Folateand VItamInB12in Blood,James - Clinical Chemistry

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Folateand VItamInB12in Blood,James. Friel, Ryna Lety, GeneHerzberg, and Wayne ... Raymond Poon and Irwin Hinberg. (Bureau of Radiation and Medical ...

Clin Chem 1983;29:1119-23. 5. Skude G, Kollberg H. Serum isoamylases in cystic fibrosis. Acts Paediatr Scand 1976;65:145-9. 6. Helfat A, Berk JE, Fridhandler L. The prevalence of macroamylasemia-further study. Am J Gastroenterol 1977;67:54-S.

ApplicatIon to Urine SpecImens of a Radloassay KIt for Measuring Folateand VItamInB12in Blood,James Friel, Ryna Lety, Gene Herzberg, and Wayne Andrews (Depth. ofBiochem. and Pediatrics, Memorial University, St. John’s, Newfoundland, A1B 3X9, Canada) Folate content in urine of intravenously fed very-low. birthweight (VLBW) infants can be assessed by the use of a radioassay kit that was originally designed for measuring folate and vitamin B12 in blood. Because the applicability of this technique to the analysis ofurine samples has not been reported, we assayed foists and B12 in urine by using the Simultrac-S Solid Phase Radioassay Kit (Becton Dickinson). Fresh urine samples were collected from three VLBW infants and analyzed according to the method of standard additions. Two sets of three 100.jL aliquots from each subject were placed in microtubes, one set for folate and the other for B12 analysis. To the first aliquot of each set we added 100 L of distilled water. The second aliquot was 7 uJ

References 1. Cooperman JM, Pesci-Bourel A, Luhby AL. Urinary


of folic acid activity in man. Clm Chem 1970;16:375-81. 2. Eisenga BH, Collins TD, McMartin KE. Differential effects c acute ethanol on urinary excretion offolate derivatives in the ral J Pharmacol Exp Ther 1989;248:916-22. 3. Ellenbogen L. Vitamin B12. In: Machlm LJ, ad. Handbook c vitamins-nutritional, biochemical and clinical aspects. Ne York: Marcel Dekker Inc., 1984:497-547.

SodIum Azide Lowers Cholesterol Readingson the “Reflotron,” Raymond Poon and Irwin Hinberg (Bureau of Radiation and Medical Devices, Health Canada, Ottawa, Canada K1A 0L2)

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While developing a stable serum-based control for th measurement of cholesterol, we found that the addition o sodium azide to serum interfered with cholesterol measure ments by the Boehringer.Mannheim Reflotron refiectano photometer. At both low and slightly increased concentra tions of cholesterol, increasing concentrations of sodiun aside in serum caused decreasing cholesterol readings (se below). At 1 g of azide per liter, a concentration common]: used to preserve biological fluids, cholesterol readings wer decreased by 12% to 15%.

-J 0 0


-J 0 LL C.’ C










Azide, 0 g/L










decroas#{149} In readings Serum B


0 10 12



0 8

the absence of azide, thecholesterolconcentration measured in serum was 6.4 mmot/L,in serum B 3.5 mmol/L.

0 0



z > 0’

_____ -60





0 B12






Fig. 1 Folate content (top) and vitamin B12content (bottom)of urine from VLBW infants (n = 3) Bats indicate± SD; y.intercept = measured value,x-intercepl calculated .


supplemented with 100 L of standard containing either ng of folate or 20 pg of B12. The third aliquot was mixe with 100 L of standard containing 2 ng of folate of 40 pj of B12. Samples were then incubated according to th manufacturer’s instructions and assayed with a standan gamma counter. All folate added to the urine samples was recovered indicating that no interferences were present (Figure] top). This kit provides an easier means of assessing folat urinary excretion in parenterally fed VLBW infants thai do either microbiological (1) or HPLC (2) techniques. The vitamin B12 added to the urine samples was no totally recovered (Figure 1, bottom), indicating interfer ences in the analysis. Because the vitamin B12 concentra tiona in urine were below the limit ofdetection for this kit a microbiological assay will be required (3).

CLINICAL CHEMISTRY, Vol. 36, No. 6, 1990

The same concentrations of sodium aside did not inter fere with serum cholesterol concentrations measured witi the Stanbio Cholesterol Colorimetric Test, a Liebermann. Burchard procedure, or with the “Roche” Reagent, ai enzymatic procedure used with the Cobas-Bio centrifttga analyzer. The specific effect of aside on cholesterol measurement with Refiotron, a dry-reagent-strip system that does no require sample dilution, demonstrates that different cho lesterol procedures may react differently to additives pros ent in specimens, calibrators, or controls. Interference fron additives should be distinguished from the better-knowi

matrix effects (1, 2), so that the individual effects can be studied and better understood. Users of other brands of reagent-strip tests for cholesterol are cautioned to investigate the effect ofazide on their results before including it in their specimens, controls, and calibrators. References 1. Boerma GJM, van Gorp I, Liem U, et al. Revised calibration of the Reflotron cholesterol assay evaluated. Clin Chem 1988; 34:1124-7. 2. Kmll MH, Chesler R, Elm RJ. EffeCt oflyophilization on results of five enzymatic methods for cholesterol. Clin Chem 1989; 35:1523-6.


Semi-MicroMethod for Llquld-Chromatographlc Determination of CyclosporlnA In Whole Bloodwith Use of a RapidExtractionProcedure,N. Sadeg, C. Huy,’2 J. R. Claude,’ and M. Hamon (‘ Lab. de Toxicol., Facult#{233} de Pharmacie, 4, Ave. de l’observatoire, 75 006 Paris; 2 Lab. de Toxicol. & Pharmacie, HApital Necker Enfants Malades, 149, Rue de S#{232}vres, 75 015 sri3 Lab. de Chim. AnaL, Facult#{233} de Pharmacie de Paris XI, Ave. J.B. Clement, 92 290 Chatenay Malabry, France)

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Cyclosporin A, a fungal metabolite with immunosuppressive activity, is widely used in the prevention of graft vs host disease in allogeneic bone marrow diseases and in the prevention of graft rejection after organ transplantation. We have developed a selective and isocratic liquidchromatographic method for the routine determination of cyclosporin A in whole blood after a rapid liquid-liquid extraction of a small volume of sample. To 300 LL of appropriate calibration standard or unknown whole blood in a glass centrifuge tube, add 100 .tL of cyclosporin D (1 mg/L in acetonitrile) as internal standard, 30Q tL of 100 mmol/L NaOH reagent, and 3 mL of diethyl ether. Vortex-mix for 2 mm and centrifuge. Draw off the ether layer, and evaporate it. Add 200 L of a mixture of methanoll0.05 moIJL HC1 (6/4, by vol) to each residue, followed by 1 mL ofn-hexane. Mix and centrifuge. A 20-iL aliquot of the extract, injected into a 25 cm x 4 mm Nucleosil octyl 5-pin-particle column heated at 72 #{176}C, is eluted with a mixture ofacetonitrile/0.01 mol/L phosphate buffer, pH 5.5 (65/35, by vol), at a flow rate of 1 mL/min. Detection is set at 210 nm.



Fig. 1 . Chromatograms of (A) drug-free whole blood and (B) wholeblood specimen from a renal transplant recipient containIng cyclosporinA(CyA)176g/L

The chromatographic run is complete within 10 mm 1). The detection limit is 25 g/L. Between-run CVs range from 2.3% to 6.2%; analytiCal recovery is 92.1% ± (Figure

6.3%. The principal advantage of our method is that the volume ofsample is 300 zL ofwhole blood instead ofi or 2 mL required in the other liquid-chromatographic methods (13) with equivalent sensitivity (25 pg/L). This semi-micro procedure is suitable for routine monitoring of cyclosporin A in whole blood in pediatric transplantations. We



for supplying cyclosporin A and D.


1. Christians U, Zimmer 0. Measurement ofcyclosponn A and of four metabolitea in whole blood by high-performance liquid chromatography. J Chromatogr Biomed Appl 1987;413:121-9. 2. Garrafo RiLapalus P. Simplified liquid chromatographic analysis for cyclosporin A in blood and plasma with use of rapid extraction. J Chromatogr Biomed App! 1985;337:416-22. 3. Moyer TS, Johnson P, Faynor SM, Sterioff S. Cyclosporine: a review of drug monitoring problems and presentation of a simple accurate liquid chromatographic procedure that solves these problems. Clin Biochem 1986;19:83-.9.

CLINICAL CHEMISTRY, Vol. 36, No. 6, 1990