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1 From the Department of Pediatric Gastroenterology (MJR) and the Laboratory for Metabolic Diseases (HWHCS, MB, and RB), the University Children's Hospital, Utrecht, Netherlands; the Department of Clinical Chemistry, Vrije Universiteit Medical Center, Amsterdam (KM); the Laboratory for Metabolic Diseases, the Department of Pediatrics, the University Hospital Groningen, Groningen, Netherlands (D-JR); and the Robert Schwartz, MD, Center for Metabolism and Nutrition, MetroHealth Medical Center, Case Western Reserve University School of Medicine, Cleveland (SCK).
2 Supported by the Foundation Speurwerk in de Kindergeneeskunde (University Children's Hospital Het Wilhelmina Kinderziekenhuis) and by the Dutch Foundation De Drie Lichten. 3 Reprints not available. Address correspondence to K de Meer, Department of Clinical Chemistry, Reception K, Vrije Universiteit Medical Center, PO Box 7057, 1007 MB Amsterdam, Netherlands. E-mail: k.demeer{at}vumc.nl.
ABSTRACT
Background: A high-fat diet has been recommended for the treatment of patients with mitochondrial myopathy due to complex I (NADH dehydrogenase) deficiency (CID).
Objective: This study evaluated the effects of intravenous infusion of isoenergetic amounts of triacylglycerol or glucose on substrate oxidation, glycolytic carbohydrate metabolism, and exercise endurance time and energy state of muscle in CID patients.
Design: Four CID patients and 15 control subjects were infused with triacylglycerol (3.7 mg·kg-1·min-1) or glucose (10 mg·kg-1·min-1) during low-intensity leg exercise. Respiratory calorimetry was used to evaluate mitochondrial substrate oxidation. The concentration and rate of appearance of plasma lactate (from dilution of [1-13C]lactate) were used to evaluate glycolytic carbohydrate metabolism. 31P magnetic resonance spectroscopy was used to determine ratios of phosphocreatine to inorganic o-phosphate in forearm muscle during exercise.
Results: In 3 patients, leg exercise endurance time was better during the triacylglycerol infusion than during the glucose infusion. In all 4 patients, whole-body oxygen consumption rates during exercise were higher during triacylglycerol infusion than during the glucose infusion. In 3 patients, the concentration and rate of appearance of plasma lactate were lower during triacylglycerol infusion than during the glucose infusion. Ratios of phosphocreatine to inorganic o-phosphate during exercise were not significantly different between the 2 infusion studies or between the patients and control subjects.
Conclusions: Triacylglycerol infusion is associated with a greater oxidation of substrates, lower rates of appearance and concentrations of plasma lactate, and greater leg exercise endurance time in myopathic CID patients than is glucose infusion. The energy state of muscle during exercise, however, was not significantly different after infusion of triacylglycerol or glucose.
Key Words: Complex I deficiency • exercise • triacylglycerol infusion • mitochondrial myopathy • stable isotopes • 31P magnetic resonance spectroscopy
INTRODUCTION
In our preceding article in this issue, we proposed a potential role for high-fat, low-carbohydrate diets in the treatment of patients with mitochondrial myopathy due to complex I (NADH dehydrogenase; EC 1.6.99.3) deficiency (CID; 1). We hypothesized that supplementation with fatty acids (triacylglycerol) might improve the oxidation of substrates in CID patients by supplying FADH2-linked reducing equivalents (electrons) to the mitochondrial respiratory chain distal to complex I. This action would make these patients' muscle cells less dependent on glycolytic carbohydrate metabolism, ie, the rate of appearance (Ra) and concentration of plasma lactate might be lower during triacylglycerol infusion than during glucose infusion. We documented that substrate oxidation rates were stimulated rather than impaired in resting, myopathic CID patients during infusion of glucose or triacylglycerol. In addition, triacylglycerol infusion did not lower the Ra or concentration of plasma lactate to resting control values. The finding that these patients showed no clinical signs or symptoms of CID at rest indicates that in vivo cellular energy balance was maintained during the triacylglycerol or glucose infusion, despite in vitro mitochondrial impairment. During exercise, when energy requirements in muscle are elevated, in vivo mitochondrial oxidation of NADH may become limiting in CID. Triacylglycerol administration then may be of benefit to these patients because fatty acid oxidation increases the relative supply of FADH2, thereby improving oxygen consumption (
SUBJECTS AND METHODS
Subjects
Details about the CID patients and healthy control subjects are provided in the preceding article (1). Briefly, the 4 CID patients had similar clinical histories, ie, easily fatiguable mild muscle weakness dating back to early childhood that remained stable over time. Therefore, exercise intolerance was the dominant clinical symptom at the time of the study. CID was diagnosed with microscopic and biochemical investigations in fresh biopsy specimens of the quadriceps (vastus lateralis) muscle, which showed markedly decreased activity of complex I in all patients (range: 5.6–23.8% of normal). Eighteen healthy control subjects matched for age (
All participants reported to the Laboratory for Metabolic Diseases (University Children's Hospital, Utrecht, Netherlands)
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TABLE 1 . Physical characteristics of individual patients with complex I deficiency (CID) and healthy control subjects at baseline1
Experimental protocol
The patients and control subjects reported to the Laboratory for Metabolic Diseases at 0800 on 2 occasions separated by 7 d. Before exercise was initiated, all subjects were infused intravenously with either glucose (10% wt:vol, 5 mg·kg-1·min-1) or a triacylglycerol emulsion (Intralipid, 20% wt:vol, 1.85 mg·kg-1 ·min-1; Fresenius Kabi, ‘s-Hertogenbosch, Netherlands) and heparin (7.5 U·kg-1·h-1; prime 14 U/kg) for 120 min, during which time the subjects remained at rest. Next, the stationary cycling exercise began at 15% Wmax (all patients and 7 control subjects) or 7 W (8 control subjects). The control subjects exercised for a nominal period of 90 min, whereas the patients exercised until exhaustion ensued. During exercise, the triacylglycerol and glucose infusion rates were doubled to 3.7 and 10 mg·kg-1·min-1, respectively.
Isotope infusion
Primed, constant infusions of [6,6-2H2]glucose (98% enriched; Mass Trace, Woburn, MA) were administered in all patients and in 9 control subjects as previously described (1). An unprimed, constant infusion of [1-13C]lactate (98% enriched; Mass Trace) was administered throughout the nominal 90-min exercise period. The [1-13C]lactate tracer infusion rates for the CID patients (n = 4) ranged from 2.50 to 5.34 µmol·kg-1·min-1; control subjects (n = 9) received 0.56 µmol·kg-1·min-1. The remaining 6 control subjects received no lactate tracer, to examine the effects of triacylglycerol or glucose infusion on the background enrichment of lactate.
Blood sampling and urine collection
Blood samples were drawn at regular intervals during exercise and handled as described previously (1). All subjects voided before and after exercise, and the urine was collected for measurement of nitrogen excretion.
Respiratory calorimetry
Open-circuit indirect calorimetry with use of a face mask was performed continuously during the 90-min exercise period. Stable
Three of the CID patients (patients 1, 2, and 3) and 3 control subjects reported to the MRS facility at 0800 for the triacylglycerol or glucose infusion on 2 separate days and received an intravenous infusion of either triacylglycerol plus heparin or glucose during a 100-min basal resting period as described above. The infusions were maintained at the same rate throughout the following 30-min study period. Peak ratios of phosphocreatine (PCr) to ß-ATP and of inorganic o-phosphate (Pi) to ß-ATP were measured at rest and during exercise at an average of 6 normalized power output levels; concentrations of Pi and PCr were calculated from each ratio, respectively, as described elsewhere (4).
The results obtained at rest were reported previously (1). Exercise consisted of bulb-squeezing at an audio signal frequency of 0.33 Hz (duration, 300 ms) with use of only the fourth and fifth digits at progressively increasing submaximal workloads in a ramp protocol with feedback of power output (details described previously; 5). Power output was measured and recorded as developed pressure x the displaced volume of air and was normalized to power output during maximal voluntary contraction (MVC). The highest implemented workload in the patients ranged from 30% MVC to 45% MVC and was 50% MVC in the control subjects. All subjects were right-handed.
Studies were conducted on the superficial mass of the flexor digitorum profundus (FDP) muscle of the right forearm, as described in detail elsewhere (5). The FDP muscle is affected in CID patients, as evidenced by observed decreases in MVC output of the muscle comparable with decreases in Wmax measured on a cycle ergometer (MJ Roef, K de Meer, unpublished observations, 1996). Radio frequency pulsing during exercise was gated to the audio signal that synchronized bulb-squeezing with a home-built audiotriggering device. To minimize motion artifacts and improve standardization of the measurements, the radio frequency pulse was set to occur 1250 ms after contraction.
Sample analysis
Blood and urine samples and MR spectra were analyzed as described previously (1).
Calculations
Whole-body glucose rate of appearance
In steady state experiments, the whole-body glucose Ra was calculated as follows (6):
RESULTS
Comparison between the CID patients and the control subjects at the group level: plasma concentrations, oxygen consumption, and substrate utilization
In control subjects exercising at 7 W, plasma glucose, plasma insulin, and blood pyruvate concentrations were significantly higher during the glucose infusion than during the triacylglycerol infusion, as expected (Table 2). Plasma fatty acid, triacylglycerol, and glycerol concentrations were significantly higher during the triacylglycerol infusion than during the glucose infusion. Plasma lactate, plasma cortisol, blood ß-hydroxybutyrate, and blood acetoacetate concentrations and lactate-pyruvate ratios were not significantly different between the 2 infusion conditions.
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TABLE 2 . Plasma substrate concentrations during cycling exercise and infusion of triacylglycerol or glucose in patients with complex I deficiency (CID) and healthy control subjects1
Plasma glucose, insulin, and lactate concentrations and lactate-pyruvate ratios were significantly lower during the glucose infusion in control subjects exercising at 15% Wmax than in control subjects exercising at 7 W (data not shown); however, plasma substrate concentrations during the triacylglycerol infusion were not significantly different between the 2 exercise conditions.
Plasma glucose and insulin concentrations were not significantly different between the CID patients and the control subjects exercising at either 7 W or 15% Wmax during infusion of either substrate. Plasma lactate concentrations were significantly higher in the patients than in the control subjects during the triacylglycerol and glucose infusions. Blood pyruvate concentrations and lactate-pyruvate ratios were significantly higher in the patients than in the control subjects during infusion of both substrates. Plasma fatty acid concentrations were significantly lower in the patients than in control subjects during the triacylglycerol infusion, but were not significantly different between the 2 subject groups during the glucose infusion. During the triacylglycerol infusion, the ratio of ß-hydroxybutyrate to acetoacetate in blood was significantly higher in the patients than in the control subjects exercising at 15% Wmax (data not shown for control subjects).
In control subjects, whole-body
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TABLE 3 . Respiratory calorimetry and the rate of appearance (Ra) of whole-body glucose during cycling exercise and infusion of triacylglycerol or glucose in patients with complex I deficiency (CID) and healthy control subjects1
Although there was no significant main effect of infusion condition, group, or condition x group interaction on whole-body
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TABLE 4 . Whole-body oxygen consumption (
Comparison of outcome parameters between the individual CID patients and the control subjects
Whole-body oxygen consumption, plasma lactate, and lactate+pyruvate Ra
Changes in
FIGURE 1. . Mean differences (
Additional effects of the triacylglycerol infusion on plasma lactate concentrations and lactate+pyruvate Ra were shown in 2 and 3 of the 4 patients, respectively. In patient 2, the triacylglycerol infusion had an additional effect on plasma lactate concentrations and lactate+pyruvate Ra.
Leg exercise endurance time
The control subjects completed the leg exercise protocol with ease, irrespective of the substrate being infused (Figure 2). Patients 1, 2, and 3 had to stop exercising prematurely (between 48 and 65 min) during the glucose infusion because of muscle fatigue and exhaustion, whereas patient 4 completed the nominal 90-min study period. All patients completed the 90-min cycling exercise during the triacylglycerol infusion.
FIGURE 2. . Leg exercise endurance time during stationary cycling exercise in 4 patients with complex I deficiency (CID) during infusion of glucose () or triacylglycerol ().
31P MRS measurements of PCr-Pi ratios at rest and during exercise
PCr-Pi ratios measured by 31P-MRS at rest and during exercise in one typical CID patient and in one typical control subject are shown in Figure 3. Relative exercise levels in the forearm exercise ramp protocol during the triacylglycerol infusion were not significantly different from those during the glucose infusion in both the patients and control subjects. At rest, PCr-Pi ratios were lower in all patients than in the control subjects during infusion of both triacylglycerol and glucose. During exercise, PCr-Pi ratios were not significantly different during the triacylglycerol infusion than during the glucose infusion in either the patients or control subjects.
FIGURE 3. . Ratios of phosphocreatine (PCr) to inorganic o-phosphate (Pi) at rest and during forearm bulb-squeeze exercise in a typical patient with complex I deficiency (A) and in a typical control subject (B) during the triacylglycerol () and glucose () infusions. MVC, maximal voluntary contraction.
DISCUSSION
Oxidative phosphorylation in the muscle of patients with mitochondrial CID is stimulated during exercise, whereas the ability of the muscle cells' mitochondria to oxidize NADH is limited. We hypothesized that fatty acid oxidation, by providing more FADH2-linked reducing equivalents to the respiratory chain distal of complex I, would enable the affected cells to bypass the metabolic defect. The finding of beneficial effects of triacylglycerol infusion in 4 myopathic patients with isolated CID provides new insights into the nutritional metabolism and cellular pathophysiology of CID patients. Results were not unequivocal in all patients however; therefore, the limitations of the study need to be discussed.
Whole-body oxygen consumption
Our assumption that substrate oxidation rates are impaired during exercise was true in only 2 of the 4 patients exercising at 7 W on the basis that absolute whole-body
In 3 of the 4 CID patients, our finding of a significant additional diminishing effect of triacylglycerol infusion on plasma lactate concentrations and the lactate+pyruvate Ra was as expected, ie, supported our hypothesis. In the fourth patient (patient 2), however, our finding was contrary to our hypothesis; this patient had a higher plasma lactate concentration and higher lactate+pyruvate Ra during the triacylglycerol infusion than during the glucose infusion. Because Steele's equation was used to calculate lactate+pyruvate Ra (see Methods), the lactate concentration and Ra in this patient were not independently measured. We have no explanation for this contrary finding, which is even more remarkable because this patient's leg exercise endurance time during triacylglycerol infusion was significantly better than that of the other 3 patients.
Leg exercise endurance time and muscle bioenergetics
The leg exercise endurance time was clearly lower during the glucose infusion than during the triacylglycerol infusion in 3 of the 4 CID patients because these patients terminated their low-intensity cycling exercise prematurely during the glucose infusion. The remaining patient (patient 4) completed the 90-min low-intensity exercise trial irrespective of the substrate infused and had the lowest plasma lactate concentration of the 4 patients during infusion of both substrates. These findings suggest that leg exercise endurance time may be related to circulating plasma lactate concentrations. Unfortunately, patient 4 was not available for the 31P-MRS studies. The finding that leg exercise endurance time in patient 2 was lower during the glucose infusion, despite lower plasma lactate concentrations during the glucose infusion than during the triacylglycerol infusion, suggests that leg exercise endurance time is not just a simple function of plasma lactate concentrations (13).
The question arises as to why 3 of the 4 patients were unable to complete the 90-min exercise trial. Although our infusion experiments were not double-blind, we believe that the patients stopped exercising because of total exhaustion. Therefore, we are confident that the observed difference in leg exercise endurance time between the 2 infusion periods has a physiologic rather than a psychological basis. Because lactate production in muscle is usually accompanied by release of [H+], differences in the release of [H+] could explain the observed differences in leg exercise endurance time. However, none of the patients became acidotic during infusion of either substrate during exercise at this low intensity level, ie, 15% Wmax (data not shown), suggesting that these patients can effectively compensate for an excess release of [H+] from their exercising muscles.
Blood bicarbonate concentrations decreased only mildly and to a comparable extent during exercise and infusion of either substrate (data not shown). These findings do not rule out a relation between exercise endurance and a decrease in intramuscular pH. The MRS data obtained from forearm muscle during exercise showed no significant differences in chemical shift of Pi resonance (reflecting differences in intramuscular pH; data not shown) between the glucose and triacylglycerol infusions, however. Thus, we can only speculate that the mechanism responsible for the early termination of leg exercise during the glucose infusion in these patients was the early depletion of muscle glycogen stores. In healthy subjects, carbohydrate administration during exercise does not spare muscle glycogen stores (14–16); in contrast, triacylglycerol infusion is associated with significant sparing of muscle glycogen during exercise (17–21). Most of these studies, however, involved exercise at moderate-to-intense exercise levels (
The finding in CID patients of no significant differences in PCr-Pi ratios during exercise between the triacylglycerol and glucose infusion studies—despite improvements in endurance time,
In the present study, the triacylglycerol infusion was associated with higher mitochondrial substrate oxidation rates and lower plasma lactate concentrations and lactate+pyruvate Ra in myopathic CID patients during exercise than was the glucose infusion. These findings were associated with improved exercise endurance times during triacylglycerol infusion in most of the patients. However, PCr-Pi ratios measured in a forearm muscle during submaximal exercise were not significantly different in the patients during the 2 infusion studies. Thus, despite the beneficial effect of triacylglycerol on mitochondrial oxidative phosphorylation and muscle function during exercise, this macronutrient does not seem to change the muscle's energy state.
REFERENCES