439
18 (1977)
Clinica Chimica Acta,
439-451 Biomedical Press
@ Elsevier/North-Holland
SCA 8692
CARRIER DETECTION IN X-LINKED RECESSIVE (DUCHENNE) MUSCULAR DYSTROPHY: PYRUVATE KINASE ISOENZYMES AND CREATINE PHOSPHOKINASE IN SERUM AND BLOOD CELLS
IAN SMITH
and W.H.S.
Research
Laboratory,
(Received
February
THOMSON
Knightswood 18th,
Hospital,
Glasgow
G13 2XG (U.K.)
1977)
Summary Allosterism allows individual assay of both isoenzymes, one abundant in muscle, of pyruvate kinase (PK), recently reported superior to serum creatine phosphokinase (CPK) in detecting patients with and female carriers of X-linked recessive (Duchenne) muscular dystrophy (DMD). Extensive comparative studies did not support these findings, and confirmed the marked superiority of CPK over variants of PK or other enzymes in sensitivity, stability and convenience. Deducting the adenylate kinase increment (AKI) further refined the CPK assay, eliminating the effect of haemolysis in diagnosis and enabling studies of blood cell content. Both leucocytes and erythrocytes liberated PK and lactate dehydrogenase (LDH) after brief chilling or disruption. Only erythrocytes showed a CPK content, however, constantly adjusted to match that of serum as if by free cell membrane passage, but less accomodating to a sudden large influx of CPK than of LDH, where an apparent buffering effect could account for differences in clinical response.
Introduction The unique abundance of creatine phosphokinase (CPK) in skeletal muscle makes its serum assay especially informative in X-linked recessive (Duchenne) muscular dystrophy (DMD), both for clinical diagnosis [3] and female carrier detection [4]; other muscle enzymes are confirmatory but far less emphatic. The 86% detection rate by CPK [4] seems to approach a theoretical maximum [5], as yet undetermined, so that still further improvement appears possible using the two forms of pyruvate kinase (PK), one found in muscle and many other tissues but the other only in liver and erythrocytes [6]. Different kinetic properties [6,7,8] allow simultaneous serum assay of considerable tissue specificity useful in muscle disease [ 91. [ 1,2]
440
In the presence of excess hexokinase and glucose, adenylate kinase (ATP : AMP phosphotransferase; EC 2.7.4.3; AK) from erythrocytes [lo] or muscle [l] is additive in a CPK assay measuring rate of phosphorylation of adenosine diphosphate (ADP) by creatine phosphate (CrP) [l]. Since suppression of serum AK by adenosine monophosphate (AMP) seems imperfect even in normal subjects [lo], this adenylate kinase increment (AKI) may be a source of diagnostic error in DMD carrier detection using standardised reagent kits. Subtraction of AK1 prevents this, and also allows studies of CPK movement across erythrocyte and other cell membranes. Methods Serum specimens were obtained as described below. None showed any haemolysis. Glassware was cleaned by immersion for a week in 30% HN03, rinsing with glass-distilled water, and drying at 125°C. Grade A pipettes were used throughout. Glucose was measured enzymatically in mg/lOO ml, and sodium and potassium by flame photometry in mequiv./l [ll]. Serum creatine phosphokinase (ATP : creatine phosphotransferase; EC 2.7.3.2; CPK), 1,6-diphosphofructoaldolase (EC 4.1.2.7; ALD), and lactate dehydrogenase (EC 1.1.1.27; LDH) were measured by semi-automatic optimal kinetic methods at 25°C and 340 nm (Unicam SP 800 B recording spectrophotometer) described elsewhere [ 121, and AK1 by substituting normal saline for CrP in the CPK assay. Values are expressed in International Units as mU/m1/25”C. Standardised reagents for glucose and enzymes were obtained as optimised kits (C.F. Boehringer and Soehne GmbH, Mannheim, G.F.R.), the latter corrected using normal saline for serum [13]. These CPK reagents (CPK-Bg; glutathione/lO mM AMP) showed measurable blank values, but those of J.T. Baker Chemicals B.V., Deventer, Holland (Diamed Diagnostics, Liverpool) for the same procedure (CPK-Bk; dithiothreitol/ll.5 mM AMP) showed none, and had the CrP solution separate, allowing AK1 to be measured. Pyruvate kinase (ATP : pyruvate phosphotransferase; EC 2.7.1.40; PK) was likewise measured at 25°C and 340 nm as the rate of conversion to lactate, by excess LDH and reduced nicotinamide-adenine dinucleotide (NADH), of pyruvate from 1.0 mM phosphoenolpyruvate (PEP) in the standardised reagent kit (Boehringer), self-corrected by reading for 10 min before adding ADP to start the PK reaction. Liver PK was inhibited [8] by adding to this reaction 2.0 mM alanine (in 0.01 ml) to give PK-Ala, and the muscle form (PK-M) measured in 0.2 mM PEP and both liver and muscle forms (PK-LM) in 2.0 mM PEP [6,7,8] by preparing different PEP solutions for otherwise unchanged assay conditions. These distinctions are possible since the muscle form is isosteric, in contrast to the allosterism cf the liver form expressed by alanine inhibition and especially substrate activation [ 91. Since this allosterism is lost on chilling but recovers rapidly at room temperature [8], all chilled or frozen sera were left at room temperature for at least two hours before assay. Procedures
Whole
blood
21-gauge
needle
specimens were obtained from an antecubital vein using a and disposable syringe, with moist alcohol skin toilet, minimal
441 venous stasis and gentle plunger traction to avoid haemolysis. Distribution before clotting into stoppered Pyrex tubes, graduated where necessary, was direct from the syringe or by a marked wide-bore Pasteur pipette. Centrifuging twice ensured cell-free serum, and was carried out forthwith unless otherwise stated. Treatment of blood specimens and serum included incubation for 24 h at 25 & O.l”C in a water bath, at 1.5 f O.l”C in a 25% ethylene glycol bath with a Grant CC15 cooling coil and 2.5 cm expanded polyurethane insulation, or successively at 1.5”C then 25°C to complete the 24-h period. Certain clotted or heparinised specimens were mechanically haemolysed by vigorous shaking for 60 set (Whirlimixer) immediately after 24 h at 1.5”C to increase erythrocyte fragility. Subjects were 48 healthy adults (24 males aged 18.14 to 61.92 years, and 24 females aged 17.41 to 65.56 years), bled in July and again in November, with many individuals common to both series. Patients included 11 boys with DMD and 2 with the similar but much milder X-linked Becker muscular dystrophy (BMD), 18 DMD and 2 BMD female carriers, 1 case of myocardial infarction and 4 cases of viral hepatitis A. Classically affected male relatives of DMD carriers are denoted by s, son; b, brother; mb, mother’s brother; ss, sister’s son; mss, mother’s sister’s son: and in BMD, where affected males may reproduce unlike DMD, additionally by f, father; mf, mother’s father. Blood cell enzyme content was determined in a healthy male subject aged 23.27 years. 56 ml blood were taken at mid-morning; 6 ml were divided in three 2-ml portions and set aside to clot, and the rest taken into ion-free heparin and divided in two 25-ml lots. Each lot was centrifuged for 15 min at 400 X g and the resulting cloudy supernatant again in an second tube for 5 min at 3200 X g to give cell-free plasma, gently returned through the packed cells using a wide-bore Pasteur pipette. Repeating four times gave a suspension of erythrocytes (RBCs) free from white cells (WBCs) in the first tube for one lot, or, by retaining the plasma in the second tube, of RBC-free WBCs for the other lot. Each suspension was divided in three portions for plasma assay forthwith, after chilling 24 h at 1.5”C, and on cell disruption after chilling. The results appear in Table XI. The three 2-ml portions of clotted blood were simultaneously treated likewise; and that evening the same subject undertook 2 h continuous severe physical exertion (press-ups, track-work etc.). He was then bled at the same time on the following five consecutive mornings, and each 10 ml specimen was divided for clotting in three portions, then treated as before. The results appear in Table XII. Statistical significance is denoted throughout by * (p < 0.05), ** Cp < O.Ol), and *** (p < 0.001). Results and discussion In DMD carriers, random X-chromosomal inactivation [ 141 before myoblast fusion [15] gives a multinuclear mosaic of binomial distribution in the mature
442
muscle fibre. The rare occurrence of severely crippled DMD carriers [ 51 suggests inactivation in a muscle adage of S-16 cells [ 161, and implies an equal rarity of phenotypically normal carriers undetectable by any means, so that complete detection may be approached even if never reached. Moreover, new observations suggest that male DMD mutants are also uncommon [17], whereby nearly all mothers of DMD boys must be carriers; and studies on the lethal X-linked recessive gene show that the proportion of new female mutants may be as high as one half [ 181. Such findings indicate a pressing need by genetic advisers for still better routine methods of carrier detection. Serum CPK alone, in optimal circumstances the simplest and most sensitive single test available, leaves 14% of carriers undetected [4], a figure improved only by a range of tests [ 191 or by complex studies of muscle ribosomal protein synthesis [20]; but such refinements are not generally available. Recently, however, serum PK has been studied in DMD, other myopathies, and myocardial infarction, and, even without recourse to its specific isoenzymes, is now reported markedly superior to CPK in diagnosing both DMD patients and carriers [9,21]. These findings merit careful examination with complete analyses throughout for tissue specificity. Normal values in healthy adults (Table I) show that in both sexes PK and PK-LM are almost identical, since a PEP concentration greater than 1 mM affects the activity of each isoenzyme only slightly [7]; and muscle PK-Ala and PK-M are likewise similar, as expected. CPK-Bk is far more sensitive than CPK-Bg, the significantly higher female standard deviation in July giving a higher upper normal limit then, with a similar (but not significant) finding in IABLE NORMAL
Sex
Male
1 VALUES Mean
Assay
Recorded range
Mean
i
2 S.D.
PK
17.55
4.09
9.37-25.19
9.36-25.73
PK-LM
18.62
4.48
10.24-27.80
9.66-27.58
PK-Ala
11.69
2.81
6.38-18.29
6.06-17.31
PK-M
10.43
2.76
4.60-15.68
4.91-15.95
33.19
5.68
22.83-43.49
21.83-44.55
% alanine Ratio
inhibition
PK-M/PK-LM
CPK-Bg
Female
Standard deviation
(July)
0.56 35.97
0.07 14.01
0.44-
0.69
17.78--69.68
CPK-Bk
(July)
44.54
16.31
21.46-81.40
CPK-Bk
(Nov.)
47.46
22.02
20.23-96.45
0.44
0.46
47.02
22.00
AK1
(Nov.)
Nett
CPK-Bk
(Nov.)
0
-
0.43-
0.69
7.95-63.99 11.92-77.16 3.42-91.51 1.73
0
-
18.50-96.20
3.01-91.04
PK
15.53
3.19
9.41-19.44
9.16-21.90
PK-LM
16.42
3.42
9.72-24.14
9.58-23.26
PK-Ala
9.45
1.99
5.43-13.48
5.48-13.43
PK-M
8.64
1.85
4.81-13.59
4.94-12.34
38.72
8.03
25.00-54.27
0.53
0.08
21.09
10.72
% analine Ratio
inhibition
PK-M/PK-LM
0.34-
22.65-54.79 0.66
1.17-45.48
0.360
0.69 -42.52
CPK-Bg
(July)
CPK-Bk
(July)
27.37
11.90
3.70-53.28
3.57-51.17
CPK-Bk
(Nov.)
24.06
6.27
14.55-34.29
11.52-36.60
0.53
0.56
23.53
6.38
AK1
(Nov.)
Nett
CPK-Bk
(Nov.)
0
-
14.31-34.04
1.36
1.48
0
-
10.77-36.29
1.66
443
males in November. Such markedly seasonal sex differences are difficult to explain. Normal AK1 is small in both sexes. Significant sex differences (Table II) occur only where there is a muscular origin (PK-Ala, PK-M, % alanine inhibition), with very large increases in male CPK, particularly in November, attributable to lack of oestrogens [ll]. Likewise, PK and PK-LM correlate very highly in both sexes (Table III) but PK-Ala and PK-M less well in females than in males; and the modest correlations between CPK and all modalities of PK in males are quite absent in females except of low significance only with PK-Ala and PK-M. No relation between age and serum enzyme values was found in either sex. In July, however, the 24 females were bled on successive days as two groups of 12, in one of which CPK showed a highly significant increase with time of day (Table IV), just losing significance in all 24; and though seasonal changes may have masked similar relationships in November, these observations tend to confirm late afternoon as the most informative time for carrier venepuncture [12]. No such relationships were found in males, other than a slight decline of AK1 with time of day in both sexes. In stored serum, all modalities of PK decay very rapidly at 25°C (Table V), surviving with slight loss at -21.5”C, while CPK is comparatively stable for 24 h at 25°C and for as long as 4 weeks at -21.5”C. Serum may thus be mailed overnight for CPK assay, and even stored thereafter; but not for PK. Ideally, enzyme assay is best in serum separated forthwith (Tables I and VI). In clotted blood spun later, however (Table VII), CPK survives 24 h at 25°C (likewise overnight mailing) and may even increase-slightly (b, c, d), especially after chilling 1 h, as if blood cells contained more CPK than serum. Frank efflux of LDH occurs after 24 h at 25”C, and progressively on chilling (a, 6) as described elsewhere [ 111, but rather less in DMD carrier d and only after prolonged chilling in DMD patient c, where lower values after 24 h at 25”C, with or without 1 h chilling, suggest actual movement into blood cells from an elevated serum value. All modalities of PK, however, decline markedly in a and b after 24 h at 25°C but show gross increases on progressive chilling, derived from blood cells as indicated by 9%alanine inhibition and ratio, while the same changes are much less marked in the already elevated values for c and d, like the behaviour of LDH and perhaps for similar reasons. Of 11 known DMD carriers examined (Table VIII) 2 were obligate (Nos. 5, 11) but hitherto always undetectable in accord with the natural distribution of
TABLE
II
% DIFFERENCES
OF
MALE
FROM
FEMALE
PK
+12.40
PK-LM
+13.39
PK-Ala
+23.63
**
PK-M
+20.65
*
% alanine Ratio
inhibition
PK-M/PK-LM
-14.28 +
**
5.66
CPK-Bg
(July)
+70.56
***
CPK-Bk
(July)
+62.75
***
CPK-Bk
(Nov.)
+97.33
***
AK1
(Nov.)
Nett
CPK-Bk
-22.22 (Nov.)
+99.79
***
MEANS
444
TABLE
III
CORRELATION
MATRICES
(22
df)
PK
PK-LM
PK-Ala
PK-M
-
CPK-Bg
CPK-Bk
(July)
(July)
Males PK
1.000
PK-LM
0.970***
1.000
PK-Ala
0.926***
0.900***
1.000
PK-M
0.901***
0.894***
0.919***
1.000
CPK-Bg
(July)
0.520**
0.504**
0.571**
0.627***
1.000
CPK-Bk
(July)
0.583**
0.583**
0.612***
o.fs44***
0.972***
1.000
Females PK PK-LM
1.000 0.945***
1.000
PK-Ala
0.799***
0.739+++
1.000
PK-M
0.820***
0.789***
0.799***
1.000
CPK-Bg
(July)
0.213
0.214
0.376*
0.377*
1.000
CPK-Bk
(July)
0.224
0.219
0.352*
0.392*
0.986***
TABLE
1.000
IV
REGRESSIONS
OF
SERUM
ENZYME
VALUES
(E’)
ON
TIME
OF
DAY
(T mm
after
9 a.m.)
Females % alanine CPK-Bg
inhibition (July)
CPK-Bk
(July)
E = 49.560-3.8809
X lo-*
T
(n = 12:
f = 2.9519;
10
df;
*)
E = 0.0042
+ 2.8729
X 10-4
T
(n = 12;
f = 4.9637;
10
df;
***)
h’ = 0.0465
+ 1.0449
X 1O-4
T
(n = 24:
t = 2.0479;
22
df)
0.0268
+ 3.5146
X 10-4
T
(n = 12;
t = 4.3056;
10 df:
X 1O-4
‘I
(n = 24:
t = 1.8999;
22 df)
0.1004-1.100
X 10-S
T
(n = 24;
t=
0.3015;
22
df)
0.0963
-3.61
X 10-h
T
(n = 24;
t = 0.0971;
22
df)
0.0041
-7.39
X 10-h
T
(n = 24;
t = 2.5722;
22
df:
*)
-
X 10-6
?’
(n = 24;
t = 2.3514;
22
df;
*)
Nett
CPK-Bk
AK1
(Nov.)
AK1
(Nov.)
E = 0.0034
CPK-Bk
(Nov.) (Nov.)
= = = = =
0.0792 + 1.2859
E E E E E
**)
M&S
TABLE
V
ENZYME Sex
STABILITIES
Age
IN
STORED
Assay
(years)
M
20.06
23.15
F
27.04
Forth
24
-with
at 25°C
h
24 h
1 week
at-21.5
at-21.5
4 weeks at -21.5
OC
“C
“C
17.0
13.2
16.5
14.5
13.8
20.8
13.3
17.5
16.4
15.5
PK-Ala
12.3
10.5
11.5
10.3
9.5
PK-M
10.7
8.2
10.1
8.2
inhibition
PK-M/PK-LM
27.6 0.51
20.6 0.61
30.1 0.58
28.8 0.50
9.7 31.1 0.63
CPK-Bg
23.6
22.2
22.2
22.0
22.7
CPK-Bk
30.3
28.2
30.0
29.5
29.5
PK
14.1
10.3
13.4
12.3
11.0
PK-LM
16.8
11.0
15.9
13.7
12.9
PK-Ala
10.5
8.2
9.6
9.3
6.7
8.8
6.9
9.1
7.7
6.6
25.6
21.2
28.1
24.6
39.1
PK-M % alanine Ratio 40.28
SERA
PK-LM
Ratio M
NORMAL
PK
% alanine
F
6.07
inhibition
PK-M/PK-LM
0.52
0.62
0.57
0.56
0.51
CPK-BB
28.1
27.8
27.0
25.7
24.8
CPK-Bk
36.8
33.3
33.5
34.3
32.7
445
TABLE
VI
NORMAL
SERUM Normal
Assay
RANGES males
1.07-
ALD
[ll] Normal
2.34
0.81-
95.17-166.13
LDH
3.55-
2.27
80.16-178.26 135.20-144.44
135.10-143.33
Na+ K+
females
3.71-
4.61
4.72
carrier manifestation [5] ; and though carrier 11 was doubtfully detected by a single barely elevated finding (ratio M/LM) all other readings in both were normal. CPK decisively detected all 9 remaining DMD carriers (3- to 20-fold elevations), LDH only 3 clearly (less than 2-fold elevations) and 4 doubtfully, while PK detected 5 clearly (about 2- to 6-fold elevations) and 4 very doubtfully, with PK-LM, PK-M and PK-Ala giving similar results in the same individuals and % alanine inhibition detecting 5 clearly but ratio M/LM only 3 marginally. Storage of clotted blood for 24 h at 25°C had little effect on the TABLE
VII
EFFECTS
OF
CHILLING
CLOTTED
BLOOD
FROM
HEALTHY
SUBJECTS,
DMD
CARRIERS
AND
PATIENTS No.
Sex
Age
DMD
Assay
(years) 22.84
Nil
16.0
31.0
17.9
38.1
74.0
PK-Ala
11.0
8.7
10.2
16.4
20.1
7.6
10.5 inhibition
33.5
PK-M/PK-LM
30.8
0.61
58.7
9.0
14.9
18.6
35.9
47.1
65.8
0.50
0.57
CPK-I3g
23.3
23.6
22.7
0.25
0.39 22.7
22.7
LDH
91.2
117.5
143.3
PK
13.2
10.9
13.8
16.8
32.9
PK-LM
13.1
10.1
14.0
21.2
43.0
PK-Ala
8.4
7.8
8.9
10.2
15.0
PK-M
5.3
6.3
7.5
8.8
12.2
inhibition
36.5
27.9
0.41
PK-M/PK-LM
12.5
35.6
0.62 15.5
231.4
39.1
0.54
255.7
54.3 0.28
0.41
16.9
15.5
15.5 202.1
106.9
127.1
165.1
PK
271
257
263
282
299
PK-LM
265
278
271
279
316
PK-Ala
246
225
232
234
240
PK-M
191
205
199
200
197
83.1
LDH
% alanine Ratio
inhibition
9.1
PK-M/PK-LM
CPK-Bg LDH 3s
-
13.8
CPK-Bg
2b;
at 1.5’~
25OC
12.5
Ratio
42.54
24 h
18h
17.0
% alanine
DMD
6 h1.5OC.
h 25°C
PK-LM
Ratio
12.56
1 h 1.5”C, 23
16.5
% alanine
Nil
24 h at 25Oc
PK
PK-M
27.26
Forth -with
12.4
0.72
11.7
17.1
0.73
0.74
19.6 0.62
0.72
1673
1759
1842
1768
1803
704
651
661
729
831
PK
23.9
18.3
22.3
23.7
28.7
PK-LM
23.3
19.0
23.9
23.9
34.1
PK-Ala
17.3
13.9
16.5
14.8
17.4
PK-M
15.4
12.2
15.0
14.6
16.2
27.5
24.0
25.8
37.5
39.4
% maline Ratio
inhibition
PK-M/PK-LM
0.66
0.64
0.63
0.47
0.61
CPK-Bg
141
143
152
146
145
LDH
165
194
210
223
224
446 TABLE
VIII
CARRIERS AND PATIENTS: AND (IN BRACKETS) AFTER Affected
male relatives: s, son: b, brother;
NO.
Age (years)
Male rrls.
1
16.58
lb
2
16.90
2b
3
21.42
lb
4
28.59
2s
5
36.98
2b:ls
6
39.77
2%
7
42.31
2b;3s
8
46.24
Is
9
52.44
2s
10
53.87
IS
11
55.13
lb: 2s
Correlations
(9 df):
PK
31.0 (31.2) 133 (130) 98.4 (88.4) 72.7 (73.7) 11.0 (10.7) 27.7 (27.1) 22.2 (21.5) 26.6 (21.1) 38.5 (34.7) 23.2 (21.5) 13.7 (13.6) PK/CPK
DMD male patients (A = ambulant: 12 6.83 Nil (A) 695 13
10.22
Nil (W)
14
12.56
Pmb(A)
15
14.53
Nil(W)
Correlations Becker 16
ENZYMES 24 HOURS
(663) 159 (185) 271 (257) 137
(136) (2 df): PK/CPK
MD carrier female 18.23 2b
Necku MD male patient 17 10.05 lb (A)
IN SERA AT 25°C
CLOTTED
mb, mother’s brother;
PK-LM
35.0 (28.6) 135
FROM
PK-Ala
25.9
(26.5) 118
(138) (117) 104 84.0 (105) (84.0) 71.5 60.8 (59.3) (70.9) 11.9 6.5 (12.4) (6.9) 21.6 27.4 (22.2) (26.4) 24.0 18.5 (24.2) (17.3) 18.4 27.7 (15.3) (20.9) 30.2 41.8 (29.3) (38.1) 25.2 15.6 (23.2) (15.6) 10.1 14.4 (11.1) (13.8) = 0.8312 **; PK/LDH
PK-M
22.1 (22.7) 106 (107) 75.9 (70.9) 52.7 (52.7) 6.0 (6.5) 12.5 (18.9) 16.9 (15.3) 17.0 (13.4) 27.4 (26.4) 15.9 (15.3) 10.1 (9.8) = +.4123;
wheclchoir) 688 623 (686) (601) 15.5 144
542 (532) 140
(192) 265
(154) 191
BLOOD
SPUN
FORTHWITH
ss. sister’s son. % Ala in- Ratio hibition MiLM
16.5 (15.1) 11.3 (10.6) 14.6 (5.0) 16.4 (19.6) 41.0 (35.3) 21.9 (18.1) 16.5 (19.4) 31.0 (27.7) 21.5 (15.7) 32.9 (27.7) 26.0 (18.5) CPK/LDH
CPK-Bg
0.63 127 (0.79) (133) 0.79 473 (0.77) (472) 0.73 852 (0.68) (745) 0.74 515 (0.74) (575) 11.7 0.50 (0.52) (11.4) 0.46 177 (0.72) (174) 0.70 155 (0.63) (148) 0.62 100 (0.64) (94.2) 0.66 280 (0.69) (278) 0.62 205 (0.66) (201) 0.70 26.5 (0.71) (29.4) = -*.7856*’
LDH
133 (145) 186 (191) 351 (284) 258 (280) 170 (150) 226 (319) 178 (169) 192 (159) 233 (237) 269 (270) 168 (170)
IV
(278) 138
(180) 246 (225) 120
(137) (123) = 0.9536 *; PK/LDH
(205) 99.3 (119) = 0.0172;
25.1 (21.1)
27.5 (23.5)
19.1 (16.9)
17.8 (15.2)
598 (588)
621 (608)
543 (528)
(472)
459
10.4 (1.3) 9.2 (2.8) 9.1 (12.4) 12.2 (9.2) CPK/LDH
23.7 (19.8)
9.1 (10.1)
0.79 (0.78) 0.91 (0.80) 0.72 (0.74) 0.72 (0.87) = 0.2147
3504 (3528) 1673 (1668) 1673 (1759) 1875 (1694)
0.65 (0.64)
70.8 (69.4)
0.74 (0.78)
3691 (3612)
613 (1337) 805 (1529) 704 (651) 441 (380)
196 (183)
1003 (614)
marked CPK elevations, but 3 marginal PK elevations became normal, while LDH behaved irregularly. The muscle-specific CPK thus emerges as far more sensitive, stable and dependable than LDH or any PK modality, although, unlike normal individuals (Table III), DMD carriers do show high correlation between CPK and PK, while LDH correlates with neither. In DMD male patients all values were grossly abnormal throughout, again with high correlation between CPK and PK but of LDH with neither. The slowly progressing BMD
447
patient 17 had values like the youngest DMD patient 12, and, as expected, CPK detected BMD carrier 16 more confidently than any other reading. The true CPK value (nett CPK-Bk) is obtained by deducting AKI. In a second series of 9 known DMD carriers (Table IX), true CPK detected 8 decisively (almost 2-fold to 50-fold elevations) and 1 barely, but ALD only 3 clearly (3to 13-fold elevations) and 2 marginally, while AK1 was slightly elevated in 4 instances. Further, CPK and ALD correlated very highly indeed, but AK1 with neither, so that a high AK1 occurring with a marginal CPK elevation could give a false carrier diagnosis unless first subtracted. True CPK and ALD were grossly elevated in DMD patients, but AK1 rather less so; CPK and ALD correlated
TABLE CPK,
IX AK1
8
ALD
IN
DMD
CARRIERS
AND
PATIENTS,
HEPATITIS-A
& MYOCARDIAL
INFARC-
TION Affected
male
mother’s
father.
NO.
relatives:
s, son;
b,
Affected
A@
brother;
mb.
mother’s
relatives
carrier
mss,
Nett
mother’s
sister’s
AK1
ALD
females
1
8.60
lb
2538
2.5
28.9
2
9.92
2b
1254
1.7
14.4
3
28.12
lb:
4
29.18
1s
118
0.74
1.5
2s
411
2.5
4.0
261
5
34.28
IS
6
38.99
IS
7
46.79
2b;
1s
8
53.16
lb;
2s
9
54.84
lmss;
Correlations DMD
male
(7 df):
patients
2.6
2.5
52.5
1.2
1.0
86.6
1.5
1.9
0.25
1.7
109 2mb;
CPK/AKI
(A = ambulant;
2s = 0.5122:
197
0.74
CPK/ALD
= 0.9875***;
6.6 ALD/AKI
= 0.4378
lli = wheelchair)
10
3.12
lb
(A)
1764
11
3.24
Nil
(A)
6857
24.9
57.7
12
3.82
Nil
(A)
4921
4.2
66.7
2.7
32.4
13
6.41
4771
4.7
68.9
14
10.12
Nil
(W)
2058
4.2
18.5
15
11.06
Nil
(W)
2081
3.2
14.7
16
12.38
Nil (W)
961
3.9
7.9
17
14.03
Nil
2162
1.5
15.0
lmb
Correlations Becker
MD
18 Becker
(6 df):
carrier
MD
malf
(A)
(W)
CPK/AKI
= 0.7573*;
CPK/ALD
= 0.8733**;
ALD/AKI
= 0.4197
female
33.23
19
1s: f
34.8
0.74
2.1
patient
10.65
mf
high
2.2
33.4
patients
20
8.44
1.7
4.2
28.3
21
16.33
7.6
2.0
29.7
22
18.01
-
7.4
3.2
19.2
23
27.39
-
4.9
3.0
26.6
24
(wry
2129
(A)
Hepatitis-A
Myocardial
son:
CPK-Bk
(years) DiUD
brother:
aminotransferase)
infarction 46.24
5 h after
onset
24 h after
onset
76.7 759
1.0
1.6
3.0
5.7
f, father;
mf,
448
highly, and AK1 only with CPK. Values in the BMD patient 19 were similar, but even CPK failed to detect his carrier mother 18. The myocardial infarction patient 24 had unremarkable AK1 values; the others were expected. Though all 4 hepatitis A patients 20-23 had very high ALD but modest AK1 elevations, true CPK was extremely low, but extensive dialysis and dilution studies gave no evidence of a dissociable inhibitor to CPK. These very low CPK values seem typical of this condition [3], and may be due to a firmly bound inhibitor. Deducting AK1 greatly clarifies the behaviour of CPK in serum and whole blood, and makes possible the assay of erythrocyte CPK content after mechanical haemolysis, since dithiothreitol (Cleland’s reagent) is used for CPK activation instead of glutathione vulnerable to erythrocyte glutathione reductase. Table X shows that brief chilling or storage at 25°C had little effect on fresh serum. Any changes in aliquots of the original clotted blood given the same treatment simultaneously must therefore arise from blood cells. Thus, compared with serum treated similarly, 24 h at 25°C caused little change except slight rises in LDH and AKI, with a fall in glucose consumed [22,23] to fuel the erythrocyte (NaK)-ATPase retaining K’ and expelling Na’ [24]. 24 h at 1.5”C, however, gave marked increases in AK1 and especially LDH [ 111, with sparing of glucose as the temperature-dependent [25] (NaK)-ATPase failed
TABLE
X
BEHAVIOUR AND NO.
OF
SERUM
AND
CLOTTED
BLOOD
FROM
HEALTHY
SUBJECTS,
DMD
CARRIERS
PATIENTS Sex
Age
DMD
Assay
Serum
Clotted
blood
(years) Sepd.
Kept
Kept
24 h
24 h
24 h
immed.
24 h
24 h
25OC,
1.5Oc,
1.5°c,
25’C
1.5Oc
sepd.
sepd.
shaken, sepd.
1
M
20.72
Nil
AK1 Nett
0.49 CPK-Bk
LDH
112
GIUCOSe Na+ 2
F
42.14
Nil
140
Kf
4.3 0.99 CPK-Bk
M
11.06
DMD
4.2 Nil
172
36.4 144
4.3
75.6 136
4.4
9.5
1.5
1.7
Nil
LDH
66.8
67.9
61.8
84.1
90.6
Glucose
76.7
75.3
74.9
28.9
71.2
142
142
142
144
11.0 34.9 340 72.4 135
27.1
136
10.5 4.9 28.5 189 65.0 133
K+
4.4
4.4
4.3
4.7
10.4
13.0
AK1
3.2
2.6
3.0
3.6
8.4
34.3
CPK-Bk
Na+ 1s
141
117
27.5
GlUCOW
34.28
141
86.8
3.9 32.6
28.5
LDH
F
115
84.7
3.0 32.8
26.5
Nett
4
110
34.0
27.6
Naf 3
0.49
32.7
89.5
AK1 Nett
0.74
33.9
2081
2036
2051
1959
2229
2159
851
841
846
851
1074
1550
85.0 140
81.1 139
84.2 139
25.4 141
65.0 135
K+
3.8
3.8
3.8
4.2
9.1
AK1
2.6
0.99
3.2
2.8
9.1
Nett
CPK-Bk
LDH Glucose Naf K+
57.9 132 10.6 25.3
261
254
264
249
263
255
150
215
190
218
392
697
94.8 142 4.3
89.8 139 4.2
82.0 140 4.3
16.9 146
76.0 134
4.5
11.9
66.9 129 14.4
449
so that serum K’ rose as Na’ fell. CPK showed little change except for a 10% rise in DMD patient 3. Mechanical haemolysis after 24 h at 1.5” C then added intact erythrocyte contents to serum, with the expected further rise of AKI, LDH and K’ and a corresponding fall in glucose and Na’ but not in CPK, as if erythrocytes contained about as much CPK as serum, though for no discernable purpose. Chilling, especially with haemolysis, seemed to produce a much greater AK1 in DMD patient 3 and carrier 4 than in normal individuals 1 and 2. Differential centrifugation of heparinised blood enables some formal assignation of these changes to erythrocytes (RBCs) or leucocytes (WBCs). In Table XI the relatively unchanged Na’ and K’ after chilling and shaking the WBC suspension indicates disruption of only a small volume of cells, with a moderate rise in LDH and a slight fall in true CPK, as if absent from WBCs, but a large rise in PK (chiefly the liver form) suggesting a very high WBC content. After the same procedures on the RBC suspension, however, large changes in Nd and K’ and very marked rises in LDH, AK1 and PK (again mostly the liver form) indicate considerable cell disruption, with expectations that true CPK should fall as much as Na’. Instead, a small increase in CPK after haemolysis demonstrates a higher content in erythrocytes than serum. Thus in whole blood any changes in serum CPK, electrolytes, and most of those in LDH seem to arise from erythrocytes, not leucocytes; while both can affect serum PK values. Determination of the origins of these changes permits in vivo examination of enzyme movements in whole blood by increasing the serum content after severe exercise. In Table XII, serum separated forthwith (procedure a) from blood taken on successive days showed little change in electrolytes, glucose or AKI, a small post-exertion rise in LDH, and a much larger immediate one in
TABLE
XI
BLOOD
CELL
Assay
CONTENTS RBC
OF
HEALTHY
MALE
(23.27
suspension
years)
WBC
suspension
Plasma
24 h 1.5OC.
24 h 1.5’C.
PlaSma
24 h 1.5OC,
24 h 1.5’C.
separated
plasma
shaken,
separated
plasma
shaken,
forthwith
separated
separated
forthwith
separated
separated
PK
14.4
25.8
PK-LM
13.8
33.0
PK-Ala
9.9
PK-M
8.2
8.9
93.5 123
12.7 14.7
12.1
_
69.7 104
18.3
9.1
31.9
10.0
24.0
8.9
25.4
65.6
80.5
28.7
54.3
% alanine inhibition Ratio
31.2
PK-M/
PK-LM CPK-Bk AK1 Nett
Glucose K+
26.1
1.7 CPK-Bk
LDH Na+
0.30
0.59 28.9 27.2 107 70.3 138 4.3
1.0 25.1 252
0.20 176 146 30.8 1808
57.3 135
67.4 125
6.3
17.7
0.60 28.9
26.7
0.74 28.2 110 76.7 140 4.3
0.24 26.9
0.41 26.3 122
1.1 25.8 352
74.9 140
74.1 142
4.6
4.9
450
TABLE
XII
EFFECT
OF
Procedures: Clotted
blood
24-h
periods
after
exercise
0
1
EXERCISE (a)
Serum
Procedure
5
forthwith;
24 h 1.5”C, AK1
BLOOD (b)
vigorously
CELL
ENZYMES
Clotted
blood
incubated
shaken,
serum
separated.
Nett
IN HEALTHY 24 h 1.5”C.
LDH
Glucose
MALE serum
NaC
(23.27
yrs)
separated;
(c)
K+
a
0.62
28.0
112
83.1
143
b
1.1
28.6
222
81.1
137
8.9
29.5
455
70.5
134
11.7 3.9
r
14.8
a
Nil 1.6 36.8
4.3
80.3
125
88.9
145
95.5
517
80.2
138
88.7
886
74.0
133
12.2 4.8
X.8
a
0.37
76.3
131
86.6
142
b
2.6
70.9
238
79.7
139
8.6
c
11.8
72.6
452
70.0
135
11.4 4.4
a
0.25
40.1
117
89.5
142
b
0.74
43.5
263
82.4
138
8.8
40.6
518
77.9
134
11.6 4.5
c 4
AND
CPK-Bk
c
3
SERUM
separated
incubated
b 2
ON
28.7
a
2.1
33.5
109
93.7
141
b
6.0
31.9
227
86.1
137
8.5
c
19.7
34.7
462
79.0
134
11.6 4.4
a
1.9
27.4
106
90.7
140
b
2.7
27.9
185
90.0
136
c
15.9
26.9
392
73.5
132
8.0 11.4
true CPK which returned smoothly to previous values by the 5th day. Simultaneous chilling of clotted blood aliquots for 24 h at 1.5”C without (procedure b) or with (procedure c) mechanical haemolysis disclosed the underlying enzyme movements. Both procedures caused very similar changes in Na+, K’ and glucose on successive days, indicating the same degree of efflux or of cell disruption. The changes in AKI were similar if less precise. The sudden two-fold increase of erythrocyte LDH on day 1, returning to normal by day 2, suggests rapid incorporation then re!ease of effluent muscle LDH (mol. wt. 135 000) in effect buffering the rise in serum values. Except for day 1, serum CPK throughout showed similar values in all 3 procedures, indicating an erythrocyte content identical to serum and arguing against simple membrane attachment, with continual equilibration by rapid movement of CPK (mol. wt. 81 000) across the cell membrane. On day 1, however, b showed marked CPK efflux from erythrocytes to serum, and simultaneously c that these erythrocytes, now disrupted, contained less CPK than the b serum into which the CPK was being discharged (though still more then the a serum). This implies active expulsion of CPK from erythrocytes tolerant only of equilibration, and also suggests that serum CPK values had been much higher earlier, which accords well with reports that the serum CPK maximum is reached some 11 h after physical exertion [26,27]. Thus an explanation is found for the prompt emphatic response of serum CPK compared with slower and lesser changes of LDH in the same circumstances. In carrier detection, therefore, serum CPK assayed in optimal circumstances [3,4,11] is far more sensitive and dependable than any variant of the much less stable and more inconvenient PK; and this is enhanced by its stability at room temperature in serum and in clotted blood, allowing overnight mailing. Further, determining true CPK after deducting AK1 refines carrier detection as well as
451
obviating false elevation due to even slight haemolysis, so irremediable in PK, ALD and LDH. The apparent equivalence of CPK in serum and erythrocytes likewise discounts the effect of efflux even without visible haemolysis, a constant but often unsuspected risk in these other enzymes present in blood cells in large amounts. Acknowledgements The authors wish to thank all who took part in this investigation, and Dr. R.W. Logan in whose department the serum electrolytes were measured. This study was supported by the Andrew Patrick Trust and the Muscular Dystrophy Group of Great Britain. References 1
Oliver.
I.T.
2
Smith,
A.F.
3
Thomson.
W.H.S.
4
Thomson,
W.H.S.
5
Thomson,
W.H.S..
6
Tanaka,
T..
Harano,
7
Tanaka,
T..
Sue,
8
Llorente.
9
Harano.
10
(1955)
G.,
11
Sweetin,
12
Thomson,
13
Smith.
14
Lyon,
15
Emery.
16
Graham,
17
Roses,
Clin. (1971)
Clin.
Chim.
Acta
35,
(1969)
Clin.
Chim.
Acta
26,
Sweetin, Y..
R..
and
I. and M.F.
A.E.H.
(1965)
J.B.,
W.
J. Med.
Barrow, Roses.
E.S. M.J..
Res.
13,
KowaI. Acta
63,
383-394
7141
Commun.
29,
444-449
45-54
J. (1973)
Clin.
Chim.
Acta 62.
Metabolism
Chem. 48,
22,
22.
493-501
1806-1811
49-3
21,469-478 i, 1038
14.135-148 2, l-7
and Elston. Miller,
Chin
Biophys.
E. (1976)
Lancet
Genet. Genet.
M. and
Clin.
Acta
Clin.
J. Biochem.
J. Biochem.
Bemt,
(1975)
J. Hum.
Eur.
(1973)
Chim.
(1975)
H. (1967)
Miller.
and
W.H.S.
W.H.S.
Am.
207-221
Biochem.
(1970)
Jr, P.J..
Clin.
Thomson.
183-191 T.E.
H. (1967) A.
Gruber,
Thomson,
(1962)
A.D.,
Vignos,
(1968)
351-359
F. and Morimura,
and Sols,
W..
39,
and Hilditch,
Morimura,
R.
Adair,
J.C.
Sue.
F. and
W.H.S.
116-122
Acta
Gerhardt, J.C.
J. 61.
Chim.
P., Marco, Y.,
Szasz.
Biochem.
(1972)
R.C.
S.E..
Hull.
Gartler,
S.M.,
(1975) K.L.
Ann. and
N.Y.
Appel,
Acad.
Sci.
240.
141-146
S.H.
(1976)
New
Eng.
Dancis,
J..
SeegmiIler,
(1968)
J. Neural.
J. Med.
294,
193-
198 18
Francke,
U.,
Nyhan,
Felsenstein,
W.L.
19
Radu.
20
Ionasescu,
21
Albert%
22
Danowski,
23
Eckel.
R.E.,
Rizzo,
24
Glynn.
T.M.
(1968)
L. and
25
Wood,
26
Nuttall,
27
King.
H..
(1976)
Migea, V.,
and
T.S.
S.W.,
S.,
Samaha.
F.J.
(1974) H. and
Bull.
E. (1967) B.E.
and
24,
J. Lab.
B. (1968)
A.
T.W. 24,
(1973)
Neurology
J.E.,
Bakay,
Sci. 23,
6, 289-300
497-502
462-464
693-705
Berggren,
A.B.
(1966)
Am.
J. Physiol.
165-169 Clin.
J. Lab.
Savory.
Radu.
Conway,
Neurology 139,
B.R..
123-137
L. and
P. and
Chem.
M&on,
28.
Bordeianu,
Lodish,
Br. Med.
and Jones.
Genet.
Shirk,
J. Biol.
S.C..
Beutler,
Z.,
H.,
(1941)
Statland,
J. Hum.
TGrak,
Zellweger,
M.C.
F.Q.
J.,
Am.
Med.
Clin.
J. (1976)
70,
287-294
Med.
71.
Clin.
Chin
847-854 Acta
72,
211-218
210.
737-743
F.
and
18 (1977)
Clinica Chimica Acta,
439-451 Biomedical Press
@ Elsevier/North-Holland
SCA 8692
CARRIER DETECTION IN X-LINKED RECESSIVE (DUCHENNE) MUSCULAR DYSTROPHY: PYRUVATE KINASE ISOENZYMES AND CREATINE PHOSPHOKINASE IN SERUM AND BLOOD CELLS
IAN SMITH
and W.H.S.
Research
Laboratory,
(Received
February
THOMSON
Knightswood 18th,
Hospital,
Glasgow
G13 2XG (U.K.)
1977)
Summary Allosterism allows individual assay of both isoenzymes, one abundant in muscle, of pyruvate kinase (PK), recently reported superior to serum creatine phosphokinase (CPK) in detecting patients with and female carriers of X-linked recessive (Duchenne) muscular dystrophy (DMD). Extensive comparative studies did not support these findings, and confirmed the marked superiority of CPK over variants of PK or other enzymes in sensitivity, stability and convenience. Deducting the adenylate kinase increment (AKI) further refined the CPK assay, eliminating the effect of haemolysis in diagnosis and enabling studies of blood cell content. Both leucocytes and erythrocytes liberated PK and lactate dehydrogenase (LDH) after brief chilling or disruption. Only erythrocytes showed a CPK content, however, constantly adjusted to match that of serum as if by free cell membrane passage, but less accomodating to a sudden large influx of CPK than of LDH, where an apparent buffering effect could account for differences in clinical response.
Introduction The unique abundance of creatine phosphokinase (CPK) in skeletal muscle makes its serum assay especially informative in X-linked recessive (Duchenne) muscular dystrophy (DMD), both for clinical diagnosis [3] and female carrier detection [4]; other muscle enzymes are confirmatory but far less emphatic. The 86% detection rate by CPK [4] seems to approach a theoretical maximum [5], as yet undetermined, so that still further improvement appears possible using the two forms of pyruvate kinase (PK), one found in muscle and many other tissues but the other only in liver and erythrocytes [6]. Different kinetic properties [6,7,8] allow simultaneous serum assay of considerable tissue specificity useful in muscle disease [ 91. [ 1,2]
440
In the presence of excess hexokinase and glucose, adenylate kinase (ATP : AMP phosphotransferase; EC 2.7.4.3; AK) from erythrocytes [lo] or muscle [l] is additive in a CPK assay measuring rate of phosphorylation of adenosine diphosphate (ADP) by creatine phosphate (CrP) [l]. Since suppression of serum AK by adenosine monophosphate (AMP) seems imperfect even in normal subjects [lo], this adenylate kinase increment (AKI) may be a source of diagnostic error in DMD carrier detection using standardised reagent kits. Subtraction of AK1 prevents this, and also allows studies of CPK movement across erythrocyte and other cell membranes. Methods Serum specimens were obtained as described below. None showed any haemolysis. Glassware was cleaned by immersion for a week in 30% HN03, rinsing with glass-distilled water, and drying at 125°C. Grade A pipettes were used throughout. Glucose was measured enzymatically in mg/lOO ml, and sodium and potassium by flame photometry in mequiv./l [ll]. Serum creatine phosphokinase (ATP : creatine phosphotransferase; EC 2.7.3.2; CPK), 1,6-diphosphofructoaldolase (EC 4.1.2.7; ALD), and lactate dehydrogenase (EC 1.1.1.27; LDH) were measured by semi-automatic optimal kinetic methods at 25°C and 340 nm (Unicam SP 800 B recording spectrophotometer) described elsewhere [ 121, and AK1 by substituting normal saline for CrP in the CPK assay. Values are expressed in International Units as mU/m1/25”C. Standardised reagents for glucose and enzymes were obtained as optimised kits (C.F. Boehringer and Soehne GmbH, Mannheim, G.F.R.), the latter corrected using normal saline for serum [13]. These CPK reagents (CPK-Bg; glutathione/lO mM AMP) showed measurable blank values, but those of J.T. Baker Chemicals B.V., Deventer, Holland (Diamed Diagnostics, Liverpool) for the same procedure (CPK-Bk; dithiothreitol/ll.5 mM AMP) showed none, and had the CrP solution separate, allowing AK1 to be measured. Pyruvate kinase (ATP : pyruvate phosphotransferase; EC 2.7.1.40; PK) was likewise measured at 25°C and 340 nm as the rate of conversion to lactate, by excess LDH and reduced nicotinamide-adenine dinucleotide (NADH), of pyruvate from 1.0 mM phosphoenolpyruvate (PEP) in the standardised reagent kit (Boehringer), self-corrected by reading for 10 min before adding ADP to start the PK reaction. Liver PK was inhibited [8] by adding to this reaction 2.0 mM alanine (in 0.01 ml) to give PK-Ala, and the muscle form (PK-M) measured in 0.2 mM PEP and both liver and muscle forms (PK-LM) in 2.0 mM PEP [6,7,8] by preparing different PEP solutions for otherwise unchanged assay conditions. These distinctions are possible since the muscle form is isosteric, in contrast to the allosterism cf the liver form expressed by alanine inhibition and especially substrate activation [ 91. Since this allosterism is lost on chilling but recovers rapidly at room temperature [8], all chilled or frozen sera were left at room temperature for at least two hours before assay. Procedures
Whole
blood
21-gauge
needle
specimens were obtained from an antecubital vein using a and disposable syringe, with moist alcohol skin toilet, minimal
441 venous stasis and gentle plunger traction to avoid haemolysis. Distribution before clotting into stoppered Pyrex tubes, graduated where necessary, was direct from the syringe or by a marked wide-bore Pasteur pipette. Centrifuging twice ensured cell-free serum, and was carried out forthwith unless otherwise stated. Treatment of blood specimens and serum included incubation for 24 h at 25 & O.l”C in a water bath, at 1.5 f O.l”C in a 25% ethylene glycol bath with a Grant CC15 cooling coil and 2.5 cm expanded polyurethane insulation, or successively at 1.5”C then 25°C to complete the 24-h period. Certain clotted or heparinised specimens were mechanically haemolysed by vigorous shaking for 60 set (Whirlimixer) immediately after 24 h at 1.5”C to increase erythrocyte fragility. Subjects were 48 healthy adults (24 males aged 18.14 to 61.92 years, and 24 females aged 17.41 to 65.56 years), bled in July and again in November, with many individuals common to both series. Patients included 11 boys with DMD and 2 with the similar but much milder X-linked Becker muscular dystrophy (BMD), 18 DMD and 2 BMD female carriers, 1 case of myocardial infarction and 4 cases of viral hepatitis A. Classically affected male relatives of DMD carriers are denoted by s, son; b, brother; mb, mother’s brother; ss, sister’s son; mss, mother’s sister’s son: and in BMD, where affected males may reproduce unlike DMD, additionally by f, father; mf, mother’s father. Blood cell enzyme content was determined in a healthy male subject aged 23.27 years. 56 ml blood were taken at mid-morning; 6 ml were divided in three 2-ml portions and set aside to clot, and the rest taken into ion-free heparin and divided in two 25-ml lots. Each lot was centrifuged for 15 min at 400 X g and the resulting cloudy supernatant again in an second tube for 5 min at 3200 X g to give cell-free plasma, gently returned through the packed cells using a wide-bore Pasteur pipette. Repeating four times gave a suspension of erythrocytes (RBCs) free from white cells (WBCs) in the first tube for one lot, or, by retaining the plasma in the second tube, of RBC-free WBCs for the other lot. Each suspension was divided in three portions for plasma assay forthwith, after chilling 24 h at 1.5”C, and on cell disruption after chilling. The results appear in Table XI. The three 2-ml portions of clotted blood were simultaneously treated likewise; and that evening the same subject undertook 2 h continuous severe physical exertion (press-ups, track-work etc.). He was then bled at the same time on the following five consecutive mornings, and each 10 ml specimen was divided for clotting in three portions, then treated as before. The results appear in Table XII. Statistical significance is denoted throughout by * (p < 0.05), ** Cp < O.Ol), and *** (p < 0.001). Results and discussion In DMD carriers, random X-chromosomal inactivation [ 141 before myoblast fusion [15] gives a multinuclear mosaic of binomial distribution in the mature
442
muscle fibre. The rare occurrence of severely crippled DMD carriers [ 51 suggests inactivation in a muscle adage of S-16 cells [ 161, and implies an equal rarity of phenotypically normal carriers undetectable by any means, so that complete detection may be approached even if never reached. Moreover, new observations suggest that male DMD mutants are also uncommon [17], whereby nearly all mothers of DMD boys must be carriers; and studies on the lethal X-linked recessive gene show that the proportion of new female mutants may be as high as one half [ 181. Such findings indicate a pressing need by genetic advisers for still better routine methods of carrier detection. Serum CPK alone, in optimal circumstances the simplest and most sensitive single test available, leaves 14% of carriers undetected [4], a figure improved only by a range of tests [ 191 or by complex studies of muscle ribosomal protein synthesis [20]; but such refinements are not generally available. Recently, however, serum PK has been studied in DMD, other myopathies, and myocardial infarction, and, even without recourse to its specific isoenzymes, is now reported markedly superior to CPK in diagnosing both DMD patients and carriers [9,21]. These findings merit careful examination with complete analyses throughout for tissue specificity. Normal values in healthy adults (Table I) show that in both sexes PK and PK-LM are almost identical, since a PEP concentration greater than 1 mM affects the activity of each isoenzyme only slightly [7]; and muscle PK-Ala and PK-M are likewise similar, as expected. CPK-Bk is far more sensitive than CPK-Bg, the significantly higher female standard deviation in July giving a higher upper normal limit then, with a similar (but not significant) finding in IABLE NORMAL
Sex
Male
1 VALUES Mean
Assay
Recorded range
Mean
i
2 S.D.
PK
17.55
4.09
9.37-25.19
9.36-25.73
PK-LM
18.62
4.48
10.24-27.80
9.66-27.58
PK-Ala
11.69
2.81
6.38-18.29
6.06-17.31
PK-M
10.43
2.76
4.60-15.68
4.91-15.95
33.19
5.68
22.83-43.49
21.83-44.55
% alanine Ratio
inhibition
PK-M/PK-LM
CPK-Bg
Female
Standard deviation
(July)
0.56 35.97
0.07 14.01
0.44-
0.69
17.78--69.68
CPK-Bk
(July)
44.54
16.31
21.46-81.40
CPK-Bk
(Nov.)
47.46
22.02
20.23-96.45
0.44
0.46
47.02
22.00
AK1
(Nov.)
Nett
CPK-Bk
(Nov.)
0
-
0.43-
0.69
7.95-63.99 11.92-77.16 3.42-91.51 1.73
0
-
18.50-96.20
3.01-91.04
PK
15.53
3.19
9.41-19.44
9.16-21.90
PK-LM
16.42
3.42
9.72-24.14
9.58-23.26
PK-Ala
9.45
1.99
5.43-13.48
5.48-13.43
PK-M
8.64
1.85
4.81-13.59
4.94-12.34
38.72
8.03
25.00-54.27
0.53
0.08
21.09
10.72
% analine Ratio
inhibition
PK-M/PK-LM
0.34-
22.65-54.79 0.66
1.17-45.48
0.360
0.69 -42.52
CPK-Bg
(July)
CPK-Bk
(July)
27.37
11.90
3.70-53.28
3.57-51.17
CPK-Bk
(Nov.)
24.06
6.27
14.55-34.29
11.52-36.60
0.53
0.56
23.53
6.38
AK1
(Nov.)
Nett
CPK-Bk
(Nov.)
0
-
14.31-34.04
1.36
1.48
0
-
10.77-36.29
1.66
443
males in November. Such markedly seasonal sex differences are difficult to explain. Normal AK1 is small in both sexes. Significant sex differences (Table II) occur only where there is a muscular origin (PK-Ala, PK-M, % alanine inhibition), with very large increases in male CPK, particularly in November, attributable to lack of oestrogens [ll]. Likewise, PK and PK-LM correlate very highly in both sexes (Table III) but PK-Ala and PK-M less well in females than in males; and the modest correlations between CPK and all modalities of PK in males are quite absent in females except of low significance only with PK-Ala and PK-M. No relation between age and serum enzyme values was found in either sex. In July, however, the 24 females were bled on successive days as two groups of 12, in one of which CPK showed a highly significant increase with time of day (Table IV), just losing significance in all 24; and though seasonal changes may have masked similar relationships in November, these observations tend to confirm late afternoon as the most informative time for carrier venepuncture [12]. No such relationships were found in males, other than a slight decline of AK1 with time of day in both sexes. In stored serum, all modalities of PK decay very rapidly at 25°C (Table V), surviving with slight loss at -21.5”C, while CPK is comparatively stable for 24 h at 25°C and for as long as 4 weeks at -21.5”C. Serum may thus be mailed overnight for CPK assay, and even stored thereafter; but not for PK. Ideally, enzyme assay is best in serum separated forthwith (Tables I and VI). In clotted blood spun later, however (Table VII), CPK survives 24 h at 25°C (likewise overnight mailing) and may even increase-slightly (b, c, d), especially after chilling 1 h, as if blood cells contained more CPK than serum. Frank efflux of LDH occurs after 24 h at 25”C, and progressively on chilling (a, 6) as described elsewhere [ 111, but rather less in DMD carrier d and only after prolonged chilling in DMD patient c, where lower values after 24 h at 25”C, with or without 1 h chilling, suggest actual movement into blood cells from an elevated serum value. All modalities of PK, however, decline markedly in a and b after 24 h at 25°C but show gross increases on progressive chilling, derived from blood cells as indicated by 9%alanine inhibition and ratio, while the same changes are much less marked in the already elevated values for c and d, like the behaviour of LDH and perhaps for similar reasons. Of 11 known DMD carriers examined (Table VIII) 2 were obligate (Nos. 5, 11) but hitherto always undetectable in accord with the natural distribution of
TABLE
II
% DIFFERENCES
OF
MALE
FROM
FEMALE
PK
+12.40
PK-LM
+13.39
PK-Ala
+23.63
**
PK-M
+20.65
*
% alanine Ratio
inhibition
PK-M/PK-LM
-14.28 +
**
5.66
CPK-Bg
(July)
+70.56
***
CPK-Bk
(July)
+62.75
***
CPK-Bk
(Nov.)
+97.33
***
AK1
(Nov.)
Nett
CPK-Bk
-22.22 (Nov.)
+99.79
***
MEANS
444
TABLE
III
CORRELATION
MATRICES
(22
df)
PK
PK-LM
PK-Ala
PK-M
-
CPK-Bg
CPK-Bk
(July)
(July)
Males PK
1.000
PK-LM
0.970***
1.000
PK-Ala
0.926***
0.900***
1.000
PK-M
0.901***
0.894***
0.919***
1.000
CPK-Bg
(July)
0.520**
0.504**
0.571**
0.627***
1.000
CPK-Bk
(July)
0.583**
0.583**
0.612***
o.fs44***
0.972***
1.000
Females PK PK-LM
1.000 0.945***
1.000
PK-Ala
0.799***
0.739+++
1.000
PK-M
0.820***
0.789***
0.799***
1.000
CPK-Bg
(July)
0.213
0.214
0.376*
0.377*
1.000
CPK-Bk
(July)
0.224
0.219
0.352*
0.392*
0.986***
TABLE
1.000
IV
REGRESSIONS
OF
SERUM
ENZYME
VALUES
(E’)
ON
TIME
OF
DAY
(T mm
after
9 a.m.)
Females % alanine CPK-Bg
inhibition (July)
CPK-Bk
(July)
E = 49.560-3.8809
X lo-*
T
(n = 12:
f = 2.9519;
10
df;
*)
E = 0.0042
+ 2.8729
X 10-4
T
(n = 12;
f = 4.9637;
10
df;
***)
h’ = 0.0465
+ 1.0449
X 1O-4
T
(n = 24:
t = 2.0479;
22
df)
0.0268
+ 3.5146
X 10-4
T
(n = 12;
t = 4.3056;
10 df:
X 1O-4
‘I
(n = 24:
t = 1.8999;
22 df)
0.1004-1.100
X 10-S
T
(n = 24;
t=
0.3015;
22
df)
0.0963
-3.61
X 10-h
T
(n = 24;
t = 0.0971;
22
df)
0.0041
-7.39
X 10-h
T
(n = 24;
t = 2.5722;
22
df:
*)
-
X 10-6
?’
(n = 24;
t = 2.3514;
22
df;
*)
Nett
CPK-Bk
AK1
(Nov.)
AK1
(Nov.)
E = 0.0034
CPK-Bk
(Nov.) (Nov.)
= = = = =
0.0792 + 1.2859
E E E E E
**)
M&S
TABLE
V
ENZYME Sex
STABILITIES
Age
IN
STORED
Assay
(years)
M
20.06
23.15
F
27.04
Forth
24
-with
at 25°C
h
24 h
1 week
at-21.5
at-21.5
4 weeks at -21.5
OC
“C
“C
17.0
13.2
16.5
14.5
13.8
20.8
13.3
17.5
16.4
15.5
PK-Ala
12.3
10.5
11.5
10.3
9.5
PK-M
10.7
8.2
10.1
8.2
inhibition
PK-M/PK-LM
27.6 0.51
20.6 0.61
30.1 0.58
28.8 0.50
9.7 31.1 0.63
CPK-Bg
23.6
22.2
22.2
22.0
22.7
CPK-Bk
30.3
28.2
30.0
29.5
29.5
PK
14.1
10.3
13.4
12.3
11.0
PK-LM
16.8
11.0
15.9
13.7
12.9
PK-Ala
10.5
8.2
9.6
9.3
6.7
8.8
6.9
9.1
7.7
6.6
25.6
21.2
28.1
24.6
39.1
PK-M % alanine Ratio 40.28
SERA
PK-LM
Ratio M
NORMAL
PK
% alanine
F
6.07
inhibition
PK-M/PK-LM
0.52
0.62
0.57
0.56
0.51
CPK-BB
28.1
27.8
27.0
25.7
24.8
CPK-Bk
36.8
33.3
33.5
34.3
32.7
445
TABLE
VI
NORMAL
SERUM Normal
Assay
RANGES males
1.07-
ALD
[ll] Normal
2.34
0.81-
95.17-166.13
LDH
3.55-
2.27
80.16-178.26 135.20-144.44
135.10-143.33
Na+ K+
females
3.71-
4.61
4.72
carrier manifestation [5] ; and though carrier 11 was doubtfully detected by a single barely elevated finding (ratio M/LM) all other readings in both were normal. CPK decisively detected all 9 remaining DMD carriers (3- to 20-fold elevations), LDH only 3 clearly (less than 2-fold elevations) and 4 doubtfully, while PK detected 5 clearly (about 2- to 6-fold elevations) and 4 very doubtfully, with PK-LM, PK-M and PK-Ala giving similar results in the same individuals and % alanine inhibition detecting 5 clearly but ratio M/LM only 3 marginally. Storage of clotted blood for 24 h at 25°C had little effect on the TABLE
VII
EFFECTS
OF
CHILLING
CLOTTED
BLOOD
FROM
HEALTHY
SUBJECTS,
DMD
CARRIERS
AND
PATIENTS No.
Sex
Age
DMD
Assay
(years) 22.84
Nil
16.0
31.0
17.9
38.1
74.0
PK-Ala
11.0
8.7
10.2
16.4
20.1
7.6
10.5 inhibition
33.5
PK-M/PK-LM
30.8
0.61
58.7
9.0
14.9
18.6
35.9
47.1
65.8
0.50
0.57
CPK-I3g
23.3
23.6
22.7
0.25
0.39 22.7
22.7
LDH
91.2
117.5
143.3
PK
13.2
10.9
13.8
16.8
32.9
PK-LM
13.1
10.1
14.0
21.2
43.0
PK-Ala
8.4
7.8
8.9
10.2
15.0
PK-M
5.3
6.3
7.5
8.8
12.2
inhibition
36.5
27.9
0.41
PK-M/PK-LM
12.5
35.6
0.62 15.5
231.4
39.1
0.54
255.7
54.3 0.28
0.41
16.9
15.5
15.5 202.1
106.9
127.1
165.1
PK
271
257
263
282
299
PK-LM
265
278
271
279
316
PK-Ala
246
225
232
234
240
PK-M
191
205
199
200
197
83.1
LDH
% alanine Ratio
inhibition
9.1
PK-M/PK-LM
CPK-Bg LDH 3s
-
13.8
CPK-Bg
2b;
at 1.5’~
25OC
12.5
Ratio
42.54
24 h
18h
17.0
% alanine
DMD
6 h1.5OC.
h 25°C
PK-LM
Ratio
12.56
1 h 1.5”C, 23
16.5
% alanine
Nil
24 h at 25Oc
PK
PK-M
27.26
Forth -with
12.4
0.72
11.7
17.1
0.73
0.74
19.6 0.62
0.72
1673
1759
1842
1768
1803
704
651
661
729
831
PK
23.9
18.3
22.3
23.7
28.7
PK-LM
23.3
19.0
23.9
23.9
34.1
PK-Ala
17.3
13.9
16.5
14.8
17.4
PK-M
15.4
12.2
15.0
14.6
16.2
27.5
24.0
25.8
37.5
39.4
% maline Ratio
inhibition
PK-M/PK-LM
0.66
0.64
0.63
0.47
0.61
CPK-Bg
141
143
152
146
145
LDH
165
194
210
223
224
446 TABLE
VIII
CARRIERS AND PATIENTS: AND (IN BRACKETS) AFTER Affected
male relatives: s, son: b, brother;
NO.
Age (years)
Male rrls.
1
16.58
lb
2
16.90
2b
3
21.42
lb
4
28.59
2s
5
36.98
2b:ls
6
39.77
2%
7
42.31
2b;3s
8
46.24
Is
9
52.44
2s
10
53.87
IS
11
55.13
lb: 2s
Correlations
(9 df):
PK
31.0 (31.2) 133 (130) 98.4 (88.4) 72.7 (73.7) 11.0 (10.7) 27.7 (27.1) 22.2 (21.5) 26.6 (21.1) 38.5 (34.7) 23.2 (21.5) 13.7 (13.6) PK/CPK
DMD male patients (A = ambulant: 12 6.83 Nil (A) 695 13
10.22
Nil (W)
14
12.56
Pmb(A)
15
14.53
Nil(W)
Correlations Becker 16
ENZYMES 24 HOURS
(663) 159 (185) 271 (257) 137
(136) (2 df): PK/CPK
MD carrier female 18.23 2b
Necku MD male patient 17 10.05 lb (A)
IN SERA AT 25°C
CLOTTED
mb, mother’s brother;
PK-LM
35.0 (28.6) 135
FROM
PK-Ala
25.9
(26.5) 118
(138) (117) 104 84.0 (105) (84.0) 71.5 60.8 (59.3) (70.9) 11.9 6.5 (12.4) (6.9) 21.6 27.4 (22.2) (26.4) 24.0 18.5 (24.2) (17.3) 18.4 27.7 (15.3) (20.9) 30.2 41.8 (29.3) (38.1) 25.2 15.6 (23.2) (15.6) 10.1 14.4 (11.1) (13.8) = 0.8312 **; PK/LDH
PK-M
22.1 (22.7) 106 (107) 75.9 (70.9) 52.7 (52.7) 6.0 (6.5) 12.5 (18.9) 16.9 (15.3) 17.0 (13.4) 27.4 (26.4) 15.9 (15.3) 10.1 (9.8) = +.4123;
wheclchoir) 688 623 (686) (601) 15.5 144
542 (532) 140
(192) 265
(154) 191
BLOOD
SPUN
FORTHWITH
ss. sister’s son. % Ala in- Ratio hibition MiLM
16.5 (15.1) 11.3 (10.6) 14.6 (5.0) 16.4 (19.6) 41.0 (35.3) 21.9 (18.1) 16.5 (19.4) 31.0 (27.7) 21.5 (15.7) 32.9 (27.7) 26.0 (18.5) CPK/LDH
CPK-Bg
0.63 127 (0.79) (133) 0.79 473 (0.77) (472) 0.73 852 (0.68) (745) 0.74 515 (0.74) (575) 11.7 0.50 (0.52) (11.4) 0.46 177 (0.72) (174) 0.70 155 (0.63) (148) 0.62 100 (0.64) (94.2) 0.66 280 (0.69) (278) 0.62 205 (0.66) (201) 0.70 26.5 (0.71) (29.4) = -*.7856*’
LDH
133 (145) 186 (191) 351 (284) 258 (280) 170 (150) 226 (319) 178 (169) 192 (159) 233 (237) 269 (270) 168 (170)
IV
(278) 138
(180) 246 (225) 120
(137) (123) = 0.9536 *; PK/LDH
(205) 99.3 (119) = 0.0172;
25.1 (21.1)
27.5 (23.5)
19.1 (16.9)
17.8 (15.2)
598 (588)
621 (608)
543 (528)
(472)
459
10.4 (1.3) 9.2 (2.8) 9.1 (12.4) 12.2 (9.2) CPK/LDH
23.7 (19.8)
9.1 (10.1)
0.79 (0.78) 0.91 (0.80) 0.72 (0.74) 0.72 (0.87) = 0.2147
3504 (3528) 1673 (1668) 1673 (1759) 1875 (1694)
0.65 (0.64)
70.8 (69.4)
0.74 (0.78)
3691 (3612)
613 (1337) 805 (1529) 704 (651) 441 (380)
196 (183)
1003 (614)
marked CPK elevations, but 3 marginal PK elevations became normal, while LDH behaved irregularly. The muscle-specific CPK thus emerges as far more sensitive, stable and dependable than LDH or any PK modality, although, unlike normal individuals (Table III), DMD carriers do show high correlation between CPK and PK, while LDH correlates with neither. In DMD male patients all values were grossly abnormal throughout, again with high correlation between CPK and PK but of LDH with neither. The slowly progressing BMD
447
patient 17 had values like the youngest DMD patient 12, and, as expected, CPK detected BMD carrier 16 more confidently than any other reading. The true CPK value (nett CPK-Bk) is obtained by deducting AKI. In a second series of 9 known DMD carriers (Table IX), true CPK detected 8 decisively (almost 2-fold to 50-fold elevations) and 1 barely, but ALD only 3 clearly (3to 13-fold elevations) and 2 marginally, while AK1 was slightly elevated in 4 instances. Further, CPK and ALD correlated very highly indeed, but AK1 with neither, so that a high AK1 occurring with a marginal CPK elevation could give a false carrier diagnosis unless first subtracted. True CPK and ALD were grossly elevated in DMD patients, but AK1 rather less so; CPK and ALD correlated
TABLE CPK,
IX AK1
8
ALD
IN
DMD
CARRIERS
AND
PATIENTS,
HEPATITIS-A
& MYOCARDIAL
INFARC-
TION Affected
male
mother’s
father.
NO.
relatives:
s, son;
b,
Affected
A@
brother;
mb.
mother’s
relatives
carrier
mss,
Nett
mother’s
sister’s
AK1
ALD
females
1
8.60
lb
2538
2.5
28.9
2
9.92
2b
1254
1.7
14.4
3
28.12
lb:
4
29.18
1s
118
0.74
1.5
2s
411
2.5
4.0
261
5
34.28
IS
6
38.99
IS
7
46.79
2b;
1s
8
53.16
lb;
2s
9
54.84
lmss;
Correlations DMD
male
(7 df):
patients
2.6
2.5
52.5
1.2
1.0
86.6
1.5
1.9
0.25
1.7
109 2mb;
CPK/AKI
(A = ambulant;
2s = 0.5122:
197
0.74
CPK/ALD
= 0.9875***;
6.6 ALD/AKI
= 0.4378
lli = wheelchair)
10
3.12
lb
(A)
1764
11
3.24
Nil
(A)
6857
24.9
57.7
12
3.82
Nil
(A)
4921
4.2
66.7
2.7
32.4
13
6.41
4771
4.7
68.9
14
10.12
Nil
(W)
2058
4.2
18.5
15
11.06
Nil
(W)
2081
3.2
14.7
16
12.38
Nil (W)
961
3.9
7.9
17
14.03
Nil
2162
1.5
15.0
lmb
Correlations Becker
MD
18 Becker
(6 df):
carrier
MD
malf
(A)
(W)
CPK/AKI
= 0.7573*;
CPK/ALD
= 0.8733**;
ALD/AKI
= 0.4197
female
33.23
19
1s: f
34.8
0.74
2.1
patient
10.65
mf
high
2.2
33.4
patients
20
8.44
1.7
4.2
28.3
21
16.33
7.6
2.0
29.7
22
18.01
-
7.4
3.2
19.2
23
27.39
-
4.9
3.0
26.6
24
(wry
2129
(A)
Hepatitis-A
Myocardial
son:
CPK-Bk
(years) DiUD
brother:
aminotransferase)
infarction 46.24
5 h after
onset
24 h after
onset
76.7 759
1.0
1.6
3.0
5.7
f, father;
mf,
448
highly, and AK1 only with CPK. Values in the BMD patient 19 were similar, but even CPK failed to detect his carrier mother 18. The myocardial infarction patient 24 had unremarkable AK1 values; the others were expected. Though all 4 hepatitis A patients 20-23 had very high ALD but modest AK1 elevations, true CPK was extremely low, but extensive dialysis and dilution studies gave no evidence of a dissociable inhibitor to CPK. These very low CPK values seem typical of this condition [3], and may be due to a firmly bound inhibitor. Deducting AK1 greatly clarifies the behaviour of CPK in serum and whole blood, and makes possible the assay of erythrocyte CPK content after mechanical haemolysis, since dithiothreitol (Cleland’s reagent) is used for CPK activation instead of glutathione vulnerable to erythrocyte glutathione reductase. Table X shows that brief chilling or storage at 25°C had little effect on fresh serum. Any changes in aliquots of the original clotted blood given the same treatment simultaneously must therefore arise from blood cells. Thus, compared with serum treated similarly, 24 h at 25°C caused little change except slight rises in LDH and AKI, with a fall in glucose consumed [22,23] to fuel the erythrocyte (NaK)-ATPase retaining K’ and expelling Na’ [24]. 24 h at 1.5”C, however, gave marked increases in AK1 and especially LDH [ 111, with sparing of glucose as the temperature-dependent [25] (NaK)-ATPase failed
TABLE
X
BEHAVIOUR AND NO.
OF
SERUM
AND
CLOTTED
BLOOD
FROM
HEALTHY
SUBJECTS,
DMD
CARRIERS
PATIENTS Sex
Age
DMD
Assay
Serum
Clotted
blood
(years) Sepd.
Kept
Kept
24 h
24 h
24 h
immed.
24 h
24 h
25OC,
1.5Oc,
1.5°c,
25’C
1.5Oc
sepd.
sepd.
shaken, sepd.
1
M
20.72
Nil
AK1 Nett
0.49 CPK-Bk
LDH
112
GIUCOSe Na+ 2
F
42.14
Nil
140
Kf
4.3 0.99 CPK-Bk
M
11.06
DMD
4.2 Nil
172
36.4 144
4.3
75.6 136
4.4
9.5
1.5
1.7
Nil
LDH
66.8
67.9
61.8
84.1
90.6
Glucose
76.7
75.3
74.9
28.9
71.2
142
142
142
144
11.0 34.9 340 72.4 135
27.1
136
10.5 4.9 28.5 189 65.0 133
K+
4.4
4.4
4.3
4.7
10.4
13.0
AK1
3.2
2.6
3.0
3.6
8.4
34.3
CPK-Bk
Na+ 1s
141
117
27.5
GlUCOW
34.28
141
86.8
3.9 32.6
28.5
LDH
F
115
84.7
3.0 32.8
26.5
Nett
4
110
34.0
27.6
Naf 3
0.49
32.7
89.5
AK1 Nett
0.74
33.9
2081
2036
2051
1959
2229
2159
851
841
846
851
1074
1550
85.0 140
81.1 139
84.2 139
25.4 141
65.0 135
K+
3.8
3.8
3.8
4.2
9.1
AK1
2.6
0.99
3.2
2.8
9.1
Nett
CPK-Bk
LDH Glucose Naf K+
57.9 132 10.6 25.3
261
254
264
249
263
255
150
215
190
218
392
697
94.8 142 4.3
89.8 139 4.2
82.0 140 4.3
16.9 146
76.0 134
4.5
11.9
66.9 129 14.4
449
so that serum K’ rose as Na’ fell. CPK showed little change except for a 10% rise in DMD patient 3. Mechanical haemolysis after 24 h at 1.5” C then added intact erythrocyte contents to serum, with the expected further rise of AKI, LDH and K’ and a corresponding fall in glucose and Na’ but not in CPK, as if erythrocytes contained about as much CPK as serum, though for no discernable purpose. Chilling, especially with haemolysis, seemed to produce a much greater AK1 in DMD patient 3 and carrier 4 than in normal individuals 1 and 2. Differential centrifugation of heparinised blood enables some formal assignation of these changes to erythrocytes (RBCs) or leucocytes (WBCs). In Table XI the relatively unchanged Na’ and K’ after chilling and shaking the WBC suspension indicates disruption of only a small volume of cells, with a moderate rise in LDH and a slight fall in true CPK, as if absent from WBCs, but a large rise in PK (chiefly the liver form) suggesting a very high WBC content. After the same procedures on the RBC suspension, however, large changes in Nd and K’ and very marked rises in LDH, AK1 and PK (again mostly the liver form) indicate considerable cell disruption, with expectations that true CPK should fall as much as Na’. Instead, a small increase in CPK after haemolysis demonstrates a higher content in erythrocytes than serum. Thus in whole blood any changes in serum CPK, electrolytes, and most of those in LDH seem to arise from erythrocytes, not leucocytes; while both can affect serum PK values. Determination of the origins of these changes permits in vivo examination of enzyme movements in whole blood by increasing the serum content after severe exercise. In Table XII, serum separated forthwith (procedure a) from blood taken on successive days showed little change in electrolytes, glucose or AKI, a small post-exertion rise in LDH, and a much larger immediate one in
TABLE
XI
BLOOD
CELL
Assay
CONTENTS RBC
OF
HEALTHY
MALE
(23.27
suspension
years)
WBC
suspension
Plasma
24 h 1.5OC.
24 h 1.5’C.
PlaSma
24 h 1.5OC,
24 h 1.5’C.
separated
plasma
shaken,
separated
plasma
shaken,
forthwith
separated
separated
forthwith
separated
separated
PK
14.4
25.8
PK-LM
13.8
33.0
PK-Ala
9.9
PK-M
8.2
8.9
93.5 123
12.7 14.7
12.1
_
69.7 104
18.3
9.1
31.9
10.0
24.0
8.9
25.4
65.6
80.5
28.7
54.3
% alanine inhibition Ratio
31.2
PK-M/
PK-LM CPK-Bk AK1 Nett
Glucose K+
26.1
1.7 CPK-Bk
LDH Na+
0.30
0.59 28.9 27.2 107 70.3 138 4.3
1.0 25.1 252
0.20 176 146 30.8 1808
57.3 135
67.4 125
6.3
17.7
0.60 28.9
26.7
0.74 28.2 110 76.7 140 4.3
0.24 26.9
0.41 26.3 122
1.1 25.8 352
74.9 140
74.1 142
4.6
4.9
450
TABLE
XII
EFFECT
OF
Procedures: Clotted
blood
24-h
periods
after
exercise
0
1
EXERCISE (a)
Serum
Procedure
5
forthwith;
24 h 1.5”C, AK1
BLOOD (b)
vigorously
CELL
ENZYMES
Clotted
blood
incubated
shaken,
serum
separated.
Nett
IN HEALTHY 24 h 1.5”C.
LDH
Glucose
MALE serum
NaC
(23.27
yrs)
separated;
(c)
K+
a
0.62
28.0
112
83.1
143
b
1.1
28.6
222
81.1
137
8.9
29.5
455
70.5
134
11.7 3.9
r
14.8
a
Nil 1.6 36.8
4.3
80.3
125
88.9
145
95.5
517
80.2
138
88.7
886
74.0
133
12.2 4.8
X.8
a
0.37
76.3
131
86.6
142
b
2.6
70.9
238
79.7
139
8.6
c
11.8
72.6
452
70.0
135
11.4 4.4
a
0.25
40.1
117
89.5
142
b
0.74
43.5
263
82.4
138
8.8
40.6
518
77.9
134
11.6 4.5
c 4
AND
CPK-Bk
c
3
SERUM
separated
incubated
b 2
ON
28.7
a
2.1
33.5
109
93.7
141
b
6.0
31.9
227
86.1
137
8.5
c
19.7
34.7
462
79.0
134
11.6 4.4
a
1.9
27.4
106
90.7
140
b
2.7
27.9
185
90.0
136
c
15.9
26.9
392
73.5
132
8.0 11.4
true CPK which returned smoothly to previous values by the 5th day. Simultaneous chilling of clotted blood aliquots for 24 h at 1.5”C without (procedure b) or with (procedure c) mechanical haemolysis disclosed the underlying enzyme movements. Both procedures caused very similar changes in Na+, K’ and glucose on successive days, indicating the same degree of efflux or of cell disruption. The changes in AKI were similar if less precise. The sudden two-fold increase of erythrocyte LDH on day 1, returning to normal by day 2, suggests rapid incorporation then re!ease of effluent muscle LDH (mol. wt. 135 000) in effect buffering the rise in serum values. Except for day 1, serum CPK throughout showed similar values in all 3 procedures, indicating an erythrocyte content identical to serum and arguing against simple membrane attachment, with continual equilibration by rapid movement of CPK (mol. wt. 81 000) across the cell membrane. On day 1, however, b showed marked CPK efflux from erythrocytes to serum, and simultaneously c that these erythrocytes, now disrupted, contained less CPK than the b serum into which the CPK was being discharged (though still more then the a serum). This implies active expulsion of CPK from erythrocytes tolerant only of equilibration, and also suggests that serum CPK values had been much higher earlier, which accords well with reports that the serum CPK maximum is reached some 11 h after physical exertion [26,27]. Thus an explanation is found for the prompt emphatic response of serum CPK compared with slower and lesser changes of LDH in the same circumstances. In carrier detection, therefore, serum CPK assayed in optimal circumstances [3,4,11] is far more sensitive and dependable than any variant of the much less stable and more inconvenient PK; and this is enhanced by its stability at room temperature in serum and in clotted blood, allowing overnight mailing. Further, determining true CPK after deducting AK1 refines carrier detection as well as
451
obviating false elevation due to even slight haemolysis, so irremediable in PK, ALD and LDH. The apparent equivalence of CPK in serum and erythrocytes likewise discounts the effect of efflux even without visible haemolysis, a constant but often unsuspected risk in these other enzymes present in blood cells in large amounts. Acknowledgements The authors wish to thank all who took part in this investigation, and Dr. R.W. Logan in whose department the serum electrolytes were measured. This study was supported by the Andrew Patrick Trust and the Muscular Dystrophy Group of Great Britain. References 1
Oliver.
I.T.
2
Smith,
A.F.
3
Thomson.
W.H.S.
4
Thomson,
W.H.S.
5
Thomson,
W.H.S..
6
Tanaka,
T..
Harano,
7
Tanaka,
T..
Sue,
8
Llorente.
9
Harano.
10
(1955)
G.,
11
Sweetin,
12
Thomson,
13
Smith.
14
Lyon,
15
Emery.
16
Graham,
17
Roses,
Clin. (1971)
Clin.
Chim.
Acta
35,
(1969)
Clin.
Chim.
Acta
26,
Sweetin, Y..
R..
and
I. and M.F.
A.E.H.
(1965)
J.B.,
W.
J. Med.
Barrow, Roses.
E.S. M.J..
Res.
13,
KowaI. Acta
63,
383-394
7141
Commun.
29,
444-449
45-54
J. (1973)
Clin.
Chim.
Acta 62.
Metabolism
Chem. 48,
22,
22.
493-501
1806-1811
49-3
21,469-478 i, 1038
14.135-148 2, l-7
and Elston. Miller,
Chin
Biophys.
E. (1976)
Lancet
Genet. Genet.
M. and
Clin.
Acta
Clin.
J. Biochem.
J. Biochem.
Bemt,
(1975)
J. Hum.
Eur.
(1973)
Chim.
(1975)
H. (1967)
Miller.
and
W.H.S.
W.H.S.
Am.
207-221
Biochem.
(1970)
Jr, P.J..
Clin.
Thomson.
183-191 T.E.
H. (1967) A.
Gruber,
Thomson,
(1962)
A.D.,
Vignos,
(1968)
351-359
F. and Morimura,
and Sols,
W..
39,
and Hilditch,
Morimura,
R.
Adair,
J.C.
Sue.
F. and
W.H.S.
116-122
Acta
Gerhardt, J.C.
J. 61.
Chim.
P., Marco, Y.,
Szasz.
Biochem.
(1972)
R.C.
S.E..
Hull.
Gartler,
S.M.,
(1975) K.L.
Ann. and
N.Y.
Appel,
Acad.
Sci.
240.
141-146
S.H.
(1976)
New
Eng.
Dancis,
J..
SeegmiIler,
(1968)
J. Neural.
J. Med.
294,
193-
198 18
Francke,
U.,
Nyhan,
Felsenstein,
W.L.
19
Radu.
20
Ionasescu,
21
Albert%
22
Danowski,
23
Eckel.
R.E.,
Rizzo,
24
Glynn.
T.M.
(1968)
L. and
25
Wood,
26
Nuttall,
27
King.
H..
(1976)
Migea, V.,
and
T.S.
S.W.,
S.,
Samaha.
F.J.
(1974) H. and
Bull.
E. (1967) B.E.
and
24,
J. Lab.
B. (1968)
A.
T.W. 24,
(1973)
Neurology
J.E.,
Bakay,
Sci. 23,
6, 289-300
497-502
462-464
693-705
Berggren,
A.B.
(1966)
Am.
J. Physiol.
165-169 Clin.
J. Lab.
Savory.
Radu.
Conway,
Neurology 139,
B.R..
123-137
L. and
P. and
Chem.
M&on,
28.
Bordeianu,
Lodish,
Br. Med.
and Jones.
Genet.
Shirk,
J. Biol.
S.C..
Beutler,
Z.,
H.,
(1941)
Statland,
J. Hum.
TGrak,
Zellweger,
M.C.
F.Q.
J.,
Am.
Med.
Clin.
J. (1976)
70,
287-294
Med.
71.
Clin.
Chin
847-854 Acta
72,
211-218
210.
737-743
F.
and
