Elkton,. MD 21921). Serum samples were prepared and were concurrently assayed in duplicate in two separate assaysby the Amerlex FT4 RIA method.
epimerase in fibroblast.s and leukocytes. The increase in the radioactivity in the
diphosphate
fraction
(6) was linearly
concentration up to 20 The epimerase reaction in fibroblasts as well as in leukocytes, exrelated smol/L.
to
pressed as the dilution of UDP-glucose with time, was linear up to 60 mm. Table 1 summarizes the activity of epimerase in various human tissues. In each case, the activity in erythrocytes, as determined with the radioactive method, was similar to that determined spectrophotometrically. Interestingly, in contrast with the values for erythrocytes, the specific epimerase activity in fibroblasts and amniotic fluid cells considerably exceeds that of unidylyltransferase (EC 2.7.7.12) (7). We previously described a sensitive radioactive method for determination of galactose 1-phosphate in various human tissues (8). Here, we first tried the same procedure for the UDP-glucose determination, using [‘4C]galactose 1-phosphate and uridylyltransferase. However, UDP-gal inhibited uridylyltransferase activity, resulting in high blank values. We then tried the UDPglucose pyrophosphorylase reaction (UDPglucose + pyrophosphate glucose 1-phosphate + UTP), using UDP[‘4C]glucose. UDP-gal does not inhibit the latter reaction, most probably because the UDP-gal is an ineffective substrate for pyrophosphorylase, even in a concentration as high as 200 smol/L.
In conclusion, we found it relatively simple to determine epimerase activity in erythrocytes as well as in cultured cells. This method should be helpful for the study of an aberration in galactose metabolism, the epimerase deficiency. References 1. Gitzelmann, R., Deficiency of uridine diphosphate galactose 4-epimerase in blood cells of an apparently healthy infant. Preliminary communication. Helu. Paediatr. Acta 27, 125-130 (1972). 2. Gitzelmann, R., and Steinmann, B., Un-
dinediphosphate galactose 4-epimerase deficiency. II. Clinical follow up, biochemical studies and family investigation. Helu. Paediatr. Acta 28, 497-510 (1973). 3. Gitzelmann, R., Steinmann, B., Mitchell, B., and Haigis, E., Uridine diphosphate galactose 4-epimerase deficiency. Report of eight cases in three families. Helu. Paediatr. Acta 31,441-452 (1976). 4. Oyanagi, K., Nakata, F., Hirano, S., et al., Uridine diphosphate galactose 4-epimerase deficiency. Ear. J. Paediatr. 135, 303-304 (1980). 5. Holton, J. B., Gullet, M. G., McFaul, R., and Young, R., Galactosemia: A new severe variant due to uridine diphosphate galactose4-epimerase deficiency. Arch. Dis. Child. 56, 885-886 (1981). 6. Shin-Buhring, V., Osang, M., Tan, T., et al., The activity of galactose-1-phosphate uridyltransferase and galactokinase in human
fetal organs. Pediatr. Res. 11, 1043-1051 (1977). 7. Shin-Buhring V., Leitner, H., Henseleit, H., et al., Characteristics of galactokinase and galactose- 1-phosphate unidyltransferase in cultivated fibroblasts and amniotic fluid cells. Hum. Genet. 48, 31-37 (1979). 8. Schaub, J., Shin-Buhring, V., Wiese, B., et al., Metabolism of galactose and accumulation of galactose-1-phosphate in various cell types of cultured fibroblasts for galactosemia.
In Models for the Study of Inborn Errors of Metabolism, F. A. Hommes, Ed., Elsevier/ North-Holland Biomedical Press, New York/Amsterdam, 1979. Yoon S. Shin’ Axel von R#{252}cker Manfred Rieth Wolf Endres Children’s Hosp. Univ. of Munich
Munich,
Address
enzyme band is seen between MM.
MB and
John Olson Janine Evancho Judi Randall Tjien 0. Oei Indiana
Univ.
Sch. of Med.
Dept. of Pathol. 926 W. Michigan St. Indianapolis, IN 46223
Siliconized vs Nonsiliconlzed Evacuated Blood-Collection Tubes for Free Thyroxln Measurements To the Editor:
F.R.G.
Kinderklinik, F.R.G.
possibility of a highglycineconcentration in the blood ifan atypical CK iso-
all correspondence
Lindwurmstr.
to V. S. Shin,
4,8 MUnchen 2,
Variant Creatine Kinase lsoenzyme Band Induced by Glycine To the Editor: A 72-year-old man was admitted to the Indiana University Hospital with symptoms of urinary retention and urethral obstruction. He had a history of insulin-dependent diabetes mellitus and
It has been reported recently (1) that serum samples prepared from blood collected in siliconized evacuated blood-collection tubes may give artefactually increased results for free thyroxin (FT4) when assayed by some solid-phase direct radioimmunoassay (RIA) methods, whereas F’T4 results for sera prepared with blood collected into equivalent nonsiliconized collection tubes were not affected. The suitability of both siliconized and nonsiliconized evacuated tubes for blood collection for serum FT4 measurements
by the Amerlex TM FT4 RIA method was examined during the development of this product, as part of its routine challenge testing program for producthypertension. The urologic workup reclaims-support purposes. Paired blood vealed an enlarged prostate with bladder samples were drawn from each of 21 dilatation and possible neurogenic euthyroid volunteers into both siliconbladderdysfunction. ized collection tubes (Corvac integrated During transurethral resection of the serum separation tubes; Sherwood prostate gland an irrigation solution Medical, St. Louis, MO 63103) and nonsiliconized tubes (Venoject; Kimble, containing glycine (15 g/L) was used. The patient developed electrocardioElkton, MD 21921). Serum samples graphic changes and decreased blood were prepared and were concurrently pressure, indicative of severe cardiac assayed in duplicate in two separate problems. Blood was drawn for several assays by the Amerlex FT4 RIA method tests, including isoenzymes of creatine (Amersham Corp., Arlington Heights, IL kinase (CK) and lactate dehydrogenase 60005). The mean FT4 value for samples (LD).
The LD pattern
showed
the
usual
isoenzymes, but the CK pattern had a large atypical band between isoenzymes MM and MB. CK isoenzymes were resolved on cellulose acetate (Helena fluorescent procedure). We suspected that glycine
absorbed
during
the irrigation
may have been the cause of this atypical band. To test this idea, we added glycine to serum that exhibited MB and MM bands and to another serum that had BB, MB, and MM bands. In both instances a band formed that corresponded to the atypical band in the patient’s serum. Irrigation with glycine solution is a standard procedure in transurethral resection, so one should consider the
collected into nonsiliconized collection tubes was 17.3 (SD 2.8) pmol/L; the
mean for sera prepared by using siliconized tubes was 16.9 (SD 2.6)pmolfL. The within-run coefficient of variation in FT4 values over the range of samples studied was 2.6% (n = 84). A withinassay comparison (paired t -test) of results for both types of blood-collection tubes revealed no statistically significant difference in FT4 values at the p = 0.05 level. To test if the FT4 results for both types of collection tube behaved in the same manner for all 21 patients, a split-plot analysisof variance was carried out. The interaction (siliconized vs nonsiliconized tubes X patients) was not statistically significant (F-test) at the p
CLINICAL CHEMISTRY,
Vol. 28, No. 11, 1982
2333
=
0.05
level,
indicating
that
the
results
were equally unaffected by choice of collection tube for all 21 patients. In summary, for the Amerlex solidphase direct F’I’4 RIA procedure, results obtained for sera prepared by using siliconized evacuated blood-collection tubes agreed well with those for corresponding serum samples obtained by using equivalent nonsiliconized tubes. In agreement with Braverman and Ingbar (1), we conclude that the two types of tube are equally suitable for blood collection for thyroid function testing by use of the Amersham FT4 RIA kit.
Table 1. Demonstration of Liposyn Interference with a Neonatal Bilirubln Assay and Correction with Persulfate Treatment Before
Liposyn a
Blllrubln Stat Analyzer reading, mg/L After After Llposyn persulf ate addn
224
107
117
87
193
99
94
85 30
192 133
99 98
93 35
97
212
102
110
80
183 190 209
100 100 97 97
83 90 66 112
87 65
Reference
163
milial euthyroid roxinemia-effects
R. R. McKiel N. Barron C. J. Needham T. A. Wilkins Amershan2 Corp. 26365. Clearbrook Arlington Heights IL 60005
100
63 64
163 163
100
63
95
185
96 92
67 93
99(3)b
89(14)b
Dr.
International
8
Adjusted for subsequent dilution.
b Mean
(and SD), n
Heights,
MA 02194) gave values of 51 (0900 hours specimen), 97 mg/L
(2000 hours
specimen),
and 59 mg/L
(0100 hours specimen) the following day. There had been no exchange transfusions during this time period. The sam-
plc
ple yielding a bilirubin value of 97 mgfL appeared lipemic. Repeat total bilirubin measurement on this lipemic sample by a manual modification (4) of the method ofJendrassik and Grof gave a value of 42 mg/L. The medical record of the neo-
White Lion Road Amers ham HP7 9LL,
85(l3)t
mg/L
and
Bucks.
100
138
tion testing. In Proc. Advanced Course on Free Hormone Assays and Neuropeptides, Venice, 1982, A. Albertini, Ed., Elsevier Biomedical Press, Amsterdam, in press.
Amersham
176 242
77 L. E., and Inbar, S. H., Fa. dysalbuminemic hyperthyon in vitro thyroid func-
U.K.
nate
revealed
that
Liposyn
10%
had
been administeredintravenously before collection of the 2000 hours specimen. Liposyn#{174} Interference with To demonstrate the interference in vitro, we added 10 tL of Liposyn 10% to Neonatal Bilirubin Measurements 0.1mL ofeachof14 serafrom neonates thathad been submitted for total biliTo the Editor: rubin determinations. Results are shown We report falsely increased values for in Table 1. The addition of Liposyn retotal bilirubin as determined by direct sults in an average positive interference spectrophotometry ina neonatewho was receivingintravenousfeedingwith Li- of 103 mg/L in total bilirubin measurement. The magnitude of this interferposyn,’a 10% fatemulsion.Although lipemia has been reportedto interfere ence approximates an in vivo effect that would be expected for a sample collected with direct spectrophotometric bilirubin at the end of a 2-h infusion of 10 mL of methods (1), specifically lipemia caused Liposyn 10% into a 1-kg neonate having by fat emulsions given intravenously (2), the use of fat emulsions for intravenous nutrition has only recently been insti1.2 tuted in the U.S. and their use in neonatal feeding is receiving increased attention (3). In this study we investigate the magnitude of this interference and report a simple procedure to correct for
2334
trademark; Abbott Chicago, IL 60064.
CLINICAL CHEMISTRY,
Labora-
=
76 142
14.
a blood volume of 100 mL, assuming no metabolism of triglyceride during the infusion. Figure 1 shows spectral curves for a neonatal serum, a 9 g/L Liposyn solution, and the same neonatal serum containing added Liposyn, 9 g/L. The Advanced Instrument Bilirubinometer measures total bilirubin on the basis of the difference in absorbance at 454 nm and 540 nm, to correct for hemoglobin interference because oxyhemoglobmn absorbs equally at the two wavelengths. However, as Figure 1 shows, the Liposyn absorbance is considerably greater at 454 nm, making this correction inadequate. The manufacturer states in the kit insert (5) that turbidity associated with a triglyceride concentration of 5 g/L will not significantly alter the instrument readings. Liposyn added to each of five nonicteric, nonlipemic samples to give a final concentration of 2.3 g/L gave a mean increase in the triglyceride value of 6480 (SD 110) mg/L. The average positive interference in bilirubin measurement was 21 (SD 1) mg/L.
1.0 0.8 0.6
0.4
the interference. Total bilirubin concentrations in serum from a neonate, measured with a Bilirubin Stat-Analyzer, Model BR II (Advanced Instruments, Inc., Needham 1 Registered tories, North
bllirubin, mg/L
112
104 1. Braverman,
Corrected
a2 325
350
400
450
500
550
600
650
Wavelength (nanometers) Fig. 1. Spectral
added Liposyn
Vol. 28, No. 11, 1982
curve of a neonatal serum, a Liposyn solution, and neonatal serum with
