ntby a GlycerolOxidaseMethod - Clinical Chemistry

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Apr 9, 1985 - measured triglyceride per 1 mmol of hydroxyurea per liter). A ... Hydroxyurea, an anti-neoplasticagent,is used in treating .... Review of the pa-.
CLIN.CHEM. 31/8,1355-1357(1985)

HydroxyureaInterleresNegativelywithTriglycerideMeasurem#{246}nt by a GlycerolOxidase Method Richard A. McPherson,1Kevin D. Brown, Raghunath P. Agarwal, Gregory A. Threatte, and Robert J. Jacobson Measured triglyceride concentrations were extremely low (‘

a.

E a) C’)

a) 0

a) a)

Time (h) after hydroxyurea FIg. 1. Time course

administration

ofapparent triglyceride contentin serum before and

aftera single oraldoseof hydroxyurea All patientswere fasting. Doses ot hydroxyurea were 0.5g (patient 1, 0-0) or 1.09 (patIent2, -A; patient3,0-0). All measurementswere made with an RA-1000 analyzer

Lipoprotein electrophoretograms of serial serum specimens (before to 4 h after medication) from patient 2 showed no qualitative or quantitative differences in any of his samples. In addition, there was essentially no change in values for cholesterol, uric acid, or phosphorus in the serum of any of the three patients during the study. Each patient did show a slight decrease in serum glucose, consistent with continuation of the fasting state. Effect of hydroxyurea added to serum and glycerol solutions (Table 1). To study this, we mixed serum with aqueous solutions of the drug to give different concentrations and analyzed the mixture in the RA-1000. Resultsfortriglyceride in serum decreased with increasing concentrations of hydroxyurea. Similarly, increasing concentrations of hydroxyurea added to a glycerol solution (as substrate) decreased the mea-

Table 1. InhibitIon of TrIglycerIde DetermInation by Hydroxyurea Added to Serum or Glycerol Solution Final

hydroxyures concn, mmot/L 0 0.04 0.08

0.21 0.42 0.85 2.1 4.2

0

E E

a, V

a, 0

>,

V

a, a) a) a,

Msasursd triglyceride centsnt mg/I. Serum

Glycerol solution

1670 1520

1440 1310

1380

1200

1120

910

650

390

70 40 40

an aqueous solution of hydroxyurea was added to normal serum or to 0.3 mL ofglycerol dilutedin saline. a10 L of

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CLINICALCHEMISTRY, Vol. 31, No. 8, 1985

10 0 0 0.3 mL of

Hydroxyurea

(mmol/L)

Fig. 2. Relationof measured triglyceride content to hydroxyurea

concentrations in solutions containing known amounts of glycerol (Gi) as substrate All measurements with an RA-1000 analyzer. The lines represent unweighted least-squares linear-regression analysisof the data

amount of product formed at equilibrium as well, we monitored Asso at 22#{176}C all during the reaction after mixing the assay reagent and glycerol substrate in the presence of various amounts of hydroxyurea. The absorbances of all these reaction mixtures became constant 6 mm after the start of the reaction (Figure 3). The final amount of product formed by the end of each reaction decreased linearly as the concentration of hydroxyurea was increased from 0 to 0.85 mmol/L. Results were similar when the Technicon assay reagent was pre-incubated with hydroxyurea before glycerol was added. This observation excluded the possibility of progressive irreversible inactivation of an enzyme component by hydroxyurea. Analysis of triglycerides onBeckmanAstra-8 system. The same mixtures of hydroxyurea with serum (Figure 1) and with glycerol (Figures 2, Table 1) that resulted in a decrease in apparent triglyceride as measured with the Technicon reagent showed no such decrease when parallel samples were analyzedintheAstra-8, inwhichtheassayisbasedon

2),concentrations of hydroxyurea in serum could be estimated from the decrease in measured triglyceride content (by a glycerol oxidase method), comparing the value before and after a dose of hydroxyurea-although this estimate would be in error if true triglyceride contentwere changing during that time interval (e.g., a postprandial increase). In that case, the hydroxyurea concentration could be estimated from the difference in triglyceride measurements for aliquotaof the same sample by a glycerol oxidasemethod vs a

glycerol-phosphate

biologicaleffect. This negative interference with triglyceride measurement by hydroxyurea could complicate proper assessment of a patient’s nutritional status during long-term administration of hydroxyurea. Furthermore, since hydroxyurea apparently passes freely into other biologicalfluids and may be cleared more slowly from them than from the circulation (9), it is important to recognizethe potential for hydroxy-

oxidase.

DIscussIon In addition to its inhibitory effect on the synthesis of DNA precursors (4), hydroxyurea evidently also interfereswith glycerol oxidase. This enzyme occurs in the mycelia of several strains of Aspergillus, Neurospora, and Penicillium (5, 7, 8). In the initial description of this enzyme (5) it was indicated that it could be inhibited by cyanide, hydroxylamine, or azide. We saw no inhibition of the glycerol oxidase-mediated assay for triglycerides by hydroxylamine or urea, even in concentrationsseveral-fold those at which hydroxyurea strongly inhibited the assay. The observed time course for inhibition of triglyceride determination in serum after a single oral dose of hydroxyurea (Figure 1) is similar to that for actual hydroxysrea concentrations in serum (9). Because this effect is quantitatively related to the amount of hydroxyurea present (Figure

glycerol-phosphatedehydrogenasemethod. Another reported method (10) for determining hydroxyurea in biological fluids is complicated. Thus the present clinical use of hydroxyurea doesnot include monitoring by chemical methods. Instead, patients who take it daily are monitored by weekly platelet or leukocyte counts (1,3). Our observations could be exploited to develop a relatively

inexpensive, and widely available procedure for hydroxyurea in the serum soon after an oral dose rather than waiting several days for emergence of its simple,

estimating

urea to interfere with triglyceride determination in pleural fluid. In that situation, a falsely low measurement of triglyceride might hinder the correct diagnosis of chylous effusion (in which the triglyceride concentration is high, owing to abnormal leakage from a lymphatic duct) vs a pseudochylous effusion (11). Finally, our observations suggest that hydroxyurea could have other, still-unrecognized biochemical effects that may be important in its mechanism of action.

References

E C 0 I0

‘0

a a

U C

a 0

a

.0

Time

(mm)

Fig. 3. Kinetics of hydroxyurea inhibition in assay of triglycerideswith

Technicon reagents Reactionswere initiatedby addingglycerol(3.4 mmol/Lequivalent 3000 mg/I oftriglycerides) arid various concentrations of hydroxyurea: 0 (0-0), 0.42 (A-A), and 0.85 (-) mmol/I

(-),

0.21

1. Kennedy BJ, Yarbro JW. Metabolicand therapeutic effects of hydroxyurea in chronic myeloid leukemia. J Am Med Assoc 195, 1038-1043 (1966). 2. Ariel IM. Therapeutic effects of hydroxyurea. Experience with 118 patients with inoperable solid tumors. Cancer 25, 705-714 (1970). 3. Bolin RW, Robinson WA, Sutherland J, Hamman RF. Busulfan versus hydroxyurea in long-term therapy of chronic myelogenous leukemia. Cancer 50, 1683-1686 (1982). 4 Theiss JC, FischerGA. Inhibition of intracellular pyrimidine ribonucleotide reduction by deoxycytidine, arabinosylcytosine and hydroxyurea. Bioc hem Pharmacol 25, 73-79 (1976). 5. Uwajima T, Akita H, Ito K, et al. Some characteristics of a new enzyme, “glycerol oxidase.” Agric Biol Chem 43, 2633-2634 (1979). 6. Megraw RE, Dunn DE, Biggs HG. Manual and continuous-flow colorimetry of triacylglycerols by a fully enzymatic method. Clin Chem 25, 273-278 (1979). 7. Uwajima T, Akita H, Ito K, et al. Formation and purification of a new enzyme, glycerol oxidase and stiochounetry of the enzyme reaction. Agric Biol Chem 44, 399-406 (1980). 8. Uwajuna T, Shimizu Y, Terada 0. Glycerol oxidase, a novel copper hemoprotein from Aspergillus japonicus. Molecular and catalytic properties of the enzyme and its application to the analysis of serum triglycerides. JBiol Chem 259, 2748-2753 (1984). 9. Beckloff GL, Lerner HJ, Frost D, et al. Hydroxyurea (NSC32065) in biologic fluids: Dose-concentration relationship. Cancer Chemother Rep 48, 57-58 (1965). 10. Davidson JD, Winter TS. A method of analyzing for hydroxyurea in biological fluids. Cancer Chemother Rep 27,97-110(1963). 11. Kjeldsberg CR, Krieg AF. Cerebrospinal fluid and other body fluids. In Clinical Diagnosis and Management by Laboratory Methods, JB Henry, Ed., Saunders, Philadelphia, PA, 1984, pp 459-492. CLINICAL CHEMISTRY, Vol. 31, No. 8, 1985

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