close

Вход

Забыли?

вход по аккаунту

?

Radioimmunoassay of serum myoglobin in polymyositis and other conditions.

код для вставкиСкачать
1514
RADIOIMMUNOASSAY OF SERUM
MYOGLOBIN IN POLYMYOSITIS AND
OTHER CONDITIONS
MASAHIKO NISHIKAl and MORRIS REICHLIN
Radioimmunoassay of serum inyoglobin was done
in 53 patients with polymyositis syndromes and other conditions. Serum myoglobin values in 33 healthy subjects
ranged from 4 to 77 [mean 33.3 f 19.8 (SD)] ng/ml.
Fifty percent o f polymyositis patients had elevated serum
myoglobin levels ( > 80 ng/ml). Serum myoglobin values
in polymyositis patients fluctuated more sensitively than
CPK and GOT. Combined estimation of myoglobin and
CPK offers advantages for the detection of musthe injury
and the prediction of disease exacerbation.
Myoglobin is localized solely to the striated
muscle cell (1-4). The estimation of myoglobin in the
serum and urine of patients with muscle diseases might
well be useful in the diagnosis and assessment of clinical
activity.
Although myoglobinuria has long been recognized in patients with fulminant polymyositis (5, 6),
From the Departments of Medicine and Biochemistry, State
University o f New York at Buffalo School of Medicine and Veterans
Administration Hospital, Buffalo, New York.
Supported by Muscular Dystrophy Association o f America,
NIH Grant AM10428 and funds from the Veterans Administration.
Masahiko Nishikai is the recipient of a Henry C . and Bertha H.
Buswell Research Fellowship.
Masahiko Nishikai, M.D.: Research Assistant Professor, Department of Medicine, State University of New York at Buffalo:
Morris Reichlin, M.D.: Professor of Medicine and Biochemistry, State
University o f New York at Buffalo.
Address reprint requests to Morris Reichlin, M . D . , Veterans
Administration Hospital, 3495 Bailey Avenue, Buffalo, New York
14215.
Submitted for publication May 23. 1977: accepted June 17.
1977.
Arthritis and Rheumatism, Vol. 20,
No. 8 (November-December 1977)
myoglobinemia in polymyositis has only recently been
reported. I n 1971 Kagen (7) studied it with the immunodiffusion method and more recently with a quantitative
microcomplement fixation technique (8).
We report here the quantitative measurement of
myoglobin with a radioimmunoassay that is more sensitive than either the immunodiffusion or complement
fixation techniques. This determination was performed
on sera of patients with polymyositis, muscular dystrophy, and myasthenia gravis.
MATERIALS AND METHODS
Preparation of Myoglobin. Human skeletal muscle
myoglobin and horse cardiac myoglobin were purified by the
modification of Bowen's method (9) as previously reported
(10).
Antimyoglobin Antiserum. Antisera to human skeletal
muscle myoglobin were prepared by immunizing adult albino
rabbits. One milligram of antigen was emulsified in an equal
volume of complete Freund's adjuvant and injected intramuscularly every 2 weeks. The rabbits were bled after each
booster injection and the serum was absorbed with normal
human serum $50 MI per ml antiserum) and stored at -20°C
until use. Sq&h sera gave a single precipitin line in double
diffusion experiments with either crude muscle extract or purified human myoglobin that formed a reaction of identity.
Labeling of l3lI to Myoglobin. This procedure was done
at room temperature. lodination of horse myoglobin was performed by the method of Hunter and Greenwood ( 1 1). Human myoglobin fails to iodinate with the chloramine T method
under a variety of conditions. Twenty microliters of 2 mg/ml
horse myoglobin solution, 0.5 mCi 1311 (Amersham-Searle
Corporation, Illinois) and 0.1 mg chloramine T in 0.05 ml in
SERUM M Y OGLO BIN RADIOIMMUNOASSAY
Table 1. Incidence of Myoglobinemia in Various Condirions
Number
Condition
Polymyositis ,syndrome
of
N um ber
Abnormal
Patients
(% 1
PM
53
27
DM
10
PM overlapped with other
connective tissue diseases
PM (or D M ) with malignancy
Progressive muscular dystrophy
Myasthenia gravis
Normal subjects
14
2
17
13
33
0.01 M phosphate buffer, pH 7.5, containing 0.02% sodium
azide were mixed. After 40 seconds the reaction was terminated by the addition of 0.3 mg of sodium metabisulfite in 0.05
ml of the same buffer. After that I .3 mg of potasium iodide in
0.04 ml of the buffer were added. The mixture was loaded onto
a Sephadex (3-25 (coarse) column (10 X 130 mm) and eluted
with 0.1 M phosphate buffer, pH 7.0. Aliquots of each fraction
were assessed for radioactivity in the autogammacounter. The
tube of the first peak with the highest concentration of labeled
myoglobin was diluted to achieve 2000 cpm/0.05 ml and
stored at -20°C until use. Incorporation of iodine into protein
varied from 50-796 and the specific activity of the labeled
myoglobin varied from 6 to 10 pCi per pg.
Radioimmunoassay Procedure. The radiommunoassay
was done in an ice bath. All reactions were performed in 11 X
75 mm glass tubes. A standard curve was constructed utilizing
cold human myoglobin at concentrations ranging from 2.0
0
5
10
20
Human Mb (nglml)
x,
40
Fig. 1. Displaret~ienr curve in which '"I horse myoglobin is displaced
from rabbit antihuman myoglobin (serum 309. B . I diluted I f lo00) by
various concenrrarions of cold human myoglobin.
1515
ng/ml to 2.0 pg/mI. For an unknown sample, 0.1 ml of human
serum and two dilutions (1:s and 1:25) of such serum in
duplicate were assayed. Approximately 2000 cpm of l 3 I I myoglobin (in 0.05 ml of buffer containing 2% bovine serum albumin) were added t o each tube. The concentration of labeled
Mb varied from 8 to 16 ng per ml or from 0.4 to 0.8 ng M b per
test. A large excess of antiserum (diluted 1/50) routinely
bound more than 90% of the labeled Mb. Background binding
of the tracer in the absence of antiserum was 5% or less of the
added counts. One tenth milliliter of antihuman myoglobin
rabbit antiserum that had been diluted 1000-fold with the
phosphate buffer solution containing 2% normal rabbit serum
was added. After an incubation at 4°C for 24 hours, 0.2 ml of
sheep antirabbit gamma globulin serum was added for the
separation of bound and free antigen. After centrifugation
(3000 rpm, 30 minutes) each precipitate was washed three
times with 0.2 ml of phosphate buffer solution and then
counted for 10 minutes in the autogammacounter.
Patients Studied. Fifty-three patients (Table 1 ) with
adult polymyositis (PM) were diagnosed by the criteria of
Medsger (12). These included 27 cases of pure PM, 10 cases of
dermatomyositis (DM), 14 cases of PM overlapping with
other connective tissue diseases (7 cases with scleroderma, 2
cases with systemic lupus erythematosus, 1 case with both, 1
case with rheumatoid arthritis, and 3 cases with Sjogren's
syndrome), and 2 cases of PM with malignancy. In addition 17
patients with muscular dystrophy ( P M D ) (16 Duchenne type
and 1 myotonic dystrophy) and I3 cases of myasthenia gravis
were studied. T o determine the normal range for serum myoglobin, serum samples of 33 healthy adult subjects were studied.
RESULTS
A typical standard curve is shown in Figure 1.
With this curve as little as 1 ng/ml of serum myoglobin
can be estimated. As can be seen 50% of the labeled
horse material is displaced at a concentration of cold
human myoglobin of 3 ng/ml. This is entirely similar to
the sensitivity reported by Stone and colleagues (13)
utilizing human myoglobin radioiodhated with the
Bolton-Hunter reagent. We have achieved exactly the
same sensitivity with our own antiserum (but diluted
I /20,000) and human myoglobin-labeled with the
Bolton-Hunter reagent (14).
The mean value of 33 healthy adults subjects was
33.3 f 19.8 (SD) ng/ml with a range of 4 t o 77 ng/ml.
This mean range is similar to those reported by other
investigators (13). A value in excess of 80 ng/ml was
taken as abnormal. Figure 2 shows serum myoglobin
levels in various conditions. Each point indicates the
highest value in an individual patient. Elevation of
serum myoglobin values was seen most frequently in
pure polymyositis, but very high values were also seen
in DM and the overlap syndromes. Interestingly, 38% of
the patients with myasthenia gravis had elevated serum
NISHIKAI A N D REICHLIN
1516
myoglobin values although none exceeded a level of 300
ng/ml. Similarly, 38% of the patients with muscular
dystrophy had elevated myoglobin values but none had
levels exceeding 350 ng/ml.
These results are presented in tabular form in
Table I . As illustrated in Figures 3 and 4, elevated serum
myoglobin was observed in the active phase of polymyositis that seems to be no different in principle from
serum muscle enzymes such as creatine phosphokinase
(CPK) and glutamic oxalacetic transaminase (GOT).
However, myoglobin values exhibited a tendency to be
elevated a few months earlier than the appearance of
clinical exacerbation or the definite elevation of the
CPK or the GOT. This prescient elevation was seen at
least once in each of the 2 patients illustrated in Figures
3 and 4. By the serial estimation of these samples, it is
seen that the elevated serum myoglobin values were
occasionally observed in the phase in which CPK and
GOT were within normal limits. On the other hand,
there are serum samples in which the CPK and GOT are
seen to be abnormal and the myoglobin level has fallen
back into the normal range.
Simultaneous estimation of myoglobin and CPK
were compared in 32 samples of patients with polymyositis. These results are tabulated in Table 2. I n 14
instances (44%) both the myoglobin and CPK values
were elevated. In 6 instances (19%) the myoglobin value
was elevated while the CPK level was in the normal
range. In 3 instances (9%) the converse was true because
the CPK was elevated at the time that the myoglobin
level was normal. In 9 instances (28%) both determinations were in the normal range. I n this small series then,
in about 25% of the instances where positive tests were
obtained with one or other of the tests, the myoglobin
value was elevated while the CPK level was normal.
DISCUSSION
Since the incidental finding of elevated serum
aldolase in cases of muscular dystrophy by Sibley and
Lehninger (15), it has been shown that several serum
enzymes, such as aldolase (16) and GOT (17) are
markedly elevated in myopathies. However, because
these enzymes reside in tissues other than skeletal
muscle, an obvious limitation of specificity in diagnosing myopathy exists. In 1959 Ebashi et al. (18) first
reported the elevated creative phosphokinase (CPK) in
myopathies. Because of the high specificity for muscle
tissue, an estimation of serum CPK is presently an established routine laboratory aid in the diagnosis of myopathies.
Recently, however, extramuscular CPK (brain
type CPK) has been described (19) and the presence of
elevated CPK in serum has been demonstrated in conditions other than myopathies. The studies of Kagen (7,8)
clearly point to the possible utility of serum myoglobin
determination in the diagnosis and management of myopathies. This report extends these findings and suggests
that the increased sensitivity and high precision of radioimmunoassay might add an additional dimension to the
utility of the measurement of serum myoglobin. In the
active phase of polymyositis, the existence of frequent
hypermyoglobinemia was confirmed. However, the intriguing finding suggested by these preliminary data is
that myoglobin levels may antedate other clinical and
enzymatic indicators of disease activity in some cases.
Kagen has also recently reported this finding in a patient
followed serially over a period of months (8).
I f these findings are confirmed and extended, it
would make the measurement of serial myoglobin levels
a useful adjunct in the diagnosis and management of
myopathies. Further study in other muscle diseases will
be necessary to see if myoglobin determinations might
be useful in assessing activity. It is of interest that small
but definite elevations were noted in 38% of patients
with myasthenia gravis.
. (17500)
- (9,385)
-
*
1500
= f
-
(12,000)
(7,308)
(3,250)
c
t
,E 1000
F
*
Y
E
2
8 500
F
Fig. 2. Plot ofserum myoglobin levels in nglml for patients with various
muscle disease. See text for explanation of abbreviations.
SERUM MYOGLOBIN RADIOIMMUNOASSAY
li
1517
-SGOT
-----CPK
I00
50
Figs. 3 and 4. Relationships of therapy, clinical weakness, and the levels of
SGOT. CPK. and myoglobin. respectively. The horizontal solid and dotted
lines represent the upper limits of normal for SGOT and CPK respectively.
The vertical lines represent the level of serum myoglobin in nglml.
0
30
PREDNISOLONE
NISHIKAI A N D REICHLIN
1518
Table 2. Simulraneous Estimation of Myoglobin and C P K Values in
32 Serum Samples in Polymyositis
Elevated
Elevated
M yoglobin
CPK
Number of
Sample (%)
One might ask what determines the presence of
an elevated myoglobin level as opposed to an elevated
CPK level. Observations in patients with uncomplicated
myocardial infarction ( 14) suggest that myoglobin values peak earlier (8-12 hours) and return to baseline
(1 8-24 hours) more promptly than CPK levels (peaking
at 20-30 hours and returning to normal at 2-3 days).
Such kinetic differences might explain how one test
could be abnormal when the other test is normal and
vice-versa.
In addition there might be states of muscle injury
when myoglobin leaks from the cell and CPK does not
(and vice-versa). Such situations are suggested by studies of certain patients with angina pectoris without EKG
or other evidence of myocardial infraction who nonetheless show modest elevations of serum myoglobin in the
presence of normal CPK values (14). Further clinical
and laboratory observations should clarify both the
mechanisms of these findings and the possible utility of
measuring serum myoglobin by radioimmunoassay for
the diagnosis and management of myopathic disease.
REFERENCES
I . Drews GA, Engel WK: An attempt at histochemical localization of myoglobin in skeletal muscle by the benzidine
peroxidase reaction. J Histochem Cytochem 9:206-207,
1961
2. Goldfischer S: The cytochemical localization of myoglobin in striated muscle of man and walrus. J Cell Biol
34:398-403, 1967
3. Kagen LT, Gurevich R: Localization of myoglobin in
human skeletal muscle using fluorescent antibody technique. J Histochem Cytochem 15:436-44 I , 1967
4. James NT: Histochemical demonstration of myoglobin
skeletal muscle fibers and muscle spindles. Nature
219:1174-1175, 1968
5. Walton JN, A d a m RD: Polymyositis. Edinburgh, Livingstone, 1958, pp 18-21
6. Kessler E, Weinberger I. Rosenfeld JB: Myoglobinuria
acute renal failure in a case of dermatomyositis. Isr J Med
Sci 8:978-983, 1972
7. Kagen LJ: Myoglobinemia and myoglobinuria in myositis
syndromes. Arthritis Rheum 14:457-464, 1971
8. Kagen LJ: Myoglobinemia in inflammatory myopathies.
JAMA 237:1448-1452, 1977
9. Bowen WJ: Note on myoglobin preparation and iron content. J Biol Chem 176:745-751, 1948
10. Reichlin M, Hay M, Levine, L: Immunochemical studies
of hemoglobin and myoglobin and their globin moities.
Biochemistry 2:971-979, 1963
I I . Hunter WM, Greenwood FC: Preparation of iodine-131
labelled human growth hormone of high specific activity.
Nature 194:495-496, 1962
12. Medsger TA Jr, Dawson WM, Masi AT: The epidemiology of polymyositis. Am J Med 48:715-723. 1970
13. Sone MJ, Willerson JT, Gomez-Sanchez CE, et al: Radioimmunoassay of myoglobin in human serum: results in
patients with acute myocardial infarction. J Clin Invest
56:1334-1339, 1975
14. Reichlin M, Visco JP, Klocke FJ: Radioimmunoassay for
human myoglobin: experience in patients with coronary
heart disease (submitted for publication)
15. Sibley JA, Legninger AL: Aldolase in the serum and tissue
of tumor-bearing animals. J Natl Cancer Inst 9:303-309,
I949
16. Dreyfus JC, Schapira G, Schapira F: Biochemical study of
muscle in progressive muscular dystrophies. J Clin Invest
33:794-797, 1954
17. Siekert RG, Fleisher GA: Serum glutamic oxalacetic
transaminase in certain neurologic and neuromuscular
diseases. Proc Staff Meet Mayo Clin 31:459-464, 1956
18. Ebashi S, Toyokura Y, Momoi, H, et al: High creatine
phosphokinase activity in sera of progressive muscular
dystrophy. J Biochem 46:103-104, 1959
19. Nevins MA, Saran M, Bright M, et al: Pitfalls in interpreting serum creative phosphokinase activity. JAMA
224:1382-1387. 1973
20. Russell DS: Histological changes in myasthenia gravis. J
Path01 65~279-289, 1953
21. Mendelow H, Genkins G: Studies in myasthenia gravis:
cardiac and associated pathology. J Mt Sinai Hosp
21:218-225, 1954
Документ
Категория
Без категории
Просмотров
0
Размер файла
372 Кб
Теги
polymyositis, serum, myoglobin, radioimmunoassay, conditions
1/--страниц
Пожаловаться на содержимое документа