- Email: [email protected]
Fasting as a provocative test in neuromuscular diseases
Fasting as a Provocative
Test in Neuromuscular
Diseases
James E. Carroll, Darryl C. DeVivo, Michael H. Brooke, G. James Planer, and James H. Hagberg A 38-hr fast was used as a provocative test in patients suspected of having defects in muscle substrate utilization. In five controls and nine patients, exercise capacity and respiratory exchange ratio were determined before and at the end of the fast. Blood was collected at intervals during the fast from ten controls and nine patients for creetine kinase (CK). free fatty acids, (FFA) @hydroxybutyrate, acetoacetate, free and total carnitine, glucose, and alanine. Two patients with myophosphorylase deficiency had increased exercise capacity, and a marked fall in CK, and one had a lesser fall in blood glucose than normal at the end of the fast. Two patients with known lipid myopathies (carnitine deficiency and carnitine palmityl transferase deficiency) had decreased exercise capacity and apparent increased dependence on carbohydrate metabolism during the fast. Carnitine concentrations became even more abnormal in the patient with carnitine deficiency during fasting. Several patients with less well-defined defects were also significantly different from the controls in several respects, indicating that the fast might be useful for finding new defects.
homeostasis dictates that to M ETABOLIC preserve body protein stores during fasting, lipids must be substituted for carbohydrates as the major substrate for oxidative metabolism.’ During caloric deprivation, serum glucose concentrations decrease and FFA concentrations rise,‘,* providing increased availability of the latter substrate. Therefore, the utility of fasting as a provocative test to identify patients suspected of defects in fatty acid metabolism3-’ is apparent. Patients with disorders of fatty acid mobilization or utilization might be expected to become symptomatic during fasting, while those with defective glycogen metabolism might actually improve. Our goal in this study has been to exploit a previously standardized protocol of fasting,* which could be used in patients thought to have defects of muscle substrate utilization. We recognize that such a test may not yield a specific diagnosis, but the results may guide subsequent biochemical investigations. In addition to identifying patients with known metabolic defects, new abnormalities might be brought to light. Accordingly, we fasted ten control subjects
Metabolism,Vol.
28, No. 6 (June), 1979
and nine patients with a variety of muscle disorders for a 38-hr period. Prior to and following fasting, exercise capacity and respiratory exchange ratio (RER) were measured. During fasting, blood was collected for various metabolites expected to reflect changes in fatty acid and carbohydrate metabolism. MATERIALS
AND
METHODS
All studies were performed in the Washington University Clinical Research Center according to protocols that had been approved by the Washington University Human Studies Committee. Informed consent was obtained from the patient and/or parents in the case of minors. The patients are shown in Table I. Ragged red fiber disease is a mitochondrial myopathy with ophthalmoplegia and mild proximal limb weakness (patients 3 and 4). The patient with central core disease (patient 9) had greater than 90% type 1 fibers on muscle biopsy. Exercise capacity and RER were determined in five normal adult controls (three males and two females, ages 25-35) and the nine neuromuscular disease patients 2-4 hr following a meal and again at the end of a 3%hr fast. An incremental exercise test9 was done on a bicycle ergometer to determine maximal oxygen consumption (VO, max) and maximum work capacity. Previous experiments have shown that those methods accurately regect exercise capacity in neuromuscular disease patients.” Data were collected after the manner of Beaver et al.” using a PerkinElmer (MGA 1100) mass spectrometer for measurement of respiratory gases, a Fleish pneumotachograph attached to a Validyne pressure transducer for the measurement of ventilatory volumes, and a PDP-I2 computer. Data were calculated on a breath-by-breath basis and averaged over IS-set intervals for oxygen consumption (VO>), carbon dioxide production (VCO,), minute ventilation (VE), and RER. Five additional postpubertal controls with various neurologic diseases were added to the five normal controls for
From the Departments of Neurology and Neurosurgery (Neurology) and Pediatrics, the Department of Neurology and Neurosurgery (Neurology), and the Department of Preventive Medicine, Washington University School of Medicine, St. Louis, MO. Supported by a center grant from the Muscular Dystrophy Association, NHLBI Training Grant S-T32-HL-0708 entitled “Multidisciplinary Heart and Vascular Diseases,” and USPHS Grant RR-00036 from the Division of Research Facilities and Resources. Address reprint requests to Dr. James E. Carroll. Department of Neurology, Washington University School of Medicine, 660 S. Euclid. Box 81 II, St. Louis, MO. 431 IO. @ 1979 by Grune & Stratton, Inc. 002s-o49S/79/2906~I 3gOI .00/O
683
664
CARROLL ET AL.
Table 1. Patients
value. If the logs resulted in less skewing,
the antilogs of their means and standard deviations were used instead of those for the absolute numbers. Correlation coefficients were determined for plasma alanine and blood ketone concentrations, FFA and acylcarnitine concentrations (total minus free carnitine concentrations), and ketones and acylcarnitine concentrations.
Age Patient
Disease
Sax
IYrl
1
Myophosphorylase deficiency
Male
23
2
Myophosphaylasa
Female
34
of patient
deficiency (sibling
1)
3
Ragged red fiber disease
Male
50
4
Ragged red fiber disease
Female
31
5
Carnitine palmityl transferase
Male
23
Female
29
RESULTS
deficiency Muscle carnitine deficiency
6 7
Limb girdle dystrophy
Female
31
6
Limb girdle dystrophy (sibling of
Female
29
9
Type 1 predominance (central core
Male
36
patient 7) disease)
blood sampling during the fast; they, however,
did not take part in the exercise testing. Fasting was begun at 1800 hr on day one following the evening meal of that day. Water was allowed and lib. during the fast. The fast ended at 0800 hr of day three. Blood samples were obtained at 1800 and 2400 hr on day one, at 0800, 1200, 1600, 2000, and 2400 hr on day two, and 0400 and 0800 hr on day three. CK, FFA,12 and total and free carnitine were determined on serum specimens. Total carnitine was assayed following a mild alkaline hydrolysis at 37°C. For the carnitine assay, the method of McGarry and Foster” was modified by the substitution of Hepes for Tris buffer.14 Glucose, @-hydroxybutyrate, and acetoacetate were determined by specific enzymatic, microfluorometric methods on perchloric acid extracts of whole blood.” Alanine was measured by an enzymatic microfluorometric procedure on heparinized plasma samples.‘6 Values from the blood samples of individual patients were taken to be significantly different if they fell outside two standard deviations from the controls. Control data were tested for skewness” before and after taking the log of each Table 2.
Exercise capacities before and after the fast are shown in Table 2. The two sisters with limb girdle dystrophy were unable to exercise, although resting respiratory gases were collected. Whereas most subjects had the same or slightly less exercise capacity at the end of the fast, the two patients with myophosphorylase deficiency improved. Conversely, the patients with carnitine palmityl transferase deficiency and carnitine deficiency were unable to exercise at the end of the fast. Changes in resting RER for the five normal controls over the 38-hr fast ranged from -0.08 to -0.22. None of the patients were outside this range. Because of diurnal variation, only the blood metabolite data from the 14- and 38-hr points (0800-0800) during the fast are presented for comparison. Figures 1 and 2 show the comparative concentrations of FFA, acetoacetate, and fl-hydroxybutyrate, total and free carnitine, and alanine at the 14- and 38-hr points for the ten controls (five normal and five disease controls) and for the nine patients. Figure 2 also shows the
Exercise Capacity VO, Max
Before Fast Subjects
(literlmin)
Work Max End of Fast
lliter/min)
Before Fast
End of Fast
(kpmlmin)
lkpmfminl
Controls 1
3.66
3.69*
1600
2
2.62
2.49
1500
1300
3
2.51
2.27
1200
1000
4
4.4 1
4.29
2200
2200
5
1.58
1.42
900
700
0.65
0.69’
225
300’
1.28
1.56.
450
525.
1.61
1.24
600
600
0.95
0.68
300
225
Myophosphorylase deficiency Ragged red fiber disease “Limb girdle dystrophy”
Did not exercise
Carnitine palmityl transferase deficiency
3.00
Camitine deficiency
0.50
--t --t
Type 1 predominance
1.59
1.64’
*Better with fasting. tCould not exercise.
1500
1500 225 525
--t --t 450
FASTING IN NEUROMUSCULAR DISEASES
t t
Fig. 1. Fatty acid. acatoacetate. and &hydroxybutyrate data for 0800 of day two and day three. (‘I Outside two standard deviations.
Fig. 2. Total and free carnitine and alanine are shown for each 0800 point. Glucose is presented as ratio of the two points. (‘1 Outside two standard deviations.
ratios of the 3%hr glucose to the 14-hr glucose for all subjects. CK activities decreased in the controls at the end of the fast to 85.1 + 14.6% (SD) of the initial value. Only the patient with carnitine palmityl transferase deficiency had a significant rise during the fast (268%). The patients with carnitine deficiency and myophosphorylase deficiency fell to 53.1% and 57% of initial values, respectively. The correlation between plasma alanine concentrations and total ketones in the controls at the 14- and 3%hr points was not significant (r = -0.24, p > 0.35). A significant correlation existed between the concentrations of acylcarnitine and FFA (r = 0,84, p < 0.01) and acylcarnitine and total ketones (r = 0.74, p < 0.01) at the 14- and 38-hr points for the ten controls.
data, indicating that elevations in these parameters during fasting should be interpreted with caution. The necessity of grouping together men and women probably contributed to the skewing effect. The patients with myophosphorylase deficiency had improved exercise capacity, and a marked fall in CK at the end of the fast, and one of the patients had a lesser decrement in blood glucose concentration that the controls. Increased dependency on FFA by these two patients was the probable mechanism underlying the salutary effects of fasting; their moderately low concentrations of FFA at the end of the test are compatible with the speculation of proportionately greater utilization of this substrate at any point in time. On the other hand, both patients with disturbances of muscle lipid metabolism were unable to exercise at the end of the fast, and they had greater than normal decrements in blood glucose. The latter observation probably indicates an increased dependence upon carbohydrate metabolism during fasting. In regard to other parameters, however, the responses of these two patients differed widely. The patient with carnitine palmityl transferase deficiency demonstrated elevated carnitine, alanine, and CK concentrations at the end of the fast. Ketone concentrations were low, consistent with the supposition that uptake of fatty acids into liver mitochondria was reduced. The patient with muscle carnitine deficiency had low concentrations of free carnitine at the beginning of the
DISCUSSION
In this study the utility of a standardized fasting protocol has been examined as a provocative screening test for metabolic diseases of muscle. Our conclusions are based on the establishment of the normal response of a control group against which individual patient values can be compared. Variations were judged to be significant if they fell outside two standard deviations from the mean. An attempt to generate a predictable response with only ten control subjects can result in considerable skewing of the data. In this study, skewing to the right was particularly evident in the ketone and fatty acid
CARROLL ET AL.
666
fast. At the end of the fast, both total and free carnitine were low. Ketone and fatty acid concentrations were high at the end of the fast, but not beyond two standard deviations. Both patients had an appropriate fall in RER during the fast, indicating that fatty acids were at least partially utilized. Among the patients with less well-defined biochemical defects, several findings stand out. The two sisters with limb girdle dystrophy had reduced concentrations of free carnitine at the end of the fast, while acylcarnitine concentrations were elevated. Blood glucose concentrations showed a greater-than-normal fall in one of the patients. Several possibilities exist for the defect in these two patients: (1) a defect similar to that in the patient with carnitine deficiency, (2) an undescribed defect of muscle lipid metabolism, or (3) decreased body stores or carnitine because of decreased muscle mass. The patient with central core disease and type 1 predominance failed to demonstrate a rise in FFA at the end of the fast, in contrast to the patients with lipid utilization defects. The most plausible explanation for the lack of rise may be increased utilization by the large numbers of type 1 fibers. In addition, plasma alanine concentrations in the central core patient were low throughout the fast. The divergent alanine responses in this patient and the patient with carnitine palmityl transferase deficiency are noteworthy in relation to the role of skeletal muscle in alanine homeostasis. Since a large fraction of plasma alanine supposedly arises from muscle, ‘* it may be that type 1 muscle fibers release less alanine. Conversely, the paradoxic increase in plasma alanine during fasting in the patient with carnitine palmityl transferase deficiency might suggest disproportionate metabolic activity in type 2 muscle fibers with increasing efflux of alanine under these conditions. Several parameters were examined for correlations. Sherwin et aLI have suggested that the decrease in plasma alanine concentration that normally occurs during fasting is caused by a feedback mechanism induced principally by rises in blood ketone concentrations. Although there was a moderate negative correlation between these values, it was not statistically significant, indicating that there are a number of other
variables influencing this process in both the controls and patients. This observation is consistent with previous studies using stable isotopes in children, where ketosis did not change alanine flux.20 Among the controls, there was a strongly positive relationship between FFA and acylcarnitine concentrations and total ketones and acylcarnitine concentrations. A similar relationship was found in the patients. Other workers have noted that these correlations exist under a variety of conditions.2’m23 The safety of fasting should also be considered in patients with metabolic defects. None of the subjects in this study had serious side effects with fasting, although the two patients with lipid myopathies experienced considerable muscle pain toward the end of the fast. The woman with carnitine deficiency developed a mild metabolic acidosis (data not shown) and a relative hypoglycemia (2.63 mM), but neither perturbation produced symptoms. Because others have reported deaths following fasting in children with undiagnosed neuromuscular diseases.24 we prefer to employ a rather brief fast. Undoubtedly, a longer fast would increase the differences between the responses of the patients and that of the controls. This 38-hr fasting protocol has been used by one of us (D.C.D.) in over one hundred children and adults with a variety of diseases without untoward events.8.20 Fasting as a provocative test acts to accentuate defects in muscle substrate utilization. Thus, abnormalities that are either not apparent or mildly abnormal under postabsorptive conditions become clearly abnormal during fasting. Most of the abnormal substrate data in this study can be interpreted as varying either because of a production or utilization defect. Specific studies of substrate turnover in the patients would be necessary to determine which aspect of metabolism is altered. Even so, the utility of this test is demonstrated by the fact that we were able to discriminate among the known metabolic defects and also suggest new defects in less well-characterized disorders.
ACKNOWLEDGMENT The authors wish to thank Dr. J. Phillip Miller for assistance in statistical analysis of the data.
FASTING
IN NEUROMUSCULAR
DISEASES
687
REFERENCES 1. Cahill GF, Herrera MG, Morgan AP, et al: Hormonefuel interrelationships during fasting. J Clin Invest 45:17511769,1966 2. Hanson PG, Johnson RE, Zaharko DS: Correlation between ketone body and free fatty acid concentrations in the plasma during early starvation in man. Metabolism 14:1037-1040, 1965 3. Engel WK, Vick NA, Glueck CJ, et al: A skeletalmuscle disorder associated with intermittent symptoms and a possible defect of lipid metabolism. N Engl J Med 282:697704, 1970 4. Bank WJ, DiMauro S, Bonilla E, et al: A disorder of muscle lipid metabolism and myoglobinuria: Absence of carnitine palmityl transferase. N Engl J Med 292:443-449, 1975 5. Hostetler KY, Hoppel CL, Romine JS, et al: Partial deficiency of muscle carnitine palmityl transferase with normal ketone production N Engl J Med 298:553-557, 1978 6. Carroll JE, Brooke MH, DeVivo DC, et al: Biochemical and physiologic consequences of carnitine palmityl transferase deficiency. Muscle Nerve 1:103-l 10, 1978 7. Reza MJ, Kar NC, Pearson CM, et al: Recurrent myoglobinuria due to muscle carnitine palmityl transferase. Ann Intern Med 88:61%615, 1978 8. Haymond MW, Karl IE, Pagliara AS: Ketotic hypoglycemia: An amino acid substrate-limited disorder. J Clin Endocrinol Metab 38:521-530, 1974 9. Wasserman K, Whipp BJ, Koyal SN, et al: Anaerobic threshold and respiratory gas exchange during exercise. J Appl Physiol 35:236-243, 1973 10. Carroll JE, Hagberg JM, Brooke MH, et al: Bicycle ergometry with computerized gas exchange measurements in neuromuscular diseases. Arch Neurol (in press) 11. Beaver WL, Wasserman K, Whipp BJ: On-line computer analysis and breath-by-breath graphical display of exercise function tests. J Appl Physiol 34: 128-l 32, 1973 12. Novak M: Calorimetric ultramicro method for the determination of free fatty acids. J Lipid Res 6:431433, 1965
13. McGarry JD, Foster DW: An improved and simplified radioisotopic assay for the determination of free and esterified carnitine. J Lipid Res 17:277-28 I, 1976 14. Christiansen RZ, Bremer J: Active transport of butyrobetaine and carnitine into isolated liver cells. Biochem Biophys Acta 4481562-577, 1976 15. DeVivo DC, Haymond MW, Leckie MP, et al: The clinical and biochemical implications of pyruvate carboxylase deficiency. J Clin Endocrinol Metab 45:1281-1296, 1977 16. Karl IE, Pagliara AS, Kipnis DM: A microfluorometric enzymatic assay for the determination of alanine and pyruvate in plasma and tissue. J Lab Clin Med 80:434-441, 1972 17. Snedecor GW, Cochran WG: Statistical Methods 6). Ames, Iowa, Iowa State University Press, 1967
(ed
18. Pozefsky T, Felig P, Tobin JD, et al: Amino acid balance across tissues of the forearm in postabsorptive man. Effects of insulin at two dose levels. J Clin Invest 48:22732282, 1969 19. Sherwin RS, Hendler RG. Felig P: Effect of ketone infusions on amino acid and nitrogen metabolism in man. J Clin Invest 55:1382-1390, 1975 20. Haymond M, Strobe1 K, Ben-Galim E, et al: Effects of ketosis on glucose and alanine fluxes in children. Clin Res 26:682A, 1978 21. Seccombe DW, Hahn P, Novak M: The effect of diet and development on blood levels of free and esterified carnitine in the rat. Biochem Biophys Acta 528:483489, 1978 22. Frolich J, Seccombe DW, Hahn P, et al: Effect of fasting on free and esterified carnitine levels in human serum and urine: Correlation with serum levels of free fatty acids and @-hydroxybutyrate. Metabolism 27:555-561, 1978 23. Brass EP, Hoppel CL: Carnitine fasting rat. J Biol Chem 253:2688-2693,
metabolism 1978
in the
24. Engei WK, Sherbert RT: Fasting-associated fatal rhabdomyolysis (familial). Neurolosy 27:347, 1977 (abstract)
Test in Neuromuscular
Diseases
James E. Carroll, Darryl C. DeVivo, Michael H. Brooke, G. James Planer, and James H. Hagberg A 38-hr fast was used as a provocative test in patients suspected of having defects in muscle substrate utilization. In five controls and nine patients, exercise capacity and respiratory exchange ratio were determined before and at the end of the fast. Blood was collected at intervals during the fast from ten controls and nine patients for creetine kinase (CK). free fatty acids, (FFA) @hydroxybutyrate, acetoacetate, free and total carnitine, glucose, and alanine. Two patients with myophosphorylase deficiency had increased exercise capacity, and a marked fall in CK, and one had a lesser fall in blood glucose than normal at the end of the fast. Two patients with known lipid myopathies (carnitine deficiency and carnitine palmityl transferase deficiency) had decreased exercise capacity and apparent increased dependence on carbohydrate metabolism during the fast. Carnitine concentrations became even more abnormal in the patient with carnitine deficiency during fasting. Several patients with less well-defined defects were also significantly different from the controls in several respects, indicating that the fast might be useful for finding new defects.
homeostasis dictates that to M ETABOLIC preserve body protein stores during fasting, lipids must be substituted for carbohydrates as the major substrate for oxidative metabolism.’ During caloric deprivation, serum glucose concentrations decrease and FFA concentrations rise,‘,* providing increased availability of the latter substrate. Therefore, the utility of fasting as a provocative test to identify patients suspected of defects in fatty acid metabolism3-’ is apparent. Patients with disorders of fatty acid mobilization or utilization might be expected to become symptomatic during fasting, while those with defective glycogen metabolism might actually improve. Our goal in this study has been to exploit a previously standardized protocol of fasting,* which could be used in patients thought to have defects of muscle substrate utilization. We recognize that such a test may not yield a specific diagnosis, but the results may guide subsequent biochemical investigations. In addition to identifying patients with known metabolic defects, new abnormalities might be brought to light. Accordingly, we fasted ten control subjects
Metabolism,Vol.
28, No. 6 (June), 1979
and nine patients with a variety of muscle disorders for a 38-hr period. Prior to and following fasting, exercise capacity and respiratory exchange ratio (RER) were measured. During fasting, blood was collected for various metabolites expected to reflect changes in fatty acid and carbohydrate metabolism. MATERIALS
AND
METHODS
All studies were performed in the Washington University Clinical Research Center according to protocols that had been approved by the Washington University Human Studies Committee. Informed consent was obtained from the patient and/or parents in the case of minors. The patients are shown in Table I. Ragged red fiber disease is a mitochondrial myopathy with ophthalmoplegia and mild proximal limb weakness (patients 3 and 4). The patient with central core disease (patient 9) had greater than 90% type 1 fibers on muscle biopsy. Exercise capacity and RER were determined in five normal adult controls (three males and two females, ages 25-35) and the nine neuromuscular disease patients 2-4 hr following a meal and again at the end of a 3%hr fast. An incremental exercise test9 was done on a bicycle ergometer to determine maximal oxygen consumption (VO, max) and maximum work capacity. Previous experiments have shown that those methods accurately regect exercise capacity in neuromuscular disease patients.” Data were collected after the manner of Beaver et al.” using a PerkinElmer (MGA 1100) mass spectrometer for measurement of respiratory gases, a Fleish pneumotachograph attached to a Validyne pressure transducer for the measurement of ventilatory volumes, and a PDP-I2 computer. Data were calculated on a breath-by-breath basis and averaged over IS-set intervals for oxygen consumption (VO>), carbon dioxide production (VCO,), minute ventilation (VE), and RER. Five additional postpubertal controls with various neurologic diseases were added to the five normal controls for
From the Departments of Neurology and Neurosurgery (Neurology) and Pediatrics, the Department of Neurology and Neurosurgery (Neurology), and the Department of Preventive Medicine, Washington University School of Medicine, St. Louis, MO. Supported by a center grant from the Muscular Dystrophy Association, NHLBI Training Grant S-T32-HL-0708 entitled “Multidisciplinary Heart and Vascular Diseases,” and USPHS Grant RR-00036 from the Division of Research Facilities and Resources. Address reprint requests to Dr. James E. Carroll. Department of Neurology, Washington University School of Medicine, 660 S. Euclid. Box 81 II, St. Louis, MO. 431 IO. @ 1979 by Grune & Stratton, Inc. 002s-o49S/79/2906~I 3gOI .00/O
683
664
CARROLL ET AL.
Table 1. Patients
value. If the logs resulted in less skewing,
the antilogs of their means and standard deviations were used instead of those for the absolute numbers. Correlation coefficients were determined for plasma alanine and blood ketone concentrations, FFA and acylcarnitine concentrations (total minus free carnitine concentrations), and ketones and acylcarnitine concentrations.
Age Patient
Disease
Sax
IYrl
1
Myophosphorylase deficiency
Male
23
2
Myophosphaylasa
Female
34
of patient
deficiency (sibling
1)
3
Ragged red fiber disease
Male
50
4
Ragged red fiber disease
Female
31
5
Carnitine palmityl transferase
Male
23
Female
29
RESULTS
deficiency Muscle carnitine deficiency
6 7
Limb girdle dystrophy
Female
31
6
Limb girdle dystrophy (sibling of
Female
29
9
Type 1 predominance (central core
Male
36
patient 7) disease)
blood sampling during the fast; they, however,
did not take part in the exercise testing. Fasting was begun at 1800 hr on day one following the evening meal of that day. Water was allowed and lib. during the fast. The fast ended at 0800 hr of day three. Blood samples were obtained at 1800 and 2400 hr on day one, at 0800, 1200, 1600, 2000, and 2400 hr on day two, and 0400 and 0800 hr on day three. CK, FFA,12 and total and free carnitine were determined on serum specimens. Total carnitine was assayed following a mild alkaline hydrolysis at 37°C. For the carnitine assay, the method of McGarry and Foster” was modified by the substitution of Hepes for Tris buffer.14 Glucose, @-hydroxybutyrate, and acetoacetate were determined by specific enzymatic, microfluorometric methods on perchloric acid extracts of whole blood.” Alanine was measured by an enzymatic microfluorometric procedure on heparinized plasma samples.‘6 Values from the blood samples of individual patients were taken to be significantly different if they fell outside two standard deviations from the controls. Control data were tested for skewness” before and after taking the log of each Table 2.
Exercise capacities before and after the fast are shown in Table 2. The two sisters with limb girdle dystrophy were unable to exercise, although resting respiratory gases were collected. Whereas most subjects had the same or slightly less exercise capacity at the end of the fast, the two patients with myophosphorylase deficiency improved. Conversely, the patients with carnitine palmityl transferase deficiency and carnitine deficiency were unable to exercise at the end of the fast. Changes in resting RER for the five normal controls over the 38-hr fast ranged from -0.08 to -0.22. None of the patients were outside this range. Because of diurnal variation, only the blood metabolite data from the 14- and 38-hr points (0800-0800) during the fast are presented for comparison. Figures 1 and 2 show the comparative concentrations of FFA, acetoacetate, and fl-hydroxybutyrate, total and free carnitine, and alanine at the 14- and 38-hr points for the ten controls (five normal and five disease controls) and for the nine patients. Figure 2 also shows the
Exercise Capacity VO, Max
Before Fast Subjects
(literlmin)
Work Max End of Fast
lliter/min)
Before Fast
End of Fast
(kpmlmin)
lkpmfminl
Controls 1
3.66
3.69*
1600
2
2.62
2.49
1500
1300
3
2.51
2.27
1200
1000
4
4.4 1
4.29
2200
2200
5
1.58
1.42
900
700
0.65
0.69’
225
300’
1.28
1.56.
450
525.
1.61
1.24
600
600
0.95
0.68
300
225
Myophosphorylase deficiency Ragged red fiber disease “Limb girdle dystrophy”
Did not exercise
Carnitine palmityl transferase deficiency
3.00
Camitine deficiency
0.50
--t --t
Type 1 predominance
1.59
1.64’
*Better with fasting. tCould not exercise.
1500
1500 225 525
--t --t 450
FASTING IN NEUROMUSCULAR DISEASES
t t
Fig. 1. Fatty acid. acatoacetate. and &hydroxybutyrate data for 0800 of day two and day three. (‘I Outside two standard deviations.
Fig. 2. Total and free carnitine and alanine are shown for each 0800 point. Glucose is presented as ratio of the two points. (‘1 Outside two standard deviations.
ratios of the 3%hr glucose to the 14-hr glucose for all subjects. CK activities decreased in the controls at the end of the fast to 85.1 + 14.6% (SD) of the initial value. Only the patient with carnitine palmityl transferase deficiency had a significant rise during the fast (268%). The patients with carnitine deficiency and myophosphorylase deficiency fell to 53.1% and 57% of initial values, respectively. The correlation between plasma alanine concentrations and total ketones in the controls at the 14- and 3%hr points was not significant (r = -0.24, p > 0.35). A significant correlation existed between the concentrations of acylcarnitine and FFA (r = 0,84, p < 0.01) and acylcarnitine and total ketones (r = 0.74, p < 0.01) at the 14- and 38-hr points for the ten controls.
data, indicating that elevations in these parameters during fasting should be interpreted with caution. The necessity of grouping together men and women probably contributed to the skewing effect. The patients with myophosphorylase deficiency had improved exercise capacity, and a marked fall in CK at the end of the fast, and one of the patients had a lesser decrement in blood glucose concentration that the controls. Increased dependency on FFA by these two patients was the probable mechanism underlying the salutary effects of fasting; their moderately low concentrations of FFA at the end of the test are compatible with the speculation of proportionately greater utilization of this substrate at any point in time. On the other hand, both patients with disturbances of muscle lipid metabolism were unable to exercise at the end of the fast, and they had greater than normal decrements in blood glucose. The latter observation probably indicates an increased dependence upon carbohydrate metabolism during fasting. In regard to other parameters, however, the responses of these two patients differed widely. The patient with carnitine palmityl transferase deficiency demonstrated elevated carnitine, alanine, and CK concentrations at the end of the fast. Ketone concentrations were low, consistent with the supposition that uptake of fatty acids into liver mitochondria was reduced. The patient with muscle carnitine deficiency had low concentrations of free carnitine at the beginning of the
DISCUSSION
In this study the utility of a standardized fasting protocol has been examined as a provocative screening test for metabolic diseases of muscle. Our conclusions are based on the establishment of the normal response of a control group against which individual patient values can be compared. Variations were judged to be significant if they fell outside two standard deviations from the mean. An attempt to generate a predictable response with only ten control subjects can result in considerable skewing of the data. In this study, skewing to the right was particularly evident in the ketone and fatty acid
CARROLL ET AL.
666
fast. At the end of the fast, both total and free carnitine were low. Ketone and fatty acid concentrations were high at the end of the fast, but not beyond two standard deviations. Both patients had an appropriate fall in RER during the fast, indicating that fatty acids were at least partially utilized. Among the patients with less well-defined biochemical defects, several findings stand out. The two sisters with limb girdle dystrophy had reduced concentrations of free carnitine at the end of the fast, while acylcarnitine concentrations were elevated. Blood glucose concentrations showed a greater-than-normal fall in one of the patients. Several possibilities exist for the defect in these two patients: (1) a defect similar to that in the patient with carnitine deficiency, (2) an undescribed defect of muscle lipid metabolism, or (3) decreased body stores or carnitine because of decreased muscle mass. The patient with central core disease and type 1 predominance failed to demonstrate a rise in FFA at the end of the fast, in contrast to the patients with lipid utilization defects. The most plausible explanation for the lack of rise may be increased utilization by the large numbers of type 1 fibers. In addition, plasma alanine concentrations in the central core patient were low throughout the fast. The divergent alanine responses in this patient and the patient with carnitine palmityl transferase deficiency are noteworthy in relation to the role of skeletal muscle in alanine homeostasis. Since a large fraction of plasma alanine supposedly arises from muscle, ‘* it may be that type 1 muscle fibers release less alanine. Conversely, the paradoxic increase in plasma alanine during fasting in the patient with carnitine palmityl transferase deficiency might suggest disproportionate metabolic activity in type 2 muscle fibers with increasing efflux of alanine under these conditions. Several parameters were examined for correlations. Sherwin et aLI have suggested that the decrease in plasma alanine concentration that normally occurs during fasting is caused by a feedback mechanism induced principally by rises in blood ketone concentrations. Although there was a moderate negative correlation between these values, it was not statistically significant, indicating that there are a number of other
variables influencing this process in both the controls and patients. This observation is consistent with previous studies using stable isotopes in children, where ketosis did not change alanine flux.20 Among the controls, there was a strongly positive relationship between FFA and acylcarnitine concentrations and total ketones and acylcarnitine concentrations. A similar relationship was found in the patients. Other workers have noted that these correlations exist under a variety of conditions.2’m23 The safety of fasting should also be considered in patients with metabolic defects. None of the subjects in this study had serious side effects with fasting, although the two patients with lipid myopathies experienced considerable muscle pain toward the end of the fast. The woman with carnitine deficiency developed a mild metabolic acidosis (data not shown) and a relative hypoglycemia (2.63 mM), but neither perturbation produced symptoms. Because others have reported deaths following fasting in children with undiagnosed neuromuscular diseases.24 we prefer to employ a rather brief fast. Undoubtedly, a longer fast would increase the differences between the responses of the patients and that of the controls. This 38-hr fasting protocol has been used by one of us (D.C.D.) in over one hundred children and adults with a variety of diseases without untoward events.8.20 Fasting as a provocative test acts to accentuate defects in muscle substrate utilization. Thus, abnormalities that are either not apparent or mildly abnormal under postabsorptive conditions become clearly abnormal during fasting. Most of the abnormal substrate data in this study can be interpreted as varying either because of a production or utilization defect. Specific studies of substrate turnover in the patients would be necessary to determine which aspect of metabolism is altered. Even so, the utility of this test is demonstrated by the fact that we were able to discriminate among the known metabolic defects and also suggest new defects in less well-characterized disorders.
ACKNOWLEDGMENT The authors wish to thank Dr. J. Phillip Miller for assistance in statistical analysis of the data.
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