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C J Van Echteld

Publications and source records attributed to C J Van Echteld.

At least 19 recordsLinked to original sources
PubMed2001

Brain death-related energetic failure of the donor heart becomes apparent only during storage and reperfusion: an ex vivo phosphorus-31 magnetic resonance spectroscopy study on the feline heart.

BACKGROUND AND OBJECTIVE: Recently, we have shown, by using localized in vivo phosphorus-31 magnetic resonance spectroscopy (31P MRS) of the anterior left ventricular wall, that brain death (BD) is not associated with reduced myocardial energy status. In this study, we applied ex vivo 31P MRS of the entire heart to study the effects of BD on the energy status of the feline donor heart following explantation. METHODS: We used cats (6 BD and 6 controls [C]) in a 26-hour protocol. After 2 hours of preparation, we induced BD by filling an intracranial balloon at t = 0 hour. At t = 6 hours, the hearts were arrested with St. Thomas' Hospital cardioplegic solution, explanted, and stored in the same solution at 4 degrees C in a 4.7 Tesla magnet for 17 hours. Subsequently, the hearts were reperfused in the Langendorff mode at 38 degrees C for 1 hour. The first 5-minute 31P MRS spectrum was obtained 1 hour after crossclamping the aorta; we obtained subsequent spectra every hour during storage and every 5 minutes during reperfusion. At the end, the hearts were dried and weighed. Phosphocreatine (PCr), gamma-adenosine triphosphate (gamma-ATP), inorganic phosphate (Pi), and phosphomonoesters (PME), were expressed per g dry heart weight. The intracellular pH (pH(i)) and the PCr/ATP ratio were calculated. RESULTS: During storage, we identified a significant but similar decrease of pH(i), PCr/ATP ratio, and PCr in both groups. During reperfusion, pH(i) and PCr/ATP ratio recovered similarly in both groups, whereas the recovery of PCr in the BD group was significantly lower (p < 0.05). The Pi and PME increased in both groups during storage but to a lesser extent in the BD group (p < 0.05). This difference disappeared during reperfusion. The gamma-ATP was already significantly lower in the BD group at the onset of storage, and this remained so throughout storage and reperfusion (p < 0.05 vs C). Contractile capacity was lost in all hearts, except for 1 heart in the BD group. CONCLUSION: Brain death-related failure of the energetic integrity of the feline donor heart becomes apparent only when using 31P MRS during ischemic preservation and subsequent reperfusion.

Animals↗
PubMed2001

Postischemic Na(+)-K(+)-ATPase reactivation is delayed in the absence of glycolytic ATP in isolated rat hearts.

Normalization of intracellular sodium (Na) after postischemic reperfusion depends on reactivation of the sarcolemmal Na(+)-K(+)-ATPase. To evaluate the requirement of glycolytic ATP for Na(+)-K(+)-ATPase function during postischemic reperfusion, 5-s time-resolution 23Na NMR was performed in isolated perfused rat hearts. During 20 min of ischemia, Na increased approximately twofold. In glucose-reperfused hearts with or without prior preischemic glycogen depletion, Na decreased immediately upon postischemic reperfusion. In glycogen-depleted pyruvate-reperfused hearts, however, the decrease of Na was delayed by approximately 25 s, and application of the pyruvate dehydrogenase (PDH) activator dichloroacetate (DA) did not shorten this delay. After 30 min of reperfusion, Na had almost normalized in all groups and contractile recovery was highest in the DA-treated hearts. In conclusion, some degree of functional coupling of glycolytic ATP and Na(+)-K(+)-ATPase activity exists, but glycolysis is not essential for recovery of Na homeostasis and contractility after prolonged reperfusion. Furthermore, the delayed Na(+)-K(+)-ATPase reactivation observed in pyruvate-reperfused hearts is not due to inhibition of PDH.

Adenosine Triphosphate↗
PubMed1999

Myocardial high-energy phosphate metabolism in heart transplant patients is temporarily altered irrespective of rejection.

A reliable, sensitive, non-invasive alternative for transvenous endomyocardial biopsy in detecting cardiac allograft rejection is desirable for optimal management of heart transplant patients. To establish whether (31)P magnetic resonance spectroscopy can become a non-invasive tool for detecting cardiac allograft rejection, the cardiac high-energy phosphate metabolism of human heart transplants was serially examined in 13 patients by means of (31)P MRS from post-operative day 13 to day 294, and compared with histologic evaluation of endomyocardial biopsies. Biopsy scores of 2 or higher, according to the Working Formulation criteria of Billingham et al., were considered to indicate rejection. Logistic regression, which was corrected for differences between the individual patients and the time after transplantation, showed no significant correlation between the occurrence of histologically detected rejection and the PCr:ATP ratio. However, using an analysis of variance, the PCr:ATP ratios of non-rejecting cases obtained within 50 days after transplantation (mean: 27 +/- 11 days) appeared to be significantly different from those obtained after post-operative day 50 [0.95 +/- 0.17 (n = 25) vs 1.17 +/- 0.17 (n = 32), mean +/- SD; p < 0.01]. No significant difference was observed between the PCr:ATP ratios obtained 100 days after transplantation (mean: 162 +/- 52 days) and the PCr:ATP ratios in the hearts of healthy volunteers [1.18 +/- 0. 18 (n = 19) and 1.23 +/- 0.17 (n = 6), mean +/- SD, respectively; p = 0.55]. The PCr:ATP ratio in transplanted human hearts is not a sensitive indicator for the detection of early acute human cardiac allograft rejection. This may be due to a temporarily altered high-energy phosphate metabolism early after transplantation irrespective of rejection.

Adenosine Triphosphate↗
PubMed1999

Bio-energetic response of the heart to dopamine following brain death-related reduced myocardial workload: a phosphorus-31 magnetic resonance spectroscopy study in the cat.

OBJECTIVE: Long-term exposure of the donor heart to high dosages of dopamine in the treatment of brain death-related hemodynamic deterioration has been shown to reduce myocardial phosphocreatine (PCr) and adenosine triphosphate (ATP) in myocardial biopsy specimens and may preclude heart donation for transplantation. Short-term exposure to the acute catecholamine release during the onset of brain death has shown an unchanged PCr/ATP ratio using in vivo phosphorus-31 magnetic resonance spectroscopy (31P MRS). In this study 31P MRS was used to evaluate in vivo myocardial energy metabolism during long-term dopamine treatment. METHODS: Twelve cats were studied in a 4.7 Tesla magnet for 360 minutes. At t = 0 minutes, brain death was induced (n = 6). At 210 minutes, when myocardial workload in the brain-death group was reduced significantly, dopamine was infused (n = 12) at 5 microg/kg/min and its dose was consecutively doubled every 30 minutes and was withheld during the last 30 minutes of the experiment. Phosphorus-31 magnetic resonance spectra were obtained from the left ventricular wall during 5-minute time frames, and PCr/ATP ratios were calculated. The hearts were histologically examined. RESULTS: Although significant changes in myocardial workload were observed after the induction of brain death and during support and withdrawal of dopamine in both groups, the initial PCr/ATP ratio of 2.00+/-0.12 and the contents of PCr and ATP did not vary significantly. Histologically identified sub-endocardial hemorrhage was observed in 3 of 6 of the brain-dead animals and in 1 of 6 of the control animals. CONCLUSIONS: High dosages of dopamine in the treatment of brain death-related reduced myocardial workload do not alter PCr/ATP ratios and the contents of PCr and ATP of the potential donor heart despite histologic damage.

Adenosine Triphosphate↗
PubMed1998

Changes of intracellular sodium T2 relaxation times during ischemia and reperfusion in isolated rat hearts.

The effect of ischemia and reperfusion on transverse relaxation (T2) of intracellular Na+ (Na+i) was measured with 5-min time resolution in isolated rat hearts. Nai T2 relaxation was biexponential with 28+/-7% fast (T2f) and 72+/-7% slow (T2s) decay. This ratio was constant throughout the protocol. During 20 min of ischemia, Na(i) T(2s) increased from 18.9+/-2.7 ms to 26.4+/-1.1 ms (P < 0.001), whereas T2f did not change significantly (3.1+/-1.8 versus 2.3+/-1.6 ms during control), and Na+i increased from 9.0+/-1.0 to 19.5+/-1.0 mmol/liter (P < 0.001). T(2s) and Na(+)i declined again during reperfusion. Changes in T2s relaxation correlated significantly (r = 0.73, P < 0.001) with the time course of Na+i.

Analysis of Variance↗
PubMed1998

Manipulation of intracellular sodium by extracellular divalent cations: a 23Na and 31P NMR study on intact rat hearts.

23Na and 31P NMR spectroscopy were used to follow intracellular [Na+] ([Na+]i) and energy metabolism in isolated, perfused rat hearts. During 30 min of Ca(2+)-free perfusion no significant change in [Na+]i could be detected, but during a subsequent 45 min period of ischemia [Na+]i rose significantly as expected, from 8.6 +/- 2.4 to 36.8 +/- 9.4 mM. In contrast, already during 30 min of Ca(2+)- and Mg(2+)-free perfusion [Na+]i rose significantly from 7.3 +/- 3.7 to 71.3 +/- 15.6 mM. During this period, the Na(+)-K+ ATPase was not limited by depletion of high energy phosphates, decrease of intracellular free Mg2+ or accumulation of inorganic phosphate. During the first 8 min of a subsequent period of ischemia, the rate of rise in [Na+]i even increased, suggesting that during the preceding period of Ca(2+)- and Mg(2+)-free perfusion, the Na(+)-K+ ATPase was indeed operative but apparently not coping with the large Na(+)-influx. Using verapamil, we could demonstrate that this large Na(+)-influx occurs through the L-type Ca2+ channels, and that both Mg2+ and verapamil can block this Na(+)-influx. Previously, we have demonstrated that [Na+]i does not play a role in the origin of the calcium paradox. The notion that an increased [Na+]i is a prerequisite for the calcium paradox to occur apparently results from experimental evidence obtained under conditions of low or absent Mg2+.

Adenosine Triphosphate↗
PubMed1998

Both Na+-K+ ATPase and Na +-H+ exchanger are immediately active upon post-ischemic reperfusion in isolated rat hearts.

Limited time resolution has hampered proper evaluation of changes in intracellular Na+ (Na+i) in whole hearts upon post-ischemic reperfusion. In isolated rat hearts perfused at 37 degrees C, we studied the contribution of the Na+-K+ ATPase and the Na+-H+ exchanger to control of Na+i during reperfusion using 23Na NMR and the shift reagent Tm(DOTP)5- with a time resolution of 5 s. To assess activities of the Na +-K+ ATPase and the Na+-H+ exchanger, 250 micro mol/l ouabain and/or 3 micro mol/l EIPA, respectively, was added to the perfusate during the first 5 min of reperfusion, following 20 min of ischemia. When used, ouabain was also present for 2 min prior to ischemia. Na+i increased during ouabain perfusion prior to ischemia (132+/-5 and 133+/-4% of the pre-ischemic control value after 2 min, in ouabain and ouabain+EIPA hearts, respectively; mean+/-s.e.m.; n=6 per group) resulting in higher end-ischemic values in ouabain and ouabain+EIPA hearts (249+/-9 and 267+/-17% of the pre-ischemic control value, respectively) than in control and EIPA hearts (207+/-21 and 199+/-10% of the pre-ischemic control value, respectively). In ouabain, hearts Na+i started to rise directly upon reperfusion and amounted to 117+/-6% of the end-ischemic value after 60 s of reperfusion. In control hearts, however, Na+i dropped immediately and was 87+/-5% of the end-ischemic value after 60 s, indicating that the Na+-K+ ATPase resumed function directly upon reperfusion. The initial steep increase of Na+i upon reperfusion in ouabain hearts, which diminished after approximately 40 s to the rate of increase observed during ischemia, was absent in ouabain + EIPA hearts. This indicates the existence, although masked by Na+-K+ ATPase activity, of a Na+-H + exchange mediated Na+ influx upon reperfusion. If only EIPA was present during reperfusion the initial decrease in Na+i was faster than in control hearts, corroborating this finding.

Amiloride↗
PubMed1998

Na+/Ca2+ exchange during Ca2+ repletion is not a prerequisite for the Ca2+ paradox in isolated rat hearts.

Ca2+ paradox damage has been suggested to be determined by Na+ entry during Ca2+ depletion and exchange of Na+ for Ca2+ during Ca2+ repletion. Since previously a Ca2+ paradox without prior increase of total intracellular [Na+] ([Na+]i) has been observed, we investigated whether local accumulation of Na+ close to the inner side of the sarcolemma during Ca2+ depletion plays a role in the Ca2+ paradox by replacing all extracellular Na+ by Li+ 5 min before and during 10 min Ca2+-free perfusion (37°C) in isolated rat hearts (group I). Subsequently, hearts were perfused with a standard, Na+- and Ca2+-containing solution. Verapamil was used to prevent contracture due to the absence of Na+/Ca2+ exchange during Na+-free perfusion in the presence of Ca2+. In group II, the Ca2+-free period was omitted, and in group III normal extracellular [Na+] was used throughout. 23Na-NMR was used to monitor intra- and extracellular Na+ signals. Total creatine kinase release was 2,977±413, 36±24 and 3170±297 IU/g dry weight in groups I, II and III respectively, indicating a full Ca2+ paradox in groups I and III. [Na+]i decreased from 11.3±0.6 mM during control perfusion to 1.2±0.4 mM after 10 min Ca2+ depletion in group I, whereas in group III [Na+]i was 10.9±2.2 mM during control perfusion and did not change significantly after 10 min Ca2+-free perfusion. It is concluded that accumulation of Na+ close to the inner side of the sarcolemma during Ca2+ depletion is not a prerequisite for the Ca2+ paradox.

Animals↗
PubMed1998

An increase in intracellular [Na+] during Ca2+ depletion is not related to Ca2+ paradox damage in rat hearts.

Ca2+ paradox damage has been suggested to be determined by Na+ entry during Ca2+ depletion and exchange of Na+ for Ca2+ during Ca2+ repletion. With the use of 23Na nuclear magnetic resonance, we previously observed a Ca2+ paradox without a prior Na+ increase. We have now demonstrated a Na+ increase during Ca2+ and Mg2+ depletion without the occurrence of the Ca2+ paradox during Ca2+ repletion. Isolated rat hearts were perfused for 20 min with a Ca(2+)-free or a Ca(2+)- and Mg(2+)-free (Ca2+/Mg(2+)-free) solution under hypothermic conditions (20 and 25 degrees C). Intracellular Na+ concentration ([Na+]i) increased from 11.9 +/- 1.2 to 26.9 +/- 5.8 mM (P < 0.001) during Ca2+/Mg(2+)-free perfusion at 20 degrees C, whereas no significant change in [Na+]i occurred during 20 min of Ca(2+)-free perfusion at 20 degrees C. In addition, we confirmed that [Na+]i did not change significantly during 20 min of normothermic Ca(2+)-free perfusion. Creatine kinase release during normothermic Ca2+ repletion in the 20 degrees C groups was approximately 10% and in the 25 degrees C groups 75% of the release in the normothermia group. Recovery of rate-pressure product was approximately 50% in the 20 degrees C groups versus 0% in the normothermia group. In conclusion, hypothermic Ca2+/Mg(2+)-free perfusion results in a significant increase of [Na+]i, which does not contribute to the extent of the Ca2+ paradox on normothermic Ca2+ repletion.

Animals↗
PubMed1998

31P NMR studies of creatine kinase flux in M-creatine kinase-deficient mouse heart.

Hearts of wild-type and cytosolic muscle creatine kinase (M-CK)-knockout mice were perfused with Krebs-Henseleit buffer containing 10 mM glucose and 5 mM pyruvate and studied during pacing at 400 and 600 beats/min and during K+ arrest. Phosphocreatine (PCr) and ATP concentrations in M-CK-deficient hearts were not significantly different from those in wild-type hearts. With the use of 31P NMR saturation transfer, the flux mediated predominantly by mitochondrial creatine kinase (Mi-CK) was clearly detected in M-CK-deficient hearts. Mi-CK flux was 4.8 +/- 0.6 and 4.5 +/- 0.6 mM/s during pacing at 400 and 600 beats/min, respectively, and was 3. 5 +/- 0.4 mM/s during cardiac arrest. In control hearts total CK flux was 7.8 +/- 1.1 and 6.6 +/- 1.3 mM/s during pacing at 400 and 600 beats/min, respectively, and decreased to 3.8 +/- 0.5 mM/s during arrest. It is suggested that the relative contribution of Mi-CK to the total NMR-measured CK flux in the wild-type heart is higher than that of the homodimeric M-CK isoform (MM-CK).

Adenosine Diphosphate↗
PubMed1996

Functional recovery after human heart transplantation is related to the metabolic condition of the hypothermic donor heart.

BACKGROUND: Although strict selection criteria are being used for the acceptance of human donor hearts for transplantation, problems with respect to functional recovery on reperfusion sometimes still occur. Therefore, evaluation of the viability of a human donor heart before implantation during heart transplantation may be of great value. METHODS AND RESULTS: In the present study, the energy metabolism of 25 excised human donor hearts arrested with St Thomas' Hospital No. 2 cardioplegic solution was evaluated noninvasively by use of 31P magnetic resonance spectroscopy (MRS) before implantation and was correlated with myocardial function measured with thermodilution in heart transplant patients. No significant correlation was observed between the cardiac index of heart transplant patients during the first hours after transplantation and the phosphocreatine/ATP (r = .13, P = .54), inorganic phosphate/ATP (r = .26, P = .21), phosphomonoesters/ATP (r = .02, P = .92), or phosphocreatine/inorganic phosphate (r = .16, P = .44) ratio or the intracellular pH (r = .06, P = .78) at the time of reperfusion. However, 1 week after transplantation, a significant correlation was observed between the cardiac index and the phosphocreatine/ATP (r = .49, P = .01), phosphomonoesters/ATP (r = .45, P = .02), and phosphocreatine/inorganic phosphate (r = .40, P = .05) ratios at the time of reperfusion. In contrast, the inorganic phosphate/ATP (r = .10, P = .63) ratio and pH (r = .31, P = .13) at the time of reperfusion showed a poor correlation with the cardiac index 1 week after transplantation. CONCLUSIONS: Functional recovery after human heart transplantation is related to the metabolic condition of the hypothermic donor heart.

Adolescent↗
PubMed1995

Quasi-linear relationship between Gibbs free energy of ATP hydrolysis and power output in human forearm muscle.

The postulated strictly linear descriptions of the rate dependence of oxidative phosphorylation in skeletal muscle on the free energy of ATP hydrolysis (delta GP) over the range of physiological steady states fail to harmonize with reported findings of identical basal respiration rates in mammalian muscles at different delta GP values. The relevance of an extension of the strictly linear description to a description deriving from enzyme kinetics that predicts a sigmoidal dependence was investigated in human finger flexor muscle using 31P-nuclear magnetic resonance spectroscopy. At constant pH 7.0, the experimental variation of adenine nucleotide concentrations with power output, which reflects the rate of oxidative phosphorylation, was compared with predictions by various formulations of adenine nucleotide control of respiration. The quasi-linear sigmoidal description was found to be statistically equivalent but physiologically superior to the strictly linear description. The predicted maximal oxidatively sustained steady-state power output and rate-dependent sensitivity of respiration to changes in delta GP were in agreement both with theoretical considerations and with experimental observations in the present study and other studies of intact mammalian skeletal muscle.

Adenosine Diphosphate↗
PubMed1993

Post-ischemic contractile dysfunction does not correlate with an elevated intracellular free [Mg2+]: a 31P-NMR study on isolated rat and rabbit hearts.

The aim of this study was to investigate whether intracellular free Mg2+ (Mgr), which increases during myocardial ischemia due to hydrolysis of ATP, remained elevated during reperfusion after a relatively short period of ischemia and thereby could account for temporary post-ischemic contractile dysfunction, often referred to as stunning. 31P-magnetic resonance (31P-NMR) spectroscopy was used to follow creatine phosphate, adenosine triphosphate, intracellular inorganic phosphate, intracellular pH and Mgr simultaneously with left ventricular developed pressure (LVDP) and coronary flow in isolated rat and rabbit hearts, which were perfused (37 degrees C) according to Langndorff. LVDP was measured in an isovolumic way by means of an intraventricular latex balloon. Rat hearts (300 beats/min) were made globally ischemic for 15 min and rabbit hearts (180 beats/min) for 15 or 20 min. All hearts were reperfused for 60 min. Control hearts were perfused for 75 min without being made ischemic. During ischemia Mgr (mmol/l) increased from 0.76 +/- 0.20 to 4.34 +2- 1.99 in the rat hearts, and from 0.72 +/- 0.22 to 2.18 +/- 1.06 (15 min) and 2.35 +/- 1.26 (20 min) in the rabbit hearts. During reperfusion Mgr in the three groups returned to the level of the control hearts within 7.5 min, and LVDP within 25 min. At the end of the reperfusion period ATP content amounted to 56 +/- 17% (rat hearts), 66 +/- 10% (rabbit hearts; 15 min ischemia group) and 61 +/- 7% (rabbit hearts; 20 min ischemia group) of the pre-ischemic levels. The results confirm that in vitro stunning is a short-lived phenomenon and indicate that an increased Mgr is not involved in this temporary mechanical dysfunction.

Adenosine Triphosphate↗
PubMed1989

Low Ca2+ reperfusion and enhanced susceptibility of the postischemic heart to the calcium paradox.

This study was designed to define the effect of postischemic low Ca2+ perfusion on recovery of high-energy phosphates, intracellular pH, and contractile function in isolated rat hearts. Phosphorus-31 nuclear magnetic resonance spectroscopy was used to follow creatine phosphate, adenosine triphosphate, intracellular inorganic phosphate, and intracellular pH during control perfusion (15 minutes), total ischemia (30 minutes), and reperfusion (30 minutes). In Group I the perfusate [Ca2+] was 1.3 mmol/l throughout the experiment, whereas in Group II the perfusate [Ca2+] was reduced to 0.05 mmol/l during the first 10 minutes of reperfusion. Hearts from Group III were not made ischemic but were subjected to 10 minutes of low Ca2+ perfusion followed by 20 minutes of normal Ca2+ perfusion. During low Ca2+ reperfusion (Group II) recovery of high-energy phosphates and pH was significantly better than in controls (Group I). However, after reexposure to normal Ca2+, metabolic recovery was largely abolished, coronary flow was suddenly impaired, and contracture developed without any rhythmic contractions. These observations indicated the occurrence of a calcium paradox rather than postponed ischemia-reperfusion damage. On the other hand, normoxic hearts (Group III) tolerated temporary perfusion with 0.05 mmol/l Ca2+ very well with respect to left ventricular developed pressure, coronary flow, and metabolic parameters. In conclusion, postischemic low Ca2+ (0.05 mmol/l) perfusion may reduce reperfusion damage, but at the same time ischemia appears to enhance the susceptibility of the heart to the calcium paradox.

Adenosine Triphosphate↗
PubMed1988

Protective effect of pretreatment with the calcium antagonist anipamil on the ischemic-reperfused rat myocardium: a phosphorus-31 nuclear magnetic resonance study.

To assess whether the prophylactic administration of anipamil, a new calcium antagonist, protects the heart against the effects of ischemia and reperfusion, rats were injected intraperitoneally twice daily for 5 days with 5 mg/kg body weight of this drug. The heart was then isolated and perfused by the Langendorff technique. Phosphorus-31 nuclear magnetic resonance spectroscopy was used to monitor myocardial energy metabolism and intracellular pH during control perfusion and 30 min of total ischemia (37 degrees C), followed by 30 min of reperfusion. Pretreatment with anipamil altered neither left ventricular developed pressure under normoxic conditions nor the rate and extent of depletion of adenosine triphosphate (ATP) and creatine phosphate during ischemia. Intracellular acidification, however, was attenuated. On reperfusion, hearts from anipamil-pretreated animals recovered significantly better than untreated hearts with respect to replenishment of ATP and creatine phosphate stores, restitution of low levels of intracellular inorganic phosphate and recovery of left ventricular function and coronary flow. Intracellular pH recovered rapidly to preischemic levels, whereas in untreated hearts a complex intracellular inorganic phosphate peak indicated the existence of areas of different pH within the myocardium. It is concluded that anipamil pretreatment protects the heart against some of the deleterious effects of ischemia and reperfusion. Because this protection occurred in the absence of a negative inotropic effect during normoxia, it cannot be attributed to an energy-sparing effect during ischemia. Therefore, alternative mechanisms of action are to be considered.

Animals↗
PubMed1982

Further aspects of the Ca2+-dependent polymorphism of bovine heart cardiolipin.

The influence of cations on the structure of aqueous dispersions of the sodium salt of bovine heart cardiolipin was investigated using binding experiments, 31P-NMR, freeze-fracture electron microscopy, small angle X-ray diffraction and batch calorimetry techniques. In the 1-3 mM concentration range, Ca2 induces a bilayer leads to hexagonal HII transition for the lipid. During this transition there is a marked increase in Ca2+ binding from a maximum of 0.35 Ca/cardiolipin in the bilayer to 1.0 Ca/cardiolipin in the hexagonal HII phase. Only when the cardiolipin liposomes are exposed to locally high Ca2+ concentrations is the bilayer leads to hexagonal HII transition accompanied by the appearance of an intermediate 'isotropic' structure characterized by an isotropic 31P-NMR signal and lipidic particles. In contrast, in mixed dioleoylphosphatidylcholine/cardiolipin (1:1) liposomes, Ca2+ concentrations as low as 100 microM will induce an 'isotropic' structure under conditions where no locally high Ca2+ concentrations can occur. In this system at higher Ca2+ concentrations (above 5 mM) the hexagonal HII phase formation occurs. At least 80% of the phosphatidylcholine can be incorporated into this phase. The Ca2+ -induced bilayer to hexagonal transition is an endothermic reaction with a delta H of approx. 1.8 kcal/mol. Removal of Ca2+ from the hexagonally organized calcium-cardiolipin (1:1) complex by dialysis is an extremely slow process with a half-time in excess of 80 h. After 23 h of dialysis at a Ca/cardiolipin ratio of 0.86 an 'isotropic' structure is observed, characterized by an isotropic 31P-NMR signal and the presence of lipidic particles. After 70 h of dialysis (Ca/cardiolipin = 0.7) a new phase is observed. This phase which is optically isotropic and highly viscous separates from a lipid-free aqueous phase and contains 111 mM cardiolipin (15.5% by weight). The phospholipid molecules undergo rapid isotropic motion and the freeze-fracture morphology indicates the presence of a highly curved interconnected bilayer network separating various aqueous compartments. No defined X-ray diffraction bands can be observed for this phase. These characteristics are typical for cubic phases. This phase is metastable as mechanical agitation immediately induces the formation of large bilayer vesicles.

Animals↗