of serum bilirubin for a given reading of Tc-BM. For example, when the reading on the Tc-BM is 20, the corresponding serum total bilirubin is. 100 mg/L, with a ...
correction of Mandel in evaluating the slope, to compensate for the error in serum bilirubin determinations (4). Standard errors of estimate (S and S) were also determined. For 33 newborns weighing more than 2500 g, the comparison between 99 Tc-BM readings (y) and the corresponding
tion
serum
bilirubin concentraper liter gave the
(x) in milligrams
regression
following 0.129(±
0.O06r
equation: y 0.2); S
+ 6.7(±
= =
S, = 10. These results are similar to those in previous reports (1, 3). It is important to note the dispersion around the line, which is evaluated by the standard error of the estimate. The S, corre1.4,
sponds approximately to the standard deviation of serum bilirubin for a given
reading of Tc-BM. For example, when the reading on the Tc-BM is 20, the corresponding serum total bilirubin is 100 mg/L, with a 95% confidence interval of 80 to 120 mgfL. For 19 low-birthweight newborns from 1500 to 2499 g, we obtained the following results (n = 57): y = 0.12(±
0.011)x 13.
+
8.0(± 0.4); S
=
1.8, S,
with a change of serum biirubin in the opposite direction. Similar evaluations
in 12
newborns
weighing
over 2500 g
who were submitted to phototherapy gave poorer results. In fact, the relation was nearly random and the use of the Tc-BM on newborns who are undergoing phototherapy cannot be recommended. We also tried to read the Tc-BM index on the temple, because the eye mask covers a wider area and the results could have been better (5). Results for the temple (not shown) were similar to those for the forehead, with or without phototherapy. Our results suggest that the Tc-BM
can only be used as a screening device, and that a limit above which serum bilirubin concentration must be determined should be established. Serum bilirubin should be measured in newborns weighing more than 2500 g when the Tc-BM
reading
reaches 20 or
more, to detect those with a serum bilirubin exceeding 120 mgfL. For lowbirthweight newborns, otherwise normal, we set the limit to 17. Because the influence of various pathological condi-
tions is poorly known, we recommend that the Tc-BM not be used on sick newborns. References 1. Yainanouchi
tional Hospital. Pediatrics 65, 195-202 (1980). 2. Hannemann RE, Shreiner RL, DeWitt DP, et al. Evaluation of the Minolta Bilirubin Meter as a screening device in white and black infants. Pediatrics 69, 107-109 (1982).
Engel RR. Nuances or nuisances for cutaneous bilirubinometry? Pediatrics 69, 126-127 (1982). 4. Westgard 10, Hunt M. Use and interpretation of common statisticaltests in method-comparison studies. Clin Chem 19, 493.
57 (1973).
5. Vogel TP. Phototherapyof neonatal hyperbilirubinemia: Bilirubin in unexposed areas of the skin. J Pediatr 85, 707-710 (1974).
Douville Michel Masson Jean-Claude Forest1 Pierre
=
The dispersion is wider than that found for the babies of normal weight.
+8
A Tc-BM reading of 20 on a 2000-g
.6
newborn gave a 95% confidence range of 74 to 126 mg/L for serum bilirubin. The dispersion is of course not exclusively imputable to the Tc-BM, because the serum bilirubin measurements are not error-free. The day-today CV for serum bilirubin
concentration in the pediatric range was 3.9%. We tested the hypothesis that every newborn has his “own line,” which would permit one to follow the newborn bilirubin concentration (increasing or decreasing) concurrently with the Tc-BM. Each newborn would serve as his own control, and differences in Tc-BM readings would correlate closely with changes in serum bilirubin. Such a relation is shown in Figure 1. Figure 1 (top) shows the correlation between changes in Tc-BM readings and the variations in serum total bilirubin in 33 newborns weighing over 2SOOg. A change of 2 or more in Tc-BM readings was usually associated with a change in the same direction of serum bilirubin, but the quantitative relation was poor. For example, for a Tc-BM change of + 1, we noted a serum bilirubin change from -10 to +30 mg/L. The dispersion was even greater for lowbirthweight infants (Figure 1, bottom). The dispersion of serum bilirubin for a given Tc-BM reading was increased, as indicated by the higher standard error of the estimate (Si, = 17 mg/L, compared with 10 mg/L). Sometimes, significant changes in the Tc-BM readings (3 or more units) were associated
/
+10
+4 +2 .0
3
N.
-2
I, Yamauchi Y, Igarashi I.
Transcutaneous bilirubinometry and preliininary studies of noninvasive transcutaneous bilirubinometer in the Okayama Na-
Services of Biochem. and Neonatology H#{244}pital Saint -Fran#{231}ois d’Assise Qu#{233}bec, Canada GiL
3L5
‘Address correspondenceto this author.
fl 66
#{149} -4 -6
Serum Catalase Activity for Detection of Hemolytlc Diseases
U
-8 -10
-80
-40 40 Difference In biHrubln Concentrations (mg/Li
80
#{149}
.10 *8
.6 .4 3
a 3
+2
0 n 38 -2
S
#{149}U
-6
-8
e
-10
-80
-40
*40
.80
DifrerenceIn bilirubin Concentrations (mg/LI
Fig. 1. Changes in Tc-BM readings vs changes in serum bilirubin in (tq) 33 new-
To the Editor:
We reported the diagnostic value of measuring serum catalase enzyme (EC 1.11.1.6) activity in acute pancreatitis (1). The hemorrhagic form yields a higher value than the edematous form. Catalase activity in various human tissues differs greatly. The highest activity is found in erythrocytes, 3600fold that in serum (2). Thus the catalase activity of blood is practically all attributable to the erythrocytes (3). Furthermore, there is a correlation between erythropoiesis and serum catalase activity, but this correlation is influenced by hemolysis (4, 5). Determination of serum catalase activity is fast, simple, and inexpensive with our polarographic method (6, 7), and we attempted to use it in the diagnosis of hemolytic diseases. We examined serum catalase activity in pernicious anemias, acquired hemolyt-
borns >2500 g and (bottom) 19 low-birth- ic anemias, and other hemolytic diseases. newborns: #{149} 2000 to 2499 g, 01500 to 1999 g For comparison we examined serum “n’ refersto numberof tests catalase activity in nonhemolytic aneweight
CLINICAL CHEMISTRY, Vol. 29, No. 4, 1983 741
mias (erythroid hypoplasia) and in polyglobulinemia and polycythemia
slightly increased serum catalase activity in polyglobulinemia, and in-
vera
creased activity in polycythemia vera. These results are in agreement with those of others (4, 5, 8). In untreated pernicious anemia we found increased serum catalase activity, a-HBDH activity, and LDH activity, and decreased profile LDH values. After seven days of treatment, the serum catalase activity returned to the normal range [89.1 (SD 27.3) kU/L, n = 111 earlier than the a-HBDH and LDH activities. In four cases the greatest serum catalase activity was observed during treatment and in three cases it started to decrease before treatment. Our results agree with those of others (8, 9). In pernicious anemia the increased activities of catalase, a-HBDH, LDH, and the decreased value of profile LDH may be explained by the increased but ineffective hemopoiesis causing increased destruction of the megaloblastic cells in the bone marrow. In different types of hemolytic anemias we found increased serum catalase, a-HBDH and LDH activities, and decreased profile LDH values. Our results are in agreement with those of others (4, 5, 8, iO) who suggested that increased serum catalase activity is an appropriate indicator of hemolysis. In Zieve’s syndrome, a disease also accompanied by hemolysis, we detected increased serum catalase, a-HBDH, and LDH activities, and decreased proifie LDH values. The serum catalase activity decreased slowly, exceeding the normal range [140.7 (SD 72.9) kU/ n = 71 on the seventh day. Increased serum catalase activity [374.8 (SD 262.7) kU/L, n = 741 was also found in acute hemorrhagic pancreatitis (1).
(erythroid
hyperplasia).
We ex-
amined 200 patients with untreated nonhemolytic anemia in whom erythroid hypoplasia was suspected (hypochromic anemia 85, protein malnutrition 27, anemia secondary to kidney disease 26, secondary to liver disease 23, secondary to cancer 22, and aplastic anemia 17). In diseases with enhanced erythropoiesis we examined 250 patients with polyglobulinemia resulting from compensatory effect of cardiopulmonary diseases and 14 patients with polycythemia vera as the primary disease of the hemopoietic system. We examined 18 patients with untreated pernicious anemia. Patients with acquired hemolytic anemia were the following: two with complications in blood transfusion, six with drug- or chemical-reagent-induced hemolysis and three with bacteria- or virus-induced hemolysis. Nine patients had Zieve’s syndrome, which is a combination of icterus, hyperlipemia, and hemolysis, together with liver cirrhosis. There were no cases of glucose-6-phosphate dehydrogenase deficiency in this group.
We measured the serum catalase activity with our fast, simple, inexpensive polarographic method (6, 7). The mean reference interval (and 2 SD) is 56.7 (42.6) kU/L (n = 111). We also measured lactate dehydrogenase activity (LDH opt. monotest; Boehringer, F.R.G.), hydroxybutyrate dehydrogenase activity (a-HBDH opt. monotest; Boehringer), LDH profile values (profile LDH test; General Diagnostics, Warner-Lanibert Co., Morris Plains, NJ 07950), blood hemoglobin value (Drabkin’s method), erythrocyte count (Celloscope, Ljunberg, Sweden), and hematocrit to compare these with the serum catalase activity. Our results (Table 1) show a slightly decreased serum catalase activity in different nonhemolytic anemias,
Serum catalase activity
in nonhemo-
lytic anemias was 0.62 of the normal mean value, but it showed increased activity in hemolytic anemias (8.3-fold) and in pernicious anemia (6.6-fold).
Therefore measurement of catalase activity in serum may help in the diagnosis of hemolytic anemias. To differentiate
among the hemolyt-
ic diseases, which yielded increased serum catalase, a-HBDH, and LDH enzyme activities, we may calculate the catalase/a-HBDH and the catalase/ LDH ratios. The normal values for these ratios are 0.58 and 0.32. In pernicious anemia these ratios are decreased (0.31 and 0.21), in hemolytic anemias they are increased (0.98 and 0.63), and in Zieve’s syndrome they are slightly more increased (1.16 and 0.64). In hemolytic diseases, determination of serum catalase activity has some advantages. The reagents are simple and cheaper than those for the traditional isoenzyme tests. The polarographic method is fast and it is uninfluenced by the increased concentration of bilirubin in serum in hemolytic anemias or by the hyperlipemia seen in Zieve’s syndrome. In hemolytic diseases the increase in serum catalase activity is persistent, and so better reflects recovery or new attacks than does serum haptoglobin. Furthermore, the catalase/a-HBDH and catalase/ LDH ratios may help differentiate the hemolytic diseases. References 1. (36th L, M#{233}sz#{225}ros I, Nemeth H. Serum
catalaseenzymeactivity in acute pancreatitis. Clin Chem 28, 1999-2000 (1982). Letter.
2. (36th L. Determination of catalase enzyme activityin human tissues by programmable polarograph. Hung Sci Instrum 53, (1982). 3. Aebi H. Catalase. In Method.s of Enzymatic Analysis (2nd English ed.) Verlag Chemie GmbH, Weinheim, 1974, pp 673. 4. Yamagata S, Seino S. Studies on blood and plasma catalase. Tohoku JExp Med 57, 101-107, 231-238 (1953). 5. Kirsch W, Burmeister W. Bestimniung 43-46
der Serum- und Plasmakatalase beim Menschen unter verschiedenenphysiologis-
Table 1. Serum Catalase and Other Measurements in Patients with Nonhemolytic and Hemolytic Diseases Erythrocytes, Catalase, kU/L
LDH, U/L
Mean
SD
34.6
13.1
-
-
109.9
46.3
-
242.7
68.8
-
Pernicious anemia n = 18
377.8
314.6
Hemolytic anemia
470.4
397.9
Nonhemolytic anemia n = 200
Polyglobulinemia n = 250 Polycythemiavera
SD
a-HBDH, U/L
Mean
SD
LDH
profile
Mean
SD
1012 L’
Hemoglobin, Hematocrlt, mmol/L
_________
Mean
SD
Mean
SD
Mean
SD
-
2.84
0.46
1.32
0.31
29.5
4.8
-
-
5.32
0.34
2.58
0.18
51.7
4.1
-
-
6.89
0.53
3.21
0.26
65.4
4.6
Mean
n = 14
n
=
1782.3
1035.1
1212.3
611.1
0.429
0.132
1.65
0.25
0.80
0.14
17.2
2.4
332.2
744.8
316.0
479.0
248.6
0.546
0.134
2.30
0.84
1.16
0.46
23.5
9,2
205.4
589.2
184.9
343.4
85.8
0.601
0.094
3.68
0.49
1.80
0.28
36.0
5.1
11
Zieve’ssyndrome n=9 Normal range
14.1-99.3
120-240
742 CLINICALCHEMISTRY, Vol. 29, No. 4, 1983
55-140
0.8-1.2
3.84-4.88
1.79-2.39
38-48
chen und pathologischen Bedingungen. Arvh Kinderheilk 174, 153-161 (1966). 6. (36th L, M#{233}sz#{225}ros I. Polarographic determination of serum catalase activity. Hung Sci Instrum 32, 13-16 (1975). 7. (36th L. Determination of serwn catalase
enzyme activity with programmable polarograph. Kdrhdz- #{233}s Orvostechnika 20, 6-9 (1982). In Hungarian. 8. Dille RS, Watkins H. Plasma catalase in hemolytic diseases and other abnormal states. J Lab Clin Med 33, 487-496 (1948). 9. Heller H. Bedeutung der Katalase fir die Praxis. Med Welt 45, 2482-2487 (1969). 10. Gianrntsis DJ, Panagopoulos DA, Timmerman A, et al. Storungen der Katalase des Serums bei verschiedenen pathologischen ZustSnden des Menschen. Enzymologin 42, 355-361 (1972).
hydroxylation
(6) and
prenolol,
influenced
the result.
Six standard solutions were preL#{225}szl#{243} Goth pared in Od mol/L hydrochloric acid Hajnalka N#{233}methand (or) glacial acetic acid, and each Istv#{225}n M#{233}sz#{225}ros was diluted with water to concentrations of 20 and 200 mgfL. The solutes Depts. of Lab. and Intern. Med. were (a) 5-HT creatinine sulfate, (b) 5Municipal Hospital, Sumeg HIAA, (c) oxprenolol hydrochloride, Hungary, H-8330 and (d) 4-hydroxyoxprenolol semioxalate. In addition, urine samples supplemented to these same compounds in Interference of an Oxprenolol the same concentrations were pre-
Metaboiltewith ScreeningTests for 5-Hydroxyindolein Urine To the Editor: Patients with metastasizing carcinoid tumors excrete increased quantities of 5-hydroxyindoles (5-hydroxytryptamine, indoleacetic
5-HT; 5-hydroxyacid, 5-HIAA; and
pared.
For the total 5-hydroxyindole test, 1 mL of each aqueous solution and urine sample, and 1 mL of water and urine
chromophore. For quantitative determinations the absorbance was mea-
sured at 540 nm. Results were identical for the aqueous solutions and the supplemented urine samples. Positive results were obtained in both diagnostic tests with concentrations of 5-liT and 5-HIAA only marginally above the upper limits of their normal range (i.e.,
0.6
5-
hydroxytryptophan) derived from tryptophan (1). Diagnostic tests for such tumors measure urinary concentrations of total 5-hydroxyindoles--or more specifically, of 5-HIAA-by reaction with 1-nitroso-2-naphthol in the presence of nitrous acid to yield a violet chromophore of undefined chemical structure (2-4). In tests involving single urine samples, results for total 5hydroxyindoles are expressed in terms of milligrams per gram of creatinine. Normal urines contain less than 10 mg, urines from patients with metastasizing tumors contain several hundred milligrams. The proportion of the individual hydroxyindoles in urine varies, but 5-HIAA usually predominates. The normal rate of excretion of 5-HIAA is less than 10 mg/24 h, but patients with carcinoid tumors excrete from 30 to 1500 mg/24 h. Several false-positive diagnostic results have been obtained in patients treated with the /3-adrenoceptor blocking drug, oxprenolol. These have been reported to the pharmaceutical company concerned but have not appeared in the literature. Several drugs are known to interfere with the screening procedures (5) but /3-adrenoceptor blocking drugs have not previously been implicated. Oxprenolol undergoes aromatic ring
were diluted with 1 mL of water, then 1 mL of a 1 g/L solution of 1nitroso-2-naphthol in ethanol was added. After shaking, the solution was mixed with 1 mL of nitrous acid reagent (0.2 mL of a 25 g/L aqueous solution of sodium nitrite + 5 mL of 1 moIJ L sulfuric acid), and then left to stand for 10 mm before being shaken with two 5-mL portions of ethyl acetate. The organic layer was discarded. For the 5-HIAA screening test, 2 mL of phosphate buffer (0.5 moIJL; pH 7) and 1 mL of the 1-nitroso-2-naphthol solution were added to 1 mL of each of the solutions (as before). After shaking, 1 mL of nitrous acid reagent was added to each and the mixture was warmed for 10 mm at 37 #{176}C. The solutions were shaken with two 5-mL portions of ethyl acetate and the organic layer was discarded. The remaining aqueous phases from each test were examined for the presence of a violet blank
significant
amounts of both 4- and 5-hydroxy metabolites have been identified in human urine, in an approximate ratio of 4:1 (7). 1-Nitroso-2-naphthol reacts with many substituted phenols under acidic conditions to produce colored derivatives; interference by hydroxylated metabolites of oxprenolol with both diagnostic tests has been proposed to explain false-positive results in patients treated with this drug. In the present investigation the total 5-hydroxyindole and 5-HIAA diagnostic tests were examined to establish whether the major ring-hydroxylated metabolite of oxprenolol, 4-hydroxyox-
0.4 0.2
0
C (0 .0 C 0 U)
.0
0.0 350
400
450
500
550
600
700
650
1.0 0;8
4 0.6 (c)
0.4 0.2 0.0 350
400
450
500
550
600
650
700
Wavelength(nm) Fig. 1. Absorption spectra of 1-nitroso-2-naplithol reactionproducts with: (a) 5-hydroxytryptamine,20mg/I.., (b)4-hydroxyoxprenolol,20 mg/L, in the total 5-hydroxyindoletest (upper graph) and (C) 5-hydroxyindole acetic acid, 30 mg/L, and (c 4-hydroxyoxprenolol, 15 mg/L, in the 5HIAA diagnostic
test (lower graph)
CLINICALCHEMISTRY, Vol. 29, No. 4,
1983
743
