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Arthritis Rheumatism - June 1987 - Valen
Arthritis Rheumatism - June 1987 - Valen
Myoadenylate deaminase (MADA) deficiency has nucleotide cycle (Figure 1) (1). A deficiency of MADA
been associated with symptoms of postexertional aches, activity, perhaps the most common cause of metabolic
cramps, weakness, and skeletal muscle dysfunction. myopathy , has been reported in association with skel-
Measurement of plasma lactate and ammonia concen- etal muscle dysfunction (2). Although the clinical
trations after forearm ischemic exercise has been sug- picture is variable, most MADA-deficient subjects
gested as a screening test for this disorder. We per- experience easy fatigue, cramps, and postexertional
formed forearm ischemic tests on 3 patients with myalgias (2-4). The precise relationship between the
histochemically defined MADA deficiency and 13 enzyme deficiency and these symptoms is not clear.
healthy control subjects, in a standardized fashion. Our Sabina et a1 have observed that significant alterations
results demonstrated that subject effort and/or perfor- in purine nucleotide content of skeletal muscle occur
mance during the exercise portion of testing is a critical with exercise in MADA-deficient individuals, both in
variable. In addition to lactate and ammonia, plasma vitro and in vivo ( 5 ) .
purine compounds (adenosine, inosine, and hypoxan- Although tissue analysis is required to secure
thine) were measured. The finding of decreased purine the diagnosis of MADA deficiency, several methods
release after exercise in MADA-deficient patients com- have been suggested as screening. tests. These involve
pared with that in normal individuals increases the sequential measurements of blood lactate and ammo-
specificity of the test and supports the hypothesis that nia concentrations in conjunction with vigorous fore-
disordered purine metabolism occurs in MADA defi- arm exercise. A normal response to the exercise is a
ciency. several-fold increase in both lactate and ammonia
concentrations (5,6). In MADA-deficient subjects, lac-
Myoadenylate deaminase (MADA) (EC 3.5.4.6) tate concentrations increase, but little or no change is
catalyzes the deamination of AMP to IMP in skeletal observed in ammonia levels (2,5-8). Such a response
muscle and plays an important role in the purine has been reported in all MADA-deficient subjects
described to date, but there have been many false-
From the Section of Rheumatology, Department of Medi-
cine, and the Department of Neurology, Medical College of Wis- positive results in normal subjects (7,9). In addition,
consin and Clement J. Zablocki Veterans Administration Medical diminished generation of adenosine, inosine, and
Center, Milwaukee, Wisconsin. hypoxanthine after exercise has been observed in
Supported in part by a grant from The Arthritis Foundation,
Wisconsin Chapter. MADA-deficient subjects compared with that seen in
Peter A. Valen, MD; Denny A. Nakayama, MD; Judith normal subjects (6,8). Methods previously described
Veum, BA; A. R. Sulaiman, MBBS, FRCP(C); Robert L. for such screening vary greatly in duration of exercise,
Wortmann, MD
Address reprint requests to Robert L. Wortmann, MD, whether exercise is performed under ischemic or
Rheumatology Division-IOCN, Medical College of Wisconsin, nonischemic conditions, and even whether blood is
Clement J. Zablocki Veterans Administration Medical Center, Mil- sampled from the exercised or nonexercised arm.
waukee, WI 53295.
Submitted for publication October 29, 1986; accepted in To establish the limitations of the test, we
revised form December 12. 1986. performed a forearm ishemic exercise test in 3 patients
Figure 2. Apparatus used to quantitate subject’s effort during exercise testing. The subject squeezed a grip
dynamometer with attached strain gauge (left) connected to strip recorder. Exercise was quantitated by
summing the areas of curves generated by each grip. Areas were measured using a planimeter (right).
inflated to, and maintained at, 10-20 mm of mercury above immediately analyzed for lactate and ammonia on a Dupont
systolic pressure for 90 seconds. While the cuff was inflated, Automated Clinical Analyzer using American Chemical As-
subjects exercised their forearms by squeezing a grip dyna- sociation analytical test packs (Dupont, Wilmington, DE).
mometer as forcefully as possible, at a rate of 1 grip every 2 Plasma from tubes B was stored at -70°C for subsequent
seconds. The dynamometer was attached to a strain gauge determination of purine nucleosides and bases.
and strip recorder so that the amplitude and duration of each Prior to purine determinations, samples were
grip was recorded (Figure 2). Exercise continued until the ultrafiltered with Amicon CV-25 cones (Amicon, Lexington,
patient was exhausted or for a maximum of 90 seconds. MA). Hypoxanthine, inosine, and adenosine were quanti-
Blood samples were collected through the intravenous line at tated on a Varian 5200 high performance liquid chromatog-
1, 3, 5, and 10 minutes after the cuff was deflated. raphy instrument, with a Varian Micropak MCH-10 reverse-
A subset of normal subjects repeated the test at least phase column, using the method described by Hartwick et a1
1 week later; they were asked to exercise at the above rate, (1 l), with modifications (12).
but at what they considered to be 50%-75% of maximum
effort. Subject performance was quantitated by summing the RESULTS
areas under the deflection curves generated with each
squeeze using a Dietzgen Model D 1806 compensating polar Subject performance was critical to the quantity
planimeter. The grip apparatus was calibrated before each of lactate, ammonia, and purine compounds generated
test and the summed areas, which reflected subject exercise after forearm ischemic exercise. Figure 3 shows re-
effort or performance, were expressed in Standardized
Planimeter Units (SPU). sults of lactate and ammonia measurements obtained
After the line was cleared of heparinized saline, 10-cc after 3 separate trials of forearm ischemic exercise in a
venous samples were collected in a plastic syringe and normal individual. As the level of effort was de-
divided equally into tubes containing lithium heparin and 250 creased, these compounds were generated to a lesser
pl of either 0.87% NaCl (tube A) or 0.1 mM EHNA (an
adenosine deaminase inhibitor) and 0.2 mM dipyridamole (as
degree, such that at 58% of maximum effort, the
inhibitor of bidirectional nucleoside transport) in 0.87% patterns of lactate and ammonia were indistinguish-
NaCl (tube B). Tubes were kept on ice and centrifuged able from the typical results for a MADA-deficient
within 10 minutes of collection. Plasma from tubes A was subject.
15290131, 1987, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/art.1780300609 by University Of Toronto Mississauga, Wiley Online Library on [28/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
VALEN ET AL
8-
-
4- d
/
/&--
--- ------
P--+-----,-_
----a
o- ' /,
1 3 5 10 1 3 5 10
MINUTES POST-EXERCISE
Figure 3. Forearm ischemic exercise test results for a normal individual exercising at different levels of
intensity on 3 separate occasions (the Standardized Planimeter Units generated by maximum performance is
called 100% effort, and subsequent performance is expressed as a percentage of that) and results from a
myoadenylate deaminase-deficient (MADD) patient exercising at maximum effort.
36 3.6 -
ma a a
28 2.8 -
a a
20 2.0 -0 0 ' a a
12 12- 0
' A
d
04
:1
0 2
I 1
4 6
1
8 10 12 14
I
I l
36
. a
28
20
a
12 A '
A r = 0.63
04
J I I I I I I l i
3 20 40 60 80
100 120 140 160
AMMONIA RISE (prnol/L )
Figure 4. Maximum levels of lactate, ammonia, and total purines (Hx = hypoxanthine, I N 0 = inosine, A 0
= adenosine) versus maximum performance for control subjects, control subjects exercising with
submaximal effort, and myoadenylate deaminase-deficient (MADD) patients. SPU = Standardized
Planimeter Units. = control subjects; A = control subjects exercising submaximally; 0 = MADD subjects.
15290131, 1987, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/art.1780300609 by University Of Toronto Mississauga, Wiley Online Library on [28/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
MADA DEFICIENCY 665
Concentrations utilized for subsequent analyses the presence of EHNA and dipyridamole, and centri-
were the maximum values obtained at a single time fuged rapidly after collection to eliminate the in vitro
point and were consistently found in the 1- or 3-minute changes in concentration that we observed in prelim-
sample for lactate and ammonia, and the 10-minute inary studies (data not shown) and that have been
samples for purines. Correlation coefficients for per- reported by others (13). Mean (+SD) baseline values
formance versus lactate, ammonia, and total purines for normal subjects were: hypoxanthine 0.52 2 0.47
(adenosine plus inosine plus hypoxanthine) were 0.71, p M , inosine 0.54 *1.27 p M , and adenosine 4.14 -+
0.63, and 0.35, respectively (Figure 4). 1.71 p M . Respective baseline values in MADA-
Measurements of peak plasma lactate and am- deficient subjects were 1.38 ? 0.01 p M , 2.04 2 2.21
monia concentrations within 10 minutes of forearm p M , and 3.09 i 0.78 p M .
ischemic exercise proved to be effective in differenti- With exercise at maximum effort, hypoxanthine
ating normal subjects from MADA-deficient subjects, concentrations in normal individuals were increased
if all individuals performed the exercise with maxi- by an average of 17.7 pM (range 6.0-32.3); the
mum effort (Figure 5). In normal subjects, lactate MADA-deficient patients had hypoxanthine concen-
concentrations were increased to 6.4 mEq/liter (range trations that were 0, 3.5, and 6.8 p M above baseline.
4.0-10.5) from a baseline of 1.0 mEqAiter (range With submaximum efforts, the increase in hypo-
1.0-1.8), and ammonia was increased to 105 pmoles/ xanthine concentration obtained in 4 of 6 normal
liter (range 42-160) from a baseline of 18 pmoles/liter subjects fell into the MADA-deficient range (Figure 5).
(range 14-61). MADA-deficient subjects had lactate No overlap between control subjects and MADA-
levels that were increased over baseline, although to a deficient subjects was observed when maximum total
lesser degree than was seen in normal subjects, but purines (hypoxanthine plus inosine plus adenosine)
their ammonia concentrations were not increased. were compared (Figure 5).
Performance levels for these tests ranged from 1.5-3.3
SPU for control subjects and were 1.1, 2.1, and 2.3
DISCUSSION
SPU for the 3 MADA-deficient subjects.
In 7 of 9 normal subjects who were retested at Muscle weakness and muscle aches are fre-
submaximal exercise levels (range 0.62.1 SPU), lac- quently encountered symptoms in medical practice.
tate and ammonia generation patterns were indistin- Possible causes of these symptoms may include
guishable from those of MADA-deficient subjects. psychoneurosis, muscular dystrophy, metabolic my-
Samples for purine analysis were collected in opathy, or inflammatory myositis. At times, no spe-
11 - 220 - 44 - 44
10 - 200 - 40 - 40 -
9- 180 - 36- 36 -
8- : 160 - 0 32 - 32-
28 -
0
7- 140- 7 28 -
120 -
5
4- 0
:
AA
loo-
80-
i
8 A
A
24-
20 -
16-
.)
AA
A
24 -
20 -
16 - ?!&
3- A 0 60- 0. 12- t 12-
2- 40- 0 8- A
8-
1- A 20- AA 0 4-
0
0 4-
d 0
A 0 A 0
- , - o 0-, I &L O - u A 0- u r n
Figure 5. Maximum rises in lactate, ammonia, total purine, and hypoxanthine in control subjects exercising
maximally (Max), control subjects exercising submaximally (Submax), and myoadenylate deaminase-
deficient (MADD) patients.
15290131, 1987, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/art.1780300609 by University Of Toronto Mississauga, Wiley Online Library on [28/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
VALEN ET AL
cific diagnosis can be made. Recent observations of a plasma lactate, ammonia, and the ATP degradation
group of patients with mild muscle weakness and product, hypoxanthine (6,8,17), These increases prob-
postexercise cramping have suggested a possible role ably result from normal MADA activity and normal
for disordered purine metabolism in the pathogenesis functioning of the purine nucleotide cycle (Figure 1).
of their symptoms. Fishbein et a1 described 5 patients Although the precise function of MADA is not com-
who had mild muscle weakness and cramping after pletely understood, it is important in the regeneration
exercise, and demonstrated diminished myoadenylate of ATP during muscular activity and recovery (17).
deaminase activity, defined histochemically , on mus- MADA catalyzes the conversion of AMP to IMP with
cle biopsy specimens (2). Further investigations have the release of ammonia (1). In the absence of MADA
shown that myoadenylate deaminase deficiency is not activity, there would be less generation of ammonia
rare and it may be the most common cause of meta- during muscle activity. This would explain the lack of
bolic myopathy . Using the histochemical staining as- rise in ammonia after exercise in MADA-deficient
say ( 2 4 , approximately 2% of muscle biopsy speci- subjects, and might also account for the smaller than
mens in large series have been shown to be deficient in normal increases in lactate observed in these individ-
this enzyme. uals, since ammonia stimulates glycolysis by activat-
Myoadenylate deaminase is a distinct isoen- ing phosphofi-uctokinase (18).
zyme of adenylate deaminase found only in skeletal Our results indicate that measurement of
muscle. It is present in both type I and type I1 fibers, venous lactate and ammonia concentrations following
with higher levels seen in the latter. MADA-deficient forearm ischemic exercise is an effective means of
patients who have been tested have normal levels of screening for MADA deficiency and that submaximal
adenylate deaminase in their lymphocytes, neutro- exercise performance, whether due to weakness, pain,
phils, and red blood cells (2). Family members of some or poor effort, can be responsible for false-positive
MADA-deficient subjects have been shown to have results. Measurements of lactate after exercise have
lowered levels of enzyme activity as well, suggesting been used for many years to test for abnormalities of
the presence of a carrier state (14). glycogen metabolism. Munsat standardized a forearm
MADA deficiency has been described in asso- ischemic exercise test for that purpose (19). Using a
ciation with many other disorders, including periodic grip dynamometer, reliable results were obtained if the
paralysis, influenza-like illness, Kugelberg-Welander subject produced a workload of 4-7 kg/meter. Initially,
syndrome, amyotrophic lateral sclerosis, spinal mus- we tried to standardize ammonia generation with
cular atrophy, facial and limb girdle myopathy, work, but found no correlation. Satisfactory results
poly myositis, dermatomyositis, systemic lupus ery- were obtained only when duration of work was added
thematosus, systemic sclerosis, diabetes, hyperthy- to the measurement. We have termed this combined
roidism, and gout (3,4,7,15,16). However, many pa- measure “performance” and expressed it in Standard-
tients with this deficiency have no other rheumatic or ized Planimeter Units. The conclusion from Munsat’s
neuromuscular disorder that could explain their symp- study and ours is that valid results depend upon the
toms. Fishbein recently reviewed 58 cases of MADA vigor of the exercise effort. Thus, failure to generate
deficiency (14). In 28 of the patients, there was no lactate or ammonia after exercise does not indicate
other neuromuscular disease. These patients tended to abnormality of lactate generation or MADA deficiency
have the lowest levels of MADA activity and normal unless an adequate exercise effort is documented. In
levels of creatine kinase and adenylate kinase, com- addition, an abnormal result should be followed by a
pared with the 30 patients who had additional muscle biopsy for confirmation of the putative enzyme
neuromuscular diagnoses, higher residual MADA ac- deficiency.
tivities, and decreased activities of creatine kinase and Our results also confirm that measurements of
adenylate kinase. It is possible, therefore, that MADA hypoxanthine and other ATP degradation products
deficiency can occur in a primary form, perhaps inher- increase the specificity of exercise testing for MADA
ited in an autosomal recessive pattern, and a second- deficiency. Patterson et a1 observed decreased ammo-
ary form, related to nonspecific pathologic muscle nia and hypoxanthine generation (6), and Sinkeler et al
damage from a variety of neuromuscular disorders. recently reported diminished changes in plasma
The exact relationship between MADA defi- adenosine, inosine, and hypoxanthine concentrations
ciency and muscular symptoms remains unclear. The in MADA-deficient subjects after ischemic and
normal response to muscular work is an increase in nonischemic exercise (8). Our results are similar,
15290131, 1987, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/art.1780300609 by University Of Toronto Mississauga, Wiley Online Library on [28/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
MADA DEFICIENCY 667
14. Fishbein WN: Myoadenylate deaminase deficiency: in- 19. Munsat TL: A standardized forearm ischemic exercise
herited and acquired forms. Biochem Med 33:15&169, test. Neurology 20: 1171-1 178, 1970
1985 20. Sabina RL, Swain JL, Olanow CW, Bradley WG,
15. Mercelis R, Martin JJ, Dehaene I, deBarsy T, van den Fishbein WN, DiMauro S, Holmes EW: Myoadenylate
Berghe G: Myoadenylate deaminase deficiency in a deaminase deficiency: functional and metabolic abnor-
patient with facial and limb girdle myopathy. J Neurol malities associated with disruption of the purine
225:157-166, 1981 nucleotide cycle. J Clin Invest 73:720-730, 1984
16. Gertler PA, Jacobs RP: Myoadenylate deaminase defi- 21. Scislowski PWD, Aleksandrowicz Z, Swierczynski J:
ciency in a patient with progressive systemic sclerosis. Purine nucleotide cycle as a possible anaplerotic process
Arthritis Rheum 27586590, 1984 in rat skeletal muscle. Experientia 38: 1035-1037, 1982
17. Sutton JR, Toews CJ, Ward GR, Fox IH: Purine metab- 22. Kushmerick MJ: Energetics of muscle contraction,
olism during strenuous muscular exercise in man. Me- Handbook of Physiology. Section 10. Skeletal Muscle.
fabolism 29:25&260, 1980 Edited by LD Peachy, R H Adrian, SR Geiger.
18. Sugden PH, Newsholme EA: The effects of ammonium, Bethesda, MD, American Physiological Society, 1983,
inorganic phosphate and potassium ions on the activity pp 189-236
of phosphofructokinase from muscle and nervous tissue 23. M O B RJ, Granger JHJ: Contribution of adenosine to
of vertebrates and invertebrates. Biochem J 150: 113- arteriolar autoregulation in striated muscle. Am J
122,1975 Physiol 244:H567-H576, 1983