Journal of the neurological Sciences, 1973, 20:297-302 297 ~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

Leucocyte Fatty Acid Oxidation in Hereditary Neuro- muscular Disorders A Preliminary Report

B R E N D A K I N G AND A. E. H. EMERY

University Department of Human Genetics, Western General Hospital, Edinburgh EH4 2HU (Great Britain)

(Received 15 May, 1973)

INTRODUCTION

The study of affected muscle in order to elucidate the biochemical defect in neuro- muscular disorders has several inherent disadvantages. Firstly, the amount of tissue available from a biopsy is very limited. Secondly, muscle is a heterogeneous tissue, and in muscular dystrophy, is infiltrated by macrophages and fibroblasts which may contribute significantly to its enzyme content (Emery 1968; Pennington 1969). Thirdly, it is difficult to obtain adequate control values.

More recently, investigators have been attracted to the idea that the neuromuscu- lar disorders are most probably generalized metabolic diseases, and that the basic biochemical defect may therefore be expressed in tissues other than muscle, e.g. skin fibroblasts (Davidson 1970) or peripheral blood leucocytes (Hsia 1970). For this reason we have studied metabolic pathways in peripheral blood leucocytes in patients suffering from various types of muscular dystrophy.

Firstly, we screened the enzymes of the glycolytic pathway in leucocytes (Emery, King and Brock 1971) by measuring the rate of conversion of glucose to lactic acid. We did not, however, find any significant difference between the patients' results and control values.

The present communication concerns the results of a study of fatty acid oxidation (t-oxidation) in leucocytes. This pathway was chosen because of the suspected diag- nosis of a "lipid myopathy" in a patient under investigation at the time and also be- cause of a report by Lin, Hudson and Strickland (1972) of decreased palmitate oxida- tion by skeletal muscle mitochondria in a patient with Duchenne muscular dystrophy.

The deficiency of an enzyme at any point in the t-oxidation pathway would be expected to result in defective conversion of fatty acid to carbon dioxide. Therefore, by studying this conversion, it should be possible to screen for a defect in this chain

This study was supported by a grant from the Muscular Dystrophy Group of Great Britain.

298 B. KING, A. E. H. EMERY

of reactions. The present study reports the measurement of the oxidation of radio- actively labelled palmitic acid to carbon dioxide, by the widely-used technique of carbon dioxide trapping and subsequent scintillation counting (Cuppy and Crevasse 1963).

MATERIALS AND METHODS

Subjects Control values were obtained from 17 healthy subjects (aged 17-60) with no family

history of any neuromuscular disease. Blood was also obtained from patients with various types of muscular dystrophy, the diagnosis in each case being established on the basis of clinical examination, electromyography and muscle histology. In all cases, venepuncture was performed after an overnight fast to minimise any dietary effect since it is known that pathways of carbohydrate metabolism interact closely with those of lipid metabolism (Buchanan, 1960; Doery, Hirsh and Cooper 1970; Elsbach, 1962; Vahouny, Katzen and Entenman 1967).

Leucocytes were isolated from 5-10 ml heparinized venous blood as previously described (Emery et al. 1971 ). The remaining red blood cells were removed by hypo- tonic shock applied for 10 sec. (Baron and Ahmed 1969). An estimation of the number of leucocytes in an aliquot of the resulting suspension was made using a Coulter counter, and approximately 10 x 106 cells were used in the incubation mixture.

Apparatus The principle of the manometric flask was employed, the apparatus consisting of

an incubation vial (inner diameter, 14 mm ;length 40 mm), an inner tube (diameter. 9 mm ; length 10 ram) suspended by stainless steel wire from the neck of the vial, and a suba-seal rubber stopper (Gallenkamp, No. 29).

Substrate preparation Fifty #Ci[ 1-14C] palmitic acid with a specific activity of 55 mCi/mM was obtained

from the Radiochemical Centre, Amersham. Since this specific activity was exces- sively high for our purposes, it was modified using unlabelled palmitate to 150 #Ci/mM. It was decided to try to mimic, as closely as possible, physiological condi- tions, by using a fatty acid concentration comparable to that found in human serum (i.e. 0.3 mM), and by complexing the fatty acid to albumin, thus facilitating its uptake by the cells.

The complex was prepared freshly when required. To bring about the fat-protein binding, the palmitate was dissolved in methylcellosolve, mixed with an equal volume of warmed 1~ albumin in Krebs-Ringer buffer D (pH 7.0), and the organic solvent was subsequently removed using a stream of nitrogen gas (modification of Vahouny et al. 1967 and Doery et al. 1970). Glucose was omitted from the buffer to avoid its sparing effect on fatty acid utilisation (Vahouny et al. 1967; Doery et al. 1970). The level of radioactivity in the complex was measured each time by placing 10/zl directly into 10 ml of scintillation fluid and counting it along with the experimental vials.

LEUCOCYTE FATTY ACID OXIDATION IN HEREDITARY NEUROMUSCULAR DISORDERS 299

Scintillation fluid Four g PPO (2,5-diphenyloxazole) and 0.5 g POPOP [p-bis-2'-(5'-phenyloxazolyl)

benzene] were dissolved in 1.0 1 of scintillation grade toluene.

Incubation The leucocytes were suspended in 0.5 ml Dulbecco A tissue culture medium, and

placed in a vial along with 10 #1 (equivalent to approximately 10,000 d.p.m.) of the substrate complex. The smaller tube was suspended from the neck of the vial and the suba-seal stopper was inserted. A blank, omitting the cells, was set up at the same time, and a control blood sample was run in parallel with each patient sample.

The vials were incubated at 37°C in a shaking water bath for 2 hr. At the end of this time, 0.2 ml hyamine hydroxide (Nuclear Enterprises), a carbon dioxide trapping agent, was injected using a hypodermic needle, gauge 23, into the inner tube. In order to stop the reaction and to release any dissolved carbon dioxide from the medium, 0.1 ml 0.25 N sulphuric acid was injected into the outer compartment.

After a further hour of incubation to ensure complete absorption of the gas by hyamine, the contents of the inner tube were transferred, using a pasteur pipette, to a scintillation vial containing 10 ml of scintillation fluid. The level of/~-radioactivity was then measured using a Nuclear Enterprises/~/~ counter, and corrected for efficiency by comparing it with an external standard.

The apparatus was checked for efficiency by placing 0.5 ml of a standard [14C] sodium bicarbonate solution in the incubation vial, inserting the stopper and injec- ting hyamine and acid. The concentration of bicarbonate was chosen so that when sufficient acid was injected to neutralise it, the amount of ~4CO2 released would be in excess of that expected from fatty acid oxidation. The volume of hyamine used to absorb the gas and also the time taken for absorption were varied. An optimum re- covery of over 90~ was obtained using 0.2 ml hyamine and 1 hr of absorption time.

RESULTS

In order to determine the optimum incubation time and the optimum number of cells required, several experiments were carried out varying these 2 parameters. The reac- tion rate was linear up to 3 hr at the substrate concentration used. An incubation time of 2 hr was chosen so that the number of counts/min recovered in the hyamine was suf- ficiently high to minimise the contribution of experimental error. The results obtained were linearly related to the number of cells in the range 5-15 × 106, an amount readily obtained from 5-10 ml blood. If sufficient cells were available, a duplicate incubation was set up.

The results were corrected for efficiency of scintillation counting using a standard graph prepared from a series of hyamine-quenched 14C standards, and an external /~-radiation source.

The results are presented in Table 1. The values were obtained by expressing the counts/min in the hyamine as a percentage of the counts/rain in 10 #1 of substrate complex.

300 t~. KING, A. E. H. EMERY

TABLt I

FATTY ACID OXIDATION (]~-OXIDATION) IN LEUCOCYTES FROM HEALTHY {.X)NTROI,S I N ::~ 17) A~I) I ' A r . ~ t";

W I T H VARIOUS NEUROMUSCULAR DISORI')ERS

Subject Age "o Counls/'min/ ( yrs) 106 cells/hr

Controls (mean + S.D) 17-60 0.394 + 0.106

Patients DMD, familial 8 0.204 DMD, sporadic 16 0.220 FSHMD 67 0.566 LGMD 50 0.516 Distal muscular dystrophy 60 0.248 "Lipid myopathy" 16 0.354 Dystrophia myotonica 31 0.600

DMD: Duchenne muscular dystrophy; FSHMD: facioscapulohumeral muscular dystrophy; LGMD: limb-girdle muscular dystrophy.

In the control series, no relationship was found with age or sex. There was no significant difference between the patients' results and those of the controls, indi- cating that there is no major defect in the t-oxidation pathway in leucocytes in any of the patients studied.

DISCUSSION

The concept that neuromuscular diseases may be generalized metabolic disorders has opened up a new approach to their study. In the present report, we have used peripheral blood leucocytes in an attempt to screen a metabolic pathway, thus obvia- ting the disadvantages encountered when using affected muscle tissue.

The decision to screen the pathway of t-oxidation was initiated by the suspected diagnosis of a possible lipid myopathy in a 16-year-old boy with a 2-year history of proximal muscle weakness. From histochemical studies, there appeared to be an accumulation of neutral lipid, possibly triglyceride, in the muscle tissue. This finding could be due to a decreased rate of degradation, or an increased rate of synthesis of lipids. It could also be a secondary effect, rather than the basic cause of the disease. We first decided to examine one of the degradative pathways in this patient but were unable to obtain any meaningful results with our initial substrate, glyceryl-tri- [1-1*C]palmitate, possibly because of the difficulty of transporting such a molecule into the cells.

We therefore decided to start at the level of fatty acid, choosing palmitate as it is one of the most commonly occurring fatty acids in the body and also because Lin et al. (1972) had reported a decreased conversion of palmitate to carbon dioxide by skeletal muscle mitochondria in a patient with Duchenne muscular dystrophy.

In the present study all the results from patients with various muscular dystrophies, including the boy with a suspected lipid myopathy, were within the range of values

LEUCOCYTE FATTY ACID OXIDATION IN HEREDITARY NEUROMUSCULAR DISORDERS 301

obtained in healthy controls. It is, however, interesting to note that, with the excep- tion of a patient with distal muscular dystrophy, the later onset dystrophies (i.e. facioscapulohumeral, limb girdle and dystrophia myotonica) had values in the upper part of the normal range whereas the results from the 2 patients with Duchenne dys- trophy were much lower. In controls, on the other hand, there was no difference in the values obtained in young individuals as compared with older individuals. There was no significant difference between the values in the familial and sporadic cases of Duchenne muscular dystrophy, which is in contrast to the findings ofLin et al. (1972) using muscle mitochondria.

These negative results do not, of course, allow any conclusions to be drawn regar- ding the aetiology of these diseases: the basic biochemical lesion could lie in the//- oxidation pathway but not be expressed in leucocytes. It is possible, however, that the lower levels of conversion observed in the 2 patients with Duchenne muscular dystro- phy may be indicative of a defective enzyme at some point in the//-oxidation pathway, which may be more fully expressed in muscle tissue in this disorder, as found by Lin et al. (1972).

ACKNOWLEDGEMENTS

We are grateful to Dr. D. J. H. Brock for helpful discussions, to Mr. D. Shirling for advice on the apparatus and use of the NE fl/y counter, and to Miss L. Shearer for technical assistance.

SUMMARY

An attempt has been made to screen for an enzyme defect in the metabolic pathway involved in fatty acid oxidation (//-oxidation) using peripheral blood leucocytes from patients with various neuromuscular disorders. The widely-used technique of 14CO2 trapping and subsequent scintillation counting was employed. In controls the values obtained were unrelated to age or sex. No significant difference was found between the values in patients with various forms of muscular dystrophy and in controls. However, in 2 patients with Duchenne muscular dystrophy the values were lower than in patients with other forms of dystrophy.

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