Immunoquantitation of carnitine palmitoyl transferase in skeletal muscle of 31 patients

Neuromusc.Disord..Vol.2. No.4, pp. 249-259. 1992 Printedin GreatBritain

0960-8966/92 $5.00 ~ 0.00 © 1992PergamonPressLtd

IMMUNOQUANTITATION OF CARNITINE PALMITOYL TRANSFERASE IN SKELETAL MUSCLE OF 31 PATIENTS GEORG1RENE D. VLADUTIU,*~"ISORA SAPONARA,* JEFFREY M. CONROY,* ROBERT E. GRIER, + LINDA BRADY§ and PAUL BRADY§ *Department of Pediatrics, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York; ~Department of Pediatrics, University of Texas Medical School, Houston, Texas; and §Department of Food Sciences and Nutrition, University of Minnesota, St. Paul, Minnesota, U.S.A.

(Received 27 November 1991; revised 18 June 1992; accepted I September 1992)

Abstract--We studied 3 i patients suspected of having muscle carnitine palmitoyl transferase 2 (CPT2) deficiency. The catalytic activity of CPT2 was measured in muscle biopsies by the isotope exchange method and CPT2 immunoreactivity was quantitated by an enzyme-linked immunosorbent assay. Nine patients had normal enzyme activity and immunoreactivity. Eight patients had significant deficiencies in catalytic activity ( > 3 S.D. below reference mean) of which six were also deficient in immunoreactivity. An additional nine patients were significantly deficient in immunoreactivity with normal catalytic activity and five patients had partial deficiencies in both. At least two categories of alterations in CPT may exist which lead to a deficiency based on the data presented: (!) a regulatory defect in CPT which only alters the enzyme active site; and (2) a structural defect due to altered synthesis, increased degradation, or changes in the immunoreactive site. It may prove to be of diagnostic importance to combine the analysis of enzyme activity and immunoreactivity in patients suspected of having a CPT deficiency and to further investigate the condition of partial CPT deficiency. Key words: Carnitine palmitoyl transferase, enzyme-linked immunosorbent assay, muscle disease, mitochondrial myopathy.

INTRODUCTION

Carnitine palmitoyl transferase (EC 2.3.1.21; CPT) deficiency of skeletal muscle was first described in 1973 [1], and by 1986, 39 patients with CPT deficiency were known or reported to have symptoms of episodic exercise-induced muscle stiffness, myalgia, weakness, rhabdomyolysis, and myoglobinuria [2]. At least 20 additional patients with CPT deficiency have been reported between 1986 and 1990 [3-13]. The occurrence of sporadic cases and familial cases suggest that the disorder follows a recessive mode of inheritance even though most of the reported cases have been males [2]. Although the residual muscle CPT activity in symptomatic patients usually ranges from 0 to 30% of normal [14], patients with clinical symptoms have been reported with as much as 45-60% of normal activity [15-17]. tAuthor to whom correspondence and reprint requests should be addressed at: Department of Pediatrics, Children's Hospital of Buffalo, 936 Delaware Avenue, Buffalo, NY 14209 U.S.A. 249

Partial deficiency of CPT has also been reported in various non-muscle cell types from affected patients, including peripheral blood leukocytes, in vitro transformed lymphoblasts, platelets and cultured skin fibroblasts [2]. One of the main difficulties in defining the causative defect(s) in CPT deficiency disorders has been in characterizing the two isoforms of CPT and determining which one or both may be altered in the muscle disorder. CPT has two functional locations within the mitochondrion. CPTj, a malonyl-CoA-sensitive enzyme that requires mitochondrial membrane association for its catalytic activity, is inactivated by detergents and is located on the inner surface of the outer mitochondrial membrane. CPT 2 is on the inner surface of the inner mitochondrial membrane, is not inhibited by malonyl-CoA, and remains functional after detergent solubilization [18]. Many investigators have studied the distinctive regulatory properties of CPT~ and CPT 2, [19, 20], however, there are currently only deductive methods of biochemical or immunochemical analyses to distinguish CPT~ from CPT2 [20]. In

250

G . D . VLADUTIL' et al.

addition to muscle CPT deficiency, believed to involve primarily CPT2, a hepatic disorder, characterized by a liver-specific CPT~ deficiency, has been described consisting of fasting hypoglycemia and inappropriately low ketogenesis [21, 22]. There have also been reports of an infantile multisystem form of CPT, deficiency in which there is impaired oxidation of long-chain fatty acids leading to a toxic accumulation of acylcarnitines in various tissues [23, 24]. In order to further characterize residual CPT in the muscle of enzyme-deficient patients, we have developed an enzyme-linked immunosorbent assay (ELISA) for quantitation of CPT2 in tissue homogenates without the necessity of purifying mitochondria or CPT. Using this immunoassay together with an isotope exchange assay to measure enzyme activity, we propose the existence of at least two categories of CPT deficiency in the muscular presentation of the disorder.

Enzymatic activity (EA) of CPT was measured in aliquots of muscle homogenate using the isotope exchange method of Norum [25]. Reaction rates were linear over the 10 min time period studied. EA was expressed as nmol of palmitoyl-'4Ccarnitine formed rain-~ g-~ wet weight of tissue. A standard reference mean of 77.8 + 13.3 nmol rain- ~g- ~was established from the analysis of 43 muscle biopsies taken mainly from the vastus lateralis of unaffected patients.

Protein determination For immunoquantitation, muscle homogenates were centrifuged at 12,000 g for 10 min at

1

2

3 !=: i!!i

MATERIALS AND M E T H O D S

Clinical material Thirty-one patients who presented clinically with complaints of exertional cramps, myalgia, muscle stiffness or weakness, with or without rhabdomyolysis and myoglobinuria, were selected for this study. An attempt was made to obtain essential clinical and laboratory data on each patient studied, either through the information provided with the referral or by additional follow-up questionnaires to the referring physician. At the conclusion of the study, collectively, 58% of the essential clinical information and 61% of the supportive laboratory data had been acquired for all of the patients. With the approval of the Children's Hospital of Buffalo institutional review board, muscle biopsies were obtained after receiving informed consent from patients of both sexes ranging in age from five to sixty years. Seventy-seven percent of the patients were males. Biopsies were obtained from the vastus lateralis muscle, snap frozen in liquid nitrogen, transported on dry ice, and stored in liquid nitrogen until analysis. Muscle homogenization and CPT enzymatic activiO" Muscle biopsy specimens were homogenized in nine volumes (10%, w/v) of 0.05 mol 1-~ Tris buffer containing 0.15 mol 1-~ KCI, pH 7.4, using an all-glass motor-driven homogenizer (Talboy Engineering Corp., Emerson, U.S.A.).

Fig. 1. Western blot analysis of CPT. Lane 1, preimmune rabbit serum + human liver extract; lane 2, human liver extract; lane 3, rat liver extract. The primary antibody was rabbit anti-rat CPT serum and the second antibody was alkaline phosphatase-labelled goat anti-rabbit serum. Blotting method used as previously described [31].

251

lmmunoquantitation of CPT

4"C, and the protein concentration of each supernatant was determined by the Coomassie blue method of Bradford [26].

Antibody production Polyclonal antibodies against purified rat liver CPT2 (MW = 68,000) were obtained by immunization of New Zealand rabbits, and were tested for specificity, as previously described [27, 28]. IgG was purified from the immune serum using a mass membrane affinity separation system (Nygene Corp., Yonkers, U.S.A.) with protein A as the affinity ligand. The eluate containing purified IgG was concentrated six-fold using a Diaflo ultrafiltration membrane (XMI00A; Amicon Corp., Danvers, U.S.A.). The antibody reacted only with mitochondrial CPT, and did not cross-react with peroxisomal CPT in Western blot analysis [29, 30]. The antibody was shown to react with human liver CPT using Western blot analysis (Fig. 1) and using previously described methodology [31 ].

CPT immunoquantitation A modified direct sandwich enzyme-linked immunosorbent assay (ELISA) was used to quantitate CPT immunoreactive protein using disposable 96 well, flat bottom polystyrene microtiter plates (Corning Glass Works, Inc., Corning, U.S.A.) [32]. All reagents used in the assay were purchased from Kirkegaard & Perry Laboratories. Inc. (Gaithersburg, MD, U.S.A.). Muscle homogenate supernatants were used for all ELISA following centrifugation at 12,000 g for 10 min at 4°C. One hundred microliters of supernatant diluted to a protein concentration of 13/ag ml-L in coating buffer (0.1 mol 1- ~sodium carbonate buffer, pH 9.6) were dispensed and serially diluted in microtiter plate wells. The plates were incubated overnight at 4°C in a humid chamber, drained, and the wells incubated with 2 x 300/~1 per well milk blocking buffer (2% non-fat dry milk in 0.19 mol 1-~ borate buffer, pH 8.0) for 4 min and 1 h, respectively, at room temperature. Fifty microliters rabbit IgG antiCPT, diluted 1:60 in milk blocking buffer were added to each well and incubated for 30 min at 37°C. The wells were washed with 4 × 300/,tl washing buffer, and then incubated at 37°C × 30 min with 50 ~1 per well of peroxidaseconjugated goat anti-rabbit IgG diluted in the Tris buffer to 1:1600. Negative controls included substitution of normal rabbit serum for immune serum and the elimination of

either the rabbit immune serum or the muscle homogenate from the reaction mixture. Plates were washed with 6 x 300 pl per well of washing buffer, then incubated with 100 /.tl per well of 3, 3', 5, 5'-tetramethylbenzidine (TMB) + 0.02% hydrogen peroxide in 0.006 mol i -t citric acid buffer, pH 2.8 for 15 min at room temperature in the dark. The reaction was terminated with 100 pl per well of I mol 1-~ H3PO4. Absorbance (450 nm) of the reaction product was measured with a Multiskan Plus MKII ELISA Microplate Reader (ICN Biomedicais, Huntsville, AL, U.S.A.).

Determination of immunoreactivity units The reference standard method as described by Peterman and Butler [33], was used for data analysis. This method provides the advantage of a relative measurement of immunoreactivity of an antigen without necessitating the titration of a purified antigen preparation. The reference standard (RS) consisted of a pool of normal muscle homogenates prepared in 0.05 mol 1-~ Tris buffer containing 0.15 mol 1-~ KC1, pH 7.4, with a protein concentration of 3.77 mg ml -~, and diluted in coating buffer to a final concentration of 13/~g ml-~. Serial dilutions of the RS were used on each microtiter plate in order to generate a standard curve. The standard curve was established by linear regression analysis of logarithmic transformed data from titration of the RS. The slope of the line approached 1.0 (range 0.8-1.1). Data derived from multiple dilutions of an unknown sample were plotted and the slope of the resulting curve was compared with that of the RS curve. The following equation provided the calculation of ELISA units (EU) for the unknown sample when the slopes of the RS and sample curves were similar: concentration (EU ml-~) = RS value x sample dilution factor/e 1~-~ ,1( where RS value = an assigned concentration of the undiluted RS (e.g. 100 EU ml-~): }" = Iog¢ [sample OD]; C = value ofy-intercept established for the standard curve; M = value of slope established from the standard curve. For an assay to be valid, there should be no trend toward increasing or decreasing values of EU ml-~ as a function of dilution [33]. The unpaired two-tailed Student's t-test was used for comparisons between groups. R ES U LTS

Figure 2 depicts the results of a typical ELISA analysis for a normal muscle homogenate

252

G . D . VLADUTIU et al.

2.0 O

x 1.0 e.-

a

0.5

-.~

o o Reference Standard • Normal Muscle 0.1

t 2.0

L 2.5

I 3.0

~\ ~k ~ 3.5

0 4.0

4.5

Logarithm of Dilution Factor Fig. 2. Logarithmic plot of CPT immunoreactivity. Referencestandard and normal musclecurves derived using the

EL1SA method (for details see Materials and Methods). Results of a typical experiment are depicted. Slope = 0.8: regression coefficient = 0.99. compared to the RS. The curve of the unknown sample was parallel to the RS curve enabling the relative EU m l - ~ of the sample to be calculated. The E U m g - ' protein was calculated using the protein concentration of the muscle homogenate supernatant. The patients were divided into five groups based on their combined CPT EA and EU results. Muscle samples with C P T EA or EU greater than 3 S.D. below their respective mean reference activities were considered to be significantly deficient. In Table 1, group A consisted of nine patients who had muscle C P T EA within the laboratory's established reference range (77.8 + 13.3 nmol m i n - ' g-~ wet weight tissue) and EU within 3 S.D. of the reference mean (26.3 + 3.4 EU rag- ~protein) with the exception of patients 1-3 who had somewhat elevated C P T EA. The reference ratio of E A / E U was 2.9. Nine patients in group B had normal muscle C P T EA but with EU values between 3 and 4 S.D. below the reference mean (p <0.001). Patients 16 and 18 had C P T EU values which were more than 5 S.D. below the reference mean (p < 0.001 ). In group C, two patients (19 and 20) had a mean muscle CPT EA more than 3 S D . below the reference mean (p<0.001) while the CPT EU fell within the reference interval. Six additional patients (group D) with significant C P T EA deficiencies, also had very low EU (3-5 S D . below the reference mean) while in group E, five patients had a moderate CPT EA and EU deficiency ( < 2 S.D. below the reference mean). Mixture experiments were performed with muscle homogenates (l:l, v/v) from CPT-defi-

cient patients and control muscle homogenates. N o evidence for inhibitors or activators of EA or of EU was obtained. These experiments did not, however, rule out the possibility of insoluble membrane-bound inhibitors or activators which may have interfered with the enzyme active site and/or immunoreactive site in situ in patients with abnormal EA or EU. Tables 2 and 3 show the clinical and laboratory features of the patients, with summaries in Tables 4 and 5, respectively. Males predominated and represented from 60 to 100% of patients in each group. A positive history did appear to be present more commonly in patients with EA > 2-5 S.D. below the reference mean (groups C, D and E) than in patients with normal EA (groups A and B; Table 4). The major clinical features in patients of all groups included cramps, pain or stiffness (25/25), exercise intolerance (20/21), weakness (15/16) and episodes of myoglobinuria (7/16). Five of the seven patients with documented myoglobinuria were in the CPT-deficient groups C, D and E (Table 4). The data on temperature extremes, stress and infection were available on only 26% of all patients studied and were mainly compiled from patients in group A. Therefore, no conclusions could be drawn from these data. The most c o m m o n abnormality among laboratory findings was an elevated creatine kinase (CK) activity ( l I / 19). All patients from CPT EAdeficient groups C, D and E were a m o n g those with elevated C K (Table 5). An abnormal E M G was found in a few patients in each group but not enough patients were tested to allow a significant analysis. For similar reasons, no conclusions could be drawn regarding ischemic exercise test results. Three patients in this study had profound enzyme deficiencies unrelated to CPT as follows: patient I, myodenylate deaminase: patient 5, glucose-6-phosphate dehydrogenase: and patient 21, cytochrome c oxidase in addition to a CPT deficiency. Additional analyses of other muscle enzymes are performed for quality assurance of the specimen when a single muscle enzyme deficiency is found. However, in the case of patient 21, no extra tissue was available for these analyses. Depending on the availability of tissue, a total of 108 additional muscle analyses were performed on patient's muscles in all groups after CPT analysis, to either rule out other muscle disorders or to assure the quality of the specimen (Table 3). Usually the inadequate size of the biopsied tissue was the primary limiting factor

Immunoquantitation of CPT

253

Table I. C a m tine palmitoyl transferase aotivitY in normal and deficient muscle

Patient

Sex

Age (yr)

Ratio

Enzyme Activity EA g- t tissue

Immunoreactivity EU rag- ~protein

EA/EU

122.7 113.9 105.5 96.9 92.0 73.5 71.9 69.2 68.2 90.4 + 19.5

24.8 16.2 18.5 26.8 19.6 16.7 24.3 34.5 19.1 22.3± 5.6

4.9 7.0 5.7 3.6 4.7 4.4 2.9 2.0 3.6 4.3 ± 1.4

12.2

7.5 5.2 5.7 6.3 5.6 4.0

Group A. Normal EA and EU I M 2 M 3 M 4 M 5 M 6 M 7 M 8 F 9 M Mean ± S.D.

13 19 36 8 13 34 31 19 29

Group B. Normal EA, EU > 3 S.D. below normal mean 10 M 19 91.5

II

M

39

81.7

15.8

12 13 14 15

F M M M

M

79.0 77.7 71.1 64.5 64.1 55.8 51.6 70.8 ± 12.2

13.8 12.4 12.7 15.9

16

5 53 43 39 39 49 57

17 F 18 M Mean ± S.D.

Group C. EA > 3 S.D. below normal mean; normal EU 19 M 38 10.1 20* M 28 (0.4) Mean ± S.D. -

8.9

7.2

13.1 7.1 12.4 + 2.7

4.2 7.3 5.9± 1.2

20.6 18.4 19.5

0.5 -

Group D. EA > 3 S.D. below normal mean; EU 3-5 S.D. below normal mean 21 M 35 39.0 14.3 22 M 36 36.8 6.9 23 M 37 28.8 14.7 24 F 26 28.6 8.1 25 F 42 26.3 15.7 26 M 37 19.3 2.1 Mean -- S.D. 31.1 ± 6.9 10.3 ± 4.9

2.7 5.3 1.9 3.5 1.7 9.2 4.0 -- 2.6

Group E. EA > 2 S.D. below normal mean: EU > 2 S.D. below normal mean 27 M 60 47.7 16.3 28 M 35 45.7 12.4 29 M 9 43.2 10.5 30 F 35 41.3 6.9 31 F 43 40.0 17.5 Mean + S.D. 43.6 ± 2.8 12.7 + 3.9

2.9 3.7 4.1 5.9 2.3 3.8 ± 1.2

Normal Reference Mean =

77.8 ± 13.3 (n = 4 3 )

26.3 -,- 3.4 (n = 5)

2.9

Note: CPT enzyme activity (EA) was measured in duplicate by the isotope exchange assay. CPT immunoreactivity was measured by an enzyme-linked immunosorbent assay (ELISA) in duplicate and expressed as ELISA units (EU) from an average of three experiments (see Materials and Methods). S.D., standard deviation: n. number of normal muscle biopsies tested. *The muscle of patient No, 20 was assayed in Dr Grier's laboratory using the backward assay for CPT EA n and was found to have an EA of 0.4 nmol rain- ~mg- ~protein (laboratory reference mean 2.3 + 0.2 nmol min - ' rag-~ protein}. The figure in the table represents these units. No tissue was available for analysis of CPT by the

isotope exchange method.

preventing further analysis. One of the most important analyses performed on 17 of the 31 patients was for citrate synthase, a tricarboxylic acid cycle enzyme known to be a reliable indicator of mitochondrial content [34]. If citrate synthase activity was significantly low in any of the patients, then an apparent deficiency of CPT EA could possibly be attributed to a low number of mitochondria. None of the patients had a significant citrate synthase deficiency (Table 3). Histopathologic changes ranging from mild to severe were found in 16 of 28 patients' muscle biopsies from all groups except for group C

in which no abnormalities were found (Table 5). Of the 16 biopsies with pathologic changes, 7 had 2 or more significant abnormalities. The most common findings observed were mild myopathic changes (5 cases), elevated glycogen (3 cases), abnormal mitochondria (3 cases), elevated lipid (3 cases), and atrophic changes (5 cases). The highest proportion of patients with abnormal histopathologic changes were in groups D and E. DISCUSSION

The analysis of CPT EA in muscle homo-

254

G. D. VLADUTIU

et al.

Table 2. Clinical features

Patient and S e x

Age at diagnosis (yr)

Family history

Weakness

Symptoms with Cramps/pain, fasting stiffness Myoglobinuria

Rhabdomyolysis

Abnormal respon~ to temp. extremes. stress, Exercise infections intolerance

Group A. Normal EA and E U IM 2M 3M 4M 5M 6M 7M 8F 9M

13 19 36 8 13 34 31 10 23

+ 0

+ + +

0 +

+

+

+ 4-

0 0

Group B. Normal E A : E U > 3 S . D . below normal mean IOM 19 + I IM 12F 13M 14M 15M 16M

17 F 18M

39 5 53 43 39 39 49 57

+ +

0 0 0

z "~+

0 ~-

0 -

* + + + + + +

0 0 0 + 0

+ 0

+

Group C . E A > 3 S . D . below normal mean: normal E U 19M 20M

38 28

~0

+ 0

+ +

+ +

Group D . E A > 3 S . D . below normal mean: E U 3 - 5 S . D . below normal mean 21M 22M 23M 24F 25F 26M

35 36 37 26 42 37

O * ~ 0

~ +

~+ +

+ + +

0 0 0

+ +

+ -

G r o u p E. E A > 2 S , D . b e l o w normal mean: E U > 2 S , D . below normal mean 27M 60 + 28M 35 + 29M 9 30F 35 0 ~31F 43 ~ +

+ 4-

+ 0

0 +

4. .,.~

~- = p r e s e n t : 0 = absent: empty spaces = no information available.

genates from CPT-deficient patients has generally been restricted to the measurement of EA using rate-limiting conditions and inhibitors with forward, backward, and isotope exchange assays [35]. Until recently, specific antibodies have been available only against rat CPT 2. CPT~ has been indirectly analyzed through its lack of reactivity with anti-CPT, antibodies [36] but it has been shown that CPT~ and CPT: probably share some epitopes in common [37]. Most recently, antibodies have been produced against CPT purified from human tissues. No apparent species-specific differences in immunoreactivity have been detected thus far [38-40]. However, the molecular weight of CPT, has been found to vary slightly among tissues derived from mouse, monkey and rat [20]. The present study of 31 patients showed that 25.8% of the patients (groups C and D) had significant deficiencies in CPT EA but only 6 of these also had deficiencies in EU suggesting that

2 patients had mutations which altered CPT EA but not the immunoreactive site. The finding of an additional 29% (group B) with deficiencies in CPT EU and normal or low normal EA suggests that a mutation altered the immunoreactive site directly or altered the configuration of the molecule which secondarily altered immunoreactivity. Examples of similar alterations of immunoreactivity in enzyme proteins have been reported, such as in cases of muscle phosphorylase deficiency [41]. Alternatively, it is possible that autoantibodies were produced which blocked the antigenic site as observed with the production of macro-CK in one reported case of CPT deficiency [4]. It was suggested that autoantibodies were produced in response to repeated episodes of rhabdomyolysis [4]. The remaining patients either had normal CPT EA and EU (29%; group A) or had moderate combined C PT EA and EU deficiencies (16.1%; group E). Since all muscle biopsies were found to

255

Immunoquantitation of CPT Table 3. Laboratory features Abnormal Abnormal muscle Patient EMG TCK carnitine ~PPL ~,PFK T G L Y C ~ G 6 P D ,~AMPDA ~AK ~CS Group A. Normal EA and E U t + 2 3

+

4 5 6 7

0 0

0

0 0

0 0

8

4-

0 0 +(4)

±

0(I,II,ni,IV)

0

+

o o

0 + (III),0(I.II,IV)

0 0

Abnormal Abnormal Abnormal exercise CT muscle test scan biopsy

0

+

9

0

0 0

0 0

+

Respiratory chain ~enzymes

±

0 ±

o

o

+

o

o

+ (5) 0 0

+

+(3)

0

+ ( 1 , 3)

Group B. Normal L#A: E U > 3 S.D. below normal mean l0

0

II 12

0 0

0 0

±

13

0

14

+

15 16 17 18

0 0

+

0

0

0

0

0 0

0 0

0

0

0

0

0

0 +

0(I,II,IV) 0

0

0

0 0

0 +(2,3,4,7)

0

0

0

0

0

0

+ 0

0

+(8) +(6,8) +(7)

0

0

0

0

0 0

0

0

0

0

0

+(1,8,9)

0

0

0 0

+

0

0

Group C. EA > 3 S.D. below normal mean; normal E U 19 20

0

+ +

0

0

0

Group D. EA > 3 S.D. below normal mean; E U 3-5 S.D. below normal mean 21 22 23 24 25 26

0 4+

+(IV) + +

0

0

0

0 o 0

0 + 0 0 0 0 + Group E. EA > 2 S.D. below normal mean; E U > S.D. below normal mean 27 0 0 28 0 29 0 0 30 0 + 0 31 + 0 0 ±

0 0 0

0 0 0 0

0 ± 0 0 00(I,II,III.IV) 0 0 0

+

0 0

+(1) +(1) +(8) ±(6,8) 0 ~(2,4) + ( 5 . I) ±(2)

l. 11, 111. IV = Respiratory chain enzyme complexes; (0) = absent; ( + ) = present; ( ± ) = partially present; empty spaces = no information available: E M G = electromyography; C K = creatine kinase; PPL = myophosphorylase; P F K = phosphofructokinase; P G K = phosphoglucomutase; G L Y C = glycogen: G 6 P D = glucose-6-phosphate dehydrogenase; A M P D A = myoadenylate deaminase; A K = adenylate kinase; CS = citrate synthase; (I) = mildly myopathic; (2) = lipid elevated; (3) = glycogen elevated; (4) = abnormal mitochondria; (5) = fiber type grouping: (6) = vacuolar inclusions: (7) = severe myopathic; (8) = atrophic changes; (9) = denervating process.

have citrate synthase activity within the laboratory's normal reference limits, it is unlikely that a reduction in the number of mitochondria could account for the partial CPT LA deficiencies in group E. Only three patients (patients I and 3 in group A and patient 21 in group D) were found to have deficiencies in enzymes other than CPT. There was no unique information in the clinical history which would have set these individuals apart from the test group. The patients with 12-50% of residual muscle CPT activity (groups C and D), were within or slightly higher than the frequently reported range of 0-30% for muscle from CPT-deficient patients [14]. Only four of seven CPT-deficient patients in groups C and D had accompanying episodes of myoglobinuria, the most commonly reported symptom associated with muscle CPT deficiency. However, myoglobinuria is not always present in cases of CPT deficiency [14] and its absence

should not be used solely as a criterion to rule out consideration of CPT deficiency. Furthermore, myoglobinuria may be associated with other metabolic disorders principally involving lipid or glycogen metabolism [42, 43]. It should be considered that patients in group E, and possibly some of those in group D whose residual CPT EA was within the 40-61% range of normal activity, were actually manifesting heterozygotes for CPT EA deficiency. A diagnosis of CPT deficiency should not be ruled out in these patients because they had more than 30% residual muscle CPT EA. An increasing number of family studies, some unpublished, have demonstrated the existence of symptomatic heterozygotes for CPT deficiency [9] based on clinical findings and activity analysis. It is possible that patients having normal CPT EA by in vitro assay but deficient CPT EU (group B) may have a significant impairment of CPT function in vivo. Altogether six patients had CPT

G . D . VLADUTIU et al.

256

Table 4. Summary of clinical features Clinical

Group A (9 cases}

Group B (9 cases)

Group C (2 cases)

Group D (6 cases)

Group E (5 cases)

Male/female

8/9

7/9

2/2

4.,6

3 '5

Mean age at diagnosis (yr) Positive

22

38

33

36

36

family history

1.5

2:6

I ."

24

12

4/4 5/'~ 1.'2 I.'2

4/5 9./9 1.2 1'5

~'" "L''~ ND 2'2

4.4 5/5 11 2'5

I I 44 ND I 2

4:5 l.I

6/6 I '2

22 0,1

4,4 3 '3

44 ND

5,'5

ND

1'1

ND

12

features

Symptoms Weakness Cramps/pain/stiffness Rhabdomyolysis Myoglobinuria

Precipitating.lactors Exercise

Fasting Temperature extremes. stress/infections ND = not determined.

Table 5. Summary of laboratory findings Test abnormalities

EMG CK G6PD

AMPDA Resp. chain (complex) Forearm ischemic exercise CT Scan Muscle biopsy

Group A (9 cases}

Group B (9 cases)

Group C (2 cases)

Group D (6 cases)

Group E (5 cases)

I/5 2'5 I/I 1,4 :i: 2'4

I/5 2/7 ND

0/1 2/2 ND

2'5 4;4 ND

12 11 ND

0/6

ND

0,3

03

12 (lit)

ND

ND

I I (IV)

ND

± 1/2

I/6

0/1

2,2

02

0/I 1./8 (abn mito)

0/4 I/9 (lipid/' glyc/abn mitoisev myop)

ND 0/2

ND 2'5 (:t: myop)

ND I 4 ( ze lipid abn mito)

1/8(± myopi glyc)

l/9(sev myop)

1/8 (glyc) I/8 (fiber type grp)

t/9 ( -,- myop/ atrophic changes.'

I/5 (atrophic changes) I/5 (vac indus atrophic changes)

1 , 4 ( ± myop fiber type grp) I ,'4 ( ~ lipid)

denerv) 1/9 (vac inclus atrophic

changes I/9 (atrophic changes) Resp = resptratory: CK = creatine kinase: AMPDA = myoadenylate deaminase; CT = computerized tomograph}: abn = abnormal: mito = mitochondria: myop = myopathy; glyc = glycogen: denerv = denervating process: vac lucius = vacuolar inclusions: ND = not determined: sev = severe: ± = mild or

sligh~

EU > 5 S.D. below the normal reference mean. Three of these patients (Nos 22, 24, 26) fell into the severely deficient CPT EA and EU group D and all three primarily had proximal muscle pain or cramping. The other three patients (Nos 16, 18, 30) either had normal CPT EA (Nos 16, 18) or had a moderate CPT EA deficiency (No. 30) and all three had predominantly distal pain or cramps in the hands or feet. A m o n g the seven patients in group B whose CPT EU was > 3 S.D. below the reference mean, only patients N o s 10 and 13 also had cramps or pain primarily in the hands and feet. While there may be an asso-

ciation between a severe reduction in immunoreactivity of CPT and the presence of pain or cramping in the extremities, many more patients need to be studied before any conclusions can be drawn. Fluctuations in CPT EA may be due to the nutritional status of the individual at the time of biopsy [14, 18]. It has been shown in rats that certain hypolipidemic drugs, such as clofibrate, and acetylsalicylic acid increase CPT activity, immunoreactive protein, and specific m R N A [29]. Anesthesia is also known to influence normal muscle CPT activity by affecting its suscept-

257

Immunoquantitation of CPT

ibility to malonyI-CoA inhibition [44]. Therefore, it may be important to note the nutritional status of the patient and the anesthesia used at the time of biopsy when assessing CPT activity in muscle [39]. Somewhat higher residual CPT activity is generally found in CPTdeficient patients' virus-transformed lymphoblasts [45] and fibroblasts [46] when compared with their muscle. This may be due to the fact that the muscle is exposed to anesthesia while cultured cells are not. The findings in this study demonstrate that at least two categories of muscle CPT deficiency exist. When both the catalytic activity and the immunoreactivity are deficient, the causative defect may result in reduced synthesis, increased degradation or conformational changes altering the entire enzyme protein. When only the catalytic activity is deficient, a regulatory defect may exist in the enzyme molecule which alters the enzyme active site e.g. an antibody specific for the active site or for the mitochondrial membrane attachment site [47]. Other categories may exist in which only the immunoreactive regions of the protein appear to be altered or in which membrane associated inhibitors impair the antigen-antibody reaction. The effect on enzyme activity in vivo in these cases is presently unknown. Demaugre et al. [48] recently reported the results of immunoquantitative measurements of CPT in cultured fibroblasts from two patients with muscle CPT deficiency (CPT:) and two patients with liver CPT deficiency (CPT~) using a polyclonal antibody against human liver CPT,. In the muscle CPT-deficient patients, a deficiency in immunoreactivity was comparable to the decrease in EA. It appears, therefore, that these investigators' patients had defects similar to our group D patients and none were found with defects similar to those in our group C. These authors have also reported a case of severe CPT: deficiency with hepatomuscular symptoms and reduced CPT 2immunoreactive protein

[23]. Woeltje et al. [49] recently isolated and characterized a complementary DNA (cDNA) encoding rat liver CPT. Finocchiaro et al. [50] have sequenced a cDNA for human liver CPT and assigned the gene to chromosome I. Continued molecular studies to determine the nature of the mutations which occur in this cDNA should provide answers to questions raised in our study such as the nature of the structure/function relationships of CPT, determination of how the

mature enzyme associates with the inner mitoclaondrial membrane, and whether more than one gene is responsible for the different forms or locations of CPT. Once these questions are answered, a delineation of the variety of possible mutations that can lead to CPT deficiencies in man will be possible. In the meantime, it will be important to measure both the EA and EU in patients suspected of having CPT deficiency. The relationship of the two determinants may provide additional important diagnostic information. Acknowledgements--We thank the many physicians who provided tissue from their patients for this study and Drs Linda Duffy, Richard Evans, Edward Fine and Mario Rattazzi for helpful discussion of the data and manuscript. We are also grateful to Drs James Nolan, Mark Ballow and Marie Talty for the loan of their ELISA platereaders and to Dr R. Ronchetti for his support in the early phase of this work. This work was supported in part by a grant from the Women's and Children's Research Foundation of the Children's Hospital of Buffalo. We thank Ms Patricia Jones for typing the manuscript. REFERENCES 1. DiMauro S, Melis-DiMauro P. Muscle carnitine palmitoyltransferase deficiency and myoglobinuria. Science 1973: 182: 929-931. 2. DiMauro S, Papadimitriou A. Carnitine palmitoyltransferase deficiency. In: Engel A and Banker B Q, eds. Myology. New York: McGraw-Hill, 1986: 2: 1697-1708. 3. Carey M P, Poulton K, Hawkins C, Murphy R P. Carnitine palmitoyltransferase deficiency with an atypical presentation and ultrastructural mitochondrial abnormalities. J. Neurol Neurosurg Psychiatry 1987; 50: 1060-1062. 4. Deguchi H, Sugiyama N, Kawamura H, Uemura T, Shimizu A, Yamamoto M. Macro creatine kinase in a case of carnitine palmitoyltransferase deficiency. Clin Chem 1990; 36: 1997-1999. 5. Galdi A P, Clark J B. An unusual case of. carnitine palmitoyl transferase deficiency. Arch Neurol 1989; 46: 819-822. 6. Heier M S, Dietrichson P, Landaas S. Familial combined deficiency of muscle carnitine and carnitine palmityl transferase (CPT). A cta Neurol Stand 1986: 74: 479-485. 7. Katsuya H, Misumi M, Ohtani Y, Milke T. Postanesthetic acute renal failure due to carnitine palmityl transferase deficiency. Anesthesiology 1988; 68: 945-948. 8. Kelly K J, Garland J S, Tang T T, Shug A L, Chusid M J. Fatal rhabdomyolysis following influenza infection in a girl with familial carnitine palmityl transferase deficiency. Pediatrics 1989: 84:312-316. 9. Kieval R I, Sotrel A, Weinblatt M E. Chronic myopathy with a partial deficiency of the carnitine palmityltransferase enzyme. Arch Neuro11989: 46: 575576. 10. Ross N S. Hoppel C L. Partial muscle carnitinepalmityltransferase A deficiency: rhabdomyolysis associated with transiently decreased muscle carnitine content after Ibuprofen therapy. J A m MedAssoc 1987; 27: 62-65. 11. Sadeh M, Gutman A. Carnitine palmitoyltransferase

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