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X-linked recessive (Duchenne) muscular dystrophy (DMD) and purine metabolism: Effects of oral allopurinol and adenylate
X-linked Recessive (Duchenne) Muscular Dystrophy (DM D) and Purine Metabolism : Effects of Oral Allopurinol and Adenylate W. H. S. Thomson and Ian Smith Data are presented which suggest that Duchenne muscular dystrophy (DMD) may have some origin in a severe deficiency of total muscle adenine nucleotides. Using double-blind techniques, this possibility was tested in 16 DMD patients by giving oral allopurinol, a synthetic inhibitor of the purine catabolic enzyme xanthine oxidase. Sublingual procaine adenylate was also briefly tested. Instances of clinical improvement quickly occurred which were statistically significant; they were ac-
companied by a significant increase in physical strength. These improvements have been maintained for more than 6 mo by administration of a small amount of allopurinol daily. Procaine adenylate had little effect. These results support the above view of DMD and seem to indicate that existing purines, retained and recycled after allopurinol, can sustain such improvement, and that additional adenylate is unnecessary.
I
N DUCHENNE MUSCULAR DYSTROPHY (DMD) skeletal muscle a variety of disorders of structure and function occur without evidence of a common underlying process. Recently, however, many of these disorders have been explained by a lack of adenosine 5’-triphosphate (ATP),’ part of a marked depletion of total adenine nucleotides? suggesting defective provision of muscle purines essential to every aspect of cell metabolism. Since allopurinol inhibits xanthine oxidase and encourages purine transport and salvage,jm5 its effects in DMD patients were examined by double-blind techniques. Sublingual procaine adenylate@ were later tested in the same way. Physical performance was measured by hand-grip manometry and by a progressive scale of clinical status. Electrocardiograms (ECG) were taken at intervals. Serum urate was measured, as was urate and creatinine excretion in 24-hr urine collections. Three of the enzymes serially assayed are plentiful in muscle and one in liver; two are tissue specific. Creatine phosphokinase (CPK) is abundant in skeletal muscle, with some in the myocardium but only traces elsewhere;“’ it is a sensitive measure of primary muscle disease,‘*” particularly in DMD.” y-Glutamyltransferase (T-GT) occurs in liver but not in muscle” and is useful in monitoring liver health.r3
From the Research Laborator_y. Knightswood Hospital, Glasgow, Scotland. Received for publication January 25, 1977. Supported by the Andrew Patrick Trust and rhe Muscular Dystrophy Group of Great Britain. The authors thank Dr. Pal Turmer of the Wellcome Foundation Ltd. for aliopurinol and placebo tablets, Dr. Rob Elton. Edinburgh, for statisrical advice, Tenovus-Scotland for the adenosine 3’-monophosphoric acid, and the Union Carbide Corporation, New York, for permission to use U. S. Parent 3,104,203. Reprint requests should be addressed to Dr. W. H. S. Thomson, Research Laboratory. Knightswood Hospital, Glasgow G13 2XG, U.K. 0 1978 by Grune & Stratton. Inc. 0026~0495/78/2702~0004$02.00/0
Metabolism, Vol. 27, No. 2 (February), 1978
151
152
THOMSON
MATERIALS
AND
SMITH
AND METHODS
Patients Of the 16 boys (aged 3.39. 14.29 yr) who took part in this study, 9 were ambulant wheelchairs. All had clinically classical DMD confirmed by serum enzymology’.” but 3) by muscle biopsy and/or genetically.
and 7 were in and (in all
Clinical Procedures In each patient, the maximum height (in centimeters) was found to which he could& using both hands in one continued exhaustive attempt--raise the mercury in a laboratory manometer by rapidly pumping air from a small rubber hand bulb into a 500-ml reservoir connected to the manometer. Clinical status was graded as shown in Table 1. Repeated examinations of the patients provided the basis for this 16-point scale of observed disabilities beginning with the worst and ending with the least. At the end of each period, the patient was placed on the scale at the point of his maximum capability; it was carefully confirmed that he could perform every action up to and including this point. but none beyond it. For each patient, a change in score merely indicated improvement (+) or no improvement (0) for statistical purposes; this system cannot quantify physical change for numerical use. ECG leads I. II, 111, V4R. VI. V4. and V6 were recorded in all patients, and aVR. aVL, and aVF were also recorded in the older boys.
Analytical Procedures The 24-hr urine collections were taken at home between 8 a.m. Sunday and 8 a.m. Monday using a polythene funnel and a 2&liter (Winchester) amber bottle containing as preservative 4 ml toluene (AR) and IO ml saturated aqueous lithium carbonate (AR). Collections were likewise obtained from 20 healthy boys of similar ages from the same locality. Parental supervision was punctilious.
1.
Table
Clinical
Status
Wheelchair 1.
Slumps
in wheelchair,
unable
2.
Slumps
in wheelchair,
holds
3.
Sits up unsteadily,
4.
Sits upright
to hold head
kicks feebly
head
with
dependent
confidently,
lifts knee from head.
5.
Raises both
arms
6.
Raises both
feet
above
7.
Can
climb
up from
8.
Con
climb
from
up.
up.
chair
leg but cannot but connot
raise
lift knees. arms.
up to seat level of wheelchair. Boor to sit on kitchen
kitchen
choir
chair.
to standing
up unsteadily,
both
hands
gripping
nearby
furni-
ture. Ambulant 9.
Frequent
collapse
10.
Walks
11.
Rises unaided
12.
Walks
less slowly,
13.
Stands
up from
with
slowly
marked
quent 14.
Con
muscle keep
cramps, 15.
Shares legs,
16.
due to knees giving
with from
goit
chair,
way,
very
and
cramps
after others
some
prefers
marked
attain
up from
position
waddle
up with
and
but cannot
con stand supine
standing
supine
up
carried unable
from
position
by climbing lordosis,
being
lordosis,
only
to walking. to rise from
supine
position
by climbing
own
legs
(Gowers’
tires quickly
and
falls
choir
without
unaided. help.
up furniture. sign),
often,
still
has sore
walks
slowly
legs and
fre-
exertion. walking
quickly,
minimal
roll
ond
lordosis,
fewer
foils
and
muscle
Gowers’ sign present. prolonged
no more
falls,
Gowers’ sign and tion.
rolling
vigorous
adventures
minimal
Gowers’
waddle
disappear,
with
friends
without
usual
rapid
fatigue
and
sore
sign. normal
activity,
no more
muscle
cramps
after
exer-
X-LINKED RECESSIVE DMD
153
Urine creatinine was measured as mg/24 hr by the alkaline picrate method modified for AutoAnalyzer. Urate in serum and urine was measured at 405 nm as a colored product of formaldehyde from the oxidation of methanol, in the presence of catalase, by Hz02 from the action of uricase on urate,14 combining simplicity and enzymatic specificity without the usual turbidity errors; results were expressed in mg/ 100 ml and mg/24 hr, respectively, and standardized reagents (“Urica-quant”) were obtained from Boehringer Mannheim GmbH. Serum enzymes were assayed forthwith in twice-centrifuged cell-free serum obtained from fresh whole blood taken from an antecubital vein using a 2l-gauge needle and disposable syringe, isopropanol skin toilet, minimal stasis, and gentle handling to avoid hemolysis. Glassware was cleaned by immersion for I wk in 30% nitric acid, rinsing with glass-distilled water, and drying at 125°C. Grade A pipettes were used throughout. CPK (ATP:creatine phosphotransferase, EC 2.7.3.2). l,6-diphosphofructoaldolase (EC 4.1.2.7; ALD), aspartate aminotransferase (glutamic-oxalacetic transaminase, EC 2.6.1.1; GOT), and *-CT (EC 2.3.2.2) were measured kinetically at 25°C and 340 nm (405 nm for y-CT), as described elsewhere,‘5.‘6 using a Unicam SP 800 B recording spectrophotometer. All values were expressed in international units as mU/ml/25”C. Standardized reagents for ALD, GOT, and y-GT wereobtained as optimized kits (Boehringer Mannheim GmbH), and ALD and GOT were corrected using normal saline for serum. ” The CPK reagents from J. T. Baker Chemicals B.V., Deventer, Holland (Diamed Diagnostics, Liverpool) gave no blank value and had the creatine phosphate substrate separate, allowing saline to be substituted, when the small serum adenylate kinase increment (despite II.5 mM adenosine monophosphate) could be deducted from each CPK '8 assay.
Materials Allopurinol (100 mg) tablets, commercial but unmarked, were allocated at random to 8 of I6 numbered bottles; identical inert placebo tablets were put in the rest. Allocations were carried out by the Wellcome Foundation Ltd.. which provided the tablets and kept the key to the numbering. Procaine adenylate tablets were made by the modified procedure of Ruskin. To 46.6 g powdered procaine base (Sigma Chemical Co.) suspended in 900 ml distilled water at room temperature was added (with stirring) an equimolecular amount (100 g) of ATP (Cambrian Chemicals) in successive portions. The pale pink opalescent solution quickly obtained was filtered and lyophilyzed in an oil-pump vacuum against EtOH-solid CO2 to a pale pink microcrystalline solid melting at 130”.-140°C with decomposition. This was ground and dried in vacua to give procaine-adenosine 5’-triphosphate (PA5) as an almost colorless powder. In the same way procaine-adenosine 3’monophosphate (PA3) was obtained as an exceedingly hygroscopic powder, of melting point 88”-93°C. from equimolecular amounts of procaine base and adenosine 3’-monophosphoric acid (BDH Chemicals Ltd.). Atomic absorption spectrophotometry confirmed the absence of toxic heavy metals in both products. Sublingual tablets, each containing 150 mg PA3 or PA5 (about 100 mg adenosine phosphate), were prepared (Arthur Cox & Co., Brighton. U.K.), together with placebo tablets matched for taste and appearance. PA3 tablets were allocated at random to 4 of 8 numbered bottles, and placebo tablets to the rest; the same procedure was followed in 8 more bottles for PA5. Allocations were made by the hospital pharmacy, which kept the key.
Experimental
Design
Patients were divided into 4 groups of 4 and were examined before noon on the same day each week for 18 wk. For the first 6 wk (period A) nothing was given. For the next 6 wk (period B) each patient, irrespective of age or weight, took one tablet daily from the allopurinol/placebo bottle allocated to him. The contents were then revealed, and for the final 6 wk (period C) all patients took one unmarked genuine allopurinol tablet daily. In addition, those who already had had allopurinol in period B received one of the PAS/placebo bottles, and the rest one of the PA3/placebo bottles; each took 4 sublingual tablets daily during period C, after which the contents were again revealed. At weekly examination, venipuncture for serum enzymes and urate was followed by hand-grip manometry and assessment of clinical status; the urine collection for urate and creatinine was also
THOMSON
154
Table
2.
Effects of Allopurinol Tablet
and
Allocotionr
AND
Adenylate Clintcal
Status’
Clinical
ot End of Perlod Periods Patient
No.
Age (yr),
Mobility
on
Allopurinol
Improvement
Adenvlote in Period
C
SMITH
A
B
C
6 vs. A
1
5.06,
ambulant
BC
Nil
13
15
16
+
2
6.67,
ambulant
BC
Nil
13
14
15
+
3
8.37,
wheelchair
BC
Nil
2
3
6
+
4
11.36,
wheelchair
BC
Nil
1
4
5
+
Cvr
B
+ + + +
5
5.28,
ambulant
BC
PA5
12
12
12
0
0
6
7.74.
ambulant
BC
PA5
13
15
16
+
+
7
8.31,
wheelchair
BC
PA5
2
5
5
+
0
8
13.39,
wheelchair
BC
PA5
5
7
8
+
+
9
3.39,
ambulant
Nil
13
15
15
+
0
10
7.42,
ambulant
Nil
11
11
12
0
+
11
8.38,
ambulant
Nil
10
11
11
+
0
12
14.29,
wheelchair
Nil
5
5
6
0
f
13
8.31,
ambulant
PA3
13
13
15
0
+
14
8.44,
ambulant
PA3
10
9
12
0
+
15
10.36,
wheelchair
PA3
2
2
5
0
+
16
11.99,
wheelchair
PA3
3
3
5
0
+
*See Table
1.
received. The ECG calculated
was recorded
at the end of each 6-wk period.
Serum and urine analyses were
only after each period.
RESULTS In health, marked serum elevations of muscle enzymes during prolonged severe exertion” suggest that muscle function, not enzyme retention,20 has prior claims on ATP. Evidence of increased muscle purine content in DMD should thus appear first as improved function, and only later as diminished enzyme elevations; conversely, overexertion without increased ATP would result in even higher elevations. In period B, percentage manual improvement (+ 16.75”,,) was significantly* greater than zero in the allopurinol group, but not in the placebo group. Furthermore, restoration of departed functions was clearly noted. In period B, of 8 patients (IH) taking allopurinol, 7 improved clinically and 1 did not: in the placebo group (9- 16) 2 improved and 6 did not (Table 2). Fisher’s exact test showed this marked difference of effect to be significant:? and since increased serum elevations (CPK,* GOT?) occurred only in the 2 improved placebo patients, overexertion is implied and this significance is enhanced. Moreoever, while serum and 24-hr urinary urate in period B fell as expected by 26.6’,,* and 27.5%,* respectively, in the allopurinol group, only there did mean 24-hr urinary creatinine output rise simultaneously (+8.7:,,t) from typically low values, consistent with increased muscular activity. Though serum enzyme elevations generally changed little, some decline (CPK,1_ ALD*) confirmed improvement in patient 4, and in period C in patients
*p < 0.01. tp < 0.05.
X-LINKED
RECESSIVE
155
DMD
5 (CPK,1_ ALDt) and 7 (ALDP). Normal y-CT values throughout (Table 3) demonstrated gene specificity as well as the absence of toxic effects on the liver. Both cardiac and skeletal muscle are affected in DM D, and 12 of the 16 boys, compared to a reported SO:;, 2’ had values higher than expected by age for the algebraic sum of the R and S waves (R-S) in lead Vl of the ECG. During periods B and C, however, these values changed only slightly and at random, giving no information, in accord with an origin probably anatomic and not metabo1ic.22 PA3 and PA5 had no obvious additional clinical effect (Table 2). Comparing period C with B did show a fall of CPKt and GOT? against zero in the PA5 but not the PA3 group; but neither group showed significant manual improvement. Thus any extra benefit seems marginal, especially since clinical improvement on allopurinol alone continued in period C (patients l-4). DISCUSSION
In DM D muscle the full effects of the dystrophic X chromosome performing alone appear as the classically rapid progressive wasting toward a fatal issue. Serum CPK elevations in DMD specifically measure active dystrophic muscle mass, and they decline exponentially in both ambulant and wheelchair patients with a half-life of 6.6 yr. ” A DMD patient ambulant at that age would thus have 50% of his muscle mass left, in the wheelchair at 13 yr old some 25% and at 20 yr of age, with death at hand, only 129/,. The fact that functioning muscle survives so long, however, suggests that even modest metabolic support in the correct area could ensure indefinite survival. In female DMD carriers, however, random fusion during early growth occurs between mononucleate myoblasts from clones with one or other X chromosome already randomly inactivated,23 giving a mature multinucleated muscle cell with a dual population of nuclei regulated by either a normal or dystrophic X chromosome. Different proportions for different individuals give a uniform X-chromosomal mosaic24 with a range of manifestation from the undetectable to grossly elevated serum enzymes and minor symptoms, though still nonprogressive and with normal life expectancy in all but rare instances.’ The carrier state thus resembles a supported DMD, protected from progression by even a small proportion of normal muscle nuclei. Thus, in the affected muscle cell some limit is implied, beyond which, as in DMD, lack of normal nuclei causes eventual cell death with clinical progression, and within which, as in the carrier, the presence of normal nuclei ensures survival by providing essential support the DMD nuclei cannot, In DMD patients such provision by other means would bring them within this limit to resemble carriers, still with marked serum enzyme elevations but no longer clinically progressive. Very many features of DMD, including the gross muscle enzyme efflux, may be wholly explained’ by chronic shortage of skeletal muscle ATP.‘,” In the reversible myopathy of vitamin E deficiency, a 22% decrease in muscle ATP is *p < 0.01. tp < 0.05.
8
1
Patient No.
1228
1440
C
C
1754
B
1348
1985
A
1322
2817
C
6
3453
B
A
2864
A
77.8
45.9
44.3
14.6
70.1
14.6*
49.2
73.1
17.7
14.9
72.2
17.3
14
89.6
107
30
36.3
33.4
133
39.4*
3670’
C
158
4598
B
142
4547
A 51.8
1173
C 47.9
1044*
B 39.4
39
10.1
1340
A
7.7
36.8
17.4
1660
C
35.5
31.9
16.4
1795
B
7.47
27.1
14.6
1677
A
3.6 2.9* 3.3
2.7 10.6 10.8 9.2
3.51
3.1
2.7
3.1
2.7
3.2
2.61
3.2
3.1
3.6
3.37
2.6
3.2
2.8
1.5
3.4
2.2 3.0
4.0
2.9 2.0
213 227 250
387 263 278
338
438
454t
276
437
310
360
371
389
308
287
297
319
270
246
2067
219 261
250
194
293
157 178
261
162
2.3
2.3 5.3
218
192
2.0
4.4
197
234
237
3.3
2.51
2.8
127
78
2.9
2.9
150
52.6
3422
C
119
64*
2.9t
34.3t
27.9
25
20.31
13.9
12.9
37.1t
33
31.2
13.4t
11.7*
9.7
22.5
22.8*
20.9
24.4
24.27
18.8
19.71
16.2t
12.3 136
120
3.9
2.3
143
54
2.2
119
49.3
3609
4082
B
200
112
2.8
4.3
160
A
19.9 23t
234
136
2.6*
3.7
210
97 87
7491
16.7
222
181
3.8
3.6
(cm)
Monometry
(mg)
6471
6955
A
Creatinine 24.hr Output
(mg)
Urote (mg/lOOml)
Urate 24.hr Output
Serum
Directly Successive Periods
C
164
85.4
Between
(mU/ml/ZYC)
y.GT
of Differences
B
(mU/ml/ZYC)
(mU/ml/ZYC)
GOT
(mU/ml/25”C)
ALD
Period
CPK
Table 3. Mean Values and Significance
Ip < 0.001.
tp < 0.01.
*p < 0.05.
16
15
14
13
12
11
10
9
310 369 287
4.0 4.6 3.71
2.9 3.7 3.5
3.0 3.3 3.2
63.9 52.6 44.8 58.3 74.7 49.8 46.4 54.8
18.9 17 18.9 24.9 29.8 15 15.8 17
2155 1966
1805
2713t
2369
1647
1400
1689
A
B
C
A
B
C
2.5
4.1 4.1
82.1* 52.9 45.6 46.8 42.1 51.8' 47
20 15.7 14 14.7 9.1 9.6 8.5
2073
1604
1357
1323
1008
1201
1076
C
A
B
C
A
B
C
7.4*
11.3'
9.1
3.8
3.3
3.1
3.6
3.3
3.4
3.0
4.1
3.9
153
190
166
330
307t
379
292
280
112
119
110
253
224*
313
269
285
24.6*
21.3
20.1
24.7
25.8*
22.9
30.6
29.9
28.8
17
1827
B
252
2.7
66.9 61.9
17.8
2065
A
294
365 2367
2.8*
3.1
59.3
22.7
1934
C 3.7
26.81
381 328
24.6
B
2.6
21.6 23.8
322
278
3.3 3.3
1.5 2.6
53.5 60.1
24.9
1907
2096
A
33.4t
27.7 377
26.2
2.21
2.8
365
257
186
3.1
2.6t
295
18.3t 21.41
215
258
3.7
16.6* 15.1
232 260
230 320
11.7 13.5
202 192
263 256
19.1*
189
129
B C
3.8 3.4 3.2
2.3*
2.1 2.7
142 52.8
64.8 15
2857 1785
7.8 15.1t
222 174
201 140
C A
3.6 3.7
3.0 2.3
92.4 147*
47.9 60.3
2279 2875
A B
is
z 8 z
@
x = Z
158
THOMSON
Table I” normal
4.
moles ond children* 18.5-96.2
mU/m1/25°C
(mean
47.02)
Serum ALD
1.07-2.34
mU/ml/25”C
(mean
1.70)
Serum GOT
6.90-15.47
mU/ml/25*C
(mean
10.56)
Serum y-GT
4.20-31.19
mU/ml/25’C
(mean
13.08)
1.2-6.8
mg/lOO
ml (mean
4.0) in 113 normal
Serum urote
2.6-4.7
mg/lOO
ml (mean
3.6) in 16 DMD
Comparisons
of 16 DMD
urote
Mea”
age
Meon
24-hi
Regressions
boys (period
9.5225 UTLIte
of mea”
Age (x) in 16 DMD Weight(x)
yr (normal),
excretion
366
24-hr-urate
Age (x) in 20 normal
boys boys
in 16 DMD
boys
mg
A) with 20 normal 8.6721
A)
boys
yr (DMD);
1.a268
t =
275 mg (DMD),
(normal),
excretion
boyst
boys (period
(34 df).
NS$
p c 0.01
(y) on
y = 0.7049x
+ 359.7320;
y = 7.6178x
+ 208.6312;f
y = 38.3715~
+ 124.4287;
f = 0.0541
(18 df),
NS
= l.O825(14df),NS t = 1.9953
(14 df),
NS
from Sweetin
tDota INS:
SMITH
Comparisons
Serum CPK
Plasma
*Data
Values and
Normal
AND
and Thomson.‘6 from Harkness and Nicol. 38
not significant.
accompanied by a 1 IQ0 increase in the much less abundant adenosine 5’-diphosphate (ADP), the small amount of adenosine 5’-monophosphate (AMP) remaining unchanged.16 In DMD, however, not only is muscle ATP reduced by 5O”i,, but also ADP is reduced by 70?;, with AMP again unchanged;2 the values in both normal and DMD muscle are measured against noncollagen nitrogen content2,25 so that such reductions cannot be ascribed to replacement of DMD muscle by fat and fibrous tissue. This serious depletion of total muscle adenine nucleotides, universally essential coenzymes, suggests some defect in DMD in muscle purine metabolism either by excessive breakdown or insufficient synthesis. Lower than normal values (Table 4) in serum and 24-hr excretion (p < 0.01) of uric acid, the end product of purine catabolism in man, indicate that the latter possibility is more likely; furthermore, since DMD muscle disappears rapidly with increasing age,” the lack of a significant relationship between age or weight and 24-hr urate excretion (Table 4) suggests its independence of body muscle mass. The DMD gene in the dystrophic nucleus may thus exert its effect by insufficient provision of essential muscle purines; this study has tested this hypothesis by using allopurinol to promote retention and recycling of whatever purines are available. Purine synthesis de novo occurs chiefly in the liver, with many peripheral tissues depending on purine salvage pathways from erythrocyte-borne hypoxanfar less ATP to run.J,j Allopurinol, thine,3 requiring a synthetic isomer of hypoxanthine, and its metabolite, oxipurinol (Fig. I), inhibit xanthine oxidase EC I .2.3.2),“.‘” thus reducing irreversible (xanthine:oxygen oxidoreductase, purine loss as uric acid (and eventually de now purine synthesis by independent feedback inhibition>), in addition to enhancing tissue salvage of available blood hypoxanthine in man.“’ The marked protective effect of allopurinol in experimental oligemrd.I ‘O is indeed associated with a considerable increase of adenine nucleotides in the liver” and other viscera)? of dogs and in the kidney of rats,?” and of ATP in the canine myocardium after operative arrest,jJ while even in
X-LINKED
RECESSIVE
159
DMD
OH
OH
Hypoxanthine
Xanthine
(Goxypurine)
Allopurinol (Qhydroxypyrazolo(3,4-d) pyrimidine) Fig. 1.
Allopurinol
(2,Sdioxypurine)
OH
Uric acid (2,6.8-trioxypurine)
Oxipurinol (4,6-dihydroxypyrazolo(3.4-d) pyrimidine) and purine degradation
in man.
healthy human subjects allopurinol induces marked increases in vivo of erythrocyte ATP. 35 In DMD skeletal muscle any similar increase of adenine nucleotide content after allopurinol should give clinical improvement, which, if maintained, must resemble the nonprogressive manifesting carrier state. Sublingual procaine-3’-adenylate is in fact reported of slight benefit in apendomysium dephosphorylates the parent DMD, 36 in which the proliferating 5’- but not the 3’-adenylate, then converted on absorption into the active continuous parts: 5’-adenylate. 37 This study thus comprises two independent periods A and B, which tested allopurinol alone using double-blind techniques; then period C, which likewise tested sublingual PA3 and PA5 superimposed on allopurinol. The duration of the study was deliberately brief in order to exclude any effect of increased growth or fibrous contraction with age, and because effective metabolic intervention should give measurable results within 6wk. The results show that the effect of PA3 and PA5 was minimal. In contrast, a small daily dose of allopurinol alone clearly produced significant physical improvement. This finding assumes particular interest since, in a condition characterized by rapidly progressive muscular weakness, all the improvements reached in period C have been maintained for more than 6 mo without regression on the same daily dose of allopurinol. These findings appear to support the view that DMD may have some basis in defective muscle purine metabolism, perhaps by failure of synthetic pathways, and suggest useful directions for further investigation. In addition, a simple and clinically effective circumvention of the prospective defect seems possible using allopurinol, though further experience alone can determine its value.
160
THOMSON
AND
SMITH
Table 5. Muscle Content (mole x 10m5/g NCN) Clinical
Subjects
(Yr)
ALPNL
*+cl+lJs
Age
Daily
Prior Now
Total
(mg)
ATP
ADP
AMP
NCN w/g
Adenylate
CrP
G-6-P
Wet Weight
(A) 5 healthy
12.51
-
-
18.29 2.82 0.38
21.50
67.19
2.79
25.99
males
20.80
-
-
19.15 2.98 0.40
22.53
67.52
1.54
28.14
38.91
-
-
17.55 2.93 0.12
20.60
57.40
1.53
23.68
48.65
-
-
19.14 2.62 0.44
22.20
58.81
0.91
19.72
56.32
-
-
17.25 2.65
1.09
20.99
54.44
3.64
22.26
la.28 2.80 0.49
21.56
61.07
2.08
23.96
0.88 0.16 0.36
0.81
5.95
1.11
3.26 la.74
Mean SD (B) 5 DMD
3.67
-
-
7.38 3.98 0.26
11.62
18.62
3.01
untreated,
boys
4.32
-
-
9.32 3.44 0.58
13.34
12.91
3.12
15.66
ambulant
4.77
-
-
3.28 2.46 0.41
6.16
7.75
0.77
27.19
-
1.98 0.85
7.57
11.24
1.32
14.91
11.57 4.60
1.04
17.22
22.23
1.92
i 1.89
M.XUl
7.26 3.29
0.63
il.18
14.55
2.03
17.68
SD
3.36 1.07 0.32
4.46
5.82
1.03
5.85
11.44 5.06 0.30
16.80
33.08
0.54
20.79
(C) 4DMD
boys
9.02
-
10.14
-
4.11
13
-
15
100
4.73
after6 mo
4.81
13
15
100
9.81 3.51
1.14
14.47
27.57
3.55
21.36
ALPNL
5.31
13
15
100
12.52 4.16
1.58
18.26
28.71
2.86
22.51
9.52
13
13
150
12.06 6.76
1.22
20.04
44.54
1.14
15.49
11.46 4.87
1.06
17.39
33.47
2.02
20.04
1.18 1.41 0.54
2.36
7.75
1.42
3.12
MeCltl
SD 4.57
13
15
100
14.76 2.76
0.63
la.15
35.88
1.40
23.82
after1 yr
6.23
13
16
100
14.87 1.96 0.24
17.07
37.61
0.69
21.82
ALPNL
7.79
13
16
100
11.74 1.71
1.16
14.62
27.19
1.01
la.79
8.83
13
15
150
16.09 3.13 0.91
20.12
45.72
1.12
20.27
9.46
13
15
100
16.86 1.32
(D) 5 DMDboys
Meon
1.62
19.80
49.69
0.60
13.55
14.87 2.18 0.91
17.95
39.22
0.96
19.65
0.52
2.24
a.81
0.33
3.89
1.95 0.75
SD ALPNL, ollopurinol.
ADDENDUM Further
that the skeletalmuscle in DMD
studies have confirmed
content both of creatine phosphate allopurinol Marked
for 6 mo greatly
physical
improvement
and of total adenylate
improved occurred
these values, early
and
shows 3 grossly diminished
due to lack of ATP.
which
increased
persisted
A small daily dose of
still further
throughout,
after
suggesting
a year. apparent
clinical arrest of the disease.
Table 6. Ratios and Significance of Differences Between Means of Subject Groups in Table 5 Comparisons ofMeans
Total ATP
ADP
AMP
Adenylate
G-6-P
NCN
0.241
CrP
0.97
0.74
WA
0.401
1.18
1.30
0.52t.
C/A
0.63t
1.74*
2.18
o.alt
0.551
0.97
0.84
D/A
o.alI
0.78
0.49
0.83$
0.641
0.46
0.82
C/B
1.58
1.48
1.68
1.56*
2.30t
1.00
1.13
D/B
2.05t
0.66
1.44
1.61*
2.701
0.48
1.11
D/C
1.30*
0.45t
0.86
1.03
1.17
0.48
0.98
Statistical notationrevised for Table 6. *p < 0.05. tp
X-LINKED
RECESSIVE
161
DMD
Table 7. Exponential in Ambulant log (CPK)
= 3.9436
log (Aldolase) The
close
identity
mass whence
these
of each elevations
=
Decline of DMD Serum Enzyme Elevations
DMD Patients -
0.005253X
1.9940
independent
-
as linear
0.005252X half-life
Regressions
(n = 22; p < 0.01;
on Age (X MO)
14 = 4.775
(n = 40; p < 0.001; (tf)
supports
its being
t+ also
yr)
= 4.776 that
yr)
of the
decaying
muscle
arise.
Procedures Muscle specimens (l-2 g) from open biopsy (vastus lateralis in all DMD boys) under general anesthesia (Nz0/02/halothane) were freeze-clamped immediately after excision and immersed in liquid Nz within 30 set of removal. The frozen specimen was weighed, powdered under liquid Nz in a chilled mortar, an aliquot weighed for noncollagen nitrogen (NCN) assay” by microKjeldahl then titration, and the remainder ground in liquid Nl with successive additions of aqueous perchloric acid, then transferred to a glass tube to thaw on ice before final homogenizing (Ultra Turrax) and centrifugation. ATP, ADP, AMP, creatine phosphate (CrP), and glucose-6 phosphate (G-6-P) were enzymatically measured2*40 forthwith in duplicate in the supernatant, and are expressed in terms of NCN content of the specimen.
Results Muscle biopsy specimens were assayed in healthy male controls undergoing surgical repair, and in ambulant boys with clinically classical DMD confirmed both histologically and by serum enzymology.9,4’ Five boys were untreated; four of this group were then given allopurinol for 6 mo, and 5 other boys for 1 yr. Each had an initial clinical status, with differences in degree, of 13 (Table 1); after 4-8 wk on allopurinol, in all but one this improved to 15 and even 16, where it has remained unchanged (Table 5). In health. muscle adenylate content was remarkably constant and unrelated to age: CrP alone showed a high negative correlation with age ( r = -0.947*) indifference was found dicating some decline in energy reserve. As in other studies,’ no significant in NCN content between healthy and DMD muscle. In untreated DMD, the CrP content of muscle was only 24% and of total adenylate (ATP + ADP + AMP) 52% of normal, the latter wholly attributable to lack of ATP (Table 6). Thereafter 6 mo of allopurinol more than doubled CrP and increased total adenylate by half without much effect on ATP. After a year on allopurinol. though CrP and total adenylate increased no further, ATP had risen to 81% of normal at the expense of ADP. The main improvements thus clearly occur in ATP and CrP. A basic adequacy of both oxidative phosphorylation and CPK activity in DMD muscle is thus implied. and the indifference throughout of G-6-P suggests a sufficient glycolysis. Discussion New data (Table 7) from refined analyses4’ Indicate a more precise half-life for DMD muscle than that quoted earlier. Thus, though a DMD patient ambulant at 4.8 yr has lost 50”” of his muscle, he reaches 9.6 yr with still some 25% left. As previously argued, such prolonged survival of functioning muscle might be made indefinite by even partial metabolic support. Our results show that adenylate conservation by allopurinol may offer such support, and that studies of muscle purine metabolism should prove rewarding. If some prospect of sustained clinical arrest and preservation of the muscle fiber population may now seem possible, neonatal diagnoses 42 and early intervention become imperative, and in older children some means, whether biochemically or by advanced orthopedic procedures, to prevent the shrinking fibrous tissue progressively impeding residual muscle action.
Acknowledgment We are greatly indebted to Dr. Alex McQueen, of the University of Glasgow Department Dermatology, for careful excision of all DMD muscle specimens; we also thank our patients colleagues, and Moira Young for skilled Kjeldahl analyses.
‘u i 0.05.
of and
162
THOMSON
AND SMITH
REFERENCES I. Thomson WHS, Sweetin JC, Hilditch TE: Studies on the carrier state in X-linked recessive (Duchenne) muscular dystrophy. Clin Chim Acta 63:383, 1975 2. Stengel-Rutkowski L. Barthelmai W: Uber den Muskel-Energie-Stoffwechsel bei Kindern mit progressiver Muskeldystrophie Typ Duchenne. Klin Wochenschr 5 1:957, 1973 3. Murray AW, Elliott DC, Atkinson MR: Nucleotide biosynthesis from preformed purines in mammalian cells: Regulatory mechanisms and biological significance, in Progress in Nucleic Acid Research and Molecular Biology, vol IO. New York, Academic, 1970, p 87 4. Seegmiller JE: Diseases of purine and pyrimidine metabolism, in Bondy PK, Rosenberg LE (eds): Duncan’s Diseases of Metabolism (ed 7). Philadelphia, Saunders. 1974, p 655 5. Fox IH: Human purine metabolism, in Meuwissen HJ, Pickering RJ, Pollara B, Porter IH (eds): Combined Immunodeticiency Disease and Adenosine Deaminase Dehciency~ -A Molecular Defect (Proceedings of a Symposium and Workshop, Albany. N.Y., 1973). New York. Academic, 1975. p 45 6. Ruskin SL: Composition of matter containing procaine adenylate. United States Patent 3,104,203, 1963 7. Colombo JP. Richterich R. Rossi E: Bestimmung Serum-Kreatin-Phosphokinase: und diagnostische Bedeutung. Klin Wochenschr 40:37. 1962 8. Smith AF: Separation of tissue and serum creatine kinase isoenzymes on polyacrylamide gel slabs. Clin Chim Acta 39:351, 1972 9. Thomson WHS: Serum enzyme studies in inherited disease of skeletal muscle. Clin Chim Acta 35:183, 1971 IO. Thomson WHS: Serum enzyme studies in acquired disease of skeletal muscle. Clin Chim Acta 35:193. 1971 Il. Thomson WHS. Sweetin JC. Elton RA: The neurogenic and myogenic hypotheses in dystrophy. human (Duchenne) muscular Nature 249:151. 1974 12. Orlowski M, Szewczuk A: Calorimetric determination of y-glutamyl transpeptidase activity in human serum and tissues with synthetic substrates. Acta Biochim Pol 8: 189. 1961 13. Zein M, Discombe G: Serum gammaglutamyl transpeptidase as a diagnostic aid. Lancet 2:748. 1970 14. Kageyama N: A direct calorimetric determination of uric acid in serum and urine with
uricase catalabe system. Clin Chim Acta 31: 421, 1971 15. Thomson WHS: Determtnation and statistical analyses of the normal ranges for five serum enzymes. Clin Chim Acta 2 1:469, 1968 16. Sweetin JC. Thomson WHS: Revised normal ranges for six serum enzymes: Further statistical analysis and the effects of diRerent treatments of blood specimens. Clin Chim Acta 48:49. 1973 17. Smith I, Thomson WHS: Serum enryme assays with commercial kits. Lancet 1:1038, 1975 18. Colowick SP: Adenylate kinase (myokinase. ADP phophomutase). in Colowick SP, Kaplan NO (eds): Methods in Enzymology. vol 2. New York. Academic, 1955, p 598 19. Grifhths PD: Serum levels of ATP: creatine phosphotransferase (creatine kinase). The normal range and effect of muscular activity. Clin Chim Acta l3:413. 1966 20. Thomson WHS. Sweetin JC. Hamilton IJD: ATP and muscle enzyme efflux after physical exertion. Clin Chim Acta 59:241, 1975 21. Emery AEH: Abnormalities of the eiectrocardiogram in hereditary myopathies. J Med Genet 9:8, 1972 22. Perloff JK. DeLeon AC, O’Doherty D: The cardiomyopathy of progresstve muscular dystrophy. Circulation 33:625. 1966 23. Lyon Ml-‘: Sex chromatrn and gene action in the mammalian X-chromosome. Am J Hum Genet 14:135. 1962 24. Emery AEH: Muscle histology riers of Duchenne muscular dystrophy. Gcnet 2: 1. I965
in carJ Med
25. Vignoc PJ, Warner JL. Glycogen. creatine. and high energy phosphate in human muscle disease. J Lab Clin Med 62:579, 1963 26. Dhalla
NS.
Fedelesova
M,
Toffler
I:
Biochemical alterations in the skeletal muscle ol vitamin E deficient rats. Can J Biochem 49: 1202. I971 27. Elion GB. Kovensky A. Hitchings GH, et al: Metabolic studies of allopurinol. an inhibitor of xanthine oxidase. Biochem Pharmacol 15:X63. 1966 28. Chalmers RA, Kramer H. Scott JT. et al: A comparative study of the xanthine oxidase inhibitors allopurinol and oxipurinol in man. Clin Sci 35:353, 1968 29. Rundles RW, Metz EN. Silberman Allopurinol in the treatment of gout. Intern Med 64:229, 1966
HR: Ann
X-LINKED
RECESSIVE
DMD
30. Crowell JW, Jones CE, Smith EE: Effect of allopurinol on hemorrhagic shock. Am J Physiol 216:744, 1969 31. Hopkins RW. Abraham J, Simenone FA, et al: Effects of allopurinol on hepatic adenosine nucleotides in hemorrhagic shock. J Surg Res 19:381, 1975 32. Keaveny TV, Cunningham SK, Fitzgerald P: Energy levels in splanchnic organs following severe haemorrhage and reinfusion. Br J Surg 62:665 1975, abstract 53 33. Cunningham SK, Keaveny TV, Fitzgerald P: Effect of allopurinol on tissue ATP, ADP and AMP concentrations in renal ischaemia. Br J Surg 61:562, 1974 34. Lindsay WC, Toledo-Pereyra LH, Foker JE, et al: Metabolic myocardial protection with allopurinol during cardiopulmonary bypass and aortic cross-clamping. Surg Forum 261259, 1975 35. Manzke H, Darner K: Effect of allopurinol on erythrocyte ATP and 2.3-diphosphoglycerate levels and hemoglobin oxygen affinity in man. IRCS Med Sci 3:107, 1975
163
36. Golarz
MN, Bourne
GH:
Use of nucleo-
tides in the treatment of muscular dystrophy. JAMA 180:761. 1962 37. Bourne GH, Golarz MN: Histochemistry and muscle disease. Rev Can Biol 21:383, 1962 38. Harkness RA, Nicol AD: Plasma uric acid levels in children. Arch Dis Child 44:773, 1969 39. Lilienthal JL. Zierler KL, Folk BP, et al: A reference base and system for analysis of muscle constituents. J Biol Chem 182:501. 1950 40. Bergmeyer HU (ed): Methods of Enzymic Analysis. New York, Academic. 1963, pp 543.573.610 41. Smith I, Thomson WHS: Carrier detection in X-linked (Duchenne) muscular dystrophy: Pyruvate kinase isoenzymes and creatine phosphokinase in serum and blood cells. Clin Chim Acta 78:439. 1977 42. Beckmann R: Detection of preclinical Duchenne muscular dystrophy and its female carriers. Isr J Med Sci 13: 102, 1977
companied by a significant increase in physical strength. These improvements have been maintained for more than 6 mo by administration of a small amount of allopurinol daily. Procaine adenylate had little effect. These results support the above view of DMD and seem to indicate that existing purines, retained and recycled after allopurinol, can sustain such improvement, and that additional adenylate is unnecessary.
I
N DUCHENNE MUSCULAR DYSTROPHY (DMD) skeletal muscle a variety of disorders of structure and function occur without evidence of a common underlying process. Recently, however, many of these disorders have been explained by a lack of adenosine 5’-triphosphate (ATP),’ part of a marked depletion of total adenine nucleotides? suggesting defective provision of muscle purines essential to every aspect of cell metabolism. Since allopurinol inhibits xanthine oxidase and encourages purine transport and salvage,jm5 its effects in DMD patients were examined by double-blind techniques. Sublingual procaine adenylate@ were later tested in the same way. Physical performance was measured by hand-grip manometry and by a progressive scale of clinical status. Electrocardiograms (ECG) were taken at intervals. Serum urate was measured, as was urate and creatinine excretion in 24-hr urine collections. Three of the enzymes serially assayed are plentiful in muscle and one in liver; two are tissue specific. Creatine phosphokinase (CPK) is abundant in skeletal muscle, with some in the myocardium but only traces elsewhere;“’ it is a sensitive measure of primary muscle disease,‘*” particularly in DMD.” y-Glutamyltransferase (T-GT) occurs in liver but not in muscle” and is useful in monitoring liver health.r3
From the Research Laborator_y. Knightswood Hospital, Glasgow, Scotland. Received for publication January 25, 1977. Supported by the Andrew Patrick Trust and rhe Muscular Dystrophy Group of Great Britain. The authors thank Dr. Pal Turmer of the Wellcome Foundation Ltd. for aliopurinol and placebo tablets, Dr. Rob Elton. Edinburgh, for statisrical advice, Tenovus-Scotland for the adenosine 3’-monophosphoric acid, and the Union Carbide Corporation, New York, for permission to use U. S. Parent 3,104,203. Reprint requests should be addressed to Dr. W. H. S. Thomson, Research Laboratory. Knightswood Hospital, Glasgow G13 2XG, U.K. 0 1978 by Grune & Stratton. Inc. 0026~0495/78/2702~0004$02.00/0
Metabolism, Vol. 27, No. 2 (February), 1978
151
152
THOMSON
MATERIALS
AND
SMITH
AND METHODS
Patients Of the 16 boys (aged 3.39. 14.29 yr) who took part in this study, 9 were ambulant wheelchairs. All had clinically classical DMD confirmed by serum enzymology’.” but 3) by muscle biopsy and/or genetically.
and 7 were in and (in all
Clinical Procedures In each patient, the maximum height (in centimeters) was found to which he could& using both hands in one continued exhaustive attempt--raise the mercury in a laboratory manometer by rapidly pumping air from a small rubber hand bulb into a 500-ml reservoir connected to the manometer. Clinical status was graded as shown in Table 1. Repeated examinations of the patients provided the basis for this 16-point scale of observed disabilities beginning with the worst and ending with the least. At the end of each period, the patient was placed on the scale at the point of his maximum capability; it was carefully confirmed that he could perform every action up to and including this point. but none beyond it. For each patient, a change in score merely indicated improvement (+) or no improvement (0) for statistical purposes; this system cannot quantify physical change for numerical use. ECG leads I. II, 111, V4R. VI. V4. and V6 were recorded in all patients, and aVR. aVL, and aVF were also recorded in the older boys.
Analytical Procedures The 24-hr urine collections were taken at home between 8 a.m. Sunday and 8 a.m. Monday using a polythene funnel and a 2&liter (Winchester) amber bottle containing as preservative 4 ml toluene (AR) and IO ml saturated aqueous lithium carbonate (AR). Collections were likewise obtained from 20 healthy boys of similar ages from the same locality. Parental supervision was punctilious.
1.
Table
Clinical
Status
Wheelchair 1.
Slumps
in wheelchair,
unable
2.
Slumps
in wheelchair,
holds
3.
Sits up unsteadily,
4.
Sits upright
to hold head
kicks feebly
head
with
dependent
confidently,
lifts knee from head.
5.
Raises both
arms
6.
Raises both
feet
above
7.
Can
climb
up from
8.
Con
climb
from
up.
up.
chair
leg but cannot but connot
raise
lift knees. arms.
up to seat level of wheelchair. Boor to sit on kitchen
kitchen
choir
chair.
to standing
up unsteadily,
both
hands
gripping
nearby
furni-
ture. Ambulant 9.
Frequent
collapse
10.
Walks
11.
Rises unaided
12.
Walks
less slowly,
13.
Stands
up from
with
slowly
marked
quent 14.
Con
muscle keep
cramps, 15.
Shares legs,
16.
due to knees giving
with from
goit
chair,
way,
very
and
cramps
after others
some
prefers
marked
attain
up from
position
waddle
up with
and
but cannot
con stand supine
standing
supine
up
carried unable
from
position
by climbing lordosis,
being
lordosis,
only
to walking. to rise from
supine
position
by climbing
own
legs
(Gowers’
tires quickly
and
falls
choir
without
unaided. help.
up furniture. sign),
often,
still
has sore
walks
slowly
legs and
fre-
exertion. walking
quickly,
minimal
roll
ond
lordosis,
fewer
foils
and
muscle
Gowers’ sign present. prolonged
no more
falls,
Gowers’ sign and tion.
rolling
vigorous
adventures
minimal
Gowers’
waddle
disappear,
with
friends
without
usual
rapid
fatigue
and
sore
sign. normal
activity,
no more
muscle
cramps
after
exer-
X-LINKED RECESSIVE DMD
153
Urine creatinine was measured as mg/24 hr by the alkaline picrate method modified for AutoAnalyzer. Urate in serum and urine was measured at 405 nm as a colored product of formaldehyde from the oxidation of methanol, in the presence of catalase, by Hz02 from the action of uricase on urate,14 combining simplicity and enzymatic specificity without the usual turbidity errors; results were expressed in mg/ 100 ml and mg/24 hr, respectively, and standardized reagents (“Urica-quant”) were obtained from Boehringer Mannheim GmbH. Serum enzymes were assayed forthwith in twice-centrifuged cell-free serum obtained from fresh whole blood taken from an antecubital vein using a 2l-gauge needle and disposable syringe, isopropanol skin toilet, minimal stasis, and gentle handling to avoid hemolysis. Glassware was cleaned by immersion for I wk in 30% nitric acid, rinsing with glass-distilled water, and drying at 125°C. Grade A pipettes were used throughout. CPK (ATP:creatine phosphotransferase, EC 2.7.3.2). l,6-diphosphofructoaldolase (EC 4.1.2.7; ALD), aspartate aminotransferase (glutamic-oxalacetic transaminase, EC 2.6.1.1; GOT), and *-CT (EC 2.3.2.2) were measured kinetically at 25°C and 340 nm (405 nm for y-CT), as described elsewhere,‘5.‘6 using a Unicam SP 800 B recording spectrophotometer. All values were expressed in international units as mU/ml/25”C. Standardized reagents for ALD, GOT, and y-GT wereobtained as optimized kits (Boehringer Mannheim GmbH), and ALD and GOT were corrected using normal saline for serum. ” The CPK reagents from J. T. Baker Chemicals B.V., Deventer, Holland (Diamed Diagnostics, Liverpool) gave no blank value and had the creatine phosphate substrate separate, allowing saline to be substituted, when the small serum adenylate kinase increment (despite II.5 mM adenosine monophosphate) could be deducted from each CPK '8 assay.
Materials Allopurinol (100 mg) tablets, commercial but unmarked, were allocated at random to 8 of I6 numbered bottles; identical inert placebo tablets were put in the rest. Allocations were carried out by the Wellcome Foundation Ltd.. which provided the tablets and kept the key to the numbering. Procaine adenylate tablets were made by the modified procedure of Ruskin. To 46.6 g powdered procaine base (Sigma Chemical Co.) suspended in 900 ml distilled water at room temperature was added (with stirring) an equimolecular amount (100 g) of ATP (Cambrian Chemicals) in successive portions. The pale pink opalescent solution quickly obtained was filtered and lyophilyzed in an oil-pump vacuum against EtOH-solid CO2 to a pale pink microcrystalline solid melting at 130”.-140°C with decomposition. This was ground and dried in vacua to give procaine-adenosine 5’-triphosphate (PA5) as an almost colorless powder. In the same way procaine-adenosine 3’monophosphate (PA3) was obtained as an exceedingly hygroscopic powder, of melting point 88”-93°C. from equimolecular amounts of procaine base and adenosine 3’-monophosphoric acid (BDH Chemicals Ltd.). Atomic absorption spectrophotometry confirmed the absence of toxic heavy metals in both products. Sublingual tablets, each containing 150 mg PA3 or PA5 (about 100 mg adenosine phosphate), were prepared (Arthur Cox & Co., Brighton. U.K.), together with placebo tablets matched for taste and appearance. PA3 tablets were allocated at random to 4 of 8 numbered bottles, and placebo tablets to the rest; the same procedure was followed in 8 more bottles for PA5. Allocations were made by the hospital pharmacy, which kept the key.
Experimental
Design
Patients were divided into 4 groups of 4 and were examined before noon on the same day each week for 18 wk. For the first 6 wk (period A) nothing was given. For the next 6 wk (period B) each patient, irrespective of age or weight, took one tablet daily from the allopurinol/placebo bottle allocated to him. The contents were then revealed, and for the final 6 wk (period C) all patients took one unmarked genuine allopurinol tablet daily. In addition, those who already had had allopurinol in period B received one of the PAS/placebo bottles, and the rest one of the PA3/placebo bottles; each took 4 sublingual tablets daily during period C, after which the contents were again revealed. At weekly examination, venipuncture for serum enzymes and urate was followed by hand-grip manometry and assessment of clinical status; the urine collection for urate and creatinine was also
THOMSON
154
Table
2.
Effects of Allopurinol Tablet
and
Allocotionr
AND
Adenylate Clintcal
Status’
Clinical
ot End of Perlod Periods Patient
No.
Age (yr),
Mobility
on
Allopurinol
Improvement
Adenvlote in Period
C
SMITH
A
B
C
6 vs. A
1
5.06,
ambulant
BC
Nil
13
15
16
+
2
6.67,
ambulant
BC
Nil
13
14
15
+
3
8.37,
wheelchair
BC
Nil
2
3
6
+
4
11.36,
wheelchair
BC
Nil
1
4
5
+
Cvr
B
+ + + +
5
5.28,
ambulant
BC
PA5
12
12
12
0
0
6
7.74.
ambulant
BC
PA5
13
15
16
+
+
7
8.31,
wheelchair
BC
PA5
2
5
5
+
0
8
13.39,
wheelchair
BC
PA5
5
7
8
+
+
9
3.39,
ambulant
Nil
13
15
15
+
0
10
7.42,
ambulant
Nil
11
11
12
0
+
11
8.38,
ambulant
Nil
10
11
11
+
0
12
14.29,
wheelchair
Nil
5
5
6
0
f
13
8.31,
ambulant
PA3
13
13
15
0
+
14
8.44,
ambulant
PA3
10
9
12
0
+
15
10.36,
wheelchair
PA3
2
2
5
0
+
16
11.99,
wheelchair
PA3
3
3
5
0
+
*See Table
1.
received. The ECG calculated
was recorded
at the end of each 6-wk period.
Serum and urine analyses were
only after each period.
RESULTS In health, marked serum elevations of muscle enzymes during prolonged severe exertion” suggest that muscle function, not enzyme retention,20 has prior claims on ATP. Evidence of increased muscle purine content in DMD should thus appear first as improved function, and only later as diminished enzyme elevations; conversely, overexertion without increased ATP would result in even higher elevations. In period B, percentage manual improvement (+ 16.75”,,) was significantly* greater than zero in the allopurinol group, but not in the placebo group. Furthermore, restoration of departed functions was clearly noted. In period B, of 8 patients (IH) taking allopurinol, 7 improved clinically and 1 did not: in the placebo group (9- 16) 2 improved and 6 did not (Table 2). Fisher’s exact test showed this marked difference of effect to be significant:? and since increased serum elevations (CPK,* GOT?) occurred only in the 2 improved placebo patients, overexertion is implied and this significance is enhanced. Moreoever, while serum and 24-hr urinary urate in period B fell as expected by 26.6’,,* and 27.5%,* respectively, in the allopurinol group, only there did mean 24-hr urinary creatinine output rise simultaneously (+8.7:,,t) from typically low values, consistent with increased muscular activity. Though serum enzyme elevations generally changed little, some decline (CPK,1_ ALD*) confirmed improvement in patient 4, and in period C in patients
*p < 0.01. tp < 0.05.
X-LINKED
RECESSIVE
155
DMD
5 (CPK,1_ ALDt) and 7 (ALDP). Normal y-CT values throughout (Table 3) demonstrated gene specificity as well as the absence of toxic effects on the liver. Both cardiac and skeletal muscle are affected in DM D, and 12 of the 16 boys, compared to a reported SO:;, 2’ had values higher than expected by age for the algebraic sum of the R and S waves (R-S) in lead Vl of the ECG. During periods B and C, however, these values changed only slightly and at random, giving no information, in accord with an origin probably anatomic and not metabo1ic.22 PA3 and PA5 had no obvious additional clinical effect (Table 2). Comparing period C with B did show a fall of CPKt and GOT? against zero in the PA5 but not the PA3 group; but neither group showed significant manual improvement. Thus any extra benefit seems marginal, especially since clinical improvement on allopurinol alone continued in period C (patients l-4). DISCUSSION
In DM D muscle the full effects of the dystrophic X chromosome performing alone appear as the classically rapid progressive wasting toward a fatal issue. Serum CPK elevations in DMD specifically measure active dystrophic muscle mass, and they decline exponentially in both ambulant and wheelchair patients with a half-life of 6.6 yr. ” A DMD patient ambulant at that age would thus have 50% of his muscle mass left, in the wheelchair at 13 yr old some 25% and at 20 yr of age, with death at hand, only 129/,. The fact that functioning muscle survives so long, however, suggests that even modest metabolic support in the correct area could ensure indefinite survival. In female DMD carriers, however, random fusion during early growth occurs between mononucleate myoblasts from clones with one or other X chromosome already randomly inactivated,23 giving a mature multinucleated muscle cell with a dual population of nuclei regulated by either a normal or dystrophic X chromosome. Different proportions for different individuals give a uniform X-chromosomal mosaic24 with a range of manifestation from the undetectable to grossly elevated serum enzymes and minor symptoms, though still nonprogressive and with normal life expectancy in all but rare instances.’ The carrier state thus resembles a supported DMD, protected from progression by even a small proportion of normal muscle nuclei. Thus, in the affected muscle cell some limit is implied, beyond which, as in DMD, lack of normal nuclei causes eventual cell death with clinical progression, and within which, as in the carrier, the presence of normal nuclei ensures survival by providing essential support the DMD nuclei cannot, In DMD patients such provision by other means would bring them within this limit to resemble carriers, still with marked serum enzyme elevations but no longer clinically progressive. Very many features of DMD, including the gross muscle enzyme efflux, may be wholly explained’ by chronic shortage of skeletal muscle ATP.‘,” In the reversible myopathy of vitamin E deficiency, a 22% decrease in muscle ATP is *p < 0.01. tp < 0.05.
8
1
Patient No.
1228
1440
C
C
1754
B
1348
1985
A
1322
2817
C
6
3453
B
A
2864
A
77.8
45.9
44.3
14.6
70.1
14.6*
49.2
73.1
17.7
14.9
72.2
17.3
14
89.6
107
30
36.3
33.4
133
39.4*
3670’
C
158
4598
B
142
4547
A 51.8
1173
C 47.9
1044*
B 39.4
39
10.1
1340
A
7.7
36.8
17.4
1660
C
35.5
31.9
16.4
1795
B
7.47
27.1
14.6
1677
A
3.6 2.9* 3.3
2.7 10.6 10.8 9.2
3.51
3.1
2.7
3.1
2.7
3.2
2.61
3.2
3.1
3.6
3.37
2.6
3.2
2.8
1.5
3.4
2.2 3.0
4.0
2.9 2.0
213 227 250
387 263 278
338
438
454t
276
437
310
360
371
389
308
287
297
319
270
246
2067
219 261
250
194
293
157 178
261
162
2.3
2.3 5.3
218
192
2.0
4.4
197
234
237
3.3
2.51
2.8
127
78
2.9
2.9
150
52.6
3422
C
119
64*
2.9t
34.3t
27.9
25
20.31
13.9
12.9
37.1t
33
31.2
13.4t
11.7*
9.7
22.5
22.8*
20.9
24.4
24.27
18.8
19.71
16.2t
12.3 136
120
3.9
2.3
143
54
2.2
119
49.3
3609
4082
B
200
112
2.8
4.3
160
A
19.9 23t
234
136
2.6*
3.7
210
97 87
7491
16.7
222
181
3.8
3.6
(cm)
Monometry
(mg)
6471
6955
A
Creatinine 24.hr Output
(mg)
Urote (mg/lOOml)
Urate 24.hr Output
Serum
Directly Successive Periods
C
164
85.4
Between
(mU/ml/ZYC)
y.GT
of Differences
B
(mU/ml/ZYC)
(mU/ml/ZYC)
GOT
(mU/ml/25”C)
ALD
Period
CPK
Table 3. Mean Values and Significance
Ip < 0.001.
tp < 0.01.
*p < 0.05.
16
15
14
13
12
11
10
9
310 369 287
4.0 4.6 3.71
2.9 3.7 3.5
3.0 3.3 3.2
63.9 52.6 44.8 58.3 74.7 49.8 46.4 54.8
18.9 17 18.9 24.9 29.8 15 15.8 17
2155 1966
1805
2713t
2369
1647
1400
1689
A
B
C
A
B
C
2.5
4.1 4.1
82.1* 52.9 45.6 46.8 42.1 51.8' 47
20 15.7 14 14.7 9.1 9.6 8.5
2073
1604
1357
1323
1008
1201
1076
C
A
B
C
A
B
C
7.4*
11.3'
9.1
3.8
3.3
3.1
3.6
3.3
3.4
3.0
4.1
3.9
153
190
166
330
307t
379
292
280
112
119
110
253
224*
313
269
285
24.6*
21.3
20.1
24.7
25.8*
22.9
30.6
29.9
28.8
17
1827
B
252
2.7
66.9 61.9
17.8
2065
A
294
365 2367
2.8*
3.1
59.3
22.7
1934
C 3.7
26.81
381 328
24.6
B
2.6
21.6 23.8
322
278
3.3 3.3
1.5 2.6
53.5 60.1
24.9
1907
2096
A
33.4t
27.7 377
26.2
2.21
2.8
365
257
186
3.1
2.6t
295
18.3t 21.41
215
258
3.7
16.6* 15.1
232 260
230 320
11.7 13.5
202 192
263 256
19.1*
189
129
B C
3.8 3.4 3.2
2.3*
2.1 2.7
142 52.8
64.8 15
2857 1785
7.8 15.1t
222 174
201 140
C A
3.6 3.7
3.0 2.3
92.4 147*
47.9 60.3
2279 2875
A B
is
z 8 z
@
x = Z
158
THOMSON
Table I” normal
4.
moles ond children* 18.5-96.2
mU/m1/25°C
(mean
47.02)
Serum ALD
1.07-2.34
mU/ml/25”C
(mean
1.70)
Serum GOT
6.90-15.47
mU/ml/25*C
(mean
10.56)
Serum y-GT
4.20-31.19
mU/ml/25’C
(mean
13.08)
1.2-6.8
mg/lOO
ml (mean
4.0) in 113 normal
Serum urote
2.6-4.7
mg/lOO
ml (mean
3.6) in 16 DMD
Comparisons
of 16 DMD
urote
Mea”
age
Meon
24-hi
Regressions
boys (period
9.5225 UTLIte
of mea”
Age (x) in 16 DMD Weight(x)
yr (normal),
excretion
366
24-hr-urate
Age (x) in 20 normal
boys boys
in 16 DMD
boys
mg
A) with 20 normal 8.6721
A)
boys
yr (DMD);
1.a268
t =
275 mg (DMD),
(normal),
excretion
boyst
boys (period
(34 df).
NS$
p c 0.01
(y) on
y = 0.7049x
+ 359.7320;
y = 7.6178x
+ 208.6312;f
y = 38.3715~
+ 124.4287;
f = 0.0541
(18 df),
NS
= l.O825(14df),NS t = 1.9953
(14 df),
NS
from Sweetin
tDota INS:
SMITH
Comparisons
Serum CPK
Plasma
*Data
Values and
Normal
AND
and Thomson.‘6 from Harkness and Nicol. 38
not significant.
accompanied by a 1 IQ0 increase in the much less abundant adenosine 5’-diphosphate (ADP), the small amount of adenosine 5’-monophosphate (AMP) remaining unchanged.16 In DMD, however, not only is muscle ATP reduced by 5O”i,, but also ADP is reduced by 70?;, with AMP again unchanged;2 the values in both normal and DMD muscle are measured against noncollagen nitrogen content2,25 so that such reductions cannot be ascribed to replacement of DMD muscle by fat and fibrous tissue. This serious depletion of total muscle adenine nucleotides, universally essential coenzymes, suggests some defect in DMD in muscle purine metabolism either by excessive breakdown or insufficient synthesis. Lower than normal values (Table 4) in serum and 24-hr excretion (p < 0.01) of uric acid, the end product of purine catabolism in man, indicate that the latter possibility is more likely; furthermore, since DMD muscle disappears rapidly with increasing age,” the lack of a significant relationship between age or weight and 24-hr urate excretion (Table 4) suggests its independence of body muscle mass. The DMD gene in the dystrophic nucleus may thus exert its effect by insufficient provision of essential muscle purines; this study has tested this hypothesis by using allopurinol to promote retention and recycling of whatever purines are available. Purine synthesis de novo occurs chiefly in the liver, with many peripheral tissues depending on purine salvage pathways from erythrocyte-borne hypoxanfar less ATP to run.J,j Allopurinol, thine,3 requiring a synthetic isomer of hypoxanthine, and its metabolite, oxipurinol (Fig. I), inhibit xanthine oxidase EC I .2.3.2),“.‘” thus reducing irreversible (xanthine:oxygen oxidoreductase, purine loss as uric acid (and eventually de now purine synthesis by independent feedback inhibition>), in addition to enhancing tissue salvage of available blood hypoxanthine in man.“’ The marked protective effect of allopurinol in experimental oligemrd.I ‘O is indeed associated with a considerable increase of adenine nucleotides in the liver” and other viscera)? of dogs and in the kidney of rats,?” and of ATP in the canine myocardium after operative arrest,jJ while even in
X-LINKED
RECESSIVE
159
DMD
OH
OH
Hypoxanthine
Xanthine
(Goxypurine)
Allopurinol (Qhydroxypyrazolo(3,4-d) pyrimidine) Fig. 1.
Allopurinol
(2,Sdioxypurine)
OH
Uric acid (2,6.8-trioxypurine)
Oxipurinol (4,6-dihydroxypyrazolo(3.4-d) pyrimidine) and purine degradation
in man.
healthy human subjects allopurinol induces marked increases in vivo of erythrocyte ATP. 35 In DMD skeletal muscle any similar increase of adenine nucleotide content after allopurinol should give clinical improvement, which, if maintained, must resemble the nonprogressive manifesting carrier state. Sublingual procaine-3’-adenylate is in fact reported of slight benefit in apendomysium dephosphorylates the parent DMD, 36 in which the proliferating 5’- but not the 3’-adenylate, then converted on absorption into the active continuous parts: 5’-adenylate. 37 This study thus comprises two independent periods A and B, which tested allopurinol alone using double-blind techniques; then period C, which likewise tested sublingual PA3 and PA5 superimposed on allopurinol. The duration of the study was deliberately brief in order to exclude any effect of increased growth or fibrous contraction with age, and because effective metabolic intervention should give measurable results within 6wk. The results show that the effect of PA3 and PA5 was minimal. In contrast, a small daily dose of allopurinol alone clearly produced significant physical improvement. This finding assumes particular interest since, in a condition characterized by rapidly progressive muscular weakness, all the improvements reached in period C have been maintained for more than 6 mo without regression on the same daily dose of allopurinol. These findings appear to support the view that DMD may have some basis in defective muscle purine metabolism, perhaps by failure of synthetic pathways, and suggest useful directions for further investigation. In addition, a simple and clinically effective circumvention of the prospective defect seems possible using allopurinol, though further experience alone can determine its value.
160
THOMSON
AND
SMITH
Table 5. Muscle Content (mole x 10m5/g NCN) Clinical
Subjects
(Yr)
ALPNL
*+cl+lJs
Age
Daily
Prior Now
Total
(mg)
ATP
ADP
AMP
NCN w/g
Adenylate
CrP
G-6-P
Wet Weight
(A) 5 healthy
12.51
-
-
18.29 2.82 0.38
21.50
67.19
2.79
25.99
males
20.80
-
-
19.15 2.98 0.40
22.53
67.52
1.54
28.14
38.91
-
-
17.55 2.93 0.12
20.60
57.40
1.53
23.68
48.65
-
-
19.14 2.62 0.44
22.20
58.81
0.91
19.72
56.32
-
-
17.25 2.65
1.09
20.99
54.44
3.64
22.26
la.28 2.80 0.49
21.56
61.07
2.08
23.96
0.88 0.16 0.36
0.81
5.95
1.11
3.26 la.74
Mean SD (B) 5 DMD
3.67
-
-
7.38 3.98 0.26
11.62
18.62
3.01
untreated,
boys
4.32
-
-
9.32 3.44 0.58
13.34
12.91
3.12
15.66
ambulant
4.77
-
-
3.28 2.46 0.41
6.16
7.75
0.77
27.19
-
1.98 0.85
7.57
11.24
1.32
14.91
11.57 4.60
1.04
17.22
22.23
1.92
i 1.89
M.XUl
7.26 3.29
0.63
il.18
14.55
2.03
17.68
SD
3.36 1.07 0.32
4.46
5.82
1.03
5.85
11.44 5.06 0.30
16.80
33.08
0.54
20.79
(C) 4DMD
boys
9.02
-
10.14
-
4.11
13
-
15
100
4.73
after6 mo
4.81
13
15
100
9.81 3.51
1.14
14.47
27.57
3.55
21.36
ALPNL
5.31
13
15
100
12.52 4.16
1.58
18.26
28.71
2.86
22.51
9.52
13
13
150
12.06 6.76
1.22
20.04
44.54
1.14
15.49
11.46 4.87
1.06
17.39
33.47
2.02
20.04
1.18 1.41 0.54
2.36
7.75
1.42
3.12
MeCltl
SD 4.57
13
15
100
14.76 2.76
0.63
la.15
35.88
1.40
23.82
after1 yr
6.23
13
16
100
14.87 1.96 0.24
17.07
37.61
0.69
21.82
ALPNL
7.79
13
16
100
11.74 1.71
1.16
14.62
27.19
1.01
la.79
8.83
13
15
150
16.09 3.13 0.91
20.12
45.72
1.12
20.27
9.46
13
15
100
16.86 1.32
(D) 5 DMDboys
Meon
1.62
19.80
49.69
0.60
13.55
14.87 2.18 0.91
17.95
39.22
0.96
19.65
0.52
2.24
a.81
0.33
3.89
1.95 0.75
SD ALPNL, ollopurinol.
ADDENDUM Further
that the skeletalmuscle in DMD
studies have confirmed
content both of creatine phosphate allopurinol Marked
for 6 mo greatly
physical
improvement
and of total adenylate
improved occurred
these values, early
and
shows 3 grossly diminished
due to lack of ATP.
which
increased
persisted
A small daily dose of
still further
throughout,
after
suggesting
a year. apparent
clinical arrest of the disease.
Table 6. Ratios and Significance of Differences Between Means of Subject Groups in Table 5 Comparisons ofMeans
Total ATP
ADP
AMP
Adenylate
G-6-P
NCN
0.241
CrP
0.97
0.74
WA
0.401
1.18
1.30
0.52t.
C/A
0.63t
1.74*
2.18
o.alt
0.551
0.97
0.84
D/A
o.alI
0.78
0.49
0.83$
0.641
0.46
0.82
C/B
1.58
1.48
1.68
1.56*
2.30t
1.00
1.13
D/B
2.05t
0.66
1.44
1.61*
2.701
0.48
1.11
D/C
1.30*
0.45t
0.86
1.03
1.17
0.48
0.98
Statistical notationrevised for Table 6. *p < 0.05. tp
X-LINKED
RECESSIVE
161
DMD
Table 7. Exponential in Ambulant log (CPK)
= 3.9436
log (Aldolase) The
close
identity
mass whence
these
of each elevations
=
Decline of DMD Serum Enzyme Elevations
DMD Patients -
0.005253X
1.9940
independent
-
as linear
0.005252X half-life
Regressions
(n = 22; p < 0.01;
on Age (X MO)
14 = 4.775
(n = 40; p < 0.001; (tf)
supports
its being
t+ also
yr)
= 4.776 that
yr)
of the
decaying
muscle
arise.
Procedures Muscle specimens (l-2 g) from open biopsy (vastus lateralis in all DMD boys) under general anesthesia (Nz0/02/halothane) were freeze-clamped immediately after excision and immersed in liquid Nz within 30 set of removal. The frozen specimen was weighed, powdered under liquid Nz in a chilled mortar, an aliquot weighed for noncollagen nitrogen (NCN) assay” by microKjeldahl then titration, and the remainder ground in liquid Nl with successive additions of aqueous perchloric acid, then transferred to a glass tube to thaw on ice before final homogenizing (Ultra Turrax) and centrifugation. ATP, ADP, AMP, creatine phosphate (CrP), and glucose-6 phosphate (G-6-P) were enzymatically measured2*40 forthwith in duplicate in the supernatant, and are expressed in terms of NCN content of the specimen.
Results Muscle biopsy specimens were assayed in healthy male controls undergoing surgical repair, and in ambulant boys with clinically classical DMD confirmed both histologically and by serum enzymology.9,4’ Five boys were untreated; four of this group were then given allopurinol for 6 mo, and 5 other boys for 1 yr. Each had an initial clinical status, with differences in degree, of 13 (Table 1); after 4-8 wk on allopurinol, in all but one this improved to 15 and even 16, where it has remained unchanged (Table 5). In health. muscle adenylate content was remarkably constant and unrelated to age: CrP alone showed a high negative correlation with age ( r = -0.947*) indifference was found dicating some decline in energy reserve. As in other studies,’ no significant in NCN content between healthy and DMD muscle. In untreated DMD, the CrP content of muscle was only 24% and of total adenylate (ATP + ADP + AMP) 52% of normal, the latter wholly attributable to lack of ATP (Table 6). Thereafter 6 mo of allopurinol more than doubled CrP and increased total adenylate by half without much effect on ATP. After a year on allopurinol. though CrP and total adenylate increased no further, ATP had risen to 81% of normal at the expense of ADP. The main improvements thus clearly occur in ATP and CrP. A basic adequacy of both oxidative phosphorylation and CPK activity in DMD muscle is thus implied. and the indifference throughout of G-6-P suggests a sufficient glycolysis. Discussion New data (Table 7) from refined analyses4’ Indicate a more precise half-life for DMD muscle than that quoted earlier. Thus, though a DMD patient ambulant at 4.8 yr has lost 50”” of his muscle, he reaches 9.6 yr with still some 25% left. As previously argued, such prolonged survival of functioning muscle might be made indefinite by even partial metabolic support. Our results show that adenylate conservation by allopurinol may offer such support, and that studies of muscle purine metabolism should prove rewarding. If some prospect of sustained clinical arrest and preservation of the muscle fiber population may now seem possible, neonatal diagnoses 42 and early intervention become imperative, and in older children some means, whether biochemically or by advanced orthopedic procedures, to prevent the shrinking fibrous tissue progressively impeding residual muscle action.
Acknowledgment We are greatly indebted to Dr. Alex McQueen, of the University of Glasgow Department Dermatology, for careful excision of all DMD muscle specimens; we also thank our patients colleagues, and Moira Young for skilled Kjeldahl analyses.
‘u i 0.05.
of and
162
THOMSON
AND SMITH
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