John D. Batjer. Lab, of Pathol. Swedish .... Bates. (3) found a com- bined range of 11 to 29. We assayed the serum from two cases of suspected sar- coidosis.
ganese precipitation procedure for estimating high density lipoprotein cholesterol. J. Lipid Res. 19,65 (1978). 5. Warnick, G. R., Cheung, M. C., and Albers, J. J., Comparison
of current
methods
high-density lipoprotein cholesterol tation. Gun. Chem. 25, 596 (1979).
for
quanti-
6. Liedtke, R. J., Busby, B., and Batjer, J. D., Use of the DuPont aca t.o measure cholesterol in high-density lipoprotein fractions prepared by the heparin/Mn2 precipitation method. Gun. Ghem. 24, 161 (1978).
Raymond
J. Liedtke Gery Kroon John D. Batjer
Lab, of Pathol. Swedish Hosp. Med. 747 Summit Ave. Seattle, WA 98104
of children with muscular dystrophy (7), examination of the amniotic fluid for the presence of CK-BB might prove useful in detecting the disease in utero. References 1. Dawson, D. M., and Fine, I. H., Creatine kinase in human tissues. Arch. Neurol. 16, 175 (1967). 2. Bayer: P. M., GabI, F., Grand itsch, G., et al, Creatine spinal fluid
kinase isoenzymes in cerebroin a case of brain damage. Gun.
Ghem. 22, 1405 (1976). 3. Tsung, S. H., Creatine kinase isoenzyme patterns in human tissue obtained at surgery. GUn. Chem. 22, 173 (1976). 4. McNeely, M., Berris, B., Papsin, F. R., et a!., Creatine kinase and its isoenzymes in the serum of women during pregnancy and the peri-partum period. Glin. Ghem. 23, 1878 (1977).
Center
5. Caq,, A., de Virgilus, S., and Falori, A., The autogenicity of creatine kinase isoenzymes.
CreatineKinaseisoenzymeBB in HumanAmnioticFluid
immuno-inhibition:
To the Editor: Most of the BB isoenzyme
of creatine to be in neural tissues such as the brain (1, 2), although some has also been demonstrated in smooth muscle, especially the uterus (3). However, it has been found in the maternal circulation during pregnancy (4) and is also present in fetal muscle (5), so we were interested in determining if it could be detected in amniotic fluid. Accordingly, we obtained amniotic fluid specimens from 20 consecutive women after 14 to 36 weeks of gestation and processed the fluids as follows within 24 h. The fluid was centrifuged at 2000 X g for 10 mm in a refrigerated centrifuge, to sediment cells
The supernate was then 50-fold in a concentrator
unit at 4 #{176}C and assayed for total CK activity (6). The concentrated solution was then applied to an agarose gel plate (Corning, Palo Alto, CA 94306) and the CK isoenzymes were separated by electrophoresis. The isoenzymes were identified by the fluorescent technique described by Corning. In every amniotic fluid supernate we examined, CK-BB was the predominant isoenzyme, with various amounts of CK-MM also present. In 70% of the samples CK-BB was the only isoenzyme species detected. Total CK activity was 50 (SD 44) U/L, with no apparent relation to gestational age. We saw no CK activity in the cellular fraction of amniotic fluid. Our finding of CK-BB in amniotic fluid is consistent with the concept that this isoenzyme predominates in fetal muscle and is not essentially restricted to neural tissue, although the possibility of some leakage of the enzyme from the uterus cannot be excluded. Because CK-BB has been detected in the serum 1756
A clinical
evaluation.
Gun. Chem. 26, 568 (1980).
kinase (EC 2.7.3.2) is considered
and debris. concentrated
Biol. Neonat. 13, 375 (1968). 6. Delahunty, T. J., and Foreback, C. C., Automated creatine-kinase-MB estimated by
CLINICAL CHEMISTRY,
Vol. 26,
7.
Yasmineh,
W. G., Ibrahim,
G. A., Abbasdistribution of creatine kinase and lactate dehydrogenase in serum and skeletal muscle in Duchenne muscular dystrophy, collagen disease, and other muscular disorders. Gun. Ghem. 24, 1985 (1978).
nezhad, M., et al., Isoenzyme
Thomas J. Delahunty Craig C. Foreback
cloudiness must be caused by lipid substances in the extract. We substituted purified acetonitrile as the solvent,
which was readily
available
ride. Two further
modifications
units/mL. References 1. Lieberman, J., Elevation of serum angiotensin-converting-enzyme (ACE) level in sarcoidosis. Am. ,J. Med. 59, 365-372 (1975).
3. Bates, H. M., Angiotensin zyme assays in sarcoidosis. 13-15 (June 1980).
converting
en-
Lab. Manage.
James Sharon
Spectrophotometryof AngiotensinConverting Enzyme in Sarcoldosis
T. Taylor Freeman
Pat hol. Labs., Ltd. Hattiesburg, MS 39401
To the Editor: Serum angiotensin-converting enzyme (ACE) can be easily and reliably assayed by Lieberman’s (1) modification of the Cushman and Cheung (2) assay for ACE in rabbit lung, if one further alteration of the original method is
made. When we followed the procedure
of
Lieberman exactly, the solution that includes 1 mol/L sodium chloride, used in the final spectrophotometry of the liberated hippuric acid, was cloudy. This cloudiness was not necessarily the same in the zero-hour blank, so that the blank could not be relied upon to compensate properly for the increased absorption due to light scattering. Because Lie-
with using water
to dissolve the extract, as prescribed by Cushman and Cheung, he used 1 mol/L sodium chloride to decrease “the chance of a precipitate forming from substances extracted from serum by the ethyl acetate.” It occurred to us that much of the
No. 12, 1980
seemed
very helpful. If we extracted the liberated hippuric acid with 3.0 mL of ethyl acetate instead of 1.5 mL, emulsions never formed. Also, we chose to evaporate 2.0 mL of the ethyl acetate extract at 60#{176}C, in a stream of nitrogen, because this takes only 5 mm rather than 15 mm (1). Sera from 13 normal women, assayed by this technique, gave a mean value of 21.0 mt. units/mL (SD 6.5). Sera from 13 normal men gave a mean of 18.0 (SD ± 6.2). The range for the 26 was 8.2 to 37.0 mt. units/mL. Bates (3) found a combined range of 11 to 29. We assayed the serum from two cases of suspected sarcoidosis and found 41.5 and 47.0 mt.
2. Cushman, D. W., and Cheung, H. S., Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochem. Pharmacol. 20, 1637-1641 (1971).
Chem. Div., Pat hol. Dept. Henry Ford Hospital Detroit, MI 48202
berman had difficulty
from our
mobile solvent stocks for high-performance liquid chromatography. We found that standard hippuric acid in acetonitrile gives the same molar absorptivity as in 1 mol/L sodium chlo-
Determinationof Serum Creatinine in the Presenceof High Concentrationsof Free Hemoglobin To the Editor: In studying the use of stroma-free hemoglobin solution as a temporary erythrocyte substitute we needed to monitor organ function during exchange transfusion in adult baboons. Creatinine clearance was of special concern because transient changes in renal function have
been reported after stroma-free hemoglobin
Our serum samples
infusion solution
contained
of a (1).
signifi-
cant amounts of free hemoglobin. Thus we tested the feasibility of using a modification of the direct creatinine procedure of Heinegard and Tinderstrom (2). Their method involves a
