and statistically tested to obtain linear .... CPK-MB. (1) indicate that âelectro- phoretic techniques used were not sensitive ... and Varat's report of CPK-MB in con-.
much more strongly (257 = 21500) than in neutral or acidic medisorb
um
(#{128}255 = 740),
a change
that
Table 1. Absorbance vs. Incubation Time
can be
Time at 37#{176}C, mm
produced repeatedly with a sample. 45
60
75
90
Creatinine-free serum
0.00
0.00
0.00
0.00
Creatinine-free serum with added creatinine, 50, 100 and 150 mg/liter
0.46 0.94
0.50
1.00
0.50 1.00
1.43
1.50
1.50
0.50 1.00 1.50
Normal serum
0.09
0.10
0.10
0.10
Sample
A quantitative modification of the TLC technique is also possible, but it requires equipment that costs about as much as that for GLC or RIA. With the chromatogram spectrophotometer (Zeiss, Oberkochen, F. R. Germany) a 0.5-gig sample of barbital can be detected. This same instrument was used in an earlier work to assess imipramine and desipramine in human blood plasma (2), and recently has been modified and statistically tested
Uremic serum
1.12
1.20
1.20
1.20
Aqueous creatinine, 50, 100 and 150
0.50 1.00
0.50 1.00 1.50
0.50
0.50 1.00
to obtain linear relationship between substance amount and instrument response (3). Several years of experience with TLC scanners in the analysis of steroids, pharmaceuticals, and drugs in biological samples suggest that these instruments deserve the clinical chemists’ attention. If properly used, they offer great sensitivity and reliability. Because detection is a nondestructive process, the samples are available for further studies after the measurement. For confirmatory reasons an appropriate reagent may be used for visualization, or subsequent GLC and RIA can be used, if the circumstances require it.
References 1. Roerig, D. L., Lewand, D. L., Mueller, M. A., and Wang, I. H., Comparison of radioimmunoassay with thin-layer chromato-
graphic
and
gas-liquid
chromatographic
methods of barbiturate detection in human urine. Clin. Chem. 21, 672 (1975). 2. Nagy, A., and Treiber, L., Quantitative determination of imipramine and desipramine in human blood plasma by direct
densitometry
of thin-layer
J. Pharm. Pharmacol.
chromatograms.
25, 599
(1973).
3. Treiber, L. R., Standardisation of the direct spectrophotometric quantitative analysis of chromatograms. IV. A comparative study on the most common mathematical treatments of thin-layer densitometric problems. J. Chromatogr. 100, 123 (1974).
mg/liter
sorbance of the second reagent measured vs. the first determines the creatinine concentration of the sample. Chromogens do not interfere. In further studies of the Jaff#{233} reaction at pH 11.50, I find that the protein interference can be completely eliminated if the alkaline picrate reagent is added after heat coagulation of the serum sample. This permits estimation of “true” creatinine by the following procedure: Transfer 0.10 ml of serum to the bottom of a 100 X 15 mm tube. Place in a boiling water bath
for 3 mm, remove, cool, and add 0.75 ml of the alkaline picrate reagent buffered at 11.50 (1, 2). Keep the tube at 37 #{176}C for 60 mm, and measure the absorbance at 500 nm (10-mm pathlength cuvet) vs. 0.10 ml of water with 0.75 ml alkaline picrate reagent also incubated at 37 #{176}C. The validation and accuracy of this procedure are presented in Table 1. Creatinine-free serum does not react at all with the alkaline picrate reagent for as long as 90 mm. This was consistently observed for 100 different sera, some of which were lipemic, icteric, or hemolyzed (protein 4.5-7.8 g/dl) that had previously been dialyzed as mentioned elsewhere (1).
Creatinine-free Laszlo
R. Treiber
Improved DeterminatIon of “True” Creatinine in Serum or Plasma
termined
“true” serum creatby a simple spectrophotometric measurement by using two alkaline picrate reagents, buffered at pH 9.65 and 11.50. The mine
can
color
produced
2),
be determined
from
the
reaction
serum with the first reagent protein alone, whereas, with the second reagent protein and creatinine.
1848
CUNICAL
of
is due to
that produced is due to both Thus, the ab-
CHEMISTRY,
serum
with added cre-
atinine and serum from normal and uremic subjects, as well as aqueous creatinine solutions, give similar reaction. Complete extraction and full development of color are attained after 60 mm and remain stable as long as 90 mm. “True” creatinine can thus be de-
Dept. of Macromolecular Science Case Western Reserve University Cleveland, Ohio 44106
To the Editor: As reported (1,
1.50
with
a
single
reagent
and
without need for a blank. The procedure also has the advantage of being equally applicable to plasma and not exclusively to serum, as the initial one was because of the appearance of turbidity when plasma is reacted with alkaline picrate reagent buffered at 9.65. No difference was recorded between creatinine in serum and heparinized plasma for 50 subjects (6-170 mg/ liter). Absorbance was linear from 5 to
Vol. 21, No. 12. 1975
200
1.00 1.50
mg of creatinine
1.50
per liter, with a
coefficient of variation and percentage of recovery ranging between 0.5-1.5% and 98-101%, respectively. References 1. Yatzidis H., New method for direct determination of “true” creatinine. Clin. Chem. 20, 1131 (1974). 2. Correction. Clin. Chem. 21, 157 (1975). Hippocrates
Yatzidis
Nephrological Center Aretaieon University Hospital Athens 611, Greece
Creatine Klnase lsoenzyme MB and Heart Disease To the Editor: Mercer and Varat in their study of CPK-MB (1) indicate that “electrophoretic techniques used were not sensitive enough to detect the low MB activity in sera with normal or slightly above-normal total CK activity.” This statement needs to be clarified in light of other studies (2). We have reported 100%
sensitivity
of CPK-MB
for
myo-
cardial infarction in a series of 100 consecutive admissions to the CCU, using a fluorometric agarose electrophoretic system. Furthermore, we reported 3% of total activity as our cutoff for CPK-MB, which agrees very well with the above authors’ results. What is most interesting is Mercer and Varat’s report of CPK-MB in congestive heart failure, atrial fibrillation and cardiomyopathy. We similarly found and reported CPK-MB (above 3%)
in cases
of coronary
insufficiency,
angina, congestive heart failure and pulmonary embolism. Our assay permits detection of significant amounts of CPK-MB
in
the clinical
course,
still
within
infarct
normal
cases total limits.
while
early
in
CPK is Clearly,
electrophoretic separation in agarose followed by fluorometric detection meets the criteria of sensitivity and
specificity jremely
to be considered accurate
diagnostic
test
NORMAL
an ex(3).
References
K
60.
K
(23-43)
1. Mercer,D. W. and Varat,M. A.,Detection
of cardiac-specific
creatine
kinase
iso-
enzyme in serawith normal or slightly increased total creatine kinase activity. Clin. Chem. 21, 1088 (1975). 2. Galen,R. S.,Reiffel, J. A., Gambino, S. R., Diagnosis of acute myocardial infarction. J. Am. Med. Assoc. 232, 145 (1975).
3. Krieg, A. F., Gambino, S. R., and Galen, R. S.,Why are clinical laboratory tests performed? When are they valid? J. Am. Med. Assoc. 233, 76 (1975). Robert
S. Raymond Department
Columbia Medical
0
0
K, K,
(17-41) 40-
0
340 (24-4)
Presbyterian Center
New York, N. Y. 10032
To the Editor:
We find that abnormal blood samples give a different yield of porphyrin in the extraction procedure used in this method. Seven to nine extractions with acidified ether were necessary to completely extract the porphyrin, i.e., obtain zero or small negative porphyrin values in further extractions. We also observed that six extractions removed 82 to 90% of the porphyrin present in the blood sample. Accordingly, we decided to assay normal and abnormal samples by using the procedure as originally described, i.e., measuring the porphyrin present in the combined first two extractions with acidified ether, designated as EP2 in to
this report. We then did four further extractions in the same way. The porphyrin present in the combined third to sixth extractions is designated EP4. All assays were done with reagents belonging to single batches. We obtained data on EP2 and EP4 for 33 heparmnized blood samples divided into two groups: 12 normal adults (10 men and two women), and 21 anemic patients (10 women in the puerperium; three women and one man with iron-deficiency anemia; and seven patients with hypoplastic anemia, of whom two were males). The samples were assayed within 0.5 to 4 h after extraction and were kept refrig1850
J
DIAGNOsIs
A BC
Fig. 1. The percentage ratio EP2/(EP2 + EP4) for 12 normal individuals and 21 anemic patients In the latter, the mean and range of the hematocrit in the three subgroups made on the basis of
the ratio are shown in the right-hand column. Diagnoses In the anemic group: A, 10 women in the puerperium; B, 4 patIents with iron-deficiency anemia; C. 7 wIth hypoplastic anemia. Sex Is Indicated by (x) for females, and (.) for males
erated. The hematocrit was estimated in Wintrobe tubes centrifuged for 30 mm (2200 X g). Table 1 summarizes our results for the normal and anemic groups. The standard deviation corresponded well with the range in the normal group but not in the anemic one. Significant differences by the t-test between groups were present in the five variables shown in Table 1. Although the
differences
hematocrit and the EPs
in
are in agreement with other reports in the literature, the group difference (t = 3.94;p = 0.0002) in the ratio EP2/ (EP2 + EP4) is unexpected and prompts
this
into with
nor etiology
three an EP/
Letter.
of the anemia
with the differences
ratio in these Technical
20
for
to 49%), and five with a higher ratio (56 to 65%). As Figure 1 shows, neither correlated
Gambino
The WHO (1) has urged research on the possibilities of using the assay of erythrocytic protoporphyrin in the study of nutritional anemias and has recommended the simplified procedure described by Heller et al. (2), a method in which total porphyrins, not just protoporphyrin, is measured.
subdivided four patients
hematocrit
C
of Pathology
ratio
(EP2 + EP4) ratio comparable to normal, 12 with intermediate values (38
#{149}
0.
the
in the ratio; they can be
er variation K,
that
normal individuals is narrow (30 to 36%) in 10 of the 12 subjects, and a single value of 46%, seen in a normal woman with a hematocrit of 50%, fell outside the limits of mean ±2 SD. In contrast, the anemic group had a larggrossly subgroups:
50-i
S. Galen
Difference in ExtractabIlity of Porphyrins from Blood of Anemic Persons
1 shows
Figure
ANEMIC
three subgroups. reasons appear
in the unlikely
as causes for the variations in the ratio: reagents used throughout the study came from single batches, and the spectrophotometer (Hitachi, Model 139) was in excellent working condition. It was also established that increasing the time or modifying the conditions of stirring when extractants were added did not alter results. The acetone/ethyl acetate mixture and the HC1 were excluded as sources of error. Delay in the separation of the HC1 layer from the acidified ether was avoided because it introduces errors that are directly proportional to time elapsed before separation. The ratio EP2/(EP2 + EP4) can be considered a measure of recovery of intrinsic porphyrins present in erythrocytes when two extractions with
acidified
ether are done. As such, our
recoveries never reach the 84 to 94% seen with two extractions by Heller et al. (2), but it should be pointed out that these authors and others (3-5) have used added (extrinsic) protoporphyrin in their recovery testing, and that decreased recovery in the presence of erythrocytes has been observed even for extrinsic protoporphynfl (5). We conclude that blood from anemic patients does not always behave
Table 1. Results for Porphyrin in Blood of 12 Normal and 21 Anemic Individuals Variable
Group
%a
Normal
Hematocrit,
Anemic EP2,pg/literb
EP4, pg/literc
EP2
+
155
33
923
507
1093
Normal
466
+
EP4) x 100
33.4
Anemic
44.5
120-220
240-2080 210-390
540-2520
488
72
330-600
821
2018
Normal
44-59 17-43
55
311
Anemic
Range
3.68 7.51
Normal
Anemic
EP2/(EP2
SD
51.6 32.5
Anemic Normal
EP4, pg/liter
Mean
780-3450
5.04
25-46
11.05
23-65
a Higher values because of Mexico b EP2, erythrocytic C
EP4, erythrocytic
CLINICAL CHEMISTRY, Vol. 21, No. 12, 1975
porphyrin
City altitude, 7350 feet. in the first two extractions with
porphyrin
in four further
extractions
acidified
ether.
with acidified
ether.
