EC over time, which was much more pronounced in the patients' erythrocytes (Fig. 4). Ouabain inclusion preserved EC values on a somewhat higher level in both patients and controls. The preserving effect of ouabain was about equal, being 7.3% in patients and 7.7% in controls (Fig. 4). The decreases in total adenylate content of erythrocytes in patients and controls were similar during preparation of erythrocytes for incubation experiments, although quantitative differences were significant at zero time (not shown) (Fig. 5). Erythrocytes of patients incubated in basic incubation medium containing 1 mmol L−1 of CaCl2 showed a rather slight increase of total adenylates at 30 min, in contrast to the sharp increase when CaCl2 was exchanged for EGTA. On the contrary, these two incubation conditions resulted in a slight decrease in adenylates of control erythrocytes at 30 min (Fig. 5). A continuous and rapid decrease of the adenylate content of patients' erythrocytes was apparent upon further incubation up to 90 min, where EGTA displayed a preserving effect, being 9.5% at 90 min (Ap in Fig. 5). The curve profile of control erythrocytes was levelled off after 30 min of incubation and the preserving effect of EGTA was slight, being only 2.2% at 90 min (Ac in Fig. 5). On the other hand, the preserving effect of ouabain was small in this regard in both patients (3.8%, Bp in Fig. 5) and controls (2.4%, Bc in Fig. 5).
The ATP concentration in erythrocytes of patients was 17% lower than in erythrocytes of controls at zero time. Furthermore, autoincubation resulted in a faster ATP hydrolysis of patients' erythrocytes compared to controls. Additionally, there was an increased rate of lactate production in the patient's erythrocytes, suggesting a metabolic coupling between the ATPase reaction and glycolysis. The distinctly lowered ADP content in patients' erythrocytes at 24 h autoincubation may reflect an increased adenylate kinase activity in these cells combined with an increased degradation rate of AMP – reflected by augmented concentrations of hypoxantine and xantine – and testifies to the accelerated need for ATP regeneration in these cells.
It is known that erythrocytes with experimentally induced ATP deficiency exhibit an increase in calcium ion content due to inadequate calcium pumping in parallel with a rise in cellular viscosity . A progressive decrease in membrane deformability also occurred, and was reflected by an increase in the negative pressure needed to deform the membrane . Finally, regeneration of ATP in depleted cells by incubation with adenosine produced a significant reversal of these changes . Thus, the changes observed represented ATP-calcium-dependent sol-gel transformations occurring at the interface between the membrane and the cell interior, the sol-gel equilibrium determining membrane deformability . Accordingly, the calcium ion gradient across the erythrocyte membrane determines erythrocyte deformability and fluidity. These phenomena were subsequently studied in greater detail by Clark et al. .
The significantly reduced fluidity of our patients' erythrocytes at 24 h of autoincubation is accordingly well in line with an enlarged metabolic pool of intracellular, free calcium ions. Since erythrocyte ATP levels were about the same in patients and controls at 24 h of autoincubation, the pronounced reduction of fluidity in patients' erythrocytes at 24 h was not the result of depressed Ca2+ transport due to inadequate fuelling of the ATPase. Instead we propose a higher degree of membrane leakiness for Ca2+ in the erythrocytes of patients with PFK-M deficiency, as a cophenomenon. This also implies an increased Ca2+-stimulated ATPase activity [15, 29] leading to an enhanced ATP turnover, with, additionally, an augmented adenylate turnover, in accordance with the reasoning above.
The significant increase of lactate production at 10 h of autoincubation in one patient's (patient 1) erythrocytes is also worth mentioning; reflecting rather, an increased glycolytic flux during autoincubation in spite of the moderate decrease of PFK activity.
The concentration of 2,3-BPG, a metabolite after the PFK step, was about half that of normal at zero time but reached very low values in both patients and control at 24 h of autoincubation. This illustrates that 2,3-BPG may be regarded as an energy source when required by the human erythrocyte and can be made use of in any state of energy crisis in this cell. Therefore, the decreased level of 2,3-BPG observed by us and by others  may not necessarily reflect the PFK-M deficiency per se but rather an increased energy demand due to an augmented Ca2+-stimulated ATPase activity as a consequence of the proposed membrane leakiness.
Metabolic exhaustion may induce shape changes in human erythrocytes registered as acquired alterations in surface and volume. A volume gain was observed in erythrocytes from patient and controls during autoincubation in the absence of Ca2+, as revealed by an MCV increase over time (and, accordingly, an MCHC decrease). A less consistent pattern between patients and controls was, however, seen during autoincubation in presence of Ca2+. The loss in volume in patients' erythrocytes at 24 h of autoincubation, as revealed by a diminished MCV, was most conspicuous and it was consistent with an increased metabolic pool of intracellular calcium ions with a selective loss of K+ due to the activation of the K+ channel by intracellular Ca2+ (Gardos effect) . Approximately 100–150 Gardos channels are present in a normal erythrocyte  and when activated, the normal discocyte turns into a xerocyte/echinocyte formation due to dehydration .
An enlarged intracellular pool of metabolically active Ca2+ in erythrocytes of PFK-M deficient patients can also be envisaged by the increased concentration of IMP at zero time. This nucleotide is the hydrolytic deamination product of AMP catalysed by 5′-AMP aminohydrolase (AMP deaminase), and this enzyme is activated by intracellular Ca2+[34, 35].
The sharp decrease of EC in patients' erythrocytes after as little as 30 min of incubation reflected an increased ATP hydrolysis also in washed cells. The most energy-demanding process in erythrocytes is ion pumping. Since the ouabain-sparing effect on EC was slight and about equal in patients and controls, the involvement of the Na+ and K+-dependent ATPase was ruled out [cf. 36]. Instead, it suggested an increased leak of calcium ions in erythrocytes of patients being the underlying cause . This in turn may stimulate the activity of the Ca2+-stimulated ATPase (in order to maintain the transmembrane calcium ion gradient) to such an extent that the normal nucleotide pattern was altered, resulting in a significantly decreased adenylate content in erythrocytes of patients at 90 min of incubation. The involvement of the Ca2+-stimulated ATPase was also emphasized by the addition of EGTA (a chelator of calcium ions) to the incubation medium, displaying a 4-fold adenylate-sparing effect compared to control erythrocytes. The energy needed for ion pumping can only be derived from glycolysis in the human erythrocyte and a coupling has been claimed to exist between glycolysis and ion pump activities in this cell [7–13]. This may have a more general significance [37, 38]. Hence, there are good reasons to believe that a coupling exists between ATP formation and ATP hydrolysis regardless of the type of ion pump involved in the human erythrocyte membrane.
The current view, that the PFK-M deficiency in these patients is primarily responsible for decreased glycolysis, and thus ATP regeneration in their erythrocytes with ensuing haemolysis, is not corroborated by data in the present study. There are different lines of evidence that four members of a family with PFK-M deficiency manifested by muscle fatigue and haemolysis have an additional defect localized in the plasma membrane of their erythrocytes. Findings of an enhanced hydrolysis of ATP, combined with an increased glycolytic flux, suggest an accelerated ATP-dependent Ca2+ extrusion machinery due to increased membrane leakiness of Ca2+ in these cells. This membrane leakiness should be understood as a cophenomenon to the PFK-M deficiency.
This work was supported by grants from the Swedish Medical Research Council (project 09940), the Swedish Heart Lung Foundation, the Medical Faculty of Uppsala University, King Gustaf V's and Queen Viktoria's Foundation and the Tore Nilsson Foundation.
The authors are grateful to Dr Bo Sandhagen who performed the viscosimetric measurements.
Received 7 March 2000; revision received 11 October 2000; accepted 11 October 2000.