JOURNAL OF PATHOLOGY, VOL. 146: 333-336 (1985)

LIPID IN THE HUMAN GALLBLADDER MUCOSA. A HISTOCHEMICAL STUDY BY

LIGHT AND ELECTRON MICROSCOPY MARTIN ENGLISH AND DAVID HOPWOOD

Department of Pathology. Ninewells Hospital and Medical School, Dundee, Scotland. U.K.

Received 14 August 1984 Accepted 17 January 1985

SUMMARY The distribution of lipids in the epithelium of 70 gallbladders removed for cholecystitis was investigated histochemi-

cally. Lipid was demonstrated in 68 gallbladders-cholesterol and its esters, triglycerides, free fatty acids and phos- pholipids. Neutral lipid was found at the bases of epithelial cells in 90 per cent and at the apex in 10 per cent of the gallbladders. Phospholipid was shown at the apex of epithelial cells in 64 per cent of the gallbladders. Electron microscopy in 12 specimens revealed cholesterol in and between the epithelial cells and in the underlying connective tissue. KEY wom--Gallbladder mucosa, surgical specimens, lipid histochemistry, electron microscopy.

INTRODUCTION

Over 100 years ago Virchow noted that lipids can accumulate in the gallbladder epithelium. More recently, it has been shown, using isotopic tech- niques in animals, that this epithelium can absorb phospholipids, cholesterol and free fatty a ~ i d s . ~ - ~ It can also synthesize sterols and triglycerides de novo.s We have recently shown by biochemical tech- niques that human gallbladder can take up free fatty acids, triglycerides and chole~tero l .~ .~ Published histochemical studies of the lipid in human gallblad- der mucosa have been confined to cholesterolotics or to a small series of patients with chronic chole- cystitis.* We decided that the histochemical dis- tribution of lipids should be described as the morphological counterpart to these biochemical investigations.

PATIENTS, MATERIALS AND METHODS

Gallbladders (70) surgically removed for chole- cystitis from patients (mean age 59 years, range 7- 92 years) were immediately opened in a theatre anteroom and fixed in phosphate buffered 4 per cent

0 1985 by John Wiley & Sons, Ltd. 0022-341 7/85/080333-04$0 1 .OO

formaldehyde. Frozen sections (8 pm) were fixed further in Baker's formal calcium. They were then stained by standard techniques for neutral lipids, phospholipids, free fatty acids, triglycerides and cholesterol e s t e r ~ . ~ J ~ At least two sections were examined from each histochemical technique. Six cystic duct lymph nodes were examined by the same techniques.

The gallbladders were divided into three groups histopathologically : (i) mild chronic cholecystitis ; (ii) marked chronic cholecystitis with significant loss of mucosal folds, thickening of muscle coat and significant inflammatory infiltrate; (iii) cholesterolosis.

To demonstrate free cholesterol ultrastruc- turally, 1 mm cubes of mucosa from 12 gallbladders received directly from theatre were fixed in the presence of digitonin." Stained thin Epon sections were examined with a Jeol lOOCX electron micro- scope at 60 kV.

Controls

Normal tissue for comparison with the diseased gallbladders is difficult to acquire as locally the number of organ donors each year is small. Sections

334 M. ENGLISH AND D. HOPWOOD

Table I-Distribution of lipid in epithelial cells ~~~

Basal Apical Free neutral neutral Apical fatty Cholesterol lipid lipid phospholipid acids esters Trig1 ycerides

Marked Positive 39 4 30 3 2 3 cholecysti tis

(n) 44 44 44 13 2 3

Mild Positive I I 0 9 2 0 1 cholecystitis

(n) 12 12 12 2 1 2

. Cholesterolosis Positive 14 3 6 3 3 0

(n) 14 14 14 4 4 3

(n)-Number ofcases examined.

from each case were examined without staining. For the digitonin-cholesterol technique, some blocks were extracted first with chloroform-methanol and others were fixed without digitonin.

RESULTS AND DISCUSSION

The results of the histochemical study of lipids are shown in Table I. Visual assessment showed no difference in the distribution of lipid between the various classes of gallbladder. This suggests that

Fig. 1 -Marked cholecystitis. A number oflipid droplets stained by Oil Red 0 can be seen mostly at the bases of the epithelial cells (arrows). Lipid droplets are also seen in various structures in the underlying lamina propria, including inflammatory cells. x 350

Fig. 2-Cystic duct lymph node showing lipid droplets in macrophages and some incorporated in residual bodies. Uranyl acetate and lead citrate. x 5000

GALLBLADDER MUCOSAL LIPIDS 335

cholesterolosis is not caused by abnormal absorp- tion of lipids. Biochemical studies (unpublished) confirmed the presence of the various lipids demon- strated histochemically. Although there was a marked increase in neutral lipids in the lamina pro- pria in severe cholecystitis (Fig. 1) inflammatory cells including macrophages may contain at least a portion of this lipid. The distribution of lipid in the epithelium corresponds with previous descriptions: including those for cholesterolosis. 12-14 The cystic duct lymph node which was not always present in the surgical specimen, showed variable amounts of neutral lipids and phospholipids in macrophages (Fig. 2).

The digitonin technique for free cholesterol showed two sorts of change compared with control tissue. Firstly, there was focal enhancement of the cell membrane in the form of arcades which has been noted in various tissues previously.’5 Secondly, we found spicules which are believed to represent the cholesterol. * These were both cytoplasmic and in the basolateral cell space from the apex to the basement membrane and across into

the subjacent lamina propria (Fig. 3). This suggests a route for the removal of absorbed cholesterol. We have seen a similar pathway in guinea pig3 in pre- liminary experiments with oleic acid.16 The lipids are then removed either through venous channels draining directly into the liver or through lymphatic channels.

The accumulation of lipids in the gallbladder may be a normal species phenomenon as is seen in the dog and cat.” Alternatively, it may arise from excessive absorption from the bile as an epi- phenomenon of cholecystitis. A similar accumula- tion of lipid may be seen in small intestinal epithelium when challenged by a large lipid load.

Finally, the lipid droplets may represent impared onward transport from the epithelium following normal absorption from the bile. This may involve an apolipoprotein.

We have recently established an Ussing chamber technique to study lipid transport across human gallbladder. l8 This apparatus consists of two com- partments, perfused with physiological saline, which are separated by an interposed disc of

Fig. 3-Tissue fixed in the presence of digitonin from a patient with marked cholecystitis. The bases of two epithelial cells (E) abut onto the reduplicated basement membrane (B). There are a number of parallel crystals or needles (arrows) in the epithelial cytoplasm and in the underlying lamina propria (C) . x 60 000

336 M. ENGLISH AND D. HOPWOOD

mucous membrane and is widely used in physiologi- cal studies of secretion and absorption. 19~20 Using this approach it should be possible to explain the accumulation of lipid in human gallbladder.

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