Apparent Km of mitochondria for oxygen computed from Vmax measured in permeabilized muscle fibers is lower in water enriched in oxygen by electrolysis than injection

Introduction

In the course of studying the possible biological effects of a water enriched in oxygen (O₂) by a new electrolytic process,¹ we have recently shown that, when compared to water enriched in O₂ by injection, intragastric administration of water enriched in O₂ by electrolysis lessened the slow decline in peripheral tissue oxygenation observed in anesthetized pigs.² This observation is consistent with the hypothesis that O₂ diffusion can be facilitated in waters enriched in O₂ by electrolysis. It has been hypothesized that this phenomenon could be due to the fact that the electrolytic process could generate water susperstructures similar to clathrates,^3–5 which could trap O₂ molecules and reduce local pressure/content relationships for O₂, thus facilitating O₂ diffusion along PO₂ gradients. Interestingly, a series of studies conducted in vitro and in animal models suggests that isotonic saline submitted to a process involving Taylor–Couette–Poiseuille flow in the presence of O₂ (electrokinetically modified water [EMW]) can interfere with cell signaling pathways involved in inflammation, cell death, and survival, and could have beneficial effects in various situations where inflammation is present.^6–11 In human, EMW tends to increase VO₂max and reduces the rate of perceived exertion during aerobic exercise,⁷ significantly reduces muscle fatigue during resistance exercise,¹² and prevents or attenuates muscle damage and inflammation in both types of exercise.^12–14 The authors hypothesized that these biological effects of EMW can also be due to the fact that this water could contain “charge-stabilized nanostructures”.

The objective of the present study was to further investigate the biological effects of water enriched in O₂ by electrolysis by comparing mitochondrial respiration (Vmax) studied in situ in permeabilized muscle fibers¹⁵ in waters enriched by injection and by the electrolytic process. The observations were made over a wide range of PO₂ and O₂ concentrations in order to compute the apparent Km of mitochondria for O₂. Since the rate of O₂ consumption by mitochondria depends on the flux of both O₂ and electrons,¹⁶ the experiments were performed with glutamate–malate, as well as with tetramethyl-p-phenylenediamine (TMPD)-ascorbate, an artificial substrate that provides electrons to complex IV of the respiratory chain at a much larger rate than glutamate–malate.¹⁷ Under the hypothesis that O₂ diffusion is facilitated in water enriched in O₂ by the electrolytic process, when compared to the water enriched in O₂ by injection, Km will be lower with both electron donors and the difference will be larger with TMPD–ascorbate than with glutamate–malate because of the higher flux of electrons. We also verified that the possible differences in mitochondrial respiration between the two waters and the two electron donors were not due to differences in the affinity of mitochondria to adenosine diphosphate (ADP) by measuring the apparent Km of mitochondria for ADP.

Materials and methods

Animal procedures were conducted in accordance with the Declaration of Helsinki and were approved by our local ethics committee Comité Régional d’Éthique en Matière d’Expérimentation Animale, (CREMEAS).

Mitochondrial respiration was studied in situ in muscle fibers permeabilized with saponin.^15,18,19 As reviewed in detail by Kuznetsov et al,¹⁵ saponin is a detergent with a large affinity for cholesterol, which specifically destroys the cholesterol-rich sarcolemma (0.5 mmol cholesterol/mmol phospholipids) without altering the cholesterol-poor mitochondrial membrane (0.07 and 0.01 cholesterol/mmol phospholipids for the outer and inner mitochondrial membranes, respectively). The end result is a preparation with intracellular structures that are intact (mitochondrion, endoplasmic reticulum, myofilaments, and cytoskelton) within an intracellular space devoid of all solutes, which have been washed out, and in equilibrium with the incubation milieu.

Male Wistar rats (body mass ~300 g) were anesthetized (sodium pentobarbitate: 0.2 g/100 g body mass, intraperitoneally) and the soleus muscles were removed and placed in solution S (as will be discussed). Muscle fibers were immediately separated under binocular microscope and permeabilized with 50 μg/mL of saponin for 30 minutes at 4°C. After being placed for 10 minutes in solution R (as will be discussed) to wash out adenine nucleotides and creatine, skinned separated fibers were transferred in a 3 mL water-jacketed oxygraphic cell (Strathkelvin Instruments Limited, North Lanarkshire, Scotland) equipped with a Clark electrode, as previously described.²⁰

Solutions R and S (prepared from tap water demineralized by reverse osmosis and then remineralized with Na₂SO₄ + Na₃PO₄, 12H₂O) both contained 2.77 mM of CaK₂EGTA, 7.23 mM of K₂EGTA (100 nM of free Ca²⁺), 6.56 mM of MgCl₂ (1 mM of free Mg²⁺), 20 mM of taurine, 0.5 mM of dithiothreitol, 50 mM of potassium methanesulfonate (160 mM ionic strength), and 20 mM of imidazole (pH 7.1). Solution S also contained 5.7 mM of Na₂ ATP and 15 mM of creatine phosphate, while solution R contained 5 mM of glutamate, 2 mM of malate, 3 mM of phosphate, and 2 mg/mL of fatty acid-free bovine serum.

Mitochondrial respiration was measured in situ in solution R at 22°C starting with an initial O₂ concentration of 240 μmol/L (PO₂=133 mmHg).²¹ In the solution used as control, this concentration was obtained by injection. In the experimental solution, this concentration was obtained by electrolysis.¹ In this process, remineralized water is pumped between two electrodes separated by a membrane permeable to electrical charges but not to gases, and the water enriched in O₂, which is recovered on the negative electrode (pH =7.1–7.2; conductivity =750–770 μS/cm; 4,375–5,000 μmol O₂/L), is used to prepare the final product. Both solutions, which were supplied by Danone Research, (Palaiseau, France), were prepared from demineralized water, which was remineralized with Na⁺ (200 mg/L), SO₄²⁻ (250 mg/L), and PO₄²⁻ (240 mg/L). It was suspected that the electrolytic process could produce ozone which, in turn, could interfere with mitochondrial function through the generation of reactive oxygen species. However, in line with the observation that ozone is quickly converted into O₂ (2 O₃ → 3 O₂, with a half-life of only about 20 minutes), and because the solution enriched by electrolysis was prepared and conserved in sealed bottles several weeks before the experiment, the level of ozone was found to be very low (6.8±5.5 μg/L).

In the first series of experiments, after the determination of basal O₂ consumption (nonstimulated respiration, V₀), Vmax was measured under continuous stirring in the presence of saturating amounts of ADP (2 mM) as a phosphate acceptor and glutamate–malate as substrates (5 mM and 2 mM). The Vmax was monitored until the O₂ content decreased from the initial value of 240 μmol/L to 10 μmol/L, corresponding to a PO₂ of 5 mmHg²¹ – ie, a value close to the range of PO₂ estimated in muscle cells (2–4 mmHg).²² In the second series of experiments, the protocol was similar, but complex III of the respiratory chain was blocked with antimycin A (6.5 μM), and N, N, N′, N′-tetramethyl-p-phenylenediamine dihydrochloride (TMPD; 0.5 mM) and ascorbate (0.5 mM) were added as artificial electron donor to cytochrome c. In the third series of experiments, mitochondrial respiration was measured at increasing concentrations of ADP (from 2.5 μmol/L to 1.5 mmol/L) and decreasing PO₂ (from the initial value of 240 μmol/L to final values <10 μmol/L at the end of titration) using glutamate–malate as substrates (5 mM and 2 mM) and without creatine. Each of the first and second series of experiments was conducted on 12 preparations, while the third series of experiments was conducted on eleven preparations. Each preparation was obtained from a different animal.

In all the preparations, at the end of each experiment, cytochrome c was added to the oxygraphic cell. No increase in O₂ consumption was observed in any of the preparations, confirming the integrity of the outer mitochondrial membrane.²³ In addition, the acceptor control ratio (Vmax/V₀) was computed for each preparation in the first series of experiments (glutamate–malate as substrates) and was found to be 3.7±0.3, which is in good agreement with values reported in the literature.^24–26

The fibers were then harvested and dried, and respiration rates were expressed as μmol O₂/min/g dry weight.

All reagents were purchased from Sigma-Aldrich Co. (St Louis, MO, USA), except ADP, which was obtained from Boehringer Ingelheim (Ingelheim, Germany).

Data are reported as the mean ± standard deviation. The apparent Km of mitochondria for O₂ (for the two types of water and with the two electron donors) and for ADP (for the two types of water and glutamate–malate) were calculated using nonlinear regression of the individual relationships between O₂ or ADP concentration and Vmax, using a Lineweaver–Burk plot. The apparent Km for O₂ (Km O₂) was compared using a two-way analysis of variance (ANOVA) (water × electron donor), while the apparent Km for ADP for the two waters was compared using one-way ANOVA. The level of significance was set at 0.05. All data are available in the Supplementary materials.

Results

Figure 1A shows the increase in Vmax with O₂ concentration measured for the two types of water and the two electron donors. As shown in Figure 1B, the Km O₂ computed from these curves was significantly lower in the solution enriched in O₂ by electrolysis than by injection, both with glutamate–malate (36.1±9.2 μmol/L versus 53.3±10.1 μmol/L, respectively; P<0.001) and TMPD–ascorbate as substrates (52.8±11.4 μmol/L and 90.3±10.7 μmol/L, respectively; P<0.001). The difference in Km O₂ between the two waters cannot be computed for each preparation and, consequently, no statistical comparisons can be made. However, the average difference was larger with TMPD–ascorbate (37.5 μmol/L or a 41% difference) than with glutamate–malate (17.2 μmol/L, or a 32% difference).

Figure 1 Vmax in solutions enriched in O2 by injection and electrolysis for O2 concentrations, as well as apparent Km of the mitochondria for O2 with the two waters and two electron donors. Notes: (A) Vmax in solutions enriched in O2 by injection and electrolysis for O2 concentrations ranging between 10 μmol/L and 240 μmol/L in rat permeabilized soleus muscle fibers with glutamate–malate and TMPD–ascorbate as substrates. (B) Apparent Km of the mitochondria for O2 with the two waters and the two electron donors. Mean ± standard deviation; n=12; #statistically different from glutamate–malate and *from injection, P<0.05. Abbreviations: dw, dry weight; TMPD, tetramethyl-p-phenylenediamine; n, number.

No significant difference was observed for the apparent Km of mitochondria for ADP: 328±67 μmol/L and 303±70 μmol/L (P=0.4) between the water enriched in O₂ by injection and electrolysis, respectively (Figure 2).

Figure 2 Vmax in solutions enriched in O2 by injection and electrolysis at increasing ADP concentrations, and apparent Km of the mitochondria for ADP with the two waters. Notes: (A) Vmax in solutions enriched in O2 by injection and electrolysis at increasing ADP concentrations in rat permeabilized soleus muscle fibers with glutamate–malate as substrates. (B) Apparent Km of the mitochondria for ADP with the two waters. Mean ± standard deviation; n=11; no significant difference was observed between the two waters. Abbreviations: dw, dry weight; ADP, adenosine diphosphate; n, number.

Discussion

Results from the present experiment show that, when compared to a solution enriched in O₂ by injection, the apparent affinity for O₂ of the mitochondria was higher in a solution enriched in O₂ by the electrolytic process, the difference being larger with TMPD–ascorbate than glutamate–malate as electron donors. This observation is consistent with the hypothesis that O₂ diffusion can be facilitated in water enriched in O₂ by electrolysis.

As expected, Vmax increased with O₂ concentration in a curvilinear fashion toward a plateau and was significantly higher with TMPD–ascorbate than with glutamate–malate, the difference decreasing slightly with O₂ concentration (~14.3 μmol O₂/min/g versus 7.8 μmol O₂/min/g at 240 μmol/L, or a 75% difference; ~2.6 μmol O₂/min/g versus 1.8 μmol O₂/min/g at 10 μmol/L, or a 45% difference) (Figure 1A). The reduction in Vmax with O₂ concentration was larger than that reported by Gnaiger and Kuznetsov²⁷ with succinate and TMPD–ascorbate in isolated mitochondria because the apparent Km of cytochrome oxidase (COX) for O₂ has been shown to be higher in saponin-skinned muscle fibers. As for the difference observed between glutamate–malate and TMPD–ascorbate, this phenomenon is known as the “cytochrome oxidase excess”¹⁶ and indicates that when O₂ availability is very high, electron supply from glutamate–malate through the respiratory chain is much lower than could be processed by COX: when electrons are directly supplied to COX at a larger rate from TMPD–ascorbate, Vmax reaches higher values. However, the difference between the two electron donors decreased with O₂ concentration. This observation is due to the fact that the control of O₂ consumption in complex IV of the respiratory chain at any O₂ concentration and over the range of electron supply by glutamate–malate and TMDP–ascorbate is shared by the availability of both O₂ and electrons; when compared to glutamate–malate, in spite of the much larger electron flux from TMPD–ascorbate, the difference in Vmax between the two substrates decreases with O₂ supply.

The main result from the present experiment is that for both electron donors, when compared to the water enriched in O₂ by injection, a left shift in O₂ consumption by the mitochondria was observed with the water enriched in O₂ by electrolysis. Vmax plateau values were similar at the highest O₂ concentration for a given electron donor (7.8 μmol O₂/min/g for both water with glutamate–malate; 14.2 μmol O₂/min/g and 14.7 μmol O₂/min/g for water enriched in O₂ by electrolysis and injection, respectively). However, for any given Vmax, consistently lower O₂ concentrations were needed with the water enriched in O₂ by electrolysis in the steepest portion of the curve, the difference between the two waters being slightly larger with TMPD–ascorbate than glutamate–malate as substrates. As a consequence, when compared to the water enriched in O₂ by injection, the shift in the apparent Km O₂ of the mitochondria with the water enriched in O₂ by electrolysis was also larger.

The higher affinity of the mitochondria for O₂ in water enriched in O₂ by electrolysis was not associated with any increase in the sensitivity of the mitochondrial respiration to ADP since the apparent Km for ADP measured with glutamate–malate remained unaffected by the type of water used to prepare the solution. In addition, this phenomenon was observed with glutamate–malate, which provides electrons to COX through complex I to III of the respiratory chain complex, but also with TMDP–ascorbate, which directly provides electrons to COX. The reduction in the apparent Km of mitochondria in water enriched in O₂ by injection thus suggests that for a given O₂ concentration, O₂ flux to COX is facilitated in the solution enriched in O₂ with the electrolytic process. This phenomenon is not observed with any of the two electron donors at high O₂ content because in these situations, the control of O₂ consumption by COX depends mainly on the availability of electrons. It is only observed at lower O₂ concentrations where the rate of O₂ consumption by COX mainly depends on the availability of O₂, and it was higher with TMPD–ascorbate, which supplies a larger flux of electrons than does glutamate–malate.

Conclusion

These findings are in line with our recent observation in pigs using intragastric administration of water enriched in O₂ that, when compared to the water enriched in O₂ by injection, the decrease in transcutaneous O₂ pressure, which develops during anesthesia, was slower with the water enriched in O₂ by electrolysis.² Although the mechanisms behind these observations have to be elucidated, they are consistent with the hypothesis that O₂ diffusion could be facilitated in water enriched in O₂ by electrolysis.

Disclosure

François Péronnet and Ruddy Richard are occasional consultants for Danone Research. Alexis Klein and Liliana Jimenez are employees of Danone Research. The authors report no further conflicts of interest in this work.

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Supplementary materials

Table S1 Individual values of oxygen consumption, in μmol O2/min/g dw, and apparent affinity for oxygen (Km O2) of mitochondria in permeabilized muscle fiber preparations at decreasing O2 content (from 240 μmol/L to 10 μmol/L) in solution enriched in O2 by injection with glutamate–malate as an electron donor Abbreviations: dw, dry weight; SD, standard deviation.

Table S2 Individual values of oxygen consumption, in μmol O2/min/g dw, and apparent affinity for oxygen (Km O2) of mitochondria in permeabilized muscle fiber preparations at decreasing O2 content (from 240 μmol/L to 10 μmol/L) in solution enriched in O2 by injection with TMPD–ascorbate as an electron donor Abbreviations: dw, dry weight; TMPD, tetramethyl-p-phenylenediamine; SD, standard deviation.

Table S3 Individual values of oxygen consumption, in μmol O2/min/g dw, and apparent affinity for oxygen (Km O2) of mitochondria in permeabilized muscle fiber preparations at decreasing O2 content (from 240 μmol/L to 10 μmol/L) in solution enriched in O2 by electrolysis with glutamate–malate as electron donor Abbreviations: dw, dry weight; SD, standard deviation.

Table S4 Individual values of oxygen consumption, in μmol O2/min/g dw, and apparent affinity for oxygen (Km O2) of mitochondria in permeabilized muscle fiber preparations at decreasing O2 content (from 240 μmol/L to 10 μmol/L) in solution enriched in O2 by electrolysis with TMPD–ascorbate as electron donor Abbreviations: dw, dry weight; TMPD, tetramethyl-p-phenylenediamine; SD, standard deviation.

Table S5 Individual values of Vmax, in μmol O2/min/g dw, and apparent affinity of mitochondria for ADP (Km ADP, in μmol/L) in permeabilized muscle fiber preparations in solution enriched in O2 by injection or electrolysis with glutamate–malate as electron donor Abbreviations: dw, dry weight; ADP, adenosine diphosphate; SD, standard deviation.