Table 1. Effect of Drugson the Determinationof ... - Clinical Chemistry

9 downloads 0 Views 483KB Size Report
Proteins concentration, g/L. OrigInal. Turbidlmetry. Ponceau Red. DruginterferenceinTurbidimetry and Colorimetryof Proteinsin. Urine. To the Editor: Colorisnetry.
and measure the drug concentration. The experiment should be repeated at least three times, and the results evaluated. Vinod

Proteins concentration, OrigInal Turbidlmetry Ponceau Red

P. Shah

Gene Knapp Jerome P. Skelly Bernard E. Cabana Office of Drugs National Center for Drugs and Biologics Food and Drug Administration Rockville, MD 20857

Drug interferencein Turbidimetry and Colorimetryof Proteinsin Urine To the Editor:

of urinary proteins based on Ponceau Red co-precipitation (1) has been applied in our laboratory for three years. Results were occasionally inconsistent with the clinical state and the Colorisnetry

medical history of the patient. A systematic duplication of the protein assays by a turbidimetric procedure (2) showed significant discrepancies for urines of patients being treated with aminoglycosides (gentamicin or kanamycin) or miconazole. Assays of urine to which these drugs were added showed that neither the Ponceau Red assay nor the turbidimetnc assay is free of interference. Table 1 showed an example of such experiments on a urine sample containing about 1 g of protein per liter. In the turbidimetric method, miconazole at concentrations usually reached during therapy increased the apparent protein concentration by a factor of 1.5 to 2.5 when the true protein concentration was higher than 0.5 g/L. It did not interfere when proteins were absent or at low concentrations. Aminoglycosides did not interfere in the turbidimetric method at any concentration of protein or of drug. The colorimetric procedure was unaffected by the presence of miconazole, but aminoglycosides at concentrations reached during therapy gave an apparent proteinuria with protein-free urines and increased the results by a factor of 2 to 4 when proteins were present. The precipitation of an insoluble salt of aminoglycosides with Ponceau Red is the cause of the interference. These observations led us to change the Ponceau Red method slightly. Uri-

nary proteins are first precipitated with trichloroacetic acid(50g/L in0.5mol/L NaC1) without Ponceau Red. The supernate is discarded. The original procedure of Pesce and Strande (1) is then carried out on the precipitated protein redissolved in 0.2 mol/L NaOH. This preliminary precipitation of 2328

Table 1. Effect of Drugson the Determinationof ProteinConcentrationIn Urineby Three DifferentMethods Drug added

None

1.00 1.10

40 mg/L

Gentamicin

120mg/L Kanamycin0.33 gIL

1.12 1.16 1.20 1.92 1.88

1.0 gIL Miconazole 20 mg/L 50 mg/L

proteins did not significantly influence the precision, accuracy (by recovery test), dure.

or linearity

of the original

proce-

References 1. Pesce, M. A., and Strande, C. S., A new micromethod for determination of protein in cerebrospinal fluid and urine. Clin. Chem. 19, 1265-1267 (1973). 2. Meulemans, 0., Determination of total protein in spinal fluid with sulphosalicylic acid and trichloroacetic acid. Clin. Chim. Acta 5,757 (1961).

M. M. Lievens P. J. Cells Lab. de Biochim. Med. Cliniques Universitaires St. Luc Avenue Hippocrate, 10/1748 1200 Bruxelles, Belgium

Determinationof Deferoxamine Chelated iron To the Editor:

Deferoxamine mesylate;

Desferal,

(desferrioxamine CIBA-Geigy)

is used

to treat iron accumulation in tissues, caused by (e.g.) iron poisoning or treatment with long-term intermittent doses of packed erythrocytes, and in therapy of diseases involving recurrent internal bleeding such as hemosiderosis. Because only traces of iron are normally present

in urine, the effectiveness of the administration of this drug can be monitored indirectly by measuring the urinary excretion of deferoxamine-chelated iron. According to WOhler (1) it seems justified to regard substantially increased iron excretion after injection of deferoxamine, together with an increased iron concentration in serum, as a sign of iron-storage disease. The better to measure iron in serum, we exchanged our former method (2), protein precipitation with trichloroacetic acid and subsequent addition of the deferoxamine-sensitive color agent, 2,4,5-tripyridyl-s -triazine (TPTZ), for a direct procedure in which deprotein-

CLINICAL CHEMISTRY, Vol. 28, No. 11, 1982

1.05

1.98 3.75 1.37

g/L Modified

Ponceau Red

1.02 1.03

t03

1.01

2.45

1.03

1.06

1.06

1.05

1.06

ization is omitted and a buffered (pH = 2) ferrozine reagent solution is used (3). Thiourea is added to this solution to

suppress the nonspecific contribution of copper (4) to the colorimetric reaction. Run samples in duplicate, with use of disposable microcuvets (Sarstedt, type 742). Place 400 zL of a buffered reducing reagent (containing, per liter, 11 g of citric acid, 9 g of NaC1, 2 g of thiourea, and, just prior to use, 4 g of ascorbic acid) in a cuvet and add 50 zL of serum or urine. Mix and record the absorbance at570 nm (blankreading). Then add 20 tL of ferrozine solution (17.5 gIL; Sigma Chemical Co., cat no. P-9762) and (for serum) again record the absorbance at 570 mm after 5 mm. For urine specimens, instead make the final absorbance reading after the reaction mixture has been incubated for 2 h at 37 #{176}C. In the calculation of original iron concentration a volumetric correction factor for the blank reading of 0.957, due to the added ferrozine reagent, must be incorporated. The standard curve is linear to at least 120 mol of Fe per liter. Using 55 serum specimens, we compared this method with our former TPTZ procedure. An excellent relationship was observed: y(ferrozine) = 1.02x(TPTZ) - 0.22 mol/L (r = 0.956, S 1.64, S = 1.67); observed concentration range: 4-30 mol of Fe per

liter. “Seronorm” control material (assigned value 27 tmol/L), included in all analytical runs, gave a mean value of 26.7 tmol/L with a day-to-day CV of 2.4% (n = 20). Addition of deferoxamine to serum up to 370 MmolIL, a concentration far beyond the therapeutic (5), produced no detectable decrease in the colorimetric signalifthe ferrozine procedure was used. However, for the TPTZ method and also for the aca discrete analyzer(Du Pont; color reagent, bathophenanthroline) a significant decrease was seen (up to 70 and 48%, respectively). The recognized underestimation of iron by the aca in the presence of deferoxamine can be obviated by the addition of extra thioglycolic acid (6). With the ferrozine method for iron a poor reproducibility was observed in

V /

2

/

‘Iine

E

of

,.-identity

i20

copper, our method is simple and reliable for the microdetermination of serum iron.

/1#

‘of

References

I’

80



/

6(

7’

r=O.996 Sy =395 sxy=3.87

/.0 20

yt.O2x -2.2 pmoIlL means:x.?4.l,y..?3.3

/ atomic

20

1.0

60

80

tOo

absogfn

120

1/.0

Fig. 1. Comparison of urinary iron (in molIL) as measured by flameiessatomic absorption

and the ferrozine method

The regression equation Is calculated accordIng to Deming’s method

results for samples from a patient being treated with deferoxamine. This was caused by slow color formation during several hours. Addition of extra reducing agent, up to 20-fold the original concentration of ascorbic acid (7), had no sufficient accelerating effect. However, the endpoint of the reaction was reached within 2 h by incubating the reaction mixture at 37 #{176}C. Investigating whether chemical equilibrium was attained after 2 h of incubation, we found 100% recovery when 13 urine specimens from this patient were so analyzed. We compared the colorimetric results with results obtained by flameless

1. Wbhler, F., Diagnosis of iron storage diseases with desferrioxamine (Desferal test). Acta Haematol. 32, 321-337 (1964). 2. O’Brien, D., Ibbott, F. A., and Rodgerson, D. 0., Laboratory Manual of Pediatric Micro-Biochemical Techniques, 4th ed. Hoeber Medical Division, Harper & Row, New York, NY, 1968, pp 195-196. 3. Klein Gebbink, J. A. G., Hoeke, J. 0. 0., and Marx, J. J. M., Influence of iron saturation as a possible source of error in the immunoturbidimetric determination of serum transferrin. Clin. Chim. Acta 121, 117-122 (1982). 4. Artiss, J. D., Vinogradov,

S., and Zak, B., Spectrophotometric study of several sensitive reagents for serum iron. Clin. Biochem. 14, 311-315 (1981). 5. Summers, M. R., Jacobs, A., Tudway, D., et al., Studies in desferrioxamine and ferrioxamine metabolism in normal and ironloaded subjects. Br. J. Haematol. 42,547-555 (1979). 6. Steinmetz, W. L., Glick, M. R., and Oei, T. 0., Modified aca method for determination of iron chelated by deferoxamine and other chelators. Clin. Chem. 26, 1593-1597 (1980). 7. Werkman, H. P. T., Trijbels, J. M. F., van Munster, P. J. J., et al., Automated determination of serum iron and ferrioxamine in urine. Clin. Chim. Acta 31, 395-401 (1971).

G. J. van Stekelenburg C. Valk G. J. de Boer

positive result is a /3 error and, conversely, a falsely negativeresult is an a error. Whether this be a clerical or a conceptual error, the fact is that the clinical biochemists can be confused on comparing literature from different authors. References 1. Strike, P. W., Medical Laboratory Statistics, Wright . PSG, Bristol, U.K., 1981, p 51.

2. Galen, R. S., and Gambino, S. R,, Beyond Normality: The Predictive Value and Elf iciency of Medical Diagnoses, Wiley, New York, NY, 1975, p 126. 3. Keller, H., and Gessner, U., Auswahl und Interpretation diagnostischer Parameters. Internist (Berlin) 21, 173-180 (1980). 4. Werner, M., Will abstract models change the practice of medicine? In Logic and Economics of Clinical Laboratory Use, E. S. Benson and M. Rubin, Eds., Elsevier, New York, NY, 1978, pp 41-46.

Javier

Fuentes

Serv. de Bioquimica C.S. Principes de Espana

Feixa Llarga s/n L’Hospitalet Barcelona,

de Llobregat Spain

Serum GlucosePhosphate isomeraseand Ornithine Carbamoyltransferase Activities Are increasedin WomenTaking ContraceptiveSteroids

atomic absorption (instrument type 300 Clin. Chem. Lab. SG, Perkin-Elmer Corp.). We injected 10 iiL of diluted urine into the heated Univ. Children’s Hosp. graphite atomizer (type HGA72, Per“Het Wilhelmina Kinderziekenhuis” kin-Elmer Corp.). The analytical specNieuwe Gracht 137 3512 LK Utrecht, The Netherlands ifications were: dry for 10 s at 100 #{176}C, ash for 15s at 100 #{176}C, atomize for 8s at 2400 #{176}C, photometric setting 248.3 nm, lamp current 30 mA, carrier gas N2. In

Serum glucosephosphate isomerase (GPI; EC 5.3.1.9) activity is increased in cancer (1), viral hepatitis (1, 2), and myocardial infarction (2). We describe here a hitherto unknown cause of high serum GPI activity, which may have a

this case the peak height obtained with a 5/10-mV recorder (Perkin-Elmer Corp. type 56) was a measure of the original iron concentration. Figure 1

Errorsin DescribingErrors

enzyme. We measured serum GPI activity with a kit (3) in 10 healthy women of reproductive age, three, six, and nine months

demonstrates the complete recovery of urinary iron, up to concentrations of 130 imol/L, if the reagent mixture is incubated before the final absorbance reading is made. The aca in this case revealed values between 0 and 0.8 imol/L

An excellent book (1) says about biochemical test results: “Some subjects who are perfectly healthy will have test results outside of the healthy range of values (a false positive result: a type I or a error) and some subjects who are ill

for all samples. For the TPTZ method recoveries ranged between 5 and 75%. Because during therapy with deferoxamine almost all urinary iron is in the chelated state, kinetics for the colonmetric reaction with ferrozine are different from those for serum samples. From our experiments we conclude

will have test results within the healthy range (a false negative result: a type II or f3 error).” These concepts about a and j3 errors are obviously coherent (the author is a clinical biochemist and Fellow of the Statistical Society in the United Kingdom) with the basic formulation of the null hypothesis for this kind of

that: (a) if the incubation conditions are optimized, urinary iron can be measured accurately and with sufficient precision, and (b) with use of citric acid in combination with ascorbic acid to free iron from transferrin and thiourea to mask

comparisons.

To the Editor:

bearing on the diagnostic

To the Editor:

Surprisingly, one can read in several relevant publications (2-4) concepts other than mentioned above. Thus, these authors hold a radically different point of view: they state that a falsely

value

of this

after they started taking an oral contraceptive (Ovulen; Searle) and in 20 age- and sex-matched controls. The data were analyzed statistically by use of Student’s t-test. Mean (and SD) serum GPI activity was 23.4 (6.6) Bodansky units in the control group, and 58.4 (16.3), 144.7 (64.1), and 192.9 (101.3) Bodansky units

after use of the oral contraceptive for three, six, and nine months, respectively. The difference at each interval was highly significant (p