SEARCH

SEARCH BY CITATION

Keywords:

  • calcium ions;
  • erythrocyte;
  • human;
  • permeability;
  • Tarui's disease

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Abstract.Waldenström A, Engström I, Ronquist G (Norrlands University Hospital, Umeå; and University Hospital, Uppsala; Sweden). Increased erythrocyte content of Ca2+ in patients with Tarui's disease. J Intern Med 2001; 249: 97–102.

Objectives. To establish by flow cytometry and fluorophores an increased calcium ion load in erythrocytes of four patients with Tarui's disease.

Design. Calcium ion levels were determined in erythrocytes of patients and controls under normal and energy-deprived conditions. Adenylates were measured to assess energy status of incubated erythrocytes.

Setting. The experiments were 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) was a 39-year-old male; patients (male, aged 46 years) 2 and 3 (female, 30 years) were his two siblings. Patient 4 (male, 16 years) was the son of patient 2.

Interventions. None.

Main outcome measures. Calcium ion homeostasis was measured under basic conditions and under energy-deprived conditions and related to cellular adenylate content.

Results. All patients showed enhanced erythrocyte calcium ion loading compared to controls under energy-deprived conditions. Under normal conditions, however, three out of the four patients showed an increased erythrocyte calcium ion level compared to controls.

Conclusions. We conclude that erythrocytes from patients with Tarui's disease have an increased Ca2+ permeability, initiating compensatory mechanisms involving increased Ca2+ pump activity and increased glycolytic flux, which are not always sufficient to keep erythrocyte calcium ion concentration within physiological range.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Phosphofructo-1-kinase (ATP- d-fructose-6-phosphate-1-phosphotransferase, EC 2.7.1.11) is the third enzyme of glycolysis, converting fructose-6-phosphate to fructose-1,6-bisphosphate [1]. It is an allosterically regulated enzyme and may therefore govern the glycolytic flux. There are three different isoenzymes: the liver, muscle, and platelet subtypes [2]. The inherited decrease or deficiency of the muscle subtype is associated with a variety of clinical manifestations including haemolysis, glycogen storage disease and myopathy [3, 4]. The most conspicuous manifestation is glycogenosis type VII, also known as Tarui's disease [3], and it appears to be prevalent amongst individuals of Ashkenazi Jewish, Italian, and Japanese ancestry, with an autosomal recessive transmission [5]. The genetic defect was recently identified in a family from northern Sweden, with affected individuals in two generations [4, 5]. We found unexpectedly strong evidence that erythrocytes from these affected individuals contained excess calcium [6].

The aim of the present investigation was to establish a calcium ion overload in erythrocytes of these individuals by a direct fluorometric method using flow cytometry.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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 phosphofructokinase deficiency of the muscle isoenzyme type (Tarui's disease). Two of his siblings were also investigated, patient 2 (male, aged 46 years) and patient 3 (female, 30 years), as well as patient 4 (male, 16 years), who was the son of patient 2. Twenty-four healthy persons (twelve males, median age: 41 years, range 23–64, and twelve females, median age: 47 years, range 33–52) were used as controls.

Reagents

All chemicals were of analytical grade. Chloroform, acetonitrile and methanol were of LiChrosol grade from Merck (Darmstadt, Germany). EGTA and the divalent cationophore A23187 (free acid, dissolved in methanol) were from Sigma Chemical Company (St.Louis, MO, USA).

Fluorophores

Fluo-3 AM and Fura Red AM were obtained from Molecular Probes Inc. (Eugene, OR, USA). The fluorophores were dissolved in dry DMSO (Sigma Chemical Company) and then thoroughly mixed in final concentrations of 250 µmol L−1, each, and stored in small aliquots at −20 °C under dry conditions.

Flow cytometry

A Coulter Epics XL-MCL flow cytometer (Coulter Corporation, Miami, FL, USA) was used for the fluorescence analysis. Fluo-3 and Fura Red were excited with an argon laser at 488 nm. The Fluo-3 emission was detected at 525 nm (FL1) and the Fura Red emission detected at 620 nm (FL3). Erythrocytes were gated from other cells by forward scatter (FS) and side scatter (SS). The optimal relationship between erythrocytes and dye concentrations was experimentally tested with respect to both number of events and detection signal. Samples were run simultaneously with 2 µL DMSO instead of fluorophores to measure background signal, which was subtracted from the fluorescence. To avoid contaminating fluorescence from extracellular calcium, 10 µmol L−1 EGTA was added after the incubations but before the readings.

Assessment of intracellular calcium ions in erythrocytes

Freshly drawn heparinized venous blood was used. As incubation media a Hepes buffer (10 mmol L−1 pH 7.4) made isotonic with NaCl, containing 0.5 mmol L−1 Na2HPO4, 5 mmol L−1 KCl, 1 mmol L−1 MgCl2, 1 mmol L−1 CaCl2 and 5 mmol L−1 glucose (RBC buffer) was utilized. In some experiments the RBC buffer also contained either A23187 (0.01–1 µmol L−1) or EGTA (10 µmol L−1). Solutions were kept at 4 °C. One microlitre of whole blood was added to 500 µL of the RBC buffer. Two microlitres of the fluorophore mixture (final concentration for each dye was 2 µmol L−1) was added to 125 µL of the erythrocyte suspension and incubated for 45 min at 37 °C. Twenty-five microlitres of the incubate were further diluted in 475 µL RBC buffer and immediately subjected to flow cytometry measurements. All samples were run in triplicate. Measurements of intracellular calcium ion content in erythrocytes (expressed as a ratio between the fluorescent probes Fluo-3 and Fura Red) were made on two different occasions with different batches of dissolved fluorophores (no significant differences in readings concerning the two batches were found, Wilcoxon signed rank test). Moreover, the within-run imprecision, expressed as coefficient of variation (CV) was calculated from 20 duplicate estimations of fluorescence ratio on day 1, and 20 duplicates on day 2. The total CV was 2.1% and the intraindividual variation (2 measurements, 4–6 weeks in between) was 6.0%. The interindividual variation (24 controls) was 7.6%. In incubation experiments, matched controls were always run concomitantly with the patients.

Preparation of samples and media for incubation experiments

Freshly drawn heparinized venous blood was used. Plasma, together with the buffy coat and part of the upper layer of erythrocytes, were removed by aspiration. Washing was done in the RBC buffer; glucose was replaced by 5 mmol L−1 deoxyglucose in some experiments. Solutions were kept at 4 °C. The washed erythrocytes were resuspended in the RBC buffer (160 µL packed erythrocytes in 20 mL RBC buffer). Incubation was carried out at 37 °C. For the adenylate incubation experiments, samples were taken after different times and terminated by addition of perchloric acid (final concentration: 0.5 mol L−1) followed by centrifugation. Samples were run in triplicate.

For the flow cytometry and adenylate incubation experiments, samples were taken concomitantly from the same erythrocyte suspension. One microlitre of the fluorophore mixture was added to 50 µL of the suspension (final concentration of each dye was 2.5 µmol L−1) and incubated for 45 min at 37 °C. After this second incubation, 3 µL of the suspension was further diluted in 300 µL of the RBC buffer and measured immediately. Samples were run in triplicate.

Biochemical methods

The supernatant (acid extract from 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. Adenylates (ATP, ADP, AMP) were determined by high performance liquid chromatography (HPLC) according to a previously described method [7, 8].

Statistics

The significance of differences was established by Welch's unpaired, two tailed t-test. Wilcoxon signed rank test was used to compare different measurements of intracellular calcium content. A P-value less than 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The ionophore A23187 is a mobile ion carrier transporting divalent cations such as Ca2+ and Mg2+. The ionophore acts as an ion-exchange shuttle, carrying two H+ out of the cell for every divalent cation that is translocated into the cell. Accordingly, when cells are exposed to the ionophore A23187, Ca2+ enters the cell interior along a steep chemical gradient. Figure 1 illustrates the increasing fluorescence ratio in erythrocytes with increasing concentrations of A23187 in the medium (0.01–1 µmol L−1). Hence, the method was suitable for measuring intracellular Ca2+ in human erythrocytes.

image

Figure 1. Changes of fluorescence ratio (concentration of calcium ions) in human erythrocytes with increasing concentrations of the divalent cationophore A23187 (0.01–1.0 µmol L−1). Freshly drawn heparinized venous blood was used. Incubation medium was Hepes buffer (10 mmol L−1, pH 7.4), made isotonic with NaCl, containing 0.5 mmol L−1 Na2HPO4, 5 mmol L−1 KCl, 1 mmol L−1 MgCl2, 1 mmol L−1 CaCl2 and 5 mmol L−1 glucose (RBC buffer). Fluorescence probes were Fluo-3 AM and Fura Red AM; final concentration for each dye was 2 µmol L−1.

Download figure to PowerPoint

The intraerythrocytic Ca2+ concentration of 12 healthy men and 12 healthy women, measured on two different occasions (4–6 weeks apart), and expressed as mean fluorescence ratio, was 0.53. No significant difference existed between sexes. Figure 2 shows the Gaussian distribution of the 24 individuals. A reference interval could therefore be determined (mean value ± 2 SD). It is evident from Fig. 3 that patients 1, 2 and 4 had a pathologically increased intraerythrocytic Ca2+ concentration, whilst patient 3 did not. These measurements were carried out in the presence of glucose. Incubating control erythrocytes and erythrocytes from patients in presence of deoxyglucose, i.e. under starved conditions, showed a faster decay of adenylates over time in the patients' cells ( Fig. 4). This indicated an augmented ATP turnover, most probably due to an enhanced Ca2+ ATPase pump activity to compensate for the increased inward flux of Ca2+ in erythrocytes of patients [cf. 7, 8]. Intraerythrocytic Ca2+ concentration, expressed as fluorescence ratio, was also determined hourly in erythrocytes of patients and controls during incubation with deoxyglucose. It is clear from Fig. 5 that the increase of intraerythrocytic Ca2+ concentration was more pronounced over time in cells of all four patients than in cells of controls, where the increase was only slight up to 7 h of incubation.

image

Figure 2. Gaussian distribution of intraerythrocytic Ca2+, expressed as mean fluorescence ratio of 24 healthy controls (12 men and 12 women, measured on two different occasions). Freshly drawn heparinized venous blood was used. Incubation medium was Hepes buffer (10 mmol L−1, pH 7.4), made isotonic with NaCl, containing 0.5 mmol L−1 Na2HPO4, 5 mmol L−1 KCl, 1 mmol L−1 MgCl2, 1 mmol L−1 CaCl2 and 5 mmol L−1 glucose (RBC buffer). Fluorescence probes were Fluo-3 AM and Fura Red AM; final concentration for each dye was 2 µmol L−1. Samples were run in triplicate.

Download figure to PowerPoint

image

Figure 3. Intraerythrocytic Ca2+, expressed as fluorescence ratio, of four patients with Tarui's disease (mean ± SD). Freshly drawn heparinized venous blood was used. Incubation medium was Hepes buffer (10 mmol L−1, pH 7.4), made isotonic with NaCl, containing 0.5 mmol L−1 Na2HPO4, 5 mmol L−1 KCl, 1 mmol L−1 MgCl2, 1 mmol L−1 CaCl2 and 5 mmol L−1 glucose (RBC buffer). Fluorescence probes were Fluo-3 AM and Fura Red AM; final concentration for each dye was 2 µmol L−1. Samples were run in triplicate.

Download figure to PowerPoint

image

Figure 4. Mean changes (± SD), over time, in adenylates of washed erythrocytes from patients 1–4 and controls 1–4, incubated at 37 °C in Hepes buffer (10 mmol L−1, pH 7.4) made isotonic with NaCl, containing 0.5 mmol L−1 Na2HPO4, 5 mmol L−1 KCl, 1 mmol L−1 MgCl2, 1 mmol L−1 CaCl2 and 5 mmol L−1 deoxyglucose. Samples were run in triplicate. Comparisons were made between patients and controls. All differences between patients 1, 2 and 4 and controls were significant, except at time zero.

Download figure to PowerPoint

image

Figure 5. Mean changes (± SD), over time, in fluorescence ratio (intraerythrocytic calcium ions) of washed erythrocytes from patients (1–4) and controls (1–4), incubated at 37 °C in Hepes buffer (10 mmol L−1 pH 7.4) made isotonic with NaCl, containing 0.5 mmol L−1 Na2HPO4, 5 mmol L−1 KCl, 1 mmol L−1 MgCl2, 1 mmol L−1 CaCl2 and 5 mmol L−1 deoxyglucose. Fluorescence probes were Fluo-3 AM and Fura Red AM; final concentration for each dye was 2.5 µmol L−1. Samples were run in triplicate. Comparisons were made between patients and controls. All differences were significant.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The main finding of the present investigation was an elevated level of Ca2+ in erythrocytes of patients with Tarui's disease. This was clearly demonstrated by the increased FL1/FL3 ratio in three of the four patients under basic conditions. The one patient (patient 3) with the normal FL1/FL3 ratio under these conditions was without any clear-cut clinical manifestations of the disease. The affliction of this patient with the disease became, however, apparent when investigating her erythrocytes under starved conditions, i.e. incubation with deoxyglycose, which is not metabolized and is therefore ruled out as a glycolytic fuel. The anaerobic glycolysis provides the erythrocytes with ATP, which is mainly used for maintenance of the electrochemical and chemical ion gradients by the activity of the Na+/K+ ATPase and the Ca2+ ATPase. Thus, the patient with the normal Ca2+ concentration under basic conditions exhibited an augmented loss of adenylates as well as an increased intracellular Ca2+ load in her erythrocytes, similar to the other three patients' erythrocytes, when under starved conditions. We therefore conclude that all four patients with Tarui's disease show signs of an enhanced calcium permeability into their erythrocytes along the calcium gradient. Apparently, patients 1, 2 and 4 were not able to compensate for this enhanced leakage in spite of an augmented Ca2+ ATPase activity even under basic conditions [6], whilst patient 3 had this capacity.

We focused our study on erythrocytes for several reasons. It is known that the erythrocytes are involved in the pathologic process since they display a compensated haemolysis. Further, the erythrocytic PFK is a heterotetramer, one dimeric part being of the muscle type. This was manifested amongst our patients with more or less pronounced decrease of erythrocytic PFK activity [6]. It should also be emphasized that the erythrocytes are easily available and unicellularly occurring, which was a prerequisite for our flow cytometric study. We determined the intraerythrocytic Ca2+ expression by the FL1/FL3 ratio, which did not permit the presentation of absolute calcium ion concentrations but which did give us a correct basis for comparison between patients and controls. Further, by using this ratio we enhanced the Ca2+ signal, which was an advantage due to the high haemoglobin concentration of the erythrocytes.

The reliability of the method was demonstrated by the dose/response relationship to the ionophore A23187 and by the low imprecision figure, CV being 2.1%.

In summary, we have shown an increased intraerythrocytic Ca2+ load in spite of an increased Ca2+-pumping activity [6] which can only be explained by an increased Ca2+ permeability across the erythrocyte membrane in patients with Tarui's disease. It is reasonable to believe that this Ca2+ leakage can also involve other cellular membranes, such as the sarcolemma. Therefore, the muscle fatigue in these patients would mainly be due to a pathological Ca2+ homeostasis rather than a lowered phosphofructokinase activity per se. This view is corroborated by the findings of others that the sarcolemma defect in Duchenne's muscle dystrophy is also found in the erythrocyte membrane of these patients [9–12]. Moreover, it is intriguing to note in this context that the PFK, Ca2+ ATPase and Ca2+ channel genes are not only located at the same chromosome, 12q, but are also in close proximity to each other [13].

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • 1
    Vora S, Seaman C, Durham S, Piomelli S. Isoenzymes of human phosphofructokinase: identification and subunit structural characterization of a new system. Proc Natl Acad Sci USA 1980; 77: 62 6.
  • 2
    Vora S, Davidson M, Seaman C et al. Heterogeneity of the molecular lesions in inherited phosphofructokinase deficiency. J Clin Invest 1983; 72: 1995 2006.
  • 3
    Tarui S, Okuno G, Ikura Y, Tanaka T, Suda M, Nishikawa M. Phosphofructokinase deficiency in skeletal muscle. Biochem Biophys Res Commun 1965; 19: 517 23.
  • 4
    Rudolphi O, Ek B, Ronquist G. Inherited phosphofructokinase deficiency associated with hemolysis and exertional myopathy. Eur J Hematol 1995; 55: 279 81.
  • 5
    Nichols RC, Rudolphi O, Ek B, Exelbert R, Plotz PH, Raben N. Glycogenosis type VII (Tarui Disease) in a Swedish family: Two novel mutations in muscle phosphofructokinase gene (PFK-M) resulting in intron retentions. Am J Hum Genet 1996; 59: 59 65.
  • 6
    Ronquist G, Rudolphi O, Engström I, Waldenström A. Familial phosphofructokinase deficiency is associated with a disturbed calcium homeostasis in erythrocytes. J Intern Med 2001; 249: 8 9 (this issue).
  • 7
    Engström I, Waldenström A, Ronquist G. Ionophore A 23187 reduces energy charge by enhanced ion pumping in suspended human erythrocytes. Scand J Clin Lab Invest 1993; 53: 239 46.
  • 8
    Engström I, Waldenström A, Nilsson-Ehle Peter Ronquist G. Dissipation of the calcium gradient in human erythrocytes results in increased heat production. Clin Chim Acta 1993; 219: 113 22.
  • 9
    Plishker GA, Gitelman HJ, Appel SH. Myotonic muscular dystrophy: altered calcium transport in erythrocytes. Science 1978; 200 (4339): 323 5.
  • 10
    Mawatari S, Schonberg M, Olarte M. Biochemical abnormalities of erythrocyte membranes in Duchenne dystrophy. Arch Neurol 1976; 33: 489 93.
  • 11
    Roses AD, Herbstreith MH, Apel SH. Membrane protein kinase alteration in Duchenne muscular dystrophy. Nature 1975; 254 (5498): 350 1.
  • 12
    Butterfield A, Roses AD, Appel SH, Chesnut DB. Electron spin resonance studies of membrane proteins in erythrocytes in myotonic muscular dystrophy. Arch Biochem Biophys 1976; 177: 226 34.
  • 13
    Howard TD, Akots G, Bowden DW. Physical and genetic mapping of the muscle phosphofructokinase gene (PFKM); reassignment to human chromosome 12q. Genomics 1996; 34: 122 7.DOI: 10.1006/geno.1996.0250

Received 7 March 2000; revision received 11 October 2000; accepted 11 October 2000.