Analytica Chimica Acta, 178 (1985) 337-339 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

Short Communication

A SENSITIVE SPECTROFLUORIMETRIC DETERMINATION OF HUMAN SERUM ALBUMIN WITH CHROME AZUROL S

YUTAKA SAITO*, YUKIKO INDEN-OKAZAKI, SHOKO WADA-YANO, ATSUKO KANETSUNA, KEI-ICHI MIYAZAKI, MASAKI MIFUNE and YOSHIMASA TANAKA

Faculty of Pharmaceutical Sciences, Okayama University, l-l-l, Tsushima-Naka, Okayama 700 (Japan)

HIDEKI OKUDA

Shionogi 8 Co. Ltd., 192 Zmafuku, Amagasaki 660 (Japan)

(Received 18th July 1985)

Summary. A simple and sensitive method for assay of human serum albumin (5-80 pg) is based on binding of chrome azurol S by the albumin and determination of the bound dye spectrofluorimetrically. The method is sufficiently sensitive for application to spinal fluid (20-100 ~1).

The bromcresol green (BCG) method [l-3], used routinely in clinical assay for human serum albumin (HSA), is not sensitive enough for samples low in HSA, such as spinal fluid. This communication shows that the complex of chrome azurol S (CAS) with HSA fluoresces at 616 nm, which enables a more sensitive fluorimetric method for HSA to be developed; it can be applied to the determination of down to one-thirtieth of the concentration of HSA determined by the BCG method.

Experimental Reagents and materials. Reagent-grade CAS (Merck) was purified by two

precipitations with hydrochloric acid [4]. The HSA (fraction V, Sigma Chemical Co.) and polyethylene glycol-p-nonylphenyl ether (PGNP-10; Tokyo Kasei Co.) were used as received. Control normal and abnormal sera (sera I and II) and spinal fluid were purchased from Hyland Diagnostics. The clinical kit for the BCG method used as control was the Albumin B-Test (Wako Junyaku Co.).

The reagent solution was a 2:2:1 (v/v) mixture of CAS (5.0 X 10m6 M), PGNP-10 (2.5 X 10s3 M) and pH 3.0 citrate buffer (0.1 M).

Apparatus. Fluorescence spectra and relative fluorescence intensities were measured on a Shimadzu RF-500 fluorescence spectrometer equipped with a R928 photomultiplier tube (Hamamatsu Photonics). Quartz cells (10 mm) were used for the measurements.

0003-2670/86/$03.30 o 1985 Elsevier Science Publishers B.V.

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Procedure. The reagent solution (5.0 ml) was added to a sample solution (20 or 100 ~1) containing 5-80 pg of HSA, and the mixture was incubated at around 27.5 for 30 min. The fluorescence intensity was measured against the reagent blank at 616 nm (excitation at 493 nm, slits 20-40 nm).

Results and discussion The conditions described above gave satisfactory results. Addition of

PGNP-10 is necessary for dissolution and stabilization of the CAS/HSA com- plex. Lactate, glycine and tartrate buffers (pH 3.0) gave almost the same intensity, but acetate buffer (pH 3.0) gave only half the intensity.

The calibration graph was linear in the range 5-80 pg of HSA (r = 0.9998, 6 points). The range of linearity depended on the concentration of CAS. The relative standard deviation was 1.3% for the determination of 40 pg of HSA (n = 10). The sensitivity was ca. 30 times better than that of the BCG method.

The change in intensity for 50 gg of HSA caused by the presence of 500 pg of calcium ion or phosphate was less than 3%. A serious decrease in intensity was caused by aluminium(II1) and iron(II1) which form chelates with CAS; this effect was negligible when the amount of these ions was <5 pg. The extent of interference of bilirubin and y-globulin was the same as in the control BCG method.

600 650 Wavelength (nm)

Fig. 1. Emission spectra for serum I and serum II (both from 20 ~1 of 5% solutions), HSA (20 rg/lOO ~1) and a blank solution.

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TABLE 1

Comparison of the chrome azurol S method with the control hromcresol green method

Sample HSA found (g dl-‘)

BCG CAP

Serum I (normal) Serum II (abnormal) Spinal fluid

4.72b 4.61b

4.73c (1.39) 4.56’ (0.59)

0.059d 0.055e (0.77)

aAverage of 10 results with standard deviations in parentheses. b-eSample amounts: b20 ~1 of serum; c20 ~1 of 5% serum; d 1.0 ml of fluid; elOO ~1 of fluid.

The emission spectra obtained for sera I and II are shown in Fig. 1, together with the reagent blank and the spectrum for standard HSA. The spectra coin- cided, indicating that other components in sera hardly affected the spectrum. Similar results were obtained for 20 and 100 ~1 of spinal fluid. As shown in Table 1, the HSA values obtained by the new procedure were in agreement with those obtained by the BCG control method, which required 20 ~1 of undiluted sera I and II and 1.0 ml of spinal fluid. The present method can therefore be regarded as convenient and sensitive enough for assay of HSA in 20-100 ~1 of spinal fluid.

REFERENCES

1 F. L. Rodkey, Clin. Chem., 11 (1965) 478. 2 0. Hemandez, L. Murray and B. T. Doumas, Clin. Chem., 13 (1967) 701. 3 B. T. Doumas, W. A. Watson and H. G. Biggs, Clin. Chim. Acta, 31 (1971) 87. 4 F. J. Langmyhr and K. S. Klausen, Anal. Chim. Acta, 29 (1963) 149.