Carbonic Anhydrase Ill in Serum in Muscular Dystrophy and - PDF by po2378


									CLIN. CHEM.37/1,        36-39 (1991)

Carbonic Anhydrase Ill in Serum in Muscular Dystrophy and Other Neurological Disorders:
Relationship with Creatine Kinase
Mitsuhlro Ohte,1Yasuko ltagakl,1NobuyuklItoh,2KyozoHayashl,3                 and
                                                            HiroshlNlshltanl,1 Klyoe Ohta1

We measured with a radioimmunoassay the concentra-                      CK would. We evaluated the applicability of CA-ill
tions of carbonic anhydrase Ill (CA-Ill, EC in sera            measurements    to the early detection and monitoring of
from 68 patients with muscular dystrophy, 10 carriers of                the progress of muscular diseases, to evaluate whether
Duchenne muscular dystrophy (DMD), and 63 patients                      the measurements of both enzymes might be useful as
with other neurological disorders. The values obtained                  specific markers of fiber abnormalities. We recently
were compared with those for creatine kinase (CK, EC                    developed a sensitive radioimmunoassay       for human Serum CA-Ill was strikingly increased in patients             muscle CA-Ill (5) and tested it on a large number of
with DM0 (mean, 274.4 IL)       and congenital (Fukuyama-               serum       samples     taken   from patients   with   various   neuro-
type)(1 82.8 g/L) and limb-girdle (203.7 p9/L) dystro-                  logical     diseases,    includingdystrophy. We also
phies and positively correlated with the activities of CK in            compared the CA-rn concentrations  with those of CK,
patients with DMD. CA-Ill concentration decreased with                  the enzyme widely used as an indicator of muscular
the subjects’ age and the severity of the disease, similar              diseases.
to the tendency observed between age or severity and the
concentration of CK. We found moderately increased                      Materials and Methods
CA-Ill in patients with polymyositis, myotonic dystrophy,                  Subjects: We examined the sera from 68 patients with
amyotrophic lateral sclerosis, spinal progressive muscular              muscular dystrophy [48 with DMD, 15 with congenital
atrophy, or Kugelberg-Welander disease and in carriers                  (Fukuyama-type), and five with limb-girdle], 10 carriers
of DMD.                                                                 of DMD (all are mothers of DMD patients),              63 patients
                                                                        with various neurological           and muscular disorders [nine
AddItIonal Keyphras.s:           radioimmunoassay . age-related ef-     with polymyositis, eight with myotonic dystrophy, 14
fects         heritable disorders
                                                                        with amyotrophic lateral sclerosis (ALS), nine with
                                                                        spinal progressive           muscular atrophy (SPMA), three
   Creatine kinase (CK; EC is widely used for
                                                                        with Kugelberg-Welander              disease, 10 with myasthenia
the monitoring    and diagnosis of Duchenne muscular
                                                                        gravis, four with neuro-Beh#{231}et’s isease, and six with
dystrophy       (DMD)       and is thought   to be the most sensitive
                                                                        polyneuropathy], and 106 normal controls. Carriers of
index of muscle breakdown.4 Of the other enzymes in
                                                                        DMD were classified into three groups according to the
use for studying DMD (e.g., enolase, aldolase, pyruvate
                                                                        concentration of CK and the family history criteria of
kinase, lactate dehydrogenase), none are as sensitive or
                                                                        Thompson        et al. (6). Serum    samples were kept at -80 #{176}C
as specific.
                                                                        until used. Hemolyzed sera were not used for this assay.
   In 1980, Carter et al. (1) first reported an isoenzyme  of
                                                                           Purification       of CA-HI: CA-HI was purified from the
carbonic anhydrase HI (CA-ifi; carbonate dehydratase,
EC that was found in significant activities only               gastrocnemius          muscle    of amputated    human   limbs by
                                                                        the modified method of Nishita and Deutsch (7). All
in skeletal muscle and was markedly increased in sera
                                                                        purification procedures were carried out at 4#{176}C. Muscle
from patients with certain neurological          diseases, in
                                                                        was homogenized      in phosphate-buffered     saline (phos-
particular in patients with DMD (2). They also reported
                                                                        phate, 10 mmoIJL, pH 6.6, containing NaC1, 150 mmoll
that CA-Ill concentrations      might be useful for making
                                                                        L), 2 mL/g of muscle. After centrifuging the homogenate
prenataldiagnosesof DMD and diagnosesof adult
                                                                        at 10 000 x g for 30 mm, we applied the supernate to a
muscular        diseases.
                                                                        2.2 x 45 cm column of CM-Sephadex          C-50 (Pharmacia
   CA-Ill in human skeletal muscle is located mainly in
                                                                        Fine Chemicals,     Uppsala, Sweden) equilibrated       with
type I fibers (3), whereas CK (4) is found in type II fibers.
                                                                        phosphate-buffered    saline. Protein was eluted by use of
Therefore, the concentration     of CA-Ill in serum might
reflect a type I fiber abnormality   more sensitively than              a linear gradient of NaC1 from 0 to 100 mmol/L           in
                                                                        phosphate-buffered    saline. Fractions containing CA-ill
                                                                        activity were pooled and concentrated,    after which they
  ‘Clinical     Research Center, Utano National Hospital, 8-Ondoya-     were passed through a 2.2 x 116cm column of Sephadex
macho, Narutaki,       Ukyo-ku, Kyoto 616, Japan.                       G-75 (Pharmacia) equilibrated with 10 mmoIJL phos-
   2Faculty     of Pharmaceutical Sciences, Kyoto University, Sakyo-
                                                                        phate buffer, pH 6.6. CA-rn was then separated by
ku, Kyoto 606, Japan.
   3Department   of Pharmaceutics, Gifu Pharmaceutical Univer-          Anipholyte displacement chromatography (Pharmacia).
sity, Mitahora, Gifu 541, Japan.                                        The purified CA-Ill showed a single band on sodium
   4Nonstandard   abbreviations: CK, creatune kinase; CA, carbonic      dodecyl sulfate-gradient polyacrylamide gel electropho-
anhydrase; DMD, Duchenne muscular dystrophy; SPMA, spinal
progressive muscular atrophy; and ALS, amyotrophic lateral scle-
                                                                        resis. The concentration of CA-rn present was estimated
rosis.                                                                  by the method of Lowry et al. (8) with bovine serum
   Received May 11, 1990; accepted October 25, 1990.                    albumin as the standard.

36 CLINICALCHEMISTRY, ol.37, No. 1, 1991
   Enzymatic   determination of CA-HI: We measured the
activity of CA-UI according to the method of Wilbur and
                                                                                    50                         Male
Anderson     (9),using a saturated  CO2 solution and 0.02
molJL barbital    buffer. The enzyme activity was calcu-
lated by measuring       the time required  for the pH to                    .30
decrease from pH 8.0 to 6.3 (the end point).                                        10            .
                                                                                             #{149}   ;              #{149}
                                                                                                                       :.           #{149}
   Preparation of specific antiserum to CA-IH: Anti-CA-
HI antisera were raised in rabbits by six subcutaneous                        10                      10          20          30    40                50            60     70
injections of 0.5 mg of purified CA-Ill emulsified with an
                                                                              ‘so                              Female
equal volume of Freund’s         complete adjuvant, given at                        40
weekly intervals. Antisera        were collected 10 days after                      30
the last injection.                                                           2O                : #{149}j1
  Radioimmunoassay         procedure for CA-Ill:       CA-Ill was                   10
radiolabeled    by the Chloramine         T method      (10). The                                     10          20          30    40                50            60     70
specific activity of the 1251-labeled CA-Ill utilized in the                                           Age (yrs)
assay was about 20 x 1012 counts/mm per gram. We
                                                              Fig. 1. Concentration of serum carbonic anhydrase III in normal
measured the concentration of CA-rn with a double-            controls: sera from 53 males and 53 females contain 12.4 ± 15 and
antibody ifiA as follows: Dilute the test and standard        11.6 ± 15 zg/L (mean ± 3 SD), respectively
serum samples 20-fold with 10 mmoIJL phosphate The broken line indscatesthe uppernormalvalue(mean + 3 SD)
buffer, pH 7.0, containing     1 g of bovine serum albumin
per liter. Mix 50 jtL of 100-fold-diluted        nonimmune
rabbit serum and 100 L of 20 000-fold-diluted anti-CA-                          DUCHE?*  -     48L                     CONGENIAL lutiSt
                                                                  4000    #{149}                            4000
rn antiserum with 100 1L of the standard or diluted test       3000                                         3000
sample, then incubate at 4#{176}C      overnight. Add 1251..                     \#{149}\
                                                               2000                                         2000
labeled CA-rn and again incubate the mixture at 4#{176}C
overnight.    Add 100 1L of 100-fold-diluted goat anti-            1000              ::..            #{149} 000

rabbit y globulin, incubate for 2 h at 37 #{176}C, centri-            C
                                                                        0          1012            20    25 0#{149}
                                                                                                                  0       10            _________.___!...#{149}.
                                                                                                                                          20 25                             -#{149}---

fuge the sample tubes at 1500 x g for 10 mm. Wash the              1400                -------1400

pellet twice with isotonic saline, then count its radioac-                                                   1000
tivity with a gamma counter. In our hands the assay
was reproducible,     with within- and between-run CVs of                                                     500
5.1% and 7.5%, respectively.
                                                                                                                    #{149} #{149}
                                                                                                                        .__           #{149}
   CK assay: CK activity was spectrophotometrically                                10t2            20    25       0       10              20 25

assayed with an N-acetylcysteine-activated         kit (Boeh-                        Ag. (yvs                               Ag. (yts)

ringer, Mannheim, F.R.G.). The normal upper activity          Fig. 2. Relationshipbetween age and the serum CA-Ill or CK
                                                              concentrations in patients with Duchenne or congenital muscular
concentration    of this enzyme    was 80 UIL in males and    dystrophy
70 U/L in females (n = 53 each). CA-Ill and CK were           The arrow indicatesthe age of 12 years,a turning point forthe DMD patients.
determined    simultaneously    on the same serum samples.    The enzymedisappearancecurves are shown by a line of bestfit through the
   We developed an RIA method for detecting CA-ill (5),.                 group, being about 10-fold those for the control groups.
The optimal conditions for this assay, have been de-                     Thus the highest CA-rn values were present at the
scribed elsewhere (11,12). In our RIA, as little CA-rn as                early onset of the disease and tended to decrease with
100 pg/tube could be detected quantitatively.     Nonspe-                age. A less-obvious tendency was found for subjects with
cific binding was <2% of the total binding. There was no                 congenital muscular dystrophy, and a similar tendency
cross-reaction with any of the other known CA isoen-                     was observed between age and the concentration of CK.
zymes (CA-I and -U) derived from erythrocytes,     up to a               The positive correlation between the concentrations of
concentration of 100 zg/L.                                               CA-rn and CK in the 68 patients with DMD is shown in
   CA-Ill concentrations in sera from 106 healthy con-                   Figure 3.
trols are shown in Figure 1. There was no significant                       Serial determinations (over as long as two years) of
difference   fortheconcentration CA-rn by age and sex
                                  of                                     the CA-rn and CK concentrations   in individual patients
in these healthy controls. CA-Ill values >27 p.gfL (the                  with DMD are shown in Figure          4. In seven of 10
mean for the normal controls plus 3 SD, 12 + 15 .tgIL)                   patients,  the concentrations of both enzymes clearly
were considered positive for DMD. Values for the                         decreased   with age. In two cases there were sporadic
healthy controls were within the normal range, except                    variations, and only one showed a concomitant increase
for two who had borderline values (32 and 33 ,.ig/L). The                for a short period; in no instance was a marked clinical
relationships between age and activity of CA-ill or CK                   change observed during this period.
for patients with DMD or congenital    types of muscular                    The CA-rn and CK concentrations in sera from 10
dystrophy are shown in Figure 2. In DMD, the increases                   carriers of DMD and 63 patients with other neurological
in CA-ill were most pronounced in those in the younger                   disorders are shown in Figure 5. Carriers of DMD had

                                                                                                      CLINICAL CHEMISTRY, Vol. 37,                                 No. 1, 1991 37
            2000                                                                           the frequency of abnormal CA-rn was higher than that
                       r:0.76(,u68)                                           .
                                                                                      .    of CK. In contrast, patients with myasthenia  gravis,
            1000                                                          .       .        neuro-Behcet’s disease, or polyneuropathy had normal
                                                          S.                          Sf
             500                                                     ..               S    values.
       N                                                                          .
        a                                                                                  Discussion
                                                                                             Carbonic anhydrase in human tissue is present in
      U                                       ..%
                                                    :t”        :                           three isoforms(CA-I, II, ifi), with high concentrations of
                  50                                                                       CA-rn being located in skeletal muscle (3,13).
                                                                                            To assess the clinical value of CA-rn measurement,
                            .         1
                                                                                                 we developed sensitive           radioimmunoassaytoquantily
                                                                                                 CA-rn, using purified CA-rn from human skeletal mus-
                                                                                                 cle (5). Sera from patients with muscular dystrophy
                     “‘20                50100               500 1000                5000
                                                                                                 showed markedly           high concentrations        of CA-rn, the
                                                 CK (U/L)                                        increases being more marked in the younger age group
 Fig. 3. Correlationbetween the concentrations of CA-Ill and CK in                               of patients with DMD, as reported previously (2,14,15).
sera from patients with muscular dystrophy
                                                                                                 The observed enzyme activities             show well the destruc-
The broken lines Indicates uppernormal values as mean + 3 SD
                                                                                                 tion of muscle that results in loss of ambulation and the
                                                                                                 flattening     of the enzyme-disappearance          curvesatabout
                                                                                        4000     age 12 years, when little muscle is left (Figure 2).
                                               0.              S.’           \,                  Patients with DMD are generally confined to a wheel-
                                                                                                 chair by age 12 years, a turning point for DM1) patients.
                                                                                        1000     From the serial determinations                in individual DM1)
                                                                                             a   patients,     we observed    that CK activities increase early
              10111211314                           8 e
      2000 o--#{176}       #{149}‘.                                                     4000
                                                                                             g   in life, reach a maximum at age five or six years, decline
                                               It.                                      3c00
                                                                                                 rapidly    until-11-13 years, and then slowly decrease
                                                                                             o   from thereon.       This trend was also reported previously
                                                                                                 (16). The decreases in CA-rn with age were similar, as
                                                                                                 was the correlation between CA-rn concentrations                and
           0  -
               234               I i 9 II 12 13 12 13 ‘.4 11 12 13                               clinical severity.     Presumedly, these changes reflect ei-
                                              A91 lit.)                                          ther a decrease of the muscle mass of the patients with
Fig. 4. Serial change in serum CA-Ill and CX concentrations In 10                                age or a change in the disease activity. The concentra-
malepatientswith DMD                                                                             tions of CA-rn in serum correlated              well with the con-
                                                                                                 centrations of CK in muscular dystrophy.
                                      CA-U (g/L)                      CCC  lU/L)                    CA-rn and CK concentrations              in sera from patients
                          0       100    200    300  400   0     200   400      000    000       with other neurological        diseases and carriers of DMD do
     CwCsr of 0             H #{149}s#{149}#{149}#{149}#{149}                                    not
                                                                #{149}#{149}:#{149}#{149}#{149}#{149} depend on age (data not shown), unlike             the case of
       Poiyniyo.ftls        .$.t       P                             #{149}#{149}#{149}#{149}
                                                                #{149}#{149}                     patients     with DM1). However, we noted that abnormal
                                                                                                 concentrations       of CA-rn are frequent in carriers            of
   MYOICI4Cdy.Iro#{216}y 4t..                                    *
                                                                                                 DM1). In patients with SPMA and ALS, CA-rn was a
        A L $                            #{149}
                                                                                                 more sensitive       indicatorfdisease than CK was. These
        SPMA                  %f%                                                                results    probably reflect the type I fiber abnormality
         KWD                #{149}.                            #{149}
                                                          #{149}                                 more predominantly          observed in SPMA and AIS, con-
Fig. 5. ConcentratIons of serumCA-IllandCX in carriers of DMDand                                 sidering that the CA-rn is located mainly in type I
in patients with other neurological disorders                                                                 m
                                                                                                 skeletal uscle fibers,        whereas CK is found in type H
The broken fine indicates the upper normal value as mean + 3 SD. KWD,                            fibers. The enzymes have different molecular masses (28
Kugelberg-.Welander            disease                                                           kDa for CA-rn, 82 kDa for CK). The extent of the
                                                                                                 release of these enzymes may be affected by the perme-
increased concentrations                        of CA-rn (mean, 100 ug/L) and                    ability the muscle membrane.
                                                                                                           of                              Hence, the specificity of
CK (mean, 319 UIL). These carriers were classified as                                            CA-ifi for skeletal muscle is an advantage in diagnos-
definite (four cases), probable (one case), and possible                                         ing.
(five cases) (6),but we found no obviouscorrelation                                                 Measurement of CA-rn in serum appears to be about
                                                                                           as valuable as that of CK for the diagnosis of muscular
between     the concentrations of CA-rn or CK and the
                                                                                           dystrophy and other neuromuscular      diseases. The CA-
classification three groups. Among patients with
                                                                                           rn determination also is of particular value in neurolog-
other neurological        disorders, CA-rn was moderately
                                                                                           ical disorders, in which the CK is normal.
increased in patients with polymyositis,      myotonic dys-
trophy, ALS, SPMA, or Kugelberg-Welander           disease.
                                                                                             This study was supported in part by the Grant 3-A from
Nearly all the patients with increased CA-ifi also had                                     National Center of Neurology and Psychiatry of the Ministry of
concomitant     increases     in CK. But in SPMA and ALS,                                  Health and Welfare, Japan.

38   CLINICAL CHEMISTRY, Vol. 37, No. 1, 1991
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                                                                                      CLINICALCHEMISTRY, ol.37, No. 1, 1991 39

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