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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 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 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
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:
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 (
SUBJECTS AND METHODS
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.
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TABLE 1 . Physical characteristics of individual patients with complex I deficiency and healthy control subjects
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. Mitochondrial DNA abnormalities (point mutations, eg, 3243AG MELAS/PEO, 8344AG MERRF, 8993TG NARP, and 11778AG LHON; deletions; duplications; or mitochondrial DNA depletion) were not detected in any of these patients, nor was a maternal pattern of inheritance. A summary of the biochemical investigations is shown in Tables 2 and 3.
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TABLE 2 . Microscopic and biochemical investigation of muscle biopsies in 3 individual patients with complex I deficiency (CID) and healthy control subjects1
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TABLE 3 . Microscopic and biochemical investigation of muscle biopsies in one patient (no. 4) with complex I deficiency (CID)1
Fifteen healthy control subjects matched for age (
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
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 (Pi) 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):
RESULTS
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.
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TABLE 4 . Plasma substrate concentrations in patients with complex I deficiency (CID) and healthy control subjects1
Whole-body
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TABLE 5 . Respiratory calorimetry and rate of appearance (Ra) of whole-body glucose and lactate+pyruvate in patients with complex I deficiency (CID) and healthy control subjects1
Comparison of outcome parameters between the individual CID patients and the control subjects
Changes in
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TABLE 6 . Whole-body oxygen consumption (
FIGURE 1. . Mean differences (
31P-MRS measurements of FDP muscle showed lower PCr-Pi ratios in the individual patients than in the control subjects during infusion of both substrates (Table 7), reflecting a lower muscle energy state due to impaired mitochondrial function. In both the patients and the control subjects, PCr-Pi ratios were not significantly different during the triacylglycerol infusion than during the glucose infusion. In addition, the triacylglycerol infusion failed to increase PCr-Pi ratios to control values.
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TABLE 7 . 31P-magnetic resonance spectroscopy measurements of the ratio of phosphocreatinine (PCr) to inorganic o-phosphate in flexor digitorum profundus muscle in patients with complex I deficiency (CID) and healthy control subjects
DISCUSSION
Whole-body oxygen consumption
Our finding of significantly higher whole-body
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-Pi 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
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.
REFERENCES