the reagent with time, after mixing ac- ... eliminated by preparing the reagent in .... Thus the active -SH of CK is blocked in human serum, protecting the enzyme.
V
1 ui
+b( IELU .V2U4l
I
b)I$4
#{247}
1
‘---
‘f::
.‘
N Ocip
1...
-+
I
i /1 0.5
1.0
- -
15
.L0
C OII8TERC
(gilJbylJ
Fig. 3. Comparison of results obtained by the methodology of Abell et al. and with the ABA 100 or manually prepared (with use of the National Bureau of Standards’ Standard Reference Material No. 911a Cholesterol), and commercial lyophilized control sera did not Dynamics/bmc)
exhibit this problem. These standards contained no esterified cholesterol and control lesterol process
sera contain less esterified chobecause of the lyophilization
and because the added choles-
terol is non-esterified. The statistical analysis using linear regression (Table 1) shows how cients, slopes,
the
correlation
coeffi-
and intercepts
proved after increasing
were imthe incubation
temperature from 37 to 42#{176}C, and after adding 0.6 mmol each of glycocholic and
taurocholic
acids
per
liter
to the
re-
agent. Additionally, in one lot of the BMC reagent we observed a deterioration of the reagent with time, after mixing according to the manufacturer’s directions. This was evidenced by a continual increase in the energy with time after reagent preparation. This problem was eliminated by preparing the reagent in two stable stock solutions, which then were mixed into a working solution on the SMA 12/60. The instrument mixes equal volumes of the buffer solution and enzyme solution, which is readily accomplished with the pump manifold on the SMA. References
Table 1. Data Resultingfrom a Linear RegressionAnalysisbefore and after IncreasingIncubation Temperatureand before and after Adng Bile Salts
3. Abell, L. L., Bevy, B. B., Brodie, B. B., et al., Cholesterol serum. Stand. Methods Clin. Chem. 2, 26in (1958).
Interc.pt
Slop. 9/liter
Incubation temperature 37 #{176}C0.894 1 0.09 13
42#{176}C1.0087-0.0015
.9934
.9912
Bile acids
Before addition After addition
0.9464
0.0061
1. Robinson, C. A., Jr., Hall, L. M., and Vasiliades, J., Evaluation of an enzymatic cholesterol method. Clin. Chem. 22, 1542 (1976). Letter to the Editor. 2. Allain, C. C., Poon, L. S., Chan, C. S. G., et a!., Enzymatic determination of total serum cholesterol. Clin. Chem. 20, 470 (1974).
.9715
4. Richmond, W., Use of cholesterol oxidase for assay of total and free cholesterol in serum by continuous-flow analysis. Clin. Chem. 22, 1579 (1976). Lynda L. Hunter Harold J. Grady Pathology
0.9839
0.0084
.9633
Baptist Memorial Hospital 6601 Rockhill Road Kansas City, Mo. 64131
NormalValuesfor Serum Dopamlne-/3-hydroxylase
Activity
To the Editor: Dopamine-9-hydroxylase
(DBH, EC blood varies over a wide range, but in a given indi1.14.17.1)
vidual
activity
the
in human
enzyme
activity
remains
constant over a long period of time (1-3). We have applied the statistical method of Hoffmann (4) to the computation of a normal value of human serum DBH activity. In principle, the method (4) is based on computer generation of a normal value by omitting values out-
side the mean ±2.2 SD range repeatedly until a constant mean and SD values are obtained. The DBH reaction was carried out at 37 #{176}C by using 50 il of human serum as
enzyme under saturated substrate
(20
mmol of tyramine) and cofactor (10 mmol of ascorbic acid) concentrations and at the optimum pH (5.0) in the incubation
mixture
of Nagatsu
and Ud-
enfriend (2). The octopamine formed was assayed by a simple, sensitive, and specific method for DBH activity by using the high-performance chromatographic technique
liquid of Fujita et
al. (5). Normal values of human serum DBH activity in 153 healthy subjects were expressed as the mean ± SD (TUB units, iimol/min per liter of serum at 37 #{176}C) both without and with the statistical computation of Hoffman (4). As shown in
Table
1, the
values
Hoffmann computation
without
the
(42.5 ± 30.9,
CLINICAL CHEMISTRY, Vol. 23, No. 10, 1977
1947
Table 1. Dopamino-9-hydroxylase(DBH) Activity in NormalHuman Serum withoutor with the Statistical Computationof Hoffmann(4) Without the Holfmann No. subj.cts
5.x
With the Hoftmann
computatton
DBH acityfty, pmof/mln per liter of serum
Age, y.a..
Range
Mean ± SO
No.
mean ± SD; range, 3.0-188.6;
n
=
153)
agree well with the previously reported normal DBH activity as assayed by a
photometric method of Nagatsu and Udenfriend (2) (42.6 ± 27.0, mean ± SD; range, 3-100; n = 54). After five counts by the Hoffmann computation, the mean and SD became lower (34.3 ± 18.2, mean ± SD; range, 3.0-73.9; n = 135). Males showed a slightly higher mean activity than females, but before or after the statistical computation by Hoffmann, the sex-related difference was not statistically significant. The computer-generated normal value for human serum DBH activity may be a suitable control value for the evaluation of serum DBH activity in
diseases.
Mechanismof Protectionand Activationof CreatineKinase Isoenzymesby Dlthiothreltolin HumanSerum We would like to comment in response to questions by readers and to the article by Morin (1) concerning creatine kinase (CK) isoenzyme MB assay with use of selective activation by dithiothreitol. This assay is only applicable to human serum and not to isolated mixtures, for
the following reasons: 1. It has been shown that the behavior of isolated CK isoenzymes differs from that of CK in human serum in (a) of activity
and
reactivation
CLINICAL CHEMISTRY,
2-72
4.0-73.9
53
1-74 1-74
3.0-72.3 3.0-73.9
per liter of serum Mean ± SO
34.3 ± 18.2
References
5 4 5
Keisuke
Fujita Maruta Ryoji Teradaira Hidehiro Beppu Mamoru Ikegame
Kazuhiro
1. Weinshilboum, R. M., and Axe!rod, J., Serum dopamine-fl-hydroxylase activity. Circ.Res.28,307 (1971).
2. Nagatsu, T., and Udpfriend, S., Photometric assay of dopamine-$-hydroxylase activity in human blood. Clin. Chem. 18, 980 (1972).
3. Horwitz, D. R., Alexander, R. W., Lovenberg, W., eta!., Human serum dopamine-hydroxylase. Circ.Res. 32,594 (1973). 4. Hoffmann, R. G., Statistics in the practice of medicine. J. Am. Med. Assoc. 185, 864 (1963). 5. Fujit.a, K., Nagatsu, T., Maruta, K., eta!., Fluorescence assay for dopamine-fl-hydroxylase activity in human serum by high-performance liquid chromatography. Anal. Biochem. 82, in press (1977).
can be reversed by adding glutathione or dithiothreitol, which deblock the active -SH of CK by binding themselves to the inhibitor(s). This effect is due to reactivation of an inactivated enzyme, not to further activation of a still-active enzyme, because the Michaelis constant for creatine and ATP do not change on adding sulfhydryl (5). Thus the active -SH of CK is blocked in human serum, protecting the enzyme from further inactivation. However, in isolated CK isoenzymes this physiological protection is not available, and this explains
Coun
36.0 ± 17.8 31.7 ± 18.7
their different
and behavior to temperature
stability
and -SH
activators.
Institute for Comprehensive Medical Science,
School of Medicine Fujita-Gakuen University Toyoake,
Aichi
470-11,
Japan
Nagatsu Takeshi Kato
Toshiharu
Laboratory of Cell Physiology Department of Life Chemistry Graduate School at Nagatsuda Tokyo Institute of Technology Yokohama 227, Japan
3. The active CK-SH sites can be de-blocked partly by fractionation on columns and electrophoresis, and fully by -SH activators. Those sulfhydryl compounds with redox potentials lower than that of the mid-point potential for reactivation or deblocking of the active site of CK-MM [-0.08 ± .005 V, 30#{176}C, pH 7.0 (ref. 7)j-namely cysteine, glutathione, N-acetyl cysteine, 2-aminoethyl isothiouronium bromide, HBr, and mercaptoacetic acid-can completely reactivate MM. CK-MB can be reactivated fully only by those -SH reducing agents that have a redox potential less than that of CK-MB (-0.31 ± .005 V), such as mercaptoethanol,
di-
by
suffliydryl (-SH) compounds (2,3); (b) stability on storage and effects of temperature and salts; and (c) effect of dilution (our unpublished data). 2. Physiological human serum contains substances (protectors/inhibitors) that can reversibly oxidize or specifically bind or block the active -SH sites of CK. These noncompetitive inhibitors include uric acid (4), oxidized glutsthione (5), alkaline phosphatase, ATPase, albumin, metal ions that can form mercaptides, inorganic anions (6), and others not yet identified. The protective effect or inactivation of active -SH 1948
82
sites of CK by inhibitors
To the Editor:
loss
unol/mln Range
135
computatIon
DRHactMty,
y.ars
sub
Male 92 2-72 4.0-188.6 44.8 ± 33.6 Female 61 1-74 3.0-1 14.9 39.1 ± 27.5 Total 153 1-74 3.0-188.6 42.5 ± 30.9 a Counts of re-computation by the methodof Hoffmann (4).
Ag.,
Vol. 23. No.
Table 1. Measurementof Free and De-blockedCK-lsoenzymesbefore and after Additionof Activators cK lso.nzym.
of total activity
measar.d
MM (freeactive)b
17.8 (8-30)
MM (free-active, deblockedreactivated)c
99.8 (98-102)
MB (free-active) b MB (deblocked-reactivated)
0.8 (0-5) 2.1 (0-8)?
c.d
#{149} Expressed as per cent dlthlothreltol activation of CK-MM and -MB (10 mmol of dlthlothreltol per lIter In serum and 9 mmol of reduced glutathione per liter In assay). b Assay medium withno activator. The CK Isoenzymes from human serum (n
=
15) are separated by
electrophoresls and quantitated by spectrophotometry afterelutlon from strip. C
Assay medium contains 9 mmol of reduced glutathione per liter. from heat inactivation experiments with human sara (MM and
I1 Obtained
10, 1977
MB)at 41 #{176}C.