Evaluation of three assays for the determination of serum total sialic acid

Clin Biochern. Vol. 26, pp. 4 4 9 - 4 5 4 . 1993 Printed in the USA. All rights reserved.

0009-9120/93 $6.00 + .00 Copyright e 1993 The Canadian Society of Clinical Chemists.

Evaluation of Three Assays for the Determination of Serum Total Sialic Acid M. C R O O K , Department

of Clinical

Chemistry,

M. H A Q , a n d P. T U T T

5th Floor Tower,

There is increasing clinical interest in the measurement of serum total sialic acid (TSA). There are a number of methods available but with little data suggesting the preferred technique. We evaluated three serum sialic acid assays: the Warren assay, the Svennerholm assay, and a commercially available enzymatic method. The analytical performances of the three assays were similar, except that the enzymatic assay had a greater analytical range than the other assays and better precision at low and high serum sialic acid concentrations. The enzymatic assay was readily automated for use on a Cobas Bio analyser but was substantially more expensive than the other two methods.

Guy's

Hospital,

London

SE1 9RT, England

present study was to compare these t hr e e assay methods for serum TSA for use in the clinical arena. Methods ASSAYS

Correspondence: Dr. M. Crook. Manuscript received February 1, 1993; revised May 19, 1993; accepted May 20, 1993.

The resorcinol method for analysis of sialic acid is that of Svennerholm (15). Briefly, this consisted of adding 50 p.L of serum to 1 mL of resorcinol re a g e n t composed of 10 mL of 2% (w/v) resorcinol, 9.75 mL of distilled water, 0.25 mL of 0.1 M copper(II) sulphate brought to a final volume of 100 mL with concentrated hydrochloric acid, in a capped tube. The mixture was vortexed and placed in a boiling water bath at 100 °C for 15 min. After cooling for 10 min in an ice bath, 2 mL of butylacetate/n-butanol m ix tu re (85:15, v/v) was added to the tube, vortexed, and then centrifuged for 10 min at 1200 × g. The extracted chromophore was then read at 580 nm. Standards ofN-acetyl neuraminic acid (type VIII Sigma Chemical Company Poole, Dorset, UK) in the range 0 - 4 . 8 mmol/L were similarly passed through the same procedure. The Warren method (16) for the analysis of serum TSA was carried out as follows. Fifty microlitres of serum was added to 0.95 mL of 0.05 M sulphuric acid and incubated at 80 °C for 1 h. After cooling in a 20 °C water bath for 5 min, a 100 ~L aliquot was transferred to a glass tube, to which was added 50 ~L of 0.2 M sodium periodate in 9 M phosphoric acid, followed after 20 min by 0.5 mL of a m i xtu re containing 0.8 M sodium arsenite, 0.5 M sodium sulphate, and 0.05 M sulphuric acid. When the yellowbrown coloration had disappeared 1.5 mL of 0.04 M thiobarbituric acid in 0.5 M sodium sulphate was added and the solution was heated in a boiling water bath (100 °C) for 15 min. After cooling to room temperature, 2.15 mL of cyclohexanone was added, the contents were vortexed and t hen centrifuged for 5 min at 2000 × g. The absorbance of the red upper cyclohexanone layer was t hen m e a s u r e d spectrophotometrically at 549 nm. Standards of N-acetyl neuraminic acid (type VIII, Sigma Chemical Co.) in the range 0 - 4 . 8 mmol/L were processed t hro u g h the same procedure. The enzymatic assay for serum TSA was per-

CLINICAL BIOCHEMISTRY,VOLUME 26, DECEMBER 1993

449

KEY WORDS: sialic acid; assay; serum.

Introduction here has been recent interest in the determina-

tion of serum total sialic acid (TSA). Serum TSA T has been shown to be a cardiovascular risk factor, with elevated levels associated with increased cardiovascular mortality (1,2) and cerebrovascular disease (3). F u r t h e r m o r e , serum TSA is raised in diabetes mellitus (4,5) particularly with certain diabetic complications including retinopathy (6). In addition, serum TSA is elevated in myocardial infarction (7,8) chronic glomerulonephritis (9), and in patients with hypertriglyceridaemia (10). Serum TSA has also been used as a t um our m a r k e r for certain cancers, including m a l i gna nt melanoma, breast carcinoma, and colon carcinoma (11-14). Although one might predict t ha t serum TSA will become increasingly measured in the clinical laboratory, there has been controversy as to the best assay for this purpose. A majority of studies to date have used colorimetric assays: either t ha t of Svennerholm (15) t h a t uses resorcinol or an adaptation of the Warren method (16) t h a t utilises thiobarbiturate. The former has been criticised on the grounds of lack of specificity for sialic acid due to interfering substances (17). A more recent innovation has been the introduction of an enzymatic assay for serum TSA, supposedly more specific for sialic acid, t hat is available as a commercial kit. The purpose of this

CROOK, HAQ, AND T U ~ Serum TSA mmol/L Warren 4

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formed using a kit supplied by Boehringer Mannheim (Lewes, UK) and made up as described by the manufacturer. Reagent A was composed of neur-

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aminidase and 4-aminoantipyrine dissolved in buffer solution. Reagent B consisted o f a solution of pyruvate oxidase, peroxidase, sialic acid aldolase,

Serum TSA mmol/L Enzymatic .

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CLINICAL BIOCHEMISTRY, VOLUME 26, DECEMBER 1993

ASSAY OF SERUM TSA

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Figure 3 -- Deming's regression for comparison of the Warren and enzymatic serum TSA assays. Y = 0.68 + 0.75X, r -0.628. FAD, and thiamine pyrophosphate also dissolved in the buffer solution. The buffer had a pH of 7.5; and c o n s i s t e d of KH2PO4, 20 mM, N - a c e t y l - N - ( 2 hydroxyethyl)-m-toluidine, 3 mM, and MgC12, 5 mM. The neuraminidase had a Vmax of 300 ~mo1/ mg/min and sialic acid aldolase one of 150 txmol/mg/ min. Standards of N-acetyl neuraminic acid (type VIII Sigma Chemical Co.) in the range of 0-6.4 mmol/L were similarly processed. Although sialic acid standards were supplied with the kit these gave identical values to those obtained with the N-acetyl neuraminic acid standards above. The method was set up for use on a Cobas Bio (Roche, Welwyn, UK) analyser as described by the manufacturer.

2.08 mmol/L) and to aliquots of this was added N-acetyl neuraminic acid at concentrations of 0.64, 1.28, and 1.92 mmol/L. The before-addition and after-addition serum sialic acid concentration was determined by each assay. The lowest measurable concentration of sialic acid for each assay was obtained by the method of MacDougall and Crummett (18) and the highest concentration of sialic acid measurable on undiluted samples by that of Buttner and co-workers (19). The effect of potential interfering substances was examined for bilirubin (0-350 ixmol/ L), haemoglobin (0-8.0 ~mol/L), and pyruvate (0318 txmol/L). Results

Samples

Forty hospital patient serum samples were randomly selected and serum TSA was assayed by each of the three methods. Linearity for each assay was compared by diluting serum samples (at least three different patient samples were used) 1/2, 1/4, and Vs with 0.15 mol/L NaC1. Inter-assay and intra-assay coefficients of variation (CV) were calculated using some of the patient samples or aqueous standards. Ten determinations were used to assess the intraassay CVs, each at three sialic acid concentrations (0.32, 2.08, 3.20 mmol/L). Inter-assay CVs for each assay were calculated using a sample sialic acid concentration of 2.46 mmol/L on three consecutive days. Recoveries for each of the assays was examined by taking a serum sample (sialic acid concentration of CLINICALBIOCHEMISTRY,VOLUME26, DECEMBER 1993

The mean serum TSA for the 40 patient samples assayed by the Svennerholm, Warren, and enzymatic methods, respectively, were 2.43 -+ 0.42 mmol/L (range 1.54-3.58), 2.53 -- 0.54 mmol/L (range 1.413.81), and 2.43 -+ 0.48 mmol/L (range 1.60-3.74). None of these results were significantly different TABLE 1 Intra-Assay Coefficients of Variation (CV%)

Sialic Acid Conc (mmol/L) Assay

0.32

2.08

3.20

Svennerholm Warren Enzymatic

12.9 19.0 4.9

1.7 2.1 2.5

6.1 8.2 3.9 451

CROOK, HAQ, A N D TUTT

sialic acid mmol/L 3.5

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Figure 4 - - Linearity data for the S v e n n e r h o l m serum TSA assay.

assay CV for the Svennerholm, Warren, and enzymatic methods were, respectively, 4.5, 3.1, and 4%. Figures 4 - 6 depict the linearity data for the three methods showing a representative patient sample. The recovery experiments for each assay showed a

from the other (Student's t-test p > 0.05). Figures 1 - 3 show regression analysis for these data calculated by the method of Deming (20). Table 1 depicts intra-assay CVs for the three sialic acid assays at three different sialic acid concentrations. The intersialic acid mmol/L 3.5

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CLINICAL BIOCHEMISTRY, VOLUME 26, DECEMBER 1993

A S S A Y OF S E R U M TSA

sialic acid mmol/L 3.5 jJ

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range of sialic acid recoveries from 96 to 105%. The lowest measurable sialic acid concentration for the Svennerholm, Warren, and enzymatic assays, respectively, was 0.12, 0.20, and 0.06 mmol/L. The highest sialic acid concentrations measurable without diluting the sample were 4.8, 4.8, and 6.4 mmoY L, respectively. The interference studies showed that for the Svennerholm method highly icteric samples (bilirubin of 350 ~moYL) gave a positive interference with.apparent serum sialic acid concentrations a b o u t 30% h i g h e r t h a n expected and for slightly icteric specimens (bilirubin concentration of 88 ~moYL) an apparent increase of 5%. The enzymatic method and the Warren method did not show such an occurrence. Furthermore, no obvious interferences was observed for any of the assays with haemolysed samples over the range studied or when the samples were spiked with pyruvate.

1.2

The data presented here show there is little to distinguish one serum sialic acid assay from another (remembering that all three assays were calibrated using a common batch of standards). The imprecision results were similar for each assay at serum sialic acid concentrations in the middle of the concentration range, but at low and high sialic acid concentrations the enzymatic assay clearly performed better than the Svennerholm and Warren assays (see Table 1). The linearity values (possibly with the exception of the Svennerholm assay) and recovery experiments were similar for the three assays. The

poor linearity of the Svennerholm assay means that caution should be exercised if diluted sialic samples are to be assayed for total sialic acid by this method. Furthermore, highly icteric samples m a y interfere with the Svennerholm method. The relatively poor precision data for the two colorimetric assays, that is, the Svennerholm and Warren methods, at low serum sialic acid concentrations, may not be important in practice as we have yet to encounter a human serum sialic acid concentration below 0.96 mmol/L. The enzymatic method has the advantage of ease of use and application to a wide range of automated equipment. However, at present prices the kit is expensive, being marketed at about $500 for 45 manual determinations, although this could be extended to about 300 determinations on the Cobas Bio. In comparison, the other methods are cheaper, although the Warren method does use sodium arseRite, a toxic compound that is becoming increasingly more difficult to purchase for safety reasons. Furthermore, both the Warren and Svennerholm methods involve the use of potentially dangerous organic solvents. It should also be noted that neuraminidase used in the enzymatic assay does not necessarily hydrolyse all sialo-conjugates, unlike acid hydrolysis that is thought to cleave all such bonds. Theoretically, therefore, assays involving n e u r a m i n i d a s e may be expected to give slightly lower total sialic acid results than those assays employing acid hydrolysis with heating. In-conclusion there is little to choose between the assays regarding their performance in measuring

CLINICAL BIOCHEMISTRY,VOLUME 26, DECEMBER 1993

453

Discussion

CROOK, HAQ, AND TUTT s e r u m sialic acid in the m i d d l e of the c o n c e n t r a t i o n range. H o w e v e r , as this a n a l y t e becomes increasingly m o r e i m p o r t a n t , in a clinical sense one would a s s u m e t h a t the a u t o m a t e d e n z y m a t i c a s s a y would be p r e f e r r e d to h a n d l e l a r g e n u m b e r s of p a t i e n t s a m ples quickly. References 1. Lindberg G, Eklund G, Gullberg B, Rastam L. Serum sialic acid concentration and cardiovascular mortality. Br Med J 1991; 302: 143-6. 2. Lindberg G, Eklund GA, Rastam L. Serum sialic acid concentration and smoking: A population based study. Br Med J 1991; 303: 1306-7. 3. Lindberg G, Rastam L, Gullberg B, Eklund GA. Serum sialic acid concentration predicts both coronary heart disease and stroke mortality: Multivariate analysis including 54385 men and women during 20.5 years follow up. Int J Epiderniol 1992; 21: 253-7. 4. Schvartz LS, Paukman LI. Diabetic angiopathies and mucopolysaccharide metabolism. Probl Endokrinol (Mosk) 1971; 17: 37-41. 5. Radhakrishnamurthy B, Berenson GS, Pargaonar PS. Serum free and protein bound sugars and cardiovascular complications in diabetes mellitus. Lab Invest 1976; 34: 159-65. 6. Crook M, Tutt P, Pickup JC. Serum sialic acid in noninsulin dependent diabetes mellitus. Diabetes Care 1993; 16: 57-60. 7. Hrncir Z, Pidrman V, Tichy M, Hamet A. Serum sialic acid in acute myocardial infarction in a dynamic follow up. Vnitr Lek 1975; 21: 436-9. (English Abstr) 8. Succari M, Foglietti MJ, Percheron F. Perchlorosoluble glycoproteins and myocardial infarct: Modifica-

454

9. 10. 11. 12.

13. 14.

15. 16. 17. 18. 19. 20.

tions of the carbohydrate moiety. Pathol Biol (Paris) 1982; 30: 151-4. Ozben T. Elevated serum and urine sialic acid in renal diseases. Ann Clin Biochem 1991; 28: 44-8. Crook M, Tutt P. Serum sialic acid concentration in patients with hypertriglyceridaemia. Clin Sci 1992; 83: 593-5. Mabry EW, Carubelli R. Sialic acid in human cancer. Experientia 1972; 28: 182-3. H o g a n - R y a n A, Fennelly JJ, Jones M, Cantwell B, Duffy MJ. Serum sialic acid and CEA concentrations in human breast cancer. Br J Cancer 1980; 41: 5 8 7 92. Horgan IE. Total and lipid-bound sialic acid levels in sera from patients with cancer. Clin Chim Acta 1982; 188: 327-31. Stefenelli N, Klotz H, Engel A, Bauer P. Serum sialic acid in malignant tumours, bacterial infections and chronic liver diseases. J Cancer Res Clin Oncol 1985; 109: 55-9. Svennerholm L. Quantitative estimation of sialic acids. Acta Chem Scand 1958; 12: 547-54. Warren L. The thiobarbituric acid assay for sialic acids. J Biol Chem 1959; 234: 1971-5. Flynn MD, Corrall RJ, Waters PJ, Pennock CA. Sialic acid and cardiovascular mortality (letter). Br Med J 1991; 302: 533-4. MacDougall D, Crummett WB. Guidelines for data acquisition and data quality evaluation in environmental chemistry. Anal Chem 1980; 52: 2242-9. Buttner J, Borth R, Boutwell JH. Provisional recommendation on quality control in Clinical Chemistry. Clin Chem 1976; 22: 532-9. Deming WE. Statistical adjustment of data. New York: John Wiley, 1943.

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