Fallet! et al.: α,-Acid glycoprotein in hepatocellular carcinoma 407

Eur. J. Clin. Chem. Clin. Biochem.Vol. 31, 1993, pp. 407-411© 1993 Walter de Gruyter & Co.

Berlin - New York

Increase of Serum αϊ-Acid GlycoproteinDespite the Decline of Liver Synthetic Functionin Cirrhotics with Hepatocellular Carcinoma

By E. Falleti1. M. Pirisi2, C. Fabris2, Nadia Bortolotti*, G. Soardo2, P. Toniulf o2, F. Gonanol and E. Bar toll2

1 Cattedra di Patologia Clinica,2 Cattedra di Medicina Interim,Universit degli Studi, Udine, Italia

(Received January I/April 15, 1993)

Summary: α,-Acid glycoprotein, an acute phase reactant synthesised by the liver, has been reported to beincreased in neoplastic conditions and reduced in chronic liver disease. We measured serum aracid glycoproteinby a nephelometric method in 186 subjects (112 males, 74 females): 55 had mild chronic liver disease (chronichepatitis and steatofibrosis), 45 cirrhosis, 38 hepatocellular carcinoma, 15 extra-hepatic malignant disease; 33healthy subjects were used as controls. Analysis of variance demonstrated a significant variability amonggroups (F = 17.08, P = 0.0000). Higher concentrations of ocj-acid glycoprotein were detected in malignantextra-hepatic disease than in all other groups (P < 0.01); concentrations of o^-acid glycoprotein were higherin hepatocellular carcinoma than in cirrhosis (P < 0.01). Multiple regression analysis by groups (dependentvariable = αϊ-acid glycoprotein; group 1 = mild chronic liver disease + cirrhosis; group 2 = hepatocellularcarcinoma) showed a significant correlation for both group 1 (r = 0.6264, F = 8.005, P = 0.0000) and group 2(r = 0.8947, F = 13.643, P = 0.0000). The significant standardised regression coefficients were: cholinesterase,C-reactive protein, γ-glutamyltransferase and iron (negative) for regression upon group 1; C-reactive protein,aj-antiproteinase, γ-glutamyltransferase, iron (negative) for regression upon group 2. A difference betweenthe 2 regression equation coefficients was detected (F = 5.209, P = 0.0002). In conclusion, a raised aracidglycoprotein concentration was found in patients with malignancies, both hepatic and extra-hepatic; however,patients with hepatocellular carcinoma had a lower aj-acid glycoprotein concentration than patients withmalignant extra-hepatic disease. In hepatocellular carcinoma, a good correlation existed between aracidglycoprotein and other serum indices of the acute phase response. In some patients with hepatocellularcarcinoma, at-acid glycoprotein was increased, despite the decrease of liver function.

Introduction

at-Acid glycoprotein is a circulating glycoprotein syn-thesized mainly in the liver. It has a relative molecularmass of about 41000 and is characterized by thepresence of five glycosylation sites (1, 2). arAcidglycoprotein has long been recognized as an acutephase reactant (3). Thus, its concentration increases2—10 fold in serum, under conditions that promotethe increase of other acute phase proteins such as arantiproteinase and C-reactive protein; moreover, itssynthesis in human adult hepatocytes is augmented

Eur. J. Clin. Chem. Clin. Biochcm. / Vol. 31,1993 / No. 7

by interleukin-1 and interleukin-6, cytokines that areconsidered the main mediators of the acute phaseresponse (4).

Previous studies have shown that the at-acid glyco-protein concentration rises in serum during the courseof several neoplastic diseases (5 — 7), including hepa-tocellular carcinoma in Asian patients (8).

However, hepatocellular carcinoma has a striking ge-ographical variation and in industrialised countries,where its incidence is relatively low, hepatocellular

408 Failed et al.: ocrAcid glycoprotein in hepatocellular carcinoma

carcinoma develops almost always in patients withlong-standing cirrhosis. In contrast, in high incidenceareas (e. g. Far-East Asia) hepatocellular carcinomais common even in the absence of cirrhosis (9). Unlikeother acute phase reactants (like arantiproteinase),aracid glycoprotein does not increase in advancedchronic liver disease and may be actually markedlydecreased in such conditions (10 — 13). In this context,the question arises as to whether the variations of aracid glycoprotein concentration in the acute phaseresponse to neoplasia is influenced by the decline ofthe hepatic synthetic function, which accompanies thedevelopment of hepatocellular carcinoma in a cir-rhotic liver. The aim of the present study was toascertain the existence or otherwise of such a patho-physiological interaction.

Materials and Methods

Pat ientsWe studied a total of 186 subjects (112 male and 74 female,mean age 54.5 ± 14.2 years), divided in 5 categories accordingto their diagnosis; their characteristics are listed in table 1. Thefollowing criteria used for the definition of the categories.Mild chronic liver disease was defined, on the histologicalevidence of percutaneous liver biopsy, as a) chronic persistenthepatitis, b) chronic active hepatitis, or c) steatofibrosis.Cirrhosis was diagnosed on the basis of clinical evidence (hy-poalbuminaemia, hypergammaglobulinaemia, ascites, oesopha-geal varices) and/or histological evidence of cirrhosis; devel-opment of primary liver cancer in patients belonging to thisgroup was excluded on the basis of negative results of diagnosticimaging and biohumoral (ocrfetoprotein) tests.Conversely, hepatocellular carcinoma, which in our patientsalways developed in the setting of long-standing liver cirrhosis,was always confirmed histologically or at autopsy.

Malignant disease of extra-hepatic origin was also diagnosedhistologically or at autopsy; more precisely, this group con-tained 5 patients with pancreatic carcinoma, 5 with gastriccarcinoma, 1 with oesophageal carcinoma, 1 with ovarian can-cer, 1 with lung cancer, 1 with high-grade non-Hodgkin lym-phoma, 1 with malignant pheochromocytoma. Eight of thesepatients had evidence of hepatic metast ses at the time serumwas collected for the present study.

Healthy blood donors were used as a control group.

Biochemical de terminat ionsSera were immediately stored at -20 °C after collection andkept frozen until use. At the time the determination of a,-acidglycoprotein was performed, sera h$d been stored for a maxi-mum of 12 months. arAcid glycoprotein was determined si-multaneously in all sera with a nephelometric method (BNA,Behring, Germany). Intra-assay and inter-assay coefficients ofvariations were 2.0% and 3.5%, respectively.All other determinations were performed with standard com-mercial kits.

Statistical analysisOne-way analysis of variance was applied to detect differencesin the population means with regard to aracid glycoproteinconcentrations. BonferronCs test was used for multiple com-parisons among the groups. One-way analysis of covariancewas used to exclude the possibility that variations of renalfunction (measured by serum creatinine) might exert a decisiveinfluence on ocracid glycoprotein concentration. The PearsonX2-test was used to test the existence of differences among thegroups with regard to categorized variables (such as normal orpathological αϊ-acid glycoprotein concentration). Multiple re-gression analysis was utilized to investigate the correlation ofserum aracid glycoprotein variations with variations of severalother biohumoral conditions. The analysis was performed onthe patients as a single group, as well as on 2 subgroups (firstsubgroup: chronic liver disease H- cirrhosis; second subgroup:hepatocellular carcinoma). In order to detect differences in theslopes or the intercepts between the regressions of the subroups,analysis of variance of the regression coefficients was per-formed. In view of their scattered distribution, the results ofoci-acid glycoprotein and C-reactive protein determinations weretransformed logarithmically when appropriate. All statisticaltests were performed with the BMDP™ statistical softwarepackage (14).

Results

Figure 1 presents the individual values of serum ocracid glycoprotein measured in the various groups ofpatients. Using a cut-off value of 1 g/1, we detected asignificant difference between the observed and theexpected frequency of pathological αϊ-acid glycopro-tein values (Pearson x24est 65.93, Ρ = 0.0000). α,-Acid glycoprotein was higher in patients with malig-nancies (both hepatic and extra-hepatic). One-wayanalysis of variance demonstrated the existence of asignificant difference among groups (F = 17.08,Ρ = 0.0000). Patients with malignant extra-hepatic

Tab. 1. Characteristics of the studied population.

Healthy controlsChronic liver diseaseLiver cirrhosisHepatocellular carcinomaExtra-hepatic malignancies

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3355453815

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1531223410

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1824230405

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41.8 ± 14.751.0 + 13.1 '58.3 ± 11.0*62.6 +11.662.8 ± 08.1

Age range(a)

20-6821-7336-7633-8043-77

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Fallet! et al.: arAcid glycoprolein in hepatocellular carcinoma 409

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Fig. 1. Individual values of aracid glycoprotein grouped according to diagnosis. The doited line represents the upper referencelimit (1 g/1).

Tab. 2. Multiple regression analysis. The analysis was performed using aracid glycoprotein as the dependent variable andcholinesterase, C-reactive protein, ocj-antiproteinase, γ-glutamyltransferase and iron as predictor variables. The regressionanalysis was performed on the total collective, as well as on separate groups.

Regression Multiple r Multiple r2

All patientsChronic non-neoplastic liver diseaseHepatocellular carcinoma

0.61400.62640.8947

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disease had higher ovacid glycoprotein concentra-tions than all other groups (P < 0.01); patients withhepatocellular carcinoma had higher concentrationsthan cirrhotics (P < 0.01) (Bonferronfs test for pair-wise comparisons). Table 2 shows the results of themultiple regression analysis, taking serum o^-acid gly-coprotein as the dependent variable, and C-reactiveprotein, oci-antiproteinase, cholinesterase,1) γ-glutamyltransferase1) and iron serum concentrationsas predictor variables. The analysis was performedfor the subjects as a single collective, as well as bygroups. In the analysis of the single collective, thestandardized regression coefficients of all predictorvariables were found to be significant: oci-antiprotei-nase (T = 3.48, P = 0.00), C-reactive protein(T = 3.60, P = 0.00), cholinesterase (T = 4.20,P = 0.00), γ-glutamyltransferase (T = 2.07,

!) Cholinesterase (EC 3.1.1.8)γ-Glutamyl transpeptidase (EC 2.3.2.2)Sialyltransferase (EC 2.4.99.6)

P = 0.04) and iron (T = -2.93, P = 0.00). In theanalysis of results from patients with chronic, non-neoplastic liver disease, significant standardized re-gression coefficients were found for iron (T = —3.12,ρ = 0.00), C-reactive protein (T = 2.51, P = 0.01),γ-glutamyltransferase (T = 2.22, P = 0.03) and cho-linesterase (T = 3.26, P = 0.00). In the analysis ofresults from patients with hepatocellular carcinoma,significant standardized regression coefficients werefound for arantiproteinase (T = 7.54, P = 0.00), C-reactive protein (T = 4.01, P = 0.00), γ-glutamyl-transferase (T = 2.12, P = 0.05) and iron(T = -2.75, P = 0.01). The analysis of variance ofregression coefficients over groups was also statisti-cally significant (F = 5.209, P = 0.0002). Finally, se-rum creatinine displayed a significant correlation withocracid glycoprotein (r = 0.170, P = 0.024). How-ever, analysis of covariance enabled us to prove thata significant variability among groups with regard tooct-acid glycoprotein concentration still existed afteradjustment for creatinine (F = 13.16, P = 0.000).

Eur. J. Clin. Chem. Clin. Biochem. / Vol. 31,1993 / No. 7

410 Fallet! et al.: arAcid glycoprotein in hepatocellular carcinoma

Discussion

In the present study, the great majority of ourpatientswith extra-hepatic malignant disease (86.7% of thisgroup) had an αϊ-acid glycoprotein concentrationabove a cut-off value of 1 g/1 (representing mean+ 2 SD of our control subjects). Moreover, this groupof patients exhibited the highest aracid glycoproteinvalues. In contrast, only 34.2% of patients with he-patocellular carcinoma showed a,-acid glycoproteinconcentrations above the cut-off. Although oci-acidglycoprotein was significantly increased in hepatocel-lular carcinoma in comparison to cirrhosis, it was alsosignificantly lower in comparison with other neoplas-tic diseases. Patients with chronic, non-malignant liverdisease did not differ from controls with respect toαϊ-acid glycoprotein.

Serum concentrations of glycoproteins can be influ-enced by the mass of functional liver in various ways.Firstly, an impairment of hepatic synthetic abilitymight lead to a decrease of their production, sincethey are mostly synthesized by the liver. In effect, ourdata indicate that the aracid glycoprotein concentra-tions of patients with chronic, non-neoplastic diseaseof the liver show a positive correlation with cholin-esterase, one of the more reliable indices of liversynthetic function. However, the liver is also the mainsite of glycoprotein catabolism, which depends onuptake through specific surface receptors which rec-ognize asialoforms of circulating glycoproteins (15).a,-Acid glycoprotein appears to have a higher clear-ance rate than other glycoproteins expressed duringthe acute phase response, like ceruloplasmin and hap-toglobin (16). Therefore, serum aracid glycoproteinconcentration might increase because less is taken upby the reduced hepatic mass (17). We found similararacid glycoprotein concentrations among patientswith chronic non-neoplastic liver disease and controlsubjects. A possible explanation of this finding mightbe the existence in the organism of a balance that ismaintained between reduced synthesis and impairedcatabolism of this glycoprotein. However, in the pres-

ence of an acute phase response, as in neoplasticconditions, a raised proportion of glycoprotein sialo-forms (not recognized by liver cell receptors whichare able to recognize only asialoforms, as notedabove) appears in the serum (18,19). Thus, one mightspeculate that the equilibrium that seems to exist inchronic, non-neoplastic liver disease could be dis-rupted by the appearance of an increased proportionof sialo-forms of ai-acid glycoprotein, possibly relatedto increased sialyltransferase1) activity (13, 19—23).In accordance with this hypothesis, aracid glycopro-tein showed a strong correlation with other acutephase indices (arantiproteinase, C-reactive proteinand iron) in the group of patients with hepatocellularcarcinoma.

Our data are only in partial agreement with previousreports (8). The incidence of pathologically elevatedoci-acid glycoprotein concentrations was not as highas that previously described in hepatocellular carci-noma in Asian patients. Geographical variations inthe clinical presentation of hepatocellular carcinomamight explain this discrepancy, since our patients wereall Italian and developed primary liver cancer alwaysin the setting of long-standing cirrhosis. A correctinterpretation of these results in relation to earlierconflicting studies is limited by the fact that we couldnot test sera of patients with hepatocellular carcinomaof different geographical origin. The existence of thesedifferences may, however, represent indirect confir-mation of the hypothesis that the decline of liversynthetic function limits the rise of aj-acid glycopro-tein serum concentration in patients with the type ofhepatocellular carcinoma commonly encountered inour country.

In conclusion, aracid glycoprotein was found to beelevated in hepatocellular carcinoma, but it was notas high as in patients with extra-hepatic tumours. Agood correlation existed between aracid glycoproteinand other serum indices of the acute phase response;in this setting, it appears that at-acid glycoprotein isable to increase despite a decline of liver function.

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Dr. Mario PirisiCattedra di Medicina TnternaUniversitä degli StudiPiazzale Santa Maria della Misericordia 11-33100 UdineItaly

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