Lentil-Lectin-Reactive Alpha-Fetoprotein inthe Differential Diagnosis ...

CLIN. CHEM. 32/11, 2083-2084

(1986)

Lentil-Lectin-Reactive Alpha-Fetoprotein Benign and Malignant Liver Disease Paul K. Buamah,1

Ronald

Oliver F.W. James,2 and Andrew

Harris,1

Affinity chromatography of serum on lentil lectin bound to Sepharose 4B has been used to identify different forms of alpha-fetoprotein in serum of patients with liver disease. We studied eight patients with non-malignant liver disease, finding that 7-15% of the serum alpha-fetoprotein bound to the lectin. In contrast, for 15 patients with malignant liver disease, 25-83% of the serum aipha-fetoprotein bound to the

lectin. Evidently, this simple technique can be used effectively to differentiate these two conditions. AddItional chronic

Keyphrases: active

affinity chromatography primary hepatocellular

hepatitis

metastatic liver disease disease

Liver

such

isoprotein as cirrhosis

cirrhosis carcinoma

cancer or chronic

active

hepatitis,

where there is no evidence of malignancy but where hepatic regeneration occurs, can lead to an increased concentration of alpha-fetoprotein (AFP) in serum. Malignant liver disease, either primary hepatocellular carcinoma or metastatic liver disease, is usually associated with a much greater increase in the AFP concentration in serum. There is no clear-cut serum AFP concentration that is characteristic of benign or malignant liver disease, and thus this assay is of little

value

(1). The serum

in

the differential

in the Differential Diagnosis of

diagnosis

of hepatic

disease

concanavalin A binding characteristics of AFP in afford a means of differentiating between the AFP

from primary hepatocellular carcinoma and metsstatic liver disease (2), but this technique is ineffective in distinguishing between the AFP produced in benign liver disease from that found in malignant liver disease. Affinity chromatography on lentil lectin, which has an affinity for glycopeptides with both a fucose residue and N-acetylglucosamine (3), has been used as a means of achieving the latter separation, and we present our preliminary findings.

of serum was loaded onto a (0.5 x 10 cm) column of lectin-Sepharose, which had been previously equilibrated with Ti-is HC1 (50 mmoIJL, pH 7.2) containing 150 mmol of NaC1, 1 mmol of MgCl2, and 1 mmol of CaCl2 per liter. The column was first eluted with 10 mL of the same buffer and then with 20 mL of Ti-is-buffered isotonic saline containing 0.5 mol of methyl-i-mannopyranoside per liter. The column was then regenerated with 50 mL of the Ti-isbuffered saline. We collected 0.5-mL fractions directly into plastic tubes and estimated the AFP concentrations in them by using an enzyme immunoassay kit from Abbott Diagnostics Limited (Wokingham, Berkshire). The non-reactive (unbound) fraction was designated I and the reactive (retarded) fraction II. Analytical recovery of AFP from the columns ranged from 95 to 106% (mean 98%). The AFP that was bound to the column was termed the “lentil-lectinaliquot lentil

reactive” total

and Method

Serum was obtained

from eight patients with benign liver chronic active hepatitis and six with cirrhosis) 15 patients with malignant liver disease (six metastatic liver disease and nine with primary hepatocellular carcinoma). The serum samples were collected disease

(two

with and from

before stored

the histology was established, and samples were at -20 #{176}C until analysis. The nature of the disease confirmed by histological examination of biopsy speci-

was

mens. We did the affinity using

lentil

Co. Limited,

‘Department

lectin

Poole,

chromatography bound to Sepharose Dorset

of Clinical

BH17

Biochemistry High Heaton,

at room temperature, 4B (Sigma Chemical 7NH, U.K.). An 0.1-mL

and2 Department of MediNewcastle upon Tyne, NE7

cine, Freeman Hospital, 7DN, U.K. 3Department of Clinical Biochemistry, The Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, U.K. Received June 17, 1986; accepted August 6, 1986.

form

and

was

expressed

of the

as a percentage

AFP.

Results Table 1 summarizes the percentage patients

with

the AFP

in the

concentration

lentil-lectin-reactive

malignant

liver

disease

disease. The AFP concentrations grams per liter, with use of IARC on Cancer)

Research

Table

arising

Patients

W. Skillen3

AFP

in serum

are expressed (International

reference

in

1. Lentli-Lectln-Reactlve AFP or Malignant

micro-

Agency

preparation

Patients with Benign

and

form for the and benign liver for

72/225

as

In Serum of 23 Liver Disease

AFP Total

Sex

LentIl

lectin

jcig/L

%

Disease

1265

65

PLC

2980 2167 1040 6308

83 39

PLC

67

1950 3600 5600 2520 14500

74

1280

780

61

PLC PLC PLC PLC

F

63

PLC

59 63 56

1466 6233 258

60

M M M

2460 7900 435

79

428

106

59 25

PLC PLC MLD

F F F M

34 40 36 65

369 130 17800 430

95 97 6372 110

26 75 36 26

MLD MLD MLD MLD

M

920 2150 161

376

41

MLD

140

7

C

F

67 83 32

23

14

CAH

M M

62 68

435 389

62 58

14 15

C

M

40

F

83

139 1710

18 253

13 15

C C

F

56

1495

F

35

145 37

10 10

CAH

Age,

M

75

M F F

71 59 49

M M

F

Concn,

reactive

y

364

PLC. primary liver cancer; and C, cirrhosis.

M LD, metastic

41 43

C

C

liver dise ase; CAN, ch ronic active

hepatitis;

CLINICAL CHEMISTRY,

Vol. 32, No. 11, 1986

2083

C

PRIMARY

HEPATOCELLULAR

CARCINOMA

APP

pill.

S

CIRRHOSIS

A,,

.10.

A

SIRONK

ACTIVO

MOTAITATIC

LIVIN

DISEAAO

‘PP

HRPATITIS

.18.

II

ISO

‘SO

S0 i0 40

a

S0

3

5

7

5

Il

13

0MM.,

IA

It

II

SI

2

24

2t

l,MM..,

#{163}1.11.,I,tMn

Fig. 1. Representative elution patterns of serum AFP from patients and (L metastatic liver disease I, non-reactive (unbound) fraction; II, reactive (retarded) fraction

with (A) chronic active hepatitis, (

cirrhosis,

(C

primary

hepatocellular

carcinoma,

the standard, the upper limit of the normal reference interval being 10 jg/L. The percentage of AFP in serum that reacted with the lentil lectin ranged from 25 to 83% (mean 51%, median 42%) in malignant liver disease and from 7 to 15% (mean 12%, median 14%) in benign liver disease. Statistical analysis (Mann-Whitney) differences in reactivity of AFP towards

lentil

showed lectin

these

liver

to be

the sugar chain as the molecular basis for this heterogeneity of AFP (6), and our own studies have shown that the test is useful in monitoring the efficacy of chemotherapy in patients with primary liver cancer (7). We believe that the estimation of this fraction may be useful in detecting primary liver cancer at an early stage, particularly in patients with chronic liver diseases associat-

highly

significant (p 0.02). Figure 1 compares representative elution patterns for serum

AFP

sorts of liver-disease

from various

(39-83%, mean 59%), this difference was not statistically significant. This preliminary study has shown that estimation of the lentil-lectin-reactive isoprotein of AFP is a useful test for differentiating between patients with benign and malignant

patients.

disease.

ed with

Previous

a malignant

work

has identified

flicosylation

of

transformation.

Discussion Several investigations have shown that there are isoproof AFP with different lectin-binding affinities. Some

thins

have reported that estimation of concanavalin A non-reactive isoprotein of AFP in amniotic fluid provides a useful means for the and Buamah nique affords with primary

prenatal detection of neural tube defects et al. (2) have demonstrated that this a means

of distinguishing

between

(4,5),

tech-

patients

hepatocellular carcinoma and hepatic secondaries. The two groups of patients could not be differentiated on the basis of their serum AFP concentrations, for there is considerable overlap of values (Table 1). The lentil-lectinreactive AFP isoprotein, however, clearly distinguishes between the two groups of patients. With patients with benign liver disease the lentil-lectin-reactive isoprotein comprised less than 16% of the total AFP, whereas with patients with malignant liver disease the lentil-lectin-reactive fraction exceeded 24% (Table 1). Although the lentil-lectin-reactive

form was a smaller proportion of the total AFP in the case of patients with metastatic liver disease (26-75%, mean 38%) than was true for those with primary hepatocellular cancer

2084

CLINICAL

CHEMISTRY,

Vol. 32, No. 11, 1986

References Y, Isemura M, et al. Differential reactivity of lectins and evaluation of its usefulness in the diagnosis of hepatocellular carcinoma. Gann 1984;75:809-15. 2. Buaniah PK, Gibb I, Bates G, Milford Ward A. Serum alpha fetoprotem heterogeneity as a means of differentiating between primary hepatocellular carcinoma and hepatic secondaries. Clin Chim Acts 1984;139:313-6. 3. Kornfeld K, Reitman ML, Kornfeld R. The carbohydrate-binding specificity of pea and lentil lectins. Fucose is an important determinant. J Biol Chem 1981;256:6633-40. 1. Aoyagi

Y, Suzuki

a-fetoprotein

4. Smith

with

(11, Kelleher PC, Belanger L, Dallaire fluid alpha-fetoprotein with concanavalin of neural tube defects. Br Med J 1979;i:920-1. amniotic

L. Reactivity of A in diagnosis

5. Buamah PK, Taylor P, Milford Ward A. Concanavalin A binding of a-fetoprotein in amniotic fluid as an aid in the diagnosis of neural

tube defects. Clin Chem 1981;27:1658-60. 6. Aoyagi Y, Isemura M, Yosizawa Z, et al. Fucosylation

of serum a-fetoprotein in patients with primary hepatocellular cancer. Biochim Biophys Acts 1985;830:217-23. 7. Buamah PK, Cornell C, Cassells-Srnith AJ, Harris AL. Fucosylation of a-fetoprotein in hepatocellular carcinomas. Lancet

1986;i:922-3.