Enzymatic assay of serum sialic acid

493

Clinica Chimica Acta, 108 (1980) 493-498 @ Elsevier/North-Holland Biomedical Press

BRIEF TECHNICAL CCA

NOTE ._

“__^_

-.

-__-

1579

ENZYMATIC

KAZUYUKI TOSHIAKI

ASSAY OF SERUM SIALIC ACID

SUGAHARA USUI

*, KATSUYA

SUGIMOTO,

OSAMU

~e~a~trnent of Laboratory Medicine, Nagasaki ~niue~ity Nagasaki City, Nagasaki (Japan) (Received

February

29th,

NOMURA

and

hospital, ~akam~tomuchi

7-1,

1980)

Serum sialic acid has been assayed enzymatically. The reaction includes neuraminidase hydrolysis of glycoprotein, cleavage of sialic acid to pyruvate by N-acetyl neuraminic acid (NANA)-aldolase, oxidation of pyruvate by pyruvate oxidase which produces hydrogen peroxide, and calorimetry of hydrogen peroxide using the peroxide-~~hl~rophenol-4-ammo-antipy~ne methbd. This method showed good correlation between a chemical method (r = 0.984), good recovery (98.8%) and good reproducibility (within-run-precision: 1.0% C.V.; day-today precision: 1.9% C.V.). Intrinsic serum pyruvate produces an equimolar positive effect. The normal value range is 1.94 + 0.29 mmol/l (mean + S.D., n = 24). --

Introduction Sialic acid in the serum has been assayed using chemical reactions such as acid condensation with resorcinol [l-3 ] or orcinol f4,5], Ehrlich’s reaction [4--6], resorcinol reaction after periodate treatment [ 7] and reaction with thiobarbiturate [ 1,891. Comb et al. [lo], Brunetti et al. [ll] and Tauchi et al. [12] assayed the compound by an enzymatic method. The method includes hydrolysis of glycoprotein by neuraminidase, cleavage of sialic acid to pyruvate by NANA-~dol~e, and finally reduction of pyruvate by lactate dehydrogenase in the presence of NADH, which is measured photometrically. In this report, an

* To whom correspondence should be addressed.

494

enzymatic method for the estimation of sialic acid has been investigated using following reactions: (l

)

glycoprotein

(2) sialic acid

(neur~~nidase) (N-acetyl

neuraminic

t sialic acid + asialo-glycoprotein acid aldolase)

’ pyruvate + N-acetyl-D-mannosamine

(3) pyruvate f phosphate + O2 (pyruvate Oxidase! acetyl phosphate + CO2 + HzOz (4) HzOz + p-chlorophenol + 4arninoantipyrine (peroxidase!(colored product) Method 1. Reagents N-acetyl neuraminie acid (NANA, molecular weight 309.3), one of the sialic acids, was purchased from Nakarai Chemicals, Ltd., Kyoto, Japan. Neuraminidase (EC 3.2.1.18, from Arthrobacter ureafaciens) and NANA-aldolase (EC 4.1.3.3, from Escherichia coli) were either purchased from Nakarai Chemicals, Ltd. or donated by Kyokuto Pharm. Co. Ltd., Tokyo, Japan. These enzymes were prepared according to the method described by Uchida et al. [ 131. Pyruvate assay reagents were given by Wako Pure Chemical Industries, Ltd., Osaka, Japan, including pyruvate oxidase (EC 1.2.3.3, from Pediococcus sp.), thiamine pyrophosphate (TTP), flavin adenine dinucleotide (FAD), peroxidase (EC 1.11.1.7, from horseradish), p-chlorophenol, 4aminoantipyrine and magnesium chloride. Reagent I. 1 vol of neuraminidase (1 U/ml), 1 vol of NANA-aldolase (5 U/ml) and 3 vol of 10 mmol/l phosphate buffer (pH 6.8) are mixed. Reagent II. 5 vol of solution A (pyruvate oxidase 100 U, TPP 14 pmol, FAD 0.36 pmol are dissolved in 15 ml of 20 mmol/l phosphate buffer, pH 7.4, containing 10 mmolfl MgClz and 3.8 mmol/l p-chlorophenol) and 2 vol of solution B (peroxidase 550 U, 4-amino~tipy~ne 89 pmol dissolved in 50 ml of 4 mmol/l phosphate buffer, pH 7.0, containing 31 mmol/l sodium azide; this reagent is stable for a week at 2 - 8°C) are mixed before use. Reagent 111. 50 mmol/l EDTA, 0.1 mol/l disodium phosphate, 0.1 mol/l sodium citrate and 3 g/l Triton X-405 are dissolved in deionized water (pH 9.0). Siatic acid standard soZut~on. 3.23 mmol/l (100 mg) of NANA is dissolved in 100 ml of water. Reagents for the chemical assay of sialic acid. The reagents were supplied from Kyokuto Pharm. Co. Ltd. An adaptation of the acid-resorcinol reaction reported by Whitehouse and Zilliken [ 31 was used. The exact contents were not described in the manual. The reagent Kit consists of two reagents; Reagent A contains resorcinol dissolved in hydrochlo~~ acid, and Reagent B contains periodic acid. Serum standard. A batch of pooled bovine serum was calibrated by the enzymatic method. 50 ~1 of the serum were digested with 0.1 ml of neuraminidase (1 U/ml) and 0.3 ml of phosphate buffer (pH 6.8) for 16 h at 37”C, the mixture was incubated again with 0.1 ml of NANA-aldolase for 30 min at 37°C

495

and then subjected to the pyruvate assay system as described below. Calibration was made against the standard NANA solution. This serum was used for the chemical assay as a standard. Biliru bin solution. 0.34. mmol/l bilirubin aqueous solution was purchased from Nissho Co. Ltd. (Tokyo, Japan). The compound is dissolved in water with bile acid as a detergent. 2. Method Enzymatic assay of sialic acid. 50 ~1 of serum are mixed with 0.5 ml of Reagent I and incubated for 30 min at 45°C. Then 1 ml of Reagent II is added to the tube and incubated again for 15 min at 37°C. After the second incubation, 3.5 ml of Reagent III are added and the mixture is allowed to stand for 10 min at room temperature. Then it is subjected to calorimetry within 60 min at 505 nm wavelength. Reagent blank is prepared exactly as above except that 50 ~1 of deionized water is pipetted instead of serum. Serum blank. For an exact assay, serum blank must be subtracted from the sialic acid assay described above, when the serum is highly hemolyzed or contains excessive pyruvate. 50 yl of serum is mixed with 0.5 ml of phosphate buffer (10 mmol/l; pH 6.8), 1.0 ml of Reagent II and 3.5 ml of Reagent III, the mixture is allowed to stand for 15 min at 37”C, and it is subjected to colorimetry at 505 nm wavelength. Pyruuate assay. Pyruvate assay was carried out enzymatically using the pyruvate oxidase-peroxidase method. The procedure is quite the same as that for the serum blank assay system described above. Hydrolysis of glycoprotein with neuraminidase. 50 ,ul of serum sample, 100 ~1 of neuraminidase (1 U/ml), 100 ~1 of NANA-aldolase and 300 ,ul of phosphate buffer (10 mmol/l, pH 6.8) are placed into each test tube, and the tube is incubated for various periods at 45°C. When incubation time is more than 30 min, the incubation is started without NANA-aldolase, and the enzyme is added to the tube 30 min before the incubation stops in order to avoid inactivation of the enzyme. After the incubation is over, the reaction mixture is treated with Reagent II and Reagent III, and assayed calorimetrically according to the method of the sialic acid assay. Recovery experiments. Recovery experiments are carried out by comparing the base material and high NANA material, which was prepared by adding 1 vol of 16.2 mmol/l NANA solution to 9 vol of the base material Sialic acid in the base material (Sbase) and the high level material (Shigh) are assayed, and recovery is calculated as follows Recovery

(%) = (Shigh - 0.9 Sbase) X loo/l.62

Chemical assay ‘of sialic acid. 20 ~1 of the serum are mixed with 0.5 ml of Reagent A and incubated for 10 min at 37°C. 4 ml of Reagent B are added to the tube, left for 5 min at room temperature, and then heated in a boiling waterbath for 10 min. The tube is cooled in tap water and assayed by colorimetry at 620 nm wavelength. The serum standard is used for calibration.

496

Results and discussion 1. Hydrolysis of glycoprotein with neuraminidase After the neuraminidase and NANA aldolase incubation at 45°C pyruvate concentration reached a maximum in 30 min and then decreased slowly. When the incubation was carried out at 37”C, the reaction required more than 2 h, or more than 2 units of the enzyme per test tube in order to achieve maximum recovery. The 45°C incubation was used in our experiments in order to conserve neuraminidase. 2. Linearity and sensitivity The calibration curve using the sialic acid standard solution was linear up to 8.0 mmol/l (Fig. l), and extinction of the reaction mixture using serum was proportional to the amount of the serum up to 7 mmol/l sialic acid concentration. The molar extinction coefficient (E) of the final color reaction in the proposed method was 1.14 X 104. The value indicates that the method is more sensitive than the enzymatic method [lo-121 in which NADH was used for UV calorimetry (E = 6.22 X lo3 at 340 nm). Although the chemical method is more sensitive than our method (E = 2.21 X 104), technical staff find it unacceptable because of the undesirable odor generated during the heating reaction at 100°C. 3. Stability of the colored product Absorbance of the final reaction temperature and room illumination.

product

was stable up to 120 min at room

4. Accuracy and precision Recovery studies using five different sera are shown in Table I. The average recovery was 98.8%, ranging from 97.4-100.4%. Within-run precision using two different sera was 1.00% for a normal level serum (mean = 1.80 mmol/l,

NANA

Concentration

Fig. 1. Calibration curve of sialic acid. Standard material. N-acetyl neuraminic acid.

497

TABLE I RECOVERY

EXPERIMENTS

FOR THE ENZYMATIC

ASSAY

OF SIALIC ACID

(The material was prepared by mixing 1 vol of 16.8 mmol/l NANA solution with 9 vol of the base sera.) No. of specimens

Recovery *

Sialic acid assay

(%)

1 2 3 4 5

*

ReCOVerY

=

(shigh

base serum Sbase (mmol/l)

spiked serum Shigh (mmol/l)

1.84 2.23 2.45 2.98 3.85

3.23 3.62 3.78 4.30 5.04

-o.gsbase)

97.4 99.8 100.4 99.0 98.4

X 100/1.68*

n = 15) and 1.04% for a high level serum (mean = 4.22 mmol/l, it = 15). Assay data from 31 consecutive days were used to calculate day-to-day variation using two batches of pooled serum, and the results were 1.91% for a pooled serum (mean = 2.23 mmol/l) and 1.24% for another pooled serum (mean = 3.34 mmol/l). 5. Correlation

with the chemical

assay

High correlation (r = 0.984, n = 100) was observed between the present enzymatic method and the chemical method (Fig. 2), and the regression equation was y = 0.932x + 0.154, where y is the enzymatic assay and x is the chemical assay. 6. Normal values Serum sialic acid concentrations in healthy persons were assayed, and the results were 1.94 + 0.29 mmol/l (mean + S.D., n = 24) for all persons, 2.03 + 0.28 mmol/l (n = 12) for males and 1.85 f 0.27 mmol/l (n = 12) for females. The values are not corrected against the serum blank. -

mmol/l

54-

0

0

I

1

I

2 Chemical

f

3

I

I

4 5 mmolll

Assay

Fig. 2. Correlation between the chemical method and the enzymatic method for sialic acid assay.

498

7. Effect of contaminants Hemoglobin. A hemolyzed

serum was prepared by the freezing-thawing method, and added to clear sera in various hemoglobin concentrations. The assay results gave apparently high values, but the effect could be eliminated using the serum blank up to 31 ~mol/l(200 mg/dl) hemoglobin concentration. Bilirubin. Bilirubin can be a reductant for the peroxidase system, since it consumes hydrogen peroxide in the presence of peroxidase, thus, this substance exerts a negative effect on the sialic acid assay system. Aqueous bilirubin solution was added to a serum so as to obtain a bilirubin concentration from 8 to 351 pmol/l, and the sialic acid was assayed enzymatically. An increment of 10 pmol/l (0.585 mg/dl) bilirubm corresponded to 0.009 mmol/l underestimation of the sialic acid value. Pyruuate. To a normal serum various concentrations of pyruvate up to 10 mmol/l were added and sialic acid concentrations were assayed with the enzymatic method. Absorbance of the reaction mixture increased proportionally to the added pyruvate concentration. Pyruvate in the serum at a level of 0.1 mmol/l caused an overestimation of sialic acid as high as 0.11 mmol/l. Pyruvate concentration of random serum samples in our laboratory were analyzed using the pyruvate oxidase system. The values were 0.104 + 0.047 mmol/l (mean f S.D.) with a range from 0.027 to 0.318 mmol/l. Many of the samples tested were less than 0.2 mmol/l, then serum blank can be omitted in routine work. However, caution must be exercised because some sera contain more than 0.3 mmol/l pyruvate, especially sera from the pediatric ward, and serum blanks are required if an exact assay is desired. References 1 2 3 4 5 6 7 8 9 10 11 12

Svennerholm. L. (1957) Biochim. Biophys. Acta 24.604-611 Cassidy. J.T., Jourdian. G.W. and Roseman, S. (1965) J. Biol. Chem. 240,3501-3506 Whitehouse, M.W. and Zilliken. F. (1960) Methods Biochem. Anal. 8.199-220 Werner. I. and Odin, L. (1952) Acta Sot. Med. Ups. 57, 230-241 Svennerholm. L. (1957) Ark. Kemi 10. 577-583 Barry, G.T.. Abbott, V. and Tsai, T. (1962) J. Gen. Microbial. 29, 335-352 Jourdian. G.W., Deane. L. and Roseman, S. (1971) J. Biol. Chem. 246. 430435 Warren, L. (1959) J. Biol. Chem. 234,1971-1975 Aminoff. D. (1961) Biochem. J. 81,384-392 Comb, D.C. and Roseman, S. (1960) J. Biol. Chem. 235,2529-2537 Brunetti, P., Swanson, A. and Roseman, S. (1963) Methods Enzymol. 6,465-473 Taniuchi. K., Miyamoto, Y., Uchida.‘Y., Chifu. K., Mukai, M.. Yamaguchi, Y.. Tsukada, Y., S T.. Doi, K. and Baba, S. (1979) Japan J. Clin. Chem. 7.403-410 (in Japanese) 13 Uchida, Y., Tsukada. Y. and Sugimori, T. (1977) J. Biochem. 82.1425-1433