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Triacylglycerol infusion does not improve hyperlactemia in resting patients with mitochondrial myopathy due to complex I deficiency^1,2,3

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 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

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Background: A high-fat diet has been recommended for correction of biochemical abnormalities and muscle energy state in patients with 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 energy state in patients with CID.

Design: Four CID patients and 15 matched control subjects were infused with triacylglycerol (1.85 mg·kg^-1·min^-1) or glucose (5 mg·kg^-1·min^-1) while at rest. Respiratory calorimetry was used to evaluate mitochondrial substrate oxidation. Metabolism of glycolytic carbohydrate was determined on the basis of the rates of appearance and concentrations of plasma lactate from dilution of [1-¹³C]lactate measurements. In addition, high-energy phosphate metabolism was measured in forearm muscle by ³¹P magnetic resonance spectroscopy.

Results: Whole-body oxygen consumption rates were higher in the patients than in the control subjects (P < 0.05). Oxygen consumption and high-energy phosphate metabolism in forearm muscle were not significantly different between the 2 infusion groups. The rates of appearance and concentrations of plasma lactate were higher in each of the 4 patients than in the control subjects (P < 0.05) and were lower during the triacylglycerol infusion than during the glucose infusion (P < 0.05); the differences were comparable in the patients and control subjects.

Conclusions: We conclude that triacylglycerol infusion, relative to glucose infusion, does not improve the oxidation of substrates or the energy state of skeletal muscle and does not lower the rates of appearance and concentrations of plasma lactate to normal values in CID patients at rest.

Key Words: Mitochondrial myopathy • hyperlactemia • complex I deficiency • triacylglycerol infusion • glucose infusion • substrate oxidation • stable isotopes • 31P magnetic resonance spectroscopy

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Complex I (NADH dehydrogenase; EC 1.6.99.3) deficiency (CID), a respiratory chain disorder characterized by impaired mitochondrial oxidation of NADH, is being recognized in an increasing number of patients (1). Clinical manifestations range from pure myopathy restricted to the extraocular muscles (2), the skeletal muscle (2, 3), or both, to multisystemic involvement in which various other tissues are also affected (4, 5). Elevated plasma lactate concentrations are a common laboratory finding in humans with CID (2–5) and are thought to reflect increased oxidation of NADH (coupled to reduction of pyruvate) in the cytosol, resulting from the impaired oxidation of NADH inside the mitochondria of these patients.

A high-fat, low-carbohydrate diet has been recommended for the treatment of CID (6) because high-carbohydrate diets may impose a metabolic challenge in these patients. The recommendation for a high-fat diet is based on the following reasons:

1. Because the mitochondrial oxidation of NADH is thought to be diminished in CID patients, FADH₂ may be an alternative carrier of reducing equivalents and may maintain oxidative phosphorylation because electrons from FADH₂ can enter the respiratory chain distal to complex I.
   
2. The supply of FADH₂ to the mitochondria can be increased (relative to NADH) by increasing the amount of triacylglycerols and fatty acids in the diet. On the basis of stoichiometry, it follows that oxidation of fatty acids yields a ratio of FADH₂ to NADH of 0.5, whereas glucose yields a much lower ratio of 0.2.
   

Therefore, we hypothesized that infusion of fatty acids to CID patients would improve mitochondrial substrate oxidation more so than would carbohydrate infusion. The hypothesis is supported by the results of in vitro studies in renal cell cultures loaded with glutamine (an NADH-linked substrate) conducted by Doctor et al (7). Addition of rotenone, a known inhibitor of complex I, in the presence of 2-deoxyglucose, a competitive inhibitor of glucose uptake, resulted in near complete depletion of ATP and a significant decrease in oxygen consumption (O₂). Subsequent addition of heptanoate completely restored ATP concentrations and O₂. This effect of heptanoate could only be shown in the absence of antimycin A (which inhibits complex II), suggesting that heptanoate oxidation can bypass complex I and that this is mediated by complex II. Thus, CID patients may benefit from fatty acid supplementation because fatty acid oxidation provides more FADH₂ that enters the respiratory chain (distal to complex I). This may result in a lower concentration and rate of appearance (Ra) of plasma lactate.

In the present study, the effects of the triacylglycerol or glucose infusion on substrate oxidation and glycolytic carbohydrate metabolism were studied in 4 CID patients and in 15 healthy control subjects. All patients had documented CID, and exercise intolerance was their main symptom. Glycolytic carbohydrate metabolism was evaluated on the basis of the Ra and concentrations of plasma lactate (from dilution of [1-¹³C] lactate), mitochondrial substrate oxidation was studied indirectly with use of respiratory calorimetry, and high-energy phosphate metabolism in forearm muscle was measured by ³¹P magnetic resonance spectroscopy (³¹P-MRS) (8, 9).

	SUBJECTS AND METHODS

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^Subjects
Four unrelated female CID patients aged 15–25 y were studied (Table 1). The patients had similar clinical histories and their weights ranged from 45 to 61 kg. All the patients had easily fatigable mild muscle weakness dating back to early childhood that had remained stable over time. Patient 1 had experienced a single strokelike episode at age 13 y, which resolved spontaneously after a few hours. Except for patient 4, in whom additional mild cerebellar atrophy and axonal neuropathy were diagnosed, none of the patients showed any signs of central nervous system involvement. Therefore, exercise intolerance was the dominant clinical symptom at the time of the study. Maximal workload performance, assessed from an incremental maximal exercise test on an electrically braked cycle ergometer (Lode Instruments, Groningen, Netherlands), as previously described (10), ranged from 50 to 60 W and was on average only 25% of control values. Baseline fasting plasma lactate concentrations ranged from 2.0 to 4.7 mmol/L (Table 1). Patients 1 and 2 had previously been prescribed riboflavin and carnitine, but discontinued this therapy because they recalled no benefit from this medication. At the time of the study, patients 3 and 4 were taking riboflavin and carnitine, although they did not recall much benefit from this therapy either. All patients were engaged in social activities. Patient 1 was a housewife and had recently given birth to a healthy son, patients 2 and 3 had service jobs, and patient 4 was a student.

^{Experimental protocol}
The patients and control subjects reported to the Laboratory for Metabolic Diseases (University Children's Hospital) at 0800 on 2 occasions separated by 7 d. Two polytetrafluoroethylene catheters were inserted, one into a dorsal hand vein for infusion and the other into a vein draining the dorsum of the contralateral hand for blood sampling. The hand was inserted in a heated box to achieve arterialization of the venous blood (12). After the blood sample was drawn, the catheter was flushed with heparin-containing saline (2.5 kU/L). Subjects were acclimatized to room conditions (temperature: 21–25°C) for 30 min before the study began. At time 0, 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) containing linoleic acid (50%), oleic acid (26%), palmitic acid (10%), linolenic acid (9%), stearic acid (3.5%), and heparin (7.5 U·kg^-1·h^-1; prime: 14 U/kg) was infused and maintained throughout the 100-min study period, during which time the subjects remained at rest in the supine position. The triacylglycerol and glucose infusions were assigned in random order. All patients and control subjects participated in both the triacylglycerol and glucose infusion studies.

Isotope infusion
Primed, constant infusions of [6,6-²H₂]glucose (98% enriched; Mass Trace, Woburn, MA) were administered in all 4 patients and in 12 control subjects: prime, 20.0 µmol [6,6-²H₂]glucose/kg; continuous infusion rate, 0.30 µmol [6,6-²H₂]glucose·kg^-1·min^-1 in the triacylglycerol infusion studies or 0.50 µmol·kg^-1·min^-1 in the glucose infusion studies. An unprimed, constant infusion of [1-¹³C]lactate (98% enriched; Mass Trace) was started 30 min into the infusion of either triacylglycerol or glucose and continued throughout the remaining 70 min of the study period. The [1-¹³C]lactate infusion rates for the CID patients (n = 4) ranged from 1.00 to 1.50 µmol·kg^-1·min^-1; the control subjects (n = 12) received 0.45 µmol·kg^-1·min^-1. The remaining 3 control subjects received no lactate or glucose tracer to examine the effects of triacylglycerol or glucose infusion on background enrichments of lactate and glucose.

Blood sampling and urine collection
Blood samples were drawn at regular intervals, placed on ice, and transferred into sodium fluoride–containing tubes for measurement of plasma glucose, lactate, and triacylglycerol or into lithium heparin–containing tubes for measurement of fatty acid, glycerol, cortisol, and insulin. Whole blood was deproteinized for measurement of blood pyruvate, ß-hydroxybutyrate, and acetoacetate. Blood samples for determination of [6,6-²H₂]glucose and [1-¹³C]lactate enrichments were centrifuged at 4°C (1000 x g, 10 min) and stored at -70°C. Urine voided at time 0 and 100 min was collected for measurement of nitrogen excretion.

Respiratory calorimetry
After the subjects had been infused with triacylglycerol or glucose for 45 min, open-circuit indirect calorimetry under a ventilated hood began and continued for 40 min while the subjects were at rest. Stable O₂ and carbon dioxide production (CO₂) values were reached within 5 min of recording. Computerized, continuous gas and air volume measurements were performed (Oxycon Champion; Jaeger, Breda, Netherlands) as previously described (13). Atmospheric pressure and temperature calibration of oxygen and carbon dioxide sensor measurements and air volume calibrations (with a standard 3000-mL cylinder) were performed before each measurement. O₂ relative to CO₂ was standardized with use of alcohol burning at regular intervals: the theoretical respiratory exchange ratio (0.67) was closely approached in all standardizations (: 0.66; CV: 1.6%; n = 10).

³¹P-MRS measurements of ratios of phosphocreatine to inorganic o-phosphate at rest
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, as described above. Triacylglycerol or glucose was infused during a 100-min basal period during which the subjects remained at rest. The infusions were maintained at the same rate throughout the following 30-min study period. Studies were conducted on the superficial mass of the flexor digitorum profundus (FDP) muscle of the right forearm. The FDP muscle is affected adversely in CID patients, as evidenced by observed decreases in maximal voluntary contraction output of the muscle comparable with decreases in maximal workload performance measured on a cycle ergometer (MJ Roef, K de Meer, unpublished observations, 1996). ³¹P-MRS spectra of the FDP were obtained at 1.5 T on an S15 HP whole-body MR spectrometer (Philips, Eindhoven, Netherlands), as described in detail elsewhere (14). Briefly, subjects were positioned prone and head first on the patient bed with their right arm extended forward, supported by cushions. Guided by palpation of the ulnar bone directly adjacent to the muscle, the forearm was placed into a support such that the FDP overlied a 2-turn 25-mm diameter surface coil tuned to a frequency of 25.86 MHz and attached with straps. Correct positioning of the FDP over the ³¹P surface coil was checked by ¹H MR imaging. Resting ³¹P-MRS spectra of the superficial region of the FDP were obtained with a frequency-modulated adiabatic 90° excitation pulse. Sixty free induction decays (1024 data points with 333-µs dwell times) were collected with a repetition time of 3 s.

Sample analysis
Plasma glucose, lactate, and triacylglycerol concentrations were measured enzymatically with autoanalyzers (Dimension AR and ACA SX, respectively; Dimension, Dade, FL). Concentrations of [6,6-²H₂]glucose and [1-¹³C]lactate used for the tracer infusions and for the standard curves (see below) were measured with the autoanalyzers with the use of calibration curves from weighed, water-free glucose (Merck, Darmstadt, Germany) and zinc lactate (Sigma, St Louis), respectively. Plasma insulin and cortisol concentrations were measured with the use of a microparticle enzyme immunoassay method (IMX analyzer; Abbott, Chicago) and a fluorescence polarization immunoassay (TDX analyzer; Abbott), respectively. Blood pyruvate, ß-hydroxybutyrate, acetoacetate, plasma fatty acids, and glycerol were measured with use of automated enzymatic colorimetric methods (COBAS FARA II; Hoffmann-La Roche, Montpellier, France). Urinary nitrogen was assayed according to a micro-Kjeldahl method (15).

The isotopic enrichments of glucose and lactate in plasma were measured with the use of gas chromatography–mass spectrometry (model 5890; Hewlett-Packard, Palo Alto, CA). Enrichment of plasma [6,6-²H₂]glucose was measured in the pentaacetate derivative with use of positive chemical ammonia ionization, selectively monitoring ions at mass-to-charge ratios of 408 and 410 (16). The peak ratios (408 and 410) were compared with those of a standard curve prepared by diluting 98% tracer [6,6-²H₂]glucose (Mass Trace) with weighed amounts of glucose and were reported as molar tracer-tracee ratios (TTRs). The enrichment of [1-¹³C]lactate in plasma was measured in the N-propylamide heptafluorobutyrate derivative with use of electron impact ionization, selectively monitoring ions at mass-to-charge ratios of 86 and 87 (17). The TTRs were likewise derived from the peak ratios and compared with a standard curve prepared by diluting 98% tracer sodium[1-¹³C]lactate (Mass Trace) with weighed amounts of zinc lactate.

Free induction decays were processed with use of the LAB ONE NMR 1 (New Methods Research, Detroit) spectroscopy processing software as described in detail elsewhere (14). Estimates of the relative peak areas of the various metabolites were obtained by curve fitting the spectrum to Lorentzian line shapes: singlets of both inorganic o-phosphate (P_i) and phosphocreatine (PCr), doublets of -ATP and -ATP, and a triplet of ß-ATP.

Calculations
Whole-body glucose Ra
In steady state experiments, the whole-body glucose Ra was calculated as follows (18):

(1)

where I is the infusion rate of the [6,6-²H₂]glucose tracer (in µmol·kg^-1·min^-1). Isotopic enrichments were considered to be at steady state when the TTRs of the 4 consecutive samples from time 70 to 100 min had a CV < 10% and a slope not significantly different from 0. When the isotopic enrichments were not in steady state, the Ra was calculated with Steele's equations for non–steady state conditions (19) as follows:

(2)

where V_d is the volume of distribution of glucose in mL/kg (200 mL/kg), (G_i + G_j)/2 is the mean plasma glucose concentration (in mmol/L) measured at time points T_i and T_j, TTR is the change in TTR from T_i to T_j, T is the time interval (T_j - T_i) in min, and (TTR_i + TTR_j)/2 is the mean TTR measured at time points T_i and T_j. Steady state [6,6-²H₂]glucose TTRs were attained in 3 of the 4 patients and in 8 of the 12 control subjects during the triacylglycerol infusion and in 3 of the 4 patients and in 5 of the 12 control subjects during the glucose infusion.

Whole-body lactate+pyruvate Ra
A single common pool approach for lactate and pyruvate was used to calculate the whole-body lactate+pyruvate Ra (in µmol·kg^-1·min^-1). Wolfe et al (20, 21) reported that the enrichment of plasma pyruvate in anesthetized dogs was 92% of the enrichment of plasma lactate at steady state when [1-¹³C]lactate was infused. The findings of Large et al (22) suggest that this approach is also applicable to humans. Therefore, we assumed that the Ra values calculated from dilution of labeled lactate in plasma represent the Ra of both lactate and pyruvate:

(3)

where I is the infusion rate of [1-¹³C]lactate (µmol·kg^-1·min^-1) and TTR is the molar ratio of tracer to tracee in lactate. Steady state conditions for lactate TTRs were defined as described for glucose. When isotopic enrichments were not at steady state, the Ra was calculated with Steele's equations for non–steady state conditions (23) as described for glucose in Equation 2. The V_d for lactate+pyruvate was assumed to be 500 mL/kg (24, 25). Steady state lactate TTRs were attained in 3 of the 4 patients and in 7 of the 12 control subjects during the triacylglycerol infusion and in all 4 patients and in 10 of the 12 control subjects during the glucose infusion.

Respiratory calorimetry
Carbohydrate oxidation was calculated according to Ferrannini (26):

(4)

where O₂ and CO₂ are expressed in mL/min, N is urinary nitrogen excretion (in mg/min), and BW is body weight (in kg). The calculated whole-body rate of substrate oxidation was assumed to reflect mitochondrial oxidation, neglecting nonmitochondrial peroxisomal and microsomal O₂. This nonmitochondrial O₂, which is insensitive to cyanide, was estimated in rats and shown to be 15–20% of the O₂ of perfused rat skeletal muscle (27). Data on nonmitochondrial O₂ in humans are not available. The relative contributions of nonmitochondrial O₂ processes to total O₂ were assumed to not be different whether triacylglycerol or glucose was infused.

Outcome parameters
Triacylglycerol or glucose infusion were expected to affect outcome parameters differently, not only in the patients but also in the control subjects. Thus, the hypothesized effects in the patients should be additional to those in the control subjects. The effects of the triacylglycerol infusion (TG) compared with those of the glucose infusion (GL) on outcome parameters in the individual patients and the control subjects is described as follows:

(5)

(6)

(7)

When the values for O₂, plasma lactate, and lactate+pyruvate Ra in the individual patients exceed the upper limit of the 95% CI for the effect of the triacylglycerol infusion (compared with the glucose infusion) in the control subjects, the effect of the triacylglycerol substrate in the patients is considered additional compared with that of glucose.

Metabolite concentrations
The average free ADP concentration in fibers within the sampled muscle mass was calculated from the creatine kinase equilibrium as follows:

(8)

where 1.66 x 10⁹ is the value of the equilibrium constant (28). It was assumed that the concentrations of ATP and total creatine ([TCr]) in the skeletal muscle of the patients were unchanged from normal values (8.2 and 42.7 mmol/L for [ATP] and [TCr], respectively; 8, 23). The concentrations of P_i and PCr were calculated from measured peak ratios of P_i to ß-ATP and of PCr to ß-ATP, respectively. Because the peak ratios of PCr to ß-ATP in the patients were well outside the normal range, an individual value for the ratio of [PCr] to [TCr] was calculated for each patient:

(9)

where (PCr/ß-ATP)_controls is the mean (±SD) peak ratio of PCr to ß-ATP in the control subjects (3.15 ± 0.25) and [PCr]/[TCr] is the peak ratio of [PCr] to [TCr] in CID patients (0.85), as described elsewhere (29).

Statistical analysis
The results are presented as means ± SDs. The data were analyzed by two-factor analysis of variance to identify main effects of group (CID patients compared with control subjects) and infusion condition (triacylglycerol or glucose) and their interaction. When there was a significant (P < 0.05) interaction, post hoc Bonferroni correction was conducted. The outcome parameters O₂, plasma lactate, and lactate+pyruvate Ra in the individual patients were compared with the 95% CIs of the control subjects. SPSS for WINDOWS (version 7.5; SPSS Inc, Chicago) was used for the analyses.

	RESULTS

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Comparison between the CID patients and the control subjects at the group level: plasma concentrations and substrate utilization
As expected, plasma glucose, insulin, and lactate and blood pyruvate concentrations in the control subjects were significantly higher during the glucose infusion than during the triacylglycerol infusion (Table 4). Likewise, plasma fatty acid, plasma triacylglycerol, blood ß-hydroxybutyrate, blood acetoacetate, and plasma glycerol concentrations were higher during the triacylglycerol infusion than during the glucose infusion. Plasma cortisol concentrations were similar after infusion of both substrates. Plasma glucose, insulin, and cortisol and blood pyruvate concentrations were not significantly different between the patients and the control subjects during infusion of either substrate, but plasma lactate concentrations and lactate-pyruvate ratios were significantly higher in the patients than in the control subjects during infusion of both substrates. During the triacylglycerol infusion, plasma fatty acids were significantly lower in the patients than in the control subjects, blood acetoacetate concentrations were lower in the patients than in the control subjects (NS), and the ratio of blood ß-hydroxybutyrate to acetoacetate was significantly higher in the patients than in the control subjects.

View this table: [in this window] [in a new window]   TABLE 6 . Whole-body oxygen consumption ( O2), plasma lactate concentrations, and rate of appearance (Ra) of lactate+pyruvate during triacylglycerol or glucose infusions in 4 patients with complex I deficiency (CID) and healthy control subjects

View larger version (37K): [in this window] [in a new window]   FIGURE 1. . Mean differences () in oxygen consumption (O2), plasma lactate, and rate of appearance (Ra) of lactate+pyruvate between the triacylglycerol and glucose infusion studies in the individual patients and the respective mean 95% CIs for differences in the control subjects (gray-shaded area). P5, lower limit of 95% CI; P95, upper limit of 95% CI. Values greater than the P95 in the individual patients indicate an effect of triacylglycerol additional to that in the control subjects according to the following hypothesis: O2 in patients > O2 in control subjects, and plasma lactate and lactate+pyruvate Ra in patients < plasma lactate and lactate+pyruvate Ra in control subjects.

	DISCUSSION

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Plasma lactate and lactate+pyruvate Ra and muscle bioenergetics
Our finding in healthy subjects that the triacylglycerol infusion was associated with lower plasma lactate concentrations than was the glucose infusion was also shown by others (31–34).

The failure of the triacylglycerol infusion to increase PCr-P_i ratios to control values is the most direct in vivo evidence that the triacylglycerol infusion does not change the energy state in the affected resting skeletal muscle of CID patients.

Relevance of the use of a high-fat diet in CID patients
The results of the present study suggest that the biochemical abnormalities observed in the 4 CID patients in the present study are not likely to improve after consumption of a high-fat diet. This lack of effect is worthy of remark. First, fatty acid availability for oxidation may have been lower in the patients than in the control subjects. However, our finding of a lower respiratory exchange ratio during the triacylglycerol infusion in the patients than in the control subjects suggests that fatty acid oxidation was unimpaired or even stimulated by the respiratory chain defect. The lower plasma fatty acid concentrations in the CID patients than in the control subjects during the triacylglycerol infusion may have been the consequence of preferential oxidation. Second, the type of fatty acids administered may have been important. In the studies by Doctor et al (7), heptanoate—a short-chain, odd-numbered fatty acid—was used to bypass rotenone-inhibited complex I in renal cell cultures. However, we administered a lipid emulsion containing only a small amount of esterified short-chain, odd-numbered fatty acids (see Methods). To address the issue, triacylglycerol infusions containing different fatty acids should be studied in a larger group of CID patients. Limited numbers of available patients make it difficult to increase the sample size, however.

Finally, the findings in the 4 CID patients do not necessarily preclude prescription of a high-fat diet to other CID patients. We selected patients with documented CID for whom exercise intolerance was a main feature of their abnormality. At rest, the CID patients showed abnormalities of a biochemical nature only; no clinical signs, such as muscle wasting, were observed. It may be that our hypothesis, the aim of which was to improve mitochondrial substrate oxidation in CID patients, was not valid in the patients that we selected, at least not in the resting condition. It is tempting to speculate, however, that in resting CID patients with clinical signs and symptoms due to impaired mitochondrial substrate oxidation, the triacylglycerol infusion may be beneficial. In addition, the triacylglycerol infusion may be useful in CID patients when energy demands are increased, such as during exercise.

Conclusion
In resting CID patients in the present study, no additional benefit of the triacylglycerol infusion over glucose infusion was shown. The triacylglycerol infusion failed to improve mitochondrial substrate oxidation, as evidenced by both whole-body O₂ and high-energy phosphate values in resting muscle, and failed to lower plasma lactate concentrations and the lactate+pyruvate Ra to values that were within the range for the healthy control subjects.

	ACKNOWLEDGMENTS

 
We thank R Ouwerkerk for his technical assistance with the ³¹P-MRS measurements and HD Bakker, JBC de Klerk, and GPA Smit for their referral of patients for the studies.

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